Combinatorial Alanine Substitution Enables Rapid Optimization of Cytochrome P450_(BM3) for Selective Hydroxylation of Large Substrates

Made for each other: Combinatorial alanine substitution of active site residues in a thermostable cytochrome P450_(BM3) variant was used to generate an enzyme that is active with large substrates. Selective hydroxylation of methoxymethylated monosaccharides, alkaloids, and steroids was thus made possible (see Scheme). This approach could be useful for improving the activity of enzymes that show only limited activity with larger substrates

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