Germinated barley provides the raw material for the malting and brewing industries. In the germinated grain, hydrolytic enzymes are secreted from the aleurone and scutellum into the starchy endosperm where they participate in the breakdown of the starch and protein reserves, and release fermentable products which are subsequently utilised in the brewing process. However, the starchy endosperm cell walls represent a physical barrier to the free diffusion of the starch and protein hydrolases to their substrates, and degradation of the starchy endosperm cell walls is therefore an important prerequisite for the efficient mobilisation of endospermic reserves. The starchy endosperm cell walls are composed mainly of (1->3,1->4)-b-glucans, and the enzymes primarily responsible for the initial degradation of these polysaccharides are the barley (1->3,1->4)-b-glucan endohydrolases.

Because of their propensity to form solutions of high viscosity, undegraded (1->3,1->4)-b-glucans in the malt can cause serious filtration problems during brewing and can also contribute to the formation of precipitates and hazes in the beer. Barley (1->3,1->4)-b-glucanases are rapidly inactivated at the elevated temperatures used during the kilning and mashing processes. Thus, if the thermostability of barley (1->3,1->4)-b-glucanases could be increased, more of the enzyme would survive the mashing and kilning processes and high molecular weight (1->3,1->4)-b-glucans would be more extensively degraded. In this study, two approaches were used in attempts to increase the thermostability of barley (1->3,1->4)-b-glucanases. The first approach involved comparing the structure of barley (1->3,1->4)-b-glucanase isoenzyme EII with that of a related barley enzyme, (1->3)-b-glucanase isoenzyme GII. Barley (1->3)-b-glucanase isoenzyme GII is a PR protein which plays a role in plant defence, and is significantly more thermostable than barley (1->3,1->4)-b-glucanase isoenzyme EII. A three-dimensional structural comparison of the two enzymes reveals that their different substrate specificities are due to a limited number of amino acid substitutions in their substrate binding grooves. Thus, an attempt was made to produce a more heat-stable (1->3,1->4)-b-glucanase by changing the specificity of barley (1->3)-b-glucanase isoenzyme GII to that of a (1->3,1->4)-b-glucanase. Four single amino acid substitutions were introduced into the substrate binding groove of barley (1->3)-b-glucanase isoenzyme GII using site-directed mutagenesis. These mutations decreased the specific activity of the enzyme but did not change its substrate specificity.

The second approach to producing a thermostable barley (1->3,1->4)-b-glucanase again involved site-directed mutagenesis, but in this case a number of rational amino acid substitutions were introduced into barley (1->3,1->4)-b-glucanase isoenzyme EII itself. The amino acid substitutions were based on a detailed structural analysis of barley (1->3,1->4)-b-glucanase isoenzyme EII, together with our knowledge of the factors which affect protein stability. Three of the resulting mutant enzymes showed increased thermostability. The largest increase in stability was observed when the histidine at position 300 was changed to a proline (mutant H300P), which effectively decreases the entropy of the unfolded state of the enzyme. The three amino acid substitutions which increased the thermostability of barley (1->3,1->4)-b-glucanase iosenzyme EII were all located in the COOH-terminus loop of the enzyme. Thus, it was proposed that this loop represents a particularly unstable region of the enzyme, and could be involved in the initiation of unfolding of the enzyme at elevated temperatures. The NH₂- and COOH-termini of barley (1->3,1->4)-b-glucanase isoenzyme EII are in close proximity, which allowed the termini to be fused in an attempt to stabilise the COOH-terminal loop region. However, the resulting mutant enzyme was inactive.

Finally, the gene encoding the thermostable barley (1->3,1->4)-b-glucanase mutant H300P was stably integrated into the genome of barley callus cells. To date, attempts to regenerate vegetative tissues from the transgenic callus have been unsuccessful. In addition, the cDNA encoding thermostable mutant enzyme was transiently expressed in barley immature embryos under the control of the constitutive rice actin promoter. The transiently expressed mutant enzyme was active, which indicated that it was expressed and processed correctly in barley cells.