Cloning And Expression Of Genes Involved In Bioconversion Of DL-ATC To L-cysteine From Pseudomonas Sp. Zjwp-14

L-cysteine was widely used in the fields of pharmaceutics, foods, cosmetics and feed additives because it was the only one amino acid which contained active sulphydryl. The production of L-cysteine by microbial transformation has been paid more and more emphasis on because of its short cycle time, low cost, high regio- and stereoselectivity, easy control of reaction condition and environment-friendly. In our laboratory, Pseudomonas sp. Zjwp-14 was screened out from soil samples, with which DL-2-amino-△²-thiazolin-4-carbonic acid （DL-ATC） could be converted to L-cysteine. On basis of deeply study on microtransformation method, two genes involved in this bioconversion process through NCC pathway were cloned, a engineering bacteria strain which harbored L-ATC hydrolase gene was constructed, and its biotransformation was also invested.L-ATC hydrolase gene （H1） and L-NCC hydrolase gene （H2） were amplified by PCR with Pseudomonas sp. Zjwp-14 genomic DNA as template. The amplified fragments were ligated to pUCm-T and transformed into E. coli DH5αfor sequencing. The result showed that the length of H1 and H2 were the same as AB070707, and the homology of nucleotide sequences were 94.6% and 95.3%, respectively. The homology of amino acid sequences were 97.1% and 95.95%, respectively.The L-ATC hydrolase gene was inserted into expression vector pET-28a（+） and transformed into E. coli BL21（DE3） to express. The molecular weight of the recombinated protein was about 20.3kD, detected by SDS-PAGE. The recombinated bacterium was fermented in LB medium at 37℃, 200r/min till liquors A₆₀₀ was between 0.25 to 0.8, added 0.05 mmol/L IPTG and incubated at 30℃for 7h, then considerable inclusion bodies were obtained. The quantity of deliquescent protein was higher in low temperature.The enzyme activity of L-ATC hydrolase was also studied. When A₆₀₀ of fermented liquors about 0.4, cells were induced by 0.05mmol/L IPTG for 5h, L-ATC hydrolase obtained the highest enzyme activity. D-sorbitol and glycerine could raise L-ATC hydrolase activity, obviously. The glycerine group enzyme activity would be 2.5 folds as contrast, while D-sorbitol group was only 70% as glycerine group. The enzyme activity was high in the initial stage of reaction, and the transformation efficiency was boosted when prolonging reaction time. The optimal concentration of substrate was 1%, the optimal enzyme concentration was 1.5 folds germ density, the optimal inversion temperature were 42℃for whole cell and 40℃for cell serum. The cell serum enzyme activity could reach 4808 U/ml in this condition detected by DTNB method, and the transformation efficiency of DL-ATC could reach 70% after 24h convention.