Purification and characterization of three extracellular (1→3)-β-d-glucan glucohydrolases from the filamentous fungus Acremonium persicinum

Abstract

Three (1-->3)-beta-D-glucanases (GNs) were isolated from the culture filtrates of the filamentous fungus Acremonium persicinum and purified by (NH4)2SO4 precipitation followed by anion-exchange and gel-filtration chromatography. Homogeneity of the purified proteins was confirmed by SDS/PAGE, isoelectric focusing and N-terminal amino acid sequencing. All three GNs (GN I, II and III) are non-glycosylated, monomeric proteins with apparent molecular masses, estimated by SDS/PAGE, of 81, 85 and 89 kDa respectively. pI values for the three enzymes are 5.3, 5.1, and 4.4 respectively. The pH optimum for GN I is 6.5, and 5.0 for GN II and III. All three purified enzymes displayed stability over the pH range 4.5-10.0. Optimum activities for GN I, II and III were recorded at 65, 55 and 60 degrees C respectively, with both GN II and III having short-term stability up to 50 degrees C and GN I up to 55 degrees C. The purified GNs have high specificity for (1-->3)-beta-linkages and hydrolysed a range of (1-->3)-beta- and (1-->3)(1-->6)-beta-D-glucans, with laminarin from Laminaria digitata being the most rapidly hydrolysed substrate of those tested. K(m) values for GN I, II, and III against L. digitata laminarin were 0.1, 0.23 and 0.22 mg/ml respectively. D-Glucono-1,5-lactone does not inhibit any of the three GNs, some metals ions are mild inhibitors, and N-bromosuccinimide and KMnO4 are strong inhibitors. All three GNs acted in an exo-hydrolytic manner, determined by the release of alpha-glucose as the initial and major product of hydrolysis of (1-->3)-beta-D-glucans, and confirmed by viscometric analysis and the inability to cleave periodate-oxidized laminarin, and may be classified as (1-->3)-beta-D-glucan glucohydrolases (EC 3.2.1.58).

Purification and characterization of an extracellular (1 → 6)-β-glucanase from the filamentous fungus Acremonium persicinum

“…Growth of Acremonium persicinum Nicot and Gams QM107a in liquid media and collection of the culture filtrates were as described previously [32].…”

“…In the absence of glucose, A. persicinum can utilize this β-glucan and others, like laminarin, carboxymethyl (CM)-pachyman and pustulan, as the sole carbon source, with the secretion of both (1 3)-β-glucanases and (1 6)-β-glucanases into the culture filtrate [31]. A recent study [32] has described the purification and characterization of three extracellular (1 3)-β-glucanases from this fungus. In the present paper the purification and characterization of a (1 6)-β-glucanase secreted by A. persicinum is reported.…”

Comparative Characterization of Laminarinases from the Filamentous Marine Fungi Chaetomium Indicum Corda and Trichoderma Aureviride Rifai

“…Chesters and Bull (1963) suggested that thiol groups were involved in the formation of the enzyme-substrate complex in fungal laminarinases: the enzyme activity was inhibited by phenylmercury nitrate, and the exo activity was inhibited to a greater extent than the endo activity. Three forms of exo-β-1,3-glucanase from A. persicinum were inhibited by p-hydroxymercury benzoate by 25-30% (Pitson et al, 1995). Our data suggest that the molecule of T. aureviride laminarinase contains a functionally significant free thiol group.…”

Transferase and hydrolytic activities of the laminarinase from rhodothermus marinus and its M133A, M133C, and M133W mutants

“…Laminarin is based on a β-1,3-glucan main chain but also has a small proportion (5.5%) of single-sugar β-1,6-branches [32], which give characteristic 1 H NMR signals. Transglycosylation with laminarin results in the appearance of 1,4-linkages not otherwise present in the native polisaccharide.…”