Cloning and Expression of Fructosyl-amine Oxidase from Marine Yeast Pichia Species N1-1

Abstract

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.

Active site analysis of fructosyl amine oxidase using homology modeling and site-directed mutagenesis

“…Based on amino acid sequence comparisons, eukaryotic FAODs appear to form a structurally related group distinct from bacterial FAODs (Ferri et al 2004). The Pichia sp.…”

“…The FAOD expression vector pTN1 was previously constructed by inserting the N1-1 FAOD gene into pTrc99A (Ferri et al 2004). Site-directed mutagenesis was carried out using the Quik-Change Ò mutagenesis kit (Stratagene) with the following oligonucleotides and their respective complementary oligonucleotides:…”

“…Employing the N1-1 FAOD, we have developed a number of biosensor systems capable of effectively measuring f-val (Tsugawa et al 2000Ogawa et al 2002). We have recently reported on the cloning of the FAOD gene from N1-1, and its recombinant production in Escherichia coli (Ferri et al 2004).…”

Crystal Structure of the Deglycating Enzyme Fructosamine Oxidase (Amadoriase II)

“…Finally, the presence of FAOX in the marine yeast Pichia sp. (37), a parasite of crabs and fishes, suggests that fructosamines can also be found in some marine ecosystems or in the animals that those marine yeasts parasitize.…”

Biomolecular Engineering of Biosensing Molecules —The Challenges in Creating Sensing Molecules for Glycated Protein Biosensing—

“…The entire structural gene of N1-1 FAOD was thus cloned and used for the recombinant expression of N1-1 FAOD in E. coli as the host microorganism. 39 Similarly, we have succeeded in the isolation, cloning, and recombinant expression of soil prokaryote-derived enzyme, FV1-1 FAOD.…”