Introduction

Species in the genus Monascus have traditionally been used in the fermentation industry in East Asia. In China, they are used to produce not only natural colorants but also red wine and red fermented soybean curd. In Okinawa, Japan, they are used for the production of tofuyo, which is a vegetable protein food made from soybean curd by the action of microorganisms. Tofuyo-making using Monascus has been studied in our laboratory [40, 43]. It was found that a large number of free amino acids (e.g., glutamic acid and alanine) are produced during the maturation of tofuyo. This is thought to be the result of the hydrolysis of soybean protein by the various proteolytic enzymes produced by Monascus.

In order to elucidate the functions of these enzymes in the maturation of tofuyo, it is necessary to purify and characterize these enzymes. In a previous study, the production, purification and properties of an acid proteinase from the genus Monascus were reported [42]. Acid proteinase is one of the endopeptidases that hydrolyze proteins at internal sites to peptides, but to only small amounts of free amino acids [41]. Therefore, the action of the acid proteinase was not sufficient to explain the occurrence of the large quantity of free amino acids during the maturation of tofuyo. On the other hand, carboxypeptidase is an exopeptidase that releases free amino acids from the carboxyl termini of peptides or proteins. It is thought to serve as a key enzyme in the production of the taste-conferring amino acids during the maturation of tofuyo [40, 43]. Although a large number of carboxypeptidases have been isolated from various species of fungi, such as Aspergillus saitoi [13, 35], Aspergillus oryzae [28–32, 34, 36], Aspergillus niger [5, 22, 23], Absidia zychae [25], Mucor racemosus [8], Penicillium janthinellum [7, 12, 44, 45], and Pycnoporus sanguineus [15], and carboxypeptidase Y from yeast [11, 17, 20], reports on Monascus enzymes are very few.

In a recent report, we described the purification and characterization of a new type of serine carboxypeptidase (MpuCP) from Monascus purpureus [26]. MpuCP is a serine carboxypeptidase purified from a submerged culture broth of this strain. We now report the purification and characterization of the enzyme from solid station fermentation of Monascus pilosus.

Materials and methods

Materials

M. pilosus IFO 4480 was obtained from the Institute for Fermentation Osaka (IFO) (Osaka, Japan). Synthetic peptides such as benzyloxycarbonyl-l-tyrosyl-l-glutamic acid (Z-Tyr-Glu), Z-Glu-Tyr and Z-Glu-Phe were purchased from the Peptide Institute, (Osaka, Japan). DEAE-cellulose was purchased from Whatman Biosystem (Kent, UK). Phenyl-Toyopearl 650 M and SP-Toyopearl 650 M were obtained from Tosoh (Tokyo, Japan). Superdex 200 HR 10/30 and Superose 6 10/300 GL were from Amersham Pharmacia (Uppsala, Sweden), and all other chemicals used were of analytical grade.

Organism and cultivation

Screening tests were performed with 28 strains of genus Monascus. M. pilosus IFO 4480 was selected for the production of carboxypeptidase. M. pilosus IFO 4480 was cultivated on steamed rice at 32°C for 8 days, obtaining red molded rice (red koji). The enzyme was extracted from red koji with three volumes of 10 mM citrate/20 mM phosphate buffer (pH 5.0) at 4°C for 24 h with stirring. The mixture was filtered through nylon cloth and then centrifuged at 10,000 g for 30 min. The supernatant was used as a crude enzyme solution for purification.

Enzyme purification

All subsequent manipulations, except HPLC, were carried out at 4°C.

Step 1

The crude enzyme solution was loaded onto a DEAE-cellulose column (2.5×30 cm) equilibrated with 10 mM citrate/20 mM phosphate buffer (pH 5.0). After washing the column thoroughly with the same buffer containing 0.1 M NaCl, the enzyme was eluted with the same buffer containing 0.3 M NaCl. Active fractions were collected and adjusted to 4 M NaCl.

Step 2

The adjusted enzyme solution was applied to a Phenyl-Toyopearl 650 M column (2.5×20 cm) equilibrated with 10 mM citrate/20 mM phosphate buffer (pH 5.0) containing 4 M NaCl. After washing the column thoroughly with the same buffer, the enzyme was eluted with a decreasing linear gradient of NaCl from 4 to 0 M in 10 mM citrate/20 mM phosphate buffer (pH 5.0). Active fractions were collected and concentrated by ultrafiltration (UHP-90 K; Advantec, Japan). The concentrate was dialyzed against 10 mM citrate/20 mM phosphate buffer (pH 3.5).

Step 3

The dialyzed enzyme solution was applied to a SP-Toyopearl 650 M column (2.5×15 cm) equilibrated with 10 mM citrate/20 mM phosphate buffer (pH 3.5). After washing the column thoroughly with the same buffer, the enzyme was eluted with an increasing linear gradient of NaCl from 0 to 0.1 M in the same buffer. Active fractions were collected and concentrated, followed by exchange with 10 mM citrate/20 mM phosphate buffer (pH 6.0) containing 0.15 M NaCl by ultrafiltration (Centriprep YM-30; Millipore, Bedford, Mass.).

Step 4

The exchanged enzyme solution was injected into a Superdex 200 HR10/30 column equilibrated with 10 mM citrate/20 mM phosphate buffer (pH 6.0) containing 0.15 M NaCl, and eluted with the same buffer at a flow rate of 0.4 ml min−1. Active fractions were pooled and concentrated, followed by exchange with 10 mM citrate/20 mM phosphate buffer (pH 6.0) by ultrafiltration (Centriprep YM-30) and stored at −20°C until used.

Enzyme and protein assay

Carboxypeptidase activity was determined by the method of Nakadai [29] using Z-Glu-Tyr as the substrate, with a slight modification. The reaction mixture, containing 40 μl enzyme solution and 160 μl 5.0×10−4 M Z-Glu-Tyr in 0.05 M acetate buffer (pH 3.5) was incubated at 37°C for 30 min. One unit of enzyme activity was defined as the amount of enzyme that liberates 1 μmol tyrosine from the substrate per minute under the conditions described above. When the other substrates were used, the appropriate amino acids were measured, for example, glutamic acid for Z-Tyr-Glu.

Protein concentration was determined with a DC protein assay kit (Bio-Rad, Japan) with bovine serum albumin as the standard. Absorbance at 280 nm was used for monitoring protein concentrations in the fractions during the purification.

Carbohydrate content assay

Carbohydrate content was measured by the phenol sulfuric acid method [9] with d(+)-mannose as standard.

Electrophoresis

Polyacrylamide gel electrophoresis (PAGE) of the enzymes was performed by the method of Laemmli [24] with 8.5% polyacrylamide gel in the presence of sodium dodecyl sulfate (SDS) and 2-mercaptoethanol (SDS-PAGE), or by the method of Davis [6] with 10% polyacrylamide gel in the absence of SDS (native-PAGE). The protein and carbohydrate bands in the gels were stained with Coomassie Brilliant Blue R-250 (CBB) and periodic acid-Schiff (PAS) reagent, respectively, according to the method of Kapitany [18]. Isoelectric focusing polyacrylamide gel electrophoresis (IEF-PAGE) was carried out with a gradient of pH 3.5–9.5 generated by ampholine pH 3.5–9.5 (Amersham Pharmacia). The isoelectric points of the purified enzymes were determined with a broad pI calibration kit (Amersham Pharmacia) used as pI marker proteins.

Enzyme characterization

The molecular masses of the native enzymes were estimated by gel filtration on a Superose 6 10/300 GL column using the methods of Whitaker [39] and San-Marina and Nicholls [33]. The column was calibrated with high and low molecular weight gel filtration calibration kits as described in the supplier’s instructions (Amersham Biosciences), and the apparent molecular masses were calculated from their elution positions relative to standard proteins. The molecular masses of the denatured enzymes were estimated by SDS-PAGE using the method of Weber et al. [38] with polypeptide SDS-PAGE standards (Broad Range; Bio-Rad, Japan) for calibration.

To estimate the pH and thermal stabilities, MpiCP-1 and MpiCP-2 were pre-incubated at different pH values at 37°C for 60 min and at different temperatures at pH 6.0 for 15 min, respectively. The residual activities were determined by the method described above.

To estimate the effects of various metal salts and compounds on enzyme activity, MpiCP-1 and MpiCP-2 were pre-incubated with 1 mM of various compounds at 37°C and pH 6.0 for 15 min. The residual activities were measured by the method described above.

The kinetic parameters of MpiCP-1 and MpiCP-2 for Z-Tyr-Glu at pH 4.0 and pH 3.5, respectively, at 37°C were calculated from Lineweaver-Burk plots.

Results

Enzyme production

A screening test of 28 strains of Monascus in solid culture revealed that Monascus sp. G-1 exhibited the highest ability to produce carboxypeptidase but a much lower ability to produce proteinase. M. pilosus IFO 4480 showed the second highest ability to produce carboxypeptidase and the highest ability to produce proteinase (Table 1). Because it was considered that it is the combined actions of proteinase and carboxypeptidase in the solid culture product, red koji, that play an important role in the formation of taste-conferring free amino acids during the maturation of tofuyo, rather than their individual actions, we selected M. pilosus IFO 4480 for production of the enzymes.

Table 1 Production of carboxypeptidase by various strains of genus Monascus in solid culture
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Enzyme purification

The enzyme purification procedures are summarized in Table 2. The two carboxypeptidases, MpiCP-1 and MpiCP-2, were purified 22-fold with yields of 5% and 1%, respectively. Both MpiCP-1 and MpiCP-2 exhibited a single protein band on SDS-PAGE gel (Fig. 1), native-PAGE (data not shown) and IEF-PAGE gel (data not shown), suggesting that both were purified to homogeneity and consisted of only a single polypeptide.

Table 2 Purification of carboxypeptidases from Monascus pilosus IFO 4480
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Fig. 1
figure 1

SDS-PAGE, stained with coomassie brilliant blue (CBB), of carboxypeptidases purified from Monascus pilosus IFO 4480. Lanes: 1 Purified MpiCP-1, 2 purified MpiCP-2, M protein markers

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Enzyme characterization

Some properties of MpiCP-1 and MpiCP-2 are summarized in Table 3. Although MpiCP-1 and MpiCP-2 had the same optimum temperature of 50°C, they had many different properties from each other. The molecular masses of native MpiCP-1 and MpiCP-2 were estimated as 125 kDa (Fig. 2a) and 2,263 kDa (Fig. 2b) by gel filtration, while the protein bands detected on SDS-PAGE gels with 2-mercaptoethanol corresponded to molecular masses of 61 and 59 kDa, respectively (Fig. 1), indicating that MpiCP-1 and MpiCP-2 are a homodimer and a homooligomer, respectively. Purified MpiCP-1 and MpiCP-2 are glycoproteins, as demonstrated by the fact that the carbohydrate bonds stained by PAS had the same mobilities as those of the protein bands stained by CBB on SDS-PAGE gels (data not shown). The carbohydrate contents of MpiCP-1 and MpiCP-2 were estimated at 11 and 33%, respectively. The isoelectric points were estimated at 3.7 and 4.0, respectively, by IEF-PAGE (data not shown). The optimum pH values for activity of MpiCP-1 and MpiCP-2 against Z-Glu-Tyr were around 4.0 and 3.5, respectively. MpiCP-1 was stable between pH 2.0 and 8.0 at 37°C for 1 h and up to 55°C at pH 6.0 for 15 min, but MpiCP-2 was stable between pH 5.5 and 6.5 at 37°C for 1 h and up to 50°C at pH 6.0 for 15 min.

Table 3 Properties of Monascus carboxypeptidases
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Fig. 2a,b
figure 2

Chromatograms of carboxypeptidases purified from M. pilosus IFO 4480 on a Superose 6 gel filtration column. The relative elution positions of blue dextran (2,000 kDa) and various standard proteins: thyroglobulin (670 kDa), ferritin (440 kDa), albumin (67 kDa), ovalbumin (43 kDa) and ribonuclease (13.7 kDa) are indicated. a MpiCP-1, b MpiCP-2

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The effects of various metal salts on the activities of MpiCP-1 and MpiCP-2 are summarized in Table 4. No metal salt activated both MpiCP-1 and MpiCP-2; MpiCP-2 was completely inhibited by Hg2+ and strongly inhibited by Fe3+, Cu+, Fe2+ and Cu2+ ions, while no metal salt significantly affected MpiCP-1.

Table 4 Effects of various metal salts on the activities of carboxypeptidases from M. pilosus IFO 4480
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The effects of various reagents on the activities of MpiCP-1 and MpiCP-2 are summarized in Table 5. MpiCP-1 was strongly, and MpiCP-2 completely, inhibited by phenylmethylsulfonylfluoride (PMSF); moreover, diisopropylfluorophosphate (DFP) also inactivated both of them to some extent, suggesting that they are both serine carboxypeptidases. MpiCP-2 was completely inhibited by chymostatin and strongly by p-chloromercuribenzoate (PCMB), MpiCP-1 was also slightly affected by these reagents, suggesting that they are both chymotrypsin-like serine carboxypeptidases and may have a cysteine residue in or near their active sites. On the other hand, neither MpiCP-1 nor MpiCP-2 were inhibited by 1,10-phenanthroline, suggesting that no metal ion is in or near their active sites. In addition, MpiCP-1 was strongly inhibited by piperastatin A and SDS, but MpiCP-2 was not significantly affected by these two reagents.

Table 5 Effects of various reagents on the activities of carboxypeptidases from Monascus pilosus IFO 4480. DFP Diisopropylfluorophosphate ; PMSF phenylmethylsulfonylfluoride; TLCK –Nα-tosyl-l-lysine-chloromethyl ketone; PCMB p-chloromercuribenzoate; MIA monoiodacetic acid; NEM N-ethylmaleimide; DTNB 5,5′-dithio-bis (2-nitrobenzoic acid); EPNP 1,2-epoxy-3-(p-nitrophenoxy) propane
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The substrate specificities of MpiCP-1 and MpiCP-2 are shown in Table 6. Both had broad substrate specificities, catalyzing the hydrolysis of various peptides. They had some similar specificities: of the substrates tested, Z-Tyr-Glu was the best, and Z-Phe-Ala and Z-Glu-Tyr were also good substrates for both enzymes. Moreover, both preferred peptides containing an aromatic or glutamic acid in the C-terminal (P1′) or penultimate (P1) position of the C-termini, but scarcely hydrolyze peptides containing an amide group or proline in the P1′ or P1 position. On the other hand, they also had some very different specificities from each other. MpiCP-1 exhibited a good reaction with Z-Glu-Phe, but MpiCP-2 did not hydrolyze this peptide at all. In addition, MpiCP-2 reacted highly to peptides containing basic amino acids (Arg and Lys) in the P1′ position, even glycine in the P1 position, and to peptides containing glycine in the P1′ position when a hydrophobic amino acid was in the P1 position, but MpiCP-1 scarcely hydrolyzed these peptides. Interestingly, like MpiCP-1, except when basic amino acid (Arg and Lys) residues were found in the P1′ position, MpiCP-2 did not hydrolyze peptides containing a glycine residue in the P1 position at all.

Table 6 Substrate specificities of carboxypeptidases from M. pilosus IFO 4480
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The kinetic parameters of MpiCP-1 and MpiCP-2 for Z-Tyr-Glu at pH 4.0 and 3.5, respectively, at 37°C were calculated from Lineweaver-Burk plots (Fig. 3). The Km, Vmax, Kcat and Kcat/Km of MpiCP-1 were 1.33 mM, 1.49 mM min−1, 723 s−1 and 545 mM−1 s−1, respectively; and the Km, Vmax, Kcat and Kcat/Km of MpiCP-2 were 1.55 mM, 1.54 mM min−1, 2,039 s−1 and 1,318 mM−1 s−1, respectively.

Fig. 3a,b
figure 3

Lineweaver-Burk plots of carboxypeptidases from M. pilosus IFO 4480 catalyzing the hydrolysis of Z-Tyr-Glu at 37°C. a MpiCP-1 at pH 4.0, b MpiCP-2 at pH 3.5

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Discussion

We recently reported that, among 28 strains of genus Monascus, M. purpureus IFO 4478 was the best producer of carboxypeptidase in submerged culture [26]. As part of a systematic study of the proteolytic enzymes produced by Monascus, we examined the carboxypeptidase produced by Monascus in solid culture. The screening test carried out using 28 strains of the genus Monascus for proteolytic enzymes activities in solid culture revealed that Monascus sp. G-1 exhibited the highest ability to produce carboxypeptidase, followed by M. pilosus IFO 4480, but that of M. purpureus IFO 4478 was much lower. However, M. pilosus IFO 4480 exhibited the highest ability to produce proteinase, but M. purpureus IFO 4478 and Monascus sp. G-1 were much lower. Furthermore, in a preliminary experiment, we also found that M. pilosus IFO 4480 was one of the six best strains for carboxypeptidase production in submerged culture among the 28 Monascus strains tested. The above results revealed that the genus Monascus has different abilities to produce proteolytic enzymes in submerged and solid cultures. We postulated that the combined actions of proteinase and carboxypeptidase in a solid culture product, red koji, not their individual actions, played an important role in the formation of tasty free amino acids during the maturation of tofuyo. Therefore, we selected M. pilosus IFO 4480, which had good ability to produce not only proteinase but also carboxypeptidase in solid culture and could produce carboxypeptidase well in submerged culture, as our study strain.

Two enzymes, MpiCP-1 and MpiCP-2, were purified to homogeneity from extracts of red koji produced by M. pilosus IFO 4480, and characterized. Inhibitor studies on the purified enzymes suggested that they are both serine carboxypeptidases. Serine carboxypeptidases are widely distributed in fungi as well as in higher plants and animal tissues, but they are particularly abundant in filamentous fungi [4]. MpiCP-1 and MpiCP-2, like most other fungal serine carboxypeptidases, had an acidic optimal pH and required serine residues, but did not require metal ions for its catalytic activity [4]. MpiCP-2 was completely inhibited by Hg2+ and strongly by PCMB. These results suggest the existence in or near the active sites of a sulfhydryl group, which is important for the tertiary structures of proteins like serine carboxypeptidases from M. purpureus (MpuCP) [26], carboxypeptidase Y from yeast [11] and carboxypeptidase S3 from P. janthinellum [7], carboxypeptidase from Paecilomyces carneus [37] and CPZ-1 and CPZ-2 from Absidia zychae [25]. However, since MpiCP-1 was only slightly inhibited by Hg2+ and PCMB, indicating a higher resistance to the actions of Hg2+ and PCMB actions than MpiCP-2 and MpuCP [26], their active centers may be slightly different structurally.

MpiCP-1 and MpiCP-2 were distinguishable by their molecular mass. MpiCP-1 had a molecular mass of 125 kDa. This value is the same as that of carboxypeptidase from A. saitoi [35] and similar to those of other most reported fungal carboxypeptidases, such as MpuCP [26] and carboxypeptidases from A. niger [14, 19, 23], carboxypeptidase Ib, I, II, and O from A. oryzae [28–30, 34], and carboxypeptidase S2 from P. janthinellum [12]. Although MpuCP was reported to be a heterodimer with a native molecular mass of 132 kDa composed of two different subunits of 64 and 67 kDa [26], most of the carboxypeptidases mentioned above [12, 23, 34, 35] are homodimers. In the present study, MpiCP-1 also seemed to be a homodimer composed of two identical subunits of 61 kDa. This distinguishes MpiCP-1 from MpuCP. Larger molecular mass forms of fungal carboxypeptidase, such as carboxypeptidase Ia from A. oryzae (230 kDa) [28] and carboxypeptidase S3 from P. janthinellum (360 kDa) [7], have also been reported. The latter is a homohexamer composed of six identical subunits of 62 kDa. MpiCP-2, like most other fungal carboxypeptidases, also seems to have a homomeric structure composed of a single polypeptide chain of about 59 kDa. However, it is a high molecular mass homooligomer with a native molecular mass of 2,263 kDa, which is remarkably higher than those of other carboxypeptidases, even that of the larger protease (900 kDa) [16], although lower than that of the largest protease (5,500 kDa) [2], and comprises about 38 identical subunits. Thus, MpiCP-2 may be the largest serine carboxypeptidase reported.

MpiCP-1 and MpiCP-2, like MpuCP and most other fungal carboxypeptidases, are acidic glycoproteins and exhibit maximal activity in the acidic pH range [4]. MpiCP-1 has an isoelectric point of 3.7 and a carbohydrate content of 11%, whereas MpiCP-2 has an isoelectric point of 4.0 and a carbohydrate content of 33%. The isoelectric point of MpiCP-1 is same as that of MpuCP [26], and similar to those of carboxypeptidase I from A. niger [5], carboxypeptidase Y from yeast [17] and carboxypeptidase S1 from P. janthinellum [12]. However, its carbohydrate content is higher than that of carboxypeptidase S1 from P. janthinellum [12], but lower than that of MpuCP [26], carboxypeptidase I from A. niger [5] and carboxypeptidase Y from yeast [17]. The isoelectric point of MpiCP-2 is similar to those of carboxypeptidase S2 from P. janthinellum [12] and the carboxypeptidase from A. niger var. macrosporus [23]; however, its carbohydrate content is higher than either of the latter. In addition, although the carbohydrate content of MpiCP-2 is similar to that of carboxypeptidase S3 from P. janthinellum [7], their isoelectric points are different.

The results of the relative hydrolysis rates of the two enzymes to N-acylpeptides revealed that both MpiCP-1 and MpiCP-2 have broad substrate specificities. Like MpuCP [26] and carboxypeptidase from A. saitoi [13], they prefer substrates with bulky amino acids, such as glutamic acid, tyrosine and phenylalanine, in the penultimate position (P1). They also prefer substrates with bulky amino acids in the C-terminal position (P1′), like MpuCP [26], but unlike carboxypeptidase from A. saitoi [13], which prefers substrates with neutral and basic amino acids, even proline, in the P1′ position. Moreover, both MpiCP-1 and MpiCP-2 only poorly hydrolyze peptides containing an amide group or proline in the P1′, proline or glycine in the P1 or P1′ position, like MpuCP [26] and carboxypeptidases from A. oryzae var. viride [28], but unlike carboxypeptidase from A. saitoi [13], carboxypeptidase O-1 from A. oryzae [36] and carboxypeptidase Y from yeast [11]. Both MpiCP-1 and MpiCP-2 exhibited the highest reactivity to Z-Tyr-Glu, MpiCP-1 also exhibited a good reaction with Z-Glu-Phe, like MpuCP [26]; MpiCP-2 did not hydrolyze the latter substrate at all. Otherwise, MpiCP-2 also exhibited high activity towards peptides containing basic amino acids (Arg and Lys) in the P1′ position, even glycine in the P1 position, or a glycine residue in the P1′ position when there was a hydrophobic amino acid in the P1 position, like the carboxypeptidase from A. saitoi [13], but MpiCP-1 scarcely hydrolyzed these peptides. Although Z-Tyr-Glu was found to be the best substrate for both MpiCP-1 and MpiCP-2, like MpuCP [26] and carboxypeptidase Ib from A. oryzae var. viride [28], and both MpiCP-1 and MpiCP-2 had similar Km and Vmax values toward Z-Tyr-Glu, MpiCP-2 had higher Kcat, and Kcat/Km values than MpiCP-1. This may be because MpiCP-2 has more catalytic sites in its macromolecule than MpiCP-1. The result that both MpiCP-1 and MpiCP-2 exhibited higher activity towards Z-Tyr-Glu, Z-Ala-Glu and Z-Phe-Ala is consistent with findings that glutamic acid and alanine were the two most abundant free amino acids in the matured tofuyo [40, 43]. Z-Glu-Tyr was reported to be the best substrate for acid carboxypeptidase III, IV, O-1 and O-2 from A. oryzae [31, 32, 36], carboxypeptidases Z-1 and Z-2 from A. zychae [25], and carboxypeptidase S1 from P. janthinellum [12], and Z-Glu-Phe was the best substrate for serine carboxypeptidase from P. carneus [37], while Z-Phe-Leu was the best for the carboxypeptidase from Mucor racemsus [8] and carboxypeptidase Y from yeast [11, 17]. In the present study, Z-Glu-Tyr and Z-Phe-Leu were also found to be good substrates for MpiCP-1 and MpiCP-2, and Z-Glu-Phe also for MpiCP-1, like the substrates of MpuCP [26]. From this point of view, it may be considered that MpiCP-1 and MpiCP-2, like MpuCP [26] and Aspergillus acid carboxypeptidase [1], play an important role in removing the bitter compounds, which are predominantly composed of hydrophobic amino acids and peptides with tyrosine or related amino acids at their C-termini [27], from soybean protein and peptides during maturation of tofuyo. Furthermore, the broad specificity of MpiCP-1 and MpiCP-2 mentioned above maybe make them applicable to sequence determination of the C-termini of peptides and proteins like carboxypeptidase Y from yeast [11] and carboxypeptidase from A. niger [5].

This is the first report on the purification and characterization of a serine carboxypeptidase from a member of the genus Monascus. Two forms of serine carboxypeptidases, MpiCP-1 and MpiCP-2, were purified form Monascus pilosus and characterized. MpiCP-2 is a macromolecule with a native molecular mass of 2,263 kDa composed of about 38 identical subunits of 59 kDa. It is the largest form of serine carboxypeptidase reported to date. Although some high molecular mass multimers of the proteasome [10], tripeptidyl peptidase [27], tricon protease [3] and metalloproteinase [2, 16], have been reported, this is the first report of a high molecular mass multimer of carboxypeptidase. In addition to having the highest molecular mass, it is also different from other forms of serine carboxypeptidase from Monascus in optimum pH, pH and thermal stability, pI and substrate specificity, and especially carbohydrate content. It remains unclear whether the differences in physicochemical and enzymatic properties of the enzymes are related to their differences in molecular mass, carbohydrate content or amino acid sequence. Therefore, cloning and sequencing of the genes encoding these enzymes will be the subject of further studies to clearly elucidate their molecular features and functions. Large-scale process studies with these enzymes and their application in the food industry are also necessary directions for further studies.