| Multifunctional chiral molecules are important blocks of many drugs.In the past few years,more and more attentions have been paid to the synthesis of chiral molecules,while the chemical synthesis of chiral molecules usually requires the use of transition metal catalysts and complex chiral ligands.Chemical synthesis not only requires harsh reaction conditions and expensive chiral ligands,but also leads to a series of environmental pollution problems.Therefore,synthesis of chiral molecules with green methods under mild conditions is still a hot topic in chemical research.Fatty acid decarboxylase FAP(WT-FAP)is a photosensitive enzyme found in recent years.It can catalyze the decarboxylation of long chain carboxylic acids under light excitation.Based on the understanding of the catalytic mechanism of the enzyme,it is considered that the selective photodecarboxylation catalyzed by FAP can realize the resolution of chiral acids,which is a new way of kinetic resolution of chiral acids.However,in the kinetic resolution of 2-hydroxyoctanoic acid,we found that WT-FAP had low catalytic activity and low stereoselectivity(ees=22%).In order to solve this problem,the protein engineering of FAP was carried out by using rational design method on the basis of molecular dynamics simulation,and the best mutant G462Y was obtained.The mutant has high catalytic activity and excellent stereoselectivity,and the R-configurational carboxylic acid with ees>99%can be obtained.G462Y also showed a wide scope of substrates,a series of chiral carboxylic acids containing hydroxyl and amino functional groups with high ee values were obtained.Compared with other enzymatic synthesis methods of a-functionalized carboxylic acids,the FAP-catalyzed resolution method does not require NADPH cycle or pre-esterification or amidation of raw materials.Candida antarctica lipase B(CALB)is one of the most widely used lipase.In this paper,we combined the mutation and the small ligand modification to regulate the stereoselectivity of CALB.The cysteine residue introducted into some key sites of the alcohol pocket and acid pocket of CALB was modified with small molecule ligand to reshape the space of the active site of CALB,thus regulating the stereoselectivity of CALB in the hydrolysis resolution of chiral carboxylic esters.Some good results were obtained in this thesis.For example,for the model reaction of 2-phenylpropionate hydrolysis,the R-configurational stereopreference of the mutant W104A/T42C was increased from eep=32%to eep=50%after the modification of small molecules.The S-configurational stereopreference of the mutant V190C/A281F(eep=14.1%)was further increased to eep=80%after binding to the ligand.The substrate expansion of a series of chiral esters was studied.It was found that the S-configurational stereoselectivity of the hydrolysis of chiral esters with large volume could be improved by modifying the acid pocket of mutant V190C/A281F with small ligand.Finally,the method of 'Modification of enzyme with small ligands to regulate the enzymatic stereoselectivity,was further applied into Bacillus Subtilis lipase A(BSLA).We first mutated some amino acids near the active site of BSLA into cysteine,which was further modified by small molecular ligands to change the structure of the enzyme to achieve the improved stereoselectivity.In the hydrolysis of 2-(p-methyl)phenyl propionic acid p-nitrophenol ester,BSLA-M78C was modified wit small ligands,and the R-configurational product stereoselectivity increased from eep=59.3%to eep=92.9%.Meanwhile,the S-configurational stereoselectivity was successfully achieved by the modification of BSLA-M137C mutant with small molecular ligands. |
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