title: Obtaining a mutant of Bacillus amyloliquefaciens xylanase A with improved catalytic activity by directed evolution.
abstract: This study aimed to obtain xylanase exhibiting improved enzyme properties to satisfy the requirements for industrial applications. The baxA gene encoding Bacillus amyloliquefaciens xylanase A was mutated by error-prone touchdown PCR. The mutant, pCbaxA50, was screened from the mutant library by using the 96-well plate high-throughput screening method. Sequence alignment revealed the identical mutation point S138T in xylanase (reBaxA50) produced by the pCbaxA50. The specific activity of the purified reBaxA50 was 9.38U/mg, which was 3.5 times higher than that of its parent expressed in Escherichia coli BL21 (DE3), named reBaxA. The optimum temperature of reBaxA and reBaxA50 were 55°C and 50°C, respectively. The optimum pH of reBaxA and reBaxA50 were pH 6 and pH 5, respectively. Moreover, reBaxA50 was more stable than reBaxA under thermal and extreme pH treatment. The half-life at 60°C and apparent melting temperature of reBaxA50 were 9.74min and 89.15°C, respectively. High-performance liquid chromatography showed that reBaxA50 released xylooligosaccharides from oat spelt, birchwood, and beechwood xylans, with xylotriose as the major product; beechwood xylan was also the most thoroughly hydrolyzed. This study demonstrated that the S138T mutation possibly improved the catalytic activity and thermostability of reBaxA50.
authors.affiliation: Key Laboratory of Marine Food Quality and Hazard Controlling Technology of Zhejiang Province, College of Life Science, China JiLiang University, Hangzhou 310018, China.
authors.forename: Xin
authors.initials: X
authors.lastname: Xu
authors.affiliation: Key Laboratory of Marine Food Quality and Hazard Controlling Technology of Zhejiang Province, College of Life Science, China JiLiang University, Hangzhou 310018, China; National & Local United Engineering Lab of Quality Controlling Technology and Instrumentation for Marine Food, China JiLiang University, Hangzhou 310018, China. Electronic address: mqliu524@163.com.
authors.forename: Ming-qi
authors.initials: MQ
authors.lastname: Liu
authors.affiliation: Key Laboratory of Marine Food Quality and Hazard Controlling Technology of Zhejiang Province, College of Life Science, China JiLiang University, Hangzhou 310018, China.
authors.forename: Wen-kang
authors.initials: WK
authors.lastname: Huo
authors.affiliation: Key Laboratory of Marine Food Quality and Hazard Controlling Technology of Zhejiang Province, College of Life Science, China JiLiang University, Hangzhou 310018, China; National & Local United Engineering Lab of Quality Controlling Technology and Instrumentation for Marine Food, China JiLiang University, Hangzhou 310018, China.
authors.forename: Xian-jun
authors.initials: XJ
authors.lastname: Dai
mesh_terms: D000069977:Bacillus amyloliquefaciens / Q000201:enzymology* / Q000235:genetics*; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism*; D019020:Directed Molecular Evolution; D043364:Endo-1,4-beta Xylanases / Q000235:genetics* / Q000378:metabolism*; D004795:Enzyme Stability; D005798:Genes, Bacterial; D006868:Hydrolysis; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D040681:Structural Homology, Protein; D013379:Substrate Specificity; D014990:Xylans / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D014990:Xylans; D043364:Endo-1,4-beta Xylanases
keywords: Catalytic activity; Error-prone PCR; Hydrolysis; Mutant; Thermostability; Xylanase