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 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 degrees 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 degrees 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.
Blinkovsky
A M
AM
Novo Nordisk Biotech, Inc., Davis, California 95616, USA. alex@nnbt.com
Byun
T
T
Brown
K M
KM
Golightly
E J
EJ
D000595:Amino Acid Sequence; D001236:Aspergillus oryzae / Q000201:enzymology* / Q000235:genetics*; D001483:Base Sequence; D002268:Carboxypeptidases / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics; D004151:Dipeptides / Q000737:chemistry; D005670:Fusarium / Q000201:enzymology* / Q000235:genetics*; D005800:Genes, Fungal; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D014170:Transformation, Genetic
D017931:DNA Primers; D018076:DNA, Complementary; D004271:DNA, Fungal; D004151:Dipeptides; D011994:Recombinant Proteins; D002268:Carboxypeptidases; C022633:serine carboxypeptidase