Quantitative determination of chitinolytic activity of lysozyme using half-deacetylated chitosan as a substrate

Abstract

In this paper, we report a novel quantitative assay for chitinolytic activity of lysozyme. Our assay is based on measuring the reducing sugar ends (RSE) released from the water-soluble macromolecular substrate, half-deacetylated chitosan. RSE reacts with 3-methyl-2-benzothiazolinonehydrazone to produce the chromogenic product that has absorption maxima at 615 and 655nm. We found that measurement of low levels of RSE at 655nm yields higher precision and better linearity than that at 615nm. The optimal procedures are: the pre-warmed half-deacetylated chitosan and lysozyme were mixed to make the final solution containing 4mgmL−1 half-deacetylated chitosan at pH 4.0 in sodium acetate buffer, and incubated at 40°C for 1h within steady state time course. The linear range of lysozyme activity was within 94–1006nMmin−1. We demonstrated that our method is simple, rapid, inexpensive and accurate, which other low-sensitive methods cannot be achieved. This method can also be used to quantitatively determine the activity and kinetic parameters of chitinase and chitosanase.