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 reesei is cellobiohydrolase Cel7A. Its three-dimensional 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 with the wild-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 3.5-5.6 (Tm, 62 +/- 2 degrees C), at which the highest enzymatic activity is also observed, and a gradual decrease in the stability at more alkaline pH values. 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 optimum [Becker, D., Braet, C., Brumer, H., III, Claeyssens, M., Divne, C., Fagerström, R.B., Harris, M., Jones, T.A., Kleywegt, G.J., Koivula, A., et al. (2001) Biochem. J.356, 19-30], destabilize the protein fold both at acidic and alkaline pH when compared with the wild-type enzyme. In addition, an interesting time-dependent fluorescence change, which was not observed by CD, was detected for the pH mutant. Our data show that in order to engineer more alkaline pH cellulases, a combination of mutations should be found, which both shift the pH optimum and at the same time improve the thermal stability at alkaline pH range.
Boer
Harry
H
VTT Biotechnology, PO Box 1500, Espoo, Finland. Harry.Boer@vtt.fi
Koivula
Anu
A
D002480:Cellulase / Q000378:metabolism*; D043366:Cellulose 1,4-beta-Cellobiosidase; D002942:Circular Dichroism; D004795:Enzyme Stability; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D014242:Trichoderma / Q000201:enzymology*
D002480:Cellulase; D043366:Cellulose 1,4-beta-Cellobiosidase