Improving the activity and stability of OPH enzyme using DNA Shuffling method

One of the effective methods for protein engineering, especially for improving the capabilities of industrial strains, is the genome shuffling method. This study is based on biocatalytic engineering techniques and DNA shuffling methods.

Pseudomonas aeruginosa wild strain was prepared under the access number JQ917006.1. Mutant strains were evaluated by DES method and compatibility with diazinon concentration. After the protoplast was prepared, the genome was mutated from the mutant library. Fused protoplasts were evaluated for activity.

The activities related to IR1.G1, IR1.D8, IR1.D4 and IR1.D5 strains are 0.234 U/ml, 0.1 U/ml, 0.098 U/ml and 0.066 U/ml, respectively, and IRL1.F2, IRL1.F3 and IRL1.F1 strains have activities of 0.541 mg/L, 0.523 mg/L and 0.509 mg/L, respectively.

The results of evaluation of the first generation of genome shuffling (first round of protoplast fusion) showed that the shuffled strains that were able to grow in the presence of toxin (3000 ml/L diazinon concentration) had better activity than the obtained strain through both methods (toxin concentration gradient and DES method).