Manganese peroxidases (MnPs) are interesting enzymes in protein engineering, aimed at maximizing industrial bioprocesses such as lignin degradation and biofuel production. cDNA of the secreted short-type of MnP from Phlebia radiata (Pr-MnP3) has been successfully engineered and amplified by polymerase chain reaction (PCR). Five mutant genes (E40H, E44H, E40H/E44H, D186H and D186N) of recombinant Phlebia radiata MnP3 (rPr-MnP3) were generated. The wild-type and the mutant genes were expressed in Escherichia coli (W3110 strain) and the resultant body proteins were lysed, purified and refolded into active enzymes. 6% - 7% recovery of pure and fully active rPr-MnP3 for wild-type and mutants were obtained and the availability of rPr-MnP3 enzymes will greatly facilitate its structure-function relationships studies. rPr-MnP3 mass was characterised using SDS-PAGE and MALDI-TOF mass spectrometry. Molecular weight of both the wild-type and mutant rPr-MnP3 enzymes was approximately 36 kDa. This describes the spectral characterization of the wild-type and mutant rPr-MnP3 enzymes with are very close similarities; substantially high spin haem enzymes. Therefore we report the engineering, cloning, expression, refolding/activation of MnP3 genes and preliminary characterization of the wild-type and mutant Phlebia radiata MnP3 enzymes.
Hatakka, A.I. and Uusi-Rauva, A.K. (1983) Degradation of it labeled poplar wood lignin by selected whiterot fungi. European Journal of Applied Microbiology Bio-technology, 17, 235-242. doi:10.1007/BF00510422
Lundell, T., Leonowicz, A., Rogalski, J. and Hatakka, A. (1990) Formation and action of lignin modifying enzymes in cultures of Phlebia radiata supplemented with veratric acid. Applied Environmental Microbiology, 56, 2623-2629.
Vares, T., Kalsi, M. and Hatakka, A. (1995) Lignin per-oxidases, manganese peroxidases, and other ligninolytic enzymes produced by Phlebia radiata during solid-state fermentation of wheat-straw. Applied Environmental Microbiology, 61, 3515-3520.
Hatakka, A., Lundell, T., Hofrichter, M. and Maijala, P. (2002) Manganese peroxidase and its role in the degradation of wood lignin. In: Mansfield, S.D. and Saddler J.N., Eds., Applications of Enzymes to Lignocellulosics, American Chemical Society Symposium Series, New York, 7-15.
Niemenmaa, O., Uusi-Rauva, A.K. and Hatakka, A. (2006) Wood stimulates the demethoxylation of [O14CH3]-labeled lignin model compounds by the white-rot fungi Phanerochaete chrysosporium and Phlebia radiata. Archives of Microbiology, 185, 307-315. doi:10.1007/s00203-006-0097-5
Lundell, T. (1993) Ligninolytic system of the white-rot fungus Phlebia radiata: Lignin model compound studies. Ph.D. Thesis, Department of Applied Chemistry and Mi- crobiology, University of Helsinki, Helsinki.
Karhunen, E., Kantelinen, A. and Niku-Paavola, M. (1990) Mn-dependent peroxidase from the lignin-degrading white rot fungus Phlebia radiata. Archives of Biochemistry and Biophysics, 279, 25-31. doi:10.1016/0003-9861(90)90458-B
Moilanen, A.M., Lundell, T., Vares, T. and Hatakka, A. (1996) Manganese and Malonate are individual regulators for the production of lignin and manganese isozymes and in the degradation of lignin by Phlebia radiata. Applied Microbiology and Biotechnology, 45, 792-799. doi:10.1007/s002530050764
Hilden, K.S., Makela, M.R., Hakala, T.K., Hatakka, A. and Lundell, T. (2006) Expression on wood, molecular cloning and characterization of three lignin peroxidase (LiP) encoding genes of the white rot fungus Phlebia radiata. Current Genetics, 49, 97-105. doi:10.1007/s00294-005-0045-y
Lundell, T. K., Makela, M.R. and Hilden, K. (2010) Lignin-modifying enzymes in filamentous basidiomy- cetes—ecological, functional and phylogenetic review. Journal of Basic Microbiology, 50, 5-20. doi:10.1002/jobm.200900338
Hofrichter, M., Ullrich, R., Pecyna, M.J., Liers, C. and Lundell, T. (2010) New and classic families of secreted fungal heme peroxidases. Applied Microbiology and Bio- technology, 87, 871-897. doi:10.1007/s00253-010-2633-0
Glenn, J.K. and Gold, M.H. (1985) Purification and Characterization of an extracellular Mn (II)-dependent per-oxidase from the lignin—degrading basidiomycete, Phanerochaete chrysosporium. Archives of Biochemistry and Biophysics, 242, 329-341. doi:10.1016/0003-9861(85)90217-6
Paszczynski, A. and Huynh, V.B. (1985) Crawford R Enzymatic activites of an extracellular, manganese- dependent peroxidase from Phanerochaete chrysosporium. FEMS Microbiology Leters, 29, 37-41. doi:10.1111/j.1574-6968.1985.tb00831.x
Hofrichter, M. (2002) Review: Lignin conversion by manganese peroxidase (MnP). Enzyme Microbial Technology, 30, 454-466. doi:10.1016/S0141-0229(01)00528-2
Kirk, T.K., Croan, S.D., Tien, M., Murtagh, K.E. and Farrell, R.L. (1986) Production of multiple ligninases by Phanerochaete chrysosporium: Effect of selected growth conditions and use of a mutant strain. Enzyme Microbial Technology, 8, 27-32. doi:10.1016/0141-0229(86)90006-2
Pease, E.A., Andrawis, A. and Tien, M. (1989) Manganese-dependent peroxidase from Phanerochaete chrysosporium. Primary structure deduced from complementary DNA sequence. Journal of Biological Chemistry, 264, 13531-13535.
Mayfield, M.B., Kishi, K., Alic, M. and Gold, M.H. (1994) Homologous expression of recombinant manganese peroxidase in Phanerochaete chrysosporium. Applied Environmental Microbiology, 60, 4303-4309.
Alic, M., Akileswaran, L. and Gold, M.H. (1997) Characterization of the gene encoding manganese peroxidase isozyme 3 from Phanerochaete chrysosporium. Biochimica et Biophysica Acta, 1338, 1-7. doi:10.1016/S0167-4838(96)00235-X
Martinez, D., et al. (2004) Genome sequence of the lignocellulose degrading fungus Phanerochaete chrysosporium strain RP78. Nature Biotechnology, 22, 695-700. doi:10.1038/nbt967
Hilden, K., Martinez, A.T., Hatakka, A. and Lundell, T. (2005) The two manganese peroxidases pr-Pr-MnP2 and Pr-MnP3 of Phlebia radiata, a lignin-degrading basidio-mycete, are phylogenetically and structurally divergent. Fungal Genetics and Biology, 42, 403-419. doi:10.1016/j.fgb.2005.01.008
Martinez, A.T. (2002) Molecular biology and structure-function of lignin-degrading heme peroxidases. Enzyme Microbial Technology, 30, 425-444. doi:10.1016/S0141-0229(01)00521-X
Forrester, I.T., Grabski, A.C., Mishra, C., Kelly, B.D., Striekl, W.N., Leatham, G.E. and Burgess, R.R. (1990) Characteristics and N-terminal amino acid sequence of a manganese peroxidase purified from Lentinula edodes culture grown on a commercial wood substrate. Applied Microbiology and Biotechnology, 33, 359-365. doi:10.1007/BF00164536
Glenn, J.K., Akileswaran, L. and Gold, M.H. (1986) Mn(II) oxidation is the principal function of extracellular Mn-peroxidase from Phanerochaete chrysosporium. Archives Biochemistry and Biophysics, 251, 688-696. doi:10.1016/0003-9861(86)90378-4
Sundaramoorthy, M., Terner, J. and Poulos, T.L. (1995) The crystal structure of chloroperoxidase: A heme per-oxidase-cytochrome P450 functional hybrid. Structure, 3, 1367-1377. doi:10.1016/S0969-2126(01)00274-X
Perez, J. and Jeffries, T.W. (1992) Roles of manganese and organic acid chelators in regulating lignin degradation and biosynthesis of Phanerochaete chrysosporium. Applied Environmental Microbiology, 58, 2402-2409.
Niku-Paavola, M.L., Karhunen, E., Salola, P. and Raunio, V. (1988) Ligninolytic enzymes of the white-rot fungus Phlebia radiata. Biochemistry Journal, 254, 877-883.
Lundell, T., Wever, R., Floris, R., Harvey, P., Hatakka, A., Brunow, G. and Schoemaker, H. (1993) Lignin per-oxidase L3 from Phlebia radiata. Pre-steady-state and steady-state studies with veratryl alcohol and a non-phenolic lignin model compound 1-(3, 4-dimethoxyphenyl)- 2-(2-methoxyphenoxy) propane-1, 3-diol. European Jour- nal of Biochemistry, 211, 391-402. doi:10.1111/j.1432-1033.1993.tb17562.x
Orth, A.B., Royse, D.J. and Tien, M. (1993) Ubiquity of lignin-degrading peroxidases among various wood-degrading fungi. Applied Environmental Microbiology, 59, 4017-4023.
Lundell, T and Hatakka, A. (1994) Participation of Mn (II) in the catalysis of laccase, manganese peroxidase and lignin peroxidase from Phlebia radiata. FEBS Letter, 348, 291-296. doi:10.1016/0014-5793(94)00627-X
Fishel, L.A., Villafranca, J.E., Maurao, J.M. and Kraut, J. (1987) Yeast cytochrome c peroxidase: mutagenesis and expression in Escherichia coli show tryptophan-51 is not the radical site in compound I. Biochemistry, 26, 351-360. doi:10.1021/bi00376a004
Smith, A.T., Santama, N., Decay, S., Edward, M., Bray, R.C., Thorneley, R.N.F. and Burke, J.F. (1990) Expression of a synthetic gene for horseradish peroxidase-C inn Escherichia coli and folding and activation of the recombinant protein with Ca2+ and haem. Journal of Biological Chemistry, 265, 13335-13343.
Doyle, W.A. and Smith, A.T. (1996) Expression of lignin peroxidase H8 in Escherichia coli: folding and activation of the recombinant enzyme with Ca2+ and haem. Bio-chemistry Journal, 315, 15-19.
Kim, S.J., Lee, J.A., Won, K., Kim, Y.H. and Song, B.K. (2009) Functional expression of Coprinus cinereus per-oxidase in Pichia pastoris. Process Biochemistry, 44, 731-735. doi:10.1016/j.procbio.2009.03.004
Pere-Boada, M., Doyle, W.A., Ruiz-Duenas, F.J., Martinez, A.T. and Smith, A.T. (2002) Expression of Pleurotus eryngii versatile peroxidase in Escherichia coli and optimization of in vitro folding. Enzyme Microbial Technology, 30, 518-524. doi:10.1016/S0141-0229(02)00008-X
Whitwam, R.E. and Tien, M. (1996) Heterologous expression and reconstitution of fungal Mn peroxidase. Archives of Biochemistry and Biophysics, 333, 439-446. doi:10.1006/abbi.1996.0413
Whitwam, R.E., Gazarian, I.G. and Tien, M. (1995) Expression of fungal manganese peroxidase in E.coli and refolding to yield active enzyme. Biochemical and Bio-physical Research Communications, 216, 1013-1017. doi:10.1006/bbrc.1995.2721
Conesa, A., Punt, P.J., and Van Den Hondel, C.A.M.J.J. (2002) Fungal peroxidases: Molecular aspects and applications. Journal Biotechnology, 93, 143-158. doi:10.1016/S0168-1656(01)00394-7
Punt, P.J. Van Biezen, N., Conesa, A., Albers, A., Mangnus, J., and Van Den Hondel, C.A.M.J.J. (2002) Filamentous fungi as cell factories for heterologous protein production. Trends Biotechnology, 20, 200-206. doi:10.1016/S0167-7799(02)01933-9
Stewart, P., Whitwam, R.E, Kersten, P.J., Cullen, D. and Tien, M. (1996) Efficient expression of a Phanerochaete chrysosporium manganese peroxidase gene in Aspergillus oryzae. Applied Environmental Microbiology, 62, 860- 864.
Kishi, K., Kusters-van Someren, M., Mayfield, M.B., Sun, J., Loehr, T.M. and Gold, M.H. (1996) Characterization of manganese (II) binding site mutants of manganese peroxidise. Biochemistry, 35, 8986-8994. doi:10.1021/bi960679c
Kishi, K., Hildebrand, D.P., Kusters-van Someren, M., Gettemy, J., Mauk, A.G. and Gold, M.H. (1997) Site-directed mutationat phenylalanine-190 of manganese per-oxidase: Effects on stability, function, and coordination. Biochemistry 36, 4268-4277. doi:10.1021/bi962627t
Whitwam, R., Brown, K.R., Musick, M., Natan, M.J. and Tien, M. (1997) Mutagenesis of the Mn2+-Binding site of manganese peroxidase affects oxidation of Mn2+ by both compound I and compound II. Biochemistry, 36, 9766- 9773. doi:10.1021/bi9708794
Andrawis, A., Pease, E. and Tien, M. (1990) Biotechnology in Pulp and Paper Manufacture. In: Kirk, T.K., Chang, H.M., Eds., Applications and Fundamental Investigations. Butterworth-Heinemann, Boston, 601.
Gelpke, M.D.S., Mayfield-Gambill, M., Cereghino, G.P. and Gold, M.H. (1999) Homologous Expression of Recombinant Lignin Peroxidase in Phanerochaete chrysosporium. Applied Environmental Microbiology, 65, 1670-1674.
Sambrook, J., Fritsch, E. and Maniatis, T. (1989) Molecular cloning: A Laboratory Manual. Corporation for Supportive Housing, New York.
Doyle, W.A., Blodig, W., Veitch, N.C., Piontex, K. and Smith, A.T. (1998) Two substrate interaction sites in lignin peroxidase revealed by site-directed mutagenesis. Bio-chemistry, 37, 15097-15105. doi:10.1021/bi981633h
Tien, M. and Kirk, K.I. (1988) Lignin peroxidase of Phanerocaete chrysosporium. Methods Enzymology, 161, 238-249. doi:10.1016/0076-6879(88)61025-1
Wariishi, H., Valli, K, Gold, M.H. (1992) Manganese peroxidase from the basidiomycete Phanerochaete chrysosporium. Kinetic mechanism and role of chelators. Journal Biology Chemistry, 267, 238-249.
Gold, M.H., Kuwahara, M., Chiu, A.A. and Glenn, J.K. (1984) Purification and characterization of an extracellular H2O2-requiring diarylpropane oxygenase from the white-rot basidiomycete Phanerocaete chrysosporium. Archives of Biochemistry and Biophysics, 234, 353-362. doi:10.1016/0003-9861(84)90280-7
Dunford, H.B. (1982) Peroxidases. Advances in Inorganic Biochemistry. Wiley-Vch, New York, 4, 41-68.
Dunford, H.B. (1991). Horseradish peroxidase: Structure and kinetic properties, Peroxidses in chemistry and biology, CRC Press, Boca Raton.
Dunford, H.B. (1999) Spectroscopy of horseradish per-oxidase. I: Optical, Resonance Raman, Magnetic circular dichroism, X-ray absorption, and Diffraction. Wiley-Vch, New York.
Wang, Y., Vazquez-Duhalt, R. and Pickard. M.A. (2002). Purification, characterization, and chemical modification of manganese peroxidase from Bjerkandera adusta UAMH 8258. Current Microbiology, 45, 77-87. doi:10.1007/s00284-001-0081-x
Johjima, T., Itoh, N., Kabuto, M., Tokimura, F., Nakagawa, T., Wariishi, H. and Tanaka, H. (1999) Direct interaction of lignin and lignin peroxidase from Phanerochaete chrysosporium. Proceedings of National Academy of Science, 96, 1989-1994. doi:10.1073/pnas.96.5.1989
Teske, J.G., Savenkova, M.I., Mauro, J.M., Erman, J.E. and Satterlee, J.D. (2000) Yeast cyrochrome c peroxidase expression in Escherichia coli and rapid isolation of various highly pure holoenzymes. Protein expression and purification. 19, 139-147. doi:10.1006/prep.2000.1220
Matsubara, M., Suzuki, J., Deguchi, T., Miura, M. and Kitaoka, Y. (1996) Characterization of manganese per-oxidases from the hyperlignolytic fungus IZU-154. Applied Environmental Microbioogy, 62, 4066-4072.
Schneegab, I., Hofrichter, M., Scheibner, K. and Fritsche, W. (1997) Purification of the main manganese peroxidase isozyme MnP2 from the white-rot fungus Nematoloma frowardii b19. Applied Microbiology and Biotechnology, 48, 602-605. doi:10.1007/s002530051102