A dienophile hydrolase mutant and use thereof

Abstract

The application discloses a kind of dien-endo-lactone hydrolase mutant and application thereof.The dien-endo-lactone hydrolase mutant disclosed in the application is as follows A1), A2) or A3): A1) the protein with amino acid sequence of sequence 4;A2) the protein with amino acid sequence of sequence 4 except for the 162th amino acid in sequence table being substituted by one or more amino acid residues and / or being deleted and / or being added and having the same function;A3) the fusion protein obtained by connecting tag at N terminal or / and C terminal of A1) or A2).The original dien-endo-lactone hydrolase is mutated by using site-directed mutagenesis technology in the application, and the mutant thereof is obtained, the problem of lower activity of the original dien-endo-lactone hydrolase is changed, the activity of degrading BHET is effectively improved, and the degradation effect is improved.The dien-endo-lactone hydrolase mutant of the application greatly improves the degradation efficiency of BHET, and has good industrial application prospect.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering, specifically a dienolactone hydrolase mutant and its applications. Background Technology

[0002] Plastic is a great invention of humankind, and the use of plastic products is becoming increasingly widespread, bringing great convenience to people's daily lives. However, at the same time, plastic pollution is becoming increasingly serious, becoming a hot environmental issue of widespread concern in countries around the world.

[0003] Statistics show that global plastic production reached 367 million tons in 2020. Aside from some usable plastics, the rest became waste. Over the past 70 years, humans have discarded more than 7 billion tons of plastic waste. Unlike metal products, plastic products are not recyclable; most plastics are single-use, with only a small portion being recyclable. Therefore, with plastic production remaining high and increasing annually, the plastic pollution problem will only worsen and be difficult to alleviate unless a complete solution is found.

[0004] Traditional disposal methods involve incineration or landfill, but both cause environmental pollution. Burning plastic produces toxic and harmful gases, polluting the air. When plastic is buried in the soil, it takes hundreds of years to completely decompose naturally. During this process, the plastic alters the soil structure and composition, leading to nutrient loss. Furthermore, plastic exposed to the elements is ingested by birds, indirectly killing many organisms.

[0005] Since scientist Kohei Oda accidentally discovered *Sakai osaka*, a microorganism that feeds on polyethylene terephthalate (PET), scientists have successively discovered several hydrolytic enzymes with PET-degrading activity, but their degradation activity is very low. At-site evolution and the addition of metal ions to improve enzyme activity have also yielded unsatisfactory results. Diethyl terephthalate (BHET) is an intermediate product of PET hydrolysis, and the accumulation of reaction intermediates is a significant factor limiting the degradation efficiency of PET hydrolytic enzymes. BHET hydrolases can break down BHET into mono(2-hydroxyethyl) terephthalic acid (MHET), which further decomposes into terephthalic acid (TPA) and ethylene glycol (EG). How to efficiently degrade BHET and break down the degradation barrier has enormous commercial value. Summary of the Invention

[0006] One object of this invention is to provide a dienolactone hydrolase (Acfe) mutant, a protein with BHET hydrolytic activity. The obtained mutant effectively enhances the activity of Acfe in degrading BHET, thereby improving the degradation effect of Acfe.

[0007] This invention first provides a protein named Acfe-D162T, wherein Acfe-D162T is as follows (A1), (A2), or (A3):

[0008] A1) The amino acid sequence is that of the protein in sequence 4;

[0009] A2) Proteins that have the same function by substituting and / or deleting and / or adding one or more amino acid residues in the amino acid sequence outside position 162 of sequence 4 in the sequence listing.

[0010] A3) is a fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of A1) or A2).

[0011] To facilitate the purification of proteins in A1), tags as shown in the table below can be attached to the amino or carboxyl terminus of the protein, which consists of the amino acid sequence shown in Sequence 4 of the sequence listing.

[0012] Table: Sequence of Labels

[0013] Poly-Arg 5-6 (usually 5) RRRRR Poly-His 2-10 (usually 6) HHHHHH FLAG 8 DYKDDDDK Strep-tag II 8 WSHPQFEK c-myc 10 EQKLISEEDL

[0014] The Acfe-D162T protein in A2) above is a protein that shares 75% or more amino acid sequence identity with the protein shown in Sequence 4 and has the same function. The 75% or more identity refers to 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity.

[0015] The Acfe-D162T protein in A2) above can be synthesized artificially, or its encoding gene can be synthesized first and then expressed biologically.

[0016] The gene encoding the Acfe-D162T protein in A2) above can be obtained by deleting one or more amino acid residues from the codons in the DNA sequence shown in Sequence 3, and / or by performing a missense mutation on one or more base pairs, and / or by attaching the coding sequence of the tag shown in the table above to its 5′ end and / or 3′ end. The DNA molecule shown in Sequence 3 encodes the Acfe-D162T protein shown in Sequence 4.

[0017] Specifically, the protein described in A2) may be the protein shown in sequence 8.

[0018] The present invention also provides biomaterials related to Acfe-D162T, said biomaterials being any one of B1) to B4) below:

[0019] B1) Nucleic acid molecules encoding Acfe-D162T;

[0020] B2) An expression cassette containing the nucleic acid molecule described in B1);

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

[0022] B4) Recombinant microorganisms containing the nucleic acid molecules described in B1), or recombinant microorganisms containing the expression cassette described in B2), or recombinant microorganisms containing the recombinant vector described in B3).

[0023] In the above-mentioned biological materials, the nucleic acid molecule described in B1) may be as follows: b11), b12), b13), or b14):

[0024] b11) The coding sequence is a cDNA molecule or DNA molecule that is sequence 3 in the sequence listing;

[0025] b12) The DNA molecule shown in sequence 3 of the sequence listing;

[0026] b13) has 75% or more identity with the nucleotide sequence defined by b11) or b12) and encodes a cDNA molecule or DNA molecule that encodes Acfe-D162T;

[0027] b14) hybridizes under strict conditions to a nucleotide sequence defined by b11) or b12) or b13) and encodes a cDNA molecule or DNA molecule that encodes Acfe-D162T.

[0028] The nucleic acid molecule can be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecule can also be RNA, such as mRNA or hnRNA.

[0029] Those skilled in the art can readily mutate the nucleotide sequence encoding the Acfe-D162T protein of the present invention using known methods, such as directed evolution and point mutation. Artificially modified nucleotides that have 75% or higher identity with the nucleotide sequence of the Acfe-D162T protein of the present invention, as long as they encode and function the Acfe-D162T protein, are derived from and equivalent to the nucleotide sequence of the present invention.

[0030] The term "identity" as used herein refers to sequence similarity to a natural nucleic acid sequence. "Identity" includes nucleotide sequences that have 75% or higher, 85% or higher, 90% or higher, or 95% or higher identity with the nucleotide sequence of a protein constituting the amino acid sequence shown in Sequence 4 of this invention. Identity can be evaluated visually or using computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.

[0031] The stringent conditions for the aforementioned biological materials can be as follows: hybridization at 50°C in a mixed solution of 7% sodium dodecyl sulfate (SDS), 0.5M NaPO4, and 1mM EDTA, followed by rinsing at 50°C in 2×SSC and 0.1% SDS; or hybridization at 50°C in a mixed solution of 7% SDS, 0.5M NaPO4, and 1mM EDTA, followed by rinsing at 50°C in 1×SSC and 0.1% SDS; or hybridization at 50°C in a mixed solution of 7% SDS, 0.5M NaPO4, and 1mM EDTA, followed by rinsing at 50°C in 0.5×SSC and 0.1% SDS; or hybridization at 50°C in a mixed solution of 7% SDS, 0.5M NaPO4, and 1mM EDTA, followed by rinsing at 50°C in 0.5×SSC and 0.1% SDS; or hybridization at 50°C in a mixed solution of 7% SDS, 0.5M NaPO4, and 1mM EDTA. Hybridization was performed in a mixed solution of EDTA, followed by rinsing at 50°C in 0.1×SSC and 0.1% SDS; alternatively, it could be performed at 50°C in 7% SDS, 0.5M NaPO4, and 1mM... Hybridization can be performed in a mixed EDTA solution, followed by rinsing at 65°C in 0.1×SSC and 0.1% SDS. Alternatively, hybridization can be performed in a solution of 6×SSC and 0.5% SDS at 65°C, followed by rinsing once each with 2×SSC and 0.1% SDS and 1×SSC and 0.1% SDS. Alternatively, hybridization can be performed in a solution of 2×SSC and 0.1% SDS at 68°C, followed by rinsing twice for 5 min each time, then hybridization in a solution of 0.5×SSC and 0.1% SDS at 68°C, followed by rinsing twice for 15 min each time. Alternatively, hybridization can be performed in a solution of 0.1×SSPE (or 0.1×SSC) and 0.1% SDS at 65°C, followed by rinsing.

[0032] The aforementioned 75% or higher degree of identity can be 80%, 85%, 90%, or 95% or higher degree of identity.

[0033] In the aforementioned biological materials, the expression cassette (Acfe-D162T gene expression cassette) containing a nucleic acid molecule encoding the Acfe-D162T protein described in B2) refers to DNA capable of expressing the Acfe-D162T protein in host cells. This DNA may include not only a promoter to initiate Acfe-D162T gene transcription but also a terminator to terminate Acfe-D162T gene transcription. Furthermore, the expression cassette may also include an enhancer sequence.

[0034] Recombinant vectors containing the Acfe-D162T gene expression cassette can be constructed using existing expression vectors.

[0035] In the aforementioned biological materials, the vector can be a plasmid, granule, bacteriophage, or viral vector. Specifically, the plasmid can be a pET32a(+) vector.

[0036] B3) The recombinant vector may specifically be pET32a-TEV-Acfe-D162T. pET32a-TEV-Acfe-D162T is a recombinant vector obtained by replacing the DNA fragment between the EcoRI and NotI recognition sequences of the pET32a(+) vector with the TEV-Acfe-D162T fusion gene shown at positions 502-1251 of sequence 7. pET32a-TEV-Acfe-D162T contains the TEV-Acfe-D162T fusion gene and can express the TEV-Acfe-D162T fusion protein shown in sequence 8.

[0037] The microorganisms mentioned above can be yeast, bacteria, algae, or fungi. Among them, bacteria can be Escherichia coli, such as Escherichia coli BL21(DE3)trxB.

[0038] The present invention also provides a method for hydrolyzing PET, the method comprising: treating PET with Acfe-D162T to achieve hydrolysis of PET.

[0039] In the above method, the treatment can be carried out at 30°C.

[0040] In the above method, PET treatment using Acfe-D162T can be carried out in 50mM PBS, pH 8.0 buffer.

[0041] The present invention also provides a method for preparing MHET, the method comprising: hydrolyzing PET using Acfe-D162T to obtain MHET.

[0042] In the above method, the treatment can be carried out at 30°C.

[0043] In the above method, the hydrolysis of PET using Acfe-D162T can be carried out in 50mM PBS, pH 8.0 buffer.

[0044] The following applications of Acfe-D162T also fall within the scope of protection of this invention:

[0045] 1) As a PET hydrolase or bis(2-hydroxyethyl) terephthalate (BHET) hydrolase;

[0046] 2) Catalytic hydrolysis of PET;

[0047] 3) Degradation of PET;

[0048] 4) Catalytic hydrolysis of PET into MHET and / or TPA;

[0049] 5) Preparation of PET degradation agent;

[0050] 6) Preparation of catalytic PET hydrolysis products;

[0051] 7) Preparation of degradable PET products;

[0052] 8) Prepare catalytic PET hydrolysis products into MHET and / or TPA products.

[0053] The following applications of the biomaterials also fall within the scope of protection of this invention:

[0054] 1) Catalytic hydrolysis of PET;

[0055] 2) Degradation of PET;

[0056] 3) Catalytic hydrolysis of PET into MHET and / or TPA;

[0057] 4) Preparation of PET degradation agent;

[0058] 5) Preparation of catalytic PET hydrolysis products;

[0059] 6) Preparation of degradable PET products;

[0060] 7) Prepare catalytic PET hydrolysis products into MHET and / or TPA products.

[0061] In the above text, PET can be BHET.

[0062] This invention utilizes site-directed mutagenesis to mutate the original dienolactone hydrolase, obtaining a mutant that overcomes the low activity of the original enzyme, effectively increasing its activity in degrading BHET and improving the degradation efficiency. BHET (short-chain PET) can alleviate the inhibitory effect of PET degradation products; the mutant of this invention significantly improves the degradation efficiency of BHET, showing promising prospects for industrial application.

[0063] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way. Attached Figure Description

[0064] Figure 1 Activity analysis of dienolactone hydrolase and its mutant proteins. WT represents wild-type dienolactone hydrolase, and D162T and P190A / L208A represent dienolactone hydrolase mutants. Detailed Implementation

[0065] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials, reagents, instruments, etc., used in the following examples are commercially available. Unless otherwise specified, in the following examples, the first position of each nucleotide sequence in the sequence listing is the 5′ terminal nucleotide of the corresponding DNA / RNA, and the last position is the 3′ terminal nucleotide of the corresponding DNA / RNA.

[0066] Example 1: Preparation, expression, purification, and activity detection of dienolactone hydrolase mutants

[0067] To increase the industrial application value of dienolactase, this invention synthesized the gene of dienolactase Acfe derived from Acidimicrobiumferrooxidans and mutated it to obtain a mutant with enhanced enzyme activity.

[0068] I. Construction of wild-type dienolactone hydrolase recombinant plasmid and its mutant recombinant plasmid

[0069] 1. Construction of recombinant plasmid for wild-type dienolactone hydrolase

[0070] The wild-type dienolactone hydrolase is Acfe derived from Acidimicrobium ferrooxidans. The nucleotide sequence of wild-type dienolactone hydrolase Acfe is sequence 1 in the sequence listing, and the amino acid sequence it encodes is sequence 2 in the sequence listing.

[0071] A wild-type dienollipase gene containing a TEV restriction site was synthesized and inserted into the EcoRI and NotI restriction sites of the pET32a(+) vector to obtain a recombinant plasmid, denoted as pET32a-TEV-Acfe. After induction and expression, the amino acid sequence encoded by this recombinant plasmid, in addition to the sequence of the wild-type dienollipase, also includes a vector sequence and a TEV restriction site at its N-terminus. The amino acid sequence after induction and expression is shown in Sequence 6. This protein is named a fusion protein containing the wild-type dienollipase (denoted as TEV-Acfe fusion protein), and its encoding gene (denoted as TEV-Acfe fusion gene) is shown in Sequence 5 of the sequence listing.

[0072] The nucleotide sequence of the TEV-Acfe fusion gene containing the TEV restriction site consists, from the 5′ end, a methionine, a His tag and a TEV restriction site (positions 502-543 of sequence 5), a non-functional amino acid encoding nucleic acid (positions 544-570 of sequence 5), and the dienolactone hydrolase gene shown in sequence 1 of the sequence listing (positions 571-1251 of sequence 5).

[0073] The amino acid sequence of the TEV-Acfe fusion protein, starting from the N-terminus, consists of the pET32a vector fragment (positions 1-167 of sequence 6), methionine, His tag and TEV restriction site (positions 168-181 of sequence 6), non-functional amino acid (positions 182-190 of sequence 6), and dienolactone hydrolase shown in sequence 2 of the sequence listing (positions 191-417 of sequence 6).

[0074] Specifically, pET32a-TEV-Acfe is a recombinant vector obtained by replacing the DNA fragment between the EcoRI and NotI recognition sequences of the pET32a(+) vector with the TEV-Acfe fusion gene. It contains the TEV-Acfe fusion gene shown in sequence 5 of the sequence listing and can express the TEV-Acfe fusion protein shown in sequence 6 of the sequence listing.

[0075] 2. Recombinant plasmid expressing dienolactone hydrolase mutant

[0076] The dienolactone hydrolase mutant is a protein obtained by mutating the dienolactone hydrolase shown in Sequence 2 as follows (denoted as Acfe-D162T): at position 162 of its amino acid sequence, aspartic acid is mutated to threonine. The amino acid sequence of the dienolactone hydrolase mutant Acfe-D162T is Sequence 4, and the nucleotide sequence of its encoding gene (i.e., the Acfe-D162T gene) is Sequence 3.

[0077] Using site-directed mutagenesis, PCR was performed with the primers shown in Table 1 using the pET32a-TEV-Acfe plasmid as a template to obtain a plasmid expressing a dienollipase mutant. The original template was then removed by adding the restriction endonuclease DpnI and reacting at 37°C. The purified reaction product was transformed into competent *E. coli* cells, and preliminary screening was performed using antibiotics. DNA sequencing was then performed to identify the successfully mutated gene, resulting in a plasmid expressing the dienollipase mutant, denoted as pET32a-TEV-Acfe-D162T.

[0078] Specifically, pET32a-TEV-Acfe-D162T is a recombinant plasmid obtained by replacing the Acfe gene in pET32a-TEV-Acfe with the Acfe-D162T gene. This recombinant plasmid contains the TEV-Acfe-D162T fusion gene (which is a DNA fragment obtained by replacing the Acfe gene in the TEV-Acfe fusion gene with the Acfe-D162T gene; the sequence of the TEV-Acfe-D162T fusion gene is sequence 7) and can express the TEV-Acfe-D162T fusion protein (which is a protein obtained by replacing the Acfe protein in the TEV-Acfe fusion protein with the Acfe-D162T protein; the sequence of the TEV-Acfe-D162T fusion protein is sequence 8).

[0079] Using site-directed mutagenesis, PCR was performed with the primers shown in Table 1 using the pET32a-TEV-Acfe plasmid as a template to obtain a plasmid expressing the dienolactone hydrolase mutant. The original template was then removed by adding the restriction endonuclease DpnI and reacting at 37°C. The purified reaction product was transformed into competent *E. coli* cells, and preliminary screening was performed with antibiotics. DNA sequencing was then performed to identify the successfully mutated gene, yielding a plasmid expressing the dienolactone hydrolase mutant, denoted as pET32a-TEV-Acfe-P190A / L208A.

[0080] Specifically, pET32a-TEV-Acfe-P190A / L208A is a recombinant plasmid obtained by replacing the Acfe gene in pET32a-TEV-Acfe with the Acfe-P190A / L208A gene. This recombinant plasmid contains the TEV-Acfe-P190A / L208A fusion gene (which is a DNA fragment obtained by replacing the Acfe gene in the TEV-Acfe fusion gene with the Acfe-P190A / L208A gene) and can express the TEV-Acfe-P190A / L208A fusion protein (which is a protein obtained by replacing the Acfe protein in the TEV-Acfe fusion protein with the Acfe-P190A / L208A protein).

[0081] The sequence of the Acfe-P190A / L208A gene is sequence 9, and the amino acid sequence of the Acfe-P190A / L208A protein is sequence 10.

[0082] Table 1. Site-directed mutagenesis primers

[0083]

[0084] In Table 1 above, D162T indicates that the 162nd amino acid in sequence 2 has been mutated from aspartic acid to threonine, and P190A / L208A indicates that the 190th amino acid in sequence 2 has been mutated from proline to alanine and the 208th amino acid has been mutated from leucine to alanine.

[0085] II. Preparation of dienolactone hydrolase mutants and wild-type dienolactone hydrolase

[0086] 1. Expression and purification of wild-type dienollipase and dienollipase mutant

[0087] The recombinant plasmid pET32a-TEV-Acfe expressing wild-type dienollipase and the recombinant plasmids pET32a-TEV-Acfe-D162T and pET32a-TEV-Acfe-P190A / L208A expressing dienollipase mutants were transformed into *E. coli* BL21(DE3)trxB competent cells, respectively. Strains were screened in LB broth containing 100 μg / ml Ampicillin. The selected strains were inoculated into 5 ml LB broth and cultured, then the culture volume was increased to 200 ml LB broth, and finally scaled up to 6 L LB broth and cultured (37℃, 220 rpm). When the OD value reached 0.6 to 0.8, the culture was cooled to 16℃, and IPTG at a final concentration of 0.4 mM was added at 16℃ and 220 rpm to induce a large expression of the enzyme protein. After 18 hours of protein induction and expression, the bacterial culture was centrifuged at 6000 rpm for 15 minutes to collect the cells. The cells were resuspended in buffer (25 mMtris, 150 mM NaCl, pH 7.5), and the cells were lysed using an ultrasonic cell disruptor. The cells were then centrifuged at 16000 rpm at 4°C for 60 minutes, and the supernatant was collected for further purification.

[0088] To obtain high-purity enzyme protein, the target protein was eluted sequentially using a fast protein liquid chromatography (FPLC) column with nickel ion chromatography substrates (buffer A: 25 mM Tris, 150 mM NaCl, 20 mM imidazole, pH 7.5; buffer B: 25 mM Tris, 150 mM NaCl, 250 mM imidazole, pH 7.5), and the target protein was collected. 300 μL of TEV protease was added to the target protein for enzymatic digestion to remove the His tag on the vector. Simultaneously, the target protein was dialyzed in 5 L of dialysate (25 mM Tris, 150 mM NaCl, pH 7.5), the dialysate was replaced, and dialyzed overnight at 4°C. The digested target protein was then passed through a nickel column again, and the His-tag-free target protein was collected. The purified target protein was dialyzed in a buffer solution of 25 mM Tris, 150 mM NaCl, and pH 7.5, concentrated, collected, and stored at -80°C for later use.

[0089] III. Comparison of relative activities between dienolactone hydrolase mutants and wild-type dienolactone hydrolases

[0090] To verify the difference between wild-type dienollipase and the dienollipase mutant, the inventors further determined their BHET degradation activities. The activity assay steps for dienollipase are as follows:

[0091] Each reaction mixture (1 mL) was placed in 50 mM PBS, pH 8.0 buffer. This mixture contained 1 mM of the substrate bis(2-hydroxyethyl) terephthalate (BHET) (an oligomer of PET, Sigma, CAS: 959-26-2) and 30 μL of enzyme (1 mg / mL of the different mutant or wild-type dienolactone hydrolase prepared above), with the balance being 100 mM PBS, pH 8.0 buffer. The resulting reaction mixtures were incubated on a shaker at 30°C and 100 rpm for 40 hours. Each reaction was performed in triplicate. After the reaction, the mixture was first heat-shocked (80℃, 10 min) to terminate the reaction, then centrifuged at 12000 rpm for 10 min. The supernatant reaction solution was collected and filtered through a 0.22 μm filter. The filtrate was collected for product determination and analysis by high performance liquid chromatography (HPLC, Agilent 1200). The analytical column was a Welch Ultimate XB-C18 column (4.6×250 mm, 5 μm, Yuexu Technology (Shanghai) Co., Ltd.). The mobile phase was 81% pure water, 18% acetonitrile, and 1% formic acid. The flow rate was 0.8 ml / min, the wavelength was 254 nm, the column temperature was 30℃, and direct elution was performed for 20 min.

[0092] High-performance liquid chromatography (HPLC) analysis of wild-type dienolactone hydrolase and mutant showed a MHET retention time of approximately 11.6 min. Mass spectrometry analysis of the elution peak (negative ion, Mass range 50-1000 m / z) confirmed it to be MHET.

[0093] The activity of wild-type and mutant enzymes is determined by comparing the peak area of ​​MHET, the hydrolysis product of wild-type dienolactone hydrolase or its mutant.

[0094] The principle of activity determination is as follows: In HPLC experiments, the amount of compound in solution is linearly related to the peak area, so the amount of compound in solution can be calculated by the peak area; in this experiment, the catalytic effect of mutant protein on substrate is defined by the peak area of ​​the product; the more product (because the substrate cannot be dissolved, it is difficult to quantify), the better the activity of the mutant protein.

[0095] The peak area of ​​MHET, the hydrolysate of wild-type dienollipase, is recorded as 100%. The peak area of ​​MHET, the hydrolysate of other dienollipase mutants, is compared with the peak area of ​​MHET, the hydrolysate of wild-type dienollipase, and recorded as the relative enzyme activity.

[0096] The test results showed that both the dienolactone hydrolase and its mutants of the present invention can hydrolyze BHET to MHET, but the mutant Acfe-D162T exhibits higher activity for dienolactone hydrolase degradation than the wild-type protein. Specifically, the mutant-catalyzed product MHET is approximately 2.4 times higher than that of the wild-type protein. In contrast, Acfe-P190A / L208A shows lower activity for dienolactone hydrolase degradation than the wild-type protein. Figure 1 As shown, this indicates that the mutant Acfe-D162T has significant application value.

[0097] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. Protein, as shown in A1 or A2): A1) The amino acid sequence is that of the protein in sequence 4; A2) is a fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of A1).

2. The biological material related to the protein of claim 1 is any one of B1) to B4) below: B1) A nucleic acid molecule encoding the protein of claim 1; B2) An expression cassette containing the nucleic acid molecule described in B1); B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4) Recombinant microorganisms containing the nucleic acid molecules described in B1), or recombinant microorganisms containing the expression cassette described in B2), or recombinant microorganisms containing the recombinant vector described in B3).

3. The biomaterial according to claim 2, characterized in that: B1) The nucleic acid molecule described is as follows (b11) or (b12)): b11) The coding sequence is the DNA molecule of sequence 3 in the sequence listing; b12) The DNA molecule shown in sequence 3 of the sequence listing.

4. The biomaterial according to claim 2, characterized in that: B1) The nucleic acid molecule described is a cDNA molecule whose coding sequence is sequence 3 in the sequence listing.

5. A method for hydrolyzing BHET, comprising: The protein described in claim 1 is used to treat BHET to achieve the hydrolysis of BHET.

6. The method according to claim 5, characterized in that: The treatment was carried out at 30°C.

7. A method for preparing MHET, comprising: MHET is obtained by hydrolyzing BHET using the protein described in claim 1.

8. The method according to claim 7, characterized in that: The hydrolysis was carried out at 30°C.

9. Any of the following applications of the protein of claim 1: 1) As a BHET hydrolase; 2) Preparation of BHET degradation agent.

10. Any of the following applications of the biomaterial according to claim 2, 3, or 4: 1) Catalytic hydrolysis of BHET; 2) Preparation of BHET degradation agent.

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