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Endo-?-1,3-glucanase (GH16 Family) from Trichoderma harzianum Participates in Cell Wall Biogenesis but Is Not Essential for Antagonism Against Plant Pathogens.

ABSTRACT: Trichoderma species are known for their ability to produce lytic enzymes, such as exoglucanases, endoglucanases, chitinases, and proteases, which play important roles in cell wall degradation of phytopathogens. ?-glucanases play crucial roles in the morphogenetic-morphological process during the development and differentiation processes in Trichoderma species, which have ?-glucans as the primary components of their cell walls. Despite the importance of glucanases in the mycoparasitism of Trichoderma spp., only a few functional analysis studies have been conducted on glucanases. In the present study, we used a functional genomics approach to investigate the functional role of the gluc31 gene, which encodes an endo-?-1,3-glucanase belonging to the GH16 family in Trichoderma harzianum ALL42. We demonstrated that the absence of the gluc31 gene did not affect the in vivo mycoparasitism ability of mutant T. harzianum ALL42; however, gluc31 evidently influenced cell wall organization. Polymer measurements and fluorescence microscopy analyses indicated that the lack of the gluc31 gene induced a compensatory response by increasing the production of chitin and glucan polymers on the cell walls of the mutant hyphae. The mutant strain became more resistant to the fungicide benomyl compared to the parental strain. Furthermore, qRT-PCR analysis showed that the absence of gluc31 in T. harzianum resulted in the differential expression of other glycosyl hydrolases belonging to the GH16 family, because of functional redundancy among the glucanases.

SUBMITTER: Suriani Ribeiro M

PROVIDER: S-EPMC6995588 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

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Endo-β-1,3-glucanase (GH16 Family) from Trichoderma harzianum Participates in Cell Wall Biogenesis but Is Not Essential for Antagonism Against Plant Pathogens.

Suriani Ribeiro Marcela M Graciano de Paula Renato R Raquel Voltan Aline A de Castro Raphaela Georg RG Carraro Cláudia Batista CB José de Assis Leandro L Stecca Steindorff Andrei A Goldman Gustavo Henrique GH Silva Roberto Nascimento RN Ulhoa Cirano José CJ Neves Monteiro Valdirene V

Biomolecules 20191126 12

Trichoderma species are known for their ability to produce lytic enzymes, such as exoglucanases, endoglucanases, chitinases, and proteases, which play important roles in cell wall degradation of phytopathogens. β-glucanases play crucial roles in the morphogenetic-morphological process during the development and differentiation processes in Trichoderma species, which have β-glucans as the primary components of their cell walls. Despite the importance of glucanases in the mycoparasit ...[more]

PMID: 31779176

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Project description:The food enzyme containing cellulase (EC 3.2.1.4), endo-1,3(4)-β-glucanase (EC 3.2.1.6) and endo-1,4-β-xylanase (EC 3.2.1.8) is produced with the non-genetically modified Trichoderma reesei strain AR-256 by AB-Enzymes GmbH. The food enzyme is considered free from viable cells of the production organism. It is intended to be used in seven food manufacturing processes: baking processes, cereal-based processes, brewing processes, fruit and vegetable processing for juice production, wine and wine vinegar production, distilled alcohol production and grain treatment for production of starch and gluten fractions. Since the residual amounts of total organic solids (TOS) are removed during grain treatment and distilled alcohol production, dietary exposure was estimated for the remaining five processes and amounted up to 3.92 mg TOS/kg body weight (bw) per day. The toxicity studies were carried out with an endo-1,4-β-xylanase from T. reesei ■■■■■, considered by the Panel as a suitable substitute, because the genetic differences between the strains are well characterised and of no concern. Additionally, several strains derived from the production strain are considered safe by EFSA and the manufacturing of both food enzymes is similar. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by a repeated dose 90-day oral toxicity rat study. The no observed adverse effect level of 939 mg TOS/kg bw per day, the highest dose tested, compared with the estimated dietary exposure, resulted in a margin of exposure above 239. In the search for the similarity of the amino acid sequences to known allergens, one match (salmon) was found. The Panel considered that, under the intended conditions of use (except distilled alcohol production), the risk of allergic reactions by dietary exposure cannot be excluded, in particular for individuals sensitised to salmon. The Panel concluded that this food enzyme does not give rise to safety concerns, under the intended conditions of use.

Project description:Endo-β-1,3-glucanase is a glycoside hydrolase (GH) that plays an essential role in the mineralization of β-glucan polysaccharides. In this study, the novel gene encoding an extracellular, non-modular GH16 endo-β-1,3-glucanase (GluH) from Hymenobacter siberiensis PAMC 29290 isolated from Arctic marine sediment was discovered through an in silico analysis of its whole genome sequence and subsequently overexpressed in Escherichia coli BL21. The 870-bp GluH gene encoded a protein featuring a single catalytic GH16 domain that shared over 61% sequence identity with uncharacterized endo-β-1,3-glucanases from diverse Hymenobacter species, as recorded in the National Center for Biotechnology Information database. The purified recombinant endo-β-1,3-glucanase (rGluH: 31.0 kDa) demonstrated peak activity on laminarin at pH 5.5 and 40°C, maintaining over 40% of its maximum endo-β-1,3-glucanase activity even at 25°C. rGluH preferentially hydrolyzed D-laminarioligosaccharides and β-1,3-linked polysaccharides, but did not degrade D-laminaribiose or structurally unrelated substrates, confirming its specificity as a true endo-β-1,3-glucanase without ancillary GH activities. The biodegradability of various substrate polymers by the enzyme was evaluated in the following sequence: laminarin > barley β-glucan > carboxymethyl-curdlan > curdlan > pachyman. Notably, the specific activity (253.1 U mg-1) and catalytic efficiency (k cat /K m : 105.72 mg-1 s-1 mL) of rGluH for laminarin closely matched its specific activity (250.2 U mg-1) and k cat /K m value (104.88 mg-1 s-1 mL) toward barley β-glucan. However, the k cat /K m value (9.86 mg-1 s-1 mL) of rGluH for insoluble curdlan was only about 9.3% of the value for laminarin, which correlates well with the observation that rGluH displayed weak binding affinity (< 40%) to the insoluble polymer. The biocatalytic hydrolysis of D-laminarioligosaccharides with a degree of polymerization between 3 and 6 and laminarin generally resulted in the formation of D-laminaribiose as the predominant product and D-glucose as the secondary product, with a ratio of approximately 4:1. These findings suggest that highly active rGluH is an acidic, cold-adapted D-laminaribiose- and D-glucose-releasing GH16 endo-β-1,3-glucanase, which can be exploited as a valuable biocatalyst for facilitating low temperature preservation of foods.

Dataset Information

Endo-?-1,3-glucanase (GH16 Family) from Trichoderma harzianum Participates in Cell Wall Biogenesis but Is Not Essential for Antagonism Against Plant Pathogens.

Publications

Endo-β-1,3-glucanase (GH16 Family) from <i>Trichoderma harzianum</i> Participates in Cell Wall Biogenesis but Is Not Essential for Antagonism Against Plant Pathogens.

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