Heterologous expression and characterization of CpI, OcpA, and novel serine-type carboxypeptidase OcpB from Aspergillus oryzae

Abstract

In the genome of Aspergillus oryzae, 12 genes have been predicted to encode serine-type carboxypeptidases. However, the carboxypeptidase activities of the proteins encoded by these genes have not yet been confirmed experimentally. In this study, we have constructed three of these 12 genes overexpressing strains using Aspergillus nidulans and characterized their overproduced recombinant proteins. Of these three genes, one was previously named cpI; the other two have not been reported yet, and hence, we named them ocpA and ocpB. The recombinant proteins released 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 serine-type carboxypeptidases. Recombinant OcpA, OcpB, and CpI were stable at 45°C, 55°C, and 55°C, respectively, at a low pH. The enzymatic properties of recombinant OcpB were different from those of any reported serine-type carboxypeptidase. On the other hand, recombinant OcpA had similar enzymatic properties to A. oryzae carboxypeptidases O1 and O2. The DNA and N-terminal amino acid sequences of carboxypeptidases O1 and O2 from A. oryzae IAM2640 were similar to those of OcpA. Result of transcriptional analysis of ocpA, ocpB, and cpI suggest differences in transcriptional regulation between these genes.

Molecular Cloning ofocpOEncoding Carboxypeptidase O ofAspergillus oryzaeIAM2640

“…Measurement of carboxypeptidase activity was performed by the ninhydrin method, as previously described. 10,19) One mM Z-Glu-Tyr dissolved in 50 mM acetate buffer (pH 3.7) was used as substrate. One katal of carboxypeptidase was defined as the amount of enzyme required to liberate 1 mole of Tyr from Z-Glu-Tyr per s at 30 C at pH 3.7.…”

“…[4][5][6][7][8] Secreted serine-type carboxypeptidases such as Aspergillus oryzae carboxypeptidase O1 and O2 degrade exogenous proteins as a nitrogen source, because the transcriptional levels of these proteinencoding genes are elevated in the presence of exogenous proteins. 9,10) A. oryzae is a filamentous fungus used in the production of traditional Japanese fermented foods, such as miso (soy bean paste), shoyu (soy sauce), and sake (rice wine). The fungus has good potential for high production of various enzymes, such as proteases and amylases, and is listed as a ''Generally Recognized As Safe (GRAS)'' organisms by the US Food and Drug Administration.…”

“…To date, several serine-type carboxypeptidases from A. oryzae have been purified and characterized. 9,10,13,14) Carboxypeptidase O is one of the enzymes purified from cultured medium of A. oryzae IAM2640. 13) This enzyme showed broad substrate specificity for N-acylpeptides in an acidic pH range.…”

Comparative Analysis of Aspergillus oryzae with Normal and Abnormal Color Conidia

“…The activities of the enzymes: acid protease, neutral protease, alkaline protease, aminopeptidase, carboxypeptidase, glutaminase, a-amylase and pectinase from A. oryzae were examined according to the previous methods [10][11][12][13][14]. To our surprise, the enzyme activity of A. oryzae with anomalous color conidium was not different from the strain with normal color conidium.…”

Serine-Type Carboxypeptidase KexA of Aspergillus oryzae Has Broader Substrate Specificity than Saccharomyces cerevisiae Kex1 and Is Required for Normal Hyphal Growth and Conidiation

“…To investigate the enzymatic properties of KexA, we attempted purification of a truncated form of KexA (amino acid residues 1 to 526 of KexA), which lacked the potential transmembrane region and the succeeding C-terminal sequence (amino acid residues 527 to 625 of KexA), by using a protein expression system with the pIECS3 vector and A. nidulans FGSC A89 (15) (Fig. 1A).…”