Optimisation of the expression of a Trametes versicolor laccase gene in Pichia pastoris

  • J O'Callaghan
  • MM O'Brien
  • K McClean
  • ADW Dobson

DOI: 10.1038/sj.jim.7000268

Cite this article as:
O'Callaghan, J., O'Brien, M., McClean, K. et al. J Ind Microbiol Biotech (2002) 29: 55. doi:10.1038/sj.jim.7000268

A cDNA encoding a laccase enzyme was isolated from a Trametes versicolor cDNA library. The gene was subcloned into the Pichia pastoris expression vector pPIC3.5 and transformed into the P. pastoris strains KM71 and GS115. Laccase-secreting transformants were selected by their ability to oxidise the substrate ABTS. No difference in laccase activity was observed between culture supernatants from GS115 (proteolytic) and KM71 (nonproteolytic) strains. The presence of at least 200 μM copper was necessary for optimal laccase activity in the culture supernatants. During growth of P. pastoris on minimal medium the pH of the medium was reduced to <3.0. If alanine was added to the medium the pH reduction was not as pronounced and at alanine concentrations >0.6% w/v the pH was kept constant for >7 days. Cultures in which the pH was maintained by alanine metabolism produced higher levels of laccase activity than those grown in the absence of alanine. This study describes the development of a medium that allows convenient pH control of P. pastoris without the need for continuous neutralisation. Journal of Industrial Microbiology & Biotechnology (2002) 29, 55–59 doi:10.1038/sj.jim.7000268

Keywords: laccase; Pichia pastoris; heterologous expression 

Copyright information

© Society for Industrial Microbiology 2002

Authors and Affiliations

  • J O'Callaghan
    • 1
  • MM O'Brien
    • 1
  • K McClean
    • 1
  • ADW Dobson
    • 1
  1. 1.Department of Microbiology and The National Food Biotechnology Centre, University College Cork, Cork, IrelandIE

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