Research On Microbial Enzymatic Resolution Of N-BOC-DL-alanine Methyl Ester

D-alanine is an important chiral unnatural ?-amino acid.It is an important chemical and pharmaceutical intermediate,which is widely used in chiral medicine,chiral additive,chiral auxiliaries and and other fields.This thesis is intended to use microbial esterase to preparation the N-BOC-D-alanine methyl ester by resolution of N-BOC-DL-alanine methyl ester.Firstly,by using N-BOC-DL-alanine methyl ester as the sole canbon source,we selected an L-configuration hydrolase-producing bacterium with best enantioselectivity toward N-BOC-DL-alanine methyl ester from soil.By morphological observation and 16S rDNA analysis,the selected bacterium was identified as Bacillus amyloliquefaciens,and thus was named as Bacillus amyloliquefaciens WZZ002.Then,by doing single factor experiment,the fermentation and enzyme producing conditions for B.amyloliquefaciens WZZ002 were investigated and the optimal formula of the fermentation medium obtained is as follows:soluble starch 0.6%,peptone 1.6%,yeast extract 0.1%,MgS04 1 mmol/L;The optimal conditions for its incubation is at pH 7.0,30 ? for 24 h.After fermentation,we using the wet bacteria as biocatalyst to catalyze the hydrolysis of N-BOC-DL-alanine methyl ester,the e.e.s value was improved from 23.8%to 49.2%,the C value was improved from 19.4%to 33.1%,the biomass was improved from 0.86 g/L to 2.21 g/L,at the same time,the E value was greater than 300.Secondly,the reaction conditions of hydrolysis resolution N-BOC-DL-alanine methyl ester by B.amyloliquefaciens WZZ002 cell were investigated and the optimal reaction conditions acquired are as follows:the substrate concentration was 2 M with the cell-mediated biocatalysis loading of 50 g/L,35 ?,pH 8.0,NH3·H20 as the neutralizer.After 10 h titrimetric reaction at optimal reaction conditions,the e.e.s value was greater than 99.9%,the e.e.p value was greater than 98%,the C value was 50.1%.Then,we immobilized the B.amyloliquefaciens WZZ002 with sodium alginate embedding method,the conditions of immobilization were investigated and the optimal conditions acquired are as follows:sodium alginate concentration was 2%,CaCl2 concentration was 4%,and the time of calcification was 4 h with 4 ?.We used the immobilized cell as biocatalyst to catalyze the hydrolysis of N-BOC-DL-alanine methyl ester ten times and the residual enzyme activity was greater than 85%,it demonstrate that the immobilized B.amyloliquefaciens WZZ002 had good reusability and stability.At last,in order to obtain the esterase produced by B.amyloliquefaciens WZZ002,which had enantioselectivity hydrolysis of N-BOC-DL-alanine methyl ester,the fermentation broth through ammonium sulfate precipitation,DEAE ion-exchange column chromatography and phenyl hydrophobic chromatography.Finally,an electrophoresis pure esterase was obtained and molecular weight of it was about 50 KDa.The specific activity was improved from 9.27 U/mg to 142.92 U/mg,the enzyme activity recovery was 9.57%and the purification fold was 15.42.Then,the enzymatic property of B.amyloliquefaciens WZZ002 esterase was investigated and the optimal conditions obtained are as follows:the optimum pH was 8.0,the optimum temperature was 55 ?,Fe2+?K+had obvious promoting effect,Al3+?EDTA had obvious inhibitory effect,twain-20 and twain-80 had certain promoting effect on enzyme activity.In addition,we also investigated the substrate specificity of B.amyloliquefaciens WZZ002 esterase.The result indicated that the enzyme preferred the esters of short-chain fatty acid as substrate.Therefore,we suggested that the enzyme produced by B.amyloliquefaciens WZZ002 involved in the enantioselective hydrolysis of N-BOC-DL-alanine methyl ester was an esterase.