Summary
The effects of protein glycosylation inhibitors were studied in Neocallimastix frontalis EB188. Low concentrations of tunicamycin and 2-deoxy-D-glucose inhibited zoospore germination, rhizoidal elongation, carbon source utilization and the production and secretion of cellulases and proteins. The carbohydrate-trimming inhibitors, deoxynojirimycin and glucono-δ-lactone, had no measurable effect on rhizoidal growth and carbon source utilization. Cellulases (intracellular or extracellular) synthesized in the presence of glycosylation inhibitors were sensitive to β-endoglycosidase H digestion, periodate modification, certain salts, changes in incubation temperature and pH, and protease. Anthrone staining of extracellular proteins confirmed the presence of glycoproteins. In N. frontalis EB188, glycosylation of protein and cellulase occurred and was important for cellular development and the production, secretion and activity of cellulases.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Barichievich EB, Calza RE (1990) Supernatant protein and cellulase activities of anaerobic ruminal fungus Neocallimastix frontalis EB188. Appl Environ Microbiol 56:43–48
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein using the principle of protein-dye binding. Anal Biochem 72:248–252
Calza RE (1990) Regulation of protein and cellulase excretion in the ruminal fungus Neocallimastix frontalis EB188. Curr Microbiol 21:109–115
Duksin D, Mahoney W (1982) Relationship of the structure and biological activity of natural homologues of tunicamycin. J Biol Chem 257:3105
Greber UF, Sode K (1988) Effect of the glycosidase inhibitor 1-deoxynojirimycin on protein secretion from Saccharomyces cerevisiae. Enzyme Microb Technol 10:246–251
Hay GW, Lewis BA, Smith F (1965) Periodate oxidation of polysaccharides: general procedures. Methods Carbohydr Chem 5:356–361
Hayashida S, Yoshioka H (1980) The role of carbohydrate moiety on thermostability of cellulases form Humicola insolens YH-8. Agric Biol Chem 44:481–487
Hodge JE, Hofreiter BT (1962) Determination of reducing sugars and carbohydrates. Methods Carbohydr Chem 1:389–390
Hungate RE (1969) A roll tube method for the cultivation of strict anaerobes. Methods Microbiol 3B:117–132
Joblin KN (1981) Isolation, enumeration, and maintenance of rumen anaerobic fungi in roll tubes. Appl Environ Microbiol 42:1119–1122
Jue CL, Lipke PN (1985) Determination of reducing sugars in the nanomole range with tetrazolium blue. J Biochem Biophys Methods 11:109–116
Kubicek CP, Panda T, Kunar G, Gruber F, Messner R (1987) O-Linked but not N-linked glycosylation is necessary for the secretion of endoglucanases I and II by Trichoderma reesei. Can J Microbiol 33:698–703
Lowe SE, Theodorou ML, Trinci APJ, Hespell RB (1985) Growth of anaerobic rumen fungi in defined and semi-defined media lacking rumen fluid. J Gen Microbiol 131:2225–2229
Merivouri H, Sands JA, Montenecourt BS (1985) Effects of tunicamycin on secretion and enzymatic activities of cellulase from Trichoderma reesei. Appl Microbiol Biotechnol 23:60–66
Mountfort DO, Asher RA (1985) Production and regulation of cellulase by two strains of the rumen anaerobic fungus Neocallimastix frontalis. Appl Environ Microbiol 49:1314–1322
Orpin CG, Letcher AJ (1979) Utilization of cellulose, starch, xylan and hemicelluloses for growth by the rumen phycomycete, Neocallimastix frontalis. Curr Microbiol 3:121–124
Sanchez A, Villaneuva J, Villa TG (1982) Effect of tunicamycin on exo-1,3 β-D-glycanase synthesis and secretion by cells and protoplasts of Saccharomyces cerevisiae. J Gen Microbiol 128:3051–3060
Trimble RB, Maley F (1984) Optimizing hydrolysis of N-linked high-mannose oligosaccharides by endo-B-N-acetyl glucosaminidase. Anal Biochem 141:515–522
Williams AG, Orpin CG (1987) Polysaccharide-degrading enzymes formed by the three species of anaerobic rumen fungi grown on a range of carbohydrate substrates. Can J Microbiol 33:419–426
Wood TM, McCrae SI, Wilson CA, Bhat KM, Gow LA (1988) Aerobic and anaerobic fungal cellulases, with special reference to their mode of attack on crystalline cellulose. In: Aubert J-P, Beguin P, Millet J (eds) Biochemistry and genetics of cellulose degradation. Academic Press, London, pp 31–52
Author information
Authors and Affiliations
Additional information
Offprint requests to: R. E. Calza
Rights and permissions
About this article
Cite this article
Li, X., Calza, R.E. Cellulases from Neocallimastix frontalis EB188 synthesized in the presence of glycosylation inhibitors: measurement of pH and temperature optima, protease and ion sensitivities. Appl Microbiol Biotechnol 35, 741–747 (1991). https://doi.org/10.1007/BF00169888
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00169888