proteins and enzymes from marine...

Journal of Amino Acids

Guest Editors: Nabil Miled, Moncef Nasri, Hideki Kishimura, and Faouzi Ben Rebah

Proteins and Enzymes from Marine Resources

Journal of Amino Acids


Guest Editors: Nabil Miled, Moncef Nasri, Hideki Kishimura,and Faouzi Ben Rebah

Copyright © 2011 SAGE-Hindawi Access to Research. All rights reserved.

This is a special issue published in volume 2011 of “Journal of Amino Acids.” All articles are open access articles distributed under theCreative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided theoriginal work is properly cited.

Editorial Board

Fernando Albericio, SpainJordi Bella, UKSimone Beninati, ItalyCarlo Chiarla, ItalyKuo-Chen Chou, USAArthur Conigrave, AustraliaArthur J. L. Cooper, USAZbigniew Czarnocki, PolandDorothy Gietzen, USAAxel G. Griesbeck, Germany

Mario Herrera-Marschitz, ChileRiccardo Ientile, ItalyHieronim Jakubowski, USAMadeleine M. Joullie, USASambasivarao Kotha, IndiaRentala Madhubala, IndiaAndrei Malkov, UKYasufumi Ohfune, JapanNorbert Sewald, GermanyH. S. Sharma, Sweden

Vadim A. Soloshonok, USAImre Sovago, HungaryHeather M. Wallace, UKPeter W. Watt, UKRobert Wolfe, USAGuoyao Wu, USAAndreas Wyttenbach, UKNigel Yarlett, USA

Contents

Proteins and Enzymes from Marine Resources, Nabil Miled, Moncef Nasri, Hideki Kishimura,and Faouzi Ben RebahVolume 2011, Article ID 594646, 1 page

Digestive Alkaline Proteases from Zosterisessor ophiocephalus, Raja clavata, and Scorpaena scrofa:Characteristics and Application in Chitin Extraction, Rim Nasri, Islem Younes, Imen Lassoued,Sofiane Ghorbel, Olfa Ghorbel-Bellaaj, and Moncef NasriVolume 2011, Article ID 913616, 9 pages

Comparative Study on Biochemical Properties and Antioxidative Activity of Cuttlefish (Sepia officinalis)Protein Hydrolysates Produced by Alcalase and Bacillus licheniformis NH1 Proteases, Rafik Balti,Ali Bougatef, Nedra El Hadj Ali, Naourez Ktari, Kemel Jellouli, Naima Nedjar-Arroume, Pascal Dhulster,and Moncef NasriVolume 2011, Article ID 107179, 11 pages

Mackerel Trypsin Purified from Defatted Viscera by Supercritical Carbon Dioxide, Byung-Soo Chun,Hideki Kishimura, Sitthipong Nalinanon, Sappasith Klomklao, and Soottawat BenjakulVolume 2011, Article ID 728082, 7 pages

Simple Preparation of Pacific Cod Trypsin for Enzymatic Peptide Synthesis, Tomoyoshi Fuchise,Haruo Sekizaki, Hideki Kishimura, Sappasith Klomklao, Sitthipong Nalinanon, Soottawat Benjakul,and Byung-Soo ChunVolume 2011, Article ID 912382, 8 pages

Dicer Functions in Aquatic Species, Yasuko Kitagishi, Naoko Okumura, Hitomi Yoshida, Chika Tateishi,Yuri Nishimura, and Satoru MatsudaVolume 2011, Article ID 782187, 5 pages

Diversified Carbohydrate-Binding Lectins from Marine Resources, Tomohisa Ogawa, Mizuki Watanabe,Takako Naganuma, and Koji MuramotoVolume 2011, Article ID 838914, 20 pages

SAGE-Hindawi Access to ResearchJournal of Amino AcidsVolume 2011, Article ID 594646, 1 pagedoi:10.4061/2011/594646

Editorial


Nabil Miled,1, 2 Moncef Nasri,3 Hideki Kishimura,4 and Faouzi Ben Rebah2

1 LBGEL, ENIS, Route Soukra, Sfax 3038, Tunisia2 Biotechnology Department, ISBS, Route Soukra, Sfax 3038, Tunisia3 LGEM, ENIS, Route Soukra, Sfax 3038, Tunisia4 Faculty of Fisheries, Hokkaido University, Hakodate 041-8611, Japan

Correspondence should be addressed to Nabil Miled, [email protected]

Received 10 October 2011; Accepted 10 October 2011

Copyright © 2011 Nabil Miled et al. This is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Sea products are valuable resources of natural substancessuch as lipids, polysaccharides, enzymes, vitamins, andproteins. In this issue, a variety of fish proteases (trypsin,alcalase, etc.) have been characterized and sometimes puri-fied. Many of these enzymes display potentially interestingnew biochemical properties for industrial applications. Theirpotential adequacy for biotechnological applications such aschitin extraction was demonstrated. Biochemical character-istics of crude alkaline protease extracts from the visceraof goby (Zosterisessor ophiocephalus), thornback ray (Rajaclavata), and scorpionfish (Scorpaena scrofa) were studied.At least four caseinolytic proteases bands were observed inzymogram of each enzyme preparation. These proteolyticpreparations were successfully used in the deproteinizationof shrimp wastes for chitin extraction. Furthermore, atrypsin was purified from the pyloric ceca of Pacific cod(Gadus macrocephalus) using chromatographic methods.The cod trypsin was successfully applied to catalyze dipeptidesynthesis using series of “inverse substrates.” A Mackereltrypsin was also purified from defatted viscera by super-critical carbon dioxide, and its biochemical properties werestudied.

Recent biotechnological progresses require the researchof new bioactive peptides from natural resources that canbe used as an alternative to chemical products in variousapplications (food, cosmetics, drugs, etc.). Sea productsproteins digestion using bacterial proteases generated proteinhydrolysates displaying interesting biochemical propertiesand antioxidant activity. Antioxidative activities and bio-chemical properties of protein hydrolysates with differenthydrolysis degrees, prepared from cuttlefish (Sepia offic-inalis) using an Alcalase and Bacillus licheniformis NH1

proteases were determined. The antioxidant activities ofcuttlefish protein hydrolysates increased with increasing thehydrolysis degree. Antioxidative activity was concentrationdependent. Furthermore, both Alcalase and NH1 proteinhydrolysates were able to retard lipid peroxidation and β-carotene-linoleic acid oxidation. In addition, cuttlefish pro-tein hydrolysates have a high percentage of essential aminoacids and could be used as supplement to poorly balanceddietary proteins. Furthermore, carbohydrate-binding lectinswere also extracted from many marine resources.

This issue highlights the potential of sea products asvaluable resources of bioactive peptides and proteins suchas hydrolytic enzymes, lectins, and antioxidants. Theseresources might be of great interest for industrial biotech-nological processes using safe and natural materials such aspeptides or enzymes.

Dicer is an RNase III enzyme with two catalytic subunits,which catalyzes the cleavage of double-stranded RNA tosmall interfering RNAs and micro-RNAs, which are mainlyinvolved in invasive nucleic acid defense and endogenousgenes regulation. In addition, Dicer is thought to be involvedin defense mechanism against foreign nucleic acids such asviruses. A paper focused on the recent progress of Dicer-related research and discussed potential RNA interferencepathways in aquatic species. Dicer is abundantly expressedin embryos, indicating the importance of the protein in earlyembryonic development.

Nabil MiledMoncef Nasri

Hideki KishimuraFaouzi Ben Rebah

SAGE-Hindawi Access to ResearchJournal of Amino AcidsVolume 2011, Article ID 913616, 9 pagesdoi:10.4061/2011/913616

Research Article

Digestive Alkaline Proteases from Zosterisessor ophiocephalus,Raja clavata, and Scorpaena scrofa:Characteristics and Application in Chitin Extraction

Rim Nasri, Islem Younes, Imen Lassoued, Sofiane Ghorbel,Olfa Ghorbel-Bellaaj, and Moncef Nasri

Laboratoire de Genie Enzymatique et de Microbiologie, Ecole Nationale d’Ingenieurs de Sfax, P.O. Box 1173, 3038 Sfax, Tunisia

Correspondence should be addressed to Moncef Nasri, mon [email protected]

Received 27 March 2011; Accepted 15 June 2011

Academic Editor: Nabil Miled

Copyright © 2011 Rim Nasri et al. This is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

The aim of this work was to study some biochemical characteristics of crude alkaline protease extracts from the viscera ofgoby (Zosterisessor ophiocephalus), thornback ray (Raja clavata), and scorpionfish (Scorpaena scrofa), and to investigate theirapplications in the deproteinization of shrimp wastes. At least four caseinolytic proteases bands were observed in zymogramof each enzyme preparation. The optimum pH for enzymatic extracts activities of Z. ophiocephalus, R. clavata, and S. scrofa were8.0-9.0, 8.0, and 10.0, respectively. Interestingly, all the enzyme preparations were highly stable over a wide range of pH from 6.0to 11.0. The optimum temperatures for enzyme activity were 50◦C for Z. ophiocephalus and R. clavata and 55◦C for S. scrofa crudealkaline proteases. Proteolytic enzymes showed high stability towards non-ionic surfactants (5% Tween 20, Tween 80, and TritonX-100). In addition, crude proteases of S. scrofa, R. clavata, and Z. ophiocephalus were found to be highly stable towards oxidizingagents, retaining 100%, 70%, and 66%, respectively, of their initial activity after incubation for 1 h in the presence of 1% sodiumperborate. They were, however, highly affected by the anionic surfactant SDS. The crude alkaline proteases were tested for thedeproteinization of shrimp waste in the preparation of chitin. All proteases were found to be effective in the deproteinization ofshrimp waste. The protein removals after 3 h of hydrolysis at 45◦C with an enzyme/substrate ratio (E/S) of 10 were about 76%,76%, and 80%, for Z. ophiocephalus, R. clavata, and S. scrofa crude proteases, respectively. These results suggest that enzymaticdeproteinization of shrimp wastes by fish endogenous alkaline proteases could be applicable to the chitin production process.

1. Introduction

Proteases constitute the most important group of industrialenzymes used in the world today, accounting for approxi-mately 50% of the total industrial enzyme market [1]. Theyhave diverse applications in a wide variety of industriessuch as detergent, food, pharmaceutical, leather, peptidesynthesis, and for the recovery of silver from used X-ray films[2, 3]. Proteases are mainly derived from animal, plant, andmicrobial sources.

Today, there is an increasing demand for fish proteolyticenzymes in food processing. Fish viscera, one of the mostimportant by-products of fishing industry, is known to bea rich source of digestive enzymes, especially proteases thathave high activity over a wide range of pH and temperature

conditions [4–6] and exhibit high catalytic activity at rela-tively low concentration [7]. These characteristics of fish pro-teases have made them suitable for some interesting newapplications in food-processing operations. In addition, fishenzymes could be utilized to produce bioactive peptides fromfish proteins [8, 9]. Considering the specific characteristicsof these enzymes, fish processing by-products are currentlyused for enzyme extraction.

The most important digestive proteolytic enzymes fromfish and aquatic invertebrates viscera are the aspartic pro-tease pepsin secreted from gastric mucosa, and the serineproteases, trypsin, and chymotrypsin secreted from the pan-creas, pyloric caeca, and intestine [10]. Acidic proteasesfrom fish stomachs display high activity between pH 2.0and 4.0, while alkaline digestive proteases, such as trypsin,

2 Journal of Amino Acids

are most active between pH 8.0 and 10.0. The distributionof proteinases varies, depending on species and organs.Digestive enzymes of several species of fish have been isolatedfrom the internal organs including gastric, intestinal, andhepatopancreas [5, 9, 11–13].

Chitin, a homopolymer of N-acetyl-D-glucosamineresidues linked by β-1,4 bonds, is the most abundant renew-able natural resource after cellulose [14]. Chitin and its de-rivatives are biomolecules of a great potential, possessingversatile biological activities, demonstrating excellent bio-compatibility and complete biodegradability. Therefore, theyhave found extensive applications in pharmacy, medicine,agriculture, food and textile industries, cosmetics, and waste-water treatment [15–17].

The main sources of raw material for the production ofchitin are cuticles of various crustaceans, principally crabsand shrimps. Chitin in biomass is closely associated withproteins, inorganic compounds (such as calcium carbonate),lipids, and pigments. They all have to be quantitativelyremoved to achieve the high purity of chitins necessary forbiological applications [18].

Conventionally, to extract chitin from crustacean shells,chemicals processing for demineralization and deproteiniza-tion have been applied. Raw materials were first treated withdilute hydrochloric acid at room temperature to removemetal salts, particularly calcium carbonate, and then withstrong bases to remove proteins [18]. However, the use ofthese chemicals may cause a partial deacetylation of thechitin and hydrolysis of the polymer, resulting in final incon-sistent physiological properties [19]. An alternative approachto these harsh chemical treatments is the use of prote-olytic microorganisms [20–23] or proteolytic enzymes [24].Bustos and Healy [25] demonstrated that chitin obtainedby the deproteinization of shrimp shell waste with variousproteolytic microorganism had higher molecular weightscompared to chemically prepared shellfish chitin.

In the present paper, we describe the extraction and char-acterization of alkaline proteases from Z. ophiocephalus, R.clavata, and S. scrofa which are suitable in the chitin produc-tion process.

2. Materials and Methods

2.1. Reagents. Casein sodium salt from bovine milk, tri-chloroacetic acid (TCA), ethylene diamine tetraacetic acid(EDTA), and bovine serum albumin were purchased fromSigma Company Co. (St Louis, Mo, USA). Hydrochloricacid and Tris(hydroxymethyl)aminomethane were procuredfrom Panreac Quimica SA (Barcelone, Spain). Sodium do-decyl sulphate (SDS), acrylamide, ammonium persul-phate, N,N,N,N′-tetramethyl ethylenediamine (TEMED),and Coomassie Brilliant Blue R-250 were from Bio-RadLaboratories (Mexico City, Mexico). All other reagents wereof analytical grade.

2.2. Materials. Goby (Z. ophiocephalus), thornback ray (R.clavata), and scorpionfish (S. scrofa) were purchased from thefish market of Sfax City, Tunisia. The samples were packed

in polyethylene bags, placed in ice with a sample/ice ratioof approximately 1 : 3 (w/w) and transported to the researchlaboratory within 30 minutes. After the fish were washedwith water, their viscera were separated, rinsed with colddistilled water, and then stored in sealed plastic bags at−20◦C until they were used for enzyme extraction.

2.3. Preparation of Crude Alkaline Proteases. Viscera (20 g)were separated and rinsed with distilled water, and thenhomogenized for 5 minutes with 20 mL of extraction buffer(10 mM Tris-HCl, pH 8.0) with the use of tissue homoge-nizer. The resulting preparations were centrifuged at 8500×gfor 30 minutes at 4◦C. The pellets were discarded and thesupernatants were collected and then frozen at −20◦C andused as crude protease extracts. All enzymatic assays wereconducted within a week after extraction.

2.4. Polyacrylamide Gel Electrophoresis. Sodium dodecyl sul-phate-polyacrylamide gel electrophoresis (SDS-PAGE) wascarried out as described by Laemmli [26], using 5% (w/v)stacking and 15% (w/v) separating gels. Samples were pre-pared by mixing the crude enzyme extracts at 1 : 5 (v/v)ratio with distilled water containing 10 mM Tris-HCl pH 8.0,2.5% SDS, 10% glycerol, 5%β-mercaptoethanol, and 0.002%bromophenol blue. The samples were heated at 100◦C for5 minutes before loading in the gel. After electrophoresis,the gel was stained with 0.25% Coomassie Brilliant Blue R-250 in 45% ethanol-10% acetic acid and destained with 5%ethanol-7.5% acetic acid.

2.5. Detection of Protease Activity of Enzyme Extracts byZymography. Protease activity staining was performed onSDS-PAGE according to the method of Garcia-Carreno et al.[27] with a slight modification. The sample was not heatedbefore loading in the gel. After electrophoresis, the gel wassubmerged in buffer A (100 mM of Tris-HCl buffer (pH 9.0))containing 2.5% Triton X-100, with shaking for 1 hour toremove SDS and allow enzyme renaturation. Triton X-100was removed by washing the gel three times with buffer A.The gel was then immersed in 100 mL of 1% (w/v) caseinin buffer A for 5 minutes at 4◦C, then further incubated for10 minutes at 50◦C to allow for the digestion of the proteinsubstrate (casein) by the active enzymes. Finally, the gel wasstained with 0.25% Coomassie Brilliant Blue R-250 in 45%ethanol-10% acetic acid and destained with 5% ethanol-7.5% acetic acid. The development of clear bands on theblue background of the gel indicated the presence of proteaseactivity.

2.6. Protease Assay. Protease activity in the crude alkalineenzyme extracts was measured by the method described byKembhavi et al. [28] using casein as a substrate. A 0.5-mLaliquot of the crude enzyme extract, suitably diluted, wasmixed with 0.5 mL of 100 mM Tris-HCl (pH 8.0) containing1% (w/v) casein, and incubated for 15 minutes at 50◦C.The reaction was stopped by the addition of 0.5 mL of TCA20% (w/v). The mixture was allowed to stand at room tem-perature for 15 minutes and then centrifuged at 10.000 ×g

Journal of Amino Acids 3

for 15 minutes to remove the precipitate. The acid-solublematerial was estimated spectrophotometrically at 280 nm. Astandard curve was generated using solutions of 0–50 mg/Ltyrosine. One unit of protease activity was defined as theamount of enzyme required to liberate 1 μg of tyrosine perminute under the experimental conditions used.

2.7. Effect of pH on Activity and Stability of Crude AlkalineProteases. The optimum pH of the crude protease extractswas studied over a pH range of 5.0–13.0, using casein as asubstrate at 50◦C. For the measurement of pH stability, thecrude enzyme extracts were incubated for 1 hour at 4◦C indifferent buffers and then the residual proteolytic activitieswere determined under standard assay conditions. Thefollowing buffer systems were used: 100 mM sodium acetatebuffer for pH 5.0-6.0, Tris-HCl buffer for pH 7.0-8.0, glycine-NaOH buffer for pH 9.0–11.0, Na2HPO4-NaOH buffer forpH 12.0, and KCl buffer for pH 13.0.

2.8. Effect of Temperature on Protease Activity and Stability.To investigate the effect of temperature, the activity wastested using casein as a substrate at the temperature rangefrom 30 to 80◦C in 100 mM Tris-HCl buffer, pH 8.0 forZ. ophiocephalus and R. clavata proteases, and pH 10.0 forS. scrofa crude alkaline proteases. Thermal stability was-examined by incubating crude enzyme extracts for 60-minutes at different temperatures from 30 to 70◦C. Aliquotswere withdrawn at desired time intervals to test the remain-ing activity at standard conditions. The nonheated crudeenzyme extracts were considered as control (100%).

2.9. Effects of Metal Ions, NaCl Concentration, Surfactants,and Oxidizing Agents on Proteolytic Activity of Crude EnzymeExtracts. The influence of various metals ions, at a concen-tration of 5 mM, on enzyme activity was investigated byadding the monovalent (Na+ or K+) or divalent (Mg2+, Hg2+,Ca2+, Zn2+, Cu2+, Co2+, Ba2+, or Mn2+) metal ions to thereaction mixture. The activity of the crude enzyme extractswithout any metallic ion was considered as 100%. The effectof NaCl concentrations on the activity of the alkaline crudeprotease extracts was studied, using casein as a substrate, byincreasing NaCl concentrations in the reaction mixture.

The effects of some surfactants (Triton X-100, Tween 80,and SDS) and oxidizing agents (sodium perborate) on alka-line crude proteases stability were studied by preincubatingenzymes for 1 hour at 30◦C. The residual activities weremeasured at optimum conditions for each crude enzyme.The activity of the crude enzyme extract without any additivewas taken as 100%.

2.10. Preparation of Shrimp Waste Powder (SWP) and Chem-ical Analysis. The SWP was prepared in our laboratory.Briefly, shrimp waste, collected from the marine food pro-cessing industry, was washed thoroughly with tap water andthen cooked 20 minutes at 90◦C. The solid material obtainedwas dried, minced to obtain a fine powder, and then stored inglass bottles at room temperature. The chemical composition(proteins, chitin, lipids, and ash) was determined.

The moisture and ash content were determined accord-ing to the AOAC standard methods 930.15 and 942.05,respectively, [29]. Total nitrogen content of shrimp proteinhydrolysates was determined by using the Kjeldahl method.Crude protein was estimated by multiplying total nitrogencontent by the factor of 6.25.

2.11. Deproteinization of Shrimp Wastes by Crude AlkalineProtease Extracts. Shrimp shell wastes (15 g) were mixedwith 100 mM Tris-HCl buffer pH 8.0 at a ratio of 1 : 3(w/v), minced and then cooked for 20 minutes at 90◦C toinactivate endogenous enzymes. The cooked sample was thenhomogenized in a Moulinex blender for about 2 minutes.The pH of the mixture was adjusted to 8.0, and thenthe shrimp waste proteins were digested with proteolyticenzymes at 45◦C using en enzyme/substrate ratio of 10/1(unit of enzyme/mg of protein). After 3-hour incubation at45◦C, the reaction was stopped by heating the solution at90◦C during 20 minutes to inactivate enzymes. The shrimpwaste protein hydrolysates were then centrifuged at 5000×gfor 20 minutes to separate insoluble and soluble fractions.The solid phase was washed, pressed manually throughfour layers of gauze, and then dried for 1 hour at 60◦C.The protein content was analyzed to measure the proteinremoval. The press cake was packed in a plastic bag andstored at −20◦C until further processing.

Deproteinization percentage (%DP) was calculated bythe following equation as described by Rao et al. [30]:

%DP = [(Po ×O)− (PR × R)]× 100Po ×O

, (1)

where PO and PR are protein concentrations (%) before andafter hydrolysis; while O and R represent the mass (grams) oforiginal sample and hydrolyzed residue in dry weight basis,respectively.

2.12. Statistical Analysis. All experiments were carried out intriplicate, and average values with standard deviation errorsare reported. Mean separation and significance were analyzedusing the SPSS software package (SPSS, Chicago, Ill). Corre-lation and regression analysis were carried out using EXCELprogram.

3. Results and Discussion

3.1. SDS-PAGE and Zymography of Crude Alkaline Proteases.In order to estimate the number of proteases in the alkalinecrude enzyme extracts, samples were separated by SDS-PAGE, and then proteolytic activities were revealed by caseinzymography activity staining. Casein zymography is a verysensitive and rapid assay method that detects nanogramof proteins, in contrast with SDS-PAGE which detectsmicrograms.

As can be observed in Figure 1, all crude enzyme extractsshowed several clear bands of protease activity with differentmolecular weights, indicating the presence of several dif-ferent proteases. It seems that goby crude enzyme extractcontained more proteolytic enzymes than the other ones


21 3

Figure 1: Activity staining of the crude alkaline protease extractfrom the viscera of R. clavata (1), Z. ophiocephalus (2), and S. scrofa(3).

as illustrated in Figure 1 by the presence of at least fiveclear bands of proteolytic activity. This result suggests thatat least five major proteinases were present in goby viscera.When comparing the different profiles, it can be observedthe presence of at least one protease common with all crudeproteases.

3.2. Biochemical Characterization of the Alkaline Crude

Protease Extracts

3.2.1. Effect of pH on Protease Activity. The activity of prote-olytic enzymes was determined at different pH values from5.0 to 13.0. The pH activity profiles of the crude alkalineproteases are shown in Figure 2(a). The proteolytic enzymesof Z. ophiocephalus displayed maximum activity at pH 8.0-9.0. The relative activities at pH 7.0 and 10.0 were 55.6%and 81.3%, respectively, of that at pH 9.0. However, proteaseactivity decreased significantly above pH 10.0. At pH 11.0,the activity was approximately 5-fold lower than that at pH9.0.

The optimum pH for the crude protease of R. clavatawas pH 8.0. The relative activity at pH 9.0 was about 94%.However, an appreciable decrease in activity was observedabove pH 9.0.

With S. scrofa crude enzyme extract, two activity peakswere observed at pH 6.0 and 10.0. The enzyme preparationwas highly active between pH 8.0 and 11.0, with an optimumat pH 10.0. The relative activities at pH 9.0, 11.0, and12.0 were about 94%, 69%, and 39%, respectively, of that

at pH 10.0. The optimum pH for S. scrofa proteases wassimilar to those reported by Esposito et al. [31] for proteasesextracted from the viscera of Colossoma macropomum andEl Hadj-Ali et al. [4] for proteases extracted from stripedseabream (Lithognathus mormyrus).

3.2.2. Effect of pH on Protease Stability. The pH stability pro-files of the three crude alkaline proteases are reported inFigure 2(b). Interestingly, the three crude enzyme extractsare highly stable over a wide broad pH range, maintainingabout 100% of their original activity between pH 5.0 and 10.0after 1 hour of incubation at 4◦C. The enzymes retained morethan 83% of their activities at pH 12.0. Our results showedthat goby proteases present a high pH stability compared tothe others crude enzyme extracts.

The enzyme preparation from scorpionfish, which ishighly active in the alkaline pH range, was also stable overa wide pH range. These results suggest that the viscera ofscorpionfish would be a potential source of alkaline proteasesfor certain industrial applications that require high alkalineconditions, such as detergents. In fact, one of the mostimportant parameters for selection proteases for detergentsis the optimum pH. Since the pH of laundry detergents iscommonly alkaline (in the range of 9.0–12.0) [32], proteaseand other enzymes currently used in detergent formulationsshould be alkaline in nature with a high optimum pH. Theseproperties were displayed by the scorpionfish proteases.

3.2.3. Effect of Temperature on Protease Activity. Optima tem-peratures for activity of crude alkaline proteases were deter-mined in order to assess their suitability for biotechnologicalapplications. The relative activities at various temperaturesusing casein as a substrate are reported in Figure 3. Thecrude proteases were active at temperatures from 30 to 70◦C.The optimum temperature for S. scrofa proteases was 55◦C,however, alkaline proteases from goby and thornback raydisplayed maximum activity at 50◦C.

The relative activities of goby proteases at 40 and 60◦Cwere 54% and 70%, respectively. However, an appreciabledecrease in enzyme activity was observed above 65◦C, due tothermal denaturation. Thornback ray proteases were moreactive at 60◦C than the other crude proteases, retaining90% of their activity after 1-hour incubation. However, therelative activities of Z. ophiocephalus and R. clavata crudeproteases were 70% and 45%, respectively.

3.2.4. Effect of Temperature on Protease Stability. Thermalstability of crude alkaline proteases is depicted in Figure 4.Enzyme preparations from goby and scorpionfish are highlyactive at temperatures below 40◦C, while that of thornbackray were stable at 30◦C. Goby crude enzyme remains fullyactive even after 60 minutes of incubation at 40◦C, indicatingthat this crude enzyme might be used under mild heatingconditions. However, at higher temperatures proteases wereinactivated.

The enzyme preparations from Z. ophiocephalus and S.scrofa retained about 24% and 45% of their initial activ-ity after 60 minutes of incubation at 50◦C, respectively.


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However, the proteolytic enzymes from R. clavata were com-pletely inactivated in the same conditions.

3.2.5. Effects of Metal Ions on Protease Activity. The effectsof various metal ions, at a concentration of 5 mM, on theactivity of the crude alkaline proteases were studied at opti-mum conditions for each crude enzyme by the addition ofthe respective cations to the reaction mixture (Table 1).

Table 1: Effects of various metal ions (5 mM) on protease activity.

Metal ionsRelative activity (%)

Goby Scorpionfish Thornback ray

Control 100 100 100

Na+ 100 91 110

K+ 100 91 80

Mg2+ 117 122 100

Mn2+ 47.5 83 37.5

Zn2+ 20 105 23

Cu2+ 17.5 67 47.5

Hg2+ 36 62 29.5

Fe2+ 0 31 0

Ca2+ 110 129 111

Ba2+ 110 97 100

The addition of CaCl2 and MgSO4 increased the activityof crude protease extracts of goby and scorpionfish. Ca2+

increased the activity of crude proteases from goby andscorpionfish to 110% and 129%, respectively. These resultsindicated that Ca2+ was very effective in improving theactivity of the crude proteases. The enhancement of proteaseactivity in the presence of calcium may be explained by thestrength of interactions inside protein molecules and thebetter stabilization of enzymes against thermal stabilization.However, the activity of R. clavata crude enzyme was notaffected by CaCl2.

The ions Ba2+ affect partially the protease activity witha relative activity between 87% and 96%. However, Fe2+

and Hg2+ affect greatly the activity of all crude enzymes.The presence of 5 mM NaCl and KCl did not affect proteaseactivity.


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70◦C

(c)

Figure 4: Effect of temperature on thermal stability of the crude alkaline proteases from goby (a), thornback ray (b) and scorpionfish (c).The temperature stability was determined by incubating the crude extract at temperatures from 30 to 70◦C for 1 hour. The residual enzymeactivity was measured under the standard conditions assay at different times. The original activity before preincubation was taken as 100%.Values are means of three independent experiments.

3.2.6. Stability of the Enzyme Extracts in the Presence of Oxi-dizing Agents and Surfactants. All the commercial detergentscontain hydrolytic enzymes such as proteases. In additionto activity and stability at high pH range and varioustemperatures [33], enzymes incorporated into detergentformulations must be compatible and stable with all com-monly used detergent components such as surfactants,perfumes, oxidizing agents, and other additives which mightbe present in the formulation [34]. Furthermore, detergentenzymes should be stable during storage and active duringwashing in the detergent solution for a long period of time[35].

The suitability of crude alkaline proteases as detergentadditive was investigated by testing their stability in the pres-ence of some surfactants and oxidizing agents. As shown inTable 2, crude protease extracts were highly stable in the pres-ence of non-ionic surfactants such as Tween 20, Tween 80,and Triton X-100. Furthermore, the activities of scorption-fish and thornback ray proteases were slightly enhanced. Forexample, the activities of scorptionfish after incubation for 1hour at 40◦C were 107%, 109%, and 107% in the presenceof 5% Triton X-100, Tween 20, and Tween 80, respectively.However, the strong anionic surfactant (SDS) at 1% caused100% inhibition proteolytic activity of R. clavata proteases.


Table 2: Stability of alkaline proteases in the presence of various surfactants and oxidizing agents.

Surfactants/oxidizing agents Concentration (%)Residual activity (%)

Goby Scorpionfish Thornback ray

None 0 100 100 100

Triton X-100 5 (v/v) 100 107 117

Tween 20 5 82 109 115

Tween 80 5 90 107 100

SDS0.1 (w/v) 40 73.5 13

0.5 33 44 0

1 14 16 0

Sodium perborate0.2 92.3 106 88

1 66 100 70

Enzyme preparations were incubated with different surfactants and oxidizing agents for 1 hour at 30◦C and the remaining activity was measured understandard conditions. The activity is expressed as a percentage of the activity level in the absence of additives.

Goby proteases



0

20

40

60

80

100

120

205 10 15 30250

[NaCl] (%)

Rel

ativ

eac

tivi

ty(%

)

Figure 5: Effect of NaCl concentration on the activity of alkalinecrude protease extracts.

In addition, we investigated the effects of oxidizing agentson the crude protease extract. Thornback ray and goby pro-teases were little influenced by oxidizing agents, retainingabout 70% and 66% of their initial activity after incubationfor 1 hour at 30◦C in the presence of 1% (w/v) sodium perbo-rate, respectively.

Interestingly, the crude enzyme of scorpionfish remainsfully active after 1 hour incubation at 40◦C. The stability ofscorpionfish enzyme extract against sodium perborate washigher than A21 protease from Bacillus mojavensis whichretained 35% of its initial activity in the presence of 1%oxidizing agent after incubation for 1 hour at 30◦C [36].The high stability of scorpionfish enzyme extract in thepresence of oxidizing agents is a very important characteristicfor its eventual use in detergent formulations. Few pub-lished reports are available on the compatibility of alkaline

proteases with oxidizing agents. Important commercial de-tergent proteases like Subtilisin Carlsberg, Subtilisin BPN,Alcalase, Esparase, and Savirase are stable in the presence ofvarious detergent components. However, most of them areunstable in the presence of oxidant agents, such as hydrogenperoxide [34].

3.2.7. Effect of NaCl. The effect of NaCl concentration onthe activity of crude alkaline proteases is shown in Figure 5.The activity of the three enzyme preparations was affected byNaCl. The activities of all crude proteases decreased graduallywith increasing NaCl concentration. The relative activitiesof goby, scorpionfish, and thornback ray at 10% NaCl wereapproximately 53%, 37%, and 14%, respectively. The resultsshowed that goby proteases exhibited a high activity in thepresence of NaCl compared to the other crude enzymes.The decrease in activity might be due to denaturation ofenzymes caused by the “salting out” effect with increasingNaCl concentrations.

3.2.8. Enzymatic Deproteinization of Shrimp Wastes by CrudeAlkaline Proteases. Chitin, a polysaccharide found in abun-dance in the shell of crustaceans, is closely associated withproteins. Therefore, deproteinization in chitin extractionprocess is crucial. Chemical treatment requires the use of HCland NaOH, which can cause deacetylation and depolymer-ization of chitin.

Few studies on the use of proteolytic enzymes for thedeproteinization of shrimp wastes have been reported. Tothe best of our knowledge, there are no available reportson the enzymatic deproteinization of shrimp wastes by fishproteases. Many factors, such as the specificity of the enzymeused for the proteolysis, E/S ratio and the conditions usedduring hydrolysis (initial temperature value and hydrolysistime) have been reported to influence the enzymatic depro-teinization process.

In the present study, alkaline proteases from Z. ophio-cephalus, R. clavata, and S. scrofa were applied for the depro-teinization of shrimp waste to produce chitin and proteinhydrolysates using an E/S ratio of 10 U/mg. As depictedin Figure 6, all fish extracts were efficient in shrimp waste


0

20

40

60

80

100

Goby Thornback ray Scorpionfish

Dep

rote

iniz

atio

nd

gree

e(%

)

Figure 6: Deproteinization degree of shrimp waste by the crude al-kaline proteases.

deproteinization, and S. scrofa crude extract was the mostefficient with a deproteinization percentage of 80%. Thedeproteinization degrees with Z. ophiocephalus and R. clavatacrude enzymes were 76%.

The deproteinization activity of crude proteases used inthis study was similar to many bacterial proteases reported inmany previous studies [20, 25].

4. Conclusion

In the present study, alkaline proteases were extracted fromthe viscera of Z. ophiocephalus, R. clavata and S. scrofa andcharacterized, and their efficiencies in deproteinization ofshrimp waste to produce chitin were investigated.

Crude alkaline proteases from Z. ophiocephalus, R. clav-ata, and S. scrofa showed optimum activity at pH 8.0-9.0,50◦C; pH 8.0, 55◦C, and pH 10.0, 55◦C, respectively. Thecrude enzyme extract showed a high activity and stability inhigh alkaline pH. These proteolytic enzymes remained fullyactive in the presence of non-ionic surfactants. They alsorevealed high resistance when incubated with 1% sodiumperborate.

The alkaline crude proteases were found to be effectivein the deproteinization of shrimp waste powder. The proteinremovals with a ratio E/S of 10 were more than 76%.

Considering their promising properties, crude proteaseextracts used in this study may find potential applicationsin the deproteinization of shrimp waste to produce chitinand chitosan. Further research is needed to purify alkalineproteases, and to determine their properties as a possiblebiotechnological tool in the fish processing and food indus-tries.

Acknowledgment

This work was funded by the Ministry of Higher Educationand Scientific Research, Tunisia.

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Research Article

Comparative Study on Biochemical Properties and AntioxidativeActivity of Cuttlefish (Sepia officinalis) Protein HydrolysatesProduced by Alcalase and Bacillus licheniformis NH1 Proteases

Rafik Balti,1 Ali Bougatef,1 Nedra El Hadj Ali,1 Naourez Ktari,1 Kemel Jellouli,1

Naima Nedjar-Arroume,2 Pascal Dhulster,2 and Moncef Nasri1

1 Laboratoire de Genie Enzymatique et de Microbiologie Ecole Nationale d’Ingenieurs de Sfax, Universite de Sfax,B P 1173, Sfax 3038, Tunisia

2 Laboratoire de Procedes Biologiques, Genie Enzymatique et Microbien, IUT A Lille, BP 179, 59653 Villeneuve d’Ascq Cedex, France

Correspondence should be addressed to Rafik Balti, [email protected]

Received 27 March 2011; Accepted 11 July 2011


Copyright © 2011 Rafik Balti et al. This is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Antioxidative activities and biochemical properties of protein hydrolysates prepared from cuttlefish (Sepia officinalis) using Alcalase2.4 L and Bacillus licheniformis NH1 proteases with different degrees of hydrolysis (DH) were determined. For the biochemicalproperties, hydrolysis by both enzymes increased protein solubility to above 75% over a wide pH range. The antioxidant activitiesof cuttlefish protein hydrolysates (CPHs) increase with increasing DH. In addition, all CPHs exhibited antioxidative activity in aconcentration-dependent manner. NH1-CPHs generally showed greater antioxidative activity than Alcalase protein hydrolysates(P < 0.05) as indicated by the higher 1,1-diphenyl-1-picryhydrazyl (DPPH) radical scavenging activity and ferrous chelatingactivity. Both Alcalase and NH1 protein hydrolysates were able to retard lipid peroxidation and β-carotene-linoleic acid oxidation.Alcalase-CPH (DH = 12.5%) and NH1-CPH (DH = 15%) contained 75.36% and 80.11% protein, respectively, with histidine andarginine as the major amino acids, followed by glutamic acid/glutamine, serine, lysine, and leucine. In addition, CPHs have a highpercentage of essential amino acids made up 48.85% and 50.04%. Cuttlefish muscle protein hydrolysates had a high nutritionalvalue and could be used as supplement to poorly balanced dietary proteins.

1. Introduction

Free radical-mediated lipid peroxidation and antioxidantsare attracting considerable research interest in many areas.Lipid oxidation is one of the major deteriorative processes inmany types of foods, leading to the changes in food qualityand nutritional value. Additionally, potentially toxic reactionproducts can be produced [1]. In particular, investigatorsreport that free radicals, generated by oxidation, play acritical role in a variety of health disorders, including theprocesses of ageing, cancer, diabetes mellitus, inflamma-tion, coronary heart, and neurological disorders, such asAlzheimer’s disease [2]. Therefore, it is important to inhibitthe oxidation and formation of free radicals occurring in theliving body and foodstuffs [3].

Some synthetic antioxidative agents, such as butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT),

and propyl gallate, are commonly used as free radicalscavengers in food and biological systems. Although, thesesynthetic antioxidants show stronger antioxidant activitythan those of natural antioxidants such as α-tocopherol andascorbic acid, the use of these chemical compounds hasbegun to be restricted because of their induction of DNAdamage and their toxicity [4]. Thus, increasing attentionhas been directed to the development of safe and effectivefunctional foods and antioxidative agents from naturalsources, especially peptides derived from hydrolyzed foodproteins.

Recently, protein hydrolysates from several fish species,such as silver carp (Hypophthalmichthys molitrix) [5],brownstripe red snapper (Lutjanus vitta) [6], sardinelle (Sar-dinella aurita) [7], smooth hound (Mustelus mustelus) [8],oyster (Crassostrea gigas) [9], yellow stripe trevally (Selaroidesleptolepis) [10], round scad (Decapterus maruadsi) [11],


yellowfin sole (Limanda aspera) [12], herring (Clupea haren-gus) [13], and mackerel (Scomber austraasicus) [14], havebeen reported to possess antioxidative activities.

The operational conditions employed in the processingof protein isolates, the type of protease, and the degree ofhydrolysis affect the antioxidant activity [15]. Especially the,proteinases used can affect both the functional propertiesand antioxidative activity of the protein hydrolysate obtained[12]. Protein hydrolysate from Alaska pollack frame preparedby mackerel intestine crude enzyme exhibited antioxida-tive activity in a linoleic acid model system [16]. Prawnhydrolysate prepared using pepsin showed the most potentantioxidative activity than those prepared by other enzymes[17]. Levels and compositions of free amino acids andpeptides were reported to determine the antioxidant activ-ities of protein hydrolysates [14]. Moreover, the utilizationof proteins or their hydrolysates for food and/or cosmeticapplications not only presents additional advantages overother antioxidants but also confers nutritional and func-tional properties [18].

Recently, protein hydrolysates from cuttlefish (Sepiaofficinalis) enriched in angiotensin I-converting enzymeinhibitory peptides have been produced successfully usingAlcalase and Bacillus licheniformis NH1 proteases [19].Nevertheless, a little information regarding the characteristicand antioxidative activity of hydrolysates prepared usingboth enzymes has been reported. The objective of thisstudy was to investigate the antioxidative activity of proteinhydrolysates from cuttlefish muscle prepared using Alcalaseand Bacillus licheniformis NH1 proteases in a model systemand the effect of concentration on their activities. Meanwhile,solubility of hydrolysates derived from cuttlefish muscle wasevaluated.


2.1. Reagents. 1,1-diphenyl-2-picrylhydrazyl (DPPH), 3-(2-pyridyl)-5,6-bis(4-phenyl-sulphonic acid)-1,2,4-triazine(Ferrozine), butylated hydroxyanisole (BHA), α-tocopherol,and linoleic acid were purchased from Sigma-Aldrich, Inc.(St. Louis, Mo, USA). All other chemicals, namely ammo-nium thiocyanate, ferric chloride, EDTA, Tween-40, andsodium hydroxide were of analytical grade.

2.2. Fish Sample. Cuttlefish (S. officinalis), in the size rangeof 8–10 cuttlefish/kg, was purchased from the fish market ofSfax city, Tunisia. The samples were packed in polyethylenebags, placed in ice with a sample/ice ratio of approximately1 : 3 (w/w), and transported to the research laboratory within30 min. The mantle was cleaned, deskinned, and evisceratedand then stored in sealed plastic bags at −80◦C until used.

2.3. Enzyme. The crude enzyme preparation from B. licheni-formis NH1 [20] and Alcalase 2.4 L obtained from NovoNordisk (Bagsverd, Denmark) was used for the productionof protein hydrolysates. Protease activity was determinedaccording to the method of Kembhavi et al. [21] using caseinas a substrate. One unit of protease activity was defined as

the amount of enzyme required to liberate 1 μg tyrosine perminute under the experimental conditions used.

2.4. Production of Protein Hydrolysates from Cuttlefish Muscle.Cuttlefish (S. officinalis) muscle (500 g), in 1000 mL distilledwater, was first minced using a grinder (Moulinex CharlotteHV3, France) and then cooked at 90◦C for 20 min toinactivate endogenous enzymes. The cooked muscle samplewas then homogenized in a Moulinex blender for about2 min and hydrolyzed with enzymes under optimal condi-tions: the crude enzyme preparation from B. licheniformisNH1 (pH 10.0 and 50◦C) and Alcalase 2.4 L (pH 8.0and 50◦C). The enzyme was added to the reaction at thesame enzyme/substrate ratio (E/S = 3 U/mg) to comparehydrolytic efficiencies. During the reaction, the pH of themixture was maintained constant by continuous addition of4 M NaOH solution. After the required digestion time, thereaction was stopped by heating the solution at 80◦C during20 min to inactivate the enzyme. The cuttlefish muscle pro-tein hydrolysates were then centrifuged at 5000×g for 20 minto separate insoluble and soluble fractions. Finally, thesoluble phase was freeze dried using freeze dryer (BioblockScientific Christ ALPHA 1-2, IllKrich-Cedex, France) andstored at −20◦C for further use.

2.5. Degree of Hydrolysis Determination (DH). The degree ofhydrolysis (DH), defined as the percent ratio of the numberof peptide bonds broken (h) to the total number of peptidebonds in the studied substrate (htot), was calculated fromthe amount of base (NaOH) added to keep the pH constantduring the hydrolysis [22] as given below:

DH (%) = h

htot× 100 = B ×Nb

MP× 1

α× 1

htot× 100, (1)

where B is the amount of NaOH consumed (mL) to keep thepH constant during the reaction, Nb is the normality of thebase, MP is the mass (g) of protein (N × 6.25), and α is theaverage degree of dissociation of the α-NH2 groups releasedduring hydrolysis expressed as:

α = 10pH−pK

1 + 10pH−pK , (2)

where pH and pK are the values at which the proteolysis wasconducted. The total number of peptide bonds (htot) in a fishprotein concentrate was assumed to be 8.6 meq/g [22].

2.6. Proximate Analysis. Moisture and ash content weredetermined according to the AOAC [23] standard methods930.15 and 942.05, respectively. Total nitrogen content of thesubstrate and selected hydrolysate products was determinedby using the Kjeldahl method. Crude protein was estimatedby multiplying total nitrogen content by the factor of6.25. Lipids were determined gravimetrically after Soxhletextraction of dried samples with hexane. All measurementswere performed in triplicate. The protein and fat contentswere expressed on a dry weight basis.


2.7. Amino Acid Analysis. For analysis of amino acids, thedry samples were dissolved in distilled water at 1 mg/mL,and 50 μL of each sample were dried and hydrolysed invacuum-sealed glass tube at 110◦C for 24 h in the presenceof constant boiling 6 N HCl containing 1% (w/v) phenoland using norleucine as internal standard. After hydrolysis,samples were again vacuum dried, dissolved in applicationbuffer and injected into a Beckman 6300 amino acid analyzer(Beckman Instruments Inc., Fullerton, Calif, USA ).

2.8. Solubility. Solubility of cuttlefish protein hydrolysateswas carried out according to Tsumura et al. [24] with slightmodifications. Briefly, 200 mg of freeze-dried hydrolysatesof cuttlefish protein were suspended in 20 mL deionizeddistilled water, and the pH of the mixture was adjusted todifferent values from 2.0 to 11.0 using either 2 N HCl or2 N NaOH solutions. The mixtures were stirred for 10 minat room temperature (25 ± 1◦C) and then centrifuged at8000×g for 10 min. After appropriate dilution, the nitrogencontent in the supernatant was determined by Biuret method[25]. The nitrogen solubility of the sample, defined as theamount of soluble nitrogen from the total nitrogen, wascalculated as follows:

Nitrogen solubility (%)

= Supernatant nitrogen concentrationSample nitrogen concentration

× 100.(3)

Solubility analysis was carried out in triplicate.

2.9. Determination of Antioxidant Activities

2.9.1. Inhibition of Linoleic Acid Autoxidation. Inhibitionactivity of in vitro lipid peroxidation of cuttlefish proteinhydrolysates was determined by assessing their ability toinhibit oxidation of linoleic acid in an emulsified modelsystem [26]. Briefly, freeze-dried hydrolysates of cuttlefishprotein (5 mg) were dissolved in 2.5 mL of 50 mM phosphatebuffer (pH 7.0) and added to a 2.5 mL of 50 mM linoleicacid in ethanol (95%). The final volume was then adjustedto 6.25 mL with distilled water. The mixture was incubatedin a 10 mL tube with silicon rubber caps at 45◦C for 12days in a dark, and the degree of oxidation was evaluatedby measuring the ferric thiocyanate values according to themethod of Mitsuda et al. [27]. Aliquot (0.1 mL) of reactionmixture was mixed with 4.7 mL of 75% ethanol followedby the addition of 0.1 mL of 30% ammonium thiocyanateand 0.1 mL of 20 mM ferrous chloride solution in 3.5% HCl.After stirring for 3 min, the degree of colour development,which represents the linoleic acid oxidation, was measuredat 500 nm. The antioxidative capacity of the inhibition ofperoxide formation in linoleic acid system was expressed asfollows:

Inhibition (%) =[

1− A500 of sampleA500 of control

]× 100, (4)

α-tocopherol, a natural antioxidant agent, and BHA, asynthetic antioxidant agent, were used as reference, anddistilled water as control.

2.9.2. 1,1-Diphenyl-2-Picrylhydrazyl (DPPH) Radical Scav-enging Activity. DPPH radical-scavenging activity was mea-sured, using the method described by Bersuder et al. [28]. A500 μL test sample was mixed with 500 μL of 99.5% ethanoland 125 μL of 99.5% ethanol containing 0.02% DPPH. Thismixture was shaken then kept in a dark at room temperaturefor 60 min before measuring absorbance at 517 nm. DPPHradical-scavenging activity was calculated according to thefollowing equation:

DPPH radical-scavenging activity (%)

=[


]× 100.

(5)

The control was conducted in the same manner exceptthat distilled water was used instead of sample. A lowerabsorbance of the reaction mixture indicated a higher DPPHscavenging activity. Butylated hydroxyanisole (BHA) wasused as a standard.

2.9.3. Ferrous Chelating Activity. The chelating activity onFe2+ was determined, using the method of Decker and Welch[29]. One millilitre of sample solution was mixed with 3.7 mLof distilled water. The mixture was then reacted with 0.1 mLof 2 mM FeCl2 and 0.2 mL of 5 mM 3-(2-pyridyl)-5,6-bis(4-phenyl-sulfonic acid)-1,2,4-triazine (ferrozine) for 20 minat room temperature. The absorbance was read at 562 nm.The control was prepared in the same manner except thatdistilled water was used instead of the sample. EDTA wasused as reference. Chelating activity (%) was then calculatedas follows [29]:

Chelating activity (%) =[


]× 100.

(6)

2.9.4. β-Carotene-Linoleic Acid Assay. Antiautooxidant activ-ity was assayed using the β-carotene bleaching method [30].In brief, 0.5 mg β-carotene in 1 mL chloroform was mixedwith 25 μL of linoleic acid and 200 μL of Tween-40. Thechloroform was evaporated under vacuum at 45◦C; then100 mL distilled water was added, and the resulting mixturewas vigorously stirred. The emulsion obtained was freshlyprepared before each experiment. An aliquot (2.5 mL) ofthe β-carotene-linoleic acid emulsion was transferred totubes containing 0.5 mL of each sample. The tubes wereimmediately placed in water bath and incubated at 50◦C for2 h. Thereafter, the absorbance of each sample was measuredat 470 nm. A control consisted of 0.5 mL of distilled waterinstead of the sample solution. BHA was used as positivestandard:

Antioxidant activity (%)

=⎡⎣1−

(Abs0

sample − Abs120sample

)(

Abs0control − Abs120

control

)⎤⎦× 100.

(7)


0

2

4

6

8

10

12

14

16

18

0 30 60 90 120 150 180 210 240 270

Hydrolysis time (min)

Deg

ree

ofhy

drol

ysis

(%)

NH1 proteasesAlcalase

Figure 1: Degree of hydrolysis (DH) of CPHs during hydrolysiswith Alcalase and NH1 proteases at 3 U/mg enzyme/substrate. Barsrepresent standard deviations from triplicate determinations.

2.10. Statistical Analysis. One-way analysis of variance(ANOVA) was used, and mean comparison was performedby Duncan’s multiple range test [31]. Statistical analyses wereperformed with Statgraphics ver. 5.1, professional edition(Manugistics Corp., Rockville, MD, USA). Differences wereconsidered significant at P < 0.05.


3.1. Preparation of Protein Hydrolysates from Cuttlefish Mus-cle. It has been demonstrated that biological activities ofproteins can be increased through hydrolysis with certainenzymes, and some peptides or fractions possess strongeractivity than others [19]. Furthermore, the specificity of theenzyme used for the proteolysis, the conditions used duringhydrolysis and the DH greatly influenced the molecularweight and amino acid composition of protein hydrolysates,and thus their biological activities [32].

The hydrolysis of the cuttlefish proteins with NH1proteases or Alcalase was characterized by a high rateof hydrolysis for the first 1 h (Figure 1). The rate ofenzymatic hydrolysis was subsequently decreased, and thenthe enzymatic reaction reached the steady-state phase whenno apparent hydrolysis took place. The shape of hydrolysiscurves is similar to those previously published for manyprotein substrates such as fish [33], whey [34], and wheatgluten [35]. The decrease in the reaction rate could beexplained by a decrease in the concentration of peptidebonds available for hydrolysis, enzyme deactivation, and/orthe inhibition of the enzyme by the products formed athigh degree of hydrolysis. These products act as effectivesubstrate competitors to the undigested or partially digestedfish proteins.

With the same E/S ratio, NH1 proteases showed higherDH values for cuttlefish protein hydrolysis than Alcalasebeyond 90 min hydrolysis period. The higher (P < 0.05) level

Table 1: Proximate composition (%) of undigested cuttlefishmuscle protein and freeze-dried CPHsa.

Compositions (%) UCMP NH1-CPH Alcalase-CPH

Moisture 5.76 ± 0.01 4.13 ± 0.02 4.96 ± 0.04

Proteinb 79.15 ± 0.48 80.11 ± 0.75 75.36 ± 0.68

Lipidsb 5.19 ± 0.24 0.68 ± 0.02 0.91 ± 0.05

Ashb 6.08 ± 0.71 12.44 ± 0.17 10.12 ± 0.53aMean ± SD from triplicate determinations.

bDry weight basis.UCMP: undigested cuttlefish muscle protein.

of DH by NH1 proteases treatment may be due to the factthat NH1 crude enzyme contains multiple proteases and,therefore, is a more efficient enzyme choice than Alcalasefor preparing cuttlefish protein hydrolysates. Therefore, thesusceptibility, to hydrolysis, of cuttlefish muscle proteinsdepends on the type of enzyme used.

3.2. Proximate Composition. Proximate composition of sol-uble fractions of freeze-dried CPHs compared to that ofundigested cuttlefish proteins is shown in Table 1. Theproximate composition of the undigested cuttlefish muscleproteins (UCMP) showed that it had high protein content(79.15 ± 0.48%). The ash and lipid contents of UCMPwere 6.08 ± 0.71% and 5.19 ± 0.24%, respectively. Alcalaseand NH1 CPHs powders had a white to light yellow colorappearance with almost no fishy odor and taste.

The protein content of CPHs varied with both enzymetreatments (Table 1). After 4 h of hydrolysis, Alcalase-CPHhad the least protein content (75.36 ± 0.68%). However,NH1-CPH showed a higher protein content (80.11 ±0.75%). During hydrolysis, proteins were solubilised, and theinsoluble nonprotein matter was removed, resulting in thehigh protein content in the resulting hydrolysate [36]. Theobtained results are similar to those of other studies on fishprotein hydrolysates that have ranged protein from 63.4 to90.8% [13, 37]. Generally, alkaline proteases exhibit a greatercapability to solubilize fish protein compared to neutral andacidic proteases, with exception of pepsin [38].

Interestingly, both dried cuttlefish protein hydrolysateshad low lipid content values 0.68± 0.02% and 0.91± 0.05%,in Alcalase-CPH and NH1-CPH, respectively. A low lipidcontents were reported in salmon [39] and herring [40]protein hydrolysates. The retention of a high amount of fatin the final products may limit the use of this ingredientin food applications, because fish protein hydrolysates withhigh lipid content can have an undesirable taste and darkenowing to changes in the lipids [41]. According to Spinelli etal. [42], the level of lipid residues in fish protein hydrolysatesmust be low.

The ash content was 10.12% and 12.44% in Alcalase-CPH, and NH1-CPH respectively, representing the saltformed during pH adjustment using alkaline solution(NaOH, 4 M). These results are similar to those of otherpublished studies on fish protein hydrolysates [39, 43].


70

75

80

85

90

95

100

2 3 4 5 6 7 8 9 10 11 12

pH

Solu

bilit

y(%

)

DH = 5.5%DH = 9.1%DH = 11.1%

DH = 13%DH = 15%

(a)

70

75

80

85

90

95

100

2 3 4 5 6 7 8 9 10 11 12

pH

Solu

bilit

y(%

)

DH = 7.6%DH = 9.6%DH = 10.8%

DH = 11.6%DH = 12.5%

(b)

Figure 2: Solubility profiles of CPHs with different degrees ofhydrolysis as influenced by pHs: (a) DH = 5.5%, DH = 9.1%,DH = 11.1%, DH = 13.0%, DH = 15.0% with proteases from B.licheniformis NH1, respectively; (b) DH = 7.6%, DH = 9.6%, DH =10.8%, DH = 11.6%, DH = 12.5% with Alcalase, respectively. Barsrepresent standard deviations from triplicate determinations.

3.3. Solubility. Functional properties influence the usefulnessof an ingredient in food and govern the physical behaviorduring preparation, processing, and storage [44]. Solubilityis one of the most important properties of proteins andprotein hydrolysates [45]. Many other functional propertiessuch as emulsification and foaming are affected by solubility.The pH solubility profiles of CPHs at different DH are shownin Figure 2. All CPHs presented typical bell-shaped solubilitycurves with minimum solubility at pH 4, which maycorrespond to the isoelectric point of protein hydrolysates,and high solubility at acidic and alkaline pH. The solubilitiesof CPHs were quite low at pH 4, whereas solubilitiesabove 78% were noticeable at other pHs tested. Undigested

Table 2: Inhibition of lipid peroxidation by CPHs at differentdegrees of hydrolysis was determined as described in the text after 8daysa.

NH1-CPH Alcalase-CPH

DH (%)Inhibition of lipidperoxidation (%)

DH (%)Inhibition of lipid

peroxidation

UCMP 2.00 ± 0.01 UCMP 2.00 ± 0.01

3.6 18.60 ± 1.27 5.6 25.30 ± 2.40

5.5 29.00 ± 1.55 7.6 37.00 ± 2.75

7.2 45.00 ± 1.83 8.7 42.00 ± 1.97

9.1 52.00 ± 2.05 9.6 46.50 ± 2.61

11.1 58.00 ± 2.68 10.8 52.00 ± 2.19

12.9 65.00 ± 1.62 11.6 56.50 ± 2.05

14.5 69.00 ± 1.55 12.3 58.00 ± 1.83

15.0 74.00 ± 1.62 12.5 58.30 ± 2.26aMean ± SD from triplicate determinations.

cuttlefish muscle protein (UCMP) was less soluble than thehydrolysates, having a solubility of 7.86 ± 0.026% and 15.86± 0.056% at pH 4.0 and 9.0, respectively (data not shown).

As shown in Figure 2, the solubility increased withincreasing protein hydrolysis. At pH 7.0, the solubility ofAlcalase-CPH (DH = 12.5%) and NH1-CPH (DH = 15%)reached about 93.8 ± 0.22 and 96.33 ± 0.7%, respectively,significantly higher (P < 0.05) than that of the UCMP(12.05 ± 0.017%). The obtained results are in line withthose of Klompong et al. [10] and Gbogouri et al. [37]who reported that hydrolysates of yellow stripe trevally meatprotein and salmon byproduct had an excellent solubility athigh degrees of hydrolysis. From these results, we can deducethat the solubility increases with the protein fraction withlower molecular mass at higher degrees of hydrolysis. Thesmaller peptides are expected to have proportionally morepolar residues, with the ability to form hydrogen bonds withwater and increase solubility [37].

In addition, the lowest solubility of CPHs observedat pH 4.0 could be attributed to both net charge ofpeptides, which increase as pH moves away from pI, andsurface hydrophobicity, that promotes the aggregation viahydrophobic interaction [46]. The pH affects the chargeon the weakly acidic and basic side chain groups, andhydrolysates generally show low solubility at their isoelectricpoints [47].

3.4. Antioxidant Activity

3.4.1. Inhibition of Linoleic Acid Autoxidation. In vitro lipidperoxidation inhibition activities of CPHs were determinedby assessing their ability to inhibit oxidation of linoleicacid in an emulsified model system. As shown in Table 2,all hydrolysates with different DH could act as significantretarders (P < 0.05) of lipid peroxidation. The hydrolysatesinhibiting lipid oxidation exhibited a nonlinear pattern. Forthe tow protease preparations used for hydrolysis reactionof cuttlefish muscle protein, the effect of inhibiting lipid


0

0.4

0.8

1.2

1.6

2

0 2 4 6 8 10 12

Incubation time (day)

Abs

orba

nce

at50

0n

m

Control

NH1 proteases (DH = 15%)

BHA (2 mM; 0.36 mg/mL)

Alcalase (DH = 12.5%)α-tocopherol (2 mM; 0.86 mg/mL)

Figure 3: Comparison of inhibition of lipid peroxidation betweenNH1-CPH (DH = 15.0%), Alcalase-CPH (DH = 12.5%), at1 mg/mL, α-tocopherol (2.0 mM; 0.86 mg/mL), and BHA (2.0 mM;0.36 mg/mL). Bars represent standard deviations from triplicatedeterminations.

oxidation increased initially and peaked on 4.0 h of hydrol-ysis. According to Dong et al. [48], the effect of inhibitinglipid oxidation of Alcalase-hydrolyzed carp protein increasedinitially and peaked on 1.5 h of hydrolysis, followed by aslight decline during the 6 h of hydrolysis. Wu et al. [14]found that the antioxidant activity of hydrolysates derivedfrom mackerel protein reached a maximum after 10 h ofhydrolysis and then declined slightly during the 25 h ofhydrolysis.

The comparative study between NH1-CPH (DH =15%) and Alcalase-CPH (DH = 12.5%) and commercialantioxidants (α-tocopherol and BHA) on the inhibition oflipid peroxidation were conducted and illustrated in Figure 3.Only NH1-CPH presents a comparable effect than naturalantioxidant α-tocopherol. However, both hydrolysates havemoderate protective effect on lipid peroxidation in compari-son with a synthetic antioxidant BHA.

Generally, the lack of a direct relationship betweenantioxidant activity and DH suggested that the specificcomposition (e.g., type of peptides, ratio of different freedamino acids) was an important factor as well [49]. Manyresearchers reported that low molecular weight peptidesshowed higher antioxidant activity [50]. In addition, Kongand Xiong [49] reported that if the hydrolysis of zein proteinwith Alcalase became too extensive (time of hydrolysis >4 h),the hydrolysate could reduce the peptide’s ability to act as aphysical barrier to prevent oxidants from reaching the lipidfraction in the liposome.

3.4.2. DPPH Radical Scavenging Activity. DPPH is a stablefree radical that shows maximum absorbance at 517 nm.When DPPH radicals encounter a proton-donating substratesuch as an antioxidant, the radicals would be scavenged, and

Table 3: DPPH radical scavenging activity of CPHs with differentDH at a sample concentration of 2.0 mg/mLa.


DH (%)DPPH scavenging

activity (%)DH (%)

DPPH scavengingactivity (%)

UCMP 1.67 ± 0.01 UCMP 1.67 ± 0.01

3.6 25.00 ± 1.32 5.6 13.00 ± 1.09

5.5 40.22 ± 1.08 7.6 28.66 ± 1.64

7.2 55.60 ± 1.30 8.7 44.40 ± 1.20

9.1 60.50 ± 1.45 9.6 53.00 ± 1.32

11.1 67.30 ± 1.54 10.8 58.30 ± 0.98

12.9 70.22 ± 1.25 11.6 63.30 ± 1.24

14.5 70.33 ± 2.26 12.3 65.80 ± 1.82


the absorbance is reduced [51]. The decrease in absorbanceis taken as a measure for radical-scavenging. Thus, theDPPH radicals were widely used to investigate the scavengingactivity of some natural compounds. Table 3 shows theDPPH radical scavenging activities of CPHs with differentDH. Both hydrolysates exhibited significant hydroxyl radicalscavenging activity (P < 0.05).

The DPPH radical scavenging activity of CPHs increasedwith increasing DH. At all designated DH, NH1-CPHshowed higher activity than did Alcalase-CPH (P < 0.05).For NH1 proteases, when DH was increased from 3.6%to 15%, the DPPH radical scavenging activity markedlyincreased from 25.0% to 71.5%. The result suggested that thepeptides in different hydrolysates might be different in termsof chain length and amino acid sequence, which contributedto varying capabilities of scavenging DPPH radicals. Theincrease in DPPH radical scavenging activity of both proteinhydrolysates was in agreement with Thiansilakul et al. [11]who reported the increase in DPPH radical scavengingactivity as the DH of the hydrolysate from round scad muscleprotein prepared using Flavourzyme and Alcalase increased.However, Klompong et al. [10] found that DPPH radicalscavenging activity of protein hydrolysate prepared fromthe muscle of yellow stripe trevally using Flavourzyme andAlcalase decreased when DH increased. Invert correlationbetween DH and DPPH radical scavenging activity wasobtained for protein hydrolysates prepared from alkaline-aided channel catfish protein isolates using Protamex [52].You et al. [53] reported that loach protein hydrolysateshowed the greater DPPH radical scavenging activity whenDH increased.

The scavenging effect of all CPHs on DPPH radical scav-enging was concentration dependent (Figure 4). The resultclearly indicated that hydrolysate produced by NH proteasesexhibited the highest radical scavenging activity (75.0 ±2.26% at 3 mg/mL). However, the hydrolysate showed a lowerradical-scavenging activity than BHA (89.87 ± 1.5%) at thesame concentration.

The IC50 values were determined. The lower IC50

indicates higher free radical scavenging ability. Hydrolysate


0

20

40

60

80

100

0.5 1 1.5 2 2.5 3Concentration (mg/mL)

Scav

engi

ng

acti

vity

(%)

NH1 proteases (DH = 15%)Alcalase (DH = 12.5%)BHA

Figure 4: DPPH scavenging activity of NH1-CPH (DH = 15.0%)and Alcalase-CPH (DH = 12.5%) at different concentrations. BHAwas used as positive control. Bars represent standard deviationsfrom triplicate determinations.

obtained by treatment with NH1 proteases showed an activeradical scavenger with IC50 about 0.68 mg/mL ± 0.017 thanAlcalase protein hydrolysate (IC50 = 0.99 mg/mL ± 0.024).DPPH is a stable free radical and can be scavenged witha proton-donating substance, such as an antioxidant [54].Therefore, protein hydrolysates from cuttlefish muscle morelikely contained peptides acting as hydrogen donors, therebyscavenging free radicals by converting them into more stableproducts.

3.4.3. Ferrous Chelating Activity. Ferrous chelating activitiesof NH1-CPH and Alcalase-CPH, determined at a sampleconcentration of 2.0 mg/mL, are presented in Table 4. Thechelating activity of hydrolysates increased with DH. NH1-CPH showed a higher chelating activity (P < 0.05) thanAlcalase-CPH at any designated DH. Ferrous ion (Fe2+)is the most powerful prooxidant among metal ions [55],leading to the initiation and acceleration of lipid oxidationby interaction with hydrogen peroxide in a Fenton reactionto produce the reactive oxygen species, hydroxyl free radical(OH•) [56]. Therefore, chelation of metal ions by peptidesin hydrolysates could retard the oxidative reaction. Theresult indicated that a higher DH rendered NH1-CPH andAlcalase-CPH with higher metal chelating activities. Theshorter chain of peptides might lose their ability to formthe complex with Fe2+. For both crude protease preparationsused, NH1-CPH (DH = 15%) and Alcalase-CPH (DH =12.5%), obtained after 4 h of hydrolysis, exhibited a higherferrous-chelating abilities 65.0 ± 1.77% and 55.6 ± 1.45%,respectively. The increased metal chelating activity could beincreased through hydrolysis with certain enzymes.

Peptides in NH1-CPH and Alcalase-CPH could effec-tively chelate the Fe2+, leading to the retardation of initiationstage. The result indicated that the excessive hydrolysis ofmuscle protein resulted in the enhanced ferrous chelatingactivity, compared with the limited hydrolysis. The higher

Table 4: Metal chelating activity of CPHs with different DH at asample concentration of 2.0 mg/mLa.


DH (%)Metal chelating

activity (%)DH (%)

Metal chelatingactivity (%)

UCMP 1.22 ± 0.02 UCMP 1.22 ± 0.02

3.6 26.00 ± 1.32 5.6 16.00± 1.25

5.5 36.00 ± 2.36 7.6 21.00 ± 1.17

7.2 44.60 ± 1.14 8.7 27.00 ± 0.81

9.1 48.80 ± 0.21 9.6 37.00 ± 1.48

11.1 56.40 ± 1.06 10.8 46.40 ± 1.16

12.9 62.00 ± 1.13 11.6 52.00 ± 1.00

14.5 64.00 ± 0.84 12.3 54.00 ± 0.65


chelating activities of both hydrolysates were coincidentalwith the higher DPPH and in vitro lipid peroxidation inhi-bition activity, as the DH increased. Fe2+ chelating activityof round scad protein hydrolysate prepared using Alcalaseshowed the increase in chelating activity with increasing DH,but those treated with Flavourzyme showed no differencein activity at all DH tested [11]. With the same enzymesused, chelating activity of protein hydrolysate prepared fromthe muscle of yellow stripe trevally increased with increasingDH [10]. Higher ferrous chelating activity was reported forhydrolysate of silver carp using Alcalase and Flavourzymewhen DH increased [48]. Apart from Fe, other transitionmetals, such as Cu and Co, can affect the rate of lipidoxidation and decomposition of hydroperoxide. Theodoreet al. [52] reported that Cu2+ chelating activity of catfishprotein hydrolysate increased with increasing DH.

Some proteins and peptides can chelate metal ions likeFe2+ due to the presence of carboxyl and amino groupsin the side chains of acidic and basic amino acids [57].Alcalase is endopeptidase capable of hydrolyzing proteinswith broad specificity for peptide bonds and is preferedfor the uncharged residue [10], whereas NH1 proteasesis a mixture of multiple proteases, which can produceboth amino acids and peptides [58]. Hydrolysates showingdifferent antioxidative activities might be attributed to thedifferences in the exposed side chains of peptides as governedby the specificity of different proteases towards peptidebonds in the proteins [59]. DH also greatly influenced thepeptide chain length. The higher DH was, the more cleavageof peptide chains took place. Peptides with various sizesand compositions had different capacities of scavenging orquenching free radicals [10, 11]. NH1 proteases and Alcalasemore likely cleaved the peptide bonds in cuttlefish muscle atdifferent positions, resulting in the different products withvarying antioxidative activities.

The ferrous chelating activity, at different concentration,was also studied with NH1-CPH and Alcalase-CPH having,respectively, a DH of 15% and 12.5%, and compared withthat of the EDTA (Figure 5). As shown in Figure 5, both


Table 5: Antioxidant activity using the β-carotene bleachingmethod of CPHs with different DH at a sample concentration of2.0 mg/mLa.


DH (%)Antioxidant activity

(%)DH (%)

Antioxidant activity(%)

UCMP 1.43 ± 0.01 UCMP 1.43 ± 0.01

3.6 16.50 ± 2.65 5.6 26.50 ± 1.22

5.5 31.50 ± 1.61 7.6 41.50 ± 1.47

7.2 44.50 ± 1.30 8.7 54.50 ± 1.60

9.1 56.50 ± 2.31 9.6 61.50 ± 1.66

11.1 67.50 ± 1.68 10.8 64.50 ± 1.47

12.9 72.00 ± 1.42 11.6 67.50 ± 1.68

14.5 72.20 ± 1.01 12.3 69.50 ± 1.10


activities increased with increasing hydrolysate concentra-tion, and reached a maximum activity with 2 mg/mL, andfurther increase in hydrolysate concentration did not affectthe activity. The IC50 values were about 0.94 ± 0.24 mg/mLand 1.25 ± 0.68 mg/mL, for NH1-CPH, and Alcalase-CPHrespectively. However, both hydrolysates showed a lowermetal chelating activity than did EDTA at all concentrationstested. For example, at 2 mg/mL the metal chelating activitiesof NH1-CPH, Alcalase-CPH, and EDTA were 74.0% ± 1.55,61.2% ± 1.34, and 98% ± 1.02, respectively.

3.4.4. Antioxidant Activity in β-Carotene Linoleic Acid Emul-sion Model System. The antioxidative activity of cuttlefishprotein hydrolysates, NH1-CPH, and Alcalase-CPH werestudied in β-carotene linoleic acid oxidation model systemas presented in Table 5. When the oxidation of linoleicacid occurs, free radicals formed are able to attack thehighly unsaturated β-carotene molecules. As a result, β-carotene is oxidized, leading to the losses in chromophoreand characteristic orange colour of β-carotene [60]. Thepresence of antioxidant in linoleic acid emulsion systemhinders β-carotene bleaching, due to the chain-breakinginhibition of lipid peroxidation by neutralizing the linoleicfree radical formed.

As shown in Table 5, NH1-CPH and Alcalase-CPHinhibited significantly (P < 0.05) the oxidation of β-caroteneto different degrees. During the first hour of hydrolysis,Alcalase-CPH achieved rapidly a higher ability to preventthe bleaching of β-carotene (61.5 ± 1.66%) than NH1-CPH (56.5 ± 2.31%). When hydrolysis reached a finalDH, both hydrolysates showed a similar antioxidant activity,approximately 70%.

The antioxidant activity of NH1-CPH (DH = 15%) andAlcalase-CPH (DH = 12.5%) at different concentrations wasalso evaluated (Figure 6). As can be seen in Figure 6, theantioxidant activity of both cuttlefish protein hydrolysatesincreased with increasing sample concentration. The NH1protein hydrolysate showed the highest ability to preventbleaching of β-carotene with 81.11% inhibition at 3 mg/mL.

0

20

40

60

80

100

0.5 1 1.5 2 2.5 3

Concentration (mg/mL)

Met

alch

elat

ing

acti

vity

(%)

NH1 proteases (DH = 15%)

EDTAAlcalase (DH = 12.5%)

Figure 5: Relative chelating activity of NH1-CPH (DH =15.0%)and Alcalase-CPH (DH = 12.5%) at different concentrations. EDTAwas used as positive control. Bars represent standard deviationsfrom triplicate determinations.

0

20

40

60

80

100

0.5 1 1.5 2 2.5 3

Concentration (mg/mL)

An

tiox

idan

tac

tivi

ty(%

)

NH1 proteases (DH = 15%)Alcalase (DH = 12.5%)BHA

Figure 6: Antioxidant activity using the β-carotene bleachingmethod of NH1-CPH (DH = 15.0%) and Alcalase-CPH (DH= 12.5%) at different concentrations. BHA was used as positivecontrol. Bars represent standard deviations from triplicate determi-nations.

Thereafter, the lower β-carotene bleaching in the systemscontaining these hydrolysates was observed, compared withthe system added with BHA. The ability of hydrolysates toprevent the bleaching of β-carotene was more likely governedby their amphiphilic properties of amino acids compositions.Localization/orientation at the interface of hydrophobicpart of peptides to oil phase and of hydrophilic portionto aqueous phase at the interface is the major principlefor emulsion stabilization [60]. The result suggested thatprotein hydrolysates tested contained peptides with bothhydrophilic and hydrophobic portions, in which the peptidesegments with hydrophobicity in nature could adsorb atthe oil droplet, while the hydrophilic domains preferablysuspended in the aqueous phase. The localization of peptideswith antioxidative activity at the oil-water interface could


Table 6: Amino acid composition (% mole) of CPHs usingAlcalase and NH1 proteases. Values shown are mean values of threemeasurements.

Amino acida Alcalase-CPH NH1-CPH

Aspartic acid 6.01 6.14

Threoninec 2.10 2.66

Serine 9.14 9.80

Glutamic acid 11.33 9.54

Glycine 3.05 3.11

Alanine 3.65 3.60

Valinec 6.01 6.89

Methioninec 2.95 3.01

Isoleucinec 3.62 3.81

Leucinec 8.78 8.90

Tyrosine 5.80 5.72

Phenylalaninec 4.01 3.99

Histidinec 11.61 11.23

Lysinec 9.77 9.55

Arginine 11.71 11.57

Cysteine 0.17 0.15

Proline 0.29 0.33

TAAd 100 100

THAAd 32.36 33.64

TEAA/TAA% 48.85 50.04aThe aspartic and glutamic acid contents include, respectively, asparagines

and glutamine.bUndigested cuttlefish muscle protein.cEssential amino acids.dTAA ≡ total amino acids; THAA ≡ total hydrophobic amino acids;TEAA ≡ total essential amino acids.

favor their antioxidative activity for oil droplet. As a result,the antioxidative activity could be maximized at the interfaceof emulsion.

3.5. Amino Acid Composition. Amino acid compositions ofCPHs are shown in Table 6. Both hydrolysates containedhistidine and arginine as the major amino acids and were alsorich in glutamic acid/glutamine, leucine, lysine, and serine.However, both hydrolysates contained low level of cysteineand proline. From the results, both hydrolysates contained alow level of proline up to 0.29% and 0.33%.

Based on total amino acids, essential amino acids madeup 48.85% and 50.04% of Alcalase-CPH and NH1-CPH,respectively. Therefore, they could serve as the excellentsource of useful nutrients. Generally, the differences in aminoacid composition between both hydrolysates depended onthe existing differences in enzyme specificity and hydrolysisconditions [47].

As presented in Table 6, the total content of hydrophobicamino acids of cuttlefish protein hydrolysates obtained atDH of 12.5% and 15% with Alcalase and NH1 proteases washigher, which accounted for 32.36% and 33.64% of the totalamino acids, respectively. Amino acids in cuttlefish proteinhydrolysates are possibly involved in antioxidative activity.Amino acids have been known to exhibit antioxidant activity;

tryptophan and histidine showed high antioxidative activityin comparison with methionine, cysteine, glycine, andalanine [61]. Antioxidative activity of histidine or a histidinecontaining peptide may be attributed to the chelating andlipid radical-trapping ability of the imidazole ring, whereasthe tyrosine residue in the peptide may act as a potenthydrogen donor [16]. Generally, aromatic amino acids areconsidered to be effective radical scavengers, because theycan donate protons easily to electron-deficient radicals. Atthe same time, their antioxidative stability can remain viaresonance structures [62]. From the results, cuttlefish proteinhydrolysate had a high nutritional value, based on its aminoacid profile.

4. Conclusions

The objective of this work was to investigate biochemicalproperties and the potential antioxidant effect of cuttlefishmuscle protein during hydrolysis. The cuttlefish proteinhydrolysed with alkaline proteases from B. licheniformis NH1(NH1-CPH) and Alcalase (Alcalase-CPH) resulted productswith an excellent solubility over a wide pH range. Theantioxidative activity of protein hydrolysate from cuttlefishmuscle was governed by enzymes used. Moreover, NH1-CPHand Alcalase-CPH exhibited high antioxidant activity, andthe highest activities were obtained with a DH of 15% and12.5%, respectively. Although both hydrolysates were lesseffective than positive controls like BHA, fish hydrolysatesin general are considered safe products, and they are notsubjected to restricted use in foods. Therefore, cuttlefishmuscle protein hydrolysate can be used in food systems asa natural additive possessing antioxidative properties.

Further works should be done to isolate and identify thespecific peptides in cuttlefish protein hydrolysates that areresponsible for the overall antioxidative capability.

Acknowledgment

This work was funded by the Ministry of Higher Educationand Scientific Research, Tunisia.

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[2] D. A. Butterfield, A. Castegna, C. B. Pocernich, J. Drake,G. Scapagnini, and V. Calabrese, “Nutritional approaches tocombat oxidative stress in Alzheimer’s disease,” Journal ofNutritional Biochemistry, vol. 13, no. 8, pp. 444–461, 2002.

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Research Article

Mackerel Trypsin Purified from Defatted Viscera bySupercritical Carbon Dioxide

Byung-Soo Chun,1 Hideki Kishimura,2 Sitthipong Nalinanon,3 Sappasith Klomklao,4

and Soottawat Benjakul5

1 Department of Food Science and Technology, Pukyong National University, Busan 608-737, Republic of Korea2 Research Faculty of Fisheries Sciences, Hokkaido University, Hakodate, Hokkaido 041-8611, Japan3 Faculty of Agro-Industry, King Mongkut’s Institute of Technology Ladkrabang, Choakhunthaharn Building,Choakhunthaharn Rd., Ladkrabang, Bangkok 10520, Thailand

4 Department of Food Technology, Faculty of Agro-Industry, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand5 Department of Food Science and Technology, Faculty of Technology and Community Development, Thaksin University,Phattalung Campus, Phattalung 93110, Thailand

Correspondence should be addressed to Hideki Kishimura, [email protected]

Received 22 February 2011; Accepted 10 May 2011

Academic Editor: Moncef Nasri

Copyright © 2011 Byung-Soo Chun et al. This is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properlycited.

Viscera of mackerel (Scomber sp.) were defatted by supercritical carbon dioxide (SCO2) treatment. Trypsin (SC-T) was thenextracted from the defatted powder and purified by a series of chromatographies including Sephacryl S-200 and Sephadex G-50.The purified SC-T was nearly homogeneous on SDS-PAGE, and its molecular weight was estimated as approximately 24,000 Da.N-terminal twenty amino acids sequence of SC-T was IVGGYECTAHSQPHQVSLNS. The specific trypsin inhibitors, soybeantrypsin inhibitor and TLCK, strongly inhibited the activities of SC-T. The pH and temperature optimums of SC-T were at aroundpH 8.0 and 60◦C, respectively, using Nα-p-tosyl-L-arginine methyl ester as a substrate. The SC-T was unstable below pH 5.0 andabove 40◦C, and it was stabilized by calcium ion. These enzymatic characteristics of SC-T were the same as those of other fishtrypsins, especially spotted mackerel (S. borealis) trypsin, purified from viscera defatted by acetone. Therefore, we concluded thatthe SCO2 defatting process is useful as a substitute for organic solvent defatting process.

1. Introduction

Fish viscera are one of the sources of digestive enzymes thatmay have some unique properties of fascinate with bothbasic research and industrial applications. Their survival inwaters required adaptation of their enzyme activity to lowtemperatures of their habitats. That is to say, fish proteinaseshave higher catalytic efficiency at low temperatures thanthose from warm-blooded animals [1, 2]. In addition, thestrong positive correlation between the habitat temperatureof marine fish and the thermostability of its trypsin has beendemonstrated [3–11]. High activity at low temperatures andinstability against heat, low pH, and autolysis of fish pro-teinases are interesting for some industrial applications [12].Cod trypsin is already practically used in food production

and cosmetics [13, 14]. Furthermore, Pacific cod and Atlanticcod trypsins were utilized as catalyst of enzymatic peptidesynthesis [9, 15].

On the other hand, lipids in the tissue prevent fromextracting, preparing, and purifying enzymes [16]. Conven-tional methods for the removal of lipids from materialsinvolve cooking, pressing, and liquid extraction. On liquidextraction for enzyme preparation, it is usually used withorganic solvents, such as hexane, ethanol, and acetone, andso forth [16, 17]. However, The removal of lipids withorganic solvents causes protein denaturation and/or loss offunctional properties [18]. Organic solvents are also highlyflammable and are toxic for human health. Considerationof such factors has led investigators to apply supercriticalfluid extraction techniques to the lipid separation [19].


Carbon dioxide (CO2) is a popular supercritical extractantparticularly in food processing, flavor and aroma isolation,and pharmaceuticals manufacture, because CO2 is nontoxicand does not leave a residue. Supercritical CO2 (SCO2) hasbeen used for extraction of oils from some marine organisms[20–22]. But, the aims of these studies were mainly thegain of oils rich in polyunsaturated fatty acids, especiallyEPA and DHA. So, the application of SCO2 for isolationof enzymes and production of quality protein meal fromdifferent sources should be examined.

Recently, we prepared a defatted powder of squid visceratreating with SCO2 and detected protease, lipase, and amy-lase activities in crude extract from the powder [18]. Next,we purified a phospholipase A2 from the starfish pyloricceca defatted by SCO2 extraction process [23]. In this study,with the aim of utilization of fish trypsin for food industry,we purified a trypsin (SC-T) from the mackerel viscerapowder treated by SCO2 defatting process and comparedits enzymatic properties with those of other fish trypsinspurified from the viscera defatted by acetone.


2.1. Materials. Mackerel (Scomber sp.) were caught offBusan, Republic of Korea. The mackerel viscera were col-lected from F & F Co., Busan, Republic of Korea, and the vis-ceral waste was brought to the laboratory in iced condition.The CO2 (99.99% pure) was supplied by KOSEM, Korea.Sephacryl S-200 and Sephadex G-50 were purchased fromGE Healthcare UK Ltd. (Amersham, UK). Nα-p-Tosyl-L-arginine methyl ester hydrochloride (TAME) and Ethylene-diaminetetraacetic acid (EDTA) were obtained from WakoPure Chemicals (Osaka, Japan). 1-(L-trans-epoxysuccinyl-leucylamino)-4-guanidinobutane (E-64), soybean trypsininhibitor, N-p-tosyl-L-lysine chloromethyl ketone (TLCK),and pepstatin A were purchased from Sigma Chemical Co.(St. Louis, Mo, USA).

2.2. Condition of Supercritical Fluid Defatting. The defattedpowder of mackerel viscera was prepared as described byChun et al. [23] using semibatch type of supercritical fluidextraction unit. The lipid extraction by SCO2 was performedat temperature of 45◦C and pressure of 25 MPa. The totalextraction time was 2.5 h. The SCO2 defatted powder wasstored at −60◦C until further analysis.

2.3. Purification of Mackerel Trypsin (SC-T) from SCO2

Defatted Powder. Trypsin was extracted by stirring from10.0 g of defatted powder in 50 volumes of 10 mM Tris-HCl buffer (pH 8.0) containing 1 mM CaCl2 at 5◦C for3 h. The extract was centrifuged (H-200, Kokusan, Tokyo,Japan) at 10,000 xg for 10 min, and then the supernatantwas concentrated by lyophilization and used as crude trypsin(50 mL). Ten milliliters of crude trypsin was applied for fourtimes to a column of Sephacryl S-200 (3.9× 64 cm) pre-equilibrated with 10 mM Tris-HCl buffer (pH 8.0) contain-ing 1 mM CaCl2,and proteins were eluted (0.8 mL/min) withthe same buffer. Each main trypsin fractions were gathered

and concentrated by lyophilization. Then the concentratedfraction (10 mL) was applied to a Sephadex G-50 column(3.9× 64 cm) pre-equilibrated with the above buffer, andproteins were eluted (0.7 mL/min) with the same buffer.A single trypsin fraction was pooled and used as purifiedtrypsin (SC-T).

2.4. Assay for Trypsin Activity. Trypsin activity was measuredby the method of Hummel [24] using TAME as a substrate.One unit of enzyme activity was defined as the amountof the enzyme hydrolyzing one micromole of TAME in aminute. The effect of inhibitors on trypsin was determinedby incubating trypsin with an equal volume of proteinaseinhibitor solution to obtain the final concentration desig-nated (0.1 mM E-64, 1 mg/mL soybean trypsin inhibitor,5 mM TLCK, 1 mM pepstatin A and 2 mM EDTA) [25]. Afterincubation of the mixture at 25◦C for 15 min, the remainingactivity was measured, and percent inhibition was thencalculated. The pH dependencies of trypsin were determinedin 50 mM buffer solutions [acetic acid-sodium acetate (pH4.0–7.0), Tris-HCl (pH 7.0–9.0), and glycine-NaOH (pH9.0–11.0)] at 30◦C. The temperature dependencies of trypsinwere determined at pH 8.0 and at various temperatures.The temperature and pH stabilities of trypsin were found byincubating enzyme at pH 8.0 for 15 min at a range of 20–80◦C and by incubating the enzyme at 30◦C for 30 min ata range of pH 4.0–11.0, respectively. The effect of CaCl2 ontrypsin activity was found by incubating the enzyme at 30◦Cand at pH 8.0 in the presence of 10 mM EDTA or 10 mMCaCl2.

2.5. Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophore-sis (SDS-PAGE). SDS-PAGE was carried out using a 0.1%SDS-13.75% polyacrylamide slab gel by the method ofLaemmli [26]. The gel was stained with 0.1% CoomassieBrilliant Blue R-250 in 50% methanol-7% acetic acid, andthe background of the gel was destained with 7% acetic acid.

2.6. Analysis of Amino Acid Sequence. To analyze the N-terminal amino acid sequence of SC-T, the enzyme was elec-troblotted to a polyvinylidene difluoride (PVDF) membraneafter SDS-PAGE. The amino acid sequence of the enzyme wasanalyzed by using a protein sequencer, Procise 492 (PerkinElmer, Foster City, Calif, USA).

2.7. Protein Determination. The protein concentration wasdetermined by the method of Lowry et al. [27] using bovineserum albumin as a standard.


3.1. Effect of SCO2 Defatting Process for Trypsin Activity. Theviscera of mackerel (Scomber sp.) were treated by SCO2 toseparate lipids on the condition of 40◦C, 25 MPa, and 2.5 h.Since SCO2 extracted almost all oil from the squid viscerain the previous study, we adopted the condition to removelipids from the mackerel viscera [18]. Trypsin was thenextracted from 10.0 g of defatted powder by SCO2, and the


Table 1: Purification of trypsin (SC-1) from mackerel visceradefamed by SCO2.

Purification Protein Total Specific Purification Yield

stages (mg) activity activity (fold) (%)

(U) (U/mg)

Crude enzyme 1,390 1,049 0.8 1 100

Sephacryl S-200 502 946 2 3 90

Sephadex G-50 15 547 36 48 50

Table 2: Effects of various inhibitors on the activity of SC-Ta.

Inhibitors Concentration % Inhibition

Control — 0

Soybean trypsin inhibitor 1 mg/mL 100

TLCK 5 mM 92

E-64 0.01 mM 0

Pepstatin A 0.01 mM 0

EDTA 2 mM 0aThe enzyme solution was incubated with the same volume of inhibitorat 25◦C for 15 min, and residual activity was analysed using TAME as asubstrate for 5 min at pH 8.0 and 30◦C.

crude enzyme was prepared. As shown in Table 1, the crudeenzyme contained 1,390 mg of total protein and 1,049 U oftotal trypsin activity. Previously, we extracted trypsin fromthe pyloric ceca powder (13.9 g) of spotted mackerel defattedby acetone, and 3,633 mg of total protein and 3,270 U of totaltrypsin activity were detected in the crude enzyme solution[5]. Although these data were not compared directly, theyields of protein and trypsin activity per weight of acetonepowder were approximately two times higher than thoseof SCO2 powder. However, the specific activity (0.8 U/mg)of crude enzyme in this study is almost the same as that(0.90 U/mg) in the previous study [5]. So, it is thought thatthe difference of total activity might come from the variationof specimen, and the defatting condition with SCO2 inthis study would not cause significant denaturation of fishtrypsin.

3.2. Purification of SC-T. The SC-T was purified from thecrude enzyme solution by two steps of chromatographiesincluding Sephacryl S-200 and Sephadex G-50, which isthe same purification procedure as that in the previousstudy [5]. The SC-T was consequently purified 48-fold witha high recovery (50%) from the crude enzyme solution(Table 1) and had a specific activity of 36 U/mg which isfairly higher than that of spotted mackerel trypsin [5].In addition, the SC-T was found nearly homogeneouson SDS-PAGE (Figure 1), and its molecular weight wasestimated as approximately 24,000, which is similar to that ofspotted mackerel trypsin [5]. Furthermore, the N-terminaltwenty amino acids sequence of SC-T was analyzed tobe IVGGYECTPYSQPWTVSLNS that accords with that ofspotted mackerel trypsin [5]. These results also show thatthe SCO2 defatting process in this study removes lipids in

45 kDa

24 kDa

18.4 kDa

14.3 kDa

1 2

Figure 1: Electrophoresis of the trypsin (SC-T) purified from themackerel (Scomber sp.) viscera defatted by SCO2. Electrophoresiswas performed using a 0.1% SDS-13.75% polyacrylamide slabgel. (1) contains protein standards; ovalbumin (molecular weight,45 kDa), bovine pancreatic trypsinogen (24 kDa), bovine milk b-lactoglobulin (18.4 kDa), and egg-white lysozyme (14.3 kDa). (2)contains mackerel trypsin (SC-T).

fish viscera as effectively as acetone defatting process forpreparation of fish trypsin.

Crude enzyme extract usually contains various proteins,and sometimes fish trypsin is composed of some isozymes.In general, the purification of fish trypsin was carried out bythe combination of some types of chromatography [28–30].However, we achieved a high purification of a trypsin fromthe mackerel viscera powder defatted by acetone using onlytwo steps of gel filtration [5]. So, in this study, we purifiedthe SC-T by gel filtration according to the previous study.

3.3. Enzymatic Properties of SC-T. The SC-T was stronglyinhibited by specific trypsin inhibitors (soybean trypsininhibitor and TLCK), but E-64 (cysteine proteinaseinhibitor), pepstatin A (aspartic proteinase inhibitor), andEDTA (metalloproteinase inhibitor) had no inhibitory effecton the activity of SC-T (Table 2).

The influence of pH on the SC-T activity is shownin Figure 2(a). The enzyme hydrolyzed TAME substrateeffectively between pH 7.0 and 9.0, with an optimum aroundpH 8.0. The optimum pH of SC-T was the same as those ofother fish trypsins [3–11, 31–38], but lower than those ofbluefish (pH 9.5) [39] and Atlantic bonito (pH 9.0) [40].Figure 2(b) shows the temperature dependencies of SC-T.The SC-T was active over a broad temperature range (20–70◦C) with the optimum at about 60◦C. Because mackerel isa temperate-zone fish, the SC-T possesses similar optimumtemperature with other trypsins from temperate-zone fish,such as anchovy [3], true sardine [4], yellow tail [6], and


100

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vity

(%)

(b)

Figure 2: Effects of pH and temperature on the activity of SC-T. (a) The activity was determined in 50 mM buffer solutions [acetic acid-sodium acetate (pH 4.0–7.0), Tris-HCl (pH 7.0–9.0), and glycine-NaOH (pH 9.0–11.0)] at 37◦C. (b) The activity was determined at pH 8.0and at various temperatures.

jacopever [7]. The optimum temperature SC-T is slightlylower than those of tropical-zone fish (around 65◦C) [33–35, 39, 40] but is evidently higher than those of frigid-zonefish trypsins (around 50◦C) [4, 6–10].

The pH stability of SC-T is shown in Figure 3(a). TheSC-T was stable at 30◦C for 30 min in the pH range frompH 6.0 to 11.0. Unlike mammalian trypsins, diminishedstability of the trypsin was more pronounced after exposureat acidic pH. Instability at acidic pH was also observedfor other fish trypsins [1, 3–11, 32–35, 37, 39, 40]. Fortemperature stability, the SC-T was stable below 40◦C, butthe activity quickly fell over 50◦C (Figure 3(b)). While theSC-T and other temperate-zone fish trypsins are relativelyless stable than tropical-zone fish trypsins, they are obviouslystable than frigid-zone fish trypsins [8, 10]. As describedpreviously, there is a strong relationship between habitattemperature of marine fish and thermostability of theirtrypsins [8, 10].

The effect of calcium ion on the stability of SC-T was theninvestigated. The stability of SC-T was enhanced by calciumion (Figure 4). Similar results have been reported for variousfish trypsins [1, 3–11, 33–35, 39, 40]. Bovine trypsinogenhas two calcium-binding sites, and the primary site, with ahigher affinity for calcium ions, is common in trypsinogenand trypsin and the secondary site is only in the zymogen[41, 42]. Occupancy of the primary calcium-binding sitestabilizes bovine trypsin toward thermal denaturation orautolysis [41, 42]. In the previous paper, we described trypsin

of arabesque greenling which also has the primary calcium-binding site [43]. The SC-T was stabilized by calcium ionfrom denaturation in this study. So, the result suggests thatthe SC-T may possess the primary calcium-binding site likebovine and arabesque greenling trypsins.

4. Conclusion

With the aim of utilization of fish trypsin for food industry,we purified a trypsin (SC-T) from mackerel (Scomber sp.)viscera powder treated by the SCO2 defatting process andcompared its enzymatic properties with those of other fishtrypsins purified from the viscera defatted by acetone. Inthis study, we adopted the condition of 40◦C, 25 MPa, and2.5 h to separate lipids from the viscera. Consequently, wecould remove most of the lipids from the viscera and couldextract considerable amount of trypsin from the defattedpowder. The characteristics of purified SC-T were nearlythe same as those of other fish trypsins, especially spottedmackerel trypsin. Therefore, we concluded that the SCO2

defatting process is useful as a substitute for the organicsolvent defatting process.

Acknowledgments

The authors wish to thank Mr. Hirose, the Center forInstrumental Analysis, Hokkaido University, for amino acidsequence analysis. This research was supported in part by


Res

idu

alac

tivi

ty(%

)

100

50

04 5 6 7 8 9 10 11

pH

(a)

20 30 40 50 60 70

Temperature (◦C)

Res

idu

alac

tivi

ty(%

)

100

50

0

(b)

Figure 3: pH and temperature stability of SC-T. (a) The enzyme was kept at 30◦C for 30 min and pH 4.0–11.0, and then the remainingactivity at 30◦C and pH 8.0 was determined. (b) The enzyme was kept at 20–70◦C for 15 min and pH 8.0, and then the remaining activity at30◦C and pH 8.0 was determined.

Res

idu

alac

tivi

ty(%

)

Time (h)

100

50

00

2 4 6 8

Figure 4: Effect of calcium ion on the stability of SC-T. The enzymewas kept at 30◦C and pH 8.0 for 0–8 h in the presence of 10 mMCaCl2 (closed symbol) or 10 mM EDTA (open symbol), and thenthe remaining activity at 30◦C and pH 8.0 was determined.

a grant of HOKUSUI Association and was also supportedby a grant from Marine Bioprocess Research Center of theMarine Biotechnology Program funded by the Ministry ofLand, Transport and Maritime, Republic of Korea. They alsoappreciate the support of the Core University Program onFisheries Sciences founded by JSPS & KOSEF.

References

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Research Article

Simple Preparation of Pacific Cod Trypsin forEnzymatic Peptide Synthesis

Tomoyoshi Fuchise,1 Haruo Sekizaki,1 Hideki Kishimura,2 Sappasith Klomklao,3

Sitthipong Nalinanon,4 Soottawat Benjakul,5 and Byung-Soo Chun6

1 Faculty of Pharmaceutical Sciences, Health Sciences University of Hokkaido, Ishikari-Tobetsu, Hokkaido 061-0293, Japan2 Laboratory of Marine Products and Food Science, Research Faculty of Fisheries Sciences, Hokkaido University, Hakodate,Hokkaido 041-8611, Japan

3 Department of Food Science and Technology, Faculty of Technology and Community Development, Thaksin University,Phattalung Campus, Phattalung 93110, Thailand

4 Faculty of Agro-Industry, King Mongkut’s Institute of Technology Ladkrabang, Choakhunthaharn Building, Choakhunthaharn Road,Ladkrabang, Bangkok 10520, Thailand

5 Department of Food Technology, Faculty of Agro-Industry, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand6 Faculty of Food Science and Biotechnology, Pukyong National University, Busan 608-737, Republic of Korea

Correspondence should be addressed to Haruo Sekizaki, [email protected] Hideki Kishimura, [email protected]

Received 24 February 2011; Revised 19 June 2011; Accepted 16 July 2011


Copyright © 2011 Tomoyoshi Fuchise et al. This is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properlycited.

Trypsin from the pyloric caeca of Pacific cod (Gadus macrocephalus) was easily prepared by affinity chromatography onBenzamidine Sepharose 6B and gel filtration on Superdex 75. Pacific cod trypsin was composed of three isozymes, and theirmolecular masses were estimated 23,756.34 Da, 23,939.62 Da, and 24,114.81 Da by desorption/ionization time-of-flight massspectroscopy (MALDI/TOF-MS) and their isoelectric points (pIs) were approximately 5.1, 6.0, and 6.2, respectively. The isolatedPacific cod trypsin showed high similarity to other frigid-zone fish trypsins. The kinetic behavior of tryptic hydrolysis towardN-p-tosyl-L-arginine methyl ester hydrochloride (TAME), N-benzoyl-L-arginine p-nitroanilide hydrochloride (BAPA), and p-amidinophenyl ester were also analyzed. In addition, the cod trypsin-catalyzed dipeptide synthesis was investigated usingtwelve series of “inverse subdtrates” that is p- and m-isomer of amidinophenyl, guanidinophenyl, (amidinomethyl)phenyl,(guanidinomethyl)phenyl, and four position isomers of guanidinonaphtyl esters derived from N-(tert-butoxycarbonyl)amino acidas acyl donor components. They were found to couple with an acyl acceptor such as L-alanine p-nitroanilide to produce dipeptidein the presence of the trypsin. All inverse substrates tested in this study undergo less enantioselective coupling reaction. The p-guanidinophenyl ester was most practical substrate in twelve series tested. The enzymatic hydrolysis of the resulting products wasnegligible.

1. Introduction

Proteinases from cold-water fish are of interest to us owingto their greater proteolytic activity towards native proteinsubstrates and lower activation energy for catalysis comparedwith proteinases from mammalian or microbial sources [1].Also, their survival in cold water required adaptation oftheir proteinase activity to low temperatures of their habitats.Proteinases from cold-adapted fish thus often have higherenzymatic activity at low temperatures than those from

warm-blooded animals [2, 3]. High activity of these fishproteinases at low temperatures may be interesting for severalindustrial applications, such as in certain food processingoperations that require low processing temperatures. Fur-thermore, proteinases from cold adapted fish inactivated atrelatively low temperatures making such enzymes potentiallyuseful in food applications where easy enzyme denaturationis desirable [4]. One of the main digestive proteinases, whichare detected in pyloric caeca and intestine of fish, is trypsin(EC 3.4.21.4). Trypsin is a member of a large family of serine


proteinases and cleaves the peptide bond on the carboxyl sideof arginine and lysine. Recently, we isolated trypsins fromthe pyloric caeca of Pacific cod (Gadus macrocephalus) andthe trypsin showed a lower optimum temperature and heatstability than those of temperate-zone fish, tropical-zone fish,and mammalian trypsins [5]. The characteristics suggestedthat the viscera of frigid-zone fish would be a potential sourceof trypsin for food processing operations.

On the other hand, peptide synthesis using protease-catalyzed reverse reaction has been extensively studied witha variety of amino acids and peptide derivatives as couplingcomponents [6–12]. It has been reported that the protease-catalyzed peptide synthesis is superior compared to thechemical coupling methods due to the requirement of lessside chain protection. The method, however, has not beenfully exploited for the possible synthesis of a number ofbiologically important peptides containing D-amino acid orother unnatural amino acid, because the enzymatic methodis subject to restriction by substrate specificity and stere-oselectivity. Previously, we reported that p-amidinophenylesters [13] and p-guanidinophenyl esters [14, 15] behave asspecific substrates for trypsin and trypsin-like enzymes. Inthese esters, the site-specific groups (charged amidinium andguanidinium) for the enzyme are included in the leaving-group portion instead of being in the acyl moiety. Suchsubstrates are termed an inverse substrate (Figure 1) [16].The inverse substrates allow the specific introduction of anacyl group carrying a nonspecific residue into the enzymeactive site. The characteristic features of inverse substratessuggested that they are useful for enzymatic peptide synthe-sis. We demonstrated the successful application of inversesubstrates for trypsin-catalyzed coupling [13, 17]. Recently,Bordusa [11] reported an enzymatic coupling reaction usinginverse substrates and a wide variety of proteases, suchas thrombin, clostripain, V8 protease, chymotrypsin andsubtilisin. We also reported that the substrate specificity andyield were different by origin of trypsin [18, 19].

In the present paper, we describe the simple separationof trypsin from the pyloric caeca of Pacific cod by affin-ity chromatography for enzymatic peptide synthesis usinginverse substrates (Scheme 1, Figure 2). Also, we reportthe selection of practical inverse substrates for Pacific codtrypsin-catalyzed peptide synthesis.


2.1. Materials. Pacific cod (Gadus macrocephalus) was caughtoff Hokkaido Prefecture, Japan. The pyloric caeca of Pacificcod were separated from other intestinal organs and werestored at −84◦C prior to homogenization.

N-p-Tosyl-L-arginine methyl ester hydrochloride(TAME), N-benzoyl-L-arginine p-nitroanilide hydrochlod-ide (BAPA), p-nitrophenyl-p′-guanidinobenzoate hydro-chloride (NPGB), tris(hydroxymethyl)aminomethane (Tris),and 3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid)ware obtained from Sigma-Aldrich (Tokyo, Japan). p-Nitrophenol, HPLC grade dimethyl sulfoxide (DMSO),acetone, and acetonitril were purchased from Kanto

Chemical Co. Inc. (Tokyo, Japan). L-Alanine p-nitroanilide(L-Ala-pNA) was purchased from Peptide Institute, Inc(Osaka, Japan). 3-(N-Morpholino)propanesulfonic acid(MOPS) was obtained from Wako Pure Chemicals (Osaka,Japan). The inverse substrate was prepared accordingto our previous paper [13, 15, 20–22]. BenzamidineSepharose 6B and Superdex 75 were purchased fromGE Healthcare (Buckinghamshire, England). Microplatereader was purchased from Tecan (Kanagawa, Japan). F96microwell plate and UV plate were purchased from Nunc(Buckinghamshire, England) and Corning (Tokyo, Japan),respectively.

2.2. Preparation of Pacific Cod Trypsin. The frozen pyloriccaeca were homogenized in three volumes of cold acetone(−20◦C) using a homogenizer (AM-8; Nihonseiki KaishaLtd., Tokyo, Japan) for 3 min, and the homogenate wasfiltrated in vacuo on ADVANTEC No. 2 filter paper. Similarlythe residue was homogenized in one volume of cold acetone,and was done twice more at same. The residue was air-driedat 4◦C, and was stored at 4◦C prior to enzyme extraction.Trypsin preparation steps below were carried out at 4◦C.

The defatted powder (40 g) was mixed with 400 mL ofbuffer A [50 mM Tris-HCl buffer (pH 7.8) containing 0.5 MNaCl and 20 mM CaCl2]. A trypsinogen in the slurry wasactivated by incubation for 46 hr at 4◦C. The slurry wascentrifuged at 11,500 rpm for 30 min. The supernatant waspooled, and the other precipitate was extracted again with200 mL of buffer A. The collected supernatant was subjectedto solid (NH4)2SO4 fractionation and the precipitate inthe 80% saturation was collected by centrifugation. Theresultant precipitate was then dissolved in 500 mL of bufferA prior to centrifugation. The supernatant was collectedand referred to as “crude enzyme.” The crude enzyme wasapplied to a Benzamidine Sepharose 6B affinity column (i.d.2.5 × 6 cm) preequilibrated with buffer A. The column waswashed with buffer A until no further decrease in absorbanceat 280 nm was observed. The main trypsin fraction waseluted with buffer A containing 20 mM benzamidine. Thefraction was concentrated on Amicon-stirred cell (YM10ultrafiltration membrane, Tokyo, Japan) and applied toSuperdex 75 column (i.d. 2.6× 100 cm) preequilibrated withbuffer A using the FPLC system (GE Healthcare). The peakcontaining TAME esterase activity was collected each and wasconcentrated on Amicon-stirred cell. The concentrate (about5 mL) was dialyzed against 1 L of 50 mM NH4HCO3 solutionfor 1 hr. The dialysate (purified Pacific cod trypsin) waslyophilized and was stored at−20◦C until used in subsequentstudies.

2.3. Protein Determination and Assay for Trypsin Activity.Protein concentrations were determined using a proteinassay kit (BIO-RAD) with bovine serum albumin as thestandard protein [23].

Trypsin activity was measured by the method of Hummel[24] using TAME as a substrate at 30◦C to compare theenzymatic properties with previous our studies [5]. One unitof enzyme activity was defined as the amount of the enzyme


L-form;D-form;

H2N

H2N

C

C

NH

NH

H2NC

NH H2NC

NH

H2NC

NH

NH

H2NC

NH

NH

NH NH

NH

H2NC

NH H2NC

NH

NH

H2NC

NH

H2N

C

NH

NH

H2N

C

NH

H2N

C

NH

NH

CH2

CH2CH2

Boc-Ala-O Boc-Ala-O Boc-Ala-O Boc-Ala-O

Boc-Ala-OBoc-Ala-OBoc-Ala-OBoc-Ala-O

Boc-Ala-O Boc-Ala-O Boc-Ala-O Boc-Ala-O

p-TsOH · p-TsOH · p-TsOH · p-TsOH ·

p-TsOH ·p-TsOH ·p-TsOH ·p-TsOH ·

p-TsOH · p-TsOH ·p-TsOH ·

p-TsOH ·

CH2

1a1b

L-form; 9aD-form; 9b





L-form; 3a

D-form; 3b

L-form; 7a

D-form; 7b


L-form; 4a

D-form; 4b



Figure 1: Structure of inverse substrates.

N-Boc-Ala-O-Ar-(CH2)m-(NH)n CNH

NH2

· TsOH + L-Ala-NH NO2 NO2

PcTN-Boc-Ala- Ala-L- NH

Ar = benzene or naphthalenem or n = 0 or 1

L-form: 1a–12a

D-form: 1b–12b

L-form: 13a

D-form: 13b

Scheme 1: Enzymatic peptide synthesis using inverse substrate.

hydrolyzing one micromole of TAME in a minute. The pHdependencies of trypsin were determined in 50 mM buffer

solutions [acetic acid-sodium acetate (pH 4.0–7.0), Tris-HCl(pH 7.0–9.0), and glycine-NaOH (pH 9.0–11.0)] at 30◦C.


Trypsin Trypsin Trypsin

Asp189 OH

OH

Ser195 OHOH

C

O

O

C

O(Inverse substrate)

C NHO

(Product)(Acyl-enzymeintermediate)

H NH(Acyl acceptor)

R1 R2

R1

R2

R1

Figure 2: Reaction process of trypsin-catalyzed peptide bond formation with inverse substrate.

The temperature dependencies of trypsin were determinedat pH 8.0 and at various temperatures. The temperature andpH stabilities of trypsin were found by incubating enzyme atpH 8.0 for 15 min at a range of 20–80◦C and by incubatingthe enzyme at 30◦C for 30 min at a range of pH 4.0–11.0,respectively.

2.4. Kinetic Measurements. First, the crystallized p-nitrophenol was utilized to determine the path lengthof the solution volume used in the microplate wells duringthe assay according to reported procedure [25]. Theconcentration of active sites of trypsins was determinedwith NPGB from the “burst” of p-nitrophenol at 405 nm(extinction coefficient = 18,200 M−1cm−1), following themethod of Chase and Shaw [26].

Amidase activity as determined by using BAPA asa substrate according to the method of Erlanger etal. [27]. The amount of p-nitroaniline liberated fromBAPA was determined by the increase in absorbance at405 nm (extinction coefficient = 10,200 M−1cm−1). Esteraseactivity was determined by using TAME as a substrateaccording to the method of Hummel [24], measuringthe change in absorbance at 247 nm (extinction coeffi-cient = 540 M−1cm−1). Kinetic parameters using N-(tert-butoxycarbonyl)-L-alanine p-amidinophenyl esters (N-Boc-L-Ala-OpAm, 5a) as inverse substrate were determinedas previously described [16]. Changes of optical den-sity were monitored at 305 nm (extinction coefficient= 16,700 M−1cm−1). Optical density changes at 305 nm(extinction coefficient = 16,700 M−1cm−1) were monitored.

The final reaction mixture (200 μL) consisted of 180 μLof 50 mM Tris-HCl (pH 8.0) containing 10 mM CaCl2, 10 μLof substrate in DMSO, and 10 μL of PcT solution in 10 mMTris-HCl (pH 8.0) containing 2 mM CaCl2. Kinetics param-eters were determined at eight substrate concentrationsin the range of 0.01–0.08 mM for BAPA, 0.004–0.039 mMfor TAME, and 0.0003–0.0038 mM for N-Boc-L-Ala-OpAm(5a) at 30◦C. Kinetic constants were obtained by least-squares fitting of initial velocity data to Lineweaver-Burktransformation of the Michaelis-Menten equation.

2.5. Polyacrylamide Gel Electrophoresis. Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was

carried out using a 0.1% SDS-12.5% polyacrylamide slab-gel by the method of Laemmli [28]. The gel was stainedwith 0.1% Coomassie Brilliant Blue R-250 (CBB) in 50%methanol-7% acetic acid and the background of the gel wasdestained with 7% acetic acid.

Isoelectric focusing (IEF) was carried out with a 111Mini IEF Cell system (BIO-RAD) according to attachedmanual. The gel was fixed with 4% sulfosalicylic acid-12.5%trichloroacetic acid-30% methanol and was stained with0.5% CuSO4-10% acetic acid-27% ethanol-0.04% CBB. Thebackground of the gel was destained with 0.5% CuSO4-7% acetic acid-12% ethanol and then 7% acetic acid-24%ethanol.

2.6. Determination of Molecular Mass. Pacific cod trypsinwas analyzed by matrix-assisted desorption/ionization time-of-flight mass spectroscopy (MALDI/TOF-MS) using a Voy-ager PK2 (Applied Biosystems). The matrix (10 mg/mL) wasprepared by dissolving sinapinic acid in 7 : 3 (acetonitrile:0.1% TFA, 1 : 1, v/v), and used the supernatant.

2.7. Analysis of Amino Acid Sequence. To analyze the N-terminal sequence, the purified enzyme was electroblot-ted on to polyvinylidene difluoride (PVDF) membrane(Immobilon-P, Millipore) after SDS-PAGE. The amino acidsequence of the enzyme was analyzed by using a proteinsequencer, Procise 492 (Perkin Elmer, Foster City, Califf,USA).

2.8. Pacific Cod Trypsin-Catalyzed Dipeptide Coupling Reac-tion. A solution of 40 μL of 50 mM MOPS buffer (containing20 mM of CaCl2, pH 8.0), 30 μL of DMSO, 10 μL of acylacceptor (200 mM L-Ala-pNA in DMSO), and 10 μL oftrypsin solution (100 μM, in 10 mM Tris buffer (pH 8.0)containing 2 mM CaCl2) were mixed. To this mixture, 10 μLof acyl donor (10 mM inverse substrate in DMSO) wasadded and incubated at 25◦C. The progress of the couplingreaction was monitored by HPLC under the followingconditions: Shim-pack CLC-ODS (M) (column i.d. 4.6 ×250 mm), isocratic elution at 1 mL/min, 50% acetonitrilecontaining 0.1% trifluoroacetic acid. An aliquot of thereaction mixture was injected and the eluate was monitoredat 310 nm (chromophore due to the p-nitroanilide moiety).


Table 1: Purification of trypsin from Pacific cod.

Purification stages Protein (mg) Total activity (U)Specific activity

(U/mg)Purity (fold) Yield (%)

Activated crude extract 503 5,281 10.5 1.0 100

80% (NH4)2SO4 fraction 424 5,217 12.3 1.2 99

Gel filtration 17.8 3,400 191 18 64

(kDa)

45

24

1 2

14.3

18.4

(a)

pH 3

4.45, 4.65, 4.75

5.1

6

6.5

6.8, 7

7.1

7.5

7.8, 8, 8.23

9.6

pH 10

1 2

(b)

Figure 3: SDS-PAGE and IEF of purified Pacific cod trypsin. (a) SDS-PAGE of purified Pacific cod trypsin. Lane 1 contains protein standards;egg albumin (molecular weight, 45.0 kDa), bovine pancreatic trypsinogen (24.0 kDa), bovine milk β-lactoglobulin (18.4 kDa), and egg whitelysozyme (14.3 kDa). (b) IEF of purified Pacific cod trypsin. Lane 1 contains purified Pacific cod trypsin. Lane 2 contains IEF isoelectric point(pI) standards: phycocyanin (pI 4.45, 4.65, 4.75), β-lactoglobulin (5.1), bovine carbonic anhydrase (6.0), human carbonic anhydrase (6.5),equine myoglobin (6.8, 7.0), human hemoglobin (7.1), lentil lectin (7.8, 8.0, 8.23), and cytochrome c (9.6).

100

50

09994

23756.34

Inte

nsi

ty(%

)

23939.62

1188

6.6

24114.81

1207

4.45

47341.45

47996.8 85999.6 124002.4

Mass (m/z)

162005.2 200008

Figure 4: MALDI/TOF-MS spectrum of purified Pacific codtrypsin.

Peak identification was made by correlating the retentiontime with that of authentic samples which were chemicallysynthesized [17]. Observed peak areas were used for theestimation of sample concentration.

Table 2: Enzymatic properties of Pacific cod trypsin.

Enzymatic properties Pacific cod trypsin

Optimum pH pH 8.0

Optimum temperature 50◦C

pH stability pH 7.0–10.0

Thermal stability <40◦C


3.1. Simple Preparation of Pacific Cod Trypsin. In this study,the stepwise purification of trypsin (TAME esterase activity)from pyloric caeca of the Pacific cod (G. macrocephalus) isshown in Table 1. Trypsin was prepared from the pyloriccaeca of Pacific cod by affinity chromatography on Ben-zamidine Sepharose 6B and gel filtration on Superdex 75.The overall purification was increased to 18 fold and therecovery was approximately 64%. In other words, about50 mg of purified trypsin was obtained from 40 g of acetonepowder. It has been reported by Ahsan and Watabe [29]


Table 3: Kinetic parameters for Pacific cod trypsin-catalyzed hydrolysis.

Substrate Parameter PcT BT PcT/BT

BAPA Km (mM) 0.037 0.57 0.063

Kcat (sec−1) 3.7 1.7 2.2

Kcat/Km (mM−1s−1) 100 2.9 35

TAME Km (mM) 0.0015 0.021 0.074

Kcat (sec−1) 108 86 1.3

Kcat/Km (mM−1s−1) 71,206 4,160 17

N-Boc-L-Ala-OpAm (4a) Km (mM) 0.0015 0.021 0.071

Kcat (sec−1) 2.0 1.2 1.6

Kcat/Km (mM−1s−1) 1,449 619 2.3

PcT, Pacific cod trypsin; BT, Bovine trypsin.

that Benzamidine Sepharose affinity chromatography usingbenzamidine in a neutral buffer is superior compared toconventional elution procedure with low pH due to theloss of activity in fish trypsin. In this case also, it wassimilar that affinity chromatography was very effective inremoving other protease to the flow-through. In addi-tion, it was considered that the separated protease wastrypsin of high purity (Figure 3(a)). Pacific cod trypsin wascomposed of three isozymes, and their molecular masseswere estimated 23,756.34 Da, 23,939.62 Da, and 24114.8 Daby desorption/ionization time-of-flight mass spectroscopy(MALDI/TOF-MS) (Figure 3(b)) and their isoelectric points(pIs), approximately 5.1, 6.0, and 6.2, were found (Figure 4).Moreover, the activity of the purified enzyme did notdeteriorate after lyophilization (data not shown).

3.2. Enzymatic Properties of Pacific Cod Trypsin. Pacificcod trypsin had the optimum pH of 8.0 and optimumtemperature of 50◦C for hydrolysis of TAME (Table 2). Theenzyme was unstable at above 40◦C and at acidic pH, andits thermal stability was highly calcium dependent (Table 2).These properties showed high similarity to other frigid-zonefish trypsins [5].

Table 3 shows the results obtained from kinetic measure-ments using two synthetic substrates (TAME and BAPA).The kinetic parameters of the Pacific cod trypsin werecompared with those of bovine trypsin. Pacific cod trypsinhydrolyzed ester bonds at a faster rate than the amide bond.This tendency was also observed for bovine trypsin and fortrypsins from fishes [2, 30] and crustaceans [31, 32]. Pacificcod trypsin showed a moderate rate of amide hydrolysis.Kinetic constants of BAPA and TAME hydrolysis by Pacificcod and bovine trypsins were compared at 30◦C (Table 3).For BAPA hydrolysis, turnover number (kcat) of Pacific codtrypsin was 2.2 times faster than that of bovine trypsin, andkm value of Pacific cod trypsin was considerably lower thanbovine enzyme. So, catalytic efficiency for amide hydrolysisexpressed as kcat/km of Pacific cod trypsin was 35 timesefficient than that of bovine trypsin. For TAME hydrolysis,Pacific cod trypsin also showed faster kcat and much lowerkm value than those of bovine counterpart and, consequently,kcat/km of Pacific cod trypsin was about 17 times higher

than that of bovine trypsin. Similar to other enzymeand protein, the industrial application for food, cosmetic,and pharmaceutical of mammalian pancreatic trypsin hasrecently been limited by the outbreak of bovine spongiformencephalopathy (BSE) or religious tradition. So, it is hopedthat the new sources of trypsin will be developed. However,there has been little basic research for trypsin from marineorganisms comparing with mammalian counterparts. Theabove results suggest that Pacific cod trypsin can be utilizedas alternative enzyme of mammalian for practical use.

In addition, the kinetics of Pacific cod trypsin hydrolysisof the inverse substrate, N-Boc-L-Ala-OpAm (5a in Figure 1)was analyzed as representative examples of the series ofcompounds. As shown in Table 3, similar results wereobtained for N-Boc-L-Ala-OpAm (5a). This result suggeststhat Pacific cod trypsin may be an efficient catalyst forpeptide synthesis.

3.3. Pacific Cod Trypsin-Catalyzed Peptide Synthesis. Previ-ously, we reported the behavior of trypsin for the sameinverse substrate was different by the origin of enzyme [18,19] and the yield of coupling reaction with same enzymewas different by the type of inverse substrate [19, 33].In this paper, twelve types inverse substrates were testedowing to selection of the most efficient substrate in thefirst using of Pacific cod trypsin for enzymatic peptidesynthesis. The reaction conditions for Pacific cod trypsin-catalyzed coupling reaction used effective same conditionfor multifarious inverse substrates by bovine trypsin asdescribed in the methods [18]. The results of the Pacific codtrypsin-catalyzed peptide coupling reaction are summarizedin Table 4. In general, Pacific cod trypsin is a moderatelyeffective catalyst for the synthesis of the peptides (entry 1–24in Table 4), toward all inverse substrates derived from L-Ala(1a–12a) and D-Ala (1b–12b).

In any event, all inverse substrates afforded the couplingproduct more or less regardless of their structures. The mosteffective substrate tested in this study was N-Boc-alaninep-guanidinophenyl esters. They (6a and 6b) afforded thecorresponded N-Boc-L-Ala-L-Ala-pNA (entry 6 in Table 4)and N-Boc-D-Ala-L-Ala-pNA (entry 18 in Table 4) in 60%and 75% yield, respectively. In the enzymatic peptide


Table 4: Yield of Pacific cod trypsin-catalyzed peptide synthesis.

Entry no. Acyl donor (no.)Reactiontime (hr)

Product (No.) Yield (%)

1 N-Boc-L-Ala-OmAm (1a) 48 N-Boc-L-Ala-L-Ala-pNA (13a) 44

2 N-Boc-L-Ala-OmGu (2a) 24 N-Boc-L-Ala-L-Ala-pNA (13a) 37

3 N-Boc-L-Ala-OmAM (3a) 24 N-Boc-L-Ala-L-Ala-pNA (13a) 55

4 N-Boc-L-Ala-OmGM (4a) 12 N-Boc-L-Ala-L-Ala-pNA (13a) 51

5 N-Boc-L-Ala-OpAm (5a) 0.2 N-Boc-L-Ala-L-Ala-pNA (13a) 58

6 N-Boc-L-Ala-OpGu (6a) 0.2 N-Boc-L-Ala-L-Ala-pNA (13a) 60

7 N-Boc-L-Ala-OpAM (7a) 5 N-Boc-L-Ala-L-Ala-pNA (13a) 59

8 N-Boc-L-Ala-OpGM (8a) 5 N-Boc-L-Ala-L-Ala-pNA (13a) 59

9 N-Boc-L-Ala-O(1-4)GN (9a) 5 N-Boc-L-Ala-L-Ala-pNA (13a) 56




13 N-Boc-D-Ala-OmAm (1b) 74 N-Boc-D-Ala-L-Ala-pNA (13b) 12

14 N-Boc-D-Ala-OmGm (2b) 74 N-Boc-D-Ala-L-Ala-pNA (13b) 52

15 Nα-Boc-D-Ala-OmAM (3b) 74 N-Boc-D-Ala-L-Ala-pNA (13b) 38

16 N-Boc-D-Ala-OmGM (4b) 74 N-Boc-D-Ala-L-Ala-pNA (13b) 58

17 N-Boc-D-Ala-OpAm (5b) 0.2 N-Boc-D-Ala-L-Ala-pNA (13b) 70

18 N-Boc-D-Ala-OpGu (6b) 0.2 N-Boc-D-Ala-L-Ala-pNA (13b) 75

19 N-Boc-D-Ala-OpAM (7b) 8 N-Boc-D-Ala-L-Ala-pNA (13b) 76

20 N-Boc-D-Ala-OpGM (8b) 48 N-Boc-D-Ala-L-Ala-pNA (13b) 74

21 N-Boc-D-Ala-O(1-4)GN (9b) 5 N-Boc-D-Ala-L-Ala-pNA (13b) 57




1 mM acyl donor, 20 mM acyl acceptor, 10 μM PcT, respectively, in MOPS (50 mM, pH 8.0, containing 20 mM CaCl2) : DMSO = 1 : 1, at 25◦C.

synthesis, secondary hydrolysis of the resulting peptide isunavoidable to some extent. This serious problem can belargely overcome by the use of inverse substrates since theresulting peptide is much less specific to the enzyme than thesubstrate—“preferential coupling to hydrolysis.”

It appears indeed that secondary hydrolysis of thecoupling product was negligible in our enzymatic procedure,since a separate experiment incubating the coupling productfor 74 h resulted in no detectable change. In this study, itwas clarified that Pacific cod trypsin, as well as chum salmontrypsin [33], is suitable for peptide synthesis. However, athermal stability of fish trypsin is lower than mammaliancounterpart, and this property would prevent a massiveand continual application of them in industrial peptidesynthesis. Now, we are investigating the optimization oftrypsin-catalyzed condensation, for example, pH, organicsolvent, and concentration of acyl acceptor. We are alsotrying the immobilization of Pacific cod trypsin to improveits stability for its practical use [34].

Acknowledgments

The authors wish to thank Mr. T. Hirose, the Center forInstrumental Analysis, Hokkaido University, for amino acid

sequence analysis, and Dr. Fumio Kondo, Department ofPharmacology, Aichi Medical University, School of Medicine,for the measurement of the MALDI/TOF-MS. This paper wassupported in part by a Grant-in-Aid for High TechnologyResearch Program and Strategic Project to Support the For-mation of Research Bases at Universities from the Ministryof Education, Culture, Sports, Science, and Technology ofJapan. This paper was also supported in part by a grant ofHOKUSUI Association.

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[14] K. Itoh, H. Sekizaki, E. Toyota, and K. Tanizawa, “Syn-thesis and properties of N-(tert-butyloxycarbonyl)peptide p-guanidinophenyl esters as trypsin substrates,” Chemical andPharmaceutical Bulletin, vol. 43, no. 12, pp. 2082–2087, 1995.

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Review Article

Dicer Functions in Aquatic Species

Yasuko Kitagishi, Naoko Okumura, Hitomi Yoshida, Chika Tateishi,Yuri Nishimura, and Satoru Matsuda

Department of Environmental Health Science, Nara Women’s University, Kita-Uoya Nishimachi, Nara 630-8506, Japan

Correspondence should be addressed to Satoru Matsuda, [email protected]

Received 23 February 2011; Accepted 2 April 2011

Academic Editor: Hideki Kishimura

Copyright © 2011 Yasuko Kitagishi et al. This is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properlycited.

Dicer is an RNase III enzyme with two catalytic subunits, which catalyzes the cleavage of double-stranded RNA to small interferingRNAs and micro-RNAs, which are mainly involved in invasive nucleic acid defense and endogenous genes regulation. Dicer isabundantly expressed in embryos, indicating the importance of the protein in early embryonic development. In addition, Diceris thought to be involved in defense mechanism against foreign nucleic acids such as viruses. This paper will mainly focus on therecent progress of Dicer-related research and discuss potential RNA interference pathways in aquatic species.

1. Introduction

In eukaryotes, small RNA-mediated RNA silencing calledRNA interference (RNAi) is able to suppress gene expression.Dicer is the key enzyme of the RNAi pathway to cleavedouble-stranded RNA (dsRNA) into small RNAs categorizedas small interfering RNAs (siRNAs) or micro-RNAs (miR-NAs), which are mainly involved in invasive nucleic aciddefense and endogenous genes regulation, respectively [1–3]. Then, Dicer is reported to participate in both the antivi-ral immune response and developmental regulation. Forexample, Drosophila harboring the Dicer mutant exhibitedenhanced disease susceptibility to cricket paralysis virus [4,5]. In addition, Caenorhabditis elegans harboring the Dicermutant had developmental phenotype defects [6–8]. ThemeRNAi is a conserved eukaryotic gene silencchanism thatworks at both the transcriptional and the posttranscriptionallevels [9]. We fortuitously cloned and sequenced the humanDicer, (initially designated as HERNA) for the first time [10].Dicer belongs to the RNase III family with ATP dependentRNA helicase, PAZ (Piwi/Argonaute/Zwille), dsRNA bind-ing, and RNase III domains (Figure 1), which is responsiblefor cleaving long dsRNAs into siRNAs or miRNAs whenassociated with other proteins likeR2D2in Drosophila orthe transactivating response RNA-binding protein in Homosapiens to recruiting Argonaute proteins. The PAZ domain

binds the single stranded 3′ end of small RNA [11], and itmight function in protein-protein interaction. Small RNAsincludes PIWI-associated RNAs (piRNAs), short single-stranded RNAs arising from a Dicer-independent pathway,which are found in germ cells and associate with the PIWIsubfamily of Argonaute proteins [12, 13]. Many zebrafishpiRNAs are derived from repetitive sequences. Mutations inthe Piwi homologue protein result either in loss of germ cellsor in defects in meiosis and chromosome segregation in eggs.However, the Dicer knockout mouse eggs raise a questionabout overlapping functions of vertebrate miRNA, RNAi,and piRNA pathways.

Most vertebrates, Urochordata, and worms are reportedto have only one Dicer-1 protein, which generates both miR-NAs and siRNAs. While insects, fungi, and plants have morethan one Dicer or Dicer-like proteins [14], Dicer enzymesin Drosophila melanogaster are classified into Dicer-1 andDicer-2 in terms of their specialized functional activities.Dicer-1 can process loop pre-miRNA to mature miRNA,while Dicer-2 can process dsRNA precursors into siRNAsmolecules [15]. Recent studies have demonstrated that Dicercan function in an RNAi pathway independent manner. TheDicer-2 of Drosophila melanogaster participated in antiviralresponses by mediating induction of antiviral gene Vago[16]. The Dicer-1 of Caenorhabditis elegans participatesin fragmenting chromosomal DNA during apoptosis, and


PAZHelicase

dsRNA binding

RNase III

Unknown

3

5

Dicer

Figure 1: Schematic representation of the predicted consensualdomain structure for the Dicer protein. Helicase: N-terminaland C-terminal helicase domains. PAZ: Pinwheel-Argonaute-Zwilledomain. RNase III: bidentate ribonuclease III domains.

undergoes a protease-mediated conversion from a ribonu-clease to a deoxyribonuclease in addition to the processingof small RNAs [17].

Since the initial discovery in 1998 by Fire et al. [18],RNAi has taken the biological community by storm. Despitemany advances, however, RNAi is still under development. Abetter understanding of the mechanism for Dicer pathway isa future goal for many scientists. This paper will mainly focuson the recent evidences of Dicer functions in aquatic species.We will also highlight the effects of RNAi in experimentalmodels of the aquatic species.

2. Expression and Developmental Functionof Dicer in Aquatic Species

Only one homolog of Dicer was identified from the seaurchin, suggesting that the sea urchin Dicer may mediateboth miRNA and siRNA-silencing pathways, similar tohumans. The Dicer mRNA accumulates asymmetricallyin one periphery of the oocyte in punctate cytoplasmicstructures [19, 20]. The asymmetric localization of DicermRNA is maintained throughout the development of blas-tula and gastrula stages. The Dicer transcript accumulationis enriched selectively in the presumptive oral ectoderm andendodermal epithelium. The transcript then decreases toundetectable levels in the larval pluteus stage. Knockdownof Dicer in sea urchin embryos results in anomalousmorphogenesis, such as impairment of gastrulation andskeletogenesis at the mesenchyme blastula stage and laterstages, suggesting that miRNA could be involved in theearly development of sea urchin [20]. Similarly, the Dicertranscripts in rainbow trout are detectable throughout theembryonic stages. Peak expression of Dicer at the time ofmaternal mRNA degradation and initiation of embryonicgenome activation could indicate its involvement in miRNAprocessing during the periods in the rainbow trout [21](Figure 2).

During the developmental stages from fertilized eggto postlarva, shrimp (Litopenaeus vannamei) Dicer-1 isconstitutively expressed at all developmental stages [22].The highest expression is observed in fertilized eggs andfollowed a decrease from fertilized egg to nauplius stage.

Dicer

Development

Immune

Shrimp

Sea urchin

Trout

VirusessiRNA

miRNA

Transposons

dsRNAs

Figure 2: Small RNAs-dependent functions of Dicer. Schematicillustrations of the tentative model for developmental and immunological functions of Dicer in aquatic species are shown.

Then, the higher levels of expression are detected at thelate nauplius and postlarva stages. The shrimp Dicer-1expression regularly increases at the upper phase of nauplius,zoea, and mysis stages than their prophase. The differentexpression in the larval stages might provide clues forunderstanding the early innate immunity in the process ofshrimp larval development. The expression level of shrimpDicer-1 mRNA varies significantly among different shrimptissues. The expression in hemocyte is significantly higherthan that in gill, muscle, brain, intestine, and pancreas. Onthe other hand, expression levels of shrimp Dicer-2 are aboutthe same in most tissues, except in muscle, which has a lowerexpression level [23].

3. Immunological Function of Dicer inAquatic Species

RNAi is a mechanism of posttranscriptional gene silencingthat functions as a natural defensive response to viralinfection in a variety of species (Figure 2). Knockdownof Dicer-1 in tiger shrimp (Penaeus mondon) resulted inincreasing mortalities and higher viral loads, suggestingthat the RNAi mechanism is active and has a powerfulimmunological function in shrimp [24]. Higher levels ofDicer-1 expression in lymphoid organs are consistent witha role in the natural defense response of shrimp. However,there is no correlation between levels of Dicer-1 expressionand the viral genetic loads in shrimp lymphoid organ tissueduring naturally acquired or persistent viral infections [25].The Dicer-1 expression might be induced at an early stage ofinfection and recovers to normal levels later.

The white shrimp (Litopenaeus vannamei) Dicer-2involves in the nonspecific antiviral immunity, and in somedegree supporting the suggested relationship between non-specific activation of antiviral immunity and induction ofRNAi [23]. The Dicer-2 might be contributed to nonspecificactivation of antiviral immunity in shrimp by enhancingRNAi potency and efficacy [26]. The shrimp Dicer-2 might


regulate the single von Willebrand factor type C domain pro-tein genes in various ways [23]. Further research is requiredto identify more components of the shrimp RNAi pathwayand investigate the exact mechanisms of genes regulation.

Targeted gene silencing and more potent, virus-specificimmunity against challenge with white spot syndrome virus(WSSV) or Taura syndrome virus (TSV) have been obtainedin Pacific white shrimp (Penaeus vannamei) by injectionof long dsRNAs corresponding to sequences encoding viralstructural proteins [27]. Similar virus-specific effects havebeen observed in Penaeus monodon shrimp using longdsRNAs targeting the yellow head virus (YHV) proteasegene. However, siRNAs have failed to induce sequence-specific antiviral protection against WSSV or TSV. Similarly,inhibition of YHV replication in primary cell culturesappears to be less efficient when short dsRNAs are employed.Although RNAi has been assumed to be the mechanismof sequence-specific gene silencing and virus inhibition inshrimp, the interplay mechanism seems to be complex.

In many unicellular organisms, they seem to haveretained only a basic set of components of the RNA-silencing machinery. Chlamydomonas reinhardtii, a unicellu-lar eukaryote, has undergone extensive duplication of Dicerand Argonaute polypeptides after the divergence of thegreen algae [28]. Chlamydomonas encodes three Dicers andone of them is uniquely involved in the posttranscriptionalsilencing of retrotransposons. Then, multiple and redundantepigenetic processes are involved in preventing transposonmobilization in this green algae [28, 29].

4. Against Dicer Function

RNA silencing is now a well-known mechanism by whichplants and invertebrates fight off viral infection; however,many plant and animal viruses possess proteins that suppresshost RNA silencing mediated by siRNA or miRNA pathways.Striped jack nervous necrosis virus (SJNNV), which infectsfish, has a bipartite genome of positive-strand RNAs. TheSJNNV protein B2 has a potent RNA-silencing suppressionactivity [30]. Betanoda viruses are small RNA viruses thatinfect teleost fish and pose large threat to marine aqua-culture production. These viruses also possess the smallprotein B2, which binds to and protects dsRNA [31]. Thisprevents cleavage of virus-derived dsRNAs by Dicer andsubsequent production of siRNA, which would otherwiseinduce an RNA-silencing response against the virus [32, 33].The B2 is able to induce apoptosis in fish cells without dsRNAbinding. The same tendency has been demonstrated using anRNA-silencing system in human HeLa cells [34].

The suppression mechanisms of RNA silencing by theviral suppressors have been widely investigated. Cucumbermosaic virus- (CMV-) encoded 2b protein (CMV2b) blocksmiRNA pathways in green alga Chlamydomonas reinhardtii[35]. The CMV2b is able to bind small RNAs, suggestingthat this may be a mechanism by which CMV2b inter-feres with RNA silencing. The CMV2b may suppress bothsiRNA and miRNA pathways in Chlamydomonas reinhardtii.The CMV2b containing an arginine-rich region has been

reported to possess the ability to bind small RNAs. So, it ispossible that CMV2b suppresses RNA-silencing pathways bydirectly interacting with small RNAs.

5. Perspectives

The miRNA pathway has been shown to be crucial inembryonic development and in embryonic stem (ES) cells,as shown by Dicer knockout analysis in mammals. Specificpatterns of miRNAs have been reported to be expressed onlyin ES cells and in early phases of embryonic development.The miRNAs have emerged as key regulators of stem cells,collaborating in the maintenance of pluripotency, controlof self-renewal, and differentiation of stem cells [36]. It hasbeen demonstrated that Dicer is essential for the regulationof chondrocyte proliferation and differentiation duringnormal skeletal development in mice. Dicer deficiency inchondrocytes results in a reduction in the number of pro-liferating chondrocytes through decreased proliferation andaccelerated differentiation into hypertrophic chondrocytes[37, 38]. Similarly, Dicer in mammals is involved in a lotof tissue-developmental stages such as limb [39], lung [40],retina [41], vessel [42], and female reproductive system [43],and so forth.

The development and use of RNAi techniques in basalmetazoan model systems such as cnidarians will helpto determine the evolutionary lineage and complexity ofhomologous pathways such as apoptosis in higher metazoans[44, 45]. The use of acasp RNAi will also enable us toanswer questions about the role of cnidarian apoptosis in theonset and breakdown of symbiosis. This technique has beenused in large-scale experiments in which manipulation ofapoptosis resulted in a marked effect on symbiosis stability.

Worldwide viral diseases by WSSV cause large-scalemortalities and substantial economic losses to shrimpaquaculture. The control of viral diseases in shrimp remainsa challenge for the shrimp aquacultural industry. As shrimpslack adaptive immunity and the typical interferon response,RNAi is thought to be an ancient and important immunemechanism against virus replication. As mentioned before,the original function of RNAi is thought to be involvedin defense mechanism against foreign nucleic acids, suchas viruses, and in endogenous transcriptional regulation[2]. Presently, RNAi is becoming attractive to develop asan important potential tool in viral disease prevention inshrimp [46]. RNAi technology shows considerable promiseas a therapeutic approach and efficient strategy for viruscontrol in insects [47, 48]. Successful use of an RNAitechnique for gene knockdown in the symbiotic sea anemone(Aiptasia pallida) has also been reported [49].

The use of RNAi technology in shrimp in vivo hasindicated that the RNAi-effective time is limited due to thevery short half-life of synthetic RNA duplexes. It is likelythat siRNAs will not be present for complete elimination ofvirus replication at an infected tissue. In order to use RNAitechnology on an effective manner to protect shrimp againstviral diseases, it would be essential that future studies focuson increasing the stability of siRNA, and the relationship


between the expression of antiviral immune genes in theimmune response and larval development could shed lighton the further practical application. However, this seemsto be expensive approach yet. Instead, making transgenicshrimp which express an anti-viral shRNA might be a feasibleway to engineer shrimp to be resistant to the bad viruses.More research on the characterization of RNAi-relatedgenes in immune response and larval development may behelpful for better understanding the antiviral mechanismand designing efficient strategies of viral disease control.And the further progress in understanding the mechanismof RNAi will definitely revolutionize therapeutic approachesfor counteracting diseases in aquatic species.

Conflict of Interests

The authors declare that they have no conflict of interests.

Acknowledgments

This work was supported by grants-in-aid from the Ministryof Education, Culture, Sports, Science, and Technology inJapan and Nara Women’s University Intramural Grant forProject Research. Part of the research had been implementedby having a grant provided by Yamada Bee Farm Grant forHoneybee Research. Y. kitagishi and S. Matsuda contributedequally to this work.

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Review Article

Diversified Carbohydrate-Binding Lectins from Marine Resources

Tomohisa Ogawa, Mizuki Watanabe, Takako Naganuma, and Koji Muramoto

Department of Biomolecular Sciences, Graduate School of Life Sciences, Tohoku University, Sendai 980-8577, Japan

Correspondence should be addressed to Tomohisa Ogawa, [email protected]

Received 12 May 2011; Accepted 13 August 2011

Academic Editor: Faouzi Ben Rebah

Copyright © 2011 Tomohisa Ogawa et al. This is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properlycited.

Marine bioresources produce a great variety of specific and potent bioactive molecules including natural organic compounds suchas fatty acids, polysaccharides, polyether, peptides, proteins, and enzymes. Lectins are also one of the promising candidates foruseful therapeutic agents because they can recognize the specific carbohydrate structures such as proteoglycans, glycoproteins, andglycolipids, resulting in the regulation of various cells via glycoconjugates and their physiological and pathological phenomenonthrough the host-pathogen interactions and cell-cell communications. Here, we review the multiple lectins from marine resourcesincluding fishes and sea invertebrate in terms of their structure-activity relationships and molecular evolution. Especially, we focuson the unique structural properties and molecular evolution of C-type lectins, galectin, F-type lectin, and rhamnose-binding lectinfamilies.

1. Introduction

Marine bioresources such as marine cyanobacteria, algae,invertebrate animals, and fishes produce a great variety ofspecific and potent bioactive molecules including naturalorganic compounds such as fatty acids, polysaccharides,polyether, peptides, proteins, and enzymes. To date, manyresearchers focused on the marine natural products and theirvarious pharmacological functions to develop new potentdrugs including antimicrobials, anti-human immunodefi-ciency virus (HIV), anticancer, and Alzheimer’s therapeutics.There are several excellent reviews that described the potenttherapeutic agents derived from marine resources and theirmedicinal applications [1–3]. In the drug discovery fromnatural resources, lectins are one of the promising candidatesfor useful therapeutic agents because carbohydrate structuressuch as proteoglycans, glycoproteins, and glycolipids havebeen implicated in certain cell types and their physiologicaland pathological functions including host-pathogen interac-tions and cell-cell communications. For example, griffithsin(GRFT), a lectin isolated from red algae Griffithsia sp.,showed a strong anti-HIV activity with half maximal effectiveconcentration (EC50) of 0.043–0.63 μM via specific bindingto gp120, which is envelop glycoprotein anchored to theHIV membrane and involved in viral entry into cells by

recognition of CD4 [4]. Cyanovirin (CN-V) isolated fromNostoc ellipsosporum cyanobacteria has been reported aspotent lectin with anti-HIV activity [5].

Lectins are group of sugar-binding proteins except forantibodies and enzymes that recognize specific carbohydratestructures, resulting in the regulation of various cells viaglycoconjugates. Thus, they can identify the cell types andcell development stages including embryonic stem (ES) andinduced pluripotent stem (iPS) cells through histochemicalapplications, flow cytometry, and lectin microarrays [6]because cells often display altered surface glycoproteins andglycolipids depending on the physiological and pathologicalconditions. Lectins are widely distributed in all taxa frommicrobial organisms, plant, and animal and are involved innumerous cellular processes that depend on their specificrecognition of complex carbohydrates. Based on the struc-tural similarity of carbohydrate recognition domain (CRD)and their characteristics, animal lectins are classified intoseveral categories: C-type lectins (CTLs), galectins, I-typelectins, pentraxins, P-type lectins, tachylectins, and so forth[7].

Intensive investigations have been carried out to clarifythe biochemical and physiological properties of humorallectins of marine resources including marine cyanobacteria,algae, and invertebrates such as barnacles [8–10], sea urchins


[11, 12], sea cucumbers [13–18], horseshoe crabs [19–30],tunicates [31–36], mollusks [37–39], and fishes [40–69] asshown in Table 1. For fish and sea invertebrate lectins, theycould be mainly classified into CTLs, galectins, F-type lectins,and rhamnose binding lectin (RBL) families in additionto the Ricin-type, Lily-type, 6x β-propeller/Tectonin-typelectins (Table 1). Here, we review these multiple lectins frommarine resources including fishes and sea invertebrate interms of their structure-activity relationships and molecularevolution.

2. C-Type Lectin Family

C-type lectins (CTLs) are one of major animal lectin family,of which members bind in a Ca2+-dependent fashion tomono- and oligosaccharides. They adopt generally multi-domain structures and contain one or more highly conservedCRD consisting of 115–130 amino acid residues [72], whichhas a unique mixed α ∼ β topology [73]. In the presence ofCa2+, CTLs initiate a broad range of biological processes suchas adhesion, endocytosis, and pathogen neutralization [74].C-type lectin domain (CTLD) superfamily is a large groupof extracellular proteins with conserved CRD sequences butdifferent function including more than a thousand identifiedmembers, most of which lacking lectin activity. Since CTLDsuperfamily has been first classified into 7 groups (I to VII)by Drickamer in 1993 [75]; the classification of CTLD wasrevised in 2002 with additional seven groups (VIII to XIV)by Drickamer and Fadden [76]; and in 2005 with three newgroups (XV to XVII) by Zelensky and Gready [77]. Group Icontains four members, versican, aggrecan, neurocan, andbrevican, which contain a proteoglycan core peptide anda single CTLD in the vicinity of the C-terminus and forwhich the name “lectican” has been proposed [78]. GroupII CTLs have a single transmembrane domain, an extracel-lular carboxyl terminus, and a cytoplasmic amino termi-nus; typical examples are the hepatocyte asialoglycoproteinreceptor (ASGR) [79], dendritic cell-specific intercellularadhesion molecule-3-grabbing nonintegrin (DC-SIGN), andmacrophage receptors. Group III CTLs are collectins, whichconsist of an amino terminal collagen domain and a carboxylterminal CTLD. They participate in the host defense mech-anism through complement activation and include serummannose binding protein (MBP) and pulmonary surfactantproteins [80]. Group IV CTLs, or selectins, are involved inthe adhesive interaction between leukocytes and vascularendothelial cells such as L-selectin (leucocytes), E-selectin(endothelial cells), and P-selectin (platelets) [81]. Group VCTLs are involved in signal transduction; typical examplesare the natural killer cell receptors [82] and the low-affinityIgE receptor (CD23) [83]. Group VI CTLs are type I trans-membrane proteins consisting of a cysteine-rich domain, afibronectin type II domain, and tandem CRDs. This groupincludes the macrophage cell surface mannose receptor anda dendritic cell surface molecule DEC-205 [84]. Group VIIincludes soluble single-CRD proteins. This group includesthe pancreatic stone protein (PSP) isolated from pancreaticstones, lithostathine, which considered an inhibitor of calcitecrystal growth [85–87]. Interestingly, CTLs isolated from

PrototypeGal-1, Gal-2, Gal-5, Gal-7,Gal-10, Gal-11, Gal-13, Gal-14

Gal-3

Tandem-repeat type Gal-4, Gal-6, Gal-8, Gal-9, Gal-12

Chimera type

Figure 1: Classification of galectins.

marine invertebrates such as acorn barnacle hemolymph[9, 88] and pearl shells [38] have also been reported tohave unique multiple functions in their biomineralization,that is, the inhibitory activity toward the crystal growth ofcalcium carbonate based on their high association constantsto calcium ions. Furthermore, fish-specific CTLD proteinshave been identified as the type II antifreeze proteins (AFPs),which inhibit the freezing by binding to ice in plasma of cold-water-living fish species including herring (Clupea harengus),rainbow smelt (Osmerus mordax), Japanese smelt (Hypome-sus nipponensis), sea raven (Hemitripterus americanus), andlongsnout poacher (Brachyopsis rostratus) [89–91].

Zelensky and Gready (2004) have reported the genome-level analysis on CTLD superfamily in Fugu rubripes genomethat demonstrate the divergence of all CTLD superfamilyproteins except for two groups V and VII present inmammals [92]. They also identified fish-specific CTLDs,AFPs, and dual-CTLD proteins, in Fugu genome. CTLsare diversified and widely distributed in animal kingdomincluding fishes as unique structural motifs and functionaldomains, regardless of whether they possess the sugar-binding properties or not.

3. Galectin Family

Galectins are family of carbohydrate-binding proteinsdefined by their Ca2+-independent affinity for β-galactosidesugar, sharing a conserved sequence motif within their CRDof about 130 amino acid residues, the lack of a signal peptideand attached carbohydrate, and predominantly cytoplasmiclocation [99]. Based on the structural features, galectinscan be classified into three types: prototype (monomeror homodimer of single carbohydrate-binding domain),tandem-repeat type (two carbohydrate-binding domains ona single chain), and chimera type (carbohydrate-bindingdomain and an extra N-terminal domain on a single chain)(Figure 1). Galectins have proposed to participate in diversephysiological phenomena such as development, differenti-ation, morphogenesis, immunity, apoptosis, metastasis ofmalignant cell, and so forth. To date, several galectinswere identified from fishes including Japanese eel Anguillajaponica [40], electric eel Electrophorus electricus [44], chan-nel catfish Ictalurus punctuates [53], windowpane flounderLophopsetta maculate [49], zebrafish Danio rerio [50], andconger eel Conger myriaster [45–47].


Table 1: Lectins from marine organisms.

Organism Lectins Binding specificity Lectin family References

[Protozoan]

Sponge

(Aphrocallistes vastus) LECCI d-Galactose C-type [93]

(Axinella polypoides)AP-I/II/III/V d-Galactose Ricin type [94]

AP-IV hexuronic acid

(Geodia cydonium) GCLT1 LacNAc Galectin [95]

(Ptilota filicina) PFL d-Galactose [96]

[Mollusk]

Tridacna

(Tridacna maxima) tridacin N-acetyl galactosamine C-type [37]

Abalone

(Haliotis laevigata) PLC d-Galactose/d-Mannose C-type [38]

Penguin wing oyster

(Pteria penguin) PPL d-Galactose RBL [39]

[Arthropod]

Barnacle

(Megabalanus rosa) BRA-1∼ 3 d-Galactose C-type [8, 9]

(Balanus rostratus) BRL d-Galactose C-type [10]

Horseshoe crab

(Tachypleus tridentatus)

TL-1 LPS (KDO), LTA 6x β-propeller/Tectonin [19]

TL-2 GlcNAc/GalNAc, LTA 5x β propeller [20–22]

TL-3 LPS (O-antigen) F-type [23]

TL-4 LPS(O-antigen), LTA, [24]

TLs-5 Fucose Ficolin [25, 26]

TL-P N-acetyl group 6x β-propeller/Tectonin [27]

GBP/PAP/LBP GlcNAc [28]

[Echinoderm]

Sea cucumber

(Cucumaria echinata)CEL-I/CEL-II GlcNAc C-type [13, 14]

CEL-IV GlcNAc-Galactose C-type [13, 15]

CEL-III d-Galactose and so forth Ricin type [13, 16]

(Stichopus japonicus)SPL-1 uronic acid C-type [17]

SPL-2 LacNAc and so forth C-type [17]

SJL-1 d-Galactose C-type [18]

Urchin

(Anthocidaris crassispina)SUEL d-Galactose RBL [11]

Echinoidin (ECH) d-Galactose C-type [12]

Starfish

(Asterina pectinifera) LacNAc C-type [97]

[Protochordate]

Ascidian

(Polyandrocarpa misakiensis) TC14 d-Galactose C-type [31]

(Botryllus schlosseri) Bs RBL-1∼5 l-Rhamnose RBL [32, 33]

(Halocynthia roretzi)P36 d-Galucose C-type [34]

P40-P50 ficolin [35]

41KD d-Glactose C-type (ficolin) [36]


Table 1: Continued.


[Fishes]

Japanese eel

(Anguilla japonica)AJL-1 β-Galactoside Galectin [40]

AJL-2 Lactose C-type [41]

eCL-1/eCL-2 Lactose C-type [42]

European eel

(Anguilla anguilla) AAA Fucose F-type [43]

Electric eel

(Electrophorus electricus) Electrolectin Lactose and so forth Galectin [44]

Conger eel

(Conger myriaster)

Congerin I Lactose and so forth Galectin [45, 46]

Congerin II Lactose and so forth Galectin [45, 47]

Congerin P Lactose/mannose Galectin unpublished

conCL-s mannose C-type [48]

Windowpane flounder

(Lophopsetta maculate) d-Galactose Galectin [49]

Zebrafish

(Danio rerio) Drgal1-L1–L3 LacNAc Galectin [50]

Shishamo smelt

(Osmerus lanceolatus)OLABL d-Galactose C-type [51]

OLL l-Rhamnose RBL [52]

Catfish

(Arius thalassinus) d-Galactose Galectin [53]

(Silurus asotus)SAL l-Rhamnose RBL [54]

saIntL d-Mannose intelectin [55]

Steelhead trout

(Oncorhynchus mykiss) STL-1–3 l-Rhamnose RBL [56, 57]

Chum salmon

(Oncorhynchus keta) CSL-1–3 l-Rhamnose RBL [58]

White-spotted charr

(Salvelinus leucomaenis) WCL-1, 3 l-Rhamnose RBL [59]

Spanish mackerel

(Scomberomorus niphonius) SML l-Rhamnose RBL [60]

Sweet fish (ayu)

(Plecoglossus altivelis) SFL l-Rhamnose RBL [61]

Ponyfish

(Leiognathus nuchalis) PFL-1,2 l-Rhamnose RBL [62]

Far-East dace

(Tribolodon brandti) TBL-1–3 l-Rhamnose RBL [63]

Striped bass

(Morone saxatilis) Fucose F-type [64]

Sea bass

(Dicentrarchus labrax) DlFBL Fucose F-type [65]

Japanese sea perch

(Lateolabrax japonicus) JspFL Fucose F-type [66]

Pufferfish

(Fugu rubripes) Pufflectin d-Mannose Lily-type [67]


Table 1: Continued.


Scorpionfish

(Scorpaena plumieri) plumieribetin d-Mannose and so forth Lily-type [68]

Carp

(Cyprinus carpio) carpFEL GlcNAc 6x β-propeller/Tectonin [69]

AAA: Anguilla anguilla agglutinin; AJL: Anguilla japonica lectin; BRA: Megabalanus rosa lectin; BRL: Balanus rostratus lectin; CEL: Cucumaria echinata lectin;CSL: chum salmon lectin; GCLT1: Geodia cydonium lectin; OLABL: Osmerus lanceolatus asialofetuin-binding lectin; OLL: Osmerus lanceolatus lectin; PFL:ponyfish lectin; PLC: perlucin; PPL: Pteria penguin lectin; SAL: Silurus asotus lectin; SFL: sweet fish (ayu) lectin; SJL: Stichopus japonicus lectin; SML: Spanishmackerel lectin; SPL: sea cucumber plasma lectin; STL: Steelhead trout lectin; SUEL: sea urchin egg lectin; TBL: Tribolodon brandti lectin; WCL: white spottedcharr lectin; KDO: 2-keto-3-deoxyoctonate; LPS: lipopolysaccharide; LTA: lipoteichoic acid; GalNAc: N-acetyl galactosamine; GlcNAc: N-acetyl glucosamine;LacNAc: N-acetyl lactosamine.

Conger eel contains two prototype galectins, congerinsI (Con I) and II (Con II). They are components of thebiological defense system: these proteins mainly exist in thefrontier organs and tissues that delineate the body fromthe outer environment, such as the epidermal club cells ofthe skin, wall of the oral cavity, pharynx, esophagus, andgills [46, 100], and agglutinate marine pathogen bacteriasuch as Vibrio anguillarum [101], and have opsonic andcytotoxic activities against cells [102–104]. It was reportedthat other fish galectin isolated from Japanese eel, AJL-1,also showed agglutinating activity against pathogenic Gram-positive bacteria, S. difficile [40]. Con I and Con II consistof 136 and 135 amino acid residues, respectively, and bothcontain several common structural characteristics: acetylatedN-termini and no cysteine residue that is related to oxidizinginactivation found in mammalian galectins. Each subunitof Con I and Con II has one carbohydrate-binding site,and they form a homodimer to exhibit divalent crosslinkingactivity. Since a gene duplication event, these genes have beenevolving at a high dN/dS (nonsynonymous/synonymoussubstitution) rate (2.6) under selection pressure, causingamino acid changes in Con I and Con II [46]. Usually, non-synonymous substitutions in coding regions are restrained,by purifying selection, to maintain protein structure orfunction, whereas synonymous substitutions and nucleotidesubstitution for noncoding regions accumulate constantlyby random genetic drift. Therefore, dN/dS ratios are nor-mally smaller than 1.0 (∼0.2 in various genes). In otherwords, Con I and Con II have evolved via acceleratedamino acid substitutions under positive selection. Similarevolutionary behavior has been observed only in severalgene families including that of biological offense and defensesystems such as snake venom isozymes and conus peptidesand reproduction systems [105]. The structure of ConI demonstrated a protein-fold evolution by swapping β-strands between subunits, which altered the β-sheet topologyat the dimer interface from entirely antiparallel to partiallyparallel to entangle two subunits, although Con I andCon II adopt the similar subunit structure with “jelly-roll”motif consisting of five-stranded and six-stranded β-sheets(Figure 2) [70, 71]. Domain swapping has been hypothesizedas a mechanism in quaternary structure formation in proteinevolution and reported several proteins [106, 107]. Oneof the important features of strand swapping seems to beincreasing the stability of the quaternary structure, which

is required for essential divalent cross-linking activity inagglutinating pathogenic bacteria (Figure 2). On the otherhands, crystal structure analysis of Con II revealed thata MES (2-(N-morpholino) ethane sulfonic acid) moleculethat corresponding to sulfonosugar bound to the cleft nearthe known CRD [71]. The similar extension of bindingsite toward the nonreducing end of lactose was observedin the fungal prototype galectins, ACG and CGL2 [108,109]. Crystal structure of Con II complex with lacto-N-fucopentaose III at 2.2 A resolution supported the extensionof carbohydrate-binding site to the position where the MESwas observed, indicating that the potent natural ligands ofCon II include the additional moieties at the nonreducingend of lactose [110]. Actually, the differences in the ther-mostability and carbohydrate specificities between Con I andCon II were also observed [47]. Furthermore, to identifythe determinants of selection pressures in the evolutionaryprocess and the structural elements associated with theunique carbohydrate-binding activities of Con I and ConII, we recently reconstructed a probable ancestral form ofcongerin (Con-anc) that corresponded to the putative aminoacid sequence at the divergence of Con I and Con II inthe phylogenic tree [111, 112]. It was found that Con-ancshowed the properties similar to those of Con II in termsof thermostability and carbohydrate-recognition specificity,although Con-anc shares the higher sequence similarity withCon I than Con II. From the differences between Con-ancand Con II on the sugar-binding specificities and mutationanalysis of Con II, Con II has been considered to acquire thebinding ability to the α2,3-sialyl galactose moieties such asGM3 and GD1a during the accelerated evolutionary eventfrom Con-anc with the replacement of Arg3 and Tyr123residues of Con II. The α2,3-sialyltransferases have beenrecently isolated and cloned from the pathogenic marinebacteria Vibrio sp. and Photobacterium phosphoreum [113,114], suggesting that α2,3-sialyl galactose-containing sugars,which are presumed to be targets for Con II, may specificallybe present in pathogenic marine bacteria. On the otherhand, Con I has evolved from the ancestral congerin Con-anc to increase the binding activity against α1,4-fucosylatedN-acetyl glucosamine [114]. These findings emphasize thatthe carbohydrate-binding ability and the specificities ofcongerins are diversified via accelerated evolution.

Cooper (2002) reviewed the genome-wide screening ofgalectin gene families (galectinomics) based on the genomic


S1 S2 S3

S4 S5F1

F5

F2

F3

F4 S6

Congerin II

Congerin I

S1 S2

N

N

S1 S2

N

N

S2

S2

S1

S1

Figure 2: Schematic presentation of the wild-type congerin I (top:PDB code 1c1f) and congerin II (bottom: PDB code 1is5) and thetopologies of β-sheet at subunit interface [70, 71].

sequence database including Arabidopsis, Drosophila, Caen-orhabditis, Xenopus, human, and zebrafish Danio [115]. Inthe zebrafish genome, homologues of mammalian galectins-1, 3, 4, 9, and HSPC159 were reported. Table 2 summarizedthe results of searching for the homologues genes encodinggalectins and related proteins in the eighth integratedassembly of the zebrafish genome, Zv8 (release 59, August2010). In the zebrafish embryo, four proto-type galectin-1-like proteins, Drgal1-L1, Drgal1-L2, Drgal1-L3, and splicevariant of Drgal1-L2, one chimera-type Drgal3, and twotandem-repeat galectins, Drgal9-L1 and Drgal9-L2, havebeen identified and characterized [50, 116]. They exhibiteddistinct phase-specific expression patterns during embryodevelopment; for example, Drgal1-L1 is maternal; Drgal1-L2 is zygotic and expressed at postbud stage in notochord,while Drgal1-L3, Drgal3, Drgal9-L1, and Drgal9-L2 are bothmaternal and zygotic, and ubiquitously in adult tissues[50]. Furthermore, knock-down experiments in zebrafishembryo showed that Drgal1-L2 plays a key role in somaticcell differentiation through the skeletal muscle formation[117]. Zebrafish genes encoding proto-type galectins containfour exons with highly conserved exon-intron boundariesto that of mammalian galectin-1. Recently, the homologueof galectin-related interfiber protein (Grifin), which is alens crystallin protein and one of galectin-related proteins(GRPs), has been also identified in zebrafish (DrGrifin)(Table 2), especially in the lens, particularly in the fiber cellsof 2 days post fertilization embryos, and adult zebrafishtissues such as oocytes, brain, and intestine [118].

Furthermore, novel galectin-related protein namedCvGal, which contains four canonical galectin CRDs, hasbeen discovered from the hemocytes of the eastern oys-ter, Crassostrea virginica [119]. CvGal can recognize bothendogenous and exogenous ligands including bacteria, algae,and Perkinsus sp. as a soluble opsonin for pathogens or as a

hemocyte surface receptor for both microbial pathogens andalgae food ingested into digestive ducts and as a modulatorfor up-regulation of CvGal itself [119]. Unique domainarchitecture for genes/proteins consist of galectin CRDs,nematogalectin, was also found in freshwater hydrozoanHydra and marine hydrozoan Clytia [120]. Nematogalectin,a 28 kDa protein with an N-terminal GlyXY domain thatcan form a collagen triple helix followed by galectin CRD,is a major component of the nematocyst tubule and istranscribed by nematocyte-specific alternative splicing [120].Thus, the galectin family proteins also diversified by uniqueevolutionary process including tandem duplication andaccelerated evolution.

4. F-Type Lectin Family

F-type lectins (fucolectin), which bind fucose and share char-acteristic sequence motif, have been identified as immuno-recognition molecules in invertebrates and vertebrates suchas horseshoe crab (Tachypleus tridentatus) [23] and Japaneseeel (Anguilla japonica) [43]. The crystal structures of singleCRD and tandem CRDs of F-type lectins with a jellyrollβ-barrel topology have been reported for Anguilla japonicaagglutinin (AAA) and MsaFBP32 from striped bass (Moronesaxatilis), respectively, [121, 122]. Bianchet et al. describedthat the fold structure of AAA, F-type lectin motifs, iswidely distributed in other proteins even with lower sequencesimilarities, for example, C1 and C2 repeats of blood coagu-lation factor V, C-terminal domain of sialidase, N-terminaldomain of galactose oxidase, APC10/DOC1 ubiquitin ligaseand XRCC1 [121]. Furthermore, it has been reported thatthe several proteins are homologous to or contained with F-type lectin CRDs, of which examples include Streptococcuspneumoniae TIGR4, furrowed receptor and CG9095 ofDrosophila melanogaster, Xenopus laevis pentraxin 1 fusionprotein, Microbulbifer degradans ZP 00065873.1, and yeastallantoises [121, 123] in addition to the tandem-repeatedtypes of F-type lectins found in modern teleosts [64–66,122], while F-type lectin CRD motifs are absent in genomesof higher vertebrates such as reptiles, birds, and mammals.

5. Rhamnose-Binding Lectin Family

The rhamnose-binding lectins (RBLs) are a family of ani-mal lectins that show the specific binding activities to l-rhamnose or d-galactose and mainly isolated from eggs andovary cells of fishes and invertebrates [39, 56, 57, 126]. Seaurchin egg lectin (SUEL) is the first example of isolatedand sequenced RBL family [11]. SUEL forms a homodimercomposed of two identical subunits, which consist of 105amino acid residues including single CRD, via intersubunitdisulfide bond, resulting in the hemagglutinating activitywith bivalent binding properties. To date, the RBL familyhas been found in over 20 species of fish, which locatedspecifically in oocytes, ovaries, and skin mucus [52, 54, 56–63]. RBLs have been also found in the mantle of penguinwing oyster [39], and ascidians [32, 33]. Except for thereproductive cells including oocyte and egg, RBLs are mainlylocated in the tissue related to the immune system such


Table 2: Zebrafish galectin-related genes.

LengthChromosome Exons Gene ID∗

Transcripts (bp) Protein (AA)

Drgal1 (proto)

lgals1 (Gal1-L1) 883 134 3 4 326706

lgals2a (Gal1-L2) 850 134 3 4 405830

lgals2b (Gal1-L3) 545 118 6 4 393486

si:ch211-10a23.2 1951 149 13 4 567812

Drgal3 (chimera)

zgc112492 (Gal3) 1993 561 3 5 550351

lgals3l (Gal3-like) 958 228 17 6 325599

lgals3bpa (Gal3-like) 2005 567 3 5 677742

lgals3bpb (Gal3-like) 1983 572 3 5 405809

si:ch211-67f24.5 1925 369 3 5 125833577

Drgal4 (tandem)

LOC567193 3981 — 18 25 567193

Drgal8 (tandem)

LOC100334749 864 287 17 7 100334749

si:ch211-199I3.2 537/402 153/133 20 9 368889

Drgal9 (tandem)

zgc92326 (Gal9-L1) 2591 321 15 11 327284

lgals9l1 1075 310 15 10 337597

LOC100148547 1017 282 15 7 100148547

zgc171951 1453 280 15 7 100124603

Others

Grifin (zgc92897) 684 139 3 4 445036

GRP (zgc136758) 3518 164 1 5 723995

GRP(si:ch211-101n13.9)

438 145 13 2 563573

LOC100535066 3984 1327 18 29 100535066

c7orf23 (zgc:112101) 1011 154 18 3 550443∗

Gene ID: ID numbers in NCBI’s database for gene-specific information [98].

as mucous cells of gill, goblet cells of intestine, spleen,thrombocyte, lymphocyte, monocyte, and neutrophil [127,128]. Moreover, RBLs were isolated from spores of themicrosporidian fish parasite, Loma salmonae, which waslocated in gill tissue [129] and Glugea plecoglossi from ayueggs [61], respectively. Thus, it is possible that RBLs partici-pate in the self-defense mechanisms. In fact, the receptor ofRBL from amago (Oncorhynchus rhodurus) was expressed onthe peritoneal macrophage after inflammatory stimulation[130], and RBLs from grass carp (Ctenopharyngodon idellus)roe induced a dose-dependent increase in phagocytic activityof seabream macrophage [131].

6. Structural Characterization of RBLs:Primary Structures and Classification

Three RBLs, named CSL1, CSL2, and CSL3, have beenisolated as rhamnose-binding lectins from chum salmon(Oncorhynchus keta) eggs [41]. The amino acid sequences

among CSLs show the 42–52% identities, while CSLs showthe 94 to 97% sequence identities compared to correspond-ing three RBLs, STL1, STL2, and STL3, from steelhead trout(Oncorhynchus mykiss) eggs, respectively. Moreover, CSL1,CSL2 and CSL3 are composed of 4, 18, and 2 subunits vianoncovalent binding, respectively.

Most RBLs are composed of two or three tandem-repeated CRDs, which consist of about 95 amino acidresidues, and share the conserved topology of four disulfidebonds. Figure 3 shows the aligned amino acid sequences ofvarious RBL-CRDs. The disulfide bond pairings of RBLs havebeen determined for Spanish mackerel lectin (SML) by pro-tein sequencing combined with peptide mapping [59] andfor SEL24K from Chinook salmon by matrix-assisted laserdesorption/ionization (MALDI) mass-spectrometry [132],respectively. Each RBL-CRD had the same disulfide bond-ing patterns: Cys(1)–Cys(3), Cys(2)–Cys(8), Cys(4)–Cys(7),and Cys(5)-Cys(6) (Figure 3). Furthermore, two charac-teristic peptide motifs, -(AN)YGR(TD)-(YGR-motif) and


β1 β2 β3 α1 α2 α3β4 β5

C1 C2 C3 C4 C5 C6 C7 C8

Figure 3: Aligned amino acid sequences of RBL-CRDs. Multiple alignment was achieved using the CLUSTAL X program. Secondarystructural elements are shown as cylinders (α helix, α1–α3) and arrows (β strand, β1–β5). Disulfide-bond paring of eight half-cystineresidues (C1–C8) is indicated at the bottom. Bold-faced amino acids indicate the residues involved in the carbohydrate binding. Thesequence data were obtained from the Entrez protein sequence database including SwissProt, PIR, PRF, and PDB. The CRDs located in the N-terminal, middle, and C-terminal regions are represented by N, M, and C, respectively. CSL: chum salmon (Oncorhynchus keta) egg lectin,OLL: shishamo smelt (Osmerus lanceolatus) egg lectin, PFL: ponyfish (Leiognathus nuchalis) egg lectin, PPL: penguin wing oyster (Pteriapenguin) lectin, SAL: catfish (Silurus asotus) egg lectin, SFL: ayu (Pleacoglossus altivelis) egg lectin, SML: Spanish mackerel (Scomberomorousniphonius) egg lectin, STL: steelhead trout (Oncorhynchus mykiss) egg lectin, SUEL: sea urchin (Anthocidaris crassispina) egg lectin, TBL: fareast dace (Tribolodon brandti) egg lectin, WCL: white spotted charr (Salvelinus leucomaenis) egg lectin. Bs: Botryllus schlosseri, Ca: snakehead(Channa argus), Ci: Ciona intestinalis, Dr: zebrafish (Danio rerio), El: northern pike (Esox lucius), Hk: spotted seahorse (Hippocampuskuda), Hm: Hydra magnipapillata, If: blue catfish (Ictalurus furcatus), Ls: humphead snapper (Lutjanus sanguineus), Mm: house mouse(Mus musculus), Ot: small-eared galago (Otolemur garnettii), Sp: Strongylocentrotus purpuratus, Ss: Atlantic salmon (Salmo salar), Tn: greenpufferfish (Tetraodon nigroviridis).


RBL-CRD7

RBL-CRD1

RBL-CRD3

RBL-CRD4

RBL-CRD5

RBL-CRD6

RBL-CRD2

0 0.25 0.5 0.75 1 1.25 1.5

0.2

(latrophilin)

Figure 4: Phylogenetic tree of CRDs of RBL family lectins. A phylogenetic tree was constructed by the neighbor-joining algorithm based onan evolutionary distance matrix constructed by Kimura’s method.

-DPCX(G)T(Y)KY(L)-(DPC-motif), which are located atthe N- and C-terminal regions in each domain, respectively,are conserved in almost RBL-CRDs. However, the structuralvariations for S–S bonds and motifs are observed in PPLand N-terminal CRDs of CSL1, STL1, WCL1, and ElRBL(Figure 3). Previously, RBLs have been classified into fivegroups (Types I to V) based on their domain structures andthe hemagglutination activity against human erythrocytesand sugar specificity against lactose (Table 3) [133]. Type I iscomposed of three tandemly repeated domains. Type II hastwo tandem-repeated domains with an extra domain. TypesIII and IV have two tandem-repeated domains, but they havedifferent hemagglutination activity and sugar specificity.Type V has only one RBL domain and exits in a homodimerwith a disulfide linkage between subunits. On the otherhand, the phylogenetic tree constructed from the amino acidsequences of CRDs derived from several RBLs revealed thatRBL-CRD can be classified into seven groups, RBL-CRD1 toRBL-CRD7, as shown in Figure 4. Based on their structuralfeatures of RBL-CRDs compositions, RBLs can be classifiedinto 13 subgroups (Ia to V) (Table 3). The subunit of CSL1is composed of 286 amino acid residues with three tandemlyrepeated domains (Type II), while the subunits of CSL2 andCSL3 are composed of 195 amino acid residues with twotandem-repeated domains (Type III).

Furthermore, recent studies including genome-widescreening revealed several variations in the RBL families. Itwas found that the genes containing the distinctive structuralmotif of RBL-CRDs broadly distributed in almost all theanimals including invertebrate (Hydra magnipapillata,Hydractinia echinata, Strongylocentrotus purpuratus, Nem-

atostella vectensis, Caenorhabditis remanei, Triatoma dimidi-ata), Chordates (Ciona intestinalis, Botryllus schlosseri,Branchiostoma floridae), and vertebrate including bony fishsuch as Danio rerio, Oncorhynchus mykiss, and so forth,amphibian Xenopus tropicalis and mammalians such asMus musculus, Rattus norvegicus, Homo sapiens, and soforth, and also in the bacterium (Flavobacterium) andplants (Arabidopsis thaliana, Medicago truncatula) whenthe genomic database was retrieved. For example, the RBLhomologues have been reported as integrated domainsinvolved in the ligand binding in membrane receptors suchas polycystic kidney disdase-1-like (PKD-1) [134], axonguidance receptor EVA-1 [135], HuC21orf63 [136], andthe adhesion-class G-protein-coupled receptor latrophilin(LPHN) [137, 138]. Figure 5 summarized the domainarchitectures of RBL superfamily proteins that containedRBL-CRDs in their sequences as a domain structure. (TSP1:thrombospondin-type 1 repeats, OLF: olfactomedin-likedomain, HormR: hormone receptor domain, GPS: G-protein-coupled receptor proteolytic site domain, 7tm 2: 7transmembrane receptor, latrophili: latrophilin cytoplasmicC-terminal region, CTLTD: C-type lectin domain. PLAT:polycystic kidney disease protein-1-like 2, PKD channel:polycystin cation channel, FAS8C: coagulation factor 5/8C-Terminal domain, discoidin domain, LCCL: limulus-clotting factor C, Coch-5b2, and Lgl1-lectin domain, IPPc:inositol polyphosphate phosphatase, catalytic domainhomologues, RhoGAP OCR: GTPase-activator protein forRho-like Small GTPases in oculocerebrorenal syndrome ofLowe-1-like protein, Prp1: proline-rich protein 1, PurA:adenylosuccinate synthase, AMN1: antagonist of mitotic


Table 3: Classification of RBL family lectins.

Type (this study) Type (Nitta et al.) No. of CRDs RBLs members

II II 3 CSL1, STL1, WCL1, EIRBL

Ia I 3 SAL, IfRBL

Ib I 3 TBL3

Ic I 3 SpRBL-like1, SpRBL-like2

IIIa III 2 CSL3, STL3, WCL3, Ot24kEL, HmRBL-like

IIIb III 2 CSL2, STL2, WCL2, SsRBL

IIIc IV 2 OLL, SML, PFL1, CaRBL, TnRBL, HkRBL, LsRBL

IIId 2 TBL1, TBL2

IIIe 2 DrBC, DrNM

IIIf 2 CiRBL-like

IVb 2 DrSTL2-like

IVa 2 PPL1

V V S-S 1 × 2 SUEL

exit network protein 1, VWD: von Willebrand factordomain, NHL: Ncl-1, HT2A, and Lin-41 proteins, PANmodule: plasminogen/hepatocyte growth factor-Appledomains of the plasma prekallikrein/coagulation factorXI-Nematode proteins module, ∗1: AAH90269/AAI22308/50383/CAM56745/56747/CAX13501/NP 001035384/-001038891/XP 692814/001922851/003199967/003200629/706941/003200654/003200655, ∗2: AAI22302/50374/51864/51941/54461/54558/55629/62646/62650/CAK11496-11501/11504/11506/11509/11515/CAM56419-56422/56424/56426/56637/56466/56467/56470-56473CAP09587/09516/CAQ13825/14198-14199/NP 001038882/001082844/001082910/001082869/001093874/001093910/001096104/001098618/001103190/001103311/001103315/001103334/001103858/001104195/001108359/001103581/001103589/001095862/001103856/001138280/001128340/001153845/002663370/002663371/002666350/XP 003199229/003199230/002663369/692138/003200629/706941/003200654-655/003200654-655/003201119/003201121/003201123/003201136/002666347/003201138-39/002667144/002666349/001333550. Beside the RBLs (TypesI–V), several groups are classifiable as an RBL superfamily:the immune recognition molecules, rhamnospondins(Rsps), LPHNs, Arabidopsis galactosidases, and others asshown in Figure 5. Rsp gene has been identified in colonialhydroid Hydractinia symbiolongicarpus and was foundto encode a secreted modular protein of 726 aminoacids composed of N-terminal serine-rich domain, eighttandem-repeated thrombospondin type 1 repeats (TSRs),and C-terminal RBL-CRD [139]. Rsps have diversified bygene duplication and predicted to act as immune recognitionmolecules from the evidence of gene structure and theirexpression profiles in the polyp’s hypostome, which face tothe external environment and pathogen. On the other hand,Caenorhabditis elegans EVA-1 forms a complex with SAX-3/roundabout (Robo) receptor and functions as a coreceptor

for shiga-like toxin 1 (SLT-1)/slit proteins in guiding celland axon migrations [135]. Furthermore, human C21orf63,an EVA-1 ortholog, has been identified from the Down’ssyndrome project [136] and reported to have specific affinityto heparin.

LPHNs are synaptic Ca2+-independent α-latrotoxin(LTX) receptor, a novel member of the secretin family ofG-protein-coupled receptors containing seven transmem-brane regions as well as long N-terminal extracellularsequences containing a 19-amino acid signal peptide, and aserine/threonine-rich glycosylation region (Figure 5) [137].LTX is a component of the venom of the black widowspider (latrodectus mactans) and stimulates exocytosis of γ-aminobutyric acid- (GABA-) containing presynaptic vesiclesvia interaction with LPHN. Recently, LPHN3, which is themost brain-specific LPHN, has been reported to be involvedin the pathogenesis of attention-deficit/hyperactivity disor-der [138]. Thus, the RBL-CRDs are diversified and widelydistributed in the functional proteins as unique structuralmotifs. Similar examples for the domain architecture ofproteins including membrane receptors have been reportedin other lectin families such as CTLD [74–79] and F-typelectin superfamilies [122, 123].

7. Gene Structure of RBL Family

More recently, cloning and characterization of a gene forsnakehead lectin (SHL) from Channa argus and its pro-moter region have been reported (Genebank accession nos.:EU693900) [140]. SHL gene, which consists of 2,382 bp fromthe transcription initiation site to the end of 3′ untranslatedregion (UTR) and includes two tandem RBL-CRDs with 35%identity, contains nine exons and eight introns. The first40 bp of exon 1 is 5′UTR, and the signal peptide is encodedby exons 1 and 2. The N-terminal CRD is encoded by exons3, 4, and 5, and C-terminal CRD is encoded by exons 6, 7,


TSP1

TSP1

TSP1

TSP1

Rhamnospondins

(Rsp)

Danio rerio

Latrophili

7tm 2GPS

DUF3497HormR

OLF

XP 002663750/691000/689120

Latrophilin

CAG02284

CAF95118

7tm 2GPS

GPS

DUF3497

HormR

7tm 2

GPSDUF3497

RBLs

Polycystic kidneydisease 1-like 2

7tm 2GPS

7tm 2

DUF3497

HormR

PKD channel

GPS

HormR

PLAT

Tetraodon nigroviridis

LCCL

FA58C XP 003200842

CAG02284

CAG01764/CAG03639/CAG00449/

CAF93692/CAF94432/CAF95311/

Accession numbers

CAG08363IPPc

OLF

OLF

HormR

I, II

III, IV

V

CAP09317AAH28296

(Homo sapiens)

XP 002631367(C. briggsae)

XP 2158747/CAJ80765ADV92627/ABD95939

ABE66341-364(Hydractinia symbiolongicarpus)

XP 2155269(H. symbiolongicarpus)

XP 2160935(H. symbiolongicarpus)

AAH94668

ADD22401-6(H. echinata)

Q7Z442(Homo sapiens)

CAP24513(C. briggsae)

XP 002606909(B. floridae)

AAT47768(D. melanogaster)

CBY23011/CBY37649(Oikopleura dioica)

CTLD

CTLD

CTLD CTLD

CTLD

CTLD CTLD CTLD CTLD CTLD CTLD CTLD CTLD CTLD

RhoGAP OCR

∗2

∗1

n =2–5

n =2–11

Others

(Mus musculus)

Figure 5: Domain architecture of RBL superfamily. The proteins with similar domain architectures have been identified using the ConservedDomain Architecture Retrieval Tool (CDART) [124].


Table 4: Zebrafish RBL-related genes.

LengthChromosome Exons Gene ID∗

Transcripts (bp) Protein (AA)

Type I/II (3CRDs)

LOC100536087 1063 274 19 10 100536087

LOC555549 1144 297 19 10 555549

Type III/IV (2CRDs)

Zgc136410 878 222 21 9 449685

LOC100536640 978 233 19 9 100536640

LOC100535118 715/868 167/209 9 8/9 100535118

LOC100536038 1050 272 19 9 100536038

LOC100536135 — (pseudo) — 19 9 798039

1CRD

LOC100333730 654/618 119/137 9 6 100333730

LOC100301575 665 128 22 6 100301575

LOC798111 668 128 22 6 798111

LOC100535660 469 129 22 7 100535660

MGC171851 677 128 22 6 798039

LOC100331690 815 186 22 7 100331690

LOC100537146 837 188 22 7 100537146

LOC100537102 804 179 22 7 100537102

LOC100536958 821 188 22 7 100536958

LOC794480 750 137 23 9 794480

Others

Latrophilin-3-like 609 131 22 6 445036

Latrophilin 2 669 128 22 7 94733430

DKEYP-98A7.10 620 168 22 7 100126138

LOC792919 652 190 2 6 792919

C21orf63 homolog 1189 380 14 7 100537109∗

Gene ID: ID numbers in NCBI’s database for gene-specific information [98].

and 8. Exon 9 includes the C-terminal region of SHL and3′UTR. These suggest that RBL-CRDs are located in threeexons; respectively, and RBL may be diverged and evolvedby gene duplication and/or exon shuffling. The 5′ flankingregions contained some unique consensus sequence for thenuclear factor of interleukin 6 (NF-IL6) and IFN-γ activationsites.

On the other hand, Rsp gene was predicted to encodea secreted protein of 726 amino acids composed of a signalpeptide, an N-terminal serine-rich domain (SRD), eightTSRs, and a RBL-CRD at C-terminal region, consisting of 13exons and 12 introns [139, 141]. However, RBL-CRD of Rspwas located in only single exon (Exon 12), suggesting that thegene structure of Rsp RBL-CRD is different from that of SHLRBL-CRD. Whole genome sequences have been determinedfor several living organisms including marine organisms suchas zebrafish, Danio rerio, of which data can be available andallow us to establish the full inventory of any particulargene family in the genome. Thus, searching the zebrafishdatabase with RBL-CRD sequences revealed that the genesencoding RBLs with tandem-repeated CRDs (Types I toIV) were located in chromosomes 9, 19, and 21 (Table 4).

Furthermore, it was found that several genes encodingsingle RBL-CRD proteins, almost all of which physiologicalfunctions are largely unknown, are located in chromosomes2, 9, 23, and especially in chromosome 22 (Table 4), althoughthe genes for latrophilins and C21orf63 homolog werelocated in chromosomes 22 and 14, respectively.

8. Sugar-Binding Specificities andPhysiological Functions of RBLs

Sugar-binding specificities of CSLs were investigated thor-oughly by frontal affinity chromatography (FAC) using 100kinds of sugar chains including N-linked and glycolipid-typeglycans [142]. Interestingly, all of CSL1, CSL2, and CSL3showed the high specific binding activity against globotriao-syl ceramide (Gb3; Galα1-4Galβ1-4Galβ1-Cer also known asCD77), which is located in lipid raft and upregulated throughimmune responses [143] and is also known as the functionalreceptor for various toxins such as Shiga toxin (Stx) [144],regardless of their low sequence homologies (42–52%) anddifferent oligomeric structures.


CSLs induced proinflammatory cytokines, including IL-1β1, IL-1 β2, TNF-α1, TNF-α2, and IL-8, by recognizingGb3 on the surface of the peritoneal macrophage cell line(RTM5) from rainbow trout and an established fibroblastic-like cell line (RTG-2) from gonadal tissue of the fish [142].RBL from catfish, SAL, has also induced the alterationsof gene expression in Burkitt’s lymphoma cells [145].Furthermore, CSLs showed the cytotoxicity against Gb3-displaying Caco-2 and Lovo cells via an apoptotic pathwaythrough the recognizing of Gb3 on the cell surfaces in a dose-dependent manner, while it was not observed with DLD-1 and HCT-15 human colonic tumor cell lines lacking Gb3

[125].RBLs from fish eggs such as STLs and CSLs also inter-

acted and agglutinate Gram-negative and Gram-positivebacteria by recognizing the cell-surface lipopolysaccharidesand lipoteichoic acid, respectively, [17, 146]. On the otherhand, PPL, an RBL from Pteria penguin pearl shell, alsoshowed the strong agglutinating activity against some Gram-negative bacteria such as Escherichia coli by recognizinglipopolysaccharides in the presence of the high concentration(500 mM) of NaCl, in which condition the oligomerizationof PPL was induced, although its carbohydrate-bindingspecificity was quite different from that of CSLs; PPL binds tod-galactose but not l-rhamnose [39]. RBLs can recognize theO-antigen, which is the immunodominant structure exposedto the environment and is highly variable among bacterialstrains, via diverse carbohydrate recognition ability. RBLsbind to glycolipids and glycoproteins of the microsporidianfish pathogens [61, 129], and the RBL receptor was expressedon peritoneal macrophages of fishes after an inflammatorystimulation [130, 147]. More recently, it was found thatCSLs induced the production of radical oxygen species (ROS)in RTM5 cells in a dose-dependent manner. This effectwas not inhibited by l-rhamnose or dl-threo-1-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP), aninhibitor of glucosyl ceramide synthesis, suggesting thatcarbohydrate recognition domains of CSLs were not involvedin the respiratory burst of RTM5 cells.

Thus, CSLs are multifunctional lectins through bindingto the carbohydrate such as Gb3 on cells and l-rhamnose/d-galactose residue of O-antigen of lipopolysaccharides. How-ever, this carbohydrate-binding specificity of RBL leads tothe interesting questions of how CSL can strongly recognizethe different sugars such as Gb3, l-rhamnose, d-galactose,all of which common structural features are the orientationof anomeric hydroxy groups at C2 and C4.

9. Structural Characterization of RBL-CRDs

More recently, the highly ordered structure of CSL3 com-posed of two subunits of 20 kDa has been determined at 1.8 Aresolution [146]. The homodimer of CSL3 revealed a kinkeddumbbell shape, in which two lobes are connected throughlinkers composed of two 5-residue peptides (-QQQET-)(Figure 6(a)). Each lobe seems to be a single globular proteinwith a pseudo-twofold axis and includes two antiparallel βsheets with two (β2 and β4) and three (β1, β3, and β5)strands and three helices (α1-3) (Figure 6(a)). The N- and

C-terminal domains, both of which share 35% sequenceidentity with the RBL-CRD for mouse latrophilin-1 (LPHN-1), their folds were similar each other and superimposedon the RBL domain of LPHN-1, which has been recentlyreported [148], with rmsds of 1.4 and 1.5 A for 94 Caatoms, respectively. These RBL domains adopt a unique α/βfold with long structured loops involved in monosacchariderecognition.

It was found that the monosaccharide (rhamnose) ornonreducing end residues (Gal1 of melibiose and Gb3) sharethe same conserved primary binding sites in CSL3 (Glu7/107,Tyr27/127, Lys86/186, and Gly83/183) and LPHN-1 (Glu42,Tyr63, Lys120, and Gly117), respectively, (Figure 6(b)).Asn74/174 and Asp79/179 are additionally used in theprimary site in CSL3. The melibiose and Gb3 complex struc-tures revealed the oligosaccharide recognition mechanism ofRBL. The Arg39/139 and Gln43/143 residues of CSL3, whichbind to the carbohydrate in the 2nd and 3rd sites, are the keyresidues in determining specificity for oligosaccharides, Gb3.The total numbers of hydrogen bonds between CSL3 andrhamnose, melibiose, and Gb3 are 7, 8, and 10, respectively,which are consistent with the observed high affinity (Kd =2.6× 10−5 M) of CSL3 to Gb3.

Interestingly, RBLs can bind to l-rhamnose and nonre-ducing d-galactose moiety of melibiose and Gb3 at thesame binding site. These specific and characteristic bindingabilities can be explained by the recognizing mechanismsinvolved in the hydrogen bonds between O2, O3, and O4

atoms of monosaccharide and the side chains of Glu7/107,Asn74/174, Asp79/179, Lys86/186, and the main chain ofGly83/183 of CSL3; that is, Glu7/107 forms hydrogen bondwith O4 atom of l-rhamnose, which correspond to O2

of inverted d-galactose, while Gly83/183 forms hydrogenbond with O2 atom of l-rhamnose and O4 of inverted d-galactose form, respectively, (Figure 6(b)). These carbohy-drate recognition mechanisms of lectins for the invertedcarbohydrates were also found in the case for F-type lectins,which bind to both α-l-fructose and 3-O-methyl-d-galactose[122].

10. Perspectives

Since lectins isolated from marine resources are highlydiversified in terms of not only structure but also functionalaspects including specific and unique carbohydrate specifici-ties as reviewed in this paper, they can be used for biomedicalapplication as drug delivery system or diagnostic markers.For example, RBL family lectins are useful for diagnosisof pathological condition involved in Gb3 ceramide suchas Burkitt’s lymphoma having high malignancy. Further-more, RBLs showed the physiological functions independentof carbohydrate-recognition ability such as ROS-inducingactivities although its molecular mechanism has not yet beenclarified. More recently, novel calcium-dependent mannose-binding lectin, intelectin, which is structurally identicalto the intestinal receptor for lactoferrin and containedfibrinogen-related domain, has been identified from theskin mucus of catfish Silurus asotus [55]. Thus, there are alarge and growing number of diversified lectins in marine


NN

C

C

Linker

(a)

Glu7/107

Asn74/174

Lys86/186

Asp79/179

Gly83/183

Thr84/184

Tyr85/185

Glu7/107

Asn74/174

Lys86/186

Asp79/179

Gly83/183

Thr84/184

Gln43/143

Tyr85/185

1

1

1 1

2

2

2 2

3

3

3 3

4

4

4 4

5

5

5 5

Gln43/143

O

O

O

OO

O

O

OO

O O

O

O

O

O

O O

O

O

OO

6

6

6

Arg39/139

D-Galactose (Gb3)

L-Rhamnose

(b)

Figure 6: Crystal structure of CSL3. (a) Pseudotetrameric structure of CSL3 dimer (PDB code: 2z×3) [125]. Each carbohydrate-bindingdomain is differently colored. The bound melibioses and phosphates are shown in stick models. (b) Comparison of the carbohydrate-bindingmanners of CSL3 complex with l-rhamnose (left) and Gb3 (right).

resources. Further study will be necessary to elucidate thedetailed structure-activity relationships of diversified marinelectins and to develop the potent therapeutic drugs.

AbbreviationsAAA: Anguilla japonica agglutininAFPs: Antifreeze proteinsASGR: Asialoglycoprotein receptor

CN-V: CyanovirinCon I: Congerin ICon II: Congerin IICRD: Carbohydrate-recognition domainCSL: Chum salmon lectinCTLs: C-type lectinsCTLD: C-type lectin domainDC-SIGN: Dendritic cell-specific intercellular adhesion

molecule-3-grabbing nonintegrin


EC50: Half maximal effective concentrationES: Embryonic stemFACT: Frontal affinity chromatography techniqueGABA: γ-Aminobutyric acidGRFT: GriffithsinGRPs: Galectin-related proteinsHIV: Human immunodeficiency virusiPS: Induced pluripotent stemLPHN: LatrophilinLTX: α-LatrotoxinMALDI: Matrix-assisted laser desorption/ionizationMBP: Mannose-binding proteinMES: 2-(N-Morpholino) ethane sulfonic acidPKD-1: Polycystic kidney disdase-1 likePPL: Pteria penguin lectinPSP: Pancreatic stone proteinRBL: Rhamnose-binding lectinRsp: RhamnospondinsSAL: Silurus asotus lectinSHL: Snakehead lectinSLT-1: Shiga-like toxin 1SML: Spanish mackerel lectinSTL: Steelhead trout lectinSUEL: Sea urchin egg lectinTSRs: Thrombospondin-type-1 repeats.

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