Abstract
Higher plants, bacteria, fungi, insects, and crustaceans all produce chitinases. Chitinase genes in many organisms are currently under investigation. Chitinase activity is usually assayed with radiolabeled or fluorogenic substrates. We developed a simple, inexpensive, nonradioactive gel-diffusion assay for chitinase that can be used to screen large numbers of samples. In this assay, chitinase diffuses from a small circular well cut in an agarose or agar gel containing the substrate glycol chitin, a soluble, modified form of chitin. Chitinase catalyzes the cleavage of glycol chitin as it diffuses through the gel, leaving a dark, unstained circular zone around the well, because the fluorescent dye calcofluor binds only to undigested chitin. Sample activities can be determined from linear regression of logstandard enzyme concentration versus the zone diameter of internal standards on each Petri dish used for a diffusion assay.
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References
Roberts, R.L., and Cabib, E. (1982) Serratia marcescens chitinase: one-step purification and use of determination of chitin. Anal. Biochem. 127, 402–412.
Otakara, A. (1961) Studies on the chitinolytic enzyme of black-koji mold. II. Purification of chitinases. Agric. Biol. Chem. 25, 54–60.
Koga, D., Jilka, J., and Kramer, K.J. (1983) Insect endochitinases: glycoproteins from moulting fluid, integument and pupal hemolymph. Insect. Biochem. 13, 295–305.
Lunt, M.R., and Kent, P.W. (1960) Chitinase system from Carcinus maenas. Biochim. Biophys. Acta. 44, 371–373.
Graham, L.S., and Sticklen, M.B. (1994) Plant chitinases. Can. J. Bot. 72, 1057–1083.
Leah, R., Tommerup, H., Svendsen, I., and Mundy, J. (1991) Biochemical and molecular characterization of three barley seed proteins with antifungal properties. J. Biol. Chem. 226, 1564–1573.
Molano, J., Polacheck, I., Duran, A., and Cabib E. (1979) An endochitinase from wheat germ. J. Biol. Chem. 254, 4901–4907.
McCreath K.J. and Gooday G.W. 1992 A rapid and sensitive microassay for determination of chitinolytic activity. J. Microbiol. Meth. 14, 229–237.
Trudel, J., and Asselin, A. (1989) Detection of chitinase activity after polyacrylamide gel electrophoresis. Anal. Biochem. 178, 362–366.
Pan, S.G., Ye, X.S., and Kuc, J. (1991) A technique for detection of chitinase, β-1, 3-glucanase, and protein patterns after a single separation using polyacrylamide gel electrophoresis or isoelectrofocusing. Phytopathology 81, 970–974.
Dingle, J., Reid, W. W., and Solomons, G.L. (1953) The enzymatic degradation of pectin and other polysaccharides. II: Application of the “cup-plate” assay to the estimation of enzymes. J. Sci. Food. Agric. 1, 149–155.
Wood, P. J., Erfle, J. D., and Teather, R. M. (1988) Use of complex formation between Congo Red and polysaccharides in detection and assay of polysaccharide hydrolases. Methods Enzymol. 160, 59–74.
Downie, B., Hilhorst, H.W.M., and Bewley, J.D. (1994) A new assay for quantifying endo-β-mannase activity using Congo Red dye. Phytochemistry 36, 829–835.
Maea, H, and Ishida, N. (1965) Specificity of binding of hexopyranosyl polysaccharides with fluorescent brightener. J. Biochem. 62, 276–278.
Boller, T., Gheri, A., Mauch, F., and Vögeli, U. (1983) Chitinase in bean leaves: induction by ethylene, purification, properties, and possible function. Planta 157, 22–31.
Tsukamoto, T., Koga, D., Ide, A., Ishibashe, T., Horino-Matsushige, M., Yagashita, K., and Imoto, T. (1984) Purification and some properties of chitinase from yam, Discorea opposita Thumb. Agric. Biol. Chem. 48, 931–939.
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Zou, X., Nonogaki, H. & Welbaum, G.E. A gel diffusion assay for visualization and quantification of chitinase activity. Mol Biotechnol 22, 19–23 (2002). https://doi.org/10.1385/MB:22:1:019
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DOI: https://doi.org/10.1385/MB:22:1:019