title: A combination of weakly stabilizing mutations with a disulfide bridge in the alpha-helix region of Trichoderma reesei endo-1,4-beta-xylanase II increases the thermal stability through synergism.
abstract: Thermal stability and other functional properties of Trichoderma reesei endo-1,4-beta-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 alpha-helix. The disulfide bridge increased the half-life of XYNII from less than 1 min to 14 min at 65 degrees C. An additional mutation at the C-terminus of the alpha-helix (Q162H or Q162Y) increased the half-life to 63 min. Mutations Q162H and Q162Y alone had a stabilizing effect at 55 degrees C but not at 65 degrees 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.
authors.affiliation: Laboratory of Bioprocess Engineering, Helsinki University of Technology, P.O. Box 6100, 02015-, HUT, Finland. ossi.turunen@hut.fi
authors.forename: O
authors.initials: O
authors.lastname: Turunen
authors.forename: K
authors.initials: K
authors.lastname: Etuaho
authors.forename: F
authors.initials: F
authors.lastname: Fenel
authors.forename: J
authors.initials: J
authors.lastname: Vehmaanperä
authors.forename: X
authors.initials: X
authors.lastname: Wu
authors.forename: J
authors.initials: J
authors.lastname: Rouvinen
authors.forename: M
authors.initials: M
authors.lastname: Leisola
mesh_terms: D020134:Catalytic Domain; D004220:Disulfides / Q000737:chemistry; D043364:Endo-1,4-beta Xylanases; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009154:Mutation; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods; D013696:Temperature; D014242:Trichoderma / Q000201:enzymology*; D014995:Xylosidases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
chemical_list: D004220:Disulfides; D014995:Xylosidases; D043364:Endo-1,4-beta Xylanases