Temporal Expression Analysis of Barley Disproportionating Enzyme 1 (DPE1) during Grain Development and Malting

Abstract

Abstract Disproportionating enzyme (D-enzyme) is a 4-α-glucanotransferase (EC 2.4.1.25) that disproportionates α-glucans and maltooligosaccharides and preferentially disproportionates maltotriose into maltopentaose and glucose. Maltotriose is a maltooligosaccharide that accumulates in barley malt and wort without being digested further by 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 providing evidence of an inherent enzymatic mechanism capable of disproportionating maltotriose into maltopentaose that can be further degraded into fermentable sugars by amylolytic enzymes such as β-amylase. A partial genomic sequence of barley disproportionating enzyme 1 (DPE1) was obtained that was comprised of 16 exons and 15 introns totaling 4680 bp. The 5’ region of the DPE1 gene was recalcitrant to Sanger sequencing owing to its localization in a highly repetitive region of the barley genome. The DPE1 gene is expressed during grain development and the protein 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 13 elite malting cultivars representing spring and winter growth habits. During grain development, DPE1 mRNA levels peak at 17 days after anthesis, which coincides with a large increase in proteins that react to anti-DPE1 polyclonal antibodies. These proteins appear as a doublet on immunoblots during initial stages of malting and as a singlet as malting progresses indicating proteolytic processing (e.g., transit peptide removal) or differential isoform/splice variant expression.