Kinetic Properties of Purified β-Mannanase from Penicillium italicum

Aim: The study aimed at purification and characterization of β-mannanase from Penicillium italicum.

Study Design: The first experiment, β-mannanase from Penicillium italicum was produced in basal medium supplemented with Locust Bean Gum (LBG). The second described the purification of crude β-mannanase, while the third experiment dealt with characterization and kinetic studies of purified β-mannanase from Penicillium italicum.

Place and Duration of Study: Microbiology Research Laboratory, Federal University of Technology, Akure Nigeria between July and August 2012.

Methodology: β-mannanase from Penicillium italicum was produced in basal medium supplemented with LBG. The enzyme was purified by ammonium sulphate precipitation, ion exchange chromatography (DEAE-Sephadex A-50) and gel filtration (Sephadex G-150). The purified enzyme was characterized to determine its optimal conditions by standard assay procedures. The kinetic parameters of the purified enzyme were determined by Lineweaver-Bulk plot.

Results: Fractionation of ammonium sulphate precipitated β-mannanase from Penicillium italicum on sephadex A-50 produced one major activity peak. Further fractionation of partially purified enzyme from ion exchange on Sephadex G-150 yielded one activity peak. A pH of 5.0 was optimum for purified enzyme activity and relatively stable between 40 to 100 min of incubation at this pH. The optimum temperature was 70ºC and 100% thermostable for 40 min after which a slight decline in activity was observed. The apparent Km for the hydrolysis of LBG from Lineweaver-Bulk plot was approximately 0.26 mg/mL, while the Vmax was 0.12 μmol/min/mL. The incubation of salts and organic compounds at 10 mM and 40 mM caused inhibition of enzyme activity. At 20 mM, enzyme activity was enhanced by FeSO4.7H2O, SDS and ZnSO4. 7H2O, while others caused inhibition of enzyme activity. The incubation of enzyme with CaCl2 and FeSO4.7H2O at 60 mM enhanced enzyme activity, while others caused inhibition.