Purification and characterization of a high molecular mass serine carboxypeptidase from Monascus pilosus.
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.
Liu
Fang
F
Department of Bioscience and Biotechnology, Faculty of Agriculture, University of the Ryukyus, 1 Senbaru, Nishihara-cho, Okinawa, 903-0213, Japan.
Tachibana
Shinjiro
S
Taira
Toki
T
Ishihara
Masanobu
M
Kato
Fumio
F
Yasuda
Masaaki
M
D002268:Carboxypeptidases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D019281:Dimerization; D004795:Enzyme Stability; D005285:Fermentation; D006863:Hydrogen-Ion Concentration; D007218:Industrial Microbiology / Q000379:methods*; D007526:Isoelectric Point; D007700:Kinetics; D008970:Molecular Weight; D032741:Monascus / Q000201:enzymology*; D012275:Oryza / Q000382:microbiology*; D013379:Substrate Specificity
D002268:Carboxypeptidases; C022633:serine carboxypeptidase