Lyoprotection and stabilization of laccase extract from Coriolus hirsutus, using selected additives

The development of stable lyophilized laccase, obtained from Coriolus hirsutus, using a wide range of temperature treatments and storage conditions, was investigated. Using selected lyoprotectants, including, dextran 6 kDa, sucrose and a mixture of sodium benzoate, potassium sorbate and sorbitol (BKSS) (1.5:1.0:98.5; w/w/w) resulted by 2.4, 1.4 and 1.8-fold increase in laccase activity after lyophilization as compared to the fresh enzyme, respectively, whereas the addition of mannitol preserved 98.2% of its activity. Using 2.5% (w/v) dextran (15-25 kDa) or mannitol appeared to be the most appropriate lyoprotectants for the laccase activity. The laccase stability of the lyophilized enzymatic extract was greatly enhanced with the presence of mannitol, with 96.2, 38.9 and 24.7% of residual activity after 4 weeks of storage at - 80, 4 and 25 °C, respectively. The inactivation constant (k_inactivation) value and the amount required to decrease 50% of the laccase activity (C_1/2) showed that Carbowax^® polyethylene glycol (PEG)-8000 was the most appropriate additive for laccase activity, followed by glycerol and CuSO₄. When the enzymatic extract was incubated at 50 °C in the presence of either CuSO₄, PEG-8000 or glycerol, the time required to decrease 50% of the laccase initial activity (t₅₀), were 52.9, 54.6, 50.2 h, respectively, as compared to that of the control trial of 38.9 h.