Thermotoga sp.RQ7内切纤维素酶TsCel12A蛋白的酶学性质和晶体学研究  

Enzymatic Characteristic and Crystallographic Analyses of Cellulase TsCel12A from Thermotoga sp. RQ7

作　　者：李媛[1] 张丽 陈安琪 季朝能[1] LI Yuan;ZHANG Li;CHEN Anqi;JI Chaoneng(School of Life Sciences,Fudan University,Shanghai 200438,China)

机构地区：[1]复旦大学生命科学学院

出　　处：《复旦学报（自然科学版）》2019年第4期489-496,共8页Journal of Fudan University：Natural Science

基　　金：国家重点研发计划“蛋白质机器与生命过程调控”(2016YFA0500600)

摘　　要：纤维素作为丰富的可再生资源,可以通过水解制取乙醇、低聚糖等多类产品,在能源与食品等领域有广泛应用,生物酶法降解因为高效环保而成为纤维素的工业应用的主要途径.内切纤维素酶可以通过水解β-1,4糖苷键将纤维素转变为短片段多糖,是起始纤维素水解的关键酶,稳定高效的内切纤维素酶是纤维素生物酶解的关键.本文成功表达、纯化了来源于嗜热菌Thermotoga sp.RQ7的内切纤维素酶TsCel12A,酶学性质实验结果显示其最适反应温度为95℃、最适反应pH为9.0,K m为(0.3602±0.0343)mmol/L,Kcat为(4.58±0.1211)s^-1,且于5 mol/LNaCl盐溶液中不失活,说明该酶具备在高温、高盐及碱性环境下工业应用的潜力.通过结晶和优化筛选到高质量晶体,X-衍射数据分析获得了分辨率为2.36?,所属空间群P622,晶胞参数为a=184.974,b=184.974,c=49.873,α=90°,β=90°,γ=120°的晶体数据,为此酶三维结构的测定及其催化机制的阐明和酶性质的改造提供了结构基础.As a rich renewable resource,Cellulose can be degraded to fuel ethanol,oligosaccharides or other products for energy,chemical and food fields.Due to the high hydrolyzation efficiency and nonpolluting nature,cellulases are being applied to the industrial production.Among cellulases,it is endoglucanase that firstly binds and degrades cellulose molecules into smaller fragments,facilitating further utilizations.Now there is an urgent need for industry to import some types of endoglucanase which can fit the high temperature or strong acid and alkali medium.Here,we reported on the construction,expression and purification of the endoglucanase Ts Cel12A from the bacteria Thermotoga sp.RQ7.And the enzymatic activity investigations showed that the optimal reaction temperature of Ts Cel12A was 95℃,the optimum reaction pH was 9.0,the K m was(0.360 2±0.034 3)mmol/L,the K cat was(4.58±0.121 1)s^-1,the activity didn t lose in the buffer containing 5 mol/L NaCl,which indicated that this enzyme was a potential candidate catalyzing efficiently in some harsh industrial reacting conditions.The crystal was diffracted to 2.36 resolution by means of crystallization and X-ray diffraction analysis and it belonged to P 6 2 2 space group,with unit cell parameters:a=184.974,b=184.974,c=49.873,α=90°,β=90°,γ=120°.We hope these results could provide a basis for endoglucanase structure studies and protein-engineering projects.

关 键 词：糖苷酶 内切纤维素酶 热稳定性 工业应用 

分 类 号：Q71[生物学—分子生物学]