Effect of adding and removing N-glycosylation recognition sites on the thermostability of barley alpha-glucosidase.

The thermostability of alpha-glucosidase is important because the conversion of starch to fermentable sugars during the industrial production of beer and fuel ethanol typically occurs at relatively high temperatures (60-75 degrees 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.