Synthesis of thermostable mutants of the Trichoderma reesei xylanase II for pulp bleaching applications

Trichoderma reesei xylanase II (TrX) has been used commercially for several years in pulp bleaching applications. Via protein engineering, the thermostability, temperature and pH optima of TrX have been increased. This was accomplished through the substitution of its (1-29) region with the corresponding sequence of the Thermomonospora fusca xylanase (TfX). The resultant chimeric xylanase showed an improvement of +10 degrees C and +0.7 unit in the optimal temperature and pH as compared to the recombinant wild-type TrX. Upstream extension from the -1 position of the new xylanase with a tripeptide G-R-R, elevated the optimal temperature and pH by 13 degrees C and 0.9 unit respectively. An improvement of thermostability by 15 degrees C was also observed. Three mutations (Asn10His, Tyr27Met and Asn29Leu) has been identified as essential for the improvement in the chimeric xylanase. Several high temperature TrX mutants were further expressed in T. reesei using the promoter of the T. reesei cellobiohydrolase I gene. One was scaled up for commercialisation. The new enzyme, BioBrite(TM) HB60C, has been in use at Weyerhauser Pulp and Paper Mill in Prince Albert, Saskatchewan since February 1997. When used in the production of softwood ECF market pulp, it resulted in chemical reductions of 8.2% in ClO2 and 21% in NaOH and has saved 7.8% in bleaching chemical costs.