Poster: Enzymology

Abs # 231: The Effect of Adding and Removing N-Glycosylation Sites on the Thermostability of Barley a-Glucosidase

Presenter:	Clark, Suzanne E., seclark@students.wisc.edu
Authors	Clark, Suzanne E. (A)   Muslin, Elizabeth H. (B) (A)  Henson, Cynthia A. (B) (A)
Affiliations:	(A): University of Wisconsin-Madison (B): USDA-ARS MWA Cereal Crops Research Unit, Madison WI

a-Glucosidase is one of the enzymes involved in the starch degradation pathway that produces sugars for the growing embryo in barley (Hordeum vulgare L.) seeds. The hydrolysis of starch to various sugars is also an important step in the alcohol production industries (e.g. beer and ethanol fuel production). The industrial process to produce fermentable sugars occurs at temperatures from 60-75°C. Unfortunately, a-glucosidase is one of the most thermolabile of the starch hydrolyzing enzymes in barley seeds so its contribution to the production of fermentable sugars at these temperatures is limited. To identify amino acids in barley a-glucosidase that may be responsible for the observed thermolability, we compared its deduced amino acid sequence to that of the sugarbeet a-glucosidase, which we showed to be more thermostable (Muslin, Clark, and Henson, 2002). N-glycosylation sites that were in the barley sequence and not in the sugarbeet sequence and vice versa were targeted for mutagenesis. One mutation that inserted a N-glycosylation recognition sequence towards the C-terminus of barley a-glucosidase resulted in a 7°C increase in the enzyme’s thermostability.