4.9.1. Assay for Chitinase Activity

Extraction of enzyme solution: 0.5 g of kiwifruit leaves was taken after two years of sulfur application, liquid nitrogen was added and then ground to the powder form, followed by the addition of 7 mL sodium acetate buffer (pH = 5.0) homogenate. The mixture was centrifuged at 4 °C, 15,000× g for 15 min, after which the activity of extracellular chitinase and intracellular chitinase was analyzed by using the supernatant collected. The enzyme activity was measured based on a slightly modified method [73].

Standard curve of production: The different concentrations of N-acetyl glucosamine (Sigma Company, Burlington, MA, USA) at a volume of 1.5 mL were taken, and 2 mL of potassium ferricyanide solution was added, following which the mixture was boiled at 100 °C in a water bath for 15 min. The measured optical density in the distilled water was considered as a control. In contrast, the optical density decreased with increasing N-acetylamino glucose concentrations displaying a positive correlation, with n-acetyl glucosamine zero optical density being about 0.85. The standard curve was thereafter plotted with the difference of optical density (0.85 minus the density of sugar) as the coordinate and N-acetylglucosamine concentration as the abscissa.

Determination of endochitinase activity: The initial reaction solution used was the same as the assay of exochitinase. After being incubated at 37 °C for 2 h and centrifuged at 1000× g for 2 min, 1.5 mL supernatant was collected and added with 0.1 mL 3% (w/v) desalted snail enzyme and 0.15 mL 1 mol L⁻¹ sodium phosphate buffer (pH = 7.1). The reaction solution was then placed in a constant temperature water bath at 37 °C for one hour to hydrolyze the various chitin fragments produced by chitin endonucleases. A reaction solution containing a substrate and an enzyme (heat-inactivated) was used as a control. The amount of N-acetylglucosamine produced was calculated by the method described above. One unit of 1 g N-acetylglucosamine per hour was produced from the decomposition of colloidal chitin.

Determination of exochitinase activity: 1.5 mL of colloidal chitin solution (containing 6 mg chitin) was absorbed, and 0.5 mL 50 mmol L⁻¹ sodium acetate buffer solution (pH = 4.5) was added. The enzyme solution 0.4 mL and 0.1mL of 75 mol L⁻¹ sodium azide solution was mixed well. After being kept at 37 °C for 2–4 h, 0.5 mL sodium borate buffer solution with a concentration of 0.8 mol L⁻¹ (pH = 9.1) was added, and centrifugation at 1000× g for 5 min was performed. Thereafter, 1.5 mL supernatant was taken to measure the N-acetylglucosamine produced by the chitin extracellular enzyme. As the standard control, 1.5 mL of the same treated solution, including substrate and enzyme (heat-inactivated), was used. The optical density was measured at 585 nm, and the amount of N-acetylglucosamine produced was calculated from the standard curve based on the difference in the optical density (the standard control optical density minus the sample liquid optical density).