Bilirubin oxidase from Magnaporthe oryzae: an attractive new enzyme for biotechnological applications

Abstract

A novel bilirubin oxidase (BOD), from the rice blast fungus Magnaporthe oryzae, has been identified and isolated. The 64-kDa protein containing four coppers was successfully overexpressed in Pichia pastoris and purified to homogeneity in one step. Protein yield is more than 100 mg for 2 L culture, twice that of Myrothecium verrucaria. The k(cat)/K(m) ratio for conjugated bilirubin (1,513 mM⁻¹ s⁻¹) is higher than that obtained for the BOD from M. verrucaria expressed in native fungus (980 mM⁻¹ s⁻¹), with the lowest K(m) measured for any BOD highly desirable for detection of bilirubin in medical samples. In addition, this protein exhibits a half-life for deactivation >300 min at 37 °C, high stability at pH 7, and high tolerance towards urea, making it an ideal candidate for the elaboration of biofuel cells, powering implantable medical devices. Finally, this new BOD is efficient in decolorizing textile dyes such as Remazol brilliant Blue R, making it useful for environmentally friendly industrial applications.

Fig. 1

Amino acid sequence alignment of potential copper coordination sites of different BOD proteins: CotA from Bacillus subtilis (accession P07788), BOD from Myrothecium verrucaria (accession Q12737), CotA from Bacillus pumilus (accession A8FAG9), BOD from Trachyderma tsunodae (accession O61263), and BOD from Pleurotus ostreatus (accession Q9UVY4). The numbers 1, 2, and 3 correspond to the coordination sites for the types 1, 2, and 3 coppers. The amino acids presumed to be involved in the copper binding are represented in red

Fig. 2

Blue Coomassie-stained SDS-PAGE gel (12%) of purified recombinant BOD from Magnaporthe oryzae

Fig. 3

UV–visible spectrum of purified BOD from Magnaporthe oryzae (0.5955 mg/ml)

Fig. 4

Dependence of the enzyme activity on the pH for the purified BOD from Magnaporthe oryzae. The activity was measured at 37 °C using 1 mM ABTS (diamonds), 22 μM SGZ (crosses), 25 μM conjugated bilirubin (circles), and 30 μM unconjugated bilirubin (triangles) in 0.1-M phosphate–citrate buffer for pH between 3 and 7.2 and Tris- H₂SO₄ for pH above 7.2

Fig. 5

Dependence of the enzyme activity on the temperature for the purified BOD from Magnaporthe oryzae. The activity was measured in a 0.1-M phosphate–citrate buffer, pH 4, in presence of 1 mM ABTS

Fig. 6

Thermostability of the purified BOD from Magnaporthe oryzae incubated either at 37 °C or 60 °C in a 0.1-M citrate–phosphate buffer, pH 7. The enzyme activity was measured at 37 °C in a 0.1-M citrate–phosphate buffer, pH 3.8, in presence of 50-μM conjugated bilirubin

Fig. 7

Dependence of the enzyme stability on the pH for the purified BOD from Magnaporthe oryzae. The residual enzyme activity was measured at 37 °C in a 0.1-M phosphate–citrate buffer, pH 4, in presence of 1 mM ABTS

Fig. 8

Dependence of the enzyme activity on the urea concentration for the purified BOD from Magnaporthe oryzae at 25 °C and 37 °C in presence of 1 mM ABTS or 50 μM conjugated bilirubin. The enzyme activity was measured in a 0.1-M phosphate–citrate buffer, pH 4, for ABTS or pH 3.8 for conjugated bilirubin

Fig. 9

Dependence of the enzyme activity on the NaCl concentration for the purified BOD from Magnaporthe oryzae in presence of either 0.1 mM SGZ in a Mcllvaine’s citrate–phosphate 0.1-M buffer, pH 7, or with 75 μM conjugated bilirubin in a Mcllvaine’s citrate–phosphate 0.1-M buffer, pH 3.8

Fig. 10

Decolorization of 80 μg ml⁻¹ Remazol brilliant blue R by the purified BOD from Magnaporthe oryzae in presence or absence of 10 μM of ABTS in a Mcllvaine’s citrate–phosphate 0.1-M buffer, pH 7