New enzymes for cleaving aromatic carbonates

Abstract

The invention relates to enzymes and enzyme-based methods for cleaving aromatic carbonates. The enzymes are particularly suitable for cleaving carbonates based on bisphenol A and can therefore be used to recover bisphenol A from polymers.

Description

[0001] Novel enzymes for the breakdown of aromatic carbonates

[0002] The present invention describes enzymes and enzyme-based processes for the cleavage of aromatic carbonates. The enzymes are particularly well suited for the cleavage of carbonates based on bisphenol A and can therefore be used for the recovery of bisphenol A from polymers.

[0003] Polycarbonates characterized by high transparency and impact strength can be obtained through the reaction of phosgene and bisphenol A. Because of these properties, such polycarbonates have found widespread use as glass substitutes, in vehicle construction, and as casings for electronic devices, such as mobile phones. Accordingly, the annual production of these polymers is very high.

[0004] Since polycarbonates are thermoplastic, recycling by melting is generally possible. However, this is complicated by the fact that polycarbonates are often mixed with additives to improve their application properties. Separating these additives may be desirable, but is technically challenging. Furthermore, repeated melting of a polycarbonate leads to partial depolymerization, which can release bisphenol A. Due to the harmful properties of bisphenol A, this is undesirable and limits the number of recycling cycles for the polymer. If polycarbonates are used in mixtures with other thermoplastics, the additional problem of separating these mixtures arises during reuse if pure polymers are required.

[0005] For this reason, there is a significant need for further processes for recycling polycarbonates. One approach, well-known from other polymers, is the targeted degradation of the polymer using enzymes to recover well-defined chemical feedstocks under gentle reaction conditions. This has already been demonstrated, for example, with polyurethanes (EP 3 587 570) and polyethylene terephthalate (Tournier (2020) “An engineered PET depolymerase to break down and recycle plastic bottles”, Nature 580: 216-219). This is also possible for aliphatic polycarbonates (Suyama & Tokiwa (1997), “Enzymatic degradation of an aliphatic polycarbonate, poly(tetramethylene carbonate)”, Enzyme and Microbial Technology, 20: 122-126).

[0006] For aromatic polycarbonates, which are technically and economically very important due to their extensive applications, especially those based on bisphenol A, technically viable enzymatic degradation processes have not yet been described. The release of bisphenol A from such compounds by a bacterium, Pseudoxanthomonas sp., has been described (Yue et al. (2021), “Biodegradation of bisphenol-A polycarbonate plastic by Pseudoxanthomonas sp. strain NY3600” Journal of Hazardous Materials, 416: 125775). However, it has not been shown which enzymes are responsible for the degradation. It is not even clear whether a single enzyme or an enzyme complex is involved. For the technical application of a recycling process, clearly defined and isolated enzymes are preferable because of their more controllable properties. This has been described (Artham et al.(2011), “Mechanistic investigations of lipase-catalyzed degradation of polycarbonate in organic solvents”, Enzyme and Microbial Technology, 48: 71-79), that lipases such as those found in Candida rugosa or Candida antarctica release bisphenol A from polycarbonates. However, it has been shown that this reaction is not catalyzed by the enzymes used there in aqueous media. The enzymatic degradation of aromatic polycarbonate in organic solvents has also been reported elsewhere (Sivalingam & Madras (2004) “Dynamics of Lipase Catalyzed Enzymatic Degradation of Poly(bisphenol-A carbonate), Journal of Polymer Science, 91: 2391-2396).

[0007] In the study underlying the present invention, enzymes were identified for the first time that enable the release of bisphenol A from aromatic polycarbonates in aqueous media. All enzymes with this catalytic capability exhibit a high degree of sequence identity. All tested enzymes from this group showed the desired activity, albeit to varying degrees.

[0008] To identify the amino acid positions crucial for activity, a consensus sequence of the amino acid sequences of all investigated enzymes was generated. This sequence is defined by SEQ ID NO.: 1. The threshold for generating the consensus sequence was set to 100%, meaning that the conserved positions represented in the consensus sequence contain the same amino acid in all investigated enzymes. Given the large number of enzymes tested, it can be assumed that these conserved positions are indeed responsible for the function according to the invention. In contrast, the positions marked with "X" as placeholders for arbitrary amino acids are considered functionally irrelevant.

[0009] In a first embodiment, the present invention relates to the use

[0010] (a) an 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

[0011] (b) an enzyme defined by an amino acid sequence according to SEQ ID NO.: 2 or 7 or a variant of one of the aforementioned enzymes defined by an amino acid sequence with at least 74% identity to SEQ ID NO.: 2 or at least 85% sequence identity to SEQ ID NO.: 7; for cleavage of an aromatic carbonate in an aqueous medium.

[0012] The aromatic carbonate is preferably a compound according to formula (I)

[0013] Where

[0014] RI is selected from the group consisting of hydrogen, hydroxyl, and any organic residues; and

[0015] R2, independently of RI, is selected from the group consisting of hydrogen, a compound according to formula (I) and any organic residues.

[0016] In a preferred embodiment of the present invention, the carbonate group to which R2 is bonded is located in the para position; more preferably, both substituents, RI and the oxygen of the carbonate group, are located in the para position. This is shown in formula (1a). The term "cleavage" refers to the hydrolytic cleavage of the carbonate group shown in formula (I). This releases a diphenylmethane derivative as an alcohol and a carbonate derivative as a carboxylic acid. Cleavage of the carbonate group at the oxygen atom bearing R2 yields a primary alcohol derived from R2 and a carboxylic acid derived from diphenylmethane. If R2 is hydrogen, carbonic acid is released. If RI is a hydroxyl group, bisphenol A is released.

[0017] An "aqueous medium" is a solvent mixture that consists of at least 50 wt% water by weight of all solvents present. The study underlying the present invention has shown that the addition of a polar organic solvent to the aqueous medium increases the degradation performance of the enzymes. Therefore, in a preferred embodiment of the present invention, the aqueous medium contains between 10 and 50 wt% of at least one polar organic solvent, more preferably 30 to 50 wt%, wherein the mass fractions of all solvents present, including the polar organic solvent, add up to 100%. It is particularly preferred that the aqueous medium consists of 80 wt%, more preferably 90 wt%, and most preferably 95 wt% water and optionally the polar organic solvent, by weight of all solvents present.A “polar organic solvent” is preferably an organic solvent that contains no carbonate or ester groups and has a log P value of at most 1.3. Preferred polar organic solvents are acetone, acetonitrile, dichloromethane, ethyl acetate, THF, and DMF. DMSO is particularly preferred as a polar organic solvent.

[0018] In a preferred embodiment of the present invention, the enzyme used according to the invention is immobilized. All techniques known to those skilled in the art, such as adsoption, covalent bonding to carrier materials, affinity immobilization, and inclusion immobilization, are suitable for immobilization.

[0019] The concentration of the aromatic carbonate, in particular the aromatic polycarbonate, in the aqueous medium is preferably 1 mg / l to 1,000 g / l, more preferably 500 mg / l to 500 g / l, even more preferably 100 mg / l to 150 g / l and most preferably 2 g / l to 70 g / l.

[0020] The enzyme concentration during use is preferably 1 pg / ml to 2,000 pg / ml, more preferably 10 pg / ml to 1,500 pg / ml, even more preferably 30 pg / ml to 1,000 pg / ml, and most preferably 70 pg / ml to 600 pg / ml. Concentrations of 120 pg / ml, 250 pg / ml, and 500 pg / ml are also conceivable. Preferably, the compound according to formula (I) is a polycarbonate. The term "polycarbonate" preferably refers to polymers obtained by reacting at least one compound selected from the group consisting of bisphenol A, bisphenol S, dihydroxydiphenyl sulfide, tetramethylbisphenol A, 1,1-bis(4-hydroxyphenyl)-3,3,5-trimethylcyclohexane (BPTMC), and 1,1,1-tris(4-hydroxyphenyl)ethane (THPE) with phosgene or diphenyl carbonate. Such polycarbonates preferably have a number-average molecular weight between 10,000 and 50,000 g / mol, more preferably between 18,000 and 40,000 g / mol.Optionally, such polycarbonates contain small amounts of tert-butylphenol and / or cumylphenol. These are used as chain terminators in the polymerization process.

[0021] Preferred polycarbonates are those obtainable by reacting bisphenol A with phosgene or diphenyl carbonate. The presence of compounds other than those mentioned above is not excluded, but less preferred. It is preferred that bisphenol A constitutes at least 80 wt.%, preferably at least 90 wt.%, and particularly preferably at least 95 wt.% of the total weight of all hydroxy-functional components of the polycarbonate. Such polycarbonates are preferably defined by formula (II).

[0022] A compound according to formula (II) is typically obtained by the reaction of bisphenol A and phosgene or by a melt condensation of bisphenol A and diphenyl carbonate.

[0023] In a further preferred embodiment, the aromatic polycarbonate is a copolymer of one of the aromatic polycarbonates defined above and at least one polyester. Preferably, it is a copolymer of a polycarbonate as defined in formula (II) and at least one polyester. Since the enzymes used according to the invention are capable of degrading polyesters as well as polycarbonates, the degradation of a polycarbonate-polyester copolymer into its monomeric components is possible. Preferred polyesters for the synthesis of such copolymers are polyethylene terephthalate, polybutylene terephthalate, polypentylene terephthalate, and polyhexylene terephthalate. To increase the accessibility of the compound according to formula (I), (1a), or of the aromatic polycarbonate, in particular the polycarbonate according to formula (II), to the enzyme when this compound is in solid form, all conventional methods can be used, especially milling.

[0024] In a preferred embodiment of the present invention, the compound is according to formula

[0025] (I), (1a) or the aromatic polycarbonate, in particular the aromatic polycarbonate according to formula

[0026] (II) as a component of a mixture of at least two different polymers. Different polymers are understood here to be those cross-linked by different functional groups. The compound according to formula (I) or (II) is therefore present in this embodiment together with at least one polymer that is not a polycarbonate. Preferably, in this embodiment, a polycarbonate as defined above is present as a mixture with at least one polymer selected from the group consisting of polyesters, styrene-acrylonitrile copolymer, and acrylonitrile-butadiene-styrene copolymer. Polyethylene terephthalate and / or polybutylene terephthalate are preferred as polyesters. It is particularly preferred that a bisphenol A-based polycarbonate is present as a mixture with at least one of the aforementioned polymers.

[0027] This makes it possible to selectively degrade the polycarbonate enzymatically, while leaving the other polymer(s) in the mixture unaffected. Aromatic polycarbonates can thus be selectively removed from polymer mixtures. Conversely, non-polycarbonate polymers can be purified in this way. This enables the recovery of pure polymers from polycarbonate-containing polymer mixtures.

[0028] Table 1: Amino acid sequences of the enzymes used * Since the enzymes were obtained from commercial suppliers, it cannot be said with absolute certainty whether their amino acid sequence was identical to the sequences obtained from public databases.

[0029] 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 the carbonate group shown in formula (1) or (1a). This is preferably demonstrated by the release of bisphenol from a polycarbonate obtained by the reaction of bisphenol A and phosgene or by melt condensation of bisphenol A and diphenyl carbonate.

[0030] In a preferred embodiment, an enzyme is used 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.: 3, 8, 2, 4, 5, 6, 7, 9, and 10. Preferably, the arbitrary amino acid positions exhibit the aforementioned sequence identities with SEQ ID NO.: 3 or 8.

[0031] 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.

[0032] In a preferred embodiment of the present invention, the variant defined above by SEQ ID NO.: 2 is characterized by a sequence identity of at least 74%, preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, and most preferably at least 95%.

[0033] In a further preferred embodiment of the present invention, the variant of an enzyme defined above by SEQ ID NO.: 7 is characterized by a sequence identity of at least 90% and preferably at least 95%. In yet another embodiment, the present invention relates to the use of an enzyme having an amino acid sequence as defined in SEQ ID NO.: 3, 8, 2, 4, 5, 6, 7, 9, 10, 11, 29 or 30, or a variant of one of the aforementioned enzymes with at least 85% sequence identity to one of the aforementioned sequences, for the cleavage of an aromatic carbonate in an aqueous medium. All definitions of the aromatic carbonate given above also apply to this embodiment.

[0034] In a preferred embodiment, the present invention relates to the use of an enzyme having an amino acid sequence as defined in SEQ ID NO.: 3, 8, 7, 9, 5 or 27, or a variant of one of the aforementioned enzymes with at least 85% sequence identity to one of the aforementioned sequences, for the cleavage of an aromatic carbonate in an aqueous medium. All the above-mentioned definitions for the aromatic carbonate also apply to this embodiment.

[0035] In a particularly preferred embodiment, the present invention relates to the use of an enzyme having an amino acid sequence as defined in SEQ ID NO. 3 or 8, or a variant of one of the aforementioned enzymes with at least 85% sequence identity to one of the aforementioned sequences, for the cleavage of an aromatic carbonate in an aqueous medium. All the above-mentioned definitions for the aromatic carbonate also apply to this embodiment.

[0036] 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.: 3, 8, 2, 4, 5, 6, 7, 9, 10, TI, 29, or 30. 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.: 3, 8, 2, 4, 5, 6, 7, 9, 10, TI, 29, or 30, which are fused at the N-terminus and / or the C-terminus with other proteins, e.g., the Green Fluorescent Protein.

[0037] 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. To ensure effective cleavage of the carbonate group, the enzymes, together with the compound according to formula (I), are incubated in the aqueous medium defined above in this application, preferably at a temperature between 15 and 80 °C, more preferably between 20 and 75 °C, and particularly preferably between 30 and 70 °C. The pH is preferably between 5 and 10. The polypeptide defined by SEQ ID NO.: 3 and its variants exhibit two pH optima, one at pH 7 and one at pH 9.Further details regarding the reaction conditions particularly suitable for the enzymes used according to the invention are given in the exemplary embodiments.

[0038] In a further embodiment, the present invention relates to a method for cleaving a carbonate bond in a compound according to formula (I) comprising the process steps a) providing an aqueous medium containing a mixture containing at least one compound according to formula (I) and

[0039] (i) at least one enzyme characterized by an amino acid sequence according to SEQ ID NO.:

[0040] 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

[0041] (ii) at least one enzyme characterized by an amino acid sequence according to SEQ ID NO.:

[0042] 2 or 7 or a variant of one of the aforementioned enzymes, defined by an amino acid sequence with at least 74% identity to SEQ ID NO.: 2 or at least 85% sequence identity to SEQ ID NO.: 7; and b) incubation of the mixture under conditions in which the enzyme cleaves aromatic carbonates.

[0043] This process is particularly suitable for the cleavage of the polycarbonates characterized by formula (II). In this process, these compounds are cleaved into fragments with lower molecular weight. Preferably, they are degraded to carbonic acid and bisphenol A. It is particularly preferred that process step b) is carried out until at least 30 wt.%, preferably at least 50 wt.%, more preferably at least 70 wt.%, and most preferably at least 90 wt.% of the total mass of the compound present at the beginning of process step b) according to formula (II) has been degraded to bisphenol A and carbonic acid. When a compound according to formula (II) is degraded by the process defined above, bisphenol A is released.Therefore, in a further embodiment, the present invention relates to a process for the production of bisphenol A comprising the steps a) providing an aqueous medium containing a mixture containing at least one compound according to formula (I) and.

[0044] (i) at least one enzyme characterized by an amino acid sequence according to SEQ ID NO.:

[0045] 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

[0046] (ii) at least one enzyme characterized by an amino acid sequence according to SEQ ID NO.:

[0047] 2 or 7 or a variant of one of the aforementioned enzymes, defined by an amino acid sequence with at least 74% identity to SEQ ID NO.: 2 or at least 85% sequence identity to SEQ ID NO.: 7; and b) incubation of the mixture under conditions in which the enzyme exhibits esterase activity.

[0048] In process step b), bisphenol A is released. Therefore, this is a process for recovering bisphenol A from polycarbonates, i.e., a recycling process.

[0049] Process step b) is preferably carried out until at least 5, preferably at least 10, more preferably at least 50 and even more preferably at least 70 wt.% of the bisphenol A bound at the beginning in the total mass of the compound according to formula (II) used is released.

[0050] In a preferred embodiment, the above-defined process for the production of bisphenol A includes a further process step for separating bisphenol A from the mixture obtained by incubation in process step b). This can advantageously be achieved by extracting the released bisphenol A with an organic solvent, preferably toluene. After phase separation, the bisphenol A can then be easily crystallized by lowering the temperature.

[0051] In yet another embodiment, the present invention relates to the use of an enzyme.

[0052] (i) that 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

[0053] (ii) that is defined by an amino acid sequence according to SEQ ID NO.: 2 or 7 or a variant of one of the aforementioned enzymes, defined by an amino acid sequence with at least 74% identity to SEQ ID NO.: 2 or at least 85% sequence identity to SEQ ID NO.: 7; for the activation of a prodrug according to formula (I) by cleavage of the carbonate group.

[0054] In yet another embodiment, the present invention relates to a composition comprising a)

[0055] (i) 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

[0056] (ii) at least one enzyme defined by an amino acid sequence according to SEQ ID NO.: 2 or 7 or a variant of one of the aforementioned enzymes defined by an amino acid sequence with at least 74% identity to SEQ ID NO.: 2 or at least 85% sequence identity to SEQ ID NO.: 7; (b) at least one aromatic carbonate; and (c) at least one aqueous medium.

[0057] The aromatic carbonate is preferably a compound according to formula (I) (I)

[0058] Where

[0059] RI is selected from the group consisting of hydrogen, hydroxyl, and any organic residues; and

[0060] R2, independently of RI, is selected from the group consisting of hydrogen, a compound according to formula (I) and any organic residues.

[0061] All further definitions given above for the enzyme and the aromatic carbonate also apply to the composition.

[0062] In yet another embodiment, the present invention relates to a kit containing a)

[0063] (i) 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

[0064] (ii) at least one enzyme defined by an amino acid sequence according to SEQ ID NO.: 2 or 7 or a variant of one of the aforementioned enzymes defined by an amino acid sequence with at least 74% identity to SEQ ID NO.: 2 or at least 85% sequence identity to SEQ ID NO.: 7; and (b) at least one aromatic carbonate.

[0065] In a particularly preferred embodiment, the kit additionally contains at least one aqueous medium.

[0066] The aromatic carbonate is preferably a compound according to formula (I)

[0067] Where

[0068] RI is selected from the group consisting of hydrogen, hydroxyl, and any organic residues; and

[0069] R2, independently of RI, is selected from the group consisting of hydrogen, a compound according to formula (I) and any organic residues.

[0070] All other definitions given above for the enzyme and the aromatic carbonate also apply to the kit.

[0071] Images

[0072] Figure 1 visualizes the sequence similarity of the enzymes investigated in the study underlying this invention.

[0073] The following exemplary embodiments serve only to illustrate the invention. They are not intended to limit the scope of protection of the patent claims in any way.

[0074] Examples of implementation

[0075] Enzymes

[0076] Commercially available enzymes with the amino acid sequences according to SEQ. ID NO.: 15 to 27 were obtained from Sigma-Aldrich (Steinheim, Germany), Strem Chemicals (Bischheim, France) or Fisher Scientific (Rockford, USA).

[0077] Plasmid construction

[0078] Genes of interest were synthesized by Eurofins Genomics (Ebersberg, Germany) and cloned into suitable Escherichia coli (E. coli) vectors using restriction cloning with Xhol and Ndel or with other suitable restriction enzymes from the multiple cloning site. For plasmid propagation, chemically competent E. coli DH5a cells were transformed. Grown single colonies were used to inoculate 4.5 ml of LB medium containing 50 pg / ml kanamycin and were either preserved in 25% glycerol stocks or prepared for sequencing at GeneWiz (Leipzig, Germany) according to the manufacturer's instructions.

[0079] Heterologous protein expression

[0080] 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 0.1% freshly grown cells and incubated at 37 °C and 200 rpm for 5 h in baffled flasks. Expression was performed overnight at 20 °C and 200 rpm. The cells were harvested at 4 °C for 10 min at 8000 x g.

[0081] Cell lysis

[0082] The harvested cells for the initial screening were resuspended in 6 ml of ammonium acetate buffer (20 mM ammonium acetate, 0.4% w / v n-dodecyl-β-maltoside, 1% w / v lysozyme, and 1 pl / ml benzoase) and incubated for 30 min at room temperature with shaking to lyse the cells. Harvested cells expressing BhrPETase, DuraPETase, ThcCutl-ACCG, SvCutl90*SS, LCC-ICCG, and LCC-WCCG for the initial screening were resuspended in 10 ml of 20 mM KPi buffer, pH 7.5. Cells for enzyme purification were resuspended in 15 ml of lysis buffer (50 mM NaH₂PO₄, 300 mM NaCl, and 10 mM imidazole). The cells were lysis twice using ultrasound (Bandelin Sonoplus HD, Sonotrode MS73, Berlin, Germany) under constant cooling in an ice bath for 2 min (50% cycle). The resulting suspension was centrifuged at 10,000 x g and 4 °C for 20 min. The supernatant was filtered through a 0.2 pm PES filter.

[0083] Lyophilization of the cell lysate: The supernatant of cells expressing BhrPETase, DuraPETase, ThcCutl-ACCG, SvCutl90*SS, LCC-ICCG, and LCC-WCCG, as well as the lysate of the other cells for the initial screening, containing all cell components, were frozen at -80 °C and lyophilized for 3 days at -68 °C and 0.1 mBar. The lyophilized cells were stored at -4 °C.

[0084] Enzyme purification

[0085] The His-tagged enzymes were purified by IMAC affinity chromatography. The columns were 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. The filtered lysate was loaded onto the column. After loading the lysate, 10 CV of wash buffer (50 mM NaHjPC, 300 mM NaCl, 20 mM imidazole) was added to the column. The specifically bound His-tagged enzyme was eluted in elution buffer (50 mM NaHjPC, 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 100 mM potassium phosphate buffer pH 7.5.

[0086] Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)

[0087] Enzyme expression and protein purity were investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). 14 µl of the lysed cell suspension, the supernatant after centrifugation, or the purified enzyme solution were mixed with 7 µl of 3x Reducing Blue Protein Loading Dye. The solutions were incubated for 5 min at 95 °C. 10 µl of the samples, along with 3 µl of EZ-Run® Prestained Rec Protein Ladder, were loaded onto a pre-poured NuPAGE 4–12% Bis-Tris gel. The proteins were separated electrophoretically at a constant voltage of 200 V for 35–40 min. The gels were stained for 1 h with 30–50 ml of Roti BlueQuick ready-to-use staining solution.

[0088] Determination of protein concentration

[0089] Protein concentration was determined colorimetrically using the Bradford assay. A standard BSA curve of 125–1000 pg / ml was prepared. The enzyme was diluted with potassium phosphate buffer pH 7.5 as needed. 250 ml of Quick Start Bradford reagent (1x) were added to 5 µL of BSA and enzyme solution. After 5 min incubation, the absorbance was measured at 595 nm.

[0090] Enzyme activity test using BPA-PC

[0091] BPA-PC (Makrolon 2408, Covestro Deutschland AG) was milled to 500 pm before use. Initial screening for hydrolysis of BPA-PC was performed using a spatula tip of lyophilized enzyme (commercially or internally heterologously expressed) dissolved in 200 pl of potassium phosphate buffer (50 mM KPi, 100 mM NaCl pH 7.5) and a spatula tip of BPA-PC. The reactions were incubated at 40 °C, 50 °C, 60 °C, and 70 °C at 800 rpm for 146 h. For poorly expressed enzymes in the lyophilisate, purified enzyme was used. 100 pg / ml of enzyme was added to 10 mg / ml of BPA-PC up to a final volume of 200 pl in 100 mM potassium phosphate buffer pH 5–8. The reactions were incubated at 40-70 °C and 800 rpm for 384 h.

[0092] All enzymes exhibiting detectable BPA-PC hydrolysis were purified. The reaction conditions with the highest BPA-PC hydrolysis from the previous assay for each enzyme were used to quantify the conversion to BPA. The four best enzymes were selected, and the conversion to BPA was measured upon addition of 50–600 pg / ml enzyme to 1, 10, and 50 mg / ml BPA-PC in 100 mM potassium phosphate buffer, CHES buffer, or 1,3-bis(tris(hydroxymethyl)methylamino)propane at pH 5–10. The reaction was run at 50–90 °C and 800 rpm for 72 h. Subsequently, DMSO was added to the reaction at a 1:1 ratio. The samples were vortexed for 10 s and centrifuged for 5 min at 11,000 x g. The supernatant was diluted 1:10 and filtered with a PVDF filter (0.2 pm).

[0093] HPLC measurements

[0094] 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). For all methods, 5 pl of sample was injected, and the column was heated to 40 °C. The flow rate was 1.0 ml / min. The method developed for the analysis and quantification of the BPA-PC hydrolysis reaction is shown in Table 2. The data were analyzed using Chromeeon Chromatography Data System 7.3.1 software (Thermo Scientific, Waltham, USA).

[0095] Table 2: HPLC method

[0096] Construction of the phylogenetic tree and consensus sequence

[0097] Sequences of various hydrolase representatives from different organisms were retrieved from the PDB and Uniprot databases and combined with sequences of interest for this study. Three hydrolase sequences from the Uniprot database have been described for Aspergillus niger and Burkholderia cepacia and were included in the analysis. ClustalW sequence alignment was performed using MEGA11 software. Phylogenetic analysis was carried out using the default settings and the maximum likelihood method. The bootstrap consensus tree represents the evolutionary history of the analyzed taxa. Original trees for heuristic searching were automatically generated by applying the Neighbor Join and BioNJ algorithms to a matrix of pairwise distances estimated using a Jones-Taylor-Thornton (JTT) model and the topology with the superior log likelihood value. The analysis encompassed 25 amino acid sequences.The sequence of alkaline phosphatase from E. coli was used as an outlier sequence.

[0098] A ClustalW alignment of the amino acid sequences of BPA-PC hydrolyzing enzymes was used to calculate consensus sequences with 100% and 85% thresholds. Two phylogenetic groups of BPA-PC hydrolyzing enzymes were used to determine sequence similarity.

[0099] Results

[0100] The hydrolysis of BPA-PC to BPA was tested using the enzymes listed in the table. Of the 26 enzymes tested, 10 hydrolyzed BPA-PC. Enzymes numbered 15-27 are commercially available. Furthermore, the cutinase from Humicola insolens, obtained as Novozym® 51032, also demonstrated hydrolysis of BPA-PC. This enzyme has an amino acid sequence as defined by SEQ. ID NO.: 27 (Ternström et al. (2005) “Unfolding and inactivation of cutinases by AOT and guanidine hydrochloride”, Biochimica et Biophysika Acta, 1748: 74-83). No conversion to BPA was detected with the lipases from Candida antarctica (CAL), Candida rugosa (CRL) and porcine pancreatic lipase (PPL) previously published (Artham et al. (2011), “Mechanistic investigations of lipase-catalyzed degradation of polycarbonate in organic solvents”, Enzyme and Microbial Technology, 48: 71-79).

[0101] Table 3: Overview of the enzymes used and their activity

[0102] *The enzymes used in the degradation experiments were obtained from commercial suppliers; the amino acid sequences listed in the sequence protocol were taken from public databases and used for sequence analysis.

[0103] The BPA-PC hydrolyzing enzymes were purified and analyzed for BPA-PC conversion (Table 3). At 70 °C in 100 mM bis(tris(hydroxymethyl)methylamino)propane at pH 7 with ThcCutl-ACCG or pH 9 for LCC, LCC-ICCG, and LCC-WCCG, conversions of 16.8–33.2% were determined. The addition of DMSO was tested with the two best variants, ThcCutl-ACCG and LCC-ICCG. Adding 20–50% DMSO to the reaction with ThcCutl resulted in 100% conversion to BPA. With LCC-ICCG, the conversion to 92.9% BPA was highest with the addition of 30% DMSO. Without the addition of DMSO, replacing the aqueous phase four times after 72 h with a fresh enzyme buffer solution resulted in 94% conversion to BPA via ThcCutl-ACCG and 51% via LCC-ICCG. For this, after 72 h, the reaction was centrifuged for 3 min at 13,300 rpm, the supernatant was transferred to a new vessel, and the same volume of enzyme buffer solution was added to the BPA-PC. The reaction was then repeated at 70 °C and 800 rpm for another 72 h.After replacing the enzyme buffer solution three times, DMSO was added to the reaction or supernatant in a 1:1 ratio. The samples were measured as described.

[0104] Incubation conditions where the highest BPA release was measured are shown in parentheses.

[0105] Table 4: Suitable reaction conditions Table 5: Quantification of BPA release determined with lyophilized lysate

[0106] Phylogenetic analysis

[0107] The obtained phylogeny shows the close relationship of the enzymes. The cluster of BPA-PC hydrolyzing enzymes shares a common ancestor with IsPETase, PET2, and PET6. No BPA-PC hydrolysis was detectable in the latter enzymes. Therefore, this function evolved later. Within the cluster of BPA-PC hydrolyzing enzymes, two groups can be distinguished. One group contains ThcCutl, ThcCutl-ACCG, TfCut2, SvCutl90*SS, and PesHl (Group 1). The other group contains LCC, LCC-ICCG, LCC-WGGC, and BhrPETase (Group 2).

[0108] The commercial enzymes (Nos. 15-27) are only distantly related to each other and to the cluster of BPA-hydrolyzing enzymes, as indicated by the longer branches. The majority of the reported relationships within the BPA-hydrolyzing enzyme cluster are validated by the bootstrap values. For early branching ancestors, bootstrap values ​​indicate a less pronounced relationship between the sequences.

[0109] Based on the alignment of the sequences of BPA-PC hydrolyzing enzymes, the consensus sequence was calculated with a 100% threshold (SEQ ID NO: 1) and an 85% threshold (SEQ ID NO: 28). The amino acid sequence shows higher sequence variability at the C-terminus compared to the N-terminus.

[0110] Table 6: Sequence identities of the active enzymes

[0111] To calculate sequence similarity, the number of identical and similar amino acid positions was added together. Then, the percentage of this sum was calculated relative to the total number of amino acid positions in the alignment.

[0112] The following amino acids were defined as similar:

[0113] Small / alipatic residues: Glycine, Alanine, Valine, Leucine, Isoleucine

[0114] Aromatic residues: phenylalanine, tyrosine, tryptophan

[0115] Sulfur-containing residues: cysteine, methionine

[0116] OH-functional or basic residues: serine, threonine, lysine, arginine, histidine

[0117] Acidic, amide-containing residues and proline: aspartic acid, glutamic acid, asparagine, glutamine,

[0118] Proline

[0119] To calculate the sequence identity, the percentage of identical amino acid positions was calculated based on the total number of amino acid positions in the alignment.

[0120] Testing of other enzymes

[0121] To verify that all amino acid positions of the consensus sequence defined by SEQ ID NO.: 1 are essential for enzyme function, two additional cutinases were tested: Kubu-P M12 (SEQ ID NO.: 29) and Mipa-P M19 (SEQ ID NO.: 30). These enzymes were described in Seo et al. (2025), “Landscape profiling of PET depolymerases using a natural sequence cluster framework”, Science, Vol. 387, eadp 5637. The enzymes were heterologously expressed as described above. The polycarbonate described above was used as the test substrate. The polycarbonate was quantified by detecting BPA using HPLC as described above.

[0122] Kubu-P M12 and the consensus sequence defined by SEQ ID NO.: 1 have a sequence similarity of 87% according to the criteria defined above. The sequence identity determined according to the criteria defined above is 24%. After 120 hours at 70°C, a conversion of 10.1% was observed in a mixture of water and DMSO containing 30% DMSO by volume.

[0123] Mipa-P M19 and the consensus sequence defined by SEQ ID NO.: 1 have a sequence similarity of 87% according to the criteria defined above. The sequence identity determined according to the criteria defined above is 26%. After 120 hours at 70°C, a conversion of 20.6% was observed in a mixture of water and DMSO containing 30% DMSO by volume (based on the total weight of the mixture).

[0124] The results show that even cutinases with very low sequence identity to the consensus sequence are able to depolymerize BPA-based polycarbonate. It can therefore be assumed that an exchange of up to 15% of the amino acid positions defined by SEQ ID NO.: 1 is possible without destroying the enzyme function according to the invention.

[0125] Detection of the degradation of low molecular weight aromatic carbonates

[0126] 0.4 mg of lyophilized crude lysate (commercially acquired or heterologously expressed enzyme) was dissolved in 400 pl KPi buffer (50 mM potassium phosphate, 100 mM NaCl pH 7.5). 5 mg / ml substrate solutions of dimethyl carbonate (DMC), benzyl(4-nitrophenyl) carbonate (BNPC), and diphenyl carbonate (DPC) were prepared in DMSO.

[0127] For the reaction with DMC, 100 µl of the enzyme solution were mixed with 250 µl of 25 mM KPi buffer containing approximately 0.1 mg / ml of phenol red, 100 µl of water, and 50 µl of substrate solution. The samples were incubated for 22 h at 40 °C and 800 rpm. The hydrolysis of dimethyl carbonate produces carbonic acid. The pH decrease due to carbonic acid formation is detected using phenol red and is visible as a color change from red to yellow.

[0128] The hydrolysis of BNPC and DPC was determined using 100 µL of the dissolved lysate mixed with 350 µL of KPi buffer and 50 µL of substrate solution. Approximately 0.1 mg / mL of phenol red was added for DPC. Samples were incubated for 20 h at 20 °C or at 20 °C followed by 40 °C, 800 rpm, for BNPC and DPC, respectively. The hydrolysis product of BNPC is p-nitrophenol (pNP), which was detected spectrophotometrically at 410 nm. A standard curve of pNP was used as a reference. For the analysis of the DPC samples, 500 µL of acetonitrile was added after the reaction to stop it, and the samples were used for HPLC measurements. A phenol standard was used as a reference. The same HPLC method used for PC was employed for the determination of phenol.

[0129] A change in color was considered an indication of degradation, without quantifying it.

[0130] Table 7: Degradation of low molecular weight substrates

[0131] Enzyme dimethyl carbonate benzyl (4-diphenyl carbonate nitrophenyl) carbonate

[0132] LCC + + +

[0133] THC-Cutl + + +

[0134] TfCut2 + + +

[0135] PesHl + + +

[0136] Novozym®51032 + + +

[0137] ThcCutl-ACCG nd 1846.3 mg N ph 1 and

[0138] Empty vector nd: not determined

[0139] The results show that the tested selection of enzymes was able to cleave even low-molecular-weight carbonates. This result is likely also applicable to all other enzymes that can degrade polycarbonate, since low-molecular-weight substrates are more accessible to the enzyme than polymers.

Claims

Patent claims 1. Use of an enzyme, (a) that 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 (b) that is defined by an amino acid sequence according to SEQ ID NO.: 2 or 7 or a variant of one of the aforementioned enzymes, defined by an amino acid sequence with at least 74% identity to SEQ ID NO.: 2 or at least 85% sequence identity to SEQ ID NO.: 7, for the cleavage of an aromatic carbonate in an aqueous medium.

2. The use according to claim 1, wherein the enzyme 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% sequence identity with the conserved amino acid positions from SEQ ID NO.: 1; and (ii) The amino acid positions homologous to the arbitrary amino acid positions from SEQ ID NO.: 1 exhibit at least 70% sequence identity to the homologous amino acid positions of one of the amino acid sequences selected from the group consisting of SEQ ID NO.: 3, 8, 2, 4, 5, 6, 7, 9 and 10.

3. The use according to claim 1, wherein the enzyme has at least 80% sequence identity to SEQ ID NO.: 2 or at least 90% sequence identity to SEQ ID NO.:

7.

4. The use according to any one of claims 1 to 3, wherein the aromatic carbonate is defined by formula (I). And whereby RI is selected from the group consisting of hydrogen, hydroxyl, and any organic residues; and R2, independently of RI, is selected from the group consisting of hydrogen, a compound according to formula (I) and any organic residues.

5. The use according to any one of claims 1 to 4, wherein the aromatic carbonate is a polycarbonate.

6. The use according to claim 5, wherein the polycarbonate is obtained or obtained by reacting at least one compound selected from the group consisting of bisphenol A, bisphenol S, dihydroxyfephenyl sulfide, tetramethylbisphenol A, 1,1-bis(4-hydroxyphenyl)-3,3,5-trimethylcyclohexane (BPTMC) and 1,1,1-tris(4-hydroxyphenyl)ethane (THPE) with phosgene or diphenyl carbonate.

7. The use according to claim 6, wherein the polycarbonate is defined by formula (II).

8. The use according to any one of claims 5 to 7, wherein the aromatic polycarbonate is present as a copolymer with at least one polyester.

9. The use according to any one of claims 5 to 7, wherein the polycarbonate is present as a mixture with at least one other polymer.

10. Method for cleaving an ester bond in a compound according to formula (I) where RI is selected from the group consisting of hydrogen, hydroxyl, and any organic residues; and R2 is selected independently of RI from the group consisting of hydrogen, a compound according to formula (I) and any organic residues; comprising the process steps a) providing an aqueous medium containing a mixture containing at least one compound according to formula (I) and (i) at least one enzyme characterized 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) at least one enzyme characterized by an amino acid sequence according to SEQ ID NO.: 2 or 7 or a variant of one of the aforementioned enzymes, defined by an amino acid sequence with at least 74% identity to SEQ ID NO.: 2 or at least 85% sequence identity to SEQ ID NO.: 7; and - TI - b) Incubation of the mixture under conditions in which the enzyme cleaves aromatic carbonates.

11. The method according to claim 10, wherein the compound is defined by formula (II).

12. The method according to claim 11, wherein the compound according to formula (II) is a mixture with at least one polymer selected from the group consisting of polyesters, styrene-acrylonitrile copolymer and acrylonitrile-butadiene-styrene copolymer.

13. The method according to claim 12, wherein the polyester is selected from the group consisting of polyethylene terephthalate and polybutylene terephthalate.

14. The method according to any one of claims 11 to 13, wherein process step b) is carried out until at least 5 wt.% of the total mass of the compound present at the beginning of process step b) is reduced according to formula (II).

15. Use of an enzyme (i) that 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) that is defined by an amino acid sequence according to SEQ ID NO.: 2 or 7 or a variant of one of the aforementioned enzymes, defined by an amino acid sequence with at least 74% identity to SEQ ID NO.: 2 or at least 85% sequence identity to SEQ ID NO.: 7; for the activation of a prodrug according to formula (I) (I) And whereby RI is selected from the group consisting of hydrogen, hydroxyl, and any organic residues; and R2 is selected independently of RI from the group consisting of hydrogen, a Compound according to formula (I) and any organic residues; by cleavage of the carbonate group.