The relationship between thermal stability and pH optimum studied with wild-type and mutant Trichoderma reesei cellobiohydrolase Cel7A.
Harry Boer,Anu Koivula +1 more
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The 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 alkali pH range.Abstract:
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 35-56 (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, Fagerstrom, RB, Harris, M, Jones, TA, Kleywegt, GJ, Koivula, A, et al (2001) Biochem J356, 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 rangeread more
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References
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TL;DR: The wealth of information provided by the recent structure determinations of many different glycosyl hydrolases shows that the substrate specificity and the mode of action of these enzymes are governed by exquisite details of their three-dimensional structures rather than by their global fold.
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Crystalline cellulose degradation : new insight into the function of cellobiohydrolases
TL;DR: Cellobiohydrolases may prove to be useful for achieving subtle modifications of fiber properties in paper or textiles without the loss of fiber strength commonly observed with other cellulases.
Journal ArticleDOI
The three-dimensional crystal structure of the catalytic core of cellobiohydrolase I from Trichoderma reesei.
Christina Divne,Jerry Ståhlberg,Tapani Reinikainen,Laura Ruohonen,Göran Pettersson,J. Knowles,TT Teeri,T.A. Jones +7 more
TL;DR: The structure of the major cellobiohydrolase, CBHI, of the potent cellulolytic fungus Trichoderma reesei has been determined and refined and may account for many of the previously poorly understood macroscopic properties of the enzyme and its interaction with solid cellulose.
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Determination of the three-dimensional solution structure of the C-terminal domain of cellobiohydrolase I from Trichoderma reesei. A study using nuclear magnetic resonance and hybrid distance geometry-dynamical simulated annealing.
TL;DR: The solution structure of a synthetic 36-residue polypeptide comprising the C-terminal cellulose binding domain of cellobiohydrolase I (CT-CBH I) from Trichoderma reesei was investigated by nuclear magnetic resonance (NMR) spectroscopy.