Bacillus pumilus bilirubin oxidase and applications thereof

Abstract

The present invention relates to a novel Bacillus pumilus bilirubin oxidase, to the method for preparing same and also to the use thereof in particular for assaying bilirubin and for using enzymatic biofuel cells.

Claims

1. A BOD electrode comprising a conductive material coated with a deposit comprising at least one bilirubin oxidase (BOD), wherein said BOD has a percentage identity of at least 97% with respect to the BOD of Bacillus pumilus of SEQ ID No. 2, and it is bound to four copper atoms.

2. The BOD electrode of claim 1 wherein said BOD is the BOD of Bacillus pumilus of SEQ ID No. 2.

3. The BOD electrode of claim 1 wherein said conductive material is selected in the group consisting of platinium, copper, silver, aluminium, gold, steel or carbon.

4. The BOD electrode of claim 1 wherein said deposit comprising at least one purified BOD also comprises a redox polymer.

5. The BOD electrode of claim 1 wherein said electrode is coated with a membrane which prevents the detachment of said BOD from said electrode.

6. Method for measuring the bilirubin concentration in solution in a liquid sample, comprising the following steps: a) measuring the absorbance at max=440 nm of said liquid sample before enzymatic reaction; b) introducing into said liquid sample the BOD electrode according to claim 1; c) measuring the absorbance at max=440 nm of said liquid sample after enzymatic reaction; d) calculating the difference in absorbances measured in steps a) and c) and comparing with differences in absorbances measured for standard solutions having a known bilirubin content; and e) determining the bilirubin concentration of said liquid sample.

7. Method for degrading the bilirubin present in a sample comprising introducing into said liquid sample the BOD electrode according to claim 1.

8. Method for oxidizing dyeing of keratin fibers comprising contacting into said keratin fibers the BOD electrode according to claim 1.

9. Method for treating wood pulp comprising contacting said wood pulp with the BOD electrode according to claim 1.

10. Method for discoloring dyes used in industrial media comprising contacting said industrial media with the BOD electrode according to claim 1.

11. Bilirubin biosensor, characterized in that it is constituted of an electrode according to claim 1.

12. Oxygen sensor, characterized in that it is constituted of an electrode according to claim 1.

13. Enzymatic biofuel cell comprising an anode on which an enzyme catalyzing an oxidation reaction is immobilized and an electrode according to claim 1 as cathode.

Description

FIGURES

(1) FIG. 1A represents schematically the operating principle for an enzymatic biofuel cell; FIG. 1B represents a glucose-based enzymatic biofuel cell.

(2) FIG. 2 represents the plasmid map of the pFD1 vector.

(3) FIG. 3 is a graph illustrating the specific activity, in U/mg, of the Bacillus pumilus BOD as a function of the ABTS concentration at 37 C.

(4) FIG. 4 is a graphic representation of the Michaelis-Menten equation (k.sub.ss in s.sup.1 as a function of the unconjugated bilirubin concentration) for the Bacillus pumilus BOD at 37 C.

(5) FIG. 5 represents the catalytic activity for oxidation of conjugated bilirubin by the Bacillus pumilus BOD at 37 C. in a 50 mM citrate/phosphate buffer, pH 4.8.

(6) FIG. 6 represents the catalytic activity for oxidation of syringaldazine (SGZ) by the Bacillus pumilus BOD at 37 C. in a 50 mM citrate/phosphate buffer, pH 6.2.

(7) FIG. 7 represents the catalytic activity for oxidation of DMP by the Bacillus pumilus BOD at 37 C. in a 50 mM citrate/phosphate buffer, pH 6.8.

(8) FIG. 8 represents the relative activity of the Bacillus pumilus BOD with respect to various substrates as a function of the pH.

(9) FIGS. 9A and 9B are graphs representing the stability as a function of pH of the Bacillus pumilus BOD on ABTS oxidation at 4 C.

(10) FIG. 10 is a histogram representing ABTS oxidation as relative activity by the Bacillus pumilus BOD as a function of temperature.

(11) FIGS. 11A and 11B represent graphically the stability (expressed as specific activity and as relative activity on ABTS oxidation) of the Bacillus pumilus BOD as a function of enzyme incubation time at 80 C.

(12) FIGS. 12A and 12B represent graphically the activity (expressed as specific activity and as relative activity on ABTS oxidation) of the Bacillus pumilus BOD as a function of urea concentration at 25 C. or 37 C. in a 100 mM citrate/phosphate buffer, pH 3.

(13) FIG. 13 represents the relative activity of the oxidation of SGZ by the Bacillus pumilus BOD as a function of NaCl concentration.

(14) FIG. 14 represents the discoloration of RBBR at 80 mg.l.sup.1 by the Bacillus pumilus BOD at 37 C. in a 50 mM potassium phosphate buffer, pH 6, in the presence or absence of 10 M ABTS.

EXAMPLE

1. Materials

1.1 Escherichia coli Bacterial Strains

(15) DH.sub.5: supE44, lacU169, (80 lacZDM15), hsdR17, recA1, endA1, gyrA96, thi-1, relA1 (Hanahan, 1983).

(16) This strain is used to amplify plasmids during the steps for constructing the protein expression vectors.

(17) BL.sub.21 Star: F-ompT hsdSB(rB-, mB-) gal dcm rne131 (DE3) (Invitrogen).

(18) This strain is used to produce the Bacillus pumilus BOD in Erlenmeyer flasks.

(19) This strain is then transformed with the pFD1 plasmid which contains the DNA sequence encoding the Bacillus pumilus BOD under the control of the T7 promoter in the pET21a vector.

1.2 Vector

(20) pFD1: pET21a plasmid containing the nucleic acid sequence SEQ ID No. 1 encoding the Bacillus pumilus BOD cloned in-frame with the 6His tag in the C-terminal position.

(21) The plasmid map of the pFD1 vector is represented in FIG. 2.

1.3 Culture Medium

(22) LB Rich Medium:

(23) 10 g/l tryptone

(24) 5 g/l yeast extract

(25) 5 g/l NaCl

(26) Distilled H.sub.2O qs 1 L

(27) pH not adjusted, autoclaved for 50 min at 1 bar.

2. Genetic Engineering Techniques

2.1 Transformation of Supercompetent Bacteria

(28) Supercompetent DH.sub.5 bacteria are prepared using the SEM method (Simple and Efficient Method) according to the protocol described by Inoue et al. (Inoue et al. 1990, Gene 96:23-28).

2.2 DNA Preparation

(29) A plasmid DNA purification kit (Quiagen) is used for the DNA preparations in small and large amounts.

2.3 Double-Stranded DNA Sequencing

(30) The double-stranded DNA is sequenced. The sequencing reactions are carried out with the BigDye Terminator v1.1 or v3.1 sequencing kit. The reagent contains the 4 ddNTPs with various fluorescent labels (BigDye Terminators), the AmpliTaq DNA polymerase, and all the other components necessary for the reaction. The extension products should be purified before being passed through an ABI 3130xl sequencer, in order to remove the unincorporated labels, the salts and the other contaminants.

2.4 Construction of the BOD Expression Vector

(31) The PCR is carried out with the Phusion HF DNA polymerase on the genomic DNA of the Bacillus pumilus bacterium, strain SAFR-032. The two oligodeoxyribonucleotides, complementary to the 3 and 5 ends of the DNA sequence of the gene encoding the Bacillus pumilus BOD (SEQ ID No. 3 and 4) will be used as primers for the DNA synthesis.

(32) The amplified product and also the pET21a plasmid are then treated with the two restriction enzymes BamH1 and Xho1, the recognition sequences of which have been introduced into the sense oligonucleotide for BamH1 and the antisense oligonucleotide for Xho1, respectively denoted SEQ ID No. 3 and 4. The digestion products are gel-purified with the Nucleospin kit (NucleoSpin Extract II, Clontech Laboratories, Inc.) and the BOD gene is then ligated into the plasmid by coincubation with T4 DNA ligase at 37 C. overnight. The newly formed plasmids are then selected and amplified by transformation of DH5 bacteria on a plate containing ampicillin.

(33) TABLE-US-00002 TABLEII Listofprimersused Primername Sequence SEQIDNo. B.pumilus_S_BamH1 CATGGATCCATGAACCTA 3 GAAAAATTTGTTGACGAG B.pumilus_AS_Xho1 TACCTCGAGAATAATATC 4 CATCGGCCTCATCATGTC

3. Production, Purification and Characterization of the Bacillus Pumilus Bilirubin Oxidase Enzyme

3.1 Production of Wild-Type BOD Enzymes

(34) The BOD enzyme is produced in the E. coli BL.sub.21 star strain by the pET21a recombinant plasmid carrying the sequence encoding wild-type BOD. A 50 ml preculture of LB medium supplemented with ampicillin (150 mg/l) (LBA) and 0.25 mM CuSO.sub.4 is inoculated with a clone isolated on an LB agar plate supplemented with ampicillin (100 mg/l), and left shaking, at 220 rpm, overnight at 37 C. Two liters of LBA medium containing 0.25 mM CuSO.sub.4, in a 5 L Erlenmeyer flask, are then inoculated at 1/100.sup.th. The latter is incubated at 37 C. with shaking (220 rpm) until an OD.sub.600nm of between 0.8 and 1 OD.sub.600nm/ml is obtained. The culture is then induced with 200 M of IPTG and left shaking (180-220 rpm) at 25 C. for 4 hours. The cells are then transferred into a sterile 2 L Schott bottle containing a magnetic bar, so as to continue, for 20 hours, the culture and the protein induction with shaking under anaerobic conditions in order to increase the incorporation of copper into the bacteria. The cells harvested by centrifugation (4000 g, 4 C.) are washed in water and stored at 20 C.

(35) It is important to emphasize that the induction of the expression of this BOD in the E. coli bacterium is carried out in only 24 hours; this represents a considerable advantage compared with the protocols for induction of the commercial enzymes currently available. This is because the production of BODs derived from Myrothecium verrucaria can require induction periods of up to 5 days.

3.2 Purification of Wild-Type BOD Enzymes

3.2.1 Rupture of Cells and Treatment with DNase I

(36) The cell pellet, derived from two liters of culture, is taken up in 40 ml of 50 mM sodium phosphate buffer containing 500 mM NaCl and 20 mM imidazole, pH 7.6, and sonicated 10 times at a sonication power of 40 W for 3 minutes by cycle of a second of ultrasound and a second of interruption. The sample obtained, called crude extract, is supplemented with a final concentration of 2 mM of MgCl.sub.2 and treated for 30 minutes at ambient temperature with DNase I (1 U/ml of crude extract). The insoluble cell debris is then removed from the crude extract by centrifugation for 60 minutes at 20 000 g.

3.2.2 Affinity Chromatography on Nickel Column

(37) The sonication supernatant filtered through a 0.22 m filter and diluted to an OD.sub.280nm of 10 is injected onto a HisPrep FF 16/10 affinity column (GC Healthcare), coupled to the AKTA purifier system (GC Healthcare), equilibrated in a 50 mM sodium phosphate buffer containing 500 mM NaCl and 20 mM imidazole, pH 7.6. The elution is carried out with a gradient of 5% to 30% of a 50 mM sodium phosphate buffer containing 500 mM NaCl and 1M imidazole, pH 7.6, at a flow rate of 1 ml/min. The fractions containing the BOD protein are identified by means of an ABTS activity test and are combined, concentrated and desalified with a 50 mM sodium phosphate buffer, pH 7.6, by centrifugation on an Amicon YM10 membrane. At this stage, the BOD protein is pure and can be stored at 20 C. in soluble form.

(38) Here again, by comparing with the commercially available BOD purification methods, the clear advantage resulting from the use of this protein can be emphasized. This is because a single purification step is necessary in order to obtain a pure enzyme, as opposed to the succession of chromatographies (size exclusion, anion or cation exchange, hydrophobic, etc.) essential for the commercial BODs.

3.2.3 Characterization of Wild-Type BOD Enzymes

3.2.3.1 Molecular Weight Determination

(39) The analysis of the weight of the whole protein was carried out on the LCQ Deca XP mass spectrometer coupled upstream of a nano liquid chromatography apparatus fitted with a C4 desalting and pre-concentrating column (-Precolumn Cartridge; Acclaim PepMap 300; internal 300 m5m; LC Packings Dionex) and of a C4 analytical column (C4 PepMap 300; internal 75 m5 cm; LC Packings Dionex).

(40) A weight of 61005.91 Da was obtained for the BOD, i.e. a difference of 130.4 Da compared with the theoretical weight of the protein; the theoretical weight is calculated for the protein truncated at the N-terminal methionine, a difference of only 0.80 Da is found, which demonstrates cleavage of this amino acid in the bacterium during the protein maturation process.

3.2.3.2 Concentration Measurement

(41) The enzyme concentration of a solution is calculated according to the Bradford technique using BSA as standard (Bradford, anal. Biochimie 72:248, 1976).

3.2.3.3 Enzymatic Assay

(42) The enzymatic assays are carried out using a Varian spectrophotometer in a 0.1M citrate/phosphate buffer at 37 C. in a volume of 3 ml, with the oxidation of ABTS being followed at 420 nm as a function of time (.sub.420nm=36 mM.sup.1 cm.sup.1). The specific activity of the enzyme is expressed in mol of ABTS oxidized per minute and per mg of protein. The standard ABTS concentration used is 1 mM. The enzyme is diluted so as to measure a slope between 0.05 and 0.3 OD.sub.420nm/min.

4. Techniques for Studying the Enzymatic Properties of the Wild-Type Bacillus pumilus BOD Enzyme

4.1 Determination of the Kinetic (kcat) and Michaelis (KM) Constants in the Stationary State

4.1.1 the Substrate is 2,2-azinobis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS)

(43) The experiments are carried out at 37 C. on a Varian spectrophotometer in a 0.1 M citrate/phosphate buffer, pH 3. The ABTS concentration varies in the test from 0 to 5 mM. The test is triggered by adding enzyme. The experimental points are analysed by nonlinear regression according to the Michaelis-Menten model using the Sigma-plot 6.0 software according to the equation below:
Michaelis-Menten model: k.sub.ss=k.sub.cat*[S]/(K.sub.M+[S])
Results:
k.sub.cat=391.3 s.sup.1 and K.sub.M=31.7 M.

(44) FIG. 3 illustrates graphically the specific activity, in U/mg, of the Bacillus pumilus BOD as a function of ABTS concentration. By way of comparison, the homologous CotA protein of Bacillus subtilis exhibits, with respect to ABTS under the same optimal activity conditions, a k.sub.cat of 322 s.sup.1 for a K.sub.M of 124 M (Martins et al., 2008. J Biol Inorg Chem, 13:183-193).

4.1.2 The Substrate is Unconjugated Bilirubin

(45) The experiments are carried out at 37 C. in a Varian spectrophotometer in a 50 mM sodium phosphate buffer, pH 7. The bilirubin concentration varies in the test from 0 to 60 M. The test, triggered by the addition of enzyme, consists in following the oxidation of the bilirubin at 450 nm by colorimetric change (.sub.450nm=32 mM.sup.1 cm.sup.1). The experimental points are analysed by nonlinear regression according to the Michaelis-Menten model using the Sigma-plot 6.0 software according to the equation below:
Michaelis-Menten model: k.sub.ss=k.sub.cat*[S]/(K.sub.M+[S])
Results:
k.sub.cat=70 s.sup.1 and K.sub.M=22 M.

(46) FIG. 4 is the graphic representation of the Michaelis-Menten equation (k.sub.ss in s.sup.1 as a function of unconjugated bilirubin concentration) for the Bacillus pumilus BOD.

4.1.3 The Substrate is Conjugated Bilirubin

(47) The experiments are carried out at 37 C. on a Varian spectrophotometer in a 50 mM sodium phosphate buffer, pH 4.8. The bilirubin concentration varies in the test from 0 to 150 M. The test, triggered by adding enzyme, consists in following the oxidation of the conjugated bilirubin at 440 nm by colorimetric change (.sub.440nm=25 mM.sup.1 cm.sup.1). The experimental points are analysed by nonlinear regression according to the Michaelis-Menten model using the Sigma-plot 6.0 software according to the equation below:
Michaelis-Menten model: k.sub.ss=k.sub.cat*[S]/(K.sub.M+[S])
Results:
k.sub.cat=66.8 s.sup.1 and K.sub.M=35.1 M.

(48) FIG. 5 represents the catalytic activity for oxidation of the conjugated bilirubin by the Bacillus pumilus BOD at 37 C. in a 50 mM citrate/phosphate buffer, pH 4.8.

4.1.4 The Substrate is Syringaldazine (SGZ)

(49) The experiments are carried out at 37 C. on a Varian spectrophotometer in a 50 mM citrate/phosphate buffer, pH 6.2. The SGZ concentration, diluted in methanol, varies in the test from 0 to 300 M. The test, triggered by adding the enzyme, consists in following the oxidation of the SGZ at 530 nm by coloroimetric change (.sub.530nm=64 mM.sup.1 cm.sup.1). The experimental points are analysed by nonlinear regression according to the Michaelis-Menten model with competitive inhibition, using the Sigma-plot 6.0 software according to the equation below:
Michaelis-Menten model with competitive inhibition:
k.sub.ss=k.sub.cat*[S]/(K.sub.M+[S]+[S].sup.2/K.sub.i)
Results:
k.sub.cat=116.1; K.sub.M=45.6 M and K.sub.i=82.9 M.

(50) FIG. 6 represents the catalytic activity for oxidation of syringaldazine by the Bacillus pumilus BOD at 37 C. in a 50 mM citrate/phosphate buffer, pH 6.2.

4.1.5 The Substrate is 2,6-Dimethoxyphenol (DMP)

(51) The experiments are carried out at 37 C. on a Varian spectrophotometer in a 50 mM sodium phosphate buffer, pH 6.8. The 2,6-dimethoxyphenol concentration varies in the test from 0 to 4000 M. The test, triggered by adding the enzyme, consists in following the oxidation of the DMP at 468 nm by coloroimetric change (.sub.468nm=14.8 mM.sup.1 cm.sup.1). The experimental points are analysed by nonlinear regression according to the Michaelis-Menten model using the Sigma-plot 6.0 software according to the equation below:
Michaelis-Menten model: k.sub.ss=k.sub.cat*[S]/(K.sub.M+[S])
Results:
k.sub.ss=57.3 s.sup.1 and K.sub.M=822 M.

(52) FIG. 7 represents the catalytic activity for oxidation of DMP by the Bacillus pumilus BOD at 37 C. in a 50 mM citrate/phosphate buffer, pH 6.8.

4.2 Study as a Function of pH

4.2.1 Activity as a Function of pH

4.2.1.1 ABTS

(53) The study of the variation in the reaction rate constant as a function of pH is carried out on a pH range of from 3 to 7 in a 0.1 M citrate/phosphate buffer, using 1 mM ABTS as substrate. The experiments are carried out at 37 C. using a Varian spectrophotometer. The activity is followed by oxidation of the ABTS resulting in a colorimetric change measured at 420 nm. The test is triggered by adding enzyme.

(54) The results of the oxidation of ABTS, as a function of pH, by the Bacillus pumilus BOD are represented as relative activity on the graph of FIG. 8.

4.2.1.2 Unconjugated Bilirubin

(55) The study of the variation in the reaction rate constant as a function of pH is carried out on a pH range of from 7 to 8.5 in a 0.2 M tris-HCl buffer, using 30 M unconjugated bilirubin as substrate. The experiments are carried out at 37 C. using a Varian spectrophotometer. The activity is followed by oxidation of the bilirubin resulting in a colorimetric change measured at 450 nm (.sub.450nm=32 mM.sup.1 cm.sup.1). The test is triggered by adding enzyme.

(56) The results of the oxidation of unconjugated bilirubin, as a function of pH, by the Bacillus pumilus BOD are represented as relative activity on the graph of FIG. 8.

4.2.1.3 Conjugated Bilirubin

(57) The study of the variation in the reaction rate constant as a function of pH is carried out on a pH range of from 3 to 7 in a 0.1 M citrate/phosphate buffer, using 100 M conjugated bilirubin as substrate. The experiments are carried out at 37 C. using a Varian spectrophotometer. The activity is followed by oxidation of the conjugated bilirubin resulting in a colorimetric change measured at 440 nm. The test is triggered by adding enzyme.

(58) The results of the oxidation of conjugated bilirubin, as a function of pH, by the Bacillus pumilus BOD are represented as relative activity on the graph of FIG. 8.

4.2.1.4 Syringaldazine (SGZ)

(59) The study of the variation in the reaction rate constant as a function of pH is carried out on a pH range of from 3 to 7.5 in a 0.1 M citrate/phosphate buffer, using 22 M syringaldazine as substrate. The experiments are carried out at 37 C. using a Varian spectrophotometer. The activity is followed by oxidation of the syringaldazine resulting in a colorimetric change measured at 530 nm. The test is triggered by adding enzyme.

(60) The results of the oxidation of syringaldazine, as a function of pH, by the Bacillus pumilus BOD are represented as relative activity on the graph of FIG. 8.

4.2.1.5 2,6-Dimethoxyphenol (DMP)

(61) The study of the variation in the reaction rate constant as a function of pH is carried out on a pH range of from 3 to 7.5 in a 0.1 M citrate/phosphate buffer, using 1 mM DMP as substrate. The experiments are carried out at 37 C. using a Varian spectrophotometer. The activity is followed by oxidation of the DMP resulting in a colorimetric change measured at 468 nm. The test is triggered by adding enzyme.

(62) The results of the oxidation of DMP, as a function of pH, by the Bacillus pumilus BOD are represented as relative activity on the graph of FIG. 8.

4.2.2 Stability as a Function of pH

(63) The stability as a function of pH, of the wild-type BOD, is determined by dilution of the enzyme, purified to homogeneity, in a mixed buffer ranging from pH 3 to 9 at ambient temperature. This mixed buffer is composed of 120 mM Tris, 30 mM imidazole and 30 mM acetic acid, the ionic strength of which is adjusted to 190 mM with NaCl. Various samples are taken as a function of time. The residual activity is measured at 4 C. using a Varian spectrophotometer, in a 0.1 M citrate/phosphate buffer, pH 3, containing 1 mM ABTS.

(64) The results of specific activity and of relative activity of the oxidation of ABTS as a function of pH at 4 C. are represented in the graphs of FIGS. 9A and 9B.

4.3 Study as a Function of the Temperature

4.3.1 Activity as a Function of Temperature

(65) The study of the variation in the reaction rate constant as a function of temperature is carried out in a 0.1 M citrate/phosphate buffer, pH 3, in the presence of 1 mM of ABTS. The temperature ranges from 10 to 85 C. The activity is followed on a temperature-regulated Varian Cary UV Biomelt spectrophotometer. The test is triggered by adding enzyme.

(66) FIG. 10 is a histogram representing the relative activity of the Bacillus pumilus BOD as a function of temperature on ABTS oxidation.

4.3.2 Stability of the Enzyme as a Function of Temperature

(67) The enzyme is preincubated at a concentration of 10 mg/ml in a dry bath at 80 C. 2 l samples are taken and the enzyme is diluted in a 50 mM sodium phosphate buffer, pH 7.6, so as to adjust the enzyme concentration for the activity test. The residual activity of the enzyme incubated at 80 C. is determined using a Varian spectrophotometer, in a 0.1 M citrate/phosphate buffer, pH 3, at 37 C., in the presence of 1 mM of ABTS. The test is triggered by adding enzyme.

(68) FIGS. 11A and 11B represent graphically the stability (expressed as specific activity and as relative activity on ABTS oxidation) of the Bacillus pumilus BOD as a function of enzyme incubation time at 80 C.

4.4 Study of the Activity as a Function of the Presence of Urea

(69) The protocol described above in point 4.1.1 was reproduced in the presence of a urea concentration ranging between 0 and 6 M. FIGS. 12A and 12B represent graphically the activity (expressed as specific activity and as relative activity on ABTS oxidation) of the Bacillus pumilus BOD as a function of urea concentration at 25 C. and at 37 C.

(70) At 25 C., an activating effect of the urea on the BOD is clearly observed. This effect could be due to a slight conformational modification of the active site of the enzyme that would be responsible for better enzymatic efficiency; this phenomenon, which is known, has already been described for other proteins (see Hong-Jie Zhang et al. Biochemical and Biophysical Research Communications 238, 382-386 (1997) and Fan et al. Biochem. J. (1996) 315, 97-102).

(71) At 37 C., this effect is not found. It is possible to put forward the hypothesis that the combined effect of the temperature and of the urea results in too great a modification of the active site, consequently leading to a decrease in the performance levels of the enzyme.

4.5 Study of the Activity as a Function of the Presence of NaCl

(72) The experiments are carried out at 37 C. on a Varian spectrophotometer in a 50 mM citrate/phosphate buffer, pH 6.2, with increasing concentrations of NaCl, from 0 mM to 1000 mM. The concentration of SGZ, diluted in methanol, is fixed in the test at 50 M. The test, triggered by adding enzyme, consists in following the oxidation of the SGZ at 530 nm by colorimetric change (.sub.530nm=64 mM.sup.1. cm.sup.1).

(73) FIG. 13 represents the relative activity of SGZ oxidation by the Bacillus pumilus BOD as a function of NaCl concentration.

4.6 Study of the Activity as a Function of the Presence of DTT or of EDTA

(74) The experiments are carried out at 37 C. on a Varian spectrophotometer in a 50 mM citrate/phosphate buffer, pH 6.2, with increasing concentrations of DTT, from 0 mM to 50 M, or else of EDTA, from 0 to 125 mM. The concentration of SGZ, diluted in methanol, is fixed in the test at 50 M. The test, triggered by adding enzyme, consists in following the oxidation of the SGZ at 530 nm by colorimetric change (.sub.530nm=64 mM.sup.1.cm.sup.1). Table III below collates the results obtained, presented in relative activity form.

(75) TABLE-US-00003 TABLE III Compound Concentration (mM) Relative activity (%) EDTA 0 100 0 0.1 97 1 1 99 5 10 95 1 25 98 3 50 99 4 75 95 2 100 95 1 125 89 3 DTT 0 100 0 0.001 99 4 0.005 93 2 0.01 93 4 0.015 94 3 0.03 85 1 0.05 81 4

4.7 Study of the Remazol Brilliant Blue R (RBBB) Discoloration Activity

(76) Like many other laccases and bilirubin oxidases, the Bacillus pumilus BOD has a discolouring activity on dyes used in the textile industry. Remazol Brilliant Blue R(RBBR) was selected as an example, and the discoloration thereof is measured over time in the presence or absence of a mediator such as ABTS.

(77) The experiments are carried out at 37 C. on a Varian spectrophotometer, in a 50 mM potassium phosphate buffer, pH 6, in the absence or presence of ABTS at 3 ml. The RBBR concentration is fixed at 80 mg.l.sup.1 in each tank. The test, triggered by adding 10 g of enzyme, consists in following, over time, the discoloration of the RBBR dye at 593 nm.

(78) FIG. 13 represents the discoloration of RBBR by the Bacillus pumilus BOD at 3.33 g.ml.sup.1 at 37 C. in a 50 mM potassium phosphate buffer, pH 6, in the absence or presence of ABTS at 10 M.

5. Verification of the Presence of the Four Coppers of the Bacillus pumilus Bilirubin Oxidase

(79) The presence of the 4 coppers is determined by means of a bioquinoline assay using a calibration range for copper concentration in order to measure the molar concentration of copper (Felsenfeld, G. 1960. Arch. Biochem. Biophys., 87, 247-251; Griffiths et al. 1961, J. Biol. Chem., 236, 1850-1856); the results are given in Table III.

(80) Each measurement, based on a colorimetric assay at 546 nm, is carried out in duplicate.

(81) This techniques makes it possible to show the presence of 15.3 M of copper for a BOD protein sample at 3.75 M, i.e. a ratio of 4.08, and clearly confirms the presence of the four copper ions associated with the enzyme.

(82) Finally, in order to confirm the presence of the 4 coppers in the BOD protein, an elemental analysis on the coppers of the protein was carried out by atomic absorption. The results clearly confirmed the presence of 4 coppers per protein.

(83) TABLE-US-00004 TABLE IV Experimental protocol for the bioquinoline assay necessary for assaying the copper of the BOD. Copper Imidazole Copper solution buffer Biquinoline concentration (solution (solution solution Total in the 2) 1) (3) volume sample Sample (l) (l) (l) (l) (M) 1 0 1200 1800 3000 0 2 0 1200 1800 3000 0 3 120 1080 1800 3000 12.59 4 120 1080 1800 3000 12.59 5 240 960 1800 3000 25.18 6 240 960 1800 3000 25.18 7 360 840 1800 3000 37.77 8 360 840 1800 3000 37.77 9 480 720 1800 3000 50.36 10 480 720 1800 3000 50.36 11 600 600 1800 3000 62.95 12 600 600 1800 3000 62.95 BOD_1 450 750 1800 3000 15.7 (3.75 M) BOD_2 450 750 1800 3000 15.3 (3.75 M)