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Collected stereoselective oxidoreductase derived from different microorganisms, and the enzyme encoding gene overexpressed in E. coli, and various prochiral carbonyl compound asymmetric catalytic reactions, to study the catalytic reactions law. Using a crude enzyme solution of cells as a biological catalyst, build the the crude enzyme aqueous phase catalytic converter system and the crude enzyme biphasic catalytic transformation system and to study cell authigenic enzyme system, to build a self-assembly coenzyme regeneration system, catalyze a variety of potential chiral carbonyl compound, which specifically includes: (1) will be derived from the connection of the seven kinds of microorganisms 13 oxidoreductase gene and expression vector pET21c, transformed into Escherichia coli BL21 (DE3), to give 13 recombinant bacteria. Expression of recombinant bacteria in IPTG induction, and 2 - hydroxyacetophenone asymmetric catalytic reduction. Studies have found that the The 17 ℃ induced expression of the recombinant enzyme specific activity was significantly higher than 30 ° C and 37 ℃ induced expression of specific activity, the optimal IPTG induction of the recombinant protein concentration of 1 mm. 13 strains of acetophenone, 2 - hydroxyacetophenone and 4 - chloro ethyl acetoacetate (COBE) catalytic asymmetric reduction. 9 bacteria capable of asymmetric reduction of 2 - hydroxyacetophenone give (R) or (S)-type product, and the optical purity of more than 90%. Recombinant strain derived from Candida parapsilosis CCTCC M203011 5 RCR, SCR, SCR1, SCR2 and SCR3 are able to asymmetric reduction of the higher concentration of 2 - hydroxyacetophenone, and the optical purity of greater than 99%, the yield is greater than 85% ; S1, ADHR, SCR1 3 bacteria to asymmetric reduction of acetophenone, wherein S1 and ADHR better, able to catalyze the reduction to give (R) -1 - phenyl ethanol, an optical purity of greater than 97%, and yields were 47.5% and 35.2%; of KRD, CR2, S1, C1, and C2 and SCR1 can be asymmetric reduction of COBE, wherein the KRD, CR2 and S1 better can be obtained an optical purity of greater than 98% of (R) - or (S The) type CHBE, and the yield is greater than 50%. (2) The constructed 13 recombinant bacteria, to SCR1 mode as a model substrate, the enzyme, 2 - hydroxyacetophenone cells autogenous enzyme systems to construct a self-assembled NADPH regeneration system, built in the form of a crude enzyme solution crude enzyme catalyzed reaction system. This crude enzyme reaction system needs glucose and a very small amount of NADP, this crude enzyme reaction system can be in the water phase Reduction of 40 mM 2 - hydroxyacetophenone, reaction time 6h, yield 93.9%, optical purity is greater than 99.9%; In view of the poor solubility characteristics the ketones substrate in water, filtering the organic solvent to construct water / twelve Ethyl two phase crude enzyme reaction system, the proportion of 12-ethyl was 20%, two-phase reaction of this crude enzyme The system can be catalytic reduction of 80 mM 2 - hydroxyacetophenone, yield 87.3%, optical purity more than 99.9%; addition, the present study using this two-phase system of the crude enzyme catalytic reduction of prochiral aryl ketones, in all 15 kinds of substrates, 2 - hydroxyacetophenone for this system the optimal substrate 2 substituents substrate the yield was significantly higher than the two substrates to be no substituent, and the 2 bit-substituted substrates, 2 - hydroxyacetophenone derivative effects 2 - bromoacetophenone derivative effect; Meanwhile, in all reactions, both high and low yield, as long as the substrate reaction, and the product The optical purity of greater than 99%; experimental results show that the catalytic asymmetric reduction effect of the crude enzyme biphasic system, without exception, the reaction system is superior to whole cells. (3) from 13 recombinant strain constructed in this study, we screened CR2 asymmetric reduction of COBE (R) -4 - chloro-3 - hydroxybutyrate ((S)-CHBE), S1 be able to asymmetric Restore COBE to give (R)-CHBE. S1 crude enzyme system is able to restore at an enzyme concentration of 40% substrate concentration of 125 mM, COBE, to give (R)-CHBE, a yield of 61.2%, an optical purity of 99.9%; CR2 is a crude enzyme system in the enzyme concentration was 20 % when the substrate concentration can be restored to 125mM of COBE to give (S)-CHBE, a yield of 68.6%, optical purity of 99.9%.
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