Aqueous Two-Phase (PEG4000/Na₂SO₄) Extraction and Characterization of an Acid Invertase from Potato Tuber (Solanum tuberosum)

Abstract

Invertases are key metabolic enzymes that catalyze irreversible hydrolysis of sucrose into fructose and glucose. Plant invertases have essential roles in carbohydrate metabolism, plant development, and stress responses. To study their isolation and purification from potato, an attractive system useful for the separation of biological molecules, an aqueous two-phase system, was used. The influence of various system parameters such as type of phase-forming salts, polyethylene glycol (PEG) molecular mass, salt, and polymer concentration was investigated to obtain the highest recovery of enzyme. The PEG4000 (12.5%, w/w)/Na₂SO₄(15%, w/w) system was found to be ideal for partitioning invertase into the bottom salt-rich phase. The addition of 3% MnSO₄ (w/w) at pH 5.0 increased the purity by 5.11-fold with the recovered activity of 197%. The K_m and V_max on sucrose were 3.95 mM and 0.143 U mL⁻¹ min⁻¹, respectively. Our data confirmed that the PEG4000/Na₂SO₄ aqueous two-phase system combined with the presence of MnSO₄ offers a low-cost purification of invertase from readily available potato tuber in a single step. The biochemical characteristics of temperature and pH stability for potato invertase prepared from an ATPS make the enzyme a good candidate for its potential use in many research and industrial applications.

Keywords:

• aqueous two-phase system (ATPS)
• bottom phase
• characterization
• invertase
• purification
• protein recovery