Controlled Production of Fructose by an Exoinulinase from Aspergillus Ficuum

• Published in: Applied biochemistry and biotechnology Vol. 159; no. 1; pp. 65 - 77
• Main Authors: Mutanda, T, Wilhelmi, B, Whiteley, C. G
• Format: Journal Article
• Language: English
• Published: New York New York : Humana Press Inc 01.10.2009; Humana Press Inc; Springer; Springer Nature B.V
• Subjects: Ammonium; Aspergillus - enzymology; Aspergillus ficuum; Biochemistry; Biological and medical sciences; Biotechnology; Chemistry; Chemistry and Materials Science; chicory; Computer Simulation; Dialysis; enzymatic hydrolysis; Enzyme Activation; enzyme activity; Enzyme Stability; Enzymes; Fractionation; fructose; Fructose - chemical synthesis; Fundamental and applied biological sciences. Psychology; Glycoside Hydrolases - chemistry; Hydrolysis; Industrial production; inulin; Inulin - chemistry; inulinase; Models, Chemical; molecular weight; optimization; purification; response surface methodology; Statistical analysis; temperature; Exoinulinase; Response surface methodology; Fructose synthesis; Fructose; Production; Enzyme; Aspergillus ficuum; Fungi; Glycosylases; Statistical method; Glycosidases; Inulinase; Hydrolases; Response surface; Fungi Imperfecti
• ISSN: 0273-2289; 1559-0291
• DOI: 10.1007/s12010-008-8479-6

An exoinulinase has been isolated, purified and characterised from a commercially available broth of Aspergillus ficuum. The enzyme was purified 4.2-fold in a 21% yield with a specific activity of 12,300 U mg⁻¹(protein) after dialysis, ammonium sulphate fractionation and Sephacryl S-200 size exclusion and ion exchange chromatography. The molecular weight of this enzyme was estimated to be 63 kDa by SDS-PAGE. It exhibited a pH and temperature optima of 5.4 and 50 °C respectively and under such conditions the enzyme remained stable with 96% and 63.8% residual activity after incubation for 12 h and 72 h respectively. The respective K m and V max values were 4.75 mM and 833.3 μmol min⁻¹ ml⁻¹, respectively. Response surface methodological statistical analysis was evaluated for the maximal production of fructose from the hydrolysis of pure commercial chicory inulin. Incubation of the dialyzed crude exoinulinase (100 U/ml, 48 h, 50 °C, 150% inulin, pH 5.0) produced the highest amount of fructose (106.4 mg/ml) under static batch conditions. The purified exoinulinase was evaluated for fructose production and the highest amount (98 mg/ml) was produced after 12 h incubation at 50 °C, 150% inulin pH 5.0. The use of a crude exoinulinase preparation is economically desirable and the industrial production of fructose from inulin hydrolysis is biotechnologically feasible.