高产青霉素酰化酶巨大芽胞杆菌的诱变选育及产酶条件优化

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

摘要:

【目的】选育高产青霉素G酰化酶(PGA)工业菌株。【方法】采用LiCl-紫外线复合诱变以及常压室温等离子体(ARTP)诱变技术对巨大芽胞杆菌(Bacillus megaterium) ATCC 14945进行处理。处理菌体涂平板后，将长出的菌落接种到液体培养基中，向培养6 h后的二代菌液中添加终浓度为0.1%的苯乙酸，28 °C、250 r/min条件下诱导培养40 h。对离心后获得的上清(粗酶液)采用NIPAB法测定PGA酶活力。以PGA酶活力最高的菌株为材料，对苯乙酸最佳添加量和最佳诱导时间进行优化，采用NIPAB法测定PGA酶活力。采用SDS-PAGE检测诱变前后巨大芽胞杆菌粗酶液中PGA的蛋白特性。【结果】从诱变菌落中筛选到PGA酶活力为39.60 U/mL的菌株12-4，酶活力比出发菌株提高了8.5倍。该菌株在液体培养6 h后添加终浓度为0.2%的苯乙酸，继续培养50 h后，PGA酶活力可达78.45 U/mL，比出发菌株提高了16.8倍。诱变前后菌株培养液中的PGA蛋白均具α、β亚基；诱变后菌株PGA α亚基的量没有明显变化，β亚基的量明显增多；α、β亚基之间的蛋白条带明显增多。【结论】采用诱变技术可提高巨大芽胞杆菌PGA活性，获得的诱变菌株12-4及培养条件对PGA工业化生产具有重要价值。

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.