title: Purification and characterization of an exoinulinase from Aspergillus fumigatus.
abstract: An extracellular exoinulinase was purified from the crude extract of Aspergillus fumigatus by ammonium sulfate precipitation, followed by successive chromatographies on DEAE-Sephacel, Sephacryl S-200, concanavalin A-linked amino-activated silica, and Sepharose 6B columns. The enzyme was purified 25-fold, and the specific activity of the purified enzyme was 171 IU/mg of protein. Gel filtration chromatography revealed a molecular weight of about 200 kDa, and native polyacrylamide gel electrophoresis (PAGE) showed an electrophoretic mobility corresponding to a molecular weight of about 176.5 kDa. Sodium dodecyl sulfate-PAGE analysis revealed three closely moving bands of about 66, 62.7, and 59.4 kDa, thus indicating the heterotrimeric nature of this enzyme. The purified enzyme appeared as a single band on isoelectric focusing, with a pI of about 8.8. The enzyme activity was maximum at pH 5.5 and was stable over a pH range of 4.0-9.5, and the optimum temperature for enzyme activity was 60 degrees C. The purified enzyme retained 35.9 and 25.8% activities after 4 h at 50 and 55 degrees C, respectively. The inulin hydrolysis activity was completely abolished with 1 mM Hg++, whereas EDTA inhibited about 63% activity. As compared to sucrose, stachyose, and raffinose, the purified enzyme had lower K(m) (0.25 mM) and higher Vmax (333.3 IU/mg) values for inulin.
authors.affiliation: Department of Biotechnogy, Guru Nanak Dev University, Amritsar-143005, Punjab, India.
authors.forename: Prabhjot Kaur
authors.initials: PK
authors.lastname: Gill
authors.forename: Rajesh Kumari
authors.initials: RK
authors.lastname: Manhas
authors.forename: Jatinder
authors.initials: J
authors.lastname: Singh
authors.forename: Prabhjeet
authors.initials: P
authors.lastname: Singh
mesh_terms: D001230:Aspergillus / Q000737:chemistry / Q000201:enzymology*; D004795:Enzyme Stability / Q000502:physiology; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008670:Metals / Q000737:chemistry; D008970:Molecular Weight; D013696:Temperature
chemical_list: D008670:Metals; D006026:Glycoside Hydrolases; C023086:inulinase