Asymmetric Reduction Of 2'-Choloracetophenone Catalyzed By Yeast And Cloning, Expression Of The Oxidoreductase

Chiral compounds have been widely applied in pharmaceuticals, food, pesticide andchemical industry due to their special optical activity. With the progress in pharmacologyof chiral pharmaceuticals and the strict regulation on the application of new racemic drugin recent years, the research and development of chiral pharmaceuticals have become thenew focus and direction. Enantiomerically pure alcohols are important intermediates forsynthesis of pharmaceuticals. Asymmetric reduction pro-chiral ketones catalyzed bymicroorganism or the oxidoreductase in the cell has been an important method ofsynthesis enantiomerically pure alcohols. In this work, o-chloroacetophenone as the modesubstrate, the reaction characteristics about reduction of aromatic ketone by yeast wholecell in aqueous phase system were studied. An oxidoreductase, carbonyl reductase (CAR),was purified from yeast cell and the recombinant strain for overexpression of CAR genewas constructed through molecular cloning and gene operation. The coenzyme NADHwas regenerated in situ successful through substrate coupling system in the reaction aboutasymmetric reduction of o-chloroacetophenone by recombinanted CAR. The maincontents of this work are as follows:1. The yeast Candida pseudotropicalis 104 (C.104) was screened from seven yeaststrains preserved in our laboratory. The strain could catalyze asymmetric synthesis of(S)-1-(2-chloro-phenyl)-ethanol from o-chloroacetophenone with high yield andstereoselectivity. After researching the effect of cosolvent (ethanol) and comparingdifferent aqueous/organic solvent two-phase reaction systems, the reaction system ofreducing high concentration o-chloroacetophenone by C.104 cell in single aqueous phasewas constructed. The high concentration of insoluble substrate was provided asmicro-droplets in the aqueous system under stirring. The yeast cell of 20 gDCW(dry cellweight)/L could catalyze 19.48 mmol/L o-chloroacetophenone at 100% conversion with20 g/L glucose as cosubstrate. The initial rate was 33.27 mmol/(h·gDCW) and thebiocatalyst(gDWC)/substrate(g) ratio (b/s) was 6.67. Yeast cell could tolerate high substrateconcentration of 233.8mmol/L in short time and the product concentration reached 58.4mmol/L during 24 h. But yeast cell would lose activity or die under high concentrationsubstrate or product for the longer time. The more toleration of yeast cell appeared to lower concentration of substrate (6.49 mmol/L). The cell could be reused 16 times with100% yield.2. The carbonyl reductase was purified through DEAE ion exchange chromatogramand blue sepharose affinity chromatogram from (C.104). The purification results were22.4% yield,15.8U/mgpr special activity and 2980 folds. Pure GAR appeared the higherstereoselectivity than C.104 cell because the enantiomeric excess (e.e.) value of(S)-1-(2-chloro-phenyl)-ethanol reached 100%.3. CAR could catalyze the reduction of ketones and the oxidation of correspondingalcohols with NAD(H) as coenzyme.The optimal temperature for enzymatic reaction was50℃. The optimalpH for enzymatic reduction and oxidation were 6.0-6.5 and 8.5respectively. CAR was stable in the temperature below 30℃or under the pH between 7.0and 8.5. Common heavy metal ions could destroy the enzyme activity, but EDAT couldnot affect the activity. The enzyme was shown abroad substrate specificity and highenantioselectivity. The substrate included many aromatic and aliphatic ketones andcorresponding alcohols. The group substituents near the carbonyl of substrate ketonesboth affected the enzymatic activity because of the steric effect and charge inducedeffects. CAR has high stereoselectivity for aromtic ketones such as acetophenone,2'-chloroacetophenone, 3'-chloroacetophenone, 4'-chloroacetophenone, 3-chloropropiophenone and2-chloro-2',4'-difluoroacetophenone. 100% e.e. values of the products were obtained. Thereactions followed Prelog rule.4. CAR gene was amplified from C.104 and inserted into expressing vectorpET28a(+). The recombinanted strain E.coli BL21 (DE3) (pET28a-CAR) wasconstructed. E. coli BL21 (DE3) (pET28a-CAR) showed the activity of 0.24 U/mgpr withinduction of IPTG, which was over 40 times than that of C.104 (0.0053 U/mgpr). Therecombinanted CAR protein was purified through Ni-NTA agrose affinity chromatogramand appeared the same characteristics of CAR from C.104. The same sequence geneswere amplified from different yeast strains (Candida pseudotropicalis 104,Saccharomyces cerevisiae B5 , Candida utilis 1257, Pichia membranaefaciens andSaccharomyces cerevisiae 4742). It proved that CAR existed in many yeast strains andthe gene sequence was a high conservative sequence.5. The substrate coupling reaction system catalyzed by recombinanted CAR wasconstructed. It can be used for asymmetric reduction of 2'-chloroacetophenone and coenzyme regeneration with 2-propanol or 2-butanol as the cosubstrate. For the reactionsystem, the optimal temperature was 30℃and the optimal pH was 7.0. The kineticparameters for CAR with the substrate of o-chloroacetophenone were as follows:K_m=0.693mmol/L,V_max=0.017μmol/min ([NADH]: 0.06mmol/L). Basing on the reactionequilibrium kinetics, the equilibrium equation in the substrate coupling reaction systemwas deducted. It was Keql·Keq2 =[P][butone]/[S][2-butanol](2-butanol as the cosubstrate).