环己酮单加氧酶的克隆表达及酶学性质分析

为研究鞘氨醇杆菌中环己酮单加氧酶的催化特性,从N.aromaticivorans DSM 12444基因组DNA克隆表达了一个编码环己酮单加氧酶的基因chmo,并分析了该基因产物的酶学性质.该基因全长1650bp,编码550个氨基酸,理论分子量为61.6kDa.将携带此基因的重组质粒pET28a-chmo转入E.coli BL21(DE3)后,获得了62kDa左右的表达产物.重组环己酮单加氧酶(CHMO)能氧化芳香族硫醚及其衍生物、链式硫醚、其他硫醚和酮类等底物,以2-氯乙基苯基硫醚为底物时活力最高(25.65U/mg).CHMO最适反应温度和pH分别为55℃和8.87,倾向于利用NADPH作辅酶.上述结果表明,重组CHMO是一个新型的环己酮单加氧酶,推测其与硫醚及酮类物质的氧化降解有关.

Cloning Expression and Enzyme Characterization of a Cyclohexanone Monooxygenase

To explore the biocatalytic properties of cyclohexanone monooxygenase(CHMO) in N.aromaticivorans,the experiment cloned the gene chmo from N.aromaticivorans DSM 12444,and analyzed the enzyme characterization of its products.The length of the gene chmo was 1650bp encoding a CHMO with 550 amino acid residues and calculated molecular mass of 61.6 kDa.The recombinant plasmid pET-28a-chmo was constructed and functionally expressed in E.coli BL21(DE3),resulting in the products with the size of 62 kDa.The recombinant CHMO could oxidize aromatic sulfide and its derivatives,chain sulfide as well as other sulfide and ketone substrates.The enzyme showed the highest activity against 2-chloro ethyl phenyl sulfide,with Vmax of 25.65 U/mg.The optimal temperature was 55 °C and optimal pH was 8.87,NADPH was inclined to be the coenzyme.These results indicate that the recombinant CHMO was a novel CHMO and might play a role in the oxidative degradation of sulfide and ketones.