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

Thermal stability and other functional properties of Trichoderma reesei end o-1,4-beta -xylanase II (XYNII; family 11) were studied by designed mutatio ns. Mutations at three positions were introduced to the XYNII mutant contai ning a disulfide bridge (S110C-N154C) in the alpha -helix. The disulfide br idge increased the half-life of XYNII from less than 1 min to 14 min at 65 degreesC. An additional mutation at the C-terminus of the alpha -helix (Q16 2H or Q162Y) increased the half-life to 63 min. Mutations Q162H and Q162Y a lone had a stabilizing effect at 55 degreesC but not at 65 degreesC. The mu tations 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 p H, 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. (C ) 2001 Elsevier Science B.V. All rights reserved.