LCC ICCG Mutant proteins and their applications

Abstract

This invention discloses LCC ICCG Mutant proteins and their applications, including: LCCs as shown in SEQ ID NO: 1 ICCG The protein amino acid sequence is subjected to any one of the following mutations a, b, and c, or a combination of two or three of a, b, and c: a. Combination mutation of any two of the amino acid residues at positions 208, 238, and 283; b. Combination mutation of the amino acid residues at positions 218 and 222; c. Mutation of one or more of the amino acid residues at positions 278, 239, 68, and 158. This invention utilizes LCC... ICCG Mutation modification was performed to obtain mutant proteins with improved thermal stability and / or catalytic activity. The resulting mutant protein M3 showed significantly enhanced affinity and catalytic rate for PET substrates, and its hydrolytic activity for Gf-PET and PCW-PET was significantly higher than that for LCC. ICCG .

Description

Technical Field

[0001] This invention belongs to the field of bio-enzymatic depolymerization of waste PET plastics, specifically LCC. ICCG Mutant proteins and their applications. Background Technology

[0002] Polyethylene terephthalate (PET) is currently the most consumed polyester plastic, widely used as a packaging material in various fields. Enzymatic depolymerization recycling allows post-consumer plastic products to be reused, providing a feasible solution to ending plastic pollution. Enzyme elements are the core of the enzymatic route. Currently, the modification strategies for PET plastic depolymerization enzymes mainly include improving the thermal stability and catalytic activity of the protein. However, thermal stability and catalytic activity are often inversely related; it is difficult to improve both thermal stability and catalytic efficiency simultaneously. Therefore, improving either thermal stability or catalytic activity is a current modification direction, but simultaneously improving both thermal stability and catalytic efficiency is a significant challenge. Summary of the Invention

[0003] The primary objective of this invention is to address the problems existing in the prior art by providing an LCC. ICCG Mutant protein.

[0004] To achieve the above technical objectives, the present invention adopts the following solution:

[0005] A type of LCC ICCG Mutant proteins, including:

[0006] For LCC ICCG The protein amino acid sequence undergoes any one of the following mutations: a, b, or c, or a combination of two or three of a, b, or c:

[0007] a. Perform a combined mutation on any two of the amino acid residues at positions 208, 238, and 283;

[0008] b. Perform combined mutations on amino acid residues at positions 218 and 222;

[0009] c. Mutate one or more of the amino acid residues at positions 278, 239, 68, and 158.

[0010] In a preferred embodiment, in a, the 208th, 238th, and 283rd amino acid residues are mutated to cysteine, aspartic acid, and serine, respectively;

[0011] In b, amino acid residues at positions 218 and 222 are mutated to serine and isoleucine, respectively.

[0012] In the c-position, amino acid residues at positions 278, 239, 68, and 158 are mutated to proline.

[0013] In 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.

[0014] In a preferred embodiment, the mutant protein is E208C / C238D, R158P, E208C / C238D / Y218S / F222I, or E208C / C238D / Y218S / F222I / R158P.

[0015] A second objective of this invention is to provide the application of the above-mentioned mutant protein in PET depolymerization.

[0016] As a preferred embodiment, the depolymerization temperature of PET is 70-75°C.

[0017] In 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).

[0018] As a preferred embodiment, the concentration of the substrate PET in the PET depolymerization system is 2-200 g / L.

[0019] As a preferred embodiment, the amount of mutant protein used in the PET depolymerization system is 1~10 mg / g PET.

[0020] The present invention has the following beneficial effects:

[0021] This invention utilizes LCC ICCG Mutation modification was performed to obtain mutant proteins with improved thermal stability and / or catalytic activity. The resulting mutant protein M3 showed significantly enhanced affinity and catalytic rate for PET substrates, and its hydrolytic activity for Gf-PET and PCW-PET was significantly higher than that for LCC. ICCG . Attached Figure Description

[0022] Figure 1 For LCC ICCG Studies on the thermal stability and hydrolytic activity of mutant proteins.

[0023] Figure 2Comparison of product release from single-strategy mutant proteins under different temperature conditions (A: mutant protein DS4; B: mutant protein GF; C: mutant protein HP4).

[0024] Figure 3 To improve the depolymerization performance of mutant protein M3 through multi-strategy combination iterative evolution (A:LCC) ICCG Iterative evolution; B: LCC ICCG and the product release and proportion of mutant proteins; C: inverse Michaelis constant analysis (LCC) ICCG and variants; D: LCC ICCG (Comparison of the depolymerization performance of M3 on different substrates).

[0025] Figure 4 For LCC ICCG Comparison of depolymerization efficiency of M3 and 200 g / L PCW-PET (A: LCC) ICCG B: M3).

[0026] Figure 5 For LCC ICCG Comparison of depolymerization efficiency of M3 and 200 g / L Gf-PET (A: LCC) ICCG B: M3). Detailed Implementation

[0027] The following embodiments will further illustrate the method provided by the present invention, but the present invention is not limited to the listed embodiments, and should also include any other known modifications within the scope of the claims of the present invention.

[0028] Example 1

[0029] This embodiment specifically illustrates the method for constructing the mutant protein in this invention, including:

[0030] (1) LCC was determined by amino acid sequence alignment. ICCG (Abbreviated as DS1) Same Ca 2+ The amino acids at the binding sites are E208, C238, and S283. Mutating any two of these three sites yields mutant proteins: E208C / C283S (DS2), C238D / C283S (DS3), and E208C / C238D (DS4).

[0031] (2) Based on the modification idea of ​​enhancing the role of non-covalent bonds, the mutant Y218S / F222I (abbreviated as GF) is introduced.

[0032] (3) Based on the accessibility of the protein surface, four mutation sites were selected: N278, N239, T268, and R158. Proline is a hydrophobic amino acid with high rigidity, which can improve the thermal stability of the protein. Therefore, this application improves the hydrophobicity of the protein surface by mutating any one of these 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), respectively.

[0033] (4) Study on the thermal stability and hydrolytic activity of mutant proteins: The melting temperature and LCC of mutant proteins obtained by the three strategies (1)-(3) were investigated. ICCG The catalytic activity at the optimal reaction temperature of 72℃ was analyzed, along with the thermal stability and the enhancement of catalytic activity of the mutant protein.

[0034] The results of the thermostability and hydrolytic activity studies of the mutant protein in Example 1 are as follows: Figure 1 , 2 As shown, the depolymerization system used was as follows: the concentration of substrate amorphous PET powder (Gf-PET) was 2 g / L, and the crystallinity of Gf-PET was 6.7% as determined by DSC.

[0035] Example 2

[0036] (1) Determining the sites for multi-strategy iterative evolution variants: In Example 1, the Tm value and depolymerization activity of the mutant protein (E208C / C238D) increased by 2.2℃ and 4%, respectively; the Tm value of the mutant protein (Y218S / F222I) decreased by 8.4℃ and the depolymerization activity increased by 45.5%; the Tm value and depolymerization activity of the mutant protein (R158P) increased by 7.3℃ and 26.8%, respectively. Therefore, DS4, GF and HP4 were selected as sites for iterative evolution.

[0037] (2) The above three mutations are iteratively combined in sequence according to the single-strategy evolution round to construct LCC. ICCG Combinatorial mutant proteins.

[0038] First, mutations were performed at sites C238 and C283 to construct the mutant protein DS4 (hereinafter referred to as M1) as described in Example 1. Then, M1 was used as the backbone for iterative mutation of GF (i.e., introducing the mutant Y218S / F222I) to obtain the mutant protein DS4+GF (M2). The addition of GF mutation improved the hydrolytic activity of PET at 72°C, increasing its average catalytic efficiency by 59.5%. Finally, M2 was used as the backbone for iterative mutation of HP4 (i.e., introducing the mutant R158P) to obtain the mutant protein DS4+GF+HP4 (M3), increasing the Tm value by 11.3°C and improving the average catalytic efficiency of PET depolymerization at 72°C by 12.1%. M3 and LCC... ICCG Compared to the previous method, Tm increased by 6.3℃, and the average catalytic efficiency increased by 78.9%.

[0039] The de-aggregation performance of the multi-strategy combined iterative evolution variant M3 in Example 2 is as follows: Figure 3 As shown.

[0040] Example 3

[0041] This example, based on Example 2, compares the depolymerization efficiency of mutant protein M3 on post-consumer recycled PET powder (PCW-PET) and amorphous PET powder (Gf-PET). The reaction temperature was 72°C, the substrate concentration was 200 g / L, and the enzyme loading was 2 mg / g PET. The DSC-detected crystallinity of PCW-PET was 15%, and that of Gf-PET was 6.7%.

[0042] Degradation results as follows Figure 4 , 5 As shown in the figure. The results indicate that when PCW-PET is used as the substrate, the mutant protein M3 exhibits an almost zero depolymerization rate in the later stages of depolymerization (8-12 h) because the substrate is almost completely consumed by hydrolysis. This suggests that the mutant protein reaches the depolymerization endpoint within 8 h, implying that M3 has a shorter depolymerization cycle compared to LCC. ICCG The reaction time was shortened by 4 h. When using Gf-PET as the substrate, the LCC was reduced after 12 h and 8 h of reaction. ICCG The depolymerization rates of M3 and M3 reached their highest points of 95.2% and 97.3% respectively, and the depolymerization products were almost entirely TPA.

[0043] The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiments. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An LCC ICCG Mutant protein, characterized by, For LCCs as shown in SEQ ID NO: 1 ICCG The mutant protein was obtained by mutating the protein's amino acid sequence as shown below: (1) Mutate amino acid residues at positions 208, 238, 218, and 222 to cysteine, aspartic acid, serine, and isoleucine, respectively; Or (2) mutate the amino acid residues at positions 208, 238, 218, 222, and 158 to cysteine, aspartic acid, serine, isoleucine, and proline, respectively.

2. The application of the mutant protein of claim 1 in PET depolymerization.

3. The application according to claim 2, characterized in that, The depolymerization temperature of PET is 70-75℃.

4. The application according to claim 2, characterized in that, The PET is in powder form.

5. The application according to claim 4, characterized in that, The PET is amorphous PET powder (Gf-PET) or post-consumer recycled PET powder (PCW-PET).

6. The application according to claim 2, characterized in that, The concentration of PET substrate in the PET depolymerization system is 2-200 g / L.

7. The application according to claim 2, characterized in that, The amount of mutant protein used in the PET depolymerization system is 1~10 mg / g PET.

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