Cloning and Expression of Fructosyl-amino Acid Oxidase Gene fromCorynebacteriumsp. 2-4-1 inEscherichia coli

Abstract

The gene encoding the fructosyl-amino acid oxidase (fructosyl-alpha-L-amino acid: oxygen oxidoreductase (defructosylating); EC 1.5.3) of Corynebacterium sp. 2-4-1 was cloned and expressed in Escherichia coli. The gene consists of 1,116 nucleotides and encodes a protein of 372 amino acids with a predicted molecular mass of 39,042. The open reading frame was confirmed as the gene of the fructosyl-amino acid oxidase by comparison with the N-terminal amino acid sequence of the purified fructosyl-amino acid oxidase from Corynebacterium sp. 2-4-1. The sequence of the AMP-binding motif, GXGXXG, was found in the deduced N-terminal region. The amino acid sequence of the fructosyl-amino acid oxidase showed no similarity to that of fungal fructosyl-amino acid oxidases. In addition, substrate specificities of this fructosyl-amino acid oxidase were different from those of other fructosyl-amino acid oxidases. The fructosyl-amino acid oxidase of Corynebacterium sp. 2-4-1 is an enzyme that has unique substrate specificity and primary structure in comparison with fungal fructosyl-amino acid oxidases.

RecombinantAgrobacteriumAgaE-like Protein with Fructosyl Amino Acid Oxidase Activity

“…The activity for Fru-Gly was not high compared with that of FAOX-C (5.4 unit W mg). 15) We found that the AgaE-like protein acted on Fru-Val and Fru-Gly, but the main substrate for this protein in Agrobacterium cells is unclear. Genetic studies suggested that an Agrobacterium sp.…”

“…This enzyme was 54z identical in sequence to that of FAOX-C, which we characterized previously. 15) Table 3 summarizes the properties of AgaE-like protein (this study), FAOX-C, 13) and fungal FAOX-A.…”

“…13) Recently, we reported the cloning and characterization of the FAOX-C gene (DDBJ AB073548). 15) With the exception of the N-terminal AMP-binding motif, GXGXXG, FAOX-C has no sequence similarity with the eukaryotic FAOXs reported to date; the enzyme is an unknown kind of fructosyl amine:oxygen oxidoreductase. Sequence alignment showed that the Agrobacterium tumefaciens AgaE-like protein (GenBank AAD41368) has 54z similarity with FAOX-C.…”

“…FAOX activity was measured spectrophotometrically at 259 C with the peroxidase-coupled reaction, as described previously. 15) The standard reaction mixtures contained 0.1 M potassium phosphate, pH 8.0, 0.5 mM TOOS, 900 U W l peroxidase, 0.45 mM 4-aminoantipyrine, and 5.0 mM N-fructosyl glycine (Fru-Gly) in aˆnal volume of 3 ml. One unit of enzyme activity was deˆned as the amount of enzyme that produced 0.5 mmol of quinoneimine dye per minute at 259 C.…”

Determination of Fructosyl Amino Acids and Fructosyl Peptides in Protease-digested Blood Sample by a Flow-Injection System with an Enzyme Reactor

“…[14][15][16][17][18] It has also been reported [19][20][21] that a fructosyl-peptide oxidase (FPO) is active for the oxidative deglycation of fructosyl amino acids and fructosyl peptides.…”