Temporal expression analysis of barley disproportionating enzyme 1 (DPE1) and its putative role in substrate production during malting and mashing

Authors: Vinje, Marcus; Walling, Jason; HENSON, CYNTHIA - Retired ARS Employee; DUKE, STANLEY - University Of Wisconsin

Submitted to: Journal of the American Society of Brewing Chemists
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/18/2022
Publication Date: 8/22/2022
Citation: Vinje, M.A., Walling, J.G., Henson, C.A., Duke, S.H. 2022. Temporal expression analysis of barley disproportionating enzyme 1 (DPE1) and its putative role in substrate production during malting and mashing. Journal of the American Society of Brewing Chemists. https://doi.org/10.1080/03610470.2022.2104060.
DOI: https://doi.org/10.1080/03610470.2022.2104060

Interpretive Summary: This research was undertaken to determine if there is a mechanism in barley that can reduce the accumulation of a three-glucose sugar, maltotriose, in barley malt and during mashing. Barley is malted to create enzymes capable of breaking down starch into fermentable sugars that are utilized by yeast during fermentation. Maltotriose is a small sugar made up of three individual glucose molecules that accumulates in barley malt and in barley wort. Brewer's yeast prefers to utilize glucose and maltose (two glucose residues) as its primary source of energy and not all yeast strains can use maltotriose for energy. D-enzyme is a protein that can combine maltotriose to produce maltopentaose (five glucose residues) and glucose. The products of this reaction yield an easily fermentable sugar (glucose) and a preferred substrate (maltopentaose) of other starch degrading enzymes such as beta-amylase. This is the first study on barley disproportionating enzyme and was undertaken to assess whether D-enzyme, specifically a D-enzyme encoded by the gene DPE1, is expressed during barley development and/or during barley malting. The DPE1 gene was expressed in barley grain development and the protein stored in the mature barley grain. Additionally, the DPE1 gene was expressed during malting with expression differences observed among elite malting cultivars. The DPE1 protein was found to be able to utilize maltotriose as a substrate to produce glucose. This research proves that DPE1 enzyme is present in malted barley and capable of utilizing maltotriose producing an easily fermentable sugar, glucose.

Technical Abstract: Disproportionating enzyme is a 4 -alpha-glucanotransferase (EC 2.4.1.25) that disproportionates alpha-glucans and maltooligosaccharides and preferentially disproportionates maltotriose into maltopentaose and glucose. Maltotriose is a maltooligosaccharide that accumulates in barley malt and wort and is not readily digested further by the four main starch degrading enzymes. Furthermore, not all brewer’s yeast strains can ferment maltotriose. This research was undertaken to determine if D-enzyme is expressed in developing and/or malting barley grains and thus, proving there is an inherent enzymatic mechanism capable of disproportionating maltotriose into maltopentaose that can be further degraded into fermentable sugars by amylolytic enzymes such as beta-amylase. A partial genomic sequence of barley disproportionating enzyme 1 (DPE1) was obtained that contained 16 exons and 15 introns for a total of 4680 bp. The 5’ region of the DPE1 gene was recalcitrant to sequencing and appears to be situated in a highly repetitive region of the barley genome. The DPE1 gene is expressed during grain development and stored in the mature grain. Additionally, the DPE1 gene is de novo expressed during malting in both a 2- and 6-row malting cultivar with significant variation observed amongst 12 elite malting cultivars representing spring and winter growth habits and 2- and 6-row spike architecture. During grain development, DPE1 mRNA levels peak at 17 days after anthesis, which coincides with a large increase in DPE1 protein levels. The DPE1 protein appears as a doublet that converts to a singlet as malting progresses indicating proteolytic processing or differential isoform expression. A D-enzyme assay was performed on both a 2-row and 6-row malting cultivar at the beginning and end of malting with glucose production observed. Heterologous DPE1 enzyme was expressed in a cell-free expression system. Production of glucose was observed when incubating the cell-free extract containing DPE1 with maltotriose suggesting disproportionating activity by barley DPE1.