Genetic Variation in Barley (1→3,1→4)-β-Glucan Endohydrolases

Abstract

Complete starchy endosperm cell wall degradation of (1→3,1→4)-β-glucan (β-glucan) during malting is essential to prevent problems associated with high wort viscosity and chill haze in beer. Cell wall β-glucan is hydrolysed by two (1→3,1→4)-β-glucanase (β-glucanase) isoforms in the early stages of germination. The EI and EII β-glucanase isoenzymes are relatively thermolabile resulting in significant activity losses during kilning and complete activity loss during mashing at high temperatures. The current study aimed to identify and characterise novel β-glucanase alleles in elite and exotic germplasm conferring increased thermostability. An allele mining approach was selected to examine 57 Hordeum vulgare ssp. spontaneum accessions from Israel and 80 elite varieties sourced globally. The exon regions of the two β-glucanase genes HvGlb1 and HvGlb2 were amplified and sequenced identifying five new EI allozymes and 13 new EII allozymes. Significantly more allelic variation was identified in HvGlb2 predominantly from H. spontaneum accessions. Changes in enzyme structure and stability caused by amino acid substitutions were examined using predictive modelling techniques to prioritise alleles for biochemical characterisation. Two EI and four EII allozymes identified in H. spontaneum were predicted as likely to possess increased thermostability and were selected for biochemical analysis. Selected alleles were heterologously expressed in Escherichia coli to produce tagged recombinant proteins that were purified for use in kinetic and thermostability assays. Allozyme characterisation identified no significant difference in catalytic efficiency for EI or EII and no improvement in EI thermostability. The four EII alleles predicted to confer increased thermostability exhibited significantly more residual β-glucanase activity following five minutes of heat treatment. However, only one EII allozyme, EII-l, exhibited increased β-glucanase activity at elevated temperatures. The functional significance of the three amino acid differences between the novel β-glucanase allozyme and the reference EII-a were examined in combinatorial mutations of EII-a using site-directed mutagenesis. Two of the three amino acids were shown to be responsible for the increase in β-glucanase thermostability. EII-a and EII-l were further examined in conditions similar to commercial processes to validate the increase in EII thermostability conferred by the EII-l allele. Recombinant β-glucanase activity was examined in a simulated barley mash at 65°C to mimic commercial mashing conditions. Additionally, the irreversible thermal inactivation of the endogenous β-glucanase activity in green malt from an F2 population derived from elite by wild barley cross was also assayed. EII-l consistently demonstrated approximately 10% more residual β-glucanase activity than the reference EII-a in both experiments. Therefore, EII-l is a promising new resource for genetic improvement of barley β-glucanase and a viable target for routine selection in the development of new malting varieties