A mutant of esterase from thermophilic bacteria and use thereof

Abstract

The application discloses a thermophilic bacteria-derived esterase mutant and application thereof. The application provides a protein, wherein the amino acid residue at the 211th position in the amino acid sequence shown in SEQ ID NO: 4 is mutated, and the amino acid residues at other positions are not changed, so that a protein with PBAT hydrolytic enzyme activity is obtained; or the amino acid residues at the 211th and 256th positions in the amino acid sequence shown in SEQ ID NO: 4 are mutated, and the amino acid residues at other positions are not changed, so that a protein with PBAT hydrolytic enzyme activity is obtained; the wild type Tcur-lipase is mutated by using structure analysis and site-directed mutagenesis technology, and the Tcur-lipase mutants N211A and N211A / D256A are obtained; the Tcur-lipase mutant improves the degradation efficiency of PBAT, and has a good industrial application prospect.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering and relates to an esterase mutant derived from thermophilic bacteria and its application. Background Technology

[0002] Plastic products are difficult to degrade and, when present in the natural environment for extended periods, pose a serious threat to ecosystems, impacting biodiversity and potentially leading to the extinction or migration of some species. When plastic products decompose in the natural environment, they release fine particles and toxic gases, negatively impacting air quality and potentially endangering human health. Polybutylene terephthalate (PBAT), an aliphatic-aromatic copolyester formed by the condensation polymerization of terephthalic acid (TA), adipic acid (AA), and 1,4-butanediol (BD), is currently one of the most widely used biodegradable polyester materials. Due to the flexible aliphatic chains and rigid aromatic rings in its molecular structure, PBAT possesses both excellent biodegradability and mechanical properties, making it suitable for film production. For example, it can be used as agricultural mulch film in agricultural production, representing an ideal alternative to address "white pollution" and showing promising development prospects.

[0003] Currently, the main methods for treating waste plastics include physical, chemical, and biological methods. Physical methods primarily involve incineration and landfill. Landfilling involves burying waste plastics in landfills to prevent their spread to the surrounding environment. However, due to the long half-life of plastics, they persist underground for extended periods, potentially causing soil and groundwater pollution and posing a potential threat to the survival of plants and animals.

[0004] Chemical methods break down polymeric plastics into monomers through reactions such as hydrolysis and glycolysis, and then synthesize entirely new plastic products. However, chemical processing of plastics typically requires strong bases or acids, which may generate environmental pollutants. Furthermore, the need for highly active catalysts and high-temperature conditions leads to high energy consumption, and the separation of product mixtures is relatively difficult. These factors limit the large-scale industrial application of this method.

[0005] In contrast, biological degradation relies on the metabolic capabilities of microorganisms in nature to break down polymeric plastics into smaller monomers. Compared to physical and chemical methods, biodegradation is more environmentally friendly. The core of microbial degradation lies in the catalytic action of enzymes; using enzymatic methods to biodegrade polyester plastics is an efficient, gentle, and environmentally friendly solution. Biodegradation of plastics is a promising sustainable solution that can more effectively degrade waste plastics and reduce adverse impacts on the ecological environment.

[0006] The triglyceride lipase family of tcur-lipases plays a crucial role in the degradation of the polyester plastic PBAT, and mutants significantly enhance their degradation activity on its membranes. This discovery provides a more solid theoretical foundation and experimental support for research on the degradation of polyester plastics, thereby having an important and positive impact on maintaining a green ecological environment. Summary of the Invention

[0007] The purpose of this invention is to provide an esterase mutant derived from thermophilic bacteria and its application.

[0008] In a first aspect, the present invention provides a protein, which is any one of the following (A1)-A6):

[0009] A1) Mutate the 211th amino acid residue in the amino acid sequence shown in SEQ ID NO: 4 and remove the N-terminal tag sequence, while leaving the amino acid residues at other positions unchanged, to obtain a protein with PBAT hydrolase activity.

[0010] A2) Mutate the amino acid residues at positions 211 and 256 of the amino acid sequence shown in SEQ ID NO: 4, and remove the N-terminal tag sequence, while leaving the amino acid residues at other positions unchanged, to obtain a protein with PBAT hydrolase activity.

[0011] A3) Mutate the 211th amino acid residue in the amino acid sequence shown in SEQ ID NO: 4, while keeping the amino acid residues at other positions unchanged, to obtain a protein with PBAT hydrolase activity.

[0012] A4) Mutate the amino acid residues at positions 211 and 256 of the amino acid sequence shown in SEQ ID NO: 4, while leaving the amino acid residues at other positions unchanged, to obtain a protein with PBAT hydrolase activity.

[0013] The protein shown in A5) is any protein shown in A1)-A4) that, except for the mutant amino acid residues, has a homology of more than 99%, more than 95%, more than 90%, more than 85%, or more than 80% of other amino acid residues, and has PBAT hydrolase activity.

[0014] A6) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of any of the proteins described in A1)-A2).

[0015] In the protein described above, the 211th amino acid residue is mutated to an asparagine residue that is mutated to alanine.

[0016] The amino acid residues at positions 211 and 256 are mutated so that asparagine at position 211 is mutated to alanine, and aspartic acid at position 256 is mutated to alanine.

[0017] Of the proteins mentioned above,

[0018] The amino acid sequence of the protein shown in A3) is SEQ ID NO: 6;

[0019] The amino acid sequence of the protein shown in A4 is SEQ ID NO: 8.

[0020] In a second aspect, the present invention provides biological materials related to the proteins described in the first aspect, which are any one of B1) to B4) below:

[0021] B1) Nucleic acid molecules that encode the proteins described in the first aspect;

[0022] B2) An expression cassette containing the nucleic acid molecule described in B1);

[0023] B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

[0024] B4) Recombinant microorganisms containing the nucleic acid molecules described in B1), or recombinant microorganisms containing the expression cassette described in B2), or recombinant microorganisms containing the recombinant vector described in B3).

[0025] In the biological materials described above, the nucleic acid molecule described in B1) is as follows: b1) or b2) or b3) or b4):

[0026] b1) The coding sequence is the cDNA molecule or DNA molecule shown in SEQ ID NO: 5 or SEQ ID NO: 7 in the sequence listing;

[0027] b2) The DNA molecule shown in SEQ ID NO: 5 or SEQ ID NO: 7 in the sequence listing;

[0028] b3) has 75% or more identity with the nucleotide sequence defined by b1) or b2) and encodes a cDNA molecule or DNA molecule that encodes the protein described in the first aspect;

[0029] b4) Hybridizes under stringent conditions to a nucleotide sequence defined by b1) or b2) or b3) and to a cDNA molecule or DNA molecule encoding the protein described in the first aspect.

[0030] Thirdly, 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.

[0031] The above-mentioned treatment was carried out at 60°C. In the embodiments of the present invention, the pH value of the treatment was 10.0 and the treatment time was 24 hours.

[0032] Fourthly, the present invention provides for the use of the protein described in the first aspect in any of the following:

[0033] C1) Catalytic hydrolysis of PBAT;

[0034] C2) Prepare PBAT degrading agent.

[0035] Fifthly, the present invention provides the use of the biomaterials described in the second aspect in any of the following:

[0036] C1) Catalytic hydrolysis of PBAT;

[0037] C2) Prepare PBAT degrading agent.

[0038] Experiments of this invention demonstrate that by utilizing structural analysis and site-directed mutagenesis to mutate wild-type Tcur-lipase, Tcur-lipase mutants N211A and N211A / D256A were obtained. This addresses the low enzyme activity of wild-type Tcur-lipase, effectively increasing its activity in degrading PBAT and enhancing its degradation efficiency. The Tcur-lipase mutants of this invention improve PBAT degradation efficiency and show promising prospects for industrial application. Attached Figure Description

[0039] Figure 1 The weight loss rate of Tcur-lipase and its mutant proteins is represented by WT, where WT represents wild-type Tcur-lipase and N211A and N211A / D256A represent mutants.

[0040] Figure 2 For the activity analysis of Tcur-lipase and its mutant proteins, WT represents wild-type Tcur-lipase, and N211A and N211A / D256A represent mutants. Detailed Implementation

[0041] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0042] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0043] The raw materials and equipment used in the specific embodiments of the present invention are all known products, obtained by purchasing commercially available products.

[0044] 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.

[0045] Terephthalic acid (TPA): Sigma, CAS: 100-21-0.

[0046] Example 1: Preparation, expression, purification, and activity detection of Tcur-lipase mutants

[0047] To increase the industrial application value of wild-type Tcur-lipase, this invention synthesizes a gene (SEQ ID NO: 1) derived from Thermomonosporacurvata, expresses and purifies it, and after studying the predicted protein structure of Tcur-lipase, selects amino acids near its active region that participate in substrate interaction for mutation to increase the enzyme's activity against the substrate PBAT.

[0048] I. Construction of wild-type Tcur-lipase recombinant plasmid and its mutant recombinant plasmid

[0049] 1. Construction of wild-type Tcur-lipase recombinant plasmid

[0050] Wild-type Tcur-lipase is derived from Thermomonospora curvata. 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.

[0051] The wild-type Tcur-lipase gene was inserted into the EcoRI and NotI restriction sites of the pET26a vector (Novagen) to obtain a recombinant plasmid, denoted as recombinant plasmid pET26a-Tcur-lipase. After induction, the amino acid sequence encoded by this recombinant plasmid, in addition to the wild-type Tcur-lipase sequence, also has a vector sequence at its N-terminus, which is a histidine tag. The amino acid sequence after induction is shown in SEQ ID No: 4, and it is named the fusion protein of wild-type Tcur-lipase. The gene encoding this recombinant protein is the wild-type Tcur-lipase fusion gene.

[0052] The nucleotide sequence of the wild-type Tcur-lipase fusion gene is SEQ ID NO: 3, which consists of M and histidine tags (SEQ ID NO: 3, positions 1-21) from the 5′ end and the Tcur-lipase gene shown in SEQ ID NO: 1 in the sequence listing (SEQ ID NO: 3, positions 22-801).

[0053] The amino acid sequence of the wild-type Tcur-lipase fusion protein is SEQ ID NO:4, which consists of the start codon and histidine tag encoding nucleic acid (SEQ ID NO:4, positions 1-7) from the N-terminus and the Tcur-lipase shown in SEQ ID NO:2 (SEQ ID NO:4, positions 8-267) in the sequence listing.

[0054] 2. Recombinant plasmids expressing the Tcur-lipase mutant

[0055] After predicting the protein structure of wild-type Tcur-lipase, site-directed mutagenesis was performed on amino acids near its active site that participate in substrate interactions to increase the enzyme's activity against the substrate PBAT. In other words, this invention utilizes structural analysis and site-directed mutagenesis to mutate Tcur-lipase, obtaining a Tcur-lipase mutant, thereby addressing the low activity of the original PBAT hydrolase and improving the PBAT degradation activity of Tcur-lipase.

[0056] The following two mutants were identified through research:

[0057] Using site-directed mutagenesis, PCR was performed with the primers shown in Table 1 using the pET26a-Tcur-lipase plasmid as a template to obtain the Tcur-lipase mutant plasmid. The restriction endonuclease DpnI was then added and reacted at 37°C to remove the original template. The purified reaction product was transformed into competent *E. coli* cells, and preliminary screening was performed with antibiotics. DNA sequencing was then performed to identify the successfully mutated gene, resulting in the plasmid representing the Tcur-lipase mutant, denoted as pET26a-Tcur-lipase-N211A.

[0058] The Tcur-lipase mutant N211A is formed by mutating asparagine at position 211 of the wild-type Tcur-lipase shown in SEQ ID NO: 4 to alanine, while other amino acid residues remain unchanged. The amino acid sequence of the Tcur-lipase mutant N211A is SEQ ID NO: 6, and the nucleotide sequence of its encoding gene is SEQ ID NO: 5.

[0059] Using site-directed mutagenesis, PCR was performed with the primers shown in Table 1 using the pET26a-Tcur-lipase-N211A plasmid as a template to obtain the Tcur-lipase mutant plasmid. The restriction endonuclease DpnI was then added and reacted at 37°C to remove the original template. The purified reaction product was transformed into E. coli competent cells, and preliminary screening was performed with antibiotics. DNA sequencing was then performed to identify the successfully mutated gene, resulting in the plasmid representing the Tcur-lipase mutant, denoted as pET26a-Tcur-lipase-N211A / D256A.

[0060] The Tcur-lipase mutant N211A / D256A is formed by mutating asparagine at position 211 to alanine and aspartic acid at position 256 of the wild-type Tcur-lipase shown in SEQ ID NO: 4, while the amino acid residues at other positions remain unchanged. The amino acid sequence of the Tcur-lipase mutant N211A / D256A is SEQ ID NO: 8, and the nucleotide sequence of its encoding gene is SEQ ID NO: 7.

[0061] The recombinant plasmid expressing the Tcur-lipase mutant is a vector obtained by inserting the coding gene of various Tcur-lipase mutants between the EcoRI and NotI restriction sites in the pET26a vector. This vector expresses various fusion proteins containing Tcur-lipase mutants (by adding a start codon and histidine tag to the N-terminus of various amino acids containing Tcur-lipase mutants).

[0062] Table 1 lists the site-directed mutagenesis primers.

[0063] mutation site Primer (5'→3') N211A AAAGCGTATCTGGAACTGgcgGGCGCGAGCCATTTTTTT N211A / D256A CCGCCGCGCCCGAGCGGCgcgATTAGCGAATATCGCGAT

[0064] In the table, N211A refers to the 211th amino acid in SEQ ID NO: 4 being mutated from asparagine to alanine, and so on.

[0065] II. Preparation of Tcur-lipase mutants and wild-type Tcur-lipase

[0066] 1. Expression and purification of wild-type Tcur-lipase and Tcur-lipase mutant

[0067] The recombinant plasmid pET26a-Tcur-lipase expressing wild-type Tcur-lipase and recombinant plasmids expressing different Tcur-lipase mutants were transformed into *E. coli* BL21(DE3) competent cells, respectively. Strains were screened in LB broth containing 50 μg / ml Kanamycin. The selected strains were inoculated into 5 ml LB broth and cultured, then the culture volume was increased to 50 ml LB broth, and finally expanded to 6 L of self-induction medium (37℃, 220 rpm). When the OD value reached 0.6 to 0.8, the culture was cooled to 16℃. After 18 hours of protein induction expression, the cells were collected by centrifugation at 6000 rpm for 15 minutes. The bacterial cells were resuspended in buffer (25 mM Tris, 300 mM NaCl, pH 8.0), and the cells were lysed using an ultrasonic cell disruptor (1000 bar, 15 min). The cells were then centrifuged at 16000 rpm at 4°C for 60 min, and the supernatant was collected for further purification.

[0068] The above self-induction culture medium was prepared as follows:

[0069] (1) Basic culture medium: 10g peptone, 2.5g yeast powder, and 460mL water.

[0070] (2) 25* Salt solution 1L: 25mM Na2HPO4, 88.75g, 25mM KH2PO4, 85g, 50mM NH4Cl, 67g, 5mM Na2SO4, 17.75g, add water to 1L.

[0071] (3) 25* Sugar solution 1L: 125mL glycerol, 12.5g glucose, 50g lactose, add water to 1L.

[0072] (4) 2M MgCl2, filter to remove bacteria (high temperature sterilization to cause precipitation).

[0073] (1)(2)(3) are sterilized separately. When using, take 20 mL of (2)(3) into (1) and 0.5 mL of (4) into (1) (1 mL / L) to obtain 500 mL of self-induction culture medium.

[0074] Note: Sugar-containing culture media should be sterilized at 115℃.

[0075] To obtain high-purity enzyme protein, the target protein was eluted sequentially using a fast protein liquid chromatography (FPLC) system with cobalt ion column substrate (buffer: 25 mM Tris, 300 mM NaCl, pH 8.0), followed by elution with buffers containing 10 mM, 30 mM, 60 mM, and 300 mM imidazole, and the target protein was collected. The target protein (eluted with 60 mM imidazole buffer) was dialyzed in 5 L of dialysate (25 mM Tris, 300 mM NaCl, balance water, pH 8.0). After 3 hours, the dialysate was replaced, and the protein was dialyzed overnight at 4 °C to remove imidazole. The protein was concentrated and collected to obtain the following solutions: Tcur-lipase fusion protein solution (concentration 10 mg / ml), Tcur-lipase mutant N211A fusion protein solution (concentration 10 mg / ml), and Tcur-lipase mutant N211A / D256A fusion protein solution (concentration 10 mg / ml). These solutions were stored at -80 °C.

[0076] SDS-PAGE analysis of each protein solution showed that the Tcur-lipase fusion protein, the Tcur-lipase mutant N211A fusion protein, and the Tcur-lipase mutant N211A / D256A fusion protein were all 28 kDa, yielding the target protein of the expected size.

[0077] III. Comparison of relative activities between wild-type Tcur-lipase and Tcur-lipase mutants

[0078] To verify the differences between wild-type Tcur-lipase and various Tcur-lipase mutants, this invention further determined their degradation activity against PBAT. The steps of the PBAT activity test are as follows:

[0079] Pretreatment of PBAT membranes (CAS: 55231-08-8, Shanghai Maclean Biochemical Co., Ltd.): The membranes were washed sequentially with 1% (w / v) SDS aqueous solution, ethanol, and ddH2O, and then dried and weighed to record the original weight.

[0080] Each reaction mixture (1 mL) was placed in 1 M Glycine pH 10.0 buffer (Glycine dissolved in water, pH adjusted to 10 with NaOH), including 4 mg of substrate PBAT membrane and 20 μg of enzyme (2 μL each of the above-prepared Tcur-lipase mutant N211A fusion protein solution (denoted as N211A in the figure), Tcur-lipase mutant N211A / D256A fusion protein solution (denoted as N211A / D256A in the figure), or Tcur-lipase fusion protein solution (denoted as WT in the figure), with the remainder being 1 M Glycine pH 10.0 buffer. The reaction was carried out at 60 °C and 1000 rpm for 24 hours. Each reaction was performed in triplicate. After 24 hours of reaction, the PBAT membrane was removed, air-dried at room temperature, and weighed using an analytical balance to calculate the weight loss rate.

[0081] The formula for calculating the weight loss rate is (W1-W2) / W1×100%, where W1 is the sample weight before the reaction and W2 is the sample weight after the reaction.

[0082] The weight loss rate of wild-type Tcur-lipase is recorded as 100%, and the weight loss rate of Tcur-lipase mutant compared with wild-type is recorded as relative enzyme activity.

[0083] Test results as follows Figure 1 and Figure 2 As shown, the weight loss rates of both mutants of the present invention are higher than those of the wild-type protein. The weight loss rate of mutant N211A is 1.75 times that of the wild-type protein, and the weight loss rate of mutant N211A / D256A is 2.2 times that of the wild-type protein. This indicates that the enzyme activity of the Tcur-lipase mutant is higher than that of the wild-type protein Tcur-lipase, and it has great application value.

[0084] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. Protein, which is any one of the following (A1)-A5): A1) Mutate the 211th amino acid residue in the amino acid sequence shown in SEQ ID NO:4 and remove the N-terminal tag sequence, while keeping the amino acid residues at other positions unchanged, to obtain a protein with PBAT hydrolase activity. A2) Mutate the amino acid residues at positions 211 and 256 of the amino acid sequence shown in SEQ ID NO: 4, and remove the N-terminal tag sequence, while leaving the amino acid residues at other positions unchanged, to obtain a protein with PBAT hydrolase activity. A3) Mutate the 211th amino acid residue in the amino acid sequence shown in SEQ ID NO: 4, while keeping the amino acid residues at other positions unchanged, to obtain a protein with PBAT hydrolase activity. A4) Mutate the amino acid residues at positions 211 and 256 of the amino acid sequence shown in SEQ ID NO: 4, while leaving the amino acid residues at other positions unchanged, to obtain a protein with PBAT hydrolase activity. A5) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of any of the proteins described in A1)-A2); The 211th amino acid residue was mutated to 211th asparagine and then to alanine. The amino acid residues at positions 211 and 256 are mutated so that asparagine at position 211 is mutated to alanine, and aspartic acid at position 256 is mutated to alanine.

2. The protein according to claim 1, characterized in that: The amino acid sequence of the protein shown in A3) is SEQ ID NO: 6; The amino acid sequence of the protein shown in A4 is SEQ ID NO:

8.

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

4. The biomaterial according to claim 3, characterized in that: B1) The nucleic acid molecule described is as follows (b1) or (b2): b1) The coding sequence is the cDNA molecule or DNA molecule shown in SEQ ID NO: 5 or SEQ ID NO: 7 in the sequence listing; b2) The DNA molecule shown in SEQ ID NO: 5 or SEQ ID NO: 7 in the sequence listing.

5. A method for hydrolyzing PBAT, comprising the following steps: treating PBAT with any of the proteins described in claims 1-2 to achieve the hydrolysis of PBAT.

6. The use of the protein according to any one of claims 1-2 in any of the following: C1) Catalytic hydrolysis of PBAT; C2) Prepare PBAT degrading agent.

7. The use of the biomaterial of claim 3 or 4 in any of the following: C1) Catalytic hydrolysis of PBAT; C2) Prepare PBAT degrading agent.

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