Characterization of the GPI-anchored endo β-1,3-glucanase Eng2 of Aspergillus fumigatus

Abstract

A GPI-anchored endo β-1,3-glucanase of Aspergillusfumigatus was characterized. The enzyme encoded by ENG2 (AFUA_2g14360) belongs to the glycoside hydrolase family 16 (GH16). The activity was characterized using a recombinant protein produced by Pichiapastoris. The recombinant enzyme preferentially acts on soluble β-1,3-glucans. Enzymatic analysis of the endoglucanase activity using Carboxymethyl-Curdlan-Remazol Brilliant Blue (CM-Curdlan-RBB) as a substrate revealed a wide temperature optimum of 24–40 °C, a pH optimum of 5.0–6.5 and a Km of 0.8 mg ml−1. HPAEC analysis of the products formed by Eng2 when acting on different oligo-β-1,3-glucans confirmed the predicted endoglucanase activity and also revealed a transferase activity for oligosaccharides of a low degree of polymerization. The growth phenotype of the Afeng2 mutant was identical to that of the wt strain.

A Novel Glycosylphosphatidylinositol-Anchored Glycoside Hydrolase from Ustilago esculenta Functions in β-1,3-Glucan Degradation

“…This suggests cooperative activity with UeBgl3A, which acts preferentially on laminarioligosaccharides with a degree of polymerization of 2 to 4 to produce glucose. Lam16A did not exhibit transglycosylation activity toward laminarioligosaccharides, unlike Eng2, possessing a GPI anchor signal peptide, a GH16 endo-␤-1,3(4)-glucanase with transglycosylation activity toward laminaritetraose (18), or Crh family proteins (7). Such a range of activity may have evolved by a process of accumulated DNA substitutions, as seen in the diversity of amino acid sequences in the GH16 phylogenetic tree (see Fig.…”

“…Total RNAs were prepared from U. esculenta and Z. latifolia by using an RNeasy plant mini kit (Qiagen, Hilden, Germany) and were treated with DNase I (Invitrogen, CA) for 15 min at 22°C. Firststrand cDNA was synthesized from total RNA with an oligo(dT) 18 primer using SuperScriptIII reverse transcriptase (Invitrogen). PCRs were performed by using a reaction mixture containing PrimeStarGXL DNA polymerase (TaKaRa Bio, Shiga, Japan), PrimeStarGXL DNA polymerase buffer, 0.1 mM each deoxynucleoside triphosphate (dNTP), 0.3 M primer pairs, and a DNA template.…”

Buscando agentes antifúngicos naturales: Ensayo sensible para detectar inhibidores de endo-1,3-β-glucanasa en extractos crudos de plantas

“…Fungal cell wall of ascomycetes, deuteromycetes, basidiomycetes and some oomycetes, are composed of β-glucans (Douglas, 2001), that are β-1,3-linked glucose homopolymers, with variable amounts of 1,6-β and 1,4-β-linked glucose side chains and chitin that are responsible for cell rigidity (Gastebois et al, 2010). The dynamic structural reorganization of fungal cell wall requires undergo partial lysis to obtain plasticity during morphological changes as cell growth (branching), cell division and germination in filamentous fungi or the cell separation in yeast, where endo-β-1,3-glucanases play an essential role during such morphogenetic events (Baladrón et al, 2002;Martin-Cuadrado, et al, 2003;Hartl et al, 2011). Therefore a strategy for discovering plant disease control agents would be to looking in plant derivatives endo-β-1,3-glucanase inhibitors…”

“…La pared celular fúngica de ascomicetos, basidiomicetos y deuteromicetos, y algunos oomicetos, están compuestos de β-glucanos (Douglas, 2001), que son homopolimeros de β-1,3-unidos a glucosas, con cantidades variables de cadenas de β-1,6 y β-1,4-unidos a glucosa y quitina que son responsables de mantener la rigidez celular (Gastebois et al, 2010). La reorganización estructural dinámica de la pared celular fúngica debe alcanzar una lisis parcial para hacerse de plasticidad durante los cambios morfológicos como el crecimiento celular (ramificación), división celular y germinación en hongos filamentosos o la separación celular en la levadura, donde las endo-β-1,3-glucanasas juegan un papel esencial durante tales eventos morfogenéticos (Baladrón et al, 2002;Martin-Cuadrado, et al, 2003;Hartl et al, 2011). Por lo tanto, una estrategia para descubrir agentes de control de enfermedades vegetales sería buscar en los compuestos de las plantas sustancias inhibidoras de endo-β-1,3-glucanasas como un sitio-objetivo específico de aquellos hongos cuyas paredes celulares contienen β-glucanos, específicamente en el enlace β-1,3-glucano.…”

Chitinases CtcB and CfcI modify the cell wall in sporulating aerial mycelium of Aspergillus niger

“…Transcription of the GH16 endo-glucanase An02g00850 was induced 13-fold. Orthologues A. fumigatus eng2 and A. nidulans xgeA hydrolyse soluble b-1,3-glucan with b-1,6-glucan branches (laminarin) and b-1,3 : 1,4-glucan (lichenan) (Bauer et al, 2006;Hartl et al, 2011). In addition, exo-glucanases bgxB and exgA were induced (Table 2b).…”