A plastic hydrolase ThPETase mutant and its application

Abstract

The present invention belongs to the field of enzyme engineering technology, and specifically relates to a plastic hydrolase ThPETase mutant and its application, wherein the plastic hydrolase ThPETase mutant is obtained by multiple mutations in the 48th, 204th, 213th, 214th and 253rd positions of the amino acid sequence shown in SEQ ID NO.1. The present invention performs molecular modification on the basis of the wild-type ThPETase. While the wild-type ThPETase has almost no hydrolysis activity for PBAT, the mutant obtained by the present invention has obvious hydrolysis ability for PBAT, and can hydrolyze a plastic film made of PBAT into powder in 2 weeks at 37°C. Moreover, the mutant obtained by the present invention has a significant improvement in thermal stability compared with the wild-type, and has great industrial application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of enzyme engineering, and in particular relates to a plastic hydrolase ThPETase mutant and application thereof. Background Art

[0002] Plastic pollution has become an important environmental problem that needs to be solved urgently worldwide, and has attracted widespread social attention. Among many plastic materials, polybutylene adipate / terephthalate (PBAT), as a new and highly-regarded biodegradable plastic, is gradually becoming a key means to solve the problem of plastic pollution. PBAT is synthesized through the polymerization reaction of 1,4-butanediol, adipic acid and terephthalic acid. Its unique molecular structure gives it excellent flexibility and biocompatibility. At the same time, its excellent processability gives PBAT a significant advantage in the field of packaging materials, especially in the production of high-quality packaging films, plastic films and disposable tableware. It shows broad application prospects.

[0003] However, although PBAT is designed to be biodegradable in the natural environment through the enzymatic action of microorganisms, thereby reducing the burden of traditional plastic pollution, the actual situation is not satisfactory. Under natural soil conditions, the degradation rate of PBAT is relatively slow, mainly because the types of microorganisms that can effectively catalyze the hydrolysis of PBAT are relatively rare. This limitation not only limits the widespread application of PBAT as a biodegradable material, but also delays its degradation process in the natural environment, which may aggravate environmental pollution problems.

[0004] Therefore, the development of efficient hydrolases for PBAT plastics has become an important issue that needs to be solved in the current field of environmental science and biotechnology. Discovering new and efficient PBAT hydrolases is not only expected to significantly accelerate the degradation rate of PBAT under natural conditions and improve the biodegradation efficiency, but also help promote the resource utilization of PBAT waste and reduce the negative impact on the natural ecology. Summary of the invention

[0005] In view of the lack of development of enzymes that can efficiently hydrolyze PBAT plastics in the prior art, the present invention provides a plastic hydrolase ThPETase mutant and its application, and the specific technical scheme is as follows:

[0006] In a first aspect, the present invention provides a mutant of plastic hydrolase ThPETase, which is obtained by mutating multiple points in the 48th, 204th, 213th, 214th and 253rd positions of the amino acid sequence shown in SEQ ID NO.1.

[0007] Furthermore, the mutant form of the plastic hydrolase ThPETase mutant is one of the following:

[0008] (1) The aspartic acid at position 204 of the amino acid sequence shown in SEQ ID NO.1 is mutated to cysteine, and the glutamic acid at position 253 is mutated to cysteine;

[0009] (2) The asparagine at position 48 of the amino acid sequence shown in SEQ ID NO.1 is mutated to aspartic acid, the aspartic acid at position 204 is mutated to cysteine, and the glutamic acid at position 253 is mutated to cysteine;

[0010] (3) the aspartic acid at position 204 of the amino acid sequence shown in SEQ ID NO.1 is mutated to cysteine, the leucine at position 213 of the amino acid sequence shown in SEQ ID NO.1 is mutated to threonine, and the glutamic acid at position 253 is mutated to cysteine;

[0011] (4) The asparagine at position 48 of the amino acid sequence shown in SEQ ID NO.1 is mutated to aspartic acid, the aspartic acid at position 204 is mutated to cysteine, the leucine at position 213 is mutated to threonine, and the serine at position 214 is mutated to proline;

[0012] (5) the aspartic acid at position 204 of the amino acid sequence shown in SEQ ID NO.1 is mutated to cysteine, the serine at position 214 of the amino acid sequence shown in SEQ ID NO.1 is mutated to proline, and the glutamic acid at position 253 is mutated to cysteine;

[0013] (6) In the amino acid sequence shown in SEQ ID NO.1, the asparagine at position 48 is mutated to aspartic acid, the aspartic acid at position 204 is mutated to cysteine, the leucine at position 213 is mutated to threonine, the serine at position 214 is mutated to proline, and the glutamic acid at position 253 is mutated to cysteine.

[0014] The present invention is derived from thermophilic microorganisms Thermobifida halotolerans The protein ThPETase (GeneBank ID: WP_243597587.1) was mutated to obtain a mutant with hydrolysis activity for PBAT (polybutylene adipate / terephthalate), while the wild-type ThPETase has almost no hydrolysis activity for PBAT. The thermal stability of the obtained mutant was tested, and it was found that the thermal stability of the ThPETase mutated by the mutation method provided in the present application was significantly improved.

[0015] In a second aspect, the present invention provides a gene encoding the above-mentioned plastic hydrolase ThPETase mutant.

[0016] In a third aspect, the present invention provides a recombinant vector comprising the above-mentioned encoding gene.

[0017] In a fourth aspect, the present invention provides a genetically engineered bacterium comprising the above-mentioned encoding gene.

[0018] In a fifth aspect, the present invention provides the use of the above-mentioned plastic hydrolase ThPETase mutant or the above-mentioned genetically engineered bacteria in degrading PBAT plastics.

[0019] Furthermore, the application method is:

[0020] After adding the plastic hydrolase ThPETase mutant to the solution containing PBAT plastic, the degradation reaction was carried out.

[0021] Furthermore, the PBAT plastic is PBAT powder or PBAT plastic film.

[0022] Furthermore, based on PBAT plastic, the addition amount of the plastic hydrolase ThPETase mutant is 0.01%~0.1% by mass; the reaction temperature is 30~55°C; the time is 1 hour to 1 month; and the pH value of the solution is 7.5~8.5.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The present invention performs molecular modification on the basis of the wild-type ThPETase. While the wild-type ThPETase has almost no hydrolysis activity for PBAT, the mutant obtained by the present invention has obvious hydrolysis ability for PBAT. At 37° C., a plastic film made of PBAT material can be hydrolyzed into powder in 2 weeks. In addition, the mutant obtained by the present invention has a significant improvement in thermal stability compared with the wild-type, and has great industrial application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The electrophoresis diagram of purified ThPETase and mutant Th_DCTPC; M represents the comparison protein, Th represents the wild-type ThPETase enzyme, and Th CC indicates mutant Th_CC, Th DCTC Denotes mutant Th_DCTC, Th DCTCP Denotes mutant Th_DCTPC, Th DCC Denotes mutant Th_DCC, Th CTC indicates mutant Th_CTC, Th CPC indicates the mutant Th_CPC.

[0026] Figure 2T of different mutants m Value analysis diagram; wherein, WT represents the wild-type enzyme of ThPETase, CC represents the mutant Th_CC, DCC represents the mutant Th_DCC, CTC represents the mutant Th_CTC, DCTC represents the mutant Th_DCTC, CPC represents the mutant Th_CPC, and DCTPC represents the mutant Th_DCTPC.

[0027] Figure 3 Comparison of the activities of ThPETase and its mutants; (a) degradation of ThPETase and its mutants Th_CC, Th_DCC, ThCTC, ThCPC, ThDCTC and Th_DCTPC p Relative activity of NPA (p-nitrophenol acetate); (b) Activity comparison of Th_DCTPC degrading PBAT powder to produce TPA, where WT represents the wild-type enzyme of ThPETase, DCTPC represents the mutant Th_DCTPC, and the vertical axis represents the amount of TPA produced by degrading PBAT.

[0028] Figure 4 This is the UPLC spectrum of PBAT powder degraded by ThPETase and mutant Th_DCTPC.

[0029] Figure 5 This is a comparison chart of the degradation effects of ThPETase and mutant Th_DCTPC on plastic films made of PBAT. DETAILED DESCRIPTION

[0030] In order to make those skilled in the art better understand the scheme of the present invention, the technical scheme of the present invention is clearly and completely described below in conjunction with specific embodiments. It should be noted that the following detailed descriptions are all exemplary and are only embodiments of a part of the present invention, rather than all embodiments.

[0031] Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative work should fall within the scope of protection of the present invention.

[0032] Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those of ordinary skill in the art to which this application belongs. The experimental materials used in the examples of the present invention are all conventional experimental materials in the art and can be purchased through commercial channels. Experimental methods without specifying detailed conditions were performed according to conventional experimental methods or according to the operating instructions recommended by the supplier.

[0033] In the present application, PBAT is the abbreviation of polybutylene adipate / terephthalate; CC to DCTPC in the mutant, wherein the first C represents the mutation mode of D204C, the second C represents the mutation mode of E253C, D represents the mutation mode of N48D, T represents the mutation mode of L213T, and P represents the mutation mode of S214P.

[0034] The amino acid sequence of wild-type ThPETase is shown in SEQ ID NO.1. ThPETase The nucleotide sequence of the gene is shown in SEQ ID NO.2.

[0035] SEQ ID NO.1:

[0036] ANPYERGPNPTNSSIEALRGPYSVSEDSVSSLVSGFGGGTIYYPTGTNETFGAVAISPGYTGTQSSISWLGPRLASQGFVVMTIDTNTTLDQPDSRASQLDAALDYMVNRSSSTVRNRIDSSRLAAMGHSM GGGGTLRLAERRPDLQAAIPLTPWHTDKTWGSVRVPTLIIGAENDTIASVRSHSEPFYNSLPGSLDKAYLELDGASHFAPNLSNTTIAKYSISWLKRFVDDDTRYTQFLCPGPSTGLFGEVEEYRSTCPF.

[0037] SEQ ID NO.2:

[0038] .

[0039] Example 1 Construction of mutants

[0040] 1. Discovery of plastic hydrolase ThPETase

[0041] Through sequence, structure and activity screening, we selected Thermobifida halotolerans The enzyme with GeneBank ID WP_243597587.1 was named ThPETase according to its source.

[0042] ThPETase The gene was synthesized by GenScript and cloned into pET21a. N I and XOt I, and finally introduced into BL21 (DE3), constructing the engineered bacteria BL21 (DE3) / pET21a- ThPETase .

[0043] 2. Construction of mutants

[0044] (1) Containing pET-21a- ThPETase The circular plasmid was used as a template for PCR amplification.

[0045] Circular plasmid PCR system: template 1 μL; primer F and primer R (primer list is shown in Table 1) 10 μM 1 μL each; dNTP (2.5 mM) 4 μL; 10× high-fidelity enzyme buffer 5 μL; high-fidelity enzyme 1 μL; add water to the reaction system to 50 μL.

[0046] Table 1 Primer sequences

[0047]

[0048] PCR thermal cycle conditions: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 s, annealing at (Tm-5)°C for 20 s; extension at 72°C for 4 min 20 s; insulation at 4°C; PCR amplification was performed for 30 cycles under the reaction conditions.

[0049] The amplified product DpN After digestion, take 5 μL and add it to 50 μL of DH5α competent cells for transformation. Incubate on ice for 20 min; heat shock at 42℃ for 45 s; place on ice for 3 min; add 800 μL LB medium, activate at 37℃, 220 rpm for 1 h, spread on the plate, culture for 14 h-16 h until mature single colonies grow, and pick bacteria for sequencing verification.

[0050] 3. Construction of mutant engineered bacteria

[0051] Th_DCTPC The genes were synthesized by GenScript and cloned into pET21a. N I and XOtI, and finally introduced into BL21 (DE3), to obtain BL21 (DE3) / pET21a-ThPETase, BL21 (DE3) / pET21a-Th_CC, BL21 (DE3) / pET21a-Th_DCC, BL21 (DE3) / pET21a-Th_CTC, BL21 (DE3) / pET21a-Th_DCTC, BL21 (DE3) / pET21a-Th_CPC, BL21 (DE3) / pET21a-Th_DCTPC. Spread on LB agar plates containing ampicillin, culture for 14 h-16 h, until mature single colonies grow, and finally obtain wild-type expression engineering bacteria and mutant expression engineering bacteria.

[0052] Example 2 Purification of ThPETase and its mutants

[0053] The constructed engineered bacteria BL21(DE3) / pET21a-ThPETase and its mutant strains BL21(DE3) / pET21a-Th_CC, BL21(DE3) / pET21a-Th_DCC, BL21(DE3) / pET21a-Th_CTC, BL21(DE3) / pET21a-Th_DCTC, BL21(DE3) / pET21a-Th_CPC, BL21(DE3) / pET21a-Th_DCTPC were inoculated into 5 mL of LB culture medium (containing ampicillin) and seed cultured for 13 h. Then 2 mL of the overnight culture was inoculated into 100 mL of fresh LB culture medium (containing antibiotics, in a 500 mL conical flask) and cultured at 37°C, 250 rpm for about 2 h until the logarithmic growth phase (OD 600 = 0.8-1). Add IPTG to a final concentration of 0.1 mM for induction, continue induction culture at 16°C for 24 hours, and harvest the cells at 7000 rpm for 10 min. Suspend the cells in 10 mL of lysis buffer solution, disrupt by ultrasound (2 s, 3 s, 30 min, 70%), and centrifuge at 8000 rpm for 15 min to obtain the supernatant.

[0054] Pre-wash the equilibrated column with two column volumes of lysis buffer containing 10 mM imidazole to allow the buffer to slowly drain out of the resin; then slowly load the supernatant after lysis centrifugation (two column volumes of sample solution); wash with lysis buffer for 20 column volumes, and then wash with wash buffer containing 20 mM imidazole for 10 column volumes; finally, elute the sample with elution buffer containing 250 mM imidazole, with an elution volume of 3 mL.

[0055] Ni-NTA purification was performed, and the purified protein was concentrated by centrifugation using a 10 kD ultrafiltration tube, and the elution buffer containing a high concentration of imidazole was replaced with a reaction buffer solution.

[0056] The formula of LB liquid medium is: yeast extract 5 g / L, tryptone 10 g / L, sodium chloride 10 g / L;

[0057] The formula of lysis buffer is: 50 mM Tris-HCl, 150 mM sodium chloride, 10 mM imidazole, pH = 7.5;

[0058] The formula of washing buffer is: 50 mM Tris-HC, 150 mM NaCl, 20 mM imidazole, pH=7.5;

[0059] The formula of elution buffer was: 50 mM Tris-HCl, 300 mM NaCl, 300 mM imidazole, pH = 7.5;

[0060] The formula of the reaction buffer is: 0.1 M potassium dihydrogen phosphate-sodium hydroxide buffer solution, pH = 8.0.

[0061] Finally, soluble proteins ThPETase, Th_CC, Th_DCC, Th_CTC, Th_DCTC, Th_CPC, and Th_DCTPC were purified and detected by SDS-PAGE electrophoresis. The results were as follows: Figure 1 shown.

[0062] Example 3 Thermal stability test of ThPETase and Th_DCTPC

[0063] The thermal stability of the enzyme was analyzed using a protein stability analyzer (nanoDSF, NanoTemper Technologies Prometheus NT.48). 10 μL of sample was loaded into a capillary tube. The concentration of the loaded protein (ThPETase protein and its mutants) was uniformly quantified as 1.0 mg / mL. The temperature was raised from 30 ℃ to 95 ℃ at a rate of 1 ℃ / min. The protein fluorescence ratio at 330 nm and 350 nm was recorded. Three parallel replicates were measured for each protein sample. The test results are shown in Figure 2. Figure 2 shown.

[0064] Depend on Figure 2 It can be seen that the T of wild-type ThPETase m The value was 41.9℃, and the T of the double-point mutant Th_CC (ThPETase_D204C / E253C) mThe T of mutant Th_DCC (ThPETase_N48D / D204C / E253C) was significantly increased to 61.9 ℃, which was about 21 ℃ higher than that of the wild type. m The mutant Th_CTC (ThPETase_D204C / L213T / E253C) had a similar T m The T of mutant Th_DCTC (Th_N48D / D204C / L213T / E253C) was increased to 64.7℃. m The value was 64.9℃, which was significantly increased by about 3.0℃ compared with Th_CC. m The value of T increased significantly to 67.3℃. m The T of wild-type ThPETase was further increased to 69.1℃, which was significantly higher than that of wild-type ThPETase. m The value increased by 28.3 ℃.

[0065] Example 4 Hydrolysis of PBAT using ThPETase and Th_DCTPC

[0066] 1. Degradation p Activity testing of NPA and PBAT plastic powders

[0067] (1) Use p-nitrophenol acetate ( p The stability of the wild-type enzyme and its mutants was evaluated by HPLC: the total volume of the reaction system was 200 μL, including 197 μL of 0.1 M potassium dihydrogen phosphate-sodium hydroxide buffer (pH 8), 2 μL of 0.1 M p NPA and 1 μL of 1.0 mg / mL enzyme solution (incubated at 50°C or 60°C for 30 minutes). Kinetics were performed and the maximum reaction rate represented the enzyme activity. For each batch, the highest activity within the group was normalized to 100% to facilitate comparison of relative activities.

[0068] like Figure 3 As shown in a, the results show that the following mutants including Th_CC, Th_DCC, Th_CTC, Th_DCTC, Th_CPC and Th_DCTPC have significantly improved performance compared with the wild type, indicating that these mutants are beneficial mutants.

[0069] (2) The mutant Th_DCTPC prepared in Example 2 was used to degrade PBAT plastic powder, and TPA (terephthalic acid) released during the depolymerization of PBAT was analyzed by ultra-high performance liquid chromatography (UPLC). MHET was measured using a C18 column (4.6×250 mm, 5µm) with a detection wavelength of 240 nm. The column temperature was maintained at 40°C, with 0.1% formic acid aqueous solution and acetonitrile as the mobile phase, the flow rate was fixed at 0.1 mL / min, the injection volume was 2 μL, and the proportion of acetonitrile in the mobile phase was reduced from 50% to 20% within 4 minutes. The peak time of TPA was about 2.8 minutes.

[0070] Reaction system: Add 5 mg / mL PBAT powder with a particle size of 100 mesh to 0.1 M potassium dihydrogen phosphate-NaOH buffer solution with a pH value of 8.0, and then add purified plastic hydrolase ThPETase and mutant Th_DCTPC with a final concentration of 0.1 mg / mL, respectively, and react at 50°C for 2 h. After the reaction is completed, centrifuge and take the supernatant, add an equal volume of acetonitrile, inject UPLC, and calculate the yield of ThPETase and Th_DCTPC products based on the peak area. The effect is shown in the following figure. Figure 3 b and Figure 4 The results showed that the wild-type ThPETase had a weak hydrolysis effect on PBAT, and the concentration of TPA produced was less than 1.5 µM. In contrast, the concentration of hydrolysis products produced by the mutant Th_DCTPC was 33 µM, which was nearly 25 times higher than that of the wild-type.

[0071] 2. Degradation of PBAT plastic film

[0072] The enzyme prepared in Example 2 was used to hydrolyze agricultural films made of PBAT.

[0073] Reaction system: Black film fragments of about 1×2 cm in size were placed in 0.1 mmol / L potassium dihydrogen phosphate-sodium hydroxide buffer (pH 8.0). Purified ThPETase and its mutant Th_DCTPC plastic hydrolase were added to make the final concentration 0.1 mg / mL. The reaction was carried out at 37°C for 2 weeks. At the same time, a group of enzyme-free controls was set as blank controls. The results were as follows: Figure 5 shown.

[0074] The results showed that compared with the control group (without enzyme), the wild-type ThPETase had a minimal hydrolysis effect on the film, and no obvious degradation was observed. In contrast, the mutant Th_DCTPC had a very obvious degradation effect, and had degraded the PBAT film into a powder state.

Claims

1. A mutant of plastic hydrolase ThPETase, characterized in that: The plastic hydrolase ThPETase mutant is obtained by mutating multiple points in the 48th, 204th, 213th, 214th and 253rd positions of the amino acid sequence shown in SEQ ID NO.1; The mutant form of the plastic hydrolase ThPETase mutant is one of the following: (1) The aspartic acid at position 204 of the amino acid sequence shown in SEQ ID NO.1 is mutated to cysteine, and the glutamic acid at position 253 is mutated to cysteine; (2) The asparagine at position 48 of the amino acid sequence shown in SEQ ID NO.1 is mutated to aspartic acid, the aspartic acid at position 204 is mutated to cysteine, and the glutamic acid at position 253 is mutated to cysteine; (3) the aspartic acid at position 204 of the amino acid sequence shown in SEQ ID NO.1 is mutated to cysteine, the leucine at position 213 of the amino acid sequence shown in SEQ ID NO.1 is mutated to threonine, and the glutamic acid at position 253 is mutated to cysteine; (4) the aspartic acid at position 204 of the amino acid sequence shown in SEQ ID NO.1 is mutated to cysteine, the serine at position 214 of the amino acid sequence shown in SEQ ID NO.1 is mutated to proline, and the glutamic acid at position 253 is mutated to cysteine; (5) In the amino acid sequence shown in SEQ ID NO.1, the asparagine at position 48 is mutated to aspartic acid, the aspartic acid at position 204 is mutated to cysteine, the leucine at position 213 is mutated to threonine, the serine at position 214 is mutated to proline, and the glutamic acid at position 253 is mutated to cysteine.

2. The encoding gene of the plastic hydrolase ThPETase mutant as described in claim 1.

3. A recombinant vector, characterized in that: Contains the coding gene as described in claim 2.

4. A genetically engineered bacterium, characterized in that: Contains the coding gene as described in claim 2.

5. Use of the plastic hydrolase ThPETase mutant as described in claim 1 or the genetically engineered bacteria as described in claim 4 in degrading PBAT plastics.

6. The use according to claim 5, characterized in that: The application pathways are: After adding the plastic hydrolase ThPETase mutant to the solution containing PBAT plastic, the degradation reaction was carried out.

7. The use according to claim 6, characterized in that: The PBAT plastic is PBAT powder or PBAT plastic film.

8. The use according to claim 6, characterized in that: Based on PBAT plastic, the addition amount of the plastic hydrolase ThPETase mutant is 0.01%~0.1% by mass; the reaction temperature is 30~55°C.

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