Purification, Characterization, and Heterologous Expression in Fusarium venenatum of a Novel Serine Carboxypeptidase from Aspergillus oryzae

Abstract

ABSTRACT A novel serine carboxypeptidase (EC 3.4.16.1 ) was found in an Aspergillus oryzae fermentation broth and was purified to homogeneity. This enzyme has a molecular weight of ca. 67,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and its specific activity is 21 U/mg for carbobenzoxy (Z)-Ala-Glu at pH 4.5 and 25°C. It has a ratio of bimolecular constants for Z-Ala-Lys and Z-Ala-Phe of 3.75. Optimal enzyme activity occurs at pH 4 to 4.5 and 58 to 60°C for Z-Ala-Ile. The N terminus of this carboxypeptidase is blocked. Internal fragments, obtained by cyanogen bromide digestion, were sequenced. PCR primers were then made based on the peptide sequence information, and the full-length gene sequence was obtained. An expression vector that contained the recombinant carboxypeptidase gene was used to transform a Fusarium venenatum host strain. The transformed strain of F. venenatum expressed an active recombinant carboxypeptidase. In F. venenatum , the recombinant carboxypeptidase produced two bands which had molecular weights greater than the molecular weight of the native carboxypeptidase from A. oryzae . Although the molecular weights of the native and recombinant enzymes differ, these enzymes have very similar kinetic parameters.