G. Gonzalez-blasco et al., Directed evolution of beta-glucosidase A from Paenibacillus polymyxa to thermal resistance, J BIOL CHEM, 275(18), 2000, pp. 13708-13712
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.