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.

1 . Purified bilirubin oxidase (BOD), charadterized in that it has a percentage identity of at least 80% with respect to the BOD of  Bacillus pumilus  of SEQ ID No. 2, in that it catalyses the reaction for oxidation of bilirubin to biliverdin and in that it is bound to four copper atoms. 
     
     
         2 . Nucleic acid molecule, characterized in that it codes an BOD according to  claim 1 . 
     
     
         3 . Nucleic acid molecUle according to  claim 2 , having a sequence chosen from SEQ ID NO. 1, SEQ ID No. 5 or SEQ ID No. 7. 
     
     
         4 . Expression vector, characterized in that it comprises a nucleic acid molecule according to  claim 2 . 
     
     
         5 . Host cell expressing a BOD characterized in that it has a percentage identity of at least 80% with respect to the BOD of  Bacillus pumilus  of SEQ ID No. 2, in that it catalyses the reaction for oxidation of bilirubin to biliverdin and in that it is bound to four copper atoms and further characterized in that it is transformed with an expression vector according to  claim 4 . 
     
     
         6 . Method for preparing a BOD characterized in that it has a percentage identity of at least 80% with respect to the BOD of  Bacillus pumilus  of SEQ ID No. 2, in that it catalyses the reaction for oxidation of bilirubin to biliverdin and in that it is bound to four copper atoms, comprising the steps of:
 a) preparing host cells according to  claim 5 ;   b) culturing host cells prepared in step a);   c) lysing said host cells;   d) treating the lysate obtained in step c) by affinity chromatography;   e) recovering said purified BOD,   characterized in that said host cell prepared in step a) is an  Escherichia coli  BL 21  Star strain transformed with the pFD1 vector and in that said culture carried out in step b) is a liquid-phase culture, with shaking, under anaerobic conditions for a period of 20 to 30 h, at a temperature between 20 and 30° C., during which the BOD expression is induced by adding isopropyl-β-D-1-thiogalactopyranoside (IPTG).   
     
     
         7 . Use of the BOD according to  claim 1 , for measuring the bilirubin concentration in a liquid sample. 
     
     
         8 . Kit for assaying bilirubin, characterized in that it comprises a BOD according to  claim 1 . 
     
     
         9 . Method for assaying the bilirubin in solution in a liquid sample, characterized in that it comprises the following steps:
 a) measuring the absorbance at λ max =440 nm of said liquid sample before enzymatic reaction;   b) introducing into said liquid sample a BOD according to  claim 1 ;   c) measuring the absorbance at λ 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;   e) determining the bilirubin concentration of said liquid sample.   
     
     
         10 . Use of a BOD according to  claim 1 , for degrading the bilirubin present in a sample. 
     
     
         11 . Use of the BOD according to  claim 1 , as a reagent in a composition for the oxidation dyeing of keratin fibres. 
     
     
         12 . Use of the BOD according to  claim 1 , for treating wood pulp. 
     
     
         13 . Use of the BOD according to  claim 1 , for discolouring dyes used in industrial media. 
     
     
         14 . BOD electrode comprising a conductive material coated with a deposit comprising at least one BOD according to  claim 1 . 
     
     
         15 . Bilirubin biosensor, characterized in that it is constituted of an electrode according to  claim 14 . 
     
     
         16 . Oxygen sensor, characterized in that it is constituted of an electrode according to  claim 14 . 
     
     
         17 . Enzymatic biofuel cell comprising an anode on which an enzyme catalysing an oxidation reaction is immobilized and an electrode according to  claim 14  as cathode.