CN117757770A - Carboxylesterase mutant and application thereof - Google Patents

Abstract

The invention discloses carboxylesterase mutants and their applications. The carboxylesterase mutant disclosed in the present invention is as follows A1), A2) or A3): A1) the amino acid sequence is the protein of sequence 4; A2) the amino acid sequence other than the 429th position in sequence 4 in the sequence listing is passed through a or A protein with the same function that has the substitution and/or deletion and/or addition of several amino acid residues; 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 carboxylesterase (Tcca) to obtain a carboxylesterase mutant, which changes the problem of low activity of the original carboxylesterase and effectively improves the activity of Tcca in degrading BHET. , improve the degradation effect of Tcca, the carboxylesterase mutant of the present invention improves the degradation efficiency of BHET, and has good industrial application prospects.

Description

Carboxylesterase mutants and their applications

Technical field

The present invention relates to carboxylesterase mutants and their applications in the field of genetic engineering.

Background technique

The polyester plastic PET (such as polyethylene terephthalate) is mainly used as a synthetic polyester fabric in the textile industry and as a packaging material for food and beverages. PET has become one of the most important mass-produced petrochemical plastics. Most PET products have a high degree of crystallinity and are durable against mechanical and chemical stress. PET is made of terephthalic acid (TPA) and ethylene glycol (EG) polymerized through ester bonds. It is stable and difficult to decompose. It is often used in mineral water bottles, polyester clothes, blister packaging and other products.

At present, the main methods for processing plastic PET waste are: landfill, incineration and recycling. Although landfilling and incineration are simple, the waste gas and waste water produced will cause secondary pollution to the environment; due to the economical cost of recycling and performance issues with recycled plastics, the current recycling rate is low. In recent years, more and more researchers have paid attention to the biodegradation of plastics. Compared with physical and chemical degradation methods, biodegradation is more environmentally friendly and ecologically friendly. For example, using enzymatic degradation to degrade PET into its component molecules, and then recycling and reusing it, is the real decomposition of plastics, and it is one of the most ideal processing methods. This method not only solves the problem of PET waste, but also can be recycled. In the past decade or so, scientists have discovered the activity of hydrolases such as esterase, lipase and cutinase in degrading PET, proving the possibility of PET biodegradation. These hydrolases all belong to the α/β hydrolase family.

Carboxylesterase is structurally similar to lipase, has a conserved Ser-Glu/Asp-His triplet structure, and belongs to the α/β hydrolase family, but there are differences in substrate specificity. Carboxyl esters Enzymes prefer to hydrolyze substrates with smaller acyl chain lengths, whereas lipases prefer to hydrolyze substrates with larger acyl chain lengths. Carboxylesterase has good degradation activity against BHET, which is an intermediate product of PET hydrolysis. The accumulation of reaction intermediate products is an important factor limiting the degradation efficiency of PET hydrolase. Therefore, improving the activity of BHET-degrading enzymes will provide more theoretical basis and experimental basis for PET degradation research, which is of positive significance for protecting the human ecological environment.

Contents of the invention

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

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

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

A2) A protein with 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 429 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 Tcca-W429A 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 Tcca-W429A protein in A2) above can be synthesized artificially, or its encoding gene can be synthesized first and then biologically expressed.

The gene encoding the Tcca-W429A 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 Tcca-W429A protein shown in sequence 4.

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

The present invention also provides biological materials related to Tcca-W429A, and the biological materials are any one of the following B1) to B4):

B1) Nucleic acid molecule encoding Tcca-W429A;

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 Tcca-W429A;

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 Tcca-W429A.

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 Tcca-W429A protein of the present invention. Those artificially modified nucleotides that have 75% or higher identity with the nucleotide sequence of the Tcca-W429A protein of the present invention, as long as they encode the Tcca-W429A protein and have the function of the Tcca-W429A protein, are all 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 Tcca-W429A protein (Tcca-W429A gene expression cassette) described in B2) refers to DNA that can express Tcca-W429A protein in host cells. This DNA can not only It includes a promoter that initiates the transcription of the Tcca-W429A gene, and may also include a terminator that terminates the transcription of the Tcca-W429A gene. Furthermore, the expression cassette may also include an enhancer sequence.

Existing expression vectors can be used to construct a recombinant vector containing the Tcca-W429A 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-Tcca-W429A. The pET32a-TEV-Tcca-W429A is a recombinant vector obtained by replacing the DNA fragment between the EcoRI and NotI recognition sequences of the pET32a(+) vector with the DNA fragment shown at positions 502-2064 of Sequence 7. The pET32a-TEV-Tcca-W429A contains the TEV-Tcca-W429A fusion gene shown in sequence 7 and can express the TEV-Tcca-W429A 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 invention also provides a method for hydrolyzing PET, which method includes: treating PET with Tcca-W429A 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 Tcca-W429A can be carried out in 50mM PBS, pH 8.0 buffer.

The invention also provides a method for preparing MHET, which method includes: hydrolyzing PET using Tcca-W429A to obtain MHET.

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

In the above method, the hydrolysis of PET using Tcca-W429A can be carried out in 50mM PBS, pH 8.0 buffer.

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

1) As PET hydrolase or 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 carboxylesterase (Tcca) to obtain a carboxylesterase mutant, which changes the problem of low activity of the original carboxylesterase and effectively improves the activity of Tcca in degrading BHET. , improve the degradation effect of Tcca. BHET (short chain PET) can relieve the product inhibition effect in PET degradation. The carboxylesterase mutant of the present invention 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 carboxylesterase and its mutant proteins. WT indicates wild-type carboxylesterase and W429A indicates the mutant.

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. The quantitative experiments in the following examples were repeated three times, and the results were averaged. 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.

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

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

In order to increase the industrial application value of carboxylesterase, the present invention synthesizes the carboxylesterase gene derived from Thermobacilluscomposti, mutates it, and obtains a mutant with improved enzyme activity.

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

1. Construction of wild-type carboxylesterase recombinant plasmid

The wild-type carboxylesterase is Tcca derived from Thermobacillus composti. The nucleotide sequence of wild-type Tcca is sequence 1 in the sequence listing, and its encoded amino acid sequence is sequence 2 in the sequence listing.

The wild-type carboxylesterase gene containing the TEV restriction site was synthesized and inserted into the EcoRI and NotI restriction sites in the pET32a(+) vector to obtain a recombinant plasmid, designated pET32a-TEV-Tcca; this recombinant plasmid In addition to the wild-type carboxylesterase sequence, the amino acid sequence encoded 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, which is named as containing wild-type carboxylesterase. The fusion protein of acid esterase (denoted as TEV-Tcca fusion protein), and its encoding gene (denoted as TEV-Tcca fusion gene) is shown in 5 in the sequence list.

The nucleotide sequence of the TEV-Tcca fusion gene containing a TEV enzyme cleavage site consists of a TEV enzyme cleavage site (positions 502-522 of Sequence 5) and a non-functional amino acid encoding nucleic acid (positions 5 of Sequence 5) starting from the 5' end. 523-537) and the Tcca gene shown in sequence 1 in the sequence listing (positions 538-2064 of sequence 5).

The amino acid sequence of the TEV-Tcca fusion protein consists of the pET32a vector partial fragment (positions 1-167 of sequence 6), TEV restriction site (positions 168-174 of sequence 6), and non-functional amino acids (positions 168-174 of sequence 6) from the N-terminus. 6) and Tcca shown in sequence 2 in the sequence listing (positions 180-687 of sequence 6).

Specifically, pET32a-TEV-Tcca replaces the DNA fragment between the EcoRI and NotI recognition sequences of the pET32a(+) vector with the wild-type carboxylesterase gene containing the TEV enzyme cleavage site (positions 502-2064 of Sequence 5 ), the recombinant vector contains the TEV-Tcca fusion gene shown in Sequence 5 in the Sequence Listing, and can express the TEV-Tcca fusion protein shown in Sequence 6 in the Sequence Listing.

2. Recombinant plasmid expressing carboxylesterase mutant

The carboxylesterase mutant is a protein obtained by mutating the wild-type carboxylesterase shown in sequence 2 as follows (the resulting protein is recorded as Tcca-W429A): position 429 of its amino acid sequence is modified by the wild-type carboxylesterase Tryptophan is mutated into alanine. The amino acid sequence of carboxylesterase mutant Tcca-W429A is sequence 4, and the nucleotide sequence of its encoding gene (ie, Tcca-W429A gene) is sequence 3.

Utilize site-directed mutagenesis, use the pET32a-TEV-Tcca plasmid as a template, and perform PCR with the primers shown in Table 1 to obtain a plasmid expressing the carboxylesterase mutant; then add the restriction endonuclease DpnI React at 37°C to remove 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 carboxylesterase mutant was obtained, which was designated 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 the TEV-Tcca-W429A fusion gene (this gene is the TEV- The Tcca gene in the Tcca fusion gene is replaced with a DNA fragment obtained by the Tcca-W429A gene. The sequence of the TEV-Tcca-W429A fusion gene is sequence 7), which can express the TEV-Tcca-W429A fusion protein (the protein is a fusion of TEV-Tcca The Tcca protein in the protein is replaced with the Tcca-W429A protein. The sequence of the TEV-Tcca-W429A fusion protein is sequence 8).

Utilize site-directed mutagenesis, use the pET32a-TEV-Tcca plasmid as a template, and perform PCR with the primers shown in Table 1 to obtain a plasmid expressing the carboxylesterase mutant; then add the restriction endonuclease DpnI React at 37°C to remove 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 carboxylesterase mutant was obtained, which was designated 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. The recombinant plasmid contains the TEV-Tcca-F325A fusion gene (this gene is the TEV- The Tcca gene in the Tcca fusion gene is replaced with a DNA fragment obtained by the Tcca-F325A gene. The sequence of the TEV-Tcca-F325A fusion gene is sequence 9), which can express the TEV-Tcca-F325A fusion protein (the protein is a fusion of TEV-Tcca Protein obtained by replacing the Tcca protein in the protein with Tcca-F325A protein).

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

Table 1. Site-directed mutagenesis primers

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

2. Preparation of carboxylesterase mutants and wild-type carboxylesterase

1. Expression and purification of wild-type carboxylesterase and mutants

The recombinant plasmids pET32a-TEV-Tcca expressing wild-type carboxylesterase and the recombinant plasmids pET32a-TEV-Tcca-W429A and pET32a-TEV-Tcca-F325A expressing carboxylesterase mutants prepared above were transformed into the large intestine respectively. Bacillus BL21(DE3)trxB competent cells, and 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, and collect the supernatant for use. Prepare for the 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 200 microliters of TEV protease to the collected target protein for enzymatic digestion in order to cut off the His tag on the carrier. At the same time, the target protein is dialyzed into 5L dialysate (25mM tris, 150mM NaCl, pH7.5) and replaced. Add the dialysate and dialyze 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. The target protein after twice purification was dialyzed in buffer (25mM Tris, 150mM NaCl, pH 7.5), concentrated and collected to obtain Tcca protein solution, Tcca-W429A protein solution, and Tcca-F325A protein solution respectively, and stored at -80°C. ,spare.

3. Comparison of relative activities between carboxylesterase mutants and wild-type carboxylesterase

In order to verify the difference between wild-type carboxylesterase and its mutant, the inventors further measured their BHET degradation activities. The steps for testing carboxylesterase activity are as follows:

The mixture (1 mL) of each reaction system is in 50mM PBS, pH 8.0 buffer. The reaction system contains 1mM BHET (first dissolved in 20% DMSO), 0.32μM enzyme (carboxylesterase mutant prepared above) or wild-type carboxylesterase), and the balance is 50mM PBS, pH 8.0 buffer. The obtained reaction system was placed on a shaker at 30°C and 300 rpm for 24 hours. Each reaction was repeated three times. After the reaction, the mixture was centrifuged at 12,000 rpm for 10 minutes, and 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 and analysis, and 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 carboxylesterase and mutants show that the peaks emerge at a retention time of about 12 minutes. The peaks are analyzed by mass spectrometry (negative ions, Mass range 50-1000m/z), which is MHET. .

Wild-type and mutant enzyme activities were determined by comparing the peak areas of the hydrolysis product MHET of carboxylesterase 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 product; the more products, the better the activity of the mutant protein.

Taking the peak area of the 24-hour hydrolysis product MHET of the wild-type carboxylesterase as 100%, the peak area of the hydrolysis product MHET of the carboxylesterase mutant is compared with the peak area of the hydrolysis product MHET of the wild-type carboxylesterase. , recorded as relative enzyme activity.

The test results show that both the carboxylesterase and its mutants of the present invention can hydrolyze BHET into MHET, but the Tcca-W429A mutant has a higher BHET degradation activity than the wild-type protein, and the product MHET produced is 1.6 times that of the wild-type. The BHET degradation activity of Tcca-F325A is much lower than that of the wild-type protein, as shown in Figure 1. It shows that the mutant Tcca-W429A 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, which is the following A1), A2) or A3): A1) The amino acid sequence is a protein of sequence 4; A2) A protein with 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 429 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). 2. Biological material related to the protein of claim 1, which is any one of the following B1) to B4): B1) 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) 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). 3. The biological material according to claim 2, characterized in that: B1) the nucleic acid molecule is 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 the protein of claim 1; 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 the protein of claim 1. 4. A method for hydrolyzing PET, comprising: using the protein of claim 1 to treat PET to achieve hydrolysis of PET. 5. The method according to claim 4, characterized in that the treatment is carried out at 30°C. 6. A method for preparing MHET, comprising: hydrolyzing PET using the protein of claim 1 to obtain MHET. 7. The method according to claim 6, characterized in that the treatment is carried out at 30°C. 8. Any of the following applications of the protein according to claim 1: 1) As PET hydrolase or 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) preparing degradable PET products; 8) Preparation of catalytic PET hydrolysis into MHET and/or TPA products. 9. Any of the following applications of the biological material according to claim 2 or 3: 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. 10. The method according to any one of claims 4-7, or the application according to claim 8 or 9, characterized in that: PET is BHET.