2-hydroxyethylterephthalate diester hydrolase mutants and encoding genes, recombinant vectors, recombinant strains, enzyme preparations and uses

Abstract

The application relates to the field of genetic engineering, and discloses a 2-hydroxyethyl terephthalate diester hydrolase mutant, a coding gene, a recombination carrier, a recombination strain, an enzyme preparation and application. The 2-hydroxyethyl terephthalate diester hydrolase mutant is obtained by mutating at least one of positions 109, 110, 138, 225, 276, 287, 312, 313, 333, 335, 351, 356, 387, 394, 395, 401 and 413 of an enzyme with the amino acid sequence shown in SEQ ID NO:1. The 2-hydroxyethyl terephthalate diester hydrolase mutant has excellent thermal stability, and has better catalytic efficiency at high temperature.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering, specifically to a 2-hydroxyethyl terephthalate diester hydrolase mutant and its encoding gene, a recombinant vector, a recombinant strain, an enzyme preparation, and their applications in the degradation of plastics and intermediates thereof. Background Technology

[0002] Since their invention, synthetic plastics have been widely used in many aspects of human life due to their plasticity and stability. Currently, synthetic plastics mainly include two categories: polyesters (e.g., polyethylene terephthalate PET, polyurethane PUR) and polyolefins (e.g., polyethylene PE, polypropylene PP, polystyrene PS, polyvinyl chloride PVC). Because synthetic plastics are high-molecular-weight compounds formed from monomers through addition or condensation polymerization, they possess characteristics such as high crystallinity, strong hydrophobicity, and stable physicochemical properties, making them highly resistant to degradation. Waste plastics decompose slowly in the natural environment, leading to the accumulation of large amounts of plastic waste in land and oceans, causing global "white pollution."

[0003] As the world's fourth largest polymer, the degradation of polyethylene terephthalate (PET) and its intermediates has always attracted much attention. Current research on PET degradation mainly focuses on the efficient depolymerization of PET, while research on PET intermediates such as bis-2-hydroxyethyl terephthalate (BHET) and mono-2-hydroxyethyl terephthalate (MHET) is limited. Compared with traditional physicochemical methods, enzymatic degradation of BHET and MHET has many advantages, including fewer reaction steps, less waste generation, environmental friendliness, lower production costs, fewer impurities, easier separation, and superior product quality. Therefore, enzymatic degradation of petroleum-based plastics is one of the trends in plastic degradation development.

[0004] 2-Hydroxyethyl terephthalate hydrolase (BsEst, EC3.1.1.1) from Bacillus subtilis PET-86, also known as carboxylesterase, is an α / β hydrolase. This enzyme exhibits excellent catalytic esterification / deesterification properties, and in the enzymatic hydrolysis of BHET, the hydrolysis reaction mainly proceeds under kinetic control. The hydrolysis system primarily involves two reactions: (1) BHET loses EG and hydrolyzes to MHET; (2) MHET further loses EG and hydrolyzes to TPA. The final conversion rate depends on the equilibrium between these two reactions.

[0005] Research on biocatalysis is increasing both domestically and internationally. One key issue is that the enzymes used often fail to meet the demands of actual industrial production. For example, while the existing 2-hydroxyethyl terephthalate diester hydrolase can hydrolyze BHET and MHET, its efficiency in hydrolyzing to the terminal product TPA is low under high-temperature conditions. Therefore, it is necessary to modify this enzyme to improve its thermal stability and catalytic efficiency under high-temperature conditions. Summary of the Invention

[0006] The purpose of this invention is to overcome the problems of low thermal stability and low catalytic efficiency at high temperatures of 2-hydroxyethyl terephthalate diester hydrolase in the prior art, and to provide a 2-hydroxyethyl terephthalate diester hydrolase mutant, its encoding gene, recombinant vector, recombinant strain and enzyme preparation, and their applications. This 2-hydroxyethyl terephthalate diester hydrolase mutant has excellent thermal stability and better catalytic efficiency at high temperatures.

[0007] To achieve the above objective, the first aspect of the present invention provides a 2-hydroxyethyl terephthalate diester hydrolase mutant, wherein the 2-hydroxyethyl terephthalate diester hydrolase mutant is formed by mutating at least one of the following positions: 109, 110, 138, 225, 276, 287, 312, 313, 333, 335, 351, 356, 387, 394, 395, 401, and 413 of the enzyme having the amino acid sequence shown in SEQ ID NO: 1.

[0008] A second aspect of the present invention provides a gene encoding a 2-hydroxyethyl terephthalate diester hydrolase mutant, the gene having a nucleotide sequence encoding the 2-hydroxyethyl terephthalate diester hydrolase mutant as described above.

[0009] A third aspect of the present invention provides a recombinant vector containing the gene as described above.

[0010] A fourth aspect of the present invention provides a recombinant strain containing the gene as described above or the recombinant vector as described above.

[0011] The fifth aspect of the present invention provides a method for preparing a 2-hydroxyethyl terephthalate diester hydrolase mutant, the method comprising: inoculating the recombinant strain as described above into a fermentation medium for fermentation.

[0012] A sixth aspect of the present invention provides an enzyme preparation comprising a 2-hydroxyethyl terephthalate diester hydrolase mutant prepared as described above.

[0013] The seventh aspect of the present invention provides the use of at least one of the following in the degradation of plastics and intermediates thereof: the 2-hydroxyethyl terephthalate diester hydrolase mutant as described above, the gene as described above, the recombinant vector as described above, the recombinant strain as described above, the 2-hydroxyethyl terephthalate diester hydrolase mutant prepared by the method as described above, and the enzyme preparation as described above.

[0014] Through the above technical solution, this invention utilizes deep learning-based rational design technology to mutate wild-type 2-hydroxyethyl terephthalate (BsEst) hydrolase derived from Bacillus subtilis PET-86, providing a variety of 2-hydroxyethyl terephthalate hydrolase mutants with high thermal stability and significantly improved catalytic efficiency for the degradation of PET plastics and their intermediates. The 2-hydroxyethyl terephthalate hydrolase mutants provided by this invention have broad prospects for industrial applications.

[0015] Furthermore, the mutant BsEst Y109R is the optimal single-point mutant. Under high-temperature conditions, compared with the wild-type BsEst, the mutant BsEst Y109R has a significantly improved degradation efficiency for 2-hydroxyethyl terephthalate. Attached Figure Description

[0016] Figure 1 SDS-PAGE electrophoresis analysis of BsEst mutant Y109R and wild-type BsEst in Example 4, M is protein marker, 1 is wild-type BsEst, and 2 is BsEst mutant Y109R.

[0017] Figure 2 This is a schematic diagram illustrating the principle of 2-hydroxyethyl terephthalate diester hydrolase hydrolyzing BHET in Example 4. Detailed Implementation

[0018] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0019] The first aspect of the present invention provides a 2-hydroxyethyl terephthalate diester hydrolase mutant, wherein the 2-hydroxyethyl terephthalate diester hydrolase mutant is formed by mutating at least one of the following positions: 109, 110, 138, 225, 276, 287, 312, 313, 333, 335, 351, 356, 387, 394, 395, 401, and 413 of the enzyme having the amino acid sequence shown in SEQ ID NO: 1.

[0020] In the research process, the inventors of this invention used AI deep learning-based enzyme engineering modification technology to mutate wild-type 2-hydroxyethyl terephthalate diester hydrolase (referred to as wild-type BsEst, amino acid sequence as shown in SEQ ID NO: 1, nucleotide sequence as shown in SEQ ID NO: 2) derived from Bacillus subtilis PET-86. Specifically, through a first round of machine learning to predict the stability of the amino acid microenvironment, 20 mutable amino acid residue sites were obtained. A second round of Gaussian process model prediction yielded 12 mutable amino acid residue sites. A third round of meta-learning model prediction yielded 12 more mutable amino acid residue sites. After excluding identical amino acid residue sites, a total of 32 mutable amino acid residue sites were obtained. Further deep learning was used to provide... Forty potential single mutants and 20 potential combined mutants were identified. The efficiency of each BsEst mutant in catalyzing the hydrolysis of 2-hydroxyethyl terephthalate was determined, and 17 mutable amino acid residue sites were screened out: positions 109, 110, 138, 225, 276, 287, 312, 313, 333, 335, 351, 356, 387, 394, 395, 401, and 413.

[0021] According to the present invention, preferably, the 2-hydroxyethyl terephthalate diester hydrolase mutant is an enzyme having the amino acid sequence shown in SEQ ID NO: 1 subjected to at least one of the mutations described in (1)-(17):

[0022] (1) The tyrosine Y at position 109 is mutated to glutamine Q, arginine R or alanine A, and is represented by Y109Q, Y109R and Y109A respectively.

[0023] (2) The leucine L at position 110 is mutated to alanine A, denoted as L100A;

[0024] (3) The leucine L at position 138 is mutated to alanine A, denoted as L138A;

[0025] (4) The glutamine Q at position 225 is mutated to arginine R or threonine T, respectively, and is represented by Q225R and Q225T.

[0026] (5) The glutamine Q at position 276 is mutated to tyrosine Y, glycine G or alanine A, and is represented by Q276Y, Q276G or Q276A respectively.

[0027] (6) The glutamic acid at position 287 is mutated from E to proline P, denoted as E287P;

[0028] (7) Tyrosine Y at position 312 is mutated to glutamine Q, arginine R or alanine A, and is represented by Y312Q, Y312R and Y312A respectively.

[0029] (8) The leucine L at position 313 is mutated to valine V, denoted as L313V;

[0030] (9) Tyrosine Y at position 333 is mutated to lysine K, denoted as Y333K;

[0031] (10) The leucine L at position 335 is mutated to glycine G, denoted as L335G;

[0032] (11) The leucine L at position 351 is mutated to alanine A or aspartic acid D, respectively denoted as L351A and L351D.

[0033] (12) Histidine H at position 356 is mutated to alanine A, denoted as H356A;

[0034] (13) The tryptophan W at position 387 is mutated to valine V, denoted as W387V;

[0035] (14) Tyrosine Y at position 394 is mutated to threonine T or proline P, denoted as Y394T and Y394P respectively.

[0036] (15) The asparagine N at position 395 is mutated to phenylalanine F, denoted as N395F;

[0037] (16) The leucine L at position 401 is mutated to isoleucine I, denoted as L401I;

[0038] (17) The leucine L at position 413 is mutated to glycine G, denoted as L413G.

[0039] The inventors have discovered that, under this preferred embodiment, it is beneficial to further improve the thermal stability and catalytic efficiency of the 2-hydroxyethyl terephthalate diester hydrolase mutant at high temperatures.

[0040] According to the present invention, preferably, the 2-hydroxyethyl terephthalate diester hydrolase mutant is mutant BsEstL313V, L110A, L401I, Q276Y, Y109Q, Y394T, L413G, L138A, L351A, Y333K, L351D, Y312A, Q276A, Q225R, N395F, Y109R; more preferably, the 2-hydroxyethyl terephthalate diester hydrolase mutant is an enzyme having the amino acid sequence shown in SEQ ID NO: 1 mutated by at least one of (1), (5), and (11). More preferably, for example, the 2-hydroxyethyl terephthalate diester hydrolase mutant is mutant BsEst Y109R, Q276A, or L351D, more preferably mutant BsEst Y109R. The inventors have discovered that, under this preferred embodiment, it is advantageous to further improve the thermal stability and catalytic efficiency at high temperatures of the 2-hydroxyethyl terephthalate diester hydrolase mutant.

[0041] According to the present invention, preferably, the 2-hydroxyethyl terephthalate diester hydrolase mutant is a combination mutation of any one of the following: (9) / (14) combination mutation, (12) / (14) combination mutation, (10) / (12) combination mutation, (6) / (13) combination mutation, and (6) / (15) combination mutation of an enzyme having the amino acid sequence shown in SEQ ID NO: 1; for example, the 2-hydroxyethyl terephthalate diester hydrolase mutant is mutant BsEst Y333K / Y394P, H356A / Y394P, L335G / H356A, E287P / W387V, or E287P / N395F. More preferably, the 2-hydroxyethyl terephthalate diester hydrolase mutant is a combination mutation of any one of the following: (12) / (14) combination mutation, (10) / (12) combination mutation, and (9) / (14) combination mutation, having the amino acid sequence shown in SEQ ID NO: 1. For example, the 2-hydroxyethyl terephthalate diester hydrolase mutant is mutant BsEst H356A / Y394P, L335G / H356A, or Y333K / Y394P. The inventors have found that, under this preferred embodiment, it is beneficial to further improve the thermal stability and catalytic efficiency of the 2-hydroxyethyl terephthalate diester hydrolase mutant at high temperatures.

[0042] The aforementioned 2-hydroxyethyl terephthalate (THT) hydrolase mutant can be obtained artificially, or its encoding gene can be synthesized first and then expressed biologically. Exemplarily, the 2-hydroxyethyl terephthalate (THT) hydrolase mutant provided in this invention uses site-directed saturation mutagenesis to mutate the encoding gene of the amino acid sequence shown in SEQ ID NO.1. After ligation into an expression vector and transformation into host *E. coli*, the mutant protein is obtained after induction at low temperature with 0.1 mM IPTG for approximately 24 hours. Using BHET as a substrate, a hydrolytic catalytic reaction is performed. Subsequently, high-performance liquid chromatography (HPLC) is used to detect the content of the substrate BHET and the products MHET and TPA, yielding the 2-hydroxyethyl terephthalate (THT) hydrolase mutant. This mutant exhibits significantly improved enzymatic catalytic activity and degradation efficiency for polyethylene terephthalate (PET) plastic derivatives, such as 2-hydroxyethyl terephthalate (BHET) and monohydroxyethyl terephthalate (MHET).

[0043] A second aspect of the present invention provides a gene encoding a 2-hydroxyethyl terephthalate diester hydrolase mutant, the gene having a nucleotide sequence encoding the 2-hydroxyethyl terephthalate diester hydrolase mutant as described above.

[0044] It is well known in the art that of the 20 different amino acids that make up proteins, except for Met (ATG) or Trp (TGG), which are encoded by a single codon, the other 18 amino acids are encoded by 2-6 codons (Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory Press, New York, USA, 2nd edition, 1989, see Appendix D on page 950). That is, due to the degeneracy of the genetic codon, most amino acids are determined by more than one codon. The substitution of the third nucleotide in a triplet codon usually does not change the amino acid composition; therefore, genes encoding the same protein can have different nucleotide sequences.

[0045] For example, the amino acid sequence of the mutant BsEst Y109R is shown in SEQ ID NO:3, and its nucleotide sequence is shown in SEQ ID NO:4.

[0046] The nucleotide sequences provided by this invention can typically be obtained using polymerase chain reaction (PCR) amplification, recombination, or artificial synthesis. Once the relevant nucleotide sequence is obtained, the relevant amino acid sequence can be obtained in large quantities using recombination. The obtained nucleotide sequence is usually cloned into a vector, then transformed into genetically engineered bacteria, and then the relevant nucleotide sequence is isolated from the proliferated host cells using conventional methods.

[0047] In addition, known methods of artificial chemical synthesis can be used to synthesize the relevant nucleotide sequences.

[0048] A third aspect of the present invention provides a recombinant vector containing the gene as described above.

[0049] In this invention, the "vector" used in the recombinant vector can be any vector known in the art, such as commercially available plasmids, granules, bacteriophages, and retroviruses. The preferred expression vector in this invention is the pET-22b plasmid. Exemplarily, the expression vector and the target fragment are ligated using seamless cloning to obtain the recombinant vector.

[0050] A fourth aspect of the present invention provides a recombinant strain containing the gene as described above or the recombinant vector as described above.

[0051] In this invention, the recombinant vector can be transformed, transduced, or transfected into host cells (strains) using conventional methods in the art, such as chemical transformation by calcium chloride method or high-voltage electroporation transformation. The host cell can be a prokaryotic cell or a eukaryotic cell, preferably Escherichia coli and / or Bacillus subtilis, and more preferably, the host cell is Escherichia coli, such as Escherichia coli BL21(DE3).

[0052] The fifth aspect of the present invention provides a method for preparing a 2-hydroxyethyl terephthalate diester hydrolase mutant, the method comprising: inoculating the recombinant strain as described above into a fermentation medium for fermentation.

[0053] In this invention, there are no particular restrictions on the fermentation conditions of the recombinant strain, as long as the fermentation process can enable the recombinant strain to proliferate in large quantities. Preferably, the fermentation process includes: firstly, seed culture of the recombinant strain to obtain a seed solution; then, inoculating the seed solution into a fermentation medium containing kanamycin and culturing it until the cell concentration OD600 is 0.5-0.7; then, adding isopropyl-β-D-thiogalactoside (IPTG) inducer at a final concentration of 0.1-0.2 mM; and culturing at a temperature of 10-20℃ for 20-30 h to obtain the fermentation broth.

[0054] According to the present invention, in a preferred embodiment, the kanamycin content in the fermentation medium is 40-60 mg / L.

[0055] More preferably, the method for preparing the seed culture includes: picking a single colony of the recombinant strain and inoculating it into a seed culture medium containing kanamycin for seed culture to obtain the seed culture. In this invention, the single colony of the recombinant strain can be selected from the freshly prepared recombinant strain or the recombinant strain that has been cryopreserved at low temperature (e.g., a recombinant strain synthesizing lipids that has been frozen in a glycerol cryovial at -80°C).

[0056] This invention does not impose any particular limitations on the seed culture method, as long as the method can activate and proliferate the recombinant strain. Preferably, the kanamycin content in the seed culture medium is 40-60 mg / L. The parameters used in the seed culture, such as temperature, pH, rotation speed, and time, can be conventional settings within the art. Preferably, the seed culture conditions include: a temperature of 30-45℃, a rotation speed of 150-250 rpm, and a time of 8-12 h.

[0057] In this invention, there are no particular limitations on the seed culture medium and fermentation culture medium, and they can be any culture media commonly used in the art. Preferably, the seed culture medium and fermentation culture medium are LB liquid medium (tryptone 8-12 g / L, yeast extract 4-6 g / L, NaCl 8-12 g / L).

[0058] A sixth aspect of the present invention provides an enzyme preparation comprising a 2-hydroxyethyl terephthalate diester hydrolase mutant prepared as described above.

[0059] In this invention, the 2-hydroxyethyl terephthalate diester hydrolase mutant can be prepared into a corresponding enzyme preparation. Specifically, the enzyme preparation can exist in solid, semi-solid or liquid form. The enzyme preparation can contain excipients or additives for preparing the enzyme preparation, which can be selected by those skilled in the art as needed, and will not be elaborated here.

[0060] In this invention, the fermentation broth obtained by the above preparation method can be used directly as an enzyme preparation, or the fermentation broth can be separated and purified to obtain a 2-hydroxyethyl terephthalate diester hydrolase mutant as an enzyme preparation. The separation and purification can be performed using conventional enzyme separation methods in the art. That is, the 2-hydroxyethyl terephthalate diester hydrolase mutant can be used in the form of whole cells of a recombinant strain, or in the form of unpurified crude enzyme or purified enzyme isolated from recombinant strain cells. If necessary, the 2-hydroxyethyl terephthalate diester hydrolase mutant of this invention can also be used to prepare immobilized enzymes or immobilized cells using immobilization techniques known in the art.

[0061] The 2-hydroxyethyl terephthalate diester hydrolase mutant provided by this invention exhibits high thermal stability and significantly improved catalytic efficiency for the degradation of PET plastics and their intermediates. A seventh aspect of this invention provides the use of at least one of the following in the degradation of plastics and their intermediates: the 2-hydroxyethyl terephthalate diester hydrolase mutant as described above, the gene as described above, the recombinant vector as described above, the recombinant strain as described above, the 2-hydroxyethyl terephthalate diester hydrolase mutant prepared by the method as described above, and the enzyme preparation as described above.

[0062] According to the present invention, preferably, the plastic and its intermediates are selected from at least one of polyethylene terephthalate, 2-hydroxyethyl terephthalate and monohydroxyethyl terephthalate.

[0063] In this invention, the method for degrading plastics and their intermediates may include: contacting at least one of the following: the 2-hydroxyethyl terephthalate diester hydrolase mutant, the gene, the recombinant vector, the recombinant strain, the 2-hydroxyethyl terephthalate diester hydrolase mutant prepared by the method described above, and the enzyme preparation described above, with at least one of polyethylene terephthalate, 2-hydroxyethyl terephthalate, and monohydroxyethyl terephthalate; wherein the contact conditions include: a temperature of 30-60°C, a rotation speed of 800-1000 rpm, and a time of 4-8 hours.

[0064] The 2-hydroxyethyl terephthalate diester hydrolase mutant has the ability to hydrolyze PET plastics and their intermediates. The BsEst mutant has great potential in the hydrolysis of PET plastics and their intermediates and has broad application prospects in biochemical engineering.

[0065] The present invention will be described in detail below through embodiments.

[0066] In the following examples, E. coli BL21(DE3) was purchased from Takara Bio Engineering (Dalian) Co., Ltd., product number 9126; pET22b was purchased from Sangon Biotech (Shanghai) Co., Ltd., product number B540183; the seamless cloning kit was purchased from Beyotime Biotechnology Co., Ltd.; MHET standards were purchased from Sigma-Aldrich; BHET and TPA standards were purchased from Maclean's Reagents Co., Ltd.; and all other reagents and raw materials were commercially available products.

[0067] LB liquid medium: tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, pH adjusted to 6.5, autoclaved for 21 min, ready for use;

[0068] LB agar plate medium: tryptone 10g / L, yeast extract 5g / L, NaCl 10g / L, agar 12g / L, pH adjusted to 7.0, autoclaved for 21 min, ready for use;

[0069] Detection of BHET, TPA and MHET: First, perform HPLC analysis with standard solutions of gradient concentrations to obtain standard curves of chromatographic peak area versus standard concentration; then, stop the reaction by taking 200 μL of sample supernatant into 200 μL of methanol, filter through a 0.22 μM filter membrane, and perform HPLC analysis.

[0070] The HPLC test conditions were as follows: column: C18 reversed-phase column; column temperature: 30℃; detection wavelength: 240nm; flow rate: 0.7mL·min -1 Injection volume: 10 μL; Mobile phase: 40% methanol (chromatographic grade), 60% water (1 / 1000 acetic acid).

[0071] Example 1

[0072] This example illustrates the site-directed mutagenesis of 2-hydroxyethyl terephthalate diester hydrolase.

[0073] The wild-type 2-hydroxyethyl terephthalate diester hydrolase (wild-type BsEst, amino acid sequence as shown in SEQ ID NO: 1, nucleotide sequence as shown in SEQ ID NO: 2) derived from Bacillus subtilis PET-86 was mutated using AI deep learning-based enzyme engineering technology. Specifically, the stability of the amino acid microenvironment was predicted in the first round of machine learning to obtain 20 mutable amino acid residue sites. A Gaussian process model was used in the second round to predict mutable amino acids, resulting in 12 mutable amino acid residue sites. A meta-learning model was used in the second round to predict mutable amino acids, again resulting in 12 mutable amino acid residue sites. After excluding identical amino acid residue sites, a total of 32 mutable amino acid residue sites were obtained. Further deep learning provided 42 potential single mutants and 20 potential combined mutants. The mutation sites of the 42 potential single mutants are as follows:

[0074] The threonine (T) at position 306 is mutated to glutamine (Q);

[0075] The threonine (T) at position 359 is mutated to glutamine (Q);

[0076] The leucine (L) at position 313 is mutated to valine (V);

[0077] The alanine (A) at position 397 is mutated to isoleucine (I);

[0078] The 401st leucine (L) is mutated to isoleucine (I);

[0079] The leucine (L) at position 361 is mutated to glycine (G);

[0080] The leucine (L) at position 403 is mutated to glycine (G);

[0081] The 110th leucine (L) is mutated to alanine (A);

[0082] The alanine (A) at position 197 is mutated to isoleucine (I);

[0083] The leucine (L) at position 413 is mutated to glycine (G);

[0084] The alanine (A) at position 112 is mutated to isoleucine (I);

[0085] The valine (V) at position 185 is mutated to glycine (G);

[0086] The leucine (L) at position 138 is mutated to alanine (A);

[0087] The 198th leucine (L) is mutated to alanine (A);

[0088] The tyrosine (Y) at position 333 is mutated to lysine (K);

[0089] The leucine (L) at position 335 is mutated to glycine (G);

[0090] The phenylalanine (F) at position 275 is mutated to alanine (A);

[0091] The histidine (H) at position 356 is mutated to alanine (A);

[0092] The leucine (L) at position 465 is mutated to valine (V);

[0093] The 117th leucine (L) is mutated to cysteine ​​(C);

[0094] The glutamine (Q) at position 272 is mutated to proline (P);

[0095] The 387th tryptophan (W) is mutated to valine (V);

[0096] The asparagine (N) at position 395 is mutated to phenylalanine (F);

[0097] The leucine (L) at position 331 is mutated to serine (S);

[0098] The glutamic acid at position 287 (E) is mutated to proline (P);

[0099] The tyrosine (Y) at position 312 is mutated to glutamine (Q), arginine (R), or alanine (A);

[0100] The tyrosine (Y) at position 109 is mutated to glutamine (Q), arginine (R), or alanine (A);

[0101] The glutamine (Q) at position 276 is mutated to tyrosine (Y), glycine (G), or alanine (A);

[0102] The glutamine (Q) at position 354 is mutated to either tyrosine (Y) or arginine (R);

[0103] The tyrosine (Y) at position 394 is mutated to either threonine (T) or proline (P);

[0104] The leucine (L) at position 351 is mutated to either alanine (A) or aspartic acid (D);

[0105] The glutamine (Q) at position 225 is mutated to arginine (R) or threonine (T);

[0106] The mutation sites of the 20 potential combined mutants are: the mutation of tyrosine (Y) at position 333 to lysine (K) and the mutation of tyrosine (Y) at position 394 to proline (P) are combined to obtain the mutant Y333K / Y394P;

[0107] The mutant L335G / Y394P was obtained by combining the mutation of leucine (L) at position 335 to glycine (G) and tyrosine (Y) at position 394 to proline (P).

[0108] The mutant L117C / Y394P is obtained by combining the mutation of leucine (L) at position 117 to cysteine ​​(C) and tyrosine (Y) at position 394 to proline (P).

[0109] The mutant H356A / Y394P is obtained by combining histidine (H) at position 356 (A) with alanine (A) and tyrosine (Y) at position 394 (P) with proline (P).

[0110] The mutant Y109A / Y394P is obtained by combining the mutation of tyrosine (Y) at position 109 (Y) to alanine (A) and the mutation of tyrosine (Y) at position 394 (Y) to proline (P).

[0111] The mutant F275A / Y333K is obtained by combining the mutation of phenylalanine (F) at position 275 to alanine (A) and the mutation of tyrosine (Y) at position 333 to lysine (K).

[0112] The mutant L335G / Y333K is obtained by combining the mutation of leucine (L) at position 335 to glycine (G) and tyrosine (Y) at position 333 to lysine (K).

[0113] The mutant L335G / L331S is created by mutating leucine (L) at position 335 to glycine (G) and leucine (L) at position 331 to serine (S).

[0114] The mutant Y333K / L351D is obtained by combining the mutation of tyrosine (Y) at position 333 (K) to lysine (K) and the mutation of leucine (L) at position 351 (D) to aspartic acid (D).

[0115] The mutant H356A / L335G is obtained by combining the mutation of leucine (L) at position 335 to glycine (G) and histidine (H) at position 356 to alanine (A).

[0116] The mutant Q276G / W387V is obtained by combining the mutation of glutamine (Q) at position 276 to glycine (G) and tryptophan (W) at position 387 to valine (V).

[0117] The mutant Q225R / Q276A is obtained by combining the mutation of glutamine (Q) at position 225 to arginine (R) and the mutation of glutamine (Q) at position 276 to alanine (A).

[0118] The mutant E287P / N395F is obtained by combining the mutation of glutamine (Q) at position 276 to glycine (G) and the mutation of asparagine (N) at position 395 to phenylalanine (F).

[0119] The mutant W387V / E287P is obtained by combining the mutation of tryptophan (W) at position 387 to valine (V) and the mutation of glutamic acid (E) at position 287 to proline (P).

[0120] The mutant E287P / N395F is obtained by combining the mutation of asparagine (N) at position 395 to phenylalanine (F) and the mutation of glutamic acid (E) at position 287 to proline (P).

[0121] The 110th leucine (L) was mutated to alanine (A) and the 413th leucine (L) was mutated to glycine (G), resulting in the mutant L110A / L413G.

[0122] The glutamine (Q) at position 276 was mutated to alanine (A) and the leucine (L) at position 413 was mutated to glycine (G), resulting in the mutant L413G / Q276A;

[0123] The mutant Y394T / Q272P was obtained by mutating tyrosine (Y) at position 394 (T) to threonine (T) and glutamine (Q) at position 272 (P) to proline (P).

[0124] The mutant L401I / W387V is obtained by combining the mutation of tryptophan (W) at position 387 to valine (V) and the mutation of leucine (L) at position 401 to isoleucine (I).

[0125] The mutant L110A / E287P is obtained by combining the mutation of leucine (L) at position 110 (A) to alanine (A) and glutamic acid (E) at position 287 (P) to proline (P).

[0126] Example 2

[0127] Blunt-end primer pairs (including primer R and primer F) were designed using Snap Gene software. Using the encoding gene of wild-type 2-hydroxyethyl terephthalate diester hydrolase of Bacillus subtilis PET-86 (amino acid sequence as shown in SEQ ID NO: 1, nucleotide sequence as shown in SEQ ID NO: 2) as a template, PCR site-directed mutagenesis was then performed.

[0128] The PCR reaction system is as follows: 25 μL PrimeSTAR Mix high-fidelity enzyme, 2 μL upstream primer (10 pmol / μL), 2 μL upstream primer (10 pmol / μL), 2 μL template, and 19 μL ddH2O;

[0129] PCR cycling process: Preheating: 95℃, 10 min; Denaturation: 95℃, 30 s; Annealing: 60℃, 30 s; Extension: 72℃, 4 min; 2-4 cycles, 25 times; Extension: 72℃, 10 min; Cool to 4℃ and store.

[0130] The PCR product was recovered and agarose gel electrophoresis was used to detect whether site-directed mutagenesis was completed. Then, Dpn I (1 μL) was added to 50 μL of the reaction mixture and kept at 37 °C for 3 h to digest the template. The mixture was then cooled to 4 °C and stored.

[0131] The pET-22b vector was mixed with the 2-hydroxyethyl terephthalate diester hydrolase mutant (BsEst mutant) obtained by the above mutation at a molar ratio of 3:1. Then, 10 μL of 2X Seamless Cloning Mix was added, and water was added to bring the volume to 20 μL. The mixture was reacted at 50 °C for 15 min. After the reaction, the reaction product was placed on ice for 5 min, and 5 μL was transformed into 100 μL of E. coli DH5α competent cells. Preliminary screening was performed with antibiotics, and DNA sequencing was performed to identify the successfully mutated gene, thus obtaining a recombinant vector containing the BsEst mutant.

[0132] The recombinant vectors containing the BsEst mutant were transformed into E. coli BL21(DE3) competent cells. Positive recombinants were screened on ampicillin-containing resistant plates, and single clones were selected. The positive clones were verified by colony PCR and DNA sequencing to obtain the recombinant E. coli with each BsEst mutant.

[0133] Example 3

[0134] This example illustrates the induction, expression, and purification of the 2-hydroxyethyl terephthalate diester hydrolase mutant.

[0135] a) Induced expression of the 2-hydroxyethylterephthalate diester hydrolase mutant

[0136] (1) The recombinant Escherichia coli of each BsEst mutant obtained in Example 2 were inoculated into LB liquid medium containing ampicillin and cultured with shaking at 37°C and 200 rpm for 12 h to obtain seed liquid;

[0137] (2) Inoculate the seed culture into a 250mL Erlenmeyer flask containing 100mL LB liquid medium at an inoculation rate of 2% (v / v), and incubate at 37℃ and 180rpm on a shaker. When the OD of the culture medium... 600 When the concentration reaches 0.6, IPTG with a final concentration of 0.1 mM is added as an inducer, and the fermentation broth is obtained by induction at 16℃ for 24 h.

[0138] (3) Centrifuge the fermentation broth, collect the cells, and wash them twice with physiological saline to obtain resting cells; suspend the resting cells in 20 mL of phosphate buffer at pH 8.0, sonicate them in an ice bath, centrifuge and collect the supernatant, which is the crude enzyme solution of BsEst mutant.

[0139] b. Purification of the 2-hydroxyethylterephthalate diester hydrolase mutant

[0140] Protein purification was performed using a Ni+ column: the collected crude BsEst mutant enzyme solution was filtered and added to a Nisepharose 6Fast Flow (FF) packing material. Impurities were eluted with 10 column volumes of buffer A (10 mM Tris-HCl pH 7.0, 250 mM NaCl, 20 mM imidazole), and the target protein was eluted with buffer B (10 mM Tris-HCl pH 7.0, 250 mM NaCl, 250 mM imidazole) to obtain the BsEst mutant enzyme solution.

[0141] The collected target proteins were subjected to SDS-PAGE protein electrophoresis. The protein electrophoresis results for the BsEst mutant Y109R (amino acid sequence as shown in SEQ ID NO: 3, nucleotide sequence as shown in SEQ ID NO: 4) are as follows: Figure 1 As shown.

[0142] Example 4

[0143] This experiment demonstrates the application of the BsEst mutant in the hydrolysis of BHET.

[0144] The principle of 2-hydroxyethyl terephthalate diester hydrolase hydrolyzing BHET is as follows: Figure 2As shown, it can both break one side of the ester bond of BHET to remove EG and generate the intermediate product MHET; and subsequently break one side of the ester bond of MHET to remove EG and generate the terminal product TPA.

[0145] The reaction system for hydrolyzing BHET was as follows: using PBS buffer at pH 7.5 as the reaction medium, the total reaction volume was 2 mL, containing 5 mM BHET. The BsEst mutant enzyme solution obtained in Example 3 was added (each BsEst mutant was added independently at 20 μg / mL). The reaction was carried out at 30 °C and 900 rpm for 6 h. Samples were taken for analysis after 6 h of reaction. The samples were diluted 10 times with a solvent (water:methanol:acetic acid mixed at a volume ratio of 60:35:5), and the content of the terminal product TPA in the samples was detected by HPLC.

[0146] The efficiency of each BsEst mutant in catalyzing the hydrolysis of BHET was determined, and 16 single mutants (Y109Q, Y109R, L110A, L138A, Q225R, Q276A, Q276Y, Y312A, L313V, Y333K, L351A, L351D, Y394T, N395F, L401I, L413G) and 5 combined mutants (Y333K / Y394P, H356A / Y394P, L335G / H356A, E287P / W387V, E287P / N395F) were screened. Compared with wild-type BsEst, these mutants showed increased enzyme activity and improved efficiency in the hydrolysis of BHET (the TPA content after 6 hours of BHET hydrolysis is shown in Table 1). The aforementioned BsEst mutants possess the ability to efficiently hydrolyze BHET, and have great potential in hydrolyzing PET plastics and their intermediates, with broad application prospects in biochemical engineering. Among them, the BsEst mutants are more preferably BsEst Y109R, Q276A, and L351D.

[0147] Comparative Example 1

[0148] (1) Mix pET-22b vector with wild-type BsEst at a molar ratio of 3:1, then add 10 μL of 2X Seamless Cloning Mix and add water to make up to 20 μL. React at 50℃ for 15 min. After the reaction, place the reaction product on ice for 5 min and take 5 μL to transform into 100 μL of E. coli DH5α competent cells. Perform preliminary screening with antibiotics and DNA sequencing to determine the successfully mutated gene to obtain a recombinant vector containing wild-type BsEst. Transform the recombinant vector containing wild-type BsEst into E. coli BL21(DE3) competent cells. Screen for positive recombinants on ampicillin-containing plates, select single clones, verify positive clones by colony PCR, and obtain recombinant E. coli containing wild-type BsEst by DNA sequencing.

[0149] (2) Recombinant wild-type BsEst Escherichia coli was inoculated into LB liquid medium containing ampicillin and cultured with shaking at 37℃ and 200 rpm for 12 h to obtain seed culture; the seed culture was inoculated into a 250 mL Erlenmeyer flask containing 100 mL of LB liquid medium at an inoculation rate of 2% (v / v) and cultured on a shaker at 37℃ and 180 rpm. 600 When the concentration reaches 0.6, IPTG with a final concentration of 0.1 mM is added as an inducer, and the fermentation broth is obtained by induction at 16℃ for 24 h.

[0150] (3) Centrifuge the fermentation broth, collect the cells, and wash them twice with physiological saline to obtain resting cells; suspend the resting cells in 20 mL of phosphate buffer at pH 8.0, sonicate them in an ice bath, centrifuge and collect the supernatant, which is the crude enzyme solution of wild-type BsEst.

[0151] The crude wild-type BsEst enzyme solution was filtered and added to a Ni sepharose 6Fast Flow (FF) packing material. Impurities were eluted with 10 column volumes of buffer A (10 mM Tris-HCl pH 7.0, 250 mM NaCl, 20 mM imidazole), and the target protein was eluted with buffer B (10 mM Tris-HCl pH 7.0, 250 mM NaCl, 250 mM imidazole) to obtain the wild-type BsEst enzyme solution.

[0152] The reaction system for hydrolyzing BHET with wild-type BsEst was as follows: PBS buffer at pH 7.5 was used as the reaction medium, with a total reaction volume of 2 mL containing 5 mM BHET. Wild-type BsEst enzyme solution was added to make the content of wild-type BsEst 20 μg / mL. The reaction was carried out at 60℃ and 900 rpm for 6 h. Samples were taken for analysis after 6 h of reaction. The samples were diluted 10 times with a solvent (water:methanol:acetic acid mixed in a volume ratio of 60:35:5). The content of the terminal product TPA after 6 h of reaction was detected by HPLC. The results are shown in Table 1.

[0153] Table 1

[0154] mutant TPA content (mM) at 6 hours Wild type BsEst 0.38 L313V 0.44 L110A 0.58 L401I 0.67 Q276Y 0.42 Y109Q 0.46 Y394T 0.69 L413G 0.52 L138A 0.57 L351A 0.45 Y333K 0.65 L351D 1.48 Y312A 0.53 Q276A 1.16 Q225R 0.86 N395F 0.94 Y109R 2.10 Y333K / Y394P 0.70 H356A / Y394P 0.75 L335G / H356A 0.75 E287P / W387V 0.39 E287P / N395F 0.6

[0155] The nucleotide sequences of the primer pairs used in the PCR reaction of the BsEst mutant (single mutant) with enhanced enzyme activity in Table 1 are shown in Table 2.

[0156] Table 2

[0157]

[0158]

[0159] Example 5

[0160] This experiment illustrates the effect of temperature on the enzymatic hydrolysis of BHET, an intermediate in PET plastics.

[0161] The reaction system was as follows: PBS buffer at pH 7.5 was used as the reaction medium, and the total reaction volume was 2 mL, containing 5 mM BHET. The BsEst mutant Y109R enzyme solution obtained in Example 3 was added to make the BsEst mutant content 20 μg / mL. The reaction was carried out at 30℃, 40℃, 50℃, and 60℃ and 900 rpm for 6 h. Samples were taken for analysis after 6 h of reaction. The samples were diluted 10 times with solvent (water:methanol:acetic acid mixed in a volume ratio of 60:35:5). The content of the terminal product TPA after 6 h of reaction was detected by HPLC. The results are shown in Table 3.

[0162] Table 3

[0163] temperature TPA content at 6h 30℃ 4.32mM 40℃ 3.56mM 50℃ 2.88mM 60℃ 2.10mM

[0164] As can be seen from the data in Table 3, the BsEst mutant provided in this application can stably carry out the catalytic hydrolysis of BHET under high temperature conditions (50℃-60℃), and has high temperature resistance, thus having broad prospects for industrial application.

[0165] Example 6

[0166] This experiment demonstrates the application of the BsEst mutant in the hydrolysis of MHET.

[0167] The reaction system was as follows: PBS buffer at pH 7.5 was used as the reaction medium, with a total reaction volume of 2 mL containing 5 mM MMHET. The BsEst mutant Y109R enzyme solution obtained in Example 3 was added to make the BsEst mutant content 20 μg / mL. The reaction was carried out at 50℃ and 900 rpm for 6 h. Samples were taken for analysis at 1 h, 3 h and 6 h. The samples were diluted 10 times with a solvent (water:methanol:acetic acid mixed in a volume ratio of 60:35:5). The content of the terminal product TPA at different time points was detected by HPLC, and the results were 0.25 mM, 1.56 mM and 2.86 mM, respectively.

[0168] Comparative Example 2

[0169] Using PBS buffer at pH 7.5 as the reaction medium, the total reaction volume was 2 mL, containing 5 mM MHET. Wild-type BsEst enzyme solution obtained in Comparative Example 1 was added to make the wild-type BsEst content 20 μg / mL. The reaction was carried out at 50℃ and 900 rpm for 6 h. Samples were taken for analysis at 1 h, 3 h and 6 h. The samples were diluted 10 times with solvent (water:methanol:acetic acid in a volume ratio of 60:35:5), and the content of the terminal product TPA at different time points was detected by HPLC. The results were 0 mM, 0.98 mM and 1.25 mM, respectively.

[0170] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A 2-hydroxyethyl terephthalate diester hydrolase mutant, characterized in that, The 2-hydroxyethyl terephthalate diester hydrolase mutant is formed by the following mutation of the enzyme with the amino acid sequence shown in SEQ ID NO: 1: (1) Tyrosine Y at position 109 is mutated to glutamine Q; (2) Tyrosine Y at position 109 is mutated to arginine R; (3) Glutamine Q at position 276 is mutated to tyrosine Y; (4) Glutamine Q at position 276 is mutated to alanine A; (5) The leucine L at position 351 is mutated to alanine A; (6) The leucine L at position 351 is mutated to aspartic acid D; (7) Tyrosine Y at position 333 is mutated to lysine K and tyrosine Y at position 394 is mutated to proline P; (8) Histidine H at position 356 is mutated to alanine A, and tyrosine Y at position 394 is mutated to proline P; (9) The leucine L at position 335 is mutated to glycine G, and the histidine H at position 356 is mutated to alanine A; Alternatively, (10) the glutamic acid E at position 287 is mutated to proline P, and the asparagine N at position 395 is mutated to phenylalanine F.

2. A gene encoding a mutant of 2-hydroxyethyl terephthalate diester hydrolase, characterized in that, The gene is a nucleotide sequence encoding the 2-hydroxyethyl terephthalate diester hydrolase mutant as described in claim 1.

3. A recombinant vector, characterized in that, The recombinant vector contains the gene described in claim 2.

4. The recombinant vector according to claim 3, characterized in that, The expression vector for the recombinant vector is the pET-22b plasmid.

5. A recombinant bacterial strain, characterized in that, The recombinant strain contains the gene of claim 2 or the recombinant vector of claim 3.

6. The recombinant strain according to claim 5, characterized in that, The recombinant strain is either Escherichia coli or Bacillus subtilis.

7. The recombinant strain according to claim 6, characterized in that, The recombinant strain is Escherichia coli.

8. A method for preparing a 2-hydroxyethyl terephthalate diester hydrolase mutant, characterized in that, The preparation method includes: inoculating the recombinant strain according to any one of claims 5 to 7 into a fermentation medium for fermentation.

9. An enzyme preparation, characterized in that, The enzyme preparation comprises the 2-hydroxyethyl terephthalate diester hydrolase mutant prepared by the method of claim 8.

10. The use of at least one of the following in the degradation of 2-hydroxyethyl terephthalate (BHET): the 2-hydroxyethyl terephthalate (BHET) hydrolase mutant of claim 1, the gene of claim 2, the recombinant vector of claim 3 or 4, the recombinant strain of any one of claims 5 to 7, the 2-hydroxyethyl terephthalate (BHET) hydrolase mutant prepared by the method of claim 8, and the enzyme preparation of claim 9.

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