title: Purification and properties of a heat-stable exoinulinase isoform from Aspergillus fumigatus.
abstract: An inducible extracellular exoinulinase (isoform II) was purified from the extracellular extract of Aspergillus fumigatus by ammonium sulphate precipitation, followed by successive chromatographies on DEAE-Sephacel, Octyl-Sepharose (HIC), Sephacryl S-200, affinity chromatography on ConA-CL Agarose and Sephacryl S-100 columns. The enzyme was purified 75-folds with 3.2% activity yield from the starting culture broth. The purified isoform II was a monomeric 62 kDa protein with a pI value of 4.5. The enzyme showed maximum activity at pH 6.0 and was stable over a pH range of 4.0-7.0, whereas the optimum temperature for enzyme activity was 60 degrees C. The inulinase isoform II showed exo-inulinolytic activity and retained 72% and 44% residual activity after 12 h at 60 degrees C and 70 degrees C, respectively. The inulin hydrolysis activity was completely abolished with 5 mM Hg2+ and Fe2+, whereas K+ and Cu2+ enhanced the inulinase activity. As compared to sucrose, stachyose and raffinose the purified enzyme had a lower Km (1.25 mM) and higher catalytic center activity (Kcat = 3.47 x 10(4) min(-1)) for inulin. As compared to exoinulinase isoform I of A. fumigatus, purified earlier, the isoform II is more thermostable and is a potential candidate for commercial production of fructose from inulin.
authors.affiliation: Department of Biotechnology, Guru Nanak Dev University, Amritsar 143 005, Punjab, India.
authors.forename: Prabhjot Kaur
authors.initials: PK
authors.lastname: Gill
authors.forename: Rajesh Kumari
authors.initials: RK
authors.lastname: Manhas
authors.forename: Prabhjeet
authors.initials: P
authors.lastname: Singh
mesh_terms: D001232:Aspergillus fumigatus / Q000201:enzymology* / Q000235:genetics; D055592:Biophysical Phenomena; D001703:Biophysics; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D002855:Chromatography, Thin Layer; D004586:Electrophoresis; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D006026:Glycoside Hydrolases / Q000032:analysis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007525:Isoelectric Focusing; D007527:Isoenzymes / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D008970:Molecular Weight; D013379:Substrate Specificity
chemical_list: D004791:Enzyme Inhibitors; D007527:Isoenzymes; D008670:Metals; D006026:Glycoside Hydrolases; C023086:inulinase