Studies On Directed Evolution Of Cyclohexanone Monooxyenase And Enzymatic Cascade Reaction

Directed evolution is an important means to improve the catalytic activity and chem-?regio-,enantio-selectivity of enzymes.Cyclohexanone monooxygenase from Acinetobacter sp.NCIMB 9871(AcCHMO)is one of the most widely studied enzymes in Baeyer-Villiger monooxygenase,which has high catalytic activity and stereoselectivity for Baeyer-Villiger reaction of many substrates.Recently,4cCHMO has been widely applied to the oxidation of various substrates including heteroatomic compounds.Chiral organic borate as an important organic intermediate with high reactivity,has been used as a precursor for the construction of chiral carbon-carbon bonds.In this paper,the stereoselectivity of the AcCHMO-catalyzed oxidization resolution of rac-borates was regulated by directed evolution,and the enzymatic resolution reaction was followed by Suzuki Coupling reaction in cascade to obtain the chiral coupling products.In addition,the substrate scope and the stereoselectivity of AcCHMO towards a series of cycloketones was further expanded and improved by directed evolution.The main research results are summarized as follows:Firstly,site-specific mutations of AcCHMO were performed on the sites L143,F246,F432 and L435 which are located in the substrates binding pocket.A highly focused mutant library was built by site-directed mutagenesis using three representative amino acids with different side chain sizes,namely alanine A,phenylalanine F and leucine L.The mutant strains with high stereoselectivity were screened by using a-phenylethyl boric acid pinacol ester as a model substrate.By analyzing the influence on the stereoselectivity in a-phenethyl borate resolution reaction,the combination of the best single mutants was carried out.Two mutants with complementary stereo-selectivity were eventually identified,L143F/F432I/L435A and F246A/L43 5F/L507A.Enantio-complementary a-benzyl ethanols were prepared in the resolution reaction catalyzed by these two mutants,with ee values of 68%and 58%respectively.As a result,enantio-complementary chiral a-phenethyl boric acid pinacol esters were also obtained with ee value up to 75%.As following,chiral products 4-(l-phenylethyl)benzene were obtained by coupling chiral a-phenethyl boric acid pinacol ester with iodobenzene as an electrophilic reagent.After optimizing the conditions of Suzuki coupling reaction as well as cascade reaction,an enzyme-catalyzed one-pot system were constructed successfully,in which the enantiomeric 4-(l-phenylethyl)benzene was obtained with 40%yield and 75%ee values.Finally,the substrate scope of AcCHMO was extended.A series of substrates from cyclobutanone to cyclooctanone were investigated,and the relationship between the size of the substrates' ring and the spatial structure of AcCHMO was analyzed.The mutant strains that could improve the conversion of each cycloketone were screened out.In addition,by comparing the results of 3-phenyl-cyclobutanone,3-phenyl-cyclopentanone and 4-phenyl-cyclohexanone,we found that sites 432 and 435 had significant effects on the oxidization enantiopreference of these bulky substrates.The F432L mutant can catalyze the oxidation of 3-phenylcyclobutanone and 4-phenylcyclohexanone to obtain enantiomers with configuration opposite to WT,respectively.After further mutation and optimization,these two substrates were oxidized to the enantiomers with>99%ee value.For 3-phenylcyclopentanone oxidization where regioselectivitive and stereoselectivitive products were formed simultaneously,the mutants F432L and L43 5G showed the regio-controlled and stereo-controlled oxidation results,completely different from WT.