Cloning, Expression And Characterization Of Short-chain Dehydrogenases From Bacteria

Short-chain dehydrogenases are ubiquitous in nature and the key enzymes for metabolisms of sugar, alcohol, lipid, amino acid, carbohydrate, cofactor and hormone. Short-chain dehydrogenases have also attracted great attention serving as potent biocatalysts in synthetic chemistry and pharceutical industry. In this study, the genes encoded short-chain dehydrogenases were amplified by PCR from the genomic DNA of three different strains (Pseudomonas fluorescens GZM1.49, Geobacillus subterraneus and Pseudomonas aeruginosa A006) and the corresponding expression plasmids were constructed. Then, those constructed plasmids were transformed into the strain E.coli BL21(DE3) and the reconbinant short-chain dehydrogenases were over-expressed, purified and characterization.1、The structural gene pfd from Pseudomonas fluorescens GZM1.49was amplified, which length was684bp, encoding a short-chain dehydrogenase whit227amino acid residues and the molecular size was28kDa. With the catalytic characteristics of psychrophilic, PFD could oxidize alcohols including4-chloro-3-hydroxbutanoate ester and reduce4-chloro-acetoacetate ester using either NAD(H) or NADP(H) as coenzyme. The enzyme showed the highest activity against4-chloro-3-hydroxbutanoate ester as substrate, which Km and Vmax were186.90mmol/L and89.56U/mg, respectively. When catalying the oxidative reaction, its optimal temperature and pH were12℃and10.5, respectively, on contrast to the values of24℃and pH10.5in the reductive reaction. The enzyme possessed high solvent tolerance and its activity was improved by a low concentration of EDTA.2、The structural gene gsd from Geobacillus subterraneus was amplified, which length was555bp, encoding a short-chain dehydrogenase whit184amino acid residues and the molecular size was24kDa. With the catalytic characteristics of thermophilic, GSD had a wide spectrum of substrate. The enzyme showed the highest activity against ethyl pyruvate as substrate, which Km and Vmax were20.25mmol/L and238.10U/mg, respectively. When catalying the oxidative reaction, its optimal temperature and pH were65℃and10.5, respectively, on contrast to the values of65℃and pH7.5in the reductive reaction. The enzyme possessed high solvent tolerance(70%V/V) and could use either NAD(H) or NADP(H) as coenzyme, so there was a great value in researching of building coenzyme regeneration system. In addition, GSD had high thermal stability, and its activity was not effected by low concentration of metal ions and EDTA.3、The structural gene pad from Pseudomonas aeruginosa A006was amplified, which length was825bp encoding a short-chain dehydrogenase with274amino acid residues and calculated molecular mass of30kDa. The BLAST search indicated that the sequence had the highest homology with a short-chain dehydrogenase from Pseudomonas aeruginosa PAO1(ABJ12131) with the identity100%.