Leaf branch compost cutinase kink and application thereof

Abstract

The invention discloses a leaf and branch compost cutinase link and application thereof, and belongs to the technical field of enzyme molecule construction. The leaf-branch compost cutinase kink is obtained by adjusting the connection sequence of fragments in leaf-branch compost cutinase, and comprises a fragment II, a connecting peptide fragment I, a fragment I, a connecting peptide fragment II and a fragment III which are connected in sequence from the N end to the C end; wherein the fragment I, the fragment II and the fragment III respectively correspond to 37th to 79th amino acids, 81st to 143rd amino acids and 150th to 293rd amino acids or homologous sequences thereof of the leaf branch compost cutinase. According to the method, protein topology engineering and an artificial intelligence assisted protein design technology are organically combined, knot topology transformation is performed on the leaf branch compost cutinase, sequence optimization is performed on a connecting peptide fragment of the knot, and the leaf branch compost cutinase knot with good biological activity is obtained; the expression quantity and the stability of the leaf and branch compost cutinase kink are improved, the problem of kinetic barriers existing in protein kink combination is solved, and the method has important application value.

Description

TECHNICAL FIELD

[0001] The present application relates to protein topological structure modification technology, in particular to the knot topological modification of leafcutter compost cutinase, and the application of leafcutter compost cutinase knot. BACKGROUND

[0002] Biodegradation of polymeric materials is one of the important ways to solve the problem of plastic pollution. Among them, polyethylene terephthalate (PET) is a widely used synthetic polymer, and its non-degradability has a lasting impact on the environment. In recent years, the discovery and modification of enzymes (such as cutinase, esterase and lipase) that can efficiently degrade PET have made it possible for the biological recycling of PET. Among them, leafcutter compost cutinase (LCC) has attracted much attention due to its high catalytic activity (Tournier, V. et al. Nature 2020, 580, 216). However, leafcutter compost cutinase still faces challenges in industrial applications, especially in terms of stability and long-term catalytic efficiency at high temperatures (~65 ℃). Therefore, improving the thermal stability of leafcutter compost cutinase through protein engineering is crucial for its practical application in the PET degradation industry.

[0003] The topological structure of a protein is one of the key factors that regulate its folding, stability and function. In nature, some proteins enhance their structural rigidity by forming complex topological structures (such as knots), thereby improving their thermodynamic stability. Knot proteins have a high kinetic barrier during folding, but once correctly folded, they often exhibit extremely high conformational stability, making them particularly suitable for functioning in extreme environments (such as high temperature, high pH or organic solvents) (Patrícia F. N. Comput. Struct. Biotechnol. 2015, 13, 459). However, due to the complexity of the folding path of knot proteins, it is extremely challenging to introduce knot structures into non-knot proteins through rational design, and it is necessary to finely optimize the target sequence to ensure that it can form a stable thermodynamic conformation while avoiding incorrect folding or aggregation due to kinetic barriers. Combined with existing artificial intelligence design methods, developing a protein backbone with high stability is very important for improving the practical application of leafcutter compost cutinase. SUMMARY

[0004] In order to solve the problems existing in the prior art, the present application is committed to determining a feasible way to split and connect the knot of leafcutter compost cutinase by combining existing artificial intelligence protein prediction and design methods, optimizing the sequence of the re-wiring loop region chain, and realizing the synthesis of leafcutter compost cutinase knot.

[0005] The linear precursor leaf branch composting cutinase (linear precursor LCC) comprises, in order from N-terminus to C-terminus, fragment one, fragment two and fragment three, as shown in Figure 1 , wherein the fragment one comprises β-strand 1 and β-strand 2 connected in order, the fragment two comprises β-strand 3, α-helix A, β-strand 4 and α-helix B connected in order, and the fragment three comprises β-strand 5, α-helix C, β-strand 6, β-strand 7, α-helix D, β-strand 8, α-helix E and β-strand 9 connected in order.

[0006] The present application provides a method for obtaining a leaf branch composting cutinase knot (LCC knot), which can be obtained by adjusting the connection order of each fragment in the linear precursor leaf branch composting cutinase and introducing a connecting peptide segment between adjacent fragments. Specifically, as shown in Figure 1 , the loop region chain between β-strand 2 and β-strand 3 and between α-helix B and β-strand 5 is split, then the C-terminus of β-strand 2 and the N-terminus of β-strand 5 are connected by connecting peptide segment two, and the N-terminus of β-strand 1 and the C-terminus of α-helix B are connected by connecting peptide segment one, the original C-terminus of the leaf branch composting cutinase remains unchanged, and the N-terminus of β-strand 3 serves as the N-terminus of the leaf branch composting cutinase knot. After adjusting the connection order of each fragment, the obtained leaf branch composting cutinase knot comprises, in order from N-terminus to C-terminus, fragment two, connecting peptide segment one, fragment one, connecting peptide segment two and fragment three.

[0007] The conserved core elements of the fragment one, the fragment two and the fragment three are determined by the method of homologous sequence search and multiple sequence alignment. Taking the amino acid sequence of the linear precursor phyllochrysen compost cutinase as the benchmark, the core elements of the fragment one are NPYQRGPNPT at 37-46, GPFSVA (β-strand 1) at 54-59, TVSRLSVSGFGGG (β-strand 2) at 62-74, IYYP (β-strand 2) at 76-79; the core elements of the fragment two are TFGGIAMSPGYTA (β-strand 3) at 85-97, SSLAWLG (α-helix A) at 100-106, RLASHGFVV (α-helix A) at 108-116, VINTNSRFD (β-strand 4) at 118-126, SRASQLSAALNYL (α-helix B) at 130-142; the core elements of the fragment three are VRARLDANRLAVAGHSMGGGGTLRIA (β-strand 5 and α-helix C) at 150-175, QNPSLKAAVPLTPW (β-strand 6) at 177-190, TDKTF (β-strand 7) at 192-196, TSVPVLIVGAE (β-strand 8) at 198-208, DTVAPVSQHA (α-helix D) at 210-219, PFYQNLPSTTPKVYVEL (α-helix D and β-strand 9) at 221-237, NASHIAPN at 239-246, NNAAIS (α-helix E) at 248-253, YTISWMK (α-helix E) at 255-261, WVDNDTRY at 263-270, QFLC at 272-275. In the case of ensuring the determination of the amino acid sequences of these core elements, the mutations of substitution, deletion or addition of the amino acid residues in the remaining part are allowed, and the knotted structure can still be formed and the function is retained after the mutation.

[0008] In some embodiments of the present application, fragment one, fragment two and fragment three correspond to the 37th-79th amino acids (SEQ ID NO: 11), the 81st-143rd amino acids (SEQ ID NO: 12) and the 150th-293rd amino acids (SEQ ID NO: 13) of the linear precursor leaf branch compost cutinase amino acid sequence, respectively. While ensuring that the core elements in the above-mentioned fragment one, fragment two and fragment three remain unchanged, the amino acid residues in the non-conserved regions can be substituted, deleted or added to obtain a plectin with more than 80% homology to the original sequence and the same function, preferably with more than 85% homology, more preferably with more than 90% homology. Among them, the sequence length of fragment one can vary in the range of 43-45 aa, the sequence length of fragment two can vary in the range of 58-63 aa, and the sequence length of fragment three can vary in the range of 122-145 aa.

[0009] In the leaf branch compost cutinase knottin of the present application, generally, the connecting peptide segment one and the connecting peptide segment two are each independently a peptide segment with 4-15 amino acid residues. Preferably, the number of amino acid residues of the connecting peptide segment one is between 8 and 13, and the number of amino acid residues of the connecting peptide segment two is between 5 and 8. In some embodiments of the present application, the connecting peptide segment one is selected from one of TSSGGSGGTGGSS, AGSRGYGGS, EGARERYSED, and the connecting peptide segment two is selected from one of SGSSA, GGSTLEE. In an embodiment of the present application, in the first constructed leaf branch compost cutinase knottin, the sequence of the connecting peptide segment one is TSSGGSGGTGGSS, and the sequence of the connecting peptide segment two is SGSSA. The amino acid sequence of this leaf branch compost cutinase knottin is shown in SEQ ID NO: 7 in the sequence listing. On this basis, the connecting peptide segments are designed and optimized by artificial intelligence protein prediction and design methods, and other leaf branch compost cutinase knottins with better comprehensive properties are screened out, and the amino acid sequences thereof are shown in SEQ ID NO: 8, 9 and 10, respectively.

[0010] Further, the recognition sequence ENLYFQG of tobacco etch virus protease (TEV enzyme) is introduced at the N-terminus of the leaf branch compost cutinase knottin, and the recognition sequence LPETG of transpeptidase A (Srt A) is introduced at the C-terminus in turn to realize the cyclization of the open-loop knottin. Further, for the convenience of purification, the histidine tag sequence HHHHHH is introduced at the N-terminus or C-terminus of the protein, and the protein is purified by nickel column affinity chromatography. In an embodiment of the present application, the amino acid sequences of the four obtained leaf branch compost cutinase knottins (k-LCC-0 and k-LCC-1 ~ k-LCC-3) are shown in SEQ ID NO: 1 and SEQ ID NO: 3-5, respectively.

[0011] The gene of the leaf branch compost cutinase knottin, the expression vector comprising the gene sequence, and the host cell comprising the expression vector are within the protection scope of the present application.

[0012] The leaf branch compost cutinase knottin provided by the present application can be applied to biodegradation of high molecular materials, especially degradation of PET, and is one of important ways to effectively solve the problem of plastic pollution.

[0013] The leaf branch compost cutinase knottin provided by the present application is obtained by organically combining protein topology engineering and artificial intelligence assisted protein design technology, and performing knotted topology modification on the leaf branch compost cutinase, and performing sequence design on the connecting peptide segment on the basis of the knotted topology structure, so that the leaf branch compost cutinase knottin with good biological activity is obtained, and the soluble expression yield and stability of the leaf branch compost cutinase knottin are improved. Therefore, the scheme provided by the present application realizes efficient synthesis of the leaf branch compost cutinase knottin, overcomes the problem of kinetic barrier in protein knotted synthesis, and has important application value. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 It is a three-dimensional structure model and secondary structure arrangement schematic diagram of the linear precursor LCC and the LCC knottin.

[0015] Figure 2 It is a sequence conservation analysis result of different LCC fragments.

[0016] Figure 3 It is a connection mode schematic diagram of the linear precursor LCC and the LCC knottin split gene fragment.

[0017] Figure 4 It is a SEC purification spectrum diagram of the LCC knottin (k-LCC-0~k-LCC-3) in Example 2, taking the linear precursor LCC (l-LCC) and the linear control LCC (l-LCC-control) as controls.

[0018] Figure 5 It is a schematic diagram of the LCC knottin and the corresponding linear control prepared into a circular product by TEV enzyme cutting and Srt A coupling in Example 3.

[0019] Figure 6 It is a SDS-PAGE characterization schematic diagram of the LCC knottin (k-LCC-1~k-LCC-3) and the corresponding linear control (l-LCC-control) prepared into a circular product by TEV enzyme cutting and Srt A coupling in Example 3.

[0020] Figure 7Schematic diagram of circular dichroism characterization of LCC knotted (k-LCC-1 ~ k-LCC-3) and corresponding linear control (l-LCC-control) in Example Four.

[0021] Figure 8 Reaction kinetics curve of LCC knotted (k-LCC-0 ~ k-LCC-3) catalyzing pNP-C2 to convert into p-nitrophenol in Example Five.

[0022] Figure 9 Schematic diagram of specific activity calculated from LCC knotted (k-LCC-0 ~ k-LCC-3) catalyzing pNP-C2 to convert into p-nitrophenol in Example Five.

[0023] Figure 10 Kinetics curve (a) of LCC knotted (k-LCC-1 ~ k-LCC-3) and corresponding linear control (l-LCC-control) catalyzing PET degradation in Example Six, and concentration of products in the system after 24 h of reaction (b). DETAILED DESCRIPTION

[0024] In order to better understand the above technical solutions, the above technical solutions will be described in detail below in combination with the drawings in the specification and specific embodiments.

[0025] The embodiment of the present application provides a method for obtaining leaf branch compost cutinase knotted, which can realize the design of the leaf branch compost cutinase knotted by adjusting the connection order between the secondary structure fragments in the linear precursor leaf branch compost cutinase (l-LCC for short). Figure 1 The three catalytic residues are S165, D210 and H242 respectively. Through the analysis of the crystal structure of the leaf branch compost cutinase, it can be found that the loop chain between the β-sheet 2 and the β-sheet 3 and the α-helix B and the β-sheet 5 is split, then the C terminal of the β-sheet 2 and the N terminal of the β-sheet 5 are connected by the connecting peptide segment two, the N terminal of the β-sheet 1 and the C terminal of the α-helix B are connected by the connecting peptide segment one, the original C terminal of the leaf branch compost cutinase remains unchanged, and the N terminal of the β-sheet 3 serves as the N terminal of the leaf branch compost cutinase knotted, so that the design of the knotted structure can be realized. The spatial position of the reconnected loop chain is far away from the active center of the protein, and the reconnected loop chain will not affect the combination of the substrate and the active pocket.

[0026] The application adjusts the connection order between each fragment of the linear precursor leaf compost cutinase, and introduces a connection peptide segment between adjacent fragments to obtain a leaf compost cutinase knotted. The connection peptide segment includes a connection peptide segment one and a connection peptide segment two, and the obtained leaf compost cutinase knotted includes, from N-terminal to C-terminal, sequentially connected fragment two, connection peptide segment one, fragment one, connection peptide segment two and fragment three.

[0027] By analyzing the amino acid sequences of fragment one, fragment two and fragment three, the core element part is determined, and the application range of fragment one, fragment two and fragment three is expanded. Sequence analysis includes homologous sequence search and multiple sequence alignment, so as to determine the sequence conservation degree of each amino acid. The initial sequence of fragment one, fragment two and fragment three is taken as input respectively, the corresponding homologous sequence is searched through the Blast function of NCBI database, the sequence with a sequence similarity of more than 70% is selected for multiple sequence alignment in Jalview software, and then the alignment results and the conservation degree of each amino acid are exported. Most of the alignment sequences are derived from mutants of leaf compost cutinase and hydrolytic enzymes derived from other species. According to the alignment results, the sequence length of fragment one can be in the range of 43-45, the sequence length of fragment two can be in the range of 58-63, and the sequence length of fragment three can be in the range of 122-145. Preferably, in the embodiments of the application, the length of fragment one is 43, the length of fragment two is 63, and the length of fragment three is 144. The calculation results of sequence conservation degree are as follows: Figure 2As shown, generally speaking, the amino acids with sequence conservation greater than 5 are considered to be relatively conservative amino acids, and when the conservation of three or more consecutive amino acids is greater than 5, the region is considered to be the core folding element of the fragment. Based on this, the core element of fragment one is NPYQRGPNPT at positions 37-46, GPFSVA (β-sheet 1) at positions 54-59, TVSRLSVSGFGGG (β-sheet 2) at positions 62-74, and IYYP (β-sheet 2) at positions 76-79; the core element of fragment two is TFGGIAMSPGYTA (β-sheet 3) at positions 85-97, SSLAWLG (α-helix A) at positions 100-106, RLASHGFVV (α-helix A) at positions 108-116, VINTNSRFD (β-sheet 4) at positions 118-126, and SRASQLSAALNYL (α-helix B) at positions 130-142; and the core element of fragment three is VRARLDANRLAVAGHSMGGGGTLRIA (β-sheet 5 and α-helix C) at positions 150-175, 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. In the case of ensuring the determination of the amino acids of these core elements, the mutation of the remaining amino acids is allowed, and the knotted structure can still be formed after the mutation. Preferably, in the embodiments of the present application, fragment one, fragment two, and fragment three correspond to the amino acids at positions 37-79, the amino acids at positions 81-143, and the amino acids at positions 150-293 of the linear precursor phylacystatin amino acid sequence, respectively.

[0028] In a preferred embodiment of the present application, the linear precursor phylacystatin amino acid sequence is shown as SEQ ID No: 2, and mainly includes three fragments: fragment one is the amino acids at positions 37-79, fragment two is the amino acids at positions 81-143, and fragment three is the amino acids at positions 150-293. The connection order of the three fragments is adjusted, and adjacent two fragments are connected by using a connection peptide segment, to obtain the following amino acid connection order of the phylacystatin knotted structure: fragment two-connection peptide segment one-fragment one-connection peptide segment two-fragment three.

[0029] The connecting peptide segment connecting the two adjacent fragments is an amino acid sequence, and the conformation and amino acid sequence of the connecting peptide segment part can be designed by the open source artificial intelligence method RFdiffusion and ABACUS-R. The RFdiffusion program is used to determine the conformation of the connecting peptide segment, and the rigid fragment with stronger non-covalent interaction of the main chain of the connecting peptide segment is very important to improve the stability of the protein. In the embodiment of the present application, before generating the conformation, it is determined by the spatial distance detection that the number of amino acids of the first connecting peptide segment is between 8 and 13, and the number of amino acids of the second connecting peptide segment is between 5 and 8. After determining the conformation of the connecting peptide segment, the amino acid sequence of the connecting peptide segment is designed by the ABACUS-R program, and the amino acid sequence matching the protein backbone is also important to improve the stability of the protein. During the design, the amino acids of the non-connecting peptide segment are fixed, and the program only optimizes the amino acids of the connecting peptide segment part. Subsequently, the amino acid sequence obtained by the design is expressed by experiment, so as to obtain the knot with improved expression amount and stability.

[0030] In the embodiment of the present application, in addition to introducing the connecting peptide segment, the recognition sequence ENLYFQG of tobacco etch virus protease (TEV enzyme) is introduced at the N terminal of the leaf branch composting cutinase knot, and the recognition sequence LPETG of transpeptidase A (Srt A) and the histidine tag HHHHHH are introduced at the C terminal in turn. The leaf branch composting cutinase knot obtained by the above method in the embodiment of the present application has an amino acid sequence as shown in SEQ ID No: 1. The sequence of the first connecting peptide segment is GGSGGTGGS, and the sequence of the second connecting peptide segment is SGSS.

[0031] The three-dimensional structure model and secondary structure arrangement of the linear precursor leaf branch composting cutinase (linear precursor LCC) and the leaf branch composting cutinase knot (LCC knot, k-LCC-0) are as shown in Figure 1

[0032] The embodiment of the present application also provides an expression vector of the leaf branch composting cutinase knot. The gene fragment encoding the amino acid sequence of k-LCC-0 is inserted into the expression vector pET15b, and after sequencing confirmation, it is used for the expression of k-LCC-0.

[0033] ​The present application also provides leaf branch composting cutinase knottins with different linker sequences. Specifically, the linkers of the constructed k-LCC-0 are designed and optimized, and the soluble expression and activity of 20 knottins are tested in total. The amino acid sequences of three LCC knottins (k-LCC-1 ~ k-LCC-3) with better comprehensive properties are shown in SEQ ID No: 3 ~ SEQ ID No: 5. The LCC knottin is converted into a corresponding linear control according to the connection sequence of the linear leaf branch composting cutinase amino acid fragment, and the amino acid sequence of one LCC knottin linear control (l-LCC-control) is shown in SEQ ID No: 6, corresponding to the LCC knottin k-LCC-1. The connection mode of the split gene fragments of the linear precursor LCC, the LCC knottin, and the corresponding LCC knottin linear control is shown in Figure 3 .

[0034] Example 1, expression of leaf branch composting cutinase knottins and corresponding linear controls

[0035] The amino acid sequence of the linear precursor LCC (l-LCC) corresponds to SEQ ID NO: 2, and a histidine tag (6×His) is added at the C-terminus. The amino acid sequence of the linear control l-LCC-control corresponds to SEQ ID NO: 6, and the N-terminus to the C-terminus correspond to a TEV enzyme recognition sequence (ENLYFQG), a flexible chain (GGELGGSGGGGSG), a linear precursor LCC sequence, a flexible chain (GSLEEGT), a transpeptidase recognition site (LPETG), and a histidine tag (6×His), respectively. The amino acid sequences of the LCC knottins (k-LCC-0 ~ k-LCC-3) correspond to SEQ ID NO: 1, SEQ ID NO: 3 ~ 5, respectively, and the N-terminus to the C-terminus correspond to a TEV enzyme recognition sequence (ENLYFQG), fragment one (amino acids 81 to 143), linker one, fragment two (amino acids 37 to 79), linker two, fragment three (amino acids 150 to 293), a transpeptidase A recognition site (LPETG), and a histidine tag (6×His), respectively. The expression vector of the linear precursor LCC is pET26b. By polymerase chain reaction (PCR), a certain flexible amino acid chain, a TEV recognition sequence, and a transpeptidase recognition site are introduced at both ends of the linear precursor LCC to construct the expression vector of the linear control LCC, and the expression vector is pET15b. The corresponding linear precursor LCC fragments are amplified by PCR, and the corresponding fragments are spliced using overlap extension PCR to construct the expression vector of the LCC knottin, and the expression vector is pET15b.

[0036] The linear precursor LCC, linear control LCC and knotted LCC were constructed into E. coli BL21 (DE3) competent cells, and were cultured overnight at 37 °C on 2xYT plates containing 100 pg / mL ampicillin sodium. Then single colonies were picked and inoculated into 5 mL 2xYT medium containing the same resistance, and were cultured at 37 °C for 10-12 hours to prepare seed bacteria liquid. The seed bacteria liquid was inoculated into 200 mL 2xYT medium containing the same resistance at a ratio of 1:100, and was cultured at 37 °C until the OD600 was between 0.5-0.7. Isopropyl-beta-D-thiogalactopyranoside was added to a final concentration of 0.5 mM, and the expression was carried out at 16 °C for 15-20 hours.

[0037] Example Two, Purification of Leaf Litter Keratinase Knotted and Corresponding Linear Control

[0038] After the end of protein expression, the bacteria were collected by high-speed refrigerated centrifuge (5000 g x 15 min), and the supernatant was discarded. The bacteria were resuspended with about 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 resuspension was ultrasonically broken by an ultrasonic cell disruptor under the condition of ice water bath, and then the supernatant was collected by centrifugation (12000 g x 45 min). The supernatant was mixed with Ni-NTA resin and incubated at 4 °C for 1 h. The mixture was poured into a purification PD-10 gravity empty column (Bio-rad), and after the lysis buffer A was completely drained, the resin was washed with 5-10 times the volume of the resin to reduce non-specific adsorption. Then elution was performed with elution buffer C (50 mM sodium dihydrogen phosphate, 300 mM sodium chloride, 250 mM imidazole, pH 8.0), and the eluate was collected.

[0039] Protein eluate was further purified using a fast purification liquid chromatography system (AKTA pure, GE Healthcare) and a size exclusion chromatography column (Superdex 200 increase 10 / 300 GL, GE Healthcare) with a flow phase of Tris-HCl buffer (20 mM Tris, 150 mM NaCl, pH 8.0) filtered by 0.22 μm filter membrane at a flow rate of 0.5 mL / min. The protein elution peak was monitored by ultraviolet absorption at 280 nm, and the sample was collected for characterization. The SEC purification spectrum (horizontal axis: retention volume, vertical axis: intensity) of a representative LCC knotted structure is shown in Figure 4 .

[0040] Example Three, Preliminary demonstration of leaf branch compost cutinase knotted topology

[0041] The strategy adopted was to use TEV enzyme digestion and SrtA coupling to cyclize the knotted construct and the corresponding linear control, as shown in Figure 5 . The products before and after cyclization were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Since they have almost identical amino acid compositions, the difference in protein band positions on the SDS-PAGE gel mainly comes from the difference in their topologies.

[0042] First, the knotted construct and the corresponding linear control were subjected to TEV enzyme digestion, exposing the N-terminal oligoglycine. The substrate concentration was 50 μM, mixed with 0.1 molar equivalent of TEV enzyme, and 20x enzyme digestion buffer (1 M Tris-HCl, 2 M NaCl, 20 mM EDTA, 100 mM DTT, pH 8) was added, and the enzyme was incubated at 4 °C overnight. After sufficient enzyme digestion, 0.1 molar equivalent of transpeptidase A and 10 mM CaCl2 were added to the above solution and reacted at 16 °C for 3-5 h. After the reaction was completed, 10 mM EDTA was added to terminate the reaction. The reaction system was added to a Ni-NTA affinity chromatography column and incubated for 1 h, and the effluent was collected as the cyclization product. The effluent was further eluted with Tris-HCl buffer and the eluate was collected in each tube, and the main component was also the cyclization product non-specifically adsorbed on the resin. The effluent and eluate were mixed and concentrated to obtain the cyclization product.

[0043] For the sample to be characterized, it 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 bring the final concentration to 1×. After heating at 98 °C for 10 min, SDS-PAGE characterization was performed. The SDS-PAGE characterization of LCC knots and corresponding linear controls prepared into cyclic products by TEV digestion and Srt A coupling is shown below. Figure 6 As shown (where those without the cyc label represent the reactant control, and those with the cyc label represent the product after coupling with Srt A). Before cyclization, the apparent molecular weight of the LCC knot was basically the same as that of its linear control. After cyclization, the apparent molecular weight of the closed-ring LCC knot was much lower than that of the corresponding cyclic LCC converted from the linear control.

[0044] Example 4: Circular dichroism spectral characterization of cutinase knots in leaf and branch compost

[0045] The protein solution purified by SEC in Example 1 was switched to potassium dihydrogen phosphate (100 mM, pH 7.4) buffer solution via a desalting column, 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, and 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) instrument. The calculation formula is as follows:

[0046] [θ] = (θ obs / 10) × (MRW / lc)

[0047] Where, θ obs The measured ellipticity is expressed 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. Circular dichroism chromatograms of LCC knots (k-LCC-1 ~ k-LCC-3) and their corresponding linear controls (l-LCC-control) (broad axis: wavelength, y-axis: ellipticity [θ]) are shown below. Figure 7 As shown, the peak shape and intensity of the circular dichroism chromatogram of the knot are basically consistent with the line type, indicating that the secondary structure components of the knot design are almost identical to the line type.

[0048] Example 5: Activity characterization of leaf and branch compost cutinase knots against p-nitrobenzene acetate substrate

[0049] LCC can catalyze the hydrolysis of a variety of small molecule ester compounds. p-nitrophenyl acetate (pNP-C2) can be hydrolyzed into acetic acid and p-nitrophenol under the action of the enzyme. The p-nitrophenol has a strong absorption at 415 nm, so the progress of the reaction can be monitored by detecting the change in absorption of the system at 415 nm. A 10 mM stock solution of p-nitrophenyl acetate substrate was prepared with acetonitrile and stored at -20 °C in the dark. Before testing, the enzyme solution was diluted to 250 nM with potassium dihydrogen phosphate (100 mM, pH 8) buffer as a concentrated stock solution of the enzyme. A certain amount of substrate stock solution and K2HPO4 buffer were diluted to 1.11 mM to prepare a working solution of the substrate. In a transparent 96-well plate, 20 μL of the concentrated stock solution of the enzyme and 180 μL of the working solution of the substrate were added in turn, mixed by blowing and sucking, and then quickly placed in an enzyme marker. The change in absorbance at 415 nm was measured under kinetic mode. Different concentrations of p-nitrophenol standard solution (0, 20, 40, 60, 80, 100, 120, 200 nmol) were prepared with potassium dihydrogen phosphate buffer, and the absorbance of the standard solution at 415 nm was measured with an enzyme marker. The standard curve was fitted. The kinetic curve obtained by measurement was plotted according to the change of time and absorbance, and the slope was obtained by linear fitting in the initial time period. The enzyme activity was calculated by combining the standard curve and the concentration of LCC and its knotted. The reaction kinetics curve of LCC knotted and the corresponding linear precursor catalyzing the hydrolysis of pNP-C2 to p-nitrophenol (horizontal axis: time Time (s), vertical axis: absorbance Abs 415 nm) and the specific activity calculated are shown in FIGS. 1 and 2. Figure 8 and Figure 9 The calculation results show that the specific activity of knotted k-LCC-0 is basically the same as that of the linear precursor, and is slightly lower than that of the linear precursor. The specific activity of the designed knotted LCC is higher than that of the original version k-LCC-0, and is closer to the specific activity of the linear precursor LCC.

[0050] Example Six, Activity Characterization of Phyllochitinase Knotted on Polyethylene Terephthalate Substrate

[0051] The polyethylene terephthalate (PET) raw material is divided into amorphous film and crystalline powder. For the amorphous film, before the experiment, it is cut into a size of 1 cm*1 cm, washed with ddH2O to clean the surface, and used. For the crystalline powder, before the experiment, a certain amount of PET powder is weighed in a 1.5 mL EP tube for standby. All the LCC samples to be tested are diluted to 3 μM with potassium dihydrogen phosphate (100 mM, pH 8) buffer to prepare a working solution. The film is placed in 1 mL of the working solution and reacted in a 65 ℃ shaking metal bath for 24 h. The reaction liquid at different time nodes is heated at 98 ℃ for 20 min to quench the reaction, then diluted by a certain multiple to ensure that the sample concentration does not exceed the UPLC detection limit, and then centrifuged at 4000 rpm for 10 min. The supernatant is taken for UPLC analysis. The sample amount of UPLC is 2 μL, and the UPLC analysis method is established by using mono (2-hydroxyethyl) terephthalate (MHET) and terephthalic acid (TPA) standard samples to identify the corresponding relationship between the 235 nm ultraviolet absorption peak and the elution time. The mobile phase is selected as a mixture of 0.05% trifluoroacetic acid aqueous solution and acetonitrile, the chromatographic column type is ZORBAX SB-C18, the column temperature is 25 ℃, and the flow rate is 0.5 mL / min. The standard solutions of MHET and TPA samples with different concentrations (0.1, 0.2, 0.5, 1.0, 2.0, 5.0 mM) are prepared respectively, and the standard curve is drawn according to the relationship between the peak area integral results of the ultraviolet absorption peak at the specified wavelength and the concentration. The product concentration changes with time Kinetics curve is obtained by converting the peak area of different product peaks measured by hydrolysis reaction. The kinetics curves of LCC knots and corresponding linear control catalyzing PET degradation and the concentration ratio of TPA and MHET in the system after 24 h of reaction are shown in FIG. 1. Figure 10 The kinetics results show that the optimized knot has very good catalytic activity, and several different LCC knots are almost identical to the linear control in the generation of TPA and MHET rate and yield.

[0052] The innovation of the present application lies in the construction of the leaf branch compost cutinase knot structure, the expansion of the linear backbone structure of the linear leaf branch compost cutinase, and the design of the conformation and sequence of the leaf branch compost cutinase connecting peptide segment. Without relying on the directed evolution strategy, high-activity and high-stability leaf branch compost cutinase knots are successfully synthesized. The main points are as follows:

[0053] 1. The present application breaks through the cognitive paradigm of protein linear backbone, and uses protein topology engineering in the design and modification of natural linear leaf branch compost cutinase to create a new type of leaf branch compost cutinase knot.

[0054] 2. Current protein design strategies are mostly applied in the case of de novo design, and their feasibility in the design process of knotted proteins has not been tested. The present application develops a reasonable process strategy and sorts out a set of feasible solutions suitable for improving the soluble expression amount of knotted proteins.

[0055] 3. The synthesis of protein knotted needs to overcome the kinetic barrier in the folding process, and inappropriate connecting peptide segments will lead to the decline of stability and activity. It is uncertain and difficult to solve this problem by relying on evolutionary strategies. The present application adopts the way of combining artificial intelligence assisted sequence design strategy to optimize the sequence of connecting peptide segment and improve the actual performance of knotted protein.

[0056] Table 1, the sequences of linear precursor LCC, LCC knotted and linear control involved in the embodiments of the present application

[0057]

[0058]

[0059]

Claims

1. A leaf litter hemicellulose knottin obtained by adjusting the order of connections between secondary structure segments in a leaf litter hemicellulose and introducing a connecting peptide segment between adjacent segments, characterized in that, The amino acid sequence of the leaf litter cutinase knottin comprises, from N-terminus to C-terminus, sequentially connected fragment two, connecting peptide segment one, fragment one, connecting peptide segment two, and fragment three, wherein the fragment one comprises, sequentially connected, beta-strand 1 and beta-strand 2 of the leaf litter cutinase, the fragment two comprises, sequentially connected, beta-strand 3, alpha-helix A, beta-strand 4, and alpha-helix B of the leaf litter cutinase, and the fragment three comprises, sequentially connected, beta-strand 5, alpha-helix C, beta-strand 6, beta-strand 7, alpha-helix D, beta-strand 8, alpha-helix E, and beta-strand 9 of the leaf litter cutinase.

2. The leaf rachis compost cutinase knotted of claim 1, wherein, The fragment one is a peptide segment with an amino acid sequence as shown in SEQ ID NO: 11 in the Sequence Listing, or a peptide segment with 80% or more homology with SEQ ID NO: 11 and a length of 43-45 aa, wherein the sequence NPYQRGPNPT, GPFSVA, TVSRLSVSGFGGG, and IYYP are kept unchanged; the fragment two is a peptide segment with an amino acid sequence as shown in SEQ ID NO: 12 in the Sequence Listing, or a peptide segment with 80% or more homology with SEQ ID NO: 12 and a length of 58-63 aa, wherein the sequence TFGGIAMSPGYTA, SSLAWLG, RLASHGFVV, VINTNSRFD, and SRASQLSAALNYL are kept unchanged; and the fragment three is a peptide segment with an amino acid sequence as shown in SEQ ID NO: 13 in the Sequence Listing, or a peptide segment with 80% or more homology with SEQ ID NO: 13 and a length of 122-145 aa, wherein the sequence VRARLDANRLAVAGHSMGGGGTLRIA, QNPSLKAAVPLTPW, TDKTF, TSVPVLIVGAE, DTVAPVSQHA, PFYQNLPSTTPKVYVEL, NASHIAPN, NNAAIS, YTISWMK, WVDNDTRY, and QFLC are kept unchanged.

3. The leaf and branch compost cutin enzyme knot as described in claim 1, characterized in that, The connecting peptide segment one and the connecting peptide segment two are peptide segments with 4-15 amino acid residues.

4. The leaf rachis compost cutinase knotted of claim 1, wherein, The connecting peptide segment one is selected from one of TSSGGSGGTGGSS, AGSRGYGGS, and EGARERYSED, and the connecting peptide segment two is selected from one of SGSSA and GGSTLEE.

5. The leaf rachis compost cutinase knotted of claim 4, wherein, The amino acid sequence of the leaf litter cutinase knottin is as shown in SEQ ID NO: 7, 8, 9, or 10 in the Sequence Listing.

6. The leaf prong compost cutinase knottin of claim 1, wherein, An N-terminal recognition sequence ENLYFQG of a tobacco etch virus protease and a C-terminal recognition sequence LPETG of a sortase A are introduced into the N-terminus or the C-terminus of the leaf litter cutinase knottin.

7. The leaf and branch compost cutin enzyme knot as described in claim 1, characterized in that, A histidine tag sequence for purification is introduced into the N-terminus or the C-terminus of the leaf litter cutinase knottin.

8. A nucleotide sequence, comprising, The nucleotide sequence comprises a gene of the leaf litter cutinase knottin according to any one of claims 1-7.

9. An expression vector comprising the nucleotide sequence of claim 8 or a host cell comprising the expression vector.

10. Use of the phyllobranchic compost cutinase knottin of any one of claims 1 to 7 in the biodegradation of polymeric materials.

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