Abstract
β-1,3-Glucanases produced by the take-all disease pathogen Gaeumannomyces graminis var. tritici (Ggt) have been suggested to be implicated in infection and colonization process of the pathogen in wheat. For further studying the role of these enzymes in pathogenesis by cytochemical technique, an extracellular β-1,3-glucanase complex (GluGgt), was purified from culture filtrate of Ggt by (NH4)2SO4 precipitation, hydrophobic-interaction, anion-exchange and size-exclusion chromatography. The complex consisted of two interconvertible isoforms (Ia and Ib), both active proteins Ia and Ib appeared to be glycosylated in native polyacrylamide gel electrophoresis (PAGE). The pI of the active proteins Ia and Ib determined by IEF-PAGE were 6.3 and 6.4, respectively. GluGgt yielded two subunits with molecular masses of 66.2 and 56.0 kDa, respectively, in 15% SDS–PAGE. The complex had a pH optimum of 5.0 and the optimal temperature was 50°C. The enzyme complex proved to be stable at a temperature up to 60°C and retained an optimal high activity in the pH range from 4.0 to 7.0. Enzyme activity of GluGgt was obviously stimulated by Mn2+ ions and moderately inhibited in the presence of Hg2+ and Co2+ ions and KMnO4. The K m of GluGgt was estimated to be 1.08 mg ml−1 for laminarin as substrate and the V max 0.20 μmol min−1.
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References
Angulo-Valadez CE, Cepeda-Palacios R, Ascencio F, Jacquiet P, Dorchies P, Romero MJ, Khelifa RM (2007) Proteolytic activity in salivary gland products of sheep bot fly (Oestrus ovis) larvae. Vet Parasitol 149:117–125
Bodenmann J, Heiniger U, Hohl HR (1985) Extracelluar enzymes of Phytophthora infestans: endo-cellulase, β-glucosidase, and β-1, 3-glucanases. Can J Microbiol 31:75–82
Carpita NC, Gibeaut DM (1993) Structural models of primary cell walls in flowering plants: consistency of molecular structure with the physical properties of the walls during growth. Plant J 3:1–30
Del Rey F, Santos T, Garcia-Acha I, Nombela C (1979) Synthesis of 1, 3-β-glucanases in Saccharomyces cerevisiae during the mitotic cycle, mating and sporulation. J Bacteriol 139:924–931
Dori S, Solel Z, Barash I (1995) Cell wall-degrading enzymes produced by Gaeumannomyces graminis var. tritici in vitro and in vivo. Physiol Mol Plant Pathol 46:189–198
Förster H, Rasched I (1985) Purification and characterization of extracellular pectinesterases from Phytophthora infestans. Plant Physiol 77:109–112
Gerard C (1990) Purification of glycoproteins. Methods Enzymol 182:529–539
Kang ZS, Buchenauer H (2002) Immunocytochemical localization of β-1, 3-glucanase and chitinase in Fusarium culmorum-infected wheat spikes. Physiol Mol Plant Pathol 60:141–153
Kang ZS, Huang LL, Buchenauer H (2000) Cytochemistry of cell wall component alteration on wheat roots infected by Gaumonomyces graminis var. tritici. J Plant Dis Protect 107:337–351
Kemp G, Botha AM, Kloppers FJ, Pretorius ZA (1999) Disease development and β-1, 3-glucanase expression following leaf rust infection in resistant and susceptible near-isogenic wheat seedlings. Physiol Mol Plant Pathol 55:45–52
Kulminskaya AA, Thomsen KK, Shabalin KA, Sidorenko IA, Eneyskaya EV, Savelev AN, Neustroev KN (2001) Isolation, enzymatic properties, and mode of action of an exo-1, 3-β-D-glucanase from Trichoderma viride. Eur J Biochem 268:6123–6131
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
Nagata S, Sawatani M, Kuriyama M, Misono H, Nagasaki S (1990) Purification and characterization of nonlytic endo-β-1, 3-glucanase I from Flavobacterium dormitator var. glucanolyticae. Agric Biol Chem 54:2107–2114
Pitson SM, Seviour RJ, McDougall BM (1993) Noncellulolytic fungal beta-glucanases: their physiology and regulation. Enzyme Microb Technol 15:178–192
Pitson SM, Seviour RJ, McDougall BM, Woodward JR, Stone BA (1995) Purification and characterization of three extracellular (1, 3)-β-D-1, 3-glucan glucohydrolases from the filamentous fungus Acremonium persicinum. Biochem J 308:733–741
Rapp P (1989) 1, 3-β-glucanase, 1, 6-β-glucanase and β-glucosidase activities of Sclerotium glucanicum: synthesis and properties. J Gen Microbiol 135:2847–2858
Sánchez M, Nombela C, Villanueva J, Santos T (1982) Purification and partial characterization of a developmentally regulated β-1, 3-glucanase from Penicillum italicum. J Gen Microbiol 128:2047–2053
Skou JP (1975) Studies on the take-all fungus Gaeumannomyces graminis. IV. Entry and growth of the fungus and significance of lignituber formation in the roots of the hosts. K Vet Landbohisk Arsskrifit 12:1–141
Stahmann KP, Schimz KL, Sahm H (1993) Purification and characterization of four extracelluar β-1, 3-glucanases of Botrytis cinerea. J Gen Microbiol 139:2833–2840
Sun L, Gurnon JR, Adams BJ, Graves MV, Van Etten JL (2000) Characterization of a β-1, 3-glucanase encoded by chlorella virus PBCV-1. Virology 276:27–36
Sun H, Yang J, Lin C, Huang X, Xing R, Zhang KQ (2006) Purification and properties of a β-1, 3-glucanase from Chaetomium sp. that is involved in mycoparasitism. Biotechnol Lett 28:131–135
Tenberge KB, Brockmann B, Tudzynski P (1999) Immunogold localization of an extracellular β-1, 3-glucanase of the ergot fungus Claviceps purpurea during infection of rye. Mycol Res 103:1103–1108
Tonon C, Guevara G, Oliva C, Daleo G (2002) Isolation of a potato acidic 39 kDa β-1, 3- glucanase with antifungal activity against Phytophthora infestans and analysis of its expression in potato cultivars differing in their degrees of field resistance. J Phytopathol 150:189–195
Van Hoof A, Leykam J, Schaeffer HJ, Walton JD (1991) A single β-1, 3-glucanase secreted by the maize pathogen Cochliobolus carbonum acts by an exolytic mechanism. Physiol Mol Plant Pathol 39:259–267
Wang XL, Huang LL, Kang ZS, Guo AG, Liu T (2003) Production and properties of β-1, 3-glucanase produced by Gaeumannomyces graminis var. tritici. Mycosystema 22:628–633 (in Chinese with english abstract)
Yu YT (2008) Purification of extracellular β-1, 3-glucanases secreted by Gaeumannomyces graminis var. Tritici and its immunolocalization in infected wheat root. Ph.D. thesis, Northwest A&F University, China
Zhen XH, Li YZ (2004) Ultrastructural changes and location of β-1, 3-glucanase in resistant and susceptible cotton callus cells in response to treatment with toxin of verticillium dahliae and salicylic acid. J Plant Physiol 161:1367–1377
Acknowledgments
This work was supported by the National Natural Science Foundation (No. 30270863), Program for Changjiang Scholars and Innovative Team in University of Education Ministry of China (No. 200558), and 111 Project from Education Ministry of China (No. B07049). We gratefully acknowledge Dr. W. N. Song and Dr. Y. Xiang for valuable advices in the paper preparation.
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Yu, Yt., Kang, Zs., Buchenauer, H. et al. Purification and characterization of a novel extracellular β-1,3-glucanase complex (GluGgt) secreted by Gaeumannomyces graminis var. tritici . World J Microbiol Biotechnol 25, 2179–2186 (2009). https://doi.org/10.1007/s11274-009-0123-2
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DOI: https://doi.org/10.1007/s11274-009-0123-2