Method for enzymatic dechlorination and depolymerization of polyvinyl chloride

Abstract

Provided herein are systems and methods for the separation of polyvinyl chloride-coated wiring into its component parts through the use of biological products to degrade the polyvinyl chloride. Methods can include the incubation of wire waste with microbes or enzymes to dechlorinate the polyvinyl chloride for enhanced separation of the polyvinyl chloride from the wire's interior.

Description

[0001] METHOD FOR ENZYMATIC DECHLORINATION AND DEPOLYMERIZATION OF POLYVINYL CHLORIDE

[0002] CROSS REFERENCE

[0003]

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 644,687, filed May 09, 2024, the contents of which are incorporated herein by reference in their entirety.

[0004] BACKGROUND

[0005]

[0002] Copper is an important industrial metal due to its resistance to corrosion and its thermal and electrical conductivities that make it useful as a conductor in electrical cables. Polyvinyl chloride (PVC) is widely used in insulating layers in electrical cables. Currently, PVC material and electrical cables are deposited in landfills, or the metal components are recovered by smelting or incinerating the insulating layers. This method is energy’ intensive and releases toxic byproducts, such as dioxins and hydrogen chloride. To reduce the environmental impact of metal recovery from electrical wiring and other PVC-containing waste, an alternative method of degrading PVC is required.

[0006] SUMMARY

[0007]

[0003] Methods and inventions presented here may improve current recycling or disposal methods for wire and polyvinyl chloride waste by degrading or dechlorinating wire waste comprising polyvinyl chloride with the aid of enzymatic degradation.

[0008]

[0004] In an aspect, the present disclosure provides a method of separating components of wire waste. The method may comprise: combining a wire waste with a medium comprising a lignin peroxidase, a catalase peroxidase, and a dehalogenase, wherein the wire waste comprises a transmitting portion encapsulated at least partially in an encapsulating layer comprising polyvinyl chloride; and at least partially separating the transmitting portion from the encapsulating layer by degrading the polyvinyl chloride of the encapsulating layer, wherein the lignin peroxidase, the catalase peroxidase, and the dehalogenase cause the degradation.

[0009]

[0005] In some embodiments, the lignin peroxidase may comprise an amino acid sequence with at least a 90% identity to SEQ ID NO: 1. In some embodiments, the catalase peroxidase may comprise an amino acid sequence with at least a 90% identity' to SEQ ID NO: 3. In some embodiments, the dehalogenase may comprise an amino acid sequence with at least a 90% identity to SEQ ID NO: 5.

[0006] In some embodiments, the degradation may comprise oxidation. In some embodiments, the degradation may comprise chain depolymerization. In some embodiments, the degradation may comprise hydrolysis. In some embodiments, the degradation may comprise dechlorination.

[0007] In some embodiments, the dehalogenase may dechlorinate the polyvinyl chloride. In some embodiments, the lignin peroxidase may dechlorinate the polyvinyl chloride.

[0010]

[0008] In some embodiments, the medium may further comprise a laccase or a monooxygenase. In some embodiments, the degradation of the polyvinyl chloride may generate a degradation product. In some embodiments, the method may further comprise mineralizing the degradation product. In some embodiments, the laccase or the monooxygenase mineralizes the degradation product.

[0011]

[0009] In some embodiments, the medium may comprise a cell lysate. In some embodiments, the medium may comprise a microorganism configured to produce the lignin peroxidase, the catalase peroxidase, or the dehalogenase. In some embodiments, the microorganism may comprise a bacterium or a yeast. In some embodiments, the bacterium comprises Escherichia coli. In some embodiments, the yeast comprises Kluyveromyces lactis. In some embodiments, the medium comprises at least one enzyme co-factor. In some embodiments, the at least one enzyme co-factor may improve the degradation of the polyvinyl chloride by the lignin peroxidase, the catalase peroxidase, or the dehalogenase.

[0012]

[0010] In some embodiments, the transmitting portion may comprise a metal portion. In some embodiments, the metal portion may comprise copper. In some embodiments, the metal portion may comprise gold, iron, nickel, tin, lead, aluminum, germanium, or zinc. In some embodiments, the metal portion may comprise a rare earth metal. In some embodiments, the rare earth metal may comprise neodymium, lanthanum, yttrium, or dysprosium.

[0013]

[0011] In some embodiments, the wire waste may comprise electrical wire waste, PVC-jacketed cable waste, wire scrap, electrical wire harness, mixed metal wire waste, fiber optic cable, cablerich e-waste, or dismantled electronics. In some embodiments, the wire waste may comprise a wire configured to transmit electricity or information.

[0014]

[0012] In some embodiments, degrading the polyvinyl chlonde may increase a brittleness of the encapsulating layer. In some embodiments, degrading the polyvinyl chloride may form cracks in the encapsulating layer.

[0015]

[0013] In some embodiments, the method may further comprise mechanically separating the metal portion from the encapsulating layer. In some embodiments, mechanically separating may comprise shredding or ball milling the wire waste. In some embodiments, mechanically separating may further comprise sieving the wire waste. In some embodiments, mechanically separating may further comprise flotation of the wire waste.

[0016]

[0014] In some embodiments, the method may further comprise treating a waste stream to produce the wire waste. In some embodiments, treating the waste stream may comprise shredding the waste stream and gravity separating the waste stream into a recycled metal portion, a free layer portion, and a portion comprising the w ire waste. In some embodiments, treating the waste stream may further comprise magnetically separating a magnetic portion of the waste stream.

[0017]

[0015] In another aspect, the present application provides a method of separating components of wire waste compnsing. The method may comprise: combining a wire waste with a medium comprising a microorganism, wherein the wire waste comprises a transmitting portion encapsulated at least partially in an encapsulating layer comprising polyvinyl chloride; and at least partially separating the transmitting portion from the encapsulating layer by degrading the polyvinyl chloride of the encapsulating layer, wherein the microorganism degrades the polyvinyl chloride by dechlorinating the polyvinyl chloride.

[0018]

[0016] In some embodiments, the dechlorination comprises an enzymatic process. In some embodiments, the enzymatic process comprises a dehalogenation step, an oxidation step, a chain depolymerization step, or a hydrolysis step.

[0019]

[0017] In some embodiments, the microorganism may be genetically modified to express an enzyme of the enzy matic process. In some embodiments, the enzyme may be secreted by the microorganism. In some embodiments, the enzy me comprises a catalase peroxidase, a lignin peroxidase, a dehalogenase, a laccase, or a monooxygenase. In some embodiments, the enzyme may comprise a mutant of a catalase peroxidase, a dehalogenase, a lignin peroxidase, a laccase, or a monooxygenase.

[0020]

[0018] In some embodiments, the enzyme may comprise a catalase peroxidase portion and a dehalogenase portion. In some embodiments, the enzyme may further comprise a lignin peroxidase portion.

[0021]

[0019] In some embodiments, the enzyme may comprises an amino acid sequence with at least 90% identity to SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5. In some embodiments, the microorganism may be genetically modified to express a second enzy me of the enz matic process. In some embodiments, the microorganism may be genetically modified to express a third enzyme of the enzymatic process.

[0020] In some embodiments, the method may further comprise mineralizing a degradation product formed by the degradation of the polyvinyl chloride. In some embodiments, the mineralization may comprise an enzymatic process. In some embodiments, a laccase or a monooxygenase may mineralize the degradation product.

[0022]

[0021] In some embodiments, the microorganism comprises a bacterium or a yeast. In some embodiments, the bacterium comprises Escherichia coli. In some embodiments, the yeast comprises Kluyveromyces lactis.

[0023]

[0022] In some embodiments, the transmitting portion may comprise a metal portion. In some embodiments, the metal portion may comprise copper. In some embodiments, the metal portion may comprise gold, iron, nickel, tin, lead, aluminum, germanium, or zinc. In some embodiments, the metal portion may comprise a rare earth metal. In some embodiments, the rare earth metal may comprise neodymium, lanthanum, yttrium, or dysprosium.

[0024]

[0023] In some embodiments, the wire waste comprises electrical wire waste, PVC -jacketed cable waste, wire scrap, electrical wire harness, fiber optic cable, mixed metal wire waste, cablerich e-waste, or dismantled electronics. In some embodiments, the wire waste may comprise a wire configured to transmit electricity or information.

[0025]

[0024] In some embodiments, degrading the polyvinyl chloride may increase a brittleness of the encapsulating layer. In some embodiments, degrading the polyvinyl chloride may form cracks in the encapsulating layer.

[0026]

[0025] In some embodiments, the method may further comprise mechanically separating the metal portion from the encapsulating layer. In some embodiments, mechanically separating comprises ball milling the wire waste. In some embodiments, mechanically separating further comprises sieving the wire waste. In some embodiments, mechanically separating further comprises flotation of the wire waste.

[0027]

[0026] In some embodiments, the method may further comprise treating a waste stream to produce the wire waste. In some embodiments, treating the waste stream may comprise shredding the waste stream and gravity’ separating the waste stream into a recycled metal portion, a free layer portion, and the wire waste portion. In some embodiments, treating the waste stream may further comprise magnetically separating a magnetic portion of the waste stream.

[0028]

[0027] In an aspect, the present application may provide a nuclei acid comprising: a genetic sequence comprising at least 90% sequence identity with SEQ ID NO: 9, wherein the genetic sequence comprises a sequence of a catalase peroxidase that is codon-optimized for expression in Escherichia coli.

[0029]

[0028] In an aspect, the present application may provide a nuclei acid comprising: a genetic sequence comprising at least 90% sequence identity with SEQ ID NO: 7, wherein the genetic sequence comprises a sequence of a lignin peroxidase that is codon-optimized for expression in Escherichia coh.

[0030]

[0029] In an aspect, the present application may provide a nuclei acid comprising: a genetic sequence comprising at least 90% sequence identity' with SEQ ID NO: 11, wherein the genetic sequence comprises a sequence of a dehalogenase that is codon-optimized for expression in Escherichia coli.

[0031]

[0030] In an aspect, the present application may provide a method of separating components of wire w aste. The method may comprise: combining a ware waste with a medium comprising a lignin peroxidase, wherein the wire waste comprises a transmitting portion encapsulated at least partially in a layer comprising polyvinyl chloride; and at least partially separating the transmitting portion from the encapsulating layer by degrading the polyvinyl chloride of the layer, wherein the lignin peroxidase degrades the polyvinyl chloride.

[0032]

[0031] In some embodiments, the lignin peroxidase may comprise an amino acid sequence with at least 90% identity to SEQ ID NO: 1.

[0033]

[0032] In an aspect, the present application may provide a method of separating components of wire waste. The method may comprise: combining a wire w aste with a medium comprising a catalase peroxidase, wherein the wire w aste comprises a transmitting portion encapsulated at least partially in an encapsulating layer comprising polyvinyl chloride; and at least partially separating the transmitting portion from the encapsulating layer by degrading the polyvinyl chloride of the encapsulating layer, wherein the catalase peroxidase degrades the polyvinyl chloride.

[0034]

[0033] In some embodiments, the catalase peroxidase may comprise an amino acid sequence with at least 90% identity’ to SEQ ID NO: 3.

[0035]

[0034] In an aspect, the present application may provide a method of separating components of wire waste. The method may comprise: combining a wire w aste with a medium comprising a dehalogenase, wherein the wire waste comprises a transmitting portion encapsulated at least partially in an encapsulating layer comprising polyvinyl chloride; and at least partially separating the transmitting portion from the encapsulating layer by degrading the polyvinyl chloride of the layer, wherein the dehalogenase degrades the polyvinyl chloride.

[0035] In some embodiments, the dehalogenase may comprise an amino acid sequence with at least 90% identity to SEQ ID NO: 5.

[0036]

[0036] In an aspect, the present application may provide a system for metal recovery. The system may comprise: a reaction vessel configured to contain a wire waste comprising a transmitting portion encapsulated at least partially in an encapsulating layer comprising polyvinyl chloride, and a medium comprising an enzyme combination configured to degrade the polyvinyl chloride of the encapsulating layer, wherein the enzyme combination comprises a lignin peroxidase, a catalase peroxidase, and a dehalogenase; and a separation vessel connected to the reaction vessel, wherein the separation vessel is configured to mechanically separate the transmitting portion from the encapsulating layer. In some embodiments, the transmitting portion may comprise a metal portion. In some embodiments, the wire waste may comprise a wire configured to transmit electricity or information.

[0037] BRIEF DESCRIPTION OF THE DRAWINGS

[0038]

[0037] The novel features of the invention are set forth in the appended claims of the present application. A better understanding of the advantages of the present invention may be obtained by reference to the following detailed description that sets forth illuminating embodiments, and in the accompanying drawings of which:

[0039]

[0038] FIG. 1A shows the chemical structure of polyvinyl chloride (PVC), and FIG. IB shows the chemical structure of lignin.

[0040]

[0039] FIG. 2 shows a process for degradation of PVC-insulated cable and analysis of conductor recovery.

[0041]

[0040] FIG. 3 shows a graphical circular map of a pET30a(+) plasmid modified to contain the catalase peroxidase enzyme (CPKv) bom Klebsiella sp. EMBL-1.

[0042]

[0041] FIG. 4 shows a graphical circular map of a pET30a(+) plasmid modified to contain the lignin peroxidase (LPPc) from Phanerochaete chrysosporium .

[0043]

[0042] FIG. 5 shows a graphical circular map of a pET30a(+) plasmid modified to contain the dehalogenase (LKv) from Klebsiella variicola.

[0044]

[0043] FIG. 6 shows the protein concentration (pg / mL) of the three enzymes as determined by the BCA assay.

[0045]

[0044] FIG. 7 shows SDS-PAGE gels for identification of LKv and LPPc enzy mes. FIG. 7A shows Lumio detection of LKv and LPPc using a UV transilluminator. FIG. 7B shows an image of a protein gel containing elution fractions of LKv and LPPc stained with Coomassie Blue.

[0045] FIG. 8 shows SDS-PAGE gels for identification of the CPKv enzyme. FIG. 8A shows Lumio detection of CPKv using a UV transilluminator. FIG. 8B shows an image of a protein gel containing elution fractions of CPKv stained with Coomassie Blue.

[0046]

[0046] FIG. 9 shows the results of a second protein extraction and purification protocol. FIG. 9A shows a microplate showing the colors of the wells after the BCA assay. FIG. 9B shows a standard curve for BCA assay analysis. FIG. 9C shows a bar plot showing the concentration results in pg / mL for each enzyme. FIG. 9D shows updated concentration measurements after desalting with the Amicon filter.

[0047]

[0047] FIG. 10 shows the SDS-PAGE gel results of a second protein extraction and purification protocol FIG. 10A shows a SDS-PAGE stained with Coomassie blue for LKv and CPKv. FIG. 10B shows a SDS-PAGE with Lumio detection for LKv and CPKv.

[0048]

[0048] FIG. 11 shows the catalase activity of CPKv samples in terms of units per liter (U / L).

[0049]

[0049] FIG. 12 shows the assay results of LKv for laccase enzyme kinetics using all fractions of the protein purification protocol.

[0050]

[0050] FIG. 13 shows the experimental design of the degradation of PVC pellets by engineered strains of E. coli. FIG. 13A shows experimental design of the degradation of PVC pellets from ACI, Mexichem, and PVC powder. FIG. 13B shows the experimental design of the degradation of PVC pellets from Westlake and Manner.

[0051]

[0051] FIG. 14 shows mass variation comparing the different strains with the 4 different PVC types.

[0052]

[0052] FIG. 15 shows the OD600 of the three engineered strains of E. coli (LKv, LPPc, and CPKv), induced and not induced, after 9 days of incubation with PVC powder.

[0053]

[0053] FIG. 16 shows the experimental design for a degradation halo test of PVC powder by the engineered strains of E. coli.

[0054]

[0054] FIG. 17 shows colonies on M9 agar plates prepared according to the experimental design in FIG. 16

[0055]

[0055] FIG. 18 shows FTIR results for PVC ACI T2-2 incubated with a lysate containing LKv+LPPc.

[0056]

[0056] FIG. 19 shows FTIR results for PVC Westlake incubated with a lysate containing LKv+CPKv.

[0057]

[0057] FIG. 20 shows FTIR results for PVC Mexichem incubated with a lysate containing LPPc.

[0058]

[0058] FIG. 21 shows FTIR results for PVC Manner incubated with a lysate containing CPKv.

[0059] FIG. 22 shows the FTIR results of Pure PVC incubated with lysates, and comparisons of FTIR results from different samples. FIG. 22A shows FTIR results for Pure PVC incubated with a lysates containing LKv+LPPc and LKv+CPKv. FIG. 22B shows the FTIR results indicating the combination of LKv and CPKv lysates can degrade the Westlake PVC powder via oxidation. FIG. 22C shows FTIR results comparing signal change between samples 2, 11, 24, and 15 of Table 2 for lysate degradation of different PVCs.

[0059]

[0060] FIG. 23 shows the FTIR results of PVC Mexichem treated with the enzyme combination LKv+LPPc+CPKv.

[0060]

[0061] FIG. 24 shows the FTIR results of PVC ACI T2-2 treated with the enzyme combination LKv+LPPc+CPKv.

[0061]

[0062] FIG. 25 shows the FTIR results of PVC Westlake treated with the enzyme combination LKv+LPPc+CPKv.

[0062]

[0063] FIG. 26 shows the FTIR results of PVC Manner treated with the enzyme combination LKv+LPPc+CPKv

[0063]

[0064] FIG. 27 shows the FTIR results of pure PVC powder treated with the enzyme combination LKv+LPPc+CPKv.

[0064]

[0065] FIG. 28 shows the experimental design for a long-term experiment comparing PVC degradation by cell extracts and purified fractions of LKv and CPKv enzy mes. FIG. 28A shows the experimental design for a long-term experiment comparing PVC powder degradation by cell extracts and purified fractions of LKv and CPKv enzymes for FTIR analysis. FIG. 28B shows the experimental design for a long-term experiment comparing PVC wire degradation by cell extracts and purified fractions of LKv and CPKv enzymes for SEM imaging and analysis.

[0065]

[0066] FIG. 29 shows the FTIR results for samples described in the experiment design of FIG. 28A. FIG. 29A shows the FTIR results for PVC powder incubated with the high concentration LKv and CPKv enzyme lysate combination across 12 weeks. FIG. 29B shows the FTIR results for PVC powder incubated with the low concentration LKv and CPKv enzyme ly sate combination across 12 weeks. FIG. 29C shows the FTIR results for PVC powder incubated with the high concentration LKv and CPKv purified enzy me combination across 12 weeks. FIG. 29D shows the FTIR results for PV C poyvder incubated with the low concentration LKv and CPKv purified enzy me combination across 12 weeks. FIG. 29E shows the FTIR results for PVC Westlake incubated with the high concentration LKv and CPKv enzyme lysate combination across 12 weeks. FIG. 29F shows the FTIR results for PVC Westlake incubated with the low concentration LKv and CPKv enzyme lysate combination across 12 yveeks. FIG. 29G shows the FTIR results for PVC Westlake incubated with the high concentration LKv and CPKv purified enzyme combination across 12 weeks. FIG. 29H shows the FTIR results for PVC Westlake incubated with the low concentration LKv and CPKv purified enzyme combination across 12 weeks.

[0066]

[0067] FIG. 30 shows the FTIR results for samples described in the experiment design of FIG. 28B. FIG. 30A shows the FTIR results for PVC wire samples incubated with the high concentration LKv and CPKv enzyme lysate combination across 12 weeks. FIG. 30B shows the FTIR results for PVC wire samples incubated with the low concentration LKv and CPKv purified enzyme combination across 12 weeks.

[0067]

[0068] FIG. 31 shows low magnification (200-250x) SEM images comparing different samples of Westlake PVC. FIG. 31A, shows no treatment. FIG. 31B was treated for 5 weeks with lysate high, FIG. 31C was treated for 5 weeks with enzy me low: FIG. 31D show s the w ater control.

[0069] FIG. 32 shows medium magnification (1000-2000x) SEM images comparing different samples of Westlake PVC. FIG. 32A shows no treatment. FIG. 32B was treated for 5 weeks with lysate high. FIG. 32C was treated for 5 weeks with enz me low. FIG. 32D shows the water control.

[0068]

[0070] FIG. 33 show s high magnification (5000x) SEM images comparing different samples of Westlake PVC. FIG. 33A shows no treatment. FIG. 33B was treated for 5 weeks with lysate high. FIG. 33C was treated for 5 weeks with enzyme low. FIG. 33D shows the water control.

[0071] FIG. 34 shows FTIR spectra of PVC incubated with E. coli expressing LPPc, CPKv, and LKv in LB Media at 0, 2 and 4 weeks. FIG. 34A show s FTIR spectra of PVC incubated with E. coli expressing LPPc, CPKv, and LKv in LB Media at 0 weeks. FIG. 34B shows FTIR spectra of PVC incubated with E. coli expressing LPPc, CPKv, and LKv in LB Media at 2 weeks. FIG. 34C shows FTIR spectra of PVC incubated with E. coli expressing LPPc, CPKv, and LKv in LB Media at 4 weeks.

[0069]

[0072] FIG. 35 show s FTIR spectra of PVC incubated with E. coli expressing LPPc, CPKv, and LKv in M9 Minimal Media with glucose at 0 and 2 weeks. FIG. 35A shows FTIR spectra of PVC incubated with E. coli expressing LPPc, CPKv, and LKv in M9 Minimal Media with glucose at 0 weeks. FIG. 35B shows FTIR spectra of PVC incubated with E. coli expressing LPPc, CPKv, and LKv in M9 Minimal Media with glucose at 2 weeks.

[0070]

[0073] FIG. 36 show s FTIR spectra of PVC incubated with E. coli expressing LPPc, CPKv, and LKv in M9 Minimal Media without glucose at 0 and 2 weeks. FIG. 36A shows FTIR spectra of PVC incubated with E. coli expressing LPPc, CPKv, and LKv in M9 Minimal Media without glucose at 0 weeks. FIG. 36B shows FTIR spectra of PVC incubated with E. coli expressing LPPc, CPKv, and LKv in M9 Minimal Media without glucose at 2 weeks.

[0071]

[0074] FIG. 37 shows polypropylene culture tubes containing neat PVC incubated with E. coli strains expressing LKv (dehalogenase), CPKv (catalase peroxidase), and LPPc (lignin peroxidase).

[0072] DETAILED DESCRIPTION

[0073]

[0075] The present application provides inventions and methods directed towards the degradation of polyvinyl chloride (PVC) through the use of microbes, enzymes, or other biological products or processes, as well as inventions and methods directed towards the separation of metal portions of wire waste from an encapsulating layer. The PVC may be a component of a wire, electrical cabling, consumer electronics, or a combination thereof. In some embodiments, the PVC may be a component of a waste product of the wire, electrical cabling, or consumer electronics, or a derivative of one or a combination of these waste products.

[0074]

[0076] In some embodiments a wire waste comprises a metal or metallic portion encapsulated at least in part by an insulating layer. The insulating layer may be polyvinyl chloride (PVC). The PVC may be standard PVC, semi-rigid PVC, or irradiated PVC. The PVC may comprise pure PVC or PVC with one or more additives. These additives may be plasticizers, such as phthalates, adipates, citrates, trimellites, dibenzoates, isosorbide diesters, or bio-based plasticizers. In some embodiments, a phthalate plasticizer is a terephthalate, a diethylhexyl phthalate (DEHP), a diisononyl phthalate (DINP), a diisodecyl phthalate (DIDP), or a di-(2-ethylhexyl) terephthalate (DEHT).

[0075]

[0077] The metal or metallic portion of the wire waste may comprise a metal such as copper, gold, iron, nickel, tin, lead, aluminum, germanium, zinc, or alloys thereof. The metal portion may comprise a rare earth metal such as neodymium, lanthanum, yttrium, or dysprosium. The metal portion may comprise germanium.

[0076]

[0078] In some embodiments, the wire waste comprises electrical wire waste. Electrical wire waste may be PVC-jacketed cable waste, insulated copper wire, wire scrap, electrical wire scrap, automotive wire scrap, multi-strand conductor waste, electrical wire harness, mixed metal wire waste, cable-rich e-waste, e-waste cable fractions, dismantled electronics, insulated wire from dismantled electronics, end-of-life wiring, discarded or damaged electrical cables, harnessed conductor waste, harness scrap, cable assemblies, wiring loom waste, copper-bearing wire scrap, post-consumer electrical wiring, or combinations thereof.

[0077]

[0079] In some embodiments, a wire waste comprises electronic waste. Electronic waste may comprise a computer, such as a desktop computer, laptop, notebook, or a server, or portions thereof. In some embodiments, electronic waste may comprise computer monitors, such as CRT, LCD, LED, and OLED monitors, or portions thereof. In some embodiments, electronic waste may comprise printers and scanners, keyboards and mice. In some embodiments, electronic waste may comprise motherboards, sound cards, hard drives, internal and external storage devices, wiring components of computers. In some embodiments, electronic waste may comprises a solar panel or a portion thereof. In some embodiments, an electronic waste comprises a telecommunication device or a portion thereof.

[0078]

[0080] In some embodiments, a wire waste may be mechanically disrupted prior to degradation by a microbe, or enzy me, or other biological product. Mechanical disruption may include shredding or separation. Shredding may be shredding such as is done by a wire shredder or crusher. Wire waste may be shredded into portions of various sizes or of a unified size. Separation may include magnetic separation or gravity separation. Gravity separation may be conducted by a gravity sorting machine that separates metal and insulating layer portions of waste by weight. Gravity separation may create a waste stream of mixed metal and insulating layer. The waste stream of mixed metal and insulating layer may be further separated by the degradation of the insulating layer by a biological product or process.

[0079]

[0081] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the claims. As used in the description of the embodiments and the appended claims, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will also be understood that the term "and / or" as used herein refers to and encompasses any and all possible combinations of one or more of the associated listed items. It will be further understood that the terms "comprises" and / or "comprising," when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof.

[0080]

[0082] As used herein, and unless otherwise specified, the term "about" or "approximately" means an acceptable error for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined. In certain embodiments, the term "about" or "approximately" means within 1, 2, 3, or 4 standard deviations. In certain embodiments, the term "about" or "approximately" means within 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, or 0.05% of a given value or range.

[0081]

[0083] As used herein, the terms "comprises", "comprising", or any other variation thereof, are intended to cover a nonexclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.

[0082]

[0084] Plastics comprise a wide range of materials, including natural and synthetic materials, comprising mostly organic polymers, also referred to as polymers, as their main ingredient. Organic polymers are chains of carbon atoms comprising repeating units or subunits. These repeating units or subunits may comprise one or more monomers. In some cases, an organic polymer may comprise two or more repeating types, such as with a copolymer. A copolymer comprises two or more monomers of differing types. These monomers may repeat each other in regular patterns, or irregularly. Organic polymers may comprise portions comprising branched polymers, linear polymers, or combinations thereof. A branched polymer is composed of a main chain with one or more side chains or branches. Organic polymers may cross-link with themselves or with other organic polymers.

[0083]

[0085] Polyvinyl chloride (PVC), the structure of which is displayed in FIG. 1A, is a petroleumbased solid polymer comprising repeating units of the vinyl chloride monomer. PVC is thermoplastic, highly hydrophobic, soluble in tetrahydrofuran (THF), and resilient to chemical abrasion, with high chemical and physical stability. PVC may be combined with a wide variety of additives such as plasticizers, stabilizers, flame retardants, and fillers. PVC is also non- hydrolyzable, with a C-C backbone that makes its degradation challenging compared to plastics with a heteroatomic backbone containing hydrolysable bonds, such as polyurethane and polyethylene terephthalate.

[0084]

[0086] PVC comprises a low biodegradability, leading to persistent plastic pollution and longterm environmental hazards when deposited in landfills. PVC can leach additives into the surrounding soil and groundwater. These additives may be toxic, contributing to environmental contamination and potential health risks. Toxic additives may include phthalates and heavy metal stabilizers. The accumulation of PVC waste in landfills may reduce landfill lifespan and necessitates costly remediation efforts. Given these consequences, sustainable alternatives such as biodegradation present a promising pathway to mitigating PVC pollution.

[0087] Biodegradation of plastics is the process of physio-chemical transformation of polymers into smaller units mediated by microorganisms or their byproducts. Some microbial enzy mes may be able to biodegrade PVC. Enzy mes are macromolecular protein compounds that act as chemical catalysts in the plastic degradation process by lowering the amount of energy required for activation and turning the substrate into the product, such that degradation may happen faster than in the absence of the enzyme. Microbial enzymes suspected to be involved in the degradation of PVC may be produced by fungi or bacteria living in the gut of xylophagous invertebrates whose diet consists primarily or solely of wood, where these potential enzy mes would assist in the breakdown of compounds that provide structural rigidi ty to the plant, such as lignin. Lignin is a non-carbohydrate natural aromatic polymer, the structure of which is displayed in FIG. IB.

[0085]

[0088] The biodegradation of PVC can involve several ty pes of reactions, including dechlorination, oxidation, chain depolymerization, and mineralization of formed intermediates. FIG. 2 shows an example schematic of a chain of reactions to degrade PVC with catalase peroxidase, dehalogenase, laccase, and monooxygenase. P VC-degrading activity has been reported for an extracellular lignin peroxidase of the fungus Phanerochaete chrysosporium, and for an extracellular catalase peroxidase of the Klebsiella sp. EMBL-1. A PVC degradation pathway of strain EMBL-1 has been proposed that suggests a class of enzymes that may be involved in the degradation of PVC.

[0086]

[0089] In some embodiments, inventions of this application may comprise recombinant enzymes comprising gene sequences encoding PVC degrading enzymes that have been cloned from original organisms into E. coli. These enzy mes may have had their expression and activities on PVC verified. These enzymes may include lignin peroxidase, catalase peroxidase, and dehalogenase. In some embodiments, recombinant enzymes can be used to degrade the PVC insulating layer in waste electric cables. This may7enable the efficient recovery7of copper or other metals from wire harnesses. In some embodiments, enzy matic degradation dechlorinates PVC and thereby facilitates downstream copper recovery, as the dechlorinated PVC can be safely introduced into a smelter alongside copper wires, behaving like an organic fuel without releasing chlorine and toxic byproducts.

[0087]

[0090] In some embodiments, PVC may be degraded by the enzy me catalase peroxidase (CPKv, SEQ ID NO: 3) from Klebsiella strain EMBL-1, dehalogenase (LKv, SEQ ID NO: 5) from Klebsiella strain EMBL-1, lignin peroxidase (LPPc, SEQ ID NO: 1) from the fungus Phanerochaete chrysosporium, or combinations thereof. Table 1 describes the suggested catalytic activity of these enzymes. In some embodiments, PVC may be degraded by a protein comprising CPKv, LKv, or LPPc, or a portion thereof. These enzymes may be expressed in a bacterial host, or a eukaryotic host. A bacterial host may be Escherichia coli (E. coli). A eukaryotic host may be a yeast, such as Kluyveromyces lactis (K. lactis). The PVC may be enzymatically degraded into byproducts, including chlorine byproducts or partially degraded PVC. Partially degraded PVC may comprise PVC monomers, PVC comprising partially- oxidized carbon backbones, or PVC molecules cleaved into two or more separate chains of PVC monomers. Chlorine byproducts or partially degraded PVC generated during enzymatic degradation may be further degraded. Further degradation may be done by additional enzymes, microbial metabolic processes, chemical treatments, or combinations thereof.

[0088]

[0091] The degradation of the PVC may cause or enhance the separation of a metal portion of a wire waste from an encapsulating layer. The degradation of the PVC may cause or enhance the separation by increasing the brittleness of the encapsulating layer, physically separating the encapsulating layer from the metal portion, warping the encapsulating layer, mechanically shifting the encapsulating layer, splitting the encapsulating layer, forming micro-fractures or cracks in the encapsulating layer, increasing the roughness of the encapsulating layer, or combinations thereof.

[0089] Table 1. Catalytic activity of enzymes selected for testing the dechlorination of polyvinyl chloride (PVC) from wire waste.

[0090]

[0092] Biodegradation of plastics is a complex physio-chemical process. In some cases, enzymes produced by microbes such as Phcmerochaete chrysosporium and Klebsiella sp. EMBL-1 can interact with the chemical linkages of plastic polymers. These interactions can include the breaking or modification of chemical bonds. In some embodiments, the chemical bond broken may be a single, double, or triple covalent bond, or a carbon-carbon bond, a carbonoxygen bond, a carbon-nitrogen bond, or a carbon-halogen bond. In some embodiments, the carbon-halogen bond may comprise a carbon-chloride bond, a carbon-fluoride bond, a carbonbromide bond, or a carbon-iodide bond. Some enzymes produced by microbes are a part of the microbe’s metabolic pathway. These enzymes may break down polymers found in nature, such as lignin. These enzymes may also break down synthetic polymers, such as polyvinyl chloride (PVC).

[0091]

[0093] In some embodiments, one or more enzymes may degrade a PVC molecule. The one or more enzymes may degrade the PVC by cleaving one or more bonds in a PVC molecule. These one or more bonds may comprise a carbon-chlorine (C-Cl) bond or a carbon-carbon (C-C) bond. The cleaving of this bond may result in or be the result of the formation of one or more new bonds, such as a double carbon (C=C), carbon-oxygen (C=O), carbon-hydrogen (C-H), or a hydroxide (O-H) bond. In some embodiments, the one or more enzy mes may comprise a lignin peroxidase, a catalase peroxidase, a dehalogenase, a laccase, a monooxygenase, or combinations thereof.

[0092]

[0094] In some embodiments, one or more enzy mes may degrade a PVC molecule by dechlorinating the PVC molecule, oxidizing the PVC molecule, or hydrolyzing the PVC molecule. In some embodiments, a PVC molecule may be resistant to enzymatic degradation as a result of chlorine atoms of the PVC molecule blocking access to the carbon backbone of the PVC molecule. In some embodiments, one or more enzymes may enhance degradation of a PVC molecule by dechlorinating a portion of the PVC such that other enzymes may access the carbon backbone. Dechlorination may be the result of directly cleaving C-Cl bonds, or of forming other bonds that disrupt C-Cl bonds. Other bonds may include C=C, C=O, C-H, or O-H bonds. In some embodiments, dechlorinating the PVC molecule may make the PVC molecule less toxic in a downstream process, such as smelting.

[0093]

[0095] In some embodiments, one or more enzymes may degrade a PVC molecule by oxidizing a portion of the PVC. The one or more enzy mes may oxidize a portion of the carbon backbone of the PVC molecule. A C-C bond may be oxidized to a C=C bond, or a C-H bond may be oxidized to a C=O bond. In some embodiments, one or more enzymes may degrade the PVC molecule through hydrolysis of a C=C bond. In some embodiments, the one or more enzymes may mineralize at least a portion of the PVC molecule. Mineralization is the process of breaking dow n a polymer molecule into carbon dioxide (CO2) and water. PVC may be mineralized into CO2, water, and Ch.

[0094]

[0096] In some embodiments, the cleavage of C-Cl bonds may result in the formation of free chloride ions (C1‘). These free chloride ions may undergo further oxidation, which may form reactive chlorinate species. Reactive chlorinate species include hypochlorous acid (HOC1), or potentially chlorine gas (CI2). Reactive chlorinate species may modify nearby chemical structures, such as amino acids, peptides, or other organic molecules. In some embodiments, the presence of free chloride ions may' result in the formation of chlorinated aromatics, quinonoid compounds, or stable, pigmented compounds. These compounds may provide a sink for chlorine atoms.

[0095]

[0097] In some embodiments, trace metals such as iron or copper may amplify radical formation and oxidative processes. Trace metals may be present in LB media through yeast extract or tryptone, and may sen e as cofactors or act through Fenton-type catalysis. In some embodiments, trace metals could contribute to the formation of chlorinated aromatics, quinonoid compounds, or stable, pigmented compounds through indirect catalysis of aromatic oxidation.

[0096]

[0098] In some embodiments, one or more enzymes may degrade a PVC molecule. In some embodiments, the one or more enzymes may comprise a lignin peroxidase, a catalase peroxidase, a dehalogenase, a laccase, a monooxygenase, or combinations thereof.

[0097]

[0099] In some embodiments, a lignin peroxidase may comprise LPPc. LPPc may comprise an amino acid sequence of SEQ ID NO: 1. In some embodiments, a lignin peroxidase may comprise a mutant or a homolog of LPPc. In some embodiments, a lignin peroxidase may comprise a protein with a sequence identity of at least 50%, 60%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or at least 100% of SEQ ID NO: 1. In some embodiments, a lignin peroxidase may comprise a protein with a sequence identity of up to 50%, 60%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or up to 100% of SEQ ID NO: 1. In some embodiments, a lignin peroxidase may comprise a codon-optimized DNA sequence, wherein the DNA sequence has been optimized for production of a protein in a microbial host. The microbial host may be bacterial or eukaryotic. A eukaryotic host may be a yeast. In some embodiments, the codon optimized DNA sequence may be SEQ ID NO: 7. In some embodiments, the codon optimized DNA sequence of the lignin peroxidase may comprise a sequence identity of at least 50%, 60%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or at least 100% of SEQ ID NO: 7. In some embodiments, the codon optimized DNA sequence of the lignin peroxidase may comprise a sequence identity of up to 50%, 60%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or up to 100% of SEQ ID NO: 7.

[0098]

[0100] In some embodiments, a catalase peroxidase may comprise CPKv. CPKv may comprise an amino acid sequence of SEQ ID NO: 3. In some embodiments, a catalase peroxidase may comprise a mutant or a homolog of CPKv. In some embodiments, a catalase peroxidase may comprise a protein with a sequence identity of at least 50%, 60%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or at least 100% of SEQ ID NO: 3. In some embodiments, a catalase peroxidase may comprise a protein with a sequence identity of up to 50%, 60%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or up to 100% of SEQ ID NO: 3. In some embodiments, a catalase peroxidase may comprise a codon-optimized DNA sequence, wherein the DNA sequence has been optimized for production of a protein in a microbial host. The microbial host may be bacterial or eukaryotic. A eukaryotic host may be a yeast. In some embodiments, the codon optimized DNA sequence may be SEQ ID NO: 9. In some embodiments, the codon optimized DNA sequence of the catalase peroxidase may comprise a sequence identity of at least 50%, 60%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or at least 100% of SEQ ID NO: 9. In some embodiments, the codon optimized DNA sequence of the catalase peroxidase may comprise a sequence identity of up to 50%, 60%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or up to 100% of SEQ ID NO: 9.

[0101] In some embodiments, a dehalogenase may comprise LKv. LKv may comprise an amino acid sequence of SEQ ID NO: 5. In some embodiments, a dehalogenase may comprise a mutant or a homolog of LKv. In some embodiments, a dehalogenase may comprise a protein with a sequence identity of at least 50%, 60%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or at least 100% of SEQ ID NO: 5. In some embodiments, a dehalogenase may comprise a protein with a sequence identity of up to 50%, 60%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or up to 100% of SEQ ID NO: 5. In some embodiments, a dehalogenase may comprise a codon-optimized DNA sequence, wherein the DNA sequence has been optimized for production of a protein in a microbial host. The microbial host may be bacterial or eukaryotic. A eukaryotic host may be a yeast. In some embodiments, the codon optimized DNA sequence may be SEQ ID NO: 11. In some embodiments, the codon optimized DNA sequence of the dehalogenase may comprise a sequence identity of at least 50%, 60%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or at least 100% of SEQ ID NO: 11 . In some embodiments, the codon optimized DNA sequence of the dehalogenase may comprise a sequence identity of up to 50%, 60%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or up to 100% of SEQ ID NO: 11.

[0099]

[0102] In some embodiments, an enzyme may comprise a combination protein comprising two or more enzymes. A combination protein may comprise a combination of two or more of catalase peroxidase, lignin peroxidase, dehalogenase, laccase, or monooxygenase. In some embodiments, a combination protein may contain one or more linker portions between the two or more enzymes.

[0100]

[0103] In some embodiments, polyvinyl chloride (PVC) may be degraded by one or more enzymes under operating conditions. The operating conditions may increase the rate or efficacy of degradation. The operating conditions can be temperature, pH, nutrient supply, cofactor presence or availability, induction signal presence or availability.

[0101]

[0104] In some embodiments, a temperature condition may be 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 °C. In some embodiments, a temperature condition may be at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or at least 50 °C.

[0105] In some embodiments, a temperature condition may be up to 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or up to 50 °C.

[0102]

[0106] In some embodiments, a pH condition may be a pH of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14. In some embodiments, a pH condition may be a pH of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or at least 14. In some embodiments, a pH condition may be a pH of up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or up to 14.

[0103]

[0107] In some embodiments, a cofactor may be present with one or more enzymes to increase the rate or efficacy of degradation. In some embodiments, a cofactor may be an organic cofactor or a metal ion cofactor. A organic cofactor can be a heme cofactor, such as a heme b group. An organic cofactor may be supplied by a cell lysate. The metal ion cofactor may be a iron, zinc, copper, calcium, magnesium, manganese, or a cobalt ion. A metal ion cofactor may be supplied by a metal salt, such as CuSC , ZnSO4, or FeSCL.

[0104]

[0108] In some embodiments, one or more enzymes are produced by a microbe. An nutrient source may be provided to the microbe to provide energy or necessary nutrients to produce the enzymes. A nutrient source may be a sugar, such as glucose. A nutrient source may be a grow th media, such as LB media. In some embodiments, the microbe may be engineered to produce a recombinant enzyme by inserting DNA into the microbe. The DNA may be inserted as a plasmid into the microbe, or it may be inserted into the chromosomal DNA of the microbe. The production of this recombinant enzyme may be basal or inducible. With basal production the enzyme may be produced constantly. With inducible production, the enzy me production may be triggered by the presence of an induction signal, such as glucose, lactose, isopropyl B-D-l- thiogalactopyranoside (IPTG), arabinose, or copper.

[0105] EXAMPLES

[0106] Example 1.1 - Enzymatic degradation of PVC

[0107]

[0109] Construction of encoding plasmid. To construct plasmids encoding referenced proteins, protein information was accessed through the NCBI / BLAST web services

[0108] (https: / / ww blast.ncbi.nlm.nih.gov), and through either the UniProt Consortium (https: / / uniprot.org) or BRENDA (https: / / w w w.brcnda-enzymes.org ). The amino acid sequence of the ligninolytic enzymes was reverse translated, and codon optimized for E. coli through SnapGene software (https: / / www.snapgene.com) (see supplementary material). The construct was designed in silico to produce the recombinant enzymes as fusion protein with nucleotides encoding for a hexahistidine tag, for downstream protein purification using Ni-NTA resin, tetracysteine LumioTM tag, as a reporter tag to confirm the protein is produced using Fluorescence, and a stop codon to determine the length of the expressed protein. The DNA vectors were manufactured and cloned into the expression vector pET30a(+) by Integrated DNA Technologies (IDT) for subsequent protein expression in transformed BL21(DE3) E. coli cell type (Intact Genomics).

[0109]

[0110] Transformation of Escherichia coli BL21(DE3). Transformation of Escherichia coli BL21(DE3) was conducted according to the following steps. Chemically competent cells of E. coli strain BL21(DE3) were transformed with the LKv, LPPc and CPKv DNA constructs. LB- agar plates containing 50 pg / mL kanamycin were prepared the day before the transformation. For each DNA construct, a 50-pL tube of competent E. coli DE3 cells was thawed on ice for 20 min. One microliter of DNA was added to the cells. The tube was tapped 4-5 times and incubated on ice for 30 min. The cells were then heat-shocked for 10s at 37 °C, followed by incubation on ice for 2 minutes. A volume of 950 pL SOC liquid medium was added to the tube and incubated at 37 °C for Ih in a rotary shaker under 210 rpm. In order to maximize the chance of obtaining individual colonies, the following volumes w ere inoculated on four LB-agar plates containing 50 pg / mL kanamycin as follows: (i) 50 pL, and (ii) 950 pL (centrifuged and resuspended in 100 pL). The plates incubated overnight at 37 °C. On the next day, the colonies were counted to calculate the transformation efficiency. Three colonies of each transformation tube were selected for subsequent experiments. To produce cryostocks, the colonies were inoculated into 3 rnL of LB + 50 pg / mL kanamycin and incubated overnight at 37 °C with shaking at 150 rpm with the tubes taped horizontally to the shaker platform. In the next day, new inocula were performed for each sample in new 15-mL Falcon tubes containing 3 mL of LB + 50 pg / mL kanamycin. The tubes were incubated for up to two hours at 37 °C with shaking at 150 rpm with the tubes taped horizontally to the shaker platform. During this period, the optical density (OD600) was monitored. When the OD600 reached a value between 0.6 and 0.8, a volume of 1.5 mL from each Falcon tube was transferred to a microcentrifuge tube and centrifuged at 8,000 rpm for 3 minutes. To obtain a cell concentrate, the supernatant was discarded, and the remaining culture from the previous step was added to the same microcentrifuge tubes and centrifuged again in the same conditions. The supernatant was discarded, and the pellet was resuspended in 250 pL of LB + 50 pg / mL kanamycin and 10% glycerol filter-sterilized in the same day. The resulting volume of each sample was distributed into 5 microcentrifuge tubes and stored at -70 °C until required. f i l l] Protein extraction and purification. Protein extraction and purification was conducted according to the following steps. The expression of the target enzymes was induced with 1% lactose added to the LB medium containing 50 pg / mL kanamycin. Cell pellets were collected from 100-mL culture after 4h of induction of protein expression. The cultures were centrifuged at 3900 rpm, 40 min, 4 °C, in 50-mL falcon tubes. The mass of each pellet was measured by weighing the tubes before and after collecting the pellets, yielding the following for the three enzymes: LKv = 0.6740g, LPPc = 0.5456g, CPKv = 0.5160g. The supernatants were transferred to new tubes and saved for further analysis. The tubes containing the pellets were frozen at -70 °C for at least 10 min. For cell lysis, the tubes containing the pellets were placed into an ice / water bath and incubated for 30 min. The previous two steps were repeated for a total of 3 times. Five milliliters of Tris-buffered saline TBS (50 mM Tris-HCl, 150 mM NaCl, pH= 7.5) were added per gram of pellet. The pellets were resuspended using a glass pipette. The pellets were incubated in ice / water bath for 60 min and the resulting samples were agitated periodically to facilitate complete mixing. The samples were distributed into 1.5-mL microcentrifuge tubes and centrifuged at 10,000 g for 15 min, 4 °C. The supernatants (clarified cell extracts) were collected in a clean 15-mL falcon tubes and at -20 °C. The pellets were also saved to check for incomplete lysis if necessary. The enzymes were purified from the total cell extract (lysate) using the HisPurTM Ni-NTA Resin from Thermo Scientific (catalog number 88221), following the manufacturer’s instructions. The enzymes were then quantified using the BCA Protein Assay Kit from Novagen (User protocol TB380) following the manufacturer’s instructions. FIG. 6 shows the protein concentration (pg / mL) of the three enzy mes as determined by the BCA assay. The protein concentrations are displayed grouped by the obtained fractions, with the concentrations of LKv, LPPc, and CPKv displayed from left to right within the groups. The fractions comprise Lysate, Supernatant, Wash 1, Wash 2, Elution 1, and Elution 2. The purified enzymes were analyzed by SDS-PAGE using Thermo Fisher's NuPAGE™ Bis-Tris XCell SureLock™ Mini-Cell Welcome Pack, and Thermo Fisher's Lumio™ Green Detection Kit to detect the presence of the cloned enzymes. After the electrophoresis, the gels were photographed with a gel imaging system and a UV transilluminator (254 nm) to detect the presence of bands stained with Lumio fluorescent detection system. Next, the gels were stained for 5 min at 25°C with Coomassie Blue (G-250) prepared in 50% methanol / 10% acetic acid, with enough volume to cover the thin gel in a small Tupperware with gentle shaking. After 5 minutes the Coomassie Blue was recovered for future use and the gel was rinsed 3 times with MiliQ water. The gels were left overnight in MiliQ water with gentle shaking to maximize de-staining and enhance contrast. On the next day, the gels with blue bands were photographed.

[0110]

[0112] The results presented in FIG. 7 and FIG. 8 show the presence of the target bands for all three enzy mes. The greatest Lumio signal detection was observed for CPKv (FIG. 8A), which is consistent with the results obtained by the protein quantification with the BCA assay (FIG. 6). For the other two enzymes (LKv and LPPc), the Lumio fluorescence in fractions '‘supernatant’’, “wash 2”, and “elution 2” was fainter. FIG. 7A shows Lumio detection using a UV transilluminator (254 nm) which indicates the presence of a band at the target molecular weight for LKv and LPPc. FIG. 7B shows an image of the gel stained with Coomassie Blue (G-250). The boxes indicate the positions of the target bands for LKv and LPPc. FIG. 8Ashows Lumio detection using a UV transilluminator (254 nm), which indicates the presence of a band at the target molecular weight for CPKv. FIG. SB shows an image of the gel stained with Coomassie Blue (G- 250). The boxes indicate the positions of the target bands for CPKv.

[0111]

[0113] LKv and CPKv were extracted again for use in larger scale and longer-term experiments. An extra purification step was introduced following extraction for this second extraction. The samples were reconstituted to their initial volume with MiliQ water. The results of a second protein extraction and purification protocol are show n in FIG. 9and FIG. 10. FIG. 9A shows a microplate showing the colors of the wells after the BCA assay. FIG. 9B shows a standard curve produced after analyzing the optical density (OD) readings at 562 nm. FIG. 9C shows a bar plot showing the concentration results in pg / mL for each enzyme. FIG. 9D shows updated concentration measurements after desalting with the Amicon filter. FIG. 10 shows a SDS-PAGE stained with Coomassie blue (FIG. 10A), and Lumio (FIG. 10B).

[0112]

[0114] Enzymatic activity assays. Enzymatic activity assays were conducted according to the following steps. To evaluate the catalase activity of the CPKv cell lysate and the different fractions of the protein purification procedure, a catalase assay kit (Sigma catalog number MAK531) was used according to the manufacturer’s instructions. Catalase is a ubiquitous antioxidant enzyme that catalyzes the decomposition of hydrogen peroxide (H2O2) to water and oxygen. The catalase assay kit directly measures catalase degradation of H2O2 using a redox dye. The change in color intensity' at 570 nm is directly proportional to the catalase activity in the samples. LKv had been suggested to be a laccase (see Zhang, Z., et al. Polyvinyl chloride degradation by a bacterium isolated from the gut of insect larvae. Nat Commun 13, 5360 (2022). To evaluate the laccase activity of the LKv cell lysates and the products of the protein purification procedure, a laccase assay kit (Abeam catalog number ab284539) was used according to the manufacturer’s instructions. In this assay, samples containing laccase oxidize the substrate to generate a colored product with a strong absorbance at 420 nm.

[0113]

[0115] FIG. 11 shows the catalase activity of CPKv samples in terms of units per liter (U / L). One unit is the amount of catalase that decomposes 1 pmole of H2O2 per minute at pH 7.0 and 25°C. The red bar represents the positive control provided by the kit. These results indicate that the lysate fraction sample has the lowest activity, suggesting it has less capacity to decompose H2O2. “Supernatant” and “wash 2” samples have an intermediate catalase activity, suggesting it has moderate capacity to decompose H2O2. The “elution 2” sample has the highest catalase activity, suggesting it has the greatest capacity to decompose H2O2. One might expect these results for a purer fraction.

[0114]

[0116] FIG. 12 shows the assay results of LKv for laccase enzyme kinetics using all fractions of the protein purification protocol (lysate, supernatant, wash 2, and elution 2), with arrow s indicating the Lysate and Positive control. The laccase assay used in this study did not indicate activity for the LKv protein purification fractions (lysate, supernatant, wash 2, and elution 2) (FIG. 6) Therefore, it w as not possible to determine their enzyme activity. Laccase activity was not demonstrated with this kit, but dechlorination was demonstrated by FTIR analysis with the LKv samples on PVC3 and PVC4, as summarized in the following sections.

[0115]

[0117] Degradation of PVC pellets containing plasticizers and P VC powder with no plasticizers. To gather data on the degradation of different types of PVC (ACI T2-2, Mexichem PV-1913, Westlake, Manner, and pure PVC pow der) by the three engineered strains of E. coli, liquid cultures were prepared from cryostocks and incubated in the presence of the plastic materials. Fifty microliters were inoculated into 3 mL of LB medium containing 50 pg / mL of kanamycin in 50-mL Falcon tubes. The cultures were incubated overnight at 37 °C, with gentle shaking (80 rpm). The tubes w ere placed horizontally in the shaker incubator. On the following day, a series of 50-mL M9 liquid media was prepared in Erlenmeyer flasks, according to the experimental design shown in FIG. 13A and FIG. 13B. FIG. 13A shows the degradation of different types of PVC (PVC1 = ACI T2- 2, PVC2 = Mexichem PV-1913, and PVC powder) by the engineered strains of E. coli (LKV, LPPc, and CPKv). Flasks 1 -9 were induced with 1 % lactose. Flasks 10-18 were not induced. FIG. 13B shows the degradation of different types of PVC (PVC3 = Westlake, and PVC4 = Manner) by the engineered strains of E. coli (LKV, LPPc, and CPKv). Flasks 19-24 were induced with 1% lactose. Flasks 25-30 were not induced.

[0116]

[0118] After sterilizing the PV C pellets and the PV C pow der by autoclaving, three PV C pellets were added into each flask numbered 1-6 and 10-15. The flasks numbered 7-9 and 16-18 The following mass of PVC powder was added for the other flasks: Flask 7 = 0. 1041g, Flask 8 = 0.1089g, Flask 9 = 0. 1105g, Flask 16 = 0.1016g, Flask 17 = 0.1086g, Flask 18 = 0.1008g. The cultures were incubated at 37 °C, 200 rpm for up to 14 days. At the end of the experiment, the mass of the PVC pellets was measured and compared to the initial mass. For the samples with PVC powder, OD600 measurements were taken every day to track the variations of optical density as a function of microbial growth and, hypothetically, the degradation of the white powder.

[0117]

[0119] The results for experiments with the different types of PVC pellets are shown in FIG. 14 , which shows mass variation comparing the different strains with the 4 different PVC types. The arrows indicate the most substantial differences between initial and final masses. Since the sizes of the pellets were very similar, 10 pellets were weighted and the average mass as well as the standard deviation was obtained for each pellet of PVC-1 (0.03033g, ±0.00769g), and PVC-2 (0.03093g, ±0.00769g). The initial masses and standard deviation values for PVC-1 and PVC-2 had the same values between the samples. For PVC-3 and PVC-4, the actual pellets used in the experiments were weighted individually, obtaining averages and standard deviations from three replicates. The initial masses and standard deviation values for PVC-3 and PVC-4 were different.

[0118]

[0120] The masses of the four types of PVC increased after 14 days of incubation with the three different strains, regardless of induction. Exceptions include the results observed for LKv not induced in PVC-1, and LPPc not induced and CPKv not induced in PVC-2. The result observed for sample LPPc not induced in PVC-2 seems to be significant because the value is slightly below the standard deviation of the initial mass. However, no standard deviation was calculated for the final mass samples because the individual pellets were not distinguishable. The PVC-2 samples showed the highest difference between initial mass and final mass for all samples, regardless of induction.

[0119]

[0121] The initial results for the liquid cultures incubated with PVC powders indicate some consistent differences in the optical absorbance at 600 nm between the different samples. FIG. 15 shows the OD600 of the three engineered strains of E. coll (LKv, LPPc, and CPKv), induced and not induced, after 9 days of incubation with PVC powder. The liquid cultures were incubated at 37 °C, 200 rpm. The LKv samples, both induced and not induced, show the lowest OD values. The LPPc and the CPKv samples, show a difference between the induced and the not induced samples. In both cases, the induced samples showed OD600 values substantially higher. These results were consistent throughout the experiment.

[0122] Halo assay using pure PVC powder (no plasticizers). The halo assay is a microbiology technique used to detect biological activity on agar plates supplemented with a test substance. In this study agar plates were supplemented with pure PVC powder. The appearance of degradation halo incubation was indicative of enzymatic activity. Agar plates containing 0.25% (w / v) PVC powder were prepared according to the experimental design shown in FIG. 16, which shows the degradation of PVC powder by the engineered strains of E. coli (LKV, LPPc, and CPKv). A volume of 1 mL of cofactors (ImM CuSO4 and 1 mM ZnSC>4 for LKv, and 1 mM FeSC for LPPc and CPKv) was spread on top of the agar plates. Three droplets of 10 pL of cultures were added in each plate. The appearance of degradation halo after 3 days was indicative of enzymatic activity. The agar plates inoculated with 10 pL droplets of the cultures were incubated for 10 days at 37 °C. After this period, one colony showed a “cloudy” halo (FIG. 17). The colony was transferred to liquid LB medium for growth and cry ostock.

[0120]

[0123] FIG. 17 shows colonies on M9 agar plates prepared according to the experimental design in FIG. 16. One colony, indicated by arrows in FIG. 17 showed a “cloudy” halo for the induced sample of the LKv culture after 10 days of incubation at 37 °C.

[0121]

[0124] Short-term degradation of PVC powders by cell extracts (lysates). The protein purification procedure resulted in different fractions for each one of the three enzymes (lysate, supernatant, wash 1, wash 2, elution 1, and elution 2), at different concentrations (FIG. 6). The purest fraction for all three samples is the elution 2 fraction, which is the result of two wash steps with 25 mM of imidazole, and two elution steps from the nickel resin using 250 mM of imidazole. Imidazole is a compound that is known to inhibit enzyme activity. This suggests that, even though the target enzyme is more concentrated in this fraction, it is not pure, and additional purification steps may be necessary to isolate the protein.

[0122]

[0125] Based on these results, experiments were prepared to test the enzyme activity of different lysates according to Table 2. The experiments were prepared in microcentrifuge tubes. The tubes were placed in the incubator at 37 °C to incubate for 2 weeks, tapping the tubes every' day to mix the contents. After that period, FTIR analysis was conducted on the samples.

[0123]

[0126] FTIR spectra of the different types of PVC with plasticizers treated with different types of lysates and their combinations are presented in FIG. 18- FIG. 21, with the sample numbers listed on the figures corresponding to their sample numbers in Table 2. Formation of different functional groups, such as hydroxyl (OH), and C=C conjugated, was observed for all PVC types indicating degradation via oxidation. The greatest effects between the different PVC types were: LKv+LPPc for PVC ACI T2-2 (FIG. 18), LKv+CPKv for PVC Westlake (FIG. 19), LPPc for PVC Mexichem (FIG. 20), and CPKv for PVC Manner (FIG. 21). Pure PVC without plasticizers also showed the same type of activity (FIG. 22A), with the greatest effects observed for LKv+LPPc, and LKv+CPKv.

[0124]

[0127] The FTIR spectra for PVC Westlake indicated the formation of new functional groups, such as additional hydroxyl (3295 cm-1) and C=C (1648 cm-1), indicating that the combination of LKv and CPKv lysates can degrade the Westlake PVC powder via oxidation (FIG. 22B). The intensities of C-H, CH2-C1, and CH-CI bands decreased after treatment with a combination of LKv and CPKv lysates. This enzyme mixture can also degrade the additives, since the intensity7of C=O band also decreased.

[0125]

[0128] A comparison between the different plastic powders with the greatest change in peak intensities revealed that the Westlake PVC powder treated with a combination of LKv and CPKv lysates exhibited the greatest change, followed by Manner treated with CPKv, Mexichem treated with LPPc, and ACI T2-2 treated with LKv+LPPc (FIG 26C).

[0126] Table 2: Experimental parameters and list of PVC samples for short term degradation 34 No substrate LKv+LPPc+CPKv Ctrl 0 67 67 67 0 201

[0127] 35 No substrate LPPc control 0 0 200 0 0 200

[0128] 36 No substrate CPKv control 0 0 0 200 0 200

[0129] 37 ACI T2-2PVC H2O control 10 0 0 0 200 200

[0130] 38 Mexichem PVC H2O control 10 0 0 0 200 200

[0131] 39 Westlake PVC H2O control 10 0 0 0 200 200

[0132] 40 H2O control 10 0 0 0 200 200

[0133] 41 PVC powder H2O control 10 0 0 0 200 200

[0134]

[0129] Short-term degradation of PVC powders by cell extracts (lysates) and purified enzymes. To concentrate the target enzymes and remove the imidazole, which could negatively impact the enzyme activity. Amicon filters with molecular weight cut-off values of 30 kDa for CPKv and 10 kDa for LPPc and LKv were used. After combining the volumes of elution 1 and elution 2 (about 0.4 mL each), approximately 9.2 mb of MiliQ water was added to make the volume up to 10 mL. We then transferred the volumes into Amicon filters and centrifuged for 20 min at 3,240g, at 15 °C. The flowthrough was discarded and the final volumes recovered from the filters were: 573 uL for LKv, 490 pL for LPPc, and 530 pL for CPKv. These fractions were resuspended in 500 pL of 300 mM NaCl / 50 mM Tris-HCl pH 8.0 and used for the treatment of PVC powders with purified enzymes according to Table 3.

[0135] Table 3. Experimental parameters and list of PVC samples treated with purified enzymes

[0136] Sample # PVC type Description PVC Mass (mg)LKvLppcCPKv H2O Total

[0137] 1 ACI T2-2 PVC LKv+LPPc+CPKv 10 67 67 67 0 201

[0138] 2 Mexichem PVC LKv+LPPc+CPKv 10 67 67 67 0 201

[0139] 3 Westlake PVC LKv+LPPc+CPKv 10 67 67 67 0 201

[0140] 4 LKv+LPPc+CPKv 10 67 67 67 0 201

[0141] 5 PVC powder LKv I LPPc I CPKv 10 67 67 67 0 201

[0142] 6 ACI T2-2 PVC H2O control 10 0 0 0 200 200

[0143] 7 Mexichem PVC H2O control 10 0 0 0 200 200

[0144] 8 Westlake PVC H2O control 10 0 0 0 200 200

[0145] 9 H2O control 10 0 0 0 200 200

[0146] 10 PVC powder H2O control 10 0 0 0 200 200

[0147]

[0130] Fourier Transform Infrared Spectroscopy (FTIR) is a technique used to obtain an infrared spectrum of absorption or emission of a solid, liquid, or gas. An FTIR spectrometer simultaneously collects high-resolution spectral data over a wide spectral range and show peaks that are characteristic of certain chemical bonds. Differences in these features between plastic samples treated or not with enzymes, such as formation of bonds C=C, C=O, and O-H, or the degradation of C-Cl bonds, may be indicative of PVC degradation and dechlorination.

[0131] FTIR analysis of the PVC powders treated with the combination of all three purified enzymes showed degradation via oxidation only for the Mexichem PVC powder, possibly because of the short term of the experiment (96h). FIG. 23 - FIG. 27 show the spectra of the different plastic powders treated with the same enzy me combination (LKv+LPPc+CPKv), with the sample numbers listed on the figures corresponding to their sample numbers in Table 3. The strong water absorption bands indicates that the samples were wet. FIG. 23 shows FTIR result for Mexichem plastic powder treated with a combination of all three enzymes, showing the formation of C C (1637 cm’1), which indicates degradation via oxidation. FIG. 24 shows the FTIR results for ACI T2-2 treated with the combination of all three enzymes (LKv+LPPc+CPKv). FIG. 25 shows the FTIR results for Westlake treated with the combination of all three enzymes (LKv+LPPc+CPKv). FIG. 26 shows the FTIR results for Manner treated with the combination of all three enzymes (LKv+LPPc+CPKv). FIG. 27 shows the FTIR results for pure PVC treated with the combination of all three enzymes (LKv+LPPc+CPKv).

[0148]

[0132] Long-term degradation of PVC powders by cell extracts (lysates) and purified enzymes. Since the FTIR results obtained with the short-term experiments revealed that LKv and CPKv had the greatest activities, a long-term experiment was prepared in the same conditions with cell extracts and purified fractions of these two enzymes, according to the experimental design shown in FIG 32A. A description of all 50 samples, their contents and time points are presented on Table 4.

[0149] Table 4: List of PVC powders treated with cell lysate and enzymes

[0150]

[0133] FTIR analysis of the pure PVC powder (no plasticizers) treated with the highest concentration of lysates (LKv+CPKv) showed the formation of C=C, C=O, and O-H bonds in all time points including the time point 0 (FIG. 29A), while the lowest concentration of lysates (LKv+CPKv) showed the same bonds in all time points except the time point 0 (FIG. 29B). This indicates that at a lower concentration requires a longer time to start producing similar results as the highest concentration. The pure PVC powder (no plasticizers) treated with both the highest and the lowest concentration of enzymes (LKv+CPKv) showed no degradation in all time points (FIG. 29C and FIG. 29D). This result suggests that the experimental conditions may not have been optimal for this type of material. The Westlake PVC powder treated with both the highest and the lowest concentration of lysates (LKv+CPKv) showed the formation of C=C, C=O, and O-H bonds in all time points (FIG. 29E and FIG. 29F). Additional C=C bonds were observed for both treatments in 4 weeks but not in 8 or 12 weeks. When treated with the highest concentration of enzyme, the Westlake PVC powder formed C=O, C=C, and O-H bonds only in time point 0 (FIG. 29G). No degradation w as observed after 2, 4, 8, and 12 weeks of treatment. It is worth noting that a high concentration of lysate also contains a high concentration of different cellular metabolites and salts that may interfere with the enzyme activity. When treated with the lowest enzyme concentration, the Westlake PVC powder formed C=O, C=C, and O-H bonds at time points 4 and 8 weeks of treatment while no degradation was observed at time point 0 (FIG. 29H). In FIG. 29, arrows indicate additional C=C bonds. M1-M4 correspond to the spectra for liquid samples. Table 4 lists sample numbers and time points for FIG. 29.

[0151]

[0134] Long-term degradation of PVC wires by cell extracts (lysates) and purified enzymes. Combinations of LKv and CPKv lysates (high concentration), as well as LKv and CPKv enzymes (low concentration), were used according to the experimental design shown in FIG. 28B and Table 5. FIG. 30 shows the FTIR results of Westlake PVC wire samples treated with the highest concentration of lysate (FIG. 30A) or lowest concentration of enzyme (FIG. 30B). FTIR analysis of the pure Westlake PVC powder treated with highest concentration of lysate (LKv+CPKv) showed the formation of C=O, C=C, and O-H bonds in time points 0, 1, and 5 weeks after treatment, indicating degradation via oxidation (FIG. 30A). Compared to the FTIR obtained with the highest lysate concentration, the lowest concentration of enzyme also showed the formation of the same bonds (C=O, C=C, and O-H) in the same timepoints (0, 1, and 5). However, the intensities of the changes relative to the w ater control were smaller, indicating a lower activity' (FIG. 30B).

[0152]

[0135] Long-term degradation of PVC wires by cell extracts (lysates) and purified enzymes and Scanning Electron Microscopy (SEM) results. An experiment was set up to test the degradation of PVC wire (Westlake) by cell extracts and purified fractions of the LKv and CPKv enzymes. The wires were cut in pieces of five millimeters and each piece was placed in clean glass vials. Combinations of LKv and CPKv lysates (high concentration), as well as LKv and CPKv enzymes (low concentration), were used according to the experimental design shown in FIG. 28B and Table 5. The samples listed in Table 5 were incubated at 37 °C (except samples from time point 0) and tapped periodically every other day. At the end of each time point, the liquid was removed and discarded, and the tubes were opened in a laminar flow hood and left drying overnight. After this period, the tubes were placed in plastic bags and shipped to YTC A for FTIR analysis.

[0153]

[0136] The SEM images obtained for the Westlake PVC wire samples treated or not for 5 weeks with lysate high or enzyme low revealed some alterations compared with the intact samples (no treatment) and the water control. At low magnification (FIG. 31) the intact sample seems smoother than the sample exposed to water for 5 weeks. The samples treated with both lysate high and enzyme low show surface features such as grooves and roughness that differ from the control samples. FIG. 31 shows low magnification (200-250x) SEM images comparing different samples of Westlake PVC. FIG. 31A, shows no treatment. FIG. 31B was treated for 5 weeks with lysate high, FIG. 31C was treated for 5 weeks with enzy me low. FIG. 3 ID shows the water control.

[0154] Table 5: List of PVC powders treated with lysate and enzymes

[0155]

[0137] At medium magnification, the differences between all four samples seem to be more pronounced (FIG. 32). The surface features of the intact sample seem to be smoother then all the others. The sample treated with lysate high showed flat sections with cracks and folds. The sample treated with enzyme low show grooves and protrusions of similar sizes. The sample exposed to water (water control) shows somewhat irregular folds. FIG. 32 shows medium magnification (1000-2000x) SEM images comparing different samples of Westlake PVC. FIG. 32A shows no treatment. FIG. 32B was treated for 5 weeks with lysate high. FIG. 32C was treated for 5 weeks with enzy me low. FIG. 32D shows the water control.

[0138] The high magnification images showed the greatest differences between the four samples (FIG. 33). The intact sample (no treatment) showed some surface irregularities. The sample treated with lysate high showed grooves, cracks and folds. The sample treated with enzyme low showed a large area with small grooves overlay ed with cracks and trenches. The water control showed small round holes and a smooth surface. FIG. 33 shows high magnification (5000x) SEM images comparing different samples of Westlake PVC. FIG. 33A shows no treatment.

[0156] FIG. 33B was treated for 5 weeks with lysate high. FIG. 33C was treated for 5 weeks with enzyme low. FIG. 33D shows the water control.

[0157]

[0139] The results suggest that the three engineered strains of E. coll containing the different enzymes were capable of gene expression and enzymatic activity on several types of plastic substrates, as revealed by Lumio detection and FTIR analysis. The halo assay results suggest microbial degradation of PVC pellets and powders was in liquid cultures and agar plates. The laccase assay did not indicate activity for the LKv strain, but PVC degradation experiments suggested PVC degradation activity for LKv. This suggests that LKv may not be a laccase, and may suggest a new venue for enzyme characterization, since the results obtained by this strain were remarkable.

[0158]

[0140] Enzymatic activity was demonstrated to some degree for all three strains in pure PVC (no plasticizers) and different types of PVC containing plasticizers. The strain CPKv produced the highest concentration of enzyme, which translated to high activity in all experiments. The lysate fractions containing total proteins of CPKv and LKv appear to have the highest enzymatic activities in all types of PVC tested, as indicated by the peak intensities in the FTIR spectra. The purified enzymes also showed enzymatic activity on the Westlake PVC, but not on pure PVC. The differences observed between Westlake wire samples treated or not with lysate high or enzyme low, as revealed by SEM images, indicate physical alterations on the wire surfaces after these treatments.

[0159]

[0141] Degradation of PVC by Live E. Coli Expressing Degradation Enzymes. To assess whether E. coli expressing lignin peroxidase, catalase peroxidase, and dehalogenase enzymes could actively degrade neat PVC (pure PVC without plasticizers), a set of experiments was conducted to evaluate both enzyme expression and potential utilization of PVC as a carbon source.

[0160]

[0142] In the first experimental condition, E. coli BL21(DE3) cells containing the constructs for LPPc, CPKv, and LKv were grown in LB medium to an optical density (OD600) of 1 to 2, ensuring sufficient biomass before enzyme induction. The cells were then washed to remove residual nutrients and transferred to M9 minimal media supplemented with galactose as an inducer, allowing the bacteria to produce and secrete their respective enzymes. In these conditions, an available, metabolizable carbon source was provided by galactose, providing a baseline for enzymatic activity under induced conditions.

[0161]

[0143] In the second condition, E. coli cultures were grown to OD600 1 to 2, washed, and then transferred into M9 minimal media containing PVC powder as the sole potential carbon source, without any additional sugars or readily metabolizable substrates. If bacterial survival or proliferation occurred in this condition, it would suggest that enzymatic breakdown of PVC led to the release of bioavailable degradation products.

[0162]

[0144] In the third condition, E. coli cultures expressing LPPc, CPKv, and LKv were grown to OD600 1 to 2 in LB medium, washed to remove residual nutrients, and then transferred into M9 minimal media without glucose or any other added carbon source. PVC powder was included in the culture tubes to assess whether the absence of a conventional carbon source would still result in any enzymatic activity or evidence of polymer transformation. This condition served to evaluate whether basal expression of the constructs or stress-induced enzyme production could lead to partial degradation of PVC. It may provide insight into the extent to which nutrient limitation alone might influence oxidative processes or the fate of PVC under star ation conditions.

[0163]

[0145] Individual sample tube conditions are listed in Table 6. Sterile 50 mL tubes were prepared for each construct, adding 2.5 pL of E. coli cryostock containing one of the constructs to 5mL of LB broth with a corresponding antibiotic. Tubes were incubated overnight at 37°C and 200-250 RPM, (approximately 12-16 hours) until turbid. 500 pL of culture from the tube was then added to 50 mL of LB broth with antibiotic in a 250 mL Erlenmeyer flask for each construct and incubated at 37°C, 200-250 RPM shaking until they reached an OD600 of approximately 1.0, about 3 to 5 hours. If OD600 was below 1.0, the culture was left to incubate longer. If the OD600 w as greater than 1.0, fresh LB broth w as added to dilute the sample until it reached an OD600 of 1.0. For each experimental condition tube, 10 mL of the culture was added to sample tubes containing a stock PVC mixture with approximately 20mg of neat PVC powder. Weighed amounts of PVC are listed in Table 6. The tubes were then spun dow n and the LB broth exchanged for the growth media of the corresponding experimental condition. Tubes w ere then incubated at 37°C, 200-250 RPM shaking. Tubes were removed for sample processing at their corresponding time points, noted in Table 6. For sample processing, culture liquid was removed and discarded and the samples dried under a laminar flow hood for 3 days. FTIR analysis was then conducted on the dried samples.

[0164] Table 6: Live Cell PVC Degradation Sampling

[0165] TUBE TRANSFORMANT TIME POINT PVC WEIGHT MEDIA GALACTOSE NUMBER (MG) INDUCTION T

[0166] NU

[0167]

[0146] Previous experiments using bacterial lysates demonstrated a dose-dependent effect on PVC degradation, as noted in Table 7. Table 7 compares the effectiveness of different enzyme delivery systems — bacterial lysate, purified enzyme, and live E. coli expressing constructs — on PVC degradation. Observations include FTIR-detected chemical changes and corresponding interpretations. Results highlight the importance of cofactor availability and cellular context in enabling enzymatic breakdown of PVC, with lysate and live-cell systems showing the most significant activity.

[0168]

[0147] When E. coli lysates were applied at high concentrations, changes were observed in the FTIR spectra, including the formation of C=O, C=C, and O-H functional groups, which indicated oxidative breakdown of the polymer. In contrast, low lysate concentrations produced similar but delayed or weaker spectral shifts, suggesting that higher enzyme titers and / or associated cofactors in the lysate may enhance the degradation process. This potential dose dependence supports the idea that multiple enzymes or supportive factors in the lysate work synergistically.

[0169]

[0148] No changes were detected in experiments using purified enzymes alone, even at high concentrations. This result suggests that essential cofactors, redox partners, or accessory proteins present in whole-cell lysates may be missing in the purified preparations, rendering them ineffective for catalyzing PVC breakdown in isolation.

[0170] Table 7: Summary of Enzymatic PVC Degradation

[0171] CONDITION SAMPLE TYPE DEGRADATION FTIR CHANGES INTERPRETATION 1 149] Similar degradation signatures were observed in the extension experiments using live E. coli expressing LPPc, CPKv, and LKv enzymes, particularly in nutrient media (both M9 + glucose and LB) (Table 8). These cultures produced C=O, C=C, and O-H peaks over time, consistent with oxidative depolymerization. This suggests that live cells, like lysates, can provide the necessary enzymatic context, cofactors, and secretion dynamics required for effective PVC transformation. This suggests that PVC degradation may depend on the enzymes and the full biochemical environment in which they operate.

[0172]

[0150] The FTIR analysis of PVC samples incubated with E. coli constructs expressing LPPc, CPKv, and LKv enzymes across various media types showed differences in degradation behavior. Cultures grown in LB (FIG. 34A - FIG. 34C) and M9 with glucose (FIG. 35A - FIG. 35B) showed the most pronounced chemical changes, including the formation of C=O, C=C, and O-H functional groups, along with a reduction in C-Cl bond intensity. These spectral shifts are consistent with oxidative depolymerization and dechlorination of PVC, suggesting that the presence of an accessible carbon source supports robust enzyme expression and metabolic activity. The M9 without glucose condition (FIG. 36A - FIG. 36B) produced only minimal spectral changes, with faint O-H signals appearing at later time points. This suggests that nutrient limitation may suppress both enzy me production and the cell’s ability7to process or utilize PVC-derived compounds. Overall, the data suggests that PVC degradation is most effective in nutrient-rich or co-metabolic conditions, where the cells may be metabolically active and capable of supporting sustained enzymatic activity.

[0173]

[0151] FIG. 34A-FIG. 34C shows FTIR spectra of PVC incubated in LB Media across 0, 2, and 4 Weeks. FTIR analysis of PVC powder treated with E. coli expressing LPPc, CPKv, and LKv in LB medium shows progressive degradation over time. Spectra from weeks 0 (FIG. 34A), 2 (FIG. 34B), and 4 (FIG. 34C) reveal increasing formation of C=C, C=O, and O-H functional groups, as well as a decline in C-Cl peaks, indicating oxidative depolymerization and dechlorination. Water and abiotic controls show minimal change. The results suggest robust enzymatic activity under nutrient-rich conditions.

[0174]

[0152] FIG. 35A and FIG. 35B show the FTIR spectra of PVC incubated in M9 Minimal Media with glucose. FTIR spectra of PVC treated with E. coli constructs in M9 minimal medium supplemented with glucose demonstrate time-dependent oxidative modifications. Samples exhibit characteristic formation of C=O, C=C, and O-H bonds from week 0 (FIG. 35A) to week 2 (FIG. 35B), especially in tubes with LKv and CPKv constructs. These changes indicate enzymatic degradation and suggest that a defined carbon source enables efficient expression and activity of the enzymes.

[0175]

[0153] FIG. 36A and FIG. 36B show FTIR spectra of PVC in M9 Minimal Media without glucose. Under nutrient-limited conditions (M9 without glucose), FTIR spectra show no evidence of PVC degradation at week 0 (FIG. 36 A). By week 2 (FIG. 36B), slight changes are detected, including weak C=O, C=C, and O-H peaks, particularly in samples with active constructs. The delay and size of these shifts suggest limited enzymatic activity under starvation conditions, suggesting that nutrient availability is important for supporting PVC biotransformation. For FIG. 34- FIG. 36, the sample numbers noted on the figures correspond to their sample numbers in Table 6. Table 8 summarizes the effect of media composition on PVC degradation for this experiment.

[0176] Table 8: Effect of Media Composition on PVC Degradation by Engineered E. coli

[0177] Media Type Nutrient Richness Observed FTIR Differences

[0178] LB (Lysogeny Broth) Rich, undefined medium Strongest changes: clear C=O. C=C, and O-H peaks; significant oxidation

[0179] M9 + Glucose Minimal, defined + glucose Strong C=O and C=C formation; pronounced oxidation and dichlorination

[0180] M9 (No Glucose) Minimal, defined, no carbon Only minor O-H formation at later stages; weak FTIR shifts overall

[0181]

[0154] During the incubation of neat PVC with E. coli strains expressing the LKv, CPKv, and LPPc enzymes, the polypropylene culture tubes developed a pronounced pink to purple coloration (FIG. 37). This discoloration was not present in control tubes lacking the enzyme constructs, despite using the same LB medium. Notably, the color extended throughout the entire inner surface of the 15 mL culture tubes, even though the liquid culture volume w as only 5 to 10 mL, indicating that the effect was not limited to areas in direct contact with the bacterial suspension. This may be consistent with colored intermediates, such as chlorinated aromatics or quinonoid compounds, which absorb light in the visible spectrum and could account for the purple / pink discoloration observed.

[0182]

[0155] FIG. 37 show s polypropylene culture tubes containing neat PVC incubated with E. coli strains expressing LKv (dehalogenase), CPKv (catalase peroxidase), and LPPc (lignin peroxidase). The tubes for LKv, CPKv, and LPPc exhibit distinct pink / purple discoloration along the inner tube walls, in contrast to the abiotic control (left), which show s no such change. All samples w ere cultured in LB medium for two weeks. The discoloration extended beyond the liquid culture volume, suggesting the formation of volatile or diffusible colored byproducts potentially associated with enzymatic dechlorination and oxidation of PVC.

[0183]

[0156] While preferred embodiments of the present disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the scope of the present disclosure. It should be understood that various alternatives to the embodiments of the present disclosure described herein may be employed in practicing the embodiments of the present disclosure. It is intended that the following claims define the scope of invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

[0184] Sequences

[0185]

[0157] SEQ ID NO: 1; Sequence Name: LPPc AA; Length: 372; Molecule t pe: AA

[0186]

[0158] Sequence:

[0187] MALKQLAAAVALALSIQAAQGAAVKEKRATCSNGATVGDASSCAWFDVLDDIQQNLFN GAQCGAEAHESIRLVFHDAIAISPALESQGKFGGGGADGSIILFDDIETNFHPNIGLDEIVN LQKPFIQKHGVTPGDFIAFAGAVAMSNCPGAPQMNFFTGRAPATQAAPDGLVPEPFHTVD QIISRVNDAGEFDELELVWMLSAHSVAAANDVDPTIQGLAFDSTPGVFDSQFFVETQLRG TAFPGSGGNQGEVESPLPGEMRLQSDSSIARDSRTACEWQSFVNNQSKLVSDFQFIFLALT QLGENPDAMTDCSDVIPISKPVPNNVPFSFFPAGKTMADVEQACAETPFPTLTTLPGPETS VQRIQPPPGA

[0188]

[0159] SEQ ID NO: 2; Sequence Name: LPPc_AA_tetracysteine; Length: 378; Molecule type : AA

[0189]

[0160] Sequence:

[0190] MALKQLAAAVALALSIQAAQGAAVKEKRATCSNGATVGDASSCAWFDVLDDIQQNLFN GAQCGAEAHESIRLVFHDAIAISPALESQGKFGGGGADGSIILFDDIETNFHPNIGLDEIVN LQKPFIQKHGVTPGDFIAFAGAVAMSNCPGAPQMNFFTGRAPATQAAPDGLVPEPFHTVD QIISRVNDAGEFDELELVWMLSAHSVAAANDVDPTIQGLAFDSTPGVFDSQFFVETQLRG TAFPGSGGNQGEVESPLPGEMRLQSDSSIARDSRTACEWQSFVNNQSKLVSDFQFIFLALT QLGENPDAMTDCSDVIPISKPVPNNVPFSFFPAGKTMADVEQACAETPFPTLTTLPGPETS VQRIQPPPGACCPGCC

[0191]

[0161] SEQ ID NO: 3; Sequence Name: CPKv AA; Length: 725 Molecule Type: AA fl 62] Sequence:

[0192] MSTSNDPSNNASAGKCPFHAETPKQSAGSGTGNRDWWPNQLRVDLLNQHSSRSNPLGE

[0193] DFNYREEFKKLDYSALKADLRALLTDSQEWWPADWGSYIGLFIRMAWHGAGTYRTVD

[0194] GRGGAGRGQQRFAPLNSWPDNVSLDKARRLLWPVKQKYGQKISWADLYMLAGNVALE

[0195] NAGFRTFGFGAGREDVWEPDLDVDWGDEKEWLAHRHPESLAKQAIGATEMGLIYVNP

[0196] EGPNASGEPLSAAAAIRATFGNMAMDDEEIVALIAGGHTLGKTHGAAETSHVGAEPEAA

[0197] PLEAQGLGWHSSYGSGAGADAITSGLEVVWTQTPTQWSNYFFENLFKYEWVQTRSPAG

[0198] AIQFEAKDAPEIIPDPFNPGKKRKPTMLVTDLTLRFDPEFEKISRRFLNDPQAFNEAFARA

[0199] WFKLTHRDMGPKSRYLGPEVPKEDLIWQDPLPAATHQPSAEDIASLKTAIAGAGLSVSEL

[0200] VSVAWASASTFRGGDKRGGANGARLALAPQKDWPVNAIASRVLPTLQAIQRASGKASL

[0201] ADIIVLAGVVGVEQAAAAAGVSVNVPFTPGRVDALPEQTDVESFDLLQPLADGFRNYR

[0202] RIEGGVSTETLLIDKAQQLTLTAPEMTVLVGGLRVLGANYDGSKHGVFTDRVGVLSNDF

[0203] FVNLLDMATVWKAADDHAELFTGSDRKTGEAKYSATRVDLVFGSNSVLRALAEVYAC

[0204] ADGQQKLVHDFVAAWTKVMNLDRFDL

[0205]

[0163] SEQ ID NO: 4; Sequence Name: CPKv_AA_tetracysteine; Length: 731; Molecule type:

[0206] AA

[0207]

[0164] Sequence:

[0208] MSTSNDPSNNASAGKCPFHAETPKQSAGSGTGNRDWWPNQLRVDLLNQHSSRSNPLGE

[0209] DFNYREEFKKLDYSALKADLRALLTDSQEWWPADWGSYIGLFIRMAWHGAGTYRTVD

[0210] GRGGAGRGQQRFAPLNSWPDNVSLDKARRLLWPVKQKYGQKISWADLYMLAGNVALE

[0211] NAGFRTFGFGAGREDVWEPDLDVDWGDEKEWLAHRHPESLAKQAIGATEMGLIYVNP

[0212] EGPNASGEPLSAAAAIRATFGNMAMDDEEIVALIAGGHTLGKTHGAAETSHVGAEPEAA

[0213] PLEAQGLGWHSSYGSGAGADAITSGLEVVWTQTPTQWSNYFFENLFKYEWVQTRSPAG

[0214] AIQFEAKDAPEIIPDPFNPGKKRKPTMLVTDLTLRFDPEFEKISRRFLNDPQAFNEAFARA

[0215] WFKLTHRDMGPKSRYLGPEVPKEDLIWQDPLPAATHQPSAEDIASLKTAIAGAGLSVSEL

[0216] VSVAWASASTFRGGDKRGGANGARLALAPQKDWPVNAIASRVLPTLQAIQRASGKASL

[0217] ADIIVLAGVVGVEQAAAAAGVSVNVPFTPGRVDALPEQTDVESFDLLQPLADGFRNYR

[0218] RIEGGVSTETLLIDKAQQLTLTAPEMTVLVGGLRVLGANYDGSKHGVFTDRVGVLSNDF

[0219] FVNLLDMATVWKAADDHAELFTGSDRKTGEAKYSATRVDLVFGSNSVLRALAEVYAC

[0220] ADGQQKLVHDFVAAWTKVMNLDRFDLCCPGCC

[0221]

[0165] SEQ ID NO: 5; Sequence Name: LKv_AA; Length: 241; Molecule type: AA

[0222]

[0166] Sequence:

[0223] MAYTSRLLNAIPGIRHAFLNVHETAAFPYAELAPVKLVHGNEVHHYQQPLPTRPHADAV FTAVAGQKVGVVTADCLPMLIASRDGRFVCSVHAGWQGLVSGIVDNSLACFRQQGVAL ADLVIAVGPHIHPCCYEVSAGFYQQLLDQPGGDRVARHRQRLFHSRSGPVSDALKAAAR GSDNLWFDLRAFAEAIFAEAGVSPASVEWLGSCTYCTPQSLGSYRRRTHFPAPKSFQYSW ILREA

[0224]

[0167] SEQ ID NO: 6; Sequence Name: LKv_AA_tetracysteine; Length: 247; Molecule Type: AA

[0225]

[0168] Sequence:

[0226] MAYTSRLLNAIPGIRHAFLNVHETAAFPYAELAPVKLVHGNEVHHYQQPLPTRPHADAV FTAVAGQKVGVVTADCLPMLIASRDGRFVCSVHAGWQGLVSGIVDNSLACFRQQGVAL ADLVIAVGPHIHPCCYEVSAGFYQQLLDQPGGDRVARHRQRLFHSRSGPVSDALKAAAR GSDNLWFDLRAFAEAIFAEAGVSPASVEWLGSCTYCTPQSLGSYRRRTHFPAPKSFQYSW ILREACCPGCC

[0227]

[0169] SEQ ID NO: 7; Sequence Name: LPPc_DNA_opt; Length: 1116; Molecule Type: DNA

[0228]

[0170] Sequence: atggcgctgaaacagctggcggcggcggtggcgctggcgctgagcattcaggcggcgcagggcgcggcggtgaaagaaaaacgcgc gacctgcagcaacggcgcgaccgtgggcgatgcgagcagctgcgcgtggtttgatgtgctggatgatattcagcagaacctgtttaacgg cgcgcagtgcggcgcggaagcgcatgaaagcattcgcctggtgtttcatgatgcgattgcgattagcccggcgctggaaagccagggca aatttggcggcggcggcgcggatggcagcattattctgtttgatgatattgaaaccaactttcatccgaacattggcctggatgaaattgtgaa cctgcagaaaccgtttattcagaaacatggcgtgaccccgggcgattttattgcgtttgcgggcgcggtggcgatgagcaactgcccgggc gcgccgcagatgaacttttttaccggccgcgcgccggcgacccaggcggcgccggatggcctggtgccggaaccgtttcataccgtgga tcagattattagccgcgtgaacgatgcgggcgaatttgatgaactggaactggtgtggatgctgagcgcgcatagcgtggcggcggcgaa cgatgtggatccgaccattcagggcctggcgtttgatagcaccccgggcgtgtttgatagccagttttttgtggaaacccagctgcgcggca ccgcgtttccgggcagcggcggcaaccagggcgaagtggaaagcccgctgccgggcgaaatgcgcctgcagagcgatagcagcattg cgcgcgatagccgcaccgcgtgcgaatggcagagctttgtgaacaaccagagcaaactggtgagcgattttcagtttatttttctggcgctg acccagctgggcgaaaacccggatgcgatgaccgattgcagcgatgtgattccgattagcaaaccggtgccgaacaacgtgccgtttagc ttttttccggcgggcaaaaccatggcggatgtggaacaggcgtgcgcggaaaccccgtttccgaccctgaccaccctgccgggcccgga aaccagcgtgcagcgcattcagccgccgccgggcgcg

[0229]

[0171] SEQ ID NO: 8; Sequence Name: LPPc_DNA_opt_tetracysteine; Length: 1134; Molecule type: DNA

[0230]

[0172] Sequence: atggcgctgaaacagctggcggcggcggtggcgctggcgctgagcattcaggcggcgcagggcgcggcggtgaaagaaaaacgcgc gacctgcagcaacggcgcgaccgtgggcgatgcgagcagctgcgcgtggtttgatgtgctggatgatattcagcagaacctgtttaacgg cgcgcagtgcggcgcggaagcgcatgaaagcattcgcctggtgtttcatgatgcgattgcgattagcccggcgctggaaagccagggca aatttggcggcggcggcgcggatggcagcattattctgtttgatgatattgaaaccaactttcatccgaacattggcctggatgaaattgtgaa cctgcagaaaccgtttattcagaaacatggcgtgaccccgggcgattttattgcgtttgcgggcgcggtggcgatgagcaactgcccgggc gcgccgcagatgaacttttttaccggccgcgcgccggcgacccaggcggcgccggatggcctggtgccggaaccgtttcataccgtgga tcagattattagccgcgtgaacgatgcgggcgaatttgatgaactggaactggtgtggatgctgagcgcgcatagcgtggcggcggcgaa cgatgtggatccgaccattcagggcctggcgtttgatagcaccccgggcgtgtttgatagccagttttttgtggaaacccagctgcgcggca ccgcgtttccgggcagcggcggcaaccagggcgaagtggaaagcccgctgccgggcgaaatgcgcctgcagagcgatagcagcattg cgcgcgatagccgcaccgcgtgcgaatggcagagctttgtgaacaaccagagcaaactggtgagcgattttcagtttatttttctggcgctg acccagctgggcgaaaacccggatgcgatgaccgattgcagcgatgtgattccgattagcaaaccggtgccgaacaacgtgccgtttagc ttttttccggcgggcaaaaccatggcggatgtggaacaggcgtgcgcggaaaccccgtttccgaccctgaccaccctgccgggcccgga aaccagcgtgcagcgcattcagccgccgccgggcgcgtgctgtccaggctgttgc

[0231]

[0173] SEQ ID NO: 9; Sequence Name: CPKv DNA opt; Length: 2175; Molecule type: DNA

[0232]

[0174] Sequence: atgagtacgtctaacgatccgagtaataatgcctcggccgggaaatgtcctttccatgctgagaccccaaaacaaagtgctggttctggaac cggaaacagagattggtggcctaatcaactgcgtgtggatctgctgaaccaacactcaagccgctcaaatccgttaggagaggattttaact atcgggaagaattcaaaaaattggattattctgctcttaaagcggatctgcgtgctctgctgactgattcccaagaatggtggccagcggact ggggatcttacataggtttatttattcgtatggcttggcatggtgccggtacataccgtactgtggatggacggggcggagctgggcgtggc cagcaacgttttgctccgttaaattcatggccggataatgtaagcctggataaagcacgtagattattatggccggtgaagcaaaagtatggc cagaaaatttcatgggcagatttatacatgctggcaggcaacgttgcgcttgaaaacgctggctttcggactttcgggttcggtgccggtcgt gaagacgtgtgggaaccggatttagatgtcgactggggcgatgagaaagaatggctggcgcatcgtcatccggagtccctggcaaaaca ggctattggagctacagaaatggggttaatatacgtgaatcctgaaggaccaaatgcaagcggtgaaccactgagcgccgcagcggcaat tagagcgacttttggaaacatggctatggatgatgaagaaattgttgcgttgatcgcgggagggcatactctgggcaaaacccacggtgcc gcggaaacttcacatgtaggtgccgaaccggaagccgcacctctggaggcacagggcctgggctggcactcttcatatggctctggggc aggcgctgatgccatcacgagcggcctggaagtagtgtggacacagactccgacccagtggtctaattacttttttgaaaatttgtttaagtac gaatgggtccagacgcggtccccggctggggccatccaatttgaagctaaagacgccccggagatcattcctgacccgtttaaccctgga aaaaagcgcaaacctaccatgttggttacagatttgacgctgcggtttgaccccgaatttgaaaaaatttcgagacgtttcctgaacgatcctc aagcgtttaatgaggcgttcgcccgtgcatggtttaaattaacccatagagatatgggcccgaaaagccgctacttgggtccagaagtccct aaagaggatctgatttggcaggatcctttaccagcggccactcatcagccaagtgcggaagatatagcgtctctgaaaaccgcgatagcag gcgcgggtctgagcgtcagcgaattggttagtgtggcttgggcaagtgcatcgacctttcggggtggcgacaagcgtggcggtgctaatg gtgcaagactggcgctggcgccgcagaaagattggccggtcaacgctatcgcaagcagagtgctgccaaccttacaggcaattcagcgc gcgagtggaaaagcatctctggcagatattatcgtcctggcgggcgtggttggcgttgagcaagccgcggcagcagctggcgtaagcgta aacgtcccgttcacaccggggcgtgtagacgccttaccggagcaaacagatgtcgaaagctttgatctgctgcaaccgttggccgatggttt tcgcaattaccgcagaatagagggcggcgtctcaaccgagactctgcttattgataaagcacagcaacttacgttaaccgcacccgaaatg accgtgctggtgggaggcctgcgtgtacttggtgctaactatgatggatctaaacacggagtttttacggatcgtgtaggcgtattaagcaac gattttttcgtcaatcttttagacatggccactgtttggaaggcagcggatgaccatgccgaattgtttaccgggtccgatagaaaaacgggtg aggcaaaatattctgcaaccagagtggacctggtgtttggaagcaactccgttttaagagctctggcggaagtgtatgcgtgcgcggatgga cagcagaaattagttcatgattttgttgctgcgtggactaaagttatgaatcttgaccgcttcgatctg

[0233]

[0175] SEQ ID NO: 10; Sequence Name: CPKv_DNA_opt_tetracysteine; Length: 2193; Molecule type; DNA

[0234]

[0176] Sequence: atgagtacgtctaacgatccgagtaataatgcctcggccgggaaatgtcctttccatgctgagaccccaaaacaaagtgctggttctggaac cggaaacagagattggtggcctaatcaactgcgtgtggatctgctgaaccaacactcaagccgctcaaatccgttaggagaggattttaact atcgggaagaattcaaaaaattggattattctgctcttaaagcggatctgcgtgctctgctgactgattcccaagaatggtggccagcggact ggggatcttacataggtttatttattcgtatggcttggcatggtgccggtacataccgtactgtggatggacggggcggagctgggcgtggc cagcaacgttttgctccgttaaattcatggccggataatgtaagcctggataaagcacgtagattattatggccggtgaagcaaaagtatggc cagaaaatttcatgggcagatttatacatgctggcaggcaacgttgcgcttgaaaacgctggctttcggactttcgggttcggtgccggtcgt gaagacgtgtgggaaccggatttagatgtcgactggggcgatgagaaagaatggctggcgcatcgtcatccggagtccctggcaaaaca ggctattggagctacagaaatggggttaatatacgtgaatcctgaaggaccaaatgcaagcggtgaaccactgagcgccgcagcggcaat tagagcgacttttggaaacatggctatggatgatgaagaaattgttgcgttgatcgcgggagggcatactctgggcaaaacccacggtgcc gcggaaacttcacatgtaggtgccgaaccggaagccgcacctctggaggcacagggcctgggctggcactcttcatatggctctggggc aggcgctgatgccatcacgagcggcctggaagtagtgtggacacagactccgacccagtggtctaattacttttttgaaaatttgtttaagtac gaatgggtccagacgcggtccccggctggggccatccaatttgaagctaaagacgccccggagatcattcctgacccgtttaaccctgga aaaaagcgcaaacctaccatgttggttacagatttgacgctgcggtttgaccccgaatttgaaaaaatttcgagacgtttcctgaacgatcctc aagcgtttaatgaggcgttcgcccgtgcatggtttaaattaacccatagagatatgggcccgaaaagccgctacttgggtccagaagtccct aaagaggatctgatttggcaggatcctttaccagcggccactcatcagccaagtgcggaagatatagcgtctctgaaaaccgcgatagcag gcgcgggtctgagcgtcagcgaattggttagtgtggcttgggcaagtgcatcgacctttcggggtggcgacaagcgtggcggtgctaatg gtgcaagactggcgctggcgccgcagaaagattggccggtcaacgctatcgcaagcagagtgctgccaaccttacaggcaattcagcgc gcgagtggaaaagcatctctggcagatattatcgtcctggcgggcgtggttggcgttgagcaagccgcggcagcagctggcgtaagcgta aacgtcccgttcacaccggggcgtgtagacgccttaccggagcaaacagatgtcgaaagctttgatctgctgcaaccgttggccgatggttt tcgcaattaccgcagaatagagggcggcgtctcaaccgagactctgcttattgataaagcacagcaacttacgttaaccgcacccgaaatg accgtgctggtgggaggcctgcgtgtacttggtgctaactatgatggatctaaacacggagtttttacggatcgtgtaggcgtattaagcaac gattttttcgtcaatcttttagacatggccactgtttggaaggcagcggatgaccatgccgaattgtttaccgggtccgatagaaaaacgggtg aggcaaaatattctgcaaccagagtggacctggtgtttggaagcaactccgttttaagagctctggcggaagtgtatgcgtgcgcggatgga cagcagaaattagttcatgattttgttgctgcgtggactaaagttatgaatcttgaccgcttcgatctgtgctgtcctggctgctgc

[0235]

[0177] SEQ ID NO: 11; Sequence name: LKv_DNA_opt; Length: 723; Moleculte type: DNA

[0236]

[0178] Sequence: atggcgtataccagccgcctgctgaacgcgattccgggcattcgccatgcgtttctgaacgtgcatgaaaccgcggcgtttccgtatgcgga actggcgccggtgaaactggtgcatggcaacgaagtgcatcatatcagcagccgctgccgacccgcccgcatgcggatgcggtgtttac cgcggtggcgggccagaaagtgggcgtggtgaccgcggattgcctgccgatgctgattgcgagccgcgatggccgctttgtgtgcagcg tgcatgcgggctggcagggcctggtgagcggcattgtggataacagcctggcgtgctttcgccagcagggcgtggcgctggcggatctg gtgattgcggtgggcccgcatattcatccgtgctgctatgaagtgagcgcgggcttttatcagcagctgctggatcagccgggcggcgatc gcgtggcgcgccatcgccagcgcctgtttcatagccgcagcggcccggtgagcgatgcgctgaaagcggcggcgcgcggcagcgata acctgtggtttgatctgcgcgcgttgcggaagcgattttgcggaagcgggcgtgagcccggcgagcgtggaatggctgggcagctgca cctattgcaccccgcagagcctgggcagctatcgccgccgcacccattttccggcgccgaaaagctttcagtatagctggattctgcgcga agcg

[0237]

[0179] SEQ ID NO: 12; Sequence Name: LKv_DNA_opt_tetracysteine; Length: 741; Molecule tvpe: DNA

[0238]

[0180] Sequence: atggcgtataccagccgcctgctgaacgcgattccgggcattcgccatgcgtttctgaacgtgcatgaaaccgcggcgtttccgtatgcgga actggcgccggtgaaactggtgcatggcaacgaagtgcatcattatcagcagccgctgccgacccgcccgcatgcggatgcggtgtttac cgcggtggcgggccagaaagtgggcgtggtgaccgcggattgcctgccgatgctgattgcgagccgcgatggccgcttgtgtgcagcg tgcatgcgggctggcagggcctggtgagcggcatgtggataacagcctggcgtgctttcgccagcagggcgtggcgctggcggatctg gtgattgcggtgggcccgcatattcatccgtgctgctatgaagtgagcgcgggcttttatcagcagctgctggatcagccgggcggcgatc gcgtggcgcgccatcgccagcgcctgtttcatagccgcagcggcccggtgagcgatgcgctgaaagcggcggcgcgcggcagcgata acctgtggtttgatctgcgcgcgtttgcggaagcgatttttgcggaagcgggcgtgagcccggcgagcgtggaatggctgggcagctgca cctattgcaccccgcagagcctgggcagctatcgccgccgcacccatttccggcgccgaaaagcttcagtatagctggatctgcgcga agcgtgctgtccaggctgttgc

Claims

CLAIMSWhat is claimed is:

1. A method of separating components of wire waste, comprising: a. combining a wire waste with a medium comprising a lignin peroxidase, a catalase peroxidase, and a dehalogenase, wherein the wire waste comprises a transmitting portion encapsulated at least partially in an encapsulating layer comprising polyvinyl chloride; and b. at least partially separating the transmitting portion from the encapsulating layer by degrading the polyvinyl chloride of the encapsulating layer, wherein the lignin peroxidase, the catalase peroxidase, and the dehalogenase cause the degradation.

2. The method of claim 1, wherein the lignin peroxidase comprises an amino acid sequence with at least a 90% identity to SEQ ID NO: 1.

3. The method of claim 1, wherein the catalase peroxidase comprises an amino acid sequence with at least a 90% identity to SEQ ID NO: 3.

4. The method of claim 1, wherein the dehalogenase comprises an amino acid sequence with at least a 90% identity to SEQ ID NO: 5.

5. The method of claim 1, wherein the degradation comprises oxidation.

6. The method of claim 1, wherein the degradation comprises chain depolymerization.

7. The method of claim 1, wherein the degradation comprises hydrolysis.

8. The method of claim 1, wherein the degradation comprises dechlorination.

9. The method of claim 8, wherein the dehalogenase dechlorinates the polyvinyl chloride.

10. The method of claim 8, wherein the lignin peroxidase dechlorinates the polyvinyl chloride.

11. The method of claim 1, wherein the medium further comprises a laccase or a monooxygenase.

12. The method of claim 11, wherein the degradation of the polyvinyl chloride generates a degradation product.

13. The method of claim 12, further comprising mineralizing the degradation product.

14. The method of claim 13, wherein the laccase or the monooxygenase mineralizes the degradation product.

15. The method of claim 1, wherein the medium comprises a cell lysate.

16. The method of claim 1, wherein the medium comprises a microorganism configured to produce the lignin peroxidase, the catalase peroxidase, or the dehalogenase.

17. The method of claim 16, wherein the microorganism comprises a bacterium or a yeast.

18. The method of claim 17, wherein the bacterium comprises Escherichia coli.

19. The method of claim 17, wherein the yeast comprises Kluyveromyces lactis.

20. The method of claim 1, wherein the medium comprises at least one enzyme co-factor.

21. The method of claim 20, wherein the at least one enzyme co-factor improves the degradation of the polyvinyl chloride by the lignin peroxidase, the catalase peroxidase, or the dehalogenase.

22. The method of claim 1, wherein the transmitting portion comprises a metal portion.

23. The method of claim 22, wherein the metal portion comprises copper.

24. The method of claim 22, wherein the metal portion comprises gold, iron, nickel, tin, lead, aluminum, germanium, or zinc.

25. The method of claim 22, wherein the metal portion comprises a rare earth metal.

26. The method of claim 25, wherein the rare earth metal comprises neodymium, lanthanum, yttrium, or dysprosium.

27. The method of claim 1, wherein the wire waste comprises a wire configured to transmit electricity or information.

28. The method of claim 1, wherein the wire waste comprises electrical wire waste, PVC- jacketed cable waste, wire scrap, electrical wire harness, mixed metal wire waste, fiber optic cable, cable-rich e-waste, or dismantled electronics.

29. The method of claim 1, wherein degrading the polyvinyl chloride increases a brittleness of the encapsulating layer.

30. The method of claim 1, wherein degrading the polyvinyl chloride forms cracks in the encapsulating layer.

31. The method of claim 1, further comprising mechanically separating the transmitting portion from the encapsulating layer.

32. The method of claim 31, wherein mechanically separating comprises shredding or ball milling the wire waste.

33. The method of claim 32, wherein mechanically separating further comprises sieving the wire waste.

34. The method of claim 33, wherein mechanically separating further comprises flotation of the wire waste.

35. The method of claim 1, further comprising treating a waste stream to produce the wire waste.

36. The method of claim 35, wherein treating the waste stream comprises shredding the waste stream and gravity separating the waste stream into a recycled transmitting portion, a free layer portion, and a portion comprising the wire waste.

37. The method of claim 36, further comprising magnetically separating a magnetic portion of the waste stream.

38. A method of separating components of wire waste, comprising: a. combining a wire waste with a medium comprising a microorganism, wherein the wire waste comprises a transmitting portion encapsulated at least partially in an encapsulating layer comprising polyvinyl chloride; and b. at least partially separating the transmitting portion from the encapsulating layer by degrading the polyvinyl chloride of the encapsulating layer, wherein the microorganism degrades the polyvinyl chloride by dechlorinating the polyvinyl chloride.

39. The method of claim 38, wherein the dechlorination comprises an enzymatic process.

40. The method of claim 39, wherein the enzymatic process comprises a dehalogenation step, an oxidation step, a chain depolymerization step, or a hydrolysis step.

41. The method of claim 39, wherein the microorganism is genetically modified to express an enzyme of the enzymatic process.

42. The method of claim 41, wherein the enzy me is secreted by the microorganism.

43. The method of claim 41, wherein the enzyme comprises a catalase peroxidase, a lignin peroxidase, a dehalogenase, a laccase, or a monooxygenase.

44. The method of claim 41, wherein the enzyme comprises a mutant of a catalase peroxidase, a dehalogenase, a lignin peroxidase, a laccase, or a monooxygenase.

45. The method of claim 41, wherein the enzyme comprises a catalase peroxidase portion and a dehalogenase portion.

46. The method of claim 45, wherein the enzyme further comprises a lignin peroxidase portion.

47. The method of any one of claims 41-46, wherein the enzy me comprises an amino acid sequence with at least 90% identity to SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5.

48. The method of claim 41, wherein the microorganism is genetically modified to express a second enzyme of the enzymatic process.

49. The method of claim 48, wherein the microorganism is genetically modified to express a third enzyme of the enzymatic process.

50. The method of claim 38, further comprising mineralizing a degradation product formed by the degradation of the polyvinyl chloride.

51. The method of claim 50, wherein the mineralization comprises an enzymatic process.

52. The method of claim 51, wherein a laccase or a monooxygenase mineralizes the degradation product.

53. The method of claim 38, wherein the microorganism comprises a bacterium or a yeast.

54. The method of claim 53, wherein the bacterium comprises Escherichia coli.

55. The method of claim 53, wherein the yeast comprises Kluyveromyces lactis.

56. The method of claim 38, wherein the wire waste comprises a wire configured to transmit electricity or information.

57. The method of claim 38, wherein the transmitting portion comprises a metal portion.

58. The method of claim 57, wherein the metal portion comprises copper.

59. The method of claim 57, wherein the metal portion comprises gold, iron, nickel, tin, lead, aluminum, germanium, or zinc.

60. The method of claim 57, wherein the metal portion comprises a rare earth metal.

61. The method of claim 60, wherein the rare earth metal comprises neodymium, lanthanum, yttrium, or dysprosium.

62. The method of claim 38, wherein the wire waste comprises electrical wire waste, PVC- jacketed cable waste, wire scrap, electrical wire harness, fiber optic cable, mixed metal wire waste, cable-rich e-waste, or dismantled electronics.

63. The method of claim 38, wherein degrading the polyvinyl chloride increases a brittleness of the encapsulating layer.

64. The method of claim 38, wherein degrading the polyvinyl chloride forms cracks in the encapsulating layer.

65. The method of claim 38, further comprising mechanically separating the transmitting portion from the encapsulating layer.

66. The method of claim 65, wherein mechanically separating comprises shredding or ball milling the wire waste.

67. The method of claim 66, wherein mechanically separating further comprises sieving the wire waste.

68. The method of claim 67, wherein mechanically separating further comprises flotation of the wire waste.

69. The method of claim 38, further comprising treating a waste stream to produce the wire waste.

70. The method of claim 69, wherein treating the waste stream comprises shredding the waste stream and gravity separating the waste stream into a recycled transmitting portion, a free layer portion, and a portion comprising the wire waste.

71. The method of claim 70, further comprising magnetically separating a magnetic portion of the waste stream.

72. A nuclei acid comprising: a. A genetic sequence comprising at least 90% sequence identity with SEQ ID NO. : 3, wherein the genetic sequence comprises a sequence of a catalase peroxidase that is codon-optimized for expression in Escherichia coli.

73. A nuclei acid comprising: a. A genetic sequence comprising at least 90% sequence identity with SEQ ID NO. :1, wherein the genetic sequence comprises a sequence of a lignin peroxidase that is codon-optimized for expression in Escherichia coli.

74. A nuclei acid comprising: a. A genetic sequence comprising at least 90% sequence identity with SEQ ID NO. : 5, wherein the genetic sequence comprises a sequence of a dehalogenase that is codon-optimized for expression in Escherichia coli.

75. A method of separating components of wire waste, comprising: a. combining a wire waste with a medium comprising a lignin peroxidase, wherein the wire waste comprises a transmitting portion encapsulated at least partially in a layer comprising polyvinyl chloride; and b. at least partially separating the transmitting portion from the encapsulating layer by degrading the polyvinyl chloride of the layer, wherein the lignin peroxidase degrades the polyvinyl chloride.

76. The method of claim 75, wherein the lignin peroxidase comprises an amino acid sequence with at least 90% identity to SEQ ID NO: 1.

77. The method of claim 75, wherein the transmitting portion comprises a metal portion.

78. A method of separating components of wire waste, comprising:a. combining a wire waste with a medium comprising a catalase peroxidase, wherein the wire waste comprises a transmitting portion encapsulated at least partially in an encapsulating layer comprising polyvinyl chloride; and b. at least partially separating the transmitting portion from the encapsulating layer by degrading the polyvinyl chloride of the encapsulating layer, wherein the catalase peroxidase degrades the polyvinyl chloride.

79. The method of claim 78, wherein the catalase peroxidase comprises an amino acid sequence with at least 90% identity to SEQ ID NO: 3.

80. The method of claim 78, wherein the transmitting portion comprises a metal portion.

81. A method of separating components of wire waste, comprising: a. combining a wire waste with a medium comprising a dehalogenase, wherein the wire waste comprises a transmitting portion encapsulated at least partially in an encapsulating layer comprising polyvinyl chloride; and b. at least partially separating the transmitting portion from the encapsulating layer by degrading the polyvinyl chloride of the layer, wherein the dehalogenase degrades the polyvinyl chloride.

82. The method of claim 81, wherein the dehalogenase comprises an amino acid sequence with at least 90% identity to SEQ ID NO: 5.

83. The method of claim 81, wherein the transmitting portion comprises a metal portion.

84. A system for metal recovery comprising: a. a reaction vessel configured to contain a wire waste comprising a transmitting portion encapsulated at least partially in an encapsulating layer comprising polyvinyl chloride, and a medium comprising an enzyme combination configured to degrade the polyvinyl chloride of the encapsulating layer, wherein the enzyme combination comprises a lignin peroxidase, a catalase peroxidase, and a dehalogenase; and b. A separation vessel connected to the reaction vessel, wherein the separation vessel is configured to mechanically separate the transmitting portion from the encapsulating layer.

85. The system of claim 84, wherein the transmitting portion comprises a metal portion.

86. The system of claim 84, wherein the wire waste comprises a wire configured to transmit electricity or information.

Images