TY - JOUR
T1 - Purification and characterization of a serine carboxypeptidase from Kluyveromyces marxianus
AU - Ramírez-Zavala, Bernardo
AU - Mercado-Flores, Yuridia
AU - Hernández-Rodríguez, César
AU - Villa-Tanaca, Lourdes
N1 - Funding Information:
This study was supported by a research grant from CONACYT (26437-N) and CGPI-IPN (20020679). B. Ramı́rez-Zavala and Y. Mercado-Flores were fellows from CONACYT and PIFI-IPN. L. Villa-Tanaca and C. Hernández-Rodrı́guez received COFAA-IPN and EDD-IPN support. L. Villa-Tanaca was hired by “Programa de Contratación para el Apoyo a la Investigación y el Posgrado del IPN”.
PY - 2004/3/15
Y1 - 2004/3/15
N2 - We purified a carboxypeptidase (CPY) from the yeast of Kluyveromyces marxianus. This enzyme was purified 170 times from a soluble extract of 100 000 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°C and was unstable at temperatures above 55°C; Michaelis constant and Vmax for N-benzoyl-L-tyrosine-p-nitroanilide were of 29 μM and 612 μM/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.
AB - We purified a carboxypeptidase (CPY) from the yeast of Kluyveromyces marxianus. This enzyme was purified 170 times from a soluble extract of 100 000 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°C and was unstable at temperatures above 55°C; Michaelis constant and Vmax for N-benzoyl-L-tyrosine-p-nitroanilide were of 29 μM and 612 μM/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.
KW - Kluyveromyces marxianus
KW - Protease
KW - Purification
KW - Serine carboxypeptidase
UR - http://www.scopus.com/inward/record.url?scp=1242338167&partnerID=8YFLogxK
U2 - 10.1016/S0168-1605(03)00409-4
DO - 10.1016/S0168-1605(03)00409-4
M3 - Artículo
SN - 0168-1605
VL - 91
SP - 245
EP - 252
JO - International Journal of Food Microbiology
JF - International Journal of Food Microbiology
IS - 3
ER -