A pet-degrading enzyme mutant and its application in pet material degradation

Abstract

The present application relates to the field of genetic engineering and PET material degradation, and particularly relates to a PET degrading enzyme mutant and its application in PET material degradation. The present application improves the activity and heat resistance of PET degrading enzyme MuPETase derived from Mumia sp., and screens to obtain mutants Mut1-3, Mut5-8 which show good degradation capacity to PET material under acidic conditions, and the amino acid sequences are shown in SEQ ID NO. 3-5, SEQ ID NO. 7-10 respectively. Compared with the original PET degrading enzyme, the heat stability and / or specific activity of the obtained mutant are significantly improved, and the degradation effect on PET material is greatly improved. The obtained mutant has important application value in the biological degradation treatment of single and mixed PET material waste.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of genetic engineering and PET material degradation, and particularly relates to a PET degrading enzyme mutant and application thereof in PET material degradation. BACKGROUND

[0002] As one of the key materials in modern industrial systems, polyester plastics are widely used in packaging, textile fibers, medical devices and other important fields. Enzyme-catalyzed biodegradation technology is considered as a potential key technology path to solve the problem of polyester plastic waste and realize recycling, because of its mild conditions, controllable products and environmental friendliness.

[0003] Since the first discovery of PET degrading enzyme in 2016, a variety of natural and engineered enzymes (such as PETase, LCCICCG variants and FAST-PETase designed by artificial intelligence) have been developed, which can efficiently depolymerize PET under mild conditions and recycle the product into useful monomers, showing good prospects for industrial application. In the current industrialization process of enzymatic degradation of PET, continuous alkali supplementation is a key operation to maintain the reaction system in the alkaline range (usually pH 7.5~9.0), which is not directly to "activate" the enzyme, but to accurately neutralize the acidic products generated in the reaction, so as to maintain the stable pH environment required by the enzyme activity, and ensure the efficient and continuous progress of the depolymerization reaction. Although this operation can maintain the reaction, it also increases the complexity of the process and the operating cost, and may introduce additional salt, which affects the separation and purification of the monomers in the subsequent process.

[0004] If PET can be efficiently depolymerized under acidic conditions, it will be possible to directly utilize the acidic environment of the reaction system, greatly reducing or even avoiding the addition of alkali, thereby simplifying the process and reducing the cost, which is more conducive to industrial continuous production. Therefore, exploring a PET degrading enzyme that can maintain high catalytic efficiency and stability under acidic conditions has become a key technical breakthrough direction to promote the industrialization of PET biological depolymerization technology and reduce processing costs. SUMMARY

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

[0006] The technical problem to be solved by the present application is to provide a nucleic acid molecule, a recombinant expression vector and a recombinant strain.

[0007] The technical problem to be solved by the present application is to provide a method for preparing the protein of the PET degrading enzyme mutant.

[0008] The present application also aims to solve the technical problem of providing the application of the PET-degrading enzyme mutant, or the recombinant expression vector, or the recombinant strain in the degradation of PET materials.

[0009] The present application also aims to solve the technical problem of providing the application of the PET-degrading enzyme mutant, or the recombinant expression vector, or the recombinant strain in the degradation of PET materials.

[0010] To solve the above technical problems, the technical scheme adopted by the present application is as follows:

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

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

[0013] Preferably, the wild-type PET-degrading enzyme is derived from Mumia sp., and belongs to the alpha / beta hydrolase family together with PET-degrading enzymes LCC, TfCut2 and IsPETase, and has the degradation activity of polyester plastic.

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

[0015] Or, the N at position 49 is mutated to F, and simultaneously the G at position 224 is mutated to F.

[0016] Or, the N at position 49 is mutated to F, and simultaneously the N at position 171 is mutated to A, and the G at position 224 is mutated to F.

[0017] Or, the N at position 49 is mutated to F, and simultaneously the N at position 171 is mutated to A, the G at position 224 is mutated to F, and the F at position 92 is mutated to Y.

[0018] Or, the N at position 49 is mutated to F, and simultaneously the N at position 171 is mutated to A, the G at position 224 is mutated to F, the F at position 92 is mutated to Y, and the S at position 97 is mutated to E.

[0019] Or, the N at position 49 is mutated to F, and simultaneously the N at position 171 is mutated to A, the G at position 224 is mutated to F, the F at position 92 is mutated to Y, and the A at position 98 is mutated to S.

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

[0021] or, the amino acid sequence of the PET-degrading enzyme mutant is obtained by mutating the amino acid sequence of wild-type PET-degrading enzyme at position 49 from N to F, at position 171 from N to A, and at position 224 from G to F.

[0022] or, the amino acid sequence of the PET-degrading enzyme mutant is obtained by mutating the amino acid sequence of wild-type PET-degrading enzyme at position 49 from N to F, at position 171 from N to A, and at position 224 from G to F.

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

[0024] or, the amino acid sequence of the PET-degrading enzyme mutant is obtained by mutating the amino acid sequence of wild-type PET-degrading enzyme at position 49 from N to F, at position 171 from N to A, and at position 224 from G to F.

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

[0026] The PET-degrading enzyme mutant can exhibit good degradation ability to PET plastics under acidic conditions, and has increased thermal stability and / or increased specific activity compared with wild-type PET-degrading enzyme.

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

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

[0029] In a second aspect, the present application provides a nucleic acid molecule encoding the PET-degrading enzyme mutant.

[0030] In a third aspect, the present application provides a recombinant expression vector containing the nucleic acid molecule.

[0031] In a fourth aspect, the present application provides a recombinant strain 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 application, the host cell is E. coli BL21 (DE3).

[0033] In the fifth aspect, the present application provides a method for preparing the protein of the PET-degrading enzyme mutant, inoculating the seed liquid of the recombinant strain into a fermentation medium for culture, so as to express the protein of the PET-degrading enzyme mutant.

[0034] In some embodiments of the present application, the seed liquid of the recombinant strain is transferred into an LB medium containing kanamycin for culture to OD 600 = 0.8~1.0, after adding IPTG with a final concentration of 0.5 mM, the culture is continued, so as to express the protein of the PET-degrading enzyme mutant.

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

[0036] In the sixth aspect, the present application provides the PET-degrading enzyme mutant, or the recombinant expression vector, or the recombinant strain for use in the degradation of PET materials.

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

[0038] In the seventh aspect, the present application provides a method for degrading PET materials by using the PET-degrading enzyme mutant, adding the enzyme solution of the PET-degrading enzyme mutant into a buffer containing PET materials, and performing depolymerization reaction.

[0039] In the seventh aspect, the present application provides a method for degrading PET materials by using the PET-degrading enzyme mutant, adding the enzyme solution of the PET-degrading enzyme mutant into a buffer containing PET materials, and performing depolymerization reaction.

[0040] In some embodiments of the present application, the depolymerization reaction has a reaction system of 2 M pH 5.0 citrate buffer, a final concentration of 5 g / L PET materials, and a final concentration of 10 mg / L enzyme solution of the PET-degrading enzyme mutant, and a reaction condition of incubation at 60℃ for 48 h.

[0041] Beneficial effects: the application constructs a MuPETase mutant library by error-prone PCR and homologous recombination on PET degrading enzyme (MuPETase), further improves the activity and heat resistance of MuPETase, and screens a mutant which shows good degradation ability to PET plastic under acidic conditions, compared with the original PET degrading enzyme, the heat stability and / or specific activity of the mutant are significantly improved, thereby enhancing the degradation effect on PET material. The application has important application value in the biological degradation treatment of PET material waste. BRIEF DESCRIPTION OF DRAWINGS

[0042] The above and / or other aspects of the present application will become apparent and more readily appreciated from the following description, taken in conjunction with the accompanying drawings, in which:

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

[0044] Figure 2 SDS-PAGE result of PET degrading enzyme MuPETase. Wherein, lane 1 is cell broken slurry, lane 2 is supernatant of broken cells, lane 3 is enzyme solution eluted by 50 mM imidazole eluent, and lane 4 is pure enzyme solution eluted by 300 mM imidazole eluent.

[0045] Figure 3 Optimum pH detection result of PET degrading enzyme MuPETase.

[0046] Figure 4 Acid resistance detection result of PET degrading enzyme MuPETase. Wherein, A is residual activity detection result under different pH conditions; B is depolymerization activity detection result under different pH conditions.

[0047] Figure 5 Depolymerization effect of PET degrading enzyme MuPETase mutant Mut7 and IsPETase on different PET materials. DETAILED DESCRIPTION

[0048] In the following examples, the experimental methods are as follows, unless otherwise specified, all are conventional methods; the reagents and materials, unless otherwise specified, can be obtained from commercial channels.

[0049] In the following examples, the detection methods used are as follows:

[0050] (1) Protein detection method

[0051] The protein concentration was detected by a protein assay kit (BSA protein assay kit), and the purity of each protein was detected by SDS-PAGE analysis. First, a 1 mg / mL bovine serum albumin (BSA) stock solution was prepared, and then phosphate buffer (50 mM, pH 7.0) was added to the stock solution to prepare a group of BSA stock solutions with a concentration range of 0.2-1 mg / L. Then, the group of solutions was diluted 10 times to obtain a group of BSA dilution solutions with a concentration range of 0.02-0.1 mg / L, i.e. BSA standard solutions. First, 20 μL of BSA standard solutions with different concentration gradients were added to a 96-well plate, and then 200 μL of coomassie brilliant blue solution was added. After mixing well, the reaction was allowed to stand for 3 min. The absorbance values of the BSA standard solutions with different concentrations at 595 nm were detected using an enzyme marker, and a standard curve was plotted with the protein concentration as the abscissa and the absorbance as the ordinate. The absorbance values of the samples were also measured by the same method, and the protein concentration was calculated by the standard curve.

[0052] (2) Enzyme activity detection method

[0053] In a 5 mL centrifuge tube, 10 μL of 10 mM p-nitrophenyl octanoate (pNPO), 10 μL of enzyme solution and 980 μL of phosphate buffer solution (50 mM, pH 7.0) were added to a final volume of 1 mL. The activity detection system was heated at 37°C for 10 min, then immediately cooled in ice, and the absorbance value at 410 nm was detected using an enzyme marker. The molar amount of p-nitrophenol was calculated from the standard curve plotted by p-nitrophenol and OD 410 The enzyme activity was defined as follows: the amount of enzyme required to generate 1 μmol of p-nitrophenol per minute at 37°C was taken as one enzyme activity unit.

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

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

[0056] The hydrolytic enzyme gene capable of degrading PET plastic in the protein data was screened, and a PET degrading enzyme MuPETase derived from Mumia sp. was obtained, the amino acid sequence of which is shown as SEQ ID NO. 1. The enzyme belongs to the α / β hydrolase family together with PET degrading enzymes LCC, TfCut2 and IsPETase, and has polyester plastic degradation activity, and its phylogenetic tree is shown in Figure 1

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

[0058] 2. Expression and purification of MuPETase

[0059] The recombinant plasmid pET29a-Mumia sp. was transformed into E. 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 liquid. 1 mL of the seed liquid was transferred to 50 mL of fresh LB medium containing 50 μg / mL kanamycin and cultured at 37°C and 220 rpm until the OD 600 = 0.8-1.0. After adding IPTG at a final concentration of 0.5 mM, the fermentation was continued for 20 h, and then stopped. The fermentation cells were collected by centrifugation.

[0060] The fermentation cells were suspended in PBS buffer. Then the cell suspension was subjected to ultrasonic treatment by FB 705 ultrasonic instrument (Fisherbrand) for 2 min, with an amplitude of 30% (2 sec ON and 1 sec OFF cycle). Then the broken supernatant and slurry were obtained by centrifugation at 10000 rpm and 4°C for 30 min, and sampled respectively. The broken supernatant was purified by Ni column affinity chromatography. First, 10 mL of 50 mM imidazole was used to elute impurities, and the eluate was sampled. Then 300 mM imidazole was used to elute to obtain the purified MuPETase enzyme solution, and the sample was subjected to SDS-PAGE detection. The results are shown in Figure 2

[0061] Example 2: Determination of the optimum pH of MuPETase

[0062] ​In 5 mL centrifuge tubes, 10 μL of 10 mM p-nitrophenyl octanoate (pNPO), 10 μL of MuPETase enzyme solution and 980 μL of buffer solution with different pH were added respectively, and 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). The enzyme activity was measured after 5 min of reaction at 37°C, and the enzyme activity under the optimal reaction pH condition was taken as 100%. The detection results are shown in Figure 3 Figure A, and pH 7.0 is the optimal pH of MuPETase.

[0063] Example 3: MuPETase acid resistance determination

[0064] By detecting the residual activity and depolymerization activity of MuPETase under different pH conditions, the specific detection method is as follows.

[0065] 1. Residual activity detection

[0066] In 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), MuPETase enzyme solution was added to a final concentration of 10 mg / L, and incubated at 37°C for 10 h. After incubation, the residual enzyme activity was measured, and the highest residual enzyme activity was taken as 100%.

[0067] The detection results are shown in Figure 4 Figure A, and the residual enzyme activity is the highest after incubation at pH 7.0 for 10 h, and the enzyme activity can still be maintained at more than 50% after incubation at pH 5.0 for 10 h relative to the condition at pH 7.0.

[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), and PET powder was added to a final concentration of 5 g / L. MuPETase was added at an enzyme to substrate ratio of 2 ‰ (m / m) to a final concentration of 10 mg / L, and the reaction system was 50 mL. After incubation at 60°C for 48 h, the reaction solution was filtered using a 0.22 μm filter, and reasonable dilution was performed according to the product concentration, and then analyzed using an Agilent 1260 Infinity II high performance liquid chromatography system through a C18 chromatographic column (Thermo Fisher Scientific, 150×4.6 mm, 5 μm). The solvent composed of 18% acetonitrile, 1% formic acid and 81% water was used as the mobile phase, the detection time was 25 min, the flow rate was 0.8 mL / min, and the detection wavelength was 254 nm. The detection results of 1 mM, 0.1 mM, 0.01 mM, 0.001 mM and 0.0001 mM TPA and MHET gradient standard solutions were used to make a standard curve. The product concentration of the enzyme hydrolysis sample was calculated by the standard curve.

[0070] The depolymerization effect is shown as B in Figure 4 At pH 7.0, MuPETase has the highest depolymerization rate for degrading PET. At pH 5.0, the depolymerization rate can still reach 77% of that at pH 7.0, indicating that MuPETase can effectively degrade PET under acidic conditions.

[0071] Example 4: Construction of a mutant library of PET degrading enzyme (MuPETase)

[0072] 1. PCR amplification

[0073] According to the sequence of PET degrading enzyme (MuPETase), error-prone PCR primers and vector PCR primers (see Table 1) were designed. The recombinant plasmid pET29a-Mumia sp was subjected to error-prone PCR amplification to amplify the MuPETase fragment with the plasmid vector homologous arm. At the same time, the plasmid pET29a fragment in the recombinant plasmid pET29a-Mumia sp was amplified by high-fidelity PCR.

[0074] Table 1 Primer sequence information

[0075]

[0076] The error-prone PCR amplification uses GeneMorph II Random Mutagenesis Kit (Stratagene), and the reaction system is as follows: 10x Mutazyme II reaction buffer 5 μL, Mutazyme II DNA polymerase 1 μL, dNTP (40 mM) 1 μL, 1 ng / μL template DNA 1 μL, 10 μM forward primer 2 μL, 10 μM reverse primer 2 μL, and add double-distilled water to 50 μL. The error-prone PCR amplification conditions are as follows: 94℃ pre-denaturation for 3 min; then 94℃ for 30 s, 55℃ for 30 s, 72℃ for 2 min, 30 cycles; finally 72℃ for 10 min.

[0077] The reaction system of the high-fidelity PCR amplification is as follows: 2x Phanta Max MasterMix 25 μL (Vazyme, item number: P515), 10 μM forward primer 2 μL, 10 μM reverse primer 2 μL, 1 ng / μL template DNA 1 μL, dNTP (40 mM) 1 μL, and add double-distilled water to 50 μL. The PCR amplification conditions are as follows: 95℃ pre-denaturation for 3 min; then 95℃ for 15 s, 61℃ for 15 s, 72℃ for 5 min, 30 cycles; finally 72℃ for 10 min.

[0078] 2. PCR amplification product purification

[0079] The above PCR amplification products are detected by 1% agarose gel electrophoresis, and after the detection is completed, 1 μL of methylation template digestion enzyme (Dpn I) is added to 50 μL of the amplification product, and the mixture is mixed by blowing and sucking with a gun head, and then reacted at 37℃ for 1 h, and then inactivated at 70℃ for 5 min to obtain the Dpn I digestion product. The Dpn I digestion product is purified using Fastpure Gel DNA Extraction Mini Kit (Vazyme), specifically, 150 μL of Buffer GDP is added to 50 μL of the digestion product, and the mixture is mixed by blowing and sucking with a gun head, and then all is transferred to the adsorption column, centrifuged at 12000 rpm for 1 min, and the waste liquid in the tube below the adsorption column is discarded, then 700 μL of Buffer GW is added to the adsorption column, centrifuged at 12000 rpm for 1 min, and the waste liquid in the tube below the adsorption column is discarded, and the above steps are repeated twice, and then the purified product is eluted with 40 μL of double-distilled water.

[0080] 3. Ligation product

[0081] The purified product of the MuPETase fragment and the purified product of the plasmid pET29a fragment were connected by a homomultimerization PCR reaction to form a linear multimeric connection product.

[0082] The homomultimerization PCR reaction system was as follows: 2x Phanta Max Master Mix 25 μL (Vazyme), plasmid pET29a fragment 2 μL (15 ng / μL), MuPETase fragment 2 μL (250 ng / μL), and double-distilled water was added to 50 μL. The PCR amplification conditions were as follows: 95 °C pre-denaturation for 3 min; then 95 °C for 15 s, 61 °C for 15 s, 72 °C for 10 min, 30 cycles; finally 72 °C for 10 min.

[0083] 4、Transformation

[0084] The linear multimeric connection product obtained in step 3 was transformed into E. 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 incubated at 37 °C for 12 h to obtain a large number of BL21 (DE3) single colonies containing MuPETase mutants.

[0085] Example 5: Screening of MuPETase mutants

[0086] The BL21 (DE3) single colonies of the original MuPETase and the large number of BL21 (DE3) single colonies containing MuPETase mutants obtained in Example 4 were inoculated into 96-deep-well plates containing 1 mL of LB medium and incubated at 37 °C for 24 h to obtain respective fermentation slurry. After resuspension with 1 mL of 50 mM citrate buffer at pH 5.0, the resuspension was subjected to ultrasonic treatment for 2 min by a FB 705 ultrasonic instrument (Fisherbrand) at an amplitude of 30% (2 s ON and 1 s OFF cycle). Then, the broken supernatant was obtained by centrifugation at 10,000 rpm and 4 °C for 30 min. The broken supernatant was subjected to enzyme activity determination, and the mutants with enzyme activity not lower than that of the original MuPETase fermentation enzyme were selected for further determination of thermal stability, residual enzyme activity, and specific activity.

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

[0088] The thermal stability of MuPETase and its mutants was compared by Tm value. Specifically, MuPETase and its mutant proteins were dialyzed in 50 mM pH 5.0 citrate buffer. The proteins were diluted to the same concentration of 1.0 mg / mL with the dialysis solution. Filtration was performed using a 0.22 μm filter membrane, and degassing was performed. Scanning was performed using a micro-heat differential scanning calorimeter (DSC) at a scanning rate of 1.0 ℃ / min in the range of 25~95°C, and the filtration pressure was 2.5 atm. A single symmetrical endothermic peak was obtained after scanning, and the peak top was the corresponding Tm value. The results are shown in Table 2.

[0089] 2. Residual enzyme activity determination

[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 was completed, samples were taken and diluted 1 to 500 times in 50 mM pH 5.0 citrate buffer, and residual enzyme activity determination was performed. The detection results are shown in Table 2.

[0091] 3. Specific activity calculation

[0092] The specific activity of MuPETase and its mutants was further calculated, and the calculation formula was as follows:

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

[0094] As can be seen from Table 2, the Tm values of N49F, L122F, G224F and N171A mutants are comparable to those of MuPETase, while the specific activity is significantly improved; the Tm values of F92Y, S97E and A98S are significantly improved while the specific activity is higher than that 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 activities and specific activities of MuPETase and mutants

[0096]

[0097] Example 6: Thermal stability and specific activity detection of combined mutants

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

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

[0100] Table 3. Primer information used in Example 6

[0101]

[0102] The thermal stability, residual enzyme activity, and specific activity of the combination mutants were determined according to the method of Example 5. The results are shown in Table 4. It can be seen that, in addition to the Tm value and specific enzyme activity of the N49F+L122F combination mutant decreasing, the Tm value and specific enzyme activity of the N49F+N171A, N49F+G224F, N49F+N171A+G224F, N49F+N171A+G224F+F92Y, N49F+N171A+G224F+F92Y+S97E, and N49F+N171A+G224F+F92Y+A98S combination mutants under acidic conditions were significantly improved. Among them, the MuPETase combination mutant N49F+N171A+G224F+F92Y+S97E exhibited the best Tm value, residual enzyme activity, and specific enzyme activity.

[0103] Table 4 Tm value, residual enzyme activity, and specific enzyme activity of MuPETase combination mutants

[0104]

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

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

[0107] The experimental results are shown in Table 6. Figure 5 It can be seen that, under acidic conditions, the depolymerization effect of IsPETase on different PET materials was much lower than that of MuPETase combination mutant Mut7. In addition, MuPETase combination mutant Mut7 produced obvious depolymerization products for all PET materials, and the amount of TPA+MHET produced was significantly higher than that of control enzyme IsPETase. Specifically, the amount of depolymerization products produced by MuPETase combination mutant Mut7 on PET fiber, PET filament, PET lunch box, PET pillow, PET fabric, and PET film, etc. after actual consumption was stable at 10-18 mM, while the amount of products produced by IsPETase under the same substrate and reaction conditions was only 3-5 mM.

[0108] The application provides a PET-degrading enzyme mutant and a method for application of the PET-degrading enzyme mutant in PET material degradation. The method and approach for realizing the technical scheme are various, and the preferred embodiments are described above. It should be noted that, for those skilled in the art, some improvements and refinements can be made without departing from the principle of the application, and these improvements and refinements should also be considered as the protection scope of the application. The components not explicitly described in the embodiments can be realized by using the prior art.

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 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. 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 through 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. 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.

9. The method according to claim 8, 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 enzyme solution of 10 mg / L PET degrading enzyme mutant at a final concentration; the reaction conditions are: incubation at 50~70℃ for 40~55 h.

Images