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 655 nm. We found that measurement of low levels of RSE at 655 nm yields higher precision and better linearity than that at 615 nm. The optimal procedures are: the pre-warmed half-deacetylated chitosan and lysozyme were mixed to make the final solution containing 4 mg mL−1 half-deacetylated chitosan at pH 4.0 in sodium acetate buffer, and incubated at 40 °C for 1 h within steady state time course. The linear range of lysozyme activity was within 94–1006 nM min−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.

A novel method for determining the degree of deacetylation of chitosan is described. This involves two-abrupt-change potentiometric titration preceded by enzymatic pretreatment (EP-TPT) with chitosanolytic enzyme (cellulase) to reduce the viscosity of chitosan solution. Acidic titration is preferred for the rapid response within the two abrupt changes of pH. Half-deacetylated chitosan (almost soluble in all pH range) was used to prove that the enzyme did not interfere with the measurement. Compared with other methods such as 1H NMR, XRD, FT-IR and linear potentiometric titration, the proposed method is simple, low-cost, accurate, reliable and easy to operate for industrial application.