ENZYMATIC DEGRADATION OF PLASTIC POLYALKENE POLYMERS BY KATG ENZYME

Abstract

The present invention relates to KatG enzymes and enzyme compositions and their uses in enzymatic degradation of plastics.

Claims

1. A composition suitable for use in the degradation of synthetic plastic polyalkene polymers, said composition comprising a KatG/EC 1.11.1.21 polyalkenase (PAase) enzyme and a suitable excipient.

2. A composition as claimed in claim 1, wherein the KatG/EC 1.11.1.21 PAase is a KatG enzyme.

3. A composition as claimed in claim 1 or 2, wherein the KatG enzyme comprises, consists of, or consists essentially of, the amino acid sequence of B2UBU5 KatG.sup.N, or a variant 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 B2UBU5 KatG.sup.N.

4. A composition as claimed in 1 or 2, wherein the KatG enzyme comprises, consists of, or consists essentially of, the amino acid sequence of B2UBU5, or a variant 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 B2UBU5.

5. A composition as claims 1 to 4, wherein the KatG enzyme is a B2UBU5 orthologue.

6. A composition as claimed in claim 5, wherein the B2UBU5 orthologue is selected from: A0A115SK77 (A0A115SK77_9 RALS) A0A0F0E7J1 (A0A0F0E7J1_9 BURK) A0A0J9DTW2 (A0A0J9DTW2_9 RALS) S9RSJ5 (S9RSJ5_9 RALS) A0A191ZZS8 (A0A191ZZS8_9 RALS) A0A5C6Y6C3 (A0A5C6Y6C3-9RALS) C6BD71 (C6BD71_RALP1) A0A2P4RN88 (A0A2P4RN88_RALPI) U3GIS7 (U3GIS7_9 RALS) A0A1COXEH7 (A0A1COXEH7_RALPI) R0CJV6 (R0CJV6_RALPI) A0A2N4TPC1 (A0A2N4TPC1_RALPI) A0A2N5LD46 (A0A2N5LD46_9RALS) A0A291E192 (A0A291E192_RALPI) A0A401K9C7 (A0A401K9C7-9RALS) UPI00046A45B6 UPI000DD31867 UPI000CEF361E UPI00073F3865 UPI00046AAE20 UPI0012AE63A9 UPI000D5F3903 UPI0004803C34 UPI000CEDCA44

7. A composition as claimed in any one of claims 1 to 6, wherein the KatG enzyme is a hyperactive KatG enzyme.

8. A composition, of any one of claims 1 to 7, wherein the KatG enzyme is a non-catalatic KatG variant.

9. A composition, of claim 8, wherein the non-catalatic KatG variant comprises a disrupted tripeptide adduct.

10. A composition of either claim 8 or 9, wherein the non-catalatic KatG variant is a non-catalatic B2UBU5 variant or a non-catalatic variant of any one of the orthologues of claim 6.

11. A composition, of claims 8 to 10, wherein the non-catalatic KatG variant comprises one or more of the following amino acid substitutions: M2481, M248V, Y221F, Y221A, Y221L, W98A, W98F and/or R410N.

12. A composition as claimed in any of claims 1 to 11, wherein the KatG enzyme is a single domain KatG enzyme.

13. A composition as claimed in claims 1 to 12 further comprising an enzyme capable of generating a peroxide.

14. A composition as claimed in claim 1 or 13 further comprising a plastic binding agent.

15. A composition as claimed in claim 13, wherein the hydrogen peroxide generating enzyme is selected from the group; formate oxidase, formate dehydrogenase, formaldehyde dismutase, methanol oxidase or combinations thereof.

16. A composition as claimed in any preceding claim, further comprising one or more adjunctive plasticase enzymes.

17. A composition as claimed in claim 16, wherein the adjunctive plasticase enzyme is selected from the group: (a) a laccase (EC 1.10.3.2.); (b) a manganese peroxidase (MnP, EC 1.11.1.13); (c) a lignin peroxidase (LiP, EC 1.11.1.14); and (d) combinations of two or more of the foregoing.

18. A composition as claimed in claim 17, wherein said one or more adjunctive plasticase enzymes comprise a complementary plasticase.

19. A composition as claimed in claim 18, wherein the complementary plasticase is selected from: (a) a laccase (EC 1.10.3.2.); (b) a manganese peroxidase (MnP, EC 1.11.1.13); (c) a lignin peroxidase (LiP, EC 1.11.1.14); (d) a versatile peroxidase; (e) a protease; (f) a lipase; (g) a carboxylesterases; (h) an esterase; and (i) combinations of two or more of the foregoing.

20. A composition of claim 19, wherein complementary plasticase comprises a cutinase and/or a PETase.

21. A composition of any one of the preceding claims, further comprising a waste remediation enzyme cocktail comprising one or more ancillary non-plasticase bioremediation enzymes.

22. A composition of claim 21, wherein said one or more ancillary bioremediation enzymes are selected from: (a) oxidoreductases; (b) oxygenases; (c) monooxygenases; (d) dioxygenases; (e) laccases; (f) peroxidases; (g) a hydrolase; (h) a peroxygenase, for example from Agrocybe aegerita; and (i) combinations of two or more of the foregoing.

23. A composition of claim 21 or claim 22, wherein the ancillary bioremediation enzymes comprise a hydrolase selected from: (a) a lipase; (b) a cellulase; (c) a protease; and (d) combinations of two or more of the foregoing.

24. A composition of any one of the preceding claims, further comprising one or more ancillary cellulose, hemicellulose and/or ligninase enzymes.

25. A composition of any one of the preceding claims, further comprising one or more cofactors.

26. A composition of claim 25, wherein said one or more cofactors comprises haem and/or iron and/or flavin mononucleotide (FMN) and/or FAD(H.sub.2) and/or NAD(H).

27. A composition of any preceding claim, wherein one or more the enzymes of the composition are produced in situ, by a recombinant host expressing and secreting said enzyme.

28. A composition as claimed in claim 27, wherein two or more enzymes are produced by expression and secretion from the host.

29. A composition as claimed in claim 27 or 28, wherein the host is selected from: Escherichia coli; Bacillus subtilis; Bacillus megaterium; Bacillus licheniformis; Lactococcus lactis; Ralstonia eutropha; Pseudomonas spp; Ralstonia pickettii; Corynebacterium spp; Saccharomyces cerevisiae; Hansenula polymorpha; Komagatella phaffii; Candida biodini; Schizosaccharomyces pombe a filamentous fungus, for example Aspergillus spp. and Trichoderma spp., and Myceliophthora thermophile, optionally in the form of a C1 expression system.

30. A composition of any one of the preceding claims, further comprises one or more microorganisms selected from the following genera: Achromobacter, Acinetobacter, Actinomadura, Alcaligines, Alcanivorax, Amycolatopsis, Aneurinibacillus, Archaeoglobus, Aromatoleum, Arthrobacter, Aspergillus, Azoarcus, Actinomadura, Bionectria, Isaria, Bacillus, Pseudomonas, Fusarium, Beauveria, Brevibacillus, Brevibacterium, Brevundimonas, Candida, Chaetomium, Citrobacter, Cladosporium, Comamonas, Coriolus, Coryneformes, Corynebacterium, Cunninghamella, Curvularia, Cycloclasticus, Delftia, Desulfococcus, Desulfosarcina, Dictyoglomus, Diplococcus, Engyodontium, Enterobacter, Exiguobacterium, Flavobacterium, Gliocladium, Gordonia, Halomonas, Hansenula, Kibdelosporangium, Klebsiella, Kluyveromyces, Leptothrix, Listeria, Marinobacter, Microbacterium, Micrococcus, Moraxella, Mortierella, Mucor, Mycobacterium, Nocardia, Ochrobactrum, Oleispira, Paecylomyces, Paenibacillus, Penicillium, Phanerochaete, Pleurotus, Proteobacterium, Proteus, Pseudomonas, Pseudozyma, Pullularia, Rahnella, Ralstonia, Rhizopus, Rhodococcus, Sphingomonas, Saccharomyces, Serratia, Sphingobacterium, Sphingomonas, Streptomyces, Staphylococcus, Stenotrophomonas, Streptococcus, Talaromyces, Thalassolituus, Thermomonospora, Trametes, Trichoderma, Tritirachium, Vibrio, Xanthomonas and combinations of two or more of the foregoing.

31. A composition as claimed in any preceding claim, wherein one or more of the enzymes are immobilised on a support.

32. A composition as claimed in any preceding claim, wherein the KatG enzyme is B2UBU5 or M1FBG9.

33. A composition as claimed in any preceding claim in the form of an aqueous solution, emulsion, suspension or dispersion.

34. A composition as claimed in any preceding claim for use in the degradation of a PA polymer.

35. A composition as claimed in claim any preceding claim for use in the bioremediation of material contaminated with a PA polymer.

36. A method for the enzymatic degradation of a PA polymer, the method comprising contacting the PA polymer with a composition of any one of claims 1 to 33.

37. A method as claimed in claim 35, wherein the enzymatic degradation of the PA polymer occurs in a reactor.

38. A method for producing fragments, oligomers, isolated monomers or repeating units, monomer or repeating unit fragments and/or monomer or repeating unit derivatives of a PA polymer, the method comprising the step of degrading said polymer by contacting it with a composition according to any of claims 1 to 33.

39. A method for producing a microbial biomass comprising the steps of: (a) providing a seed culture comprising at least one microorganism expressing a KatG/EC 1.11.1.21 PAase; (b) providing a feedstock comprising a PA polymer; (c) mixing said seed culture with said feedstock to form a seeded feedstock; and (d) incubating the seeded feedstock under conditions whereby C—C bonds in the PA polymer are cleaved to produce degradation products of said polymer which are metabolized by said microorganism to yield said microbial biomass.

40. A method for producing a bioproduct comprising the steps of: (a) providing a seed culture comprising at least one microorganism expressing a KatG/EC 1.11.1.21 PAase; (b) providing a feedstock comprising a PA polymer; (c) mixing said seed culture with said feedstock to form a seeded feedstock; and (d) incubating the seeded feedstock under conditions whereby C—C bonds in the PA polymer are cleaved by said enzyme to produce degradation products of said polymer which are metabolized by said microorganism to yield said bioproduct.

41. A method of claim 40, wherein the bioproduct comprises a biofuel.

42. A method of claim 40 or 41, wherein the bioproduct comprises one or more carboxylic acids, aldehydes, or dicarboxylic acids.

43. A method of claims 40 to 42, wherein the bioproduct comprises one or more compounds selected from the group: oxalic (ethanedioic), malonic (propanedioic), succinic (butanedioic), glutaric (pentanedioic), adipic(hexanedioic), pimelic (heptanedioic), suberic (octanedioic), azelaic (nonanedioic), sebacic (decanedioic), undecanedioc and dodecanedioic acids, glycolic acid, glyoxylic acid, citric acid, furoic acid and phenylpyruvic acids.

44. A method as claimed in claims 40 to 43, wherein the PA polymer is pre-treated abiotically.

45. A method as claimed in claim 44, wherein the abiotic pre-treatment is selected from mechanical, physical and chemical treatments or a combination of two or more thereof.

46. Use of a composition as claimed in any of claims 1 to 33, for the degradation of a PA polymer.

47. Use of a composition as claimed in any of claims 1 to 33, for producing fragments, oligomers, isolated monomers or repeating units, monomer or repeating unit fragments and/or monomer or repeating unit derivatives of a PA polymer.

48. Use of a composition as claimed in any of claims 1 to 33, for the bioremediation of material contaminated with a PA polymer.

49. A KatG prodegradant comprising a KatG/EC 1.11.1.21 polyalkenase enzyme (and/or a microorganism expressing and/or secreting a KatG/EC 1.11.1.21 polyalkenase enzyme), for use as a biotic prodegradant plastic additive, optionally wherein the PAase is present in the form of an inactive proenzyme.

50. A composite KatG plastic comprising a plastic polymer in combination with the KatG prodegradant of claim 50.

51. An autodegradative PA polymer comprising a PA polymer in combination with the KatG prodegradant of claim 50.

52. A composite KatG plastic or autodegradative polymer of claim 51 or 52, wherein the KatG prodegradant is contained within or disposed on the surface of the polymer.

53. A plastic article comprising the KatG prodegradant, composite KatG plastic or autodegradative PA polymer of any one of claims 51 to 53.

54. A KatG EC 1.11.1.21 enzyme comprising one or more of the following amino acid substitutions: M2481, M248V, Y221F, Y221A, Y221L, W98A, W98F and/or R410N.

55. A composition as claimed herein wherein the KatG/EC1.11.1.21 enzyme is the enzyme of claim 53.

Description

FIGURE LEGENDS

[0537] FIG. 1: Mass loss of polyethylene (grey) and polypropylene (black) for 100 mg (panel A) and 200 mg (panel B) incubations with Ralstonia pickettii (ATCC 27511) over a 28 day period.

[0538] FIG. 2: Biodegradation of LDPE film after incubation with Ralstonia pickettii (ATCC 27511).

[0539] FIG. 3: Effect of Ralstonia pickettii culture supernatant on the biodegradation of polyethylene beads.

[0540] FIG. 4: Biodegradation of low-density polyethylene (LDPE) films with Ralstonia pickettii supernatant.

[0541] FIG. 5: Biodegradation of polyethylene beads by a microbial extract containing recombinant Ralstonia pickettii KatG

[0542] FIG. 6: Biodegradation of polyethylene beads by purified recombinant His-tagged KatG

[0543] FIG. 7: High-throughput flow cytometric assay of degradation of fluorescent PS bead by recombinant KatG

[0544] FIG. 8: Plastic biodegradation using KatG-helper enzyme system

[0545] FIG. 9: Production of malonic acid from various polymers incubated with KatG and H.sub.2O.sub.2.

[0546] FIG. 10: Production of various compounds from polyethylene incubated with KatG and H.sub.2O.sub.2.

[0547] FIG. 11: Production of malonic acid from polyethylene incubated with KatG but without H.sub.2O.sub.2.

[0548] FIG. 12: Production of various compounds from polyethylene incubated with KatG but without H.sub.2O.sub.2.

[0549] FIG. 13: Mass spectrum of malonic acid as produced by incubation of polyethylene with KatG, and the mass spectrum of an authentic standard.

[0550] FIG. 14: Mass spectrum of sebacic acid as produced by incubation of polyethylene with KatG, and the mass spectrum of an authentic standard.

[0551] FIG. 15: Mass spectrum of azelaic acid as produced by incubation of polyethylene with KatG, and the mass spectrum of an authentic standard.

EXAMPLE 1: GROWTH OF RALSTONIA PICKETTII (ATCC 27511) ON PLASTIC FILMS

[0552] Liquid Carbon-Free Basal Medium (LCFBM) is a medium lacking any added carbon source and is described by Yang et al. (2014) Evidence of polyethylene biodegradation by bacterial strains from the guts of plastic-eating waxworms Environ Sci Technol 48: 13776-13784). Parallel 50 mL LCFBM cultures of R. picketti were set up with either 100 mg or 200 mg of sterilised PP or PE powders (Goonvean Fibres HM20/70P and Goodfellow ET316031). Cultures were inoculated with an existing stock of R. pickettii, and incubated at 30° C. for 3, 7, 10, 14, 21 and 28 days. Upon removal, each culture was spun down to remove the cells. The plastic was poured off, strained, and washed in 2% SDS solution for 4 h. The plastics were then washed in distilled water 4 times and left to dry overnight. They were then weighed to record gravimetric change.

[0553] Controls were prepared with polymers but without cells, using the same washing and drying process as all other cultures. No growth was observed nor polymer degradation.

[0554] FIG. 1 shows the % mass loss of polyethylene (grey) and polypropylene (black) for the 100 mg (A) and 200 mg (B) incubations.

[0555] LDPE film (Goodfellow ET311126) was cut into 50 mm×50 mm squares, disinfected in 70% ethanol, and dried overnight in a sterile hood. LCFBM agar was prepared (Yang et al. (2014) op. cit.) and 50 μL of a Ralstonia picketii culture was pipetted into the middle of the plate, before being spread by a sterile loop. An LDPE square was then placed over the inoculum, and the plate was sealed with Parafilm™ before incubation for 3 days at 30° C. Large areas of biodegradation are clearly observable (FIG. 2).

EXAMPLE 2: BIODEGRADATION OF PE BEADS WITH RALSTONIA PICKETTII CULTURE SUPERNATANT

[0556] 2.24 mg of 32-38 μm Cospheric PE Microspheres (UVPMS-BY2-1.00) were placed in a glass vial, and suspended in 1 mL of LCFBM (as above), and incubated for 2 days at 30° C. with 5 mM H.sub.2O.sub.2. Samples were vortexed and pipetted onto a glass slide before visualization under a benchtop microscope. The beads were unchanged (FIG. 3A)

[0557] R. pickettii cultures were grown with sterilised polypropylene at a concentration of 100 mg per 100 mL (Goonvean Fibres HM20/70P) in LCFBM (as above) for 3 days at 30° C. Cultures were centrifuged for 3 minutes at 10,000×g. The supernatant was removed, and was concentrated using an Amicon Stirred Cell Concentrator. 1 mL of 120-fold concentrated supernatant was added to 2.24 mg of 32-38 μm Cospheric microspheres (UVPMS-BY2-1.00 32-38 μm) together with 5 mM H.sub.2O.sub.2 and incubated for 2 days at 30° C. Samples were vortexed and pipetted onto a glass slide before visualization under a benchtop microscope. Much degradation had evidently occurred (FIG. 3B)

[0558] R. pickettii cultures were grown, and the supernatant obtained and concentrated, precisely as described above, save that before adding to the Cospheric microspheres the supernatant was boiled first for 10 min. The beads were unchanged (FIG. 3C).

EXAMPLE 3: BIODEGRADATION OF LOW-DENSITY POLYETHYLENE (LDPE) FILMS WITH RALSTONIA PICKETTII SUPERNATANT

[0559] 100 mg of LDPE film (Goodfellow ET311126) was sterilised by incubation in 0.1M Sodium Hydroxide for 1 hour followed by 5× washes with sterile water and 1 wash with LCFBM before drying in a sterile hood. The films were added to 50 mL of LCFBM, and incubated for 3 days. The films were imaged using a Keyence VHX-7000 microscope.

[0560] The films remained intact (FIG. 4A).

[0561] 100 mg of LDPE film (Goodfellow ET311126) was sterilised as described above. The films were added to 50 mL of R. pickettii supernatant prepared as described in Example 2 and incubated for 3 days. The films were imaged using a Keyence VHX-7000 microscope.

[0562] Extensive fragmentation of the polymer film was observed (FIG. 4B).

EXAMPLE 4: BIODEGRADATION OF POLYETHYLENE BEADS BY A MICROBIAL EXTRACT CONTAINING RECOMBINANT RALSTONIA PICKETTII KATG

[0563] KatG from R. picketii was cloned into a pET vector, E. coli BL21 cells were transformed to incorporate this plasmid. Lysates were prepared by growing 50 ml of culture at 30° C. in Auto Induction Terrific Broth (ForMedium) and resuspending the centrifuged bacterial pellet in 2 ml Acetate buffer (sodium acetate 45 mM, Acetic acid 55 mM, 50 mM sodium chloride, pH 4.5) supplemented with 0.1 mg/ml lysozyme. Samples were subjected to bead beating at 29.9 KHz for 2 min with approx. 30 mg of 0.5 mm Zirconia beads in a Tissue Lyser II (Qiagen) and subsequent incubation at 37° C. for 1h with shaking at 500 rpm. Cell free lysates were obtained by centrifugation of cell debris at 10,000 g for 10 min.

[0564] 2.24 mg of 32-38 μm Cospheric Microspheres (UVPMS-BY2-1.00 32-38 μm) were placed in a glass vial, and suspended in 200 μm of LCFBM (as above). H.sub.2O.sub.2 was added to a final concentration of 5 mM, and the sample was incubated for 2 days at 30° C. Samples were vortexed and visualised under a Keyence VHX-7000 microscope. No degradation was observed (FIG. 5A).

[0565] 2.24 mg of 32-38 μm Cospheric Microspheres (UVPMS-BY2-1.00 32-38 μm) were placed in a glass vial. 200 uL of recombinant KatG lysate was extracted from cells, and added to the beads. H.sub.2O.sub.2 was added to a final concentration of 5 mM. The sample was incubated for 2 days at 30° C. Samples were vortexed and visualised under a Keyence VHX-7000 microscope. Considerable degradation was observed (FIG. 5B).

[0566] 2.24 mg of 32-38 um Cospheric Microspheres (UVPMS-BY2-1.00 32-38 um) were placed in a glass vial. 200 uL of recombinant KatG lysate was extracted from cells, and boiled for 10 mins at 99° C. The lysate was cooled to room temperature, and added to the beads. H.sub.2O.sub.2 was added to a final concentration of 5 mM. The sample was incubated for 2 days at 30° C. Samples were vortexed and visualised under a Keyence VHX-7000 microscope. No degradation was observed (FIG. 5C).

EXAMPLE 5: BIODEGRADATION OF POLYETHYLENE BEADS BY PURIFIED RECOMBINANT HIS-TAGGED KATG

[0567] 2.24 mg of 32-38 μm Cospheric Microspheres (UVPMS-BY2-1.00 32-38 μm) were placed in a glass vial, and suspended in 200 μm of LCFBM (as above). H.sub.2O.sub.2 was added to a final concentration of 5 mM, and the sample was incubated for 2 days at 30° C. Samples were vortexed and visualised under a Keyence VHX-7000 microscope. No degradation was observed (FIG. 6A).

[0568] 2.24 mg of 32-38 μm Cospheric Microspheres (UVPMS-BY2-1.00 32-38 μm) were placed in a glass vial. 2.24 mg of 32-38 μm Cospheric Microspheres (UVPMS-BY2-1.00 32-38 μm) were placed in a glass vial. 10 mL of recombinant KatG lysate was extracted from cells using B-Per lysis buffer (Thermo) and purified using His-Pur Ni-NTA Magnetic Beads (Thermo) according to manufacturer's instructions. 200 μL of purified enzyme solution was added to the beads, and H.sub.2O.sub.2 was added to a final concentration of 5 mM. The sample was incubated for 2 days at 30° C. Samples were vortexed and visualised under a Keyence VHX-7000 microscope. Considerable degradation was observed (FIG. 6B).

EXAMPLE 6: BIODEGRADATION OF POLYPROPYLENE AND POLYETHYLENE BEADS BY PURIFIED RECOMBINANT HIS-TAGGED KATG AND KATG VARIANTS

[0569] Mass loss attendant on incubation with recombinant KatG and derivatives was evaluated using polypropylene (PP; Goonvean Fibres HM20/70P) and polyethylene (PE; Cospherics CPMS-0.96 38-45 μm) beads.

[0570] Pre-weighed 1.5 ml Eppendorf centrifugation tubes were supplemented with PP or PE beads and the tube weight rechecked to determine initial bead weight. 256-3 and 256-6 are BL21 E. coli lysates expressing recombinant R. pickettii KatG enzyme. 256-3 is the wild-type sequence, while 256-6 is a Y221F/R410N variant of the wild-type sequence. All recombinant KatG enzymes tested included a C-terminal His tag.

[0571] Lysates were prepared by growing 50 ml of culture at 37° C. in Auto Induction Terrific Broth (ForMedium) and resuspending the centrifuged bacterial pellet in 2 ml acetate buffer (sodium acetate 45 mM, Acetic acid 55 mM, 50 mM sodium chloride, pH 4.5) supplemented with 0.1 mg/ml lysozyme. Samples were subjected to bead beating at 29.9 kHz for 2 min with approx. 30 mg of 0.5 mm Zirconia beads in a Tissue Lyser II (Qiagen) and subsequent incubation at 37° C. for 1h with shaking at 500 rpm. Cell-free lysates were obtained by centrifugation of cell debris at 10,000 g for 10 min at 4° C.

[0572] A type strain of R. pickettii (ATCC 275111) was used to inoculate 500 ml of Liquid Carbon Free Basal Medium (see above) with 20 g of Cling film as carbon source and incubated at 30° C. for 5 days on a shaker. The supernatant was recovered by centrifugation of the cell pellet and concentration via an Amicon stirred cell concentrator and buffer exchange into the above acetate buffer to a final volume of approximately 2 ml.

[0573] For assay 400 μl of lysate/supernatant supplemented with H.sub.2O.sub.2 (5 mM final concentration) was vortexed with the beads and incubated at 30° C. for 24h at 200 RPM. Aqueous solution was separated from beads by centrifugation at 8000 g for 5 min and beads subsequently washed with water then ethanol with separation again being achieved by centrifugation. Residual ethanol was removed by vacuum centrifugation (SpeedVac, Thermo). The weight of the dry beads and tube was again checked to determine mass loss.

[0574] Table 1 (below) shows the mass loss after just 24h of incubation. Notable mass loss was observed for both polypropylene and polyethylene beads relative to incubation in buffer/H.sub.2O.sub.2 only.

TABLE-US-00007 TABLE 1 start end Mass Bead weight weight change % average Sample ID type (mg) (mg) (mg) loss % 256-3 PP 41.97 39.91 −2.06 4.91 6.47 256-3 PP 20.92 19.24 −1.68 8.03 256-6 PP 17.91 15.77 −2.14 11.95 11.62 256-6 PP 30.21 26.8 −3.41 11.29 R. Pickettii SN PP 20.29 15.36 −4.93 24.30 17.41 R. Pickettii SN PP 30.25 27.07 −3.18 10.51 Acetate Buffer PP 12.87 11.64 −1.23 9.56 5.35 Acetate Buffer PP 17.47 17.27 −0.20 1.14 256-3 PS 19.6 19.17 −0.43 2.19 3.35 256-3 PS 12.42 11.86 −0.56 4.51 256-6 PS 20.36 19.36 −1.00 4.91 6.21 256-6 PS 10.51 9.72 −0.79 7.52 R. Pickettii SN PS 11.14 11.03 −0.11 0.99 1.81 R. Pickettii SN PS 15.97 15.55 −0.42 2.63 Acetate Buffer PS 17.4 16.94 −0.46 2.64 1.64 Acetate Buffer PS 9.52 9.46 −0.06 0.63

EXAMPLE 7: HIGH-THROUGHPUT FLOW CYTOMETRIC ASSAY OF DEGRADATION OF FLUORESCENT PE BEAD BY RECOMBINANT KATG

[0575] Cell extract 256-3 was prepared as in Example 6. 200 μl was then used to desalt into PBS adjusted to pH 5 via a Zeba spin desalt column, to which 1.2 mg of Cospherics beads as described in Example 6 were added. The fluorescent beads consist of polymer spheres through which are dispersed individual ‘rocks’ of a yellow, water-insoluble fluorophore. 150 ul was used in the above assay.

[0576] Samples were analysed using an Intellicyt iQue Screener Plus; the data shown used excitation at 488 nm and emission at 675±30 nm. FIG. 7A shows a dot plot of side scatter (indicating size) vs fluorescence of particles detected (the BL4 detection channel on this instrument). The initial size of the beads means that their side scatter is actually off the scale (>10.sup.7 units). The discretised size of the fluorescence signal reflects the discrete nature of the fluorescent inclusions in the beads, as well as plastic-free fluorophore particles. Many of the events between 2,000 and 20,000 are background noise. It is very obvious that extremely extensive degradation of the beads has occurred, as observed in both the light scattering and fluorescence channels. FIG. 7B shows a subset of the data of A as a histogram of fluorescence, emphasising its discrete nature.

EXAMPLE 8: PLASTIC BIODEGRADATION USING KATG-HELPER ENZYME SYSTEM

[0577] An H.sub.2O.sub.2-regenerating system was employed using glucose (D+) and glucose oxidase from Aspergillus niger (both from Sigma). Assays were supplemented with 0.1M glucose and 2U glucose oxidase, but were otherwise performed with cell extract 256-3 as in Example 7 for a period of 4 days. FIG. 8A shows the control without enzyme, while FIG. 8B shows the degradative effect of the enzyme system.

EXAMPLE 9: PRODUCTION OF CHEMICALS BY PURIFIED KATG INCUBATED WITH POLYALKENE PLASTICS, WITH AND WITHOUT H.SUB.2.O.SUB.2

Lysate

[0578] Version 256-6. A Y221F/R410N katG mutant was transformed into BL21 E. coli and selected colonies were grown on in LB including carbenicillin. The LB culture was used to inoculate 1 L of Terrific Broth Auto Induction and grown for 24h at 30 C. The resulting bacterial growth was recovered by resuspension in 10 ml of 50 mM Sodium phosphate dibasic and 0.1 mg/ml Lysosyme and 100 mg/ml DNAse I and incubated with shaking for 1h at 30 C. The suspension was subject to cell lysis via a Constant Cell Disruption System (Constant Systems) and the disrupted lysate subject to centrifugation at 12000 g for 15 min. The resulting supernatant of the lysate was recovered and used in subsequent assays. The pH of this lysate was 6.4.

Purified KatG

[0579] Sumo Y221F/R410N katG is a His Tagged protein with a C-terminal peptide sequence recognized by a His-tagged Sumo protease to leave a katG enzyme with traceless tags after digestion. The coding sequence for katG mutant (Y221F/R410N) 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. The clone was prepared as described for 256-6 (as in Example 6) with the exception that the bacterial cells were resuspended in 20 mM 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 Äkta Start Chromatography system with fraction collector. The recombinant enzyme was purified by gradient elution with 20 mM 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 200 ml of 7 mg/ml Sumo protease overnight at 25° C. with dialysis using Snakeskin® dialysis tubing (Thermo Scientific) against 50 mM Tris-HCl, pH 8.0/150 mM NaCl/1 mM DTT. The dialysed, Sumo protease-digested KatG was then returned to the His Trap column and the tag-free mutant (Y221F/R410N) collected by flow through off column with the His tags retained on the column until gradient elution with imidazole.

[0580] The purified KatG mutant (Y221F/R410N) in 20 mM sodium phosphate buffer pH 7.4, 300 mM NaCl was then subjected to buffer exchange using PD-10 desalting columns into 0.1M sodium acetate pH 5 and used in subsequent assays. In this example, this preparation is used in the experiments below, and referred to as KatG.

Incubation Conditions

[0581] Ultra high molecular weight polyethylene (UHMWPE) powder (Sigma 434272), 14-20 μm diameter polystyrene microspheres (Cospheric PSMS-1.07), polyvinyl chloride (PVC) powder (Goodfellow 996-673-33), polypropylene (PP) powder (Goonvean Fibres HM20/70P) and Polytetrafluoroethylene (PTFE) powder (Goodfellow 531-672-48) were weighed into separate glass vials. Purified KatG mutant (Y221F/R410N) enzyme and 0.1 M acetate buffer pH5, were added to vials to give final enzyme and polymer concentrations of 1.25 μM (.sup.˜0.1 mg.Math.mL.sup.−1) and 5 mg/mL, respectively, in a total reaction volume of 2.5 mL. For each polymer two biological replicate reactions were set up alongside controls using purified KatG denatured by incubating with 10 mM DTT (REF) at 80° C. for 30 minutes and an enzyme negative control. All samples were incubated at 30° C. and 250 RPM. Every hour samples were removed and dosed with 2 μL 9.8 M hydrogen peroxide (Sigma H1009) diluted 1000× into the reaction mixture.

[0582] Aliquots were taken, quenched and stored at −80° C. for analysis by mass spectrometry (MS) prior to the first dose with hydrogen peroxide and incubation. For all samples excluding PTFE, aliquots were then taken after 5 and 10 hours of incubation. Only one aliquot was taken from the PTFE reaction after 5 hours of incubation. Aliquots were added to MS grade methanol pre-cooled to −80° C. Samples in methanol were then vortexed and centrifuged for 15 minutes at 4° C. and 14,000 RPM before transferring supernatants to microfuge tubes which were stored at −80° C. Specifically, the sample is diluted 1 in 4.5 (100 mL sample+350 mL methanol). Then 75 mL of that mix is dried and resuspended in 40 mL prior to injection.

[0583] This process was then repeated for UHMWPE without the addition of hydrogen peroxide. Aliquots were taken and quenched for MS as described above after 5 hours and 57 hours of incubation.

[0584] Mass Spectrometry

[0585] Mass spectra of samples incubated as above were obtained in both positive and negative mode on an Orbitrap instrument using the methods described and published in Open Access form in Wright Muelas M, Roberts I, Mughal F, O'Hagan S, Day P J, Kell D B: An untargeted metabolomics strategy to measure differences in metabolite uptake and excretion by mammalian cell lines. Metabolomics 2020; 16:107. Those displayed here were obtained in using negative ionisation.

Proton NMR Spectroscopy

[0586] .sup.1H 1D NMR spectra were acquired on a Bruker 700 MHz Avance IIIHD spectrometer equipped with a TCI cryoprobe, using standard vendor-supplied pulse sequences (noesygppr1d and cpmgpr1d) acquired with 4s interscan delay 32 transients and 64k points at 298K (25° C.). Temperature calibrated using 99.8% deuterated methanol standard and 3D shimming on standard 2 mM Sucrose reference for quality assurance. Sample quality control for all spectra was performed on the linewidth of acetic acid.

[0587] Compounds identified in various extracts, that typically differed in molecular weight by 14, 28 or 56 amu, included oxalic (ethanedioic), malonic (propanedioic), succinic (butanedioic), glutaric (pentanedioic), adipic(hexanedioic), pimelic (heptanedioic), suberic (octanedioic), azelaic (nonanedioic), sebacic (decanedioic), undecanedioc and dodecanedioic acids. On occasion, glycolic acid, glyoxylic acid, citric acid, furoic and phenylpyruvic acids were also observed.

[0588] Malonic and citric acids were specifically confirmed by NMR spectroscopy.

EQUIVALENTS

[0589] The foregoing description details presently preferred embodiments of the present invention. Numerous equivalents, modifications and variations in practice thereof are expected to occur to those skilled in the art upon consideration of these descriptions. Those modifications and variations are, or are intended to be, encompassed by the following claims.