Purification and characterization of a high molecular mass serine carboxypeptidase from Monascus pilosus

Abstract

Two serine carboxypeptidases, MpiCP-1 and MpiCP-2, were purified to homogeneity from Monascus pilosus IFO 4480. MpiCP-1 is a homodimer with a native molecular mass of 125 kDa composed of two identical subunits of 61 kDa, while MpiCP-2 is a high mass homooligomer with a native molecular mass of 2,263 kDa composed of about 38 identical subunits of 59 kDa. This is unique among carboxypeptidases and distinguishes MpiCP-2 as the largest known carboxypeptidase. The two purified enzymes were both acidic glycoproteins. MpiCP-1 has an isoelectric point of 3.7 and a carbohydrate content of 11%, while for MpiCP-2 these values were 4.0 and 33%, respectively. The optimum pH and temperature were around 4.0 and 50 degrees C for MpiCP-1, and 3.5 and 50 degrees C for MpiCP-2. MpiCP-1 was stable over a broad range of pH between 2.0 and 8.0 at 37 degrees C for 1 h, and up to 55 degrees C for 15 min at pH 6.0, but MpiCP-2 was stable in a narrow range of pH between 5.5 and 6.5, and up to 50 degrees C for 15 min at pH 6.0. Phenylmethylsulfonylfluoride strongly inhibited MpiCP-1 and completely inhibited MpiCP-2, suggesting that they are both serine carboxypeptidases. Of the substrates tested, benzyloxycarbonyl-L: -tyrosyl-L: -glutamic acid (Z-Tyr-Glu) was the best for both enzymes. The Km, Vmax, Kcat and Kcat/Km values of MpiCP-1 for Z-Tyr-Glu at pH 4.0 and 37 degrees C were 1.33 mM, 1.49 mM min(-1), 723 s(-1) and 545 mM(-1) s(-1), and those of MpiCP-2 at pH 3.5 and 37 degrees C were 1.55 mM, 1.54 mM min(-1), 2,039 s(-1) and 1,318 mM(-1) s(-1), respectively.

Debittering effect of Monascus carboxypeptidase during the hydrolysis of soybean protein

“…MpiCP-1, like most of the other known fungal carboxypeptidases [4], such as Aspergillus acid carboxypeptidase [2], is a serine carboxypeptidase with broad substrate specificity [11]. Besides acidic amino acids at the C-termini [11], MpiCP-1 especially prefers peptides containing the hydrophobic and aromatic amino acids.…”

“…Besides acidic amino acids at the C-termini [11], MpiCP-1 especially prefers peptides containing the hydrophobic and aromatic amino acids. As, based on our data, the bitter peptides that resulted from the pepsin hydrolysis of soybean protein are apparently the appropriate substrates of MpiCP-1 [12], we suggest that MpiCP-1 might be able to lessen the bitterness of the pepsin hydrolyzate of soybean protein by decomposing the C-terminal structures of the bitter peptides, as has been shown for Aspergillus acid carboxypeptidase [2] and wheat carboxypeptidase [13].…”

“…Umetsu reported that wheat carboxypeptidase was also able to eliminate the bitter taste in the peptic hydrolyzate of soybean protein [13]. Liu et al [11] reported that a carboxypeptidase 1 that had isolated from Monascus pilosus (MpiCP-1) was in fact a serine carboxypeptidase with a broad specificity similar to that of other fungal carboxypeptidases [4], such as Aspergillus acid carboxypeptidase [2]. In the investigation reported here, we investigated the action of MpiCP-1 during the hydrolysis of soybean protein.…”

Application of an acid proteinase from Monascus purpureus to reduce antigenicity of bovine milk whey protein

“…Among the reported fungal acid proteinases only the penicillopepsin (EC 3.4.23.20), aspergillopepsin (EC 3.4.23.18), rhizopuspepsin (EC 3.4.23.21) and mucorpepsin (EC 3.4.23.23) have been extensively studied [8]. Genus Monascus is one of the emerging sources of enzymes and the species of this genus are frequently utilized in the fermentation industry in East Asia [13,14]. Yasuda and co-workers studied the various enzymes produced by the genus Monascus that is used for the microbial fermentation of tofuyo which is a cheese-like vegetable protein food in Okinawa, Japan [29,[34][35][36].…”

Purification and Characterization of Heterogeneous Glucoamylases fromMonascus purpureus

“…[1][2][3][4][5] This fungus produced proteinases, carboxypeptidases, and amylases, and their enzymatic properties were examined. [6][7][8][9][10] These enzymes play important roles not only in the ripening of tofuyo, but also in the formation of the good taste, and physical properties of this product. Especially, glucoamylase is notable as a key enzyme in the formation of the good taste of the product.…”