Purification and extreme thermostabilization of glucoamylase by zinc produce of novel fungus Gymnoascella citrina

Abstract

Abstract 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.