LCCICCG mutant protein and application thereof

Abstract

The invention discloses an LCCICCG mutant protein and application thereof. The LCCICCG mutant protein is prepared by carrying out any one mutation of a, b and c or combined mutation of two or three of a, b and c on an LCCICCG protein amino acid sequence shown as SEQ ID NO: 1: a, carrying out combined mutation on any two of 208th, 238th and 283th amino acid residues; b, performing combined mutation on the 218th and 222th amino acid residues; and c, mutating one or more of the 278th, 239th, 68th and 158th amino acid residues. The mutant protein with improved thermal stability and / or catalytic activity is obtained by performing mutation modification on LCCICCG, the affinity and catalytic rate of the obtained mutant protein M3 to a PET substrate are obviously improved, and the hydrolytic activity of the mutant protein M3 to Gf-PET and PCW-PET is obviously higher than that of the LCCICCG.

Description

Technical Field

[0001] The present invention belongs to the field of bioenzymatic depolymerization of PET waste plastics, specifically LCC ICCG Mutant proteins and their applications. Background Art

[0002] Polyethylene terephthalate (PET) is the most consumed polyester plastic currently, and is widely used as a packaging material in various fields. Enzymatic depolymerization recycling allows post-consumer plastic products to be recycled and reused, providing a feasible solution to end plastic pollution. Enzyme elements are the core of the enzymatic route. At present, the modification strategy of PET plastic depolymerase mainly includes improving the thermal stability and catalytic activity of the protein. However, thermal stability and catalytic activity are often in a "trade-off" relationship. It is difficult to improve thermal stability while also improving catalytic efficiency. Therefore, improving thermal stability or catalytic activity is the current modification direction, but improving thermal stability while also improving catalytic efficiency is even more difficult. Summary of the invention

[0003] The first object of the present invention is to provide a LCC ICCG Mutant protein.

[0004] In order to achieve the above technical objectives, the present invention adopts the following scheme: A LCC ICCG Mutated proteins, including: About LCC ICCG The protein amino acid sequence undergoes any one of the following mutations a, b, c, or a combination of two or three of a, b, c: a. Combining mutations in any two of the amino acid residues at positions 208, 238, and 283; b. Combined mutation of amino acid residues 218 and 222; c. Mutate one or more of the amino acid residues at positions 278, 239, 68, and 158.

[0005] As a preferred embodiment, in said a, the amino acid residues at positions 208, 238, and 283 are mutated to cysteine, aspartic acid, and serine, respectively; In b, the amino acid residues at positions 218 and 222 are mutated to serine and isoleucine, respectively; In said c, the amino acid residues at positions 278, 239, 68 and 158 are mutated to proline.

[0006] As a preferred embodiment, the mutant protein is E208C / C238D, E208C / C283S, Y218S / F222I, N239P, T268P, R158P, E208C / C238D / Y218S / F222I or E208C / C238D / Y218S / F222I / R158P.

[0007] As a preferred embodiment, the mutant protein is E208C / C238D, R158P, E208C / C238D / Y218S / F222I or E208C / C238D / Y218S / F222I / R158P.

[0008] The second object of the present invention is to provide the use of the mutant protein in PET depolymerization.

[0009] As a preferred embodiment, the temperature for depolymerization of PET is 70-75°C.

[0010] As a preferred embodiment, the PET is in powder form. Further, the PET is amorphous PET powder Gf-PET or post-consumer recycled PET powder PCW-PET.

[0011] As a preferred embodiment, the concentration of substrate PET in the PET depolymerization system is 2-200 g / L.

[0012] As a preferred embodiment, the amount of mutant protein used in the PET depolymerization system is 1-10 mg / g PET.

[0013] The present invention has the following beneficial effects: The present invention is through LCC ICCG Mutation modification was performed to obtain mutant proteins with improved thermal stability and / or catalytic activity. The obtained mutant protein M3 had significantly improved affinity and catalytic rate for the PET substrate, and its hydrolysis activity for Gf-PET and PCW-PET was significantly higher than that of LCC ICCG . BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 For LCC ICCG And study on the thermal stability and hydrolytic activity of mutant proteins.

[0015] Figure 2 Comparison of product release of single-strategy mutant proteins under different temperature conditions (A: mutant protein DS4; B: mutant protein GF; C: mutant protein HP4).

[0016] Figure 3Iterative evolution of the disaggregation performance of mutant protein M3 by combining multiple strategies (A: LCC ICCG Iterative evolution; B: LCC ICCG And the product release and proportion of mutant proteins; C: reverse Michaelis constant analysis LCC ICCG and variants; D: LCC ICCG Comparison of the depolymerization performance of M1 and M3 on different substrates).

[0017] Figure 4 For LCC ICCG Comparison of depolymerization efficiency of 200 g / L PCW-PET with M3 (A: LCC ICCG ; B:M3).

[0018] Figure 5 For LCC ICCG Comparison of depolymerization efficiency of 200 g / L Gf-PET with M3 (A: LCC ICCG ; B:M3). DETAILED DESCRIPTION

[0019] The following examples will further illustrate the method provided by the present invention, but the present invention is not limited to the listed examples, and should also include any other known changes within the scope of the rights claimed by the present invention.

[0020] Example 1 This example specifically describes the method for constructing the mutant protein of the present invention, including: (1) Determined by amino acid sequence alignment, LCC ICCG (abbreviated as DS1) same Ca 2+ The amino acids of the binding sites are E208, C238 and S283. Any two of the three sites are mutated to obtain mutant proteins: E208C / C283S (abbreviated as DS2), C238D / C283S (abbreviated as DS3) and E208C / C238D (abbreviated as DS4).

[0021] (2) Based on the idea of ​​improving the effect of non-covalent bonds, the mutation Y218S / F222I (abbreviated as GF) was introduced.

[0022] (3) Four mutation sites were selected based on the accessibility of the protein surface, namely N278, N239, T268 and R158. Proline is a hydrophobic amino acid with strong rigidity, which can improve the thermal stability of the protein. Therefore, this application improves the surface hydrophobicity of the protein by mutating any of the four amino acids to proline, and obtains mutant proteins N278P (abbreviated as HP1), N239P (abbreviated as HP2), T268P (abbreviated as HP3) and R158P (abbreviated as HP4).

[0023] (4) Study on thermal stability and hydrolysis activity of mutant proteins: The melting temperature and hydrolysis activity of mutant proteins obtained by the three strategies (1)-(3) were investigated. ICCG The catalytic activity at the optimal reaction temperature of 72°C was used to analyze the thermal stability and catalytic activity improvement of the mutant protein.

[0024] The results of the study on the thermal stability and hydrolytic activity of the mutant protein in Example 1 are as follows Figure 1 , 2 As shown, the depolymerization system used was: the concentration of substrate amorphous PET powder (Gf-PET) was 2 g / L, and the DSC-detected crystallinity of Gf-PET was 6.7%.

[0025] Example 2 (1) Determining variant sites for multi-strategy iterative evolution: In Example 1, the Tm value and dissociation activity of the mutant protein (E208C / C238D) increased by 2.2°C and 4%, respectively; the Tm value of the mutant protein (Y218S / F222I) decreased by 8.4°C, and the dissociation activity increased by 45.5%; the Tm value and dissociation activity of the mutant protein (R158P) increased by 7.3°C and 26.8%, respectively. Therefore, DS4, GF and HP4 were selected as sites for iterative evolution.

[0026] (2) According to the single strategy evolution rounds, the above three mutations are iteratively combined in sequence to construct LCC ICCG Combination mutant proteins.

[0027] First, the C238 and C283 sites were mutated, and the mutant protein DS4 (hereinafter referred to as M1) was constructed in the manner of Example 1. Then, M1 was used as the next mutant skeleton to iteratively mutate GF (i.e., introduce mutations Y218S / F222I) to obtain the mutant protein DS4+GF (referred to as M2). After adding the GF mutation, the hydrolysis activity of PET at 72°C was improved, and its average catalytic efficiency was increased by 59.5%. Finally, M2 was used as the next mutant skeleton to iteratively mutate HP4 (i.e., introduce mutation R158P) to obtain the mutant protein DS4+GF+HP4 (referred to as M3), which increased the Tm value by 11.3°C and increased the average catalytic efficiency of depolymerizing PET at 72°C by 12.1%. M3 and LCC ICCG In comparison, Tm increased by 6.3℃ and the average catalytic efficiency increased by 78.9%.

[0028] The depolymerization performance of the multi-strategy combination iterative evolution variant M3 in Example 2 is as follows: Figure 3 shown.

[0029] Example 3 Based on Example 2, this example compares the depolymerization efficiency of mutant protein M3 on post-consumer recycled PET powder (PCW-PET) and amorphous PET powder (Gf-PET), with a reaction temperature of 72°C, a substrate concentration of 200 g / L, and an enzyme loading of 2 mg / g PET. The DSC-detected crystallinity of PCW-PET is 15%, and the DSC-detected crystallinity of Gf-PET is 6.7%.

[0030] Degradation results such as Figure 4 , 5 The results showed that when PCW-PET was used as the substrate, the depolymerization rate of the mutant protein M3 was almost 0 in the late depolymerization period (8-12h) because the hydrolyzable substrate was exhausted, indicating that the mutant protein reached the depolymerization end point in 8 h, which means that M3 has a shorter depolymerization cycle than LCC. ICCG The reaction time was shortened by 4 h. When Gf-PET was used as substrate, the LCC ICCG The depolymerization rates of M1 and M3 reached the highest points of 95.2% and 97.3%, respectively, and the depolymerization products were almost all TPA.

[0031] The above is an explanation of the embodiments of the present invention. However, the present invention is not limited to the above embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A LCC ICCG A mutant protein, characterized in that include: For the LCC shown in SEQ ID NO: 1 ICCG The protein amino acid sequence undergoes any one of the following mutations a, b, c, or a combination of two or three of a, b, c: a. Combining mutations in any two of the amino acid residues at positions 208, 238, and 283; b. Combined mutation of amino acid residues 218 and 222; c. Mutate one or more of the amino acid residues at positions 278, 239, 68, and 158.

2. The mutant protein according to claim 1, characterized in that In a, the amino acid residues at positions 208, 238, and 283 are mutated to cysteine, aspartic acid, and serine, respectively; In b, the amino acid residues at positions 218 and 222 are mutated to serine and isoleucine, respectively; In said c, the amino acid residues at positions 278, 239, 68 and 158 are mutated to proline.

3. The mutant protein according to claim 1, characterized in that The mutant protein is E208C / C238D, E208C / C283S, Y218S / F222I, N239P, T268P, R158P, E208C / C238D / Y218S / F222I or E208C / C238D / Y218S / F222I / R158P.

4. The mutant protein according to claim 3, characterized in that The mutant protein is E208C / C238D, R158P, E208C / C238D / Y218S / F222I or E208C / C238D / Y218S / F222I / R158P.

5. Use of the mutant protein according to any one of claims 1 to 4 in PET depolymerization.

6. The use according to claim 5, characterized in that: The temperature for PET depolymerization is 70-75°C.

7. The use according to claim 5, characterized in that: The PET is in powder form.

8. The use according to claim 7, characterized in that The PET is amorphous PET powder Gf-PET or post-consumer recycled PET powder PCW-PET.

9. The use according to claim 5, characterized in that: The concentration of substrate PET in the PET depolymerization system is 2-200 g / L.

10. The use according to claim 5, characterized in that The amount of mutant protein used in the PET depolymerization system is 1~10 mg / g PET.

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