Dienolide hydrolase mutant and application thereof

Abstract

The invention discloses a diene lactone hydrolase mutant and an application thereof. The protein provided by the invention is a diene lactone hydrolase mutant, the 184th amino acid residue on an amino acid sequence shown as SEQ ID NO: 2 is mutated, and the amino acid residues at other positions are not changed, so that the protein with PBAT hydrolase activity is obtained; the PmDLH mutant T184H is obtained by mutating wild type PmDLH by utilizing a structural analysis and site-directed mutagenesis technology, and compared with the wild type PmDLH, the PmDLH mutant T184H has the advantages that the problem of low enzyme activity of the wild type PmDLH is solved, the activity of the PmDLH in degrading polyester plastic PBAT is effectively improved, the degradation effect of the PmDLH is improved, and the industrial application prospect is good.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering and relates to a diene lactone hydrolase mutant and its application. Background Art

[0002] Plastic products are widely used, and the resulting white pollution problem has received extensive attention worldwide. The large accumulation of plastic waste in the environment has had a serious impact on the ecological environment. Biodegradable aliphatic-aromatic copolyesters represented by Polybutylene adipate terephthalate (PBAT) have advantages such as good flexibility, good thermal stability, and biocompatibility. They are widely used in agricultural films, food packaging, and the textile industry and are an ideal alternative material to solve "white pollution" with good development prospects.

[0003] Polybutylene adipate terephthalate (PBAT) is an aliphatic-aromatic copolyester formed by the polycondensation of terephthalic acid, adipic acid, and 1,4-butanediol and is one of the most widely used biodegradable polyester materials. Due to the presence of flexible aliphatic chains and rigid aromatic rings in the molecular structure of PBAT, it has both good biodegradability and mechanical properties and is suitable for film production, such as being used as an agricultural film in agricultural production.

[0004] Currently, the main methods for treating waste plastics are physical methods, chemical methods, and biological methods. Physical methods mainly use incineration and landfill. Landfill is to bury plastic waste in landfills to prevent it from spreading into the environment. However, due to the long half-life of plastics, they will exist underground for a long time, leading to the pollution of soil and groundwater by plastic waste and posing a great hidden danger to the survival conditions of animals and plants. Chemical methods refer to depolymerizing high-molecular plastics into monomers through hydrolysis, glycolysis, etc., and then synthesizing and processing them into new plastic products. The principle of chemically degrading polyester plastics is to utilize the instability of ester bonds and degrade them into small-molecule substances through chemical reactions. Chemical treatment usually requires strong alkali or strong acid treatment, generating environmental pollutants; moreover, it also requires relatively expensive highly active catalysts and high-temperature conditions, with high energy consumption during the treatment process, and it is difficult to separate the resulting product mixture. These disadvantages limit the large-scale application of this method in industry. Biological methods rely on the metabolism of microorganisms in nature to depolymerize the high-molecular structure of plastics into small-molecule monomers. Compared with physical and chemical methods, biodegradation is more environmentally friendly to the ecological environment. The essence of microbial degradation of plastics is an enzyme-catalyzed process, and using enzymatic methods for biodegradation of polyester plastics is a relatively efficient, mild, and friendly solution.

[0005] The PmDLH of the dienelactone hydrolase family has a degradation effect on the polyester plastic PBAT, improving its activity in degrading PBAT films, providing more theoretical basis and experimental evidence for degrading polyester plastics, and having a positive significance for protecting the green ecological environment. Summary of the Invention

[0006] The object of the present invention is to provide a dienelactone hydrolase mutant and its application.

[0007] In the first aspect, the present invention provides a protein, which is a dienelactone hydrolase mutant, and is any one of the following A1), A2) or A3):

[0008] A1) Mutate the 184th amino acid residue on the amino acid sequence shown in SEQ ID NO: 2, and keep other amino acid residues unchanged to obtain a protein with PBAT hydrolase activity;

[0009] A2) The protein shown is a protein with a homology of more than 99%, more than 95%, more than 90%, more than 85% or more than 80% of other amino acid residues except the mutated amino acid residue in the protein shown in A1), and having PBAT hydrolase activity;

[0010] A3) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of any one of the proteins in A1)-A2).

[0011] In the above-mentioned protein, the 184th amino acid residue is mutated from threonine at the 184th position to histidine.

[0012] In the above-mentioned protein, the amino acid sequence of the protein shown in A1) is SEQ ID NO: 4.

[0013] In the embodiment of the present invention, the A3) is a fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of the protein shown in SEQ ID NO: 4, specifically the TEV-PmDLH-T184H fusion protein, which consists of a start codon and a histidine tag (the 1st - 7th positions of SEQ ID NO: 6), a TEV cleavage site (the 8th - 13th positions of SEQ ID NO: 6), a non-functional amino acid (the 14th - 23rd positions of SEQ ID NO: 6), a starting amino acid (the 24th position of SEQ ID NO: 6) and the PmDLH mutant T184H shown in SEQ ID NO: 4 in the sequence listing from the N-terminus in sequence.

[0014] In the second aspect, the present invention provides a biological material related to the protein in the first aspect, which is any one of the following B1) to B4):

[0015] B1) A nucleic acid molecule encoding the protein in the first aspect;

[0016] B2) An expression cassette containing the nucleic acid molecule described in B1);

[0017] B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

[0018] 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).

[0019] Among the biological materials described above, the nucleic acid molecule described in B1) is any one of the following b1), b2), b3), or b4):

[0020] b1) The coding sequence is the cDNA molecule or DNA molecule shown as SEQ ID NO: 3 in the sequence listing;

[0021] b2) The DNA molecule shown as SEQ ID NO: 3 in the sequence listing;

[0022] b3) A cDNA molecule or DNA molecule that has 75% or more identity with the nucleotide sequence defined by b1) or b2) and encodes the protein described in the first aspect;

[0023] b4) A cDNA molecule or DNA molecule that hybridizes with the nucleotide sequence defined by b1), b2), or b3) under stringent conditions and encodes the protein described in the first aspect.

[0024] In the third aspect, the present invention provides a method for hydrolyzing PBAT, comprising the following steps: treating PBAT with the protein described in the first aspect to achieve hydrolysis of PBAT.

[0025] The above-mentioned treatment is carried out at 50°C.

[0026] In the fourth aspect, the present invention provides a method for preparing TPA, comprising the following steps: hydrolyzing PBAT with the protein described in the first aspect to obtain TPA.

[0027] The above-mentioned treatment is carried out at 50°C.

[0028] In the fourth aspect, the present invention provides the use of the protein described in the first aspect in any one of the following:

[0029] C1) Catalyzing the hydrolysis of PBAT;

[0030] C2) Catalyzing the hydrolysis of PBAT to TPA;

[0031] C3) Preparing a PBAT degrading agent;

[0032] C4) Preparation of a product for catalyzing the hydrolysis of PBAT into TPA.

[0033] In a fifth aspect, the present invention provides the use of the biomaterial described in the second aspect in any one of the following:

[0034] C1) Catalyzing the hydrolysis of PBAT;

[0035] C2) Catalyzing the hydrolysis of PBAT into TPA;

[0036] C3) Preparation of a PBAT degrading agent;

[0037] C4) Preparation of a product for catalyzing the hydrolysis of PBAT into TPA.

[0038] Experimental evidence of the present invention shows that by using structural analysis and site-directed mutagenesis techniques, the wild-type PmDLH was mutated to obtain the PmDLH mutant T184H. Compared with the wild-type PmDLH, the problem of low enzyme activity of the wild-type PmDLH was solved, the activity of PmDLH in degrading the polyester plastic PBAT was effectively improved, the degradation effect of PmDLH was enhanced, and it has good prospects for industrial application. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 Activity analysis of PmDLH and its mutant proteins; WT represents wild-type PmDLH, and T184H represents the PmDLH mutant T184H. DETAILED DESCRIPTION OF THE INVENTION

[0040] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods.

[0041] Unless otherwise specified, all materials, reagents, etc. in the following examples can be obtained from commercial sources.

[0042] The raw materials and equipment used in the specific embodiments of the present invention are all known products and are obtained by purchasing commercially available products.

[0043] In the following examples, unless otherwise specified, 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.

[0044] Terephthalic acid (TPA): sigma, cas: 100-21-0.

[0045] Example 1: Preparation, expression purification and activity detection of PmDLH mutants

[0046] To increase the industrial application value of wild-type PmDLH, the present invention synthesized the gene (SEQ ID NO: 1) derived from Pyrinomonas methylalipatogenes, expressed and purified it. After studying the predicted protein structure of PmDLH, the amino acids involved in substrate interaction near its active region were selected for mutation to increase the enzyme activity towards the substrate PBAT.

[0047] I. Construction of recombinant plasmids of wild-type PmDLH and its mutant

[0048] 1. Construction of recombinant plasmid of wild-type PmDLH

[0049] Wild-type PmDLH is derived from Pyrinomonas methylalipatogenes. The nucleotide sequence of this enzyme is SEQ ID NO: 1 in the sequence listing, and the amino acid sequence it encodes is SEQ ID NO: 2 in the sequence listing.

[0050] The nucleotide of wild PmDLH gene containing TEV cleavage site (SEQ ID NO: 5) was inserted into the EcoRI and NotI cleavage sites of pET32a vector (Novagen, 69015-3) to obtain a recombinant plasmid (denoted as pET32a-TEV-PmDLH); the amino acid sequence encoded by this recombinant plasmid after induced expression, in addition to the sequence of wild-type PmDLH, has a vector sequence, a histidine tag and a TEV cleavage site at its N-terminus. The amino acid sequence after induced expression is as shown in SEQ ID No: 6, named the fusion protein of wild-type PmDLH, and the encoding gene of this protein is named the fusion gene of wild-type PmDLH.

[0051] The nucleotide sequence of the fusion gene of wild-type PmDLH (denoted as TEV-PmDLH fusion gene) is SEQ ID NO: 5, which consists of a start codon and a histidine tag (positions 1-21 of SEQ ID NO: 5), a TEV cleavage site (positions 22-39 of SEQ ID NO: 5), nucleic acids encoding non-functional amino acids (positions 40-69 of SEQ ID NO: 5), a start codon (positions 70-72 of SEQ ID NO: 5) and the PmDLH gene shown in SEQ ID NO: 1 in the sequence listing (positions 73-879 of SEQ ID NO: 5) from the 5′ end in sequence.

[0052] The amino acid sequence of the fusion protein of wild-type PmDLH (denoted as TEV-PmDLH fusion protein) is SEQ ID NO: 6, which consists of, from the N-terminus, a start codon and a histidine tag (positions 1-7 of SEQ ID NO: 6), a TEV cleavage site (positions 8-13 of SEQ ID NO: 6), non-functional amino acids (positions 14-23 of SEQ ID NO: 6), a start amino acid (position 24 of SEQ ID NO: 6), and the PmDLH protein shown in SEQ ID NO: 2 in the sequence listing (positions 25-293 of SEQ ID NO: 6).

[0053] 2. Recombinant plasmid expressing PmDLH mutant

[0054] After studying the predicted protein structure of wild-type PmDLH, amino acids involved in substrate interaction near its active region were selected for site-directed mutagenesis to increase the enzyme's activity towards the substrate PBAT. That is, in the present invention, PmDLH was mutated using structure analysis and site-directed mutagenesis techniques to obtain a PmDLH mutant, thus changing the problem of low original PBAT hydrolase activity and improving the activity of PmDLH in degrading PBAT.

[0055] The following PmDLH mutants were determined through research:

[0056] Using the site-directed mutagenesis technique, with the pET32a-TEV-PmDLH plasmid as a template, PCR was performed using the primers shown in Table 1 to obtain the plasmid of the PmDLH mutant; then, the restriction enzyme DpnI was added and reacted at 37 °C to remove the original template. The purified reaction product was transformed into Escherichia coli competent cells, and preliminary screening was carried out with antibiotics. DNA sequencing was performed to determine the successfully mutated gene, and the plasmid expressing the PmDLH mutant T184H, denoted as pET32a-TEV-PmDLH-T184H, was obtained. This plasmid contains the TEV-PmDLH-T184H fusion gene and expresses the TEV-PmDLH-T184H fusion protein.

[0057] The TEV-PmDLH-T184H fusion gene consists of, from the 5′ end, a start codon and a histidine tag (positions 1-21 of SEQ ID NO: 5), a TEV cleavage site (positions 22-39 of SEQ ID NO: 5), nucleic acids encoding non-functional amino acids (positions 40-69 of SEQ ID NO: 5), a start codon (positions 70-72 of SEQ ID NO: 5), and the PmDLH mutant T184H gene shown in SEQ ID NO: 3 in the sequence listing.

[0058] The TEV-PmDLH-T184H fusion protein consists of, starting from the N-terminus, a start codon and a histidine tag (positions 1-7 of SEQ ID NO: 6), a TEV protease cleavage site (positions 8-13 of SEQ ID NO: 6), non-functional amino acids (positions 14-23 of SEQ ID NO: 6), a starting amino acid (position 24 of SEQ ID NO: 6), and the PmDLH mutant T184H shown in SEQ ID NO: 4 in the sequence listing.

[0059] The PmDLH mutant T184H is obtained by mutating the 184th threonine of the wild-type PmDLH shown in SEQ ID NO: 2 to histidine, with the amino acid residues at other positions remaining unchanged. The amino acid sequence of the PmDLH mutant T184H is SEQ ID NO: 4, and the nucleotide sequence of its encoding gene is SEQ ID NO: 3;

[0060] The recombinant plasmid expressing the PmDLH mutant T184H is obtained by replacing the fragment between the EcoRI and NotI cleavage sites in the pET32a vector with the TEV-PmDLH-T184H fusion gene, resulting in a recombinant plasmid (designated pET32a-TEV-PmDLH-T184H). This recombinant plasmid contains the TEV-PmDLH-T184H fusion gene and can express the TEV-PmDLH-T184H fusion protein.

[0061] Table 1 shows the site-directed mutagenesis primers

[0062] Mutation site Primer (5’→3’) T184H GTGATTTTTTATGGCCGTcacCCGCTGGAACTGGTGGAT

[0063] In the table, T184H means that the 184th amino acid in SEQ ID NO: 2 is mutated from threonine to histidine.

[0064] II. Preparation of the PmDLH mutant T184H and wild-type PmDLH

[0065] 1. Expression and purification of wild-type PmDLH and the PmDLH mutant T184H

[0066] The above-prepared recombinant plasmid pET32a-TEV-PmDLH expressing wild-type PmDLH and the recombinant plasmid pET32a-TEV-PmDLH-T184H expressing the PmDLH mutant T184H were separately transformed into Escherichia coli BL21(DE3) competent cells, and the strains were screened on LB plates containing 100 μg / ml Ampicillin. The screened strains were inoculated into 5 ml of LB for culture, and then the bacterial volume was amplified to 200 ml of LB for culture, and finally amplified to 6 L of LB medium for culture (37 °C, 220 rpm). When the OD value reached 0.6 to 0.8, the culture was cooled to 16 °C, and at 16 °C and 220 rpm, IPTG with a final concentration of 0.4 mM was added to induce the overexpression of the enzyme protein. After 18 hours of protein induction expression, the bacterial solution was centrifuged at 6000 rpm for 15 minutes to collect the cells. The cells were resuspended with a buffer (25 mM tris, 300 mM NaCl, pH 8.0), and the bacteria were lysed using a sonicator (1000 bar, 15 min), and then centrifuged at 16000 rpm at 4 °C for 60 minutes to collect the supernatant for the next purification step.

[0067] To obtain highly pure enzyme protein, the supernatant was subjected to fast protein liquid chromatography (FPLC) to elute the target protein successively using a nickel ion chromatography column substrate (buffer A: 25 mM Tris, 300 mM NaCl, 20 mM imidazole, pH 8.0; buffer B: 25 mM Tris, 300 mM NaCl, 250 mM imidazole, pH 8.0), and the buffer B gradient eluate was collected to obtain the target protein. 200 μl of TEV protease (Guangzhou Elabscience Biotechnology Co., Ltd., YVV16201-100ug) was added to the target protein for enzymatic cleavage, aiming to cut off the His tag on the vector, and at the same time, the target protein was dialyzed in 5 L of dialysis buffer (25 mM tris, 150 mM NaCl, pH 8.0). After 3 hours, the dialysis buffer was changed, and dialysis was carried out overnight at 4 °C. The target protein after enzymatic cleavage was passed through the nickel column again to collect the target protein without the His tag that flowed through. The target protein after two purifications was dialyzed in a buffer (25 mM Tris, 150 mM NaCl, the balance being water, pH 8.0), concentrated and collected, and stored at -80 °C to obtain a wild-type PmDLH solution (concentration 20 mg / ml) and a PmDLH mutant T184H solution (concentration 20 mg / ml).

[0068] The protein sizes in the wild-type PmDLH solution and the PmDLH mutant T184H solution were detected by SDS-PAGE to be 30KD respectively, which was consistent with the expectation.

[0069] III. Comparison of the relative activities of wild-type PmDLH and PmDLH mutant T184H

[0070] To verify the differences between wild-type PmDLH and PmDLH mutant T184H, the present invention further determined their degradation activities towards PBAT. The steps of the PBAT activity test method are as follows:

[0071] The mixture (1 mL) for each reaction was in a buffer of 50 mM PBS pH 8.0, including the substrate PBAT membrane (CAS: 55231-08-8, Shanghai Macklin Biochemical Co., Ltd., 0.8 cm × 0.8 cm) and 10 mM enzyme (the PmDLH mutant T184H or wild-type PmDLH prepared above), and the balance was the buffer of 50 mM PBS pH 8.0. It was placed on a shaker and reacted at 50 °C and 300 rpm for 24 hours. Each reaction was repeated 3 times. After the reaction, the mixture was first centrifuged at 12000 rpm for 10 minutes, the supernatant was taken and an equal volume of ice methanol was added, and after mixing, the reaction solution was filtered through a 0.22 μm filter; high performance liquid chromatography (HPLC, Agilent 1200) was used for product determination and analysis, and the analytical column was Welch Ultimate XB-C18 column (4.6 × 250 mm, 5 μm, Yuexu Technology (Shanghai) Co., Ltd.). The mobile phase was 0.1% formic acid water and acetonitrile, the flow rate was 0.8 ml / min, and gradient elution was carried out at 240 nm: 0 - 9 min: 25% acetonitrile + 75% formic acid water; 10 - 20 min: 55% acetonitrile + 45% formic acid water), and the column oven temperature was 30 °C.

[0072] The high performance liquid chromatography (HPLC) detection results of wild-type PmDLH and PmDLH mutant T184H showed a peak at about 6 min of retention time, and the retention time of the standard product TPA was 6 min. It could be judged that the substances produced by wild-type PmDLH and the mutant were TPA. The results showed that the hydrolysis products of wild-type PmDLH or its mutant of the present invention were all TPA.

[0073] By comparing the peak areas of the hydrolysis product TPA of wild-type PmDLH and PmDLH mutant T184H, the enzyme activities of the wild-type and mutant were determined.

[0074] The principle of activity determination is as follows: In the HPLC experiment, the amount of the compound in the solution is linearly related to the peak area. Therefore, the amount of the compound in the solution can be calculated by the peak area. In this experiment, the catalytic effect of the mutant protein on the substrate is defined by the peak area of the product. The more the product (since the substrate is insoluble and difficult to quantify), the better the activity of the mutant protein.

[0075] Taking the peak area of the hydrolysis product TPA of wild-type PmDLH as 100%, the peak area of the hydrolysis product TPA of the PmDLH mutant T184H is compared with the peak area of the hydrolysis product TPA of wild-type PmDLH, and is denoted as the relative enzyme activity.

[0076] The detection results are as Figure 1 shown. Both PmDLH of the present invention (denoted as WT in the figure) and the PmDLH mutant T184H (denoted as T184H in the figure) can hydrolyze PBAT into TPA. The degradation activity of T184H on PBAT is higher than that of the wild-type protein. The product TPA catalyzed by the mutant T184H is 3.4 times that of the wild-type, indicating that the PmDLH mutant T184H has good application value.

[0077] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention and without unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to cover any modification, use or improvement of the present invention, including changes made by using conventional techniques known in the art that are outside the scope disclosed in this application. Some basic features can be applied according to the scope of the appended claims below.

Claims

1. A protein, which is any one of A1), A2) or A3) as follows: A1) A protein with PBAT hydrolase activity is obtained by mutating the 184th amino acid residue in the amino acid sequence shown in SEQ ID NO: 2, while keeping other amino acid residues unchanged; A2) The protein as described is a protein with PBAT hydrolase activity, in which, except for the mutated amino acid residue in the protein described in A1), the homology of other amino acid residues is greater than 99%, 95%, 90%, 85% or 80% homology, and; A3) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of any one of the proteins described in A1)-A2).

2. The protein according to claim 1, wherein: The 184th amino acid residue is mutated from threonine at the 184th position to histidine.

3. The protein according to claim 1 or 2, characterized in that: The amino acid sequence of the protein described in A1) is SEQ ID NO:

4.

4. A biological material related to the protein according to any one of claims 1-3, which is any one of the following B1) to B4): B1) A nucleic acid molecule encoding the protein according to any one of claims 1-3; 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).

5. The biomaterial according to claim 4, wherein: The nucleic acid molecule described in B1) is any one of the following b1), b2), b3) or b4): b1) The coding sequence is the cDNA molecule or DNA molecule shown in SEQ ID NO: 3 in the sequence listing; b2) The DNA molecule shown in SEQ ID NO: 3 in the sequence listing; b3) A cDNA molecule or DNA molecule having 75% or more identity with the nucleotide sequence defined in b1) or b2) and encoding the protein according to claim 1; b4) A cDNA molecule or DNA molecule that hybridizes with the nucleotide sequence defined in b1), b2) or b3) under stringent conditions and encodes the protein according to any one of claims 1-3.

6. A method for hydrolyzing PBAT, comprising the following steps: treating PBAT with the protein according to any one of claims 1-3 to achieve hydrolysis of PBAT.

7. A method for preparing TPA, comprising the following steps: hydrolyzing PBAT with the protein according to any one of claims 1-3 to obtain TPA.

8. Use of the protein according to any one of claims 1-3 in any one of the following: C1) Catalyzing the hydrolysis of PBAT; C2) Catalyzing the hydrolysis of PBAT to TPA; C3) Preparing a PBAT degrading agent; C4) Preparing a product for catalyzing the hydrolysis of PBAT to TPA.

9. Use of the biological material according to claim 4 or 5 in any one of the following: C1) Catalyzing the hydrolysis of PBAT; C2) Catalyzing the hydrolysis of PBAT to TPA; C3) Preparing a PBAT degrading agent; C4) Preparing a product for catalyzing the hydrolysis of PBAT to TPA.

Images