Synthesis of organic compounds by the enzyme with modified substrate specificity

L乳酸脱氢酶催化丙酮酸立体定向还原为L乳酸。对在大肠杆菌中克隆的双歧杆菌LDH、水热杆菌LDH和钙叶热杆菌LDH的底物特异性进行了测定。水热菌LDH能催化烷基比丙酮酸更长的2-酮酸的还原，而双歧杆菌LDH不能。通过对LDH三维结构的计算机分析，筛选出与底物识别有关的氨基酸残基，并通过定点突变将这些残基替换为其他氨基酸。对得到的改性LDHs的酶活性进行了测定。结果表明，几种改性LDH的K&lt；CAT&gt；/S&lt；0.5&gt；值是野生型LDH的100~500倍，并测定了它们在双歧杆菌LDH和水热杆菌LDH几种易水有机溶剂中的酶活性。

L-Lactate dehydrogenase (LDH) catalyzes stereo-specific reduction of pyruvate into L-lactate. Substerate specificity of Bifidobacterium LDH, Thermus aquaticus LDH and Thermus calophylus LDH, which were cloned in Esherichia coli, was measured. Thermus aquaticus LDHs can catalyze ther reduction of 2-keto acids having longer alkyl group than pyruvate, although Bifidobacterium LDH cannot.Amino acid residues relating to substrate recognition in the active site of Bifidobacterium LDH were selected using computer analysis of 3-dimensional structure of the LDH.These residues were replaced to other amino acids by site-directed mutagenesis. Enzyme activity of the resulting modified LDHs were measured. k_<cat>/S_<0.5> Values of several modified LDHs were found to be 100 to 500 times higher than that of wild-type LDH.Enzyme activity in several water-missible organic solvents of Bifidobacterium LDH and Thermus aquaticus LDHs was measured.