title: Improvement of alkali stability and thermostability of Paenibacillus campinasensis Family-11 xylanase by directed evolution and site-directed mutagenesis.
abstract: The extreme process condition of high temperature and high alkali limits the applications of most of natural xylanases in pulp and paper industry. Recently, various methods of protein engineering have been used to improve the thermal and alkalic tolerance of xylanases. In this work, directed evolution and site-directed mutagenesis were performed to obtain a mutant xylanase improved both on alkali stability and thermostability from the native Paenibacillus campinasensis Family-11 xylanase (XynG1-1). Mutant XynG1-1B43 (V90R/P172H) with two units increased in the optimum pH (pH 7.0-pH 9.0) and significant improvement on alkali stability was selected from the second round of epPCR library. And the further thermoduric mutant XynG1-1B43cc16 (V90R/P172H/T84C-T182C/D16Y) with 10 °C increased in the optimum temperature (60-70 °C) was then obtained by introducing a disulfide bridge (T84C-T182C) and a single amino acid substitution (D16Y) to XynG1-1B43 using site-directed mutagenesis. XynG1-1B43cc16 also showed higher thermostability and catalytic efficiency (k cat /K m ) than that of wild-type (XynG1-1) and XynG1-1B43. The attractive improved properties make XynG1-1B43cc16 more suitable for bioleaching of cotton stalk pulp under the extreme process condition of high temperature (70 °C) and high alkali (pH 9.0).
authors.affiliation: Key Laboratory of Industrial Fermentation Microbiology, Education Ministry of China, Tianjin, 300457, China.
authors.forename: Hongchen
authors.initials: H
authors.lastname: Zheng
authors.forename: Yihan
authors.initials: Y
authors.lastname: Liu
authors.forename: Mingzhe
authors.initials: M
authors.lastname: Sun
authors.forename: Yang
authors.initials: Y
authors.lastname: Han
authors.forename: Jianling
authors.initials: J
authors.lastname: Wang
authors.forename: Junshe
authors.initials: J
authors.lastname: Sun
authors.forename: Fuping
authors.initials: F
authors.lastname: Lu
mesh_terms: D000468:Alkalies / Q000737:chemistry; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D019020:Directed Molecular Evolution; D004220:Disulfides / Q000737:chemistry; D043364:Endo-1,4-beta Xylanases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D004795:Enzyme Stability / Q000235:genetics; D015723:Gene Library; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D056507:Paenibacillus / Q000201:enzymology*; D016415:Sequence Alignment
chemical_list: D000468:Alkalies; D004220:Disulfides; D043364:Endo-1,4-beta Xylanases