Purification and Characterization of Thermostable Alkaline Xylanase by Aspergillus terreus from Elephant Dung

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Aims: Purification and characterization of xylanase by Aspergillus terreus isolated from elephant dung.

Study Design: Xylanase enzyme produced by A. terreus was extracted from the fermented solid medium using solid state fermentation and purified using chromatographic techniques. The purified enzyme was characterized for physio-chemical and kinetic properties.

Place and Duration of Study: Experiments were performed at the School of Biotechnology, Devi Ahilya University, Indore, INDIA and Maharaja Ranjit Singh College of Professional Sciences, Indore, INDIA, between June, 2019 and September, 2021.

Methodology: The enzyme was extracted and purified using ammonium sulphate precipitation, ion exchange chromatography using DEAE cellulose and gel filtration chromatography (GFC) using Sephadex G-200. The molecular weight of the purified enzyme was determined using native poly acrylamide gel electrophoresis (Native PAGE) and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The purified enzyme was characterized to determine pH and temperature optima. Thermostability, pH stability and substrate kinetics were studied for purified xylanase. Effect of several metal ions and detergents were also studied.

Results: The alkali-thermo xylanase of Aspergillus terreus was purified to 14.6-fold with a 39.48% recovery through IEC on DEAE-cellulose and GFC using Sephadex G-200. The purified enzyme had a specific activity, 111.6 U/mg. The SDS-PAGE revealed that the enzyme was monomeric with a molecular weight of 87 kDa. The optimum pH and temperature were 8 and 60⁰C, respectively. The Km and Vmax values of the partially purified xylanase were found to be 0.016 mg/l and 102.64 U/mg protein, respectively with birch wood xylan as substrate.

Conclusion: The enzyme was active over a wide pH range of 6-11 and temperature range of 40⁰C to 80ºC, indicating its alkali tolerant and thermostable characteristics which is the requirement of different industries. Several ions namely Zn2+ enhanced xylanase activity by 187% followed by Ca2+ (129%), Mg2+ (123%), NH4+ (146%) and Fe3+ (118%) at 10 mM concentration. However, Cu2+ and Hg2+ completely inhibited xylanase activity. This research provides the basis for application of xylanase and improves its biotechnological potential in mainly in paper pulp industry, biofuel generation and in bread baking.