Purification and characterization of angiotensin I-converting enzyme inhibitory peptide from enzymatic hydrolysates of Styela clava flesh tissue

Process Biochemistry 47 (2012) 34–40

Contents lists available at SciVerse ScienceDirect

Process Biochemistry journal homepage: www.elsevier.com/locate/procbio

Purification and characterization of angiotensin I-converting enzyme inhibitory peptide from enzymatic hydrolysates of Styela clava flesh tissue Seok-Chun Ko a , Jung-Kwon Lee b , Hee-Guk Byun b , Seung-Cheol Lee c , You-Jin Jeon a,∗ a

Department of Marine Life Sciences, Jeju National University, Jeju 690-756, Republic of Korea Faculty of Marine Bioscience and Technology, Gangneung-Wonju National University, Gangneung 210-720, Republic of Korea c Division of Food Science and Technology, Kyungnam University, Masan 631-701, Republic of Korea b

a r t i c l e

i n f o

Article history: Received 12 May 2011 Received in revised form 17 September 2011 Accepted 7 October 2011 Available online 14 October 2011 Keywords: Styela clava Angiotensin I-converting enzyme (ACE) Enzymatic hydrolysate Inhibitory activity

a b s t r a c t Angiotensin I-converting enzyme (ACE) inhibitory peptide was isolated from the Styela clava flesh tissue. Nine proteases (Protamex, Kojizyme, Neutrase, Flavourzyme, Alcalase, pepsin, trypsin, ␣-chymotrypsin and papain) were used, and their respective enzymatic hydrolysates and an aqueous extract were screened to evaluate their potential ACE inhibitory activity. Among all of the test samples, Protamex hydrolysate possessed the highest ACE inhibitory activity, and the Protamex hydrolysate of flesh tissue showed relatively higher ACE inhibitory activity compared with the Protamex hydrolysate of tunic tissue. We attempted to isolate ACE inhibitory peptide from the Protamex hydrolysate of S. clava flesh tissue using ultrafiltration, gel filtration on a Sephadex G-25 column and high performance liquid chromatography (HPLC) on an ODS column. The purified ACE inhibitory peptide exhibited an IC50 value of 37.1 ␮M and was identified as non-competitive inhibitor of ACE. Amino acid sequence of the peptide was identified as Ala-His-Ile-Ile-Ile, with a molecular weight 565.3 Da. The results of this study suggested that the peptides derived from enzymes-assisted extracts of S. clava would be useful new antihypertension compounds in functional food resource. © 2011 Elsevier Ltd. All rights reserved.

1. Introduction Recently, hypertension has become a major problem, and has come to pose a major worldwide threat to human health. Hypertension has been estimated to affect approximately 20% of the world’s adult population [1]. It is the most common serious chronic health problem, because it is a high risk factor for arteriosclerosis, stroke, and myocardial infarction [2]. In cases in which hypertension presents with complications such as cerebral hemorrhage, heart disease, and nephropathy, the lethality of this condition can prove to be quite high [1]. Angiotensin I-converting enzyme (ACE, EC 3.4.15.1) performs a pivotal function in the regulation of blood pressure [3]. ACE is a zinc-containing exopeptidase enzyme discovered in vascular, heart, lung and brain tissue, and cleaves dipeptides at the C-terminus of oligopeptides [4]. ACE converts an inactive form of decapeptide, angiotensin I (AspArg-Val-Tyr-Ile-His-Pro-Phe-His-Leu), to octapeptide angiotensin II (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe), a potent vasoconstrictor, and inactivates bradykinin, which exerts a depressor effect [2,5]. ACE inhibition has been used extensively in therapeutic strategies for

∗ Corresponding author. Tel.: +82 64 754 3475; fax: +82 64 756 3493. E-mail address: [email protected] (Y.-J. Jeon). 1359-5113/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2011.10.005

the prevention and treatment of hypertension, and the literature regarding ACE inhibitory compounds is also rather extensive. Since the discovery of an ACE inhibitor in snake venom, several synthetic ACE inhibitors have been developed, including alacepril, captopril, benazepril, enalapril, fosinopril, ramipril, and zofenopril, all of which are currently extensively used in the treatment of essential hypertension and heart failure in humans [4,6]. However, these synthetic ACE inhibitors are believed to exert certain side effects, including cough, taste disturbances, and skin rashes [7,8]. Therefore, the development of ACE inhibitors from natural products has become a major area of research. Recently, many ACE inhibitory peptides have been isolated from various food proteins such as sheep milk yogurt [9], peanut [10], egg white [11], soybean [12] and yak milk casein [13]. Additionally, some ACE inhibitory peptides have also been reported in certain marine bioresource proteins such as rotifer [3], hard clam [14], tuna frame [15], sea cucumber [16] and skate skin [17]. These peptides are less effective than synthetic ones, but do not exhibit known side effects [18]. Potent bioactive peptides have been produced via the enzymatic hydrolysis of food proteins [19,20]. While these peptides are inactive within the sequence of the parent protein, bioactive peptides can be released via enzymatic hydrolysis during the food manufacturing process [17]. Additionally, enzymatic hydrolysates are also a source of bioactive peptides, which are short peptides released from proteins by hydrolysis that have been shown to exert

S.-C. Ko et al. / Process Biochemistry 47 (2012) 34–40 Table 1 Optimum conditions of enzymatic hydrolysis for various enzymes. Enzyme

Protamex Kojizyme Neutrase Flavourzyme Alcalase ␣-Chymotrypsin Trypsin Papain Pepsin

Optimum conditions pH

Temp. (◦ C)

Buffer

6.0 6.0 6.0 7.0 8.0 8.0 8.0 6.0 2.0

40 40 50 50 50 37 37 37 37

50 mM sodium phosphate 50 mM sodium phosphate 50 mM sodium phosphate 50 mM sodium phosphate 50 mM sodium phosphate 50 mM sodium phosphate 50 mM sodium phosphate 50 mM sodium phosphate 20 mM glycine–HCl

biological effects such as antihypertensive [18], antioxidative [21], and antimicrobial [22] effects. The Styela clava used in this study, a valuable marine resource, is found in Korea and Far East Asia. It is a traditional remedy for healing various internal conditions, and is believed to have profound curative properties. Additionally, in our previous study, S. clava was shown to reduce blood pressure in spontaneously hypertensive rats (SHRs). Several studies of the bioactivities of S. clava, such as its ACE inhibitory activity [23], as well as its antioxidant and anticancer activities [23,24] have been carried out previously. However, the above studies employed solvent extraction methods, and there remains a lack of data regarding the biological activities of S. clava protein by enzymatic hydrolysis. The principal objective of the present study was to attempt an enzymatic hydrolysis technique to prepare enzymatic hydrolysates, such as smaller peptides, from S. clava, and to characterize the purified peptide with regard to ACE inhibitory activity. 2. Materials and methods 2.1. Materials S. clava used was kindly donated by Miduduk Corporated Association (Masan, Korea). Commercial food grade proteases including Protamex, Kojizyme 500 MG, Neutrase 0.8 L, Flavourzyme 500 MG and Alcalase 2.4 L FG were purchased from Novo Co. (Novozyme Nordisk, Bagasvaerd, Denmark). Other proteases containing pepsin, trypsin, ␣-chymotrypsin and papain, as well as angiotensin I converting enzyme (from rabbit lung) and N-Hippuryl-His-Leu tetrahydrate (HHL) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). The other chemicals and reagents used were of analytical grade. 2.2. Approximate chemical composition of S. clava Approximate chemical composition of S. clava was determined according to AOAC method [25]. Crude carbohydrate was determined by phenol–sulfuric acid reaction (absorbance at 480 nm; using glucose as the calibration standard), crude lipid was performed by Soxhlet method and crude ash was prepared at 550 ◦ C in the dry-type furnace. Moisture was determined by keeping the sample in a dry oven and the crude protein was determined by Kjeldahl method. 2.3. Preparation of enzymatic hydrolysates and aqueous extract of S. clava S. clava was washed three times with tap water to remove salt, epiphytes, and sand attached to the surface of the samples. Finally, the sample was carefully rinsed using fresh water and stored at −20 ◦ C. The frozen samples were then lyophilized and homogenized in a grinder prior to hydrolysis. Enzymatic hydrolysates of S. clava were performed according to the method used by Jung et al. [18] and Heo et al. [26] and the different enzymatic hydrolysates were obtained. The hydrolytic enzymes used in preparation of enzymatic hydrolysates, nine proteases (Protamex, Kojizyme, Neutrase, Flavourzyme, Alcalase, pepsin, trypsin, ␣-chymotrypsin and papain) digested the powder of S. clava under the respective optimal conditions for 24 h. One gram of the dried ground S. clava powder was homogenized with buffer (100 ml), and was hydrolyzed with enzymes in a substrate to enzyme ratio of 10:1 (w/w). The pH of the homogenates was adjusted to its optimal pH value before the enzymatic hydrolysis. The enzymatic reactions were performed for 24 h to achieve optimal degree of enzymatic hydrolysis (Table 1). Then the hydrolysates were boiled for 10 min at 100 ◦ C in water bath to inactivate the enzyme reaction. Each enzymatic hydrolysate was clarified by centrifugation (3500 rpm, for 20 min at 4 ◦ C) to remove the residue. And for the preparation of aqueous extract, one gram of the ground dried

35

S. clava powder was homogenized for 24 h with water (100 ml) at room temperature and was clarified by centrifugation (3500 rpm, for 20 min at 4 ◦ C) to remove the residue. The yields of enzymatic hydrolysates and aqueous extract were determined by subtracting the dried weight of the residue from one gram of extracts dried and was expressed as a percentage. Enzymatic hydrolysates and aqueous extract were kept −20 ◦ C for the further experiments. 2.4. ACE inhibitory activity The ACE inhibitory activity was assayed by measuring the concentration of hippuric acid liberated from HHL by the method of Cushman and Cheung [27]. For each assay, a 50 ␮l of the sample solution with 50 ␮l of ACE solution (25 mU/ml) was pre-incubated at 37 ◦ C for 10 min, and then incubated with 100 ␮l of substrate (25 mM HHL in 50 mM sodium borate buffer containing 500 mM NaCl at pH 8.3) at 37 ◦ C for 60 min. The reaction stopped by adding 250 ␮l of 1 N HCl. Hippuric acid was extracted with 500 ␮l of ethyl acetate. Then a 200 ␮l aliquot of the extract was removed by evaporation in a dry-oven at 80 ◦ C. The residue was dissolved in 1 ml distilled water and its UV spectra absorbance was measured at 228 nm. The IC50 value, defined as the concentration required for 50% inhibition of ACE activity, was determined. The ACE inhibitory activity was calculated as follows: Inhibition (%) =

Ac − As Ac − Ab

Ac = absorbance of control sample, As = absorbance of sample solution, and Ab = absorbance of blank solution. 2.5. Purification of ACE inhibitory peptide The enzymatic hydrolysate was fractionated using an ultrafiltration (UF) with 5 and 10 kDa molecular weight cut-off UF membranes (Millipore Corporation, Bedford, MA, USA). Three fractions with molecular weights of <5 kDa (5 kDa or smaller), 5–10 kDa (between 5 and 10 kDa), and >10 kDa (10 kDa or larger) were gathered and lyophilized. The molecular size distribution profile was analyzed by 15% SDS-PAGE. The highest ACE inhibitory fraction was applied to a Sephadex G-25 gel filtration column (2.5 cm × 75 cm), equilibrated with distilled water. The active fraction was eluted with distilled water at a flow rate of 2 ml/min. The active fractions obtained were then applied to a preparative reverse-phase high performance liquid chromatography (HPLC) on a YMC-Pack ODS-A (5 ␮m, 4.6 mm × 250 mm) with a linear gradient of acetonitrile (0–15% in 50 min) containing 0.1% trifluoroacetic acid (TFA) at a flow rate of 1.0 ml/min. Elution peaks were detected at a 220 nm. Finally, the fraction with the ACE inhibitory activity was collected and lyophilized; this was followed by identification of the amino acid sequence. 2.6. Determination of molecular weight and amino acid sequence Accurate molecular weights of ACE inhibitory peptide were determined with a Waters Synapt high definition mass spectrometer (HDMS) coupled with electrospray ionization (ESI) source (Waters Corporation, Milford, MA, USA). The instrument was operated in positive-ion mode with a capillary voltage of 2.8 kV unless stated otherwise. The cone voltage was maintained at 30 V for intact mass analysis. The quadrupole was operated in nonresolving mode to transmit a wide m/z range. Sequencing of ACE inhibitory activity peptide was obtained over the m/z range 60–640 and sequenced using the MassLynx 4.1 software (Waters Corp.). 2.7. Determination of ACE inhibition pattern A Lineweaver–Burk plot was drawn to estimate the ACE-inhibitory types of the enzyme-assistant extraction. Different concentrations of ACE-inhibitory peptide were added to each reaction mixture according to the method of Kim et al. [28]. Enzyme activity was measured with different concentrations of substrate. ACE inhibitory pattern in presence of the inhibitor was obtained with Lineweaver–Burk plot. 2.8. Statistical analysis All data were represented as the mean ± SD. Statistical comparisons of the mean values were performed by analysis of variance (ANOVA), followed by Duncan’s multiple-range test using SPSS (11.5) software. Statistical significance was considered at p < 0.05.

3. Results and discussion 3.1. Approximate chemical composition The approximate chemical composition of S. clava is shown in Table 2. The major chemical component of the tested S. clava whole tissue was found to be carbohydrate; carbohydrate contents

36

S.-C. Ko et al. / Process Biochemistry 47 (2012) 34–40 Table 4 ACE inhibitory activity of Protamex extracts from flesh and tunic tissue of S. clava.

Table 2 Chemical compositions of S. clava. Composition

Content (%) Whole

Moisture Ash Protein Carbohydrate Lipid

9.34 10.77 33.12 42.52 4.25

Flesh ± ± ± ± ±

0.21 0.33 0.29 0.41 0.43

1.84 7.05 67.80 16.77 6.54

Tunic ± ± ± ± ±

0.18 0.32 0.22 0.07 0.21

1.78 3.57 31.51 60.38 2.76

± ± ± ± ±

0.37 0.25 0.21 0.21 0.11

accounted for more than 40% of the total dry weight. The moisture, ash, protein, and lipid contents of S. clava whole tissue were 9.34, 10.77, 33.12, and 4.25%. Additionally, the carbohydrate content (60.38%) of S. clava tunic tissue was the highest among all components. However, S. clava flesh tissue evidenced a higher protein content (67.80%) than was observed in the other samples. 3.2. Preparation of S. clava enzymatic hydrolysates and aqueous extract and their ACE inhibitory activity To produce ACE inhibitory peptides, tuna frame protein was separated and hydrolyzed using a variety of commercial digestive enzymes. The enzymatic hydrolysates and aqueous extract yield generated from S. clava is shown in Table 3. All of the enzymatic hydrolysates evidenced different yields and the enzymatic hydrolysates exhibited relatively higher yields, as compared with the aqueous extract. The highest yield was recorded in the Protamex hydrolysate (43.21%) from S. clava, whereas the lowest yield was noted in the aqueous extract (19.35%) from S. clava. These results demonstrate that proteases function more effectively than water in the preparation of enzymatic hydrolysates from S. clava. The enzymes appear to work primarily by macerating the tissues and breaking down the cell walls and complex interior storage materials [21]. We screened the ACE inhibitory activity of the enzymatic hydrolysates and the aqueous extract from S. clava. The ACE inhibitory activities of the different enzymatic hydrolysates and aqueous extract are provided in Table 3. The IC50 value for the aqueous extract differed profoundly from those obtained for the enzymatic hydrolysates. The IC50 values of the protease hydrolysates were less than 2.5 mg/ml, whereas the IC50 for aqueous extract was over 6 mg/ml. Among all of the hydrolysates, the Protamex hydrolysate, in particular, evidenced the highest level of activity relative to the other hydrolysates. In terms of the activation of the ACE inhibitory effect, the highest IC50 value was observed with the Protamex hydrolysate at a concentration of 1.023 mg/ml. Thus, Protamex was selected for the effective hydrolysis of S. clava. We evaluated the effects of the ACE inhibitory activity of Protamex hydrolysate from the flesh and tunic tissues of S. clava. The Protamex hydrolysate of flesh

Tissue

IC50 value (mg/ml)

Whole Flesh Tunic

1.023 ± 0.003 0.455 ± 0.011 2.060 ± 0.007

tissues evidenced relatively higher levels of ACE inhibitory activity (Table 4; IC50 flesh tissue of 0.455 ± 0.011 mg/ml compared to IC50 tunic tissue of 2.060 ± 0.007 mg/ml). Compared to what has been observed in previous reports, the ACE inhibitory activities of enzymatic hydrolysates were more effective than those of the organic solvents and aqueous extracts from S. clava [23]. Many previous reports have demonstrated that enzymes are capable of producing bioactive properties when they are incorporated to hydrolyze natural resources [3,21,30]. 3.3. Purification of ACE inhibitory peptide Fractionation with different molecular weights of the enzymatic hydrolysates was conducted using an ultra-filtration (UF) membranes of different pore sizes [31]. The principal advantage of the UF system is that the molecular weight distribution of the desired digests can be controlled by adopting an appropriate UF membrane [32]. In this study, in order to identify the ACE inhibitory peptide in the Protamex hydrolysates of flesh tissues, the sample was fractionated with an UF system into three individual fractions with molecular weights of <5, 5–10, and >10 kDa. In the SDS-PAGE gel, various band patterns were seen among the fractions, according to molecular size (Fig. 1). Molecular size of fractions became smaller,

Table 3 Yield and ACE inhibitory activity of enzymatic hydrolysates and aqueous extract from S. clava whole tissue. Enzyme

Yield (%)

Kojizyme Flavourzyme Neutrase Alcalase Protamex Pesin Trypsin ␣-Chymotrypsin Papain Aqueous

28.28 35.87 38.11 40.19 43.21 30.19 31.73 33.98 35.71 19.35

a

± ± ± ± ± ± ± ± ± ±

IC50 value (mg/ml)a 0.37 0.77 0.16 0.52 0.43 0.13 0.65 0.47 0.55 0.11

2.481 2.343 2.234 1.781 1.023 2.147 2.427 2.263 2.282 6.887

± ± ± ± ± ± ± ± ± ±

0.032 0.022 0.019 0.018 0.047 0.051 0.033 0.028 0.042 0.031

The concentration of an inhibitor required to inhibit 50% of the ACE activity. The values of IC50 were determined by at triplicate individual experiments.

Fig. 1. SDS-PAGE of molecular weight fractions from Protamex hydrolysate. A: Marker, B: Protamex hydrolysate, C: >10 kDa fraction, D: 5–10 kDa fraction, and E: <5 kDa fraction.

S.-C. Ko et al. / Process Biochemistry 47 (2012) 34–40

37

Fig. 2. Sephadex G-25 gel filtration chromatogram of <5 kDa fraction of Protamex hydrolysate from flesh tissue. (A) Separation was performed with 2 ml/min and collected at a fraction volume (10 ml). The fractions isolated by Sephadex G-25. Gel column were separated into four fractions (F1–F4). (B) ACE inhibitory activity of each fraction.

and evidenced a low extent as compared with molecular weight size. ACE inhibitory activity was widely observed in all of the fractions, thus suggesting that many ACE inhibitory substances with various molecular weight ranges were contained in the Protamex

hydrolysate of flesh tissue. However, the most potent ACE inhibition was noted at a less than 5 kDa fraction, and evidenced an IC50 value of 0.281 mg/ml (Table 5). This result was consistent with the previous studies of ACE inhibitory peptides, in which the low

Fig. 3. RP-HPLC chromatogram of the potent ACE inhibitory activity fraction F1 isolated from Sephadex G-25. (A) Separation into sub-fractions (F1-A and F1-B) was performed with linear gradient of acetonitrile from 0% to 15% at a flow rate of 1.0 ml/min and an YMC-Pack ODS-A column (5 ␮m, 4.6 mm × 250 mm). The elution was monitored at 220 nm. (B) ACE inhibitory activity of each fraction.

38

S.-C. Ko et al. / Process Biochemistry 47 (2012) 34–40

Table 5 ACE inhibitory activity of molecular weight fractions of Protamex hydrolysate from S. clava flesh tissue. Fraction

IC50 value (mg/ml)a

Unfractionated >10 kDa 5–10 kDa <5 kDa

0.455 0.325 0.552 0.281

± ± ± ±

0.047 0.008 0.005 0.003

a The concentration of an inhibitor required to inhibit 50% of the ACE activity. The values of IC50 were determined by at triplicate individual experiments.

Table 6 Purification of ACE inhibitory peptide of Protamex hydrolysate from S. clava flesh tissue. Purification step

IC50 value (mg/ml)a

Protamex extract Ultrafiltration (<5 kDa) Gel filtration chromatography RP-HPLC

0.455 0.281 0.162 0.021

± ± ± ±

0.047 0.003 0.009 0.012

Purification foldb 1 1.62 2.81 21.67

a The concentration of an inhibitor required to inhibit 50% of the ACE activity. The values of IC50 were determined by at triplicate individual experiment. b Relative value of reciprocal of ACE IC50 .

molecular weight fraction had more potent ACE inhibitory activity than the high molecular weight fraction when analyzed with a UF membrane [13,33]. The <5 kDa fraction was loaded onto a Sephadex G-25 gel filtration column to separate according to its molecular size, and obtained four fractions (Fig. 2(A)). Among them, fraction F1 evidenced the highest ACE inhibitory activity, with an IC50 value of 0.162 mg/ml (Fig. 2(B)). This active fraction was further separated via RP-HPLC on a YMC-Pack ODS-A (5 ␮m, 4.6 mm × 250 mm) reversed phase analytical column using a linear gradient of acetonitrile (0–15%) containing 0.1% TFA, and the fractions were divided into two fractions (Fig. 3(A)). Fraction F1-B exhibited the most potent ACE inhibitory activity, with an IC50 value of 0.021 mg/ml (Fig. 3(B)). Table 6 summarizes the results of the purification of the ACE inhibitory peptide obtained from S. clava. The ACE inhibitor was purified at 21.67-fold from the enzymatic hydrolysate of S. clava flesh tissue (0.455 mg/ml) via a three-step purification procedure.

3.4. Amino acid sequences and inhibition pattern of purified peptide The amino acid sequences were identified using a Waters Synapt high-definition mass spectrometer (HDMS) and identified as Ala-His-Ile-Ile-Ile for the fraction evidencing an ACE inhibitory IC50 value of 37.1 ␮M and a molecular weight of 565.3 Da (Fig. 4). Several ACE inhibitory peptides derived from enzymatic hydrolysates of various natural resources. The identified ACE inhibitory peptides are Tyr-Asn (IC50 value = 51 ␮M) from hard clam [14], Pro-Gly-Pro-Leu-Gly-Leu-Thr-Gly-Pro (IC50 value = 95 ␮M) from skate skin [17], Met-Ile-Phe-Pro-Gly-AlaGly-Gly-Pro-Glu-Leu (IC50 value = 22.1 ␮M) from yellowfin sloe frame [18], Leu-Val-Gln-Gly-Ser (IC50 value = 43.7 ␮M) from fermented soybean [33], and Ala-Ile-Tyr-Lys (IC50 value = 213 ␮M) from wakame [34]. In this study, the isolated ACE inhibitory peptide exhibited higher or similar activity, but evidenced lower activity than that of captopril (6.9 nM), currently the most widely used antihypertensive drug [14]. As reported by Cushman et al. [35], the active sites of two domains of somatic ACE are functionally and structurally homologous as dipeptidyl carboxypeptidases, and the relevant zinc coordination geometry is critical to their hydrolytic action. However, the two catalytic sites are differentially activated by chloride ions, and the physiological substrate angiotensin I binds preferentially to the C-domain catalytic site. Additionally, the substrate contributes to the chloride-induced activation of the active site. Therefore, these differences indicate that, despite the higher level of primary sequence, structural, homology and functional differences do indeed exist between the two active sites [18]. Analysis of the structure–activity relationships among different peptide inhibitors of ACE demonstrates that binding to ACE is strongly affected by the C-terminal tripeptide sequence of the substrate, and it has been proposed that peptides, which include hydrophobic amino acids at these positions, function as potent inhibitors [3]. The majority of ACE peptides thus far identified are generally short peptides harboring a proline residue at the C-terminal end. Proline is known to prevent enzyme digestion, and may pass from the capillaries into the blood circulation in the short peptide sequences [29,30]. Compared with our results, differences in substrate

Fig. 4. Identification of molecular mass and amino acid sequence of the purified peptide from S. clava flesh tissue Protamex hydrolysate by RP-HPLC.

S.-C. Ko et al. / Process Biochemistry 47 (2012) 34–40

39

Fig. 5. The ACE inhibition pattern of purified peptide was estimated using Lineweaver–Burk plots. 1/[V] (␮M/min) and 1/[S] (mM) represents the double-reciprocal plot of reaction rate (V) and substrate concentration (S), respectively.

preparation and method purification might have resulted in different peptide sequences [31]. The ACE inhibition pattern of the purified peptide was estimated using Lineweaver–Burk plots, and was shown to be a non-competitive inhibition pattern (Fig. 5). This means that the peptide can combine with an enzyme molecule to generate a deadend complex, regardless of whether or not a substrate molecule is bound.

4. Conclusion In this study, we evaluated the ACE inhibitory activity of the purified peptide from an enzymatic hydrolysate of S. clava flesh tissue. Via consecutive chromatographic methods, the ACE inhibitory peptide was found to exhibit potent inhibitory activity, with an IC50 value of 37.1 ␮M. The ACE inhibitory pattern of the purified peptide from S. clava flesh tissue was shown by the Lineweaver–Burk plots to be a non-competitive inhibition pattern. Based on the results of this study, it appears that this peptide may be beneficial to the bioactivity of some materials and functional foods. Acknowledgments This research was supported by a grant from the Korea Institute of Planning & Evaluation for Technology of Food, Agriculture, Forestry & Fisheries funded by Ministry of Food, Agriculture, Forestry & Fisheries, Korea. References [1] Unger T. The role of renin–angiotensin system in the development of cardiovascular disease. Am J Cardiol 2002;89:3–9. [2] Je JY, Park JY, Jung WK, Park PJ, Kim SK. Isolation of angiotensin I converting enzyme (ACE) inhibitor from fermented oyster sauce Crassostrea gigas. Food Chem 2005;90:809–14. [3] Lee JK, Hong S, Jeon JK, Kim SK, Byun HG. Purification and characterization of angiotensin I converting enzyme inhibitory peptides from the rotifer Brachionus rotundufirmis. Bioresour Technol 2009;100:5255–9.

[4] Je JY, Park PJ, Byun HG, Jung WK, Kim SK. Angiotensin I converting enzyme (ACE) inhibitory peptide derived from the sauce of fermented blue mussel Mytilus edulis. Bioresour Technol 2005;96:1624–9. [5] Lapointe N, Rouleau JL. Activation of vascular tissue angiotensin-converting enzyme (ACE) in heartfailure. J Am Coll Cardiol 2002;39:776–9. [6] Ondetti MA. Design of specific inhibitors of angiotensin converting enzyme: new class of orally active antihypertensive agents. Science 1977;196: 441–4. [7] Kato H, Suzuki T. Bradykinin-potentiating peptides from the venom of Agkistrodon halys blomhoffii: isolation of five bradykinin potentiators B and C. Toxicon 1972;10:313. [8] Thurman JM, Schrier RW. Comparative effects of angiotensin-converting enzyme inhibitors and angiotensin receptor blockers on blood pressure and the kidney. Am J Med 2003;114:588–98. [9] Papadimitriou GC, Vafopoulou MA, Silva VS, Gomes A, Malcata XF, Alichanidis E. Identification of peptides in traditional and probiotic sheep milk yoghurt with angiotensin I-converting enzyme (ACE)-inhibitory activity. Food Chem 2007;105:647–56. [10] Wang NF, Li CY, Yan Z. Purification and characterization of angiotensin Iconverting enzyme inhibitory peptide from lactic acid fermented peanut flour. J Biotechnol 2008;136:S723. [11] Miguel M, Alonso JM, Salaices M, Aleixandre A, Lopez FR. Antihypertensive ACEinhibitory and vasodilator properties of an egg white hydrolysate: effect of a simulated intestinal digestion. Food Chem 2007;104:163–8. [12] Chem JR, Okada T, Muramoto K. Identification of angiotensin I-converting enzyme inhibitory peptides derived from the peptic digest of soybean protein. J Food Biochem 2002;6:543–4. [13] Maoa X, Ni J, Sun W, Hao P, Fan L. Value-added utilization of yark milk casein for the production of angiotensin I-converting enzyme inhibitory peptides. Food Chem 2007;103:1282–7. [14] Tsai JS, Chen JL, Pan BS. ACE-inhibitory peptides identified from the muscle protein hydrolysate of hard clam (Meretrix lusoria). Process Biochem 2008;43:743–7. [15] Lee SH, Qian ZJ, Kim SK. A novel angiotensin I converting enzyme inhibitory peptide from tuna frame protein hydrolysate and its antihypertensive effect in spontaneously hypertensive rats. Food Chem 2010;118:96–102. [16] Zhao Y, Li B, Liu Z, Dong S, Zhao X, Zeng M. Antihypertensive effect and purification of an ACE inhibitory peptide from sea cucumber gelatin hydrolysate. Process Biochem 2007;42:1586–91. [17] Lee JK, Jeon JK, Byun HG. Effect of angiotensin I converting enzyme inhibitory peptide purified from skate skin hydrolysate. Food Chem 2011;125: 495–9. [18] Jung WK, Mendis E, Je JY, Park PJ, Son BW, Kim HC, et al. Angiotensin Iconverting enzyme inhibitory peptide from yellowfin sole (Limanda aspera) frame protein and its antihypertensive effect in spontaneously hypertensive rats. Food Chem 2006;94:26–32. [19] Fahmi A, Morimura S, Guo HC, Shigematsu T, Kida K, Ueyama Y. Production of angiotensin I converting enzyme inhibitory peptides from sea bream scales. Process Biochem 2004;39:1195–200.

40

S.-C. Ko et al. / Process Biochemistry 47 (2012) 34–40

[20] Lahl WJ, Braun SD. Enzymatic production of protein hydrolysate for food use. Food Technol 1994;48:68–71. [21] Bougatef A, Nedjar-Arroume N, Manni L, Ravallec R, Barkia A, Guillochon D, et al. Purification and identification of novel antioxidant peptides from enzymatic hydrolysates of saldinelle (Sardinella aurita) by-products proteins. Food Chem 2010;118:559–65. [22] Kim JB, Iwamuro S, Knoop FC, Conlon JM. Antimicrobial peptides from the skin of the Japanese mountain brown frog, Rona ornativentris. J Pept Res 2001;58:349–56. [23] Lee DW, You DH, Yang EK, Jang IC, Bae MS, Jeon YJ, et al. Antioxidant and ACE inhibitory activities of Styela clava according to harvesting time. J Food Sci Nutr 2010;39:331–6. [24] Kim JJ, Kim SJ, Kim SH, Park HR, Lee SC. Antioxidant and anticancer activities of extracts from Styela clava according to the processing methods and solvents. J Korean Soc Food Sci Nutr 2006;35:278–83. [25] AOAC Official Methods of Analysis. 16th ed. Virginia, USA: Association of Official Analytical Chemists; 1990. [26] Heo SJ, Park EJ, Lee KW, Jeon YJ. Antioxidant activities of enzymatic extracts from brown seaweeds. Bioresour Technol 2005;96:1613–23. [27] Cushman DW, Cheung HS. Spectrophotometric assay and properties of the angiotensin-converting enzyme of rabbit lung. Biochem Pharmacol 1970;20:1637–48. [28] Kim SK, Byun HG, Park PJ, Shahidi F. Angiotensin I converting enzyme inhibitory peptides purified from bovine skin gelatin hydrolysate. J Agric Food Chem 2001;49:2992–7.

[29] Siriwardhana N, Jeon YJ, Kim SH, Ha JH, Heo SJ, Lee KW. Enzymatic hydrolysis for effective extraction of antioxidative compounds from Hizikia fusiformis. Algae 2004;19:59–68. [30] Li GH, Wan JZ, Le GW, Shi YH. Novel angiotensin I-converting enzyme inhibitory peptides isolated from alcalase hydrolysate of mung bean protein. J Pept Sci 2006;12:509–14. [31] Byun HG, Kim SK. Purification and characterization of angiotensin I converting enzyme (ACE) inhibitory peptides from Alaska pollock (Theragra chalogramma) skin. Process Biochem 2001;36:1155–62. [32] Jeon YJ, Byun HG, Kim SK. Improvement of functional properties of cod frame protein hydrolysate using ultrafiltration membranes. Process Biochem 2000;35:471–8. [33] Rho SJ, Lee JS, Chung YI, Kim YW, Lee HG. Purification and identification of an angiotensin I-converting enzyme inhibitory peptide from fermented soybean extract. Process Biochem 2009;44:490–3. [34] Suetsuna K, Nakano T. Identification of an antihypertensive peptide from peptic digest of wakame (Undaria pinnatifida). J Nutr Biochem 2000;11: 450–4. [35] Cushman DW, Cheung HS, Sabo EF, Ondetti MA. Angiotensin converting enzyme inhibitor: evaluation of a new class of antihypertensive drugs. In: Horovitz ZP, editor. Angiotensin converting enzyme inhibitors: mechanism of action and clinical implications. Baltimore/Munich: Urban & Schwarzenberg; 1981. p. 1–25; Korhonen H, Pihlanto A. Bioactive peptides: production and functionality. Int Dairy J 2006;16:945–60.