Characterization Of The Chitinase Produced By Coniothyrium Minitans And Its Effect On Sclerotinia Sclerotiorum

Coniothyrium minitans is an important mycoparasite and biocontrol agent of Sclerotinia sclerotiorum. In order to disclose the mechanisms underlining the parasitism of C. minitans upon S. sclerotioum, one of the exocellular enzymes, chitinase, produced by this mycoparasite was characterized in this study.Medium formulation for the production of chitinase by C. minitans was conducted by comparison of the effect of different carbon and nitrogen sources on the yield of this enzyme. The results showed that conventional potato broth amending with 0.5% (w/v) glucose and 0.1% (w/v) potassium nitrate was the best medium when the pH value was adjusted to 6.5. It also indicated that N-acetyl-glucosamine (the end product of the hydrolysis by chitinase ) could induce the production of chitisase more efficiently than chitin (the substrate for the chitinase reaction). When oxalic acid (the phytotoxin produced by 5. sclerotiorum) was added to the media at the concentrations lower than 3000 wg/mL, the chitinase production were enhanced and pH values of these cultures were increased to about 8.0.Using the medium, inoculum dosage of C. minitans and chitinase-secreting dynamics was rationalized. The results revealed that inoculation of 107 conidia of the fungus per 50 mL media and shaking-incubation (200 r/min) for 15d at 20C were the appropriate controllable conditions. Addition of Tween 80 and SDS did not show any effect on the chitinase production by C. minitans.The crude chitinase of C. minitans was prepared by saturated (NJ4)2SO4 precipitation and dialysis in a buffer and used for characterization of the properties of this enzyme. It was demonstrated that the optimum temperature was 50 C and the optimum pH value was 8.0. The enzymatic activity of was completely inhibited chitinase when the enzyme was treated at 60 C. Different metal ions were found to show different effects on the chitinase activity. The chitinase activity was enhanced by the ions including Mg2+, Fe2+, Ca2+, Ba2+ and Mn2+, whereas Zn2+ and Cu2+ could strongly inhibit the enzymaticreaction. Boric acid (100 mmol/L) and oxalic acid (<8mmol/L) were also found to improve the chitinolytic reaction.The chitinase was purified by ammonium sulfate fraction, DEAE-Sepharose FF chromatography and Sephacrys S-300HR chromatography. The enzyme fractions were isolated by the DEAE-Sepharose FF chromatography, but not by the Sephacrys S-300HR chromatography.The chitinase activity in the scleratia parasited by C. minitans was determined. However, no activity was detected. No inhibition of the mycelial growth of S. sclerotiorum by the chitinase preparations of various levels of purification were demonstrated.