A plant-derived plastic-degrading enzyme and a method for degrading pp plastic in vitro

Abstract

The application discloses a plant-derived plastic-degrading enzyme and a method for degrading PP plastic in vitro, and belongs to the technical field of enzyme engineering. The plant-derived plastic-degrading enzyme is a plant-derived HIS1 protease. The method for degrading PP plastic in vitro comprises the following steps: cloning a HIS1 gene from a plant by using an RT-PCR technology; connecting the gene to a prokaryotic expression plasmid by using a blunt-end cloning technology; introducing the gene into an expression platform of Bl21 (DE3) E. coli to express and purify the HIS1 protein; and degrading PP plastic in an enzyme reaction environment constructed in vitro. The HIS1 protein provided by the application can effectively degrade PP plastic, provides a new idea for degrading plastic by using plant enzymes, and has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of enzyme engineering, specifically relating to a plant-derived plastic-degrading enzyme and a method for in vitro degradation of PP plastic. Background Technology

[0002] Since 1950, 6.3 billion tons of plastic waste have been generated (Geyer et al., 2017). Due to its low price, plastic has become one of the most widely used materials. Plastic persists in the environment for a long time and is widely dispersed because the degradative enzymes that can break down its plastic components are absent or have low activity. In particular, polypropylene (PP) is a polymer made by polymerizing propylene. Its main ring is a hydrocarbon structure composed of only carbon atoms, which is chemically inert and resistant to microbial degradation, making it difficult to be directly degraded. Polypropylene (PP) is the second largest plastic after polyethylene (PE), accounting for about 18.85% of the total plastic production. It is expected to rise to 21.65% by the end of 2050, making it an important reason for academic attention (Statista, 2022).

[0003] The degradation of polypropylene (PP) is typically achieved through photodegradation or chemical degradation, with few reports on its biodegradation or its use to support microbial growth. Shimpi et al. studied the degradation of pure PP using a mixed strain of *Aspergillus niger* and *Paecilomyces variotii*, finding a degradation rate of only 0.62% after 30 days. Sinosh Skariyachan et al. screened three bacteria capable of degrading PP from cow dung samples, with a new species of *Enterobacter* achieving a degradation rate (weight loss) of over 60% for PP membranes at 37°C. Microorganisms possessing the enzymatic mechanisms required for PP degradation could serve as environmental remediation strategies and as degradation and / or fermentation platforms for the biorecycling of PP waste.

[0004] Recently, the rice HIS1 (HPPD inhibitor-sensitive) gene was identified. HIS1 encodes an iron(II) / 2-oxoglutarate (2OG)-dependent oxygenase that detoxifies five different β-trione herbicides (bTHs) by catalyzing their hydroxylation. Natsuki Takamura et al., in evaluating the effects of plant growth regulators on HIS1 enzyme function, found that HIS1 plays a crucial role in the detoxification of iron (II) / 2-oxoglutarate (2OG)-dependent oxygenase. 2+ The hydroxylation of anti-tack esters (TE) is mediated in the presence of 2-OG. bTHs and TE are complex compounds with different chemical structures, and there is currently no research on the plastic degradation function of HIS1 enzymes.

[0005] The relevant reference information is as follows:

[0006] Geyer,R.,Jambeck,JR,and Law,KL2017.Production,use,and fate of allplastics ever made.3,e1700782.

[0007] Statista,2022.Production forecast of thermoplastics worldwidefrom2020to 2050,by

[0008] tyPP.https: / / www.statista.com / statistics / 1192886 / thermoplastics-productionvolume bytyPP-globally / . Summary of the Invention

[0009] Given the lack of existing technologies for plastic biodegradation, the purpose of this invention is to develop a plant-derived plastic-degrading enzyme. This invention first abandons the traditional thinking of microbial degradation of plastics and uses a broad-spectrum enzyme from plants to degrade plastics. A plant-derived oxygenase was discovered, and the protein was expressed and purified using prokaryotic expression technology. A catalyst was then added in vitro to catalyze the degradation of PP plastics.

[0010] Another objective of this invention is to provide a method for in vitro degradation of PP plastic using a plant-derived plastic-degrading enzyme, which involves mixing the enzyme with a desired buffer solution and then co-bathing the enzyme with the PP plastic at 30°C to achieve in vitro enzymatic hydrolysis of the PP plastic.

[0011] The above-mentioned objectives of the present invention are achieved through the following technical means:

[0012] A plant-derived plastic-degrading enzyme, specifically the plant-derived HIS1 protease.

[0013] The plant-derived HIS1 protease is preferably a rice-derived HIS1 protease.

[0014] Furthermore, the rice-derived HIS1 protease is preferably a HIS1 protease derived from japonica rice.

[0015] Further, the nucleic acid sequence of the HIS1 gene described in step 1) is shown in SEQ ID NO. 1.

[0016] A method for in vitro degradation of PP plastic by a plant-derived plastic-degrading enzyme, comprising the following steps:

[0017] 1) The HIS1 gene was cloned from plants using RT-PCR technology;

[0018] 2) Genes are ligated into prokaryotic expression plasmids using blunt-end cloning technology;

[0019] 3) The HIS1 protein was expressed and purified using the Bl21(DE3) E. coli expression platform;

[0020] 4) Degradation of PP plastic by constructing an enzyme reaction environment in vitro.

[0021] The plant mentioned in step 1) is preferably rice. In this invention, the HIS1 gene is derived from a plant, unlike traditional microbial proteins that can degrade plastics.

[0022] Further, the cloning of the HIS1 gene from plants using RT-PCR technology described in step 1) is carried out as follows: 3-week-old japonica rice is ground in liquid nitrogen, and total RNA is extracted using Trizol reagent. The entire process is carried out in a low-temperature, RNase-free environment. The CDS sequence of the HIS1 gene is obtained using RT-PCR technology, purified by gel recovery, and the HIS1 gene is cloned.

[0023] The primer sequences used in the blunt-end cloning described in step 2) are shown in SEQ ID NO. 2 and SEQ ID NO. 3.

[0024] The prokaryotic expression plasmid mentioned in step 2) is preferably... E1 plasmid. The sequence of the E1 plasmid is the same as that of PET-30a.

[0025] The Bl21(DE3) E. coli expression platform mentioned in step 3) is for introducing Bl21(DE3) E. coli for expression. This invention utilizes a prokaryotic expression system, which is more convenient than obtaining proteins from plants; furthermore, Bl21(DE3) contains tRNAs corresponding to rare codons, which can optimize plant gene expression.

[0026] The expression and purification of HIS1 protein described in step 3) are as follows: The specific steps are as follows: [The text abruptly ends here, likely due to an incomplete sentence or missing information.] E1 was introduced into Bl21(DE3) E. coli. After the transgenic Bl21(DE3) E. coli was cultured to an OD600 of 0.5, IPTG was added to a final concentration of 0.6 mM to induce expression. The mixture was then cultured in a shaker at 28°C for 24 h. The HIS1 active protein was purified using the his-tag purification kit.

[0027] The in vitro enzyme reaction environment described in step 4) is constructed in 2-oxoglutarate (2-OG) and Fe... 2+Catalytic environment. Based on the fact that the HIS1 protease is a 2-oxoglutarate / Fe(II)-dependent dioxygenase (HIS1).

[0028] Further, in step 4), the in vitro enzyme reaction environment is constructed with the following final concentrations of the reaction environment auxiliaries: 0.1 mM MFeSO4, 0.6 mM 2-oxoglutarate, and HEPES-KOH at a concentration of 100 mM and pH 7.0 as the solvent.

[0029] The specific steps for constructing an in vitro enzyme reaction environment to degrade PP plastic as described in step 4) are as follows: HIS1 protein (final reaction concentration of 1-5 mg / ml) is mixed with PP film and catalysts with a final reaction concentration of 0.1 mM FeSO4 and 0.6 mM 2-oxoglutarate, and then added to a solvent with a concentration of 100 mM and pH 7.0 HEPES-KOH; the reaction mixture is then incubated at 30°C for 24 h or 48 h to achieve the degradation of PP plastic.

[0030] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows:

[0031] 1. This invention is the first to demonstrate the novel application of plant-derived oxygenases in degrading PP plastics. HIS1 protein can effectively degrade PP plastics, providing a new approach to the biodegradation of PP plastics from plants with promising application prospects; it also provides an effective research approach for solving current plastic pollution.

[0032] 2. This invention utilizes a prokaryotic expression system to express and purify plant-derived proteins, which is highly targeted, yields extremely high protein output, and optimizes the process of extracting plant proteins; furthermore, the selected prokaryotic expression system Bl21(DE3) contains tRNA corresponding to rare codons, thus optimizing the expression of plant genes.

[0033] 3. The HIS1 protein of this invention has a good effect on the degradation of PP plastic. Compared with ordinary microorganisms, HIS1 has a faster degradation rate and is easier to operate.

[0034] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments. Attached Figure Description

[0035] Figure 1 Image of nucleic acid gel electrophoresis results for cloning the CDS region of the HIS1 gene (a. total RNA from rice, b. RT-PCR amplification of HIS1).

[0036] Figure 2 for The validation results of vector construction are shown in the figure (a. screening of positive clones on antibiotic plates, b. validation by colony PCR).

[0037] Figure 3 The images show the SDS-PAGE results of protein expression (a. Investigation of protein expression conditions, b. Protein purification).

[0038] Figure 4 A protein standard curve for detecting protein concentration using the BCA kit.

[0039] Figure 5 The images show the SEM (Series Electron Microscopy) results of the enzyme-catalyzed mixture after reacting at 30°C for 24 h and 48 h.

[0040] Figure 6 The image shows the Fourier transform infrared (FTIR) analysis results of the enzyme-catalyzed mixture after reacting at 30℃ for 48 hours. Detailed Implementation

[0041] The following experimental examples are further illustrative of the present invention and are not intended to limit the invention. Specific experimental conditions and methods are not specified in the following examples, and the techniques used are generally conventional methods well known to those skilled in the art.

[0042] Example 1: Cloning the HIS1 gene from a plant to... E1, the specific steps are as follows:

[0043] Three-week-old japonica rice was ground in liquid nitrogen, and total RNA was extracted using Trizol reagent. Figure 1 a) The entire process was carried out in a low-temperature, RNase-free environment; total RNA was reverse transcribed into cDNA using a reverse transcription kit, and the CDS sequence of the HIS1 gene was amplified using high-fidelity polymerase. The sequence was then separated by agarose gel electrophoresis, the target fragment was excised, and purified using a DNA recovery kit. Figure 1 b). Purified HIS1 gene and linearized E1 plasmid was mixed at a molar ratio of 7:1 and ligated at 25°C for 5 min. The ligation product was transformed into DH5α Escherichia coli by heat shock and screened on Amp resistance plates at 37°C. Figure 2 a) Colony PCR verification ( Figure 2 b) Sequencing verification confirmed the absence of mutations, yielding the product described in this invention.

[0044] Example The specific steps for expressing and purifying the protein by introducing it into E. coli Bl21(DE3) are as follows:

[0045] Extracted from DH5α Escherichia coli Plasmid (50 ng / μl) was heat-shocked at 42℃ and transformed into Bl21(DE3) E. coli containing rare codon tRNA. After selection by antibiotic plates and PCR verification, the HIS1-transformed Bl21(DE3) E. coli was cultured in 50 ml LB liquid medium at 37℃ and 180 rpm in a shaker until the OD600 reached 0.5. IPTG was then added at final concentrations of 0, 0.3, 0.6, and 0.9 mM to induce expression. The cultures were then shaken for 24 h at 22, 25, and 28℃ and 180 rpm, respectively. Total protein samples from the bacterial cell pellet were collected for protein quantification. 5×SDS-PAGE protein loading buffer was added, and the mixture was incubated in a boiling water bath for 8 minutes. 12% SDS-PAGE electrophoresis was performed, and after staining and descaling, the expression of the target HIS1 protein could be detected. Figure 3 (a) The molecular weight of the his::HIS1 protein is approximately 44.6 kDa. The active HIS1 protein was purified using a his-tag purification kit. Figure 3 b) The purified protein was used to prepare a protein standard curve using a BCA kit. Figure 4 The absorbance at A562 nm of the purified protein was measured, and the protein concentration was calculated based on the protein standard curve. Experimental results show that the protein constructed in this invention... It can effectively achieve normal expression of plant proteins in the E. coli expression system and purify 10 mg / ml of protein.

[0046] Example 3: Degradation of PP plastic by HIS1 protein. The specific steps are as follows:

[0047] HIS1 protein (final reaction concentration 1 mg / ml) was mixed with PP film and catalysts at final reaction concentrations of 0.1 mM FeSO4 and 0.6 mM 2-oxoglutarate, and then added to a solvent of 100 mM HEPES-KOH at pH 7.0. The reaction mixture was then incubated at 30°C for 24 h or 48 h, with the reaction solution and protein being replaced every 12 h to prevent protein inactivation. The effects of different catalytic incubation times on the plastic film were observed using scanning electron microscopy (SEM). Figure 5 ) and Fourier transform infrared (FTIR) analysis were used to analyze the changes in the functional groups of the thin film after 48 h of catalytic degradation. Figure 6Numerous severe cracks and deep pores were observed on the surface and even inside the PP film, indicating that the HIS1 protein degraded the film. Although the FTIR spectrum of the PP film treated with HIS1 showed no significant change after 48 hours, new raised bands were observed at wavelengths of 3365 and 1647 nm, indicating that some structural changes occurred in the PP film during degradation, producing -OH and -C=C- functional groups. The peak changes in the FTIR test of the film were limited, which may be due to the relatively weak degradation effect of the HIS1 protein on the PP film. Therefore, the spectrum was similar to the control. In conclusion, the HIS1 protein has limited degradative activity on PP plastic.

[0048] The polymer polypropylene structure of the above-mentioned PP film is shown in Formula I:

[0049]

[0050] Where: in formula I, n>2;

[0051] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows:

[0052] 1. This invention is the first to demonstrate the novel application of plant-derived oxygenases in degrading PP plastics. HIS1 protein can effectively degrade PP plastics, providing a new approach to the biodegradation of PP plastics from plants with promising application prospects; it also provides an effective research approach for solving current plastic pollution.

[0053] 2. This invention utilizes a prokaryotic expression system to express and purify plant-derived proteins, which is highly targeted, yields extremely high protein output, and optimizes the process of extracting plant proteins; furthermore, the selected prokaryotic expression system Bl21(DE3) contains tRNA corresponding to rare codons, thus optimizing the expression of plant genes.

[0054] 3. The HIS1 protein of this invention has a good effect on the degradation of PP plastic. Compared with ordinary microorganisms, HIS1 has a faster degradation rate and is easier to operate.

[0055] The above embodiments are only used to explain the inventive concept of the present invention, and are not intended to limit the protection of the present invention. Any non-substantial modifications and substitutions made to the relevant details of the present invention using this concept should fall within the protection scope of the present invention.

Claims

1. A method for in vitro degradation of PP plastic by a plant-derived plastic-degrading enzyme, characterized in that: The steps are as follows: 1) The HIS1 gene was cloned from plants using RT-PCR technology; 2) Genes are ligated into prokaryotic expression plasmids using blunt-end cloning technology; 3) The HIS1 protein was expressed and purified using the Bl21(DE3) E. coli expression platform; 4) Degrading PP plastic by constructing an in vitro enzyme reaction environment; The plant-derived plastic-degrading enzyme is a plant-derived HIS1 protease, and the nucleic acid sequence of the HIS1 protease is shown in SEQ ID NO.

1. Step 4) describes the in vitro construction of the enzyme reaction environment. The final concentrations of the reaction environment auxiliaries are as follows: 0.1 mM MFeSO4, 0.6 mM 2-oxoglutarate, and the solvent is 100 mM HEPES-KOH at pH 7.

0.

2. The method for in vitro degradation of PP plastic using plant-derived plastic-degrading enzymes according to claim 1, characterized in that: The specific steps for cloning the HIS1 gene from plants using RT-PCR technology described in step 1) are as follows: Three-week-old japonica rice was ground in liquid nitrogen, and total RNA was extracted using Trizol reagent. The entire process was carried out in a low-temperature, RNase-free environment. The CDS sequence of the HIS1 gene was obtained using RT-PCR technology, purified by gel recovery, and the HIS1 gene was cloned.

3. The method for in vitro degradation of PP plastic using plant-derived plastic-degrading enzymes according to claim 1, characterized in that: The primer sequences used in the blunt-end cloning described in step 2) are shown in SEQ ID NO.2 and SEQ ID NO.

3.

4. The method for in vitro degradation of PP plastic using plant-derived plastic-degrading enzymes according to claim 1, characterized in that: The prokaryotic expression plasmid mentioned in step 2) is Plasmid.

5. The method for in vitro degradation of PP plastic using plant-derived plastic-degrading enzymes according to claim 1, characterized in that: The expression and purification of HIS1 protein described in step 3) are as follows: The specific steps are as follows: [The text abruptly ends here, likely due to an incomplete sentence or missing information.] Transgenic Bl21(DE3) Escherichia coli was introduced and cultured until the OD600 reached 0.

5. Then, IPTG was added to a final concentration of 0.6 mM to induce expression. The mixture was cultured in a shaker at 28°C for 24 h. The HIS1 active protein was purified using the his-tag purification kit.

6. The method for in vitro degradation of PP plastic using plant-derived plastic-degrading enzymes according to claim 1, characterized in that: The specific steps for constructing an in vitro enzyme reaction environment to degrade PP plastic as described in step 4) are as follows: HIS1 protein is mixed with PP film and a catalyst with a final reaction concentration of 0.1 mM FeSO4 and 0.6 mM 2-oxoglutarate, and then added to a solvent of 100 mM HEPES-KOH at pH 7.0; the final concentration of HIS1 protein in the reaction is 1-5 mg / ml; then the reaction mixture is incubated at 30°C for 24 h or 48 h or longer to achieve the degradation of PP plastic.

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