荧光假单胞菌短链脱氢酶的克隆、表达及酶学性质分析

Cloning,expression and characterization of a short-chain dehydrogenase from Pseudomonas fluorescens

为了研究荧光假单胞菌中短链脱氢酶的生理角色和催化特性,从荧光假单胞菌Pseudomonas fluorescens GIM1.49基因组DNA克隆表达了一个短链脱氢酶的编码基因pfd,并分析了该基因产物的酶学性质。基因pfd全长684 bp,编码227个氨基酸,推算分子量为24.2 kDa。将携带短链脱氢酶基因的重组质粒pET28b-pfd转入大肠杆菌BL21(DE3)进行表达,得到了28 kDa的表达产物。重组荧光假单胞菌短链脱氢酶(PFD)能氧化4-氯-3-羟基丁酸乙酯、1-苯乙醇、苯甲醇、仲丁醇和还原4-氯-乙酰乙酸乙酯、2-溴-苯乙酮、4-溴-苯乙酮等底物。以4-氯-3-羟基丁酸乙酯为底物时活力最高,Km值为186.40 mmol/L,Vmax为89.56 U/mg。氧化4-氯-3-羟基丁酸乙酯时,最适反应温度和pH分别为12℃和10.5,倾向于利用NAD+作辅酶;而还原4-氯-乙酰乙酸乙酯时,最适温度和pH为24℃和8.8,倾向于利用NADPH作辅酶。重组PFD能耐受50%(V/V)的甲醇等有机助溶剂,Ca2+(1 mmol/L)和EDTA(5 mmol/L)对其酶活有一定的促进作用。上述结果表明,重组PFD是一个新型的短链脱氢酶,其代谢角色推测与卤代次级醇的氧化降解有关。

To explore the physiological role and biocatalytic properties of short-chain dehydrogenases from Pseudomonas fluorescens GIM1.49,we cloned the structural gene pfd and characterized its over-expressed product.The length of gene pfd was 684 bp encoding a short-chain dehydrogenase with 227 amino acid residues and calculated molecular mass of 24.2 kDa.The recombinant plasmid pET28b-pfd was constructed and functionally expressed in Escherichia coli BL21（DE3）,resulting in the over-production of recombinant short-chain dehydrogenase PFD with a size of 28 kDa.The enzyme could oxidize alcohols including 4-chloro-3-hydroxbutanoate ester and reduce 4-chloro-acetoacetate ester using either NAD（H） or NADP（H） as coenzyme.The enzyme showed the highest activity against 4-chloro-3-hydroxbutanoate ester as substrate,with Km of 186.40 mmol/L and Vmax of 89.56 U/mg.When catalying the oxidative reaction,its optimal temperature was 12 ？ムand optimal pH was 10.5,in contrast to the values of 24 ？ムand pH 8.8 in the reductive reaction.The enzyme had high solvent tolerance and its activity was improved by the addition of Ca2＋（1 mmol/L） or EDTA（5 mmol/L）.These results indicated that the enzyme from Pseudomonas fluorescens GIM1.49 was a novel short-chain dehydrogenase and might play a role in oxidative degradation of halogenated secondary alcohols.