NOVEL URETHANASES FOR THE ENZYMATIC DEGRADATION OF POLYURETHANES

Abstract

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

Claims

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

2. A method of breaking down a polyurethane, comprising enzymatically cleaving urethane linkages with a polypeptide having an amino acid sequence as defined by SEQ ID NO.: 1, 2 or 3, or a variant thereof, wherein the variant is obtained by the addition, deletion or exchange of up to 15% of the amino acids present in the respective polypeptide defined by SEQ ID NO.: 1, 2 or 3, and wherein the polypeptide has urethanase activity.

3. The method as claimed in claim 2, wherein the urethane linkage is aromatically attached.

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

5. A process comprising treating a low-molecular-weight urethane with a polypeptide having urethanase activity and having an amino acid sequence as defined by SEQ ID No. 1, 2 or 3, or a variant thereof, wherein the variant is obtained by the addition, deletion or exchange of up to 15% of the amino acids present in the respective polypeptide defined by SEQ ID NO.: 1, 2 or 3, thereby cleaving the low-molecular-weight urethane.

6. The process as claimed in claim 5, wherein at least one polyamine selected from the group consisting of 4,4-diaminodiphenylmethane, 2,4-diaminodiphenylmethane, 2,2-diaminodiphenylmethane, a multiring derivative of 4,4-diaminodiphenylmethane, a multiring derivative of 2,4-diaminodiphenylmethane, a multiring derivative of 2,2-diaminodiphenylmethane, tolylene-2,4-diamine, tolylene-2,6-diamine, naphthylene-1,4-diamine, naphthylene-1,5-diamine and tolylene-2,6-diamine is formed.

7. The process as claimed in claim 5, wherein an alcohol having at least two hydroxyl groups and a molecular weight of not more than 700 g/mol is released.

8. The process as claimed in claim 7, wherein at least one alcohol selected from the group consisting of ethylene glycol, diethylene glycol, propylene glycol, dipropylene glycol, methyl glycol, triethylene glycol, glycerol, 2-methyl-1,3-propanediol, 1,4-butanediol, 1,5-pentanediol, 1,6-hexanediol and polyethylene glycol 400 is released.

9. A method of breaking down a low molecular weight urethane, comprising cleaving a urethane linkage with at least one polypeptide having urethanase activity and having an amino acid sequence as defined by SEQ ID NO.: 1, 2 or 3 or having an amino acid sequence with at least 85% sequence identity thereto.

10. A process for breaking down a polyester polyurethane into low-molecular-weight breakdown products, comprising: a) cleaving the ester groups present in the polyester polyurethane; and b) treating the polyurethane with a polypeptide having urethanase activity and selected from the group consisting of polypeptides as defined by SEQ ID NO.: 1, 2 and 3 and variants of said polypeptides, wherein the variants are obtained by the addition, deletion or exchange of up to 15% of the amino acids present in the respective polypeptide defined by SEQ ID NO.: 1, 2 or 3; and with the proviso that process steps a) and b) are carried out in either order or in parallel.

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

Description

[0087] FIG. 1 shows: Enzyme substrates used. pNPB: 4-nitrophenyl butyrate; ENPC: ethyl 4-nitrophenyl carbamate; MDA-BA: carbamate of 4,4-MDI reacted with benzyl alcohol; TDA-ethoxyethanol: carbamate of toluene diisocyanate (TDI) reacted with ethoxyethanol; TDA-DEG: carbamate of TDI reacted with diethylene glycol; NDA-MEG: carbamate of NDI reacted with ethylene glycol; EMACC: carbamate from the reaction of 7-amino-4-methylcoumarin with ethyl chloroformate.

[0088] 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

Example 1: Determination of Activity Using EMACC

Enzyme Preparation

[0089] Urethanase genes were cloned into the pET-26 expression vector directly after the NdeI cleavage site. For expression, E. coli BL21 (DE3) were transformed with the relevant plasmids. A single colony was used for inoculation of the overnight culture in LB medium containing 1% (w/v) glucose and 50 g/mL kanamycin. For the main culture, 200 ml of ZYP-5052 containing 50 g/mL kanamycin were inoculated with 1 mL of the overnight culture and incubated in baffled flasks at 37 C. at 100 rpm for 4 h. The temperature was then reduced to 20 C. before the cultures were harvested by centrifugation at 4500 g and 4 C. for 20 min. Cell pellets were stored at 20 C. before the proteins were purified.

[0090] For purification, 15 mL of lysis buffer (50 mM sodium phosphate (NaPi), pH 8.0, 300 mM NaCl, 10 mM imidazole) were added to the cell pellet. Disruption was carried out by ultrasound on ice for two cycles (3 min, 50% pulse, 50% power). The lysate was cleared by centrifugation at 10 000 g and 4 C. for 40-60 min. The supernatants were stored on ice before the IMAC column was loaded. IMAC resin (1 ml, ROTI Garose His Beads, nickel form) was equilibrated with lysis buffer. The lysate was then applied to the columns followed by a wash step with at least ten column volumes of wash buffer (50 mM NaPi, pH 8.0, 300 mM NaCl, 20 mM imidazole). The proteins were then eluted with 15 mL of elution buffer (50 mM NaPi, pH 8.0, 300 mM NaCl, 250 mM imidazole). The volume of the elution fraction was reduced to 2.5 mL using Vivaspin 20 ultrafiltration units (10 kDa MWCO). The proteins were then rebuffered into storage buffer (10 mM NaPi, pH 8.0, 100 mM NaCl) using PD10 columns.

EMACC Assay

[0091] To determine urethanase activity, a fluorogenic substrate was synthesized from 7-amino-4-methylcoumarin (AMC) and ethyl chloroformate. The ethyl carbamate of AMC thus formed (EMACC) was in the form of a fine powder. Specific activity was determined under standard conditions (50 mM NaPi, pH 8.0, 30 C., 100 UM EMACC). EMACC was freshly added from a 50 mM DMSO stock. Purified urethanases were diluted in storage buffer before addition to the assay, such that there was a linear increase in absorption at 365 nm over 10-30 min. Measurement was carried out in 96-well plates in a plate photometer preheated to 30 C. and specific activity was calculated from the linear increase in absorption in the first few minutes. The results are shown in table 1.

TABLE-US-00001 TABLE 1 Specific activity of urethanase with respect to EMACC Specific activity toward EMACC Urethanase [mol/min/mg enzyme] UMG-SP-8 0.749 0.012 (SEQ ID NO.: 1) UMG-SP-11.2 0.377 0.020 (SEQ ID NO.: 2) UMG-SP-16 0.651 0.064 (SEQ ID NO.: 3)

Example 2: Screening Against Further Substrates

Enzyme Preparation To prepare the enzyme preparations, single colonies of E. coli BL21 (DE3) pET26b_UMG-8, BL21 (DE3) pET26b_UMG-11.2 and BL21 (DE3) pET26b_UMG-16 were each used to inoculate 4.5 mL of LB medium containing 1% (w/v) glucose, and they were incubated overnight in an incubation shaker at 37 C. and 200 rpm. All the cultures were admixed with 50 g/mL kanamycin. Thereafter, 200 L of each overnight culture was used to inoculate 200 ml of ZYP-5052 medium. Cultivation was carried out in an incubation shaker for 4 h at 37 C. and 200 rpm, followed by 24 h at 20 C. and 200 rpm. The cells were removed by centrifugation in a high-capacity centrifuge at 20 425 g and 4 C. for 20 min. The cell pellets were each suspended in 6 ml of disruption buffer (20 mM ammonium acetate, 0.4% n-dodecyl--maltoside, 1% lysozyme, 1 L/mL benzonase (Sigma-Aldrich Chemie GmbH, Taufkirchen)) and incubated on an orbital shaker at room temperature for 30 min. The total cell extract solution was then frozen at 80 C. and lyophilized. The lyophilizate was stored at 4 C.

Enzyme Reactions

[0092] The substrates ENPC, MDA-BA, NDA-MEG, TDA-ethoxyethanol and TDA-DEG were synthesized from the corresponding isocyanates and alcohols by reacting the isocyanates with an excess of alcohol. EMACC and 4-nitrophenyl butyrate (pNPB) were used to confirm urethanase activity. The enzyme substrates are shown in FIG. 1.

[0093] One spatula tip (3 mg) each of the lyophilized total cell extracts was suspended in 800 L of 100 mM KPi containing 100 mM NaCl, pH 7.5. The enzyme solution was diluted 1:10 in the reaction mixture containing the same buffer. Substrate stocks of the carbamates were prepared in DMSO at a concentration of 5-340 mg/mL. These were diluted 1:10 in the reaction mixture, such that the final DMSO concentration was 10% (v/v). The reactions were prepared with a total volume of 500 L. Incubation was carried out in a thermal shaker for one day at 30 C. and 800-1000 rpm, followed by one day at 40 C. and 800-1000 rpm. Following incubation, 500 L of acetonitrile were added to improve the solubility of substrates and products. The samples were filtered through a 0.2 m PVDF filter plate by centrifugation and used for HPLC analysis. For the substrates EMACC and pNPB, the total volume of the reaction was 200 L, and fluorescence (EMACC, excitation at 365 nm, emission at 440 nm) or absorption (pNPB, 410 nm) was measured at various times in a Varioskan Lux plate photometer (Thermo Fisher Scientific Inc., Waltham, Massachusetts, USA).

HPLC Analysis

[0094] High-pressure liquid chromatography was carried out on an 1260 Infinity II series instrument from Agilent Technologies (Santa Clara, USA), equipped with a multisampler and DAD (diode array detector) for UV and the visible light region. All measurements were carried out using a Zorbax Eclipse Plus C18 column having a particle size of 5 m and dimensions of 4.6150 mm (Agilent Technologies, Santa Clara, USA) with an appropriate precolumn. In all methods, a 5 L sample was injected into a column heated to 40 C. The flow was generally 1.0 mL/min.

[0095] Aromatic amines were detected by using the ER_Juni2021-2 method. This method allowed the quantification of aromatic amines at 210 nm and 232 nm without derivatization on account of their high intrinsic absorption. ddH.sub.2O containing 5% (v/v) acetonitrile was used as eluent A, and acetonitrile containing 5% (v/v) ddH.sub.2O was used as eluent B. The data were analyzed using the OpenLAB CDS 2.4 software, version 2.204.0.661 (Agilent Technologies, Santa Clara, USA).

TABLE-US-00002 TABLE 2 HPLC gradient t [min] Eluent A [%] Eluent B [%] 0.00 100.00 0.00 2.00 100.00 0.00 10.00 0.00 100.00 11.00 0.00 100.00 11.50 100.00 0.00 15.00 100.00 0.00

Results

[0096] The hydrolytic activity of the urethanases with respect to the carbamates tested was classified into the categories no activity (), little activity (+), medium activity (+) and high activity (+) on the basis of the peak area in the chromatogram of the products formed. It was found that the urethanases UMG-8, UMG11.2 and UMG 16 accept a broad spectrum of substrates. The substrates tested are shown in FIG. 1.

TABLE-US-00003 TABLE 3 Urethanase activity with respect to various carbamates. UMG-8 UMG-11.2 UMG-16 (SEQ ID (SEQ ID (SEQ ID Substrate NO.: 1) NO.: 2) NO.: 3) EMACC +++ +++ +++ pNPB +++ +++ +++ ENPC ++ +++ ++ MDA-BA ++ ++ + NDA-MEG ++/+++ ++/+++ ++ TDA-ethoxyethanol +++ +++ +++ TDA-DEG ++ +++ ++ Activity is divided into the categories no activity (), little activity (+), medium activity (++) and high activity (+++) on the basis of HPLC peak area.

Example 3: Optimization of Reaction Conditions

[0097] To determine pH optimums, enzyme activity was determined analogously to example 1, with the modification of carrying out the reaction in different buffers (100 mM). The buffers used were buffers of pH 4.0, 5.0 and 6.0 (citrate), 7.0, 8.0 and 9.0 (bis-tris propane) and 10.0 (CHES). In the case of pH values 10, 11, 12 and 13, the buffer used was a buffer composed of 100 mM sodium phosphate and 100 mM sodium carbonate andas a departure from example 1containing 200 mM EMACC and 10% (v/v) DMSO.

[0098] Temperature optimums were determined by measuring the release of AMC from 100 M EMACC in 50 mM NaPi (pH 8.0) at 30 C., 35 C., 40 C., 45 C., 50 C., 55 C., 60 C., 65 C. and 70 C. Solvent stability was determined by quantifying the hydrolysis of EMAC under the conditions specified in example 1 at 0%, 10%, 20%, 30%, 40% and 50% (v/v) DMSO. The activity optimums determined herein for pH, temperature and DMSO concentration are

TABLE-US-00004 pH T Optimal DMSO Urethanase optimum optimum ( C.) concentration (% v/v) UMG-SP-8 10 70 0 (SEQ ID NO.: 1) UMG-SP-11.2 10 70 10 (SEQ ID NO.: 2) UMG-SP-16 10 35 10 (SEQ ID NO.: 3)
summarized in table 4. The dependence of the activity is in

TABLE-US-00005 TABLE 4 Optimal reaction conditions for urethanase. pH T Optimal DMSO Urethanase optimum optimum ( C.) concentration (% v/v) UMG-SP-8 10 70 0 (SEQ ID NO.: 1) UMG-SP-11.2 10 70 10 (SEQ ID NO.: 2) UMG-SP-16 10 35 10 (SEQ ID NO.: 3)

TABLE-US-00006 TABLE 5 pH dependence of urethanase activity. In both data sets, activity was highest at pH 10, and so normalization was carried out to this value. The activity data of pH 12 and pH 13 were not shown because the fluorescence of AMC is constant only in the range between pH 3 and pH 12. pH 4 5 6 7 8 9 10 11 UMG-SP-8 0% 0% 0% 41% 77% 82% 100% 86% (SEQ ID No. 1) UMG-SP-11.2 0% 0% 0% 5% 15% 25% 100% 20% (SEQ ID NO.: 2) UMG-SP-16 0% 0% 0% 5% 7% 11% 100% 34% (SEQ ID NO.: 3)

Temperature Stability

[0099] To determine long-term temperature stability, solutions of the purified urethanases having a protein concentration of 0.5 mg/ml in 100 mM KPi, pH 7.5, were prepared. 100 L in each case were incubated in a Biometra TAdvanced Basis thermal cycler (Analytik Jena GmbH, Jena) at 20 C., 25.7 C., 29.4 C., 36.9 C., 40.6 C., 44.3 C. and 50 C. for 12 h and then at 4 C. before measurement of activity. The temperature of the lid heater was adjusted to 99 C. A reference sample was incubated at 4 C. The residual activity of the enzymes was determined using a pNPB assay. To this end, 21 L of pNPB were mixed with 4.979 mL of DMSO. This solution was diluted 1:10 in 100 mM KPi, pH 7.5. 20 L of enzyme solution (UMG-8 diluted 1:5 in 100 mM KPi, PH 7.5, UMG-11.2 diluted 1:20 in 100 mM KPi, pH 7.5, UMG-16 diluted 1:20 in 100 mM KPi, pH 7.5) were loaded in a microplate, and the reaction was started by adding 180 L of pNPB in 100 mM KPi, pH 7.5, containing 10% DMSO. When the pNPB is hydrolyzed by the urethanases, para-nitrophenol is released and this was quantified by measuring the absorption at 410 nm in a Varioskan Lux plate reader (Thermo Fisher Scientific Inc., Waltham, Massachusetts, USA). The results are shown in table 6.

TABLE-US-00007 TABLE 6 Specific urethanase activity with respect to pNPB in U/mg following incubation for 12 h at 20 C., 25.7 C., 29.4 C., 36.9 C., 40.6 C., 44.3 C. and 50 C. and at 4 C. as reference. Incubation temperature 4 C. 20 C. 25.7 C. 29.4 C. 36.9 C. 40.6 C. 44.3 C. 50 C. Enzyme Specific activity [mol/min/mg enzyme] UMG-8 6.73 6.50 7.12 6.42 3.23 0 0 0 (SEQ ID NO.: 1) UMG-SP-11.2 22.81 8.51 21.44 10.00 0 0 0 0 (SEQ ID NO.: 2) UMG-SP-16 27.86 0 0 0 0 0 0 0 (SEQ ID NO.: 3)