Highly thermostable fungal cellobiohydrolase I (Cel7A) engineered using predictive methods.

Building on our previous efforts to generate thermostable chimeric fungal cellobiohydrolase I (CBH I, also known as Cel7A) cellulases by structure-guided recombination, we used FoldX and a 'consensus' sequence approach to identify individual mutations present in the five homologous parent CBH I enzymes which further stabilize the chimeras. Using the FoldX force field, we calculated the effect on ΔG(Folding) of each candidate mutation in a number of CBH I structures and chose those predicted to be stabilizing in multiple structures. With an alignment of 41 CBH I sequences, we also used amino acid frequencies at each candidate position to calculate predicted effects on ΔG(Folding). A combination of mutations chosen using these methods increased the T(50) of the most thermostable chimera by an additional 4.7°C, to yield a CBH I with T(50) of 72.1°C, which is 9.2°C higher than that of the most stable native CBH I, from Talaromyces emersonii. This increased stability resulted in a 10°C increase in the optimal temperature for activity, to 65°C, and a 50% increase in total sugar production from crystalline cellulose at the optimal temperature, compared with native T.emersonii CBH I.

Water dynamics and salt-activation of enzymes in organic media: mechanistic implications revealed by NMR spectroscopy.

Deuterium spin relaxation was used to examine the motion of enzyme-bound water on subtilisin Carlsberg co-lyophilized with inorganic salts for activation in different organic solvents. Spectral editing was used to ensure that the relaxation times were associated with relatively mobile deuterons, which were contributed almost entirely by D(2)O rather than hydrogen-deuteron exchange on the protein. The results indicate that the timescale of motion for residual water molecules on the biocatalyst, (tau(c))(D(2)O), in hexane decreased from 65 ns (salt-free) to 0.58 ns (98% CsF) as (k(cat)/K(M))(app) of the biocatalyst preparation increased from 0.092 s(-1) x M(-1) (salt-free) to 1,140 s(-1) x M(-1) (98% CsF). A similar effect was apparent in acetone; the timescale decreased from 24 ns (salt-free) to 2.87 ns (98% KF), with a corresponding increase in (k(cat)/K(M))(app) of 0.140 s(-1) x M(-1) (salt-free) to 12.8 s(-1) x M(-1) (98% KF). Although a global correlation between water mobility and enzyme activity was not evident, linear correlations between ln[(k(cat)/K(M))(app)] and (tau(c))(D(2)O) were obtained for salt-activated enzyme preparations in both hexane and acetone. Furthermore, a direct correlation was evident between (k(cat)/K(M))(app) and the total amount of mobile water per mass of enzyme. These results suggest that increases in enzyme-bound water mobility mediated by the presence of salt act as a molecular lubricant and enhance enzyme flexibility in a manner functionally similar to temperature. Greater flexibility may permit a larger degree of local transition-state mobility, reflected by a more positive entropy of activation, for the salt-activated enzyme compared with the salt-free enzyme. This increased mobility may contribute to the dramatic increases in biocatalyst activity.