Recycling polyamides

Abstract

The present invention relates to a method for recycling polyamide, the method comprising: (i) subjecting the polyamide to acid hydrolysis in an aqueous liquid to produce oligomers of the polyamide that are soluble in the aqueous liquid; and (ii) using an amidase to convert the oligomers into their monomeric constituent component(s).

Description

RECYCLING POLYAMIDESFIELD OF THE INVENTION

[0001] The present invention relates to recycling polyamides.BACKGROUND OF THE INVENTION

[0002] The management of waste plastic products continues to be a significant problem for modem day society. Some modelling predicts as much as 90 million metric tonnes of plastic waste could end up being discarded annually into the environment by 2030 if current global trends of poor waste management continue.

[0003] It is now well documented that waste plastic is causing numerous deleterious effects to the environment.

[0004] The manufacture of polyamides has been steadily increasing since the development of nylon in the late 1930's. It is estimated that the global production of polyamides now exceeds about 6 million metric tons per year, with a large majority of that production being allocated to nylon 6 and nylon 6, 6. Other common commercial polyamides produced 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 that make them so highly desirable for use in, for example, textile, automotive and sportswear applications, unfortunately also renders those polymers with poor biodegradation properties. Accordingly, discarded polyamides will persist in the environment and remain a problem for many generations to come.

[0006] Most polyamide products are produced from virgin (i.e. non-recycled) resin. Some polyamides are now being produced using bio / plant-derived monomers (as opposed to petrochemical derived monomers). Irrespective of the source of monomers from which the polyamides are derived, a majority of the polyamides produced still ends up in the waste stream. Such poor recycling management is in part caused by the inefficiency of current polyamide recycling technologies.

[0007] Most waste polyamide products can in theory be repurposed into recycled products by melt processing (for example by melt extrusion).

[0008] While melt processed recycled polyamide products certainly have a number of useful applications, recycling polyamides in that way inherently promotes a reduction in the polymers physical and chemical properties. For example, the molecular weight of the polyamides progressively reduces the more times it undergoes melt processing resulting in a reduction in the polymers intrinsic viscosity (IV). The reduced IV of such recycled polyamide will limit its use in numerous applications. Using a feedstock of coloured waste polyamides inherently affords a coloured recycled product, which will also limit its use in numerous applications. Recycling polyamides by melt processing is also very energy intensive, which in turn reduces the economic viability of the technique.

[0009] So-called chemical recycling is an alternative pathway for recycling polyamides. Unlike the melt processing approach, chemical recycling aims to depolymerise the polyamide back into its monomeric constituent component(s). The specific monomers produced will vary depending on the type of polyamide being processed. The so formed monomer(s) can then be repurposed, for example by again being polymerised to produce virgin polyamide. That virgin polyamide will be colour / contaminant free and can be used once again to produce consumer products.

[0010] An advantage of chemical recycling is the resulting polyamide produced from the repurposed monomer(s) will for the most part be essentially indistinguishable from virgin polyamide produced from monomer(s) derived from conventional petrochemical or bio-based resources. Furthermore, chemical recycling can be undertaken indefinitely without compromising the chemical or physical properties of the polyamide produced from the repurposed monomer(s).

[0011] The chemical recycling approach therefore effectively uses waste polyamide as a feedstock for producing monomer(s) that are then polymerised to produce virgin polyamide.

[0012] Various chemical recycling technologies have been developed over the years. However, such processes typically make use of relatively hazardous chemicals and harsh reaction conditions. For example, common chemical recycling approaches are often performed under rather harsh and energy consuming conditions such as with high catalyst loadings and at high temperature and pressure.

[0013] Enzymatic depolymerisation of polymers is a growing area of both academic and industrial interest. In a similar manner to chemical recycling, the enzymatic recycling pathway breaks down the polymer into its monomeric constituent component(s). Enzymatic depolymerisation does not require the use of the relatively hazardous chemicals and harsh reactionconditions of its chemical depolymerisation counterpart.

[0014] However, there are only limited examples of enzymes suitable for depolymerising commercial polymers, many of which only demonstrate activity toward polyesters such as polyethylene terephthalate (PET). Furthermore, such enzymes typically exhibit relatively poor activity on the crystalline form of most polymer waste, including polyamide waste.

[0015] An opportunity therefore remains to develop new methodologies for recycling polyamides that address one or more of the problems associated with conventional recycling techniques, or at least provide a useful 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 oligomers of the polyamide that are soluble in the aqueous liquid; and (ii) using an amidase to convert the oligomers into their monomeric constituent component(s).

[0017] It has now been found that polyamides can be depolymerized to provide for their monomeric constituent component(s) using a combination of relatively mild chemical depolymerisation via acid hydrolysis and enzymatic depolymerisation. In particular, the polyamide is first partially depolymerised into oligomeric species under relatively mild chemical conditions, with the so formed oligomeric polyamide then being depolymerized into their monomeric constituent component(s). The combination of the mild chemical depolymerisation to produce oligomeric species and then the subsequent enzymatic depolymerisation of those oligomers has been found to be a highly efficient and an effective means of producing polyamide monomer feedstock.

[0018] The monomers produced in accordance with the method of the invention can then advantageously be used to produce virgin polyamide.

[0019] The method in accordance with the invention can advantageously be scaled up into industrial production and can operate batch wise or continuously.

[0020] In one embodiment, the acid hydrolysis is performed at a temperature of less than 200°C, for example at a temperature ranging from about 100°C to about 180°C.

[0021] In a further embodiment, the amidase is immobilised on a substrate, for example a resin.

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

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

[0024] In another embodiment, the amidase capable of hydrolysing an amide bond in aqueous soluble oligomers of the polyamide.

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

[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 that has at least 80% sequence identity thereto.

[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 that has at least 70% sequence identity thereto.

[0028] In another embodiment the amidase comprises an amino acid sequence of amino acid residues 2-398 of SEQ ID NO: 1 or SEQ IDNO:95. In another embodiment the amidase comprises an amino acid the amidase comprises an 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 that has at least 75% sequence identity thereto.

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

[0031] Further aspects and embodiments of the invention are discussed in more detail below.BRIEF DESCRIPTION OF DRAWINGS

[0032] The present invention will herein be described with reference to the following non-limiting drawings in which:

[0033] Figure 1A illustrates the product distribution of the nylon 6,6 oligomers under the conditions described in Example 1 and analysed by UPLC. Figure IB illustrates the analysis by HRMS of the different oligomers formed in the depolymerization reaction descried in Example 1.

[0034] Figure 2 illustrates the product distribution in the water after depolymerization using the conditions described in Example 2 Part A, analysed by UPLC.

[0035] Figure 3 illustrates whole-cell activity assays demonstrating the activity of variant polypeptides produced in Example 3, having amino acid sequence of SEQ ID NOs:2-15 and 87 in hydrolysing an amide bond in a polyamide, in comparison to a selection of extant / ancestral NylB polypeptides. A colorimetric assay was used to detect free amines in solution (absorbance at 335 nm). Increased concentration of free amine groups in the reaction solution corresponds to hydrolysis of Nylon-6, 6 trimer substrate to hexamethylenediamine and adipic acid.

[0036] Figure 4 illustrates an alignment of polypeptides of SEQ ID NOs:2, 4-15, demonstrating consensus SEQ ID NO: 1 (see Example 3).

[0037] Figure 5 illustrates activity of a selection of variant polypeptides generated from further engineering (Phase II). Purified candidate enzymes were analysed for activity against nylon 6,6, dimers, trimers and tetramers (produced in the nylon 6,6 acid hydrolysis of Example 3) using UHPLC (see Example 3). Y-axis values correspond to % normalised conversion of nylon 6,6 trimer in comparison to no-enzyme control. C4 corresponds to SEQ ID NO:89; D3 corresponds to a polypeptide comprising SEQ ID NO:90; E3 corresponds to a polypeptide comprising SEQ ID NO:94; F3 corresponds to a polypeptide comprising SEQ ID NO:91; F3 corresponds to apolypeptide comprising SEQ ID NO:91 and G3 corresponds to a polypeptide comprising SEQ ID NO:92).

[0038] Figure 6 illustrates UHPLC assay of Phase 1 candidates (SEQ ID NOs 4, 47, 87, 62 and 3) and Phase II (SEQ ID NOs: 88, 89, 90, 91, 92, 93) in comparison to extant sequences (SEQ ID NOs 74, 75, 78, 79, 80, 81, 82, 83, 85) on the nylon 6,6 oligomers (dimers (A); tetramers (B) and trimers (C)) from Example 3. Y-axis values correspond to nylon 6,6 oligomer peak area as measured using LC-MS.

[0039] Figure 7 illustrates an alignment of polypeptides of SEQ ID NOs:2, 4-15, 88, 90-94, with consensus II (SEQ ID NO:95) (see Example 3).

[0040] Figure 8 illustrates the activity of immobilised enzyme of SEQ ID NO:4 on the nylon 6,6 dimer, trimer and tetramer produced in the nylon 6,6 acid hydrolysis of Example 3, when incubated with the immobilised enzyme for (B) 1 hour at 40 °C (see Example 4). Conversion of the substrate was quantified by UHPLC and compared to a control (A) - no enzyme at time 0 hour.

[0041] Figure 9 illustrates the activity of SEQ ID NOs: 100-115 and 121-123 in comparison to extant sequence of SEQ ID NO: 75 on nylon 6,6, trimers. A no-enzyme control is included.

[0042] Figure 10 illustrates an alignment of SEQ ID NOs: 2, 4-15, 88-94, and 101-126 in generating consensus sequence of consensus IV (SEQ ID NO: 129).DETAILED DESCRIPTION OF THE INVENTION

[0043] The present invention provides a method for recycling polyamides. As used herein, the term "recycling" is intended to mean using in the method of the invention polyamide that is no longer considered to be of practical use. While such polyamide will often be in the form of post consumer waste, it may also be in the form of manufacturing waste or simply excess stock.

[0044] According to the method, the polyamide provides a source of monomer(s) that are constituent component(s) of the polyamide. Such monomers include diacid, diamine and amino acid compounds. Those compounds / monomers can be reused / recycled in various applications, for example in the manufacture of polyamides.

[0045] There is no particular limitation on the type of polyamide that may be used in accordance with the invention. The polyamide may have any molecular weight and have a crystalline or amorphous morphology.

[0046] Polyamides are a family of synthetic polymers characterised by repeating units linked by amide groups. Polyamides that are substantially aliphatic are more commonly known in the art as nylons.

[0047] Polyamides are typically made through the condensation polymerisation of a diacid and a diamine, self-condensation polymerisation of an amino acid (often an omega amino acid), or through ring opening polymerisation of a lactam form of an amino acid.

[0048] Common nylons 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.

[0049] As those skilled in the art will appreciate, the numbering system associated with nylons relates to the number of carbon atoms in the monomer(s) used to produce the nylon.

[0050] Where the designating number contains a single number (e.g. nylon 6 or nylon 10), the nylon is produced using a single monomer (i.e. it is a homopolymer). That monomer may be an amino acid that undergoes self-condensation polymerisation to produce the nylon, or a lactam of an amino acid that undergoes ring opening polymerisation to produce the nylon.

[0051] Where the designating number contains two numbers (e.g. nylon 6, 6 or nylon 10, 10), the nylon is produced using two monomers (i.e. it is a copolymer), namely a diacid and a diamine.

[0052] 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 produced by the condensation polymerisation of hexamethylenediamine (HMD) and adipic acid (AA).

[0053] In one embodiment, the polyamide comprises an aliphatic polyamide.

[0054] In another embodiment, the polyamide comprises an aliphatic copolymer polyamide.

[0055] In a further embodiment, the polyamide is of general formula (I):where x is an integer ranging from 2 to about 10, y is an integer ranging from 2 to about 10 and n is an integer ranging from about 50 to about 250.

[0056] In one embodiment, x in formula (I) is 2, 4, 8 or 10 and y is 2, 4, 8 or 10

[0057] In a further embodiment, the polyamide comprises an aliphatic homopolymer polyamide.

[0058] In another embodiment, the polyamide is of general formula (II):where x is an integer ranging from 2 to about 9 and n is an integer ranging from about 100 to about 800.

[0059] In one embodiment, x in formula (II) is 3, 8, or 9.

[0060] Those skilled in the art will appreciate the features of formula (I) and (II) located within the square brackets ([ ]) represent the repeat unit of the polyamide, which in the case of formula (I) is made up from the condensed residues of a diamine and a diacid and in the case of formula (I I) is made up of an amino acid. The polyamide or oligomer of the polyamide will of course be terminated with functionality reflective its monomeric constituent components. In the case of the subject polyamides, those end groups will be independently selected from an acid or amine group. In other words, the polyamide or oligomer of the polyamide will have either acid end groups, amine end groups or an acid end group and an amine end group.

[0061] In another embodiment, the polyamide comprises a mixture of aliphatic copolymer polyamide and aliphatic homopolymer polyamide.

[0062] 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.

[0063] There is no particular limitation on the physical form of polyamide that may be used in accordance with the invention. To facilitate process handling and / or the rate of acid hydrolysis, it may be desirable for the polyamide to be in a comminuted form, for example in the form of polyamide shreds, pellets, agglomerates, fibres, flake or powder.

[0064] Conventional techniques and equipment can advantageously be used in comminuting polyamide for use in accordance with the invention.

[0065] The method comprises subjecting the polyamide to acid hydrolysis in an aqueous liquid.That stage of the method will typically be performed in a reaction vessel.

[0066] The acid hydrolysis of polyamides is a known technique in the art and the present invention can advantageously use conventional equipment and reagents for that step of the method.

[0067] Suitable reaction vessels include those used in conventional polyamide chemical depolymerisation technologies. Such reaction vessels will typically be non-reactive to the acidic conditions employed and often made from glass, inconel, hastelloy or stainless steel.

[0068] The volume of the reaction vessel will be tailored to meet the scale of the intended operation, such as lab scale, pilot scale through to industrial scale. If required, the reaction vessel may comprise one or more agitation or stirring devices to assist with movement and mixing of reagents within the vessel.

[0069] The acid hydrolysis will of course be performed using an acid. There is no particular limitation on the type of acid that can be used.

[0070] In one embodiment, the acid hydrolysis is performed using a mineral acid.

[0071] Examples of suitable mineral acids include, but are not limited to, one or more of hydrochloric acid, nitric acid, phosphoric acid and sulphuric acid.

[0072] In one embodiment, the acid hydrolysis is performed using sulphuric acid.

[0073] In another embodiment, the acid hydrolysis is performed using an organic acid.

[0074] An example of a suitable organic acid include carboxylic acids, for example dicarboxylic acids such as adipic acid, sebacic acid, terephthalic acid and dodecandioic acid.

[0075] The acid hydrolysis will typically be performed at a pH in the range of about 0 to about 5, or about 0 to 3.

[0076] Provided the aqueous soluble oligomers are formed, there is no particular limitation on the amount of acid that can be used. Those skilled in the art can suitably select the amount of acid to use. For example, the acid may be used in an amount ranging from about 0.5 to about 10 equivalents on a molar basis relative to the polyamide.

[0077] The acid hydrolysis is undertaken or performed in an aqueous liquid. That aqueous liquidmay also be described as being an aqueous reaction medium within which the acid hydrolysis takes place. That aqueous liquid will of course be acidic.

[0078] The method may therefore be described as comprising subjecting the polyamide to acid hydrolysis in an acidic aqueous liquid to produce oligomers of the polyamide that are soluble in the acidic aqueous liquid.

[0079] If desired, the aqueous liquid may be stirred to facilitate the acid hydrolysis of the polyamide.

[0080] As those skilled in the art will appreciate, conventional polyamide chemical depolymerisation typically has as its end goal the depolymerisation of the polyamides back to its monomeric constituent component(s). That full conversion of the polyamides back to its monomeric constituent component(s) typically requires rather harsh and energy consuming conditions. The acid hydrolysis performed in accordance with the invention is not intended to proceed to an extent that depolymerises the polyamide into its monomeric constituent component(s). Rather, the acid hydrolysis need only proceed to an extent that produces oligomers of the polyamide that are soluble in the aqueous liquid. The method of the invention can therefore advantageously be performed under relatively mild reaction conditions and proceeds with high conversion.

[0081] In one embodiment, the acid hydrolysis is performed at a temperature of less than 200 °C, for example of less than about 190 °C, or less than about 180 °C, or less than about 170 °C, or less than about 160 °C, or less than about 150 °C.

[0082] In another embodiment, the acid hydrolysis is performed at a temperature of 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.

[0083] In a further embodiment, the acid hydrolysis is performed at a temperature ranging from about 75 °C to about 200 °C, or from about 75 °C to less than 200 °C, or from about 75 °C to about 180 °C, or from about 100 °C to about 180 °C, or from about 130 °C to about 175 °C.

[0084] Heat may be applied to the acid hydrolysis reaction using conventional means.

[0085] For example, the reaction component(s) may be combined in a reaction vessel and heatapplied to the reaction vessel to increase the temperature of the reaction component(s) to a desired temperature.

[0086] The acid hydrolysis can be performed at atmospheric pressure or pressures up to about 40 atm, or about 30 atm, or about 20 atm, or about 15 atm.

[0087] In one embodiment, the acid hydrolysis is performed at atmospheric pressure.

[0088] The acid hydrolysis is conducted for a period of time suitable to produce oligomers of the polyamide that are soluble in the aqueous liquid. The required time will of course depend on variables such as temperature, pressure, surface area of the polyamide and the ratio of the acid: polyamide.

[0089] The acid hydrolysis can advantageously be performed in a time efficient manner.

[0090] For example, the acid hydrolysis may be performed with high conversion over a period of time ranging from only about 30 mins to about 8 hour(s).

[0091] In accordance with the method of the invention the polyamide undergoes depolymerisation by acid hydrolysis to produce oligomers that are soluble in the aqueous liquid. For a given particle of polyamide in the reaction process, the acid hydrolysis is in practice complete when that particle of polyamide has undergone sufficient depolymerisation to dissolve in the aqueous liquid. When the method of the invention is performed batch wise, the acid catalysed hydrolysis will be complete when all of the polyamide present has in effect dissolved in the aqueous liquid. When the method of the invention is performed continuously, it will be appreciated that polyamide within the reaction system may be present at various stages of depolymerisation.

[0092] By the oligomers of the polyamide being soluble in the aqueous liquid is meant those oligomers are at least soluble in the aqueous liquid at the temperature at which the acid hydrolysis is undertaken.

[0093] In other words, the method comprises subjecting the polyamide to acid hydrolysis in an aqueous liquid to produce oligomers of the polyamide that are soluble in the aqueous liquid at least at a temperature at which the acid hydrolysis is performed.

[0094] For example, if the acid hydrolysis is performed at a temperature of about 150° C, then the oligomers of the polyamide produced will be soluble in the aqueous liquid at least at about150° C.

[0095] The oligomers of the polyamide that are soluble in the aqueous liquid may or may not be soluble in the aqueous liquid at room temperature.

[0096] In some embodiments, the oligomers of the polyamide produced are not soluble in the aqueous liquid at room temperature.

[0097] Provided the oligomers of the polyamide produced in accordance with the method invention are soluble in the aqueous liquid, there is no particular limitation on the number of monomer repeat units that make up the oligomers. The solubility of those oligomers relative to the number of monomer repeat units that make up the oligomers may vary depending upon the nature of the monomer repeat units per se.

[0098] In some embodiments, the oligomers of the polyamide that are soluble in the aqueous liquid comprise 1 to about 15, or about 1 to about 12, or about 1 to about 10 monomeric repeat units, or about 1 to about 8, or about 1 to about 6 monomeric repeat units.

[0099] In some embodiments, the oligomers of the polyamide that are soluble in the aqueous liquid are derived from polyamide homopolymer and comprise 1 to about 15, or about 1 to about 12, or about 1 to about 10 monomeric repeat units.

[0100] In some embodiments, the oligomers of the polyamide that are soluble in the aqueous liquid are derived from polyamide copolymer and comprise 1 to about 5, or about 1 to about 4, or about 1 to about 3 monomeric repeat units.

[0101] The acid hydrolysis of polyamide copolymers in accordance with the method of the invention may be represented by reaction scheme 1.Reaction scheme 1 : Acid hydrolysis of polyamide copolymer in accordance with the invention to provide for oligomers soluble in the aqueous reaction media; where x is an integer ranging from 2 to about 10, y is an integer ranging from 2 to about 10, n is an integer ranging from about 50 to about 250 and m is an integer ranging from 1 to about 5.

[0102] The acid hydrolysis of polyamide homopolymers in accordance with the method of the invention may be represented by reaction scheme 2.Reaction scheme 2: Acid hydrolysis of polyamide homopolymer in accordance with the invention to provide for oligomers soluble in the aqueous reaction media; where x is an integer ranging from 2 to about 9, n is an integer ranging from about 100 to about 800, and m is an integer ranging from 1 to about 15.

[0103] The oligomers of the polyamide produced in accordance with the method of the invention will be present in an acidic aqueous liquid as a result of the polyamide being subjected to acidhydrolysis.

[0104] In other words, the method comprises subjecting the polyamide to acid hydrolysis in an acidic aqueous liquid to produce oligomers of the polyamide that are soluble in the acidic aqueous liquid.

[0105] Before undertaking the next enzymatic stage of the method, the acidic aqueous liquid comprising the oligomers that are soluble in the aqueous liquid may be neutralised with base.

[0106] In one embodiment, before using the amidase to convert the oligomers into their monomeric constituent component(s), the aqueous liquid comprising the oligomers of the polyamide is neutralised with base.

[0107] In another embodiment, the acid hydrolysis produces an acidic aqueous liquid comprising the oligomers of the polyamide that is neutralised with base before using the amidase to convert the oligomers into their monomeric constituent component(s).

[0100] In a further embodiment, the aqueous liquid comprising the oligomers of the polyamide is neutralised with base to a pH of at least about 7 or 8, or ranging from about 7 or about 8 to about 12.

[0101] Examples of a base for that neutralisation include, but are not limited to, an alkali metal base, an alkaline earth metal base and an organic amine base.

[0102] Examples of suitable alkali metal bases include, but are not limited to, alkali metal hydroxides.

[0103] Examples of suitable alkali metal hydroxides include, but are not limited to, lithium hydroxide, sodium hydroxide and potassium hydroxide.

[0104] Examples of suitable alkaline earth metal bases include, but are not limited to, alkaline earth metal hydroxides.

[0105] Examples of suitable alkaline earth metal hydroxides include, but are not limited to, magnesium hydroxide and calcium hydroxide.

[0106] 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.

[0107] As a variation on performing such a neutralisation step before undertaking the enzymatic stage of the method, the aqueous liquid comprising the oligomers of the polyamide that are soluble in the aqueous liquid may be cooled (e.g. from a temperature at which acid hydrolysis took place) to promote the precipitation of organic acid, for example dicarboxylic acids such as those described herein, that may be present in the aqueous liquid. The origin of any precipitated organic acid may be as a hydrolysis product of the polyamide and / or organic acid used to promote the hydrolysis. The precipitated organic acid may then be separated from the aqueous liquid which contains soluble oligomers, with the aqueous liquid then being neutralised as required with base as described herein.

[0108] In one embodiment, before using the amidase to convert the oligomers into their monomeric constituent component(s), the aqueous liquid comprising the oligomers of the polyamide that are soluble in the aqueous liquid is reduced in temperature to promote precipitation of organic acid, for example dicarboxylic acids such as those described herein, in the aqueous liquid.

[0109] In a further embodiment, the precipitated organic acid is separated from the aqueous liquid.

[0110] In another embodiment, the oligomers of the polyamide that are soluble in the aqueous liquid are neutralised with base at a pH of at least about 7 or about 8, or ranging from about 7 or about 8 to about 12.

[0111] In a further embodiment, the oligomers of the polyamide that are soluble in the aqueous liquid are neutralized by the addition of base, for example an organic amine base as described herein, before undertaking the enzymatic stage of the method.

[0112] As a further variation on performing such a neutralisation step before undertaking the enzymatic stage of the method, the aqueous liquid comprising the oligomers of the polyamide that are soluble in the aqueous liquid may be cooled (e.g. from a temperature at which acid hydrolysis took place) to promote precipitation of at least some of the oligomers in the aqueous liquid. For example, depending on the nature of the feedstock polyamide, oligomers of the polyamide having 6 repeat units or more may be precipitated and oligomers of the polyamide having less than 6 repeat units can remain solubilised in the aqueous liquid. The precipitated oligomers may then be separated from the aqueous liquid. If desired, those separated oligomers may then be neutralised as described herein and optionally subjected to a second acid hydrolysis as described herein forprocessing in accordance with the invention. The oligomers that remain solubilised in the aqueous liquid may then be neutralised as described herein and further processed in accordance with the invention.

[0113] In one embodiment, before using the amidase to convert the oligomers into their monomeric constituent component(s), the aqueous liquid comprising the oligomers of the polyamide that are soluble in the aqueous liquid is reduced in temperature to promote precipitation of at least some of the oligomers in the aqueous liquid.

[0114] In a further embodiment, the precipitated oligomers are separated from the aqueous liquid to provide a further aqueous liquid comprising oligomers of the polyamide that are soluble in the aqueous liquid.

[0115] In another embodiment, the further aqueous liquid comprising oligomers of the polyamide that are soluble in the aqueous liquid is neutralised with base as described herein.

[0116] In another embodiment, the amidase is then used to convert the oligomers from the further aqueous liquid into their monomeric constituent component(s).

[0117] In a further embodiment, the separated precipitated oligomers are neutralised with base.

[0118] In another embodiment, the oligomers are neutralised with base to a pH of at least about 7 or about 8, or ranging from about 7 or about 8 to about 12.

[0119] Examples of a base for neutralisation are as herein described.

[0120] In a further embodiment, the method includes before using the amidase to convert the oligomers into their monomeric constituent component(s), steps of (i) reducing the temperature of the aqueous liquid comprising the oligomers of the polyamide that are soluble in the aqueous liquid to promote precipitation of the oligomers in the aqueous liquid, (ii) separating the precipitated oligomers from the aqueous liquid and reusing the separated aqueous liquid in the method to undertake acid catalysed hydrolysis of more polyamide, and (iii) neutralising the separated oligomers with base.

[0121] Once the aqueous liquid comprising the oligomers or the separated oligomers have been neutralised with base to form an alkaline aqueous liquid, the oligomers become soluble in the alkaline aqueous liquid. The oligomers can advantageously soluble in that base neutralised liquid at room temperature. The pH of that base neutralised liquid will generally be neutral or alkaline,for example in the range of about 7 or about 8 to about 12.

[0122] In one embodiment, before using the amidase to convert the oligomers into their monomeric constituent component(s), the oligomers of the polyamide that are soluble in the aqueous liquid are provided in a neutral or alkaline aqueous liquid, wherein the oligomers are soluble in the neutral or alkaline aqueous liquid

[0123] Depending upon the feedstock of polyamide used in accordance with the invention, the neutral or alkaline aqueous liquid comprising the oligomers may contain some colour contamination. If desired, the neutral or alkaline aqueous liquid may be passed through activated charcoal to assist with removing any colour contamination

[0124] Upon producing the oligomers of the polyamide, the method according to the invention comprises using an amidase to convert the oligomers into their monomeric constituent component(s)

[0125] As those skilled in the art will appreciate, the monomeric constituent component(s) of the oligomers are essentially the same monomeric constituent component(s) of the polyamide from which the oligomers are derived. The form and composition of the monomeric constituent component(s) will vary depending upon the type of polyamide used in the method of the invention. For example, if nylon 6 is used in the method of the invention, the monomeric constituent component will be 6-6-aminocaproic acid. If nylon 6, 6 is used in the method of the invention, the monomeric constituent component(s) will be HMD and AA. If a mixture of nylon 6 and nylon 6, 6 is used in the method of the invention, the monomeric constituent component(s) will be HMD, AA and 6-aminocaproic acid. The so formed monomeric constituent component(s) may also be in the form of an ammonium carboxylate salt (e.g. hexamethylenediamine adipate also known as hexamethylene diammonium adipate).

[0126] As those skilled in the art will appreciate, nylon 6 and other homopolymer polyamides may be produced by ring opening polymerisation of a lactam of a given amino acid. The repeat monomeric unit of those polyamides is of course reflective of the amino acid and not the corresponding lactam per se. Accordingly, in the context of homopolymer polyamides it is entirely appropriate to refer to the amino acid as the monomeric constituent component even though the polyamide may have been produced using a lactam of the amino acid

[0127] The amidase depolymerisation of oligomers derived from polyamides in accordance withthe invention has advantageously been found to provide for a highly efficient and effective path to producing amino acids, diacids, diamines and ammonium carboxylate salts (e.g. hexamethylenediamine adipate also known as hexamethylene diammonium adipate).

[0128] Collectively, the combined chemical and enzymatic methodology adopted in accordance with the invention has been found to provide for a scalable and economically viable means for recycling polyamide

[0129] The amidase depolymerisation of oligomers derived from polyamide homopolymers in accordance with the method of the invention may be represented by reaction scheme 3.Reaction scheme 3: Amidase depolymerisation of oligomers derived from polyamide homopolymer in accordance with the invention to provide for amino acids that can, if desired, be subsequently cyclised into their lactam; where x is an integer ranging from 2 to about 9 and m is an integer ranging from 1 to about 15.

[0130] As shown in reaction scheme 3, the amidase depolymerisation of oligomers derived from polyamide homopolymers affords an amino acid. That amino acid represents the monomeric constituent component of the polyamide homopolymer. The amino acid may be subsequently used in a given application in its native form or, as shown in reaction scheme 3, subjected to a cyclisation reaction to afford the corresponding lactam. Methodology for converting such amino acids into the corresponding lactam are known in the art. For example, by treating the solution of the amino acid (e.g. 6-aminocaproic acid) with steam at elevated temperatures in the presence of a catalyst. The so formed lactam may then be used in a given application.

[0131] In one embodiment, amino acid produced by the method is converted into a lactam.

[0132] The amidase depolymerisation of oligomers derived from polyamide copolymers in accordance with the method of the invention may be represented by reaction scheme 4.Reaction scheme 4: Amidase depolymerisation of oligomers derived from polyamide copolymer in accordance with the invention to provide for diacids and diamines; where x is an integer ranging from 2 to about 10, y is an integer ranging from 2 to about 10, and m is an integer ranging from 1 to about 5.

[0133] The enzymatic depolymerisation step of the method may be performed using techniques and equipment known to those skilled in the art.

[0134] Generally, the oligomers derived from the polyamide will be provided in combination with a solvent / reaction media and the amidase in a reaction vessel and incubated for a suitable period of time for undertaking conversion of the oligomers into their monomeric constituent component(s).

[0135] In one embodiment, the oligomers derived from the polyamide are provided in combination with the amidase and a suitable liquid in a reaction vessel and incubated for a suitable period of time for undertaking conversion of the oligomers into their monomeric constituent component(s).

[0136] Examples of suitable liquids that may be used include, but are not limited to, aqueous liquids, including aqueous buffers.

[0137] In one embodiment, the oligomers derived from the polyamide are provided in a neutralor alkaline aqueous liquid in combination with the amidase for converting the oligomers into their monomeric constituent component(s). That aqueous liquid may have a pH ranging from about 7 or about 8 to about 12.

[0138] The time taken for conversion of the oligomers into their monomeric constituent component(s) using the amidase will vary depending upon at least temperature and the ratio of enzyme to oligomers. Generally, conversion of the oligomers into their monomeric constituent component(s) using the amidase will require an incubation period ranging from about 5 mins to about 24 hours. Generally, the incubation period may range from about 5 minutes to about 20 hours, from about 5 minutes to about 18 hours, from about 5 minutes to about 16 hours, from about 5 minutes to about 14 hours, from about 5 minutes to about 12 hours, from about 5 minutes to about 10 hours, from about 5 minutes to about 8 hours, from about 5 minutes to about 6 hours. In further examples, the incubation period may be from about 5 minutes to about 5 hours, 5 minutes to about 4 hours, from about 5 minutes to about 3 hours, from about 5 minutes to about 2 hours, and from about 5 minutes to about 1 hour.

[0139] Those skilled in the art will be able to select a suitable incubation temperature for use with the particular amidase. Generally, incubation may be performed at a temperature ranging from about 0°C to about 80 °C, for example from about 2°C to about 75 °C, from about 4°C to about 70°C, from about 6°C to about 70°C; from about 8°C to about 65°C , from about 10°C to about 65°C, from about 20°C to about 60°C, from about 25°C to about 55°C, from about 30°C to about 55 °C, from about 30°C to about 50°C, from about 35°C to about 50 °C, or from about 40°C to about 50°C

[0140] The amidase used in accordance with invention may be a polypeptide capable of hydrolysing an amide bond in a polyamide. In one embodiment, the polyamide is a substantially aliphatic polyamide commonly known in the art as nylons. In one embodiment, the polyamide is a homopolymer polyamide. In one embodiment, the polyamide is a copolymer polyamide.

[0141] The term "amidase" typically refers to hydrolase enzymes, illustrative examples of which include hydrolase enzymes classified as EC 3.5.1 or 3.5.2 according to Enzyme Nomenclature which catalyses the hydrolysis of an amide by acting on carbon-nitrogen bonds, other than peptide bonds

[0142] Amidases capable of hydrolysing an amide bond in a polyamide nylon were first discovered in a strain of bacteria that can digest certain by-products of nylon manufacture(Kinoshita et al., 1975 and Kinoshita et al., 1977).

[0143] In one embodiment, amidases capable of hydrolysing an amide bond in a polyamide include• 6-aminohexanoate -cyclic -dimer hydrolases / NylA; e.g. UniProt Accession No: P13398.2 (SEQ ID NO: 96) and P13397.2 (SEQ ID NO: 97);• 6-aminohexanoate-dimer hydrolases / NylB; e.g. UniProt Accession Nos: P07061 (SEQ ID NO: 72) and P07062.1 (SEQ ID NO: 86);• 6-aminohexanoate-oligomer endohydrolases / NylC; e.g. UniProt Accession Nos: Q79F77.1 (SEQ ID NO: 98); Q1EPR5.2 (SEQ ID NO: 99) and Q1EPR4.1 (SEQ ID NO: 100).

[0144] In an embodiment, the oligomers of the polyamide are soluble in an aqueous liquid. Further, it is desirable that the amidase is a polypeptide that is capable of converting the oligomers into their monomeric constituent component(s).

[0145] As disclosed elsewhere herein, the oligomers of the polyamide that are soluble in the aqueous liquid comprise from 1 to about 15, or from about 1 to about 12, or from about 1 to about 10 monomeric repeat units.

[0146] In an embodiment, the oligomers of the polyamide that are soluble in the aqueous liquid are derived from polyamide homopolymer and comprise from 1 to about 15, or from about 1 to about 12, or from about 1 to about 10 monomeric repeat units. In some embodiments, the oligomers of the polyamide that are soluble in the aqueous liquid are derived from polyamide copolymer and comprise from 1 to about 5, or from about 1 to about 4, or from about 1 to about 3 monomeric repeat units.

[0147] In one embodiment, the amidase is a polypeptide that is capable of hydrolysing an amide bond in a nylon 6 oligomer. In another embodiment, the amidase is a polypeptide that is capable of hydrolysing an amide bond in a nylon 6,6 oligomer. In some embodiments, the amidase is a polypeptide that is capable of hydrolysing an amide bond in a nylon 6 oligomer and an amide bond in a nylon 6, 6 oligomer. In some embodiments, one or more amidases may be used in accordance with the invention.

[0148] In an embodiment, the amidase comprises: a) an amino acid sequence of amino acid residues 2-398 of SEQ ID NO: 1 or SEQ ID NO:95;b) an amino acid sequence of amino acid residues 2-398 of SEQ ID NO: 2 or an amino acid sequence that has at least 70% sequence identity thereto; c) an amino acid sequence of amino acid residues 2-394 of SEQ ID NO:87 or an amino acid sequence that has at least 75% sequence identity thereto, d) an amino acid sequence of amino acid residues 2-391 of SEQ ID NO:44 or an amino acid sequence that has at least 61% sequence identity thereto; or e) an amino acid sequence of any one of SEQ ID NOs: 72-86 and 96-100 or an amino acid sequence that has at least 70% sequence identity thereto.

[0149] As used herein, the term "sequence identity" or "identity" refers to the number (or fraction expressed as a percentage %) of matches (identical amino acid residues) between two polypeptide sequences. In a preferred embodiment, the sequence identity is determined by comparing the sequences when aligned so as to maximize overlap and identity while minimizing sequence gaps. Sequence identity may be determined using any of a number of mathematical global or local alignment algorithms known to persons skilled in the art, depending on the length of the two sequences. Sequences of similar lengths may be aligned using a global alignment algorithms (e.g., Needleman and Wunsch algorithm; Needleman and Wunsch, 1970), which aligns the sequences optimally over the entire length, while sequences of substantially different lengths are preferably aligned using a local alignment algorithm (e.g., Smith and Waterman algorithm (Smith and Waterman, 1981) or Altschul algorithm (Altschul et al., 1997; Altschul et al., 2005)). Alignment for the purposes of determining percent amino acid sequence identity can be achieved by any means available to persons skilled in the art, illustrative examples of which include publicly available computer software, such as is available ator Persons skilled in the art can readily determine appropriateparameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. As used herein, % sequence identity typically refers to values generated using pair wise sequence alignment that creates an optimal global alignment of two sequences (e.g., using the Needleman-Wunsch algorithm), where all search parameters are set to default values, e.g., Scoring matrix = BLOSUM62, Gap open = 10, Gap extend = 0.5, End gap penalty = false, End gap open = 10 and End gap extend = 0.5.In an embodiment, the amidase the amidase is a polypeptide that is capable of hydrolysing an amide bond in a nylon 6,6 oligomer. In one embodiment, the amidase comprises an amino acid sequence of SEQ ID NO:95 or an amino acid sequence having at least 80% sequence identity thereto.SEQ ID NO: 95:MTXITX2LMQGX3PPAPEQX4VTLANWRX5X6PFX7RWX8FHHVREX9XIOPTAXHIPRGPGX12X13X14PLPXl5Xl6PRDLX17Xl8IAX19EGX20DGX21X22X23TVX24EMLX25EX26YTDX27FLVX28HRGRIVX29EX3OYANGMTPHX3IPHIX32FSVSKSITGX33LAGILVX34RGQLDPDAPVTX35YIPEX36X37GSAYGDATVRHVLDMTVX38IDFX39EDYLDPDGDFARYRX4oAX4iGWNPX42X43DGX44TPSDLRSFLX45TLX46X47X48DGX49HGETFHYX5oSPNSDLLGWIX5iERASGQRFAX52LLSEX53lWX54PMGAEX55DAYITVDRLGAPRTAGGX56CATX57RDLARFGX58MMX59NRGVANGRQX6OVPX6IX62WIDDIX63X64X65GDX66EAWARGDFAX67X68X69PX7OGRYRSKWYVTGNARGAFCX7 1IGIHGQWIYX72DPAAEVVIX73KX74S SQPX75PVDDAMDRLX76LAAFX77AIARALX78X79(where X1-X79 is any amino acid)

[0150] 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 that has at least 80% sequence identity thereto comprises an amino acid sequence wherein i. the amino acid at position 3 is 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 Q or E; v. the amino acid at position 25 is Q or T; vi. the amino acid at position 26 is A or P; vii. the amino acid at position 29 is N or S; viii. the amino acid at position 32 is 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; xi. the amino acid at position 44 is N or Q; xii. the amino acid at position 51 is A or P; xiii. the amino acid at position 52 is A or V; xiv. the amino acid at position 53 is S or W; xv. the amino acid at position 57 is A or R; xvi. the amino acid at position 58 is A or S; xvii. the amino acid at position 63 is 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 xx. the amino acid at position 70 is P or deleted; 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 A or G; xxv. the amino acid at position 82 is A or E; xxvi. the amino acid at position 84 is S or T; xxvii. the amino acid at position 88 is A or G; xxviii. the amino acid at position 92 is L or M; xxix. the amino acid at position 99 is A or S; xxx. the amino acid at position 101 is W or H; xxxi. the amino acid at position 110 is 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 D or E; xxxv. the amino acid at position 143 is H, R or D; xxxvi. the amino acid at position 148 is A or V; xxxvii. the amino acid at position 149 is A or K; xxxviii.the amino acid at position 167 is S or G; xxxix. the amino acid at position 171 is E or D; xl. the amino acid at position 186 is R, E or Q; xli. the amino acid at position 188 is T or M; xlii. the amino acid at position 193 is A or P; xliii. the amino acid at position 194 is S or P; xliv. the amino acid at position 197 is A or E; xlv. the amino acid at position 207 is 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 deleted; xlviii. the amino acid at position 212 is S, D or G; xlix. the amino acid at position 215 is P or E;1. the amino acid at position 223 is A or V; li. the amino acid at position 234 is L or I;lii. the amino acid at position 244 is D or E; liii. the amino acid at position 249 is H or R; liv. the amino acid at position 252 is Q or R;Iv. the amino acid at position 258 is H or A;Ivi. the amino acid at position 276 is L or I;Ivii. the amino acid at position 280 is L or A;Iviii. the amino acid at position 288 is Q or E; lix. the amino acid at position 291 is L or R; lx. the amino acid at position 301 is I or V;Ixi. the amino acid at position 304 is E, A or G;Ixii. the amino acid at position 305 is A or W;Ixiii. the amino acid at position 311 is L or R;Ixiv. the amino acid at position 312 is Q or T;Ixv. the amino acid at position 313 is N or G;Ixvi. the amino acid at position 316 is R, K or P;Ixvii. the amino acid at position 326 is K or H;Ixviii. the amino acid at position 327 is F or L;Ixix. the amino acid at position 328 is F or L;Ixx. the amino acid at position 330 is N or G;Ixxi. the amino acid at position 349 is A or G;Ixxii. the amino acid at position 359 is I or V;Ixxiii. the amino acid at position 368 is A or V;Ixxiv. the amino acid at position 370 is L or F;Ixxv. the amino acid at position 375 is L or E;Ixxvi. the amino acid at position 385 is C, M or N;Ixxvii. the amino acid at position 390 is D, E or R;Ixxviii. the amino acid at position 397 is G or A; and / orIxxix. the amino acid at position 398 is G or deleted, wherein the numbering is relative to the amino acid positions of SEQ ID NO: 1 or SEQ ID NO:95.

[0151] In an embodiment, the amidase comprises an amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 80% sequence identity thereto.SEQ ID NO: 1:MTX1TX2LMQGFPPAPEQQVTLANWRX3X4PFNRWX5FHHVREIX6PTANIPRGPGX7X8X9 PLPX10X11PRDLX12X13IAFEGX14DGX15X16X17TVX18EMLX19EX20YTDX21FLVX22HRGRIV X23EX24YANGMTPHX25PHIX26FSVSKSITGX27LAGILVX28RGQLDPDAPVTX29YIPEX30X3 1GSAYGDATVRHVLDMTVX32IDFX33EDYLDPDGDFARYRX34ATGWNPX35X36DGX37TPSDLRSFLX38TLX39X40X41DGX42HGETFHYX43SPNSDLLGWIX44ERASGQRFAX45LLSEHI WQPMGAEHDAYITVDRLGAPRTAGGX46CATX47RDLARFGX48MMX49NRGVANGRQX5 0VPX51X52WIDDIX53X54X55GDX56EAWARGDFAKFX57PX58GRYRSKWYVTGNARGAFCX59lGIHGQWIYIDPAAEWIX6oKX6iSSQPX62PVDDAMDRLX63LAAFX64AIARALX65G(where Xi-XA is any amino acid)

[0152] In one embodiment, the amidase comprises an amino acid sequence having amino acid residues 2-398 of SEQ ID NO:95, amino acid residues 2-398 of SEQ ID NO: 1; or an amino acid sequence that has at least 80% sequence identity thereto, comprises an amino acid sequence wherein i. the amino acid at position 3 is Q or T; ii. the amino acid at position 5 is N or D; iii. the amino acid at position 25 is Q or T; iv. the amino acid at position 26 is A or P; v. the amino acid at position 32 is A or S; vi. the amino acid at position 40 is I or V; vii. the amino acid at position 51 is A or P; viii. the amino acid at position 52 is A or V; ix. the amino acid at position 53 is S or W; x. the amino acid at position 57 is A or R; xi. the amino acid at position 58 is A or S; xii. the amino acid at position 63 is 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 deleted; xv. the amino acid at position 73 is R or K; xvi. the amino acid at position 74 is S or E; xvii. the amino acid at position 75 is T, M or W; xviii. the amino acid at position 78 is A or G; xix. the amino acid at position 82 is A or E; xx. the amino acid at position 84 is S or T;xxi. the amino acid at position 88 is A or G; xxii. the amino acid at position 92 is L or M; xxiii. the amino acid at position 99 is A or S; xxiv. the amino acid at position 101 is W or H; xxv. the amino acid at position 110 is S or T; xxvi. the amino acid at position 114 is L or V; xxvii. the amino acid at position 124 is I or T; xxviii. the amino acid at position 131 is D or E; xxix. the amino acid at position 143 is H, R or D; xxx. the amino acid at position 148 is A or V; xxxi. the amino acid at position 149 is A or K; xxxii. the amino acid at position 167 is S or G; xxxiii. the amino acid at position 171 is E or D; xxxiv. the amino acid at position 186 is R, E or Q; xxxv. the amino acid at position 193 is A or P; xxxvi. the amino acid at position 194 is S or P; xxxvii. the amino acid at position 197 is A or E; xxxviii. the amino acid at position 207 is A or V; xxxix. the amino acid at position 210 is R or K; xl. the amino acid at position 211 is R, K or deleted; xli. the amino acid at position 212 is S, D or G; xlii. the amino acid at position 215 is P or E; xliii. the amino acid at position 223 is A or V; xliv. the amino acid at position 234 is L or I; xlv. the amino acid at position 244 is 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 Q or E; xlix. the amino acid at position 291 is L or R;1. the amino acid at position 301 is I or V; li. the amino acid at position 304 is E, A or G; lii. the amino acid at position 305 is A or W; liii. the amino acid at position 311 is L or R; liv. the amino acid at position 312 is Q or T;Iv. the amino acid at position 313 is N or G;Ivi. the amino acid at position 316 is R, K or P;Ivii. the amino acid at position 328 is F or L;Iviii. the amino acid at position 330 is N or G; lix. the amino acid at position 349 is A or G; lx. the amino acid at position 368 is A or V;Ixi. the amino acid at position 370 is L or F;Ixii. the amino acid at position 375 is L or E;Ixiii. the amino acid at position 385 is C or N;Ixiv. the amino acid at position 390 is D, E or R; and / orIxv. the amino acid at position 397 is G or A, wherein the numbering is relative to the amino acid positions of SEQ ID NO: 1 or SEQ ID NO:95.

[0153] By "at least 80%" is meant that the amidase shares 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 to SEQ ID NO: 1 or SEQ ID NO:95.

[0154] In an embodiment, the amidase is a polypeptide capable of hydrolysing an 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 that has at least 70% sequence identity thereto. In an embodiment, the amidase comprises at least one amino acid modification at a position selected from the group consisting of 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, with numbering relative to SEQ ID NO: 95 or SEQ ID NO: 1. In another embodiment, the 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 of T84; A88 or G88; L92 or M92; A99 or S99; W101 or H101; SI 10 or T110; L114 or V114; 1124 or T124; D131 of E131; H143, D143 or R143; A148 or V148; A149 or K149; S167 of 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 21 Idel; S212, G212 or D212; P215 or E215; A223 or V223; L234 or 1234; D244or 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, with numbering relative to SEQ ID NO: 95 or SEQ ID NO: 1.

[0155] In an embodiment, the amidase is a polypeptide capable of hydrolysing an 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 that has at least 70% sequence identity thereto. In an embodiment, the amidase comprises at least one amino acid modification at a position selected from the group consisting of 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, with numbering relative toSEQ ID NO: 129. In another embodiment, the 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; S84 of T84; 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; D131 of E131; 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 21 Idel; 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 orA379; C385, L384 or E384; N385 or M385; D390, E390 or R390; R394 or A394; G397 or A397 and G398 or 398del, with numbering relative to SEQ ID NO: 95 or SEQ ID NO: 129.

[0156] The amidase used in accordance with the invention is a polypeptide capable of hydrolysing an amide bond in a polyamide, having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:2. There is also provided a polypeptide capable of hydrolysing an amide bond in a polyamide, having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:2. There is also provided a polypeptide capable of hydrolysing an amide bond in a polyamide, having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:2. There is also provided a polypeptide capable of hydrolysing an amide bond in a polyamide, having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:2.

[0157] In one embodiment, the amidase comprises the amino acid sequence having amino acid residues 2-397 of SEQ ID NOT; 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 NOTO; 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; the amino acid sequence having amino acid residues 2-398 of SEQ ID NO: 14; the amino acid sequence having amino acid residues 2-398 of SEQ ID NO: 88; the amino acid sequence having amino acid residues 2-398 of SEQ ID NO:6; the amino acid sequence having amino acid residues 2-398 of SEQ ID NO: 89; the amino acid sequence having amino acid residues 2-398 of SEQ ID NO:9; the amino acid sequence having amino acid residues 2-398 of SEQ ID NO:7; the amino acid sequence having amino acid residues 2-398 of SEQ ID NO: 11 or the amino acid sequence having amino acid residues 2-398 of SEQ ID NO: 13.

[0158] In another embodiment; the polypeptide consists of the amino acid sequence of SEQ ID NOT; SEQ ID NO: 15; SEQ ID NO:5; SEQ ID NOTO; 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 NOT; SEQ ID NO: 11; or SEQ ID NO: 13.

[0159] The polyamide may be a polypeptide capable of hydrolysing an amide bond in a polyamide, wherein the amidase comprises an amino acid sequence of amino acid residues 2-394 of SEQ ID NO:87 or an amino acid sequence that has more than 75% sequence identity thereto. In one embodiment, the polypeptide at comprises at least 80% sequence identity to SEQ ID NO:87. In one embodiment, the polypeptide at comprises at least 85% sequence identity to SEQ ID NO: 87. In one embodiment, the polypeptide at comprises at least 90% sequence identity to SEQ ID NO: 87. In one embodiment, the polypeptide at comprises at least 95% sequence identity to SEQ ID NO: 87.

[0160] In an embodiment, the amidase comprises the amino acid sequence having amino acid residues 2-392 of SEQ ID NO:3; the amino acid sequence having amino acid residues 2-394 of SEQ ID NO:62; or the amino acid sequence having amino acid residues 2-398 of SEQ ID NO:47. In further embodiments, the polypeptide consists of the amino acid sequence of SEQ ID NO:3, SEQ ID NO:62 or SEQ ID NO:47. In an embodiment, the amidase comprises the amino acid sequence having amino acid residues 2-394 of SEQ ID NO: 127. In an embodiment, the amidase consists of the amino acid sequence having amino acid residues of SEQ ID NO: 127.

[0161] The amidase used in accordance with the invention may be a polypeptide capable of hydrolysing an amide bond in a polyamide, wherein the amidase comprises an amino acid sequence of amino acid residues 2-391 of SEQ ID NO:44 or an amino acid sequence that has at least 61% sequence identity thereto. In one embodiment, the polypeptide at comprises at least 70% sequence identity to SEQ ID NO:44. In one embodiment, the polypeptide at comprises at least 75% sequence identity to SEQ ID NO:44. In one embodiment, the polypeptide at comprises at least 80% sequence identity to SEQ ID NO:44. In one embodiment, the polypeptide at comprises at least 85% sequence identity to SEQ ID NO: 44 In one embodiment, the polypeptide at comprises at least 90% sequence identity to SEQ ID NO:44. In one embodiment, the polypeptide at comprises at least 95% sequence identity to SEQ ID NO:44.

[0162] In one embodiment, the polypeptide at comprises the amino acid sequence having amino acid residues 2-391 of SEQ ID NO:24 or the 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 one embodiment, the polypeptide consists of the amino acid sequence of SEQ ID NO: 24, SEQ ID NO: 62 or SEQ ID NO:21.

[0163] In one embodiment, the amidase is a polypeptide capable of hydrolysing an amide bondin a polyamide, wherein the amidase comprises an amino acid sequence of amino acid residues 2-392 of SEQ ID NO:73

[0164] The amidase used in accordance with the invention may be a polypeptide that is capable of hydrolysing an amide bond in a nylon 6 oligomer.

[0165] In one embodiment, the amidase may be a polypeptide an amino acid sequence of any one of SEQ ID NOs: 72-86 and 96-100 or an amino acid sequence that has at least 70% sequence identity thereto.

[0166] In one embodiment, the amidase is a polypeptide capable of hydrolysing an amide bond in a polyamide, wherein the amidase comprises an 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 hydrolysing an amide bond in a polyamide, wherein the amidase comprises an amino acid sequence of amino acid residues 2-392 of SEQ ID NO:73. In one embodiment, the amidase is a polypeptide capable of hydrolysing an amide bond in a polyamide, wherein the amidase comprises an amino acid sequence of amino acid residues 2-392 of SEQ ID NO: 75. In one embodiment, the amidase is a polypeptide capable of hydrolysing an amide bond in a polyamide, wherein the amidase comprises an amino acid sequence of amino acid residues 2-392 of SEQ ID NO:76. In one embodiment, the amidase is a polypeptide capable of hydrolysing an amide bond in a polyamide, wherein the amidase comprises an amino acid sequence of amino acid residues 2-391 of SEQ ID NO:77. In one embodiment, the amidase is a polypeptide capable of hydrolysing an amide bond in a polyamide, wherein the amidase comprises an amino acid sequence of amino acid residues 2-389 of SEQ ID NO:78. In one embodiment, the amidase is a polypeptide capable of hydrolysing an amide bond in a polyamide, wherein the amidase comprises an amino acid sequence of amino acid residues 2-394 of SEQ ID NO:79. In one embodiment, the amidase is a polypeptide capable of hydrolysing an amide bond in a polyamide, wherein the amidase comprises an amino acid sequence of amino acid residues 2-401 of SEQ ID NO: 80. In one embodiment, the amidase is a polypeptide capable of hydrolysing an amide bond in a polyamide, wherein the amidase comprises an amino acid sequence of amino acid residues 2-406 of SEQ ID NO:81. In one embodiment, the amidase is a polypeptide capable of hydrolysing an amide bond in a polyamide, wherein the amidase comprises an amino acid sequence of amino acid residues 2-407 of SEQ ID NO:82. In one embodiment, the amidase is a polypeptide capable of hydrolysing an amide bond in a polyamide, wherein the amidase comprises an amino acid sequence of amino acid residues 2-401 of SEQ ID NO:83. In one embodiment, theamidase is a polypeptide capable of hydrolysing an amide bond in a polyamide, wherein the amidase comprises an amino acid sequence of amino acid residues 2-391 of SEQ ID NO: 84. In one embodiment, the amidase is a polypeptide capable of hydrolysing an amide bond in a polyamide, wherein the amidase comprises an amino acid sequence of amino acid residues 2-391 of SEQ ID NO: 85. In one embodiment, the amidase is a polypeptide capable of hydrolysing an amide bond in a polyamide, wherein the amidase comprises an amino acid sequence of amino acid residues 2-392 of SEQ ID NO:86. In one embodiment, the amidase is a polypeptide capable of hydrolysing an amide bond in a polyamide, wherein the amidase comprises an amino acid sequence of amino acid residues 2-391 of SEQ ID NO:84. In one embodiment, the amidase is a polypeptide capable of hydrolysing an amide bond in a polyamide, wherein the amidase comprises an amino acid sequence of amino acid residues 2-493 of SEQ ID NO:96. In one embodiment, the amidase is a polypeptide capable of hydrolysing an amide bond in a polyamide, wherein the amidase comprises an amino acid sequence of amino acid residues 2-492 of SEQ ID NO:97. In one embodiment, the amidase is a polypeptide capable of hydrolysing an amide bond in a polyamide, wherein the amidase comprises an amino acid sequence of amino acid residues 2-355 of SEQ ID NO: 98. In one embodiment, the amidase is a polypeptide capable of hydrolysing an amide bond in a polyamide, wherein the amidase comprises an amino acid sequence of amino acid residues 2-355 of SEQ ID NO:99. In one embodiment, the amidase is a polypeptide capable of hydrolysing an amide bond in a polyamide, wherein the amidase comprises an amino acid sequence of amino acid residues 2-355 of SEQ ID NO: 100.

[0167] In one embodiment, the amidase is a polypeptide capable of hydrolysing an amide bond in a polyamide, wherein the polypeptide consists of an amino acid sequence of SEQ ID NO:72 or SEQ ID NO:74. In one embodiment, the amidase is a polypeptide capable of hydrolysing an amide bond in a polyamide, wherein the polypeptide consists of an amino acid sequence SEQ ID NO:73. In one embodiment, the amidase is a polypeptide capable of hydrolysing an amide bond in a polyamide, wherein the polypeptide consists of an amino acid sequence of SEQ ID NO:75. In one embodiment, the amidase is a polypeptide capable of hydrolysing an amide bond in a polyamide, wherein the polypeptide consists of an amino acid sequence of SEQ ID NO:76. In one embodiment, the amidase is a polypeptide capable of hydrolysing an amide bond in a polyamide, wherein the polypeptide consists of an amino acid sequence of SEQ ID NO:77. In one embodiment, the amidase is a polypeptide capable of hydrolysing an amide bond in a polyamide, wherein the polypeptide consists of an amino acid sequence of SEQ ID NO: 78. In one embodiment, the amidase is a polypeptide capable of hydrolysing an amide bond in a polyamide,wherein the polypeptide consists of an amino acid sequence of SEQ ID NO:79. In one embodiment, the amidase is a polypeptide capable of hydrolysing an amide bond in a polyamide, wherein the polypeptide consists of an amino acid sequence of SEQ ID NO: 80. In one embodiment, the amidase is a polypeptide capable of hydrolysing an amide bond in a polyamide, wherein the polypeptide consists of an amino acid sequence of SEQ ID NO:81. In one embodiment, the amidase is a polypeptide capable of hydrolysing an amide bond in a polyamide, wherein the polypeptide consists of an amino acid sequence of SEQ ID NO: 82. In one embodiment, the amidase is a polypeptide capable of hydrolysing an amide bond in a polyamide, wherein the polypeptide consists of an amino acid sequence of SEQ ID NO: 83. In one embodiment, the amidase is a polypeptide capable of hydrolysing an amide bond in a polyamide, wherein the polypeptide consists of an amino acid sequence of amino acid residues 2-391 of SEQ ID NO: 84. In one embodiment, the amidase is a polypeptide capable of hydrolysing an amide bond in a polyamide, wherein the polypeptide consists of an amino acid sequence of SEQ ID NO:85. In one embodiment, the amidase is a polypeptide capable of hydrolysing an amide bond in a polyamide, wherein the polypeptide consists of an amino acid sequence of SEQ ID NO: 86. In one embodiment, the amidase is a polypeptide capable of hydrolysing an amide bond in a polyamide, wherein the polypeptide consists of an amino acid sequence of amino acid residues 2- 391 of SEQ ID NO: 84. In one embodiment, the amidase is a polypeptide capable of hydrolysing an amide bond in a polyamide, wherein the polypeptide consists of an amino acid sequence of SEQ ID NO:96. In one embodiment, the amidase is a polypeptide capable of hydrolysing an amide bond in a polyamide, wherein the polypeptide consists of an amino acid sequence of SEQ ID NO:97. In one embodiment, the amidase is a polypeptide capable of hydrolysing an amide bond in a polyamide, wherein the polypeptide consists of an amino acid sequence of SEQ ID NO:98

[0168] In one embodiment, the amidase is a polypeptide capable of hydrolysing an amide bond in a polyamide, wherein the polypeptide consists of an amino acid sequence of SEQ ID NO:99. In one embodiment, the amidase is a polypeptide capable of hydrolysing an amide bond in a polyamide, wherein the polypeptide consists of an amino acid sequence of SEQ ID NO: 100.

[0169] In some embodiments, the amidase is a polypeptide that is capable of hydrolysing an amide bond in a nylon 6 oligomer and an amide bond in a nylon 6, 6 oligomer. In an embodiment, the amidase is a polypeptide capable of hydrolysing an amide bond in a polyamide, wherein the amidase comprises an amino acid sequence of amino acid residues 2-392 of SEQ ID NO:73. In an embodiment, the amidase is a polypeptide capable of hydrolysing an amide bond in a polyamide, wherein the polypeptide consists of an amino acid sequence of SEQ ID NO:73

[0170] Illustrative examples of amidases that can be used in accordance with the invention is provided I Table 1.

[0171] In one embodiment, the polypeptides disclosed herein are capable of hydrolysing a polyamide oligomer. In another embodiment, the polypeptides disclosed herein has adipic acid mono- and di-N-alkyl amide hydrolase activity.Table 1: Amino acid sequences of amidase enzymes

[0172] As noted elsewhere herein, the amidase, including variants of extant amidases, will suitably retain at least some amidase, adipic acid mono- and / or di-N-alkyl amide hydrolase activity , irrespective of any modifications made, including to its amino acid sequence. Suitable methods of determining or measuring amidase activity will be familiar to persons skilled in the art, including methods that are described herein. Illustrative examples of which 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 which are incorporated herein by reference in their entirety. In an embodiment, the amidase activity is determined by UV absorbance assay to monitor the amount of monomeric constituent component(s) produced from using polyamide oligomers as substrate. Another method useful for determining or measuring the amidase / hydrolase activity is by measuring the amount of monomeric constituent component(s) produced using LC-MS. The amidase activity of the polyppetide may be assigned an absolute value or a value relative to the amidase activity of a comparator (e.g., an extant amidase). In an embodiment, the amidase activity is measured as the rate of monomers and / or oligomers (e.g., in mg or mol) released per hour and per mg or mol of enzyme under suitable conditions of temperature, pH and buffer.

[0173] Those skilled in the art will be able to select a suitable incubation temperature for use with the particular amidase. Generally, incubation may be performed at a temperature ranging from about 0°C to about 80 °C, for example from about 2°C to about 75 °C, from about 4°C to about 70°C, from about 6°C to about 70°C; from about 8°C to about 65°C , from about 10°C to about 65°C, from about 20°C to about 60°C, from about 25°C to about 55°C, from about 30°C to about 55 °C, from about 30°C to about 50°C, from about 35°C to about 50 °C, or from about 40°C to about 50°C

[0174] In an embodiment, the amidase exhibits measurable amidase activity at least in a range of pH from about 5 to about 11, or in a range of pH from about 6 to about 10, or in a range of pH from about 7 to about 10, or in a range of pH from about 7.5 to about 9.5, or in a range of pH from about 7.5 to about 8

[0175] The amount of amidase used will of course depend upon the amount of the one or both of nylon polyamide substrate or nylon oligomer to be converted into their monomeric constituent component(s)). Generally, the amount of amidase used will range from 1: 1000 to about 1: 10 (enzyme mass to nylon substrate)

[0176] Once the enzymatic conversion into monomeric constituent component(s) is complete, it may be desirable to subject the composition comprising the products of enzymatic conversion to one or more purification procedures to increase the purity of the monomeric constituent component(s), as described elsewhere herein.

[0177] The purity of the monomeric constituent component(s) produced may be increased using techniques well-known to those skilled in the art. For example, the composition comprising monomeric constituent component(s) produced may be subjected to one or more techniques selected from washing, solvent extraction, filtration, distillation, solvent evaporation, column chromatography and crystallisation.

[0178] In one embodiment, the composition comprising the monomeric constituent component(s) is subjected to one or more techniques to increase the purity of the so formed monomeric constituent component(s).

[0179] In another embodiment, the one or more techniques used to increase the purity of the so formed monomeric constituent component(s) are selected from washing, solvent extraction, filtration, distillation, solvent evaporation, column chromatography and crystallisation.

[0180] In a further embodiment, the monomeric constituent component(s) produced by enzymatic conversion of the polyamide oligomers is purified and isolated.

[0181] It may be convenient to perform the method of the invention using the amidase enzymes that is immobilised on a substrate. Using immobilised amidase may be beneficial when performing the method of the invention in a semicontinuous or continuous manner.

[0182] In one embodiment, the amidase is immobilised on a substrate.

[0183] The amidase can be immobilised on any suitable substrate using techniques known to those skilled in the art. For example, the amidase may be immobilised on a support resin by ion exchange, adsorption (e.g. hydrophobic adsorption), or covalent coupling.

[0184] In one embodiment, the amidase is immobilised on a support resin.

[0185] In one embodiment, the amidase is immobilised on an ion exchange resin. In another embodiment, the amidase is immobilised on an adsorption resin. In another embodiment the amidase is immobilised on a nickel-affmity resin. In an embodiment the amidase is immobilised on a covalent resin.

[0186] Those skilled in the art will be familiar with the general principle of enzymatic immobilisation technology and that principle can advantageously be applied in the context of immobilising the amidase on a substrate in accordance with the present invention.

[0187] Suitable ion-exchange resins for immobilising the amidase will generally comprise a polymer matrix or a polymer / ceramic hybrid matrix. An example of such a resin includes, but is not limited to, CM Ceramic HyperD® Ion Exchange Chromatography Resin.

[0188] In one embodiment, the ion exchange resin is a cationic exchange resin.

[0189] The method in accordance with the invention can advantageously be performed in a batch wise, semicontinuous or continuous manner.

[0190] For continuous operation of the method in accordance with the invention, the amidase will typically be immobilised on a support resin and loaded into a column. The composition of soluble polyamide oligomers produced in step (i) might then be continuously passed through the column so as to promote conversion of oligomers into their monomeric constituent component(s) using the immobilised amidase.

[0191] Prior to being passed through the column, the composition of soluble polyamide oligomers produced in step (i) might first be diluted in a solvent such as one herein described and be combined with one or more additives for adjusting / stabilising pH and / or ion content. Such additives might include, for example, pH buffer and / or an ionic compound such as NaCl.

[0192] Step (ii) in accordance with the method of the invention produces a composition comprising the monomeric constituent component(s). The specific monomers produced will vary depending on the type of polyamide being processed. As those skilled in the art will appreciate, the monomeric constituent component(s) of the oligomers are essentially the same monomeric constituent component(s) of the polyamide from which the oligomers are derived. For example, if nylon 6 is used in the method of the invention, the monomeric constituent component will be 6-6-aminocaproic acid. If nylon 6, 6 is used in the method of the invention, the monomeric constituent component(s) will be HMD and AA. If a mixture of nylon 6 and nylon 6, 6 is used in the method of the invention, the monomeric constituent component(s) will be HMD, AA and 6- aminocaproic acid.

[0193] The method of the invention can readily produce a variety of amino acids through the amidase conversion of oligomers derived from polyamide homopolymer and / or a variety ofdiacids and diamines through the amidase conversion of oligomers derived from polyamide copolymer.

[0194] Once the enzymatic conversion is complete, the method provides for a liquid comprising the so formed monomeric constituent component(s).

[0195] The method may therefore be described as using one or more amidase to convert the oligomers into their monomeric constituent component(s) to provide for a liquid comprising the monomeric constituent component(s).

[0196] It may be desirable to subject the resulting liquid to one or more purification procedures to increase the purity of the so formed monomeric constituent component(s) and assist with their separation and isolation.

[0197] The purity of the monomeric constituent component(s) may be increased and / or the monomeric constituent component(s) separated and isolated using techniques well-known to those skilled in the art. For example, the liquid comprising monomeric constituent component(s) may be subjected to one or more techniques selected from washing, solvent extraction, filtration, distillation, solvent evaporation, column chromatography, crystallisation, stripping process, separation by aqueous solution, steam selective condensation, filtration and concentration of the medium after the bioprocess, separation, vacuum evaporation, extraction, electrodialysis, adsorption, ion exchange, precipitation, concentration and acid addition dehydration and precipitation, nanofiltration, acid catalyst treatment, semi continuous mode distillation or continuous mode distillation, solvent extraction, evaporative concentration, evaporative crystallization, liquid / liquid extraction, hydrogenation, azeotropic distillation process, acid or heat catalysed lactamisation, adsorption, simple vacuum distillation and microfiltration, combined or not.

[0198] In one embodiment, the so formed monomeric constituent component(s) are subjected to one or more purifying techniques.

[0199] In a further embodiment, the so formed monomeric constituent component(s) are purified and isolated.

[0200] In one embodiment, use of the amidase provides for a liquid comprising of the monomeric constituent component(s) and the monomeric constituent component(s) are purified by adjusting the pH of the liquid comprising the monomeric constituent component(s).

[0201] In one embodiment, the pH of the liquid comprising the monomeric constituent component(s) is rendered acidic (for example to a pH of about 1 to about 4) to assist with purifying and isolating a diacid monomeric constituent component.

[0202] In another embodiment, the pH of the liquid comprising the monomeric constituent component(s) is rendered alkaline (for example to a pH of about 8 to about 13) to assist with purifying and isolating a diamine monomeric constituent component.

[0203] In one embodiment of the pH of the liquid comprising the monomeric constituent component(s) is first rendered acidic to assist with purifying and isolating a diacid monomeric constituent component, and then the resulting liquid is rendered alkaline to assist with purifying and isolating a diamine monomeric constituent component.

[0204] Suitable reagents for rendering the liquid acidic include the acids described herein.

[0205] Suitable reagents for rendering the liquid alkaline include the alkali metal bases and alkaline earth metal bases described herein.

[0206] In another embodiment, the pH of the liquid comprising the monomeric constituent component(s) is rendered neutral to assist with purifying and isolating an ammonium carboxylate salt, for example a salt comprising a diamine and diacid such as hexamethylene diammonium adipate.

[0207] Suitable reagents for rendering the liquid comprising the monomeric constituents neutral include the acid and bases described herein, for example organic amine bases and organic acids. In one embodiment the base used is an organic diamine base, for example hexamethylene diamine. In another embodiment the acid used is an organic acid, for example a dicarboxylic acid such as adipic acid.

[0208] The recovery of the ammonium carboxylate salt may involve a concentration step using techniques well-known to those skilled in the art comprising, but not limited to, membrane distillation, reverse osmosis or solvent evaporation.

[0209] The purification of an ammonium carboxylate salt may be achieved using techniques well- known to those skilled in the art comprising, but not limited to, precipitation, activated carbon treatment, or recrystallization.

[0210] Production of the monomeric constituent component(s) can be confirmed using techniqueswell-known to those skilled in the art. For example, the formation of amino acids, diacids, diamines and ammonium carboxylate salts can be readily determined using NMR spectroscopy.EXAMPLESExample 1: Acid hydrolysis of PA66 with different mineral acids

[0211] In a round bottomed flask, 10 g PA66, 7.2 mL of H2SO4, 3.7 mL of water was heated to reflux for 3 h. The mixture was cooled to room temperature, neutralised with NaOH, fdtered, and the remaining solid was dried under vacuum and weighed to give a percentage of conversion to PA66 oligomers. The soluble oligomers were analysed by UPLC and the results are shown in Figure 1A. The analysis by mass spectrometry of each oligomer is illustrated in Figure IB.

[0212] In a round bottomed flask, 10 g PA66, 9.4 mL of 37% aqueous hydrochloric acid was heated to reflux for 3 h. The mixture was cooled to room temperature, neutralised with NaOH, fdtered, and the remaining solid was dried under vacuum and weighed to give a percentage of conversion to PA66 oligomers.

[0213] In a round bottomed flask, 10 g PA66, 9.1 mL of 85% aqueous H3PO4, 5.0 mL of water was heated to reflux for 3 h. The mixture was cooled to room temperature, neutralised with NaOH fdtered, and the remaining solid was dried under vacuum and weighed to give a percentage of conversion to PA66 oligomers.Sulfuric acid = 100% conversionHydrochloric acid = 100% conversionPhosphoric acid = 100% conversionExample 2: Acid hydrolysis of PA66 using adipic acidPart A

[0214] In a 2 L pressure reactor, 250 g of PA66 was suspended in 500 mL of water, followed by the addition of 116 g of adipic acid. The reactor was sealed and pressurize with 6 bars of nitrogen. The reaction mixture was then heated to 195 C under 20 bars of pressure for 2h. The reaction mixture was cooled to 100 C and the pressure was released through the vent valve of the reactor. Any insoluble adipic acid was fdtered, the fdtrate containing oligomers, hexamethylenediammonium adipate and adipic acid was filtered through am activated carbon filter. UPLC analyses indicated the formation of adipic acid, dimers, trimers, tetramers, pentamers and hexamers (Figure 2).Part B

[0215] In a 10 L pressure reactor, 1 kg of PA66 was suspended in 3 L of water, followed by the addition of 125 g of adipic acid. The reaction mixture was then heated to 180 C for 6h. The reaction mixture was cooled to 100 C and the pressure was released. Any insoluble adipic acid and long chain oligomers were filtered, the filtrate containing oligomers, hexamethylene diammonium adipate and adipic acid was used as a feedstock for enzymatic hydrolysis. UPLC analyses indicated the formation of adipic acid, dimers, trimers, tetramers, pentamers and hexamers, analysis of the solids suggests longer chain oligomers: heptamers and higher molecular weight oligomers, similar to that observed in Figure 2 derived from Example 2 Part A.Example 3: Engineered polypeptides for the hydrolysis of nylon oligomers

[0216] Variant enzyme sequences designed by ancestral sequence reconstruction using NylB sequences, P07061 from Flavobacterium sp. (strain K172), P07062 from Flavobacterium sp. (strain KI 72) and related extant sequences were synthesised and tested for the ability to hydrolyse amide bonds in polyamides. Amino acid sequences are provided in Table 1.

[0217] A number of variant polypeptides (SEQ ID NOs: 2-15 and 87) were surprisingly found to have increased amidase activity in hydrolysing amide bonds in nylon 6,6 polyamide oligomers when compared to most extant sequences (Figure 3).

[0218] At least one extant sequence (SEQ ID NO: 73; corresponding to 1WYC_A, Chain A, 6- aminohexanoate-dimer hydrolase of Flavobacterium sp.) was able to increased activity in hydrolysing amide bonds in nylon 6,6 polyamide oligomers when compared to most extant sequences and no the enzyme control.

[0219] Sequence analysis indicated that many of the engineered polypeptides that had increased ability to hydrolyse amide bonds in nylon 6,6 oligomers showed high sequence identity that is represented by the consensus sequence of SEQ ID NO: 1 (see Figure 4):MTX1TX2LMQGFPPAPEQQVTLANWRX3X4PFNRWX5FHHVREIX6PTANIPRGPGX7X8X9 PLPXioXiiPRDLXi2Xi3lAFEGXi4DGXi5Xi6Xi7TVXi8EMLXi9EX2oYTDX2iFLVX22HRGRIVX23EX24YANGMTPHX25PHIX26FSVSKSITGX27LAGILVX28RGQLDPDAPVTX29YIPEX30X3 1GSAYGDATVRHVLDMTVX32IDFX33EDYLDPDGDFARYRX34ATGWNPX35X36DGX37TP SDLRSFLX38TLX39X40X41DGX42HGETFHYX43SPNSDLLGWIX44ERASGQRFAX45LLSEHI WQPMGAEHDAYITVDRLGAPRTAGGX46CATX47RDLARFGX48MMX49NRGVANGRQX5 0VPX51X52WIDDIX53X54X55GDX56EAWARGDFAKFX57PX58GRYRSKWYVTGNARGAFC X59lGIHGQWIYIDPAAEWIX6oKX6iSSQPX62PVDDAMDRLX63LAAFX64AIARALX65G(where Xi-XA is any amino acid)

[0220] Some of these sequences were selected as a base for further engineering. This second round of engineering identified a number of new sequences with improved enzyme activity on nylon 6,6 oligomers (see Figure 5 where C4 corresponds to SEQ ID NO:89; D3 corresponds to a polypeptide comprising SEQ ID NO:90; E3 corresponds to a polypeptide comprising SEQ ID NO:94; F3 corresponds to a polypeptide comprising SEQ ID NO:91; F3 corresponds to a polypeptide comprising SEQ ID NO:91 and G3 corresponds to a polypeptide comprising SEQ ID NO:92).

[0221] The activity of a selection of these engineered polypeptides from Phase 1 and Phase II is shown in Figure 6.

[0222] Analysis of these sequences having improved activity in hydrolysing amide bonds in nylon 6,6 polyamide oligomers indicated many they shared a broad consensus sequence SEQ ID NO:95 (Figure 7). Table 2 provides the amino acid substitutions at the specified positions of the variant polypeptides, wherein the numbering is relative to the amino acid positions of SEQ ID NO: 1 or SEQ ID NO: 95.SEQ ID NO: 95MTXITX2LMQGX3PPAPEQX4VTLANWRX5X6PFX7RWX8FHHVREX9XIOPTAXIIIPRGPG X12X13X14PLPX15X16PRDLX17X18IAX19EGX20DGX21X22X23TVX24EMLX25EX26YTDX27FLV X28HRGRIVX29EX30YANGMTPHX31PHIX32FSVSKSITGX33LAGILVX34RGQLDPDAPVTX 35YIPEX36X37GSAYGDATVRHVLDMTVX38IDFX39EDYLDPDGDFARYRX40AX41GWNPX 42X43DGX44TPSDLRSFLX45TLX46X47X48DGX49HGETFHYX50SPNSDLLGWIX51ERASGQR FAX52LLSEX53IWX54PMGAEX55DAYITVDRLGAPRTAGGX56CATX57RDLARFGX58MMX 59NRGVANGRQX6OVPX6IX62WIDDIX63X64X65GDX66EAWARGDFAX67X68X69PX7OGRYRSKWYVTGNARGAFCX71IGIHGQWIYX72DPAAEVVIX73KX74S SQPX75PVDDAMDRLX76LAAFX77AIARALX78X79(where X1-X79 is any amino acid)Table 2: Amino acid substitutions across variant polypeptidesExample 4: Acid hydrolysis of nylon 6,6 oligomers by immobilized enzyme

[0223] An engineered polypeptide was immobilised on an adsorption-based polymeric resin at an enzyme loading of 10% w / w. The immobilised enzyme was incubated with nylon 6,6 dimer, trimer and tetramer produced in the nylon 6,6 acid hydrolysis of Example 3. The reaction solution was sampled after 1 hour for analysis by UHPLC and was compared to a control reaction containing no enzyme (time 0 hr). The data are shown in Figure 8.Example 7: Acid hydrolysis of PA6 with H2SO4

[0224] In a round botomed flask, 10 g PA6, 3.6 mL of H2SO4, 3.7 mL of water was heated to reflux for 3 h. The mixture was cooled to room temperature, neutralised with NaOH, filtered, andthe remaining solid was dried under vacuum and weighed to give a percentage of conversion to PA6 oligomers. 100% conversion was achieved. UHPLC indicated the presence of dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer and nonomeric units of 6-aminohexanoate.Example 8: Isolation of adipic acid and hexamethylenediamine

[0225] The aqueous solution from the enzyme reactor (from Example 4) is cooled to 0 °C and acidified to pH 1 using 50% aqueous H2SO4. After 30 mins, the resulting suspension was filtered to isolate AA as a white, crystalline solid. The filtrate was basified to pH 13 using Ca(OH)2 and filtered to give a solution of HMD in water which is further purified using vacuum distillation to give colourless HMD which solidified on standing.Example 9: Further enzyme engineering

[0226] Further rounds of enzyme engineering identified variant polypeptides that demonstrated activity in hydrolysing amide bonds in a polyamide, including nylon 6,6, oligomers. Assays of enzymatic activity against nylon 6,6 oligomers (dimer, trimer and tetramer) were conducted at 40 °C for 2 hours. Reactions were stopped by removing the enzyme using 10 kDa MWCO centrifugal filters (15 min, 13,000 x g). The reactions were analysed using ultra high performance liquid chromatography (UHPLC).

[0227] Polypeptide variants comprising amino acid sequences of any one of SEQ ID NOs: 101- 127 were capable of hydrolysing nylon 6,6 oligomers. The activity of some of these polypeptides in hydrolysing nylon 6,6 trimers is demonstrated in Figure 9.

[0228] A significant proportion of polypeptides capable of hydrolysing nylon 6,6 oligomers shared a consensus sequence of SEQ ID NO: 128 / consensus III.SEQ ID NO: 128MTQTNLXIQX2X3PPAX4EX5EVTLANWRQAPFSRWSFHHVRELVPTAQIPRX6PG PASPLPAAPX7X8LGEIALEGPDX9KEGTVAEMLEESYTDXIOFLVLHRGRIVAEHY ANGMTPHX11PHIVFSVSKSITX12TLAGILVEX13GQLDPX14APVTDYIPEVAGSAY GX15ATVRHVLDMTVX16IDFEEX17YLDPX18X19X2OFARYRRAMGWNPPSX21GET PSDLX22SFX23ATLKKGX24GPHGETFHYX25SPNSDLLGWILERASGQRFADLLSE RIWRPMGAEADAYX26TVDRLGAPRTAGGICX27TX28RDLARFGEMMRNRGVAN GRQIVPEAWIX29DILTNGDX30EAWAX31GDFAHX32LPNGRYRX33KWYVTGNARGAFCAIGIHGQWIYX34DPAAEVVIVKLSSQPLPVDX35AMDRX36MLAAFRAIAX37 ALG(where X1-X37 is any amino acid)

[0229] Analysis of the polypeptides disclosed herein as capable of hydrolysing amide bonds in a polyamide showed significant sequence conservation that can be represented by consensus sequence IV of SEQ ID NO: 129 (Figure 10). This includes SEQ ID NOs: 2, 4-15, 88-94, and 101-126.

[0230] Table 3 shows the amino acid substitutions at the specified positions of the variant polypeptides, where the amino acid numbering is relative to the amino acid positions of SEQ ID NO: 1, SEQ ID NO:95 or SEQ ID NO: 129.Table 3: Amino acid substitutions across variant polypeptides

[0231] The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

[0232] Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

Claims

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS1. A method for recycling polyamide, the method comprising:(i) subjecting the polyamide to acid hydrolysis in an aqueous liquid to produce oligomers of the polyamide that are soluble in the aqueous liquid; and(ii) using an amidase to convert the oligomers into their monomeric constituent component(s) .

2. The method according to claim 1, wherein the polyamide comprises an aliphatic copolymer polyamide.

3. The method according to claim 1 or 2, wherein the polyamide comprises an aliphatic homopolymer polyamide.

4. The method according to claim 1, wherein the polyamide comprises 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 performed using one or more mineral acids selected from hydrochloric acid, nitric acid, phosphoric acid and sulphuric acid.

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

7. The method according to any one of claims 1 to 6, wherein the acid hydrolysis is performed at a temperature of less than 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 oligomers of the polyamide that is neutralised with base before using the amidase to convert the oligomers into their monomeric constituent component(s).

9. The method according to any one of claims 1 to 7, wherein before using the amidase to convert the oligomers into their monomeric constituent component(s), the method comprises the steps of (i) reducing the temperature of the aqueous liquid comprising the oligomers of the polyamide that are soluble in the aqueous liquid to promote precipitation of the oligomers in theaqueous liquid, and (ii) neutralising the separated oligomers with base.

10. The method according to any one of claims 1 to 7, wherein before using the amidase to convert the oligomers into their monomeric constituent component(s), the method comprises the steps of (i) reducing the temperature of the aqueous liquid comprising the oligomers of the polyamide that are soluble in the aqueous liquid to promote precipitation of any organic acid in the aqueous liquid, (ii) separating any precipitated organic acid from the aqueous liquid to provide a purified aqueous liquid comprising the oligomers of the polyamide that are soluble in the aqueous liquid, and (iii) neutralising the purified aqueous liquid with base.

11. The method according to any one of claims 1 to 10, wherein the amidase is immobilised on a support resin.

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

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

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

15. The method according to any one of claims 1 to 14, wherein the amidase comprises an 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 an 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 an amino acid sequence of amino acid residues 2-394 of SEQ ID NO:87 or an amino acid sequence that has at least 75% sequence identity thereto.

18. The method according to claim 17, wherein the amidase comprises a) an amino acid sequence of amino acid residues 2-392 of SEQ ID NO:3; b) the amino acid sequence having amino acid residues 2-394 of SEQ ID NO:62; or c) the amino acid sequence having amino acid residues 2-398 of SEQ ID NO:47.

19. The method according to claim 17, wherein the amidase comprises an amino acid sequence of amino acid residues 2-394 of SEQ ID NO: 127.

20. The method according to claim 12, wherein the amidase comprises an amino acid sequence of any one of SEQ ID NOs: 72-86 and 96-100 or an amino acid sequence that has at least 70% sequence identity thereto.

21. The method according to any one of claims 1 to 20, wherein the monomeric constituent component(s) is isolated and comprises an ammonium carboxylate salt.

22. The method according to claim 21, wherein the ammonium carboxylate salt is hexamethylenediamine adipate.

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