Purification And Characterization Of Cutinase From Alternaria Longipes And Its Role In Pathogenesis

The tobacco brown spot, caused by Alternaria longipes, is one of the major tobacco diseases in our country. In recent years, this disease was widespread and caused economic impact. In order to further reveal the important role of cutinase in plant-microbe interactions and provide new theory basis for controlling the disease, the liquid fermentation conditions of the extracellular cutinase produced by A. longipes were optimized, the cutinase was purified and its role in pathogenesis was studied. The main results and conclusions are as follows:1. Alternaria longipes is the causal agent of tobacco brown spot, a world-wide tobacco disease. Based on Czapek medium supplemented with CaCO3 and apple cutin, the liquid fermentation conditions were optimized in order to acquire higher cutinase activity. Optimum culture time, temperature and pH were 8 d, 25℃and 5.5, respectively; and optimum carbon and nitrogen sources were dextrose and maltose instead of NaNO3. According to this fermentation and orthogonal test, the optimum composition of culture medium included dextrose 3%, casein 1%, Tween-80 0.3%. A 11-fold higher cutinase activity was obtained compared to the basal medium.2. After fermentation, the cutinase was purified through ammonium sulphate precipitation, DEAE (diethylaminoethyl) Sepharose, Sephacryl S-100 HR, and Phenyl-Sepharose 6 fast flow (high sub). A single band was observed by SDS-PAGE, corresponding to a molecular weight of 59.5 kDa. The yield and purification fold of the cutinase were 3.52% and 26.44 times, respectively.3. Using p-nitrophenyl butyrate as a substrate to test the optimum temperature and the optimum pH for the cutinase activity. The two parameters were found to be 35℃and pH 9.0, respectively; the enzyme activity was stable from 20 to 40℃and within pH 4.0～10.0; Na+, Cu2+ and Mn2+ could partially inhibit its activity. Partial inhibition was observed in the presence of Ca2+ at concentration of 2.5 and 5 mmol/L, while Mg2+and Zn2+ had no effect on the enzyme activity at concentration of 2.5 or 5 mmol/L. Unexpectedly, the enzymatic activity could be promoted effectively at higher concentration of the two ions, i.e. in the presence of 10 mmol/L MgSO4 and 10 mmol/L ZnSO4.4. Tobacco leaves were inoculated with conidial suspension of TBA001 compensated with the purified cutinase (cutinase absent as a control). According to the emergence of lesion formation and the severity of the disease, we could draw a conclusion that cutinase played an effective role in pathogenesis. By the pathogenicity test of the 9 strains, TBA003 was a strong one, TBA002 was a middle one and TBA001 was a weak one. Then use the three strains to inoculate the fementation medium and compare the difference of ability in producing cutinase. As a result, the pathogenicity ability of A. longipes was coincident with the ability of producing cutinase.