Use of alkali-resistant broad-spectrum plastic-degrading enzymes

Abstract

The application discloses application of an alkali-resistant broad-spectrum plastic-degrading enzyme, which is derived from Bacillus lipase LipA, and has a degradation rate of more than 95% on polycaprolactone within 5 hours under the condition of 45 DEG C and pH 11, a degradation rate of more than 4% on polylactic acid within 5 days, a degradation rate of more than 2% on polybutylene succinate within 5 days, a degradation rate of more than 5.5% on polyurethane within 5 days, a degradation rate of more than 2.5% on polyethylene terephthalate within 5 days, and a degradation rate of more than 7.5% on polybutylene adipate / terephthalate plastic within 5 days. The degradation products are mainly monomers and dimers, which can be recycled to resynthesize plastics. The alkali-resistant broad-spectrum plastic-degrading enzyme can not only be effectively applied to the biodegradation treatment of mixed plastic waste of the above-mentioned various plastics, so as to reduce the environmental problems caused by plastic pollution, but also can realize monomer recycling, and has a wide application prospect.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This invention claims priority to Chinese patent application No. CN202211387050.0, filed on November 7, 2022, the entire text of which is incorporated herein by reference. Technical Field

[0003] The present invention relates to the technical field of enzyme engineering, in particular to the application of an alkali-resistant broad-spectrum plastic-degrading enzyme. Background Art

[0004] Plastic products are widely used in human production and life due to their excellent properties. However, due to their resistance to degradation in the environment, the accumulation of plastic waste has caused serious environmental pollution, necessitating the development of appropriate methods and technologies for the treatment of waste plastics. Unlike landfill, incineration, physical, and chemical treatment methods, the use of bioenzymatic degradation of plastics is not only mild and environmentally friendly, but also allows for the recycling of plastic synthesis monomers, making it a promising technology for the treatment of waste plastics. The key to achieving this bioenzymatic degradation is the development of highly efficient plastic-degrading enzymes.

[0005] There are reports on plastic-degrading enzymes, such as lipase from the fungus Mucor miehei, which can degrade polycaprolactone (PCL) powder by 74% within 24 hours at 40°C and pH 7.7 (Pastorino et al., Lipase-catalyzed degradation of poly(ε-caprolactone). Enzyme and Microbial Technology, 2004, 35(4): 321-326.). Lipase from Lactobacillus brevis can reduce the weight of PCL membrane by 60% within 10 days at 37°C and pH 8.1 (Khan et al., Lactobacillus sp. lipase mediated poly(ε-caprolactone) degradation. International Journal of Biological Macromolecules, 2017, 95: 126-131.).

[0006] The currently discovered polylactic acid (PLA) degrading enzymes are mainly derived from the lipase of Paenibacillus amylolyticus (Akutsu-Shigeno et al., Cloning and sequencing of a poly(DL-lactic acid) depolymerase gene from Paenibacillus amylolyticus strain TB-13 and its functional expression in Escherichia coli. Applied and Environmental Microbiology, 2003, 69: 2498-2504.) and the cutinase-like enzyme of yeast Cryptococcus sp. S-2 (Masaki et al., Cutinase-like enzyme from the yeast Cryptococcus sp. strain S-2 hydrolyzes polylactic acid and other biodegradable plastics. Applied and Environmental Microbiology, 2005, 71(11): 7548-7550.).

[0007] The currently discovered poly(butylene succinate) (PBS) degrading enzyme is derived from the cutinase of Fusarium solani. Studies have found that under the condition of 37°C, pH 7.4, the enzyme can complete the degradation of more than 90% of the PBS film within 48 hours (Zhangyong Wang et al. Enzymatic degradation of by cutinase cloned from Fusarium solani. Polymer Degradation and Stability. 2016, 134, 211-219.).

[0008] The currently discovered polyurethane (PU) degrading enzymes are mainly derived from the lipases PueA and PueB of Pseudomonas chlororaphis (Stern et al., The polyester polyurethanase gene (pueA) from Pseudomonas chlororaphis encodes a lipase. FEMS Microbiology Letters, 2000, 185(2): 163-168; Howard et al., Cloning, nucleotide sequencing and characterization of a polyurethanase gene (pueB) from Pseudomonas chlororaphis. International Biodeterioration & Biodegradation, 2001, 47(3): 141-149.) and the esterase pulA of Pseudomonas fluorescens (Howard et al., Nucleotide sequencing of a polyurethanase gene (pulA) from Pseudomonas fluorescens [J]. International Biodeterioration & Biodegradation, 1999, 44(2-3): 127-131.). These enzymes can hydrolyze the ester bonds of polyurethane plastics, thereby achieving the degradation of polyurethane plastics.

[0009] Poly(ethylene terephthalate) plastic (PET) degrading enzymes have been discovered. For example, Muller et al. found that the cutinase TfH from the thermophilic actinomycete Thermobifida fusca could cause PET with a crystallinity of 9% to lose 50% of its weight in 21 days at 55°C (Müller et al., Enzymatic Degradation of Poly(ethylene terephthalate): Rapid Hydrolyse using a Hydrolase from T. fusca [J]. Macromolecular Rapid Communications, 2005, 26(17): 1400-1405). In 2009, Ronkvist et al. found that cutinase HiC from Humilica insolens could cause 7% crystallinity PET to lose 97% of its weight within 96 hours at 70°C and pH 8 (Ronkvist et al., Cutinase-Catalyzed Hydrolysis of Poly (ethylene terephthalate) [J]. Macromolecules, 2009, 42 (14): 5128–5138.).

[0010] Currently found poly(butylene adipate-co-terephthalate) (PBAT) degrading enzymes, mainly reported esterase Chath_Est1 from Anaerobic Clostridium, which can hydrolyze PBAT oligomer-BaETaEBa (wherein BaE represents benzoic acid dihydroxyethyl ester, ETaE: terephthalic acid bis-2-hydroxyethyl ester, Ba: benzoic acid) into smaller monomer polymers at 37℃, pH 7.0 (Perz et al., An Esterase from Anaerobic Clostridium hathewayi Can Hydrolyze Aliphatic-Aromatic Polyesters. [J]. Environmental science & technology, 2016, 50(6): 2899-2907.). In addition, Wallace Paal et al. found that esterase PpEst from Pseudomonas pseudoalcaligenes can degrade PBAT film and release small molecule monomers TA (terephthalic acid) and BuTA (4,4-hydroxy-butyl carbonyl benzoic acid) at 65℃, pH 7 (Wallace Paal et., PpEst is a novel PBAT degrading polyesterase identified by proteomic screening of Pseudomonas pseudoalcaligenes. [J]. Applied microbiology and biotechnology, 2017, 101(6): 2291-2303.).

[0011] The above studies provide a basis and foundation for the development of biocatalytic degradation of plastics. However, the currently reported plastic degrading enzymes can only degrade one kind of plastic, and there are few reports on multi-substrate enzymes that can simultaneously degrade two or more kinds of plastics. Plastic waste in actual life garbage usually exists in mixed form and is diverse in species. The sorting and pretreatment of mixed plastic waste is very difficult and complex. Therefore, the development of multi-substrate plastic degrading enzymes that can simultaneously degrade multiple plastics can save the sorting process, thereby realizing the degradation of mixed plastic waste in garbage. In addition, many actually used plastic products are usually plastic alloys prepared by blending two or more kinds of plastics, and to achieve effective degradation and treatment of these plastic alloys, multi-substrate plastic degrading enzymes that can simultaneously degrade multiple plastics need to be developed.

[0012] In addition, since plastic degradation produces acidic products, alkaline conditions can buffer these acidic products, which is important for maintaining the continuity and efficiency of enzyme degradation reactions. Currently, the plastic-degrading enzymes discovered are basically active under neutral or weakly alkaline conditions (pH 7-8) and do not have good alkali resistance. Therefore, developing a broad-spectrum plastic-degrading enzyme that can simultaneously degrade multiple plastics and has good alkali resistance has important value for realizing the enzymatic degradation of mixed plastic waste. SUMMARY

[0013] The purpose of the present application is to provide the use of an alkali-resistant broad-spectrum plastic-degrading enzyme.

[0014] In order to achieve the purpose of the present application, in a first aspect, the present application provides the use of an alkali-resistant broad-spectrum plastic-degrading enzyme as follows:

[0015] 1) for plastic degradation.

[0016] 2) for preparing a plastic-degrading agent.

[0017] The alkali-resistant broad-spectrum plastic-degrading enzyme comprises or consists of an amino acid sequence as follows:

[0018] i) an amino acid sequence as shown in SEQ ID NO: 1 from Bacillus lipase LipA; or

[0019] ii) an amino acid sequence obtained by connecting a tag to the N-terminus and / or C-terminus of i); or

[0020] iii) an enzyme with the same function obtained by substituting, deleting and / or adding one or more amino acids of the amino acid sequence of i) or ii).

[0021] Further, the alkali-resistant broad-spectrum plastic-degrading enzyme degrades plastics in a solution system at pH 4-12 (preferably pH 11).

[0022] Further, the alkali-resistant broad-spectrum plastic-degrading enzyme degrades plastics under conditions of a temperature of 25-45°C (preferably 45°C).

[0023] In the present application, the plastics include, but are not limited to, polycaprolactone (PCL), polylactic acid (PLA), polybutylene succinate (PBS), polyurethane (PU), polyethylene terephthalate (PET), polybutylene adipate / terephthalate (PBAT), etc.

[0024] In a second aspect, the present application provides a method for degrading plastics, the method comprising: soaking a plastic product in a buffer containing the alkali-resistant broad-spectrum plastic-degrading enzyme for degradation.

[0025] The buffer can be a Citrate-Na2HPO4 buffer with pH 4-6, a NaH2PO4-Na2HPO4 buffer with pH 7-8, or a Glycine-NaOH buffer with pH 9-12, etc. Preferably, the Glycine-NaOH buffer with pH 9-12, more preferably, the Glycine-NaOH buffer with pH 11.

[0026] The preparation method of the Citrate-Na2HPO4 buffer with pH 4-6 is as follows:

[0027] A solution: 100 mM Citrate; B solution: 200 mM Na2HPO4.

[0028] The buffer with pH 4.0 is prepared by mixing 30.7 mL of the A solution and 19.4 mL of the B solution, and then adding water to 100 mL.

[0029] The buffer with pH 5.0 is prepared by mixing 24.3 mL of the A solution and 25.7 mL of the B solution, and then adding water to 100 mL.

[0030] The buffer with pH 6.0 is prepared by mixing 17.9 mL of the A solution and 32.1 mL of the B solution, and then adding water to 100 mL.

[0031] The preparation method of the NaH2PO4-Na2HPO4 buffer with pH 7-8 is as follows:

[0032] A solution: 200 mM NaH2PO4, B solution: 200 mM Na2HPO4.

[0033] The buffer with pH 7.0 is prepared by mixing 39.0 mL of the A solution and 61.0 mL of the B solution, and then adding water to 200 mL.

[0034] The buffer with pH 8.0 is prepared by mixing 5.3 mL of the A solution and 94.7 mL of the B solution, and then adding water to 200 mL.

[0035] The preparation method of the Glycine-NaOH buffer with pH 9-12 is as follows: adjust 50 mM Glycine to the required pH with 6 M NaOH solution.

[0036] The aforementioned method, the temperature condition for degradation is 25-45°C, preferably 45°C.

[0037] The amino acid sequence encoding the alkali-resistant, broad-spectrum plastic degrading enzyme provided by the present invention is shown in SEQ ID NO:1, and the encoding gene sequence is shown in SEQ ID NO:2. An engineered Escherichia coli bacterium constructed using the encoding gene sequence can effectively express the alkali-resistant, broad-spectrum plastic degrading enzyme. The nucleotide sequence encoding the enzyme was cloned into the expression vector pET28a and then transformed into the expression host Escherichia coli BL21(DE3) to express the target protein. After shake flask fermentation and nickel column purification, the protein concentration can reach 400 μg / mL.

[0038] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0039] The alkali-resistant, broad-spectrum plastic-degrading enzyme provided by the present invention degrades polycaprolactone (PCL) at a rate of over 95% within 5 hours at a strongly alkaline pH of 11 at 45°C. The degradation products are primarily polycaprolactone monomers and dimers. The degradation rate of polylactic acid (PLA) is over 4% within 5 days, with lactic acid monomers being the primary degradation products. The degradation rate of polybutylene succinate (PBS) is over 2% within 5 days, with succinic acid monomers and succinic acid oligomers being the primary degradation products. The degradation rate of polyurethane (PU) is over 5.5% within 5 days, with aniline, diethylene glycol, and 4,4-methylenedianiline caprolactone being the primary degradation products. The degradation rate of polyethylene terephthalate (PET) is over 2.5% within 5 days, with terephthalic acid and monohydroxyethyl terephthalic acid being the primary degradation products. The degradation rate of polybutylene adipate / terephthalate plastic (PBAT) reached over 7.5% within 5 days, with the main degradation products being adipic acid, terephthalic acid, butylene adipate, and butylene terephthalate. This alkali-resistant, broad-spectrum plastic-degrading enzyme can not only be effectively used in the biodegradation of mixed plastic waste, alleviating environmental issues caused by plastic pollution, but also enables monomer recycling, with broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 Figure 2 is a map of the pET-28a-BsEst4 recombinant expression plasmid (without a signal peptide) in a preferred embodiment of the present invention.

[0041] Figure 2 This is an SDS gel image of the recombinant protein BsEst4 in a preferred embodiment of the present invention. M: molecular weight marker; Su: supernatant after recombinant expression cells were disrupted; Pr: precipitate after ultrasonic disruption of recombinant expression cells; Pu: BsEst4 protein after nickel column purification from the supernatant of ultrasonic disruption of recombinant expression cells.

[0042] Figure 3Optimal reaction conditions for the BsEst4 protein in preferred embodiments of the present invention: (a) Optimal reaction pH, (b) pH stability, (c) Optimal reaction temperature, and (d) Temperature stability assay. Enzyme activity was measured using 4-nitrophenyllaurate (pNPL) as the substrate.

[0043] Figure 4 Degradation of PCL plastic in a preferred embodiment of the present invention. (a) Chemical structure of PCL, (b) Optical image of the degraded morphology, (c) Weight loss, (d) Mass spectrum of the degradation products. CK: Control group.

[0044] Figure 5 The degradation effect of PLA plastic in a preferred embodiment of the present invention. (a) Chemical structure of PLA, (b) SEM image of the degraded morphology, (c) weight loss, and (d) mass spectrum of the degradation products. CK: Control group.

[0045] Figure 6 The degradation effect of PBS in a preferred embodiment of the present invention is shown. (a) Chemical structure of PBS, (b) SEM image of the morphology after degradation, (c) weight loss, and d) mass spectrum of the degradation products. CK: Control group.

[0046] Figure 7 The degradation effect of PU plastics in a preferred embodiment of the present invention. (a) Chemical structure of PU, (b) SEM image of the degraded morphology, (c) weight loss, and (d) mass spectrum of the degradation products. CK: Control group.

[0047] Figure 8 The degradation effect of PET plastic in a preferred embodiment of the present invention. (a) Chemical structure of PET, (b) SEM image of the morphology after degradation, (c) weight loss, and (d) mass spectrum of the degradation products. CK: Control group.

[0048] Figure 9 Degradation of PBAT plastics in a preferred embodiment of the present invention. (a) Chemical structure of PBAT, (b) SEM image of the degraded morphology, (c) weight loss, and (d) mass spectrum of the degradation products. CK: Control group. DETAILED DESCRIPTION

[0049] The present invention aims to provide an alkaline-resistant, broad-spectrum plastic-degrading enzyme. The present invention first discovered that an expression vector constructed using the nucleotide sequence shown in SEQ ID NO: 2 can secrete an alkaline-resistant, broad-spectrum plastic-degrading enzyme with an amino acid sequence such as SEQ ID NO: 1 after expression in a microorganism, capable of degrading a variety of plastics.

[0050] The first aspect of the present application provides an alkali-resistant broad-spectrum plastic-degrading enzyme, which has:

[0051] The enzyme provided by the present application is named BsEst4, and is a protein as described in 1) or 2) or 3) below:

[0052] 1) a protein consisting of the amino acid sequence shown in SEQ ID NO: 1;

[0053] 2) a protein consisting of the amino acid sequence from the 31st to the 212th amino acid at the end of the amino acid sequence derived from 1);

[0054] 3) a protein in which one or more amino acids are substituted, deleted and / or added to the amino acid sequence shown in SEQ ID NO: 1 without changing the function.

[0055] The sequence SEQ ID NO: 1 consists of 212 amino acids.

[0056] In order to facilitate the purification of the protein in 1) or 2) or 3), a corresponding His tag (His6) can be added at the end of the amino acid sequence shown in SEQ ID NO: 1. The imidazole ring on the histidine can have an affinity interaction with nickel ions, so that the target protein with a histidine tag is specifically bound to a nickel column, realizing the separation and purification of the protein.

[0057] The sequence of the protein in the above 3) is a substitution, deletion and / or addition of no more than 10 amino acid residues.

[0058] The protein in the above 3) can be artificially synthesized, or a gene sequence encoding the protein can be synthesized first, and then biologically expressed.

[0059] The gene sequence encoding the protein in the above 3) can be obtained by deleting one or several amino acid codons of the sequence in 1), and / or performing one or several base pair missense mutations, and / or connecting the sequence encoding the histidine tag shown in the table at the 5' end or the 3' end.

[0060] The nucleic acid molecule encoding the protein also belongs to the protection scope of the present application.

[0061] The nucleic acid molecule encoding the protein can be a DNA molecule as described in 1) or 2) below.

[0062] 1) a DNA molecule consisting of the nucleotide sequence shown in SEQ ID NO: 2.

[0063] 2) A DNA molecule that has 75% or more identity with the nucleotide sequence shown in 1) and encodes the protein.

[0064] The nucleotide molecule can be DNA, genomic DNA or recombinant DNA.

[0065] Among them, the sequence SEQ ID NO: 1 consists of 212 amino acids, and the nucleotide sequence in the sequence SEQ ID NO: 2 encodes the amino acid sequence shown in the sequence SEQ ID NO: 1.

[0066] Those skilled in the art can readily mutate the nucleotide sequences of the proteins obtained in the present invention using known methods, such as site-directed mutagenesis and rational modification of the gene nucleotide sequence. Artificially modified nucleotide sequences that are 75% or higher similar to the nucleotide sequences encoding the proteins isolated in the present invention are derived from and are equivalent to the nucleotide sequences of the present invention, as long as the encoded enzymes are active.

[0067] The identity described herein refers to similarity to a natural nucleotide sequence, and includes nucleotide sequences having a nucleotide sequence similarity of 75% or greater, or 80% or greater, or 85% or greater, or 90% or greater, or 95% or greater to the nucleotide sequence of the protein consisting of the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing encoded by the present invention. Identity can be calculated and evaluated using relevant software. When using computer software to evaluate sequence identity, the identity between two or more nucleotide sequences can be expressed as a percentage (%), and numerical values ​​can be used to evaluate the identity between related nucleotide sequences.

[0068] The expression cassette, recombinant plasmid, recombinant cloning strain and recombinant expression strain containing the nucleic acid molecule encoding the protein also fall within the scope of protection of the present invention.

[0069] The expression vector can insert the nucleotide sequence shown in SEQ ID NO: 2 (without the sequence at positions 1-93, which is the signal peptide) into the multiple cloning sites EcoRI (GAATTC) and NotI (GCGGCCGC) of the vector pET28a to obtain the recombinant plasmid pET-28a-BsEst4.

[0070] The recombinant expression vector can be introduced into a host microorganism.

[0071] The host microorganism can be bacteria, fungi or yeast, etc. The bacteria can be Escherichia coli BL21 (DE3) or the like.

[0072] The recombinant expression vector pET28a-BsEst4 was introduced into Escherichia coli BL21 (DE3) to obtain the recombinant expression bacteria BL21 (DE3)-BsEst4.

[0073] Another object of the present invention is to provide a method for producing a protein:

[0074] The method comprises the steps of fermenting and inducing the recombinant expression bacteria to obtain the protein.

[0075] The fermentation induction culture is carried out through the following steps:

[0076] 1) Seed culture: The recombinant expression bacteria with correct sequencing were inoculated into 5 mL of LB liquid culture medium containing Kan antibiotic (50 μg / mL), and cultured overnight at 37°C and 220 rpm until the OD 600 =2-6.

[0077] 2) Expansion culture: The seed solution cultured in step 1) was inoculated into a 500 mL conical flask containing 100 mL LB liquid medium at a ratio of 1:100, and cultured at 37°C and 220 rpm until the OD 600 =0.6-0.8.

[0078] 3) Induction culture: Add IPTG inducer with a final concentration of 0.8 mM to the bacteria expanded in step 2), and induce culture at 16° C. and 180 rpm for 21-24 hours.

[0079] The degradation of various plastics such as PCL, PLA, PBS, PU, ​​PET and PBAT by the above-mentioned proteins also falls within the scope of protection of the present invention.

[0080] The application provides an alkali-resistant broad-spectrum plastic-degrading enzyme derived from Bacillus sp. BIT-YP1 and a coding gene thereof. After the coding gene of the enzyme is cloned into a vector pET-28a, the vector is transformed into an expression host BL21 (DE3), and then the recombinant Escherichia coli BL21 (DE3)-BsEst4 is subjected to IPTG-induced fermentation culture in a flask, the bacterial body is collected and broken, and then the target protein with a His tag is subjected to nickel column purification, so that the concentration of the enzyme can reach 400 μg / mL, and the efficient expression of the alkali-resistant broad-spectrum plastic-degrading enzyme is realized. The optimal reaction pH of the enzyme is 11, the enzyme activity basically remains unchanged under the condition of pH 4-12 for 48 hours, and the enzyme has good alkali resistance and pH stability. The optimal reaction temperature of the enzyme is 45 DEG C, and the enzyme activity can still remain above 80% under the condition of 25-45 DEG C for 30 min. The alkali-resistant broad-spectrum plastic-degrading enzyme can degrade six kinds of polyester plastics including PCL, PLA, PBS, PU, PET and PBAT, and it is found through mass spectrometry that the degradation products of PCL are polycaprolactone monomer and dimer; the degradation product of PLA is lactic acid monomer; the degradation product of PBS is succinic acid monomer and succinic acid butylene glycol succinic acid oligomer; the degradation product of PU is aniline, diethylene glycol and 4,4-methylene diphenylamine caprolactone; the degradation product of PET is terephthalic acid and monohydroxyethyl terephthalic acid; and the degradation product of PBAT is terephthalic acid, adipic acid, terephthalic acid butylene glycol and adipic acid butylene glycol. After the products are recovered, they can be used as raw materials for synthesizing plastics, and therefore the alkali-resistant broad-spectrum plastic-degrading enzyme has great application potential and prospect in the degradation treatment and recycling of mixed plastic waste.

[0081] The following examples are intended to illustrate the application but not to limit the scope of the application. If not specifically indicated, the examples are carried out according to the conventional experimental conditions, such as Sambrook et al. Molecular Cloning: a Laboratory Manual (2001), or the conditions suggested in the manufacturer's manual.

[0082] Example 1 Construction of a recombinant expression plasmid pET-28a-BsEst4 of an alkali-resistant broad-spectrum plastic-degrading enzyme

[0083] The BsEst4 gene (excluding the signal peptide sequence) was amplified using the complete genome of Bacillus sp. YP1 (Yu Yang et al. Complete genome sequence of Bacillus sp. YP1, a polyethylene-degrading bacterium from waxworm's gut [J]. Journal of Biotechnology, 2015, 200:77–78.) as a template using primers F' (5'-TGCGAGCGGCCGCTTAGTTTGTATTCTGGCCC-3') and R' (5'-TGCGAGAATTCGCTGAACACAATCCAGTCGTTATGG-3'). The amplification procedure was a pre-denaturation at 98°C for 30 seconds, followed by 35 cycles of denaturation at 98°C for 5 seconds, annealing at 56°C for 5 seconds, and extension at 72°C for 1 minute, followed by a post-extension at 72°C for 2 minutes.

[0084] The target gene with enzyme cutting sites was placed in the Thermo Scientific FastDigest buffer and the restriction endonuclease EcoRI (Thermo Scientific TM FastDigest EcoRI, Cat. No. FD0274) and NotI (Thermo Scientific TM Digest with FastDigest NotI (Cat. No. FD0595) at 37°C for 15 min. The digestion system includes: 15 μL of PCR product, 1 μL each of EcoRI and NotI, 2 μL of 10× FastDigest buffer, and add water to 20 μL.

[0085] Using pET-28a plasmid as template, restriction enzyme EcoRI (Thermo Scientific TM FastDigest EcoRI, Cat. No. FD0274) and NotI (Thermo Scientific TM FastDigest NotI, catalog number FD0595) was digested in a universal buffer FastDigest at 37°C for 15 min to obtain the plasmid backbone. The digestion system was as follows: 15 μL of pET-28a plasmid (concentration approximately 150 ng / μL), 1 μL each of EcoRI and NotI, 2 μL of 10× FastDigest buffer, and water was added to 20 μL.

[0086] Mix 50 μL of the target gene product (target gene or plasmid) obtained after enzyme digestion with 10 μL of 6× DNA Loading buffer and load it on a 1% agarose gel (preparation method: weigh 0.3 g of agar powder and add it to 30 mL of 0.5× TAE buffer, heat and boil in a microwave oven, cool to about 60°C, add 1 μL of Golden View, and pour it into a gel plate with a comb inserted). After electrophoresis at 120 V for 20 minutes, the target band was cut under the UV light of a gel illuminator and then gel-extracted using the Thermo Scientific GeneJET Gel Extraction Kit (#K0692).

[0087] After gel recovery of the digested product, the remaining template plasmid was digested with DpnI (Thermo Scientific FastDigest DpnI, #FD1703). The reaction was incubated in a 37°C water bath for 30 min. The reaction system consisted of 2 μL of 10× FastDigest buffer, 1 μL of FastDigest DpnI, 1 μg of DNA, and ddH2O added to 20 μL.

[0088] The target gene fragment and plasmid backbone after gel recovery were ligated with T4 ligase (Thermo Scientific TM Ligation was performed using T4 DNA ligase (Cat. No. EL0012) at 30°C for 1 hour. The reaction system consisted of: 10 μL of plasmid, 5 μL of target gene fragment, and 2 μL of 10× T4 DNA ligase buffer. The ligation product was then diluted to 20 μL. The ligation product was used in subsequent experiments.

[0089] A tube of DH5a competent cells was taken out from -80℃ refrigerator and placed on ice until incubation. 10 μL of the above ligation product was added to 80 μL of E. coli DH5a competent cells, mixed gently, and then placed on ice for 30 min. During this time, avoid shaking. Then, the mixture was heated at 42℃ for 90 sec, and immediately placed on ice. During this time, avoid shaking. 1 mL of LB liquid was added to the competent cells, and then the mixture was incubated at 37℃ and 220 rpm for 45 min. Then, 200 μL of the recovered culture was taken out in a sterile environment, and then spread on an LB plate containing kanamycin (50 μg / mL). The plate was sealed with a sealing film, and then incubated at 37℃ overnight. The single colony strain grown on the plate was subjected to colony PCR using primers T7 (5'-TAATACGACTCACTATAGGG-3') and T7t (5'-GCTAGTTATTGCTCAGCGG-3'). The PCR reaction program was as follows: pre-denaturation at 94℃ for 5 min; denaturation at 94℃ for 30 sec, annealing at 56℃ for 30 sec, extension at 72℃ for 1 min, for a total of 35 cycles; and post-extension at 72℃ for 5 min. The PCR product was subjected to sequencing verification, and a positive transformant plasmid (pET-BSest4) was obtained. Figure 1

[0090] Example 2: Construction of a recombinant strain for producing an alkaline broad-spectrum plastic-degrading enzyme

[0091] The positive recombinant plasmid in Example 1 was extracted (the extraction steps refer to the TIANGEN plasmid extraction kit DP103), and finally 60 μL of water was used to elute the adsorption column to obtain the target plasmid. The target plasmid was verified using 1% agarose gel, and the size of the target band was confirmed to be correct. Then, 1 μL (about 200 ng / μL) of the plasmid was mixed with E. coli BL21(DE3) competent cells, and the transformation method and conditions were the same as those for DH5a. Colony PCR was performed on the strain grown on the plate using primers T7 and T7t. The PCR reaction program was as follows: pre-denaturation at 94℃ for 5 min; denaturation at 94℃ for 30 sec, annealing at 56℃ for 30 sec, extension at 72℃ for 1 min, for a total of 35 cycles; and post-extension at 72℃ for 5 min. The strain with the correct band size was a positive recombinant strain.

[0092] Example 3: Fermentation of strain Escherichia coli BL21(DE3)-BsEst4 to produce alkaline broad-spectrum plastic-degrading enzyme BsEst4

[0093] ​The recombinant bacteria BL21 (DE3)-BsEst4 was inoculated in 5 mL of LB liquid medium containing 50 μg / mL of Kan antibiotic, and was cultured at 37°C with 220 rpm overnight to prepare a seed solution. The seed solution was inoculated in a new 100 mL of LB medium at a ratio of 1:100, and was cultured at 37°C with 220 rpm until the OD 600 was 0.6-0.8. Then, IPTG was added to the bacterial solution at a final concentration of 0.8 mM, and the solution was cultured at 16°C with 180 rpm for 21 h to induce the expression of the protein. The induced bacterial solution was centrifuged at 4°C with 6000 rpm for 15 min to harvest the bacteria, and 50 mL was obtained per tube.

[0094] The bacteria harvested from the 50 mL bacterial solution in the previous step were resuspended in 30 mL of Lysis buffer (50 mM Tris-HCl, 300 mM NaCl, prepared by first weighing 6.06 g of Tris and 17.53 g of NaCl on a balance, dissolving the Tris in 900 mL of distilled water in a beaker, stirring until uniform, and finally adjusting the pH to 7.5 with HCl and diluting to 1 L in a volumetric flask, and storing in a 4°C refrigerator), and the bacteria were disrupted using a cell disrupter on ice at a power of 180 w with 3 s of ultrasonic and 4 s of interval for a total of 20 min until the bacterial solution became clear. The disrupted bacterial solution was centrifuged at 4°C with 10000 rpm for 20 min to obtain the supernatant, and the precipitate was resuspended in 30 mL of Lysis buffer.

[0095] 1 mL of Ni-NTA was added to a 12 mL chromatography column, and the nickel column was washed with 10-20 times the column volume of Lysis buffer to equilibrate the resin. The supernatant of the cell disruption solution (the protein was detected by SDS-PAGE, and was mainly expressed in the supernatant) was slowly combined with the equilibrated nickel column on ice for 30 min. The combined solution was slowly transferred to an open column in a 4°C chromatography cabinet, and the column was opened. After the combined solution flowed out, the flow-through was collected, and the column wall was repeatedly washed with the flow-through. In this process, the target protein was bound to the nickel ion resin, and most of the impurities flowed down with the flow-through. The column was washed with 10-15 times the column volume of Wash buffer (50 mM Tris-HCl, 300 mM NaCl, 30 mM imidazole, and the pH was adjusted to 7.5 with HCl) to remove non-specifically bound impurities. The target protein bound to the resin was eluted with 10 times the column volume of Elution buffer (50 mM Tris-HCl, 300 mM NaCl, 250 mM imidazole, and the pH was adjusted to 7.5 with HCl). In this step, a high concentration of imidazole competitively binds to the nickel column, thereby eluting the target protein.

[0096] The desalting column was used to remove the high concentration of imidazole remaining in the purified target protein. The bovine serum was used as the standard protein, and the concentration of the protein after desalting was determined by the Coomassie brilliant blue method. According to the requirements of the experiment, the obtained protein can be concentrated to a final concentration of 1 mg / mL using a 3 kDa ultrafiltration tube. 40 μL of the purified protein after desalting was mixed with 10 μL of 5x protein loading buffer, boiled at 100°C for 10 min, and 15 μL of the sample was subjected to polyacrylamide gel electrophoresis. The results showed that after 12.5% SDS-PAGE gel (Beijing Botaike Biotechnology Co., Ltd., WB1103) electrophoresis, the molecular weight of the target protein (SEQ ID NO: 1) band was 20.0 kDa ( Figure 2 ), which was consistent with the expected value.

[0097] Example 4 Enzymatic properties of the alkali-resistant broad-spectrum plastic-degrading enzyme BsEst4

[0098] 1) Optimal reaction pH and pH stability

[0099] A BsEst4 enzyme solution with a final concentration of 0.45 μg / mL was prepared, and a small molecule esterase substrate, 4-nitrophenyl laurate (pNPL), was used as the reaction substrate. The pNPL reaction substrate was prepared at a concentration of 4 mM with DMSO, and the enzyme activity was measured in the environment of pH 4-12. The enzyme activity reaction system was 100 μL: 90 μL of enzyme solution (diluted to the appropriate concentration), 10 μL of pNPL substrate, three replicates per group, and the change in absorbance at 410 nm was measured within 5 min. The reaction temperature was 30°C, and one point every 5 seconds. The buffer solutions were Citrate-Na2HPO4 (pH 4-6), NaH2PO4-Na2HPO4 (pH 7-8), and Glycine-NaOH (pH 9-12).

[0100] The enzyme was incubated at different pH for 48 h at 4°C, and then the enzyme activity was tested by an enzyme marker under the optimal pH conditions at 30°C. The enzyme activity of the unincubated enzyme tested under the same conditions was taken as 100%, and the remaining enzyme activity after incubation at different pH was converted. The pH was taken as the abscissa, and the relative residual enzyme activity was taken as the ordinate to draw a curve to determine the pH stability of the enzyme.

[0101] The results showed that the optimal reaction pH of the esterase was 11 ( Figure 3 a), and it had excellent stability under the conditions of pH 4-12, and the enzyme activity was basically unchanged after 48 h of incubation ( Figure 3 b). This indicated that BsEst4 had excellent alkali resistance and pH stability.

[0102] 2) Optimal reaction temperature and temperature stability

[0103] The small molecule esterase substrate, 4-nitrophenyl laurate (pNPL), was used as the reaction substrate. The pNPL substrate was dissolved in DMSO to prepare a 4 mM pNPL solution. Enzyme activity was tested at the optimal pH of 11 and at different reaction temperatures. The relative enzyme activity at other temperature conditions was converted with the maximum enzyme activity as 100%. A curve was plotted with temperature as the horizontal axis and relative enzyme activity as the vertical axis to compare the changes in enzyme activity with temperature.

[0104] The enzyme was incubated at the optimum pH of 11 and different temperatures for 30 minutes, and then the residual enzyme activity was tested at the optimum pH of 11 and 30°C. The enzyme activity of the unincubated enzyme under the same conditions was taken as 100%, and the relative enzyme activity after incubation at different temperatures was converted. The temperature stability curve was plotted with temperature as the horizontal axis and the relative residual enzyme activity as the vertical axis.

[0105] The results showed that the optimum temperature of the enzyme was 45℃( Figure 3 c) It has good activity at 25℃-45℃, and still maintains more than 80% residual enzyme activity after incubation for 30 minutes ( Figure 3 d).

[0106] Example 5 Application of Alkali-Resistant Broad-Spectrum Plastic Degrading Enzyme BsEst4 in Degrading PCL Plastics

[0107] In the Gly-NaOH buffer with the optimal pH of 11, the optimal temperature of 45 °C, 1 mL reaction system with different enzyme concentrations was configured in a test tube, and the enzyme concentrations were 0, 1, 2.5, 5, 10, 20, and 30 μM, respectively. Then, one piece of PCL film with a size of 1.2 cm x 1.2 cm was placed in the prepared reaction system, and the incubation was stopped after 1 h 40 min (BsEst4) at 45 °C. The film was cleaned with 1% SDS, anhydrous ethanol, and distilled water, and then dried in a constant temperature oven at 45 °C. The degradation rate under different enzyme concentrations was calculated, and the optimal enzyme concentration for degradation was explored. Under the optimal pH of 11, the optimal temperature of 45 °C, and the optimal enzyme concentration of 5 μM, one piece of film with a size of 1.2 cm x 1.2 cm was placed in a test tube. The corresponding test tube was taken out at 0 h, 0.5 h, 1 h, 1.5 h, 2 h, 3 h, 4 h, and 5 h, and the PCL film was taken out. The film was cleaned with 1% SDS, anhydrous ethanol, and distilled water, and then dried in a constant temperature oven at 45 °C. The weight of the degraded PCL film was measured, the degradation rate of the film was calculated, and the optimal degradation time was explored. At the end of the degradation experiment, the reaction was terminated on ice, the reaction solution was filtered with a 0.22 μm filter membrane to remove insoluble solids, and then the filtered filtrate was centrifuged with a 3 kDa ultrafiltration tube to remove the protein component. The filtrate in the ultrafiltration tube was collected for mass spectrometry test. The degradation products were scanned by mass spectrometry, and the mass spectrometer was SHIMADZU triple quadrupole liquid chromatography mass spectrometry LCMS-8050, and the loading amount was 1 μL.

[0108] Under the optimal reaction conditions of pH 11 and 45 °C, the degradation efficiency of the PCL film was the highest when the enzyme concentration was 5 μM. When the enzyme concentration was higher than 5 μM, the degradation rate did not increase significantly. Therefore, the optimal enzyme concentration was 5 μM. Under the optimal conditions of pH 11 and temperature of 45 °C, the enzyme concentration was 5 μM, and the surface morphology of the PCL film also changed significantly during the degradation process. The plastic film first became white and thin, then cracked at the weak place, and finally achieved complete degradation. Figure 4 b) The degradation efficiency of the PCL film reached 95% within 5 h Figure 4 c) The degradation products of the enzyme were identified by mass spectrometry, and peaks with mass ratios of 131 and 245 were found. Because the mass spectrum was a negative ion peak, the actual mass number of the corresponding substance should be plus one (or the original mass number), which corresponded to the mass number of PCL monomer polycaprolactone monomer (m / z: 132) and dimer (m / z: 246) Figure 4 d)

[0109] Application of alkali-resistant broad-spectrum plastic-degrading enzyme BsEst4 in degrading PLA plastic

[0110] Under the conditions of Gly-NaOH buffer at the optimum pH of 11, the optimum temperature of 45°C, 1 mL reaction system with enzyme concentrations of 0, 1, 2.5, 5, 10, 20, and 30 μM was respectively configured, then a piece of 0.7 cm x 0.7 cm PLA film was placed in the test tube, after 5 days of reaction, the film was taken out, washed with 1% SDS, anhydrous ethanol, and distilled water, dried in a constant temperature oven at 45°C, and weighed. The degradation rate under different enzyme concentrations was calculated, and the optimum enzyme concentration was explored.

[0111] Under the conditions of Gly-NaOH buffer at the optimum pH of 11, the optimum temperature of 45°C, and the optimum enzyme concentration of 5 μM, a piece of 0.7 cm x 0.7 cm film was placed in the test tube, and the corresponding PLA film was taken out at 0, 1, 2, 3, 4, and 5 days, washed with 1% SDS, anhydrous ethanol, and distilled water, dried in a constant temperature oven at 45°C, and weighed. The degradation rate of the film was calculated. The changes in the surface of the degraded PLA film were observed by scanning electron microscopy (SEM). First, the degraded film was taken out with tweezers, soaked in 2% SDS for 3 hours, then washed with clean water, placed on filter paper, and dried overnight in a 50°C incubator. The dried sample was placed in a clean sterilized 1.5 mL EP tube. Then conductive adhesive was attached to the sample stage, the degraded film was cut into a suitable size (0.15 cm x 0.15 cm, which can be determined according to the sample stage and the actual number of samples to be placed) and attached to the conductive adhesive, ensuring that the film is tightly attached to the conductive adhesive to ensure subsequent conductivity. Then the prepared sample was sputtered for 60 s, and finally placed in the sample slot of the scanning electron microscope, ensuring that the tray is slightly lower than the sample slot plane, vacuum, SED (secondary electron) mode, 5 kv, and adjusting the contrast, magnification, and focusing number to observe the surface structure of the degraded PLA film.

[0112] At the end of the degradation experiment, the reaction was terminated on ice, the reaction solution was filtered with a 0.22 μm filter membrane to remove insoluble solids, then the filtered filtrate was centrifuged with a 3 kDa ultrafiltration tube to remove protein components, and the filtrate in the ultrafiltration tube was collected for mass spectrometry test. The degradation products were scanned by mass spectrometry, and the mass spectrometer was a SHIMADZU triple quadrupole liquid chromatography mass spectrometry LCMS-8050, and the sample amount was 1 μL.

[0113] Under the optimal reaction conditions of pH 11 and 45℃, and an enzyme concentration of 5μM, the PLA surface morphology showed obvious irregular pits ( Figure 5 b, 5000×). The weight loss of PLA film during degradation is about 4% ( Figure 5 c). Mass spectrometry was used to identify the degradation products of the enzyme. A peak with a relatively high mass-to-nuclear ratio of 89 was found in the experimental group. Because the mass spectrum is a negative ion peak, the actual mass number of the substance should be increased by one (or the original mass number), corresponding to the mass number of the polylactic acid monomer (m / z: 90). In addition, peaks with mass-to-nuclear ratios of 156 and 228, 166 and 238, and 210 and 282 were also found. Calculations showed that the mass number difference between the two corresponding peaks was 72, which just corresponds to the repeating unit of polylactic acid (-OCHCH3CO-) ( Figure 5 d), it can be inferred that the bonds of PLA were broken during the degradation process, generating oligomers.

[0114] Example 7 Degradation of PBS Plastics by Alkali-Resistant Broad-Spectrum Plastic Degrading Enzyme BsEst4

[0115] In a Gly-NaOH buffer solution at an optimal pH of 11 and a temperature of 45°C, 1 mL of the reaction system was prepared with enzyme concentrations of 0, 1, 2.5, 5, 10, 20, and 30 μM. A 0.7 cm × 0.7 cm PBS membrane was then placed in a test tube. After 5 days of reaction, the membrane was removed, cleaned with 1% SDS, anhydrous ethanol, and distilled water, dried in a 45°C oven, and weighed. The degradation rate at different enzyme concentrations was calculated as (initial membrane weight - degraded membrane weight) / initial membrane weight%, to explore the optimal enzyme concentration for enzyme degradation.

[0116] Under the optimal conditions of pH 11 Gly-NaOH buffer, 45°C temperature, and 5 μM enzyme concentration, a 0.7 cm × 0.7 cm membrane sheet was placed in a test tube. At six time points (0, 1, 2, 3, 4, and 5 days), the corresponding PBS membrane sheet was removed and cleaned with 1% SDS, anhydrous ethanol, and distilled water. The sheet was then dried in a 45°C oven. The degraded PBS membrane sheet was weighed and the degradation rate was calculated. Scanning electron microscopy (SEM) was used to observe changes in the surface of the degraded PBS membrane. The degraded membrane sheet was first removed with tweezers and soaked in 2% SDS for 3 hours. The sheet was then rinsed with clean water, placed on filter paper, and dried overnight in a 50°C incubator. The dried sample was then placed in a clean, sterile 1.5 mL EP tube. Double-sided conductive tape was then applied to the sample stage. The degraded membrane was cut into pieces of appropriate size (0.15 cm x 0.15 cm, depending on the sample stage and the number of samples to be placed). These pieces were then placed on the conductive tape, ensuring the membrane adhered tightly to the adhesive to maintain subsequent conductivity. The prepared sample was then sprayed with gold for 60 seconds and placed in the sample well of a scanning electron microscope (SEM), ensuring the tray was slightly below the sample well. The microscope was vacuumed, and the SED (secondary electron) mode was set at 5 kV. The contrast, magnification, and focus factor were adjusted to observe the surface structure of the membrane after PBS degradation.

[0117] At the end of the degradation experiment, the reaction was terminated on ice. The reaction solution was filtered through a 0.22 μm filter to remove insoluble solids. The filtrate was then centrifuged through a 3 kDa ultrafiltration tube to remove protein components. The filtrate in the ultrafiltration tube was collected for mass spectrometry analysis. Degradation products were analyzed using a full-scan mass spectrometer on a Shimadzu triple quadrupole liquid chromatography-mass spectrometer (LCMS-8050). A 1 μL sample volume was loaded.

[0118] Under the optimal reaction conditions of pH 11 and 45℃, and an enzyme concentration of 5μM, the reaction lasted for 5 days. Scanning electron microscopy showed that obvious reticular pores appeared on the surface of PBS ( Figure 6 b, 5000×). The weight loss of PBS membrane during degradation was about 2% ( Figure 6 c). Mass spectrometry was used to identify the degradation products of the enzyme. It was found that the experimental group had relatively high peaks with a mass-to-nuclear ratio of 117 and a mass-to-nuclear ratio of 289. Because the mass spectrum is a negative ion peak, the corresponding actual mass number of the substance should be increased by one (or the original mass number), corresponding to the mass numbers of succinic acid (m / z: 118) and succinic acid butanediol succinate oligomers (m / z: 290). ( Figure 6 d).

[0119] Example 8 Degradation of PU Plastics by Alkali-Resistant Broad-Spectrum Plastic Degrading Enzyme BsEst4

[0120] In a Gly-NaOH buffer solution at an optimal pH of 11 and a temperature of 45°C, 1 mL of the reaction system was prepared with enzyme concentrations of 0, 1, 2.5, 5, 10, 20, and 30 μM. A 0.7 cm × 0.7 cm PU membrane was then placed in the test tube. After 5 days of reaction, the membrane was removed, cleaned with 1% SDS, anhydrous ethanol, and distilled water, dried in a 45°C oven, and weighed. The degradation rate at different enzyme concentrations was calculated as (initial membrane weight - membrane weight after degradation) / initial membrane weight%, exploring the optimal enzyme concentration for enzyme degradation.

[0121] Under the optimal conditions of pH 11 Gly-NaOH buffer, 45°C temperature, and 5 μM enzyme concentration, a 0.7 cm × 0.7 cm membrane sheet was placed in a test tube. At six time points (0, 1, 2, 3, 4, and 5 days), the corresponding PU membrane sheet was removed and cleaned with 1% SDS, anhydrous ethanol, and distilled water. The sheet was then dried in a 45°C oven. The degraded PU membrane sheet was weighed, and the degradation rate was calculated. Scanning electron microscopy (SEM) was used to observe changes in the surface of the degraded PU membrane. The degraded membrane sheet was first removed with tweezers and soaked in 2% SDS for 3 hours. It was then rinsed with clean water, placed on filter paper, and dried overnight in a 50°C incubator. The dried sample was then placed in a clean, sterile 1.5 mL EP tube. Double-sided conductive tape was then applied to the sample stage. The degraded film was cut into pieces of appropriate size (0.15 cm x 0.15 cm, depending on the sample stage and the number of samples to be placed). These pieces were then placed on the conductive tape, ensuring the film adhered tightly to the adhesive to maintain subsequent conductivity. The prepared sample was then sprayed with gold for 60 seconds and placed in the sample well of a scanning electron microscope (SEM), ensuring the tray was slightly below the sample well. The microscope was vacuumed, and the SED (secondary electron microscope) mode was set to 5 kV. The contrast, magnification, and focus factor were adjusted to observe the surface structure of the degraded PU film.

[0122] At the end of the degradation experiment, the reaction was terminated on ice. The reaction solution was filtered through a 0.22 μm filter to remove insoluble solids. The filtrate was then centrifuged through a 3 kDa ultrafiltration tube to remove protein components. The filtrate in the ultrafiltration tube was collected for mass spectrometry analysis. Degradation products were analyzed using a full-scan mass spectrometer on a Shimadzu triple quadrupole liquid chromatography-mass spectrometer (LCMS-8050). A 1 μL sample volume was loaded.

[0123] Under the optimal reaction conditions of pH 11 and 45°C, and an enzyme concentration of 5 μM, the reaction lasted for 5 days, and cracks appeared on the surface of the PU film ( Figure 7b, 5000x). The weight loss of the PU film was about 5.5% during the degradation process Figure 7 c) The degradation products of the enzyme were identified using mass spectrometry, and peaks with mass-to-charge ratios of 93, 105, and 355 were found. Since the mass spectrum was a negative ion peak, the actual mass number of the corresponding substance should be plus one (or the original mass number), which corresponded to the mass numbers of aniline (m / z: 93), diethylene glycol (m / z: 106), and butyl 4,4-methylene diphenylamine caprolactone (m / z: 356) Figure 7 d).

[0124] Example 9 Broad-spectrum plastic-degrading enzyme BsEst4 degrades PET plastic

[0125] Under the conditions of the optimal pH 11 Gly-NaOH buffer and the optimal temperature 45°C, 1 mL of reaction system was configured with enzyme concentrations of 0, 1, 2.5, 5, 10, 20, 30 μM, respectively, and then a piece of 0.7 cm x 0.7 cm PET film was placed in the test tube. After 5 days of reaction, the film was taken out, washed with 1% SDS, absolute ethanol, and distilled water, and then dried in a constant temperature oven at 45°C. The degradation rate under different enzyme concentrations was calculated, and the calculation method was: (initial film weight - degraded film weight) / initial film weight %. The optimal enzyme concentration for degradation was explored.

[0126] Under the conditions of the optimal pH 11 Gly-NaOH buffer, the optimal temperature 45°C, and the optimal enzyme concentration 5 μM, a piece of 0.7 cm x 0.7 cm film was placed in the test tube, and the corresponding PET film was taken out at 0, 1, 2, 3, 4, 5 days, respectively. The film was washed with 1% SDS, absolute ethanol, and distilled water, and then dried in a constant temperature oven at 45°C. The weight of the degraded PET film was measured, and the degradation rate of the film was calculated. The changes in the surface of the degraded PET film were observed using a scanning electron microscope (SEM). First, the degraded film was taken out with tweezers and soaked in 2% SDS for 3 hours, then washed with clean water, and placed on filter paper in a 50°C incubator for overnight drying. The dried sample was placed in a clean sterilized 1.5 mL EP tube. Then conductive adhesive was attached to the sample stage, and the degraded film was cut into appropriate size blocks (0.15 cm x 0.15 cm, which can be determined according to the sample stage and the actual number of samples to be placed) and attached to the conductive adhesive, ensuring that the film is tightly attached to the conductive adhesive to ensure subsequent conductivity. Next, the prepared sample was sputtered for 60 s, and finally it was placed in the sample slot of the scanning electron microscope, ensuring that the tray was slightly lower than the sample slot plane, vacuum, SED (secondary electron) mode, 5 kv, and the contrast, magnification, and focus were adjusted to observe the surface structure of the degraded PET film.

[0127] At the end of the degradation experiment, the reaction was terminated by placing it on ice, the reaction solution was filtered with a 0.22 pm filter membrane to remove insoluble solids, and then the filtered filtrate was centrifuged using a 3 kDa ultrafiltration tube to remove the protein component, and the filtrate in the ultrafiltration tube was collected for mass spectrometry testing. The degradation products were scanned by mass spectrometry mass number, and the mass spectrometer was a SHIMADZU triple quadrupole liquid chromatography mass spectrometer LCMS-8050, and the loading amount was 1 pL.

[0128] Under the optimal reaction conditions of pH 11 and 45°C, the enzyme concentration was 5 pM, and the reaction was observed for 5 days. Electron microscopy showed that fine pore structures appeared on the surface of the PET film Figure 8 b, 5000x), and the weight loss of the PET film during the degradation process was about 2.5% Figure 8 c) The degradation products of the enzyme were identified by mass spectrometry, and it was found that the peaks with relatively high intensity in the experimental group had mass-to-charge ratios of 166 and 209. Because the mass spectrum was a negative ion peak, the actual mass number of the corresponding substance should be plus one (or the original mass number), which corresponded to the mass number of terephthalic acid (m / z: 166) and monohydroxyethyl terephthalic acid (m / z: 210) Figure 8 d).

[0129] Example 10 Broad-spectrum plastic-degrading enzyme BsEst4 degrades PBAT plastic

[0130] Under the optimal conditions of pH 11 Gly-NaOH buffer and optimal temperature 45°C, 1 mL reaction system with enzyme concentrations of 0, 1, 2.5, 5, 10, 20, and 30 pM was configured, respectively, and then a piece of 0.7 cm x 0.7 cm PBAT film was placed in the test tube. After 5 days of reaction, the film was removed, washed with 1% SDS, anhydrous ethanol, and distilled water, and then dried in a constant temperature oven at 45°C. The degradation rate under different enzyme concentrations was calculated, and the calculation method was: (initial film weight - degraded film weight) / initial film weight%. The optimal enzyme concentration for enzyme degradation was explored.

[0131] Under the optimal pH 11 of Gly-NaOH buffer, the optimal temperature of 45℃ and the optimal enzyme concentration of 5 μM, a piece of 0.7 cm x 0.7 cm film was placed in a test tube, and the corresponding PET film was taken out at 0, 1, 2, 3, 4, 5 days, respectively, and washed with 1% SDS, absolute ethanol and distilled water, then washed with distilled water in a 45℃ constant temperature oven, dried at 45℃, weighed the weight of the degraded PBAT film, and calculated the degradation rate of the film. The changes of the surface of the degraded PBAT film were observed by scanning electron microscope SEM. First, the degraded film was taken out with tweezers, soaked in 2% SDS for 3 hours, then washed with clean water, placed on filter paper, and dried overnight in a 50℃ incubator. The dried sample was placed in a clean sterilized 1.5 mL EP tube. Then the double-sided conductive adhesive was stuck on the sample stage, the degraded film was cut into appropriate size blocks (0.15 cm x 0.15 cm, which can be determined according to the sample stage and the actual number of samples to be placed) and stuck on the conductive adhesive, ensuring that the film is tightly stuck on the conductive adhesive to ensure its subsequent conductivity. Then the prepared sample was sputtered for 60 s, and finally it was placed in the sample slot of the scanning electron microscope, ensuring that the tray was slightly lower than the sample slot plane, vacuum, SED (secondary electron) mode, 5kv, adjusting the contrast, magnification and focusing number to observe the surface structure of the PBAT degraded film.

[0132] At the end of the degradation experiment, it was placed on ice to terminate the reaction, and the reaction solution was filtered with a 0.22 μm filter membrane to remove insoluble solids, and then the filtered filtrate was centrifuged with a 3 kDa ultrafiltration tube to remove protein components, and the filtrate in the ultrafiltration tube was collected for mass spectrometry test. The degradation products were scanned by mass spectrometry, and the mass spectrometer was SHIMADZU triple quadrupole liquid chromatography mass spectrometry LCMS-8050, and the loading amount was 1 μL.

[0133] Under the optimal reaction conditions of pH 11 and 45℃, and enzyme concentration of 5 μM, the PBAT film surface was observed to have cracks by electron microscopy after 5 days of reaction Figure 9 b, 5000x), the weight change of the PBAT film during the degradation process was about 7.5% Figure 9 c) The degradation products of the enzyme were identified by mass spectrometry, and it was found that there were peaks with relatively high relative intensity with mass-to-charge ratios of 145, 166, 217 and 237 in the experimental group. Because the mass spectrum is a negative ion peak, the actual mass number of the corresponding substance should be plus one (or the original mass number), which corresponds to the mass number of adipic acid (m / z: 145), terephthalic acid (m / z: 166), adipic acid butanediol (m / z: 218) and terephthalic acid butanediol (m / z: 238) Figure 9 d).

[0134] While the application has been described in detail and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope thereof. It is therefore intended that whatever lies within the scope of the application be covered by the appended claims.

Claims

1. Any of the following applications of the alkaline-resistant broad-spectrum plastic degrading enzyme: 1) Used for plastic degradation; 2) Used to prepare plastic degradation agents; The amino acid sequence of the alkali-resistant broad-spectrum plastic degrading enzyme is shown in SEQ ID NO: 1; The alkali-resistant broad-spectrum plastic-degrading enzyme degrades plastic in a solution system with a pH of 11.

2. The use according to claim 1, characterized in that The alkali-resistant broad-spectrum plastic degrading enzyme degrades plastics at a temperature of 25-45°C.

3. The use according to claim 2, characterized in that The alkali-resistant broad-spectrum plastic-degrading enzyme degrades plastics at a temperature of 45°C.

4. The use according to any one of claims 1 to 3, characterized in that The plastics include polycaprolactone, polylactic acid, polybutylene succinate, polyurethane, polyethylene terephthalate, and polybutylene adipate / terephthalate.

5. A method for degrading plastics, characterized in that: The method comprises: immersing the plastic product in a buffer solution containing an alkali-resistant broad-spectrum plastic degrading enzyme for degradation, wherein the amino acid sequence of the alkali-resistant broad-spectrum plastic degrading enzyme is shown in SEQ ID NO: 1; The buffer solution is a Glycine-NaOH buffer solution with a pH of 9-12.

6. The method according to claim 5, characterized in that The degradation temperature condition is 25-45℃.

7. The method according to claim 6, characterized in that The degradation temperature condition is 45°C.

8. The method according to any one of claims 5 to 7, characterized in that: The plastics include polycaprolactone, polylactic acid, polybutylene succinate, polyurethane, polyethylene terephthalate, and polybutylene adipate / terephthalate.

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