|Universität Bielefeld > Department of Chemistry > Research Groups > Prof. Dr. Gabriele Fischer von Mollard > Biochemistry III|
Appl. Microbiol. Biotechnol. (2019) https://doi.org/10.1007/s00253-018-09609-7
Expression, characterization and site-specific covalent immobilization of an L-amino acid oxidase from the fungus Hebeloma cylindrosporum.
Bloess, S., Beuel, T., Krüger, T., Sewald, N., Dierks, T., Fischer von Mollard, G.
l-Amino acid oxidases (LAAOs) areflavoproteins, which use oxygen to deaminate l-amino acids and produce the corresponding α-keto acids, ammonia and hydrogen peroxide. Here we describe the heterologous expression of LAAO4 from the fungus Hebeloma cylindrosporum without signal sequence as fusion protein with a 6His tag in E. coli and its purification. 6His-hcLAAO4 could be activated by exposure to acidic pH, the detergent sodium dodecyl sulfate, or freezing. The enzyme converted 14 proteinogenic l-amino acids with l-glutamine, l-leucine, l-methionine, l-phenylalanine, l-tyrosine, and l-lysine being the best substrates. Methyl esters of these l-amino acids were also accepted. Even ethyl esters were converted but with lower activity. Km values were below 1 mM and vmax values between 19 U mg-1 and 39 U mg-1 for the best substrates with the acid-activated enzyme. The information for an N-terminal aldehyde tag was added to the coding sequence. Co-expressed formylglycine generating enzyme was used to convert a cysteine residue in the aldehyde tag to a Cα-formylglycine residue. The aldehyde tag did not change the properties of the enzyme. Purified Ald-6His-hcLAAO4 was covalently bound to a hexylamine resin via the Cα-formylglycine residue. The immobilized enzyme could be reused repeatedly to generate phenylpyruvate from l-phenylalanine with a total turnover number of 17,600 and was stable for over 40 days at 25 °C.