Microbial Screening For Carbonyl Reductase Following The Anti-prelog's Rule And Its Application In The Synthesis Of Statin Side Chain

Atorvastatin(ATS)is the best-selling cholesterol-lowering medicine in the market.It plays a role as competitive inhibitor of HMG-CoA reductase in the cholesterol synthesize,as a result of the similar structure of the key perssad and the 3-hydroxy-3-methylglutaryl,the substrate of HMG-CoA reductase.Since strict chirality purity is required in the synthetic process of ATS's chiral intermediate t-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate,the biocatalytic approach has great potential for its excellent stereoselectivity.The screening for carbonyl reductase with efficient activity and excellent stereoselectivity is a indispensable process in the reconstruction of t-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate.In this thesis,an efficient screening model for carbonyl reductase was built for the first time.This model is based on the coloration variation when the 2,4-dinitrophenylhydrazine reacts with carbonyl group in the t-butyl 6-cyano-(5R)-hydroxyl-3-oxohexanoate,measuring carbonyl reductase activity indirectly from the consumption of substrate.Then we confirmed the acitivity and stereoselectivity of carbonyl reductase using HPLC.More than 60 strains following the anti-prelog's rule were isolated from nature with this screening model.Strain ZJB-09225 displayed outstanding activity,and was choosen for the following research,which was identified as Pichia caribbic according to morphology,physiological tests and 18S rDNA sequence analysis.P.caribbic ZJB-09225 was preserved at CCTCC under accession number of CCTCC M2012411.Medium composition was optimized for P.caribbic ZJB-09225 at first.The optimized medium composition was composed of malt extract 30.0 g/L,glucose 20.0 g/L,(NH4)2HP04 1.0 g/L,K2HPO4-3H20 2.28 g/L,NaCl 1.0 g/L,CuSO4 3.0 mg/L,pH adjuste to 7.5.Further,fermentation condition was investigated.Results revealed that peak biomass dens:ity and ketoreductase activity was obtainded under the conditions as follows:seed age 15 h,inoculation size 3.0%(v/v).As the result of cultivation optimization,the volumetric activity increased from 4.70 U/L to 7.17 U/L.Biocatlytic parameters were evaluated for P.caribbic ZJB-09225 in terms of t-butyl 6-cyano-(5R)-hydroxyl-3-oxohexanoate yield and d.e.value of t-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate.Outcomes demonstrated that the optimized bioconversion system were as follow:K2HP04-KH2PO4(50.0 mM),pH7.5,5.0 g/L sodium gluconate,14.0 g/L t-butyl 6-cyano-(5R)-hydroxyl-3-oxohexanoate,P.caribbic ZJB-0922525.0 g DCW/L,reaction temperature 35?.Twelve hrs reduction of 50.0 g/L t-butyl 6-cyano-(5R)-hydroxyl-3-oxohexanoate gave t-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate in a yield of 19.4%,d.e.value of 94.3%.In addition,neutral red and surfactant can insignificantly affect P.caribbic ZJB-09225 carbonyl reductase activity 0.4‰(w/v)Neutral red improved initial reaction rate by 0.24 fold,and 1%(w/v)SDS improved the initial reaction rate by 0.54 fold.Finally,a two phase reaction system was established to weaken t-butyl 6-cyano-(5R)-hydroxyl-3-oxohexanoate inhibition.The initial reaction rate was improved 1.38 fold in paraxylene/water two phase,compared with the aqueous reaction system.The concentration of t-butyl 6-cyano-(3R,5R)-dhydroxyhexanoate accumulated to 10.53 g/L after 10h reaction,1.35 times higher than that of aqueous reaction system.