High-activity high-thermal-stability MHET hydrolase mutant and application thereof

Abstract

The invention discloses a high-activity and high-thermal-stability MHET hydrolase mutant and application thereof. The invention provides a mutant protein of thermophilic carboxylesterase, which is shown as 1) or 2): 1) the protein shown as 1) is a protein obtained by mutating amino acid residues at the 130th site and the 171th site of wild thermophilic carboxylesterase shown as SEQ ID NO.2 or a sequence corresponding to the SEQ ID NO.2 and keeping other amino acid residues unchanged; (2) the protein is obtained by mutating amino acid residues at the 127th site, the 130th site and the 171th site of wild thermophilic carboxylesterase and keeping other amino acid residues unchanged. Experiments prove that the activity of the modified mutant for catalyzing MHET hydrolysis at the temperature of 50 DEG C is improved by 95 times compared with that of a wild type, and the modified mutant has higher enzyme activity and thermal stability and is the first enzyme capable of efficiently catalyzing MHET hydrolysis at the temperature of 50 DEG C at present.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a high-activity and high-thermal-stability MHET hydrolase mutant and an application thereof. Background Art

[0002] Polyethylene terephthalate (PET) is the most important thermoplastic polyester polymerized from terephthalic acid (TPA) and ethylene glycol (EG). More than 82 million tons are produced worldwide each year and are widely used in films, packaging bottles, clothing, electronic products, and mechanical equipment. However, PET degrades slowly in the environment, causing white pollution to the ecological environment. At present, the main methods for treating PET waste include landfill, incineration, and recycling. Although landfill and incineration are the simplest, the waste gas and wastewater generated will cause secondary pollution to the environment. PET can be degraded into small molecules by biological methods (enzymatic degradation or microbial degradation), and then recycled and reused. Compared with chemical catalytic recycling with harsh reaction conditions, biodegradation can be carried out under milder conditions, is green and environmentally friendly, and conforms to the concept of sustainable development. In 2016, Yoshida et al. isolated Ideonella sakaiensis 201-F6 (Yoshida, S. et al. Abacterium that degrades and assimilates poly(ethylene terephthalate). Science 351, 1196–1199 (2016).), which 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 1 shown.

[0003] As the main intermediate product of PET degradation, MHET is more difficult to degrade than BHET. Almost all PET degrading enzymes are inhibited by MHET, resulting in reduced degradation efficiency. The introduction of a suitable MHET hydrolase to construct a dual enzyme system can eliminate the product inhibition of MHET on the PET degrading enzyme, thereby improving the efficiency of the PET degrading enzyme and the purity of TPA in the product, which is required for the enzymatic recovery process of PET. However, there are currently few research reports on the design and modification of MHET hydrolases, and the reported enzymes cannot simultaneously meet the requirements of high activity and high thermal stability. The natural MHETase expressed by the Ideonella sakaiensis 201-F6 strain has a very high catalytic efficiency in hydrolyzing MHET at 30°C, and its catalytic efficiency k cat / K m For 10 6 s - 1 M -1 However, there is no report on the improvement of the thermal stability of MHETase, which results in MHETase only being able to hydrolyze MHET at 30°C (Palm, GJ et al. Structure of the plastic-degrading Ideonellasakaiensis MHETase bound to a substrate. Nat Commun 10, 1717 (2019).). Other enzymes that can degrade MHET, such as TfCa from Thermobifida fusca and BsCE from Bacillus subtilis, have a catalytic efficiency of MHET hydrolysis at 50°C. cat / K m Only 12.72s -1 M -1 and 43.1s -1 M -1 , which is much lower than MHETase ( JA, Borch, K., Jensen, K., Brask, J. & Westh, P. Comparative Biochemistry of Four Polyester (PET) Hydrolases**. ChemBioChem 22, 1627–1637 (2021).). The thermostable carboxylesterase TTCE from Thermus thermophilus and the mutant TfCa-WA of TfCa from Thermobifida fusca can also hydrolyze MHET at 50°C, but their catalytic efficiency is at least 5 orders of magnitude lower than that of the natural MHETase.

[0004] In summary, there is currently no report on enzymes that can stably and efficiently hydrolyze MHET at temperatures above 50°C, which limits the development of a dual-enzyme degradation system for PET. With the increasing severity of white pollution, it is particularly important and urgent to develop highly active and highly thermally stable MHET hydrolases that can work together with compatible PET degradation enzymes for the degradation and recycling of PET. Summary of the invention

[0005] The technical problem to be solved by the present invention is that the existing mono(2-hydroxyethyl)terephthalate (MHET) hydrolase cannot achieve both high thermal stability and high activity. Through de novo screening and site-directed mutagenesis guided by computational enzyme design, a highly active and highly thermally stable MHET hydrolase mutant is provided. The modified mutant has high enzyme activity at 50°C. When used in combination with a highly active and highly thermally stable PET degradation enzyme, the inhibitory effect of MHET can be reduced, and the purity and yield of the recyclable monomer TPA in the product can be further improved.

[0006] In a first aspect, the present invention provides a mutant protein of a thermophilic carboxylesterase, which is as follows 1) or 2):

[0007] 1) The protein shown is a protein obtained by mutating the 130th and 171st amino acid residues of the wild-type thermophilic carboxylesterase shown in SEQ ID NO.2 or its corresponding sequence, while keeping other amino acid residues unchanged;

[0008] 2) The protein shown is a protein obtained by mutating the 127th, 130th and 171st amino acid residues of the wild-type thermophilic carboxylesterase shown in SEQ ID NO.2 or its corresponding sequence, while keeping other amino acid residues unchanged.

[0009] In the above method, each amino acid residue is mutated as follows:

[0010] The amino acid residue at position 127, Met, mutated to Ser;

[0011] The amino acid residue Gly at position 130 was mutated to Leu or Phe;

[0012] The amino acid residue at position 171, Ile, was mutated to Lys.

[0013] In the above method, the mutant protein is any of the following, named Est30_M1, Est30_M2, Est30_M3:

[0014] 1) A protein consisting of the amino acid sequence shown in SEQ ID NO.4, SEQ ID NO.6 or SEQ ID NO.8 in the sequence listing;

[0015] 2) A protein derived from 1) having the activity of hydrolyzing MHET, wherein one or more amino acid residues of the amino acid sequence of SEQ ID NO.4 or SEQ ID NO.6 or SEQ ID NO.8 in the sequence list are substituted and / or deleted and / or added;

[0016] 3) A protein whose sequence contains the amino acid sequence defined in 1) or 2);

[0017] 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).

[0018] Compared with the wild-type thermophilic carboxylesterase, the mutant protein has high activity and high thermal stability, which is specifically reflected in the stable and efficient hydrolysis of MHET above 50°C, that is, the activity of catalyzing the hydrolysis of MHET at 50°C is increased by 95 times compared with the wild-type.

[0019] In a second aspect, the present invention provides a nucleic acid encoding the mutant protein of the first aspect.

[0020] The encoding nucleic acid described above is a DNA molecule described in any one of the following 1)-4):

[0021] 1) A DNA molecule whose coding region includes a nucleotide sequence as shown in SEQ ID NO.3, SEQ ID NO.5 or SEQ ID NO.7;

[0022] 2) A DNA molecule having a nucleotide sequence as shown in SEQ ID NO.3, SEQ ID NO.5 or SEQ ID NO.7;

[0023] 3) A DNA molecule that hybridizes with the nucleotide sequence described in 1) or 2) under stringent conditions and encodes the mutant protein described in the first aspect.

[0024] 4) A DNA molecule that has more than 90% homology with the nucleotide sequence of 1) or 2) and encodes the mutant protein described in the first aspect.

[0025] In a third aspect, the present invention provides a recombinant vector or a transgenic cell line or a recombinant bacterium containing the coding gene described in the second aspect.

[0026] In the above, the recombinant vectors containing the above-mentioned genes (named pET22b(+)-Est30_M1, pET22b(+)-Est30_M2, pET22b(+)-Est30_M3) are recombinant expression vectors obtained by inserting the encoding gene described in the second aspect of the present invention between the multiple cloning sites of the pET22b(+) vector.

[0027] The recombinant bacteria are recombinant bacteria containing the recombinant vector. Specifically, the recombinant bacteria are recombinant Escherichia coli containing the recombinant vector.

[0028] In a fourth aspect, the present invention provides the use of the mutant protein described in the first aspect in preparing a protein having at least one of the following functions 1)-4);

[0029] Or the present invention provides the use of the encoding nucleic acid of the second aspect or the recombinant vector or transgenic cell line or recombinant bacteria of the third aspect in preparing a product having at least one of the following functions 1)-4);

[0030] Or the present invention provides the use of the mutant protein and PET degrading enzyme described in the first aspect in the preparation of a product having at least one of the following functions 1)-4);

[0031] 1) Hydrolysis of MHET;

[0032] 2) Degradation of PET;

[0033] 3) Increase TPA production;

[0034] 4) Improve PET conversion rate.

[0035] The above PET degrading enzyme is a highly efficient PET degrading enzyme FAST-PETase, and in the embodiment of the present invention, its amino acid sequence is SEQ ID NO.9.

[0036] In an embodiment of the present invention, the substrate concentration of PET is greater than or equal to 1.6 g / L.

[0037] In a fifth aspect, the present invention provides a product comprising the mutant protein described in the first aspect and a PET degrading enzyme.

[0038] In the above, the mutant protein and PET degrading enzyme can be packaged independently and act together.

[0039] The above product is a PET dual enzyme degradation system.

[0040] In the above product, in an embodiment of the present invention, the molar ratio of the mutant protein to the PET degrading enzyme is 35:11-33.

[0041] In a sixth aspect, the present invention provides a method for degrading PET, wherein the product described in the fifth aspect is used to act on PET to achieve degradation.

[0042] In an embodiment of the present invention, the substrate concentration of PET is greater than or equal to 1.6 g / L.

[0043] In the seventh aspect, a method for hydrolyzing MHET is provided, wherein the mutant protein described in the first aspect or the product described in the fifth aspect is used to act on MHET to achieve hydrolysis.

[0044] Compared with the prior art, the present invention has the following advantages:

[0045] (1) The thermophilic carboxylesterase Est30 of the present invention has high thermal stability, with a melting temperature of 74°C, and the melting temperatures of mutants M1, M2, and M3 are 67.6, 63.3, and 63.2°C, which are much higher than the thermal stability of the natural MHET hydrolase MHETase (melting temperature 50.6°C);

[0046] (2) The catalytic efficiency of the mutants M1, M2, and M3 in the present invention for catalytic hydrolysis of MHET at 50°C reached 4.70, 12.58, and 8.11 mM, respectively. -1 s -1 , compared with the catalytic efficiency of wild-type Est30 of 0.13 mM -1 s -1 The results showed that the hydrolysis of MHET at 50°C was improved by 35, 95 and 61 times, respectively, which is comparable to the 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;

[0047] (3) The present invention further compounded the mutant M1 with the highly efficient PET degrading enzyme FAST-PETase reported in the literature (Lu, H. et al. Machine learning-aided engineering of hydrolases for PET depolymerization. Nature 604, 662–667 (2022).) at 50°C. The dual-enzyme system constructed increased the TPA yield generated by degrading post-consumer PET cola bottle powder within 48 hours to about 1.3 times compared with the single FAST-PETase, and the purity of TPA in the product reached more than 98.0%, which is expected to be applied to the industrialization of enzymatic recovery of PET. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 The reaction route is the enzyme-catalyzed degradation of PET into BHET and MHET, and the hydrolysis of MHET to produce terephthalic acid TPA and ethylene glycol EG.

[0049] Figure 2 This is the plasmid map of pET22b(+)-Est30.

[0050] Figure 3HPLC peak time diagram of TPA, MHET and BHET standards.

[0051] Figure 4 The chromatograms of samples after HPLC detection of wild-type enzyme Est30 and mutant hydrolyzing MHET.

[0052] Figure 5 The kinetic fitting curves of the hydrolysis of MHET by wild-type and mutant Est30 are shown in the figure. The results of the Michaelis-Menten equation fitting are given in the figure.

[0053] Figure 6 Schematic diagram of the change over time of the concentration of products generated by the degradation of PET by the combination of mutant Est30_M1 with different enzyme amounts and FAST-PETase. DETAILED DESCRIPTION

[0054] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0055] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0056] Example 1. Preparation and purification of carboxylesterase Est30 mutant

[0057] 1. Obtaining the gene encoding the carboxylesterase Est30 mutant

[0058] The coding gene of wild carboxylesterase Est30 (Genebank: AY186197.1) was obtained from the NCBI database, and the nucleotide sequence of the gene is SEQ ID NO.1; the amino acid sequence of the wild carboxylesterase Est30 encoded by the gene is SEQ ID NO.2.

[0059] First, the gene of wild carboxylesterase Est30 is obtained by total gene synthesis, and then the coding gene of wild carboxylesterase Est30 is subjected to site-directed mutagenesis by Overlap PCR to obtain the coding gene of carboxylesterase Est30 mutant.

[0060] The above-mentioned carboxylesterase Est30 mutants are Est30_M1 mutant protein, Est30_M2 mutant protein and Est30_M3 mutant protein.

[0061] The Est30_M1 mutant protein is a mutant obtained by mutating the 130th Gly to Leu, the 171st Ile to Lys of the wild-type thermophilic carboxylesterase Est30 shown in SEQ ID NO.2, and keeping other amino acid residues unchanged; the amino acid sequence of the Est30_M1 mutant protein is SEQ ID NO.4;

[0062] The Est30_M2 mutant protein is a mutant obtained by mutating the 127th Met to Ser, the 130th Gly to Leu, the 171st Ile to Lys of the wild-type thermophilic carboxylesterase Est30 shown in SEQ ID NO.2, and leaving other amino acid residues unchanged; the amino acid sequence of the Est30_M2 mutant protein is SEQ ID NO.6;

[0063] The Est30_M3 mutant protein is a mutant obtained by mutating the 127th Met to Ser, the 130th Gly to Phe, the 171st Ile to Lys of the wild-type thermophilic carboxylesterase Est30 shown in SEQ ID NO.2, while keeping other amino acid residues unchanged; the amino acid sequence of the Est30_M3 mutant protein is SEQ ID NO.8.

[0064] The Est30_M1 mutant protein encoding gene is a mutant protein encoding gene obtained by mutating the codon encoding Gly at position 130 to a codon encoding Leu, and the codon encoding Ile at position 171 to a codon encoding Lys in the wild-type thermophilic carboxylesterase Est30 encoding gene shown in SEQ ID NO.2, while keeping other nucleotides unchanged; the nucleotide sequence of the Est30_M1 mutant protein encoding gene is SEQ ID NO.3;

[0065] The Est30_M2 mutant protein is a mutant protein encoding gene obtained by mutating the codon encoding Met at position 127 to a codon encoding Ser, the codon encoding Gly at position 130 to a codon encoding Leu, and the codon encoding Ile at position 171 to a codon encoding Lys in the encoding gene of the wild-type thermophilic carboxylesterase Est30 shown in SEQ ID NO.2, while other nucleotides remain unchanged; the nucleotide sequence of the Est30_M2 mutant protein encoding gene is SEQ ID NO.5;

[0066] The Est30_M3 mutant protein is a mutant protein encoding gene obtained by mutating the codon encoding Met at position 127 in the encoding gene of the wild-type thermophilic carboxylesterase Est30 shown in SEQ ID NO.2 into a codon for Ser, the codon encoding Gly at position 130 into a codon for Phe, and the codon encoding Ile at position 171 into a codon for Lys, while keeping other nucleotides unchanged; the nucleotide sequence of the Est30_M3 mutant protein encoding gene is SEQ ID NO.7.

[0067] 2. Construction of recombinant expression vector

[0068] The gene of wild carboxylesterase Est30 with NdeI and XhoI restriction recognition sites at both ends was obtained as the target gene Est30 by the whole gene synthesis method.

[0069] The target gene Est30 was double-digested with NdeI and XhoI, and then connected with the expression vector pET22b(+) (containing the ampicillin resistance gene) double-digested with the same endonucleases overnight, and then transformed into competent cells BL21(DE3) to obtain the recombinant expression plasmid pET22b(+)-Est30. pET22b(+) was purchased from BGI, and the product catalog number is 69744-3. The plasmid map is shown in Figure 2 The pET22b(+) plasmid carries a carboxyl-terminal histidine tag His6 coding sequence at the XhoI-cleaved end, making the target protein easier to purify.

[0070] After sequencing, the sequence of pET22b(+)-Est30 was correct. The plasmid was composed of the recombinant plasmid pET22b(+)-Est30, in which the Est30 gene shown in positions 1-747 of SEQ ID NO.1 in the sequence list replaced the fragment between the NdeI and XhoI sites of pET22b(+), and the Est30 gene was fused with the His6 tag on the vector for expression.

[0071] The recombinant expression vectors pET22b(+)-Est30_M1, pET22b(+)-Est30_M2 and pET22b(+)-Est30_M3 were prepared according to the above method.

[0072] The recombinant expression vector pET22b(+)-Est30_M1 is obtained by replacing the Est30_M1 gene shown in SEQ ID NO.3 with the fragment between the NdeI and XhoI sites of pET22b(+), thereby forming the recombinant plasmid pET22b(+)-Est30_M1, and the Est30_M1 gene is fused with the His6 tag on the vector for expression.

[0073] The recombinant expression vector pET22b(+)-Est30_M2 is obtained by replacing the Est30_M2 gene shown in SEQ ID NO.5 with the fragment between the NdeI and XhoI sites of pET22b(+), thereby forming the recombinant plasmid pET22b(+)-Est30_M2, and the Est30_M2 gene is fused with the His6 tag on the vector for expression.

[0074] The recombinant expression vector pET22b(+)-Est30_M3 is obtained by replacing the Est30_M3 gene shown in SEQ ID NO.7 with the fragment between the NdeI and XhoI sites of pET22b(+), thereby forming the recombinant plasmid pET22b(+)-Est30_M3, and the Est30_M3 gene is fused with the His6 tag on the vector for expression.

[0075] 3. Obtaining recombinant bacteria

[0076] The pET22b(+)-Est30, pET22b(+)-Est30_M1, pET22b(+)-Est30_M2 and pET22b(+)-Est30_M3 obtained in step 2 were transformed into Escherichia coli BL21(DE3) competent cells (competent cells purchased from Beijing Bomade Gene Technology Co., Ltd., catalog number BC201-02) to obtain recombinant bacteria BL21(DE3) / pET22b(+)-Est30, BL21(DE3) / pET22b(+)-Est30_M1, BL21(DE3) / pET22b(+)-Est30_M2, BL21(DE3) / pET22b(+)-Est30_M3. The extracted plasmids were sequenced to verify the correctness.

[0077] 4. Obtaining carboxylesterase Est30 and its mutants

[0078] 1. Inducible expression of enzyme mutants

[0079] Streak each recombinant bacterium constructed in step 3 on an LB plate containing ampicillin (75 μg / mL), culture overnight at 37°C (more than 14 hours), pick a single colony with good growth and inoculate it into 50 mL LB liquid medium (containing 75 μg / mL ampicillin), and culture it at 37°C and 200 rpm for 7 hours;

[0080] 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 the mixture was cultured at 18° C. and 120 rpm for 16 hours to induce the expression of carboxylesterase Est30 or its mutant gene.

[0081] 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 with 100mL phosphate buffer (50mM sodium phosphate, pH 7.5) to obtain a resuspension. The resuspension is then subjected to ultrasonic disruption to extract soluble proteins (ultrasonic time 4s, interval time 6s, 60% power, 30min) to obtain a lysate. The lysate is then centrifuged at 4°C, 10,000g for 30min, and the supernatant is the crude enzyme solution containing each target protein.

[0082] 2. Purification of enzyme mutants

[0083] The crude enzyme solution containing each target protein prepared in 1 above was loaded onto a Ni-NTA column, and the target protein was eluted using a gradient of different imidazole concentrations using nickel ions as affinity ions. Since carboxylesterase Est30 and its mutants have a specific absorption peak at 280 nm, detecting the protein peak at 280 nm during the purification process can effectively prevent interference from foreign proteins.

[0084] 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 Ultra ultrafiltration centrifuge tubes (Millipore) were used to concentrate the target protein and desalted twice to remove the imidazole and NaCl components in the target protein. The purified target protein was stored in phosphate buffer (50 mM sodium phosphate, pH 7.5) and refrigerated at 4°C to obtain purified Est30, Est30_M1, Est30_M2 and Est30_M3 protein solutions, which were the target protein solutions.

[0085] The purity of the target protein was tested by SDS-PAGE (5% stacking gel, 12% separation gel). According to the SDS-PAGE results, the purity of the collected protein was greater than 90%. The sizes of the purified Est30, Est30_M1, Est30_M2 and Est30_M3 proteins were all 29.5 kDa, which was consistent with the expected size.

[0086] The protein concentration of each purified target protein solution was determined by detecting the absorbance (A562 nm) of the mixture using a BCA protein detection kit (Beyotime Biotechnology) and an Infinite M200 PRO microplate reader (Tecan). The concentration of the purified Est30, Est30_M1, Est30_M2 and Est30_M3 protein solutions was 5-10 mg / ml.

[0087] Example 2: Determination of catalytic activity and thermal stability of enzyme mutants in hydrolyzing MHET

[0088] 1. Determination of the catalytic activity of Est30 wild type and mutants in hydrolyzing MHET

[0089] The MHET conversion products were analyzed by HPLC (LC-20AT, Shimadzu Corporation) to determine the catalytic activity and kinetic parameters of the purified Est30, Est30_M1, Est30_M2 and Est30_M3 protein mutants prepared in Example 1 as target proteins for hydrolyzing MHET.

[0090] During the reaction, the ratio of enzyme mutant to substrate was: 0.3 mL of purified target protein solution (final concentration of 200-300 nM, stored in 50 mM sodium phosphate buffer, pH 7.5) and 0.3 mL of MHET solution (MHET dissolved in 50 mM sodium phosphate buffer, pH 7.5, concentration gradient of 2-20 mM), and the reaction was carried out at 50°C for 10 min. After the reaction was completed, 0.6 mL of methanol was added to terminate the reaction.

[0091] 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 3 As shown, the selected chromatographic column is an Inertsil C18 reverse phase column (GLSciences, 5μm, 150×4.6mm). The mobile phase A of HPLC is distilled water containing 0.1% formic acid, and the mobile phase B is chromatographically pure acetonitrile. From 0 minutes to 5 minutes, the acetonitrile is 5%; from 5 minutes to 12 minutes, the acetonitrile increases to 44%; then increases to 70% at the 15th minute, and maintains the 70% ratio for 3 minutes; from 18 minutes to 24 minutes, it gradually decreases to 5%. The flow rate is 0.8mL / min, the detection wavelength is 254nm, and the column oven is constant at 30°C.

[0092] The HPLC spectra of Est30, Est30_M1, Est30_M2 and Est30_M3 proteins are shown in Figure 4As shown, Figure (A) is the spectrum of the wild-type enzyme Est30 (denoted as WT in the figure) hydrolyzing 5mM substrate MHET at 50 degrees for 10 minutes; Figure (B) is the spectrum of the mutant Est30_M1 (denoted as M1 in the figure) hydrolyzing 5mM substrate MHET at 50 degrees for 10 minutes; Figure (C) is the spectrum of the mutant Est30_M2 (denoted as M2 in the figure) hydrolyzing 5mM substrate MHET at 50 degrees for 10 minutes; Figure (D) is the spectrum of the mutant Est30_M3 (denoted as M3 in the figure) hydrolyzing 5mM substrate MHET at 50 degrees for 10 minutes; It can be seen that the mutant has a high retention time at retention time t R = 17.2min (consistent with the peak time of TPA standard), the chromatographic peak of the hydrolysis product TPA appears at retention time t R = 17.8min (consistent with the elution time of the MHET standard) shows a chromatographic peak of the substrate MHET. Compared with the spectrum (A), the peak area and peak height of the hydrolysis product TPA at around 17.2min in the spectra (B), (C) and (D) are greatly increased, while the peak area and peak height of the hydrolysis substrate MHET are greatly decreased, indicating that the ability of the mutants Est30_M1, Est30_M2 and Est30_M3 to hydrolyze the substrate MHET to generate TPA is greatly improved compared with the wild-type Est30.

[0093] 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 As shown in Table 1, WT is the wild-type enzyme Est30, and M1-M3 are Est30_M1 protein mutant, Est30_M2 protein mutant, and Est30_M3 protein mutant, respectively. The results of the fitted parameters such as the turnover number, Michaelis constant, and catalytic efficiency improvement factor are shown in Table 1. It can be seen that the k of the carboxylesterase Est30 mutants Est30_M1, Est30_M2, and Est30_M3 in the present invention for hydrolyzing MHET is cat / K m 4.70, 12.58, 8.11 mM respectively -1 s -1 , compared with the wild-type enzyme (k cat / K m =0.13mM -1 s -1 ) were increased to 36.0, 96.3 and 62.1 times of the original ones.

[0094] 2. Thermal stability determination of Est30 wild type and mutants

[0095] The thermal stability of the enzyme is also an important property that affects the application performance of the enzyme. The higher the thermal stability of the enzyme, the higher the temperature at which the enzyme can be used. Differential scanning calorimetry DSC (Malvern Panalytical, Malvern, UK) was used to determine the thermal stability of the wild type and mutants of Est30. Each target protein purified in Example 1 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. Using the matching PEAQ-DSC analysis software, the baseline value of the buffer solution without protein was deducted from the DSC curve of the protein to obtain the melting temperature (T m ).

[0096] The results of the melting temperature determination are shown in Table 1. Among them, the melting temperatures of the mutants Est30_M1, Est30_M2, and Est30_M3 were 67.6, 63.2, and 63.3°C, respectively, which were 6.5, 10.9, and 10.8°C lower than the melting temperature of the wild-type Est30 (74.1°C). The significant improvement in the catalytic efficiency of the mutants led to a decrease in the thermal stability of the wild-type thermophilic enzyme. In the subsequent application of compounding with FAST-PETase to degrade PET, the use of a mutant with a similar structure to FAST-PETase (T m =67.8°C) was used to perform the experiment with the mutant Est30_M1, which has a thermal stability matching that of the control.

[0097] Table 1 Evaluation of the properties of Est30 and its mutants

[0098]

[0099] In the above table, the catalytic efficiency improvement factor is the multiple of the catalytic efficiency of the mutant compared to the wild type.

[0100] Example 3: Application of Est30 carboxylesterase mutant Est30_M1 in constructing a dual enzyme complex system for degrading post-consumer PET

[0101] Post-consumer PET bottles (Nongfu Spring) were cut into 1 cm × 1 cm pieces, washed and dried for later use. The PET bottle pieces were crushed using a high-speed rotary grinder (Ant Technology, AM500S) equipped with 0.1 mm and 0.2 mm trapezoidal hole screens to obtain PET bottle powder with a particle size of about 0.1-0.2 mm.

[0102] The Est30_M1 mutant and the highly efficient PET degrading enzyme FAST-PETase reported in the literature were used to construct a dual enzyme complex system to compare the degradation effect of the FAST-PETase single enzyme system. The amino acid sequence of the FAST-PETase protein is SEQ ID NO.9, and the FAST-PETase protein was obtained by prokaryotic expression.

[0103] The phosphate buffer used below was a 100 mM NaH2PO4-NaOH aqueous solution, pH 8.0.

[0104] Single enzyme experimental group: weigh about 5 mg of post-consumer PET bottle powder into a 5 ml reaction bottle, add 3 ml of pH 8.0 phosphate buffer containing 0.35 μM purified FAST-PETase protein (FAST-PETase is dissolved in phosphate buffer, and the concentration of FAST-PETase is 0.35 μM).

[0105] Compound experimental group: weigh about 5 mg of post-consumer PET bottle powder into a 5 ml reaction bottle, add 3 ml of phosphate buffer with a pH of 8.0 containing 0.35 μM purified FAST-PETase and 0.11 μM purified mutant Est30_M1 (FAST-PETase and purified mutant Est30_M1 were dissolved in phosphate buffer, and the concentration of FAST-PETase was 0.35 μM, and the concentration of mutant Est30_M1 was 0.11 μM);

[0106] Alternatively, weigh about 5 mg of post-consumer PET bottle powder into a 5 ml reaction bottle, and add 3 ml of phosphate buffer with a pH of 8.0 containing 0.35 μM purified FAST-PETase and 0.33 μM purified mutant Est30_M1 (the purified FAST-PETase and the purified mutant Est30_M1 are dissolved in phosphate buffer, and the concentration of purified FAST-PETase is 0.35 μM, and the concentration of mutant Est30_M1 is 0.33 μM).

[0107] The above groups of substances were placed in a constant temperature mixer at 50°C and 800 rpm for reaction for 48 hours. After 4, 8, 12, 24, 36 and 48 hours, samples were taken and diluted to appropriate multiples, and an equal volume of 100% methanol was added to terminate the reaction. The supernatant was collected by centrifugation (13,000 rpm, 2 min), and the product was analyzed by high performance liquid chromatography HPLC (LC-20AT, Shimadzu Corporation).

[0108] 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 12 minutes, the acetonitrile increased to 44%; then increased to 70% at 15 minutes, and maintained at 70% for 3 minutes; and gradually decreased to 5% from 18 to 24 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.

[0109] The reaction products of each group are at retention time t R =17.2min, the chromatographic peak of the degradation product TPA appeared (consistent with the peak time of TPA standard), and at the retention time t R =17.8min, the chromatographic peak of the degradation product MHET appeared (consistent with the peak time of the MHET standard), and at the retention time t R = The chromatographic peak of the degradation product BHET appeared around 20.0 min (consistent with the elution time of the BHET standard).

[0110] The calculation formula for conversion rate (or degradation rate) and TPA purity is:

[0111] Conversion rate = total product concentration × dilution factor × reaction volume / (1000 × PET mass / MHET monomer molecular weight) × 100%;

[0112] TPA purity = TPA concentration / (total product concentration)×100%.

[0113] 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.

[0114] The results of product content and conversion rate after degradation of dual enzyme complex system and single enzyme system are as follows Figure 6 As shown, the horizontal axis is the concentration of mutant Est30_M1 added, 0.00uM represents the single enzyme experimental group. It can be seen that in the dual enzyme experimental group, when the enzyme amount of FAST-PETase is fixed at 0.35μM and the enzyme amount of Est30_M1 is 0.11μM, the reaction temperature is 50°C, the reaction pH is 8.0, and the PET substrate used is bottle-grade PET powder (100-200μm, concentration 1.66g / L). After 48 hours, the degradation rate of PET reaches 88.7%, which is equivalent to the degradation rate of the single enzyme system. At the same time, the TPA production is 1.3 times that of the single enzyme system, and the TPA purity is increased to more than 98.0%.

Claims

1. A mutant protein of a thermophilic carboxylesterase, which is as follows 1) or 2): 1) The protein shown is a protein obtained by mutating the 130th and 171st amino acid residues of the wild-type thermophilic carboxylesterase shown in SEQ ID NO.2 or its corresponding sequence, while keeping other amino acid residues unchanged; 2) The protein shown is a protein obtained by mutating the 127th, 130th and 171st amino acid residues of the wild-type thermophilic carboxylesterase shown in SEQ ID NO.2 or its corresponding sequence, while keeping other amino acid residues unchanged.

2. The mutant protein according to claim 1, characterized in that The mutation patterns of each amino acid residue are as follows: The amino acid residue at position 127, Met, mutated to Ser; The amino acid residue Gly at position 130 was mutated to Leu or Phe; The amino acid residue at position 171, Ile, was mutated to Lys.

3. The mutant protein according to claim 1 or 2, characterized in that The mutant protein is any of the following: 1) A protein consisting of the amino acid sequence shown in SEQ ID NO.4, SEQ ID NO.6 or SEQ ID NO.8 in the sequence listing; 2) A protein derived from 1) having the activity of hydrolyzing MHET, wherein one or more amino acid residues of the amino acid sequence of SEQ ID NO.4 or SEQ ID NO.6 or SEQ ID NO.8 in the sequence list 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. A nucleic acid encoding a mutant protein according to any one of claims 1 to 3.

5. The coding nucleic acid according to claim 4, characterized in that: The coding gene is a DNA molecule described in any one of the following 1)-4): 1) A DNA molecule whose coding region includes a nucleotide sequence as shown in SEQ ID NO.3, SEQ ID NO.5 or SEQ ID NO.7; 2) A DNA molecule having a nucleotide sequence as shown in SEQ ID NO.3, SEQ ID NO.5 or SEQ ID NO.7; 3) A DNA molecule that hybridizes with the nucleotide sequence described in 1) or 2) under stringent conditions and encodes the mutant protein 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 mutant protein according to any one of claims 1-3.

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 mutant protein according to any one of claims 1 to 3 in preparing a protein having at least one of the following functions 1) to 4); 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 at least one of the following functions 1) to 4); Or use of the mutant protein and PET degrading enzyme described in any one of claims 1 to 3 in the preparation of a product having at least one of the following functions 1) to 4); 1) Hydrolysis of MHET; 2) Degradation of PET; 3) Increase TPA production; 4) Improve PET conversion rate.

8. A product comprising the mutant protein according to any one of claims 1 to 3 and a PET degrading enzyme.

9. A method for degrading PET, comprising using the product of claim 8 to act on PET to achieve degradation.

10. A method for hydrolyzing MHET, comprising using the mutant protein according to any one of claims 1 to 3 or the product according to claim 8 to act on MHET to achieve hydrolysis.

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