Purification and Analytical Application of Vigna mungo Chitinase for Determination of Total Fungal Load of Stored Cereals
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A novel chitinase from urd bean (Vigna mungo) seeds was purified up to homogeneity and optimized with respect to its optimum working conditions of pH, temperature, and substrate concentration. Overall, 145-fold purification with 70% yield of the purified chitinase was achieved. The notable features of the purified enzyme were its appreciative substrate affinity as well as catalytic efficiency, high thermo stability (70% retention of initial activity at 70 °C after 60 min of continuous exposure), and pretty good storage stability (half-life of 45 days at 5 °C). The enzyme was used for determination of total chitin contents of the stored cereals that in the absence of any insect infestation, were considered to be directly proportional to the total fungal load of the tested samples. The method was linear up to 7.0 mM with 0.04 mM as the limit of detection. Percent recoveries of added chitin were < 90.0% and within-day and between-day coefficients of variations were > 3.0% for all the samples. Chitin values in stored cereal samples obtained by the present method and the popular DNS method showed good correlation, the value for coefficient of determination (R2) being < 0.98. Overall, the method yielded acceptable sensitivity, reproducibility, and accuracy.
KeywordsChitinase Purification Vigna mungo Optimization Chitin determination
This work was supported by SERB, Department of Science and Technology (DST; File no. SB/YS/LS-67/2013) and Haryana State Council for Science and Technology (HSCST/688).
Compliance with Ethical Standards
Conflict of Interest
The authors declare that they have no conflict of interest.
- 1.Bothast, R. (1978). Fungal deterioration and related phenomena in cereals, legumes and oilseeds. Post-harvest biology and Biotechnology, 210–243.Google Scholar
- 2.Miller, J. D. (1995). Fungi and mycotoxins in grain: implications for stored product research. Proceedings of the 6th International Working Conference on Stored-product Protection.Google Scholar
- 3.Newton, A. C., Lucas, A., & Ainsworth, M. (2005). Fungi. New Forests, 1, 112–122.Google Scholar
- 5.Kern, M. F., Maraschin, S. D. F., Vom Endt, D., Schrank, A., Vainstein, M. H., & Pasquali, G. (2010). Expression of a chitinase gene from Metarhizium anisopliae in tobacco plants confers resistance against Rhizoctonia solani. Applied Biochemistry and Biotechnology, 160(7), 1933–1946.CrossRefPubMedGoogle Scholar
- 14.Mauch, F., & Staehelin, L. A. (1989). Functional implications of the subcellular localization of ethylene-induced chitinase and ß-1,3-glucanase in bean leaves. The Plant Cell, 447–457.Google Scholar
- 20.Steven, S. Z. (1993). Chemistry (3rd ed.). Lexington, Mass: D. C. Health & Co..Google Scholar
- 24.Chang, Y. M., Chen, L. C., Wang, H. Y., Chiang, C. L., Chang, C. T., & Chung, Y. C. (2014). Characterization of an acidic chitinase from seeds of black soybean (Glycine max (L) Merr Tainan no. 3). PLoS One, 9(12), 1–15.Google Scholar
- 26.Chen-Tien, C., Yi-Ling, H., & Hsien-Yi, S. (1996). Purification and properties of chitinase from cabbage stems with roots. Biochemistry and Molecular Biology International, 40(2), 417–425.Google Scholar
- 37.Pareek, S. S., Ravi, I., & Sharma, V. (2013). Induction of β-1,3-glucanase and chitinase in Vigna aconitifolia inoculated with Macrophomina phaseolina. Journal of Plant Interactions, 9(1), 1–6.Google Scholar