Journal of CerealScience 28 (1998) 301-309 Article No. jc980209 Thermostability Variation in Alleles of Barley Beta-Amylase J. K. Eglinton, P. Langridgeand D. E. Evans The Univers#yof Adelaide, Waite Campus, Departmentof PlantScience, Glen Osmond, SA 5064, Australia Received 19 May, 1998 ABSTRACT Thermostability assays in conjunction with IEF and molecular mapping were used to identify three beta-amylase alleles (Bmyl-Sdl, -Sd2L, -Sd2H) in cultivated barley and an additional allele (Bmyl-Sd3) in an accession of wild barley Hordeum vu~gare ssp. spontaneum. The four forms of beta-amylase exhibit different rates of thermal inactivation in barley extracts. This variation was shown to persist after the proteolytic processing of the enzyme that occurs during germination. Three forms of beta-amylase representing the range of thermostabilities were purified and shown to have Ts0 temperatures of 56-8°C for the Sd2L enzyme, 58"5°C for the Sdl enzyme, and 60'8°C for the Sd3 beta-amylase from wild barley. Analysis of the relationship between beta-amylase thermostability and fermentability, i.e. the yield of fermentable sugars obtained from starch hydrolysis during brewing in 42 commercial malt samples suggests that increased thermostability results in more efficient starch degradation. Screening for specific beta-amylase alleles is proposed as a method for increasing fermentability in malting barley. © 1998 Academic Press K(ywords: beta-amylase, thermostability, mapping, fermentability. ® INTRODUCTION Beta-amylase (1,4-0t-glucan maltohydrolase; EC 3.2.1.2) is a key enzyme in the degradation of starch in germinated barley (Hordeum vulgare L.), and catalyses the liberation of [3,maltose from the non-reducing ends of 1,4-0t-glucans. Beta-amylase is synthesised during the development of the barley grain j, in contrast to other major hydrolytic en- zymes which are synthesised de novo during ger- mination. The primary structure of barley beta-amylase has been deduced from full length cDNA clones2'3. In mature grain the enzyme consists of a single ABBREVIATIONS USED: AAL = apparent attenuation limit; EBC = European Brewing Convention; IOB = Institute of Brewing; IEF=isoelectric focusing; BSA--bovine serum albumin; Q TL=quantitati've trait loci; SDS= sodium dodecyl sulphate; PAGE=polyacrylamide gel electrophoresis; DP = diastatic power. polypeptide chain of Mr 59"7 kDa which is con- verted during germination to an isoform of Mr 56"0 kDa, and the reduction in molecular weight is accompanied by an increase in pI of the complex band pattern of beta-amylase detected by isoelectric focusing (IEF) 4. The conversion of isoforms is mediated by limited proteolysis of the C-terminal region of the enzyme by malt endopeptidase 5. Two alleles for the beta-amylase gene (Bmyl) have been identified in H. vulgare by electrophoretic techniques and are termed Bmyl-Sdl and Bmyl- Sd26'7. The corresponding enzymes, referred to as Sdl and Sd2, have the same apparent molecular mass and exhibit similar patterns of charge hetero- geneity, although the Sd2 enzyme pattern is more basic. The two forms of beta-arfiylase can be differ- entiated by IEF in both the intact enzyme and the proteolytically cleaved form of the enzyme generated during germination 7. Starch hydrolysis during germination is achieved primarily by the action of four major 0733-5210/98/060301 + 09 $30.00/0 © 1998 Academic Press