连接肽对β-木糖苷酶HJ14GH43热适应性的影响  

Effects of Linker on the Thermal Adaptability ofβ-Xylosidase HJ14GH43

作　　者：曹丽娟 李娜 刘钰 张蕊[1,2,3,4] 黄遵锡 周峻沛[1,2,3,4] Cao Lijuan;Li Na;Liu Yu;Zhang Rui;Huang Zunxi;Zhou Junpei(College of Life Science,Yunnan Normal University,Kunming 650500;Engineering Research Center of Sustainable Development and Utilization of Biomass Energy,Ministry of Education,Yunnan Normal University,Kunming 650500;Key Laboratory of Yunnan for Biomass Energy and Biotechnology of Environment,Kunming 650500;Key Laboratory of Yunnan Provincial Education Department for Plateau Characteristic Food Enzymes,Yunnan Normal University,Kunming 650500)

机构地区：[1]云南师范大学生命科学学院,昆明650500 [2]云南师范大学生物能源持续开发利用教育部工程研究中心,昆明650500 [3]云南省生物质能与环境生物技术重点实验室,昆明650500 [4]云南师范大学云南省高校高原特色食品酶重点实验室,昆明650500

出　　处：《中国食品学报》2023年第6期81-91,共11页Journal of Chinese Institute Of Food Science and Technology

基　　金：国家自然科学基金项目(31960459);云南省基础研究计划重点项目(202001AS070022);云南省万人计划青年拔尖人才项目(YNWR-QNBJ-2018-383)。

摘　　要：为揭示连接肽对β-木糖苷酶热适应性的影响机制,本文通过替换β-木糖苷酶HJ14GH43的连接肽序列获得突变子Mut LK10,利用大肠杆菌BL21(DE3)对其进行重组异源表达。表达后对Mut LK10纯酶进行酶学特性和蛋白质结构分析。结果表明:突变酶Mut LK10的最适温度为20℃,相比野生酶HJ14GH43降低5℃。突变酶Mut LK10在20℃和10℃下处理60 min后分别保持约28%和69%的相对酶活,而野生酶HJ14GH43在20℃和10℃下处理60 min后分别保持约70%和88%的相对酶活。突变酶的最适温度和热稳定性与野生酶相比均降低。野生酶和突变酶的连接肽是位于蛋白质表面的一段无规则卷曲区域。蛋白质结构分析表明:突变后该区域内的负电势面积增大,由此带来的是该区域内亲水性的增加。结论:增大连接肽中的酸性氨基酸比例,使β-木糖苷酶通过增加表面负电势面积来竞争水合作用,从而增加酶与溶剂的相互作用并最终使酶能适应低温环境。本研究结果可为β-木糖苷酶及其它类型酶的热适应性改性研究提供参考。In this study,the mutant MutLK10 was constructed by replacing the linker ofβ-xylosidase HJ14GH43 in order to reveal the mechanism of the linker on thermal adaptability of theβ-xylosidase.MutLK10 was successfully expressed by Escherichia coli BL21(DE3).Then,the enzymatic characteristics and 3D structure of MutLK10 were analyzed.The results showed that the optimum temperature of mutant MutLK10 was 20℃,which was 5℃lower than that of the wild-type enzyme HJ14GH43.MutLK10 retained approximately 28%and 69%relative activities after being incubated at 20℃and 10℃for 60 min,respectively.Whereas HJ14GH43 retained approximately 70%and 88%relative activities after incubation at 20℃and 10℃for 60 min,respectively.Thus,MutLK10 had lower optimum temperature and worse thermalstability than HJ14GH43.The linkers of HJ14GH43 and MutLK10 were constructed as coil regions on the surfaces of proteins.Structural analysis showed that the negative potentials of the coil regions increased after the mutation,which led to the increase of hydrophilicity.It indicates that increasing the proportion of acidic amino acids in the linker can help theβ-xylosidase compete for hydration by increasing the surface negative electrostatic potentials,that may result in the increase of the interactions between the enzyme and solvents and finally enables the enzyme to adapt to low-temperature environments.This study provides a reference for thermal adaptability modification ofβ-xylosidases and other industrial enzymes.

关 键 词：Β-木糖苷酶 连接肽 热适应性 低温 机制 

分 类 号：TS201.2[轻工技术与工程—食品科学]