Mutants of pet hydrolytic enzyme de7, methods of making and using same

Abstract

The application discloses a mutant of PET hydrolytic enzyme DE7 and a preparation method and application thereof. A classification model of PET hydrolytic enzyme and a regression model of lipase B are constructed respectively, and a PET hydrolytic enzyme DE7 with high activity to BHET or PET is obtained according to the classification model and the regression model. An omnidirectional multi-point mutagenesis strategy is used to transform the PET hydrolytic enzyme DE7, and eight mutants with high catalytic activity to PET are selected, and the amino acid sequences of the eight mutants are shown in SEQ ID No. 2-9. According to the determination of hydrolytic activity, the eight mutants are all improved in the PET hydrolysis efficiency compared with DE7. According to the determination results of thermal stability, the values of four mutants in the mutants with improved catalytic activity are improved compared with the wild type DE7, and the thermal stability of the four mutants is effectively improved. The application has important application prospects in the efficient degradation and recycling of PET or BHET. T m ​

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 PETase for representative cutinase. 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. SUMMARY

[0003] One of the purposes of the present application is to provide a mutant of PET hydrolytic enzyme DE7.

[0004] The second purpose of the present application is to provide a coding gene of the mutant of PET hydrolytic enzyme DE7.

[0005] 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.

[0006] 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).

[0007] The above purposes of the present application are achieved by the following technical solutions:

[0008] In one aspect of the present application, a mutant of PET hydrolase DE7 is provided, 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.

[0009] The amino acid sequence of the PET hydrolase DE7 described in the present application is shown in SEQ ID No. 1.

[0010] In another aspect of the present application, a gene encoding the mutant of PET hydrolase DE7 is provided.

[0011] In still another aspect of the present application, an expression cassette containing the gene encoding the mutant of PET hydrolase DE7, a recombinant expression vector or a recombinant host cell containing the recombinant expression vector are provided; wherein the recombinant expression vector can be a recombinant prokaryotic expression vector or a recombinant eukaryotic vector.

[0012] The present application further provides a method for preparing any one of the mutants of PET hydrolase DE7, comprising:

[0013] (1) constructing a recombinant expression vector by operably linking the gene encoding the mutant of PET hydrolase DE7 with an expression regulatory element;

[0014] (2) transforming the recombinant expression vector into a host cell, culturing the host cell, inducing the expression of the recombinant protein, purifying the obtained crude protein, and obtaining the mutant of PET hydrolase DE7.

[0015] In a preferred embodiment of the present application, the method for inducing the expression of the recombinant protein in step (2) comprises adding 0.3 mM IPTG and inducing for 18-20 h at 16 ℃ to obtain the soluble protein.

[0016] In a preferred embodiment of the present application, the purification in step (2) comprises purifying the crude protein by Ni-NTA affinity chromatography; more preferably, loading the lysate to 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 a 200 mM imidazole buffer; and dialyzing the eluate against a Tris-HCl buffer to remove the imidazole.

[0017] Still another aspect of the present application is to apply the mutant of the PET hydrolytic enzyme DE7, the encoding gene thereof, the expression cassette containing the encoding gene of the unit point 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).

[0018] In a preferred embodiment of the present application, the present application provides the application of the mutant of the PET hydrolytic enzyme DE7 to degrade polyethylene terephthalate or bis (2-hydroxyethyl) terephthalate, which comprises: performing an enzymatic hydrolysis reaction with the mutant of the PET hydrolytic enzyme DE7 as a degradation enzyme and polyethylene terephthalate or bis (2-hydroxyethyl) terephthalate as a substrate.

[0019] The present application aims at the problems of poor thermal stability and low catalytic efficiency of natural PET hydrolytic enzymes, which are difficult to meet the actual application requirements, etc. Based on the Transformer network of BERT architecture, a pre-training model CEMP integrating protein and substrate information is constructed, and a classification model of PET hydrolytic enzyme and a regression model of lipase B are constructed through transfer learning. The two models are used to screen the public database, and finally a PET hydrolytic enzyme DE7 with high activity to BHET or PET is obtained. On this basis, the identified hydrolytic enzyme DE7 is further modified by using the omni-directional multi-point mutagenesis (ODM) strategy: first, the homologous sequence of DE7 is retrieved in the UniProt-TrEMBL database, and an ODM multi-point mutation model is constructed based on this to screen potential high-efficiency mutants; then, the candidate mutants are functionally screened by combining the PET degradation classification model and the lipase B activity prediction model, and 8 mutants with high catalytic activity to PET are selected. According to the hydrolysis activity determination results, it can be seen that the 8 mutants all show higher product yield than DE7, indicating that they have been improved to different degrees in PET hydrolysis efficiency, among which 2 mutants are more obvious: mutant 600071 has an enzyme activity 27.6% higher than DE7, and mutant 338226 has an enzyme activity 29.4% higher than DE7. According to the thermal stability determination results, it can be seen that among the mutants with improved catalytic activity, the values of 4 mutants are higher than that of the wild type DE7, indicating that the thermal stability of these mutants has been improved; among them, the value of mutant 600071 is the highest, reaching 86.31℃, which is 6.5℃ higher than that of the wild type DE7, followed by mutants 1929, 680538 and 467130, which are 5.9℃, 5.6℃ and 5.4℃ higher than that of the wild type DE7, respectively. T m T m T m ​​​T m Value promotion 4.39℃, 4.09℃ and 1.81℃. The present application has important application prospects in PET or BHET high-efficiency degradation and recycling, etc.

[0020] Definitions of terms involved in the present invention

[0021] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which this application belongs. Although any methods, devices and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods, devices and materials are now described.

[0022] The term "polynucleotide" or "nucleotide" means deoxyribonucleotides, deoxyribonucleosides, ribonucleosides or ribonucleotides in either single- or double-stranded form, and polymers thereof. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless specifically indicated otherwise, the term also means oligonucleotide analogs, including PNAs (peptide nucleic acids), DNA analogs used in antisense technology (phosphorothioates, phosphoramidates, etc.). Unless otherwise specified, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (including, but not limited to, simplifying degenerate codon substitutions) and Mol Cell. Probes 8:91-98 (1994).

[0023] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to mean a polymer of amino acid residues. That is, a description in terms of a polypeptide equally applies to a description in terms of a peptide and to a description in terms of a protein, and vice versa. The terms apply to naturally-occurring amino acid polymers as well as to amino acid polymers in which one or more of the amino acid residues is a non-naturally encoded amino acid. As used herein, the terms encompass amino acid chains of any length including full-length proteins (i.e., antigens) in which the amino acid residues are connected via covalent peptide bonds.

[0024] The terms "mutation" and "mutant" have their usual meaning herein and refer to genetic, naturally occurring or introduced changes in nucleic acid or polypeptide sequences, in the sense commonly known to persons skilled in the art.

[0025] The term "recombinant host cell strain" or "host cell" means a cell which contains a polynucleotide of the application, whether or not the polynucleotide is integrated into the host cell genome, or maintained in the host cell as a non-integrated vector, such as a plasmid. The host cell can be a prokaryotic or eukaryotic cell.

[0026] The term "operably linked" means functional linkage between two or more elements, such as a promoter and a coding sequence, wherein the elements are in a relationship permitting them to interact, either directly or indirectly.

[0027] The term "transformed" means the genetic transformation of a host cell with a polynucleotide or polypeptide.

[0028] The term "expression" refers to the transcription and / or translation of an endogenous gene or a transgene in a host cell. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 Results of the preliminary determination of the PET hydrolytic activity of hydrolytic enzyme DE7.

[0030] Figure 2 Results of the determination of the optimum temperature and optimum pH value of hydrolytic enzyme DE7.

[0031] Figure 3 Results of the determination of the PET hydrolytic activity of hydrolytic enzyme DE7 and its eight mutants. DETAILED DESCRIPTION

[0032] The present application will be further described in conjunction with specific test examples, and the advantages and features of the present application will become more apparent from the description. However, these test examples are merely exemplary and do not limit the scope of the present application in any way. Those skilled in the art will understand that modifications or substitutions can be made to the details and forms of the present application without departing from the spirit and scope of the present application, and such modifications and substitutions fall within the scope of the present application.

[0033] Test Example 1: Excavation of PET hydrolytic enzyme DE7, determination of PET hydrolytic activity and basic properties

[0034] 1. Excavation of PET hydrolytic enzyme DE7

[0035] 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:

[0036] MTERGLAPTASNITGNGSYSVTSASITGQSGFGGGMVYYPTANEKFPVVAISPGYTERWAPFAWMGPRLASWGFVVVGIETNSTLDQPDSRGTQLLKALDWAGSSAPAAVRDRVDSTRQGVAGHSMG GGGTLSAMNQRPSVRAGVPLAPWHTTTSWPGVKNPVLIMGGQNDTIAPPSQHAIPMYNGVASSEKEYVELAGAGHNFPNSSNPDVSKALVSWFKRFLDDDTRFSPFACDWTGSAYSQVRSTCPV (SEQ ID No.1).

[0037] 2. Determination of PET hydrolytic activity and basic properties of DE7 PET hydrolase

[0038] 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.

[0039] 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.

[0040] 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.

[0041] Design, screening, expression and enzymatic performance determination of DE7 mutants

[0042] 1 Experimental method

[0043] 1.1 Design and modification of DE7

[0044] In this experiment, DE7 was modified by using the strategy of omni-directional multi-point mutagenesis (ODM). First, the homologous sequences of DE7 were searched in the UniProt-TrEMBL database, and an ODM multi-point mutation model was constructed based on this to screen potential high-efficiency mutants. Subsequently, the candidate mutants were functionally screened by combining the PET degradation classification model and the lipase B activity prediction model, and 8 mutant sequences with high catalytic activity for PET were selected, i.e., 680538, 600071, 540571, 506821, 467130, 358963, 338226 and 1929.

[0045] The amino acid sequence of mutant 680538 is as follows:

[0046] MTERGLAPTASNITGNGSYSVTSASITGGSGFGGGMVYYPTANEKFPVVAISPGYTERWASFAWMGPRLASWGFVVIGIETNSTLDQPDSRGTQLLKALDWAGSSAPAAVRDRVDSTRQGVAGHSMGGGGTLSAMNQRPSVRAGVPLAPWHTTKSWPGVKNPVLIMGGQNDTIAPPSQHAIPMYNGVASSEKAYVELAGAGHNFPNSSNPDVSKALVSWFKRFLDDDTRFSPFACDWTGSAISQVRSTCPV (SEQ ID No. 2).

[0047] The amino acid sequence of mutant 600071 is as follows:

[0048] MTERGLAPTASNITGNGSYSVTSASITGQSGFGGGMVYYPTANEKFPVVAISPGYTERWASFSWMGPRLASWGFVVVGIETNSTLDQPDSRGTQLLKALDWAGSSAPAAVRDRVDSTRQGVAGHSMGGGGTLSAMNQRPSVRAGVPLAPWHTTKSWPGVKNPVLIMGGQNDTIAPPSQHAIPMYNGVASSEKAYVELAGAGHNFPNSSNPDVSKALVSWFKRFLDDDTRFSPFACDWTGSAYSQVRSTCPV (SEQ ID No. 3).

[0049] The amino acid sequence of mutant 540571 is shown below:

[0050] MTERGLAPTASNITGNGSYSVTSASITGQSGFGGGMVYYPTANEKFPVVAISPGFTERWASFAWMGPRLASWGFVVVGIETNSTLDQPDSRGTQLLKALDWATSSAPAAVRDRVDSSRQGVAGHSMGGGGTLSAMNQRPSVRAGVPLAPWHTTKSWPGVKNPVLIMGGQNDTIAPPSQHAIPMYNGVASSEKEYVELAGAGHNFPNSSNPDVSKALVSWFKRFLDDDTRFSPFACDWTGSAYSQYRSTCPV (SEQ ID No. 4).

[0051] The amino acid sequence of mutant 506821 is shown below:

[0052] MTERGLAPTASNITGNGSYSVTSASITGQSGFGGGMVYYPTANEKFPVVAISPGYTERWASFAWMGPRLASWGFVVVGIETNSTLDQPASRGTQLLKALDWAGSSAPAAVRDRVDSSRQGVAGHSMGGGGTLSAMNQRPSLRAGVPLAPWHTTTSWPGVKNPVLIMGGQNDTIAPPSQHAIPMYNGVASSEKEYVELAGAGHNFPNSSNPDVSKALVSWFKRFLDDDTRFSPFACDWTGSAYSQVRSTCPY (SEQ ID No. 5).

[0053] The amino acid sequence of mutant 467130 is shown below:

[0054] MTERGLAPTASNITGNGSYSVTSASITGQNGFGGGMVYYPTANEKFPVVAISPGYTERWASFAWMGPRLASWGFVVVGIETNSTLDQPDSRGTQLLKALDWAGSSAPAAVRDRVDSTRQGVAGHSMGGGGTLSAMNQRPSVRAGVPLAPWHTTKSWPGVKNPTLIMGGQNDTIAPPSQHAIPMYNGVASSEKAYVELAGAGHNFPNSSNPTVSKALVSWFKRFLDDDTRFSPFACDWTGSAYSQVRSTCPV (SEQ ID No. 6).

[0055] The amino acid sequence of mutant 358963 is shown below:

[0056] MAERGLAPTASNITGNGSFSVTSASITGQSGFGGGMVYYPTANEKFPVVAISPGYTERWASFSWMGPRLASWGFVVVGIETNSTLDQPDSRGTQLLKALDWAGSSAPAAVRDRVDSTRQGVAGHSMGGGGTLSAMNQRPSVRAGVPLAPWHTTKSWPGVKNPVMIMGGQNDTIAPPSQHAIPMYNGVASSEKAYVELAGAGHNFPNSSNPDVSKALVSWFKRFLDDDTRFSPFACDWTGSAYSQVRSTCPV (SEQ ID No. 7).

[0057] The amino acid sequence of mutant 338226 is shown below:

[0058] MTERGLAPTASNITGNGSYSVTSASITGQSGFGGGMVYYPTANEKFPVVAISPGYTERWASFAWMGPRLASWGFVVVGIETNSTLDQPDSRGTQLLKALDWAGSSAPAAVRDRVDSSRQGVAGHSMGGGGTLSAMNQRPSVRAGVPLAPWHTTTSWPGVKNPVLIMGGQNDTIAPPSQHAIPMYNGVASSEKEYVELAGAGHNFPNSSNPDVSKALVSWFKRFLDDDTRFSPFACDWTGSAYSQVRSTCPV (SEQ ID No. 8).

[0059] The amino acid sequence of mutant 1929 is shown below:

[0060] MTERGLAPTASNITGNGSYSVTSASITGGSGFGGGMVYYPTANEKFPVVAISPGYTERWASFSWMGPRLASWGFVVVGIETNSTLDQPDSRGTQLLKALDWAGSSAPAAVRDRVDSTRQGVAGHSMGGGGTLSAMNQRPSVRAAVPLAPWHTTKSWPGVKNPVLIMGGQNDTIAPPSQHAIPMYNGVASSEKAYVELAGAGHNFPNSSNPDVSKALVSWFKRFLDDDTRFSPFACDWTGSAYSQVRSTCPV (SEQ ID No. 9).

[0061] 1.2 Expression and purification of DE7 and its mutants

[0062] Expression and purification of DE7 and its mutants were performed using E. coli BL21(DE3) system. First, the strain carrying recombinant plasmid was cultured in LB medium containing kanamycin (50 μg / mL) to OD600 about 0.6-0.8, then 0.3 mM IPTG was added to induce for 18-20 h at 16 ℃ to obtain soluble protein. The bacteria were collected by centrifugation and resuspended in 20 mM Tris-HCl (pH 8.0) buffer, and then lysed by ultrasonic on ice. After centrifugation, the supernatant was used as crude enzyme solution. Subsequently, the Ni-NTA affinity chromatography was used for purification: the lysate was loaded into the pre-equilibrated Ni-NTA column, and the impurities were eluted with buffer containing different concentrations of imidazole (20 mM, 40 mM, 60 mM) in turn, and finally the target protein was eluted with 200 mM imidazole buffer. The eluate was dialyzed against Tris-HCl buffer to remove imidazole, and concentrated to 1 mg / mL by PEG8000.

[0063] 1.3 Activity detection of DE7 and its mutants

[0064] A 6.04% crystallinity PET film was punched into 6 mm diameter discs as substrate, and placed in 2 mL centrifuge tubes; the reaction system was 20 mM Tris-HCl (pH 8.0) 800 µL with enzyme protein solution (1 mg / mL) 100 µL, total volume 900 µL, and reacted at 60 ℃ for 12 h. After filtration through a 0.45 µm filter, the sample was injected into HPLC (Eclipse Plus C18, 4.6 x 250 mm, 5 µm; 0.5% acetic acid in water / methanol gradient elution; flow rate 1 mL / min; injection volume 10 µL; column temperature 35 ℃; detection wavelength 240 nm), and the product was quantified by external standard method according to the standard curve (0.25-3 mM) of MHET and TPA. The 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 amount). Each sample was set in two technical replicates and the enzyme-free blank was deducted, and the results were reported as mean ± standard deviation;

[0065] 1.4 Optimum temperature and pH detection

[0066] Under the condition of keeping the same substrate and enzyme dosage (0.1 mg / mL), the hydrolysis activity of DE7 on PET was determined by changing the temperature or pH, respectively. The temperature gradient was set at 30 ℃, 40 ℃, 50 ℃, 60 ℃, 70 ℃ and 80 ℃, and each temperature was reacted in 20 mM Tris-HCl (pH 8.0) buffer for 12 h; the pH gradient was set at 3-9, and pH 3-7 used citric acid-disodium hydrogen phosphate buffer and pH 7-9 used Tris-HCl buffer, and reacted at 60 ℃ for 12 h. After the reaction was completed, the substrate was isolated and filtered, and the TPA and MHET products were quantitatively detected by HPLC, and the relative activity under each condition was calculated, and the optimum temperature and pH value were determined accordingly. Each condition was set in triplicate, and the results were expressed as mean ± standard deviation.

[0067] 1.5 DE7 and its mutants T m value detection

[0068] The purified protein was prepared to about 0.5 mg / mL in PBS pH 7.4, and was ultrasonicated and degassed; the same buffer was used as a blank to set the baseline. The sample was placed in a DSC cell, and was scanned at a temperature program of 25-100 ℃ at a rate of 1 ℃ / min, and the apparent heat capacity (Cp)-temperature curve was recorded; the Tm was taken as the temperature at the top of the thermal denaturation endothermic peak or the midpoint temperature (when the two-state model was fitted, the midpoint was taken). Each sample was determined in two independent experiments, and the average value ± standard deviation was reported; DE7 T m can be used as a control for comparison with the Tm Difference (Δ T m ) comparison.

[0069] 2 Test results

[0070] 2.1 DE7 optimum temperature and optimum pH determination results

[0071] In order to systematically evaluate the enzymatic properties of DE7, its hydrolysis activity was detected under different temperature and pH conditions. In terms of temperature adaptability, low crystallinity PET film was used as the substrate and reacted at 30°C, 40°C, 50°C, 60°C, 70°C and 80°C for 12 h respectively; according to the optimum temperature determination results of Figure 2 DE7 showed the highest hydrolysis activity at 60°C, which was its optimum reaction temperature. At the same time, the thermal stability of DE7 was determined by differential scanning calorimetry (DSC), and the results showed that its T m value was 79.84°C, indicating that the enzyme could maintain good structural stability at higher temperatures.

[0072] In the pH dependence analysis Figure 2 ), citric acid-disodium hydrogen phosphate buffer was used in the pH 3-7 range, and Tris-HCl buffer was used in the pH 7-9 range, and the results showed that DE7 had the highest activity at pH 8, which was its optimum reaction pH.

[0073] In summary, DE7 not only has high thermal stability and tolerance, but also exhibits excellent hydrolysis performance in a near neutral to weak alkaline environment, which provides a good foundation for its application in PET degradation under environmental conditions.

[0074] 2.2 DE7 and its mutant hydrolysis activity determination results

[0075] To further improve the catalytic efficiency of DE7, the mutants of DE7 were designed by using ODM omnidirectional mutagenesis strategy combined with model screening, and then the hydrolysis activity of DE7 and its mutants was determined under the same conditions. The specific process is as follows: low crystallinity PET film was used as the substrate and reacted with enzyme solution (final concentration 1 mg / mL) at 60°C for 12 h, and after the reaction was terminated by isolating the substrate, it was filtered by 0.45 μm filter membrane. Then the concentrations of main products TPA and MHET in the reaction solution were detected by high performance liquid chromatography (HPLC), and the sum of the two was used as the enzyme activity index.

[0076] Figure 1 , Figure 3Tables 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.

[0077] Table 1 Raw data of product formation amount (mM) detected by HPLC

[0078]

[0079] Note: The unit for the amount of all products generated in the table is mM.

[0080] 2.3 Results of thermal stability determination of DE7 and its mutants

[0081] To further evaluate the heat resistance of these mutants, differential scanning calorimetry (DSC) was used to study DE7 and its mutants. T m The 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.

[0082] Table 2 DE7 and its mutants T m (°C) value determination

[0083]

[0084] 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 Value increase 4.39, 4.09 and 1.81 °C.

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. 6 or SEQ ID No.

9.

2. The mutant according to claim 1, characterized in that, The amino acid sequence of the mutant is shown in SEQ ID No.

3.

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

4. An expression cassette or recombinant expression vector containing the coding gene as described in claim 3.

5. A recombinant host cell containing the expression cassette or recombinant expression vector as described in claim 4.

6. A method for preparing the mutant of 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.

7. The method according to claim 6, 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.

8. The use of the mutant of claim 1, the encoding gene of claim 3, the expression cassette or recombinant expression vector of claim 4, and the recombinant host cell of claim 5 in the degradation of polyethylene terephthalate or bis(2-hydroxyethyl) terephthalate.

9. 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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