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.

Efficient screening of fungal cellobiohydrolase class I enzymes for thermostabilizing sequence blocks by SCHEMA structure-guided recombination.

We describe an efficient SCHEMA recombination-based approach for screening homologous enzymes to identify stabilizing amino acid sequence blocks. This approach has been used to generate active, thermostable cellobiohydrolase class I (CBH I) enzymes from the 390 625 possible chimeras that can be made by swapping eight blocks from five fungal homologs. Constructing and characterizing the parent enzymes and just 32 'monomeras' containing a single block from a homologous enzyme allowed stability contributions to be assigned to 36 of the 40 blocks from which the CBH I chimeras can be assembled. Sixteen of 16 predicted thermostable chimeras, with an average of 37 mutations relative to the closest parent, are more thermostable than the most stable parent CBH I, from the thermophilic fungus Talaromyces emersonii. Whereas none of the parent CBH Is were active >65°C, stable CBH I chimeras hydrolyzed solid cellulose at 70°C. In addition to providing a collection of diverse, thermostable CBH Is that can complement previously described stable CBH II chimeras (Heinzelman et al., Proc. Natl Acad. Sci. USA 2009;106:5610-5615) in formulating application-specific cellulase mixtures, the results show the utility of SCHEMA recombination for screening large swaths of natural enzyme sequence space for desirable amino acid blocks.

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.

Hydrothermal growth of mesoporous SBA-15 silica in the presence of PVP-stabilized Pt nanoparticles: synthesis, characterization, and catalytic properties.

A novel high surface area heterogeneous catalyst based on solution phase colloidal nanoparticle chemistry has been developed. Monodisperse platinum nanoparticles of 1.7-7.1 nm have been synthesized by alcohol reduction methods and incorporated into mesoporous SBA-15 silica during hydrothermal synthesis. Characterization of the Pt/SBA-15 catalysts suggests that Pt particles are located within the surfactant micelles during silica formation leading to their dispersion throughout the silica structure. After removal of the templating polymer from the nanoparticle surface, Pt particle sizes were determined from monolayer gas adsorption measurements. Infrared studies of CO adsorption revealed that CO exclusively adsorbs to atop sites and red-shifts as the particle size decreases suggesting surface roughness increases with decreasing particle size. Ethylene hydrogenation rates were invariant with particle size and consistent with a clean Pt surface. Ethane hydrogenolysis displayed significant structure sensitivity over the size range of 1-7 nm, while the apparent activation energy increased linearly up to a Pt particle size of approximately 4 nm and then remained constant. The observed rate dependence with particle size is attributed to a higher reactivity of coordinatively unsaturated surface atoms in small particles compared to low-index surface atoms prevalent in large particles. The most reactive of these unsaturated surface atoms are responsible for ethane decomposition to surface carbon. The ability to design catalytic structures with tunable properties by rational synthetic methods is a major advance in the field of catalyst synthesis and for the development of accurate structure-function relationships in heterogeneous reaction kinetics.