MHETase-EstD9 mutant and its application

Abstract

The present invention belongs to the field of enzyme engineering technology, and discloses a MHETase-EstD9 mutant and its application. The present invention provides a MHETase-EstD9 mutant, whose amino acid sequence is shown in SEQ ID No.2. On the basis of the mutant, five MHETase-EstD9 mutants are obtained by further mutating its amino acids. At 70°C, compared with the wild-type MHETase-EstD9, the BHET degradation activity of the MHETase-EstD9 mutant provided by the present invention is increased by more than 15%, and can be increased by up to 24 times; at the same time, the thermal stability of the MHETase-EstD9 mutant is also improved to a certain extent, and the Tm value is increased by up to 18.0°C. The present invention can be applied to the degradation of PET, MHET or BHET, the preparation of degradation agents for PET and its degradation products, and the recovery of TPA, and has important environmental, economic and social benefits.

Description

Technical Field

[0001] The present invention belongs to the technical field of enzyme engineering, and relates to an MHET degrading enzyme, in particular to an MHETase-EstD9 mutant and an application thereof. Background Art

[0002] Polyethylene terephthalate (PET) is a widely used plastic, commonly found in beverage bottles, food packaging, and textiles. Due to its high chemical stability, PET is difficult to degrade in the natural environment. With the increasing use of PET plastic, PET waste has accumulated in the environment, causing serious environmental pollution. Enzymatic degradation is an environmentally friendly solution to PET pollution.

[0003] In the process of bioenzymatic degradation of PET, the typical reaction pathway includes the following two key steps: the first step: PET hydrolase catalyzes the cleavage of PET ester bonds to produce MHET (Mono(2-hydroxyethyl) terephthalate, i.e., mono(2-hydroxyethyl) terephthalate, abbreviated as MHET); the second step: MHET is hydrolyzed by MHET degrading enzyme to break the remaining ester bonds, ultimately producing terephthalic acid (TPA) and ethylene glycol (EG).

[0004] MHET degradation is a key step in PET biodegradation. By degrading MHET, PET plastics can be converted into reusable monomers, thereby achieving closed-loop recycling of plastics. Currently, enhanced mutants have been discovered and obtained through molecular engineering of various PET-degrading enzymes. These mutants exhibit excellent catalytic efficiency under medium and high temperature conditions. However, compared with engineered PETases, the degradation activity of MHETases needs to be further improved, and research on improving their thermal stability remains limited. Therefore, a highly active MHET-degrading enzyme is urgently needed to improve the efficiency of PET degradation. Summary of the Invention

[0005] In view of the above-mentioned problems existing in the prior art, the present invention provides an MHETase-EstD9 mutant with significantly improved degradation activity by modifying MHETase-EstD9, which can be further applied to PET degradation or MHET degradation, providing support for the development of biodegradable PET.

[0006] To achieve the above-mentioned purpose, the embodiment of the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a MHETase-EstD9 mutant, the amino acid sequence of which is shown in SEQ ID No. 2, and is named MHETase-EstD9 mutant KLC.

[0008] The present invention provides a MHETase-EstD9 mutant, KLC, comprising the wild-type MHETase-EstD9 amino acid sequence shown in SEQ ID No. 1, wherein proline at position 7 is site-directedly mutated to cysteine, glycine at position 55 is site-directedly mutated to cysteine, glycine at position 130 is site-directedly mutated to leucine, and isoleucine at position 171 is site-directedly mutated to lysine, while amino acid residues at other positions remain unchanged. Compared to wild-type MHETase-EstD9, the MHETase-EstD9 mutant KLC exhibits 31.8-fold increased BHET degradation activity at 50°C and 9.6-fold increased BHET degradation activity at 60°C.

[0009] Secondly, based on the above-mentioned MHETase-EstD9 mutant KLC, the present invention site-directedly mutates the glutamic acid at position 209 in the amino acid sequence shown in SEQ ID No. 2 to arginine, while the amino acid residues at other positions remain unchanged, which is recorded as MHETase-EstD9 mutant KLCR.

[0010] Compared with the wild-type MHETase-EstD9, the MHETase-EstD9 mutant KLCR provided by the present invention not only increases the yield of TPA by 15.6 times when degrading BHET at 70°C, but also increases the Tm value from 70.25°C of the wild-type enzyme to 74.50°C, a Tm value increase of 4.3°C.

[0011] In the third aspect, based on the above-mentioned MHETase-EstD9 mutant KLC, the present invention further replaces the amino acid residues at at least two positions in the amino acid sequence shown in SEQ ID No. 2 to obtain four MHETase-EstD9 mutants, whose amino acid sequences are any one of the following (1) to (4):

[0012] (1) The isoleucine at position 121 of the amino acid sequence shown in SEQ ID No. 2 was site-directedly mutated to glutamic acid and the glutamic acid at position 209 was site-directedly mutated to arginine, while the amino acid residues at other positions remained unchanged, and the mutant was designated as MHETase-EstD9 mutant KLCRE;

[0013] (2) The serine at position 29 of the amino acid sequence shown in SEQ ID No. 2 was site-directedly mutated to proline, the methionine at position 34 was site-directedly mutated to glutamic acid, the isoleucine at position 121 was site-directedly mutated to glutamic acid, and the glutamic acid at position 209 was site-directedly mutated to arginine, while the amino acid residues at other positions remained unchanged. This mutant was designated as MHETase-EstD9 mutant KLCREPE;

[0014] (3) The serine at position 29 of the amino acid sequence shown in SEQ ID No. 2 was site-directedly mutated to proline, the methionine at position 34 was site-directedly mutated to glutamic acid, the serine at position 100 was site-directedly mutated to alanine, the isoleucine at position 121 was site-directedly mutated to glutamic acid, the isoleucine at position 178 was site-directedly mutated to leucine, and the glutamic acid at position 209 was site-directedly mutated to arginine. The amino acid residues at other positions remained unchanged. The mutant was named MHETase-EstD9 mutant KLCREPEAL.

[0015] (4) The serine at position 29 of the amino acid sequence shown in SEQ ID No. 2 was site-directedly mutated to proline, the methionine at position 34 was site-directedly mutated to glutamic acid, the serine at position 100 was site-directedly mutated to alanine, the isoleucine at position 121 was site-directedly mutated to glutamic acid, the threonine at position 163 was site-directedly mutated to phenylalanine, the isoleucine at position 178 was site-directedly mutated to leucine, and the glutamic acid at position 209 was site-directedly mutated to arginine. The amino acid residues at other positions remained unchanged. The mutant was named MHETase-EstD9 mutant KLCREPEALF.

[0016] Testing revealed that compared to wild-type MHETase-EstD9, the four mutants not only significantly enhanced their BHET degradation activity at 70°C but also significantly increased their Tm values. Specifically, TPA yields increased by 10.4-24.2 times, and Tm values ​​increased by 4.3°C-17.2°C. Notably, the MHETase-EstD9 mutant KLCREPEALF exhibited a 17.2°C higher Tm value and a 24.2-fold increase in BHET degradation activity compared to wild-type MHETase-EstD9.

[0017] In a fourth aspect, the present invention provides a recombinant plasmid, which can express the MHETase-EstD9 mutant described in the first to third aspects.

[0018] In a fifth aspect, the present invention provides an MHETase-EstD9 mutant engineered strain, which comprises the recombinant plasmid described in the fourth aspect.

[0019] In a sixth aspect, the present invention provides use of the MHETase-EstD9 mutant described in the first to third aspects above in degrading PET, MHET or BHET.

[0020] In a seventh aspect, the present invention provides use of the MHETase-EstD9 mutant described in the first to third aspects above in the preparation of a PET degrader, an MHET degrader, or a BHET degrader.

[0021] In an eighth aspect, the present invention provides use of the MHETase-EstD9 mutant described in the first to third aspects above in recovering TPA, a PET degradation product.

[0022] The present invention mutated MHETase-EstD9 through rational molecular evolution to obtain a MHETase-EstD9 mutant with significantly improved BHET degradation activity and / or thermal stability. Compared with the wild-type MHETase-EstD9, the six MHETase-EstD9 mutants protected by the present invention have a BHET degradation activity at 70°C that is at least 15% higher, and up to 24.2 times higher, while the Tm value is basically the same or higher. Compared with the wild-type MHETase-EstD9, the Tm value of the MHETase-EstD9 mutant KLCREPEALF is increased by 18.0°C, and the BHET degradation activity is increased by 24.2 times. The MHETase-EstD9 mutant provided by the present invention can be applied to the degradation of PET, MHET or BHET, the preparation of degradation agents for PET and its degradation products, and the recovery of TPA, and has important environmental, economic and social benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0024] Figure 1 is the plasmid map of the recombinant plasmid pET-22b-MHETase-EstD9 in Example 1 of the present invention;

[0025] Figure 2 Schematic diagram of the plasmid map of the recombinant plasmid pET-22b-G130L-I171K in Example 1 of the present invention;

[0026] Figure 3The results of the determination of BHET degradation activity and Tm of the wild-type MHETase-EstD9 and its mutants at 50°C in Example 1 of the present invention are shown;

[0027] Figure 4 The results of the determination of BHET degradation activity and Tm of the wild-type MHETase-EstD9 and its mutants at 60°C in Example 1 of the present invention are shown;

[0028] Figure 5 This is the plasmid map of the recombinant plasmid pET-22b-P7C-G55C-G130L-I171K in Example 2 of the present invention;

[0029] Figure 6 The results of the determination of BHET degradation activity and Tm of the wild-type MHETase-EstD9 and its mutants at 50°C in Example 2 of the present invention are shown;

[0030] Figure 7 The results of the determination of BHET degradation activity and Tm of the wild-type MHETase-EstD9 and its mutants at 60° C. in Example 2 of the present invention are shown;

[0031] Figure 8 These are the results of measuring the BHET degradation activity and Tm of the wild-type MHETase-EstD9 and its mutants at 70°C in Example 3 of the present invention. DETAILED DESCRIPTION

[0032] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0033] The mutants described in the present invention are named according to the conventional naming method of those skilled in the art. For example, the MHETase-EstD9 mutant G130L-I171K indicates that in the amino acid sequence of the wild-type MHETase-EstD9, the glycine (G) at position 130 is site-directedly mutated to leucine (L), and the isoleucine (I) at position 171 is site-directedly mutated to lysine (K).

[0034] Example 1

[0035] This example provides a method for preparing, expressing, purifying, and detecting the activity of an MHETase-EstD9 mutant. The specific contents are as follows:

[0036] The present invention aims to further enhance the degradation activity of MHETase-EstD9 against the PET intermediates BHET and MHET, without reducing or increasing its thermal stability, thereby achieving complete PET degradation. The target gene and expression vector are obtained through polymerase chain reaction (PCR). Recombinant plasmids are prepared using molecular biology methods such as DMTase (GD111 from Quanshijin Biotechnology Co., Ltd.) and seamless cloning. These recombinant plasmids are then transformed into competent Escherichia coli BL21(DE3) (CD601 from Quanshijin Biotechnology Co., Ltd.) cells. Recombinant E. coli expressing the target protein heterologously is then cultured. The specific process is as follows:

[0037] 1. Construction of recombinant plasmid MHETase-EstD9

[0038] 1. Construction of wild-type MHETase-EstD9 recombinant plasmid

[0039] The present invention commissioned Jinweizhi Company to synthesize the recombinant plasmid. The amino acid sequence of wild-type MHETase-EstD9 is shown in SEQ ID No. 1. After codon optimization, its encoding gene was obtained, and its encoding gene is shown in SEQ ID No. 3. The Escherichia coli pET-22b plasmid carrying the wild-type MHETase-EstD9 encoding gene is denoted as the recombinant plasmid pET-22b-MHETase-EstD9. The schematic diagram of its plasmid map is shown in Figure 1 , the construction method can be found in the record of Chinese patent CN202311211045.9.

[0040] 2. Screening of single-point or double-point mutants of wild-type MHETase-EstD9 and construction of MHETase-EstD9 mutant recombinant plasmids

[0041] The present invention screens mutants based on MHETase-EstD9 based on rational design methods such as evolutionary analysis and free energy calculation, and obtains MHETase-EstD9 mutants E40M, E78L, K83N, E124P, T163F, T185V, E209R, E220N, I121E and G130L-I171K.

[0042] Among them, the MHETase-EstD9 mutant E40M is a mutant in which the glutamic acid at position 40 in the amino acid sequence shown in SEQ ID No. 1 is site-directedly mutated to methionine, while the amino acid residues at other positions remain unchanged;

[0043] The MHETase-EstD9 mutant E78L is a mutant in which the glutamic acid at position 78 in the amino acid sequence shown in SEQ ID No. 1 is site-directedly mutated to leucine, while the amino acid residues at other positions remain unchanged;

[0044] The MHETase-EstD9 mutant K83N is a site-directed mutation of lysine at position 83 in the amino acid sequence shown in SEQ ID No. 1 to asparagine, while the amino acid residues at other positions remain unchanged;

[0045] The MHETase-EstD9 mutant E124P is a mutant in which the glutamic acid at position 124 in the amino acid sequence shown in SEQ ID No. 1 is site-directedly mutated to proline, while the amino acid residues at other positions remain unchanged;

[0046] The MHETase-EstD9 mutant T163F is a mutant in which the threonine at position 163 in the amino acid sequence shown in SEQ ID No. 1 is site-directedly mutated to phenylalanine, while the amino acid residues at other positions remain unchanged;

[0047] The MHETase-EstD9 mutant T185V is a site-directed mutation of the threonine at position 185 in the amino acid sequence shown in SEQ ID No. 1 to valine, while the amino acid residues at other positions remain unchanged;

[0048] The MHETase-EstD9 mutant E209R is a mutant in which the glutamic acid at position 209 in the amino acid sequence shown in SEQ ID No. 1 is site-directedly mutated to arginine, while the amino acid residues at other positions remain unchanged;

[0049] The MHETase-EstD9 mutant E220N is a mutant in which the glutamic acid at position 220 in the amino acid sequence shown in SEQ ID No. 1 is site-directedly mutated to asparagine, while the amino acid residues at other positions remain unchanged;

[0050] The MHETase-EstD9 mutant I121E is a mutant in which the isoleucine at position 121 in the amino acid sequence shown in SEQ ID No. 1 is site-directedly mutated to glutamic acid, while the amino acid residues at other positions remain unchanged;

[0051] The MHETase-EstD9 mutant G130L-I171K is a mutant in which the glycine at position 130 in the amino acid sequence shown in SEQ ID No. 1 is site-directedly mutated to leucine, and the isoleucine (I) at position 171 is site-directedly mutated to lysine, while the amino acid residues at other positions remain unchanged.

[0052] The statistics of site-directed mutagenesis primers used in the construction of the above-mentioned MHETase-EstD9 mutant recombinant plasmid are shown in Table 1 below.

[0053] Table 1

[0054]

[0055] Using site-directed mutagenesis, PCR was performed using the recombinant plasmid pET-22b-MHETase-EstD9 constructed above as a template and the primer pairs listed in Table 1 to obtain linearized plasmid fragments. The PCR reaction system was 20 μL, consisting mainly of 1 μL template (plasmid), 1 μL forward primer (F), 1 μL reverse primer (R), 10 μL high-fidelity amplification reagent, and the remainder enzyme-free water. The PCR product was digested with DMT enzyme (GD111 from Quanshijin Co., Ltd.) and 10 recombinant plasmids, including pET-22b-G130L-I171K, pET-22b-E40M, pET-22b-E78L, pET-22b-K83N, pET-22b-E124P, pET-22b-T163F, pET-22b-T185V, pET-22b-E209R, pET-22b-E220N, and pET-22b-I121E, were obtained by molecular biological methods such as seamless cloning. The plasmid map of the recombinant plasmid pET-22b-G130L-I171K is shown in the figure. Figure 2 shown.

[0056] The PCR reaction conditions in this step were as follows: pre-denaturation at 98°C for 3 min; denaturation at 98°C for 15 s; annealing at 68°C for 15 s; extension at 72°C for 3 min; and final extension at 72°C for 5 min, for 30 cycles.

[0057] The amino acid sequence of wild-type MHETase-EstD9 is shown in SEQ ID No. 1, and is as follows:

[0058] MMKIVSPKPFTFEAGERAVLLLHGFTGNSSDVRMLGRFLESKGYTCHAPIYKGHGVPPEELVHTGPDDWWQDVMNAYEFLREKGYQKIAVVGLSLGGVFSLKLGYTVPVVGIVPMCAPMYIKSE ETMYQGVLDYAREYKKREGKAPEQIEKEMEEFRKTPMKTLKALQALIAEVRNHIDLIYAPTFVVQARHDDMINTDSANIIYNGVESPIKQIKWYEESGHVITLDKEKEQLHEDIYAFLESLDW.

[0059] The encoding gene of wild-type MHETase-EstD9 is shown in SEQ ID No. 3, specifically:

[0060] ATGATGAAAATTGTGAGCCCGAAACCGTTTACCTTTGAAGCGGGCGAACGCGCGGTGCTGTTACTGCATGGCTTTACCGGCAACAGCAGCGATGTGCGCATGCTGGGCCGCTTCCTGGAAAGCAAAGGTTATACCTGCCATGCGCCGATTTATAAAGGCCATGGCGTGCCGCCGGAAGAACTGGTGCATACCGGCCCGGATGATTGGTGGCAAGATGTGATGAACGCGTATGAATTTCTGCGCGAAAAAGGCTATCAGAAAATTGCGGTGGTGGGCCTGAGCCTGGGCGGCGTGTTTAGCCTGAAACTGGGCTATACCGTGCCGGTGGTGGGCATTGTGCCGATGTGCGCGCCGATGTATATTAAAAGCGAAGAAACCATGTATCAAGGCGTGCTGGATTATGCGCGCGAATATAAAAAACGCGAAGGCAAAGCGCCGGAACAGATTGAAAAAGAAATGGAAGAATTTCGCAAAACCCCGATGAAAACCCTGAAAGCGCTGCAAGCGCTGATTGCGGAAGTGCGCAACCATATTGATCTGATTTATGCGCCGACCTTTGTGGTGCAAGCGCGCCATGATGATATGATTAACACCGATAGCGCGAACATTATTTATAACGGCGTGGAAAGCCCGATTAAACAGATTAAATGGTATGAAGAAAGCGGCCATGTGATTACCCTGGATAAAGAAAAAGAACAGCTGCATGAAGATATTTATGCGTTTCTGGAAAGCCTGGATTGG。

[0061] II. Construction of MHETase-EstD9 Mutant Engineering Strains

[0062] The recombinant plasmid pET-22b-MHETase-EstD9 and its ten mutant recombinant plasmids were introduced into competent Escherichia coli BL21 (DE3) cells (Quanshijin Co., CD601), gently mixed, and placed on ice for 30 minutes. The cells were then heat-shocked in a 42°C water bath for 45 seconds, quickly transferred to an ice bath for 2 minutes, and 500 μL of sterile LB medium (without antibiotics) was added. The cells were mixed thoroughly and incubated at 37°C and 200 rpm for 1 hour to allow bacterial recovery. Following this, the cells were centrifuged at 6000 rpm for 90 seconds, and 450 μL of the supernatant was removed. The remaining supernatant was added to LB agar, and the cells were evenly spread until the liquid was absorbed. The plates were then inverted and incubated at 37°C for 12 hours. Then, single clone colonies were picked and transferred to LB liquid culture medium containing ampicillin resistance and cultured for 12 hours. Positive single clone strains were selected and verified, and the wild-type MHETase-EstD9 engineered strain and ten MHETase-EstD9 mutant engineered strains including the MHETase-EstD9 mutant E40M engineered strain were obtained accordingly.

[0063] 3. Preparation and purification of MHETase-EstD9 mutant

[0064] The 10 engineered strains obtained above were cultured in 5 mL of LB medium at 37°C and 220 rpm for 12 h, and then inoculated into a 100 mL LB medium shake flask at a 1% inoculum for fermentation. The culture was shaken at 37°C and 220 rpm for 3 h to achieve a bacterial concentration of OD600nm between 0.8 and 1. Then, isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM, and expression was induced at 16°C and 160 rpm for 14 h to 18 h to obtain a bacterial solution rich in MHETase.

[0065] The bacterial suspension was centrifuged at 8000g for 5 minutes in a high-speed refrigerated centrifuge to collect the cells. The cells were resuspended in 10 mL of lysis buffer (50 mM Tris-HCl, 150 mM NaCl, and 10 mM imidazole per liter, pH 7.5). The collected cells were then disrupted using a high-pressure disruptor. After disruption, the cells were centrifuged at 10,000 rpm for 1 hour to remove cell debris. The supernatant contained the complete protein solution containing MHETase-EstD9 and its mutants.

[0066] The whole protein solution was passed through a 0.45 μm membrane to remove impurities, and then purified using a Ni-NTA packed column and gradient elution to obtain the target protein. The specific steps were as follows: first, equilibrate with lysis buffer for 2 minutes, then repeatedly apply the whole protein solution after the membrane to the column three times, wash three times with wash buffer (50 mM Tris-HCl, 150 mM NaCl, and 80 mM imidazole per liter, pH = 7.5) to remove impurities; then elute with elution buffer (50 mM Tris-HCl, 300 mM NaCl, and 300 mM imidazole per liter, pH = 7.5) to obtain a protein eluate; further concentrate the protein, and dilute the imidazole in the protein eluate to 1‰ of the original concentration by changing the wash buffer three times (20 mM Tris-HCl and 300 mM NaCl per liter, pH = 7.5), thereby obtaining a concentrated protein solution.

[0067] 4. Performance Characterization Methods

[0068] 1. Determination of BHET degradation activity

[0069] In the present invention, BHET is used as a substrate and dissolved in dimethyl sulfoxide to prepare a BHET solution with a concentration of 600 mM.

[0070] The concentrated protein solution was added to a 300 μL reaction solution (100 mM potassium phosphate buffer, pH 8) at the corresponding concentration (500 nM). 1.5 μL of BHET solution was then added to the reaction solution and allowed to react in a water bath at different temperatures for 1 hour. Acetonitrile was added to terminate the reaction, and the levels of TPA, MHET, and BHET produced were determined by high-performance liquid chromatography (HPLC). The percentage of TPA in the system was used as an indicator of BHET degradation activity, which was then used to characterize the degradation activity.

[0071] The HPLC assay conditions are as follows:

[0072] Chromatographic column: ZORBAX Eclipse Plus C18 reverse phase column; column temperature: 30°C;

[0073] Mobile phase: Mobile phase A is 0.1% formic acid-distilled water, and mobile phase B is acetonitrile;

[0074] Flow rate: 0.8 mL / min;

[0075] Gradient elution: the mobile phase changed from 95% mobile phase A to 30% mobile phase A within 20 min;

[0076] Detection wavelength: 240nm.

[0077] 2. Tm value determination method

[0078] Protein melting temperatures were determined using differential scanning fluorimetry (DSF). Protein samples were loaded into a 96-well plate, with each well containing 25 μL of the sample containing 15 μL of enzyme stock solution (containing 20 mM Tris-HCl and 300 mM NaCl per liter), 9 μL of protein solution (0.4 mg / mL), and 1 μL of SYPRO Orange dilution solution. DSF experiments were performed using a real-time fluorescence quantitative PCR system with a 465 nm excitation and 580 nm emission filter. Samples were heated from 25°C to 100°C at a rate of 0.3°C / s, and fluorescence was measured every 0.03 s. Tm was determined from the first-order derivative curve.

[0079] 5. Experimental Results

[0080] The above-mentioned assay method was used to determine the BHET degradation activity and Tm value of the wild-type MHETase-EstD9 and its mutants obtained in this example. The results of the BHET degradation activity and Tm value of the wild-type MHETase-EstD9 and its mutants at 50°C are shown in the following table. Figure 3 The results of the determination of BHET degradation activity and Tm of wild-type MHETase-EstD9 and its mutants at 60°C are shown in Figure 4 The black star corresponds to the right vertical axis representing the Tm value. The control group in the figure used 100 mM potassium phosphate buffer without enzyme as a blank control, D9 represents wild-type MHETase-EstD9, and E40M to I171K-G130L correspond to MHETase-EstD9 mutants E40M to I171K-G130L, respectively.

[0081] Depend on Figures 3 and 4 The 10 mutants exhibited varying degrees of improvement in BHET substrate degradation compared to wild-type MHETase-EstD9 at both 50°C and 60°C. At 50°C, the TPA yields of the MHETase-EstD9 mutants increased by 1.38- to 38.87-fold, with the MHETase-EstD9 mutant I171K-G130L exhibiting the best catalytic performance. At 60°C, the TPA yields of the MHETase-EstD9 mutants increased by 15% to 342%, with the MHETase-EstD9 mutant I171K-G130L also exhibiting the best catalytic performance.

[0082] Compared with wild-type MHETase-EstD9, the MHETase-EstD9 mutant I171K-G130L showed 38.87-fold and 3.42-fold increases in BHET degradation activity at 50°C and 60°C, respectively, but its Tm decreased by approximately 6.3°C. Compared with wild-type MHETase-EstD9, the MHETase-EstD9 mutant E209R showed 4.68-fold and 1.54-fold increases in BHET degradation activity at 50°C and 60°C, respectively, and its Tm increased by 4.0°C.

[0083] Example 2

[0084] Based on Example 1, in this example, the MHETase-EstD9 mutant I171K-G130L (named MHETase-EstD9 mutant KL) with significantly improved BHET degradation activity was further subjected to site-directed mutagenesis to obtain 18 mutants of MHETase-EstD9 mutants: KL-S29P, KL-C46V, KL-I50L, KL-S100A, KL-G104A, KL-I178L, KL-T198P, KL-P7C-G55C (abbreviated as KL-7-55C), KL-V110E, KL-H177K, KL-H47M, KL-Q129L, KL-H192R, KL-Y239F, KL-M34E, KL-M195L, KL-R33L and KL-H63L.

[0085] The statistics of site-directed mutagenesis primers used to construct recombinant plasmids based on single-point or double-point mutation of MHETase-EstD9 mutant KL are shown in Table 2-3 below.

[0086] Table 2

[0087]

[0088] Table 3

[0089]

[0090] Using site-directed mutagenesis technology, the recombinant plasmid pET-22b-G130L-I171K constructed in Example 1 was used as a template and the primer pairs shown in Table 2-3 were used for PCR to prepare pET-22b-KL-S29P, pET-22b-KL-C46V, pET-22b-KL-I50L, pET-22b-KL-S100A, pET-22b-KL-G104A, pET-22b-KL-I178L, pET-22b-KL-T198P, pET-22b-KL-7-55C, pET Eighteen MHETase-KL mutant recombinant plasmids, including pET-22b-KL-V110E, pET-22b-KL-H177K, pET-22b-KL-H47M, pET-22b-KL-Q129L, pET-22b-KL-H192R, pET-22b-KL-Y239F, pET-22b-KL-M34E, pET-22b-KL-M195L, pET-22b-KL-R33L, and pET-22b-KL-H63L, were constructed using the same method as in Example 1. The plasmid map of the recombinant plasmid pET-22b-P7C-G55C-G130L-I171K (designated as recombinant plasmid pET-22b-KLC) is shown in FIG. Figure 5 shown.

[0091] Using the recombinant plasmid constructed in this example, the corresponding MHETase-EstD9 mutant engineered strain was further constructed according to the method described in Example 1. The corresponding mutants were prepared and the BHET degradation activity and Tm of different MHETase-EstD9 mutants were measured. The results of the BHET degradation activity and Tm of the wild-type MHETase-EstD9 and its mutants at 50°C are shown in Figure 2. Figure 6 The results of the determination of BHET degradation activity and Tm of wild-type MHETase-EstD9 and its mutants at 60°C are shown in Figure 7 shown.

[0092] Depend on Figure 6-7It can be seen that at 50°C, compared with the MHETase-EstD9 mutant KL, the BHET degradation activity of the other MHETase-EstD9 mutants constructed in this example, except for the mutant KL-7-55C, was increased by 13.3% to 53.4%; compared with the wild-type MHETase-EstD9, the BHET degradation activity of the other MHETase-EstD9 mutants constructed in this example, except for the mutant KL-7-55C, was increased by 31.8 times to 58.8 times, respectively. At 60°C, compared with the MHETase-EstD9 mutant KL, the BHET degradation activities of 13 MHETase-EstD9 mutants, including KL-S29P, KL-C46V, KL-S100A, KL-G104A, KL-I178L, KL-T198P, KL-7-55C, KL-H47M, KL-Q129L, KL-H192R, KL-M34E, KL-R33L and KL-H63L, were significantly improved.

[0093] In terms of thermal stability, the Tm values ​​of MHETase-EstD9 mutants KL-S29P, KL-M34E, KL-S100A, KL-I178L and KL-7-55C were all increased by more than 2°C compared with MHETase-EstD9 mutant KL. Among them, the Tm value of MHETase-EstD9 mutant KL-7-55C (named MHETase-EstD9 mutant KLC) was increased by 5.47°C compared with MHETase-EstD9 mutant KL.

[0094] The amino acid sequence of the MHETase-EstD9 mutant KLC is shown in SEQ ID No. 2, specifically:

[0095] MMKIVSCKPFTFEAGERAVLLLHGFTGNSSDVRMLGRFLESKGYTCHAPIYKGHCVPPEELVHTGPDDWWQDVMNAYEFLREKGYQKIAVVGLSLGGVFSLKLGYTVPVVGIVPMCAPMYIKSE ETMYQLVLDYAREYKKREGKAPEQIEKEMEEFRKTPMKTLKALQALKAEVRNHIDLIYAPTFVVQARHDDMINTDSANIIYNGVESPIKQIKWYEESGHVITLDKEKEQLHEDIYAFLESLDW.

[0096] Example 3

[0097] On the basis of Example 2, this example comprehensively considered further mutation of the MHETase-EstD9 mutant KLC to obtain MHETase-EstD9 mutant KLC-E209R (named MHETase-EstD9 mutant KLCR), MHETase-EstD9 mutant KLC-I121E-E209R (named MHETase-EstD9 mutant KLCRE), MHETase-EstD9 mutant KLC-S29P-M34E-I121E-E209R (named The amino acid sequences of the five mutants are as follows:

[0098] The amino acid sequence of the MHETase-EstD9 mutant KLCR is as follows: the amino acid sequence is site-directedly mutated from glutamic acid at position 209 of the amino acid sequence shown in SEQ ID No. 2 to arginine, and the amino acid residues at other positions remain unchanged;

[0099] The amino acid sequence of the MHETase-EstD9 mutant KLCRE is as follows: the isoleucine at position 121 of the amino acid sequence shown in SEQ ID No. 2 is site-directedly mutated to glutamic acid, and the glutamic acid at position 209 is site-directedly mutated to arginine, while the amino acid residues at other positions remain unchanged;

[0100] The amino acid sequence of the MHETase-EstD9 mutant KLCREPE is as follows: the serine at position 29 of the amino acid sequence shown in SEQ ID No. 2 is site-directedly mutated to proline, the methionine at position 34 is site-directedly mutated to glutamic acid, the isoleucine at position 121 is site-directedly mutated to glutamic acid, and the glutamic acid at position 209 is site-directedly mutated to arginine, while the amino acid residues at other positions remain unchanged;

[0101] The amino acid sequence of the MHETase-EstD9 mutant KLCREPEAL is as follows: the serine at position 29 of the amino acid sequence shown in SEQ ID No. 2 is site-directedly mutated to proline, the methionine at position 34 is site-directedly mutated to glutamic acid, the serine at position 100 is site-directedly mutated to alanine, the isoleucine at position 121 is site-directedly mutated to glutamic acid, the isoleucine at position 178 is site-directedly mutated to leucine, and the glutamic acid at position 209 is site-directedly mutated to arginine, while the amino acid residues at other positions remain unchanged;

[0102] The amino acid sequence of the MHETase-EstD9 mutant KLCREPEALF is as follows: the serine at position 29 of the amino acid sequence shown in SEQ ID No. 2 is site-directedly mutated to proline, the methionine at position 34 is site-directedly mutated to glutamic acid, the serine at position 100 is site-directedly mutated to alanine, the isoleucine at position 121 is site-directedly mutated to glutamic acid, the threonine at position 163 is site-directedly mutated to phenylalanine, the isoleucine at position 178 is site-directedly mutated to leucine, and the glutamic acid at position 209 is site-directedly mutated to arginine, while the amino acid residues at other positions remain unchanged.

[0103] Based on the site-directed mutagenesis of the MHETase-EstD9 mutant, the statistics of the site-directed mutagenesis primers used to construct the corresponding recombinant plasmids of the mutants KLCR, KLCRE, KLCREPE, KLCREPEAL and KLCREPEALF are shown in Table 4 below.

[0104] Table 4

[0105]

[0106] Using site-directed mutagenesis technology, the recombinant plasmid pET-22b-P7C-G55C-G130L-I171K constructed in Example 2 was used as a template and PCR was performed with the primers shown in Table 4 to prepare five MHETase mutant recombinant plasmids, including recombinant plasmids pET-22b-KLCR, pET-22b-KLCRE, pET-22b-KLCREPE, pET-22b-KLCREPEAL and pET-22b-KLCREPEALF. The method for constructing the recombinant plasmids was the same as in Example 1.

[0107] Furthermore, after constructing five MHETase-EstD9 mutant strains, including KLCR, KLCRE, KLCREPE, KLCREPEAL, and KLCREPEALF, using the method described in Example 1, the corresponding mutants were prepared and the BHET degradation activity and Tm of the different MHETase-EstD9 mutants were measured. The results of the BHET degradation activity and Tm of the wild-type MHETase-EstD9 and its mutants at 70°C are shown in Figure 2. Figure 8 shown.

[0108] Depend on Figure 8 Further mutations of the MHETase-EstD9 mutant KLC significantly increased the Tm values ​​and BHET degradation activities of the MHETase-EstD9 mutants KLCR, KLCRE, KLCREPE, KLCREPEAL, and KLCREPEALF. The Tm values ​​increased by 4.3°C to 17.2°C, and TPA yields increased by 10.4-fold to 24.2-fold. Compared with the MHETase-EstD9 mutant KLC, the MHETase-EstD9 mutant KLCREPEALF exhibited an 18.0°C increase in Tm value and a 21.0-fold increase in BHET degradation activity at 70°C. Compared with wild-type MHETase-EstD9, the KLCREPEALF mutant exhibited a 17.1°C increase in Tm value and a 24.2-fold increase in BHET degradation activity. Based on a comprehensive analysis of activity and thermal stability, the MHETase-EstD9 mutant KLCREPEALF exhibited the best performance.

[0109] Example 4

[0110] Given that the MHETase-EstD9 mutant provided by the present invention can increase BHET degradation activity by more than 15%, with a maximum increase of 24.2-fold, while maintaining a substantially unchanged or significantly improved Tm value, it can significantly promote the complete degradation of PET and facilitate the recovery of TPA. Therefore, the MHETase-EstD9 mutant provided by the present invention, used alone or in combination with a PET degrader, has great application potential in the fields of degrading PET, MHET, or BHET, preparing degradation agents for PET and its degradation products, or recovering TPA.

[0111] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A MHETase-EstD9 mutant, characterized in that: The proline at position 7 in the amino acid sequence of the wild-type MHETase-EstD9 shown in SEQ ID No.1 is site-directedly mutated to cysteine, the glycine at position 55 is site-directedly mutated to cysteine, the glycine at position 130 is site-directedly mutated to leucine, and the isoleucine at position 171 is site-directedly mutated to lysine, while the amino acid residues at other positions remain unchanged; the amino acid sequence of the MHETase-EstD9 mutant is shown in SEQ ID No.

2.

2. A MHETase-EstD9 mutant, characterized in that: The method comprises the following steps: site-directed mutation of the glutamic acid at position 209 of the amino acid sequence shown in SEQ ID No. 2 as claimed in claim 1 to arginine, while the amino acid residues at other positions remain unchanged.

3. A MHETase-EstD9 mutant, characterized in that: The amino acid sequence is obtained by replacing the amino acid residues at at least two positions in the amino acid sequence shown in SEQ ID No. 2 as claimed in claim 1, and the amino acid sequence is any one of the following (1) to (4): (1) The isoleucine at position 121 of the amino acid sequence shown in SEQ ID No. 2 was site-directedly mutated to glutamic acid and the glutamic acid at position 209 was site-directedly mutated to arginine, while the amino acid residues at other positions remained unchanged; (2) The serine at position 29 of the amino acid sequence shown in SEQ ID No. 2 was site-directedly mutated to proline, the methionine at position 34 was site-directedly mutated to glutamic acid, the isoleucine at position 121 was site-directedly mutated to glutamic acid, and the glutamic acid at position 209 was site-directedly mutated to arginine, while the amino acid residues at other positions remained unchanged; (3) The serine at position 29 of the amino acid sequence shown in SEQ ID No. 2 was site-directedly mutated to proline, the methionine at position 34 was site-directedly mutated to glutamic acid, the serine at position 100 was site-directedly mutated to alanine, the isoleucine at position 121 was site-directedly mutated to glutamic acid, the isoleucine at position 178 was site-directedly mutated to leucine, and the glutamic acid at position 209 was site-directedly mutated to arginine, while the amino acid residues at other positions remained unchanged; (4) The serine at position 29 of the amino acid sequence shown in SEQ ID No. 2 was site-directedly mutated to proline, the methionine at position 34 was site-directedly mutated to glutamic acid, the serine at position 100 was site-directedly mutated to alanine, the isoleucine at position 121 was site-directedly mutated to glutamic acid, the threonine at position 163 was site-directedly mutated to phenylalanine, the isoleucine at position 178 was site-directedly mutated to leucine, and the glutamic acid at position 209 was site-directedly mutated to arginine. The amino acid residues at other positions remained unchanged.

4. A recombinant plasmid, characterized in that: The recombinant plasmid can express the MHETase-EstD9 mutant according to any one of claims 1 to 3.

5. An engineered MHETase-EstD9 mutant strain, characterized in that: It comprises the recombinant plasmid according to claim 4.

6. Use of the MHETase-EstD9 mutant according to any one of claims 1 to 3 in degrading PET, MHET or BHET.

7. Use of the MHETase-EstD9 mutant according to any one of claims 1 to 3 in the preparation of a PET degrader, an MHET degrader or a BHET degrader.

8. Use of the MHETase-EstD9 mutant according to any one of claims 1 to 3 in recovering terephthalic acid, a PET degradation product.

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