Microbial Enzymatic Preparation Of N-BOC-L-homoserine Lactone And Its Application In The Sythesisi Of L-selenomethionine

L-homoserine lactone is an important chiral non-natural a-amino acid lactone.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 esterase for the preparation of L-homoserine lactone and its derivatives,and then apply it to the synthesis of L-selenomethionine.Firstly,by using N-Boc-L-homoserine lactone as the sole canbon source,more than 130 strains with good enantioselectivity toward N-Boc-L-homoserine lactone were screened from soil.Amongset them,most are(R)-configuration esterase producing strains.An(R)-configuration esterase producing bacterial strain which shows the best enantioselectivity toward substrate was screened as the target strain for further study.By morphological observation and 16S rDNA analysis,the selected bacterial strain was finally identified as Arthrobacter nicotinovorans,thus this strain was named as Arthrobacter nicotinovorans WYG001.Then,by doing single factor experiment,the fermentation and enzyme producing conditions for Arthrobacter nicotinovorans WYG001 were detailed investigated and the optimal composition of the fermentation medium obtained is as follows:yeast extract 1.2%,lactose 1.0%,MgSO4 2 mmol/L,KH2PO4 1 mmol/L,NaCl 1.0%,initial pH at 7.0;sterilized in 1210 C for 20 min;The optimal conditions for its incubation is at 30?,200 rmp in 30 L fermenter for 24 hours;After fermentation,the biomass was improved to 8.55 g/L,which is four times as the un-optimized 2.06 g/L.By using the wet mycelium as biocatalyst to catalyze the hydrolysis of N-Boc-L-homoserine lactone,the enzyme activity was improved from 3.48 U/L to 75.55 U/L,which is 22 times better after optimization than un-optimization.At the same time,the enantiomeric excess of N-Boc-L-homoserine lactone(e.e.s)was improved to 82.98%from the initial 7.67%and the convertion(C)was improved to 47.9%from 4.9%.And then,the reaction conditions of enzymatic kinetic resolution of N-Boc-DL-homoserine lactone by Arthrobacter nicotinovorans WYG001 were investigated and the optimal reaction conditions acquired are as follows:DMSO(substrate/DMSO=1:2,w/v)as co-solvent,0.1 M,pH 7.2 potassium phosphate buffer(KPB),substrate 2.5%(125 mmol/L),Arthrobacter nicotinovorans WYG001 5%(0.05 g/mL),30?,200 rpm under water-bath.After 11 h reaction,optically pure N-Boc-L-homoserine lactone(e.e.s>99.9%)was abtained in yield of 48.39%with conversion of 51.09%and E value of 278.Under the best optimal reaction conditions,the enzymetic activity of Arhrobacter nicotinovorans WYG001 was still 85%remained as the initial activity after 8 times biocatalyzation.At last,the kinetic resolution system was enlarged to 200 mL.The reaction was complete in 11 h and then by treatment of the reaction solution,2.4 g optically pure N-Boc-L-homoserine lactone was obtained in yield of 48%and followed with examination by chiral GC proving that its enantiomeric excess is more than 99%.Then,based on the optically pure N-Boc-L-homoserine lactone obtained by microbial selective catalytic hydrolysis process,optically pure L-selenomethionine was successfully synthesized through two or three sequential chemical reactionsm,and synthesized L-selenomethionine structures were identified.