High-activity PET hydrolase mutant and application thereof

Abstract

The invention belongs to the field of biological catalysis, and discloses a high-activity PET hydrolase mutant and application thereof. The mutant of the thermophilic PET hydrolase provided by the invention is as shown in 1), 2) or 3): 1) a protein corresponding to an amino acid sequence SEQ ID NO.4; 2) a protein corresponding to the amino acid sequence of SEQ ID NO.6; and 3) a protein corresponding to the amino acid sequence of SEQ ID NO.8. Experiments prove that the modified mutant shows higher catalytic efficiency on PET hydrolysis and can keep activity for a long time at the optimal temperature for PET depolymerization. The activity of the mutant protein BhrM3 for catalyzing PET hydrolysis is improved by 1.82 times compared with that of a wild type and is 1.22 times that of efficient PET hydrolase LCC-ICCG, and the protein melting temperature reaches 92.9 DEG C.

Description

Technical Field

[0001] The present invention belongs to the field of biocatalysis, and relates to a highly active PET hydrolase mutant and its application. Background Art

[0002] Due to its heat resistance, good mechanical properties, stable chemical properties and other characteristics, plastics are widely used globally. However, the recycling rate of plastics is only 9%, and most plastics can only be finally disposed of by incineration or landfill, causing huge pollution to the environment. Polyethylene terephthalate (PET) is the most abundant synthetic polyester in circulation. Due to its stable chemical properties, a large amount of waste PET is difficult to be naturally degraded and finally flows into the ocean, soil, and even affects the food chain, causing serious damage to the ecological environment. Traditional chemical methods (methanolysis, hydrolysis, aminolysis, glycolysis) for degrading PET require hazardous chemicals and the reaction conditions are under high temperature and high pressure, which greatly limits their industrial applications. Compared with chemical degradation methods, the enzymatic depolymerization of PET has milder reaction conditions and purer products, which is more conducive to the recycling and industrial application of PET.

[0003] Cutinase (EC 3.1.1.74) belongs to the serine esterase family and has an α / β hydrolase fold structure. As early as 2005, Müller et al. found that after incubating for three weeks at 55 °C, the cutinase TfH from Thermobifida fusca could erode up to 17 μM of PET film (a 54% reduction in PET mass). (Müller, R.J., Schrader, H., Profe, J., Dresler, K. & Deckwer, W.D. Enzymatic degradation of poly(ethylene terephthalate): rapid hydrolyse using a hydrolase from T. fusca. Macromolecular rapid communications 26, 1400 - 1405 (2005).) Although the thermophilic native cutinase found in nature has high thermal stability, its catalytic efficiency is low, making enzymatic catalysis for PET recycling not economically feasible in industrial applications. Compared with cutinase, in 2016, the PET hydrolase IsPETase (T m(= 46 °C) showed higher activity. IsPETase was detected to have obvious hydrolysis activity towards amorphous PET at 30 °C (Yoshida, Shosuke, et al. "A bacterium that degrades and assimilates poly(ethyleneterephthalate)." Science 353.6278 (2016): 759.). However, its thermal stability is too low, making it difficult for the enzyme to maintain activity at the optimal depolymerization temperature of PET.

[0004] In summary, to achieve the economic feasibility of the enzyme-catalyzed PET recycling process, PET hydrolases must possess excellent thermal stability and high catalytic activity. However, most engineered PET hydrolases are difficult to meet these two key performance indicators simultaneously. To address the increasingly serious plastic pollution problem, it is particularly important to develop PET hydrolases with high activity and high thermal stability. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a PET hydrolase that can maintain activity for a long time at the optimal temperature (68 - 72 °C) for PET depolymerization, aiming at the problem of insufficient activity of current poly(ethylene terephthalate) (PET) hydrolases at temperatures close to the glass transition temperature of PET.

[0006] To solve the above technical problem, in the first aspect, the present invention provides a mutant protein of a PET hydrolase, which is any one of the following:

[0007] The mutant protein shown in A1) is obtained by mutating the 210th amino acid residue of SEQ ID NO.2 to obtain a protein with the same function;

[0008] The mutant protein shown in A2) is obtained by mutating the 189th and 210th amino acid residues of SEQ ID NO.2 to obtain a protein with the same function;

[0009] The mutant protein shown in A3) is obtained by mutating the 185th, 189th, and 210th amino acid residues of SEQ ID NO.2 to obtain a protein with the same function;

[0010] In the mutant protein shown in any of A1) - A3), except for the mutated amino acid residues, the other amino acid residues have a homology of more than 99%, more than 95%, more than 90%, more than 85%, or more than 80%, and have the same function;

[0011] A5) A protein obtained by connecting a tag to the N-terminus and / or C-terminus of the mutant protein shown in any of A1) - A4).

[0012] In the mutant proteins described above, the mutation modes of each amino acid residue are as follows:

[0013] The amino acid residue His at position 185 is mutated to Asn;

[0014] The amino acid residue Phe at position 189 is mutated to Met;

[0015] The amino acid residue Phe at position 210 is mutated to Thr.

[0016] In the mutant proteins described above,

[0017] The amino acid sequence of the mutant protein shown in A1) is SEQ ID NO.4, named Bhr_M1;

[0018] The amino acid sequence of the mutant protein shown in A2) is SEQ ID NO.6, named Bhr_M2;

[0019] The amino acid sequence of the mutant protein shown in A3) is SEQ ID NO.8, named Bhr_M3.

[0020] Compared with the wild-type thermophilic hydrolase (SEQ ID NO.2), the mutant protein Bhr_M3 has high activity and high thermal stability for PET hydrolysis, specifically manifested as being stable and highly catalytic for PET hydrolysis above 68°C, that is, the degradation rate of PET at 68°C for 12 hours is 187% of that of the wild type.

[0021] In a second aspect, the present invention provides biological materials related to the mutant proteins described in the first aspect, which are any one of the following B1) to B4):

[0022] B1) A nucleic acid molecule encoding the mutant protein described in the first aspect;

[0023] B2) An expression cassette containing the nucleic acid molecule described in B1);

[0024] B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

[0025] 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).

[0026] Among the biological materials described above, the nucleic acid molecule described in B1) is any one of the following b1)-b6):

[0027] b1) The DNA molecule shown in SEQ ID NO.3;

[0028] b2) The DNA molecule shown in SEQ ID NO.5;

[0029] b3) The DNA molecule shown in SEQ ID NO.7;

[0030] b4) A DNA molecule having 75% or more identity with the nucleotide sequence defined in any one of b1)-b3) and encoding the mutant protein described in the first aspect;

[0031] b5) A DNA molecule that hybridizes with the nucleotide sequence defined in any one of b1)-b3) under stringent conditions and encodes the mutant protein described in the first aspect.

[0032] As mentioned above, the recombinant vector (named pET-22b(+)-Bhr_M1, pET-22b(+)-Bhr_M2 or pET-22b(+)-Bhr_M3) is a recombinant expression vector obtained by inserting the coding gene described in the second aspect of the present invention between the NdeI and XhoI of the multiple cloning site of the pET-22b(+) vector.

[0033] As mentioned above, the recombinant microorganism is a recombinant Escherichia coli containing the above recombinant vector.

[0034] In the third aspect, the present invention provides the use of the mutant protein described in the first aspect in any one of the following;

[0035] C1) Degrading PET;

[0036] C2) Accelerating the degradation rate of PET;

[0037] C3) Increasing the total degradation rate of PET;

[0038] C4) Preparing a product for degrading PET;

[0039] C5) Preparing a product for accelerating the degradation rate of PET;

[0040] C6) Preparing a product for increasing the total degradation rate of PET.

[0041] In the fourth aspect, the present invention provides the use of the biomaterial described in the second aspect in any one of the following:

[0042] C1) Degrading PET;

[0043] C2) Accelerating the conversion rate of PET;

[0044] C3) Increasing the total degradation rate of PET;

[0045] C4) Preparing a product for degrading PET;

[0046] C5) Preparing a product for accelerating the conversion rate of PET;

[0047] C6) Preparing a product for increasing the total degradation rate of PET.

[0048] In a fifth aspect, the present invention provides a product comprising the mutant protein described in the first aspect or the biological material described in the second aspect.

[0049] In a sixth aspect, the present invention provides a method for degrading PET, comprising the following steps: degrading PET with the mutant protein described in the first aspect to achieve degradation.

[0050] In the above, the degradation temperature can be 68 °C or 72 °C.

[0051] In the above, the mass ratio of the mutant protein to PET can be 1:1000.

[0052] In the above, the concentration of the above PET in the degradation system can be greater than or equal to 1.6 g / L.

[0053] The present invention performs site-directed mutagenesis on Bhr-PETase from the thermophilic bacterium bacterium HR29. The modified mutant has an 87% higher activity than the wild type and has the same thermal stability as the wild type, and can maintain its activity for a long time at the optimal temperature for PET depolymerization (68 - 72 °C). The mutant enzyme can almost completely degrade PET within 24 hours with only one-thousandth of the enzyme added based on the mass of PET, showing great application value in the industrialization of PET recycling.

[0054] Compared with the prior art, the present invention has the following advantages:

[0055] (1) The mutants Bhr_M1, Bhr_M2, and Bhr_M3 of the thermophilic PET hydrolase Bhr-PETase in the present invention have extremely high thermal stability, and their melting temperatures are 95.9 °C, 98.3 °C, and 92.9 °C respectively, and can maintain their activity for a long time at the optimal temperature for PET depolymerization;

[0056] (2) The catalytic activities of the mutants Bhr_M1, Bhr_M2, and Bhr_M3 in the present invention are significantly improved. Under the conditions of 68 °C, a PET substrate concentration of 1.6 g / L, and an enzyme amount of one-thousandth of the mass of PET (enzyme concentration of 0.057 μM), the PET degradation rates of the mutants Bhr_M1, Bhr_M2, and Bhr_M3 in 12 hours are 50.3%, 45.9%, and 67.9% respectively, which are 1.35, 1.23, and 1.82 times that of the wild-type PET hydrolase Bhr-PETase.

[0057] (3) Under the optimal temperature for PET depolymerization, the mutant Bhr_M3 in the present invention has a significantly better catalytic efficiency than the highly efficient PET hydrolase LCC-ICCG reported in the literature, and has good industrial application prospects in the field of enzymatic catalysis for PET recycling. Under the conditions of 68 °C or 72 °C, a PET substrate concentration of 1.6 g / L, and an enzyme amount of one-thousandth of the PET mass (enzyme concentration of 0.057 μM), the degradation rates of the mutant Bhr_M3 in 12 hours are 67.9% and 68.8% respectively, which are 1.22 times and 1.09 times that of the PET hydrolase LCC-ICCG under the same conditions. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 It is a reaction route diagram for the enzymatic catalysis of PET to degrade into ethylene terephthalate (BHET), mono-hydroxyethyl terephthalate (MHET), terephthalic acid (TPA), and ethylene glycol (EG).

[0059] Figure 2 It is a map of the recombinant plasmid pET-22b(+)-Bhr_M3.

[0060] Figure 3 It is a melting curve diagram of the mutants Bhr_M1, Bhr_M2, and Bhr_M3.

[0061] Figure 4 It is an HPLC peak time diagram of the TPA, MHET, and BHET standards.

[0062] Figure 5 It is a product time curve diagram of the mutant Bhr_M3 and the PET hydrolase catalyzing PET depolymerization at a low substrate concentration.

[0063] Figure 6 It is a product time curve diagram of the mutant Bhr_M3 and the PET hydrolase catalyzing PET depolymerization at a high substrate concentration. DETAILED DESCRIPTION OF THE INVENTION

[0064] The present invention will be further described in detail below in conjunction with the specific embodiments. The provided embodiments are only for clarifying the present invention, rather than limiting the scope of the present invention. The following provided embodiments can be used as a guide for those of ordinary skill in the art to make further improvements, and do not limit the present invention in any way.

[0065] The experimental methods in the following embodiments are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0066] Unless otherwise specified, in the following examples, quantitative tests were all set up with three replicate experiments, and the results were averaged.

[0067] Example 1: Preparation and Purification of the Hydrolyase Bhr-PETase Mutant from Thermophilic Bacterium Bacterium HR29

[0068] I. Obtaining the Encoding Gene of the Hydrolyase Bhr-PETase Mutant

[0069] The nucleotide sequence of the wild-type hydrolyase Bhr-PETase in thermophilic bacterium Bacterium HR29 obtained from the NCBI database (Genebank: GBD22443.1) is SEQ ID NO.1; the amino acid sequence of the encoded Bhr-PETase is SEQ ID NO.2.

[0070] First, the gene of the wild thermophilic PET hydrolase Bhr-PETase was obtained by total gene synthesis. Then, site-directed mutagenesis was performed on the encoding gene of Bhr-PETase to obtain the gene of the Bhr-PETase mutant.

[0071] The above Bhr-PETase mutants are Bhr_M1 mutant protein, Bhr_M2 mutant protein, and Bhr_M3 mutant protein.

[0072] The Bhr_M1 mutant protein is a mutant obtained by mutating the 210th Phe of the wild thermophilic PET hydrolase Bhr-PETase shown in SEQ ID NO.2 to Thr, with other amino acid residues unchanged; the amino acid sequence of the Bhr_M1 mutant protein is SEQ ID NO.4;

[0073] The Bhr_M2 mutant protein is a mutant obtained by mutating the 189th Phe of the wild thermophilic PET hydrolase Bhr-PETase shown in SEQ ID NO.2 to Met and the 210th Phe to Thr, with other amino acid residues unchanged; the amino acid sequence of the Bhr_M2 mutant protein is SEQ ID NO.6;

[0074] The Bhr_M3 mutant protein is a mutant obtained by mutating the 185th His of the wild thermophilic PET hydrolase Bhr-PETase shown in SEQ ID NO.2 to Asn, the 189th Phe to Met, and the 210th Phe to Thr, with other amino acid residues unchanged; the amino acid sequence of the Bhr_M3 mutant protein is SEQ ID NO.8;

[0075] II. Construction of the Recombinant Expression Vector

[0076] First, the Bhr-PETase gene (with a nucleotide sequence shown in SEQ ID NO.1, containing 786 bases) was obtained by total gene synthesis, and the gene was constructed into the pET-22b(+) vector (containing the ampicillin resistance gene) using NdeI and XhoI restriction enzymes. Then, this recombinant plasmid was transformed into competent cells BL21(DE3) to obtain the recombinant expression plasmid pET-22b(+)-BhrPETase. pET-22b(+) was purchased from BGI, and the product catalog number is 69744-3. The pET-22b(+) plasmid carries a carboxyl-terminal histidine tag His6 coding sequence at the XhoI digestion end, facilitating the purification of the target protein.

[0077] After sequencing, the sequence of pET-22b(+)-BhrPETase was correct. This plasmid is a recombinant plasmid pET-22b(+)-BhrPETase formed by replacing the fragment between the NdeI and XhoI sites of pET-22b(+) with the Bhr-PETase gene shown at positions 1-786 of SEQ ID NO.1 in the sequence listing, and the Bhr-PETase gene is fused and expressed with the His6 tag on the vector.

[0078] According to the above method, the coding genes of mutants Bhr_M1 (SEQ ID NO.3), Bhr_M2 (SEQ ID NO.5), and Bhr_M3 (SEQ ID NO.7) were respectively inserted between the NdeI and XhoI digestion sites of pET-22b(+) to obtain recombinant plasmids pET-22b(+)-Bhr_M1, pET-22b(+)-Bhr_M2, and pET-22b(+)-Bhr_M3. The map of the recombinant plasmid pET-22b(+)-Bhr_M3 is as Figure 2 shown.

[0079] III. Construction method of engineering bacteria

[0080] The pET-22b(+)-BhrPETase, pET-22b(+)-Bhr_M1, pET-22b(+)-Bhr_M2, and pET-22b(+)-Bhr_M3 obtained in Step 2 were separately transformed into the Escherichia coli competent cell Escherichia coli BL21(DE3) (the competent cell was purchased from Beijing Solarbio Science & Technology Co., Ltd., product number C1400), to obtain the engineered bacteria E. coli BL21(DE3) / pET-22b(+)-BhrPETase, E. coli BL21(DE3) / pET-22b(+)-Bhr_M1, E. coli BL21(DE3) / pET-22b(+)-Bhr_M2, and E. coli BL21(DE3) / pET-22b(+)-Bhr_M3. The plasmids were extracted and verified to be correct by sequencing.

[0081] IV. Obtaining of Thermophilic Hydrolase Bhr-PETase and Its Mutants

[0082] 1. Induced Expression of Engineered Bacteria

[0083] Each of the engineered bacterial strains constructed in Step 3 was streaked and cultured on an LB agar plate containing 100 μg / mL ampicillin, and then cultured at 37 °C for 14 hours. Subsequently, well-grown single colonies were selected and inoculated into 5 mL of LB liquid medium (containing 100 μg / mL ampicillin), and continued to be cultured at 37 °C and 200 rpm for 7 hours until the OD 600 reached 1.2 - 2.0;

[0084] Each engineered bacterium was transferred at an inoculation amount of 1% volume to two bottles of 200 mL of LB liquid medium (containing 100 μg / mL ampicillin), and continued to be cultured at 37 °C and 200 rpm until the OD 600 was 0.6 - 0.8. Subsequently, 0.2 g / mL of L-arabinose was added to a final concentration of 0.2% (w / v, g:ml), and cultured at 20 °C and 180 rpm for 14 hours and 30 minutes to induce the expression of the Bhr-PETase or its mutant gene.

[0085] After the cultivation, the bacterial solution was centrifuged at 4 °C and 6000 rpm for 20 minutes. The obtained cell pellet was resuspended by shaking with 50 mL of protein lysis buffer (100 mM potassium dihydrogen phosphate, 300 mM sodium chloride, the balance being water, pH 8.0). Then, an ultrasonic cell disruptor was used to disrupt the resuspended solution in an ice-water bath, working for 4 seconds and resting for 6 seconds, using 60% of the maximum power, and the cell disruption was carried out for a total of 50 minutes. The obtained liquid was centrifuged at 4 °C and 7000 rpm for 15 minutes, and then the supernatant was centrifuged at 4 °C and 12000 rpm for 10 minutes. The finally obtained supernatant was the crude enzyme solution containing Bhr-PETase or its mutant.

[0086] The formula of the above LB liquid medium is: 5 g / L of yeast extract, 10 g / L of tryptone, 5 g / L of sodium chloride, and the balance being water.

[0087] The formula of the above LB agar plate is: 5 g / L of yeast extract, 10 g / L of tryptone, 5 g / L of sodium chloride, 15 g / L of agarose, and the balance being water.

[0088] 2. Purification of Thermophilic PET Hydrolase Bhr-PETase and Its Mutant Enzymes

[0089] The crude enzyme solution containing PET hydrolase Bhr-PETase or its mutant was loaded onto a Ni-NTA column, with nickel ions as the affinity ions, and eluted with imidazole solutions of different concentrations to obtain the pure target protein. Proteins have specific absorption peaks under ultraviolet light at 280 nm. The absorbance of the eluates with different imidazole concentrations was measured under ultraviolet light at 280 nm to remove non-target proteins in the solution. Specifically as follows:

[0090] The crude enzyme solution was purified using a 20 mL nickel agarose gel column (Beijing Weishi Bohui Chromatography Technology Co., Ltd., FF) and a nucleic acid and protein detector (Shanghai Huxi Analytical Instrument Factory, HD-21-1). First, the column bed was pre-equilibrated with binding buffer A (100 mM potassium dihydrogen phosphate, pH 8.0, containing 300 mM sodium chloride, with the balance being water). Subsequently, the contaminating proteins were eluted using binding buffer B containing imidazole (100 mM potassium dihydrogen phosphate, pH 8.0, containing 300 mM sodium chloride, 50 mM imidazole, with the balance being water) until the absorbance of the eluate at 280 nm no longer changed. Then, the eluted protein was collected using elution buffer (100 mM potassium dihydrogen phosphate, pH 8.0, containing 300 mM sodium chloride, 200 mM imidazole, with the balance being water). To further improve the purity of the target protein, the collected liquid was heated in a water bath at 60 °C for 2 hours, and then centrifuged at 4 °C and 12,000 rpm for 30 minutes to remove the contaminating proteins. The supernatant after centrifugation was poured into an ultrafiltration centrifugal tube with a molecular weight cut-off of 10 kD, and the target protein was centrifuged at 4 °C and 6,000 rpm for 20 minutes, and centrifuged 3 times in total to remove the imidazole in the liquid. The purified target protein was stored in phosphate buffer (100 mM potassium dihydrogen phosphate, 300 mM sodium chloride, with the balance being water, pH 8.0) to obtain the target protein solutions (PET hydrolase Bhr-PETase solution, mutant Bhr_M1 solution, mutant Bhr_M2 solution, and mutant Bhr_M3 solution) respectively, and stored in a refrigerator at 4 °C.

[0091] The presence and purity of the target protein were examined by SDS-PAGE (5% stacking gel, 10% separating gel). According to the SDS-PAGE results, the purity of the collected protein was greater than 90%.

[0092] The concentration of the target protein in the purified target protein solution was determined by the Coomassie brilliant blue method using an ultraviolet spectrophotometer (Shanghai Muxi Instrument Co., Ltd., UV-1200) with bovine serum albumin as the standard reagent at an absorbance of 595 nm. As a result, in each of the target protein solutions, the concentrations of the corresponding PET hydrolase Bhr-PETase protein, mutant Bhr_M1 protein, mutant Bhr_M2 protein, and mutant Bhr_M3 protein were in the range of 2 - 10 mg / mL.

[0093] Example 2. Determination of the ability of PET hydrolase Bhr-PETase mutants to catalyze the depolymerization of post-consumer bottle-grade PET

[0094] I. Thermal stability test of PET hydrolase Bhr-PETase mutants

[0095] The optimal reaction temperature for enzyme-catalyzed PET depolymerization is at the glass transition temperature (T g) Near (65 - 71 °C), which requires that the PET hydrolase has sufficient thermal stability to maintain its activity at this temperature. By using micro differential scanning fluorimetry (NanoDSF) and a protein stability analyzer (Prometheus NT.48, NanoTemper Technologies, Germany), the melting temperature (T m ) of PET hydrolase Bhr-PETase and its mutants Bhr_M1, Bhr_M2, and Bhr_M3 in a protein buffer (100 mM potassium dihydrogen phosphate, 300 mM sodium chloride, the balance being water, pH 8.0) was measured. During the measurement, the sample was heated from 25 °C to 110 °C at a rate of 2 °C per minute. Each enzyme was analyzed in triplicate, and its T m was obtained from the DSF curve of the protein, and the measured mean value and standard deviation were calculated as shown in Figure 3 .

[0096] The T Figure 3 of mutant Bhr_M1 ( Figure 3 (a)), mutant Bhr_M2 ( Figure 3 (b)), and mutant Bhr_M3 ( m (c)) were 95.9 °C, 98.3 °C, and 92.9 °C, respectively, all exceeding the T g of PET by more than 15 °C, ensuring that the activity of the enzyme would not be lost at the optimal reaction temperature for PET depolymerization.

[0097] II. Activity test of the mutants of PET hydrolase Bhr-PETase in catalyzing the depolymerization of PET at low substrate concentrations

[0098] Post-consumer PET for bottles is from the body of post-consumer beverage bottles produced by The Coca-Cola Company (removing the bottle caps, bottle necks, bottle bottoms, and packaging stickers). First, the Coke bottles were cut into 1 cm × 1 cm small pieces, then washed successively with water, surfactant, and ethanol, and then dried. The post-consumer PET bottle chips were pulverized using a high-speed rotary grinder (AM500S, Antsource Scientific Instruments Co., Ltd., Beijing) equipped with a 0.5 mm trapezoidal hole sieve ring. The rotational speed of the grinder was 8000 rpm. The pulverized powder was filtered through sieves with pore sizes of 200 μm and 355 μm respectively to obtain post-consumer PET bottle powder with a particle size of 200 - 355 μm.

[0099] The post-consumer PET bottle powder described below refers to the pre-treated post-consumer PET bottle powder with a particle size of 200 - 355 μm.

[0100] Buffer 1 is a solution with a pH of 8.0 composed of 100 mM potassium dihydrogen phosphate, 300 mM sodium chloride, and water.

[0101] 1. Activity test for comparing the catalytic depolymerization of wild-type PET hydrolase Bhr-PETase and Bhr-PETase mutants

[0102] Experimental group of wild-type PET hydrolase Bhr-PETase (3 mL): Weigh approximately 5 mg of post-consumer PET bottle powder into a 5 mL glass reaction flask, and then add 3 mL of Buffer 1 with pH 8.0 containing 0.057 μM PET hydrolase Bhr-PETase (obtained by dissolving the PET hydrolase Bhr-PETase solution (protein content: 5 μg) in Buffer 1, where the concentration of Bhr-PETase is 0.057 μM).

[0103] Experimental group of Bhr-PETase mutants (3 mL): Weigh approximately 5 mg of post-consumer PET bottle powder into a 5 mL glass reaction flask, and then add 3 mL of Buffer 1 with pH 8.0 containing 0.057 μM Bhr-PETase mutant M1 or Bhr-PETase mutant M2 or Bhr-PETase mutant M3 (obtained by dissolving each mutant protein solution (protein content: 5 μg) in Buffer 1, where the concentration of each mutant protein is 0.057 μM).

[0104] Put the above substances into a thermostatic shaking mixer (DH300, Hangzhou Ruicheng Instrument Co., Ltd.), react at 68 °C and a rotation speed of 400 rpm for 12 hours. At the end of the reaction, add an equal volume of 100% methanol to terminate the reaction, obtain the sample, dilute the sample 10 times by adding Buffer 1, centrifuge (12000 rpm, 2 minutes) and collect the supernatant. Then, use a syringe to take the supernatant through a 0.22 μm filter membrane to remove the inactivated enzyme in the solution, and obtain each hydrolysis product sample.

[0105] The process of PET depolymerization is as Figure 1 shown. Only terephthalic acid (TPA), mono(2-hydroxyethyl) terephthalate (MHET), and bis(2-hydroxyethyl) terephthalate (BHET) in its products can be detected by liquid chromatography.

[0106] Use high-performance liquid chromatography (HPLC) to measure the peak areas of standard products TPA (Shanghai Macklin Biochemical Co., Ltd.), MHET (Bide Pharmaceutical Technology Co., Ltd.), and BHET (Bide Pharmaceutical Technology Co., Ltd.) with different concentrations respectively. Take the concentration (X) as the abscissa and the peak area (Y) as the ordinate to draw the standard curve. The standard curve functions corresponding to the three products are as follows:

[0107] TPA peak area = concentration(TPA) × 7.35188 × 10 6

[0108] Peak area of MHET = Concentration (MHET) × 8.81652 × 10 6

[0109] Peak area of BHET = Concentration (BHET) × 1.00305 × 10 7

[0110] The sample to be tested is analyzed by high performance liquid chromatography (HPLC). According to the corresponding peak emergence time and peak area, substituting them into the corresponding standard curve can obtain the contents of TPA, MHET and BHET in the sample.

[0111] For the above HPLC (Shimadzu Corporation, LC - 20AD) analysis, the chromatographic column selected is SunFire TM C18 reversed - phase column (GL Sciences, 5μm, 250×4.6mm). The sample loading volume for each sample is 10 μL. The mobile phase A of HPLC is distilled water containing 0.1% formic acid (v / v), and the mobile phase B is chromatographically pure acetonitrile. From 0 minutes to 5 minutes, the acetonitrile concentration remains constant at 5%; from 5 minutes to 13 minutes, the acetonitrile concentration increases from 5% to 44%; from 13 minutes to 18 minutes, the acetonitrile concentration increases from 44% to 70%; from 18 minutes to 23 minutes, the acetonitrile concentration remains constant at 70%; from 23 minutes to 24 minutes, the acetonitrile concentration drops from 70% to 5%; from 24 minutes to 28 minutes, the acetonitrile concentration remains at 5%. The flow rate is 0.8 mL / minute, the detection wavelength is 254 nm, and the column oven is kept constant at 40°C.

[0112] The HPLC chromatogram results of the standards TPA, MHET and BHET are as Figure 4 shown. A chromatographic peak of the degradation product TPA appears near the retention time t R = 14.88 minutes, a chromatographic peak of the degradation product MHET appears near the retention time t R = 15.46 minutes, and a chromatographic peak of the degradation product BHET appears near the retention time t R = 15.76 minutes.

[0113] The concentrations of TPA, MHET and BHET in the hydrolysis products of wild - type Bhr - PETase, mutant Bhr_M1, mutant Bhr_M2 and mutant Bhr_M3 determined by HPLC, as well as the calculated degradation rates of the substrates, are presented in Table 1 respectively.

[0114] The calculation formula for the degradation rate in Table 1 is as follows. The total product concentration is the sum of the concentrations of TPA, MHET, and BHET detected by HPLC. The reaction volume is 3 mL in this example, and the dilution factor is 10 in this example; the PET mass is 5 mg, and the molecular weight of the PET monomer is 182.14 Da.

[0115] Degradation rate = total product concentration × dilution factor × reaction volume / (PET mass / PET monomer molecular weight) × 100%

[0116] It can be seen that compared with the wild-type Bhr-PETase, the degradation rates of the mutants Bhr_M1, Bhr_M2, and Bhr_M3 at 12 hours are 1.35, 1.23, and 1.82 times that of the wild-type, respectively, proving that the mutation has significantly improved the activity of the enzyme-catalyzed PET depolymerization.

[0117] Table 1 shows the PET hydrolysis products catalyzed by the PET hydrolase Bhr-PETase and the mutants Bhr_M1, Bhr_M2, and Bhr_M3

[0118] enzyme TPA (mM) MHET (mM) BHET (mM) degradation rate wild-type Bhr-PETase 1.703 1.513 0.023 37.29％ mutant Bhr_M1 2.255 2.072 0.039 50.34％ mutant Bhr_M2 1.899 2.036 0.046 45.91％ mutant Bhr_M3 3.025 2.812 0.054 67.85％

[0119] III. Activity evaluation of the mutant Bhr_M3 and the engineered PET hydrolase LCC-ICCG in catalyzing PET depolymerization at low substrate concentrations

[0120] To compare the ability of the mutant Bhr_M3 and the reported engineered PET hydrolase LCC-ICCG in catalyzing PET depolymerization, PET depolymerization time-course experiments were carried out on the two enzymes at 68 °C for 24 hours.

[0121] The PET substrate for the reaction was from the body of a post-consumer beverage bottle produced by Coca-Cola. First, the body of the Coke bottle was placed in an aluminum foil paper box and then put into a muffle furnace (Hefei Kejing Materials Technology Co., Ltd., KSL-1200X), heated at 290 °C for 15 minutes until completely melted, and then the aluminum foil paper box was placed in an ice-water bath to cool and solidify. The solidified PET was washed with water and then dried and put into a high-speed rotary grinder (Antiyuan Scientific Instruments Beijing Co., Ltd., AM500S) equipped with a 0.5-mm trapezoidal hole sieve ring to crush the post-consumer PET bottle chips. The rotation speed of the grinder was 8000 rpm. The crushed powder was filtered through sieves with pore sizes of 200 μm and 355 μm respectively to obtain post-consumer PET bottle powder with a particle size of 200 - 355 μm.

[0122] Mutant Bhr_M3 experimental group (3 mL): Weigh approximately 5 mg of the treated PET bottle powder into a 5 mL glass reaction flask, and then add 3 mL of Buffer 1 with pH 8.0 containing 0.029 μM mutant Bhr_M3 (obtained by dissolving the mutant Bhr_M3 protein solution (protein content: 2.5 μg) in Buffer 1, where the concentration of mutant Bhr_M3 protein is 0.029 μM).

[0123] Engineered PET hydrolase LCC-ICCG experimental group (3 mL): Weigh approximately 5 mg of the treated PET bottle powder into a 5 mL glass reaction flask, and then add 3 mL of Buffer 1 with pH 8.0 containing 0.029 μM PET hydrolase LCC-ICCG (obtained by dissolving the PET hydrolase LCC-ICCG solution (protein content: 2.5 μg; Tournier, V., et al. "An engineered PET depolymerase to break down and recycle plastic bottles." Nature 580.7802 (2020): 216 - 219.) in Buffer 1, where the concentration of hydrolase LCC-ICCG is 0.029 μM).

[0124] Each group of the above substances was placed in a thermostatic shaker mixer (DH300, Hangzhou Ruicheng Instrument Co., Ltd.) and reacted at 68 °C and a rotation speed of 400 rpm for 4, 8, and 12 hours respectively. At the end of the reaction, an equal volume of 100% methanol was added to terminate the reaction. The samples were diluted 10 times by adding Buffer 1, centrifuged (12000 rpm, 2 minutes), and the supernatant was collected. Then, the supernatant was taken with a syringe and passed through a 0.22 μm filter membrane to obtain the hydrolysis product sample for HPLC analysis.

[0125] According to the HPLC detection method described above, quantitative analysis was performed on the products of PET depolymerization, and the results are presented in Figure 5 . At 68 °C, the maximum reaction rate (reaction for 4 hours) of mutant Bhr_M3 catalyzing PET depolymerization is 1.24 times that of PET hydrolase LCC-ICCG. Its degradation rate at 8 hours reached 71.7%, which is 7.8% higher than that of PET hydrolase LCC-ICCG. The final degradation rate at 12 hours reached 90.7%; while the 12-hour degradation rate of PET hydrolase LCC-ICCG was only 77.2%. These results indicate that mutant Bhr_M3 has a stronger ability to catalyze PET depolymerization than PET hydrolase LCC-ICCG and can maintain its activity at the optimal reaction temperature for PET depolymerization for a long time.

[0126] IV. Activity Evaluation of Mutant Bhr_M3 and Engineered PET Hydrolase LCC-ICCG in Catalyzing PET Depolymerization at High Substrate Concentrations

[0127] To compare the ability of mutant Bhr_M3 and the reported engineered PET hydrolase LCC-ICCG to catalyze PET depolymerization at industrial-grade substrate concentrations, PET depolymerization experiments were conducted on the two enzymes at 70 °C for 24 hours respectively.

[0128] Experimental group of mutant Bhr_M3 (50 mL): Weigh approximately 8.25 g of treated PET bottle powder into a 50 mL three-necked flask, and then add 44 mL of Buffer 1 with pH 8.0 containing 2.22 μM mutant Bhr_M3 (obtained by dissolving the mutant Bhr_M3 protein solution (protein content 4.125 mg) in Buffer 1, where the concentration of mutant Bhr_M3 protein is 2.22 μM).

[0129] Experimental group of engineered PET hydrolase LCC-ICCG (50 mL): Weigh approximately 8.25 g of treated PET bottle powder into a 50 mL three-necked flask, and then add 44 mL of Buffer 1 with pH 8.0 containing 2.23 μM PET hydrolase LCC-ICCG (obtained by dissolving the PET hydrolase LCC-ICCG solution (protein content 4.125 mg) in Buffer 1, where the concentration of hydrolase LCC-ICCG is 2.23 μM).

[0130] Place the corresponding three-necked flasks of the above substances in a heating water bath constant temperature magnetic stirrer (Shanghai Yuhua Instrument Co., Ltd., ZNCL-G), stir the reaction solution to keep the reaction temperature at 70 °C. Use a pH meter FE28 (Mettler Toledo, FE28) to detect the pH value of the reaction solution in real time and control the pH value of the reaction system to be maintained between 7.8 - 8.2 by adding 5 M sodium hydroxide solution. Take 1 mL of the sample at 3, 6, 9, 12, 18, and 24 hours respectively, dilute the sample 800 times by adding Buffer 1, centrifuge (12000 rpm, 2 minutes) and collect the supernatant, then use a syringe to take the supernatant through a 0.22 μm filter membrane to remove the inactivated enzyme in the solution. Obtain the hydrolyzate sample. Calculate the degradation rate of PET based on the added amount of sodium hydroxide solution and the HPLC quantitative analysis results of the sample. The results are presented in Figure 6 in.

[0131] The mutant Bhr_M3 had already converted 59% of PET after 3 hours of reaction, which was 1.17 times that of the PET hydrolase LCC-ICCG at the same time. After 18 hours, the degradation rate of the mutant Bhr_M3 reached 90%, and its final degradation rate after 24 hours was 93%. While the final degradation rate of the PET hydrolase LCC-ICCG was only 85%.

[0132] The present invention has been described in detail above. For those skilled in the art, without departing from the gist and scope of the present invention and without unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to cover any modifications, uses or improvements of the present invention, including those that depart from the scope disclosed in this application and are made by conventional techniques known in the art. The application of some basic features can be made according to the scope of the appended claims below.

Claims

1. A mutant protein of a PET hydrolase, which is any one of the following: A1) The mutant protein shown is obtained by mutating the 210th amino acid residue of SEQ ID NO.2 to obtain a protein with the same function; A2) The mutant protein shown is obtained by mutating the 189th and 210th amino acid residues of SEQ ID NO.2 to obtain a protein with the same function; A3) The mutant protein shown is obtained by mutating the 185th, 189th and 210th amino acid residues of SEQ ID NO.2 to obtain a protein with the same function; A4) In any of the mutant proteins shown in A1)-A3), except for the mutated amino acid residues, the other amino acid residues have a homology greater than 99%, 95%, 90%, 85% or 80% homology, and have the same function; A5) A protein obtained by connecting a tag to the N-terminus and / or C-terminus of any of the mutant proteins shown in A1)-A4).

2. The mutant protein according to claim 1, wherein The mutation mode of each amino acid residue is as follows: The 185th amino acid residue His is mutated to Asn; The 189th amino acid residue Phe is mutated to Met; The 210th amino acid residue Phe is mutated to Thr.

3. The mutant protein according to claim 1 or 2, wherein The amino acid sequence of the mutant protein shown in A1) is SEQ ID NO.4; The amino acid sequence of the mutant protein shown in A2) is SEQ ID NO.6; The amino acid sequence of the mutant protein shown in A3) is SEQ ID NO.

8.

4. A biological material related to the mutant protein according to any one of claims 1-3, 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 1-3; 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: The nucleic acid molecule described in B1) is any one of the following b1)-b6): b1) The DNA molecule shown in SEQ ID NO.3; b2) The DNA molecule shown in SEQ ID NO.5; b3) The DNA molecule shown in SEQ ID NO.7; b4) A DNA molecule having 75% or more identity with the nucleotide sequence defined in any of b1)-b3) and encoding the mutant protein according to any one of claims 1-3; b5) A DNA molecule that hybridizes with the nucleotide sequence defined in any of b1)-b3) under stringent conditions and encodes the mutant protein according to any one of claims 1-3.

6. The application of the mutant protein according to any one of claims 1-3 in any of the following; C1) Degrading PET; C2) Accelerating the degradation rate of PET; C3) To increase the total degradation rate of PET; C4) To prepare a product for degrading PET; C5) To prepare a product for accelerating the degradation rate of PET; C6) To prepare a product for increasing the total degradation rate of PET.

7. Use of the biomaterial according to claim 4 or 5 in any of the following: C1) Degrading PET; C2) Accelerating the degradation rate of PET; C3) Increasing the total degradation rate of PET; C4) Preparing a product for degrading PET; C5) Preparing a product for accelerating the conversion rate of PET; C6) Preparing a product for increasing the total degradation rate of PET.

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

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

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