Statistical optimization of medium components for chitinase production by Pseudomonas fluorescens strain HN1205: role of chitinase on egg hatching inhibition of root-knot nematode

In the present research, statistical Plackett-Burman design (PBD) and central composite design (CCD) were used to optimize the medium's components in order to enhance the chitinase activity of Pseudomonas fluorescens strain HN1205. Among the seven nutritional elements that were studied, crab shell powder, CaCl2 center dot 2H(2)O and yeast extract were proved using PBD to have significant effect on chitinase activity. An optimal medium was obtained by applying a three-factor central composite design, which consisted of crab shell powder (1.0 g/L), CaCl2 (0.5 g/L) and yeast extract (0.5 g/L), with the highest chitinase activity of 1.03 U/mL. This value was 2.87-fold higher than the activity obtained in the lowest productive medium in the design matrix. The chitinase produced by the HN1205 strain was partially purified with 80% ammonium sulphate and anion-exchange chromatography and assessed for inhibition of hatching of nematode eggs. The partially purified chitinase significantly reduced the hatch of Meloidogyne incognita eggs (8.1%), as compared to the effect of 10 mmol/L Tris-HCl buffer (pH 8.0) (49.4%) and boiled chitinase (54.7%). This study demonstrated the role of chitinolytic enzymes produced by P. fluorescens strain HN1205. The obtained optimal activity proved that the enzymes could be potential biological control agents.