Purification, Characterization and Immobilization of an acid stable invertase from Lagenaria siceraria stem for the Production of Invert Syrup
Abhishek Mukherjee
Published: November 01, 2021.
Abstract
A 67 kDa acid invertase (specific activity = 780 U/mg protein), appreciably present in Lagenaria siceraria stem (2,600 + 200 U /100 g fresh wt.; 17,500 + 300 U/100 g of lyophilized stem powder) was purified by (NH4)2SO4 precipitation, ion exchange chromatography (DEAE Sephadex A50), size exclusion chromatography (Sephacryl S-300-HR) and HPLC. The enzyme was optimally active in the pH range of 4-5 (retaining 52 + 1 % of its activity at pH 3) and stable in the pH range of 3-7 (retaining 37 + 1% and 53 + 1% of its activity at pH 2 and 8 respectively), up to 55°C, with a Km of 5.84 mM (2 mg/ml) sucrose. Hg2+> Ag+>Cu2+>Pb2+>Cd2+ inhibited the enzyme activity. DTNB, iodoacetic acid, iodoacetamide, N-ethylmaleimide did not affect the activity suggesting the non-thiol nature of the enzyme. The enzyme hydrolyzed sucrose and raffinose, could slightly hydrolyze inulin, but was completely inactive upon maltose, levan, melezitose and trehalose. 8 U/ml of acid invertase almost completely hydrolyzed 10% (w/v) sucrose solution to invert syrup in 5 h at 50°C. Immobilization of the enzyme on oxidized bagasse (dialdehyde cellulose) increased its temperature optima (by 10°C) and thermo-stability (retaining 41 + 1% and 30 + 1 % of its activity at 70°C and 80°C respectively). Immobilized enzyme system efficiently produced invert syrup from sucrose, remaining 83 + 1 % and 72 + 1 % active after 15th and 25th cycles respectively.
Keywords: Lagenaria siceraria; Acid invertase; Sucrose; Invert syrup; Cellulose-dialdehyde