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

Zheng Zhou, Ai-Hui Zhang, Jing-Ru Wang, Mao-Lin Chen, Ren-Bao Li, Sheng Yang, Zhong-Yi Yuan
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Abstract

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.

利用DNA家族洗牌提高青霉素酰化酶的合成活性。
利用改进的DNA家族洗牌方法,对盘尼西林G酰基蛋白(PGA)进行了进化。大肠埃希菌、嗜雪克鲁菌和雷氏普罗维登菌的pga基因同源性在62.5% ~ 96.9%之间。将上述3个物种的pga基因进行重组和洗牌,建立种间pga基因融合文库。通过将组装好的嵌合体替换为pETPPGA的相应区域,构建不同的重组体并在大肠杆菌JM109(DE3)中表达。从培养皿中选择具有明显β -内酰胺合成活性的突变体,测定合成与水解比(S/H)。结果表明,各文库间所选阳性基因的主要结构存在显著差异。最佳突变体比野生型PrPGA酶的合成活性高40%。本研究进一步证明α亚基结构域对提高合成比活性的作用更大。结果表明,采用DNA洗牌法获得了具有较高合成活性的重组PGA。
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