Production of β -1,3 glucanase from Penicillium oxalicum T3.3 for biocontrol against Colletotrichum gloeosporioides and Neoscytalidium dimidiatum

β-1,3 glucanase produced from fungi is an important hydrolytic enzymes which can been used as biocontrol agent against plant-pathogenic fungi. The objectives of this study are to determine the best substrates for β-1,3 glucanase production, to evaluate the cultural conditions which stimulate in vitro production of β-1,3 glucanase and to characterize the β-1,3 glucanase activity and then study the effect of crude β-1,3 glucanase towards fungal hypahe of C. gleosporioides and N. dimidiatum. β-1,3 glucanase production by Penicillium oxalicum T3.3 was investigated using shake flasks under various culture conditions such as different types of carbon and nitrogen sources, initial medium pH, agitation speed and different types of surfactants. The determination of best cultural conditions was carried out by varying and optimizing one variable at a time. The best cultural condition obtained from the shake flask experiment was used for β-1,3 glucanase production in a 2 L stirred tank fermenter where the effect of different impeller speed was investigated. Furthermore, the crude β-1,3 glucanase was characterized to study the enzyme stability. The role of this enzyme in antagonism action against Colletotrichum gloeosporioides and Neoscytalidium dimidiatum, a well-known fungal plant pathogens causing anthracnose disease in dragon fruit also was examined. In this study, seaweed Undaria pinnatifida was chosen as the best substrates for β- 1,3 glucanase production. The highest production of β-1,3 glucanase of 84.73 U/mL was obtained at substrate concentration of 1% (w/v), 0.3% (w/v) peptone and 0.2% (w/v) yeast extract as nitrogen source, initial medium pH 5, temperature at 30ᵒC, agitation speed at 200 rpm and with addition of sodium dodecyl sulfate as surfactant in shake flask condition. The β-1,3 glucanase activity was increased by 38.61% when optimized media and process parameters were used in shake flask culture. The best cultural condition obtained from shake flask was further used for production β- 1,3 glucanase in bioreactor. In bioreactor, the highest production of β-1,3 glucanase was obtained at 250 rpm. However, β-1,3 glucanase production in bioreactor was at par as compared with production in shake flask culture. The crude β-1,3 glucanase obtained was then concentrated by ammonium sulphate precipitation method. Concentrated enzyme was characterized and it was found that the optimum temperature and pH for the enzyme activity were 50ºC and pH 5, respectively. This enzyme can also retain 93.4% and 83.6% of its activity after incubation for 2 h and 4 h, respectively, at 50°C and pH 5. In addition, fungal plant pathogen inhibition assay showed that the crude β-1,3 glucanase produced by P. oxalicum T3.3 was able to hydrolyze the fungal hyphae of C. gloeosporioides and N. dimidiatum.

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