Discovery of a new tyrosinase-like enzyme family lacking a C-terminally processed domain: production and characterization of an Aspergillus oryzae catechol oxidase

A homology search against public fungal genome sequences was performed to discover novel secreted tyrosinases. The analyzed proteins could be divided in two groups with different lengths (350–400 and 400–600 residues), suggesting the presence of a new class of secreted enzymes lacking the C-terminal domain. Among them, a sequence from Aspergillus oryzae (408 aa, AoCO4) was selected for production and characterization. AoCO4 was expressed in Trichoderma reesei under the strong cbh1 promoter. Expression of AoCO4 in T. reesei resulted in high yields of extracellular enzyme, corresponding to 1.5 g L−1 production of the enzyme. AoCO4 was purified with a two-step purification procedure, consisting of cation and anion exchange chromatography. The N-terminal analysis of the protein revealed N-terminal processing taking place in the Kex2/furin-type protease cleavage site and removing the first 51 amino acids from the putative N-terminus. AoCO4 activity was tested on various substrates, and the highest activity was found on 4-tert-butylcatechol. Because no activity was detected on L-tyrosine and on l-dopa, AoCO4 was classified as a catechol oxidase. AoCO4 showed the highest activity within an acidic and neutral pH range, having an optimum at pH 5.6. AoCO4 showed good pH stability within a neutral and alkaline pH range and good thermostability up to 60°C. The UV–visible and circular dichroism spectroscopic analysis suggested that the folding of the protein was correct.

[1]  Kenji Matsuura,et al.  Reconstructing the early evolution of Fungi using a six-gene phylogeny , 2006, Nature.

[2]  B. Krebs,et al.  The crystal structure of catechol oxidase: new insight into the function of type-3 copper proteins. , 2002, Accounts of chemical research.

[3]  Y. Fujita,et al.  Molecular cloning and nucleotide sequence of the protyrosinase gene, melO, from Aspergillus oryzae and expression of the gene in yeast cells. , 1995, Biochimica et biophysica acta.

[4]  E. Record,et al.  Comparison of the characteristics of fungal and plant tyrosinases. , 2007, Journal of biotechnology.

[5]  P. Goloboff.  Analyzing Large Data Sets in Reasonable Times: Solutions for Composite Optima , 1999, Cladistics : the international journal of the Willi Hennig Society.

[6]  J. Buchert,et al.  Production and characterization of a secreted, C‐terminally processed tyrosinase from the filamentous fungus Trichoderma reesei , 2006, The FEBS journal.

[7]  Heinz Decker,et al.  The first crystal structure of tyrosinase: all questions answered? , 2006, Angewandte Chemie.

[8]  J. Buchert,et al.  Oxidation of peptides and proteins by Trichoderma reesei and Agaricus bisporus tyrosinases. , 2008, Journal of biotechnology.

[9]  Rolf Apweiler,et al.  InterProScan: protein domains identifier , 2005, Nucleic Acids Res..

[10]  H. Decker,et al.  Bacterial tyrosinases. , 2006, Systematic and applied microbiology.

[11]  B. Krebs,et al.  Biochemical and spectroscopic characterization of catechol oxidase from sweet potatoes (Ipomoea batatas) containing a type‐3 dicopper center 1 , 1998, FEBS letters.

[12]  R. Varón,et al.  A review on spectrophotometric methods for measuring the monophenolase and diphenolase activities of tyrosinase. , 2007, Journal of agricultural and food chemistry.

[13]  J. Sigoillot,et al.  Characterization of a new tyrosinase from Pycnoporus species with high potential for food technological applications , 2005, Journal of applied microbiology.

[14]  L. Viikari,et al.  Purification and characterisation of a novel laccase from the ascomycete Melanocarpus albomyces , 2002, Applied Microbiology and Biotechnology.

[15]  Jennifer K Inlow,et al.  Comparative analysis of polyphenol oxidase from plant and fungal species. , 2006, Journal of inorganic biochemistry.

[16]  B. Kwon,et al.  Isolation and sequence of a cDNA clone for human tyrosinase that maps at the mouse c-albino locus. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[17]  R. T. Biesebeke,et al.  Scientific Advances with Aspergillus Species that Are Used for Food and Biotech Applications. , 2008, Microbes and environments.

[18]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[19]  Rodrigo Lopez,et al.  Clustal W and Clustal X version 2.0 , 2007, Bioinform..

[20]  C. Kubicek,et al.  Role of Endoproteolytic Dibasic Proprotein Processing in Maturation of Secretory Proteins inTrichoderma reesei , 1998, Applied and Environmental Microbiology.

[21]  Jennifer K Inlow,et al.  Proteolytic processing of polyphenol oxidase from plants and fungi. , 2008, Journal of inorganic biochemistry.

[22]  John R. Whitaker,et al.  The biochemistry and control of enzymatic browning , 1995 .

[23]  S. Brunak,et al.  Locating proteins in the cell using TargetP, SignalP and related tools , 2007, Nature Protocols.

[24]  J. Buchert,et al.  Elucidating the mechanism of laccase and tyrosinase in wheat bread making. , 2007, Journal of agricultural and food chemistry.

[25]  K. Haghbeen,et al.  Activity and structural changes of mushroom tyrosinase induced by n-alkyl sulfates. , 2005, Colloids and surfaces. B, Biointerfaces.

[26]  F. Solano,et al.  Molecular anatomy of tyrosinase and its related proteins: beyond the histidine-bound metal catalytic center. , 2002, Pigment cell research.

[27]  Anton J. Enright,et al.  An efficient algorithm for large-scale detection of protein families. , 2002, Nucleic acids research.

[28]  A. Koivula,et al.  The relationship between thermal stability and pH optimum studied with wild-type and mutant Trichoderma reesei cellobiohydrolase Cel7A. , 2003, European journal of biochemistry.

[29]  A. Mayer.  Polyphenol Oxidases in Plants and Fungi: Going Places? A Review , 2007 .

[30]  Narmada Thanki,et al.  CDD: a conserved domain database for interactive domain family analysis , 2006, Nucleic Acids Res..

[31]  Tianhong Chen,et al.  Enzymatic grafting of peptides from casein hydrolysate to chitosan. Potential for value-added byproducts from food-processing wastes. , 2004, Journal of agricultural and food chemistry.

[32]  M Penttilä,et al.  A versatile transformation system for the cellulolytic filamentous fungus Trichoderma reesei. , 1987, Gene.

[33]  G. Freddi,et al.  Tyrosinase-catalyzed grafting of sericin peptides onto chitosan and production of protein-polysaccharide bioconjugates. , 2007, Journal of biotechnology.

[34]  G. Freddi,et al.  Tyrosinase-catalyzed modification of Bombyx mori silk fibroin: grafting of chitosan under heterogeneous reaction conditions. , 2006, Journal of biotechnology.

[35]  K. Lerch.  Primary structure of tyrosinase from Neurospora crassa. II. Complete amino acid sequence and chemical structure of a tripeptide containing an unusual thioether. , 1982, The Journal of biological chemistry.

[36]  James C. Sacchettini,et al.  Crystal structure of a plant catechol oxidase containing a dicopper center , 1998, Nature Structural Biology.

[37]  J. Sigoillot,et al.  Fungal tyrosinases: new prospects in molecular characteristics, bioengineering and biotechnological applications , 2006, Journal of applied microbiology.

[38]  O. Akita,et al.  Cloning of a novel tyrosinase-encoding gene (melB) from Aspergillus oryzae and its overexpression in solid-state culture (Rice Koji). , 2004, Journal of bioscience and bioengineering.

[39]  K. V. van Holde,et al.  Crystal structure of a functional unit from Octopus hemocyanin. , 1998, Journal of molecular biology.

[40]  B. Ranjbar,et al.  Activity, structural and stability changes of mushroom tyrosinase by sodium dodecyl sulfate , 2003 .

[41]  B. Ranjbar,et al.  Stability, structural and suicide inactivation changes of mushroom tyrosinase after acetylation by N-acetylimidazole. , 2004, International journal of biological macromolecules.

[42]  F. Tuczek,et al.  Purification and spectroscopic studies on catechol oxidases from Lycopus europaeus and Populus nigra: Evidence for a dinuclear copper center of type 3 and spectroscopic similarities to tyrosinase and hemocyanin , 1999, JBIC Journal of Biological Inorganic Chemistry.

[43]  M. Beltramini,et al.  Fluorescence properties of Neurospora tyrosinase. , 1982, The Biochemical journal.

[44]  V. Hearing,et al.  Enzymatic control of pigmentation in mammals , 1991, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[45]  J. Buchert,et al.  Tyrosinase-aided protein cross-linking: effects on gel formation of chicken breast myofibrils and texture and water-holding of chicken breast meat homogenate gels. , 2007, Journal of agricultural and food chemistry.

[46]  P Ferrara,et al.  In-gel digestion of proteins for internal sequence analysis after one- or two-dimensional gel electrophoresis. , 1992, Analytical biochemistry.

[47]  Y. Matoba,et al.  Crystallographic Evidence That the Dinuclear Copper Center of Tyrosinase Is Flexible during Catalysis* , 2006, Journal of Biological Chemistry.

[48]  D. Ussery,et al.  Comparison of protein coding gene contents of the fungal phyla Pezizomycotina and Saccharomycotina , 2007, BMC Genomics.

[49]  Korbinian Strimmer,et al.  APE: Analyses of Phylogenetics and Evolution in R language , 2004, Bioinform..