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Directed evolution of a beta-galactosidase from Pyrococcus woesei resulting in increased thermostable beta-glucuronidase activity

文献类型： 外文期刊

作者： Xiong, Ai-Sheng 1 ; Peng, Ri-He 2 ; Zhuang, Jing 2 ; Li, Xian 2 ; Xue, Yong 2 ; Liu, Jin-Ge 2 ; Gao, Feng 2 ; Cai, Bin 3 ; C 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; Yangzhou Univ, Coll Biosci & Biotechnol, Yangzhou 225009, Peoples R China

3.Shanghai Acad Agr Sci, Biote

关键词： beta-galactosidase;beta-glucuronidase;Pyrococcus woesei;directed evolution;enzyme properties;structure-function analysis;GREEN FLUORESCENT PROTEIN;HIGH-LEVEL EXPRESSION;ESCHERICHIA-COLI;TRANSGENIC PLANTS;PICHIA-PASTORIS;SATURATION MUTAGENESIS;SCREENING STRATEGIES;MOLECULAR EVOLUTION;ISOLATED ENZYME;REPORTER GENE

期刊名称：APPLIED MICROBIOLOGY AND BIOTECHNOLOGY （ 影响因子：4.813； 五年影响因子：4.697 ）

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收录情况： SCI

摘要： We performed directed evolution on a chemically synthesized 1,533-bp recombinant beta-galactosidase gene from Pyrococcus woesei. More than 200,000 variant colonies in each round of directed evolution were screened using the pYPX251 vector and host strain Rosetta-Blue (DE3). One shifted beta-galactosidase to beta-glucuronidase mutant, named YG6762, was obtained after four rounds of directed evolution and screening. This mutant had eight mutated amino acid residues. T29A, V213I, L217M, N277H, I387V, R491C, and N496D were key mutations for high beta-glucuronidase activity, while E414D was not essential because the mutation did not lead to a change in beta-glucuronidase activity. The amino acid site 277 was the most essential because mutating H back to N resulted in a 50% decrease in beta-glucuronidase activity at 37 degrees C. We also demonstrated that amino acid 277 was the most essential site, as the mutation from N to H resulted in a 1.5-fold increase in beta-glucuronidase activity at 37 degrees C. Although most single amino acid changes lead to less than a 20% increase in beta-glucuronidase activity, the YG6762 variant, which was mutated at all eight amino acid sites, had a beta-glucuronidase activity that was about five and seven times greater than the wild-type enzyme at 37 and 25 degrees C, respectively.

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