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 degrees C. Kinetic constants: K-m and K-cat for starch breakdown were 0.021 mg mL(-1) and 21.42 s(-1). Metals stripped GA (EDTA treated) had half life (t1/2) = 16.7 min at 62 degrees C. Surprisingly, refolded forms of GA bound with Zinc at temperatures 57-82 degrees C showed activation of the enzyme; while at 100 degrees C the GA had half-life (t1/2) of 43.8 h (2626 min). Calcium bound refolded form of GA at 80 degrees C also presented activation trend and doubling time (t(d)) was 462 min. Thermodynamic parameters of metals stripped GA at 62 degrees C were: Delta G* = 102.44 kJ mol(-1), Delta H* = 245.97 kJ mol(-1); while Delta G* and Delta H* of Zinc bound GA at 100 degrees 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 (Delta G*), which stabilized the transition state.