Silk material for degrading plastic products as well as preparation method and application of silk material
By recombinantly expressing Fast-PETase and MHETase enzymes in home silk, the problem of low enzyme utilization rate during PET plastic degradation in the prior art is solved, and efficient and rapid degradation of PET plastics is achieved.
Patent Information
- Application Number
- CN202411608273.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-12
- Publication Date
- 2025-02-11
AI Technical Summary
In the prior art, when degrading PET plastics, the utilization rate of enzyme liquid is not high and needs to be continuously replaced, resulting in cumbersome and inefficient.
By recombinantly expressing Fast-PETase and MHETase enzymes in home silk, using home silk glands as bioreactors, and combining genetic engineering technology, silk materials that can efficiently degrade PET plastics were developed.
Efficient degradation of PET plastics is achieved, reducing degradation time from centuries to hours to days, and improving enzyme utilization.
Smart Images
Figure CN119410604A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the field of biotechnology, in particular to a silk material for degrading plastic products, a preparation method of the silk material, and applications of the prepared silk. Background Art
[0002] Polyethylene terephthalate (PET) is a common plastic material. It is widely used in the production of mineral water bottles, beverage bottles and other products because of its lightness and durability. However, these PET plastic products are not easy to degrade, which allows them to exist in the natural environment for a long time. The degradation time may exceed 500 years, which not only causes "white pollution", but also accumulates in the soil, water and ocean, and ultimately endangers human health. In addition, during their degradation, they will also release some endocrine disruptors or toxic and harmful chemicals, such as bisphenol A, phthalates, perfluorinated compounds, dioxins, heavy metals lead and cadmium, etc., which may interfere with the reproduction and development of organisms and pollute soil and water sources. It can be seen that the development of a mild and environmentally friendly biodegradation method to treat PET waste is of great significance to promoting the sustainable development of society.
[0003] In recent years, the bioenzymatic degradation of plastics has attracted the attention of many researchers due to its advantages such as environmental friendliness, high efficiency, specificity, low cost and recyclability. In 2016, Japanese researchers discovered an enzyme called IsPETase, which can effectively degrade low-crystallinity PET plastics at 30°C, but the stability of this enzyme is poor and cannot meet the needs of actual biodegradation. IsPETase can decompose PET to produce soluble products, including mono(2-hydroxyethyl) terephthalate (MHET), while MHETase further cleaves MHET into terephthalic acid (TPA) and ethylene glycol (EG). In order to improve the efficiency of PET degradation, researchers developed a PETase / MHETase dual enzyme system. In 2022, American researchers used machine learning technology to transform IsPETase, the key rate-limiting step in PET degradation, and synthesized Fast-PETase, an enzyme with better plastic degradation activity. Using PET degradation enzymes recombinantly expressed by prokaryotes such as Escherichia coli, the plastic degradation time can be shortened from centuries to hours to days, bringing new hope to PET degradation. However, in the process of plastic degradation, it is necessary to constantly replace new enzyme solutions, which is not only cumbersome, but also the utilization rate of the enzyme solution is not high.
[0004] Bombyx mori silk, as a natural protein long fiber, is known for its safe, non-toxic and environmentally friendly properties. In recent years, with the advancement of silkworm genetic manipulation technology, researchers have been able to design and recombinantly express specific target genes as needed to give silk new functions. For example, by expressing human acidic fibroblast growth factor, human basic fibroblast growth factor and human platelet-derived growth factor in the sericin layer of Bombyx mori silk, silk materials with the ability to promote cell proliferation and accelerate wound healing have been successfully developed. In addition, by expressing human lactoferrin and human transforming growth factor in the sericin layer of silk, silk with enhanced anti-inflammatory activity has also been created, which is expected to be used in the treatment of colitis. Similarly, by overexpressing antimicrobial peptides such as BmCecropinB6, Cecropin B or Moricin in Bombyx mori silk, new silk materials with antibacterial activity have also been prepared.
[0005] Therefore, we continue to develop a new silk material that can degrade PET plastic, and then develop a method that can efficiently degrade plastic waste. Summary of the invention
[0006] In view of this, one of the objects of the present invention is to provide a material for degrading plastic products, a second object of the present invention is to provide a method for preparing the material for degrading plastic products, and a third object of the present invention is to provide an application of the material for degrading plastic products in materials for degrading plastic waste.
[0007] In order to achieve the above object, the present invention provides the following technical solutions: 1. A material for degrading plastic products, wherein the material is a silk material containing recombinant Fast-PETase bioenzyme and recombinant MHETase enzyme, wherein the amino acid sequence of the Fast-PETase bioenzyme is shown in SEQ ID NO.1, and the amino acid sequence of the MHETase enzyme is shown in SEQ ID NO.2.
[0008] Preferably, the recombinant Fast-PETase bioenzyme and recombinant MHETase enzyme are obtained by using the silk gland of the silkworm as a bioreactor and regulating the expression of the silkworm FibH gene promoter FibH-P and the Sericin1 gene 3'-UTR termination sequence Ser1PA.
[0009] 2. The method for preparing the material for degrading plastic products comprises recombinantly expressing genes encoding Fast-PETase bioenzyme and MHETase enzyme in silkworms, and the silk secreted by the obtained transgenic silkworms is the material for degrading plastic products.
[0010] Preferably, the genes encoding Fast-PETase enzyme and MHETase enzyme are shown in SEQ ID NO.3 and SEQ ID NO.4.
[0011] Preferably, the recombinant expression method of the present invention is to connect the genes encoding Fast-PETase biological enzyme and MHETase enzyme into expression frames containing Bombyx mori nuclear polyhedrosis virus enhancer Hr3, Bombyx mori FibH gene promoter FibH-P and Sericin1 gene 3'-UTR termination sequence Ser1PA, respectively, and then connect them into pBac[3xp3Redaf] transgenic vector to obtain transgenic expression vectors pBac-R and pBac-G, respectively, transfer the transgenic vectors into silkworm eggs, screen transgenic silkworms, and secrete silk as the material for recombinantly expressed Fast-PETase biological enzyme and MHETase enzyme.
[0012] 3. Application of the material for degrading plastic products in materials for degrading plastic waste.
[0013] The beneficial effect of the present invention is that: the present invention provides a material for degrading plastic products, by taking the Fast-PETase bioenzyme capable of degrading PET and the MHETase enzyme capable of degrading MHET monomer as research objects, using the silk gland of the silkworm as a bioreactor, combining genetic engineering technology, obtaining transgenic silkworm silk having the characteristic of being able to degrade PET plastic, and then developing a method for efficiently degrading plastic waste. BRIEF DESCRIPTION OF THE DRAWINGS
[0014] In order to make the purpose, technical solution and beneficial effects of the present invention clearer, the present invention provides the following drawings for illustration: Figure 1 The process of constructing the transgenic expression vector (A: the process of constructing the Fast-PETase gene expression vector; B: the process of constructing the MHETase gene expression vector).
[0015] Figure 2For identification of expression vectors (A: Enzyme digestion identification of T-Fast-PETase and T-MHETase vectors; lanes 1 and 2 are the products of BamHI and NotI digestion of T-Fast-PETase vector, and lanes 3 and 4 are the products of BamHI and NotI digestion of T-MHETase vector; B: Enzyme digestion identification of pMD19T[Fast-PETase] and pMD19T[Fast-MHETase]; lanes 1 and 3 are pMD19T[Fast-PETase] and pMD19T[Fast-MHETase] control vectors, respectively, and lane 2 and 4 are the products of pMD19T[Fast-PETase] and pMD19T[Fast-MHETase] vectors digested with BamHI and NotI, respectively; C: Enzyme digestion identification of pBac[Fast-PETase] and pBac[Fast-MHETase] vectors; lanes 1 and 3 are pBac[Fast-PETase] and pBac[Fast-MHETase] control vectors, respectively, and lanes 2 and 4 are the products of pBac[Fast-PETase] and pBac[Fast-MHETase] vectors digested with AscI, respectively).
[0016] Figure 3 For the screening of transgenic silkworms, the eyes of the eggs, larvae and adults of the silkworms carrying the Fast-PETase gene showed red fluorescent signals, and the eyes of the eggs, larvae and adults of the silkworms carrying the MHETase gene showed green fluorescent signals.
[0017] Figure 4 Detection of silkworm cocoons, silk powder and recombinant proteins carrying the Fast-PETase and MHETase genes (A: Silkworm individuals, cocoons and silk powder containing both Fast-PETase and MHETase genes were obtained by hybridization strategy; B: Silkworm cocoons and silk powder containing Fast-PETase and MHETase proteins; C: Electron microscopic observation of the inner cocoon shell and silk powder containing Fast-PETase and MHETase proteins; D: Detection of Fast-PETase and MHETase proteins in silk; RG is silk material containing both Fast-PETase and MHETase proteins; the red and green arrows indicate Fast-PETase protein and MHETase protein, respectively).
[0018] Figure 5 This is the experimental process of degrading plastic products using genetically modified RG silk powder.
[0019] Figure 6 The transgenic RG silk powder has the activity of degrading plastic products (A and B are the processes of transgenic RG silk powder degrading plastic products A and B respectively).
[0020] Figure 7 SEM observation of plastic products A and B after 5 days of degradation.
[0021] Figure 8 The effect of transgenic RG silk powder on degrading plastic products (A: degradation rate and thinning rate of plastic products A and B; B: degradation process of plastic products A and B). DETAILED DESCRIPTION
[0022] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.
[0023] Example 1: Construction of gene expression vector Based on the amino acid sequences of T-Fast-PETase and T-MHETase genes (shown in SEQ ID NO.1 and SEQ ID NO.2, respectively), T-Fast-PETase (SEQ ID NO.3) and T-MHETase (SEQ ID NO.4) genes optimized with silkworm codon preference, suitable for expression in silkworm, and with 8 histidine His tags at the N-terminus were artificially synthesized. At the same time, BamH I and Not I restriction endonuclease cleavage sites were fused to both sides of Fast-PETase and MHETase genes, respectively. Then, the target gene was double-digested with BamH I and Not I, and the Fast-PETase and MHETase gene fragments were subcloned into the pMD19T [Hr3 FibH-P gene Ser1PA] intermediate vector (the vector is based on the pMD19T vector and uses a seamless cloning method to successively clone the regulatory elements Bombyx mori nuclear polyhedrosis virus enhancer Hr3 (N- and C-terminal restriction sites are AscI-AccI and AccI, respectively), Bombyx mori FibH gene promoter FibH-P (N- and C-terminal restriction sites are AccI and BamHI), and Sericin1 gene 3'-UTR termination sequence Ser1PA (N- and C-terminal restriction sites are NotI and AscI) to form a complete expression cassette, and the exogenous gene is regulated by the above expression cassette). The termination sequence Ser1PA was added to obtain pMD19T[Hr3 FibH-P Fast-PETase Ser1PA] and pMD19T[Hr3 FibH-P MHETase Ser1PA], respectively.
[0024] Afterwards, the intermediate vector was digested with AscI and the gene fragments of [Hr3 FibH-P Fast-PETase Ser1PA] and [Hr3 FibH-P MHETase Ser1PA] were subcloned into the pBac[3xp3Redaf] (Aichun Zhao. et al 2000) and pBac [3xp3 EGFPaf] transgenic expression vectors (Carsten Horn. et al 2000), respectively, to obtain the transgenic expression vectors p[3xp3 Redaf; Hr3 Fast-PETase Ser1PA] and p[3xp3 Red af; Hr3 FibH-PMHETase Ser1PA], named pBac-R and pBac-G, respectively. The construction process and vector structure are shown in the following figure. Figure 1 The transgenic expression vectors p[3xp3 Redaf;Hr3 FibH-P Fast-PETase Ser1PA] and p[3xp3 Red af;Hr3 FibH-PMHETase Ser1PA] contain a 3xp3-DsRed-SV40 and 3xp3 EGFP SV40 transcriptional regulatory unit, respectively, which can specifically express red and green fluorescent proteins in the eyes and nervous system of silkworms, emitting red and green fluorescent signals.
[0025] The constructed vector was verified by enzyme digestion. Figure 2 The results showed that the enzyme cleavage bands of the expression vectors pMD19T[Fast-PETase], pMD19T[Fast-MHETase], pBac[Fast-PETase], and pBac[Fast-MHETase] were consistent with expectations, indicating that the expression vectors were successfully constructed.
[0026] Example 2: Silkworm embryo injection and positive strain screening First, the transgenic expression vectors pBac-R and pBac-G were mixed with the helper plasmid Helper in a ratio of 1:1, and then microinjected into 200 non-diapause silkworm eggs that had been laid within 2 hours. The G0 generation silkworm larvae that had been successfully hatched after being accelerated were carefully raised with mulberry leaves, and the G1 generation silkworm eggs were obtained by self-pollination. The positive transgenic individuals were selected by observing the red or green fluorescent signals in the eyes of the G1 generation silkworm embryos, and were named R and G respectively. The positive transgenic silkworms R and G were hybridized, and the individuals with both red and green fluorescent signals in their eyes were selected in the offspring. The results are as follows. Figure 3As shown, the results showed that the eggs, larvae and eyes of the silkworms with the Fast-PETase gene showed red fluorescence signals, and the eggs, larvae and eyes of the silkworms with the MHETase gene showed green fluorescence signals, indicating that the transgenic silkworms with the Fast-PETase and MHETase genes were successfully constructed, and were named R and G, respectively. The secreted silk contained both recombinant Fast-PETase and MHETase proteins, and the silk was named RG.
[0027] Will Figure 3 By hybridizing the transgenic silkworms R and G screened in the experiment, transgenic silkworm individuals with both red and green fluorescent signals in their eyes were obtained. Their silk contained both Fast-PETase and MHETase proteins, which were named RG. RG silk powder ( Figure 4 , AB). Scanning electron microscopy (SEM) observations revealed that the morphological characteristics of transgenic RG silkworm cocoon shells and silk powder were not significantly different from those of wild-type WT silkworm cocoon shells and silk powder ( Figure 4 , C). The particle size of WT and RG silk powders was analyzed to be in the range of 8-150 μm.
[0028] Example 3: Release of recombinant silk protein from RG silk The release of recombinant Fast-PETase and MHETase proteins in RG silk was analyzed by continuous extraction of recombinant proteins in RG silk using KH2PO4-NaOH buffer. First, 30 mg of RG silk sample was weighed and mixed with 1 mL of KH2PO4-NaOH buffer (100 mM KH2PO4-NaOH, pH = 8.0), and then extracted at 37°C for 1 to 11 days. Then, the protein sample was collected by centrifugation at 13,400 rpm for 5 min at 4°C for testing.
[0029] SDS-PAGE and Western blotting were used to analyze the content of recombinant Fast-PETase and MHETase proteins in RG silk extract. The concentration of total cocoon shell protein samples was determined using an enhanced BCA protein concentration assay kit, and then 12% SDS-PAGE electrophoresis was performed and stained with Coomassie Brilliant Blue R-250. In the Western blotting experiment, the protein samples in the SDS-PAGE gel were transferred to a polyvinylidene fluoride (PVDF) membrane, which was blocked with 5% skim milk powder buffer for 1 h, incubated with Flag primary antibody buffer (Beyotime, China) for 1 h, washed 5 times with TBST buffer, incubated with IgG secondary antibody buffer (Beyotime, China) for 1 h, and washed 5 times with TBST buffer. The results were recorded by automatic exposure using a Chemiscope Series instrument (ClinxScience Instruments, China). The results are shown in Figure 2. Figure 4 As shown in D. The results showed that RG silk contained both Fast-PETase and MHETase proteins; the release of Fast-PETase protein was high in the first 5 days, and its release decreased during the 6th to 11th day due to protein degradation; MHETase protein reached the highest release in the first day, then gradually degraded, and the protein could not be detected after two days. This indicates that transgenic RG silk can continuously release Fast-PETase protein within 11 days and MHETase protein within 2 days ( Figure 4 , D).
[0030] Example 4: Degradation of PET plastic by RG silk powder (1) Preparation of RG silk powder First, the RG silk cocoon shells were cut into pieces using scissors, and then the RG silk cocoon shells were ground in liquid nitrogen to obtain RG silk powder.
[0031] 5) Degradation effect of PET plastic The PET plastic products used in this experiment are opaque fruit container A (referred to as plastic product A) (PET) (crystallinity is 10.55%) and transparent container B (PET) (referred to as plastic product B) (crystallinity is 5.59%) commonly used in the laboratory. Plastic products A and B are uniformly made into plastic sheets of standard size with a diameter of 4.5 mm using a punch as experimental samples.
[0032] The thickness of the cocoon powder was measured with a vernier caliper and recorded as H0. The cocoon powder was then immersed in a KH2PO4-NaOH buffer solution containing an equal mass of RG cocoon powder, where the amount of cocoon powder added was 30 mg / mL, and the cocoon powder was treated continuously at 50°C for 15 days. The experimental process is as follows: Figure 5 The degradation effect was observed on the 0th, 5th, 7th, 10th and 15th day of degradation. The results are shown in Figure 6 shown.
[0033] SEM observation of plastic products A and B after 5 days of degradation. The results are as follows Figure 7 shown.
[0034] Plastic products A and B with a diameter of 4.5 mm were weighed respectively using an analytical balance, and the degradation rate, thinning rate and surface morphology changes of plastic products A and B were analyzed to detect the effect of RG silk powder on degrading PET plastic.
[0035] Degradation rate (%) = ((M0-Mx) / M0) * 100. M0: the original weight of the plastic product; Mx: the weight of the plastic product after 15 days of degradation.
[0036] Thinning rate (%) = ((H0-Hx) / M0) * 100. H0: the original thickness of the plastic product; Hx: the thickness of the plastic product after 15 days of degradation.
[0037] The above results show that after 7 days of silk powder treatment of plastic product A and 5 days of plastic product B, compared with the WT group, plastic products A and B in the transgenic silk RG group were significantly degraded, with obvious notches and dissolution on their surfaces; after 15 days of silk powder treatment of plastic product A and 10 days of plastic product B, compared with the WT group, plastic products A and B in the transgenic silk RG group were completely dissolved ( Figure 6 ). SEM results showed that compared with the WT group, the surface and interior of plastic product A after 7 days of treatment with transgenic silk RG powder and plastic product B after 5 days showed rough and porous structures, forming a large number of holes (Figure 7). This shows that transgenic silk RG powder has the ability to degrade plastic products. Further analysis showed that after 5 days of treatment with transgenic silk RG powder, the degradation rate of plastic product A was 35.1%, the thinning rate was 40.9%, and it could be completely degraded within 15 days; the degradation rate and thinning rate of plastic product B were 44.3% and 48.1%, respectively, and it could be completely degraded within 10 days (Figure 8).
[0038] The above-described embodiments are only preferred embodiments for fully illustrating the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or changes made by those skilled in the art based on the present invention are within the protection scope of the present invention. The protection scope of the present invention shall be subject to the claims.
Claims
1. A material for degrading plastic products, characterized in that: The material is a silk material containing recombinant Fast-PETase bioenzyme and recombinant MHETase enzyme. The amino acid sequence of the Fast-PETase bioenzyme is shown in SEQ ID NO.1, and the amino acid sequence of the MHETase enzyme is shown in SEQ ID NO.
2.
2. The material for degrading plastic products according to claim 1, characterized in that: The recombinant Fast-PETase bioenzyme and the recombinant MHETase enzyme are obtained by using the silk gland of the silkworm as a bioreactor and regulating the expression of the silkworm FibH gene promoter FibH-P and the Sericin1 gene 3'-UTR termination sequence Ser1PA.
3. The method for preparing the material for degradable plastic products according to claim 1, characterized in that: The genes encoding Fast-PETase and MHETase are recombinantly expressed in silkworms, and the silk secreted by the transgenic silkworms is the material for degrading plastic products.
4. The method for preparing the material for degrading plastic products according to claim 3, characterized in that: The genes encoding Fast-PETase bioenzyme and MHETase enzyme are shown in SEQ ID NO.3 and SEQ ID NO.
4.
5. The method for preparing the material for degrading plastic products according to claim 3, characterized in that: The recombinant expression method comprises the following steps: respectively connecting genes encoding Fast-PETase biological enzyme and MHETase enzyme into expression frames containing Bombyx mori nuclear polyhedrosis virus enhancer Hr3, Bombyx mori FibH gene promoter FibH-P and Sericin1 gene 3'-UTR termination sequence Ser1PA, and then connecting into pBac[3xp3Redaf] transgenic vectors to obtain transgenic expression vectors pBac-R and pBac-G, respectively, injecting the transgenic vectors into silkworm eggs, screening transgenic silkworms, and secreting silk as materials for recombinantly expressed Fast-PETase biological enzyme and MHETase enzyme.
6. Use of the material for degrading plastic products according to claim 1 or 2 in materials for degrading plastic waste.