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- Title
Expression, characterization, and site-specific covalent immobilization of an L-amino acid oxidase from the fungus Hebeloma cylindrosporum.
- Authors
Bloess, Svenja; Beuel, Tobias; Krüger, Tobias; Sewald, Norbert; Dierks, Thomas; Fischer von Mollard, Gabriele
- Abstract
L-Amino acid oxidases (LAAOs) are flavoproteins, 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 Escherichia 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 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.
- Subjects
COVALENT bonds; THERAPEUTIC immobilization; AMINO acids; OXIDASES; HEBELOMA
- Publication
Applied Microbiology & Biotechnology, 2019, Vol 103, Issue 5, p2229
- ISSN
0175-7598
- Publication type
Article
- DOI
10.1007/s00253-018-09609-7