Pet hydrolytic enzyme mutants and uses thereof

Abstract

The present application relates to PET hydrolytic enzyme mutant and its application, the amino acid sequence of the PET hydrolytic enzyme mutant is shown as SEQ ID NO:1.The present application is through the structure research of PET hydrolytic enzyme LSPET4, combines the directed evolution technology, carries out mutation to the 96th, 127th, 130th, 241st, 209th, 238th site of LSPET4 protein, constructs mutant enzyme LSPET.The experimental results show that the mutant enzyme greatly increases the hydrolytic activity to PET, and using the mutant enzyme for degrading PET reaction can make the yield of degrading PET reaction increase, and has good industrial application value.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of enzyme engineering and biotechnology, and particularly relates to a PET hydrolytic enzyme mutant and application thereof. BACKGROUND

[0002] Polyethylene terephthalate (PET) is a common plastic, which is composed of terephthalic acid (TPA) and ethylene glycol (EG) connected by ester bonds. PET products have been widely used in food, drug, cosmetic packaging and other fields due to their plasticity and high stability, and the amount of waste generated is huge, which is one of the main sources of white pollution. Due to its high stability, PET waste needs hundreds of years to completely decompose under natural conditions, which accumulates in the global ecosystem and invades the human food chain, seriously threatening the ecosystem and human health.

[0003] Biological enzyme method for recycling PET plastics is a green and ideal treatment method, which provides a new opportunity to solve PET waste pollution. At present, many esterases, lipases or cutinases in the form of PET hydrolytic enzymes have been identified, among which IsPETase and LCC are the most widely studied plastic degradation enzymes. Many research teams have developed more stable enzyme mutants based on these two wild-type PET hydrolytic enzymes, such as DuraPETase, HotPETase, FastPETase, DepoPETase, and LCC ICCG However, due to the structural limitations of IsPETase and LCC, the ability of their mutants to degrade PET has reached a bottleneck, and there is still a lack of more excellent PET hydrolytic enzyme modification templates. Moreover, the reported PET hydrolytic enzymes only contain one PET hydrolysis domain, and lack of substrate binding domain, which is an important factor limiting the activity of PET hydrolytic enzymes. Recently, our team discovered a new type of PET hydrolytic enzyme LSPET4 in Micromonospora sp. HM5-17, which contains a carbohydrate binding module (CBM) in addition to the PET hydrolysis domain. Compared with common PET hydrolytic enzymes, the substrate binding conformation of LSPET4 hydrolytic enzyme is linear, which is different from the common PET hydrolytic enzyme and substrate binding conformation of right angle. However, the degradation efficiency of LSPET4 is still low, and there is still a gap from commercial application. SUMMARY

[0004] The first purpose of the present application is to provide a PET hydrolytic enzyme mutant by studying the structure of PET hydrolytic enzyme LSPET4 and combining directed evolution technology, which has better enzyme activity and can improve the industrial application value of PET hydrolytic enzyme.

[0005] To achieve the above technical purposes, the technical scheme adopted by the present application is as follows:

[0006] A PET hydrolytic enzyme mutant, the amino acid sequence of which is shown in SEQ ID NO: 1.

[0007] A second object of the present application is to provide a nucleotide sequence encoding the PET hydrolytic enzyme mutant, an alternative of which is shown in SEQ ID NO: 2.

[0008] A third object of the present application is to provide a recombinant vector comprising the nucleotide sequence. Preferably, the recombinant vector is constructed using pET-22b plasmid.

[0009] A fourth object of the present application is to provide an engineered strain comprising the recombinant vector. Preferably, the engineered strain is constructed using Escherichia coli as the host bacteria.

[0010] A fifth object of the present application is to provide the use of the PET hydrolytic enzyme mutant in hydrolyzing PET.

[0011] As a preferred embodiment, the use comprises adding the PET hydrolytic enzyme mutant to a solution of PET to hydrolyze the PET.

[0012] As a preferred embodiment, the solvent of the solution of PET is a mixture of phosphate buffer and organic solvent. Preferably, the organic solvent is dimethyl sulfoxide.

[0013] As a preferred embodiment, the PET hydrolytic enzyme mutant hydrolyzes PET at a temperature ranging from 30℃ to 55℃; preferably, the temperature ranges from 45℃ to 55℃.

[0014] As a preferred embodiment, the PET hydrolytic enzyme mutant hydrolyzes PET at a pH ranging from 7.4 to 8.0.

[0015] In the study of the structure of the existing LSPET4, the inventors believe that the four sites of the 96th, 127th, 130th and 241st sites of the existing LSPET4 protein are located in the catalytic pocket of the enzyme, and thus the directional mutation of the amino acids at the four sites is beneficial to the linear combination of the substrate and the active pocket of the enzyme, thereby improving the enzyme activity of LSPET4; the 209th and 238th sites are located on the surface of the enzyme molecule, and the mutation of the two amino acids is beneficial to the formation of salt bridge, thereby increasing the stability of the protein; based on the above six sites, the LSPET4 mutant enzyme is named as LSPET. The experimental results show that the mutant enzyme increases the hydrolytic activity of PET, and the use of the mutant enzyme for degrading PET reaction can improve the yield of the degrading PET reaction, and has good industrial application value. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 Figure 1 is a schematic diagram of the recombinant plasmid pET-22b-LSPET4.

[0017] Figure 2 Figure 2 is a schematic diagram of the recombinant plasmid pET-22b-LSPET.

[0018] Figure 3 Figure 3 is a schematic diagram of PETase hydrolyzing PET.

[0019] Figure 4 Figure 4 is a yield of TPA and MHET obtained by the PET hydrolase LSPET4 and LSPET catalyzing degradation of polyethylene terephthalate.

[0020] Figure 5 Figure 5 is a yield of TPA and MHET obtained by LSPET and the reported high-efficiency PET hydrolases DuraPETase and FastPETase catalyzing degradation of polyethylene terephthalate at different temperatures. Embodiment

[0021] The present application will be further explained in conjunction with the accompanying drawings and embodiments. Embodiment 1

[0022] Since the obtaining of the existing PET hydrolase LSPET4 gene is the basis for related sequence analysis and directed evolution mutation, the present embodiment first briefly introduces the cloning process of the existing PET hydrolase LSPET4 gene as follows.

[0023] The amino acid sequence of the PET hydrolase LSPET4 is as Genbank Sequence ID: ROT25881.1.

[0024] First, the LSPET4 gene (nucleotide sequence as shown in SEQ ID NO: 3, containing 1278 bases) is obtained by the method of whole gene synthesis, and the gene is constructed into the pET-22b vector using NdeI and XhoI restriction enzymes, respectively, and then the recombinant plasmid is transformed into the competent cell BL21(DE3) to obtain the LSPET4 recombinant plasmid.

[0025] In order to increase the PET hydrolysis activity, the present application uses point mutation technology, and the mutation sequence is performed according to D130P, N127F, Y96F, Q209E, A238K, and D241S, using the LSPET4 gene before modification as a template, and the primers are designed as follows:

[0026] D130P-F: 5'-AATGACTTCCCGGAAGCACGCGGCACCCAGCT-3' (introducing mutation at site 130)

[0027] D130P-R: 5'-GCGTGCTTCCGGGAAGTCATTACGACTATTGG-3' (introducing mutation at position 130)

[0028] N127F-F: 5'-AATAGTCGTTTCGACTTCCCGGAAGCACGC-3' (introducing mutation at position 127)

[0029] N127F-R: 5'-CGGGAAGTCGAAACGACTATTGGTTTCAATACC-3' (introducing mutation at position 127)

[0030] Y96F-F: 5'-ATTAGTCCGGGCTTCACCGCCCTGTTCAGCGCAG-3' (introducing mutation at position 96)

[0031] Y96F-R: 5'-GAACAGGGCGGTGAAGCCCGGACTAATTGCAATG-3' (introducing mutation at position 96)

[0032] Q209E-F: 5'-TTCGCCGGTGAACAGGATACCGTTGTTAG-3' (introducing mutation at position 209)

[0033] Q209E-R: 5'-CGGTATCCTGTTCACCGGCGAACACCATT-3' (introducing mutation at position 209)

[0034] A238K-F: 5'-TATCTGGAAATTAAAGGCGGCGATCATGGCTT-3' (introducing mutation at position 238)

[0035] A238K-R: 5'-ATGATCGCCGCCTTTAATTTCCAGATATGCGCTTTCGG-3' (introducing mutation at position 238)

[0036] D241S-F: 5'-AATGGCGGCAGTCATGGCTTCCCGGTTGGTC-3' (introducing mutation at position 241)

[0037] D241S-R: 5'-GGGAAGCCATGACTGCCGCCTTTAATTTCCAG-3' (introducing mutation at position 241)

[0038] According to the reverse PCR technology, the recombinant plasmid containing the mutant gene is constructed, and the original template DNA is removed by DpnI. Then the mutant plasmid is transformed into the DH5α competent cells, and the mutant plasmid pET-22b-LSPET4 is obtained through the screening of ampicillin plate culture medium, plasmid extraction and sequencing verification D130P、N127F、Y96F、Q209E、A238K、D241S .

[0039] The formula of the ampicillin plate culture medium is: yeast extract 5 g / L, tryptone 10 g / L, sodium chloride 10 g / L, agar powder 15 g / L, ampicillin 50 mg / L.

[0040] The LSPET4 obtained after mutation D130P、N127F、Y96F、Q209E、A238K、D241S The gene is named LSPET, and the LSPET amino acid sequence is shown in SEQ ID NO: 1, and the nucleotide sequence is shown in SEQ ID NO: 2. Hereinafter, the mutant plasmid is referred to as pET-22b-LSPET. Example 2

[0041] This embodiment specifically illustrates the construction method of the BL21(DE3) / pET-22b-LSPET engineering bacteria.

[0042] The mutant plasmid pET-22b-LSPET is transformed into the BL21(DE3) competent cells, and is coated on the ampicillin plate culture medium to obtain the positive recombinant BL21(DE3) / pET-22b-LSPET, that is, the expression engineering bacteria containing the recombinant plasmid pET-22b-LSPET is successfully constructed; Example 3

[0043] This embodiment specifically illustrates the induction expression and the target protein purification method of the BL21(DE3) / pET-22b-LSPET.

[0044] The BL21(DE3) / pET-22b-LSPET engineering bacteria is cultured in the LB culture medium at 37℃ and 200 r / min overnight; the overnight culture liquid is inoculated into the LB culture medium at 37℃ and 200 r / min at a 1% inoculation amount, until the OD600 is 0.6, 1 M IPTG is added to a final concentration of 0.5 mM, and is induced at 20℃ for 16 h.

[0045] After the induction is completed, the bacterial liquid is centrifuged at 8000 rpm to collect the bacterial body, 3 mL of phosphate buffer is added, and is resuspended by shaking, and is ultrasonically broken for 10 min at a power of 300 W.

[0046] The broken liquid is ultracentrifuged at 12000 rpm in an environment of 4°C for 10 min, and the supernatant is taken to be purified in a Ni-NTA resin, the impure proteins are washed by using a washing buffer, and the target protein is eluted by using an elution buffer, and the eluate is concentrated by a protein concentration tube.

[0047] The formula of the LB liquid culture medium is: yeast extract 5 g / L, tryptone 10 g / L, sodium chloride 10 g / L.

[0048] The formula of the phosphate buffer is: 8 mM Na2HPO4, 2 mM NaH2PO4, 136 mM NaCl, 2.6 mM KCl, pH = 7.4.

[0049] The formula of the washing buffer is: 30 mM imidazole, 300 mM NaCl, 45 mM Na2HPO4, 4.25 mM NaH2PO4, pH = 7.8. 4，

[0050] The formula of the elution buffer is: 300 mM imidazole, 300 mM NaCl, 45 mM Na2HPO4, 4.25 mM NaH2PO4, pH = 7.8. 4，

[0051] After the engineering bacteria BL21 (DE3) / pET-22b-LSPET4 of LSPET4 is induced and expressed according to the above method, the protein is purified and concentrated under the same conditions. Example 4

[0052] The PET hydrolytic enzyme LSPET4 is used to catalyze the degradation of polyethylene terephthalate (PET).

[0053] Dimethyl sulfoxide is added to the phosphate buffer with a pH value of 7.4 prepared in Example 3 to make the final concentration 20% (v / v); polyethylene terephthalate is added to make the final concentration 40 mg / mL; LSPET4 is added to make the final concentration 500 nM; after incubation at 45°C and 200 rpm for 2 days, an equal volume of methanol is used for inactivation. HPLC detection shows that the concentration of the PET degradation product in the reaction solution is 19.68 mg / L.

[0054] The PET degradation product includes terephthalic acid (TPA), monohydroxyethyl terephthalate (MHET) and bis(2-hydroxyethyl) terephthalate (BHET), wherein BHET is an intermediate product and has a very small content in the final product, so the yield is the sum of the yields of TPA and MHET. Example 5

[0055] ​​The PET hydrolytic enzyme LSPET4 mutant enzyme LSPET catalyzes the degradation of polyethylene terephthalate (PET).

[0056] Dimethyl sulfoxide was added to the phosphate buffer prepared in Example 3 to a final concentration of 20% (v / v); polyethylene terephthalate was added to a final concentration of 40 mg / mL; LSPET was added to a final concentration of 500 nM; after incubation at 45°C, 200 rpm for 2 days, an equal volume of methanol was added to inactivate the enzyme. The concentration of PET degradation products in the reaction solution was determined by HPLC to be 762.41 mg / L.

[0057] The detection conditions of the HPLC were as follows: ultraviolet detector, characteristic absorption peak at 260 nm, C18 chromatographic column (4 μL, 150 mm x 4.6 mm), mobile phase A: 0.1% formic acid aqueous solution, mobile phase B: acetonitrile, mobile phase: 80% mobile phase A, 20% mobile phase B, column temperature: 25°C, injection volume: 10 μL, flow rate: 0.5 mL / min. Example 6

[0058] The PET hydrolytic enzymes DuraPETase and FastPETase catalyze the degradation of polyethylene terephthalate (PET).

[0059] In order to compare the differences in activity between LSPET and existing excellent PET hydrolytic enzyme mutants, the abilities of DuraPETase and FastPETase to catalyze the degradation of PET were measured, as follows.

[0060] First, the DuraPETase and FastPETase genes were obtained by whole gene synthesis, and the genes were constructed into the pET-22b vector using NdeI and XhoI restriction enzymes, respectively. The recombinant plasmid was then transformed into competent cells BL21 (DE3) to obtain the corresponding engineering bacteria, and the corresponding PET hydrolytic enzymes were obtained using the protein purification method of Example 3.

[0061] Dimethyl sulfoxide was added to the phosphate buffer at pH 8.0 (the optimal pH of LSPET4 is 7.4, and the optimal pH of mutant LSPET is 8.0) to a final concentration of 20% (v / v); polyethylene terephthalate was added to a final concentration of 40 mg / mL; three experimental groups were set up, and LSPET, DuraPETase or FastPETase was added to a final concentration of 500 nM; each experimental group was incubated at 30°C, 37°C, 45°C, 50°C, 55°C, 200 rpm for 2 days, and an equal volume of methanol was added to inactivate the enzyme, and the concentration of PET degradation products in the reaction solution was determined by HPLC.

[0062] The formulation of the pH 8.0 phosphate buffer is: 94.7 mL of 100 mM Na2HP04mixed with 5.3 mL of 100 mM NaH2P04.

[0063] A comparison of LSPET and DuraPETase, FastPETase activity is shown in FIG. 2. LSPET showed higher activity than DuraPETase at a temperature range of 30 °C to 55 °C. Compared to FastPETase, LSPET showed higher PET hydrolysis activity at a range of 45 °C to 55 °C. These results suggest that LSPET has potential as a heat-resistant candidate enzyme for industrial PET hydrolysis. Figure 5 A comparison of LSPET and DuraPETase, FastPETase activity is shown in FIG. 2. LSPET showed higher activity than DuraPETase at a temperature range of 30 °C to 55 °C. Compared to FastPETase, LSPET showed higher PET hydrolysis activity at a range of 45 °C to 55 °C. These results suggest that LSPET has potential as a heat-resistant candidate enzyme for industrial PET hydrolysis.

Claims

1. A PET hydrolase mutant, characterized in that Its amino acid sequence is shown in SEQ ID NO:

1.

2. A nucleic acid encoding the PET hydrolase mutant according to claim 1.

3. A recombinant vector comprising the nucleic acid of claim 2.

4. An engineered strain comprising the recombinant vector according to claim 3.

5. Use of the PET hydrolase mutant according to claim 1 in hydrolyzing PET.

6. The use according to claim 5, characterized in that include: The PET hydrolase mutant is added to the PET solution to hydrolyze PET.

7. The use according to claim 6, characterized in that The solvent of the PET solution is a mixture of phosphate buffer and an organic solvent.

8. The use according to claim 7, characterized in that The organic solvent is dimethyl sulfoxide.

9. The use according to claim 5, characterized in that The PET hydrolase mutant hydrolyzes PET within the range of 30°C-55°C.

10. The use according to claim 9, characterized in that The PET hydrolase mutant hydrolyzes PET within the range of 45-55°C.

11. The use according to claim 5, characterized in that The PET hydrolase mutant hydrolyzes PET at pH 8.0.

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