title: Recombinant expression and characterization of a L-amino acid oxidase from the fungus Rhizoctonia solani.
abstract: L-Amino acid oxidases (L-AAOs) catalyze the oxidative deamination of L-amino acids to the corresponding α-keto acids, ammonia, and hydrogen peroxide. L-AAOs are homodimeric enzymes with FAD as a non-covalently bound cofactor. They are of potential interest for biotechnological applications. However, heterologous expression has not succeeded in producing large quantities of active recombinant L-AAOs with a broad substrate spectrum so far. Here, we report the heterologous expression of an active L-AAO from the fungus Rhizoctonia solani in Escherichia coli as a fusion protein with maltose-binding protein (MBP) as a solubility tag. After purification, it was possible to remove the MBP-tag proteolytically without influencing the enzyme activity. MBP-rsLAAO1 and 9His-rsLAAO1 converted basic and large hydrophobic L-amino acids as well as methyl esters of these L-amino acids. The progress of the conversion of L-phenylalanine and L-leucine into the corresponding α-keto acids was determined by HPLC and1H-NMR analysis of reaction mixtures, respectively. Enzymatic activity was stimulated 50-100-fold by SDS treatment. Kmvalues ranging from 0.9-10 mM and vmaxvalues from 3 to 10 U mg-1were determined after SDS activation of 9His-rsLAAO1 for the best substrates. The enzyme displayed a broad pH optimum between pH 7.0 and 9.5. In summary, a successful overexpression of recombinant L-AAO in E. coli was established that results in a promising enzymatic activity and a broad substrate spectrum for biotechnological application.
authors.affiliation: Biochemie III, Fakultät für Chemie, Universität Bielefeld, Universitätsstrasse 25, 33615, Bielefeld, Germany.
authors.forename: Katharina
authors.initials: K
authors.lastname: Hahn
authors.affiliation: Biochemie III, Fakultät für Chemie, Universität Bielefeld, Universitätsstrasse 25, 33615, Bielefeld, Germany.
authors.forename: Katrin
authors.initials: K
authors.lastname: Neumeister
authors.affiliation: Anorganische Chemie und Strukturchemie, Fakultät für Chemie, Universität Bielefeld, Universitätsstrasse 25, 33615, Bielefeld, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Mix
authors.affiliation: Physikalische und Biophysikalische Chemie, Fakultät für Chemie, Universität Bielefeld, Universitätsstrasse 25, 33615, Bielefeld, Germany.
authors.forename: Tilman
authors.initials: T
authors.lastname: Kottke
authors.affiliation: Organische Chemie I, Fakultät für Chemie, Universität Bielefeld, Universitätsstrasse 25, 33615, Bielefeld, Germany.
authors.forename: Harald
authors.initials: H
authors.lastname: Gröger
authors.affiliation: Biochemie III, Fakultät für Chemie, Universität Bielefeld, Universitätsstrasse 25, 33615, Bielefeld, Germany. gabriele.mollard@uni-bielefeld.de.
authors.forename: Gabriele
authors.initials: G
authors.lastname: Fischer von Mollard
mesh_terms: D000595:Amino Acid Sequence; D001709:Biotechnology / Q000379:methods; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D007651:Keto Acids / Q000378:metabolism; D007700:Kinetics; D050830:L-Amino Acid Oxidase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D007930:Leucine / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D058619:Maltose-Binding Proteins / Q000235:genetics; D010649:Phenylalanine / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D012232:Rhizoctonia / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity
chemical_list: D007651:Keto Acids; D058619:Maltose-Binding Proteins; D011993:Recombinant Fusion Proteins; D010649:Phenylalanine; D050830:L-Amino Acid Oxidase; D007930:Leucine
keywords: E. coli; Heterologous expression; L-Amino acid oxidase; Maltose-binding protein; Solubility tag