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Kiyosu, Japan

Yoshino-Yasuda S.,Food Research Center | Nakamura E.,Food Research Center | Ono N.,Food Research Center | Hasegawa O.,Food Research Center | And 9 more authors.
Food Science and Technology Research | Year: 2014

Single disruption of seven acid phosphatase genes (aphB-H) demonstrated that the aphC gene is mainly responsible for acid phosphatase production in the soybean-koji culture of the miso koji mold, A. oryzae KBN630. None of the single disruptions of aphB-H affected growth in the culture. Both the acid phosphatase activity and 5'-IMP dephosphorylation activity of the aphC disruptant decreased by 95% compared to the wild-type strain. Utilizing the promoter of the A. oryzae taaG2 gene, AphC was successfully overproduced in A. oryzae and secreted into liquid culture medium. The purified AphC had a molecular mass of 69.0 kDa, a pH optimum of 4.5, and a temperature optimum of 50?. AphC exhibited high dephosphorylation activity towards the umami flavor enhancers, 5'-IMP and 5'-GMP, supporting the idea that AphC is involved in degradation of disodium 5'-ribonucleotides supplemented in miso products. Copyright © 2014, Japanese Society for Food Science and Technology. Source


Marui J.,Japan National Agriculture and Food Research Organization | Tada S.,Japan National Agriculture and Food Research Organization | Fukuoka M.,Japan National Agriculture and Food Research Organization | Wagu Y.,Bioc | And 6 more authors.
International Journal of Food Microbiology | Year: 2013

Miso (fermented soybean paste) is a traditional Japanese fermented food, and is now used worldwide. The solid-state culture of filamentous fungus, Aspergillus oryzae, grown on rice is known as rice-koji, and is important as a starter for miso fermentation because of its prominent hydrolytic enzyme activities. Recently, commercial miso products have been supplemented with purinic ribonucleotides, such as inosine monophosphate (IMP) and guanine monophosphate, to enhance the characteristic umami taste of glutamate in miso. Because the purinic ribonucleotides are degraded by enzymes such as acid phosphatases in miso, heat inactivation is required prior to the addition of these flavorings. However, heat treatment is a costly process and reduces the quality of miso. Therefore, an approach to lower acid phosphatase activities in koji culture is necessary. Transcriptional analysis using an A. oryzae KBN8048 rice-koji culture showed that eight of the 13 acid phosphatase (aph) genes were significantly down-regulated by the addition of phosphoric acid in the preparation of the culture in a concentration-dependent manner, while aphC expression was markedly up-regulated under the same conditions. The eight down-regulated genes might be under the control of the functional counterpart of the Saccharomyces cerevisiae transcriptional activator Pho4, which specifically regulates phosphatase genes in response to the ambient phosphate availability. However, the regulatory mechanism of aphC was not clear. The IMP dephosphorylation activities in rice-koji cultures of KBN8048 and the aphC deletion mutant (δ. aphC) were reduced by up to 30% and 70%, respectively, in cultures with phosphoric acid, while protease and amylase activity, which is important for miso fermentation, was minimally affected. The miso products fermented using the rice-koji cultures of KBN8048 and δ. aphC prepared with phosphoric acid had reductions in IMP dephosphorylation activity of 80% and 90%, respectively, without any adverse effects on amylase and protease activities. Thus, preparing the A. oryzae rice-koji culture under phosphate-sufficient conditions preferentially produces a fermentation starter of miso exhibiting low purinic ribonucleotide dephosphorylation activity. Moreover, aphC is a potential breeding target to reduce purinic ribonucleotide degradation activity further in commercial miso products. © 2013 Elsevier B.V. Source


Marui J.,Japan National Food Research Institute | Tada S.,Japan National Food Research Institute | Fukuoka M.,Japan National Food Research Institute | Suzuki S.,Japan National Food Research Institute | And 6 more authors.
Food Science and Technology Research | Year: 2012

Aspergillus oryzae KBN8048 was selected as a strain exhibiting low level acid phosphatase activity in solid-state rice and soybean cultures for miso brewing. The activity was particularly low in the latter case, and was correlated with decreased dephosphorylation activity of IMP, an enhancer of miso palatability. Based on transcriptional properties, 13 acid phosphatase genes in the fungus were classified into type R and type S, which exhibited higher expression in solid-state rice and soybean cultures, respectively. Type R genes appeared to be up-regulated in response to acidic pH and limited phosphate availability in the culture via a pH-dependent transcriptional factor, PacC, and a functional counterpart of Saccharomyces cerevisiae Pho4p, which transcriptionally activates phosphatase genes under phosphate-limited conditions, respectively. On the other hand, existence of type S genes suggested an unknown mechanism that responds to inductive regulatory cue(s) other than pH and phosphate availability in solid-state soybean culture. Source


Yoshino-Yasuda S.,Aichi Institute of Technology | Hasegawa O.,Aichi Institute of Technology | Iga Y.,Bioc | Shiraishi Y.,Bioc | And 6 more authors.
Food Science and Technology Research | Year: 2012

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°C. 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. Source

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