Aim: Purification and characterization of pectin methylesterase produced by Aspergillus tubingensis in solid state fermentation.
Study Design: Pectin methylesterase enzyme produced by A. tubingensis was extracted from the fermented solid medium and purified using chromatographic techniques. The purified enzyme was characterized for physico-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 October, 2014 and August, 2015.
Methodology: The enzyme was extracted and purified using ammonium sulphate fractionation, ion exchange chromatography (IEC) using CM- cellulose and gel filtration chromatography (GFC) using Sephadex G-100. The molecular weight of the purified enzyme was determined using native polyacrylamide gel electrophoresis (Native PAGE) and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS–PAGE). The purified enzyme was characterized to determine the pH and temperature optima. Thermostability, pH stability and substrate kinetics were studied for purified pectin methylesterase.
Results: The acidic pectin methylesterase of Aspergillus tubingensis was purified to 20.3 fold with a 47.7% recovery through IEC on CM- cellulose and GFC using Sephadex G-100. The purified enzyme had a specific activity, 112.6 U/mg. The SDS-PAGE revealed that the enzyme was monomeric with a molecular weight of 45.7 kDa. The optimum pH and temperature were 4.6 and 50°C, respectively. This enzyme was stable over a wide pH range (3.0–8.0) and at relatively high temperature at 50°C for 1 h. The Km and Vmax values of pectin methylesterase towards citrus pectin were 33.3 mg/l and 251.2 µmol/ml/min, respectively. In addition, the enzyme activity increased by about 16% in the presence of 5 mM Mg2+.
Conclusion: The pectin methylesterase enzyme of A. tubingensis has been purified up to homogeneity and found to be monomeric on SDS-PAGE. Enzyme characterization revealed that purified enzyme worked optimally in acidic conditions and was stable at wider pH range.
Dr. Mukesh Kumar Patidar
Department of Biosciences, Maharaja Ranjit Singh College of Professional Sciences, Indore, India.
Dr. Anand Nighojkar
Department of Biosciences, Maharaja Ranjit Singh College of Professional Sciences, Indore 452001, India.
Dr. Sadhana Nighojkar
Department of Biotechnology, Mata Gujri College of Professional Studies, Indore 452001 India.
Dr. Anil Kumar
School of Biotechnology, Devi Ahilya University, Khandwa Road, Indore 452001, India.
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