Recovery of polyamides

Abstract

The present invention relates to a process for recovering a polyamide, the process comprising: (i) subjecting the polyamide to acid hydrolysis in an aqueous liquid to produce polyamide oligomers soluble in the aqueous liquid; and (ii) converting the oligomer into its monomer component using an amidase.

Description

[0001] Field of the invention The present invention relates to recycling polyamides. BACKGROUND

[0002] The management of waste plastic products remains a significant problem for modern society. Models predict that if current global waste management trends continue, up to 90 million metric tons of plastic waste could be ultimately discarded into the environment each year by 2030.

[0003] There is ample evidence that waste plastics are having a number of deleterious effects on the environment.

[0004] The production of polyamides has been steadily increasing since the advent of nylon in the late 1930s. It is estimated that the global annual production of polyamides is now in excess of about 60 million metric tons, the vast majority of which is nylon 6 and nylon 6,6. Other common polyamides that have been commercialized include nylon 10,10, nylon 11, nylon 12, nylon 6,10, nylon 4,6, and nylon 6,12.

[0005] The excellent durability and strength of polyamides make them well suited for use in areas such as textiles, automotive, and sportswear, but unfortunately this also results in poor biodegradability of these polymers. As a result, waste polyamides will persist in the environment and become a problem for many generations to come.

[0006] Most polyamide products are produced from virgin (i.e., non-recycled) resins. Currently, some polyamides are produced using bio / plant-derived monomers (as opposed to petrochemical-derived monomers). Regardless of the source of the monomers used to produce the polyamides, the majority of polyamide products ultimately end up in the waste stream. This poor recycling management is, in part, due to the inefficiency of existing polyamide recycling technologies.

[0007] In theory, most waste polyamide products can be converted into recycled products through melt processing (e.g., by melt extrusion).

[0008] While recycled polyamide products obtained through melt processing do have a number of uses, this method itself promotes a decrease in the physical and chemical properties of the polymer. For example, the more times a polyamide is subjected to melt processing, the lower its molecular weight becomes, which results in a decrease in the intrinsic viscosity (IV) of the polymer. This decrease in the IV of the recycled polyamide will limit its use in many applications. The use of colored waste polyamides as feedstock will result in a colored recycled product, which will also limit its use in many applications. The recycling of polyamides through melt processing is also very energy intensive, which in turn decreases the economic viability of this technology.

[0009] So-called chemical recycling is another approach to recovering polyamides. Unlike melt processing methods, chemical recycling aims to depolymerize the polyamide, returning it to its monomeric components. The specific monomers produced will vary depending on the type of polyamide being processed. The monomers thus obtained can be reused, for example, by repolymerization to produce virgin polyamide. This virgin polyamide will be colorless / polluting and can be reused in the manufacture of consumer products.

[0010] One advantage of chemical recycling is that polyamides produced from recycled monomers are virtually indistinguishable in quality from virgin polyamides produced from conventional petrochemical or bio-based monomer resources in most respects. Furthermore, the chemical recycling process can be repeated an unlimited number of times without impairing the chemical or physical properties of the polyamides produced from recycled monomers.

[0011] Therefore, the chemical recycling method essentially uses waste polyamide as a raw material for producing monomers, which are then polymerized to produce virgin polyamide.

[0012] Various chemical recovery technologies have been developed over the years. However, these processes typically use highly hazardous chemicals and harsh reaction conditions. For example, common chemical recovery methods are often carried out under rather harsh and energy-intensive conditions, such as high catalyst loading, high temperature, and high pressure environments.

[0013] Enzymatic depolymerization of polymers is an area of ​​increasing interest from both academia and industry. Similar to chemical recycling, enzymatic recycling breaks down polymers into their monomeric components. Enzymatic depolymerization does not require the use of highly hazardous chemicals, the chemical depolymerization counterparts, nor does it need to be carried out under harsh reaction conditions.

[0014] However, there are limited examples of commercially available depolymerization polymerases, and most of them are only active against polyesters such as polyethylene terephthalate (PET). Furthermore, these enzymes typically exhibit relatively poor activity against the crystalline forms of most polymer wastes, including polyamide waste.

[0015] Therefore, developing new methods for polyamide recycling remains an opportunity that can address one or more problems with conventional recycling technologies, or at least provide a practical alternative. Summary of the Invention

[0016] The present invention provides a method for recycling polyamide, the method comprising: (i) subjecting the polyamide to acid hydrolysis in an aqueous liquid to produce a polyamide oligomer soluble in the aqueous liquid; and (ii) using an amidase to convert the oligomer into its monomeric components.

[0017] By combining relatively mild chemical depolymerization via acid hydrolysis with enzymatic depolymerization, polyamides can be depolymerized to obtain their monomeric components. Specifically, polyamides are first partially depolymerized into oligomers under relatively mild chemical conditions, and then the resulting oligomeric polyamides are depolymerized to obtain their monomeric components. This combined method of first generating oligomers through mild chemical depolymerization and then enzymatically depolymerizing the oligomers is a highly efficient and effective means of producing polyamide monomer raw materials.

[0018] The monomers produced according to the method of the invention can then be advantageously used to manufacture virgin polyamides.

[0019] The method of the present invention can be advantageously scaled up to industrial production scale and can be operated in batch or continuous manner.

[0020] In one embodiment, acid hydrolysis is carried out at a temperature below 200°C, for example, in a temperature range of about 100°C to about 180°C.

[0021] In another embodiment, the amidase is immobilized on a substrate, such as a resin.

[0022] In another embodiment, the method is performed continuously.

[0023] In one embodiment, the amidase is immobilized on the substrate, and the method is performed continuously.

[0024] In another embodiment, amidases are capable of hydrolyzing the amide bonds in water-soluble oligomers of polyamides.

[0025] In one embodiment, the amidase comprises: a) The amino acid sequence of amino acid residues 2-398 of SEQ ID NO:1 or SEQ ID NO:95; b) The amino acid sequence of amino acid residues 2-398 of SEQ ID NO:2, or an amino acid sequence having at least 70% sequence identity with it; c) The amino acid sequence of amino acid residues 2-394 of SEQ ID NO:87, or an amino acid sequence having at least 75% sequence identity with it. d) The amino acid sequence of amino acid residues 2-391 of SEQ ID NO:44, or an amino acid sequence having at least 61% sequence identity with it; or e) An amino acid sequence of any one of SEQ ID NO:72-86 and 96-100, or an amino acid sequence having at least 70% sequence identity with it.

[0026] In another embodiment, the amidase comprises: an amino acid sequence of amino acid residues 2-398 of SEQ ID NO:129, or an amino acid sequence having at least 80% sequence identity with it.

[0027] In another embodiment, the amidase comprises: an amino acid sequence of amino acid residues 2-398 of SEQ ID NO:2, or an amino acid sequence having at least 70% sequence identity with it.

[0028] In another embodiment, the amidase comprises the amino acid sequence of amino acid residues 2-398 of SEQ ID NO:1 or SEQ ID NO:95. In another embodiment, the amidase comprises the amino acid sequence of amino acid residues 2-398 of SEQ ID NO:128 or 129.

[0029] In another embodiment, the amidase comprises: an amino acid sequence of amino acid residues 2-394 of SEQ ID NO:87, or an amino acid sequence having at least 75% sequence identity with it.

[0030] In a further embodiment, the amidase comprises the amino acid sequence of any one of SEQ ID NO:72-86 and 96-100, or an amino acid sequence having at least 70% sequence identity with it. In another embodiment, the amidase comprises the amino acid sequence having amino acid residues 2-392 of SEQ ID NO:73. In yet another embodiment, the amidase comprises the amino acid sequence having amino acid residues 2-394 of SEQ ID NO:127.

[0031] Other aspects and embodiments of the invention are discussed in more detail below.

[0032] Brief description of the attached figures The invention will be described herein with reference to the following non-limiting drawings, in which: Figure 1A The product distribution of nylon 6,6 oligomers obtained under the conditions described in Example 1 and by UPLC analysis is shown. Figure 1B The analysis of the different oligomers formed in the depolymerization reaction described in Example 1 is shown using HRMS.

[0033] Figure 2 The product distribution in water after depolymerization under the conditions described in Part A of Example 2 is shown by UPLC analysis.

[0034] Figure 3Whole-cell activity assay results are shown, demonstrating the activity of the variant peptide with the amino acid sequence SEQ ID NOs:2-15 and 87 generated in Example 3 in hydrolyzing the amide bonds in polyamides compared to a selected group of extant / ancestral NylB peptides. Free amines in the solution (absorbance at 335 nm) were detected using a colorimetric assay. An increase in the concentration of free amine groups in the reaction solution corresponds to the hydrolysis of the nylon-6,6 trimer substrate into hexamethylenediamine and adipic acid.

[0035] Figure 4 The sequence alignment of the peptides SEQ ID NOs:2, 4-15 is shown, confirming the consensus sequence SEQ ID NO:1 (see Example 3).

[0036] Figure 5 The activities of a group of variant peptides derived from further engineering (Phase II) are shown. The activities of the purified candidate enzymes against nylon 6,6 dimers, trimers, and tetramers (generated in the acid hydrolysis of nylon 6,6 in Example 3) were analyzed using UHPLC (see Example 3). Y-axis values ​​correspond to the normalized percentage conversion of the nylon 6,6 trimer compared to the enzyme-free control. C4 corresponds to SEQ ID NO:89; D3 corresponds to the peptide including SEQ ID NO:90; E3 corresponds to the peptide including SEQ ID NO:94; F3 corresponds to the peptide including SEQ ID NO:91; and G3 corresponds to the peptide including SEQ ID NO:92.

[0037] Figure 6 shows the UHPLC analysis results of the Phase I candidates (SEQ ID NOs 4, 47, 87, 62, and 3) and Phase II candidates (SEQ ID NOs: 88, 89, 90, 91, 92, and 93) compared with those of known sequences (SEQ ID NOs 74, 75, 78, 79, 80, 81, 82, 83, and 85) on nylon 6,6 oligomers (dimer (A), tetramer (B), and trimer (C)) from Example 3. The Y-axis values ​​represent the peak areas of nylon 6,6 oligomers as measured by LC-MS.

[0038] Figure 7 The comparison of peptides SEQ ID NOs:2, 4-15, 88, 90-94 with consensus II (SEQ ID NO:95) (see Example 3) is shown.

[0039] Figure 8The activity of the immobilized enzyme of SEQ ID NO:4 against nylon 6,6 dimers, trimers, and tetramers generated by acid hydrolysis of nylon 6,6 in Example 3 is shown after co-incubation at 40°C for 1 hour (B) (see Example 4). The substrate transformation was quantified by UHPLC and compared with the control (A) enzyme-free sample after 0 hours.

[0040] Figure 9 The activity of SEQ ID NOs:100-115 and 121-123 on nylon 6,6 trimer is shown compared with that of the known sequence SEQ ID NO:75. An enzyme-free control is included in the figure.

[0041] Figure 10 The comparison of SEQ ID NOs:2, 4-15, 88-94 and 101-126 in generating the consensus sequence of consensus IV (SEQ ID NO:129) is shown.

[0042] Detailed Description of the Invention This invention provides a method for recycling polyamide. In this application, the term "recycled" means polyamide used in the method of this invention that is no longer of practical use. While such polyamide is typically in the form of post-consumer waste, it may also be in the form of manufacturing waste or simply excess stock.

[0043] According to this method, polyamides can serve as a source of their own monomeric components. These monomers include diacids, diamines, and amino acid compounds. These compounds / monomers can be reused / recycled in a variety of applications, such as for the manufacture of new polyamides.

[0044] There are no particular limitations on the type of polyamide that can be used in this invention. The polyamide can have any molecular weight and can be crystalline or amorphous.

[0045] Polyamides are a class of synthetic polymers characterized by repeating units linked by amide groups. Polyamides that are primarily aliphatic are more commonly referred to in the art as nylon.

[0046] Polyamides are typically produced by the condensation polymerization of diacids and diamines, the self-condensation polymerization of amino acids (usually ω amino acids), or the ring-opening polymerization of amino acids in the form of lactams.

[0047] Common types of nylon include, but are not limited to, nylon 6,6, nylon 6, nylon 10,10, nylon 11, nylon 12, nylon 6,10, nylon 4,6 and nylon 6,12.

[0048] Those skilled in the art will understand that the numbering system related to nylon is related to the number of carbon atoms in the monomers used to produce nylon.

[0049] When the specified number includes a single digit (such as Nylon 6 or Nylon 10), nylon is produced using a single monomer (i.e., it is a homopolymer). This monomer can be an amino acid that undergoes a self-condensation reaction to produce nylon, or it can be a lactam of an amino acid that undergoes a ring-opening polymerization reaction to produce nylon.

[0050] When the specified number includes two numbers (e.g., nylon 6,6 or nylon 10,10), nylon is produced using two monomers (i.e., it is a copolymer), namely a dicarboxylic acid and a diamine.

[0051] The first number represents the number of carbon atoms in the diamine, and the second number represents the number of carbon atoms in the diacid. For example, nylon 6,6 is formed by the condensation polymerization of hexamethylenediamine (HMD) and adipic acid (AA).

[0052] In one embodiment, the polyamide includes an aliphatic polyamide.

[0053] In another embodiment, the polyamide includes aliphatic copolymer polyamide.

[0054] In another embodiment, the polyamide has the general formula (I): (I) Where x is an integer ranging from 2 to 10, y is an integer ranging from 2 to 10, and n is an integer ranging from approximately 50 to 250.

[0055] In one embodiment, x in equation (I) is 2, 4, 8 or 10, and y is 2, 4, 8 or 10.

[0056] In another embodiment, the polyamide includes aliphatic homopolymer polyamide.

[0057] In another embodiment, the polyamide has the general formula (II): (II) Where x is an integer ranging from 2 to 9, and n is an integer ranging from approximately 100 to approximately 800.

[0058] In one embodiment, x in equation (II) is 3, 8, or 9.

[0059] Those skilled in the art will understand that the features of formulas (I) and (II) enclosed in square brackets ([...]) represent repeating units of a polyamide, which in the case of formula (I) consist of condensed residues of a diamine and a diacid, and in the case of formula (II) consist of amino acids. Polyamides or oligomers thereof will, of course, have the corresponding functional groups of their monomeric components as end groups. With regard to the polyamides to which this subject matter relates, those end groups will be independently selected from acid or amine groups. In other words, polyamides or polyamide oligomers will have acid end groups, or amine end groups, or both acid and amine end groups.

[0060] In another embodiment, the polyamide includes a mixture of aliphatic copolymer polyamide and aliphatic homopolymer polyamide.

[0061] In a further embodiment, the polyamide is selected from nylon 6,6, nylon 6, nylon 10,10, nylon 11, nylon 12, nylon 6,10, nylon 4,6, nylon 6,12 and combinations thereof.

[0062] There are no particular limitations on the physical form of the polyamide that can be used in this invention. To facilitate processing and / or improve the acid hydrolysis rate, the polyamide is preferably in a pulverized form, such as fragments, granules, agglomerates, fibers, flakes, or powder.

[0063] Conventional techniques and equipment can be advantageously used to pulverize polyamides for use in this invention.

[0064] This method involves acid hydrolysis of the polyamide in an aqueous liquid. This stage of the method is typically carried out in a reaction vessel.

[0065] Acid hydrolysis of polyamides is a technique known in the art, and the present invention can advantageously utilize conventional equipment and reagents for this step of the method.

[0066] Suitable reaction vessels include those used in conventional polyamide chemical depolymerization techniques. These vessels typically do not react under the acidic conditions employed and are usually made of glass, Inconel, Hastelloy, or stainless steel.

[0067] The volume of the reaction vessel can be adjusted according to the expected scale of operation, such as laboratory scale, pilot scale, or even industrial scale. If necessary, the reaction vessel may include one or more agitators or stirring devices to assist in the movement and mixing of reagents within the vessel.

[0068] Of course, acid hydrolysis must be performed. There are no particular restrictions on the types of acids that can be used.

[0069] In one embodiment, an inorganic acid is used for acid hydrolysis.

[0070] Examples of suitable inorganic acids include, but are not limited to, one or more of hydrochloric acid, nitric acid, phosphoric acid, and sulfuric acid.

[0071] In one embodiment, sulfuric acid is used for acid hydrolysis.

[0072] In another embodiment, an organic acid is used for acid hydrolysis.

[0073] Examples of suitable organic acids include carboxylic acids, such as dicarboxylic acids like adipic acid, sebacic acid, terephthalic acid, and dodecanoic acid.

[0074] Acid hydrolysis typically occurs in a pH range of about 0 to about 5, or about 0 to 3.

[0075] There are no particular restrictions on the amount of acid used, as long as a water-soluble oligomer can be formed. Those skilled in the art can appropriately select the amount of acid used. For example, the amount of acid used can be about 0.5 equivalents to about 10 equivalents of the molar number of polyamide.

[0076] Acid hydrolysis takes place or is performed in an aqueous liquid. This aqueous liquid can also be described as an aqueous reaction medium in which acid hydrolysis occurs. This aqueous liquid is, of course, acidic.

[0077] Therefore, this method can be summarized as follows: subjecting polyamide to acid hydrolysis in an acidic aqueous solution to produce polyamide oligomers soluble in the acidic aqueous solution.

[0078] If necessary, the aqueous liquid can be stirred to promote the acid hydrolysis of the polyamide.

[0079] Those skilled in the art will understand that the ultimate goal of conventional polyamide chemical depolymerization is usually to depolymerize the polyamide back to its monomeric components. Completely converting polyamide back to its monomeric components typically requires rather harsh and energy-intensive conditions. The acid hydrolysis performed in this invention is not intended to completely depolymerize the polyamide to its monomeric components. Rather, the acid hydrolysis is only required to proceed to the extent that it produces polyamide oligomers soluble in aqueous liquids. Therefore, the method of this invention can advantageously be carried out under relatively mild reaction conditions and with high conversion rates.

[0080] In one embodiment, acid hydrolysis is carried out at a temperature below 200°C, for example below about 190°C, or below about 180°C, or below about 170°C, or below about 160°C, or below about 150°C.

[0081] In another embodiment, the acid hydrolysis is carried out at a temperature greater than about 100°C, for example, greater than about 110°C, or greater than about 120°C, or greater than about 130°C, or greater than about 140°C, or greater than about 150°C, or greater than about 160°C.

[0082] In a further embodiment, the acid hydrolysis is carried out at a temperature of about 75°C to about 200°C, or about 75°C to below 200°C, or about 75°C to about 180°C, or about 100°C to about 180°C, or about 130°C to about 175°C.

[0083] The acid hydrolysis reaction can be heated using conventional methods.

[0084] For example, reaction components can be assembled in a reaction vessel and the reaction vessel can be heated to raise the temperature of the reaction components to the desired temperature.

[0085] Acid hydrolysis can be carried out at atmospheric pressure, or at pressures up to about 40 atm, or about 30 atm, or about 20 atm, or about 15 atm.

[0086] In one embodiment, acid hydrolysis is carried out at atmospheric pressure.

[0087] Acid hydrolysis proceeds for a period of time to produce polyamide oligomers that are soluble in water. The required time, of course, depends on variables such as temperature, pressure, the surface area of ​​the polyamide, and the ratio of acid to polyamide.

[0088] Advantageously, acid hydrolysis can be carried out in an efficient manner.

[0089] For example, acid hydrolysis can be carried out with high conversion rates in a time span of only about 30 minutes to 8 hours.

[0090] According to the method of the present invention, polyamide is depolymerized by acid hydrolysis to produce oligomers soluble in an aqueous liquid. For a given polyamide particle in the reaction process, acid hydrolysis is practically complete when the polyamide particle has undergone sufficient depolymerization to dissolve in the aqueous liquid. When the method of the present invention is carried out in batches, acid-catalyzed hydrolysis will be completed when all the polyamide present has practically dissolved in the aqueous liquid. When the method of the present invention is carried out continuously, it should be understood that polyamide in the reaction system may exist at various stages of depolymerization.

[0091] The statement that polyamide oligomers are soluble in water means that those oligomers are soluble in water at least at the temperature at which acid hydrolysis occurs.

[0092] In other words, the method includes: acid hydrolyzing the polyamide in an aqueous liquid to produce a polyamide oligomer that is soluble in the aqueous liquid at least at the temperature at which the acid hydrolysis is carried out.

[0093] For example, if acid hydrolysis is carried out at a temperature of about 150°C, the resulting polyamide oligomers will dissolve in aqueous liquids at least at about 150°C.

[0094] Polyamide oligomers that are soluble in water may or may not be soluble in water at room temperature.

[0095] In some embodiments, the resulting polyamide oligomers are insoluble in aqueous liquids at room temperature.

[0096] As long as the polyamide oligomers prepared according to the method of the present invention are soluble in aqueous liquid, there is no particular limitation on the number of monomer repeating units constituting the oligomers. The solubility of these oligomers relative to the number of monomer repeating units constituting the oligomers may vary depending on the properties of the monomer repeating units themselves.

[0097] In some embodiments, the water-soluble polyamide oligomer comprises 1 to 15, or about 1 to 12, or about 1 to 10 monomer repeating units, or about 1 to 8, or about 1 to 6 monomer repeating units.

[0098] In some embodiments, the water-soluble polyamide oligomer is derived from the polyamide homopolymer and includes about 1 to about 15, or about 1 to about 12, or about 1 to about 10 monomer repeating units.

[0099] In some embodiments, the water-soluble polyamide oligomer is derived from a polyamide copolymer and includes one to about five, or about one to about four, or about one to about three monomer repeating units.

[0100] According to the method of the present invention, the acid hydrolysis of polyamide copolymers can be represented by reaction scheme 1.

[0101]

[0102] Reaction scheme 1: According to the present invention, the polyamide copolymer is acid-hydrolyzed to provide an oligomer soluble in an aqueous reaction medium; wherein x is an integer from 2 to about 10, y is an integer from 2 to about 10, n is an integer from about 50 to about 250, and m is an integer from 1 to about 5.

[0103] According to the method of the present invention, the acid hydrolysis of polyamide homopolymer can be represented by reaction scheme 2.

[0104]

[0105] Reaction scheme 2: According to the present invention, the polyamide homopolymer is acid-hydrolyzed to provide an oligomer soluble in an aqueous reaction medium; wherein x is an integer from 2 to about 9, n is an integer from about 100 to about 800, and m is an integer from 1 to about 15.

[0106] The polyamide oligomers generated according to the method of the present invention will exist in acidic aqueous liquids due to the acid hydrolysis of polyamide.

[0107] In other words, the method includes subjecting the polyamide to acid hydrolysis in an acidic aqueous liquid to produce a polyamide oligomer soluble in the acidic aqueous liquid.

[0108] Before proceeding to the next enzymatic stage of this method, the acidic aqueous liquid containing soluble oligomers can be neutralized with alkali.

[0109] In one embodiment, the aqueous liquid containing the polyamide oligomer is neutralized with an alkali before the oligomer is converted to its monomeric components using an amidase.

[0110] In another embodiment, acid hydrolysis produces an acidic aqueous liquid comprising polyamide oligomers, which is neutralized with alkali before the oligomers are converted to their monomeric components using an amidase.

[0111] In another embodiment, the aqueous liquid comprising the polyamide oligomer is neutralized with an alkali to a pH of at least around 7 or 8; or a pH range of around 7 to around 12, or around 8 to around 12.

[0112] Examples of bases used for neutralization include, but are not limited to, alkali metal bases, alkaline earth metal bases, and organic amine bases.

[0113] Examples of suitable alkali metal bases include, but are not limited to, alkali metal hydroxides.

[0114] Examples of suitable alkali metal hydroxides include, but are not limited to, lithium hydroxide, sodium hydroxide, and potassium hydroxide.

[0115] Examples of suitable alkaline earth metal bases include, but are not limited to, alkaline earth metal hydroxides.

[0116] Examples of suitable alkaline earth metal hydroxides include, but are not limited to, magnesium hydroxide and calcium hydroxide.

[0117] Examples of suitable organic amine bases include, but are not limited to, diamines such as hexamethylenediamine, 1,4-diaminobutane, 1,5-diaminopentane, and 1,10-diaminodecane.

[0118] As a variation of performing such a neutralization step prior to the enzymatic phase, the aqueous liquid comprising the polyamide oligomer soluble in water may be cooled (e.g., cooled from the temperature at which acid hydrolysis occurs) to promote the precipitation of organic acids (e.g., dicarboxylic acids as described herein) that may be present in the liquid. The source of any precipitated organic acid may be the hydrolysis product of the polyamide and / or the organic acid used to promote hydrolysis. The precipitated organic acid can then be separated from the aqueous liquid containing the soluble oligomer, and the aqueous liquid can then be neutralized with the alkali as described herein, if necessary.

[0119] In one embodiment, before using an amidase to convert the oligomer into its monomeric components, the temperature of the aqueous liquid containing the polyamide oligomer soluble in the aqueous liquid is lowered to promote the precipitation of organic acids (such as dicarboxylic acids as described herein) in the aqueous liquid.

[0120] In another embodiment, the precipitated organic acid is separated from the aqueous liquid.

[0121] In another embodiment, the polyamide oligomer soluble in water is neutralized with an alkali to bring the pH to at least around 7 or around 8; or the pH range is from around 7 to around 12, or from around 8 to around 12.

[0122] In a further embodiment, prior to the enzymatic phase of the method, the water-soluble polyamide oligomer is neutralized by adding a base (such as an organic amine base described herein).

[0123] As a further variation of performing such a neutralization step prior to the enzymatic stage, the aqueous liquid comprising the polyamide oligomers soluble in the water can be cooled (e.g., from the temperature at which acid hydrolysis occurs) to promote precipitation of at least some of the oligomers in the aqueous liquid. For example, depending on the properties of the feed polyamide, polyamide oligomers having six or more repeating units can precipitate, while polyamide oligomers having fewer than six repeating units can remain dissolved in the aqueous liquid. The precipitated oligomers can then be separated from the aqueous liquid. If desired, those separated oligomers can then be neutralized as described herein, and optionally, a second acid hydrolysis as described herein can be performed for processing according to the invention. The oligomers remaining dissolved in the aqueous liquid can then be neutralized as described herein and further processed according to the invention.

[0124] In one embodiment, before using an amidase to convert the oligomer into its monomeric components, the temperature of the aqueous liquid containing the polyamide oligomer soluble in the aqueous liquid is lowered to promote the precipitation of at least a portion of the oligomer in the aqueous liquid.

[0125] In a further embodiment, the precipitated oligomers are separated from the aqueous liquid to obtain a separate aqueous liquid comprising polyamide oligomers soluble in the aqueous liquid.

[0126] In another embodiment, an additional aqueous liquid comprising polyamide oligomers soluble in aqueous liquid is neutralized with an alkali as described herein.

[0127] In another embodiment, an amidase is then used to convert oligomers from another aqueous liquid into their monomeric components.

[0128] In another embodiment, the separated precipitated oligomers are neutralized with alkali.

[0129] In another embodiment, the oligomer is neutralized with an alkali to make its pH at least around 7 or around 8, or the pH range from around 7 to around 12, or from around 8 to around 12.

[0130] Examples of bases used for neutralization are described in this article.

[0131] In a further embodiment, the method includes: (i) lowering the temperature of an aqueous liquid to promote precipitation of the oligomer in the aqueous liquid, which includes polyamide oligomers soluble in the aqueous liquid, before using an amidase to convert the oligomer into its monomeric components; (ii) separating the precipitated oligomer from the aqueous liquid and reusing the separated aqueous liquid in the method for further acid-catalyzed hydrolysis of the polyamide; and (iii) neutralizing the separated oligomer with a base.

[0132] Once an aqueous liquid containing oligomers or isolated oligomers is neutralized with an alkali to form an alkaline aqueous liquid, the oligomers will dissolve in the alkaline aqueous liquid. At room temperature, the oligomers are advantageously soluble in this alkali-neutralized liquid. The pH of this alkali-neutralized liquid is typically neutral or alkaline, for example, in the range of around 7 to around 12, or around 8 to around 12.

[0133] In one embodiment, before using an amidase to convert the oligomer into its monomeric components, the water-soluble polyamide oligomer is placed in a neutral or alkaline aqueous solution, wherein the oligomer is soluble in the neutral or alkaline aqueous solution.

[0134] The polyamide raw materials used in this invention, including oligomers, may contain some colored contaminants in neutral or alkaline aqueous solutions. If necessary, activated carbon can be used to help remove any colored contaminants from the neutral or alkaline aqueous solution.

[0135] In the production of polyamide oligomers, the method of the present invention includes using an amidase to convert the oligomers into their monomeric components.

[0136] It will be understood by those skilled in the art that the monomer composition of the oligomer is substantially the same as that of the polyamide derived from the oligomer. The form and composition of the monomer composition will depend on the type of polyamide used in the method of the present invention. For example, if nylon 6 is used in the method of the present invention, the monomer composition will be 6-6-aminohexanoic acid. If nylon 6,6 is used in the method of the present invention, the monomer composition will be HMD and AA. If a mixture of nylon 6 and nylon 6,6 is used in the method of the present invention, the monomer composition will be HMD, AA, and 6-aminohexanoic acid. The monomer composition thus formed can also be in the form of an ammonium carboxylate (e.g., hexamethylenediamine adipate, also known as hexamethylenediammonium adipate).

[0137] Those skilled in the art will understand that nylon 6 and other homopolymer polyamides can be produced by ring-opening polymerization of lactams containing given amino acids. The repeating monomer units of these polyamides naturally reflect the amino acids, not the corresponding lactams themselves. Therefore, in the context of homopolymer polyamides, it is perfectly appropriate to refer to amino acids as monomeric components, even if the polyamide may be produced using lactams containing amino acids.

[0138] Advantageously, the amidease hydrolysis of oligomers derived from polyamides according to the present invention provides an efficient and effective route for the production of amino acids, diacids, diamines and ammonium carboxylate salts (e.g., hexamethylenediamine adipic acid, also known as hexamethylenediamine adipic acid).

[0139] In summary, the chemical and enzymatic combination method employed in this invention provides a scalable and economically feasible method for recycling polyamides.

[0140] According to the method of the present invention, the amide hydrolysis of oligomers derived from polyamide homopolymers can be represented by reaction scheme 3.

[0141]

[0142] Reaction scheme 3: According to the present invention, oligomers derived from polyamide homopolymers are subjected to amidase depolymerization to obtain amino acids, which, if desired, can then be cyclized to form lactams; wherein x is an integer from about 2 to 9, and m is an integer from about 1 to 15.

[0143] As shown in reaction scheme 3, the amino acid is produced by the enzymatic hydrolysis of an oligomer derived from a polyamide homopolymer. This amino acid represents a monomeric component of the polyamide homopolymer. The amino acid can then be used in its natural form for a given application, or, as shown in reaction scheme 3, undergo a cyclization reaction to produce the corresponding lactam. Methods for converting such amino acids into the corresponding lactams are known in the art. For example, an amino acid solution (e.g., 6-aminohexanoic acid) can be treated with high-temperature steam in the presence of a catalyst. The lactam thus formed can then be used in a given application.

[0144] In one embodiment, the amino acid produced by this method is converted into a lactam.

[0145] According to the method of the present invention, the amidease depolymerization of oligomers derived from polyamide copolymers can be represented by reaction scheme 4.

[0146]

[0147] Reaction scheme 4: According to the present invention, oligomers derived from polyamide copolymers are hydrolyzed by an amide enzyme to obtain a dicarboxylic acid and a diamine; wherein x is an integer from 2 to about 10, y is an integer from 2 to about 10, and m is an integer from 1 to about 5.

[0148] The enzymatic depolymerization step of this method can be performed using techniques and equipment known to those skilled in the art.

[0149] Typically, oligomers derived from polyamides are placed in a reaction vessel with a solvent / reaction medium and an amidase, and incubated for an appropriate time to convert the oligomers into their monomeric components.

[0150] In one embodiment, an oligomer derived from polyamide is placed in a reaction vessel with an amidase and a suitable liquid combination and incubated for an appropriate time to convert the oligomer into its monomeric components.

[0151] Examples of suitable liquids that may be used include, but are not limited to, aqueous liquids, including aqueous buffer solutions.

[0152] In one embodiment, an oligomer derived from polyamide is combined with an amidase and placed in a neutral or alkaline aqueous solution to convert the oligomer into its monomeric components. The pH range of the aqueous solution may be approximately 7 to approximately 12, or approximately 8 to approximately 12.

[0153] The time required to convert oligomers into their monomeric components using amidase depends at least on the temperature and the enzyme-to-oligomer ratio. Typically, the conversion of oligomers into their monomeric components using amidase requires an incubation period ranging from approximately 5 minutes to approximately 24 hours. Incubation periods can typically range from approximately 5 minutes to approximately 20 hours, 5 minutes to approximately 18 hours, 5 minutes to approximately 16 hours, 5 minutes to approximately 14 hours, 5 minutes to approximately 12 hours, 5 minutes to approximately 10 hours, 5 minutes to approximately 8 hours, and 5 minutes to approximately 6 hours. In further examples, incubation periods can be approximately 5 minutes to approximately 5 hours, 5 minutes to approximately 4 hours, 5 minutes to approximately 3 hours, 5 minutes to approximately 2 hours, and 5 minutes to approximately 1 hour.

[0154] Those skilled in the art will be able to select an incubation temperature suitable for a specific amidase. Typically, incubation can be carried out at temperatures ranging from approximately 0°C to approximately 80°C, for example, approximately 2°C to approximately 75°C, approximately 4°C to approximately 70°C, approximately 6°C to approximately 70°C; approximately 8°C to approximately 65°C, approximately 10°C to approximately 65°C, approximately 20°C to approximately 60°C, approximately 5°C to approximately 55°C, approximately 30°C to approximately 55°C, approximately 30°C to approximately 50°C, approximately 35°C to approximately 50°C, or approximately 40°C to approximately 50°C.

[0155] The amidase used in this invention can be a polypeptide capable of hydrolyzing the amide bonds in a polyamide. In one embodiment, the polyamide is a basic aliphatic polyamide commonly referred to in the art as nylon. In one embodiment, the polyamide is a homopolymer polyamide. In one embodiment, the polyamide is a copolymer polyamide.

[0156] The term "amidase" generally refers to a hydrolase, exemplary examples of which include hydrolases classified as EC 3.5.1 or 3.5.2 according to enzyme nomenclature, which catalyze the hydrolysis of amides by acting on carbon-nitrogen bonds rather than peptide bonds.

[0157] Amideases, which can hydrolyze the amide bonds in polyamide nylon, were first discovered in a bacterial strain that can digest certain byproducts of nylon manufacturing (Kinoshita et al., 1975, and Kinoshita et al., 1977).

[0158] In one embodiment, an amidase capable of hydrolyzing the amide bond in a polyamide includes: ●6-Aminohexanoic acid-cyclic-dimer hydrolase / NylA; e.g. UniProt accession numbers: P13398.2 (SEQ ID NO: 96) and P13397.2 (SEQ ID NO: 97); ● 6-Aminohexanoic acid dimer hydrolase / NylB; for example, UniProt accession numbers: P07061 (SEQ ID NO: 72) and P07062.1 (SEQ ID NO: 86); ●6-Aminohexanoic acid oligomer endohydrolase / NylC; for example, UniProt accession number: Q79F77.1 (SEQ ID NO: 98); Q1EPR5.2 (SEQ ID NO: 99) and Q1EPR4.1 (SEQ ID NO: 100).

[0159] In one embodiment, the polyamide oligomer is soluble in an aqueous liquid. Furthermore, ideally, the amidase is a polypeptide capable of converting the oligomer into its monomeric components.

[0160] As disclosed elsewhere herein, water-soluble polyamide oligomers comprise approximately 1 to approximately 15, or approximately 1 to approximately 12, or approximately 1 to approximately 10 repeating monomer units.

[0161] In one embodiment, the water-soluble polyamide oligomer is derived from a polyamide homopolymer and comprises approximately 1 to approximately 15, or approximately 1 to approximately 12, or approximately 1 to approximately 10 repeating monomer units. In some embodiments, the water-soluble polyamide oligomer is derived from a polyamide copolymer and comprises approximately 1 to approximately 5, or approximately 1 to approximately 4, or approximately 1 to approximately 3 repeating monomer units.

[0162] In one embodiment, the amidase is a polypeptide capable of hydrolyzing the amide bonds in nylon 6 oligomers. In another embodiment, the amidase is a polypeptide capable of hydrolyzing the amide bonds in nylon 6,6 oligomers. In some embodiments, the amidase is a polypeptide capable of hydrolyzing both the amide bonds in nylon 6 oligomers and the amide bonds in nylon 6,6 oligomers. In some embodiments, one or more amidases may be used according to the present invention.

[0163] In one embodiment, the amidase comprises: a) The amino acid sequence of amino acid residues 2-398 of SEQ ID NO:1 or SEQ ID NO:95; b) The amino acid sequence of amino acid residues 2-398 of SEQ ID NO:2, or an amino acid sequence having at least 70% sequence identity with it; c) The amino acid sequence of amino acid residues 2-394 of SEQ ID NO:87, or an amino acid sequence having at least 75% sequence identity with it. d) The amino acid sequence of amino acid residues 2-391 of SEQ ID NO:44, or an amino acid sequence having at least 61% sequence identity with it; or e) An amino acid sequence of any one of SEQ ID NOs:72-86 and 96-100, or an amino acid sequence having at least 70% sequence identity with it.

[0164] As used herein, the term "sequence identity" or "identity" refers to the number (or a percentage) of pairs (identical amino acid residues) between two polypeptide sequences. In a preferred embodiment, sequence identity is determined by comparing sequences at alignment to maximize overlap and identity while minimizing sequence gaps. Sequence identity can be determined using any of a number of mathematical global or local alignment algorithms known to those skilled in the art, depending on the length of the two sequences. Global alignment algorithms (e.g., the Needleman and Wunsch algorithm; Needleman and Wunsch, 1970) can be used to align sequences of similar length, which best align sequences across their entire length, while sequences of substantially different lengths are preferably aligned using local alignment algorithms (e.g., the Smith and Waterman algorithm (Smith and Waterman, 1981) or the Altschul algorithm (Altschul et al., 1997; Altschul et al., 2005)). To determine the percentage of amino acid sequence identity, alignments can be performed using any method available to those skilled in the art, illustrative examples of which include publicly available computer software, such as those available at http: / / blast.ncbi.nlm.nih.gov / or http: / / www.ebi.ac.uk / Tools / emboss / . Those skilled in the art can readily determine appropriate parameters for measuring the alignment, including any algorithm required to achieve maximum alignment of the full length of the compared sequences. As used herein, the percentage of sequence identity generally refers to a value generated using pairwise sequence alignments that create an optimal global alignment of two sequences (e.g., using the Needleman-Wunsch algorithm), where all search parameters are set to default values, such as score matrix = BLOSUM62, void open = 10, void extension = 0.5, terminal void penalty = false, terminal void open = 10, and terminal void extension = 0.5.

[0165] In one embodiment, the amidase is a polypeptide capable of hydrolyzing the amide bonds in nylon 6,6 oligomers. In one embodiment, the amidase comprises the amino acid sequence of SEQ ID NO:95, or an amino acid sequence having at least 80% sequence identity with it.

[0166] SEQ ID NO:95: MTX1TX2LMQGX3PPAPEQX4VTLANWRX5X6PFX7RWX8FHHVREX9X 10PTAX 11 IPRGPGX 12 X 13 X 14 PLPX 15 X 16 PRDLX 17 X 18 IAX 19 EGX 20 DGX 21 X 22 X 23 TVX 24 EMLX 25 EX 26 YTDX 27 FLVX 28 HRGRIVX 29 EX 30 YANGMTPHX 31 PHIX 32 FSVSKSITGX 33 LAGILVX 34 RGQLDPDAPVTX 35 YIPEX 36 X 37 GSAYGDATVRHVLDMTVX 38 IDFX 39 EDYLDPDGDFARYRX 40 AX 41 GWNPX 42 X 43 DGX 44 TPSDLRSFLX 45 TLX 46 X 47 X 48 DGX 49 HGETFHYX 50 SPNSDLLGWIX 51 ERASGQRFAX 52 LLSEX 53 IWX 54 PMGAEX 55 DAYITVDRLGAPRTAGGX 56 CATX 57 RDLARFGX 58 MMX 59 NRGVANGRQX 60 VPX 61 X 62 WIDDIX 63 X 64 X 65 GDX66 EAWARGDFAX 67 X 68 X 69 PX 70 GRYRSKWYVTGNARGAFCX 71 IGIHGQWIYX 72 DPAAEVVIX 73 KX 74 SSQPX 75 PVDDAMDRLX 76 LAAFX 77 AIARALX 78 X 79 (where X1-X) 79 (It can be any amino acid) In one embodiment, the amidase comprises an amino acid sequence having amino acid residues 2-398 of SEQ ID NO:95, or an amino acid sequence having at least 80% sequence identity with it, including the following amino acid sequence, wherein i. The amino acid at position 3 is either Q or T; ii. The amino acid at position 5 is N or D; iii. The amino acid at position 10 is S or F; iv. The amino acid at position 17 is either Q or E; v. The amino acid at position 25 is either Q or T; vi. The amino acid at position 26 is either A or P; vii. The amino acid at position 29 is N or S; viii. The amino acid at position 32 is either A or S; ix. The amino acid at position 39 is I or L; x. The amino acid at position 40 is I or V; The amino acid at position 44 is N or Q; xii. The amino acid at position 51 is either A or P; xiii. The amino acid at position 52 is either A or V; xiv. The amino acid at position 53 is S or W; xv. The amino acid at position 57 is either A or R; xvi. The amino acid at position 58 is either A or S; xvii. The amino acid at position 63 is either D or G; xviii. The amino acid at position 64 is G, E, or R; xix. The amino acid at position 67 is F or L. The amino acid at position 70 is P or is missing; xxi. The amino acid at position 73 is R or K; xxii. The amino acid at position 74 is S or E; xxiii. The amino acid at position 75 is T, M, G, or W; xxiv. The amino acid at position 78 is either A or G; The amino acid at position 82 in xxv is either A or E; xxvi. The amino acid at position 84 is either S or T; xxvii. The amino acid at position 88 is either A or G; xxviii. The amino acid at position 92 is L or M; The amino acid at position 99 in xxix is ​​either A or S. The amino acid at position 101 is either W or H. The amino acid at position 110 of xxxi is either S or T; xxxii. The amino acid at position 114 is L or V; xxxiii. The amino acid at position 124 is I or T; xxxiv. The amino acid at position 131 is either D or E; The amino acid at position 143 in xxxv is H, R, or D; The amino acid at position 148 of xxxvi is either A or V; xxxvii. The amino acid at position 149 is either A or K; xxxviii. The amino acid at position 167 is S or G; The amino acid at position 171 of xxxix is ​​either E or D; xl. The amino acid at position 186 is R, E, or Q; The amino acid at position 188 of xli is either T or M; The amino acid at position 193 of xlii is either A or P; xliii. The amino acid at position 194 is S or P; The amino acid at position 197 in xliv. is either A or E; The amino acid at position 207 in xlv. is either A or V. xlvi. The amino acid at position 210 is R or K; xlvii. The amino acid at position 211 is R, K, or missing; xlviii. The amino acid at position 212 is S, D, or G; The amino acid at position 215 of xlix is ​​either P or E; 1. The amino acid at position 223 is either A or V; The amino acid at position 234 in li is either L or I. The amino acid at position 244 in lii is either D or E; liii. The amino acid at position 249 is H or R; The amino acid at position 252 of liv. is either Q or R; The amino acid at position 258 is H or A; lvi. The amino acid at position 276 is L or I; lvii. The amino acid at position 280 is L or A; lviii. The amino acid at position 288 is either Q or E; The amino acid at position 291 of lix is ​​either L or R; The amino acid at position 301 in lx is I or V; The amino acid at position 304 in lxi is E, A, or G; lxii. The amino acid at position 305 is either A or W; lxiii. The amino acid at position 311 is L or R; The amino acid at position 312 in lxiv is either Q or T; lxv. The amino acid at position 313 is N or G; lxvi. The amino acid at position 316 is R, K, or P; lxvii. The amino acid at position 326 is K or H; 1xviii. The amino acid at position 327 is F or L; lxix. The amino acid at position 328 is either F or L; The amino acid at position 330 in lxx is N or G; lxxi. The amino acid at position 349 is either A or G; lxxii. The amino acid at position 359 is I or V; lxxiii. The amino acid at position 368 is either A or V; lxxiv. The amino acid at position 370 is L or F; The amino acid at position 375 in lxxv is either L or E; lxxvi. The amino acid at position 385 is C, M, or N; lxxvii. The amino acid at position 390 is D, E, or R; lxxviii. The amino acid at position 397 is G or A; and / or The amino acid at position 398 of lxxix is ​​G or is missing. The numbering method is related to the amino acid position of SEQ ID NO:1 or SEQ ID NO:95.

[0167] In one embodiment, the amidase comprises the amino acid sequence of SEQ ID NO:1, or an amino acid sequence having at least 80% sequence identity with it.

[0168] SEQ ID NO:1: MTX1TX2LMQGFPPAPEQQVTLANWRX3X4PFNRWX5FHHVREIX6PTANIPRGPGX7X8X9PLPX 10 X 11 PRDLX 12 X 13 IAFEGX 14 DGX 15 X 16 X 17 TVX 18 EMLX 19 EX 20 YTDX 21 FLVX 22 HRGRIVX 23 EX 24 YANGMTPHX 25 PHIX 26 FSVSKSITGX 27 LAGILVX 28 RGQLDPDAPVTX 29 YIPEX 30 X 31 GSAYGDATVRHVLDMTVX 32 IDFX 33 EDYLDPDGDFARYRX 34 ATGWNPX 35 X 36 DGX 37 TPSDLRSFLX 38 TLX 39 X 40 X 41 DGX 42 HGETFHYX 43 SPNSDLLGWIX 44 ERASGQRFAX 45 LLSEHIWQPMGAEHDAYITVDRLGAPRTAGGX 46 CATX 47 RDLARFGX 48 MMX 49NRGVANGRQX 50 VPX 51 X 52 WIDDIX 53 X 54 X 55 GDX 56 EAWARGDFAKFX 57 PX 58 GRYRSKWYVTGNARGAFCX 59 IGIHGQWIYIDPAAEVVIX 60 KX 61 SSQPX 62 PVDDAMDRLX 63 LAAFX 64 AIARALX 65 G (where X1-X) 65 (It can be any amino acid) In one embodiment, the amidase comprises an amino acid sequence having amino acid residues 2-398 of SEQ ID NO:95, an amino acid sequence having amino acid residues 2-398 of SEQ ID NO:1; or, an amino acid sequence having at least 80% sequence identity with it, comprising the following amino acid sequence, wherein i. The amino acid at position 3 is either Q or T; ii. The amino acid at position 5 is N or D; iii. The amino acid at position 25 is either Q or T; iv. The amino acid at position 26 is either A or P; v. The amino acid at position 32 is either A or S; vi. The amino acid at position 40 is I or V; vii. The amino acid at position 51 is either A or P; viii. The amino acid at position 52 is either A or V; ix. The amino acid at position 53 is S or W; x. The amino acid at position 57 is either A or R; The amino acid at position 58 is either A or S. xii. The amino acid at position 63 is either D or G; xiii. The amino acid at position 64 is G, E, or R; xiv. The amino acid at position 70 is P or is missing; xv. The amino acid at position 73 is R or K; xvi. The amino acid at position 74 is either S or E; xvii. The amino acid at position 75 is T, M, or W; xviii. The amino acid at position 78 is either A or G; xix. The amino acid at position 82 is either A or E; The amino acid at position 84 is either S or T. xxi. The amino acid at position 88 is either A or G; xxii. The amino acid at position 92 is L or M; xxiii. The amino acid at position 99 is either A or S; xxiv. The amino acid at position 101 is W or H; The amino acid at position 110 in xxv is either S or T; The amino acid at position 114 in xxvi. is either L or V; xxvii. The amino acid at position 124 is I or T; xxviii. The amino acid at position 131 is either D or E; The amino acid at position 143 in xxix is ​​H, R, or D; The amino acid at position 148 is either A or V. The amino acid at position 149 of xxxi is either A or K; xxxii. The amino acid at position 167 is S or G; xxxiii. The amino acid at position 171 is either E or D; xxxiv. The amino acid at position 186 is R, E, or Q; The amino acid at position 193 in xxxv is either A or P; The amino acid at position 194 of xxxvi is either S or P; xxxvii. The amino acid at position 197 is either A or E; xxxviii. The amino acid at position 207 is either A or V; The amino acid at position 210 of xxxix is ​​either R or K; xl. The amino acid at position 211 is R, K, or missing; The amino acid at position 212 of xli is S, D, or G; The amino acid at position 215 of xlii is either P or E; xliii. The amino acid at position 223 is either A or V; The amino acid at position 234 of xliv. is either L or I. The amino acid at position 244 in xlv. is either D or E; xlvi. The amino acid at position 276 is L or I; xlvii. The amino acid at position 280 is L or A; xlviii. The amino acid at position 288 is either Q or E; The amino acid at position 291 in xlix is ​​either L or R; l. The amino acid at position 301 is I or V; The amino acid at position 304 in li is E, A, or G; The amino acid at position 305 in lii is either A or W; iii. The amino acid at position 311 is L or R; The amino acid at position 312 in liv. is either Q or T; The amino acid at position 313 is N or G; lvi. The amino acid at position 316 is R, K, or P; lvii. The amino acid at position 328 is either F or L; lviii. The amino acid at position 330 is N or G; The amino acid at position 349 of lix is ​​either A or G; 1x. The amino acid at position 368 is either A or V; The amino acid at position 370 in lxi is either L or F; lxii. The amino acid at position 375 is either L or E; lxiii. The amino acid at position 385 is either C or N; The amino acid at position 390 in lxiv is D, E, or R; and / or The amino acid at position 397 of lxv is G or A. The numbering method is related to the amino acid position of SEQ ID NO:1 or SEQ ID NO:95.

[0169] "At least 80%" means that the amidase has at least 80%, or at least 85%, or at least 90%, or at least 92%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 95.

[0170] In one embodiment, the amidase is a polypeptide capable of hydrolyzing the amide bond in a polyamide, wherein the amidase comprises: an amino acid sequence of amino acid residues 2-398 of SEQ ID NO: 2, or an amino acid sequence having at least 70% sequence identity with it. In one embodiment, the amidase includes at least one amino acid modification at positions selected from: amino acid positions 3, 5, 10, 17, 25, 26, 29, 32, 39, 40, 44, 51, 52, 53, 57, 58, 63, 64, 67, 70, 73, 74, 75, 78, 82, 84, 88, 92, 99, 101, 110, 114, 124, 131, 143, 148, 149, 167, 171, 186, 188, 193, 194, 197, 207, 210, 211, 212, 215, 223. 234, 244, 249, 252, 258, 276, 280, 288, 291, 301, 304, 305, 311, 312, 313, 316, 326, 327, 328, 330, 349, 359, 368, 370, 375, 385, 390, 397, and 398, wherein the numbering method is related to SEQ ID NO: 95 or SEQ ID NO: 1. In another embodiment, at least one amino acid modification is selected from the group consisting of: Q3 or T3; N5 or D5; S10 or F10; Q17 or E17; Q25 or T25; A26 or P26; N29 or S29; A32 or S32; 139 or L39; 140 or V40; N44 or Q44; A51 or P51; A52 or V52; S53 or W53; A57 or R57; A58 or S58; D63 or G63; G64, E64 or R64; F67 or L67; P70 or 70del; R73 or K73; S74 or E74; T75, M75, G75 or W75; A78 or G78; A82 Or E82; S84 or T84; A88 or G88; L92 or M92; A99 or S99; W101 or H101; SI 10 or T110; L114 or V114; 1124 or T124; D131 or E131; H143, D143 or R143; A148 or V148; A149 or K149; S167 or G167; E171 or D171; R186, Q186 or E186; T188 or M188; A193 or P193; S194 or P194; A197 or E197; A207 or V207; R210 or K210;R211, K211, or 21Idel; S212, G212, or D212; P215, or E215; A223, or V223; L234, or 1234; D244, or E244; H249, or R249; Q252, or R252; H258, or A258; 1276, or L276; L280, or A280; E288, or Q288; R291, or L291; 1301, or V301; G304, E304, or A304; A305, or W305; L311, or R311; Q312, or T312; N313, or G313; R316, P316, or K316; K326 Or H326; F327 or L327; F328 or L328; N330 or G330; A349 or G349; 1359 or V359; V368 or A368; L370 or F370; L375 or E375; C385, N385 or M385; D390, E390 or R390; G397 or A397 and G398 or 398del, wherein the numbering scheme is related to SEQ ID NO: 95 or SEQ ID NO: 1.

[0171] In one embodiment, the amidase is a polypeptide capable of hydrolyzing the amide bond in a polyamide, wherein the amidase comprises: an amino acid sequence of amino acid residues 2-398 of SEQ ID NO: 2, or an amino acid sequence having at least 70% sequence identity with it. In one embodiment, the amidase includes at least one amino acid modification at positions selected from: amino acid positions 3, 5, 7, 9, 10, 14, 16, 17, 25, 26, 29, 32, 39, 40, 44, 48, 51, 52, 53, 57, 58, 60, 61, 63, 64, 67, 70, 72, 73, 74, 75, 78, 82, 84, 88, 92, 99, 101, 110, 114, 123, 124, 131, 132, 138, 143, 148, 149, 155, 167, 171, 173, ... 178, 179, 180, 186, 188, 193, 194, 195, 197, 203, 206, 207, 210, 211, 212, 213, 215, 223, 234, 244, 249, 252, 258, 262, 276, 278, 280, 288, 291, 301, 304, 305, 308, 311, 312, 313, 316, 321, 326, 327, 328, 330, 335, 349, 359, 368, 370, 375, 379, 384, 385, 390, 394, 397, and 398, wherein the numbering method is related to SEQ ID NO: 129. In another embodiment, at least one amino acid modification is selected from the group consisting of: Q3 or T3; N5 or D5; M7 or F7; G9 or T9; S10 or F10; P14 or A14; Q16 or G16; Q17 or E17; Q25 or T25; A26 or P26; N29 or S29; A32 or S32; 139 or L39; 140 or V40; N44 or Q44; G48 or A48; A51 or P51; A52 or V52; S53 or W53; A57 or R57; A58 or S58; R60 or A60; D61, A61 or G61; D63 or G63; G64, E64 or R64; F67 or L67. P70 or 70del; G72 or A72; R73 or K73; S74 or E74; T75, M75, G75 or W75; A78 or G78; A82 or E82; T84 or S84; A88 or G88; L92 or M92; A99 or S99; W101 or H101; SI 10 or T110; LI 14 or VI 14; G123 or A123;1124 or T124; E131 of D131; R132 or D132; D138 or A138; H143, D143 or R143; A148 or V148; A149 or K149; D155 or G155; S167 or G167; E171 or D171; D173 or A173; D178 or A178; G179 or S179; D180 or A180; R186, Q186 or E186; T188 or M188; A193 or P193; S194 or P194; D195 or P195; A197 or E197; R203 or Y203; L206 Or 1206; A207 or V207; R210 or K210; R211, K211 or 21Idel; S212, G212 or D212; D213 or A213; P215 or E215; A223 or V223; L234 or 1234; D244 or E244; H249 or R249; Q252 or R252; H258 or A258; 1262 or V262; 1276 or L276; A278 or C278; L280 or A280; E288 or Q288; R291 or L291; 1301 or V301; G304, E304 or A304; A305 Or W305; D308 or A308; L311 or R311; Q312 or T312; N313 or G313; R316, P316 or K316; R321 or Q321; K326 or H326; F327 or L327; F328 or L328; N330 or G330; S335 or N335; A349 or G349; 1359 or V359; V368 or A368; L370 or F370; L375 or E375; D379 or A379; C385, L384 or E384; N385 or M385; D390, E390 or R390; R394 Or A394; G397 or A397 and G398 or 398del, wherein the numbering scheme is related to SEQ ID NO:95 or SEQ ID NO:129.

[0172] The amidase used in this invention is a polypeptide capable of hydrolyzing the amide bond in polyamide, which has at least 70% sequence identity with the amino acid sequence of SEQ ID NO:2. A polypeptide capable of hydrolyzing the amide bond in polyamide is also provided, which has at least 80% sequence identity with the amino acid sequence of SEQ ID NO:2. A polypeptide capable of hydrolyzing the amide bond in polyamide is also provided, which has at least 90% sequence identity with the amino acid sequence of SEQ ID NO:2. A polypeptide capable of hydrolyzing the amide bond in polyamide is also provided, which has at least 95% sequence identity with the amino acid sequence of SEQ ID NO:2.

[0173] In one embodiment, the amidase comprises the amino acid sequence having amino acid residues 2-397 of SEQ ID NO:2; the amino acid sequence having amino acid residues 2-398 of SEQ ID NO:15; the amino acid sequence having amino acid residues 2-398 of SEQ ID NO:5; the amino acid sequence having amino acid residues 2-398 of SEQ ID NO:10; the amino acid sequence having amino acid residues 2-397 of SEQ ID NO:8; the amino acid sequence having amino acid residues 2-398 of SEQ ID NO:4; the amino acid sequence having amino acid residues 2-398 of SEQ ID NO:12; the amino acid sequence having amino acid residues 2-397 of SEQ ID NO:90; the amino acid sequence having amino acid residues 2-397 of SEQ ID NO:91; the amino acid sequence having amino acid residues 2-397 of SEQ ID NO:92; the amino acid sequence having amino acid residues 2-397 of SEQ ID NO:93; the amino acid sequence having amino acid residues 2-397 of SEQ ID NO:94; and the amino acid sequence having amino acid residues 2-397 of SEQ ID NO:92. The amino acid sequence of amino acid residues 2-398 of SEQ ID NO:14; the amino acid sequence of amino acid residues 2-398 of SEQ ID NO:88; the amino acid sequence of amino acid residues 2-398 of SEQ ID NO:6; the amino acid sequence of amino acid residues 2-398 of SEQ ID NO:89; the amino acid sequence of amino acid residues 2-398 of SEQ ID NO:9; the amino acid sequence of amino acid residues 2-398 of SEQ ID NO:7; the amino acid sequence of amino acid residues 2-398 of SEQ ID NO:11; or the amino acid sequence of amino acid residues 2-398 of SEQ ID NO:13.

[0174] In another embodiment, the polypeptide consists of the following amino acid sequences: SEQ ID NO:2; SEQ ID NO:15; SEQ ID NO:5; SEQ ID NO:8; SEQ ID NO:4; SEQ ID NO:12; SEQ ID NO:90; SEQ ID NO:91; SEQ ID NO:92; SEQ ID NO:93; SEQ ID NO:94; SEQ ID NO:14; SEQ ID NO:88; SEQ ID NO:6; SEQ ID NO:89; SEQ ID NO:9; SEQ ID NO:7; SEQ ID NO:11; or SEQ ID NO:13.

[0175] The polyamide can be a polypeptide capable of hydrolyzing the amide bond in the polyamide, wherein the amidase comprises the amino acid sequence of amino acid residues 2-394 of SEQ ID NO:87, or an amino acid sequence having more than 75% sequence identity with it. In one embodiment, the polypeptide has at least 80% sequence identity with SEQ ID NO:87. In one embodiment, the polypeptide has at least 85% sequence identity with SEQ ID NO:87. In one embodiment, the polypeptide has at least 90% sequence identity with SEQ ID NO:87. In one embodiment, the polypeptide has at least 95% sequence identity with SEQ ID NO:87.

[0176] In one embodiment, the amidase comprises an amino acid sequence having amino acid residues 2-392 of SEQ ID NO:3; an amino acid sequence having amino acid residues 2-394 of SEQ ID NO:62; or an amino acid sequence having amino acid residues 2-398 of SEQ ID NO:47. In a further embodiment, the polypeptide consists of an amino acid sequence of SEQ ID NO:3, SEQ ID NO:62, or SEQ ID NO:47. In one embodiment, the amidase comprises an amino acid sequence having amino acid residues 2-394 of SEQ ID NO:127. In one embodiment, the amidase consists of an amino acid sequence having amino acid residues of SEQ ID NO:127.

[0177] The amidase used in this invention can be a polypeptide capable of hydrolyzing the amide bond in polyamide, wherein the amidase comprises the amino acid sequence of amino acid residues 2-391 of SEQ ID NO:44 or an amino acid sequence having at least 61% sequence identity with it. In one embodiment, the polypeptide has at least 70% sequence identity with SEQ ID NO:44. In one embodiment, the polypeptide has at least 75% sequence identity with SEQ ID NO:44. In one embodiment, the polypeptide has at least 80% sequence identity with SEQ ID NO:44. In one embodiment, the polypeptide has at least 85% sequence identity with SEQ ID NO:44. In one embodiment, the polypeptide has at least 90% sequence identity with SEQ ID NO:44. In one embodiment, the polypeptide has at least 95% sequence identity with SEQ ID NO:44.

[0178] In one embodiment, the polypeptide comprises an amino acid sequence having amino acid residues 2-391 of SEQ ID NO:24 or an amino acid sequence having amino acid residues 2-391 of SEQ ID NO:21. In one embodiment, the polypeptide consists of the amino acid sequence of SEQ ID NO:24 or SEQ ID NO:21. In another embodiment, the polypeptide consists of the amino acid sequence of SEQ ID NO:24, SEQ ID NO:62, or SEQ ID NO:21.

[0179] In one embodiment, the amidase is a polypeptide capable of hydrolyzing the amide bond in a polyamide, wherein the amidase comprises the amino acid sequence of amino acid residues 2-392 of SEQ ID NO:73.

[0180] The amidase used in this invention can be a polypeptide capable of hydrolyzing the amide bonds in nylon 6 oligomers.

[0181] In one embodiment, the amidase may be an amino acid sequence of any one of SEQ ID NOs:72-86 and 96-100 or a polypeptide having at least 70% sequence identity with it.

[0182] In one embodiment, the amidase is a polypeptide capable of hydrolyzing the amide bond in a polyamide, wherein the amidase comprises the amino acid sequence of amino acid residues 2-392 of SEQ ID NO:72 or amino acid residues 2-392 of SEQ ID NO:74. In one embodiment, the amidase is a polypeptide capable of hydrolyzing the amide bond in a polyamide, wherein the amidase comprises the amino acid sequence of amino acid residues 2-392 of SEQ ID NO:73. In one embodiment, the amidase is a polypeptide capable of hydrolyzing the amide bond in a polyamide, wherein the amidase comprises the amino acid sequence of amino acid residues 2-392 of SEQ ID NO:75. In one embodiment, the amidase is a polypeptide capable of hydrolyzing the amide bond in a polyamide, wherein the amidase comprises the amino acid sequence of amino acid residues 2-392 of SEQ ID NO:76. In one embodiment, the amidase is a polypeptide capable of hydrolyzing the amide bond in a polyamide, wherein the amidase comprises the amino acid sequence of amino acid residues 2-391 of SEQ ID NO:77. In one embodiment, the amidase is a polypeptide capable of hydrolyzing the amide bond in a polyamide, wherein the amidase comprises the amino acid sequence of amino acid residues 2-389 of SEQ ID NO:78. In one embodiment, the amidase is a polypeptide capable of hydrolyzing the amide bond in a polyamide, wherein the amidase comprises the amino acid sequence of amino acid residues 2-394 of SEQ ID NO:79. In one embodiment, the amidase is a polypeptide capable of hydrolyzing the amide bond in a polyamide, wherein the amidase comprises the amino acid sequence of amino acid residues 2-401 of SEQ ID NO:80. In one embodiment, the amidase is a polypeptide capable of hydrolyzing the amide bond in a polyamide, wherein the amidase comprises the amino acid sequence of amino acid residues 2-406 of SEQ ID NO:81. In one embodiment, the amidase is a polypeptide capable of hydrolyzing the amide bond in a polyamide, wherein the amidase comprises the amino acid sequence of amino acid residues 2-407 of SEQ ID NO:82. In one embodiment, the amidase is a polypeptide capable of hydrolyzing the amide bond in a polyamide, wherein the amidase comprises the amino acid sequence of amino acid residues 2-401 of SEQ ID NO:83. In one embodiment, the amidase is a polypeptide capable of hydrolyzing the amide bond in a polyamide, wherein the amidase comprises the amino acid sequence of amino acid residues 2-391 of SEQ ID NO:84. In one embodiment, the amidase is a polypeptide capable of hydrolyzing the amide bond in a polyamide, wherein the amidase comprises the amino acid sequence of amino acid residues 2-391 of SEQ ID NO:85. In one embodiment, the amidase is a polypeptide capable of hydrolyzing the amide bond in a polyamide, wherein the amidase comprises the amino acid sequence of amino acid residues 2-392 of SEQ ID NO:86. In one embodiment, the amidase is a polypeptide capable of hydrolyzing the amide bond in a polyamide, wherein the amidase comprises the amino acid sequence of amino acid residues 2-391 of SEQ ID NO:84.In one embodiment, the amidase is a polypeptide capable of hydrolyzing the amide bond in a polyamide, wherein the amidase comprises the amino acid sequence of amino acid residues 2-493 of SEQ ID NO:96. In one embodiment, the amidase is a polypeptide capable of hydrolyzing the amide bond in a polyamide, wherein the amidase comprises the amino acid sequence of amino acid residues 2-492 of SEQ ID NO:97. In one embodiment, the amidase is a polypeptide capable of hydrolyzing the amide bond in a polyamide, wherein the amidase comprises the amino acid sequence of amino acid residues 2-355 of SEQ ID NO:98. In one embodiment, the amidase is a polypeptide capable of hydrolyzing the amide bond in a polyamide, wherein the amidase comprises the amino acid sequence of amino acid residues 2-355 of SEQ ID NO:99. In one embodiment, the amidase is a polypeptide capable of hydrolyzing the amide bond in a polyamide, wherein the amidase comprises the amino acid sequence of amino acid residues 2-355 of SEQ ID NO:100.

[0183] In one embodiment, the amidase is a polypeptide capable of hydrolyzing the amide bond in a polyamide, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:72 or SEQ ID NO:74. In one embodiment, the amidase is a polypeptide capable of hydrolyzing the amide bond in a polyamide, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:73. In one embodiment, the amidase is a polypeptide capable of hydrolyzing the amide bond in a polyamide, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:75. In one embodiment, the amidase is a polypeptide capable of hydrolyzing the amide bond in a polyamide, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:76. In one embodiment, the amidase is a polypeptide capable of hydrolyzing the amide bond in a polyamide, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:77. In one embodiment, the amidase is a polypeptide capable of hydrolyzing the amide bond in a polyamide, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:78. In one embodiment, the amidase is a polypeptide capable of hydrolyzing the amide bond in a polyamide, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:79. In one embodiment, the amidase is a polypeptide capable of hydrolyzing the amide bond in a polyamide, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 80. In one embodiment, the amidase is a polypeptide capable of hydrolyzing the amide bond in a polyamide, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 81. In one embodiment, the amidase is a polypeptide capable of hydrolyzing the amide bond in a polyamide, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 82. In one embodiment, the amidase is a polypeptide capable of hydrolyzing the amide bond in a polyamide, wherein the polypeptide comprises the amino acid sequence of amino acid residues 2-391 of SEQ ID NO: 84. In one embodiment, the amidase is a polypeptide capable of hydrolyzing the amide bond in a polyamide, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 85. In one embodiment, the amidase is a polypeptide capable of hydrolyzing the amide bond in a polyamide, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 86. In one embodiment, the amidase is a polypeptide capable of hydrolyzing the amide bond in a polyamide, wherein the polypeptide consists of the amino acid sequence of amino acid residues 2-391 of SEQ ID NO:84. In one embodiment, the amidase is a polypeptide capable of hydrolyzing the amide bond in a polyamide, wherein the polypeptide consists of the amino acid sequence of SEQ ID NO:96. In one embodiment, the amidase is a polypeptide capable of hydrolyzing the amide bond in a polyamide, wherein the polypeptide consists of the amino acid sequence of SEQ ID NO:97.In one embodiment, the amidase is a polypeptide capable of hydrolyzing the amide bond in a polyamide, wherein the polypeptide consists of the amino acid sequence of SEQ ID NO:98.

[0184] In one embodiment, the amidase is a polypeptide capable of hydrolyzing the amide bond in a polyamide, wherein the polypeptide consists of the amino acid sequence of SEQ ID NO:99. In another embodiment, the amidase is a polypeptide capable of hydrolyzing the amide bond in a polyamide, wherein the polypeptide consists of the amino acid sequence of SEQ ID NO:100.

[0185] In some embodiments, the amidase is a polypeptide capable of hydrolyzing amide bonds in nylon 6 oligomers and nylon 6,6 oligomers. In one embodiment, the amidase is a polypeptide capable of hydrolyzing amide bonds in polyamides, wherein the amidase comprises the amino acid sequence of amino acid residues 2-392 of SEQ ID NO:73. In one embodiment, the amidase is a polypeptide capable of hydrolyzing amide bonds in polyamides, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:73.

[0186] Table 1 provides illustrative examples of amidases that can be used in this invention.

[0187] In one embodiment, the polypeptide disclosed herein is capable of hydrolyzing polyamide oligomers. In another embodiment, the polypeptide disclosed herein has adipic acid mono- and di-N-alkylamide hydrolase activity.

[0188] Table 1 : Amino acid sequences of amidases

[0189] As described elsewhere herein, the amidase (including variants of existing amidases) will suitably retain at least some amidase, adipic acid mono- and / or di-N-alkylamide hydrolase activity, regardless of any modifications made, including modifications to its amino acid sequence. Suitable methods for determining or measuring amidase activity are well known to those skilled in the art, including those described herein. Illustrative examples are described in: Kiumarsi and Parvinzadeh, 2010 J Appl Polymer Sci, 116:3140, and Gashti et al., 2013 Preparative Biochemistry & Biotechnology, 43:798. The contents of these references are incorporated herein by reference in their entirety. In one embodiment, amidase activity is determined by UV absorbance assay to monitor the amount of monomeric components generated using polyamide oligomers as substrates. Another method for determining or measuring amidase / hydrolase activity is to measure the amount of monomeric components generated using LC-MS. The amidase activity of the polypeptide can be specified as an absolute value or a value relative to the amidase activity of a comparison group (e.g., an existing amidase). In one embodiment, amidase activity is measured as the rate at which monomers and / or oligomers (e.g., in mg or mol) of enzyme are released per hour, per mg or mol, under suitable temperature, pH, and buffer conditions.

[0190] Those skilled in the art will be able to select an incubation temperature suitable for a specific amidase. Typically, incubation can be carried out at temperatures ranging from approximately 0°C to approximately 80°C, for example, approximately 2°C to approximately 75°C, approximately 4°C to approximately 70°C, approximately 6°C to approximately 70°C; approximately 8°C to approximately 65°C, approximately 10°C to approximately 65°C, approximately 20°C to approximately 60°C, approximately 5°C to approximately 55°C, approximately 30°C to approximately 55°C, approximately 30°C to approximately 50°C, approximately 35°C to approximately 50°C, or approximately 40°C to approximately 50°C.

[0191] In one embodiment, the amidase exhibits measurable amidase activity at least in a pH range of approximately 5 to approximately 11, approximately 6 to approximately 10, approximately 7 to approximately 10, approximately 7.5 to approximately 9.5, or approximately 7.5 to approximately 8.

[0192] The amount of amidase used will, of course, depend on the amount of one or both of the nylon polyamide substrate or nylon oligomer to be converted into its monomeric components. Typically, the amount of amidase used will be in the range of 1:1000 to approximately 1:10 (enzyme mass to nylon substrate).

[0193] Once the enzymatic conversion to the monomeric components is complete, it may be desirable to subject the composition including the enzymatic conversion product to one or more purification procedures to increase the purity of the monomeric components, as described elsewhere in this document.

[0194] The purity of the resulting monomeric components can be improved using techniques well known to those skilled in the art. For example, compositions containing the resulting monomeric components can be subjected to one or more techniques selected from washing, solvent extraction, filtration, distillation, solvent evaporation, column chromatography, and crystallization.

[0195] In one embodiment, the composition comprising monomeric components is subjected to one or more techniques to increase the purity of the monomeric components thus formed.

[0196] In another embodiment, one or more techniques for improving the purity of the monomeric components thus formed are selected from washing, solvent extraction, filtration, distillation, solvent evaporation, column chromatography, and crystallization.

[0197] In another embodiment, the monomeric components produced by the enzymatic conversion of polyamide oligomers are purified and isolated.

[0198] Using an amidase immobilized on a substrate may be convenient for carrying out the method of the present invention. The use of an immobilized amidase may be advantageous when carrying out the method of the present invention in a semi-continuous or continuous manner.

[0199] In one embodiment, the amidase is immobilized on the substrate.

[0200] Amideases can be immobilized on any suitable substrate using techniques known to those skilled in the art. For example, amidases can be immobilized on a support resin by ion exchange, adsorption (e.g., hydrophobic adsorption), or covalent coupling.

[0201] In one embodiment, the amidase is immobilized on a carrier resin.

[0202] In one embodiment, the amidase is immobilized on an ion exchange resin. In another embodiment, the amidase is immobilized on an adsorption resin. In yet another embodiment, the amidase is immobilized on a nickel affinity resin. In one embodiment, the amidase is immobilized on a covalent resin.

[0203] Those skilled in the art will be familiar with the general principles of enzyme immobilization techniques, and these principles can be advantageously applied to the immobilization of amidases on substrates according to the present invention.

[0204] Suitable ion exchange resins for immobilizing amidases typically include polymer matrices or polymer / ceramic hybrid matrices. Examples of such resins include, but are not limited to, CM ceramic HyperD® ion exchange chromatography resin.

[0205] In one embodiment, the ion exchange resin is a cation exchange resin.

[0206] The method according to the invention can be advantageously carried out in a batch, semi-continuous or continuous manner.

[0207] For continuous operation of the method according to the invention, the amidase is typically immobilized on a carrier resin and loaded into a column. The composition of the soluble polyamide oligomers produced in step (i) can then be continuously passed through the column to facilitate the conversion of the oligomers into their monomeric components using the immobilized amidase.

[0208] Before passing through the column, the composition of the soluble polyamide oligomers generated in step (i) may first be diluted in a solvent (such as those described herein) and combined with one or more additives to adjust / stabilize the pH and / or ion content. Such additives may include, for example, pH buffers and / or ionic compounds such as NaCl.

[0209] According to the method of the present invention, step (ii) produces a composition comprising monomeric components. The specific monomers produced will vary depending on the type of polyamide being processed. It will be understood by those skilled in the art that the monomeric components of the oligomer are substantially the same as those of the polyamide derived from the oligomer. For example, if nylon 6 is used in the method of the present invention, the monomeric components will be 6-6-aminohexanoic acid. If nylon 6,6 is used in the method of the present invention, the monomeric components will be HMD and AA. If a mixture of nylon 6 and nylon 6,6 is used in the method of the present invention, the monomeric components will be HMD, AA, and 6-aminohexanoic acid.

[0210] The method of the present invention can readily generate a variety of amino acids by converting oligomers derived from polyamide homopolymers with amidase and / or readily generate a variety of dicarboxylic acids and diamines by converting oligomers derived from polyamide copolymers with amidase.

[0211] Once the enzymatic conversion is complete, the method provides a liquid comprising the monomer components thus formed.

[0212] Therefore, this method can be described as using one or more amidases to convert oligomers into their monomeric components in order to provide a liquid containing the monomeric components.

[0213] One or more purification procedures may be required on the resulting liquid to improve the purity of the monomeric components thus formed and to aid in their separation and exfoliation.

[0214] Techniques well known to those skilled in the art can be used to improve the purity of monomeric components and / or to separate and isolate them. For example, liquids comprising monomeric components may be subjected to one or more techniques selected from the following, which may be used in combination or not: washing, solvent extraction, filtration, distillation, solvent evaporation, column chromatography, crystallization, stripping, separation by aqueous solution, selective vapor condensation, filtration and concentration of bioprocess media, separation, vacuum evaporation, extraction, electrodialysis, adsorption, ion exchange, precipitation, concentration and acid addition dehydration and precipitation, nanofiltration, acid catalyst treatment, semi-continuous or continuous distillation, solvent extraction, evaporation concentration, evaporation crystallization, liquid / liquid extraction, hydrogenation, azeotropic distillation, acid or thermal catalytic lactamation, adsorption, simple vacuum distillation, and microfiltration.

[0215] In one embodiment, the monomeric components thus formed are subjected to one or more purification techniques.

[0216] In another embodiment, the monomeric components thus formed are purified and isolated.

[0217] In one embodiment, an amidase is used to provide a liquid comprising monomeric components, and the monomeric components are purified by adjusting the pH of the liquid.

[0218] In one embodiment, the pH of the liquid comprising the monomeric components is made acidic (e.g., adjusted to around 1 to around 4) to aid in the purification and separation of the dicarboxylic acid monomeric components.

[0219] In another embodiment, the pH of the liquid containing the monomer components is made alkaline (e.g., adjusted to around 8 to around 13) to aid in the purification and separation of the diamine monomer components.

[0220] In one embodiment, the pH of the liquid containing the monomer components is first made acidic to aid in the purification and separation of the dicarboxylic acid monomer components; then the resulting liquid is made alkaline to aid in the purification and separation of the diamine monomer components.

[0221] Reagents suitable for making liquids acidic include the acids described herein.

[0222] Reagents suitable for making liquids alkaline include alkali metal bases and alkaline earth metal bases as described herein.

[0223] In another embodiment, the pH of the liquid comprising the monomeric components is made neutral to aid in the purification and separation of ammonium carboxylate salts, such as salts containing diamines and dicarboxylic acids, like hexamethylenediamine adipic acid.

[0224] Suitable reagents for neutralizing liquids comprising monomeric components include acids and bases described herein, such as organic amine bases and organic acids. In one embodiment, the base used is an organic diamine base, such as hexamethylenediamine. In another embodiment, the acid used is an organic acid, such as a dicarboxylic acid, like adipic acid.

[0225] The recovery of ammonium carboxylate salts may involve a concentration step using techniques well known to those skilled in the art, including but not limited to membrane distillation, reverse osmosis, or solvent evaporation.

[0226] Purification of ammonium carboxylate salts can be achieved using techniques well known to those skilled in the art, including but not limited to precipitation, activated carbon treatment, or recrystallization.

[0227] The formation of monomeric components can be confirmed using techniques well known to those skilled in the art. For example, the formation of amino acids, diacids, diamines, and ammonium carboxylates can be readily determined using NMR spectroscopy.

[0228] Example Example 1: Acid hydrolysis of PA66 using different inorganic acids In a round-bottom flask, 10 g of PA66, 7.2 mL of H2SO4, and 3.7 mL of water were heated under reflux for 3 hours. The mixture was cooled to room temperature, neutralized with NaOH, filtered, and the remaining solid was vacuum dried and weighed to obtain the conversion rate of the PA66 oligomers. UPLC analysis of the soluble oligomers yielded the following results: Figure 1A As shown. Mass spectrometry analysis of each oligomer is as follows. Figure 1B As shown.

[0229] In a round-bottom flask, 10 g of PA66 and 9.4 mL of 37% hydrochloric acid aqueous solution were heated under reflux for 3 hours. The mixture was cooled to room temperature, neutralized with NaOH, filtered, and the remaining solid was dried under vacuum and weighed to obtain the conversion rate of PA66 oligomers.

[0230] In a round-bottom flask, 10 g of PA66, 9.1 mL of 85% H3PO4 aqueous solution, and 5.0 mL of water were heated under reflux for 3 hours. The mixture was cooled to room temperature, neutralized with NaOH, filtered, and the remaining solid was dried under vacuum and weighed to obtain the conversion rate of PA66 oligomers.

[0231] Sulfuric acid: 100% conversion rate.

[0232] Hydrochloric acid: 100% conversion rate.

[0233] Phosphoric acid: 100% conversion rate.

[0234] Example 2: Acid hydrolysis of PA66 using adipic acid Part A In a 2 L pressure reactor, 250 g of PA66 was suspended in 500 mL of water, and then 116 g of adipic acid was added. The reactor was sealed and pressurized with nitrogen at 6 bar. The reaction mixture was then heated to 195 °C at 20 bar and maintained for 2 hours. The reaction mixture was cooled to 100 °C, and then the pressure was released through the reactor's vent valve. Any insoluble adipic acid was filtered off, and the resulting filtrate (containing oligomers, hexamethylenediammonium adipic acid, and adipic acid) was then filtered through an activated carbon filter. UPLC analysis showed the formation of adipic acid, dimers, trimers, tetramers, pentamers, and hexamers. Figure 2 ).

[0235] Part B In a 10 L pressure reactor, 1 kg of PA66 was suspended in 3 L of water, and then 125 g of adipic acid was added. The reaction mixture was then heated to 180 °C for 6 h. The reaction mixture was cooled to 100 °C and the pressure was released. Any insoluble adipic acid and long-chain oligomers were filtered off, and the resulting filtrate (containing oligomers, hexamethylenediammonium adipic acid, and adipic acid) was used as a feedstock for enzymatic hydrolysis. UPLC analysis showed the formation of adipic acid, dimers, trimers, tetramers, pentamers, and hexamers; analysis of the solids showed the presence of longer-chain oligomers: heptamers and oligomers with higher molecular weights, consistent with Part A of Example 2. Figure 2 The results shown are similar.

[0236] Example 3: Engineered peptides for hydrolyzed nylon oligomers Variant enzyme sequences were designed by reconstructing ancestral sequences, including the NylB sequence and sequences from the genus Flavobacterium. Flavobacterium sp., strain K172 P07061, from the genus Flavobacterium ( Strain K172 The known sequences of P07062 and related polyamides were obtained; these variant sequences were synthesized and their ability to hydrolyze amide bonds in polyamides was tested. The amino acid sequences are listed in Table 1.

[0237] Unexpectedly, several variant peptides (SEQ ID NO: 2-15 and 87) exhibited higher amidase activity in the hydrolysis of amide bonds in nylon 6,6 polyamide oligomers compared to most known sequences. Figure 3 ).

[0238] At least one known sequence (SEQ ID NO:73; corresponding to) Flavobacterium The 1WYC_A, chain A, 6-aminohexanoic acid dimer hydrolase also showed higher activity in hydrolyzing amide bonds in nylon 6,6 polyamide oligomers compared to most known sequences and the enzyme-free control.

[0239] Sequence analysis revealed that many engineered peptides with enhanced amide bond capacity in hydrolyzed nylon 6,6 oligomers exhibited high sequence identity, as evidenced by the consensus sequence of SEQ ID NO:1 (see [link to SEQ ID NO:1]). Figure 4 ): MTX1TX2LMQGFPPAPEQQVTLANWRX3X4PFNRWX5FHHVREIX6PTANIPRGPGX7X8X9PLPX 10 X 11 PRDLX 12 X 13 IAFEGX 14 DGX 15 X 16 X 17 TVX18 EMLX 19 EX 20 YTDX 21 FLVX 22 HRGRIVX 23 EX 24 YANGMTPHX 25 PHIX 26 FSVSKSITGX 27 LAGILVX 28 RGQLDPDAPVTX 29 YIPEX 30 X 31 GSAYGDATVRHVLDMTVX 32 IDFX 33 EDYLDPDGDFARYRX 34 ATGWNPX 35 X 36 DGX 37 TPSDLRSFLX 38 TLX 39 X 40 X 41 DGX 42 HGETFHYX 43 SPNSDLLGWIX 44 ERASGQRFAX 45 LLSEHIWQPMGAEHDAYITVDRLGAPRTAGGX 46 CATX 47 RDLARFGX 48 MMX 49 NRGVANGRQX 50 VPX 51 X 52 WIDDIX 53 X 54 X 55 GDX 56 EAWARGDFAKFX 57 PX 58 GRYRSKWYVTGNARGAFCX 59 IGIHGQWIYIDPAAEVVIX 60 KX 61 SSQPX 62 PVDDAMDRLX 63 LAAFX 64 AIARALX 65 G (where X1 - X 65 are arbitrary amino acids) Selected sequences were chosen as the basis for further engineering. This second round of engineering identified many new sequences with improved enzymatic activity against nylon 6,6 oligomers (see [link to enzymatic analysis]). Figure 5 Where C4 corresponds to SEQ ID NO:89; D3 corresponds to the polypeptide including SEQ ID NO:90; E3 corresponds to the polypeptide including SEQ ID NO:94; F3 corresponds to the polypeptide including SEQ ID NO:91; F3 corresponds to the polypeptide including SEQ ID NO:91; and G3 corresponds to the polypeptide including SEQ ID NO:92.

[0240] The activity of some engineered peptides from Phase I and Phase II is shown in Figure 6.

[0241] Analysis of the sequences with increased amide bond activity in these hydrolyzed nylon 6,6 polyamide oligomers revealed that many of these sequences share a broad consensus sequence SEQ ID NO:95 ( Figure 7 Table 2 provides the amino acid substitutions at specified positions for the variant peptides, where the numbering is relative to the amino acid positions of SEQ ID NO:1 or SEQ ID NO:95.

[0242] SEQ ID NO:95 MTX1TX2LMQGX3PPAPEQX4VTLANWRX5X6PFX7RWX8FHHVREX9X 10 PTAX 11 IPRGPGX 12 X 13 X 14 PLPX 15 X 16 PRDLX 17 X 18 IAX 19 EGX 20 DGX 21 X 22 X 23 TVX 24 EMLX 25 EX 26 YTDX 27 FLVX 28 HRGRIVX 29 EX 30 YANGMTPHX 31 PHIX 32 FSVSKSITGX 33 LAGILVX 34 RGQLDPDAPVTX 35 YIPEX 36 X37 GSAYGDATVRHVLDMTVX 38 IDFX 39 EDYLDPDGDFARYRX 40 AX 41 GWNPX 42 X 43 DGX 44 TPSDLRSFLX 45 TLX 46 X 47 X 48 DGX 49 HGETFHYX 50 SPNSDLLGWIX 51 ERASGQRFAX 52 LLSEX 53 IWX 54 PMGAEX 55 DAYITVDRLGAPRTAGGX 56 CATX 57 RDLARFGX 58 MMX 59 NRGVANGRQX 60 VPX 61 X 62 WIDDIX 63 X 64 X 65 GDX 66 EAWARGDFAX 67 X 68 X 69 PX 70 GRYRSKWYVTGNARGAFCX 71 IGIHGQWIYX 72 DPAAEVVIX 73 KX 74 SSQPX 75 PVDDAMDRLX 76 LAAFX 77 AIARALX 78 X 79 (where X1-X) 79 (It can be any amino acid) Table 2: Amino acid substitutions between variant polypeptides

[0243] Example 4: Immobilized enzyme acid hydrolysis of nylon 6,6 oligomers The engineered peptide was immobilized on an adsorption-based polymer resin at a 10% w / w enzyme loading. The immobilized enzyme was incubated with nylon 6,6 dimers, trimers, and tetramers generated in the acid hydrolysis of nylon 6,6 in Example 3. The reaction solution was sampled after 1 hour for analysis by UHPLC and compared with a control reaction without the enzyme (time 0 hours). Data are as follows. Figure 8 As shown.

[0244] Example 7: Acid hydrolysis of PA6 with H2SO4 In a round-bottom flask, 10 g of PA6, 3.6 mL of H2SO4, and 3.7 mL of water were heated under reflux for 3 hours. The mixture was cooled to room temperature, neutralized with NaOH, filtered, and the remaining solid was dried under vacuum and weighed to obtain the conversion of PA6 oligomers. A 100% conversion was achieved. UHPLC analysis showed the presence of dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer, and nonamer units of 6-aminohexanoate.

[0245] Example 8: Separation of adipic acid and hexamethylenediamine The aqueous solution from the enzyme reactor (from Example 4) was cooled to 0°C and acidified to pH 1 using 50% H2SO4 aqueous solution. After 30 minutes, the resulting suspension was filtered to separate AA, yielding a white crystalline solid. The filtrate was alkalized to pH 13 using Ca(OH)2 and filtered to obtain an aqueous solution of HMD, which was further purified by vacuum distillation to obtain colorless HMD, which solidified upon standing.

[0246] Example 9: Further Enzyme Engineering Further rounds of enzyme engineering identified variant peptides that exhibited activity in hydrolyzing amide bonds in polyamides, including nylon 6,6 oligomers. Enzyme activity assays against nylon 6,6 oligomers (dimers, trimers, and tetramers) were performed at 40 °C for 2 h. The reaction was terminated by removing the enzyme using a 10 kDa MWCO centrifugal filter (15 min, 13000 × g). The reaction was analyzed using ultra-high performance liquid chromatography (UHPLC).

[0247] Peptide variants including any of the amino acid sequences in SEQ ID NOs: 101-127 are capable of hydrolyzing nylon 6,6 oligomers. Some of these peptides exhibit activities such as hydrolysis of nylon 6,6 trimers. Figure 9 As shown.

[0248] Peptides capable of hydrolyzing a significant proportion of nylon 6,6 oligomers share the consensus sequence of SEQ ID NO:128 / Consensus III.

[0249] SEQ ID NO:128 MTQTNLX1QX2X3PPAX4EX5EVTLANWRQAPFSRWSFHHVRELVPTAQIPRX6PGPASPLPAAPX7X8LGEIALEGPDX9KEGTVAEMLEESYTDX 10 FLVLHRGRIVAEHYANGMTPHX 11 PHIVFSVSKSITX 12 TLAGILVEX 13 GQLDPX 14 APVTDYIPEVAGSAYGX 15 ATVRHVLDMTVX 16 IDFEEX 17 YLDPX 18 X 19 X 20 FARYRRAMGWNPPSX 21 GETPSDLX 22 SFX 23 ATLKKGX 24 GPHGETFHYX 25 SPNSDLLGWILERASGQRFADLLSERIWRPMGAEADAYX 26 TVDRLGAPRTAGGICX 27 TX 28 RDLARFGEMMRNRGVANGRQIVPEAWIX 29 DILTNGDX 30 EAWAX 31 GDFAHX 32 LPNGRYRX33KWYVTGNARGAFCAIGIHGQWIYX 34 DPAAEVVIVKLSSQPLPVDX 35 AMDRX 36 MLAAFRAIAX 37 ALG (where X1-X) 37 (It can be any amino acid) Analysis of the polypeptides disclosed in this paper that can hydrolyze the amide bonds in polyamides shows that they exhibit significant sequence conservation, which can be determined by the consensus sequence IV of SEQ ID NO:129 ( Figure 10 These polypeptides include SEQ ID NOs: 2, 4-15, 88-94, and 101-126.

[0250] Table 3 shows the amino acid substitutions at specific positions in the variant peptides, where the amino acid numbers are determined with reference to the amino acid positions of SEQ ID NO:1, SEQ ID NO:95, or SEQ ID NO:129.

[0251] Table 3: Amino acid substitutions between variant polypeptides

[0252] References to any prior publications (or information derived therefrom) or any known matters in this specification do not constitute, nor should be construed as, an acknowledgment, endorsement, or any form of implication that such prior publications (or information derived therefrom) or known matters constitute part of the general knowledge in the technical field to which this specification pertains.

[0253] Throughout this specification and the appended claims, unless the context clearly requires otherwise, the word “comprise” and its variations, such as “comprises” and “comprising”, shall be understood to indicate the presence of the stated element, step, or group of elements / steps, but do not exclude the presence of one or more other elements, steps, or groups of elements / steps.

Claims

1. A method for recycling polyamide, the method comprising: (i) subjecting the polyamide to acid hydrolysis in an aqueous liquid to produce a polyamide oligomer soluble in the aqueous liquid; as well as (ii) Using an amidase to convert the oligomer into its monomeric components.

2. The method according to claim 1, wherein, The polyamide includes aliphatic copolymer polyamide.

3. The method according to claim 1 or 2, wherein, The polyamide includes aliphatic homopolymer polyamide.

4. The method according to claim 1, wherein, The polyamides include nylon 6,6, nylon 6, nylon 10,10, nylon 11, nylon 12, nylon 6,10, nylon 4,6, nylon 6,12 and combinations thereof.

5. The method according to any one of claims 1 to 4, wherein, The acid hydrolysis is carried out using one or more inorganic acids selected from hydrochloric acid, nitric acid, phosphoric acid, and sulfuric acid.

6. The method according to any one of claims 1 to 4, wherein, The acid hydrolysis is carried out using one or more carboxylic acids.

7. The method according to any one of claims 1 to 6, wherein, The acid hydrolysis is carried out at a temperature below 200°C.

8. The method according to any one of claims 1 to 7, wherein, The acid hydrolysis produces an acidic aqueous liquid comprising the polyamide oligomer, which is then neutralized with alkali before the oligomer is converted into its monomeric components using the amidase.

9. The method according to any one of claims 1 to 7, wherein, Before using the amidase to convert the oligomer into its monomeric components, the method includes the following steps: (i) Lowering the temperature of the aqueous liquid to promote the precipitation of the oligomer in the aqueous liquid, wherein the aqueous liquid comprises the polyamide oligomer soluble in the aqueous liquid; and (ii) Neutralize the separated oligomers with alkali.

10. The method according to any one of claims 1 to 7, wherein, Before using the amidase to convert the oligomer into its monomeric components, the method includes the following steps: (i) Lowering the temperature of the aqueous liquid to promote the precipitation of any organic acid in the aqueous liquid, the aqueous liquid comprising the polyamide oligomer soluble in the aqueous liquid; (ii) Separating any precipitated organic acid from the aqueous liquid to provide a purified aqueous liquid comprising the polyamide oligomer soluble in the aqueous liquid; and (iii) Neutralize the purified aqueous liquid with alkali.

11. The method according to any one of claims 1 to 10, wherein, The amidase is immobilized on a carrier resin.

12. The method according to any one of claims 1 to 11, wherein, The amidase includes: f) The amino acid sequence of amino acid residues 2-398 of SEQ ID NO:1 or SEQ ID NO:95; g) The amino acid sequence of amino acid residues 2-398 of SEQ ID NO:2, or an amino acid sequence having at least 70% sequence identity with it; h) The amino acid sequence of amino acid residues 2-394 of SEQ ID NO:87, or an amino acid sequence having at least 75% sequence identity with it; i) The amino acid sequence of amino acid residues 2-391 of SEQ ID NO:44, or an amino acid sequence having at least 61% sequence identity with it; or j) An amino acid sequence of any one of SEQ ID NO:72-86 and 96-100, or an amino acid sequence having at least 70% sequence identity with it.

13. The method according to any one of claims 1 to 12, wherein, The amidase comprises: the amino acid sequence of amino acid residues 2-398 of SEQ ID NO:129, or an amino acid sequence having at least 80% sequence identity with it.

14. The method according to any one of claims 1 to 13, wherein, The amidase comprises: the amino acid sequence of amino acid residues 2-398 of SEQ ID NO:2, or an amino acid sequence having at least 70% sequence identity with it.

15. The method according to any one of claims 1 to 14, wherein, The amidase comprises the amino acid sequence of amino acid residues 2-398 of SEQ ID NO:1 or SEQ ID NO:

95.

16. The method according to any one of claims 1 to 14, wherein, The amidase comprises the amino acid sequence of amino acid residues 2-398 of SEQ ID NO:128 or 129.

17. The method according to any one of claims 1 to 12, wherein, The amidase comprises: the amino acid sequence of amino acid residues 2-394 of SEQ ID NO:87, or an amino acid sequence having at least 75% sequence identity with it.

18. The method according to claim 17, wherein, The amidase includes: a) The amino acid sequence of amino acid residues 2-392 of SEQ ID NO:3; b) An amino acid sequence having amino acid residues 2-394 of SEQ ID NO:62; or c) An amino acid sequence having amino acid residues 2-398 of SEQ ID NO:

47.

19. The method of claim 17, wherein, The amidase comprises the amino acid sequence of amino acid residues 2-394 of SEQ ID NO:

127.

20. The method of claim 12, wherein, The amidase comprises: an amino acid sequence of any one of SEQ ID NO:72-86 and 96-100, or an amino acid sequence having at least 70% sequence identity with it.

21. The method according to any one of claims 1 to 20, wherein, The monomer components are separated, and include ammonium carboxylate salts.

22. The method according to claim 21, wherein, The ammonium carboxylate salt is hexamethylenediamine adipic acid salt.

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