Abstract
Seven fractions rich in endoglucanase activity were separated from the extracellular cellulase system of the anaerobic rumen fungus Neocallimastix frontalis. The fractions (ES1, ES3, ES2U1, ES2U2, ES2U4, ES2U3C1 and ES2U3C2) were separated from each other and from a fraction that could solubilize crystalline cellulose (the so-called crystalline-cellulose-solubilizing component, CCSC) by the sequential use of differential adsorption on the microcrystalline cellulose Avicel, gel filtration and affinity chromatography on concanavalin-A—Sepharose. The molecular masses of the endoglucanase fractions, when determined by gel filtration, were 64, 30, 61, 113, 17, 38 and 93 kDa respectively. Each enzyme degraded carboxymethylcellulose and was rich in activity to cellulose swollen in phosphoric acid to break the hydrogen bonding: cellobiose, cellotriose and cellotetraose were released in differing proportions. Each fraction showed a characteristic gradient when the capacity of each enzyme to increase the fluidity of a solution of carboxymethylcellulose was plotted against the increase in reducing power of the solution. Although neither endoglucanase fraction, acting in isolation, could degrade crystalline cellulose, three of the fractions (ES1, ES3 and ES2U1) could act synergistically with the CCSC fraction in this regard. Remarkably, the same three fractions also acted in synergism with the cellobiohydrolase (CBH I and CBH II) of the aerobic fungus Penicillium pinophilum in degrading crystalline cellulose, but only when both cellobiohydrolase enzymes were present in the solution along with any one of the three endoglucanases. These observations support the conclusion that the mechanism of action of the cellulase system of N. frontalis in degrading crystalline cellulose may be similar to that operating in the aerobic fungi.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Beguin P, Aubert J-P (1994) The biological degradation of cellulose. FEMS Microbiol Rev 13:25–58
Bhat KM, McCrae SI, Wood TM (1989) The endo-1,4-β-d-glucanase system of Pencillium pinophilum cellulase: isolation, purification, and characterisation of five major endoglucanase components. Carbohydr Res 190:279–297
Corbett WM (1963) Purification of cotton cellulose. Methods Carbohydr Chem 3:3–4
Goyal A, Ghosh B, Eveleigh D (1991) Characterization of fungal cellulases. Bioresource Technol 36:37–50
Hébraud M, Fèvre M (1988) Characterization of glycoside hydrolases secreted by the rumen anaerobic fungi Neocallimastix frontalis, Sphaeromonas communis and Piromonas communis. J Gen Microbiol 134:1123–1129
Klyosov AA (1990) Trends in biochemistry and enzymology of cellulose degradation. Biochemistry 29:10577–10585
Li X, Calza RE (1991) Fractionation of cellulases from the ruminal fungus Neocallimastix frontalis EB 188. Appl Environ Microbiol 57:3331–3336
Lowe SE, Theodorou MK, Trinci APJ, Hespell RB (1985) Growth of anaerobic fungi on defined medium and semi-defined medium lacking rumen fluid. J Gen Microbiol 31:2225–2229
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275
Mountford DO (1987) The rumen anaerobic fungi. FEMS Microbiol Rev 46:401–408
Mountford 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
Nelson N (1952) A photometric adaptation of the Somogyi method for the determination of glucose. J Biol Chem 19:19–23
Teunison MJ, Kort GVM de, Op Den Camp HJM, Huis In `T Veld JHJ (1992) Production of cellulolytic and xylanolytic enzymes during growth of the anaerobic fungus Piromonas sp. on different substrates. J Gen Microbiol 138:1657–1664
Wallace RJ, Joblin KN (1985) Proteolytic activity of a rumen anaerobic fungus. FEMS Microbiol Lett 29:19–25
Wilson CA, Wood TM (1992a) The anaerobic fungus Neocallimastix frontalis: isolation and properties of a cellulosome-type enzyme fraction with the capacity to solubilize hydrogen bond-ordered cellulose. Appl Microbiol Biotechnol 37:125–129
Wilson CA, Wood TM (1992b) Studies on the cellulase of the rumen anaerobic fungus Neocallimastix frontalis, with special reference to the capacity of the enzyme to degrade crystalline cellulose. Enzyme Microbiol Technol 14:258–264
Wilson CA, McCrae SI, Wood TM (1994) Characterization of a β-d-glucosidase from the anaerobic fungus Neocallimastix frontalis with particular reference to attack on cello-oligosaccharides. J Biotechnol 37:217–227
Wood TM (1971) The cellulase of Fusarium solani. Purification and specificity of the β-(1 → 4)-glucanase and the β-d-glucosidase components. Biochem J 121:353–362
Wood TM (1992) Fungal cellulases. Biochem Soc Trans 20:46–53
Wood TM, Garcia-Campayo V (1993) Enzymes and mechanisms involved in microbial celluloysis. In: Ratledge C (ed) Biochemistry of microbial degradation. Kluwer, The Netherlands, pp 197–232
Wood TM, McCrae SI (1978) The cellulose of Trichoderma koningii. Purification and properties of some endoglucanase components with special reference to their action or cellulose when acting alone and in synergism with the cellobiohydrolase. Biochem 171:61–72
Wood TM, McCrae SI (1986) The cellulase of Penicillium pinophilum. Synergism between enzyme components in solubilizing cellulose with special reference to the involvement of two immunologically-distinct cellobiohydrolases. Biochem J 234:93–99
Wood TM, Wilson CA (1995) Studies on the capacity of the cellulase of the anaerobic rumen fungus P. communis P to degrade hydrogen bond-ordered cellulase. Appl Microbiol Biotechnol 43:572–578
Wood TM, Wilson CA, McCrae SI, Joblin KN (1986) A highly active extracellular cellulase from the anaerobic rumen fungus Neocallimastix frontalis. FEMS Microbiol Lett 34:37–40
Wood TM, McCrae SI, Wilson CA, Bhat KM, Gow LA (1988) Aerobic and anaerobic fungal cellulases, with special reference to the mode of attack on crystalline cellulose. In: Aubert JP, Beguin P, Millet J (eds) Biochemistry and genetics of cellulose degradation. Academic Press, London, pp 31–52
Wood TM, McCrae SI, Bat KM (1989) The mechanism of fungal cellulase action. Synergism between enzyme components of Penicillium pinophilum cellulase in solubilizing hydrogen bond-ordered cellulose. Biochem J 260:37–43
Wood TM, Wilson CA, McCrae SI (1994) Synergism between cellulase components of he anaerobic rumen fungus Neocallimastix frontalis and those of the aerobic fungi Penicillium pinophilum and Trichoderma koningii in degrading crystalline cellulose. Appl Microbiol Biotechnol 41:257–261
Xue G-P, Orpin CG, Gobius KS, Aylward JH, Simpson GD (1992a) Cloning and expression of multiple cellulase cDNAs from the anaerobic rumen fungus Neocallimastix patriciarum in Escherichia coli. J Gen Microbiol 138:1413–1420
Xue G-P, Gobius KS, Orpin CG (1992b) A novel polysaccharide hydrolase cDNA (cel d from Neocallimastix patriciarum encoding three multi-functional catalytic domains with high endoglucanase, cellobiohydrolase and xylanase activities. J Gen Microbiol 138:2397–2403
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Wood, T.M., Wilson, C.A. & McCrae, S.I. The cellulose system of the anaerobic rumen fungus Neocallimastix frontalis: studies on the properties of fractions rich in endo-(1→4)-β-d-glucanase activity. Appl Microbiol Biotechnol 44, 177–184 (1995). https://doi.org/10.1007/BF00164499
Received:
Revised:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00164499