A Simple Turbidimetric Assay Using Chitin Nanofibers as the Substrate for Determinination of Chitinase Activity

Abstract

A simple turbidimetric assay using chitin nanofiber as the substrate was employed to measure chitinase activity. The higher dispersive properties of chitin nanofibers enabled the degradation of chitin to be monitored turbidimetrically. When non-processive chitinases, a family GH18 chitinase from the tobacco plant and a GH19 chitinase from rye seeds, were added to the β-chitin nanofiber suspension, no significant changes were observed in the turbidity of the suspension, however, the amounts of reducing sugars produced were significantly high and small amounts of GlcNAc and (GlcNAc)2 were detected by HPLC in the reaction mixture. However, the addition of a processive family GH18 chitinase, Serratia marcescence chitinase B or chitinase from Autographa californica multiple nucleopolyhedrovirus, resulted in a significant decrease in the turbidity of the chitin nanofiber suspension, and produced larger amounts of reducing sugars including GlcNAc and (GlcNAc)2. The rate of decreases in turbidity was clearly dependent upon the enzyme concentration. We concluded that the turbidimetric assay using β-chitin nanofibers as the substrate was useful for measuring the activities of processive chitinases.