The Discovery And Evolution Of Enzymes For Chiral Synthesis Based On The Protein Structures Of Lipases And So On

Candida antarctica lipase B(CALB)is one of the most frequently used lipases with many different applications,but CALB shows poor enantioselectivity towards profen derivatives.As the well studied enzyme,CALB has been engineered or redesigned to modify many properties by different strategies,however,in which only a few works were reported with enhanced activity and selectivity toward chiral carboxylic acids.In the first part of this thesis,CALB was subjected to directed evolution by CAST and ISM based on its structure for improving the enantioselectivity toward profens derivatives.The CALB variants showed remarkable enhancement of activity and enantioselectivity toward three profen esters.The best variants with 4 substitutions had E values of 31,37 and>200,with preference for the(S)-enantiomer of ibuprofen,flurbiprofen and ketoprofen esters respectively.Molecular dynamics simulation suggested that the cooperative effect of beneficial residues might contribute to these improvements.Ketoreductases are important biocatalysts of pharmaceutical interest.Recently,two ketoreductases from Candida glabrata were reported,but the substrate scopes and enantioselectivity of CgKRs have not been fully evaluated.In the second part of this thesis,the activity and enantioselectivity of CgKR1 and CgKR2 toward a series of prochiral substituted acetophenones were evaluated.CgKRs displayed excellent enantioselectivity and structure related activity toward these substrates.It was interesting to find that CgKRs showed opposite selectivity to para-,meta-halogen substituted acetophenones following the Prelog's rule and substrates with halogen substituents in ortho position following the anti-Prelog's rule.Homology modeling and docking analysis were in conformity with this interested enantiopreference obtained from experimental tests.Recently we have found that the natural product curdione could be hydroxylated by Aspergillus niger AS 3.739 and cytochrome P450 monooxygenases in A.niger played an import role for the hydroxylation,also the curdione could be synthesized to curcumalactone in the acid condition.In the third part of this thesis,three stereoisomers of curdione were used as substrates for hydroxylation by A.niger and for synthesis of curcumalactone.The results showed that the reaction pathways of two pairs of enantiomers were also enantiomeric.The hydroxylation of enantiomeric curdiones by A.niger would generate the enantiomeric 3-OH-curdiones,while synthesis from enantiomeric curdiones in the acid condition would generate the enantiomeric curcumalactones.Through these reactions,four stereoisomers of 3-OH-curdione and four stereoisomers of curcumalactones were obtained.We also found some P450s in A.niger by genome data mining,and they displayed related hydroxylation activity.