Purification and characterization of a new type of serine carboxypeptidase from Monascus purpureus

Abstract

Carboxypeptidase produced by Monascus purpureus IFO 4478 was purified to homogeneity. The purified enzyme is a heterodimer with a molecular mass of 132 kDa and consists of two subunits of 64 and 67 kDa. It is an acidic glycoprotein with an isoelectric point of 3.67 and 17.0% carbohydrate content. The optimum pH and temperature were 4.0 and 40 degrees C, respectively. The enzyme was stable between pH 2.0 and 8.0 at 37 degrees C for 1 h, and up to 50 degrees C at pH 5.0 for 15 min. The enzyme was strongly inhibited by piperastatin A, diisopropylfluoride phosphate (DFP), phenylmethylsulfonylfluoride (PMSF), and chymostatin, suggesting that it is a chymotrypsin-like serine carboxypeptidase. Monascus purpureus carboxypeptidase was also strongly inhibited by p-chloromercuribenzoic acid (PCMB) but not by ethylenediaminetetraacetic acid (EDTA) and 1,10-phenanthroline, indicating that it requires cysteine residue but not metal ions for activity. Benzyloxycarbonyl- l-tyrosyl- l-glutamic acid (Z-Tyr-Glu), among the substrates tested, was the best substrate of the enzyme. The K(m), V(max), K(cat), and K(cat) /K(m) values of the enzyme for Z-Tyr-Glu at pH 4.0 and 37 degrees C were 0.86 mM, 0.917 mM min(-1), 291 s(-1), and 339 mM(-1 )s(-1), respectively.

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

“…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][29][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.…”

“…MpiCP-2 was completely inhibited by Hg 2+ 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 Hg 2+ and PCMB, indicating a higher resistance to the actions of Hg 2+ and PCMB actions than MpiCP-2 and MpuCP [26], their active centers may be slightly different structurally.…”

“…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.…”

“…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.…”

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.…”