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PEDS Advance Access published online on March 29, 2004
Protein Engineering Design and Selection, doi:10.1093/protein/gzh028
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Received December 16, 2003
Revised February 25, 2004
Accepted March 1, 2004
Oxford University Press 1741-0134

Article

The Effect of Adding and Removing N-Glycosylation Recognition Sites on the Thermostability of Barley {alpha}-Glucosidase

S. E. Clark 1, E. H. Muslin 1, and C. A. Henson 2*

1 Department of Agronomy, University of Wisconsin-Madison, 1575 Linden Drive, Madison, WI, 53706, U.S.A
2 Department of Agronomy, University of Wisconsin-Madison, 1575 Linden Drive, Madison, WI, 53706, U.S.A; Cereal Crops Research Unit, Agricultural Research Service, U. S. Department of Agriculture, 1575 Linden Drive, Madison, WI, 53706, U.S.A


  Abstract

The thermostability of {alpha}-glucosidase is important because the conversion of starch to fermentable sugars during industrial production of beer and fuel ethanol typically occurs at relatively high temperatures (60-75°C). Barley (Hordeum vulgare) {alpha}-glucosidase is unstable at these elevated temperatures; however, the {alpha}-glucosidase from sugar beet (Beta vulgaris) is stable at these temperatures. An alignment of the deduced amino acid sequences of barley and sugar beet {alpha}-glucosidases revealed considerable differences in the number and position of N-glycosylation recognition sites (NGRS). Other researchers have shown that additions or removals of NGRS resulted in either the stabilization or destabilization of the enzymes at elevated temperatures. NGRS present in the barley sequence and absent in the sugar beet sequence were removed via site-directed mutagenesis from the barley protein. Recognition sites absent in the barley sequence and present in the sugar beet sequence were added via mutagenesis into the barley {alpha}-glucosidase. Two mutations significantly increased thermostability, one mutation significantly decreased thermostability and five mutations had little effect on {alpha}-glucosidase thermostability.

Keywords: Hordeum vulgare/Maltase/Peptide bond cleavage


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