Abstract
Novel protease inhibitors were isolated from liquid-cultured mycelia of the white rot fungus Trametes versicolor. Two bands of antiproteinase K activity, TvPI-A and TvPI-B, were detected in the crude cell extract by native polyacrylamide gel electrophoresis (PAGE). Proteins corresponding to TvPI-A were purified by heat treatment, anion-exchange chromatography, and gel filtration. Sodium dodecyl sulfate (SDS)-PAGE demonstrated the presence of three proteins with molecular masses of 14.5, 16.6, and 20 kDa, respectively. T. versicolor protease inhibitors suppressed the activity of proteinase K and, to a smaller extent, of Carlsberg subtilisin, whereas trypsin and chymotrypsins were not inhibited. The inhibitors were acidic proteins and showed remarkable heat stability. To our knowledge, this is the first report about proteinase K inhibitors from fungi.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Literature Cited
Bode W, Huber R (1992) Natural protein proteinase inhibitors and their interaction with proteinases. Eur J Biochem 204:433–451
Bode W, Huber R (2000) Structural basis of the endoproteinase-protein inhibitor interaction. Biochim Biophys Acta 1477:241–252
Rawlings ND, Tolle DP, Barrett AJ (2004) Evolutionary families of peptidase inhibitors. Biochem J 378:705–716
Krowarsch D, Cierpicki T, Jelen F, Otlewski J (2003) Canonical protein inhibitors of serpine proteases. Cell Mol Life Sci 60:2427–2444
Gettins PGW (2002) Serpin structure, mechanism, and function. Chem Rev 102:4751–4803
Schu P, Suarez Rendueles P, Wolf DH (1991) The proteinase yscB inhibitor (P B12) gene of yeast and studies on the function of its protein product. Eur J Biochem 197:1–7
Dohmae N, Takio K, Tsumuraya Y, Hashimoto Y (1995) The complete amino acid sequences of two serine proteinase inhibitors from the fruiting bodies of a basidiomycete, Pleurotus ostreatus. Arch Biochem Biophys 316:498–506
Odani S, Tominaga S, Kondou S, et al. (1999) The inhibitory properties and primary structure of a novel serine proteinase inhibitor from the fruiting body of the basidiomycete, Lentinus edodes. Eur J Biochem 262:915–923
Tian M, Huitema E, da Cunha L, Torto-Alalibo T, Kamoun S (2004) A Kazal-like extracellular serine protease inhibitor from Phytophthora infestans targets the tomato pathogenesis-related protease P69B. J Biol Chem 279:26,370–26,377
Schu P, Wolf DH (1991) The proteinase ysc-A inhibitor, IA 3, gene; studies of cytoplasmic proteinase inhibitor deficiency on yeast physiology. FEBS Lett 283:78–84
Tian Y-P, Zhang K-C (2005) Purification and characterization of a novel proteinase A inhibitor from Ganoderma lucidum by submerged fermentation. Enzyme Microb Technol 36:357–361
Brzin J, Rogelj B, Popovič T, Štrukelj B, Ritonja A (2000) Clitocypin, a new type of cysteine proteinase inhibitor from fruit bodies of mushroom Clitocybe nebularis. J Biol Chem 275:20,104–20,109
Schwarze FWMR, Engels J, Mattheck C (2000) Fungal strategies of wood decay in trees. Springer-Verlag, Berlin
Shah V, Nerud F (2002) Lignin degrading system of white-rot fungi and its exploitation for dye decolorization. Can J Microbiol 48:857–870
Staszczak M, Nowak G, Grzywnowicz K, Leonowicz A (1996) Proteolytic activities in cultures of selected white-rot fungi. J Basic Microbiol 36:193–203
Rancaño G, Lorenzo M, Molares N, Rodriguez Cuoto S, Sanromán MA (2003) Production of laccase by Trametes versicolor in an airlift fermentor. Process Biochem 39:467–473
Fahreus G, Reinhammar B (1967) Large scale production and purification of laccase from cultures of the fungus Polyporus versicolor and properties of laccase A. Acta Chem Scand 21:2367–2378
Lee T-M, Lin Y-H (1995) Trypsin inhibitor and trypsin-like protease activity in air- or submergence-grown rice (Oryza sativa L.) coleoptiles. Plant Sci 106:43–54
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
Uriel J, Berges J (1968) Characterization of natural inhibitors of trypsin and chymotrypsin by electrophoresis in acrylamide-agarose gels. Nature 218:578–580
Shevchenko A, Wilm M, Vorm O, Mann M (1996) Mass spectrometric sequencing of proteins from silver-stained polyacrylamide gels. Electrophoresis 68:850–858
Schacterle GR, Pollack RL (1973) A simplified method for the quantitative assay of small amounts of protein in biologic material. Anal Biochem 51:654–655
Eguchi M, Itoh M, Chou L-Y, Nishino K (1993) Purification and characterization of a fungal protease specific protein inhibitor (FPI-F) in the silkworm haemolymph. Comp Biochem Physiol 104B:537–543
Jelínková P, Maňásková P, Tichá M, Jonáková V (2003) Proteinase inhibitors in aggregated forms of boar seminal plasma proteins. Int J Biol Macrom 32:99–107
Staszczak M, Zdunek E, Leonowicz A (2000) Studies on the role of proteases in the white-rot fungus Trametes versicolor: Effect of PMSF and chloroquine on ligninolytic enzymes activity. J Basic Microbiol 40:51–63
Grzywnowicz K (1997) Habilitation thesis. Maria Curie-Sklodowska University
Acknowledgment
This work was partly supported by the grant of the European Social Fund.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Zuchowski, J., Grzywnowicz, K. Partial Purification of Proteinase K Inhibitors from Liquid-Cultured Mycelia of the White Rot Basidiomycete Trametes versicolor . Curr Microbiol 53, 259–264 (2006). https://doi.org/10.1007/s00284-005-0386-2
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00284-005-0386-2