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

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 degreesC, respectively. The enzyme was stable between pH 2.0 and 8.0 at 37 degreesC for 1 h, and up to 50 degreesC 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. Benzyloxvcarbonyl-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 degreesC were 0.86 mM. 0.917 mM min(-1), 291 s(-1), and 339 mM(-1) s(-1), respectively.