Isolation and Purification of Enzymes from Ligninolytic Complex of the Basidial Fungus Trametes pubescens (Schumach.) Pilat and Study of Their Properties

Abstract

A method for purification of enzymes from the ligninolityc complex of the basidiomycete Trametes pubescens (Schumach.) Pilat has been elaborated. Two homogeneous isoforms of laccases (laccase 1 and laccase 2) as well as a homogeneous preparation of lignin peroxidase were isolated. Basic biochemical parameters of the enzymes were determined, such as the molecular weights (67, 67, and 45 kD, respectively), isoelectric points (5.3, 5.1, and 4.2, respectively), as well as content and composition of the carbohydrate moiety of the laccases (N-acetylglucosamine, mannose, and xylose). The pH dependences and thermal stabilities of the laccases were investigated. The kinetic parameters of the enzymatic reactions catalyzed by the laccases were determined using different substrates, such as catechol, hydroquinone, 2,2 -azinobis-(3-ethylbenzthiazoline-6-sulfonate), and K4Fe(CN)6. The structure of the active sites of both laccases and the lignin peroxidase were studied by EPR, CD, and UV-VIS spectroscopy, as well as using fluorescence analysis. Our studies showed similarity of the spectral characteristics of the two laccases, whereas their kinetic properties were found to be different.

Simultaneous production of laccase and decolouration of the diazo dye Reactive Black 5 in a fixed-bed bioreactor

“…The main band likely corresponds to laccase, since no other ligninolytic enzymes were detected in the culture extract, with a molecular weight of about 63 kDa as visualised by Coomassie Brilliant Blue staining. This molecular weight is similar to those found for other laccases from fungal species belonging to the genus Trametes [22][23][24][25][26].…”

Laccase‐Mediated Oxidation of Totarol

“…However, the pH of the reaction mixture did affect the bioconversion of totarol, the best performances being achieved at pH 4.5-5 (a result consistent with other reports on this laccase). [10] The oxidation of totarol at various temperatures was also investigated, and it was shown to increase with temperature up to an optimum temperature 30 8C, beyond which the conversion decreased. Under these optimized reaction conditions a 62.6 % conversion of totarol was observed after 24 h, as demonstrated by HPLC.…”

Production of Trametes pubescens Laccase under Submerged and Semi-Solid Culture Conditions on Agro-Industrial Wastes

“…Two isoforms have been identified in T . pubescens strains; two monomeric (Lac1 and Lac2) enzymes of 67 kDa but with different pI (5.3 and 5.1) [50], and two laccase-activity fractions (Lap1 and Lap2) with different pI (> 3.0 and 2.6) from which Lap2 is a monomeric enzyme of 65 kDa [17]. In this study, we identify two laccase isoenzymes from T .…”