Purification, thermodynamics and kinetic characterization of fungal endoinulinase for the production of fructooligosaccharides from inulin

Abstract

Three-step purification technique (isopropanol precipitation, ion-exchange and size-exclusion chromatography) was used for the purification of an endoinulinase from the culture broth of Aspergillus tritici BGPUP6. The molecular mass of purified endoinulinase was found to be 53.45 kDa and 53.70 kDa by denatured protein gel (SDS-PAGE) and size-exclusion (Sephadex G-100) chromatographic analysis, respectively. Higher Km (0.90 mM), Vmax (19.60 mM/min·mg), Kcat (1.3 × 10−3/min) and Vmax/Km ratio (21.77/min·mg) of purified endoinulinase for inulin than stachyose depicts its higher affinity towards inulin. Purified enzyme was found stable in a pH range 4.0–7.0 with an optimal pH 5.5. The optimal temperature of purified biocatalyst was 55 °C with thermostability in the range of 50–70 °C. D-value and Z-value for endoinulinase at 55 °C was found to be 100.08 h and 11.62 °C, respectively. Thermodynamics inactivation parameters (ΔG, ΔH and ΔS) of endoinulinase shows its wide range thermal stability. Endoinulinase activity was enhanced by CaCl2 and MnSO4, while CuSO4, CoCl2, AgNO3, CdCl2, NiCl2, ZnSO4, BaCl2, HgCl2 and EDTA inhibited the activity of enzyme. Purified endoinulinase was successfully used for the production of fructooligosaccharides from inulin.