A composite for plastic degradation and its applications

Abstract

The present invention discloses a complex for plastic degradation and its application, belonging to the technical field of enzyme gene engineering. Based on the enzyme complex provided by the present disclosure, an in vitro degradation system is constructed. Through in vitro self-assembly, plastic degradation reactions are carried out in the form of a multi-enzyme complex, especially for plastic substrates with high crystallinity, further expanding the substrate adaptability of the complex. Finally, the plastic degradation under the participation of the complex has achieved a substantial improvement in degradation efficiency, realizing the efficient degradation of plastics and having great industrialization value.

Description

Technical Field

[0001] The present disclosure belongs to the technical field of enzyme gene engineering, and particularly relates to a complex capable of degrading plastics and its applications. Background Art

[0002] Plastics, especially polyethylene terephthalate (PET), have excellent toughness, tensile strength, impact resistance, wear resistance, electrical insulation, etc. Due to their plasticity and high stability, plastic products have been widely used in the fields of fibers, packaging, containers, electrical components, etc. The amount of waste generated is huge, making it one of the main sources of white pollution. The resulting white pollution has had a serious impact on soil and water sources, and PET can also enter the human body through the food chain in the form of microplastics, causing harm to the human body.

[0003] Traditional plastic (such as PET) degradation technologies can be divided into physical degradation and chemical degradation. Physical degradation mainly involves, without changing the structure of the PET polymer chain, crushing, cleaning, sorting, drying, and granulating plastic products, and then injection molding, stretching, and blow molding to make packaging containers, or producing short fibers, non-woven fabrics, polyester filaments, etc. through melt extrusion for making clothing and household items. Physical degradation has a simple process and low cost, and is the main method for plastic waste degradation in China. However, this method has high requirements for the purity of plastics, and the mechanical properties of the degraded plastic products decline, with low product added value, and can only be used as secondary-grade plastics, and can only be added to chemical fiber products in extremely small amounts. In addition, when blown and drawn at high temperatures, some plastics (such as PET) are prone to decompose and produce acetaldehyde, so such packaging is not suitable for direct use in products such as food and medicine.

[0004] Chemical degradation uses methods such as alcoholysis, hydrolysis, ammonia / amineolysis to depolymerize plastics into monomers or intermediates. For PET, it is degraded into dimethyl terephthalate (DMT), terephthalic acid (TPA), bis-hydroxyethyl terephthalate (BHET), etc., and then monomers or intermediates are polymerized to prepare raw materials or products. Using chemical methods to degrade PET, such as for producing food-grade PET packaging such as "bottle-to-bottle", has high requirements for the purity of raw materials and can only be applied to transparent PET bottles. Therefore, it is necessary to develop more economical and green and efficient degradation technologies.

[0005] Ecologically friendly, highly efficient, and thorough degradation by enzymatic methods provides more options for the recycling of polyethylene terephthalate (PET) plastic waste. Many studies have used machine learning, random mutagenesis, etc. to modify plastic-degrading enzymes to improve their enzymatic properties. Among them, the FAST-PETase designed using machine learning has a high PET plastic degradation ability; the engineered protein ICCG suitable for large-scale fermenters enhances the substrate adaptability to PET waste and shows the degradation ability for highly crystalline plastics. However, a large number of studies mainly focus on the optimization of single plastic-degrading enzymes. Exploring and optimizing more plastic enzymatic degradation methods is of great significance for improving the degradation efficiency of plastic-degrading enzymes. Summary of the Invention

[0006] To solve one of the above technical problems existing in the prior art, the present invention provides an enzyme complex and its application to solve at least one of the above problems. Using the enzyme complex provided by the present disclosure can improve the degradation efficiency of plastics.

[0007] According to one aspect of the present disclosure, there is provided a complex for degrading plastics with an enzyme complex, the complex for degrading plastics comprising: at least one biodegradation module, each biodegradation module independently comprising an enzyme capable of degrading plastics or a functional variant or a fragment thereof, and a member of a polypeptide interaction pair; and, at least one scaffold protein, each scaffold protein independently comprising one or more other members of the polypeptide interaction pair, optionally further comprising one or more hydrophobic domains.

[0008] In some embodiments, one member of the polypeptide interaction pair specifically binds to the other member of the polypeptide interaction pair. The at least one biodegradation module and the at least one scaffold protein self-assemble through the protein interaction pair to form a complex for degrading plastics, which includes at least one enzyme capable of degrading plastics.

[0009] In some embodiments, the enzymes or functional variants or fragments thereof included in the at least one biodegradation module are the same or different from each other.

[0010] In some embodiments, the members of the polypeptide interaction pair included in the at least one biodegradation module are the same or different from each other.

[0011] In some embodiments, the plastics include any one or more of polylactic acid (PLA), polyethylene, polypropylene (PP), polystyrene (PS), polyester, polyurethane (PU).

[0012] In some embodiments, the polyester includes any one or more of polyethylene terephthalate (PET), polytrimethylene terephthalate (PTT), polybutylene terephthalate (PBT), polyethylene isosorbide terephthalate (PEIT), acrylonitrile butadiene styrene (ABS), polymethyl methacrylate (PMMA), cellulose acetate (CA), polyvinyl chloride (PVC), polycarbonate (PC), thermoplastic elastomer (TPE), polyhydroxyalkanoate (PHA), polybutylene succinate (PBS), poly(butylene succinate-co-adipate) (PBSA), poly(butylene adipate-co-terephthalate) (PBAT), polyethylene furandicarboxylate (PEF), polycaprolactone (PCL), polyethylene naphthalate (PEN), polyester amide (PEA), poly(1,4-cyclohexanedimethylene terephthalate) (PCT), polyethylene terephthalate-1,4-cyclohexanedimethanol ester (PETG), poly(butylene terephthalate-co-butylene succinate) (PBST).

[0013] In some embodiments, the plastic may be polyethylene terephthalate (PET).

[0014] In some embodiments, the enzymes capable of degrading plastics include laccase, esterase, lipase, cutinase, and the like.

[0015] In some embodiments, the enzymes capable of degrading plastics include one or more of PET hydrolase (PETase) or its mutants, leaf compost cutinase (LCC) or its mutants, cutinase TfC or its mutants, cutinase Est119 or its mutants, cutinase BTA-1 or its mutants, cutinase Tcur0390 or its mutants, polyhydroxybutyrate (PHB) depolymerase and its mutants, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) depolymerase and its mutants, PLA depolymerase and its mutants, PBSA depolymerase and its mutants.

[0016] In some embodiments, the enzymes capable of degrading plastics include one or more of the following: PETase, IsPETase, FAST-PETase, ThermoPETase, DuraPETase, DepoPETase, BhrPETase, TurboPETase, CaPETaseM9, HotPETase, LCC, LCC-A2, ICCG, WCCG, ICCM, WCCM, MHETase, KLMHETase, and optiMHETase.

[0017] Those skilled in the art should understand that the protein pairs in the complexes for the present disclosure can be protein pairs that can specifically bind to each other and are commonly used in the art. In some embodiments, the polypeptide pairs are selected from one or more of ankyrin - integrin pairs, growth factor and growth factor receptor pairs, cytokine and cytokine receptor pairs, chemokine and chemokine receptor pairs, antibody - ligand pairs, and enzyme - substrate pairs.

[0018] In some embodiments, the polypeptide pairs are selected from one or more of the following: CelK - CipA, CelF - CipC, ScaA - ScaF, Cel44A - ScaA, Ce3B - ScaC, XDoc - ScaE, docCipA - olpB, ScaC - ScaI, ScaH - ScaE, ScaF - ScaJ, ScaG - ScaJ, Cel44A - CipA, Xyn10B - CipA, SH3 D -SH3 L , ScaA - ScaB or Xyn11A - CipA.

[0019] In some embodiments, the one or more hydrophobic domains are disposed between or on one side of the other member of the plurality of polypeptide pairs.

[0020] In some embodiments, the hydrophobic domains include one or more of the following: hydrophobins (HFB), carbohydrate binding modules (CBM); and polyhydroxyalkanoate binding modules (PBM).

[0021] In some embodiments, the HFB includes one or more of HFBI, HFBII, HFBIII, HFBIV, HFBVIII, SRHI, SC3 or HGFI.

[0022] In some embodiments, the CBM includes one or more of CBM1, CBM2, CBM3, CBM3a, CBM4, CBM5, CBM6, CBM9, CBM10, CBM11, CBM12, CBM14, CBM15, CBM17, CBM18, CBM19, CBM20, CBM21, CBM25, CBM27, CBM28, CBM33, CBM48, CBM49, CBM50, CBM51, CBM52, CBM53, CBM54, CBM55, CBM56, CBM57, CBM58, CBM59, CBM60, CBM61, CBM62, CBM63, CBM64, CBM65, CBM66, CBM67, CBM68, CBM69, CBM70, CBM71, CBM72, CBM73, CBM74, CBM75, CBM76, CBM77, CBM78, CBM79, CBM80, CBM81, CBM82, CBM83, CBM84, CBM85, CBM86, CBM87, or CBM88.

[0023] In some embodiments, the complex for degrading plastics includes: a first biodegradation module, which includes a first enzyme capable of degrading plastics or a functional variant or a fragment thereof, and a member of a first polypeptide interaction pair; a second biodegradation module, which includes a second enzyme capable of degrading plastics or a functional variant or a fragment thereof, and a member of a second polypeptide interaction pair; and a scaffold protein, which includes the other member of the first polypeptide interaction pair and the other member of the second polypeptide interaction pair, and optionally further includes one or more hydrophobic domains.

[0024] In some embodiments, the complex for degrading plastics includes: a first biodegradation module, which includes a first enzyme capable of degrading plastics or a functional variant or a fragment thereof, and a member of a first polypeptide interaction pair; a second biodegradation module, which includes a second enzyme capable of degrading plastics or a functional variant or a fragment thereof, and a member of a second polypeptide interaction pair; a third biodegradation module, which includes a second enzyme capable of degrading plastics or a functional variant or a fragment thereof, and a member of a third polypeptide interaction pair; and a scaffold protein, which includes the other member of the first polypeptide interaction pair, the other member of the second polypeptide interaction pair, and the other member of the third polypeptide interaction pair, and optionally further includes one or more hydrophobic domains.

[0025] In some embodiments, the first biodegradation module, the second biodegradation module, and / or the third biodegradation module may sequentially include, from the N-terminus to the C-terminus: an enzyme capable of degrading plastics or a functional variant or a fragment thereof, and a member of a polypeptide interaction pair.

[0026] In some embodiments, the first biodegradation module, the second biodegradation module, and / or the third biodegradation module may sequentially include, from the N-terminus to the C-terminus: a member of a polypeptide pair interaction, and an enzyme capable of degrading plastics or a functional variant or a fragment thereof.

[0027] In some embodiments, the scaffold protein may sequentially include, from the N-terminus to the C-terminus: the other member of the first polypeptide pair interaction and the other member of the second polypeptide pair interaction.

[0028] In some embodiments, the scaffold protein may sequentially include, from the N-terminus to the C-terminus: the other member of the first polypeptide pair interaction, the other member of the second polypeptide pair interaction, and a hydrophobic domain.

[0029] In some embodiments, the scaffold protein may sequentially include, from the N-terminus to the C-terminus: the other member of the first polypeptide pair interaction, the hydrophobic domain, and the other member of the second polypeptide pair interaction.

[0030] In some embodiments, the scaffold protein may sequentially include, from the N-terminus to the C-terminus: the hydrophobic domain, the other member of the first polypeptide pair interaction, and the other member of the second polypeptide pair interaction.

[0031] In some embodiments, the scaffold protein may sequentially include, from the N-terminus to the C-terminus: the other member of the first polypeptide pair interaction, the other member of the second polypeptide pair interaction, a member of the third polypeptide pair interaction, and a hydrophobic domain.

[0032] In some embodiments, the scaffold protein may sequentially include, from the N-terminus to the C-terminus: the hydrophobic domain, the other member of the first polypeptide pair interaction, the other member of the second polypeptide pair interaction, and a member of the third polypeptide pair interaction;

[0033] In some embodiments, the scaffold protein may sequentially include, from the N-terminus to the C-terminus: the other member of the first polypeptide pair interaction, the hydrophobic domain, the other member of the second polypeptide pair interaction, and a member of the third polypeptide pair interaction.

[0034] In some embodiments, the scaffold protein may sequentially include, from the N-terminus to the C-terminus: the other member of the first polypeptide pair interaction, the other member of the second polypeptide pair interaction, the hydrophobic domain, and a member of the third polypeptide pair interaction.

[0035] In some embodiments, the first enzyme capable of degrading plastics, the second enzyme capable of degrading plastics, and / or the third enzyme capable of degrading plastics are each independently selected from FAST-PETase, ICCG, and / or MHETase.

[0036] In some embodiments, the hydrophobic domain includes CBM.

[0037] The first polypeptide pair, the second polypeptide pair, and the third polypeptide pair are each independently selected from CelK-CipA, CelF-CipC, SH3 D -SH3 L and ScaA-ScaB.

[0038] In some embodiments, in the biodegradation module, the plastic-degrading enzyme or its functional variant or its fragment is directly linked or linked via a linker to a member of the polypeptide pair.

[0039] In some embodiments, in the scaffold protein, the other members of multiple polypeptide pairs and / or the other member of the polypeptide pair and the hydrophobic domain are directly linked or linked via a linker.

[0040] In some embodiments, the linker includes one or more of the amino acid sequences shown by (G n S) m , (G) n or (EA 3 K) n , where n and m are each independently selected from integers of 0 to 5.

[0041] In some embodiments, the plastic-degrading enzyme or its functional variant or its fragment is selected from the amino acid sequences shown in any one of SEQ ID NO: 1-4, or an amino acid sequence having at least 85% sequence identity therewith.

[0042] In some embodiments, the biodegradation module is selected from the amino acid sequences shown in any one of SEQ ID NO: 9-14, or an amino acid sequence having at least 85% sequence identity therewith.

[0043] In some embodiments, the scaffold protein is selected from the amino acid sequences shown in any one of SEQ ID NO: 24-26, or an amino acid sequence having at least 85% sequence identity therewith.

[0044] In some embodiments, the plastic-degrading complex includes the amino acid sequences shown in SEQ ID NO: 9, 10, and 24, or an amino acid sequence having at least 85% sequence identity therewith.

[0045] In some embodiments, the plastic-degrading complex includes the amino acid sequences shown in SEQ ID NO: 9, 13, and 25, or an amino acid sequence having at least 85% sequence identity therewith.

[0046] In some embodiments, the complex of the degradable plastic comprises the amino acid sequences shown in SEQ ID NO: 9, 13, 14, and 26, or amino acid sequences having at least 85% sequence identity therewith.

[0047] In some embodiments, in the complex, the at least one biodegradation module and the at least one scaffold protein have a molar ratio of (0.1 - 1):1. In some embodiments, in the complex, the at least one biodegradation module and the at least one scaffold protein have a molar ratio of 0.1:1, 0.11:1, 0.12:1, 0.13:1, 0.14:1, 0.15:1, 0.16:1, 0.17:1, 0.18:1, 0.19:1, 0.2:1, 0.21:1, 0.22:1, 0.23:1, 0.24:1, 0.25:1, 0.26:1, 0.27:1, 0.28:1, 0.29:1, 0.3:1, 0.31:1, 0.32:1, 0.33:1, 0.34:1, 0.35:1, 0.36:1, 0.37:1, 0.38:1, 0.39:1, 0.4:1, 0.45:1, 0.5:1, 0.55:1, 0.6:1, 0.65:1, 0.7:1, 0.75:1, 0.8:1, 0.85:1, 0.9:1, 0.95:1, or 1:1.

[0048] In some embodiments, in the complex, when the at least one biodegradation module comprises two or more biodegradation modules, the biodegradation modules can be mixed in equal proportions.

[0049] According to another aspect of the present disclosure, there is provided a nucleic acid molecule capable of encoding the complex of the degradable plastic.

[0050] According to another aspect of the present disclosure, there is provided an expression vector comprising the nucleic acid molecule or expressing the complex of one or more of the degradable plastics.

[0051] According to another aspect of the present disclosure, there is provided a host cell carrying the nucleic acid molecule or expressing the complex of one or more of the degradable plastics.

[0052] In some embodiments, the host cell is selected from prokaryotic cells or eukaryotic cells.

[0053] In some embodiments, the host cell is selected from bacteria, fungi, insect cells, plant cells, or animal cells.

[0054] In some embodiments, the host cell is selected from Escherichia coli, Bacillus subtilis, or Saccharomyces cerevisiae.

[0055] According to another aspect of the present disclosure, a method for degrading plastics is provided, and the method includes the following steps: taking a complex of the degradable plastics or a host cell or its culture and contacting it with the plastics in a reaction medium.

[0056] In some embodiments, the complex or the host cell or its culture is contacted with the plastics at 30°C to 70°C. In some embodiments, the complex or the host cell or its culture is contacted with the plastics at 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C or 70°C.

[0057] In some embodiments, the pH of the reaction medium is 5.0 to 9.0. In some embodiments, the pH of the reaction medium is 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5 or 9.0.

[0058] In some embodiments, the method further includes a step of recovering the degradation products, preferably recovering terephthalic acid monomers and / or ethylene glycol monomers.

[0059] According to another aspect of the present disclosure, a method for producing terephthalic acid monomers and / or ethylene glycol monomers is provided, including the following steps: taking a complex of the degradable plastics and contacting it with a plastic product-containing material in a reaction medium for a degradation reaction, and recovering and optionally purifying the terephthalic acid monomers and / or the ethylene glycol monomers.

[0060] According to another aspect of the present disclosure, uses of the complex of the degradable plastics, the nucleic acid molecule, the expression vector, the host cell or its culture in degrading plastics are provided.

[0061] In some embodiments, the plastics include any one or more of polyethylene terephthalate, polylactic acid, polyethylene, polypropylene, polystyrene, polyester, polyurethane.

[0062] In some embodiments, the polyester includes any one or more of polyethylene terephthalate (PET), polytrimethylene terephthalate (PTT), polybutylene terephthalate (PBT), polyethylene isophthalate (PEIT), polylactic acid (PLA), polyhydroxyalkanoate (PHA), polybutylene succinate (PBS), polybutylene succinate adipate (PBSA), polybutylene adipate terephthalate (PBAT), polyethylene furanoate (PEF), polycaprolactone (PCL), polyethylene naphthalate (PEN), polyethylene adipate (PEA), poly(1,4-cyclohexanedimethylene terephthalate) (PCT), glycol-modified polyethylene terephthalate (PETG), poly(butylene terephthalate-co-butylene adipate) (PBST).

[0063] In some embodiments, the polyester is polyethylene terephthalate (PET).

[0064] In some embodiments, the plastic includes one or more of plastic sheets, tubes, rods, profiles, shapes, films, blocks or fibers.

[0065] In some embodiments, the crystallinity range of the film-like plastic includes ≥20%.

[0066] In some embodiments, the crystallinity range of the film-like plastic includes <20%.

[0067] Advantages of the present invention:

[0068] Using the complex of degradable plastics provided by the present disclosure can improve the degradation efficiency of plastics.

[0069] The complex of degradable plastics provided by the present disclosure includes a biodegradation module containing an enzyme capable of degrading plastics and at least one scaffold protein. Through in vitro self-assembly, the plastic degradation reaction is carried out in the form of a multi-enzyme complex. The plastic degradation in the presence of the multi-enzyme complex has achieved a significant improvement in degradation efficiency, such as achieving the efficient degradation of plastic products, especially highly crystalline plastic substrates that are difficult to degrade. Compared with the prior art, the biodegradation efficiency has been significantly improved. Further expanding the substrate adaptability of the enzyme complex, the enzyme complex provided by the present disclosure has great industrial value. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Figure 1 Shows a schematic diagram of the scaffold protein-multi-enzyme complex design.

[0071] Figure 2 Shows the expression optimization process of MHETase.

[0072] Figure 3Shows the optimization results of MHETase expression.

[0073] Figure 4 Shows the affinity of the CBM-containing scaffold protein (Scaf.2) for the substrate (compared to Scaf.1).

[0074] Figure 5 Shows the binding ability of the corresponding enzyme MHETase-CelF to the scaffold protein Scaf.2.

[0075] Figure 6 Shows the binding ability of the corresponding enzyme FAST-PETase-CelK to the scaffold protein Scaf.2.

[0076] Figure 7 Shows the corresponding enzyme SH3 L -GS linker-OptiMHETase binding ability to the scaffold protein Scaf.3.

[0077] Figure 8 Shows the corresponding enzyme optiMHETase-SH3 L Binding ability to the scaffold protein Scaf.3.

[0078] Figure 9 Shows the binding ability of the corresponding enzyme FAST-PETase-CelK to the scaffold protein Scaf.3.

[0079] Figure 10 Shows the corresponding enzyme optiMHETase-SH3 L Binding ability to the scaffold protein Scaf.4.

[0080] Figure 11 Shows the binding ability of the corresponding enzyme FAST-PETase-CelK to the scaffold protein Scaf.4.

[0081] Figure 12 Shows the binding ability of the corresponding enzyme ICCG-ScaA to the scaffold protein Scaf.4.

[0082] Figure 13 Shows the degradation results of complex A containing FAST, MHETase, Scaf.1, complex B containing FAST, MHETase, Scaf.2 and the control on PET plastics.

[0083] Figure 14 Shows the degradation results of complex C containing FAST, MHETase, Scaf.3 and the control on PET plastics.

[0084] Figure 15Shows the degradation results of PET plastics by complex D containing FAST, MHETase, ICCG, Scaf.4 and the control.

[0085] Figure 16 Shows the comparison results of the ratio of enzyme to scaffold protein in the PET plastic degradation reaction.

[0086] Figure 17 Shows the comparison results of the ratio of enzyme to scaffold protein with and without Scaf.4 in the PET plastic degradation reaction.

[0087] Figure 18 Shows the comparison of PET plastic degradation yields under different temperature conditions.

[0088] Figure 19 Shows the comparison of PET plastic degradation yields under different pH conditions.

[0089] Figure 20 Shows the degradation results of scaffold protein - multi - enzyme (complex D) on PET powder. The degradation reaction was carried out at 40°C and pH 7.0, with enzyme:scaffold protein = 1:2 (molar ratio, 1 nmol scaffold protein).

[0090] Figure 21 Shows the degradation results of scaffold protein - multi - enzyme (complex D) on low - crystallinity PET film. The degradation reaction was carried out at 40°C and pH 7.0, with enzyme:scaffold protein = 1:2 (molar ratio, 1 nmol scaffold protein).

[0091] Figure 22 Shows the surface morphology analysis of the degradation of PET plastics by scaffold protein - multi - enzyme (complex D). The control group was the enzyme - free control group. Detailed implementation mode

[0092] To make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the following further details the present invention in conjunction with embodiments. The specific embodiments described herein are only used to explain the present invention and do not constitute any limitation to the present invention. In addition, in the following description, the descriptions of well - known structures and technologies are omitted to avoid unnecessarily confusing the concepts of the present disclosure. Such structures and technologies have also been described in many publications.

[0093] Through research and screening, the present disclosure has creatively designed an in - vitro plastic degradation system based on purified proteins. Figure 1Schematically shows the plastic degradation system of the present disclosure. In some embodiments, the in vitro plastic degradation system of the present disclosure may include a scaffold protein, the scaffold protein comprising a polypeptide interaction pair, such as CipA-CipC (Scaf.1); optionally further comprising a carbohydrate binding module (CBM), such as CipA-CBM-CipC (Scaf.2), CipA-CBM-SH3 D (Scaf.3), CipA-CBM-SH3 D -ScaB (Scaf.4). In some embodiments, the plastic degradation system of the present disclosure further includes a biodegradation module, the biodegradation module including FAST-PETase fused and expressed with CelK, ICCG fused and expressed with ScaA, MHETase fused and expressed with CelF or SH3 L fused and expressed. The amino acid sequences of the above-mentioned scaffold protein and biodegradation module are shown in Table 1.

[0094] Table 1. Amino acid sequences of each component in the PET degradation system

[0095]

[0096] The present disclosure has found through research that in the enzyme complex, the introduction of the heat-resistant cutinase ICCG improves the plastic degradation efficiency; MHETase reduces the proportion of the intermediate product MHET and increases the content of the end product terephthalic acid (TPA); the combination of the plastic degradation enzyme and MHETase improves the plastic degradation efficiency; moreover, compared with only adding enzymes, the participation of the scaffold protein can arrange multiple enzymes in close proximity, which can trigger the substrate channel effect, thereby realizing the efficient utilization and cascade catalysis of enzymes, and significantly improving the plastic degradation efficiency. Hydrophobic modules such as CBM proteins have a hydrophobic protein bilayer structure, can form an amphiphilic membrane at the hydrophobic / hydrophilic interface, and improve the contact area with the substrate as a biosurfactant, thereby improving the degradation efficiency.

[0097] The present disclosure forms a multi-enzyme complex in a self-assembled manner by mixing plastic-degrading enzymes and polypeptide interactions. In the complex, FAST or ICCG can degrade plastics (such as PET) as substrates, and MHETase degrades the intermediate products mono(2-hydroxyethyl) terephthalate (MHET) and bis(2-hydroxyethyl) terephthalate (BHET), reducing the inhibitory effect of the intermediate products on the activity of plastic-degrading enzymes. In addition, ICCG has a high degradation activity for highly crystalline plastic substrates, further expanding the substrate adaptability of the complex, thereby effectively biodegradating plastics and having broad application prospects.

[0098] Definitions

[0099] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly used in the field to which this invention belongs. For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural, and vice versa.

[0100] Unless the context clearly indicates otherwise, the expressions "a" and "an" used herein include plural referents.

[0101] The expression "about" as used herein is as understood by those of ordinary skill in the art and varies within a certain range depending on the context in which it is used. If those of ordinary skill in the art do not understand the use of this term according to the context in which it is used, "about" will mean up to plus or minus 10% of a particular value.

[0102] The term "plastic" or "plastic product" as used herein refers to any article or product that contains at least one polymer, such as plastic sheets, tubes, rods, profiles, shapes, films, blocks, fibers, etc. Preferably, plastic products are manufactured products, such as rigid or flexible packaging (bottles, trays, cups, etc.), agricultural films, bags and sacks, disposable items, etc., carpet waste, fabrics, textiles, etc. Plastic products may contain additional substances or additives, such as plasticizers, minerals, organic fillers or dyes. In the context of the present disclosure, plastic products may contain a mixture of semi-crystalline and / or amorphous polymers and / or additives.

[0103] The term "polymer" as used herein refers to a chemical compound or mixture of compounds whose structure is composed of multiple repeating units (i.e., "monomers") connected by covalent chemical bonds. In the context of the present disclosure, the term "polymer" refers to such compounds used in the composition of plastic products.

[0104] As used herein, the term "polyethylene terephthalate (PET)" or "PET and derivatives" includes high molecular weight polymers produced by condensing ethylene glycol with dimethyl terephthalate or terephthalic acid, regardless of how they are prepared. In addition, these terms are meant to include known polyethylene terephthalate polymers modified by including a small amount, e.g., less than about 20 wt% of a comonomer or other known modifiers, such as polyethylene terephthalate polymers. Such comonomers or modifiers include aromatic and aliphatic diols and polyols; aromatic and aliphatic carboxylic acids; or a single molecule containing both carboxyl and alcohol functionalities. Examples of diols include 1,4-butanediol, cyclohexanedimethanol, diethylene glycol, and / or 1,3-propanediol. Examples of dicarboxylic acids include isophthalic acid, adipic acid, 2,6-naphthalenedicarboxylic acid, and p-hydroxybenzoic acid. Small amounts of chain branching agents and / or chain terminators may also be used. The chain branching agents include, for example, polyfunctional acids and / or polyfunctional alcohols, such as trimethylolpropane and pentaerythritol. Chain terminators include monofunctional alcohols and / or monofunctional carboxylic acids, such as stearic acid and benzoic acid. Mixtures of the chain branching and chain terminating agents may also be used. In some embodiments, PET and derivatives include, but are not limited to, PET powder, PET bottles, PET films, PET fibers, etc.

[0105] As used herein, the term "degradation" includes depolymerization, which refers to the process of converting a polymer into its final monomers and / or degrading it into smaller molecules (such as monomers and / or oligomers and / or any degradation products). The term "degradation" more generally describes the cleavage of polymer chains by at least one enzyme in an enzyme, resulting in shorter polymer chains. For example, PET can be degraded by PETase, and intermediate products mono(2-hydroxyethyl) terephthalate (MHET) and bis(2-hydroxyethyl) terephthalate (BHET) are produced during the degradation process, and the end products are terephthalic acid (TPA) and ethylene glycol (EG). Among them, the intermediate products MHET and BHET will competitively inhibit the degradation activity of PET degrading enzymes.

[0106] As used herein, the term "PETase" is a PET plastic depolymerase found from Ideonella sakaiensis A series of PET plastic depolymerases including ThermoPETase, IsPETase, DuraPETase, FastPETase, DepoPETase, BhrPETase, TurboPETase, CaPETase M9 and HotPETase have been developed based on the engineering modification of PETase.

[0107] As used herein, the term "LCC" refers to a cutinase found in compost, called LCC (leaf-branch compost cutinase), which can cleave ester bonds and decompose PET into terephthalic acid and ethylene glycol. However, the rate of PET degradation by LCC is too low. Genetically modified LCCs can efficiently degrade polyethylene terephthalate (PET), such as LCC-A2, ICCG, ICCM, WCCG, and WCCM.

[0108] As used herein, the term "mutant" refers to a protein disclosed in the present invention, in which one or more amino acids are added, and / or substituted, and / or removed (deleted), and / or incorporated (inserted) into the N-terminus, and / or C-terminus, and / or the native amino acid sequence within the protein.

[0109] As used herein, the term "active fragment" has the same or similar meaning as that conventionally understood by those skilled in the art, and refers to a fragment whose amino acid sequence is part of the amino acid sequence of a full-length protein or polypeptide, but the fragment has the same or similar function or activity as the full-length protein or polypeptide. Specifically, in the present invention, an "active fragment" refers to any amino acid fragment having the activity of an enzyme obtained from any of the enzymes described in the present disclosure.

[0110] As used herein, the term "codon optimization" refers to modifying the codons of a nucleic acid molecule in a gene or coding region without changing the polypeptide encoded by the nucleic acid molecule to reflect the typical codon usage of the host organism. Such optimization includes replacing at least one, or more than one, or a large number of codons with one or more codons more frequently used in the genes of the host organism. In some embodiments, codon optimization is used to fine-tune the expression level of a construct of interest. Codon optimization includes, but is not limited to, the process of selecting codons for a coding sequence to accommodate the codon preference of an expression host organism. Many organisms show a bias or preference for using specific codons to encode the insertion of specific amino acids into the growing polypeptide chain. Codon preference or codon bias, which varies in codon usage among organisms, is permitted by the degeneracy of the genetic code and is well documented among many organisms. Codon bias is generally related to the translational efficiency of messenger RNA (mRNA), which in turn is thought to depend particularly on the properties of the codons being translated and the availability of specific transfer RNA (tRNA) molecules. The predominance of the tRNAs selected in a cell is usually a reflection of the codons most frequently used in peptide synthesis. Thus, genes can be customized for optimal gene expression in a given organism based on codon optimization.

[0111] As used herein, the term "scaffold protein" includes more than one adhesion protein that can concentrate more than one enzyme on the scaffold protein.

[0112] As used herein, the terms "cohesin" and "dockerin" are from two bacterial species Clostridium thermocellum and Clostridium clarifavum , as well as archaea Archaeoglobus fulgidus Thermostable Cohesin-Dockerin pairs have been found in different species, with an affinity between the non-covalent interaction modules Cohesin-Dockerin. In this article, the Dockerin-cohesin interaction can pair an enzyme with a specific scaffold to form a complex.

[0113] As used herein, the term "CipA" is the scaffold protein CipA of the cellulosome of Clostridium thermocellum ( Clostridium thermocellum ), a hydrophobic protein that is prone to aggregation to form protein crystal inclusion bodies (PCIs) under conditions of high protein expression, and its formation mechanism is similar to that of general inclusion bodies. Secondly, CipA can act as a scaffold to assemble co-expressed fusion proteins into subcellular structures without affecting the activity of the proteins.

[0114] As used herein, the term "CelK" is the dockerin (anchoring protein module) of a cellulase CelK that specifically binds to CipA. The enzyme has a size of 98000 Da, of which the anchoring protein module has a size of 11000 Da and is a major component of the cellulosome of Clostridium thermocellum. The enzyme's domain consists of a cellulose-binding domain (CBD) of family IV, a glycosyl hydrolase domain of family 9, and a dockerin domain.

[0115] As used herein, the term "CipC" is a scaffold protein whose organizational arrangement is similar to that of the scaffold protein of Clostridium cellulolyticum ( Clostridirm cellulovorans ). It has a size of 160000 and is composed of a series of oval modules connected like a pearl necklace. It has one CBM3a module at its N-terminus, followed by one X2 module of unknown function and seven hydrophobic cohesin modules. Its C-terminus contains a second X2 module of unknown function, followed by the eighth cohesin module, but does not contain any docking protein modules. This module may be involved in the process of anchoring to the cell surface, similar to the scaffold protein CipA of the cellulosome of Clostridium thermocellum ( Clostridium thermocellum ).

[0116] As used herein, the term "CelF" is the dockerin (anchoring protein module) of cellulase CelF, which can specifically bind to CipC. The enzyme has a size of 80,000 Da, and the anchoring protein module has a size of 13,000 Da. It exists in the genomic DNA of Clostridium cellulolyticum ( Clostridium cellulolyticum ), and forms a cellulosome together with the scaffold protein. The genes encoding the enzyme and the anchoring protein module are located between CipC (the gene encoding the cellulosome scaffold protein) and CelC (the gene encoding endoglucanase C), and the corresponding enzyme belongs to the cellulase L family (glycoside hydrolase family).

[0117] As used herein, the term "SH3 D " refers to the SH3 domain (Src homology domain 3 from the adaptor protein CRK), which is a protein-protein interaction domain from metazoan cells (mouse SH3) and can specifically bind to SH3 L .

[0118] As used herein, the term "SH3 L " refers to the SH3 ligand (Src homology domain 3 from the adaptor protein CRK), which is a protein-protein interaction ligand from metazoan cells (mouse SH3) and can specifically bind to SH3 D .

[0119] As used herein, the terms "SacA" and "ScaB": R. flavefaciens FD-1 encodes more than 10 putative scaffold proteins and more than 200 proteins containing dockerins. The genes encoding 4 major scaffold proteins (termed ScaA, B, C, and E) and a remarkable cellulose-binding protein (CttA) are arranged contiguously on the genome in the form of a scaffold protein gene cluster. The interconnectivity of these scaffold proteins determines the overall cellulosome structure and the arrangement of enzyme subunits. ScaA has 2 adhesin and dockerin modules and can bind to the adhesin of the larger, independent scaffold protein ScaB.

[0120] The term "substrate channeling effect" as used herein refers to the channeling effect experienced by substrates during the diffusion and reaction processes on the surface of a catalyst. In a catalytic reaction, substrate molecules first need to diffuse through the surface of the catalyst to the active sites, then react at the sites, and finally the products diffuse away through the surface of the catalyst. The substrate channeling effect is mainly affected by pore size and shape, adsorption mode, adsorption capacity, distribution of active sites, etc. For example: the size and distribution of pores directly affect the diffusion rate of substrate molecules. Larger pores can provide larger channels, accelerating the diffusion rate of substrate molecules and thus increasing the reaction rate; the stronger the adsorption capacity of the catalyst, the longer the residence time of substrate molecules on the catalyst surface, and the reaction rate will also increase; if the active sites are evenly distributed, substrate molecules can be more evenly adsorbed on the catalyst surface and are more likely to react with the active sites.

[0121] The term "cellulose-binding module (CBM)" as used herein, also known as carbohydrate binding module, is a non-catalytic domain of glycoside hydrolases and other proteins that recognize and bind polysaccharides. CBMs are often found in fungal and bacterial proteins containing glycoside hydrolases that degrade insoluble polysaccharides. However, CBMs have also been identified in proteins that do not contain glycoside hydrolase domains but are involved in the degradation of insoluble polysaccharides such as cellulose. There are at least 59 CBM families currently. Among these CBMs, different members have been shown to recognize crystalline cellulose, amorphous cellulose, chitin, β-glucan, xylan, mannan, galactan, and starch. CBMs of family 1 have a high binding affinity for crystalline cellulose, while CBMs of other families have a high binding affinity for amorphous cellulose or single-chain polysaccharides.

[0122] The term "linker" as used herein refers to a (peptide) linker of natural and / or synthetic origin, composed of linear amino acids. The domains in the bispecific fusion polypeptides of the present invention can be linked by linkers, where each linker is fused and / or otherwise linked (e.g., via peptide bonds) to at least two polypeptides or domains. Linkers are classified, for example, as flexible linkers, rigid linkers, etc. A "rigid linker" is composed of amino acid residues that easily form stable secondary structures. In many cases, because it can form relatively stable secondary structures, it can more effectively separate functional domains and maintain their independent functions compared to flexible linkers. When the spatial separation of functional domains is crucial for the stability and biological activity of the fusion protein, a rigid linker can be selected. Many natural rigid linkers form α-helical structures. A commonly used rigid α-helical linker is (E 3 K) n(n ≤ 6). Due to the presence of hydrogen bonds and a tight backbone within it, the α-helical structure is rigid and stable. The "flexible linker" is composed of small non-polar amino acids (such as glycine) or polar amino acids (such as serine or threonine). Small amino acids can provide more flexibility, enabling the linked functional domains not to interfere with each other and thus better play their roles. Polar amino acids such as serine and threonine can form hydrogen bonds with water molecules, so it can ensure the stability of the linker in aqueous solution while reducing the adverse reactions between the linker and the protein region.

[0123] The linker should have a length suitable for linking two or more modules. The linker can ensure that the different modules it links fold correctly and are properly presented, thus playing their biological activity functions. In different embodiments, the linker has a flexible conformation. Suitable flexible linkers include, for example, those having glycine, glutamine, and / or serine residues. In some embodiments, the linker can be selected from (G n S) m , (G) n , (EA 3 K) n or (XP) n , where n and m are each independently selected from integers from 0 to 5. For example, n is selected from 0, 1, 2, 3, 4, or 5, and m is selected from 1, 2, 3, 4, or 5.

[0124] As used herein, the term "sequence identity" refers to the "percent sequence identity" or "percent sequence identity" between two polynucleotides, that is, the number of identical matching positions shared by the sequences within a comparison window, taking into account the additions or deletions (i.e., gaps) that must be introduced for the optimal alignment of the two sequences. A matching position is any position where the same nucleotide exists in both the target sequence and the reference sequence. Since gaps are not nucleotides, the gaps present in the target sequence are not counted. Similarly, since the nucleotides of the target sequence are counted and the nucleotides from the reference sequence are not counted, the gaps present in the reference sequence are not counted. At least 60% sequence identity includes continuous fragments having at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity over the full length of the sequence.

[0125] The percent sequence identity can be calculated by the following process: Determine the number of positions in which the same amino acid residue or nucleic acid base occurs in both of two sequences to obtain the number of matching positions, divide the number of matching positions by the total number of positions in the comparison window, and multiply the result by 100 to obtain the percent sequence identity. The comparison of sequences and the determination of the percent sequence identity between two sequences can be accomplished using software readily available for online use and download. Suitable software programs are available from a variety of sources for aligning protein and nucleotide sequences. One suitable program for determining the percent sequence identity is bl2seq, which is part of the BLAST program suite available from the BLAST website (blast.ncbi.nlm.nih.gov) of the National Center for Biotechnology Information of the U.S. government. Bl2seq uses the BLASTN or BLASTP algorithm to compare two sequences. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. Other suitable programs are, for example, Needle, Stretcher, Water, or Matcher, which are part of the EMBOSS suite of bioinformatics programs and are also available from the European Bioinformatics Institute (EBI) at www.ebi.ac.uk / Tools / psa.

[0126] Examples and figures are provided below to assist in understanding the present invention. It should be understood, however, that these examples and figures are for illustrative purposes only and do not constitute any limitation. The actual scope of protection of the present invention is set forth in the claims. It should be understood that any modifications and changes can be made without departing from the spirit of the present invention.

[0127] Examples

[0128] 1. Experimental Instruments

[0129] Applied Biosystems VeritiPro PCR instrument (Thermo Fisher), PowerPac™ basic electrophoresis instrument (Bio-Rad), horizontal electrophoresis tank (Beijing Liuyi), ChemiDoc™ MP gel imaging system (Bio-Rad), low-temperature centrifuge (Thermo), blue light gel cutter (MIULAB), electric constant temperature incubator (Shanghai Jinghong), horizontal shaker (Qilin Bell), manual pipette (reserch plus, Eppendorf), pH meter (Mettler), NanoDrop micro-spectrophotometer (NanoDrop OneC, Thermo), ÄKTA pure chromatography system (Cytiva), isothermal titration calorimeter (MicroCalPEAQ-ITC Automated, Malvern), precision balance (Mettler), ultra-low temperature refrigerator (Haier), low-temperature -20℃ refrigerator (Haier), clean bench (Sujing Antai), water bath (Shanghai Jinghong), full-band microplate reader (Synergy H1, BioTek), Milli-Q® Direct Water Purification System (Merck).

[0130] 2. Experimental Reagents

[0131] Competent cells: E. coli DH5α (Kangti Life Science), E. coli BL21 (DE3) (Kangti Life Science), E. coli C41 (DE3) (Zhuangmeng Biotechnology).

[0132] For protein purification: HisTrap HP purification columns (Cytiva), 10 kDa ultrafiltration tubes (Millipore).

[0133] Reagents used for PCR and electrophoresis: PrimeSTAR® Max DNA Polymerase (TaKaRa), agarose (Yisheng), TAE (Shengang), 1 kb Plus DNA Ladder (NEB), Purple Loading Dye (NEB), Hyper Gel Red (APE×BIO).

[0134] Reagents used for enzyme digestion: NdeI (NEB), XhoI (NEB), SacI-HF (NEB), rCutSmart Buffer (NEB).

[0135] Reagents used for enzyme ligation: T4 DNA Ligase, T4 DNA Ligase Reaction Buffer (NEB).

[0136] Kits: Quick Plasmid Extraction Kit (Tiangen), ClonExpress Ultra One Step Cloning Kit (Vazyme), Gel Extraction Kit (omega BIO-TEK), BCA Protein Quantification Kit (Beyotime).

[0137] Reagents for SDS-PAGE: FuturePAGE 4-20% 15-well precast gel (ACE), Color Prestained Protein Marker (Genestar).

[0138] Buffers, organic reagents and salts: PBS buffer (Sangon Biotech), glycerol (Sangon Biotech), 10% SDS (Sangon Biotech), absolute ethanol (Sangon Biotech), sodium dihydrogen phosphate monohydrate (Bidepharm), disodium hydrogen phosphate anhydrous (BBI), IPTG (Beyotime), imidazole (Beyotime), sodium chloride (Bidepharm), calcium chloride anhydrous (Greagent).

[0139] Media and antibiotics: LB broth medium (Sangon Biotech), agar powder (Sangon Biotech), sodium ampicillin (Sangon Biotech), kanamycin sulfate (Sangon Biotech).

[0140] Example 1. Plasmid construction

[0141] The recombinant plasmids containing the target protein sequences were amplified and stored using E. coli DH5α. The pET28a vector was used for the expression of FAST-PETase (FAST), ICCG and scaffold proteins CipA-CipC (Scaf.1), CipA-CBM-CipC (Scaf.2), and the pColdII vector was used for the expression of MHETase enzyme. The synthesized genes FAST-PETase-CelK, ICCG-ScaA, scaffold protein Scaf.1, scaffold protein Scaf.2 and the pET28a vector were respectively digested with NdeI and XhoI at 37°C to obtain linear DNAs. After gel extraction of the digestion products, they were ligated by T4 DNA ligase at room temperature to obtain the recombinant expression vectors pET28a-FAST-PETase-CelK, pET28a-CipA-CBM-CipC, pET28a-CipA-CipC, pET28a-ICCG-ScaA, which were used for protein expression and purification after sequencing verification.

[0142] To construct SH3 L-GS linker-OptiMHETase (codon-optimized MHETase) expression vector. Use primers M1-F / M-R to perform polymerase chain reaction (PCR) to amplify optiMHETase, and use the gel-extracted fragment from the previous round of PCR as a template, and sequentially use primer pairs M2-F / M-R and M3-F / M-R for amplification to obtain fragment SH3 L -GS linker-OptiMHETase is used for subsequent restriction enzyme digestion to construct an expression vector. Use primers his-F / his-R to amplify MHETase to add a his tag to the C-terminus of MHETase. Use NdeI and XbaI to perform double digestion on MHETase-CelF, SH3 L -GS linker-OptiMHETase and the pColdII vector respectively at 37 °C. After the digestion products are recovered by gel extraction, they are ligated by T4 DNA ligase at room temperature to obtain recombinant expression vectors pColdII-MHETase-CelF and pColdII-SH3 L -GS linker-OptiMHETase. The expression vector is transformed into E. coli DH5α competent cells. The transformed cells are spread on a resistant LB plate (the pET28a series vector is a kanamycin-resistant LB plate, and the pColdII series vector is an ampicillin-resistant LB plate). After sequencing verification, it is used for protein expression and purification.

[0143] According to the different protein interaction modules fused to MHETase, the scaffold protein is adjusted to a sequence that can bind to the corresponding MEHTase. Use E. coli DH5α for plasmid amplification and preservation. The pET28a vector is used for the expression of other scaffold proteins CipA-CBM-SH3 D (Scaf.3), CipA-CBM-SH3 D -ScaB (Scaf.4). Use NdeI and XhoI to perform double digestion on the scaffold proteins Scaf.3, Scaf.4 and the pET28a vector respectively at 37 °C. After the digestion products are recovered by gel extraction, they are ligated by T4 DNA ligase at room temperature to obtain recombinant expression vectors pET28a-CipA-CBM-SH3 D 、pET28a-CipA-CBM-SH3 D -ScaB and transform them into E. coli DH5α competent cells. After sequencing verification, they are used for protein expression and purification.

[0144] The nucleotide sequences involved are shown in Table 2.

[0145] Table 2. DNA sequences used in plasmid construction

[0146]

[0147] Example 2 Protein Expression and Purification

[0148] According to Figure 2 the steps exemplified, protein expression and purification were carried out.

[0149] The recombinant expression vectors containing the target protein sequences of the pET28a series and the pColdII series were respectively transformed into competent cells of E. coli BL21(DE3) and E. coli C41(DE3) for protein expression. Single colony transformants were picked and pre-cultured overnight at 37°C and 220 rpm in LB resistant medium (containing 100 μg / mL ampicillin or 50 μg / mL kanamycin). The next day, 1 mL of the pre-cultured bacterial solution was taken into 1 L of sterile LB resistant medium and cultured at 37°C and 220 rpm until OD600 = 0.6 - 0.7. Isopropyl β-D-thiogalactoside (IPTG) was added to a final concentration of 0.5 mM, and the culture was continued at 16°C and 220 rpm for 20 h for the expression of the target protein. The expression product (bacterial cells) was collected by centrifugation at 4000 rpm / min for 30 min at 4°C and used for subsequent protein purification.

[0150] The collected expression product was resuspended in 20 mL of lysis buffer and then sonicated in an ice-water bath. The cell lysate mixture was centrifuged at 12000 rpm / min for 30 min at 4°C. The supernatant obtained by centrifugation was filtered through a 0.45 μm filter membrane, and then the sample was loaded onto an AKTA automatic purification system (equipped with a HisTrap HP protein purification column) for protein purification.

[0151] The purification results showed that the target proteins (enzyme and scaffold protein) could be solubly expressed in the supernatant of the solution treated with an ultrasonic crusher, and could be purified using a pre-packed HisTrap HP purification column according to the his tags carried at the N-terminus and C-terminus of the target proteins. Among them, after purification, the purity of the target proteins was relatively high and could be used for subsequent experiments. Table 3 andFigure 3 The purification results of proteins containing MHETase or optiMHETase are shown. After codon optimization and replacement of the dockerin module, SH3L-GS linker-OptiMHETase has a lower protein molecular weight, and the expression-purification efficiency is significantly improved, reaching about 3.58 times that of the initial MHETase-CelF protein.

[0152] Table 3. Purification efficiency of MHETase

[0153]

[0154] Example 3. Detection of the affinity between scaffold protein and PET substrate

[0155] In this example, the protein expressed in Example 2 was used to detect its affinity for the PET plastic substrate. The PET plastic substrate (Xingxiang New Materials, 200 mesh, final concentration 1 w / v% (0.1 g / 10 mL)) was washed successively with 10% SDS solution and phosphate buffer (SP buffer, pH 7.0) (repeated three times). 100 mg of PET plastic that had been dried and cleaned at room temperature was incubated overnight at 4 °C in phosphate buffer (SP buffer) with scaffold proteins Scaf.1 and Scaf.2 for subsequent affinity detection. Among them, the total volume of the co-incubation system was 200 μL, and the concentrations of the scaffold proteins were 2.5 μM, 5 μM, and 7.5 μM. The co-incubated samples were centrifuged at 12,000 rpm / min for 1 min at 4 °C and washed twice with sodium hydrogen phosphate buffer, and all the supernatants obtained during the washing process were collected and the protein concentration was detected using a BCA protein quantification kit. The experiment of detecting the affinity between the scaffold protein and the PET plastic substrate was repeated three times. The calculation of the protein-substrate affinity is shown in the following formula:

[0156]

[0157] As shown in the binding experimental results Figure 4 shown, as the CBM content in the system increases, the affinity of the scaffold protein for the PET substrate also gradually increases. Compared with scaffold protein Scaf.1, the affinity of scaffold protein Scaf.2 for PET plastic is 1.13 - 1.55 times higher than that of Scaf.1. The results show the positive effect of CBM on enhancing the binding ability between the protein and the PET substrate.

[0158] Example 4. Detection of self-assembly of scaffold protein-PET degrading enzyme

[0159] Using a 10 kDa ultrafiltration tube, centrifuged at 4000 rpm / min for 30 min at 4 °C multiple times to replace the purified product into a solution containing 0.5 mM CaCl 2In PBS buffer (pH 7.2 - 7.5), adjust the protein concentration so that the concentration of the scaffold protein is 120 μM, and the concentrations of PET-degrading enzymes FAST and MHETase are 20 μM. Load the scaffold protein into a titration syringe, and load the enzymes into the sample cell to be titrated. Use an isothermal titration calorimeter MicroCal PEAQ-ITC Automated to detect protein binding (titration procedure: 25°C, 18 injections, 2 μL for each injection, injection interval 150 s). The titration heat reaction detection results are analyzed using a single-site binding model in the MicroCal PEAQ-ITC Analysis software.

[0160] To construct a scaffold protein-multi-enzyme complex, the scaffold protein and the enzymes are mixed at a molar ratio of 5:1. Under the conditions of room temperature and pH 7.0, the cohesin of the scaffold protein binds to the dockerin of the corresponding enzyme, realizing the self-assembly of the enzyme-scaffold protein multi-enzyme complex in vitro. The binding detection results of the scaffold protein and the enzymes are as Figures 5 - 12 shown. The number of binding sites N and the KD value in the binding verification experiment results indicate that the scaffold protein can form complexes with the corresponding enzymes through the protein-protein interaction pairs of CipA-CelK, CipC-CelF, SH3 D -SH3 L , ScaA-ScaB, but MHETase-SH3 L with SH3 fused to the C-terminus L cannot bind to the corresponding scaffold protein Scaf.3 (as Figure 8 shown). For other enzyme-scaffold protein pairs except this pair, the number of binding sites is 1, and they have strong binding.

[0161] Example 5. Degradation of PET plastics by complexes formed by different scaffold proteins and PET-degrading enzymes

[0162] To verify the degradation of PET plastics by the multi-enzyme complexes formed by different scaffold proteins and enzymes and compare the efficiency of the complexes in degrading PET plastics, HPLC is used to detect the intermediate products MHET and BHET and the end product TPA of the PET degradation reaction.

[0163] Prepare a degradation reaction system using a sodium hydrogen phosphate buffer at pH 7.0, with the amount of PET substrate being 100 mg. In the degradation reaction system containing the scaffold protein, the molar ratio of enzyme to scaffold protein = 0.2:1, and the amount of scaffold protein used is 0.5 nmol; in the degradation reaction system without the scaffold protein, the amount of enzyme used is 0.1 nmol.

[0164] The detection results are as Figures 13 - 15As shown, different types of enzymes have different selectivities for substrates. FAST and ICCG can directly utilize PET as a substrate, degrading it to produce TPA and a small amount of MHET, while MHETase has no degradation activity towards PET (the degradation reaction products MHET, BHET, and TPA were not detected). The degradation efficiency of ICCG for PET powder (200 mesh, 40.82% crystallinity, Xingxiang New Materials), low-crystallinity PET film (Film-L, 17.99% crystallinity, Goodfellow, ES30-FM-000145), and high-crystallinity PET film (Film-H, 43.31% crystallinity, Goodfellow, ES30-FM-000250) is higher than that of FAST; compared with each treatment group without MHETase, in the presence of MHETase, the proportion of MHET in the final product is significantly reduced, and the content of TPA is significantly increased. Compared with the treatment groups using only FAST, ICCG, FAST and ICCG, the combined use of PET-degrading enzyme and MHETase further improves the PET degradation efficiency; the introduction of ICCG improves the PET degradation efficiency. Compared with complex C, the degradation reaction efficiency of complex D containing ICCG for PET powder, low-crystallinity film, and high-crystallinity film is increased by 1.8 times, 2.1 times, and 1.5 times respectively. In addition, compared with adding only enzymes, the participation of scaffold proteins significantly improves the PET plastic degradation efficiency. After 96 h of degradation reaction, the total amounts of degradation products of PET powder in complex A containing Scaf.1, complex B containing Scaf.2, complex C containing Scaf.3, and complex D containing Scaf.4 are 22.77 mM, 24.73 mM, 24.19 mM, and 44.66 mM respectively; the total amounts of degradation products of low-crystallinity PET film are 15.85 mM, 17.29 mM, 23.49 mM, and 49.03 mM respectively; the total amounts of degradation products of high-crystallinity PET film are 0.71 mM, 0.73 mM, 0.89 mM, and 1.32 mM respectively. The composition of the complexes is shown in Table 4.

[0165] Table 4. Composition of Complexes

[0166]

[0167] Example 6. Comparative Experiment on the Ratio of Enzyme to Scaffold Protein in the PET Plastic Degradation Reaction

[0168] In this example, complex D was used as the experimental object to further optimize the ratio of the biodegradation module to the scaffold protein in the PET plastic degradation reaction.

[0169] The degradation reaction system contains 100 mg of PET powder (200 mesh, 40.82% crystallinity, Xingxiang New Materials), 1.0 mM of scaffold protein, biodegradation modules FAST-CelK, MHETase-CelF, ICCG-ScaA (a 1:1:1 mixture of the three biodegradation modules) at a corresponding molar ratio (biodegradation module:scaffold protein = 0.2:1 to 1:1), and reaction buffer (50 mM sodium phosphate, 0.5 mM CaCl 2 , 10% DMSO (v / v), pH 7.0), with a total reaction volume of 500 μL. After pre-mixing the biodegradation modules and the scaffold protein at room temperature at different molar ratios (biodegradation module:scaffold protein = 0.2:1, 0.25:1, 0.33:1, 0.5:1, 1:1), let it stand for 1 h to achieve self-assembly of the scaffold protein-multienzyme complex. The complex and PET powder (before the reaction, the PET powder was pretreated by washing it three times with 10% SDS, ddH 2 O, and sodium hydrogen phosphate buffer at pH 7.0 and then air-dried naturally) were reacted at pH 7.0 and 30 °C for 96 h to explore the optimal enzyme-scaffold protein molar ratio for achieving the highest PET degradation efficiency under this condition.

[0170] The above reactions were carried out in three independent replicates to reduce experimental error, and the composition and content of the reaction supernatant were detected by high performance liquid chromatography (HPLC).

[0171] The results are shown in Figure 16 and Figure 17 indicating that the degradation effect is the best when the enzyme:scaffold protein = 0.5:1.

[0172] Example 7. Optimization experiment of PET plastic degradation reaction conditions

[0173] In this example, this complex D was used as the experimental object to further optimize the PET plastic degradation reaction conditions.

[0174] A degradation reaction system was prepared with an enzyme:scaffold protein molar ratio of 0.5:1. It was reacted at different temperatures (30 °C, 40 °C, 50 °C, 60 °C, 70 °C) at pH 7.0 for 96 h, and the others were the same as in Example 6, to explore the optimal reaction temperature under this condition, or the degradation reaction was carried out in reaction buffers with different pH values (pH 5.0, 6.0, 7.0, 8.0, 9.0) to explore the optimal reaction pH.

[0175] The above reactions were carried out in three independent replicates to reduce experimental error, and the composition and content of the reaction supernatant were detected by high performance liquid chromatography (HPLC).

[0176] After optimizing the above reaction conditions, the results are as follows Figure 18 and Figure 19 shown, and the optimal reaction conditions are obtained: 40 °C, pH 7.0.

[0177] Example 8. Degradation effect test of different PET plastics

[0178] Under the optimal reaction conditions (40 °C, pH 7.0, enzyme:scaffold protein = 0.5:1), the complex D was used to carry out degradation reactions with PET powder (crystallinity of 40.82%), low-crystallinity PET film (Film-L, crystallinity of 17.99%), and high-crystallinity PET film (Film-H, crystallinity of 43.31%) as substrates. The reaction was carried out for 17 days, and the reaction supernatant was taken every 24 h for component and content detection to explore the degradation effect of the scaffold protein-multienzyme complex on different PET substrates.

[0179] The above reactions were carried out in three independent replicate experiments to reduce experimental errors, and the components and contents of the reaction supernatant were detected by high-performance liquid chromatography (HPLC).

[0180] The degradation results of the scaffold protein-multienzyme (complex D) on different PET substrates are as follows Figures 20 - 21 shown. After reacting for 17 days under the optimal reaction conditions (40 °C, pH 7.0), the complex D has degradation activity on both PET powder and two PET films with different crystallinities: the total amount of degradation products of PET powder ( Figure 20 ) is 174.24 mM; the total amount of degradation products of the low-crystallinity PET film ( Figure 21 ) is 201.89 mM; the total amount of degradation products of the high-crystallinity PET film is 9.51 mM. Among them, after reacting for 96 h, the total amount of degradation products produced by the complex D for degrading PET powder is as high as 143.76 mM, the content of the complex D is 0.25 nmol, and the reaction efficiency = total product amount (TPA + MHET + BHET) / 0.25 = 696.96 mM / nmol complex, demonstrating the great potential of this complex for degrading PET plastics.

[0181] The results of the surface morphology analysis of the scaffold protein-multienzyme (complex D) for degrading PET plastics are as follows Figure 22As shown, after 96 hours of reaction, the surface of the low-crystallinity PET film showed different morphological differences. Compared with the control group without any enzyme and the treatment group with only MHETase added, the addition of FAST, ICCG, or the combination of FAST, MHETase, and ICCG all had an impact on the surface of the PET film, and a large number of depressions caused by the degradation reaction appeared on the surface of the PET film. When FAST, MHETase, and ICCG were present in the degradation system simultaneously, obvious erosion occurred on the surface of the PET film; the complex D containing the scaffold protein degraded the PET film more deeply, expanding the holes generated by the surface erosion and increasing the surface roughness of the cross-section.

[0182] The technical solution of the present invention is not limited to the limitations of the above specific embodiments. Any technical deformation made according to the technical solution of the present invention falls within the protection scope of the present invention.

Claims

1. A composite of degradable plastics, characterized in that: The complex comprises: at least one biodegradation module, each biodegradation module independently comprising an enzyme capable of degrading plastic and a member of a polypeptide interaction pair; and at least one scaffold protein, each scaffold protein independently comprising one or more other members of said polypeptide interaction pair, and optionally further comprising one or more hydrophobic domains; wherein one member of the polypeptide interaction pair specifically binds to another member of the polypeptide interaction pair, and the biodegradation module is selected from the amino acid sequence shown in any one of SEQ ID NOs: 9, 10, 12 and 14; The scaffold protein is selected from the amino acid sequence shown in any one of SEQ ID NOs: 24 to 26; The degradable plastic complex comprises the amino acid sequences shown in SEQ ID NOs: 9, 10 and 24; and / or, The degradable plastic complex comprises the amino acid sequences shown in SEQ ID NOs: 9, 12 and 25; and / or, The degradable plastic complex comprises amino acid sequences as shown in SEQ ID NOs: 9, 12, 14 and 26.

2. The composite body according to claim 1, characterized in that The plastic is polyethylene terephthalate.

3. The composite according to claim 1 or 2, characterized in that In the complex, the at least one biodegradable module and the at least one scaffold protein have a molar ratio of (0.1~1):

1.

4. A nucleic acid molecule, which is capable of encoding the complex according to any one of claims 1 to 3.

5. An expression vector comprising the nucleic acid molecule of claim 4, or expressing the complex of any one of claims 1 to 3.

6. A host cell carrying the nucleic acid molecule of claim 4, or expressing the complex of any one of claims 1 to 3.

7. Use of the complex according to any one of claims 1 to 3, the host cell according to claim 6 or a culture thereof in degrading plastics; the plastic is polyethylene terephthalate.

8. A method for degrading plastics, characterized in that: The method comprises the step of contacting the complex according to any one of claims 1 to 3 or the host cell or culture thereof according to claim 6 with plastic in a reaction medium; the plastic is polyethylene terephthalate.

9. The method according to claim 8, characterized in that The complex or the host cell or culture thereof is contacted with the plastic at 30°C to 70°C; and / or The pH of the reaction medium is 5.0 to 9.0; and / or The method further comprises the step of recovering the degradation products.

Images