Ds petase 01 plastic-degrading enzyme mutant and applications thereof

Abstract

This invention discloses a mutant of the dsPETase01 plastic degrading enzyme and its applications, relating to the field of bioengineering technology. This invention designs and modifies the amino acid sequence of the wild-type plastic degrading enzyme (dsPETase01), providing 8 single mutation sites or 19 combined mutation sites for dsPETase01. The obtained dsPETase01 mutants exhibit approximately 4-21°C higher thermal stability and nearly 1.2-3 times higher activity compared to wild-type dsPETase01. Considering the significant improvements in thermal stability and activity from the mutation sites, this invention also provides 4 preferred mutant proteins that, compared to wild-type dsPETase01, exhibit higher yield, activity, and thermal stability, have broader application conditions, and are more suitable for degrading PET plastics, thus facilitating large-scale production and industrial applications.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, specifically to a dsPETase01 plastic degrading enzyme mutant and its applications. Background Technology

[0002] Polyethylene phthalate (PET) is the most important type of thermoplastic polyester, commonly known as polyester resin. It is white or light yellow in color with a smooth and glossy surface. It possesses excellent physical and mechanical properties over a wide temperature range, with a service temperature up to 120℃. It also exhibits excellent electrical insulation properties, maintaining good electrical performance even at high temperatures and frequencies. Therefore, it is widely used in packaging, electronics, medical and health, construction, and automotive industries, accounting for one-sixth of all plastics. However, with the increasing use of PET, the resulting pollution problem is significant. These plastics are difficult to degrade in the natural environment, leading to the accumulation of large amounts of plastic waste and causing long-term and profound impacts on the ecological environment and human health.

[0003] More and more researchers are discovering the potential of certain microorganisms and enzymes in the degradation of plastics. Enzymes capable of degrading PET are relatively recent discoveries. The most classic PET-degrading enzyme, LCC, was discovered in the cuticle of leaf and branch compost, and it has already achieved some industrial applications. Other PET-degrading enzymes, such as isPETase (isolated from food plastics in 2016) and PHL7 (isolated in 2022), also have the potential to decompose PET. However, due to their relatively low thermal stability, industrial-scale degradation of PET is difficult to achieve. Summary of the Invention

[0004] The purpose of this invention is to provide a dsPETase01 plastic degrading enzyme mutant and its application.

[0005] The present invention achieves the above objectives through the following technical solutions:

[0006] As a first aspect of the present invention, a dsPETase01 plastic degrading enzyme mutant is provided, which is obtained by introducing disulfide bonds into the sequence of wild-type dsPETase01 plastic degrading enzyme as shown in SEQ ID NO.1 through single-point mutation or combination mutation, or by introducing disulfide bonds into the single-point mutated sequence or combination mutated sequence.

[0007] The single mutation site includes at least one of Q171L, G191E, G191D, G191R, G191Q, S215P, A242V or F249A;

[0008] The combined mutation sites include at least (Q171L, G191E), (K142R, Q171L), (E2P, Q171L), (Q171L, S215P), (Q171L, A242V), (K142R, G191E), (E2P, G191E), (G191E, A242V), (K142R, S215P), (K142R, A242V), (E2P, A242V), (S215P, A242V), (E2P, K142R, Q171L). One of the following combinations: (G191E), (K142R, Q171L, G191E), (E2P, K142R, Q171L, G191E, A242V), (E2P, K142R, Q171L, G191E, S215P), (E2P, K142R, Q171L, G191E, S215P, A242V), (Q171L, G191E, S215P, Q171L, S215P, K142R), or (Q171L, G191E, S215P, A242V).

[0009] As a further optimization of the present invention, the mutation sites of the dsPETase01 plastic degrading enzyme mutant are (Q171L, G191E) or (Q171L, G191E, S215P) or (Q171L, G191E, S215P, A242V), and the amino acid sequence of the dsPETase01 plastic degrading enzyme mutant obtained by the above mutation is shown in SEQ ID NO.2-4.

[0010] As a further optimization of the present invention, the amino acid sequence of the dsPETase01 plastic degrading enzyme mutant obtained by introducing disulfide bonds into the sequence shown in SEQ ID NO.2 is shown in SEQ ID NO.5.

[0011] As a second aspect of the invention, a polynucleotide is also provided, said polynucleotide encoding a dsPETase01 plastic degrading enzyme mutant as described above.

[0012] As a further optimization of the present invention, the polynucleotide sequences encoding the amino acid sequences of the dsPETase01 plastic degrading enzyme mutants as shown in SEQ ID NO.2-5 are shown in SEQ ID NO.6-9, respectively.

[0013] As a third aspect of the present invention, a recombinant plasmid is also provided, wherein the recombinant plasmid is an expression vector capable of correspondingly translating and expressing the dsPETase01 plastic degrading enzyme mutant as described above, and the expression vector is the pET-28a vector.

[0014] As a fourth aspect of the invention, the application of the dsPETase01 plastic degrading enzyme mutant as described above in the degradation or recycling of PET plastics is also provided.

[0015] As a further optimization of the present invention, the application is to decompose PET plastic using the dsPETase01 plastic degrading enzyme mutant and generate terephthalic acid (TPA), the main component of PET plastic, during the decomposition process.

[0016] The present invention has the following beneficial effects:

[0017] This invention uses the wild-type dsPETase01 plastic degrading enzyme from deep sea (Chen, J., Jia, Y., Sun, Y. et al. Global marine microbial diversity and its potential in bioprospecting. Nature 633, 371–379 (2024).) as the research object, and designs and modifies its amino acid sequence to provide a series of single-point mutations and combination mutations targeting the wild-type dsPETase01 plastic degrading enzyme. These mutant proteins have improved thermostability by approximately 4–21 °C and activity by nearly 1.2–3 times compared to the wild-type dsPETase01 plastic degrading enzyme. The dsPETase01 plastic degrading enzyme mutants have higher yield, activity, and thermostability than the wild-type dsPETase01 plastic degrading enzyme, making them more suitable for industrial degradation of plastics and beneficial for large-scale production and industrial applications. Attached Figure Description

[0018] Figure 1 The image shows the modified pET-28a vector.

[0019] Figure 2A , Figure 2B , Figure 2C The results of a small-scale purification of the dsPETase01 single mutant protein;

[0020] Figure 3A , Figure 3B The results of a small-scale purification of the dsPETase01 double mutant protein;

[0021] Figure 4 The results of a small-scale purification of the dsPETase01 combinatorial mutant protein;

[0022] Figure 5 The chemical structural formulas of PET and BHET-OH are shown below.

[0023] Figure 6A , Figure 6BThe results show the activity assay results for dsPETase01 single mutant protein, dsPETase01 double mutant protein, and dsPETase01 combined mutant protein.

[0024] Figure 7 The results of affinity chromatography purification of the high-quality mutant protein dsPETase01;

[0025] Figure 8A , Figure 8B , Figure 8C The results of quality testing for the dsPETase01 high-quality mutant protein.

[0026] Figure 9 The results show the activity identification of the high-quality mutant protein dsPETase01. Detailed Implementation

[0027] The present application will now be described in further detail with reference to the accompanying drawings. It should be noted that the following specific embodiments are only used to further illustrate the present application and should not be construed as limiting the scope of protection of the present application. Those skilled in the art can make some non-essential improvements and adjustments to the present application based on the above application content.

[0028] 1. Materials and Reagents

[0029] Unless otherwise specified, all methods used in this invention are conventional methods known to those skilled in the art. Where specific conditions are not specified, they shall be performed according to conventional conditions or conditions recommended by the manufacturer. Where the manufacturers of reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0030] 2. Method

[0031] 2.1 Construction of the dsPETase01 mutant plasmid

[0032] The gene sequences of wild-type dsPETase01 and its mutants were obtained through gene synthesis. The protein sequence of wild-type dsPETase01 is shown in SEQ ID NO.1. All mutants were constructed using molecular cloning methods based on corresponding mutant primers designed from the wild-type mutants. These mutants included 27 single-point mutations: D4P, Q171L, Q109Y, G191E, G191D, H44Y, G191R, G191Q, T143A, K142R, E2P, F33S, S13A, E148R, E6L, E6V, S215P, Q259E, T154A, E67Q, A242V, S26A, F249A, K160R, S254A, E139Y, and G191A.

[0033] Fifteen mutant proteins were identified, including double mutations (Q171L, G191E), (K142R, Q171L), (E2P, Q171L), (Q171L, S215P), (Q171L, A242V), (K142R, G191E), (E2P, G191E), (G191E, S215P), (G191E, A242V), (E2P, K142R), (K142R, S215P), (K142R, A242V), (E2P, S215P), (E2P, A242V), and (S215P, A242V).

[0034] Combinatorial mutations: (E2P, K142R, Q171L, G191E), (K142R, Q171L, G191E), (E2P, K142R, Q171L, G191E, A242V), (E2P, K142R, Q171L, G191E, S215P), (E2P, K142R, Q171L, G191E, S215P, A242V), (Q171L, G191E, S215P), (Q171L, S215P, K142R), (Q171L, G191E, S215P, A242V), a total of 8 combined mutations.

[0035] Wild-type dsPETase01 and its mutant proteins were constructed into the modified pET-28a vector (GenScript). This vector has an 8His-strepII-TEV-GG tag sequence fused to the T7 promoter. The tag sequence is shown in SEQ ID NO. 10 (where 8His and strep II are tag sequences used for affinity purification, "TEV" is the TEV protease cleavage site used for tag removal during subsequent purification, and "GG" is the tag sequence). The gene sequences of the constructed recombinant proteins were verified to be correct by the sequencing company. The vector map is shown below. Figure 1 .

[0036] 2.2. Small-scale expression and purification of dsPETase01 mutant protein

[0037] Using standard molecular biology techniques, the constructed dsPETase01 mutant plasmid was transformed into BL21(DE3) *E. coli* competent cells in a clean bench and cultured overnight at 37°C. Single colonies from the overnight culture were picked and transferred to 5 ml of LB broth and incubated at 37°C until the bacterial culture showed an OD value. 600When the pH is 0.6-0.8, a small amount of bacterial culture is fixed with loading buffer, and a small amount of bacterial culture is added to glycerol and frozen to -80℃. The remaining bacterial culture is added to 0.5mM IPTG and induced at 15℃ for 16 hours. After induction, the bacterial cells are collected and the induced bacterial culture is analyzed by SDS-PAGE. The collected bacterial cells are dissolved in lysis buffer (50mM Tris-HCl (pH 8.0), 500mM NaCl, 5% glycerol), sonicated, centrifuged at 12000 rpm at 4℃ for 10 minutes, and the supernatant is collected. 50 μL of Strep-2 buffer (prepared with 50mM Tris-HCl (pH 8.0), 500mM NaCl, 5% glycerol) is added to the supernatant. XT packing material was incubated at 4°C for 30 minutes. After incubation, the sample was centrifuged at 12000 rpm at 4°C for 10 minutes. 1 mL of buffer was added, and the sample was washed 3 times. Then, 100 μL of elution buffer (50 mM Tris-HCl (pH 8.0), 500 mM NaCl, 5% glycerol, 75 mM biotin) was added, and the sample was centrifuged at 12000 rpm at 4°C for 5 minutes. The eluted sample was collected. A small amount of sample was retained from each step of the process and fixed with loading buffer, and then analyzed by SDS-PAGE.

[0038] The experimental results are shown in Figure 2A , Figure 2B and Figure 2C Of the 27 single-point mutations, all single-point mutant proteins were clearly expressed. Purification of all expressed samples revealed that all mutant proteins could be eluted with high purity.

[0039] The small-scale results for double mutants and combined mutants were similar to those for single mutants; all double mutant and combined mutant proteins could be eluted with high purity. Small-scale results for double mutants are shown below. Figure 3A and 3B The elution results for the combined mutations are shown in [the table]. Figure 4 .

[0040] 2.3 Detection of the thermal stability of dsPETase01 mutant protein

[0041] The thermostability assay for the dsPETase01 mutant protein was performed using nano-differential scanning fluorescence (nanoDSF). The specific procedure is as follows:

[0042] 20 μL of protein at a concentration of 0.5 mg / ml was added to each well of a 384-well experimental plate. After shaking and centrifugation (to avoid sample inhomogeneity or air bubbles during aspiration), the plate was placed on the sampling rack, and the sample was aspirated using a Nano DSF capillary tube, ensuring the entire capillary was filled. The capillary tube was placed in the nano DSF instrument, and the initial temperature was set to 20°C. The temperature was increased at a rate of 2.0°C per minute until it reached 90°C. The instrument would perform the temperature increase and real-time monitoring according to the set parameters. The specific Tm values ​​for all single mutant proteins are shown in Table 1.

[0043] Table 1. Tm values ​​of dsPETase01 single mutant protein

[0044]

[0045] As shown in Table 1, among the 27 single mutant proteins, the Tm values ​​of Q171L, G191E, G191D, G191R, G191Q, S215P, A242V, and F249A increased by 2.02℃-9.27℃, with the mutations at the four G191 positions significantly increasing the Tm value. Therefore, this invention screened out eight mutants—Q171L, G191E, G191D, G191R, G191Q, S215P, A242V, and F249A—that can improve the thermal stability of dsPETase01.

[0046] After combining the single mutants with significantly increased Tm values, 15 double mutant proteins were obtained by expressing them in small amounts according to the single mutant protocol. The Tm values ​​of the double mutant proteins are shown in Table 2.

[0047] Table 2. Tm values ​​of dsPETase01 double mutant protein

[0048]

[0049] As can be seen from Table 2, except for the three double mutations (G191E, S215P), (E2P, K142R) and (E2P, S215P) whose Tm values ​​decreased, the Tm values ​​of the other double mutations were significantly increased, ranging from 3.85℃ to 16.03℃. Therefore, a total of 12 double mutant proteins with improved thermostability were obtained, namely (Q171L, G191E), (K142R, Q171L), (E2P, Q171L), (Q171L, S215P), (Q171L, A242V), (K142R, G191E), (E2P, G191E), (G191E, A242V), (K142R, S215P), (K142R, A242V), (E2P, A242V), and (S215P, A242V). Among them, the (Q171L, G191E) double mutation showed the best effect, with a Tm increase of 16.03℃.

[0050] To obtain mutations with higher stability, single or double mutations that significantly increased Tm values ​​were combined to construct a total of 8 combined mutations. The Tm values ​​of the combined mutant proteins were specifically tested and are shown in Table 3.

[0051] Table 3. Tm values ​​of dsPETase01 combined mutant proteins

[0052]

[0053] As can be seen from Table 3, the Tm values ​​of all combined mutations were 10.27℃-21.17℃ higher than those of the wild type. Among them, the Tm values ​​of the (E2P, K142R, Q171L, G191E, S215P, A242V) and (Q171L, G191E, S215P, A242V) mutants were increased by more than 21℃.

[0054] 2.4 Detection of dsPETase01 mutant protein activity

[0055] PET plastic is actually a type of polyethylene terephthalate, and its structural formula is shown below. Figure 5 On the left, the present invention uses bis(2-hydroxyethyl)-2-hydroxyterephthalate (BHET-OH), the smallest unit of which has the structural formula shown in [see diagram]. Figure 5 Using Biortus as the substrate, an assay protocol for the dsPETase01 enzyme was established. The specific steps are as follows:

[0056] Preparation buffer: 75 mM PBS, pH 8.0; substrate: 2.5 mM BHET-OH (dissolved in DMSO, then 10 mM sodium carbonate solution was added); reaction temperature: 30 °C. Dilute the dsPETase01 mutant protein obtained in step 2.2 to 2 μM using the buffer. Transfer 20 μL of substrate to a 384-well plate with two replicates. Transfer 40 μL of the dsPETase01 mutant protein to be tested to the corresponding wells. Immediately centrifuge and vortex to mix. Collect the fluorescence signal values ​​generated by the reaction using a TECAN F200 microplate reader. Analyze the data using GraphPad Prism9 software to obtain the enzyme activity parameters of the test protease.

[0057] Among them, the activity results of single mutants are as follows Figure 6A As shown. Among the 27 single mutations detected, the E148R and E148K mutants lost about 50% of their activity, while the activity of the other mutants was not significantly different from that of the wild-type protein, generally ranging from 80% to 127%.

[0058] Activity data for double mutations and combined mutations can be found in [link to relevant data]. Figure 6B The activity of the double mutant was generally 10%-15% lower than that of the wild type, but still maintained a high level of activity. The activity of the combined mutant was basically equivalent to that of the wild type, indicating that these mutants with improved thermal stability still maintained good activity.

[0059] 2.5 Expression and purification of high-quality mutant protein dsPETase01

[0060] To further investigate the function of high-quality mutant proteins of dsPETase01, double mutant proteins (Q171L, G191E) with significantly improved Tm values ​​and activities were selected. Their amino acid sequences are shown in SEQ ID NO.2 and their nucleotide sequences are shown in SEQ ID NO.6. Combined mutant proteins (Q171L, G191E, S215P) and (Q171L, G191E, S215P, A242V) are also selected. Their amino acid sequences are shown in SEQ ID NO.3-SEQ ID NO.4 and their nucleotide sequences are shown in SEQ ID NO.7-SEQ ID NO.8.

[0061] Introducing disulfide bonds to improve protein stability is a common method. This invention also provides the protein dsPETase01-dis M1 obtained by modifying the disulfide bonds of wild-type dsPETase01 and introducing the Q171L, G191E double mutation. Its amino acid sequence is shown in SEQ ID NO.5 and its nucleotide sequence is shown in SEQ ID NO.9.

[0062] The protein performance of the above proteins was compared. All plasmids were heterologously expressed in E. coli, and the expressed proteins were purified.

[0063] 2.5.1 Purification of high-quality mutant protein dsPETase01

[0064] (1) Affinity chromatography

[0065] The collected bacterial blocks were weighed and added to the appropriate volume of lysis buffer (50mM Tris-HCl (pH 7.5), 500mM NaCl, 5% glycerol) at a 1:10 ratio. The bacterial cells were homogenized using a high-pressure homogenizer, and the supernatant was collected by high-speed centrifugation at 16,000 rpm. All recombinant dsPETase01 high-quality mutant proteins were tagged with StrepII. The proteins were enriched and purified using a Strep-Tactin XT affinity chromatography column. The specific procedure was as follows: the Strep-Tactin XT affinity chromatography column was first washed and equilibrated with lysis buffer to 10 column volumes. Then, the lysis supernatant was loaded onto the Strep-Tactin XT FF affinity chromatography column and eluted with lysis buffer containing 75mM biotin. The eluted protein was collected for SDS-PAGE analysis, and the protein concentration was determined using Nanodrop to calculate the protein yield.

[0066] Protein purification results are as follows Figure 7 The wild-type yield was 6 mg / L, while the yields of (Q171L, G191E), (Q171L, G191E, S215P), and (Q171L, G191E, S215P, A242V) were 13 mg / L, 15.2 mg / L, and 15.6 mg / L, respectively; the yield of dsPETase01-disM1 was 10 mg / L. All mutants showed yields approximately 2-2.6 times higher than the wild-type.

[0067] (2) Enzyme digestion and reverse affinity chromatography

[0068] To obtain a protein with higher purity, a certain amount of TEV enzyme was added to the sample after affinity chromatography. After overnight digestion at 4°C, the supernatant was further purified using a HisFF chromatography column. Since wild-type dsPETase01 and its mutant protease do not have an affinity tag after digestion, they will not bind to the affinity column and will flow out of the column (referred to as the permeate). Therefore, the permeate was collected.

[0069] (3) Gel filtration chromatography and QC detection

[0070] The permeate was concentrated to approximately 2 mL and then subjected to gel filtration chromatography. The gel chromatography column was a Super rdex 200 Increase 10 / 300GL, and the buffer consisted of 20 mM Tris–HCl pH 7.5, 150 mM NaCl, and 1 mM DTT. The gel filtration samples were collected and subjected to protein content determination, specifically SDS-PAGE purity analysis, mass spectrometry analysis, and analytical molecular sieve analysis.

[0071] SDS-PAGE results showed that the purity of both the wild-type and dsPETase01 mutant proteins was greater than 99%. Mass spectrometry analysis also indicated that the molecular weight of the tested samples was essentially consistent with the target protein, confirming that the purified protein was indeed the target protein. Furthermore, analytical molecular sieve analysis showed that all proteins remained in monomeric form in solution. The detection results are shown in Figure 8.

[0072] 2.5.3 Detection of the activity of dsPETase01 high-quality mutant protein

[0073] To further verify the plastic degradation ability of the purified dsPETase01 high-quality mutant protein, the purified protein was subjected to activity testing again, using the same method as in step 2.4. The difference was that the protein used for testing was unlabeled and of higher purity. The activity results are as follows: Activity was tested at 30℃ and 60℃. The results showed that at 30℃, the activity of most dsPETase01 high-quality mutants was comparable to that of the wild type, with (Q171L, G191E, S215P) being approximately 2.1 times higher than the wild type. At 60℃, the activity of the mutants was higher than that of the wild type, with most being approximately 1.5 times higher, and (Q171L, G191E, S215P) showing approximately 3 times higher activity than the wild type. Figure 9 Industrial degradation of plastics requires high temperatures. These mutants, by improving both their thermal stability and activity, are more suitable for the degradation of industrial plastics.

[0074] 3. Conclusion

[0075] The above description shows that the dsPETase01 mutant protein provided by the present invention has higher protein yield, higher enzyme activity and better thermal stability, has broader application conditions and stronger practical application value, and is more suitable for large-scale production and industrial use.

[0076] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the protection scope of the present invention.

Claims

1. A dsPETaseOl plastic-degrading enzyme mutant, characterized in that, The dsPETase01 plastic degrading enzyme mutants are the following three types: The mutation sites of the first mutant are Q171L and G191E, and its amino acid sequence is shown in SEQ ID NO.2; The mutation sites of the second mutant are Q171L, G191E and S215P, and its amino acid sequence is shown in SEQ ID NO.3; The mutation sites of the third mutant are Q171L, G191E, S215P and A242V, and its amino acid sequence is shown in SEQ ID NO.

4.

2. The dsPETaseO1 plastic-degrading enzyme mutant of claim 1, wherein, The amino acid sequence of the dsPETase01 plastic degrading enzyme mutant obtained by introducing disulfide bonds into the sequence shown in SEQ ID NO.2 is shown in SEQ ID NO.

5.

3. A polynucleotide, characterized in that, The polynucleotide encodes the dsPETase01 plastic degrading enzyme mutant as described in any one of claims 1-2.

4. A polynucleotide according to claim 3, characterized in that, The polynucleotide sequences encoding the amino acid sequences of the dsPETase01 plastic degrading enzyme mutants shown in SEQ ID NO.2-5 are shown in SEQ ID NO.6-9, respectively.

5. A recombinant plasmid, characterized in that, The recombinant plasmid is an expression vector capable of correspondingly translating and expressing the dsPETase01 plastic degrading enzyme mutant as described in any one of claims 1-2, and the expression vector is the pET-28a vector.

6. The application of the dsPETase01 plastic degrading enzyme mutant as described in any one of claims 1-2 in the degradation or recycling of PET plastics.

7. The application according to claim 6, characterized in that, The application involves using the dsPETase01 plastic degrading enzyme mutant to decompose PET plastic and generate terephthalic acid (TPA), the main component of PET plastic, during the decomposition process.

Images