CN117757770A - Carboxylesterase mutant and application thereof - Google Patents

Abstract

The invention discloses carboxylesterase mutants and application thereof. The carboxylesterase mutants disclosed in the present invention are the following A1), A2) or A3): a1 Amino acid sequence is a protein of sequence 4; a2 A protein which has the same function and is obtained by substituting and/or deleting and/or adding one or more amino acid residues for an amino acid sequence except 429 th site in the sequence 4 in the sequence table; a3 A fusion protein obtained by ligating a tag to the N-terminal or/and the C-terminal of A1) or A2). The invention uses the site-directed mutagenesis technology to mutate the original carboxylesterase (Tcca) to obtain a carboxylesterase mutant, thereby changing the problem of lower activity of the original carboxylesterase, effectively improving the activity of the Tcca in degrading BHET and improving the degradation effect of the Tcca.

Description

Carboxylesterase mutant and application thereof

Technical Field

The invention relates to carboxylesterase mutant and application thereof in the field of genetic engineering.

Background

Polyester-based plastics PET (such as polyethylene terephthalate) has become one of the most important mass-produced petrochemical plastics, mainly as synthetic polyester fabrics for the textile industry, as well as packaging materials for foods and beverages. Most PET products have a high degree of crystallinity and durability against mechanical and chemical stresses. PET is formed by polymerizing terephthalic acid (TPA) and Ethylene Glycol (EG) through ester bonds, has stable properties and is not easy to decompose, and is commonly used for mineral water bottles, polyester clothes, plastic uptake packages and other products.

The current treatment method for plastic PET waste mainly comprises the following steps: landfill, incineration, recycling, and the like. Although landfill and incineration are simple, the generated waste gas and waste water can cause secondary pollution to the environment; recycling is lower in recycling rate at the present stage due to economical efficiency of recycling cost and performance problem of recycled plastics. In recent years, more and more researchers focus on the biodegradation of plastics, and compared with a physicochemical degradation method, the biodegradation is more environment-friendly and ecologically friendly. If the PET is degraded into constituent molecules by enzyme degradation, then the PET is recycled to be used as a true decomposed plastic, and the method is one of the most ideal treatment methods. The method not only solves the problems of PET waste and can be recycled. Scientists have discovered their activity on PET degradation from hydrolases such as esterases (esterases), lipases (lipases) and cutinases (cutinases) over the last decade, demonstrating the possibility of PET biodegradation, which are all of the alpha/beta hydrolase family.

Carboxylesterase (carboxylesterase), similar in structure to lipases with a conserved Ser-Glu/Asp-His triplet structure belongs to the alpha/beta hydrolase family, but differs in substrate specificity, is more prone to hydrolyze substrates with smaller acyl chain lengths, whereas lipases tend to hydrolyze substrates with larger acyl chain lengths. Carboxylesterase has good degradation activity on BHET, which is an intermediate product of PET hydrolysis, and accumulation of reaction intermediates is an important factor limiting the degradation efficiency of PET hydrolytic enzymes. Therefore, the activity of degrading BHET enzyme is improved, more theoretical basis and experimental basis are provided for PET degradation research, and positive significance is provided for protecting human ecological environment.

Disclosure of Invention

The technical problem to be solved by the invention is how to degrade polyethylene terephthalate (PET).

In order to solve the technical problems, the invention firstly provides a protein, the name of the protein is Tcca-W429A, and Tcca-W429A is A1), A2) or A3):

a1 Amino acid sequence is a protein of sequence 4;

a2 A protein which has the same function and is obtained by substituting and/or deleting and/or adding one or more amino acid residues for an amino acid sequence except 429 th site in the sequence 4 in the sequence table;

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

In order to facilitate purification of the protein of A1), a tag as shown in the following Table may be attached to the amino-terminal or carboxyl-terminal of the protein consisting of the amino acid sequence shown in the sequence 4 in the sequence Listing.

Table: tag sequence

Label (Label)	Residues	Sequence(s)
			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 Tcca-W429A protein in A2) is a protein which has 75% or more identity with the amino acid sequence of the protein shown in the sequence 4 and has the same function. The identity of 75% or more is 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity.

The Tcca-W429A protein in the A2) can be synthesized artificially or can be obtained by synthesizing the coding gene and then biologically expressing.

The coding gene of the Tcca-W429A protein in the A2) can be obtained by deleting one or a plurality of amino acid residues in the DNA sequence shown in the sequence 3 and/or carrying out one or a plurality of base pair missense mutation and/or connecting the coding sequences of the labels shown in the table at the 5 'end and/or the 3' end. Wherein the DNA molecule shown in the sequence 3 codes for the Tcca-W429A protein shown in the sequence 4.

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

The present invention also provides a biomaterial associated with Tcca-W429A, which biomaterial is any one of the following B1) to B4):

b1 A nucleic acid molecule encoding Tcca-W429A;

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).

In the above biological material, the nucleic acid molecule of B1) may be B11) or B12) or B13) or B14) 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 Tcca-W429A;

b14 Under stringent conditions with a nucleotide sequence defined in b 11) or b 12) or b 13) and encoding a cDNA molecule or DNA molecule of Tcca-W429A.

Wherein the nucleic acid molecule may be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc.

The nucleotide sequence encoding the Tcca-W429A protein of the invention can be easily mutated by a person skilled in the art using known methods, such as directed evolution and point mutation. Those artificially modified nucleotides having 75% or more identity to the nucleotide sequence of the Tcca-W429A protein of the present invention are all derived from and are equivalent to the nucleotide sequence of the present invention as long as they encode the Tcca-W429A protein and function as the Tcca-W429A protein.

The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "identity" includes a nucleotide sequence having 75% or more, or 85% or more, or 90% or more, or 95% or more identity with the nucleotide sequence of a protein consisting of the amino acid sequence shown in the coding sequence 4 of the present invention. Identity can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to evaluate the identity between related sequences.

In the above biological material, the stringent conditions may be as follows: 50℃in 7% Sodium Dodecyl Sulfate (SDS), 0.5M NaPO ₄ Hybridization with 1mM EDTA, rinsing in 2 XSSC, 0.1% SDS at 50 ℃; the method can also be as follows: 50℃in 7% SDS, 0.5M NaPO ₄ Hybridization with 1mM EDTA, rinsing in 1 XSSC, 0.1% SDS at 50 ℃; the method can also be as follows: 50℃in 7% SDS, 0.5M NaPO ₄ Hybridization with 1mM EDTA, rinsing in 0.5 XSSC, 0.1% SDS at 50 ℃; the method can also be as follows: 50℃in 7% SDS, 0.5M NaPO ₄ Hybridization with 1mM EDTA, rinsing in 0.1 XSSC, 0.1% SDS at 50 ℃; the method can also be as follows: 50℃in 7% SDS, 0.5M NaPO ₄ Hybridization with 1mM EDTA, rinsing in 0.1 XSSC, 0.1% SDS at 65 ℃; the method can also be as follows: hybridization was performed in a solution of 6 XSSC, 0.5% SDS at 65℃and then washed once with 2 XSSC, 0.1% SDS and 1 XSSC, 0.1% SDS; the method can also be as follows: hybridization and washing the membrane 2 times at 68℃in a solution of 2 XSSC, 0.1% SDS for 5min each time, and hybridization and washing the membrane 2 times at 68℃in a solution of 0.5 XSSC, 0.1% SDS for 15min each time; the method can also be as follows: hybridization and washing of membranes were performed at 65℃in 0.1 XSSPE (or 0.1 XSSC), 0.1% SDS solution.

The 75% or more identity may be 80%, 85%, 90% or 95% or more identity.

In the above biological material, the expression cassette (Tcca-W429A gene expression cassette) described in B2) containing a nucleic acid molecule encoding the Tcca-W429A protein means a DNA capable of expressing the Tcca-W429A protein in a host cell, and the DNA may include not only a promoter for initiating transcription of the Tcca-W429A gene but also a terminator for terminating transcription of the Tcca-W429A gene. Further, the expression cassette may also include an enhancer sequence.

Recombinant vectors containing the Tcca-W429A gene expression cassette can be constructed using existing expression vectors.

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

B3 The recombinant vector may specifically be pET32a-TEV-Tcca-W429A. The pET32a-TEV-Tcca-W429A is a recombinant vector obtained by replacing a DNA fragment between EcoRI and NotI recognition sequences of a pET32a (+) vector with the DNA fragment shown in the 502 th-2064 th positions of the sequence 7. The pET32a-TEV-Tcca-W429A contains a TEV-Tcca-W429A fusion gene shown in a sequence 7, and can express a TEV-Tcca-W429A fusion protein shown in a sequence 8.

In the above biological material, the microorganism may be yeast, bacteria, algae or fungi. The bacterium may be E.coli, such as E.coli BL21 (DE 3) trxB.

The invention also provides a method of hydrolyzing PET, the method comprising: hydrolysis of PET was achieved by treatment of PET with Tcca-W429A.

In the above method, the treatment may be performed at 30 ℃.

In the above method, treatment of PET with Tcca-W429A can be performed in 50mM PBS, pH8.0 buffer.

The invention also provides a method of preparing MHET, the method comprising: PET is hydrolyzed by Tcca-W429A to obtain MHET.

In the above method, the treatment may be performed at 30 ℃.

In the above method, hydrolysis of PET using Tcca-W429A can be performed in 50mM PBS, pH8.0 buffer.

Any of the following applications of Tcca-W429A also fall within the scope of the present invention:

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.

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

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.

Above, the PET may be BHET.

The invention uses the site-directed mutagenesis technology to mutate the original carboxylesterase (Tcca) to obtain a carboxylesterase mutant, thereby changing the problem of lower activity of the original carboxylesterase, effectively improving the activity of the Tcca in degrading BHET and improving the degradation effect of the Tcca. The carboxylesterase mutant of the invention improves the degradation efficiency of BHET (short chain PET) and has good industrial application prospect when the BHET (short chain PET) can relieve the effect of inhibiting the products in PET degradation.

The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.

Drawings

FIG. 1 is an activity analysis of carboxylesterase and mutant proteins thereof. WT represents wild-type carboxylesterase and W429A represents mutant.

Detailed Description

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents, instruments and the like used in the examples described below are commercially available unless otherwise specified. The quantitative tests in the following examples were all set up in triplicate and the results averaged. In the following examples, unless otherwise specified, the 1 st 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.

Bis (2-hydroxyethyl) terephthalate (BHET): oligomers of PET, sigma, cas:959-26-2.

EXAMPLE 1 preparation, expression purification and Activity detection of carboxylesterase mutant

In order to increase the industrial application value of carboxylesterase, the invention synthesizes the gene of the carboxylesterase from Thermobacillus composti and mutates the gene to obtain the mutant with improved enzyme activity.

1. Construction of wild carboxylesterase recombinant plasmid and mutant recombinant plasmid thereof

1. Construction of wild carboxylesterase recombinant plasmid

The wild carboxylesterase is Tcca from Thermobacillus composti, the nucleotide sequence of the wild Tcca is sequence 1 in the sequence table, and the coded amino acid sequence is sequence 2 in the sequence table.

Synthesizing a wild carboxylesterase gene containing a TEV restriction enzyme site, and inserting the wild carboxylesterase gene into EcoRI and NotI restriction enzyme sites in a pET32a (+) vector to obtain a recombinant plasmid, which is marked as pET32a-TEV-Tcca; the amino acid sequence encoded by the recombinant plasmid after induced expression is shown as a sequence 6, namely fusion protein (marked as TEV-Tcca fusion protein) containing wild carboxylesterase, and the encoding gene (marked as TEV-Tcca fusion gene) is shown as a sequence table 5.

The nucleotide sequence of the TEV-Tcca fusion gene containing the TEV enzyme cutting site sequentially comprises the TEV enzyme cutting site (502-522 th site of the sequence 5), nonfunctional amino acid encoding nucleic acid (523-537 th site of the sequence 5) and the Tcca gene (538-2064 th site of the sequence 5) shown in the sequence 1 in the sequence table from the 5' end.

The amino acid sequence of the TEV-Tcca fusion protein consists of a pET32a vector partial fragment (1 st to 167 th positions of a sequence 6), a TEV enzyme cleavage site (168 th to 174 th positions of the sequence 6), nonfunctional amino acid (175 th to 179 th positions of the sequence 6) and Tcca (180 th to 687 th positions of the sequence 6) shown in a sequence 2 in a sequence table from the N end.

Specifically, pET32a-TEV-Tcca is a recombinant vector obtained by replacing a DNA fragment between EcoRI and NotI recognition sequences of pET32a (+) vector with a wild carboxylesterase gene (502 th to 2064 th positions of sequence 5) containing a TEV enzyme cutting site, wherein the recombinant vector contains a TEV-Tcca fusion gene shown as sequence 5 in a sequence table, and can express the TEV-Tcca fusion protein shown as sequence 6 in the sequence table.

2. Recombinant plasmid expressing carboxylesterase mutant

The carboxylesterase mutant was a protein obtained by mutating the wild-type carboxylesterase shown in sequence 2 as follows (the obtained protein was designated Tcca-W429A): the 429 th position of the amino acid sequence is mutated from tryptophan (trpophan) of wild-type carboxylesterase to alanine (alanine). The carboxylesterase mutant Tcca-W429A has a sequence 4 and the nucleotide sequence of the coding gene (i.e. Tcca-W429A gene) has a sequence 3.

PCR was performed using the site-directed mutagenesis technique (site-directed mutagenesis) and the pET32a-TEV-Tcca plasmid as a template, using the primers shown in Table 1, to obtain plasmids expressing carboxylesterase mutants; further, restriction enzyme DpnI was added to react at 37℃to remove the original template. The purified reaction product is transformed into competent cells of escherichia coli, preliminary screening is carried out by antibiotics, DNA sequencing is carried out to determine the successfully mutated gene, and the plasmid for expressing carboxylesterase mutant is obtained and is marked as pET32a-TEV-Tcca-W429A.

Specifically, pET32a-TEV-Tcca-W429A is a recombinant plasmid obtained by replacing the Tcca gene in pET32a-TEV-Tcca with the Tcca-W429A gene, the recombinant plasmid contains a TEV-Tcca-W429A fusion gene (the gene is a DNA fragment obtained by replacing the Tcca gene in the TEV-Tcca fusion gene with the Tcca-W429A gene, the sequence of the TEV-Tcca-W429A fusion gene is sequence 7), and the recombinant plasmid can express the TEV-Tcca-W429A fusion protein (the protein is a protein obtained by replacing the Tcca protein in the TEV-Tcca fusion protein with the Tcca-W429A protein, and the sequence of the TEV-Tcca-W429A fusion protein is sequence 8).

PCR was performed using the site-directed mutagenesis technique (site-directed mutagenesis) and the pET32a-TEV-Tcca plasmid as a template, using the primers shown in Table 1, to obtain plasmids expressing carboxylesterase mutants; further, restriction enzyme DpnI was added to react at 37℃to remove the original template. The purified reaction product is transformed into competent cells of escherichia coli, preliminary screening is carried out by antibiotics, DNA sequencing is carried out to determine the successfully mutated gene, and the plasmid expressing carboxylesterase mutant is obtained and is marked as pET32a-TEV-Tcca-F325A.

Specifically, pET32a-TEV-Tcca-F325A is a recombinant plasmid obtained by replacing the Tcca gene in pET32a-TEV-Tcca with the Tcca-F325A gene, and the recombinant plasmid contains a TEV-Tcca-F325A fusion gene (the gene is a DNA fragment obtained by replacing the Tcca gene in the TEV-Tcca fusion gene with the Tcca-F325A gene, the sequence of the TEV-Tcca-F325A fusion gene is sequence 9), and can express the TEV-Tcca-F325A fusion protein (the protein is a protein obtained by replacing the Tcca protein in the TEV-Tcca fusion protein with the Tcca-F325A protein).

Wherein the sequence of the TEV-Tcca-F325A gene is sequence 9, and the amino acid sequence of the TEV-Tcca-F325A protein is sequence 10.

TABLE 1 site-directed mutagenesis primer

In the above table, W429A refers to the mutation of tryptophan to alanine at amino acid 429 in sequence 2, and F325A refers to the mutation of phenylalanine to alanine at amino acid 325 in sequence 2.

2. Preparation of carboxylesterase mutants and wild-type carboxylesterases

1. Expression purification of wild-type carboxylesterase and mutants

The recombinant plasmids pET32a-TEV-Tcca and pET32a-TEV-Tcca-W429A, pET a-TEV-Tcca-F325A were transformed into competent cells of E.coli BL21 (DE 3) trxB, respectively, and strains were selected in LB dishes containing 100. Mu.g/ml Ampicillin. The strain thus selected was inoculated into 5ml of LB medium for cultivation, and the amount of the strain was further amplified to 200ml of LB medium, and finally amplified to 6L of LB medium for cultivation (37 ℃ C., 220 rpm). When the OD reached 0.6 to 0.8, the culture was cooled to 16 ℃ and the large expression of the enzyme protein was induced by adding IPTG at a final concentration of 0.4mM at 16 ℃ and 220 rpm. After 18 hours of protein-induced expression, the bacterial liquid was centrifuged at 6000rpm for 15 minutes to collect the cells. The cells were resuspended in buffer (25mM tris,150mM NaCl,pH7.5), disrupted by an ultrasonic cell disrupter (sonicator), centrifuged at 16000rpm for 60 minutes at 4℃and the supernatant collected for ready purification in the next step.

In order to obtain a high-purity enzyme protein, a target protein (buffer A:25mM Tris,150mM NaCl,20mM imidazole, pH7.5; buffer B:25mM Tris,150mM NaCl,250mM imidazole, pH 7.5) was eluted with a rapid protein liquid chromatograph (fast protein liquid chromatography; FPLC) sequentially using a nickel ion column substrate, and the target protein was collected. To the collected target protein, 200. Mu.l of TEV protease was added for cleavage, the His tag on the carrier was cleaved off, and the target protein was dialyzed in 5L of dialysate (25mM tris,150mM NaCl,pH7.5), and the dialysate was changed and dialyzed overnight at 4 ℃. And (3) passing the digested target protein through a nickel column again, and collecting the target protein which does not contain His tag and flows out. Dialyzing the purified target protein in buffer (25mM Tris,150mM NaCl,pH 7.5), concentrating, collecting to obtain Tcca protein solution, tcca-W429A protein solution and Tcca-F325A protein solution, and storing at-80deg.C.

3. Comparison of the relative Activity of carboxylesterase mutants and wild-type carboxylesterases

To verify the differences between wild-type carboxylesterase and its mutant, the inventors further determined the degradation activity of both on BHET. The carboxylesterase activity test steps were as follows:

each reaction mixture (1 mL) was in 50mM PBS, pH8.0 buffer, and the reaction system contained 1mM BHET (first dissolved in 20% DMSO), 0.32. Mu.M enzyme (carboxylesterase mutant or wild-type carboxylesterase prepared as described above), the balance 50mM PBS, pH8.0 buffer. The resulting reaction system was allowed to react at 300rpm at 30℃on a shaker for 24 hours. Each reaction was repeated 3 times. After the reaction, the mixture was centrifuged at 12000rpm for 10 minutes, and the supernatant was filtered through a 0.22 μm filter; the filtrate was collected for high performance liquid chromatography (HPLC, agilent 1200) product determination and analysis, analytical column Welch Ultimate XB-C18 column (4.6X105 mm,5 μm, yuehu Seiki Co., ltd.). The mobile phase is 81% pure water, 18% acetonitrile, 1% formic acid, the flow rate is 0.8ml/min, the wavelength is 254nm, and the column temperature is 30 ℃, and the direct elution is carried out for 20min.

The High Performance Liquid Chromatography (HPLC) detection result of the wild carboxylesterase and the mutant shows that the peak appears about 12min in retention time, and Mass spectrometry detection analysis (anion, mass range 50-1000 m/z) is carried out on the peak, which is MHET.

Wild-type and mutant enzyme activities were determined by comparing peak areas of the hydrolysate MHET of carboxylesterase or mutants thereof.

The principle of activity measurement is as follows: in HPLC experiments, the amount of compound in solution is linear with peak area, so the amount of compound in solution can be calculated from the peak area; in the experiment, the catalytic effect of the mutant protein on the substrate is defined by the peak area of the product; the more products, the better the activity of the mutant protein.

The peak area of the 24-hour hydrolysate MHET of the wild-type carboxylesterase was designated as 100%, and the peak area of the hydrolysate MHET of the carboxylesterase mutant was designated as relative enzyme activity as compared with the peak area of the hydrolysate MHET of the wild-type carboxylesterase.

The detection result shows that the carboxylesterase and the mutant thereof can hydrolyze BHET into MHET, but the degradation activity of the Tcca-W429A mutant on the BHET is higher than that of the wild type protein, the generated product MHET is 1.6 times of that of the wild type protein, and the degradation activity of the Tcca-F325A on the BHET is far lower than that of the wild type protein, as shown in figure 1. The mutant Tcca-W429A has good application value.

The present invention is described in detail above. It will be apparent to those skilled in the art that the present invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with respect to specific embodiments, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.

Claims (10)

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

a1 Amino acid sequence is a protein of sequence 4;

a2 A protein which has the same function and is obtained by substituting and/or deleting and/or adding one or more amino acid residues for an amino acid sequence except 429 th site in the sequence 4 in the sequence table;

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.