Research On Microbial Enzymatic Preparation Of Chiral L-2-aminobutyric Acid And Its Derivatives

L-2-aminobutyric acid is an important chiral non-natural ?-amino acid.It is an important chemical and pharmaceutical intermediate,which is widely used in the synthesis of chiral drugs.This thesis is intended to use microbial lipase for the preparation of L-2-aminobutyric acid and its derivatives.Firstly,by using N-Boc-2-aminobutyric acid methyl ester as the sole canbon source,more than 30 strains with good enantioselectivity toward N-Boc-2-aminobutyric acid methyl ester were screened from soil.Amongset them,most are(S)-configuration lipase producing strains.We selected an(S)-configuration lipase producing fungi strain which shows the best enantioselectivity toward substrate for further study.By morphological observation and 18S rDNA analysis,the selected fungi strain was finally identified as Aspergillus tamari,and.thus was named as Aspergillus tamarii ZJUT ZQ013.Then,by doing single factor experiment,the fermentation and enzyme producing conditions for Aspergillus tamarii ZJUT ZQ013 were detailed investigated and the optimal composition of the fermentation medium obtained is as follows:soluble starch 2%,peptone 1.6%,MgS041 mmol/L,NaCl 1%,K2HP04 0.3%,KH2PO4 0.1%;The optimal conditions for its incubation is at pH 7.0,30?,200 rpm for 46 h.After fermentation,the biomass was improved to 9.9 g/L from 4.9 g/L.By using the wet mycelium as biocatalyst to catalyze the hydrolysis of N-Boc-2-aminobutyric acid methyl ester,the E value was improved from 9.3 to 78.2,at the same time,the yield of the product N-Boc-L-2-aminobutyric acid(e.e.>99%)was increased from 30.8%to 42.4%.And then,the reaction conditions of enzymatic kinetic resolution of N-Boc-2-aminobutyric acid methyl ester by' Aspergillus tamarii ZJUT ZQ013 were investigated and the optimal reaction conditions acquired are as follows:acetone(substrate/acetone=1:8,v/v)as co-solvent,0.1 M,pH 7.2 potassium phosphate buffer(KPB),substrate 1%(52.2 mmol/L),A.tamarii ZJUT ZQ013 2%(0.02 g/mL),30?,200 rpm under water-bath.After 6 h reaction,optically pure N-Boc-L-2-aminobutyric acid(e.e.>99.9%)was abtained in yield of 45.7%,the e.e.s is above 99%with E value of 257.At last,the kinetic resolution system was enlarged to 200 mL.The reaction was complete in 6 h and then by treatment of the reaction solution,0.83 g optically pure N-Boc-L-2-aminobutyric acid and 0.79 g optically pure N-Boc-D=2-aminobutyric acid methyl ester were obtained and followed with examined by chiral GC proving that both of them are in more than 99%e.e.In order to obtain the catalytically active lipase produced by Aspergillus tamarii ZJUT ZQ013,Aspergillus tamarii ZJUT ZQ013 was treated by sonication to acquire the crude enzyme,followed with ammonium sulfate precipitation and low pressure hydrophobic interaction chromatography for further separation and purification.At last,a relatively pure while still contain some impurities Aspergillus tamarii ZJUT ZQ013 lipase was obtained.