Isolation and Characterization of Angiotensin I-Converting Enzyme

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Bioactive Constituents, Metabolites, and Functions

Isolation and Characterization of Angiotensin I-Converting Enzyme (ACE) Inhibitory Peptides from the Enzymatic Hydrolysate of Carapax Trionycis (the shell of the turtle Pelodiscus sinensis) Pengying Liao, Xiongdiao Lan, Dankui Liao, LiXia Sun, Liqin Zhou, Jianhua Sun, and Zhangfa Tong J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01558 • Publication Date (Web): 19 Jun 2018 Downloaded from http://pubs.acs.org on June 20, 2018

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Journal of Agricultural and Food Chemistry

Isolation and Characterization of Angiotensin I-Converting Enzyme (ACE) Inhibitory Peptides from the Enzymatic Hydrolysate of Carapax Trionycis (the shell of the turtle Pelodiscus sinensis)

Pengying Liao, Xiongdiao Lan, Dankui Liao*, Lixia Sun*, Liqin Zhou, Jianhua Sun, Zhangfa Tong

Guangxi Colleges and Universities Key Laboratory of New Technology and Application in Resource Chemical Engineering, School of Chemistry and Chemical Engineering, Guangxi University, Nanning 530004, Guangxi, P.R. China

* Corresponding authors. Tel: +86 771 327 2702 Fax: +86 771 323 3718 E-mail: [email protected]; [email protected]

Dankui Liao: School of Chemistry & Chemical Engineering, Guangxi University, 100 Daxue East Road, Nanning 530004, Guangxi, P.R. China.

Lixia Sun: School of Chemistry & Chemical Engineering, Guangxi University, 100 Daxue East Road, Nanning, 530004, Guangxi, P.R. China.

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ABSTRACT

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Carapax Trionycis (the shell of the turtle Pelodiscus sinensis) was hydrolyzed by six different

3

commercial proteases. The hydrolysate prepared from papain showed stronger inhibitory activity

4

against angiotensin I-converting enzyme (ACE) than other extracts. Two noncompetitive ACE

5

inhibitory peptides were purified successively by ultrafiltration, gel filtration chromatography, ion

6

exchange column chromatography, and high-performance liquid chromatography (HPLC). The

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amino acid sequences of them were identified as KRER and LHMFK, with IC50 values of 324.1 and

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75.6 μM, respectively, confirming that Carapax Trionycis is a potential source of active peptides

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possessing ACE inhibitory activities. Besides, both enzyme kinetics and isothermal titration

10

calorimetry (ITC) assay showed that LHMFK could form more stable complex with ACE than KRER,

11

which is in accordance with the better inhibitory activity of LHMFK.

12 13

KEYWORDS: Carapax Trionycis; ACE inhibitory peptides; isolation and characterization; enzyme

14

kinetics; isothermal titration calorimetry.

15 16 17 18 19 20 21 22 23 2

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INTRODUCTION

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Angiotensin I-converting enzyme (ACE), a dipeptidyl carboxypeptidase containing a zinc ion in

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the active site, plays an important role in the renin–angiotensin–aldosterone system (RAAS), which

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is crucial in regulating body fluids and electrolyte balance. ACE can convert the decapeptide

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angiotensin I (Ang I) to the octapeptide angiotensin II (Ang II) and inactivate the vasodilator

29

bradykinin, both of which can induce blood pressure enhancement.1 Thus, synthetic ACE inhibitors

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(ACEI) such as perindopril, captopril, and enalapril are believed to be pivotal drugs in the treatment

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of hypertension. However, adverse effects, such as coughing, allergic reactions, taste disturbances,

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renal injury, and skin rashes, have always been associated with synthetic ACEI.2

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The hypotensive effects of the ACEI peptides deriving from various sources including plant and

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animals are usually weaker than synthetic ACEI, but they are milder and safer.3 Thus, the natural

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peptides have been used in the development of new therapeutics or nutraceuticals. One important

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method to obtain these active peptides is protein digestion using commercially available proteases.4

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Various proteases have been used in acquiring oligopeptides owing to the different specific cleavage

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sites of them, and the activities of the hydrolysates are the dominant factors in protease screening.5–7

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The amino acid compositions of the peptides from the hydrolysates are totally dependent on the

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substrate and the proteases, whereas the activity of the peptide is mainly influenced by amino acid

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compositions and sequences.8 Numerous principles concerning the relationship between the

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structures and the activities of the ACEI peptides have been summarized. The short sequences and

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hydrophobic amino acid residues or charged amino acid residues at C-terminal are the common

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characteristics of most known active peptides.2, 9

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Soft-shelled turtle (SST) (Pelodiscus sinensis) is a type of edible aquatic animal possessing

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antihypertensive effect in spontaneously hypertensive rats (SHR).10 The ACE inhibitory activity of 3

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the hydrolysate of SST treated by gastrointestinal enzymes (pepsin, trypsin, and chymotrypsin) has

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been reported to be better than SST.11 Besides, three ACEI peptides have been purified from the

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enzymatic hydrolysates of egg from SST.12–14 These results demonstrate that SST is a valuable source

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of ACEI peptides. Carapax Trionycis, the shell of SST, is rich in amino acids and peptides.15, 16 Active

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peptides associated with hepatic diseases have been isolated from the water extracts of Carapax

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Trionycis.16–18 However, the potential ACE inhibition of this source has never been reported.

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This work is focused on the isolation and characterization of ACEI peptides from the hydrolysate

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of Carapax Trionycis. The extracts of Carapax Trionycis were hydrolyzed by six different proteases,

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and the papain hydrolysates possessing the strongest ACE inhibitory activities were further purified

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by series of chromatographic methods. After purification, matrix-assisted laser desorption/ionization

57

time-of-flight tandom mass spectrometry (MALDI-TOF/TOF) was used to identify the ACEI

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peptides, whose inhibitory activities were further confirmed by synthetic authentic samples.

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Moreover, the inhibitory mechanism of the ACEI peptides was proposed according to the results of

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enzyme kinetics and ITC analysis.

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MATERIALS AND METHODS

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Materials and Chemical Regents. Carapax Trionycis was purchased in Nanning, China, and

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authenticated by Professor Songji Wei from Guangxi University of Chinese Medicine as the shell of

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P. sinensis. ACE (from rabbit lung) and hippuryl-L-histidyl-L-leucine (HHL) were both purchased

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from Sigma-Aldrich (St. Louis, MO, USA). Alcalase (300 000 U/g), neutral protease (400 000 U/g),

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papain (300 000 U/g), and bromelain (20 000 U/g) were all provided by Pangbo Biological

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Engineering Co., Ltd. (Nanning, China). Trypsin (300 000 U/g) and pepsin (10 000 U/g) were

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purchased from Sinopharm Medicine Holding Co., Ltd. (Shanghai, China). Acetonitrile (HPLC grade)

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was purchased from Fisher Scientific Inc. (Hudson, NH, USA). Other reagents were of analytical 4

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grade and purchased from Sinopharm Chemical Reagent Company (Shanghai, China).

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Preparation of enzymatic hydrolysates and screening of the proteases. Carapax Trionycis was

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crushed in a grinder and sieved through a 40 mesh. The powder (1 Kg) was then mixed with 10 L of

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distilled water and heated at 60 °C for 2 h. The extract (WEP) was filtered and lyophilized as raw

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material for enzymatic hydrolysis. The protein content of WEP was 77.02% ± 1.60% as determined

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by Kjeldahl method, using 6.25 as protein conversion factor.19 WEP was dissolved in ultrapure water

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at a protein concentration of 2% in a 500 mL glass reactor, and was digested by six different enzymes

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(papain, neutral protease, trypsin, bromelain, alcalase, and pepsin) under their optimal conditions

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(shown in Table 1), respectively. During enzymatic reactions, 0.1 M NaOH was added to maintain

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pH at the optimal level, and the solution was continuously stirred for 7 h. Afterwards, the hydrolysate

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was then placed in a boiling water bath for 10 min to inactivate the enzyme and the pH of the solution

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was adjusted to 7.0. The hydrolysates prepared using six different proteases were centrifuged at 4 000

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rpm for 30 min at 4 °C (Model 5810R, Eppendorf), and the supernatant was lyophilized to evaluate

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the ACE inhibitory activities.

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Determination of ACE inhibitory activities. ACE inhibitory activity was evaluated by

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determining the amount of hippuric acid (HA) generated from HHL using a previously described

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method with a few modifications.7 ACE was dissolved into the 100 mM borate buffer (pH 8.3)

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containing 300 mM NaCl at the concentrations of 0.10 U/mL, and the substrate HHL was also

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dissolved into the same buffer at the concentration of 5.40 mM. 20 μL of ACE solution was mixed

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with 140 μL of sample/blank buffer for 10 min at 37 °C bath, and 40 μL of HHL solution was then

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added and incubated for 15 min at 37 °C. At last, the reaction was terminated by adding 150 μL of 1

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M HCl. 20 μL of the reaction solution was injected into HPLC (Agilent 1260) equipped with Zorbax

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SB C18 column (4.6 mm × 150 mm, 5 μm, Agilent, USA) to detect the content of HA . The IC50 5

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value of the sample was defined as the concentration when 50% ACE was inhibited under the same

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conditions.

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Preparation of the papain hydrolysate and ultrafiltration. WEP (equivalent to 20 g of protein)

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was mixed with 1 L of ultrapure water, and papain was added to begin the hydrolysis at an

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enzyme/substrate ratio of 2:100 (w/w). The reaction was conducted at 37 °C and pH 7.0 for 7 h. The

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hydrolysates (WEPH) were filtered through a 0.45 μm filtrate membrane (Shanghai Xingya Purifying

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Materials Factory, China), and the filtrate was fractioned by ultrafiltration (UF) using a Labscale

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system (Labscale TFF System, Millipore Co., Billerica, MA, USA) with molecular weight cut-off

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(MWCO) membranes of 10 KDa (Pelllicon XL Biomax 10) to obtain the retentate (WEPH-I, Mw >

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10 KDa) and the permeate (WEPH-II, Mw < 10 KDa). WEPH, WEPH-I and WEPH-II were

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lyophilized and used for further study.

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Purification of ACEI peptides. WEPH-II was loaded onto a Sephadex G-15 (Pharmacia Fine

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Chemicals, Uppsala, Sweden) column (Φ 2 cm × 40 cm) pre-equilibrated with ultrapure water. After

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the sample was loaded, the column was eluted with ultrapure water for 270 min at a flow rate of 0.5

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mL/min, and the eluted solution was monitored at 280 nm. Each fraction was collected, reduced, and

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lyophilized to investigate the ACE inhibitory activity. The fraction showing the strongest activity

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against ACE was further purified using strong anion exchange chromatography HiPrep Q FF 16/10

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(16 × 100 mm; GE Healthcare, Buckinghamshire, UK) coupled with an ÄKTA purifier system (GE

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Healthcare) at 20 °C. The sample was dissolved into buffer A (10 mM pH 8.3 borate buffer) at 5

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mg/mL, and 2 mL was loaded onto the column, which was equilibrated with 5 column volumes (CV)

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of buffer A previously. The flow rate was set at 2 mL/min. The elution condition was as follows: 0–

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85 min: 0% to 20% of buffer B (10 mM pH 8.3 borate buffer containing 1 M NaCl); 85–100 min:

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20%–100% of buffer B; and 100–115 min: 100% of buffer B. The chromatograms were monitored at 6

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214 nm. The fractions of each major peak were collected, reduced, and lyophilized for the analysis

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of ACE inhibitory activities.

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The active fractions were further separated by semi-preparative RP-HPLC on a YMC-Pack ODS-

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A column (10 × 250 mm, 5 μm, YMC Co., Japan) under the following chromatographic conditions:

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flow rate, 1.8 mL/min; UV detector, λ 214 nm; mobile phase, 0.1% trifluoroacetic acid (TFA) in

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acetonitrile (solvent A) and 0.1% TFA in water (solvent B), gradient elution from 0% to 50% A for

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150 min. Two active fractions were further subjected repeatedly chromatographic runs by analytical

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ODS-A column (4.6 × 250 mm, 5 μm (YMC); flow rate, 1 mL/min; detection and eluent, as above).

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After removing the solvent under reduced pressure and freeze-drying, the samples were stored at

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-20 °C before further analysis.

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Characterization of ACEI peptides. The molecular mass and amino acid sequences of the

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purified peptides were determined using a 4800 plus MALDI-TOF/TOF Analyzer (Applied

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Biosystems, Beverly, MA, USA). The sample was mixed with matrix solution (a-cyano-4-

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hydroxycinnamic acid solution) and dried on a conventional steel matrix-assisted laser

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desorption/ionization target. The sample was ionized at 337 nm and operated in the positive ion

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delayed extraction reflector mode. Spectra were recorded over the mass/charge (m/z) range of 0–1500

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Da. Tandem mass spectrometry (MS) experiments were conducted by collision-induced dissociation,

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and sequence information was obtained by tandem MS analysis.

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Determination of inhibitory pattern of ACEI peptides. The ACE inhibitory kinetics of the active

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peptides were investigated at different concentrations of HHL (0.25, 0.50, 1.00, and 2.00 mM), and

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the ACE activities were measured in the absence and presence of ACEI peptides.13 The inhibitory

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pattern was determined by the Lineweaver–Burk plot of the reciprocal of the production speed of HA

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(y-axis) versus the reciprocal of the substrate concentration (x-axis). 7

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Simulated gastrointestinal digestion of ACEI peptides. To evaluate the stability of ACEI

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peptides,

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(http://web.expasy.org/peptide_cutter/) was used to evaluate the potential cleavage sites of the

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gastrointestinal (GI) tract enzymes in the ACEI peptides. The pepsin (pH 1.3 and pH > 2.0), trypsin

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and chymotrypsin (high and low specificity) were chosen to predict the peptides stability.

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in

silico

analysis

was

performed.

The

program

Expasy

Peptide

Cutter

In silico prediction of toxicity of ACEI peptides. The ACEI peptides were assessed for potential toxicity in silico using ToxinPred, available at http://crdd.osdd.net/raghava//toxinpred/design.php.

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Isothermal Titration Calorimetry (ITC) analysis of ACEI peptides. To measure the

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thermodynamic parameters (enthalpy change ΔH, entropy change ΔS, the association constant Ka, the

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dissociation constant Kd, and binding stoichiometry n) of the interactions between the active peptides

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and ACE, ITC experiments were performed on the Nano ITC Low Volume (TA Instrument, USA) at

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25 °C. All solutions were degassed thoroughly to avoid the bubble. Both the active peptides and ACE

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were prepared in 50 mM N-[(2-amino-2-oxoethyl) amino] ethanesulfonic acid buffer solution (pH

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7.0, containing 300 mM NaCl) (ACES). The reference cell was filled with the blank buffer solution,

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and the sample cell was injected with 4.89 nM ACE solution (1.97 U/mg). The peptides concentration

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in the syringe was 30.0 mM for KRER and 1.2 mM for LHMFK, respectively. While achieving the

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baseline stability, the peptide solution was titrated into ACE via 20 individual injections, the volume

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of each injection was 2.5 µL with a 300 s interval, and the stirring speed was fixed at 250 rpm. A plot

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of the corrected heat rate (µJ/s) against time (s) was achieved after subtracting the dilution heat in the

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blank experiment by using the Nano Analyze Software (TA Instrument, New Castle, DE 19720, USA).

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The independent binding model was used to analyze the thermodynamic parameters of the binding

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action.

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Statistical analysis. All assays were conducted in triplicate. Data were presented as the mean ± 8

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standard deviation. Statistical analysis was performed using ANOVA one-way analysis by SPSS 19.0

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Statistics software (IBM SPSS, Armonk, NY) with post-hoc least significant difference (LSD)

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analysis. Significant difference was taken into account when p < 0.05.

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RESULTS AND DISCUSSION

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The screening of the proteases for Carapax Trionycis. Six proteases were used in the hydrolysis

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of Carapax Trionycis protein. The IC50 values of the hydrolysates from papain, neutral protease,

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trypsin, bromelain, alcalase, pepsin and the WEP were 0.99 ± 0.06 (a), 1.27 ± 0.08 (a), 1.30 ± 0.09

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(a), 1.34 ± 0.11 (a), 1.35 ± 0.09 (a), 7.11 ± 0.57 (b), and 7.94 ± 0.38 (c) mg/mL respectively. The

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papain hydrolysate possessed the lowest IC50 value, even if no significant differences were observed

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among the neutral protease, trypsin, bromelain, and alcalase digestion (Data with the same letter

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indicated that the differences were not significant) (p > 0.05). The results imply that Carapax

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Trionycis protein was cleaved at different sites during hydrolysis by different enzymes, producing

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different peptides with varying ACE inhibitory activities. The broad cleavage site of papain, which

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originates from Carica papaya, shows a preference for Lys–Arg and Phe–X–COOH at the terminal

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amino acid, whereas the basic amino acids at C-terminal is one of the preferred characteristics for

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ACEI peptides.20, 21 The result demonstrated the effectiveness of papain for generating the ACE

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inhibitory hydrolysates from Carapax Trionycis, which was in agreement with the screening results

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from threadfin bream frames, bovine fibrinogen, and bovine brisket.21–23 Owing to the utilization as

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a food-grade enzyme and a tenderizer in meat industry, papain was finally chosen for the protein

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digestion of Carapax Trionycis.22, 23

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Purification of ACEI peptides. MW is an important factor for ACEI peptides, and UF with

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different MWCO membranes has always been used in the first step of purification to enrich the low-

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molecular active fractions.5, 6, 12 WEPH were fractioned using an UF system with 10 KDa MWCO. 9

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The ACE inhibitory activities of the fractions WEPH, WEPH-I (MW > 10 KDa), and WEPH-II (MW

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< 10 KDa) were assayed. The results showed that WEPH-II with an IC50 value of 0.72 ± 0.03 (a)

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mg/mL presented significantly stronger inhibitory activity than WEPH and WEPH-I with IC50 values

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of 0.99 ±0.06 (b) and 1.23 ±0.06 (c) mg/mL, respectively (p < 0.05).

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WEPH-II was subsequently applied to Sephadex G-15 column chromatography, and three primary

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fractions (A, B, and C) were collected and lyophilized. Fraction A exhibited the strongest ACE

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inhibitory activity with IC50 value of 0.46 ± 0.06 mg/mL (a), whereas fractions B and C showed

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activities with IC50 values of 0.68 ± 0.07 (b) and 0.61 ± 0.05 (b) mg/mL, respectively (p < 0.05).

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Fraction A, which was consecutively separated using anion exchange column chromatography

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(Hiprep Q FF 16/10), was divided into five main fractions (A1–A5) (Figure 1). The A2 fraction

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showed the lowest IC50 value of 0.17 ±0.04 mg/mL, compared with the fractions A1 and A3–A5 with

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IC50 values of 0.34 ±0.05, 0.64 ±0.10, 0.50 ±0.07 and 0.94 ±0.12 mg/mL, respectively (p < 0.05).

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The A2 fraction was loaded onto semi-preparative HPLC with ODS-A column for further

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purification. Fractions A21–A28 were collected respectively, and the ACE inhibitory activities of

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fractions A21 and A22 were both higher than 85% at the concentration of 0.30 mg/mL, and the IC50

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value of fraction A22 (0.05 ± 0.01 mg/mL) was the lowest among them (the IC50 values of fractions

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A21, and A23–A28 were 0.19 ±0.02, 0.24 ±0.05, 0.27 ±0.04, 0.28 ±0.04, 0.59 ±0.08, 0.64 ±0.06,

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and 0.48 ± 0.09 mg/mL, respectively) (p < 0.05) (Figure 2). Both of A21 and A22 were further

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purified on analytical HPLC with ODS-A column and fractions A21-1 and A22-1 were obtained

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whose IC50 values were determined as 0.19 ±0.00 and 0.05 ±0.00 mg/mL. The ACEI peptides from

205

papain hydrolysates of Carapax Trionycis were purified 5-fold for fraction A21-1 and 20-fold for

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fraction A22-1 by using a five-step purification procedure. Consecutive chromatography methods

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including ion-exchange chromatography, gel-filtration chromatography, and HPLC have been usually 10

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adopted to purify the ACEI peptides, and the purification fold ranged between 15–30 folds, like 15-

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fold for tilapia hydrolysates, 19-fold for hydrolysates of marine sponge Stylotella aurantium, and 24-

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fold for hydrolysate of duck skin.6, 24–26

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Identification of ACEI peptides by MALDI-TOF/TOF MS. Peptide sequences of fractions A21-

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1 and A22-1 were identified by MALDI-TOF/TOF Analyzer (Figure 3). The molecular weight of

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fraction A21-1 was 587.4 Da (588.3652 [M + H]+), and the amino acid sequence was identified as

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KRER with IC50 value of 324.1 ± 2.5 μM. The molecular weight of fraction A22-1 was 674.3 Da

215

(675.3256 [M + H]+), and the amino acid sequence was identified as LHMFK with IC50 value of 75.6

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±1.5 μM. Peptides with the same sequences were synthesized (98% purity) by Shanghai GL Biochem.

217

Ltd., China, and their ACE inhibitory activities were investigated. The IC50 values of synthetic KRER

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and LHMFK were 328.1 ±3.5 and 72.9 ±1.3 μM, respectively. Some ACE inhibitory peptides derived

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from Pelodiscus sinensis egg have been reported. The peptides were IVR, IVRDPNGMGAW,

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DPNGMGAW AKLPSW and ILTKQDPYHGPF with the IC50 values of 0.8 μM, 4.4 μM, 14.4 μM,

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15.3 μM, and 231.7 μM, respectively, which differ greatly from the two novel peptides KRER and

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LHMFK.12–14 This may be due to the differences in protein sources and digestive enzymes. Moreover,

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the IC50 values of two novel peptides were within 0.3–1500.0 μM, which is the rang of IC50 values of

224

ACE inhibitory peptides derived from foodstuffs.24 Thus, KRER and LHMFK have the prospects for

225

application in preventing hypertension and for therapeutic purposes.

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To our knowledge, these two peptides have never been reported to be ACEI peptides before in the

227

literature by searching in different peptides databases, including AHTPDB, BIOPEP and EROP-

228

Moscow (last search on March 26th, 2018).

229

Structure-activity correlations of ACEI peptides. One of the hydrophobicity trends of the 20

230

amino acids was as follows: F > L = I > W = Y > V > M > P > C > A > G > T > S > K > Q > N > H > 11

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E > D > R.9 Most of the ACEI peptides from nature are composed of 2–20 amino acid residues and

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possess hydrophobic amino acids residues at the C-terminal, however, the active peptides in this work

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both possessed hydrophilic amino acids residues at the C-terminal. The positive charge of the amino

234

acids R and K are the relative factors contributing ACE inhibitory activities.27

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Table 2 shows part of the purified ACEI peptides possessing similar C-terminals to KRER and

236

LHMFK. Log P values of the peptides, the logarithm (base 10) of the oil-water partition coefficient

237

(P), which can be calculated in silico by ACE/ChemSketch (Freeware 2017.2.1), have also been listed

238

out. Obviously, for the ACEI peptides purified in this work, Log P is correlated with the inhibitory

239

activity. LHMFK (1.45) assessing the hydrophobic character showed much stronger inhibitory

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activity against ACE than KRER (-4.37) possessing the hydrophilic character. But the Log P value is

241

not always proportional to ACE inhibitory activity, e.g. the IC50 value of APER (-2.72), ER (-2.38)

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and PER (-2.66) shown in Table 2 are all higher than KRER, and the ACE inhibitory activity of FK

243

(0.28) is stronger than VFK (1.36). Besides, the IC50 values of LHMFK, FK, and VFK were in a wide

244

range despite possessing the same dipeptidyl sequence, and the length of the peptides may be one of

245

the main reasons for the differences in ACE inhibitory activities of them.

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Determination of inhibitory pattern of ACEI peptides. The inhibition patterns of KRER and

247

LHMFK against ACE were estimated by Lineweaver–Burk plot analysis. Both peptides exhibited

248

noncompetitive inhibition pattern, as shown in Figure 4. A great many of ACEI peptides purified

249

from food origin acted in noncompetitive manner. The peptide AKLPSW from SST yolk exhibited a

250

noncompetitive pattern towards ACE, while the other two active peptides IVRDPNGMGAW and

251

IVR from egg white exhibited a competitive pattern.12–14

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The Vmax values decreased in a dose-dependent pattern for KRER and LHMFK, whereas less

253

change was observed in the Km value, as shown in Table S1 of Supporting Information. According to 12

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the Michaelis–Menten model, a noncompetitive inhibitor is marked by the Vmax value but exerts a

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minimum effect on Km. Noncompetitive inhibitors interact with the non-catalytic site of the enzyme

256

and combine with the enzyme to produce an enzyme–inhibitor complex regardless of whether the

257

substrate is bound.28 The Ki value of KRER (1.11 mM) is about 9 times of LHMFK (0.12 mM),

258

indicating the stronger affinity of LHMFK to ACE, which is in line with the better inhibitory activity

259

of LHMFK.

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Simulated GI tract digestion of ACEI peptides. Some of the ACEI peptides fail to show the

261

hypotensive activity after oral administration in vivo, and the stability against GI enzymes needs

262

evaluation. The stability of ACEI peptides during a simulated GI digestion were predicted by using

263

Peptide Cutter, a useful and efficient tool for predicting potential cleavage sites of the proteases in

264

given peptides in silico22, 28. The peptide KRER would be cleaved by trypsin into the di-peptide ER,

265

while the peptide LHMFK would be cleaved into tetra-peptide LHMF by chymotrypsin (high

266

specificity), and di-peptides HM and FK by chymotrypsin (low specificity) and pepsin.

267

As shown in Table 2, the IC50 values of ER and FK are 667.0 μM and 265.4 μM, both of which are

268

larger than the peptides KRER and LHMFK, respectively. The degradation of the peptides may lead

269

to the decreased ACE inhibitory activities, but the in vivo studies of the stability of the peptides in

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animal model are required to certificate the judgement. The resistance of the peptides against the

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digestive proteases is fatal for the development as food ingredients, for the weakened activity in vivo

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may interfere the bioavailability of the peptides.

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Determination of toxicity of ACEI peptides using in silico method. In recent decades, peptides

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have been developed as potential therapeutic molecules against many diseases, while toxicity has

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become one of the bottlenecks in the development of peptide-based functional food/drugs. ToxinPred

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has been proved to be a useful in silico method to predict toxicity of peptides/proteins.29 KRER, 13

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LHMFK and their possible GI digestive products ER, LHMF and FK were analyzed to estimate their

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potential toxicity by using ToxinPred. The support vector machine (SVM) score of them were -0.71,

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-0.82, -0.80, -0.81, and -0.80, respectively, where the negative value reflects nontoxic.30 The results

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suggested that the two peptides had the potential to be used as a potent natural ingredient for the

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manufacture of functional foods or pharmaceuticals against for hypertension treatment.

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Binding of the active peptides to ACE characterized by ITC. ITC method has been thoroughly

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used to study the binding mechanism between some inhibitors and ACE.31, 32 The ITC diagrams of

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the active peptides KRER and LHMFK binding to ACE were shown in Figure 5. Both of the reaction

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equilibrium was achieved at the 10th injection. The fitting curve of the integrated injection heat versus

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the injection number is like a typical “S” curve and has been fitted to the independent binding model

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which means that there existed a set of identical peptide binding sites on ACE (Other binding models

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including multiple sites, dimer dissociation, and cooperative have also been explored, but the

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independent model has the best fit).33

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The upward ITC titration peaks indicated that the interaction between the active peptides and ACE

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was an endothermic reaction. The thermodynamic parameters from the ITC experiments were all

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summarized in Table 3. The ΔG value of KRER are mainly contributed by -TΔS, indicating that the

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reaction was mainly driven by entropy. For the active peptide LHMFK, the ΔH and -TΔS values are

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almost the same, indicating that the reaction was both enthalpically and entropically driven. The

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positive entropy change (ΔS) can be explained by a strong hydrophobic effect, which means that the

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interactions between apolar groups from inhibitors and ACE requiring the dehydration.34 KRER

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possessing strong hydrophilic character cannot interact with the active site of ACE fluently. For

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forming the stable complex with ACE, the interfacial water is transferred to the bulk solvent, and the

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entropy change is obtained. However, the hydrophobic groups of KRER is hard to interact with ACE, 14

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proved by the phenomenon that the corrected heat value was too little to detect at the low

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concentration of KRER in the syringe (data not shown). Only by increasing the concentration of

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KRER, the typical ITC profile could be obtained. LHMFK, possessing some typical characters of

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ACEI peptides, may interact with ACE by hydrophobic effect, electrostatic interactions and some

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other actions derived from conformation change which need further exploring.

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The dissociation constant Kd values can reflect the binding affinity of the active peptides and

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ACE.31 The Kd value of KRER is about 8 times of LHMFK, which means that LHMFK could form

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more stable complex with ACE. This is well accordance with the Ki value derived from enzyme

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kinetics study, also showing the stronger affinity of LHMFK to ACE.

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In summary, this is the first investigation of the ACE inhibitory activities of the papain hydrolysates

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from Carapax Trionycis. Two novel noncompetitive inhibitory peptides KRER and LHMFK were

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purified successively by sequential chromatographic separation. Both enzyme kinetics and ITC assay

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confirmed that LHMFK could bind more tightly to ACE. Combining enzyme kinetics and ITC

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experiments in present work to study the interactions mechanism between the inhibitor and the

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enzyme open up a new way for elucidating the in vitro inhibitory mechanism. More comprehensive

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and thorough research on the interaction mechanism is in progress and will be reported. In silico study

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predicted that the peptides in this work were not stable against the digestive enzymes. However, in

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silico study is not enough to prove the stability and effectiveness of the peptides in vivo, and the

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further study investigating the bioavailability of the peptides after ingestion in animal models is

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required. The results obtained in this work demonstrated the potential of Carapax Trionycis for

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generation of ACEI peptides, and have broaden up its application in nutraceuticals against

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hypertention and other related diseases.

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Supporting information 15

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Kinetics Constants of ACE catalyzed reaction at different concentrations of the active peptides.

324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 16

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Funding

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This work was supported by National Natural Science Foundation of China (51372043), Natural

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Science Foundation of Guangxi Province (2017GXNSFDA198052, 2017GXNSFBA198215,

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2017GXNSFAA198289) and Project of Guangxi Department of Education (KY2016YB206,

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KY2016YB035).

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Figure captions: Figure 1. Chromatography of fraction A on Hiprep Q FF 16/10 and the ACE inhibitory activities of the fractions A1–A5 (upper panel). Different letters (a–d) above the bar represents the significant differences (p < 0.05). Figure 2. Chromatography of the fraction A2 by using semi-prep HPLC on ODS-A column and the ACE inhibitory activities of fractions A21–A28 (upper panel). Different letters (a–d) above the bar represents the significant differences (p < 0.05). Figure 3. Characterization of molecular mass and amino acid sequences of the purified peptides from Carapax Trionycis hydrolysate: (a) MS/MS spectrum of molecular ion m/z 587.4 Da of fraction A211 and (b) m/z 674.3 Da of fraction A22-1. Figure 4. Lineweaver–Burk plot for the determination of the inhibitory pattern of the active peptides (a) KRER and (b) LHMFK purified from Carapax Trionycis hydrolysate. The ACE activities were measured in the absence (control) or presence of the purified peptides. ●, absence of the active peptides; ▲, 256 μM for KRER and 148 μM for LHMFK; ■, 1022 μM for KRER and 296 μM for LHMFK. 1/V and 1/S represent the reciprocals of velocity and substrate, respectively. Figure 5. ITC diagram of ACEI peptides binding to ACE. (a) KRER; (b) LHMFK. The upper panel shows the corrected heat data of calorimetric titration of the ACEI peptides binding to ACE, and the lower panel shows the integrated injection heat from the upper panel. The integrated heat data were fit to an independent model.

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Table 1. Optimum Conditions of Enzymatic Hydrolysis for Various Enzymes enzyme pH 7.0 7.0 8.0 6.0 9.5 2.0

papain neutral protease trypsin bromelain alcalase pepsin

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optimum conditions temperature (°C) 37.0 50.0 37.0 45.0 45.0 37.0

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Table 2. IC50 Values of ACEI Peptides from Various Sources sources antarctic krill (Euphausia superba)24 this study bromelain digest of trevally proteins5 pepsin and pancreatin digest of pork meat35 skeletal muscle gated chloride channel35 soybean treated by protease from Bacillus subtilis36 soybean protein hydrolysate by protease D337 this study trypsin digest of sweet-potato tuber defensin38 milk39

sequences of the ACEI peptides VFER KRER APER ER PER PGTAVFK YVVFK LHMFK FK VFK

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IC50 (μM)

Log P

152.8 324.1 530.2 667.0 > 1000.0 26.5 44.0 75.6 265.4 1029.0

-0.19 -4.37 -2.72 -2.38 -2.66 -0.31 2.76 1.45 0.28 1.36

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peptides KRER LHMFK

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Table 3. Thermodynamic Parametres for ACEI Peptides Binding to ACE Ka (1/µM) Kd (µM) -TΔS (KJ/mol) ΔH (KJ/mol) ΔG (KJ/mol) 0.05 19.36 -24.34 -2.55 -26.89 0.41 2.42 -15.88 -15.63 -31.51

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Figure 1.

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Figure 2.

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Figure 3.

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Figure 4.

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Figure 5.

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Table of Contents Graphic (TOC)

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