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

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

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

Ai-Sheng Xiong,Ri-He Peng,Jing Zhuang,Xian Li,Yong Xue,Jin-Ge Liu,Feng Gao,Bin Cai,Jian-Min Chen,Quan-Hong Yao,Ai-Sheng Xiong,Ri-He Peng,Jing Zhuang,Xian Li,Yong Xue,Jin-Ge Liu,Feng Gao,Bin Cai,Jian-Min Chen,Quan-Hong Yao,Ai-Sheng Xiong,Ri-He Peng,Jing Zhuang,Xian Li,Yong Xue,Jin-Ge Liu,Feng Gao,Bin Cai,Jian-Min Chen,Quan-Hong Yao,

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.

Directed evolution of a beta-galactosidase from Pyrococcus woesei resulting in increased thermostable beta-glucuronidase activity. Publishing Authors By Initials

AS Xiong,RH Peng,J Zhuang,X Li,Y Xue,JG Liu,F Gao,B Cai,JM Chen,QH Yao,AS Xiong,RH Peng,J Zhuang,X Li,Y Xue,JG Liu,F Gao,B Cai,JM Chen,QH Yao,AS Xiong,RH Peng,J Zhuang,X Li,Y Xue,JG Liu,F Gao,B Cai,JM Chen,QH Yao,

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

PUBLICATION TYPE: Journal Article

Journal: Applied microbiology and biotechnology

VOLUME: 77

Page Numbers: 569-78

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ISSN: 0175-7598

DAY: 18

MONTH: 09

YEAR: 2007

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

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LANGUAGE: eng

NlmUniqueID: 8406612

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Grant and Affiliation Information for Directed evolution of a beta-galactosidase from Pyrococcus woesei resulting in increased thermostable beta-glucuronidase activity.

AFFILIATION: Biotechnology Research Institute, Shanghai Academy of Agricultural Sciences, 2901 Beidi Road, Shanghai, 201106, China, yaoquanhongcn@yahoo.com.cn.

Country: Germany

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MEDLINETA: Appl Microbiol Biotechnol

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