Fred Fenel

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

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.

Engineering the thermotolerance and pH optimum of family 11 xylanases by site-directed mutagenesis.

Publisher Summary This chapter describes the engineering of the thermotolerance and pH optimum of family 11 xylanases by site-directed mutagenesis. The potential of using xylanases in industrial applications generated an intensive academic and industrial activity to hunt xylanases with desired properties from all kinds of natural sources. The thermostability of mesophilic xylanases has been increased considerably by designed mutations. T. reesei xylanase II (XYNII) is stable at 40–45°C, whereas temperatures above 50°C cause conformational changes and the enzyme activity is lost quite quickly. A combination of various mutations has often a cumulative effect on the thermostability of family 11 xylanases. It is observed that when mutations with only a small stabilizing effect were combined with the disulfide bridge engineered into α helix, a considerable increase in thermostability was achieved. It is found that xylanase activity is determined by using the 3,5-dinitrosalicylic acid assay to measure the amount of reducing sugars liberated from 1% birchwood xylan.