A combination of weakly stabilizing mutations with a disulfide bridge in the α-helix region of Trichoderma reesei endo-1,4-β-xylanase II increases the thermal stability through synergism

• Published in: Journal of biotechnology 2001-06, Vol.88 (1), p.37-46
• Main Authors: Turunen, Ossi, Etuaho, Kirsikka, Fenel, Fred, Vehmaanperä, Jari, Wu, Xiaoyan, Rouvinen, Juha, Leisola, Matti
• Format: Article
• Language: English
• Subjects: Biological and medical sciences; Biotechnology; Catalytic Domain; Designed mutations; Disulfides - chemistry; Endo-1,4-beta Xylanases; Endo-1,4-β-xylanase; Enzyme Stability; Family 11; Fundamental and applied biological sciences. Psychology; Hydrogen-Ion Concentration; Kinetics; Methods. Procedures. Technologies; Mutation; pH activity; Protein Conformation; Protein engineering; Protein Engineering - methods; Temperature; Thermal stability; Trichoderma - enzymology; Trichoderma reesei; Xylosidases - chemistry; Xylosidases - genetics; Xylosidases - metabolism
• ISSN: 0168-1656; 1873-4863
• DOI: 10.1016/S0168-1656(01)00253-X

Thermal stability and other functional properties of Trichoderma reesei endo-1,4-β-xylanase II (XYNII; family 11) were studied by designed mutations. Mutations at three positions were introduced to the XYNII mutant containing a disulfide bridge (S110C–N154C) in the α-helix. The disulfide bridge increased the half-life of XYNII from less than 1 min to 14 min at 65°C. An additional mutation at the C-terminus of the α-helix (Q162H or Q162Y) increased the half-life to 63 min. Mutations Q162H and Q162Y alone had a stabilizing effect at 55°C but not at 65°C. The mutations N11D and N38E increased the half-life to about 100 min. Due to the stabilizing mutations the pH stability increased in a wide pH range, but at the same time the activity decreased both in acidic and neutral–alkaline pH, the pH optimum being at pH region 5–6. There was no essential difference between the specific activities of the mutants and the wild-type XYNII.