tailieunhanh - Báo cáo khoa học: The relationship between thermal stability and pH optimum studied with wild-type and mutant Trichoderma reesei cellobiohydrolase Cel7A

The major cellulase secreted by the filamentous fungus Trichoderma reeseiis cellobiohydrolase Cel7A. Its three-dimensional structure has been solved and various mutant enzymes produced. In order to study the potential use of T. reeseiCel7A in the alkaline pHrange, the thermal sta-bilityofCel7Awas studiedas a functionof pHwith thewild-type and two mutant enzymes using different spectroscopic methods. Tryptophan fluorescence and CD measurements of the wild-type enzyme show an optimal thermostability between – (Tm,62 ± 2 C), at which the highest enzymatic activity is alsoobserved, andagradual decrease in the stability at more alkaline pHvalues. . | Eur. J. Biochem. 270 841-848 2003 FEBS 2003 doi The relationship between thermal stability and pH optimum studied with wild-type and mutant Trichoderma reesei cellobiohydrolase Cel7A Harry Boer and Anu Koivula VTT Biotechnology Espoo Finland The major cellulase secreted by the filamentous fungus Trichoderma reesei is cellobiohydrolase Cel7A. Its threedimensional structure has been solved and various mutant enzymes produced. In order to study the potential use of T. reesei Cel7A in the alkaline pH range the thermal stability of Cel7A was studied as a function of pH wt th th e wikl-type and two mutant enzymes using different spectroscopic methods. Tryptophan fluorescence and CD measurements of the wild-type enzyme show an optimal thermostability between pH Tm 62 2 C at which the highest enzymatic activity is also observed and a gradual decrease in the stability at more alkaline pHvalues. A soluble substrate cellotetraose was shown to stabilize the protein fold both at optimal and alkaline pH. In addition unfolding of the Cel7A enzyme and the release of the substrate seem to coincide at both acidic and alkaline pH demonstrated by a change in the fluorescence emission maximum. CD measurements were used to show that the five point mutations E223S A224H L225V T226A D262G that together result in a more alkaline pH opiimum Becker D. Braiet. c. Brumer H. III Claeyssens M. Divne C. Fagerstrom . Hanis. M. Jones . Kleyweglt GJ. Koivula. A. et al. 2001 Biochem. J. 356 19-30 destabilize the protein fold both at acidic and alkaline pHwhen compared with the wild-type enzyme. In addition an interesting time-dependent fluorescence change which was not observed by CD was detected for the pHmutant. Our data show that in order to engineer more alkaline pHcellulases a combination of mutations should be found which both shift the pHopti-mum and at the same time improve the thermal stability at alkaline pHrange. Keywords cellulase circular .