Novel urethanases for the enzymatic degradation of polyurethanes

Abstract

The invention relates to novel urethanases for the enzymatic degradation of polyurethanes and to an enzymatic process for the complete degradation of polyurethanes in defined monomers.

Description

[0001] New urethanases for the enzymatic degradation of polyurethanes

[0002] The present invention relates to novel urethanases for the enzymatic degradation of polyurethanes and to an enzymatic process for the complete degradation of polyurethanes into defined monomers.

[0003] Polyurethanes are established in many areas of everyday life. They can be found, for example, in flexible foams (mattresses, sponges, upholstered furniture), rigid foams (insulation materials, building materials), thermoplastics (athletic shoes), and coatings (varnishes, paints, adhesives). Due to the constantly rising demand for these products, increasingly large quantities are being produced. At the same time, there is a growing need for methods for the most sustainable recycling of no longer needed polyurethane products, allowing the reuse of the polymer building blocks. To achieve this, the bonds in the polyurethanes must be specifically cleaved to obtain defined degradation products, thus making them recyclable.

[0004] In addition to the physiological functions that enzymes perform in living organisms, they can be used in a variety of ways to catalyze chemical reactions outside of this context. Compared to conventional chemical processes, the reactions can be carried out under milder conditions such as lower temperatures, neutral pH, and without the use of aggressive chemicals. This saves energy, minimizes the formation of byproducts, and protects the environment, which contributes to reducing operating costs. The use of enzymes makes it possible, in some cases, to convert labile reactants (Jaeger, K.-E. & Reetz, MT (1998) Microbial lipases form versatile tools for biotechnology. Trends in biotechnology, 16, 396-403).Furthermore, enzymes are often chemo-, regio-, stereo- and enantioselective, which significantly facilitates the purification of the products and can thus enable the efficient synthesis of difficult-to-access products (Hasan, F., Shah, AA & Hameed, A. (2006) Industrial applications of microbial lipases. Enzyme and Microbial Technology, 39, 235-251).

[0005] Today, polyurethanes are rarely recycled. Disposal occurs via landfilling or, alternatively, complete incineration, which generates energy but does not allow for efficient reuse of the polymer building blocks.

[0006] It is known that polyurethanes can be degraded to a certain extent by bacteria and fungi. Polyester polyurethanes are significantly more susceptible to microbial or enzymatic degradation than polyether polyurethanes (Nakajima-Kambe, T., Shigeno-Akutsu, Y., Nomura, N., Onuma, F. & Nakahara, T. (1999) Microbial degradation of polyurethane, polyester polyurethanes and polyether polyurethanes. Applied microbiology and biotechnology, 51, 134-140). The degradation of polyester polyurethanes is readily achieved via hydrolysis of the ester bonds. The relatively simple degradation of polyesters is not surprising, since ester bonds in hydrophobic substrates must also be cleaved during the degradation of lipids in nature and even polyesters without urethane bonds can be degraded relatively easily by esterases and lipases (Marten, E., Müller, R.-J. & Deckwer, W.-D. (2003) Studies on the enzymatic hydrolysis of polyesters I.Low molecular mass model esters and aliphatic polyesters. Polymer degradation and stability, 80, 485-501; Marten, E., Müller, R.-J. & Deckwer, W.-D. (2005) Studies on the enzymatic hydrolysis of polyesters. II. Aliphatic-aromatic copolyesters. Polymer degradation and stability, 88, 371-381.). In verschiedenen Literaturstellen wurden zum Abbau von Polyurethan verwendete Enzyme als Esterasen charakterisiert (Allen, A. B., Hilliard, N. P. & Howard, G. T. (1999) Purification and characterization of a soluble polyurethane degrading enzyme from Comamonas acidovorans. International biodeterioration & biodegradation, 43, 37-41; Blake, R., Norton, W. & Howard, G. (1998) Adherence and growth of a Bacillus species on an insoluble polyester polyurethane. International biodeterioration & biodegradation, 42, 63-73; Crabbe, J. R., Campbell, J. R., Thompson, L., Walz, S. L. & Schultz, W. W. (1994) Biodegradation of a colloidal ester-based polyurethane by soil fungi.International biodeterioration & biodegradation, 33, 103-113; Darby, R. T. & Kaplan, A. M. (1968) Fungal susceptibility of polyurethanes. Applied microbiology, 16, 900-905; Howard, G. T., Norton, W. N. & Burks, T. (2012) Growth of Acinetobacter gerneri P7 on polyurethane and the purification and characterization of a polyurethanase enzyme. Biodegradation, 23, 561-573; Kaplan, A. M., Darby, R. T., Greenberger, M. & Rodgers, M. (1968) Microbial deterioration of polyurethane systems. Dev Ind Microbiol, 82, 362-371; Kay, M., Morton, L. & Prince, E. (1991) Bacterial degradation of polyester polyurethane. International biodeterioration, 27 , 205-222; Vega, R. E., Main, T. & Howard, G. T. (1999) Cloning and expression in Escherichia coli of a polyurethane-degrading enzyme from Pseudomonas fluorescens. International biodeterioration & biodegradation, 43, 49-55).There is no clear evidence of cleavage of the urethane bond, since the enzyme characterization was never carried out based on the cleavage of a molecule with a urethane group.

[0007] The degradation of poly(ester)urethanes with fungi or bacteria is described in many publications and patents. However, the degradation usually only targets the relatively easily cleaved ester bonds and is usually only documented by macroscopic observation of the polymer degradation. There is no controlled degradation of ester and urethane bonds, as in the present invention, and long degradation times often result. These publications show that urethanases are widely used enzymes, but they do not document their specific capabilities, applications, and group affiliation, as used in the present invention. (JP09192633, Tang, YW, Labow, RS, Santerre, JP (2003) Enzyme induced biodegradation of polycarbonate-polyurethanes: dose dependence effect of cholesterol esterase. Biomaterials 24 (12), 2003-2011, Vega, RE, Main, T. & Howard, GT(1999) Cloning and expression in Escherichia coli of a polyurethane-degrading enzyme from Pseudomonas fluorescens. International biodeterioration & biodegradation, 43, 49-55) A degradation process is known for the enzymatic degradation of poly(ester)urethanes. In the first step, an esterase is obtained from a culture of Comamonas acidovorans, using only poly(ester)urethane as the carbon source. In a complex processing step, the esterase is separated and used for the degradation of poly(ester)urethanes in batch mode. This results in long degradation times in a multi-step process and no evidence for specific cleavage of the urethane bonds. (JP 09201192, JP 10271994).

[0008] Various patents and publications describe the degradation of poly(ester)urethanes using cutinases, esterases, and / or lipases. However, the degradation here also only aims at the relatively simple cleavage of the ester bonds, not specifically at the urethane bonds. Furthermore, no targeted combination of enzymes that cleave ester and urethane bonds to specifically control the degradation is described. It can be assumed that the described processes lead to little or no cleavage of the urethane bond. Thus, diamines used in the synthesis of polyurethanes cannot be efficiently recycled. (EP 0968300, US 6,180,381)

[0009] WO 2013 / 134801 describes the degradation of aromatic polyurethanes based on polyether polyols using an enzyme of class EC 3. No specific enzyme sequences are specified, so the specificity of the process in the degradation of certain urethane bonds, as well as the controlled cleavage of ester bonds and, separately, the cleavage of urethane bonds, as shown in the present invention, is not documented in the cited patent.

[0010] WO 2006 / 019095 describes a urethanase and variants of this enzyme obtained by protein engineering. The enzyme can cleave TDA- or MDA-based urethane oligomers. Furthermore, WO 2019 / 243293 describes newly identified urethanases.

[0011] The present invention was therefore based on the object of providing further enzymes that can be used for the enzymatic cleavage of urethane bonds and preferably for use in the complete enzymatic degradation of polyurethanes. Furthermore, an enzymatic process was to be provided that enables the degradation of polyurethanes into defined monomers.

[0012] This object is achieved by the embodiments disclosed in the patent claims and in the description below.

[0013] In a first embodiment, the present invention relates to a polypeptide having an amino acid sequence as defined by SEQ ID NO.: 1, 2 or 3, or a variant thereof, wherein the variant is obtained by the addition, deletion or replacement of up to 15% of the amino acids contained in the respective polypeptide defined by SEQ ID NO.: 1, 2 or 3, characterized in that the polypeptide has urethanase activity.

[0014] Preferably, a polypeptide having a sequence according to SEQ ID NO.: 1, 2 or 3 consists of the amino acid sequence defined by SEQ ID NO.: 1, 2 or 3.

[0015] polypeptide

[0016] The term "polypeptide" is well known to those skilled in the art. It refers to a chain of at least 50, preferably at least 70 amino acids linked by peptide bonds. A polypeptide can contain both naturally occurring and synthetic amino acids. Preferably, it contains the known proteinogenic amino acids and optionally selenocysteine, pyrrolysine, and hydroxyproline. More preferably, it consists of the known proteinogenic amino acids and optionally additionally selenocysteine, pyrrolysine, and hydroxyproline.

[0017] A variant is obtained from the amino acid sequences according to the invention defined by SEQ ID NO.: 1, 2 or 3, preferably by adding, deleting or exchanging up to 10%, and even more preferably up to 5%, of the amino acids contained in the respective polypeptide. The basis for calculating the sequence identity is preferably the amino acid sequence defined by SEQ ID NO.: 1, 2 or 3. The person skilled in the art is aware that fusion proteins are formed by enzymes with other proteins without this impairing the activity of the enzyme. Therefore, in a further embodiment of the invention, the term "variant" also encompasses those amino acid sequences derived from the polypeptide defined by SEQ ID NO.: 1, 2 or 3 that are fused at the N-terminus and / or C-terminus with other proteins, e.g., the Green Fluorescent Protein.

[0018] Particularly preferred variants of the polypeptides according to the invention are obtained by adding, deleting, or exchanging up to 20, preferably up to 10, and even more preferably up to 5 amino acids of the disclosed sequences. In principle, the aforementioned modifications can be made continuously or discontinuously at any position of the polypeptide in question. However, they preferably occur only at the N-terminus and / or C-terminus of the polypeptide. However, any variant according to the invention obtained by adding, exchanging, or deleting amino acids is characterized by urethanase activity as defined further below in this application.

[0019] Urethanase activity The term "urethanase activity" refers to the ability of a polypeptide to enzymatically catalyze the cleavage of a urethane group. For each mole of urethane group, one mole of amine, one mole of alcohol, and one mole of CO2 is produced.

[0020] The urethane group can be aromatic, cycloaliphatic, or aliphatically bonded. In an aromatically bonded urethane group, the nitrogen atom is directly bonded to an aromatic ring. In an aliphatically bonded urethane group, the nitrogen atom is bonded to an alkyl radical that is part of an open-chain alkyl radical. It is preferably an unbranched alkyl radical having at least one, more preferably at least two, and most preferably at least three carbon atoms. In a cycloaliphatically bonded urethane group, the nitrogen atom is bonded to a carbon atom that is part of an aliphatic ring. This ring can be unsaturated at one or more positions, as long as it does not acquire aromatic character due to the presence of double bonds.In a preferred embodiment of the present invention, the polypeptide with urethanase activity is capable of enzymatically cleaving an aromatically bound urethane group.

[0021] Whether a polypeptide possesses urethanase activity can be tested by cleavage of suitable model substrates. These are preferably the substrates whose cleavage was demonstrated in the working examples.

[0022] For the ability to cleave aromatically bound urethane groups, ethyl 4-nitrophenylcarbamate (ENPC) or the carbamate obtainable by the reaction of 7-amino-4-methylcoumarin with ethyl chloroformate (EMACC) are particularly preferred model substrates. The cleavage of ENPC is demonstrated by determining the increase in the concentration of 4-nitroaniline. This is preferably done photometrically at a wavelength of 405 nm. The enzyme activity is preferably determined in a reaction buffer containing 100 mM K2HPO4 / KH2PO4, pH 7, with 6.25 vol% ethanol in the presence of 0.2 mg / L ENPC as the substrate. The enzyme is incubated in the reaction buffer with ENPC preferably at room temperature and preferably for 24 hours. CO2 + EtOH

[0023] The cleavage of EMACC is detected by determining the increase in the concentration of 7-amino-4-methylcoumarin (AMC). This is preferably done photometrically at a wavelength of 365 nm. Enzyme activity is preferably determined in a reaction buffer containing 50 mM Na2HPO4 / NaH2PO4, pH 8.0, with 0.2 vol% DMSO in the presence of 100 pM EMACC as substrate. The enzyme is incubated in the reaction buffer with ENPC preferably at 30 °C and preferably for up to 24 hours.

[0024] Ethyl phenethylcarbamate (EPEC) is preferably used as a model substrate for the ability to cleave aliphatically bound urethane groups. Cleavage is demonstrated by determining the increase in phenethylamine concentration. This is preferably done by HPLC. The reaction buffer and reaction conditions used preferably correspond to the parameters described above for ENPC.

[0025] + CO2 + EtOH

[0026] The urethane bonds that can be cleaved by the urethanases according to the invention are formally based on the addition of a polyisocyanate with aliphatically, cycloaliphatically, araliphatically or aromatically bound isocyanate groups and an alcohol.

[0027] In an isocyanate with aliphatically bound isocyanate groups, all isocyanate groups are bonded to a carbon atom that is part of an open carbon chain. This chain may be unsaturated at one or more positions. The aliphatically bound isocyanate group, or—in the case of polyisocyanates—the aliphatically bound isocyanate groups, are preferably bonded to the terminal carbon atoms of the carbon chain.

[0028] Preferred polyisocyanates with aliphatically bound isocyanate groups are 1,4-diisocyanatobutane (BDI), 1,5-diisocyanatopentane (PDI), 1,6-diisocyanatohexane (HDI), 2-methyl-1,5-diisocyanatopentane, 1,5-diisocyanato-2,2-dimethylpentane, 2,2,4- or 2,4,4-trimethyl-1,6-diisocyanatohexane and 1,10-diisocyanatodecane.

[0029] In an isocyanate with cycloaliphatically bonded isocyanate groups, all isocyanate groups are bonded to carbon atoms that are part of a closed ring of carbon atoms. This ring can be unsaturated at one or more positions, as long as the presence of double bonds does not impart aromatic character.Preferred polyisocyanates with cycloaliphatically bound isocyanate groups are 1,3- and 1,4-diisocyanatocyclohexane, l,4-diisocyanato-3,3,5-trimethylcyclohexane, l,3-diisocyanato-2-methylcyclohexane, l,3-diisocyanato-4-methylcyclohexane, l-isocyanato-3,3,5-trimethyl-5-isocyanatomethyl-cyclohexane isophorone diisocyanate; (IPDI), l-isocyanato-l-methyl-4(3)-isocyanatomethylcyclohexane, 2,4'- and 4,4'-diisocyanatodicyclohexylmethane (H12MDI), 1,3- and 1,4-bis(isocyanatomethyl)cyclohexane, bis-(isocyanatomethyl)-norbornane (NBDI), 4,4'-Diisocyanato-3,3'-dimethyldicyclohexylmethane, 4,4'-Diisocyanato-3,3',5,5'-tetramethyl-dicyclohexylmethane, 4,4'-Diisocyanato-l,l'-bi(cyclohexyl), 4,4'-Diisocyanato-3,3'-dimethyl-l,l'-bi(cyclohexyl), 4,4'-Diisocyanato-2,2',5,5'-tetra-methyl-l,l'-bi(cyclohexyl), 1,8-Diisocyanato-p-menthane, 1,3-Diisocyanato-adamantane and l,3-Dimethyl-5,7-diisocyanatoadamantane.

[0030] In an isocyanate with araliphatically bound isocyanate groups, all isocyanate groups are bound to methylene residues, which in turn are bound to an aromatic ring.

[0031] Preferred polyisocyanates with araliphatically bound isocyanate groups are 1,3- and 1,4-bis-(isocyanatomethyl)benzene (xylylene diisocyanate); XDI), 1,3- and l,4-bis(l-isocyanato-l-methylethyl)benzene (TMXDI) and bis(4-(l-isocyanato-l-methylethyl)phenyl) carbonate.

[0032] In an isocyanate with an aromatically bound isocyanate group, all isocyanate groups are directly bonded to carbon atoms that are part of an aromatic ring.

[0033] Preferred isocyanates with aromatically bound isocyanate groups are tolylene diisocyanate (TDI), methylene diphenyl isocyanate (MDI) and naphthylene diisocyanate.

[0034] The term "toluene diisocyanate" refers to toluene-2,4-diisocyanate (2,4-TDI), toluene-2,6-diisocyanate (2,6-TDI), and any mixtures of the two isomers. The term "methylenediphenyl isocyanate" refers to all isomers of MDI, in particular 2,2'-diphenylmethane diisocyanate, 2,4'-diphenylmethane diisocyanate, 4,4'-diphenylmethane diisocyanate, all mixtures containing at least two of the aforementioned isomers, and polynuclear derivatives of MDI.

[0035] The term "naphthylene diisocyanate" refers to 1,4-naphthylene diisocyanate, 1,5-naphthylene diisocyanate and 1,6-naphthylene diisocyanate as well as any mixtures of the aforementioned isomers.

[0036] The skilled person is aware that all of the above-mentioned polyisocyanates can also exist as oligomers, so that the urethane bond to be cleaved is formed between an oligomeric polyisocyanate based on one of the above-mentioned monomeric diisocyanates and an alcohol. The term "oligomeric polyisocyanate" refers to polyisocyanates composed of at least two of the above-mentioned diisocyanates. According to the invention, the oligomeric polyisocyanates can, in particular, have uretdione, isocyanurate, allophanate, biuret, iminooxadiazinedione, and / or oxadiazinetrione structures. The oligomeric polyisocyanates preferably have at least one of the following oligomeric structure types or mixtures thereof:

[0037] Uretdione Isocyanurate Allophanate Biuret Iminooxadiazindione Oxadiazinetrione

[0038] Preferred substrates of the urethanase of the present invention are urethanes which can be obtained from the aromatic polyisocyanates defined above.

[0039] Enzymatic cleavage

[0040] The term "enzymatic cleavage of a urethane group" indicates that the above-described cleavage of a urethane group occurs more rapidly in the presence of a polypeptide with urethanase activity than when incubated with the reaction buffer without enzyme under the same reaction conditions or when incubated with the reaction buffer under the same conditions in the presence of an inactive polypeptide. Bovine serum albumin is preferred as a model for an inactive polypeptide. If, in the presence of a polypeptide to be tested, the cleavage of the urethane group occurs more rapidly than in an otherwise identical control containing BSA, said polypeptide possesses urethanase activity as understood in this application.

[0041] nucleic acid

[0042] In a further embodiment, the present invention relates to a nucleic acid encoding a polypeptide as defined in SEQ ID NO.: 1, 2, or 3 or one of the above-defined variants of these polypeptides, wherein the encoded polypeptide exhibits urethanase activity. Preferably, the encoding nucleic acid sequence is under the control of a promoter that enables expression of the polypeptide in a fungus, yeast, or bacterium.

[0043] Use In a further embodiment, the present invention relates to the use of a polypeptide having an amino acid sequence as defined by SEQ ID NO.: 1, 2 and 3, or one of the variants of these polypeptides defined above in this application, characterized in that the polypeptide has urethanase activity, for the enzymatic cleavage of urethane bonds.

[0044] All definitions given above also apply to this embodiment, unless explicitly defined otherwise. The use preferably consists in bringing the enzyme according to the invention into contact with a compound containing at least one urethane bond. This preferably takes place under conditions under which the enzyme exhibits its urethanase activity. Such conditions can be found in the working examples of this application. Furthermore, the person skilled in the art is easily able to adjust the reaction conditions in a system, such as temperature, ionic strength, solvent, and pH, through simple series of experiments so that the enzyme is active.

[0045] For the urethanase according to the invention, the temperature during use is preferably in the range between 10 °C and 70 °C. A higher temperature range between 30 °C and 70 °C is preferred if the reaction rate is more important than the stability of the enzyme. Conversely, a temperature range between 10 °C and 40 °C is preferred if the reaction rate is less important.

[0046] When using the urethanases defined by SEQ ID NO. 1 and 2 and their variants, the pH is preferably between 7 and 12, more preferably between 7 and 11, and most preferably between 8 and 11. When using the urethanase defined by SEQ ID NO. 3 or one of its variants, the pH is preferably between 7 and 12, more preferably between 7 and 11, and most preferably between 9 and 11.

[0047] Cleavage of low molecular weight urethanes

[0048] In yet another embodiment, the present invention relates to a method comprising the step of treating a low molecular weight urethane with a polypeptide having urethanase activity and selected from the group consisting of a polypeptide defined by SEQ ID NOS. 1, 2 and 3 and the variants of these polypeptides defined above in this application, thereby cleaving the low molecular weight urethane.

[0049] The term "low molecular weight" in this context means that the urethane contains a maximum of four, preferably a maximum of three, more preferably a maximum of one or two urethane groups. Preferably, the structures of the low molecular weight urethanes are formally based on the formation of one or more urethane bonds between a polyisocyanate and one or more low molecular weight alcohols.

[0050] Preferred polyisocyanates are those already mentioned above as partners for the urethane bonds to be cleaved by the urethanases of the invention. Aromatic polyisocyanates, in particular toluene 2,4-diisocyanate (2,4-TDI), toluene 2,6-diisocyanate (2,6-TDI), 2,2'-diphenylmethane diisocyanate (2,2'-MDI), 2,4'-diphenylmethane diisocyanate (2,4'-MDI), 4,4'-diphenylmethane diisocyanate (4,4'-MDI), the polynuclear derivatives of the aforementioned diphenylmethane diisocyanates, 1,4-naphthylene diisocyanate, 1,5-naphthylene diisocyanate, and 1,6-naphthylene diisocyanate are particularly preferred.

[0051] In principle, any compound with at least one hydroxyl group per molecule is suitable as a low-molecular-weight alcohol. It preferably has a molecular weight of no more than 700 g / mol, more preferably no more than 500 g / mol, and most preferably no more than 200 g / mol.

[0052] Furthermore, low-molecular-weight alcohols with a higher polarity are preferred. The low-molecular-weight alcohol particularly preferably contains one or two hydroxyl groups per molecule. Furthermore, the low-molecular-weight alcohol preferably has a melting point of at most 45°C, more preferably at most 20°C.

[0053] Therefore, alcohols with two hydroxyl groups, a molecular weight of not more than 500 g / mol and a melting point of not more than 45 °C are more preferred.

[0054] In a particularly preferred embodiment of the present invention, the low-molecular-weight alcohol is selected from the group consisting of ethylene glycol, diethylene glycol, propylene glycol, dipropylene glycol, methyl glycol, triethylene glycol, glycerin, 2-methyl-1,3-propanediol, 1,4-butanediol, 1,5-pentanediol, 1,6-hexanediol, and polyethylene glycol 400. More preferably, it is selected from the group consisting of ethylene glycol, diethylene glycol, propylene glycol, dipropylene glycol, methyl glycol, and triethylene glycol. Most preferably, the low-molecular-weight alcohol is diethylene glycol.

[0055] A particularly preferred low molecular weight urethane for the process according to the invention is obtainable by forming a urethane bond between one of the low molecular weight alcohols mentioned in the preceding section, in particular diethylene glycol, and.

[0056] The low-molecular-weight urethanes can be obtained in various ways. In a preferred embodiment of the present invention, the ester bonds of a polyester-polyurethane are cleaved to release carboxylic acids and low-molecular-weight urethanes. In a preferred embodiment of the invention, the cleavage of the ester groups is preferably carried out enzymatically. Lipases (EC 3.1.1.3) or cutinases (EC 3.1.1.74) are preferably used.

[0057] In a further preferred embodiment of the present invention, the low-molecular-weight urethanes are preferably those obtained by the chemical cleavage of a polyether or polyester polyurethane. This chemical cleavage is preferably a so-called "alcoholysis" or "glycolysis," in which the polyether or polyester polyurethane is reacted with one of the above-mentioned low-molecular-weight alcohols, so that the ether component of the polyether polyurethane or the polyester polyol of a polyester polyurethane is replaced by the low-molecular-weight alcohol.

[0058] A particularly preferred embodiment for providing low-molecular-weight urethanes from polyether polyurethanes is described in WO 2021 / 032513. In this process, polyether polyurethanes are reacted with a low-molecular-weight alcohol in such a way that the polyether component of the polyether polyurethane is released and a urethane bond is formed between the low-molecular-weight alcohol used and the isocyanates used to construct the polyether polyurethane. Thus, the low-molecular-weight urethanes to be cleaved according to the invention are compounds formally obtained by forming a urethane bond between a polyisocyanate, preferably one of the aliphatic, aromatic, araliphatic, or cycloaliphatic polyisocyanates listed above in this application, and a low-molecular-weight alcohol.

[0059] Polyester-polyurethane

[0060] The term "polyester-polyurethane" refers to a polyurethane whose polyol component contains polyester polyols. Preferably, at least 40 wt.% of the hydroxyl groups contained in the polyol component are components of polyester polyols. More preferably, this is at least 60 wt.%, even more preferably at least 80 wt.%, and most preferably at least 95 wt.%. The polyester-polyurethane can be foamed or unfoamed.

[0061] The polyester-polyurethane contains at least one aromatic, aliphatic, or cycloaliphatic isocyanate as the isocyanate component. The polyurethane preferably contains aromatic isocyanates. The at least one aromatic polyisocyanate is particularly preferably selected from the list given above.

[0062] The term "polyester polyol" is known to those skilled in the art; it refers to polyesters that contain, on average, at least 1.5, preferably at least 1.8, and more preferably at least 2.0 hydroxyl groups per molecule. Particularly preferably, the polyester polyols contained in the polyurethane to be degraded have a functionality between 1.5 and 6.0. They contain, as structural components, aromatic and / or aliphatic polyols and aromatic and / or aliphatic polycarboxylic acids in any desired combination. Polyester polyols obtained by ring-opening polymerization of a lactone are also preferred.

[0063] Polyether polyurethane

[0064] The term "polyether polyurethane" refers to a polyurethane whose polyol component contains polyether polyols. Preferably, at least 40 wt.% of the hydroxyl groups contained in the polyol component are constituents of polyether polyols. More preferably, this is at least 60 wt.%, even more preferably at least 80 wt.%, and most preferably at least 95 wt.%. According to the invention, it is possible for a polyether polyurethane to also contain further polyols as structural components while maintaining the aforementioned proportions of the polyether polyols. These are preferably polyester polyols.

[0065] The term "polyether polyol" is well known to those skilled in the art. These are polyethers with an average hydroxyl functionality between 1.5 and 6.0. Preferably, the polyether polyol contained in the polyether polyurethane is a polyaddition product of one or more alkylene oxides having 2 to 4 carbon atoms using at least one starter molecule containing 2 to 8, preferably 2 to 6, reactive hydrogen atoms.

[0066] Preferred alkylene oxides are styrene oxide, ethylene oxide, propylene oxide, tetrahydrofuran, butylene oxide, and epichlorohydrin. More preferred are 1,3-propylene oxide, 1,2- or 2,3-butylene oxide, and styrene oxide. Ethylene oxide and 1,2-propylene oxide are particularly preferred. The alkylene oxides can be used individually, alternating one after the other, or as mixtures.

[0067] The "treatment" of the low-molecular-weight urethane takes place under conditions under which the enzyme used exhibits urethanase activity. Under these conditions, the reaction products of the enzymatic cleavage of the low-molecular-weight urethane are a low-molecular-weight alcohol or a mixture of low-molecular-weight alcohols and an amine or a mixture of amines. The chemical structure of the resulting amines depends on the type of polyisocyanate used to synthesize the urethane. Amines are released, which can be derived from the polyisocyanates used by addition of water and subsequent release of CO2.

[0068] In yet another embodiment, the present invention relates to the use of at least one of the polypeptides of the invention for cleaving a urethane bond in low-molecular-weight urethanes. Degradation of polyester urethanes into low-molecular-weight degradation products

[0069] In yet another embodiment, the present invention relates to a process for degrading polyester-polyurethanes into low-molecular-weight degradation products, comprising the steps of a) cleavage of the ester groups contained in the polyester-polyurethane; and b) treatment of the polyurethane with a polypeptide which has urethanase activity and is selected from the group consisting of polypeptides as defined in SEQ. ID NO.: 1, 2, and 3 and the variants of these polypeptides defined above in this application; with the proviso that process steps a) and b) can be carried out in any order or simultaneously.

[0070] Preferably, process step a) is carried out before process step b).

[0071] Unless otherwise stated, all definitions given above in this application also apply to this embodiment. This applies in particular to the treatment of the polyurethane with a urethanase in process step b).

[0072] In a preferred embodiment of the invention, process step a) is carried out using a lipase or cutinase. More preferably, a lipase capable of cleaving tributyrin is used. Process step a) is preferably carried out under reaction conditions under which the lipase or cutinase used exhibits activity. Such conditions can be determined by routine experiments using standard biochemical methods.

[0073] In another, also preferred embodiment, process step a) is carried out chemically.

[0074] The polyurethane contains at least one aromatic, aliphatic, or cycloaliphatic isocyanate as the isocyanate component. The polyurethane preferably contains aromatic isocyanates.

[0075] The low molecular weight degradation products of the polyester-based polyurethanes preferably have a molecular weight of at most 1,000 g / mol. They are preferably

[0076] (i) amines derived from the isocyanates used in the manufacture of the polyurethane in question, for example, in the case of 2,4-toluene diisocyanate, 2,4-toluene diamine; and (ii) alcohols and carboxylic acids used to synthesize the polyester polyols used in the synthesis of the polyurethane in question.

[0077] A "polyol" resulting as a low-molecular-weight degradation product in the process defined in this section is understood in this application to mean any compound having at least two hydroxyl groups. Said polyol preferably has a molecular weight of at most 300 g / mol. Preferred polyols that are low-molecular-weight degradation products of the polyester-based polyurethanes are selected from the group consisting of ethylene glycol, diethylene glycol, triethylene glycol, propylene glycol, 1,3-pentanediol, 1,4-butanediol, 1,6-hexanediol, 1,2-dipropylene glycol, neopentyl glycol, glycerol, 1,1,1-trimethylolpropane, sucrose, sorbitol, and pentaerythritol.

[0078] A "polycarboxylic acid" formed as a low-molecular-weight degradation product in the process defined in this section is understood in this application to mean any compound containing at least two carboxyl groups. Said polycarboxylic acid preferably has a molecular weight of at most 300 g / mol. Preferred polycarboxylic acids, which are low-molecular-weight degradation products of the polyester-based polyurethane foams, are selected from the group consisting of succinic acid, glutaric acid, adipic acid, itaconic acid, phthalic acid, isophthalic acid, terephthalic acid, and benzenetricarboxylic acid.

[0079] A "polyamine" which is formed as a low-molecular degradation product in the process defined in this section is understood in this application to mean any compound which contains at least two amino groups. Said polyamine preferably has a molecular weight of at most 300 g / mol. Preferred polyamines which are low-molecular degradation products of the polyester-based polyurethane foams are selected from the group consisting of 4,4'-diaminodiphenylmethane, 2,4'-diaminodiphenylmethane, 2,2'-diaminodiphenylmethane, the polynuclear derivatives of the aforementioned diaminodiphenylmethane compounds, 2,4-

[0080] Toluenediamine, 2,6-toluenediamine, 1,4-naphthylenediamine, 1,5-naphthylenediamine, 1,6-

[0081] Naphthylenediamine, hexamethylenediamine, isophoronediamine, xylylenediamine, pentamethylenediamine, para-phenylenediamine, butyldiamine, and H12-methylenediamine. Particularly preferred polyamines are those selected from the group consisting of 4,4'-methylenediamine, 2,4'-methylenediamine, 2,2'-methylenediamine, 1,5-naphthylenediamine, 2,4-toluenediamine, and 2,6-toluenediamine.

[0082] Degradation of polyether polyurethane

[0083] In yet another embodiment, the present invention relates to a process comprising the steps of a) transurethanization of a polyether polyurethane with at least one low-molecular-weight alcohol, whereby polyether polyols and low-molecular-weight urethanes are formed; and b) the enzymatic cleavage of the low-molecular-weight urethanes formed in process step a) with a polypeptide which has urethanase activity and is selected from the group consisting of polypeptides as defined in SEQ. ID NO.: 1, 2, and 3 and the variants of these polypeptides defined above in this application.

[0084] Process step a) has two objectives: (i) The polyether polyol used to synthesize the polyurethane should be released from the polyurethane as an isolable compound. (ii) The isocyanate used to synthesize the polyurethane should be present as a component of a low-molecular-weight urethane. In contrast to the polyurethane with its high molecular weight, this low-molecular-weight urethane is well suited as a substrate for the enzymatic cleavage occurring in process step b) due to its lower molecular weight and the resulting better solubility.

[0085] Unless otherwise stated, all definitions given above in this application also apply to this embodiment. This applies in particular to the enzymatic cleavage of the low-molecular-weight urethanes in process step b).

[0086] In process step b), enzymatic cleavage of the low-molecular-weight urethanes releases an amine and the low-molecular-weight alcohol used for transurethanization in process step a). CO2 is also released in this process step. These compounds can be isolated by suitable separation processes and subsequently reused. It is preferable to reuse the released low-molecular-weight alcohol for the transurethanization carried out in process step a). The released amine is available as a pure and well-defined starting material for new syntheses.

[0087] The generally suitable reaction conditions and catalysts for the transurethanization in process step a) are described in Simon et al. (2018), Waste Management, 76: 147-171. Process step a) is carried out at temperatures between 140 °C and 300 °C, preferably between 160 °C and 270 °C. The weight ratio of the low molecular weight alcohol to the polyetherurethane is between 2:1 and 1:17. Particularly suitable catalysts are alkali metal hydroxides, alkaline earth metal hydroxides, alkali metal salts of carboxylic acids (especially acetates), alkaline earth metal salts of carboxylic acids (especially acetates), Lewis acids (such as, in particular, dibutyltin dilaurate), organic amines (such as, in particular, diethanolamine), organometallic compounds (such as, in particular, titanium tetrabutoxide), and tin compounds (such as, in particular, tin octoate).The transurethanization is preferably carried out at temperatures in the range of 160 °C to 270 °C in the presence of 0.1 mass% to 5 mass% catalyst, based on the mass of the added polyurethane product.

[0088] Figure 1 shows the enzyme substrates used. pNPB: 4-nitrophenylbutyrate; ENPC: ethyl 4-nitrophenylcarbamate; MDA-BA: carbamate of 4,4'-MDI reacting with benzyl alcohol; TDA-ethoxyethanol: carbamate of toluene diisocyanate (TDI) reacting with ethoxyethanol; TDA-DEG: carbamate of TDI reacting with diethylene glycol; NDA-MEG: carbamate of NDI reacting with ethylene glycol; EMACC: carbamate from the reaction of 7-amino-4-methylcoumarin with ethyl chloroformate.

[0089] The following embodiments serve only to illustrate the invention. They are not intended to limit the scope of the patent claims in any way.

[0090] Examples

[0091] Example 1: Activity determination with EMACC

[0092] Enzyme production

[0093] Urethanase genes were cloned into the pET-26 expression vector directly behind the NdeI site. E. coli BL21(DE3) were transformed with the corresponding plasmids for expression. A single colony was used to inoculate the overnight culture in LB medium containing 1% (w / v) glucose and 50 pg / mL kanamycin. For the main culture, 200 ml of ZYP-5052 containing 50 pg / mL kanamycin was inoculated with 1 mL of the overnight culture and incubated for 4 h at 37°C and 100 rpm in baffled flasks. The temperature was then reduced to 20°C before the cultures were harvested by centrifugation for 20 min at 4500 g and 4°C. Cell pellets were stored at -20°C until protein purification.

[0094] For purification, 15 mL of lysis buffer (50 mM sodium phosphate (NaPi), pH 8.0, 300 mM NaCl, 10 mM imidazole) was added to the cell pellet. Disruption was performed using ultrasonication on ice for two cycles (3 min, 50% pulse, 50% power). To clarify the lysate, it was centrifuged at 10,000 g and 4 °C for 40–60 min. The supernatants were stored on ice until loading onto the IMAC column.

[0095] IMAC resin (1 ml, ROTI®Garose His Beads, nickel form) was equilibrated with lysis buffer. The lysate was then applied to the columns, followed by a wash step with at least ten column volumes of wash buffer (50 mM NaPi, pH 8.0, 300 mM NaCl, 20 mM imidazole). The proteins were then eluted with 15 mL of elution buffer (50 mM NaPi, pH 8.0, 300 mM NaCl, 250 mM imidazole). The volume of the elution fraction was reduced to 2.5 mL using Vivaspin-20 ultrafiltration units (10 kDa MWCO). The proteins were then buffered into storage buffer (10 mM NaPi, pH 8.0, 100 mM NaCl) using PDIO columns.

[0096] EMACC Assay

[0097] To determine urethanase activity, a fluorogenic substrate was synthesized from 7-amino-4-methylcoumarin (AMC) and ethyl chloroformate. The resulting ethyl carbamate of AMC (EMACC) was available as a fine powder. Specific activity was determined under standard conditions (50 mM NaPi, pH 8.0, 30 °C, 100 pM EMACC). EMACC was added fresh from a 50 mM stock in DMSO. Purified urethanases were diluted in storage buffer prior to addition to the assay, resulting in a linear increase in absorbance at 365 nm over 10–30 min. The measurement was performed in 96-well plates in a plate photometer preheated to 30 °C, and the specific activity was calculated from the linear increase in absorbance during the first few minutes. The results are shown in Table 1. Table 1: Specific activity of urethanase toward EMACC

[0098] Example 2: Screening against other substrates

[0099] Enzyme production

[0100] To prepare the enzyme preparations, 4.5 mL of LB medium containing 1% (w / v) glucose was inoculated with single colonies of E. coli BL21(DE3) pET26b_UMG-8, BL21(DE3) pET26b_UMG-11.2, or BL21(DE3) pET26b_UMG-16 and incubated overnight at 37°C and 200 rpm in an incubator shaker. All cultures were treated with 50 pg / mL kanamycin. Subsequently, 200 mL of ZYP-5052 medium were inoculated with 200 pL of the overnight cultures. Cultivation was carried out for 4 h at 37°C and 200 rpm, followed by 24 h at 20°C and 200 rpm in an incubator shaker. The cells were separated by centrifugation at 20,425 g and 4 °C for 20 min in a large-capacity centrifuge. The cell pellets were suspended in 6 mL of disruption buffer (20 mM ammonium acetate, 0.4% n-dodecyl-ß-maltoside, 1% lysozyme, 1 pL / mL benzonase (Sigma-Aldrich Chemie GmbH, Taufkirchen)) and incubated for 30 min at room temperature on an orbital shaker.The whole-cell extract solution was then frozen at -80 °C and freeze-dried. The lyophilisate was stored at 4 °C.

[0101] Enzyme reactions

[0102] The substrates ENPC, MDA-BA, NDA-MEG, TDA-ethoxyethanol, and TDA-DEG ​​were synthesized from the corresponding isocyanates and alcohols by reacting the isocyanates with an excess of alcohol. EMACC and 4-nitropenyl butyrate (pNPB) were used to confirm the activity of the urethanases. The enzyme substrates are shown in Figure 1.

[0103] A spatula tip (~3 mg) of each lyophilized whole-cell extract was suspended in 800 μL of 100 mM KPi with 100 mM NaCl (pH 7.5). The enzyme solution was diluted 1:10 into the reaction mixture containing the same buffer. Substrate stocks of the carbamates were prepared at a concentration of 5–340 mg / mL in DMSO. These were diluted 1:10 into the reaction mixture so that the final DMSO concentration was 10% (v / v). The mixtures were prepared with a total volume of 500 μL. Incubation was carried out for one day at 30°C and 800–1000 rpm, followed by one day at 40°C and 800–1000 rpm in a thermoshaker. After incubation, 500 μL of acetonitrile was added to improve the solubility of substrates and products. The samples were filtered by centrifugation through a 0.2 pm PVDF filter plate and used for HPLC analysis.For the substrates EMACC and pNPB, the total reaction volume was 200 pL and the fluorescence (EMACC, excitation 365 nm, emission 440 nm) or absorbance (pNPB, 410 nm) was measured at different time points in the Varioskan Lux plate photometer (Thermo Fisher Scientific Inc., Waltham, Massachusetts, USA).

[0104] H PLC -Analytics

[0105] High-pressure liquid chromatography was performed on a 1260 Infinity II series instrument from Agilent Technologies (Santa Clara, USA) equipped with a multisampler and DAD (diode array detector) for UV and visible light. For all measurements, a Zorbax Eclipse Plus-CIS column with a particle size of 5 pm and dimensions of 4.6 x 150 mm (Agilent Technologies, Santa Clara, USA) with an appropriate guard column was used. For all methods, 5 pL of sample was injected, and the column was heated to 40 °C. The flow rate was generally 1.0 mL / min.

[0106] The method "ER_June2021-2" was used to detect aromatic amines. Aromatic amines could be quantified at 210 nm and 232 nm without derivatization due to their high intrinsic absorption. Mobile phase A was ddHjO with 5% (v / v) acetonitrile, and mobile phase B was acetonitrile with 5% (v / v) ddHjO. Data were analyzed using the software "OpenLAB CDS 2.4" version 2.204.0.661 (Agilent Technologies, Santa Clara, USA).

[0107] Table 2: HPLC gradient

[0108] Results

[0109] The hydrolytic activity of the urethanases toward the tested carbamates was classified based on the peak area of ​​the formed products in the chromatogram into the categories no activity (-), low activity (+), medium activity (++), and high activity (+++). It was shown that the urethanases UMG-8, UMG11.2, and UMG 16 accept a broad spectrum of substrates. The tested substrates are shown in Table 3. Activity of the urethanase toward various carbamates. Activity is categorized into "no activity" (-), "low activity" (+), "medium activity" (++), and "high activity" (+++) based on the HPLC peak area.

[0110] Example 3: Optimization of reaction conditions

[0111] To determine the pH optima, enzyme activity was determined analogously to Example 1, with the adjustment that the reaction was carried out in different buffers (100 mM). Buffers with pH 4.0, 5.0, and 6.0 (citrate), 7.0, 8.0, and 9.0 (Bis-Tris-Propane), and 10.0 (CHES) were used. For pH values ​​10, 11, 12, and 13, a buffer consisting of 100 mM sodium phosphate and 100 mM sodium carbonate was used. In contrast to Example 1, a buffer consisting of 200 mM EMACC and 10% (v / v) DMSO was used. Temperature optima were determined by measuring the release of AMC from 100 pM EMACC in 50 mM NaPi (pH 8.0) at 30, 35, 40, 45, 50, 55, 60, 65, and 70 °C. Solvent stability was determined by quantifying the hydrolysis of EMAC under the conditions specified in Example 1 at 0, 10, 20, 30, 40, and 50% (v / v) DMSO. The activity optima determined here for pH, temperature, and DMSO concentration are shown in

[0112] Table 4 summarizes the activity dependence.

[0113] Table 4: Optimal reaction conditions for urethanase.

[0114] Table 5: Dependence of urethanase activity on pH. In both data sets, activity was highest at pH 10, so normalization was performed to this value. Activity data from pH 12 and pH 13 were not shown because AMC fluorescence is only constant between pH 3 and pH 12.

[0115] Temperature stability

[0116] To determine long-term temperature stability, solutions of the purified urethanases were prepared with a protein concentration of 0.5 mg / mL in 100 mM KPi pH 7.5. 100 pL each were incubated in the Biometra TAdvanced Basis thermocycler (Analytik Jena GmbH, Jena) at 20°C, 25.7°C, 29.4°C, 36.9°C, 40.6°C, 44.3°C, and 50°C for 12 h and then at 4°C until activity was measured. The lid heater was set to 99°C. A reference sample was incubated at 4°C. The residual activity of the enzymes was determined using the pNPB assay. For this purpose, 21 pL of pNPB was mixed with 4.979 mL of DMSO. This solution was diluted 1:10 in 100 mM KPi pH 7.5. 20 pL of enzyme solution (UMG-8 diluted 1:5 in 100 mM KPi pH 7.5, UMG-11.2 diluted 1:20 in 100 mM KPi pH 7.5, UMG-16 diluted 1:20 in 100 mM KPi pH 7.5) were placed in a microtiter plate, and the reaction was started by adding 180 pL of pNPB in 100 mM KPi pH 7.5 with 10% DMSO.Hydrolysis of pNPB by urethanases releases para-nitrophenol, which was quantified by measuring the absorbance at 410 nm in a Varioskan Lux plate reader (Thermo Fisher Scientific Inc., Waltham, Massachusetts, USA). The results are presented in Table 6.

[0117] Table 6: Specific activity of urethanase against pNPB in U / mg after incubation for 12 h at 20°C, 25.7°C, 29.4°C, 36.9°C, 40.6°C, 44.3°C and 50°C and at 4°C as reference.

Claims

Patent claims 1. A polypeptide having an amino acid sequence as defined by SEQ ID NO.: 1, 2 or 3, or a variant thereof, wherein the variant is obtained by the addition, deletion or exchange of up to 15% of the amino acids contained in the respective polypeptide defined by SEQ ID NO.: 1, 2 or 3, characterized in that the polypeptide exhibits urethanase activity.

2. Use of a polypeptide with an amino acid sequence as defined by SEQ ID NO.: 1, 2 or 3, or a variant of one of these polypeptides, wherein the variant is obtained by the addition, deletion or exchange of up to 15% of the amino acids contained in the respective polypeptide defined by SEQ ID NO.: 1, 2 or 3, characterized in that the polypeptide has urethanase activity for the enzymatic cleavage of urethane bonds.

3. Use according to claim 2, wherein the urethane group is aromatically bonded.

4. Nucleic acid encoding a polypeptide as defined by SEQ ID NO. 1, 2 or 3, or a variant of one of the aforementioned polypeptides, exhibiting urethanase activity, wherein the variant is obtained by the addition, deletion or substitution of up to 15% of the amino acids contained in the respective polypeptide defined by SEQ ID NO.: 1, 2 or 3.

5. A process comprising the step of treating a low molecular weight urethane with a polypeptide exhibiting urethanase activity and having an amino acid sequence as defined by SEQ ID No. 1, 2 or 3, or with a variant thereof, wherein the variant is obtained by adding, deleting or exchanging up to 15% of the amino acids contained in the respective polypeptide defined by SEQ ID No. 1, 2 or 3, thereby cleaving the low molecular weight urethane.

6. The method according to claim 5, wherein at least one polyamine selected from the group consisting of 4,4'-diaminodiphenylmethane, 2,4'-diaminodiphenylmethane, 2,2'-diaminodiphenylmethane, the polynuclear derivatives of the aforementioned diaminodiphenylmethane compounds, 2,4-toluenediamine, 2,6-toluenediamine, 1,4-naphthylenediamine, 1,5-naphthylenediamine and 2,6-toluenediamine. 23 CORRECTED SHEET (RULE 91) ISA / EP The process of claim 5 or 6, wherein an alcohol having at least two hydroxyl groups and a molecular weight of at most 700 g / mol is released. The process of claim 7, wherein at least one alcohol selected from the group consisting of ethylene glycol, diethylene glycol, propylene glycol, dipropylene glycol, methyl glycol, triethylene glycol, glycerol, 2-methyl-1,3-propanediol, 1,4-butanediol, 1,5-pentanediol, 1,6-hexanediol, and polyethylene glycol 400 is released. Use of at least one polypeptide exhibiting urethanase activity and having an amino acid sequence as defined by SEQ ID NO. 1, 2, or 3, or having an amino acid sequence with at least 85% sequence identity to the aforementioned sequence, for cleavage of a urethane bond in low molecular weight urethanes.A process for the degradation of polyester polyurethanes into low-molecular-weight degradation products, comprising the steps of: a) cleavage of the ester groups contained in the polyester polyurethane; and b) treatment of the polyurethane with a polypeptide exhibiting urethanase activity and selected from the group consisting of polypeptides as defined by SEQ ID No. 1, 2, and 3, and variants of these polypeptides, wherein the variants are obtained by the addition, deletion, or substitution of up to 15% of the amino acids contained in the respective polypeptide defined by SEQ ID No. 1, 2, or 3; provided that process steps a) and b) may be carried out in any order or simultaneously.A process comprising the steps a) urethanization of a polyether polyurethane with at least one low molecular weight alcohol, yielding polyether polyols and low molecular weight urethanes; and b) enzymatic cleavage of the low molecular weight urethanes obtained in process step a) with a polypeptide exhibiting urethanase activity and selected from the group consisting of polypeptides as defined by SEQ ID NO.: 1, 2 and 3 and variants of these polypeptides. 24 CORRECTED SHEET (RULE 91) ISA / EP the variants are obtained by adding, deleting or exchanging up to 15% of the amino acids contained in the respective polypeptide defined by SEQ. ID NO.: 1, 2 or 3. 25 CORRECTED SHEET (RULE 91) ISA / EP

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