The Study On Rational Regulation Of Lipase-catalyzed Asymmetric Organic Reaction

Candida antarctica lipase B(CalB)is widely used in chiral medicines,chiral pesticides and other intermediates synthesis due to its high enantioselectivity,high catalytic activity,mild reaction conditions and simple post-processing.Sometimes,the substrate of the enzyme-catalyzed reaction does not belong to the category of natural substrates,thus the result of the reaction is not ideal.The desired result can be obtained by rationally regulating enzyme-catalyzed asymmetric organic synthesis reactions through media engineering,protein engineering and substrate engineering strategies.(S)-N-(2-ethyl-6-methylphenyl)alanine((S)-NEMPA)is the key chiral precursor compound for lots of herbicides.For this important intermediate,lipase CalB is employed to catalyze(R,S)-N-(2-ethyl-6-methylphenyl)alanine methyl ester((R,S)-NEMPA-ME)enantioselectivity hydrolysis reaction to obtain(S)-NEMPA.Regarding the undesirable catalytic performance of lipase CalB,this paper uses media engineering,additive strategies,enzyme modification strategy based on additive regulation and protein engineering strategy to rationalize and optimize the reaction to improve the enantioselectivity of(S)-NEMPA chiral molecules catalyzed by lipase CalB.In this paper,the commercial lipase CalB(immobilized form,Novozym 435)was first used to catalyze the enantioselective hydrolysis reaction of(R,S)-NEMPA-ME.Novozym 435 exhibits low enantioselectivity although fast reaction speed,so we use media engineering strategies to regulate the reaction system.Under the condition of 35%(v/v)tetrahydrofuran(THF)co-solvent,amine additives including amides and amino acid compounds were continuously introduced.Finally,the highest enantioselectivity(E value)of Novozym 435 was increased to 6.1 times and the enantiomeric excess of the product reached 96.6%under the 0.3 mol/L of added formamide.When the amount of added histidine and lysine was 0.1 mol/L,E value was increased to 4.4 and 3.6 times,respectively.The enantiomeric excess of the product reached 94.6%and 93.8%,respectively.The method of adjusting the reaction medium and the introduction of amine additives improved the enantioselectivity of Novozym 435 successfully,and the reasonable regulation of the asymmetric organic reaction catalyzed by lipase through the medium engineering strategy was realized.In our previous part of work,it was found that amino acids and amide molecules can significantly improve the enantioselectivity of the enzyme.Next,we modified the enzyme based on the additive strategy to further improve the catalytic performance.Our group fused histidine and lysine tags at both terms of CalB to construct a series of recombinant lipase n His-CalB-n Lys(rCalBs).The target CalB in the form of inclusion bodies(rCalBs-IBs)was also obtained while obtaining the target soluble rCalBs.The reaction confirmed that both soluble rCalBs and rCalBs-IBs can catalyze the hydrolysis of(R,S)-NEMPA-ME,which present better enantioselectivity compared with those of the commercial Novozym 435.This method not only realizes the soluble expression of rCalBs in E.coli,but also the catalytically active rCalBs-IBs were obtained while maintaining the higher enantioselectivity.This provides a reference for the subsequent construction and modification of lipase CalB.We continued to explore whether rCalBs-IBs,which have the catalytic ability and exist in the form of precipitation,can be used as the immobilized enzyme to evaluate the stability.It was found that rCalBs-IBs exhibit good enantioselectivity but poor stability after being applied to the model reaction.Therefore,the oxidized dextran solution and magnetic nanoparticles Fe₃O₄ were applied to modify 6His-CalB-5Lys-IBs(rCalB-IBs)to prepare CLEAs(rCalB-cl IBs)and magnetic CLEAs(rCalB-mcl IBs).The best cross-linking condition is:rCalB-IBs(50 mg/m L),Fe₃O₄(50 mg/m L),the oxidized dextran solution(10%,v/v),Triton X-100(0.25?mol/L),and shaken for 3 h during the cross-linking.As a result,the final cross-linking efficiency and the activity recovery of rCalB-mcl IBs reached 98.0%and 113.9%,respectively.After 10 repeated reactions,the activity recovery of rCalB-mcl IBs was 85.4%,and the enantiomeric excess of the product was 84.7%.In addition,rCalB-mcl IBs were extended to the MBH reaction of C-C addition.In the scale-up reaction,the yields of MBH and Aldol product catalyzed by rCalB-mcl IBs were 17.8%and 13.9%,respectively.The rCalB-mcl IBs with higher enantioselectivity obtained after cross-linking modification,further improved the stability in the reaction,which confirmed the catalytic performance and application value of rCalB-mcl IBs.In order to further improve the enantioselectivity of lipase,protein engineering strategy is the most direct and preferred method.We used the obtained soluble form of6His-CalB-10Lys(rCalB)as the research object and rationally modified it.Finally,three single-point positive mutants were obtained,namely V161L,I196V,and L285V.The E values increased to 2.1,2.3 and 2.6 times of the wild rCalB,respectively.The second-generation mutant V161L/I196V and the third-generation mutant V161L/I196V/L285V continued to increase the E value to 2.3 and 2.7 times after the iterative mutation.The enantiomeric excess of the product increased from 75.8%to90.4%under the optimal catalytic conditions of the mutant V161L/I196V/L285V,which shows that the adopted rational design to improve the enantioselectivity of rCalB is effective.In conclusion,this thesis successfully improved the enantioselectivity of(R,S)-NEMPA-ME catalyzed by Novozym 435 through media engineering and additive strategies.Based on the additive strategy,our research group constructed a series of lipase rCalBs fused with polyamine tags,which not only obtained the soluble form of rCalBs,but also obtained rCalBs-IBs,both of which have higher enantioselectivity.The stability was further improved through the modification of cross-linking,and the obtained rCalB-mcl IBs were extended and applied to the MBH reaction of C-C addition.Using the rational design method of protein engineering strategy,the enantioselectivity of soluble rCalB was successfully improved,and the rational regulation of the asymmetric organic reaction catalyzed by lipase was realized.