Cloning and expression of fructosyl-amine oxidase from marine yeast Pichia species N1-1.
The gene encoding the fructosyl-amine oxidase (FAOD) from the marine yeast Pichia sp. N1-1 was cloned and expressed in Escherichia coli. Partial amino acid sequence analysis of the Pichia sp. N1-1 FAOD allowed the design of oligonucleotide primers for the amplification of the gene by inverse polymerase chain reaction. The FAOD gene was found to be devoid of introns and to encode a 48-kDa protein composed of 429 amino acid residues. The FAD-binding consensus sequence GXGXXG: and the FAD covalent attachment-site cysteine residue have been identified within the predicted amino acid sequence. Comparisons with the amino acid sequences of other eukaryotic FAODs showed only 30% to 40% identities, establishing that the isolated Pichia N1-1 gene encodes a unique FAOD. Recombinant FAOD expression levels in E. coli reached 0.48 U/mg of soluble protein, which is considerably greater than native expression levels by inducing Pichia sp. N1-1 with fructosyl-valine (f-Val). The kinetic properties of the recombinant enzyme were almost indistinguishable from those of the native enzyme. We previously reported on the construction of a number of effective Pichia sp. N1-1 FAOD-based biosensors for measuring f-Val, a model compound for glycated hemoglobin. The further development of these biosensor systems can now greatly benefit from protein engineering and recombinant expression of the FAOD from Pichia N1-1.
Ferri
Stefano
S
Department of Biotechnology, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo, 184-8588, Japan.
Miura
Seiji
S
Sakaguchi
Akane
A
Ishimura
Fumimasa
F
Tsugawa
Wakako
W
Sode
Koji
K
D000594:Amino Acid Oxidoreductases / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D016000:Cluster Analysis; D017931:DNA Primers; D004926:Escherichia coli; D015870:Gene Expression; D008969:Molecular Sequence Data; D010802:Phylogeny; D010843:Pichia / Q000201:enzymology* / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D017385:Sequence Homology
D017931:DNA Primers; D011994:Recombinant Proteins; D000594:Amino Acid Oxidoreductases; C103012:amadoriase