Application of mutation site as target to improvement of enzymatic activity, thermal stability and / or long-term effectiveness of PET (polyethylene terephthalate) plastic degrading enzyme

Abstract

The invention relates to the field of bioengineering, in particular to application of a mutation site as a target to improvement of enzymatic activity, thermal stability and / or long-term effectiveness of PET (polyethylene terephthalate) plastic degrading enzyme. The invention provides application of a mutation site as a target spot in improvement of enzymatic activity and / or thermal stability of a PET plastic degrading enzyme. The mutation sites comprise one or more of the 114 site, the 205 site, the 233 site and the 269 site of the amino acid sequence of the wild PET plastic degrading enzyme; the PET plastic degrading enzyme is prepared from one or more of FAST-PETase enzyme, PA-PETase enzyme and DepoPETase enzyme. The invention further discloses a preparation method of the PET plastic degrading enzyme. According to the present invention, the site mutation is applied to the most excellent variants known at present, such that the short-term activity problem of FAST-PETase is compensated, and the activity and the thermal stability of PA-PETase and DepoPETase are improved;

Description

Technical Field

[0001] The invention relates to the field of bioengineering, and in particular to the application of mutation sites as targets in improving the enzyme activity, thermal stability and / or long-term effect of PET plastic degrading enzymes. Background Art

[0002] Plastic waste increased from 156 million tons in 2000 to 353 million tons in 2019, of which 150 million to 200 million tons (or even more) were discarded in landfills or natural environments such as oceans. Microplastics have even been found in human feces, indicating that plastic pollution has entered the food chain and begun to directly threaten human health.

[0003] Polyethylene terephthalate (PET) is a common plastic made by dehydration condensation esterification of terephthalic acid (TPA) and ethylene glycol (EG). Due to the chain mobility, crystallinity and surface hydrophobicity of PET, and the fact that it does not undergo complete mineralization, PET plastics naturally degrade very slowly and are prone to accumulation, causing white pollution. However, its ester bond is a feasible cleavage site for many hydrolases, so researchers have discovered many biological enzymes with PET hydrolysis activity and optimized their performance in a targeted manner.

[0004] IsPETase was first discovered in 2016. As the PET hydrolase with the best degradation activity at ambient temperature, PETase has great degradation potential and prospects. However, after PETase is taken out of the body, its enzyme activity and thermal stability are very poor, making it difficult to achieve industrialization. Therefore, researchers have actively carried out research work mainly around improving activity and thermal stability, and have made relatively outstanding progress. In 2022, Hongyuan Lu et al. used a structure-based machine learning algorithm to design a powerful and active PET hydrolase variant FAST-PETase, which ultimately made it possible for untreated post-consumer PET from 51 different thermoformed products to be almost completely degraded by FAST-PETase within 1 week; in 2023, Lixia Shi et al. used PETaseWT as the starting point to construct an error-prone PCR library, and used BHET-OH fluorescence detection technology to screen 10,000 clones in 3 rounds of directed evolution, and finally obtained a variant DepoPETase with an activity 1,407 times higher than that of the wild type; in 2024, Qingdian Ying et al. used semi-saturation mutations to stabilize the most flexible region in IsPETase, and constructed a heat-resistant S121P / D186A variant PA-PETase with a Tm value of 70.8°C (ΔTm=24.1C), which increased the depolymerization products of amorphous polyester at 40°C by 109.3 times.

[0005] However, the long-term effectiveness of FAST-PETase is very poor, and the enzyme activity is almost 100% lost after working at 50°C for 24 hours; the optimal reaction temperature of DepoPETase and FAST-PETase is 50°C, but the activity of DepoPETase at 50°C for 24 hours is not as good as that of FAST-PETase; the optimal reaction temperature of PA-PETase is 40°C, and its thermal stability is poor. Summary of the invention

[0006] In view of this, the present invention provides the use of mutation sites as targets in improving the enzymatic activity and / or thermal stability of PET plastic degrading enzymes. The present invention applies four-site mutations to several of the best variants known so far, which makes up for the short-term activity problem of FAST-PETase and improves the activity and thermal stability of PA-PETase and DepoPETase.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides the use of mutation sites as targets in improving the enzyme activity and / or thermal stability of PET plastic degrading enzymes;

[0009] The mutation sites include: one or more of the 114th, 205th, 233rd and 269th positions of the amino acid sequence of the wild-type PET plastic degrading enzyme;

[0010] The PET plastic degrading enzyme includes one or more of FAST-PETase enzyme, PA-PETase enzyme and DepoPETase enzyme.

[0011] The present invention also provides a mutant, which, based on the wild-type PET plastic degrading enzyme, has one or more of the following amino acid site mutations: position 114, position 205, position 233, and position 269, and the amino acid sequence of the wild-type PET plastic degrading enzyme is shown in any of SEQ ID NO: 1 to SEQ ID NO: 3.

[0012] In some embodiments of the present invention, in the above mutants, the nucleotide sequence encoding the wild-type PET plastic degrading enzyme is shown as SEQ ID NO:10 to SEQ ID NO:12.

[0013] In some embodiments of the present invention, in the above mutant, the mutation at position 114 includes: N114I;

[0014] The mutation at position 205 includes: N205K;

[0015] The mutation at position 233 includes: N233K;

[0016] The mutation at position 269 includes: S269V.

[0017] In some embodiments of the present invention, the mutants described above have:

[0018] (1) the amino acid sequence shown in SEQ ID NO: 4 to SEQ ID NO: 6; or

[0019] (2) an amino acid sequence obtained by replacing, deleting or adding one or more amino groups of the amino acid sequence shown in (1), and having the same or similar function as the amino acid sequence shown in (1); or

[0020] (3) An amino acid sequence that is at least 80% identical to the amino acid sequence shown in (1) or (2).

[0021] The present invention also provides a nucleic acid molecule encoding the mutant.

[0022] In some embodiments of the present invention, the above-mentioned nucleic acid molecule has:

[0023] (4) the nucleotide sequence shown in SEQ ID NO:7 to SEQ ID NO:9; or

[0024] (5) A nucleotide sequence obtained by modifying, replacing, deleting or adding one or more bases to the nucleotide sequence described in (4); or

[0025] (6) a sequence having at least 80% homology with the nucleotide sequence described in (4) or (5); or

[0026] (7) A complementary sequence of the nucleotide sequence described in (4), (5) or (6).

[0027] The present invention also provides an expression vector, comprising: the above nucleic acid molecule.

[0028] The present invention also provides a host for transforming and / or transfecting the above expression vector.

[0029] The present invention also provides a method for preparing the mutant, which comprises culturing the host, centrifuging, collecting the supernatant, and purifying to obtain the mutant.

[0030] The present invention also provides a product, including: the mutant, the nucleic acid molecule, the expression vector, the host and / or the mutant obtained by the preparation method.

[0031] The present invention also provides the mutant, the nucleic acid molecule, the expression vector, the host, the mutant obtained by the preparation method and / or the use of the mutant in degrading plastics.

[0032] The invention improves the activity and long-term effect of FAST-PETase, and improves the activity and thermal stability of PA-PETase and DepoPETase. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0034] Figure 1 The results show that FAST-PETase and M4 were expressed and purified intracellularly, and the precise yield was determined by HPLC after reaction at 45℃, 50℃ and 55℃ for 24h and 72h;

[0035] Figure 2 The intracellular expression and purification of PA-PETase and M5 were shown, and the precise yield was determined by HPLC after reaction at 40℃, 45℃ and 50℃ for 24h and 72h;

[0036] Figure 3The intracellular expression and purification of DepoPETase and M6 were shown, and the precise yield was determined by HPLC after reaction at 45°C, 50°C and 55°C for 24h and 72h. DETAILED DESCRIPTION

[0037] The invention discloses the application of mutation sites as targets in improving the enzyme activity, thermal stability and / or long-term effect of PET plastic degrading enzyme.

[0038] It should be understood that the expression "one or more of..." includes each of the items recited after the expression individually and in various different combinations of two or more of the recited items, unless otherwise understood from the context and usage. The expression "and / or" in combination with three or more recited items should be understood to have the same meaning, unless otherwise understood from the context.

[0039] The use of the terms "comprising", "having" or "containing", including their grammatical synonyms, should generally be understood as open and non-restrictive, for example not excluding other unrecited elements or steps, unless otherwise specifically stated or otherwise understood from the context.

[0040] It should be understood that the order of steps or the order in which certain actions are performed is not important as long as the present invention remains operable. In addition, two or more steps or actions may be performed simultaneously.

[0041] The use of any and all examples or exemplary language, such as "for example" or "including", herein is intended only to better illustrate the invention and does not limit the scope of the invention unless otherwise claimed. No language in this specification should be construed as indicating that any non-claimed element is essential to the practice of the invention.

[0042] In addition, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise expressly stated, it should be understood that all ranges, quantities, values ​​and percentages used in this disclosure are modified by "about". Here, "about" generally means that the actual value is within plus or minus 10%, 5%, 1% or 0.5% of a specific value or range.

[0043] The present invention improves different properties of the scaffold mutants by site mutation: ① activity; ② thermal stability; ③ long-term effect. It was finally found that the activity of the enzymes was improved, and at the same time, ① the long-term effect of FAST-PETase was improved; ② the thermal stability of PA-PETase and DepoPETase was improved.

[0044] In Examples 1 to 3 of the present invention, all the raw materials and reagents used can be purchased from the market.

[0045] The present invention will be further described below in conjunction with embodiments:

[0046] Example 1 Construction and verification of M4 mutants - adding site mutations to the FAST-PETase scaffold

[0047] 1. Acquisition of experimental materials

[0048] Acquisition of chassis strains: TOP10 and BL21 competent strains were purchased from Bomed Company.

[0049] The gene FAST-PETase (pET_28a(+)) involved in this experiment was synthesized by Qingke Biotechnology Co., Ltd.

[0050] Amino acid sequence of FAST-PETase (261): NPYARGPNPTAASLEASAGPF TVRSFTVSRPSGYGAGTVYYPTNAGGTVGAIAIVPGYTARQSSIKWWGPRLASHGFVVITIDTNSTLDQPESRSSQQMAALRQVASLNGTSSSPIYGKVDTARMGVMGWSMGGGGSLISAAN NPSLKAAAPQAPWHSSTNFSSVTVPTLIFACENDSIAPVNSSALPIYDSMSQNAKQFLEIKGGSHSCANSGNSNQALIGKKGVAWMKRFMDNDTRYSTFACENPNSTAVSDFRTANCS (such as SEQ ID NO:1).

[0051] Nucleotide sequence of FAST-PETase (optimized for E. coli as the host): AATCCGTACGCA CGTGGTCCGAATCCAACCGCAGCATCTCTGGAAGCAAGTGCTGGTCCATTCACCGTTCGTTCTTTCACCGTTTCTCGTCCATCTGGTTACGGTGCAGGTACCGTTTACTATCCGACCAACGCTGGTGGTACTGTTGGTGCAATCGCTATCGTTCCAGGTTACACCGCTCGTCAGAGCTCCATCAAGTGGTGGGGTCCACGTTTGGCATCTCACGGTTTCGTTGTCATCACCATCGACACCAACTCCACTCTGGATCAGCCGgaaAGCCGTTCTTCTCAGCAGATGGCTGCTCTGCGTCAGGTTGCTTCTCTGAACGGTACCAGCTCTTCTCCAATCTACGGTAAAGTTGATACCGCTCGTATGGGTGTTATGGGTTGGTCTATGGGTGGTGGTGGTAGCCTGATCTCTGCTGCGAACAATCCAAGCCTGAAAGCAGCTGCACCGCAAGCTCCATGGcacTCTTCCACCAACTTCAGCTCTGTTACCGTTCCGACTCTGATCTTCGCATGTGAGAACGACTCTATCGCACCAGTTAACTCTTCTGCACTGCCGATCTACGACTCCATGTCTcagAACGCGAAGCAGTTTCTGGAAATCaaaGGTGGTTCTCATAGCTGTGCAAACTCTGGTAACTCCAACCAGGCACTGATCGGTAAGAAAGGTGTTGCGTGGATGAAGCGTTTCATGGACAACGACACTCGTTACTCTACCTTCGCGTGTGAGAACCCGAACTCTACTGCGGTTTCTGACTTCCGTACCGCTAACTGCTCT (as shown in SEQ ID NO: 10).

[0052] Obtaining the substrate for the enzyme activity reaction: Good fellow film.

[0053] 2. Experimental methods:

[0054] LB medium: 10 g / L TPYPONE, 5 g / L YEAST EXTRACT, 10 g / L NaCl;

[0055] LB solid medium: 10 g / L TPYPONE, 5 g / L YEAST EXTRACT, 10 g / L Na Cl, 15 g / L agarose;

[0056] Kanamycin stock solution: 50 mg / mL;

[0057] IPTG solution: 1 M, 0.238 g / mL;

[0058] Lysis buffer (LE Buffer): 50 mM Na2HPO4, 100 mM NaCl, 10 mM imidazole, pH = 7.5;

[0059] Eluent: 50 mM Na2HPO4, 100 mM NaCl, 500 mM imidazole, pH = 7.5;

[0060] Protein preservation solution: 50mM Na2HPO4, 100mM NaCl, pH=7.5;

[0061] Reaction buffer: 50 mM glycine-NaOH, glycine 3.74 g / L, pH = 9.0;

[0062] PMSF (serine protease inhibitor) solution: 100 mM;

[0063] Coomassie brilliant blue staining solution: 2.4 g / L Coomassie brilliant blue R250 dissolved in methanol: glacial acetic acid: water = 4:1:5;

[0064] Decolorizing solution: methanol: glacial acetic acid: water = 4:1:5;

[0065] Protein electrophoresis buffer: Dissolve the purchased MOPS-SDS Running Buffer in 1000 mL of water, stir well and set aside.

[0066] Construction of the M4 mutant: Using FAST-PETase as a template, single-point mutations N205K, N114I, and S269V were added in sequence to finally obtain the M4 mutant.

[0067] Amino acid sequence of M4 mutant (261): NPYARGPNPTAASLEASAGPFTV RSFTVSRPSGYGAGTVYYPTNAGGTVGAIAIVPGYTARQSSIKWWGPRLASHGFVVITIDTISTLDQPESRSSQQMAALRQVASLNGTSSSPIYGKVDTARMGVMGWSMGGGGSLISAANNP SLKAAAPQAPWHSSTNFSSVTVPTLIFACEKDSIAPVNSSALPIYDSMSQNAKQFLEIKGGSHSCANSGNSNQALIGKKGVAWMKRFMDNDTRYVTFACENPNSTAVSDFRTANCS (such as SEQID NO:4 shown).

[0068] Nucleotide sequence of the M4 mutant (after optimization for large intestine as host):

[0069] (as shown in SEQ ID NO:7).

[0070] Mutant transformation: ① Add 2μL mutant plasmid to 50μL BL21 competent medium and place on ice for 15-25min; ② Heat shock in a 42℃ water bath for 1min; ③ Place on ice for 2min; ④ Add 500μL LB non-antibiotic medium and culture in a shaker at 37℃, 220rpm for 1h; ⑤ Centrifuge the EP tube at 4000rpm for 2min, pour off the supernatant, resuspend the precipitate and evenly apply it on a plate with kanamycin resistance added; ⑥ Incubate inverted in a 37℃ incubator overnight.

[0071] Cultivation and collection of intracellular expression bacteria: ① Pick a single colony on the plate and add it to 5mL LB medium, then add 5μL kana mother solution, shake the tube and place it in a shaker at 37℃, 220rpm for 9-12h; ② Measure OD, shake the bacterial solution in advance, dilute 5-10 times to 2mL in a cuvette, use 2mL LB + 2μL kana as a blank control, and measure the absorbance at λ=600nm; ③ Transfer: calculate the final OD after transfer to be 0.05-0.1, shake the bacterial solution, and transfer it to a sterilized LB conical flask (100mL) according to the mL number calculated by the OD value, add 100μL kana mother solution to each flask, and place it in a shaker at 37℃, 220rpm for 2.5-3h; ④ Measure OD, shake the test tube in advance, take 2mL in a cuvette without dilution, and use 2mL LB + 2μL kanamycin was used as the blank control. The absorbance was measured at λ = 600nm. When the estimated OD was 0.6-0.8, the conical flask was placed on ice to quickly cool down. ⑤ 0.1mM IPTG was added to the conical flask and cultured in a shaker at 17℃ and 180rpm for 20-24h. ⑥ The conical flask was placed on ice, balanced, and incubated at 4200rpm, 4℃, for 40min to collect the bacterial solution and freeze it in a -80℃ refrigerator for later use.

[0072] Enzyme purification: ①Take the bacteria from the refrigerator and thaw them naturally on ice. After thawing, add 30-40mL LEBuffer, 50mg / L DNase, lysozyme, and 1% PMSF, and resuspend them; ②Break the bacteria by ultrasonic disruption, transfer the resuspended bacteria to a beaker, place the beaker in ice water, and disrupt them at 350W for 30min; ③After the disrupted bacteria are balanced, place them in a high-speed centrifuge at 11500rpm, 4℃, 45min, and collect the supernatant; ④Pre-cool LE Buffer and eluent, and use LE Buffer was used to balance the Ni column; ⑤ The supernatant obtained by centrifugation and the Ni column filler were mixed and placed on an ice shaker at a low speed of 70-80 rpm for 1 hour; ⑥ The mixed solution was returned to the gravity column and waited for the liquid to flow out; ⑦ The Ni column was washed with 80 mM imidazole as clean as possible; ⑧ The target protein was eluted with 250 mM imidazole and the effluent was collected; ⑨ The eluate was added to an ultrafiltration tube and concentrated at 4000 rpm, 4°C, for 20 min, and washed with protein preservation solution for 2-3 times, and finally concentrated to 1.5-2 mL; ⑩ The absorbance at λ = 280 nm was measured to indicate the concentration (mg / mL).

[0073] Enzyme activity reaction: The enzyme addition amount was 500 nM, which was calculated based on the enzyme concentration, and then the reaction buffer was added to 600 μL. The substrate was gf-PET cut into small discs with a diameter of 6 mm. The best mutant in the previous round was used as a control. Multiple parallel and multiple temperature conditions were set for iterative verification.

[0074] Initial screening of absorbance: The absorption peaks of PET products TPA and MHET reach their highest values ​​at around λ=245nm, which can quantitatively characterize the degree of hydrolysis of PET.

[0075] HPLC: Accurately quantify the yields of TPA and MHET.

[0076] 3. Experimental results

[0077] The same batch of FAST-PETase and M4 were expressed and purified in cells for verification. The reaction was carried out at 45°C, 50°C, and 55°C for 24h and 72h respectively. The results are as follows: Figure 1 shown.

[0078] pass Figure 1 It can be seen that at 24h, the activity of FAST-PETase at 45℃ and 50℃ was higher than that of M4; only at 55℃, the activity of M4 was slightly higher than that of FAST-PETase. At 72h, the activity of M4 was higher than that of FAST-PETase at three temperatures; M4 reached the optimum temperature at 45℃, and the yield of M4 reached 7688μM after 72h of reaction at 45℃. It can be seen that adding site mutations to FAST-PETase can improve the activity, thermal stability and long-term effect of the enzyme.

[0079] Example 2 Construction and verification of M5 mutants - adding site mutations to the PA-PETase scaffold 1. Acquisition of experimental materials

[0080] Acquisition of chassis strains: TOP10 and BL21 competent strains were purchased from Bomed Company.

[0081] The gene PA-PETase (pET_28a(+)) involved in this experiment was synthesized by Qingke Biotechnology Co., Ltd.

[0082] Amino acid sequence of PA-PETase (261): NPYARGPNPTAASLEASAGPFTV RSFTVSRPSGYGAGTVYYPTNAGGTVGAIAIVPGYTARQSSIKWWGPRLASHGFVVITIDTNSTLDQPPSRSSQQMAALRQVASLNGTSSSPIYGKVDTARMGVMGWSMGGGGSLISAANNP SLKAAAPQAPWASSTNFSSVTVPTLIFACENDSIAPVNSSALPIYDSMSRNAKQFLEINGGSHSCANSGNSNQALIGKKGVAWMKRFMDNDTRYSTFACENPNSTRVSDFRTANCS (such as SEQID NO:2 shown).

[0083] Nucleotide sequence of PA - PETase (optimized for E. coli as the host): AATCCGTACGCACG TGGTCCGAATCCAACCGCAGCATCTCTGGAAGCAAGTGCTGGTCCATTCACCGTTCGTTCTTTCACCGTTTCTCGTCCATCTGGTTACGGTGCAGGTACCGTTTACTATCCGACCAACGCTGGTGGTACTGTTGGTGCAATCGCTATCGTTCCAGGTTACACCGCTCGTCAGAGCTCCATCAAGTGGTGGGGTCCACGTTTGGCATCTCACGGTTTCGTTGTCATCACCATCGACACCAACTCCACTCTGGATCAGCCGccgAGCCGTTCTTCTCAGCAGATGGCTGCTCTGCGTCAGGTTGCTTCTCTGAACGGTACCAGCTCTTCTCCAATCTACGGTAAAGTTGATACCGCTCGTATGGGTGTTATGGGTTGGTCTATGGGTGGTGGTGGTAGCCTGATCTCTGCTGCGAACAATCCAAGCCTGAAAGCAGCTGCACCGCAAGCTCCATGGgctTCTTCCACCAACTTCAGCTCTGTTACCGTTCCGACTCTGATCTTCGCATGTGAGAACGACTCTATCGCACCAGTTAACTCTTCTGCACTGCCGATCTACGACTCCATGTCTCGTAACGCGAAGCAGTTTCTGGAAATCAACGGTGGTTCTCATAGCTGTGCAAACTCTGGTAACTCCAACCAGGCACTGATCGGTAAGAAAGGTGTTGCGTGGATGAAGCGTTTCATGGACAACGACACTCGTTACTCTACCTTCGCGTGTGAGAACCCGAACTCTACTCGTGTTTCTGACTTCCGTACCGCTAACTGCTCT (as shown in SEQ ID NO: 11).

[0084] Obtaining the substrate for the enzyme activity reaction: Good fellow film.

[0085] 2. Experimental methods:

[0086] LB medium: 10 g / L TPYPONE, 5 g / L YEAST EXTRACT, 10 g / L NaCl;

[0087] LB solid medium: 10 g / L TPYPONE, 5 g / L YEAST EXTRACT, 10 g / L NaCl, 15 g / L agarose;

[0088] Kanamycin stock solution: 50 mg / mL;

[0089] IPTG solution: 1 M, 0.238 g / mL;

[0090] Lysis buffer (LE Buffer): 50 mM Na2HPO4, 100 mM NaCl, 10 mM imidazole, pH = 7.5;

[0091] Eluent: 50 mM Na2HPO4, 100 mM NaCl, 500 mM imidazole, pH = 7.5;

[0092] Protein preservation solution: 50mMNa2HPO4, 100mMNaCl, pH=7.5;

[0093] Reaction buffer: 50 mM glycine-NaOH, glycine 3.74 g / L, pH = 9.0;

[0094] PMSF (serine protease inhibitor) solution: 100 mM;

[0095] Coomassie brilliant blue staining solution: 2.4 g / L Coomassie brilliant blue R250 dissolved in methanol: glacial acetic acid: water = 4:1:5;

[0096] Decolorizing solution: methanol: glacial acetic acid: water = 4:1:5;

[0097] Protein electrophoresis buffer: Dissolve the purchased MOPS-SDS Running Buffer in 1000 mL of water, stir well and set aside.

[0098] Construction of the M5 mutant: Using PA-PETase as a template, single-point mutations N233K, N205K, N114I, and S269V were added in sequence to finally obtain the M5 mutant.

[0099] Amino acid sequence of M5 mutant (261): NPYARGPNPTAASLEASAGPFTV RSFTVSRPSGYGAGTVYYPTNAGGTVGAIAIVPGYTARQSSIKWWGPRLASHGFVVITIDTISTLDQPPSRSSQQMAALRQVASLNGTSSSPIYGKVDTARMGVMGWSMGGGGSLISAANNPSLKAAAPQAPWASSTNFSSVTVPTLIFACEKDSIAPVNSSALPIYDSMSRNAKQFLEIKGGSHSCANSGNSNQALIGKKGVAWMKRFMDNDTRYVTFACENPNSTRVSDFRTANCS (as shown in SEQ ID NO: 5).

[0100] The nucleotide sequence of the M5 mutant (after optimization with the large intestine as the host) is: AATCCGTACGCACG (as shown in SEQ ID NO: 8).

[0101] The steps of mutant transformation, intracellular expression bacterial cell culture and collection, enzyme purification, enzyme activity reaction, absorbance initial screening, and HPLC measurement are shown in Example 1.

[0102] 3. Experimental results

[0103] The same batch of PA-PETase and M5 were expressed and purified in cells for verification. The reaction was carried out at 40℃, 45℃, and 50℃ for 24h and 72h respectively. The results are as follows: Figure 2 shown.

[0104] pass Figure 2 It can be seen that after 24h and 72h of reaction at 45℃ and 50℃, the activity of M5 is much higher than that of PA-PETase. After 72h of reaction at 50℃, the yield of M5 reaches 7317μM, which is 9.4 times that of PA-PETase. It can be seen that adding site mutations to PA-PETase can improve the activity and thermal stability of the enzyme.

[0105] Example 3 Construction and verification of M6 mutants - adding site mutations to the DepoPETase scaffold

[0106] 1. Acquisition of experimental materials

[0107] Acquisition of chassis strains: TOP10 and BL21 competent strains were purchased from Bomed Company.

[0108] The gene DepoPETase (pET_28a(+)) involved in this experiment was synthesized by Qingke Biotechnology Co., Ltd.

[0109] Amino acid sequence of DepoPETase (261): NPYARGPNPTAASLEASAGPFT VRSFTVSRPSGYGAGTVYYPTNAGGTVGAIAIVPGYIARQSSIKWWGPRLASHGFVVITIDTNSTLDQPSSRSSQQMAALRQVASLNGTSSSPIYGKVDTARMGVMGWSMGGGGSLISAANN PSLKAAAPQAPWHSSTNFSSVTVPTLIFACENDSIAPVNSSALPIYNSMSRNAKQFLEIKGGSHSCANSGNSDQALIGKKGVAWMKYFMDNDTRYSTFACENPNSTRVSDFRTANCS (such as SEQ ID NO:3).

[0110] Nucleotide sequence of DepoPETase (optimized for E. coli as the host): AATCCGTACGCAC GTGGTCCGAATCCAACCGCAGCATCTCTGGAAGCAAGTGCTGGTCCATTCACCGTTCGTTCTTTCACCGTTTCTCGTCCATCTGGTTACGGTGCAGGTACCGTTTACTATCCGACCAACGCTGGTGGTACTGTTGGTGCAATCGCTATCGTTCCAGGTTACATCGCTCGTCAGAGCTCCATCAAGTGGTGGGGTCCACGTTTGGCATCTCACGGTTTCGTTGTCATCACCATCGACACCAATTCCACTCTGGATCAGCCGTCTAGCCGTTCTTCTCAGCAGATGGCTGCTCTGCGTCAGGTTGCTTCTCTGAACGGTACCAGCTCTTCTCCAATCTACGGTAAAGTTGATACCGCTCGTATGGGTGTTATGGGTTGGTCTATGGGTGGTGGTGGTAGCCTGATCTCTGCTGCGAACAATCCAAGCCTGAAAGCAGCTGCACCGCAAGCTCCATGGCATTCTTCCACCAACTTCAGCTCTGTTACCGTTCCGACTCTGATCTTCGCATGTGAGAATGACTCTATCGCACCAGTTAACTCTTCTGCACTGCCGATCTACAACTCCATGTCTCGTAACGCGAAGCAGTTTCTGGAAATCAAGGGTGGTTCTCATAGCTGTGCAAACTCTGGTAACTCCGACCAGGCACTGATCGGTAAGAAAGGTGTTGCGTGGATGAAGTACTTCATGGACAACGACACTCGTTACTCTACCTTCGCGTGTGAGAACCCGAACTCTACTCGTGTTTCTGACTTCCGTACCGCTAACTGCTCT (shown as SEQ ID NO:12).

[0111] Obtaining the substrate for the enzyme activity reaction: Good fellow film.

[0112] 2. Experimental method:

[0113] LB medium: 10 g / L TPYPONE, 5 g / L YEAST EXTRACT, 10 g / L NaCl;

[0114] LB solid medium: 10 g / L TPYPONE, 5 g / L YEAST EXTRACT, 10 g / L NaCl, 15 g / L agarose;

[0115] Kanamycin stock solution: 50 mg / mL;

[0116] IPTG solution: 1 M, 0.238 g / mL;

[0117] Lysis buffer (LE Buffer): 50 mM Na2HPO4, 100 mM NaCl, 10 mM imidazole, pH = 7.5;

[0118] Eluent: 50 mM Na2HPO4, 100 mM NaCl, 500 mM imidazole, pH = 7.5;

[0119] Protein preservation solution: 50mMNa2HPO4, 100mMNaCl, pH=7.5;

[0120] Reaction buffer: 50 mM glycine-NaOH, glycine 3.74 g / L, pH = 9.0;

[0121] PMSF (serine protease inhibitor) solution: 100 mM;

[0122] Coomassie brilliant blue staining solution: 2.4 g / L Coomassie brilliant blue R250 dissolved in methanol: glacial acetic acid: water = 4:1:5;

[0123] Decolorizing solution: methanol: glacial acetic acid: water = 4:1:5;

[0124] Protein electrophoresis buffer: Dissolve the purchased MOPS-SDS Running Buffer in 1000 mL of water, stir well and set aside.

[0125] Construction of the M6 ​​mutant: Using DepoPETase as a template, single-point mutations N205K, N114I, and S269V were added in sequence to finally obtain the M6 ​​mutant.

[0126] Amino acid sequence of M6 mutant (261): NPYARGPNPTAASLEASAGPFTV RSFTVSRPSGYGAGTVYYPTNAGGTVGAIAIVPGYIARQSSIKWWGPRLASHGFVVITIDTISTLDQPSSRSSQQMAALRQVASLNGTSSSPIYGKVDTARMGVMGWSMGGGGSLISAANNPSLKAAAPQAPWHSSTNFSSVTVPTLIFACEKDSIAPVNSSALPIYNSMSRNAKQFLEIKGGSHSCANSGNSDQALIGKKGVAWMKYFMDNDTRYVTFACENPNSTRVSDFRTANCS (as shown in SEQ ID NO: 6).

[0127] The nucleotide sequence of the M6 ​​mutant (after optimization with the large intestine as the host) is: AATCCGTACGCACG (as shown in SEQ ID NO: 9).

[0128] The steps of mutant transformation, intracellular expression bacterial cell culture and collection, enzyme purification, enzyme activity reaction, absorbance initial screening, and HPLC measurement are shown in Example 1.

[0129] 3. Experimental results

[0130] The same batch of DepoPETase and M6 were expressed and purified in cells for verification. The reaction was carried out at 45℃, 50℃, and 55℃ for 24h and 72h respectively. The results are as follows: Figure 3 shown.

[0131] pass Figure 3 It can be seen that after reacting at 55℃ for 24h and 72h, the activity of M6 is much higher than that of DepoPETase. The yield of M6 after reacting at 55℃ for 72h reaches 7964μM, which is twice that of DepoPETase. This shows that adding site mutations to DepoPETase can improve the activity and thermal stability of the enzyme.

[0132] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Application of mutation sites as targets in improving the enzymatic activity, thermal stability and / or long-term effectiveness of PET plastic degrading enzymes; The mutation sites include: One or more of positions 114, 205, 233 and 269 of the amino acid sequence of a wild-type PET plastic degrading enzyme; The PET plastic degrading enzyme includes one or more of FAST-PETase enzyme, PA-PETase enzyme and DepoPETase enzyme.

2. A mutant, characterized in that Based on the wild-type PET plastic degrading enzyme, it has one or more of the following amino acid site mutations: position 114, position 205, position 233, and position 269. The amino acid sequence of the wild-type PET plastic degrading enzyme is shown in any of SEQ ID NO: 1 to SEQ ID NO:

3.

3. The mutant according to claim 2, characterized in that The mutation at position 114 includes: N114I; The mutation at position 205 includes: N205K; The mutation at position 233 includes: N233K; The mutation at position 269 includes: S269V.

4. The mutant according to claim 2 or 3, characterized in that It has: (1) the amino acid sequence shown in SEQ ID NO: 4 to SEQ ID NO: 6; or (2) an amino acid sequence obtained by replacing, deleting or adding one or more amino groups of the amino acid sequence shown in (1), and having the same or similar function as the amino acid sequence shown in (1); or (3) An amino acid sequence that is at least 80% identical to the amino acid sequence shown in (1) or (2).

5. A nucleic acid molecule encoding a mutant according to any one of claims 2 to 4, characterized in that: It has: (4) the nucleotide sequence shown in SEQ ID NO:7 to SEQ ID NO:9; or (5) A nucleotide sequence obtained by modifying, replacing, deleting or adding one or more bases to the nucleotide sequence described in (4); or (6) a sequence having at least 80% homology with the nucleotide sequence described in (4) or (5); or (7) A complementary sequence of the nucleotide sequence described in (4), (5) or (6).

6. An expression vector, characterized in that include: The nucleic acid molecule according to claim 5.

7. A host, characterized in that Transformation and / or transfection is carried out using the expression vector according to claim 6.

8. A method for preparing a mutant, characterized in that: The host cell according to claim 7 is cultured, centrifuged, the supernatant is collected, and purified to obtain the mutant.

9. A product, characterized in that include: The mutant according to any one of claims 2 to 4, the nucleic acid molecule according to claim 5, the expression vector according to claim 6, the host according to claim 7 and / or the mutant obtained by the preparation method according to claim 8.

10. Use of the mutant according to any one of claims 2 to 4, the nucleic acid molecule according to claim 5, the expression vector according to claim 6, the host according to claim 7, the mutant obtained by the preparation method according to claim 8 and / or the product according to claim 9 in degrading plastics.

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