Characterization of endo-1,3–1,4-β-glucanases in GH family 12 from Magnaporthe oryzae

Abstract

We have cloned three putative endoglucanase cDNAs, designated MoCel12A, MoCel12B, and MoCel12C, from Magnaporthe oryzae. The deduced peptide sequences of both MoCel12A and MoCel12B contain secretion signal peptides and a catalytic core domain that classify them into GH subfamily 12-1. In contrast, the deduced peptide sequence of MoCel12C consists of a signal peptide, a catalytic core domain, and a fungal-type carbohydrate binding module belonging to GH subfamily 12-2. Although most GH family 12 endoglucanases hydrolyze β-1,4-glucans such as carboxymethylcellulose or phosphoric acid-swollen cellulose, MoCel12A that was prepared by overexpression in M. oryzae and Brevibacillus choshinensis hydrolyzed specifically 1,3-1,4-β-glucans, such as barley β-glucan and lichenan. The specific activity of MoCel12A overexpressed in M. oryzae was about 20 times higher than that prepared from B. choshinensis. Furthermore, MoCel12B prepared by overexpression in B. choshinensis also revealed preferential hydrolysis of endo-1,3-1,4-β-glucans with limited hydrolysis on carboxymethylcellulose. In comparison with MoCel12A, the activity of MoCel12B was more stable under alkaline conditions. Levels of mRNA encoding MoCel12A were constitutively high during infection and spore formation. The overexpression and disruption of the MoCel12A gene did not affect germination, appressorium formation, or invasion rate; however, M. oryzae overexpressing MoCel12A produced larger numbers of spores than the wild type or a mutant in which the MoCel12A gene was disrupted. These results suggest that MoCel12A functions in part to hydrolyze 1,3-1,4-β-glucan during infection and spore formation.

Matrix solubilization and cell wall weakening by β‐expansin (group‐1 allergen) from maize pollen

“…Enzyme sources were as follows: XynC, a recombinant xylanase from Bacillus subtilis , expressed in E. coli and purified by HPLC (Kato and Nevins, 1984a; St John et al. , 2006); recombinant pectate lyase from Cellvibrio japonicus (E‐PLYCJ, Megazyme); recombinant (1,3),(1,4)‐β‐glucan endo‐4‐glucanase from Magnaportha oryzae (MoCel12A, Takeda et al. , 2010), and xylanase XynM4 from Aspergillus niger (E‐XYAN4, Megazyme).…”

Degradation and Synthesis of β-Glucans by a Magnaporthe oryzae Endotransglucosylase, a Member of the Glycoside Hydrolase 7 Family

“…Preparation of Recombinant Enzyme-Overexpression of a recombinant MoCel7B fused with seven contiguous histidine residues at the carboxyl terminus in M. oryzae, purification using a polyhistidine-binding resin (TALON metal affinity resin, Clontech), and immunoblotting using a horseradish peroxidase-conjugated monoclonal antibody against the histidine tag (Qiagen) were conducted as described previously (4). Protein concentration was determined using a Bradford protein assay kit (ThermoScientific) with BSA (Sigma) as a standard.…”

“…The mixtures were divided into soluble and insoluble fractions by centrifugation at 22,000 ϫ g for 5 min. The amount of insoluble material was measured using 0.5% (w/v) anthrone in H 2 SO 4 after the insoluble material was dissolved in ice-cold 72% (v/v) H 2 SO 4 .…”

“…Enhanced activity of 1,3-1,4-glucanase (MoCel12A) caused increased spore formation of Magnaporthe oryzae (4). Deletion of genes encoding polygalacturonases in Botrytis cinerea reduced its virulence on tomato (17,18).…”

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

“…Culture filtrates obtained after 3 days of growth of the A. oryzae transformant expressing Lam16A-His 7 were used to purify Lam16A-His 7 by using polyhistidinebinding resin (Talon metal affinity resin; Clontech, CA) as described previously (42). Purified Lam16A-His 7 was concentrated and equilibrated in 20 mM sodium phosphate buffer (pH 6.0) by ultrafiltration before use.…”