Screening For Anti-prelog Carbonyl Reductase Producer And Its Application In Chiral Alcohol Synthesis

As lipid-lowering medicines,statins are intense inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A reductase?HMGR?,which catalyze the limiting step of cholesterol synthesis.Owning to the outstanding efficiency and high safty,atorvastatin has become the best selling drugs all over the world.In this article,biocatalytic routine for t-butyl 6-cyano-?3R,5R?-dihydroxyhexanoate,a vital chiral block of atorvastatin,was established.Firstly,a strain displaying anti-prelog carbonyl reductase activity was isolated from oil samples.Through morphological identification,physiological and biochemical identification and molecular identification,the strain was indentified as Debaryomyces castellii.Further,optimization of cultiviation conditions for Debaryomyces castellii ZJB-12032's growth and carbonyl reductase activity were conducted,including carbon source type and concentration,nitrogen source type and concentration,phosphorus source type and concentration,initial media pH value,inoculation size,inocula age,fermentation time and fermentation temperature.Results indicated that the optimized fermentation medium consisted of 50 g/L maltose,15 g/L NaNO3,0.5 g/L NaH2PO4·2H2O,0.5 g/L Na2HPO4·12H2O,initial pH value 7.0,and under the ideal cultivation conditions,10%?v/v?inoculation size,inocula age of 8 h.Under these conditions,after grown at 30 C for 24 h,D.castellii ZJB-12032 exhbited peak activity of 10.2 U/L,specific activity of 1.20 U/g DCW,with t-butyl 6-cyano-?3R,5R?-dihydroxyhexanoate d.e.value over 99.5%,displaying excellent diastereoselectivity towards t-butyl?R?-6-cyano-5-hydroxyl-3-carbonylhexanoate.Finally,effects of cosubstrate,temperature,pH,metal ion,organic solvent,substrate concentration on synthesis of t-butyl 6-cyano-?3R,5R?-dihydroxyhexanoate were investigated.Under the optimized conditions,which were 30 ?,pH 7,0,glycerol supplemented as cosubstrate,1mM CoCl₂,maximal t-butyl 6-cyano-?3R,5R?-dihydroxyhexanoate yield reached 96.7%in the case of 5 g/L t-butyl?R?-6-cyano-5-hydroxyl-3-carbonylhexanoate.10 g/L t-butyl?R?-6-cyano-5-hydroxyl-3-carbonyl-hexanoate gave 75.0%yield.Besides,operation in the fed-batch mode was evaluated to raise substrate loading and eliminate the waste of material.It was found that peak yield of 91.2%was obtained via adding 5 g/L t-butyl?R?-6-cyano-5-hydroxyl-3-carbonylhexanoate instead of glycerol after 5 g/L t-butyl?R?-6-cyano-5-hydroxyl-3-carbonylhexanoate was completely converted.