Studies on Acetone Powder and Purified Rhus Laccase Immobilized on Zirconium Chloride for Oxidation of Phenols

Rhus laccase was isolated and purified from acetone powder obtained from the exudates of Chinese lacquer trees (Rhus vernicifera) from the Jianshi region, Hubei province of China. There are two blue bands appearing on CM-sephadex C-50 chromatography column, and each band corresponding to Rhus laccase 1 and 2, the former being the major constituent, and each had an average molecular weight of approximately 110？kDa. The purified and crude Rhus laccases were immobilized on zirconium chloride in ammonium chloride solution, and the kinetic properties of free and immobilized Rhus laccase, such as activity, molecular weight, optimum pH, and thermostability, were examined. In addition, the behaviors on catalytic oxidation of phenols also were conducted. 1. Introduction Rhus laccase (EC.1.10.3.2) is a copper-containing glycoprotein occurring in the exudates of lacquer trees. Yoshida [1] first discovered the enzyme in 1883. Since then, many studies of the enzyme have been conducted. However, the results obtained so far in different laboratories frequently show considerable discrepancies. For example, the molecular weight reported varies from 100 to 141？kDa [2–4], and the properties of coppers differ considerably depending on the origin of the laccase preparations [5, 6]. Previously, when the Rhus laccase from Japanese lacquer trees was used to oxidize urushiol, the formation of semiquinone radicals, C–C or C–O coupling products, and dibenzofuran compounds were detected [7]. The enzyme laccase, whether obtained from a lacquer tree or fungus, is active in the oxidation of monophenolic compounds such as eugenol and isoeugenol [8]. The laccase-catalyzed oxidation of O-phenylenediamine [9], coniferyl alcohol [10], catechol [11], phenylpropanoid [12], and lignocatechol [13] were also demonstrated. Studies of the effects of proteins and polysaccharides in the activities of Rhus laccase showed that most proteins and polysaccharides, except laccase proteins, are not only incapable of catalyzing the oxidation of urushiol but can inhibit the activity of laccase to varying extents [14]. Recently, we immobilized Rhus laccase from acetone powder obtained from the exudates of lacquer trees grown in the Maoba region, Hubei province of China, on water-soluble chitosan and chitosan microspheres, and their properties were compared with transitional metal (Fe3+)-immobilized laccase by chelation [15]. The results showed that, compared with the free Rhus laccase, immobilized Rhus laccase displayed a lower specific activity but has a similar substrate affinity with improved stability