Variation in barley (1 → 3, 1 → 4)-ß-glucan endohydrolases reveals novel allozymes with increased thermostability.

KEY MESSAGE: Novel barley (1 â†’ 3, 1 â†’ 4)-ß-glucan endohydrolases with increased thermostability. Rapid and reliable degradation of (1 â†’ 3, 1 â†’ 4)-ß-glucan to produce low viscosity wort is an essential requirement for malting barley. The (1 â†’ 3, 1 â†’ 4)-ß-glucan endohyrolases are responsible for the primary hydrolysis of cell wall ß-glucan. The variation in ß-glucanase genes HvGlb1 and HvGlb2 that encode EI and EII, respectively, were examined in elite and exotic germplasm. Six EI and 14 EII allozymes were identified, and significant variation was found in ß-glucanase from Hordeum vulgare ssp. spontaneum (wild barley), the progenitor of modern cultivated barley. Allozymes were examined using prediction methods; the change in Gibbs free energy of the identified amino acid substitutions to predict changes in enzyme stability and homology modelling to examine the structure of the novel allozymes using the existing solved EII structure. Two EI and four EII allozymes in wild barley accessions were predicted to have improved barley ß-glucanase thermostability. One novel EII candidate was identified in existing backcross lines with contrasting HvGlb2 alleles from wild barley and cv Flagship. The contrasting alleles in selected near isogenic lines were examined in ß-glucanase thermostability analyses. The EII from wild barley exhibited a significant increase in ß-glucanase thermostability conferred by the novel HvGlb2 allele. Increased ß-glucanase thermostability is heritable and candidates identified in wild barley could improve malting and brewing quality in new varieties.

Novel Barley (1→3,1→4)-ß-Glucan Endohydrolase Alleles Confer Increased Enzyme Thermostability.

Barley (1→3,1→4)-ß-glucan endohydrolases (ß-glucanases; EI and EII) are primarily responsible for hydrolyzing high molecular weight (1→3,1→4)-ß-glucans (ß-glucan) during germination. Incomplete endosperm modification during malting results in residual ß-glucan that can contribute to increased wort viscosity and beer chill haze. Four newly identified forms of EI and EII and the reference enzymes EI-a and EII-a were expressed in Escherichia coli, and the recombinant proteins were characterized for enzyme kinetics and thermostability. EI and EII variants that exhibited higher residual ß-glucanase activity than EI-a and EII-a after heat treatment also exhibited increased substrate affinity and decreased turnover rates. The novel EII-l form exhibited significantly increased thermostability compared with the reference EII-a when activity was measured at elevated temperature. EII-l exhibited a T50 value, which indicates the temperature at which 50% of ß-glucanase activity remains, 1.3 °C higher than that of EII-a. The irreversible thermal inactivation difference between EII-a and EII-l after 5 min of heat treatment at 56 °C was 11.9%. The functional significance of the three amino acid differences between EII-a and EII-l was examined by making combinatorial mutations in EII-a using site-directed mutagenesis. The S20G and D284E amino acid substitutions were shown to be responsible for the increase in EII-1 thermostability.