Screening and Characterization of Fructosyl-Valine-Utilizing Marine Microorganisms

Abstract

We describe the isolation of microorganisms utilizing fructosyl-amine (Amadori compound) from the marine environment and of fructosyl-amine oxidase from a marine yeast. Using fructosyl-valine (Fru-Val), a model Amadori compound for glycated hemoglobin, we isolated 12 microbial strains that grow aerobically in a minimal medium supplemented with Fru-Val as the sole nitrogen source. Among these strains, a yeast strain identified as Pichia sp. N1-1, produced a Fru-Val-oxidizing enzyme. The enzyme was purified in its active form, a single-polypeptide water-soluble protein of 54 kDa by gel electrophoresis, producing H(2)O(2) with the oxidation of Fru-Val. By its substrate specificity, the enzyme was categorized as a novel fructosyl-amine oxidase. This is the first study on the isolation of microorganisms utilizing fructosyl-amine in the marine environment and of fructosyl-amine oxidase from budding yeast.

Enzymes used for the determination of HbA1C

“…and Pichia sp. [6][7][8][9][10]. Bacterial FAOXs showed strict specificity toward a-glycated amino acids and had sequence similarity with opine-catabolizing enzymes in Agrobacterium [11].…”

Development of a flow-injection analysis (FIA) enzyme sensor for fructosyl amine monitoring

“…N1-1 [15] and the construction of FAO-based fructosyl valine sensors using either an electron mediator or direct detection of hydrogen peroxide [16], a product of the FAO reaction. Because of the high sensitivity of the hydrogen peroxide electrode-based enzyme sensor, we have developed an enzymatic sensor system that can be used for consecutive multiple analyses.…”

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

“…By following the above screening process, we have succeeded in isolating a marine budding yeast strain, Pichia N1-1, producing FAOD. 20 The FAOD enzyme from Pichia N1-1 was then purified and the amino acid sequence of a proteolytic digestion was determined. A set of degenerated oligonucleotide primers were designed based on the partial amino acid sequence to carry out inverse PCR using as template a cDNA library prepared from mRNA isolated from Pichia N1-1.…”