BGN16.3, a novel acidic β‐1,6‐glucanase from mycoparasitic fungus Trichoderma harzianum CECT 2413

Abstract

Trichoderma harzianum is a filamentous fungus that has been proposed as a potential biocontrol agent against phytopathogenic fungi [1] and more recently as opportunistic, avirulent plant symbiont [2]. The antagonism by T. harzianum has been explained by different mechanisms [3]. One of them, mycoparasitism, involves the production of several hydrolytic enzymes for the local degradation of the host fungal cell wall and further penetration inside its hyphae as main steps [1].Several mycoparasitic strains included in different taxonomic groups in the Trichoderma genus [4,5] secrete complex sets of enzymes [6]. Within these enzymes we can find hydrolytic activities able to degrade most components of fungal cell walls (chitinases, glucanases, proteases, lipases, etc.). These are usually present as isozyme groups composed by proteins with the same activity but different catalytic and molecular properties [7][8][9][10][11][12].Chitinases and b-1,3-glucanases are considered the main enzymes responsible for the degradation of the host cell walls, as chitin and b-1,3-glucan are their two major components. However, other enzymes hydrolyzing less abundant, but structurally important components (as b-1,6-glucan), can contribute to the efficient disorganization and further degradation of the cell wall by Trichoderma. b-1,6-glucan has been described in budding yeasts as the link between cell wall proteins and the main b-1,3-glucan ⁄ chitin polysaccharide [13] supporting an important role for this polymer in the structure of the fungal cell wall.Although b-1,6-glucanases are widely distributed among filamentous fungi, few of them have been purified and characterized [10,[14][15][16][17] and few gene sequences have been published [18][19][20][21][22].We have previously described two b-1,6-glucanases in T. harzianum CECT 2413: BGN16.1 and BGN16.2 A new component of the b-1,6-glucanase (EC 3.2.1.75) multienzymatic complex secreted by Trichoderma harzianum has been identified and fully characterized. The protein, namely BGN16.3, is the third isozyme displaying endo-b-1,6-glucanase activity described up to now in T. harzianum CECT 2413. BGN16.3 is an acidic b-1,6-glucanase that is specifically induced by the presence of fungal cell walls in T. harzianum growth media. The protein was purified to electrophoretical homogenity using its affinity to b-1,6-glucan as first purification step, followed by chomatofocusing and gel filtration. BGN16.3 has a molecular mass of 46 kDa in SDS ⁄ PAGE and a pI of 4.5. The enzyme only showed activity against substrates with b-1,6-glycosidic linkages, and it has an endohydrolytic mode of action as shown by HPLC analysis of the products of pustulan hydrolysis. The expression profile analysis of BGN16.3 showed a carbon source control of the accumulation of the enzyme, which is fast and strongly induced by fungal cell walls, a condition often regarded as mycoparasitic simulation. The likely involvement b-1,6-glucanases in this process is discussed.

Senescence of the Lentinula edodes Fruiting Body After Harvesting

“…Some mycoparasitic fungi such as Trichoderma species produce an extracellular β-1,6-glucanase, member of GH30, for attack and degradation of host cell walls during their mycoparasitic action (De la Cruz and Llobell, 1999;Djonović et al, 2006;Montero et al, 2005). However, little information is known about the physiological function and role of the fungal β-1,6-glucanases.…”

Consolidation of glycosyl hydrolase family 30: A dual domain 4/7 hydrolase family consisting of two structurally distinct groups

“…An inclusive set of GH30 amino acid sequences with E-values as high as 10 À20 were collected using BLASTp [22] with the amino acid sequences of the newly assigned GH30 glucuronoxylan xylanohydrolase XynC of B. subtilis (ACC Q45070) [4,6], a protein of unknown function also being reassigned to GH30 from Bacteroides fragilis (ACC Q5LF82), Gba1 (glucosylceramidase) from H. sapiens (ACC P04062) [14,23], a B. breve b-xylosidase (ACC B1P195) [16] and a Trichoderma harzianum endo-b-1,6-glucanase (ACC Q8J0I9) [17]. In each case, sequences having low amino acid sequence identity levels ($27%) were used in secondary rounds of BLASTp.…”

“…The chemical characteristics of saponins may be considered analogous to glucosylceramides, both having glycosyl moieties O-linked to large hydrophobic molecules. A clear distinction is observed between these b-glucosidase/b-xylosidase like enzymes that have exo-function and the phylogenetically related endo-b-1,6-glucanase [17,18]. These later enzymes function in an endo-manner and have been characterized to degrade b-1, 6-glucan, a polysaccharide component of fungal cell walls [19,20].…”

Tvbgn3, a β-1,6-Glucanase from the Biocontrol Fungus Trichoderma virens , Is Involved in Mycoparasitism and Control of Pythium ultimum

“…Thus, ␤-1,6-glucanase may contribute to the efficient disorganization and further degradation of the host cell walls that occur during mycoparasitism. The lytic activity of ␤-1,6-glucanases against yeast (63) and cell walls of filamentous fungi in combination with other lytic enzymes has been described (9,39). Recently, a ␤-1,6-glucanase has been shown to be a virulence factor in the interaction between the mycopathogen Verticillium fungicola and its host, Agaricus bisporus (1).…”