Article ID Journal Published Year Pages File Type
69739 Journal of Molecular Catalysis B: Enzymatic 2014 9 Pages PDF
Abstract

•The RIL activity at low pH value is controlled by an ionizable residue present in its active site.•DEPC derivatization of accessible His of RlL affect the kinetic behavior of RlL with pH and ionic strength.•Main role of His-457 in the control of the catalytic activity of laccase from Rigidoporus lignosus.

Laccases (benzendiol:oxygen oxidoreductases; EC 1.10.3.2) catalyze the oxidation of a broad range of substrates, such as polyphenols, dyes and pollutants, and thus these enzymes are widely applied in industrial, biotechnological and environmental fields. In order to improve their biotechnological applications, a deep knowledge of structural factors involved in controlling their activity, in various experimental conditions and on different substrates, is required. In the present study, a laccase from the mushroom Rigidoporus lignosus was kinetically characterized. In particular, the stability, the effects of pH, ionic strength and fluoride ion concentration on the kinetic parameters were investigated, using three di-hydroxy-benzene isomers (1,2-dihydroxy-benzene, 1,3-dihydroxy-benzene and 1,4-dihydroxy-benzene) as substrates. The catalytic constant values of the laccase showed a bell-shaped pH profile, with the same optimum pH and pKa values for all tested substrates. This behavior appears to be due to the presence of an ionizable residue in the enzyme active site. To identify this residue, the enzyme was derivatized with diethyl-pyrocarbonate to modify accessible histidine residues, which, according to structural data, are present in the active site of this enzyme. The kinetic behavior of the derivatized laccase was compared with that of the native enzyme and the derivatized residues were identified by mass spectrometry. Mass spectrometry and kinetic results suggest the main role of His-457 in the control of the catalytic activity of laccase from R. lignosus.

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Physical Sciences and Engineering Chemical Engineering Catalysis
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