CN117757771A - A diene lactone hydrolase mutant and its application - Google Patents

Abstract

The invention discloses a dienolide hydrolase mutant and its application. The dienolide hydrolase mutant disclosed by the present invention is as follows A1), A2) or A3): A1) the amino acid sequence is a protein of sequence 4; A2) the amino acid sequence other than the 162nd position in sequence 4 in the sequence listing A protein that has the same function after one or several amino acid residues have been substituted and/or deleted and/or added; A3) a fusion protein obtained by connecting a tag to the N-terminus or/and C-terminus of A1) or A2). The present invention uses site-directed mutation technology to mutate the original dienolide hydrolase to obtain its mutant, which changes the problem of low activity of the original dienolide hydrolase, effectively improves the activity of degrading BHET, and improves the degradation effect. The dienolide hydrolase mutant of the present invention greatly improves the degradation efficiency of BHET and has good industrial application prospects.

Description

A diene lactone hydrolase mutant and its application

Technical field

The invention relates to a diene lactone hydrolase mutant and its application in the field of genetic engineering.

Background technique

Plastic is a great invention of mankind. The use of plastic products is becoming increasingly widespread, bringing great convenience to people's daily lives. But at the same time, the problem of plastic pollution is becoming increasingly serious and has become a hot environmental issue of widespread concern around the world.

According to statistics, global plastic production reached 367 million tons in 2020. Except for some plastics that can be used, the rest have become garbage discarded by people. In the past 70 years, the weight of plastic waste discarded by humans has exceeded 7 billion tons. Unlike metal products, plastic products can be recycled. Most plastics are disposable, and only a small part can be recycled. Therefore, when plastic production remains high and increases every year, the problem of plastic pollution will only become more and more serious and will be difficult to alleviate unless people can find a complete solution to plastic pollution.

The traditional treatment methods are incineration or landfill, but both methods will cause environmental pollution. After plastic is burned, it will produce toxic and harmful gases and pollute the air. After plastic is buried in the soil, it will take hundreds of years to completely degrade naturally. During this process, the plastic will change the soil structure and composition, and soil nutrients will be lost. In addition, plastics exposed in nature can be accidentally eaten by birds, indirectly killing many organisms.

Since scientist Oda Kohei accidentally discovered a microorganism that can feed on polyethylene terephthalate (PET), Sakai bacterium Osaka, scientists have successively discovered some hydrolytic enzymes with the activity of degrading PET, but the degradation Vitality is low. Methods such as fixed-site evolution and adding metal ions were used to improve enzyme activity, but the results were not ideal. Bishydroxyethyl terephthalate (BHET) is an intermediate product of PET hydrolysis. The accumulation of reaction intermediates is an important factor limiting the degradation efficiency of PET hydrolase. BHET hydrolase can decompose BHET into mono(2-hydroxyethyl) ) terephthalic acid (MHET), and then further decompose it into terephthalic acid (TPA) and ethylene glycol (EG). How to efficiently degrade BHET and break the degradation barrier has huge commercial value.

Contents of the invention

One object of the present invention is to provide a diene lactone hydrolase (Acfe) mutant, a protein with BHET hydrolysis activity. The obtained mutant effectively improves the BHET-degrading activity of Acfe and improves the degradation effect of Acfe.

The present invention first provides a protein, the name of the protein is Acfe-D162T, and Acfe-D162T is as follows A1), A2) or A3):

A1) The amino acid sequence is a protein of sequence 4;

A2) A protein that has the same function by substituting and/or deleting and/or adding one or several amino acid residues to the amino acid sequence other than position 162 in Sequence 4 in the sequence listing;

A3) A fusion protein obtained by connecting a tag to the N-terminus or/and C-terminus of A1) or A2).

In order to facilitate the purification of the protein in A1), a tag as shown in the table below can be connected to the amino terminus or carboxyl terminus of the protein consisting of the amino acid sequence shown in Sequence 4 in the sequence listing.

Table: Sequence of tags

Label Residues sequence 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

The Acfe-D162T protein in A2) above is a protein that has 75% or more identity with the amino acid sequence of the protein shown in Sequence 4 and has the same function. The said having 75% or more identity means having 75%, having 80%, having 85%, having 90%, having 95%, having 96%, having 97%, having 98% or having 99% identity. .

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

The gene encoding the Acfe-D162T protein in A2) above can be obtained by deleting the codon of one or several amino acid residues in the DNA sequence shown in Sequence 3, and/or performing a missense mutation of one or several base pairs. , and/or obtained by connecting the coding sequence of the tag shown in the above table to its 5′ end and/or 3′ end. Among them, the DNA molecule shown in sequence 3 encodes the Acfe-D162T protein shown in sequence 4.

Specifically, the protein in A2) may be the protein shown in sequence 8.

The present invention also provides biomaterials related to Acfe-D162T, which biomaterials are any one of the following B1) to B4):

B1) Nucleic acid molecule encoding Acfe-D162T;

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

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

B4) A recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3).

In the above biological materials, the nucleic acid molecules described in B1) may be the following b11) or b12) or b13) or b14):

b11) The coding sequence is a cDNA molecule or DNA molecule of sequence 3 in the sequence listing;

b12) The DNA molecule shown in sequence 3 in the sequence listing;

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

b14) A cDNA molecule or DNA molecule that hybridizes to the nucleotide sequence defined by b11) or b12) or b13) under stringent conditions and encodes Acfe-D162T.

Wherein, 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, etc.

Those of ordinary skill in the art can easily use known methods, such as directed evolution and point mutation methods, to mutate the nucleotide sequence encoding the Acfe-D162T protein of the present invention. Those 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 the Acfe-D162T protein and have the function of the Acfe-D162T protein, are derived from Nucleotide sequences of the invention and are equivalent to sequences of the invention.

The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "Identity" includes 75% or higher, or 85% or higher, or 90% or higher, or 95% or Nucleotide sequences of higher identity. Identity can be assessed with the naked eye or with 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.

In the above biological materials, the stringent conditions can be as follows: 50°C, hybridization in a mixed solution of 7% sodium dodecyl sulfate (SDS), 0.5M NaPO ₄ and 1mM EDTA, 2×SSC at 50°C, Rinse in 0.1% SDS; alternatively: 50°C, hybridize in a mixed solution of 7% SDS, 0.5M NaPO ₄ and 1mM EDTA, rinse in 50°C, 1×SSC, 0.1% SDS; alternatively: 50 ℃, hybridize in a mixed solution of 7% SDS, 0.5M NaPO ₄ and 1mM EDTA, rinse in 50℃, 0.5×SSC, 0.1% SDS; also: 50℃, in 7% SDS, 0.5M NaPO ₄ Hybridize in a mixed solution of 1mM EDTA and 50°C, rinse in 0.1×SSC, 0.1% SDS; alternatively: 50°C, hybridize in a mixed solution of 7% SDS, 0.5M NaPO ₄ and 1mM EDTA, rinse at 65 ℃, rinse in 0.1×SSC, 0.1% SDS; also: hybridize in 6×SSC, 0.5% SDS solution at 65℃, then use 2×SSC, 0.1% SDS and 1×SSC, 0.1% Wash the membrane once with each SDS; it can also be: hybridize and wash the membrane twice at 68°C in a solution of 2×SSC, 0.1% SDS, 5 min each time, and then in a solution of 0.5×SSC, 0.1% SDS, at 68°C. Hybridize and wash the membrane twice at 68°C, 15 minutes each time; it can also be hybridized and washed at 65°C in a solution of 0.1×SSPE (or 0.1×SSC) and 0.1% SDS.

The above-mentioned 75% or above identity may be 80%, 85%, 90% or 95% or above identity.

Among the above biological materials, the expression cassette containing the nucleic acid molecule encoding the Acfe-D162T protein (Acfe-D162T gene expression cassette) described in B2) refers to DNA that can express the Acfe-D162T protein in host cells. This DNA can not only It includes a promoter that starts the transcription of the Acfe-D162T gene, and may also include a terminator that terminates the transcription of the Acfe-D162T gene. Furthermore, the expression cassette may also include an enhancer sequence.

Existing expression vectors can be used to construct a recombinant vector containing the Acfe-D162T gene expression cassette.

In the above biological materials, the vector can be a plasmid, cosmid, phage or viral vector. The plasmid may specifically be pET32a(+) vector.

B3) The recombinant vector can specifically be pET32a-TEV-Acfe-D162T. The 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 in positions 502-1251 of Sequence 7 . The pET32a-TEV-Acfe-D162T contains the TEV-Acfe-D162T fusion gene and can express the TEV-Acfe-D162T fusion protein shown in sequence 8.

In the above biological material, the microorganism may be yeast, bacteria, algae or fungi. The bacterium may be Escherichia coli, such as Escherichia coli BL21(DE3)trxB.

The present invention also provides a method for hydrolyzing PET, which comprises: treating PET with Acfe-D162T to achieve hydrolysis of PET.

In the above method, the treatment can be performed at 30°C.

In the above method, the treatment of PET with Acfe-D162T can be carried out in 50mM PBS, pH 8.0 buffer.

The invention also provides a method for preparing MHET, which method includes: using Acfe-D162T to hydrolyze PET to obtain MHET.

In the above method, the treatment can be performed at 30°C.

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

Any of the following applications of Acfe-D162T also falls within the protection scope of the present invention:

1) As PET hydrolase or bis(2-hydroxyethyl)terephthalate (BHET) hydrolase;

2) Catalyze PET hydrolysis;

3) Degradation of PET;

4) Catalyze the hydrolysis of PET into MHET and/or TPA;

5) Prepare PET degradation agent;

6) Preparation of catalytic PET hydrolysis products;

7) Preparation of degraded PET products;

8) Preparation of catalytic PET hydrolysis into MHET and/or TPA products.

Any of the following applications of the biological material also falls within the protection scope of the present invention:

1) Catalyze PET hydrolysis;

2) Degradation of PET;

3) Catalyze the hydrolysis of PET into MHET and/or TPA;

4) Prepare PET degradation agent;

5) Preparation of catalytic PET hydrolysis products;

6) Preparation of degraded PET products;

7) Preparation of catalytic PET hydrolysis into MHET and/or TPA products.

In the above, PET can be BHET.

The present invention uses site-directed mutation technology to mutate the original dienolide hydrolase to obtain its mutant, which changes the problem of low activity of the original dienolide hydrolase, effectively improves the activity of degrading BHET, and improves the degradation effect. BHET (short-chain PET) can relieve the inhibition of products in PET degradation. The mutant of the present invention greatly improves the degradation efficiency of BHET, and has good industrial application prospects.

The present invention will be described in further detail below in conjunction with specific embodiments. The examples given are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for those of ordinary skill in the art to make further improvements, and do not limit the present invention in any way.

Description of the drawings

Figure 1 shows the activity analysis of dienolide hydrolase and its mutant proteins. WT represents wild-type dienolide hydrolase, and D162T and P190A/L208A represent dienolide hydrolase mutants.

Detailed ways

The experimental methods in the following examples, unless otherwise specified, are all conventional methods and are carried out in accordance with the techniques or conditions described in literature in the field or in accordance with product instructions. The materials, reagents, instruments, etc. used in the following examples can all be obtained from commercial sources unless otherwise specified. In the following examples, unless otherwise specified, the first position of each nucleotide sequence in the sequence list is the 5' terminal nucleotide of the corresponding DNA/RNA, and the last position is the 3' terminal core of the corresponding DNA/RNA. glycosides.

Example 1. Preparation, expression, purification and activity detection of dienolide hydrolase mutants

In order to increase the industrial application value of dienolide, the present invention synthesizes the gene of dienolide hydrolase Acfe derived from Acidimicrobium ferrooxidans, mutates it, and obtains a mutant with improved enzyme activity.

1. Construction of wild-type dienolide hydrolase recombinant plasmid and its mutant recombinant plasmid

1. Construction of wild-type dienolide hydrolase recombinant plasmid

The wild-type dienelide hydrolase is Acfe derived from Acidimicrobiumferrooxidans. The nucleotide sequence of the wild-type dienelide hydrolase Acfe is sequence 1 in the sequence listing, and its encoded amino acid sequence is sequence 2 in the sequence listing.

The wild-type dienolide hydrolase gene containing the TEV enzyme cleavage site was synthesized and inserted into the EcoRI and NotI enzyme cleavage sites in the pET32a(+) vector to obtain a recombinant plasmid, designated as pET32a-TEV-Acfe; In addition to the sequence of the wild-type dienelide hydrolase, the amino acid sequence encoded by the recombinant plasmid after induced expression also has a vector sequence and a TEV enzyme cleavage site at its N-terminus. The amino acid sequence after induced expression is shown in Sequence 6, named The fusion protein containing wild-type dienolide hydrolase (denoted as TEV-Acfe fusion protein), and its encoding gene (denoted as TEV-Acfe fusion gene) is shown in 5 in the sequence list.

The nucleotide sequence of the TEV-Acfe fusion gene containing the TEV enzyme cleavage site consists of methionine, His tag and TEV enzyme cleavage site (positions 502-543 of sequence 5), starting from the 5' end, and has no function. It consists of an amino acid encoding nucleic acid (positions 544-570 of Sequence 5) and the dienolide hydrolase gene shown in Sequence 1 in the sequence listing (positions 571-1251 of Sequence 5).

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

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, and contains the TEV-Acfe fusion shown in sequence 5 in the sequence list. The gene can express the TEV-Acfe fusion protein shown in sequence 6 in the sequence listing.

2. Recombinant plasmid expressing dienolide hydrolase mutant

The dienelide hydrolase mutant is a protein obtained by mutating the dienelide hydrolase shown in Sequence 2 as follows (the resulting protein is recorded as Acfe-D162T): the 162nd position of its amino acid sequence is changed from the wild-type diene The Aspartic acid of lactis hydrolase is mutated to Threonine. The amino acid sequence of dienolide hydrolase mutant Acfe-D162T is Sequence 4, and the nucleotide sequence of its encoding gene (ie, Acfe-D162T gene) is Sequence 3.

Utilize site-directed mutagenesis, use the pET32a-TEV-Acfe plasmid as a template, and perform PCR with the primers shown in Table 1 to obtain a plasmid expressing the diene lactone hydrolase mutant; then add restriction endonuclease The enzyme DpnI reacts at 37°C to remove the original template. The purified reaction product was transformed into E. coli competent cells, and antibiotics were used for preliminary screening. DNA sequencing was performed to determine the successfully mutated genes, and a plasmid expressing the diene lactone hydrolase mutant was obtained, which was designated as pET32a-TEV-Acfe. -D162T.

Specifically, pET32a-TEV-Acfe-D162T is a recombinant plasmid obtained by replacing the Acfe gene in pET32a-TEV-Acfe with the Acfe-D162T gene. The recombinant plasmid contains the TEV-Acfe-D162T fusion gene (this gene is the TEV- The Acfe gene in the Acfe fusion gene is replaced with the DNA fragment obtained by the Acfe-D162T gene. The sequence of the TEV-Acfe-D162T fusion gene is sequence 7), which can express the TEV-Acfe-D162T fusion protein (the protein is a fusion of TEV-Acfe The Acfe protein in the protein is replaced with the Acfe-D162T protein. The sequence of the TEV-Acfe-D162T fusion protein is sequence 8).

Utilize site-directed mutagenesis, use the pET32a-TEV-Acfe plasmid as a template, and perform PCR with the primers shown in Table 1 to obtain a plasmid expressing the diene lactone hydrolase mutant; then add restriction endonuclease The enzyme DpnI reacts at 37°C to remove the original template. The purified reaction product was transformed into Escherichia coli competent cells, and antibiotics were used for preliminary screening. DNA sequencing was performed to determine the successfully mutated genes, and a plasmid expressing the diene lactone hydrolase mutant was obtained, which was recorded as pET32a-TEV-Acfe. -P190A/L208A.

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. The recombinant plasmid contains the TEV-Acfe-P190A/L208A fusion gene ( This gene 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 (this protein is a DNA fragment obtained by replacing the Acfe gene in the TEV-Acfe fusion protein). Protein obtained by replacing Acfe protein with Acfe-P190A/L208A protein).

Among them, the sequence of Acfe-P190A/L208A gene is sequence 9, and the amino acid sequence of Acfe-P190A/L208A protein is sequence 10.

Table 1. Site-directed mutagenesis primers

In Table 1 above, D162T refers to the mutation of the 162nd amino acid in sequence 2 from aspartic acid to threonine, and P190A/L208A refers to the mutation of the 190th amino acid in sequence 2 from proline to alanine, and the 208th amino acid in sequence 2. An amino acid is mutated from leucine to alanine.

2. Preparation of dienolide hydrolase mutants and wild-type dienolide hydrolase

1. Expression and purification of wild-type dienolide hydrolase and dienolide hydrolase mutants

The above-prepared recombinant plasmid pET32a-TEV-Acfe expressing wild-type dienolide hydrolase and the recombinant plasmids pET32a-TEV-Acfe-D162T and pET32a-TEV-Acfe-P190A expressing dienolide hydrolase mutants were prepared. /L208A were transformed into E. coli BL21(DE3)trxB competent cells, and the strains were screened in LB culture dishes containing 100 μg/ml Ampicillin. Inoculate the selected strains into 5 ml LB for culture, then amplify the bacterial volume to 200 ml LB for culture, and finally amplify it into 6L LB medium for culture (37°C, 220 rpm). When the OD value reaches 0.6 to 0.8, the culture is cooled to 16°C, and IPTG at a final concentration of 0.4mM is added to induce large expression of the enzyme protein at 16°C and 220 rpm. After 18 hours of protein induction, the bacterial solution was centrifuged at 6000 rpm for 15 minutes to collect the cells. Resuspend the bacteria in buffer (25mM tris, 150mM NaCl, pH 7.5), disrupt the bacteria using an ultrasonic cell disrupter (sonicator), and then centrifuge at 16,000 rpm at 4°C for 60 minutes. Collect the supernatant for preparation. Next step of purification.

In order to obtain high-purity enzyme protein, fast protein liquid chromatography (FPLC) was used to sequentially elute the target protein using a nickel ion chromatography column substrate (buffer A: 25mM Tris, 150mM NaCl, 20mM imidazole, pH 7 .5; buffer B: 25mM Tris, 150mM NaCl, 250mM imidazole, pH7.5), collect the target protein. Add 300 microliters of TEV protease to the target protein for enzymatic digestion in order to cut off the His tag on the carrier, and at the same time dialyze the target protein into 5L dialysate (25mM tris, 150mM NaCl, pH7.5), and replace the dialysis solution and dialyzed overnight at 4°C. The target protein after digestion is passed through the nickel column again, and the target protein that does not contain the His tag that flows through is collected. Dialyze the twice-purified target protein into a buffer of 25mM Tris, 150mM NaCl, pH 7.5, concentrate and collect, and store at -80°C for later use.

3. Comparison of the relative activities of dienolide hydrolase mutants and wild-type dienolide hydrolase

In order to verify the difference between the wild-type dienolide hydrolase and the dienolide hydrolase mutant, the inventors further determined the BHET degradation activities of the two. The test steps for the activity of dienolide hydrolase are as follows:

Mixtures (1 mL) of each reaction containing 1 mM of the substrate bis(2-hydroxyethyl)terephthalate (BHET) (oligomeric form of PET) were in 50 mM PBS, pH 8.0 buffer. substance, sigma, cas: 959-26-2) and 30 μL enzyme (1 mg/mL different mutants or wild-type diene lactone hydrolase prepared in the above two preparations), the balance is 100 mM PBS, pH 8.0 buffer . The obtained reaction system was placed on a shaker at 30°C and 100 rpm for 40 hours. Each reaction was repeated three times. After the reaction, the mixture was first heat-shocked (80°C, 10 min) to terminate the reaction, and then centrifuged at 12,000 rpm for 10 minutes. The supernatant reaction solution was filtered through a 0.22 μm filter; the filtrate was collected for high-performance liquid chromatography (HPLC, Agilent 1200) product determination. For analysis, the analytical column was Welch Ultimate XB-C18 column (4.6×250mm, 5μm, Yuexu Technology (Shanghai) Co., Ltd.). The mobile phase is 81% pure water, 18% acetonitrile, 1% formic acid, flow rate 0.8ml/min, wavelength 254nm, column oven 30°C, direct elution, 20min.

The high-performance liquid chromatography (HPLC) detection results of wild-type dienolide hydrolase and mutants are that the MHET retention time is about 11.6 minutes, and the peaks are analyzed by mass spectrometry (negative ions, Mass range 50-1000m/z), which is MHET.

The activities of the wild-type and mutant enzymes were determined by comparing the peak areas of the hydrolysis product MHET of the wild-type dienolide hydrolase or its mutants.

The principle of activity measurement is as follows: In HPLC experiments, the amount of compounds in the solution has a linear relationship with the peak area, so the amount of compounds in the solution can be calculated by the peak area; in this experiment, the peak area of the product is used to define the response of the mutant protein to the bottom The catalytic effect of the substance; the more products (because the substrate cannot be dissolved, so it is difficult to quantify), the better the activity of the mutant protein.

Taking the peak area of the hydrolysis product MHET of the wild-type dienolide hydrolase as 100%, the peak area of the hydrolysis product MHET of other dienolide hydrolase mutants is the same as the peak area of the hydrolysis product MHET of the wild-type dienolide hydrolase. The peak area was compared and recorded as relative enzyme activity.

The test results show that both the dienolide hydrolase and its mutants of the present invention can hydrolyze BHET into MHET, but the mutant Acfe-D162T has a higher activity of dienolide hydrolase degradation than the wild-type protein. Among them, the mutant The catalyzed product MHET is approximately 2.4 times higher than that of the wild type; while Acfe-P190A/L208A has lower dienolactone hydrolase degradation activity than the wild type protein, as shown in Figure 1. It shows that the mutant Acfe-D162T has good application value.

The present invention has been described in detail above. For those skilled in the art, the present invention can be implemented in a wider range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the invention and without performing unnecessary experiments. Although specific embodiments of the present invention have been shown, it should be understood that further modifications can be made to the invention. In short, based on the principles of the present invention, this application is intended to include any changes, uses, or improvements to the present invention, including changes that depart from the scope disclosed in this application and are made using conventional techniques known in the art. Some essential features may be applied within the scope of the appended claims below.

Claims (10)

1. Protein, A1), A2) or A3) as follows:

a1 Amino acid sequence is a protein of sequence 4;

a2 A protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues for an amino acid sequence except 162 th position in a sequence 4 in a sequence table and has the same function;

a3 A fusion protein obtained by ligating a tag to the N-terminal or/and the C-terminal of A1) or A2).

2. A biological material related to the protein of claim 1, which is any one of the following B1) to B4):

b1 A nucleic acid molecule encoding the protein of claim 1;

b2 An expression cassette comprising the nucleic acid molecule of B1);

b3 A recombinant vector comprising the nucleic acid molecule of B1) or a recombinant vector comprising the expression cassette of B2);

b4 A recombinant microorganism comprising the nucleic acid molecule of B1), a recombinant microorganism comprising the expression cassette of B2), or a recombinant microorganism comprising the recombinant vector of B3).

3. The biomaterial according to claim 2, characterized in that: b1 The nucleic acid molecule is b 11) or b 12) or b 13) or b 14) as follows:

b11 A cDNA molecule or a DNA molecule of which the coding sequence is a sequence 3 in a sequence table;

b12 A DNA molecule shown in a sequence 3 in a sequence table;

b13 A cDNA or DNA molecule having 75% or more identity to the nucleotide sequence defined in b 11) or b 12) and encoding the protein according to claim 1;

b14 A cDNA molecule or a DNA molecule which hybridizes under stringent conditions to the nucleotide sequence defined in b 11) or b 12) or b 13) and which codes for a protein according to claim 1.

4. A method of hydrolyzing PET comprising: hydrolysis of PET is achieved by treating PET with the protein of claim 1.

5. The method according to claim 4, wherein: the treatment is carried out at 30 ℃.

6. A method of making an MHET comprising: the use of the proteolytic PET according to claim 1 to obtain MHET.

7. The method according to claim 6, wherein: the treatment is carried out at 30 ℃.

8. Use of the protein of claim 1 for any of the following:

1) As PET hydrolase or BHET hydrolase;

2) Catalyzing hydrolysis of PET;

3) Degrading PET;

4) Catalyzing hydrolysis of PET to MHET and/or TPA;

5) Preparing a PET degradation agent;

6) Preparing a catalytic PET hydrolysis product;

7) Preparing a degraded PET product;

8) Preparation of a product that catalyzes the hydrolysis of PET to MHET and/or TPA.

9. Use of the biomaterial of claim 2 or 3 for any of the following:

1) Catalyzing hydrolysis of PET;

2) Degrading PET;

3) Catalyzing hydrolysis of PET to MHET and/or TPA;

4) Preparing a PET degradation agent;

5) Preparing a catalytic PET hydrolysis product;

6) Preparing a degraded PET product;

7) Preparation of a product that catalyzes the hydrolysis of PET to MHET and/or TPA.

10. The method according to any one of claims 4-7, or the use according to claim 8 or 9, characterized in that: PET is BHET.