Improving the specific synthetic activity of a penicillin g acylase using DNA family shuffling.

Penicillin G Acylas (PGA) of Providencia rettgeri (ATCC 25599) was evolved using a modified DNA family shuffling method. The identity of pga genes from Escherichia coli, Kluyvera citrophila and Providencia rettgeri ranges from 62.5% to 96.9%. The pga genes from above three species were recombined and shuffled to create interspecies pga gene fusion libraries. By substituting assembled chimaeras for corresponding region of pETPPGA, different recombinants were constructed and expressed in E. coli JM109(DE3). Mutants with obvious beta-lactam synthetic activity were selected from the plates and the ratios of synthesis to hydrolysis (S/H) were determined subsequently. It was shown that the primary structures of selected positives exhibited significant diversity among each library. The best mutant possessed 40% higher synthetic activity than the wild type enzyme of PrPGA. It was further proved in this study that the domain of alpha subunit contributed much more to improve the specific activity of synthesis. Results showed a recombinant PGA with higher synthetic activity was acquired by the method of DNA shuffling.

Purification and characterization of Alcaligenes faecalis penicillin G acylase expressed in Bacillus subtilis.

The Alcaligenes faecalis PGA gene encoding heterodimeric penicillin G acylase (PGA) was cloned and successfully expressed in Escherichia coli and Bacillus subtilis respectively. In contrast to E.coli hosts where the enzymes were retained in the periplasm, B. subtilis cell secreted the recombinant enzyme into the medium. Contrary to limited expression yield of E. coli (pETAPGA), PGA extracellularly expressed by B. subtilis (pBAPGA) and B. subtilis (pMAPGA) reached the highest yield of 653 u/L. This yield increased 109-fold higher than the native expression of A. faecalis CICC AS1.767. The enzyme was fractionated with (NH(4))(2)SO(4) and purified by DEAE-Sepharose CL-6B with a yield of 81%. The purified enzyme had a specific activity of 1.469 u/mg. Furthermore, some enzyme characteristics, such as the pH and temperature optimum, the stability against organic solvent and the ratio of cepholexin synthesis to hydrolysis were determined. By overexpressing A. faecalis PGA in B. subtilis and purifying secreted enzyme from culture medium one could readily obtain a large amount of an alternative source of PGA.