The doctoral dissertations of the former Helsinki University of Technology (TKK) and Aalto University Schools of Technology (CHEM, ELEC, ENG, SCI) published in electronic format are available in the electronic publications archive of Aalto University - Aaltodoc.
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Characterization and Heterologous Production of a Novel Laccase from Melanocarpus albomyces

Laura-Leena Kiiskinen

Dissertation for the degree of Doctor of Science in Technology to be presented with due permission of the Department of Chemical Technology for public examination and debate in Auditorium KE2 (Komppa Auditorium) at Helsinki University of Technology (Espoo, Finland) on the 28th of January, 2005, at 12 noon.

Overview in PDF format (ISBN 951-38-6430-8)   [851 KB]
VTT Publications 556, ISSN 1455-0849

Dissertation is also available in print (ISBN 951-38-6429-4)
Copyright © 2005 VTT Technical Research Centre of Finland
VTT Publications 556, ISSN 1235-0621
VTT-PUBS-556
TKK-DISS-1954

Abstract

Laccases are copper-containing enzymes that oxidize a variety of industrially relevant substrates. In order to fully exploit laccases in industrial processes, novel laccases with high stability and activity at elevated temperatures and pH values are needed. This work focused on identifying and characterizing novel fungal laccases having potential for the applications as well as on development of efficient production methods for laccases.

Laccase-producing fungi were screened from various environmental samples by plate tests, and a total of 26 positive fungal strains were isolated. Four novel fungal laccases were preliminarily characterized, and these enzymes were found to be rather typical basidiomycete laccases.

A novel laccase from the ascomycete Melanocarpus albomyces was purified and biochemically characterized. Interestingly, M. albomyces laccase showed good thermostability and activity at neutral to alkaline pH values. The crystal structure of M. albomyces laccase was resolved and the overall structure was shown to be similar to other blue copper oxidases. However, novel features were discovered at the active site and at the C-terminal end of the enzyme.

The gene encoding M. albomyces laccase was isolated, and the amino acid sequence of the enzyme was shown to be about 60-70% identical with laccases from other ascomycetes. M. albomyces laccase cDNA was expressed in Saccharomyces cerevisiae. Very low laccase production levels were significantly improved by replacing the native signal and propeptide sequences of laccase cDNA by the prepro-sequence of the S. cerevisiae α-factor gene.

M. albomyces laccase was also expressed in the filamentous fungus Trichoderma reesei. The laccase was expressed as a non-fused laccase and as a fusion protein with the T. reesei hydrophobin I. The unmodified recombinant M. albomyces laccase was produced in a laboratory-scale fermentor and the production level reached 920 mg l-1. Recombinant M. albomyces laccase was purified and biochemically characterized and it was shown to be very similar to the native laccase.

M. albomyces laccase was shown to bind to lignocellulose and purified cellulose. The adsorption parameters indicated that M. albomyces laccase binds to cellulose very efficiently but with a relatively low binding capacity. No binding was detected with other laccases, which suggests that binding to cellulose is not a common feature among laccases.

This thesis consists of an overview and of the following 6 publications:

  1. Kiiskinen, L.-L., Rättö, M. and Kruus, K. (2004) Screening for novel laccase-producing microbes. Journal of Applied Microbiology 97:640-646.
  2. Kiiskinen, L.-L., Viikari, L. and Kruus, K. (2002) Purification and characterisation of a novel laccase from the ascomycete Melanocarpus albomyces. Applied Microbiology and Biotechnology 59:198-204.
  3. Hakulinen, N., Kiiskinen, L.-L., Kruus, K., Saloheimo, M., Paananen, A., Koivula, A. and Rouvinen, J. (2002) Crystal structure of a laccase from Melanocarpus albomyces with an intact trinuclear copper site. Nature Structural Biology 9:601-605.
  4. Kiiskinen, L.-L. and Saloheimo, M. (2004) Molecular cloning and expression in Saccharomyces cerevisiae of a laccase gene from the ascomycete Melanocarpus albomyces. Applied and Environmental Microbiology 70:137-144.
  5. Kiiskinen, L.-L., Kruus, K., Bailey, M., Ylösmäki, E., Siika-aho, M. and Saloheimo, M. (2004) Expression of Melanocarpus albomyces laccase in Trichoderma reesei and characterization of the purified enzyme. Microbiology 150:3065-3074.
  6. Kiiskinen, L.-L., Palonen, H., Linder, M., Viikari, L. and Kruus, K. (2004) Laccase from Melanocarpus albomyces binds effectively to cellulose. FEBS Letters 576:251-255.

Keywords: enzyme, laccase, screening, characterization, Melanocarpus albomyces, heterologous expression, Saccharomyces cerevisiae, Trichoderma reesei, cellulose

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© 2005 Helsinki University of Technology

Last update 2011-05-26