<i>Myxococcus</i> sp.V11海藻糖合酶TreS II分子改造 <sup>*</sup>

doi:10.13523/j.cb.1907049

中国生物工程杂志

2020, Vol. 40

Issue (3): 79-87 DOI: 10.13523/j.cb.1907049

技术与方法

Myxococcus sp.V11海藻糖合酶TreS II分子改造 ^*

赵晓艳¹,陈允妲¹,章雅倩¹,吴晓玉^1,²,王飞^1,^2,^**(

),陈金印²

1 江西农业大学生物科学与工程学院南昌 330045
2 江西省果蔬保鲜与质量安全创新中心南昌 330045

Site-directed Mutagenesis Improves the Thermostability of Trehalose Synthase TreS II from Myxococcus sp.V11

ZHAO Xiao-yan¹,CHEN Yun-da¹,ZHANG Ya-qian¹,WU Xiao-yu^1,²,WANG Fei^1,^2,^**(

),CHEN Jin-yin²

1 College of Bioscience and Bioengineering, Jiangxi Agriculture University, Nanchang 330045, China
2 Collaborative Innovation Center of Postharvest Key Technology and Quality Safety of Fruits and Vegetables in Jiangxi Province, Nanchang 330045, China

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摘要：

来源于黏细菌Myxococcus sp.V11的海藻糖合酶(trehalose synthase, EC 2.4.1.245)TreS II可通过转糖苷作用将麦芽糖转化成为海藻糖,在酶法生产海藻糖上显示出一定的应用潜力,但TreS II对热敏感,在60℃保温3h,酶活性丧失,限制了其应用范围.目的:拟探索TreS II影响热稳定性的氨基酸残基构成,通过对可能的氨基酸位点进行定点突变,以期获得耐热性的突变子,扩大TreS II应用范围.方法: 通过PCR介导的方法对TreS II可能影响到热稳定性的氨基酸Q3,A283,W374,R449和Y537进行定点突变,以野生型重组酶为对照,比较突变型与野生型的最适反应温度和最适反应pH,通过测定不同温度下保存不同时间后的残留酶活,检测突变子的耐热效果.结果: 研究表明突变子Q3D,A283R,W374D,R449Q和Y537H的比酶活与野生型无显著差异,且最适pH 和最适反应温度也未发生改变;A283R,Y537H在60℃条件下,3h后活性剩余68%;Q3D,W374D,R449Q在温度60℃时,3h后活性剩余35%.结论: TreS II分子结构中与金属离子结合的几个氨基酸残基的改变对蛋白质分子的耐热性具有显著影响.

关键词： Myxococcus; sp.V11; 海藻糖合酶; 定点突变; 热稳定性

Abstract:

The trehalose synthase (EC 2.4.1.245) from Myxococcus sp.V11 (TreS II) catalyzes the reversible interconversion of maltose and trehalose. The high catalytic activity and high conversion rate of maltose into trehalose of TreS II indicate that it has potential application in industrial production of trehalose. However, the thermal instability of TreS II limits its wide application in trehalose production. Objective:The effects of amino acid residues mutations on the thermal stability, optima of pH and temperature, and specific activity of TreS II were studied by site-directed mutagenesis. Methods: Site-directed mutation experiment of the two possible metal ion-binding sites (A283 and Y537) and the three sites (Q3, W374 and R449) in two regions which may correlate with thermostability by using overlapping PCR were performed. Mutants of A283R, Y537H, Q3D, W374D and R449Q were heterogeneous expressed in E.coli BL21(DE3). At the same time the specific activity, the optimum reaction temperature, the optimum pH and the thermal stability of mutants were compared with wild-type strain. Results: Mutation of Q3D, W374D, R449Q, A283R and Y537H enhanced the thermal stability, but did not affect the pH and temperature optima. Only the mutant R449Q reduced the specific activity. The modified enzymes A283R and Y537H showed 68% and the mutants Q3D, R449Q, W374D showed 35% of maximal activity after incubating in maltose substrate for 3h at 60℃ compared to only 20% activity for wild-type enzyme. Conclusion: These factors may render TreS II relatively more thermostable among mesophilic trehalose synthases. The thermophilic amino acid residues provided herein may provide guidance for further protein engineering in the design of stabilized enzymes.

Key words: Myxococcus sp.V11 Trehalose synthase Site-directed mutagenesis Thermostability

收稿日期: 2019-07-27 出版日期: 2020-04-18

ZTFLH:

Q814

基金资助: * 国家自然科学基金地区科学项目(31560031);江西省自然科学基金(20161BAB204178);江西省教育厅科学技术研究项目(GJJ160387)

通讯作者: 王飞 E-mail: wangfei179@163.com

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引用本文:

赵晓艳,陈允妲,章雅倩,吴晓玉,王飞,陈金印. Myxococcus sp.V11海藻糖合酶TreS II分子改造 ^*[J]. 中国生物工程杂志, 2020, 40(3): 79-87.

ZHAO Xiao-yan,CHEN Yun-da,ZHANG Ya-qian,WU Xiao-yu,WANG Fei,CHEN Jin-yin. Site-directed Mutagenesis Improves the Thermostability of Trehalose Synthase TreS II from Myxococcus sp.V11. China Biotechnology, 2020, 40(3): 79-87.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.1907049 或 https://manu60.magtech.com.cn/biotech/CN/Y2020/V40/I3/79

表1 TreS II 定点突变PCR扩增所用引物

图1 TreS II 和已知海藻糖合酶氨基酸序列的比较

图2 PCR介导定点突变tresII

图3 突变重组酶TreS II SDS-PAGE图谱

图4 突变型TreS II最适反应温度

图5 突变型TreS II最适反应pH

图6 突变型TreS II与野生型比酶活的比较

图7 突变型TreS II与野生型热稳定性比较

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