Radiation mutagenesis and purification of xylanase produced by soil fungi

The production, optimization, radiation mutagenesis and purification of extracellular xylanase enzymeproduced by some fungal species isolated from three soil sites located in Jeddah governorate, SaudiArabia were studied. Ten fungal species (Alternaria alternata, Aspergillus candidus, Aspergillusochrochus, Aspergillus flavus, Botrytis cinerea, Fusarium roseum, Penicillium chrsogenum, Penicilliumitalicum, Penicillium canescens and Rhizopus microsporus) constituting 188 colonies were isolated. Thehighest significant value of extracellular xylanase enzyme (0.60 unit/ml) was shown in the culture filtrateof the Aspergillus candidus. Optimization of some nutritional and physical factors in order to intensifythe 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 andorganic nitrogen sources, respectively. The optimum pH and temperature for extracellular xylanaseactivities were 9.0 and 55 °C, respectively. The effect of ultraviolet radiation on A. candidus xylanaseindicated that the maximum stimulation of A. candidus xylanase (1.20 unit/ml) was attained on exposureto 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 passagethrough chromatography resins (Sephadex G-100 column and Diethylaminoethyl sephadex column). Theirradiated purified xylanase enzyme resulted in 1.301 fold of purification over the crude extract, exhibiteda specific activity of 2.258 unit/mg with the recovery of 90.5 %. Test for the purity by Sodium dodecylsulfate polyacrylamide gel electrophoresis technique (SDS-PAGE) resulted in a single protein band ofthe pure irradiated xylanase. Studying factors affecting the activity of the irradiated purified xylanasewas 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.