Radiation mutagenesis and purification of xylanase produced by soil fungi

Amera Naser, Al-Qahtani Neveen, Saleh Geweely, Prof Fahed, Abdel-Rahman Al-Fasi
unpublished
The production, optimization, radiation mutagenesis and purification of extracellular xylanase enzyme produced by some fungal species isolated from three soil sites located in Jeddah governorate, Saudi Arabia were studied. Ten fungal species (Alternaria alternata, Aspergillus candidus, Aspergillus ochrochus, Aspergillus flavus, Botrytis cinerea, Fusarium roseum, Penicillium chrsogenum, Penicillium italicum, Penicillium canescens and Rhizopus microsporus) constituting 188 colonies were isolated.
more » ... nies were isolated. The highest significant value of extracellular xylanase enzyme (0.60 unit/ml) was shown in the culture filtrate of the Aspergillus candidus. Optimization of some nutritional and physical factors in order to intensify the production of A. candidus extracellular xylanase was carried out. The highest productivity of A. candidus extracellular xylanase (0.60 and 0.62 unit/ml) occurred on xylan and peptone as carbon and organic nitrogen sources, respectively. The optimum pH and temperature for extracellular xylanase activities were 9.0 and 55 °C, respectively. The effect of ultraviolet radiation on A. candidus xylanase indicated that the maximum stimulation of A. candidus xylanase (1.20 unit/ml) was attained on exposure to UV after 50 min. The radio-stimulated xylanase from the most xylanase producing organism (A. candidus) was purified to homogeneity by salting out with ammonium sulphate, dialysis and passage through chromatography resins (Sephadex G-100 column and Diethylaminoethyl sephadex column). The irradiated purified xylanase enzyme resulted in 1.301 fold of purification over the crude extract, exhibited a specific activity of 2.258 unit/mg with the recovery of 90.5 %. Test for the purity by Sodium dodecyl sulfate polyacrylamide gel electrophoresis technique (SDS-PAGE) resulted in a single protein band of the pure irradiated xylanase. Studying factors affecting the activity of the irradiated purified xylanase was carried out. The optimum reaction temperature and pH for maximum purified xylanase activities (1.82 and 2.10 unit/ml) were 60°C and 8, respectively. Thermostability of the irradiated purified A. candidus xylanase showed that it was stable (35% loss of activity) a 60°C after 60 min. exposure time.
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