Expression, characterization, and site-specific covalent immobilization of an L-amino acid oxidase from the fungus Hebeloma cylindrosporum
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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.
KeywordsL-amino acid oxidase Heterologous expression E. coli Aldehyde tag Formylglycine Formylglycine-generating enzyme Enzyme immobilization
We thank Marco Wißbrock and Annalena Lausch for excellent technical assistance. The pET14b-mtbFGE-His6 plasmid was kindly provided by David Rabuka (Redwood Bioscience, Emeryville, USA).
This study was funded by Universität Bielefeld as a strategic project.
Compliance with ethical standards
Conflict of interest
All authors declare that they have no conflict of interest.
This article does not contain any studies with human participants or animals performed by any of the authors.
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