Substrate Specificities of Variants of Barley (1,3)- and (1,3;1,4)-β-d-Glucanases Resulting from Mutagenesis and Segment Hybridization.

Barley (1,3;1,4)-β-d-glucanase is believed to have evolved from an ancestral monocotyledon (1,3)-β-d-glucanase, enabling the hydrolysis of (1,3;1,4)-β-d-glucans in the cell walls of leaves and germinating grains. In the present study, we investigated the substrate specificities of variants of the barley enzymes (1,3;1,4)-β-d-glucan endohydrolase [(1,3;1,4)-β-d-glucanase] isoenzyme EII (EII) and (1,3)-β-d-glucan endohydrolase [(1,3)-β-d-glucanase] isoenzyme GII (GII) obtained by protein segment hybridization and site-directed mutagenesis. Using protein segment hybridization, we obtained three variants of EII in which the substrate specificity was that of a (1,3)-β-d-glucanase and one variant that hydrolyzed both (1,3)-β-d-glucans and (1,3;1,4)-β-d-glucans; the wild-type enzyme hydrolyzed only (1,3;1,4)-β-d-glucans. Using substitutions of specific amino acid residues, we obtained one variant of EII that hydrolyzed both substrates. However, neither protein segment hybridization nor substitutions of specific amino acid residues gave variants of GII that could hydrolyze (1,3;1,4)-β-d-glucans; the wild-type enzyme hydrolyzed only (1,3)-β-d-glucans. Other EII and GII variants showed changes in specific activity and their ability to degrade the (1,3;1,4)-β-d-glucans or (1,3)-β-d-glucans to larger oligosaccharides. We also used molecular dynamics simulations to identify amino-acid residues or structural regions of wild-type EII and GII that interact with (1,3;1,4)-β-d-glucans and (1,3)-β-d-glucans, respectively, and may be responsible for the substrate specificities of the two enzymes.