Fusion enzyme for efficiently degrading PET (polyethylene terephthalate) plastic and application of fusion enzyme

Abstract

The invention discloses a fusion enzyme capable of efficiently degrading PET (polyethylene terephthalate) plastic and application of the fusion enzyme. The invention provides a recombinant fusion enzyme. The recombinant fusion enzyme comprises a mutant KL-MHETase of thermophilic carboxylesterase Est30 and a PET (Polyethylene Terephthalate) degrading enzyme FAST-PETase, the amino acid sequence of the mutant KL-MHETase of the thermophilic carboxylesterase Est30 is as shown in SEQ ID NO. 3; the amino acid sequence of the FAST-PETase is as shown in the 284th to 546th amino acids of SEQ ID NO.1 (Sequence Identifier Number 1). The fusion enzyme is used as a biocatalyst, the activity of catalyzing hydrolysis of MHET at 50 DEG C is 52.6 times that of FAST-PETase, and the fusion enzyme has high enzyme activity and thermal stability. PET degradation is catalyzed at the temperature of 50 DEG C, the conversion rate of PET is 90% after 48 hours of reaction, and meanwhile, the TPA content in the product is close to 100%.

Description

Technical Field

[0001] The invention belongs to the field of biocatalysis and relates to a fusion enzyme for efficiently degrading PET plastic and application thereof. Background Art

[0002] Polyethylene terephthalate (PET) plastics account for 18% of the world's total polymer production, with nearly 82 million tons produced annually worldwide, and are an important source of white pollution. PET is stable and difficult to decompose. It is widely used in films, packaging bottles, clothing, electronic products and mechanical equipment. The amount of waste it produces is huge. A large amount of difficult-to-decompose plastic waste is dumped in the environment, which brings a serious burden to the global ecosystem. The current methods for treating PET waste are: landfill, incineration and recycling. Although landfill and incineration are the simplest, the waste gas and wastewater produced will cause secondary pollution to the environment. Traditional chemical recovery methods such as glycolysis, hydrolysis and alcoholysis require high temperature and high pressure, toxic catalysts and other conditions, and the monomer yield is low. The use of biological methods (enzymatic degradation or microbial degradation) can degrade PET into small molecules for recycling and reuse. Compared with chemical catalytic recovery with harsh reaction conditions, biodegradation can be carried out under milder conditions, and is green and environmentally friendly, in line with the concept of sustainable development. In 2016, Yoshida et al. isolated Ideonella sakaiensis 201-F6 (Yoshida, S. et al. A bacterium that degrades and assimilates poly(ethylene terephthalate). Science 351, 1196–1199 (2016).) that can effectively utilize PET as a carbon source. This bacterium can secrete two synergistic enzymes, including a PET hydrolase called PETase and a MHET hydrolase called MHETase. PETase degrades PET into mono(2-hydroxyethyl) terephthalate (MHET) and bis(hydroxyethyl) terephthalate (BHET), and MHETase hydrolyzes MHET into recyclable monomers TPA and EG, which is a key step in the recycling of PET, such as Figure 1As shown. In 2020, Tournier et al. modified leaf and branch compost cutinase (LCC) and obtained a four-point mutant LCC-ICCG, which degraded 90% of PET in just 10 hours at 70 degrees. This is the most efficient PET degrading enzyme reported so far (Tournier, V. et al. An engineered PET depolymerase to break down and recycle plastic bottles. Nature 580, 216–219 (2020).). Other PET degrading enzymes, such as ThermoPETase and DuraPETase, which have greatly improved thermal stability obtained by modifying PETase, and the cutinase TfCut2 from Thermobifida fusa KW3 and its mutants, can degrade PET at high temperatures above 50 degrees, but their activity is generally low. In 2022, Lu et al., under the guidance of machine learning, introduced five-point mutations into PETase. The resulting mutant FAST-PETase was able to degrade various post-consumer PET wastes at a medium temperature of 50°C, exceeding the activity of other PET degrading enzymes, and is expected to achieve in situ degradation of PET wastes at ambient temperature (Lu, H. et al. Machine learning-aided engineering of hydrolases for PET depolymerization. Nature 604, 662–667 (2022).). However, the problem with these single enzyme systems for degrading PET is that the MHET content in the product is high, resulting in the need to increase expensive separation costs in order to obtain pure monomer TPA that can be recycled. At the same time, MHET, as the main intermediate product of PET degradation, has a product inhibitory effect on PET degrading enzymes such as LCC-ICCG and FAST-PETase, resulting in reduced degradation efficiency.

[0003] At present, relevant research has eliminated the product inhibition of MHET on PET degradation enzyme by introducing appropriate MHET hydrolase to construct a dual enzyme system, thereby improving the efficiency of PET degradation enzyme and the purity of TPA in the product. In 2020, Knott et al. covalently linked MHETase and PETase through Gly-Ser peptide. The constructed fusion protein significantly improved the efficiency of PET film degradation compared with single enzyme and unlinked dual enzyme (Knott, BC et al. Characterization and engineering of a two-enzyme system for plastics depolymerization. Proc Natl Acad Sci USA 117, 25476–25485 (2020).). In 2022, Mrigwani et al. combined a heat-resistant carboxylesterase from Thermus thermophilus that can degrade MHET with leaf and branch compost cutinase LCC that degrades PET. Compared with LCC single enzyme, the degradation rate of PET film by the dual enzyme system was increased to 1.3-2 times, and the purity of TPA in the product was greater than 99% (Mrigwani, A., Thakur, B. & Guptasarma, P. Conversion of polyethylene terephthalate into pure terephthalic acid through synergy between a solid-degrading cutinase and a reaction intermediate-hydrolysing carboxylesterase. Green Chem. 10.1039. D2GC01965E (2022) doi: 10.1039 / D2GC01965E.).Haugwitz et al. (2022) modified the carboxylesterase TfCa from Thermobifida fusca, and the resulting mutant TfCa I69W / V376A increased the MHET hydrolysis activity by 2.6 times, and produced 14 times more TPA than the single enzyme system after complexing with PET degrading enzyme (von Haugwitz, G. et al. Structural Insights into (Tere)phthalate-Ester Hydrolysis by a Carboxylesterase and Its Role in Promoting PET Depolymerization. ACS Catal. 15259–15270 (2022) doi: 10.1021 / acscatal.2c03772.). Chen et al. co-immobilized natural MHETase and PETase mutant DuraPETase in calcium phosphate nanocrystals, and the resulting fusion enzyme system had a degradation efficiency of PET at 40 degrees and 50 degrees that was 6.1 times and 1.5 times higher than that of the free enzyme, but its PET conversion rate within 10 days was only 50.3% (Chen, K., Dong, X. & Sun, Y. Sequentially co-immobilized PET and MHEThydrolases via Spy chemistry in calcium phosphate nanocrystals present high-performance PET degradation. Journal of Hazardous Materials 438, 129517 (2022).). In general, the current reports on dual enzyme systems or fusion enzymes for PET degradation have the problems of low thermal stability and low PET degradation efficiency, which are far from the level of industrial application.

[0004] The key problem is that the currently reported MHET hydrolases cannot have both high activity and high thermal stability, and cannot be used in combination with existing high-efficiency PET degradation enzymes at higher temperatures. With the increasing severity of white pollution, it is particularly important and urgent to develop highly active and highly thermally stable MHET hydrolases and couple them with compatible PET degradation enzymes to develop a dual enzyme system to degrade and recycle PET. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a recombinant fusion enzyme of a PET degrading enzyme and a heat-stable MHET hydrolase in order to address the problem that the current PET degrading enzyme is inhibited by the product of MHET and the TPA content in the product is low, and to utilize the fusion enzyme to catalyze the degradation of PET into monomeric TPA, thereby improving the efficiency of PET enzymatic degradation and the purity and yield of monomeric TPA that can be recovered and reused in the product.

[0006] In a first aspect, the present invention provides a recombinant fusion enzyme comprising an MHET hydrolase KL-MHETase and a PET degrading enzyme FAST-PETase;

[0007] The MHET hydrolase KL-MHETase is a mutant of the thermophilic carboxylesterase Est30 derived from Geobacillus stearothermophilus, named KL-MHETase. The thermophilic carboxylesterase mutant is a mutant of the wild-type thermophilic carboxylesterase Est30 shown in SEQ ID NO.5, wherein the glycine at position 130 is mutated to leucine, and the isoleucine at position 171 is mutated to lysine (G130L / I171K); preferably, the amino acid sequence of the MHET hydrolase KL-MHETase is shown in SEQ ID NO.1, positions 1-247 or SEQ ID NO.3; the nucleotide sequence of the KL-MHETase encoding gene is shown in SEQ ID NO.4;

[0008] The amino acid sequence of the PET degrading enzyme FAST-PETase is shown in positions 284-546 of SEQ ID NO.1.

[0009] In the above-mentioned recombinant fusion enzyme, the recombinant fusion enzyme further comprises a connecting peptide connecting the KL-MHETase and the FAST-PETase;

[0010] The connecting peptide is a flexible peptide chain composed of 36 glycine and serine residues, named L36. Preferably, its amino acid sequence is shown in SEQ ID NO.6; the nucleotide sequence encoding the connecting peptide gene is shown in SEQ ID NO.7.

[0011] The PET degrading enzyme FAST-PETase is a five-point mutant of PETase with greatly improved PET degradation activity and thermal stability reported in the literature (Lu, H. et al. Machine learning-aided engineering of hydrolases for PET depolymerization. Nature 604, 662–667 (2022).). Preferably, its amino acid sequence is shown in the 284th to 546th amino acids of SEQ ID NO.1, and its nucleotide sequence is shown in the 850th to 1638th bases of SEQ ID NO.2.

[0012] In the above-mentioned recombinant fusion enzyme, the recombinant fusion enzyme is any one of the following proteins:

[0013] 1) A protein consisting of the amino acid sequence shown in SEQ ID NO.1 in the sequence listing;

[0014] 2) A protein derived from 1) with the same function as SEQ ID NO. 1 in the sequence list, wherein one or more amino acid residues are substituted and / or deleted and / or added;

[0015] 3) A protein whose sequence contains the amino acid sequence defined in 1) or 2);

[0016] 4) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of a protein defined in any one of 1) to 3).

[0017] In a second aspect, the present invention provides a nucleic acid encoding the recombinant fusion enzyme described in the first aspect.

[0018] The nucleotide sequence encoding the nucleic acid described above is a DNA molecule described in any one of the following 1)-4):

[0019] 1) A DNA molecule whose coding region includes the nucleotide sequence shown in SEQ ID NO.2;

[0020] 2) a DNA molecule having a nucleotide sequence as shown in SEQ ID NO.2;

[0021] 3) A DNA molecule that hybridizes with the nucleotide sequence described in 1) or 2) under stringent conditions and encodes the recombinant fusion enzyme described in any one of claims 1 to 3;

[0022] 4) A DNA molecule having more than 90% homology with the nucleotide sequence of 1) or 2) and encoding the recombinant fusion enzyme according to any one of claims 1 to 3.

[0023] In a third aspect, the present invention provides a recombinant vector or a transgenic cell line or a recombinant bacterium containing the encoding nucleic acid described in the second aspect.

[0024] The above-mentioned recombinant vector (pET22b(+)-KL36F) is a recombinant expression vector obtained by inserting the fusion enzyme gene between the multiple cloning sites NdeI and XhoI of the pET22b(+) vector.

[0025] Furthermore, the above-mentioned recombinant bacteria are recombinant bacteria containing the above-mentioned recombinant vector.

[0026] Specifically, the above-mentioned recombinant bacteria are obtained by transforming the above-mentioned recombinant vector into Escherichia coli E. coli BL21 (DE3) to obtain the recombinant genetic engineering bacteria containing the recombinant fusion enzyme gene, which is named E. coli BL21 (DE3) / pET22b (+)-KL36F.

[0027] In a fourth aspect, the present invention provides the use of the recombinant fusion enzyme described in the first aspect in any of the following 1)-4) or in the preparation of a product having any of the following functions;

[0028] Or the present invention provides the use of the encoding nucleic acid described in the second aspect or the recombinant vector or transgenic cell line or recombinant bacteria described in the third aspect in producing a product having any one of the following functions 1)-4);

[0029] 1) Hydrolysis of MHET;

[0030] 2) Degradation of PET;

[0031] 3) Increase TPA production;

[0032] 4) Improve PET conversion rate.

[0033] The substrate concentration of PET is greater than or equal to 1.6 g / L.

[0034] In a fifth aspect, the present invention provides a method for degrading PET, wherein the recombinant fusion enzyme described in the first aspect is used to act on PET to achieve degradation.

[0035] The substrate concentration of PET is greater than or equal to 1.6 g / L.

[0036] In a sixth aspect, the present invention provides a method for hydrolyzing MHET, wherein the recombinant fusion enzyme described in the first aspect acts on MHET to achieve hydrolysis.

[0037] Compared with the prior art, the present invention has the following advantages:

[0038] (1) The thermophilic carboxylesterase Est30 of the present invention has high thermal stability, with a melting temperature of 74°C, the melting temperature of the mutant KL-MHETase is 67.6°C, and the melting temperature of the recombinant fusion enzyme KL36F after connecting with FAST-PETase is 68.7°C, which is much higher than the thermal stability of the natural MHET hydrolase MHETase (melting temperature 50.6°C);

[0039] (2) The catalytic efficiency of the recombinant fusion enzyme in the present invention for catalyzing the hydrolysis of MHET at 50°C reached 3.64 mM -1 s -1 , which is 52.6 times more efficient than FAST-PETase in hydrolyzing MHET, and comparable to existing enzymes such as TfCa (0.013 mM -1 s -1 )、TTCE(0.020mM -1 s -1 ) compared with the catalytic efficiency is greatly improved;

[0040] (3) The recombinant fusion enzyme KL36F of the present invention catalyzes the degradation of post-consumer bottle-grade PET powder at 50°C, and the conversion rate reaches 90% after 48 hours, and the TPA content in the product is close to 100%. Compared with the single enzyme catalysis of FAST-PETase, the TPA production at 12 hours and 24 hours is increased to 2.3 and 1.6 times, respectively, and the PET conversion rate is increased to 1.3 and 1.1 times, respectively. It is an excellent candidate enzyme for achieving efficient degradation and recovery of PET. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 The reaction route is that PET is degraded into BHET and MHET by dual enzyme catalysis, and MHET is hydrolyzed to generate terephthalic acid TPA and ethylene glycol EG.

[0042] Figure 2 The schematic diagram of the recombinant fusion enzyme KL36F provided by the present invention consists of three parts: (1) MHET hydrolase KL-MHETase, comprising 247 amino acid residues at positions 1-247; (2) connecting peptide segment L36, comprising 36 glycine and serine residues at positions 248-283; (4) FAST-PETase, comprising 263 amino acid residues at positions 284-546.

[0043] Figure 3 The three-dimensional structure of the fusion enzyme KL36F predicted for AlphaFold2.

[0044] Figure 4 It is the peak time of PET degradation product TPA, MHET, BHET standard in HPLC spectrum.

[0045] Figure 5 Comparison of the Michaelis-Menten fitting curves of MHET hydrolysis by the recombinant fusion enzyme KL36F and FAST-PETase.

[0046] Figure 6 The results of HPLC detection of recombinant fusion enzyme KL36F and FAST-PETase after 24 hours of PET degradation are compared. Figure (A) shows the product spectrum of PET degradation by FAST, and (B) shows the product spectrum of PET degradation by KL36F.

[0047] Figure 7 This is a comparison of the product concentration and conversion rate of PET degradation by the recombinant fusion enzyme KL36F and the single enzyme FAST-PETase within 48 hours. DETAILED DESCRIPTION

[0048] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0049] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0050] Example 1. Preparation and purification of recombinant fusion enzyme

[0051] 1. Construction and analysis of recombinant fusion enzyme

[0052] The fusion enzyme is composed of the C-terminus of a MHET hydrolase (KL-MHETase) and the N-terminus of a PET degrading enzyme (FAST-PETase) connected by a connecting peptide (L36), named KL36F, as shown in the schematic diagram Figure 2 The three-dimensional structure of the fusion enzyme KL36 was predicted by AlphaFold2, and the results are shown in Figure 3 As shown, structural analysis shows that the enzyme binding pockets of KL-MHETase (KL) and FAST-PETase (FAST) do not affect each other. At the same time, the flexible peptide chain L36 (linker36) ensures that the two enzyme domains have greater fluidity and flexibility without affecting the binding with the substrate.

[0053] The amino acid sequence of the fusion enzyme KL36F is shown in SEQ ID NO.1, wherein positions 1-247 in SEQ ID NO.1 are MHET hydrolase KL-MHETase, positions 248-283 are connecting peptide (L36), and positions 284-546 are PET degrading enzyme (FAST-PETase).

[0054] The nucleotide sequence of the fusion enzyme KL36F encoding gene is shown in SEQ ID NO.2, wherein positions 1-741 of SEQ ID NO.2 are the MHET hydrolase KL-MHETase encoding gene, positions 742-849 are the connecting peptide (L36) encoding gene, and positions 850-1638 are the PET degrading enzyme (FAST-PETase) encoding gene.

[0055] 2. Construction of recombinant expression vector

[0056] First, the coding gene (SEQ ID NO.2) of the fusion enzyme KL36F with NdeI and XhoI restriction sites at both ends was obtained as the target gene by the method of total gene synthesis, and then the target gene was double-digested with NdeI and XhoI restriction enzymes, and then connected with the expression vector pET22b(+) (containing ampicillin resistance gene) double-digested with the same endonuclease overnight, and then transformed into competent cells E.coli BL21(DE3), and the recombinant expression plasmid pET22b(+)-KL36F was obtained. pET22b(+) was purchased from BGI, and the product catalog number is 69744-3. The pET22b(+) plasmid carries a carboxyl-terminal histidine tag His6 coding sequence at the XhoI restriction end, which makes the target protein easy to purify.

[0057] After sequencing, the pET22b(+)-KL36F sequence was correct. The plasmid was a recombinant vector obtained by inserting the fusion enzyme KL36F encoding gene shown in SEQ ID NO.2 in the sequence table into the NdeI and XhoI double restriction sites of the pET22b(+) vector, and the fusion enzyme KL36F encoding gene was fused with the His6 tag on the vector for expression.

[0058] 3. Obtaining the recombinant fusion enzyme KL36F

[0059] 1. Induced expression of recombinant bacteria

[0060] The pET22b(+)-KL36F obtained in step 2 was transformed into the competent Escherichia coli cells BL21(DE3) to obtain the recombinant bacteria Escherichia coli BL21(DE3) / pET22b(+)-KL36F. The obtained recombinant bacteria were streaked on an LB plate containing ampicillin (75 μg / mL), cultured overnight at 37°C (more than 14 hours), and then a single colony with good growth was picked and inoculated into 50 mL LB liquid culture medium (containing 75 μg / mL ampicillin), and cultured at 37°C and 200 rpm for more than 7 hours.

[0061] The above 1:100 inoculum was transferred to three bottles of 200 mL fresh LB liquid medium (containing 75 μg / mL ampicillin) and cultured at 37°C and 200 rpm until OD 600 Then IPTG (isopropanol-β-thiogalactoside) was added at a final concentration of 0.5 mM, and cultured at 18°C ​​and 120 rpm for more than 16 hours to induce the expression of the fusion enzyme KL36F gene.

[0062] After the induction expression is completed, the three bottles of 200mL bacterial solution are centrifuged at 4°C, 10,000rpm for 10min, and the bacterial precipitate obtained by centrifugation is resuspended in 100mL phosphate buffer (100mM sodium phosphate, pH 7.5). The resuspended solution is subjected to ultrasonic disruption to extract soluble protein (ultrasonic time 4s, interval time 6s, 60% power, 30min). The lysate is centrifuged at 4°C, 10,000rpm (11,000g) for 30min, and the supernatant is the crude enzyme solution containing the target protein.

[0063] 2. Purification of recombinant fusion enzyme

[0064] The crude enzyme solution containing the target protein is loaded onto the Ni-NTA column, and the target protein is eluted using different imidazole concentration gradients using nickel ions as affinity ions. Since the recombinant fusion enzyme has a specific absorption peak at 280nm, detecting the protein peak at 280nm during the purification process can effectively prevent interference from foreign proteins.

[0065] A 5 mL HisTrap HP (Cytiva) nickel column and Purification chromatography system is used to purify the crude enzyme solution. First, pre-equilibrate the column bed with binding buffer A (20mM sodium phosphate, pH 7.4, containing 500mM NaCl and 20mM imidazole). Then, gradually increase the concentration ratio of elution buffer B (20mM sodium phosphate, pH 7.4, containing 500mM NaCl and 500mM imidazole) linearly to elute the impurities. According to the absorption peak curve, the target protein is collected when the imidazole concentration is greater than 300mM, and finally the column is thoroughly washed with elution buffer B. The collected pure enzyme solution is washed with 10kDa The target protein was concentrated in an Ultra filtration centrifuge tube (Millipore) and desalted twice to remove the imidazole and NaCl components in the target protein. The purified target protein was stored in a phosphate buffer (100 mM sodium phosphate, pH 7.5) and refrigerated at 4°C to obtain a purified fusion enzyme KL36F solution, which was the target protein solution.

[0066] The purity of the target protein was tested by SDS-PAGE (5% stacking gel, 12% separation gel). According to the results of SDS-PAGE, the purity of the collected protein was greater than 90%. The size of the fusion enzyme KL36F was 59 kDa, which was consistent with the expected size.

[0067] The protein concentration of the purified target protein solution was determined by detecting the absorbance (A562 nm) of the mixture using a BCA protein assay kit (Beyotime Biotechnology) and an Infinite M200 PRO microplate reader (Tecan). The concentration of the purified fusion enzyme KL36F solution was 8-10 mg / ml.

[0068] Example 2: Determination of catalytic activity of recombinant fusion enzyme hydrolyzing MHET

[0069] The MHET conversion product was analyzed by HPLC (LC-20AT, Shimadzu Corporation) to determine the catalytic activity and kinetic parameters of the recombinant fusion enzyme KL36F in hydrolyzing MHET. FAST-PETase (SEQ ID NO.1, positions 284-546) was used as a control. FAST-PETase was obtained by prokaryotic expression.

[0070] The sodium phosphate buffer used below was a 100 mM NaH2PO4-NaOH aqueous solution, pH 7.5.

[0071] During the reaction, the ratio of FAST-PETase or recombinant fusion enzyme KL36F to substrate was: 0.3 mL of purified FAST-PETase solution or KL36F solution (10 mg / ml, stored in 100 mM phosphate buffer) and 0.3 mL of MHET solution (dissolved in 100 mM phosphate buffer, concentration of 20 mM), and the reaction was carried out at 50 ° C for 10 min. After the reaction, 0.6 mL of methanol was added to terminate the reaction. After the reaction sample was centrifuged (13,000 rpm, 2 min), the supernatant was taken with a syringe and filtered through a 0.22 μm filter membrane, and then detected by HPLC.

[0072] The chromatographic column used in HPLC was an Inertsil C18 reverse phase column (GL Sciences, 5 μm, 150×4.6 mm). The mobile phase A of HPLC was distilled water containing 0.1% formic acid, and the mobile phase B was chromatographically pure acetonitrile. From 0 to 5 minutes, the acetonitrile was 5%; from 5 to 13 minutes, the acetonitrile increased to 44%; then increased to 70% at 18 minutes, and maintained at 70% for 5 minutes; from 23 to 24 minutes, it gradually decreased to 5%, and then maintained at 5% for 4 minutes. The flow rate was 0.8 mL / min, the detection wavelength was 254 nm, and the column oven was kept constant at 30°C.

[0073] The HPLC spectrum of the mixed standard TPA (TCI, product number T0166), MHET (BiDe Pharmaceutical, product number BD00777875-1g), and BHET (TCI, product number B3429) is as follows: Figure 4 As shown, at retention time t R =14.8min, the chromatographic peak of the hydrolysis product TPA appears at retention time t R The chromatographic peak of substrate MHET appears around =15.3min.

[0074] Enzyme kinetic parameters (k cat , K m , k cat / K m ) is obtained by fitting the experimental data through the Michaelis-Menten equation. The Michaelis-Menten fitting curve results are shown in Figure 5 The parameters such as the conversion number, Michaelis constant and catalytic efficiency obtained after fitting are shown in Table 1. It can be seen that the k of the recombinant fusion enzyme in the present invention for hydrolyzing MHET is cat / K m 3.64mM -1 s -1 , is the catalytic efficiency of FAST-PETase (k cat / K m =0.07mM -1 s -1 ) is 52.6 times.

[0075] The melting temperature of the fusion enzyme KL36F was determined using differential scanning calorimetry (DSC) (Malvern Panalytical, Malvern, UK) (the melting temperature of FAST-PETase was reported in the literature). The purified fusion enzyme KL36F was diluted to 0.5-1.0 mg / mL and degassed by centrifugation before testing. During the measurement, the sample was heated from 30°C to 120°C at a rate of 3°C / min. The baseline value of the buffer solution without protein was subtracted from the DSC curve of the protein using the matching PEAQ-DSC analysis software to obtain the melting temperature (T m The measurement results are shown in Table 1. It can be seen that after the fusion enzyme KL36F is fused with the protein KL-MHETase, the melting temperature is 68.67°C, which is basically consistent with the melting temperature of FAST-PETase.

[0076] Table 1 shows the kinetic parameters and melting temperatures of MHET hydrolysis catalyzed by FAST-PETase and KL36F

[0077]

[0078] The catalytic efficiency improvement factor in the above table is the multiple of the catalytic efficiency of the fusion enzyme compared to the catalytic efficiency of FAST.

[0079] Example 3: Application of recombinant fusion enzyme in degradation of post-consumer PET

[0080] Bottle-grade PET comes from the post-consumer Coca-Cola bottles (excluding the bottle head and bottle bottom) produced by Coca-Cola Company. First, the Coke bottles are cut into 1cm*1cm pieces, then washed with water, surfactant and ethanol, dried, put into a grinder, and crushed at 8000rpm. The crushed powder is filtered with a 100μm and 200μm pore size screen to obtain 100-200μm PET bottle powder.

[0081] The comparison fusion enzyme KL36F uses the highly efficient PET degrading enzyme FAST-PETase (SEQ ID NO.1, positions 284-546) reported in the literature to degrade PET.

[0082] The phosphate buffer used below was a 100 mM NaH2PO4-NaOH aqueous solution, pH 8.0.

[0083] Single enzyme experimental group: weigh about 5 mg of post-consumer PET bottle powder into a 5 ml reaction bottle, and then add 3 ml of phosphate buffer with a pH of 8.0 containing 0.35 μM purified FAST-PETase (FAST-PETase is dissolved in phosphate buffer, and the concentration of FAST-PETase is 0.35 μM).

[0084] Fusion enzyme experimental group: About 5 mg of post-consumer PET bottle powder was weighed into a 5 ml reaction bottle, and then 3 ml of phosphate buffer with a pH of 8.0 containing 0.35 μM purified recombinant fusion enzyme KL36F was added (KL36F was dissolved in phosphate buffer, and the concentration of KL36F was 0.35 μM).

[0085] The above groups of substances were placed in a constant temperature mixer at 50°C and 800 rpm. When the reaction time was 12, 24, 36, and 48 hours, equal volumes of 100% methanol were added to terminate the reaction. After appropriate dilution, the supernatant was collected by centrifugation (13,000 rpm, 2 min). The supernatant was taken with a syringe and filtered through a 0.22 μm filter membrane.

[0086] The product was analyzed by high performance liquid chromatography HPLC (LC-20AT, Shimadzu Corporation), and the selected chromatographic column was Inertsil C18 reverse phase column (GL Sciences, 5 μm, 150×4.6 mm). The mobile phase A of HPLC was distilled water containing 0.1% formic acid, and the mobile phase B was chromatographically pure acetonitrile. From 0 to 5 minutes, the acetonitrile was 5%; from 5 to 13 minutes, the acetonitrile increased to 44%; then increased to 70% at 18 minutes, and maintained at 70% for 5 minutes; from 23 to 24 minutes, it gradually decreased to 5%, and then maintained at 5% for 4 minutes. The flow rate was 0.8 mL / min, the detection wavelength was 254 nm, and the column oven was kept constant at 30°C.

[0087] The HPLC peak spectrum of the standard product is as follows Figure 4 As shown, at retention time t R =14.8min, the chromatographic peak of the degradation product TPA appeared. R =15.3min, the chromatographic peak of the degradation product MHET appeared. R The chromatographic peak of the degradation product BHET appeared around =15.8min.

[0088] The HPLC chromatograms of the products of the single enzyme experimental group and the fusion enzyme experimental group after 24 hours of degradation of PET are shown in the figure. Figure 6 As shown, the HPLC spectrum of the product after FAST-PETase degraded PET for 24 hours is as follows Figure 6 As shown in (A), the HPLC spectrum of the product after KL36F degradation of PET for 24 hours is as follows Figure 6 As shown in (B), it can be seen that the target product TPA is obtained.

[0089] The calculation formula for conversion rate (or degradation rate) and TPA purity is:

[0090] Conversion rate = total product concentration × dilution factor × reaction volume / (1000 × PET mass / MHET monomer molecular weight) × 100%; wherein 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, and the molecular weight of MHET monomer is 210.14.

[0091] The results of the product content and conversion rate of PET degradation in the single enzyme experimental group and the fusion enzyme experimental group within 48 hours are as follows Figure 7As shown, the PET conversion rate of the 48-hour fusion enzyme experimental group was 90.4%, and the TPA content in the aromatic product was 99.7%. Compared with the FAST-PETase single enzyme experimental group, the TPA production of the fusion enzyme experimental group at 12 hours and 24 hours increased to 2.3 and 1.6 times, respectively, and the TPA proportion increased from 53.8% and 67.8% to 98.8% and 99.5%, and the PET conversion rate increased to 1.3 and 1.1 times, respectively.

Claims

1. A recombinant fusion enzyme comprising MHET hydrolase KL-MHETase and PET degrading enzyme FAST-PETase; The amino acid sequence of the MHET hydrolase KL-MHETase is shown in SEQ ID NO.1, positions 1-247; The amino acid sequence of the PET degrading enzyme FAST-PETase is shown in positions 284-546 of SEQ ID NO.

1.

2. The recombinant fusion enzyme according to claim 1, characterized in that: The recombinant fusion enzyme also includes a connecting peptide connecting the KL-MHETase and the FAST-PETase; The connecting peptide is a flexible peptide chain composed of 36 glycine residues and serine residues.

3. The recombinant fusion enzyme according to claim 1 or 2, characterized in that: The recombinant fusion enzyme is any one of the following proteins: 1) A protein consisting of the amino acid sequence shown in SEQ ID NO.1 in the sequence listing; 2) A protein derived from 1) with the same function as SEQ ID NO. 1 in the sequence list, wherein one or more amino acid residues are substituted and / or deleted and / or added; 3) A protein whose sequence contains the amino acid sequence defined in 1) or 2); 4) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of a protein defined in any one of 1) to 3).

4. The nucleic acid encoding the recombinant fusion enzyme according to any one of claims 1 to 3.

5. The coding nucleic acid according to claim 4, characterized in that: The nucleotide sequence of the encoding nucleic acid is a DNA molecule described in any one of the following 1)-4): 1) A DNA molecule whose coding region includes the nucleotide sequence shown in SEQ ID NO.2; 2) a DNA molecule having a nucleotide sequence as shown in SEQ ID NO.2; 3) A DNA molecule that hybridizes with the nucleotide sequence described in 1) or 2) under stringent conditions and encodes the recombinant fusion enzyme described in any one of claims 1 to 3; 4) A DNA molecule having more than 90% homology with the nucleotide sequence of 1) or 2) and encoding the recombinant fusion enzyme according to any one of claims 1 to 3.

6. A recombinant vector, transgenic cell line or recombinant bacterium containing the encoding nucleic acid according to claim 4 or 5.

7. Use of the recombinant fusion enzyme according to any one of claims 1 to 3 in any one of the following 1) to 4) or in the preparation of a product having any one of the following functions; Or use of the encoding nucleic acid according to claim 4 or 5 or the recombinant vector or transgenic cell line or recombinant bacteria according to claim 6 in producing a product having any of the following functions 1)-4); 1) Hydrolysis of MHET; 2) Degradation of PET; 3) Increase TPA production; 4) Improve PET conversion rate.

8. A method for degrading PET, comprising: using the recombinant fusion enzyme according to any one of claims 1 to 3 to act on PET to achieve degradation.

9. A method for hydrolyzing MHET, comprising: using the recombinant fusion enzyme according to any one of claims 1 to 3 to act on MHET to achieve hydrolysis.

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