Mutant dura mhetaase and use thereof
By mutating specific amino acids in the MHET hydrolase IsMHETase, a heat-resistant mutant, DuraMHETase, was constructed, which solved the problem of insufficient activity of the wild-type enzyme at high temperatures and significantly improved the PET degradation efficiency.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- INST OF MICROBIOLOGY CHINESE ACAD OF SCI
- Filing Date
- 2025-01-02
- Publication Date
- 2026-07-03
AI Technical Summary
Wild-type MHET hydrolase cannot tolerate PET degradation under high temperature conditions, and MHET, as a competitive inhibitor, hinders the degradation process of PET.
The thermostable mutant DuraMHETase was obtained by modifying the amino acid sites of the MHET hydrolase IsMHETase, specifically Ala66Pro, Ser136Glu, Thr159Val, Met192Phe, Ala330Lys, Ala333Arg, Thr328Arg, Gln410Phe, Ser413Asn, Asp434Thr, Ala501Ile, Ala509Lys, and Ser561Val.
The mutant DuraMHETase has an increased apparent melting temperature of 7°C and maintains significant MHET hydrolytic activity at high temperatures, thereby improving PET degradation efficiency.
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Figure CN122326569A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the fields of enzyme engineering and bioengineering, and particularly to the field of MHET hydrolase bioengineering. Background Technology
[0002] Polyethylene terephthalate (PET) is one of the most widely used plastics with a high recycling rate, and the biodegradation of recycled PET materials by bio-enzymes has always been a hot research topic. Currently, various engineered PET hydrolytic enzymes (such as LCC) are being developed. ICCG TurboPETase has been developed, maximizing degradation efficiency under high-temperature conditions and advancing research on large-scale PET degradation reactions. In these reactions, MHET, as a competitive inhibitor of PET, significantly hinders the degradation process. To overcome this obstacle, constructing a two-enzyme system for PET degradation by introducing MHET hydrolases is considered an ideal approach. However, wild-type MHET hydrolases cannot tolerate large-scale PET degradation reactions under high-temperature conditions, thus requiring thermostability modification. Summary of the Invention
[0003] In view of this, the present invention provides an MHET hydrolase mutant, wherein the 66th amino acid of IsMHETase is mutated from Ala to Pro, the 136th amino acid from Ser to Glu, the 159th amino acid from Thr to Val, the 192nd amino acid from Met to Phe, the 330th amino acid from Ala to Lys, the 333rd amino acid from Ala to Arg, the 328th amino acid from Thr to Arg, the 410th amino acid from Gln to Phe, the 413th amino acid from Ser to Asn, the 434th amino acid from Asp to Thr, the 501st amino acid from Ala to Ile, the 509th amino acid from Ala to Lys, and the 561st amino acid from Ser to Val; the amino acid composition of the MHET hydrolase IsMHETase is shown in SEQ ID No. 1 of the sequence listing.
[0004] The heat-resistant mutant MHET hydrolase provided by this invention has an apparent melting temperature that is 7°C higher than that of the wild-type enzyme, and still exhibits significant MHET hydrolytic activity under high-temperature conditions. Attached Figure Description
[0005] Figure 1 HPLC comparative chromatograms of TPA yields from the wild-type MHET hydrolase IsMHETase and the mutant DuraMHETase catalyzed by the hydrolysis of ethylene terephthalate at 60 °C. Detailed Implementation
[0006] Example
[0007] This invention obtains a heat-resistant mutant, DuraMHETase, by protein engineering the MHET hydrolase IsMHETase derived from Ideonella sakaiensis. Compared with the wild-type enzyme, the mutant DuraMHETase has an apparent melting temperature increased by 7°C and still exhibits significant MHET hydrolytic activity under high-temperature conditions.
[0008] The MHET hydrolase mutant (named DuraMHETase) provided by this invention is obtained by mutating the following amino acids from MHET hydrolase IsMHETase: amino acid 66 from Ala to Pro, amino acid 136 from Ser to Glu, amino acid 159 from Thr to Val, amino acid 192 from Met to Phe, amino acid 330 from Ala to Lys, amino acid 333 from Ala to Arg, amino acid 328 from Thr to Arg, amino acid 410 from Gln to Phe, amino acid 413 from Ser to Asn, amino acid 434 from Asp to Thr, amino acid 501 from Ala to Ile, amino acid 509 from Ala to Lys, and amino acid 561 from Ser to Val. The amino acid composition of IsMHETase is as shown in the sequence listing SEQ ID. As shown in No.1, the nucleic acid sequence of its gene is shown in SEQ ID NO.2 in the sequence listing.
[0009] 1. Construction of engineered strains of wild-type MHET hydrolase IsMHETase
[0010] The wild-type MHET hydrolase IsMHETase gene was codon optimized using Escherichia coli as the host cell to obtain the optimized MHET hydrolase gene (i.e., the target gene). The recombinant plasmid pET21a-IsMHETase was constructed using chemical synthesis. Its nucleotide sequence is shown in SEQ ID No. 2 in the sequence listing, and the expressed amino acid sequence is shown in SEQ ID No. 1 in the sequence listing.
[0011] The chemically synthesized pET21a-IsMHETase plasmid was transformed into E. coli C41 competent cells and plated on ampicillin agar plates to obtain the positive recombinant C41 / pET21a-IsMHETase, thus successfully constructing the engineered strain C41 / pET21a-IsMHETase containing the recombinant plasmid pET21a-IsMHETase.
[0012] 2. Expression and purification of MHET hydrolase protein IsMHETase
[0013] Select a portion of engineered bacteria C41 / pET21a-IsMHETase colonies and inoculate them into 5 mL of Luria-Bertani liquid medium (containing Amp). Incubate at 37°C with shaking at 200 rpm for 4-6 hours. Then, inoculate 1% of the culture onto 2YT medium (containing Amp) and incubate at 37°C with shaking at 200 rpm for approximately 5 hours (OD). 600 Add 0.1% (w / v) IPTG (0.8-1.0%) and induce at 20℃ for 18-20h.
[0014] Centrifuge the overnight induced bacterial culture at 10,000 rpm for 10 min at 4 °C. Discard the supernatant and collect the engineered bacterial wet cells. Resuspend the cells in protein purification buffer A, sonicate at low temperature for 15-30 min, centrifuge at 10,000 rpm for 1 h at 4 °C to remove cell debris, collect the supernatant, filter it through a 0.22 μm filter, and place it on ice.
[0015] Purification was performed using a His Trap HP column. After washing with 5 column volumes of deionized water, the column was equilibrated with 5-10 column volumes of protein purification buffer A. The filtered supernatant was loaded at a flow rate of 5 mL / min. Unbound proteins were washed with protein purification buffer A, followed by washing with low-concentration imidazole protein purification buffer B to remove impurities. The target protein was then eluted with high-concentration imidazole protein purification buffer B and collected.
[0016] The target protein buffer was replaced with protein purification Buffer C using a HiPrep desalting column to remove imidazole. The collected protein was concentrated using an ultrafiltration concentrator with a molecular cutoff of 30 kDa to concentrate the enzyme solution. The protein concentration was determined using the BCA method and stored at 4°C for later use.
[0017] The formulation of the 2YT liquid culture medium is as follows: yeast extract 10 g / L, tryptone 16 g / L, sodium chloride 5 g / L; the formulation of the lysis buffer is as follows: Buffer A: 20 mM Tris, 300 mM NaCl, 10 mM Imidazole, pH = 8.0; the formulation of the washing buffer is as follows: Buffer B: 20 mM Tris, 300 mM NaCl, 300 mM Imidazole, pH = 8.0; the formulation of the elution buffer is as follows: Buffer C: 20 mM Tris, 100 mM NaCl, pH = 7.5.
[0018] 3. Degradation of polyethylene terephthalate (MHET) using MHET hydrolase (IsMHETase):
[0019] MHET was added to 100 mM phosphate buffer (pH 8.0) to a final concentration of 40 mM; engineered bacteria C41 / pET21a-IsMHETase were added to a final concentration of 39 nM; and the mixture was incubated at 60 °C for 3 h. HPLC analysis showed that the TPA yield in the reaction solution was 2 mM. Figure 1 ).
[0020] 4. Construction of the MHET hydrolase mutant DuraMHETase:
[0021] Using MHET hydrolase IsMHETase as a template, its nucleotide sequence is SEQ ID No. 2, and the expressed amino acid sequence is SEQ ID No. 1.
[0022] Using a pair of complementary oligonucleotides with mutation sites as amplification primers, the whole plasmid was amplified by purchasing PrimeSTAR high-fidelity enzyme (Takara) to obtain a recombinant plasmid with specific mutation sites.
[0023] The amplification primer pairs used are:
[0024] (1) In the amplification primer pair for the A66P mutation site, the upstream amplification primer is shown in SEQ ID No. 3, and the downstream amplification primer is shown in SEQ ID No. 4. Specifically, the sequence shown in SEQ ID No. 3 is: 5'-ttggccgaatccggccacggttgtagag-3'. The sequence shown in SEQ ID No. 4 is: 5'-attcggccaaaccatgtcgc-3'.
[0025] (2) In the amplification primer pair for the mutation site S136E, the upstream amplification primer is shown in SEQ ID No. 5, and the downstream amplification primer is shown in SEQ ID No. 6. Specifically, the sequence shown in SEQ ID No. 5 is: 5'-acgaacggcgagctctcagcggcgaccggaagt-3'. The sequence shown in SEQ ID No. 6 is: 5'-gccgttcgtaccactgcca-3'.
[0026] (3) In the amplification primer pair for the T159V mutation site, the upstream amplification primer is shown in SEQ ID No. 7, and the downstream amplification primer is shown in SEQ ID No. 8. Specifically, the sequence shown in SEQ ID No. 7 is: 5'-cgtaactttgcagtgattgctaccgacggagga-3'. The sequence shown in SEQ ID No. 8 is: 5'-tgcaaagttacgactcagcgc-3'.
[0027] (4) The amplification primer pair for the mutation site M192F is shown in SEQ ID No. 9, and the downstream amplification primer is shown in SEQ ID No. 10. Specifically, the sequence shown in SEQ ID No. 9 is: 5'-gcacgcttagactttggctacaactcctatgatc-3'. The sequence shown in SEQ ID No. 10 is: 5'-gtctaagcgtgcctggggatcg-3'.
[0028] (5) The amplification primer pair for the mutation site T328R-A330K-A333R is shown in SEQ ID No. 11, and the downstream amplification primer is shown in SEQ ID No. 12. Specifically, the sequence shown in SEQ ID No. 11 is: 5'-tttgatccggcgcgcgcaaaaaacccaagaaatggccaagccctg-3'. The sequence shown in SEQ ID No. 12 is: 5'-cgccggatcaaaagccgctt-3'.
[0029] (6) In the amplification primer pair for the mutation site Q410F-S413N, the upstream amplification primer is shown in SEQ ID No. 13, and the downstream amplification primer is shown in SEQ ID No. 14. Specifically, the sequence shown in SEQ ID No. 13 is: 5'-cggcgaataacgcattccgtgtaaacggtttctcagcgcggagct-3'. The sequence shown in SEQ ID No. 14 is: 5'-tgcgttattcgccgagctgttaaa-3'.
[0030] (7) The amplification primer pair for the T434D mutation site has the upstream amplification primer shown in SEQ ID No. 15 and the downstream amplification primer shown in SEQ ID No. 16. Specifically, the sequence shown in SEQ ID No. 15 is: 5'-ccgatgcccatggatcaagtcgccgcccgt-3'. The sequence shown in SEQ ID No. 16 is: 5'-catgggcatcggctccggcggggt-3'.
[0031] (8) The amplification primer pair for the A501I mutation site has the upstream amplification primer shown in SEQ ID No. 17 and the downstream amplification primer shown in SEQ ID No. 18. Specifically, the sequence shown in SEQ ID No. 17 is: 5'-gcactagatacaattgattattatgaacgcctgggt-3'. The sequence shown in SEQ ID No. 18 is: 5'-tgtatctagtgcagagaatgcggcatcg-3'.
[0032] (9) The amplification primer pair for the A509K mutation site is shown in SEQ ID No. 19, and the downstream amplification primer is shown in SEQ ID No. 20. Specifically, the sequence shown in SEQ ID No. 19 is: 5'-gaacgcctgggtaaggcaatgccgggcgcc-3'. The sequence shown in SEQ ID No. 20 is: 5'-acccaggcgttcataataatcaattgtatcta-3'.
[0033] (10) The amplification primer pair for the S561V mutation site is shown in SEQ ID No. 21 for the upstream amplification primer and in SEQ ID No. 22 for the downstream amplification primer. Specifically, the sequence shown in SEQ ID No. 21 is: 5'-attagcgcctgggttggcacccccggctactt-3'. The sequence shown in SEQ ID No. 22 is: 5'-ccaggcgctaatttggtcagggg-3'.
[0034] 5. Induction and expression of the engineered strain C41 / pET21a-IsMHETase mutant and purification of the target protein:
[0035] The plasmid of the constructed MHET hydrolase mutant was transduced into E. coli C41 competent cells. A portion of the colonies were picked and inoculated into 5 mL of LB liquid medium (containing Amp), and cultured at 37°C with shaking at 200 rpm for 4-6 h. Then, 1% of the culture was inoculated into 2YT medium (containing Amp) and cultured at 37°C with shaking at 200 rpm for approximately 5 h (OD). 600 Add 0.1% (w / v) IPTG (0.8-1.0%) and induce at 20℃ for 18-20h.
[0036] The expression and purification methods for the wild-type MHET hydrolase IsMHETase were followed.
[0037] 6. Enzymatic characterization of the MHET hydrolase mutant DuraMHETase:
[0038] MHET was added to 100 mM phosphate buffer (pH 8.0) to a final concentration of 40 mM; the engineered strain C41 / pET21a-IsMHETase mutant was added to a final concentration of 39 nM; and the mixture was incubated at 60 °C for 3 h. HPLC analysis showed that the TPA yield in the reaction solution was 13 mM. Figure 1 ).
[0039] Degradation of ethylene terephthalate (MHET) using the MHET hydrolase mutant DuraMHETase at different temperatures: MHET was added to 100mM phosphate buffer at pH 8.0 to a final concentration of 40mM; the MHET hydrolase mutant DuraMHETase was added to a final concentration of 39nM; and the mixture was incubated at different temperatures for 3 hours.
[0040] The MHET hydrolase mutant of this invention exhibits optimal activity at pH 8.0, and its relative enzymatic activity for MHET significantly exceeds that of the wild-type hydrolase at high temperatures. In combined applications with other PET hydrolases, especially at 60°C, its degradation efficiency for PET is effectively enhanced. It is worth emphasizing that the MHET hydrolase mutant of this invention demonstrates significant superiority in the direct degradation of PET, further enhancing its potential for industrial applications.
[0041] The above-described embodiments are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. The technical problem to be solved is to address the shortcomings of the prior art by providing an IsMHETase mutant enzyme, a recombinant vector, a recombinant engineered bacterium, and its applications.
Claims
1. A mutant MHET hydrolase enzyme, DuraMHETase, characterized in that, The MHET hydrolase IsMHETase was obtained by mutating the following amino acids: amino acid 66 from Ala to Pro, amino acid 136 from Ser to Glu, amino acid 159 from Thr to Val, amino acid 192 from Met to Phe, amino acid 330 from Ala to Lys, amino acid 333 from Ala to Arg, amino acid 328 from Thr to Arg, amino acid 410 from Gln to Phe, amino acid 413 from Ser to Asn, amino acid 434 from Asp to Thr, amino acid 501 from Ala to Ile, amino acid 509 from Ala to Lys, and amino acid 561 from Ser to Val. The MHET hydrolase IsMHETase has the amino acid sequence shown in SEQ ID No. 1 of the sequence listing.
2. An engineered strain, characterized in that, Contains the MHET hydrolase mutant DuraMHETase as described in claim 1.
3. Composition, characterized in that, Contains the MHET hydrolase mutant DuraMHETase as described in claim 1.
4. The use of the MHET hydrolase mutant DuraMHETase as described in claim 1 in the preparation of a bacterial agent for the catalytic degradation of ethylene terephthalate.
5. Use of the MHETase mutant DuraMHETase according to claim 4 for the preparation of a bacterial agent for the catalytic degradation of ethylene glycol terephthalate, characterized in that, The degradation temperature is 60°C.
6. The use of the composition of claim 3 in the preparation of a catalytic degradative agent for polyethylene terephthalate.
7. Use of the composition according to claim 6 for the preparation of a bacterial agent for the catalytic degradation of ethylene glycol terephthalate, characterized in that, The degradation temperature is 60°C.
8. The use of the engineered strain according to claim 2 in the preparation of a bacterial agent for the catalytic degradation of ethylene terephthalate.
9. Use of the engineered strain according to claim 8 for the preparation of a bacterial agent for the catalytic degradation of ethylene glycol terephthalate, characterized in that, The degradation temperature is 60°C.