A transglycosylating 1,3(4)‐β‐glucanase from Rhodothermus marinus

Abstract

The enzymatic hydrolysis of polysaccharides by the 1,3(4)-b-glucanase (LamR) from Rhodothermus marinus has been explored. The enzyme cleaves the 1,3-b-linkages of 3-O-substituted glucose units in 1,3-b-glucans such as laminarin and curdlan, and also the 1,4-b-linkages of 3-O-substituted b-glucose in b-glucans such as lichenin and 1,3-1,4-b-glucan from the cell walls of barley endosperm. The polysaccharide substrates (laminarin, curdlan and barley b-glucan) were characterised using NMR spectroscopy. The reaction of LamR with its substrates was followed by recording one-dimensional and two-dimensional 1 H-NMR and 13 C-NMR spectra at suitable time intervals after addition of the enzyme. It is shown that hydrolysis occurs with retention of the anomeric configuration and that LamR performs transglycosylation to generate both 1,3-b-glycosidic and 1,4-b glycosidic linkages. The transglycosylation results in, e.g. formation of the trisaccharide 4-O-glucosyl-laminaribiose from exclusively 1,3-b-oligoglucosides. When barley 1,3-1,4-b-glucan was incubated with LamR the b-1,4-linkages of 3-O-substituted b-glycosyl residues were rapidly hydrolysed. Simultaneously de novo formation of 1,3-b-glycosidic linkages was observed which, however, were cleaved during prolonged incubations. It is shown that a laminaribiosyl unit is the minimum requirement for formation of an enzyme±substrate complex and subsequent hydrolysis/transglycosylation.

Comparative spectroscopic and rheological studies on crude and purified soluble barley and oat β-glucan preparations

“…3. The resonance at 4.75 ppm is assigned to anomeric β-(1 → 3) and the resonance at 4.55 ppm is assigned to β-(1 → 4) in accordance with Petersen et al (2000). The OBG Mz and BBG Mz samples were confirmed to be very pure with no additional anomeric carbohydrate signals, besides those of β-(1→ 3) and β-(1→ 4)-linkages.…”

“…Nuclear magnetic resonance (NMR) spectroscopy has been widely used for the overall structure and linkage sequence analysis of cereal β-glucans (Cui, Wood, Blackwell, & Nikiforuk, 2000;Johansson, Tuomainen, Ylinen, Ekholm, & Virkki, 2004;Lazaridou et al, 2004;Morgan, Roberts, Tendler, Davies, & Williams, 1999;Petersen, Krah, Duus, & Thomsen, 2000;Roubroeks, Mastromauro, Andersson, Christensen, & Aman, 2000;Seefeldt, Larsen, Viereck, Wollenweber, & Engelsen, 2008;Wood et al, 1994). Liquid state 1 H NMR allows estimation of the ratio of β-(1 → 3) to β-(1→ 4) linkages in the β-glucan polymers as well as the residual constituents present in the β-glucan extract.…”

Structure of β-Glucan Oligomer from Laminarin and Its Effect on Human Monocytes to Inhibit the Proliferation of U937 Cells

“…The assignments of 1 H and 13 C signals were based on the two-dimensional COSY, NOESY, and HSQC experiments in comparison with the data on laminarin from Laminaria digitata and related compounds, reported previously. 26) The 1 H signals at 5.28, 4.78, 4.68, and 4.56 ppm were assigned to be H-1 of glucopyranose attributed to the -anomer of the reducing end, the -1,3-linkage, the -anomer of the reducing end, and the -1,6-linkage respectively. The 13 C signals at 104.0, 97.1, and 93.4 ppm were assigned to be C-1 of glucopyranose attributed to the -1,3-and -1,6-linkages, the -anomer of the reducing end, and the -anomer of the reducing end respectively.…”

Enzymatic properties and the primary structure of a β-1,3-glucanase from the digestive fluid of the Pacific abalone Haliotis discus hannai

“…The tetrasaccharide is considered to be 6-O-glycosyl-laminaritriose derived from the -1,6-linked glucose branching region of laminarin since the branching regions are hardly degraded by -1,3-glucanases (Petersen et al, 2000;Kawai et al, 2006). From these results, we may conclude that HdLam33 is a typical endo-1,3;4--glucanase (EC 3.2.1.6).…”