Screening And Application In Resolution Of N-(2-ethyl-6-methyl-phenyl)-alanine Methyl Ester Of Lipase-producing Microorganisms

Lipase is the third largest industrial enzyme,after protease and amylase with huge application prospect,extensive sources,mature screening method,good catalytic effect?hydrolysis reactions of esters,transesterification,esterification?,ect.The aim of this paper is to screen strains which can produce lipase for resolution of N-?2-ethyl-6-methyl-phenyl?-alanine methyl ester?NEMPAME?with good stereoselectivity and catalytic activity.Fifteen microbial strains used for resolution of N-?2-ethyl-6-methyl-phenyl?-ala-nine methyl ester were found from soils,a strain to hydrolyze R-N-?2-ethyl-6-methyl-phenyl?-alanine methyl ester with ee_p value is greater than95%,which was identified as Pseudomonas by physiological identification and 16Sr RNA sequence analysis was named styj.Optimization of lipase production was carried out through single factor and orthogonal test.After optimization,the composition of the optimized medium used for lipase production was as followed:glucose 10 g/L,yeast extract 20 g/L,MgSO₄ 0.5g/L,ZnCl₂ 0.5 g/L,Olive oil 10 g/L,pH7.5,fermentation at 37?for 48h,and relative enzyme activity increased nearly to 308.98%,lipase activity increased to 5.61U/mL.With 70%ammonium sulfate precipitation,dialysis,ultrafiltration,ion-exchange chromatography and gel filtration,the purified target protein was about 35kDa.We used 0.2M phosphate buffer?pH=7.0?as solvent for resolution of NEMPAME.The optimum temperature and pH for enzymatic activity were about37?and pH 7.0.The enzyme was stable at 20?,it fall sharply at 50?.It was the most stable at pH 5.0-9.0,when it fall sharply in less than pH 5.0.K⁺can slightly improve the activity,but most laboratory metal ions inhibited it.Its enzymatic activity was inhibited when presenting in some organic solvents,such as DMSO?acetonitrile?acetone?ethyl ether?diisopropyl ether?cyclohexane?n-hexane.