Expression, characterization, and site-specific covalent immobilization of an L-amino acid oxidase from the fungus Hebeloma cylindrosporum.

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. K