Urethanases for the enzymatic degradation of polyurethanes

Abstract

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.

Claims

1. A method comprising utilizing a polypeptide for cleaving a urethane group, the 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, SEQ ID No. 7, and variants of said polypeptides, characterized in that the polypeptide has urethanase activity in the enzymatic cleavage of urethane linkages, wherein variants is defined as an amino acid sequence having at least 90% sequence identity with a polypeptide to which it refers.

2. The method according to claim 1, wherein the urethane group is aromatically attached, wherein aromatically attached is defined as a nitrogen atom of the urethane group being directly attached to an aromatic ring.

3. The method according to claim 1, wherein the polypeptide has 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, SEQ ID No. 7, and variants of said polypeptides, wherein variants is defined as an amino acid sequence having at least 95% sequence identity with a polypeptide to which it refers.

4. The method according to claim 1, wherein the polypeptide has 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 SEQ ID No. 7.

5. The method according to claim 1, wherein the polypeptide has 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.

6. The method according to claim 1, wherein the polypeptide has an amino acid sequence in accordance with SEQ ID No. 7 or a variant thereof.

7. The method according to claim 1, wherein the polypeptide has an amino acid sequence in accordance with SEQ ID No. 2 or a variant thereof.

8. The method according to claim 1, wherein the polypeptide has an amino acid sequence in accordance with SEQ ID No. 3 or a variant thereof.

9. The method according to claim 1, wherein the polypeptide has an amino acid sequence in accordance with SEQ ID No. 4 or a variant thereof.

10. The method according to claim 1, wherein the polypeptide has an amino acid sequence in accordance with SEQ ID No. 5 or a variant thereof.

11. The method according to claim 1, wherein the polypeptide has an amino acid sequence in accordance with SEQ ID No. 6 or a variant thereof.

12. The method according to claim 1, wherein the polypeptide has an amino acid sequence in accordance with SEQ ID No. 8 or a variant thereof.

Description

OVERVIEW OF THE FIGURES

(1) FIG. 1: Result of the phylogenetic analysis of the amino acid sequences disclosed in the present application

(2) The working examples that follow serve merely to elucidate the invention. They are not intended to limit the scope of the claims in any way.

EXAMPLES

Test of Enzyme Activity with ENPC

(3) 0.2 mg/ml of ENPC was incubated for 24 hours in 100 mM KH.sub.2PO.sub.4/K.sub.2HPO.sub.4 at pH 7.0 containing 6.25% by volume of ethanol at room temperature and 900 rpm on the MTS 2/4 plate shaker (IKA, Staufen).

(4) After filtering the samples, 100 L of each was transferred to transparent flat-bottom 96-well UV-Star plates (Greiner Bio-One, Frickenhausen) and the absorbance at 405 and 480 nm determined. The value at 480 nm was measured, because 4-nitroaniline does not show any significant absorption and, if high values are observed at both wavelengths, it is highly likely that is not 4-nitroaniline but another substance that was responsible for the absorbance at 405 nm.

(5) Hydrolysis by urethanases causes cleavage of the almost colorless ENPC into 4-nitroaniline, CO.sub.2, and ethanol, resulting in the detection of 4-nitroaniline at 405 nm in the Infinite M1000PRO microtiter plate photometer (Tecan, Mannedorf, Switzerland). The photometer was controlled using the i-control software (Tecan, Mannedorf, Switzerland), version 3.4.2.0. 4-Nitroaniline was additionally detected by HPLC using the dabsylamine method.

High Pressure Liquid Chromatography (HPLC)

(6) 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 4.675 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.

(7) 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.sub.2O 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.sub.2O 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).

(8) Dabsylamine: Eluent: AcN and 10 mM Na.sub.2HPO.sub.4/NaH.sub.2PO.sub.4, pH 7.0

(9) TABLE-US-00002 t [min] AcN 0 5 6.5 85 8.0 5 10.0 5

(10) Dabsylamin95: Eluent: AcN containing 5% (v/v) dd H.sub.2O and 10 mM Na.sub.2HPO.sub.4/NaH.sub.2PO.sub.4, pH 7.0

(11) TABLE-US-00003 % AcN (+5% t [min] (v/v) dd H.sub.2O) 0 5 6.5 90 8.0 5 10.0 5

(12) Dabsylannin-12-MeOH-lang: Eluent: Methanol and dd H.sub.2O containing 0.1% (v/v) methanoic acid

(13) TABLE-US-00004 t [min] % methanol 0 5 2.5 35 8.0 70 8.5 85 10.0 5 12.0 5

(14) TABLE-US-00005 Designation Hydrolysis SEQ ID No. in study of ENPC 1 GatA61 + 2 Aes70 + 3 Aes72 + 4 Aes170 + 5 Aes174 + 6 Aes175 + 7 GatA197 + 8 Aes214 + 9 GatA250 + 10 AesG56 + 11 SB12 + 12 SB23 +

Test of Enzyme Activity with EPEC

(15) The test was carried out as described for ENPC. The phenethylamine formed was detected by HPLC as described above.

(16) TABLE-US-00006 Designation Hydrolysis SEQ ID No. in study of EPEC 1 GatA61 + 2 Aes70 3 Aes72 + 4 Aes170 5 Aes174 + 6 Aes175 7 GatA197 + 8 Aes214 + 9 GatA250 + 10 AesG56 11 SB12 + 12 SB23 +

Phylogenetic Analysis of the Enzymes

(17) 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.

(18) Alignments of the different proteins were created using the Clustal Omega software (Sievers et al., 2011).

(19) Database searches for protein sequences were carried out using BLASTP (Altschul et al., 1990).

(20) Open reading frames (ORFs) in sequenced metagenome sequences were located using the online application ORF Finder from the NCBI (Wheeler et al., 2007).

(21) 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.

(22) In order to compare the individual members of the two identified urethanase groups (GatA and Aes), an alignment was in each case created with the Clustal Omega software and a phylogenetic tree additionally created with the MegAlign software, with a common alignment of the two groups created for the phylogenetic tree. The sequence comparison also included the sequences for the enzymes from the literature (Ure, Ana, and NfpolyA), which all showed similarity with GatA.

(23) The phylogenetic tree is shown in FIG. 1. This shows that the two groups are located in different branches, the similarities within the two groups being not so clear in some instances, as can be seen from the lower bootstrapping values at the nodes. 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. In particular 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.

Production of the Polyurethane Foam for the Breakdown Tests

(24) The starting materials listed below were reacted in the manner of processing customary for the production of polyurethane foams in the one-step process.

(25) The bulk density was 38 kg/m.sup.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)

Formulation

(26) TABLE-US-00007 100 parts Desmophen 2200B 3 parts water 19 parts Desmodur T80 19 parts Desmodur T65 0.7 parts N,N-dimethylpiperazine 1 part Tegostab 8325

Raw Materials

(27) 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.

(28) Desnnodur T80, Covestro Deutschland AG; isomer mixture comprising tolylene 2,4- and 2-6-diisocyanate in a mixture ratio of approx. 80:20.

(29) Desnnodur T65, Covestro Deutschland AG; isomer mixture comprising tolylene 2,4- and 2-6-diisocyanate in a mixture ratio of approx. 67:33.

(30) N,N-Dimethylpiperazine, catalyst from abcr GmbH

(31) TegostabB 8325, foam stabilizer, from Evonik

(32) Water; deionized water

(33) 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.

Breakdown of Polyurethane Foam

(34) 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.

(35) In the first step, 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.

(36) For further 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. After three days, 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.

(37) After the reaction in the mixture containing the CalB lyophilizate, it was already macroscopically evident by comparison with a negative control without enzyme that the foam had lost all structure and was present as a turbid suspension containing small particles of foam. The buffer, which had been almost completely absorbed by the foam at the start of the experiment, subsequently contained the entire foam mass in the form of broken-down particles. HPLC analysis showed clear peaks that were assigned to the oligomers formed, but no peaks pointing to the formation of tolylenediamine (TDA) (data not shown).

(38) 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. In addition, in contrast to the other mixtures, the oligomer peaks for these two enzymes showed changes and a general reduction in size. In the case of SEQ ID No. 7, 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. The fact that the product peaks identified as oligomer peaks from the hydrolysis with SEQ ID No. 12 were dramatically smaller after further treatment with urethanases, this being accompanied by significant TDA formation, confirmed that these were oligomer peaks.

(39) It was also demonstrated that insoluble TDI-based polyester-polyurethane foam can be cleaved into its monomers by a combination of two reaction steps. In a first step, the PU foam was predigested using the lipase CaIB 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.

(40) In conclusion, it can be seen that a combination of hydrolytic cleavage of the ester linkages by means of lipases, neutralization of the oligomer solution, and subsequent hydrolytic cleavage of the urethane linkages permits the complete breakdown of polyurethanes into defined monomers.