CN119040295B

CN119040295B - Plastic hydrolase ThPETase mutant and application thereof

Info

Publication number: CN119040295B
Application number: CN202411540951.8A
Authority: CN
Inventors: 徐盛春; 李新佳; 陶晓园
Original assignee: Xianghu Laboratory Agricultural Zhejiang Provincial Laboratory
Current assignee: Xianghu Laboratory Agricultural Zhejiang Provincial Laboratory
Priority date: 2024-10-31
Filing date: 2024-10-31
Publication date: 2025-02-11
Anticipated expiration: 2044-10-31
Also published as: CN119040295A

Abstract

The invention belongs to the technical field of enzyme engineering, and particularly relates to a plastic hydrolase ThPETase mutant and application thereof, wherein the plastic hydrolase ThPETase mutant is obtained by carrying out mutation on multiple points in 48 th, 204 th, 213 th, 214 th and 253 th positions of an amino acid sequence shown in SEQ ID NO. 1. The invention carries out molecular transformation on the basis of a wild type ThPETase, under the condition that the wild type ThPETase has little hydrolytic activity to PBAT, the obtained mutant has obvious hydrolytic capacity to PBAT, the plastic film made of PBAT can be hydrolyzed into powder after 2 weeks at 37 ℃, and compared with the wild type, the obtained mutant has great improvement on thermal stability and huge industrial application prospect.

Description

Plastic hydrolase ThPETase mutant and application thereof

Technical Field

The invention belongs to the technical field of enzyme engineering, and particularly relates to a plastic hydrolase ThPETase mutant and application thereof.

Background

Plastic pollution has become an important environmental problem to be solved in the global world, and has attracted extensive social attention. Among the numerous plastic materials, poly (butylene adipate/terephthalate) (PBAT) is becoming a new and attractive biodegradable plastic, and is becoming a key means to solve the plastic contamination problem. The PBAT is synthesized by polymerization reaction of the 1, 4-butanediol, the adipic acid and the terephthalic acid, the unique molecular structure of the PBAT endows the PBAT with excellent flexibility and biocompatibility, and meanwhile, the excellent processability of the PBAT enables the PBAT to have remarkable advantages in the field of packaging materials, and particularly has wide application prospects in the aspects of producing high-quality packaging films, plastic films, disposable tableware and the like.

However, although PBAT is designed to be able to achieve biodegradation by enzymatic action of microorganisms in natural environment, thereby reducing the burden of pollution to conventional plastics, the actual situation is not satisfactory. Under natural soil conditions, the degradation rate of PBAT is relatively slow, mainly because of the relatively rare types of microorganisms that are able to effectively catalyze the hydrolysis of PBAT. This limitation not only limits the wide application of PBAT as a biodegradable material, but also delays its degradation process in natural environment, thereby possibly exacerbating the environmental pollution problem.

Therefore, development of efficient hydrolytic enzymes for PBAT plastics is an important topic to be solved in the current environmental science and biotechnology fields. The development of new and efficient PBAT hydrolase is expected to remarkably accelerate the degradation speed of PBAT under natural conditions, improve the biodegradation efficiency, facilitate the resource utilization of PBAT wastes and reduce the negative influence on natural ecology.

Disclosure of Invention

Aiming at the defect of insufficient development of enzyme capable of efficiently hydrolyzing PBAT plastics in the prior art, the invention provides a plastic hydrolase ThPETase mutant and application thereof, and the specific technical scheme is as follows:

In a first aspect, the invention provides a mutant of plastic hydrolase ThPETase, wherein the mutant of plastic hydrolase ThPETase is obtained by mutating a plurality of points in 48 th, 204 th, 213 th, 214 th and 253 th of an amino acid sequence shown in SEQ ID NO. 1.

Further, the mutant form of the plastic hydrolase ThPETase mutant is one of the following:

(1) Aspartic acid at position 204 of the amino acid sequence shown in SEQ ID NO.1 is mutated into cysteine, and glutamic acid at position 253 is mutated into cysteine;

(2) Asparagine at position 48 of the amino acid sequence shown in SEQ ID NO.1 is mutated into aspartic acid, aspartic acid at position 204 is mutated into cysteine, and glutamic acid at position 253 is mutated into cysteine;

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

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

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

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

According to the application, through carrying out mutation on protein ThPETase (GeneBank ID: WP_ 243597587.1) from thermophilic microorganism Thermobifida halotolerans, a mutant with hydrolytic activity on PBAT (polybutylene adipate/terephthalate) is obtained, but wild ThPETase almost has no hydrolytic activity on PBAT, and the obtained mutant is subjected to thermal stability test, so that ThPETase after mutation in the mutation mode provided by the application is found, and the thermal stability is greatly improved.

In a second aspect, the present invention provides a gene encoding the plastid hydrolase ThPETase mutant described above.

In a third aspect, the present invention provides a recombinant vector comprising the coding gene described above.

In a fourth aspect, the present invention provides a genetically engineered bacterium comprising the coding gene described above.

In a fifth aspect, the invention provides the use of the plastic hydrolase ThPETase mutant, or the genetically engineered bacterium, as described above, in degrading PBAT plastics.

Further, the application method comprises the following steps:

After adding the plastic hydrolase ThPETase mutant to the solution containing the PBAT plastic, a degradation reaction is performed.

Further, the PBAT plastic is PBAT powder or a PBAT plastic film.

Further, 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 ℃, the reaction time is 1 hour to 1 month, and the pH value of the solution is 7.5-8.5.

Compared with the prior art, the invention has the following beneficial effects:

The invention carries out molecular transformation on the basis of a wild type ThPETase, under the condition that the wild type ThPETase has little hydrolytic activity to PBAT, the obtained mutant has obvious hydrolytic capacity to PBAT, the plastic film made of PBAT can be hydrolyzed into powder after 2 weeks at 37 ℃, and compared with the wild type, the obtained mutant has great improvement on thermal stability and huge industrial application prospect.

Drawings

FIG. 1 shows the electrophoresis patterns of ThPETase and mutant Th_ DCTPC after purification, wherein M represents a control protein, th represents ThPETase wild-type enzyme, th _CC represents mutant Th_CC, th _DCTC represents mutant Th_DCTC, th _DCTCP represents mutant Th_ DCTPC, th _DCC represents mutant Th_DCC, th _CTC represents mutant Th_CTC, and Th _CPC represents mutant Th_CPC.

FIG. 2 is a graph showing analysis of T _m values of different mutants, wherein WT represents ThPETase wild-type enzyme, CC represents mutant Th_CC, DCC represents mutant Th_DCC, CTC represents mutant Th_CTC, DCTC represents mutant Th_DCTC, CPC represents mutant Th_CPC, and DCTPC represents mutant Th_ DCTPC.

FIG. 3 is a graph showing comparison of ThPETase and mutant activities, wherein (a) ThPETase and mutants Th_CC, th_ DCC, thCTC, thCPC, thDCTC and Th_ DCTPC are relative to each other in terms of activity for degrading pNPA (p-nitrophenylacetate), and (b) Th_ DCTPC is a graph showing comparison of activity of the degradation of PBAT powder to TPA, wherein WT represents ThPETase wild-type enzyme, DCTPC represents mutant Th_ DCTPC, and the ordinate represents the amount of TPA produced by degradation of PBAT.

FIG. 4 is a UPLC spectrum of ThPETase and mutant Th_ DCTPC after degradation of PBAT powder.

FIG. 5 is a graph showing the comparison of the degradation effects of ThPETase and mutant Th_ DCTPC on plastic films of PBAT material degradation.

Detailed Description

In order to make the present invention more well understood by those skilled in the art, the following description of the present invention will be made with reference to specific embodiments. It should be noted that the following detailed description is exemplary and is merely an example of a portion, but not all, of the present invention.

All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, shall fall within the scope of the invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The experimental materials used in the embodiment of the application are all conventional in the field and can be purchased through commercial channels. Experimental methods without specifying detailed conditions were performed according to conventional experimental methods or according to the instructions recommended by the suppliers.

In the application, PBAT is short-term of poly (adipic acid)/butylene terephthalate, and CC-DCTPC are mutants, wherein the first C represents a mutation mode of D204C, the second C represents a mutation mode of E253C, D represents a mutation mode of N48D, T represents a mutation mode of L213T, and P represents a mutation mode of S214P.

The amino acid sequence of the wild ThPETase is shown as SEQ ID NO.1, and the nucleotide sequence of the ThPETase gene is shown as SEQ ID NO. 2.

SEQ ID NO.1:

ANPYERGPNPTNSSIEALRGPYSVSEDSVSSLVSGFGGGTIYYPTGTNETFGAVAISPGYTGTQSSISWLGPRLASQGFVVMTIDTNTTLDQPDSRASQLDAALDYMVNRSSSTVRNRIDSSRLAAMGHSMGGGGTLRLAERRPDLQAAIPLTPWHTDKTWGSVRVPTLIIGAENDTIASVRSHSEPFYNSLPGSLDKAYLELDGASHFAPNLSNTTIAKYSISWLKRFVDDDTRYTQFLCPGPSTGLFGEVEEYRSTCPF.

SEQ ID NO.2:

GCTAATCCTTATGAAAGGGGACCCAACCCAACGAACAGCTCTATCGAAGCGCTGCGTGGTCCGTATTCTGTTTCCGAAGATTCCGTGAGCTCCCTGGTTAGCGGTTTCGGTGGCGGCACGATCTACTACCCGACTGGCACGAACGAAACCTTCGGCGCGGTCGCAATTTCGCCGGGCTACACCGGCACCCAAAGCTCGATCAGCTGGTTAGGTCCACGTTTGGCGTCACAGGGTTTTGTCGTGATGACCATCGACACCAACACCACATTGGACCAGCCGGATTCCAGAGCAAGCCAACTGGATGCTGCGCTGGACTACATGGTTAACCGTTCTTCCTCTACTGTACGTAATCGTATTGATAGCAGTCGCTTGGCGGCTATGGGTCATAGCATGGGTGGTGGCGGAACCCTCCGTCTTGCGGAGCGCCGTCCGGACCTGCAGGCAGCGATTCCGCTGACGCCTTGGCATACCGATAAGACCTGGGGTTCCGTTCGCGTGCCGACCCTGATTATTGGTGCCGAGAACGATACTATCGCCAGCGTGCGCAGCCACAGCGAGCCGTTCTATAACAGCTTGCCGGGTAGCCTGGACAAAGCATATCTGGAGCTGGACGGCGCGAGCCACTTTGCTCCGAATCTGAGCAATACCACCATCGCCAAATACAGCATCAGCTGGCTCAAGCGCTTCGTGGATGACGACACCCGTTATACCCAATTTCTGTGTCCGGGCCCAAGCACCGGTCTGTTTGGCGAAGTTGAAGAGTACCGTTCGACGTGCCCGTTC.

EXAMPLE 1 construction of mutants

1. Development of Plastic hydrolase ThPETase

Through sequence, structure and activity selection, the enzyme with the GeneBank ID WP_243597587.1 from the thermophilic microorganism Thermobifida halotolerans was selected and named ThPETase according to the source.

The ThPETase gene is synthesized by Jinsri, cloned to pET21a, cut into NdeI and XhoI sites, and finally introduced into BL21 (DE 3) to construct engineering bacterium BL21 (DE 3)/pET 21a-ThPETase.

2. Construction of mutants

(1) PCR amplification was performed using a circular plasmid containing pET-21a-ThPETase as a template.

The circular plasmid PCR system consisted of 1. Mu.L of template, 1. Mu.L of each of primer F and primer R (primer list shown in Table 1), 4. Mu.L of dNTP (2.5 mM), 5. Mu.L of 10 XHi-Fi buffer, 1. Mu.L of Hi-Fi, and a reaction system for adding water to 50. Mu.L.

TABLE 1 primer sequences

The PCR thermal cycle conditions were 94℃for 3 min, 94℃for 30s ℃for annealing at (Tm-5) ℃for 20 s, 72℃for 4 min for 20 s, 4℃for incubation, and the reaction conditions were subjected to PCR amplification for 30 cycles.

After digestion with Dpn I, 5. Mu.L of the amplified product was added to 50. Mu.L of DH 5. Alpha. Competent cells for transformation. Incubating on ice for 20 min, heat-shocking at 42 ℃ for 45 s, placing on ice for 3: 3min, adding 800 μL LB culture medium, activating at 37 ℃ for 220 rpm for 1: 1h, coating a flat plate, culturing for 14: 14 h-16: 16 h until mature single colony grows out, and performing sequencing verification by picking.

3. Construction of mutant engineering bacteria

Th_ DCTPC gene is synthesized by Jinsri, cloned to pET21a respectively, and enzyme cutting sites NdeI and XhoI, and finally introduced into BL21 (DE 3) 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., which is coated on LB agar plate containing ampicillin, and cultured to 14 h-16 h until mature single colony grows, finally obtaining wild type expression engineering bacteria and mutant expression engineering bacteria.

Example 2 ThPETase purification of mutants thereof

The engineering bacteria BL21 (DE 3)/pET 21a-ThPETase and its mutant strain 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 are inoculated into LB culture solution (ampicillin) of 5mL, and the seeds are cultured to 13 h. The overnight broth of 2mL was then inoculated into 100mL fresh LB medium (containing antibiotics, in 500 mL conical flasks) at 37 ℃,250 rpm for about 2h to logarithmic growth phase (OD ₆₀₀ = 0.8-1). The cells were harvested by adding 0.1 mM IPTG final concentration for induction, and then continuing the induction culture at 16℃for 24 hours at 7000 rpm for 10 minutes. 10 The cells were suspended in the mL of lysis buffer, sonicated (2 s,3 s,30 min,70%), centrifuged at 8000 rpm for 15 min, and the supernatant was obtained.

The equilibrated column was rinsed with twice the column volume of lysis buffer containing 10 mM imidazole to allow the buffer to drain out the resin slowly, then the supernatant after the lysis centrifugation was slowly loaded (twice the column volume of sample solution), 20 column volumes were washed with lysis buffer, 10 column volumes were washed with wash buffer containing 20 mM imidazole, and finally the sample was eluted with elution buffer containing 250 mM imidazole, elution volume being 3 mL.

The Ni-NTA purification was performed, and the protein obtained after purification was concentrated by centrifugation using an ultrafiltration tube of 10 kD, and the elution buffer containing imidazole at a high concentration was replaced with the reaction buffer.

Wherein, the formula of the LB liquid medium comprises 5 g/L of yeast extract, 10 g/L of tryptone and 10 g/L of sodium chloride;

the lysis buffer formulation was 50 mM Tris-HCl,150 mM sodium chloride, 10 mM imidazole, ph=7.5;

The formulation of the washing buffer is 50 mM Tris-HC,150 mM sodium chloride, 20 mM imidazole, pH=7.5;

the formulation of elution buffer was 50 mM Tris-HCl,300 mM sodium chloride, 300 mM imidazole, ph=7.5;

the reaction buffer was formulated as a 0.1M potassium dihydrogen phosphate-sodium hydroxide buffer solution, ph=8.0.

Finally, soluble proteins ThPETase, th_CC, th_DCC, th_CTC, th_DCTC, th_CPC, th_DCTPC and SDS-PAGE electrophoresis were purified, and the results are shown in FIG. 1.

Example 3 ThPETase and Th_ DCTPC thermal stability experiment

The enzyme was analyzed for thermal stability using a protein stability analyzer (nanoDSF, nanoTemper Technologies Prometheus NT.48), 10. Mu.L was loaded using a capillary, the concentration of protein loaded (ThPETase protein and its mutants) was quantified uniformly at 1.0 mg/mL, the temperature was increased from 30℃to 95℃at a rate of 1℃per minute, the protein fluorescence ratios of 330 nm and 350 nm were recorded, and three replicates were measured per protein sample. The test results are shown in fig. 2.

As can be seen from fig. 2, the T _m value of wild type ThPETase was 41.9 ℃, the T _m of the double point mutant th_cc (ThPETase _d204C/E253C) was significantly raised to 61.9 ℃ and about 21 ℃ higher than the wild type, the T _m of the mutant th_dcc (ThPETase _n48d/D204C/E253C) was substantially the same as the th_cc, the T _m of the mutant th_ctc (ThPETase _d204C/L213T/E253C) was raised to 64.7 ℃, the T _m value of the mutant th_dctc (th_n48d/D204C/L213T/E253C) was 64.9 ℃ and significantly raised by about 3.0 ℃ as compared to the th_cc. The mutant Th_CPC (Th_D204C/S214P/E253C) had a T _m value that was again significantly elevated to 67.3 ℃. T _m of mutant Th_ DCTPC (Th_N48D/D204C/L213T/S214P/E253C) was further raised to 69.1℃compared to the T _m value of wild type ThPETase by 28.3 ℃.

Example 4 hydrolysis of PBAT Using ThPETase and Th_ DCTPC

1. Activity test for degrading pNPA and PBAT plastic powder

(1) The stability of the wild-type enzyme and its mutants was evaluated using p-nitrophenylacetate (pNPA, benzene ring containing esters as in PBAT) in a total reaction volume of 200. Mu.L containing 197. Mu.L of 0.1M potassium dihydrogen phosphate-sodium hydroxide buffer (pH 8), 2. Mu.L of 0.1 MpNPA and 1. Mu.L of 1.0 mg/mL enzyme solution (incubated at 50℃or 60℃for 30 minutes). Kinetic measurements were performed representing enzyme activity at maximum reaction rate. For each batch, the highest activity within the group was normalized to 100% in order to compare the relative activities.

As shown in a of fig. 3, the results indicate that the following mutants, including th_cc, th_dcc, th_ctc, th_dctc, th_cpc, and th_ DCTPC, all had significantly improved properties relative to the wild type, indicating that these mutants were all beneficial mutants.

(2) The mutant Th_ DCTPC prepared in example 2 was used to degrade PBAT plastic powder and the 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 (4.6x250 mm,5 μm) with a detection wavelength of 240 nm. The column temperature is kept at 40 ℃, a 0.1% formic acid aqueous solution and acetonitrile are taken as mobile phases, the flow rate is fixed at 0.1 mL/min, the sample injection amount is 2 mu L, the acetonitrile proportion of the mobile phases in 4 min is reduced from 50% to 20%, and the peak time of TPA is about 2.8 min.

The reaction system comprises adding 5 mg/mL of PBAT powder with particle size of 100 meshes into 0.1M potassium dihydrogen phosphate-NaOH buffer solution with pH value of 8.0, then adding purified plastid hydrolase ThPETase with final concentration of 0.1 mg/mL and mutant Th_ DCTPC respectively, and reacting at 50 ℃ for 2 h. After the reaction is finished, centrifuging to obtain a supernatant, adding acetonitrile with the same volume, sampling by UPLC, and calculating ThPETase and the yield of the Th_ DCTPC product according to the peak area. The effect is shown in b in fig. 3 and fig. 4. The results show that wild type ThPETase has a weaker hydrolysis of PBAT, resulting in TPA concentrations below 1.5 μm. In contrast, mutant Th DCTPC produced a hydrolysate concentration of 33. Mu.M, which was approximately 25-fold increased compared to the wild type.

2. Plastic film for degrading PBAT material

Agricultural films made from PBAT were hydrolyzed using the enzyme prepared in example 2.

Reaction System Black film fragments of about 1X 2 cm in size were incubated in 0.1 mmol/L potassium dihydrogen phosphate-sodium hydroxide buffer (pH 8.0). To this was added purified ThPETase and its mutant Th_ DCTPC plastic hydrolase to a final concentration of 0.1 mg/mL. The reactions were run at 37 ℃ for 2 weeks while a set of controls without enzyme was set up as a blank, the effect is shown in figure 5.

The results show that the wild type ThPETase has very little hydrolysis of the film compared to the control (no enzyme added) and no significant degradation is seen. In contrast, the degradation of mutant th_ DCTPC was very pronounced, and the PBAT film had been degraded to powder state.

Claims

1. A mutant of plastic hydrolase ThPETase, characterized in that the mutant of plastic hydrolase ThPETase is obtained by mutating multiple points in the amino acid sequence shown in SEQ ID NO.1 at positions 48, 204, 213, 214 and 253;

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 it comprises the coding gene as described in claim 2.

4. A genetically engineered bacterium, characterized in that it 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 application according to claim 5, characterized in that the application is carried out by:

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 percentage; and the reaction temperature is 30~55°C.