[Cloning, expression and the application of human, rat alcohol dehydrogenase and aldo-keto reductase].

ABSTRACT

This study is aimed to clone and express human, rat alcohol dehydrogenase (ADH) and aldo-keto reductase. Then the enantioselective metabolism of mandelic acid (MA) was studied. Human alcohol dehydrogenase 2, rat alcohol dehydrogenase 1, human and rat aldo-keto reductase 1A1 were amplified using RT-PCR from human and rat liver samples. Then subcloned into pET-28a (+) and expressed in E. coli BL21 (DE3) stably. The protein was induced with IPTG and purified by affinity chromatography. Then the enzyme activities were measured. MA enantiomers were incubated with rat, human ADH and phenylglyoxylic acid (PGA) with AKR1A1, respectively. The metabolism was analyzed with HPLC. The proper genes were cloned and purified and proteins were obtained. All of the proteins obtained showed good activity. Stereoselective-metabolism of MA was observed in human ADH2, which favors for S-MA metabolism. The expression plasmids are constructed and the recombinant proteins are expressed successfully. The recombinant alcohol dehydrogenase and aldo-keto reductase have been employed to study MA metabolism.