Enzymatic characterization of six recombinant serine-type carboxypeptidases of Aspergillus oryzae RIB40

Hiroto Morita[1] Ayako Okamoto[1] Youhei Yamagata[1] Ken-Ichi Kusumoto[2] Yoshinao Koide[3] Hiroki Ishida[4] Michio Takeuchi[

Author address: 

1Tokyo University of Agriculture and Technology, 2National Food Research Institute, 3Amano enzyme Inc., 4Gekkeikan Sake Co. Ltd

Abstract: 

Serine-type carboxypeptidase (SCP) is an exopeptidase that has Ser, His, and Asp residues as a catalytic triad construct and can sequentially release C-terminal amino acid residues of peptides and proteins. In the genome of Aspergillus oryzae RIB40, 12 genes have been predicted to encode SCPs. However, the carboxypeptidase activities of the gene products have not yet been confirmed experimentally. Therefore, we have constructed those gene overexpressing strains using Aspergillus nidulans and characterized their overproduced recombinant proteins. Here, we report enzymatic character of six of the gene products. The recombinant proteins were able to release amino acid residues from the C terminus of peptides, and the activity of the enzymes was inhibited by phenylmethylsulfonyl fluoride, indicating the enzymes to be SCPs. The enzymes were stable at lower than 40-55°C, at low and neutral pH. The optimum pHs of the enzymes except for AOCP16 were around pH 4. That of AOCP16 was pH5.5. The substrate specificities of each enzyme for N-acyl-peptides were different. Result of transcriptional analysis of these genes suggested differences in transcriptional regulation between these genes. The enzymatic properties of AOCP6 and AOCP9 were different from those of any reported SCP. AOCP4 and AOCP13 had similar enzymatic properties to carboxypeptidases O1 and O2 and carboxypeptidase O from A. oryzae IAM2640. Results of sequence analysis of DNA and N-terminal amino acid sequences showed AOCP4 correspond to carboxypeptidases O1 and O2, and AOCP13 correspond to carboxypeptidase O. This study was supported by the Program for Promotion of Basic Research Activities for Innovative Biosciences.
2010

abstract No: 

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Full conference title: 

10th EUROPEAN CONFERENCE ON FUNGAL GENETICS
    • ECFG 10th (2010)