Synthesis of organic compounds by the enzyme with modified substrate specificity

l -乳酸脱氢酶（LDH）催化丙酮酸立体特异性还原成l -乳酸。测定了在大肠杆菌中克隆的双歧杆菌LDH、水热杆菌LDH和钙热杆菌LDH的底酯特异性。水生热菌的LDH可以催化丙酮酸还原烷基较长的2-酮酸，而双歧杆菌的LDH不能。利用计算机对双歧杆菌LDH的三维结构进行分析，筛选出与LDH活性位点底物识别相关的氨基酸残基。这些残基通过定点诱变被其他氨基酸取代。测定所得改性ldl的酶活性。k_<cat>/S_<0.5>几种改性LDH的值比野生型LDH高100 ~ 500倍。测定了双歧杆菌LDH和水热菌LDH在几种可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.