Identification Of The Chitinase-producing Bacterium L03 And Purification, Characterization Of The Chitinase

Chitinase could hydrolyze the cell wall of the fungi with pathogenicity to plant, so that could inhibite the growth of those fungi. It was clear that the microorganisms that secreted chitinase to extracellular could be used as the factor of biocontrol plant diseases. The L03 strain screened from soil by author could produce chitinase and the chitinase activity was 0.702U/mL. Identification of L03 strain was achieved by the sequence analysis of 16SrDNA from L03 and the comparison with the approximative species in morphology, physiology and biochemics. The results exhibited that the overall similarity values between L03 and Sinorhizobium sp. etc were 99% and the characterizations of L03 were similar to Sinorhizobium sp. On the basis of above experimentations, consequently, L03 strain was identified to be Sinorhizobium sp..The chitinase of L03 could be precipitated by 30% amonium sulfate and the crude protein could be obtained after dialysis to the precipitate with buffer. The chitinase protein was purificated by chromatography. Firstly througy Phenyl-Sepharose 6 Fast Flow hydrophobic chromatography, the crude protein displayed 7 absorption peaks at 280nm. Absorption peak 4 with higher content in the crude protein exhibited the activity of hydrolysis chitin. The peak 4 protein was collected and purificated by DEAE-Sephrose Fast Flow ion-exchange chromatography. The result displaied 7 peaks and peak 4-5 protein possessed chitinase activity. Through Sephadex G-75 gel filtration, the protein of peak 4-5 was separated into 3 protein peaks, which the peak 4-5-1 with activity was collected as the protein of L03 chitinase. The molecular weight was determined by SDS-PAGE to be about 41.3kDa.Characterizations of the chitinase were detected in this paper. The range of the optimum temperature for the enzyme activity was between 40 45℃and the optimum pH was of 6. The chitinase activity was stable at the treatment 1h with temperature 10℃ 40℃respectively and in the solution with pH 4-8, while the activity was distinct declining at 50℃persisting for 1h. The some metal ions could inhibit the chitinase activity when the enzyme solution amended with metal ions respectively, such as Ca²⁺,Zn²⁺,Cu²⁺,Fe³⁺, while others significantly increased on the enzyme activity, such as Mg²⁺,Ba²⁺.L03 strain chitinase possessed obvious degradation to the cell wall of many plant pathogens, such as Rhizoctonia solani,Pyricularia oryzae,Fusarium moniliforme,Rhizoctonia cerealis,Fusarium graminearum,Fusarium oxysporum f. sp. vasinfectum,Botrytis cinerea,Glomerella cingulata et al. It was observed that the outline of hyphal cells became illegibility, the colour of hypha became depigmentation, and even the some hypha represented abnormality or collapsed completely when the pathogens were treated with the chitinase solution.It were optimized that the culture conditions for L03 strain produced the chitinase on 5 factors in 6 level respectively by Uniform Design. The test data were analysed with DPS. The analysis results indicated that appropriate carbon nutrition was colloidal chitin and the concentration was 7%, nitrogen nutrition was yeast extract juice and the concentration was 0.2% (other nutrition substances in medium: 0.15% NaNO₃, 0.05% MgSO₄·7H₂O, 0.3% KH₂PO₄, 0.015% FeSO₄·7H₂O), the medium primary pH value was 7.0, the culture temperature was 28℃and the time of incubation L03 was 7d.