The relationship between thermal stability and pH optimum studied with wild‐type and mutant Trichoderma reesei cellobiohydrolase Cel7A

Abstract

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 with the wildtype 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 (T m , 62 ± 2°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., Fagerstro¨m, 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.

Model-Based Fed-Batch for High-Solids Enzymatic Cellulose Hydrolysis

“…Performing the saccharification reaction at high insoluble solids introduces a new set of process-related problems associated with slurry mixing and cellulase enzyme effectiveness. Enzyme-dependent factors include sugar inhibition [14] and reaction temperature [15]. At increasing levels of solids, sugar inhibition of enzymes may become more important due to the increasing difficulty in diffusion of sugars away from the catalytic site.…”

Computational Investigation of the pH Dependence of Loop Flexibility and Catalytic Function in Glycoside Hydrolases

“…H. jecorina Cel6A and Cel7A function optimally at pH ϳ5 on cellotetraose (Cel6A) (22), 3,4-dinitrophenyl lactoside (Cel7A) (16), and 4-nitrophenyl ␣-D-glucopyranoside (Cel7A) (23), and their activities decrease dramatically at other pH values. Experimental studies using directed evolution have revealed that certain mutants of H. jecorina Cel7A and Cel5A have higher pH optima compare with the wild type (16,19). Carboxyl-carboxylate pair mutations have been used to stabi-lize H. jecorina Cel6A at neutral or alkaline pH (21).…”

Small Angle Neutron Scattering Reveals pH-dependent Conformational Changes in Trichoderma reesei Cellobiohydrolase I

“…In particular, pH values between 4 and 5 are optimal for Cel7A (17,18). Further, an acidic pH confers thermal stability to Cel7A (19). The pI values calculated from the amino acid sequences of the three domains (20) indicate that the catalytic core has a pI of 4.3, whereas those of the cellulose binding domain and linker are 6.4 and 12.3, respectively.…”