Directed evolution of beta-glucosidase A from Paenibacillus polymyxa to thermal resistance

The beta-glucosidase encoded by the bglA gene from Paenibacillus polymyxa h as a half-life time of 15 min at 35 degrees C and no detectable activity at 55 degrees C, We have isolated random mutations that enhance the thermores istance of the enzyme. Following a directed evolution strategy, we have com bined some of the isolated mutations to obtain a beta-glucosidase with a ha lf-life of 12 min at 65 degrees C, in the range of resistance of thermophil ic enzymes. No significant alteration of the kinetic parameters of the enzy me was observed. One of the mutants isolated in the screening for thermores istant beta-glucosidase had the same resistance to denaturation as the wild type. This mutation caused the accumulation of enzyme in E. coli, probably due to its lower turnover. The structural changes responsible for the prop erties of the mutant enzymes have been analyzed. The putative causes increa sing thermoresistance are as follows: the formation of an extra salt bridge , the replacement of an Asn residue exposed to the solvent, stabilization o f the hydrophobic core, and stabilization of the quaternary structure of th e protein.