| The chiral compounds have been widely applied in pharmaceuticals, agrochemicals, materials, essence and other fine chemicals because of their specific biological activity. Recently, chiral technology has highly attracted attentions from industry and academia, and the asymmetrical synthesis of chiral compounds and their chiral intermediates have become an active field in research and development. Because of mild reaction conditions, high conversion ratio and outstanding stereochemical specificity, biocatalysis has become the most promising and first choice method in the asymmetric synthesis. In this work, two genes of NADP-dependent aldehyde reductase(ALR) and NADP-dependent carbonyl reductase(CAR) were cloned and and expressed by the recombinant E. coli M15 (pQE30-ALR) and E. coli M15 (pQE30-CAR), respectively, which were applied to catalyze the asymmetric reduction of prochiral P-oxer ester, ethyl 4-chloro-3-oxobutanoate(COBE), to its corresponding L-(R)- and D-(S)- ethyl R-4-chloro-3- hydroxybutanoate(CHBE). In order to solve co-enzyme NADPH regeneration during reaction, a recombinant E. coli M15 (pQE30-gdh223) was constructed to expressing glucose dehydrogenase, which is able to regenerate NADP+ to NADPH coupled with the oxidation of glucose. The two-strain systems, M15 (pQE30-ALR) or M15 (pQE30-CAR) coupled with M15 (pQE30-gdh223) were studied for asymmetric reduction of COBE,respectively. The major progresses in the research work are as followings.1. The gene of NADP-dependent aldehyde reductase(ALR) was cloned from genome of Sporobolomyces salmonicolor ZJU010, and the recombinant E. coli M15 (pQE30-ALR) was constructed. After optimization of culture conditions and inducing method, the specific ALR activity of E. coli M15 (pQE30-ALR) was 7.28 U/mg, which was 14 times higher than that of S. salmonicolor ZJU010. The asymmetric reduction of COBE to R-CHBE catalyzed by recombinant E. coli in aqueous phase was investigated and the results indicated that the product was optically pure R-CHBE, and both the yield and the stereo-selectivity of R-CHBE were much higher than that by S. salmonicolor ZJU010. During COBE reduction, co-enzyme regenerating enzyme, NADPH and glucose were required to regenerate the co-enzyme NADPH. The reaction was inhibited by high concentration of COBE and CHBE, but high density of recombinant cells could eleviate the inhibitory effect. Under appropriate reaction conditions, R-CHBE yield and ee value can reach 98.5 % and 100 %, respectively.2. in order to solve the co-enzyme regeneration, two genes of glucose dehydrogenase (GDH, E.C. 1.1.1.47), gdh223 and gdh151, were cloned from Bacillus megaterium AS 1.223 and AS1.151, respectively. The DNA sequences of gdh223 and gdh151 arein 94.7% homology with reported coding sequence of gdh gene of B. megaterium IAM1030. Only one amino acid residue difference between gdh223 and gdhl51was observed, Arg39 in gdh223 and Ser39 in gdhl51. The expression vectors, pQE30-gdh223 and pQE30-gdhl51, were constructed and transformed into E. coli Ml 5 respectively, and the expressed GDH activity by using pQE30-gdh223 was 2.16 U/mg, which was about 11 times higher than that by using pQE30-gdhl51. After optimization of cultivation and inducing conditions, the specific GDH activity expressed by E. coli Ml5 (pQE30-gdh223) was increased up to 4.45 U/mg. The whole cells of recombinant E. coli Ml5 (pQE30-gdh223) were used as the system for NADPH regeneration, and the remarkable increase in extracellular NADPH concentration was observed. The recombinant E. coli Ml5 (pQE30-gdh223) coupled with Ml5 (pQE30-ALR) or acetone-dried yeast cells was successfully applied to catalyze asymmetric reduction of COBE to ^-CHBE. This simple and cheap method of regenerating co-enzyme NADPH might have potential application in other bioreduction systems.3. The two-strain coupled system of M15 (pQE30-ALR) and M15 (pQE30-gdh223) was studied in the biotransformation of COBE to .tf-CHBE. The system can be operated without any addition of co-enzyme NADPH and glucose dehydrogenase. The optical purity and yield of .tf-CHBE reached 100% (e.e.) and 98.5%, respectively, when initial COBE concentration was 60mmol/L. The optimized bioreduction conditions for two-strain coupled system are: 34°C, 100 r/min of rotate speed and the 4:1 in weight ratio of wet cells for Ml5 (pQE30-ALR) to Ml5 (pQE30-gdh223). The bioreduction was also inhibited by high concentrations of COBE and CHBE, and the fed-batch operation was favorable to decrease the inhibitory effect, the final product of i?-CHBE reached 124mmol/L in aqueous phase. Two-phase system was also attractive to reduce the inhibitory effect caused by both substrate and product. In a two-phase system consisted of dibutyl o-phthalate and potassium phosphate buffer, the productivity of J?-CHBE reached 283mmol/L and the yield was 78.6% from 360mmol/L of substrate, whereas the ee value was lowered to 87%.4. The aldehyde reductase(ALR) and glucose dehydrogenase(GDH) was purified from recombinant M15 (pQE30-ALR) and M15 (pQE30-gdh223) cells, respectively, and the kinetics of enzyme catalyzed reaction was studied. The ordered BiBi reaction mechanism was adopted to the systems with two-substrate participating in the reaction, one is the substrate and the another is the co-enzyme, the kinetic equations of ALR and GDH were obtained as followings, respectively.<sub><sub><sub><sub><sub><sub><sub><sub><sub><sub><sub><sub><sub><sub>l.85[NADPH][COBE]<sub><sub><sub><sub><sub><sub><sub><sub><sub><sub><sub><sub><sub>Valr [NADPH][COBE] + 0.273[COBE] + 0.2 \[NADPH] + 0.218<sub><sub><sub><sub><sub><sub><sub><sub><sub><sub><sub><sub><sub>22327[NADP+][glu]<sub><sub><sub><sub><sub><sub><sub><sub><sub><sub><sub><sub>V°D" [NADP+][glu] + 0.U5[glu] + 0.m[NADP+] + 0M73 These equations were applied in the two-enzyme coupled system successfully. 5. The gene of NADP-dependent carbonyl reductase(CAR) was cloned from genome of Candida magnolia ZJU106, and the recombinant E. coli Ml5 (pQE30-CAR) was constructed. After the optimization of cultivation and inducing conditions, the specific CAR activity of E. coli Ml5 (pQE30-CAR) was 5.78 U/mg, which was 8.4 times higher than that of C. magnolia ZJU106. Asymmetric reduction of COBE to S-CHBE catalyzed by E. coli Ml5 (pQE30-CAR) in aqueous phase was investigated. The product was optically pure S-CHBE, and both the yield and the stereo-selectivity of S-CHBE were higher than that by C. magnolia ZJU106 catalyzed. The two-strain coupled system of Ml5 (pQE30-CAR) and Ml5 (pQE30-gdh223) for S-CHBE production from COBE in aqueous phase was also investigated. The bioreduction was completed effectively without any addition of co-enzyme NADPH and glucose dehydrogenase. The optical and the yield of S-CHBE reached 100%(e.e.) and 99.5%, respectively, from 50mmol/L of COBE. Similarly, in the two-phase system of dibutyl o-phthalate and potassium phosphate buffer, the final S-CHBE concentration reached 208.9mmol/L and the yield was 69.7% from 300 mmol/L of substrate.
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