Purification, characterization, and heterologous expression in Fusarium venenatum of a novel serine carboxypeptidase from Aspergillus oryzae

A novel serine carboxypeptidase (EC 3.4.16.1) was found in an Aspergillus o ryzae fermentation broth and was purified to homogeneity, This enzyme has a molecular weight of ca. 67,000, as determined by sodium dodecyl sulfate-po lyacrylamide gel electrophoresis, and its specific activity is 21 U/mg for carbobenzoxy (Z)-Ala-Glu at pH 4.5 and 25 degrees C, It has a ratio of bimo lecular constants for Z-Ala-Lys and Z-Ala-Phe of 3.75. Optimal enzyme activ ity occurs at pH 4 to 4.5 and 58 to 60 degrees C for Z-Ala-Ile. The N termi nus of this carboxypeptidase is blocked, Internal fragments, obtained by cy anogen 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 carboxypeptid ase gene was used to transform a Fusarium venenatum host strain. The transf ormed strain off. venenatum expressed an active recombinant carboxypeptidas e. In F. venenatum, the recombinant carboxypeptidase produced two bands whi ch had molecular weights greater than the molecular weight of the native ca rboxypeptidase from A. oryzae, Although the molecular weights of the native and recombinant enzymes differ, these enzymes have very similar kinetic pa rameters.