|Universität Bielefeld > Department of Chemistry > Research Groups > Prof. Dr. Gabriele Fischer von Mollard > Biochemistry III|
Appl. Microbiol. Biotechnol. (2017) 101, 2853-2864
Recombinant expression and characterization of a L-amino acid oxidase from the fungus Rhizoctonia solani.
Hahn, K., Neumeister, K., Mix, A., Kottke, T. Gröger, H., Fischer von Mollard, G.
l-Amino acid oxidases (l-AAO) 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 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 E. 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 and 1H-NMR analysis of reaction mixtures, respectively. Enzymatic activity was stimulated 50 - 100 fold by SDS treatment. Km values ranging from 0.9 - 10 mM and vmax values from 3 - 10 U mg-1 were 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.