Purification and characterization of a serine carboxypeptidase from Kluyveromyces marxianus.
We purified a carboxypeptidase (CPY) from the yeast of Kluyveromyces marxianus. This enzyme was purified 170 times from a soluble extract of 100000 x g. Purification consisted in a fractionated precipitation with ammonium sulfate and two chromatographic steps consisting of anion exchange chromatography and hydrophobic interactions chromatography. The native enzyme depicted a molecular mass of 67 kDa by gel filtration. This serine carboxypeptidase depicted an optimal pH of 8.5 and was stable at a pH ranging from 6.0 to 9.0, its optimal temperature was of 45 degrees C and was unstable at temperatures above 55 degrees C; Michaelis constant and Vmax for N-benzoyl-l-tyrosine-p-nitroanilide were of 29 microM and 612 microM/min mg of protein, respectively. The enzyme was strongly inhibited by phenylmethylsufonyl fluoride (PMSF) and, to a lesser degree, by trans-epoxysuccinyl-l-leucylamido-(4-guanidine)-butane. This study indicated that K. marxianus carboxypeptidase could be an alternative to other animal-source carboxypeptidases in the industry.
Ramírez-Zavala
Bernardo
B
Departamento de Microbiología, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, México D.F., Mexico.
Mercado-Flores
Yuridia
Y
Hernández-Rodríguez
César
C
Villa-Tanaca
Lourdes
L
D002268:Carboxypeptidases / Q000302:isolation & purification* / Q000378:metabolism*; D002852:Chromatography, Ion Exchange; D004587:Electrophoresis, Agar Gel; D006863:Hydrogen-Ion Concentration; D007218:Industrial Microbiology; D007716:Kluyveromyces / Q000201:enzymology*; D008970:Molecular Weight; D013696:Temperature
D002268:Carboxypeptidases; C022633:serine carboxypeptidase