title: Directed evolution of a beta-galactosidase from Pyrococcus woesei resulting in increased thermostable beta-glucuronidase activity.
abstract: 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.
authors: Biotechnology Research Institute, Shanghai Academy of Agricultural Sciences, 2901 Beidi Road, Shanghai, 201106, China.
authors: Ai-Sheng
authors: AS
authors: Xiong
authors: Ri-He
authors: RH
authors: Peng
authors: Jing
authors: J
authors: Zhuang
authors: Xian
authors: X
authors: Li
authors: Yong
authors: Y
authors: Xue
authors: Jin-Ge
authors: JG
authors: Liu
authors: Feng
authors: F
authors: Gao
authors: Bin
authors: B
authors: Cai
authors: Jian-Min
authors: JM
authors: Chen
authors: Quan-Hong
authors: QH
authors: Yao
mesh_terms: D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D019020:Directed Molecular Evolution; D004795:Enzyme Stability; D015964:Gene Expression Regulation, Bacterial; D005966:Glucuronidase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D015202:Protein Engineering; D019711:Pyrococcus / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D001616:beta-Galactosidase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D001616:beta-Galactosidase; D005966:Glucuronidase