PROPERTIES OF LACCASE IN HUMUS-ENZYME COMPLEXES

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Abstract
An enzymatic system that catalyzes the oxidation of a number of phenols and aromatic amines was extracted from soil with neutral sodium pyrophosphate solution and partially purified by Sephadex G-25 gel chromatography. Enzymatic activity was assayed polarographically by measuring the O2 consumption during the oxidation reaction. From HPLC [high performance liquid chromatography] gel chromatography and the properties of soil extract, it has been suggested that the enzyme associated with organic matter to form a humus-enzyme complex. The best substrates were quinol and p-phenylenediamine. Other diphenols and polyphenols were slowly oxidized and monophenols were not oxidized. On the basis of substrate specificity study, the enzyme is a laccase, probably of microbial origin. This soil enzyme had its optimum activity located on a narrow plateau between pH 7.0 and 7.5 and its partial inactivation began at temperatures above 65.degree. C. The enzyme was very stable, being denatured only at a temperature of 90.degree. C. For temperatures ranging from 30.degree. to 60.degree. C, the Arrhenius activation energy (Ea) and the average temperature coefficient (Q10) were 5.7 kcal mol-1 and 1.33, respectively. Dependence of activity on quinol, p-phenylenediamine, catechol and 4-methylcatechol concentrations indicated that the humus-laccase enzyme followed Michaelis-Menten kinetics. This conclusion was based on 2 methods of analysis (Lineweaver-Burk and Eadie-Hofstee plots) of data determined over a wide range of substrate concentrations. The kinetic parameters for the 4 substrates were calculated. An unusual feature of the enzyme was that the oxidation reaction of quinol showed a significant inhibition at a substrate concentration above 20 mM. The percentage inhibition up to 80 mM quinol concentration was also calculated. The influence of a humic microenvironment on enzyme activity was discussed. Moreover, the properties examined were critically compared with the reports of other workers.