Affordable Access

Access to the full text

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
Type
Published Article
Journal
Applied Microbiology and Biotechnology
Publisher
Springer-Verlag
Publication Date
Jan 11, 2019
Volume
103
Issue
5
Pages
2229–2241
Identifiers
DOI: 10.1007/s00253-018-09609-7
Source
Springer Nature
Keywords
License
Yellow

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.

Report this publication

Statistics

Seen <100 times