Purification and properties of a heat-stable exoinulinase isoform from Aspergillus fumigatus.
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.
Gill
Prabhjot Kaur
PK
Department of Biotechnology, Guru Nanak Dev University, Amritsar 143 005, Punjab, India.
Manhas
Rajesh Kumari
RK
Singh
Prabhjeet
P
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
D004791:Enzyme Inhibitors; D007527:Isoenzymes; D008670:Metals; D006026:Glycoside Hydrolases; C023086:inulinase