Preparation of immobilized Trametes pubescens laccase on a cryogel-type polymeric carrier and application of the biocatalyst to apple juice phenolic compounds oxidation

摘要

A new biocatalyst was prepared by the immobilization of a Trametes pubescens laccase, into a wide-pore poly(vinyl alcohol) cryogel. The known enzyme was produced and purified by the previously described procedure. The resulted laccase (yield 40%) has an activity of 46.4 U mg−1 and 12.51 mg mL−1 protein content. The enzyme was subsequently immobilized in a functionalized macroporous cryogel beads by a covalent immobilization technique. The time dependence of the immobilization process and the enzyme loading of the carrier material (5.2 mg g−1 cryogel) were determined by measuring the decrease of protein amount in the enzyme solution. In conversion experiments, a higher stability of the immobilized biocatalyst compared to the free enzyme was evidenced. Steady-state kinetic characterization of four phenols (catechol, caffeic and chlorogenic acids, and catechin) has been performed with free and immobilized laccase, the catalytic parameters being determined and compared. The effect of both laccases (free and immobilized) on the phenol content of retailed apple juice samples, having the same initial composition, was also investigated by working in batch conversion. The variation in phenolic compound content has been compared with that of an untreated apple juice sample having initially the same content of phenolic compounds. A number of advantages resulted in using the immobilized laccase for the apple juice treatment (conservation to some extent of enzyme activity, higher content of phenols preserved, easy separation of the enzyme from the apple juice, therefore avoiding the possible unhealthy effects due to the remaining protein, etc.).