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WO-2026096862-A1 - PURIFICATION OF ACRYLAMIDE AND RECYCLING BIOCATALYST USING MEMBRANE FILTRATION

WO2026096862A1WO 2026096862 A1WO2026096862 A1WO 2026096862A1WO-2026096862-A1

Abstract

The present invention provides methods for the manufacture of amides from their corresponding nitriles using a biocatalyst and purification of the manufactured amides using membrane filtration. The invention also relates to methods of using membrane filtration for isolating and reusing biocatalysts. More specifically, the inventive method produces a crude aqueous acrylamide (AMD) by feeding acrylonitrile (AN), water, and a biocatalyst with nitrile hydratase activity into a reactor. After complete conversion of AN to AMD, the crude reaction is directly purified by membrane filtration to produce a purified AMD monomer (without nitrile hydratase activity) and a recovered biocatalyst with nitrile hydratase activity. Purified monomer (e.g., AMD without enzyme activity) in the permeate may be stored, transported, and used for polymerization. Biocatalyst in the retentate retains enzymatic activity and may be reused.

Inventors

  • OKOLI, Samuel
  • Hesampour, Mehrdad
  • SIMELL, Jaakko
  • Stok, Michel
  • VUORI, Vesa

Assignees

  • KEMIRA OYJ
  • KEMIRA WATER SOLUTIONS, INC.

Dates

Publication Date
20260507
Application Date
20251031
Priority Date
20241210

Claims (16)

  1. 1. A method for producing a chemical product from a substrate, the method comprising: (a) contacting the substrate with a biocatalyst comprising an initial enzyme activity to form a reaction mixture; (b) converting the substrate into the chemical product; and (c) performing a membrane filtration step comprising filtering the reaction mixture through at least one membrane filter to form a retentate comprising a recovered biocatalyst and a permeate comprising a purified chemical product.
  2. 2. The method of claim 1, wherein: (i) the initial enzyme activity comprises an initial nitrile hydratase activity; (ii) the substrate comprises a monomer precursor, a nitrile, or acrylonitrile; and/or (iii) the chemical product comprises a monomer, an amide, a monomer comprising an amide, or acrylamide.
  3. 3. The method of claim 1 or 2, wherein the method further comprises: (i) prior to step (a), optionally thawing said biocatalyst; (ii) prior to step (a), optionally adjusting said biocatalyst to a pH of 3-10, 4-10, 5-9.5, 6-9, 7-9, 7.5-8.5 or 8-8.5; (iii) prior to step (a), adjusting said biocatalyst to a temperature of 5-30 °C, 5-25 °C, 5-20 °C, or 5-15 °C; (iv) after step (a), optionally adjusting the reaction mixture to a pH of 3-10, 4- 10, 5-9.5, 6-9, 7-9, 7.5-9, 7.5-8.5 or 8-8.5; (v) after step (a), optionally adjusting the reaction mixture to an initial temperature of 10-35 °C, 10-30 °C, 15-25 °C, or 20-25 °C and then maintaining the reaction mixture at a temperature of 20-30 °C for the duration of step (b); (vi) during step (b), analyzing the reaction mixture to determine a concentration of the substrate remaining in the reaction and/or a mol% conversion of the substrate to the chemical product; (vii) during step (b), allowing said reaction mixture to react until the concentration of the substrate remaining in the reaction ranges from 0- 3000 ppm, 0-1000 ppm, 0-300 ppm, 0-200 ppm, 0-100 ppm, 1-80 ppm, 0-60 ppm, 0-40 ppm, 0-20 ppm, or less than 100 ppm, and/or the mol% conversion of the substrate to the chemical product ranges from 90-100%, 95-100%, 98-100%, or 99-100%; or (viii) any combination of (i)-(vii).
  4. 4. The method of claim 1, 2 or 3, wherein the method further comprises one or more of the following: (i) prior to step (c), optionally treating the reaction mixture by acidification, addition of adsorption media, addition of activated carbon, acidification and addition of adsorption media followed by filtration, sedimentation, centrifugation, filtration, or any combination thereof to remove a partial amount of the biocatalyst, optionally 1-90 wt%, 1-80 wt%, 1-60 wt%, 1-40 wt%, or 1-20 wt% of the biocatalyst, from the reaction mixture; (ii) prior to step (c), adjusting the reaction mixture to a pH of 3-10, 4-10, 5-10, 6-9.5, 6.5-9, preferably 7-9, 7.5-9, 7.5-8.5 or 8.0-8.5 when said recovered biocatalyst is to be discarded or to a pH of 5-10, 6-10, 7-9, 7.5-9, 7.5-8.5 or 8.0-8.5 when said recovered biocatalyst is to be reused; (iii) prior to step (c), adjusting the reaction mixture to a temperature of 10-40 °C, 10-35 °C, 15-30 °C, 15-25 °C, 20-30 °C, or 20-25 °C; (iv) after step (c), optionally post-treating the permeate comprising the purified chemical product by addition of activated carbon, addition of adsorption media, acidification, acidification and addition of adsorption media followed by filtration, sedimentation, centrifugation, filtration, at least one additional membrane filtration step, or any combination thereof, wherein said post-treatment removes a residual biocatalyst and/or residual activated carbon from the purified chemical product; (v) after step (c), washing said membrane filter with an amount of water, a water solution, a chemical product solution, acid solution, alkaline solution, surfactant, cleaner, or any combination of the foregoing at a temperature of 20-50 °C, 20-40 °C, or 20-30 °C; or (vi) any combination of (i)-(v).
  5. 5. The method of any one of the foregoing claims, wherein the method further comprises one or more of the following: (i) after step (b), optionally stabilizing the chemical product by addition of a polymerization inhibitor; (ii) after step (c) adjusting the purified chemical product to a pH of 5-10, 6-9.5, 6-8, 7-9, 7.5-9, 7.75-8.7, or 8-8.5; (iii) after step (c) analyzing the purified chemical product to determine a wt% removal of the biocatalyst, optionally by spectrophotometric analysis, wherein said wt% removal of the biocatalyst from the chemical product comprises 90-100 wt%, 95-100 wt%, 98-100 wt%, 99-100 wt% or 99.9-100 wt%; (iv) after step (c) analyzing the purified chemical product to determine a residual enzyme activity, wherein said residual enzyme activity comprises 0- 10%, 0-5%, 0-2%, 0-1%, or 0-0. l%of said initial enzyme activity; (v) after step (c), storing said purified chemical product for 0.1-24 h, 1-24 h, 1-2 days, 1-10 days, or 1-30 days; (vi) after step (c), transporting said purified chemical product; (vii) after step (c), using said purified chemical product in a subsequent polymerization reaction to produce a polymer comprising the chemical product; or (viii) any combination of (i)-(vii).
  6. 6. The method of any one of the foregoing claims, wherein the method further comprises one or more of the following: (i) after step (c), washing said recovered biocatalyst with an amount of water, a mixture of water and the chemical product, or an aqueous solution; (ii) after step (c), analyzing said recovered biocatalyst to determine a recovered enzyme activity, wherein said recovered enzyme activity optionally is 10- 100%, 20-90%, 30-80%, 40-75%, 50-70% of said initial enzyme activity; (iii) after step (c), storing said recovered biocatalyst for 0.01-48 h, 0.1-24 h, 0.5- 24 h, 1-24 h, or at least 24 h; (iv) after step (c), reusing said recovered biocatalyst in a subsequent method for producing a chemical product from a substrate; (v) after step (c), combining said recovered biocatalyst with an additional amount of unused biocatalyst, thereby forming a recovered biocatalyst mixture and then using the recovered biocatalyst mixture in a subsequent method for producing a chemical product from a substrate; or (vi) any combination of (i)-(v).
  7. 7. The method of any one of the foregoing claims, wherein: (a) said substrate comprises a monomer precursor, the nitrile, a substituted nitrile, a substituted aliphatic nitrile having 1 to 10 carbon atoms, acrylonitrile, or methacrylonitrile; and/or (b) said polymer comprising the chemical product comprises a homopolymer comprising the chemical product or the amide; a copolymer comprising the chemical product or the amide and one or more neutral monomers, cationic monomers, anionic monomers, or any combination thereof; a polyacrylamide homopolymer; a polyacrylamide copolymer; a cationic polyacrylamide (CPAM); an anionic polyacrylamide (APAM), an amphoteric polyacrylamide (AMPAM); a dry polyacrylamide (DPAM); an emulsion polyacrylamide (EPAM); or a glyoxalated polyacrylamide (GPAM).
  8. 8. The method of any one of the foregoing claims, wherein the biocatalyst: (a) comprises at least one nitrile hydratase enzyme, at least one crude nitrile hydratase enzyme, at least one purified nitrile hydratase enzyme, or any combination thereof; (b) comprises whole cells, cell lysate, live cells, dead cells, broken cells, cell fragments, or any combination thereof; (c) comprises a freshly fermented biocatalyst, a frozen or thawed biocatalyst, a lysed biocatalyst, a hydrated biocatalyst, a dried biocatalyst, a rehydrated biocatalyst, or any combination thereof; (d) comprises a buffered aqueous solution, a cellular growth media, at least one stabilizer, glycerol, or any combination thereof; or (e) any combination of (a)-(d).
  9. 9. The method of any one of the foregoing claims, comprising one or more of the following: (a) said membrane filter comprises a pore size comprising a molecular weight cutoff (MWCO) with at least 90% efficiency of 0.1-150 kDa, 1-125 kDa, 2-100 kDa, 5-90 kDa, 10-80 kDa, 10-70 kDa, 10-60 kDa, 10-50 kDa, 10-40 kDa, or 20- 30 kDa; (b) said membrane filter comprises an average pore size of 0.1-500 nm, 0.5-490 nm, 1-475 nm, 1-450 nm, 1-400 nm, 1-300 nm, 1-200 nm, 1-100 nm, 1-80 nm, 1-60, 1-50 nm, 1-40 nm, 1-30 nm, 1-20 nm, or 1-10 nm; (c) said membrane filter comprises a membrane configuration selected from the group consisting of a spiral wound element (SWE), a hollow fiber (HF) membrane, a tubular membrane, a flat membrane, or multiple membranes comprising any combination of the foregoing; (d) said membrane filter comprises a polymer membrane, a polyacrylonitrile (PAN) membrane, a polyethersulfone (PES) membrane, a ceramic membrane, a metal membrane, or multiple membranes comprising any combination of the foregoing; (e) said steps comprising filtering said biocatalyst and/or said reaction mixture through said at least one membrane filter comprise a transmembrane permeability of 10-500 kg/(m 2 h bar), 20-250 kg/(m 2 h bar), 30-100 kg/(m 2 h bar), 40-90 kg/(m 2 h bar), 45-80 kg/(m 2 h bar), or 50-60 kg/(m 2 h bar); (f) said steps comprising filtering said biocatalyst and/or said reaction mixture through said at least one membrane filter comprise a transmembrane pressure of 0.1-10 bar, 1-8 bar, 1.5-7 bar, 2-6 bar, or 3-5 bar; or (g) any combination of (a)-(f).
  10. 10. The method of any one of the foregoing claims, comprising one or more of the following: (a) the reaction mixture comprises an aqueous medium; (b) the reaction mixture is formed in a batch reactor, a semi-continuous reactor, or a continuous reactor; (c) the membrane filter comprises said spiral wound element (SWE), said hollow fiber (HF) membrane, or multiple membranes comprising any combination of the foregoing; (d) the membrane filter comprises said polyacrylonitrile (PAN) membrane, said polyethersulfone (PES) membrane, or multiple membranes comprising any combination of the foregoing; (e) the membrane filter comprises said pore size comprising said molecular weight cutoff (MWCO) with at least 90% efficiency of 10-50 kDa, 10-40 kDa, or 10-30 kDa; (f) the membrane filter comprises said spiral wound element (SWE), said polyacrylonitrile (PAN) or said PES membrane, and said MWCO of 20-40 kDa, 25-30 kDa, or 30 kDa; (g) the membrane filter comprises said hollow fiber (HF) membrane, said polyethersulfone (PES) or said PAN membrane, and said MWCO of 1-20 kDa, 5- 15 kDa, or 10 kDa; or (h) any combination of (a)-(g).
  11. 11. The method of any one of the foregoing claims, wherein the method results in: (a) a reduced down time and/or a reduced amount of filter clogging compared to the same method or an alternative method that does not comprise at least one membrane filtration step; (b) an increased wt% removal of the biocatalyst from the purified chemical product compared to the same method or an alternative method that does not comprise at least one membrane filtration step; (c) a decreased residual enzyme activity in the purified chemical product compared to the same method or an alternative method that does not comprise at least one membrane filtration step; (d) an increased recovered enzyme activity of the recovered biocatalyst compared to the same method or an alternative method that does not comprise at least one membrane filtration step; (e) a recovered biocatalyst yield of 90-100 wt%, 95-100 wt%, or 99-100 wt %; (f) an increased stability of the purified chemical product over 1-15 days, 1-14 days, 1-13 days, 1-12 days, 1-11 days, 1-10 days, 1-5, or 1-4 days at 75 °C compared to the same method or an alternative method that does not comprise at least one membrane filtration step, optionally wherein said stability of the purified chemical product increases as said wt% removal of the biocatalyst from the purified chemical product increases; (g) a reduced auto-polymerization of the purified chemical product over 1-15 days, 1-14 days, 1-13 days, 1-12 days, 1-11 days, 1-10 days, 1-5, or 1-4 days at 75 °C compared to the same method or an alternative method that does not comprise at least one membrane filtration step, optionally wherein said autopolymerization of the purified chemical product decreases as said wt% removal of the biocatalyst from the purified chemical product increases; (h) a positive correlation between said stability of the purified chemical product and said wt% removal of the biocatalyst from the purified chemical product; or (i) any combination of (a)-(h).
  12. 12. A method for producing a chemical product from a substrate, the method comprising: (a) contacting the substrate with a biocatalyst comprising an initial enzyme activity to form a reaction mixture; (b) converting the substrate into the chemical product; (c) performing a membrane filtration step comprising filtering the reaction mixture through at least one membrane filter to form a retentate comprising a recovered biocatalyst and a permeate comprising a purified chemical product; and (d) reusing the recovered biocatalyst in a subsequent method for producing the chemical product from the substrate.
  13. 13. The method of claim 12, wherein: (i) the initial enzyme activity comprises an initial nitrile hydratase activity; (ii) the substrate comprises a monomer precursor, a nitrile, or acrylonitrile; and/or (iii) the chemical product comprises a monomer, an amide, a monomer comprising an amide, or acrylamide.
  14. 14. The method of claim 12 or 13, wherein the method further comprises: (i) after step (c), storing said purified chemical product for 0.1-24 h, 1-24 h, 1-2 days, 1-10 days, or 1-30 days; (ii) after step (c), transporting said purified chemical product; (iii) after step (c), using the purified chemical product in a subsequent polymerization reaction to produce a polymer comprising the chemical product, wherein said polymer comprises a homopolymer comprising the chemical product or the amide, a copolymer comprising the chemical product or the amide and one or more neutral monomers, cationic monomers, anionic monomers, or any combination thereof, a polyacrylamide homopolymer, a polyacrylamide copolymer, a cationic polyacrylamide (CPAM), an anionic polyacrylamide (APAM), an amphoteric polyacrylamide (AMPAM), a dry polyacrylamide (DPAM), a glyoxalated polyacrylamide (GPAM), or an emulsion polyacrylamide (EPAM); (iv) after step (c), washing said membrane filter with an amount of water, a mixture of water and the chemical product, or an aqueous solution, or any combination of the foregoing at a temperature of 20-50 °C, 20-40 °C, or 20- 30 °C; (v) after step (d), washing said recovered biocatalyst with an amount of water, a mixture of water and the chemical product, or an aqueous solution, or any combination of the foregoing at a temperature of 20-50 °C, 20-40 °C, or 20-30 °C; (vi) storing said recovered biocatalyst for 0.01-48 h, 0. 1-24 h, 0.5-24 h, 1-24 h, or at least 24 h; (vii) after step (d), analyzing the recovered biocatalyst to determine a recovered enzyme activity, wherein said recovered enzyme activity is 10-100%, 20- 90%, 30-80%, 40-75%, 50-70% of said initial enzyme activity; or (viii) any combination of (i)-(vii).
  15. 15. The method of claim 12 or 13 or 14, wherein: (i) said substrate comprises a monomer precursor, the nitrile, a substituted nitrile, a substituted aliphatic nitrile having 1 to 10 carbon atoms, acrylonitrile, or methacrylonitrile; (ii) said biocatalyst comprises at least one nitrile hydratase enzyme, at least one crude nitrile hydratase enzyme, at least one purified nitrile hydratase enzyme, or any combination thereof; (iii) said biocatalyst comprises whole cells, cell lysate, live cells, dead cells, broken cells, cell fragments, or any combination thereof; (iv) said biocatalyst comprises a freshly fermented biocatalyst, a frozen or thawed biocatalyst, a lysed biocatalyst, a hydrated biocatalyst solution, a dried biocatalyst, a rehydrated biocatalyst, or any combination thereof; (v) said biocatalyst comprises a buffered aqueous solution, a cellular growth media, at least one stabilizer, glycerol, or any combination thereof; (vi) said membrane filter comprises a pore size comprising a molecular weight cutoff (MWCO) with at least 90% efficiency of 0.1-150 kDa, 1-125 kDa, 2- 100 kDa, 5-90 kDa, 10-80 kDa, 10-70 kDa, 10-60 kDa, 10-50 kDa, 10-40 kDa, or 20-30 kDa; (vii) said membrane filter comprises an average pore size of 0.1-500 nm, 0.5-490 nm, 1-475 nm, 1-450 nm, 1-400 nm, 1-300 nm, 1-200 nm, 1-100 nm, 1-80 nm, 1-60, 1-50 nm, 1-40 nm, 1-30 nm, 1-20 nm, or 1-10 nm; (viii) said membrane filter comprises a membrane configuration selected from the group consisting of a spiral wound element (SWE), a hollow fiber (HF) membrane, a tubular membrane, a flat membrane, or multiple membranes comprising any combination of the foregoing; (ix) said membrane filter comprises a polymer membrane, a polyacrylonitrile (PAN) membrane, a polyethersulfone (PES) membrane, a ceramic membrane, a metal membrane, or multiple membranes comprising any combination of the foregoing; (x) said membrane filtration step comprises a transmembrane permeability of 10-500 kg/(m 2 h bar), 20-250 kg/(m 2 h bar), 30-100 kg/(m 2 h bar), 40-90 kg/(m 2 h bar), 45-80 kg/(m 2 h bar), or 50-60 kg/(m 2 h bar); (xi) said membrane filtration step comprises a transmembrane pressure of 0.1- 15 bar, 0.5-10 bar, 1-8 bar, 1.5-7 bar, 2-6 bar, or 3-5 bar; or (xii) any combination of (i)-(xi).
  16. 16. A composition comprising: (a) a recovered biocatalyst obtainable by a method according to any one of the foregoing claims; (b) a purified chemical product, monomer, amide monomer, or acrylamide obtainable by a method according to any one of the foregoing claims; or (c) a polymer comprising said purified chemical product, monomer, amide monomer, or acrylamide obtainable by a method according to any one of the foregoing claims.

Description

PURIFICATION OF ACRYLAMIDE AND RECYCLING BIOCATALYST USING MEMBRANE FILTRATION RELATED APPLICATIONS The present invention relates to and claims benefit of priority to U.S. Provisional Application Number 63/715,166, filed on November 1, 2024, and Finnish Application Number Fl 20246447, filed on December 10, 2024, the contents of both are which are incorporated by reference in their entirety herein. FIELD OF THE INVENTION [0001] The present invention relates to methods for the manufacture of amides from the corresponding nitriles using a biocatalyst and to processes for purifying the manufactured amides using membrane filtration. The invention also relates to methods of using membrane filtration for isolating and reusing biocatalysts. BACKGROUND OF THE INVENTION [0002] Industrial scale reactions are typically accomplished using biocatalysts, such as microorganisms that contain enzymes, for catalyzing chemical reactions. Nitrile hydratase enzymes and nitrile hydratase producing microorganisms are biocatalysts for increasing the rate of hydration of nitriles directly to their corresponding amides. [0003] Nitrile hydratase producing microorganisms include various eukaryotes and prokaryotes, such as Rhodococcus rhodochrous. Substrates for these nitrile hydratase biocatalysts include variously substituted aliphatic and aromatic nitriles. A well-known commercial example of nitrile bioconversion is the manufacture of acrylamide (AMD) from acrylonitrile (AN). [0004] Bioconversion of acrylonitrile (AN) to acrylamide (AMD) is typically performed by feeding AN, water, and a biocatalyst with nitrile hydratase activity into a reaction vessel, such as a bioreactor. The biocatalyst converts acrylonitrile (AN) to acrylamide (AMD). Purification (removal of biocatalyst from the AMD product) is necessary before storage, shipment, or polymerization of the AMD. The purified AMD is then used, e.g., as a starting material for polyacrylamide (PAM) homopolymers and copolymers. [0005] After the reaction is complete, purification of the produced acrylamide may be accomplished by acidifying the reaction and adding an adsorption media. The biocatalyst adsorbs onto the surface of the adsorption media, which is thenty pically removed by filtering through a coarse filter. Purified AMD (without enzyme activity) remains in the filtrate. The biocatalyst and adsorption media are discarded as waste. [0006] This existing method causes several operational challenges, including generating significant amounts of classified hazardous waste (spent carbon-biocatalyst complex with monomer residues) and frequent downtime due to blocking of the coarse filter pores. The method also requires acidification and addition of filtration media, both of which deactivate the biocatalyst. Therefore, the deactivated biocatalyst cannot be reused. The operational costs of downtime, remediation methods for clogged filters, and hazardous waste disposal are significant. [0007] Efficient methods for removal of biocatalyst from crude bioconversion reactions remains a significant challenge in the industry. Certain types of biocatalysts can be removed by centrifugation; however some are not amenable to centrifugation, depending on the host cell. For example, Gram-positive bacteria can typically be removed with a centrifuge due to their rigid cell walls. By contrast, Gram-negative host cells cannot be removed with a centrifuge due to their more easily breakable cell walls, breakage of which may result in leaking of cell constituents. [0008] Therefore, the technical problem underlying the present invention is to provide improved methods for removing biocatalyst from bioconversion reactions, which improved methods: (i) do not require acidification or addition of adsorption media, (ii) allow for isolation of purified monomer for use in polymerization reactions, and (iii) allow for isolation and reuse of recovered biocatalyst in subsequent reactions. [0009] The present invention addresses this technical problem by using membrane filtration for purification of monomers, such as acrylamide (AMD), from bioconversion reactions (e.g., from crude aqueous AMD) comprising active biocatalyst. After membrane filtration, active biocatalyst is recovered as the retentate and purified monomer is recovered as the permeate. After membrane filtration, recovered biocatalyst in the retentate retains enzymatic activity and may be reused. Purified monomer (e.g., AMD) in the permeate contains minimal residual biocatalyst, and are therefore safely stored, transported, and used for polymerization. SUMMARY OF THE INVENTION [0010] The present invention relates to methods for the manufacture of chemical products from their corresponding substrates using a biocatalyst and purification of the manufactured chemical products using membrane filtration. The invention also relates to methods of using membrane filtration for isolating and reusing biocatalysts. [0011] The inventive method produces a crude aqueous