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

摘要:

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186.90 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。

Abstract:

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 186.90 mmol/L and Vmax of 89.56 U/mg. When catalying the oxidative reaction, its optimal temperature was 12 °C and optimal pH was 10.5, in contrast to the values of 24 °C 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.