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J Biol Chem, Vol. 275, Issue 18, 13708-13712, May 5, 2000

Directed Evolution of -Glucosidase A from Paenibacillus polymyxa to Thermal Resistance^*

Gracia González-Blasco, Juliana Sanz-Aparicio^§, Beatriz González^§, Juan A. Hermoso^§, and Julio Polaina^¶

From the  Instituto de Agroquímica y Tecnología de Alimentos, CSIC, E46980 Paterna (Valencia) and ^§ Departamento de Cristalografía, CMBE, Instituto de Química Física Rocasolano, CSIC, E28006 Madrid, Spain

The -glucosidase encoded by the bglA gene from Paenibacillus polymyxa has a half-life time of 15 min at 35 °C and no detectable activity at 55 °C. We have isolated random mutations that enhance the thermoresistance of the enzyme. Following a directed evolution strategy, we have combined some of the isolated mutations to obtain a -glucosidase with a half-life of 12 min at 65 °C, in the range of resistance of thermophilic enzymes. No significant alteration of the kinetic parameters of the enzyme was observed. One of the mutants isolated in the screening for thermoresistant -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 properties of the mutant enzymes have been analyzed. The putative causes increasing thermoresistance are as follows: the formation of an extra salt bridge, the replacement of an Asn residue exposed to the solvent, stabilization of the hydrophobic core, and stabilization of the quaternary structure of the protein.

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