Research On Enzymatic Preparation For Crizotinib Key Chiral Precursor(S)-1-(2,6-Dichloro-3-fluorophenyl) Ethanol

(S)-1-(2,6-dichloro-3-fluorophenyl) ethanol, a chiral precursor of Crizotinib, can be prepared by catalytic reduction of 2,6-dichloro-3 fluoroacetophenone. Due to the participation of coenzyme in the reaction, the expensive coenzyme is the limited factor to industrial application. To solve the problem of coenzyme regeneration, a genetic engineering method to coexpress alcohol dehydrogenase(ADH) and glucose dehydrogenase(GDH) in E.coli BL21(DE3) were established in this eassay.The gene fragment of ADH and GDH were obtained by gene synthesis. The fragments were inserted into pET-28 a separately and obtained pET-28a-gdh and pET-28a-adh, they were transformed into Host bacteria, formed the recombinant strains E.coli BL21-GDH and E.coli BL21-ADH.After inducded by IPTG, expression protein were observed at the site of 30 kDa and 29 kDa from the SDS-PAGE analysis. The optimize expression results demonstrated that E.coli BL21-GDH gained the highest expression under the conditions of 20℃ within 0.1 mmol/L IPTG for 8h. The activity of GDH in crude enzyme solution was 9.8U/ml, and the activity of total protein was 9.61U/mg. E.coli BL21-ADH had the highest expression under the conditions of 16℃ within 0.1mmol/L IPTG for 10 h. The activity of ADH in crude enzyme solution was 2.3U/ml, and the activity of total protein was 1.91U/mg.The recombinant E.coli BL21-ADH/GDH was constructed to realize the co expression of glucose dehydrogenase and alcohol dehydrogenase. The optimize expression result illustrated that E.coli BL21-ADH/GDH got a optimal soluble expression with 0.2mmol/L IPTG for 12 h at 25℃.For the catalytic reaction, E.coli BL21-ADH/GDH could significantly transfer 2,6-Dichloro-3-fluoroacetophenone to(S)-1-(2,6-two chlorine-3-fluorine phenyl) ethanol under the condition of 30℃, pH7, and 7% feeding amount. The transform rate was 90.35% and the e.e. value was more than 99%.Finally, in order to further expand the reaction system, the co-expression recombinant strains were cultured in high density fermentation. OD600 was beyond 90 after 30 hours culture. The enzyme catalytic reaction system was amplified from 40 ml to 2L, the transform rate was 98% after 20 hours of catalytic reaction.