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Originally published In Press as doi:10.1074/jbc.M003036200 on June 27, 2000

J. Biol. Chem., Vol. 275, Issue 37, 28843-28848, September 15, 2000
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Increased Thermal Resistance and Modification of the Catalytic Properties of a beta -Glucosidase by Random Mutagenesis and in Vitro Recombination*

María Jesús Arrizubieta and Julio PolainaDagger

From the Instituto de Agroquímica y Tecnología de los Alimentos, Consejo Superior de Investigaciones Cientifícas, Apdo. 73, E46100-Burjassot, Valencia, Spain

The bglB gene from Paenibacillus polymyxa was subjected to random mutagenesis mediated by error prone polymerase chain reaction amplification and DNA shuffling. After this treatment, mutant variants of the encoded beta -glucosidase with enhanced thermal resistance were selected. We identified five amino acid substitutions at four different positions of the sequence that increased the resistance of the enzyme to heat denaturation. Four of the mutations, H62R, M319V, M319I, and M361I, did not change the kinetic parameters of the enzyme. However, mutant N223Y, which caused only a marginal increase in thermoresistance, showed an 8-fold decrease in Km. Copies of the bglB gene carrying each one of the individual mutations were recombined in vitro by DNA shuffling. As a result, we obtained an enzyme that simultaneously exhibited a 20-fold increase in heat resistance and an 8-fold increase in the catalytic efficiency. The structural basis of the properties conferred by the mutations was analyzed using homology-based structural models. The four mutations causing a more pronounced effect on thermoresistance were located in loops, on the periphery of the (alpha /beta )8 barrel that conforms the structure of the protein. Mutation N223Y, which modifies the catalytic properties of the enzyme, was on one of the barrel beta -strands that shape the active center.

* This work was supported by Comision Interministerial de Ciencia y Tecnología, Spain Grant ALI97-0362.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed. Tel.: 34-96-3900022; Fax: 34-96-3636301; E-mail: jpolaina@iata.csic.es.

Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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