Improvement of thermostability and activity of Trichoderma reesei endo-xylanase Xyn III on insoluble substrates
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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.
KeywordsXylanase Trichoderma reesei Biomass Xylan Thermostability Xylan-binding domain
We thank Yuko Chujo and Akiko Yamanaka (AIST) for assistance in screening of thermostable Xyn III mutants and protein purification. This work was supported in part by grants from the New Energy and Industrial Technology Development Organization (NEDO).
Compliance with ethical standards
This article does not contain any studies with human participants or animals performed by any of the authors.
Conflict of interest
The authors declare that they have no conflicts of interest.
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