title: 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.
authors.affiliation: Department of Bioscience and Biotechnology, Faculty of Agriculture, University of the Ryukyus, 1 Senbaru, Nishihara-cho, 903-0213, Okinawa, Japan.
authors.forename: Fang
authors.initials: F
authors.lastname: Liu
authors.forename: Shinjiro
authors.initials: S
authors.lastname: Tachibana
authors.forename: Toki
authors.initials: T
authors.lastname: Taira
authors.forename: Masanobu
authors.initials: M
authors.lastname: Ishihara
authors.forename: Masaaki
authors.initials: M
authors.lastname: Yasuda
mesh_terms: D002268:Carboxypeptidases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D005285:Fermentation; D007218:Industrial Microbiology; D008970:Molecular Weight; D032741:Monascus / Q000201:enzymology*
chemical_list: D002268:Carboxypeptidases; C022633:serine carboxypeptidase