title: Characterization and application of a novel class II thermophilic peroxidase from Myceliophthora thermophila in biosynthesis of polycatechol.
abstract: A peroxidase from the thermophilic fungus Myceliophthora thermophila that belongs to ascomycete Class II based on PeroxiBase classification was functionally expressed in methylotrophic yeast Pichia pastoris. The putative peroxidase from the genomic DNA was successfully cloned in P. pastoris X-33 under the transcriptional control of the alcohol oxidase (AOX1) promoter. The heterologous production was greatly enhanced by the addition of hemin with a titer of 0.41 U mL(-1) peroxidase activity at the second day of incubation. The recombinant enzyme was purified to homogeneity (50 kDa) and characterized using a series of phenolic substrates that indicated similar characteristics with those of generic peroxidases. In addition, the enzyme was found thermostable, retaining its activity for temperatures up to 60 °C after eight hours incubation. Moreover, the enzyme displayed remarkable H2O2 stability, retaining more than 80% of its initial activity after 24h incubation in 5000-fold molar excess of H2O2. The ability of the peroxidase to polymerize catechol at high superoxide concentrations, together with its high thermostability and substrate specificity, indicate a potential commercial significance of the enzyme.
authors.affiliation: Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens, 5 Iroon Polytechniou Str., Zografou Campus, Athens 15780, Greece.
authors.forename: Anastasia
authors.initials: A
authors.lastname: Zerva
authors.affiliation: Biochemical and Chemical Process Engineering, Division of Sustainable Process Engineering, Department of Civil, Environmental and Natural Resources Engineering, Luleå University of Technology, SE-97187 Luleå, Sweden.
authors.forename: Paul
authors.initials: P
authors.lastname: Christakopoulos
authors.affiliation: Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens, 5 Iroon Polytechniou Str., Zografou Campus, Athens 15780, Greece. Electronic address: vtopakas@chemeng.ntua.gr.
authors.forename: Evangelos
authors.initials: E
authors.lastname: Topakas
mesh_terms: D002396:Catechols / Q000737:chemistry / Q000378:metabolism*; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D005800:Genes, Fungal; D006861:Hydrogen Peroxide / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007218:Industrial Microbiology; D007700:Kinetics; D009195:Peroxidase / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D010843:Pichia / Q000201:enzymology / Q000235:genetics; D059808:Polyphenols / Q000096:biosynthesis* / Q000737:chemistry; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D020037:Sordariales / Q000201:enzymology* / Q000235:genetics; D017550:Spectroscopy, Fourier Transform Infrared; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D002396:Catechols; D005656:Fungal Proteins; D059808:Polyphenols; D011994:Recombinant Proteins; D006861:Hydrogen Peroxide; D009195:Peroxidase
keywords: Class II peroxidase; Heterologous production; Myceliophthora thermophila; Pichia pastoris; Polycatechol; Thermostability