Study On Biocatalytic Enantioselective MBH Reactions

Morita-Baylis-Hillman reaction(MBH reaction)is an important carbon carbon single bond formation reaction.In the past few decades,MBH Products have attracted extensive attention.It can be used as a valuable component of complex natural Products containing allyl Propanol.There are many problems in traditional chemical catalysis,such as environmental pollution and complex reaction conditions.At present,there are many reports about MBH reaction catalyzed by enzyme.we used esterase(yitV),alanine racemase 1(alr1)and enoate reductase(ER)to catalyze the MBH reaction.We mainly use nitrobenzaldehyde and cyclohexanone as substrates and phosphoric acid buffer as solvent to catalyze MBH reaction.After optimizing several important parameters of three kinds of enzyme catalysis,it is found that ER has better catalytic effect.Then,additives were introduced to assist ER to catalyze the MBH reaction,and a variety of mutants were prepared to further improve the catalytic effect of the enzyme.We cloned and expressed esterase and alanine racemase and purified them.The putative esterase yitv and alanine racemase gene alr1 were obtained from the genome of Bacillus subtilis 168 and successfully expressed in E.coli BL21(DE3).The optimal induction temperature of yitV was 16 ℃,the final concentration of IPTG was 0.01 mM,the optimal induction temperature of alr1 was 25 ℃,and the final concentration of IPTG was 0.1 mM.The protein was purified by nickel column to obtain pure alr1 and yitV.yitV,alr1 and ER were successfully constructed to catalyze the MBH reaction.The results showed that yitV,alr1 and ER could catalyze MBH reaction.The optimal yields of MBH products were 32.5%,29.4%,36.0%,respectively.The optimal yields of aldol products were 28.5%,28.7%,30.1%,respectively and ee value were 6.0%,2.1% and 6.5%,respectively.Then four kinds of additives were introduced to assist ER and prepare ER mutants to catalyze MBH reaction.According to the experimental results,it was found that ammonia water and small molecular alcohol can promote the reaction to acertain extent.ER mutants were prepared to catalyze MBH reaction.On the surface of ER,tyrosine at position 28 changed to histidine,phenylalanine at position 315 changed to serine,arginine at position 195 changed to serine,lysine at position 12 changed to isoleucine,and arginine at position 215 changed to serine.The glycine at position 51 in ER mutated to serine.These mutants were purified to catalyze the reaction.The results showed that 12 Lys and 215 Arg on the ER surface were mutated to other amino acids,and the yield of MBH Products was reduced by about 10%,while the other amino acid mutants had no significant change.According to this phenomenon,the MBH reaction was catalyzed by a single amino acid.It was found that the MBH reaction can take place except for the acid based acid,and more aldol products can be produced at the same time.The three basic amino acids,L-Lys,L-Arg and L-His,catalyze the MBH reaction.The yield of MBH product,ee value,aldol product and L-His product are 46.9%,5.8%,36.8%,29.2%,2.1% and 12.4% respectively.According to the experimental results,the catalytic effect of L-Lys is better than that of L-His.It is possible that lysine plays a better role in the enzyme catalyzed reaction.The yields MBH products were 43.0%,43.7%,19.9%,respectively and ee values were 0.9%,15.1%(s type)and 51.0%,respectively,using L-Gly,L-Ala and L-Pro as catalysts.L-Gly and L-Ala preferred to promote the formation of MBH products,and L-Pro preferred to increase the enantioselectivity of MBH products.The catalytic effect of L-Ser and L-Trp has no obvious advantage.In this paper,we expand the application of allyl reductase,alanine racemase and esterase in the organic reaction by using allyl reductase,alanine racemase and esterase to catalyze MBH reaction.The introduction of additives and the preparation of ER mutants can improve the catalytic effect of the enzyme and provide more methods for catalytic reaction.Using amino acids to catalyze the MBH reaction provides a new way to improve the yield of MBH products.