Directed evolution of thermotolerant malic enzyme for improved malate production(ENZYMOLOGY, PROTEIN ENGINEERING, AND ENZYME TECHNOLOGY)
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概要
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The directed evolution of the thermotolerant NADP(H)-dependent malic enzyme from Thermococcus kodakarensis was conducted to alter the cofactor preference of the enzyme from NADP(H) to NAD(H). The construction and screening of two generations of mutant libraries led to the isolation of a triple mutant that exhibited 6-fold higher k_<cat>/K_m with NAD^+ than the wild type. We serendipitously found that, in addition to the change in the cofactor preference, the reaction specificity of the mutant enzyme was altered. The reductive carboxylation of pyruvate to malate catalyzed by the wild type enzyme is accompanied by HCO_3^- -independent reduction of pyruvate and gives lactate as a byproduct. The reaction specificity of the triple mutant was significantly shifted to malate production and the mutant gave a less amount of the byproduct than the wild type. When the triple mutant enzyme was used as a catalyst for pyruvate carboxylation with NADH, the enzyme gave 1.2 times higher concentration of malate than the wild type with NADPH. Single-point mutation analysis revealed that the substitution of Arg221 with Gly is responsible for the shift in reaction specificity. This finding may shed light on the catalytic mechanisms of malic enzymes and other related CO_2- and/or HCO_3^- -fixing enzymes.
- 公益社団法人日本生物工学会の論文
著者
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Ohtake Hisao
Department of Biotechnology, Graduate School of Engineering, Osaka University
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Ohtake Hisao
Department Of Biotechnology Graduate School Of Engineering Osaka University
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Honda Kohsuke
Department Of Biotechnology Graduate School Of Engineering Osaka University
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Okano Kenji
Department Of Biotechnology Graduate School Of Engineering Osaka University
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YE Xiaoting
Department of Biotechnology, Graduate School of Engineering, Osaka University
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Morimoto Yumi
Department of Biotechnology, Graduate School of Engineering, Osaka University
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