Leaf branch compost cutinase ethofencarb and preparation method thereof

Abstract

The invention discloses a leaf and branch compost cutinase Soxhlet and a preparation method thereof, and belongs to the technical field of enzyme molecule construction. The leaf branch compost cutinase Soxhlet comprises a first unit ring and a second unit ring which are interlocked, the first unit ring comprises a first fragment, a first connecting peptide fragment, a third fragment and a second connecting peptide fragment which are sequentially connected to form a ring, and the second unit ring comprises a second fragment and a third connecting peptide fragment which are sequentially connected to form a ring; the fragment I, the fragment II and the fragment III respectively correspond to 36th to 64th amino acids, 72th to 151st amino acids and 159th to 293th amino acids or homologous sequences of the 36th to 64th amino acids, the 72th to 151st amino acids and the 159th to 293th amino acids of the linear leaf and branch compost cutinase amino acid sequence. The Soxhdrocarbon design is constructed by splitting a linear leaf branch compost cutinase structure and introducing an artificially designed connecting peptide fragment, the sequence of the connecting peptide fragment is optimized by an artificial intelligence assisted protein design method, and based on an orthogonal cyclization element mediated coupling reaction, the leaf branch compost cutinase Soxhdrocarbon with good biological activity is obtained.

Description

Technical Field

[0001] This invention belongs to the field of enzyme molecule construction technology, and relates to protein topology modification technology, and in particular to a leaf and branch compost cutinase hydrocarbon and its preparation method. Background Technology

[0002] As a crucial parameter for regulating macromolecular properties, topology significantly influences the properties and functions of proteins. In nature, some proteins with atypical topologies often exhibit unique functional advantages, such as better thermal stability, resistance to chemical denaturation, and resistance to enzymatic degradation. Therefore, developing efficient artificial topological protein synthesis techniques is essential. Currently, researchers have developed a one-step intracellular synthesis method for protein hydrocarbons mediated by isolated integrins and applied it to the synthesis of various protein hydrocarbons (Liu, Y. et al, Angew. Chem. Int. Ed. 2020, 59, 19153; Qu, Z. et al, Nat. Commun. 2023, 14, 3480; Fang, J. et al, Natl. Sci. Rev. 2023, 10, nwad304.). Although this method has been successfully applied to the synthesis of tocopheryl groups for dihydrofolate reductase and green fluorescent protein, the uncontrollable rate during assembly and reaction results in problems such as low product purity and decreased protein activity. Therefore, developing rational design strategies to improve the tocopherylation efficiency in protein tocopherylation modification while preserving functional activity as much as possible is crucial for enhancing the performance of synthesized topological proteins.

[0003] Leaf and branch compost cutinase (LCC), a natural ester hydrolase, has the ability to catalyze the degradation of polyethylene terephthalate (PET). Its engineered mutant, LCC... ICCG It exhibits very high thermal stability and PET hydrolytic activity, making it highly industrially viable (Tournier, V. et al. Nature 2020, 580, 216.). Furthermore, it possesses clear crystal structure information, making it well-suited for structure-based topology modification. Modification of this type of keratinase holds promise for yielding topoprotein variants with even greater stability and activity. Summary of the Invention

[0004] To address the problems existing in the prior art, this invention aims to determine a feasible method for splitting and linking leaf and branch compost cutinase (LCC) cord hydrocarbons by combining existing AI protein prediction and design methods, and to optimize the sequence of its reconnected loop region chain to improve the actual performance of the LCC cord hydrocarbons.

[0005] The fragments in the linear leaf and branch compost cutinase, linked sequentially from the N-terminus to the C-terminus, include fragment one, fragment two, and fragment three, respectively. See [link to relevant documentation]. Figure 1 The first segment includes a β-folded sheet 1; the second segment includes a β-folded sheet 2, a β-folded sheet 3, an α-helix A, a β-folded sheet 4, and an α-helix B connected in sequence; and the third segment includes a β-folded sheet 5, an α-helix C, a β-folded sheet 6, a β-folded sheet 7, an α-helix D, a β-folded sheet 8, an α-helix E, and a β-folded sheet 9 connected in sequence.

[0006] The first aspect of this invention provides a method for designing foliar compost cutinase hydrocarbons, which can be obtained by adjusting the connection order between fragments in linear foliar compost cutinase and introducing linking peptides between adjacent fragments. Specifically, the loop chains of the linear foliar compost cutinase located between β-sheet 1 and β-sheet 2, and between α-helix B and β-sheet 5, are split. Then, the C-terminus of β-sheet 1 and the N-terminus of β-sheet 5, as well as the original N and C-termini of the foliar compost cutinase, are connected to obtain unit loop one. Unit loop two is obtained by connecting the N-terminus of β-sheet 2 and the C-terminus of α-helix B. By ensuring that these linking peptides have appropriate lengths and spatial relationships, foliar compost cutinase hydrocarbons can be obtained.

[0007] Specifically, the leaf and branch compost cutinase hydrocarbon of this invention comprises mechanically interlocked unit ring one and unit ring two. Unit ring one comprises a ring segment one, a linking peptide one, a ring segment three, and a linking peptide two, sequentially linked together. Unit ring two comprises a ring segment two and a linking peptide three, sequentially linked together. Conserved core elements are identified through amino acid sequence analysis of segments one, two, and three. Based on the amino acid sequence of the linear leaf and branch compost cutinase, the core elements of fragment 1 are NPYQRGPNPT at positions 37-46 and GPFSV at positions 54-58 (β-sheet 1); the core elements of fragment 2 are GGG at positions 72-74 (β-sheet 2), IYYPT at positions 76-80 (β-sheet 2), TFGGIAMSPGYTA at positions 85-97 (β-sheet 3), SSLAWLG at positions 100-106 (α-helix A), RLASHGFVV at positions 108-116 (α-helix A), VINTNS at positions 118-123 (β-sheet 4), and SRASQLSAALNYL at positions 130-142 (α-helix B); the core element of fragment 3 is LAVAGH at positions 159-175. SMGGGGTLRIA (β-folded layer 5 and α-helix C), QNPSLKAAVPLTPW (β-folded layer 6) at positions 177-190, TDKTF (β-folded layer 7) at positions 192-196, TSVPVLIVGAE (β-folded layer 8) at positions 198-208, DTVAPVSQHA (α-helix D) at positions 210-219, PFYQNLPSTTPKVYVEL (α-helix D and β-folded layer 9) at positions 221-237, NASHIAPN at positions 239-246, NNAAIS (α-helix E) at positions 248-253, YTISWMK (α-helix E) at positions 255-261, WVDNDTRY at positions 263-270, and QFLC at positions 272-275. While ensuring that the amino acid sequences of these core components are determined, mutations such as substitution, deletion, or addition of the remaining amino acid residues are allowed, and the resulting structures still form and retain their functions.

[0008] In some embodiments of the present invention, fragment one, fragment two, and fragment three correspond to amino acids 36-64, 72-151, and 159-293 of the amino acid sequence of linear leaf branch compost cutinase, respectively, and the mutated nephrolith can still be formed. While ensuring that the core elements in fragment one, fragment two, and fragment three remain unchanged, amino acid residues in non-conserved regions can be replaced, deleted, or added to obtain a nephrolith protein with more than 80% homology and the same function as the original sequence, preferably more than 85% homology, and more preferably more than 90% homology. The sequence length of fragment one can vary in the range of 24-30 aa, the sequence length of fragment two can vary in the range of 72-81 aa, and the sequence length of fragment three can vary in the range of 118-136 aa.

[0009] In the leaf and branch compost cutinase hydrocarbon of the present invention, the first linker peptide of the first unit ring is optimized in conformation and sequence through an artificial intelligence design process, and its length is set to 12 to 18 amino acid residues. In some preferred leaf and branch compost cutinase hydrocarbons constructed in the present invention, the amino acid sequence of the first linker peptide is AAELAKGGSGSGGSTR. The second and third linker peptides are independent and are flexible linker peptides with 4 to 20 amino acid residues each. In some embodiments of the present invention, the amino acid sequence of the second linker peptide is GTESGGSGKGS or GTESGGSGKENLYFQGGS, and the amino acid sequence of the third linker peptide is GGTSCFNHHHHHHEL.

[0010] A second aspect of the present invention provides a method for preparing the aforementioned leaf and branch compost cutinase hydrocarbon, comprising the following steps:

[0011] (1) Design the protein precursor sequence of leaf and branch compost cutinase hydrocarbon, and synthesize the coding gene sequence of the protein precursor sequence; wherein the protein precursor sequence includes at least: the sequence of unit loop one and unit loop two, and two pairs of orthogonal cyclization motifs that mediate the cyclization of unit loop one and unit loop two in the cell.

[0012] (2) The coding gene sequence synthesized in step (1) is introduced into the expression vector to obtain the recombinant expression vector;

[0013] (3) The recombinant expression vector is transferred into host cells for expression to obtain the fusion protein;

[0014] (4) The fusion protein was purified to obtain the leaf and branch compost cutinase hydrocarbon.

[0015] In step (1) above, the two pairs of orthogonal cyclization units can be selected from any of the following combinations: (i) two orthogonal isolated integrins; (ii) two orthogonal polypeptide-protein reaction pairs; (iii) a combination of polypeptide-protein reaction pairs and isolated integrins.

[0016] In some specific embodiments of the present invention, the protein precursor sequence in step 1) includes, from the N-terminus to the C-terminus, the following sequence: Int C 1. Linking peptide II C-terminus, Fragment 1, Linking peptide I, Fragment 3, Linking peptide II N-terminus, Int N Int′ C , connecting peptide C-terminus, fragment 3, connecting peptide C-terminus and Int′ N ;Int N and Int C Int′ represents the N-terminal and C-terminal portions of the isolated inteptide Int, respectively. N and Int′ C The N-terminal and C-terminal portions of the isolated inteptide Int′ are represented respectively, and the two isolated inteptides Int and Int′ are orthogonal.

[0017] Preferably, the two orthogonal separable intepids are VidaL and Npu. Specifically, the intepid VidaL... C The amino acid sequence is shown in SEQ ID NO: 5 in the sequence listing, containing the peptide VidaL. N The amino acid sequence is shown in SEQ ID NO: 6; it contains the peptide Npu C The amino acid sequence is shown in SEQ ID NO: 7, containing the peptide Npu N The amino acid sequence is shown in SEQ ID NO: 8.

[0018] The amino acid sequence of the linear leaf and branch compost cutinase is shown in SEQ ID No:2. In some embodiments of the present invention, the amino acid sequence of the precursor protein for synthesizing leaf and branch compost cutinase is shown in SEQ ID NO:1, and the linear precursor protein sequence containing the recognition sequence ENLYFQG of tobacco etch virus protease is shown in SEQ ID NO:3.

[0019] A third aspect of this invention provides a gene sequence for expressing and synthesizing the foliage compost cutinase hydrocarbon, an expression vector containing the gene sequence, and a host cell containing the expression vector, all of which are within the scope of protection of this invention. The gene sequence encodes a protein precursor sequence of the foliage compost cutinase hydrocarbon.

[0020] The leaf and branch compost cutinase hydrocarbon provided by this invention can be applied to the biodegradation of polymer materials, especially PET, and is one of the important ways to effectively solve the problem of plastic pollution.

[0021] The innovation of this invention lies in constructing a cutinase hydrocarbon structure from leaf and branch compost, expanding the polypeptide backbone structure of linear leaf and branch compost cutinase, and designing the conformation and sequence of the linker peptides of leaf and branch compost cutinase. This allows for the successful synthesis of highly active leaf and branch compost cutinase hydrocarbons without relying on directed evolution strategies. The main innovations are as follows:

[0022] 1. This invention breaks through the cognitive paradigm of linear protein backbones and applies protein topology engineering to the design and modification of natural linear leaf and branch compost cutinase, creating a novel leaf and branch compost cutinase hydrocarbon.

[0023] 2. Currently, most protein design strategies are applied in de novo design cases, and their feasibility in topological protein design has not yet been tested. This invention develops a reasonable process strategy and compiles a set of feasible solutions applicable to topological protein design.

[0024] 3. Existing technologies for protein topology modification all rely on directed evolution strategies to restore enzyme activity, which is time-consuming and has high uncertainty. The present invention adopts the following approach: combining existing artificial intelligence-assisted protein design strategies to achieve one-step synthesis of leaf and branch compost cutinase hydrocarbon with high expression level, high main product ratio and high activity.

[0025] In summary, the beneficial effects of this invention are as follows: The leaf and branch compost cutinase hydrocarbon design provided by this invention organically combines protein topology engineering and artificial intelligence-assisted protein design technology to perform hydrocarbon topological modification on leaf and branch compost cutinase and design the sequence of the linking peptides of the hydrocarbon structure, resulting in leaf and branch compost cutinase hydrocarbon designs with high main product ratio and good biological activity. Therefore, the solution provided by this invention achieves efficient design and synthesis of leaf and branch compost cutinase hydrocarbons, overcomes the problem of low proportion caused by uncontrollable factors during the synthesis of protein hydrocarbons, provides a guiding scheme for the modification process of hydrocarbon proteins, and further provides highly active leaf and branch compost cutinase hydrocarbon variants. Attached Figure Description

[0026] Figure 1 This is a three-dimensional structural model and a schematic diagram of the secondary structure arrangement of linear LCCs and LCC-linked hydrocarbons.

[0027] Figure 2 The results show the sequence conservation analysis of different segments of LCC.

[0028] Figure 3This is a schematic diagram illustrating the synthesis of LCC hydroxyl groups.

[0029] Figure 4 The SEC purification spectra of linear LCC (l-LCC) and LCC filament (cat-LCC) in Example 2 are shown.

[0030] Figure 5 The images shown are SDS-PAGE characterization images of the LCC-linked peptide before and after optimization in Example 3, where cat-LCC-nopt represents the peptide before optimization and cat-LCC represents the peptide after optimization.

[0031] Figure 6 The images show the SDS-PAGE characterization (left) and LC-MS characterization (right) diagrams of LCC cat-LCC-L after TEV digestion in Example 3.

[0032] Figure 7 This is a schematic diagram of circular dichroism characterization of linear LCC and LCC filaments in Example 4.

[0033] Figure 8 The figures above show the reaction kinetics curves (top) and the calculated specific activity diagram (bottom) of the linear LCC and LCC-based catalytic conversion of pNP-C2 to p-nitrophenol in Example 5.

[0034] Figure 9 The graph above shows the kinetic curves of PET degradation catalyzed by linear LCC and LCC-based hydrocarbons in Example 6, and the concentration of the products in the system after 24 h of reaction (bottom). Detailed Implementation

[0035] To better understand the above technical solutions, the following will provide a detailed description of the technical solutions in conjunction with the accompanying drawings and specific implementation methods.

[0036] This invention provides a method for obtaining phyllodes compost cutinases with hydrocarbons. The design of phyllodes compost cutinases with hydrocarbons can be achieved by adjusting the connection sequence between secondary structure fragments in linear phyllodes compost cutinases. Figure 1The two-dimensional projected structure shown has three catalytic residues: S165, D210, and H242. Analysis of the crystal structure of the leaf-branch compost cutinase reveals that a cord structure can be designed by disassembling the loop chains located between β-sheet 1 and β-sheet 2, and between α-helix B and β-sheet 5, followed by connecting the C-terminus of β-sheet 1 to the N-terminus of β-sheet 5, the N-terminus of β-sheet 2 to the C-terminus of α-helix B, and the original N and C-termini of the leaf-branch compost cutinase. The designed reconnected loop chains are spatially located away from the protein's active site, and the reconnected loop chains do not affect the binding of the substrate to the active pocket.

[0037] The amino acid sequence of the linear leaf and branch compost cutinase is shown in SEQ ID No: 2. After deleting some amino acids from the amino acid sequence of the linear leaf and branch compost cutinase, it can be divided into the following three segments: the amino acid sequence of segment one is amino acids 36-64, the amino acid sequence of segment two is amino acids 72-151, and the amino acid sequence of segment three is amino acids 159-293.

[0038] By analyzing the amino acid sequences of fragments one, two, and three, the core components were identified, expanding the applicability of fragments one, two, and three. Sequence analysis included homology sequence searching and multiple sequence alignment to determine the sequence conservation of each amino acid. Using the initial sequences of fragments one, two, and three as input, corresponding homology sequences were searched using the BLAST function in the NCBI database. Sequences with a similarity of over 70% were selected for multiple sequence alignment in Jalview software, and the alignment results and conservation status of each amino acid were then exported. Most of the aligned sequences originated from mutants of leaf and branch compost cutinase and hydrolases from other species. According to the alignment results, the sequence length of fragment one is in the range of 24-30, the sequence length of fragment two is in the range of 72-81, and the sequence length of fragment three is in the range of 118-136. The sequence conservation analysis results of the three fragments are as follows: Figure 2As shown. Generally speaking, amino acids with a sequence conservation greater than 5 are considered relatively conserved amino acids. When three or more consecutive amino acids have a conservation greater than 5, the region is considered a core folding element of the fragment. Based on this, the core elements of fragment 1 are determined to be NPYQRGPNPT (bits 37-46) and GPFSV (bits 54-58), which is a β-folded layer 1; the core elements of fragment 2 are GGG (bits 72-74), IYYPT (bits 76-80), TFGGIAMSPGYTA (bits 85-97), SSLAWLG (bits 100-106), RLASHGFVV (bits 108-116), VINTNS (bits 118-123), and SRASQLSAALNYL (bits 130-142); the core element of fragment 3 is LAVAGHSMGGGGT (bits 159-175). The amino acids in the core components are: LRIA (β-sheet 5 and α-helix C), QNPSLKAAVPLTPW (β-sheet 6) at positions 177-190, TDKTF (β-sheet 7) at positions 192-196, TSVPVLIVGAE (β-sheet 8) at positions 198-208, DTVAPVSQHA (α-helix D) at positions 210-219, PFYQNLPSTTPKVYVEL (α-helix D and β-sheet 9) at positions 221-237, NASHIAPN at positions 239-246, NNAAIS (α-helix E) at positions 248-253, YTISWMK (α-helix E) at positions 255-261, WVDNDTRY at positions 263-270, and QFLC at positions 272-275. While ensuring that these core amino acids are defined, mutations in the remaining amino acids are allowed, and the resulting amino acid chain can still be formed.

[0039] This invention provides an expression and synthesis method for foliar compost cutinase hydrocarbons. The foliar compost cutinase hydrocarbons include interlocking unit ring one and unit ring two. Unit ring one includes a first segment, a first linking peptide, a third segment, and a second linking peptide, sequentially linked together. Unit ring two includes a second segment and a third linking peptide. The reconnected loop region mentioned above is divided into three parts. The synthesis method is based on an intracellular synthesis method mediated by isolated integrins. This method can covalently couple a segment of the reconnected loop region from each of the two unit rings of the hydrocarbon through isolated integrins. Considering that two segments of the reconnected loop region are located on the same unit ring (unit ring one) of the hydrocarbon, two different expression and synthesis methods can be generated. Synthesis Method 1 uses the N and C terminals of the cutinase from linear leaf and branch compost as cyclization sites, with the looped chain between β-sheet 1 and β-sheet 5 linked by a flexible linker peptide. Synthesis Method 2 uses the looped chain between β-sheet 1 and β-sheet 5 as the cyclization site, with the original N and C terminals linked by a flexible linker peptide. Considering that the cyclization sites of the two unit rings in Synthesis Method 1 are spatially close, which may cause steric hindrance and affect the synthesis of the cyclinase, Synthesis Method 2 is preferred as the synthetic strategy for the cyclinase from leaf and branch compost. Figure 3 ).

[0040] This invention provides a deep learning-based protein loop chain design workflow. The connecting peptide sequence between β-sheet layer 1 and β-sheet layer 5 in synthesis method 2 is optimized to improve protein soluble expression and the proportion of nematicides. First, based on the loop chain end distance, loop chain orientation, and nematicide structure characteristics predicted by AlphaFold2, a roughly reasonable range of amino acid numbers for the loop chain is given, determined to be between 12 and 18 amino acids. Next, the predicted structure is input into the RFdiffusion deep learning method for protein conformation generation, setting the number of amino acids in the reconnecting loop chain to 12 to 18, generating different loop chain conformations. The generated conformations are clustered, and one loop chain conformation is determined as the fixed conformation for sequence design. The ABACUS-R method is used to design the loop chain sequence, and one of the designed sequences is determined as the expression sequence for nematicide design.

[0041] In this embodiment of the invention, in addition to the designed linker peptide, a histidine tag HHHHHH for purification is introduced at the N-terminus of the second unit ring of the leaf-branch compost cutinase hydrocarbon, named cat-LCC. Furthermore, the recognition sequence ENLYFQG of the tobacco etching virus protease (TEV enzyme) is introduced into the first linker peptide of the first unit ring of the leaf-branch compost cutinase hydrocarbon, resulting in a design for verifying the topological structure, named cat-LCC-L. The amino acid sequences of the leaf-branch compost cutinase hydrocarbon designed by the above method in this embodiment of the invention are shown in SEQ ID NO:1 and SEQ ID NO:3, respectively.

[0042] The three-dimensional structural models and secondary structure arrangements of linear leaf-branch compost cutinase (l-LCC) and leaf-branch compost cutinase filament hydrocarbons (cat-LCC) are shown below. Figure 1 As shown.

[0043] This invention also provides an expression vector for cutinase hydrocarbons in leaf and branch compost. A gene fragment encoding the cat-LCC amino acid sequence was inserted into the expression vector pQE80L, and after sequencing confirmation, it was used for cat-LCC expression. The gene pack construction method is as follows... Figure 3 As shown, the expressed polypeptide chain achieves cat-LCC synthesis through non-covalent interaction assembly and cyclization mediated by a dissociative inteptide. Among them, the inteptide VidaL, which mediates the monocyclization of the unit ring, is involved. C The corresponding amino acid sequence is shown in SEQ ID NO: 5 in Table 1, containing the peptide VidaL. N The corresponding amino acid sequence is shown in Table 1 as SEQ ID NO: 6; the in-peptide Npu mediating the bicyclization of the unit ring. C The corresponding amino acid sequence is shown in SEQ ID NO: 7 in Table 1, containing the peptide Npu. N The corresponding amino acid sequences are shown in Table 1 as SEQ ID NO: 8. In the cyclized cat-LCC, the amino acid sequence corresponding to linker peptide one is AAELAKGGSGSGGSTR (SEQ ID NO: 9), the amino acid sequence corresponding to linker peptide two is GTESGGSGKGS (SEQ ID NO: 10), and the amino acid sequence corresponding to linker peptide three is GGTSCFNHHHHHHEL (SEQ ID NO: 11). Compared to cat-LCC, the amino acid sequence corresponding to linker peptide two in cat-LCC-L is GTESGGSGKENLYFQGGS (SEQ ID NO: 12). Compared to cat-LCC, the amino acid sequence corresponding to linker peptide one in the unoptimized LCC nodule (cat-LCC-nopt) is RLSGGSGTGSGST (SEQ ID NO: 13).

[0044] Example 1: Design and Expression of Cutinase Hydrocarbons in Leaf and Branch Compost

[0045] The amino acid sequence of the linear LCC is shown in SEQ ID NO: 2. For ease of purification, a histidine tag (6×His) is linearly attached to its C-terminus. The amino acid sequence of the cyclophosphamide LCC is shown in SEQ ID NO: 1. It contains the intipeptide VidaL sequentially from the N-terminus to the C-terminus. C The following are related to peptide 2: SGKGS region, LCC fragment 1 (amino acids 36-64), peptide 1, LCC fragment 3 (amino acids 159-293), GTESG region of peptide 2, and VidaL (intended peptide). N Intended peptide Npu C The CFNHHHHHHEL portion connecting peptide three, LCC fragment two (amino acids 72 to 151), the GGT portion connecting peptide three, and the intima-peptide Npu N Compared to cat-LCC, cat-LCC-L (SEQ ID NO: 3) replaces the sequence linking peptide 2 SGKGS with SGK. ENLYFQG GS, which contains the TEV enzyme recognition sequence. Compared to cat-LCC, cat-LCC-nopt (Seq ID NO:4) replaces the linker peptide sequence with RLSGGSGTGSGST. The corresponding linear LCC fragment was amplified by PCR, and the corresponding fragment was spliced ​​using overlap extension PCR to construct the expression vector of the hydroxyl group, pQE80L.

[0046] Expression vectors encoding the linear LCC and cyclophosphamide LCC genes were constructed and transformed into *E. coli* BL21(DE3) competent cells. The cells were cultured overnight at 37°C on 2×YT plates containing 100 µg / mL ampicillin sodium. Single colonies were then selected and inoculated into 5 mL of 2×YT medium containing the same antibiotic, and cultured with shaking at 37°C for 10–12 hours to prepare a seed culture. This seed culture was then inoculated at a 1:100 ratio into 200 mL of 2×YT medium containing the same antibiotic, and cultured with shaking at 37°C until the OD600 reached 0.5–0.7. Isopropyl-β-D-thiopyranoside was added to a final concentration of 0.5 mM, and the cells were transferred to 16°C for expression for 15–20 hours.

[0047] Example 2: Design and purification of cutinase hydrocarbons from leaf and branch compost and linear control

[0048] After protein expression, bacterial cells were collected by centrifugation using a high-speed refrigerated centrifuge (5000 g × 15 min), and the supernatant was discarded. The bacterial cells were resuspended in approximately 30 mL of lysis buffer A (50 mM sodium dihydrogen phosphate, 300 mM sodium chloride, 10 mM imidazole, pH 8.0), and β-mercaptoethanol was added to a final concentration of 10 mM. The resuspended cells were sonicated in an ice-water bath using an ultrasonic cell disruptor, followed by centrifugation to collect the supernatant (12000 g × 45 min). The supernatant was mixed thoroughly with Ni-NTA resin and incubated at 4 °C for 1 h. This mixture was poured into a PD-10 gravity column (Bio-rad) for purification. After the lysis buffer A had drained, the resin was washed with 5–10 times the resin volume of wash buffer B (50 mM sodium dihydrogen phosphate, 300 mM sodium chloride, 20 mM imidazole, pH 8.0) to reduce nonspecific adsorption. Elution was then performed using elution buffer C (50 mM sodium dihydrogen phosphate, 300 mM sodium chloride, 250 mM imidazole, pH 8.0), and the eluent was collected.

[0049] Protein eluent was further purified using a rapid purification liquid chromatography system (ÄKTA pure, GE Healthcare) and a size exclusion column (Superdex 200 increase 10 / 300GL, GE Healthcare). The mobile phase was Tris-HCl buffer (20 mM Tris, 150 mM sodium chloride, pH 8.0) filtered through a 0.22 μm membrane at a flow rate of 0.5 mL / min. The protein elution peak was monitored by UV absorption at 280 nm, and samples were collected for characterization. A representative LCC cat-LCC purification chromatogram (x-axis: retention volume, y-axis: intensity) is shown below. Figure 4 As shown.

[0050] Example 3: Optimization results and topological proof of the design of cutinase hydrocarbons in leaf and branch compost.

[0051] Enzymatic digestion with tobacco etch virus protease (TEV) was used to verify the topological structure of LCC filaments. During the digestion characterization experiment, the concentration of LCC filament substrate was 20 μM, the molar ratio of TEV to substrate was 10:1, and the reaction conditions were Tris buffer (20 mM Tris, 100 mM NaCl, pH 8.0), digestion at 30 ℃ for 5 h. Subsequently, the reaction solution before and after digestion was diluted to a 10 μM × 20 μL system, and 5× SDS loading buffer (250 mM Tris, 50% glycerol, 10% SDS, 250 mM β-mercaptoethanol, 0.05% bromophenol blue) was added to a final concentration of 1×. After heating at 98 ℃ for 10 min, SDS-PAGE characterization was performed. The remaining reaction solution was filtered through a 0.22 μm filter membrane, and the final protein concentration was controlled at 0.5 mg / mL for liquid chromatography-mass spectrometry characterization. The liquid chromatography instrument was a UPLCHClass / SQD2 / Acquity QDa. A BEH C4 column (Waters 300, 1.7 μm; 2.1 × 50 mm) was used for protein separation. The mobile phase consisted of acetonitrile containing 0.1% formic acid and aqueous solution. The molecular weight of the proteins was calculated using MassLynx V 4.1 software. A comparison of the purified products before and after sequence optimization of the linking peptide is shown below. Figure 5 As shown, compared to the construction of unoptimized linker peptide sequences, the proportion of the main product from the optimized cat-LCC design was significantly increased. The SDS-PAGE and LC-MS characterization results of the circular and linear products of the LCC cat-LCC-L design containing the TEV recognition sequence after TEV digestion are shown below. Figure 6 As shown, before enzyme digestion, the apparent molecular weight of the designed cord containing the TEV recognition sequence was approximately 25 kDa. After digestion, a linear product (l-LCC-L-R1) with an apparent molecular weight of approximately 20 kDa and a cyclic product (c-LCC-L-R2) with an apparent molecular weight of approximately 8 kDa were generated. LC-MS results confirmed that the molecular weight of the cord before digestion was 30892 Da, and the molecular weights of the linear and cyclic products after digestion were 20886 Da and 10024 Da, respectively. These results demonstrate the advantages of cord sequence optimization and the correct formation of the cord structure.

[0052] Example 4: Circular dichroism spectral characterization of cutinase hydrocarbons designed from leaf and branch compost

[0053] The protein solution was switched from a desalting column to a potassium dihydrogen phosphate (100 mM, pH = 7.4) buffer solution, and the protein was diluted to 0.15 mg / mL with this buffer solution. 200 μL of the solution was added to a cuvette with a 1 mm path length. The molar ellipticity in the range of 190–260 nm was scanned at room temperature with a step size of 1 nm and a data collection interval of 1 s. The test was repeated three times. K₂HPO₄ buffer solution was used as a blank sample to subtract background absorption. The average molar ellipticity [θ] of the protein residues was determined using a circular dichroism spectroscopy (CDS) spectrometer. The formula is: [θ] = (θ obs / 10) × (MRW / lc), where θ obs The measured ellipticity is given in mdeg; MRW is the average molecular weight of residues in Da; l is the optical path length of the cuvette in cm; c is the protein concentration in mg / mL. The circular dichroism chromatograms of LCC solenoid design and corresponding linear controls (b) are shown below. Figure 7 As shown, the circular dichroism chromatogram of the designed cyclohexane is basically consistent with the linear control, with a slight decrease in intensity.

[0054] Example 5: Activity Characterization of Leaf and Branch Compost Cutinase Hydrocarbon Design on p-Nitrophenyl Acetate Substrate

[0055] LCC can catalyze the hydrolysis of various small molecule ester compounds. p-Nitrophenyl acetate (pNP-C2) can be hydrolyzed by this enzyme to produce acetic acid and p-nitrophenol. p-Nitrophenol exhibits strong absorption at 415 nm, so the reaction progress can be monitored by detecting changes in the absorption at 415 nm. A concentrated stock solution of 10 mM p-nitrophenyl acetate substrate was prepared using acetonitrile and stored at –20 °C protected from light. Before testing, the enzyme solution was diluted to 250 nM with potassium dihydrogen phosphate (100 mM, pH 8) buffer to prepare the concentrated enzyme stock solution. A certain amount of the concentrated substrate stock solution was diluted to 1.11 mM with K₂HPO₄ buffer to prepare the working substrate solution. 20 μL of the concentrated enzyme stock solution and 180 μL of the working substrate solution were added sequentially to a clear 96-well plate, mixed thoroughly by pipetting, and then quickly placed in a microplate reader. The change in absorbance at 415 nm was measured in kinetic mode. Different concentrations of p-nitrophenol standard solutions (0, 20, 40, 60, 80, 100, 120, 200 nmol) were prepared using potassium dihydrogen phosphate buffer. The absorbance of the standard solutions at 415 nm was measured using a microplate reader, and a standard curve was fitted. The obtained kinetic curves were plotted according to the change in absorbance over time. A linear fit was performed on the initial time period to obtain the slope, and the enzyme activity was calculated by combining the standard curve and LCC concentration. The reaction kinetic curves of the LCC-based design and corresponding linear control catalyzing the hydrolysis of pNP-C2 to p-nitrophenol (x-axis: time (s), y-axis: absorbance at 340 nm) and the calculated specific activity are shown below. Figure 8 As shown in the figure. The calculation results indicate that the specific activity of the linear design is basically consistent with that of the linear control.

[0056] Example 6: Activity Characterization of Polyethylene Terephthalate Substrate Based on the Design of Cutinase Hydrocarbons from Leaf and Branch Compost

[0057] Polyethylene terephthalate (PET) raw materials are available in amorphous films and crystalline powders. For amorphous films, they are cut into 1 cm × 1 cm sheets and cleaned with ddH2O before the experiment. For crystalline powders, a certain amount of PET powder is weighed into a 1.5 mL EP tube before the experiment. All LCC samples to be tested are diluted to 3 μM with potassium dihydrogen phosphate (100 mM, pH 8) buffer to prepare working solutions. The films are placed in 1 mL of working solution and reacted in a shaking metal bath at 65 ℃ for 48 h. The reaction solutions at different time points are collected, and the reaction is quenched by heating at 98 ℃ for 20 min. The solutions are then diluted by a certain factor to ensure that the sample concentration does not exceed the UPLC detection limit. After centrifugation at 4000 rpm for 10 min, the supernatant is collected for UPLC analysis. The sample loading volume for UPLC was 2 μL. A UPLC analytical method was established using methyl terephthalate (MHET) and terephthalic acid (TPA) standards, and the correspondence between the 235 nm UV absorption peak and elution time was determined. The mobile phase was a mixture of 0.05% trifluoroacetic acid aqueous solution and acetonitrile. A ZORBAX SB-C18 column was used, with a column temperature of 25 ℃ and a flow rate of 0.5 mL / min. Standard solutions of MHET and TPA samples at different concentrations (0.1, 0.2, 0.5, 1.0, 2.0, 5.0 mM) were prepared, and standard curves were plotted based on the peak area integral of the UV absorption peak at the specified wavelength and the relationship between concentration. Kinetic curves of product concentration versus time were obtained by converting the peak areas of different product peaks measured from the hydrolysis reaction. The kinetic curves of PET degradation catalyzed by LCC catalysis and corresponding linear controls, and the ratio of TPA and MHET concentrations in the system after 24 h of reaction were also presented. Figure 9 As shown, the kinetic results indicate that the catalytic design still has very good catalytic activity. Although the TPA product formation rate is slightly lower than that of the linear LCC, after 24 h of reaction, the total concentration of TPA and MHET in the catalytic system is basically consistent with that of the linear control, indicating that the degradation rate of PET by the catalytic design is basically consistent with that of the linear control.

[0058] Table 1. Sequences of linear LCCs and LCC filaments involved in the embodiments of the present invention.

[0059]

[0060]

[0061]

Claims

1. A leaf and branch compost cutinase hydrocarbon, comprising mechanically interlocked unit ring one and unit ring two, characterized in that, Unit ring one includes a ring segment one, a linking peptide one, a ring segment three, and a linking peptide two connected in sequence; unit ring two includes a ring segment two and a linking peptide three connected in sequence; wherein, the ring segment one contains a β-sheet 1 of leaf-branch compost cutinase, the ring segment two contains a ring segment 2, a ring segment 3, an α-helix A, a ring segment 4, and an α-helix B connected in sequence, and the ring segment three contains a ring segment 5, an α-helix C, a ring segment 6, a ring segment 7, an α-helix D, a ring segment 8, an α-helix E, and a ring segment 9 connected in sequence of wild-type leaf-branch compost cutinase.

2. The leaf and branch compost cutinase hydrocarbon as described in claim 1, characterized in that, The first fragment is a peptide with the amino acid sequence shown in SEQ ID NO: 14 of the sequence listing, or a peptide with a length of 24-30 aa that has more than 80% homology with SEQ ID NO: 14 and retains the sequences NPYQRGPNPT and GPFSV unchanged; the second fragment is a peptide with the amino acid sequence shown in SEQ ID NO: 15 of the sequence listing, or a peptide with a length of 72-81 aa that has more than 80% homology with SEQ ID NO: 15 and retains the sequences GGG, IYYPT, TFGGIAMSPGYTA, SSLAWLG, RLASHGFVV, VINTNS, and SRASQLSAALNYL unchanged; the third fragment is a peptide with the amino acid sequence shown in SEQ ID NO: 16 of the sequence listing, or a peptide with the amino acid sequence shown in SEQ ID NO: 14 that has more than 80% homology with SEQ ID NO: 14 and retains the sequences GGG, IYYPT, TFGGIAMSPGYTA, SSLAWLG, RLASHGFVV, VINTNS, and SRASQLSAALNYL unchanged.

16. Peptides with over 80% homology and maintaining the sequences LAVAGHSMGGGGTLRIA, QNPSLKAAVPLTPW, TDKTF, TSVPVLIVGAE, DTVAPVSQHA, PFYQNLPSTTPKVYVEL, NASHIAPN, NNAAIS, YTISWMK, WVDNDTRY, and QFLC for a length of 118-136 aa.

3. The leaf and branch compost cutinase hydrocarbon as described in claim 1, characterized in that, The first linker peptide is a peptide with 12 to 18 amino acid residues; the second and third linker peptides are each independently flexible linker peptides with 4 to 20 amino acid residues.

4. The leaf and branch compost cutinase hydrocarbon as described in claim 3, characterized in that, The amino acid sequence of the first linker peptide is AAELAKGGSGSGGSTR; and / or the amino acid sequence of the second linker peptide is GTEGSGKGS or GTEGSGKENLYFQGGS; and / or the amino acid sequence of the third linker peptide is GGTSCFNHHHHHHEL.

5. A method for preparing the leaf and branch compost cutinase hydrocarbon according to any one of claims 1 to 4, comprising the following steps: 1) Design the protein precursor sequence of leaf and branch compost cutinase hydrocarbon, and synthesize the coding gene sequence of the protein precursor sequence; wherein, the protein precursor sequence includes at least: the sequence of unit loop one and unit loop two, and two pairs of orthogonal cyclization motifs mediating the cyclization of unit loop one and unit loop two in the cell. 2) The coding gene sequence synthesized in step 1) is introduced into the expression vector to obtain the recombinant expression vector; 3) The recombinant expression vector is transferred into host cells for expression to obtain the fusion protein; 4) The fusion protein was purified to obtain the leaf and branch compost cutinase hydrocarbon.

6. The preparation method according to claim 5, characterized in that, The two pairs of orthogonal cyclization units mentioned in step 1) are selected from any of the following combinations: i. two orthogonal isolated intekines; ii. two orthogonal polypeptide-protein reaction pairs; iii. a combination of polypeptide-protein reaction pairs and isolated intekines.

7. The preparation method according to claim 6, characterized in that, The protein precursor sequence described in step 1) includes, from the N-terminus to the C-terminus, Int C Fragment 1, linking peptide 2 (C-terminus), Fragment 3, linking peptide 2 (N-terminus), Int N Int′ C , connecting the C-terminal portion of peptide 3, fragment 3, connecting the C-terminal portion of peptide 3, Int′ N ;Int N and Int C Int′ represents the N-terminal and C-terminal portions of the isolated inteptide Int, respectively. N and Int′ C The N-terminal and C-terminal portions of the isolated inteptide Int′ are represented respectively, and the two isolated inteptides Int and Int′ are orthogonal.

8. The preparation method according to claim 7, characterized in that, The two orthogonal isolated integrins are VidaL and Npu.

9. The preparation method according to claim 8, characterized in that, Step 1) The protein precursor sequence is shown in SEQ ID NO: 1 or SEQ ID NO: 3 in the sequence listing.

10. A gene sequence expressing the synthesis of leaf and branch compost cutinase hydrocarbons, characterized in that, The gene sequence encodes the protein precursor sequence as described in any one of claims 5 to 9.

11. An expression vector comprising the gene sequence of claim 10 for expressing synthetic leaf and branch compost cutinase hydrocarbon, or a host cell comprising the expression vector.

12. The application of the leaf and branch compost cutinase hydrocarbon according to any one of claims 1 to 4 in the biodegradation of polymer materials.

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