WO2020055369A2

WO2020055369A2 - Method for preparing recombinant petase and mhetase enzymes for use in decomposition of plastics

Info

Publication number: WO2020055369A2
Application number: PCT/TR2019/050737
Authority: WO
Inventors: Turan DEMIRCAN; Emine KESKIN
Original assignee: Istanbul Medipol Universitesi
Priority date: 2018-09-11
Filing date: 2019-09-09
Publication date: 2020-03-19
Also published as: WO2020055369A3

Abstract

The present invention is related to methods used in obtaining recombinant PETase and MHETase enzymes that are used in decomposition of plastics, for example plastic wastes and PETase and MHETase enzymes that are obtained using this method and the use of the PETase and MHETase enzymes that have been obtained by this method in decomposition of plastics.

Description

METHOD FOR PREPARING RECOMBINANT PETase AND MHETase ENZYMES FOR USE IN DECOMPOSITION OF PLASTICS

Technical Field

The present invention is related to methods used in obtaining recombinant PETase and MHETase enzymes that are used in decomposing of plastics, for example plastic wastes and PETase and MHETase enzymes that are obtained using this method and the usage of the PETase and MHETase enzymes that have been obtained by this method in decomposition of plastics.

Prior Art

The increasing of industrialization together with the development of communities, has brought about, besides several benefits, environmental pollution which affects our natural habitats. Together with industrialization and increasing consumption, solid wastes occupy an important place in the creation of environmental pollution. One of the issues that has started to attract attention in environmental politics and in the current and strategic planning of several developed countries is the fact that the solid waste problem has reached a crisis point in many countries. In many countries, particularly in industrialized countries, besides preventing environmental pollution these wastes have been causing efforts are being made to recover these wastes by using recycling technologies and to provide contribution to the economy.

One of the principle elements that have contributed to this pollution problem is polyethylene terephthalate, or in short PET. People produce millions of tons of PET waste every year. The situation is not different in our country either. In the sector monitoring report of 2013, prepared by the Plastic Industrialists Federation, relating to plastic packaging products, it has been reported that Turkey ranked the ninth country in the world in plastic packaging production, and third in Europe, following Germany and Italy and that production increased up to 13% in yield when compared to the previous year and has reached up to 3,25 million tons. According to the calculations made, the amount of plastic waste in the world is around 9 billion tons and more than half of this amount was produced after the year 2004, which indicates increased PET production and usage prevalence. Plastic wastes whose usage area and quantities have been increasing every day due to said plastics being economic, easily applicable and having increasing characteristics, are very difficult to decompose and break down in nature because of their chemical structures and as they do not decompose in nature for many years, they cause a significant amount of environmental pollution. Although the basic solutions we have brought to the problem of plastic packaging wastes are trying to control them by incineration and burying, these approaches also lead to different environmental problems. For this reason, recycling and re-functionalization of plastic wastes as a sustainable action plan is being carried out in many developed countries. The most important problem we face here is that the production costs of the plastic are lower than the recycling costs and therefore the manufacturers prefer to produce from scratch instead of recycling products. For this reason, large piles of plastic garbage are formed both on land and in the seas, which cause great harm to nature, to biological diversity and human life.

In prior art, plastic wastes are basically dealt with as follows: a) Incineration of plastics (in energy production centers or solid waste dump sites) and disposal of plastics by means of incineration

b) Burying plastic waste, therefore preventing the accumulation of waste on land

c) Usage of plastics in recycling in order to form large plastic materials by breaking down plastics into small parts, using mechanical methods

d) Trying to disintegrate PET into basic units of matter using hydrolysis enzymes (esterase, lipase and cutinase)

The most significant disadvantages of these solution suggestions can be listed as follows: a) A different and multi layered environmental pollution is created due to incineration of plastic waste.

b) Although accumulation on land is prevented when plastics are buried, the decomposition rate of plastics in nature is very slow and due to increased plastic usage, it is not possible to hide all of the accumulated plastic in future by burying them underground. Moreover when it rains, several toxic materials find their way initially to groundwaters and then to seas via this route and finally to the food chain. c) Disintegrating plastic by mechanical methods in order to recycle plastics, is a much more expensive process in comparison to production of plastics from scratch; moreover it is not always possible to obtain the basic units of matter of said plastics.

d) PET decomposition using hydrolysis enzymes is a very inefficient and time consuming process due to the chemical characteristics of PET.

When these reasons are taken into consideration it can be seen that a method which provides a radical solution to the increasing plastic waste problem is required.

In the recent years it has been shown in studies that the enzyme called PETase, which is a cutinase enzyme that is isolated from the Ideonella sakaiensis 201-F6 bacteria strain decomposes plastic more efficiently in comparison to present methods.

MHETase enzyme separates the intermediate product ((mono(2-hydroxyethyl) terephthalate)) produced as a result of plastic decomposition of the PETase enzyme into its units of matter (terephthalate and ethylene glycol); in other words it plays a more efficient role in the decomposition of polyethylene terephthalate, also known as PET.

In these studies of the prior art, the genes that encode the PETase enzyme are transferred to E.coli bacteria via plasmids and as a result the PETase enzyme is expressed as surface protein. However PETase enzymes that are produced with this method can only be produced in small quantities and this causes yield to be low.

Based on these problems present in the prior art, the inventors have aimed to develop novel methods which enable to produce higher yields of PETase and/or MHETase enzymes which can be purified.

Detailed Description

The invention is generally related to a production method to be used for the production of PETase and/or MHETase enzymes, wherein said method, comprises the steps of sending the genes encoding the PETase and/or MHETase enzymes to the E.coli and B.subtilis cells in a plasmid, or inserting them into the genome and releasing the formed PETase and/or MHETase enzymes outside the cell and obtaining recombinant PETase and/or MHETase enzymes.

In an embodiment of the invention, the method subject to the invention is a production method used for producing PETase and/or MHETase enzymes, (Method 1), comprising the steps of; a. Obtaining Recombinant DNA1 specific to the PETase enzyme or Recombinant DNA2 specific to the MHETase enzyme by cloning the recombinant genes comprising secretion signals at the N-terminus and marker peptides at the C-terminus after the ‘T7’ or‘trc’ promoter for E.coli and‘T7’ or‘PVT promoter for B.subtilis b. Inserting the Recombinant DNA1 or Recombinant DNA2 into the genomic DNA of the E. coli or B. subtilis bacteria,

c. Excreting out the PETase or MHETase enzyme formed from the cell,

d. Purification of the PETase or MHETase enzymes that have been transported out of the cell.

By means of this method (Method 1), enzyme production is carried out by means of the genes that have been inserted into the bacteria genome, different from the known enzyme production techniques and high yields are obtained as a result of the strong promoter used (‘T7’, ‘trc’ or‘PVl’). Moreover by means of the secretion signal added to the obtained enzymes, the enzymes are transported out of the cell and they can easily be purified by means of the peptide markers located thereon.

The transportation of the PETase or MHETase enzymes out of the cell, mentioned in step C, is performed by means of the secretion signal located on the enzyme.

The purification method mentioned in step D, is carried out by collection of the enzymes released into the medium from the bacteria by means of the antibodies specific to the marker peptides thereon, and separation of said enzymes from other materials comprised in the medium.

From another aspect the present invention is a method to be used in the production of PETase and/or MHETase enzymes (Method 2); comprising the steps of cloning the recombinant DNA’s which encode the PETase and/or MHETase enzymes as a plasmid, then transferring the obtained recombinant plasmid into E. coli or B. subtilis, following this obtaining recombinant PETase and/or MHETase enzymes by excreting the obtained PETase and MHETase enzymes out of the cell.

In an embodiment of the invention, the method according to the invention is a production method used for producing PETase and/or MHETase enzymes, (Method 2), comprising the steps of; a. Obtaining Recombinant DNA1 specific to the PETase enzyme or Recombinant DNA2 specific to the MHETase enzyme by cloning the recombinant genes that comprise secretion signals at the N-terminus and marker peptides at the C-terminus after the ‘T7’ or ‘trc’ promoter for E.coli and‘T7’ or‘PVT promoter for B. subtilis, b. Obtaining a Recombinant plasmid by cloning the Recombinant DNA1 or Recombinant DNA2 into a pET-3a vector or derivative thereof,

c. Transferring the recombinant plasmid to E.coli or B. subtilis,

d. Excreting obtained PETase or MHETase enzymes out of the cell,

e. Purification of the PETase or MHETase enzymes that have been excreted out of the cell.

By this means, unlike the known methods which depend on inserting the plasmids into E.coli or B. subtilis, the PETase and MHETase enzymes that are formed in the E.coli or B. subtilis do not remain on the surface but they are excreted out of the cell. Moreover these enzymes that are excreted out of the cell can easily be purified by means of the marker peptides located thereon.

The excretion of the PETase or MHETase enzymes out of the cell, mentioned in step D, is performed by means of the secretion signal located on the enzyme.

The purification method mentioned in step E, is carried out by collection of the enzymes released into the medium from the bacteria by means of the antibodies specific to the marker peptides thereon, and separation of said enzymes from other materials located in the medium.

The secretion signals used in the methods subject to the invention (Method 1 and Method 2) are selected from the secretion signals of AbnA, AmyE, AprE, BglC, BglS, Bpr, Csn, Epr, Ggt, GlpQ, HtrA, LipA, LytD, MntA, Mpr, NprE, OppA, PbpA, PbpX, Pel, PelB, PenP, PhoA, PhoB, PhoD, PstS, TasA, Vpr, WapA, WprA, XynA, XynD, YbdN, Ybxl, YcdH, YclQ, YdhF, YdhT, YfkN, YflE, YfmC, Yfnl, YhcR, YlqB, YncM, YnfF, YoaW, YocH, YolA, YqiX, Yqxl, YrpD, YrpE, YuaB, Yurl, YvcE, YvgO, YvpA, YwaD, YweA, YwoF, YwtD, YwtF, YxaLk, YxiA, YxkC. This signal is found at the N-terminus of recombinant genes.

The marker peptide used in the methods (Method 1 and Method 2) subject to the invention is selected from; AviTag (GLNDIFEAQKIEWHE), Calmodulin-tag ( KRRWKKN F 1 AV S AAN RF K KISSSGAL), polyglutamate tag (EEEEEE), E-tag (GAPVPYPDPLEPR), HA-tag (YPYDVPDYA), His-tag 5-10 histidines (HHHHHH), Myc-tag (EQKLISEEDL), NE-tag (TKENPRSNQEESYDDNES), S-tag (KETAAAKFERQHMDS), SBP-tag (MDEKTTGWRG GHWEGLAGELEQLRARLEHHPQGQREP), Softag 3 (TQDPSRVG), Spot tag (PDRVRA VSHWSS), Strep-tag (WSHPQFEK), TC tag (CCPGCC), Ty tag (EVHTNQDPLD), V5 tag (GKPIPNPLLGLDST), VSV-tag (YTDIEMNRLGK), Xpress tag (DLYDDDDK), GFP tag, Nus Tag, FC tag. MBP tag and GST tag or from a group consisting of different peptide sequences that provide the same function as these peptides.

The term“HA” used herein is Hemaagglutinin; the term“HIS” is polyhistidine; the term “FLAG” is a polypeptide formed of the DYKDDDDK octapeptide sequence; the term“MBP” is a maltose binder protein, the term“GST” is glutathione S -transferase; the term“GFP” is green fluorescent protein; the expression“Nus” is a monoclonal antibody specific to the 495- aa NusA protein; the term“FC” is the fixed region of the immunoglobulin heavy chain (region 3 and 4); the term“MBP” is the maltose binding protein.

The“A” term used above to express amino acids represents alanine;“R” represents arginine; “N” represents aspargine;“D” represents aspartic acid;“C” represents cystein;“Q” represents glutamine; Έ” represents glutamic acid;“G” represents glycine;“H” represents histidine;“I” represents isoleucine; “L” represents leucine; “K” represents lysine; “M” represents methionine;“F” represents phenylalanine;“P” represents proline;“S” represents serine;“T” represents threonine;“W” represents tryptophan;“Y” represents thyrosine;“V” represents valine.

The term“T7 promoter” used herein, refers to the promoter obtained from bacteriophage T7, the term‘trc promoter’ refers to the promoter produced synthetically as a hybrid of T7 and Lac promoters, the term‘PV1 promoter’ refers to the promoter mutated from the P_srfA promoter.

The term“vector” used herein expresses the DNA molecule that is used to transport a foreign genetic material artificially to another cell. Vectors can be exemplified as plasmids, viral vectors, cosmids and artificial chromosomes.

The term“plasmid” used herein, is the name given to small DNA molecules that can reproduce independently and that is physically separate from the chromosomal DNA inside a cell.

The term“recombinant plasmid” used herein, has the same meaning as the term“vector” and they can be used alternately with each other.

Another embodiment of the invention is the PETase enzyme obtained by using Method 1.

Another embodiment of the invention is the MHETase enzyme obtained by using Method 1

Another embodiment of the invention is the PETase enzyme obtained by using Method 2.

Another embodiment of the invention is the MHETase enzyme obtained as a result of using Method 2.

According to another aspect of the invention, the invention is a recombinant DNA (Recombinant DNA1) having the secretion signal-PETase-marker peptide sequence comprising a secretion signal at the forward primer, selected from the group of preferably AbnA, AmyE, AprE, BglC, BglS, Bpr, Csn, Epr, Ggt, GlpQ, HtrA, LipA, LytD, MntA, Mpr, NprE, OppA, PbpA, PbpX, Pel, PelB, PenP, PhoA, PhoB, PhoD, PstS, TasA, Vpr, WapA, WprA, XynA, XynD, YbdN, Ybxl, YcdH, YclQ, YdhF, YdhT, YfkN, YflE, YfmC, Yfnl, YhcR, YlqB, YncM, YnfF, YoaW, YocH, YolA, YqiX, Yqxl, YrpD, YrpE, YuaB, Yurl, YvcE, YvgO, YvpA, YwaD, YweA, YwoF, YwtD, YwtF, YxaLk, YxiA, YxkC and a marker peptide at the reverse primer, preferably selected from AviTag (GLNDIFEAQKIEWHE), Calmodulin-tag ( KRRWKKN F 1 AV S AAN RFKK1SSSGAL), polyglutamate tag (EEEEEE), E-tag (GAPVPYPDPLEPR), HA-tag (YPYDVPDYA), His-tag 5-10 histidines (HHHHHH), Myc-tag (EQKLISEEDL), NE-tag (TKENPRSNQEESYDDNES), S-tag (KETAAAKFERQHMDS), SBP- tag (MDEKTTGWRGGHWEGLAGELEQLRARLEHHPQGQREP), Softag 3 (TQDPSRVG), Spot-tag (PDRVRAVSHWSS), Strep-tag (WSHPQFEK), TC tag (CCPGCC), Ty tag (EVHTNQDPLD), V5 tag (GKPIPNPLLGLDST), VSV-tag (YTDIEMNRLGK), Xpress tag (DLYDDDDK), GFP tag, Nus Tag, FC tag.MBP tag and GST tag.

According to another aspect of the invention, the invention is a recombinant DNA (Recombinant DNA2) having the secretion signal-MHETase-marker peptide sequence comprising a secretion signal at the forward primer, which is selected from the group of preferably, AbnA, AmyE, AprE, BglC, BglS, Bpr, Csn, Epr, Ggt, GlpQ, HtrA, LipA, LytD, MntA, Mpr, NprE, OppA, PbpA, PbpX, Pel, PelB, PenP, PhoA, PhoB, PhoD, PstS, TasA, Vpr, WapA, WprA, XynA, XynD, YbdN, Ybxl, YcdH, YclQ, YdhF, YdhT, YfkN, YflE, YfmC, Yfnl, YhcR, YlqB, YncM, YnfF, YoaW, YocH, YolA, YqiX, Yqxl, YrpD, YrpE, YuaB, Yurl, YvcE, YvgO, YvpA, YwaD, YweA, YwoF, YwtD, YwtF, YxaLk, YxiA, YxkC and a marker peptide at the reverse primer, preferably selected from AviTag (GLNDIFEAQKIEWHE), Calmodulin-tag ( KRRWKKN F 1 AV S AAN RFKK1SSSGAL), polyglutamate tag (EEEEEE), E-tag (GAPVPYPDPLEPR), HA-tag (YPYDVPDYA), His-tag 5-10 histidines (HHHHHH), Myc-tag (EQKLISEEDL), NE-tag (TKENPRSNQEESYDDNES), S-tag (KETAAAKFERQHMDS), SBP- tag (MDEKTTGWRGGHWEGLAGELEQLRARLEHHPQGQREP), Softag 3 (TQDPSRVG), Spot-tag (PDRVRAVSHWSS), Strep-tag (WSHPQFEK), TC tag (CCPGCC), Ty tag (EVHTNQDPLD), V5 tag (GKP1PNPLLGLDST), VSV-tag (YTD1EMNRLGK), Xpress tag (DLYDDDDK), GFP tag, Nus Tag, FC tag.MBP tag and GST tag.

The expressions “recombinant DNA having a secretion signal-PETase-marker peptide sequence” and the“Recombinant DNAl”mentioned herein, have the same meaning, and they can be used alternately with each other within the scope of the invention.

The espressions “recombinant DNA having a secretion signal -MHETase marker peptide sequence” and the“Recombinant DNA2” mentioned herein, have the same meaning, and they can be used alternately with each other within the scope of the invention.

Another aspect of the invention is the PETase enzyme (Enzyme 1) comprising a secretion signal at the forward primer and a marker peptide at the reverse primer. Another aspect of the invention is the MHETase enzyme (Enzyme2) comprising a secretion signal at the forward primer and a marker peptide at the reverse primer.

The secretion signal mentioned herein is selected from the group comprising AbnA, AmyE, AprE, BglC, BglS, Bpr, Csn, Epr, Ggt, GlpQ, HtrA, LipA, LytD, MntA, Mpr, NprE, OppA, PbpA, PbpX, Pel, PelB, PenP, PhoA, PhoB, PhoD, PstS, TasA, Vpr, WapA, WprA, XynA, XynD, YbdN, Ybxl, YcdH, YclQ, YdhF, YdhT, YfkN, YflE, YfmC, Yfnl, YhcR, YlqB, YncM, YnfF, YoaW, YocH, YolA, YqiX, Yqxl, YrpD, YrpE, YuaB, Yurl, YvcE, YvgO, YvpA, YwaD, YweA, YwoF, YwtD, YwtF, YxaLk, YxiA, YxkC.

The marker peptide mentioned herein is selected from the group comprising AviTag (GLNDIFEAQKIEWHE), Calmodulin- tag ( KRRWKKN F 1 AV S AAN RF KK1 S S S G AL) , polyglutamate tag (EEEEEE), E-tag (GAPVPYPDPLEPR), HA-tag (YPYDVPDYA), His-tag 5- 10 histidines (HHHHHH), Myc-tag (EQKLISEEDL), NE-tag (TKENPRSNQEESYDDNES), S- tag (KETAAAKFERQHMDS), SBP-tag (MDEKTTGWRGGHWEGLAGELEQLRARLEHHP QGQREP), Softag 3 (TQDPSRVG), Spot-tag (PDRVRAVSHWSS), Strep-tag (WSHPQFEK), TC tag (CCPGCC), Ty tag (EVHTNQDPLD), V5 tag (GKPIPNPLLGLDST), VSV-tag (YTDIEMNRLGK), Xpress tag (DLYDDDDK), GFP tag, Nus Tag, FC tag.MBP tag and GST tag.

The Enzymel and Enzyme2 according to the invention are enzymes that comprise secretion signals and marker peptides and are expressed in high yields under the T7, trc or PV1 promoter. By means of these enzymes according to the invention, the enzymes are excreted out of the bacteria rather than remaining on their surface, and can be easily purified with the help of the marker peptides thereon.

Another aspect of the invention is related to kits comprising Recombinant DNA1 or Recombinant DNA2. The kits according to the invention are suitable for use in the oanufacture of Enzymel or Enzyme2, and preferably they can comprise at least one excipient and/or solvent.

Another aspect of the invention is that Recombinant DNA1 is used in manufacturing of Enzyme 1.

Another aspect of the invention is that Recombinant DNA2 is used in manufacturing of Enzyme 2.

Another aspect of the invention is that Enzyme 1 is used in decomposition of aromatic polyesters.

Another aspect of the invention is that Enzyme 2 is used in the decomposition of the degradation products that are created due to the use of Enzyme 1 in the decomposition of aromatic polyesters.

The degradation product mentioned above can be mono(2-hydroxyethyl)terephthalate.

Another aspect of the invention is that Enzyme 1 and Enzyme 2 are used at the same time or sequentially to decompose aromatic polyesters.

According to another aspect, the invention is related to chemical compositions comprising Enzyme 1 and/or Enzyme 2.

Said compositions can be used in decomposing aromatic polyesters.

According to a preferred embodiment of the invention the chemical compositions comprising Enzyme 1 and/or Enzyme 2 can also comprise at least an excipient and/or solvent besides Enzyme 1 and/or Enzyme 2.

Although the aromatic polyesters mentioned herein can be selected from Polyethylene terephthalate (PET), Polybutylene terephthalate (PBT), Polytrimethylene terephthalate (PTT), Polyethylene naphthalate (PEN), it is particularly preferred to be Polyethylene terephthalate (PET).

From here-on, the invention shall be described by referring to the below mentioned examples which should not be construed to limit the scope of the invention in any way and which should be considered as illustration of the invention. EXAMPLES:

Examples 68-134 are the same as Examples 1-67 respectively in terms of secretion signal and marker peptide, as the enzyme MHETase enzyme has been used instead of PETase enzyme.

Examples 135-201 are the same as Examples 1-67 respectively in terms of secretion signal and enzyme, as marker peptide Calmodulin-tag (KRRWKKNFIAVSAANRFKKISSSGAL) has been used.

Examples 202-268 are the same as Examples 1-67 respectively in terms of secretion signal, MHETase enzyme has been used as enzyme and Calmodulin-tag (KRRWKKNFIAVSAANRFKKISSSGAL) has been used as marker peptide.

Examples 269-335 are the same as Examples 1-67 respectively in terms of secretion signal and enzyme, polyglutamate tag (EEEEEE) has been used as a marker peptide.

Examples 336-392 are the same as Examples 1-67 respectively in terms of secretion signal, as enzyme MHETase enzyme has been used and as marker peptide polyglutamate tag (EEEEEE) has been used.

Examples 393-495 are the same as Examples 1-67 respectively in terms of secretion signal and enzyme, and as marker peptide E-tag (GAPVPYPDPLEPR) has been used.

Examples 460-466 are the same as Examples 1-67 respectively in terms of secretion signal, as enzyme MHETase enzyme has been used and as marker peptide E-tag (GAPVPYPDPLEPR) has been used.

Examples 467-533 are the same as Examples 1-67 respectively in terms of secretion signal and enzyme, as marker peptide HA-tag (YPYDVPDYA) has been used. Examples 534-600 are the same as Examples 1-67 respectively in terms of secretion signal, as enzyme MHETase enzyme has been used and as marker peptide HA-tag (YPYDVPDYA) has been used.

Examples 601-667 are the same as Examples 1-67 respectively in terms of secretion signal and enzyme, as marker peptide His-tag 5-10 histidines (HHHHHH) has been used.

Examples 668-734 are the same as Examples 1-67 respectively in terms of secretion signal, as enzyme MHETase enzyme has been used and as marker peptide His-tag 5-10 histidines (HHHHHH) has been used.

Examples 735-801 are the same as Examples 1-67 respectively in terms of secretion signal and enzyme, as marker peptide Myc-tag (EQKL1SEEDL) has been used.

Examples 802-868 are the same as Examples 1-67 respectively in terms of secretion signal, as enzyme MHETase enzyme has been used and as marker peptide Myc-tag (EQKL1SEEDL) has been used.

Examples 869-935 are the same as Examples 1-67 respectively in terms of secretion signal and enzyme, as marker peptide NE-tag (TKENPRSNQEESYDDNES) has been used.

Examples 936-1002 are the same as Examples 1-67 respectively in terms of secretion signal, as enzyme MHETase enzyme has been used and as marker peptide NE-tag (TKENPRSNQEESYDDNES) has been used.

Examples 1003-1069 are the same as Examples 1-67 respectively in terms of secretion signal and enzyme, as marker peptide S-tag (KETAAAKFERQHMDS) has been used.

Examples 1070-1136 are the same as Examples 1-67 respectively in terms of secretion signal, as enzyme MHETase enzyme has been used and as marker peptide S-tag (KETAAAKFERQHMDS) has been used.

Examples 1137-1203 are the same as Examples 1-67 respectively in terms of secretion signal and enzyme, as marker peptide SBP-tag (MDEKTTGWRGGHWEGLAGELEQLRA RLEHHPQGQREP) has been used.

Examples 1204-1270 are the same as Examples 1-67 respectively in terms of secretion signal, as enzyme MHETase enzyme has been used and as marker peptide SBP-tag (MDEKTTGWRGGHWEGLA GELEQLRARLEHHPQGQREP) has been used.

Examples 1271-1337 are the same as Examples 1-67 respectively in terms of secretion signal and enzyme, as marker peptide Softag 3 (TQDPSRVG) has been used.

Examples 1338-1404 are the same as Examples 1-67 respectively in terms of secretion signal, as enzyme MHETase enzyme has been used and as marker peptide Softag 3 (TQDPSRVG) has been used.

Examples 1405-1471 are the same as Examples 1-67 respectively in terms of secretion signal and enzyme, as marker peptide Spot-tag (PDRVRAVSHWSS) has been used.

Examples 1472-1538 are the same as Examples 1-67 respectively in terms of secretion signal, as enzyme MHETase enzyme has been used and as marker peptide Spot-tag (PDRVRAVSHWSS) has been used.

Examples 1539-1605 are the same as Examples 1-67 respectively in terms of secretion signal and enzyme, as marker peptide Strep-tag (WSHPQFEK) has been used.

Examples 1606-1672 are the same as Examples 1-67 respectively in terms of secretion signal, as enzyme MHETase enzyme has been used and as marker peptide Strep-tag (WSHPQFEK) has been used.

Examples 1673-1739 are the same as Examples 1-67 respectively in terms of secretion signal and enzyme, as marker peptide TC tag (CCPGCC) has been used.

Examples 1740-1806 are the same as Examples 1-67 respectively in terms of secretion signal, as enzyme MHETase enzyme has been used and as marker peptide TC tag (CCPGCC) has been used. Examples 1807-1873 are the same as Examples 1-67 respectively in terms of secretion signal and enzyme, as marker peptide Ty tag (EVHTNQDPLD) has been used.

Examples 1874-1940 are the same as Examples 1-67 respectively in terms of secretion signal, as enzyme MHETase enzyme has been used and as marker peptide Ty tag (EVHTNQDPLD) has been used.

Examples 1941-2007 are the same as Examples 1-67 respectively in terms of secretion signal and enzyme, as marker peptide V5 tag (GKP1PNPLLGLDST) has been used.

Examples 2008-2074 are the same as Examples 1-67 respectively in terms of secretion signal, as enzyme MHETase enzyme has been used and as marker peptide V5 tag (GKP1PNPLLGLDST) has been used.

Examples 2075-2141 are the same as Examples 1-67 respectively in terms of secretion signal and enzyme, as marker peptide VSV-tag (YTD1EMNRLGK) has been used.

Examples 2142-2208 are the same as Examples 1-67 respectively in terms of secretion signal, as enzyme MHETase enzyme has been used and as marker peptide VSV-tag (YTD1EMNRLGK) has been used.

Examples 2209-2275 are the same as Examples 1-67 respectively in terms of secretion signal and enzyme, as marker peptide Xpress tag (DLYDDDDK) has been used.

Examples 2276-2343 are the same as Examples 1-67 respectively in terms of secretion signal, as enzyme MHETase enzyme has been used and as marker peptide Xpress tag (DLYDDDDK) has been used.

Examples 2344-2410 are the same as Examples 1-67 respectively in terms of secretion signal and enzyme, as marker peptide GFP tag has been used.

Examples 2411-2477 are the same as Examples 1-67 respectively in terms of secretion signal, as enzyme MHETase enzyme has been used and as marker peptide. GFP tag has been used

Examples 2478-2544 are the same as Examples 1-67 respectively in terms of secretion signal and enzyme, as marker peptide Nus Tag has been used..

Examples 2545-2611 are the same as Examples 1-67 respectively in terms of secretion signal, as enzyme MHETase enzyme has been used and as marker peptide Nus Tag has been used.

Examples 2612-2678 are the same as Examples 1-67 respectively in terms of secretion signal and enzyme, as marker peptide FC tag has been used.

Examples 2679-2746 are the same as Examples 1-67 respectively in terms of secretion signal, as enzyme MHETase enzyme has been used and as marker peptide FC tag has been used.

Examples 2747-2813 are the same as Examples 1-67 respectively in terms of secretion signal and enzyme, as marker peptide MBP tag been used.

Examples 2814-2880 are the same as Examples 1-67 respectively in terms of secretion signal, as enzyme MHETase enzyme has been used and as marker peptide MBP tag has been used.

Examples 2881-2947 are the same as Examples 1-67 respectively in terms of secretion signal and enzyme, as marker peptide GST been used.

Examples 2948-3016 are the same as Examples 1-67 respectively in terms of secretion signal, as enzyme MHETase enzyme has been used and as marker peptide GST has been used.

The secretion signals, PETase and MHETase enzymes and the amino acid sequences of marker peptides used within the scope of the invention and which have been provided in the above mentioned specific combination examples, shall be apparent to those skilled in the art without performing an additional inventive step to the prior art. Therefore even though they have not been mentioned in this description document, the specific combinations given to illustrate enzyme 1 and enzyme 2 according to the invention in the example sections and the amino acid sequences thereof are included within the scope of the invention.

Within these basic concepts, it is possible for several other embodiments to be carried out in relation to the subject matter of the invention, wherein the invention cannot be limited to the examples mentioned herein, and it is actually as described in the claims.

It is apparent that the person skilled in the art can provide the novelty disclosed in the invention using similar embodiments and/or can apply this embodiment to other fields having similar aims used in the related technique. As a result it is obvious that these kind of embodiments are void of the novelty and especially surpassing of the prior art, criteria.

Claims

1. A production method for use in producing PETase and/or MHETase enzymes (Method 1), comprises the steps of;

a. Obtaining Recombinant DNA1 specific to the PETase enzyme or Recombinant DNA2 specific to the MHETase enzyme by cloning recombinant genes comprising secretion signals in the N-terminus and marker peptides in the C-terminus after the T7, trc or PV1 promoter , b. Inserting the E. coli or B. subtilis bacteria into the genomic DNA of Recombinant DNA1 or Recombinant DNA2,

c. Excreting the formed PETase or MHETase enzyme out of the cell,

d. Purifying the PETase or MHETase enzymes that have been transported out of the cell.

2. A method for use in the production of PETase and/or MHETase enzyme (Method 2) comprises the steps of; a. Obtaining Recombinant DNA1 specific to the PETase enzyme or Recombinant DNA2 specific to the MHETase enzyme by cloning recombinant genes comprising secretion signals in the N-terminus and marker peptides in the C-terminus after the T7, trc or PV1 promoter , b. Obtaining Recombinant plasmid by cloning Recombinant DNA1 or Recombinant DNA2 to pET-3a vector or a derivative thereof,

c. Transferring the Recombinant plasmid to E.coli or B. subtilis,

d. Excreting the formed PETase or MHETase enzyme out of the cell

e. Purifying the PETase or MHETase enzymes that have been transported out of the cell.

3. A production method according to Claim 1 or 2, wherein the secretion signal is selected from the group comprising AbnA, AmyE, AprE, BglC, BglS, Bpr, Csn, Epr, Ggt, GlpQ, HtrA, LipA, LytD, MntA, Mpr, NprE, OppA, PbpA, PbpX, Pel, PelB, PenP, PhoA, PhoB, PhoD, PstS, TasA, Vpr, WapA, WprA, XynA, XynD, YbdN, Ybxl, YcdH, YclQ, YdhF, YdhT, YfkN, YflE, YfmC, Yfnl, YhcR, YlqB, YncM, YnfF, YoaW, YocH, YolA, YqiX, Yqxl, YrpD, YrpE, YuaB, Yurl, YvcE, YvgO, YvpA, YwaD, YweA, YwoF, YwtD, YwtF, YxaLk, YxiA, YxkC.

4. A production method according to Claim 1 or 2, wherein the marker peptide is selected from one of AviTag (GLNDIFEAQKIEWHE), Calmodulin-tag (KRRWKKN FIAVSAANRFKKISSSGAL), polyglutamate tag (EEEEEE), E-tag (GAPVPYPDPLEPR), HA- tag (YPYDVPDYA), His-tag 5-10 histidines (HHHHHH), Myc-tag (EQKLISEEDL), NE-tag (TKENPRSNQEESYDDNES), S-tag (KETAAAKFERQHMDS), SBP-tag (MDEKT TGWRGGHWEGLAGELEQLRARLEHHPQGQREP), Softag 3 (TQDPSRVG), Spot-tag (PDRVRAVSHWSS), Strep-tag (WSHPQFEK), TC tag (CCPGCC), Ty tag (EVHTNQDP LD), V5 tag (GKPIPNPLLGLDST), VSV-tag (YTDIEMNRLGK), Xpress tag (DLYDDDDK), GFP tag, Nus Tag, FC tag.MBP tag and GST tag or from a group comprised of different peptide sequences that provide the same function as these peptides.

5. PETase and/or MHETase enzyme obtained by using Method 1 according to claim 1.

6. PETase and/or MHETase enzyme obtained by using Method 2 according to claim 2.

7. Recombinant DNA (Recombinant DNA1) having a secretion signal-PETase-marker peptide sequence, comprising a secretion signal at the forward primer and a marker peptide at the reverse primer.

8. Recombinant DNA (Recombinant DNA2) having a secretion signal-MHETase-marker peptide sequence, comprising a secretion signal at the forward primer and a marker peptide at the reverse primer.

9. Recombinant DNA according to Claim 7 or 8 wherein the secretion signal is selected from a group comprising AbnA, AmyE, AprE, BglC, BglS, Bpr, Csn, Epr, Ggt, GlpQ, HtrA, LipA, LytD, MntA, Mpr, NprE, OppA, PbpA, PbpX, Pel, PelB, PenP, PhoA, PhoB, PhoD, PstS, TasA, Vpr, WapA, WprA, XynA, XynD, YbdN, Ybxl, YcdH, YclQ, YdhF, YdhT, YfkN, YflE, YfmC, Yfnl, YhcR, YlqB, YncM, YnfF, YoaW, YocH, YolA, YqiX, Yqxl, YrpD, YrpE, YuaB, Yurl, YvcE, YvgO, YvpA, YwaD, YweA, YwoF, YwtD, YwtF, YxaLk, YxiA, YxkC.

10. Recombinant DNA according to Claim 7 or 8 wherein the marker peptide is selected from AviTag (GLNDIFEAQKIEWHE), Calmodulin-tag ( KRRWKKN F 1 AV S AAN RF KK1 SSSGAL), polyglutamate tag (EEEEEE), E-tag (GAPVPYPDPLEPR), HA-tag (YPYDVPDYA), His-tag 5-10 histidines (HHHHHH), Myc-tag (EQKL1SEEDL), NE-tag (TKENPRSNQEESYDDNES), S-tag (KETAAAKFERQHMDS), SBP-tag (MDEKTTGWR GGHWEGLAGELEQLRARLEHHPQGQREP), Softag 3 (TQDPSRVG), Spot-tag (PDRV RAVSHWSS), Strep-tag (WSHPQFEK), TC tag (CCPGCC), Ty tag (EVHTNQDPLD), V5 tag (GKPIPNPLLGLDST), VSV-tag (YTDIEMNRLGK), Xpress tag (DLYDDDDK), GFP tag, Nus Tag, FC tag.MBP tag and GST tag or from a group comprised of different peptide sequences that provide the same function as these peptides.

11. PETase enzyme (Enzymel) comprising a secretion signal at the forward primer and a marker peptide at the reverse primer.

12. MHETase enzyme (Enzyme 2) comprising a secretion signal at the forward primer and a marker peptide at the reverse primer.

13. An enzyme according to claim 11 or 12 wherein the secretion signal is selected from the group comprising AbnA, AmyE, AprE, BglC, BglS, Bpr, Csn, Epr, Ggt, GlpQ, HtrA, LipA, LytD, MntA, Mpr, NprE, OppA, PbpA, PbpX, Pel, PelB, PenP, PhoA, PhoB, PhoD, PstS, TasA, Vpr, WapA, WprA, XynA, XynD, YbdN, Ybxl, YcdH, YclQ, YdhF, YdhT, YfkN, YflE, YfmC, Yfnl, YhcR, YlqB, YncM, YnfF, YoaW, YocH, YolA, YqiX, Yqxl, YrpD, YrpE, YuaB, Yurl, YvcE, YvgO, YvpA, YwaD, YweA, YwoF, YwtD, YwtF, YxaLk, YxiA, YxkC.

14. Enzyme according to Claim 11 or 12 wherein the marker peptide is selected from

AviTag (GLNDIFEAQKIEWHE), Calmodulin-tag (KRRWKKNFIAVSAANRFKKISSSGAL), polyglutamate tag (EEEEEE), E-tag (GAPVPYPDPLEPR), HA-tag (YPYDVPDYA), His-tag 5- 10 histidines (HHHHHH), Myc-tag (EQKLISEEDL), NE-tag (TKENPRSNQEESYDDNES), S- tag (KETAAAKFERQHMDS), SBP-tag (MDEKTTGWRGGHWEGLAGELEQLRARLEHHP QGQREP), Softag 3 (TQDPSRVG), Spot-tag (PDRVRAVSHWSS), Strep-tag

(WSHPQFEK), TC tag (CCPGCC), Ty tag (EVHTNQDPLD), V5 tag

(GKPIPNPLLGLDST), VSV-tag (YTDIEMNRLGK), Xpress tag (DLYDDDDK), GFP tag, Nus Tag, FC tag.MBP tag and GST tag or from a group comprised of different peptide sequences that provide the same function as these peptides.

15. Kits comprising Recombinant DNA1 or Recombinant DNA2 according to Claim 7 or 8.

16. A kit according to Claim 15, comprising at least an excipient and/or a solvent.

17. Use of Recombinant DNA1 according to Claim 7 for the manufacture of Enzyme 1 according to Claim 11.

18. Use of Recombinant DNA2 according to Claim 8 for the manufacture of Enzyme 2 according to Claim 12.

19. Use of Enzyme 1 according to Claim 11 for decomposition of aromatic polyesters.

20. Use of Enzyme 2 according to Claim 12, for decomposition of the degradation products created by the use of Enzyme 1 in the decomposition of aromatic polyesters.

21. Use according to Claim 20, wherein the degradation product is mono(2- hydroxyethyl)terephthalate.

22. Use of Enzyme 1 according to Claim 11 and Enzyme 2 according to Claim 12 at the same time or sequentially for decomposing aromatic polyesters.

23. Use according to Claim 19-22, wherein the aromatic polyesters are selected from polyethylene terephthalate (PET), polybutylene terephthalate (PBT), Polytrimethylene terephthalate (PTT), polyethylene naphthalate (PEN).

24. Chemical composition comprising Enzyme 1 according to Claim 11.

25. Chemical composition comprising Enzyme 2 according to Claim 12.

26. A chemical composition according to Claims 24-25 which comprises at least an excipient and/or solvent.