Optimization Of Fermentation Condition And Properties Of Chitinase From Gliocladium Virens

Gliocladium virens has resistance to several pathogens,it is a biocontrol fungus withtremendous potential,and play an important role in the biocontrol of disease and insect offorest.Now the domestic reaserch of chitinase produced by Gliocladium virens and its trait ofbiocontrol are few, mostly, it is around the parasitics, compete and antimicrobial metabolitessuch as gliotoxin.With the Gliocladium virens F051 as the experimental material, the paper studied the bestcarbon source, nitrogen source and culture condition of Gliocladium virens F051 producedchitinase;separated and purified the chitinase,studied the property of chitinase and observedthe effect of the chitinase to three pathogens Rhizoctonia solani,Fusarium spp and Alternariaspp.The results as follows:With measuring reducing sugar, the fermented broth was mixed with colloidal chitin,thenadded 3.5—dinitrosalicylic acid reagent(DNS),the mixture produced brown and redproducts,this showed that Gliocladium virens F051 could produce chitinase.The fungus couldproduce chitinase only in the cuimre medium with the chitin as inducement, this proved thatthe chitinase was an derivational enzyme.With glucose,sucrose,maltose and lactose as the four carbon source, ammonium sulfate,ammonium nitrate, peptone and yeast powder as the nitrogen source;by mensuration,maltoseand yeast powder were the best carbon and nitrogen source,the activity of the chitinase wastiptop.Multiple factor design was used for optimizing cultural condition of chitinase productionfrom Gliocladium virens F051, we mensurated a number of factors on the production ofchitinase,including temperature,pH,bottled volume,inoculation volume, the volume of carbonand nitrogen.The result showed that the production of chitinase was effected most by thevolume of carbon,temperature was the next,again it was bottled volume; we got the best fermentation condition was cultural temperature:28, pH 7,100ml medium volume in 300mltriangle flask bulk, 1 ml inoculation volume, 1.5% maltose and 0.2% yeast powder.Separated and purified chitinase by 60% ammonium sulfate and High Q Fast Flowchromatography,then mensurated the molecular weight by SDS—PAGE.The result showedthat the molecular weight of the chitinase was 40KD.The study on the property of chitinase showed that the best reaction temperature was50℃between 20℃and 80℃,sith,activity of chitinase gradually descended with highertemperature;this chitinase preserved heat for lhour between 20℃and 40℃,the activity keptsteadily on the whole,activity descended if the temperature overran this range,the chitinaseactivity lost when the temperature was 80℃.The chitinase activity reached the most when pHamounted to 4;the chitinase was disposed for 1hour in the different buffer solution with pH at2～8,the activity of chitinase kept steadily between 3～8.The effect of metal irons onchitinase activity showed that Ba²⁺,Mn²⁺,Sn²⁺had stimulative function to the chitinase fromGliocladium virens F051,thereinto, Sn²⁺ was the best; however,Na⁺,K⁺,Mg²⁺,Ca²⁺,Fe²⁺ hadsuppressive function to the chitinase activity, Fe²⁺ restrained the chitinase activityentirely.The effect of different concentration of organic solvents on chitinase showed that 5%and 20% acetone,5% glycol and n_propyl alcohol had stimulative function to thechitinase,else organic solvents restrained the chitinase activity.The effect of crude chitinase on three pathogens showed that it could degrade the cell-wall of thepathogens.The crude chitinase could make Fusarium spp and Alternaria spp's cell-wall thin andrupture,cell-wall and bioplasm separate,the bioplasm of Rhizoctonia solani distribute asymmetry andbecome vacuole,the cell-wall thin and rupture also. The experiment didn't discover that the crudechitinase can affect the spore's bourgeon of Fusarium spp and Alternaria spp.