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Successive digestion of tilapia collagen by serine proteinase and proline specifc endopeptidase to produce novel angiotensin I-converting enzyme inhibitory peptides

  • Corresponding author: Minjie Cao, mjcao@jmu.edu.cn
  • Received Date: 2019-08-24
    Accepted Date: 2019-10-08
    Published online: 2020-04-14
  • Edited by Xin Yu.
  • Serine proteinase, purified from the hepatopancreas of Pacific white shrimp (Litopenaeus vannamei), was used to hydrolyze acid solubilized collagen (ASC) isolated from Nile tilapia (Oreochromis sp.) skin to produce angiotensin I-converting enzyme (ACE) inhibitory peptides (ACEIPs). A series of column chromatography assays were used to separate the ACEIPs. A peptide, NPARTCR, was isolated as it exhibited high ACE inhibition potential. Further digestion of this peptide by a proline specific endopeptidase (PSEP), produced a pentapeptide ARTCR with ACE inhibitory activity (IC50) of 77.0 μmol/L. Both NPARTCR and ARTCR inhibited ACE in a non-competitive manner. An in vivo study in rats demonstrated that ARTCR has ACE inhibitory activity via lowering systolic blood pressure in spontaneously hypertensive rats (SHRs). These results suggest that processing by-products from shrimp and tilapia are ideal raw materials for the production of serine proteinase and collagen, respectively. Serine proteinase and collagen are both ideal raw materials that can be used to derive ACE inhibitory active peptides against hypertension.
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Successive digestion of tilapia collagen by serine proteinase and proline specifc endopeptidase to produce novel angiotensin I-converting enzyme inhibitory peptides

    Corresponding author: Minjie Cao, mjcao@jmu.edu.cn
  • 1. College of Food and Biological Engineering, Jimei University, Xiamen 361021, China
  • 2. Collaborative Innovation Center of Marine Food Deep Processing, Dalian Polytechnic University, Dalian 116034, China
  • 3. Department of Biological and Chemical Engineering, Shaoyang University, Shaoyang 422000, China
  • 4. Faculty of Fisheries, Nagasaki University, Nagasaki 852-8521, Japan

Abstract: Serine proteinase, purified from the hepatopancreas of Pacific white shrimp (Litopenaeus vannamei), was used to hydrolyze acid solubilized collagen (ASC) isolated from Nile tilapia (Oreochromis sp.) skin to produce angiotensin I-converting enzyme (ACE) inhibitory peptides (ACEIPs). A series of column chromatography assays were used to separate the ACEIPs. A peptide, NPARTCR, was isolated as it exhibited high ACE inhibition potential. Further digestion of this peptide by a proline specific endopeptidase (PSEP), produced a pentapeptide ARTCR with ACE inhibitory activity (IC50) of 77.0 μmol/L. Both NPARTCR and ARTCR inhibited ACE in a non-competitive manner. An in vivo study in rats demonstrated that ARTCR has ACE inhibitory activity via lowering systolic blood pressure in spontaneously hypertensive rats (SHRs). These results suggest that processing by-products from shrimp and tilapia are ideal raw materials for the production of serine proteinase and collagen, respectively. Serine proteinase and collagen are both ideal raw materials that can be used to derive ACE inhibitory active peptides against hypertension.

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Abbreviations
  • ACEIP ACE inhibitory peptide
    PSEP Proline specifc endopeptidase
    SHRs Spontaneously hypertensive rats
    SBP Systolic blood pressure
    HBP High blood pressure
    NPARCTR Crude peptide isolate
    ARTCR N-terminal cleavage pentapeptide
Introduction
  • Hypertension, a growing public health problem worldwide, is a major risk factor for cardiovascular disease (CVD) (Sharashova et al. 2019). According to a World Health Organization (WHO) report, hypertensive heart disease causes an estimated 9.4 million deaths, accounting for about 16.5% of total deaths (WHO 2013). Unfortunately, the population with hypertension is rapidly growing. Thus, safe and effective antihypertensive drugs are needed to minimize the number of deaths.

    In pharmacology studies, antihypertensive drugs are classified based on their mechanisms of action, such as angiotensin I-converting enzyme (ACE) inhibitors, beta-blockers, calcium channel blockers, and diuretics (Law et al. 2009). ACE is a carboxydipeptidase involved in the renin-angiotensin system (RAS) and kinin nitric oxide system (KNOS). ACE acts as an exopeptidase that converts angiotensin-Ⅰ to angiotensin-Ⅱ, which is a stronger vasopressor and is responsible for the elevation of blood pressure (Tu et al. 2018). ACE also inactivates bradykinin, a vasodilator peptide (Eriksson et al. 2002). Inhibition of ACE has been regarded as an effective strategy for the prevention and treatment of hypertension. Effective ACE inhibitory synthetic compounds, such as captopril, enalapril, and lisinopril, are all used as drugs. However, there is growing concern about the possible side effect of drugs developed from non-food sources. Therefore, using novel ACE inhibitors derived from food sources is highly desirable. Recent studies have reported enzymatic hydrolysates with ACE inhibitory activity from different marine food sources, including oysters (Zhang et al. 2019), thornback ray (Lassoued et al. 2015), and sea cucumber Stichopus horrens (Forghani et al. 2016). Processing by-products from abalone gonads have also been used as a potential source of ACE inhibitory peptides (ACEIP) preparation (Wu et al. 2015a, b).

    Efficient utilization of raw materials from aquatic resources has been an important topic for several decades. Nile tilapia (Oreochromis sp.), is widely cultured in Asian countries. In 2017, its production in China was 1.6 million tons (Anonymous 2018). Processing of tilapia produces a massive amount of by-products, such as fish skin, which is rich in collagen. Hydrolysis of collagen has been widely used for the production of bioactive peptides, including ACE inhibitory peptides (Choonpicharn et al. 2015).

    The choice of proteinase is crucial for bioactive peptide preparation. It is feasible to produce various peptides by digestion of collagen with a combination of different proteinases. Even though different types of proteinase are commercially available and have been used for digestion of collagen into peptides (Choonpicharn et al. 2015), production of new peptides is becoming increasingly difficult. Thus, the selection of proteinases is critical in the preparation of novel bioactive peptides.

    Pacific white shrimp (Litopenaeus vannamei) is one of the most common commercial shrimp species in the world. In 2017, its production in China reached 1.7 million tons (Anonymous 2018). Such massive production leads to a considerable amount of waste during processing. One study revealed that proteolytic enzymes from the hepatopancreas of northern shrimp (Pandalus eous) exhibited collagen or gelatin degradation ability (Aoki et al. 2003). To date, the potential for using shrimp processing by-products as an enzyme resource for industrial applications has not been documented.

    In the present study, serine proteinase was purified to homogeneity from the digestive gland of Pacific white shrimp and used for collagen degradation. ACE inhibitory peptides were purified and further characterized. In addition, the potential influence of proline specific endopeptidase (PSEP) on ACE inhibitory activity of peptides during digestion was investigated. Our present study aimed to provide a reference for the industrial recycling of proteinase from marine processing byproducts and its potential application in the preparation of bioactive peptides with therapeutic potential for high blood pressure (HBP) treatment.

Results and discussion

    Purification of serine proteinase

  • Hepatopancreas from fresh shrimp was used as a raw material for serine proteinase purification. The enzyme was purified to homogeneity by ammonium sulfate fractionation followed by a series of column chromatography assays. Active enzyme peak was observed at 0.4 mol/L NaCl during linear gradient elution on the DEAE-Sepharose column (Fig. 1a). The most positive peak was pooled, concentrated by membrane filtration, and further purified by passing through Sephacryl S-200 HR gel filtration (Fig. 1b) and HiTrap DEAE-Sepharose Fast Flow column (Fig. 1c). The whole purification process resulted in a purification fold of 23.5 and a yield of 6.4%, respectively (Table 1). The enzyme revealed a single band with a molecular weight of approximately 28 kDa on SDS-PAGE and a single active band on gelatin zymography (Fig. 1c), suggesting the target protein was highly purified.

    Figure 1.  Column chromatography purification of trypsin from Pacific white shrimp. a DEAE-Sepharose chromatography (4 ml per tube). b Sephacryl S-200 HR from DEAE-Sepharose chromatography (2 ml per tube). c DEAE-Sepharose Fast Flow column from Sephacryl S-200 HR (2 ml per tube). (dashed line) Spectrometry absorbance at 280 nm; (filled circle) Boc-Phe-Ser-Arg-MCA hydrolyzing activity. Fractions under the bars were pooled. The SDS-PAGE and Gelatin zymography of finally purified trypsin are shown in the inset of c. Lane M, Protein marker; Lane 1, SDS-PAGE of purified trypsin; Lane 2, Gelatin zymography of purified trypsin. The gels were stained with Coomassie Brilliant Blue

    Purification steps Total protein (mg) Total activity (U) Specific activity (U/mg) Purification (fold) Yield (%)
    Crude enzyme 2823 188, 667 66.8 1.0 100
    Ammonium sulfate precipitation 258 101, 172 392.8 5.9 53.6
    DEAE sepharose fast flow 61.9 27, 011 436.4 6.5 14.3
    Sephacryl S-200 HR 44.0 26, 141 594.1 8.9 13.8
    HiTrap DEAE Sepharose fast flow 7.7 12, 103 1572 23.5 6.4

    Table 1.  Purification of serine proteinase from Pacific white shrimp

  • N-terminal amino acid sequence of the serine proteinase and peptide mass fingerprinting

  • The N-terminal amino acid sequence of the purified serine proteinase was determined to be IVGGTDAKPGELPYQLSFQDI, sharing 95.2%, 76.2% and 95.2% identities to trypsinogen 1 (JQ277721.1), trypsinogen 2 (JQ304272.1), and trypsin (CAA60129) from Litopenaeus vannamei, respectively. High identity to trypsins from Panulirus argus (ADB667115) (95.2%) and Fenneropenaeus chinensis (ACQ45455) (90.4%) were also obtained (Fig. 2a). The purified protein was further analyzed by MALDI-TOF/TOF mass spectrometry. Several peptide fragments were observed in the m/z range of 800-4000 Da, which was compared to the NCBI non-redundant protein sequence database. Subsequently, peaks having signal-to-noise ratios (SNR) > 50 were analyzed by MS/MS. Four peptide fragments with 89 amino acid residues in total were obtained (Fig. 2b), which were 100% identical to trypsinogen (JQ277721.1) from Pacific white shrimp (Litopenaeus vannamei), strongly suggesting the purified enzyme is a trypsin (Fig. 2c). The enzyme was active at a broad alkaline pH range from 7.0 to 12.0 with an optimum temperature of 40 ℃ and pH of 9.0 (data not shown).

    Figure 2.  Sequence analysis of Pacific white shrimp trypsin. a Alignment of the N-terminal amino acid sequences of trypsin from different species. The trypsin sequence of Pacific white shrimp was compared with those of Litopenaeus vannamei (tryp 1 JQ277721.1; tryp 2 JQ304272.1; Try CAA60129), Panulirus argus (ADB66715), Fenneropenaeus chinensis (ACQ45455), Marsupenaeus japonicus (ACE80257), and Pontastacus leptodactylus (AAX98287). A chart of the amino acid sequences is shown in the box. b The mass spectrometry (MS/MS) map of the trypsin and active peptides from MS compared with standard data in NCBInr. c Protein sequence alignment of the target serine proteinase (trypsin) was carried out, followed by comparison with the complete sequence of trypsin from Pacific white shrimp (Litopenaeus vannamei). Identical amino acid residues are shown in black shadow

  • Preparation of enzymatic hydrolysate

  • Recently, different methods were proposed to enable a better understanding of the utilization of aquatic processing by-products. The most recognized one is enzymatic hydrolysis, which is used to produce bioactive peptides. Acid-solubilized collagen (ASC) hydrolysates were obtained by treatment with trypsin, purified from the hepatopancreas of Pacific white shrimp. Gel filtration chromatography analysis showed that small peptides (< 3 kDa) occupied 99.7% in the trypsin hydrolysate (data not shown), suggesting enzymolysis by trypsin can be initially used to break down collagen into peptides. The prepared hydrolysate revealed its ACE inhibitory activity with an IC50 value of 2.34 mg/ml.

  • Separation and purification of the ACE inhibitory peptide

  • Ultrafiltration is a fast, simple, and reliable way for an enrichment of bioactive peptides and is suitable for industrial-scale applications (Yan et al. 2019). In the separation and purification steps, ASC hydrolysates were fractionated according to their molecular weights by ultrafiltration, obtaining fractions < 3 kDa, and > 3 kDa. The permeate fraction (< 3 kDa), which represented low molecular weight peptides, revealed 74.3% ACE inhibitory activity, which was higher than that (58.5%) of the retentive fraction (> 3 kDa). Thus, ultrafiltration could be used as a processing technique to generate a crude pool of bioactive peptides. The low molecular weight fraction (< 3 kDa) was collected for further assay.

    The permeate fraction was lyophilized and loaded to SP-Sepharose (Fig. 3a). The major active peak was pooled and further purified by passing through Sephadex G-15 gel filtration (Fig. 3b). The fractions that revealed highest ACE inhibitory activity (84.7% inhibition) were lyophilized for further purification by reverse-phase high-performance liquid chromatography (RP-HPLC), using a Zorbax SB-C18 column. Figure 3c displays active fraction, separated into 14 peaks (designated as fractions a-n), with most fractions revealing ACE inhibitory activity. Among them, fraction g (Fig. 3d) was the most potent, with an IC50 value of 532.5 μg/ml, thus was further characterized.

    Figure 3.  Purification of ACE inhibitory peptides. a SP-Sepharose purification of ACE inhibitory fractions (4.0 ml per tube). b Sephadex G-15 gel filtration purification of active fractions (1.8 ml per tube). c Purification chromatogram of active fractions. d ACE inhibitory activity of each fraction from Reversed-phase HPLC

  • Identification and synthesis of the ACE inhibitory peptides

  • Peptide sequence using mass spectrometry showed fraction g was not composed of a single peptide, but three peptides, having amino acid sequences NPARTCR, GEAGTPGENGTPGAMGPR, and GEAGTPGENGTPGAMGPRGLPGER (Fig. 4). When searching in our protein database, we discovered all three peptides were identical to Nile tilapia (Oreochromis niloticus) collagen alpha-1 (I) chain precursor (NP_001266373.1), strongly suggesting these three peptides are derivatives of tilapia collagen.

    Figure 4.  Peptide profile of Fraction g performed by mass spectrometry analysis and the peptide sequences

    Gastrointestinal hydrolysis is of particular importance in the bioavailability of ACE inhibitory peptides. After oral intake, peptides with high molecular weights may be easily digested by gastrointestinal enzymes. Studies have shown that ACE inhibitory peptides are mainly associated with low molecular weight peptides (Barbana and Boye 2011; He et al. 2013; Yu et al. 2006). Therefore, comparing with the other two peptides (GEAGTPGENGTPGAMGPR and GEAGTPGENGTPGAMGPRGLPGER), the heptapeptide NPARTCR would be more resistant against digestion in the gastrointestinal tract and is thus synthesized and used for following experiments. The ACE inhibitory activity of the synthesized NPARTCR was determined with IC50 value of 61.4 μmol/L.

    On the other hand, in our previous study, a proline specific endopeptidase (PSEP) was purified to homogeneity from the skeletal muscle of common carp (Wang et al. 2012). We identified this endopeptidase as having a unique ability to hydrolyse peptide bonds on the carboxyl side of a proline residue, consistent with properties of such enzymes (Gass and Khosla. 2007). PSEP was characterized as a unique serine proteinase that contains a peptidase domain with an alpha/beta hydrolase fold. Its catalytic triad (Ser-554, His-680, Asp-641) is shielded by the central tunnel of an unusual beta propeller (Fulop et al. 1998). Further digestion of the heptapeptide NPARTCR by PSEP produced a pentapeptide ARTCR and a dipeptide NP. Both peptides, ARTCR and NP, were then synthesized for biological activity assay. The pentapeptide ARTCR showed ACE inhibitory activity with IC50 of 77.0 μmol/L, while the dipeptide NP did not reveal any inhibitory activity, indicating the two amino acid residues (Asn-Pro) in the N-terminal of NPARTCR did not contribute to ACE inhibition.

    Biological functions of peptides are closely related to their sequences. Several reports suggest peptides with high ACE-inhibitory activity contain alphatic amino acid(s) at the N-terminal. Two examples of such peptides have been identified in cuttlefish muscle (Ala-Phe-Val-Gly-Tyr-Val-Leu-Pro, IC50 of 18.02 μmol/L) (Balti et al. 2015) and hen egg white lysozyme (Ala-Met-Lys, IC50 of 2.8 μmol/L) (Rao et al. 2012). In the present study, ARTCR exhibited higher ACE inhibitory activity with IC50 of 77.0 μmol/L. Though its inhibitory activity was lower than peptides derived from cuttlefish and egg white, its inhibitory activity was higher than a peptide (Ala-Met-Asn) from abalone gonads (IC50 of 378.7 μmol/ml) (Wu et al. 2015a, b). All these peptides share the same amino acid residue Ala at the N-terminal. In contrast, peptides MNPPK, VPAAPPK, and PPK, which share similar peptide sequence at their C-terminals, possess far different inhibition capacities with IC50 values of 945.5, 0.45 and > 1000 μmol/L, respectively (Forghani et al. 2016). In the present study, ARTCR, an N-terminal truncated form of NPARTCR, showed better ACE inhibitory activity. This inhibitory effect may be because ARTCR has alphatic amino acids at the N-terminal, which promotes its ACE inhibitory activity.

  • Kinetic study of ACE inhibition

  • Figure 5 shows Lineweaver-Burk plots, obtained by examining the effect of NPARTCR and ARTCR on ACE inhibition as a function of peptide concentration. Lines with the coinciding intercept at the x axis were generated, indicating both NPARTCR and ARTCR are non-competitive inhibition types. The inhibition constant values are shown in the inner table of Fig. 5. The Km of the inhibitors did not change, but Vmax decreased. This suggests the peptides were unable to bind to the active site of ACE but may instead bind to other sites on the ACE molecule to produce an inactive complex, irrespective of substrate binding. It has been reported that peptide TPTQQS, which showed non-competitive inhibition pattern, could bind to the catalytic area of ACE, as well as, chelate zinc ion away from the ACE active site (Ni et al. 2012). In the present study, the ACE inhibitory peptides NPARTCR and ARTCR exhibited a non-competitive inhibition, which could be attributed to the same potent amino acid sequence (Ala-Arg-Thr-Cys-Arg) while the two amino acid residues (Asn-Pro) in the N-terminus of heptapeptide (Asn-Pro-Ala-Arg-Thr-Cys-Arg) are unnecessary.

    Figure 5.  Kinetics study of the inhibitory activity of peptides against ACE. Lineweaver-Burk plot analysis of the ACE inhibitory activity was determined in the absence or presence of different concentrations of the peptides. a NPARTCR: 120 μmol/L (purple colored open triangle), 90 μmol/L (green colored filled triangle), 60 μmol/L (red colored open circle), 0 μmol/L (blue colored filled circle), using value of 1/v against 1/[S]; b ARTCR: 150 μmol/L (purple colored open triangle), 100 μmol/L (green colored filled triangle), 60 μmol/L (red colored open circle), 0 μmol/L (blue colored filled circle), using value of 1/v against 1/[S]

    It is of interest to notice that ACE inhibitory peptides (ACEIPs) isolated from porcine hemoglobin (Yu et al. 2006) and small red bean (Rui et al. 2013) fit the competitive inhibition pattern. In contrast, peptides obtained from lentil protein hydrolysates exhibited non-competitive inhibition (Barbana and Boye 2011). Furthermore, ACEIPs isolated from rapeseed hydrolysates exhibited uncompetitive inhibition (Mäkinen et al. 2012), suggesting that ACEIP isolated from different sources has the potential to display different substrate inhibition patterns, despite having inhibitory properties.

  • Antihypertensive effect of ACE inhibitory peptides on SHRs

  • Antihypertensive activity of NPARTCR and ARTCR were evaluated by measuring the change in systolic blood pressure (SBP) at 0, 2, 4, 6, 12 and 24 h after oral administration of 10 mg/kg of body weight. In the negative control group, no significant changes were found in the SBP 24 h after administration (Fig. 6). Administration of NPARTCR, however, also did not cause a decrease in SBP during the investigation period. This suggests that gastrointestinal enzymes had further digested NPARTCR in the digestive tract producing smaller peptides or amino acids that had no antihypertensive effect. In vitro digestion tests were performed and the results revealed that NPARTCR could be further cleaved by trypsin to form two peptides, NPAR and TCR, and completely lose its ACE inhibitory activity. On the other hand, after oral administration of ARTCR, SBP clearly decreased and was maintained for about 12 h which is comparable to the positive control captopril (Fig. 6). The maximal decrements in SBP after oral administration of ARTCR and captopril were 25 and 31 mmHg at 4 h, respectively. Blood pressure gradually recovered in 24 h for all groups. Furthermore, no allergic reactions or coughing were observed on the day of the experiment or the following day. The results showed that pentapeptide ARTCR had a substantial effect on reduction of SBP in SHRs, suggesting it could be directly absorbed in the digestive tract.

    Figure 6.  Time course of oral NPARTCR and ARTCR administration in SHR. Captopril was used as a positive control. Single oral administration of the drug was performed with a dose of 10 mg/kg of body weight, and SBP was measured at 0, 2, 4, 6, 12 and 24 h after administration. Indicates significant difference between test and control, P < 0.05

Conclusions
  • A serine proteinase from the hepatopancreas of Pacific white shrimp was prepared and its ability to hydrolyze tilapia collagen for bioactive peptide production was tested. After screening the hydrolysates, a novel peptide NPARTCR was recovered from which an active pentapeptide, ARTCR, was derived, after proline specific endopeptidase (PSEP) digestion. ARTCR exhibited a non-competitive substrate inhibition with an IC50 value of 77.0 μmol/L. Thus, serine proteinase recovered from the by-products of Pacific white shrimp is a promising enzyme for the production of ACE inhibitory peptides. Administration of the active form of pentapeptide ARTCR to spontaneously hypertensive rats significantly decreased their systolic HBP to an extent comparable to the experimental captopril positive control, suggesting ARTCR has a therapeutic potential for HBP treatment.

Materials and methods

    Materials and chemicals

  • Nile tilapia (Oreochromis sp.) skin was freshly obtained from a local fish processing factory. Fresh Pacific white shrimp (Litopenaeus vannamei) were purchased from a local market, mixed with ice, and transferred to our laboratory within 1 h. The hepatopancreas from each sample was collected and immediately examined. DEAE-Sepharose, SP-Sepharose, Sephadex G-15, HiTrap DEAE-Sepharose Fast Flow and Sephacryl S-200 HR were purchased from GE Healthcare (Piscataway, NJ, USA). Fluorogenic peptide substrate t-Butyloxy-carbonyl-Phe-Ser-Arg-4-methyl-coumaryl-7-amide (Boc-Phe-Ser-Arg-MCA) and other MCA substrates were from Peptide Institute, Inc. (Osaka, Japan). ACE, captopril, hippuryl-histidyl-leucine (HHL), and proline specific endopeptidase were purchased from Sigma-Aldrich Corp. (St Louis, MO, USA). Protein markers for SDS-PAGE were from Bio-Rad Corp. (Richmond, VA, USA). Three peptides were synthesized by Cellmano Biotech Corp (Hefei, China)—peptides NP, with purity of 88.55%; NPARTCR with purity of 95.99%; and ARTCR with purity of 98.23%. All reagents used were of analytical grade.

  • Purification of serine proteinase

  • The hepatopancreas of Pacific white shrimp (18 g) were homogenized in 4 folds of ice-cold 20 mmol/L Tris-HCl buffer (pH 8.0) using a homogenizer (Kinematica, PT-2100, Switzerland) then centrifuged at 10, 000 g for 20 min in a centrifuge (Beckman Coulter, Avanti J-25, USA). The supernatant was collected as crude enzyme and subjected to ammonium sulfate from 40 to 80% (w/v) saturation. After centrifugation at 12, 000 g for 30 min, the resulting precipitate was subsequently dissolved in a minimum volume of 20 mmol/L Tris-HCl buffer (pH 8.0) and extensively dialyzed against the same buffer. All procedures were performed at 4 ℃.

    The dialyzed fraction was then applied to a DEAE-Sepharose column (2.6cm ×12 cm). After washing, binding proteins were eluted with a linear gradient of NaCl from 0 to 0.5 mol/L in 20 mmol/L Tris-HCl buffer (pH 8.0). Active fractions were pooled and concentrated by ultrafiltration using a membrane of YM-10 (Millipore, MA, USA). The concentrated sample was then applied to a Sephacryl S-200 HR gel filtration column (1.5cm ×98 cm) and subsequently pre-equilibrated with 20 mmol/L Tris-HCl buffer (pH 8.0) containing 0.2 mol/L NaCl. Active fractions were pooled and extensively dialyzed against 20 mmol/L Tris-HCl buffer (pH 8.0) and applied to HiTrap DEAE-Sepharose Fast Flow column (1 ml), connected with AKTA purifier (GE Healthcare, Piscataway, NJ, USA). Binding proteins were eluted with a linear gradient of 0-0.5 mol/L NaCl. Active fractions were then collected as purified serine proteinase and used for experiments.

  • Assay of enzyme activity

  • To monitor the target enzyme during purification, enzyme activity was routinely assayed using the fluorogenic substrate Boc-Phe-Ser-Arg-MCA. The reaction was initiated by adding 50 μl of 10 μmol/L substrate to a solution containing 900 μl of 0.1 mol/L Tris-HCl buffer (pH 8.0) and 50 L of the appropriately diluted enzyme. After incubation at 37 ℃ for 10 min, 1.5 ml of stopping solution (methyl alcohol: isopropyl alcohol: distilled water, 35:30:35, v/v) was added to terminate the reaction. The fluorescence intensity of the released 7-amino-4-methyl-coumarin (AMC) was measured by a fluorescence spectrophotometer (JASCO, FP-8200, Japan) at the excitation wavelength (Ex) of 380 nm and emission wavelength (Em) of 450 nm. One unit (U) of proteinase activity was defined as the amount of enzyme to release one nmol of AMC per min.

  • SDS-PAGE and gelatin zymography

  • Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out to determine the purity and molecular mass of the protein. The purity of the proteinase was also evaluated by gelatin zymography. Samples were mixed with a quarter of SDS sample buffer (200 mmol/L Tris-HCl, pH 6.8 containing 8% SDS, 0.4 bromophenol blue and 40% glycerol) then applied to 12% polyacrylamide gel with 1 mg/ml gelatin in the gel and electrophoresed at 4 ℃. After electrophoresis, the gel was washed with 2.5% (v/v) Triton X-100 for 30 min by gently shaking to remove SDS, followed by rinsing with deionized water. The gels were then incubated at 37 ℃ for 13 h in 50 mmol/L Tris-HCl, pH 8.0 (buffer B) and stained with Coomassie Brilliant Blue R-250 (CBB). The enzyme active site appeared as a clear band on the CBB-stained dark blue background. The brightness of the band is in positive correspondence to enzyme activity.

  • Characterization of purified serine proteinase

    N-terminal amino acid sequence assay
  • For the determination of N-terminal amino acid sequence, purified protein was applied to SDS-PAGE and transferred to a polyvinylidene difluoride (PVDF) membrane. After a brief staining with CBB, the target protein band was excised and submitted to determine the amino acid sequence using a protein sequencer (Shimadzu, PPSQ-33A, Kyoto, Japan).

MALDI-TOF/TOF-MS/MS analysis
  • To obtain the protein primary structure information, purified protein was applied to 12% SDS-PAGE, stained with silver nitrate and the target protein collected for mass spectrometry analysis. MALDI-TOF mass spectra results were obtained using a 4800 Plus MALDI-TOF/TOF-MS/MS Analyzer (Applied Biosystems, Carlsbad, CA, USA) by Shanghai Zhongke New Life Biotechnology Co. Ltd, China.

  • Purification of acid solubilized collagen (ASC) from tilapia skin

  • ASC was extracted according to the method of Yan et al. (2014) with slight modification. Tilapia skin was mixed with tenfold of 0.1 mol/L NaOH for 48 h to remove non-collagenous proteins then washed with distilled water. The samples were defatted with 10% butyl alcohol, followed by washing with distilled water. The insoluble matter was extracted with 0.5 mol/L acetic acid for 72 h then centrifuged at 20, 000g for 30 min. The supernatant was salted out by adding NaCl to a final concentration of 0.9 mol/L, followed by dissolving in 0.5 mol/L acetic acid. The sample was dialyzed against 0.1 mol/L acetic acid for 48 h then lyophilized.

  • Preparation of enzymatic hydrolysate from ASC

  • ASC (1.4 g) was homogenized in tenfold of 20 mmol/L Tris-HCl buffer (pH 8.0) using a PT-2100 homogenizer. Serine proteinase purified from the hepatopancreas of Pacific white shrimp was added to the homogenate with an enzyme dosage to the substrate of 17, 000 U/g ASC. Digestion was carried out at 35 ℃ for 90 min and terminated by heating the reaction mixture at 95 ℃ for 10 min. The collagen hydrolysate was centrifuged at 10, 000g for 20 min to remove insoluble materials. The supernatant was then ultrafiltrated through a 3 kDa molecular weight cutoff (MWCO) membrane (Millipore, Boston, USA) and the ACE inhibitory activity of the filtrate measured.

  • Angiotensin-converting enzyme (ACE) inhibition activity assay

  • ACE inhibitory activity was determined according to the method of Wu et al.(2015a, b) with slight modification. A 50 μl of the substrate (6.5 mmol/L HHL in 50 mmol/L sodium borate buffer containing 0.3 mol/L NaCl, pH 8.3) was mixed with 20 μl of sample. The reaction was initiated by adding 20 μl of ACE solution (25 mU/ml), then the mixture was incubated at 37 ℃ for 60 min. Reaction termination was performed by addition of 50 μl 1 mol/L HCl. The hippuric acid (HA) liberated from HHL after 60 min was extracted with 300 μl of ethyl acetate. After centrifugation (3000 g, 3 min), 200 μl of the upper layer was collected and evaporated at room temperature for 60 min in a vacuum concentration system (Thermo Fisher, Fair Lawn, USA). The HA was then dissolved in 600 μl distilled water and absorbance was measured at 228 nm using UV-visible spectrophotometer (PerkinElmer, Shelton, USA). ACE inhibitory activity was determined according to the formula:

    where A1 indicates the content of HA generated without ACE inhibitor, A2 is the content of HA generated in the presence of ACE inhibitor and A0 is the content of HA generated without ACE. The concentration of the sample required for 50% inhibition of ACE was defined as IC50. Each determination was performed in triplicate.

  • Isolation of ACE inhibitory peptides

  • The sample obtained from lyophilization was dissolved in 20 mmol/L NaAc-HAc buffer (pH 4.0) then applied to a SP-Sepharose column (2.6 cm × 12 cm) pre-equilibrated with 20 mmol/L NaAc-HAc buffer (pH 4.0). Binding peptides were eluted with a linear gradient of NaCl from 0 to 2.0 mol/L in 20 mmol/L NaAc-HAc buffer (pH 4.0). The eluent was monitored at 220 nm and tested for ACE inhibitory activity. The fractions containing ACE inhibitory activity were lyophilized, dissolved in distilled water then applied to Sephadex G-15 gel filtration column (1.5cm ×85 cm), which was eluted with distilled water at a flow rate of 0.8 ml/min. The fraction demonstrating the highest ACE inhibitory activity was collected and further fractionated using a reversed-phase HPLC (RP-HPLC) on a Zorbax SB-C18 column (4.6 mm I.D. × 250 mm, Agilent, USA). The sample was eluted with 0.1% trifluoroacetic acid for 10 min, followed by a linear gradient of acetonitrile containing 0.1% trifluoroacetic acid from 0 to 20% for 50 min at a flow rate of 1.0 ml/min. The separation was monitored by UV-visible spectrophotometry at 220 nm. The resulting purified peptide showing the highest ACE inhibitory activity was collected for mass spectrometry analysis.

  • Identification of the amino acid sequence of ACE inhibitory peptides

  • The sequence of ACEIP was determined using 4800 Plus MALDI-TOF/TOF-MS/MS Analyzer (Applied Biosystems, Carlsbad, CA, USA).

  • Kinetic study of ACE inhibition

  • The inhibition kinetics was conducted as described (Forghani et al. 2016) with slight modifications. Substrate (HHL) concentrations (0, 0.5, 1.0, 2.5, 3.5, 6.5 mmol/L), were incubated with ACE solution in the presence of different concentrations of peptide solution at 37 ℃. The type of inhibition was determined by plotting Lineweaver-Burk graphs of the reciprocal of HA absorbance versus the reciprocal of HHL concentrations. The kinetic parameters, maximum initial velocity (Vmax) and Michaelis-Menten constant (Km), were calculated by the x-axis intercept of the Lineweaver-Burk plot.

  • Antihypertensive action in spontaneously hypertensive rats

  • Spontaneously hypertensive rats (SHRs, 11 weeks old, male, specific pathogen-free, 230-280 g of body weight) with tail-cuff systolic blood pressure (SBP) over 180 mmHg were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). Conscious SHRs were housed individually in steel cages in a room kept at 25 ℃ with a 12 h light-dark cycle and fed a standard laboratory diet and water ad libitum. The SHRs were split into four groups of four rats each. Synthesized peptides (NPARTCR and ARTCR) were dissolved in saline at a dose of 10 mg/kg of body weight and injected orally using a metal gastric zoned in SHR. Antihypertensive activity of NPARTCR and ARTCR were evaluated by measuring change in systolic blood pressure (SBP) at 0, 2, 4, 6, 12 and 24 h after oral administration of 10 mg/kg body weight. The lowering efficacy of two peptides on SBP was compared to that of the commercial antihypertensive drug (captopril, 10 mg/kg), and the same volume of saline solution was administered to the negative control group. Following oral administration, SBP was measured by a tail-cuff method with a Softron BP system (Softron BP-98AL, Tokyo, Japan) after warming up SHRs in a chamber maintained at 37 ℃ for 10 min.

  • Statistical analysis

  • All experiments for activity assays were conducted in triplicate. Statistical analysis was performed using the Statistical Package for Social Science (SPSS, software version 17.0, IBM SPSS, Chicago, IL, USA). The least significant difference test was used to determine the difference between means. A P-value of < 0.05 was taken as the level of statistical significance. The data were expressed as mean±standard deviations.

Acknowledgements
  • This work was sponsored by the National Key R & D Program of China (2018YFD0901004), the National Natural Scientifc Foundations of China (31471640, 31702372).

Authors Contributions
  • XH contributed to the presented idea and design. LS implemented the computational and statistical analysis and took the lead in writing the manuscript. CZ and AY assisted with data analysis. MC and GL supervised the fndings of this work. All authors provided critical feedback and helped to conduct the research, analysis, and preparation of the manuscript.

Compliance with ethical standards

    Conflict of Interest

  • The authors declare that they have no confict of interest.

  • Animal and human rights statement

  • This article does not include results from any study involving human subjects performed by any of the authors. All study procedures that involved animals were in accordance with the ethical standards of the institution or practice where the study was conducted.

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