Disruption and Overexpression of Acid Phosphatase Gene (aphA) from a Miso Koji Mold, Aspergillus oryzae KBN630, and Characterization of the Gene Product

Abstract

An acid phosphatase gene (aphA gene) from an industrial miso koji mold strain, A. oryzae KBN630, was disrupted by using the recently developed homologous gene replacement system for this strain. The aphA gene disruption did not affect growth on steamed soybean. Acid phosphatase production decreased by approximately 20% in the aphA gene disruptants compared with that of the wild-type strain. Utilizing the promoter of the A. oryzae TEF1 gene, AphA expressed in A. oryzae was successfully secreted into the culture medium. AphA had a molecular mass of 58.0 to 65.0 kDa, a pH optimum of 4.0, and a temperature optimum of 40℃. AphA had the ability to release inorganic phosphate from GMP and IMP. This is the first report to show directly that an A. oryzae acid phosphatase has the ability to hydrolyze GMP and IMP.

Characterization of Acid Phosphatase (AphC) from the Miso Koji Mold, Aspergillus oryzae KBN630: AphC is Mainly Responsible for Both Acid Phosphatase Activity and 5′-IMP Dephosphorylation Activity in Soybean-Koji Culture

“…The methods used to construct A. oryzae PDE1 will be published elsewhere. A. oryzae ΔaphA was used as an aphA gene disrupted strain (Yoshino-Yasuda et al, 2012 oryzae was carried out as described previously (Kitamoto et al, 1995).…”

“…The results of AphK1 were taken from the report by Shimizu (Shimizu 1993 The results of AphA were taken from our previous report (Yoshino-Yasuda et al 2012). The results of ACP-I, ACP-II and ACP-III were taken from the report by Fujita et al (Fujita et al 2003a).…”

“…Further studies are contemplated to verify the effectiveness of the aphC disruptant in laboratory scale miso brewing using soybean-koji culture. It is also necessary to consider the pos- The results of AphA were taken from our previous report (Yoshino-Yasuda et al 2012). …”

“…niger phyA gene were identified in the A. oryzae genome database by BLAST searches (Yoshino-Yasuda et al, 2012, Marui et al, 2012. In our previous report, we directly showed that acid phosphatase A encoded by the aphA gene (GenBank accession number AB042805 / AP007157) in A. oryzae possesses some level of 5'-IMP and 5'-GMP dephosphorylation (Yoshino-Yasuda et al, 2012).…”

“…In our previous report, we directly showed that acid phosphatase A encoded by the aphA gene (GenBank accession number AB042805 / AP007157) in A. oryzae possesses some level of 5'-IMP and 5'-GMP dephosphorylation (Yoshino-Yasuda et al, 2012). Disruption of the aphA gene in A. oryzae KBN630 resulted in only a 20% reduction in acid phosphatase activity in soybean-koji culture, a starter culture for soybean-miso brewing unique to the Chukyo area of Japan (Yoshino-Yasuda et al, 2012).…”