Identification of a novel lactose oxidase in Myrmecridium flexuosum NUK-21

Abstract

Lactobionic acid (O-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, Myrmecridium flexuosum 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 ^-1 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.

Keywords: Myrmecridium flexuosum; Zn cofactor; lactobionic acid; lactose oxidase.

Figure 1

SDS/PAGE analysis of LOD. The gel concentration was 10%. (A) Lane M, molecular mass markers; lane S, 20 μg of the major aggregations of proteins after Fractogel HW‐50 column chromatography with 0.2% SDS. (B) Lane S, 6 μg of purified enzyme.

Figure 2

Effects of pH on LOD activity and stability. (A) Enzyme activity was assayed in various buffers at the pH values indicated by oxygraph method, as described in the text. (B) Enzyme (15 μg·mL⁻¹) was incubated in various buffers at the pH values indicated. After incubation at 30 °C for 1 h, the residual activity was estimated at pH 7.8 using the peroxidase‐4AA method, as described in the text. The buffer systems (50 mm) used were acetate buffer (pH 4.5–5.5) (●), phosphate buffer (pH 5.5–8.0) (○), Tris–HCl buffer (pH 7.0–9.0) (▼) and carbonate buffer (pH 9.0–11.0) (▵).

Figure 3

Effects of temperature on LOD activity and stability. (A) Enzyme activity was assayed by the oxygraph method, as described in the text, at various temperatures in buffer A. (B) Enzyme (15 μg·mL⁻¹ in buffer A) was incubated at the temperature indicated for 1 h, and the remaining activity was estimated at 30 °C using the peroxidase‐4AA method, as described in the text.

Figure 4

TLC analysis of the product. Lane M, 50 μg lactose; lane S, 50 μg of complete conversion product from lactose.