Activity of tyrosinase immobilized on hydroxyaluminum-montmorillonite complexes

Abstract

Montmorillonite was coated with varying levels of hydroxyaluminum, and the influence of degree of coatings on the adsorption, immobilization and activity of tyrosinase (Cu-containing polyphenoloxidase) was investigated in the absence of buffers. Tyrosinase was considered immobilized on the clay surfaces when after repeated washings with deionized distilled water no protein was detected in the supernatant. The immobilization of tyrosinase on Ca-montmorillonite (CaMte) was not evident at pH 6.5, whereas tyrosinase was immobilized on hydroxyaluminum-montmorillonite (Al(OH) x Mte) complexes. The amount of tyrosinase immobilized on the clay surfaces increased with increasing level of hydroxyaluminum coatings from 1 to 5 mmol Al/g clay. The specific activity of the immobilized tyrosinase ranged from 19.3 to 62.1% of the free enzyme, indicating some perturbation in the conformation of the protein molecules bound to the coated clay surfaces and the hindrance of some enzyme active sites after binding. Both free and immobilized tyrosinase had an optimum pH of 6.0 and an optimum temperature of 30°C. Immobilized tyrosinase was less sensitive to the change in temperature, indicating the role of hydroxyaluminum coatings in protecting tyrosinase molecules against denaturation. After 30 d of storage at 4 and 25°C, the free enzyme retained 63% and 33% of its initial activity, whereas the immobilized enzyme retained 91% and 49%, respectively, of its initial activity. This indicated the higher stability of tyrosinase after immobilization on the Al-coated clay. Infrared analysis of the tyrosinase-Al(OH) x Mte complex after reaction with catechol indicated the presence of humic-like polymers adsorbed on the clay surfaces.