Protein Engineering, Vol. 15, No. 1, 29-33,
January 2002
© 2002 Oxford University Press
The effect of proline insertions on the thermostability of a barley 
-glucosidase
1 Department of Agronomy, University of Wisconsin, 1575 Linden Drive, Madison, WI 53706 and 2 Cereal Crops Research Unit, Agricultural Research Service, U.S. Department of Agriculture, USA
E-mail: cahenson{at}facstaff.wisc.edu
The thermal stability of 
-glucosidase is important because the conversion of starch to fermentable sugars during industrial production of ethanol (e.g. brewing, fuel ethanol production) typically takes place at temperatures of 65–73°C. In this study we investigate the thermostability of -glucosidases from four plant species, compare their deduced amino acid sequences, and test the effect of substituting a proline for the residue present in the wild-type enzyme on the thermostability of -glucosidase. The -glucosidase from barley (Hordeum vulgare) was significantly less thermostable than the other three -glucosidases. A comparison of the published deduced amino acid sequences of these four -glucosidases revealed conserved proline residues in the three most thermostable -glucosidases that were not found in the barley enzyme. Site-directed mutagenesis was done on recombinant barley -glucosidase to create proteins with prolines at these conserved positions. The thermostability (T50) of one of these mutant enzymes, T340P, was 10°C higher than the non-mutated enzyme.
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