ORIGINAL PAPER

Fang Liu Shinjiro Tachibana Toki TairaMasanobu Ishihara Masaaki Yasuda

Purification and characterization of a new type of serinecarboxypeptidase from Monascus purpureus

Received: 4 August 2003 / Accepted: 8 December 2003 / Published online: 29 January 2004 Society for Industrial Microbiology 2004

Abstract Carboxypeptidase produced by Monascuspurpureus IFO 4478 was puried to homogeneity. Thepuried enzyme is a heterodimer with a molecular massof 132 kDa and consists of two subunits of 64 and67 kDa. It is an acidic glycoprotein with an isoelectricpoint of 3.67 and 17.0% carbohydrate content. Theoptimum pH and temperature were 4.0 and 40 C,respectively. The enzyme was stable between pH 2.0 and8.0 at 37 C for 1 h, and up to 50 C at pH 5.0 for15 min. The enzyme was strongly inhibited by piper-astatin A, diisopropyluoride phosphate (DFP), phen-ylmethylsulfonyluoride (PMSF), and chymostatin,suggesting that it is a chymotrypsin-like serine car-boxypeptidase. Monascus purpureus carboxypeptidasewas also strongly inhibited by p-chloromercuribenzoicacid (PCMB) but not by ethylenediaminetetraacetic acid(EDTA) and 1,10-phenanthroline, indicating that itrequires cysteine residue but not metal ions for activity.Benzyloxycarbonyl-L-tyrosyl-L-glutamic acid (Z-Tyr-Glu), among the substrates tested, was the best substrateof the enzyme. The Km, Vmax, Kcat, and Kcat/Km valuesof the enzyme for Z-Tyr-Glu at pH 4.0 and 37 C were0.86 mM, 0.917 mM min)1, 291 s)1, and 339 mM)1 s)1,respectively.

Keywords Monascus purpureus Chymotrypsin-likeenzyme Serine carboxypeptidase Heterodimer Glycoprotein

Introduction

Monascus species have been traditionally used in thefermentation industry in East Asia. In China, they areused to produce not only natural colorants but also redwine and red fermented soybean curd. In Okinawa,Japan, they are used for the production of tofuyo, whichis a vegetable protein food made from soybean curd bythe action of microorganisms. Tofuyo-making byMonascus has been studied in our laboratory [27, 29]. Itwas found that a large number of free amino acids (e.g.glutamic acid and aspartic acid) were produced duringthe maturation of tofuyo. This was considered to be theresult of proteolytic enzymes produced by Monascushydrolyzed soybean protein. However, knowledge ofthis process is very limited.

In order to elucidate the functions of Monascusenzymes in the maturation of tofuyo, they must bepuried and characterized. In a previous study, theproduction, purication, and properties of an acidproteinase from Monascus were reported [28]. The acidproteinase is an endopeptidase that hydrolyzes proteinsfrom their internal chain to peptides, but only to smallamounts of free amino acids [30]. Thus, the action ofacid proteinase is not enough to explain the occur-rences of a large number of free amino acids during thematuration of tofuyo. By contrast, carboxypeptidase isan exopeptidase that releases free amino acids from thecarboxyl termini of peptides or proteins. It is consid-ered to serve as a key enzyme in the production ofavorful amino acids during the maturation of tofuyo.Although a large number of carboxypeptidases havebeen isolated from various species of fungi such asAspergillus saitoi [10], A. oryzae [17, 19, 20, 21, 22,23, 24], A. niger [4, 14], Absidia zychae [16], Mucorracemsus [7], Penicillium janthinellum [6, 9, 31], andPycnoporus sanguineus [12], the Monascus enzyme hasnot yet been reported. In this study, we describe thepurication and characterization of carboxypeptidaseproduced by M. purpureus IFO 4478.

F. Liu S. Tachibana T. Taira M. Ishihara M. Yasuda (&)Department of Bioscience and Biotechnology,Faculty of Agriculture, University of the Ryukyus,1 Senbaru, Nishihara-cho,9030213 Okinawa,JapanE-mail: yasuda@agr.u-ryukyu.ac.jpTel.: +81-98-895-8807Fax: +81-98-895-8734

J Ind Microbiol Biotechnol (2004) 31: 2328DOI 10.1007/s10295-004-0107-z

Materials and methods

Organism and cultivation

M. purpureus IFO 4478 was used for the production of carboxy-peptidase. The fungus was cultured at 28 C for 6 days withshaking in a medium containing 2% soybean protein isolate (SPI),1% glucose, 0.5%MgSO47H2O, 0.5% KH2PO4 (pH 6.0), with 1%(v/v) mineral solution (per ml: 15 mg CaCl2, 15 mg FeSO47H2O,7.8 lg CuSO45H2O, 10 lg H3BO3, 10 lg MnSO45H2O, 70 lgZnSO4, and 10 lg MoO3). The culture broth was ltered throughNo. 2 lter paper (Advantec, Japan). The ltrate was adjusted topH 6.0 with 0.1 M citric acid and centrifuged at 10,000 g for30 min. The supernatant was used as a crude enzyme solution forthe purication.

Purication of the enzyme

All subsequent manipulations, except HPLC, were carried out at4 C.

Step 1. The crude enzyme solution was put on a DEAE-cellu-lose column (5.035 cm) equilibrated with 10 mM citrate/20 mMphosphate buer (pH 6.0). After the column had been washedthoroughly with the same buer but containing 0.1 M NaCl, theenzyme was eluted with the equilibrating buer containing 0.2 MNaCl. Active fractions were collected, combined, concentrated byultraltration (UHP-90 K, Advantec), and then dialyzed against10 mM citrate/20 mM phosphate buer (pH 6.0).

Step 2. The dialyzed enzyme solution was applied to a DEAE-Toyopearl 650 M column (1.520 cm) equilibrated with 10 mMcitrate/20 mM phosphate buer (pH 6.0). After the column hadbeen washed thoroughly with the same buer, the enzyme waseluted with a NaCl linear gradient from 0 to 0.4 M in the samebuer. Active fractions were collected, combined, concentrated byultraltration (UHP-90 K, Advantec), and then dialyzed against10 mM citrate/20 mM phosphate buer (pH 6.0).

Step 3. The dialyzed enzyme solution was placed on aQ-Sepharose Fast Flow column (1.010 cm) equilibrated with10 mM citrate/20 mM phosphate buer (pH 6.0). After the columnhad been washed thoroughly with the same buer, the enzyme waseluted with a NaCl linear gradient from 0 to 0.5 M in the samebuer. Active fractions were collected, combined, concentrated,and the sample buer exchanged with 10 mM citrate/20 mMphosphate buer (pH 6.0) containing 0.15 M NaCl by ultraltra-tion (Centriprep YM-30, Millipore, USA).

Step 4. The exchanged enzyme solution was injected into aSuperdex 200 HR10/30 column equilibrated with 10 mM citrate/20 mM phosphate buer (pH 6.0) containing 0.15 M NaCl, andeluted with the same buer at a ow rate of 0.5 ml/min. Activefractions were pooled, concentrated, and the sample buerexchanged with 25 mM 1-methylpiperazine buer (pH 5.70) byultraltration (Centriprep YM-30, Millipore, USA).

Step 5. The exchanged enzyme solution was injected into aMono P HR 5/5 column. Fractions were eluted with a pH gradientfrom 5.0 to 4.0 generated by 10% Polybuer 74 (pH 3.97), fol-lowed by 20 ml of a NaCl gradient from 0 to 0.4 M in the samebuer at a ow rate of 0.5 ml/min. Active fractions were pooled,dialyzed against 10 mM citrate/20 mM phosphate buer (pH 6.0),and stored at )20C before use.

Enzyme and protein assay

Carboxypeptidase activity was determined using a modication ofthe method of Nakadai [18] with Z-Glu-Tyr as the substrate. Thereaction mixture, containing 40 ll of enzyme solution and 160 ll of5.010)4 M Z-Glu-Tyr in 0.05 M acetate buer (pH 3.5), wasincubated at 37 C for 30 min. One unit of enzyme activity isdened as the amount of enzyme liberating 1 lmol tyrosine fromthe substrate per min under the applied conditions.

Protein concentrations were determined by the method ofBradford [2] with bovine serum albumin as the standard. Absor-bance at 280 nm was used for monitoring protein concentrations inthe fractions during the purication.

Carbohydrate content assay

Carbohydrate content was measured by the phenol sulfuric acidmethod [8] with D-(+)-mannose as standard.

Electrophoresis

PAGE of the enzyme was carried out by the method of Laemmli[15] using an 8.5% polyacrylamide gel in the presence of SDS(SDS-PAGE), or by the method of Davis [5] using a 10% poly-acrylamide gel in the absence of SDS (native PAGE). The proteinand carbohydrate bands in the gels were stained with CoomassieBrilliant blue R-250 and periodic acid-Schi (PAS) reagentaccording to the method of Zacharius [32], respectively. Isoelectricfocusing (IEF)-PAGE was carried out with a pH gradient of 3.59.5 generated by ampholine pH 3.59.5 (Amersham PharmaciaBiotech, USA). The isoelectric point of the puried enzyme wasdetermined using pI marker proteins from a broad pI calibrationkit (Amersham Pharmacia Biotech, USA).

Characterization of the enzyme

The molecular mass of the native and denatured enzyme was esti-mated by gel ltration on a Superdex 200 HR 10/30 column usingthe method of Andrews [1] and by SDS-PAGE using the method ofWeber [26]. Molecular mass marker proteins (Oriental Yeast,Japan) and polypeptide SDS-PAGE standards (Broad Range,Bio-Rad, Japan), respectively were used for calibration.

To estimate the pH and thermal stabilities, the enzyme was pre-incubated at dierent pHs at 37 C for 60 min and at dierenttemperatures at pH 5.0 for 15 min, respectively. Residual activitieswere determined by the above-described method.

To estimate the eects of various compounds on enzymeactivity, the enzyme was pre-incubated with 1 mM of these com-pounds at 37 C for 15 min (DFP and PMSF for 2 h), and theresidual enzyme activity was measured by the above-describedmethod.

The kinetic parameters of the enzyme for Z-Tyr-Glu at pH 4.0and 37 C were calculated from a Lineweaver-Burk plot.

Materials

Monascus purpureus IFO 4478 was obtained from the Institute forFermentation, OSAKA (Osaka, Japan). Synthetic peptides such asZ-Tyr-Glu, Z-Glu-Tyr and Z-Glu-Phe were purchased from thePeptide Institute, Inc. (Osaka, Japan). DEAE-cellulose and DEAE-Toyopearl 650 M were obtained from Whatman Biosystems (Kent,England) and TOSOH (Tokyo, Japan), respectively. Q-SepharoseFast Flow, Superdex 200 HR 10/30, and Mono P HR 5/5 werefrom Amersham Pharmacia Biotech (Uppsala, Sweden). All otherchemicals used were of analytical grade.

Results

Production of the enzyme

Preliminarily screening, carried out using 28 strains ofMonascus, revealed that M. purpureus IFO 4478 exhib-ited the highest ability to produce carboxypeptidase

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(data not shown); therefore, this strain was chosen forproduction of the enzyme. The eect of various nitrogensources in the medium on enzyme production by strainIFO 4478 was examined (Table 1). The enzyme was in-duced by most organic nitrogen sources (SPI, 64.2 mU/ml; tryptone, 53.3 mU/ml; polypeptone, 32.6 mU/ml;peptone, 32.2 mU/ml), but not by urea or inorganicnitrogen sources. SPI exhibited the highest ability toinduce carboxypeptidase, and the addition of SPI at aninitial concentration of 2.0% resulted in maximalactivity.

Purication of the enzyme

The purication procedures are summarized in Table 2.The enzyme was puried to homogeneity, correspond-ing to a 213-fold increase compared to the crude en-zyme, and a 4% yield. The nal enzyme preparationexhibited a single protein band following native-PAGE(Fig. 1B) and IEF-PAGE gel in the absence of urea(native IEF-PAGE, data not shown). However, twoprotein bands were detected following SDS-PAGE(Fig. 1A) and IEF-PAGE in the presence of urea(denatured IEF-PAGE, data not shown). These resultssuggested that the enzyme consists of two dierentpolypeptides.

Characterization of the enzyme

The molecular mass of the native enzyme was estimatedas 132 kDa by gel ltration. The two protein bandsdetected on SDS-PAGE gel with (Fig. 1A) or without2-mercaptoethanol (data not shown) corresponded tomolecular mass of 64 and 67 kDa, respectively. Theseresults indicated that the nal enzyme preparation is aheterodimer with two subunits linked by a non-disuldebond.

PAS staining on the native-PAGE gel showed thatthe puried enzyme is a glycoprotein (Fig. 1C) with17.0% carbohydrate content. The isoelectric point of theenzyme is estimated to be 3.67 by slab IEF electropho-resis. The optimum pH and temperature of the enzymefor Z-Glu-Tyr were around 4.0 and 40 C, respectively.The enzyme is stable between pH 2.0 and 8.0 at 37 Cfor 1 h, and up to 50 C at pH 5.0 for 15 min.

The eects of various metal salts and reagents on theenzyme activity were examined at pH 4.0 as shown inTable 3. The enzyme was completely inhibited by Sn2+

and SDS, strongly inhibited by Pb2, Fe3+, DFP, PMSF,piperastatin A, chymostatin, and PCMB, partiallyinhibited by Hg2+ and TPCK, but not inhibited byEDTA and 1,10-phenanthroline. In addition, the en-zyme was not activated by metal ions, indicating that itis a chymotrypsin-like serine carboxypeptidase, and thatthere may be cysteine and histidine residues but no metalion in or near its active site.

Table 1 Eects of various nitrogen sources on the production ofcarboxypeptidase from Monascus purpureus IFO 4478. Values arethe means of those from three independent assays

Nitrogen source (2% w/v) Activity (mU/mlltrate)

Peptone 32.2Meat extract 19.0Yeast extract 10.1Urea 0Casein 25.2Polypeptone 32.6Tryptone 53.3Soybean protein isolate 64.2Ammonium sulfate 1.5Ammonium phosphatemonobasic

0.7

Ammonium carbonate 0Ammonium chloride 0Ammonium nitrate 0Sodium nitrate 0Potassium nitrate 0

Table 2 Purication ofcarboxypeptidase fromMonascus purpureus IFO 4478

Step Total protein(mg)

Totalactivity (U)

Specic activity(U/mg)

Purication(-fold)

Yield(%)

Crude enzyme 4675 42.4 0.01 1 100DEAE-cellulose 1046 39.0 0.04 4 92DEAE-Toyopearl 650 M 69.5 19.1 0.27 27 45Q-Sepharose Fast Flow 14.7 10.2 0.69 69 24Superdex 200 HR 10/30 3.7 4.7 1.27 127 11Mono P HR 5/5 0.8 1.7 2.13 213 4

Fig. 1AC Electrophoresis of the puried serine carboxypeptidasefrom Monascus purpureus. A SDS-PAGE, B Native-PAGE. Thegels were stained with CBB. C Native-PAGE followed by stainingwith periodic acid-Schi. Lane M Protein markers, lane S puriedenzyme

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The substrate specicity of the enzyme is shown inTable 4. The enzyme has a broad substrate specicitycatalyzing the hydrolysis of various peptides. Among thesubstrates tested, Z-Tyr-Glu was the best substrate ofthe enzyme, followed by Z-Glu-Phe and Z-Glu-Tyr. Theenzyme prefers peptides containing an aromatic or glu-tamic acid in the C-terminal position (P1) or penulti-mate position (P1) of the C-terminus, suggesting thatit possesses chymotrypsin-like activity. The enzyme didnot nearly hydrolyze peptides containing an amide

group in the C-terminal position (P1) or proline orglycine in the penultimate position (P1).

The kinetic parameters of the enzyme for Z-Tyr-Gluat pH 4.0 and 37 C were calculated from a Lineweaver-Burk plot (Fig. 2). The Km, Vmax, kcat, and kcat/Km of theenzyme were 0.86 mM, 0.917 mM min)1, 291 s)1, and339 mM)1 s)1, respectively.

Discussion

In preliminary experiments, we found that M. purpureusIFO 4478 exhibited the highest ability to produce car-boxypeptidase among 28 strains of the genus Monascus.Further experiments on induction of the enzyme byvarious nitrogen compounds suggested that SPI was themost potent inducer of carboxypeptidase from M. pur-pureus IFO 4478. It has also been reported that carb-oxypeptidases from Aspergillus saitoi and Penicilliumjanthinellum are induced by defatted soybean [11, 31].

The M. purpureus IFO 4478 enzyme was puried tohomogeneity and characterized. Inhibitory and speci-city studies on the puried enzyme suggested that it is achymotrypsin-like serine carboxypeptidase. Serinecarboxypeptidases are widely distributed in fungi as wellas in higher plants and animal tissues, but they areparticularly abundant in lamentous fungi [3]. Car-boxypeptidase from M. purpureus IFO 4478, like mostother fungal serine carboxypeptidases, has an acidicoptimal pH and requires a serine residue, but not metalions for its catalytic activity [3]. Serine carboxypeptidasefrom M. purpureus IFO 4478 was strongly inhibited byPCMB, partially by TPCK, indicating that cysteine andhistidine residues are located in or near its active site,similar to the serine carboxypeptidase from yeast [13].

SDS-PAGE and molecular mass analysis of thepuried enzyme revealed that it is a heterodimer with anative molecular mass of 132 kDa. The enzyme com-prises two dierent subunits, of 64 and 67 kDa, linkedby a non-disulde bond. Although carboxypeptidasesfrom other fungi, such as those from A. saitoi [10] andA. niger [14], carboxypeptidase Ib, I, II, and O fromA. oryzae [17, 19, 20, 24], and carboxypeptidase S2 from

Table 3 Eects of various metal salts and reagents on the activityof carboxypeptidase from Monascus purpureus IFO 4478. DTNB5,5-Dithio-bis (2-nitrobenzoic acid), EPNP 1,2-epoxy-3-(p-nitro-phenoxy) propane

Metal salt orreagent (nalconcentration1 mM)

Relativeactivity(%)

Metal salt orreagent (nalconcentration1 mM)

Relativeactivity(%)

None 100 DFP 37MgCl2 108 PMSF 28CaCl2 84 Piperastatin A 30MnCl2 86 TLCK 87FeCl2 90 TPCK 43FeCl3 8 Chymostatin 11CoCl2 93 Elastatinal 113NiCl2 103 STI 88CuCl 63 Leupeptin 104CuCl2 61 PCMB 5ZnSO4 99 MIA 90HgCl2 46 NEM 29PbCl2 5 DTNB 104CdCl2 103 E-64 109SnCl2 0 SDS 0EDTA 98 DTT 1011,10-Phenanthroline 98 2-Mercaptoethanol 90r,r-Dipyridyl 100 Pepstatin A 92Phosphoramidon 107 EPNP 104

Table 4 Substrate specicity of the enzyme

Substrate (nalconcentration 0.5 mM)

Relativeactivity (%)

Z-Glu-Tyr 100Z-Glu-Phe 119Z-Tyr-Glu 134Z-Phe-Ala 77Z-Phe-Gly 24Z-Phe-Leu 21Z-Phe-Tyr 18Z-Ala-Glu 76Z-Gly-Pro-Leu-Gly 59Z-Gly-Leu 8Z-Gly-Phe 3Z-Gly-Pro 3Z-gly-Pro-Leu 0Bz-Gly-Arg 3Bz-Gy-Lys 0Z-Gy-Leu-NH2 4Z-Gly-Phe-NH2 2Bz-Tyr-pNA 0Bz-L-Arg-pNA-HCl 3Leu-Gly-Gly 2

Fig. 2 Lineweaver-Burk plot of M. purpureus IFO 4478 carboxy-peptidase catalyzing the hydrolysis of Z-Glu-Tyr at pH 4.0, 37 C

26

P. janthinellum [9] have similar molecular masses, theyare not heterodimers, but either homodimers or homo-trimers [10] whose subunits are made up of a singlepolypeptide chain linked by disulde bridge. The serinecarboxypeptidase from M. racemosus is also a hetero-dimer [7], but with a lower native molecular mass of52 kDa. That enzyme comprises two dierent subunits,of 31and 18 kDa, linked by a disulde bridge.

Serine carboxypeptidase from M. purpureus, likemost other fungal carboxypeptidases, is an acidic gly-coprotein with an isoelectric point of 3.67 and 17.0%carbohydrate content. The isoelectric point is similar tothat of carboxypeptidase I from A. niger [4] and car-boxypeptidase S1 from P. janthinellum [9]; however, thecarbohydrate content is lower than that of carboxy-peptidase I from A. niger [4] and higher than that ofcarboxypeptidase S1 from P. janthinellum [9].

The relative hydrolysis rates of N-acylpeptides indi-cated that the enzyme, like acid carboxypeptidase fromA. saitoi [10], prefers substrates with bulky amino acids,such as glutamic acid, tyrosine, and phenylalanine, in thepenultimate position (P1) from the C-terminus, but italso prefers substrates with bulky amino acids in theC-terminal position (P1), in contrast to the enzyme fromA. saitoi [10], which prefers substrates with neutral andbasic amino acids, even proline, in the C-terminal posi-tion (P1). The M. purpureus enzyme, like acid carboxy-peptidases from A. oryzae var. viride [17], does nothydrolyze peptides with glycine or proline in the penul-timate position (P1). Z-Tyr-Glu was the best substrate ofcarboxypeptidase from M. purpureus, also similar tocarboxypeptidase Ib from A. oryzae var. viride [17]. Thisis consistent with the ndings that glutamic acid is theone of the most abundant free amino acids found inmatured tofuyo [27, 29]. Z-Glu-Tyr was also reported tobe the best substrate of acid carboxypeptidases III, IV,O-1, and O-2 from A. oryzae [21, 22, 23], carboxypep-tidases Z-1 and Z-2 from A. zychae [16], and carboxy-peptidase S1 from P. janthinellum [9], while Z-Glu-Phewas the best substrate of serine carboxypeptidase fromP. carneus [25]. In the present study, both Z-Glu-Tyr andZ-Glu-Phe were good substrates of carboxypeptidasefrom M. purpureus. Thus, carboxypeptidase fromM. purpureus appears to play an important role inremoving the bitter compounds from soybean proteinsand peptides during the maturation of tofuyo.

This is the rst report on the purication and char-acterization of a serine carboxypeptidase from the genusMonascus. Although it is similar to other fungal serinecarboxypeptidases in its molecular mass, glycoprotein,pI, optimum pH, and substrate specicity, it diers in itsprotein structure (heterodimer, the two subunits ofwhich are not linked by a disulde bond). Therefore, itseems to be a new type of fungal serine carboxypepti-dase. Cloning and sequencing of the gene encoding theM. purpureus serine carboxypeptidase will elucidate themolecular features of the enzyme. In addition, the large-scale synthesis of this enzyme and its application in thefood industry will be the subjects of further studies.

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