Abstract

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

O. V. Nikitina¹, S. V. Shleev¹, E. S. Gorshina², T. V. Rusinova², V. A. Serezhenkov³, D. Sh. Burbaev³, L. V. Belovolova⁴, and A. I. Yaropolov^1*

¹Bach Institute of Biochemistry, Russian Academy of Sciences, Leninsky pr. 33, 119071 Moscow, Russia; fax: (7-095) 954-2732; E-mail: yaropolov@inbi.ras.ru

²Moscow State University of Environmental Engineering, ul. Staraya Basmannaya 21/4, 105006 Moscow, Russia; fax: (7-095) 267-0728; E-mail: gorshina@msuie.ru

³Semenov Institute of Chemical Physics, Russian Academy of Sciences, ul. Kosygina 4, 119977 Moscow, Russia; fax: (7-095) 939-7382; E-mail: mikoyan@center.ch.ph.ras.ru

⁴Prokhorov Institute of General Physics, Russian Academy of Sciences, ul. Vavilova 38, 119991 Moscow, Russia; E-mail: giv@smp.gphi.ru

^* To whom correspondence should be addressed.

Received October 14, 2004; Revision received December 30, 2004
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.
KEY WORDS: Trametes pubescens, laccase, lignin peroxidase, spectral properties

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

O. V. Nikitina1, S. V. Shleev1, E. S. Gorshina2, T. V. Rusinova2, V. A. Serezhenkov3, D. Sh. Burbaev3, L. V. Belovolova4, and A. I. Yaropolov1*

O. V. Nikitina¹, S. V. Shleev¹, E. S. Gorshina², T. V. Rusinova², V. A. Serezhenkov³, D. Sh. Burbaev³, L. V. Belovolova⁴, and A. I. Yaropolov^1*