Screening and culture optimization of Bacillus megaterium strains for penicillin G acylase production

Abstract:

[Objective] The aim of this study was to screen strains with high penicillin G acylase (PGA) activity. [Methods] Induced mutation of Bacillus megaterium ATCC 14945 was performed by LiCl-ultraviolet composite mutagenesis and atmospheric and room temperature plasma (ARTP) mutagenesis. Strains treated by two mutation methods were spread on solid plate culture medium. The colony growing on plate was inoculated into liquid medium and cultured at 28 °C, 250 r/min. Continuing culture was performed via transferring bacterium broth to fresh liquid medium with 10% of inoculum size to obtain second generation broth. After 6 h, phenylacetic acid was added with 0.1% final concentration into second generation broth and cultured for 40 h. The bacterium broth was centrifuged for 5 minutes with 8 000 r/min. The supernatants are crude enzyme and their PGA activities were assayed with NIPAB method. The enzyme-producing conditions, including the addition quantity and timing of phenylacetic acid were optimized using the strain with the best PGA enzyme of activity from the mutants. The protein properties of PGA from the strains induced before and after was analyzed by SDS-PAGE. [Results] The strains numbered 12-4 had the highest PGA of 39.60 U/mL. PGA activity increases by 8.5 times than that of original strains. After culturing strains12-4 for 6 h and adding phenylacetic acid into broth with quantity of 0.2% (W/V), continuing culture for 50 h, the PGA activity from the supernatants reached to 78.45 U/mL, which is 16.8 times than that of original strains. PGA from the strains induced before and after all has α and β subunit. The amount of α subunit in PGA from the induced strains was no significant change. However, the amount of β subunit significantly increased, also protein bands locating between α and β subunit markedly increased. [Conclusion] The activity of PGA from Bacillus megaterium could be increased by mutation. The obtained strains and PGA-producing conditions in this study are of important value to industrial production.