PROCESS FOR OXIDISING A SUBSTRATE

Abstract

A process for oxidising a substrate selected from hydroxymethylfurfural (HMF), diformylfuran (DFF), hydroxymethylfurancarboxylic acid (HMFCA) and formylfurancarboxylic acid (FFCA). Said process comprises mixing said substrate with catalase, one or more further enzymes and hydrogen peroxide to form a reaction mixture. Said one or more further enzymes have the ability to catalyse oxidation of said substrate. Said hydrogen peroxide is provided at a total molar ratio of at least about 0.1:1 hydrogen peroxide to substrate.

Claims

1. A process for oxidising a substrate selected from hydroxymethylfurfural (HMF), diformylfuran (DFF), hydroxymethylfurancarboxylic acid (HMFCA) and formylfurancarboxylic acid (FFCA), said process comprising: mixing said substrate with catalase, one or more further enzymes and hydrogen peroxide to form a reaction mixture; wherein said one or more further enzymes have the ability to catalyse oxidation of said substrate and wherein said hydrogen peroxide is provided at a total molar ratio of at least about 0.1:1 hydrogen peroxide to substrate.

2. The process as claimed in claim 1, wherein said hydrogen peroxide is provided at a total molar ratio of at least about 1:1 hydrogen peroxide to substrate, optionally at least about 2:1 hydrogen peroxide to substrate.

3. The process as claimed in any preceding claim, wherein said hydrogen peroxide is provided at a total molar ratio of at least about 3:1 hydrogen peroxide to substrate.

4. The process as claimed in any preceding claim, wherein said hydrogen peroxide is provided at a total molar ratio of up to about 20:1 hydrogen peroxide to substrate, optionally about 15:1, optionally about 10:1 hydrogen peroxide to substrate.

5. The process as claimed in any preceding claim, wherein said mixing comprises mixing said substrate with catalase and one or more further enzymes to form a mixture, the process comprising adding hydrogen peroxide to the mixture to form the reaction mixture.

6. The process as claimed in any preceding claim, wherein said mixing comprises mixing said substrate with catalase and one or more further enzymes to form a mixture, the process comprising providing a flow of said mixture and adding hydrogen peroxide to the mixture flow to form the reaction mixture.

7. The process as claimed in claim 6, wherein said flow follows a tortuous passageway having one or more bends.

8. The process as claimed in claim 6 or 7, wherein said hydrogen peroxide is added at the bend, a subset of said bends or each bend.

9. The process as claimed in any one of claims 5 to 8, wherein said adding hydrogen peroxide comprises adding hydrogen peroxide at an amount sufficient to bring the molar ratio of the hydrogen peroxide in the reaction mixture to within about 50% of a predetermined molar ratio, optionally within about 40%, optionally within about 30%, optionally within about 20%, optionally within about 10%, optionally within about 5%, optionally within about 5% of said predetermined molar ratio, optionally approximately to said predetermined ratio.

10. The process as claimed in any one of claims 5 to 9, wherein said adding comprises pumping hydrogen peroxide.

11. The process as claimed in any one of claims 5 to 10, wherein said adding comprises adding multiple individual streams of hydrogen peroxide.

12. The process as claimed in any one of claims 5 to 11, wherein said adding comprises adding at least 5 individual streams of hydrogen peroxide, optionally at least 7, optionally at least 10, optionally at least 11 individual streams of said hydrogen peroxide.

13. The process as claimed in any one of claims 5 to 12, wherein said adding comprises continuously adding or periodic adding.

14. The process as claimed in claim 13, wherein said periodic adding comprises adding 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (or more) portions of said hydrogen peroxide.

15. The process as claimed in claim 13 or 14, wherein said periodic adding comprises adding two or more portions of hydrogen peroxide, the addition of each portion being separated by a time interval of from about 1 second to about 60 minutes, optionally about 5 minutes to about 25 minutes, optionally about 10 minutes to about 20 minutes, optionally about 15 minutes.

16. The process as claimed in any one of claims 6 to 15, wherein said flow has a velocity and wherein the process is configured to maintain a predetermined flow velocity along a path of the flow.

17. The process as claimed in claim 16, wherein said provision of hydrogen peroxide is configured to maintain the predetermined flow velocity.

18. The process as claimed in any preceding claim, wherein said one or more further enzymes comprise metal.

19. The process as claimed in any preceding claim, wherein said one or more further enzymes comprise iron, molybdenum and/or copper metal.

20. The process as claimed in any preceding claim, wherein the substrate is selected from 2,5-diformylfuran (DFF), 5-hydroxymethyl-2-furancarboxylic acid (HMFCA) and/or 5-formylfuran-2-carboxylic acid (FFCA).

21. The process as claimed in any preceding claim, wherein the process for the formation of furandicarboxylic acid, optionally 2,5-furandicarboxylic acid (2,5-FDCA).

22. The process as claimed in any preceding claim, wherein said one or more further enzymes comprise xanthine oxidoreductase (XOR), aldehyde oxidase, aldehyde dehydrogenase (ALD), alcohol oxidase, galactose oxidase variant (such as galactose oxidase variant M.sub.3-5, GOase M.sub.3-5), ketoreductase (KRED) and/or nicotinamide oxidase (NOX).

23. The process as claimed in claim 22, wherein said xanthine oxidoreductase is selected from E. coli XDH, Rhodococcus capsulatus xanthine dehydrogenase (XDH) single variant E232V, and double mutant XDH E232 V/R310, and periplasmic aldehyde oxidase (PaoABC).

24. The process as claimed in claim 22 or 23, wherein the xanthine oxidoreductase is periplasmic aldehyde oxidase (PaoABC).

25. The process as claimed in any preceding claim, wherein said one or more further enzymes comprise galactose oxidase variant M.sub.3-5 (GOase M.sub.3-5).

26. The process as claimed in any preceding claim, wherein said one or more further enzymes comprise (a) galactose oxidase variant (such as galactose oxidase variant M.sub.3-5 (GOase M.sub.3-5)] and/or ketoreductase (KRED); and (b) one or more of xanthine oxidoreductase (XOR), aldehyde dehydrogenase (ALD) and nicotinamide oxidase (NOX).

27. The process as claimed in any preceding claim, wherein said catalase is immobilised.

28. The process as claimed in any preceding claim, wherein said one or more further enzymes comprise one or more immobilised enzymes.

29. The process as claimed in claim 28, wherein said one or more further enzymes comprise immobilised periplasmic aldehyde oxidase (PaoABC).

30. The process as claimed in claim 28 or 29, wherein said one or more further enzymes comprise immobilised galactose oxidase variant [such as galactose oxidase variant M.sub.3-5 (GOase M.sub.3-5)].

31. The process as claimed in any preceding claim, wherein said mixing further comprises mixing with horseradish peroxidase (HRP) and/or metal complex.

32. The process as claimed in any preceding claim, wherein said mixing further comprises mixing with nicotinamide adenine dinucleotide phosphate (NADP.sup.+) and/or nicotinamide adenine dinucleotide (NAD.sup.+).

33. The process as claimed in any preceding claim, wherein said one or more further enzymes comprises nicotinamide oxidase (NOX) and wherein said mixing further comprises mixing with nicotinamide adenine dinucleotide phosphate (NADP.sup.+) and/or nicotinamide adenine dinucleotide (NAD.sup.+).

34. The process as claimed in any preceding claim, wherein one or more further enzymes comprises ketoreductase (KRED) and wherein said mixing comprises mixing with nicotinamide adenine dinucleotide phosphate (NADP.sup.+) and/or nicotinamide adenine dinucleotide (NAD.sup.+).

35. The process as claimed in any preceding claim, wherein said diformylfuran (DFF), hydroxymethylfurancarboxylic acid (HMFCA) and/or formylfurancarboxylic acid (FFCA) is/are obtained by oxidation of hydroxymethylfurfural (HMF).

36. The process as claimed in any preceding claim, wherein said hydroxymethylfurfural (HMF) is obtained from glucose and/or fructose.

37. The process as claimed in claim 36, wherein said glucose and/or fructose is obtained from cellulose.

38. The process as claimed in claim 37, wherein said cellulose is obtained from lignocellulose.

39. An apparatus for a flow process of oxidising a substrate, said apparatus comprising: a primary tube defining a primary fluid passageway for flow of a first fluid; secondary tubing defining a secondary passageway for adding one or more further fluids to the primary fluid passageway, said secondary tubing having one or more apertures to permit fluid communication of the secondary passageway with the primary passageway.

40. The apparatus according to claim 39, wherein the first fluid is liquid.

41. The apparatus according to claim 39 or 40, wherein the total cross-sectional area of the aperture(s) of the secondary tubing is about 10 to 30 times smaller than the cross-sectional area of the primary passageway, optionally about 15 to 25 times smaller, optionally about 20 times smaller than the cross-sectional area of the primary passageway.

42. The apparatus as claimed in any one of claims 39 to 41, wherein said apparatus is configured to control flow of fluid through said primary passageway and/or flow of fluid through said secondary passageway based on a molar ratio of components in the first fluid of the primary passageway.

43. The apparatus according to any one of claims 39 to 42, wherein the primary tube is configured to provide a tortuous passageway, having one or more bends.

44. The apparatus as claimed in claim 43, wherein the or each aperture of the secondary tubing is provided at the bend, a subset of said bends or each bend in the primary passageway.

45. The apparatus according to any one of claims 39 to 44, wherein the primary tube is provided with one or more flow disruptors.

46. The apparatus according to any one of claims 39 to 45, wherein the flow disruptor(s) are particles, such as inert particles, optionally glass beads.

47. The apparatus according to any one of claims 39 to 46, wherein the flow disruptor(s) may be sized to closely fit inside the primary tube.

48. The apparatus as claimed in any one of claims 39 to 47, wherein said secondary tubing comprises one or more main secondary tubes, the or each main secondary tube defining an individual fluid passageway and being in fluid communication with a series of further subordinate secondary tubes, said secondary tubes each having the said apertures for fluid communication with the primary passageway, said further subordinate secondary tubes fluidly connecting the primary passageway and the fluid passageway of the main secondary tube.

49. The apparatus as claimed in any one of claims 39 to 48, wherein said secondary tubing comprises at least 5 individual fluid passageways, optionally at least 7, optionally at least 10, optionally at least 11 individual fluid passageways.

50. The apparatus as claimed in any one of claims 39 to 49, wherein a cross-sectional area and/or length of tubes in the secondary tubing may be configured to maintain a predetermined flow velocity along the length of the primary tube.

51. The apparatus as claimed in any one of claims 39 to 50, wherein a cross-sectional area of the primary tube may be configured to maintain a predetermined flow velocity along the length of the primary tube.

52. A process for the formation of a mono- or diester of furandicarboxylic acid from furandicarboxylic acid, the process comprising mixing furandicarboxylic acid, an alcohol and a catalyst, wherein the furandicarboxylic acid is obtained by a process as defined in any one of claims 1 to 38.

53. The process as claimed in claim 52, wherein said alcohol is selected from methanol and ethanol.

54. The process as claimed in claim 52 or 53, wherein the catalyst is an organic acid or inorganic acid.

55. The process as claimed in any one of claims 52 to 54, wherein the catalyst is sulphuric acid.

56. A process for the formation of a copolymer comprising the copolyester of: (a) furandicarboxylic acid (FDCA) and/or a mono- or diester of furandicarboxylic acid; and (b) at least one diol; wherein the process comprises polymerising components (a) and (b) wherein the furandicarboxylic acid is obtained by a process as defined in any one of claims 1 to 38, and/or wherein the mono- or diester of furandicarboxylic acid is obtained by a process as defined in any one of claims 52 to 55.

57. The process as claimed in claim 56, wherein the furandicarboxylic acid, or mono- or diester of furandicarboxylic acid is selected from ##STR00017## and a combination thereof.

58. The process as claimed in claim 56 or 57, wherein the mono- or diester of furandicarboxylic acid is selected from ##STR00018## and a combination thereof.

59. The process as claimed in any one of claims 56 to 58, comprising said at least one mono- or diester of furandicarboxylic acid.

60. The process as claimed in any one of claims 56 to 59, wherein said at least one diol comprises an aliphatic diol.

61. The process as claimed in any one of claims 56 to 60, wherein the at least one diol comprises: ##STR00019## wherein R.sup.2 is a straight, branched or cyclic C.sub.2 to C.sub.10 alkylene.

62. The process as claimed in any one of claims 56 to 61, wherein the at least one diol comprises a diol selected from 1,2-ethanediol, 1,4-butanediol, and a combination thereof.

63. The process as claimed in any one of claims 56 to 62, wherein the copolymer comprises the copolyester of: (a) at least one furandicarboxylic acid (FDCA) or a mono- or diester of furandicarboxylic acid; (b) at least one diol; and (c) at least one dicarboxylic acid or a mono- or diester derivative thereof.

64. The process as claimed in claim 63, wherein said at least one dicarboxylic acid, or mono- or diester derivative thereof acid, comprises an aliphatic, heteroaromatic and/or aromatic dicarboxylic acid, or mono- or diester derivative thereof.

65. The process as claimed in claim 63 or 64, wherein said at least one dicarboxylic acid, or mono- or diester derivative thereof acid, comprises an aliphatic dicarboxylic acid, or mono- or diester derivative thereof.

66. The process as claimed in claim 65, wherein the aliphatic dicarboxylic acid, or mono- or diester derivative thereof, is: ##STR00020## wherein R.sup.3 is a straight, branched or cyclic, C.sub.1 saturated or C.sub.2 to C.sub.10 saturated or unsaturated alkylene, and wherein each R.sup.4 independently represents H or a straight, branched or cyclic, C.sub.1 to C.sub.8 (optionally C.sub.1 to C.sub.6) alkyl group.

67. The process as claimed in claim 65 or 66, wherein the aliphatic dicarboxylic acid, or mono- or diester derivative thereof, is selected from adipic acid, adipic acid monomethyl ester, adipic acid dimethyl ester, adipic acid monoethyl ester, adipic acid diethyl ester, succinic acid, succinic acid monomethyl ester, succinic acid dimethyl ester, succinic acid monoethyl ester, succinic acid diethyl ester, and a combination thereof.

68. The process as claimed in any one of claims 64 to 67, wherein said at least one dicarboxylic acid, or mono- or diester derivative thereof, comprises an aromatic dicarboxylic acid, or mono- or diester derivative thereof.

69. The process as claimed in any one of claims 56 to 68, wherein the copolymer comprises the copolyester of: (A) the dimethyl ester or diethyl ester of furandicarboxylic acid (FDCA) (optionally 2,5-furandicarboxylic acid (2,5-FDCA)); (B) 1,4-butanediol; and (C) dimethyl ester or diethyl ester of adipic acid.

70. The process as claimed in any one of claims 56 to 69, comprising the diethyl ester of furandicarboxylic acid.

71. The process as claimed in any one of claims 56 to 70, wherein the copolymer comprises the copolyester of (a) from 1 to 98 mol % of at least one 2,5-furandicarboxylic acid or a mono- or diester of 2,5-furandicarboxylic acid (2,5-FDCA); (b) from 1 to 98 mol % of at least one diol; and (c) when present, from 1 to 98 mol % of at least one aliphatic dicarboxylic acid or a mono- or diester derivative thereof.

72. A copolymer obtainable by a process as defined in any one of claims 56 to 71.

73. A process or copolymer substantially as hereinbefore described, with reference to the figures.

Description

DESCRIPTION OF THE FIGURES

[0244] FIG. 1 shows the .sup.1H NMR spectra for 2,5-polybutyrate adipate furandicarboxylate (2,5-PBAF), i.e. a copolymer of the invention.

[0245] FIG. 2 shows the 1H NMR spectra for polybutyrate adipate terephthalate (PBAT) (Comparative Example 8).

[0246] FIG. 3 shows the .sup.1H NMR spectra for commercial PBAT.

[0247] FIG. 4 shows the Simultaneous Thermal Analysis (STA) trace for 2,5-PBAF.

[0248] FIG. 5 shows the STA trace for PBAT (Comparative Example 8).

[0249] FIG. 6 shows the STA trace for commercial PBAT.

[0250] FIG. 7 shows Differential Scanning calorimetry (DSC) traces for 2,5-PBAF, PBAT (Comparative Example 8) and commercial PBAT.

[0251] FIG. 8 shows the Gel Permeation Chromatography (GPC) spectra for 2,5-PBAF.

[0252] FIG. 9 shows the Gel Permeation Chromatography (GPC) spectra for Comparative Example 8.

[0253] FIG. 10 shows the GPC spectra for commercial PBAT.

[0254] FIG. 11 shows the relative amount of DFF, FFCA and 2,5-FDCA as a function of time during the process described in Example 1.

[0255] FIG. 12 shows the relative amount of DFF, FFCA and 2,5-FDCA as a function of time during the process described in Example 2.

[0256] FIG. 13 shows the relative amount of DFF, FFCA and 2,5-FDCA as a function of time during the process described in Example 3.

[0257] FIG. 14 shows the relative amount of DFF, FFCA and 2,5-FDCA as a function of time during the process described Example 4.

[0258] FIG. 15 shows a comparison of the amount of 2,5-FDCA produced as a function of time during the processes described in Examples 1 to 4.

[0259] FIG. 16 shows that under the biodegradation test conditions outlined in Example 12, 2,5-PBAF result in a carbon loss of 29.3% after 40 days. The 90% level set for biodegradation in the test accounts for a +1-10% statistical variability of the experimental measurement, which one would expect virtually complete biodegradation in the composting environment of the test.

[0260] FIG. 17 shows that under the biodegradation test conditions outlined in Example 12, 2,5-PBAF loses carbon at a steady rate for over 60 days. The 90% level is as defined for FIG. 16 above.

[0261] FIG. 18 shows the attenuated total reflectance Fourier transform infrared spectra (ATR-FTIR) of 2,5-polybutyrate adipate furandicarboxylate (2,5-PBAF) using a Thermo Nicolet Nexus FT-IR spectrometer coupled with a Continuum IR microscope.

[0262] FIG. 19 shows the attenuated total reflectance Fourier transform infrared spectra (ATR-FTIR) of commercial PBAT using a Thermo Nicolet Nexus FT-IR spectrometer coupled with a Continuum IR microscope.

[0263] FIG. 20 is a perspective view of an apparatus for a continuous flow process of oxidising a substrate, as explained in Example 13.

[0264] FIG. 21 is a front view of an apparatus for a continuous flow process of oxidising a substrate, as explained in Example 14.

[0265] FIG. 22 shows calibration curves for quantitative gas chromatography (GC) and high-performance liquid chromatography (HPLC) analysis of hydroxymethylfurfural (HMF) and diformylfuran (DFF), as explained in Example 15.

[0266] FIG. 23 shows three GC traces for continuous-flow oxidation of 5-hydroxymethylfurfural, as explained in Example 15.

[0267] FIG. 24 shows results of a continuous run of HMF conversion using a trickle bed reactor, as explained in Example 16.

[0268] FIG. 25 shows a graphical representation of suitable flow velocity for use in the apparatus depicted in FIG. 20.

[0269] The following examples are merely illustrative examples of the invention described herein and are not intended to be limiting upon the scope of the invention.

EXAMPLES

General Experimental Information and Materials

[0270] The E. coli TP1000 mutant strain used for PaoABC expression is a derivative of MC4100 with a kanamycin cassette inserted in the mobAB gene region. E. coli xanthine dehydrogenase and catalase were sourced from Sigma-Aldrich. Starting materials were purchased from Alfa Aesar and Sigma-Aldrich and used as received. HPLC analysis was performed on an Agilent 1200 system equipped with a G1379A degasser, G1312A binary pump, a G1329 autosampler unit, a G1315B diode array detector and a G1316A temperature controlled column compartment. The columns used were Thermofisher Hypurity C18 (5 m particle size, 4.6 mm diameter250 mm), Thermofisher ODS Hypersil C18 (5 m particle size, 4.6 mm diameter250 mm) and Bio-Rad Aminex HPX-87H, 300 mm7.8 mm pre-packed column. GC analysis was performed on an Agilent 7890A chromatograph using an Alltech SE-30, 30.0 m320 m0.25 m column. Conditions are indicated separately for each compound. .sup.1H NMR spectra were recorded on a Bruker Avance 400 or 500 without additional internal standard.

Preparation of Enzymes

[0271] Galactose Oxidase Variant M.sub.3-5 (GOase M.sub.3-5)

[0272] GOase mutant M.sub.3-5 (Escalettes; Turner J. ChemBioChem 2008, 9, 857-860) was transformed into E. coli BL21 Star (DE3) cells (Invitrogen) according to manufacturer's specifications. A single colony was picked from an overnight LB plate containing 1 L of kanamycin of a 30 mg/mL stock solution per mL of agar and used to inoculate 5 mL LB medium supplemented with 5 L kanamycin and grown overnight at 37 C. and 250 rpm. 500 L of the overnight culture was used to inoculate 250 mL of an autoinduction medium (8ZY-4LAC) as described by Deacon and McPherson (Deacon; McPherson J. ChemBioChem. 2011, 12, 593-601) and supplemented with 250 L of kanamycin in a 2-L-baffled Erlenmeyer flask. The cells were grown at 26 C. and 250 rpm for 60 hour. Cells were harvested by centrifugation at 6,000 rpm and 40 C. for 20 min and subsequently prepared for protein purification.

Purification of GOase M.SUB.3-5

[0273] The cell pellet from a 250-mL-culture was resuspended in 30 mL lysis buffer containing 50 mM piperazine-N,N-bis(2-ethanesulfonic acid) (PIPES), 25% sucrose (w/v), 1 mg mL.sup.1 lysozyme, 5 mM MnCl.sub.2 and 1% Triton X-100 (v/v). The suspension was gently shaken at 4 C. for 20 min. Afterwards, cells were mechanically disrupted via ultrasonication (30 sec. on, 30 sec. off; 20 cycles) followed by ultracentrifugation (20,000g, 30 min, 4 C.). The cleared crude extract was transferred into a flexible tubing (30 kDa cut-off), dialysed into buffer C (50 mM NaPi buffer, 300 mM NaCl, pH 8.0) for 12 hours at 4 C. and subsequently passed through a syringe filter with a 0.22 m pore size. Protein purification was accomplished with a peristaltic tubing pump (Thermo Scientific) equipped with a 5-mL-Strep-Tag-II column (GE Healthcare) pre-equilibrated with buffer C. After loading with crude extract, the column was washed with 5 column volumes of buffer C followed by protein elution with 70 mL of buffer D (50 mM NaPi buffer, 300 mM NaCl, 5 mM desthiobiotin, pH 8.0).

[0274] For copper-loading, GOase Mm-containing fractions were pooled and subsequently transferred into flexible dialysis tubing (30 kDa cut-off) and dialysed twice for 12 hours into buffer E (50 mM NaPi buffer saturated with CuSO.sub.4, pH 7.4) at 4 C. Removal of excess CuSO.sub.4 was attained by two cycles of dialysis into buffer E (without CuSO.sub.4) for 12 hours at 4 C. and protein samples concentrated to approximately 3 mg/mL using a Sartorius Vivaspin 6 spin column (30 kDa mass cut-off). The protein samples were aliquoted and the aliquots were frozen in liquid nitrogen prior to storage at 80 C.

E. coli Perisplasmic Aldehyde Oxidase (PaoABC)

[0275] For PaoABC expression (Neumann et al. FEBS Journal 2009, 276, 2762-2774), the plasmid pMN100 derived from pTrcHisA (Invitrogen), containing the PaoABC genes with a His6 tag fused to the N-terminus of PaoA, was used. For heterologous expression in E. coli, pMN100 was transformed into E. coli TP1000 cells, containing a deletion in the mobAB genes responsible for Moco dinucleotide formation. One litre of LB supplemented with 1 mM sodium molybdate and 10 M isopropyl thio--D-galactoside was inoculated with 2 mL of an overnight culture and incubated for 24 hours at 22 C. and 100 rpm. The cells were harvested by centrifugation at 4,000g for 15 min.

Purification of PaoABC

[0276] The cell pellet was resuspended in 8 volumes of 50 mM sodium phosphate, 300 mM NaCl, pH 8.0, 10 mM imidazole and cell lysis was achieved by sonication (MSE Soniprep) with cooling on ice (20 bursts of 20 s on/off at 14 u). After addition of DNase I, the lysate was incubated for 30 min. After centrifugation at 17,000g for 25 min the supernatant was filtered through 0.45 and 0.2 M membranes before loading onto Ni.sub.2-nitrilotriacetic agarose (HiTrap 1 mL column (GE Healthcare)). The column was washed with 2 column volumes of 10 mM imidazole, 50 mM sodium phosphate, 300 mM NaCl, pH 8.0, followed by a wash with 10 column volumes of the same buffer with 20 mM imidazole. His-tagged PaoABC was eluted with 20 mL of 100 mM imidazole in 50 mM sodium phosphate, 300 mM NaCl, pH 8.0. Fractions containing PaoABC were buffer exchanged into 50 mM Tris, 1 mM EDTA, pH 7.5. The yield of protein was about 13 mg/L of E. coli culture.

Xanthine Dehydrogenase Variants E232V and E232VR310 (XDH E232V, XDH E232VR310)

[0277] For expression of XDH mutants, the plasmid pSL207 derived from pTrcHisA (Invitrogen), containing the xdh genes with a His6 tag fused to the N-terminus of XDHA, was used. For heterologous expression in E. coli, pSL207 was transformed into E. coli TP1000 cells, containing a deletion in the mobAB genes responsible for Moco dinucleotide formation. The enzyme was expressed in 500-mL-cultures of TP1000 cells carrying plasmid pSL207 grown at 30 C. in LB medium supplemented with 150 g/mL ampicillin, 1 mM molybdate, and 0.02 mM isopropyl-D-thiogalactopyranoside until the OD.sub.600nm=1. This culture was then transferred to a bottle containing 8 L of supplemented LB medium and subsequently grown at 30 C. for 18 to 20 hours. Cells were harvested by centrifugation at 5000g at 4 C. and subsequently prepared for protein purification.

Purification of XDH E232V and XDH E232VR310

[0278] The cell pellet was resuspended in eight volumes of 50 mM sodium phosphate, 300 mM NaCl, pH 8.0, and cell lysis was achieved by several passages through a French press. After addition of DNase I, the lysate was incubated for 30 min. After centrifugation at 17,000g for 25 min, imidazole was added to the supernatant to a final concentration of 10 mM. The supernatant was mixed with 2 mL of Ni.sub.2-nitrilotriacetic agarose (Qiagen) per litre of cell growth, and the slurry was equilibrated with gentle stirring at 4 C. for 30 min. The slurry was poured into a column, and the resin was washed with two column volumes of 10 mM imidazole, 50 mM sodium phosphate, 300 mM NaCl, pH 8.0, followed by a wash with ten column volumes of the same buffer with 20 mM imidazole. His-tagged XDH was eluted with 100 mM imidazole in 50 mM sodium phosphate, 300 mM NaCl, pH 8.0. Fractions containing XDH were combined and dialyzed against 50 mM Tris, 1 mM EDTA, 2.5 mM dithiothreitol, pH 7.5. The dialyzed sample was applied to a Q-Sepharose fast protein liquid chromatography column and eluted with a linear gradient of 0-250 mM NaCl. To the pool of fractions containing XDH, 15% ammonium sulphate was added, and the protein was then applied to a phenyl-Sepharose column equilibrated with 50 mM Tris, 1 mM EDTA, 2.5 mM dithiothreitol, 15% ammonium sulphate, pH 7.5. XDH E232V was eluted from the column with a linear gradient of from 15 to 0% ammonium sulphate. During purification, fractions were monitored using SDS-PAGE, whereas enzyme activity was measured spectrophotometrically as described above.

Screening of Xanthine Oxidoreductases for Oxidation of HMF, DFF and FFCA

[0279] XORs were screened using potassium phosphate buffer (50 mM, pH 7.6), 3 L 0.1 M HMF (in MeCN), 30 L 0.01 M DCPIP (aq.) final volume 300 L, 36 C. When DCPIP was the oxidant, activity was determined by the colour change from blue to colourless; when O.sub.2 was the oxidant, activity was detected using NBT assay (Agarwal; Banerjee Open Biotech J. 2009, 3, 46-49). The results can be found in Table 1.

TABLE-US-00001 TABLE 1 Screening of xanthine oxidoreductases Enzyme HMF DFF FFCA Oxidant E. coli XDH.sup.a Active O.sub.2 XDH E232V.sup.b Active Active Active DCPIP XDH E232V R310.sup.c Active Active Active DCPIP PaoABC.sup.d Active Active Active O.sub.2 .sup.aE. coli XDH (1.1 mg/mL); .sup.bXDH E232V (25.4 mg/mL); .sup.cXDH E232V/R310M (23 mg/mL); .sup.dPaoABC (13.3 mg/mL); .sup.ePaoABC (13.3 mg/mL).

Example 1No Initial Shaking of Buffer

[0280] To 490 L of 0.2 M KPi buffer pH 7.0 was added DFF (final concentration 100 mM) and 1 mg catalase. 10 L of a 100 M PaoABC was then added and the reaction was left in a shaking incubator. 5 L of the reaction mixture was extracted, diluted with 80 L water and quenched with 15 L 1 M HCl. The aliquots were analysed by RP HPLC. The results are shown in FIG. 11.

Example 2Initial Shaking of Buffer

[0281] To 490 L of 0.2M KPi buffer pH 7.0 was added DFF (final concentration 100 mM) and 1 mg catalase. The Eppendorf was vigorously shaken. 10 L of a 100 M PaoABC was then added and the reaction was left in a shaking incubator. 5 L of the reaction mixture was extracted, diluted with 80 L water and quenched with 15 L 1M HCl. The aliquots were analysed by RP HPLC. The results are shown in FIG. 12.

Example 3Oxygen Sparged Buffer

[0282] To 490 L of 0.2 M KPi pH 7.0 (previously sparged with compressed air (HPLC filter) for 5 hours) was added DFF (final concentration 100 mM) and 1 mg catalase. 10 L of a 100 M PaoABC was then added and the reaction was left in a shaking incubator. 5 L of the reaction mixture was extracted, diluted with 80 L water and quenched with 15 L 1 M HCl. The aliquots were analysed by RP HPLC. The results are shown in FIG. 13.

Example 4Periodic Hydrogen Peroxide Addition

[0283] To 490 L of 0.2 M KPi pH 7.0 was added 0.05 mmol DFF and 1 mg catalase. 10 L of a 100 M PaoABC was then added and the reaction was left in a shaking incubator. About 0.0003 mmol H.sub.2O.sub.2 was added every 15 minutes (1 l of a 1% solution). 5 L of the reaction mixture was extracted, diluted with 80 L water and quenched with 15 L 1 M HCl. The aliquots were analysed by RP HPLC. The results are shown in FIGS. 14 and 15.

Example 5AEntrapment of PaoABC in SiO.SUB.2 .Hydrogel

[0284] Tetramethyl orthosilicate (TMOS) (0.450 g) was placed in a small vial, cooled in an ice bath, and stirred at about 600 rpm. HCl (108 L, 2.44 mM) was added, and the solution was stirred for 10 min. The solution was adjusted to pH 5.1 by adding 60 L of 20 mM sodium phosphate buffer (pH 7.4). In a separate small vial, 1 mg of PaoABC, 540 L of 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (sodium salt) buffer solution (pH 7.5), and, when polymer was added, 60 L of 20 mg/mL aqueous PVI or PEI were mixed. The PaoABC-containing solution was added to the TMOS-containing solution, and the resulting mixture was stirred for 1 min. A vacuum was applied to the stirred mixture until a gel formed. The vacuum was released, and the gel was rinsed with 2 mL of distilled water three times. The gel was then soaked in 2 mL of distilled water overnight at 4 C. The water was removed from the vial, and the gel was allowed to dry at room temperature overnight in a vial. The dried gel was first collected and then ground to a powder with a mortar and pestle. 750 mg of hydrogel was produced with 1 mg of PaoABC, as determined by Bradford assay of the supernatant.

Example 5BEntrapment of PaoABC in Ni-Sepharose

[0285] 30 mL binding buffer (400 mM NaCl, 20 mM Imidazole, 50 mM KPi pH 7.0), 3 g of PaoABC CFE (cell free extract) and 5 g Ni.sup.+2 5 g of resin was stirred for 40 minutes. The slurry was centrifuged at 500 RPM and the supernatant removed. This was repeated twice with washing with binding buffer.

Example 5CEntrapment of PaoABC in Eupergit EC

[0286] 35 l of PaoABC was dissolved in 1 mL 1M KPi buffer with a pH as specified in Table 2. The concentration of PaoABC before immobilisation was recorded. 10 mg of Eupergit EC was added and the mixture was left in a shaking incubator at 150 rpm and 25 C. for 24 hours. The amount of enzyme absorbed onto the resin was determined by UV spectroscopy (428 nm). Blocking buffer (0.2M ethanolamine in 0.1M KPi pH 7) was added and the mixture was left to shake for 30 minutes. The immobilised enzyme was filtered through a sinter funnel and washed with KPi buffer pH 7 and then KPi buffer pH 7 with 1M NaCl. The immobilised beads were tested for activity.

TABLE-US-00002 TABLE 2 Enzyme Immobilisation Enzyme Immobilized Conversion.sup.[a] Entry pH (mg/mL) (mg) (%).sup.[b] 1 6 0.85 0.7 44 2 7 1 0.97 48 3 8 0.94 0.8 60 .sup.[a]Reaction conditions: 0.1 mg PaoABC (on resin), 2.5 l benzaldehyde, 500 l 100 mM KPi pH 7.6, 37 C., 5 Hr .sup.[b]Conversion calculated by RP HPLC

Example 5DEntrapment of PaoABC in Eupergit CM

[0287] 70 L (2 mg) of paoABC was dissolved in 1 mL 1M KPi buffer pH 7. The concentration before immobilisation was recorded. 200 mg of Eupergit EC was added and left in a shaking incubator on 150 rpm at 25 C. for 24 Hr. The amount of enzyme absorbed onto the resin was determined by UV spectroscopy (428 nm). After this time the pH was increased to pH 8.5 to facilitate multipoint attachment to the resin (5 hr). Blocking buffer (3M Glycine pH 8.5) was added and left shake for 5 hours. The immobilised enzyme was filtered through a filter paper and washed with 100 mM KPi Buffer pH 7 and, 100 mM KPi buffer pH 7 with 1M NaCl. The immobilised beads were tested for activity.

TABLE-US-00003 Conversion Entry Resin Amount Run (%) 1 Eupergit CM 50 mg 1 100 2 2 100 Reaction conditions: 100 mM KPi phosphate buffer pH 7.0, 100 mM DFF, 5 hr, 37 C.

Example 6

Synthesis of 2,5-diethyl-2,5-furandicarboxylate

[0288] ##STR00015##

[0289] 2,5-furandicarboxylic acid (25.18 g; 160 mmol) was added to ethanol (1,800 mL). Aqueous sulfuric acid (1.32 mL) was added. The mixture was heated at reflux (about 78 C.) for 67 hours, during which time water was removed from the reaction by the use of a Dean-Stark apparatus. The reaction progress was monitored using NMR spectroscopy. After the 2,5-diethyl-2,5-furandicarboxylate had been formed in >97% purity by NMR, the reaction mixture was allowed to cool to ambient temperature and was extracted with 2-methyltetrahydrofuran. The combined organic layers were washed with a saturated aqueous brine solution and deionised water, and dried (MgSO.sub.4). The organics were filtered and the volatiles were removed in vacuo to afford the title compound (26.77 g; 130 mmol; >98% conversion).

Example 7

General Methodology for the Formation of Copolymers

[0290] A 250 mL flange flask with 5 quick-fit ports was used in connection with a Dean-Stark apparatus. Stirring was achieved via a magnetic stirrer using a large precious metal stirrer bar. The rates of stirring were gradually decreased from the initial 400 rpm down to 200 rpm to avoid issues as a result of the increasing viscosity of the reaction mixture. All reagents were added to the reactor and warmed to 110 to 130 C. as described below to allow total melting and achieve miscibility. A flow of N.sub.2 gas was applied for 20 minutes to purge the reagents and reactor of oxygen. The temperature was then increased to the desired point as stated below. After a further four hours of very low N.sub.2 flow the gas line was removed, the Dean-Stark drained and a vacuum pump turned on, initially at a low vacuum (200 mbar) but slowly increased as stated below.

Synthesis of 2,5-polybutyrate adipate furandicarboxylate (2,5-PBAF)

[0291] ##STR00016##

[0292] 2,5-Diethyl-2,5-furandicarboxylate (21.20 g; 100 mmol), diethyl adipate (20.23 g; 100 mmol), 1,4-butane diol (22.53, 250 mmol) and T titanium(IV) tert-butoxide (0.77 mL; cat.) were combined. The reaction mixture was heated at 110 C. for 4 hours at atmospheric pressure with stirring at 400 rpm, 180 C. for 17 hours at 200 mbar and 350 rpm, and at 180 C. for 3 hours at 25 mbar and 250 rpm. The polymer was formed (37.20 g). The .sup.1H NMR spectra for 2,5-PBAF can be found at FIG. 1.

[0293] The molar ratio of 2,5-furandicarboxylate:adipate was determined by .sup.1H NMR to be 0.90:1. The molecular weight of the 2,5-PBAF was estimated using end-group analysis, wherein the ratio of end groups to those of the bulk polymer were calculated using .sup.1H NMR to give the number of constitutional repeating units (CRU), which was estimated to be 20.71. One ideal CRU is 410.43 gmol.sup.1. Therefore, the molecular weight of the 2,5-PBAF was estimated to be 8,497.5 gmol.sup.1.

Comparative Example 8

Synthesis of Polybutyrate Adipate Terephthalate (PBAT)

[0294] Diethyl terephthalate (22.22 g; 100 mmol), diethyl adipate (20.23 g; 100 mmol), 1,4-butane diol (22.73, 230 mmol) and titanium(IV) tert-butoxide (0.77 mL; cat.) were combined. The reaction mixture was heated at 130 C. for 2 hours at atmospheric pressure with stirring at 400 rpm, 180 C. for 2 hours at atmospheric pressure and 400 rpm, 180 C. for 17 hours at 200 mbar and 350 rpm, and at 180 C. for 3 hours at 25 mbar and 250 rpm. The copolymer was formed (40.51 g). The .sup.1H NMR spectra for PBAT can be found at FIG. 2.

[0295] The molecular weight of the PBAT was estimated by .sup.1H NMR using end-group analysis as described for 2,4-PBAP. The molar ratio of terephthalate:adipate was determined to be 1.047:1. The number of CRUs was estimated to be 16.4. One ideal CRU is 420.45 gmol.sup.1. Therefore, the molecular weight of the PBAT was estimated to be 6,893 gmol.sup.1.

[0296] PBAT is available commercially under a range of trade names. The molecular weight of one particular commercial PBAT was estimated by .sup.1H NMR using end-group analysis as described for 2,4-PBAP. The molar ratio of terephthalate:adipate was determined to be 0.93:1. The number of CRUs was estimated to be 25.7. One ideal CRU is 420.45 gmol.sup.1. Therefore, the molecular weight of the commercial PBAT was estimated to be 10,809 gmol.sup.1. The .sup.1H NMR spectra for commercial PBAT can be found at FIG. 3.

Example 9

Thermal Analysis of Polymers Using (STA and DSC)

[0297] The thermal stability of cured copolymer was analysed using Simultaneous Thermal Analysis (STA) using a Stanton Redcroft STA 625. Approximately 10-20 mg of copolymer was heated from ambient temperature to 625 C. at a heating rate of 10 C. min.sup.1 under nitrogen. The decomposition may be that of the copolymer backbone. The results can be found in Table 3.

TABLE-US-00004 TABLE 3 STA analysis of polymers Temperature of Temperature of 5 wt % loss decomp. Copolymer C. C. STA trace 2,5-PBAF 315.0 391.7 FIG. 4 Comparative Example 8 289.5 406.0 FIG. 5 Commercial PBAT 341.5 409.5 FIG. 6

[0298] The glass transition temperature (T.sub.g) and melting point (T.sub.m) of the copolymers were obtained by Differential Scanning calorimetry (DSC) analysis using a TA Instruments Q2000 DSC. Indium was used as the standard to calibrate the temperature and heat capacity. Copolymer samples (7-10 mg) were sealed in Tzero aluminum hermetic DSC pans. The method was carried out under a constant flow of dry nitrogen of 50 mL/min, at 10 C./min over a temperature range of 80 C. to 250 C. The results can be found in Table 4. The DSC traces can be found at FIG. 7.

TABLE-US-00005 TABLE 4 DSC analysis of copolymers T.sub.g1 T.sub.g2 T.sub.m Copolymer C. C. C. 2,5-PBAF 30.4 40.5 87.2 Comparative Example 8 39.5 42.4 134.6 Commercial PBAT 30.1 45.4 122.2

Example 10

[0299] The molecular weight (M.sub.n and M.sub.w) and polydispersity (Pd.sub.i) data as generated by GPC can be found in Table 5. GPC was conducted on an Agilent SECurity GPC System 1260 Infinity using THF as the solvent, a polystyrene standard, and a light scattering detector.

TABLE-US-00006 TABLE 5 GPC analysis of copolymers GPC Copolymer M.sub.n M.sub.w Pd.sub.i chromatogram 2,5-PBAF 862.5 6,121 7.097 FIG. 8 Comparative Example 8 5,582 8,615 1.544 FIG. 9 Commercial PBAT 42,190 113,100 2.680 FIG. 10

Example 11

Tensile Strength Measurement

[0300] Mechanical properties including tensile strength, elongation at break and Young's modulus of samples are summarised in Table 6. Film samples were prepared by heating about 8 g of copolymer in a fan-assisted oven at 160 C. for 15 min (180 C. for PBAT). The resulting films were cut into standard dumb-bell shapes (60 mm10 mm). Film thickness was in the region of 1.5-2.0 mm. Tensile studies were conducted in triplicate using an Instron 3367 universal testing machine fitted with 1000 N capacity load cell. The initial grip separation was set at 35 mm and the crosshead speed was 20 mm/min. The results reported were the average of the three measurements (the elongation at break was obtained automatically from the software). Commercially PBAT is a typical elastomer with elongation over 293%. It has the highest tensile strength over 19.5 MPa and good Young's modulus of 100.8 MPa.

TABLE-US-00007 TABLE 6 Tensile strength measurement of copolymers Elongation Young's Tensile strength at break Modulus Copolymer MPa % MPa 2,5-PBAF 2.2 0.4 4.7 0.8 75.3 2.0 Comparative Example 8 4.8 0.5 2.3 0.2 269.8 0.2 Commercial PBAT >19.5 >293.1 100.8

[0301] The 2,5-PBAF copolymer produced is soft like that of the commercial PBAT. The expected ratio of FDCA to adipate of about 1:1 has been incorporated into the copolymer. The observed molecular weight of 2,5-PBAF and Comparative Example 8 (PBAT) are significantly lower than that of commercial PBAT. This is expected given the relatively small scale on which the copolymerisations were conducted and will be higher in a full scale production process. The NMR data provides an indication of the relative number of constitutional repeating units (CRU) and hence an indication of molecule weight, though the GPC provides more accurate values.

[0302] The differences in the data obtained for the copolymers of the invention and the commercial BPAT may be attributed to a lack of branching in 2,5-PBAF.

Example 12

[0303] Stabilised green waste compost is matured in a composting bin under controlled aeration conditions. Before use, the mature compost is sieved on a screen of 5 mm. The fine fraction forms the inoculum with a total solids content of approximately 50-55% and the volatile content of the total solids is more than 30%.

[0304] The standard and control materials are mixed with the inoculum in a ratio of approximately 1 to 1.5 parts of total solids to 6 parts of total solids and introduced into a reactor. These reactors are closed and put into an incubator. The temperature of the reactors is maintained at 58 C.+1-2 C. Pressurised air is pumped through a gas flow controller and blown into the composting vessel at the bottom through a porous plate. During biodegradation, solid carbon of the test sample is converted into CO.sub.2.

[0305] The gas leaving each individual reactor is analysed at regular intervals for CO.sub.2 and O.sub.2 concentrations. As the flow rate is continually measured, the cumulative CO.sub.2 production can be determined. The percentage of biodegradation is determined as the percentage of solid carbon of the test compound that is converted into CO.sub.2.

Example 13

[0306] FIG. 20 depicts a perspective view of an apparatus 1 suitable for conducting a continuous flow process for oxidising a substrate (e.g. according to the present invention) on a laboratory scale.

[0307] The apparatus 1 comprises a primary tube 3 defining a primary fluid passageway (also labelled 3 herein) for flow of a fluid (such as a liquid reaction mixture). The apparatus further comprises secondary tubing 5 to provide flow of one or more further fluids to the primary fluid passageway 3.

[0308] The secondary tubing 5 is in fluid communication with the primary fluid passageway 3. In this way, one or more further fluids can be added to the primary fluid passageway 3 via the secondary tubing 5 (e.g. to permit mixture and hence reaction between components in fluid flowing through the primary passageway 3 and further fluids in the secondary tubing 5).

[0309] Apparatus 1 comprises a polytetrafluoroethylene (PTFE) primary tube 3 sandwiched between two generally square-shaped Perspex blocks 7a, 7b. The primary tube 3 comprises a tortuous primary passageway 3 having an inlet end 3a and an outlet end 3b, the passageway 3 extending in an undulating fashion between edges of the Perspex blocks 7a, 7b. The primary tube 3 comprises a total of eleven bends (collectively labelled 9) representing peaks and troughs provided at the upper and lower edges (respectively) of the Perspex blocks 7a, 7b.

[0310] The primary passageway 3 is provided with an inlet port 11 at one end for injecting fluid thereinto and an outlet port 13 for discharge at the other end. As shown, the ports 11, 13 comprise syringes 11a, 13a for input and outlet (respectively) of fluids.

[0311] The secondary tubing 5 comprises two horizontal main tubes 13 provided at the upper and lower edges of the Perspex blocks 7a, 7b, said main tubes 13 being provided with a supply of fluid thereinto (not shown). The secondary tubing further comprises a series of eleven secondary tubes (not visible) depending from one or other of the main tubes 13 (five upper, six lower). Said secondary tubes are in fluid communication with the primary passageway 3 by means of an aperture (not visible) in each secondary tube, each aperture defining an opening between each secondary tube and the primary passageway 3. Each of said eleven openings/apertures is provided at a corresponding bend 9 in the primary fluid passageway 3. Thus, the secondary tubes permit fluid communication between the main tube of the secondary tubing and the primary fluid passageway.

[0312] Such an apparatus may be useful for admitting a relatively low local concentration of one or more components (such as hydrogen peroxide) into a fluid flowing through the primary passageway, while providing a sufficient amount of that component on a bulk basis.

[0313] FIG. 25 shows a graphical representation of flow velocity through the primary fluid passageway 3, horizontal main tubes 13 and secondary tubing 5 in apparatus 1. Flow velocity was simulated with computational fluid dynamics.

[0314] For clarity, only the first (5A) and last (5F) tubes forming part of the secondary tubing 5 are labelled. Numbering of the tubes is in sequence (A-K) along the primary fluid passageway from inlet end 3a to outlet end 3b.

[0315] The pressure within tubes of the secondary tubing 5 was generally constant between different tubes, as shown in the table below.

TABLE-US-00008 Secondary tube mL/min 5A 0.0028 5B 0.0019 5C 0.0071 5D 0.0132 5E 0.0212 5F 0.0323 5G 0.0027 5H 0.0076 5I 0.0131 5J 0.02 5K 0.0295

[0316] The flow velocity in the primary fluid passageway 3 varies from the inlet end 3a to outlet end 3b. Approximations of the flow velocity are shown in the table below for various sections of the primary tube. Sections with similar velocity are labelled with the same letter (A-I) in the figure.

TABLE-US-00009 Velocity mms.sup.1 A (inlet end, 3a) 0.6 B 0.8 C 1 D 1.1 E 1.3 F 1.5 G 1.7 H 1.9 I 2.1

[0317] In general, velocity increases along the length of the primary fluid passageway 3 (from inlet end 3a to outlet 3b).

[0318] It will be appreciated that modifications can be made so as to balance flow rate through the apparatus 1 (e.g. to maintain a flow rate along the length of the primary fluid passageway 3). For example, the width (e.g. cross-sectional area) and/or length of tubes in the secondary tubing 5 can be configured to maintain a predetermined (e.g. constant) flow velocity along the length of the primary fluid passageway 3. Alternatively or additionally, the cross-sectional area of the primary fluid passageway 3 could similarly be configured to maintain a predetermined (e.g. constant) flow velocity along the length of the primary fluid passageway 3.

[0319] Configuration in these ways may be useful so that reagents admitted into the primary fluid passageway 3 via the secondary tubing 5 have sufficient residency time in the apparatus to undergo substantial reaction.

Example 14

[0320] FIG. 21 depicts a front view of an apparatus 101 suitable for conducting a continuous flow process for oxidising a substrate (e.g. according to the present invention) on a laboratory scale.

[0321] The apparatus 101 is similar to the apparatus in FIG. 20 and only differences between the apparatuses will be explained below. In FIG. 21, features equivalent to features of the apparatus in FIG. 20 take the same reference number but elevated by 100.

[0322] The apparatus 101 comprises a primary tube 103 defining a primary fluid passageway (also labelled 103 herein) for flow of a fluid (such as a liquid reaction mixture). The primary passageway 103 is provided with a plurality of flow disruptors in the form of glass beads 115. The glass beads 115 disrupt the flow of liquid passing through the primary fluid passageway and thereby improve liquid mixing.

Example 15

[0323] Calibration curves with an internal standard were created for quantitative gas chromatography (GC) and high-performance liquid chromatography (HPLC) analysis of hydroxymethylfurfural (HMF) and diformylfuran (DFF). The results are shown in FIG. 22. The identity of the products was additionally confirmed by GC/HPLC co-injection of reaction mixtures with chemically synthesized authentic products, or by NMR spectroscopic analysis of products obtained from reactions performed on preparative scale.

[0324] GC traces were obtained for samples of a reaction mixture during continuous-flow oxidation of 5-hydroxymethylfurfural, using GOase M.sub.3-5 biocatalyst. The results are shown in FIG. 23. Traces indicate solution composition after 1 reactor volume (RV; A), 3 RVs (B) and at steady-state (C). Peak shown at 4.20 minutes=1,3,5-trimethoxybenzene as internal standard (ISTD).

Example 16

[0325] A trickle bed reactor with 5 successive stages and 5 separate H.sub.2O.sub.2 side feeds was used for conversion of HMF to DFF in flow. The reactor was filled with glass beads, 2 mm diameter. Enzymes (GOase M.sub.3-5 CFE, 4.80 g; CuSO.sub.4, 0.028 g; catalase, 0.352 g; and horseradish peroxidase (HRP), 0.228 g), HMF (48 mM), and H.sub.2O.sub.2, 5% (w/v) (H.sub.2O.sub.2/HMF=50:1 mol/mol) were dissolved in potassium phosphate buffer 0.1 M and pumped through the reactor in the ratio of 1:1:1.

[0326] Reaction parameters were as follows: [0327] Residence time=13.6 min. [0328] Room temperature (21 C.) [0329] Room pressure (101 kPa) [0330] Total volume of the reactor=110 mL (22 mL per stage)

[0331] Reactor volumes were collected and analysed on HLPC. The results are shown in FIG. 24.

[0332] 311 reactor volumes were collected over 3 days of continuous run, which yielded a product solution of 934 g. HMF was fully converted into DFF and HMFCA as main products of reaction. FDCA and HMFCA were also formed in smaller quantities.

[0333] The product solution of all the reactor volumes collected at 100% conversion had the following composition as measured by HPLC: [0334] 50.2% DFF [0335] 45.2% FFCA [0336] 3.6% HMFCA [0337] 1.0% FDCA

[0338] Process productivity was 1.99 g of product/L of solution/day.

Example 17

[0339] A MultiPoint-Injection Reactor (MPIR) (See FIG. 15) was used to convert HMF to DFF in flow. This MPIR was fitted with 2H.sub.2O.sub.2 distributor channels, which results in 11 addition ports. Enzymes (GOase M.sub.3,5 CFE, 0.65 g; CuSO.sub.4, 0.013 g; catalase, 0.013 g; and Horseradish Peroxidase (HRP), 0.010 g), HMF (30 mM), and H.sub.2O.sub.2 (H.sub.2O.sub.2/HMF at a 2:1 mol/mol) are dissolved in NaPi buffer at pH 7.4 and pumped through the reactor.

[0340] Reaction parameters are as follows: [0341] Residence time=15 min. [0342] Room temperature (21 C.) [0343] Pressure=40 psi (276 kPa [0344] Total volume of the reactor=2.6 mL

[0345] Reactor volumes were collected and analysed on HLPC. At steady state, 86% conversion was achieved with 100% selectivity for DFF. Process productivity was 144 g of product/L of solution/day.

[0346] Any listing or discussion of an apparently prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or common general knowledge. All references disclosed herein are to be considered to be incorporated herein by reference.

[0347] Those skilled in the art will recognise or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments described herein. The scope of the present embodiments described herein is not intended to be limited to the above description, but rather is as set forth in the appended claims. Those of ordinary skill in the art will appreciate that various changes and modifications to this description may be made without departing from the spirit or scope of the present invention, as defined in the following claims.