Study of Laccase Adsorption Isotherms on Montmorillonite K10 and Zeolite Minerals

Laccases are potent enzymes that are capable of oxidizing various phenolic and non-phenolic compounds as well as resistant environmental pollutants. One of the most effective methods for improving their properties, such as increasing the stability of these enzymes and even increasing their activity, is the immobilization of laccases on different carriers. In the process of immobilization, the enzyme is bonded to a solid carrier which is insoluble in the reaction mixture. In this process, the movement of the enzyme in space is severely restricted, while its catalytic activity is still maintained. One of the carriers used to create recyclable biocatalyst systems is mineral. Minerals as inorganic carriers are inexpensive, abundant in nature, readily available, and also have high biocompatibility. The objective of the present study was to investigate the adsorption properties of Laccase enzyme from T. versicolor fungus on montmorillonite K10 and zeolite minerals using Langmuir, Freundlich, Temkin and Dubinin–Radushkevich isotherms. For this study, the pure laccase enzyme (> 10U mg-1), 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid (ABTS) substrate and montmorillonite K10 mineral (with a specific surface area of 220-270 m2/g and a cation exchange capacity (CEC) equal to 30 meq 100 g-1) were purchased from Sigma-Aldrich. Zeolite mineral was provided from a mine located in southeast Semnan province. Scanning electron microscopy (SEM) images of both minerals, CEC of zeolite with sodium acetate solution (pH=8.2) and zeolite surface area were determined. X-ray Diffraction (XRD) and X-ray Fluorescence (XRF) analyzes of zeolite mineral were also done. In order to immobilize laccase on the minerals, 200 mg of both minerals were activated by shaking with 0.5N HNO3 for 2 hours and a solution of 2% 3-aminopropyltriethoxylane in acetone. The activated minerals were treated by a 5% solution of glutaraldehyde in a 0.1M sodium acetate buffer (pH=5) and were shaken for 24 hours with 0.25-2.0 mg of the laccase dissolved in the buffer. Langmuir, Freundlich, Temkin and Dubinin–Radushkevich isotherms were determined. The experiment was carried out at a constant temperature of 20°C. The results were analyzed using the MSTATC software and the means of the data were compared using Duncan’s multiple range test. Based on the results, the zeolite type was clinoptilolite with a chemical formula of (Na,K,Ca)2.5Al3(Al,Si)2Si13O36.12H2O. Moreover, BET Surface Area, Langmuir Surface Area, t-Plot Micropore Area and t-Plot External Surface Area of zeolite were 40.2712, 645.4780, 3.5188 and 36.7524 m2/g, respectively. Laccase absorption on montmorillonite K10 showed the highest compliance first with the Dubinin–Radushkevich model (R2=0.97) and then with the Langmuir adsorption isotherm model (R2=0.96). Based on the D-R model, the theoretical monolayer sorption capacity (qm) and the constant of the sorption energy (ß) of montmorillonite K10 were 3 mg/g and 0.62 (×103 mol2/J2), respectively. According to the Langmuir isotherm, there was probably a homogeneous distribution of active sites on the montmorillonite K10 mineral surface. On the other hand, laccase adsorption on zeolite showed the best compliance with the Freundlich model (R2=0.87). Accordingly, sorption capacity (KF) of zeolite was 0.05 mg/g (L/mg)1/n. The amount of n parameter as an indicator of the favorability of sorption process was 1.49 demonstrating favorable absorption condition. The values of R2 obtained for Temkin isotherm model were, however, equal in both minerals (R2=0.62 for montmorillonite K10 and R2 = 0.61 for zeolite), and based on this model, the adsorption process was likely to be exothermic. According to the values of the equilibrium parameter (RL) of montmorillonite K10, the absorption was favorable. However, with increasing the initial concentration of laccase, the amount of RL approached zero indicating the laccase adsorption on the mineral is more favorable at higher initial concentrations of laccase. Based on % Removal parameter, the highest percentage of laccase adsorption on montmorillonite K10 and zeolite was related to concentrations of 250 and 125 mg/L, respectively, which showed a statistically significant difference with other concentrations. In general, laccase absorption on montmorillonite K10 showed the best fit with Dubinin–Radushkevich and Langmuir adsorption isotherm models. On the other hand, adsorption of laccase on zeolite mineral showed the best fit with Freundlich model. A higher degree of steric hindrance and conformational changes in the enzyme structure is likely to occur and subsequently, the catalytic efficiency of the enzyme complexes may decrease. Therefore, montmorillonite is more suited to be used as a carrier of laccase enzymes. However, complementary studies such as kinetic tests will help to make final decisions.