Abstract
Determination of the apparent pK a's of purified carboxymethylcellulases fromAspergillus niger andCellulomonas biazotea at different temperatures and in the presence of dioxane indicated two side chain carboxyl groups which controlled the limiting rate in both organisms. The thermostability of both enzymes slightly decreased with increasing pH from 5 to 7.5 but was unaffected in the presence of 0.5 mmol/L Mn2+. The CMCase fromC. biazotea had an activation energy of 35 kJ/mol and a half-life of 89 min in the presence of 8 mol/L urea at 40°C. The half-life of CMCase fromA. niger in 8 mol/L urea and at 37°C was 125 min as determined by a 0–9 mol/L transverse urea gradient PAGE. The CMCases fromA. niger andC. biazotea had the same thermostabilities in the absence of CMC although the enzyme from the former was more thermostable in the presence of the substrate. The CMCase fromA. niger was also more efficient in hydrolyzing CMC than the enzyme fromC. biazotea.
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Akiba S., Kimura Y., Yamamoto K., Kumagai H.: Purification and characterization of a protease-resistant cellulase fromAspergillus niger.J. Ferment. Bioeng. 79, 125–130 (1995).
Beguin P., Joliff G., Juy M., Amit A.G., Millet J., Poliak R.J., Aubert J.P.: Crystalline endoglucanase D ofClostridium thermocellum overproduced inEscherichia coli.Methods Enzymol. 160, 355–362 (1988).
Calza R.E., Irwin D.C., Wilson D.B.: Purification and characterization of two β-1,4-endoglucanases fromThermomonospora fusca.Biochemistry 24, 7797–7804 (1985).
Clarke A.J., Bray M.R., Starting H.: β-Glucosidase, β-glucanases and xylanases: their mechanism of catalysis, pp. 27–41 inβ-Glucosidase, Biochemistry and Molecular Biology (A. Esen, Ed.). American Chemical Society, Washington (DC) 1993.
Clarke A.J., Yaguchi M.: The role of carboxyl groups in the function of endo-β-1,4-glucanase fromSchizophyllum commune.Eur. J. Biochem. 149, 233–238 (1985).
Coughlan M.P.: Staining techniques for the detection of the individual components of cellulolytic enzyme system.Methods Enzymol. 160, 135–144 (1988).
Creighton T.E.: Detection of intermediates using urea-gradient electrophoresis.Methods Enzymol. 131, 156–172 (1986).
Fersht A.: The pH dependence of enzyme catalysis, pp. 155–175 inEnzyme Structure and Mechanism, W.H. Freeman and Co., New York 1985.
Godfrey T., West S.:Industrial Enzymology. Macmillan Press Ltd., New York 1996.
Goldenberg D.P.: Analysis of protein conformation by gel electrophoresis, pp. 225–250 inProtein Structure: A Practical Approach (T.E. Creighton, Ed.). IRL Press, Oxford (UK) 1989.
Hurst P.L., Sullivan P.A., Shepherd M.G.: Chemical modification of a cellulase fromAspergillus niger.Biochem. J. 167, 549–556 (1977).
Kundu R.K., Dube S., Dube D.K.: Extracellular cellulolytic enzyme system ofAspergillus japonicus. 3. Isolation, purification and characterization of multiple forms of endoglucanase.Enzyme Microb. Technol. 10, 100–109 (1988).
Macarron R., Acebal C., Castillon M.P., Dominguez I., Pettersson G., Tomme P., Claeyssens M.: Mode of action of endoglucanase-III fromTrichoderma reesei.Biochem. J. 32, 867–873 (1993).
Nakamura K., Kitamura K.: Purification and some properties of a cellulase active on crystalline cellulose fromCellulomonas uda.J. Ferment. Technol. 61, 379–382 (1983).
Ng T.K., Zeikus J.G.: Endoglucanase fromClostridium thermocellum.Methods Enzymol. 160, 351–355 (1988).
Rangarajan M., Asboth B., Hartley B.S.: Stability ofArthrobacter d-xylose isomerase to denaturants and heat.Biochem. J. 285, 889–898 (1992).
Rangarajan M., Hartley B.S.: Mechanism ofd-fructose isomerization byArthrobacter d-xylose isomerase.Biochem. J. 283, 223–233 (1992).
Sami A.J., Akhtar M.W.: Purification and characterization of two low-molecular weight endoglucanases ofCellulomonas flavigena.Enzyme Microb. Technol. 15, 586–592 (1993).
Sarkar A., Upadhyay S.N.: Purification and characterization of cellulase fromBacillus thermoalcaliphilus isolated from a termite mound.Folia Microbiol. 38, 29–32 (1993).
Siddiqui K.S., Azhar M.J., Rashid M.H., Rajoka M.I.: Activity and thermostability of carboxymethylcellulase fromA. niger is strongly influenced by non-covalently attached polysaccharides.World J. Biotechnol. Microbiol. 12, 213–216 (1996).
Siddiqui K.S., Rashid M.H., Rajoka M.I.: Kinetic analysis of the active site of an intracellular β-glucosidase fromCellulomonas biazotea.Folia Microbiol. 42, 53–58 (1997a).
Siddiqui K.S., Azhar M.J., Rashid M.H., Ghuri T.M., Rajoka M.I.: Purification and the effect of manganese ion on the activity of carboxymethylcellulase fromAspergillus niger andCellulomonas biazotea. Folia Microbiol. 42, (1997b, in press).
Stoll V.S., Blanchard J.S.: Buffers: principles and praetice.Methods Enzymol. 182, 24–38 (1990).
Tipton K.F., Dixon H.B.F.: Effect of pH on enzymes.Methods Enzymol. 63, 183–234 (1979).
Wood T.M., Bhat K.M.: Methods for measuring cellulase activities.Methods Enzymol. 160, 87–112 (1988).
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Siddiqui, K.S., Azhar, M.J., Rashid, M.H. et al. Stability and identification of active-site residues of carboxymethylcellulases fromAspergillus niger andCellulomonas biazotea . Folia Microbiol 42, 312–318 (1997). https://doi.org/10.1007/BF02816941
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DOI: https://doi.org/10.1007/BF02816941