Patent Publication Number: US-2023151340-A1

Title: Plastic degrading fusion proteins and methods of using the same

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application claims priority from U.S. Provisional Patent Application No. 63/022,784 filed on May 11, 2020, the contents of which is incorporated herein by reference in their entirety. 
    
    
     CONTRACTUAL ORIGIN 
     This invention was made with government support under Contract No. DE-AC36-08G028308 awarded by the Department of Energy. The government has certain rights in the invention. 
    
    
     SEQUENCE LISTING 
     The instant application contains a Sequence Listing which has been submitted via EFS-web and is hereby incorporated by reference in its entirety. The ASCII copy created on 7 May2021, is named NREL PCT 20-86_ST25.txt and is 61 kilobytes in size. 
     BACKGROUND 
     Poly (ethylene terephthalate) (PET) is one of the most abundant manmade synthetic polyesters. Crystalline PET is being widely used for production of single-use beverage bottles, clothing, packaging, and carpeting materials. PET resistance to biodegradation due to limited accessibility to ester linkage, and disposal of PET products into the environment pose a serious threat to biosphere, particularly to marine environment. PET can be chemically recycled. However, the extra costs in chemical recycling are not justified when converting PET back to PET. Thus, there remains a need for alternative strategies for recycling/recovering/reusing plastics, for example, polyesters such as PET. 
     SUMMARY 
     An aspect of the present disclosure is a non-naturally occurring enzyme that includes a first polypeptide that catalyzes the hydrolysis of a polyester to produce mono-(2-hydroxyethyl) terephthalate (MHET), a second polypeptide that catalyzes the cleavage of MHET to produce at least one of terephthalic acid or ethylene glycol, and a third polypeptide that links the first polypeptide with the second polypeptide. In some embodiments of the present disclosure, the enzyme may have a sequence identity that is greater than 80% to SEQ ID NO: 36. 
     In some embodiments of the present disclosure, the enzyme may have a turnover rate of up to 69 s −1 . In some embodiments of the present disclosure, the third polypeptide may have about 8 amino acids. In some embodiments of the present disclosure, the enzyme may have a sequence identity that is greater than 80% to SEQ ID NO: 38. In some embodiments of the present disclosure, the enzyme may have a turnover rate of up to 77 s −1 . In some embodiments of the present disclosure, the third polypeptide may have about 12 amino acids. In some embodiments of the present disclosure, the enzyme may have a sequence identity that is greater than 80% to SEQ ID NO: 40. In some embodiments of the present disclosure, the enzyme may have a turnover rate of up to 56 −1 . In some embodiments of the present disclosure, the third polypeptide may have about 20 amino acids. 
     In some embodiments of the present disclosure, the polyester may include at least one of polyethylene terephthalate (PET), polyglycolic acid, polylactic acid, polycaprolactone, polyhydroxyalkanoate, polyhydroxybutyrate, polyethylene adipate, polybutylene succinate, poly(3-hydroxybutyrate-co-3-hydroxyvalerate), polybutylene terephthalate, polytrimethylene terephthalate, and/or polyethylene naphthlate. In some embodiments of the present disclosure, the third polypeptide may have between 1 and 100 amino acids. In some embodiments of the present disclosure, the third polypeptide may include at least one of glycine, serine, proline, and/or threonine. In some embodiments of the present disclosure, the third polypeptide may covalently link the C-terminus of the second polypeptide to the N-terminus of the first polypeptide. 
     In some embodiments of the present disclosure, the enzyme may further include a fourth polypeptide capable of catalyzing hydrolysis of a polyester to produce mono-(2-hydroxyethyl) terephthalate (MHET) and a fifth polypeptide, where the fifth polypeptide covalently links the fourth polypeptide with the second polypeptide. In some embodiments of the present disclosure, the enzyme may further include a mutation of at least one of a S to G, a T to L, F, or Y, a E to N, T, D, Q, or G, a R to F, E, T, A, Y, I, S, W, L, V, Q, G, M, or N, a F to P, D, L, A, S, T, E, N, G, or V, a S to A, G, Q, P, E, D, or V, a S to R, A, K, Q, or G, a T to V or L, and/or a F to I. In some embodiments of the present disclosure, the mutation may occur in the second polypeptide. 
     An aspect of the present disclosure is a genetically modified organism that expresses the enzyme as described herein. In some embodiments of the present disclosure, the organism may include at least one of  Pseudomonas putida  and/or  Escherichia coli.    
     An aspect of the present disclosure is a method for degrading a polyester, where the method includes contacting an organism as described herein with the polyester. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       Some embodiments are illustrated in referenced figures of the drawings. It is intended that the embodiments and figures disclosed herein are to be considered illustrative rather than limiting. 
         FIG.  1    illustrates fusion proteins, according to some embodiments of the present disclosure. 
         FIG.  2 A  illustrates a heatmap of synergistic degradation by PETase and MHETase on amorphous PET film over 96 hours at 30° C., according to some embodiments of the present disclosure. Total product release in mM (sum of BHET, MHET, and TPA), x-axis: PETase loading (mg/g PET), y-axis: MHETase loading (mg/g PET). 
         FIG.  2 B  illustrates three PETase-MHETase fusion proteins, according to some embodiments of the present disclosure. Linkers composed of glycine (orange) and serine (yellow) residues connecting the C-terminus of MHETase to the N-terminus of PETase. 
         FIGS.  2 C and  2 D  illustrate a comparison of depolymerization performance of PETase alone, MHETase alone, PETase and MHETase at equimolar loading, and the three fusion proteins on amorphous PET film after 96 h at 30° C., according to some embodiments of the present disclosure. Product release in mM resulting from hydrolysis by  FIG.  2 C  0.08 mg PETase/g PET or 0.16 mg MHETase/g PET and  FIG.  2 D  0.25 mg PETase/g PET or 0.5 mg MHETase/g PET. All comparisons are statistically significant with p-values £ 0.0001 based on 2way ANOVA analysis and Tukey&#39;s multiple comparisons test. 
         FIG.  2 E  illustrates MHET turnover rate by each fusion protein compared to MHETase alone using 250 μM MHET and 5 nM enzyme, according to some embodiments of the present disclosure. Asterisks indicate statistically significant comparisons between MHETase and each chimera enzyme with p-values £ 0.01 (*), 0.001 (**), and 0.0005 (***). 
         FIG.  3    illustrates SEM images of amorphous PET film after 96 hours of enzyme treatment at 30° C., according to some embodiments of the present disclosure. Digestion conditions represent treatment with no enzyme, treatment with 0.4 mg MHETase/g PET, treatment with 0.4 mg PETase/g PET, simultaneous treatment with 0.4 mg PETase and 0.4 mg MHETase/g PET, and treatment with each fusion protein corresponding to the samples presented in  FIG.  2 D . 
         FIG.  4    illustrates a conservation analysis of 6,671 tannase family sequences, according to some embodiments of the present disclosure. Panel A) illustrates conservation scores (relative entropy) of positions in tannase family sequences plotted against the 603 positions in MHETase. A higher relative entropy implies a greater level of amino acid conservation in the site. Panel B) illustrates conservation scores of active-site residues in MHETase within 6 A of the MHET substrate. Conservation scores are shown as percentiles. Arg411, Phe415, and Ser416 are the least conserved active-site positions in the active site and are more variable than 81% of all positions in MHETase. Panel C) illustrates the closest distance between atoms of MHETase active-site residues and the MHET substrate. The molecular coordinates for MHETase bound with MHET are the same as those in the model from which the molecular simulations were started. 
         FIGS.  5 A- 5 D  illustrate amino acid frequencies of active-site positions in MHETase within 6 A of the MHET substrate, according to some embodiments of the present disclosure. The frequency of amino acids for each position was determined from a MAFFT multiple sequence alignment of 6,671 tannase family sequences. The positions are named using Is MHETase numbering, and the red bars indicate the amino acids in Is MHETase. 
         FIG.  6 A  illustrates the conservation of Cys positions forming five disulfide bonds in MHETase, according to some embodiments of the present disclosure. Conservation scores are shown as percentiles. Ao FAEB-1 has a 6 th  disulfide bond between Cys76 and Cys129 which are very variable positions and are less conserved than 98% of positions in multiple sequence alignment. 
         FIG.  6 B  illustrates a histogram of Cys occurrence in tannase family sequences showing the rarity of a 6 th  disulfide bond, according to some embodiments of the present disclosure. Assuming, all Cys form disulfide bonds, less than 8% of tannase family sequences have six disulfide bonds. 
         FIG.  7 A  illustrates the sequence identity of 6,671 tannase family sequences retrieved by PSI-BLAST compared to MHETase, according to some embodiments of the present disclosure. 
         FIGS.  7 B and  7 C  illustrate a conservation analysis of residue positions  131  ( FIG.  7 B ) and 415 ( FIG.  7 C ) (using MHETase numbering), according to some embodiments of the present disclosure. Frequency of each amino acid is based on a multiple sequence alignment of the 6,671 tannase family sequences. The residue found in MHETase at each position is indicated in black. 
         FIG.  7 D  illustrates a homology model of the MHET-bound active site within 6 A of the bound substrate comparing MHETase to homology models of the  C. thiooxydans  and  Hydrogenophaga sp . PML113 homologs (generated by SWISS-MODEL), according to some embodiments of the present disclosure. 
         FIGS.  7 E and  7 F  illustrate the rate of enzymatic turnover of enzymes described herein, according to some embodiments of the present disclosure. 
         FIGS.  7 G through  7 J  show the initial enzyme reaction velocity as a function of substrate concentration for MHETase,  C. thiooxydans, Hydrogenophaga sp . PML113, and the MHETase S131G mutant, respectively, according to some embodiments of the present disclosure. Solid lines represent the Michaelis-Menten kinetic model fit with substrate inhibition. 
     
    
    
     REFERENCE NUMBERS 
     
         
         
           
               100  fusion protein 
               110  first polypeptide 
               120  second polypeptide 
               130  third polypeptide 
               140  fourth polypeptide 
               150  fifth polypeptide 
           
         
       
    
     DETAILED DESCRIPTION 
     The present disclosure may address one or more of the problems and deficiencies of the prior art discussed above. However, it is contemplated that some embodiments as disclosed herein may prove useful in addressing other problems and deficiencies in a number of technical areas. 
     Therefore, the embodiments described herein should not necessarily be construed as limited to addressing any of the particular problems or deficiencies discussed herein. 
     References in the specification to “one embodiment”, “an embodiment”, “an example embodiment”, “some embodiments”, etc., indicate that the embodiment described may include a particular feature, structure, or characteristic, but every embodiment may not necessarily include the particular feature, structure, or characteristic. Moreover, such phrases are not necessarily referring to the same embodiment. Further, when a particular feature, structure, or characteristic is described in connection with an embodiment, it is submitted that it is within the knowledge of one skilled in the art to affect such feature, structure, or characteristic in connection with other embodiments whether or not explicitly described. 
     As used herein the term “substantially” is used to indicate that exact values are not necessarily attainable. By way of example, one of ordinary skill in the art will understand that in some chemical reactions 100% conversion of a reactant is possible, yet unlikely. Most of a reactant may be converted to a product and conversion of the reactant may asymptotically approach 100% conversion. So, although from a practical perspective 100% of the reactant is converted, from a technical perspective, a small and sometimes difficult to define amount remains. For this example of a chemical reactant, that amount may be relatively easily defined by the detection limits of the instrument used to test for it. However, in many cases, this amount may not be easily defined, hence the use of the term “substantially”. In some embodiments of the present invention, the term “substantially” is defined as approaching a specific numeric value or target to within 20%, 15%, 10%, 5%, or within 1% of the value or target. In further embodiments of the present invention, the term “substantially” is defined as approaching a specific numeric value or target to within 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.10% of the value or target. 
     As used herein, the term “about” is used to indicate that exact values are not necessarily attainable. Therefore, the term “about” is used to indicate this uncertainty limit. In some embodiments of the present invention, the term “about” is used to indicate an uncertainty limit of less than or equal to ±20%, ±15%, +10%, ±5%, or ±1% of a specific numeric value or target. In some embodiments of the present invention, the term “about” is used to indicate an uncertainty limit of less than or equal to ±1%, ±0.9%, ±0.8%, ±0.7%, ±0.6%, ±0.5%, ±0.4%, ±0.3%, ±0.2%, or ±0.1% of a specific numeric value or target. 
     A “vector” or “recombinant vector” is a nucleic acid molecule that is used as a tool for manipulating a nucleic acid sequence of choice or for introducing such a nucleic acid sequence into a host cell. A vector may be suitable for use in cloning, sequencing, or otherwise manipulating one or more nucleic acid sequences of choice, such as by expressing or delivering the nucleic acid sequence(s) of choice into a host cell to form a recombinant cell. Such a vector typically contains heterologous nucleic acid sequences not naturally found adjacent to a nucleic acid sequence of choice, although the vector can also contain regulatory nucleic acid sequences (e.g., promoters, untranslated regions) that are naturally found adjacent to the nucleic acid sequences of choice or that are useful for expression of the nucleic acid molecules. 
     A vector can be either RNA or DNA, either prokaryotic or eukaryotic, and typically is a plasmid. The vector can be maintained as an extrachromosomal element (e.g., a plasmid) or it can be integrated into the chromosome of a recombinant host cell. The entire vector can remain in place within a host cell, or under certain conditions, the plasmid DNA can be deleted, leaving behind the nucleic acid molecule of choice. An integrated nucleic acid molecule can be under chromosomal promoter control, under native or plasmid promoter control, or under a combination of several promoter controls. Single or multiple copies of the nucleic acid molecule can be integrated into the chromosome. A recombinant vector can contain at least one selectable marker. 
     The term “expression vector” refers to a recombinant vector that is capable of directing the expression of a nucleic acid sequence that has been cloned into it after insertion into a host cell or other (e.g., cell-free) expression system. A nucleic acid sequence is “expressed” when it is transcribed to yield an mRNA sequence. In most cases, this transcript will be translated to yield an amino acid sequence. The cloned gene is usually placed under the control of (i.e., operably linked to) an expression control sequence. The phrase “operatively linked” refers to linking a nucleic acid molecule to an expression control sequence in a manner such that the molecule can be expressed when introduced (i.e., transformed, transduced, transfected, conjugated or conduced) into a host cell. 
     Vectors and expression vectors may contain one or more regulatory sequences or expression control sequences. Regulatory sequences broadly encompass expression control sequences (e.g., transcription control sequences or translation control sequences), as well as sequences that allow for vector replication in a host cell. Transcription control sequences are sequences that control the initiation, elongation, or termination of transcription. Suitable regulatory sequences include any sequence that can function in a host cell or organism into which the recombinant nucleic acid molecule is to be introduced, including those that control transcription initiation, such as promoter, enhancer, terminator, operator and repressor sequences. Additional regulatory sequences include translation regulatory sequences, origins of replication, and other regulatory sequences that are compatible with the recombinant cell. The expression vectors may contain elements that allow for constitutive expression or inducible expression of the protein or proteins of interest. Numerous inducible and constitutive expression systems are known in the art. 
     Typically, an expression vector includes at least one nucleic acid molecule of interest operatively linked to one or more expression control sequences (e.g., transcription control sequences or translation control sequences). In one aspect, an expression vector may comprise a nucleic acid encoding a recombinant polypeptide, as described herein, operably linked to at least one regulatory sequence. It should be understood that the design of the expression vector may depend on such factors as the choice of the host cell to be transformed and/or the type of polypeptide to be expressed. As used herein, a “non-natural” polypeptide is synonymous with a “recombinant” polypeptide. 
     Expression and recombinant vectors may contain a selectable marker, a gene encoding a protein necessary for survival or growth of a host cell transformed with the vector. The presence of this gene allows growth of only those host cells that express the vector when grown in the appropriate selective media. Typical selection genes encode proteins that confer resistance to antibiotics or other toxic substances, complement auxotrophic deficiencies, or supply critical nutrients not available from a particular media. Markers may be an inducible or non-inducible gene and will generally allow for positive selection. Non-limiting examples of selectable markers include the ampicillin resistance marker (i.e., beta-lactamase), tetracycline resistance marker, neomycin/kanamycin resistance marker (i.e., neomycin phosphotransferase), dihydrofolate reductase, glutamine synthetase, and the like. The choice of the proper selectable marker will depend on the host cell, and appropriate markers for different hosts as understood by those of skill in the art. 
     Suitable expression vectors may include (or may be derived from) plasmid vectors that are well known in the art, such as those commonly available from commercial sources. Vectors can contain one or more replication and inheritance systems for cloning or expression, one or more markers for selection in the host, and one or more expression cassettes. The inserted coding sequences can be synthesized by standard methods, isolated from natural sources, or prepared as hybrids. Ligation of the coding sequences to transcriptional regulatory elements or to other amino acid encoding sequences can be carried out using established methods. A large number of vectors, including bacterial, yeast, and mammalian vectors, have been described for replication and/or expression in various host cells or cell-free systems, and may be used with the sequences described herein for simple cloning or protein expression. 
     Nucleic acids referred to herein as “isolated” are nucleic acids that have been removed from their natural milieu or separated away from the nucleic acids of the genomic DNA or cellular RNA of their source of origin (e.g., as it exists in cells or in a mixture of nucleic acids such as a library), and may have undergone further processing. Isolated nucleic acids include nucleic acids obtained by methods described herein, similar methods or other suitable methods, including essentially pure nucleic acids, nucleic acids produced by chemical synthesis, by combinations of biological and chemical methods, and recombinant nucleic acids that are isolated. 
     Nucleic acids referred to herein as “recombinant” are nucleic acids which have been produced by recombinant DNA methodology, including those nucleic acids that are generated by procedures that rely upon a method of artificial replication, such as the polymerase chain reaction (PCR) and/or cloning or assembling into a vector using restriction enzymes. 
     Recombinant nucleic acids also include those that result from recombination events that occur through the natural mechanisms of cells but are selected for after the introduction to the cells of nucleic acids designed to allow or make probable a desired recombination event. Portions of isolated nucleic acids that code for polypeptides having a certain function can be identified and isolated by, for example, the method disclosed in U.S. Pat. No. 4,952,501. 
     A nucleic acid molecule or polynucleotide can include a naturally occurring nucleic acid molecule that has been isolated from its natural source or produced using recombinant DNA technology (e.g., polymerase chain reaction (PCR) amplification, cloning) or chemical synthesis. Isolated nucleic acid molecules can include, for example, genes, natural allelic variants of genes, coding regions or portions thereof, and coding and/or regulatory regions modified by nucleotide insertions, deletions, substitutions, and/or inversions in a manner such that the modifications do not substantially interfere with the nucleic acid molecule&#39;s ability to encode a polypeptide or to form stable hybrids under stringent conditions with natural gene isolates. An isolated nucleic acid molecule can include degeneracies. As used herein, nucleotide degeneracy refers to the phenomenon that one amino acid can be encoded by different nucleotide codons. Thus, the nucleic acid sequence of a nucleic acid molecule that encodes a protein or polypeptide can vary due to degeneracies. 
     Unless so specified, a nucleic acid molecule is not required to encode a protein having enzyme activity. A nucleic acid molecule can encode a truncated, mutated, or inactive protein, for example. In addition, nucleic acid molecules may also be useful as probes and primers for the identification, isolation and/or purification of other nucleic acid molecules, independent of a protein-encoding function. 
     Suitable nucleic acids include fragments or variants that encode a functional enzyme. For example, a fragment can comprise the minimum nucleotides required to encode a functional enzyme. Nucleic acid variants include nucleic acids with one or more nucleotide additions, deletions, substitutions, including transitions and transversions, insertion, or modifications (e.g., via RNA or DNA analogs). Alterations may occur at the 5′ or 3′ terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among the nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence. 
     Embodiments of the nucleic acids include those that encode the polypeptides that function as an O-dealkylase or a reductase or functional equivalents thereof. A functional equivalent includes fragments or variants of these that exhibit the ability to function as an O-dealkylase or a reductase. As a result of the degeneracy of the genetic code, many nucleic acid sequences can encode a given polypeptide with a particular enzymatic activity. Such functionally equivalent variants are contemplated herein. 
     Nucleic acids may be derived from a variety of sources including DNA, cDNA, synthetic DNA, synthetic RNA, or combinations thereof. Such sequences may comprise genomic DNA, which may or may not include naturally occurring introns. Moreover, such genomic DNA may be obtained in association with promoter regions or poly (A) sequences. The sequences, genomic DNA, or cDNA may be obtained in any of several ways. Genomic DNA can be extracted and purified from suitable cells by means well known in the art. Alternatively, mRNA can be isolated from a cell and used to produce cDNA by reverse transcription or other means. 
     Also disclosed herein are recombinant vectors, including expression vectors, containing nucleic acids encoding polypeptides and/or enzymes. A “recombinant vector” is a nucleic acid molecule that is used as a tool for manipulating a nucleic acid sequence of choice or for introducing such a nucleic acid sequence into a host cell. A recombinant vector may be suitable for use in cloning, assembling, sequencing, or otherwise manipulating the nucleic acid sequence of choice, such as by expressing or delivering the nucleic acid sequence of choice into a host cell to form a recombinant cell. Such a vector typically contains heterologous nucleic acid sequences not naturally found adjacent to a nucleic acid sequence of choice, although the vector can also contain regulatory nucleic acid sequences (e.g., promoters, untranslated regions) that are naturally found adjacent to the nucleic acid sequences of choice or that are useful for expression of the nucleic acid molecules. 
     The nucleic acids described herein may be used in methods for production of enzymes and enzyme cocktails through incorporation into cells, tissues, or organisms. In some embodiments, a nucleic acid may be incorporated into a vector for expression in suitable host cells. The vector may then be introduced into one or more host cells by any method known in the art. One method to produce an encoded protein includes transforming a host cell with one or more recombinant nucleic acids (such as expression vectors) to form a recombinant cell. The term “transformation” is generally used herein to refer to any method by which an exogenous nucleic acid molecule (i.e., a recombinant nucleic acid molecule) can be inserted into a cell but can be used interchangeably with the term “transfection.” 
     Non-limiting examples of suitable host cells include cells from microorganisms such as bacteria, yeast, fungi, and filamentous fungi. Exemplary microorganisms include, but are not limited to, bacteria such as  E. coli ; bacteria from the genera  Pseudomonas  (e.g.,  P. putida  or  P. fluorescens ),  Bacillus  (e.g.,  B. subtilis, B. megaterium  or  B. brevis ),  Caulobacter  (e.g.,  C. crescentus ), Lactoccocus (e.g.,  L. lactis ),  Streptomyces  (e.g.,  S. coelicolor ),  Streptococcus  (e.g.,  S. lividans ), and Corynybacterium (e.g.,  C. glutamicum ); fungi from the genera  Trichoderma  (e.g.,  T. reesei, T. viride, T. koningii , or  T. harzianum ),  Penicillium  (e.g.,  P. funiculosum ),  Humicola  (e.g.,  H. insolens ),  Chrysosporium  (e.g.,  C. lucknowense ),  Gliocladium, Aspergillus  (e.g.,  A. niger, A. nidulans, A. awamori , or  A. aculeatus ),  Fusarium, Neurospora , Hypocrea (e.g.,  H. jecorina ), and  Emericella ; yeasts from the genera  Saccharomyces  (e.g.,  S. cerevisiae ),  Pichia  (e.g.,  P. pastoris ), or  Kluyveromyces  (e.g.,  K. lactis ). Cells from plants such as  Arabidopsis , barley, citrus, cotton, maize, poplar, rice, soybean, sugarcane, wheat, switch grass, alfalfa,  miscanthus , and trees such as hardwoods and softwoods are also contemplated herein as host cells. 
     Host cells can be transformed, transfected, or infected as appropriate by any suitable method including electroporation, calcium chloride-, lithium chloride-, lithium acetate/polyene glycol-calcium, phosphate-, DEAE-dextran-, liposome-mediated DNA uptake, spheroplasting, injection, microinjection, microprojectile bombardment, phage infection, viral infection, or other established methods. Alternatively, vectors containing the nucleic acids of interest can be transcribed in vitro, and the resulting RNA introduced into the host cell by well-known methods, for example, by injection. Exemplary embodiments include a host cell or population of cells expressing one or more nucleic acid molecules or expression vectors described herein (for example, a genetically modified microorganism). The cells into which nucleic acids have been introduced as described above also include the progeny of such cells. 
     Vectors may be introduced into host cells such as those from bacteria or fungi by direct transformation, in which DNA is mixed with the cells and taken up without any additional manipulation, by conjugation, electroporation, or other means known in the art. Expression vectors may be expressed by bacteria or fungi or other host cells episomally or the gene of interest may be inserted into the chromosome of the host cell to produce cells that stably express the gene with or without the need for selective pressure. For example, expression cassettes may be targeted to neutral chromosomal sites by recombination. 
     Host cells carrying an expression vector (i.e., transformants or clones) may be selected using markers depending on the mode of the vector construction. The marker may be on the same or a different DNA molecule. In prokaryotic hosts, the transformant may be selected, for example, by resistance to ampicillin, tetracycline or other antibiotics. Production of a particular product based on temperature sensitivity may also serve as an appropriate marker. 
     Host cells may be cultured in an appropriate fermentation medium. An appropriate, or effective, fermentation medium refers to any medium in which a host cell, including a genetically modified microorganism, when cultured, is capable of growing or expressing the polypeptides described herein. Such a medium is typically an aqueous medium comprising assimilable carbon, nitrogen, and phosphate sources, but can also include appropriate salts, minerals, metals and other nutrients. Microorganisms and other cells can be cultured in conventional fermentation bioreactors and by any fermentation process, including batch, fed-batch, cell recycle, and continuous fermentation. The pH of the fermentation medium is regulated to a pH suitable for growth of the particular organism. Culture media and conditions for various host cells are known in the art. A wide range of media for culturing bacteria or fungi, for example, are available from ATCC. Exemplary culture/fermentation conditions and reagents are provided in the Examples that follow. Media may be supplemented with aromatic substrates like guaiacol, guaethol or anisole for dealkylation reactions. 
     As used herein, the terms “protein” and “polypeptide” are synonymous. “Peptides” are defined as fragments or portions of polypeptides, preferably fragments or portions having at least one functional activity as the complete polypeptide sequence. “Isolated” proteins or polypeptides are proteins or polypeptides purified to a state beyond that in which they exist in cells. In certain embodiments, they may be at least 10% pure; in others, they may be substantially purified to 80% or 90% purity or greater. Isolated proteins or polypeptides include essentially pure proteins or polypeptides, proteins or polypeptides produced by chemical synthesis or by combinations of biological and chemical methods, and recombinant proteins or polypeptides that are isolated. Proteins or polypeptides referred to herein as “recombinant” are proteins or polypeptides produced by the expression of recombinant nucleic acids. 
     Polypeptides may be retrieved, obtained, or used in “substantially pure” form, a purity that allows for the effective use of the protein in any method described herein or known in the art. For a protein to be most useful in any of the methods described herein or in any method utilizing enzymes of the types described herein, it is most often substantially free of contaminants, other proteins and/or chemicals that might interfere or that would interfere with its use in the method (e.g., that might interfere with enzyme activity), or that at least would be undesirable for inclusion with a protein. In an embodiment, a non-naturally occurring enzyme may also be referred to as a recombinant protein Among other things, the present disclosure relates to fusion proteins, chimeric enzymes, for depolymerizing plastics, for example, polyethylene terephthalate (PET). As described herein, fusion proteins are disclosed having at least a two-enzyme system of a first enzyme (i.e., a first polypeptide) for deconstructing PET (i.e., a PETase) to its constituent monomers, including mono-(2-hydroxyethyl) terephthalate (MHET), and a second enzyme (i.e., a second polypeptide), a MHETase, which cleaves the MHET to yield terephthalic acid (TPA) and ethylene glycol (EG). 
       FIG.  1    illustrates fusion proteins  100 , according to some embodiments of the present disclosure. Referring to Panel A), a fusion protein  100  may include a first polypeptide  110 , for example an enzyme capable of degrading a polyester to an intermediate, smaller molecular weight molecule. This first polypeptide  100  may be covalently linked to a second polypeptide  120 , for example an enzyme capable of further degrading (e.g., cleaving) the intermediate to even smaller molecular weight components. The first polypeptide  100  may be covalently linked to the second polypeptide by a third polypeptide  130 , for example a flexible chain of amino acids. Panel B) illustrates that, according to some embodiments of the present disclosure, a fusion protein may include three or more catalytically active polypeptides covalently linked together by one or more linker polypeptides. In some embodiments of the present disclosure, a linker polypeptide may have between 1 and 100 amino acids, or between 20 and 100 amino acids, or between 10 and 50 amino acids. Panel B) illustrates that in some embodiments of the present disclosure, a fusion protein  100  may include two linking molecules ( 130  and  150 ), linking two enzymes ( 110  and  140 ) to a third enzyme  120 . In this example, two enzymes ( 110  and  140 ) capable of degrading a polyester to an intermediate may be covalently linked by two separate flexible amino acid chains ( 130  and  150 , respectively) to a polypeptide  120  capable of further degrading (e.g., cleaving) the intermediate to even smaller molecular weight components. In some embodiments of the present disclosure, the three or more enzymes may be covalently bound in linear fashion along an unbranched polypeptide chain. For example, using the reference numbers of Panel B) of  FIG.  1   :  110  to  130  to  140  to  150  to  120 . 
     In some embodiments of the present disclosure, a fusion protein  100  may include a first polypeptide  110  capable of catalyzing hydrolysis of a polyester to produce a first intermediate covalently linked to a second polypeptide  120  capable of catalyzing cleavage of the first intermediate to produce smaller molecular weight compounds. The first polypeptide  110  may be covalently linked to the second polypeptide  120  by a third polypeptide, for example a flexible chain of amino acids. For the example where the polyester includes polyethylene terephthalate (PET), a first polypeptide  110  capable of catalyzing hydrolysis of the PET to produce at least mono-(2-hydroxyethyl) terephthalate (MHET) is referred to herein as a PETase and the second polypeptide  120  capable of further degrading the MHET to at least one of terephthalic acid and/or ethylene glycol is referred to herein as a MHETase. 
     In some embodiments of the present disclosure, a fusion protein  100  may be capable of degrading a plastic such as a polyester to smaller molecular weight compounds that may be reused to produce valuable materials. Examples of polyesters that may be degraded using the enzymes, organisms, and methods described herein include at least one of polyethylene terephthalate (PET), polyglycolic acid, polylactic acid, polycaprolactone, polyhydroxyalkanoate, polyhydroxybutyrate, polyethylene adipate, polybutylene succinate, poly(3-hydroxybutyrate-co-3-hydroxyvalerate), polybutylene terephthalate, polytrimethylene terephthalate, and/or polyethylene naphthlate. 
     In some embodiments of the present disclosure, at least one of the first polypeptide (e.g., PETase) and/or the second polypeptide (e.g., MHETase) may be derived from at least one of a bacterium and/or a fungus. In some embodiments of the present disclosure, the first polypeptide and/or the second polypeptide may be derived from a fungus such as  Fusarium solani . In some embodiments of the present disclosure, the first polypeptide and/or the second polypeptide may be derived from a bacterium from a family that includes at least one of Comamonadaceae and/or Nocardiopsaceae. In some embodiments of the present disclosure, the first polypeptide and/or the second polypeptide may be derived from a bacterium from a genus that includes at least one of  Ideonella, Comamonas, Hydrogenophaga , and/or  Thermobifida . In some embodiments of the present disclosure, the first polypeptide and/or the second polypeptide may be derived from a bacterium that includes at least one of  Ideonella  sakaiensis, and/or  Comamonas thiooxydans.    
     In some embodiments of the present disclosure, a third polypeptide  130  that covalently links a first polypeptide  110  to a second polypeptide may include between 1 amino acid and 100 amino acids, inclusively. In an embodiment, a third peptide is from about 10 to about 50 amino acids. In an embodiment, a third peptide is from about 20 to about 50 amino acids. In an embodiment, a third peptide is from about 10 to about 80 amino acids. In an embodiment, a third peptide is from about 20 to about 80 amino acids. In an embodiment, a third peptide is from about 10 to about 90 amino acids. In an embodiment, a third peptide is from about 20 to about 90 amino acids. In some embodiments of the present disclosure a third polypeptide  130 , i.e., a linking protein chain, may include at least 2 amino acids, at least 5 amino acids, at least 8 amino acids, at least 11 amino acids, at least 14 amino acids, at least 17 amino acids, or at least 20 amino acids. In some embodiments of the present disclosure a third polypeptide  130 , i.e., a linking protein chain, may include up to 25 amino acids, up to 50 amino acids, up to 75 amino acids, or up to 100 amino acids. A linking protein may be constructed of amino acids that include, among others, at least one of glycine, serine, proline, and/or threonine. In some embodiments of the present disclosure, a third polypeptide  130  (i.e., a linking protein chain) may covalently link the C-terminus of the second polypeptide  120  to the N-terminus of the first polypeptide  110 . In some embodiments of the present disclosure, a first polypeptide  110  may be covalently linked to a third polypeptide  130  by a maleimide crosslinker, provided each polypeptide has a sulfhydryl group (—SH). Examples of a maleimide include bis-maleimidoethane and 1,4-di(maleimido)butane. 
     In some embodiments of the present disclosure, at least one of the first polypeptide  110  and/or the second polypeptide  120  may include a mutation to at least one amino acid, resulting in improved catalytic activity by the mutated polypeptide, as described herein. In some embodiments of the present disclosure at least one of the amino acids of a MHETase as described herein, may be mutated at least one of the following locations along the MHETase polypeptide:  131  (S to G),  133  (T to L, F, or Y),  226  (E to N, T, D, Q, or G),  411  (R to F, E, T, A, Y, I, S, W, L, V, Q, G, M, or n),  415  (F to P, D, L, A, S, T, E, N, G, or V),  416  (S to A, G, Q, P, E, D, or V),  419  (S to R, A, K, Q, or G),  494  (a TO V or L), or  495  (F to I). (See  FIGS.  5 A through  5 D .) In some embodiments of the present disclosure, a fusion protein may also include a secretion signal peptide. 
     In an embodiment, additional enzymes are contemplated herein that at least 80% sequence identity to the enzymes disclosed herein. In other embodiments, additional enzymes are contemplated herein that at least 85%, 90%, 95%, 98%, 99%, and up to 100% sequence identity to the enzymes disclosed herein. 
     As described herein, an organism may be genetically modified to manufacture the fusion proteins described herein. In some embodiments of the present disclosure, an organism for producing a fusion protein may include a bacterium such as at least one of a  Pseudomonas putida  and/or  Escherichia coli . Further, as described herein, a plastic (e.g., PET) may be degraded to smaller molecular weight compounds by mixing and/or contacting at least one of the fusion proteins and/or organisms producing the fusion proteins with the plastic, where the mixing/contacting results in the degrading of the plastic to smaller molecular weight components. 
     As shown herein, fusion proteins (i.e., chimeric proteins) of MHETase and PETase can improve PET degradation and MHET hydrolysis rates. As described below in more detail, in view of the synergistic relationship between PETase and MHETase on amorphous PET, the relationship between the proximity of the two enzymes and hydrolytic activity was examined. Chimeric proteins covalently linking the C-terminus of MHETase to the N-terminus of PETase using flexible glycine-serine linkers of 8, 12, and 20 total glycine and serine residues were generated and tested for degradation of amorphous PET (see  FIG.  2 B ). Varying linker lengths were explored to understand the effect of increased mobility between the two domains. Furthermore, for comparison to the fusion protein, two loadings of the individual, non-fused enzymes were compared—the lower loading corresponding to approximately 0.08 mg PETase/g PET and 0.16 mg MHETase/g PET, and the higher enzyme loading corresponding to 0.25 mg PETase/g PET and 0.5 mg MHETase/g PET (see  FIGS.  2 C and  2 D ). At both loadings, when comparing the extent of degradation achieved by PETase alone, MHETase alone, and an equimolar mix of PETase and MHETase, the fusion proteins outperformed PETase, as well as the mixed reaction containing both PETase and MHETase. Furthermore, the fusion proteins demonstrated a higher catalytic activity on MHET (see  FIG.  2 E ). Fusion proteins linking the C-terminus of PETase to the N-terminus of MHETase did not successfully express protein (see  FIG.  2 B ). SEM analysis of digested amorphous PET film confirms degradation of PET by the fusion proteins described herein (see  FIG.  3   ). 
     In addition, as shown herein, PETase and MHETase act synergistically during PET depolymerization. While MHET is susceptible to hydrolysis by a number of PET-degrading cutinases, I. sakaiensis favors the action of two enzymes for PET degradation to liberate TPA and EG. To better understand this two-enzyme system, the extent of hydrolysis was measured of a commercial amorphous PET substrate over 96 hours at 30° C. using PETase and MHETase at varying concentrations (see  FIG.  2 A  and Table 1). As expected, MHETase alone has no activity on PET film. Over the range of enzyme loadings tested (between 0 and 2.0 mg enzyme/g PET), degradation by PETase alone, as determined by concentration of product released (the sum of BHET, MHET, and TPA), scaled with enzyme loading and then plateaued. An optimal ratio of PETase:MHETase loading was observed at 0.4 mg each enzyme/g PET, corresponding to an approximately 2:1 molar ratio (see  FIG.  2 A ). The presence of MHETase in concentrations ranging between 0.2 and 0.4 mg enzyme/g PET in the reaction enhanced degradation as compared to that observed for the same loading of PETase without MHETase. At higher levels of MHETase loading, however, degradation was negatively impacted and resulted in less product release than in reactions containing the same loading of PETase with MHETase. 
                     TABLE 1                  Synergistic degradation of amorphous PET film over 96 hours at 30° C.                                                         Sum total of       PETase loading   MHETase loading   TPA   MHET   BHET   product release       (mg enzyme/g PET)   (mg enzyme/g PET)   (mM)   (mM)   (mM)   (mM)                                             0   0   0.00 ± 0.00   0.00 ± 0.00   0.00 ± 0.00   0.00 ± 0.00       0   0.4   0.00 ± 0.00   0.00 ± 0.00   0.00 ± 0.00   0.00 ± 0.00       0   0.8   0.00 ± 0.00   0.00 ± 0.00   0.00 ± 0.00   0.00 ± 0.00       0.2   0.2   2.42 ± 0.11   0.00 ± 0.00   0.00 ± 0.00   2.43 ± 0.12       0.2   0.6   1.47 ± 0.21   0.00 ± 0.00   0.00 ± 0.00   1.47 ± 0.21       0.2   1.0   0.10 ± 0.02   0.00 ± 0.00   0.00 ± 0.00   0.10 ± 0.02       0.4   0   0.94 ± 0.09   1.15 ± 0.13   0.00 ± 0.00   2.08 ± 0.22       0.4   0.4   3.50 ± 0.25   0.00 ± 0.00   0.01 ± 0.01   3.50 ± 0.26       0.4   0.8   1.31 ± 0.18   0.00 ± 0.00   0.00 ± 0.00   1.31 ± 0.18       0.6   0.2   2.34 ± 0.03   0.02 ± 0.03   0.01 ± 0.00   2.37 ± 0.07       0.6   0.6   1.74 ± 0.05   0.00 ± 0.00   0.01 ± 0.00   1.75 ± 0.05       0.6   1.0   1.05 ± 0.26   0.00 ± 0.00   0.00 ± 0.00   1.05 ± 0.27       0.8   0   1.47 ± 0.04   0.58 ± 0.04   0.00 ± 0.00   2.05 ± 0.08       0.8   0.4   2.53 ± 0.19   0.03 ± 0.06   0.00 ± 0.00   2.56 ± 0.25       0.8   0.8   1.31 ± 0.22   0.00 ± 0.00   0.00 ± 0.00   1.31 ± 0.22       1.0   0.2   2.62 ± 0.17   0.07 ± 0.12   0.00 ± 0.00   2.69 ± 0.29       1.0   0.6   2.52 ± 0.33   0.00 ± 0.00   0.00 ± 0.00   2.53 ± 0.33       1.0   1.0   1.61 ± 0.21   0.00 ± 0.00   0.00 ± 0.00   1.61 ± 0.21       1.2   0   1.30 ± 0.03   0.70 ± 0.01   0.00 ± 0.00   2.00 ± 0.03       1.2   0.4   2.57 ± 0.13   0.05 ± 0.09   0.00 ± 0.00   2.62 ± 0.22       1.2   0.8   1.47 ± 0.11   0.00 ± 0.00   0.00 ± 0.00   1.47 ± 0.11       1.4   0.2   2.39 ± 0.21   0.01 ± 0.03   0.00 ± 0.00   2.40 ± 0.23       1.4   0.6   1.67 ± 0.40   0.00 ± 0.00   0.00 ± 0.00   1.67 ± 0.40       1.4   1.0   1.84 ± 0.15   0.00 ± 0.00   0.00 ± 0.00   1.84 ± 0.15       1.6   0   1.74 ± 0.07   0.69 ± 0.06   0.00 ± 0.00   2.43 ± 0.14       1.6   0.4   2.80 ± 0.13   0.00 ± 0.00   0.00 ± 0.00   2.81 ± 0.13       1.6   0.8   2.21 ± 0.09   0.00 ± 0.00   0.00 ± 0.00   2.21 ± 0.09       1.8   0.2   2.12 ± 0.17   0.07 ± 0.06   0.00 ± 0.00   2.19 ± 0.23       1.8   0.6   2.23 ± 0.26   0.00 ± 0.00   0.01 ± 0.00   2.24 ± 0.26       1.8   1.0   1.42 ± 0.11   0.00 ± 0.00   0.01 ± 0.00   1.43 ± 0.11       2.0   0   1.84 ± 0.25   0.21 ± 0.04   0.00 ± 0.00   2.05 ± 0.30       2.0   0.4   2.32 ± 0.59   0.06 ± 0.05   0.00 ± 0.00   2.38 ± 0.64       2.0   0.8   1.55 ± 0.29   0.00 ± 0.00   0.01 ± 0.00   1.56 ± 0.29                    
Further, using the multiple sequence alignment of 6,671 tannase family sequences, conservation analysis was performed with MHETase sequence positions as a reference (see  FIGS.  4  and  5 A through  5 D ), which shows that most positions in the active site are highly conserved. Notable exceptions are positions  257 ,  411 ,  415 , and  416 , which exhibit low conservation scores and are less conserved than 80% of MHETase positions overall (see Panels B and C of  FIG.  4   ). It is noteworthy that position  131  is a well-conserved glycine in 91% o of tannase family sequences but serine appears at position  131  in MHETase. Furthermore, the ten cysteine positions in MHETase that form five disulfide bonds are highly conserved in the tannase family (see  FIGS.  6 A and  6 B ). Although a sixth disulfide bond exists in AoFaeB, less than 8% of tannase family sequences exhibit this sixth disulfide bond, and the sixth 68.91+/−8.66 Mutation of this lipase box residue to threonine (E226T) yielded a ˜50% reduction in MHET activity relative to the wild-type MHETase. Mutation of the catalytic serine (S225A), as expected, produced an inactive enzyme.  FIGS.  7 A through  7 J  illustrated other aspects according to some embodiments of the present disclosure.
 
     In an embodiment, a MHETase-8 amino acid linker-PETase chimeric enzyme was created having a DNA sequence of SEQ ID NO: 35 and an expressed polypeptide sequence of SEQ ID NO: 36. The expressed chimeric enzyme with an 8 aa linker (SEQ ID NO: 36) exhibited a turnover number of 68.91+/−8.66 −1 . In an embodiment, a MHETase-12 amino acid linker-PETase chimeric enzyme was created having a DNA sequence of SEQ ID NO: 37 and an expressed polypeptide sequence of SEQ ID NO: 38. The expressed chimeric enzyme with a 12 aa linker (SEQ ID NO: 38) exhibited a turnover number of 76.94+/−12.89 s −1 . In an embodiment, a MHETase-20 amino acid linker-PETase chimeric enzyme was created having a DNA sequence of SEQ ID NO: 39 and an expressed polypeptide sequence of SEQ ID NO: 40. The expressed chimeric enzyme with a 20 amino acid linker (SEQ ID NO: 40) exhibited a turnover number of 56.25+/−4.27 s −1 . 
     Methods: 
     Plasmid construction (see Table 2 for plasmid construction, Table 3 for synthesized DNA fragments and (where applicable) translated polypeptide sequences, and Table 3 for primers): pET-21b(+) (EMD Millipore)-based plasmids for expression of the various  Ideonella  sakaiensis PETase and MHETase enzymes, as well as homologous, and mutant proteins were either synthesized by Twist Bioscience or constructed using NEBuilder® HiFi DNA Assembly Master Mix (New England Biolabs) and/or the Q5® Site-Directed Mutagenesis Kit (New England Biolabs) such that each protein has a C-terminal hexa-histidine epitope tag. For DNA assembly, DNA fragments were either amplified using Q5® High-Fidelity 2X Master Mix (New England Biolabs) or synthesized by Integrated DNA Technologies. Kits and master mixes were used according to the manufacturer&#39;s instructions. Plasmids were initially transformed into NEB® 5-alpha F′Iq Competent  E. coli  (New England Biolabs) and confirmed using Sanger sequencing by GENEWIZ, Inc. 
     Protein expression and purification: For initial screening for soluble protein expression of the proteins of interest, various cell lines and induction methods were used to purify protein for kinetic assays. For expression and purification, OverExpress™  E. coli  C41 (DE3) (Lucigen) cells were transformed with pET21b(+) plasmid constructed with the gene of interest. Single colonies from transformation were then inoculated into a starter culture of Luria Broth (LB) media containing 100 μg/mL ampicillin and grown at 37° C. overnight. The starter culture was inoculated at a 100-fold dilution into a 2xYT medium containing 100 μg/mL ampicillin and grown at 37° C. until the optical density measured at 600 nM (OD600) reached between 0.6 and 0.8. Protein expression was then induced by addition of isopropyl β-D-1-thiogalactopyranoside (IPTG) to a final concentration of 1 mM. Cells were maintained at 20° C. for 18 to 24 hours following IPTG induction, harvested by centrifugation, and stored at −80° C. until purification. Harvested cells were resuspended in a lysis buffer (300 mM NaCl, 10 mM imidazole, 20 mM Tris HCl, pH 8.0) and lysed using a bead beater (BioSpec Products, Inc.). Lysate was clarified by centrifugation at 40,000×g for 45 minutes. Clarified lysate was then applied to a 5 mL HisTrap HP (GE Healthcare) Ni-NTA column using an ÄKTA Pure chromatography system (GE Healthcare) and eluted using 300 mM NaCl, 300 mM imidazole, 20 mM Tris HCl, pH 8.0. Resulting fractions containing proteins of interest were applied to a Sephacryl S-100 26/60 HR (GE Healthcare) size exclusion column equilibrated with 100 mM NaCl, 20 mM Tris HCl, pH 7.5 for biochemical assays, or the fractions were applied to a Superdex 75 pg 16/60 (GE Healthcare) size exclusion column equilibrated with 100 mM NaCl, 20 mM Tris HCl, pH 7.5 for crystallography. Protein in eluted fractions from Ni-NTA and size exclusion columns were assessed using SDS-PAGE with Coomassie staining and Western blot using primary antibody against the hexa-histidine epitope tag (Invitrogen). Total protein was assessed by BCA assay. For proteins that did not express, or expressed in inclusion bodies, using the above-described expression protocol, additional  E. coli  expression cell lines were tested, including Rosetta 2 (DE3) (Novagen), BL21 (DE3), and Lemo21 (DE3) (New England Biolabs), as was expression by autoinduction at 30° C. in ZYP-5052 media. 
     MHETase:PETase fusion proteins: Fusion proteins were expressed and purified as described above with the following noted exceptions: Single colonies from transformation into C41 (DE3) competent cells were used to inoculate a starter culture of 200 mL Terrific Broth (TB) media containing 100 μg/mL ampicillin for overnight outgrowth at 37° C. From the starter culture, 50 mL was used to inoculate 1 L of TB media containing 100 μg/mL ampicillin. For purification, cells were disrupted by sonication. In the final chromatography step a Superdex 200 pg 16/600 (GE Healthcare) size exclusion column equilibrated with 100 mM NaCl, 20 mM Tris HCl, pH 7.5 was used. 
     Crystallography. After purification, as described above, MHETase protein was concentrated to a range of concentrations (9-14 mg/mL) and dialyzed into 100 mM NaCl, 10 mM Tris, pH 7.0 for crystallography. 
     For seleno-methionine labeling of MHETase, K-MOPS minimal media was used. Cells were grown to an OD600 of 0.5 after which 100 mg/L of DL-seleno-methionine (Sigma), 100 mg/L lysine, threonine and phenylalanine, leucine, isoleucine and valine were added as solids. IPTG (1 mM final concentration) was then added after 20 min and cells were grown for a further 16 h at 20° C. Seleno-methionine labeled protein was purified as described above. MHETase was crystallized at a range of concentrations from 9-14 mg/mL by sitting-drop vapor diffusion. Several conditions yielded crystals, four of which were used to obtain datasets, one of which contained seleno-methionine labelled protein. The crystals were cryo-cooled in liquid nitrogen after the addition of glycerol to 20% (v/v) while leaving the other components of the mother liquor at the same concentration. Seleno-methionine MHETase crystals belonging to space group P22121 were used to obtain phase information using the 103 beamline at the Diamond Light Source (Oxford, UK). Data were obtained from 3600 images collected at 0.9795 A with 0.1° increments. All images were integrated using XDS (4) and scaled using SCALA. Phases were obtained using PHASERSAD in the CCP4i software in combination with PARROT and SHELXD. The initial output was subsequently built using BUCCANEER and further refined using iterative rounds of COOT and PHENIX. One molecule of MHETase was observed in the asymmetric unit of the P22121 seleno-methionine SAD dataset. Three additional native datasets, each containing 1800 images collected at 0.1° increments, were collected at beamline 103 of the Diamond Light Source. The structure of native MHETase were obtained using molecular replacement from a refined molecule of MHETase obtained initially from the seleno-methionine SAD data. All structures were refined using iterative rounds of COOT and PHENIX. 
     Determination of enzyme turnover rates. Comparative assays for each enzyme were performed at the same enzyme and substrate concentration. Reactions were performed in triplicate over a 15 min time course using 5 nM enzyme concentration and 250 μM MHET in 90 mM NaCl, 10% (v/v) DMSO, 45 mM sodium phosphate, pH 7.5, at 30° C. Reactions were terminated using an equal volume of 100% methanol followed by heat treatment at 85° C. for 10 min. Product and substrate were quantified by HPLC. Apparent turnover rate (kcat) was determined by terephthalic acid (TPA) produced. 
     Michaelis-Menten kinetics of MHETase and variants. Reactions were performed in triplicate over a 10 min time course using 5 nM enzyme and substrate concentrations ranging from 10 μM to 250 μM MHET in 90 mM NaCl, 10% (v/v) DMSO, 45 mM sodium phosphate, pH 7.5, at 30° C. Each reaction was terminated using an equal volume of 100% methanol and heat treatment at 85° C. for 10 min. Product and substrate were quantified by HPLC. Initial reaction velocities were calculated from TPA produced over time and kinetic parameters were determined by nonlinear regression of the initial velocities fit to the Michealis-Menten equation with substrate inhibition using GraphPad Prism version 8.4.1 for MacOS (GraphPad Software, San Diego, Calif. USA), as follows: 
     
       
         
           
             
               
                 
                   v 
                   = 
                   
                     
                       
                         V 
                         max 
                       
                       [ 
                       S 
                       ] 
                     
                     
                       
                         K 
                         m 
                       
                       + 
                       
                         
                           [ 
                           S 
                           ] 
                         
                         ⁢ 
                         
                           ( 
                           
                             1 
                             + 
                             
                               
                                 [ 
                                 S 
                                 ] 
                               
                               
                                 K 
                                 i 
                               
                             
                           
                           ) 
                         
                       
                     
                   
                 
               
               
                 
                   ( 
                   
                     Eq 
                     . 
                         
                     1 
                   
                   ) 
                 
               
             
           
         
       
     
     While both substrate inhibition and product inhibition are possible in these reactions, the relationship between initial reaction velocity and initial substrate concentration indicates substrate inhibition predominates in these reaction conditions. Low substrate concentrations were considered in these kinetic studies in order to minimize the effect of substrate inhibition. 
     Enzymatic degradation of PET film. Amorphous PET film (2-3% crystallinity, Goodfellow, UK) was incubated with enzyme of interest in polypropylene tubes containing 90 mM NaCl, 10% (v/v) DMSO, 45 mM sodium phosphate, pH 7.5, at 30° C. for 96 hours. The reaction was terminated by addition of equal volume 100% methanol and PET coupons were removed from the reaction solution. The reaction solution was heat treated at 85° C. for 10 minutes. PET coupons were washed with consecutive rinses of 1% SDS, 100% DMSO, ultrapure water, and 95% ethanol. Coupons were then vacuum dried for 24 h at 60° C. for scanning electron microscopy. 
     Activity assay of MHETase with non-MHET substrates. Evaluation of MHETase activity was performed in triplicate using 5 nM enzyme concentration and 25 μM, 50 μM, and 250 μM substrate concentration at 30° C. for 24 h in a 0.5 mL reaction volume. The reaction was carried out in 90 mM NaCl, 10% (v/v) DMSO, 45 mM sodium phosphate, pH 7.5, reaction buffer with three concentrations of each substrate (MHET, MHEI, or MHEF). Reactions commenced upon addition of enzyme or an equal volume of reaction buffer for the no enzyme controls. At the end of 24 h the reactions were terminated using an equal volume of 100% DMSO and heat treatment at 85° C. for 10 min. Product and substrate were analyzed by HPLC. Values reported as percentage of substrate hydrolyzed into product. 
     HPLC method. Standards of BHET, TPA, 2,5-furandicarboxylic acid, and isophthalate were obtained from Sigma Aldrich. MHET, MHEI, and MHEF were synthesized as described above. Analyte analysis of samples was performed on an Agilent 1260 LC system (Agilent Technologies, Santa Clara, Calif.) equipped with a G1315A diode array detector (DAD). Each sample and standard were injected using a volume of 10 μL onto a Phenomenex Luna C18(2) column, 5 μm, 4.6×150 mm (Phenomenex, Torrance, Calif.). The column temperature was maintained at 40° C. and the mobile phase used to separate the analytes of interest was composed of 20 mM phosphoric acid in water (A) and 100% methanol (B). The separation was carried out using a constant flow rate of 0.6 mL/min and a gradient program of: at t=0 min (A)=80% and (B)=20%; at t=15 min (A)=35% and (B)=65%; at t=15.01 min through 20 min (A) =80% and (B)=20% for a total run time of 20 min. The calibration curve for each analyte was evaluated between concentrations of 0.1-200 mg/L. DAD detection at a wavelength of 240 nm was performed for each analyte. Ten calibration standards were used with an r2 coefficient of 0.995 or better and a calibration verification standard (CVS) at 100 mg/L for each analyte was analyzed every 18 samples to ensure the integrity of the initial calibration. Samples were diluted with an equal volume of ultrapure water for analysis. 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Plasmid Construction. 
               
            
           
           
               
               
               
               
            
               
                 Protein 
                 Plasmid 
                 Plasmid description 
                 Construction details, reference, and other notes 
               
               
                   
               
               
                 PETase 
                 pCJ135 
                 pET-21b(+) based plasmid for expression 
                 Described previously in Austin, H. P., Allen, M. D., Donohoe, B. S., 
               
               
                   
                   
                 of PETase from  Ideonella sakaiensis  201- 
                 Rorrer, N. A., Kearns, F. L., Silveira, R. L., Pollard, B. C., Dominick, 
               
               
                   
                   
                 F6 (Genbank GAP38373.1), codon 
                 G., Duman, R., Omari, El, K., Mykhaylyk, V., Wagner, A., Michener, 
               
               
                   
                   
                 optimized for expression in  E. coli  K12, 
                 W. E., Amore, A., Skaf, M. S., Crowley, M. F., Thorne, A. W., 
               
               
                   
                   
                 with C-terminal His tag. Deposited to 
                 Johnson, C. W., Woodcock, H. L., McGeehan, J. E., Beckham, G. T., 
               
               
                   
                   
                 addgene as pET-21b(+)-Is-PETase 
                 2018. Characterization and engineering of a plastic-degrading 
               
               
                   
                   
                 (Plasmid 112202). 
                 aromatic polyesterase. Proc. Natl. Acad. Sci. U.S.A. 39, 
               
               
                   
                   
                   
                 201718804-8. 
               
               
                 MHETase 
                 pCJ136 
                 pET-21b(+) based plasmid for expression 
                 pCJ136 was constructed by assembling the DNA fragment 
               
               
                   
                   
                 of MHETase from  Ideonella sakaiensis   
                 CJ_MHETase_opt_Ec (synthesized by IDT), which omitted the 
               
               
                   
                   
                 201-F6 (Genbank GAP38911.1), codon 
                 stop codon to enable a C-terminal His tag, into pET-21b(+) 
               
               
                   
                   
                 optimized for expression in  E. coli  K12, 
                 digested with Ndel and Xhol. 
               
               
                   
                   
                 with C-terminal His tag. 
               
               
                 Lidded PETase 
                 pCJ208 
                 pET-21b(+) based plasmid for expression 
                 pCJ208 was constructed by assembling the DNA fragment 
               
               
                   
                   
                 of PETase from  Ideonella sakaiensis  201- 
                 CJ_MHETLid (synthesized by IDT), which omitted the stop codon 
               
               
                   
                   
                 F6 (Genbank GAP38373.1) incorporating 
                 to enable a C-terminal His tag, into pCJ135 digested with with 
               
               
                   
                   
                 the MHETase lid, codon optimized for 
                 Ncol and Agel. 
               
               
                   
                   
                 expression in  E. coli  K12, with C-terminal 
               
               
                   
                   
                 His tag. 
               
               
                 Lidless MHETase 
                 pCJ209 
                 pET-21b(+) based plasmid for expression 
                 pCJ209 was constructed by site-directed mutagenesis of pCJ136 
               
               
                   
                   
                 of MHETase from  Ideonella sakaiensis   
                 using NEB&#39;s Q5 ® Site-Directed Mutagenesis Kit according to the 
               
               
                   
                   
                 201-F6 (Genbank GAP38911.1) with the 
                 manufacturer&#39;s instructions. pCJ136 was amplified using primer 
               
               
                   
                   
                 lid removed, codon optimized for 
                 pair oCJ787/oCJ788, incorporating the lid replacement from 
               
               
                   
                   
                 expression in  E. coli  K12, with C-terminal 
                 PETase. The resulting PCR product was treated with NEB&#39;s 
               
               
                   
                   
                 His tag. 
                 Kinase, Ligase, and Dpnl (KLD) enzyme mix. 
               
               
                 MHETase 
                 pCJ205 
                 pET-21b(+) based plasmid for expression 
                 pCJ205 was constructed by site-directed mutagenesis of pCJ136 
               
               
                 C224A/C529A 
                   
                 of MHETase from  Ideonella sakaiensis   
                 using NEB&#39;s Q5 ® Site-Directed Mutagenesis Kit according to the 
               
               
                   
                   
                 201-F6 (Genbank GAP38911.1), codon 
                 manufacturer&#39;s instructions. pCJ136 was amplified using primer 
               
               
                   
                   
                 optimized for expression in  E. coli  K12, 
                 pair oCJ756/oCJ757 to generate a Cys224Ala mutation in the 
               
               
                   
                   
                 with C-terminal His tag, incorporating 
                 MHETase. The resulting PCR product was treated with NEB&#39;s 
               
               
                   
                   
                 C224A and C529A mutations. 
                 Kinase, Ligase, and Dpnl (KLD) enzyme mix. This plasmid was 
               
               
                   
                   
                   
                 used as template for amplification with primer pair 
               
               
                   
                   
                   
                 oCJ758/oCJ759 to generate a Cys529Ala mutation in the 
               
               
                   
                   
                   
                 MHETase gene and the resulting PCR product was treated with 
               
               
                   
                   
                   
                 NEB&#39;s Kinase, Ligase, and Dpnl (KLD) enzyme mix. 
               
               
                 MHETase 
                 pCJ201 
                 pET-21b(+) based plasmid for expression 
                 pCJ201 was constructed by site-directed mutagenesis of pCJ136 
               
               
                 C224W/C529S 
                   
                 of MHETase from  Ideonella sakaiensis   
                 using NEB&#39;s Q5 ® Site-Directed Mutagenesis Kit according to the 
               
               
                   
                   
                 201-F6 (Genbank GAP38911.1), codon 
                 manufacturer&#39;s instructions. pCJ136 was amplified using primer 
               
               
                   
                   
                 optimized for expression in  E. coli  K12, 
                 pair oCJ756/oCJ760 to generate a Cys224Trp mutation in the 
               
               
                   
                   
                 with C-terminal His tag, incorporating 
                 MHETase gene. The resulting PCR product was treated with 
               
               
                   
                   
                 C224W and C529SS mutations. 
                 NEB&#39;s Kinase, Ligase, and Dpnl (KLD) enzyme mix. This plasmid 
               
               
                   
                   
                   
                 was used as template for amplification with primer pair 
               
               
                   
                   
                   
                 oCJ758/oCJ761 to generate a Cys529Ser mutation in the 
               
               
                   
                   
                   
                 MHETase gene. The resulting PCR product was treated with 
               
               
                   
                   
                   
                 NEB&#39;s Kinase, Ligase, and Dpnl (KLD) enzyme mix. 
               
               
                 MHETase 
                 pCJ204 
                 pET-21b(+) based plasmid for expression 
                 pCJ204 was constructed by site-directed mutagenesis of pCJ136 
               
               
                 C224H/C529F 
                   
                 of MHETase from  Ideonella sakaiensis   
                 using NEB&#39;s Q5 ® Site-Directed Mutagenesis Kit according to the 
               
               
                   
                   
                 201-F6 (Genbank GAP38911.1), codon 
                 manufacturer&#39;s instructions. pCJ136 was amplified using primer 
               
               
                   
                   
                 optimized for expression in  E. coli  K12, 
                 pair oCJ756/oCJ762 to generate a Cys224His mutation in the 
               
               
                   
                   
                 with C-terminal His tag, incorporating 
                 MHETase gene. The resulting PCR product was treated with 
               
               
                   
                   
                 C224H and C529F mutations. 
                 NEB&#39;s Kinase, Ligase, and Dpnl (KLD) enzyme mix. This plasmid 
               
               
                   
                   
                   
                 was used as template for amplification with primer pair 
               
               
                   
                   
                   
                 oCJ758/oCJ763 to generate a Cys529Phe mutation in the 
               
               
                   
                   
                   
                 MHETase gene. The resulting PCR product was treated with 
               
               
                   
                   
                   
                 NEB&#39;s Kinase, Ligase, and Dpnl (KLD) enzyme mix. 
               
               
                 PETase 
                 pCJ202 
                 pET-21b(+) based plasmid for expression 
                 pCJ202 was constructed by site-directed mutagenesis of pCJ135 
               
               
                 W159C/S238C 
                   
                 of PETase from  Ideonella sakaiensis  201- 
                 using NEB&#39;s Q5 ® Site-Directed Mutagenesis Kit according to the 
               
               
                   
                   
                 F6 (Genbank GAP38373.1), codon 
                 manufacturer&#39;s instructions. pCJ135 was amplified using primer 
               
               
                   
                   
                 optimized for expression in  E. coli  K12, 
                 pair oCJ764/oCJ765 to generate a Trp159Cys mutation in the 
               
               
                   
                   
                 with C-terminal His tag, incorporating 
                 PETase gene. The resulting PCR product was treated with NEB&#39;s 
               
               
                   
                   
                 W159C and S238C mutations. 
                 Kinase, Ligase, and Dpnl (KLD) enzyme mix. This plasmid was 
               
               
                   
                   
                   
                 used as template for amplification with primer pair 
               
               
                   
                   
                   
                 oCJ766/oCJ767 to generate a Ser238Cys mutation in the PETase 
               
               
                   
                   
                   
                 gene. The resulting PCR product was treated with NEB&#39;s Kinase, 
               
               
                   
                   
                   
                 Ligase, and Dpnl (KLD) enzyme mix. 
               
               
                 MHETase S225A 
                 pCJ196 
                 pET-21b(+) based plasmid for expression 
                 pCJ196 was constructed by site-directed mutagenesis of pCJ136 
               
               
                   
                   
                 of MHETase from  Ideonella sakaiensis   
                 using NEB&#39;s Q5 ® Site-Directed Mutagenesis Kit according to the 
               
               
                   
                   
                 201-F6 (Genbank GAP38911.1), codon 
                 manufacturer&#39;s instructions. pCJ136 was amplified using primer 
               
               
                   
                   
                 optimized for expression in  E. coli  K12, 
                 pair oCJ756/oCJ757 to generate a Ser225Ala mutation. The 
               
               
                   
                   
                 with C-terminal His tag, incorporating 
                 resulting PCR product was treated with NEB&#39;s Kinase, Ligase, and 
               
               
                   
                   
                 catalytic mutation, S225A 
                 Dpnl (KLD) enzyme mix. 
               
               
                 
                   Comamonas 
                 
                 pCJ199 
                 pET-21b(+) based plasmid for expression 
                 pCJ199 was synthesized by Twist Bioscience. 
               
               
                 
                   thiooxydans 
                 
                   
                 of the putative MHETase from 
               
               
                   
                   
                   Comamonas thiooxydans  (Genbank 
               
               
                   
                   
                 WP_080747404.1) with signal peptide 
               
               
                   
                   
                 and C-terminal His tag, codon optimized 
               
               
                   
                   
                 for expression in  E. coli  K12. 
               
               
                 
                   Comamonas 
                 
                 pCJ203 
                 pET-21b(+) based plasmid for expression 
                 pCJ203 was constructed by removing the signal peptide from 
               
               
                   thiooxydans  with 
                   
                 of the putative MHETase from 
                 pCJ199 using NEB&#39;s Q5 ® Site-Directed Mutagenesis Kit according 
               
               
                 truncated signal 
                   
                   Comomonas thiooxydans  (Genbank 
                 to the manufacturer&#39;s instructions. pCJ199 was amplified with 
               
               
                 peptide (Δ75aa) 
                   
                 WP_080747404.1) without signal 
                 oCJ770/oCJ771 to exclude the 75-residue signal peptide. The 
               
               
                   
                   
                 peptide, with C-terminal His tag, codon 
                 resulting PCR product was treated with NEB&#39;s Kinase, Ligase, and 
               
               
                   
                   
                 optimized for expression in  E. coli  K12. 
                 Dpnl (KLD) enzyme mix. 
               
               
                 
                   Hydrogenophaga 
                 
                 pCJ207 
                 pET-21b(+) based plasmid for expression 
                 pCJ207 was synthesized by Twist Bioscience. 
               
               
                 sp. PML113 
                   
                 of the putative MHETase from 
               
               
                   
                   
                   Hydrogenophaga  sp. PML113 (Genbank 
               
               
                   
                   
                 WP_083293388.1) with signal peptide 
               
               
                   
                   
                 and C-terminal His tag, codon optimized 
               
               
                   
                   
                 for expression in  E. coli  K12. 
               
               
                 
                   Hydrogenophaga 
                 
                 pCJ211 
                 pET-21b(+) based plasmid for expression 
                 pCJ211 was constructed by removing the signal peptide from 
               
               
                 sp. PML113 with 
                   
                 of the putative MHETase from 
                 pCJ207 using NEB&#39;s Q5 ® Site-Directed Mutagenesis Kit according 
               
               
                 truncated signal 
                   
                   Hydrogenophaga  sp. PML113 (Genbank 
                 to the manufacturer&#39;s instructions. pCJ207 was amplified using 
               
               
                 peptide ((Δ19aa) 
                   
                 WP_083293388.1) without signal 
                 primer pair oCJ771/oCJ772 to exclude the 19-residue signal 
               
               
                   
                   
                 peptide, with C-terminal His tag, codon 
                 peptide. The resulting PCR product was treated with NEB&#39;s 
               
               
                   
                   
                 optimized for expression in  E. coli  K12. 
                 Kinase, Ligase, and Dpnl (KLD) enzyme mix. 
               
               
                 Lidless MHETase 
                 pCJ220 
                 pET-21b(+) based plasmid for expression 
                 pCJ220 was constructed by site-directed mutagenesis of pCJ201 
               
               
                 C224W/C529S 
                   
                 of MHETase from  Ideonella sakaiensis   
                 using NEB&#39;s Q5 ® Site-Directed Mutagenesis Kit according to the 
               
               
                   
                   
                 201-F6 (Genbank GAP38911.1), codon 
                 manufacturer&#39;s instructions. pCJ201 was amplified with primer 
               
               
                   
                   
                 optimized for expression in  E. coli  K12, 
                 pair oCJ788/oCJ787 on Jan. 24, 2019 to generate a lid replacement 
               
               
                   
                   
                 with C-terminal His tag, incorporating lid 
                 from PETase. The resulting PCR product was treated with NEB&#39;s 
               
               
                   
                   
                 deletion and C224W and C529S 
                 Kinase, Ligase, and Dpnl (KLD) enzyme mix. 
               
               
                   
                   
                 mutations from PETase active site. 
               
               
                 Lidless MHETase 
                 pCJ221 
                 pET-21b(+) based plasmid for expression 
                 pCJ221 was constructed by site-directed mutagenesis of pCJ204 
               
               
                 C224H/C549F 
                   
                 of MHETase from  Ideonella sakaiensis   
                 using NEB&#39;s Q5 ® Site-Directed Mutagenesis Kit according to the 
               
               
                   
                   
                 201-F6 (Genbank GAP38911.1), codon 
                 manufacturer&#39;s instructions. pCJ204 was amplified with primer 
               
               
                   
                   
                 optimized for expression in  E. coli  K12, 
                 pair oCJ788/oCJ787 on Jan. 24, 2019 to generate a lid replacement 
               
               
                   
                   
                 with C-terminal His tag, incorporating lid 
                 from wtPETase. The resulting PCR product was treated with 
               
               
                   
                   
                 deletion and C224H and C529F 
                 NEB&#39;s Kinase, Ligase, and Dpnl (KLD) enzyme mix. 
               
               
                   
                   
                 mutations from PETase active site. 
               
               
                 MHETase S131G 
                 pCJ197 
                 pET-21b(+) based plasmid for expression 
                 pCJ197 was constructed by site-directed mutagenesis of pCJ136 
               
               
                   
                   
                 of MHETase from  Ideonella sakaiensis   
                 using NEB&#39;s Q5 ® Site-Directed Mutagenesis Kit according to the 
               
               
                   
                   
                 201-F6 (Genbank GAP38911.1), codon 
                 manufacturer&#39;s instructions. pCJ136 was amplified using primer 
               
               
                   
                   
                 optimized for expression in  E. coli  K12, 
                 pair oCJ773/oCJ774 to generate a Ser131Gly mutation in the 
               
               
                   
                   
                 with C-terminal His tag, incorporating 
                 MHETase gene. The resulting PCR product was treated with 
               
               
                   
                   
                 S131G mutation. 
                 NEB&#39;s Kinase, Ligase, and Dpnl (KLD) enzyme mix. 
               
               
                 MHETase F495I 
                 pCJ198 
                 pET-21b(+) based plasmid for expression 
                 pCJ198 was constructed by site-directed mutagenesis of pCJ136 
               
               
                   
                   
                 of MHETase from  Ideonella sakaiensis   
                 using NEB&#39;s Q5 ® Site-Directed Mutagenesis Kit according to the 
               
               
                   
                   
                 201-F6 (Genbank GAP38911.1), codon 
                 manufacturer&#39;s instructions. pCJ136 was amplified using primer 
               
               
                   
                   
                 optimized for expression in  E. coli  K12, 
                 pair oCJ775/oCJ776 to generate a Phe495Ile mutation in the 
               
               
                   
                   
                 with C-terminal His tag, incorporating 
                 MHETase gene. The resulting PCR product was treated with 
               
               
                   
                   
                 F495I mutation. 
                 NEB&#39;s Kinase, Ligase, and Dpnl (KLD) enzyme mix. 
               
               
                 MHETase with 6 th   
                 pCJ200 
                 pET-21b(+) based plasmid for expression 
                 pCJ200 was synthesized by Twist Bioscience. 
               
               
                 Disulfide as 
                   
                 of MHETase from  Ideonella sakaiensis   
               
               
                 AoFaeB 
                   
                 201-F6 (Genbank GAP38911.1), codon 
               
               
                   
                   
                 optimized for expression in  E. coli  K12, 
               
               
                   
                   
                 with C-terminal His tag, incorporating 
               
               
                   
                   
                 mutations to introduce a 6th disulfide 
               
               
                   
                   
                 bond as in AoFaeB-2. 
               
               
                 MHETase E226T 
                 pCJ206 
                 pET-21b(+) based plasmid for expression 
                 pCJ206 was constructed by site-directed mutagenesis of pCJ136 
               
               
                   
                   
                 of MHETase from  Ideonella sakaiensis   
                 using NEB&#39;s Q5 ® Site-Directed Mutagenesis Kit according to the 
               
               
                   
                   
                 201-F6 (Genbank GAP38911.1), codon 
                 manufacturer&#39;s instructions. pCJ136 was amplified using primer 
               
               
                   
                   
                 optimized for expression in  E. coli  K12, 
                 pair oCJ777/oCJ778 to generate a Glu226Thr mutation in the 
               
               
                   
                   
                 with C-terminal His tag, incorporating 
                 MHETase gene. The resulting PCR product was treated with 
               
               
                   
                   
                 the E226T mutation to the putative 
                 NEB&#39;s Kinase, Ligase, and Dpnl (KLD) enzyme mix. 
               
               
                   
                   
                 lipase box. 
               
               
                 MHETase 
                 pCJ217 
                 pET-21b(+) based plasmid for expression 
                 pCJ217 was constructed by site-directed mutagenesis of pCJ136 
               
               
                 G489C/S530C 
                   
                 of MHETase from  Ideonella sakaiensis   
                 using NEB&#39;s Q5 ® Site-Directed Mutagenesis Kit according to the 
               
               
                   
                   
                 201-F6 (Genbank GAP38911.1), codon 
                 manufacturer&#39;s i]nstructions. pCJ136 was amplified using primer 
               
               
                   
                   
                 optimized for expression in  E. coli  K12, 
                 pair oCJ779/oCJ780 to generate Gly489Cys mutation in the 
               
               
                   
                   
                 with C-terminal His tag, incorporating 
                 MHETase gene. The resulting PCR product was treated with 
               
               
                   
                   
                 two point mutations, G489C and S530C, 
                 NEB&#39;s Kinase, Ligase, and Dpnl (KLD) enzyme mix. This plasmid 
               
               
                   
                   
                 to introduce a 6th disulfide bond (from 
                 was used as template for amplification with primer pair 
               
               
                   
                   
                 PETase). 
                 oCJ781/oCJ782 to generate a Ser530Cys mutation in the 
               
               
                   
                   
                   
                 MHETase gene. The resulting PCR product was treated with 
               
               
                   
                   
                   
                 NEB&#39;s Kinase, Ligase, and Dpnl (KLD) enzyme mix. 
               
               
                 MHETase with 6 th   
                 pCJ210 
                 pET-21b(+) based plasmid for expression 
                 pCJ210 was constructed by site-directed mutagenesis of pCJ200 
               
               
                 and 7 th  Disulfide 
                   
                 of MHETase from  Ideonella sakaiensis   
                 using NEB&#39;s Q5 ® Site-Directed Mutagenesis Kit according to the 
               
               
                 as AoFaeB 
                   
                 201-F6 (Genbank GAP38911.1), codon 
                 manufacturer&#39;s instructions. pCJ200 was amplified using primer 
               
               
                   
                   
                 optimized for expression in  E. coli  K12, 
                 pair oCJ779/oCJ780 to generate Gly489Cys mutation in the 
               
               
                   
                   
                 with C-terminal His tag, incorporating 
                 MHETase gene. The resulting PCR product was treated with 
               
               
                   
                   
                 mutations to introduce a 6th and 7th 
                 NEB&#39;s Kinase, Ligase, and Dpnl (KLD) enzyme mix. This plasmid 
               
               
                   
                   
                 disulfide bond as in AoFaeB-2. 
                 was used as template for amplification with primer pair 
               
               
                   
                   
                   
                 oCJ781/oCJ782 to generate a Ser530Cys mutation in the 
               
               
                   
                   
                   
                 MHETase gene. The resulting PCR product was treated with 
               
               
                   
                   
                   
                 NEB&#39;s Kinase, Ligase, and Dpnl (KLD) enzyme mix. 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Synthesized DNA Fragments. 
               
            
           
           
               
               
               
            
               
                 Fragment 
                 Sequence (5′-3′) 
                 Description 
               
               
                   
               
               
                 CJ_MHETase_ 
                 ctttaagaaggagatataCATATGcagaccaccgtgacca 
                 The MHETase from 
               
               
                 opt_Ec 
                 ccatgctcctcgcgtccgtagcattagcggcttgcgccgg 
                 
                   Ideonella sakaiensis 
                 
               
               
                 (SEQ ID NO: 1) 
                 aggaggttccactcctctgcctctaccgcagcagcagccg 
                 strain 201-F6 
               
               
                   
                 cctcagcaggaaccgccacctcctcctgttccgctagcca 
                 (Genbank GAP38911.1) 
               
               
                   
                 gtcgcgccgcgtgtgaggcgctcaaagatggtaatggcga 
                 was codon optimized for 
               
               
                   
                 catggtttggccgaatgccgccacggttgtagaggttgca 
                 expression in  E. coli   
               
               
                   
                 gcctggcgtgatgcagcaccggccacggcatcagccgcag 
                 K12 MG1655 (Highly 
               
               
                   
                 ccctgccggagcattgcgaagtatcaggcgcgattgccaa 
                 Expressed Genes) 
               
               
                   
                 gcgtactgggattgatgggtacccgtatgaaattaagttt 
                 using the codon 
               
               
                   
                 cgcctgcgcatgcccgctgagtggaacggccgttttttca 
                 optimizer at 
               
               
                   
                 tggagggtggcagtggtacgaacggctctctctcagcggc 
                 http://genomes.urv.es/ 
               
               
                   
                 gaccggaagtatcggcggcggtcagatcgcctcagcgctg 
                 OPTIMIZER/ (guided random 
               
               
                   
                 agtcgtaactttgcaacaattgctaccgacggaggacatg 
                 method) (CAI: 0.658, 
               
               
                   
                 acaatgcggtgaatgataatccggatgcgctcggtaccgt 
                 ENc: 52, %GC: 58.5). 
               
               
                   
                 cgcatttggtctcgatccccaggcacgcttagacatgggc 
                 The stop codon was 
               
               
                   
                 tacaactcctatgatcaggtgactcaggccggcaaagccg 
                 omitted and overlaps 
               
               
                   
                 ccgttgcacgcttttatggtcgcgcagccgacaagagcta 
                 were added for assembly 
               
               
                   
                 cttcatcggctgttcggagggcggccgcgagggcatgatg 
                 into the expression 
               
               
                   
                 ctgtcccagcgctttccatcacattacgatggcattgtgg 
                 vector pET-21b(+) 
               
               
                   
                 cgggcgcaccgggatatcagttgccgaaggccggaattag 
                 digested with NdeI and 
               
               
                   
                 tggcgcgtggaccacccagagcttagcgcccgccgccgtt 
                 XhoI such that the 
               
               
                   
                 ggcctggatgcccagggagtgccgctgattaataagagct 
                 assembly would results 
               
               
                   
                 tttctgacgcagacctccatttactgtcgcaggcgattct 
                 in the addition of a 
               
               
                   
                 cggaacatgcgacgccttggatggcctggccgacggcatc 
                 C-terminal 6 His tag. 
               
               
                   
                 gttgacaactaccgagcgtgccaagcggcttttgatccgg 
                 The 6 His sequence in 
               
               
                   
                 cgactgcagccaacccagcgaatggccaagccctgcagtg 
                 pET-21b(+) is the same 
               
               
                   
                 cgtgggcgcaaagacagccgattgcttatcgcccgtccaa 
                 codon (CAC) repeated 
               
               
                   
                 gttacggcgattaaacgagcgatggccggtccggtaaata 
                 6 times and so one of 
               
               
                   
                 gcgcgggtacgccgttatataatagatgggcctgggacgc 
                 the His codons in the 
               
               
                   
                 aggtatgagcggtcttagtggtaccacttacaatcagggt 
                 synthesized DNA 
               
               
                   
                 tggcgcagctggtggctgggatcgtttaacagctcggcga 
                 fragment was changed 
               
               
                   
                 ataacgcacaacgtgtatctggtttctcagcgcggagctg 
                 to CAT to enable 
               
               
                   
                 gctggtggactttgctaccccgccggagccgatgcccatg 
                 assembly. 
               
               
                   
                 acccaagtcgccgcccgtatgatgaaatttgatttcgata 
                   
               
               
                   
                 tcgatcctctgaaaatatgggctacttcgggccaatttac 
                   
               
               
                   
                 ccagagtagtatggactggcacggtgccactagcaccgac 
                   
               
               
                   
                 cttgctgcctttcgggaccgcggcggtaaaatgattctgt 
                   
               
               
                   
                 atcacggaatgagcgatgccgcattctctgcactagatac 
                   
               
               
                   
                 agcagattattatgaacgcctgggtgccgcaatgccgggc 
                   
               
               
                   
                 gccgcgggctttgctcgtctgttcttggttccgggaatga 
                   
               
               
                   
                 accattgctccgggggtccaggtaccgaccgctttgatat 
                   
               
               
                   
                 gctaacaccgttagttgcatgggttgaacgtggggaagcc 
                   
               
               
                   
                 cctgaccaaattagcgcctggagcggcacccccggctact 
                   
               
               
                   
                 ttggtgtggccgcccgcactcgaccgttatgtccctatcc 
                   
               
               
                   
                 gcagattgcgcgctataagggatcaggcgatatcaatacc 
                   
               
               
                   
                 gaagcaaattttgcgtgtgccgctccaccgCTCGAGcacc 
                   
               
               
                   
                 accatcaccaccactgagatccggct 
                   
               
               
                   
               
               
                 C_MHETase_Lid 
                 tccgcgcttagctagccatggctttgtggttattaccatc 
                 Lid region from 
               
               
                 (SEQ ID NO: 2) 
                 gatacgaacagcactctagaccagcccagcagccgtagct 
                 the  Ideonella   
               
               
                   
                 cgcaacagatggccgcgcttcgtcaagttgcgagcttgaa 
                 
                   sakaiensis 
                 
               
               
                   
                 cgggaccagcagtagcccgatttacggaaaggtcgatact 
                 strain 
               
               
                   
                 gcccgcatgggtgtgatgggctggtcaatggggggcggcg 
                 201-F6 (Genbank 
               
               
                   
                 gttcacttattagcgccgcgaacaacccgagtttaaaagc 
                 GAP38911.1) MHETase, 
               
               
                   
                 agcggcaccgcaggcgccaggatatcagttgccgaaggcc 
                 with 
               
               
                   
                 ggaattagtggcgcgtggaccacccagagcttagcgcccg 
                 overlaps for assembly 
               
               
                   
                 ccgccgttggcctggatgcccagggagtgccgctgattaa 
                 into pCJ135. Assembly 
               
               
                   
                 taagagcttttctgacgcagacctccatttactgtcgcag 
                 will generate PETase 
               
               
                   
                 gcgattctcggaacatgcgacgccttggatggcctggccg 
                 with the MHETase lid. 
               
               
                   
                 acggcatcgttgacaactaccgagcgtgccaagcggcttt 
                   
               
               
                   
                 tgatccggcgactgcagccaacccagcgaatggccaagcc 
                   
               
               
                   
                 ctgcagtgcgtgggcgcaaagacagccgattgcttatcgc 
                   
               
               
                   
                 ccgtccaagttacggcgattaaacgagcgatggccggtcc 
                   
               
               
                   
                 ggtaaatagcgcgggtacgccgttatataatagatgggcc 
                   
               
               
                   
                 tgggacgcaggtatgagcggtcttagtggtaccacttaca 
                   
               
               
                   
                 atcagggttggcgcagctggtggctgggatcgtttaacag 
                   
               
               
                   
                 ctcggcgaataacgcacaacgtgtatctggtttctcagcg 
                   
               
               
                   
                 cggagctggctggtggactttgctaccccgccggagccga 
                   
               
               
                   
                 tgcccatgacccaagtcgccgcccgtatgatgaaatttga 
                   
               
               
                   
                 tttcgatatcgatcctctgaaaatatgggctacttcgggc 
                   
               
               
                   
                 caatttacccagagtagtatggactggcacggtgccacta 
                   
               
               
                   
                 gcaccagcagtgttaccgtgccgacgctgattttcgcgtg 
                   
               
               
                   
                 cgagaatgatagcattgcaccggtgaacagcagcgcgct 
                   
               
               
                   
               
               
                 pCJ199 
                 CATATGttcgtacgcaacgccgaccgtgccaagaattgta 
                 pCJ199 was synthesized 
               
               
                 (SEQ ID NO: 3) 
                 tgcgcgcacctttacgcgtattcccactcaaggatacttt 
                 by Twist Bioscience. 
               
               
                   
                 tagcgcccagtgtgcgaatgtttcggtctggattaccagc 
                 Only the sequence 
               
               
                   
                 agcgtaccaccgctccgcgagcgtcacatggatcgccgcg 
                 integrated between the 
               
               
                   
                 tgacgcgccgcgatttaatgcaaactcgcatcttattaat 
                 NdeI and XhoI sites 
               
               
                   
                 gctcattgcagccactggcgtggcagcgtgtggcggagac 
                 in pET-21b(+) is shown. 
               
               
                   
                 ggtggttccacacctgccgcgcaaaatccccctttgcccc 
                   
               
               
                   
                 tggccagtcgtgcggcttgcgaagcttttcaaggcaatag 
                   
               
               
                   
                 caatagtatcgcgtggccccatcgcgcaaccgttgtggaa 
                   
               
               
                   
                 gtggccacttggcacgaagcagagcctgcgaatgccacag 
                   
               
               
                   
                 cagcggcgacgcccgagcactgtgagatttccggcgccat 
                   
               
               
                   
                 tgctcgccgcaccggaattgatggatatccttacgagatt 
                   
               
               
                   
                 aagtttcgcttacgtatgccctcagaatggaatggtcgct 
                   
               
               
                   
                 tctttatggaaggggggggtggaaccaatgggtcattgag 
                   
               
               
                   
                 tgccgctacagggtcccttggcggtggacaaactgcgtcg 
                   
               
               
                   
                 gccttgagtcgtaattttgcaactattgcaaccgatggtg 
                   
               
               
                   
                 gtcatgataatgctgtcaacaataatcctgatgcgctcgg 
                   
               
               
                   
                 cactgtcgcttttggcatggaccctcaagcgcgcattgat 
                   
               
               
                   
                 atgggatataattcctacgaccaggtgacccaagcgggaa 
                   
               
               
                   
                 aggcggccgtagcgcaattttatggccgtgccccggataa 
                   
               
               
                   
                 aagctattttattggctgtagcgaaggtgggcgcgagggg 
                   
               
               
                   
                 atgatgttgtcccaacgctttccgagtcattatgacggaa 
                   
               
               
                   
                 ttgtggcgggagcaccgggctaccaactcccaaaggcggg 
                   
               
               
                   
                 catttctggcgcatggacgacgcaaagtctggcaccagca 
                   
               
               
                   
                 gcggtgggcgtggatgctcaaaatgtacctttgatcaata 
                   
               
               
                   
                 aggcgttttcggatgtcgatttacatcttctttcacgcgg 
                   
               
               
                   
                 cattcttggtacttgcgatgccttggatggactcagcgat 
                   
               
               
                   
                 ggaattgtgaacgacttccgtgcctgtcaagccgcctttg 
                   
               
               
                   
                 accctgccactgcgttgaatcccgacaccagtcaaccctt 
                   
               
               
                   
                 acaatgcactggtgctaagacgcctgattgcttaagtgcc 
                   
               
               
                   
                 gcccaagtcactggcattaaacgcgccatgggtgggcctg 
                   
               
               
                   
                 tggacagcgccggtgcggcattatataatcgctgggcatg 
                   
               
               
                   
                 ggaccctggcatgtcggggctcaatggcacctcttacaat 
                   
               
               
                   
                 cagggatggcgctcttggtggttagggagctacgcatcca 
                   
               
               
                   
                 gcactaataatgcccaacgcgtcaatggcttttccgcccg 
                   
               
               
                   
                 ctcttggttggttgattttgcgacgccgcctgaaccaatg 
                   
               
               
                   
                 ccagtcacacaggttgctgctcgcatgatgaacttcaact 
                   
               
               
                   
                 ttgacaccgacccgcctaagattcgcgcgactagtggccc 
                   
               
               
                   
                 ctttactccatcgtctatggagtggcatggtgcaacgagc 
                   
               
               
                   
                 actaatcttgcggccttccgcgaccgcggtgggaagctga 
                   
               
               
                   
                 tgctctatcatggcatgtcagatgccgcgttctccgcatt 
                   
               
               
                   
                 agacaccgcggattactacgagcgtttaggtgccgcgatg 
                   
               
               
                   
                 ccgggcgccgccggcttcgcacgtcttttcttagttccag 
                   
               
               
                   
                 gtatgaatcattgtagtggtggacccggcactgatcgttt 
                   
               
               
                   
                 tgatatgcttactccccttgtggcctgggtggaacgtgat 
                   
               
               
                   
                 aaggcgccagatcaagttagcgcttgggcaggcacaccgg 
                   
               
               
                   
                 gctatttcggcgcaaccgcccgtacacgccccctttgtcc 
                   
               
               
                   
                 atacccccaaatcgcacgttataagggctctggtgatatc 
                   
               
               
                   
                 aatgccgaggcatcgtttgtgtgtgtggccccaCTCGAG 
                   
               
               
                   
               
               
                 pCJ200 
                 CATATGcagaccaccgtgaccaccatgctcctcgcgtccg 
                 pCJ200 was synthesized 
               
               
                 (SEQ ID NO: 4) 
                 tagcattagcggcttgcgccggaggaggttccactcctct 
                 by Twist Bioscience. 
               
               
                   
                 gcctctaccgcagcagcagccgcctcagcaggaaccgcca 
                 Only the sequence 
               
               
                   
                 cctcctcctgttccgctagccagtcgcgccgcgtgtgagg 
                 integrated between the 
               
               
                   
                 cgctcaaagatggtaatggcgacatggtttggccgaatgc 
                 NdeI and XhoI sites 
               
               
                   
                 cgccacggttgtagaggttgcagcctacgtgccggcaggc 
                 in pET-21b(+) is shown. 
               
               
                   
                 gttaacatcagcatggcggataacccgagcatctgtggtg 
                   
               
               
                   
                 gcgacgaggacccgattacttccaccttcgcgttctgcga 
                   
               
               
                   
                 agtatcaggcgcgattgccaagcgtactgggattgatggg 
                   
               
               
                   
                 tacccgtatgaaattaagtttcgcctgcgcatgcccgctg 
                   
               
               
                   
                 agtggaacggccgttttttcatggagggtggcagtggtac 
                   
               
               
                   
                 gaacggctgcctctcagcggcgaccggaagtatcggcggc 
                   
               
               
                   
                 ggtcagatcgcctcagcgctgagtcgtaactttgcaacaa 
                   
               
               
                   
                 ttgctaccgacggaggacatgacaatgcggtgaatgataa 
                   
               
               
                   
                 tccggatgcgctcggtaccgtcgcatttggtctcgatccc 
                   
               
               
                   
                 caggcacgcttagacatgggctacaactcctatgatcagg 
                   
               
               
                   
                 tgactcaggccggcaaagccgccgttgcacgcttttatgg 
                   
               
               
                   
                 tcgcgcagccgacaagagctacttcatcggctgttcggag 
                   
               
               
                   
                 ggcggccgcgagggcatgatgctgtcccagcgctttccat 
                   
               
               
                   
                 cacattacgatggcattgtggcgggcgcaccgggatatca 
                   
               
               
                   
                 gttgccgaaggccggaattagtggcgcgtggaccacccag 
                   
               
               
                   
                 agcttagcgcccgccgccgttggcctggatgcccagggag 
                   
               
               
                   
                 tgccgctgattaataagagcttttctgacgcagacctcca 
                   
               
               
                   
                 tttactgtcgcaggcgattctcggaacatgcgacgccttg 
                   
               
               
                   
                 gatggcctggccgacggcatcgttgacaactaccgagcgt 
                   
               
               
                   
                 gccaagcggcttttgatccggcgactgcagccaacccagc 
                   
               
               
                   
                 gaatggccaagccctgcagtgcgtgggcgcaaagacagcc 
                   
               
               
                   
                 gattgcttatcgcccgtccaagttacggcgattaaacgag 
                   
               
               
                   
                 cgatggccggtccggtaaatagcgcgggtacgccgttata 
                   
               
               
                   
                 taatagatgggcctgggacgcaggtatgagcggtcttagt 
                   
               
               
                   
                 ggtaccacttacaatcagggttggcgcagctggtggctgg 
                   
               
               
                   
                 gatcgtttaacagctcggcgaataacgcacaacgtgtatc 
                   
               
               
                   
                 tggtttctcagcgcggagctggctggtggactttgctacc 
                   
               
               
                   
                 ccgccggagccgatgcccatgacccaagtcgccgcccgta 
                   
               
               
                   
                 tgatgaaatttgatttcgatatcgatcctctgaaaatatg 
                   
               
               
                   
                 ggctacttcgggccaatttacccagagtagtatggactgg 
                   
               
               
                   
                 cacggtgccactagcaccgaccttgctgcctttcgggacc 
                   
               
               
                   
                 gcggcggtaaaatgattctgtatcacggaatgagcgatgc 
                   
               
               
                   
                 cgcattctctgcactagatacagcagattattatgaacgc 
                   
               
               
                   
                 ctgggtgccgcaatgccgggcgccgcgggctttgctcgtc 
                   
               
               
                   
                 tgttcttggttccgggaatgaaccattgctccgggggtcc 
                   
               
               
                   
                 aggtaccgaccgctttgatatgctaacaccgttagttgca 
                   
               
               
                   
                 tgggttgaacgtggggaagcccctgaccaaattagcgcct 
                   
               
               
                   
                 ggagcggcacccccggctactttggtgtggccgcccgcac 
                   
               
               
                   
                 tcgaccgttatgtccctatccgcagattgcgcgctataag 
                   
               
               
                   
                 ggatcaggcgatatcaataccgaagcaaattttgcgtgtg 
                   
               
               
                   
                 ccgctccaccgCTCGAG 
                   
               
               
                   
               
               
                 pCJ207 
                 CATATGaagtctagcattccgattagcgtaggtatgctgg 
                 pCJ207 was synthesized 
               
               
                 (SEQ ID NO: 5) 
                 ctaccgctctgatttccggttgcggtagcgttccggataa 
                 by Twist Bioscience. 
               
               
                   
                 caccagcaatgaaccgacggttccgctggcatccaaagaa 
                 Only the sequence 
               
               
                   
                 ttgtgcgagggcatcgcgtctggtgcgaccaaagtaaact 
                 integrated between 
               
               
                   
                 ggccgaaccagaacaccgtcgtaaaagcttcagtttggca 
                 the NdeI and XhoI 
               
               
                   
                 cgctgttaccccggcaaccgccaacgccccggaactgccg 
                 sites in pET-21b(+) 
               
               
                   
                 gaacattgcgaggtcactggctctatcaaccagcgtactg 
                 is shown. 
               
               
                   
                 gcgtggacggctatccgtatgaaatcaaaatgcgtctgcg 
                   
               
               
                   
                 catgccggcagattggaacggccgtttcttcatggaaggc 
                   
               
               
                   
                 ggtggaggtactaacgggagcctgtctgccgctctgggtt 
                   
               
               
                   
                 cgctgggcggtggtcagaccagcaatgctctgagccgtcg 
                   
               
               
                   
                 tttcgctaccgtttctaccgatggtggtcatgataacgca 
                   
               
               
                   
                 gtgaacaacaatccggcggcgctgggttcggtcgctttcg 
                   
               
               
                   
                 gcatggacccgcaggctcgcctggatcatggttacaactc 
                   
               
               
                   
                 atacgatcaggttaccctggcgggcaagtcagcagtaagc 
                   
               
               
                   
                 actttttacgggcgcggcccggacaaatcatacttcatcg 
                   
               
               
                   
                 gctgttccgaaggcggtcgtgagggcatgatgttcagcca 
                   
               
               
                   
                 gcgtttcccggcgcactatgacggcatcgtcgccggtgca 
                   
               
               
                   
                 ccgggctaccagctgccgaaagcaggcatcagcggtgcat 
                   
               
               
                   
                 ggacaacgcaatctctggcaccagcggccgttggtgttga 
                   
               
               
                   
                 cccggacggtgcaccgctggtgaacaaatccttcagcgat 
                   
               
               
                   
                 ccggacctgtatctgctgactcaggcaatcctgggcagct 
                   
               
               
                   
                 gcgacgctctggacggtctggctgacggtatcgtcggcaa 
                   
               
               
                   
                 ttattccgcgtgtcagtcgctgtttgacccgtctaccgcc 
                   
               
               
                   
                 ttgaaccctgcgacgggacaaccactgcgttgcacgggcg 
                   
               
               
                   
                 ctaagaccgacgactgcctaagcccggttcaggtggatgc 
                   
               
               
                   
                 gatcaaacgtgctatgtccggtccagttgatactgccgga 
                   
               
               
                   
                 accgccctgtataacaaatggccgtgggataccggtatgt 
                   
               
               
                   
                 cgggcctgaacggcaccacttatttccagggctggcgcag 
                   
               
               
                   
                 ctggtggctgggctcctacgacagctctactaacaacgcg 
                   
               
               
                   
                 cagcgtgttaacggcagcagcgcacgctcttggctggtag 
                   
               
               
                   
                 atttcgctactccgccggaacctgtaccgctgaaccaggt 
                   
               
               
                   
                 ggccactcgtatgatgaactttgattttgatgttgacccg 
                   
               
               
                   
                 ccgaaaatctttgctacctctggtctctttacccagccgt 
                   
               
               
                   
                 ccatgcaatggcacggtgccacctcaaccgatctgaacgc 
                   
               
               
                   
                 ttttcgctctcgcggtggcaagctgatgctgtaccacggc 
                   
               
               
                   
                 atggctgacgcggcattcagcgcactggataccattgctt 
                   
               
               
                   
                 attatgagcgcctgagcaccgcaatgccttccgtgtccga 
                   
               
               
                   
                 cttttctcgcctgtttctggtgcctggtatggggcactgt 
                   
               
               
                   
                 tccggcggtccgggcaccgatcgctttgatatgctgactc 
                   
               
               
                   
                 cgctggtggcgtgggttgagaacggtactgcaccggctcg 
                   
               
               
                   
                 cgtcgaagcgtcgtcctccactccgggttacttcggtgtt 
                   
               
               
                   
                 tcggcccgcagccgccccctgtgcccgcatccgcagattg 
                   
               
               
                   
                 cacgttataccgggtccggcgacattaacgaagccaccaa 
                   
               
               
                   
                 ctttgtatgcggtaacccgCTCGAG 
                   
               
               
                   
               
               
                 MP8 DNA 
                 atgcagaccaccgtgaccaccatgctcctcgcgtccgtag 
                 DNA sequence for 
               
               
                 (SEQ ID NO: 35) 
                 cattagcggcttgcgccggaggaggttccactcctctgcc 
                 MHETase-8 aa linker 
               
               
                   
                 tctaccgcagcagcagccgcctcagcaggaaccgccacct 
                 PETase 
               
               
                   
                 cctcctgttccgctagccagtcgcgccgcgtgtgaggcgc 
                   
               
               
                   
                 tcaaagatggtaatggcgacatggtttggccgaatgccgc 
                   
               
               
                   
                 cacggttgtagaggttgcagcctggcgtgatgcagcaccg 
                   
               
               
                   
                 gccacggcatcagccgcagccctgccggagcattgcgaag 
                   
               
               
                   
                 tatcaggcgcgattgccaagcgtactgggattgatgggta 
                   
               
               
                   
                 cccgtatgaaattaagtttcgcctgcgcatgcccgctgag 
                   
               
               
                   
                 tggaacggccgttttttcatggagggtggcagtggtacga 
                   
               
               
                   
                 acggctctctctcagcggcgaccggaagtatcggcggcgg 
                   
               
               
                   
                 tcagatcgcctcagcgctgagtcgtaactttgcaacaatt 
                   
               
               
                   
                 gctaccgacggaggacatgacaatgcggtgaatgataatc 
                   
               
               
                   
                 cggatgcgctcggtaccgtcgcatttggtctcgatcccca 
                   
               
               
                   
                 ggcacgcttagacatgggctacaactcctatgatcaggtg 
                   
               
               
                   
                 actcaggccggcaaagccgccgttgcacgcttttatggtc 
                   
               
               
                   
                 gcgcagccgacaagagctacttcatcggctgttcggaggg 
                   
               
               
                   
                 cggccgcgagggcatgatgctgtcccagcgctttccatca 
                   
               
               
                   
                 cattacgatggcattgtggcgggcgcaccgggatatcagt 
                   
               
               
                   
                 tgccgaaggccggaattagtggcgcgtggaccacccagag 
                   
               
               
                   
                 cttagcgcccgccgccgttggcctggatgcccagggagtg 
                   
               
               
                   
                 ccgctgattaataagagcttttctgacgcagacctccatt 
                   
               
               
                   
                 tactgtcgcaggcgattctcggaacatgcgacgccttgga 
                   
               
               
                   
                 tggcctggccgacggcatcgttgacaactaccgagcgtgc 
                   
               
               
                   
                 caagcggcttttgatccggcgactgcagccaacccagcga 
                   
               
               
                   
                 atggccaagccctgcagtgcgtgggcgcaaagacagccga 
                   
               
               
                   
                 ttgcttatcgcccgtccaagttacggcgattaaacgagcg 
                   
               
               
                   
                 atggccggtccggtaaatagcgcgggtacgccgttatata 
                   
               
               
                   
                 atagatgggcctgggacgcaggtatgagcggtcttagtgg 
                   
               
               
                   
                 taccacttacaatcagggttggcgcagctggtggctggga 
                   
               
               
                   
                 tcgtttaacagctcggcgaataacgcacaacgtgtatctg 
                   
               
               
                   
                 gtttctcagcgcggagctggctggtggactttgctacccc 
                   
               
               
                   
                 gccggagccgatgcccatgacccaagtcgccgcccgtatg 
                   
               
               
                   
                 atgaaatttgatttcgatatcgatcctctgaaaatatggg 
                   
               
               
                   
                 ctacttcgggccaatttacccagagtagtatggactggca 
                   
               
               
                   
                 cggtgccactagcaccgaccttgctgcctttcgggaccgc 
                   
               
               
                   
                 ggcggtaaaatgattctgtatcacggaatgagcgatgccg 
                   
               
               
                   
                 cattctctgcactagatacagcagattattatgaacgcct 
                   
               
               
                   
                 gggtgccgcaatgccgggcgccgcgggctttgctcgtctg 
                   
               
               
                   
                 ttcttggttccgggaatgaaccattgctccgggggtccag 
                   
               
               
                   
                 gtaccgaccgctttgatatgctaacaccgttagttgcatg 
                   
               
               
                   
                 ggttgaacgtggggaagcccctgaccaaattagcgcctgg 
                   
               
               
                   
                 agcggcacccccggctactttggtgtggccgcccgcactc 
                   
               
               
                   
                 gaccgttatgtccctatccgcagattgcgcgctataaggg 
                   
               
               
                   
                 atcaggcgatatcaataccgaagcaaattttgcgtgtgcc 
                   
               
               
                   
                 gctccaccgggtggtggttctggtggttctggtcagacca 
                   
               
               
                   
                 atccgtatgcgcgcggccccaaccctaccgccgcctcgtt 
                   
               
               
                   
                 ggaagccagcgcgggaccctttaccgttcgtagctttacc 
                   
               
               
                   
                 gttagccgtccgtccggatatggtgcagggaccgtctatt 
                   
               
               
                   
                 acccaaccaatgcaggcggcaccgttggcgcgattgcaat 
                   
               
               
                   
                 cgtccccgggtacaccgcgcgtcaaagcagcattaagtgg 
                   
               
               
                   
                 tggggtccgcgcttagctagccatggctttgtggttatta 
                   
               
               
                   
                 ccatcgatacgaacagcactctagaccagcccagcagccg 
                   
               
               
                   
                 tagctcgcaacagatggccgcgcttcgtcaagttgcgagc 
                   
               
               
                   
                 ttgaacgggaccagcagtagcccgatttacggaaaggtcg 
                   
               
               
                   
                 atactgcccgcatgggtgtgatgggctggtcaatgggggg 
                   
               
               
                   
                 cggcggttcacttattagcgccgcgaacaacccgagttta 
                   
               
               
                   
                 aaagcagcggcaccgcaggcgccatgggactcttcaacca 
                   
               
               
                   
                 acttcagcagtgttaccgtgccgacgctgattttcgcgtg 
                   
               
               
                   
                 cgagaatgatagcattgcaccggtgaacagcagcgcgctg 
                   
               
               
                   
                 ccgatttatgatagcatgtcccgcaacgcaaaacagtttc 
                   
               
               
                   
                 tggaaattaacggcggtagccactcttgtgccaactctgg 
                   
               
               
                   
                 gaacagcaaccaggcactgatcggaaaaaaaggggttgca 
                   
               
               
                   
                 tggatgaaacgattcatggataatgacacccgttactcaa 
                   
               
               
                   
                 ccttcgcctgtgagaatcccaacagcacacgcgtgtcgga 
                   
               
               
                   
                 ttttcgcaccgcgaactgttccctcgagcaccaccatcac 
                   
               
               
                   
                 caccactga 
                   
               
               
                   
               
               
                 MP8 aa 
                 MQTTVTTMLLASVALAACAGGGSTPLPLPQQQPPQQEPPP 
                 Amino acid sequence 
               
               
                 (SEQ ID NO: 36) 
                 PPVPLASRAACEALKDGNGDMVWPNAATVVEVAAWRDAAP 
                 for MHETase- 
               
               
                   
                 ATASAAALPEHCEVSGAIAKRTGIDGYPYEIKFRRMPAEW 
                 8 aa linker-PETase 
               
               
                   
                 NGRFFMEGGSGTNGSLSAATGSIGGGQIASALSRNFATIA 
                   
               
               
                   
                 TDGGHDNAVNDNPDALGTVAFGLDPQARLDMGYNSYDQVT 
                   
               
               
                   
                 QAGKAAVARFYGRAADKSYFIGCSEGGREGMMLSQRFPSH 
                   
               
               
                   
                 YDGIVAGAPGYQLPKAGISGAWTTQSLAPAAVGLDAQGVP 
                   
               
               
                   
                 LINKSFSDADLHLLSQAILGTCDALDGLADGIVDNYRACQ 
                   
               
               
                   
                 AAFDPATAANPANGQALQCVGAKTADCLSPVQVTAIKRAM 
                   
               
               
                   
                 AGPVNSAGTPLYNRWAWDAGMSGLSGTTYNQGWRSWWLGS 
                   
               
               
                   
                 FNSSANNAQRVSGFSARSWLVDFATPPEPMPMTQVAARMM 
                   
               
               
                   
                 KFDFDIDPLKIWATSGQFTQSSMDWHGATSTDLAAFRDRG 
                   
               
               
                   
                 GKMILYHGMSDAAFSALDTADYYERLGAAMPGAAGFARLF 
                   
               
               
                   
                 LVPGMNHCSGGPGTDRFDMLTPLVAWVERGEAPDQISAWS 
                   
               
               
                   
                 GTPGYFGVAARTRPLCPYPQJARYKGSGDINTEANFACAA 
                   
               
               
                   
                 PPGGGSGGSGQTNPYARGPNPTAASLEASAGPFTVRSFTV 
                   
               
               
                   
                 SRPSGYGAGTVYYPTNAGGTVGAIAIVPGYTARQSSIKWW 
                   
               
               
                   
                 GPRLASHGFVVITIDTNSTLDQPSSRSSQQMAALRaVASL 
                   
               
               
                   
                 NGTSSSPIYGKVDTARMGVMGWSMGGGGSLISAANNPSLK 
                   
               
               
                   
                 AAAPQAPWDSSTNFSSVTVPTLIFACENDSIAPVNSSALP 
                   
               
               
                   
                 IYDSMSRNAKQFLEINGGSHSCANSGNSNQALIGKKGVAW 
                   
               
               
                   
                 MKRFMDNDTRYSTFACENPNSTRVSDFRTANCSLEHHHHH 
                   
               
               
                   
                 H 
                   
               
               
                   
               
               
                 MP12 DNA 
                 atgcagaccaccgtgaccaccatgctcctcgcgtccgtag 
                 DNA sequence 
               
               
                 (SEQ ID NO: 37) 
                 cattagcggcttgcgccggaggaggttccactcctctgcc 
                 for MHETase- 
               
               
                   
                 tctaccgcagcagcagccgcctcagcaggaaccgccacct 
                 12 aa linker- 
               
               
                   
                 cctcctgttccgctagccagtcgcgccgcgtgtgaggcgc 
                 PETase 
               
               
                   
                 tcaaagatggtaatggcgacatggtttggccgaatgccgc 
                   
               
               
                   
                 cacggttgtagaggttgcagcctggcgtgatgcagcaccg 
                   
               
               
                   
                 gccacggcatcagccgcagccctgccggagcattgcgaag 
                   
               
               
                   
                 tatcaggcgcgattgccaagcgtactgggattgatgggta 
                   
               
               
                   
                 cccgtatgaaattaagtttcgcctgcgcatgcccgctgag 
                   
               
               
                   
                 tggaacggccgttttttcatggagggtggcagtggtacga 
                   
               
               
                   
                 acggctctctctcagcggcgaccggaagtatcggcggcgg 
                   
               
               
                   
                 tcagatcgcctcagcgctgagtcgtaactttgcaacaatt 
                   
               
               
                   
                 gctaccgacggaggacatgacaatgcggtgaatgataatc 
                   
               
               
                   
                 cggatgcgctcggtaccgtcgcatttggtctcgatcccca 
                   
               
               
                   
                 ggcacgcttagacatgggctacaactcctatgatcaggtg 
                   
               
               
                   
                 actcaggccggcaaagccgccgttgcacgcttttatggtc 
                   
               
               
                   
                 gcgcagccgacaagagctacttcatcggctgttcggaggg 
                   
               
               
                   
                 cggccgcgagggcatgatgctgtcccagcgctttccatca 
                   
               
               
                   
                 cattacgatggcattgtggcgggcgcaccgggatatcagt 
                   
               
               
                   
                 tgccgaaggccggaattagtggcgcgtggaccacccagag 
                   
               
               
                   
                 cttagcgcccgccgccgttggcctggatgcccagggagtg 
                   
               
               
                   
                 ccgctgattaataagagcttttctgacgcagacctccatt 
                   
               
               
                   
                 tactgtcgcaggcgattctcggaacatgcgacgccttgga 
                   
               
               
                   
                 tggcctggccgacggcatcgttgacaactaccgagcgtgc 
                   
               
               
                   
                 caagcggcttttgatccggcgactgcagccaacccagcga 
                   
               
               
                   
                 atggccaagccctgcagtgcgtgggcgcaaagacagccga 
                   
               
               
                   
                 ttgcttatcgcccgtccaagttacggcgattaaacgagcg 
                   
               
               
                   
                 atggccggtccggtaaatagcgcgggtacgccgttatata 
                   
               
               
                   
                 atagatgggcctgggacgcaggtatgagcggtcttagtgg 
                   
               
               
                   
                 taccacttacaatcagggttggcgcagctggtggctggga 
                   
               
               
                   
                 tcgtttaacagctcggcgaataacgcacaacgtgtatctg 
                   
               
               
                   
                 gtttctcagcgcggagctggctggtggactttgctacccc 
                   
               
               
                   
                 gccggagccgatgcccatgacccaagtcgccgcccgtatg 
                   
               
               
                   
                 atgaaatttgatttcgatatcgatcctctgaaaatatggg 
                   
               
               
                   
                 ctacttcgggccaatttacccagagtagtatggactggca 
                   
               
               
                   
                 cggtgccactagcaccgaccttgctgcctttcgggaccgc 
                   
               
               
                   
                 ggcggtaaaatgattctgtatcacggaatgagcgatgccg 
                   
               
               
                   
                 cattctctgcactagatacagcagattattatgaacgcct 
                   
               
               
                   
                 gggtgccgcaatgccgggcgccgcgggctttgctcgtctg 
                   
               
               
                   
                 ttcttggttccgggaatgaaccattgctccgggggtccag 
                   
               
               
                   
                 gtaccgaccgctttgatatgctaacaccgttagttgcatg 
                   
               
               
                   
                 ggttgaacgtggggaagcccctgaccaaattagcgcctgg 
                   
               
               
                   
                 agcggcacccccggctactttggtgtggccgcccgcactc 
                   
               
               
                   
                 gaccgttatgtccctatccgcagattgcgcgctataaggg 
                   
               
               
                   
                 atcaggcgatatcaataccgaagcaaattttgcgtgtgcc 
                   
               
               
                   
                 gctccaccgggtggtggttctggtggttctggtggtggtt 
                   
               
               
                   
                 ctggtcagaccaatccgtatgcgcgcggccccaaccctac 
                   
               
               
                   
                 cgccgcctcgttggaagccagcgcgggaccctttaccgtt 
                   
               
               
                   
                 cgtagctttaccgttagccgtccgtccggatatggtgcag 
                   
               
               
                   
                 ggaccgtctattacccaaccaatgcaggcggcaccgttgg 
                   
               
               
                   
                 cgcgattgcaatcgtccccgggtacaccgcgcgtcaaagc 
                   
               
               
                   
                 agcattaagtggtggggtccgcgcttagctagccatggct 
                   
               
               
                   
                 ttgtggttattaccatcgatacgaacagcactctagacca 
                   
               
               
                   
                 gcccagcagccgtagctcgcaacagatggccgcgcttcgt 
                   
               
               
                   
                 caagttgcgagcttgaacgggaccagcagtagcccgattt 
                   
               
               
                   
                 acggaaaggtcgatactgcccgcatgggtgtgatgggctg 
                   
               
               
                   
                 gtcaatggggggcggcggttcacttattagcgccgcgaac 
                   
               
               
                   
                 aacccgagtttaaaagcagcggcaccgcaggcgccatggg 
                   
               
               
                   
                 actcttcaaccaacttcagcagtgttaccgtgccgacgct 
                   
               
               
                   
                 gattttcgcgtgcgagaatgatagcattgcaccggtgaac 
                   
               
               
                   
                 agcagcgcgctgccgatttatgatagcatgtcccgcaacg 
                   
               
               
                   
                 caaaacagtttctggaaattaacggcggtagccactcttg 
                   
               
               
                   
                 tgccaactctgggaacagcaaccaggcactgatcggaaaa 
                   
               
               
                   
                 aaaggggttgcatggatgaaacgattcatggataatgaca 
                   
               
               
                   
                 cccgttactcaaccttcgcctgtgagaatcccaacagcac 
                   
               
               
                   
                 acgcgtgtcggattttcgcaccgcgaactgttccctcgag 
                   
               
               
                   
                 caccaccatcaccaccactga 
                   
               
               
                   
               
               
                 MP12 aa 
                 MQTTVTTMLLASVALAACAGGGSTPLPLPQQQPPQQEPPP 
                 Amino Acid sequence 
               
               
                 (SEQ ID NO: 38) 
                 PPVPLASRAACEALKDGNGDMVWPNAATVVEVAAWRDAAP 
                 for MHETase-12 aa 
               
               
                   
                 ATASAAALPEHCEVSGAIAKRTGIDGYPYEIKFRLRMPAE 
                 linker-PETase 
               
               
                   
                 WNGRFFMEGGSGTNGSLSAATGSIGGGQIASALSRNFATI 
                   
               
               
                   
                 ATDGGHDNAVNDNPDALGTVAFGLDPQARLDMGYNSYDQV 
                   
               
               
                   
                 TQAGKAAVARFYGRAADKSYFIGCSEGGREGMMLSQRFPS 
                   
               
               
                   
                 HYDGIVAGAPGYQLPKAGISGAWTTQSLAPAAVGLDAQGV 
                   
               
               
                   
                 PLINKSFSDADLHLLSQAILGTCDALDGLADGIVDNYRAC 
                   
               
               
                   
                 QAAFDPATAANPANGQALQCVGAKTADCLSPVQVTAIKRA 
                   
               
               
                   
                 MAGPVNSAGTPLYNRWAWDAGMSGLSGTTYNQGWRSWWLG 
                   
               
               
                   
                 SFNSSANNAQRVSGFSARSWLVDFATPPEPMPMTQVAARM 
                   
               
               
                   
                 MKFDFDIDPLKIWATSGQFTQSSMDWHGATSTDLAAFRDR 
                   
               
               
                   
                 GGKMILYHGMSDAAFSALDTADYYERLGAAMPGAAGFARL 
                   
               
               
                   
                 FLVPGMNHCSGGPGTDRFDMLTPLVAWVERGEAPDQISAW 
                   
               
               
                   
                 SGTPGYFGVAARTRPLCPYPQIARYKGSGDINTEANFACA 
                   
               
               
                   
                 APPGGGSGGSGGGSGQTNPYARGPNPTAASLEASAGPFTV 
                   
               
               
                   
                 RSFTVSRPSGYGAGTVYYPTNAGGTVGAIAIVPGYTARQS 
                   
               
               
                   
                 SIKWWGPRLASHGFWITIDTNSTLDQPSSRSSQQMAALRQ 
                   
               
               
                   
                 VASLNGTSSSPIYGKVDTARMGVMGWSMGGGGSLISAANN 
                   
               
               
                   
                 PSLKAAAPQAPWDSSTNFSSVTVPTLIFACENDSIAPVNS 
                   
               
               
                   
                 SALPIYDSMSRNAKQFLEINGGSHSCANSGNSNQALIGKK 
                   
               
               
                   
                 GVAWMKRFMDNDTRYSTFACENPNSTRVSDFRTANCSLEH 
                   
               
               
                   
                 HHHHH 
                   
               
               
                   
               
               
                 MP20 DNA 
                 atgcagaccaccgtgaccaccatgctcctcgcgtccgtag 
                 DNA sequence 
               
               
                 (SEQ ID NO: 39) 
                 cattagcggcttgcgccggaggaggttccactcctctgcc 
                 for MHETase- 
               
               
                   
                 tctaccgcagcagcagccgcctcagcaggaaccgccacct 
                 20 aa linker- 
               
               
                   
                 cctcctgttccgctagccagtcgcgccgcgtgtgaggcgc 
                 PETase 
               
               
                   
                 tcaaagatggtaatggcgacatggtttggccgaatgccgc 
                   
               
               
                   
                 cacggttgtagaggttgcagcctggcgtgatgcagcaccg 
                   
               
               
                   
                 gccacggcatcagccgcagccctgccggagcattgcgaag 
                   
               
               
                   
                 tatcaggcgcgattgccaagcgtactgggattgatgggta 
                   
               
               
                   
                 cccgtatgaaattaagtttcgcctgcgcatgcccgctgag 
                   
               
               
                   
                 tggaacggccgttttttcatggagggtggcagtggtacga 
                   
               
               
                   
                 acggctctctctcagcggcgaccggaagtatcggcggcgg 
                   
               
               
                   
                 tcagatcgcctcagcgctgagtcgtaactttgcaacaatt 
                   
               
               
                   
                 gctaccgacggaggacatgacaatgcggtgaatgataatc 
                   
               
               
                   
                 cggatgcgctcggtaccgtcgcatttggtctcgatcccca 
                   
               
               
                   
                 ggcacgcttagacatgggctacaactcctatgatcaggtg 
                   
               
               
                   
                 actcaggccggcaaagccgccgttgcacgcttttatggtc 
                   
               
               
                   
                 gcgcagccgacaagagctacttcatcggctgttcggaggg 
                   
               
               
                   
                 cggccgcgagggcatgatgctgtcccagcgctttccatca 
                   
               
               
                   
                 cattacgatggcattgtggcgggcgcaccgggatatcagt 
                   
               
               
                   
                 tgccgaaggccggaattagtggcgcgtggaccacccagag 
                   
               
               
                   
                 cttagcgcccgccgccgttggcctggatgcccagggagtg 
                   
               
               
                   
                 ccgctgattaataagagcttttctgacgcagacctccatt 
                   
               
               
                   
                 tactgtcgcaggcgattctcggaacatgcgacgccttgga 
                   
               
               
                   
                 tggcctggccgacggcatcgttgacaactaccgagcgtgc 
                   
               
               
                   
                 caagcggcttttgatccggcgactgcagccaacccagcga 
                   
               
               
                   
                 atggccaagccctgcagtgcgtgggcgcaaagacagccga 
                   
               
               
                   
                 ttgcttatcgcccgtccaagttacggcgattaaacgagcg 
                   
               
               
                   
                 atggccggtccggtaaatagcgcgggtacgccgttatata 
                   
               
               
                   
                 atagatgggcctgggacgcaggtatgagcggtcttagtgg 
                   
               
               
                   
                 taccacttacaatcagggttggcgcagctggtggctggga 
                   
               
               
                   
                 tcgtttaacagctcggcgaataacgcacaacgtgtatctg 
                   
               
               
                   
                 gtttctcagcgcggagctggctggtggactttgctacccc 
                   
               
               
                   
                 gccggagccgatgcccatgacccaagtcgccgcccgtatg 
                   
               
               
                   
                 atgaaatttgatttcgatatcgatcctctgaaaatatggg 
                   
               
               
                   
                 ctacttcgggccaatttacccagagtagtatggactggca 
                   
               
               
                   
                 cggtgccactagcaccgaccttgctgcctttcgggaccgc 
                   
               
               
                   
                 ggcggtaaaatgattctgtatcacggaatgagcgatgccg 
                   
               
               
                   
                 cattctctgcactagatacagcagattattatgaacgcct 
                   
               
               
                   
                 gggtgccgcaatgccgggcgccgcgggctttgctcgtctg 
                   
               
               
                   
                 ttcttggttccgggaatgaaccattgctccgggggtccag 
                   
               
               
                   
                 gtaccgaccgctttgatatgctaacaccgttagttgcatg 
                   
               
               
                   
                 ggttgaacgtggggaagcccctgaccaaattagcgcctgg 
                   
               
               
                   
                 agcggcacccccggctactttggtgtggccgcccgcactc 
                   
               
               
                   
                 gaccgttatgtccctatccgcagattgcgcgctataaggg 
                   
               
               
                   
                 atcaggcgatatcaataccgaagcaaattttgcgtgtgcc 
                   
               
               
                   
                 gctccaccgggtggtggttctggtggttctggtggtggtt 
                   
               
               
                   
                 ctggtggtggtggttctggtggttctggtcagaccaatcc 
                   
               
               
                   
                 gtatgcgcgcggccccaaccctaccgccgcctcgttggaa 
                   
               
               
                   
                 gccagcgcgggaccctttaccgttcgtagctttaccgtta 
                   
               
               
                   
                 gccgtccgtccggatatggtgcagggaccgtctattaccc 
                   
               
               
                   
                 aaccaatgcaggcggcaccgttggcgcgattgcaatcgtc 
                   
               
               
                   
                 cccgggtacaccgcgcgtcaaagcagcattaagtggtggg 
                   
               
               
                   
                 gtccgcgcttagctagccatggctttgtggttattaccat 
                   
               
               
                   
                 cgatacgaacagcactctagaccagcccagcagccgtagc 
                   
               
               
                   
                 tcgcaacagatggccgcgcttcgtcaagttgcgagcttga 
                   
               
               
                   
                 acgggaccagcagtagcccgatttacggaaaggtcgatac 
                   
               
               
                   
                 tgcccgcatgggtgtgatgggctggtcaatggggggcggc 
                   
               
               
                   
                 ggttcacttattagcgccgcgaacaacccgagtttaaaag 
                   
               
               
                   
                 cagcggcaccgcaggcgccatgggactcttcaaccaactt 
                   
               
               
                   
                 cagcagtgttaccgtgccgacgctgattttcgcgtgcgag 
                   
               
               
                   
                 aatgatagcattgcaccggtgaacagcagcgcgctgccga 
                   
               
               
                   
                 tttatgatagcatgtcccgcaacgcaaaacagtttctgga 
                   
               
               
                   
                 aattaacggcggtagccactcttgtgccaactctgggaac 
                   
               
               
                   
                 agcaaccaggcactgatcggaaaaaaaggggttgcatgga 
                   
               
               
                   
                 tgaaacgattcatggataatgacacccgttactcaacctt 
                   
               
               
                   
                 cgcctgtgagaatcccaacagcacacgcgtgtcggatttt 
                   
               
               
                   
                 cgcaccgcgaactgttccctcgagcaccaccatcaccacc 
                   
               
               
                   
                 actga 
                   
               
               
                   
               
               
                 MP20 aa 
                 MQTTVTTMLLASVALAACAGGGSTPLPLPQQQPPQQEPPP 
                 Amino acid sequence 
               
               
                 (SEQ ID NO: 40) 
                 PPVPLASRAACEALKDGNGDMVWPNAATVVEVAAWRDAAP 
                 for MHETase-20 aa 
               
               
                   
                 ATASAAALPEHCEVSGAIAKRTGIDGYPYEIKFRLRMPAE 
                 linker-PETase 
               
               
                   
                 WNGRFFMEGGSGTNGSLSAATGSIGGGQIASALSRNFATI 
                   
               
               
                   
                 ATDGGHDNAVNDNPDALGTVAFGLDPQARLDMGYNSYDQV 
                   
               
               
                   
                 TQAGKAAVARFYGRAADKSYFIGCSEGGREGMMLSQRFPS 
                   
               
               
                   
                 HYDGIVAGAPGYQLPKAGISGAWTTQSLAPAAVGLDAQGV 
                   
               
               
                   
                 PLINKSFSDADLHLLSQAILGTCDALDGLADGIVDNYRAC 
                   
               
               
                   
                 QAAFDPATAANPANGQALQCVGAKTADCLSPVQVTAIKRA 
                   
               
               
                   
                 MAGPVNSAGTPLYNRWAWDAGMSGLSGTTYNQGWRSWWLG 
                   
               
               
                   
                 SFNSSANNAQRVSGFSARSWLVDFATPPEPMPMTQVAARM 
                   
               
               
                   
                 MKFDFDIDPLKIWATSGQFTQSSMDWHGATSTDLAAFRDR 
                   
               
               
                   
                 GGKMILYHGMSDAAFSALDTADYYERLGAAMPGAAGFARL 
                   
               
               
                   
                 FLVPGMNHCSGGPGTDRFDMLTPLVAWVERGEAPDQISAW 
                   
               
               
                   
                 SGTPGYFGVAARTRPLCPYPQIARYKGSGDINTEANFACA 
                   
               
               
                   
                 APPGGGSGGSGGGSGGGGSGGSGQTNPYARGPNPTAASLE 
                   
               
               
                   
                 ASAGPFTVRSFTVSRPSGYGAGTVYYPTNAGGTVGAIAIV 
                   
               
               
                   
                 PGYTARQSSIKWWGPRLASHGFWITIDTNSTLDQPSSRSS 
                   
               
               
                   
                 QQMAALRQVASLNGTSSSPIYGKVDTARMGVMGWSMGGGG 
                   
               
               
                   
                 SLISAANNPSLKAAAPQAPWDSSTNFSSVTVPTLIFACEN 
                   
               
               
                   
                 DSIAPVNSSALPIYDSMSRNAKQFLEINGGSHSCANSGNS 
                   
               
               
                   
                 NQALIGKKGVAWMKRFMDNDTRYSTFACENPNSTRVSDFR 
                   
               
               
                   
                 TANCSLEHHHHHH 
                   
               
               
                   
               
               
                 PM8 DNA 
                 atgaacttcccccgtgcctcgcgccttatgcaggctgctg 
                 DNA sequence for PETase- 
               
               
                 (SEQ ID NO: 41) 
                 tgctgggcggccttatggccgtttccgcagcggccaccgc 
                 8 aa linker- 
               
               
                   
                 gcagaccaatccgtatgcgcgcggccccaaccctaccgcc 
                 MHETase 
               
               
                   
                 gcctcgttggaagccagcgcgggaccctttaccgttcgta 
                   
               
               
                   
                 gctttaccgttagccgtccgtccggatatggtgcagggac 
                   
               
               
                   
                 cgtctattacccaaccaatgcaggcggcaccgttggcgcg 
                   
               
               
                   
                 attgcaatcgtccccgggtacaccgcgcgtcaaagcagca 
                   
               
               
                   
                 ttaagtggtggggtccgcgcttagctagccatggctttgt 
                   
               
               
                   
                 ggttattaccatcgatacgaacagcactctagaccagccc 
                   
               
               
                   
                 agcagccgtagctcgcaacagatggccgcgcttcgtcaag 
                   
               
               
                   
                 ttgcgagcttgaacgggaccagcagtagcccgatttacgg 
                   
               
               
                   
                 aaaggtcgatactgcccgcatgggtgtgatgggctggtca 
                   
               
               
                   
                 atggggggcggcggttcacttattagcgccgcgaacaacc 
                   
               
               
                   
                 cgagtttaaaagcagcggcaccgcaggcgccatgggactc 
                   
               
               
                   
                 ttcaaccaacttcagcagtgttaccgtgccgacgctgatt 
                   
               
               
                   
                 ttcgcgtgcgagaatgatagcattgcaccggtgaacagca 
                   
               
               
                   
                 gcgcgctgccgatttatgatagcatgtcccgcaacgcaaa 
                   
               
               
                   
                 acagtttctggaaattaacggcggtagccactcttgtgcc 
                   
               
               
                   
                 aactctgggaacagcaaccaggcactgatcggaaaaaaag 
                   
               
               
                   
                 gggttgcatggatgaaacgattcatggataatgacacccg 
                   
               
               
                   
                 ttactcaaccttcgcctgtgagaatcccaacagcacacgc 
                   
               
               
                   
                 gtgtcggattttcgcaccgcgaactgttccggtggtggtt 
                   
               
               
                   
                 ctggtggttctggttgcgccggaggaggttccactcctct 
                   
               
               
                   
                 gcctctaccgcagcagcagccgcctcagcaggaaccgcca 
                   
               
               
                   
                 cctcctcctgttccgctagccagtcgcgccgcgtgtgagg 
                   
               
               
                   
                 cgctcaaagatggtaatggcgacatggtttggccgaatgc 
                   
               
               
                   
                 cgccacggttgtagaggttgcagcctggcgtgatgcagca 
                   
               
               
                   
                 ccggccacggcatcagccgcagccctgccggagcattgcg 
                   
               
               
                   
                 aagtatcaggcgcgattgccaagcgtactgggattgatgg 
                   
               
               
                   
                 gtacccgtatgaaattaagtttcgcctgcgcatgcccgct 
                   
               
               
                   
                 gagtggaacggccgttttttcatggagggtggcagtggta 
                   
               
               
                   
                 cgaacggctctctctcagcggcgaccggaagtatcggcgg 
                   
               
               
                   
                 cggtcagatcgcctcagcgctgagtcgtaactttgcaaca 
                   
               
               
                   
                 attgctaccgacggaggacatgacaatgcggtgaatgata 
                   
               
               
                   
                 atccggatgcgctcggtaccgtcgcatttggtctcgatcc 
                   
               
               
                   
                 ccaggcacgcttagacatgggctacaactcctatgatcag 
                   
               
               
                   
                 gtgactcaggccggcaaagccgccgttgcacgcttttatg 
                   
               
               
                   
                 gtcgcgcagccgacaagagctacttcatcggctgttcgga 
                   
               
               
                   
                 gggcggccgcgagggcatgatgctgtcccagcgctttcca 
                   
               
               
                   
                 tcacattacgatggcattgtggcgggcgcaccgggatatc 
                   
               
               
                   
                 agttgccgaaggccggaattagtggcgcgtggaccaccca 
                   
               
               
                   
                 gagcttagcgcccgccgccgttggcctggatgcccaggga 
                   
               
               
                   
                 gtgccgctgattaataagagcttttctgacgcagacctcc 
                   
               
               
                   
                 atttactgtcgcaggcgattctcggaacatgcgacgcctt 
                   
               
               
                   
                 ggatggcctggccgacggcatcgttgacaactaccgagcg 
                   
               
               
                   
                 tgccaagcggcttttgatccggcgactgcagccaacccag 
                   
               
               
                   
                 cgaatggccaagccctgcagtgcgtgggcgcaaagacagc 
                   
               
               
                   
                 cgattgcttatcgcccgtccaagttacggcgattaaacga 
                   
               
               
                   
                 gcgatggccggtccggtaaatagcgcgggtacgccgttat 
                   
               
               
                   
                 ataatagatgggcctgggacgcaggtatgagcggtcttag 
                   
               
               
                   
                 tggtaccacttacaatcagggttggcgcagctggtggctg 
                   
               
               
                   
                 ggatcgtttaacagctcggcgaataacgcacaacgtgtat 
                   
               
               
                   
                 ctggtttctcagcgcggagctggctggtggactttgctac 
                   
               
               
                   
                 cccgccggagccgatgcccatgacccaagtcgccgcccgt 
                   
               
               
                   
                 atgatgaaatttgatttcgatatcgatcctctgaaaatat 
                   
               
               
                   
                 gggctacttcgggccaatttacccagagtagtatggactg 
                   
               
               
                   
                 gcacggtgccactagcaccgaccttgctgcctttcgggac 
                   
               
               
                   
                 cgcggcggtaaaatgattctgtatcacggaatgagcgatg 
                   
               
               
                   
                 ccgcattctctgcactagatacagcagattattatgaacg 
                   
               
               
                   
                 cctgggtgccgcaatgccgggcgccgcgggctttgctcgt 
                   
               
               
                   
                 ctgttcttggttccgggaatgaaccattgctccgggggtc 
                   
               
               
                   
                 caggtaccgaccgctttgatatgctaacaccgttagttgc 
                   
               
               
                   
                 atgggttgaacgtggggaagcccctgaccaaattagcgcc 
                   
               
               
                   
                 tggagcggcacccccggctactttggtgtggccgcccgca 
                   
               
               
                   
                 ctcgaccgttatgtccctatccgcagattgcgcgctataa 
                   
               
               
                   
                 gggatcaggcgatatcaataccgaagcaaattttgcgtgt 
                   
               
               
                   
                 gccgctccaccgctcgagcaccaccatcaccaccactga 
                   
               
               
                   
               
               
                 PM12 DNA 
                 atgaacttcccccgtgcctcgcgccttatgcaggctgctg 
                 DNA sequence for PETase- 
               
               
                 (SEQ ID NO: 42) 
                 tgctgggcggccttatggccgtttccgcagcggccaccgc 
                 12 aa linker- 
               
               
                   
                 gcagaccaatccgtatgcgcgcggccccaaccctaccgcc 
                 MHETase 
               
               
                   
                 gcctcgttggaagccagcgcgggaccctttaccgttcgta 
                   
               
               
                   
                 gctttaccgttagccgtccgtccggatatggtgcagggac 
                   
               
               
                   
                 cgtctattacccaaccaatgcaggcggcaccgttggcgcg 
                   
               
               
                   
                 attgcaatcgtccccgggtacaccgcgcgtcaaagcagca 
                   
               
               
                   
                 ttaagtggtggggtccgcgcttagctagccatggctttgt 
                   
               
               
                   
                 ggttattaccatcgatacgaacagcactctagaccagccc 
                   
               
               
                   
                 agcagccgtagctcgcaacagatggccgcgcttcgtcaag 
                   
               
               
                   
                 ttgcgagcttgaacgggaccagcagtagcccgatttacgg 
                   
               
               
                   
                 aaaggtcgatactgcccgcatgggtgtgatgggctggtca 
                   
               
               
                   
                 atggggggcggcggttcacttattagcgccgcgaacaacc 
                   
               
               
                   
                 cgagtttaaaagcagcggcaccgcaggcgccatgggactc 
                   
               
               
                   
                 ttcaaccaacttcagcagtgttaccgtgccgacgctgatt 
                   
               
               
                   
                 ttcgcgtgcgagaatgatagcattgcaccggtgaacagca 
                   
               
               
                   
                 gcgcgctgccgatttatgatagcatgtcccgcaacgcaaa 
                   
               
               
                   
                 acagtttctggaaattaacggcggtagccactcttgtgcc 
                   
               
               
                   
                 aactctgggaacagcaaccaggcactgatcggaaaaaaag 
                   
               
               
                   
                 gggttgcatggatgaaacgattcatggataatgacacccg 
                   
               
               
                   
                 ttactcaaccttcgcctgtgagaatcccaacagcacacgc 
                   
               
               
                   
                 gtgtcggattttcgcaccgcgaactgttccggtggtggtt 
                   
               
               
                   
                 ctggtggttctggtggtggttctggttgcgccggaggagg 
                   
               
               
                   
                 ttccactcctctgcctctaccgcagcagcagccgcctcag 
                   
               
               
                   
                 caggaaccgccacctcctcctgttccgctagccagtcgcg 
                   
               
               
                   
                 ccgcgtgtgaggcgctcaaagatggtaatggcgacatggt 
                   
               
               
                   
                 ttggccgaatgccgccacggttgtagaggttgcagcctgg 
                   
               
               
                   
                 cgtgatgcagcaccggccacggcatcagccgcagccctgc 
                   
               
               
                   
                 cggagcattgcgaagtatcaggcgcgattgccaagcgtac 
                   
               
               
                   
                 tgggattgatgggtacccgtatgaaattaagtttcgcctg 
                   
               
               
                   
                 cgcatgcccgctgagtggaacggccgttttttcatggagg 
                   
               
               
                   
                 gtggcagtggtacgaacggctctctctcagcggcgaccgg 
                   
               
               
                   
                 aagtatcggcggcggtcagatcgcctcagcgctgagtcgt 
                   
               
               
                   
                 aactttgcaacaattgctaccgacggaggacatgacaatg 
                   
               
               
                   
                 cggtgaatgataatccggatgcgctcggtaccgtcgcatt 
                   
               
               
                   
                 tggtctcgatccccaggcacgcttagacatgggctacaac 
                   
               
               
                   
                 tcctatgatcaggtgactcaggccggcaaagccgccgttg 
                   
               
               
                   
                 cacgcttttatggtcgcgcagccgacaagagctacttcat 
                   
               
               
                   
                 cggctgttcggagggcggccgcgagggcatgatgctgtcc 
                   
               
               
                   
                 cagcgctttccatcacattacgatggcattgtggcgggcg 
                   
               
               
                   
                 caccgggatatcagttgccgaaggccggaattagtggcgc 
                   
               
               
                   
                 gtggaccacccagagcttagcgcccgccgccgttggcctg 
                   
               
               
                   
                 gatgcccagggagtgccgctgattaataagagcttttctg 
                   
               
               
                   
                 acgcagacctccatttactgtcgcaggcgattctcggaac 
                   
               
               
                   
                 atgcgacgccttggatggcctggccgacggcatcgttgac 
                   
               
               
                   
                 aactaccgagcgtgccaagcggcttttgatccggcgactg 
                   
               
               
                   
                 cagccaacccagcgaatggccaagccctgcagtgcgtggg 
                   
               
               
                   
                 cgcaaagacagccgattgcttatcgcccgtccaagttacg 
                   
               
               
                   
                 gcgattaaacgagcgatggccggtccggtaaatagcgcgg 
                   
               
               
                   
                 gtacgccgttatataatagatgggcctgggacgcaggtat 
                   
               
               
                   
                 gagcggtcttagtggtaccacttacaatcagggttggcgc 
                   
               
               
                   
                 agctggtggctgggatcgtttaacagctcggcgaataacg 
                   
               
               
                   
                 cacaacgtgtatctggtttctcagcgcggagctggctggt 
                   
               
               
                   
                 ggactttgctaccccgccggagccgatgcccatgacccaa 
                   
               
               
                   
                 gtcgccgcccgtatgatgaaatttgatttcgatatcgatc 
                   
               
               
                   
                 ctctgaaaatatgggctacttcgggccaatttacccagag 
                   
               
               
                   
                 tagtatggactggcacggtgccactagcaccgaccttgct 
                   
               
               
                   
                 gcctttcgggaccgcggcggtaaaatgattctgtatcacg 
                   
               
               
                   
                 gaatgagcgatgccgcattctctgcactagatacagcaga 
                   
               
               
                   
                 ttattatgaacgcctgggtgccgcaatgccgggcgccgcg 
                   
               
               
                   
                 ggctttgctcgtctgttcttggttccgggaatgaaccatt 
                   
               
               
                   
                 gctccgggggtccaggtaccgaccgctttgatatgctaac 
                   
               
               
                   
                 accgttagttgcatgggttgaacgtggggaagcccctgac 
                   
               
               
                   
                 caaattagcgcctggagcggcacccccggctactttggtg 
                   
               
               
                   
                 tggccgcccgcactcgaccgttatgtccctatccgcagat 
                   
               
               
                   
                 tgcgcgctataagggatcaggcgatatcaataccgaagca 
                   
               
               
                   
                 aattttgcgtgtgccgctccaccgctcgagcaccaccatc 
                   
               
               
                   
                 accaccactga 
                   
               
               
                   
               
               
                 PM20 DNA 
                 atgaacttcccccgtgcctcgcgccttatgcaggctgctg 
                 DNA sequence for PETase- 
               
               
                 (SEQ ID NO: 43) 
                 tgctgggcggccttatggccgtttccgcagcggccaccgc 
                 20 aa linker- 
               
               
                   
                 gcagaccaatccgtatgcgcgcggccccaaccctaccgcc 
                 MHETase 
               
               
                   
                 gcctcgttggaagccagcgcgggaccctttaccgttcgta 
                   
               
               
                   
                 gctttaccgttagccgtccgtccggatatggtgcagggac 
                   
               
               
                   
                 cgtctattacccaaccaatgcaggcggcaccgttggcgcg 
                   
               
               
                   
                 attgcaatcgtccccgggtacaccgcgcgtcaaagcagca 
                   
               
               
                   
                 ttaagtggtggggtccgcgcttagctagccatggctttgt 
                   
               
               
                   
                 ggttattaccatcgatacgaacagcactctagaccagccc 
                   
               
               
                   
                 agcagccgtagctcgcaacagatggccgcgcttcgtcaag 
                   
               
               
                   
                 ttgcgagcttgaacgggaccagcagtagcccgatttacgg 
                   
               
               
                   
                 aaaggtcgatactgcccgcatgggtgtgatgggctggtca 
                   
               
               
                   
                 atggggggcggcggttcacttattagcgccgcgaacaacc 
                   
               
               
                   
                 cgagtttaaaagcagcggcaccgcaggcgccatgggactc 
                   
               
               
                   
                 ttcaaccaacttcagcagtgttaccgtgccgacgctgatt 
                   
               
               
                   
                 ttcgcgtgcgagaatgatagcattgcaccggtgaacagca 
                   
               
               
                   
                 gcgcgctgccgatttatgatagcatgtcccgcaacgcaaa 
                   
               
               
                   
                 acagtttctggaaattaacggcggtagccactcttgtgcc 
                   
               
               
                   
                 aactctgggaacagcaaccaggcactgatcggaaaaaaag 
                   
               
               
                   
                 gggttgcatggatgaaacgattcatggataatgacacccg 
                   
               
               
                   
                 ttactcaaccttcgcctgtgagaatcccaacagcacacgc 
                   
               
               
                   
                 gtgtcggattttcgcaccgcgaactgttccggtggtggtt 
                   
               
               
                   
                 ctggtggttctggtggtggttctggtggtggtggttctgg 
                   
               
               
                   
                 tggttctggttgcgccggaggaggttccactcctctgcct 
                   
               
               
                   
                 ctaccgcagcagcagccgcctcagcaggaaccgccacctc 
                   
               
               
                   
                 ctcctgttccgctagccagtcgcgccgcgtgtgaggcgct 
                   
               
               
                   
                 caaagatggtaatggcgacatggtttggccgaatgccgcc 
                   
               
               
                   
                 acggttgtagaggttgcagcctggcgtgatgcagcaccgg 
                   
               
               
                   
                 ccacggcatcagccgcagccctgccggagcattgcgaagt 
                   
               
               
                   
                 atcaggcgcgattgccaagcgtactgggattgatgggtac 
                   
               
               
                   
                 ccgtatgaaattaagtttcgcctgcgcatgcccgctgagt 
                   
               
               
                   
                 ggaacggccgttttttcatggagggtggcagtggtacgaa 
                   
               
               
                   
                 cggctctctctcagcggcgaccggaagtatcggcggcggt 
                   
               
               
                   
                 cagatcgcctcagcgctgagtcgtaactttgcaacaattg 
                   
               
               
                   
                 ctaccgacggaggacatgacaatgcggtgaatgataatcc 
                   
               
               
                   
                 ggatgcgctcggtaccgtcgcatttggtctcgatccccag 
                   
               
               
                   
                 gcacgcttagacatgggctacaactcctatgatcaggtga 
                   
               
               
                   
                 ctcaggccggcaaagccgccgttgcacgcttttatggtcg 
                   
               
               
                   
                 cgcagccgacaagagctacttcatcggctgttcggagggc 
                   
               
               
                   
                 ggccgcgagggcatgatgctgtcccagcgctttccatcac 
                   
               
               
                   
                 attacgatggcattgtggcgggcgcaccgggatatcagtt 
                   
               
               
                   
                 gccgaaggccggaattagtggcgcgtggaccacccagagc 
                   
               
               
                   
                 ttagcgcccgccgccgttggcctggatgcccagggagtgc 
                   
               
               
                   
                 cgctgattaataagagcttttctgacgcagacctccattt 
                   
               
               
                   
                 actgtcgcaggcgattctcggaacatgcgacgccttggat 
                   
               
               
                   
                 ggcctggccgacggcatcgttgacaactaccgagcgtgcc 
                   
               
               
                   
                 aagcggcttttgatccggcgactgcagccaacccagcgaa 
                   
               
               
                   
                 tggccaagccctgcagtgcgtgggcgcaaagacagccgat 
                   
               
               
                   
                 tgcttatcgcccgtccaagttacggcgattaaacgagcga 
                   
               
               
                   
                 tggccggtccggtaaatagcgcgggtacgccgttatataa 
                   
               
               
                   
                 tagatgggcctgggacgcaggtatgagcggtcttagtggt 
                   
               
               
                   
                 accacttacaatcagggttggcgcagctggtggctgggat 
                   
               
               
                   
                 cgtttaacagctcggcgaataacgcacaacgtgtatctgg 
                   
               
               
                   
                 tttctcagcgcggagctggctggtggactttgctaccccg 
                   
               
               
                   
                 ccggagccgatgcccatgacccaagtcgccgcccgtatga 
                   
               
               
                   
                 tgaaatttgatttcgatatcgatcctctgaaaatatgggc 
                   
               
               
                   
                 tacttcgggccaatttacccagagtagtatggactggcac 
                   
               
               
                   
                 ggtgccactagcaccgaccttgctgcctttcgggaccgcg 
                   
               
               
                   
                 gcggtaaaatgattctgtatcacggaatgagcgatgccgc 
                   
               
               
                   
                 attctctgcactagatacagcagattattatgaacgcctg 
                   
               
               
                   
                 ggtgccgcaatgccgggcgccgcgggctttgctcgtctgt 
                   
               
               
                   
                 tcttggttccgggaatgaaccattgctccgggggtccagg 
                   
               
               
                   
                 taccgaccgctttgatatgctaacaccgttagttgcatgg 
                   
               
               
                   
                 gttgaacgtggggaagcccctgaccaaattagcgcctgga 
                   
               
               
                   
                 gcggcacccccggctactttggtgtggccgcccgcactcg 
                   
               
               
                   
                 accgttatgtccctatccgcagattgcgcgctataaggga 
                   
               
               
                   
                 tcaggcgatatcaataccgaagcaaattttgcgtgtgccg 
                   
               
               
                   
                 ctccaccgctcgagcaccaccatcaccaccactga 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 Primers. 
               
            
           
           
               
               
               
            
               
                 Oligo 
                 Sequence (5′ -&gt; 3′) 
                 Description 
               
               
                   
               
               
                 oC756 
                 teggagggcggccg 
                 For linear amplification of  Ideonella sakaiensis   
               
               
                 (SEQ ID 
                   
                 MHETase expression plasmid, 
               
               
                 NO: 6) 
                   
                 pCJ136, at Ser225 F 
               
               
                   
               
               
                 oC757 
                 ggcgccgatgaagta 
                 For linear amplification of  Ideonella sakaiensis   
               
               
                 (SEQ ID 
                 gctcttgtcggc 
                 MHETase expression plasmid, 
               
               
                 NO: 7) 
                   
                 pCJ136, at Ser225 R with Cys224Ala mutation 
               
               
                   
               
               
                 oC758 
                 tccgggggtccaggt 
                 For linear amplification of  Ideonella sakaiensis   
               
               
                 (SEQ ID 
                 acc 
                 MHETase expression plasmid, 
               
               
                 NO: 8) 
                   
                 pCJ136, at Ser530 F 
               
               
                   
               
               
                 oCJ759 
                 ggcatggttcattcc 
                 For linear amplification of  Ideonella sakaiensis   
               
               
                 (SEQ ID 
                 cggaaccaagaacag 
                 MHETase expression plasmid, 
               
               
                 NO: 9) 
                   
                 pCJ136, at Ser530 R with Cys529Ala mutation 
               
               
                   
               
               
                 oCJ760 
                 ccagccgatgaagta 
                 For linear amplification of  Ideonella sakaiensis   
               
               
                 (SEQ ID 
                 gctcttgtcggc 
                 MHETase expression plasmid, 
               
               
                 NO: 10) 
                   
                 pCJ136, at Ser225 R with Cys224Trp mutation 
               
               
                   
               
               
                 oCJ761 
                 gctatggttcattcc 
                 For linear amplification of  Ideonella sakaiensis   
               
               
                 (SEQ ID 
                 cggaaccaagaacag 
                 MHETase expression plasmid, 
               
               
                 NO: 11) 
                   
                 pCJ136, at Ser530 R with Cys529Ser mutation 
               
               
                   
               
               
                 oCJ762 
                 gtggccgatgaagta 
                 For linear amplification of  Ideonella sakaiensis   
               
               
                 (SEQ ID 
                 gctcttgtcggc 
                 MHETase expression plasmid, 
               
               
                 NO: 12) 
                   
                 pCJ136, at Ser225 R with Cys224His mutation 
               
               
                   
               
               
                 oC763 
                 gaaatggttcattcc 
                 For linear amplification of  Ideonella sakaiensis   
               
               
                 (SEQ ID 
                 cggaaccaagaacag 
                 MHETase expression plasmid, 
               
               
                 NO: 13) 
                   
                 pCJ136, at Ser530 R with Cys529Phe mutation 
               
               
                   
               
               
                 oC764 
                 tcaatggggggcggc 
                 For linear amplification of  Ideonella sakaiensis   
               
               
                 (SEQ ID 
                 g 
                 PETase expression plasmid, 
               
               
                 NO: 14) 
                   
                 pCJ135, at Ser160 F 
               
               
                   
               
               
                 oC765 
                 gcagcccatcacacc 
                 For linear amplification of  Ideonella sakaiensis   
               
               
                 (SEQ ID 
                 catgcgg 
                 PETase expression plasmid, 
               
               
                 NO: 15) 
                   
                 pCJ135, at Ser160 R with Trypl59Cys mutation 
               
               
                   
               
               
                 oC766 
                 tgtgccaactctggg 
                 For linear amplification of  Ideonella sakaiensis   
               
               
                 (SEQ ID 
                 aacagc 
                 PETase expression plasmid, 
               
               
                 NO: 16) 
                   
                 pCJ135, at Cys239 F 
               
               
                   
               
               
                 oC767 
                 gcagtggctaccgcc 
                 For linear amplification of  Ideonella sakaiensis   
               
               
                 (SEQ ID 
                 gttaatttccag 
                 PETase expression plasmid, 
               
               
                 NO: 17) 
                   
                 pCJ135, at Cys239 R with Ser238Cys mutation 
               
               
                   
               
               
                 oC768 
                 gagggcggccgcga 
                 For linear amplification of  Ideonella sakaiensis   
               
               
                 (SEQ ID 
                   
                 MHETase expression plasmid, 
               
               
                 NO: 18) 
                   
                 pCJ136, at Glu226 F 
               
               
                   
               
               
                 oCJ769 
                 ggcacagccgatgaa 
                 For linear amplification of  Ideonella sakaiensis   
               
               
                 (SEQ ID 
                 gtagctcttgtcg 
                 MHETase expression plasmid, 
               
               
                 NO: 19) 
                   
                 pCJ136, at Glu226 R with Ser225Ala mutation 
               
               
                   
               
               
                 oC770 
                 tgtggcggagacggt 
                 For linear amplification of  Comamonas thiooxydans   
               
               
                 (SEQ ID 
                 g 
                 expression plasmid, 
               
               
                 NO: 20) 
                   
                 pCJ199, at Cys76 F 
               
               
                   
               
               
                 oCJ771 
                 catatgtatatctcc 
                 For linear amplification of putative  Comomonas   
               
               
                 (SEQ ID 
                 ttctta 
                   thiooxydans  expression 
               
               
                 NO: 21) 
                 aagttaaacaaaatt 
                 plasmid, pCJ199, and  Hydrogenophaga  sp. PML113 
               
               
                   
                 atttcta 
                 expression plasmid, 
               
               
                   
                   
                 pCJ211, at MetIR 
               
               
                   
               
               
                 oCJ772 
                 tgcggtagcgttccg 
                 For linear amplification of  Hydrogenophaga  sp. 
               
               
                 (SEQ ID 
                 g 
                 PML113 expression plasmid, 
               
               
                 NO: 22) 
                   
                 pCJ211, at Cys20 F 
               
               
                   
               
               
                 oC773 
                 ggcggtacgaacggc 
                 For linear amplification of  Ideonella sakaiensis   
               
               
                 (SEQ ID 
                 tctctctcag 
                 MHETase expression plasmid, 
               
               
                 NO: 23) 
                   
                 pCJ136, at Ser131 F with Ser131Gly mutation 
               
               
                   
               
               
                 oCJ774 
                 gccaccctccatgaa 
                 For linear amplification of  Ideonella sakaiensis   
               
               
                 (SEQ ID 
                 aaaacgg 
                 MHETase expression plasmid, 
               
               
                 NO: 24) 
                   
                 pCJ136, at Ser131 R 
               
               
                   
               
               
                 oC775 
                 atctctgcactagat 
                 For linear amplification of  Ideonella sakaiensis   
               
               
                 (SEQ ID 
                 acagcagattattat 
                 MHETase expression plasmid, 
               
               
                 NO: 25) 
                 gaac 
                 pCJ136, at Phe495 F with Phe495Ile mutation 
               
               
                   
               
               
                 oC776 
                 tgcggcatcgctcat 
                 For linear amplification of  Ideonella sakaiensis   
               
               
                 (SEQ ID 
                 tcc 
                 MHETase expression plasmid, 
               
               
                 NO: 26) 
                   
                 pCJ136, at Phe495 R 
               
               
                   
               
               
                 oC777 
                 ggcggccgcgagg 
                 For linear amplification of  Ideonella sakaiensis   
               
               
                 (SEQ ID 
                   
                 MHETase expression plasmid, 
               
               
                 NO: 27) 
                   
                 pCJ136, atGly227 F 
               
               
                   
               
               
                 oC778 
                 ggtcgaacagccgat 
                 For linear amplification of  Ideonella sakaiensis   
               
               
                 (SEQ ID 
                 gaagtagctcttgtc 
                 MHETase expression plasmid, 
               
               
                 NO: 28) 
                   
                 pCJ136, at Gly227 R with Glu226Thr mutation 
               
               
                   
               
               
                 oC779 
                 tgcatgagcgatgcc 
                 For linear amplification of  Ideonella sakaiensis   
               
               
                 (SEQ ID 
                 gcattctctg 
                 MHETase expression plasmid, 
               
               
                 NO: 29) 
                   
                 pCJ136, at Gly489 F with Gly489Cys mutation 
               
               
                   
               
               
                 oC780 
                 gtgatacagaatcat 
                 For linear amplification of  Ideonella sakaiensis   
               
               
                 (SEQ ID 
                 tttaccgccgcg 
                 MHETase expression plasmid, 
               
               
                 NO: 30) 
                   
                 pCJ136, atGly489 R 
               
               
                   
               
               
                 oC781 
                 gggggtccaggtacc 
                 For linear amplification of  Ideonella sakaiensis   
               
               
                 (SEQ ID 
                 gac 
                 MHETase expression plasmid, 
               
               
                 NO: 31) 
                   
                 pCJ136, at Ser530 F 
               
               
                   
               
               
                 oC782 
                 gcagcaatggttcat 
                 For linear amplification of  Ideonella sakaiensis   
               
               
                 (SEQ ID 
                 tcccggaacc 
                 MHETase expression plasmid, 
               
               
                 NO: 32) 
                   
                 pCJ136, at Ser530 R with Ser530Cys mutation 
               
               
                   
               
               
                 oC787 
                 caaccaacttcgacc 
                 For linear amplification of  Ideonella sakaiensis   
               
               
                 (SEQ ID 
                 ttgctgcctttcggg 
                 MHETase expression plasmid, 
               
               
                 NO: 33) 
                 ac 
                 pCJ136, F 
               
               
                   
               
               
                 oC788 
                 aagagtcccacggtg 
                 For linear amplification of  Ideonella sakaiensis   
               
               
                 (SEQ ID 
                 cgcccgcc 
                 MHETase expression plasmid, 
               
               
                 NO: 34) 
                   
                 pCJ136, R 
               
               
                   
               
            
           
         
       
     
     Table 5 depicts the Michaelis-Menten kinetic parameters of fitting initial reaction velocities of enzymatic turnover for Is MHETase, Is MHETase S131G,  Comamonas thiooxydans  MHETase, and  Hydrogenophaga sp . PML113 MHETase at MHET substrate concentrations between 10.M and 250.M using the Michaelis-Menten model with substrate inhibition. Non-linear regression was performed using GraphPad Prism (8.4.1) along with 95 confidence intervals for each parameter and R 2  value given for fit of the model to the data. 
     
       
         
           
               
               
               
               
               
               
             
               
                 TABLE 5 
               
               
                   
               
               
                   
                 K m   
                 V max   
                 K i   
                   
                 k cat /K m   
               
               
                 Enzyme 
                 (μM) 
                 (μM s −1 ) 
                 (μM) 
                 R 2   
                 (μM −1  s −1 ) 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 Is MHETase 
                 23.17 ± 1.65 
                 0.252 ± 0.045 
                 307.3 ± 20.65 
                 0.9027 
                 2.17 
               
               
                 Is MHETase 
                 995.10 ± 19.58 
                 0.455 ± 0.071 
                 102.7 ± 6.05  
                 0.9174 
                 0.09 
               
               
                 S131G 
               
               
                 
                   Comamonas 
                 
                 174.70 ± 4.75  
                 0.203 ± 0.047 
                 78.8 ± 3.04 
                 0.9328 
                 0.23 
               
               
                 
                   thiooxydans 
                 
               
               
                 MHETase 
               
               
                 
                   Hydrogenophaga 
                 
                 41.09 ± 3.38 
                 0.013 ± 0.003 
                 221.5 ± 19.01 
                 0.9269 
                 0.13 
               
               
                 sp. PML113 
               
               
                 MHETase 
               
               
                   
               
            
           
         
       
     
     The foregoing discussion and examples have been presented for purposes of illustration and description. The foregoing is not intended to limit the aspects, embodiments, or configurations to the form or forms disclosed herein. In the foregoing Detailed Description for example, various features of the aspects, embodiments, or configurations are grouped together in one or more embodiments, configurations, or aspects for the purpose of streamlining the disclosure. The features of the aspects, embodiments, or configurations, may be combined in alternate aspects, embodiments, or configurations other than those discussed above. This method of disclosure is not to be interpreted as reflecting an intention that the aspects, embodiments, or configurations require more features than are expressly recited in each claim. Rather, as the following claims reflect, inventive aspects lie in less than all features of a single foregoing disclosed embodiment, configuration, or aspect. While certain aspects of conventional technology have been discussed to facilitate disclosure of some embodiments of the present invention, the Applicants in no way disclaim these technical aspects, and it is contemplated that the claimed invention may encompass one or more of the conventional technical aspects discussed herein. Thus, the following claims are hereby incorporated into this Detailed Description, with each claim standing on its own as a separate aspect, embodiment, or configuration.