Identification of a novel lactose oxidase in NUK-21.

Lactobionic acid (-β-galactosyl-(1-4)-gluconic acid) (LBA) is a high-value lactose derivative, produced via oxidation of the reducing terminal of lactose. LBA can be produced by fermentation using certain microorganisms, although subsequent purification is challenging. Therefore, we have attempted to identify an enzyme for possible use in LBA production. Here, we purified a novel lactose oxidase (LOD) to homogeneity from a wheat bran culture of a soil-isolated fungal strain, NUK-21. Maximal activity was observed on the wheat bran solid culture after 3 days of NUK-21 growth, following release from cells at 0.66 unit·mL culture filtrate. This new sugar oxidase was composed of a single polypeptide chain with a molecular mass of 47.2 kDa and was found to contain 2.0 zinc ions per mole of enzyme but no flavin adenine dinucleotide or heme. This enzyme was stable in the pH range 5.5-9.0, with an optimal reaction pH of 7.5. Its optimal reaction temperature was 40 °C, and it was stable up to 50 °C for 1 h at pH 7.5. LOD oxidized disaccharides with reducing-end glucosyl residues linked by an α or β-1,4 glucosidic bond. The relative activity of LOD toward lactose, cellobiose and maltose was 100 : 83 : 4, respectively. To the best of our knowledge, this is the first report on the discovery of an LOD based on coenzyme moiety and enzyme substrate specificity.