A recombinant protein of PET enzyme-spider silk protease and its application in the degradation of PET microplastics

Abstract

This invention discloses a recombinant protein of PETase-spider silk protease and its application in the degradation of PET microplastics. The recombinant protein is obtained by seamlessly cloning the spider silk protein R gene to the C-terminus of the PETase gene. Combining the PET digestive enzyme with spider silk protease improves the degradation efficiency of the PET digestive enzyme in wastewater, overcoming the shortcomings of PET microplastics, such as large dispersion area and low probability of enzyme contact, making them difficult to degrade. SDS-PAGE analysis, p-NP determination, and high-performance liquid chromatography confirmed that the recombinant protein is more effective than PETase alone in degrading PET plastics. This invention not only solves the problem of low degradation efficiency of PET microplastics in wastewater but also provides an innovative biochemical solution for environmental protection and plastic pollution control, possessing significant practical value and broad application prospects.

Description

Technical Field

[0001] This invention relates to the field of environmental protection, and in particular to a PET enzyme-spider silk protease recombinant protein and its application in the degradation of PET microplastics. Background Technology

[0002] Plastic pollution has become a global environmental crisis. Polyethylene terephthalate (PET), as one of the most common synthetic polymers, is widely used in packaging, textiles, and other fields, making it a major source of microplastic pollution in water bodies. Large quantities of discarded PET products enter water bodies through natural decomposition or anthropogenic discharge, forming microplastic particles that are difficult to remove. These microplastics, due to their small size, wide distribution, and ability to readily adsorb toxic substances, pose a serious threat to aquatic ecosystems and may harm human health through the food chain.

[0003] Currently, the main technologies for treating PET microplastics include physical filtration, chemical degradation, and enzymatic hydrolysis. Among these, enzymatic hydrolysis has attracted much attention due to its environmental friendliness and high specificity. For example, PETase can catalyze the hydrolysis of ester bonds in PET plastics, breaking them down into monomers terephthalic acid (TPA) and ethylene glycol (EG). However, in practical applications, the degradation efficiency of PETase is significantly limited: on the one hand, the high dispersion of microplastics in water bodies reduces the probability of enzyme-substrate contact; on the other hand, PETase is unstable in complex aquatic environments and is prone to inactivation. Although studies have attempted to improve its activity by optimizing enzyme structure or immobilization techniques, these methods still suffer from high cost, complex operation, or limited applicability. In other words, using PETase alone results in low degradation efficiency for dispersed microplastics, and there is a lack of low-cost, high-stability methods to enhance enzyme-plastic contact.

[0004] Therefore, there is an urgent need to develop an efficient and sustainable biodegradation technology to address the increasingly serious problem of PET microplastic pollution. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing technologies by providing a PET enzyme-spider silk protease recombinant protein and a method for degrading PET microplastics in water using this recombinant protein. By combining the PET enzyme (PET digestive enzyme, BBa_K4804002) and spider silk protein (R, i.e., the repeating region of the pear-shaped silk gene PySp1, BBa_K4804003), PET plastics can be degraded more efficiently.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides a PET enzyme-spider silk protease recombinant protein, wherein the recombinant protein is formed by gene fusion of PET digestive enzyme (PETase) and spider silk protein (R), wherein the spider silk protein (R) is a protein sequence encoded by the repeating region of the pear-shaped silk gene PySp1.

[0008]

[0009] Preferably, the gene fusion is achieved through a prokaryotic expression system comprising a recombinant plasmid of the PETase gene and the spider silk protein gene, the recombinant plasmid being transformed into Escherichia coli BL21 (DE3).

[0010] Preferably, the recombinant plasmid is PETase-R_pET-21a (+), wherein the PETase gene and the spider silk protein gene are linked by seamless cloning technology.

[0011] Preferably, the PETase is combined with spider silk protease and expressed by IPTG induction to obtain recombinant protein PETase-R.

[0012] Secondly, the present invention provides a method for preparing the above-mentioned recombinant protein, specifically prepared through a prokaryotic expression system, comprising the following steps:

[0013] a. Construct a fusion expression vector containing the PETase gene and the spider silk protein (R) gene;

[0014] b. Transform the vector into Escherichia coli BL21(DE3) for expression;

[0015] c. Induce the expression of recombinant proteins using IPTG and then purify them.

[0016] Preferably, the fusion expression vector connects the spider silk protein (R) gene to the C-terminus of the PETase gene using seamless cloning technology.

[0017] Thirdly, the present invention provides a method for degrading PET microplastics in water using recombinant proteins, the method comprising:

[0018] Spider silk protein (R) and PET digestive enzyme (PETase) were fused together through genetic engineering to form PETase-R recombinant protein;

[0019] The recombinant protein was placed in an aquatic environment containing PET microplastics. The adhesion of spider silk protein enhanced the contact area between PETase and microplastics, thereby improving the degradation efficiency of PET plastics.

[0020] Fourthly, the present invention provides the application of the above-mentioned PET enzyme-spider silk protease recombinant protein in the degradation of PET microplastics.

[0021] Therefore, the present invention has the following beneficial effects:

[0022] This invention provides a novel recombinant protein. First, R (a repeating region of the spider silk gene PySp1) was amplified and then inserted into the C-terminus of the PETase gene using seamless cloning technology. The recombinant plasmid was transformed into *E. coli* strain BL21(DE3), successfully constructing a prokaryotic expression system. The recombinant protein was obtained through IPTG induction. p-NP assays showed that the recombinant protein could degrade p-nitrophenylbutyrate (p-NPB). Furthermore, HPLC analysis of protein degradation on actual PET plastics showed that the recombinant protein was more effective at degrading PET than PETase alone, and the expression of spider silk protein-R in the recombinant protein did not affect the activity of PETase.

[0023] In summary, this invention expands the contact area with the substrate by combining PETase with spider silk protease (R), thus solving the problems of slow natural degradation rate of PET microplastic pollutants in wastewater and low degradation efficiency of PET digestive enzymes alone. Attached Figure Description

[0024] Figure 1 It is the structure of the T7 promoter-RBS-PETase-R-T7 terminator gene circuit.

[0025] Figure 2 This is the flowchart of the project.

[0026] Figure 3 It is achieved through a seamless cloning reaction of PETase-R_pET-21a (+).

[0027] Figure 4 This is a map of the PETase-R_pET-21a (+) recombinant plasmid. The pET-21a (+) vector contains an ampicillin resistance fragment for screening positive clones.

[0028] Figure 5 This refers to the PCR amplification program settings.

[0029] Figure 6 This is an electrophoresis image of the PCR product amplified from the PETase_pET-21a (+) plasmid. 1: DNA marker; 2: PCR amplification band of the vector fragment (product size: 6283bp).

[0030] Figure 7 This is an electrophoresis image of the PCR product amplified from the R_pET-21a (+) plasmid. 1: DNA molecular weight standard; 2-5: PCR amplification bands of R (product size: 675bp).

[0031] Figure 8The correct clone was identified. (A) Colony PCR electrophoresis diagram. 1: DNA marker; 2-13: Colony PCR. (B) Sequencing peak diagram shows the sequencing results.

[0032] Figure 9 This is an SDS-PAGE analysis of PETase-R. 1: Label; 2: Simple before IPTG induction; 3: Simple after IPTG induction; 4: Precipitate after ultrasonic disruption; 5: Eluent after nickel column affinity chromatography; 6: 20 mM imidazole elution buffer.

[0033] Figure 10 This is an activity assay for PETase-R. (A) The mechanism of pNPB degradation; (B) Loading the sample into a 96-well plate for detection using a microplate reader; (C) OD405 value of pNPB hydrolysis by overexpressed PETase-R; (D) OD405 value of pNPB hydrolysis by overexpressed PETase and PETase-R.

[0034] Figure 11 These are high-performance liquid chromatography (HPLC) results of the relative concentration of TPA after protein normalization in real sample testing. Detailed Implementation

[0035] The present invention will be further described below with reference to specific embodiments.

[0036] This invention designs a prokaryotic expression system to obtain the recombinant PETase-R protein. The components (PETase-R, BBa_K4804000) include PETase (BBa_K4804002) and R (BBa_K4804003) (e.g., ...). Figure 1 As shown in SEQ ID NO.1, the successfully constructed PETase-R vector was transformed into Escherichia coli BL21 (DE3), and the sequence of its encoding gene is shown in SEQ ID NO.1.

[0037] Then proceeded as follows Figure 2 The following tests are shown:

[0038] 1. SDS-PAGE analysis of PETase-R.

[0039] 2. The proteolytic substrate activity of the recombinant protein was analyzed using the p-NP assay.

[0040] 3. The ability to degrade actual PET plastics was analyzed using high performance liquid chromatography.

[0041] Specifically, it includes the following steps:

[0042] First, the basic components (PETase and R) were designed, then the sequences were synthesized using GenScript and cloned into the pET-21a(+) vector. Using seamless cloning technology, the R sequence was subcloned into the C-terminus of PETase to construct the combinatorial component (…). Figure 3 Finally, the PETase-R_pET-21a (+) plasmid was successfully recombined. Figure 4 ), and transformed it into Escherichia coli BL21(DE3) strain.

[0043] Vector fragments containing PETase were subjected to PCR ( Figure 5 The vector fragment was cloned from plasmid PETase_pET-21a(+) and detected by 0.8% agarose gel electrophoresis. The vector fragment size was 6283 bp. Figure 6 Simultaneously, the R fragment was amplified by PCR, and the product was detected by 0.8% agarose gel electrophoresis. Figure 7 The target fragment was recombined with the vector using a seamless cloning kit (SangoBiotech, Seamless Cloning Master Mix). The upstream homologous arm was derived from the C-terminal sequence of the PETase gene; the downstream homologous arm was derived from the N-terminal sequence of the spider silk protein R gene, with homologous sequence lengths of 15-25 bp. After recombination, the cells were transformed (E. coli, DH5α competent cells). Colony PCR results were obtained (…). Figure 8 A in the middle) and sequencing results ( Figure 8 B) shows that R was successfully inserted into the C-terminus of PETase. The correctly sequenced plasmid was then transferred into E. coli BL21(DE3).

[0044] Example 1: Protein Expression

[0045] PETase-R expression was detected by SDS-PAGE after IPTG induction. Figure 9 As shown, compared with the uninduced simple case (lane 2), the PETase-R induced by IPTG (lanes 3-6) had a protein band at close to 45 kDa, indicating that the protein was successfully expressed in BL21(DE3).

[0046] Example 2: Activity test of PETase-R

[0047] The p-NP method is a common method for quantifying the enzymatic activity of PETase, used to determine its hydrolytic activity. p-nitrophenylbutyrate (p-NPB) was chosen as the substrate, as it can be hydrolyzed to p-nitrophenol (p-NP). Figure 10 (A in the middle).

[0048] p-NPs were spectrally tracked by monitoring the increase in absorbance at 405 nm (A405) using a microplate reader (BioTek Synergy H1). Figure 10 (B) The supernatant of the ultrasonically disrupted bacteria was mixed with p-NPB substrate, and absorbance was measured at a series of time points. Figure 10 As shown in Figure C, the OD405 value increases with increasing reaction time, indicating the degradation activity of PETase-R. Furthermore, the degradation activity of PETase-R alone was further tested using the p-NP method. Figure 10 The D-display shows that the OD405 value increases over time. The OD405 value of the PETase-R system is higher than that of PETase alone after 40 minutes.

[0049] In other words, R can be used to improve the degradation activity of PETase.

[0050] Example 3: Degradation Test of Actual Samples

[0051] To accurately quantify the degradation product TPA, high-performance liquid chromatography (HPLC) was first used to determine the concentration of TPA standards at different concentrations, and a standard curve was established between its concentration and peak area. The chromatographic data are shown in Table 1 below.

[0052] Table 1

[0053] substance Retention time (min) Peak area (mAU*s) Peak height (mAU) Peak area ratio (%) 0.005 mM terephthalic acid standard 7.795 21.30 2.14 100.0000 0.05 mM terephthalic acid standard 7.734 1228.58887 52.85714 100.0000 0.067 mM terephthalic acid standard 7.704 7125.69336 316.95465 100.0000

[0054] The results showed a good linear relationship between TPA concentration and peak area, R 2 = 0.995, which can be used for accurate quantification of TPA in subsequent actual samples.

[0055] Based on this, to verify the degradation ability of the system constructed in this invention on actual PET plastic, the supernatant after ultrasonic disruption was co-cultured with PET plastic fragments overnight, and the TPA content in the culture medium was determined by high performance liquid chromatography. Under the same experimental conditions, degradation experiments were conducted using engineered strains overexpressing PETase, engineered strains overexpressing PETase-R, and mixed systems of strains expressing PETase and R respectively, with LB medium and solutions containing only R serving as control groups. The TPA chromatographic data under degradation by different strains are shown in Table 2:

[0056] Table 2

[0057] substance Retention time (min) Peak area (mAU*s) Peak height (mAU) Peak area ratio (%) LB + PET fragments + strain expressing PETase-R 7.621 289.09 14.05 100.0000 LB + PET fragments + PETase-expressing strain 7.792 24.22 2.56 100.0000 LB + PET fragments + strains expressing PETase and R respectively 7.789 14.07 1.48 100.0000

[0058] After normalizing the TPA peak area of ​​each group of samples to reflect protein content, the resulting relative TPA concentrations were plotted as shown in Figure 11. Figure 11 The results showed that the relative TPA content in the PETase-R treatment group was significantly higher than that in the PETase-only group and the mixed protein (PETase and R) group. This result confirms that the fusion expression of PETase and R (PETase-R) has better degradation efficiency than simply mixing the two proteins.

[0059] The scope of this invention is not limited to the specific embodiments described, which are intended as a single illustration of various aspects of this disclosure, and any functionally equivalent compositions or methods are included within the scope of this disclosure. It will be apparent to those skilled in the art that various modifications and variations can be made to the methods and compositions of this invention without departing from the spirit or scope of the invention.

Claims

1. A recombinant protein of PET enzyme-spider silk protease, characterized in that, The recombinant protein is formed by gene fusion of PET digestive enzyme PETase and spider silk protein R, wherein spider silk protein R is a protein sequence encoded by the repeating region of the pear-shaped silk gene PySp1; the nucleotide sequence of the gene encoding the recombinant protein is shown in SEQ ID NO.

1.

2. The PET enzyme-spider silk protease recombinant protein according to claim 1, characterized in that, The gene fusion was achieved through a prokaryotic expression system, which included recombinant plasmids of the PETase gene and the spider silk protein gene, and the recombinant plasmids were transformed into Escherichia coli BL21 (DE3).

3. The PET enzyme-spider silk protease recombinant protein according to claim 2, characterized in that, The recombinant plasmid is PETase-R_pET-21a (+), wherein the PETase gene and the spider silk protein gene are linked by seamless cloning technology.

4. The PET enzyme-spider silk protease recombinant protein according to claim 3, characterized in that, The PETase was combined with spider silk protease and expressed by IPTG induction to obtain recombinant protein PETase-R.

5. A method for preparing the PET enzyme-spider silk protease recombinant protein as described in claim 4, comprising the following steps: a. Construct a fusion expression vector containing the PETase gene and the spider silk protein R gene; b. Transform the vector into Escherichia coli BL21(DE3) for expression; c. Induce the expression of recombinant proteins using IPTG and then purify them.

6. The method according to claim 5, characterized in that, The fusion expression vector connects the spider silk protein R gene to the C-terminus of the PETase gene using seamless cloning technology.

7. A method for degrading PET microplastics in water using the recombinant protein as described in any one of claims 1-4, characterized in that, The method includes: The recombinant protein was placed in an aquatic environment containing PET microplastics to degrade the PET microplastics.

8. The use of the recombinant protein as described in any one of claims 1-4 in the degradation of PET microplastics.

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