Production and biochemical characterization of Pleurotus ostreatus NRC 620 laccase and evaluation of its efficacy in apple juice clarification

Abstract

The highest activity of the Pleurotus ostreatus NRC620 laccase enzyme occurred in the broth of mushroom growth after 25 days of incubation at 28 °C and static conditions. The optimum pH and temperature of the enzyme activity were revealed at pH 3.0, and 70 °C, respectively, and retained 68.33 and 59.61% of its activity after incubation at 40 and 50 °C for 2 h, respectively. The enzyme retained 100% activity after 2 h of incubation in citrate-phosphate buffer (pH 7.0). The addition of MgSO₄ and CuSO₄ with concentrations of 10 mM caused about 21% and 35% increase in enzyme activity, while NaCl, MnCl₂, KCl, and CaCl₂ inhibited the enzyme. The values of the kinetic parameters (K_m and V_max) of the Pleurotus ostreatus NRC 620 laccase were 1.99 mM and 16,217 µmol Min^-1 L^-1 respectively, using ABTS as a substrate. The apple juice enzyme-treated sample showed a significant reduction in pH, as well as viscosity, after enzyme treatment along with storage time. Apple juice treatment with laccase exhibited slight degradation in total phenolic; however, this observation was not found in antioxidant activity.

Keywords: Pleurotus ostreatus; Biochemical characterization; Juice clarification; Laccase.

Fig. 1

Effect of incubation time on the production of Pleurotus ostreatus NRC 620 laccase. Three (12-mm) disks of fungal mycelia were used to inoculate 50 mL sterile media and then incubated at 28 °C for different incubation periods.

Fig. 2

Effect of reaction temperature (a) and pH (b) on the activity of Pleurotus ostreatus NRC 620 laccase. A range of temperatures from 20 to 90 °C was employed through prior mixture temperature maintaining (5 min.) at required different temperatures before introducing the enzyme and initiating the reaction. The influence of pH values on laccase activity was assessed by using ABTS as a substrate in a buffer containing 0.1 M citrate–phosphate buffer, with pH values ranging from 2.5 to 7.0.

Fig. 3

Thermal (a) and pH (b) stability of Pleurotus ostreatus NRC 620 laccase. The thermal stability of the enzyme was assessed by incubating it for two hours at various temperatures of 40, 50, 60, and 70 °C in a 0.05 M sodium phosphate buffer (pH 7.0). For two hours, the enzyme’s pH stability was assessed by incubating the enzyme solution in 0.1 M citrate and Tris buffers (pH 3, 4, 6, and 7) at 40 °C. After incubation, the residual activity using ABTS as a substrate was computed.

Fig. 4

Metal ions effect on Pleurotus ostreatus NRC 620 laccase activity. Laccase was incubated for 10 min in a sodium phosphate buffer (0.05 M, pH 7.0) that included various metal ions at concentrations of 2.5 and 10 mM. After that, the reaction was initiated by adding the substrate (ABTS), and then the relative activity was assessed.

Fig. 5

Effect of ABTS concentration on Pleurotus ostreatus NRC 620 laccase activity and Lineweaver-Bulk plot of the reciprocal of initial velocities and ABTS concentrations. The oxidation of ABTS by laccase at several doses (0.025–3.0 mM) was measured at pH 4.5 to determine the kinetic parameters (V_max and K_m). The Michaelis–Menten equation’s kinetic constants were calculated using the Lineweaver–Burk plots of the reciprocal of reaction velocities and substrate concentrations. Using the Lineweaver–Burk plot, the kinetic constants were obtained using GraphPad Prism version 6.01.