Production of angiotensin I-converting enzyme ...

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Process Biochemistry 40 (2005) 2191–2196 www.elsevier.com/locate/procbio

Production of angiotensin I-converting enzyme inhibitory peptides from soybean protein with Monascus purpureus acid proteinase M. Kuba, C. Tana, S. Tawata, M. Yasuda* Department of Bioscience and Biotechnology, Faculty of Agriculture, University of the Ryukyus, 1 Senbaru, Nishihara-cho, Okinawa 903-0213, Japan Received 27 May 2004; received in revised form 12 July 2004; accepted 21 August 2004

Abstract Soybean proteins, b-conglycinin and glycinin were hydrolysed by an acid proteinase from Monascus purpureus. The degree of hydrolysis and inhibitory activities of angiotensin I-converting enzyme (ACE) increased with increasing proteolysis time. After 10 h of incubation, the IC50 values of the b-conglycinin and glycinin hydrolysates were determined as 0.126 mg/ml and 0.148 mg/ml, respectively. Four ACE inhibitory peptides were isolated from the soybean protein hydrolysates and identified by protein sequencer. ACE inhibitory peptides isolated from the b-conglycinin hydrolysate were identified as LAIPVNKP (IC50 = 70 mM) and LPHF (670 mM), and those from the glycinin hydrolysate as SPYP (850 mM) and WL (65 mM). The inhibitory activity of SPYP markedly increased after successive digestion by pepsin, chymotrypsin and trypsin in vitro. # 2004 Elsevier Ltd. All rights reserved. Keywords: Angiotensin I-converting enzyme inhibitor; Acid proteinase; Soybean protein hydrolysate; Monascus Purpureus; Tofuyo

1. Introduction Angiotensin I-converting enzyme (ACE, EC 3.4.15.1.) is a dipeptidyl carboxy peptidase that plays an important role in the regulation of blood pressure. It converts angiotensin I into a powerful vasoconstrictor, angiotensin II, and also inactivates the vasodilator bradykinin [1,2]. ACE inhibitors in various types of foods have been studied and show the ability to prevent and alleviate hypertension. Soybean is a valuable source of ACE inhibitors, and some ACE inhibitory peptides have already been isolated from its hydrolysate [3,4] and fermented soybean food products [5–7]. ACE inhibitory peptides (WL and IFL) were isolated recently from tofuyo, which is a soybean curd fermented by fungi such as Monascus and Aspergillus [7]. It has been considered that these ACE inhibitory peptides were liberated from soybean protein during fermentation. It was previously reported that the acid proteinase from Monascus purpureus * Corresponding author. Tel.: +81 98 895 8807; fax: +81 98 895 8734. E-mail address: [email protected] (M. Yasuda). 0032-9592/$ – see front matter # 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2004.08.010

gradually degraded soybean protein and that enzyme activity remained even in the presence of 15–20% ethanol that is necessary for tofuyo fermentation [8]. These findings indicated that M. purpureus acid proteinase might greatly contribute to the ripening of tofuyo and the formation of the functional peptides [7,8]. The enzyme is considered to be an aspartic proteinase similar to pepsin (unpublished data), and it should be useful for processing foodstuffs and other materials because it hydrolyses animal proteins like milk casein and bovine serum albumin as well as vegetable proteins like soybean protein and wheat gluten [9]. Proteinases such as pepsin, chymotrypsin and trypsin are frequently used in hydrolysis to obtain ACE inhibitory peptides. Microbial alkaline proteinases are also utilized in the production of ACE inhibitors from food proteins such as sardine [10,11], sea bream scales [12] and rapeseed [13]. However, there are a small number of reports on the use of microbial acid proteinase in the production of ACE inhibitor. In this paper, we describe digestion of soybean proteins (b-conglycinin and glycinin) by M. purpureus acid proteinase, and isolation and identification of the ACE

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inhibitory peptides from the hydrolysates. We also include the investigation on the resistance of the ACE inhibitory peptides against gastrointestinal proteinases in vitro.

hydrolysate. The degree of hydrolysis was determined by trinitrobenzensulphonic acid [15]. 2.5. Purification of ACE inhibitors

2. Materials and methods 2.1. Preparation of acid proteinase from M. purpureus The microorganism used in this study was M. purpureus No. 3403, which was isolated from red-mould rice in this laboratory. Preparation of red-mould rice and purification of acid proteinase were carried out as previously reported [9]. The purified enzyme was used for soybean protein hydrolysis. 2.2. Preparation of ACE from pig lung The ACE extract from pig lung was prepared by the modified method described by Andujar-Sanchez et al. [14]. The ACE extract was fractionated with ammonium sulphate between the limits of 35–65%, and the resulting precipitate was dissolved in 10 mM sodium phosphate buffer (pH 7.8) and dialyzed against the same buffer. The fraction was applied to a DE52 column (Whatman Biosystems Ltd., 1.5 cm 7.8 cm) and eluted with 0–0.2 M NaCl in 10 mM sodium phosphate buffer in a linear gradient profile. Active fractions were combined and concentrated by ultrafiltration. The concentrate was dialyzed against 50 mM sodium phosphate buffer (pH 7.8) containing 0.15 M NaCl. The enzyme solution was subjected to a Sephacryl S-200 column (Amersham Bioscience, 1.5 cm 114 cm) and eluted with the same buffer. Active fractions were pooled, concentrated by ultrafiltration, and dialyzed against 20 mM borate buffer (pH 8.3) containing 0.1 M NaCl. The final ACE solution had no activities of alkaline protease and carboxy peptidase.

b-Conglycinin or glycinin hydrolysate was fractionated with DIAION HP21SS resin (Mitsubishi Chemical Co.) by a batch method. After washing the resin with deionized water, the inhibitors were eluted with 10–70% ethanol by step-wise gradient method. Each fraction was evaporated in a rotary evaporator (Yamato RE-46), and the resulting residue was dissolved in deionized water. The active fraction was successively ultrafiltered with a membrane UK-10 (molecular weight cut-off (MWCO) 10,000, Advantec), YM3 (MWCO 3000, Millipore) and YC05 (MWCO 500, Millipore). Subsequently, the active fraction was subjected to reversephaseHPLC with Cosmosil 5C18-AR-300 column. Theelution was carried out with a linear gradient of 0–60% acetonitrile in 0.05% trifluoroacetic acid (TFA) for 60 min at 0.5 ml/min and monitored at 220 nm. The fraction corresponding to an active peak was rechromatographed in the same column or in a Cosmosil 5Ph-AR-300 column. Elution on Cosmosil 5C18AR-300 column was carried out with a linear gradient of acetonitrile (0–32% for 10 min, 32–33% for 50 min) in 0.05%TFA at 0.25 ml/min. Elution on Cosmosil 5Ph-AR-300 column was performed with a linear gradient of acetonitrile (0–30% for 60 min) in 0.05% TFA at 0.25 ml/min. Peptide concentration in a sample throughout the purification was determined with a DC protein assay reagent kit (Bio-Rad), with bovine serum albumin used as the standard. 2.6. Amino acid sequence analysis The amino acid sequences of the ACE inhibitors were determined by automated Edman degradation [16] with a gas/ liquid-phase protein sequencer (Applied Biosystems 473A).

2.3. Assay for ACE inhibitory activity 2.7. Digestion test ACE inhibitory activity was assayed with the pig pulmonary ACE and hippuryl-L-histidyl-L-leucine (Peptide Institute Inc., Japan) as previously described [7]. IC50 value was defined as the sample concentration required to inhibit 50% of the ACE activity under the assay conditions. 2.4. Hydrolysis of b-conglycinin and glycinin by acid proteinase from M. purpureus b-Conglycinin and glycinin were provided by Fuji Oil Co. Ltd., Japan. One gram of b-conglycinin or glycinin was suspended in 50 ml of 0.1 M lactate buffer (pH 3.3, the optimum pH for the enzyme activity) and hydrolysed by 100 units of M. purpureus acid proteinase for 10 h at 37 8C. After incubation, the mixture was boiled for 10 min to stop the hydrolysis and then centrifuged at 12,000 g for 10 min. The resulting supernatant was used as soybean protein

The stability of each purified ACE inhibitory peptide against gastrointestinal proteinases was assessed in vitro. Each inhibitor solution (1.5–30 mM) was successively digested with pepsin (porcine stomach mucosa), chymotrypsin (bovine pancreas) and trypsin (bovine pancreas) as previously described [7]. The digest was used for measuring ACE inhibition.

3. Results and discussion 3.1. ACE inhibitory activity in b-conglycinin and glycinin hydrolysates Each soybean protein, b-conglycinin and glycinin, was hydrolysed by M. purpureus acid proteinase, and degree of

M. Kuba et al. / Process Biochemistry 40 (2005) 2191–2196

Fig. 1. Hydrolysis of soybean proteins with M. purpureus acid proteinase. The concentration of protein in each sample was 0.2 mg/ml in the reaction mixture for ACE inhibition assay.

hydrolysis and ACE inhibition were measured (Fig. 1). Both proteins were rapidly hydrolysed in the first 2 h and ACE inhibitory activities in the both hydrolysates gradually increased with the increasing proteolysis time. Attempts were made to purify the ACE inhibitors in 10 h-hydrolysates that had the highest ACE inhibitory activities under these hydrolysis conditions. The IC50 values of b-conglycinin and glycinin hydrolysates were 0.126 mg/ml and 0.148 mg/ml, respectively. These values were lower than that of soybean protein hydrolysed by alcalase (0.34 mg/ml) and of enzymic hydrolysates of other proteins [3,10,13,17]. This result indicated that the acid proteinase from M. purpureus would be effective in producing strong inhibitors for the ACE reaction. 3.2. Purification of the ACE inhibitory peptides from soybean protein hydrolysates Each hydrolysate was subjected to DIAION HP21SS resin that adsorbed mainly hydrophobic substances and eluted with a step-wise gradient of ethanol. Fig. 2 shows that all fractions have inhibitory activities and 50% ethanol fractions of both hydrolysates have the highest activities. It

Fig. 2. Separation of ACE inhibitory peptides in soybean protein hydrolysates by DIAION HP21SS. The final concentration of protein in each fraction was 0.1 mg/ml in the reaction mixture for ACE inhibition assay.

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is generally accepted that hydrophobic peptides seem to have high ACE inhibitory activities. However, it was difficult to purify ACE inhibitors in 50% ethanol fractions further because peptide yields of the fractions were very low (b-conglycinin, 0.7%; glycinin, 1.4%). We have previously isolated hydrophobic peptides, WL and IFL, as ACE inhibitors in 20% ethanol fraction from tofuyo extract with SEPABEADS SP-825 resin that has an almost the same property as DIAION HP21SS [7]. This result indicated that the 20% ethanol fraction should contain hydrophobic peptides with predominant ACE inhibitory activity. In addition, Matsui et al. [11] reported that 10% ethanol fraction of sardine hydrolysate separated by ODS column had strong ACE inhibitory activity and would contain the polar peptides with potent inhibitory activities. Therefore we decided to continue purification of ACE inhibitors in the 10% ethanol fraction of b-conglycinin hydrolysate and the 20% ethanol fraction of glycinin hydrolysate. The peptide yield of each fraction was 32.8% and 16.4%, respectively. Each active fraction of b-conglycinin and glycinin from DIAION HP21SS was successively separated using stepwise ultrafiltration into four fractions—molecular weight (MW) less than 500, 500–3000, 3000–10,000 and more than 10,000 (Fig. 3). Fractions from both hydrolysates that had MW less than 500 had the highest activities. These fractions would include small peptides that had two to seven amino acid residues. Fractions that had a MW of less than 500 were applied to a reverse-phase HPLC (Figs. 4 and 5) and some peaks had inhibitory activities. The peaks depicted as A and B in Fig. 4 had high inhibitory activities (60.3% and 69.8%, respectively, at 20 mg/ml). Although inhibitory activities of the peaks depicted as C and D in Fig. 5 were lower than A and B, they were relatively high compared to other peaks in glycinin hydrolysate (28.9% and 19.8% at 20 mg/ml). These

Fig. 3. Molecular weight (MW) fractionation of ACE inhibitory peptides by ultrafiltraion. The active fractions obtained from DIAION HP21SS were successively ultrafiltered with a membrane UK-10, YM3 and YC05 (MW cut-off 10,000, 3000 and 500), respectively. The final concentration of protein in each fraction was 40 mg/ml in the reaction mixture for ACE inhibition assay.

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M. Kuba et al. / Process Biochemistry 40 (2005) 2191–2196 Table 1 ACE inhibitory peptides isolated from soybean protein hydrolysates Peaka

A B C D

Fig. 4. Elution profile of active fraction separated from b-conglycinin hydrolysate on Cosmosil 5C18-AR-300. Peaks A and B had high inhibitory activities, and they were subjected to further purification.

four peaks were then re-chromatographed on a 5C18-AR300 column or a 5Ph-AR-300 column with a moderate acetonitrile gradient. As a result of separation of the potent peaks on the reverse-phase HPLC, four ACE inhibitory peptides were isolated. 3.3. Identification of the ACE inhibitory peptides from soybean protein hydrolysates Each ACE inhibitory peptide isolated from soybean protein hydrolysates was identified as LAIPVNKP, LPHF, SPYP and WL by protein sequencer (Table 1). Comparison with the SWISS-PROT protein sequence database showed that the amino acid sequences of these peptides exist in the primary structure of b-conglycinin or glycinin. Synthesized peptides were used to evaluate ACE inhibitory activities based on the amino acid sequences, and the results showed that WL and LAIPVNKP had high ACE inhibitory activities. The three peptides, except WL, were newly identified as natural ACE inhibitory peptides. It should be noted that WL has been purified from tofuyo extract previously [7]. Therefore, the result from this study suggested that ACE inhibitory peptides in tofuyo might have been derived from soybean protein by the action of M. purpureus acid

Fig. 5. Elution profile of active fraction separated from glycinin hydrolysate on Cosmosil 5C18-AR-300. Peaks C and D had high activities, and they were subjected to further purification.

Columnb/Retention time (min) 1st

2nd

O/31.9 O/32.5 O/23.9 O/35.9

P/48.1 O/19.2 P/36.7 P/64.3

Amino acid sequence

IC50 (mM)

Yield (%)

LAIPVNKP LPHF SPYP WL

70 670 850 65

1.50 10 6.42 10 2.69 10 1.45 10

3 4 3 3

a Peak indicate the active peaks isolated from fractions of b-conglycinin and glycinin hydrolysates that have MW of less than 500 by a reverse-phase HPLC. b O and P represent Cosmosil 5C18-AR-300 column and Cosmosil 5PhAR-300 column, respectively.

proteinase that is considered to be the key enzyme for tofuyo fermentation [8]. The other ACE inhibitory peptide purified from tofuyo, i.e. IFL, was not isolated in this study. It may be that 10% ethanol fraction was used in the purification scheme in this study, which was different from the purification of ACE inhibitors in tofuyo. There were two peptides whose MW were over 500, although they were isolated from the hydrolysate fractions of the MW less than 500 fractions. This could be due to the MW cut-off size of ultrafiltration membrane that is generally nominal, and the passage property of a peptide probably depends on its conformation. In this study, we used pig pulmonary ACE because pig is a closer species to human than rabbit. The IC50 value of WL from pig pulmonary ACE (65 mM) in this study was higher than that of the rabbit pulmonary ACE (30 mM) reported previously [7]. This may be explained as the substrate specificity and inhibitory characteristics of pig pulmonary ACE being different from that of rabbit pulmonary ACE. The IC50 value of LAIPVNKP was 70 mM, which was also higher than that of the rabbit pulmonary ACE (35 mM). WL had the highest activity in the purified peptides (IC50 = 65 mM). YL [10] and FL [18] have already been reported as potent inhibitors (IC50 = 82 mM and 16 mM, respectively), indicating that dipeptides that contain aromatic amino acid (W, Y and F) and L would exhibit relatively high activities. Cheung et al. [19] concluded that ACE is highly specific with terminal dipeptide residues of inhibitors and the Cterminal amino acid is the most important to the overall binding to the active site of ACE. The most favorable Cterminal residues were W, Y, P, or F. Consistently with this report, LAIPVNKP that has P at the C-terminal residue showed strong inhibitory activities. KP [20], IKP and LKP [21] whose two C-terminal residues are the same as LAIPVNKP have been reported as strong inhibitors with IC50 values of 22 mM, 1.7 mM and 1.6 mM, respectively. Further studies must be performed to determine the antihypertensive effect of soybean protein hydrolysates and purified ACE inhibitory peptides in vivo.

M. Kuba et al. / Process Biochemistry 40 (2005) 2191–2196 Table 2 Digestive stability of ACE inhibitory peptides isolated from soybean protein hydrolysates IC50 (mM) LAIPVNKP LPHF SPYP WL

Before digestion

After digestion

70 670 850 65

376 610 60 77

Each inhibitor solution was successively treated with pepsin, chymotrypsin and trypsin as previously described [7]. The digests were used for measuring the ACE inhibitions.

3.4. Digestion test on the inhibitors The stability of each ACE inhibitory peptide from soybean protein hydrolysates against gastrointestinal proteinases in vitro was examined in order to predict their anti-hypertensive effects in vivo (Table 2). The ACE inhibitory activities of LPHF and WL were almost preserved after the digestion. The cleavage sites of protein for typical gastrointestinal proteinases have been well studied. Pepsin A from porcine stomach mucosa preferentially cleaves the C-terminal to F, L and E. a-Chymotrypsin hydrolyses peptide bonds with aromatic or large hydrophobic side chains (Y, W, F and M) on the carboxyl end of the bond. Trypsin hydrolyses peptide bond with R or K on the carboxyl end of the bond. These cleavage sites existed in the ACE inhibitory peptides whose IC50 values were altered after digestion, i.e. LAIPVNKP and SPYP (Table 2). The IC50 value of LAIPVNKP increased significantly after digestion. The N- and C-terminal amino acid residues of this peptide (L and P, respectively) may be cleaved by pepsin and trypsin. The loss of C-terminal amino acid residue (P), which was predominant for inhibition, may cause the reduction in the inhibitory activity of LAIPVNKP. In contrast, the IC50 value SPYP decreased significantly after digestion. This peptide may be split into SPYand P by trypsin. The activity of the split peptide SPY that had Yat C-terminus was expected to increase after digestion. Additional study is required to prove the inhibitory activities of liberated-peptide fragments from hydrolysed or synthetic peptides. Susceptibility to absorption as well as resistance to digestion by gastrointestinal proteases is essential for the anti-hypertensive effect of ACE inhibitory peptides in vivo. Matsui [4] found that 18 di- and tri-peptides derived from bconglycinin, including WL, were absorbed intact through small intestine membrane of rats in the study on intestinal membrane transport. WL isolated from glycinin hydrolysate and tofuyo [7] is expected to show the anti-hypertensive effect in vivo.

4. Conclusions Acid proteinase from M. purpureus was used for hydrolysis of soybean proteins, b-conglycinin and glycinin,

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and the IC50 values of the hydrolysates were determined to be 0.126 and 0.148 mg/ml, respectively. The hydrolysates would contain many ACE inhibitory peptides because all fractions separated by adsorption resin and ultrafiltration had ACE inhibitory activities. The molecular weight of the active fraction seemed to be relatively short (MW < 500). Four ACE inhibitory peptides were isolated from the hydrolysates and identified as LAIPVNKP, LPHF, SPYP and WL. The ACE inhibitory activities of LPHF and WL were preserved after digestion in vitro. Moreover, the inhibitory activity of SPYP markedly increased after the digestive treatment. These results indicated that the acid proteinase from M. purpureus is a useful enzyme for production of ACE inhibitory peptides. The enzyme is expected to produce not only ACE inhibitory peptides but also other bioactive peptides from various proteins.

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