Fluorimetric chitinase assay kit

Chitinase activity was analyzed by using the substrate and standard solutions of the fluorimetric chitinase assay kit (Sigma-Aldrich Chemie GmbH, Munich, Germany). Substrates 4-MU-(GlcNAc)₂ (exochitinase activity) and 4-MU-(GlcNAc)₃ (endochitinase activity) were adjusted to 1.1 mM in DMSO and diluted 1 : 20 in assay buffer (100 mM citric acid, 200 mM sodium phosphate, pH 5.6) immediately prior to starting the assay by adding 50 μl of diluted substrate solution to 10 μl of protein KFD48490.1 (Ts-Chit) (5 μg/ml). The reaction mix was incubated for 30 min at 37°C and then quenched by adding 500 μl stop solution (500 mM sodium carbonate, 500 mM sodium bicarbonate, pH 10.7). 250 μl of the reaction solution was transferred in duplicate into a black 96-well plate, and fluorescence of liberated 4-MU was measured using an excitation wavelength of 360 nm and recording emission at 450 nm. The effect of pH on KFD48490.1/Ts-Chit chitinase activity was determined by using assay buffers adjusted to pH = 2, 3, 4, 5, 6, 7, 8, 9, and 10. Heat stability of Ts-Chit chitinase was assessed by preincubation of the protein for 60 min at different temperatures (4°C–90°C) followed by the chitinase assay.

To obtain proteins for the enzymatic assay, the coding sequences of native PR3b and alternatively spliced PR3b were amplified with the same 5′-end primer 5′-AAAGGATCCATGAGCATTAAGCTATCTT-3′ (restriction site is italicized) and specific 3′-end primers 5′-AACAGCACCCCTGATAGC-3′ (for native PR3b) and 5′-TTCCAAAGCATGACACCTC-3′ (for spliced PR3b), and cloned in-frame with the glutathione S-transferase (GST) tag coding region in pGEX-4T-2 vector (Novagen) through restriction sites BamHI and SmaI. Then, the protein expression vectors were transformed into E. coli BL21 cells to induce prokaryotic protein expression by treating cells with 1mM IPTG for 3h at 37 °C. The recombinant proteins were purified using GST affinity column chromatography according to the manufacturer’s protocol (Invitrogen) and dialysed against the dialysing buffer (50mM sodium phosphate, 10% glycerol, pH 6.5). The empty pGEX-4T-2 vector was used to produce control GST protein in the same procedure. The enzyme-specific activity was determined with a fluorimetric Chitinase Assay Kit (Sigma CS1030) with 4-methylumbelliferyl β-d-N, N′, N′-triacetylchitotriose [4MU-GlcNAc₃] as substrate (Brotman et al., 2012 (link)) according to the manufacturer’s introduction. After incubation for 1h at 37 °C, fluorescence of the reaction mixture was measured by a fluorescence spectrophotometer (excitation at 360nm, emission at 450nm).