immobilization of alkaline collagenase from bacillus subtilis onto...

Research ArticleImmobilization of Alkaline Collagenase from Bacillussubtilis onto Sulfonated Polystyrene Nanospheres forHydrolysis of Tilapia Collagen

Ling Zhang12 Xiaocui Yang1 Kaijun Xiao 1 Yuyi Lu2 Chunhai Li2 and Zijin Zhang2

1School of Food Sciences and Engineering South China University of Technology Guangzhou 510640 Guangdong China2College of Biological and Food Engineering Guangdong University of Petrochemical TechnologyFood Science Innovation Team of Guangdong Higher Education Institutes Maoming 525000 Guangdong China

Correspondence should be addressed to Kaijun Xiao fekjxiaoscuteducn

Received 10 April 2019 Accepted 17 July 2019 Published 26 September 2019

Academic Editor Antoni Szumny

Copyright copy 2019 Ling Zhang et al is is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

e structure of an alkaline protease from Bacillus subtilis used by a tilapia collagen peptide manufacturer was analyzed and thetechnology of the enzyme immobilized by sulfonated polystyrene (SPS) nanoparticles was studied e particle size distributionSEM EDS TEM and FT-IR spectroscopy of the carrier before and after immobilization were analyzed e results showed thatthe molecular weight of the puried enzyme protein was 310 kDae amino acid sequence with a consistency of 6404 and onethree-dimensional structure simulation diagram of the puried enzyme protein were obtained by LC-MS-MS which suggestedthat the protein might belong to subtilisin e optimal immobilization conditions were as follows the volume ratio of theimmobilization carrier to the enzyme was 3 50 (mL mL) the immobilized temperature was 25degC and the system pH was 45Under this condition the immobilization ratio of collagenase was 7348 the specic activity was 27405Uμg and the specicactivity of the immobilized enzyme was about 5374 that of the free enzyme e average particle size of SPS nanospheres was1551 nm e characterization results of SEM EDS TEM and FT-IR spectroscopy showed that the collagenase was successfullyimmobilized onto SPS nanospheres e experimental results also showed that the collagenase could be immobilized ecentectivelyunder the optimal conditions by using SPS nanospheres and the operation process was simple feasible and of low cost with goodprospect of industrial application

1 Introduction

In recent years collagen hydrolysate and polypeptide havebeen widely used in biology medicine food cosmetics andso on whose function has been increasingly recognized andhighly valued because of the continuous study of thestructure and properties of collagen [1ndash3] At present en-zyme technology is considered to be the most ecentective wayto hydrolyze collagen [4 5] Collagenase which can beproduced by many tissues and cells of various microor-ganisms and animals especially under pathological condi-tions is a kind of enzyme that specically degrades thecollagen helix or gelatin without acting on other proteins [6]A large amount of collagenase can be obtained by microbialfermentation as compared to the low yield extracted from

the animal or sh viscera which is thought to be an ecentectiveway to solve the growing industrial application demand[7 8] Bacillus subtilis is a traditional high-yield strain forproducing alkaline protease and the enzyme has become ahot spot in protease research for the characteristics of hightemperature alkali resistance etc [9 10] No matter whatthe source of the enzyme is generally speaking the crudeenzyme is obtained at the beginning A series of separationand purication methods are needed to get pure enzymes

Immobilized enzyme technology is an ecentective way toovercome deciencies of the free enzyme which can im-prove the stability and recycling of the enzyme save the use-cost of the enzyme and improve the feasibility of the ap-plication of the enzyme in scale [11] As a new carrier of theimmobilized enzyme nanomaterials have the characteristics

HindawiJournal of Food QualityVolume 2019 Article ID 7521895 14 pageshttpsdoiorg10115520197521895

of minor pore size large specific surface area large surfacebinding energy and easy integration with the enzymesteadily which can effectively improve the enzyme load andthe stability of enzymes [12] In recent years polystyrene hasbeen not only widely used in light industry daily decorationlighting indication fields and so on but also applied as a newcarrier to enzyme immobilization technology [13 14]

When the team worked with a local tilapia collagenpeptide producer to study how to reduce the productioncost we decided to use immobilized enzyme technology tosave the cost of the enzyme We found that sulfonatedpolystyrene (SPS) nanospheres are a good immobilizedmaterial with simple processing low cost good corrosionresistance small pore size large specific surface area andgood biocompatibility [15] but also there are few studies onSPS nanospheres at present and especially their applicationin food industry is very rare -erefore we chose SPSnanoparticles as a carrier and carried out some relatedcharacterization works It is a new attempt which canprovide more basic data for further study of the enzymeimmobilization

2 Materials and Methods

21 Materials and Reagents Collagen hydrolase (alkalineprotease fermented by Bacillus subtilis) and tilapia skincollagen were provided by Guangdong Baiwei Bio Ma-terial Co Ltd Maoming China All reagents were ofanalytical pure grade and purchased from the marketsuch as styrene sodium dodecyl sulfate (SDS) Coo-massie Brilliant Blue G-250 potassium persulfate con-centrated sulfuric acid sodium hydroxide citric acidtrichloroacetic acid (TCA) and potassium sodiumtartrate

22 Isolation and Purification of Collagen HydrolaseAmmonium sulfate precipitation is divided into twosteps the first one is to remove the impure protein (inprecipitation) and the second one is to retain the pre-cipitate to obtain the purified enzyme protein [16] -einitial enzyme solution of 25mL was extracted andprecipitated by ammonium sulfate with a saturation of20 and 40 respectively -e enzyme protein wasprecipitated by centrifugation then dialyzed many timeswith a dialysis bag with a cutoff molecular weight of8000ndash14000 Da and dehydrated by polyethylene glycol20000 [17 18] -e concentrated enzyme protein waspurified by a full-automatic polysaccharide and proteinpurification system (PS-P China) with glucan gel G-759as a column chromatography filler afterwards using10mmolL pH 55 phosphate buffer as equilibrium andelution liquid and 20 ethanol for rinsing When theelution peak began to appear the peak time was recordedand the eluted enzyme was collected (3 mLtube) -eenzyme collected repeatedly was first concentrated withpolyethylene glycol 20000 and then freeze-dried intopowder and stored hermetically at low temperature forfurther use

23 Determination of Molecular Weight and Structure Sim-ulation of the PurifiedEnzymeProtein -emolecular weightof the purified enzyme protein was determined by SDS-PAGE (1645050 US) [19]

-e amino acid sequence of the enzyme protein wasdetermined by LC-MS-MS (Q Exactive US) using the bandsseparated by SDS-PAGE gel electrophoresis and hydrolyzedby trypsin [20 21] Liquid chromatographic column in-formation 300 μm idtimes 5mm packed with Acclaim PepMapRSLC C18 5μm 100 A nanoViper mobile phase A 01formic acid mobile phase B 01 formic acid and 80 CANflow rate 300 nLmin and analysis time of each group60min -e separated peptide entered the Q Exactive massspectrometer directly for online detection Parameters of MS1 resolution 70000 AGC target 3e6 maximum IT 40msand scan range 350 to 1800mz Parameters of MS 2 res-olution 17500 AGC target 1e5 maximum IT 60ms TopN20 and NCEstepped NCE 27 -e three-dimensionalstructure of the enzyme protein was modeled by MM FileConversion online (httpswwwproteinmodelportalorgqueryuniprot) and retrieved by Mascot Finally the blastcomparison was used on the NCBI database to analyze thehomology of the protein-e secondary structure and tertiarystructure of the enzyme protein were analyzed by Phyre2NCBI SWISS-MODEL and PyMOL

24 Preparation of Sulfonated Polystyrene -e styrene (St)was first soaked in NaOH aqueous solution (10wt) andafter 24 hours it was washed several times with distilledwater -e treated St was rotary evaporated under reducedpressure at 60degC SDS (02 g) and Na2CO3 (01 g) wereweighed and dissolved in 300mL distilled water -eresulting solution was transferred to the three-neck flask andpurged with nitrogen for 30min Subsequently 30mL oftreated St was added to the above solution under strongstirring and the solution was heated to 60degC After 30minpotassium persulfate aqueous solution (15mL 023mmolL)was introduced into it [22] -e polymerization reaction wasconducted at 75degC for 20 h After cooling to room tem-perature the product was alternately washed with 95ethanol and distilled water Afterwards the product waspurified by reduced-pressure rotary evaporation at 60degC anddried at 60degC for further use [23]

-e above polystyrene (PS) powder (10 g) was dispersedinto concentrated H2SO4 (40mL 98) by ultrasonicationAn ultrasonic cleaning tank was used for ultrasonication for20min at room temperature -e sulfonation reaction wasconducted at room temperature for 24 h -e product wasthoroughly washed with distilled water and ethanol anddried at 60degC using a vacuum oven After drying the productwas dispersed into distilled water by ultrasonication and thesulfonated polystyrene emulsion (40mgmL) was made

25 Study on Immobilization of Enzymes

251 Immobilization Method SPS nanosphere emulsionwas treated under ultrasonication for 30min and a certainamount of it was diluted 100 times with phosphate buffer

2 Journal of Food Quality

-e enzyme was diluted 20 times with phosphate buffer -ediluted enzyme solution and SPS emulsion were oscillatedand mixed in a test tube After a certain reaction time atroom temperature the reactants were separated by centri-fugation at the speed of 16000 rmin for 15min the su-pernatant was collected and stored at 4degC to determine theimmobilization ratio and the precipitate was collected andfreeze-dried to measure immobilized enzyme activity [24]

252 Optimization of Immobilized Process A single-factorexperiment was carried out and other conditions weredetermined -e effects of four factors such as the volumeratio of the immobilization carrier to the enzyme immo-bilization time temperature and pH were studied by usingimmobilization ratio and specific activity of protein as in-dicators According to the results of the single-factor testorthogonal experiments were used to optimize the immo-bilization conditions

(1) Single-Factor Experiment

(1) Effects of the Volume Ratio of the ImmobilizationCarrier to the Enzyme In the test tubes 1 SPSemulsion (2mL 3mL 4mL 5mL 6mL and7mL) after ultrasonic treatment was added re-spectively to 5 enzyme solution (10mL)reacting for 8min after shaking at 25degC Aftercentrifugation immobilizing effects could becompared

(2) Effects of Immobilization Time In the test tubes1 SPS emulsion (4mL) after ultrasonic treat-ment was added to 5 enzyme solution (10mL)reacting for 2ndash10min after shaking at 25degC Aftercentrifugation immobilizing effects could becompared

(3) Effects of Immobilization Temperature In the testtubes 1 SPS emulsion (4mL) after ultrasonictreatment was added to 5 enzyme solution(10mL) reacting for 8min after shaking at 15degC20degC 25degC 30degC and 35degC After centrifugationimmobilizing effects could be compared

(4) Effects of Immobilization pH -e enzyme andSPS emulsion were diluted 20 times and 100times with phosphate buffer of different pH(32sim64) respectively In the test tubes 1 SPSemulsion (4mL) after ultrasonic treatment wasadded to 5 enzyme solution (10mL) reactingfor 8min after shaking at 25degC After centrifu-gation immobilizing effects could be compared

(2) Orthogonal Test

According to the results of single-factor experi-ments three factors such as the volume ratio of theimmobilization carrier to the enzyme immobili-zation temperature and immobilization systempH were chosen for study and L9(3)3 orthogonaltests were carried out to obtain the optimal im-mobilization conditions -e factor-level table isshown in Table 1

26 Determination of the Enzyme Immobilization Ratio-e content of enzyme protein was determined by theCoomassie Brilliant Blue method and the immobilizationratio (Y) of protein was calculated using the followingequation

Y m0 minus m1( 1113857

m01113890 1113891 times 100 (1)

wherem0 (mg) is the total protein quality of enzyme solutionbefore immobilization andm1 (mg) is the total protein massof residual liquid after immobilization

27 Determination of Enzyme Activity First the modifiedbiuret method was used to determine the concentration ofpeptides in this experiment and the operation was based onthe patent ldquoA Method for the Determination of CollagenPeptide in Tilapia by Biuret Methodrdquo [25] -e peptidecontent was calculated according to the following equation

M A

btimes c times d times V1 (2)

where M (mg) is the peptide content in the reaction systemA is the absorbance b is the coefficient of the standard curvec (111) is the dilution multiple of the reacting solution afterprecipitating protein d is the dilution multiple of thereacting solution after adjusting pH and V1 (mL) is the totalvolume in the reaction system

-e second step was to determine the activity of theenzyme -e reaction conditions for the determination ofenzyme activity were as follows at 60degC the concentration ofcollagen in the reaction system was 10 and the volumeconcentration of the original enzyme solution was 100 ppmreacting for 10min -e enzyme activity was defined as thatunder the above conditions one enzyme activity unit (U)was required for 1 μg polypeptide produced by hydrolyzingcollagen of fish skin with the original enzyme solution perminute -e enzyme activity was calculated according to thefollowing equation

Q M times 103

Ttimes

1V2

(3)

where Q (U) is the enzyme activity M (mg) is the peptidecontent in the reaction system T (min) is the reaction timeand V2 (mL) is the volume of the original enzyme solutionadded in the reaction system

28 Determination of SpecificActivity of Immobilized Enzymes-e specific activity of immobilized enzymes was calculatedusing the following equation

S Q

m0 minus m1( 1113857 (4)

where S (Uμg) is the specific activity Q (U) is the enzymeactivity m0 (mg) is the total protein quality of enzymesolution before immobilization and m1 (mg) is the totalprotein mass of residual liquid after immobilization

Journal of Food Quality 3

29 Characterization of Sulfonated Polystyrene Nanospheresand Immobilized Enzymes

291 Analysis of Particle Size Distribution of SPSNanospheres-e particle size distribution of SPS nanospheres was de-termined by a nanoparticle analyzer (SZ-100Z Japan) Itworked under the condition that the scattering angle was 90degthe stent temperature was 252degC and the dispersion me-dium was water

292 Scanning Electron Microscopy (SEM) and EnergySpectrumAnalysis -emorphology of SPS nanospheres andimmobilized enzymes was studied by a scanning electronmicroscope (JSM-6510LV Japan) -e SPS emulsion wasdiluted 100 times with distilled water dropped on tin foildried and then subjected to gold spray treatment -esamples were observed under a scanning electron micro-scope of which the working voltage was set to 1 kV [26] -efreeze-dried immobilized enzyme was dispersed in distilledwater and determined by the same method

293 Transmission Electron Microscope (TEM) -e solu-tion of SPS nanospheres was dialyzed and the produceobtained was dried at 50degC -e samples were observed by atransmission electron microscope (H7650 Japan) and theoperating voltage was 200 kV -e immobilized enzyme wasdissolved in distilled water and determined by the samemethod

294 Fourier Transform Infrared (FT-IR) Spectroscopy -edry SPS nanoparticles and immobilized enzyme were re-spectively made into suitable sized wafers by the KBrpowder compaction method In the measurement range of4000ndash450 cmminus 1 the wafers were measured by a Fouriertransform infrared spectrometer (Nicolet 6700 US) -einfrared spectrum of the prefixed material and immobilizedenzyme was obtained [27]

210 Experimental Data Plotting and Mathematical Statis-tical Methods Single-factor and orthogonal experimentswere done three times in parallel taking the mean value -eSPSS 20 0 statistical analysis software was used to analyzethe variance of data and Origin 85 was applied to draw

3 Results and Discussion

31 Molecular Weight Determination of the Purified Enzyme-e result of gel electrophoresis bands is shown in Figure 1As seen in the chart the purified enzyme solution has only

one distinct single zone which indicates that the purificationmethod adopted in the experiment achieved the ideal pu-rification effect According to the relative mobility rate andstandard curve of the sample the molecular weight of en-zyme protein after separation and purification is calculatedto be 310 kDa

32 Amino Acid Sequence and Structure Simulation of Col-lagen Hydrolase Referring to documents [28 29] theprotein sequence of the purified collagen hydrolase is ob-tained after sequencing and then the mass number ofcollagen protein hydrolysate is retrieved from the Mascotdatabase to obtain several suspicious proteins After onlineblast matching a protein with 100 homology was obtainedand identified as the Bacillus subtilis protease (accessionnumber Q45299-BACLI) Its amino acid sequence asshown in Figure 2 consists of 379 amino acids with amolecular weight of 39082Da -e molecular formula of theenzyme protein is C1715H2730N472O548S11 the total numberof atoms is 5476 and the isoelectric point is 866 Accordingto the amino acid sequence the enzyme was identified asalkaline protease because the amino acid residue (Arg Lys)was 32 and the acidic amino acid residue (Asp Glu) was 29 Itis a hydrophilic protease for the total hydrophobic average(GRAVY) of minus 0030 Its aliphatic amino acid index is 8182According to the criterion of stable protein (unstableindexlt 40) it can also be judged as the stable protein as itsunstable index (II) is 1563 -e N-terminal of the enzyme isM (Met)-e content of alanine glycine valine and serine is116 113 111 and 108 respectively -e content ofcysteine is the lowest (03)

-en using c3whiA as the template the Phyre2 serverwas used to predict the secondary structure of the enzymeprotein molecule As shown in Figure 3 the consistencybetween the predicted structure and the submitted targetprotein amino acid sequence is 6404 the informationabout the possible locations of α-helix and β-fold can beobtained and the result is credible

-e secondary structure of the enzyme was analyzedby the SOPMA online program As shown in Figure 4 thesecondary structure of the enzyme contains 3298α-helix structures 2058 β-fold structures 976 β-turnstructures and 3668 irregular curl structures -eα-helix structure and irregular crimping structure existalternately which are the main part of the wholestructure of the protease and the extended long chain isuniformly distributed in the whole chain -is distri-bution is beneficial to the stability of the enzymestructure

Table 1 Factors and levels of the orthogonal test

LevelA B C

Volume ratio of the immobilization carrier to theenzyme (mL mL) Immobilization temperature (degC) pH of the immobilized system

1 3 50 20 352 4 50 25 403 5 50 30 45


-e amino acid sequence of the protein was analyzed bythe Related Structures (Summary) function in NCBI andthe result showed that the protease molecule of Bacillussubtilismainly contained a functional domain As shown inFigure 5 the active sites of the enzyme are in the domain of133thndash325th amino acids -e specific active sites are the137th 168th 211th 230th 259th and 325th amino acids -esites with catalytic activity are the 137th 168th and 325thamino acids

Using c3whiA as the template SWISS-MODEL (httpswissmodelexpasyore) was used to predict the tertiarystructure of the enzyme and we found that the range of theresidues of the enzyme molecule was from the 36th to the379th amino acid -e simulated structure diagram is shownin Figure 6 and the performance is displayed in differentcolors in the structure with N-terminals above and C-ter-minals below Figure 6(b) is a structure diagram based onhydrophilic labeling showing that most of the proteinmolecules are hydrophilic fragments and only a few arehydrophobic fragments Figures 6(c) and 6(d) show that thepositive charge of the protease residue is more than thenegative charge mainly on the surface according to thecharge-marked structure diagram

Figure 7 is about catalytic activity sites showing that theactive site is located in the middle of the structure and is a

triplet with catalytic hydrolysis of a-peptide bonds in in-ternal peptide chains composed of aspartic acid (137th) withan acidic residue histidine (168th) with an alkaline residueand serine (325th) with the nucleophilic property

33 Conditions for Immobilization of CollagenHydrolase ontoSulfonated Polystyrene Nanospheres

331 Single-Factor Experimental Result Analysis -e in-fluence of the volume ratio of the immobilization carrier tothe enzyme time temperature and pH on immobilization isshown in Figure 8 It can be seen from Figure 8(a) thatunder the condition of a certain amount of collagenase theimmobilization ratio of the enzyme increases with the im-provement of the amount of the SPS nanosphere but thespecific activity of the immobilized enzyme is continuouslydecreased When the ratio of the amount of the SPS to thequantity of the emulsion reaches 5 50 the immobilizationratio is largest and basically stable and the maximum valueis 9730 which indicates that when the volume of SPSemulsion is 10 of the collagenase volume the SPS nano-spheres can adsorb collagenase to the greatest extent Whenthe ratio reaches 2 50 the specific activity is largesthowever when the ratio reaches 7 50 the specific activity issmallest -is may be due to the influence of SPS on theactivity of collagenase Considering comprehensively it isadvisable to determine the effect of volume ratio of 4 50

It can be seen from Figure 8(b) that the change of theimmobilization ratio is very weak all up to 97 in 2min to10min but the activity of the immobilized enzyme decreasesslightly When immobilized for 6min the immobilizationratio reaches the maximum which is 9776 Whenimmobilized for 4min the specific activity is the highestand after 8min the specific activity decreases significantly-ese indicate that the SPS has a strong adsorption capacityfor collagenase and can quickly complete the immobilizationprocess So 8min can be chosen as the best immobilizationtime

It can be seen from Figure 8(c) that the immobilizationratio changes little with the increase of temperature whilethe specific activity is greatly influenced When the tem-perature is 25degC the immobilization ratio is the highest andwhen the temperature is between 15 and 25degC the loss ofspecific activity is less but the specific activity decreasessignificantly when the temperature continues to rise whichmay be because the high-temperature environment willdestroy the active site of the enzyme resulting in a decreasein specific activity -erefore 25degC can be determined as thebest immobilization temperature

It can be seen from Figure 8(d) that the system pH hasgreat influence on the immobilization ratio and specificactivity With the increase of pH the immobilization ratiodecreases while the specific activity increases first and thendecreases When the pH is 32 the immobilization ratioreaches 9625 and the specific activity decreases to theminimum and when the pH is 56 the immobilization ratiodecreases to the minimum but the specific activity reachesthe maximum which may be due to the decreased activity of

S2MarkerkD

350

250

184

144

Figure 1 Gel electrophoresis bands of marker and purified col-lagenase (S2)

Figure 2 Amino acid sequence of the Bacillus subtilis protease(accession number Q45299-BACLI matched peptides are shownin bold red)


the alkaline protease in a peracid environment -ereforepH 40 is considered to be an appropriate condition

332 Orthogonal Experimental Result Analysis According tothe single-factor experimental results considering the im-mobilization time has little effect on immobilization taking8min as the best immobilization time the L9(3)3 orthogonaloptimization experiment is carried out by selecting threefactors (the volume ratio of the immobilization carrier to theenzyme immobilization temperature and pH) from foursingle factors -e orthogonal experimental results are

shown in Table 2 and the variance analysis is shown inTable 3

According to the orthogonal experimental results andvariance analysis if the immobilization ratio is used as theevaluation index the optimal process combination isA3B1C1 -e influence degree of each factor on the im-mobilization ratio is as follows pHgt volume ratio of theimmobilization carrier to the enzymegt immobilizationtemperature in which the pH and the volume ratio of thematerial and enzyme liquid are extremely significantHowever if the specific activity of the immobilized enzymeis used as the evaluation index the best combination is

Figure 3 Secondary structure prediction of Bacillus subtilis by the Phyre2 Server


A1B2C3 -e influence degree of each factor on thespecific activity of the immobilized enzyme is as followsvolume ratio of the immobilization carrier to theenzyme gt pH gt immobilized temperature and only the

influence of volume ratio of the immobilization carrier tothe enzyme is extremely significant

-e two combinations of A3B1C1 and A1B2C3 areconducted in parallel for three validation tests and the

Figure 4 Secondary structure prediction of Bacillus subtilis by the SOPMA server (h alpha-helix e beta-strand t beta-turn c randomcoil)

Figure 5 Characteristic fields of the Bacillus subtilis molecular sequence

(a) (b) (c) (d)

Figure 6 Tertiary structure of the Bacillus subtilis protein (andashd) Structure diagrams marked with different colors (a) Rainbow(N-terminus⟶C-terminus) (b) Hydrophobic

(least hydrophobic⟶most hydrophobic) (c d) Charged red (negative) and blue (positive)


(a) (b)

Figure 7 Active site of the Bacillus subtilis molecule

Imm

obili

zatio

n ra

tio o

f pro

tein

()

100

90

80

70

60

400

350

300

250

200

150

100

50

0

Spec

ific a

ctiv

ity (U

μg)

2 50 350 450 550 650 750Volume ratio of the nanomaterial colloid to the

enzyme solution (mLmL)

Immobilization ratio of proteinSpecific activity

(a)

150

120

90

60

Spec

ific a

ctiv

ity (U

μg)

2 4 6 8 10Immobilization time (min)

Imm

obili

zatio

n ra

tio o

f pro

tein

()

100

90

80

70

50

60


(b)

Imm

obili

zatio

n ra

tio o

f pro

tein

()

100

90

80

70

60

300

250

200

150

100

50

Spec

ific a

ctiv

ity (U

μg)

15 20 25 30 35Immobilization temperature (degC)


(c)

350

300

250

200

150

100

50

Spec

ific a

ctiv

ity (U

μg)

30 35 40 45 50 55 60 65pH

Imm

obili

zatio

n ra

tio o

f pro

tein

()

100

80

60

40

20


(d)

Figure 8 Influence of the volume ratio of the nanomaterial to the enzyme solution (a) immobilization time (b) temperature (c) and pH (d)on immobilization


results are shown in Table 4 -e immobilization ratio ofA3B1C1 is 9478 and the specific activity of A1B2C3 is26895Uμg which is larger than the maximum valueappearing in the orthogonal nine sets of experiments -eresults prove that the orthogonal optimization is reliable Inaddition this experiment pays more attention to the specificactivity of the immobilized enzyme -erefore the bestcombination is A1B2C3 in which the volume ratio of thematerial to the enzyme is 3 50 (mL mL) the immobili-zation temperature is 25degC and the pH is 45 At this timethe protein immobilization ratio is 7348 and theimmobilized enzyme specific activity is 27405UμgCompared with the specific activity of the free enzyme of

50990Uμg the specific activity of the immobilized enzymeis about 5374 that of the free enzyme indicating that theimmobilization of SPS nanospheres would reduce the en-zymatic activity of collagenase

34 Characterization of Sulfonated Polystyrene Nanoparticlesbefore and after Immobilization of Collagen Protease

341 Particle Size Distribution Analysis Determination ofparticle size distribution of SPS nanospheres is shown inFigure 9 -eir average particle size is 1551 nm and themultidispersion index is 0296

Table 2 Orthogonal experimental results and analysis

Experiment number A B C Immobilization ratio of protein () Specific activity (Uμg)1 1 (3 50) 1 (20) 1 (35) 8727plusmn 042 20329plusmn 14552 1 2 (25) 2 (40) 7933plusmn 049 22659plusmn 15143 1 3 (30) 3 (45) 7291plusmn 038 26733plusmn 13024 2 (4 50) 1 2 8608plusmn 087 16955plusmn 14835 2 2 3 8051plusmn 052 18014plusmn 14256 2 3 1 9139plusmn 063 15830plusmn 15547 3 (5 50) 1 3 8288plusmn 054 14351plusmn 19958 3 2 1 9400plusmn 078 15408plusmn 15469 3 3 2 8917plusmn 044 11021plusmn 1944k1-Ir 7984 8541 9089k2-Ir 8599 8461 8486k3-Ir 8868 8449 7877k1-Sa 23240 17212 17189k2-Sa 16933 18694 18341k3-Sa 13593 17861 19699R-Ir 884 092 1212R-Sa 9647 1482 2510Note k-Ir means k value of the immobilization ratio of protein R-Ir means range value of the immobilization ratio of protein k-Sa means k value of specificactivity R-Sa means range value of specific activity

Table 3 Analysis of variance

Indicator Source of variation SS df MS F

Immobilization ratio of protein

Interblock 129717013 1 129717013 12180163A 237015 2 118508 111276lowastlowastB 2688 2 1344 1262 F005(211) 398C 440833 2 220416 206966lowastlowast F001(211) 721

Error 11715 11 1065Total variation 692251 17

Specific activity

Interblock 578160136 1 578160136 812812A 28801349 2 14400674 20245lowastlowastB 662364 2 331182 0466 F005(211) 398C 2871830 2 1435915 2019 F001(211) 721


Note lowastsignificant difference Fge F005(211) lowastlowastextremely significant difference Fge F001(211)

Table 4 Results of confirmatory experiments

Parameter combination Immobilization ratio of protein () Specific activity (Uμg)A3B1C1 9478plusmn 078 16226plusmn 1428A1B2C3 7348plusmn 026 27405plusmn 1552


0 150 300 450 600 750 900

0

2

4

6

8

10

12

Freq

uenc

y (

)

Diameter (nm)

Figure 9 Granularity diagram of SPS nanoparticles

(a)

I

50 μm

10

5

0

cps

eV

0 5 10 15

(b)

Figure 10 (a) SEM diagram of SPS nanoparticles before immobilization (b) EDS spectrogram of SPS nanoparticles before immobilization


342 SEMObservation and Energy Spectrum Analysis A setof scanning electron microscopy and energy spectrum testsabout SPS nanoparticles before and after immobilization areshown in Figures 10 and 11 It can be observed from thefigures that the SPS nanoparticles have an obvious sphericalshape uniform size uniform distribution and good mon-odispersity -e surface of the nanospheres is smooth beforeimmobilization and rough after immobilization [30] whichindicates that collagen hydrolase has been successfullyimmobilized onto the surface of SPS nanospheresAccording to the energy spectrum test results in Table 5 theSPS nanospheres before immobilization contain C O and S

elements of which the content of C is the highest and thecontent of O and S is significantly less -e S element iscaused by the introduction of a sulfonic acid group when theconcentrated sulfuric acid is sulfonating polystyrene in-dicating that the immobilized material is successfullysulfonated polystyrene -e nanoparticles after immobili-zation contain C N O and S elements Compared withthose before immobilization the C element content is de-creased while the N and O contents are significantly in-creased and the S content is less changed of the immobilizednanospheres [31] -is further indicates that collagenase hasbeen successfully immobilized onto SPS nanospheres

(a)

40

cps

eV

20

00 5 10 15

II

5 μm

(b)

Figure 11 (a) SEM diagram of SPS nanoparticles after immobilization (b) EDS spectrogram of SPS nanoparticles after immobilization

Table 5 Energy spectrum elements of sulfonated polystyrene nanospheres before and after immobilization

Element (before immobilization) wt at Element (after immobilization) wt atC 9524 9684 C 7365 7824N 000 000 N 1009 919O 352 268 O 1527 1218S 124 047 S 099 039Total 10000 10000 Total 10000 10000


343 TEM Analysis -e TEM images of the SPS nano-spheres before and after immobilization of collagen hy-drolase are shown in Figures 12 and 13 It can be clearly seenfrom Figure 12 that the SPS nanospheres before immobi-lization have regular spherical shapes with uniform sizesmooth surface no impurities homogeneous distributionand good monodispersity According to Figure 13 thesurface of SPS nanospheres adsorbed some particles andbecame rough which also indicates that the collagen hy-drolase is successfully immobilized onto the surface of theSPS nanospheres

344 FT-IR Spectroscopy Analysis It can be observed fromFigure 14 that the infrared spectrum of the SPS nanospheresbefore and after the collagen hydrolase immobilization hassimilarities and obvious differences -e infrared spectrashow that the characteristic absorption peaks of O-H bondsof aromatic compounds appear at about 3400 cmminus 1 thestretching vibration peaks of C-H bonds appear at2900 cmminus 1 and the vibration absorption peaks of C-O ap-pear at 1 645 cmminus 1 In addition the characteristic peaks ofsulfonic acid groups appear at 1122 and 1029 cmminus 1 [32]

However only the infrared spectrum of the immobilizedenzyme shows a characteristic absorption peak of the aminogroup at 1520 cmminus 1 and the peak at 1647 cmminus 1 of the fixedenzyme is significantly enhanced [33] -erefore the in-frared suggests that collagen hydrolase has been successfully

(a) (b) (c)

Figure 12 Transmission electron microscopy (TEM) before immobilization of collagenase onto SPS nanospheres

Figure 13 Transmission electron microscopy (TEM) after immobilization of collagenase onto SPS nanospheres

4000 3500 3000 2500 2000 1500 1000 5000

20

40

60

80

100

T (

)

Wavenumber (cmndash1)

334578

292445

164571

112203102943

88185

66832

344135

296116

164787

152059

1224

a

b

Figure 14 Infrared spectrum of SPS nanospheres (a) andimmobilized enzymes (b)


immobilized onto SPS nanospheres which is consistent withthe expected results

4 Conclusions

(1) In this paper an alkaline collagen from fermentationof Bacillus subtilis was isolated purified and iden-tified and its structure was simulated -e molecularweight of the purified enzyme protein was 310 kDadetermined by SDS-PAGE gel electrophoresis ByLC-MS-MS sequencing and structure simulationonline the amino acid sequence and one three-di-mensional structure simulation diagram of the pu-rified enzyme protein were obtained -e sequenceconsistency was 6404 which suggested that theprotein might belong to the subtilisin proteasefamily Homologous modeling is the mainmethod toconstruct the three-dimensional structure of theprotein Analysis of the protein database PDB leadsto the conclusion that any pair of proteins if theirsequence consistency is more than 30 has a similarthree-dimensional structure that is the basic foldingof the two proteins is the same and only some of thedetails in the nonspiral and nonfolded lamellar re-gions are different For the sequences consistent with60 parts the 3D models determined by the ho-mologous modeling method are very accurate Iftheir sequence consistency exceeds 60 the con-struction results will be close to the test resultsobtained from the experiment [34] -e purpose ofthree-dimensional structure simulation for the en-zyme protein using the resources in the proteindatabase in this paper is to obtain some importantreference information for the study of the structureand function of the enzymes On the basis of thisstudy combining with the classical biochemicalknowledge we will analyze the enzyme protein fromthe essence predict its function from the structurecompare the function with the similar enzymeprotein and hope to find a substitute enzyme

(2) -e SPS nanospheres before and after immobiliza-tion were analyzed by particle size distribution SEMEDS TEM and Fourier transform infrared spec-troscopy indicating that the average particle size ofthe SPS nanospheres was 1551 nm and collagenhydrolase was successfully immobilized onto SPSnanospheres

(3) -e optimum immobilization conditions obtainedby the orthogonal test are as follows volume ratio ofthe immobilization carrier to the enzyme was 3 50(mL mL) the immobilization temperature was25degC and the pH was 45 At this time the proteinimmobilization ratio was 7348 and the immo-bilized enzyme specific activity was 27405 UμgCompared with the specific activity (50990Uμg)of the free enzyme the specific activity of theimmobilized enzyme was about 5374 that of thefree enzyme indicating that the immobilization of

SPS nanospheres under the optimum conditionswould reduce the enzymatic activity of collagenaseAs a new immobilization carrier SPS nanospherescan immobilize collagen hydrolase effectively with asimple and easy operation process which has goodpractical application prospect and significance forlarge-scale industrial production with mildimmobilized conditions and with low cost Al-though only the conditions of the enzyme immo-bilized by SPS nanospheres have been studied inthis paper we have also systematically studied theapplication conditions and properties of immobi-lized enzymes and the results will be reported in thenear future

Data Availability

-e data used to support the findings of this study areavailable from the corresponding author upon request

Conflicts of Interest

-e authors declare that they have no conflicts of interest

Acknowledgments

-is work was financially supported by the Science andTechnology Planning Project of Guangdong Province(2015B020230001) the Project of Food Science In-novation Team of Guangdong Higher Education Institutes(2016KCXTD020) and the Guangdong University ofPetrochemical Technology 2018 University-Level Culti-vation Project for College Studentsrsquo Innovation and En-trepreneurship (2018pyA039)

References

[1] P Banerjee L Suguna and C Shanthi ldquoWound healingactivity of a collagen-derived cryptic peptiderdquo Amino Acidsvol 47 no 2 pp 317ndash328 2015

[2] E J Lee J Hur S A Ham et al ldquoFish collagen peptideinhibits the adipogenic differentiation of preadipocytes andameliorates obesity in high fat diet-fed micerdquo InternationalJournal of Biological Macromolecules vol 104 pp 281ndash2862017

[3] Y Bu J Elango J Zhang et al ldquoImmunological effects ofcollagen and collagen peptide from blue shark cartilage on 6T-CEM cellsrdquo Process Biochemistry vol 57 pp 219ndash227 2017

[4] P Selvakumar T C Ling A D Covington and A LyddiattldquoEnzymatic hydrolysis of bovine hide and recovery of collagenhydrolysate in aqueous two-phase systemsrdquo Separation andPurification Technology vol 89 pp 282ndash287 2012

[5] L Ma Isolation and Enzyme Characteristics of a StrainProducing Collagenase Dalian Polytechnic University DalianChina 2012

[6] J D Maclennan I Mandl and E L Howes ldquoBacterial di-gestion of collagenrdquo Journal of Clinical Investigation vol 32no 12 pp 1317ndash1322 1953

[7] R A Cai H Li L Y Li and L P Zhang ldquoPhysiologicalfunction property and applications of collagen proteinrdquoMeatIndustry vol 1 pp 7ndash9 2010


[8] W B Sun Marine Microorganisms Source Collagenase En-zyme System Research Dalian Polytechnic University DalianChina 2014

[9] J Betbel and G R Naik ldquoStudies on production of ther-mostable alkaline protease from thermophilic and alkaliphilicBacillus sp JB-99 in a chemically defined mediumrdquo ProcessBiochemistry vol 37 no 2 pp 139ndash144 2001

[10] X Yang X Xiao D Liu et al ldquoOptimization of collagenaseproduction by Pseudoalteromonas sp SJN2 and application ofcollagenases in the preparation of antioxidative hydrolysatesrdquoMarine Drugs vol 15 no 12 p 377 2017

[11] M K Walsh and R Rastal ldquoImmobilized enzyme technologyfor food applicationsrdquo in Novel Enzyme Technology For FoodApplications pp 60ndash84 Taylor amp Francis London UK 2007

[12] J Jiang P Wang and D Hou ldquo-e mechanism of cesiumions immobilization in the nanometer channel of calciumsilicate hydrate a molecular dynamics studyrdquo PhysicalChemistry Chemical Physics vol 19 no 41 pp 27974ndash279862017

[13] K Zhao J Zhao CWu et al ldquoFabrication of silver-decoratedsulfonated polystyrene microspheres for surface-enhancedRaman scattering and antibacterial applicationsrdquo RSC Ad-vances vol 5 no 85 pp 69543ndash69554 2015

[14] J Wang D Wu G Zhao et al ldquoReversible immobilization ofglucoamylase onto magnetic polystyrene beads with multi-functional groupsrdquo Process Biochemistry vol 49 no 5pp 845ndash849 2014

[15] A M Al-Sabagh Y M Moustafa A Hamdy H M Killa andR E Morsi ldquoPreparation and characterization of sulfonatedpolystyrenemagnetite nanocomposites for organic dye ad-sorptionrdquo Egyptian Journal of Petroleum vol 27 no 3pp 403ndash413 2017

[16] L Chen Purification and Characterization of a Collagenasefrom Bacillus Pumilus Sichuan Agricultural University YaanShi China Master degree 2008

[17] Z L Zeng R H Xu and T Xiong ldquoPurification of Alliinaseand determination of its enzymatic propertiesrdquo Food Sciencevol 29 no 12 pp 431ndash434 2008

[18] Y Li Purify the Collagenase from Bacillus Cereus R75E and ItsRecombinant Rxpression in Pichia pastoris Tianjin Universityof Commerce Tianjin China 2016

[19] N Ahmadifard J H C Murueta A Abedian-KenariA Motamedzadegan and H Jamali ldquoComparison the effectof three commercial enzymes for enzymatic hydrolysis of twosubstrates (rice bran protein concentrate and soy-been pro-tein) with SDS-PAGErdquo Journal of Food Science and Tech-nology vol 53 no 2 pp 1279ndash1284 2016

[20] F Lottspeich ldquoProteomics-an unexpected journey into thecomplexity of protein structures and functionsrdquo EuPA OpenProteomics vol 21 pp 1-2 2018

[21] A Fiser ldquoProtein structure modeling in the proteomics erardquoExpert Review of Proteomics vol 1 no 1 pp 97ndash110 2004

[22] L W Ding H Li C X Wang P Li L Zhang and Z B Wenldquo-e preparation and characterization of polystyrene sub-microspheresrdquo Journal of Jiangxi Normal University vol 42no 2 pp 155ndash159 2018

[23] A Matin E Elena T Laptinskaya and S Klimonsky ldquoSelf-assembly of polystyrene microspheres into two-level hierar-chical structuresrdquo Superlattices and Microstructures vol 120pp 806ndash811 2018

[24] Y G Tao S X Zheng Z Y Jiang L B Zhu G C Du andM D Liang ldquoInterfacial characterization between biomimeticadhesion nano TiO2 and lipaserdquo New Chemical Materialsvol 44 no 8 pp 156ndash158 2016

[25] L Zhang C W Shi C H Li W X Wang J Q Mo andY B Shao A Method for the Determination of CollagenPeptide in Tilapia by Biuret Method CN 108827945A 2018httpdbpubcnkinetgrid2008dbpubdetailaspxdbcode

SCPDampdbnameSCPD2018ampfilenameCN108827945Aampuid

WEEvREdxOWJmbC9oM1NjYkZCbDdrdVRVRFFQajIwelgvalNqM25pWS9GeVk$R1yZ0H6jyaa0en3RxVUd8df-oHi7XMMDo7mtKT6mSmEvTuk11l2gFA

[26] H T Gao and X H Ma ldquoEmulsion polymerization ofpolystyrene (PS) polymer microspheres and characteriza-tionrdquo New Chemical Materials vol 41 no 7 pp 91-92 2013

[27] X Y Du and L P Geng ldquoPreparation and characterization ofFe3O4Polystyrene composite particlesrdquo Material Reviewvol 24 no 1 pp 170ndash176 2010

[28] W Han X Q Xu X Y Ye and J Lin ldquoPurification andcharacterization of the alginate lyase isolated from marinerdquoJournal of Fuzhou University vol 46 no 1 2018

[29] J Yang Y J Zou R Y Zhang and Q X Hu ldquoPurification andproperty of laccase from Pleurotus eryngii var tuoliensisrdquoMycosystema vol 34 no 3 pp 456ndash464 2015

[30] Q Qiu J H Cha Y W Choi J H Choi J Shin and Y S LeeldquoPreparation of polyethylene membranes filled with cross-linked sulfonated polystyrene for cation exchange andtransport in membrane capacitive deionization processrdquoDesalination vol 417 pp 17ndash19 2017

[31] M A Abdel-Naby ldquoImmobilization of Paenibacillus mac-erans NRRL B-3186 cyclodextrin glucosyltransferase andproperties of the immobilized enzymerdquo Process Biochemistryvol 34 no 4 pp 103ndash107 1999

[32] J Wang F M Zhu J C Liu H M Li and S G LinldquoCharacterization of sulfonated syndiotactic polystyrenerdquoActa Macromolecule vol 22 no 2 pp 250ndash253 2001

[33] K Zhao Q J Di L Deng and F Wang ldquoSynthesis andcharacterization of the immobilized lipase with adsorptionand cross-linkingrdquoModern Chemical Industry vol 36 no 12pp 63ndash68 2016

[34] H B Wen W R Jing D L Yang L P Liu and Z Q WangldquoScreening and characterization of an thermophilic alkalinecrude protease from an isolated strain of Bacillus subtilis I15rdquoJournal of Zhengzhou University vol 31 no 1 pp 70ndash732010


Hindawiwwwhindawicom

International Journal of

Volume 2018

Zoology

Hindawiwwwhindawicom Volume 2018

Anatomy Research International

PeptidesInternational Journal of



Journal of Parasitology Research

GenomicsInternational Journal of


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The Scientific World Journal

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BioinformaticsAdvances in

Marine BiologyJournal of



Neuroscience Journal


BioMed Research International

Cell BiologyInternational Journal of



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ArchaeaHindawiwwwhindawicom Volume 2018


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Enzyme Research



MicrobiologyHindawiwwwhindawicom

Nucleic AcidsJournal of

Volume 2018

Submit your manuscripts atwwwhindawicom

of minor pore size large specific surface area large surfacebinding energy and easy integration with the enzymesteadily which can effectively improve the enzyme load andthe stability of enzymes [12] In recent years polystyrene hasbeen not only widely used in light industry daily decorationlighting indication fields and so on but also applied as a newcarrier to enzyme immobilization technology [13 14]

When the team worked with a local tilapia collagenpeptide producer to study how to reduce the productioncost we decided to use immobilized enzyme technology tosave the cost of the enzyme We found that sulfonatedpolystyrene (SPS) nanospheres are a good immobilizedmaterial with simple processing low cost good corrosionresistance small pore size large specific surface area andgood biocompatibility [15] but also there are few studies onSPS nanospheres at present and especially their applicationin food industry is very rare -erefore we chose SPSnanoparticles as a carrier and carried out some relatedcharacterization works It is a new attempt which canprovide more basic data for further study of the enzymeimmobilization

2 Materials and Methods

21 Materials and Reagents Collagen hydrolase (alkalineprotease fermented by Bacillus subtilis) and tilapia skincollagen were provided by Guangdong Baiwei Bio Ma-terial Co Ltd Maoming China All reagents were ofanalytical pure grade and purchased from the marketsuch as styrene sodium dodecyl sulfate (SDS) Coo-massie Brilliant Blue G-250 potassium persulfate con-centrated sulfuric acid sodium hydroxide citric acidtrichloroacetic acid (TCA) and potassium sodiumtartrate

22 Isolation and Purification of Collagen HydrolaseAmmonium sulfate precipitation is divided into twosteps the first one is to remove the impure protein (inprecipitation) and the second one is to retain the pre-cipitate to obtain the purified enzyme protein [16] -einitial enzyme solution of 25mL was extracted andprecipitated by ammonium sulfate with a saturation of20 and 40 respectively -e enzyme protein wasprecipitated by centrifugation then dialyzed many timeswith a dialysis bag with a cutoff molecular weight of8000ndash14000 Da and dehydrated by polyethylene glycol20000 [17 18] -e concentrated enzyme protein waspurified by a full-automatic polysaccharide and proteinpurification system (PS-P China) with glucan gel G-759as a column chromatography filler afterwards using10mmolL pH 55 phosphate buffer as equilibrium andelution liquid and 20 ethanol for rinsing When theelution peak began to appear the peak time was recordedand the eluted enzyme was collected (3 mLtube) -eenzyme collected repeatedly was first concentrated withpolyethylene glycol 20000 and then freeze-dried intopowder and stored hermetically at low temperature forfurther use

23 Determination of Molecular Weight and Structure Sim-ulation of the PurifiedEnzymeProtein -emolecular weightof the purified enzyme protein was determined by SDS-PAGE (1645050 US) [19]

-e amino acid sequence of the enzyme protein wasdetermined by LC-MS-MS (Q Exactive US) using the bandsseparated by SDS-PAGE gel electrophoresis and hydrolyzedby trypsin [20 21] Liquid chromatographic column in-formation 300 μm idtimes 5mm packed with Acclaim PepMapRSLC C18 5μm 100 A nanoViper mobile phase A 01formic acid mobile phase B 01 formic acid and 80 CANflow rate 300 nLmin and analysis time of each group60min -e separated peptide entered the Q Exactive massspectrometer directly for online detection Parameters of MS1 resolution 70000 AGC target 3e6 maximum IT 40msand scan range 350 to 1800mz Parameters of MS 2 res-olution 17500 AGC target 1e5 maximum IT 60ms TopN20 and NCEstepped NCE 27 -e three-dimensionalstructure of the enzyme protein was modeled by MM FileConversion online (httpswwwproteinmodelportalorgqueryuniprot) and retrieved by Mascot Finally the blastcomparison was used on the NCBI database to analyze thehomology of the protein-e secondary structure and tertiarystructure of the enzyme protein were analyzed by Phyre2NCBI SWISS-MODEL and PyMOL

24 Preparation of Sulfonated Polystyrene -e styrene (St)was first soaked in NaOH aqueous solution (10wt) andafter 24 hours it was washed several times with distilledwater -e treated St was rotary evaporated under reducedpressure at 60degC SDS (02 g) and Na2CO3 (01 g) wereweighed and dissolved in 300mL distilled water -eresulting solution was transferred to the three-neck flask andpurged with nitrogen for 30min Subsequently 30mL oftreated St was added to the above solution under strongstirring and the solution was heated to 60degC After 30minpotassium persulfate aqueous solution (15mL 023mmolL)was introduced into it [22] -e polymerization reaction wasconducted at 75degC for 20 h After cooling to room tem-perature the product was alternately washed with 95ethanol and distilled water Afterwards the product waspurified by reduced-pressure rotary evaporation at 60degC anddried at 60degC for further use [23]

-e above polystyrene (PS) powder (10 g) was dispersedinto concentrated H2SO4 (40mL 98) by ultrasonicationAn ultrasonic cleaning tank was used for ultrasonication for20min at room temperature -e sulfonation reaction wasconducted at room temperature for 24 h -e product wasthoroughly washed with distilled water and ethanol anddried at 60degC using a vacuum oven After drying the productwas dispersed into distilled water by ultrasonication and thesulfonated polystyrene emulsion (40mgmL) was made

25 Study on Immobilization of Enzymes

251 Immobilization Method SPS nanosphere emulsionwas treated under ultrasonication for 30min and a certainamount of it was diluted 100 times with phosphate buffer









(2) Orthogonal Test



Y m0 minus m1( 1113857

m01113890 1113891 times 100 (1)



M A




Q M times 103

Ttimes

1V2

(3)



S Q

m0 minus m1( 1113857 (4)
















LevelA B C


1 3 50 20 352 4 50 25 403 5 50 30 45











S2MarkerkD

350

250

184

144















(a) (b) (c) (d)




(a) (b)


Imm

obili

zatio

n ra

tio o

f pro

tein

()

100

90

80

70

60

400

350

300

250

200

150

100

50

0

Spec

ific a

ctiv

ity (U

μg)




(a)

150

120

90

60

Spec

ific a

ctiv

ity (U

μg)


Imm

obili

zatio

n ra

tio o

f pro

tein

()

100

90

80

70

50

60


(b)

Imm

obili

zatio

n ra

tio o

f pro

tein

()

100

90

80

70

60

300

250

200

150

100

50

Spec

ific a

ctiv

ity (U

μg)



(c)

350

300

250

200

150

100

50

Spec

ific a

ctiv

ity (U

μg)

30 35 40 45 50 55 60 65pH

Imm

obili

zatio

n ra

tio o

f pro

tein

()

100

80

60

40

20


(d)














Specific activity







0 150 300 450 600 750 900

0

2

4

6

8

10

12

Freq

uenc

y (

)

Diameter (nm)


(a)

I

50 μm

10

5

0

cps

eV

0 5 10 15

(b)





(a)

40

cps

eV

20

00 5 10 15

II

5 μm

(b)








(a) (b) (c)



4000 3500 3000 2500 2000 1500 1000 5000

20

40

60

80

100

T (

)


334578

292445

164571

112203102943

88185

66832

344135

296116

164787

152059

1224

a

b




4 Conclusions





Data Availability




Acknowledgments


References









































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018









(2) Orthogonal Test



Y m0 minus m1( 1113857

m01113890 1113891 times 100 (1)



M A




Q M times 103

Ttimes

1V2

(3)



S Q

m0 minus m1( 1113857 (4)
















LevelA B C


1 3 50 20 352 4 50 25 403 5 50 30 45











S2MarkerkD

350

250

184

144















(a) (b) (c) (d)




(a) (b)


Imm

obili

zatio

n ra

tio o

f pro

tein

()

100

90

80

70

60

400

350

300

250

200

150

100

50

0

Spec

ific a

ctiv

ity (U

μg)




(a)

150

120

90

60

Spec

ific a

ctiv

ity (U

μg)


Imm

obili

zatio

n ra

tio o

f pro

tein

()

100

90

80

70

50

60


(b)

Imm

obili

zatio

n ra

tio o

f pro

tein

()

100

90

80

70

60

300

250

200

150

100

50

Spec

ific a

ctiv

ity (U

μg)



(c)

350

300

250

200

150

100

50

Spec

ific a

ctiv

ity (U

μg)

30 35 40 45 50 55 60 65pH

Imm

obili

zatio

n ra

tio o

f pro

tein

()

100

80

60

40

20


(d)














Specific activity







0 150 300 450 600 750 900

0

2

4

6

8

10

12

Freq

uenc

y (

)

Diameter (nm)


(a)

I

50 μm

10

5

0

cps

eV

0 5 10 15

(b)





(a)

40

cps

eV

20

00 5 10 15

II

5 μm

(b)








(a) (b) (c)



4000 3500 3000 2500 2000 1500 1000 5000

20

40

60

80

100

T (

)


334578

292445

164571

112203102943

88185

66832

344135

296116

164787

152059

1224

a

b




4 Conclusions





Data Availability




Acknowledgments


References









































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018















LevelA B C


1 3 50 20 352 4 50 25 403 5 50 30 45











S2MarkerkD

350

250

184

144















(a) (b) (c) (d)




(a) (b)


Imm

obili

zatio

n ra

tio o

f pro

tein

()

100

90

80

70

60

400

350

300

250

200

150

100

50

0

Spec

ific a

ctiv

ity (U

μg)




(a)

150

120

90

60

Spec

ific a

ctiv

ity (U

μg)


Imm

obili

zatio

n ra

tio o

f pro

tein

()

100

90

80

70

50

60


(b)

Imm

obili

zatio

n ra

tio o

f pro

tein

()

100

90

80

70

60

300

250

200

150

100

50

Spec

ific a

ctiv

ity (U

μg)



(c)

350

300

250

200

150

100

50

Spec

ific a

ctiv

ity (U

μg)

30 35 40 45 50 55 60 65pH

Imm

obili

zatio

n ra

tio o

f pro

tein

()

100

80

60

40

20


(d)














Specific activity







0 150 300 450 600 750 900

0

2

4

6

8

10

12

Freq

uenc

y (

)

Diameter (nm)


(a)

I

50 μm

10

5

0

cps

eV

0 5 10 15

(b)





(a)

40

cps

eV

20

00 5 10 15

II

5 μm

(b)








(a) (b) (c)



4000 3500 3000 2500 2000 1500 1000 5000

20

40

60

80

100

T (

)


334578

292445

164571

112203102943

88185

66832

344135

296116

164787

152059

1224

a

b




4 Conclusions





Data Availability




Acknowledgments


References









































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018











S2MarkerkD

350

250

184

144















(a) (b) (c) (d)




(a) (b)


Imm

obili

zatio

n ra

tio o

f pro

tein

()

100

90

80

70

60

400

350

300

250

200

150

100

50

0

Spec

ific a

ctiv

ity (U

μg)




(a)

150

120

90

60

Spec

ific a

ctiv

ity (U

μg)


Imm

obili

zatio

n ra

tio o

f pro

tein

()

100

90

80

70

50

60


(b)

Imm

obili

zatio

n ra

tio o

f pro

tein

()

100

90

80

70

60

300

250

200

150

100

50

Spec

ific a

ctiv

ity (U

μg)



(c)

350

300

250

200

150

100

50

Spec

ific a

ctiv

ity (U

μg)

30 35 40 45 50 55 60 65pH

Imm

obili

zatio

n ra

tio o

f pro

tein

()

100

80

60

40

20


(d)














Specific activity







0 150 300 450 600 750 900

0

2

4

6

8

10

12

Freq

uenc

y (

)

Diameter (nm)


(a)

I

50 μm

10

5

0

cps

eV

0 5 10 15

(b)





(a)

40

cps

eV

20

00 5 10 15

II

5 μm

(b)








(a) (b) (c)



4000 3500 3000 2500 2000 1500 1000 5000

20

40

60

80

100

T (

)


334578

292445

164571

112203102943

88185

66832

344135

296116

164787

152059

1224

a

b




4 Conclusions





Data Availability




Acknowledgments


References









































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018













(a) (b) (c) (d)




(a) (b)


Imm

obili

zatio

n ra

tio o

f pro

tein

()

100

90

80

70

60

400

350

300

250

200

150

100

50

0

Spec

ific a

ctiv

ity (U

μg)




(a)

150

120

90

60

Spec

ific a

ctiv

ity (U

μg)


Imm

obili

zatio

n ra

tio o

f pro

tein

()

100

90

80

70

50

60


(b)

Imm

obili

zatio

n ra

tio o

f pro

tein

()

100

90

80

70

60

300

250

200

150

100

50

Spec

ific a

ctiv

ity (U

μg)



(c)

350

300

250

200

150

100

50

Spec

ific a

ctiv

ity (U

μg)

30 35 40 45 50 55 60 65pH

Imm

obili

zatio

n ra

tio o

f pro

tein

()

100

80

60

40

20


(d)














Specific activity







0 150 300 450 600 750 900

0

2

4

6

8

10

12

Freq

uenc

y (

)

Diameter (nm)


(a)

I

50 μm

10

5

0

cps

eV

0 5 10 15

(b)





(a)

40

cps

eV

20

00 5 10 15

II

5 μm

(b)








(a) (b) (c)



4000 3500 3000 2500 2000 1500 1000 5000

20

40

60

80

100

T (

)


334578

292445

164571

112203102943

88185

66832

344135

296116

164787

152059

1224

a

b




4 Conclusions





Data Availability




Acknowledgments


References









































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018


(a) (b)


Imm

obili

zatio

n ra

tio o

f pro

tein

()

100

90

80

70

60

400

350

300

250

200

150

100

50

0

Spec

ific a

ctiv

ity (U

μg)




(a)

150

120

90

60

Spec

ific a

ctiv

ity (U

μg)


Imm

obili

zatio

n ra

tio o

f pro

tein

()

100

90

80

70

50

60


(b)

Imm

obili

zatio

n ra

tio o

f pro

tein

()

100

90

80

70

60

300

250

200

150

100

50

Spec

ific a

ctiv

ity (U

μg)



(c)

350

300

250

200

150

100

50

Spec

ific a

ctiv

ity (U

μg)

30 35 40 45 50 55 60 65pH

Imm

obili

zatio

n ra

tio o

f pro

tein

()

100

80

60

40

20


(d)














Specific activity







0 150 300 450 600 750 900

0

2

4

6

8

10

12

Freq

uenc

y (

)

Diameter (nm)


(a)

I

50 μm

10

5

0

cps

eV

0 5 10 15

(b)





(a)

40

cps

eV

20

00 5 10 15

II

5 μm

(b)








(a) (b) (c)



4000 3500 3000 2500 2000 1500 1000 5000

20

40

60

80

100

T (

)


334578

292445

164571

112203102943

88185

66832

344135

296116

164787

152059

1224

a

b




4 Conclusions





Data Availability




Acknowledgments


References









































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018













Specific activity







0 150 300 450 600 750 900

0

2

4

6

8

10

12

Freq

uenc

y (

)

Diameter (nm)


(a)

I

50 μm

10

5

0

cps

eV

0 5 10 15

(b)





(a)

40

cps

eV

20

00 5 10 15

II

5 μm

(b)








(a) (b) (c)



4000 3500 3000 2500 2000 1500 1000 5000

20

40

60

80

100

T (

)


334578

292445

164571

112203102943

88185

66832

344135

296116

164787

152059

1224

a

b




4 Conclusions





Data Availability




Acknowledgments


References









































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018


0 150 300 450 600 750 900

0

2

4

6

8

10

12

Freq

uenc

y (

)

Diameter (nm)


(a)

I

50 μm

10

5

0

cps

eV

0 5 10 15

(b)





(a)

40

cps

eV

20

00 5 10 15

II

5 μm

(b)








(a) (b) (c)



4000 3500 3000 2500 2000 1500 1000 5000

20

40

60

80

100

T (

)


334578

292445

164571

112203102943

88185

66832

344135

296116

164787

152059

1224

a

b




4 Conclusions





Data Availability




Acknowledgments


References









































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018




(a)

40

cps

eV

20

00 5 10 15

II

5 μm

(b)








(a) (b) (c)



4000 3500 3000 2500 2000 1500 1000 5000

20

40

60

80

100

T (

)


334578

292445

164571

112203102943

88185

66832

344135

296116

164787

152059

1224

a

b




4 Conclusions





Data Availability




Acknowledgments


References









































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018





(a) (b) (c)



4000 3500 3000 2500 2000 1500 1000 5000

20

40

60

80

100

T (

)


334578

292445

164571

112203102943

88185

66832

344135

296116

164787

152059

1224

a

b




4 Conclusions





Data Availability




Acknowledgments


References









































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



4 Conclusions





Data Availability




Acknowledgments


References









































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018


































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018


immobilization of alkaline collagenase from bacillus subtilis onto...

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