PET degrading enzyme mutant and application thereof in PET material degradation

Abstract

The invention relates to the field of genetic engineering and PET material degradation, in particular to a PET degrading enzyme mutant and application thereof in PET material degradation. According to the present invention, the activity and the heat resistance of the PET degrading enzyme MuPETase derived from Mumia sp. Are modified, such that mutants Mut1-3 and Mut5-8 having good degradation ability on the PET material under the acidic condition are obtained through screening, and the amino acid sequences of the mutants Mut1-3 and Mut5-8 are sequentially represented by SEQ ID NO.3-5 and SEQ ID NO.7-10; compared with the original PET degrading enzyme, the thermal stability and / or the specific activity of the obtained mutant are / is remarkably improved, and the degradation effect on a PET material is greatly improved. The mutant obtained by the invention has important application value in the aspect of biodegradation treatment of single and mixed PET material wastes.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering and PET material degradation, specifically to a PET degrading enzyme mutant and its application in PET material degradation. Background Technology

[0002] Polyester plastics, as one of the key materials in modern industrial systems, are widely used in many important fields such as packaging, textile fibers, and medical devices. Enzyme-catalyzed biodegradation technology, due to its mild conditions, controllable products, and environmentally friendly characteristics, is considered a potential key technological path to solve the waste problem of polyester plastics and achieve recycling.

[0003] Since the first discovery of PET-degrading enzymes in 2016, various natural and engineered enzymes (such as PETase, LCCICCG variants, and AI-designed FAST-PETase) have been developed. These enzymes can efficiently depolymerize PET under mild conditions and recover the products as usable monomers, demonstrating promising prospects for industrial applications. In the current industrialization of enzymatic PET degradation, continuous alkali supplementation to maintain the reaction system within a slightly alkaline range (typically pH 7.5–9.0) is a crucial operation. The main reason is not to directly "activate" the enzyme, but to precisely neutralize the acidic products generated in the reaction, thereby maintaining the stable pH environment required for enzyme activity and ensuring the efficient and continuous depolymerization reaction. While continuous alkali supplementation sustains the reaction, it also increases process complexity and operating costs, and may introduce additional salts, thus affecting the subsequent separation and purification of monomers.

[0004] If efficient depolymerization of PET can be achieved under acidic conditions, the acidic environment of the reaction system can be directly utilized, significantly reducing or even eliminating the need for alkali solutions. This would simplify the process, lower costs, and facilitate continuous industrial production. Therefore, developing a PET-degrading enzyme that maintains high catalytic efficiency and stability under acidic conditions is a key technological breakthrough for advancing the industrialization of PET biopolymerization technology and reducing processing costs. Summary of the Invention

[0005] The technical problem to be solved by the present invention is that the existing technology requires continuous alkali replenishment to maintain the reaction system in industrial operation, which increases the complexity of the process and the operating cost, and may introduce additional salt, thereby affecting the subsequent separation and purification of monomers. The present invention provides a PET degrading enzyme mutant.

[0006] The technical problem that this invention also aims to solve is to provide a nucleic acid molecule, a recombinant expression vector, and a recombinant bacterial strain.

[0007] Another technical problem to be solved by the present invention is to provide a method for preparing the protein of the PET degradation enzyme mutant.

[0008] Another technical problem to be solved by the present invention is to provide the application of the PET degrading enzyme mutant, the recombinant expression vector, or the recombinant strain in the degradation of PET materials.

[0009] The final technical problem to be solved by the present invention is to provide a method for degrading PET materials using the PET degrading enzyme mutant.

[0010] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0011] In a first aspect, the present invention provides a PET degrading enzyme mutant, wherein the amino acid sequence of the PET degrading enzyme mutant is obtained by mutating N to F at position 49 of the amino acid sequence of the wild-type PET degrading enzyme;

[0012] The amino acid sequence of the wild-type PET degrading enzyme is shown in SEQ ID NO.1.

[0013] The wild-type PET degrading enzyme is derived from Mumia sp. and belongs to the α / β hydrolase family along with PET degrading enzymes LCC, TfCut2 and IsPETase, and has degradation activity for polyester plastics.

[0014] Furthermore, the present invention provides a PET degrading enzyme mutant, wherein the amino acid sequence of the PET degrading enzyme mutant is obtained by mutating N at position 49 to F and N at position 171 to A in the amino acid sequence of the wild-type PET degrading enzyme.

[0015] Alternatively, it can be obtained by mutating N at position 49 to F and simultaneously mutating G at position 224 to F;

[0016] Alternatively, it can be obtained by mutating N at position 49 to F, mutating N at position 171 to A, and mutating G at position 224 to F;

[0017] Alternatively, the 49th N position mutates to F, the 171st N position mutates to A, the 224th G position mutates to F, and the 92nd F position mutates to Y.

[0018] Alternatively, the mutation at position 49 (N) is changed to F, while at position 171 (N) it is changed to A, position 224 (G) it is changed to F, position 92 (F) it is changed to Y, and position 97 (S) it is changed to E.

[0019] Alternatively, the 49th N position mutates to F, the 171st N position mutates to A, the 224th G position mutates to F, the 92nd F position mutates to Y, and the 98th A position mutates to S.

[0020] Preferably, the amino acid sequence of the PET degrading enzyme mutant is obtained by mutating N at position 49 to F, N at position 171 to A, G at position 224 to F, F at position 92 to Y, and S at position 97 to E from the amino acid sequence of the wild-type PET degrading enzyme.

[0021] Alternatively, the 49th N position mutates to F, the 171st N position mutates to A, the 224th G position mutates to F, and the 92nd F position mutates to Y.

[0022] Alternatively, it can be obtained by mutating N at position 49 to F, while mutating N at position 171 to A and mutating G at position 224 to F.

[0023] More preferably, the amino acid sequence of the PET degrading enzyme mutant is obtained by mutating N at position 49 to F, N at position 171 to A, G at position 224 to F, F at position 92 to Y, and S at position 97 to E in the amino acid sequence of the wild-type PET degrading enzyme.

[0024] Alternatively, the 49th N position mutates to F, the 171st N position mutates to A, the 224th G position mutates to F, and the 92nd F position mutates to Y.

[0025] Most preferably, the amino acid sequence of the PET degrading enzyme mutant is obtained by mutating N at position 49 to F, N at position 171 to A, G at position 224 to F, F at position 92 to Y, and S at position 97 to E from the amino acid sequence of the wild-type PET degrading enzyme.

[0026] The PET degrading enzyme mutant exhibits good degradation ability of PET plastic under acidic conditions, and shows increased thermal stability and / or increased specific activity compared with wild-type PET degrading enzyme.

[0027] In some embodiments of the present invention, the acidic environment is a pH 4.0 to 6.0 environment.

[0028] In some embodiments of the present invention, the acidic environment is a pH 5.0 environment.

[0029] Secondly, the present invention provides a nucleic acid molecule that encodes the gene encoding the PET degradation enzyme mutant.

[0030] Thirdly, the present invention provides a recombinant expression vector containing the aforementioned nucleic acid molecule.

[0031] Fourthly, the present invention provides a recombinant strain, which is obtained by introducing the nucleic acid molecule into a host cell, or by introducing the nucleic acid molecule into a host cell through the recombinant expression vector.

[0032] In some embodiments of the present invention, the host cell is Escherichia coli BL21(DE3).

[0033] Fifthly, the present invention provides a method for preparing the protein of the PET degrading enzyme mutant, wherein the seed culture of the recombinant strain is inoculated into a fermentation medium and cultured to express the protein of the PET degrading enzyme mutant.

[0034] In some embodiments of the present invention, the seed culture of the recombinant strain is transferred to LB medium containing kanamycin and cultured until OD200. 600 =0.8~1.0, after adding IPTG to a final concentration of 0.5 mM, continue culturing to express the protein of the PET degrading enzyme mutant.

[0035] In a sixth aspect, the present invention provides the application of the PET degrading enzyme mutant, the recombinant expression vector, or the recombinant strain in the degradation of PET materials.

[0036] The PET material is either PET plastic or PET fiber.

[0037] Specifically, the PET plastic includes any one or a combination of two of PET lunch boxes and PET films, and the PET fiber includes any one or a combination of several of PET spinning, PET pillow cores, and PET fabrics.

[0038] In a seventh aspect, the present invention provides a method for degrading PET material using the PET degrading enzyme mutant, wherein an enzyme solution of the PET degrading enzyme mutant is added to a buffer containing PET material to carry out a depolymerization reaction.

[0039] The depolymerization reaction is carried out in the following reaction system: 2 M pH 5.0 citrate buffer, 5 g / L PET material at a final concentration, and 10 mg / L PET degrading enzyme mutant enzyme solution at a final concentration; the reaction conditions are: incubation at 50~70℃ for 40~55h.

[0040] In some embodiments of the present invention, the depolymerization reaction is carried out in a reaction system consisting of 2 M pH 5.0 citrate buffer, 5 g / L PET material at a final concentration, and an enzyme solution of 10 mg / L PET degrading enzyme mutant at a final concentration; the reaction conditions are: incubation at 60°C for 48 h.

[0041] Beneficial Effects: This invention constructs a MuPETase mutant library through error-prone PCR and homologous recombination of the PET-degrading enzyme (MuPETase). Further modifications to the activity and heat resistance of MuPETase lead to the screening of mutants exhibiting good degradation ability against PET plastics under acidic conditions. Compared to the original PET-degrading enzyme, the mutants show significantly improved thermal stability and / or specific activity, thereby enhancing the degradation effect on PET materials. This invention has significant application value in the biodegradation treatment of PET waste. Attached Figure Description

[0042] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0043] Figure 1 Phylogenetic tree of MuPETase, a PET degrading enzyme.

[0044] Figure 2 The results are shown in the SDS-PAGE of MuPETase, a PET-degrading enzyme. Lane 1 contains cell-broken bacterial sludge, lane 2 contains cell-broken supernatant, lane 3 contains enzyme solution eluted with 50 mM imidazole, and lane 4 contains pure enzyme solution eluted with 300 mM imidazole.

[0045] Figure 3 The optimal pH result for the PET-degrading enzyme MuPETase.

[0046] Figure 4 The results show the acid resistance of the PET-degrading enzyme MuPETase. In this diagram, A represents the residual activity under different pH conditions; B represents the depolymerization activity under different pH conditions.

[0047] Figure 5 The depolymerization effects of PET degrading enzymes MuPETase mutants Mut7 and IsPETase on different PET materials. Detailed Implementation

[0048] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; the reagents and materials described are commercially available unless otherwise specified.

[0049] The detection methods used in the following embodiments are as follows:

[0050] (1) Protein detection methods

[0051] Protein concentrations were determined using a BSA protein assay kit, and the purity of each protein was analyzed by SDS-PAGE. First, a 1 mg / mL bovine serum albumin (BSA) stock solution was prepared. Then, phosphate buffer (50 mM, pH 7.0) was added to the stock solution to prepare a series of BSA stock solutions with concentrations ranging from 0.2 to 1 mg / L. These solutions were then diluted 10-fold to obtain a series of BSA dilution solutions with concentrations ranging from 0.02 to 0.1 mg / L, i.e., BSA standard solutions. 20 μL of each concentration gradient of BSA standard solution was added to a 96-well plate, followed by 200 μL of Coomassie Brilliant Blue solution. After thorough mixing, the mixture was allowed to stand for 3 min. The absorbance of different concentrations of BSA standard solutions at 595 nm was measured using a microplate reader, and a standard curve was plotted with protein concentration on the x-axis and absorbance on the y-axis. The absorbance of the samples could be measured using the same method, and the protein concentration could be calculated from the standard curve.

[0052] (2) Enzyme activity detection method

[0053] Add 10 μL of 10 mM p-nitrophenol octanoate (pNPO), 10 μL of enzyme solution, and 980 μL of phosphate buffer (50 mM, pH 7.0) to a 5 mL centrifuge tube, bringing the final volume to 1 mL. Heat the assay system at 37 °C for 10 min, then immediately cool on ice. Measure the absorbance at 410 nm using a microplate reader. The absorbance is determined by p-nitrophenol and OD... 410 The standard curve was plotted to calculate the molar amount of p-nitrophenol. Enzyme activity is defined as the amount of enzyme required to produce 1 μmol of p-nitrophenol per minute at 37°C.

[0054] Example 1: Expression and purification of PET-degrading enzyme (MuPETase)

[0055] 1. Construction of recombinant plasmid pET29a-Mumia sp

[0056] Screening for hydrolytic enzyme genes capable of degrading PET plastic in protein data revealed MuPETase, a PET degrading enzyme derived from Mumiasp. with the amino acid sequence shown in SEQ ID NO. 1. This enzyme, along with PET degrading enzymes LCC, TfCut2, and IsPETase, belongs to the α / β hydrolase family and exhibits degradation activity against polyester plastics. Its phylogenetic tree is shown below. Figure 1 As shown.

[0057] The nucleotide sequence of MuPETase was chemically synthesized (as shown in SEQ ID NO.2), and the obtained gene was ligated with the pET29a plasmid (Fenghui Biotechnology Co., Ltd.) using the Cloneexpress II One Step Cloning Kit (Novazan Biotechnology Co., Ltd., Vazyme). The ligation product was then transformed into Escherichia coli JM109 (Takara), and the transformation product was plated on LB solid medium and cultured at 37°C for 12–14 h. Four transformants were picked from the LB solid medium and inoculated into LB liquid medium. After culturing at 37°C for 12 h, the plasmid was extracted for enzyme digestion and sequencing verification. If the verification was correct, the recombinant plasmid pET29a-Mumia sp was obtained.

[0058] 2. Expression and purification of MuPETase

[0059] Recombinant plasmid pET29a-Mumia sp was transformed into Escherichia coli BL21(DE3) competent cells to obtain recombinant E. coli. The recombinant E. coli was inoculated into LB medium containing 50 μg / mL kanamycin and cultured overnight at 37°C and 220 rpm to obtain a seed culture. 1 mL of the seed culture was transferred to 50 mL of fresh LB medium containing 50 μg / mL kanamycin and cultured at 37°C and 220 rpm until OD (October Expiratory Time) was reached. 600 =0.8~1.0, add IPTG to a final concentration of 0.5 mM, continue culturing for 20 h, then stop fermentation and collect fermented cells by centrifugation.

[0060] Fermentation cells were suspended in PBS buffer. The cell suspension was then sonicated for 2 minutes using an FB 705 (Fisherbrand) sonicator with an amplitude of 30% (2 seconds ON and 1 second OFF cycles). The mixture was then centrifuged at 10,000 rpm for 30 min at 4°C to obtain the lysed supernatant and bacterial sludge, which were sampled separately. The lysed supernatant was purified by Ni column affinity chromatography. First, 10 mL of 50 mM imidazole was used to remove impurities, and the eluent was sampled. Then, 300 mM imidazole was used to elute the purified MuPETase enzyme solution, and samples were taken for SDS-PAGE analysis. The results are shown below. Figure 2 As shown, the purified MuPETase band size is 30 kDa (lane 4).

[0061] Example 2: Determination of the optimal pH for MuPETase

[0062] Add 10 μL of 10 mM p-nitrophenol octanoate (pNPO), 10 μL of LuPETase enzyme solution, and 980 μL of buffer solutions at different pH values ​​to 5 mL centrifuge tubes. The buffer solutions were 50 mM citrate buffer (pH 4.0, 5.0), 50 mM phosphate buffer (pH 6.0, 7.0, 8.0), and 50 mM glycine-NaOH buffer (pH 9.0, 10.0). React at 37℃ for 5 min, then measure enzyme activity. Enzyme activity under the optimal reaction pH conditions was considered 100%. The results are as follows: Figure 3 As shown, pH 7.0 is the optimal pH for MuPETase.

[0063] Example 3: MuPETase acid resistance test

[0064] The residual activity and depolymerization activity of MuPETase under different pH conditions were detected by the following specific detection methods.

[0065] 1. Residual activity detection

[0066] MuPETase enzyme solution was added to 5 mL of 50 mM citrate buffer (pH 4.0, 5.0), 5 mL of 50 mM phosphate buffer (pH 6.0, 7.0, 8.0), and 5 mL of 50 mM glycine-NaOH buffer (pH 9.0, 10.0) to a final concentration of 10 mg / L, and incubated at 37°C for 10 h. After incubation, residual enzyme activity was measured, with the highest residual enzyme activity taken as 100%.

[0067] Test results as follows Figure 4 As shown in A, the residual enzyme activity was highest after 10 h of incubation at pH 7.0. Compared to pH 7.0, more than 50% of the enzyme activity could still be maintained after 10 h of incubation at pH 5.0.

[0068] 2. Depolymerization activity detection

[0069] The purified and concentrated MuPETase was placed in 2 M citrate buffer (pH 4.0, 5.0), 2 M PBS buffer (pH 6.0, 7.0, 8.0), and 2 M glycine-NaOH buffer (pH 9.0, 10.0), respectively. PET powder was added to a final concentration of 5 g / L, and MuPETase was added at a ratio of 2‰ (m / m) to a final concentration of 10 mg / L. The reaction volume was 50 mL. After incubation at 60 °C for 48 h, the reaction solution was filtered through a 0.22 μm filter. The product was diluted appropriately according to its concentration, and then analyzed using an Agilent 1260 Infinity II high-performance liquid chromatography system with a C18 column (Thermo Fisher Scientific, 150 × 4.6 mm, 5 μm). The mobile phase consisted of 18% acetonitrile, 1% formic acid, and 81% water. The detection time was 25 min, the flow rate was 0.8 mL / min, and the detection wavelength was 254 nm. Standard curves were constructed using the detection results of TPA and MHET gradient standard solutions at concentrations of 1 mM, 0.1 mM, 0.01 mM, 0.001 mM, and 0.0001 mM, respectively. The product concentrations of the enzymatically digested samples were calculated from the standard curves.

[0070] Depolymerization effect Figure 4 As shown in Figure B, MuPETase exhibits the highest depolymerization rate of PET at pH 7.0. Even at pH 5.0, the depolymerization rate still reaches 77% of that at pH 7.0, indicating that MuPETase can effectively degrade PET under acidic conditions.

[0071] Example 4: Construction of a MuPETase mutant library

[0072] 1. PCR amplification

[0073] Based on the sequence of the PET-degrading enzyme (MuPETase), error-prone PCR primers and vector PCR primers were designed (primers are shown in Table 1). Error-prone PCR was performed on the recombinant plasmid pET29a-Mumia sp to amplify the MuPETase fragment containing the homologous arm of the plasmid vector. Simultaneously, high-fidelity PCR was performed to amplify the pET29a fragment of the recombinant plasmid pET29a-Mumia sp.

[0074] Table 1 Primer sequence information

[0075]

[0076] Error-prone PCR amplification was performed using the GeneMorph II Random Mutagenesis Kit (Stratagene). The reaction mixture consisted of: 5 μL of 10×Mutazyme II reaction buffer, 1 μL of Mutazyme II DNA polymerase, 1 μL of dNTP (40 mM), 1 μL of 1 ng / μL template DNA, 2 μL of 10 μM forward primer, 2 μL of 10 μM reverse primer, and double-distilled water to a final volume of 50 μL. Error-prone PCR amplification conditions were: pre-denaturation at 94℃ for 3 min; followed by 30 cycles of 94℃ for 30 s, 55℃ for 30 s, and 72℃ for 2 min; and a final incubation at 72℃ for 10 min.

[0077] The high-fidelity PCR amplification reaction system consisted of: 25 μL of 2×Phanta Max MasterMix (Vazyme, catalog number: P515), 2 μL of 10 μM forward primer, 2 μL of 10 μM reverse primer, 1 μL of 1 ng / μL template DNA, 1 μL of dNTPs (40 mM), and double-distilled water to a final volume of 50 μL. PCR amplification conditions were: 95℃ pre-denaturation for 3 min; followed by 30 cycles of 95℃ for 15 s, 61℃ for 15 s, and 72℃ for 5 min; and a final incubation at 72℃ for 10 min.

[0078] 2. Purification of PCR amplification products

[0079] All PCR amplification products were detected by 1% agarose gel electrophoresis. After detection, 1 μL of methylation template digesting enzyme (Dpn I) was added to 50 μL of amplification products, mixed by pipetting, and reacted at 37℃ for 1 h, followed by inactivation at 70℃ for 5 min to obtain the Dpn I digested product. The Dpn I digested product was purified using the Fastpure Gel DNA Extraction Mini Kit (Vazyme). Specifically, 150 μL of Buffer GDP was added to 50 μL of digested product, mixed by pipetting, and then transferred to an adsorption column. The column was centrifuged at 12000 rpm for 1 min, and the waste liquid at the bottom of the adsorption column was discarded. Then, 700 μL of Buffer GW was added to the adsorption column, centrifuged at 12000 rpm for 1 min, and the waste liquid at the bottom of the adsorption column was discarded. This process was repeated twice. Finally, the product was eluted with 40 μL of double-distilled water.

[0080] 3. Connecting products

[0081] The purified product of the MuPETase fragment and the purified product of the plasmid pET29a fragment were ligated by homologous polymer PCR to form a linear multimer ligation product.

[0082] The homologous polymerase PCR reaction system consisted of: 25 μL of 2×Phanta Max Master Mix (Vazyme), 2 μL of pET29a plasmid fragment (15 ng / μL), 2 μL of MuPETase fragment (250 ng / μL), and double-distilled water to a final volume of 50 μL. The PCR amplification conditions were: 95℃ pre-denaturation for 3 min; followed by 30 cycles of 95℃ for 15 s, 61℃ for 15 s, and 72℃ for 10 min; and a final incubation at 72℃ for 10 min.

[0083] 4. Transformation

[0084] The linear multimer ligation product obtained in step 3 was transformed into Escherichia coli BL21(DE3) competent cells, and the transformation product was then plated on LB solid medium containing a final concentration of 50 mg / L kanamycin and cultured at 37°C for 12 h to obtain a large number of BL21(DE3) single colonies containing the MuPETase mutant.

[0085] Example 5: Screening for MuPETase mutants

[0086] Single colonies of the original MuPETase BL21(DE3) and a large number of single colonies of BL21(DE3) containing MuPETase mutants obtained in Example 4 were inoculated into 96-well plates containing 1 mL of LB medium and fermented at 37°C for 24 h to obtain their respective fermentation sludge. The sludge was resuspended in 1 mL of 50 mM pH 5.0 citrate buffer and then sonicated for 2 minutes using an FB 705 (Fisherbrand) ultrasonic instrument with an amplitude of 30% (2 seconds ON and 1 second OFF cycles). The mixture was then centrifuged at 10,000 rpm at 4°C for 30 min to obtain the supernatant. Enzyme activity was measured in the supernatant. Mutants with enzyme activity in the supernatant not lower than that of the original MuPETase fermentation enzyme were selected for further thermostability, residual enzyme activity, and specific activity determination.

[0087] 1. Thermal stability determination (Tm value determination)

[0088] The thermal stability of MuPETase and its mutants was compared using the Tm value. Specifically, MuPETase and its mutant proteins were dialyzed in 50 mM citrate buffer (pH 5.0). The proteins were diluted to the same concentration of 1.0 mg / mL using dialysate. The proteins were filtered through a 0.22 μm filter and degassed. Differential scanning calorimetry (DSC) was used to scan the temperature range of 25–95°C at a scan rate of 1.0 °C / min and a filtration pressure of 2.5 atm. After scanning, a single symmetrical endothermic peak was obtained, and the peak value was the corresponding Tm value. The results are shown in Table 2.

[0089] 2. Residual enzyme activity assay

[0090] MuPETase and its mutants were placed in 50 mM pH 5.0 citrate buffer to a final concentration of 10 mg / mL and incubated at 70 °C for 10 h. After the reaction, samples were taken and diluted 1 to 500 times in 50 mM pH 5.0 citrate buffer for residual enzyme activity assay. The results are shown in Table 2.

[0091] 3. Specific activity calculation

[0092] The specific enzyme activities of MuPETase and its mutants were further calculated using the following formula:

[0093] Protein specific enzyme activity (U / mg) = Total enzyme activity of purified protein solution (U / mL) / Total protein content of purified protein solution (mg / L). The specific enzyme activity of MuPETase was used as a reference and considered as 100% degradation activity. The results of the specific enzyme activity calculation are shown in Table 2.

[0094] As shown in Table 2, the N49F, L122F, G224F, and N171A mutants exhibited significantly higher specific enzyme activities while having Tm values ​​comparable to MuPETase; the F92Y, S97E, and A98S mutants showed significantly higher Tm values ​​even with specific enzyme activities exceeding those of MuPETase. Therefore, N49F, F92Y, S97E, A98S, L122F, N171A, and G224F are preferred for further screening of subsequent combined mutations.

[0095] Table 2. Tm values, residual enzyme activity, and specific enzyme activity of MuPETase and mutants

[0096]

[0097] Example 6: Detection of thermal stability and specific activity of combined mutants

[0098] The positive mutation sites N49F, F92Y, S97E, L122F, N171A, and G224F of MuPETase in Example 5 were combined with mutations to obtain combined mutants (Table 3). The specific implementation method is as follows: According to the gene sequence of MuPETase (as shown in SEQ ID NO. 2), primers for introducing mutations of L122F, N171A, G224, F92Y, S97E, and A98S were designed and synthesized respectively, as shown in Table 3. Using the MuPETase-N49F plasmid as a template, PCR amplification was performed with the corresponding primers. The amplified PCR products were digested and transformed with enzymes, and verified by sequencing. The plasmids were extracted using a plasmid extraction kit to obtain the recombinant expression vectors pET29a-MuPETase-N49F / N171A, pET29a-MuPETase-N49F / L122F, and pET29a-MuPETase-N49F / G224 containing the MuPETase mutant gene. The amino acid sequences of the combined mutants N49F / N171A, N49F / L122F, and N49F / G224F obtained by mutation are shown in SEQ ID NO.4~6.

[0099] Using pET29a-MuPETase-N49F / G224 as a template and N171A-F and N171R as primers, the recombinant expression vector pET29a-MuPETase-N49F / N171A / G224N was obtained according to the above method. The amino acid sequence of the combined mutant N49F / N171A / G224N is shown in SEQ ID NO.7. Using pET29a-MuPETase-N49F / N171A / G224N as a template and F92Y-F and F92Y-R as primers, the recombinant expression vector pET29a-MuPETase-N49F / N171A / G224N / F92Y was obtained according to the above method. The amino acid sequence of the combined mutant N49F / N171A / G224N / F92Y is shown in SEQ ID NO.8. Using the recombinant expression vector pET29a-MuPETase-N49F / N171A / G224N / F92Y as a template, and with S97E-F, S97-R and A98S-F, A98S-R as primers, the recombinant expression vectors pET29a-MuPETase-N49F / N171A / G224N / F92Y / S97E and pET29a-MuPETase-N49F / N171A / G224N / F92Y / A98S were obtained according to the above method. The amino acid sequences of the combined mutants N49F / N171A / G224N / F92Y / S97E and N49F / N171A / G224N / F92Y / A98S are shown in SEQ ID NO. 9~10. The recombinant expression vector obtained above was transformed into Escherichia coli BL21(DE3) competent cells, and the lysate supernatant of the combined mutant was obtained according to the method in Example 5.

[0100] Table 3 Primer information used in Example 6

[0101]

[0102] The thermostability, residual enzyme activity, and specific activity of the combined mutants were determined according to the method in Example 5. The results are shown in Table 4. It can be seen that, except for the N49F+L122F combined mutant, which showed a decrease in Tm value and specific enzyme activity, the N49F+N171A, N49F+G224F, N49F+N171A+G224F, N49F+N171A+G224F+F92Y, N49F+N171A+G224F+F92Y+S97E, and N49F+N171A+G224F+F92Y+A98S combined mutants all showed significant increases in Tm value and specific enzyme activity under acidic conditions. Among them, the MuPETase combined mutant N49F+N171A+G224F+F92Y+S97E exhibited the best performance in terms of Tm value, residual enzyme activity, and specific enzyme activity.

[0103] Table 4. Tm values, residual enzyme activity, and specific enzyme activity of MuPETase combinatorial mutants

[0104]

[0105] Example 7: Evaluation of the depolymerization ability of the MuPETase combined mutant Mut7 on different PET materials

[0106] 50 mL reaction system: Different PET materials (final concentration 5 g / L) were added to 2 M pH 5.0 citrate buffer. MuPETase combination mutant Mut7 and IsPETase enzyme solutions (final concentration 10 mg / L) were added at an enzyme ratio of 2‰ (m / m). The reaction was incubated at 60℃ for 48 h. The depolymerization ability of MuPETase combination mutant Mut7 and IsPETase on different PET materials was evaluated and compared.

[0107] Experimental results are as follows Figure 5 As shown, under acidic conditions, IsPETase's depolymerization effect on different PET materials is far lower than that of the MuPETase hybrid mutant Mut7. Furthermore, the MuPETase hybrid mutant Mut7 produced significant depolymerization products for all PET materials, with its TPA+MHET production significantly higher than that of the control enzyme IsPETase. Specifically, the MuPETase hybrid mutant Mut7 consistently produced depolymerization products of 10–18 mM for post-consumer PET materials such as PET fibers, PET spindles, PET lunch boxes, PET pillow cores, PET fabrics, and PET films, while IsPETase, under the same substrate and reaction conditions, only produced 3–5 mM of products.

[0108] This invention provides a PET-degrading enzyme mutant and its application in the degradation of PET materials, along with a method. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. A PET-degrading enzyme mutant, characterized in that, The amino acid sequence of the PET degrading enzyme mutant is obtained by mutating N to F at position 49 of the amino acid sequence of the wild-type PET degrading enzyme; The amino acid sequence of the wild-type PET degrading enzyme is shown in SEQ ID NO.

1.

2. A PET-degrading enzyme mutant, characterized in that, The amino acid sequence of the PET degrading enzyme mutant is obtained by mutating N at position 49 to F and N at position 171 to A in the amino acid sequence of the wild-type PET degrading enzyme. Alternatively, it can be obtained by mutating N at position 49 to F and simultaneously mutating G at position 224 to F; Alternatively, it can be obtained by mutating N at position 49 to F, mutating N at position 171 to A, and mutating G at position 224 to F; Alternatively, the 49th N position mutates to F, the 171st N position mutates to A, the 224th G position mutates to F, and the 92nd F position mutates to Y. Alternatively, the mutation at position 49 (N) is changed to F, while at position 171 (N) it is changed to A, position 224 (G) it is changed to F, position 92 (F) it is changed to Y, and position 97 (S) it is changed to E. Alternatively, the mutation can be obtained by changing N at position 49 to F, N at position 171 to A, G at position 224 to F, F at position 92 to Y, and A at position 98 to S. The amino acid sequence of the wild-type PET degrading enzyme is shown in SEQ ID NO.

1.

3. A nucleic acid molecule, characterized in that, The encoding gene of the PET degrading enzyme mutant as described in claim 1 or 2.

4. A recombinant expression vector, characterized in that, It contains the nucleic acid molecule as described in claim 3.

5. A recombinant bacterial strain, characterized in that, The recombinant strain is obtained by introducing the nucleic acid molecule of claim 3 into a host cell, or by introducing the nucleic acid molecule of claim 3 into a host cell using the recombinant expression vector of claim 4.

6. A method for preparing the protein of the PET-degrading enzyme mutant according to claim 1 or 2, characterized in that, The seed culture of the recombinant strain of claim 5 is inoculated into a fermentation medium and cultured to express the protein of the PET degrading enzyme mutant.

7. The application of the PET degrading enzyme mutant of claim 1, or the PET degrading enzyme mutant of claim 2, or the recombinant expression vector of claim 4, or the recombinant strain of claim 5 in the degradation of PET materials.

8. The application according to claim 7, characterized in that, The PET material is PET plastic or PET fiber; wherein, the PET plastic includes any one or a combination of two of PET lunch boxes and PET films, and the PET fiber includes any one or a combination of several of PET spinning, PET pillow cores, and PET fabrics.

9. A method for degrading PET material using the PET-degrading enzyme mutant according to claim 1 or 2, characterized in that, An enzyme solution of the PET degradation enzyme mutant was added to a buffer solution containing PET material to carry out a depolymerization reaction.

10. The method according to claim 9, characterized in that, The depolymerization reaction is carried out in the following reaction system: 2 M pH 5.0 citrate buffer, 5 g / L PET material at a final concentration, and 10 mg / L PET degrading enzyme mutant enzyme solution at a final concentration; the reaction conditions are: incubation at 50~70℃ for 40~55 h.

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