A novel urethanase for the enzymatic degradation of polyurethanes.

Abstract

The present invention relates to novel uretanases for the enzymatic degradation of polyurethanes and to an enzymatic method for the complete degradation of polyurethanes into defined monomers.

Description

[Technical field]

[0001] The present invention relates to novel uretanases for the enzymatic degradation of polyurethanes and to an enzymatic method for the complete degradation of polyurethanes into defined monomers. [Background technology]

[0002] Polyurethanes have established themselves in many areas of daily life. They can be found, for example, in flexible foams (mattresses, sponges, upholstered furniture), rigid foams (thermal insulation, building materials), thermoplastics (sports shoes) or coatings (varnishes, paints, adhesives). Due to the constantly increasing demand for the products, the production volumes are also increasing. At the same time, there is an increasing need for methods that maximize the sustainable recycling of polyurethane products that are no longer needed and allow the reuse of the components of the polymer. For this, the bonds in the polyurethanes must be selectively cleaved so that defined decomposition products can be obtained, thereby making the polyurethanes recyclable.

[0003] In addition to the physiological functions that enzymes perform in living organisms, enzymes can be used in a variety of ways for catalysis of chemical reactions other than physiological functions. Such reactions can be carried out under milder conditions than traditional chemical methods, e.g., lower temperatures, neutral pH, and without the use of harsh chemicals. This allows to save energy, minimize the production of by-products, protect the environment, and contribute to lower operating costs. In some cases, only by using enzymes can unstable starting materials be used as reaction materials (Jaeger, K.-E. & Reetz, MT (1998) Microbial lipases form versatile tools for biotechnology. Trends in biotechnology, 16, 396-403). Furthermore, enzymes often possess chemo-, regio-, stereo- and enantioselectivity, which can make product purification much easier and enable efficient synthesis of products that are otherwise difficult to obtain (Hasan, F., Shah, AA & Hameed, A. (2006) Industrial applications of microbial lipases. Enzyme and Microbial Technology, 39, 235-251).

[0004] Today, there are only a few cases of polyurethane recycling: disposal involves landfilling or complete incineration, in the latter case obtaining energy but without efficient reuse of the polymer components.

[0005] It is known that polyurethanes can be degraded to some extent by bacteria and fungi, and polyester polyurethanes are significantly more susceptible to such microbial / 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).

[0006] The degradation of polyester polyurethane can be easily achieved by hydrolysis of ester bonds. The relatively simple degradation of polyester is not surprising, considering that when lipids are degraded, ester bonds in essentially hydrophobic substrates must be cleaved, and polyesters without urethane bonds can be degraded relatively easily by esterases and lipases as well (Marten, E., Mueller, 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., Mueller, R.-J. & Deckwer, W.-D. (2005) Studies on the enzymatic hydrolysis of polyesters. II. Aliphatic-aromatic copolyesters. Polymer degradation and stability, 88, 371-381). The enzymes used to degrade polyurethanes have been characterized as esterases in various publications (Allen, AB, Hilliard, NP & Howard, GT (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, JR, Campbell, JR, Thompson, L.,Walz,SL&Schultz,WW(1994)Biodegradation of a colloidal ester-based polyurethane by soil fungi.International biodeterioration&biodegradation,33,103-113;Darby,RT&Kaplan,AM(1968)Fungal susceptibility of polyurethanes.Applied microbiology,16,900-905;Howard,GT,Norton,WN&Burks,T.(2012)Growth of Acinetobacter gerneri P7 on polyurethane and the purification and characterization of a polyurethanase enzyme.Biodegradation,23,561-573;Kaplan,AM,Darby,RT,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, 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). Since there have been no examples of enzyme characterization based on the cleavage of molecules with urethane groups, the cleavage of urethane bonds has not been clearly demonstrated.

[0007] Fungal or bacterial degradation of poly(ester urethane) has been described in many publications and patents. However, the degradation mainly targets only the relatively easily cleaved ester bonds and is mostly only demonstrated by macroscopic observation of polymer degradation. Here, controlled degradation of ester and urethane bonds is not performed as in the present invention, and long degradation times often occur. These publications indicate that urethanase is a commonly found enzyme, but do not demonstrate the specific capabilities, potential applications and groupings of urethanase 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).

[0008] Degradation methods for the enzymatic degradation of poly(ester urethane) are known, the first step of which is to obtain esterase from a culture of Comamonas acidovorans by using only poly(ester urethane) as a carbon source. In a complex purification step, the esterase is isolated and used to degrade poly(ester urethane) in a batch process. This results in long degradation times in a multi-step process, and there is no demonstration of specific cleavage of the urethane bond (JP09201192, JP10271994).

[0009] The degradation of poly(ester urethane) by cutinase, esterase, and / or lipase has been described in various patents and publications. However, the degradation here targets only the relatively simple cleavage of ester bonds, but does not specifically target urethane bonds. Furthermore, a specific combination of enzymes that cleave ester and urethane bonds has not been described for selective control of degradation. It can be assumed that the described methods result in little or no cleavage of urethane bonds. This means that the diamines used in the synthesis of polyurethanes cannot be efficiently recovered (EP0968300, US6,180,381).

[0010] WO2013 / 134801 describes the degradation of aromatic polyurethanes based on polyether polyols using enzymes of class EC3. No specific enzyme sequence is described, and therefore neither the specificity of the method in decomposing specific urethane bonds, nor the controlled cleavage of ester bonds and separate cleavage of urethane bonds, as shown in the present invention, is demonstrated in the cited patent.

[0011] WO2006 / 019095 describes urethanase and mutants of this enzyme obtained by protein engineering. The enzyme is capable of cleaving urethane oligomers based on TDA or MDA. Furthermore, WO2019 / 243293 describes a newly identified urethanase. [Prior art documents] [Patent documents]

[0012] [Patent Document 1] Japanese Patent Application Publication No. 09-192633 [Patent Document 2] Japanese Patent Application Publication No. 09-201192 [Patent Document 3] Japanese Patent Publication No. 10-271994 [Patent Document 4] European Patent No. 0968300

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Non-licensed literature

[0013] [Non-licensed document 1] Jaeger,K.-E.&Reetz,MT(1998)Trends in biotechnology,16,396-403 [Non-licensed document 2] Hasan,F.,Shah,AA&Hameed,A.(2006)Enzyme and Microbial Technology,39,235-251 [Non-licensed document 3] Nakajima-Kambe,T.,Shigeno-Akutsu,Y.,Nomura,N.,Onuma,F.&Nakahara,T.(1999)Applied microbiology and biotechnology,51,134-140

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[0014] It was therefore an object of the present invention to provide further enzymes which can be used for the enzymatic cleavage of urethane bonds, preferably for the complete enzymatic degradation of polyurethanes. A further object was to provide an enzymatic process which makes it possible to degrade polyurethanes into defined monomers. [Means for solving the problem]

[0015] This object is achieved by the embodiments disclosed in the claims and in the following description.

[0016] In a first embodiment, the present invention relates to a polypeptide having an amino acid sequence defined by SEQ ID NO: 1, 2 or 3, or a variant thereof, wherein the variants are obtained by addition, deletion or exchange of up to 15% of the amino acids present in each of the polypeptides defined by SEQ ID NO: 1, 2 or 3, The polypeptide is characterized in that it has urethanase activity.

[0017] Preferably, the 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.

[0018] Polypeptides 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 together by peptide bonds. A polypeptide may contain both natural and synthetic amino acids. It preferably contains the known proteinogenic amino acids, and optionally also selenocysteine, pyrrolysine and hydroxyproline. It more preferably consists of the known proteinogenic amino acids, and optionally also selenocysteine, pyrrolysine and hydroxyproline.

[0019] The variants are obtained from the amino acid sequences according to the invention defined by SEQ ID NO: 1, 2 or 3 by adding, deleting or exchanging preferably up to 10%, more preferably up to 5% of the amino acids present in the respective polypeptide. The basis for calculating the sequence identity is preferably in each case the amino acid sequence defined by SEQ ID NO: 1, 2 or 3. It is known to those skilled in the art to form fusion proteins of enzymes with other proteins without affecting the activity of the enzyme. Thus, the term "variant" in a further embodiment of the invention also includes amino acid sequences derived from the polypeptides defined by SEQ ID NO: 1, 2 or 3, which are fused at the N-terminus and / or C-terminus with other proteins, for example green fluorescent protein.

[0020] Particularly preferred variants of the polypeptides according to the invention are obtained by adding, deleting or exchanging up to 20, preferably up to 10, more preferably up to 5 amino acids of the disclosed sequences. The aforementioned modifications can in principle be carried out continuously or discontinuously at any desired point in the polypeptide in question. However, they are preferably only carried out at the N-terminus and / or C-terminus of the polypeptide. However, each variant according to the invention obtained by adding, exchanging or deleting amino acids is characterized by uretase activity as defined in the present application below.

[0021] Urethanase activity The term "urethanase activity" refers to the ability of a polypeptide to enzymatically catalyze the cleavage of urethane groups. In the process, one mole of urethane group produces one mole of amine, one mole of alcohol, and one mole of CO2.

[0022] The urethane group may be aromatic, alicyclic or aliphatically bound. In the case of aromatically bound urethane groups, the nitrogen atom is directly bound to the aromatic ring. In the case of aliphatically bound urethane groups, the nitrogen atom is bound to an alkyl radical that is part of an open chain alkyl radical. The alkyl radical is preferably unbranched and consists of at least one, preferably at least two, more preferably at least three carbon atoms. In the case of alicyclically bound urethane groups, the nitrogen atom is bound to a carbon atom that is part of an aliphatic ring. This ring may be unsaturated at one or more positions, but does not acquire aromaticity as a result of the presence of a double bond. In a preferred embodiment of the present invention, the polypeptide with urethanase activity is capable of enzymatically cleaving aromatically bound urethane groups.

[0023] Whether a polypeptide has urethanase activity can be determined by cleavage of suitable model substrates, preferably those substrates which are shown to be cleaved in the examples.

[0024] Model substrates that are particularly preferably used due to their ability to cleave aromatically-linked urethane groups are ethyl 4-nitrophenylcarbamate (ENPC) or the carbamate obtained by reaction of 7-amino-4-methylcoumarin with ethyl chloroformate (EMACC). The cleavage of ENPC is detected by determining the increase in the concentration of 4-nitroaniline. This is preferably carried out photometrically at a wavelength of 405 nm. The enzyme activity is preferably measured in a buffer containing 100 mM K2HPO4 / KH2PO4, pH 7, 6.25% by volume of ethanol, in the presence of 0.2 mg / L ENPC as substrate. The incubation of the enzyme with ENPC in the reaction buffer is preferably carried out at room temperature, preferably for 24 hours. [ka]

[0025] The cleavage of EMACC is detected by determining the increase in the concentration of 7-amino-4-methylcoumarin (AMC). This is preferably performed photometrically at a wavelength of 365 nm. The enzyme activity is preferably measured in a buffer containing 50 mM Na2HPO4 / NaH2PO4, pH 8.0, 0.2% by volume of DMSO, in the presence of 100 μM EMACC as substrate. Incubation of the enzyme with EMACC in the reaction buffer is preferably performed at 30° C., preferably for up to 24 hours. [ka]

[0026] A model substrate for the ability to cleave aliphatically bound urethane groups is preferably ethyl phenethyl carbamate (EPEC). Cleavage is detected by measuring the increase in the concentration of phenethylamine. This is preferably carried out by HPLC. The reaction buffer and reaction conditions used preferably correspond to the parameters described above for ENPC. [ka]

[0027] The urethane bonds which can be cleaved by the urethanase according to the invention are based formally on the addition of polyisocyanates and alcohols which have aliphatically, cycloaliphatically, araliphatically or aromatically bound isocyanate groups.

[0028] In isocyanates with aliphatically bound isocyanate groups, all the isocyanate groups are bound to carbon atoms that are part of an open carbon chain, which may be unsaturated in one or more positions. The aliphatically bound isocyanate group or - in the case of polyisocyanates - the aliphatically bound isocyanate groups are preferably bound to the terminal carbon atoms of the carbon chain.

[0029] Preferred polyisocyanates having 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.

[0030] In isocyanates with cycloaliphatically bound isocyanate groups, all of the isocyanate groups are attached to carbon atoms that are part of a closed ring of carbon atoms. This ring may be unsaturated at one or more positions, but does not acquire aromaticity as a result of the presence of a double bond.

[0031] Preferred polyisocyanates having cycloaliphatically bound isocyanate groups are 1,3- and 1,4-diisocyanatocyclohexane, 1,4-diisocyanato-3,3,5-trimethylcyclohexane, 1,3-diisocyanato-2-methylcyclohexane, 1,3-diisocyanato-4-methylcyclohexane, 1-isocyanato-3,3,5-trimethyl-5-isocyanatomethylcyclohexane (isophorone diisocyanate; IPDI), 1-isocyanato-1-methyl-4(3)-isocyanatomethylcyclohexane, 2,4'- and 4,4'-diisocyanatodicyclohexylmethane (H12MDI), 1,3- and 1,4-bis(isocyanato bis(isocyanatomethyl)cyclohexane, bis(isocyanatomethyl)norbornane (NBDI), 4,4'-diisocyanato-3,3'-dimethyldicyclohexylmethane, 4,4'-diisocyanato-3,3',5,5'-tetramethyldicyclohexylmethane, 4,4'-diisocyanato-1,1'-bi(cyclohexyl), 4,4'-diisocyanato-3,3'-dimethyl-1,1'-bi(cyclohexyl), 4,4'-diisocyanato-2,2',5,5'-tetramethyl-1,1'-bi(cyclohexyl), 1,8-diisocyanato-p-menthane, 1,3-diisocyanatoadamantane, and 1,3-dimethyl-5,7-diisocyanatoadamantane.

[0032] In isocyanates having araliphatically bonded isocyanate groups, all of the isocyanate groups are bonded to methylene radicals which are attached to an aromatic ring.

[0033] Preferred polyisocyanates having araliphatically bound isocyanate groups are 1,3- and 1,4-bis(isocyanatomethyl)benzene (xylylene diisocyanate; XDI), 1,3- and 1,4-bis(1-isocyanato-1-methylethyl)benzene (TMXDI), and bis(4-(1-isocyanato-1-methylethyl)phenyl)carbonate.

[0034] In isocyanates having aromatically bound isocyanate groups, all of the isocyanate groups are attached directly to carbon atoms that are part of an aromatic ring.

[0035] Preferred isocyanates having aromatically bound isocyanate groups are tolylene diisocyanate (TDI), methylene diphenyl isocyanate (MDI) and naphthylene diisocyanate.

[0036] The term "tolylene diisocyanate" refers to tolylene 2,4-diisocyanate (2,4-TDI), tolylene 2,6-diisocyanate (2,6-TDI), and any desired mixture of the two isomers. The term "methylene diphenylisocyanate" refers to all isomers of MDI, in particular diphenylmethane 2,2'-diisocyanate, diphenylmethane 2,4'-diisocyanate, diphenylmethane 4,4'-diisocyanate, all mixtures containing at least two of the aforementioned isomers, and polycyclic derivatives of MDI.

[0037] The term "naphthylene diisocyanate" refers to naphthylene 1,4-diisocyanate, naphthylene 1,5-diisocyanate and naphthylene 1,6-diisocyanate, as well as any desired mixtures of the foregoing isomers.

[0038] It is known to those skilled in the art that all the above polyisocyanates may exist as oligomers, and thus the urethane bond that is cleaved is formed between an oligomeric polyisocyanate based on one of the above monomeric diisocyanates and an alcohol.

[0039] The term "oligomeric polyisocyanates" refers to polyisocyanates composed of at least two of the above diisocyanates. The oligomeric polyisocyanates according to the invention may in particular have uretdione, isocyanurate, allophanate, biuret, iminooxadiazinedione and / or oxadiazinetrione structures. Preferably, the oligomeric polyisocyanates have at least one of the following oligomeric structure types or mixtures thereof: [ka]

[0040] A preferred substrate for the urethanease of the present invention is urethane obtainable from the aromatic polyisocyanates defined above.

[0041] enzymatic cleavage The expression "enzymatic cleavage of urethane group" indicates that the cleavage of said urethane group proceeds more quickly in the presence of a polypeptide having urethanase activity than in the presence of an enzyme-free reaction buffer under the same reaction conditions, or in the presence of an inactive polypeptide and reaction buffer under the same conditions. A preferred model of an inactive polypeptide is bovine serum albumin. If the cleavage of the urethane group proceeds more quickly in the presence of the polypeptide to be tested than in the otherwise identical control with BSA, then the polypeptide has urethanase activity, as understood in the present application.

[0042] nucleic acid 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 has urethanase activity. Preferably, the encoding nucleic acid sequence is under the control of a promoter allowing expression of the polypeptide in fungi, yeast or bacteria.

[0043] Purpose 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 as defined above in the present application, for the enzymatic cleavage of a urethane bond, characterized in that it has urethanase activity for the enzymatic cleavage of a urethane bond.

[0044] Unless otherwise expressly defined, all definitions given above also apply to this embodiment. The use preferably consists of contacting the enzyme according to the invention with a compound containing at least one urethane bond. This is preferably carried out under conditions under which the enzyme exhibits its urethanase activity. Such conditions can be found in the examples of this application. Moreover, the skilled person can easily adjust the reaction conditions in the system, such as temperature, ionic strength, solvent and pH, by a simple series of experiments, so that the enzyme is active.

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

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

[0047] Cleavage of low molecular weight urethanes. In yet a further embodiment, the present invention relates to a method comprising a process step of treating low molecular weight urethane with a polypeptide having urethanase activity, said polypeptide being selected from the group consisting of the polypeptides defined by SEQ ID NOs: 1, 2 and 3, and variants of these polypeptides as defined above in the present application, thereby cleaving the low molecular weight urethane.

[0048] In this context, the term "low molecular weight" means that the urethane contains no more than 4, preferably no more than 3, more preferably no more than 1 or 2 urethane groups.

[0049] Preferably, the structure of the low molecular weight urethane is formally based on the formation of one or more urethane bonds between a polyisocyanate and one or more low molecular weight alcohols.

[0050] Here, the polyisocyanates already mentioned above as partners in the urethane bond cleaved by the urethanase according to the invention are preferred. Particular preference is given to aromatic polyisocyanates, in particular tolylene 2,4-diisocyanate (2,4-TDI), tolylene 2,6-diisocyanate (2,6-TDI), diphenylmethane 2,2'-diisocyanate (2,2'-MDI), diphenylmethane 2,4'-diisocyanate (2,4'-MDI), diphenylmethane 4,4'-diisocyanate (4,4'-MDI), the polycyclic derivatives of the aforementioned diphenylmethane diisocyanates, naphthylene 1,4-diisocyanate, naphthylene 1,5-diisocyanate and naphthylene 1,6-diisocyanate.

[0051] In principle, any compound having at least one hydroxyl group per molecule is suitable as a low molecular weight alcohol, which preferably has a molecular weight of less than or equal to 700 g / mol, more preferably less than or equal to 500 g / mol, most preferably less than or equal to 200 g / mol.

[0052] Furthermore, low molecular weight alcohols having relatively high polarity are preferred. Particularly preferred are low molecular weight alcohols containing one or two hydroxyl groups per molecule. The melting point of the low molecular weight alcohol is preferably 45°C or less, more preferably 20°C or less.

[0053] Thus, alcohols having two hydroxyl groups, a molecular weight of less than or equal to 500 g / mol and a melting point of less than or equal to 45° C. are particularly preferred.

[0054] In a particularly preferred embodiment of the 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, glycerol, 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. Very particular preference is given to the low molecular weight alcohol diethylene glycol.

[0055] Low molecular weight urethanes which are particularly preferred for the process according to the invention can be obtained by forming a urethane bond with one of the low molecular weight alcohols mentioned in the previous paragraph, in particular with diethylene glycol.

[0056] Low molecular weight urethanes can be obtained in a variety of ways: In a preferred embodiment of the invention, the ester bonds of the polyester polyurethane are cleaved to release a carboxylic acid and a low molecular weight urethane.

[0057] In a preferred embodiment of the invention, the ester group is cleaved, preferably enzymatically, preferably using lipases (EC 3.1.1.3) or cutinases (EC 3.1.1.74).

[0058] In a further preferred embodiment of the present invention, low molecular weight urethanes are preferred which are obtained by chemical cleavage of polyether polyurethanes or polyester polyurethanes, said chemical cleavage being preferably the so-called "alcohololysis" or "glycolysis", in which the polyether polyurethanes or polyester polyurethanes are reacted with one of the abovementioned low molecular weight alcohols, so that the ether component of the polyether polyurethanes or the polyester polyol of the polyester polyurethanes are replaced by the low molecular weight alcohol.

[0059] A particularly preferred embodiment for providing low molecular weight urethane from polyether polyurethane is described in WO2021 / 032513. In this method, polyether polyurethane is reacted with low molecular weight alcohol so 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 isocyanate used in the synthesis of the polyether polyurethane. Thus, the low molecular weight urethane to be cleaved according to the present invention is a compound that is formally obtained by forming a urethane bond between a polyisocyanate, preferably one of the aliphatic, aromatic, araliphatic or cycloaliphatic polyisocyanates listed further above in this application, and a low molecular weight alcohol.

[0060] Polyester Polyurethane The term "polyester polyurethane" refers to a polyurethane whose polyol component contains polyester polyol. Preferably, at least 40% by weight of the hydroxyl groups present in the polyol component are constituents of polyester polyol. More preferably, this is at least 60% by weight, even more preferably at least 80% by weight, and most preferably at least 95% by weight. The polyester polyurethane may be foamed or non-foamed.

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

[0062] The term "polyester polyol" is known to those skilled in the art and describes polyesters containing an average of at least 1.5, preferably at least 1.8, more preferably at least 2.0 hydroxyl groups per molecule. The polyester polyols present in the polyurethane to be decomposed particularly preferably have a functionality of 1.5 to 6.0. They contain as structural elements aromatic and / or aliphatic polyols and aromatic and / or aliphatic polycarboxylic acids in any combination. Also preferred are polyester polyols obtained by ring-opening polymerization of lactones.

[0063] Polyether Polyurethane The term "polyether polyurethane" refers to a polyurethane, the polyol component of which contains polyether polyol. Preferably, at least 40% by weight of the hydroxyl groups present in the polyol component are constituents of polyether polyol. More preferably, this is at least 60% by weight, even more preferably at least 80% by weight, and most preferably at least 95% by weight. According to the present invention, the polyether polyurethane can also contain further polyols as structural components, while maintaining the above-mentioned proportions of polyether polyols. These are preferably polyester polyols.

[0064] The term "polyether polyols" is well known to those skilled in the art. These are polyethers with an average hydroxyl functionality of 1.5 to 6.0. The polyether polyols present in the polyether polyurethanes are preferably polyaddition products 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, bonded reactive hydrogen atoms.

[0065] 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, alternately in succession or as mixtures.

[0066] The "treatment" of the low molecular weight urethanes is carried out under conditions in which the enzymes used exhibit urethanase activity. Under these conditions, the reaction products of the enzymatic cleavage of the low molecular weight urethanes formed are low molecular weight alcohols or mixtures of low molecular weight alcohols and amines or mixtures of amines. The chemical structure of the amines formed depends on the nature of the polyisocyanate used to synthesize the urethanes. The amines liberated are amines that can be derived from the polyisocyanates used by the addition of moisture and subsequent elimination of CO2.

[0067] In yet a further embodiment, the present invention relates to the use of at least one polypeptide according to the invention for the cleavage of urethane bonds in low molecular weight urethane.

[0068] Decomposition of polyester urethane into low molecular weight degradation products. In yet another embodiment, the present invention provides a method for degrading a polyester polyurethane into low molecular weight degradation products, comprising the steps of: a) cleaving the ester groups present in the polyester polyurethane; and b) treating the polyurethane with a polypeptide having urethanase activity and selected from the group consisting of the polypeptides as defined in SEQ ID NOs: 1, 2 and 3 and the variants of said polypeptides as defined above in the present application; Including, However, it relates to a method, wherein process steps a) and b) can be carried out sequentially or in parallel.

[0069] Preferably, process step a) is carried out before process step b).

[0070] Unless otherwise stated, all definitions given further above in the present application also apply to this embodiment, in particular to the treatment of polyurethane with urethanase in process step b).

[0071] In a preferred embodiment of the present invention, the process step a) is carried out using a lipase or cutinase. It is particularly preferred to use a lipase capable of cleaving tributyrin. The process step a) is preferably carried out under reaction conditions under which the lipase or cutinase used is active. Such conditions can be determined by routine experimentation using common biochemical methods.

[0072] In another preferred embodiment, process step a) is carried out chemically.

[0073] The polyurethane comprises, as the isocyanate component, at least one aromatic, aliphatic or cycloaliphatic isocyanate. The polyurethane preferably comprises an aromatic isocyanate.

[0074] The low molecular weight degradation products of the polyester-based polyurethanes preferably have a molecular weight of 1000 g / mol or less. They are preferably (i) an amine derived from the isocyanate used in the preparation of the polyurethane, e.g., in the case of tolylene 2,4-diisocyanate, tolylene-2,4-diamine; and (ii) The alcohols and carboxylic acids used to form the polyester polyols used in the synthesis of the polyurethanes.

[0075] "Polyol" formed as a low molecular weight decomposition product in the process defined in this section is understood in the present application to mean any compound having at least two hydroxyl groups. Said polyol preferably has a molecular weight of 300 g / mol or less. Preferred polyols that are low molecular weight decomposition products of 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.

[0076] The "polycarboxylic acid" formed as a low molecular weight decomposition product in the process defined in this section is understood in the present application to mean any compound containing at least two carboxyl groups. Said polycarboxylic acid preferably has a molecular weight of 300 g / mol or less. Preferred polycarboxylic acids that are low molecular weight decomposition products of 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.

[0077] "Polyamine" formed as a low molecular weight decomposition product in the process defined in this section is understood in the present application to mean any compound containing at least two amino groups. Said polyamine preferably has a molecular weight of 300 g / mol or less. Preferred polyamines that are low molecular weight decomposition products of polyester-based polyurethane foams are selected from the group consisting of 4,4'-diaminodiphenylmethane, 2,4'-diaminodiphenylmethane, 2,2'-diaminodiphenylmethane, polycyclic derivatives of the above diaminodiphenylmethane compounds, tolylene-2,4-diamine, tolylene-2,6-diamine, naphthylene-1,4-diamine, naphthylene-1,5-diamine, naphthylene-1,6-diamine, hexamethylenediamine, isophoronediamine, xylylenediamine, pentamethylenediamine, paraphenylene diamine, butyldiamine, and H12-methylenediamine. Particularly preferred are polyamines selected from the group consisting of 4,4'-diaminodiphenylmethane, 2,4'-diaminodiphenylmethane, 2,2'-diaminodiphenylmethane, naphthylene-1,5-diamine, tolylene-2,4-diamine, and tolylene-2,6-diamine.

[0078] Decomposition of polyether polyurethane In yet another embodiment, the present invention relates to a method for producing a method for the treatment of a disease comprising the steps of: a) transurethanizing a polyether polyurethane with at least one low molecular weight alcohol to form a polyether polyol and a low molecular weight urethane; and b) enzymatically cleaving the low molecular weight urethane formed in process step a) with a polypeptide having urethanase activity and selected from the group consisting of the polypeptides as defined in SEQ ID NOs: 1, 2 and 3 and the variants of said polypeptides as defined above in the present application. The present invention relates to a method comprising the steps of:

[0079] Process step a) has two objectives: (i) the polyether polyol used to synthesize the polyurethane should be liberated as an isolatable compound from the polyurethane; (ii) the isocyanate used to synthesize the polyurethane should be present as a constituent of a low molecular weight urethane. In contrast to polyurethane with its high molecular weight, said low molecular weight urethane has its low molecular weight and, as a result, better solubility, which is well suited as a substrate for the enzymatic cleavage that takes place in process step b).

[0080] Unless otherwise stated, all definitions given further above in the application also apply to this embodiment, in particular to the enzymatic cleavage of the low molecular weight urethane in process step b).

[0081] In process step b), the amine and low molecular weight alcohol used in the transurethanization in process step a) are liberated by enzymatic cleavage of low molecular weight urethane. In addition, CO2 is generated in this process step. These compounds can be isolated by suitable separation methods and then further used. Here, it is preferred that the liberated low molecular weight alcohol is reused for the transurethanization carried out in process step a). The liberated amine is available as a pure and well-defined starting material for new synthesis.

[0082] Reaction conditions and catalysts suitable in principle 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 low molecular weight alcohol to polyether urethane is between 2:1 and 1:17. Particularly suitable as 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 (especially dibutyltin dilaurate, etc.), organic amines (especially diethanolamine, etc.), organometallic compounds (especially titanium tetrabutoxide, etc.) and tin compounds (especially tin octoate, etc.). The transurethanization is preferably carried out at temperatures in the range of 160°C to 270°C in the presence of 0.1% by weight to 5% by weight of catalyst, based on the weight of the polyurethane product added. [Brief description of the drawings]

[0083] [Figure 1] FIG. 1 shows the enzyme substrates used: pNPB: 4-nitrophenyl butyrate; ENPC: ethyl 4-nitrophenyl carbamate; MDA-BA: carbamate of 4,4'-MDI reacted with benzyl alcohol; TDA-ethoxyethanol: carbamate of toluene diisocyanate (TDI) reacted with ethoxyethanol; TDA-DEG: carbamate of TDI reacted with diethylene glycol; NDA-MEG: carbamate of NDI reacted with ethylene glycol; EMACC: carbamate obtained from reaction of 7-amino-4-methylcoumarin with ethyl chloroformate.

[0084] The following examples merely serve to elucidate the invention and are not intended to limit the scope of the claims in any way. EXAMPLES

[0085] Working Example Example 1: Activity determination using EMACC Preparation of enzymes The urethanase gene was cloned into the pET-26 expression vector immediately following the NdeI cleavage site. For expression, E. coli BL21(DE3) was transformed with the relevant plasmid. A single colony was used to inoculate an overnight culture in LB medium containing 1% (w / v) glucose and 50 μg / mL kanamycin. For the main culture, 200 ml of ZYP-5052 containing 50 μg / mL kanamycin was inoculated with 1 mL of the overnight culture and incubated at 37° C., 100 rpm in a baffled flask for 4 h. The temperature was then reduced to 20° C. before the culture was harvested by centrifugation at 4500 g for 20 min at 4° C. The cell pellet was stored at −20° C. before the protein was purified.

[0086] 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 by sonication on ice for 2 cycles (3 min, 50% pulse, 50% power). The lysate was clarified by centrifugation at 10000g, 4 °C for 40-60 min. The supernatant was stored on ice before loading onto the IMAC column.

[0087] IMAC resin (1 ml, ROTI® Garose His Beads, nickel form) was equilibrated with lysis buffer. The lysate was then applied to the column, followed by a washing step with at least 10 column volumes of wash buffer (50 mM NaPi, pH 8.0, 300 mM NaCl, 20 mM imidazole). The protein was then eluted with 15 mL of elution buffer (50 mM NaPi, pH 8.0, 300 mM NaCl, 250 mM imidazole). The volume of the eluted fraction was reduced to 2.5 mL using a Vivaspin 20 ultrafiltration unit (10 kDa MWCO). The protein was then rebuffered in storage buffer (10 mM NaPi, pH 8.0, 100 mM NaCl) using a PD10 column.

[0088] EMACC assay To determine urethanase activity, a fluorescent substrate was synthesized from 7-amino-4-methylcoumarin (AMC) and ethyl chloroformate. The ethyl carbamate of AMC (EMACC) thus formed was in the form of a fine powder. The specific activity was measured under standard conditions (50 mM NaPi, pH 8.0, 30 °C, 100 μM EMACC). EMACC was added freshly from a 50 mM DMSO stock. Purified urethanase was diluted in storage buffer before addition to the assay to give a linear increase in absorbance at 365 nm over 10-30 min. Measurements were performed in a 96-well plate in a plate photometer preheated to 30 °C, and the specific activity was calculated from the linear increase in absorbance in the first few minutes. The results are shown in Table 1. [Table 1]

[0089] Example 2: Screening for additional substrates Preparation of enzymes To prepare the enzyme preparations, a single colony of E. coli BL21(DE3)pET26b_UMG-8, BL21(DE3)pET26b_UMG-11.2 and BL21(DE3)pET26b_UMG-16 was used to inoculate 4.5 mL of LB medium containing 1% (w / v) glucose, respectively, which were incubated overnight in an incubation shaker at 37°C and 200 rpm. All cultures were mixed with 50 μg / mL of kanamycin. Then, 200 μL of each overnight culture was used to inoculate 200 mL of ZYP-5052 medium. The cultures were grown in an incubation shaker at 37°C and 200 rpm for 4 h, followed by 20°C and 200 rpm for 24 h. The cells were removed by centrifugation in a large-capacity centrifuge at 20425 g and 4°C for 20 min. Each cell pellet was suspended in 6 mL of disruption buffer (20 mM ammonium acetate, 0.4% n-dodecyl-β-maltoside, 1% lysozyme, 1 μL / mL benzonase (Sigma-Aldrich Chemie GmbH, Taufkirchen)) and incubated for 30 min at room temperature on an orbital shaker. Total cell extracts were then frozen at -80°C and lyophilized. Lyophilizates were stored at 4°C.

[0090] Enzyme reaction 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-nitrophenyl butyrate (pNPB) were used to confirm urethanase activity. The enzyme substrates are shown in Figure 1.

[0091] One spatula tip (~3 mg) of lyophilized whole cell extract was suspended in 800 μL of 100 mM KPi (pH 7.5) containing 100 mM NaCl. The enzyme solution was diluted 1:10 in the reaction mixture containing the same buffer. Carbamate substrate stocks were prepared in DMSO at concentrations ranging from 5 to 340 mg / mL. These were diluted 1:10 in the reaction mixture to a final DMSO concentration of 10% (v / v). Reactions were prepared in a total volume of 500 μL. Incubations were performed in a thermal shaker at 30 °C and 800-1000 rpm for 1 day, followed by 1 day at 40 °C and 800-1000 rpm. After incubation, 500 μL of acetonitrile was added to improve the solubility of the substrate and product. Samples were filtered through 0.2 μm PVDF filter plates by centrifugation and used for HPLC analysis. For the substrates EMACC and pNPB, the total reaction volume was 200 μL, and fluorescence (EMACC, excitation at 365 nm, emission at 440 nm) or absorbance (pNPB, 410 nm) was measured at various times in a Varioskan Lux plate photometer (Thermo Fisher Scientific Inc., Waltham, Massachusetts, USA).

[0092] HPLC analysis 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 the UV and visible light regions. All measurements were performed using a "Zorbax Eclipse Plus C18" column (Agilent Technologies, Santa Clara, USA) with a particle size of 5 μm and dimensions of 4.6 × 150 mm, with an appropriate pre-column. In all methods, 5 μL of sample was injected into the column heated to 40 °C. The flow rate was typically 1.0 mL / min.

[0093] Aromatic amines were detected using the “ER_Juni2021-2” method, which allowed quantification of aromatic amines at 210 nm and 232 nm without derivatization due to their high intrinsic absorption. ddH2O containing 5% (v / v) acetonitrile was used as eluent A, and acetonitrile containing 5% (v / v) ddH2O was used as eluent B. Data were analyzed using “OpenLAB CDS 2.4” software, version 2.204.0.661 (Agilent Technologies, Santa Clara, USA). [Table 2]

[0094] result The hydrolytic activity of uretases towards the tested carbamates was classified into the categories of "no activity" (-), "low activity" (+), "moderate activity" (++) and "high activity" (+++) based on the peak area in the chromatogram of the products formed. Uretanases UMG-8, UMG11.2 and UMG16 were found to accept a wide range of substrates. The substrates tested are shown in Figure 1. [Table 3]

[0095] Example 3: Optimization of reaction conditions To determine the pH optimum, the enzyme activity was determined as in Example 1 and reactions were carried out in different buffers (100 mM). The buffers used were those with pH 4.0, 5.0 and 6.0 (citrate), 7.0, 8.0 and 9.0 (bis-trispropane) and 10.0 (CHES). For pH values ​​of 10, 11, 12 and 13, the buffers used were those composed of 100 mM sodium phosphate and 100 mM sodium carbonate and, as a deviation from Example 1, a buffer containing 200 mM EMACC and 10% (v / v) DMSO.

[0096] Temperature optima were determined by measuring AMC release from 100 μM EMACC in 50 mM NaPi (pH 8.0) at 30° C., 35° C., 40° C., 45° C., 50° C., 55° C., 60° C., 65° C. and 70° C. Solvent stability was determined by quantifying EMAC hydrolysis under the conditions specified in Example 1 at 0%, 10%, 20%, 30%, 40% and 50% (v / v) DMSO. The activity optima determined herein for pH, temperature and DMSO concentration are summarized in Table 4. Activity dependencies are in Table 5. [Table 4-1] [Table 4-2] [Table 5]

[0097] temperature stability To determine the long-term temperature stability, a solution of purified uretase with a protein concentration of 0.5 mg / mL in 100 mM KPi, pH 7.5 was prepared. In each case 100 μL was incubated for 12 h at 20°C, 25.7°C, 29.4°C, 36.9°C, 40.6°C, 44.3°C and 50°C in a Biometra TAdvanced Basis thermal cycler (Analytik Jena GmbH, Jena) and then brought to 4°C for activity measurement. The temperature of the lid heater was adjusted to 99°C. A reference sample was incubated at 4°C. The residual activity of the enzyme was determined using the pNPB assay. For this purpose, 21 μL of pNPB were mixed with 4.979 mL of DMSO. The solution was diluted 1:10 in 100 mM KPi, pH 7.5. 20 μL 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) was loaded onto a microplate and the reaction was initiated by adding 180 μL of pNPB in 100 mM KPi, pH 7.5 containing 10% DMSO. When pNPB was hydrolyzed by urethanase, para-nitrophenol was released, 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 were as shown in Table 6. [Table 6]

Claims

1. A polypeptide having an amino acid sequence defined by SEQ ID NO: 1, 2 or 3, or a variant thereof, wherein the variants are obtained by addition, deletion or exchange of up to 15% of the amino acids present in the respective polypeptides defined by SEQ ID NO: 1, 2 or 3, A polypeptide characterized in that the polypeptide has urethanase activity.

2. Use of a polypeptide having the amino acid sequence defined by SEQ ID NO: 1, 2 or 3, or a variant of one of these polypeptides, wherein the variants are obtained by addition, deletion or exchange of up to 15% of the amino acids present in the respective polypeptides defined by SEQ ID NO: 1, 2 or 3, 2. The use according to claim 1, characterized in that the polypeptide has urethanase activity for the enzymatic cleavage of urethane bonds.

3. 3. The use according to claim 2, wherein the urethane groups are aromatically bound.

4. A nucleic acid encoding a polypeptide as defined by SEQ ID NO: 1, 2 or 3, or a variant of any of the aforementioned polypeptides, having uretase activity, wherein the variant is a nucleic acid obtained by addition, deletion or exchange of up to 15% of the amino acids present in the respective polypeptide defined by SEQ ID NO: 1, 2 or 3.

5. 1. A method comprising the process step of treating low molecular weight urethane with a polypeptide having urethanase activity and having an amino acid sequence as defined by SEQ ID NO: 1, 2 or 3, or a variant thereof, wherein the variants are obtained by addition, deletion or exchange of up to 15% of the amino acids present in each of the polypeptides defined by SEQ ID NO: 1, 2 or 3, whereby the low molecular weight urethane is cleaved.

6. 6. The method of claim 5, wherein at least one polyamine selected from the group consisting of 4,4'-diaminodiphenylmethane, 2,4'-diaminodiphenylmethane, 2,2'-diaminodiphenylmethane, polycyclic derivatives of said diaminodiphenylmethane compounds, tolylene-2,4-diamine, tolylene-2,6-diamine, naphthylene-1,4-diamine, naphthylene-1,5-diamine and tolylene-2,6-diamine is formed.

7. 7. The method according to claim 5 or 6, wherein an alcohol having at least two hydroxyl groups and a molecular weight of 700 g / mol or less is released.

8. 8. The method 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.

9. Use of at least one polypeptide having urethanase activity and having an amino acid sequence defined by SEQ ID NO: 1, 2 or 3, or having at least 85% sequence identity with the aforementioned sequences, for cleaving urethane bonds in low molecular weight urethanes.

10. 1. A method for degrading a polyester polyurethane into low molecular weight degradation products, comprising the steps of: a) cleaving ester groups present in the polyester polyurethane; and b) treating the polyurethane with a polypeptide having urethanase activity and selected from the group consisting of the polypeptides defined by SEQ ID NOs: 1, 2 and 3 and variants of said polypeptides, wherein the variants are obtained by addition, deletion or exchange of up to 15% of the amino acids present in each of the polypeptides defined by SEQ ID NO: 1, 2 or 3, Including, The method, however, wherein process steps a) and b) can be carried out sequentially or in parallel.

11. Steps below: a) transurethanizing a polyether polyurethane with at least one low molecular weight alcohol to form a polyether polyol and a low molecular weight urethane; and b) enzymatically cleaving the low molecular weight urethane formed in process step a) with a polypeptide having urethanase activity and selected from the group consisting of the polypeptides defined by SEQ ID NOs: 1, 2 and 3 and variants of said polypeptides, wherein the variants are obtained by addition, deletion or exchange of up to 15% of the amino acids present in each of the polypeptides defined by SEQ ID NO: 1, 2 or 3, A method comprising:

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