Improving the Pyrophosphate-inosine Phosphotransferase Activity of Escherichia blattae Acid Phosphatase by Sequential Site-directed Mutagenesis

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概要

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• Escherichia blattae acid phosphatase/phosphotransferase (EB-AP/PTase) exhibits C-5′-position selective pyrophosphate-nucleoside phosphotransferase activity in addition to its intrinsic phosphatase. Improvement of its phosphotransferase activity was investigated by sequential site-directed mutagenesis. By comparing the primary structures of higher 5′-inosinic acid (5′-IMP) productivity and lower 5′-IMP productivity acid phosphatase/phosphotransferase, candidate residues of substitution were selected. Then a total of 11 amino acid substitutions were made with sequential substitutions. As the number of substituted amino acid residues increased, the 5′-IMP productivity of the mutant enzyme increased, and the activity of the 11 mutant phosphotransferases of EB-AP/PTase reached the same level as that of Morganella morganii AP/PTase. This result shows that Leu63, Ala65, Glu66, Asn69, Ser71, Asp116, Thr135, and Glu136, whose relevance was not directly established by structural analysis alone, also plays an important role in the phosphotransferase activity of EB-AP/PTase.

• 2004-05-23

著者

• Asano Yasuhisa

  Biotechnology Research Center, Toyama Prefectural University

• Asano Yasuhisa

  Biotechnology Res. Center Toyama Prefectural Univ.

• Suzuki E

  Institute Of Life Sciences Ajinomoto Co. Inc.

• Suzuki Ei-ichiro

  Institute Of Life Sciences Ajinomoto Co Inc

• Suzuki Ei-ichiro

  Cnetral Research Laboratories Ajinomoto Co. Inc

• Ishikawa Kohki

  Institute Of Life Sciences Ajinomoto Co. Inc.

• MIHARA Yasuhiro

  Applied Microbiology Laboratory, Fermentation and Biotechnology Laboratories, Ajinomoto Co., Inc.

• Mihara Y

  Fermentation And Biotechnology Laboratories Ajinomoto Co. Inc.

• Asano Yasuhisa

  Biotechnology Res. Center And Dep. Of Biotechnology Toyama Prefectural Univ.

• Asano Yasuhisa

  Biotechnology Research Center, Faculty of Engineering, Toyama Prefectural University

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