High-activity PET hydrolase mutant and industrial grade depolymerization application thereof

Abstract

The invention relates to the field of biological catalysis, and discloses a high-activity PET hydrolase mutant and industrial-grade depolymerization application thereof. The invention provides a PET hydrolase mutant, which is obtained by mutating amino acid residues at the 182nd site and the 186th site in SEQ ID NO.2 and has proteins with the same functions. According to the invention, through calculation design and site-directed mutagenesis, the PET hydrolase mutant Kubu-PM12-NM with high thermal stability and catalytic efficiency is successfully obtained. Experiments prove that the fusion temperature of the modified mutant protein reaches 92.9 DEG C; in a 165 g / L industrial grade PET substrate system, the maximum reaction speed of catalyzing PET hydrolysis is increased by 1.29 times compared with that of a template enzyme, and is 1.43 times of that of efficient PET hydrolase LCC-ICCG; the depolymerization rate of high-immobilization-capacity PET under low enzyme loading capacity can reach 90% or above, and the mutant shows excellent application performance under harsh industrial conditions.

Description

Technical Field

[0001] The invention belongs to the field of biocatalysis and relates to a highly active PET hydrolase mutant and an industrial-grade depolymerization application thereof. Background Art

[0002] Plastics are widely used in industry, agriculture and medicine due to their excellent heat resistance, mechanical properties and chemical stability, but only 9% of plastics are effectively recycled worldwide. Polyethylene terephthalate (PET), a synthetic polyester with an annual output of over 82 million tons (China's output will reach 65 million tons in 2023), is difficult to degrade in the natural environment due to its stable aromatic structure and crystalline region characteristics. It enters the ecological cycle through soil infiltration, ocean migration and other pathways, causing persistent pollution. Traditional physical recycling methods (landfill, incineration, mechanical regeneration) have the disadvantages of land occupation by landfill, high pollution by incineration, and decreased quality of mechanically recycled products; traditional chemical degradation methods (methanolysis, hydrolysis, aminolysis, glycolysis) require the use of dangerous catalysts under high temperature and high pressure, and face problems such as difficult catalyst separation, harsh reaction conditions and high costs, which limit its industrial application. Enzymatic depolymerization of PET can be achieved at temperatures close to the glass transition temperature (T g =75°C), the product is highly pure and requires no toxic reagents. It combines environmental friendliness with resource recycling advantages, providing a sustainable solution for plastic pollution control.

[0003] In recent years, the bioenzymatic degradation technology of PET has made significant breakthroughs. In 2016, Yoshida et al. isolated IsPETase (T m =46℃) can catalyze the hydrolysis of amorphous PET at 30℃ (Yoshida, Shosuke, et al. "A bacterium that degrades and assimilates poly(ethylene terephthalate)." Science 353.6278(2016):759.), but its insufficient thermal stability limits its sustained activity at the optimal depolymerization temperature of PET (>75℃). In comparison, the engineered cutinase LCC-ICCG from the branch and leaf compost metagenome introduced a disulfide bond to hydrolyze T mBy increasing the temperature by 9.5°C, a 90% depolymerization rate was achieved within 10 hours at a substrate concentration of 200 g / L (Tournier, V., et al. "An engineered PET depolymerase to break down and recycle plastic bottles." Nature 580.7802(2020):216-219.), but 0.3% of the enzyme amount needed to be added to compensate for the activity deficiency.

[0004] Given the severity of plastic pollution, the development of highly active and thermostable PET hydrolases and their modification to enhance degradation activity are urgent. Modified PET hydrolases are of great significance in the degradation of industrial PET plastics and the treatment of environmental plastic waste, contributing to the reduction of plastic pollution and aligning with global trends in sustainable development and environmental protection. Summary of the Invention

[0005] The technical problem solved by the present invention is how to efficiently degrade PET.

[0006] In order to solve the above technical problems, in a first aspect, the present invention provides a PET hydrolase mutant, which is any one of the following:

[0007] A1) The protein shown includes a protein having the same function obtained by mutating the amino acid residues 182 and 186 of SEQ ID NO. 2;

[0008] A2) a protein in which, except for the mutated amino acid residue, the remaining amino acid residues in the protein of A1) have a homology of greater than 99%, greater than 95%, greater than 90%, greater than 85%, or greater than 80%, and has the same function;

[0009] A3) comprises connecting a tag to the N-terminus and / or C-terminus of the protein shown in A1) or A2) to obtain the protein shown in the sequence.

[0010] In the mutants described above, the mutation patterns of the amino acid residues are as follows:

[0011] The amino acid residue at position 182, His, was mutated to Asn;

[0012] The amino acid residue at position 186, Phe, was mutated to Met.

[0013] Among the mutants described above, the nucleotide sequence of the mutant protein shown in A1) includes SEQ ID NO.4.

[0014] Among the mutants described above, the nucleotide sequence of the mutant protein shown in A1) can be SEQ ID NO. 4. The mutant protein is named Kubu-PM12 -NM:

[0015] The activity of the mutant protein is greater than that of the protein shown in SEQ ID NO.2.

[0016] With template PET hydrolase Kubu-P M12 In comparison, the mutant protein Kubu-P M12 -NM has high activity and high thermal stability for PET hydrolysis, which is specifically reflected in its stable and efficient catalytic PET hydrolysis above 70°C. That is, at 70°C and a low enzyme loading of 0.5%, the depolymerization rate of PET for 165g / L industrial-grade PET reached 72.75% in 3 hours, which is 129% of the template enzyme and 143% of the PET hydrolase LCC-ICCG.

[0017] In a second aspect, the present invention provides a biological material related to the mutant protein described in the first aspect, which is any one of the following B1) to B4):

[0018] B1) a nucleic acid molecule encoding the mutant protein of the first aspect;

[0019] B2) an expression cassette containing the nucleic acid molecule described in B1);

[0020] B3) a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

[0021] B4) A recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3).

[0022] In the biological material described above, the nucleic acid molecule in B1) is any one of the following b1) to b6):

[0023] b1) DNA molecule shown in SEQ ID NO.3;

[0024] b2) a DNA molecule that has 75% or more identity with the nucleotide sequence defined in b1) and encodes the mutant protein described in the first aspect;

[0025] b3) A DNA molecule that hybridizes with the nucleotide sequence defined in b1) under stringent conditions and encodes the mutant protein described in the first aspect.

[0026] In the biological materials described above, the recombinant vector is a recombinant vector containing the above gene (named pET22b(+)-Kubu-P M12 -NM is a recombinant expression vector obtained by inserting the encoding gene described in the second aspect of the present invention between the multiple cloning sites NdeI and XhoI of the pET22b(+) vector.

[0027] In the biological material described above, the recombinant bacteria are recombinant Escherichia coli containing the recombinant vector.

[0028] In a third aspect, the present invention provides the use of the mutant protein described in the first aspect in any of the following:

[0029] C1) Degradation of PET;

[0030] C2) increasing the depolymerization rate of catalytic PET hydrolysis;

[0031] C3) preparing a product of degraded PET;

[0032] C4) preparing a product that increases the depolymerization rate of catalyzed PET hydrolysis.

[0033] In a fourth aspect, the present invention provides use of the biomaterial according to the second aspect in any of the following:

[0034] C1) Degradation of PET;

[0035] C2) increasing the depolymerization rate of catalytic PET hydrolysis;

[0036] C3) preparing a product of degraded PET;

[0037] C4) preparing a product that increases the depolymerization rate of catalyzed PET hydrolysis.

[0038] In the above, the degradation of PET or the improvement of the depolymerization rate of catalytic PET hydrolysis is carried out under the conditions of high substrate and low enzyme loading, and can also be carried out under the conditions of high substrate and high enzyme loading.

[0039] In the above, the high substrate may be the PET concentration in industrial bioprocessing, and specifically the PET concentration is greater than or equal to 165 g / L.

[0040] In a fifth aspect, the present invention provides a product comprising the mutant protein according to the first aspect or the biomaterial according to the second aspect.

[0041] In a sixth aspect, the present invention provides a method for degrading PET, comprising the following steps: using the mutant protein described in the first aspect to degrade PET to achieve degradation.

[0042] The above degradation temperature is 50°C-75°C, further 68°C-72°C, and more specifically 70°C.

[0043] The mass ratio of the above mutant protein to PET was 5:10,000 or 3:10,000.

[0044] In the above, the degradation of PET or the improvement of the depolymerization rate of catalytic PET hydrolysis is carried out under the conditions of high substrate and low enzyme loading, and can also be carried out under the conditions of high substrate and high enzyme loading.

[0045] In the above, the high substrate may be the PET concentration in industrial bioprocessing, and specifically the PET concentration is greater than or equal to 165 g / L.

[0046] The present invention aims to modify PET hydrolase to improve its catalytic activity at the optimal temperature for PET depolymerization (68-72°C), and to achieve a depolymerization efficiency of more than 90% for industrial-grade highly loaded PET under low enzyme loading conditions.

[0047] The present invention is a PET hydrolase Kubu-P from Kutzneria buriramensis reported in the literature. M12 Perform site-directed mutagenesis to transform the mutant Kubu-P M12 -NM exhibits 29% higher activity than the template and comparable thermal stability to the wild-type, maintaining activity for extended periods at the optimal temperature for PET depolymerization (68-75°C). At industrial-grade PET substrate concentrations, the mutant enzyme achieves near-complete PET depolymerization within 9 hours with only 0.05% enzyme addition. Even at the more stringent enzyme dosage of 0.03%, depolymerization rates exceeding 90% can be achieved within 18 hours, demonstrating its significant value in industrial PET recycling applications.

[0048] Compared with the prior art, the present invention has the following advantages:

[0049] (1) PET hydrolase Kubu-P in the present invention M12 The mutant Kubu-P M12 -NM has a melting temperature of 92.9°C and can remain active for a long time at the optimal temperature for PET depolymerization;

[0050] (2) The mutant Kubu-P of the present invention M12 The catalytic activity of -NM was significantly enhanced. Under the conditions of a substrate concentration of 1.6 g / L PET and an enzyme dosage of 1 / 1000th the mass of PET (enzyme concentration of 0.057 μM), after 8 hours of reaction, depolymerization rates reached 32.8%, 64.6%, 77.2%, 80.6%, and 80.17% at temperatures of 50°C, 60°C, 68°C, 72°C, and 75°C, respectively.

[0051] (3) The mutant Kubu-P of the present invention M12 -NM shows significant industrial application potential in industrial-grade PET depolymerization system, and its catalytic efficiency is significantly better than that of the template enzyme Kubu-P M12The depolymerization rate reached 72.8% in 3 hours (a 1.29-fold increase compared to the template enzyme) and 93.8% in 9 hours (a 1.11-fold increase compared to the template enzyme), respectively, 1.43 times and 1.31 times that of the PET hydrolase LCC-ICCG under the same conditions, demonstrating excellent catalytic activity and application value.

[0052] (4) The mutant Kubu-P of the present invention M12 -NM also has significantly better catalytic efficiency than the template enzyme Kubu-P in the harsh low enzyme loading PET depolymerization system. M12 At 70°C and a substrate concentration of 165 g / L, when the enzyme addition amount was 0.03% of the substrate mass (1.67 μM), the depolymerization rate reached 91.1% in 24 hours, which was better than that of the template enzyme Kubu-P under the same conditions. M12 83.1% and 71.1% of PET hydrolase LCC-ICCG BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 The reaction route of enzymatic degradation of PET to ethylene terephthalate (BHET), monohydroxyethyl terephthalate (MHET), terephthalic acid (TPA) and ethylene glycol (EG) was presented.

[0054] Figure 2 The recombinant plasmid pET22b(+)-Kubu-P M12 -NM's map.

[0055] Figure 3 Kubu-P mutant M12 -Analysis of the DSF characteristic curve of NM.

[0056] Figure 4 PET hydrolase Kubu-P M12 (a) Mutant Kubu-P M12 -HPLC chromatograms of PET depolymerization products catalyzed by NM (b) and PET hydrolase LCC-ICCG (c) at 0.05% enzyme loading and high PET substrate concentration.

[0057] Figure 5 PET hydrolase Kubu-P M12 (a) Mutant Kubu-P M12 -NM (b) and PET hydrolase LCC-ICCG (c) catalyze the depolymerization of PET at different time points at 0.05% enzyme loading and high PET substrate concentration.

[0058] Figure 6 PET hydrolase Kubu-P M12 , mutant Kubu-P M12 -NM and PET hydrolase LCC-ICCG catalyze PET depolymerization at 0.3% enzyme loading and high PET substrate concentration. DETAILED DESCRIPTION

[0059] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0060] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0061] Unless otherwise specified, the quantitative tests in the following examples were performed in triplicate and the results were averaged. M12 Preparation and purification of mutants I. Hydrolase Kubu-P M12 Acquisition of mutant encoding genes

[0062] The template PET hydrolase Kubu-P was obtained from the published literature (Seo H, et al. "Landscape profiling of PET depolymerases using a natural sequence cluster framework." Science, 2025, 387(6729): eadp5637.) M12 The nucleotide sequence is SEQ ID NO.1; the encoded Kubu-P M12 The amino acid sequence is SEQ ID NO.2.

[0063] First, the template PET hydrolase Kubu-P was obtained by whole gene synthesis. M12 Then, Kubu-P M12 The coding gene of Kubu-P was subjected to site-directed mutagenesis to obtain M12 Mutant genes.

[0064] Kubu-P M12 The mutant gene is mutant Kubu-PM12 -NM coding gene, the nucleotide sequence of which is SEQ ID NO.3.

[0065] The above Kubu-P M12 The mutant is Kubu-P M12 -NM mutant protein.

[0066] Kubu-P M12 -NM mutant protein is a template PET hydrolase Kubu-P shown in SEQ ID NO.2 M12 The mutant was obtained by mutating His at position 182 to Asn and Phe at position 186 to Met, while other amino acid residues remained unchanged; Kubu-P M12 The amino acid sequence of the -NM mutant protein is SEQ ID NO.4.

[0067] 2. Construction of recombinant expression vector

[0068] First, Kubu-P was obtained by whole gene synthesis. M12 The gene (nucleotide sequence is shown in SEQ ID NO.1, containing 762 bases) was constructed into the pET-22b(+) vector (containing the ampicillin resistance gene) using NdeI and XhoI restriction enzymes, and then the recombinant plasmid was transformed into competent cells BL21(DE3) to obtain the recombinant expression plasmid pET22b(+)-Kubu-P M12 pET-22b(+) was purchased from BGI, catalog number 69744-3. The pET22b(+) plasmid carries a carboxyl-terminal histidine tag encoding sequence, His6, at the XhoI-cleaved end, facilitating purification of the target protein.

[0069] After sequencing, pET22b(+)-Kubu-P M12 The sequence is correct. The plasmid is Kubu-P shown in positions 1-762 of SEQ ID NO.1 in the sequence list. M12 The gene was replaced into the fragment between NdeI and XhoI sites of pET-22b(+), forming the recombinant plasmid pET22b(+)-Kubu-P M12 , and Kubu-P M12 The gene is fused with the His6 tag on the vector to express recombinant Kubu-P M12 Protein (in Kubu-P M12 The protein was obtained by connecting a HIS tag to the N-terminus.

[0070] According to the above method, the mutant Kubu-P M12The coding gene of -NM (SEQ ID NO.3) was inserted between the NdeI and XhoI restriction sites of pET-22b(+) to obtain the recombinant expression vector pET22b(+)-Kubu-P M12 -NM, and Kubu-P M12 -NM gene was fused with the His6 tag on the vector to express the recombinant mutant Kubu-P M12 -NM protein (in mutant Kubu-P M12 -NM (protein obtained by connecting the N-terminus of the protein to a HIS tag).

[0071] Recombinant plasmid pET22b(+)-Kubu-P M12 -NM (denoted as Kubu-P in the figure) M12 -NM) spectrum as Figure 2 shown.

[0072] 3. Construction Methods of Engineered Bacteria

[0073] The pET22b(+)-Kubu-P obtained in step 2 was M12 , pET22b(+)-Kubu-P M12 -NM were transformed into Escherichia coli BL21 (DE3) competent cells (competent cells purchased from Beijing Solebow Technology Co., Ltd., catalog number C1400) to obtain recombinant engineering bacteria E. coli BL21 (DE3) / pET22b (+) -Kubu-P M12 , E.coli BL21(DE3) / pET22b(+)-Kubu-P M12 -NM. The extracted plasmid was sequenced to verify its correctness.

[0074] PET hydrolase Kubu-P M12 and the acquisition of its mutants

[0075] 1. Induced expression of engineered bacteria

[0076] Each recombinant engineered strain obtained in step 3 was inoculated on an LB agar plate (containing 100 μg / mL ampicillin), cultured by plate streak method, and incubated in a 37°C constant temperature incubator for 14 hours. After the colony morphology is fully developed, a well-grown monoclonal colony is selected and inoculated into 5 mL of LB liquid medium (containing 100 μg / mL ampicillin), and shaken in a 37°C shaker at 200 rpm for 7 hours until the culture reaches OD 600Reaching the range of 1.2-2.0; then transfer each recombinant engineered bacteria to two bottles of 200mL LB liquid culture medium (containing 100μg / mL ampicillin) at a 1% (v / v) inoculation volume, and maintain 200rpm in a constant temperature shaking incubator at 37℃ for expansion culture. 600 After reaching the range of 0.6-0.8, isopropyl-β-D-thiogalactopyranoside (IPTG) was added at a final concentration of 1 mM to obtain a culture system. The culture system was then transferred to a low-temperature induction at 20°C, and the shaking rate was adjusted to 180 rpm for 16 hours to induce Kubu-P M12 and expression of its mutant proteins. After terminating the culture, the culture medium was centrifuged at 4°C and 6000rpm for 20 minutes to collect the bacteria, and the resulting precipitate was vortexed and resuspended with 50mL of protein lysis buffer (100mM potassium dihydrogen phosphate, 300mM sodium chloride, the balance being water, pH 8.0) to obtain a resuspended bacterial solution. The resuspended bacterial solution was then crushed for 50 minutes using an ultrasonic crusher (working / intermittent cycle 4 seconds / 6 seconds, power intensity 60%) under ice bath conditions. The crushed product was centrifuged at 4°C, first at 7000rpm for 15 minutes to remove cell debris, and the collected supernatant was further centrifuged at 12000rpm for 10 minutes. The clarified supernatant finally obtained was the product containing recombinant Kubu-P M12 Kubu-P M12 -NM protein crude enzyme solution.

[0077] The formula of the LB liquid culture medium is as follows: 5 g / L yeast extract, 10 g / L tryptone, 5 g / L sodium chloride, and the balance is water.

[0078] The formula of the LB agar plate is as follows: 5 g / L yeast extract, 10 g / L tryptone, 5 g / L sodium chloride, 15 g / L agarose, and the balance is water.

[0079] 2. PET hydrolase Kubu-P M12 Purification of its mutant enzymes

[0080] Based on the specific binding principle of histidine tag and immobilized metal ions, nickel ions were used as the coordinating metal ions and gradient concentration of imidazole elution buffer was used to achieve the elution of recombinant Kubu-P M12 or recombinant mutant Kubu-P M12 -NM target protein is separated from the crude enzyme solution. The elution peak is monitored by 280nm UV absorption. The absorbance of different elution components under 280nm UV light is dynamically measured by a UV spectrophotometer to achieve effective removal of non-target proteins.

[0081] The specific operation process is as follows: A 20 mL nickel-based agarose gel column (Beijing Weishi Bohui Chromatography Technology Co., Ltd., FF) and a nucleic acid protein detector (Shanghai Huxi Analytical Instrument Factory, HD-21-1) were used for crude enzyme chromatography purification. First, the column bed was pre-equilibrated with binding buffer A (100 mM potassium dihydrogen phosphate, 300 mM sodium chloride, the balance being water, pH 8.0) until the baseline was stable. Then, the sample (i.e., containing recombinant Kubu-P M12 Kubu-P M12 -NM protein crude enzyme solution), use binding buffer B containing imidazole (100mM potassium dihydrogen phosphate, 300mM sodium chloride, 50mM imidazole, the balance is water, pH8.0) to elute non-target binding proteins until the UV absorbance value no longer changes. Subsequently, elution buffer (100mM potassium dihydrogen phosphate, 300mM sodium chloride, 200mM imidazole, the balance is water, pH 8.0) is used to elute the target protein and collect it. In order to further improve the purity of the protein, the collected elution fractions are placed in a 60°C water bath for 2 hours of thermal stability differential purification, and then centrifuged at 12000rpm for 30 minutes at 4°C to remove heat-denatured miscellaneous proteins. The supernatant is centrifuged at 4°C, 6000rpm for 20 minutes through an ultrafiltration concentration tube with a 10kDa molecular weight cutoff, and desalted three times to completely remove residual imidazole. The final purified protein (recombinant Kubu-P M12 Protein, recombinant mutant Kubu-P M12 -NM protein) was stored in phosphate buffer (100 mM potassium dihydrogen phosphate, 300 mM sodium chloride, the balance being water, pH 8.0) and stored at 4°C for later use. The purified recombinant Kubu-P M12 Protein solution, recombinant mutant Kubu-P M12 -NM protein solution.

[0082] Purity of the target protein was verified using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, 5% stacking gel, 10% separating gel), which showed a purity exceeding 90%. Protein concentration was then quantified using the Coomassie brilliant blue method: a standard curve was constructed using bovine serum albumin (BSA), and colorimetric determination was performed using a UV-visible spectrophotometer (Shanghai Meixi Instrument Co., Ltd., UV-1200) at a wavelength of 595 nm.

[0083] The results showed that the purified recombinant Kubu-P M12 Protein solution, recombinant mutant Kubu-P M12 -NM protein solution concentration distribution is in the range of 2-10 mg / mL.

[0084] Example 2, PET hydrolase Kubu-P M12Determination of the ability of mutants to catalyze the depolymerization of post-consumer bottle-grade PET

[0085] 1. PET Hydrolase Kubu-P M12 Thermal stability test of mutants

[0086] The optimal reaction temperature for enzyme-catalyzed PET depolymerization is at the glass transition temperature (T g ) (65-71°C), which places strict demands on the thermal stability of the enzyme.

[0087] NanoDSF was used to determine the stability of the recombinant mutant Kubu-P using a protein stability analyzer (Prometheus NT.48, Germany). M12 -NM in protein buffer (100 mM potassium dihydrogen phosphate, 300 mM sodium chloride, the balance is water, pH 8.0) thermal denaturation temperature (T m The experiment was set up with a linear temperature ramp of 2°C per minute from 25°C to 110°C.

[0088] After three independent repeated measurements, the DSF characteristic curve analysis showed that the Tm value of the recombinant mutant reached 92.9℃( Figure 3 ), more than PET material T g 15℃, ensuring that the enzyme can maintain activity at the optimal reaction temperature for PET depolymerization.

[0089] PET hydrolase Kubu-P at low substrate concentration M12 -Testing the activity of NM mutants in catalyzing PET depolymerization

[0090] The experiment used bottle-grade post-consumer PET (derived from the main body of Coca-Cola beverage bottles, minus the cap, neck, bottom, and label sticker). The pretreatment process was as follows: The original bottle flakes were mechanically cut into 3 cm × 3 cm pieces, ultrasonically cleaned with deionized water, a surfactant, and anhydrous ethanol, and then dried. The PET flakes were placed in a ceramic crucible covered with aluminum foil and placed on the foil. The crucible was heated in a muffle furnace (Hefei Kejing Materials Technology Co., Ltd., KSL-1200X) at a rate of 10°C / min from 30°C to 310°C until the PET flakes were completely melted, and the temperature was maintained for 30 minutes. After heating, the muffle furnace was immediately opened, the crucible was removed and placed in an ice-water mixture to rapidly cool the PET to room temperature. The solidified PET solid was removed, rinsed with deionized water, and then dried to obtain de-crystallized PET flakes. The PET flakes were then pulverized using a high-speed rotary mill (Ant Source Scientific Instruments Beijing Co., Ltd., AM500S) equipped with a 0.5 mm trapezoidal screen at 8000 rpm. The obtained product was sieved through a standard sieve (200–355 μm) to obtain post-consumer PET bottle powder with uniform particle size.

[0091] The post-consumer PET powder described below specifically refers to the 200–355 μm particle size obtained through the aforementioned pretreatment. Buffer 1 used in the reaction system was a phosphate standard solution (100 mM potassium dihydrogen phosphate, 300 mM sodium chloride, the balance being water, pH 8.0).

[0092] 1. PET hydrolase mutant Kubu-P M12 -Test of the activity of NM in catalyzing PET depolymerization at different temperatures

[0093] About 5 mg of post-consumer PET bottle powder was weighed into a 5 mL glass reaction bottle, and 3 mL of pH 8.0 containing 0.057 μM purified recombinant mutant Kubu-P was added. M12 -NM buffer 1 (containing 5 μg of protein purified recombinant mutant Kubu-P M12 -NM protein solution was dissolved in buffer 1 to obtain a total volume of 3 mL, and the concentration of the mutant protein was 0.057 μM).

[0094] The reaction system was placed in a thermostatic shaker (DH300, Hangzhou Ruicheng Instrument Co., Ltd.) and incubated at 50°C, 60°C, 68°C, 72°C, and 75°C for 12 hours, followed by an 8-hour enzymatic hydrolysis reaction at 400 rpm. Upon completion, the reaction was terminated by the addition of pre-chilled methanol (v:v = 1:1). The resulting mixture was diluted 10-fold with buffer 1 and centrifuged at 12,000 rpm for 2 minutes at 4°C. The supernatant was collected and filtered through a 0.22 μm pore size microporous membrane to effectively remove heat-denatured enzyme proteins and particulate matter, yielding PET hydrolyzate samples obtained under different conditions.

[0095] The process of PET depolymerization is as follows Figure 1 As shown, among the products, only terephthalic acid (TPA), mono(2-hydroxyethyl) terephthalate (MHET) and bis(2-hydroxyethyl) terephthalate (BHET) can be detected by liquid chromatography.

[0096] Standard products TPA (Shanghai MacLean Biochemical Technology Co., Ltd.), MHET (BiDe Pharmaceutical Technology Co., Ltd.) and BHET (BiDe Pharmaceutical Technology Co., Ltd.) with different concentrations were measured by high performance liquid chromatography (HPLC). The peak areas were measured and the standard curves were plotted with the concentration (X) as the abscissa and the peak area (Y) as the ordinate. The standard curve functions corresponding to the three products are shown below:

[0097] TPA peak area = concentration (TPA) × 7.35188 × 10 6

[0098] MHET peak area = concentration (MHET) × 8.81652 × 10 6

[0099] BHET peak area = concentration (BHET) × 1.00305 × 10 7

[0100] The concentration and peak area curves were used to calculate the concentrations of the three substances in the product. The samples were tested by HPLC, and the corresponding peak times and peak areas were substituted into the corresponding standard curves to determine the contents of TPA, MHET, and BHET in the samples.

[0101] High performance liquid chromatography (HPLC) (Shimadzu LC-20AD, Japan) was used for quantitative analysis of the products, and SunFire was used for chromatographic separation. TMA C18 reversed-phase column (GL Sciences, 5 μm, 250 × 4.6 mm) was used with an injection volume of 10 μL. The mobile phase consisted of phase A (0.1% formic acid in water, v / v) and phase B (chromatographic-grade acetonitrile). A multistep gradient elution program was used: isocratic elution with 5% phase B (0–5 min); linear gradient to 44% phase B (5–13 min); gradient increase to 70% phase B (13–18 min); isocratic elution (18–23 min); gradient return to the initial ratio (23–24 min); and system equilibration (24–28 min). Detection parameters were set as follows: flow rate 0.8 mL / min, detection wavelength 254 nm, column temperature 40°C, corresponding to the characteristic UV absorption band of PET degradation products.

[0102] After HPLC analysis, at the retention time t R =14.88 minutes, the chromatographic peak of the degradation product TPA appeared. R =15.46 minutes, the chromatographic peak of the degradation product MHET appeared. R The chromatographic peak of the degradation product BHET appeared around 15.76 minutes.

[0103] Based on the external standard quantitative analysis, the recombinant mutant Kubu-P M12 The concentrations of TPA, MHET, and BHET generated by -NM at different temperatures and their corresponding substrate conversion rates are shown in Table 1.

[0104] The PET hydrolase recombinant mutant Kubu-P was determined by HPLC. M12 The concentrations of TPA, MHET, and BHET in the -NM hydrolysates, as well as the calculated depolymerization rates of the substrates, are presented in Table 1 , respectively.

[0105] The calculation formula for the depolymerization rate in Table 1 is as follows, where the total product concentration is the sum of the concentrations of TPA, MHET, and BHET detected by HPLC, the reaction volume in this example is 3 mL, the mass of PET is 5 mg, the molecular weight of the PET monomer is 182.14 Da, and the dilution factor is 10.

[0106] Depolymerization rate = total product concentration × dilution factor × reaction volume / (PET mass / PET monomer molecular weight) × 100%

[0107] It can be seen that the recombinant mutant Kubu-P M12 -NM has catalytic PET depolymerization activity at 50°C, 60°C, 68°C, 72°C and 75°C, and the depolymerization rate of PET catalyzed hydrolysis at 68°C-75°C for 8 hours is greater than 75%.

[0108] Table 1 shows the PET hydrolase recombinant mutant Kubu-P at 50-75°C. M12-Products of PET hydrolysis catalyzed by NM

[0109]

[0110] PET hydrolase Kubu-P at high substrate concentration M12 , mutant Kubu-P M12 - Activity test of PET depolymerization catalyzed by NM and efficient PET hydrolase LCC-ICCG

[0111] In order to evaluate the effect of PET hydrolase mutants on improving the degradation efficiency of industrial grade PET, the template PET hydrolase Kubu-P M12 , mutant Kubu-P M12 -NM and the highly efficient PET hydrolase LCC-ICCG were used to carry out PET depolymerization tests in a high solid content system (substrate loading 165 g / L) simulating industrial bioprocessing conditions.

[0112] 1. Comparison of PET hydrolase Kubu-P M12 , mutant Kubu-P M12 Activity test of PET depolymerization catalyzed by NM and high-efficiency PET hydrolase LCC-ICCG at 0.5% enzyme dosage

[0113] PET hydrolase Kubu-P M12 Experimental group: About 8.25 g of post-consumer PET bottle powder was weighed into a 100 mL three-necked flask, and 44 mL of pH 8.0 buffer 1 containing 2.78 μM purified recombinant Kubu-PM12 was added (the purified recombinant Kubu-PM12 containing 4.125 mg protein was added). M12 The protein solution was dissolved in buffer 1 to obtain a total volume of 44 mL, and the PET hydrolase recombinant Kubu-P M12 The protein concentration was 2.78 μM).

[0114] Mutant Kubu-P M12 -NM experimental group: About 8.25 g of post-consumer PET bottle powder was weighed into a 100 mL three-necked flask, and then 44 mL of pH 8.0 buffer 1 containing 2.78 μM purified mutant Kubu-PM12-NM was added (the purified recombinant mutant Kubu-PM12 containing 4.125 mg protein was added). M12 -NM protein solution was dissolved in buffer 1 to obtain a total volume of 44 mL, and the recombinant mutant Kubu-P M12 -NM protein concentration was 2.78 μM).

[0115] PET Hydrolase LCC-ICCG Experimental Group: Approximately 8.25 g of post-consumer PET bottle powder was weighed into a 100 mL three-necked flask. Then, 44 mL of pH 8.0 buffer 1 containing 2.23 μM purified template PET hydrolase recombinant LCC-ICCG was added. (A solution of purified PET hydrolase recombinant LCC-ICCG containing 4.125 mg of protein (the recombinant LCC-ICCG protein solution was prepared by dissolving the LCC-ICCG protein with the amino acid sequence of SEQ ID NO. 5 according to the method previously described for recombinant Kubu-PM12) was dissolved in buffer 1. The total volume was 44 mL, and the concentration of PET hydrolase LCC-ICCG was 2.23 μM.)

[0116] The three-necked flask containing the above reaction system was placed in a thermostatic magnetic stirring reaction system (Shanghai Yuhua Instrument Co., Ltd., ZNCL-G). The water bath temperature was set at 70°C with continuous mechanical stirring. The pH of the reaction solution was measured in real time using a pH meter (Mettler-Toledo, FE28). Standardized NaOH solution (5 mol / L) was added to maintain a stable pH range of 7.8-8.2. The volume of alkali solution consumed was also recorded. 1000 μL of the reaction solution was sampled 3, 6, 9, 12, 18, and 24 hours after the start of the reaction. The sample was diluted 800-fold by adding buffer 1. The reaction was terminated by adding pre-chilled methanol (v:v = 1:1). The supernatant was collected by centrifugation (12,000 rpm, 2 minutes) and filtered through a 0.22 μm filter with a syringe to remove inactivated enzymes. Hydrolyzate samples were obtained. The experiment was repeated in duplicate for each enzyme to ensure data accuracy.

[0117] The PET depolymerization products were quantitatively analyzed using the HPLC detection method described above, and the depolymerization rate of PET was calculated accordingly.

[0118] PET hydrolase recombinant Kubu-P M12 Protein, recombinant mutant Kubu-P M12 The HPLC chromatograms of the products of -NM protein and highly efficient PET hydrolase recombinant LCC-ICCG protein after 3 hours of reaction time are shown in Figure 4 (a) Figure 4 (b) and Figure 4 (c). From Figure 4 It can be observed that the three enzymes have the highest retention time t R The chromatographic peak of the hydrolysis product TPA appeared around t = 14.9 min, which was consistent with the peak time of the TPA standard. R The chromatographic peak of substrate MHET appeared around t = 15.5 min, which was consistent with the peak time of MHET standard. R=15.8min, the chromatographic peak of substrate BHET appeared at the same time as the peak of BHET standard. Figure 4 The chromatographic peak of MHET in (b) was significantly higher in terms of peak height and peak area than Figure 4 (a) and Figure 4 (c).

[0119] PET hydrolase recombinant Kubu-P M12 Protein, recombinant mutant Kubu-P M12 -NM protein and efficient PET hydrolase recombinant LCC-ICCG protein at different time points Figure 5 As shown in the figure, the recombinant mutant Kubu-P M12 -NM depolymerization rate at 3 h was the same as that of PET hydrolase recombinant Kubu-P M12 At 6 hours, the recombinant mutant Kubu-P M12 -NM depolymerization rate has exceeded 90%, compared with the PET hydrolase recombinant mutant Kubu-P at the same reaction time. M12 -NM had a 9% higher depolymerization rate and 23% higher than that of the PET hydrolase recombinant LCC-ICCG protein, and the depolymerization rate of the mutant exceeded that of the latter two.

[0120] 2. Comparison of PET hydrolase Kubu-P M12 , mutant Kubu-P M12 Activity test of PET depolymerization catalyzed by NM and high-efficiency PET hydrolase LCC-ICCG at 0.3% enzyme dosage

[0121] To systematically evaluate the mutant Kubu-P M12 -NM's catalytic advantage allowed the degradation of high-solid-content PET (165 g / L) to be carried out under low-dose conditions with the enzyme loading reduced to 0.3% (w / w).

[0122] PET hydrolase Kubu-P M12 Experimental group: About 8.25 g of post-consumer PET bottle powder was weighed into a 100 mL three-necked flask, and 44 mL of pH 8.0 solution containing 1.67 μM purified template PET hydrolase recombinant Kubu-P was added. M12 Buffer 1 (containing 2.475 mg of protein purified PET hydrolase recombinant Kubu-P M12 The protein solution was dissolved in buffer 1 to obtain a total volume of 44 mL, and the PET hydrolase recombinant Kubu-P M12 The protein concentration was 1.67 μM).

[0123] Mutant Kubu-P M12 -NM experimental group: About 8.25 g of post-consumer PET bottle powder was weighed into a 100 mL three-necked flask, and then 44 mL of pH 8.0 containing 1.67 μM purified recombinant mutant Kubu-P was added. M12 -NM buffer 1 (containing 2.475 mg of protein purified recombinant mutant Kubu-P M12 -NM protein solution was dissolved in buffer 1 to obtain a total volume of 44 mL, and the recombinant mutant Kubu-P M12 -NM protein concentration was 1.67 μM).

[0124] PET Hydrolase LCC-ICCG Experimental Group: Approximately 8.25 g of post-consumer PET bottle powder was weighed into a 100 mL three-necked flask. 44 mL of pH 8.0 buffer 1 containing 1.34 μM purified template PET hydrolase recombinant LCC-ICCG protein was added (prepared by dissolving 2.475 mg of purified recombinant LCC-ICCG protein in buffer 1, resulting in a total volume of 44 mL. The concentration of PET hydrolase LCC-ICCG was 1.34 μM).

[0125] The three-necked flask containing the above reaction system was placed in a thermostatic magnetic stirring reaction system (Shanghai Yuhua Instrument Co., Ltd., ZNCL-G). The water bath temperature was set at 70°C with continuous mechanical stirring. The pH of the reaction solution was measured in real time using a pH meter (Mettler-Toledo, FE28). Standardized NaOH solution (5 mol / L) was added to maintain a stable pH range of 7.8-8.2. The volume of alkali solution consumed was also recorded. 1000 μL of the reaction solution was sampled 3, 6, 9, 12, 18, and 24 hours after the start of the reaction. The sample was diluted 800-fold by adding buffer 1. The reaction was terminated by adding pre-chilled methanol (v:v = 1:1). The supernatant was collected by centrifugation (12,000 rpm, 2 minutes) and filtered through a 0.22 μm filter with a syringe to remove inactivated enzymes. Hydrolyzate samples were obtained. The experiment was repeated in duplicate for each enzyme to ensure data accuracy.

[0126] The PET depolymerization products were quantitatively analyzed using the HPLC detection method described above, and the depolymerization rate of PET was calculated based on the analysis. M12 , mutant Kubu-P M12 -NM and the efficient PET hydrolase LCC-ICCG at different time points. Figure 6 As shown in the figure, the mutant Kubu-P M12 -NM depolymerization rate at 3 h was PET hydrolase Kubu-P M12At 9 hours, the mutant Kubu-P M12 -NM depolymerization rate has exceeded 80%, compared with the PET hydrolase Kubu-P at the same reaction time. M12 The depolymerization rate of the mutant Kubu-P was 8.3% higher than that of the PET hydrolase LCC-ICCG, which was 21.4% higher. M12 -NM depolymerization rate reached 91.1%, while PET hydrolase Kubu-P M12 The depolymerization rates of the ET hydrolase LCC-ICCG did not reach 90%.

[0127] These results suggest that the mutant Kubu-P M12 -NM catalyzes PET depolymerization faster than PET hydrolase Kubu-P M12 and the highly efficient PET hydrolase LCC-ICCG, and its 24-hour depolymerization rate can still reach more than 90% under low enzyme loading and high substrate concentration, which has good application value in the industrialization of PET recycling.

[0128] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be practiced over a wide range of parameters, concentrations, and conditions without departing from the spirit and scope of the present invention and without unnecessary experimentation. Although specific embodiments have been given herein, it should be understood that further modifications may be made to the present invention. In summary, this application is intended to encompass any variations, uses, or improvements to the present invention, including those made by conventional techniques known in the art that depart from the scope of the present invention. Applications of the essential features may be made within the scope of the following claims.

Claims

1. A PET hydrolase mutant, any of the following: A1) The protein shown includes a protein having the same function obtained by mutating the amino acid residues 182 and 186 of SEQ ID NO. 2; A2) a protein in which, except for the mutated amino acid residue, the remaining amino acid residues in the protein of A1) have a homology of greater than 99%, greater than 95%, greater than 90%, greater than 85%, or greater than 80%, and has the same function; A3) comprises connecting a tag to the N-terminus and / or C-terminus of the protein shown in A1) or A2) to obtain the protein shown in the sequence.

2. The mutant according to claim 1, characterized in that The mutation patterns of each amino acid residue are as follows: The amino acid residue at position 182, His, was mutated to Asn; The amino acid residue at position 186, Phe, was mutated to Met.

3. The mutant protein according to claim 1 or 2, characterized in that The nucleotide sequence of the mutant protein shown in A1) includes SEQ ID NO.

4.

4. The biological material related to the mutant protein according to any one of claims 1 to 3, which is any one of the following B1) to B4): B1) a nucleic acid molecule encoding the mutant protein according to any one of claims 1 to 3; B2) an expression cassette containing the nucleic acid molecule described in B1); B3) a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4) A recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3).

5. The biomaterial according to claim 4, characterized in that: B1) The nucleic acid molecule is any one of the following b1) to b6): b1) DNA molecule shown in SEQ ID NO.3; b2) a DNA molecule that has 75% or more identity with the nucleotide sequence defined in b1) and encodes the mutant protein according to any one of claims 1 to 3; b3) A DNA molecule that hybridizes with the nucleotide sequence defined in b1) under stringent conditions and encodes the mutant protein according to any one of claims 1 to 3.

6. Use of the mutant protein according to any one of claims 1 to 3 in any of the following; C1) Degradation of PET; C2) increasing the depolymerization rate of catalytic PET hydrolysis; C3) preparing a product of degraded PET; C4) preparing a product that increases the depolymerization rate of catalyzed PET hydrolysis.

7. Use of the biomaterial according to claim 4 or 5 in any of the following: C1) Degradation of PET; C2) increasing the depolymerization rate of catalytic PET hydrolysis; C3) preparing a product of degraded PET; C4) preparing a product that increases the depolymerization rate of catalyzed PET hydrolysis.

8. A product comprising the mutant protein according to any one of claims 1 to 3 or the biomaterial according to claim 4 or 5.

9. A method for degrading PET, comprising the following steps: degrading PET using the mutant protein according to any one of claims 1 to 3 to achieve degradation.

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