The Characterization Of Coli BL21(DE3)/pET28a(+)-cr Carbonyl Reductase And Its Application In Synthesis Of Statin Chiral Building Blocks

t-butyl 6-cyano-?5R?-hydroxy-3-oxo-hexanoate?CHOHB?and Ethyl 4-chloro-3-oxobutanoate?CHBE?are the crucial building blocks and pharmacophore of atorvastatin,whose synthesis is always challenging on account of high requirement for high chemical and stereochemical purity.Herin,biocataytic methods for?S?-4-cyano-3-hydroxybutanoate and 6-cyano-?3R,5R?-dihydroxylhexanoate were established,which displayed low cost,high conversion rate and excellent selectivity.Bast on the previous research,a NADP?H?-dependent carbonyl reductase,produced by E.coli BL21?DE3?/pET28a?+?-cr was purified to homogeneity through Macro-prep High S chromatography followed by Macro-prep t-butyl HIC chromatography.It was revealed that the purified carbonyl reductase had a relative molecular mass of 35.4 kDa by LC-MS-QTOF.The purified enzyme exhibited maximum activity at 30? and pH 7.5,and it is a non-metal-dependent enzyme.The thermostability of enzyme is poor,and the enzyme has a weide pH stability range over 6.0-8.0.The carbonyl reductase showed high active to CHOHB and ^COBE,meanwhile it exhibit reduction activity on dicarbonyl compounds substrate like ethyl pyruvate and butanedione simultaneously,besides it was also active against aromaticketone,aliphaticketone,aldehyde and ketonic ester.100 mmol·L^{-1 COBE} was reduced to CHBE in 15 min by carbonyl reductase of E.coli BL21?DE3?/pET28a?+?-cr in KH2PO4-K2HPO4 buffer,in 100%yield and e.e.value>99.9%.And the glucose dehydrogenase produced by E.coliBL21?DE3?/pET28a-gdh-cr was introduced to build a coenzyme regeneration system.However,in case of increased substrate concentration,the inhibition of both substrate and product were serious to catalyst.To alleviate these problems,a water/dibutyl phthalate?phase radio of 1:1?two phase system was construced,in which 300 mmol·L^{-1 COBE} was reduced in 80 min under the conditions of 26 pH 7.0,agitation speed 200 rpm,yielding CHBE up to 294.2 mmol·L^-1 with e.e.value over 99.9%.Through the investigation of the influence of substrate concentration to the enzyme,dissociation constants?KiNADPH?from E-NADPH was gained as 0.019 mmol·L^-1 using Lineweaver-Burke plotting.For t-butyl 6-cyano-?5R?-hydroxy-3-oxo-hexanoate?CHOHB?,the maximum reaction rate and apparent Michaelis-Menten constant K_m CHOHB of the purified carbonyl reductase are 54.3 ?mol·mg-1·min^-1 and 4.4 mmol·L^-1 respectively,the kinetic equation was gained as:v=54.3[CHOBH][NADPH]/?0.0836+0.043[CHOBH]+4.4[NADPH]+[CHOBH][NADPH]?For ethyl 4-chloro-3-oxobutanoate,its maximum reaction rate Vmax and apparent Michaelis-Menten constant K_m^COBEare 36.5 ?mol·mg-1·min^-1 and 1.2 × 10-1 mmol·L^-1,the kinetic equation was gained as:v=36.5[^COBE][NADPH]/(0.00276+0.0022[^COBE]+0.12[NADPH]+[^COBE][NADPH])The results demonstrated that the strain E.coli BL21?DE3?/pET28a?+?-cr was a ideal biocatalyst regarding the biosynthesis of 6-cyano-?3R,5R?-dihydroxylhexanoate and?S?-4-cyano-3-hydroxybutanoate,contributing to its great potential industrial applications.