High-activity and high-stability PET hydrolase mutant and application thereof

Abstract

The invention belongs to the technical field of enzyme engineering, and discloses a high-activity and high-stability PET hydrolase mutant and application thereof. The high-activity and high-stability PET hydrolase mutant is a protein A, a protein B or a protein C, wherein the protein A is obtained by mutating the 132 amino acid of wild cutinase TfCut from glutamine Q to lysine K; the protein B is obtained by mutating the 244th and 293rd amino acids into cysteine C on the basis of the protein A; protein C is a fusion protein containing protein A or protein B. The modified enzyme breaks through a trade-off mechanism of enzyme catalytic activity and stability, and the activity and thermal stability of the modified enzyme are greatly improved, so that the industrial application value of the PET hydrolase in the aspect of PET degradation is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of enzyme engineering, and particularly relates to a PET hydrolase mutant with high activity and stability and application thereof. Background Art

[0002] With the widespread use of plastic products around the world, plastic pollution has become an environmental problem that needs to be solved urgently. 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 microorganisms or physical and chemical methods in nature, resulting in 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 (biorecycling) of post-consumer plastics 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 and has no lid structure. 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 will become rubbery at temperatures above the glass transition temperature (Tg), the overall conformation becomes soft. At this temperature, the polymer chains become flexible and accessible to the active sites of thermophilic hydrolases, which helps the degradation of PET. The high Tg value of PET makes the thermal stability of PET degrading enzymes essential for the effective depolymerization of PET. In recent years, some thermophilic enzymes, including LCC (leaf branch compost cutinase from metagenomic sources), HiC (from Humicola insolens), TfCut (from thermophilic ascomycetes), BhrPETase (from bacteria HR 29) and other PET hydrolases, have attracted much attention because they have been proven to meet the thermal stability requirements above 60°C, but their thermal stability and catalytic activity are still insufficient to meet industrial needs. 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 the application of the above-mentioned PET hydrolase mutant with high activity and high stability.

[0007] The purpose of the present invention is achieved by the following technical solution: A highly active and highly 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 registration number: AAZ54921.1, with a total of 301 amino acids, including a signal peptide and an active protein. Positions 1-40 are signal peptides, 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 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 PET hydrolase mutant with high activity and high stability.

[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 contains 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, compares the differences in PET capture clamp ring dynamics between different PET hydrolases, determines the key residues regulating the flexibility of the ring, and designs mutant TfCut 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 showed significant improvement in both activity and stability. In the PET degradation reaction at 80°C for 18h, its reaction efficiency was 78 times higher than that of the wild type, and the performance improvement effect was significant. The present invention uses simulation calculations to adjust the conformational dynamics of the PET capture clamp of the PET hydrolase as an entry point to conduct a reasonable mutation transformation design, effectively overcome the enzyme activity-stability trade-off mechanism, and design a highly active and highly stable PET hydrolase mutant, showing better industrial application potential. 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 For TfCut and TfCut Q132K Comparison of the yields of PET hydrolysis products at different temperatures among the 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 is a comparison of the yield of PET hydrolysis products by wild-type and mutant TfCut at 60°C.

[0030] Figure 5 This is a comparison of the yields of PET hydrolysis products of wild-type and mutant TfCut under different high temperatures and different reaction times. DETAILED DESCRIPTION

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

[0032] If no specific experimental conditions are specified in the following embodiments, conventional experimental conditions or experimental conditions recommended by the reagent company are generally followed. The materials and reagents used, unless otherwise specified, are all reagents and materials obtained from commercial sources.

[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 Biosai Biotechnology Co., Ltd. Guangzhou Branch; PET powder was purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd., catalog number 429252, and the powder was crushed and screened with a particle size between 80-100 mesh by a pulverizer; all other chemicals were purchased from reagent companies such as McLean / 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 in Figure 2), we identified the key residues regulating loop flexibility and designed the mutant TfCut Q132K .

[0036] First, the TfCut gene without a signal peptide was obtained by total gene synthesis, and the nucleotide sequence was 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 primer 1 / primer 2 was added for polymerase chain reaction (PCR). The PCR reaction program was: pre-denaturation at 98°C for 3 min; 98°C for 10 s, 69.5°C for 5 s, 72°C for 2 min, 28 cycles; 72°C for 5 min; 4°C storage. 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 place in a static 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 LB liquid culture medium containing ampicillin (100μg / mL) for expansion and culture, extract the plasmid for use, and the mutant plasmid pET21b-TfCut is obtained. Q132K .

[0038] pET21b-TfCut Q132K As templates, primers 3 / primer 4 and primers 5 / primer 6 were used for PCR respectively. After the amplified products were purified, fragments F1 and V1 were obtained. 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 1.5447W 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 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 for 20 min using an ultrasonic disruptor at 350 W power, on for 3 s, off for 5 s. The supernatant and precipitate were separated by centrifugation at 4 °C and 12,000 rpm for 30 min. The supernatant was filtered through a 0.45 μm filter membrane and added to a pre-equilibrated HISTrapHP nickel column, followed by washing with a wash buffer (20 mmol L -1 Tris-HCl, 300 mmol L -1 NaCl and 75mmol 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 300mmol L -1 Imidazole, pH 8.0) 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 The concentrated target protein solution was diluted with 4% NaCl and centrifuged again. The above steps were repeated several times to remove imidazole. The purity of the target protein was analyzed by SDS-PAGE, and the protein concentration was determined by BCA method. The obtained pure enzyme solution was stored at 4°C for 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 , pH 9.0), wherein the final concentration of the enzyme is 10 μg / mL unless otherwise specified, and the reaction is shaken at 200 rpm. The reaction is terminated by heating at 100° C. for 10 minutes. After centrifugation at 12000 rpm for 10 minutes, the supernatant of the sample is filtered through a 0.22 μm filter using a 1 mL syringe to prepare the sample for 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 product 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 experimental results of PET hydrolysis reaction show that:

[0050] (1) Under the condition of 18 h reaction time, 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 are analyzed by molecular dynamics simulation (such as Figure 3 ), through TfCut and TfCut Q132K Comparison of the RMSF values ​​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 show that compared with the wild-type enzyme, TfCut Q132K The mutant had a 2.3-fold increased hydrolysis activity on PET. Q132K / D244C / E293CThe hydrolysis activity of the mutant 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 did not increase its hydrolysis activity on PET as much 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, and showed hydrolysis performance far higher than that of the wild-type enzyme under high temperature reaction conditions such as 60°C, 70°C and 80°C. Among them, in the PET degradation reaction at 70°C and 18h, its reaction efficiency was 37 times higher than that of the wild-type, and in the PET degradation reaction at 80°C and 18h, its reaction efficiency was 78 times higher than that of the wild-type. It can be seen that the present invention greatly improves the PET hydrolysis performance of TfCut, and the high activity and high stability of the mutant are more in line with 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 equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A highly active and highly stable PET hydrolase mutant, characterized in that: The highly active and highly stable PET hydrolase mutant is protein A, protein B or protein C: Protein A is a wild-type cutinase TfCut in which the 132nd amino acid is mutated from glutamine Q to lysine K; Protein B is prepared by mutating the 132nd amino acid of the wild-type cutinase TfCut from glutamine Q to lysine K, the 244th amino acid from aspartic acid D to cysteine ​​C, and the 293rd amino acid from glutamic acid E to cysteine ​​C; Protein C is a fusion protein containing either Protein A or Protein B; The amino acid sequence of the wild-type cutinase TfCut active protein is shown in SEQ ID NO.

1.

2. The highly active and highly stable PET hydrolase mutant according to claim 1, characterized in that: The amino acid sequence of the protein A is shown in SEQ ID NO.2; The amino acid sequence of protein B is shown in SEQ ID NO.3; The fusion protein also contains a fusion peptide that is beneficial to expression and / or a tag for separation and purification.

3. The highly active and highly stable 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 separation and purification tag is a histidine tag.

4. A nucleic acid molecule, characterized in that: A PET hydrolase mutant encoding the high-activity and high-stability PET hydrolase mutant according to any one of claims 1 to 3.

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

6.

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

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

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

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

10. Use of the highly active and highly stable PET hydrolase mutant according to any one of claims 1 to 3, the recombinant vector according to claim 6 or 7, or the recombinant engineered bacteria according to claim 8 or 9 in PET degradation.

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