Colloidal chitin was prepared by the method of Wen et al [46 (link)]. One gram of chitin from shrimp (Sigma Aldrich) powder was added to 12 mL of HCl and kept overnight at 4 °C with continuous stirring. The mixture was added to 400 mL of ice-cold 95% ethanol with rapid stirring and was incubated at 25 °C overnight. The mixture was centrifuged at 5000× g for 20 min at 4 °C to collect the precipitate and was washed with sterile distilled water until it reached neutral pH. The prepared colloidal chitin was stored at 4 °C until the enzyme assay.
The chitinase activity was assayed by measuring the reducing ends of sugars. The assay was carried out by mixing 0.5 mL of both 1% colloidal chitin and the crude enzyme. After the incubation for 1 h, 3 mL of DNS were added to the mixture to terminate the reaction [41 (link)]. The mixture was boiled for 15 min and centrifuged to remove the insoluble chitin. Of the supernatant 200 µL were taken in the 96 well flat bottomed plate and the absorbance was measured at 540 nm using the Synergy HTX Multi-Mode reader. All reactions were carried out in triplicates. The reducing sugar was quantified from a standard curve using various concentrations of N-acetyl-D-glucosamine.
The chitinase activity was assayed by measuring the reducing ends of sugars. The assay was carried out by mixing 0.5 mL of both 1% colloidal chitin and the crude enzyme. After the incubation for 1 h, 3 mL of DNS were added to the mixture to terminate the reaction [41 (link)]. The mixture was boiled for 15 min and centrifuged to remove the insoluble chitin. Of the supernatant 200 µL were taken in the 96 well flat bottomed plate and the absorbance was measured at 540 nm using the Synergy HTX Multi-Mode reader. All reactions were carried out in triplicates. The reducing sugar was quantified from a standard curve using various concentrations of N-acetyl-D-glucosamine.