FIELD OF THE INVENTION

The present invention relates to the fields of life sciences, enzymes, micro organisms and degradation of plastics or synthetic polymers. Specifically, the in vention relates to a method of degrading a polyester or polyesters with a specific micro-organism or enzyme or fragment of the enzyme comprising polyethylene 2,5-furandicarboxylate (PEF) degrading activity. Also, the present invention relates to an isolated enzyme or a fragment thereof comprising PEF degrading activity and capable of degrading a polyester, and a micro-organism or host cell capable of degrading a polyester and comprising an enzyme or a fragment thereof com prising PEF degrading activity. And still, the present invention relates to a polynu cleotide, an expression vector or plasmid, and use of the enzyme, fragment, mi cro-organism or host cell of the present invention for degrading a polyester.

BACKGROUND OF THE INVENTION

Plastics are widely used in our economy because of for example low costs and ease of manufacturing. Due to increasing production and use of plastics as well as poor recycling of plastics, millions of tons accumulate annually in terrestrial or ma rine environments. The total amount of virgin plastics produced from 1950 through 2015 is around 8300 million tonnes. A major share of this amount has been manufactured in the 2000’s and it will be increasing. E.g. annual plastic production in 2016 was 335 million tonnes. To date, globally around 6300 million tonnes of plastic waste has been generated in total. Of the plastic waste generated around 9% has been recycled, 12% incinerated, and the rest 79% landfilled or accumulat ed in the natural environment. (Geyer et al. 2017, Science Advances, 3(7), p.e1700782)

It is well known that plastics have adverse effects in all ecosystems and they are of particular concern to our health.

Plastic waste is a challenging material for recycling. Usually, plastic waste is a mixture of several kind of plastics like including e.g. polyesters. Also, different kind of biodegradable plastics (which amount will increase in the future) cause chal lenges into plastic waste recycling. Removal of plastics from the environment using microbes or microbial enzymes is of high interest. In general, biotechnical plastic degradation is not common yet. In biotechnological systems microbes or enzymes degrade specific plastic waste to different products. The benefit of biotechnical plastic waste recycling compared to mechanical or chemical recycling is that plastic waste can be impure and it can contain several different kind of plastics. In biotechnical plastic recycling all plas tics could be recycled and the quality of a starting material does not limit or pre vent the use of micro-organisms or enzymes for degradation. Microbes and en- zymes degrade specific plastics from the mixture and furthermore, microbes and/or enzymes can also degrade organic material. With biotechnical recycling it can be possible to degrade plastics to smaller units (e.g. having a smaller particle size and/or unit size, or e.g. shorter chain length or molecular weight of a polymer) when compared to the existing mechanical recycling systems, or to larger units (e.g. having a longer chain length or molecular weight of a polymer) compared to chemical recycling systems. Furthermore, high volumes of plastics are not needed for biotechnical degradation, e.g. because energy demand will be lower in bio technical systems compared to chemical and mechanical recycling. For example, CN108004166 (A) describes a specific microbial flora capable of ef ficiently degrading a polyester film (polybutylene adipate terephthalate (PBAT) film). Yoshida et al. (2016, Science, 351 (6278), 1196-1199) describe a bacterium Ideonella sakaiensis 201 -F6 that is able to degrade a polyester polyethylene ter ephthalate (PET). WO2019/168811 A1 describes a genetically modified Ideonella sakaiensis enzyme comprising very specific modifications and capable of degrad ing PET polyesters. Micro-organisms and enzymes acting on polyesters are need ed for rapid degradation and recycling of said high molecular weight polymers. Thus, there remains a need for effective micro-organisms or enzymes for degrad ing polyesters of one type or a combination of different types of polyesters.

BRIEF DESCRIPTION OF THE INVENTION

By biotechnical degradation and tools of the present invention it is possible to de grade and recycle polyesters including mixtures of polyesters and other synthetic polymers or plastics, and dirty/impure polyesters. Biotechnical degradation of dif ferent plastics can be carried out e.g. simultaneously or sequentially. Furthermore, the tools of the present invention can be used e.g. for upcycling polyesters i.e. for modifying a non-biodegradable polyester to a biodegradable polyester (such as polyhydroxyalkanoate (PHA)) by micro-organisms or enzymes.

The objects of the invention, namely methods and tools for degrading a polyester are achieved by utilizing a specific enzyme or enzymes, or a specific micro organism or micro-organisms (e.g. a bacterium/bacteria or fungus/fungi). Actually, the present invention provides specific tools for biotechnical degradation of a poly ester.

The methods and tools of the present invention provide surprising degradation ef fects on a polyester, a combination of different polyesters, or a combination of a polyester and other one or more specific plastic or synthetic polymer. Also, the present invention can overcome the problems of the prior art including but not lim ited to a slow biotechnical degradation speed.

Also, the inventors of the present disclosure found out that unique or specific deg radation products can be obtained from a polyester with the present invention. In other words, degradation end products of interest (e.g. a plastic monomer or pol ymer, a chemical,) can be tailored by biotechnical degradation of the present in vention.

The present invention relates to a method of degrading a polyester, said method comprising providing a polyester containing material and an enzyme comprising or hav ing polyethylene 2,5-furandicarboxylate (PEF) degrading activity, wherein said en zyme is capable of degrading the polyester and said enzyme has at least 70% (e.g. at least 80%) sequence identity to SEQ ID NO: 5, or a fragment of the en zyme comprising PEF degrading activity, and allowing said enzyme or fragment to degrade the polyester.

Also, the present invention relates to an isolated enzyme comprising or having PEF degrading activity, wherein said enzyme is capable of degrading a polyester and said enzyme has at least 70% (e.g. at least 80%) sequence identity to SEQ ID NO: 5, or a fragment thereof comprising PEF degrading activity.

And also, the present invention relates to a micro-organism or host cell comprising an enzyme comprising or having PEF degrading activity, wherein said enzyme is capable of degrading a polyester and said enzyme has at least 70% (e.g. at least 80%) sequence identity to SEQ ID NO: 5, or comprising a fragment of the enzyme comprising PEF degrading activity.

Still, the present invention relates to an isolated enzyme or fragment, or a micro organism or host cell of the present invention for degrading a polyester.

Still, the present invention relates to a polynucleotide encoding the enzyme of the present invention or a fragment thereof.

Still, the present invention relates to an expression vector or plasmid comprising the polynucleotide of the present invention.

Still furthermore, the present invention relates to use of the enzyme, fragment, mi cro-organism, host cell, polynucleotide, expression vector or plasmid of the pre sent invention or any combination thereof for degrading a polyester.

And still furthermore, the present invention relates to a method of producing the enzyme or fragment of the present invention, wherein a recombinant micro organism or host cell comprising a polynucleotide encoding the enzyme or frag ment of the present invention is allowed to express said enzyme or fragment.

Other objects, details and advantages of the present invention will become appar ent from the following drawings, detailed description and examples.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows PEF polymer degradation products with Acidovorax delafieldii PBS(A) depolymerase and Ideonella sakaiensis PETase in reverse-phase HPLC.

Figure 2 shows the amount of FDCA released (mM) in enzyme reaction with Aci dovorax delafieldii PBS(A) depolymerase and Ideonella sakaiensis PETase.

Figure 3 shows PEF degradation products with Acidovorax delafieldii PBS(A) de polymerase and Ideonella sakaiensis PETase in GC-MS analysis.

Figure 4 shows a PBS degradation product with Acidovorax delafieldii PBS(A) de polymerase in GC-MS analysis. Figure 5 shows a PET dagradation product with Acidovorax delafieldii PBS(A) de polymerase in GC-MS analysis.

Figure 6 shows a PFIBV degradation product with Acidovorax delafieldii PBS(A) depolymerase in GC-MS analysis.

Figure 7 shows an alignment of Ideonella sakaiensis PETase (IsPETase) and Aci dovorax delafieldii PBS(A) depolymerase (AdPETase) amino acid sequences.

Figure 8 shows PEF degradation products with Rhizobacter sp. dienelactone hy drolase (RhPETase) in GC-MS analysis.

Figure 9 shows PEF degradation products with Cytophagales bacterium dienelac tone hydrolase (CbPETase) in GC-MS analysis.

SEQUENCE LISTING

SEQ ID NO: 1 shows a A. delafieldii PBS(A) depolymerase amino acid sequence with a signal sequence.

SEQ ID NO: 2 shows a mature A. delafieldii PBS(A) depolymerase nucleotide se quence codon optimized to E. coli (the sequence comprises a His tag).

SEQ ID NO: 3 shows a I. sakaiensis PETase amino acid sequence with a signal sequence.

SEQ ID NO: 4 shows a mature I. sakaiensis PETase nucleotide sequence codon optimized to E. coli (the sequence comprises a His tag).

SEQ ID NO: 5 shows a A. delafieldii PBS(A) depolymerase amino acid sequence without a signal sequence.

SEQ ID NO: 6 shows a Thermobifida alba Cutinase 1 (E9LVH7) amino acid se quence with a signal sequence.

SEQ ID NOs: 7 - 100 show amino acid sequences of specific amino acid motifs. SEQ ID NO: 101 shows a Rhizobacter sp. dienelactone hydrolase amino acid se quence with a signal sequence.

SEQ ID NO: 102 shows a mature a Rhizobacter sp. dienelactone hydrolase nucle otide sequence codon optimized to E. coli (the sequence comprises a His tag).

SEQ ID NO: 103 shows a Rhizobacter sp. dienelactone hydrolase amino acid se quence without a signal sequence.

SEQ ID NO: 104 shows a Cytophagales bacterium dienelactone hydrolase amino acid sequence with a signal sequence.

SEQ ID NO: 105 shows a mature Cytophagales bacterium dienelactone hydrolase nucleotide sequence codon optimized to E. coli (the sequence comprises a His tag).

SEQ ID NO: 106 shows a Cytophagales bacterium dienelactone hydrolase amino acid sequence without a signal sequence.

DETAILED DESCRIPTION OF THE INVENTION

The present invention concerns a method of degrading a polyester, wherein a specific enzyme or micro-organism of the present invention is used for degrading said polyester. In one embodiment a polyester or polyester containing material (such as plastics or polymers of fossil origin, bio-based plastic material, polymer composite, packaging material, textile, plastics or synthetic polymers (e.g. oil- based and/or biobased) containing waste material) is allowed to contact with an enzyme or micro-organism capable of degrading said polyester or polyester con taining material.

As used herein, “a plastic” refers to a material consisting of synthetic and/or semi synthetic organic compounds and having the capability of being molded or shaped. As used herein “a synthetic polymer” refers to a human-made polymer. Synthetic polymers can be classified into four main categories: thermoplastics, thermosets, elastomers, and synthetic fibers. Thermoplastics are a type of synthet ic polymers that become moldable and malleable past a certain temperature, and they solidify upon cooling. Thermosets become hard and cannot change shape once they have set. Elastomers are flexible polymers. Synthetic fibers are fibers made by humans through a chemical synthesis.

As used herein polyester refers to a polymer comprising an ester functional group in the main chain. Ester is derived from an acid (organic or inorganic) in which at least one -OH (hydroxyl) group is replaced by an -O-alkyl (alkoxy) group. Polyes ters include both naturally occurring chemicals as well as synthetic polymers. Nat ural polyesters and some synthetic ones are biodegradable, but most synthetic polyesters are not. Polyesters, which may be degraded by the method, enzyme, fragment or micro-organism of the present invention, include any polyesters such as naturally occurring polyesters, bio-polyesters or biobased polyesters, polyesters produced from renewable resources, synthetic polyesters, oil-based polyesters, co-polymers comprising a polyester, biodegradable polyesters, and any combina tion thereof. In one embodiment, the polyester to be degraded by the present in vention is included in a material or part of a material comprising both oil-based and biobased polymers, e.g. as a copolymer. Any copolymers can be suitable for the present invention, e.g. including but not limited to starch-PLA, starch-PBAT, starch-PHA, PBS-PLA, PET-PLA, PCL-PLA, PEG-PLA, PGA-PLA, PCL-PBS, or any combination thereof. In one embodiment of the invention the polyester is a re cycled polyester or from a recycled plastic material.

In one embodiment the polyester is an aliphatic polyester (e.g. a homopolymer such as polyglycolic acid (PGA), polylactic acid (PLA), polycaprolactone (PCL), polyhydroxyalkanoate (PHA) or polyhydroxybutyrate (PHB), or a copolymer such as polyethyleneadipate (PEA), polybutylene succinate (PBS) or poly(3- hydroxybutyrate-co-3-hydroxyvalerate) (PHBV)), semi-aromatic polyester (e.g. a copolymer such as PET, PBT, PTT, PEF, PBF, PPF, PBAT, PPTF or PEN) or an aromatic polyester (e.g. a copolymer such as vectran). In one embodiment the polyester is any combination of an aliphatic polyester, semi-aromatic polyester and aromatic polyester.

In one embodiment the polyester is polyethylene 2,5-furandicarboxylate (PEF), poly(butylene 2,5-furandicarboxylate) (PBF), polypropylene 2,5- furandicarboxylate) (PPF), polyglycolic acid (PGA), polylactic acid (PLA), poly caprolactone (PCL), polyhydroxyalkanoate (PHA), polyhydroxybutyrate (PHB), polyethyleneadipate (PEA), polybutylene adipate terephthalate (PBAT), polybutyl ene succinate (PBS), poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), poly ethylene terephthalate (PET), polybutylene terephthalate (PBT), polytrimethylene terephthalate (PTT), polyethylene naphthalate (PEN), polypropylene 2,5- thiophenedicarboxylate) (PPTF), vectran, or any combination thereof; or the poly ester is PEF, PLA, PET, PBS, PFIBV, or PFIA, or any combination thereof. In a specific embodiment the polyester is a combination of one or more from the group comprising PEF, PLA and PBS.

Polyethylene terephthalate (PET) comprises repeating units of the aromatic ter- ephthalic acid and ethylene glycol. The PET monomers are designated bis(2- hydroxyethyl) terephthalate and/or 2-hydroxyethyl terephthalate.

Polyethylene 2,5-furandicarboxylate (PEF) (also known as polyethylene 2,5- furandicarboxylate, poly(ethylene 2,5-furandicarboxylate), polyethylene furanoate and polyethylene furanoate)) can be produced by polycondensation of 2,5- furandicarboxylic acid (FDCA) and ethylene glycol. The PEF monomers are desig nated bis(2-hydroxyethyl) furandicarboxylate and/or 2-hydroxyethyl furandicarbox- ylate.

Polyethylene naphthalate (PEN) is a polyester derived from naphthalene-2, 6- dicarboxylic acid and ethylene glycol. The PEN monomers are designated dihy- droxyethyl-2,6-naphthalenedicarboxylate and/or 2,6-naphthalenedicarboxylic acid.

Poly(butylene 2,5-furandicarboxylate) (PBF) can be synthesized from 2,5- furandicarboxylic acid (2,5-FDCA) and 1 ,4-butanediol (1,4-BD).

Polypropylene 2,5-furandicarboxylate) (PPF) can be produced e.g. from diethyl fumarate and 1,2-propanediol, and can be degraded into propylene glycol and fu- maric acid

Polyglycolic acid (PGA) can be prepared starting from glycolic acid e.g. by means of polycondensation or ring-opening polymerization.

Polylactic acid (PLA) can be prepared from monomers lactic acid and the cyclic di ester lactide or from lactic acid monomers.

Polycaprolactone (PCL) can be prepared from e- caprolactone e.g. by ring-opening polymerization. Polyhydroxyalkanoate (PHA) can be e.g. a po!y(HA SCL) produced from hydroxy fatty acids with short chain lengths including three to five carbon atoms, poly(HA MCL) produced from hydroxy fatty acids with medium chain lengths including six to 14 carbon atoms, poly-3-hydroxybutyrate (P3HB) consisting of 1000 to 30000 hydroxy fatty acid monomers, poly-4-hydroxybutyrate (P4HB), polyhydroxyvalerate (PHV), polyhydroxyhexanoate (PHH), polyhydroxyoctanoate (PHO) or poly(3- hydroxybutyrate-co-3-hydroxyvalerate) (PHBV).

Polyhydroxybutyrate (PHB) is an example of PHA. E.g. poly-3-hydroxybutyrate (P3HB), poly-4-hydroxybutyrate (P4HB) are included within the group of PHBs.

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) is an example of PHA. It can be obtained from 3-hydroxybutanoic acid and 3-hydroxypentanoic acid by the copolymerization.

Polyethyleneadipate (PEA) can be produced from ethylene glycol and adipic acid e.g. by a polycondensation reaction.

Polybutylene adipate terephthalate (PBAT) is a copolyester of adipic acid, 1,4- butanediol and terephthalic acid.

Polybutylene succinate (PBS) (also called as poly(1 ,4-butylene succinate) can be produced from succinic acid and 1-4-butanediol e.g. by polycondensation.

Polybutylene terephthalate (PBT) can be produced from butanediol and tereph thalic acid by the polymerization.

Polytrimethylene terephthalate (PTT) can be produced from 1,3-propanediol and terephthalic acid or dimethyl terephthalate.

Polypropylene 2,5-thiophenedicarboxylate) (PPTF) can be produced from 2,5- thiophenedicarboxylic acid (TFDCA) and 1,3-propanediol.

Vectran can be produced from 4-hydroxybenzoic acid and 6-hydroxynaphthalene- 2-carboxylic acid e.g. by polycondensation.

In one embodiment of the invention the enzyme capable of degrading a polyester or a polyester containing material is from a bacterium (gram-positive or gram- negative) or fungus, and/or the micro-organism capable of degrading a polyester or a polyester containing material is a bacterium (gram-positive or gram-negative) or fungus.

As used herein, “degradation” of a plastic, synthetic or non-synthetic polymer or polyester refers to either partial or complete degradation of a plastic, synthetic or non-synthetic polymer or polyester to oligomers and/or monomers. Said degrada tion can also include lowering of the molecular weight of a polymer and/or reduc tion in polydispersity of a polymer. Indeed, any loss in the chain length of a poly mer e.g. lowers tensile strength. “Microbial degradation” refers to a degradation caused by a micro-organism. According to some hypothesis, in the microbial deg radation the larger polymers are initially degraded by secreted exoenzymes into smaller subunits (multimers, dimers) that can be incorporated into the cells of mi cro-organisms and further degraded through the classical degradation pathways to yield energy and/or suit as building blocks for catabolism or metabolism.

Many plastics, textiles or other materials are mixtures comprising synthetic, semi synthetic and non-synthetic polyesters, or any combination thereof, and further more solubilizers and/or optionally other chemical agents or compounds for alter ing the mechanical and physical properties of said plastics or materials. The solu bilizers and other chemical compounds or agents may also be targets of microbial biodegradation.

In one embodiment of the invention the enzyme (or a fragment thereof), micro organism or host cell comprises PEF degrading activity and further PBF, PPF, PGA, PLA, PCL, PHA, PHB, PEA, PBAT, PBS, PHBV, PET, PBT, PTT, PEN, PPTF or vectran degrading activity, or any combination thereof. In one em bodiment the enzyme (or a fragment thereof), micro-organism or host cell com prises PEF degrading activity and one or more selected from the group consisting of PLA, PET, PBS, PHBV, and PHA degrading activity, or any combination there of. In one embodiment the enzyme or a fragment thereof, micro-organism or host cell comprises PEF and PLA degrading activity. In one embodiment the enzymes, fragments, micro-organisms or host cells of the present invention can be capable of utilizing short, medium-sized and/or long substrates (such as 40 - 70 kDa or 45 - 55 kDa substrates including but not limited to about 49 kDa PEF) and/or can be capable of degrading both aromatic and aliphatic polyesters. Degradation of a polyester can result in at least one or more degradation products. In one embodiment of the invention, at least one or more degradation products se lected from the group consisting of an alkane, diacid, acid, fatty acid, alcohol, a compound comprising a benzene ring, ethylene glycol, diethylene glycol, ethylene, 1 ,4-butanedioi, 3-hydroxybutyrate, 3-hydroxyva!erate, adipic acid, propylene, poly- hydroxyalkanoate, 2,5-furandicarboxylic acid, 2,5-dimethylfurandicarboxylate, ter- ephtalic acid, ethyl terephthalic acid monomer, dimer, trimer, tetramer, pentamer, hexamer, bis(2-hydroxyethyl) terephthalic acid dimer, hydroxyethyl [bis(2- hydroxyethyl)] terephthalic acid monomer, dimer, trimer, tetramer, pentamer, hex amer, ethyl furandicarboxylic acid monomer, dimer, trimer, tetramer, pentamer, hexamer, bis(2-hydroxyethyl) furandicarboxylic acid monomer, dimer, ethyl [bis(2- hydroxyethyl)] furandicarboxylic acid monomer, dimer, trimer, tetramer, pentamer, hexamer succinic acid, lactic acid monomer, dimer, trimer, tetramer, pentamer, glycolic acid monomer, dimer, trimer, tetramer, pentamer, caprolactone monomer, dimer, trimer, tetramer, pentamer, heksamer, and butanediol are obtained or ob tainable by the degradation of a polyester. For example, polyethylene tereph- thalate (PET) can be degraded to terephtalic acid, ethylene glycol, bis(2- hydroxyethyl) terephthalic acid, hydroxyethyl terephthalic acid, ethyl terephthalic acid monomer, dimer, trimer, tetramer, pentamer, hexamer, bis(2-hydroxyethyl) terephthalic acid monomer, dimer, ethyl [bis(2-hydroxyethyl)] terephthalic acid monomer, dimer, trimer, tetramer, pentamer, and/or hexamer; polyethylene furanoate (PEF) can be degraded to 2,5-furandicarbozylic acid (FDCA), ethylene glycol, bis(2-hydroxyethyl) furandicarboxylic acid, 2,5-dimethylfurandicarboxylate, ethyl furandicarboxylic acid monomer, dimer, trimer, tetramer, pentamer, hexamer, bis(2-hydroxyethyl) furandicarboxylic acid monomer, dimer, hydroxyethyl [bis(2- hydroxyethyl)] furandicarboxylic acid monomer, dimer, trimer, tetramer, pentamer, hexamer, and/or hydroxyethyl furandicarboxylic acid.

In one embodiment of the invention only the micro-organism(s) or enzyme(s) or a combination thereof is(are) needed for a biotechnical or enzymatic degradation of a polyester or a combination of different types of polyesters. In other words, no other degradation methods such as UV light or mechanical disruption or chemical degradation are needed in said embodiment. In other embodiments, biotechnical, microbial or enzymatic degradation can be combined with one or more other deg radation methods (e.g. non-enzymatic degradation methods) including but not lim ited to UV light, gamma irradiation, microwave treatment, mechanical disruption and/or chemical degradation. In one embodiment of the invention the method is a biotechnical method, or the method comprises degradation of the polyester by non-enzymatic methods or means. Non-enzymatic, non-microbial or non- biotechnical degradation methods or steps including pretreatments can be carried out sequentially (e.g. before or after) or simultaneously with the biotechnical, mi crobial or enzymatic degradation.

In one embodiment the method of degrading a polyester is a biotechnical method.

The present invention concerns an isolated enzyme comprising PEF degrading ac tivity, wherein said enzyme is capable of degrading a polyester and said enzyme has at least 70% sequence identity to SEQ ID NO: 5. Also, the present invention concerns a micro-organism or host cell comprising an enzyme comprising PEF degrading activity, wherein said enzyme is capable of degrading a polyester and said enzyme has at least 70% sequence identity to SEQ ID NO: 5.

The enzyme of the present invention refers to not only fungal or bacterial but also to any other enzyme homologue from any micro-organism, organism or mammal. Also, all isozymes, isoforms and variants are included with the scope of said en zyme. In one embodiment of the method, enzyme, fragment, micro-organism or host cell of the present invention, the enzyme is an enzyme of a bacterium select ed from the group comprising or consisting of Acidovorax, Ideonella, Gammapro- teobacteria, Saccharothrix, Rhizobacter, Candidatus, Candidatus Muproteobacte- ria, Actinomadura, Lechevalieria, Asanoa, Actinobacteria, Burkholderiales, Co- mamonadaceae, Burkholderia, Rhizobacter, and Cytophagales ; or the enzyme is an enzyme of a bacterium selected from the group comprising or consisting of Ac idovorax delafieldii, Ideonella sakaiensis, Gammaproteobacteria bacterium, Sac charothrix sp., Rhizobacter gummiphilus, Candidatus Muproteobacteria bacterium, Actinomadura meyerae, Lechevalieria fradiae, Asanoa hainanensis, Actinobacteria bacterium, Actinobacteria bacterium OV320, Burkholderiales bacterium, Coma- monadaceae bacterium, Burkholderia cepacia, Rhizobacter sp., and Cytophagales bacterium. In one embodiment “an enzyme of a bacterium” refers to a situation, wherein the amino acid sequence of the enzyme has the same amino acid se quence as a wild type enzyme of a bacterium (e.g. any of the above listed bacte ria) or the amino acid sequence of the enzyme has a high sequence identity (e.g. 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, or 95% or more) to an amino acid sequence of a wild type bacterial enzyme (e.g. of any of the above listed bacteria). In other words, the amino acid sequence of the enzyme used in the present invention can be modified (e.g. genetically mod ified). In one embodiment, the enzyme, micro-organism or host cell is a genetically modi fied enzyme, micro-organism or host cell. In a specific embodiment the enzyme, fragment, micro-organism or host cell has an increased ability to degrade a poly- ester compared to the corresponding unmodified enzyme, fragment, micro organism or host cell, respectively. In one embodiment the enzyme, micro organism or host cell comprises a genetic modification increasing an enzyme ac tivity or the amount of a specific enzyme in a micro-organism or host cell. Genetic modifications (e.g. resulting in increased enzyme activity, increased expression of an enzyme, or increased or faster degradation of a polyester) include but are not limited to genetic insertions, deletions, disruptions or substitutions of one or more genes or a fragment(s) thereof or insertions, deletions, disruptions or substitutions of one or more nucleotides (e.g. insertion of a polynucleotide encoding an en zyme), or addition of plasmids. For example, one or several polynucleotides en- coding an enzyme of interest can be integrated to the genome of a micro-organism or host cell. As used herein "disruption" refers to insertion of one or several nucle otides into a gene or polynucleotide sequence resulting in a lack of the corre sponding polypeptide or enzyme or presence of non-functional polypeptide or en zyme with lowered activity. Methods for making any genetic modifications or modi- fying micro-organisms or host cells by adaptive evolution strategy are generally well known by a person skilled in the art and are described in various practical manuals describing laboratory molecular techniques.

As used herein "increased degradation (activity/ability/capability) of a polyester" or “faster degradation (activity/ability/capability) of a polyester” of an enzyme or mi cro-organism refers to the presence of higher activity or more activity of an en zyme or micro-organism, when compared to another enzyme or micro-organism, e.g. a genetically unmodified (wild type) enzyme or micro-organism. “Increased or faster degradation” may result e.g. from the presence of a specific enzyme in a micro-organism or an up-regulated gene or polypeptide expression in a micro organism. Also, “increased or faster degradation” may result e.g. from the pres ence of (enhancing) mutations of a specific enzyme having degradation capability.

As used herein "up-regulation of the gene or polypeptide expression" refers to ex- cessive expression of a gene or polypeptide by producing more products (e.g. mRNA or polypeptide, respectively) than an unmodified micro-organism. For ex ample, one or more copies of a gene or genes may be transformed to a cell (e.g. to be integrated to the genome of the cell) for upregulated gene expression. The term also encompasses embodiments, where a regulating region such as a pro moter or promoter region has been modified or changed or a regulating region (e.g. a promoter) not naturally present in the micro-organism has been inserted to allow the over-expression of a gene. Also, epigenetic modifications such as reduc ing DNA methylation or histone modifications as well as classical mutagenesis are included in "genetic modifications", which can result in an upregulated expression of a gene or polypeptide. As used herein "increased or up-regulated expression" refers to an increased expression of the gene or polypeptide of interest compared to a wild type micro-organism without the genetic modification. Expression or in creased expression can be proved for example by western, northern or southern blotting or quantitative PCR or any other suitable method known to a person skilled in the art.

In one embodiment the genetically modified enzyme, micro-organism, host cell or polynucleotide is a recombinant enzyme, micro-organism, host cell or polynucleo tide. As used herein, "a recombinant enzyme, micro-organism, host cell or polynu cleotide" refers to any enzyme, micro-organism, host cell or polynucleotide that has been genetically modified to contain different genetic material compared to the enzyme, micro-organism, host cell or polynucleotide before modification (e.g. comprise a deletion, substitution, disruption or insertion of one or more nucleic ac ids or amino acids e.g. including an entire gene(s) or parts thereof). The recombi nant micro-organism may also contain other genetic modifications than those spe cifically mentioned or described in the present disclosure. Indeed, the micro organism may be genetically modified to produce, not to produce, increase pro duction or decrease production of e.g. other polynucleotides, polypeptides, en zymes or compounds than those specifically mentioned in the present disclosure. In certain embodiments, the genetically modified micro-organism includes a heter ologous polynucleotide. The micro-organism can be genetically modified by trans forming it with a heterologous polynucleotide sequence that encodes a heterolo gous polypeptide. Alternatively, for example heterologous promoters or other regu lating sequences can be utilized in the micro-organisms, polypeptides or polynu cleotides of the invention. As used herein "a heterologous polynucleotide or en zyme" refers to a polynucleotide or enzyme, which does not naturally occur in a cell or micro-organism. In one embodiment of the method, enzyme, fragment, mi cro-organism or host cell of the present invention, the enzyme or fragment is en coded by a heterologous polynucleotide sequence and optionally expressed by a micro-organism or host cell. Genetic modifications may be carried out using conventional molecular biological methods. Genetic modification (e.g. of a micro-organism) can be accomplished in one or more steps via the design and construction of appropriate vectors and transformation of the micro-organism cell with those vectors. For example, elec troporation, protoplast-PEG and/or chemical (such as calcium chloride or lithium acetate based) transformation methods can be used. Also, any commercial trans formation methods are appropriate. Suitable transformation methods are well known to a person skilled in the art.

The term "vector" refers to a nucleic acid compound and/or composition that transduces, transforms, or infects a micro-organism or a host cell, thereby causing the cell to express nucleic acids and/or proteins other than those native to the cell, or in a manner not native to the cell. An "expression vector" contains a sequence of nucleic acids to be expressed by the modified micro-organism. Optionally, the expression vector also comprises materials to aid in achieving entry of the nucleic acids into the micro-organism, such as a virus, liposome, protein coating, or the like. The expression vectors contemplated for use in the present invention include those into which a nucleic acid sequence (i.e. polynucleotide) can be inserted, along with any preferred or required operational elements. Further, the expression vector must be one that can be transferred into a micro-organism or host cell and replicated therein. Vectors can be circularized or linearized and may contain re striction sites of various types for linearization or fragmentation. In specific embod iments expression vectors are plasmids, particularly those with restriction sites that have been well documented and that contain the operational elements preferred or required for transcription of the nucleic acid sequence. Such plasmids, as well as other expression vectors, are well known to those of ordinary skill in the art. Useful vectors may for example be conveniently obtained from commercially available micro-organism, yeast or bacterial vectors. Successful transformants can be se lected using the attributes contributed by the marker or selection gene. Screening can be performed e.g. by PCR or Southern analysis to confirm that the desired genetic modifications (e.g. deletions, substitutions or insertions) have taken place, to confirm copy number or to identify the point of integration of nucleic acids (i.e. polynucleotides) or genes into the micro-organism cell's genome.

Indeed, the present invention also relates to a polynucleotide encoding the en zyme of the present invention or a fragment thereof, and an expression vector or plasmid comprising said polynucleotide of the present invention. Nucleic acid and amino acid databases (e.g., GenBank) can be used to identify a polynucleotide sequence that encodes a polypeptide having an enzymatic activity. Sequence alignment software such as BLASTP (polypeptide), BLASTN (nucleo tide) or PASTA can be used to compare various sequences. Briefly, any amino ac id sequence having some homology to a polypeptide having enzymatic activity, or any nucleic acid sequence having some homology to a sequence encoding a pol ypeptide having enzymatic activity can be used as a query to search e.g. Gen- Bank. Percent identity of sequences can conveniently be computed using BLAST software with default parameters. Sequences having an identities score and a pos itive score of a given percentage, using the BLAST algorithm with default parame ters, are considered to be that percent identical or homologous. In a specific em bodiment of the invention the entire sequence of an enzyme or polynucleotide is compared to the entire sequence presented in the present application (e.g. with or without a signal sequence) (e.g. SEQ ID NO: 1, 5 or 2) to find out specific identi ties.

For example, an enzyme comprising polyethylene 2,5-furandicarboxylate (PEF) degrading activity, wherein said enzyme is capable of degrading the polyester and said enzyme has at least 70% sequence identity to SEQ ID NO: 5, can be found as described in example 7 or 8. In brief, in example 7 sequence searches were carried out utilizing SEQ ID NO: 3 (/. sakaiensis PETase) and 6 ( Thermobifida alba Cutinase 1 (E9LVH7)) and detected amino acid sequences were studied and op tionally aligned for detecting consensus sequences.

The enzyme can comprise one or more specific amino acid motifs for example af fecting a polyester degrading activity (e.g. enabling different substrates). In one embodiment of the method, enzyme, fragment, micro-organism or host cell of the present invention, the enzyme or fragment comprises one or several amino acid motifs shown in figure 7; or the enzyme or fragment comprises one or several of the following amino acid motifs:

..YARGPNPTAASLEASAGPF..

..FRRGPAPTNSSIEASRGPF..

..YQKGPEPTAALLEAGTGPF..

..FEKGPDPTKTMLEASTGPF..

..FQRGPEPTPTSLQASSGPF..

..YERGPAPTNSSIEATRGPF..

..YQKGPDPTVSGLEAARGPF..

..YERGPAPTTSILDATRGPY.. ..VDPNRLGWGWSYGGGGTL..

..VDPNRLGVMGWSMGGGGTL..

..VDGTRRGVMGWSMGGGGSL.

..IDASRLGVMGHSMGGGGTL.. ..VDATRIGVSGHSMGGGATL.. ..VDATRLAVGGHSMGGGGTL.. ..IDATRLGVMGHSMGGGGTL.. ..IDSSRLAVMGHSMGGGGTL.. ..VDTSRLGVMGWSMGGGGSL.. ..LKAAAPQAP..

..LQAAIPLTG..

..LKAAMALAP..

..LKAAIPFAP..

..IKAAVPLAP.. ..LQAAIPLTG..

..IKGAFPLTP..

..LQAAIPLAP..

..LQAAIPLTP..

..LKAAIPLTP.. JKAAAPQAP..

..DSIAP..

..DSVAP..

..DTTAA..

..DSTAP.. ..DSTAP..

..DSVAP..

..DTVAA..

..DSVAP..

..DTIAP..

..KKGVAWMKRFMDNDTRYSTFACENP..

..KYSIAWLKRFIDNDTRYEQFLCPSP..

..KYGVAWLKRFMDEDTRYAPFLCGAP..

.. KYGVSWM KRFMDNDTRFS PYLCGAP .. .. KYGVSWM KRFLDDDLRFGPYLCDAP..

..KYSISWLKRFIDNDTRYEQFLCPGP.. ..KYAVAWFKRFVDNDERYAPFLSGAL.. ..RQMVAWLKRFVDNDTRYEQFLCPGP.. ..KYSISWLKRYIDNDTRYDQFLCPPP..

..KYSVAWLKRFVDNDTRYTQFLCPGP.., and ..KKGVAWMKRFMDNDRRYTSFACSNP...; or the enzyme or fragment thereof comprises one or more amino acid motifs selected from the group consisting of an amino acid motif of AXE1 (e.g. Pfam database number PF05448.12; INTERPRO number IPR008391, SO 0000417), an amino acid motif of Abhydrolase_5 (e.g. Pfam database number PF12695.7; INTERPRO number IPR029059; SO 0000417) and an amino acid motif of DLFI (e.g. Pfam da tabase number PF01738.18; INTERPRO number IPR002925; SO 0000417).

AXE1 i.e. Acetyl xylan esterase 1 hydrolyzes ester linkages of acetyl groups. Ab- hydrolase 5 belongs to a family of alpha/beta hydrolases. DLFI i.e. Dienelactone hydrolase is an enzyme from the beta-ketoadipate pathway and catalyzes the hy drolysis of dienelactone to maleylacetate.

In one embodiment of the invention the enzyme does not have a narrowed binding cleft (e.g. as described in WO2019/168811) compared to an unmodified enzyme; and/or the enzyme comprises an amino acid W at position 159 of figure 7 and/or an amino acid S at position 238 of figure 7 (A. delafieldii PBS(A) depolymer- ase/PETase and/or I. sakaiensis PETase).

An A. delafieldii PBS(A) depolymerase can comprise one or more of the following amino acids (e.g. compared to I. sakaiensis PETase) (the first amino acids men tioned in brackets can be the amino acids of the I. sakaiensis PETase; the later amino acid is the amino acid residue of the A. delafieldii PBS(A) depolymerase: (Y)87F, (D)186S, (S)214H, (G)234N, (R)280N (for amino acid numbering see fig ure 7). In one embodiment one or more of said amino acids affect the degrading activity (e.g. by increasing the degrading activity) of polyesters (e.g. aliphatic poly esters or PET), cause a more hydrophobic environment (e.g. (Y)87F), increase possible interaction with substrates (e.g. (G)234N improving hydrophobic interac tion with substrates, or (D)186S), or extend substrate binding site (e.g. (R)280N).

In one embodiment of the method, enzyme, fragment, micro-organism or host cell of the present invention, the enzyme or fragment comprises a signal sequence. The signal sequence can be any signal sequence i.e. a short polypeptide present at the N-terminus of synthesized polypeptides that are destined towards the secre tory pathway, said polypeptides including but not limited to those polypeptides that are targeted inside specific organelles, secreted from the cell, or inserted into cel- lular membranes. One example of a specific signal sequence comprises amino ac ids 1 - 39 of SEQ ID NO: 1 or is described in SEQ ID NO: 1 (amino acids 1 - 39).

In a specific embodiment an enzyme of the present invention comprises a se quence having a sequence identity of at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 82.5%, 83%, 83.5%, 84%, 84.5%, 85%, 85.5%, 86%, 86.5%, 87%, 87.5%, 88%, 88.5%, 89%, 89.5%, 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 100% to SEQ ID NO: 1, 2, 3, 4, 5, 6, 101, 102, 103, 104, 105 and/or 106, or an enzymatically active fragment or variant thereof. Said enzyme can be genetically modified (i.e. differs from the wild type enzyme) or unmodified. In a specific embodiment an enzyme is an isolated enzyme.

In one embodiment of the invention the enzyme has at least 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 82.5, 83, 83.5, 84, 84.5, 85, 85.5, 86, 86.5, 87, 87.5, 88,

88.5, 89, 89.5, 90, 90.5, 91, 91.5, 92, 92.5, 93, 93.5, 94, 94.5, 95, 95.5, 96, 96.5, 97, 97.5, 98, 98.5, or 99 % (e.g. 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8 or 99.9 %) sequence identity to SEQ ID NO: 5 (SEQ ID NO: 5 is an A. delafieldii PBS(A) depolymerase amino acid sequence without a signal sequence); and/or the enzyme has at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 82.5, 83, 83.5, 84, 84.5, 85, 85.5, 86, 86.5, 87, 87.5, 88, 88.5, 89, 89.5, 90, 90.5, 91, 91.5, 92, 92.5, 93, 93.5, 94, 94.5, 95, 95.5, 96,

96.5, 97, 97.5, 98, 98.5, or 99 % (e.g. 99.1 , 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8 or 99.9 %) sequence identity to SEQ ID NO: 1 (SEQ ID NO: 1 is an A. delafieldii PBS(A) depolymerase amino acid sequence with a signal sequence). In one em bodiment of the invention the enzyme has 100 % sequence identity to SEQ ID NO: 5; or the enzyme has 100 % sequence identity to SEQ ID NO: 1.

For example, sequence identities of amino acid sequences SEQ ID NO: 103 (a Rhizobacter sp. dienelactone hydrolase amino acid sequence without a signal se quence) and SEQ ID NO: 106 (a Cytophagales bacterium dienelactone hydrolase amino acid sequence without a signal sequence) to SEQ ID NO: 5 are at least 82%, at least 82.5% or at least 82.8%.

A polynucleotide of the present invention encodes the polypeptide of the present invention. In a specific embodiment a polynucleotide comprises a sequence hav ing a sequence identity of at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% to SEQ ID NO: 2, 4, 102 and/or 105, or a variant thereof. Said polynucleotide can be genetically modified (i.e. differs from the wild type polynucleotide) or unmodified. In a specific embodiment a polynucleotide is an isolated polynucleotide.

In one embodiment the micro-organism, polypeptide and/or enzyme have been genetically modified and optionally have an increased ability to degrade a synthet ic polymer compared to the corresponding unmodified micro-organism, polypep tide and/or enzyme, respectively.

In one embodiment of the method, enzyme, fragment, micro-organism or host cell of the present invention the enzyme is selected from the group comprising or con sisting of a protease, esterase, lipase, cutinase, laccase, depolymerase, oxidase, peroxidase, carboxylesterase, hydrolase, dienelactone hydrolase, petase and mhetase; and/or the enzyme comprises protease, esterase, lipase, cutinase, lac case, depolymerase, oxidase, peroxidase, carboxylesterase, hydrolase, dienelac tone hydrolase, petase and/or mhetase activity, or any combination thereof. As an example, a protease, esterase, cutinase, depolymerase, carboxylesterase, hydro lase, dienelactone hydrolase, petase, and/or mhetase can be used for PET degra dation. The enzyme(s) involved in the degradation of polyesters can be selected from one or several of the following: a protease (EC 3.4.21.14), hydrolase or es terase (EC 3.1.1.X, e.g. dienelactone hydrolase (EC 3.1.1.45) or carboxylesterase (EC 3.1.1.1)), lipase (EC 3.1.1.3, a subclass of the esterases), cutinase (EC 3.1.1.74, a subclass of the esterases), laccase (EC 1.10.3.2), oxidase (EC 1.2.3.X, e.g. glyoxal oxidase EC 1.2.3.15), peroxidase (EC 1.11.1.X), petase (EC 3.1.1.101, a subclass of the esterases) and mhetase (EC 3.1.1.102, a subclass of the esterases). The presence, absence or amount of specific enzyme activities can be detected by any suitable method known in the art. Specific examples of studying enzyme activities of interest are well known to a person skilled in the art. Non-limiting examples of suitable detection methods include commercial kits on market, enzymatic assays, immunological detection methods (e.g., antibodies specific for said proteins), PCR based assays (e.g., qPCR, RT-PCR), and any combination thereof.

In one embodiment, the enzymes of the present invention have high turnover rates when degrading one or more polyesters, e.g. when compared to prior art en- zymes. In specific embodiments the activity of an enzyme to degrade a polyester is determined by monitoring the absorbance after allowing the enzyme or micro organism comprising said enzyme to contact with a polyester e.g. as described in any of the examples 2 - 6, and/or determining or measuring the degradation prod ucts of a polyester. In one embodiment, the enzyme or micro-organism comprising the enzyme of the present invention (e.g. a protease, esterase, lipase, cutinase, laccase, depolymerase, oxidase, peroxidase, carboxylesterase, hydrolase, dienealkene hydrolase, petase or mhetase, or any combination thereof) can be used for producing one or more degradation products of one or more polyesters. In one embodiment one or more degradation products selected from the group comprising or consisting of an alkane, diacid, acid, fatty acid, alcohol, a compound comprising a benzene ring, ethylene glycol, diethylene glycol, ethylene, 1,4- butanediol, 3-hydroxybutyrate, 3-hydroxyvalerate, adipic acid, propylene, polyhy- droxyalkanoate, 2,5-furandicarboxylic acid, terephtalic acid, succinic acid, lactic acid monomer, dimer, trimer, tetramer, pentamer, heksamer, glycolic acid mono mer, dimer, trimer, tetramer, pentamer, heksamer, caprolactone monomer, dimer, trimer, tetramer, pentamer, heksamer, 2,5-dimethylfurandicarboxylate, ethyl ter- ephthalic acid monomer, dimer, trimer, tetramer, pentamer, hexamer, bis(2- hydroxyethyl) terephthalic acid dimer, hydroxyethyl [bis(2-hydroxyethyl)] tereph- thalic acid monomer, dimer, trimer, tetramer, pentamer, hexamer, ethyl furandicar- boxylic acid monomer, dimer, trimer, tetramer, pentamer, hexamer, bis(2- hydroxyethyl) furandicarboxylic acid monomer, dimer, ethyl [bis(2-hydroxyethyl)] furandicarboxylic acid monomer, dimer, trimer, tetramer, pentamer, hexamer and butanediol are obtained or obtainable by the degradation of a polyester.

Degradation of a polyester can be measured by any suitable method known in the field. The presence, absence or level of degradation products of a polyester, e.g. degraded by an enzyme, micro-organism or host cell, can be detected or meas ured by any suitable method known in the art. Non-limiting examples of suitable detection and/or measuring methods include liquid chromatography, gas chroma tography, mass spectrometry or any combination thereof (e.g. RP-HPLC, GC-MS or LC-TOF-MS) of samples, optionally after cultivating a micro-organism e.g. 1 - 11 hours, or 12 hours - 12 months (e.g. one, two, three, four, five, six, seven, eight, nine, ten or 11 months) or even longer in the presence of plastics or synthet ic polymers or after allowing a micro-organism, polypeptide or enzyme to contact with plastics or synthetic polymers. Other examples of suitable detection and/or measuring methods (including methods of fractionating, isolating or purifying deg radation products) include but are not limited to filtration, solvent extraction, cen- trifugation, affinity chromatography, ion exchange chromatography, electrophore sis, hydrophobic interaction chromatography, gel filtration chromatography, re verse phase chromatography, chromatofocusing, differential solubilization, prepar ative disc-gel electrophoresis, isoelectric focusing, HPLC, gel permeation chroma- tography (GPC), fourier-transform infrared spectroscopy (FT-IR), NMR and/or re- versed-phase FIPLC.

For degradation, polyesters or a material comprising polyesters can be contacted with an enzyme, micro-organism or host cell (or any combination thereof) at a ra- tio, concentration and/or temperature for a time sufficient for the degradation of in terest. Suitable time for allowing the enzyme, micro-organism, or host cell to de grade a polyester or polyesters can be selected e.g. from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, and 24 hours, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, and 31 days, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12 weeks, and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12 months. The degradation may take place in liquid, semi-solid, moist or dry conditions. The degradation is conven iently conducted aerobically, microaerobically and/or anaerobically. If desired, specific oxygen uptake rate can be used as a process control. The degradation can be conducted continuously, batch-wise, feed batch-wise or as any combina tion thereof.

In some embodiments of the invention a micro-organism, host cell, micro organisms (e.g. a combination of different micro-organisms) or host cells can pro- duce material (e.g. degradation products (such as alkane, fatty acids or acyl es ters) or modified material) for micro-organisms of other type(s) or enzymes to fur ther degrade or modify said material (e.g. to PFIA or diacids). On the other hand, in some embodiments of the invention an enzyme and/or enzymes (e.g. a combina tion of different polypeptides or enzymes) can produce material (e.g. degradation products (such as alkane, fatty acids or acyl esters) or modified material) for en zymes of other type(s) or micro-organisms to further degrade or modify said mate rial (e.g. to PHA or diacids).

In some embodiments of the present invention the micro-organisms, host cells or genetically modified micro-organisms or host cells are cultured under conditions in which the cultured micro-organism or host cell produce polypeptides, enzymes or compounds or interest (e.g. the micro-organisms are cultured for producing poly peptides or enzymes of interest or for degrading polyesters). The (genetically mod- ified) micro-organisms or host ceils can be cultivated in a medium containing ap propriate carbon sources together with other optional ingredients seiected from the group consisting of nitrogen or a source of nitrogen (such as amino acids, pro teins, inorganic nitrogen sources such as nitrate, ammonia, urea or ammonium salts), yeast extract, peptone, minerals and vitamins, such as KH2P04, Na2HPO, MgSO, CaC!2, FeC!s, ZnSO, citric acid, MnSO, COCI2, CuSO, Na2Mo04, FeS04, HsB04, D-biotin, Ca-Pantothenate, nicotinic acid, myoinositol, thiamine, pyridoxine, p-amino benzoic acid. Suitable cultivation conditions, such as tempera ture, cell density, selection of nutrients, and the like are within the knowledge of a skilled person and can be selected to provide an economical process with the mi cro-organism in question. Temperatures may range from above the freezing tem perature of the medium to about 50°C, although the optimal temperature will de pend somewhat on the particular micro-organism. In a specific embodiment the temperature is from about 25 to 35°C. The pH of the cultivation process may or may not be controlled to remain at a constant pH, but is usually between 4 and 9, depending on the production organism. Optimally the pH is controlled to a con stant pH of 7 and 8. Suitable buffering agents include, for example, calcium hy droxide, calcium carbonate, sodium hydroxide, potassium hydroxide, potassium carbonate, hydrogen chloride, sodium carbonate, ammonium carbonate, ammo nia, ammonium hydroxide and/or the like. In general, those buffering agents that have been used in conventional cultivation methods are also suitable here.

The micro-organisms or host cells can be normally separated from the culture me dium after cultivation or after contacting with polyesters. The separated micro organisms, host cells or a liquid (e.g. culture medium) comprising micro-organisms or host cells can be used for contacting the synthetic polymers.

Polypeptides or enzymes can be secreted outside of the cells or they can stay in the cells. Furthermore, the polypeptides or enzymes can be recovered from the cells or directly from the culture medium. In some embodiments both intracellular and extracellular polypeptides or enzymes are recovered. Prior to recovering, cells can be disrupted. Isolation and/or purification of polypeptides or enzymes can in clude one or more of the following: size exclusion, desalting, anion and cation ex change, based on affinity, removal of chemicals using solvents, extraction of the soluble proteinaceous material e.g. by using an alkaline medium (e.g. NaOH, Bo- rate-based buffers or water is commonly used), isoelectric point-based or salt- based precipitation of proteins, centrifugation, and ultrafiltration. In one embodi ment of the method, polypeptide or enzyme of the present invention, said polypep- tide or enzyme is a purified or partly purified polypeptide or enzyme. If the poly peptide or enzyme is secreted outside of the cell it does not necessarily need to be purified.

Polyester(s) degrading enzymes can be expressed in any suitable host (cell). Ex amples of suitable host cells include but are not limited to cells of micro-organisms such as bacteria, yeast, fungi and filamentous fungi, as well as cells of plants and animals (such as mammals). Specific examples of host cells include but are not limited to Escherichia, Escherichia coli, Pichia, Pichia pastoris, Trichoderma, Trichoderma reesei, Aspergillus, Aspergillus nidulans, Aspergillus niger, Bacillus, Bacillus subtilis, Bacillus licheniformis.

In one embodiment of the invention the micro-organism or host cell is a bacterium, or the micro-organisms or host cells are bacteria selected from the group compris ing or consisting of Acidovorax, Ideonella, Gammaproteobacteria, Saccharothrix, Rhizobacter, Candidatus, Candidatus Muproteobacteria, Actinomadura, Lecheva- lieria, Asanoa, Actinobacteria, Burkholderiales, Comamonadaceae, Burkholderia, Rhizobacter, and Cytophagales ; or the micro-organism is a bacterium or the mi cro-organisms are bacteria selected from the group comprising or consisting of Ac idovorax delafieldii, Ideonella sakaiensis, Gammaproteobacteria bacterium, Sac charothrix sp., Rhizobacter gummiphilus, Candidatus Muproteobacteria bacterium, Actinomadura meyerae, Lechevalieria fradiae, Asanoa hainanensis, Actinobacteria bacterium, Actinobacteria bacterium OV320, Burkholderiales bacterium, Coma monadaceae bacterium, Burkholderia cepacia, Rhizobacter sp., and Cytophagales bacterium, or micro-organisms or host cells are any combination thereof. Also, the micro-organism or host cell of the present invention can be used in a combination with any other micro-organism (simultaneously or consecutively), e.g. micro organisms can be a population of different micro-organisms degrading different synthetic polymers or micro-organisms can be a combination of a bacterium and fungus (to be used simultaneously or consecutively).

The inventors have been able to isolate enzymes of the present invention capable of degrading polyesters from micro-organisms, and use said enzymes or micro organisms (e.g. suitable cultivation conditions for micro-organisms) so that they can degrade polyesters and/or produce degradation products of interest. For ex ample, the inventors of the present disclosure have revealed that an enzyme hav ing e.g. at least 70% (e.g. 70% or more, 80% or more, or 82% or more) identity to an Acidovorax, e.g. A. delafieldii polypeptide (and optionally characterized to have specific enzyme activities, e.g. PBS(A) depolymerase activities) can degrade PEF and optionally other polyesters (see e.g. examples 2 - 6). In one embodiment said enzyme can be more effective in degrading polyesters (e.g. PEF) than /. sa- kaiensis PETase.

The present invention further relates to use of the enzyme, micro-organism, host cell, polynucleotide, expression vector or plasmid of the present invention or any combination thereof for degrading a polyester or polyesters of different types, and optionally other synthetic polymers and/or plastics.

Also, the present invention concerns a method of producing the enzyme of the present invention, wherein a recombinant micro-organism or host cell comprising the polynucleotide encoding the enzyme of the present invention expresses or is allowed to express said enzyme. For example, a vector or plasmid comprising the polynucleotide of interest can be transfected to a host cell, and the host cell can be used for expressing the enzyme of the present invention. In one embodiment said expression of the enzyme can be controlled for example through inducible ele ments of promoters, vectors or plasmids.

As used in the present disclosure, the terms "polypeptide" and "protein" are used interchangeably to refer to polymers of amino acids of any length. As used herein “an enzyme” refers to a protein or polypeptide which is able to accelerate or cata lyze (bio)chemical reactions.

As used herein "polynucleotide" refers to any polynucleotide, such as single or double-stranded DNA (genomic DNA or cDNA or synthetic DNA) or RNA (e.g. mRNA or synthetic RNA), comprising a nucleic acid sequence encoding a poly peptide in question or a conservative sequence variant thereof. Conservative nu cleotide sequence variants (i.e. nucleotide sequence modifications, which do not significantly alter biological properties of the encoded polypeptide) include variants arising from the degeneration of the genetic code and from silent mutations.

As used herein “isolated” polypeptides, proteins or polynucleotides refer to poly peptides, proteins or polynucleotides purified to a state beyond that in which they exist in cells. Isolated polypeptides, proteins or polynucleotides include e.g. sub stantially purified (e.g. purified to at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% purity) or pure polypeptides, proteins or polynucleotides. It is well known that deletion, addition or substitution of one or a few amino acids does not necessarily change the catalytic properties of an enzyme. Therefore, the invention also encompasses variants and fragments of the enzymes of the present invention or given amino acid sequences having the stipulated enzyme activity. The term "variant" as used herein refers to a sequence having minor changes in the amino acid sequence as compared to a given sequence. Such a variant may occur naturally e.g. as an allelic variant within the same strain, species or genus, or it may be generated by mutagenesis or other gene modification. It may com- prise amino acid substitutions, deletions or insertions, but it still functions in sub stantially the same manner as the given enzymes, in particular it retains its catalyt ic function as an enzyme. In one embodiment of the invention a fragment of the enzyme is an enzymatically active fragment or variant thereof. A "fragment" of a given enzyme or polypeptide sequence means part of that se quence, e.g. a sequence that has been truncated at the N- and/or C-terminal end. It may for example be the mature part of a protein comprising a signal sequence, or it may be only an enzymatically active fragment of the mature protein. It will be obvious to a person skilled in the art that, as the technology advances, the inventive concept can be implemented in various ways. The invention and its embodiments are not limited to the examples described below but may vary within the scope of the claims. EXAMPLES

Example 1. Expression of I. sakaiensis PETase and Acidovorax delafieldii PBS(A) depolymerase in Escherichia coli The genes encoding Ideonella sakaiensis PETase (Genbank GAP38373.1, SEQ ID NO: 3) and Acidovorax delafieldii PBS(A) depolymerase (GenBank AB066349.1 , SEQ ID NO: 1) amino acids were commercially (Geneart) synthe sized with codon optimization for expression in Escherichia coli cells (SEQ ID NO: 4 and 2, respectively). The sequence encoding the N-terminal signal peptide was excluded from the constructs, starting IsPETase and AdPETase constructs at res idues 27 and 40 respectively. C-terminal His6-tag encoding sequence (CATCACCATCATCATCAC) was introduced before stop codon. Nco\ and Hind\\\ restriction sites were included at 5’ and 3’ ends of construct for restriction digestion cloning. The constructs were cloned into E. coli expression vector pBAT4 by re striction digestion cloning and expressed in E. coli strain SHuffle T7 Express (New England Biolabs).

Constructs were expressed in E. coli SHuffle T7 Express grown at 28°C in SB (30 g tryptone, 20 g yeast extract, 10 g MOPS (3-[A/-morpholino3-propanesu!fonic acid) per liter) media containing 100 pg/ml ampicill in . Protein expression was induced by the addition of 1mM isopropyl b-D-l-thiogalactopyranoside (IPTG), and induced cultures were further incubated at 16 °C for 24 h. Cells were harvested by centrif ugation (1500g, 15min, RT), and pellet was stored at -80°C until purification.

Lysate was prepared by suspending pellet in Buffer A (50mM Na ₂ HP0 ₄ -HCI, 100mM NaCI pH 8.0) with 1x EDTA-Free Complete Protease Inhibitor Cocktail (Roche). Cells were lysed by sonication and cell debris was removed by centrifu gation (13500 g, 20 min 4 °C). Supernatant was filtered through 0.22pm filter and Buffer B (50mM Na ₂ HP0 ₄ -HCI, 100mM NaCI, 500mM imidazole pH 8.0) was add ed to lysate to 10mM imidazole concentration. Enzymes were purified by affinity chromatography with 5ml HisTrap column (GE Healthcare). Supernatant was ap plied to column equilibrated with 10 mM imidazole and column was washed with 30mM imidazole. A linear imidazole gradient from 30mM to 500mM was used to elute bound proteins. Peak fractions were concentrated and imidazole was re moved by exchanging the elution buffer to Buffer A with salt exchange column (5ml Desalting column, GE Healthcare).

The quality of purified protein was assessed by SDS-PAGE, to verify high enough (>95%) homogeneity of protein samples for upstream applications. Protein con centration was determined by Bio-Rad Bradford protein assay with BSA as stand ard by using the standard microplate assay. Samples were made in triplicate and were incubated for 15 minutes and A ⁵⁹⁵ was measured with Varioskan Flash (Thermo Fischer).

Example 2. Polymer degradation with I. sakaiensis PETase and A. delafieldii PBS depolymerase

Purified enzyme produced in Example 1 and described in Examples 1 was incu bated in 50 mM Na ₂ HP0 ₄ -HCI pH 8.0, with a small amount of powdered polymer substrate (see Table 1). The reaction was stopped by addition of 5 mM H ₂ SO ₄ containing 10 % (v/v) DMSO in 1 :1 ratio, followed by heat treatment (85°C, 10 min). The supernatant obtained by centrifugation (15,000 x g, 10 min) was ana lyzed by GC-MS (example 3) or LC-MS (example 4). Control samples were incu bated without enzymes. PEF was polymerised from FDCA butyl ester and eth ylene glycol according to protocol described in Ma et al. 2012. J. Mater. Chem 22:3457, DOI: 10.1039/c2jm 15457a.

Table 1. Used polymers

Example 3. Gas chromatography - mass spectrometry (GC-MS) analysis of degradation products of polymers with I. sakaiensis PETase and A. delafield- ii PBS(A) depolymerase

Samples from example 2 were filtered through 0.22 pm filter and transferred to GC-MS vials. Samples were evaporated to dryness. 20ul of labelled 3- hydroxybutyric acid was added to the sample. Next, 100 pi pyridine and 50 pi

MSTFA (L/-T rimethylsilyl-A/-methyl trifluoroacetamide) + 1% TMCS (Trimethylsilyl chloride) silylation reagent were added to the sample, and samples were incubat ed at 70 °C for 1 hour. The GC-MS analyses were carried out using Agilent 6890 Series Gas Chromatograph coupled to an Agilent 5973 mass selective detector. The column used was DB-5ms column, 30m ^c 0.25 mm ^c 0.25 pm (Agilent Tech nologies). Flelium was the carrier gas at 1.2 ml/min constant flow. The injector temperature was 250 °C, with 20:1 split. The furnace temperature was 50 °C for 4 min, increased to 310 °C at 15 °C/min and kept at 310 °C for 4 min. The ion source temperature was maintained at 230 °C and the mass spectra were record ed over 30-700 atomic mass unit range. Identification was done using relative re tention indices with NIST08 Mass Spec Library.

In the GC-MS analysis with both enzymes PEF and PBS degradation products FDCA and succinic acid, respectively, could be seen (Figures 3 and 4). With A. delafieldii PBS(A) depolymerase PET and PHBV degradation products terephthal- ic acid and butanediol like compound, respectively, could be seen (Figures 5 and 6). None of these compounds could be seen with the control sample.

Example 4. Liquid chromatography/electrospray/time-of-flight mass spectrometry (LC/TOF-MS) analysis of degradation products of polymers with I. sakaiensis PETase and A. delafieldii PBS(A) depolymerase

Reverse-phase HPLC system consisted of a Waters e2695 separation module with Waters 2996 photodiode array detector. Separation was performed on a Hypersil BDS-C18 column (4.6 ^c 150 mm, 5 pm) at 25 °C with a nonlinear gradient of 0.1% formic acid (A) and acetonitrile (B) described in Table 2 at a flow rate of 1 ml/min. The injection volume was 10pl . Analytes were detected with the PDA de tector from 190nm to 600 nm. Detected peaks were identified by running same samples with LC/TOF-MS with RP-HPLC corresponding gradient as described in article Hoppe M, De Voogt P, Franz R 2018. Food Additives and Contaminants: Part A, 35:2244-2255. DOI: 10.1080/19440049.2018.1523576. Released FDCA amounts were calculated based on FDCA standard curve.

With reverse-phase HPLC and LC/TOF-MS PEF degradation products could be detected with both I. sakaiensis PETase and A. delafieldii PBS(A) depolymerase compared to the control (Figure 1). Additionally, release of FDCA was linked to in cubation time showing enzymatic degradation of PEF (Figure 2).

Table 2. RP-HPLC gradient used for the analysis of polyester degradation prod ucts.

Example 5. Preparation of PLA and PBS polymer emulsions

2 grams of PBS (BioPBS, PTT MCC Biochem) and PLA (NatureWorks 3051 D) granules were dissolved in 50 ml of dichloromethane. 100 ml of distilled water and 2 ml of 2% Sarkosyl NL were added and the mixture was blended with a homoge- nizer (10000 rpm, 4 min). The dichloromethane was evaporated by stirring at room temperature overnight. The emulsion was filled to 400 ml with distilled water, and pH was adjusted to 7 with KOH.

Example 6. Degradation of emulsified PLA and PBS by I. sakaiensis PETase and A. delafieldii PBS(A) depolymerase enzymes

The substrate solutions were prepared by mixing polymer emulsion of example 5 with 50 mM Na ₂ HP0 ₄ -HCI pH 8.0 in 4:1 ratio (emulsion :buffer). Mixtures of purified enzymes (10ug/ml) and substrate solutions were then incubated at 30 °C with gen tle agitation. Control samples were prepared without enzyme. The decrease in tur bidity was measured at 630 nm using a spectrophotometer (Ultrospec 2100 Pro, GE Healthcare) at 0 h, 24 h and 48 h. Samples were prepared in triplicate. With enzyme samples I. sakaiensis PETase and A. delafieldii PBS(A) depolymerase decrease in absorbance was detected indicating PLA degradation activity (see Table 3).

Table 3. Enzyme samples I. sakaiensis PETase and A. delafieldii PBS(A) depoly- merase revealed decrease in absorbance indicating PLA degradation activity.

When the experiment was carried out with PLA and PBS with different enzyme amounts (1, 10 and 20 pg/ml) degrease in absorbance could be detected after 24 hours incubation with both enzymes with PBS and PLA (see Table 4).

Table 4. Enzyme samples I. sakaiensis PETase and A. delafieldii PBS(A) depoly merase revealed decrease in absorbance after 24 hours incubation indicating PLA and PBS degradation activity. Example 7. Characterisation of amino acid sequence motifs of PET, PEF, PBS, PLA, PHA degrading enzymes

A sequence search based on HMMER (Robert D. Finn, Jody Clements, Sean R. Eddy (2011) HMMER web server: interactive sequence similarity searching. Nu- deic Acids Research, Volume 39, Issue suppl_2, 1 July 2011 , Pages W29-W37, https://doi.org/10.1093/nar/qkr367) was done using the amino acid SEQ ID NO: 3 (Is PEtase) and Thermobifida alba Cutinase 1 (E9LVH7) (SEQ ID NO: 6) sepa rately. The results of the both searches were combined. The searches resulted in nine novel amino acid sequences in addition to sequences SEQ ID N:0 1 and 3. Detected amino acid sequences were aligned with Geneious programme. In the alignment following consensus sequences could be detected from the aligned se quences.

..YARGPNPTAASLEASAGPF.. (SEQ ID NO: 7) ..FRRGPAPTNSSIEASRGPF.. (SEQ ID NO: 8) ..YQKGPEPTAALLEAGTGPF.. (SEQ ID NO: 9) ..FEKGPDPTKTMLEASTGPF.. (SEQ ID NO: 10) ..FQRGPEPTPTSLQASSGPF.. (SEQ ID NO: 11) ..YERGPAPTNSSIEATRGPF.. (SEQ ID NO: 12) ..YQKGPDPTVSGLEAARGPF.. (SEQ ID NO: 13) ..YERGPAPTTSILDATRGPY.. (SEQ ID NO: 14) ..YERGPAPTVSSIEALRGPF.. (SEQ ID NO: 15) ..YERGPNPTDALLEASSGPF.. (SEQ ID NO: 16) ..YERGPAPTTSSLEASRGPF.. (SEQ ID NO: 17)

Consensus sequence:

Y/F-X-R/K-G-P-X-P-T-X-X-X-l/L-X-A-X-X-G-P-F/Y (SEQ ID NO: 18)

..GYGAGTVYYP.. (SEQ ID NO: 19) ..GFGGGTIYYP.. (SEQ ID NO: 20)

..GFGGGTIHYP.. (SEQ ID NO: 21)

..GYRQGTIYHP.. (SEQ ID NO: 22)

..GYGGGTIYYP.. (SEQ ID NO: 23)

..GFGGGTIYYP.. (SEQ ID NO: 24) ..GFGGGTLHYP.. (SEQ ID NO: 25)

..GFGGGVIYYP.. (SEQ ID NO: 26)

..GFGGGTIYYP.. (SEQ ID NO: 27)

..GFGGGTIYYP.. (SEQ ID NO: 28)

..GYRAGTVYYP.. (SEQ ID NO: 29) Consensus sequence:

G-Y/F-G/R-X-G-T/V-X-Y/H-Y/H-P (SEQ ID NO: 30)

..GAIAIVPGYTARQSSIKWWGPRLASHGFVV.. (SEQ ID NO: 31) ..GAIAISPGFTGTQSTISWLGPRIASQGFVV.. (SEQ ID NO: 32) ..AAVAWPGYLAAESTIAWWGPRLASHGFW.. (SEQ ID NO: 33) ..AAVAWPGYLASQSSINWWGPRLASHGFW.. (SEQ ID NO: 34) ..APIAVVPGYLAAQSSIQSWGPRLASWGFW.. (SEQ ID NO: 35) ..GAWISPGFTGTQSSIDWLGPRIASQGFW.. (SEQ ID NO: 36) ..GLIAVAPPFIAQSSSIGWLGPRIASHGFW.. (SEQ ID NO: 37) ..GAIALSPGYTAAWSSISWLGPRIASHGFW.. (SEQ ID NO: 38) ..GAVAVAPGFTADQSSMAWLGPRLASQGFVI.. (SEQ ID NO: 39) ..GAVAISPGYTGTEASIAWLGERIASHGFVV.. (SEQ ID NO: 40) ..GAIAIVPGFTARQSSINWWGPRLASHGFVV.. (SEQ ID NO: 41) Consensus sequence:

G/A-X-l/V-A/V-X-X-P-X-Y/F-X-A/G-X-X-S/A-S/T-l/M-X-W/S-G-P /E-R-L/l-A-S-X-G- F-V-V/l (SEQ ID NO: 42) ..NSTLDQPSSRSSQQMAAL.. (SEQ ID NO: 43)

..NSTLDQPDSRASQLLAAL.. (SEQ ID NO: 44)

..NNTLDLPASRSAQLTAAL.. (SEQ ID NO: 45)

..NSTSDQPPSRATQLMAAL.. (SEQ ID NO: 46)

..NSSTDDPSQRATQLVAAL.. (SEQ ID NO: 47) ..ITIYDQPDSRASQLLAAL.. (SEQ ID NO: 48)

..NSTLDFPESRSRQQLAAI.. (SEQ ID NO: 49)

..NSRFDQPASRGRQLLAAL.. (SEQ ID NO: 50)

..NTRLDQPDSRSRQLLAAL.. (SEQ ID NO: 51)

JTTLDQPDSRAEQLNAAL. (SEQ ID NO: 52) ..NSTLDQPDSRSRQQMAAL. (SEQ ID NO: 53)

Consensus sequence: l/N-S/T-X-X-D-X-P-X-S/Q-R-X-X-Q-L/Q-X-A-A-L/l (SEQ ID NO: 54)

..VDTARMGVMGWSMGGGGSL. (SEQ ID NO: 55) JDASRLGLMGHSMGGGGTM.. (SEQ ID NO: 56) ..VDPNRLGWGWSYGGGGTL. (SEQ ID NO: 57) ..VDPNRLGVMGWSMGGGGTL.. (SEQ ID NO: 58) ..VDGTRRGVMGWSMGGGGSL.. (SEQ ID NO: 59) ..IDASRLGVMGHSMGGGGTL.. (SEQ ID NO: 60) ..VDATRIGVSGHSMGGGATL. (SEQ ID NO: 61 ) ..VDATRLAVGGHSMGGGGTL. (SEQ ID NO: 62) JDATRLGVMGHSMGGGGTL. (SEQ ID NO: 63) JDSSRLAVMGHSMGGGGTL. (SEQ ID NO: 64) ..VDTSRLGVMGWSMGGGGSL. (SEQ ID NO: 65) Consensus sequence:

V/l-D-X-X-R-X-G/A-V/L-X-G-W/H-S-M/Y-G-G-G-G/A-T/S-L/M (SEQ ID NO: 66)

..LKAAAPQAP.. (SEQ ID NO: 67)

..LQAAIPLTG.. (SEQ ID NO: 68) ..LKAAMALAP.. (SEQ ID NO: 69)

..LKAAIPFAP.. (SEQ ID NO: 70)

JKAAVPLAP.. (SEQ ID NO: 71)

..LQAAIPLTG.. (SEQ ID NO: 72) ..IKGAFPLTP.. (SEQ ID NO: 73)

..LQAAIPLAP.. (SEQ ID NO: 74)

..LQAAIPLTP.. (SEQ ID NO: 75)

..LKAAIPLTP.. (SEQ ID NO: 76) ..IKAAAPQAP.. (SEQ ID NO: 77)

Consensus sequence:

L/l - K/Q- A/ G - A-X- P/A-X-T/A- P/G (SEQ ID NO: 78)

..DSIAP.. (SEQ ID NO: 79) ..DSVAP.. (SEQ ID NO: 80)

..DTTAA.. (SEQ ID NO: 81)

..DSTAP.. (SEQ ID NO: 82)

..DSTAP.. (SEQ ID NO: 83)

..DSVAP.. (SEQ ID NO: 84) ..DTVAA.. (SEQ ID NO: 85)

..DSVAP.. (SEQ ID NO: 86)

..DTIAP.. (SEQ ID NO: 87)

Consensus sequence:

D-S/T-X-A-P/A (SEQ ID NO: 88)

..KKGVAWMKRFMDNDTRYSTFACENP.. (SEQ ID NO: 89) ..KYSIAWLKRFIDNDTRYEQFLCPSP.. (SEQ ID NO: 90) ..KYGVAWLKRFMDEDTRYAPFLCGAP.. (SEQ ID NO: 91) ..KYGVSWMKRFMDNDTRFSPYLCGAP.. (SEQ ID NO: 92) ..KYGVSWMKRFLDDDLRFGPYLCDAP.. (SEQ ID NO: 93) ..KYSISWLKRFIDNDTRYEQFLCPGP.. (SEQ ID NO: 94) ..KYAVAWFKRFVDNDERYAPFLSGAL. (SEQ ID NO: 95) ..RQMVAWLKRFVDNDTRYEQFLCPGP.. (SEQ ID NO: 96) ..KYSISWLKRYIDNDTRYDQFLCPPP.. (SEQ ID NO: 97) ..KYSVAWLKRFVDNDTRYTQFLCPGP.. (SEQ ID NO: 98) ..KKGVAWMKRFMDNDRRYTSFACSNP.. (SEQ ID NO: 99)

Consensus sequence:

K/R-X-X-V/l-S/A-W-X-K-R-F/Y-X-D-X-D-X-R-Y-X-X-L/A-C/S-X-X -P/L (SEQ ID NO: 100)

Example 8. Protein motif search from databases based on PET, PEF, PBS, PLA, PH A degrading enzyme amino acid sequences Protein motif search was carried out with amino acid sequences detected in Ex ample 7 in Genome database (https://www.qenome.ip/tools/motif/Database) with E-value = 1. In the search following motif was detected to have significant p-value from all sequences tested: AXE1 (INTERPRO; IPR008391, SO; 0000417), Abhy- drolase_5 (PF12695.7, INTERPRO; IPR029059) and DLH (PF01738.18).

For alignment of I. sakaiensis PETase and A. delafieldii PBS(A) depolymerase amino acid sequences see figure 7.

Example 9. Expression of Rhizobacter sp. and Cytophagales bacterium dienelactone hydrolase in Escherichia coii

The genes encoding Rhizobacter sp. dienelactone hydrolase (Genbank MBC7995986.1 , SEQ ID NO: 101, RhPETase) and Cytophagales bacterium (GenBank MBC7956640.1, SEQ ID NO: 104, CbPETase) amino acids were com mercially (Genescript) synthesized with codon optimization for expression in Escherichia coli cells (SEQ ID NOs: 102 and 105, respectively). The sequence en coding the N-terminal signal peptide was excluded from the constructs, starting RhPETase and CbPETase constructs at residues 29 and 30, respectively. C- terminal His6-tag encoding sequence (CATCACCATCACCACCAT) was intro duced before stop codon. Nco\ and Hind III restriction sites were included at 5’ and 3’ ends of construct for restriction digestion cloning. The constructs were cloned into E. coli expression vector pBAT4 by restriction digestion cloning and expressed in E. coli strain SHuffle T7 Express (New England Biolabs).

Constructs were expressed in E. coli SHuffle T7 Express grown at 37°C in SB (30 g tryptone, 20 g yeast extract, 10 g MOPS (3-[A/-morpholino]-propanesulfonic acid) per liter) media containing 100 pg/ml ampicillin. Protein expression was induced by the addition of 1mM isopropyl b-D-l-thiogalactopyranoside (IPTG), and induced cultures were further incubated at 30 °C for 24 h. Cells were harvested by centrif ugation (1500g, 15min, RT), and pellet was stored at -80°C until purification.

Lysate was prepared by suspending pellet in Buffer A (50mM Na ₂ HP0 ₄ -HCI, 100mM NaCI pH 8.0) with 1x EDTA-Free Complete Protease Inhibitor Cocktail (Roche). Cells were lysed by homogenisation with glass beads and cell debris was removed by centrifugation (13500 g, 20 min 4 °C). After centrifugation Buffer B (50mM Na ₂ HP0 ₄ -HCI, 100mM NaCI, 500mM imidazole pH 8.0) was added to ly sate to 10mM imidazole concentration. Enzymes were purified by affinity chroma- tography with 5ml HisTrap column (GE Healthcare). Supernatant was applied to column equilibrated with 10 mM imidazole and column was washed with 30mM im idazole. Bound proteins were eluted with Buffer B (500 mM Imidatzole). The quali ty of purified protein was assessed by SDS-PAGE, to verify high enough (>80%) homogeneity of protein samples for enzyme assays.

Example 10. Polymer degradation with Rhizobacter sp. and Cytophagales bacterium dienelactone hydrolases Purified enzymes from Example 9 were incubated seven days with PEF polymer (Table 1) as described in Example 2. After enzyme reaction was stopped GC-MS analysis was carried out as described in Example 3.

In the GC-MS analysis with both enzymes FDCA and ethylene glycol like com- pounds could be detected (Figures 8 and 9) indicating degradation of PEF. None of these compounds could be seen with the control sample.

Example 11. Degradation of emulsified PLA by Rhizobacter sp. dienelactone hydrolase

The substrate solutions were prepared by mixing PLA polymer emulsion of exam ple 5 with 50 mM Na ₂ HP0 ₄ -HCI pH 8.0 in 4:1 ratio (emulsio buffer). Mixture of partially purified Rhizobacter sp. enzyme from Example 9 and substrate solution was then incubated at 30 °C with gentle agitation. Control sample was prepared without enzyme. The decrease in turbidity was measured at 630 nm using a spec trophotometer (Ultrospec 2100 Pro, GE Healthcare) at 0 h and 69 h. With enzyme sample Rhizobacter sp. dienelactone hydrolase decrease in absorbance was de tected indicating PLA degradation activity (see Table 5). Table 5. Enzyme sample Rhizobacter sp. dienelactone hydrolase revealed de-