| Co-Investigator(Kenkyū-buntansha) |
YOSHIDA Toyokazu Gifu University, Faculty of Engineering, Associate Professor, 工学部, 助教授 (90220657)
MITSUKURA Koichi Gifu University, Faculty of Engineering, Research Associate, 工学部, 助手 (70324283)
MATSUDA Tomoko Tokyo Institute of Technology, Associate Professor, 大学院生命理工学研究科, 講師 (10319494)
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| Budget Amount *help |
¥3,400,000 (Direct Cost: ¥3,400,000)
Fiscal Year 2006: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 2005: ¥2,700,000 (Direct Cost: ¥2,700,000)
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| Research Abstract |
We focused on two kinds of decarboxylases, 4-hydroxybenzoate decarboxylase and 2,6-dihydroxybenzoate decarboxylase, which catalyze reverse carboxylation reaction. We isolated two facultative anaerobes, Enterobacter cloacae P240 and Enterobacter cloacae P241 which produce 4-hydroxybenzoate decarboxylase and 3,4-dihydroxybenzoate decarboxylase, respectively. Agrobacterium tumefaciens IAM12048 and Pandoraea sp. 12B-2 were isolated as 2,6-didydroxybenzoate decarboxylase-producing bacteria. We purified and characterized each enzyme, and analyzed the genes encoding of each enzyme. We optimized the reaction conditions for the reverse carboxylation reaction and applied for the efficient synthesis of 2,6-dihydroxybenzoate an useful chemical, using the whole cells of Pandoraea sp. 12B-2. The enzymes catalyzing the Kolbe-Schmitt carboxylation seem to occur ubiquitously. Some of them, such as 2,6-dihydroxybenzoate decarboxylase catalyzes efficiently the reverse carboxylation reaction and accumulat
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e high concentration of 2,6-dihydroxybenzoate from 1,3-dihydroxybenzene in the presence of 3 M KHCO_3. The facts rouse us to explore the possible application of biological Kolbe-Schmitt carboxylation for the production of aromatic acids. The biological Kolbe-Schmitt carboxylation is a promising new tool for the regiospecific introduction of carboxy groups in precursors of pharmaceuticals and agrochemicals in order to functionalize them and, thus, alter biological activities. Unfortunately, so far, aromatic acid decarboxylases are highly specific for each aromatic substrate, and a number of its analogs are not carboxylated. The gene resources of various aromatic decarboxylases have been gathered and preserved. Since there are various other valuable targets for regiospecific carboxylations, the finding of novel CO_2-fixing enzymes with a wider substrate spectrum is desirable. The screening for other reversible decarboxylases, showing wide substrate specificity and catalyzing regiospecific carboxylation, is significant and potential. Further survey of microbial resources having aromatic decarboxylases will be continued. From now on, for the further application of the reverse carboxylation of decarboxylase, the most important thing is to develop new excellent techniques to shift the reaction equilibrium to the direction of carboxylation. Less
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