CHITINASE ASSAY PROTOCOL

Chitinase assay: - Chitinase assay was performed by the method of Reissig et al.(1955) and modified by Boller et al (1983). Reagents: 1.Extraction buffer (0.1M, pH 5.0): Sodium citrate buffer was used for the extraction of the enzyme. The buffer was prepared as described for the glucanase assay. 2.Sodium azide (0.3M): The stock solution of sodium azide was prepared by dissolving 9.75 gm of sodium azide in 100 ml of distilled water. I00 l of this stock was diluted to 500 ml with and this solution (0.3 M) was used as the working solution. 3.Sodium acetate buffer (0.3m, pH 4.5) The stock solution was prepared by dissolving 5mg of sodium acetate in distilled water, making up the final volume to 10ml with distilled water. The pH was adjusted to 4.5 with acetic acid and the solution was stored at 2oC. Working buffer solution was prepared by diluting 1ml of this stock to 10ml. 4.Borate buffer (0.8M, pH 9.1): This buffer was prepared by mixing solution A and solution B Solution A: This solution was prepared by dissolving 7.63g of di-sodium tetraborate in distilled water, then adjusting the final volume to 100ml Solution B: This solution was prepared by dissolving 4.96g of boric acid in distilled water and then by adding distilled water to a final volume of 100ml The buffer was

prepared by mixing 83ml of solution A and 50ml of solution B, adjusting the pH to 9.1 and making up the volume to 200 ml. 5.DMAB reagent: This reagent was prepared by dissolving 10gm of p-

dimethylaminobenzaldehyde in 100ml of glacial acetic acid containing 12.5ml of hydrochloric acid (AR grade). This solution was diluted ten times with glacial acetic and this diluted solution was used as the working reagent.

6.Stock solution of colloidal chitin (substrate solution): The substrate stock was prepared by homogenizing 500mg of crab shell chitin in 50ml of distilled water. 200 l of this stock solution containing 1 mg of colloidal chitin was used for enzyme assay. Procedure: Enzyme extraction: The enzyme, chitinase was extracted from the frozen tissues as described above using the same sodium citrate buffer. Assay: The assay mixture was prepared by adding 100 l of sodium acetate buffer (pH 4.5), 100 l of sodium azide solution, 200 l of colloidal chitin and 100 l of enzyme extract. The volume of the enzyme mixture was adjusted to 1 ml by extraction buffer and incubated at 37 oC for 3 hours. After incubation, 100 l of sodium acetate buffer (pH 9.1) was added to the reaction mixture and heated in a boiling water bath for 3 minutes. The mixture was cooled and centrifuged at 1000 rpm for 5 minutes. The clear supernatant was collected and 3 ml of DMAB reagent was added to it. The mixture was incubated at 37oC for 20 minutes. The absorbance was recorded at 585 nm against a blank containing enzyme and all reagents except chitin.

Preparation of standard curve: For preparation of standard curve, different concentrations of N-acetyl d-glucosamine ranging from 1-100mg were prepared. They were incubated with 3 ml of DMAB reagent at 37 oC for 20 minutes and the absorbance was recorded at 585 nm. The absorbance was plotted against concentration to prepare the standard curve. From this standard curve, the quantity of N-acetyl dglucosamine in the samples, formed as a result of chitinase activity was calculated. The total protein content was estimated by the protein-dye binding method .The chitinase activity was expressed in Kats/mg protein. The chitinase activity was calculated as Chitinase activity = 407.3 x Quantity of N-acetyl d-glucosamine ( Kats/mg protein ) Amount of protein /gm of sample