Bioconversion DL-ATC To L-cysteine By Escherichia Coli Whole-cell

L-cysteine is an important sulfur-containing amino acid that has been widely applied to food additives, medicines, cosmetic and fodder and so on. In 1980s, it was reported that Pseudomonas putida has the ability to convert DL-2-amino-?2-thiazoline-4-carboxylic acid(DL-ATC) to L-cysteine, which received attention and research of majority of shorlars.L-ATC hydrolase gene atcB and N-carbamyl-L-cysteine amidohydrolase gene atcC in Pseudomonas.sp TS1138 genome were cloned to expression vector and expressed in Escherichia coli. Bioconversion DL-ATC to L-cysteine was carried out by the recombinant Escherichia coli whole-cell in this study. The optimation conditions for conversion DL-ATC to L-cysteine was determined. At the same time, different recombinant plasmids were constructed to investigate the abilities of conversion DLATC to L-cysteine.Firstly, the recombinant plasmid pET-his/atcBC was constructed, and transformed into E.coli BL21 to overexpress L-ATC hydrolase and N-carbamyl-L-cysteine (L-NCC)amidohydrolase.Secondly, the gene tnaA was knocked out in BL21 to investigate the effect of L-cysteine desulfhydrase on L-cysteine accumulation. The results indicated that L-cysteine production was increased by 46.2% by the recombinant strain BL21?tnaA/pET-his/atcBC whole-cell to bioconvert DL-ATC to L-cysteine. This suggested that deleted of tnaA contributes to reduction degradation of L-cysteine, and enhances the production of L-cysteine. The catalytic conditions of conversion DL-ATC to L-cysteine with BL21-pET-his/atcBC were explored, the optimizd pH and temperature was 7.0 and 37?,respectively. Based on the above researchs, effects of metal icons, Dithiothreitol (DTT),glycerol and sorbitol on L-cysteine biosynthesis were investigated. The results showed that different metal icons have dfferent levels of inhibition on catalytic activities, especially.Cu2+ and Fe2+ have strongly effects on inhibited catalytic activities. The yield of L-cysteine was enhanced by 57.98% and 24.4% with addition 200 g/L sorbitol and 2 g/L DTT in catalytic mixture, respectively. The yield of L-cysteine was enhanced by 16.1% by addition 10% glycerol (v/V) to dissolve DL-ATC in reaction mixture.Thirdly, the recombinant plasmid pET-his/atcB-linker-atcC was constructed to enhance the solubility and expression of target proteins by series connection atcB and atcC with linker, which was based on recombinant plasmid pET-his/atcBC. Recombinant plasmid pET-duet/atcABC was constructed to enhance the utilization ratio of substrate DL-ATC by introducing L-ATC racemase gene atcA. The rencombinant strains BL21 -pET-his/atcB-linker-atcC and BL21 -pET-duet/atcABC were constructed in succession to investigate the abilities of conversion DL-ATC to L-cysteine. The results showed that the strain BL21- pET-duet/atcABC can more efficiently convert DL-ATC to L-cysteine, the yield of L-cysteine was enhanced by 9.0% compared to control. In addition,the effect of immol/Lobilized cells on accumulation of L-cysteine was investigated in this study, and the enzymes activities of free cells was 69.8% more than immol/Lobilized cells of 20% gelatin.Finally, the cultivation process of catalytic system was investigated with shaking flask fermentation to increase the catalytic activities of BL-pET-his/atcBC. The optimal addition time and induction temperature of IPTG was about 5 h (OD600, 1.0-1.2) and 25?,respectively. The optimal addition concentration and induction time of IPTG was 0.1 mmol/L and 9 h, respectively. The catalytic activities reached to maximum under the optimal conditions. At the same time, the cultivation process of catalytic system was investigated with fed-batch fermentation to increase the catalytic activities of BL-pET-his/atcBC. The optimal addition time and induction temperature of lactose was 4-6 h (OD600, 20-25) and 25?, respectively. The optimal induction time of lactose was 8 h,the maximum catalytic activities reached 112U/mg, and the accumulation of L-cysteine was 44.5 mmol/L.