Recombinatorial and Predictive Methods to Increase Cellulase Thermostability and Structural Analysis of a Thermostable P450

To address the world's need for improved biomass breakdown for the production of renewable fuel, we sought to improve cellulase thermostability and thereby enzyme lifetime, operating temperature, and specific activity. We created an eight block SCHEMA recombination library based on five fungal cellobiohydrolase class I (CBHI) enzymes. By characterizing this library, we identified several stabilizing sequence blocks and combined these to produce a set of well-expressed, thermostable CBHI chimeras. To further increase the stability of these chimeras, we used a combination of the chimera thermostability screening data, a consensus analysis of 40 naturally occurring CBHI sequences, and FoldX ΔΔG predictions to identify individual mutations for testing. Our final enzyme has a T50 9.3 °C greater than that of the most stable parental CBHI, resulting in a 10 °C increase in optimal temperature and a 50% increase in total sugar production at the optimal temperature.;To produce an ideal parent for directed evolution for improved activity on varied compounds, we increased the thermostability of a P450BM3 enzyme with broad substrate specificity to produce enzyme 9-10ATS. Directed evolution libraries based on 9-10ATS produced variants with improved activity on a number of structurally diverse compounds. We determined the structure of 9-10ATS using x-ray crystallography and compared it to other P450BM3 structures. Examination of the stucture shows clear structural basis for the thermostabilizing mutations and broad substrate specificity.