CN115975983A

CN115975983A - Application of alkali-resistant broad-spectrum plastic degrading enzyme

Info

Publication number: CN115975983A
Application number: CN202310028588.0A
Authority: CN
Inventors: 杨宇; 张敬雅; 胡琳
Original assignee: Beijing Institute of Technology BIT
Current assignee: Beijing Institute of Technology BIT
Priority date: 2022-11-07
Filing date: 2023-01-09
Publication date: 2023-04-18

Abstract

The invention discloses an application of an alkali-resistant broad-spectrum plastic degrading enzyme, wherein the degrading enzyme is derived from a bacillus lipase LipA, the degrading speed of the degrading enzyme on polycaprolactone within 5 hours reaches more than 95%, the degrading speed on polylactic acid within 5 days reaches more than 4%, the degrading speed on poly (butylene succinate) within 5 days reaches more than 2%, the degrading speed on polyurethane within 5 days reaches more than 5.5%, the degrading speed on polyethylene terephthalate within 5 days reaches more than 2.5%, and the degrading speed on poly (adipic acid)/butylene terephthalate plastic within 5 days reaches more than 7.5% under the condition of 45 ℃ and pH 11. The degradation products are mainly monomers and dimers, and can be recycled to synthesize plastics again. The alkali-resistant broad-spectrum plastic degrading enzyme can be effectively applied to biodegradation treatment of mixed plastic wastes of various plastics, so that the environmental problem caused by plastic pollution is reduced, monomer recycling can be realized, and the application prospect is wide.

Description

Application of alkali-resistant broad-spectrum plastic degrading enzyme

Cross Reference to Related Applications

The present invention claims priority from chinese patent application No. CN202211387050.0, filed on 7/11/2022, which is incorporated herein by reference in its entirety.

Technical Field

The invention relates to the technical field of enzyme engineering, in particular to application of alkali-resistant broad-spectrum plastic degrading enzyme.

Background

Plastic products are widely applied to human production and life due to excellent characteristics, and meanwhile, the accumulation of plastic wastes in the environment causes serious environmental pollution problems because the plastics are not easy to degrade in the environment, so that the development of a proper waste plastic treatment method and technology is urgently needed. Different from landfill, incineration, physical and chemical treatment methods, the method for treating plastics by using biological enzyme degradation not only has mild conditions but also is green and environment-friendly, can realize the recycling of plastic synthetic monomers, and is a promising waste plastic treatment technology. The key to realizing the biological enzyme degradation treatment is to develop high-efficiency plastic degrading enzyme.

There have been reports of plastics degrading enzymes, such as Lipase from the fungus Mucor miehei (Mucor miehei), which can achieve 74% degradation of Polycaprolactone (PCL) powder in 24 hours at 40 ℃ and pH7.7 (Pasorino et al, lipase-catalyzed degradation of poly (. Epsilon. -captorifice.) Enzyme and Microbial Technology,2004,35 (4): 321-326.). Lipases from Lactobacillus (Lactobacillus brevis) can weightlessness of PCL lamellae by 60% in 10 days at 37 ℃, ph8.1 (Khan et al, lactobacillus sp. Lipase mediated poly (epsilon-callactone) degradation. International Journal of Biological Macromolecules,2017, 95.

Polylactic acid (PLA) degrading enzymes found to date are derived mainly from lipases of the species Bacillus amyloliquefaciens Paenibacillus amyloliquefaciens (Akutsu-Shigeno et al, cloning and sequencing of enzymatic (DL-lactic acid) polymerization gene from Paenibacillus amyloliquefaciens strain TB-13and essential functional expression in Escherichia coli applied and Environmental microbiology,2003, 2498-2504), and cutinases of the yeast species Cryptococcus sp.S-2 (Masaki et al, cutinase-promoter strain sp.S-2. Strain S-2. Lipase and biological enzyme (7548, 7511. Radial 71, 7548. D. A. Lipase and strain).

The presently discovered degradation enzyme of poly (butylene succinate), PBS, is derived from the cutinase of Fusarium solani. It was found that the enzyme could complete Degradation of 90% or more of PBS membrane within 48 hours at 37 ℃ and pH7.4 (Zhang curing Wang et al enzymatic Degradation of by cutting enzyme bound from Fusarium solani. Polymer Degradation and stability.2016,134, 211-219.).

The Polyurethane-Plastic (PU) degrading enzymes found to date are mainly derived from The lipases Pu A and Pu B of Pseudomonas chlororaphis (Stern et al), the polyester Polyurethane-enzyme (pueA) from Pseudomonas chlororaphs enzymes codes a liquids, FEMS Microbiology Letters,2000,185 (2): 163-168 Howard et al, cloning, nucleotide sequencing and catalysis of a Polyurethane-enzyme (pueB) from Pseudomonas chlororaphs International BioDegree & Biogradation, 2001,47 (3-141-149) and from Pseudomonas fluorescens (phosphor) lipase, 127-127 family J, (18-44) biological esterase derived from Pseudomonas chlororaphs, jet & biochemicals, 18-149). These enzymes can hydrolyze ester bonds of polyurethane plastics, thereby achieving degradation of the polyurethane plastics.

Currently, polyethylene terephthalate Plastic (PET) degrading enzymes have been discovered. For example, muller et al found that TfH, a cutinase derived from Thermobifida fusca, a Thermobifida thermophila, can lose 50% of PET with a crystallinity of 9% at 55 ℃ over 21 days (Muller et al, enzymatic Degradation of Poly (ethylene terephthalate): rapid hydrolysis using a hydrosol 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 lose 97% of PET at 70 deg.C, pH8 over 96h (Ronkvistat, cutinase-Catalyzed Hydrolysis of Poly [ J ]. Macromolecules,2009,42 (14): 5128-5138.).

The currently discovered polybutylene adipate/terephthalate Plastic (PBAT) degrading enzyme, mainly reported as Esterase Chath _ Est1 from Anaerobic Clostridium, can Hydrolyze oligomer of PBAT, baETaEBa (wherein BaE represents dihydroxy ethyl benzoate, ETaE: bis-2-hydroxyethyl terephthalate, ba: benzoic acid), into smaller monomer polymers (Perz et al, anesterase from Anaerobic Clostridium hydrothora hydro-esterification-Aromatic Polyesters) [ J. Furthermore, wallace Paal et al found that an esterase Ppost derived from Pseudomonas pseudoalcaligenes could degrade PBAT films and release small monomers TA (terephthalic acid) and ButA (4, 4 hydroxy-butylcarbonylbenzoic acid) at 65 ℃, pH7 (Wallace Paal et al, ppt is a novel PBAT degrading polymeric formed by a proteinaceous screening of Pseudomonas pseudoalcaligenes [ J ] Applied microbiology and biotechnology,2017,101 (6): 2291-2303.).

The research provides basis and foundation for developing the biological enzyme degradation treatment technology of plastics. However, the plastic degrading enzymes reported at present can only degrade one of the plastics, and there are few reports of multi-substrate enzymes that can degrade two or more plastics simultaneously. The plastic waste in the real life garbage generally exists in a mixed form, and the variety is various. Sorting pre-treatment of mixed plastic waste is very difficult and complicated. Therefore, the development of multi-substrate plastic degrading enzymes capable of degrading various plastics simultaneously can save the sorting process, thereby realizing the degradation of mixed plastic wastes in garbage. In addition, in many plastic products in practical use, usually plastic alloys prepared by blending two or more plastics, in order to achieve effective degradation treatment of these plastic alloys, it is also necessary to develop multi-substrate plastic degrading enzymes capable of degrading a plurality of plastics simultaneously.

In addition, because the degradation of plastics can generate acidic products, the alkaline condition can buffer the acidic products, and the method has important significance for maintaining the continuity and the high efficiency of the enzymatic degradation reaction. At present, the discovered plastic degrading enzymes basically have good activity under neutral or weak alkaline conditions (pH 7-8) and do not have good alkali resistance. Therefore, the development of broad-spectrum plastic degrading enzyme capable of degrading various plastics simultaneously has good alkali resistance, and has important value for realizing the enzyme degradation treatment of mixed plastic wastes.

Disclosure of Invention

The invention aims to provide application of the alkali-resistant broad-spectrum plastic degrading enzyme.

To achieve the object of the present invention, in a first aspect, the present invention provides any one of the following uses of an alkaline-resistant broad-spectrum plastic-degrading enzyme:

1) Used for plastic degradation.

2) Is used for preparing plastic degradation agent.

The alkali-resistant broad-spectrum plastic degrading enzyme comprises or consists of the following amino acid sequence:

i) An amino acid sequence of LipA from bacillus as shown in SEQ ID NO 1; or

ii) an amino acid sequence obtained by connecting a label at the N end and/or the C end of the i); or

iii) i) or ii) by substitution, deletion and/or addition of one or more amino acids.

Further, the alkali-resistant broad-spectrum plastic degrading enzyme degrades plastics in a solution system with pH4-12 (preferably pH 11).

Further, the alkali-resistant broad-spectrum plastic degrading enzyme degrades plastic under the condition of temperature of 25-45 ℃ (preferably 45 ℃).

In the present invention, the plastic includes, but is not limited to, polycaprolactone (PCL), polylactic acid (PLA), polybutylene succinate (PBS), polyurethane (PU), polyethylene terephthalate (PET), poly adipic acid/butylene terephthalate (PBAT), and the like.

In a second aspect, the present invention provides a method of degrading a plastic, the method comprising: and soaking the plastic product in a buffer solution containing the alkali-resistant broad-spectrum plastic degrading enzyme for degradation.

The buffer may be Citrate-Na at pH4-6 ₂ HPO ₄ Buffer, naH of pH7-8 ₂ PO ₄ -Na ₂ HPO ₄ Buffer or Glycine-NaOH buffer at pH9-12, and the like. Preferably a Glycine-NaOH buffer at pH9-12, more preferably a Glycine-NaOH buffer at pH 11.

Wherein, citrate-Na with pH4-6 ₂ HPO ₄ The preparation method of the buffer solution comprises the following steps:

solution A: 100mM Citrate; and B, liquid B: 200mM Na ₂ HPO ₄ 。

Buffer at ph 4.0: 30.7mL of the solution A and 19.4mL of the solution B were mixed and the volume was adjusted to 100mL.

Buffer at ph 5.0: 24.3mL of the solution A and 25.7mL of the solution B were mixed and the mixture was made to 100mL.

Buffer at ph 6.0: after mixing 17.9mL of the solution A and 32.1mL of the solution B, the volume was adjusted to 100mL.

NaH at pH7-8 ₂ PO ₄ -Na ₂ HPO ₄ The preparation method of the buffer solution comprises the following steps:

solution A: 200mM NaH ₂ PO ₄ And liquid B: 200mM Na ₂ HPO ₄ 。

Buffer at ph 7.0: 39.0mL of the solution A and 61.0mL of the solution B were mixed and the mixture was made to 200mL.

Buffer at pH 8.0: 5.3mL of solution A and 94.7mL of solution B were mixed and the mixture was made to volume of 200mL.

The preparation method of the Glycine-NaOH buffer solution with the pH value of 9-12 comprises the following steps: 50mM Glycine was adjusted to the desired pH with 6M NaOH solution.

In the above method, the temperature condition for degradation is 25-45 deg.C, preferably 45 deg.C.

The amino acid sequence of the alkali-resistant broad-spectrum plastic degrading enzyme code is shown as SEQ ID NO. 1, and the coding gene sequence is shown as SEQ ID NO. 2. The engineering bacterium of colibacillus constructed by the coding gene sequence can effectively express the alkali-resistant broad-spectrum plastic degrading enzyme. The nucleotide sequence for coding the enzyme is cloned to an expression vector pET28a, then is transformed into an expression host escherichia coli BL21 (DE 3) for expression of target protein, and after shake flask fermentation and nickel column purification, the concentration of the protein can reach 400 mu g/mL.

By the technical scheme, the invention at least has the following advantages and beneficial effects:

the degradation rate of the alkali-resistant broad-spectrum plastic degrading enzyme provided by the invention on Polycaprolactone (PCL) within 5 hours at 45 ℃ under the condition of strong alkalinity and pH11 reaches more than 95%, and degradation products are mainly polycaprolactone monomers and dimers; the degradation rate of polylactic acid (PLA) in 5 days reaches more than 4 percent, and the degradation product is mainly lactic acid monomer. The degradation rate of poly (butylene succinate) (PBS) in 5 days reaches more than 2 percent, and degradation products mainly comprise succinic acid monomers and succinic acid oligomers of succinic acid, butanediol and succinic acid. The degradation rate of the Polyurethane (PU) in 5 days reaches more than 5.5 percent, and the degradation products mainly comprise aniline, diethylene glycol and 4,4 methylene diphenylamine caprolactone. The degradation rate of polyethylene terephthalate (PET) in 5 days reaches more than 2.5 percent, and degradation products mainly comprise terephthalic acid and monohydroxyethyl terephthalic acid. The degradation rate of poly adipic acid/butylene terephthalate Plastic (PBAT) in 5 days reaches more than 7.5 percent, and degradation products mainly comprise adipic acid, terephthalic acid, butanediol adipate and butanediol terephthalate. The alkali-resistant broad-spectrum plastic degrading enzyme can be effectively applied to biodegradation treatment of mixed plastic wastes of the plastics, so that the environmental problem caused by plastic pollution is reduced, monomer recycling can be realized, and the application prospect is wide.

Drawings

FIG. 1 is a diagram of a recombinant expression plasmid of pET-28a-BsEst4 (without signal peptide) in a preferred embodiment of the present invention.

FIG. 2 is a SDS gel of the recombinant protein BsEst4 in a preferred embodiment of the present invention. M: a molecular weight Marker; su: the supernatant after the recombinant expression thallus is crushed; pr: carrying out ultrasonic crushing on the recombinant expression thallus to obtain a precipitate; pu: bsEst4 protein is obtained after the supernatant fluid of the recombinant expression thallus after ultrasonic disruption is purified by a nickel column.

FIG. 3 shows the optimal reaction conditions for BsEst4 protein in a preferred embodiment of the present invention. (a) optimum reaction pH, (b) pH stability, (c) optimum reaction temperature, and (d) temperature stability. The enzyme activity of the reagent is measured by taking p-nitrophenol laurate (4-nitrophenyllaurate, pNPL) as a reaction substrate.

FIG. 4 shows the effect of degradation on PCL plastic in the preferred embodiment of the present invention. Chemical structure of PCL, (b) optical photo of appearance after degradation, (c) weight loss, and (d) mass spectrum of degradation product. CK is a control group.

FIG. 5 shows the effect of the present invention on the degradation of PLA plastic. Chemical structure of PLA, (b) appearance SEM photo after degradation, (c) weight loss, (d) mass spectrum of degradation product. CK is a control group.

FIG. 6 is a graph showing the effect of degradation on PBS according to the preferred embodiment of the present invention. Chemical structure of PBS, (b) appearance SEM picture after degradation, (c) weight loss, d) mass spectrum of degradation product. CK is a control group.

FIG. 7 shows the effect of the present invention on the degradation of PU plastics. Chemical structure of PU, (b) appearance SEM photo after degradation, (c) weight loss, and (d) mass spectrum of degradation product. CK is a control group.

FIG. 8 shows the effect of the present invention on the degradation of PET plastic. Chemical structure of PET, (b) appearance SEM picture after degradation, (c) weight loss, (d) mass spectrum of degradation product. CK is a control group.

FIG. 9 shows the effect of the preferred embodiment of the invention on the degradation of PBAT plastic. Chemical structure of PBAT, (b) SEM picture of appearance after degradation, (c) weight loss, and (d) mass spectrum of degradation product. CK is a control group.

Detailed Description

The invention aims to provide an alkali-resistant broad-spectrum plastic degrading enzyme. The invention discovers for the first time that an expression vector is constructed by using a nucleotide sequence shown in SEQ ID NO. 2, and the expression vector secretes an alkali-resistant broad-spectrum plastic degrading enzyme which has an amino acid sequence shown in SEQ ID NO. 1 and can degrade various plastics after being expressed in microorganisms.

In a first aspect the present invention provides an alkali-resistant broad-spectrum plastics degrading enzyme having:

the enzyme provided by the invention is named as BsEst4, and is the protein as 1) or 2) or 3) as follows:

1) 1, as shown in SEQ ID NO;

2) A protein consisting of 1) an amino acid sequence derived from the 31 st to 212 th amino acid terminal;

3) 1, one or more amino acids are substituted, deleted and/or added under the condition of not changing the function.

Wherein the sequence SEQ ID NO 1 consists of 212 amino acids.

In order to facilitate the purification of the protein in 1) or 2) or 3), a corresponding His tag (His 6) can be added at the end of the amino acid shown in the sequence SEQ ID NO. 1, and the imidazole ring on the histidine can perform affinity interaction with nickel ions, so that the target protein with the histidine tag can be specifically bound to a nickel column, and the separation and purification of the protein can be realized.

The sequence of the protein of 3) above, which shows an amino acid sequence in which one or more amino acids are substituted, deleted and/or added to not more than 10 amino acid residues without changing the function.

The protein in 3) above may be artificially synthesized, or may be obtained by synthesizing a gene sequence encoding the protein and then performing biological expression.

The gene sequence encoded by the protein of 3) above can be obtained by subjecting the gene sequence of 1) to a codon that is missing one or several amino acids in the nucleotide sequence, and/or to a missense mutation of one or several base pairs, and/or to a sequence encoded by the histidine tag shown in the above table attached to the 5 'end or to the 3' end.

Nucleic acid molecules encoding proteins are also within the scope of the invention.

The nucleic acid molecule encoding the protein may be a DNA molecule as described in 1) or 2) below.

1) 2, and the DNA molecule consisting of the nucleotide sequence shown in SEQ ID NO.

2) A DNA molecule which has 75 percent or more than 75 percent of identity with the nucleotide sequence shown in the sequence of 1) and codes the protein.

Wherein the nucleotide molecule can be DNA, genomic DNA or recombinant DNA.

Wherein, the sequence SEQ ID NO. 1 consists of 212 amino acids, and the nucleotide sequence in the sequence SEQ ID NO. 2 encodes an amino acid sequence shown in the sequence SEQ ID NO. 1.

The protein nucleotide obtained by the present invention can be easily mutated by a person of ordinary skill in the art using a known method, for example, by site-directed mutagenesis and rational modification of the nucleotide sequence of a gene. Those nucleotide sequences which are artificially modified and have a similarity of 75% or more to the nucleotide sequence encoding the protein isolated according to the present invention are derived from and identical to the nucleotide sequence of the present invention as long as the encoded enzyme has activity.

The identity refers to the similarity with a natural nucleotide sequence, and the identity comprises a nucleotide sequence with the similarity of 75 percent or higher, or 80 percent or higher, or 85 percent or higher, or 90 percent or higher, or 95 percent or higher with the nucleotide sequence of the protein consisting of the amino acid sequence shown in SEQ ID NO. 1 in the sequence table coded by the invention. Identity can be evaluated by calculation using relevant software, and when sequence identity is evaluated using computer software, identity between two or more nucleotide sequences can be expressed in (%) and numerical values can be used to evaluate identity between related nucleotide sequences.

Expression cassettes, recombinant plasmids, recombinant clonal strains and recombinant expression strains containing nucleic acid molecules encoding said proteins also belong to the scope of protection of the present invention.

The expression vector can insert a nucleotide sequence (without a sequence of 1-93 bits, the segment is a signal peptide) shown by a sequence SEQ ID NO. 2 into EcoRI (GAATTC) and NotI (GCGGCCGC) of a multiple cloning site of the vector pET28a to obtain a recombinant plasmid pET-28a-BsEst4.

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

The host microorganism can be bacteria, fungi or yeast. The bacteria are Escherichia coli BL21 (DE 3) and the like.

The recombinant expression vector pET28a-BsEst4 is introduced into escherichia coli BL21 (DE 3) to obtain recombinant expression bacterium BL21 (DE 3) -BsEst4.

Another object of the present invention is to provide a method for producing a protein, which comprises:

the method is to perform fermentation induction culture on the recombinant expression bacteria to obtain the protein.

The fermentation induction culture comprises the following steps:

1) Culturing a seed solution: the correctly sequenced recombinant expression bacteria were inoculated into a liquid medium containing 5mL of LB containing Kan antibiotics (50. Mu.g/mL), and cultured overnight at 37 ℃ and 220rpm with shaking to OD ₆₀₀ ＝2-6。

2) And (3) amplification culture: inoculating the seed solution cultured in the step 1) into a medium containing 100mL of L according to the proportion of 1B liquid Medium in 500mL Erlenmeyer flask, 37 ℃,220rpm culture to OD ₆₀₀ ＝0.6-0.8。

3) And (3) induction culture: adding IPTG inducer with final concentration of 0.8mM into the bacteria in the enlarged culture in the step 2), and carrying out induced culture at 16 ℃ and 180rpm for 21-24 hours.

The degradation of the protein on various plastics such as PCL, PLA, PBS, PU, PET and PBAT also belongs to the protection scope of the invention.

The invention provides an alkali-resistant broad-spectrum plastic degrading enzyme derived from Bacillus sp.BIT-YP1 and a coding gene thereof. The coding gene of the enzyme is cloned to a vector pET-28a and then is transformed into an expression host BL21 (DE 3), recombinant escherichia coli BL21 (DE 3) -BsEst4 is subjected to IPTG induced fermentation culture in a shake flask, thalli are collected and crushed to obtain target protein, then the target protein with His label is purified by a nickel column, and finally the concentration of the obtained enzyme can reach 400 mu g/mL, so that the high-efficiency expression of the alkali-resistant broad-spectrum plastic degrading enzyme is realized. The optimum reaction pH of the enzyme is 11, the enzyme activity is basically kept unchanged after 48 hours of incubation under the condition of pH4-12, and the enzyme has better alkali resistance and pH stability. The optimal reaction temperature of the enzyme is 45 ℃, and the enzyme still can keep more than 80 percent of enzyme activity after incubation for 30min at the temperature of 25-45 ℃. The alkali-resistant broad-spectrum plastic degrading enzyme can degrade 6 polyester plastics such as PCL, PLA, PBS, PU, PET, PBAT and the like, and mass spectrometry tests show that products obtained after PCL degradation include polycaprolactone monomers and dimers; the product after PLA degradation is lactic acid monomer; degradation products of the PBS are succinic acid monomers and succinic acid-butanediol-succinic acid oligomers; the degradation products of PU are aniline, diethylene glycol and 4,4 methylene dianiline caprolactone; the degradation products of PET are terephthalic acid and monohydroxyethyl terephthalic acid; among the degradation products of PBAT are terephthalic acid, adipic acid, butylene terephthalate and butylene adipate. After being recovered, the products can be used as raw materials for synthesizing plastics again, so the alkali-resistant broad-spectrum plastic degrading enzyme has great application potential and prospect in the aspects of degradation treatment and recycling of mixed plastic wastes.

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise indicated, the examples follow conventional experimental conditions, such as the Molecular Cloning handbook, sambrook et al (Sambrook J & Russell DW, molecular Cloning: a Laboratory Manual, 2001), or the conditions as recommended by the manufacturer's instructions.

EXAMPLE 1 construction of recombinant expression plasmid pET-28a-BsEst4 for an alkaline-resistant broad-spectrum Plastic degrading enzyme

BsEst4 gene (without signal peptide sequence) was amplified using the whole genome of strain Bacillus sp.YP1 (Yu Yang et al. Complete genome sequence of Bacillus sp.YP1, a polyethylene-degrading bacterium from wall word's gut [ J ]. Journal of Biotechnology,2015,200: 77-78.) as a template with primers F' (5 '-TGCGAGCGGCCGCTTAGTTTGTATTCTGGCCC-)) and R' (5 '-TGCGAGAATTCGCTGACAATCCAGTTATCGG-) -3'). The amplification program is pre-denaturation at 98 ℃ for 30sec; denaturation at 98 ℃ for 5sec, annealing at 56 ℃ for 5sec, extension at 72 ℃ for 1min, for 35 cycles; stretching for 2min at 72 ℃.

The obtained target gene with the restriction enzyme site was placed in Saimerfin universal buffer FastDiget using restriction enzyme EcoRI (Thermo Scientific) ^TM Fastdigest EcoRI, cat No. FD 0274) and NotI (Thermo Scientific) ^TM Fastdigest NotI, cat No. FD 0595) was digested at 37 ℃ for 15min, the digestion system: PCR product 15. Mu.L, ecoRI and NotI each 1. Mu.L, 10 XFastdigest buffer 2. Mu.L, water was added to 20. Mu.L.

Using pET-28a plasmid as a template, the restriction enzyme EcoRI (Thermo Scientific) ^TM Fastdigest EcoRI, cat No. FD 0274) and NotI (Thermo Scientific) ^TM FastDigest NotI, cat No. FD 0595) in universal buffer FastDigest, at 37 ℃ for 15min, to obtain a plasmid backbone, digestion system: mu.L of pET-28a plasmid (concentration: about 150 ng/. Mu.L), 1. Mu.L each of EcoRI and NotI, and 2. Mu.L of 10 XFastdigest buffer were added with water to 20. Mu.L.

Mixing 50 μ L of the target gene product (target gene or plasmid) obtained after the enzyme digestion with 10 μ L of 6 × DNA Loading buffer, loading, heating and boiling with 1% agarose Gel (preparation method: 0.3g of agar powder is weighed and added into 30mL of 0.5 × TAE buffer solution, microwave oven, cooling to about 60 ℃, adding 1 μ L of Golden View, pouring into a Gel plate inserted with a comb), performing electrophoresis for 20min under 120V voltage, cutting the target band under a Gel-irradiating instrument ultraviolet lamp, and performing Gel recovery with a Thermo Scientific Gene JET Gel Extraction Kit, # K0692).

After recovering the digestion product, the gel was digested with DpnI (Thermo Scientific Fastdigest DpnI, # FD 1703) to recover the original template plasmid remaining in the gel system, and incubated in a 37 ℃ water bath for 30min. The reaction system was 2. Mu.L of 10 XFastdigest buffer, 1. Mu.L of Fastdigest DpnI, 1. Mu.g of DNA, supplemented with ddH2O to 20. Mu.L.

Recovering the target gene fragment and plasmid backbone from the gel using T4 ligase (Thermo Scientific) ^TM T4 DNA ligase, cat # EL 0012), ligation was performed for 1h at 30 ℃. The reaction system is as follows: mu.L of plasmid, 5. Mu.L of the target gene fragment, 2. Mu.L of 10 XT 4 DNA ligase buffer, supplemented with water to 20. Mu.L, and the ligation product was used in the subsequent experiments.

A tube of DH 5. Alpha. Competent cells was removed from a freezer at-80 ℃ and incubated on ice, 10. Mu.L of the ligation product was added to 80. Mu.L of E.coli DH 5. Alpha. Competence, mixed by gentle inversion and then placed on ice for 30min while avoiding shaking, and heat shock was carried out at 42 ℃ for 90sec and immediately on ice while avoiding shaking. 1mL of LB liquid was added to the competent cells and allowed to resuscitate for 45min at 37 ℃ on a 220rmp shaker. Then, 200. Mu.L of the recovery culture medium was taken out from the sterile environment and applied to LB plate containing kanamycin antibiotic (50. Mu.g/mL), the plate was sealed with a sealing film, and was inverted overnight cultured in a 37 ℃ incubator, and colony PCR was performed on the single clone strain grown on the plate using primer pairs T7 (5': pre-denaturation at 94 ℃ for 5min; denaturation at 94 ℃ 30sec, annealing at 56 ℃ 30sec, extension at 72 ℃ for 1min for 35 cycles; stretching for 5min at 72 ℃. Sequencing verification of the PCR product was performed to obtain positive transformant plasmids (FIG. 1).

Example 2 construction of an alkaline broad-spectrum Plastic-degrading enzyme-producing recombinant Strain

The positive recombinant plasmid in example 1 was extracted (see TIANGEN plasmid mini-extraction kit DP103 in the extraction step), and the target plasmid was finally obtained by eluting the column with 60. Mu.L of water. The target plasmid was verified by using 1% agarose gel to confirm that the size of the band of interest was correct, and then 1. Mu.L (about 200 ng/. Mu.L) of the plasmid was taken and mixed with E.coli BL21 (DE 3) to be competent, and transformation method and conditions were the same as those for DH 5. Alpha. Transformation. The strains grown on the plates were subjected to colony PCR validation using primer pairs T7 and T7T. The reaction procedure for PCR was: pre-denaturation at 94 ℃ for 5min; denaturation at 94 ℃ 30sec, annealing at 56 ℃ 30sec, extension at 72 ℃ for 1min for 35 cycles; stretching for 5min at 72 ℃. Strains with the correct band size were positive recombinant strains.

EXAMPLE 3 fermentation of the Strain Escherichia coli BL21 (DE 3) -BsEst4 to produce the alkali-resistant broad-spectrum Plastic degrading enzyme BsEst4

The recombinant strain BL21 (DE 3) -BsEst4 was inoculated in 5mL of LB liquid medium (containing 50. Mu.g/mL Kan antibiotic) and cultured overnight with shaking at 220rpm at 37 ℃ to prepare a seed solution. The seed solution is inoculated into a new 100mL LB culture medium according to the proportion of 1 ₆₀₀ Is 0.6-0.8. Then, IPTG was added to the bacterial solution at a final concentration of 0.8mM, and the mixture was cultured at 16 ℃ and 180rpm for 21 hours with shaking to induce expression of the protein. The induced expression bacterial liquid was centrifuged at 6000rpm for 15min at 4 ℃ to obtain 50mL of cells per tube.

30mL of lysine buffer (50 mM Tris-HCl,300mM NaCl, preparation method is that 6.06g Tris,17.53g NaCl are weighed by a balance, 900mL distilled water is added to dissolve Tris in a beaker, the mixture is stirred evenly, finally, the pH value is adjusted to 7.5 by HCl, the volume is determined to be 1L in a volumetric flask, and the mixture is stored in a refrigerator at 4 ℃) to re-suspend the thalli, a cell crusher is used for crushing the re-suspension on ice, the power is 180w, the ultrasonic is carried out for 3s, the interval is 4s, the total time is 20min, and the crushed thalli is clear. The crushed bacterial liquid is centrifuged at 10000rpm for 20min at 4 ℃ to obtain a supernatant, and the precipitate is resuspended by using 30mL lysine buffer.

1mL of Ni-NTA was added to a 12mL column, and the resin was equilibrated by washing the nickel column with 10-20 column volumes of lysine buffer. Supernatant (SDS-PAGE protein, mainly expressed in supernatant) from the cell disruption solution was taken and combined with the equilibrated nickel column on ice with slow shaking for 30min. And (3) slowly transferring the binding solution to an open column in a chromatography cabinet at 4 ℃, opening a column port, collecting the flow-through solution after the binding solution flows out, repeatedly cleaning the residual resin on the wall of the beaker by using the flow-through solution, wherein the target protein is bound on the nickel ion resin in the process, and most of the foreign protein flows down along with the flow-through solution. The retained resin in the column was washed with 10-15 column volumes of Wash buffer (50 mM Tris-HCl,300mM NaCl,30mM imidazole, pH adjusted to 7.5 using HCl) to remove non-specifically bound contaminating proteins. Elution buffer (50 mM Tris-HCl,300mM NaCl,250mM imidazole, pH adjusted to 7.5 using HCl) was used in 10 column volumes to wash out the target protein bound to the resin, where high concentrations of imidazole could competitively bind to the nickel column, thereby eluting the target protein.

The high concentration imidazole remaining in the purified target protein was removed by using a desalting column, and the concentration of the desalted protein was measured by using a Coomassie brilliant blue method using bovine serum as a standard protein. The resulting protein can be concentrated using a 3kDa ultrafiltration tube to give a final concentration of 1mg/mL protein, depending on the experimental requirements. 40 μ L of the desalted purified protein was mixed with 10 μ L of 5 × protein loading buffer, and the mixture was boiled at 100 ℃ for 10min, and 15 μ L of the sample was subjected to polyacrylamide electrophoresis, and the result showed that the molecular weight of the band of the target protein (SEQ ID NO: 1) was 20.0kDa (FIG. 2) after electrophoresis on 12.5% SDS-PAGE gel (WB 1103, begoni Patais Biotech Co., ltd., beijing), which was in agreement with the expected molecular weight.

EXAMPLE 4 enzymatic Properties of the alkaline-resistant broad-spectrum Plastic degrading enzyme BsEst4

1) Optimum reaction pH and pH stability

BsEst4 enzyme solution with the final concentration of 0.45 mu g/mL is prepared, small molecular esterase substrate p-nitrophenyl laurate (pNPL) is used as a reaction substrate, the reaction substrate of the pNPL with the concentration of 4mM is prepared by DMSO, and the enzyme activity is measured under the environment with the pH value of 4-12. The enzyme activity reaction system is 100 mu L:90 μ L of enzyme solution (diluted to the appropriate concentration), 10 μ L of substrate for pNPL, triplicate in each group, were assayed over 5min at 410nmThe change in absorbance was 30 ℃ at a point of every 5 sec. Wherein Citrate-Na ₂ HPO ₄ Buffer (pH 4-6), naH ₂ PO ₄ -Na ₂ HPO ₄ Buffer (pH 7-8), glycine-NaOH buffer (pH 9-12).

The enzyme is incubated for 48h at 4 ℃ under different pH values, and then the enzyme activity is tested by an enzyme-labeling instrument under the condition of 30 ℃ and the optimum pH value. And (3) converting residual enzyme activity after incubation at different pH values by taking the enzyme activity tested under the same condition of the non-incubated enzyme as 100%, drawing a curve by taking the pH value as a horizontal coordinate and taking the relative residual enzyme activity as a vertical coordinate, and judging the pH stability of the enzyme.

The results show that: the esterase has the optimum reaction pH of 11 (figure 3 a), has excellent stability under the condition of pH4-12, and has basically unchanged enzyme activity after incubation for 48 hours (figure 3 b). Indicating that BsEst4 has excellent alkali resistance and pH stability.

2) Optimum reaction temperature and temperature stability

A small-molecule esterase substrate p-nitrophenyl laurate (pNPL) is used as a reaction substrate, and the pNPL substrate is dissolved in DMSO to prepare a 4mM pNPL solution. The enzyme activity is tested under the reaction conditions of the optimum pH value of 11 and different temperatures. Converting relative enzyme activity under other temperature conditions by taking the maximum enzyme activity as 100 percent, drawing a curve by taking the temperature as an abscissa and the relative enzyme activity as an ordinate, and comparing the change of the enzyme activity along with the temperature

Incubating the enzyme for 30min at the optimum pH of 11 and different temperatures, testing the residual enzyme activity at the optimum pH of 11 and at 30 ℃, converting the relative enzyme activity after incubation at different temperatures by taking the enzyme activity result under the same conditions of the non-incubated enzyme as 100%, and drawing a temperature stability curve by taking the temperature as an abscissa and the relative residual enzyme activity as an ordinate.

The results show that the enzyme has the optimum temperature of 45 ℃ (figure 3 c), has better activity under the condition of 25 ℃ -45 ℃, and still maintains more than 80% of residual enzyme activity after incubation for 30min (figure 3 d).

Example 5 application of the alkaline-resistant broad-spectrum Plastic degrading enzyme BsEst4 to degradation of PCL plastic

1mL of reaction systems with different enzyme concentrations are prepared in a test tube in Gly-NaOH buffer solution with optimal pH11 at the optimal temperature of 45 ℃, 1mL of reaction systems with the enzyme concentrations of 0,1, 2.5, 5, 10, 20 and 30 mu M are prepared, then 1.2cm multiplied by 1.2cm PCL membrane pieces are respectively put into the prepared reaction systems, incubation is carried out for 1h 40min (BsEst 4) at 45 ℃, then the membrane pieces are taken out, cleaned by using 1% SDS, absolute ethyl alcohol and distilled water, dried in a constant temperature oven at 45 ℃ and weighed. Calculating the degradation rate under different enzyme concentration conditions, wherein the calculation method comprises the following steps: (initial membrane weight-membrane weight after degradation)/initial membrane weight%, the optimum enzyme concentration for enzymatic degradation was explored. Under the conditions of Gly-NaOH buffer solution with the optimal pH of 11, the optimal temperature of 45 ℃ and the optimal enzyme concentration of 5 mu M, a 1.2 cm-by-1.2 cm membrane is placed in a test tube, the corresponding test tube is taken out at eight time points of 0h,0.5h,1h,1.5h,2h,3h,4h and 5h, the PCL membrane is taken out, cleaned by using 1% SDS, absolute ethyl alcohol and distilled water and dried in a constant-temperature oven at the temperature of 45 ℃, the weight of the degraded PCL membrane is weighed, the degradation rate of the membrane is calculated, and the optimal degradation time of enzyme degradation is searched. At the end of the degradation experiment, the reaction was stopped by placing it on ice, the reaction solution was filtered through a 0.22 μm filter to remove insoluble solids, the filtered filtrate was centrifuged using a 3kDa ultrafiltration tube to remove protein components, and the filtrate in the ultrafiltration tube was collected for mass spectrometry. The degradation product is subjected to mass spectrum mass number full scan, the mass spectrometer is Shimadzu (SHIMADZU) triple quadrupole liquid chromatography-mass spectrometer LCMS-8050, and the loading amount is 1 μ L.

Under the conditions of optimal reaction conditions of pH11 and 45 ℃ and the enzyme concentration of 5 mu M, the degradation efficiency of the PCL membrane is highest. When the enzyme concentration is higher than 5 mu M, the degradation rate is not obviously improved any more. Therefore, the optimal enzyme concentration is 5. Mu.M. Under the conditions of optimal pH11 and temperature of 45 ℃ and enzyme concentration of 5 mu M, the surface morphology of the PCL membrane also changes obviously in the degradation process, and the plastic membrane is whitened and thinned firstly, then is cracked in a weak place, and finally realizes full degradation (figure 4 b). The membrane degradation efficiency of PCL was up to 95% within 5h (fig. 4 c). The degradation products of the enzyme were identified using mass spectrometry and peaks with a mass to nuclear ratio of 131 and 245 were found, since the mass spectrum is a negative ion peak, the corresponding actual mass numbers of the species should be increased by one (or the original mass number), corresponding to the mass numbers of the PCL monomer polycaprolactone monomer (m/z: 132) and the dimer (m/z: 246), respectively (FIG. 4 d).

Example 6 application of the alkali-resistant broad-spectrum Plastic degrading enzyme BsEst4 to degradation of PLA plastics

Preparing 1mL reaction systems with enzyme concentrations of 0,1, 2.5, 5, 10, 20 and 30 mu M respectively in Gly-NaOH buffer solution with the optimal pH value of 11 and at the optimal temperature of 45 ℃, then putting a 0.7 cm-0.7 cm PLA membrane into a test tube, taking out the membrane after 5 days of reaction, cleaning the membrane by using 1% SDS, absolute ethyl alcohol and distilled water, drying the membrane in a constant-temperature oven at 45 ℃, and weighing the membrane. Calculating the degradation rate under different enzyme concentration conditions, wherein the calculation method comprises the following steps: (initial membrane weight-membrane weight after degradation)/initial membrane weight%, the optimum enzyme concentration for enzymatic degradation was explored.

Putting a 0.7cm x 0.7cm film in a test tube under the conditions of Gly-NaOH buffer solution with the optimum pH value of 11, the optimum temperature of 45 ℃ and the optimum enzyme concentration of 5 mu M, respectively at 6 time points of 0,1, 2, 3, 4 and 5 days, taking out the corresponding PLA film, cleaning the PLA film by using 1% SDS, absolute ethyl alcohol and distilled water, drying the PLA film in a constant-temperature oven at 45 ℃, weighing the weight of the degraded PLA film, and calculating the degradation rate of the film. And observing the change of the surface of the degraded PLA film by adopting a scanning electron microscope SEM. The degraded membrane was first removed with tweezers, soaked in 2% SDS for 3 hours, rinsed clean with clear water, placed on filter paper, oven dried overnight in an incubator at 50 deg.C, and the oven dried sample was placed in a clean, sterile 1.5mL EP tube. Then the double-sided conductive adhesive is adhered on a sample table, the degraded membrane is cut into blocks with proper size (0.15 cm multiplied by 0.15cm, which can be determined according to the sample table and the number of samples to be placed actually) and is adhered on the conductive adhesive, so that the membrane is ensured to be tightly adhered on the conductive adhesive, and the subsequent conductivity of the membrane is ensured. And then, performing metal spraying on the prepared sample for 60s, finally placing the sample in a sample groove of a scanning electron microscope, ensuring that the tray is slightly lower than the plane of the sample groove, vacuumizing, performing SED (secondary electron device) mode, performing 5kv, and adjusting contrast, magnification and focusing number to observe the surface structure of the film after PLA (polylactic acid) degradation.

At the end of the degradation experiment, the reaction was stopped by placing it on ice, the reaction solution was filtered through a 0.22 μm filter to remove insoluble solids, the filtered filtrate was centrifuged using a 3kDa ultrafiltration tube to remove protein components, and the filtrate in the ultrafiltration tube was collected for mass spectrometry. The degradation product is subjected to mass spectrum mass number full scan, the mass spectrometer is Shimadzu (SHIMADZU) triple quadrupole liquid chromatography-mass spectrometer LCMS-8050, and the loading amount is 1 μ L.

Under the conditions of the optimal reaction conditions of pH11 and 45 ℃ and the enzyme concentration of 5 mu M, the reaction is carried out for 5 days, and obvious irregular recesses are formed on the surface morphology of the PLA by the observation of a scanning electron microscope (figure 5b, 5000X). The weight loss of PLA film sheets during degradation was about 4% (fig. 5 c). The degradation products of the enzyme were identified by mass spectrometry, and it was found that a peak with a relatively high intensity of a mass-to-nuclear ratio of 89 appeared in the experimental group, and since the mass spectrum is a negative ion peak, the corresponding actual mass number should be added by one (or the original mass number), corresponding to the mass number of the polylactic acid monomer (m/z: 90), and in addition, peaks with mass-to-nuclear ratios of 156 and 228, 166 and 238, and 210 and 282 were also found, and it was found by calculation that the difference in mass number between the corresponding two peaks is 72, which exactly corresponds to the repeating unit (-OCHCH) of polylactic acid ₃ CO-) (FIG. 5 d), from which it can be concluded that cleavage of bonds occurs during degradation of PLA, resulting in oligomers.

Example 7 degradation of PBS Plastic by the alkali-resistant broad-spectrum Plastic degrading enzyme BsEst4

Preparing 1mL reaction systems with enzyme concentrations of 0,1, 2.5, 5, 10, 20 and 30 mu M respectively in Gly-NaOH buffer solution with the optimal pH value of 11 and at the optimal temperature of 45 ℃, then putting a piece of PBS membrane with the thickness of 0.7cm multiplied by 0.7cm into a test tube, taking out the membrane after 5 days of reaction, cleaning the membrane by using 1% SDS, absolute ethyl alcohol and distilled water, drying the membrane in a constant-temperature oven at the temperature of 45 ℃, and weighing the membrane. Calculating the degradation rate under different enzyme concentration conditions, wherein the calculation method comprises the following steps: (initial membrane weight-membrane weight after degradation)/initial membrane weight%, the optimum enzyme concentration for enzymatic degradation was explored.

Putting a piece of 0.7cm multiplied by 0.7cm membrane in a test tube under the conditions of Gly-NaOH buffer solution with the optimal pH value of 11, the optimal temperature of 45 ℃ and the optimal enzyme concentration of 5 mu M, respectively taking out the corresponding PBS membrane at 6 time points of 0 day, 1 day, 2 day, 3 day, 4 day and 5 day, cleaning the PBS membrane by using 1 percent SDS, absolute ethyl alcohol and distilled water, drying the cleaned PBS membrane in a constant-temperature oven at 45 ℃, weighing the weight of the degraded PBS membrane, and calculating the degradation rate of the membrane. The change of the surface of the degraded PBS membrane was observed by SEM. The degraded membrane was first removed with tweezers, soaked in 2% SDS for 3 hours, rinsed clean with clear water, placed on filter paper, oven dried overnight in an incubator at 50 deg.C, and the oven dried sample was placed in a clean, sterile 1.5mL EP tube. Then the double-sided conductive adhesive is adhered on a sample table, the degraded membrane is cut into blocks with proper size (0.15 cm multiplied by 0.15cm, which can be determined according to the sample table and the number of samples to be placed actually) and is adhered on the conductive adhesive, so that the membrane is ensured to be tightly adhered on the conductive adhesive, and the subsequent conductivity of the membrane is ensured. And then, performing gold spraying on the prepared sample for 60s, finally placing the sample in a sample groove of a scanning electron microscope, ensuring that the tray is slightly lower than the plane of the sample groove, vacuumizing, performing SED (secondary electron device) mode, performing 5kv, and adjusting contrast, magnification and focusing number to observe the surface structure of the membrane after PBS degradation.

The reaction was carried out under the conditions of optimum reaction conditions of pH11 and 45 ℃ and an enzyme concentration of 5. Mu.M for 5 days, and the surface of PBS was observed to have a large number of network pores by a scanning electron microscope (FIG. 6b, 5000X). The weight loss of the PBS membrane during degradation was about 2% (fig. 6 c). The degradation products of the enzyme are identified by using mass spectrum, and a peak with a relatively high intensity of a mass-to-nuclear ratio of 117 and a peak with a mass-to-nuclear ratio of 289 are found in an experimental group, and because a mass spectrum is a negative ion peak, the corresponding actual mass number is added by one (or is the original mass number) and corresponds to the mass numbers of succinic acid (m/z: 118) and succinic acid butanediol succinic acid oligomer (m/z: 290). (FIG. 6 d).

Example 8 degradation of PU plastics by BsEst4, an alkali-resistant broad-spectrum Plastic degrading enzyme

Preparing 1mL reaction systems with enzyme concentrations of 0,1, 2.5, 5, 10, 20 and 30 mu M respectively in Gly-NaOH buffer solution with the optimal pH value of 11 and at the optimal temperature of 45 ℃, then putting a PU membrane with the thickness of 0.7cm multiplied by 0.7cm into a test tube, taking out the membrane after 5 days of reaction, cleaning the membrane by using 1% SDS, absolute ethyl alcohol and distilled water, drying the membrane in a constant-temperature oven at the temperature of 45 ℃, and weighing the membrane. Calculating the degradation rate under different enzyme concentration conditions, wherein the calculation method comprises the following steps: (initial membrane weight-membrane weight after degradation)/initial membrane weight%, the optimum enzyme concentration for enzymatic degradation was explored.

Under the conditions of Gly-NaOH buffer solution with the optimal pH value of 11, the optimal temperature of 45 ℃ and the optimal enzyme concentration of 5 mu M, putting a piece of 0.7cm multiplied by 0.7cm membrane in a test tube, respectively at 6 time points of 0 day, 1 day, 2 days, 3 days, 4 days and 5 days, taking out the corresponding PU membrane, cleaning the PU membrane by using 1% SDS, absolute ethyl alcohol and distilled water, drying the PU membrane in a constant-temperature oven at 45 ℃, weighing the weight of the degraded PU membrane, and calculating the degradation rate of the PU membrane. And observing the change of the surface of the degraded PU film by adopting a scanning electron microscope SEM. The degraded membrane was first removed with tweezers, soaked in 2% SDS for 3 hours, rinsed clean with clear water, placed on filter paper, oven dried overnight in an incubator at 50 deg.C, and the oven dried sample was placed in a clean, sterile 1.5mL EP tube. Then the double-sided conductive adhesive is adhered on a sample table, the degraded membrane is cut into blocks with proper size (0.15 cm multiplied by 0.15cm, which can be determined according to the sample table and the number of samples to be placed actually) and is adhered on the conductive adhesive, so that the membrane is ensured to be tightly adhered on the conductive adhesive, and the subsequent conductivity of the membrane is ensured. And then, carrying out metal spraying on the prepared sample for 60s, finally placing the sample in a sample groove of a scanning electron microscope, ensuring that the tray is slightly lower than the plane of the sample groove, vacuumizing, carrying out SED (secondary electron device) mode, carrying out 5kv, and debugging contrast, magnification and focusing number to observe the surface structure of the membrane after PU degradation.

The reaction was carried out under optimum conditions of pH11 and 45 ℃ at an enzyme concentration of 5. Mu.M for 5 days, and cracks were observed on the surface of the PU film by electron microscopy (FIG. 7b, 5000X). The weight loss of the PU membranes during degradation was about 5.5% (FIG. 7 c). The degradation products of the enzyme were identified using mass spectrometry and peaks with mass-to-nuclear ratios of 93, 105 and 355 were found, since the mass spectrum is a negative ion peak, the corresponding actual mass numbers should be incremented by one (or the original mass number), corresponding to the mass numbers of aniline (m/z: 93), diethylene glycol (m/z: 106) and

butane

4,4 methylene dianiline caprolactone (m/z: 356), respectively (FIG. 7 d).

Example 9 degradation of PET Plastic by BsEst4, an alkali-resistant broad-spectrum Plastic degradation enzyme

Preparing 1mL reaction systems with enzyme concentrations of 0,1, 2.5, 5, 10, 20 and 30 mu M respectively in Gly-NaOH buffer solution with the optimal pH value of 11 and at the optimal temperature of 45 ℃, then putting a piece of PET membrane with the thickness of 0.7cm multiplied by 0.7cm into a test tube, taking out the membrane after 5 days of reaction, cleaning the membrane by using 1% SDS, absolute ethyl alcohol and distilled water, drying the membrane in a constant-temperature oven at the temperature of 45 ℃, and weighing the membrane. Calculating the degradation rate under different enzyme concentration conditions, wherein the calculation method comprises the following steps: (initial membrane weight-membrane weight after degradation)/initial membrane weight%, the optimum enzyme concentration for enzymatic degradation was explored.

Putting a piece of 0.7cm multiplied by 0.7cm membrane in a test tube under the conditions of Gly-NaOH buffer solution with the optimal pH value of 11, the optimal temperature of 45 ℃ and the optimal enzyme concentration of 5 mu M, respectively at 6 time points of 0 day, 1 day, 2 days, 3 days, 4 days and 5 days, taking out the corresponding PET membrane, cleaning the PET membrane by using 1% SDS, absolute ethyl alcohol and distilled water, drying the cleaned PET membrane in a constant-temperature oven at 45 ℃, weighing the weight of the degraded PET membrane, and calculating the degradation rate of the membrane. And observing the change of the surface of the degraded PET film by adopting a scanning electron microscope SEM. The degraded membrane was first removed with tweezers, soaked in 2% SDS for 3 hours, rinsed clean with clear water, placed on filter paper, oven dried overnight in an incubator at 50 deg.C, and the oven dried sample was placed in a clean, sterile 1.5mL EP tube. Then the double-sided conductive adhesive is adhered on a sample table, the degraded membrane is cut into blocks with proper size (0.15 cm multiplied by 0.15cm, which can be determined according to the sample table and the number of samples to be placed actually) and is adhered on the conductive adhesive, so that the membrane is ensured to be tightly adhered on the conductive adhesive, and the subsequent conductivity of the membrane is ensured. And then, carrying out metal spraying on the prepared sample for 60s, finally placing the sample in a sample groove of a scanning electron microscope, ensuring that the tray is slightly lower than the plane of the sample groove, vacuumizing, carrying out SED (secondary electron device) mode, carrying out 5kv, and debugging contrast, magnification and focusing number to observe the surface structure of the film after PET degradation.

The reaction was carried out under the conditions of pH11 and 45 ℃ under optimum reaction conditions and an enzyme concentration of 5. Mu.M for 5 days, and it was observed by electron microscopy that a fine pore structure appeared on the surface of the PET film (FIG. 8b, 5000X) and the weight loss of the PET film sheet during degradation was about 2.5% (FIG. 8 c). The degradation products of the enzyme were identified using mass spectrometry and it was found that the experimental group had peaks with relatively high intensity mass-to-nuclear ratios of 166 and 209, and that the actual mass numbers of the corresponding species should be increased by one (or the original mass number) because the mass spectrum was a negative ion peak, corresponding to the mass numbers of terephthalic acid (m/z: 166) and monohydroxyethyl terephthalic acid (m/z: 210), respectively (fig. 8 d).

Example 10 degradation of PBAT Plastic by the broad-spectrum alkaline-resistant Plastic degrading enzyme BsEst4

1mL reaction systems with enzyme concentrations of 0,1, 2.5, 5, 10, 20 and 30 μ M are prepared respectively in Gly-NaOH buffer solution with the optimum pH value of 11 and at the optimum temperature of 45 ℃, then a PBAT membrane with the thickness of 0.7cm multiplied by 0.7cm is put into a test tube, after 5 days of reaction, the membrane is taken out, cleaned by using 1% SDS, absolute ethyl alcohol and distilled water, dried in a constant temperature oven at 45 ℃ and weighed. Calculating the degradation rate under different enzyme concentration conditions, wherein the calculation method comprises the following steps: (initial membrane weight-membrane weight after degradation)/initial membrane weight%, the optimum enzyme concentration for enzymatic degradation was explored.

Putting a 0.7cm multiplied by 0.7cm membrane in a test tube under the conditions of Gly-NaOH buffer solution with the optimal pH value of 11, the optimal temperature of 45 ℃ and the optimal enzyme concentration of 5 mu M, respectively at 6 time points of 0,1, 2, 3, 4 and 5 days, taking out the corresponding PET membrane, washing the PET membrane by using 1% SDS, absolute ethyl alcohol and distilled water, washing the PET membrane in a constant-temperature oven at 45 ℃ by using distilled water, drying the PET membrane at 45 ℃, weighing the weight of the degraded PBAT membrane, and calculating the degradation rate of the membrane. Changes in the surface of the degraded PBAT film were observed using a scanning electron microscope SEM. The degraded membrane was first removed with tweezers, soaked in 2% SDS for 3 hours, rinsed clean with clear water, placed on filter paper, oven dried overnight in an incubator at 50 deg.C, and the oven dried sample was placed in a clean, sterile 1.5mL EP tube. Then the double-sided conductive adhesive is adhered on a sample table, the degraded membrane is cut into blocks with proper size (0.15 cm multiplied by 0.15cm, which can be determined according to the sample table and the number of samples to be placed actually) and is adhered on the conductive adhesive, so that the membrane is ensured to be tightly adhered on the conductive adhesive, and the subsequent conductivity of the membrane is ensured. And then, performing metal spraying on the prepared sample for 60s, finally placing the sample in a sample groove of a scanning electron microscope, ensuring that a tray is slightly lower than the plane of the sample groove, vacuumizing, performing SED (secondary electron) mode, performing 5kv, and adjusting contrast, magnification and focusing number to observe the surface structure of the membrane after PBAT degradation.

When the reaction was carried out under optimum reaction conditions of pH11 and 45 ℃ and at an enzyme concentration of 5. Mu.M for 5 days, cracks were observed on the surface of the PBAT membrane as observed by electron microscopy (FIG. 9b, 5000X), and the weight change of the PBAT membrane during degradation was about 7.5% (FIG. 9 c). The degradation products of the enzyme were identified using mass spectrometry and it was found that the experimental group had peaks with relatively high intensity of the nuclei ratios 145, 166, 217 and 237, and that the actual mass numbers of the species corresponding to the mass numbers of adipic acid (m/z: 145), terephthalic acid (m/z: 166), butanediol adipate (m/z: 218) and butanediol terephthalate (m/z: 238), respectively, were added (or the original mass numbers, respectively) because the mass spectra were negative ion peaks (fig. 9 d).

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims

1. Any one of the following applications of the alkali-resistant broad-spectrum plastic degrading enzyme:

1) The method is used for plastic degradation;

2) Used for preparing plastic degradation agent;

i) An amino acid sequence of LipA from bacillus as shown in SEQ ID NO 1; or

2. The use according to claim 1, wherein the alkali-resistant broad-spectrum plastic degrading enzyme degrades plastic in a solution system with pH 4-12.

3. The use according to claim 2, wherein the alkali-resistant broad-spectrum plastic degrading enzyme degrades plastic in a solution system at pH 11.

4. The use according to claim 1, wherein the alkali-resistant broad-spectrum plastic degrading enzyme degrades plastic at a temperature of 25-45 ℃.

5. The use according to claim 4, wherein the broad spectrum alkaline-resistant plastic-degrading enzyme degrades plastic at a temperature of 45 ℃.

6. Use according to any one of claims 1 to 5, wherein the plastic comprises polycaprolactone, polylactic acid, polybutylene succinate, polyurethane, polyethylene terephthalate, polybutylene adipate/terephthalate.

7. A method of degrading a plastic, the method comprising: and (2) soaking the plastic product in a buffer solution containing an alkali-resistant broad-spectrum plastic degrading enzyme for degradation, wherein the alkali-resistant broad-spectrum plastic degrading enzyme is the same as the alkali-resistant broad-spectrum plastic degrading enzyme described in claim 1.

8. The method of claim 7, wherein the buffer is Citrate-Na at pH4-6 ₂ HPO ₄ Buffer, naH of pH7-8 ₂ PO ₄ -Na ₂ HPO ₄ Buffer or Glycine-NaOH buffer at pH 9-12.

9. The method according to claim 7, characterized in that the temperature conditions for degradation are 25-45 ℃, preferably 45 ℃.

10. The method according to any one of claims 7-9, wherein the plastic comprises polycaprolactone, polylactic acid, polybutylene succinate, polyurethane, polyethylene terephthalate, polybutylene adipate/terephthalate.