Optimizing Conditions Of Chitinase Production From Paecilomyces Lilacinus And Characterization Of The Chitinase From Strain FZ-0289

Parasite nematodes are critical plants pathogens and have caused serious loss in economical plants. Paecilomyces lilacinus is a common egg parasite of plant parasitic nematodes, and has been considered as a hopeful biocontrol agent. Chitinases produced by nematocidal fungus play an important role in destroying the egg wall of nematodes. One strain with highest ability of producing chitinase was screened from 123 strains of Paecilomyces lilacinus through culturing on chitin agar plants and fermenting in chitin liquid medium. The effects of parasitism in vitro showed that over 95 percentages of eggs of Meloidogyne incognita had been parasitized by Paecilomyces lilacinus strain FZ-0289 after 7d and Ditylenchus destructor also had been parasitized by the strain FZ-0289 in some degree. Single-factor design and multiple factor designs were used for optimizing cultural conditions of chitinase production from Paecilomyces lilacinus strain FZ-0289. Single-factor design found that chitin powder (165μm) was the best media for inducing the chitinase from the strain FZ-0289, and adding peptone with chitin powder led to an earliest and highest chitinase production. Multiple factor designs showed that optimal cultural medium for chitinase production included 2g·L-1chitin powder (165μm), 2g·L^-1 peptone, 1 g·L^-1 KH₂PO₄, 0.5 g·L^-1 MgSO4, 0.2 g·L^-1 CaCl₂ and 0.2 g·L^-1 NaCl. The strain FZ-0289 was activated on potato sugar agar (PSA) plates for 10 days and then inoculated with 6×10⁹spores·L^-1 in 150mL optimal liquid medium. After fermented at 28 ℃with the revolving speed of 150 r·min^-1 in 500mL-conical-flask, the chitinase activity could reach 0.11 U·mL^-1 on the third day of incubation period. A Chitinase was isolated from the culture fluid of Paecilomyces lilacinus FZ-0289 and purified by chitin power affinity and DEAE sepharose ion-exchange chromatography. The molecular mass of the purified chitinase was 45.4KD. The two steps give overall purification of 28-fold for chitinase activity, and the yield of the purified chitinase was 3.4%, with special activity of 4.14U·mg-1. Chitinase was optimally actived at the pH of 4.0 and at the temperature from 35℃to 45℃. The enzyme was stable from pH4 to pH6, and the temperature up to 40℃. Among the metals and inhibitors that were tested, Hg2+intensively inhibited the chitinase activity even the concentration lowers to 0.05mmol·L-1. Ag+ and Cu2+ obviously inhibited the chitinase activity at 5mmol·L-1, while the Fe2+ would enhance the chitinase activity.