Purification of bacterial inulinase in aqueous two-phase systems

In this study, extracellular inulinase from Bacillus sp. 11/3 was partially purified and concentrated using aqueous two-phase system (ATPS). Two different phase forming salts and four types of polyethylene glycol (PEG) were used. Binodal curves and tie-length lines (TLLs) for eight ATPS were developed. For inulinase purification, concentrations of PEG and salt according to binodal curves (between 17 and 26%) were chosen. All ATPSs for inulinase purification were characterized. An ATPS consisted of 26% PEG1000 and 26% MgSO₄ was found to be the most suitable for inulinase purification. This ATPS has 28.47% TLL, 1.03 of volume ratio, purification factor of 4.65 fold and recovery yield of 66.17%. On the SDS-PAGE electrophoresis two protein bands with molecular weight of around 24 and 56 kDa were observed. The partially purified enzymes had optimal activity at pH 8.0 and 6.5, optimal temperature at 30 and 70°C and kinetic parameters K_m = 26.32 mmol and V_max = 526 mmol/min.