Purification of acrylamide and recycling biocatalyst using membrane filtration

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.

Description

PURIFICATION OF ACRYLAMIDE AND RECYCLING BIOCATALYST USING MEMBRANE FILTRATIONRELATED APPLICATIONSThe 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 withmonomer 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 chemical product, such as acrylamide by feeding a substrate, such as acrylonitrile, water, and a biocatalyst with enzyme activity, such as nitrile hydratase activity, into a reactor. After complete conversion of substrate to chemical product , the crude reaction is directly purified by membrane filtration to produce a purified chemical product (without enzyme activity) and a recovered biocatalyst with enzyme activity. Purified chemical product (e.g., AMD monomer withoutenzyme activity) in the permeate may be stored, transported, and used for polymerization. Biocatalyst in the retentate retains enzymatic activity and may be reused.

[0012] In one aspect, the present invention provides a method for producing a chemical product from a substrate, the method comprising at least steps (a)-(c):

[0013] (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.

[0014] In certain embodiments of the method:

[0015] (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.

[0016] In certain embodiments the method further comprises:

[0017] (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 8.0-8.5; (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 optionally 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 / orthe 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).

[0018] In certain embodiments the method further comprises:

[0019] (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, 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 tostep (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, water-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).

[0020] In certain embodiments the method further comprises:

[0021] (i) after step (b), optionally stabilizing the chemical product by addition of a polymerization inhibitor, such as 4-methoxyphenol (MEHQ) or a copper-ion in the form of a copper salt; (ii) after step (c) adjusting the purified chemical product to a pH of 5-10, 6-9.5, 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 0-0.1% 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 the amide; or (viii) any combination of (i)-(vii).

[0022] In certain embodiments the method further comprises:

[0023] (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 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 the unused biocatalyst, thereby forming a recovered biocatalyst mixture and then reusing the recovered biocatalyst mixture in a subsequent method for producing a chemical product from a substrate; or (vi) any combination of (i)-(v).

[0024] In certain embodiments of the method:

[0025] (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 amide comprises a homopolymer comprising the chemical product or the amide; a copolymer comprising the chemical product orthe 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).

[0026] In certain embodiments of the method the biocatalyst comprises:

[0027] (a) 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) whole cells, cell lysate, live cells, dead cells, broken cells, cell fragments, or any combination thereof; (c) 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) a buffered aqueous solution, a cellular growth media, at least one stabilizer, glycerol, or any combination thereof; or (e) any combination of (a)-(d).

[0028] In certain embodiments of the method:

[0029] (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) comprises a transmembrane permeability of 10-500 kg / (m2h bar), 10-250 kg / (m2h bar), 10- 100 kg / (m2h bar), 10-90 kg / (m2h bar), 10-80 kg / (m2h bar), or 10-60 kg / (m2h bar); or (f) any combination of (a)-(e).

[0030] In certain embodiments of the method said membrane filtration step and / or said additional membrane filtration step comprises:

[0031] (a) a transmembrane permeability of 10-500 kg / (m2h bar), 10-250 kg / (m2h bar), 10- 100 kg / (m2h bar), 10-90 kg / (m2h bar), 10-80 kg / (m2h bar), or 10-60 kg / (m2h bar); and / or(b) 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.

[0032] In certain embodiments of the method:

[0033] (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; (h) the membrane filter comprises a transmembrane permeability of 10-500 kg / (m2h bar), 10-250 kg / (m2h bar), 10- 100 kg / (m2h bar), 10-90 kg / (m2h bar), 10-80 kg / (m2h bar), or 10-60 kg / (m2h bar); or (i) any combination of (a)-(h).

[0034] In certain embodiments the method results in:

[0035] (a) a reduced down time and / or a reduced amount of filter clogging compared to a similar method which 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 which 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 which 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 which 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 thebiocatalyst 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).

[0036] In another aspect, the present invention provides a method for producing a chemical product from a substrate, the method comprising at least steps (a)-(d):

[0037] (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.

[0038] In certain embodiments the method:

[0039] (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.

[0040] In certain embodiments the method further comprises:

[0041] (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 orthe 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 water-chemical product solution, an acid solution, an alkaline solution, a surfactant, a cleaner, 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).

[0042] In certain embodiments of the method:

[0043] (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, at least one crude nitrile hydratase enzyme, at least one purified nitrile hydratase enzyme, or any combination thereof; (iv) said biocatalyst comprises a freshly fermented biocatalyst, a frozen or thawed biocatalyst, a hydrated biocatalyst solution, a lysed biocatalyst, 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 filter comprises a transmembrane permeability of 10-500 kg / (m2 h bar), 10-250 kg / (m2 h bar), 10-100 kg / (m2 h bar), 10-90 kg / (m2 h bar), 10-80 kg / (m2 h bar), or 10-60 kg / (m2 h bar); (xi) said membrane filtration step comprises a transmembrane permeability of 10-500 kg / (m2h bar), 10-250 kg / (m2h bar), 10-100 kg / (m2h bar), 10-90 kg / (m2h bar), 10-80 kg / ( m2h bar), or 10-60 kg / (m2h bar); (xii) 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 (xiii)-(xii).

[0044] In certain embodiments of the method said membrane filtration step and / or said additional membrane filtration step comprises:

[0045] (a) a transmembrane permeability of 10-500 kg / (m2h bar), 10-250 kg / (m2h bar), 10- 100 kg / (m2h bar), 10-90 kg / (m2h bar), 10-80 kg / (m2h bar), or 10-60 kg / (m2h bar); and / or (b) 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.

[0046] In another aspect, the present invention provides a composition comprising:

[0047] (a) a recovered biocatalyst obtainable by any of the foregoing methods; (b) a purified chemical product, monomer, amide monomer, or acrylamide obtainable by any of theforegoing methods; or (c) a polymer comprising said purified chemical product, monomer, amide monomer, or acrylamide obtainable by any of the foregoing methods.BRIEF DESCRIPTION OF THE DRAWINGS

[0048] The invention will be described in more detail with reference to appended drawings, described in detail below.

[0049] FIG 1 shows an exemplary schematic of a standard process for acrylamide (AMD) synthesis and purification using adsorption media followed by filtration.

[0050] FIG 2 shows an exemplary schematic of acrylamide (AMD) synthesis and purification by membrane filtration enabling reuse of the recovered biocatalyst according to Example 1.

[0051] FIG 3 shows an exemplary schematic of a reactor for acrylamide (AMD) synthesis with an inline membrane filtration unit for initial screening of membrane filters for separation of biocatalyst from AMD product according to Example 1.

[0052] FIG 4 shows an exemplary schematic of a pilot scale reactor setup for larger scale acrylamide (AMD) synthesis with a larger scale inline membrane filtration unit for evaluation of membrane filters for separation of biocatalyst from AMD product according to Example 2.DETAILED DESCRIPTION OF THE INVENTION

[0053] Before describing the invention, the following definitions are provided. Unless stated otherwise all terms are to be construed as they would be by a person skilled in the art.DEFINITIONS

[0054] As used herein, all technical and scientific terms have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs unless clearly indicated otherwise.

[0055] As used herein, the singular forms "a," "an," and "the" may mean "one" but also include plural referents such as "one or more" and "at least one" unless the context clearly dictates otherwise. All technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs unless clearly indicated otherwise.

[0056] As used herein, the term "or" in the claims is used to mean "and / or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and / or."

[0057] As used herein the term "or combinations thereof" as used herein refers to all permutations and combinations of the listed items preceding the term unless stated otherwise.Filtration and Membrane Filtration

[0058] As used herein the term "filtration" refers to filtration under gravity, pressure, or vacuum that is used to remove dissolved and / or suspended solid particles (i.e., micrometer scale and larger) from a slurry.

[0059] As used herein, "feed stream" refers to a solution, suspension, or culture that flows into a membrane filter to be separated into two separate streams, e.g., a permeate and a retentate. For the present invention, a "feed stream" may refer to a crude reaction mixture; a crude bioconversion reaction mixture; an aqueous solution of cellular material, enzymes, organic molecules, and / or salts; a crude aqueous reaction mixture containing an amide, biocatalysts, stabilizers, buffers, salts, and optionally unreacted nitrile; or a crude aqueous reaction mixture containing AMD, biocatalysts, stabilizers, buffers, salts, and optionally unreacted AN.

[0060] As used herein the term "membrane configuration" refers to the geometry of the membrane and its position in space in relation to the flow of the feed fluid and of the permeate. Membrane configuration also refers the manner in which the membrane is packed inside the modules. For the present invention, membrane configurations may be chosen from 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. Other conventional membrane configurations may also be used.

[0061] As used herein "membrane filter" refers to a physical barrier through which only certain desired components in a feed stream can pass. For the present invention, the principle mechanism of separation by membrane filtration is based on membrane pore size, i.e., only molecules smaller than the pores of the membrane filter can pass through into the permeate and larger molecules remain in the retentate.

[0062] As used herein "membrane filtration" refers to the pressure driven passing a "feed stream" through a membrane filter system, which separates the feed stream into two separate streams, e.g., a "permeate" and a "retentate". The "permeate" is a "flow through" or "filtrate" that passes through the pores of the membrane filter and the "retentate" contains material that is retained by the membrane and does not passthrough.

[0063] For the present invention, the principle mechanism of separation by membrane filtration is based on membrane pore size. In certain embodiments, average membrane pore sizes range from 0.1-500 nm, which require transmembrane pressures high enough to force aqueous material and solutes through the pores.

[0064] Filtration is achieved when the pores of the membrane are smallerthan the diameter of a substance in the feed stream. In certain embodiments of the present invention, membrane filtration uses nanometer sized pores through which particles smaller than about 500 nm, 490 nm, 475 nm, 450 nm, 400 nm, 300 nm, 200 nm, 100 nm, 80 nm, 60, 50 nm, 40nm, 30 nm, 20 nm, or 0.1-10 nm can pass. In other embodiments, the membrane filter contains pores through which only particles with molecular weights smallerthan 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 10-30 kDa can pass.

[0065] In addition to pore size, membrane material, membrane configuration, membrane flux, and permeability also play important roles in filtration efficiency. In certain embodiments, the membrane filters may be polymeric, ceramic, metal or any combination thereof. In other embodiments, the membrane filters comprise a polymer membrane, polyacrylonitrile (PAN) membrane, a polyethersulfone (PES) membrane, a ceramic membrane, a metal membrane such as aluminum, or multiple membranes comprising any combination of the foregoing. Other conventional membrane materials, such as polyethylene terephthalate, PTFE, cellulose, nylon, polycarbonate, cellulose acetate, glass fiber, silver, MCE, polypropylene, PVDF, and nitrocellulose may also be used. Membrane filter material can be altered to optimize wettability, transmembrane flux, permeability and flow rates, to minimize membrane fouling and / or clogging, and to increase reusability of the membrane.

[0066] In certain embodiments, the membrane filters comprise 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.

[0067] In certain embodiments, the membrane filters comprise a transmembrane permeability of 10-500 kg / (m2h bar), 10-250 kg / (m2h bar), 10-100 kg / (m2h bar), 10-90 kg / (m2h bar), 10-80 kg / (m2h bar), or 10-60 kg / (m2h bar). It is highly desirable to maximize transmembrane permeability and flow rates and to increase reusability of the membrane.

[0068] As used herein the terms "flux", "membrane flux", "transmembrane flux", or "total membrane flux" refer to the membrane permeate throughput (i.e., the mass (kg) or volume (L) of water, solutes, and particles) that flows through a membrane having a surface area (m2) over time (h). Units for flux are L / (m2h) or kg / (m2h). Membrane flux varies with pressure (i.e., transmembrane pressure). To avoid misinterpretation of data, flux can be normalized to pressure. This is called permeability.

[0069] As used herein the term "permeability" or "transmembrane permeability" refers to the mass (kg) or volume (L) of water, solutes, and particles that flows through a membrane having a fixed surface area (m2) over time (h) at a certain operating transmembrane pressure (bar). Transmembrane permeability is reported in units of kg / (m2h bar). Alternatively, the permeability units may be presented using volume, e.g., L / (m2h bar).

[0070] As used herein the term "molecular weight cutoff" or "MWCO" refers to a nominal molecular weight cut-off, with units in Dalton or kDa, of a membrane filter. MWCO isdefined as the minimum molecular weight of a globular molecule that is retained to 90% by the membrane and which remains in the retentate. Molecules with molecular weights smaller than the MWCO can pass through the filter and are found in the permeate. MWCO is highly dependent on membrane pore size and is also dependent to a lesser extent on membrane material and configuration.

[0071] As used herein the term "recovered biocatalyst" refers to biocatalyst that is isolated from the reaction mixture, such as by a membrane filtration step. Recovered biocatalyst is found in the retentate of a membrane filtration step.

[0072] As used herein the term "residual nitrile hydratase activity" or "residual enzyme activity" refers to the reaction velocity (reaction speed) of enzymatically catalyzed conversion of a substrate to a product (e.g., a nitrile to an amide) that remains in a solution of purified product (e.g., amide). Ideally, for embodiments of the present invention, a purified amide solution, such as a membrane filter permeate, contains little to no residual biocatalyst and, therefore, displays little to no residual nitrile hydratase activity.

[0073] As used herein the term "transmembrane pressure" or "TMP" refers to the average pressure difference between the pressure applied to the feed stream side of the membrane and the pressure on the permeate side (i.e., the opposite side) of the membrane. TMP [bar] = [(PF + PR) / 2] - PP. In this equation, TMP represents the transmembrane pressure, PF represents the feed stream inlet pressure, PR represents the retentate stream pressure, and PP represents the permeate stream pressure.Biotransformation

[0074] As used herein, "amide" refers to a compound produced by Nitrile Hydratase from a nitrile compound. An amide compound has the functional group RnC(O)xNR'2, wherein R and R' refer to H or organic groups, for organic amides n=l, x=l. Examples of an amide compounds include methacrylamide, acetamide or nicotinamide and preferably comprise acrylamide.

[0075] As used herein, "auto-polymerization" refers to the extent to which a monomer polymerizes during production, storage, or transport, without being intentionally subjected to polymerization reaction conditions.

[0076] As used herein the term "biocatalyst" refers to any biological material having enzymatic activity for catalyzing conversion of a chemical or an organic starting material, i.e., "substrate", into its corresponding "product", optionally by catalyzing a chemical reaction with one or more reagents with the substrate. The enzyme component of a biocatalyst binds to a substrate to form an enzyme-substrate complex, thereby decreasing the activation energy required to convert the substrate into its corresponding product. In certain embodiments, "biocatalyst" refers to any biological material having enzymatic activity wherein the enzyme comprises one or more hydrolases, amidases, ketoreductases,dehydrogenases, lipases, transaminases, oxygenases, monooxygenases, peroxygenases, hydratases, hydrogenases, lyases, oxidases, reductases, reductive aminases, aldolases, esterases, decarboxylases, transferases, ligases, glycosyltransferases, transglycosidases, phosphorylases, isomerases, synthases, or any enzyme used for organic synthesis or for the synthesis polymers or biopolymers. More specifically, the term "biocatalyst" refers to any biocatalyst having nitrile hydratase activity. The biocatalyst capable of converting acrylonitrile to acrylamide may be a microorganism which encodes an enzyme having nitrile hydratase activity or any part of said microorganism having nitrile hydratase activity. The biocatalyst may be selected from said microorganism, lysed cells of said microorganism, a cell lysate of said microorganism, or any combination of these.

[0077] As used herein the term "substrate" refers to any chemical or organic starting material that binds to an enzyme or the enzyme component of a biocatalyst to form an enzyme-substrate complex, thereby accelerating a chemical reaction to form a corresponding "product", optionally by reacting the substrate with one or more reagents. As used herein, the term "substrate" refers to a solid substrate, a liquid substrate, a slurry, a suspension, or a solution of the substrate in water or any appropriate solvent or carrier liquid known in the art, optionally comprising additional salts and / or buffers. Exemplary embodiments of a substrate include a monomer precursor, a nitrile, an amide, a ketone, an amine, an ester, an aldol, a nitrile, a carboxylate, an alkene, or any suitable substrate for any enzyme More specifically, as used herein, "substrate" refers to a nitrile, such as acrylonitrile, a nitrile solution, or a nitrile slurry.

[0078] As used herein the terms "product" and "chemical product" are used interchangeably to refer to any chemical that is formed by bioconversion or enzymatic conversion from its corresponding substrate. In exemplary embodiments, a "chemical product" comprises a monomer, an amide, a carboxylate, an alcohol, an amine, an alkene, or any suitable product formed from a substrate by any enzyme, wherein the enzyme catalyzes conversion of the substrate into the product. More specifically, as used herein, "chemical product" refers to an amide, such as acrylamide, an amide solution, or an amide slurry.

[0079] As used herein, the term "microorganism(s)", when used herein encompasses enzyme producing organisms or "Nitrile hydratase producing microorganism(s)", wherein said microorganisms endogenously express and / or are engineered to express a variant Nitrile Hydratase. Such microorganism in the context of the present invention is preferably a bacterium, fungus or yeast. Within the present invention "Nitrile Hydratase producing microorganisms" are used, or are for use, as a biocatalyst for converting a nitrile compound into the corresponding amide compound.

[0080] As used herein, the term "nitrile" is one converted by a nitrile hydratase according to the invention or a microorganism which expresses a Nitrile Hydratase according to thepresent invention into an amide compound by the action of said Nitrile Hydratase. A nitrile compound is any organic compound that has a -C=N functional group such as methacrylonitrile, acetonitrile or 3-cyanopyridine and preferably acrylonitrile.

[0081] As used herein, the term "Nitrile Hydratase activity" or "enzyme activity" or "activity" refers to the ability of biocatalyst to convert one or more substrates, such as nitriles or AN, to their corresponding products, such as amides or AMD) per unit of time with units of mol of amide / second or pmol of amide / minute.

[0082] As used herein, the term "enzyme producing organism" or "Nitrile Hydratase producing microorganism" may be any microorganism which is able to produce an enzyme, such as Nitrile Hydratase. In the context of the present invention, "Nitrile Hydratase producing microorganisms" include both naturally encoding Nitrile Hydratase microorganisms and microorganisms which are genetically engineered to contain a gene or polynucleotide encoding a Nitrile Hydratase (e.g., via transformation, transduction, transfection, conjugation, or other methods suitable to transfer or insert a polynucleotide into a cell as known in the art; cf. Sambrook and Russell 2001, Molecular Cloning: A Laboratory Manual, CSH Press, Cold Spring Harbor, N.Y., USA), thus enabling the microorganisms to produce and stably maintain the Nitrile Hydratase enzyme. For this purpose, it may further be required to insert additional polynucleotides which may be necessary to allow transcription and translation of the Nitrile Hydratase gene or mRNA, respectively. Such additional polynucleotides may comprise, inter alia, promoter sequences, or replication origins or other plasmid-control sequences. In this context, such genetically engineered microorganisms which naturally do not contain a gene encoding a Nitrile Hydratase but which have been manipulated such as to contain a polynucleotide encoding a Nitrile Hydratase may be prokaryotic or eukaryotic microorganisms. Examples of such prokaryotic microorganisms include, e.g., Escherichia coli and Rhodococcus species. Examples for such eukaryotic microorganisms include, e.g., yeast (e.g., Saccharomyces cerevisiae or Pichia pastoris).

[0083] "Nitrile Hydratase producing microorganisms" which (naturally or non-naturally) encode Nitrile Hydratase are in some embodiments capable of producing and stably maintaining Nitrile Hydratase. However, in accordance with the present invention, it is also possible that such microorganisms only produce Nitrile Hydratase during cultivation (or fermentation) of the microorganisms. Such microbia include, inter alia, bacteria of the genus Rhodococcus, Aspergillus, Acidovorax, Agrobacterium, Bacillus, Bradyrhizobium, Brevibacterium, Burkholderia, Escherichia, Geobacillus, Klebsiella, Mesorhizobium, Moraxella, Pantoea, Pseudomonas, Rhizobium, Rhodopseudomonas, Serratia, Amycolatopsis, Arthrobacter, Brevibacterium, Corynebacterium, Microbacterium, Micrococcus, Nocardia, Pseudonocardia, Trichoderma, Myrothecium, Aureobasidium,Candida, Cryptococcus, Debaryomyces, Geotrichum, Hanseniaspora, Kluyveromyces, Pichia, Rhodotorula, Comomonas, and Pyrococcus.In exemplary embodiments of the invention, the microorganism may be selected from bacteria of the genus Rhodococcus, Pseudomonas, Escherichia and Geobacillus. Also, "Nitrile Hydratase producing microorganism" include, inter alia, the following species Rhodococcus rhodochrous, Rhodococcus pyridinovorans, Rhodococcus erythropolis, Rhodococcus equi, Rhodococcus ruber, Rhodococcus opacus, Aspergillus niger, Acidovorax avenae, Acidovorax facilis, Agrobacterium tumefaciens, Agrobacterium radiobacter, Bacillus subtilis, Bacillus pallidus, Bacillus smithii, Bacillus sp BR449, Bradyrhizobium oligotrophicum, Bradyrhizobium diazoefficiens, Bradyrhizobium japonicum, Burkholderia cenocepacia, Burkholderia gladioli, Escherichia coli, Geobacillus sp. RAPcS, Klebsiella oxytoca, Klebsiella pneumonia, Klebsiella variicola, Mesorhizobium ciceri, Mesorhizobium opportunistum, Mesorhizobium sp F28, Moraxella, Pantoea endophytica, Pantoea agglomerans, Pseudomonas chlororaphis, Pseudomonas putid, Rhizobium, Rhodopseudomonas palustris, Serratia liquefaciens, Serratia marcescens, Amycolatopsis, Arthrobacter, Brevibacterium sp CHI, Brevibacterium sp CH2, Brevibacterium sp R312, Brevibacterium imperiale, Corynebacterium nitrilophilus, Corynebacterium pseudodiphteriticum, Corynebacterium glutamicum, Corynebacterium hoffmanii, Microbacterium imperiale, Microbacterium smegmatis, Micrococcus luteus, Nocardia globerula, Nocardia rhodochrous, Pseudonocardia thermophila, Trichoderma, Myrothecium verruca ria, Aureobasidium pullulans, Candida famata, Candida guilliermondii, Candida tropicalis, Cryptococcus flavus, Cryptococcus sp UFMG-Y28, Debaryomyces hanseii, Geotrichum candidum, Geotrichum sp JRl, Hanseniaspora, Kluyveromyces thermotolerans, Pichia kluyveri, Rhodotorula glutinis, Comomonas testosterone, Pyrococcus abyss!, Pyrococcus furiosus, Pyrococcus horikoshii, Brevibacterium casei, or Nocardia sp. 163.

[0084] In the context of the present invention, "Nitrile hydratase" or "NHase" refers to a microbial enzyme that catalyzes the hydration of nitriles to their corresponding amides (IUBMB Enzyme Nomenclature EC 4.2.1.84. The terms "Nitrile hydratase" and "Nitrile Hydratase" as used herein also encompass modified or enhanced enzymes which are, e.g., capable of converting a nitrile compound (e.g. acrylonitrile) to an amide compound (e.g. acrylamide) more quickly, or which can be produced at a higher yield / time-ratio, or which are more stable, as long as they are capable to catalyze conversion (i.e. hydration) of a nitrile compound (e.g. acrylonitrile) to an amide compound (e.g. acrylamide).

[0085] As used herein the terms "Nitrile Hydratase stability" or "enzyme stability" or "stability" refer to how well the biocatalyst tolerates contact with substrate and / or product, such as acrylonitrile and / or acrylamide, under specific reaction conditions (e.g., temperature, pH, reagents such as acrylonitrile, products such as acrylamide, etc.), i.e., a good stability means under specific reaction conditions means that the Nitrile Hydratase has a lower deactivation rate than another Nitrile Hydratase under the same under specificreaction conditions (since both acrylonitrile and acrylamide are known to deactivate endogenous Nitrile Hydratase biocatalysts).

[0086] As used herein the terms "amide stability" or "stability of the purified amide" refers to the amount of time that the amide or purified amide (such as acrylamide) can be stored or transported (at room temperature such as 20-30 °C, or 25 °C, or at elevated temperature such as 30-80 °C, 60-80 °C, 70-80 °C, or 75 °C) without significant degradation of the amide, such as by hydrolysis, oxidation, reduction, auto-polymerization, or gelation. In certain embodiments, amide stability refers to less 20 wt%, 0-20 wt%, 0-15 wt%, 0-10 wt%, 0-8 wt%, 0-6 wt%, 0-4 wt%, 0-2 wt%, 0-1 wt%, or 0-0.1 wt% degradation of the amide during storage for up to 1-20, 1-18, 1-15, or 1-13 days at 75 °C.Polymers

[0087] As used herein "acrylamide" refers to a neutral monomer of molecular formula: C3H5NO and a molecular weight of 71.08 g / mol.

[0088] As used herein, the term "amphoteric polymer" refers to polymers containing anionic and cationic monomers and optionally neutral monomers such as acrylamide. Amphoteric polymers contain both anionic and cationic groups on their macromolecular chains. These polymers exhibit both attraction and repulsion in their electrostatic intermolecular interactions (resulting in anti-polyelectrolyte association called "Amphoteric Effect") and they exhibit excellent salt tolerance, especially in high Ca+2 aqueous compositions.

[0089] As used herein, "anionic monomer" refers to a monomer which possesses a negative charge in aqueous solution at a pH above a certain threshold depending on the pKa values of acidic protons contained therein. The "anionic monomers" may be neutral at low pH (e.g., from a pH of about 0-1, 0-2, or 0-3) and become anionic as the pH of solution is increased. Non-limiting representative anionic monomers include acrylic acid, sodium acrylate, ammonium acrylate, methacrylic acid, 2-acrylamido-2-methylpropanesulfonic acid (AMPS), vinyl sulfonic acid, styrene sulfonic acid, maleic acid, sulfopropyl acrylate or methacrylate or other water-soluble forms of these or other polymerizable carboxylic or sulphonic acids, sulfomethylated acrylamide, ally sulfonate, itaconic acid, acrylamidomethylbutanoic acid, fumaric acid, vinylphosphonic acid, allylphosphonic acid, phosphonomethylated acrylamide, methacrylate, itaconate, 2-acrylamido 2-methyl propane sulphonate, sulfoalkyl(meth)acrylic acids, sulfonated styrenes, unsaturated dicarboxylic acids, sulfoalkyl(meth)acrylamides, vinyl acetate, n-vinylformamide, n-vinylacetamide, n- vinylcaprolactam, n-vinylimidazole, n-vinylpyridine, n-vinylpyrolidone, acrylamidopropyltrimonium chloride, salts of said acids and the like, or another anionic ethylenically unsaturated compound.

[0090] As used herein, the term "cationic monomer" generally refers to a monomer that possesses a positive charge. Examples thereof include acryloyloxy ethyl trimethylammonium chloride (Q9) monomers. Cationic monomers may also be selected from 2-(acryloyloxy)ethyl trimethylammonium chloride ("AETAC" or "Q9"), methacryloyloxyethyltrimethylammonium chloride ("MAETAC"), methacrylamidopropyltrimethylammonium chloride ("MAPTAC"), acrylamidopropyltrimethylammonium chloride ("APTAC"), methacryloyloxyethyldimethylammonium sulfate, diallyldimethylammonium chloride ("DADMAC"); dialkylaminoalkyl acrylates and dialkylaminoalkyl methacrylates and their quaternary or acid salts, including but not limited to, dimethylaminoethyl acrylate ("DMAEA"), dimethylaminoethyl methacrylate ("DMAEA"), dimethylaminoethyl acrylate methyl chloride quaternary salt, dimethylaminoethyl acrylate methyl sulfate quaternary salt, dimethylaminoethyl acrylate benzyl chloride quaternary salt, dimethylaminoethyl acrylate sulfuric acid salt, dimethylaminoethyl acrylate hydrochloric acid salt, diethylaminoethyl acrylate methyl chloride quaternary salt, dimethylaminoethyl methacrylate methyl chloride quaternary salt, dimethylaminoethyl methacrylate methyl sulfate quaternary salt, dimethylaminoethyl methacrylate benzyl chloride quaternary salt, dimethylaminoethyl methacrylate sulfuric acid salt, dimethylaminoethyl methacrylate hydrochloric acid salt, dimethylaminoethyl methacryloyl hydrochloric acid salt; dialkylaminoalkylacrylamides and methacrylamides and their quaternary or acid salts, including but not limited to, acryloylamidopropyltrimethylammonium chloride, dimethylaminopropyl acrylamide, dimethylaminopropyl acrylamide methyl sulfate quaternary salt, dimethylaminopropyl acrylamide sulfuric acid salt, dimethylaminopropyl acrylamide hydrochloric acid salt, methacrylamidopropyltrimethylammonium chloride, dimethylaminopropyl methacrylamide, dimethylaminopropyl methacrylamide methyl sulfate quaternary salt, dimethylaminopropyl methacrylamide sulfuric acid salt, dimethylaminopropyl methacrylamide hydrochloric acid salt, diethylaminoethylacrylate, diethylaminoethylmethacrylate; and diallyldialkylammonium halides, including but not limited to, diallyldiethylammonium chloride and diallyldimethylammonium chloride ("DADMAC"), and any combination thereof.

[0091] As used herein, "nonionic monomer" refers to a monomer which possesses a net charge of zero in aqueous solution. Non-limiting examples of nonionic monomers include acrylamide, N-alkylacrylamides, N,N-dialkylacrylamides, methacrylamide, N- vinylmethylacetamide or formamide, vinyl acetate, vinyl pyrrolidone, alkyl methacrylates, acrylonitrile, N-vinylpyrrolidone other acrylic (or other ethylenically unsaturated) ester or other water insoluble vinyl monomers such as styrene or acrylonitrile.

[0092] As used herein, the terms "polyacrylamide" or "PAM" generally refer to polymers and co-polymers comprising acrylamide moieties, and the terms encompass any polymers or copolymers, including terpolymers, comprising acrylamide moieties, e.g., one or more acrylamide (co)polymers of acrylamide and additional monomers capable of copolymerizingwith acrylamide. PAMs described herein may be produced in one of various forms, including, for example, dry (powder ) form (e.g., DPAM), emulsion polyacrylamide (EPAM), or liquid polyacrylamide (aqueous solution). Amphoteric polyacrylamides (AmPAM) may be formulated in dry (powder ) form (e.g., AmDPAM), or emulsion form (AmEPAM).

[0093] As used herein, the term "cationic glyoxalated polyacrylamides (GPAMs)" or "GPAM" generally refers to a polymer obtained by reacting glyoxal and a polyacrylamide "base polymer" to form a glyoxalated polymer backbone (see e.g., U.S. Pat. No. 3,556,932 which first disclosed the synthesis of a GPAM composition prepared by reacting glyoxal with a cationic polyacrylamide). The base polymer may contain acrylamide, optionally cationic monomers, and optionally anionic monomers. The base polymer may comprise a PAM, a CPAM, an APAM, or an AmPAM. Cationic charge on the polyacrylamide backbone of the GPAM renders the polymer self-retaining on fibers. In general, GPAM comprises a reactive polymer that can covalently bond with cellulose upon dehydration. Exemplary GPAMS are reactive cationic polymers containing reactive aldehydes, which react with carboxylate and - OH moieties on cellulosic and lignocellulosic fibers to form covalent bonds. Exemplary GPAMS are used as wet and / or dry strength additives and as retention aids and drainage aids in papermaking. GPAM wet strength is temporary since it decreases overtime. GPAM increases paper strength through covalent bond formation between its aldehyde groups and cellulose hydroxyl groups. This covalent bond is reversible in water, resulting in rapid wet strength decay over time. This rapid decrease in wet strength is desirable for flushed paper products, which must disintegrate rapidly after use

[0094] As used herein, the term "anionic polyacrylamide (APAM)" refers to a copolymer of acrylamide and anionic monomers, such as acrylic acid or ATBS, and also encompasses acrylamide polymers which are partially or completely hydrolyzed following polymerization to form acrylate or acrylic acid side chains.

[0095] As used herein, the term "cationic polyacrylamide (CPAM)" refers to a copolymer of acrylamide and cationic monomers, such as DADMAC or Q9.

[0096] As used herein, the term "amphoteric polyacrylamide (AmPAM)" refers to a terpolymer (or higher order polymer) of acrylamide, cationic monomers, and anionic monomers, which may also encompass acrylamide groups which are partially or completely hydrolyzed following polymerization to form acrylate or acrylic acid side chains.

[0097] As used herein, the terms "polymer" or "polymeric additives" and similar terms are used in their ordinary sense as understood by one skilled in the art, and thus may be used herein to refer to or describe a large molecule (or group of such molecules) that may comprise recurring units. Polymers may be formed in various ways, including by polymerizing monomers and / or by chemically modifying one or more recurring units of a precursor polymer. Unless otherwise specified, a polymer may comprise a "homopolymer" that may comprise substantially identical recurring units that may be formed by, forexample, polymerizing a particular monomer. Unless otherwise specified, a polymer may also comprise a "copolymer" that may comprise two or more different recurring units that may be formed by, for example, copolymerizing, two or more different monomers, and / or by chemically modifying one or more recurring units of a precursor polymer. Unless otherwise specified, a polymer or copolymer may also comprise a "terpolymer" or a "tetrapolymer" which generally refer to polymers that comprise three, four, or more different recurring monomer units. The term "polymer" as used herein is intended to include both the acid form of the polymer as well as its various salts. Polymers may be amphoteric in nature, that is, containing both anionic and cationic substituents, although not necessarily in the same proportions. Polymer molecular weights may be measured by various methods known to persons of skill in the art. For example, weight average molecular weight may be measured using gel permeation chromatography (GPC). Polymer molecular weights may be measured by various methods known to persons of skill in the art. For example, weight average molecular weight may be measured using gel permeation chromatography (GPC). Additionally, polymer molecular weights may be measured by GPC / Light Scattering / Viscometry also known as Triple Detection GPC which employs Refractive Index Detector (with or without UV Detector), Dilute Solution Viscometry and Light Scattering all in series to determine molecular weights, distribution and related solution parameters.

[0098] As uses herein, the phrase "polymerization inhibitor" refers to substances that prevent or slow down the polymerization chain reaction of monomers. In the context of acrylamide, which readily polymerizes, these inhibitors play a crucial role in stabilizing its solution. Exemplary polymerization inhibitors used with acrylamide and other monomers include hydroquinone, copper (II) ions (Cu2+), sodium metabisulfite (Na2S2O5), and phenothiazine derivatives. Hydroquinone effectively suppresses polymerization by reacting with free radicals formed during the process. Typically, concentrations of 0.01% to 0.05% hydroquinone are added to acrylamide solutions. In certain embodiments hydroquinone, may be used in stabilizing acrylonitrile in certain production steps. In other embodiments, such as the final production step, hydroquinone is removed and replaced with 4- methoxyphenol (MEHQ), which also acts as a polymerization inhibitor. Copper ions act as inhibitors by binding to the free radicals and disrupting the polymerization chain. Copper sulfate (CuS04) is commonly used for this purpose. Sodium metabisulfite releases sulfur dioxide (SO2) when dissolved in water. SO2scavenges free radicals, preventing them from initiating polymerization. Sodium metabisulfite is often used in combination with other inhibitors. Phenothiazine derivatives inhibit acrylamide polymerization by capturing free radicals. They are effective at low concentrations.Terms And Units

[0099] As used herein, the term "% by wt" or "wt%" denotes the dry mass of additive per total dry mass of solids in a formulation, solution, suspension, slurry, or solid, multiplied by 100%.DESCRIPTION OF THE INVENTIONStandard process for synthesis and purification of acrylamide (AMD)

[0100] Acrylamide (AMD) is typically manufactured by feeding acrylonitrile (AN), water, and a biocatalyst with nitrile hydratase activity into a batch reactor. The biocatalyst converts AN to AMD. Purification (removal of biocatalyst from AMD) is necessary before shipment or polymerization. The biocatalyst may be deactivated during the reaction after being in contact with AN and AMD. Purification may be accomplished by reducing the pH and then adding adsorption media. The biocatalyst adsorbs onto the surface of the adsorption media, which is then typically removed by filtering through a coarse filter. Purified AMD remains in the filtrate. The biocatalyst and adsorption media are discarded as waste.

[0101] FIG 1 shows an exemplary schematic of a standard process for acrylamide (AMD) synthesis and purification using acidification and addition of adsorption media followed by filtration.

[0102] This existing method causes several operational challenges, including generating significant amounts of classified hazardous waste and frequent downtime due to blocking of the filter pores. The method also requires acidification and addition of adsorption media, both of which deactivate the biocatalyst. The deactivated biocatalyst cannot be reused. The operational costs of downtime, remediation methods for clogged filters, and hazardous waste disposal are significant.Membrane Filtration

[0103] For the present invention, purification (removal of biocatalyst from AMD) was accomplished by using membrane filtration for purification of biocatalyst from bioconversion reactions (e.g., from crude aqueous AMD). After membrane filtration, active biocatalyst is recovered as the retentate and purified monomer is recovered as the permeate. Biocatalyst in the retentate retains enzymatic activity and may be reused.Purified monomer (e.g., AMD without enzyme activity) in the permeate may be stored, transported, and used for polymerization.

[0104] For the best performing membranes, enzyme removal and thermal stability were similar or better than commercial AMD treated with adsorption media. This invention provides reduced operating costs, possibility to recycle biocatalyst, increased sustainability, and reduced amount of hazardous.Methods for Bioconversion of Substrates to their Corresponding Chemical Products

[0105] In one aspect, the present invention provides a method for producing a chemical product from a substrate, the method comprising at least steps (a)-(c):

[0106] (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.

[0107] In certain embodiments of the method:

[0108] (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.

[0109] In other embodiments of the method:

[0110] (a) the biocatalyst comprises at least one enzyme selected from the group consisting of hydratases, nitrile hydratases, hydrolases, amidases, ketoreductases, dehydrogenases, lipases, transaminases, oxygenases, monooxygenases, peroxygenases, hydratases, hydrogenases, lyases, oxidases, reductases, reductive aminases, aldolases, esterases, decarboxylases, transferases, ligases, glycosyltransferases, transglycosidases, phosphorylases, isomerases, synthases, and proteases; (b) the substrate comprises a monomer precursor, a nitrile, an amide, a ketone, an amine, an ester, an aldol, a nitrile, a carboxylate, an alkene, or any suitable substrate for any one or more of said enzymes and the product comprises a monomer, an amide, a carboxylate, an alcohol, an amine, an alkene, or any suitable product from any one or more of said enzymes, wherein the substrate is converted into the product by the enzyme; (c) the biocatalyst comprises at least one nitrile hydratase enzyme, the substrate comprises at least one nitrile, and the product comprises at least one amide, wherein the nitrile is converted into the amide by the nitrile hydratase enzyme; and / or (d) the biocatalyst comprises at least one nitrile hydratase enzyme, the substrate comprises acrylonitrile, and the product comprises acrylamide.

[0111] In another aspect, the present invention provides a method for producing an amide from a nitrile, the method comprising at least steps (a)-(c):

[0112] (a) contacting the nitrile with a biocatalyst comprising an initial nitrile hydratase activity to form a reaction mixture; ( b) converting the nitrile into the amide; 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 amide.

[0113] In certain embodiments the method further comprises:

[0114] (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, 7.5-9, 7.5-8.5 or 7.5-8 8-8.5; (iii) prior to step (a), adjusting said biocatalyst to a temperature of 5-30 °C, 5-25 °C, 5-20 °C, 5-15 °C, or 5-10 °C; (iv) after step (a), optionally adjusting the reaction mixture to a pH of 3- 10, 4-9.5, 5-9, 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, 10-25 °C, 15-25 °C, or 20- 25 °C and then optionally maintaining the reaction mixture at a temperature of 20-30 °C or 20-25 °C for the duration of step (b); (vi) during step (b) analyzing the reaction mixture to determine a concentration of the nitrile remaining in the reaction and / or a mol% conversion of the nitrile to the amide; (vii) during step ( b), allowing said reaction mixture to react until concentration of the nitrile remaining in the reaction ranges from 0-50,000 ppm, 0-40,000 ppm, 0-30,000 ppm, 0-20,000 ppm, 0-10,000 ppm, 0-8000 ppm, 0-6000 ppm, 0-5000 ppm, 0-4000 ppm, 0-3000 ppm, 0-2500 ppm, 0-2000 ppm, 0-1500 ppm, 0-1000 ppm, 0-800 ppm, 0-600 ppm, 0-500 ppm, 0-400 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 and / or the mol% conversion of the nitrile to the amide ranges from 90-100%, 95-100%, 98-100%, or 99-100%; or (viii) any combination of (i)-(vii).

[0115] In certain embodiments the method further comprises:

[0116] (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 (e.g., 1-90 wt%, 1-80 wt%, 1-60 wt%, 1-40 wt%, or 1-20 wt%) from the reaction mixture; (ii) prior to step (c), adjusting the reaction mixture to a pH of 3-10, 4-10, 5-10, 6-10, 6.5-9.5, 7-9, 7.5-9, 7.5-8.5 or 8-8.5 when said recovered biocatalyst is to be discarded or to a pH of 6-10, 7-9, 7.5-9, 7.5-8.5 or 8-8.5 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 amide 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 amide; (v) after step (c), washing said membrane filter with an amount of water, water-amide 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).

[0117] In certain embodiments the method further comprises:

[0118] (i) after step (b), optionally stabilizing the amide by addition of a polymerization inhibitor, which may comprise hydroquinone, a copper salt, copper (II) ions (Cu2+), sodium metabisulfite (Na2S2O5), phenothiazine derivatives, or 4-methoxyphenol (MEHQ); (ii) after step (c) adjusting the purified amide to a pH of 5-10, 6-9.5, 7-9, 7.5-9, 7.75-8.7, 8-8.5, or preferably 6-8; (iii) after step (c) analyzing the purified amide to determine a wt% removal of the biocatalyst, optionally by spectrophotometric analysis, wherein said wt% removal of the biocatalyst from the amide is 90-100 wt%, 95-100 wt%, 98-100 wt%, 99-100 wt% or 99.9-100 wt%; (iv) after step (c) analyzing the purified amide to determine a residual nitrile hydratase activity, wherein said residual nitrile hydratase activity is 0-10%, 0-5%, 0-2%, 0- 1%, or 0-0.1%; (v) after step (c), storing said purified amide 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 amide; (vii) after step (c), using said purified amide in a subsequent polymerization reaction to produce a polymer comprisingthe amide; or (viii) any combination of (i)-(vii).

[0119] In certain embodiments the method further comprises:

[0120] (i) after step (c), washing said recovered biocatalyst with an amount of water, a mixture of water and the amide, or an aqueous solution; (ii) after step (c), analyzing said recovered biocatalyst to determine a recovered nitrile hydratase activity, wherein said recovered nitrile hydratase activity is 5-100%, 10-100%, 20-90%, 30-80%, 40-75%, 50-70% of said initial nitrile hydratase 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 an amide from a nitrile; (v) after step (c), combining said recovered biocatalyst with an additional amount of the unused biocatalyst and then reusing the recovered biocatalyst in a subsequent method for producing an amide from a nitrile; or (vi) any combination of (i)-(v).

[0121] In certain embodiments of the method: (a) said nitrile comprises a monomer precursor, a substituted nitrile, a substituted aliphatic nitrile having 1 to 10 carbon atoms, acrylonitrile, or methacrylonitrile; and / or (b) said polymer comprising the amide comprises a homopolymer comprising the amide; a copolymer comprising 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).

[0122] In certain embodiments of the method the biocatalyst comprises:

[0123] (a) 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) whole cells, cell lysate, live cells, dead cells, broken cells, cell fragments, or any combination thereof; (c) a freshly fermented biocatalyst, a frozen or thawed biocatalyst, a lysed biocatalyst, ahydrated biocatalyst, a dried biocatalyst, a rehydrated biocatalyst, or any combination thereof; (d) a buffered aqueous solution, a cellular growth media, at least one stabilizer, glycerol, or any combination thereof; or (e) any combination of (a)-(d).

[0124] In certain embodiments of the method said membrane filter:

[0125] (a) 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 10-30 kDa ; (b) 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) 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) 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) comprises a transmembrane permeability of 10-500 kg / (m2 h bar), 10-250 kg / (m2 h bar), 10-100 kg / (m2 h bar), 10-90 kg / (m2 h bar), 10-80 kg / (m2 h bar), or 10-60 kg / (m2 h bar); or (f) any combination of (a)-(e).

[0126] In other embodiments of the method said membrane filtration comprises a total membrane surface area of 0.1-10000 m2, 1-1000 m2, 10-900 m2, 50-800 m2, 100-600 m2, 200-500 m2, or 300-400 m2.

[0127] In certain embodiments of the method said membrane filtration step and / or said additional membrane filtration step comprises:

[0128] (a) said transmembrane permeability of 10-500 kg / (m2h bar), 20-250 kg / (m2h bar), 30-100 kg / (m2h bar), 40-90 kg / (m2h bar), 45-80 kg / (m2h bar), or 50-60 kg / (m2h bar); and / or (b) 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.

[0129] In certain embodiments of the method:

[0130] (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 membranefilter 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; (h) the membrane filter comprises a said transmembrane permeability of 10-500 kg / (m2 h bar), 10-250 kg / (m2 h bar), 10-100 kg / (m2 h bar), 10-90 kg / (m2 h bar), 10-80 kg / (m2 h bar), or 10-60 kg / (m2 h bar); or (i) any combination of (a)-(h).

[0131] In certain embodiments the method results in:

[0132] (a) a reduced down time and / or a reduced amount of filter clogging compared to a similar method which does not comprise at least one membrane filtration step; (b) an increased wt% removal of the biocatalyst from the purified amide compared to a similar method which does not comprise at least one membrane filtration step; (c) a decreased residual nitrile hydratase activity in the purified amide compared to a similar method which does not comprise at least one membrane filtration step; (d) an increased recovered nitrile hydratase activity of the recovered biocatalyst compared to a similar method which 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 amide 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 amide increases as said wt% removal of the biocatalyst from the purified amide increases; (g) a reduced auto-polymerization of the purified amide 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 auto-polymerization of the purified amide decreases as said wt% removal of the biocatalyst from the purified amide increases; (h) a positive correlation between said stability of the purified amide and said wt% removal of the biocatalyst from the purified amide; or (i) any combination of (a)-(h).

[0133] In another aspect, the present invention provides a method for producing a chemical product from a substrate, the method comprising at least steps (a)-(d):

[0134] (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.

[0135] In certain embodiments the method:

[0136] (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.

[0137] In other embodiments of the method:

[0138] (a) the biocatalyst comprises at least one enzyme selected from the group consisting of hydratases, nitrile hydratases, hydrolases, amidases, ketoreductases, dehydrogenases, lipases, transaminases, oxygenases, monooxygenases, peroxygenases, hydratases, hydrogenases, lyases, oxidases, reductases, reductive aminases, aldolases, esterases, decarboxylases, transferases, ligases, glycosyltransferases, transglycosidases, phosphorylases, isomerases, synthases, and proteases; (b) the substrate comprises a monomer precursor, a nitrile, an amide, a ketone, an amine, an ester, an aldol, a nitrile, a carboxylate, an alkene, or any suitable substrate for any one or more of said enzymes and the product comprises a monomer, an amide, a carboxylate, an alcohol, an amine, an alkene, or any suitable product from any one or more of said enzymes, wherein the substrate is converted into the product by the enzyme; (c) the biocatalyst comprises at least one nitrile hydratase enzyme, the substrate comprises at least one nitrile, and the product comprises at least one amide, wherein the nitrile is converted into the amide by the nitrile hydratase enzyme; and / or (d) the biocatalyst comprises at least one nitrile hydratase enzyme, the substrate comprises acrylonitrile, and the product comprises acrylamide.

[0139] In another aspect, the present invention provides a method for producing an amide from a nitrile, the method comprising at least steps (a)-(d):

[0140] (a) contacting the nitrile with a biocatalyst comprising an initial nitrile hydratase activity to form a reaction mixture; (b) converting the nitrile into the amide; (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 amide; and (d) reusing the recovered biocatalyst in a subsequent method for producing the amide from the nitrile.

[0141] In certain embodiments the method further comprises:

[0142] (i) after step (c), storing said purified amide 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 amide; (iii) after step (c), using the purified amide in a subsequent polymerization reaction to produce a polymer comprising the amide, wherein said polymer comprises a homopolymer comprisingthe amide, a copolymer comprising 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); (iv) after step(c), washing said membrane filter with an amount of water, a water-amide 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; (v) after step (d), washing said recovered biocatalyst with an amount of water, a mixture of water and the amide, or an aqueous solution; (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 nitrile hydratase activity, wherein said recovered nitrile hydratase activity is 5- 100%, 10-100%, 20-90%, 30-80%, 40-75%, 50-70% of said initial nitrile hydratase activity; or (viii) any combination of (i)-(vii).

[0143] In certain embodiments of the method:

[0144] (a) said nitrile comprises a monomer precursor, a substituted nitrile, a substituted aliphatic nitrile having 1 to 10 carbon atoms, acrylonitrile, or methacrylonitrile; (b) 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; (c) said biocatalyst comprising nitrile hydratase activity comprises whole cells, cell lysate, live cells, dead cells, broken cells, cell fragments, or any combination thereof; (c) said biocatalyst comprising nitrile hydratase activity 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; (d) said biocatalyst comprising nitrile hydratase activity comprises a buffered aqueous solution, a cellular growth media, at least one stabilizer, glycerol, or any combination thereof; (e) 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; (f) 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; (g) 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; (h) 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; (i) said membrane filter comprises a transmembrane permeability of 10-500 kg / (m2 h bar), 20-250 kg / (m2 h bar), 30-100 kg / (m2 h bar), 40-90 kg / (m2 h bar), 45-80 kg / (m2 h bar), or 50-60 kg / (m2 h bar); (j) said membrane filtration step optionally comprises a transmembrane permeability of 10- 500 kg / (m2h bar), 10-250 kg / (m2h bar), 10-100 kg / (m2h bar), 10-90 kg / (m2h bar), 10-80 kg / ( m2h bar), or 10-60 kg / (m2h bar), wherein said transmembrane permeability is optionally maintained over a filtration time ranging from 0.1-24 h, 1-12 h, 1-10 h, 1-8 h, 1-6 h, 1-4 h, or 1-2 h; (k) said membrane filtration step comprises a transmembrane pressure of0.1-15 bar, 0.5-10 bar, 1-8 bar, 1.5-7 bar, 2-6 bar, or 3-5 bar; or (I) any combination of (a)- (k).Methods for Producing Recovered Biocatalyst, Purified Amide, Polymers

[0145] In another aspect, the present invention provides a composition comprising:

[0146] (a) a recovered biocatalyst obtainable by any of the foregoing methods; (b) a purified chemical product, monomer, amide monomer, or acrylamide obtainable by any of the foregoing methods; or (c) a polymer comprising said purified chemical product, monomer, amide monomer, or acrylamide obtainable by any of the foregoing methods.

[0147] The methods and compositions illustratively disclosed herein suitably may be practiced in the absence of any element which is not specifically disclosed herein and / or any element specifically disclosed herein. Exemplary embodiments of the invention and its advantages are further disclosed in the following examples.EXAMPLES

[0148] The examples provided herein are for illustrative purposes so that the invention may be more fully understood. These examples should not be construed as limiting the invention in any way.Example 1: Initial evaluation of membrane filters for removing biocatalyst

[0149] For the present invention, purification (removal of biocatalyst from AMD) was accomplished by membrane filtration of the crude reaction. FIG 2 shows a schematic of acrylamide (AMD) synthesis and purification by membrane filtration, which enables reuse of the recovered biocatalyst.

[0150] By contrast, certain existing processes for acrylamide (AMD) purification shown in FIG 1 require acidification and / or addition of adsorption media, both of which deactivate the biocatalyst. The deactivated biocatalyst cannot be reused. These processes also generate significant amounts of hazardous waste, which is expensive to handle and causes frequent downtime due to blocking of the filter pores adsorption media-biocatalyst complex.

[0151] Preparation of crude aqueous acrylamide (AMD)

[0152] Crude aqueous acrylamide (AMD) solution was prepared in a reactor setup. Acrylonitrile (AN), water, and a biocatalyst were fed into the reactor at 25-30 °C and pH 7.0- 8.5. The biocatalyst was a crude cell lysate with nitrile hydratase activity, which contained a mixture of whole cells and small cellular fragments in a modified aqueous growth media or in an aqueous solution. In representative experiments, less than 0.1% of the AN remained after conversion of AN to AMD. After 95%, 99%, 99.9%, or 100% conversion of AN to AMD, the crude acrylamide (AMD) solution was used for initial screening of the membrane filters.

[0153] The reactor setup included a reaction vessel, which may be a batch reactor, a semi- continuous reactor, a bioreactor, feed tank, or any suitable reactor known in the art, equipped with jacket to control temperature and a pump, which fed through a pressure gauge (Pl) to a membrane filtration module (Cell 1). Permeate (membrane filtrate) was fed through a pressure gauge (Fp) into a sample collector. Retentate, which retained biocatalyst, was capable of being fed through a pressure gauge (P2) and returned through a pressure regulator to the reaction vessel for reuse. In certain processes, including but not limited to batch processes, the retentate is recirculated back to the feed vessel until a desired concentration is obtained then the enzyme in the feed vessel is reused. FIG 3 shows a schematic of the reactor with inline membrane filtration unit for separation of biocatalyst from AMD product.

[0154] Membrane filtration of crude aqueous acrylamide solution

[0155] Membrane screening experiments were conducted on the crude acrylamide (AMD) in once-through mode with and without pH adjustment. Membrane filters with a range of pore sizes having a molecular weight cutoff (MWCO) of 20-100 kDa, polymeric materials (polyacrylonitrile (PAN), polyethersulfone (PES), Polyamide (PI) and flat sheet configuration were screened. Total surface area of the screening membranes was about 0.1 m2.

[0156] Membrane permeate throughput (flux) at different transmembrane pressure (TMP) was monitored over 1-2 hours of filtration. Permeate samples were collected and assayed for biocatalyst removal efficiency. Membrane characteristics and results from initial membrane screening are shown in Table 1.

[0157] Table 1: Membrane characteristics and their performance from initial screening*PAN-polyacrylonitrile; PES-polyethersulfone; PI: Polyamide, Screening Test: Pass when the biocatalyst rejection is >90%

[0158] Results indicated that ultrafiltration membrane with pore size of 20-30 kDa performed the best for membrane throughput (flux) and biocatalyst removal efficiency. Both PAN and PES membranes with two different configurations (spiral wound SWE and hollow fiber HF) were chosen for pilot scale validation.

[0159] These results provide initial proof of concept that membrane filtration can be used for purification of acrylamide (AMD) product from crude biotransformation reaction mixtures containing biocatalyst to obtain purified acrylamide and recovered biocatalyst. Acidification and addition of adsorption media were not necessary. After membrane filtration, the biocatalyst was in the retentate and purified AMD (without enzyme activity) was in the permeate. The recovered biocatalyst retained nitrile hydratase activity and was reusable in subsequent reactions.

[0160] The results suggest that membrane filtration may be used industrially for removal of biocatalyst from acrylamide (AMD) product, which may lower operational costs and allow for recycling of biocatalyst, which was not possible with the previous adsorption media based purification method.Example 2: Pilot scale evaluation of membrane filtration for removing biocatalyst

[0161] Selected membrane filters from Example 1 were evaluated for removing biocatalyst from crude AMD in a pilot scale reactor setup.

[0162] Preparation of crude aqueous acrylamide (AMD)

[0163] Crude aqueous acrylamide (AMD) solution was prepared as described in Example 1 in a pilot scale reactor setup for AMD preparation and purification by membrane filtration.In representative experiments, less than 1 ppm of AN remained after conversion of AN to AMD. After 95%, 99%, 99.9%, or 100% conversion of AN to AMD, the crude acrylamide (AMD) solution was used for pilot scale evaluation of the selected membrane filters.

[0164] The pilot scale reactor setup was a larger scale version of the reactor setup described in Example 1. FIG 4 shows a schematic of the pilot scale reactor setup with a larger scale inline membrane filtration unit for separation of biocatalyst from AMD product.

[0165] Membrane filtration of crude aqueous acrylamide solution

[0166] Larger scale versions of selected membrane filters (total membrane surface area of 1 m2) from Example 1 were evaluated for removing biocatalyst from AMD in a pilot scale reactor setup. PAN and PES membrane filters with spiral wound element (SWE), and hollow fiber (HF), and flat were used.

[0167] Membrane characteristics and performance are shown in Table 2.

[0168] Membrane screening experiments were conducted on the crude acrylamide (AMD) in once-through mode with and without pH adjustment. Membrane permeate throughput (flux) at different transmembrane pressures (TMP) was monitored over 2 hours of filtration.

[0169] Permeate samples containing purified acrylamide (AMD) were collected and assayed for biocatalyst removal efficiency and for thermal stability, determined by the extent of auto-polymerization during storage. Results from pilot scale membrane evaluation are shown in Table 2.

[0170] Permeate samples from the best experiments were also collected and subjected to polymerization testing. The purified AMD was used to prepare polyacrylamide. The polymerization of AMD was successful and similar polymers were obtained from the inventive AMD purified by membrane filtration compared to benchmark technology.

[0171] Table 2: Membrane filter characteristics and results of pilot scale filtration of biocatalyst from crude aqueous acrylamide (AMD).

[0172] Results indicate that three types of membrane, PAN-SWE at pH 5 (Experiment 2), PES-HF at pH 8 (Experiment 4), and PAN-Flat at pH 8 (Experiment 5), were able to remove biocatalyst from AMD with high retention. A third membrane type, PES-SWE, also gave results were rated as successful (data not shown).

[0173] It was surprisingly found that the highest permeability was achieved with the 30 kDa PAN-SWE membrane at pH 8 (Experiment 6). Permeability was maintained over 2 h of use. This membrane displayed surprisingly small amounts of clogging and surprisingly successful membrane performance.

[0174] Without being bound to theory, it can be rationalized that certain combinations of membrane filter pore size, material, and configuration are optimal for retaining a high amount of biocatalyst while maintaining high permeability over time, and therefore providing high biocatalyst removal efficiency. Pore size is important for biocatalyst removal efficiency and for maintaining flux. Spiral wound (SWE) and hollow fiber (HF) configurations provide high surface areas for maintaining flux.

[0175] Best throughputs were observed at pH 8, which is the same pH as the crude aqueous AMD. Biocatalyst recovered at pH= 8, was tested in a subsequent AMD synthesis reaction and was found to be still active.

[0176] Experiments performed at pH 5 suggest that the final AMD quality is somewhat improved. However, pH 5 gave much lowerthroughput (30-50% of that @pH=8). Results of polymerization tests indicate that permeates (containing purified AMD) from Experiment 4 (PES HF, 10 kDa) and Experiment 6 (PAN SWE, 30 kDa) can be polymerized with good endproduct quality. AMD quality was confirmed by polymerizing AMD purified with PES HF, 10kDa and with PAN SWE, 30 kDa membranes, both resulting in polymers that were determined to be within specification.

[0177] Taken together, these results provide proof of concept that PAN-SWE and PES-HF having pore sized (MWCO) between 10-30 kDa are effective for removing biocatalyst from crude AMD product at a rate of 50 metric ton of AMD within 2 hours. The membrane filters provided a sustained permeability over 2 h of use. Permeates containing purified AMD were successfully used in polymerization reactions to produce polyacrylamides. These results also provide proof of concept that approximately 100% of the biocatalyst can be recovered at pH 7.0-8.5 and can then be reused.Example 3: Pilot scale evaluation of membrane filter performance after use and cleaning

[0178] Selected experiments from Example 2 were repeated over multiple (n) trials. A clean in place (CIP) protocol was performed between each trial. CIP was performed at pH=8 by flushing with circulating water for 5-10 minutes. This short amount of time was sufficient to restore flux. Results of CIP experiments are shown in Table 3

[0179] Table 3: Membrane filter performance during filtration of AMD .CWF- Clean water flux (CWF) at 25 °C; Maximum achieved yield = [permeate vol / initial tota vol]*100%; Enzyme removal efficiency based on rejection rate of biocatalyst

[0180] These results indicate that the HF-1 membrane provided the best performance over multiple trials (e.g., minimum % CWF loss, best enzyme removal efficiency, and best yield). The SWE-3 membrane provided the second best results.

[0181] These results provide proof of concept that the HF-1 and SWE-3 membranes can be used for multiple purifications of crude AMD with high reproducibility and highly efficient CIP between filtrations.

[0182] In the preceding disclosure which includes the examples, different procedures and various steps have been described. It will, however, be evident that various modifications and changes may be made thereto, and additional procedures may be implemented,without departing from the broader scope of the procedures as set forth in the claims that follow.

Claims

CLAIMSWhat is claimed is:

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. 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. 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. 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. 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. 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. 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. 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. 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 / (m2h bar), 20-250 kg / (m2h bar), 30-100 kg / (m2h bar), 40-90 kg / (m2h bar), 45-80 kg / (m2h bar), or 50-60 kg / (m2h 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. The method of any one of the foregoing claims, comprising one or more of thefollowing:(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. 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 notcomprise 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. 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. 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. 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, cationicmonomers, 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. 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, 0.5-490nm, 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 / (m2h bar), 20-250 kg / (m2h bar), 30-100 kg / (m2h bar), 40-90 kg / (m2h bar), 45-80 kg / (m2h bar), or 50-60 kg / (m2h 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. 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.

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