A highly active and stable PET hydrolase mutant and its application

Abstract

The present invention belongs to the field of enzyme engineering technology and discloses a highly active and stable PET hydrolase mutant and its application. The highly active and stable PET hydrolase mutant is Protein A, Protein B, or Protein C: Protein A is a wild-type cutinase TfCut in which amino acid position 132 is mutated from glutamine Q to lysine K; Protein B is a protein A modified by further mutating amino acids 244 and 293 to cysteine ​​C; and Protein C is a fusion protein containing Protein A or Protein B. The modified enzyme breaks the trade-off between catalytic activity and stability, significantly improving both its activity and thermal stability, thereby enhancing the industrial application value of PET hydrolase in PET degradation.

Description

Technical Field

[0001] The invention belongs to the technical field of enzyme engineering, and particularly relates to a highly active and highly stable PET hydrolase mutant and application thereof. Background Art

[0002] With the widespread use of plastic products around the world, plastic pollution has become an urgent environmental problem that needs to be addressed. Polyethylene terephthalate (PET) is a synthetic polymer in the polyester family. PET is known for its excellent mechanical properties, chemical resistance, and transparency. However, its high hydrophobicity and high molecular weight also make PET highly resistant to degradation by wild-type microorganisms or physical and chemical methods in nature, leading to the accumulation of plastic waste in the environment. Traditional plastic degradation relies on physical and chemical methods, but these methods are costly, energy-intensive, and may produce secondary pollution. In recent years, biodegradation technology has gradually attracted people's attention. Enzymatic hydrolysis of post-consumer plastics (biorecycling) is an emerging strategy for closed-loop recycling of PET. Among them, PET hydrolase has gradually become a research hotspot due to its potential in decomposing plastics.

[0003] PET hydrolase belongs to the α / β hydrolase superfamily and has a catalytic triad composed of serine, histidine, and aspartic acid residues. The substrate binding pocket of PET hydrolase is large, without a lid structure, and the active site is directly exposed to the solvent, which is conducive to the binding of PET to the active center. PET hydrolase mainly degrades plastic polymers by hydrolyzing ester bonds in plastics. It decomposes PET polymers into different products, such as terephthalic acid (TPA), ethylene glycol (EG), bis(hydroxyethyl) terephthalate (BHET), and mono(2-hydroxyethyl) terephthalate (MHET).

[0004] Considering that polymer molecules transform into a rubbery state and their overall conformation becomes soft at temperatures above the glass transition temperature (Tg), at which point the polymer chains become flexible and accessible to the active sites of thermophilic hydrolases, facilitating PET degradation, the high Tg of PET makes the thermal stability of PET-degrading enzymes crucial for effective PET depolymerization. In recent years, several thermophilic enzymes, including LCC (metagenomic-derived leaf compost cutinase), HiC (from Humicola insolens), TfCut (from Thermoascus ascomycetes), and BhrPETase (from the bacterium HR 29), have garnered significant attention due to their demonstrated thermal stability above 60°C. However, their thermal stability and catalytic activity remain insufficient to meet industrial requirements. Summary of the Invention

[0005] The primary purpose of the present invention is to overcome the defects and shortcomings of the prior art and provide a PET hydrolase mutant with high activity and high stability.

[0006] Another object of the present invention is to provide an application of the above-mentioned highly active and highly stable PET hydrolase mutant.

[0007] The purpose of the present invention is achieved through the following technical solution: A highly active and stable PET hydrolase mutant is protein A, protein B or protein C:

[0008] Protein A is a wild-type cutinase TfCut in which the 132nd amino acid is mutated from glutamine Q to lysine K (abbreviated as TfCut Q132K );

[0009] Protein B is a wild-type cutinase TfCut in which the 132nd amino acid is mutated from glutamine Q to lysine K, the 244th amino acid is mutated from aspartic acid D to cysteine ​​C, and the 293rd amino acid is mutated from glutamic acid E to cysteine ​​C (abbreviated as TfCut Q132K / D244C / E293C );

[0010] Protein C is a fusion protein containing either Protein A or Protein B.

[0011] The amino acid sequence of the wild-type cutinase TfCut is shown in gene accession number: AAZ54921.1, which has a total of 301 amino acids, including a signal peptide and an active protein. Positions 1-40 are the signal peptide, the active protein is shown in SEQ ID NO.1, and the signal peptide is shown in SEQ ID NO.7.

[0012] The amino acid sequence of the protein A is preferably as shown in SEQ ID NO.2.

[0013] The amino acid sequence of the protein B is preferably as shown in SEQ ID NO.3.

[0014] The fusion protein preferably further contains a fusion peptide that is beneficial to expression and / or a tag for separation and purification.

[0015] The fusion peptide that is beneficial to expression is preferably a signal peptide suitable for the host cell.

[0016] The separation and purification tag is preferably a histidine tag.

[0017] A nucleic acid molecule encodes the above-mentioned highly active and highly stable PET hydrolase mutant.

[0018] The nucleotide sequence of the above nucleic acid molecule is preferably as shown in SEQ ID NO.4 or SEQ ID NO.5

[0019] A recombinant vector containing the above nucleic acid molecule. The recombinant vector includes a vector capable of replicating the above nucleic acid molecule and a vector capable of expressing the above nucleic acid molecule.

[0020] The vector framework of the recombinant vector is preferably a pET series vector; more preferably pET-21b(+).

[0021] A recombinant engineering bacterium contains the above-mentioned recombinant vector.

[0022] The starting strain of the recombinant engineered bacteria is preferably Escherichia coli; more preferably Escherichia coli BL21 (DE3).

[0023] Application of the highly active and highly stable PET hydrolase mutant, the recombinant vector, and the recombinant engineered bacteria in PET degradation.

[0024] Compared with the existing technology, the present invention has the following advantages and beneficial effects:

[0025] The present invention uses thermophilic PET hydrolase TfCut (gene accession number: AAZ54921.1) as the research object. By comparing the differences in the PET capture clamp ring dynamics between different PET hydrolases, the key residues regulating the flexibility of the ring are determined, and mutant TfCut is designed. Q132K Compared with the wild type, TfCut Q132K The PET degradation activity was continuously enhanced in the temperature range of 40°C to 80°C, and the mutant TfCut was obtained by incorporating an additional disulfide bond (D244C / E293C). Q132K / D244C / E293C This variant exhibits significant improvements in both activity and stability. In a PET degradation reaction at 80°C for 18 hours, its reaction efficiency is 78-fold higher than that of the wild-type, demonstrating significant performance improvements. Through simulations and calculations, the present invention utilizes the conformational dynamics of the PET capture clamp of the PET hydrolase as a starting point for rational mutational engineering. This effectively overcomes the enzyme's activity-stability trade-off, resulting in the design of a highly active and stable PET hydrolase mutant with enhanced potential for industrial application. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a conformational comparison analysis of the flexible ring of the PET capture clamp of different PET hydrolases.

[0027] Figure 2 TfCut and TfCut Q132K Comparison of PET hydrolysis product yields at different temperatures among variants.

[0028] Figure 3 For TfCut and TfCut Q132K Comparison of RMSF at 300 K; the two loops containing residues 99 to 104 and residues 125 to 133 are highlighted.

[0029] Figure 4 This figure compares the yield of PET hydrolysis products by wild-type and mutant TfCut at 60°C.

[0030] Figure 5 This is a comparison of the PET hydrolysis product yields of wild-type and mutant TfCut under different high temperature and reaction time conditions. DETAILED DESCRIPTION

[0031] The present invention will be described in further detail below with reference to the embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0032] If specific experimental conditions are not specified in the following embodiments, conventional experimental conditions or those recommended by the reagent company will generally be used. Materials and reagents used were commercially available unless otherwise specified.

[0033] Materials and reagents used in the following examples: TfCut gene was synthesized by Sangon Biotech (Shanghai) Co., Ltd.; empty plasmid pET-21b (+) was stored in the applicant's laboratory and can be purchased from Novagen; Escherichia coli BL21 (DE3) competent cells were purchased from Sangon Biotech (Shanghai) Co., Ltd.; plasmid extraction kit was purchased from Sangon Biotech (Shanghai) Co., Ltd.; various enzymes involved in molecular biology experiments were purchased from Shanghai Baisai Biotechnology Co., Ltd. Guangzhou Branch; PET powder was purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd., catalog number 429252, and was crushed and screened with a powder with a particle size between 80 and 100 mesh using a grinder; all other chemicals were purchased from reagent companies such as Maclean / Aladdin.

[0034] Example 1: Construction of wild-type PET hydrolase recombinant plasmid and its mutant recombinant plasmid

[0035] The present invention uses thermophilic PET hydrolase TfCut (gene accession number: AAZ54921.1) as the research object, and compares the differences in PET capture clamp ring dynamics between different PET hydrolases (such as Figure 1 As shown), we determined the key residues regulating loop flexibility and designed mutant TfCut Q132K .

[0036] First, the TfCut gene without a signal peptide was obtained by whole gene synthesis, the nucleotide sequence of which is shown in SEQ ID NO.6; and the gene was constructed into the pET-21b(+) vector using NdeI and XhoI restriction enzymes to obtain the recombinant plasmid pET21b-TfCut.

[0037] Using site-directed mutagenesis technology, the recombinant plasmid pET21b-TfCut was used as a template and primers 1 and 2 were added for polymerase chain reaction (PCR). The PCR reaction procedure was as follows: pre-denaturation at 98°C for 3 minutes; 28 cycles of 98°C for 10 seconds, 69.5°C for 5 seconds, and 72°C for 2 minutes; 72°C for 5 minutes; and storage at 4°C. The amplified PCR product was purified using a PCR product purification kit to obtain the mutant plasmid pET21b-TfCut. Q132K Linearized fragment. Add 100ng of linearized fragment to 50μL E. coli DH5α competent cells, place in an ice bath for 30min, heat shock in a 42℃ water bath for 1min, and then keep in an ice bath for 3min. Then, evenly spread it on LB agar medium containing ampicillin (100μg / mL) and culture at 37℃ for 10-12h. Pick a single colony for DNA sequencing verification, inoculate the colony with the correct sequencing result into 2mL of LB liquid medium containing ampicillin (100μg / mL) for expansion and culture, extract the plasmid for use, and thus obtain the mutant plasmid pET21b-TfCut Q132K .

[0038] pET21b-TfCut Q132K As templates, primers 3 / primer 4 and primers 5 / primer 6 were used for PCR respectively. The amplified products were purified to obtain fragments F1 and V1. The two fragments were assembled by Gibson. The subsequent experimental steps of the assembled products were the same as those of the linearized fragments above. In this way, the mutant plasmid pET21b-TfCut was obtained. Q132K / D244C / E293C The PCR reaction system is shown in Table 1 below, and the primer sequences and their usage are shown in Table 2.

[0039] Table 1

[0040]

[0041] Table 2

[0042]

[0043] Example 2: Preparation of PET hydrolase mutants and wild-type PET hydrolase

[0044] The recombinant plasmid was transformed into the overexpression strain Escherichia coli BL21 (DE3) and evenly spread on a plate containing 100 μg mL -1 After the plate was incubated at 37°C for 12 h, a single colony was selected and inoculated into 10-20 mL of LB agar containing 100 μg mL -1The LB liquid medium containing ampicillin was cultured at 37°C and 210 rpm for 12-16 hours to obtain seed solution. The seed solution was then inoculated into 400 mL of 100 μg mL -1 The cells were cultured in LB medium containing ampicillin at 37°C and 210 rpm until the OD 600 To induce protein expression, isopropyl-β-D-1-thiogalactopyranoside (IPTG) was added at 0.4 mmol L -1 The final concentration of 100 μg / mL was added to the culture medium, and then cultured at 16°C and 180 rpm for 20 h.

[0045] After the induction of expression, the cells were harvested by centrifugation at 8000 rpm for 5 min and washed twice with physiological saline. The cell pellet was resuspended in loading buffer (20 mmol L -1 Tris-HCl, 300 mmol L -1 NaCl and 20 mmol L -1 Imidazole, pH 8.0) and the cells were disrupted using an ultrasonic disruptor at 350W power, on for 3s and off for 5s for 20min. The supernatant and precipitate were separated by centrifugation at 4°C and 12000rpm for 30min. The supernatant was filtered through a 0.45μm filter membrane and added to a pre-equilibrated HISTrapHP nickel column. The column was then washed with a washing buffer (20mmol L -1 Tris-HCl, 300 mmol L -1 NaCl and 75 mmol L -1 Imidazole, pH 8.0) was used to elute weakly bound proteins until no further proteins were eluted from the column. Finally, elution buffer (20 mmol L -1 Tris-HCl, 300 mmol L -1 NaCl and 300 mmol L -1 Imidazole, pH 8.0) was used to elute the target protein. The collected target protein solution was concentrated using an Ultra-15 ultrafiltration tube (molecular cutoff 10 kDa) at 4°C and 3200 × g, and then filtered with a buffer (20 mmol L -1 Tris-HCl, 300 mmol L -1 Dilute the concentrated target protein solution with 1% NaCl and centrifuge again. Repeat this process several times to remove the imidazole. Analyze the purity of the target protein by SDS-PAGE and determine the protein concentration using the BCA assay. Store the resulting pure enzyme solution at 4°C until use.

[0046] Example 3: Comparison of relative activities of wild-type and mutant PET hydrolases

[0047] PET hydrolysis reaction conditions: The enzyme purified in Example 2 was added to 1 mL of glycine-NaOH (50 mmol L -1 The cells were incubated with 4% paraformaldehyde (pH 9.0) at a final enzyme concentration of 10 μg / mL unless otherwise noted, and shaken at 200 rpm. The reaction was terminated by heating at 100°C for 10 minutes. After centrifugation at 12,000 rpm for 10 minutes, the supernatant was filtered through a 0.22 μm filter using a 1 mL syringe to prepare the sample for later use.

[0048] Product detection method: The reaction product mixture is terephthalic acid TPA or its esters (monohydroxyethyl terephthalate MHET, bis(hydroxyethyl terephthalate) BHET, etc.), which has strong ultraviolet absorption at 240nm. The average molar extinction coefficient of these product combinations is λ = 17000 Lmol -1 cm -1 Based on this molar extinction coefficient, the sample absorbance value can be converted into the total amount of soluble PET hydrolysis products according to the Lambert-Beer law. Therefore, the method for detecting the hydrolysis reaction products is to measure the absorbance of the sample after the hydrolysis reaction on a UV-visible spectrophotometer (Shimadzu, Japan). If necessary, the sample can be diluted in glycine-NaOH (50 mmol L -1 , pH 9.0) solution before absorbance detection.

[0049] The results of PET hydrolysis reaction experiments show that:

[0050] (1) Under the reaction time of 18 h, the mutant TfCut Q132K The PET degradation activity in TfCut was continuously enhanced in the temperature range of 40°C to 80°C (e.g. Figure 2 ), and the reasons were analyzed by molecular dynamics simulation (such as Figure 3 ), through TfCut and TfCut Q132K Comparison of the RMSF values ​​between them revealed that the flexibility of the ring containing Tyr100 was increased, confirming the initial mutant design concept. The design of certain flexible ring sites of the PET capture clamp can enhance the activity of PET hydrolase and effectively overcome the enzyme activity-stability trade-off mechanism.

[0051] (2) Figure 4 The results of PET hydrolysis at 60℃ with an enzyme concentration of 10μg / mL are shown. It can be seen that compared with the wild-type enzyme, TfCut Q132K The hydrolysis activity of the mutant towards PET was increased by 2.3 times. Q132K / D244C / E293CThe mutant's hydrolysis activity towards PET was further increased to 2.7 times. Figure 5 The results showed that when the temperature was increased to 70℃ and 80℃, TfCut Q132K The mutant's hydrolysis activity on PET was not as improved as at 60°C, which may be due to its insufficient stability; TfCut was obtained by incorporating an additional disulfide bond (D244C / E293C). Q132K / D244C / E293C The variant showed significant improvements in both activity and stability, exhibiting significantly higher hydrolysis performance than the wild-type enzyme under high-temperature reaction conditions such as 60°C, 70°C, and 80°C. In the PET degradation reaction at 70°C for 18 hours, its reaction efficiency was 37-fold higher than that of the wild-type, and in the PET degradation reaction at 80°C for 18 hours, its reaction efficiency was 78-fold higher than that of the wild-type. This shows that the present invention significantly improves the PET hydrolysis performance of TfCut, and the high activity and high stability of the mutant better meet actual industrial needs.

[0052] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A PET hydrolase mutant, characterized in that: The PET hydrolase mutant is protein A, protein B or protein C: The amino acid sequence of protein A is shown in SEQ ID NO. 2; The amino acid sequence of protein B is shown in SEQ ID NO. 3; Protein C is a fusion protein composed of a fusion peptide that is advantageous for expression and / or a tag for separation and purification and protein A, or a fusion protein composed of a fusion peptide that is advantageous for expression and / or a tag for separation and purification and protein B.

2. The PET hydrolase mutant according to claim 1, characterized in that: The fusion peptide that is conducive to expression is a signal peptide suitable for the host cell; The tag for separation and purification is a histidine tag.

3. A nucleic acid molecule, characterized in that: Encoding the PET hydrolase mutant according to claim 1 or 2.

4. The nucleic acid molecule according to claim 3, characterized in that: The nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO.5 or SEQ ID NO.

6.

5. A recombinant vector, characterized in that: Containing the nucleic acid molecule according to claim 3 or 4.

6. The recombinant vector according to claim 5, characterized in that: The vector framework of the recombinant vector is a pET series vector.

7. A recombinant engineered bacterium, characterized in that: Contains the recombinant vector according to claim 5 or 6.

8. The recombinant engineered bacterium according to claim 7, characterized in that: The starting strain of the recombinant engineering bacteria is Escherichia coli.

9. Use of the PET hydrolase mutant according to any one of claims 1 to 2, the recombinant vector according to any one of claims 5 to 6, or the recombinant engineered bacteria according to any one of claims 7 to 8 in PET degradation.

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