Field of the invention

The invention relates to a method for oxidising a non-polymeric organic molecule or a natural polymer comprising only a C-C backbone, wherein the natural polymer comprising only a C-C backbone has a molecular weight of less than 5000, 2500, 1000, 500, 250, 150 Da.

Background to the invention

There is an increasing need to process domestic and commercial waste, particularly mixed waste, in order to reduce environmental damage and to recycle the waste back into the supply chain. There is therefore considerable interest in enzymatic processes for polymer degradation, particularly for mixed polymers.

Co-pending PCT application number PCT/GB2021/050844 (published as W02021205160 - Mellizyme Biotechnology Limited) describes a method for the enzymatic degradation of a synthetic polyalkene polymer, the method comprising contacting the synthetic polyalkene polymer with a composition suitable for use in the degradation of synthetic plastic polyalkene polymers, said composition comprising a KatG enzyme or a catalase-peroxidase EC 1.11.1.21 enzyme.

Commercial polymers and articles formed therefrom contain various additives, such as acid scavengers, prodegradants, antioxidants, antistatic agents, flame -retardants, light stabilizers, thermal stabilizers, lubricants, non-slip compounds, plasticizers, pigments, dyes and mineral or organic fillers (including metals, such as cadmium, chromium, lead, mercury, cobalt and zinc), which may be released during enzymatic degradation of the polymers. There is therefore a pressing need for enzymes capable of both degrading polymers and at least some of the various aforementioned additives and other organic non- polymeric molecules or small natural polymers comprising only a C-C backbone, for example, waste.

Adipic acid is one of the two main components (along with butane- 1,4-diol) of the polymeric ester known as Nylon-6. It is a very high volume chemical (> 3 million tonnes per annum) and is mainly made by an environmentally damaging process in which an initial oxidation of cyclohexane with molecular O2 leads to a mixture of cyclohexanol and cyclohexanone known as K7A oil. In a second step, the K7A oil is oxidised with nitric acid to adipic acid, along with a considerable production of nitrous oxide (N2O), a greenhouse gas that is nearly 300 times more potent than is CO2. Consequently there is a great desire for a process that does not have these environmentally damaging consequences.

The need is to provide a bio-based alternative (Bart JCJ, Cavallaro S, ‘Transiting from Adipic Acid to Bioadipic Acid. 1, Petroleum-Based Processes’, Ind. Eng. Chem. Res., 2015; 54:1-46). A ‘one-pot’ reaction is perfectly feasible thermodynamically (Ishii Y, Sakaguchi S, Iwahama T, ‘Innovation of hydrocarbon oxidation with molecular oxygen and related reactions’, Adv. Synth. Catal., 2001; 343:393-427), and might be catalysed by haem- or porphyrin-containing substances (Noack H, Georgiev V, Blomberg MR, Siegbahn PE, Johansson AJ., ‘Theoretical insights into heme-catalyzed oxidation of cyclohexane to adipic acid’, Inorg. Chem., 2011; 50:1194-1202). However, most processes contemplated involve the production and then reduction of cis-cis-muconate (Bart JCJ, Cavallaro S., ‘Transiting from Adipic Acid to Bioadipic Acid. 2, Biosynthetic pathways’, Ind. Eng. Chem. Res., 2015; 54:567-576).

The present invention is based, at least in part, on the discovery that KatG enzymes are able to cleave C-C bonds and/or C-aryl bonds and/or C-heteroatom bonds in a non-polymeric organic molecule and/or cleave C-C bonds in a natural polymer comprising only a C-C backbone, wherein the natural polymer comprising only a C-C backbone has a molecular weight of less than 5000 Da as well as cleave backbone and side group C-C bonds in organic polymers formed from alkene monomers.

Summary of the invention

In a first aspect of the invention, a method for oxidising a non-polymeric organic molecule and/or a natural polymer comprising only a C-C backbone, wherein the natural polymer comprising only a C-C backbone has a molecular weight of less than 5000, 2500, 1000, 500, 250, 150 Da is provided, the method comprising the step of treating the non-polymeric organic molecule and/or a natural polymer comprising only a C-C backbone with a composition comprising a KatG or a catalase -peroxidase EC 1.11.1.21 enzyme in the presence of oxygen and/or hydrogen peroxide.

A KatG enzyme is a structural sub-class of phylogenetically-related enzymes which fall within Class I of the haem peroxidase -catalase (Px-Ct) superfamily (Passardi et al. Genomics 89 (2007) 567-579). The KatG enzymes, in the presence of oxygen and/or hydrogen peroxide, cleave C-C bonds and/or C- heteroatom bonds and/or C-aryl bonds in a non-polymeric organic molecule and/or cleave C-C bonds in a natural polymer comprising only a C-C backbone, wherein the natural polymer comprising only a C-C backbone has a molecular weight of less than 5000 Da thereby enzymatically degrading the non- polymeric organic molecule and/or a natural polymer comprising only a C-C backbone, wherein the natural polymer comprising only a C-C backbone has a molecular weight of less than 5000 Da into: (a) fragments; and/or (b) oligomers; and/or (c) isolated monomers or repeating units; and/or (d) monomer or repeating unit fragments; and/or (e) monomer or repeating unit derivatives.

The C-C backbone of the non-polymeric organic molecule and/or the natural polymer may be linear, branched, cyclic or bicyclic. A cyclic or bicyclic structure may or may not be branched. The non-polymeric organic molecule and/or the natural polymer can be C2-20 alkanes. The C-C backbone of the non-polymeric organic molecule and/or the natural polymer may, for example, comprise n number of carbons, wherein n may be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

The term catalase -peroxidase EC 1.11.1.21 enzyme defines a functional sub-class of enzymes which exhibit dual peroxidase and catalase activity. The Enzyme Commission number (EC number) is a numerical classification scheme for enzymes, based on the chemical reactions they catalyze. Thus EC numbers do not specify enzymes per se but rather enzyme -catalyzed reactions (i.e. the EC number defines a functional class). It is believed that naturally-occurring catalase-peroxidase EC 1.11.1.21 enzymes are KatG enzymes.

The KatG enzyme or the catalase-peroxidase EC 1.11.1.21 enzyme is preferably present in the compositions in isolated or purified form. The KatG enzyme or the catalase-peroxidase EC 1.11.1.21 enzyme may be expressed, derived, secreted, isolated, or purified from a microorganism (for example from Ralstonia pickettii). for example, the type strain ATCC 27511. The KatG enzyme or the catalase- peroxidase EC 1.11.1.21 enzyme may then be purified by biochemical techniques known in the art.

The KatG enzyme or the catalase-peroxidase EC 1.11.1.21 enzyme may also be conveniently produced by recombinant techniques using a cellular expression system (see e.g. Johnsson et al. (1997) J. Biol. Chem., 272: 2834-2840; Zamocky et al. (2012) Biochimie, 94: 673-683; Doyle and Smith (1996) Biochem. J., 315 (Pt 1): 15-19). Any suitable expression system may be used, including those involving prokaryotic cells and eukaryotic cells. Suitable methodologies have been recently reviewed by Gomes et al. (2016) Advances in Animal and Veterinary Sciences 4(4): 346-356. As used herein, the term recombinant is used to define material which has been produced by that body of techniques collectively known as "recombinant DNA technology" (for example, using the nucleic acid, vectors and or host cells described infra).

The KatG enzyme or the catalase-peroxidase EC 1.11.1.21 enzyme may be a naturally-occurring enzyme, or may be a variant of a naturally-occurring enzyme, and so the KatG enzyme or the catalase- peroxidase EC 1.11.1.21 enzyme is to be understood as including the various natural and artificial KatG enzymes or catalase-peroxidase EC 1.11.1.21 enzymes or enzyme variants described below.

The degradation products are typically one or more of a peroxide, an alcohol, a carboxylic acid, a dicarboxylic acid, an aldehyde, or a dialdehyde. Typical carboxylic acid degradation products are preferably selected from the group consisting of glycolic acid, glyoxylic acid, citric acid, furoic acid, and phenylpyruvic acid. Typical dicarboxylic acids degradation products are C1-12 dicarboxylic acids such as oxalic (ethanedioic), malonic (propanedioic), succinic (butanedioic), glutaric (pentanedioic), adipic (hexanedioic), pimelic (heptanedioic), suberic (octanedioic), azelaic (nonanedioic), sebacic (decanedioic), undecanedioc, dodecanedioic acid, and mixtures thereof.

The use of such compounds as high value speciality chemicals are known to those skilled in the art, and include a number of manufacturing processes such as the electronics, flavour and fragrance, speciality solvents, polyesters, polymer cross-linking, food additives and pharmaceutical industries. For example, malonic acid (and malonate) is used as a building block chemical to produce diverse valuable compounds. Malonic acid and chemical derivatives of malonic acid (such as, for example, monoalkyl malonate, dialkyl malonate, and 2, 2-dimethyl-l, 3 -dioxane-4, 6-dione ("Meldrum's acid")) are used for the production of many industrial and consumer products, including polyesters, protective coatings, solvents, electronic products, flavours, fragrances, pharmaceuticals, surgical adhesives, and food additives.

In a second aspect of the invention, a method for producing a feedstock comprising one or more of a peroxide, an alcohol, carboxylic acid, a dicarboxylic acid, an aldehyde, or a dialdehyde is provided, the method comprising the step of treating a non-polymeric organic molecule and/or a natural polymer comprising only a C-C backbone with a composition comprising a KatG or a catalase -peroxidase EC

1.11.1.21 enzyme in the presence of oxygen, wherein the natural polymer comprising only a C-C backbone has a molecular weight of less than 5000, 2500, 1000, 500, 250, 150 Da.

In a third aspect of the invention, a method for producing adipic acid is provided, the method comprising the step of treating a cyclohexane with a composition comprising a KatG or a catalase-peroxidase EC

1.11.1.21 enzyme in the presence of oxygen.

In a fourth aspect of the invention, a composition comprising a KatG or a catalase-peroxidase EC

1.11.1.21 enzyme and a detergent, for example, a laundry or household cleaning composition is provided.

The composition may additionally comprise an anionic detergent, a cationic detergent, a non-ionic detergent or an amphoteric detergent. The detergent can be selected from, but not limited to, the following: surfactants, foam regulators, builders, bleach, bleach activators, other enzymes, dyes, fragrances, cleavable detergents, dispersant, green cleaning, hard-surface cleaner, laundry detergent or cleaning products. Brief description of the figures

The invention is now described in more detail with reference to:

Figure 1(A) petri dishes of A. baylyi ADP1 (wild type strain) grown on M9 agar plates (with or without the addition of glucose) with 100 pl of hydrocarbon impregnated into squares of filter paper adhered to the lid of the petri dish. When cyclohexane (left hand plate) is used as the sole carbon source no growth of A. baylyi ADP1 can be observed. A. baylyi ADP1 is known to be able to metabolise longer chain hydrocarbons and consequently, under the same conditions, A. baylyi ADP1 shows growth with dodecane as the sole carbon source (right hand plate). (B) When KatG is expressed on the cell surface of A. baylyi a change in optical density (OD) at 600 nm versus culture time in days can be seen with cyclohexane (circles) versus without (triangles), suggesting the addition of KatG confers the ability to utilise cyclohexane;

Figure 2(A) change in optical density (OD) at 600 nm versus culture time in days for A. baylyi ADP1 expressing KatG on the cell surface when supplemented with cyclohexane. Cells were first grown on adipic acid. Once this had been consumed the culture was supplemented with cyclohexane (day 2) which triggered a further increase in OD , and (B) After 15 days of growth on cyclohexane (including a further addition on day 9), the same culture plated onto agar plates (either neat or diluted 4 times) showed actively growing cells even following 2 weeks on cyclohexane.

Figure 3(A) a schematic of the predicted oxidation of cyclohexane by a KatG enzyme yielding adipic acid, (B) overlaid liquid chromatograph mass spectroscopy (LCMS) ion chromatograms for three assays containing either just the KatG enzyme (from Ralstonia pickettii, recombinantly expressed and purified from E. coli B121(DE3), cyclohexane only, and the combination of the KatG enzyme with cyclohexane. The extracted ion chromatogram for the predicted adipic acid product is shown; and (C) LCMS peak area of adipic acid for each of the three assays.

Detailed description of the invention

In a first aspect of the invention, a method for oxidising a non-polymeric organic molecule and/or a natural polymer comprising only a C-C backbone, wherein the natural polymer comprising only a C-C backbone has a molecular weight of less than 5000, 2500, 1000, 500, 250, 150 Da is provided, the method comprising the step of treating the non-polymeric organic molecule and/or a natural polymer comprising only a C-C backbone with a composition comprising a KatG or a catalase -peroxidase EC 1.11.1.21 enzyme in the presence of oxygen and/or hydrogen peroxide. The KatG enzymes, in the presence of oxygen and/or hydrogen peroxide, cleave C-C bonds and/or C- heteroatom bonds and/or C-aryl bonds in non-polymeric organic molecule or a natural polymer comprising only a C-C backbone, wherein the natural polymer comprising only a C-C backbone has a molecular weight of less than 5000 thereby enzymatically degrading the organic polymer into: (a) fragments; and/or (b) oligomers; and/or (c) isolated monomers or repeating units; and/or (d) monomer or repeating unit fragments; and/or (e) monomer or repeating unit derivatives.

The terms fragmentation, oligomerization and depolymerisation are to be understood as the generation of: (a) fragments; and/or (b) oligomers; and/or (c) isolated monomers, monomer fragments or monomer derivatives, respectively.

Preferably the step of treating the organic polymer with a composition comprising an enzyme for degrading the non-polymeric organic molecule and/or the natural polymer comprising only a C-C backbone, wherein the natural polymer comprising only a C-C backbone has a molecular weight of less than 5000 is carried out in the presence of oxygen and in the absence of hydrogen peroxide

In one embodiment of the first aspect of the invention, the KatG enzyme or the catalase-peroxidase EC 1.11.1.21 enzyme is the sole enzyme for degrading the non-polymeric organic molecule and/or a natural polymer comprising only a C-C backbone, wherein the natural polymer comprising only a C-C backbone has a molecular weight of less than 5000.

In another embodiment, the KatG enzyme or the catalase-peroxidase EC 1.11.1.21 enzyme is a KatG enzyme.

KatG enzymes are widely (but not universally) distributed among archaea, eubacteria, and lower eukaryotes. Despite this wide distribution, there is little sequence diversity among KatG enzymes from different sources and multiple sequence alignments show that these are very closely related enzymes despite their diverse origins (Passardi et al. Gene, 397 (2007) 101-113).

KatG enzymes in their wild-type forms are typically multimeric (homodimers or homotetramers), and in most cases the KatG enzyme subunit is a two domain structure (with distinct N-terminal and C- terminal domains where each domain has a typical peroxidase scaffold).

The KatG enzyme may comprise, consist of, or consist essentially of, the amino acid sequence of SEQ ID NO. 1, or a variant enzyme thereof which comprises, consists of, or consists essentially of, an amino acid sequence which is at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99 % identical to SEQ ID NO. 1. SEQ ID NO: 1 is the amino acid sequence for the KatG enzyme of Ralstonia pickettii (strain 12J) (UniProt Accession Number: B2UBU5, entry name KATG_RALPJ, release 2020_01, entry version 76) and is a 721 amino acid protein comprising two domains, each homologous to haem peroxidases: an N- terminal domain (77-391) and a C-terminal domain (398-691). The former is referenced herein as the KatG ^N of the KatG enzyme of amino acid SEQ ID NO. 1 and the latter as the KatG ^c of the KatG enzyme of amino acid SEQ ID NO. 1. The KatG enzyme has the following amino acid sequence:

SEQ ID NO: 1

MTTEAKCPFSGHAPAASHAFGGGTANKDWWPNQLRVDLLNQHSEKSDPLGSNFNYRK SFN AIDYDALKADLRRLMTDSQDWWPADFGHYGPQFIRMAWHAAGTYRTGDGRGGAGRGQQR FAPLNSWPDNVNIDKSRRLLWPIKQKYGQAISWADLLILTGNVALETMGFRTFGFAAGRE DT WEPDNDVYWGNETKWEEATRYSGERNEANPEAAVQMGEIYVNPEGPEHAHGDPEAAAKDI RETFARMAMDDEETVAEIAGGHTFGKTHGAGPASHVGADVEAAPEEAQGEGWASTFGTGK GADAITSGLEVTWTQTPAQWSNFFFENLFKYEWVQEKSPAGALQWVAKDAEAIIPGPTPD SP

KRRPTMETTDESERFDPAYEKISRRFEDNPQAFAEAFARAWFKETHRDEGPKSRYEG PEVPRE DLIWQDPLPTATHKPTEADIADLKAKIAASGLSASELVAVAWASASTFRGSDKRGGANGA RI RLAPQKDWAVNQPVAGTLARLEEIQRASGKASLADVIVLAGSVGIELAAKAAGTTITVPF TP GRVDATAEQTDATSFSVEEPVADGFRNYQKTQFAVPGEVEEEDRAQEETETAPEETVEIG GE RAININ ADGSQHGVFTSTPGAETNDFFVNEEDMNTEWKPAGDIYEGYDRKTGERKWTGTRV DLVFGSNSILRALAEVFGSADGKERFISEFVAAWVKVMNLDRFDLAAA

Identity in relation to an amino acid sequence defines the number (or fraction when expressed as a percentage, %) of identical amino acid residues between two polypeptide sequences. The sequence identity is determined by comparing the sequences when aligned so as to maximize overlap and identity while minimizing sequence gaps, and may be determined using any of a number of alignment algorithms known to those skilled in the art. In particular, sequence identity may be determined using the public domain BEAST (2.10.0) program.

The KatG enzyme may also comprise, consist of, or consist essentially of, the amino acid sequence of the N-terminal domain amino acids 77-391 of SEQ ID NO. 1, or a variant enzyme thereof which comprises, consists of, or consists essentially of, an amino acid sequence which is at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99 % identical to the N-terminal domain amino acids 77-391 of SEQ ID NO. 1.

Preferred variants of the KatG enzyme comprise, consist of, or consist essentially of, an amino acid sequence which is at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99 % identical to either the KatG enzyme of amino acid SEQ ID NO. 1 or to the amino acid sequence of the N-terminal domain amino acids 77-391 of SEQ ID NO. 1 and which have one or more amino acid substitutions, deletions or insertions which:

(a) increase the enzymatic degradation activity to a non-polymeric organic molecule and/or a natural polymer comprising only a C-C backbone, wherein the natural polymer comprising only a C-C backbone has a molecular weight of less than 5000, in particular the enzymatic activity to cleave C-C bonds and/or C-heteroatom bonds and/or C-aryl bonds in a non-polymeric organic molecule and/or a natural polymer comprising only a C-C backbone, wherein the natural polymer comprising only a C-C backbone has a molecular weight of less than 5000; and/or

(b) increase thermostability; and/or

(c) increase psychrophilicity; and/or

(d) increase stability in relation to organic solvents, contaminants, detergents, pH and/or oxidants (including hydrogen peroxide); all relative to the KatG enzyme of amino acid SEQ ID NO. 1.

Such variants can be produced by methods that are well-established in the art, including directed evolution and site-directed mutagenesis (see Zeymer and Hilvert (2018) Annual Review of Biochemistry 87:131-157; Watkins et al. Nat. Commun., 2017; 8:358; Watanabe and Nakajima, Methods Enzymol., 2016; 580:455-470; Lichtenstein et al. Biochem. Soc. Trans., 2012; 40:561-566; Anderson et al. Chem. Sci., 2014; 5:507-514; and Grayson and Anderson, Curr. Opin. Struct. Biol., 2018; 51:149-155.

An increase in thermostability indicates an increased ability to resist changes to the chemical and/or physical structure of the KatG enzyme at high temperatures, and particularly at temperature between 50 and 90°C.

Thermostability can be determined by measurement of the melting temperature (Tm) of the KatG enzyme according to methods known in the art, for example, differential scanning fluorimetry (DSF) may be used to quantify the change in thermal denaturation temperature of the KatG enzyme or circular dichroism can be used by analyse protein folding. In this context, the “melting temperature” refers to the temperature at which half of the enzyme population assayed is unfolded or misfolded. An increase in psychrophilicity indicates an increase in the enzymatic degradation activity to a non- polymeric organic molecule and/or a natural polymer comprising only a C-C backbone, wherein the natural polymer comprising only a C-C backbone has a molecular weight of less than 5000, in particular the enzymatic activity to cleave C-C bonds and/or C-heteroatom bonds and/or C-aryl bonds in a non- polymeric organic molecule and/or a natural polymer comprising only a C-C backbone, wherein the natural polymer comprising only a C-C backbone has a molecular weight of less than 5000, at low temperatures, and particularly at temperatures below 20, below 15, below 10 or below 5°C.

The KatG enzyme may be an orthologue enzyme of SEQ ID NO. 1. As used herein, the term orthologue in relation to a protein defines a species variant of that protein which has diverged in different organisms following a speciation event. Orthologue proteins may perform the same, or a different, role in each organism in which they are found.

Preferred orthologue enzymes of the enzyme of SEQ ID NO. 1 are KatG enzymes from a Ralstonia spp/strain other than Ralstonia pickettii (strain 12J), more preferably from a Ralstonia species/strain selected from: Ralstonia sp. NFACC01; Burkholderiaceae bacterium 26; Ralstonia sp. MD27; Ralstonia sp. AU12-08; Ralstonia insidiosa; Ralstonia sp. TCR112; Ralstonia pickettii (strain 12D); Ralstonia pickettii (Burkholderia pickettii); Ralstonia sp. 5_2_56FAA; Ralstonia pickettii OR214; Ralstonia mannitolilytica; Ralstonia sp. SET104; Ralstonia sp. GX3-BWBA; Ralstonia sp. NT80; Ralstonia sp. GV074; and Ralstonia sp. UNC404CL21Col.

Orthologue enzymes having at least 90 % amino acid sequence identity to the KatG enzyme of amino acid SEQ ID NO. 1 are suitable for use according to the invention including the following KatG enzymes (UniProt Accession Number followed by the entry name, if allocated, in parentheses): A0A1I5SK77 (AOA1I5SK77_9RALS);

A0A0F0E7J1 (A0A0F0E7Jl_9BURK);

A0A0J9DTW2 (A0A0J9DTW2_9RALS);

S9RSJ5 (S9RSJ5_9RALS);

A0A191ZZS8 (A0A191ZZS8_9RALS);

A0A5C6Y6C3 (A0A5C6Y6C3_9RALS);

C6BD71 (C6BD71_RALP1);

A0A2P4RN88 (A0A2P4RN88_RALPI);

U3GIS7 (U3GIS7_9RALS);

A0A1C0XEH7 (AOA1COXEH7_RALPI);

R0CJV6 (R0CJV6_RALPI);

A0A2N4TPC1 (AOA2N4TPC1_RALPI); A0A2N5LD46 (A0A2N5LD46_9RALS);

A0A291EI92 (A0A291EI92_RALPI);

A0A401K9C7 (A0A401K9C7_9RALS);

UPI00046A45B6;

UPI000DD31867;

UPI000CEF361E;

UPI00073F3865;

UPI00046AAE20;

UPI0012AE63A9;

UPI000D5F3903;

UPI0004803C34; and

UPI000CEDCA44.

Preferred orthologue enzyme variants comprise, consist of, or consist essentially of, an amino acid sequence which is at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 99 % identical to any one of the above-mentioned KatG orthologue enzymes.

Also contemplated for use are orthologue enzyme variants which comprise, consist of, or consist essentially of, an amino acid sequence which is at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99 % identical to any one of the above KatG orthologue enzymes and which have one or more amino acid substitutions, deletions or insertions which:

(a) increase the enzymatic degradation activity to a non-polymeric organic molecule and/or a natural polymer comprising only a C-C backbone, wherein the natural polymer comprising only a C-C backbone has a molecular weight of less than 5000, in particular the enzymatic activity to cleave C-C bonds and/or C-heteroatom bonds and/or C-aryl bonds in a non-polymeric organic molecule and/or a natural polymer comprising only a C-C backbone, wherein the natural polymer comprising only a C-C backbone has a molecular weight of less than 5000; and/or

(b) increase thermostability; and/or

(c) increase psychrophilicity; and/or

(d) increase stability in relation to organic solvents, contaminants, detergents, pH and/or oxidants (including hydrogen peroxide); all relative to the KatG enzyme of amino acid SEQ ID NO. 1.

As explained above, such variants can be produced by methods that are well-established in the art.

The KatG enzyme may be a non-catalatic KatG enzyme variant. In one embodiment, the non-catalatic KatG enzyme variant comprises a disrupted tripeptide adduct. The non-catalatic KatG enzyme variant may be an enzyme variant of the enzyme of SEQ ID NO. 1 or an orthologue enzyme variant of the enzyme of SEQ ID NO. 1.

Native KatG enzymes are post-translationally modified by an autocatalytic crosslinking event which yields a crosslinked Met-Tyr-Trp (MYW) tripeptide (also known as the tripeptide adduct or cofactor). The tripeptide adduct is located in, or near, the active site of the protein opposite the prosthetic haem iron atom. In the absence of the intact cross-linked tripeptide, KatG catalase activity is greatly attenuated, while the peroxidase activity may be increased (Paul R. Ortiz de Montellano, Chapter 1 : Self-processing of Peroxidases in Heme Peroxidases, 2015, pp. 1-30).

The above principle applies mutatis mutandis to catalase-peroxidase EC 1.11.1.21 enzymes (which, in their native form, also exhibit the MYW tripeptide), and such non-catalatic catalase-peroxidase EC 1.11.1.21 enzyme variants may exhibit improved degradation activity to organic polymers solely formed from non-alkene monomers.

Thus, the catalase and peroxidase activities of the KatG enzymes or catalase-peroxidase EC 1.11.1.21 enzymes can be separated, and non-catalatic variants can be derived (see e.g. Ghiladi et al. (2005) J Biol Chem 280: 22651-22663; Ghiladi et al. (2005) Biochemistry 44: 15093-15105; Viasits et al. (2010) J Inorg Biochem 104: 648-656; Viasits et al. (2010) Biochim Biophys Acta 2010b; 1804:799-805; Ortiz de Montellano (2010) Biocatalysis based on heme peroxidases'. 79-107; Jakopitsch et al. (2003) J Biol Chem 278: 20185-20191; Jakopitsch et al. (2004) J Biol Chem 279: 46082-46095; Jakopitsch et al. (2003) FEBS Lett 552: 135-140; Santoni et al. (2004) Biopolymers 74: 46-50; Yu et al. (2003) J Biol Chem 278: 44121-44127; and Bernroitner et al. (2009) J Exp Bot 60: 423-440).

As used herein, the term non-catalatic, as applied in relation to both KatG enzymes or catalase- peroxidase EC 1.11.1.21 enzymes, defines an enzyme in which the catalase activity is reduced or eliminated but which retains degradation activity to a non-polymeric organic molecule and/or a natural polymer comprising only a C-C backbone, wherein the natural polymer comprising only a C-C backbone has a molecular weight of less than 5000. Preferred non-catalatic KatG enzymes or catalase- peroxidase EC 1.11.1.21 enzymes contain one or more substitutions of the MYW tripeptide residues. Such non-catalatic KatG enzymes or catalase-peroxidase EC 1.11.1.21 enzymes may exhibit an increase in the enzymatic degradation activity to a non-polymeric organic molecule and/or a natural polymer comprising only a C-C backbone, wherein the natural polymer comprising only a C-C backbone has a molecular weight of less than 5000, in particular the enzymatic activity to cleave C-C bonds and/or C- heteroatom bonds and/or C-aryl bonds in a non-polymeric organic molecule and/or a natural polymer comprising only a C-C backbone, wherein the natural polymer comprising only a C-C backbone has a molecular weight of less than 5000.

In the case of the KatG enzyme of amino acid SEQ ID NO. 1, the MYW tripeptide residues are M248, Y221 and W98. Suitable substitutions therefore include variants comprising one or more of the following amino acid substitutions: M248I, M248V, Y221F, Y221A, Y221L, W98A, W98F and/or R410N. Preferred is a Y221F and/or R410N substitution, particularly a combination of Y221F and R410N substitutions.

Other preferred non-catalatic KatG enzymes or catalase-peroxidase EC 1.11.1.21 enzymes contain amino acid substitutions in amino acids other than the MYW residues (including those which impair the function of the MYW tripeptide). For example, substitution of the arginine residue corresponding to R439 of the Synechocystis KatG (R410) may also be employed to generate non-catalatic variants (see for example Jakopitsch et al. (2004) J. Biol. Chem., 279: 46082-46095; Carpena et al. (2005) EMBO Rep. 2005, 6:1156-1162).

In one embodiment, the KatG enzyme is a single domain KatG enzyme which comprises, consists of, or consists essentially of, an amino acid sequence which is at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99 or 100 % identical to the KatG enzyme of the dinoflagellate Prorocentrum minimum (UniProt Accession Number: M1FBG9, entry name M1FBG9_PROMN, release 2019_l 1, entry version 16) of amino acid SEQ ID NO: 2, or an orthologue enzyme of the KatG enzyme of the dinoflagellate Prorocentrum minimum of amino acid SEQ ID NO: 2.

MNSLIAALLPIGVFGSCPFMDIGTPPSLKAEVISDSDFKTALEGLDIEALEQDLRIL MTDSQTC WPADDGHYGGFMIRLAWHCAGTFRTSDQKGGCGGAGIRFPPESDWEDNGNLDKARALLVP I KQKYGDALSWGDLISFAGTVAIRDMGGPTNPHCFGRVDDADGNKSDIFGVTDSWQDTDCV VQGNCQEPMGAVKVGLIYVNPEGPLNDPNDLNSGQNPDPEKSAVEIREVFGRMGMNDSET A SLIAGGHAFGKCHGAGVMTSGFEGPWTTTPSQWTNQFLTGMLDEEWEQVATPSGSAVQWQ TKDRTSILAGTMRLTADLALVNDDAYLALAKHWVCDQQKLDIAFAASWKKLVESGGGWLP VEDRRCEPESKATGQQTDPQKNFHSVCATTTTDDSQEASTATKFCLSFLAVVLTPFSLGV HS WLA

KatG enzymes comprise two peroxidase-like domains. The N-terminal domain (KatG ) contains the haem group and is catalytically active. The C-terminal domain (KatG ^c ) lacks the haem cofactor, has no catalytic activity, and is separated from the active site by > 30 A. However, KatG is typically not redundant (the only reported KatG lacking the C-terminal domain is that from the dinoflagellate Prorocentrum minimum - see Guo and Ki, J. Phycol. (2013) 49(5): 1011-6). KatG truncates containing only the KatG ^N domain were constructed and shown to exhibit neither catalase nor peroxidase activity. However, the isolated KatG ^N domain retained gross secondary structure, and both activities were restored by adding back separately expressed and isolated KatG ^c (Baker et al. Biochem. Biophys. Res. Commun., 2004, 320(3): 833-9; Baker et al. Biochemistry 2006, 45(23): 7113-7121). Thus, KatG ^c functions as a platform for the folding of the N-terminal domain and as a scaffold for stabilization of the molecular dimer which can be supplied in trans as a separate moiety.

More particularly preferred are variants of the single domain KatG enzyme of amino acid sequence SEQ. ID. NO. 2 which comprises, consists of, or consists essentially of, an amino acid sequence which is at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99 % identical to the enzyme of amino acid sequence SEQ. ID. NO. 2 and which have one or more amino acid substitutions, deletions or insertions which:

(a) increase the enzymatic degradation activity to a non-polymeric organic molecule and/or a natural polymer comprising only a C-C backbone, wherein the natural polymer comprising only a C-C backbone has a molecular weight of less than 5000, in particular the enzymatic activity to cleave C-C bonds and/or C-heteroatom bonds and/or C-aryl bonds in organic polymers formed with a heteroatomic backbone; and/or

(b) increase thermostability; and/or

(c) increase psychrophilicity; and/or

(d) increase stability in relation to organic solvents, contaminants, detergents, pH and/or oxidants (including hydrogen peroxide); all relative to the single domain KatG enzyme of amino acid sequence SEQ. ID. NO. 2.

Such variants can be produced by methods that are well-established in the art, including directed evolution and site-directed mutagenesis (see Zeymer and Hilvert (2018) Annual Review of Biochemistry, 87:131-157; Watkins et al. Nat. Commun., 2017; 8:358; Watanabe and Nakajima, Methods Enzymol., 2016; 580:455-470; Lichtenstein et al. Biochem. Soc. Trans., 2012; 40:561-566; Anderson et al. Chem. Sci., 2014; 5:507-514; and Grayson and Anderson, Curr. Opin. Struct. Biol., 2018; 51:149-155. More generally, the KatG enzyme may consist, or consist essentially, of a KatG ^N domain, also known as a KatG ¹⁴ truncate. In such embodiments, the KatG ¹⁴ truncate may be used alone or together with a separate KatG ^c (or synthetic analogues thereof).

The KatG enzyme may comprise a KatG ¹⁴ domain fused to a heterologous KatG ^c (or a synthetic analogue thereof) and are known KatG ^N fusions.

Suitable synthetic analogues of KatG ^c for use in combination with KatG ^N truncates (or as part of a KatG ^N fusion protein) may be produced by synthetic biological methods that are well-established in the art (see e.g. Watkins et al. Nat. Commun., 2017; 8:358; Watanabe and Nakajima, Methods Enzymol., 2016; 580:455-470; Lichtenstein et al. Biochem. Soc. Trans., 2012; 40:561-566; Anderson et al. Chem. Sci., 2014; 5:507-514; and Grayson and Anderson, Curr. Opin. Struct. Biol., 2018; 51:149-155).

The KatG enzyme may comprise a KatG enzyme or a KatG ^N domain, fused to a non-enzymatic protein which promotes or mediates the adsorption of the KatG enzyme onto the non-polymeric organic molecule and/or the natural polymer comprising only a C-C backbone, wherein the natural polymer comprising only a C-C backbone has a molecular weight of less than 5000.

The C-C backbone of the non-polymeric organic molecule and/or the natural polymer may be linear, branched, cyclic or bicyclic. A cyclic or bicyclic structure may or may not be branched. The C-C backbone of the non-polymeric organic molecule and/or the natural polymer may comprise n number of carbons, wherein n may be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

Examples of non-polymeric organic molecule suitable for the invention include surfactants, antioxidants, antistatic agents, flame -retardants, light stabilizers, thermal stabilizers, lubricants, plasticizers, organic pigments, dyes, organic fillers, lipids such as phospholipids, cyclohexane, linear alkanes for example hexane, octane, dodecane, benzylic substrates such as xylene, styrene and lindane. Other non-polymeric organic molecules leading to products such as 5-(hydroxymethyl)furfural (HMF) for the production of 2,5-Furandicarboxylic Acid could be used.

Non-polymeric organic molecules suitable for the invention include linear or branched alkanes. Linear alkanes include, a C2-20 linear alkanes, more specifically, a C5-18 linear alkane. For example, ethane, propane, butane, pentane, hexane, heptane, octane, nonane, decane, undecane, dodecane, tridecane, tetradecane, pentadecane, cetane (hexadecane), heptadecane, octadecane, nonadecane or eicosane.

In one example, the linear or branched alkanes above include those in C2-20, prepferably Ce is, more preferably C12-18 fatty acids and lipids Branched alkanes include C4-20 branched alkanes, more specifically, a C4-18 branched alkane. Most specifically a C4-15 branched alkane. For example, the branched alkane may be selected from, but not limited to, the following compounds: methylpropane (isobutane), dimethylpropane (neopentane), methylpentane, dimethylbutane, methylhexane, dimethyl pentane, trimethylbutane, ethylbutane, trimethylpentane, ethylpentane, dimethylhexane.

Non-polymeric organic molecules further include cyclic or bicyclic alkanes, wherein the cyclic or bicyclic alkane may or may not be branched such molecules include for example, (where possible), C3 20 , cyclic or bicyclic alkanes preferably, C3-18 cyclic or bicyclic alkane, particularly C5 to C10, preferably C5 to Cs cylcoalkanes. For example, the cyclic or bicyclic alkane may be selected from, but not limited to, the following compounds: cyclopropane, cyclobutane, cyclopentane, cyclohexane, cycloheptane, cyclooctane, cyclononane or cyclodecane.

Benzylic substrates include alkyl benzenes and dialkyl benzenes, such as C(i-6)alkyl and dialkyl benzenes including toluene, ethyl benzene, toluene and xylene.

An example of a natural polymer comprising only a C-C backbone has a molecular weight of less than 5000 suitable for the invention is a paraffin.

The amount of the KatG enzyme or the catalase-peroxidase EC 1.11.1.21 enzyme present in the composition can be readily determined by the skilled person by reference inter alia to the nature of the nature and (relative amounts) of the non-polymeric organic molecule and/or the natural polymer comprising only a C-C backbone, wherein the natural polymer comprising only a C-C backbone has a molecular weight of less than 5000 and/or the number of any additional enzymes present in the composition. For example, in preferred embodiments, the KatG enzyme or the catalase-peroxidase EC 1.11.1.21 enzyme is used in an amount up to 5 % by weight of the organic polymer formed from nonalkene and/or optionally alkene monomers, more preferably up to 1 %, even more preferably up to 0.1 %, and yet more preferably up to 0.05 % by weight of the non-polymeric organic molecule and/or the natural polymer comprising only a C-C backbone, wherein the natural polymer comprising only a C-C backbone has a molecular weight of less than 5000. For example, the amount of the KatG enzyme or the catalase-peroxidase EC 1.11.1.21 may be in a range of 0.001 to 5 % by weight of the non-polymeric organic molecule and/or the natural polymer comprising only a C-C backbone, wherein the natural polymer comprising only a C-C backbone has a molecular weight of less than 5000, preferably in the range of 0.001 to 1 %, more preferably in the range of 0.001 to 0.1 %, and even more preferably in the range of 0.001 to 0.05 % by weight of the non-polymeric organic molecule and/or the natural polymer comprising only a C-C backbone, wherein the natural polymer comprising only a C-C backbone has a molecular weight of less than 5000. The amount of the KatG enzyme or the catalase-peroxidase EC 1.11.1.21 enzyme present in the composition is therefore selected to conveniently facilitate the above dosing ratios, and so in preferred embodiments, the composition preferably comprises at least 0.01, 0.1, 1, 5, 10, 15, 20, 25 or 30 % by weight KatG enzyme or catalase-peroxidase EC 1.11.1.21 enzyme, based on the total weight of the composition. In some embodiments, the composition preferably comprises up to 0.01, 0.1, 1, 5, 10, 15, 20, 25 or 30 % by weight KatG enzyme or catalase-peroxidase EC 1.11.1.21 enzyme, based on the total weight of the composition. In some preferred embodiments, the composition comprises between 0.1 and 50 %, or between 0.1 and 40 %, or between 0.1 and 30 %, or between 0.1 and 20 %, or between 0.1 and 10 %, KatG enzyme or catalase-peroxidase EC 1.11.1.21 enzyme (based on the total weight of the composition).

The composition comprising the KatG enzyme or the catalase-peroxidase EC 1.11.1.21 enzyme is preferably substantially anhydrous, for example in the form of granules or a powder. Anhydrous compositions may be prepared by any suitable technique known to those skilled in the art, including lyophilisation, freeze-drying, spray-drying, supercritical drying, down-draught evaporation, thin-layer evaporation, centrifugal evaporation, conveyer drying, fluidized bed drying, drum drying and combinations of the foregoing techniques. Preferably, the KatG enzyme or the catalase-peroxidase EC 1.11.1.21 enzyme is freeze-dried or spray-dried.

In the case of substantially anhydrous compositions, the KatG enzyme or catalase-peroxidase EC 1.11.1.21 enzyme preferably further comprises an inert bulking agent selected from starch, dextrin (e.g. cyclodextrin and/or maltodextrin), sugars (e.g. sorbitol, trehalose and/or lactose), carboxymethylcellulose, poly-electrolytes, trehalose, and compatible solutes selected from proline, betaine, glutamate and glycerol.

In preferred embodiments, the KatG enzyme or the catalase-peroxidase EC 1.11.1.21 enzyme is in a stabilized form. For example, it may be in the form of a cross-linked enzyme, crystallized cross-linked enzyme and/or an aggregated cross-linked enzyme (see e.g. Velasco-Lozano et al. (2015) Biocatalysis, 1: 166-177 for a review of suitable technologies).

The KatG enzyme or the catalase-peroxidase EC 1.11.1.21 enzyme may be stabilized by a coating. In such cases, the composition comprising the KatG enzyme or the catalase-peroxidase EC 1.11.1.21 enzyme may take the form of coated enzyme granules (for example as described in WO 00/01793, WO 2004/003188, WO 2004/067739, WO 99/32595, WO 2006/034710 and WO 2007/044968. In other embodiments, the KatG enzyme or the catalase-peroxidase EC 1.11.1.21 enzyme is in the form of a solution, suspension, emulsion or dispersion, optionally in the form of an aqueous solution, suspension, emulsion or dispersion. In certain embodiments, the KatG enzyme or the catalase- peroxidase EC 1.11.1.21 enzyme may be present in the composition in an immobilized from, for example being bound to cell membranes, within lipid vesicles, or attached to solid supports, matrices or particles (for example selected from glass beads and mineral, polymer or metallic particles or matrices).

The KatG enzyme or the catalase-peroxidase EC 1.11.1.21 enzyme may be present in the composition together with one or more excipients. Such excipients include buffers, preservatives (for example selected from sodium benzoate, sodium sorbate and sodium ascorbate), bulking, protective and/or stabilizing agents (for example selected from carboxymethylcellulose, starch, dextrin, arabic gum, salts, sugars (e.g. sorbitol, trehalose and/or lactose), glycerol, polyethyleneglycol, polypropylene glycol, propylene glycol, sequestering agent (e.g. EDTA), reducing agents, amino acids, carriers (such as an aqueous or organic solvent or suspension medium), poly-electrolytes, trehalose, and compatible solutes such as proline, betaine, glutamate and/or glycerol.

The KatG enzyme or catalase-peroxidase EC 1.11.1.21 enzyme may also be present in the composition together with various reactants and/or cofactors. Such reactants and cofactors may be used by the KatG enzyme or the catalase-peroxidase EC 1.11.1.21 enzyme.

In one embodiment, oxidative power is provided by molecular dioxygen (O2), including from air, rather than or in addition to hydrogen peroxide, providing degradation products in the same families as described above. Thus the step of treating the non-polymeric organic molecule and/or a natural polymer comprising only a C-C backbone with a composition comprising an enzyme for degrading the organic polymer is carried in the presence of oxygen and in the absence of hydrogen peroxide.

In a second aspect of the invention, a method for producing a feedstock comprising one or more of a peroxide, an alcohol, carboxylic acid, a dicarboxylic acid, an aldehyde, or a dialdehyde is provided, the method comprising the step of treating a non-polymeric organic molecule and/or a natural polymer comprising only a C-C backbone with a composition comprising a KatG or a catalase-peroxidase EC 1.11.1.21 enzyme in the presence of oxygen, wherein the natural polymer comprising only a C-C backbone has a molecular weight of less than 5000, 2500, 1000, 500, 250, 150 Da., for example octane or decane

The carboxylic acid is preferably selected from the group consisting of glycolic acid, glyoxylic acid, citric acid, furoic acid, phenylpyruvic acid, and mixtures thereof, and the dicarboxylic acid is preferably selected from the group consisting of oxalic, malonic, succinic, glutaric, adipic, pimelic, suberic, azelaic, sebacic, undecanedioc, dodecanedioic acid, and mixtures thereof.

The foregoing methods can be carried out in batch or continuous reactors. Suitable reactor configurations therefore include the following: stirred tank batch reactors; packed bed reactors, also called plug-flow reactor, containing a settled bed of immobilised enzyme particles; continuous flow stirred tank reactors; and fluidised bed reactors, where the flow of gas and/or polymers and articles formed therefrom for enzymatic degradation keeps the immobilised enzyme particles in a fluidised state.

Preferred reactors are airlift reactors, which are reactors in which the reaction mixture is kept mixed and gassed by introduction of air or another gas (or mixture of gases) at the base of a column-like reactor element equipped either with a draught tube or another device (e.g. external tube) by which the reactor volume is separated into gassed and un-gassed (or relatively less gassed) regions, thus generating a vertically circulating flow.

The reactors may also comprise a source of peroxide (for example a source of hydrogen peroxide) or means for generating peroxide (for example means for generating hydrogen peroxide). Alternatively and as previously described, the source of peroxide comprises an enzyme which produces peroxide within the reactor vessel in conjunction with suitable substrates, water and a source of oxygen (such as compressed air). The methods may be practised without the provision of peroxide, provided oxygen is present.

The reactors for use according to the invention preferably further comprise a heating and/or a cooling coil, and may also further comprise means for monitoring and controlling the pH of the reaction mix. The reactors for use according to the invention preferably further comprise an aerator and/or a stirrer.

The KatG enzyme or catalase-peroxidase EC 1.11.1.21 enzyme and the polymers and articles formed therefrom for enzymatic degradation may form part of a reaction mixture for use in the reactors.

The polymers and articles formed therefrom for enzymatic degradation are preferably processed before incorporation into the reaction mixture.

Thus the polymers and articles formed therefrom for enzymatic degradation may be mechanically processed in order to increase the surface area available for contact with the KatG enzyme or the catalase-peroxidase EC 1.11.1.21 enzyme. Thus, shredding, cutting, shearing, maceration, micronization, pelletization, and granulation are preferred and grinding a particularly preferred mechanical processing step(s). Such mechanical processing may promote, accelerate or increase the extent of enzymatic degradation of the polymers and articles formed therefrom for enzymatic degradation.

In preferred embodiments, the methods are carried out at a temperature of 20 to 90°C, preferably 40 to 80°C, more preferably 50 to 70°C, even more preferably 60 to 70°C.

In preferred embodiments, the method is carried out at a neutral or acid pH, more preferably at an acid pH. For example, the method is preferably carried out at a pH of 2 to 7, preferably at a pH of 3 to 6, more preferably at a pH of 3.5 to 5.5, even more preferably at a pH of about 4.5 to 5.

In preferred embodiments, the methods further comprise agitating the reaction mixture to improve contact between the KatG enzyme or the catalase-peroxidase EC 1.11.1.21 enzyme and the polymers and articles formed therefrom for enzymatic degradation and the non-polymeric organic molecule and/or the natural polymer comprising only a C-C backbone, wherein the natural polymer comprising only a C-C backbone has a molecular weight of less than 5000 Da, released therefrom and so promote adsorption of the enzyme thereon. This step may comprise continuous stirring, for example at a rate of 100 to 5000 rpm. Alternatively, continuous agitation can be achieved by running the method in an airlift reactor.

Those skilled in the art will be able to determine an appropriate reaction time by reference to the nature of the polymers and articles formed therefrom for enzymatic degradation and the non-polymeric organic molecule and/or the natural polymer comprising only a C-C backbone, wherein the natural polymer comprising only a C-C backbone has a molecular weight of less than 5000 Da, the KatG enzyme or the catalase-peroxidase EC 1.11.1.21 enzyme selected and the extent of degradation required. In preferred embodiments, the enzymatic degradation may be carried out over a reaction time of 5 to 72 hours. Alternatively, the method may be run continuously in circumstances where continuous reactors are employed.

The method may further comprise the step of isolating the degradation product(s) produced. Suitable isolation steps include stripping, separation by aqueous solution, steam selective condensation, filtration, separation, distillation, vacuum evaporation, extraction, electrodialysis, adsorption, ion exchange, precipitation, crystallization, concentration and acid addition dehydration and precipitation, nanofiltration, acid catalyst treatment, semi continuous mode distillation or continuous mode distillation, solvent extraction, evaporative concentration, evaporative crystallization, liquid/liquid extraction, hydrogenation, azeotropic distillation process, adsorption, column chromatography, simple vacuum distillation and microfiltration and combinations of two or more of the foregoing. Alternatively, or in addition, the plastic products may be physically processed by a treatment selected from: irradiation, for example drying, UV irradiation, amorphization, agglomeration, heating (e.g. by microwaves), cooling, freezing, dessication, and a combination of two or more of the foregoing. Alternatively, or in addition, the plastic product may be treated directly without being physically processed by a treatment selected from: irradiation, for example drying, UV irradiation, amorphization, agglomeration, heating (e.g. by microwaves), cooling, freezing, dessication, and a combination of two or more of the foregoing. Specifically, the plastic products may be treated directly without any UV irradiation.

In a third aspect of the invention, a method for producing adipic acid is provided, the method comprising the step of treating a cyclohexane with a composition comprising a KatG or a catalase-peroxidase EC 1.11.1.21 enzyme in the presence of oxygen.

The various embodiments, and optional and preferred technical features of the first aspect of the invention are also relevant to the second aspect of the invention where applicable.

A fourth aspect of the invention provides a cleaning composition, such as a laundry or household cleaning composition comprising a KatG or a catalase-peroxidase EC 1.11.1.21 enzyme as described herein. The composition may additionally comprise a suractand such as an anionic or amphoteric surfactant and may additionally comprise one or more further components selected from cationic surfactants, non-ionic surfactants foam regulators, builders, bleaching agents, bleach activators, other enzymes (such as lipases or proteases), dyes, fragrances, cleavable detergents and dispersants.

The invention will now be illustrated by one or more examples and figures, further embodiments of the invention will be apparent to the skilled worker in light of these.

Example la: KatG A. baylyi Surface display

The coding sequence of KatG wild type enzyme was cloned into an E. coli/A. baylyi shuttle vector containing Gentamycin resistance gene and a broad compatibility origin of replication (pRG1600). katG was cloned downstream of a PelB signal peptide leader sequence, targeting the produced protein for transport to the cell surface. A C-terminally-fused autotransporter (AIDA1) then allows for katG display on the cell surface. A. baylyi ADP1 (wild type strain) was then transformed with this vector, which contains an inducible promoter to allow tuneable levels of protein expression when grown in the presence of arabinose.

Example lb: KatG activity towards adipic acid A. baylyi ADP1 (wild type strain) was grown on M9 agar plates (with or without the addition of glucose) with 100 pl of hydrocarbon (cyclohexane or dodecane) impregnated into squares of filter paper adhered to the lid of the petri dish. The plates were sealed with parafilm to allow hydrocarbon vapours to accumulate within the dish and incubated for 24 hours at 30 °C. The results are illustrated in figure 1(A). When cyclohexane vapour was used as the sole carbon source, no growth of A. baylyi ADP1 was seen. Under the same conditions on M9 agar supplemented with glucose, growth can be seen suggesting no toxicity arising from gaseous cyclohexane. A filter paper soaked with dodecane (a carbon source A. baylyi ADP1 is known to utilise), in the absence of glucose, showed evidence of A. baylyi ADP1 growth, indicating that hydrocarbon in a gaseous phase is accessible for use.

Six 50 ml cultures of modified A. baylyi ADP1, displaying KatG on the cell surface, as described below, were initially grown in A. baylyi minimal media (AMM) with 6 mM of adipic acid as the sole carbon source at 30 °C and 250 rpm shaking and production of the recombinant katG was induced with 0.2% arabinose. AMM contained 20 mM sodium succinate, mineral solution (50 ml per litre) and phosphate buffer (50 ml per litre). Mineral solution contained 20 g/L NH4CI, 11.6 g/L MgSO^VJUO, 2 g/L KNO3, 1.34 g/L CaC12-2H2O, 40 mg/L (NJDeMovOzrAJUO and 20 mL/L SL9 trace elements. SL9 trace elements contained 12.8 g/L nitrilotriacetic acid, 2 g/L FeSO^VJUO, 104 mg/L C0CI2, 70 mg/L ZnCL. 36 mg/L Na2MoO4-2H2O, 13 mg/L NiCL. 6 mg/L H3BO3 and 2 mg/L CUCI2.2H2O. Phosphate buffer contained 136 g/L KH2PO4 and 265 g/L Na2HPO4-H2O with a final pH of 06.9. Once all the cells had reached a stationary phase and all the adipic acid was consumed (within 24 hours, reaching a final OD of approximately 0.13), three of the cultures were supplemented with 300 pl of cyclohexane and returned to incubation. The remaining 3 flasks were given no additional nutrients. The results are illustrated in figure 1(B). The graph shows the average change in optical density (OD) at 600 nm over 11 days in the flasks fed with cyclohexane versus the flasks that were given no additional nutrients. The cultures supplemented with cyclohexane reached a higher OD than those without, indicating the possibility that cyclohexane was being converted into an energy source in the presence of KatG, leading to increased cell growth. Error bars represent the standard error between the three flasks.

Example 2: A. baylyi ADP1 (+KatG) growth on cyclohexane

A 50 ml culture of A. baylyi ADP1, displaying KatG on the cell surface (see above), was grown in AMM minimal media with adipic acid (6 mM) to stationary phase (48 hours). The flask was then supplemented with 1 ml of cyclohexane and the optical density (OD) change monitored daily. The result is shown in figure 2(A). After an initial increase in measured OD, it was observed to drop after 5 days. Fresh cyclohexane (0.5 ml) was dosed in on day 9 which stabilised the decrease. The OD remained stable for the remaining 12 days of the experiment. When the 15 day old culture was plated onto LB agar plates (with antibiotic selection for the KatG plasmid) colonies could be seen on plates spread with 50 pl of neat culture and also on LB agar plated with a 4x diluted sample of culture as illustrated in figure 2(B), indicating sustained cell viability even after growing in cyclohexane as the sole carbon source for two weeks.

Example 3a: KatG activity towards adipic acid

2 pl of cyclohexane was added to a 1.5 ml eppendorf tube containing 150 pl of phosphate buffer (at pH 7.5) and 16 pM of purified KatG enzyme (recombinantly expressed and purified from E. coli BL21(DE3) as described below). Control reactions containing either no KatG, or no cyclohexane were set up in parallel. The reactions were incubated at 30 degrees Celsius for 20 hours before they were quenched and the enzyme removed with the addition of an equal volume of MeOH (150 pl). The reactions were centrifuged (13000 rpm, 10 mins) to remove the precipitated protein. 75 pl of the supernatants were dried and each resuspended in 40 pl of MeOH and analysed directly by LCMS. Figure 3(A) illustrates the predicted reaction of the oxidation of cyclohexane by KatG, yielding the dicarboxylic adipic acid.

3b KatG enzyme purification

Sumo wild type katG is a His Tagged protein with a peptide sequence recognized by a His-tagged Sumo protease to leave a katG enzyme with traceless tags after digestion. The coding sequence for R. pickettii katG wild type enzyme with an N-terminal cleavable his-SUMO tag was cloned into PE-SUMOPro (LifeSensors, PE-1000-K100) vector with KANAMYCIN resistance marker and transformed into BL21 (DE3) E. coli cells. Cells expressing recombinant KatG were grown at 30 °C in Auto Induction Terrific Broth (For Medium) for 24 hours. Cells were harvested by centrifugation and the bacterial pellet resuspended in 20mM sodium phosphate buffer pH 7.4, 300 mM NaCl to allow direct loading of the lysate supernatant onto a His Trap HP purification column (Cytivia) initialised in same buffer using an Akta Start Chromatography system with fraction collector. The recombinant enzyme was purified by gradient elution with 20mM sodium phosphate buffer pH 7.4, 300 mM NaCl, 500 mM imidazole. The fractionated His-tagged KatG enzyme (approx. 12 ml) was then subject to incubation with 200microlitres of 7mg/ml Sumo protease overnight at 25°C with dialysis using Snakeskin® dialysis tubing (Thermo Scientific) against 50 mM Tris-HCl, pH 8.0 /150mM NaCl /ImM DTT. The dialysed, Sumo protease-digested KatG was then returned to the His Trap column and the tag-free KatG enzyme was collected by flow through off column with the His tags retained on the column until gradient elution with imidazole."

Figure 3(B) illustrates the overlaid liquid chromatography mass spectroscopy (LCMS) extracted ion chromatograms from three assays. One containing just KatG enzyme (from Ralstonia pickettii, recombinantly expressed and purified from E. coli B121(DE3), one with cyclohexane only, and a third where recombinant and purified KatG was mixed with cyclohexane. The extracted ion chromatogram target ions consistent with the expected mass of adipic acid (m/z 145.0506 [M-H]-). There is a noticeably higher abundance of adipic acid ions when KatG and cyclohexane are mixed. Figure 3(C) illustrates the relative abundance of adipic acid in the three samples as shown by the comparisons of peak area, showing an increase in abundance of detected adipic acid when KatG was mixed with cyclohexane.