Improvement of the Activity of Cephalexin Semi-synthesizing Enzyme was attempted by the NTG mutagenesis. Two systems of Cephalexin acylase were used.
In Xanthomonas citri IF03835, The conditions for Mutant screening were adjusted. Two mutants having higher activity of cephalexin semi-synthesis than that of the- wild type were selected.
In E. coli SH2 having a gene for cephalexin acylase from Bacillus megaterium ATCC14945 on pUC19 plasmid, two tester organisms, E. coli KL16-21 and S. aureus ATCC6583P, with different sensitivity cephalexin (KL16-21 > S. aureus) were used for soft-agar bioassay. Two mutants SH2B and SH2D were obtained from the first round mutagenesis. And using SH2B as parent strain, two more mutants SH2B1, SH2B7 were selected. In Soft-agar Bioassay and Penicillin G hydrolysis assay, the mutants showed higher activity than wild type. In vitro recombination mapping showed that the mutated sites of SH2B, SH2B1 and SH2B7 were located in the C-terminal region of BCA gene. Using shuttle-vector, the BCA genes of SH2B, SH2D, SH2B1 and SH2B7 were transferred to Bacillus subtilis DB104. Higher activity than that of the wild type was detected in B. subtilis having mutated acylase than wild type.
The partially purified enzyme from B. subtilis also showed that the mutated acylase had higher activity than wild type enzyme.
