Pseudomonas sp.B-3 is a promising industrial strain for bioconversion of DL-2-amino-A2-thiazoline-4-carboxylic acid (DL-ATC) to L-cysteine.In order to further enhance its bioconversion ability, nitrogen ion beam implantation was applied in strain B-3 and a powerful mutant with highest enzyme activity designated as Pseudomonas sp.C-25 was obtained, of which L-cysteine enzyme activity achieved 2748 U/mL, an increase of 26.3% compared to that of initial strain B-3 (2176 U/mL).Meanwhile the yield of L-cysteine improved from 11.85 g/L to 14.12 g/L.The mutant strain exhibited favorable property about producing L-cysteine over the original strain.The maximum enzyme activity of strain C-25 in 7 L fermentor reached 3925 U/mL, and the cultivation time with maximum enzyme activity in the fermentor was shorten for 11 h than in the Erlenmeyer flasks, and the yield of L-cysteine attained 16.53 g/L in 7 L fermentor.Moreover, the genes of N-carbamyl-L-cysteine (L-NCC) hydrolase were cloned from strain B-3 and mutant strain C-25 respectively and their protein sequence were analyzed.Subsequently, online server Ⅰ-TASSER was used to predict its 3-dimensional (3D)structure and understand the function sites.The results showed that the first mutant site was 212th nucleotide, which changed from T to C and the 71th amino acid changed from Ile to Thr, the second mutant site was 1052th nucleotide, which changed from C to T and the 351th amino acid changed from His to Tyr.The protein 3D structure of L-NCC hydrolase was predicted and has a high confidence.