| L-cysteine, an aliphatic amino acid, has been widely applied in some fields of pharmaceuticals, foods, cosmetics, and other biotechnology areas. It is also an important component for synthesizing some biochemical products, such as glutathione, thiamine, biotin, etc. Currently, production of L-cysteine mainly depends on hydrolysis of hair, chemical synthesis, these processes not only had low yield with poor quality but also polluted environment. Biosynthesis of L-cysteine, as a new process, has been paid more and more emphasis on owing to its more economical, efficient, and environmental-friendly.On basis of last research in our lab, initial strain (pseudomonas sp. B-3), biosynthesize from DL-2-amino-Δ2-thiazoline-4-carboxylic acid (DL-ATC) to L-cysteine, was mutated by low energy ion implantation, comparing and analyzing the gene sequence and protein sequence about L-cysteine-production enzymes between initial strain and the mutant one, and optimizing its enzyme-production medium and fermentation conditions in fermentor.Pseudomonas sp.B-3was mutated by N+ion implantation,and a mutant strain with high enzyme activity and genetic stability designated as Pseudomonas sp.C-25was obtained,of which L-cysteine enzyme activity improved to2748U/mL,namely increased by26.3%compared with the original strain.In order to research the molecule mechanism of high enzyme activity, genes of L-ATC hydrolase,N-carbamyl-L-cysteine(L-NCC)hydrolase and L-cysteine desulfhydrase were cloned from strain B-3and C-25respectively,and analyzed their DNA sequence and amino acid sequence. I-TASSER online system was used to predict their protein3-dimensional (3D)structure.The results showed that,after mutation,the mutant site of L-ATC hydrolase gene was the203th nucleotide,which changed from A to G and the68th amino acid changed from Asp to Gly,it is also a enzyme binding site.It can be inferred that the enzyme binding site was changed by mutation,and enzyme acti.,ity was enhanced.The mutant sites of L-NCC hydrolase gene showed as following:the212th nueleotide changed from T to C and the71th amino acid changed from Ⅱe to Thr,the second mutant site was the1052th nucleotide changed from C to T,while the351th amino acid changed from His to Tyr,and the amino acid mutant site71th was close to binding site Arg70,threonine and tyrosine are polar amino acid,which changed the hydrophilia of L-NCC hydrolase,thus changed the enzyme activity of L-NCC hydrolase. The mutant sites of L-cysteine desulfhydrase gene showed as following:the930h nucleotide changed from C to G and the301th amino acid changed from Cys to Try, the second mutant site was the465th nucleotide changed from G to T, while its amino was not changed. The mutant site was not close to enzyme binding sites, it can be inferred that this mutant site not influence the enzyme activity significantly. It was a foundation for study the mechanism of L-cysteine production enzyme system.Subsequently, single-factor experiment was used to determine the levels of each medium component and then, response surface methodology (RSM) was applied to optimize the components of fermentation medium for enhance enzyme activity for L-cysteine production by Pseudomonas sp. C-25. The optimum fermentation medium contained:DL-ATC·3H2O4.7g/L, glycerol16.0g/L, beef extract6.5g/L, yeast extract4.0g/L, peptone5.0g/L, NaCl3.0g/L, MnSO4·H2O0.06g/L, MgSO4·7H2O0.5g/L. Under such fermentation conditions, the maximum enzyme activity attained3301U/mL, with an increase of18.6%, compared with the original fermentation medium components.Finally, on the basis of shake culture, the mutant strain was cultured in7L NBS BioFlo415fermentor, the agitate speed and ventilatory capacity were controlled by dissolved oxygen (DO), the optimal DO was kept in the range of30%to40%. After fermented2h, which is the best time to add inducer (DL-ATC·3H2O), the enzyme activity was reached4668U/mL, with an increase of41.4%, compared with cultured in shake buttle, and cut down the cultivation time from16h in shake flask to6h in7L fermentor. The L-cysteine production reached a maximum of15.1g/L... |
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