US20210269834A1 - Novel urethanases for the enzymatic degradation of polyurethanes - Google Patents

Novel urethanases for the enzymatic degradation of polyurethanes Download PDF

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US20210269834A1
US20210269834A1 US17/254,701 US201917254701A US2021269834A1 US 20210269834 A1 US20210269834 A1 US 20210269834A1 US 201917254701 A US201917254701 A US 201917254701A US 2021269834 A1 US2021269834 A1 US 2021269834A1
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diamine
group
polypeptide
acid
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Natalie BITTNER
Hartmut Nefzger
Gesa Behnken
Gernot Jäger
Swantje Behnken
Lukas REISKY
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Covestro Intellectual Property GmbH and Co KG
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
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    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J11/00—Recovery or working-up of waste materials
    • C08J11/04—Recovery or working-up of waste materials of polymers
    • C08J11/10—Recovery or working-up of waste materials of polymers by chemically breaking down the molecular chains of polymers or breaking of crosslinks, e.g. devulcanisation
    • C08J11/105—Recovery or working-up of waste materials of polymers by chemically breaking down the molecular chains of polymers or breaking of crosslinks, e.g. devulcanisation by treatment with enzymes
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • C12N9/20—Triglyceride splitting, e.g. by means of lipase
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C12N9/80—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)
    • C—CHEMISTRY; METALLURGY
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    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/001—Amines; Imines
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/02—Amides, e.g. chloramphenicol or polyamides; Imides or polyimides; Urethanes, i.e. compounds comprising N-C=O structural element or polyurethanes
    • C—CHEMISTRY; METALLURGY
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    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/18—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic polyhydric
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/44—Polycarboxylic acids
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    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/01—Carboxylic ester hydrolases (3.1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/01—Carboxylic ester hydrolases (3.1.1)
    • C12Y301/01003—Triacylglycerol lipase (3.1.1.3)
    • C—CHEMISTRY; METALLURGY
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    • C12Y—ENZYMES
    • C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
    • C12Y305/01—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in linear amides (3.5.1)
    • C12Y305/01075—Urethanase (3.5.1.75)
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J2375/00—Characterised by the use of polyureas or polyurethanes; Derivatives of such polymers
    • C08J2375/04—Polyurethanes
    • C08J2375/06—Polyurethanes from polyesters

Definitions

  • the present invention relates to new urethanases for the enzymatic breakdown of polyurethanes and to an enzymatic process for the complete breakdown of polyurethanes into defined monomers.
  • Polyurethanes are established in many areas of normal life. They can be found, for example, in soft foams (mattresses, sponges, upholstered furniture), hard foams (insulation materials, building materials), thermoplastics (sports shoes) or coatings (varnishes, paints, adhesives).
  • soft foams mattresses, sponges, upholstered furniture
  • hard foams insulation materials, building materials
  • thermoplastics sportss shoes
  • coatings varnishes, paints, adhesives
  • enzymes can be used in a diversity of ways for the catalysis of chemical reactions outside this context. Such reactions can be carried out under milder conditions than conventional chemical processes, for example lower temperature, neutral pH, and without the use of aggressive chemicals. Through this it is possible to save on energy, minimize the formation of by-products, and protect the environment, which helps to reduce operating costs. In some cases, it is only through the use of enzymes that it is possible for labile starting materials to be used as reaction feedstocks (Jaeger, K.-E. & Reetz, M. T. (1998) Microbial lipases form versatile tools for biotechnology. Trends in biotechnology, 16, 396-403).
  • enzymes are often regio-, stereo- and enantioselective, which makes the purification of the products substantially easier, which can permit the efficient synthesis of products that are otherwise difficult to obtain (Hasan, F., Shah, A. A. & Hameed, A. (2006) Industrial applications of microbial lipases. Enzyme and Microbial Technology, 39, 235-251).
  • the recycling of polyurethanes is primarily carried out through thermal recycling. This process generally takes place at very high temperatures and with very long reaction times in a batch process, as well as involving the use of catalysts. What can happen in such processes is that thermal breakdown of the polymer chains in cracking reactions leads to undesired and undefined breakdown products or else the formation of epoxy rings occurs, which results in a high odor nuisance and disadvantageous crosslinking of the chains in the recycled raw material, which can make it impossible to reuse said materials in products in particular with close human contact, particularly in the production of foams for use in furniture and mattresses.
  • An alternative option is for complete combustion and thus energy recovery to be carried out, which generates energy, but does not allow efficient reuse of the polymer building blocks.
  • polyester polyurethanes can be broken down to a certain degree by bacteria and fungi.
  • Polyester polyurethanes are considerably more susceptible to such microbial/enzymatic breakdown 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).
  • polyester polyurethanes can be readily accomplished by hydrolysis of the ester linkages.
  • the relatively simple breakdown of polyesters is not surprising, given that ester linkages in hydrophobic substrates in nature must also be cleaved when lipids are broken down and polyesters without urethane linkages can likewise be broken down relatively easily by esterases and lipases (Marten, E., Müller, R.-J. & Deckwer, W.-D. (2003) Studies on the enzymatic hydrolysis of polyesters I. Low molecular mass model esters and aliphatic polyesters. Polymer degradation and stability, 80, 485-501; Marten, E., Müller, R.-J. & Deckwer, W.-D.
  • a breakdown process for the enzymatic breakdown of poly(ester urethane)s is known, the first step of which is to obtain an esterase from a culture of Comamonas acidovorans strains by using only poly(ester urethane) as the carbon source.
  • the esterase is separated and used for the breakdown of poly(ester urethane)s in a batch process. This gives rise to long breakdown times in a multistage process and no demonstration of specific cleavage of the urethane linkages (JP 09201192, JP 10271994).
  • WO 2013/134801 describes the breakdown of aromatic polyurethanes based on polyether polyols using an enzyme of class EC 3. No specific enzyme sequences are stated, consequently neither the specificity of the process in the breakdown of particular urethane linkages, nor the controlled cleavage of ester linkages and separate cleavage of urethane linkages, as shown in the present invention, are demonstrated in the cited patent. Moreover, there is no description of the regulation of the pH of the mixture during polymer breakdown in order to maintain urethanase activity. Moreover, no regioselective breakdown is described, nor breakdown of aliphatic poly(ester urethane)s.
  • WO 2006/019095 describes a urethanase and variants of this enzyme obtained by protein engineering.
  • the enzyme can cleave urethane oligomers based on TDA or MDA.
  • bonds are not cleaved regioselectively here, neither is there any application in combination with esterases for the breakdown of polymers.
  • no other urethanases from the GatA or Aes families or any other group are described.
  • the present invention relates to a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID No. 3, SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10 and variants of said polypeptides or to a polypeptide having an amino acid sequence in accordance with SEQ ID No. 7 or a variant thereof, characterized in that the polypeptide has urethanase activity.
  • 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 to one another by peptide linkages.
  • a polypeptide may comprise both naturally occurring and synthetic amino acids. It preferably comprises the known proteinogenic amino acids.
  • a variant is obtained by adding, deleting or exchanging up to 10%, preferably up to 5%, of the amino acids present in the respective polypeptide.
  • a preferred variant of SEQ ID No. 7 is obtained by adding, deleting or exchanging up to 5% of the amino acids defined in SEQ ID No. 7.
  • Particularly preferred variants of the abovementioned polypeptides are obtained by adding, deleting or exchanging up to 20, preferably up to 10, and more preferably up to 5, amino acids of the disclosed sequences.
  • Preferred variants of SEQ ID No. 6 and SEQ ID No. 8 are obtained by adding, deleting or exchanging up to 3, more preferably up to 2, amino acids.
  • polypeptides as defined by SEQ ID No. 3, SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 8, and SEQ ID No. 10. form a group that is phylogenetically different from the sole enzyme having urethanase activity that is known to date, Ure (see FIG. 1 ). No enzymes having corresponding activity were previously known from this group.
  • This group of polypeptides is also referred to herein below as “Aes-like”.
  • urethanase activity refers to the ability of a polypeptide to enzymatically catalyze the cleavage of a urethane group. In this process, each mole of urethane group gives rise to one mole of amine, one mole of alcohol, and one mole of CO 2 .
  • the urethane group may be an aromatically or aliphatically attached urethane group.
  • the nitrogen atom is attached directly to an aromatic ring.
  • the nitrogen atom is attached to an alkyl radical.
  • the alkyl radical is preferably unbranched and composed of at least one, more preferably at least two, and most preferably at least three, carbon atoms.
  • the polypeptide having urethanase activity is capable of enzymatically cleaving an aromatically attached urethane group.
  • Whether a polypeptide has urethanase activity can be checked through the cleavage of suitable model substrates.
  • the model substrate for the ability to cleave aromatically attached urethane groups is preferably ethyl 4-nitrophenyl carbamate (ENPC). Cleavage is demonstrated by determining the increase in the concentration of 4-nitroaniline. This is done preferably photometrically at a wavelength of 405 nm.
  • the enzyme activity is determined preferably in a reaction buffer containing 100 mM of K 2 HPO 4 /KH 2 PO 4 , pH 7 with 6.25% by volume of ethanol in the presence of 0.2 mg/L of ENPC as substrate. Incubation of the enzyme with ENPC in the reaction buffer is carried out preferably at room temperature and preferably for 24 hours.
  • the model substrate for the ability to cleave aliphatically attached urethane groups is preferably ethyl phenethyl carbamate (EPEC). Cleavage is demonstrated by determining the increase in the concentration of phenethylamine. This is done preferably by HPLC.
  • the reaction buffer used and the reaction conditions preferably correspond to the parameters described above for ENPC.
  • enzyme cleavage of a urethane group indicates that the cleavage of a urethane group described above proceeds more rapidly in the presence of a polypeptide having urethanase activity than it does when incubated with the reaction buffer without enzyme under the same reaction conditions or when incubated with the reaction buffer under the same conditions in the presence of an inactive polypeptide.
  • the preferred model for an inactive polypeptide is bovine serum albumin. If, in the presence of a polypeptide being tested, the cleavage of the urethane group proceeds more rapidly than in an otherwise identical control with BSA, said polypeptide possesses urethanase activity as understood in this application.
  • the present invention relates to the use of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID No. 3, SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10 and variants of said polypeptides or to a GatA-similar polypeptide having an amino acid sequence in accordance with SEQ ID No. 7 or a variant thereof, characterized in that the polypeptide has urethanase activity in the enzymatic cleavage of urethane linkages.
  • the present invention relates to a process for breaking down polyester polyurethanes into low-molecular-weight breakdown products, comprising the steps of
  • peptides having urethanase activity are the peptides described in this application having amino acid sequences as defined in the group consisting of SEQ ID Nos. 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 and amino acid sequences having at least 90% sequence identity with the abovementioned sequences. Very particular preference is given to peptides having amino acid sequences as defined in SEQ ID No. 3 or 7 and amino acid sequences having at least 90% sequence identity with the abovementioned sequences.
  • the present invention relates to a process for breaking down polyester polyurethanes into low-molecular-weight breakdown products, comprising the steps of
  • Process step a) is preferably carried out with a lipase.
  • This lipase is preferably water-soluble and not present in an immobilized form.
  • “Immobilized” here refers to the attachment of peptides that is generally known in biotechnology, particularly the attachment of antibodies or enzymes, to the surface of vessels or to water-insoluble particles.
  • a lipase capable of cleaving tributyrin particularly preference is given to using a lipase capable of cleaving tributyrin. Even more particular preference is given to using a polypeptide that has an amino acid sequence as defined in SEQ ID No. 11 or SEQ ID No. 12 or that has an amino acid sequence having at least 90%, preferably at least 95%, sequence identity with one of the two abovementioned sequences and which is capable of cleaving tributyrin.
  • Process step a) is preferably carried out under reaction conditions in which the employed lipase shows activity. Such conditions can be determined by routine experiments using common biochemical methods.
  • process step b) is preferably carried out at a pH between 6.0 and 10.0, preferably between 6.0 and 8.0.
  • the pH may be adjusted using all suitable bases known to those skilled in the art.
  • polyester polyurethane refers to a polyurethane formed from one or more polyester polyols and one or more isocyanates.
  • the polyurethane may be foamed or non-foamed. It is preferably foamed. To increase the specific surface area, it is preferable to comminute the polyurethane before carrying out process steps a) and b). This is particularly preferable when polyurethane is to be used in non-foamed form. Comminution may be done in any way familiar to those skilled in the art, preferably by milling, slicing, tearing or cutting.
  • the polyurethane comprises as the isocyanate component at least one aromatic, aliphatic or cycloaliphatic isocyanate.
  • the polyurethane preferably comprises only aromatic isocyanates.
  • Preferred aromatic isocyanates are methylene diphenyl isocyanate (MDI), MDI variants having three or more aromatic rings, naphthylene diisocyanate, and tolylene diisocyanate.
  • Particularly preferred aromatic isocyanates are methylene diphenyl isocyanate (MDI), MDI variants having three or more aromatic rings, and tolylene diisocyanate.
  • MDI variants having three or more aromatic rings are synthesis by-products and may also be present in polyurethanes.
  • the polyurethane to be broken down particularly preferably comprises tolylene 2,4-diisocyanate and tolylene 2,6-diisocyanate.
  • 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, and more preferably at least 2.0, hydroxyl groups per molecule.
  • the polyester polyols present in the polyurethane to be broken down particularly preferably have functionality of between 1.5 and 6.0. They contain as structural elements aromatic and/or aliphatic polyols and also aromatic and/or aliphatic polycarboxylic acids in any combination.
  • the low-molecular-weight breakdown products of the polyester-based polyurethane foams preferably have a molecular weight of not more than 1000 g/mol. These are preferably
  • polyol is in this application understood as meaning any compound having at least two hydroxyl groups. Said polyol preferably has a molecular weight of not more than 300 g/mol.
  • Preferred polyols that are low-molecular-weight breakdown products of polyester-based polyurethane foams are selected from the group consisting of ethylene glycol, diethylene glycol, 1,4-butanediol, triethylene glycol, propylene glycol, 1,2-dipropylene glycol, neopentyl glycol, glycerol, 1,1,1-trimethylolpropane, sucrose, sorbitol, and pentaerythritol.
  • polycarboxylic acid is in this application understood as meaning any compound containing at least two carboxyl groups. Said polycarboxylic acid preferably has a molecular weight of not more than 300 g/mol.
  • Preferred polycarboxylic acids that are low-molecular-weight breakdown products of polyester-based polyurethane foams are selected from the group consisting of succinic acid, glutaric acid, adipic acid, phthalic acid, terephthalic acid, benzenetricarboxylic acid, oleic acid, and ricinoleic acid.
  • Particularly preferred polycarboxylic acids that are low-molecular-weight breakdown products of polyester-based polyurethane foams are selected from the group consisting of succinic acid, glutaric acid, adipic acid, phthalic acid, terephthalic acid, and benzenetricarboxylic acid.
  • a “polyamine” is in this application understood as meaning any compound containing at least two amino groups. Said polyamine preferably has a molecular weight of not more than 300 g/mol.
  • Preferred polyamines that are low-molecular-weight breakdown products of the polyester-based polyurethane foams are selected from the group consisting of methylene-4,4′-diamine, methylene-2,4′-diamine, methylene-2,2′-diamine, tolylene-2,4-diamine, tolylene-2,6-diamine, hexamethylenediamine, isophorone diamine, xylylenediamine, pentamethylenediamine, para-phenylenediamine, butyldiamine, and H12-methylenediamine.
  • polyamines selected from the group consisting of methylene-4,4′-diamine, methylene-2,4′-diamine, methylene-2,2′-diamine, naphthylene-1,4-diamine, naphthylene-1,5-diamine, naphthylene-1,6-diamine, tolylene-2,4-diamine, and tolylene-2,6-diamine.
  • the process according to the invention allows effective recycling of polyurethanes in two ways: (i) The process itself operates under mild reaction conditions and so does not require a high input of energy and (ii) it allows the polyurethane to be recycled, because defined breakdown products are formed that are themselves valuable chemical raw materials.
  • thermal glycolysis which is currently the most common chemolysis for recycling polyurethane and has already been put into practice industrially, is carried out at very high temperatures.
  • the focus here is on extraction of the polyols, whereas the amines are separated as an interfering species and are not recovered.
  • non-enzymatic hydrolysis both polyols and amines are obtained as products.
  • this process is carried out at high temperatures and high ambient pressures.
  • FIG. 1 Result of the phylogenetic analysis of the amino acid sequences disclosed in the present application
  • High-pressure liquid chromatography was carried out on an Agilent Technologies (Santa Clara, USA) 1100 series instrument equipped with an autosampler and DAD (diode array detector) for UV and the visible light region. All measurements were carried out using a “Zorbax XDB-C18” column having a particle size of 3.5 ⁇ m and dimensions of 4.6 ⁇ 75 mm (Agilent Technologies, Santa Clara, USA). In all methods, a 5 ⁇ L sample was injected into a column heated to 40° C. The flow was generally 1.5 ml/min. The use of a reverse-phase column means that elution in all methods is with increasing concentrations of organic solvent.
  • Detection and quantification of dabsylated aliphatic amines and urethanes was done using the “dabsylamine” method. This method allows the quantification of aromatic amines and urethanes without derivatization on account of their high intrinsic absorption.
  • Also used as eluent in addition to AcN was 10 mM sodium phosphate buffer pH 7.0, to which 0.005% (w/v) sodium azide was added to protect against microbial growth. In order to prevent pressure problems caused by contaminated pump valves, 5% (v/v) of dd H 2 O was later added to the AcN and the method adjusted (“Dabsylamin95”).
  • the MDEC formed from the enzyme-catalyzed reactions of 4,4′-MDA with EC was quantified using the “Dabsylamin-12-MeOH” method, in which the aqueous component is acidified and the protonated aromatic amines thereby generated elute very early.
  • the reactions of 4,4′-MDA with DMC, 2,4-TDA with DMC, and 2,4-TDA with EC were investigated using the “Dabsylamin95-H2O” method, which differs from “Dabsylamin95” only in that pure dd H 2 O is used instead of buffer.
  • the data were analyzed using the “OpenLAB CDS ChemStationLC” software, version A.02.09 [017] (Agilent Technologies, Santa Clara, USA).
  • Dabsylamine Eluent: AcN and 10 mM Na 2 HPO 4 /NaH 2 PO 4 , pH 7.0
  • Phylogenetic trees showing the relatedness of the urethanases were created using the “MegAlign” software (DNASTAR, Madison, USA), version 10.1.0. The phylogenetic trees were created with the default settings using “ClustalW”.
  • ORFs Open reading frames in sequenced metagenome sequences were located using the online application “ORF Finder” from the NCBI (Wheeler et al., 2007).
  • Identical hydrolase genes were reduced to a single representative and all sequences examined with ORFs in order to obtain the complete sequences of the genes.
  • Alternative start codons were also allowed in the search. It was evident here that the gene from pLip214 included an N-terminal region with similarity to aes, but without a start codon having been identified. This gene segment was not located on the edge of the insert of the metagenome vector, which could explain a truncated gene. For further analyses, the region with similarity to aes but without a start codon was used as the sequence for this gene.
  • the identified putative urethanase genes were translated in silico and compared with the NCBI database using BLASTP. The putative urethanases were named on the basis of their number in the lipase bank and the similarity to GatA or Aes.
  • the phylogenetic tree is shown in FIG. 1 .
  • Within the GatA group there seem to be greater differences than within the Aes group, as can be seen from the longer branch lengths in this group.
  • Aes70 and Aes72 and also Aes175 and Aes214 show very high similarity, as manifested both by the relatively short branches in the phylogenetic tree and by the same protein with greatest similarity having been found in the BLASTP search.
  • the bulk density was 38 kg/m 3 (DIN EN ISO 845 in the version of October 2009), the compressive strength at 40% compression was 3.5 kPa (DIN EN ISO 3386-1 in the version of October 2015)
  • Desmophen® 2200B Covestro Deutschland AG; branched polyester polyol based on adipic acid, diethylene glycol and 1,1,1-trimethylolpropane having a hydroxyl value of approx. 60 mg KOH/g.
  • the formulation may be executed with indices of 90 to 115.
  • the index is defined as the molar ratio of isocyanate groups to isocyanate-reactive groups multiplied by 100.
  • the substrate used was a polyester polyurethane produced with tolylene diisocyanate. Breakdown took place in two reaction steps. First, the foam was incubated with a lipase. The resulting oligomers were neutralized and then cleaved into monomers with a urethanase.
  • 1 g of the foam was transferred to a 50 ml centrifuge tube with 20 ml of potassium phosphate buffer pH 7.0 and approx. 30 mg of CalB lyophilizate (“Chirazyme L2” from Roche, Basel, Switzerland) (here referred to as SEQ ID No. 12) and incubated at 37° C. and 200 rpm for 5 days. Fragments of the foam residues were photographed with a “MH2” microscope (Olympus, Hamburg) by comparison with a negative control without enzyme. The turbid solution was then centrifuged for 10 minutes at 25° C. and 4000 rpm in a large-capacity centrifuge. The clear supernatant was adjusted to pH 7.0 with 1 M NaOH. After about 6 hours at room temperature, the slight fall in pH was retitrated to 7.0 and the solution underwent a sterilizing filtration. The soluble oligomers were stored at 4° C. until use.
  • the soluble oligomers were transferred to 1.5 mL reaction vessels and mixed with 20 ⁇ L of DMF and 150 ⁇ L of the optimal buffer for the respective urethanase (100 mM sodium phosphate buffer, adjusted to the respective optimal pH for the urethanase in the pH 6.0 to pH 8.0 range). To each was then added 30 ⁇ L of the undiluted, purified urethanase and the mixtures were shaken on the heating block at 30° C. and 1000 rpm. A mixture containing enzyme storage buffer was used as the negative control.
  • the batches were filtered through filter plates with a PVDF membrane and a pore size of 0.2 ⁇ m (Corning, Kaiserslautern) and the filtrate was analyzed by HPLC using the “Dabsylamine95” method in respect of the tolylene 2,4- and 2-6-diisocyanate formed.
  • the oligomer solution was treated with all of the expressed urethanases and with SEQ ID No. 12 and then examined by HPLC for the formation of TDA. This was demonstrated for the mixtures containing SEQ ID No. 7 and SEQ ID No. 3, with the measured amount of 2,6-TDA being approximately the same in the two mixtures and the formation of 2,4-TDA in the mixture containing Enz03 found to be markedly more pronounced.
  • SEQ ID No. 3 afforded 0.057 g/L of 2,4-TDA and 0.025 g/L of 2,6-TDA, whereas SEQ ID No. 7 resulted in the formation of 0.0075 g/L of 2,4-TDA and 0.024 g/L of 2,6-TDA.
  • the oligomer peaks for these two enzymes showed changes and a general reduction in size.
  • TDA was cleaved from the polyester PU foam even without prior pretreatment, whereas in the case of SEQ ID No. 3 this was possible only by providing neutralized oligomers after prior ester cleavage.
  • insoluble TDI-based polyester-polyurethane foam can be cleaved into its monomers by a combination of two reaction steps.
  • a first step the PU foam was predigested using the lipase CalB through hydrolysis of the ester linkages. After neutralization, the liberated oligomers served as a substrate for the overexpressed urethanases. This was accompanied by hydrolysis of the urethane linkages and the detection of TDA in monomeric form.

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