Abstract
TheSomogyi—Nelson colorimetric method is applied in a new manner which is more suitable for following the kinetics of cellobiose hydrolysis catalyzed by β-glucosidase (EC 3.2.1.21). TheSomogyi—Nelson colour reagent, which is a mixture of the solutions of the reagent ofSomogyi and that ofNelson in a volume ratio of 1:1, is added to the enzyme-substrate solution at the very start of the reaction. The colour reagent reacts with the product (D-glucose). Under the reaction conditions (0.1M acetate buffer,pH = 5.0 and temperature 37°C) the colour reagent does not affect the enzyme activity. The method excludes any inhibition of the product, owing to the continuous removal of the latter by the colour reagent. The method suggested has been applied to monitor cellobiose hydrolysis with β-glucosidase, contained in four cellulase enzyme preparations from various fungal sources. The values of theMichaelis parameters (Km, V) were determined.
Zusammenfassung
Die kolorimetrische Methode nachSomogyi undNelson wird nach einem neuen Verfahren zur Verfolgung der Kinetik der hydrolytischen Spaltung von Cellobiose, katalysiert durch β-Glucosidase (EC 3.2.1.21), angewandt. Das Farbreagenz nachSomogyi undNelson (Mischung der Reagenzien vonSomogyi undNelson im Volumenverhältnis 1:1) wird der Enzym-Substrat-Lösung zu Beginn der Reaktion hinzugefügt. Das Farbreagenz tritt mit derD-Glukose in Reaktion, wobei unter den gegebenen Reaktionsbedingungen (0,1M Azetatpuffer,pH = 5,0 und 37°C) die Enzymaktivität nicht beeinflußt wird. Die entwickelte Methode wurde zur Verfolgung der Hydrolyse von Cellobiose durch ß-Glucosidasen, die in vier Enzympräparaten aus verschiedenen Pilzstämmen enthalten waren, angewandt. Es wurden dieMichaelis-Parameter (Km, V) bestimmt.
Similar content being viewed by others
References
IUPAC and IUB. Enzyme Nomenclature. Amsterdam: Elsevier. 1973.
Mandels M., Reese E. T., Dev. Ind. Microbiol.5, 5 (1964).
Berghem L. E. R., Pettersson L. G., Eur. J. Biochem.46, 295 (1974).
Gong C. S., Ladish M. R., Tsao G. T., Biotech. Bioeng.19, 957 (1977).
Deshpande V. V., Eriksson K.-E., Pettersson B., Eur. J. Biochem.90, 191 (1978).
Klyosov A. A., Rabinovitch M. L., Sinitsyn A. P., Churilova I. V., Grigorash S. Yu., Bioorg. Chem.6, 1225 (1980).
Lee Y.-H., Fan L. T., Fan L.-Sh., in: Adv. in Biochem. Eng. (Fiechter, A., ed.), Vol. 17, p. 132. Berlin-Heidelberg-New York: Springer. 1980.
Somogyi M., J. Biol. Chem.160, 61 (1945).
Somogyi M., J. Biol. Chem.195, 19 (1952).
Nelson N., J. Biol. Chem.153, 376 (1944).
Lineweaver H., Burk D., J. Amer. Chem. Soc.56, 658 (1934).
Umezurike G. M., Biochem. J.177, 9 (1979).
Hirayama T., Nagayama H., Matsuda K., J. Biochem.87, 1203 (1980).
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Chetkarov, M.L., Hatour, F.D. & Kolev, D.N. A kinetic method for the direct determination of cellobiose hydrolysis by β-glucosidases. Monatsh Chem 115, 1321–1327 (1984). https://doi.org/10.1007/BF00798310
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00798310