D194G突变对<em>meso</em>-2,3-丁二醇脱氢酶催化特性的影响

doi:10.13523/j.cb.20160107

中国生物工程杂志

2016, Vol. 36

Issue (1): 47-54 DOI: 10.13523/j.cb.20160107

研究报告

D194G突变对meso-2,3-丁二醇脱氢酶催化特性的影响

郝文博^1,2, 姬芳玲¹, 王静云¹, 张悦¹, 王天琪¹, 车文实², 包永明¹

1. 大连理工大学生命科学与技术学院大连 116024;
2. 黑河学院物理化学系黑河 164300

Effects of D194G Mutant on meso-2, 3-Butanediol Dehydrogenase Catalytic Properties

HAO Wen-bo^1,2, JI Fang-ling¹, WANG Jing-yun¹, ZHANG Yue¹, WANG Tian-qi¹, CHE Wen-shi², BAO Yong-ming¹

1. School of Life Science and Biotechnology, Dalian University of Technology, Dalian 116024, China;
2. Department of Physics and Chemistry, Heihe University, Heihe 164300, China

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摘要：

目的:比较来源于Enterobacter aerogenes CICC10293和Bacillus subtilis的meso-2,3-丁二醇脱氢酶(E. a-BDH和D194G B. s-BDH)活性和动力学参数,分析D194氨基酸对BDH催化特性的影响。方法:利用E. coli BL21(DE3)原核表达E. a-BDH和D194G B. s-BDH,经HiTrap Q FF阴离子交换柱和Superdex 75凝胶柱纯化后,用MALDI-TOF MS确定其分子质量;检测NADH/NAD⁺氧化还原的吸光度变化确定BDH活性、辅酶和底物的特异性、最适pH、温度及动力学参数。结果:重组表达E. a-BDH和D194G B. s-BDH是同源四聚体蛋白,基因序列有两处碱基不同(g.27A/T和g.581A/G),其中g.581A/G导致BDH的一处氨基酸发生改变(p.D194G)。D194G B. s-BDH的活性约为E. a-BDH的2.3%,并且丧失了氧化meso-2,3-丁二醇的能力。二者均以乙偶姻/NADH为最适底物,但D194G B. s-BDH的K_m是E. a-BDH的5.63倍。结论:D194G氨基酸突变降低了BDH的活性。

关键词： meso-2,3-丁二醇脱氢酶; 乙偶姻; 枯草芽孢杆菌

Abstract:

Objective: To compare the activity and kinetic parameters of meso-2,3-Butanediol dehydrogenase (BDH) from Enterobacter aerogenes (E. a-BDH) and Bacillus subtilis (B. s-BDH), and analysis the influences of the residue D194 on catalytic properties of BDH. Methods: E. a-BDH and D194G B. s-BDH were expressed in E. coli BL21 (DE3), and purified by HiTrap Q FF anion-exchange and Superdex 75 gel column. MALDI-TOF MS was used to determine the molecular weight. The enzyme activity, coenzyme and substrate specificity, optimum pH, temperature, and kinetic parameters of BDH were investigated by monitoring changes in absorbance of NADH/NAD⁺ redox reaction. Results: The recombinant E. a-BDH and D194G B. s-BDH are homo-tetramer. Their nucleotide sequences exhibit two different bases (g.27A/T and g.581A/G), and g.581A/G results in an amino acid change (p.D194G). D194G B. s-BDH activity is about 2.3% of E. a-BDH, and lost the ability of oxidation of meso-2, 3-butanediol. Acetoin/NADH is the optimal substrate of BDH, but K_m of D194G B. s-BDH is 5.63 times greater than that of E. a-BDH. Conclusion: D194G mutation reduces the BDH activity.

Key words: meso-2 3-butanediol dehydrogenase Acetoin Bacillus subtilis

收稿日期: 2015-08-04 出版日期: 2016-01-11

ZTFLH:

Q78

基金资助:

黑龙江省青年科学基金资助项目(QC2012C009)

通讯作者: 包永明 E-mail: biosci@dlut.edu.cn

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引用本文:

郝文博, 姬芳玲, 王静云, 张悦, 王天琪, 车文实, 包永明. D194G突变对meso-2,3-丁二醇脱氢酶催化特性的影响[J]. 中国生物工程杂志, 2016, 36(1): 47-54.

HAO Wen-bo, JI Fang-ling, WANG Jing-yun, ZHANG Yue, WANG Tian-qi, CHE Wen-shi, BAO Yong-ming. Effects of D194G Mutant on meso-2, 3-Butanediol Dehydrogenase Catalytic Properties. China Biotechnology, 2016, 36(1): 47-54.

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https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20160107 或 https://manu60.magtech.com.cn/biotech/CN/Y2016/V36/I1/47

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