Method of reducing aromatic nitro compounds

1. A method of reducing an aromatic nitro compound comprising the step of:

(i) contacting an aromatic nitro compound with a catalyst,

wherein, the catalyst comprises:

(a) disproportionation agent comprising a transition metal selected from vanadium, chromium, molybdenum, iron, nickel, cobalt, and copper;

(b) a biocatalyst that is a nitroreductase, wherein the biocatalyst comprises a polypeptide having at least 70% sequence identity to SEQ ID NO: 1 [NR-4], SEQ ID NO:2 [NR-14], SEQ ID NO:3 [NR-17] or SEQ ID NO:4 [NR-24]; and

(c) a co-substrate which provides hydride for the reduction.

2. The method of claim 1 , wherein the disproportionation agent is selected from CuCl 2 , Cu(OAc) 2 , CuO 2 , copper metal, FeCl 2 , FeCl 3 , Fe(acac) 3 , FeCl 2 , FeSO 4 , iron metal, NaMoO 4 , NH 4 VO 3 , VOSO 4 , V(acac) 3 , V 2 O 5 , vanadium metal, and CoCl 2 .

3. The method of claim 1 , wherein the biocatalyst is or comprises a polypeptide having the following motif (1)

(1)

A-x(3,4)-G-x-[ADEGQST]-x(4)-[ADEGNQST]-

[AEGNQST]

wherein:

x denotes any amino acid residue,

x(n) denotes a segment consisting of any amino acid residues of length n;

[ab] denotes a single residue selected from a or b; and

amino acids are denoted by their single letter codes.

4. The method of claim 1 , wherein the biocatalyst is or comprises a polypeptide having at least 9 amino acid residues that are identical to or represent a conservative substitution for those of SEQ ID NO: 1 [NR-4] at the positions corresponding to:

15, 39, 40, 41, 42, 64, 65, 67, 112, 132, 135, 136, 138, 220, 224, 229, 230 of SEQ ID NO: 1 [NR-4].

5. The method of claim 1 , wherein the biocatalyst is or comprises a polypeptide having at least 15 amino acid residues that are identical to or represent a conservative substitution for those of SEQ ID NO: 1 [NR-4] at the positions corresponding to:

13, 15, 38, 39, 40, 41, 42, 43, 64, 65, 67, 69, 104, 112, 132, 133, 134, 135, 136, 137, 138, 139, 172, 220, 221, 224, 225, 229, 230, 233

of SEQ ID NO: 1 [NR-4].

6. The method of claim 1 , wherein the biocatalyst is or comprises a polypeptide having at least 70% similarity or identity to SEQ ID NO: 1 [NR-4] and having the following amino acids at the residues corresponding to the following positions in SEQ ID NO: 1 [NR-4]:

41

[SIMVAHNTWL];

136

[GSAN];

224

[KATYFREGIQV];

229

[SKRYAQCGHNTV];

230

[RKYE],

wherein:

[ab] denotes a single residue selected from a or b; and

amino acids are denoted by their single letter codes.

7. The method of claim 1 , wherein the method is a method of reducing an aromatic nitro compound to produce an aromatic hydroxylamine compound or the method is a method of reducing an aromatic nitro compound to produce an aromatic amine compound.

8. The method of claim 1 , further comprising the step(s) of:

(i) isolating the product; and/or

(ii) isolating the catalyst; and/or

(iii) contacting the biocatalyst with a co-substrate.

9. The method of claim 8 , wherein when the method comprises step (iii) the co-substrate is a cofactor, and optionally further comprising the step of regenerating the cofactor using a reduced cofactor regenerating system.

10. The method of claim 1 , wherein the step of contacting the aromatic nitro compound with the catalyst comprises adding discreet portions of the aromatic nitro compound to the catalyst or continuously adding the aromatic nitro compound to the catalyst.

11. The method of claim 1 , wherein the temperature is maintained in the range 0 to 100° C. and/or the pH is maintained in the range 3.0 to 9.0.

12. A kit comprising:

(a) a disproportionation agent comprising a transition metal selected from vanadium, chromium, molybdenum, iron, nickel, cobalt, and copper;

(b) a biocatalyst that is a nitroreductase, wherein the biocatalyst comprises a polypeptide having at least 70% sequence identity to SEQ ID NO: 1 [NR-4], SEQ ID NO:2 [NR-14], SEQ ID NO: 3 [NR-17] or SEQ ID NO:4 [NR-24]; and

(c) a co-substrate which provides hydride for the reduction.

13. The method according to claim 1 , wherein the co-substrate is a co-factor.

14. The method according to claim 1 , wherein the co-substrate is a co-factor selected from NADH, and NADPH.