Cloning, Expression, And Characterization Of Alcohol Dehydrogenase From Lactobacillus Plantarum

Optically active alcohols are important intermediates for the synthesis of many pharmaceuticals and agrochemicals. Dehydrogenases, especially the alcohol dehydrogenase can be used in the synthesis of these compounds. For example(3R,5R))-tert-butyl-6-cyano-3,5-dihydroxyhexanoate(A7), which is a key building block for the cholesterol-lowering drug named lipitor, can be synthesized by reducingβ-ketones. In our studies, Lactobacillus plantarum showed the ability to reduceβ-ketones into chiral alcohols in a whole cell format. For this reason, the alcohol dehydrogenases encoded by the genome of Lactobacillus plantarum were systematically investigated. First, the degenerate primers were designed according to the amino acid sequence alignment of the members of short-chain dehydrogenase/reductase(SDR) and middle-chain dehydrogenase/reductase(MDR). Five genes encoding alcohol dehydrogenases were cloned from the genome DNA of Lactobacillus plantarum by using hiTAIL-PCR. Two of them encode two short-chain dehydrogenases(named 1s and 3s), while the other three encode three middle-chain dehydrogenases(named 2m,3m, and 4m). Because of the complete genome sequence of Lactobacillus plantarum is available, so we selected five dehydrogenases according to the related documents( including 1s,3s,2m and two novel alcohol deydrogenase 1a and 2a) and cloned them to pBAD and pEASY-E1 vectors, resulting in five recombinant expression vectors, MC1061-pBAD-3s,TransB(DE3)-pEASY-E1-1a,BL21(DE3)-pEASY-E1-2a,BL21(DE3)-pEASY-E1-2m and BL21(DE3)-pEASY-E1-1s. Finally, they were expressed and purified, and the corresponding activities were preliminarily tested.The results showed that:Soluble dehydrogenases were successfully expressed for1 a, 2a, and 3s. While the other two proteins 1s and 2m were expressed as inclusion body in E. coli. 3s was subcloned into plasmid pBAD and expressed in E. coli MC1061 with0.05mg/mL L-ara at 25 ℃(180rpm). The induction time was 12 h. By using ion exchange chromatography, 150 mg of 3s with a purity of 60% was obtained from 1L culture of MC1061/PBAD. The enzyme showed the activity toward β-ketones, such as ethyl acetoacetate with the enzyme activity of 16.4U/mg and 5R-tert-butyl5-cyano-3-oxohexanoate(A6). The ORF of 3s is 744 bp, coding for 247 amino acids with a molecular mass of 26 kDa. 1a and 2a were subcloned into plasmid pEASY-E1 andwere expressed in E. coliTransB(DE3) and BL21(DE3) respectively. The optimal expression condition of 1a was as follows: 200 mM inoculated LB culture was grown at37℃(180 rpm) until the OD600 reached to 0.4. The culture was kept at 16℃ for 90 min and subsequently inducted by 0.1mM IPTG followed by continuously growing at 16℃(180rpm) for 5h. Using Ni2+-chelating affinity chromatography, 15 mg of 1a with a purity of 95% was obtained from 1L culture of TransB(DE3)/pEASY-E1. The optimal expression condition of 2a was same as that of 1a except for the induction time and temperature. The induction time of 2a was 12-14 h and the induction temperature was25 ℃. By using Ni2+-chelating affinity chromatography, 13.8mg of 2a with a purity of95% was obtained from 1L culture of BL21(DE3)/pEASY-E1. The ORF of 1a is 1071 bp,coding for 356 amino acids with a molecular mass of 42 kDa. The ORF of 2a is 1074 bp,coding for 357 amino acids with a molecular mass of 42 kDa. The two enzymes showed a relatively higher activity toward ketones with bulky side chains compared to that ofβ-ketones. 1s and 2m were subcloned to pEASY-E1 vector and expressed as inclusion body in E. coli. The ORF of 1s is 795 bp, coding for 264 amino acids with a molecular mass of 30 kDa and the ORF of 2m is 1029 bp, coding for 342 amino acids with a molecular mass of 38 kDa. The study on correct refolding of the two enzymes should be done in the further.