PU depolymerase mutant with high catalytic activity and its application

Abstract

The present invention belongs to the field of genetic engineering, and particularly relates to highly catalytically active PU depolymerase mutants and their applications. Using structural analysis and site-directed mutagenesis, the present invention mutates amino acids 29, 89, 141, 196, 224, and 276 in the amino acid sequence of the PU depolymerase shown in SEQ ID NO:2 to generate multiple mutants with enhanced catalytic activity. These PU depolymerase mutants enhance the hydrolysis efficiency of carbamate bonds in PU, thereby improving their ability to degrade PU plastics. In the future, they could be applied to the enzymatic depolymerization of various types of PU plastics in daily life.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering, in particular to a highly active PU depolymerase mutant and application thereof. Background Art

[0002] Polyurethane (PU) is the world's sixth most common plastic, boasting excellent physical and chemical properties such as wear resistance, tear resistance, oil resistance, and aging resistance. It is primarily used in thermoset flexible and rigid foams for home furnishings, construction, and transportation, as well as thermoplastic polyurethanes for footwear and apparel. However, the widespread use of PU plastics has led to the generation of large amounts of waste, which is difficult to degrade in the natural environment, significantly impacting the environment. According to statistics, in 2020, my country's polyurethane waste reached 12 million tons, nearly half of which was landfilled or discarded, causing microplastic pollution in soil, water, and even human bodies.

[0003] At present, the main methods for treating polyurethane (PU) waste include direct landfill, incineration, mechanical recycling and chemical recycling. Landfill, as the most primitive treatment method, not only occupies land, but also pollutes water and soil resources, and consumes a lot of manpower and material resources; the incineration process produces toxic and harmful substances, such as nitrogen oxides, benzene, formaldehyde, etc., which pose a potential threat to human health; mechanical recycling mainly involves grinding, crushing or cutting PU waste into small pieces, and then using them to make new PU products, but the recycling process requires high temperature and pressure conditions, which has great limitations. Chemical recycling requires complex processes and equipment, resulting in high recycling costs. In contrast, the use of biological methods (microorganisms or enzymes) to degrade PU plastics not only avoids the harmful gases or by-products produced by traditional physical or chemical methods, but also better meets environmental sustainability requirements and reduces negative impacts on the environment.

[0004] Esterases PueA and PueB from Pseudomonas chlororaphis were isolated and diluted in 1.5% (v / v) After 6 hours of reaction at 23°C, a distinct hydrolysis zone appeared on the surface of a DLN plate. The lipase CALB from Candida Antarctica caused a 25% weight loss in PCL-based polyester PU foam after 24 hours of reaction at 37°C. Magnin et al. developed a laccase-mediated system (LMS) that can degrade different types of PU, depolymerizing PU by hydrolyzing both ester and ether bonds. After 18 days of incubation at 37°C, the foam lost 12.1% of its mass. Most PU depolymerases identified to date hydrolyze only ester bonds in the soft segment of polyester PU, and few have been reported to hydrolyze carbamates. While the lipase Aes72 from Comamonas acidophilus has been reported to hydrolyze oligomers containing carbamate bonds, after 9 hours of reaction, this esterase (4 mg) released only a minimal amount of diamine monomer (MDA), indicating low degradation efficiency. While this esterase has some depolymerization activity against solid PU, its effectiveness needs to be improved. Summary of the Invention

[0005] Purpose of the invention: The technical problem to be solved by the present invention is to provide a highly active PU depolymerase mutant and its application in view of the deficiencies in the prior art.

[0006] In order to solve the above technical problems, the present invention discloses the following technical solutions:

[0007] In a first aspect, the present invention discloses a mutant protein having high depolymerization activity on PU plastics, which is any one of the following 1)-8):

[0008] The protein shown in 1) is a protein having PU depolymerase activity obtained by mutating the tryptophan at position 196 in the PU depolymerase amino acid sequence shown in SEQ ID NO: 2 to alanine, while the amino acid residues at other positions remain unchanged;

[0009] 2) The protein shown is a protein having PU depolymerase activity obtained by mutating the phenylalanine at position 276 in the amino acid sequence of PU depolymerase shown in SEQ ID NO: 2 to alanine, while the amino acid residues at other positions remain unchanged;

[0010] 3) The protein shown is a protein having PU depolymerase activity obtained by mutating the histidine at position 224 in the PU depolymerase amino acid sequence shown in SEQ ID NO: 2 to tyrosine, while the amino acid residues at other positions remain unchanged;

[0011] 4) The protein shown is a protein having PU depolymerase activity obtained by mutating the leucine at position 141 in the amino acid sequence of PU depolymerase shown in SEQ ID NO: 2 to isoleucine, while the amino acid residues at other positions remain unchanged;

[0012] 5) The protein shown is a protein having PU depolymerase activity obtained by mutating the glutamic acid at position 29 in the PU depolymerase amino acid sequence shown in SEQ ID NO: 2 to alanine, while the amino acid residues at other positions remain unchanged;

[0013] 6) The protein shown is a protein having PU depolymerase activity obtained by mutating the histidine at position 89 in the PU depolymerase amino acid sequence shown in SEQ ID NO: 2 to alanine, while the amino acid residues at other positions remain unchanged;

[0014] 7) The protein is a protein obtained by removing the mutation site from any of the proteins in 1) to 6) and having a homology of more than 99%, more than 95%, more than 90%, more than 85% or more than 80% in other amino acid residues and having PU depolymerase activity;

[0015] The protein shown in 8) is a protein having PU depolymerase activity obtained by adding a 6xHis tag sequence to the C-terminus of the amino acid sequence of any one of the proteins in 1) to 7).

[0016] The nucleotide sequence of the wild-type PU depolymerase (Aes72) is shown in SEQ ID No. 1, and the amino acid sequence is shown in SEQ ID No. 2.

[0017] The mutant proteins 1)-6) with high depolymerization activity against PU plastics were obtained by mutating amino acid residues at positions 196, 276, 224, 141, 29, and 89 in the amino acid sequence of the wild-type PU depolymerase. The resulting mutant proteins were named W196A, F276A, H224YL, 141I, E29A, and H89A, respectively. Compared to wild-type Aes72, the mutants W196A, F276A, H224YL, 141I, E29A, and H89A showed significantly improved activity.

[0018] In a second aspect, the present invention discloses a coding gene, which is a gene encoding the protein described in the first aspect.

[0019] In a second aspect, the present invention discloses a recombinant plasmid containing the coding gene described in the second aspect.

[0020] In a third aspect, the present invention discloses the use of the protein described in the first aspect, the encoding gene described in the second aspect, and the recombinant vector or recombinant bacteria described in the third aspect.

[0021] The application is at least one of the following a) and b);

[0022] a) Degradation of polyether PU;

[0023] b) Degradable polyester PU.

[0024] The polyether PU and polyester PU include substrate polyether PU and polyester PU, and also include post-consumer PU products such as discarded pillows, discarded car mats, etc.

[0025] The application specifically comprises the following steps: adding the crude enzyme solution or purified protein of the protein described in the first aspect to the PU degradation system to achieve PU degradation.

[0026] The amount of crude enzyme solution or purified protein added is such that, based on the amount of protein, the protein concentration in the PU degradation system is 10 wt % to 20 wt %, such as 15 wt %, of the substrate concentration.

[0027] Beneficial effects:

[0028] This study uses structural analysis and site-directed mutagenesis to mutate a wild-type polyurethane depolymerase (Aes72), generating six mutants that effectively enhance Aes72's activity in degrading polyurethane (PU). PU is an insoluble polymer that is difficult to degrade. These six mutants significantly enhance the degradation efficiency of PU plastics and hold promise for industrial applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more apparent.

[0030] Figure 1 Analysis of the hydrolytic activity of six mutant proteins.

[0031] Figure 2 Comparison of the concentration of diamine products MDA released by the hydrolysis of polyether PU by six mutants.

[0032] Figure 3 HPLC liquid phase diagram of the diamine product MDA released during the hydrolysis of polyether PU by six mutant proteins. a is the MDA standard, b is the wild-type depolymerase, c is the mutant H89A, d is the mutant E29A, e is the mutant L141I, f is the mutant H224Y, g is the mutant F276A, and h is the mutant W196A. DETAILED DESCRIPTION

[0033] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials are commercially available unless otherwise specified.

[0034] The raw materials and equipment used in the specific embodiments of the present invention are all known products and are obtained by purchasing commercial products.

[0035] Example 1. Construction of PU depolymerase mutant plasmid

[0036] In order to increase the industrial application value of PU depolymerase Aes72, Aes72 was cloned, expressed and purified. By studying the structure of this PU depolymerase, the amino acids in its active region involved in substrate interaction were mutated to increase the enzyme's catalytic activity on the substrate PU.

[0037] Construction of wild-type PU depolymerase recombinant plasmid and its mutant recombinant plasmid

[0038] The nucleotide sequence of the wild-type PU depolymerase is Sequence 1 in the sequence listing, and the amino acid sequence encoded by it is Sequence 2 in the sequence listing.

[0039] The Aes72 gene was inserted into the plasmid pET-29a(+) using the Nde I and Xho I restriction sites and then transformed into E. coli DH5α. 1 μL of the pure pET-29a(+)-Aes72 plasmid was added to E. coli BL21(DE3) competent cells for transformation. Single clones were selected and transferred to a tube containing LB liquid containing kanamycin sulfate resistance. The plasmid was extracted and used as a genomic template. PCR was performed using the above primers to obtain plasmids expressing different PU depolymerase mutants. The purified reaction products were transformed into competent E. coli cells, and preliminary screening was performed with kanamycin sulfate. DNA sequencing was performed to confirm the successful mutation of the gene, thereby obtaining plasmids expressing different PU depolymerase mutants.

[0040] Rationally designed site-directed mutagenesis primers were used to perform site-directed mutagenesis on six sites of Aes72. The mutants were E29A, H89A, L141I, W196A, H224Y and F276A, respectively. The specific enzyme activities were 0.0164, 0.0049, 0.0258, 0.0155, 0.0180 and 0.0230 U / mg, respectively. The specific enzyme activity of Aes72 was 0.0132 U / mg. The enzyme activity was defined as the amount of enzyme required to generate 1 μmol of aniline substances per minute at 60°C as one unit of enzyme activity.

[0041] Table 1 Site-directed mutagenesis primers

[0042]

[0043] Example 2. Preparation of PU depolymerase mutants and wild-type PU depolymerase

[0044] Upstream and downstream primers were designed and synthesized based on the nucleotide sequence corresponding to the premutation site. Site-directed mutagenesis was then performed using plasmid pET-29a(+)-Aes72 as a template to obtain a PCR product. The PCR procedure was as follows: (1) pre-denaturation: 95°C, 3 min; (2) denaturation: 95°C, 30 s; annealing: 55°C, 30 s; extension: 68°C, 3 min; this step was cycled 20 times; (3) final extension: 68°C, 10 min. After the PCR procedure was completed, the PCR product was digested with the digestion enzyme Dpn I at a ratio of 2%. The digested PCR products were transformed into E. coli DH5α competent cells, and then spread on LB plates containing kanamycin sulfate to screen for positive clones and culture. The selected positive clones were transferred to 5mL of liquid LB containing 50ng / mL of kanamycin sulfate for test tube culture. The plasmids were extracted and verified. The plasmids with correct mutations were transformed into E. coli BL21 (DE3) competent cells, and then spread on LB plates containing kanamycin sulfate to screen for positive clones and culture. The selected positive clones were transferred to 5mL of liquid LB containing 50ng / mL of kanamycin sulfate for test tube culture. The seed liquid was transferred to 1L of LB liquid medium for fermentation. The optimal conditions for PU depolymerase induction expression were: 18°C, 0.1mM IPTG induction for 20-24h, and crude enzyme was obtained by ultrasonic cell disruption.

[0045] To obtain a highly pure enzyme protein, the bacterial suspension was ultrasonically disrupted and centrifuged at 9000 rpm for 10 minutes. The supernatant was filtered through a 0.22 μm filter and purified using an AKTApure protein purifier and a Ni-NTA column. The target protein was eluted using a buffer containing 50 mM Na₂HPO₄, 50 mM KH₂PO₄, 300 mM NaCl, and varying concentrations of imidazole. The eluted mutant protein was concentrated using an ultrafiltration centrifuge tube and dialyzed to remove the imidazole. It was then stored at 4°C.

[0046] Example 3. Relative enzyme activity analysis of PU depolymerase mutants and wild-type mutant Aes72

[0047] To verify the difference between the wild-type PU depolymerase and the PU depolymerase mutant, this example further measured the PU degradation activity of the two. The activity test method of PU depolymerase is mainly:

[0048] Dissolve the PU-based substrate bis(4-hydroxybutyl)(methylenebis(4,1-phenyl))dicarbamate (BMC) in dimethyl sulfoxide (DMSO) to prepare a 2 g / L test solution. Add 50 μL of this solution and 50 μL of pure enzyme to 900 μL of 50 mM PB buffer (pH 7.0). Incubate at 60°C for 10 minutes. Then, add 10 μL of 4-aminoantipyrine and 10 μL of potassium ferrocyanide and react at room temperature for 5 minutes. Measure the absorbance at 515 nm using a microplate reader to calculate the amount of product produced. Enzyme activity is defined as the amount of enzyme required to generate 1 μmol of aniline per minute at 60°C.

[0049] The results are as follows Figure 1 As shown, except for H89A, the relative enzymatic activities of the carbamate bond enzymatic activity of the other mutants were improved compared with the wild-type Aes72, among which the activity of L141I was increased by 2 times, and the carbamate bond enzymatic activity of F276A was increased by 1.8 times.

[0050] Example 4: Degradation of polyether PU by PU depolymerase mutants and wild-type Aes72 depolymerase

[0051] The reaction system consisted of 8 mL of enzymatic depolymerization: the reaction temperature for enzymatic depolymerization was 50°C, and the buffer was 50 mM phosphate buffer at pH 7.0. The PU substrate was added at 3 mg / mL, and the enzyme was added at 15 wt% of the substrate. Therefore, the final enzymatic depolymerization system consisted of 8 mL of phosphate buffer, 4 mg of pure enzyme, and 30 mg of PU powder (Mw = 94,600) as substrate. Enzymatic degradation reactions were performed in a metal bath at different temperatures and a speed of 200 rpm for 9 hours. After the reaction, the mixture was centrifuged at 12,000 rpm for 10 minutes, and the supernatant was filtered through a 0.22 μm filter. The products were then analyzed by high-performance liquid chromatography (HPLC, Agilent 1260 Series) using an Agilent Polaris C18 A (4.6 mm × 250 mm, 5 μm) analytical column. Detection conditions: column temperature 30°C; mobile phase 65% acetonitrile and 35% water; flow rate 0.5 mL / min; detection wavelength 240 nm; injection volume 10 μL.

[0052] The activity of wild-type and mutant PU depolymerases was determined by comparing the peak areas of their hydrolysis products, MDA, and their mutants. During HPLC analysis, the amount of compound in solution is linearly related to the peak area, so the peak area can be used to calculate the concentration of the hydrolysis product in the solution.

[0053] The results are as follows Figure 2As shown, it can be seen that the 6 listed mutants have higher PU depolymerization activity than the wild-type protein, among which W196A has the highest activity, and the concentration of the catalytic product MDA monomer is nearly 2 times higher than that of the wild-type WT. Figure 3 The HPLC test results of the six activity-enhanced mutant proteins show that the corresponding peak time of MDA in liquid chromatography is 13.7 min. It can be seen from the figure that the other wild-type and mutant proteins all peak at 13.7 min, indicating that both the wild-type and mutant proteins have degradation activity on PU and MDA monomers were detected after degradation, and the MDA produced by the mutants is greater than that of the wild-type.

[0054] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended claims.

Claims

1. A protein, which is any one of the following 1)-2): The protein shown in 1) is a protein having PU depolymerase activity obtained by mutating the tryptophan at position 196 in the PU depolymerase amino acid sequence shown in SEQ ID NO: 2 to alanine, while the amino acid residues at other positions remain unchanged; The protein shown in 2) is a protein having PU depolymerase activity obtained by adding a 6xHis tag sequence to the C-terminus of the amino acid sequence of the protein described in 1).

2. A gene encoding the protein according to claim 1.

3. A recombinant plasmid containing the coding gene according to claim 2.

4. Use of the protein according to claim 1 in the degradation of polyether PU.

5. The use according to claim 4, characterized in that The crude enzyme solution or purified protein of the protein according to claim 1 is added to the PU degradation system to achieve PU degradation.

6. The use according to claim 5, characterized in that The amount of crude enzyme solution or purified protein added is: calculated on a protein basis, the protein concentration in the PU degradation system is 10-20 wt % of the substrate concentration.

7. The use according to claim 5, characterized in that The amount of crude enzyme solution or purified protein added is: calculated on the basis of protein amount, the protein concentration in the PU degradation system is 15 wt % of the substrate concentration.

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