Mutant of PET hydrolase DE7 as well as preparation method and application of mutant

Abstract

The invention discloses a PET hydrolase DE7 mutant as well as a preparation method and application thereof. According to the invention, a classification model of the PET hydrolase and a regression model of lipase B are respectively constructed, so that the PET hydrolase DE7 with relatively high activity to BHET or PET is obtained through mining and identification according to the classification model and the regression model; an omni-directional multipoint mutagenesis strategy is adopted to modify and optimize eight mutants with relatively high catalytic activity on PET, and the amino acid sequences of the eight mutants are respectively shown as SEQ ID No.2-9; according to hydrolytic activity determination, compared with DE7, the eight mutants are improved in the aspect of PET (Polyethylene Terephthalate) hydrolysis efficiency. A thermal stability determination result shows that in the mutants with improved catalytic activity, Tm values of four mutants are improved compared with that of wild type DE7, so that the thermal stability of the mutants is effectively improved. The method disclosed by the invention has an important application prospect in the aspects of efficient degradation and cyclic utilization of PET or BHET and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to PET hydrolytic enzyme mutants, in particular to mutants of PET hydrolytic enzyme DE7 and their application in degrading polyethylene terephthalate (PET) or bis (2-hydroxyethyl) terephthalate (BHET), and belongs to the field of PET hydrolytic enzyme mutants and their applications. BACKGROUND

[0002] Polyethylene terephthalate (PET) as a widely used synthetic polyester, its environmental accumulation has become a global pollution problem. In recent years, the discovery of PET hydrolytic enzyme provides an important way for plastic biodegradation, among which the cutinase represented by PETase has attracted widespread attention. However, the natural PET hydrolytic enzyme generally has poor thermal stability and low catalytic efficiency, which is difficult to meet the actual application requirements. If the PET hydrolytic enzyme sequence is excavated and directionally modified, an enzyme variant with higher stability and activity will be obtained, which will have very important application prospects in PET efficient degradation and recycling, etc. Is SUMMARY

[0003] One of the purposes of the present application is to provide a mutant of PET hydrolytic enzyme DE7. The second purpose of the present application is to provide a coding gene of the mutant of PET hydrolytic enzyme DE7.

[0004] The third purpose of the present application is to provide an expression cassette containing the coding gene of the mutant, a recombinant expression vector or a recombinant host cell containing the recombinant expression vector. The fourth purpose of the present application is to apply the mutant of PET hydrolytic enzyme DE7, its coding gene, the expression cassette containing the coding gene of the mutant, the recombinant expression vector or the recombinant host cell containing the recombinant expression vector, etc. to degrade polyethylene terephthalate (PET) or bis (2-hydroxyethyl) terephthalate (BHET).

[0005] The above purposes of the present application are achieved by the following technical solutions: ​One aspect of the present invention provides a mutant of PET hydrolase DE7, wherein the amino acid sequence of the mutant is selected from any one of the amino acid sequences shown in SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8, or SEQ ID No. 9; preferably, the amino acid sequence of the mutant is selected from any one of the amino acid sequences shown in SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 6, SEQ ID No. 8, or SEQ ID No. 9; most preferably, the amino acid sequence of the mutant is shown in SEQ ID No. 3.

[0006] The amino acid sequence of the PET hydrolase DE7 described in this invention is shown in SEQ ID No. 1.

[0007] Another aspect of the present invention is to provide the encoding gene of a mutant of PET hydrolase DE7.

[0008] Another aspect of the present invention is to provide an expression cassette containing the coding gene of the mutant of the PET hydrolase DE7, a recombinant expression vector, or a recombinant host cell containing the recombinant expression vector; wherein the recombinant expression vector may be a recombinant prokaryotic expression vector or a recombinant eukaryotic vector.

[0009] The present invention further provides a method for preparing a mutant of any of the PET hydrolase DE7, comprising: (1) The coding gene of the mutant of the PET hydrolase DE7 was operatively linked with an expression regulatory element to construct a recombinant expression vector; (2) Transform the recombinant expression vector into host cells, culture the host cells, induce the expression of recombinant protein, and purify the crude protein obtained.

[0010] In a preferred embodiment of the present invention, the method for inducing the expression of recombinant protein in step (2) includes: adding 0.3 mM IPTG and inducing at 16 °C for 18-20 h to obtain soluble protein.

[0011] In a preferred embodiment of the present invention, the purification in step (2) includes: purifying the crude protein using Ni-NTA affinity chromatography; more preferably, loading the lysis buffer into a pre-equilibrated Ni-NTA column, eluting the impurities with buffers containing 20 mM, 40 mM, and 60 mM imidazole in sequence, and finally eluting the target protein with 200 mM imidazole buffer; the eluent is dialyzed with Tris-HCl buffer to remove imidazole.

[0012] Another aspect of the present invention is to apply the mutant of the PET hydrolase DE7, its encoding gene, an expression cassette containing the encoding gene of the single point mutant, a recombinant expression vector, or a recombinant host cell containing the recombinant expression vector to the degradation of polyethylene terephthalate (PET) or bis(2-hydroxyethyl) terephthalate (BHET).

[0013] In a preferred embodiment of the present invention, the present invention provides the application of the mutant of the PET hydrolase DE7 to degrade polyethylene terephthalate or bis(2-hydroxyethyl) terephthalate, comprising: using polyethylene terephthalate or bis(2-hydroxyethyl) terephthalate as a substrate, and using the mutant of the PET hydrolase DE7 as a degrading enzyme to carry out an enzymatic hydrolysis reaction.

[0014] This invention addresses the common drawbacks of natural PET hydrolases, such as poor thermal stability and low catalytic efficiency, which make them unsuitable for practical applications. Based on the BERT architecture and Transformer network, a pre-trained model CEMP integrating protein and substrate information was constructed. Through transfer learning, a classification model for PET hydrolases and a regression model for lipase B were built. These two models were used to screen public databases, ultimately obtaining the PET hydrolase DE7, which exhibits high activity towards BHET or PET. Furthermore, this invention employs an omnidirectional multi-point mutagenesis (ODM) strategy to modify the identified hydrolase DE7: First, DE7 was retrieved from the UniProt-TrEMBL database. Homologous sequences were obtained and an ODM multi-point mutation model was constructed based on this to screen for potential high-efficiency mutants. Subsequently, the candidate mutants were functionally screened using a PET degradation classification model and a lipase B activity prediction model, and eight mutants with high catalytic activity to PET were selected. According to the hydrolysis activity test results, all eight mutants showed higher product yields than DE7, indicating that they were improved in PET hydrolysis efficiency to varying degrees. Among them, two mutants showed more significant improvements: mutant 600071 showed a 27.6% increase in enzyme activity compared to DE7, and mutant 338226 showed a 29.4% increase in enzyme activity compared to DE7. According to the thermal stability test results, among the mutants with improved catalytic activity, four mutants showed higher product yields than DE7. T m The values ​​were higher than those of the wild-type DE7, indicating that the thermal stability of these mutants was improved; among them, T m The highest value was found in mutant 600071, reaching 86.31℃, which is higher than that of the wild type. T m The value increased by 6.5℃, followed by mutants 1929, 680538, and 467130, which were significantly higher than the wild type. Tm The values ​​increased by 4.39℃, 4.09℃, and 1.81℃. This invention has significant application prospects in the efficient degradation and recycling of PET or BHET.

[0015] Definitions of terms involved in the present invention Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While any methods, apparatus, and materials similar to or equivalent to those described herein may be used in the practice or testing of this invention, preferred methods, apparatus, and materials are now described.

[0016] The terms "polynucleotide" or "nucleotide" refer to deoxyribonucleotides, deoxyribonucleosides, ribonucleosides, or ribonucleotides and their polymers in single-stranded or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides, which have binding properties similar to a reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specifically limited, the term also refers to oligonucleotide analogs, including PNAs (peptide nucleic acids), DNA analogs (phosphate thioesters, phosphoramidites, etc.) used in antisense techniques. Unless otherwise specified, specific nucleic acid sequences implicitly encompass variants of their conserved modifications (including (but not limited to) degenerate codon substitutions) and complementary sequences, as well as explicitly specified sequences. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the 3rd position of one or more selected (or all) codons is substituted with mixed bases and / or deoxyinosine residues. Mol Cell. Probes 8:91-98 (1994)).

[0017] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers of amino acid residues. That is, the description of a polypeptide is equally applicable to the description of a peptide and the description of a protein, and vice versa. The terms apply to naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues are non-naturally encoded amino acids. As used herein, the terms cover amino acid chains of any length, including full-length proteins (i.e., antigens), wherein the amino acid residues are linked by covalent peptide bonds.

[0018] The terms “mutation” and “mutant” have their common meanings here, referring to genetic, naturally occurring or introduced changes in a nucleic acid or polypeptide sequence, and their meanings are the same as those commonly known to those skilled in the art.

[0019] The terms "recombinant host cell line" or "host cell" refer to a cell containing the polynucleotides of the present invention, regardless of the method used for insertion to produce the recombinant host cell, such as direct uptake, transduction, f-pairing, or other methods known in the art. The exogenous polynucleotides may be maintained as, for example, non-integrating vectors of plasmids or may be integrated into the host genome. The host cell may be a prokaryotic or eukaryotic cell.

[0020] The term "operable connection" refers to a functional connection between two or more elements, which can be adjacent or non-adjacent.

[0021] The term "transformation" refers to the genetic conversion of polynucleotides or polypeptides into host cells by introducing gene-coding molecules into the host cells.

[0022] The term "expression" refers to the transcription and / or translation of endogenous genes or transgenes in host cells. Attached Figure Description

[0023] Figure 1 These are preliminary results of the PET hydrolysis activity of the hydrolase DE7.

[0024] Figure 2 The results show the optimal temperature and optimal pH value for the hydrolase DE7.

[0025] Figure 3 The results show the PET hydrolytic activity assay of the hydrolase DE7 and its eight mutants. Detailed Implementation

[0026] The present invention will be further described below with reference to specific experimental examples, and the advantages and features of the present invention will become clearer with the description. However, these experimental examples are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions to the details and form of the present invention can be made without departing from the spirit and scope of the present invention, but all such modifications and substitutions fall within the protection scope of the present invention.

[0027] Experimental Example 1: Discovery of PET hydrolase DE7, determination of PET hydrolytic activity and basic properties. 1. Discovery of DE7, a PET hydrolase A pre-trained model, CEMP, integrating protein and substrate information, was constructed using the Transformer network based on the BERT architecture. Transfer learning was then used to build a classification model for PET hydrolases and a regression model for lipase B. Using these two models, public databases were screened to identify DE7 (UniProt ID A0A8J3TDE7), a PET hydrolase derived from *Plasmodium mesodeans*, exhibiting high activity against BHET or PET. The amino acid sequence of DE7 is shown below: MTERGLAPTASNITGNGSYSVTSASITGQSGFGGGMVYYPTANEKFPVVAISPGYTERWAPFAWMGPRLASWGFVVVGIETNSTLDQPDSRGTQLLKALDWAGSSAPAAVRDRVDSTRQGVAGHSMG GGGTLSAMNQRPSVRAGVPLAPWHTTTSWPGVKNPVLIMGGQNDTIAPPSQHAIPMYNGVASSEKEYVELAGAGHNFPNSSNPDVSKALVSWFKRFLDDDTRFSPFACDWTGSAYSQVRSTCPV (SEQ ID No.1).

[0028] 2. Determination of PET hydrolytic activity and basic properties of DE7 PET hydrolase Commercial PET plastic sheets with a degree of polymerization of 6.04% were selected as substrates and subjected to long-term reaction at a constant temperature of 50 °C to compare the degradation performance of different PET hydrolases.

[0029] The specific process is as follows: an equal amount of enzyme protein is added to the reaction system containing PET plastic sheets, and the reaction is continued at 50 °C for 72 h. After the reaction is terminated, the concentrations of the main hydrolysis products TPA and MHET are quantitatively detected by HPLC, and the total amount of products generated is calculated.

[0030] according to Figure 1 The experimental results show that DE7 exhibits the strongest hydrolytic ability under these conditions, with a total product yield of 4648.85 μM, which is higher than that of other reported high-activity products. Is DE7 significantly outperformed PETase (3389.9 μM) and LCC (4564.9 μM), and was significantly superior to TFcut2 (1448.5 μM). This result indicates that DE7 not only maintained stable activity during the long-term hydrolysis of low-polymerization-degree PET sheets, but also surpassed the classic LCC and LCC in terms of product accumulation levels. Is PETase shows superior PET degradation potential.

[0031] Experiment 2: Design, screening, expression, and enzymatic performance assay of DE7 mutants. 1. Experimental Methods 1.1 Design and Modification of DE7 This experiment employed an omnidirectional multi-point mutagenesis (ODM) strategy to modify DE7. First, homologous sequences for DE7 were retrieved from the UniProt-TrEMBL database, and an ODM multi-point mutagenesis model was constructed to screen for potential high-efficiency mutants. Subsequently, a PET degradation classification model and a lipase B activity prediction model were used to functionally screen candidate mutants, selecting eight mutant sequences with high catalytic activity against PET: 680538, 600071, 540571, 506821, 467130, 358963, 338226, and 1929.

[0032] The amino acid sequence of mutant 680538 is shown below: MTERGLAPTASNITGNGSYSVTSASITGGSGFGGGMVYYPTANEKFPVVAISPGYTERWASFAWMGPRLASWGFVVIGIETNSTLDQPDSRGTQLLKALDWAGSSAPAAVRDRVDSTRQGVAGHSMG GGGTLSAMNQRPSVRAGVPLAPWHTTKSWPGVKNPVLIMGGQNDTIAPPSQHAIPMYNGVASSEKAYVELAGAGHNFPSSNPDVSKALVSWFKRFLDDDTRFSPFACDWTGSAISQVRSTCPV (SEQ ID No.2).

[0033] The amino acid sequence of mutant 600071 is shown below: MTERGLAPTASNITGNGSYSVTSASITGQSGFGGGMVYYPTANEKFPVVAISPGYTERWASFSWMGPRLASWGFVVVGIETNSTLDQPDSRGTQLLKALDWAGSSAPAAVRDRVDSTRQGVAGHSMG GGGTLSAMNQRPSVRAGVPLAPWHTTKSWPGVKNPVLIMGGQNDTIAPPSQHAIPMYNGVASSEKAYVELAGAGHNFPSSNPDVSKALVSWFKRFLDDDTRFSPFACDWTGSAYSQVRSTCPV (SEQ ID No. 3).

[0034] The amino acid sequence of mutant 540571 is shown below: MTERGLAPTASNITGNGSYSVTSASITGQSGFGGGMVYYPTANEKFPVVAISPGFTERWASFAWMGPRLASWGFVVVGIETNSTLDQPDSRGTQLLKALDWATSSAPAAVRDRVDSSRQGVAGHSMG GGGTLSAMNQRPSVRAGVPLAPWHTTKSWPGVKNPVLIMGGQNDTIAPPSQHAIPMYNGVASSEKEYVELAGAGHNFPNSSNPDVSKALVSWFKRFLDDDTRFSPFACDWTGSAYSQYRSTCPV (SEQ ID No.4).

[0035] The amino acid sequence of mutant 506821 is shown below: MTERGLAPTASNITGNGSYSVTSASITGQSGFGGGMVYYPTANEKFPVVAISPGYTERWASFAWMGPRLASWGFVVVGIETNSTLDQPASRGTQLLKALDWAGSSAPAAVRDRVDSSRQGVAGHSMG GGGTLSAMNQRPSLRAGVPLAPWHTTTSWPGVKNPVLIMGGQNDTIAPPSQHAIPMYNGVASSEKEYVELAGAGHNFPNSSNPDVSKALVSWFKRFLDDDTRFSPFACDWTGSAYSQVRSTCPY (SEQ ID No.5).

[0036] The amino acid sequence of mutant 467130 is shown below: MTERGLAPTASNITGNGSYSVTSASITGQNGFGGGMVYYPTANEKFPVVAISPGYTERWASFAWMGPRLASWGFVVVGIETNSTLDQPDSRGTQLLKALDWAGSSAPAAVRDRVDSTRQGVAGHSMG GGGTLSAMNQRPSVRAGVPLAPWHTTKSWPGVKNPTLIMGGQNDTIAPPSQHAIPMYNGVASSEKAYVELAGAGHNFPNSSNPTVSKALVSWFKRFLDDDTRFSPFACDWTGSAYSQVRSTCPV (SEQ ID No. 6).

[0037] The amino acid sequence of mutant 358963 is shown below: MAERGLAPTASNITGNGSFSVTSASITGQSGFGGGMVYYPTANEKFPVVAISPGYTERWASFSWMGPRLASWGFVVVGIETNSTLDQPDSRGTQLLKALDWAGSSAPAAVRDRVDSTRQGVAGHSMG GGGTLSAMNQRPSVRAGVPLAPWHTTKSWPGVKNPVMIMGGQNDTIAPPSQHAIPMYNGVASSEKAYVELAGAGHNFPSSNPDVSKALVSWFKRFLDDDTRFSPFACDWTGSAYSQVRSTCPV (SEQ ID No.7).

[0038] The amino acid sequence of mutant 338226 is shown below: MTERGLAPTASNITGNGSYSVTSASITGQSGFGGGMVYYPTANEKFPVVAISPGYTERWASFAWMGPRLASWGFVVVGIETNSTLDQPDSRGTQLLKALDWAGSSAPAAVRDRVDSSRQGVAGHSMG GGGTLSAMNQRPSVRAGVPLAPWHTTTSWPGVKNPVLIMGGQNDTIAPPSQHAIPMYNGVASSEKEYVELAGAGHNFPNSSNPDVSKALVSWFKRFLDDDTRFSPFACDWTGSAYSQVRSTCPV (SEQ ID No.8).

[0039] The amino acid sequence of mutant 1929 is shown below: MTERGLAPTASNITGNGSYSVTSASITGGSGFGGGMVYYPTANEKFPVVAISPGYTERWASFSWMGPRLASWGFVVVGIETNSTLDQPDSRGTQLLKALDWAGSSAPAAVRDRVDSTRQGVAGHSMG GGGTLSAMNQRPSVRAAVPLAPWHTTKSWPGVKNPVLIMGGQNDTIAPPSQHAIPMYNGVASSEKAYVELAGAGHNFPSSNPDVSKALVSWFKRFLDDDTRFSPFACDWTGSAYSQVRSTCPV (SEQ ID No.9).

[0040] 1.2 Expression and purification of DE7 and its mutants Expression and purification of DE7 and its mutants were performed using the *E. coli* BL21(DE3) system. First, strains carrying the recombinant plasmid were cultured in LB medium containing kanamycin (50 μg / mL) until the OD600 reached approximately 0.6–0.8. Then, 0.3 mM IPTG was added, and the mixture was induced at 16 °C for 18–20 h to obtain soluble protein. The bacterial cells were collected by centrifugation and resuspended in 20 mM Tris-HCl (pH 8.0) buffer. The cells were then sonicated on ice for lysis, and the supernatant was used as the crude enzyme solution after centrifugation. Purification was then performed using Ni-NTA affinity chromatography: the lysate was loaded onto a pre-equilibrated Ni-NTA column, and contaminating proteins were eluted sequentially with buffers containing different concentrations of imidazole (20 mM, 40 mM, 60 mM). Finally, the target protein was eluted with 200 mM imidazole buffer. The eluent was dialyzed against Tris-HCl buffer to remove imidazole and concentrated to 1 mg / mL using PEG8000.

[0041] 1.3 Activity detection of DE7 and its mutants 6.04% crystallinity PET film was cut into 6 mm diameter discs and used as substrates, placed in 2 mL centrifuge tubes. The reaction system consisted of 800 µL of 20 mM Tris-HCl (pH 8.0) and 100 µL of enzyme protein solution (1 mg / mL), for a total volume of 900 µL, and reacted at 60 °C for 12 h. After filtration through a 0.45 µm filter, the sample was injected into HPLC (Eclipse Plus C18, 4.6 × 250 mm, 5 µm; 0.5% acetic acid aqueous solution / methanol gradient elution; flow rate 1 mL / min; injection volume 10 µL; column temperature 35 °C; detection wavelength 240 nm). The product was quantified using the external standard method based on the MHET and TPA standard curve (0.25–3 mM). Activity was expressed as the sum of released MHET and TPA (which can be normalized to relative activity or converted to specific activity per unit protein mass). Each sample was tested twice, with the enzyme-free blank subtracted. Results were reported as mean ± standard deviation. 1.4 Optimal Temperature and Optimal pH Detection Under the condition of maintaining the same substrate and enzyme dosage (0.1 mg / mL), the hydrolytic activity of DE7 on PET was determined with different temperatures or pH values ​​as variables. Temperature gradients were set at 30℃, 40℃, 50℃, 60℃, 70℃, and 80℃, with each temperature reacted for 12 h in 20 mM Tris–HCl (pH 8.0) buffer. pH gradients were set at 3–9, with pH 3–7 using citrate–disodium hydrogen phosphate buffer and pH 7–9 using Tris–HCl buffer, reacting at 60℃ for 12 h. After the reaction, the reaction was terminated by substrate isolation and filtered. TPA and MHET products were quantitatively detected by HPLC, and the relative activities under each condition were calculated to determine the optimal temperature and pH. Triple replicates were performed for each condition, and results are expressed as mean ± standard deviation.

[0042] 1.5 DE7 and its mutants T m Value detection The purified protein was prepared to approximately 0.5 mg / mL with PBS pH 7.4, and degassed by sonication. A baseline was established using the same buffer as a blank. Samples were placed in a DSC cell and scanned at 1 °C / min increments according to a 25–100 °C program, recording the apparent heat capacity (Cp)-temperature curve. Tm was taken as the endothermic peak temperature of thermal denaturation or the midpoint temperature of the transition (the midpoint was used for two-state model fitting). Each sample was measured twice independently, and the mean ± standard deviation was reported. DE7 values ​​measured in the same batch were also recorded. T m It can be used as a control group to compare with various mutants. T m Difference (Δ) T m )Compare.

[0043] 2. Experimental Results 2.1 Results of DE7 Optimal Temperature and Optimal pH Measurement To systematically evaluate the enzymatic properties of DE7, its hydrolytic activity was tested under different temperature and pH conditions. Regarding temperature adaptability, low-crystallinity PET films were used as substrates and reacted for 12 h at 30℃, 40℃, 50℃, 60℃, 70℃, and 80℃, respectively. Figure 2 The optimal temperature determination results show that DE7 exhibits the highest hydrolytic activity at 60 °C, which is its optimal reaction temperature. Simultaneously, the thermal stability of DE7 was determined using differential scanning calorimetry (DSC), and the results indicate that its... T m The value was 79.84 °C, indicating that the enzyme can maintain good structural stability at higher temperatures.

[0044] In pH-dependent analysis (Figure 2 The assay was performed using citrate-disodium hydrogen phosphate buffer in the pH range of 3–7 and Tris-HCl buffer in the pH range of 7–9. The results showed that DE7 had the highest activity at pH 8, which was its optimal reaction pH.

[0045] In summary, DE7 not only has high thermal stability and tolerance, but also exhibits excellent hydrolytic performance in near-neutral to weakly alkaline environments. These characteristics provide a good foundation for its application in PET degradation under environmental conditions.

[0046] 2.2 Results of hydrolytic activity assay of DE7 and its mutants To further improve the catalytic efficiency of DE7, a DE7 mutant was designed using an ODM omnidirectional mutagenesis strategy combined with model screening. The hydrolytic activity of DE7 and the mutant was then measured under the same conditions. The specific procedure is as follows: A low-crystallinity PET film was used as the substrate and reacted with the enzyme solution (final concentration 1 mg / mL) at 60 °C for 12 h. After the reaction, the substrate was isolated to terminate the reaction, and the mixture was filtered through a 0.45 μm filter membrane. The concentrations of the main products TPA and MHET in the reaction solution were then detected by high-performance liquid chromatography (HPLC), and their sum was used as the enzyme activity indicator.

[0047] Figure 1 , Figure 3 Tables 1 and 2 show the hydrolytic activity assay results of DE7 and its mutants, respectively. According to the hydrolytic activity assay results, all eight constructed mutants exhibited higher product yields than DE7, indicating varying degrees of improvement in PET hydrolysis efficiency. The four mutants showed the most significant improvements: mutant 600071 showed a 27.53% increase in enzyme activity compared to DE7, mutant 338226 showed a 29.23% increase, mutant 506821 showed a 24.07% increase, and mutant 467130 showed a 28.46% increase.

[0048] Table 1 Raw data of product formation amount (mM) detected by HPLC

[0049] Note: The unit for the amount of all products generated in the table is mM.

[0050] 2.3 Results of thermal stability determination of DE7 and its mutants To further evaluate the heat resistance of these mutants, differential scanning calorimetry (DSC) was used to study DE7 and its mutants. T mThe values ​​were determined. Differential scanning calorimetry (DSC) was used to determine DE7 and its mutants within the range of 25–100 °C at a heating rate of 1 °C / min. T m Value. 20 mM PBS was used as a control buffer, and each sample was measured in parallel.

[0051] Table 2 DE7 and its mutants T m (°C) value determination

[0052] According to Table 2 T m The results of the value determination show that among the mutants with enhanced catalytic activity, there are 4 mutants (i.e., 600071, 1929, 680538, and 467130). T m The values ​​were higher than those of the wild-type DE7, indicating that the thermal stability of these mutants was improved. T m The highest value was found in mutant 600071, reaching 86.31℃, which is higher than that of the wild type. T m The value increased by 6.47℃, followed by mutants 1929, 680538, and 467130, which were significantly higher than the wild type. T m The values ​​increased by 4.39, 4.09 and 1.81℃.

Claims

1. A mutant of PET hydrolase DE7, characterized in that, The amino acid sequence of the mutant is selected from any one of the amino acid sequences shown in SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8 or SEQ ID No.

9.

2. The mutant according to claim 1, characterized in that, The amino acid sequence of the mutant is selected from any one of the amino acid sequences shown in SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 6, SEQ ID No. 8 or SEQ ID No.

9.

3. The mutant according to claim 2, characterized in that, The amino acid sequence of the mutant is shown in SEQ ID No.

3.

4. The coding gene of the mutant according to claim 1.

5. An expression cassette or recombinant expression vector containing the coding gene as described in claim 4.

6. A recombinant host cell containing the expression cassette or recombinant expression vector as described in claim 5.

7. A method for preparing any mutant according to claim 1, characterized in that, include: (1) The coding gene of the mutant is operatively linked to the expression regulatory element to construct a recombinant expression vector; (2) Transform the recombinant expression vector into host cells, culture the host cells, induce the expression of recombinant protein, and purify the crude protein obtained.

8. The method according to claim 7, characterized in that, The method for inducing the expression of recombinant protein in step (2) includes: adding 0.3 mM IPTG and inducing at 16 °C for 18–20 h to obtain soluble protein; The purification described in step (2) includes: purifying the crude protein using Ni-NTA affinity chromatography; wherein, the lysis buffer is loaded into a pre-equilibrated Ni-NTA column, and impurities are eluted sequentially with buffers containing 20 mM, 40 mM, and 60 mM imidazole, and finally the target protein is eluted with 200 mM imidazole buffer; the eluent is dialyzed with Tris-HCl buffer to remove imidazole.

9. The use of the mutant of claim 1, the encoding gene of claim 4, the expression cassette or recombinant expression vector of claim 5, and the recombinant host cell of claim 6 in the degradation of polyethylene terephthalate or bis(2-hydroxyethyl) terephthalate.

10. A method for degrading polyethylene terephthalate or bis(2-hydroxyethyl) terephthalate, characterized in that, Using polyethylene terephthalate or bis(2-hydroxyethyl) terephthalate as substrates, and the mutant described in claim 1 as the degrading enzyme, an enzymatic hydrolysis reaction is carried out.

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