title: Improvement of thermostability and activity of Trichoderma reesei endo-xylanase Xyn III on insoluble substrates.
abstract: Trichoderma reesei Xyn III, an endo-β-1,4-xylanase belonging to glycoside hydrolase family 10 (GH10), is vital for the saccharification of xylans in plant biomass. However, its enzymatic thermostability and hydrolytic activity on insoluble substrates are low. To overcome these difficulties, the thermostability of Xyn III was improved using random mutagenesis and directed evolution, and its hydrolytic activity on insoluble substrates was improved by creating a chimeric protein. In the screening of thermostable Xyn III mutants from a random mutagenesis library, we identified two amino acid residues, Gln286 and Asn340, which are important for the thermostability of Xyn III. The Xyn III Gln286Ala/Asn340Tyr mutant showed xylanase activity even after heat treatment at 60 °C for 30 min or 50 °C for 96 h, indicating a dramatic enhancement in thermostability. In addition, we found that the addition of a xylan-binding domain (XBD) to the C-terminal of Xyn III improved its hydrolytic activity on insoluble xylan.
authors.affiliation: Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki, 305-8566, Japan.
authors.forename: Tomohiko
authors.initials: T
authors.lastname: Matsuzawa
authors.affiliation: Department of Subtropical Biochemistry and Biotechnology, Faculty of Agriculture, University of the Ryukyus, 1 Senbaru, Nishihara, Okinawa, 903-0213, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Kaneko
authors.affiliation: Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki, 305-8566, Japan. k-yaoi@aist.go.jp.
authors.forename: Katsuro
authors.initials: K
authors.lastname: Yaoi
mesh_terms: D019943:Amino Acid Substitution; D019020:Directed Molecular Evolution; D043364:Endo-1,4-beta Xylanases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability; D006358:Hot Temperature; D006868:Hydrolysis; D016296:Mutagenesis; D015202:Protein Engineering / Q000379:methods*; D055550:Protein Stability; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors; D014242:Trichoderma / Q000201:enzymology*
chemical_list: D011994:Recombinant Proteins; D043364:Endo-1,4-beta Xylanases
keywords: Biomass; Thermostability; Trichoderma reesei; Xylan; Xylan-binding domain; Xylanase