purification of angiotensin-i-converting enzyme...

Research ArticlePurification of Angiotensin-I-Converting Enzyme InhibitoryPeptides Derived from Camellia oleifera Abel SeedMeal Hydrolysate

Guang-long Yao12 Wei He2 You-gen Wu 1 Jian Chen 2 Xin-wen Hu1 and Jing Yu1

1Institute of Tropical Agriculture and Forestry Hainan University Haikou 570228 China2College of Food Science and Technology Hainan University Haikou 570228 China

Correspondence should be addressed to You-gen Wu wygeng2003163com and Jian Chen chenjian19850702163com

Received 31 July 2018 Revised 24 December 2018 Accepted 14 January 2019 Published 11 March 2019

Academic Editor Teresa Zotta

Copyright copy 2019 Guang-long Yao et al is 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 isproperly cited

China is a large country that produces Camellia oleiferaAbel seed meal (COASM) a by-product of tea-seed oil which is only usedas an organic fertilizer resulting in a serious waste of high-quality resources e preparation of the ACE inhibitory peptide fromCOASM and the study of its functional properties are of practical importance in improving the comprehensive utilization ofCOASM Our manuscript presents an optimized preparation of ACE inhibitory peptides with alkaline protease and enzymekinetics parameters Ultraltration gel chromatography and RP-HPLC purication were conducted for ACE inhibitory peptidesand peptide molecular weight distribution and amino acid composition were analyzed in the enzymolysis liquid e followingwere the conditions of the optimized enzymatic hydrolysis to obtain ACE inhibitory peptides fromCOASM15 times of hydrolysisin distilled water for 35 h at 50degC pH 85 substrate concentration of 17mgg and addition of 6 (ww) alkaline protease Underthis condition the peptides produced exhibited an ACE inhibition rate of 7924 and the reaction kinetics parameters are asfollows Km 0152mgmL and Vmax 0130mgmLmiddotmin e majority of ACE inhibitory peptides from COASM have molecularweight below 1 kDa and a high ACE inhibitory rate was achieved after dextran gel chromatography separation and purication(whose IC50 was 0678mgmL) e hydrophobic amino acid content in this fraction reached 5121

1 Introduction

Hypertension has become a global killer Patients with highblood pressure also gradually appear younger [1 2] us asafe and ebrvbarective treatment for high blood pressure must bedeveloped Angiotensin-I-converting enzyme (ACE) is adipeptide carboxyl protease that plays an important role inblood pressure regulation Ebrvbarectively inhibiting the ACErate can help reduce blood pressure [3 4] With the de-velopment in biological active peptide research lowering theblood pressure and obtaining functional peptides fromnatural resources have become a hot spot in the study ofmedicine [4ndash6]

e methods of preparing ACE inhibitory peptidesmainly include enzyme hydrolysis and microbial fermenta-tion Enzymatic hydrolysis refers to the hydrolysis by

proteases to obtain all kinds of bioactive peptides some ofwhich can inhibit ACE [2 7 8]ismethod is simple and canbe used to easily control digestion conditions e obtainedACE inhibitory peptides still require ultraltration macro-porous resin adsorption gel chromatography RP-HPLC andpreparative chromatography purication [9 10] Otte was therst to study the ebrvbarects of using ve kinds of protease hy-drolysis and nine kinds of milk protein to produce ACEinhibitory peptides Gel chromatography and RP-HPLC wereapplied to separate and purify the ACE inhibitory peptidesfrom the lactalbumin protease hydrolysate Five high rates ofACE inhibitory peptides with IC50 values of 1ndash5microM wereachieved [11] Minervini used Lactobacillus helveticus PR4 forthe protease enzymolysis of casein from six species of animal(eg cattle sheep goats pigs water bubrvbaralo and people) andthus produced a variety of ACE inhibitory peptides [12]

HindawiJournal of Food QualityVolume 2019 Article ID 7364213 9 pageshttpsdoiorg10115520197364213

C oleifera which belongs to the 1eaceae family isregarded as one of the four largest woody oil species in theworld and has high economic values and ecological benefits[13 14] COASM is a by-product of oil extraction and itsutilization ratio is relatively low For a long time COASMhas been used as a fertilizer and fuel resulting in a seriouswaste of resources [15] C oleifera contains 10ndash20protein and some small peptides derived from food proteinsare easily digested and absorbed by the human bodyCOASM can provide nutrients for human growth and de-velopment and exhibit strong antioxidant and lipid-loweringproperties and other health care functions 1us it is apromising functional factor worldwide [16 17]

In this study COASM was used as the raw materialand the response surface method was used to determinethe best enzymolysis conditions for alkaline protease 1eenzymolysis kinetic parameters were obtained accordingto the MichaelisndashMenten equation Ultrafiltration gelchromatography and RP-HPLC were conducted to pro-duce a component with high ACE inhibitory rate 1eamino acid composition and in vitro digestion of thiscomponent were subsequently analyzed Our experimentcan provide the basis for further research to increase thevalue of C oleifera

2 Materials and Methods

21Materials andReagents COASM samples were providedby Lv YuanC oleifera processing Co (Qionghai city HainanChina) Angiotensin-I-converting enzyme (ACE) N-[3-(2-furylacryloyl)]-L-phenyalanyl-glycyl-glycine (FAPGG) andSephadex G-25 glucan gel were purchased from Sigma-Aldrich Co (USA) Trypsin (250Umg) neutral protease(50Umg) alkaline protease (2000Umg) and papain(6000Umg) were purchased from Novozymes BiologicalTechnology Co (Beijing China) Tris-HCl buffer NaClNaOH formic acid ethanol and phenol reagents were ofanalytical grade and purchased from Beijing Chemical Re-agent Co (Beijing China) Deionized water was usedthroughout the experiment

22 Preparation of ACE Inhibitory Peptides from COASMCOASM samples underwent lyophilization crushingscreening with 40-mesh size using petroleum ether assolvent with a SL ratio of 1 8 (gml) and 65degC extractionfor 4 h to remove fat In brief 15 g of defatted COASMpowder was accurately weighted in a beaker 1e optimalpH and temperatures for four kinds of protease were asfollows trypsin (pH 80 37degC) neutral protease (pH 7050degC) alkaline protease (pH 95 60degC) and papain (pH57 60degC) 1ese proteases were set to digest for 4 hEnzymatic hydrolysis conditions are as follows a SL ratioof 1 15 (gml) and enzyme amount for 50 (ww) 1eenzymes were inactivated via a boiling water bath for10min After centrifugation for 10min at 4degC and10000 rpm the supernatant fluid was subjected to evap-oration and freeze drying Finally the ACE inhibitorypeptides were obtained [18 19]

23 Determination of the Degree of Hydrolysis (DH) fromCOASM Enzymolysis DH was determined according to thepH-stat method which is based on the protein hydrolysisprocess related to the pH value of the solution 1e pHsystem was maintained by adding acid or alkaline whosequantity must be based on the calculated DH from theenzymolysis of COASM using the following equation[18 20 21]

DH() Vb times Nb

Zp times 732231113888 1113889 times 100 (1)

where Vb is the NaOH volume (mL) Nb is the sodiumhydroxide concentration (01molL) Zp is the proteinweight (g) and 723 is the number of peptide bonds in per gproteins (mmol) and that of COASM was 723

24 Determination of ACE Inhibitory Rate ACE inhibitionrate of COASM hydrolysate was evaluated by the methodestablished in our laboratory [22] In brief 10 microL of ACEaqueous solution and 10 microL of ACE inhibitory peptides(concentration of 1mgmL) were added to an enzymemicroplate 1e substrate (10mmolL FAPGG dissolved in50mmolL Tris-HCl containing 03molLmiddotNaCl pH 75) waspreheated for 37degC for 5min added to the microplate andallowed to react for 2min 1e absorbance at 340 nm wasrecorded and labeled as A1 After a reaction time of 30minthe absorbance at 340 nm was determined and labeled as A21e control group comprised 10 microL of substrates instead ofACE inhibitory peptides 1e initial absorbance of the blanksolution was recorded as A01 and that after the reaction wasrecorded as A02 Changes in the absorbance value (ΔAInhibitorA1minusA2 ΔBlankA01minusA02) were calculated toobtain the ACE inhibitory rate

ACE inhibitory rate 1minusΔA inhibitorΔblank

1113888 1113889 times 100 (2)

25 Response Surface Design Experiment under EnzymaticHydrolysis Conditions According to the results of thesingle-factor experiment the experimental design was op-timized using response surface methodology [23] 1eBoxndashBehnken center combination experimental principlewas also applied [24 25] In this principle we selected fivefactors namely substrate concentration pH value reactiontemperature enzyme quantity and reaction time whichaffect the ACE inhibitory rate 1ree levels per factor wereused to optimize the test and the ACE inhibitory rate wasused as the response variable

26 Determination of the Maximum Reaction Rate of MIConstant and Enzyme-Promoting Reaction 1e substrateconcentration of COASM protein solution with 002 004006 008 and 01mgmL was prepared respectively al-kaline protease was added and hydrolysated for pH 95 at60degC 005molLmiddotNaOH solution was used to maintain aconstant pH value in COASM protein solution 1e average

2 Journal of Food Quality

velocity of the generated peptides speed was calculated in-stead of the initial velocity MI constant and maximumreaction rate were obtained according to the followingformula [1 10]

1Vo

1113888 1113889 Km

Vmax[S]+

1Vmax

1113888 1113889 (3)

where Vo is the average velocity of the generated peptidesspeed [S] is the substrate concentration Km is the MIconstant and Vmax is the maximum reaction rate

27 Separation and Identification of ACE Inhibitory Peptides

271 Ultrafiltration of ACE Inhibitory Peptides 1eCOASM protein hydrolytic liquid was filtered using three(10 5 and 1 kDa) ultrafiltration membranes and separatedby a Vivaflow ultrafiltration system [22 26] 1e fractionswere collected and freeze-dried and their IC50 values weremeasured 1e fraction with a highest ACE inhibition rateand minimum mean IC50 value was selected for subsequentpurification

272 Gel Chromatography of ACE Inhibitory Peptides1e desalination Sephadex G-15 resin was loaded in a16 cmtimes 60 cm glass chromatographic column Samples of10 25 and 50mgmL solution were prepared with ultra-pure water and 3mL was eluted each time at a flow rate of1mLmin ultrapure water Samples were collected by using atube every 10min and 50 samples were finally obtained1eabsorbance of these 50 samples was measured at 280 nm1eobtained fractions were collected and then freeze-dried andpreserved at 4degC [27 28]

273 Preparation of RP-HPLC Method to Purify ACE In-hibitory Peptides After isolating Sephadex G-25 thecomponent with highest ACE inhibitory rate was dissolvedin ultrapure water After 022 microm membrane filtering RP-HPLC was conducted for further purification with thefollowing conditions chromatographic column C18 col-umn (standard 46times100mm 5 μm) detection wavelengthof 215 nm and mobile phases A (acetonitrile) and B (ul-trapure water containing 010 trifluoroacetic acid) elu-tion conditions were 0 A for 5min and 0ndash40 A for5ndash30min sample quantity of 10 μl and column temper-ature at 35degC [21 29]

274 Amino Acid Analysis of ACE Inhibitory PeptidesType and content of amino acids in ACE inhibitory peptidesfrom COASM was determined by the method established inour laboratory [22] 1mL of the filtrate was taken into a 5mLvolumetric flask and dried in a vacuum drier 1e residuewas dissolved in deionized water then dried repeated fortwo times and dissolved in 1mL of pH 22 in the Tris-HClbuffer Accurately absorbing 02mL mixed standard aminoacids diluting to 5mL with pH22 Tris-HCl buffer to theconcentration of 10 nmol10 μl which as the standardamino acid for determination the amino acid automatic

analyzer with the external standard method to determine thetype and content of amino acids in ACE inhibitory peptidesfrom COASM

28 Statistical Analysis Final data were expressed as themeanplusmn standard deviation (x plusmn s) ANOVA was employedto assess the differences among groups Statistical analysiswas performed using SPSS 210 Significant differences weredenoted by Plt 005

3 Results and Discussion

31 Screening of Protease for COASM Figure 1(b) showsthat during the different protein hydrolyses of COASMthe ACE inhibition rates all showed a trend of an initialincrease and then decrease with the increasing hydrolysistime 1e ACE inhibitory rate in alkaline protease hy-drolysate was the highest with a maximum of 615 at 25 hhydrolysis time and the DH was also the largest but after35 h of hydrolysis By comparing the DH and corre-sponding hydrolysate of ACE inhibition rate the resultsshowed that the DH was not positively correlated with ACEinhibition rate 1e DH was the third for papain proteasehydrolysate (Figure 1(a)) but this hydrolysate exhibited thelowest ACE inhibition rate (Figure 1(b))1is phenomenonoccurred because the ACE inhibition rate was associatedwith enzyme loci 1e more the ACE inhibitory peptidesare cut down from COASM proteins the higher their ACEinhibition rate Hydrolysates with a high DH does notnecessarily exhibit a high ACE inhibitory activity [2 30]Alkaline protease is a kind of serine enzyme that tends toform the peptide with hydrophobic amino acids at the end[9 31] Furthermore alkaline protease can cut -CO-NHinternal peptide which makes protein converted intopeptides with a smaller molecular weight [7 29] Vasquez-Villanueva et al used alkaline protease to enzymolyze cornprotein and found that the alkaline protease hydrolysatehas a high ACE inhibitory rate [30] Minervini et al se-lected alkaline protease to hydrolyse whey protein andproduce ACE inhibitory peptides 1is selection is due tothe high amount of alkaline protease hydrolysis locuswhich has particularity for selecting hydrophobic aminoacids on the protein carboxyl terminal [12] 1ereforebased on the above data reported and our results shown inFigure 1 our study selected alkaline protease hydrolysatefor further purification

32 Various Factors on Preparation of ACE InhibitoryPeptides by Alkaline Protease Hydrolysis COASM Proteins1e basis of parameters for alkaline protease hydrolysissolution was set as follows COASM protein concentrationwas 9 the ratio of the enzyme and substrate (E S) was 5and pH was 950 and hydrolysed at 55degC for 3 h A single-factor experiment was used to study the temperature (40degC45degC 50degC 55degC and 60degC) pH (85 90 95 10 and 105)adding amounts of ES (4 5 6 7 and 8) COASMprotein concentrations (3 5 7 9 and 11) andhydrolysis time (15 20 25 30 35 and 40 h) which verify

Journal of Food Quality 3

the effect of these factors on the ACE inhibitory peptides ofalkaline protease for COASM protein 1e results are shownin Figure 2

Figures 2(a) and 2(b) show that with the DH and ACEinhibitory rate of COASM hydrolysates initially increasedand then decreased with the increase in the hydrolysistemperature With the increase in the hydrolysis tempera-ture and pH the alkaline protease activity was also graduallyenhanced hence the DH and ACE inhibitory rate increaseduntil reaching the maximum1e continuous increase in thetemperature and pH led to the change in the enzymestructure influenced the combination of the substrategradually inactivated the enzyme and reduced the DH andACE inhibition rate

According to Figures 2(c) and 2(d) with the increasinghydrolysis time and alkaline protease amount the DH ofCOASM hydrolysates gradually increased whereas the ACEinhibition rate initially increased and then decreased 1eseresults confirmed that the DH and ACE inhibition rate werenot necessarily related Figure 2(e) shows that with theincreasing substrate concentration the DH and ACE in-hibitory rate of COASM hydrolysates were all rapidly in-creased at the substrate concentration before 9 and thenwere remained in a stable state

33 Optimization of ACE Inhibitory Peptide Preparationfor Alkaline Protease Enzymolysis of COASM ProteinAccording to the screening test results of the main factorsthe response surface method optimized the preparation ofACE inhibitory peptides for the enzymolysis of alkalineprotease from COASM protein 1e BoxndashBehnken designand results are shown in Table 1 the response value was ACEinhibition rate

Table 1 shows the multiple regression analysis data fromSAS software 1e quadratic polynomial fitting equation was

DH 1248 + 017A + 028Bminus 0026C + 0062AB

minus 00025ACminus 00005BCminus 055A2 minus 053B2 minus 064C2

(4)

where A B and C are the hydrolysis temperature pH andtime respectively 1e quadratic polynomial regressionequation prediction model was significant (plt 005)whereas the lost item was not significant (p 0073gt 005)All these results showed the reliability of the model De-termination coefficient R2 9148 showed that the ex-perimental data were well-fitted on the equation 1us thismodel could be used to simulate the optimization of theenzymatic hydrolysis of COASM powder

34 Reaction Kinetics in the Preparation of ACE InhibitoryPeptides from COASM Protein Using Alkaline ProteaseCOASM protein was added into different concentrationsolutions for the hydrolysis using alkaline protease under theoptimum conditions 1e reciprocal of the substrate con-centration is the abscissa whereas that of the generatedpeptides speed is the ordinate1e double bottom diagram isshown in Figure 3 Based on the LineweaverndashBurk methodKm and Vmax were 0152mgmL and 0130mgmLmiddotminrespectively as calculated using the formulation in Figure 3

35 Analysis of ACE Inhibitory Rate from COASM Peptideafter Ultrafiltration Membrane Filtration COASM peptideswere prepared using the optimized process and wereultrafiltrated by molecular weights of 10 5 and 1 kDa ul-trafiltration membrane 1e fractions were obtained andlyophilized to determine the ACE inhibitory rate and IC50value concentration 1e results are shown in Table 2

Table 2 shows that with the gradual decrease in themolecular weight of the ultrafiltration membrane the ACE

3456789

1011121314151617

15 20 25 30 35 40

Deg

ree o

f hyd

roly

sis (

)

Hydrolysis time (h)

Neutral proteaseAlkaline protease

10 45

TrypsinPapain

(a)

30

35

40

45

50

55

60

65

ACE

inhi

bitio

n ra

te (

)

Hydrolysis time (h)15 20 25 30 35 4010 45


TrypsinPapain

(b)

Figure 1 Effect of enzymes and hydrolysis time on properties of protein from COASM (a) DH (b) ACE inhibition rate


9

10

11

12

13

14

15

16

10152025303540455055606570

40 45 50 55 60

Deg

ree o

f hyd

roly

sis (

)

ACE

inhi

bitio

n ra

te (

)

Hydrolysis temperature (degC)

ACE inhibition rate ()Degree of hydrolysis ()

(a)

Deg

ree o

f hyd

roly

sis (

)

ACE

inhi

bitio

n ra

te (

)

4

5

6

7

8

9

10

11

20

25

30

35

40

45

50

55

60

65

70

8580 90 95 100 110105pH


(b)

Deg

ree o

f hyd

roly

sis (

)

ACE

inhi

bitio

n ra

te (

)

6

8

10

12

14

16

10

20

30

40

50

60

70

1510 20 25 30 35 4540Hydrolysis time (h)


(c)

Deg

ree o

f hyd

roly

sis (

)

ACE

inhi

bitio

n ra

te (

)

9

10

11

12

13

14

15

16

10

20

30

40

50

60

70

4 5 6 7 8Protease amount ()


(d)

4

6

8

10

12

14

16

10

20

30

40

50

60

70

3 5 7642 98 1110 12Substrate concentration ()

Deg

ree o

f hyd

roly

sis (

)

ACE

inhi

bitio

n ra

te (

)


(e)

Figure 2 Influence of the selected factors on the DH and ACE inhibitory rate hydrolysate by alkaline protease (a) hydrolysis temperature(b) pH (c) hydrolysis time (d) protease amount (e) substrate concentration


inhibition rate of samples also gradually increased and theIC50 value concentrations gradually reduced 1ese resultssuggest that ultrafiltration allowed to obtain a fraction withmolecular weight peptides lower than 1 kDa exhibitinghigher ACE inhibitory activity Our results are similar tothose of Korhonen et al who used ultrafiltration to separateACE inhibitory peptides from whey protein hydrolysate1ey found that the IC50 value is the minimum for thepeptide in 1 kDa membrane ultrafiltration [20] Our labo-ratory also demonstrates that the cashew nut peptidesexhibiting ACE inhibitory activity had molecular weightlower than 3000Da [22] 1e above results also verified the

conclusion that ACE inhibitory peptides are mainly com-posed of peptides below 1 kDa

36 COASM Peptide Purified by Gel ChromatographyFigure 4 shows the peptide profile of hydrolysate solutionswith three different concentrations 1e separation effect foreach peak was ineffective for 10mgmL concentrationGiven the low and diluted concentration of the sample theseparation effect between the components was not idealWhen the sample concentration was 25mgmL the sepa-ration of the three peaks became apparent and it was moreeffective

When the sample concentration was increased to 50mgmL the chromatographic column separation effect wassignificantly reduced Samples were continuously dilutedduring gel chromatography separationWhen the separationefficiency is affected the sample concentration should beincreased as soon as possible However the viscosity ofsamples was also increased when the sample concentrationwas extremely high which would affect the flow of com-ponents in the gel and finally reduce the resolution of the gelchromatography 1erefore 25mgmL was selected as thebest sample concentration

Peaks I II and III from the 25mgmL COASM peptideafter Sephadex G-15 separation were collected and freeze-dried and then their IC50 value concentrations were de-termined 1e results were in the following order I(0145mgmL)lt II (0202mgmL)lt III (0213mgmL) 1eIC50 value concentration of peak I was lower than those ofthe other two components which might be because peak Icontains free amino acids Je et al used gel chromatographyto purify ACE inhibitory peptides from Alaska pollock(=eragra chalcogramma) frame protein hydrolysate theIC50 value reached 0066mgmL [18] 1erefore peak I wascollected as the subsequent test samples after freeze drying

37 COASMPeptide Purified by RP-HPLC 1e component Iof the COASM peptide was solubilized in ultrapure water and

Table 1 BoxndashBehnken design and results for ACE inhibitory peptides by alkaline protease

Number Hydrolysis temperature (degC) pH Hydrolysis time (h) ACE inhibitory peptides1 45 95 30 57072 45 10 30 59473 55 95 30 54144 55 100 30 60645 50 95 25 56556 50 90 35 61217 50 100 25 60058 50 100 35 58589 50 90 25 590110 55 100 30 578811 55 95 35 605312 50 90 35 580313 50 95 30 616514 55 95 25 564515 50 100 35 586216 50 95 30 595817 50 95 30 6098

10

80

70

60

50

40

30

20

10 504030200

1V

(mLmiddot

min

mg)

1S (mLmg)

y = 09943x + 72342

R2 = 09352

Figure 3 Equation curve of 1V to 1S for LineweaverndashBurk(alkaline protease)

Table 2 ACE inhibition rate and IC50 of the ultrafiltration frac-tions in the sample

Molecular weight ACE inhibition rate () IC50 (mgmL)Mlt 10 kDa 7378plusmn 114 0874Mlt 5 kDa 7727plusmn 089 0728Mlt 1 kDa 7924plusmn 114 0678


injected into the RP-HPLC after 022 μm filter membranetreatment Peak I purity was determined after RP-HPLCpurification and the results are shown in Figures 5 and 6

Figure 5 shows that component I of COASM ACE in-hibitory peptides has two main peaks and its purity wasdetermined by analytical RP-HPLC after collection 1eresults are shown in Figure 6

According to Figure 6(a) and 6(b) the chromatogramhad two peaks Based on calculations the peak concentra-tions of A were 8217 and 1783 whereas those of B were9024 and 976

38 Amino Acid Composition Analysis of COASM PeptideIt has been reported that ACE inhibitory peptides containhydrophobic amino acids residues and also that the threeamino acid sequence of C-terminal in these peptides canaffect the ability of peptides to combine with ACE If thesesequences contain hydrophobic amino acids it is highlylikely that they had high ACE inhibitory activity [16 26]Table 3 shows that the hydrophobic and aromatic aminoacids in peak I account for 5121 and 959 of the totalamino acid respectively 1is finding agrees with the studyby Aleman et al who isolated the highest ACE inhibitory

05

04

03

02

01

00 50 100 150 200

Elution time (min)

Abso

rban

ce

215nm

(a)

05

04

03

02

01

00 50 100 150 200

Elution time (min)

Abso

rban

ce I

II III

215nm

(b)

05

04

03

02

01

00 50 100 150 200

Elution time (min)

Abso

rban

ce

215nm

(c)

Figure 4 Separation of Sephadex G-15 at sample concentrations of 10mgmL (a) 25mgmL (b) and 50mgmL (c)

3 4 5 6 7 8 9 10 11 12 13 14Time (min)

300250200150100

500

mV

A

B

Figure 5 Peptide mapping of fraction I from COASM separated by RP-HPLC

200

160

120

80

40

0

mV

50 55 60 65 70 75 80Time (min)

(a)

50

40

30

20

10

0

mV

11 12 13 14 15Time (min)

(b)

Figure 6 Purity analysis of fractions (a) and (b) from COASM hydrolysate


rate component (molecular weight was less than 1000Da)from squid skin collagen with the hydrophobic amino acidcontent of up to 546 [31] Table 3 also indicates that peak Ihad high glycine alanine leucine and valine contentsSeveral studies have found that the ACE inhibitory peptidesfrom sea stings sea cucumbers and shrimps contain glu-tamic acid glycine alanine and leucine [29 31]

4 Conclusions

DH was not positively correlated with the ACE inhibitionrate in COASM protein hydrolysates After conducting theresponse surface method to optimize the hydrolytic processalkaline protease enzymolysis of COASM protein was foundto exhibit the highest ACE inhibitory rate 1e molecularweight of the ACE inhibitory peptide was less than 1 kDaafter ultrafiltration 1e components of ACE inhibitorypeptides in COASM protein could be separated by SephadexG-15 Component I had the strongest inhibitory rate thatreached 7924 and its IC50 was 0678mgmL RP-HPLCcan be used to prepare ACE inhibitory peptides fromCOASM Purity analysis of the single component revealedthat the separated component I has high alanine glutamicacid glycine and leucine contents

Data Availability

1e data used to support the findings of this study are in-cluded within the article

Conflicts of Interest

1e authors declare that there are no conflicts of interest

Acknowledgments

1is research was funded by the Natural Science Foundationof Hainan Province (318QN203) Hainan Province

Innovation Research Project of Graduate Students(Hyb2017-28 and Hys2017-36) Hainan Province MajorScience and Technology Project (SQ2017GXNY0010) andHainan Province Key Research and Development Plan(ZDYF2016157)

References

[1] G Boschin G M Scigliuolo D Resta and A ArnoldildquoOptimization of the enzymatic hydrolysis of Lupin (Lupinus)proteins for producing ACE-inhibitory peptidesrdquo Journal ofAgricultural and Food Chemistry vol 62 pp 1846ndash1851 2014

[2] T Kleekayai P A Harnedy M B OrsquoKeeffe A A PoyarkovW CunhaNeves and R J FitzGerald ldquoExtraction of anti-oxidant and ACE inhibitory peptides from 1ai traditionalfermented shrimp pastesrdquo Food Chemistry vol 176pp 441ndash447 2015

[3] E Escudero L Mora and F Toldra ldquoStability of ACE in-hibitory ham peptides against heat treatment and in vitrodigestionrdquo Food Chemistry vol 161 pp 305ndash311 2014

[4] B Zhang Q Sun H J Liu S Z Li and Z Q JiangldquoCharacterization of actinidin from Chinese kiwifruit culti-vars and its applications in meat tenderization and productionof angiotensin I-converting enzyme (ACE) inhibitory pep-tidesrdquo LWT-Food Science and Technology vol 78 pp 1ndash72017

[5] C Wang W Song L Jiang and M Du ldquoPurification andidentification of an ACE-inhibitory peptide from walnutprotein hydrolysaterdquo European Food Research and Technol-ogy vol 239 pp 333ndash338 2014

[6] S Rayaprolu N Hettiarachchy R Horax E SatchithanandamP Chen and A Mauromoustakos ldquoAmino acid profiles of 44soybean lines and ACE-I inhibitory activities of peptide frac-tions from selected linesrdquo Journal of the American Oil ChemistsSociety vol 92 pp 1023ndash1033 2015

[7] R E Cian A G Garzon D Betancur Ancona L ChelGuerrero and S R Drago ldquoHydrolyzates from Pyropiacolumbina seaweed have antiplatelet aggregation antioxidantand ACE I inhibitory peptides which maintain bioactivityafter simulated gastrointestinal digestionrdquo LWT-Food Scienceand Technology vol 64 pp 881ndash888 2015

[8] R Vasquez-Villanueva M Luisa Marina and M ConcepcionGarcia ldquoRevalorization of a peach (Prunus persica (L) Batsch)byproduct extraction and characterization of ACE-inhibitorypeptides from peach stonesrdquo Journal of Functional Foodsvol 18 pp 137ndash146 2015

[9] I Jemil L Mora R Nasri et al ldquoA peptidomic approach forthe identification of antioxidant and ACE-inhibitory peptidesin sardinelle protein hydrolysates fermented by Bacillussubtilis A26 and Bacillus amyloliquefaciens An6rdquo Food Re-search International vol 89 pp 347ndash358 2016

[10] B Wang G G Atungulu R Khir et al ldquoUltrasonic treatmenteffect on enzymolysis kinetics and activities of ACE-inhibitorypeptides from oat-isolated proteinrdquo Food Biophysics vol 10pp 244ndash252 2015

[11] J Otte S Shalabya and M Zakora ldquoAngiotensin-convertingenzyme inhibitory activity of milk protein hydrolysates effectof substrate enzyme and time of hydrolysisrdquo InternationalDairy Journal vol 17 pp 488ndash503 2007

[12] F Minervini F Algaron C G Rizzello P F Fox andM Monnet ldquoI-converting-enzyme-inhibitory and antibac-terial peptides from Lactobacillus helveticus PR4 proteinase-hydrolyzed caseins of milk from six speciesrdquo Applied and

Table 3 Amino acid compositions of fraction I () (g100 gprotein)

Amino acid ContentAsp 3141r 317Ser 189Glu 348Gly 943Ala 734Val 378Met 278Iso 275Leu 599Tyr 228Phe 356Lys 378His 178Arg 301Pro 287Trp 004Cys 028


Environmental Microbiology vol 69 no 9 pp 5297ndash53052003

[13] X Fang M Du F Luo and Y Jin ldquoPhysicochemicalproperties and lipid composition of Camellia seed oil (Ca-mellia oleifera Abel) extracted using different methodsrdquo FoodScience and Technology Research vol 21 no 6 pp 779ndash7852015

[14] J Hu W Wu Z Cao J Wen Q Shu and S Fu ldquoMor-phological physiological and biochemical responses of ca-mellia oleifera to low-temperature stressrdquo Pakistan Journal ofBotany vol 48 pp 899ndash905 2016

[15] J Wu J Zhao Y Liu et al ldquoFive new triterpenoid saponinsfrom the roots of Camellia oleifera C Abel with cytotoxicactivitiesrdquo Phytochemistry Letters vol 13 pp 379ndash385 2015

[16] S Zhang and X Li ldquoHypoglycemic activity in vitro ofpolysaccharides from Camellia oleifera Abel seed cakerdquo In-ternational Journal of Biological Macromolecules vol 115pp 811ndash819 2018

[17] T M Di S L Yang F Y Du L Zhao T Xia and X F ZhangldquoCytotoxic and hypoglycemic activity of triterpenoid saponinsfrom camellia oleifera Abel Seed pomacerdquoMolecules vol 22no 10 p 1562 2017

[18] J Y Je P J Park J Y Kwon and S K Kim ldquoA novel an-giotensin I converting enzyme inhibitory peptide from Alaskapollack (=eragra chalcogramma) frame protein hydrolysaterdquoJournal of Agricultural and Food Chemistry vol 52pp 7842ndash7845 2004

[19] M B OrsquoKeeffe R Norris M A Alashi R E Aluko andR J FitzGerald ldquoPeptide identification in a porcine gelatinprolyl endoproteinase hydrolysate with angiotensin con-verting enzyme (ACE) inhibitory and hypotensive activityrdquoJournal of Functional Foods vol 34 pp 77ndash88 2017

[20] H Korhonen and A Pihlanto ldquoFood-derived bioactivepeptides--opportunities for designing future foodsrdquo Currentpharmaceutical design vol 9 pp 1297ndash1308 2003

[21] M Mosquera B Gimenez P Montero and M CarmenGomez-Guillen ldquoIncorporation of liposomes containingsquid tunic ACE-inhibitory peptides into fish gelatinrdquo Journalof the Science of Food and Agriculture vol 96 pp 769ndash7762016

[22] G L Yao Y Chai J Chen and Y G Wu ldquoSeparation andidentification of ACE inhibitory peptides from cashew nut(Anacardium occidentale Linnaeus) proteinrdquo InternationalJournal of Food Properties vol 20 no 1 pp S981ndashS991 2017

[23] R E Cian J Vioque and S R Drago ldquoStructure-mechanismrelationship of antioxidant and ACE I inhibitory peptidesfrom wheat gluten hydrolysate fractionated by pHrdquo FoodResearch International vol 69 pp 216ndash223 2015

[24] S K Chaudhary A De S Bhadra and P K MukherjeeldquoAngiotensin-converting enzyme (ACE) inhibitory potentialof standardized Mucuna pruriens seed extractrdquo Pharma-ceutical Biology vol 53 pp 1614ndash1620 2015

[25] C Zhou H Ma Q Ding et al ldquoUltrasonic pretreatment ofcorn gluten meal proteins and neutrase effect on proteinconformation and preparation of ACE (angiotensin con-verting enzyme) inhibitory peptidesrdquo Food and BioproductsProcessing vol 91 no 4 pp 665ndash671 2013

[26] S G Wu X Z Feng X D Lan Y J Xu and D K LiaoldquoPurification and identification of Angiotensin-I ConvertingEnzyme (ACE) inhibitory peptide from lizard fish (Sauridaelongata) hydrolysaterdquo Journal of Functional Foods vol 13pp 295ndash299 2015

[27] M Gallego C Grootaert L Mora M Concepcion AristoyJ Van Camp and F Toldra ldquoTransepithelial transport of dry-

cured ham peptides with ACE inhibitory activity through aCaco-2 cell monolayerrdquo Journal of Functional Foods vol 21pp 388ndash395 2016

[28] O Martinez-Alvarez I Batista C Ramos and P MonteroldquoEnhancement of ACE and prolyl oligopeptidase inhibitorypotency of protein hydrolysates from sardine and tuna by-products by simulated gastrointestinal digestionrdquo Food ampFunction vol 7 no 4 pp 2066ndash2073 2016

[29] S Mane and S N Jamdar ldquoPurification and identification ofAce-inhibitory peptides from poultry viscera protein hy-drolysaterdquo Journal of Food Biochemistry vol 41 articlee12275 2017

[30] R Vasquez-Villanueva M Luisa Marina and M ConcepcionGarcia ldquoPeptide with angiotensin I-converting enzyme in-hibitory activity from hydrolyzed corn gluten mealrdquo Journalof Agricultural and Food Chemistry vol 55 pp 7891ndash78952007

[31] A Aleman M C Gomez-Guillen and P Montero ldquoIden-tification of ACE-inhibitory peptides from squid skin collagenafter in vitro gastrointestinal digestionrdquo Food Research In-ternational vol 54 pp 790ndash795 2013


Hindawiwwwhindawicom

International Journal of

Volume 2018

Zoology

Hindawiwwwhindawicom Volume 2018

Anatomy Research International

PeptidesInternational Journal of



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GenomicsInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018


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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Virolog y Stem Cells International



Enzyme Research



MicrobiologyHindawiwwwhindawicom

Nucleic AcidsJournal of

Volume 2018

Submit your manuscripts atwwwhindawicom

C oleifera which belongs to the 1eaceae family isregarded as one of the four largest woody oil species in theworld and has high economic values and ecological benefits[13 14] COASM is a by-product of oil extraction and itsutilization ratio is relatively low For a long time COASMhas been used as a fertilizer and fuel resulting in a seriouswaste of resources [15] C oleifera contains 10ndash20protein and some small peptides derived from food proteinsare easily digested and absorbed by the human bodyCOASM can provide nutrients for human growth and de-velopment and exhibit strong antioxidant and lipid-loweringproperties and other health care functions 1us it is apromising functional factor worldwide [16 17]

In this study COASM was used as the raw materialand the response surface method was used to determinethe best enzymolysis conditions for alkaline protease 1eenzymolysis kinetic parameters were obtained accordingto the MichaelisndashMenten equation Ultrafiltration gelchromatography and RP-HPLC were conducted to pro-duce a component with high ACE inhibitory rate 1eamino acid composition and in vitro digestion of thiscomponent were subsequently analyzed Our experimentcan provide the basis for further research to increase thevalue of C oleifera

2 Materials and Methods

21Materials andReagents COASM samples were providedby Lv YuanC oleifera processing Co (Qionghai city HainanChina) Angiotensin-I-converting enzyme (ACE) N-[3-(2-furylacryloyl)]-L-phenyalanyl-glycyl-glycine (FAPGG) andSephadex G-25 glucan gel were purchased from Sigma-Aldrich Co (USA) Trypsin (250Umg) neutral protease(50Umg) alkaline protease (2000Umg) and papain(6000Umg) were purchased from Novozymes BiologicalTechnology Co (Beijing China) Tris-HCl buffer NaClNaOH formic acid ethanol and phenol reagents were ofanalytical grade and purchased from Beijing Chemical Re-agent Co (Beijing China) Deionized water was usedthroughout the experiment

22 Preparation of ACE Inhibitory Peptides from COASMCOASM samples underwent lyophilization crushingscreening with 40-mesh size using petroleum ether assolvent with a SL ratio of 1 8 (gml) and 65degC extractionfor 4 h to remove fat In brief 15 g of defatted COASMpowder was accurately weighted in a beaker 1e optimalpH and temperatures for four kinds of protease were asfollows trypsin (pH 80 37degC) neutral protease (pH 7050degC) alkaline protease (pH 95 60degC) and papain (pH57 60degC) 1ese proteases were set to digest for 4 hEnzymatic hydrolysis conditions are as follows a SL ratioof 1 15 (gml) and enzyme amount for 50 (ww) 1eenzymes were inactivated via a boiling water bath for10min After centrifugation for 10min at 4degC and10000 rpm the supernatant fluid was subjected to evap-oration and freeze drying Finally the ACE inhibitorypeptides were obtained [18 19]

23 Determination of the Degree of Hydrolysis (DH) fromCOASM Enzymolysis DH was determined according to thepH-stat method which is based on the protein hydrolysisprocess related to the pH value of the solution 1e pHsystem was maintained by adding acid or alkaline whosequantity must be based on the calculated DH from theenzymolysis of COASM using the following equation[18 20 21]

DH() Vb times Nb

Zp times 732231113888 1113889 times 100 (1)

where Vb is the NaOH volume (mL) Nb is the sodiumhydroxide concentration (01molL) Zp is the proteinweight (g) and 723 is the number of peptide bonds in per gproteins (mmol) and that of COASM was 723

24 Determination of ACE Inhibitory Rate ACE inhibitionrate of COASM hydrolysate was evaluated by the methodestablished in our laboratory [22] In brief 10 microL of ACEaqueous solution and 10 microL of ACE inhibitory peptides(concentration of 1mgmL) were added to an enzymemicroplate 1e substrate (10mmolL FAPGG dissolved in50mmolL Tris-HCl containing 03molLmiddotNaCl pH 75) waspreheated for 37degC for 5min added to the microplate andallowed to react for 2min 1e absorbance at 340 nm wasrecorded and labeled as A1 After a reaction time of 30minthe absorbance at 340 nm was determined and labeled as A21e control group comprised 10 microL of substrates instead ofACE inhibitory peptides 1e initial absorbance of the blanksolution was recorded as A01 and that after the reaction wasrecorded as A02 Changes in the absorbance value (ΔAInhibitorA1minusA2 ΔBlankA01minusA02) were calculated toobtain the ACE inhibitory rate

ACE inhibitory rate 1minusΔA inhibitorΔblank

1113888 1113889 times 100 (2)

25 Response Surface Design Experiment under EnzymaticHydrolysis Conditions According to the results of thesingle-factor experiment the experimental design was op-timized using response surface methodology [23] 1eBoxndashBehnken center combination experimental principlewas also applied [24 25] In this principle we selected fivefactors namely substrate concentration pH value reactiontemperature enzyme quantity and reaction time whichaffect the ACE inhibitory rate 1ree levels per factor wereused to optimize the test and the ACE inhibitory rate wasused as the response variable

26 Determination of the Maximum Reaction Rate of MIConstant and Enzyme-Promoting Reaction 1e substrateconcentration of COASM protein solution with 002 004006 008 and 01mgmL was prepared respectively al-kaline protease was added and hydrolysated for pH 95 at60degC 005molLmiddotNaOH solution was used to maintain aconstant pH value in COASM protein solution 1e average



1Vo

1113888 1113889 Km

Vmax[S]+

1Vmax

1113888 1113889 (3)


















DH 1248 + 017A + 028Bminus 0026C + 0062AB


(4)





3456789

1011121314151617

15 20 25 30 35 40

Deg

ree o

f hyd

roly

sis (

)

Hydrolysis time (h)


10 45

TrypsinPapain

(a)

30

35

40

45

50

55

60

65

ACE

inhi

bitio

n ra

te (

)



TrypsinPapain

(b)



9

10

11

12

13

14

15

16

10152025303540455055606570

40 45 50 55 60

Deg

ree o

f hyd

roly

sis (

)

ACE

inhi

bitio

n ra

te (

)



(a)

Deg

ree o

f hyd

roly

sis (

)

ACE

inhi

bitio

n ra

te (

)

4

5

6

7

8

9

10

11

20

25

30

35

40

45

50

55

60

65

70

8580 90 95 100 110105pH


(b)

Deg

ree o

f hyd

roly

sis (

)

ACE

inhi

bitio

n ra

te (

)

6

8

10

12

14

16

10

20

30

40

50

60

70



(c)

Deg

ree o

f hyd

roly

sis (

)

ACE

inhi

bitio

n ra

te (

)

9

10

11

12

13

14

15

16

10

20

30

40

50

60

70



(d)

4

6

8

10

12

14

16

10

20

30

40

50

60

70


Deg

ree o

f hyd

roly

sis (

)

ACE

inhi

bitio

n ra

te (

)


(e)











10

80

70

60

50

40

30

20

10 504030200

1V

(mLmiddot

min

mg)

1S (mLmg)

y = 09943x + 72342

R2 = 09352









05

04

03

02

01

00 50 100 150 200

Elution time (min)

Abso

rban

ce

215nm

(a)

05

04

03

02

01

00 50 100 150 200

Elution time (min)

Abso

rban

ce I

II III

215nm

(b)

05

04

03

02

01

00 50 100 150 200

Elution time (min)

Abso

rban

ce

215nm

(c)


3 4 5 6 7 8 9 10 11 12 13 14Time (min)

300250200150100

500

mV

A

B


200

160

120

80

40

0

mV

50 55 60 65 70 75 80Time (min)

(a)

50

40

30

20

10

0

mV

11 12 13 14 15Time (min)

(b)




4 Conclusions


Data Availability




Acknowledgments



References








































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



1Vo

1113888 1113889 Km

Vmax[S]+

1Vmax

1113888 1113889 (3)


















DH 1248 + 017A + 028Bminus 0026C + 0062AB


(4)





3456789

1011121314151617

15 20 25 30 35 40

Deg

ree o

f hyd

roly

sis (

)

Hydrolysis time (h)


10 45

TrypsinPapain

(a)

30

35

40

45

50

55

60

65

ACE

inhi

bitio

n ra

te (

)



TrypsinPapain

(b)



9

10

11

12

13

14

15

16

10152025303540455055606570

40 45 50 55 60

Deg

ree o

f hyd

roly

sis (

)

ACE

inhi

bitio

n ra

te (

)



(a)

Deg

ree o

f hyd

roly

sis (

)

ACE

inhi

bitio

n ra

te (

)

4

5

6

7

8

9

10

11

20

25

30

35

40

45

50

55

60

65

70

8580 90 95 100 110105pH


(b)

Deg

ree o

f hyd

roly

sis (

)

ACE

inhi

bitio

n ra

te (

)

6

8

10

12

14

16

10

20

30

40

50

60

70



(c)

Deg

ree o

f hyd

roly

sis (

)

ACE

inhi

bitio

n ra

te (

)

9

10

11

12

13

14

15

16

10

20

30

40

50

60

70



(d)

4

6

8

10

12

14

16

10

20

30

40

50

60

70


Deg

ree o

f hyd

roly

sis (

)

ACE

inhi

bitio

n ra

te (

)


(e)











10

80

70

60

50

40

30

20

10 504030200

1V

(mLmiddot

min

mg)

1S (mLmg)

y = 09943x + 72342

R2 = 09352









05

04

03

02

01

00 50 100 150 200

Elution time (min)

Abso

rban

ce

215nm

(a)

05

04

03

02

01

00 50 100 150 200

Elution time (min)

Abso

rban

ce I

II III

215nm

(b)

05

04

03

02

01

00 50 100 150 200

Elution time (min)

Abso

rban

ce

215nm

(c)


3 4 5 6 7 8 9 10 11 12 13 14Time (min)

300250200150100

500

mV

A

B


200

160

120

80

40

0

mV

50 55 60 65 70 75 80Time (min)

(a)

50

40

30

20

10

0

mV

11 12 13 14 15Time (min)

(b)




4 Conclusions


Data Availability




Acknowledgments



References








































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018







DH 1248 + 017A + 028Bminus 0026C + 0062AB


(4)





3456789

1011121314151617

15 20 25 30 35 40

Deg

ree o

f hyd

roly

sis (

)

Hydrolysis time (h)


10 45

TrypsinPapain

(a)

30

35

40

45

50

55

60

65

ACE

inhi

bitio

n ra

te (

)



TrypsinPapain

(b)



9

10

11

12

13

14

15

16

10152025303540455055606570

40 45 50 55 60

Deg

ree o

f hyd

roly

sis (

)

ACE

inhi

bitio

n ra

te (

)



(a)

Deg

ree o

f hyd

roly

sis (

)

ACE

inhi

bitio

n ra

te (

)

4

5

6

7

8

9

10

11

20

25

30

35

40

45

50

55

60

65

70

8580 90 95 100 110105pH


(b)

Deg

ree o

f hyd

roly

sis (

)

ACE

inhi

bitio

n ra

te (

)

6

8

10

12

14

16

10

20

30

40

50

60

70



(c)

Deg

ree o

f hyd

roly

sis (

)

ACE

inhi

bitio

n ra

te (

)

9

10

11

12

13

14

15

16

10

20

30

40

50

60

70



(d)

4

6

8

10

12

14

16

10

20

30

40

50

60

70


Deg

ree o

f hyd

roly

sis (

)

ACE

inhi

bitio

n ra

te (

)


(e)











10

80

70

60

50

40

30

20

10 504030200

1V

(mLmiddot

min

mg)

1S (mLmg)

y = 09943x + 72342

R2 = 09352









05

04

03

02

01

00 50 100 150 200

Elution time (min)

Abso

rban

ce

215nm

(a)

05

04

03

02

01

00 50 100 150 200

Elution time (min)

Abso

rban

ce I

II III

215nm

(b)

05

04

03

02

01

00 50 100 150 200

Elution time (min)

Abso

rban

ce

215nm

(c)


3 4 5 6 7 8 9 10 11 12 13 14Time (min)

300250200150100

500

mV

A

B


200

160

120

80

40

0

mV

50 55 60 65 70 75 80Time (min)

(a)

50

40

30

20

10

0

mV

11 12 13 14 15Time (min)

(b)




4 Conclusions


Data Availability




Acknowledgments



References








































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018


9

10

11

12

13

14

15

16

10152025303540455055606570

40 45 50 55 60

Deg

ree o

f hyd

roly

sis (

)

ACE

inhi

bitio

n ra

te (

)



(a)

Deg

ree o

f hyd

roly

sis (

)

ACE

inhi

bitio

n ra

te (

)

4

5

6

7

8

9

10

11

20

25

30

35

40

45

50

55

60

65

70

8580 90 95 100 110105pH


(b)

Deg

ree o

f hyd

roly

sis (

)

ACE

inhi

bitio

n ra

te (

)

6

8

10

12

14

16

10

20

30

40

50

60

70



(c)

Deg

ree o

f hyd

roly

sis (

)

ACE

inhi

bitio

n ra

te (

)

9

10

11

12

13

14

15

16

10

20

30

40

50

60

70



(d)

4

6

8

10

12

14

16

10

20

30

40

50

60

70


Deg

ree o

f hyd

roly

sis (

)

ACE

inhi

bitio

n ra

te (

)


(e)











10

80

70

60

50

40

30

20

10 504030200

1V

(mLmiddot

min

mg)

1S (mLmg)

y = 09943x + 72342

R2 = 09352









05

04

03

02

01

00 50 100 150 200

Elution time (min)

Abso

rban

ce

215nm

(a)

05

04

03

02

01

00 50 100 150 200

Elution time (min)

Abso

rban

ce I

II III

215nm

(b)

05

04

03

02

01

00 50 100 150 200

Elution time (min)

Abso

rban

ce

215nm

(c)


3 4 5 6 7 8 9 10 11 12 13 14Time (min)

300250200150100

500

mV

A

B


200

160

120

80

40

0

mV

50 55 60 65 70 75 80Time (min)

(a)

50

40

30

20

10

0

mV

11 12 13 14 15Time (min)

(b)




4 Conclusions


Data Availability




Acknowledgments



References








































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018










10

80

70

60

50

40

30

20

10 504030200

1V

(mLmiddot

min

mg)

1S (mLmg)

y = 09943x + 72342

R2 = 09352









05

04

03

02

01

00 50 100 150 200

Elution time (min)

Abso

rban

ce

215nm

(a)

05

04

03

02

01

00 50 100 150 200

Elution time (min)

Abso

rban

ce I

II III

215nm

(b)

05

04

03

02

01

00 50 100 150 200

Elution time (min)

Abso

rban

ce

215nm

(c)


3 4 5 6 7 8 9 10 11 12 13 14Time (min)

300250200150100

500

mV

A

B


200

160

120

80

40

0

mV

50 55 60 65 70 75 80Time (min)

(a)

50

40

30

20

10

0

mV

11 12 13 14 15Time (min)

(b)




4 Conclusions


Data Availability




Acknowledgments



References








































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018






05

04

03

02

01

00 50 100 150 200

Elution time (min)

Abso

rban

ce

215nm

(a)

05

04

03

02

01

00 50 100 150 200

Elution time (min)

Abso

rban

ce I

II III

215nm

(b)

05

04

03

02

01

00 50 100 150 200

Elution time (min)

Abso

rban

ce

215nm

(c)


3 4 5 6 7 8 9 10 11 12 13 14Time (min)

300250200150100

500

mV

A

B


200

160

120

80

40

0

mV

50 55 60 65 70 75 80Time (min)

(a)

50

40

30

20

10

0

mV

11 12 13 14 15Time (min)

(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


purification of angiotensin-i-converting enzyme...

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