采用随机突变方法对β-琼胶酶YM01-3活性位点的研究  

Exploration on the active sites of β-agarase YM01-3 with random mutation

作　　者：曲雯雯 吴蕾蕾 周顺 史晓翀[1,2] 张晓华 QU Wen-Wen;WU Lei-Lei;ZHOU Shun;SHI Xiao-Chong;ZHANG Xiao-Hua(College of Marine Life Sciences,Ocean University of China,Qingdao,Shandong 266003,China;Laboratory for Marine Ecology and Environmental Science,Pilot National Laboratory for Marine Science and Technology（Qingdao）,Qingdao,Shandong 266071,China)

机构地区：[1]中国海洋大学海洋生命学院,山东青岛266003 [2]青岛海洋科学与技术试点国家实验室海洋生态与环境科学功能实验室,山东青岛266071

出　　处：《微生物学通报》2018年第9期2000-2005,共6页Microbiology China

基　　金：国家重点研发计划(2016YFA0601303;2017YFA0603200);国家自然科学基金(41206117;41476112)~~

摘　　要：【背景】琼胶酶是一种多糖水解酶,在保健食品、医药、科研及化妆品等行业极具价值。本实验室发现来源自嗜琼胶卵链菌(Catenovulumagarivorans)的β-琼胶酶YM01-3具有较高的酶活性,在最适条件下的比酶活可达到1.14×10~4U/mg。【目的】探讨不同位点的突变对β-琼胶酶YM01-3酶活力的作用,发现影响其酶活力的新位点。【方法】通过易错PCR在短芽孢杆菌(Bacillus brevis)表达系统中构建随机突变文库,从约10 000个克隆中筛选出227株有效突变体,从中选取80株进行测序。【结果】对突变体序列进行分析和定点突变验证发现,137位和237位氨基酸发生突变后酶活力丧失90%以上。【结论】位于催化腔内的137位和237位氨基酸,对于维持β-琼胶酶YM01-3酶活力具有重要作用。该研究结果为β-琼胶酶的催化机理研究及分子改造提供了借鉴。[Background] Agarases belong to glycoside hydrolases with broad application potentials in scientific research, health-food, pharmaceutical and cosmetic industries. Although a β-agarase YM01-3 from Catenovulum agarivorans YM01T was obtained in previous studies with an activity up to 1.14×104 U/mg, its catalytic mechanism is still unclear. [Objective] In order to clarify the catalytic mechanism of YM01-3, the key sites affecting the activity should be identified. [Methods] A random mutation library in Bacillus brevis was constructed by error-prone PCR to select mutants that completely lost enzyme activity. In total 227 mutants showing lower activity were selected from about 10 000 clones and 80 mutants were sequenced and analyzed. The putative key sites were confirmed by mutation construction. [Results] Compared to the original enzyme YM01-3, two mutants at 137 and 237 sites lost more than 90% catalytic activity. Y137 and D237, located in the catalytic cavity, were proved to be the novel key sites closely related to YM01-3 hydrolytic activity. [Conclusion] Our findings provide reference for further studying catalytic mechanism and molecular modification of β-agarase.

关 键 词：β-琼胶酶YM01-3 随机突变 定点突变 

分 类 号：Q55[生物学—生物化学]