利用DNA家族重排提高青霉素G酰化酶合成活力(英文)  

Improving the Specific Synthetic Activity of a Penicillin G Acylase Using DNA Family Shuffling

作　　者：周政[1] 张爱晖[1] 王晶茹[2] 陈茂林[2] 李仁宝[1] 杨晟[2] 袁中一[1] 

机构地区：[1]中国科学院上海生命科学院研究院生物化学与细胞生物学研究所,上海200031 [2]中国科学院上海生命科学研究院植物生理生态研究所,上海200032

出　　处：《生物化学与生物物理学报》2003年第6期573-579,共7页

基　　金：国家自然科学基金资助项目 (No .3 0 10 0 0 2 9);国家高技术研究发展计划 ( 863计划 )资助项目 (No .2 0 0 1AA2 3 5 0 81)~~

摘　　要：NA家族重排技术是酶定向进化的有力工具 ,已在实际应用中获得了巨大成功。来源于Providenciarettgeri、Escherichiacoli和Kluyveracitrophila的青霉素酰化酶基因序列同源性为 6 2 .5 %～ 96 .9%。在Providenciarettgeri青霉素酰化酶基因克隆和表达的基础上 ,利用DNA家族重排技术构建了上述基因的嵌合体突变库。通过平板初筛获得有活力的阳性克隆 ,表达提取突变酶测定其合成水解活力比。对突变酶进行随机测序的结果表明多基因嵌合体突变库显示出明显的多样性。通过一轮重排及筛选 ,获得了合成活力提高 4 0 %的突变酶 ,并且发现α亚基的重排对酶合成活力的提高更加有效。上述方法的应用有望获得合成活力进一步提高的青霉素G酰化酶。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 β-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 α 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.

关 键 词：青霉素酰化酶 DNA家族重排 合成水解比 合成活力 酶定向进化 

分 类 号：Q556.4[生物学—生物化学]