Abstract
•Glucoamylase purified to homogeneity level on FPLC (Fast Protein Liquid Chromatography).•Molecular mass 88 kDa was determined on PAGE (Polyacrylamide gel electrophoresis).•Enzyme remained thermostable at 100 °C with zinc attachment.•Optimum pH 4.5 while temperature 55 °C was observed.•Thermodynamic parameters also support enzyme stabilization.
Extreme thermostabilization of glucoamylase (GA) from novel fungus Gymnoascella citrina was achieved first time through metals binding by using high temperature-unfolding- refolding technique. Crude GA (16.7 U mg−1 protein) produced by G. citrina was purified to homogeneity by using FPLC purification system. The molecular mass on SDS-PAGE was 88 kDa; optimum pH 4.5 and temperature was 55 °C. Kinetic constants: Km and Kcat for starch breakdown were 0.021 mg mL−1 and 21.42 s−1. Metals stripped GA (EDTA treated) had half life (t½) = 16.7 min at 62 °C. Surprisingly, refolded forms of GA bound with Zinc at temperatures 57–82 °C showed activation of the enzyme; while at 100 °C the GA had half-life (t½) of 43.8 h (2626 min). Calcium bound refolded form of GA at 80 °C also presented activation trend and doubling time (td) was 462 min. Thermodynamic parameters of metals stripped GA at 62 °C were: ΔG* = 102.44 kJ mol−1, ΔH* = 245.97 kJ mol−1; while ΔG* and ΔH* of Zinc bound GA at 100 °C was 130.32 and 44.39 kJ mol−1, respectively. We concluded thermostabilization conferred by Zinc was entropically driven and contributed to improve the functional energy (ΔG*), which stabilized the transition state.