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. Graphical Abstract ᅟ.
Keywords: Chitin determination; Chitinase; Optimization; Purification; Vigna mungo.