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Concurrent mutations in six amino acids in beta-glucuronidase improve its thermostability

文献类型： 外文期刊

作者： Xiong, Ai-Sheng 1 ; Peng, Ri-He 2 ; Cheng, Zong-Ming 2 ; Li, Yi 3 ; Liu, Jin-Ge; Zhuang, Jing; Gao, Feng; Xu, Fa 1 ;

作者机构： 1.Shanghai Acad Agr Sci, Biotechnol Res Inst, Shanghai 201106, Peoples R China

2.Shanghai Acad Agr Sci, Biotechnol Res Inst, Shanghai 201106, Peoples R China; Univ Tennessee, Dept Plant Sci, Knoxville, TN 37996 USA; Univ Connecticut, Dept Plant Sci, Storrs, CT 06269 USA; Nanjing Agr Univ, Coll Horticulture, Nanjing 210095, Peoples R China; Yangzhou Univ, Coll Biosci & Biotechnol, Yangzhou 225009, Peoples R China

3.Shanghai Acad Agr Sci, Biotechnol Res Inst, Shanghai 201106, Peoples R China; Univ Tennessee, Dept Plant Sci, Knoxville, TN 37996 USA; Univ Connecticut, Dept Plant Sci, Storrs, CT 06269 USA; Nanjing Agr Univ, Coll Horticulture, Nanjing 210095, Peoples R China; Yangzhou Univ

关键词： beta-glucuronidase;directed evolution;enzyme properties;structure-function analysis;thermostability

期刊名称：PROTEIN ENGINEERING DESIGN & SELECTION （ 影响因子：1.65； 五年影响因子：2.103 ）

ISSN： 1741-0126

年卷期： 2007 年 20 卷 7 期

页码：

收录情况： SCI

摘要： To achieve a thermostable beta-glucuronidase (GUS) and identify key mutation sites, we applied in vitro directed evolution strategy through DNA shuffling and obtained a highly thermostable mutant GUS gene, gus-tr, after four rounds of DNA shuffling and screening. This variant had mutations in 15 nucleic acid sites, resulting in changes in 12 amino acids (AAs). Using gus-tr as the template, we further performed site-directed mutagenesis to reverse the individual mutation to the wild-type protein. We found that six sites (Q493R, T509A, M532T, N550S, G559S and N566S) present in GUS-TR3337, were the key AAs needed to confer its high thermostability. Of these, Q493R and T509A were not reported previously as important residues for thermostability of GUS. Furthermore, all of these six mutations must be present concurrently to confer the high thermostability. We expressed the gus-tr3337 gene and purified the GUS-TR3337 protein that contained the six AA mutations. Compared with the wild-type protein which lost its activity completely after 10 min at 70 degrees C, the mutant GUS-TR3337 protein retained 75% of its activity when heated at 80 degrees C for 10 min. The GUS-TR3337 exhibited high activity even heated at 100 degrees C for 30 min on nitrocellulose filter. The comparison of molecular models of the mutated and wild-type enzyme revealed the relation of protein function and these structural modifications.

  • 相关文献

[1]Directed evolution of a beta-galactosidase from Pyrococcus woesei resulting in increased thermostable beta-glucuronidase activity. Xiong, Ai-Sheng,Peng, Ri-He,Zhuang, Jing,Li, Xian,Xue, Yong,Liu, Jin-Ge,Gao, Feng,Cai, Bin,Chen, Jian-Min,Yao, Quan-Hong.

[2]Directed evolution of beta-galactosidase from Escherichia coli into beta-glucuronidase. Xiong, Ai-Sheng,Peng, Ri-He,Zhuang, Jing,Liu, Jin-Ge,Xu, Fang,Cai, Bin,Guo, Zhao-Kui,Qiao, Yu-Shan,Chen, Jian-Min,Zhang, Zhen,Yao, Quan-Hong. 2007

[3]High efficiency and throughput system in directed evolution in vitro of reporter gene. Xiong, Ai-Sheng,Peng, Ri-He,Liu, Jin-Ge,Zhuang, Jing,Qiao, Yu-Shan,Xu, Fang,Cai, Bing,Zhang, Zhen,Chen, Jian-Min,Yao, Quan-Hong.

[4]Semi-rational site-directed mutagenesis of phyI1s from Aspergillus niger 113 at two residue to improve its phytase activity. Tian, Yong-Sheng,Peng, Ri-He,Xu, Jing,Zhao, Wei,Gao, Feng,Fu, Xiao-Yan,Xiong, Ai-Sheng,Yao, Quan-Hong.

[5]A semi-rational design strategy of directed evolution combined with chemical synthesis of DNA sequences. Xiong, Ai-Sheng,Peng, Ri-He,Zhuang, Jing,Liu, Jin-Ge,Gao, Feng,Xu, Fang,Cai, Bin,Yao, Quan-Hong.

[6]Expression and Function of a Modified AP2/ERF Transcription Factor from Brassica napus Enhances Cold Tolerance in Transgenic Arabidopsis. Xiong, Ai-Sheng,Wang, Feng,Jiang, Hai-Hua,Peng, Ri-He,Jin, Xiao-Fen,Zhu, Bo,Yao, Quan-Hong,Zhuang, Jing,Zhang, Jian.

[7]Characterization of a thermostable beta-glucuronidase from Thermotoga maritima expressed in Arabidopsis thaliana. Xu, Jing,Tian, Yong-Sheng,Peng, Ri-He,Zhu, Bo,Gao, Jian-Jie,Yao, Quan-Hong. 2012

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