Biodegradable polyester polyurethanes by enzyme incorporation

Abstract

The present invention relates to polyester polyurethanes containing active esterases. These polymers are characterized by particularly good biodegradability under environmental conditions.

Description

[0001] 2024PF30157 Abroad

[0002] BIODEGRADABLE POLYESTER POLYURETHANES THROUGH INSTALLATION OF ENZYMES

[0003] The present invention relates to polyester polyurethanes containing active esterases. These polymers are characterized by particularly good biodegradability under environmental conditions.

[0004] Polyester polyurethanes are polymers used in many applications. In many cases, it is unavoidable that the products made from them, or parts thereof, are released into the environment instead of being disposed of properly. Due to the high persistence of these materials, this can be a problem.

[0005] Therefore, it would be desirable to improve the biodegradability of conventional polyester polyurethanes. Appropriately modified polyurethanes would not accumulate in the environment after their release. One factor limiting the biodegradability of polyester polyurethanes is their proportion of urethane groups, which form crystalline domains within the polymer. The presence of such crystalline domains is known to inhibit the enzymatic degradation of polyesters (Thomsen et al. (2024) “Enzymatic degradation of pol(ethylene terephthalate) (PET): Identifying the cause of the hypersensitive enzyme kinetic response to increased PET crystallinity”, Enzyme and Microbial Technology, 173: 110353). Since the biodegradation of polymers in the environment is often enzyme-based, this poses a problem.

[0006] The study underlying the present invention surprisingly demonstrated that the incorporation of esterases into polyester polyurethanes leads to the at least partial degradation of these polymers when incubated under conditions that allow the incorporated enzymes to be active. Model experiments have shown that modified polyester polyurethanes containing esterases that enable partial depolymerization of the polyester polyurethane are significantly more biodegradable under environmental conditions than polyester polyurethanes without esterases or with inactive esterases.

[0007] This makes it possible to modify conventional polyester polyurethanes so that they exhibit significantly lower persistence under environmental conditions. This is particularly advantageous for applications where these materials are intentionally released into the environment, e.g., in agriculture, or where unintentional release cannot be reliably prevented.

[0008] Bisceglie et al. (2022), “Cutinase-Catalyzed Polyester-Polyurethane Degradation: Elucidation of the Hydrolysis Mechanism”, Polymers, 14: 411, describe the partial degradation of a film formed by thermoplastic polyurethane. The enzyme was added to the finished film. The results shown do not indicate that the incorporation of an enzyme into a polyurethane of which

[0009] Biodegradability is improved. In particular, it is not shown that an enzyme incorporated into the polyurethane remains active despite exposure to heat or solvents.

[0010] EP 3 517 808 describes the cutinase defined by SEQ ID.: 1. Its activity is described only against polyethylene terephthalate. Incorporation into a polymer is mentioned in general terms. However, it is not shown that the enzyme can be used in this way without loss of activity. Heat stability is only shown up to temperatures of 75 °C.

[0011] Therefore, the present patent application discloses in the patent claims and the following description polyester polyurethanes with improved biodegradability.

[0012] In a first embodiment, the present invention relates to a polyester polyurethane composition comprising at least one polyester polyurethane and

[0013] (a) at least one enzyme defined by an amino acid sequence according to SEQ ID NO.: 1 or a variant of the aforementioned enzyme defined by an amino acid sequence with at least 85% identity to the conserved amino acids defined in SEQ ID NO.: 1; or

[0014] (b) at least one enzyme defined by an amino acid sequence according to SEQ ID NO.: 4 or 8 or a variant of one of the aforementioned enzymes defined by an amino acid sequence with at least 64% identity to SEQ ID NO.: 4 or at least 85% sequence identity to SEQ ID NO.: 8.

[0015] A polyester polyurethane composition is a composition containing at least one polyester polyurethane and at least one incorporated enzyme as defined above. It may optionally contain additives or other polymers. If the polyester polyurethane composition contains other polymers, it is preferred that, based on the total weight of all polymers contained therein, it consists of at least 80 wt.%, more preferably at least 90 wt.%, and most preferably at least 95 wt.% of one or more polyester polyurethanes. Enzymes are not treated as polymers in this calculation.

[0016] Polyester polyurethane

[0017] The polyester polyurethane is preferably a thermoplastic polyester polyurethane (TPU). TPUs suitable according to the invention are obtained by reacting at least one isocyanate component with at least one polyester polyol. They are particularly preferably obtained by reacting at least one isocyanate component with at least one polyester polyol and with at least one chain extender having a molecular weight between 60 and 500 g / mol. [2024PF30157 Ausland]

[0018] The molar ratio of isocyanate groups to isocyanate-reactive groups in the reaction mixture is preferably between 0.9 to 1.0 and 1.1 to 1.0. Isocyanate-reactive groups within the meaning of the present application are in particular hydroxyl, amino and thiol groups.

[0019] Those skilled in the art know that TPUs are block copolymers. These contain low-polarity segments composed of the isocyanates of the polyisocyanate component and the polyester polyol, and high-polarity segments composed of the isocyanates of the polyisocyanate component and the low-molecular-weight (60 to 500 g / mol) chain extender. The polar segments of different molecules are cross-linked via hydrogen bonds. Since these bonds can be reversibly broken at elevated temperatures, TPU can be melted and processed like ordinary thermoplastics. After cooling, the polar segments cross-link the different molecules, thus producing properties similar to those of an elastomer.These are therefore polymers whose melting points are significantly above room temperature, preferably between 120 and 250 °C, more preferably between 140 and 220 °C, and which are not dispersible in water.

[0020] For the present invention, this means that the enzyme is already contained in the polyurethane product during its use, e.g., as a film, and is not added to it subsequently. This is also not possible, since TPU is a solid that is insoluble in water.

[0021] TPUs are preferably obtained from reaction mixtures containing at least one polyester polyol between 50 wt.% and 70 wt.% by weight. A TPU as defined in this application may contain other polyols besides the polyester polyol, in particular polycarbonate polyols and / or polyether polyols. However, these other polyols preferably have a weight fraction of the total weight of the reaction mixture that does not exceed 20 wt.%.

[0022] The term "isocyanate component" refers to the entirety of all compounds used for the synthesis of a TPU and containing at least one isocyanate group per molecule. Particularly preferably, the isocyanate component contains at least one compound with an average functionality of at least two isocyanate groups per molecule, hereinafter also referred to as "polyisocyanate". Suitable polyisocyanates can contain aliphatic, cycloaliphatic, or aromatically bound isocyanate groups.

[0023] Suitable polyisocyanates with aromatically bonded isocyanate groups are 2,4- and 2,6-diisocyanatotoluene (TDI), 2,4'-, 4,4'-diisocyanatodiphenylmethane (MDI), their polynuclear homologues, 1,5-diisocyanatonaphthalene, and oligomeric polyisocyanates based on the aforementioned monomeric polyisocyanates. 2024PF30157 Abroad

[0024] Suitable polyisocyanates with aliphatic 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, 1,10-diisocyanatodecane and the oligomeric polyisocyanates based on the aforementioned monomeric polyisocyanates.

[0025] Suitable polyisocyanates with cycloaliphatic 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-isocyanatomethyl-cyclohexane isophorone diisocyanate; (IPDI) 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, 1,3-Dimethyl-5,7-diisocyanatoadamantane and the oligomeric polyisocyanates based on the aforementioned diisocyanates.

[0026] The term "oligomeric polyisocyanates" refers to reaction products obtained by crosslinking at least two monomeric isocyanates to form at least one uretdione, isocyanurate, allophane, biuret, iminooxadiazindione, and / or oxadiazinetrione structure. Preferably, oligomeric polyisocyanates contain two to four molecules of a monomeric polyisocyanate.

[0027] The polyester polyols used are preferably composed of dicarboxylic acids with 2 to 12, preferably 4 to 6 carbon atoms, and polyhydric alcohols. Preferred dicarboxylic acids are succinic acid, adipic acid, and glutaric acid. It is also possible to use mixtures of different dicarboxylic acids. Instead of dicarboxylic acids, dicarboxylic acid esters can also be used for the synthesis. These preferably have 1 to 4 carbon atoms in the alcohol residue. Alternatively, carboxylic anhydrides or carboxylic acid chlorides can also be used for the synthesis. Preferred polyhydric alcohols are glycols with 2 to 10, preferably 2 to 6 carbon atoms, in particular ethylene glycol, diethylene glycol, 1,4-butanediol, 1,5-pentanediol, 1,6-hexanediol, 1,10-decanediol, 2,2-dimethyl-1,3-propanediol, 1,3-propanediol, and dipropylene glycol. Depending on the desired properties of the TPU, the polyhydric alcohols can be used alone or, if necessary, in mixtures with each other.Also suitable are esters of carbonic acid with the aforementioned diols, in particular those with 4 to 6 carbon atoms, especially 1,4-butanediol or 1,6-hexanediol, condensation products of hydroxycarboxylic acids, preferably hydroxycaproic acid, and polymerization products of lactones, preferably caprolactones. 2024PF30157 Foreign.

[0028] Caprolactones may be substituted. The polyester polyols preferably have a number-average molecular weight between 500 and 4,000 g / mol.

[0029] Preferred polyester polyols for the production of TPUs are ethanediol polyadipates, 1,4-butanediol polyadipates, ethanediol-1,4-butanediol polyadipates, 1,6-hexanediol neopentylglycol polyadipates, 1,6-hexanediol-1,4-butanediol polyadipates and polycaprolactones.

[0030] TPUs particularly suitable according to the invention have a number-average molecular weight between 500 and 4,000 g / mol, preferably between 10,000 and 200,000 g / mol. They particularly preferably contain polyester polyols containing 1,4-butanediol. Their polyisocyanate component particularly preferably contains methylene diphenyl diisocyanate (MDI) or hexamethylene diisocyanate (HDI).

[0031] Preferred chain extenders with a molecular weight between 60 and 500 g / mol have two isocyanate-reactive groups. Aliphatic diols with 2 to 14 carbon atoms are particularly preferred. Particularly preferred are ethanediol, propane-1,3-diol, pentane-1,5-diol, hexane-1,6-diol, octane-1,8-diol, decane-1,10-diol, dodecane-1,12-diol, diethylene glycol, dipropylene glycol, and butane-1,4-diol.Also suitable are diesters of terephthalic acid with glycols containing 2 to 4 carbon atoms, preferably bis(ethylene glycol) terephthalate or bis(butane-1,4-diol) terephthalate, hydroxyalkylene ethers of hydroquinone, preferably 1,4-di(β-hydroxyethyl)hydroquinone, ethoxylated bisphenols, preferably 1,4-di(β-hydroxyethyl)bisphenol A, (cyclo)aliphatic diamines, preferably isophoronediamine, ethylenediamine, propylene-1,2-diamine, propylene-1,3-diamine, N-methylpropylene-1,3-diamine, N,N'-dimethylethylenediamine and aromatic diamines such as tolylene-2,4-diamine, tolylene-2,6-diamine, 3,5-diethyltolylene-2,4-diamine and / or 3,5-Diethyltolylene-2,6-diamine and primary mono-, di-, tri-, and / or tetraalkyl-substituted 4,4'-diaminodiphenylmethanes. Mixtures of the aforementioned chain extenders can also be used. Small amounts of trioiens can also be added, provided the resulting product retains thermoplastic properties.

[0032] Enzymes

[0033] Table 1: Amino acid sequences of the enzymes used

[0034]

[0035] 2024PF30157 Abroad

[0036]

[0037] Enzymes with sequence identities defined elsewhere in this application, corresponding to the enzymes defined by SEQ ID NO.: 2 to SEQ ID NO.: 10 or to the consensus sequence defined by SEQ ID NO.: 1, can be obtained by adding, exchanging, or deleting amino acids. Such enzymes with amino acid sequences that differ from those of SEQ ID NO.: 1 to 10 are also referred to as "variants." Each variant according to the invention is preferably characterized in that it can cleave ester groups in a polyester polyurethane. This enzyme activity is preferably detected by a decrease in pH caused by the cleavage of the polyester polyurethane component. This can be done using any suitable method, such as those described in the exemplary embodiments. It is particularly preferably done by chromatographic detection of the monomers released after ester hydrolysis.

[0038] In a preferred embodiment, the polyester polyurethane composition contains at least one enzyme whose amino acid sequence is defined as follows: (i) The amino acid positions of the enzyme homologous to the conserved amino acid positions according to SEQ ID NO.: 1 exhibit at least 90%, more preferably at least 95%, sequence identity with the conserved amino acid positions from SEQ ID NO.: 1, and (ii) the amino acid positions homologous to any amino acid positions from SEQ ID NO.: 1 exhibit at least 70%, preferably at least 80%, sequence identity with the homologous amino acid positions of one of the amino acid sequences selected from the group consisting of SEQ ID NO.: 5, 2, 3, 4, 6, 7, 8, 9, and 10. Preferably, the arbitrary amino acid positions exhibit the aforementioned sequence identities with SEQ ID NO.: 4 or 8.

[0039] In other words, the enzyme in this embodiment is characterized in that its functionally important amino acid positions have a high degree of similarity to the consensus sequence, while the non-conserved and functionally less relevant positions with greater sequence deviation are derived from one of the enzymes identified as active in the study underlying this application.

[0040] In a preferred embodiment of the present invention, the variant defined above of an enzyme defined by SEQ ID NO.: 4 is characterized by a sequence identity of at least 74%, preferably at least 80%, more preferably at least 85%, and even more preferably 2024PF30157 Abroad

[0041] at least 90% and most preferably at least 95% characterized by the amino acid sequence defined by SEQ ID NO: 4.

[0042] In a further preferred embodiment of the present invention, the variant defined above of an enzyme defined by SEQ ID NO.: 8 is characterized by a sequence identity of at least 90% and preferably at least 95% with the amino acid sequence defined by SEQ ID NO. 8.

[0043] In yet another embodiment, the polyester polyurethane composition contains at least one enzyme having an amino acid sequence as defined in SEQ ID NO.: 5, 2, 3, 4, 6, 7, 8, 9 or 10, or a variant of one of the aforementioned enzymes with at least 85% sequence identity to one of the aforementioned sequences.

[0044] In a preferred embodiment, the polyester polyurethane composition contains at least one enzyme having an amino acid sequence as defined in SEQ ID NO.: 5, 3, 4 or 10, or a variant of one of the aforementioned enzymes with at least 85% sequence identity to one of the aforementioned sequences.

[0045] In a particularly preferred embodiment, the polyester polyurethane composition contains at least one enzyme having an amino acid sequence as defined in SEQ ID NO.: 5 or a variant of this enzyme with at least 85% sequence identity to the aforementioned sequence.

[0046] A variant is preferably obtained from the amino acid sequences according to the invention by adding, deleting, or exchanging the respective defined proportion of the amino acids contained in the respective enzyme. The basis for calculating the sequence identity is preferably the amino acid sequence defined by SEQ ID NO.: 2, 3, 4, 5, 6, 7, 8, 9, or 10. It is known to those skilled in the art that fusion proteins are formed by enzymes with other proteins without this affecting the activity of the enzyme. Therefore, the term "variant" also includes amino acid sequences derived from the polypeptides defined by SEQ ID NO.: 2, 3, 4, 5, 6, 7, 8, 9, or 10, which are fused at the N-terminus and / or the C-terminus with other proteins, e.g., the Green Fluorescent Protein.

[0047] Particularly preferred variants of the enzymes 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 carried out continuously or discontinuously at any desired position of the polypeptide in question. Preferably, however, they are carried out only at the N-terminus and / or the C-terminus of the polypeptide. 2024PF30157 Abroad

[0048] In a preferred embodiment of the present invention, the polyester polyurethane composition contains more than one enzyme, in particular mixtures of at least two or three different enzymes. This makes it possible to exploit synergistic effects of enzymes with different activity optima or different substrate specificity.

[0049] The polyester polyurethane composition according to the invention contains between 0.1 wt% and 2 wt% of the enzyme(s) based on the total weight of the polyester polyurethanes contained therein. A concentration of 0.1 wt% to 1.0 wt% is more preferred.

[0050] The study underlying the present application has surprisingly shown that the purity of the enzyme preparation used has a significant influence on the enzyme activity in polyester polyurethane and, in particular, favorably affects the enzyme's thermostability. Purer enzymes have higher thermostability and can therefore be incorporated into the polyester polyurethane at higher temperatures than less pure enzymes. The production costs for an enzyme-containing polyester polyurethane composition are lower when the enzymes are used in the form of a so-called "crude extract." This is the mixture of proteins and other soluble cell components, especially nucleic acids, sugars, and amino acids, obtained by lysis of the cells used for enzyme expression. Lysis is preferably carried out by ultrasound.The separation of insoluble cell components after lysis is preferably carried out by centrifugation.

[0051] A “purified enzyme composition” within the meaning of the present application is preferably a composition in which the proportion of other soluble cell components and / or proteins that do not have the amino acid sequence of the enzymes to be used according to the invention is reduced compared to the crude extract. Conversely, this means that the proportion of the enzyme(s) to be used according to the invention is increased in a purified enzyme composition compared to the crude extract.

[0052] It is particularly preferred that the proportion of the enzyme(s) to be used according to the invention in the total protein content of the composition is at least 25 wt.%. Preferably it is at least 35 wt.%, more preferably at least 50 wt.%, and even more preferably at least 70 wt.%. It is most particularly preferred that, while adhering to the aforementioned threshold values, the proportion of the enzyme to be used according to the invention in the total protein content of the composition in the purified enzyme composition is increased by a factor of at least 2 compared to the crude extract.

[0053] It is further particularly preferred that the proportion of soluble cell components that are not proteins in the purified enzyme composition is reduced by at least 80 wt.% compared to the crude extract, 2024PF30157 Abroad

[0054] more preferably, the concentration is reduced by at least 90 wt.%. These values ​​are based on the total mass of the insoluble cell components originally present in the crude extract. "Insoluble cell components" are preferably sugars, nucleic acids, oligopeptides, and organic molecules with a molecular weight of no more than 2,500 g / mol. In this application, "oligopeptide" is understood to mean peptides containing no more than 20 amino acids. Peptides with more than 20 amino acids are defined here as proteins.

[0055] It is particularly preferred that a purified enzyme composition is characterized both by a proportion of the enzyme to be used according to the invention in the total protein content as defined above, and by a reduction in the proportion of soluble cell components as defined above.

[0056] In a preferred embodiment of the present invention, the polyester polyurethane composition according to the invention contains the enzyme to be used according to the invention or a plurality of the enzymes to be used according to the invention as component(s) in the form of a purified enzyme composition as defined in the preceding paragraph.

[0057] The polyester polyurethane compositions according to the invention are characterized by improved biodegradability. "Biodegradability" here refers to the decomposition of the polyester polyurethanes contained in the polyester polyurethane compositions by microorganisms. The products of decomposition are preferably microbial biomass, carbon dioxide, and water.

[0058] In a further preferred embodiment of the present application, the polyester polyurethane composition contains at least one acid scavenger. Preferably, the acid scavenger is an inorganic salt selected from the group consisting of carbonates, hydrogen carbonates, phosphates, and hydroxides. Preferred carbonates are sodium carbonate, potassium carbonate, and calcium carbonate. Preferred hydrogen carbonates are sodium hydrogen carbonate, potassium hydrogen carbonate, and calcium hydrogen carbonate. Preferred phosphates are sodium phosphate, disodium hydrogen phosphate, and sodium dihydrogen phosphate. A preferred hydroxide is magnesium hydroxide. Acid scavengers, in particular the aforementioned inorganic salts, preferably have a weight fraction of between 5 wt.% and 25 wt.% of the polyester polyurethane composition. These values ​​are based on the total weight of the polyester polyurethanes contained in the polyester polyurethane composition.

[0059] In yet another embodiment, the present application relates to the use of an enzyme, which 2024PF30157 foreign

[0060] (i) is defined by an amino acid sequence according to SEQ ID NO.: 1 or a variant of the aforementioned enzyme defined by an amino acid sequence with at least 85% identity to the conserved amino acids defined in SEQ ID NO.: 1; or

[0061] (ii) is defined by an amino acid sequence according to SEQ ID NO.: 4 or 8 or a variant of one of the aforementioned enzymes defined by an amino acid sequence with at least 64% identity to SEQ ID NO.: 4 or at least 85% sequence identity to SEQ ID NO.: 8;

[0062] to improve the biodegradability of a polyester polyurethane.

[0063] All definitions given above in this application, in particular concerning the enzyme and the polyester polyurethane, also apply to this embodiment.

[0064] The use of the enzyme consists of incorporating the enzyme into a composition containing at least one polyester polyurethane as defined above. Preferably, this is a polyester polyurethane composition contained in polymer products that are intentionally released into the environment. However, it can also be a polyester polyurethane composition contained in polymer products where it cannot be ruled out that they will remain in the environment, even if this is not intended. The latter applies to products, e.g., packaging materials, that are generally intended for regular waste disposal, but where it cannot be ruled out that parts of them will remain in the environment, e.g., due to carelessness. In this application, "environment" preferably refers to soil.

[0065] Training

[0066] To obtain a polyester polyurethane composition containing at least one of the enzymes that can be used according to the invention, the at least one enzyme is preferably incorporated into the previously produced polymer. In other words, the enzyme is preferably not introduced into the reaction mixture during the synthesis of the polyester polyurethane.

[0067] There are basically two preferred methods for incorporating an enzyme into the finished polyester polyurethane.

[0068] In a first embodiment, at least one polyester polyurethane is dissolved in a suitable solvent, and the at least one enzyme is added to the dissolved polymer. Evaporation of the solvent then yields the polyester polyurethane containing the at least one enzyme. This process is advantageous because it does not subject the enzymes to any temperature stress.

[0069] Suitable solvents include, in particular, tetrahydrofuran (THF), acetone, methylethylketone, toluene, cyclohexanone, trichloroethylene, and n-hexane. THF is especially preferred.

[0070] In another embodiment, at least one TPU is melted and the enzyme is added to the melt. The liquid TPU can then be shaped using any method known to those skilled in the art. This can be done particularly advantageously by casting, especially injection molding, or by extrusion. After solidification, a thermoplastic polyester polyurethane containing the at least one enzyme is present. Since this method of incorporation requires heating the TPU above its melting point, the enzyme is also exposed to the corresponding temperatures. Therefore, for this embodiment, it is preferred to use thermostable enzymes that largely retain their activity even after treatment at the required temperatures. Preferred thermostable enzymes are HiC (SEQ ID No. 5), ThcCutl-ACCG (SEQ ID No. 3), SvCutl90*SS (SEQ ID No. 4), and LCC-ICCG (SEQ ID No. 10) and their variants defined above.

[0071] The incorporation of the enzymes according to the invention into at least one polyester polyurethane results in a polyester polyurethane composition that degrades up to 30% faster under environmental conditions than a composition without an active enzyme. Therefore, if the corresponding polyester polyurethane compositions are released into the environment – ​​intentionally or accidentally – there is a low risk that they will accumulate there and have harmful effects.

[0072] In a further embodiment, the present invention relates to a polymer product containing or consisting of the polyurethane composition according to the invention. The polymer product is preferably selected from the group consisting of packaging materials, plant fasteners, in particular trellises, plastic components of fireworks, broom bristles, animal marking tapes, and waste bags, in particular for biowaste, which contains the polyester polyurethane composition according to the invention.

[0073] Proceedings

[0074] In a further embodiment, the present invention relates to a method for producing a polyester polyurethane composition comprising the steps

[0075] a) Provision of at least one polyester polyurethane and at least one enzyme which

[0076] (i) is defined by an amino acid sequence according to SEQ ID NO.: 1 or a variant of the aforementioned enzyme defined by an amino acid sequence 2024PF30157 Abroad

[0077] defined with at least 85% identity to the conserved amino acids defined in SEQ ID NO.: 1; or

[0078] (ii) is defined by an amino acid sequence according to SEQ ID NO.: 4 or 8 or a variant of one of the aforementioned enzymes defined by an amino acid sequence with at least 64% identity to SEQ ID NO.: 4 or at least 85% sequence identity to SEQ ID NO.: 8;

[0079] b) Mixing the at least one polyester polyurethane with the at least one enzyme, wherein the at least one polyester polyurethane is present either as a melt or dissolved in a suitable solvent; and

[0080] c) Curing of the polyester polyurethane composition by cooling the melt or by removing the solvent.

[0081] All the definitions given above also apply to this embodiment.

[0082] In yet another embodiment, the present invention relates to a polyester polyurethane composition obtained or obtainable by the method defined above.

[0083] The following embodiments serve only to illustrate the invention. They are not intended to limit the scope of protection of the patent claims in any way. 2024PF30157 Abroad

[0084] Examples

[0085] Enzymes

[0086] The commercially available enzyme, which presumably has the amino acid sequence according to SEQ. ID NO.: 5, was obtained from Strem Chemicals (Bischheim, France).

[0087] Plasmid construction

[0088] The gene for SvCutl90*SS was synthesized by Eurofins Genomics (Ebersberg, Germany) and cloned into the pET26b(+) vector from Merck (Darmstadt, Germany) using restriction cloning with Xhol and Ndel. The genes for LCC-ICCG and ThcCutl were also cloned into the pET26b(+) vector from Merck (Darmstadt, Germany) using restriction cloning with Xhol and Ndel. ThcCutl-ACCG was generated from the ThcCutl gene contained in the pET26b(+) vector using mutation PCR. The plasmids were propagated by transforming chemically competent E. coli DH5a cells. Grown single colonies were used for inoculation of 4.5 ml LB medium containing 50 pg / ml kanamycin and either preserved in 20% glycerol stocks or prepared for sequencing at GeneWiz (Leipzig, Germany) according to the manufacturer's instructions.

[0089] Heterologous protein expression

[0090] Heterologous expression of the enzymes was performed in E. coli BL21 (DE3) cells transformed with the plasmid construct. 4.5 ml of LB medium containing 1% w / v glucose and 50 pg / ml kanamycin were inoculated from a glycerol stock culture and incubated overnight at 37 °C and 200 rpm. 200 ml of ZYP-5052 autoinduction medium containing 50 pg / ml kanamycin were inoculated with 0.2% freshly grown cells and incubated in flasks at 37 °C and 200 rpm for 4 hours. Expression was performed for 24 hours at 20 °C and 200 rpm. The cells were harvested at 4 °C for 20 minutes at 8,000 x g.

[0091] Cell lysis

[0092] The harvested cells were resuspended in 10 ml of 20 mM KPi buffer, pH 7.5. The cells were disrupted three times using ultrasound (Bandelin Sonoplus HD, Sonotrode MS73, Berlin, Germany) under constant cooling in an ice bath for 2 min (amplitude: 50%). The resulting suspension was centrifuged at 10,000 x g and 4 °C for 40 min. The supernatant was filtered through a 0.2 pm PES filter.

[0093] Lyophilization of the cell-free expression culture supernatant

[0094] The filtered supernatant of the lysed cells was frozen at -80 °C. Subsequently, the frozen cell-free supernatant was lyophilized for 3 days at a refrigerator temperature of -65 °C and a pressure of 0.1 mbar. The enzyme lyophilisate was stored at -4 °C.

[0095] Production of Novozym® 51032 Lyophilisate 2024PF30157 Abroad

[0096] The commercially available liquid preparation Novozym® 51032 was rebuffered via crossflow filtration using a Vivaflow 50 MWCO 10000 cassette to replace the glycerol in the stock solution with ammonium acetate buffer (20 mM). The rebuffered enzyme was then lyophilized for 3 days at a cooler temperature of -65 °C and a pressure of 0.1 mbar. The resulting enzyme lyophilisate was stored at 4 °C.

[0097] Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)

[0098] Enzyme expression and protein purity were investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). For this purpose, a culture volume equivalent to 1 ml with an optical density of 5 (at 600 nm) was taken and centrifuged at 13,000 rpm for 2 min. The supernatant was discarded, and the cell pellet was resuspended in 200 µl of 1x PBS buffer. 200 µl of BugBuster® protein extraction reagent containing 1 µl / ml benzoase was added to each cell pellet. For cell lysis, the samples were incubated at room temperature for 20 min with occasional inversion. After lysis, 14 µl of the lysed cell suspension was mixed with 7 µl of 3x reducing blue protein loading dye (= whole cell extract). The remainder of the lysed cell suspension was centrifuged at 13,000 rpm for 5 min. From the resulting supernatant, 14 pl were mixed with 7 pl of 3x Reducing Blue Protein Loading Dye (= soluble enzyme fraction). Both mixtures were incubated for 5 min at 95 °C.15 pl of the samples were loaded onto a pre-cast NuPAGE 4-12% Bis-Tris gel along with 3 pl of EZ-Run® Prestained Rec Protein Ladder. The proteins were separated electrophoretically at a constant voltage of 200 V for 40 min. The gels were stained for 2 hours with 30-50 ml of ready-to-use Roti BlueQuick staining solution and then destained with ultrapure water for 30-60 min.

[0099] Determination of protein concentration

[0100] Protein concentration was determined colorimetrically using the Bradford assay. A standard bovine serum albumin (BSA) curve of 125–1,000 pg / ml was prepared. The lyophilized enzymes and the blank vector control were dissolved in ultrapure water and diluted with ultrapure water as needed. 250 pl of Quick Start Bradford reagent, 1x, was added to 5 µL of BSA and enzyme solution. After 5 min incubation, the absorbance was measured at 595 nm.

[0101] Enzyme activity test using p-nitrophenylbutyrate (pNPB)

[0102] Enzyme activity and that of the empty vector control were determined using the ester hydrolysis substrate p-nitrophenylbutyrate (pNPB), whose hydrolysis leads to the release of p-nitrophenol (pNP), which was measured photometrically. For this purpose, a pNP standard curve of 0.001–0.05 mg / ml was prepared, and the absorbance at 410 nm was measured (pNP, Sigma-Aldrich). For enzyme activity measurements, the lyophilized enzymes were dissolved in ultrapure water and, if necessary, diluted with ultrapure water. The pNPB substrate solution was prepared by adjusting the commercially available pNPB preparation (Sigma-Aldrich) to a concentration of 5 mg / ml with DMSO. Subsequently, the pNPB solution was diluted 1:10 with KPi buffer 2024PF30157 (foreign countries).

[0103] (100 mM, pH 7.0) to a final concentration of 0.5 mg / ml. 180 pl of 0.5 mg / ml pNPB solution were added to 20 pl of enzyme solution. The absorbance of pNP was measured every 15 seconds at 410 nm and 30°C over a period of 15 minutes. The activity was calculated from the initial slope.

[0104] Thermoplastic polyester polyurethanes

[0105] TPU 1 consists of 63.01 wt% polyester polyol (based on adipic acid and 1,4-butanediol) with a number-average molecular weight of 2250 g / mol, as well as 1,4-butanediol and MDI.

[0106] TPU 2 consists of 59.07 wt% polyester polyol (based on adipic acid and 1,4-butanediol) with a number-average molecular weight of 2250 g / mol, as well as 1,4-butanediol, HDI and a lactide-based polyol.

[0107] Incorporation of enzymes via melt extrusion

[0108] A twin-screw micro-compounder, model Xplore MC 15 (DSM), was used for incorporating enzymes into TPUs. In the case of TPU 1, the TPU granules were milled to a particle size of < 1 mm with the addition of liquid nitrogen, while TPU 2 was used unmilled with a particle size of 3–5 mm. In both cases, the TPU was dried at 70°C for 48 hours before extrusion. The enzymes to be incorporated were prepared as a lyophilisate for extrusion, consisting of the cell-free supernatant from an expression culture. The commercially available Novozym® 51032 preparation was incorporated after buffering and freeze-drying. To ensure homogeneous enzyme distribution, a premix of TPU and enzyme lyophilisate was prepared, containing 0.3 wt% enzyme by weight of TPU. For TPU 2, an additional formulation was prepared containing 20 wt% calcium carbonate and 0.3 wt% enzyme content based on the TPU weight.Furthermore, preparations for rinsing the extruder between each sample change were made using both TPUs, with TPU also being mixed with 0.3 wt% enzyme lyophilisate. The sample filling time of the extruder was less than 1 minute. After the entire sample was filled into the mixing section of the extruder, compounding was carried out for 1:30 minutes at the set melting temperature (Table 2) with a rotation speed of 50 rpm for both extruder screws, before the TPU exited the extruder as a strand. The samples were cooled at room temperature and dried for 24 hours at 70°C before degradation studies were performed.

[0109] Table 2: Overview of the extrusion conditions for incorporating the enzymes into TPUs 1 and 2.

[0110]

[0111] 2024PF30157 Abroad

[0112]

[0113] Incorporation of enzymes using a solvent-based method

[0114] For the solvent-based incorporation of the cutinases Novozym® 51032, ThcCutl, ThcCutl-ACCG, SvCutl90*SS, LCC ICCG, and the blank vector control pET26b_LV into TPU 1, a 5 wt% TPU solution in THF was prepared. The TPU granules were dissolved under magnetic stirrer for 48 hours. After the TPU was almost completely dissolved, the lyophilisates were added to suitable glass dishes at concentrations of 0.3 wt% and 0.5 wt% based on the polymer weight, the corresponding amount of TPU solution was added, and everything was mixed by pipetting.

[0115] For the solvent-based incorporation of the enzymes LCC, LCC-WCCG, TfCut2, and PesHl into TPU 1, a 4 wt% TPU solution in THF was prepared. The TPU granules were dissolved in a magnetic stirrer for 24 hours. After the TPU was completely dissolved, the lyophilisates were added to suitable glass dishes at concentrations of 0.5 wt% based on the polymer weight, the corresponding amount of TPU solution was added, and everything was mixed by pipetting.

[0116] For the negative control without enzyme addition, pure TPU solution was transferred to a glass dish. In all cases, the volumes were chosen so that the films had the same thickness. The THF was evaporated for 2 days at room temperature, and the dried films were used for the degradation studies.

[0117] PU degradation studies under laboratory conditions in an aqueous system

[0118] TPU degradation studies were performed using a standard 200 mg TPU sample. The TPU samples (film or strand) were dried overnight at 70°C in a convection oven. Subsequently, the samples were cut into equal-sized pieces and, in the case of TPU films, weighed into 15 ml centrifugation tubes, or, in the case of TPU strands, into the wells of a deep-well plate. The degradation study of samples with incorporated enzymes was initiated by adding 5 ml of KPi buffer (200 mM, pH 8.0). Control reactions were prepared by adding external enzymes and KPi, ensuring that the final volume did not exceed 5 ml. For the external enzyme addition, the respective enzyme lyophilisates and the blank vector lyophilisate were dissolved in ultrapure water, sterile-filtered through a 0.2 pm PES filter, and added to each TPU sample at final concentrations of 0.3 wt% or 0.5 wt%. For the negative controls, only 5 ml of KPi buffer was added to the samples.All samples were incubated at 37°C for a period of at least 14,2024PF30157 abroad.

[0119] The standard duration was 21 days, and in some cases 33 days, at 150-200 rpm. For adipic acid quantification, 150 pl of supernatant were taken at specific time points and stored at -20°C.

[0120] HPLC measurements

[0121] HPLC measurements were performed on an Agilent 1260 Infinity IL system (Santa Clara, USA). The LC system was equipped with a multisampler (G7167A), a DAD (diode array detector) for UV and visible light, and a Zorbax Eclipse Plus C18 column (4.6 mm x 150 mm, 5 pm, Agilent, Santa Clara, USA). Adipic acid was measured using a gradient method consisting of aqueous mobile phase (0.085% v / v phosphoric acid in demineralized water) and 99.8% acetonitrile at a column temperature of 35°C (Table 3). The mobile phase flow rate was 1.5 ml / min, and 5 pl of sample was injected. The data were analyzed using Chromeeon Chromatography Data System 7.3.1 software (Thermo Scientific, Waltham, USA).

[0122] Table 3: HPLC method with mobile phase gradient for adipic acid quantification.

[0123]

[0124] TPU biodegradability studies in the biological system (according to OECD 301 F)

[0125] The biodegradability studies were conducted according to OECD (Organisation for Economic Co-operation and Development) guideline 301 F on the "manometric respiratory determination of the biodegradability of chemical test materials." The Lovibond BD 600 system was used. For the biodegradability study, a predetermined quantity of the substance to be tested was dissolved in the test medium (minimal salt medium with sewage sludge). The solution was incubated in a closed bottle in the dark, with stirring, at a constant temperature of 20°C for 90 days. The biodegradation of the test substance was measured by determining the biochemical oxygen demand (BOD). The following steps were performed:

[0126] 1. Calculation of the amount of test substance required (Annex IV in OECD 301)

[0127] a) Theoretical oxygen demand (ThOD):

[0128] Test substance: C c HhClciNnNanaOoPpS s 2024PF30157 Abroad

[0129] mg O216[2C + 0.5 (H - Cl - 3N) + 3S + 2.5P + 0.5 Na - O] ThOD

[0130] mg test substance MW

[0131] This calculation implies that C is mineralized to CO2, H to H2O, P to P2O5, and Na to Na2O. The halogen (Cl = chlorine) is eliminated as hydrogen halide, and nitrogen as ammonia. Note: A nitrification inhibitor can be added to the samples (see step 7) to ensure that nitrogen is eliminated as ammonia and no nitrification occurs.

[0132] b) Calculation of the required amount of the test substance taking into account the volume of the test medium (157 ml) and the maximum BOD value in the measuring range of the equipment used (400 mg / l):

[0133] 400 mg O21 mg test substance

[0134] mg test substance = - * 0.157 l water *

[0135] 11 Water ThOD mg O2

[0136] 2. The sewage sludge used in the degradation study is subjected to a flow of compressed air at a constant temperature (23 °C) for at least 24 hours.

[0137] 3. The minimum salt medium was set according to the specifications in OECD 301 A, section 5.

[0138] 4. Composition of 11 test media:

[0139] a. 10 ml of concentrated sewage sludge (deposit at the bottom of the sewage sludge container)

[0140] b. 15 ml diluted sewage sludge (supernatant from the sewage sludge container) c. 970 g demineralized water

[0141] d. 1 ml of the salt and trace element solutions AD from OECD 301 A, Section 5

[0142] 5. The test medium was saturated at 20°C by aerating it with clean compressed air while stirring for 20 minutes.

[0143] 6. The degradability study was carried out in duplicates, with sodium acetate and a blank sample (without test substance or sodium acetate) included as references.

[0144] 7. Composition of the reaction mixture: Calculated amount of the test substance added to 157 ml of test medium and addition of 5 drops of nitrification inhibitor (allylthiourea)

[0145] 8. Calculation of BOD for complete biodegradability according to the following formula: 2024PF30157 Abroad

[0146] / mg 0.2\ ThOD mg 0.21

[0147] BOD at complete degradation - = mg test substance * - * - \ I / 1 mg test substance 0.1571 water

[0148] 10. Calculation of the biodegradability rate:

[0149] , mg O2.

[0150] BOD measured ( — — - )

[0151] Biodegradability ( '%) f = -

[0152]

[0153] 100

[0154] BOD upon complete degradation ( — ® — -)

[0155] Construction of the phylogenetic tree and consensus sequence

[0156] For the analysis of sequence identities, a sequence alignment was performed using the software Geneious Prime® 2023.2.1 with the Clustal Omega 1.2.2 algorithm.

[0157] The phylogenetic analysis was performed using the MEGAll software and the maximum likelihood method.

[0158] The phylogenetic tree was generated using the statistical bootstrap method, with a replicate count of 100. Neighbor-Join and BioNJ algorithms were used to automatically generate primordial trees on a matrix of pairwise distances for heuristic searching. These distances were estimated using the Jones-Taylor-Thornton (JTT) model and topology with the superior log-likelihood value. The phylogenetic analysis included 10 amino acid sequences. The sequence of alkaline phosphatase from E. coli was used as an outgroup.

[0159] Results

[0160] Example 1

[0161] Degradation of the solvent-based TPU / enzyme composition under laboratory conditions in an aqueous system

[0162] The enzymes listed in Table 1 were incorporated into TPU 1 using a solvent-based method. The hydrolysis activity of the enzymes after incorporation was measured under laboratory conditions in an aqueous buffer system based on monomer release (adipic acid). The results are shown in Table 4. Novozym® 51032 showed the strongest hydrolysis of TPU 1 after incorporation. This enzyme likely has an amino acid sequence as defined by SEQ ID NO.: 5. Furthermore, all other tested cutinases also showed hydrolysis of TPU 1 after incorporation, which differed significantly from the background activity of the blank vector control. Any adipic acid release exceeding 0.25% was considered hydrolytic activity by the incorporated cutinases. All measured adipic acid values ​​were verified by Blankwerte2024PF30157 Ausland.

[0163] The autohydrolysis and background activity were corrected for by the empty vector control. Thus, the values ​​listed in Table 4 reflect the amount of adipic acid released solely by the hydrolytic activity of the incorporated cutinases. In all cases, a higher enzyme concentration resulted in a higher adipic acid release. The conversion to adipic acid is given in % in Table 4 and refers to the amount released from the total amount of adipic acid contained in the TPU sample.

[0164] Table 4; Quantification of adipic acid release from TPU 1 films after 21 days of incubation at 37°C. All films contained enzymes incorporated by a solvent-based process in an amount of 0.3 or 0.5 wt%.

[0165]

[0166] ** Information based on literature review

[0167] *** Value corrected for empty vector and negative control

[0168] **** Value was corrected for negative control

[0169] Degradation of the TPU / enzyme composition produced by melt extrusion under in vitro laboratory conditions

[0170] The residual activity of the enzymes incorporated into TPU 1 and 2 by melt extrusion was tested under laboratory conditions in an aqueous system and evaluated by detecting the adipic acid monomer released during degradation. For all enzymes listed in Table 5, hydrolytic activity after incorporation into TPU 2 was demonstrated by adipic acid release, while no monomer release was detected in the empty vector control (pET26b_LV). The combination of calcium carbonate and Novozym® 51032 improved the hydrolysis of TPU 2 compared to the reaction containing only Novozym® 51032 but no calcium carbonate. [2024PF30157 Ausland]

[0171] Incorporation into TPU 1 was carried out exclusively with the enzymes Novozym® 51032 and SvCutl90*SS, whereby hydrolytic activity was demonstrated for both enzymes after incorporation via melt extrusion, while no monomer release was demonstrated in the empty vector control (pET26b_LV) (Table 6).

[0172] Table 5; Quantification of adipic acid release from TPU 2 with cutinases incorporated via melt extrusion after 21 days of incubation at 37°C. All TPU samples contained 0.3 wt% enzyme or blank vector lyophilisate and were incorporated at a melt temperature of 150°C. 20 wt% calcium carbonate was incorporated along with Novozym® 51032. The values ​​are given as mean values ​​of triplicates, except for Novozym® 51032 + calcium carbonate, which was measured as a single sample.

[0173]

[0174] ** Information based on literature review

[0175] *** Value corrected for empty vector and negative control

[0176] **** Value was corrected for negative control

[0177] Table 6: Quantification of adipic acid release from TPU 1 with cutinases incorporated via melt extrusion after 21 days of incubation at 37°C. All TPU samples contained 0.3 wt% enzyme or blank vector lyophilisate and were incorporated at a melt temperature of 190°C, or 195°C for the blank vector. Values ​​are given as mean values ​​of triplicates.

[0178]

[0179] ** Information based on literature review

[0180] *** Value corrected for empty vector and negative control

[0181] **** Value was corrected for negative control

[0182] Degradation of the TPU / enzyme composition produced by melt extrusion under near-environmental conditions

[0183] The biodegradation of TPU 2 was tested in vivo using sewage sludge according to OECD 301 F. The biodegradation rates after 90 days of incubation are summarized in Table 7. The TPU combination with Novozym® 51032 showed the highest biodegradation rate, with the combination of TPU, Novozym® 51032, and calcium carbonate resulting in a further improvement. For the blank vector control, the biodegradation rate was compared to all combinations of TPU and [2024PF30157 foreign].

[0184] The lowest biodegradation rate was measured for the enzyme. Additionally, a TPU combination containing enzymatically inactive protein was tested. This also showed no significant increase in biodegradation. These results confirmed the hydrolytic activity of all enzymes listed in Table 7 after incorporation into TPU 2 via melt extrusion, resulting in improved biodegradability compared to TPU combinations without hydrolytically active enzymes.

[0185] Table 7: Biodegradation rates of TPU 2 with enzyme lyophilisates incorporated via melt extrusion or with a blank vector lyophilisate as a reference at 0.3 wt% based on the TPU weight. 20 wt% calcium carbonate was incorporated together with Novozym® 51032. The values ​​are given as mean values ​​of duplicates.

[0186]

[0187] Example 2: Thermostability of a purified enzyme

[0188] Enzyme purification

[0189] The His-tagged version of SvCutl90*SS was purified by IMAC affinity chromatography. The filtered cell-free expression culture supernatant after cell lysis was used for purification. The purification column was packed with the required column volume of Ni-NTA (HisPur™ Ni-NTA Resin, Fisher Scientific, Rockford, USA) and equilibrated with 8 column volumes (CV) of lysis buffer (50 mM NaH₂PO₄, 300 mM NaCl, 10 mM imidazole). The filtered lysate was loaded onto the column. After loading the lysate, 10 CV of wash buffer (50 mM NaH₂PO₄, 300 mM NaCl, 20 mM imidazole) were added to the column. The specifically bound His-tagged enzyme was eluted in elution buffer (50 mM NaH₂PO₄, 300 mM NaCl, and 250 mM imidazole, pH 8). If necessary, the proteins were concentrated to 2.5 ml using Amicon® ultracentrifugal filters (10 kDa MWCO).The proteins were desalted using the Gravity protocol for PD-10 columns according to the manufacturer's instructions and stored in 20 mM potassium phosphate buffer pH 7.5.

[0190] After purification, the target enzyme comprised approximately 49% of the total protein content. This represented an enrichment of approximately 2 times the other proteins present in the crude extract. The entire purification process removed at least 70% by weight of the soluble, non-protein cellular components. 2024PF30157 Abroad

[0191] For comparison, TPU, which was extruded with crude extract as described above, was used in the same way in the degradation test.

[0192] Incorporation of the purified SvCutl90*SS by melt extrusion

[0193] The purified enzyme was freeze-dried for incorporation into TPU 2 (unmilled) via melt extrusion. The general incorporation procedure was carried out as described for the unpurified enzymes and Novozym® 51032, using a twin-screw micro-compounder, model Xplore MC 15 (DSM). In the case of the purified SvCutl90*SS, 0.18 wt% enzyme content, based on the TPU weight, was incorporated. This enzyme content of the purified enzyme corresponds to the incorporated activity for the unpurified SvCutl90*SS, allowing for a comparison of the two approaches. The sample filling time of the extruder was a maximum of 1 minute.After the entire sample was loaded into the mixing chamber of the extruder, compounding was carried out for 1 minute 30 seconds at the set melting temperature (200-150°C, with 10°C increments) at a rotation speed of 50 rpm for both extruder screws, before the TPU exited the extruder as a strand. The samples were cooled at room temperature and dried for 24 hours at 70°C before degradation studies were performed.

[0194] Results

[0195] Degradation of the TPU / enzyme composition produced by melt extrusion under in vitro laboratory conditions with purified SvCutl90*SS

[0196] The residual activity of the purified SvCutl90*SS incorporated into TPU 2 by melt extrusion was tested under laboratory conditions in an aqueous system and evaluated by detecting the adipic acid monomer released during degradation. Compared to the unpurified enzyme, the purified enzyme showed increased adipic acid release from TPU 2 after 21 days of incubation (Table 8). Furthermore, the purified enzyme exhibited higher overall thermostability compared to the unpurified enzyme from the cell-free expression supernatant, as adipic acid release was still measurable even at an incorporation temperature of 200°C.

[0197] Table 8: Quantification of adipic acid release from TPU 2 with purified SvCutl90*SS (SEQ ID NO.: 4) incorporated via melt extrusion after 21 days of incubation at 37°C. All TPU samples contained 0.18 wt% enzyme lyophilisate and were incorporated at melt temperatures ranging from 200°C to 150°C. Values ​​are given as mean values ​​of triplicates.

[0198]

[0199] 2024PF30157 Abroad

[0200]

[0201] ** Value corrected for negative control (TPU without enzyme addition) *** Value corrected for empty vector control

Claims

2024PF30157 Abroad Patent claims 1. A process for producing a polyester polyurethane composition comprising the steps a) Provision of at least one thermoplastic polyester polyurethane and at least one enzyme which (i) is defined by an amino acid sequence according to SEQ ID NO.: 1 or a variant of the aforementioned enzyme defined by an amino acid sequence with at least 85% identity to the conserved amino acids defined in SEQ ID NO.: 1; or (ii) is defined by an amino acid sequence according to SEQ ID NO.: 4 or 8 or a variant of one of the aforementioned enzymes defined by an amino acid sequence with at least 64% identity to SEQ ID NO.: 4 or at least 85% sequence identity to SEQ ID NO.: 8; b) Mixing the at least one polyester polyurethane with the at least one enzyme, wherein the at least one polyester polyurethane is present either as a melt or dissolved in a suitable solvent; and c) Curing of the polyester polyurethane composition by cooling the melt or by removing the solvent.

2. A polyester polyurethane composition obtained or obtainable by the method according to claim 1.

3. A polyester polyurethane composition comprising at least one thermoplastic polyester polyurethane and a) at least one enzyme defined by an amino acid sequence according to SEQ ID NO.: 1 or a variant of the aforementioned enzyme defined by an amino acid sequence with at least 85% identity to the conserved amino acids defined in SEQ ID NO.: 1; or b) at least one enzyme defined by an amino acid sequence according to SEQ ID NO.: 4 or 8, or a variant of one of the aforementioned enzymes defined by an amino acid sequence with at least 64% identity to SEQ ID NO.: 4 or at least 85% sequence identity to SEQ ID NO.:

8. 2024PF30157 Abroad 4. The polyester polyurethane composition according to claim 2 or 3, wherein the thermoplastic polyester polyurethane contains between 50 and 70 wt.% polyester polyol based on its total weight.

5. The polyester polyurethane composition according to any one of claims 2 to 4, wherein the thermoplastic polyester polyurethane has a number-average molecular weight between 10,000 g / mol and 200,000 g / mol.

6. The polyester polyurethane composition according to any one of claims 2 to 5, wherein the variant of the enzyme defined by SEQ ID NO.: 1 is characterized by an amino acid sequence which has at least 95% identity to the conserved amino acids defined in SEQ ID NO.:

1.

7. The polyester polyurethane composition according to any one of claims 2 to 6, additionally comprising an acid scavenger.

8. The polyester polyurethane composition according to claim 7, wherein the acid scavenger is an inorganic salt selected from the group consisting of carbonates, hydrogen carbonates, phosphates and hydroxides.

9. The polyester polyurethane composition according to any one of claims 2 to 8, wherein the at least one enzyme or its variant is included as a component of an enzyme composition purified from a crude extract.

10. The polyester polyurethane composition according to claim 9, wherein the proportion of the at least one enzyme is at least 25 wt.% based on the total protein content of the purified enzyme composition.

11. The polyester polyurethane composition according to claim 9 or 10, wherein the purified enzyme composition is characterized in that the proportion of soluble cell components in the purified enzyme composition is reduced by at least 80 wt.% compared to the total mass of the non-protein soluble cell components contained in the crude extract.

12. The polyester polyurethane composition according to claim 11, wherein the soluble cell components, which are not proteins, consist of sugars, nucleic acids, oligopeptides and organic molecules with a molecular weight of at most 2,500 g / mol. 2024PF30157 Abroad 13. A polymer product comprising the polyurethane composition as defined by any one of claims 2 to 12.

14. Use of an enzyme that (i) is defined by an amino acid sequence according to SEQ ID NO.: 1 or a variant of the aforementioned enzyme defined by an amino acid sequence with at least 85% identity to the conserved amino acids defined in SEQ ID NO.: 1; or (ii) is defined by an amino acid sequence according to SEQ ID NO.: 4 or 8 or a variant of one of the aforementioned enzymes defined by an amino acid sequence with at least 64% identity to SEQ ID NO.: 4 or at least 85% sequence identity to SEQ ID NO.: 8; To increase the biodegradability of a polyester polyurethane composition containing a thermoplastic polyester polyurethane.

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