Submitted to: Protein Engineering
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/3/2003
Publication Date: 6/3/2004
Citation: Clark, S.E., Muslin, E.H., Henson, C.A. 2004. The effect of adding and removing n-glycosylation recognition sites on the thermostability of barley alpha-glucosidase. Protein Engineering, Design and Selection. 17:245-249. Interpretive Summary: The thermostability of malt carbohydrases is important because the conversion of starch to fermentable sugars during mashing typically takes place at temperatures of 60-75 degrees Celsius. Of the four enzymes known to be involved in the degradation of malt starch, only alpha-amylase is sufficiently thermostable to function throughout the starch conversion phase of mashing. In this study, we developed a recombinant alpha-glucosidase with enhanced thermostability by site directed mutagenesis of the alpha-glucosidase gene previously cloned from barley. Our strategy for selection of mutagenesis targets involved comparing the deduced amino acid sequences of alpha-glucosidases from two plant species and the thermostability of the enzymes encoded, followed by identification of key amino acids that were present in the thermostable enzymes but absent in the barley alpha-glucosidase. Additional mutagenesis targets identified were key amino acids present in the barley enzyme that were absent in the thermostable enzyme. A total of ten mutant enzymes were created to assess the roles of these amino acids in stabilizing barley alpha-glucosidase. The thermostability (T(50)) of one of these mutant enzymes was 7 degrees Celsius higher than the non-mutated enzyme. The development of a mutant gene that encodes an alpha-glucosidase with significantly enhanced thermostability provides researchers with a better gene for insertion into cereal crops than presently exists in their genomes. Additionally, because this gene was created by a single base change and because this mutation naturally exists in dicots, screening a diverse collection of monocots may discover this mutation in a genotype which would allow the use of traditional breeding methods to incorporate this gene into commercially viable germplasm.
Technical 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. Barley alpha-glucosidase is unstable at these elevated temperatures. Other researchers have shown that additions or removals of N-glycosylation recognition sites (NGRS) resulted in either the stabilization or destabilization of the enzymes at elevated temperatures. In this study we compared the deduced amino acid sequences of alpha-glucosidases from barley and sugar beet. We identified NGRS present in the thermostable sugar beet enzyme that were absent in the enzyme from barley. Additionally, we identified NGRS present in the barley enzyme that were absent in the sugar beet enzyme. We investigated the effects of these NGRS differences by changing the barley sequence, one NGRS at a time, to resemble the sugar beet alpha-glucosidase sequence. One mutant alpha-glucosidase with a NGRS inserted and one mutant with a NGRS removed were shown to be significantly more thermostable (increases of 7 degrees celsius) than the barley wild type enzyme. The removal of another NGRS significantly destabilized the enzyme and five other mutations had little effect on enzyme thermostability.