A transglycosylating 1,3(4)-beta-glucanase from Rhodothermus marinus NMR analysis of enzyme reactions

The enzymatic hydrolysis of polysaccharides by the 1,3(4)-beta-glucanase (L amR) from Rhodothermus marinus has been explored. The enzyme cleaves the 1, 3-beta-linkages of 3-O-substituted glucose units in 1,3-beta-glucans such a s laminarin and curdlan, and also the 1,4-beta-linkages of 3-O-substituted beta-glucose in beta-glucans such as lichenin and 1,3-1,4-beta-glucan from the cell walls of barley endosperm. The polysaccharide substrates (laminari n, curdlan and barley P-glucan) were characterised using NMR spectroscopy. The reaction of LamR with its substrates was followed by recording one-dime nsional and two-dimensional H-1-NMR and C-13-NMR spectra at suitable time i ntervals after addition of the enzyme. It is shown that hydrolysis occurs w ith retention of the anomeric configuration and that LamR performs transgly cosylation to generate both 1,3-beta-glycosidic and 1,4-beta glycosidic lin kages. The transglycosylation results in, e.g. formation of the trisacchari de 4-O-glucosyl-laminaribiose from exclusively 1,3-beta-oligoglucosides. Wh en barley 1,3-1,4-beta-glucan was incubated with LamR the beta-1,4-linkages of 3-O-substituted beta-glycosyl residues were rapidly hydrolysed. Simulta neously de novo formation of 1,3-beta-glycosidic linkages was observed whic h, however, were cleaved during prolonged incubations. It is shown that a l aminaribiosyl unit is the minimum requirement for formation of an enzyme-su bstrate complex and subsequent hydrolysis/transglycosylation.