Esterase and application thereof in plastic degradation

Abstract

The invention belongs to the technical field of enzyme engineering and plastic degradation, and particularly relates to esterase as well as a preparation method and application thereof in plastic degradation. According to the invention, genome sequencing analysis is carried out on the screened polyester type plastic degradation strain, and the esterase 8GL004290 is successfully screened and belongs to an alpha / beta hydrolase family. Experiments prove that the polyester degrading enzyme provided by the invention not only can degrade a polyester model micromolecule substrate, but also shows good degradation capacity for different polyester plastics. The esterase 8GL004290 has the highest degradation activity on polyurethane substrates, and meanwhile, the coverage of the types of the degraded substrates is wide. Therefore, the method disclosed by the invention has an extremely important application value in biodegradation treatment of the mixed plastic wastes.

Description

Technical Field

[0001] The invention belongs to the technical field of enzyme engineering and plastic degradation, and particularly relates to an esterase and application thereof in plastic degradation. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Plastics are widely used in modern society, and the market size is growing. Among them, polyester plastics are a type of polymer material formed by ester polymerization, mainly including polyethylene terephthalate (PET), polyurethane (PU), polylactic acid (PLA), polybutylene adipate / terephthalate (PBAT), polybutylene succinate (PBS) and polyhydroxyalkanoate (PHA). They have excellent mechanical properties, transparency and chemical resistance, and are widely used in packaging, textiles, agriculture and medical treatment. However, most polyester plastics are difficult to degrade quickly under natural conditions. Recycling through landfill, incineration and other methods has problems such as environmental pollution and waste of resources. The use of enzymes to degrade polyester plastics has great application potential.

[0004] Esterases are widely present in nature and belong to the α / β hydrolase superfamily. They play an important role in cellular lipid metabolism and protein biosynthesis. They have high similarities in core domains and catalytic mechanisms. They catalyze the hydrolysis of ester bonds by forming a catalytic triad of serine, histidine and negatively charged amino acids (usually aspartic acid or glutamic acid). At present, some esterases can degrade ester bonds in polyester plastics. The cutinase LCC (Leaf-branch Compost Cutinase) derived from leaf-branch compost has degradation activity for PET and PBAT. In 2017, PotockiVeronese isolated 26 strains showing esterase activity from the bovine rumen microbiome, and isolated a new esterase CE_Ubrb with polyurethane degradation activity from strain 44I12. In 2019, Avérous selected esterase E3576 from 50 hydrolases to degrade polyester polyurethane. However, the inventors have found that the types of polyester-type plastics degraded by the esterases reported above are relatively single and do not have the ability to degrade polyester-type plastics on a broad spectrum. Summary of the invention

[0005] In view of the problems existing in the prior art, the present invention provides an esterase and its application in plastic degradation. Specifically, the present invention performs genome sequencing analysis on the screened polyester-type plastic-degrading strains and successfully screens out an esterase 8GL004290, which belongs to the α / β hydrolase family and has a broad-spectrum degradation activity of polyester-type plastics. Based on the above research, the present invention is completed.

[0006] Specifically, the present invention relates to the following technical solutions:

[0007] The first aspect of the present invention provides an esterase, which is named 8GL004290, and the esterase 8GL004290 has:

[0008] a1) the amino acid sequence shown in SEQ ID NO.1; or,

[0009] a2) an amino acid sequence having at least 90% sequence homology with SEQ ID NO.1.

[0010] The second aspect of the present invention provides a nucleic acid molecule, wherein the nucleic acid molecule encodes the esterase described in the first aspect.

[0011] The third aspect of the present invention provides a recombinant expression vector, wherein the recombinant expression vector contains the nucleic acid molecule described in the second aspect.

[0012] The fourth aspect of the present invention provides a host cell, wherein the host cell contains the vector described in the third aspect or the chromosome has the nucleic acid molecule described in the second aspect integrated therein or expresses the esterase described in the first aspect.

[0013] The fifth aspect of the present invention provides a method for preparing the above-mentioned esterase, comprising the steps of: culturing the host cell described in the fourth aspect to express the esterase; and isolating and purifying to obtain the esterase.

[0014] The sixth aspect of the present invention provides the use of the esterase described in the first aspect, the nucleic acid molecule described in the second aspect, the recombinant expression vector described in the third aspect, and the host cell described in the fourth aspect in the field of plastic hydrolysis, depolymerization, degradation and / or catalysis.

[0015] Wherein, the plastic is a polyester plastic, and the polyester plastic includes any one or more of PET oligomer plastic, polyurethane plastic, bis(hydroxyethyl terephthalate) (BHET) plastic, PBAT plastic and PBS plastic.

[0016] The seventh aspect of the present invention provides a method for degrading polyester plastics, the method comprising: applying the esterase described in the first aspect or the host cell described in the fourth aspect to the polyester plastics for reaction.

[0017] Beneficial technical effects of one or more of the above technical solutions:

[0018] The above technical solution provides an esterase and a preparation method thereof and an application in plastic degradation. Specifically, the polyester degrading enzyme provided by the above technical solution can not only degrade polyester-type model small molecule substrates, but also show good degradation ability for different polyester-type plastics. Esterase 8GL004290 has the highest degradation activity for polyurethane substrates and covers a wide range of degradation substrate types. Therefore, the present invention has extremely important application value in the biodegradation management of mixed plastic waste. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0020] Figure 1 This is the phylogenetic tree of the esterase 8GL004290 of the present invention.

[0021] Figure 2 The figure is the SDS-PAGE result of the esterase 8GL004290 of the present invention.

[0022] Figure 3 It is the substrate specificity of the esterase 8GL004290 of the present invention.

[0023] Figure 4 The esterase 8GL004290 of the present invention decomposes the polyurethane film to produce adipic acid and butanediol.

[0024] Figure 5 The product concentrations of different polyester plastics produced by the esterase 8GL004290 of the present invention are as follows: (a) PUR; (b) PBS; (c) PBAT; (d) BHET; and (e) PET oligomers. DETAILED DESCRIPTION

[0025] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0026] It should be noted that the terms used here are only for describing specific embodiments, and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should also be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof. If the experimental methods of specific conditions are not indicated in the following specific embodiments, the conventional methods and conditions of molecular biology in the art are usually followed, and such techniques and conditions are fully explained in the literature. See, for example, Sambrook et al., the techniques and conditions described in "Molecular Cloning: Laboratory Manual", or according to the conditions recommended by the manufacturer.

[0027] In a typical embodiment of the present invention, an esterase is provided, which is named 8GL004290, and the esterase 8GL004290 has:

[0028] a1) the amino acid sequence shown in SEQ ID NO.1; or,

[0029] a2) an amino acid sequence having at least 90% sequence homology with SEQ ID NO.1.

[0030] In another embodiment of the present invention, the amino acid sequence of the esterase has at least 95% homology with SEQ ID NO.1; such as at least 95%, 96%, 97%, 98%, 99% homology.

[0031] In another specific embodiment of the present invention, a nucleic acid molecule is provided, wherein the nucleic acid molecule encodes the above-mentioned esterase.

[0032] In another specific embodiment of the present invention, a recombinant expression vector is provided, wherein the recombinant expression vector contains the above-mentioned nucleic acid molecule of the present invention.

[0033] In another specific embodiment of the present invention, the recombinant expression vector is obtained by effectively connecting the above-mentioned nucleic acid molecule to an expression vector, and the expression vector is any one or more of a viral vector, a plasmid, a phage, a cosmid or an artificial chromosome; the viral vector may include an adenovirus vector, a retrovirus vector or an adeno-associated virus vector, and the artificial chromosome includes a bacterial artificial chromosome, a phage P1-derived vector, a yeast artificial chromosome or a mammalian artificial chromosome; preferably, the expression vector is a plasmid, and the plasmid may be pET28a.

[0034] In another specific embodiment of the present invention, a host cell is provided, wherein the host cell contains the vector or chromosome described in the third aspect of the present invention in which the above nucleic acid molecule is integrated or is capable of expressing the above esterase.

[0035] The host cell may be a prokaryotic cell or a eukaryotic cell.

[0036] In another specific embodiment of the present invention, the host cell is any one or more of a bacterial cell or a fungal cell;

[0037] wherein the bacterial cell is any species within the genus Escherichia, Agrobacterium, Bacillus, Streptomyces, Pseudomonas or Staphylococcus;

[0038] In another specific embodiment of the present invention, the bacterial cell is Escherichia coli (such as Escherichia coli BL21 (DE3)), Agrobacterium tumefaciens (such as GV3101), Agrobacterium rhizogenes, Bacillus subtilis, Bacillus cereus or Pseudomonas, etc.

[0039] The fungal cells include yeast (such as Pichia pastoris) and the like.

[0040] In another specific embodiment of the present invention, a method for preparing the above-mentioned esterase is provided, comprising the steps of: culturing the above-mentioned host cell of the present invention to express the esterase; and isolating and purifying to obtain the esterase.

[0041] In another specific embodiment of the present invention, the use of the above-mentioned esterase, nucleic acid molecule, recombinant expression vector, host cell in the field of plastic hydrolysis, depolymerization, degradation and / or catalysis is provided.

[0042] Wherein, the plastic is a polyester plastic, and the polyester plastic includes any one or more of PET oligomer plastic, polyurethane plastic, bis(hydroxyethyl terephthalate) (BHET) plastic, PBAT plastic and PBS plastic.

[0043] In another specific embodiment of the present invention, a method for degrading polyester plastic is provided, the method comprising: applying the above-mentioned esterase or host cell to the polyester plastic for reaction.

[0044] The polyester plastic includes any one or more of PET oligomer plastic, polyurethane plastic, bis(hydroxyethyl) terephthalate (BHET) plastic, PBAT plastic and PBS plastic.

[0045] The present invention is further explained by the following examples, but they are not intended to limit the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0046] Example 1

[0047] The esterase 8GL004290 (its amino acid sequence is shown in SEQ ID NO.1) was screened out by genome sequencing analysis of the polyester-type plastic-degrading strains. Esterase 8GL004290, polyurethane-degrading enzymes Tcur_1278, Tcur0390, and PET-degrading enzymes LCC, TfCut2, and IsPETase all belong to the α / β hydrolase family and have polyester-type plastic-degrading activity. The phylogenetic tree is shown in Figure 1 shown.

[0048] The esterase 8GL004290 gene was constructed into the pET28a plasmid and transformed into E. coli BL21 (DE3) cells for expression. The E. coli expressing 8GL004290 was inoculated into LB medium containing 50 μg / ml kanamycin and cultured overnight at 37°C and 220 rpm. 1 ml of the culture was transferred to 50 ml of fresh LB medium containing 50 μg / ml kanamycin and cultured at 37°C and 220 rpm until the OD 600 to 0.8-1.0. After adding IPTG at a final concentration of 0.5 mM, the strain was cultured for 16 h to allow protein expression and secretion to the extracellular space. After purification and concentration, SDS-PAGE was performed. Figure 2 As shown, the esterase 8GL004290 band size is 35 KDa.

[0049] The substrate specificity of esterase 8GL004290 for acyl chains of different lengths was tested using the p-nitrophenol substrate pNPEs (C2–C16). Figure 3 As shown in the figure, esterase 8GL004290 showed a preference for short acyl chains (C2–C6), with the highest activity against p-nitrophenol hexanoate (C6), while the lowest activity against p-nitrophenol palmitate (C16). This suggests that 8GL004290, as an esterase, prefers to degrade low molecular weight ester-containing substrates.

[0050] After IPTG induction, Escherichia coli BL21 (DE3) expressing the esterase 8GL004290 gene was able to completely degrade the polyurethane film within 14 hours and accumulate the degradation products adipic acid and butanediol. Figure 4 shown.

[0051] Example 2

[0052] The protein concentration of esterase 8GL004290 concentrated to 1 mL by ultrafiltration was measured using a BCA kit, and the purified concentration of esterase 8GL004290 was 2.124 g / L. Take 500 μL of the reaction system (enzyme + Buffer), the buffer is a 20 mM Tris-HCl buffer at pH = 8, and the final enzyme concentration is 400 nmol / L. The substrates are PET oligomer powder (molecular weight 27,000), polyurethane powder (molecular weight 60,000), BHET powder, PBAT powder (molecular weight 120,000) and PBS powder (molecular weight: 100,000). The amount of PET oligomer powder, polyurethane powder, BHET powder, PBAT powder and PBS powder added to the substrate is 5 g / L, that is, 5 mg of substrate powder is added to each 1 mL of the reaction system. After incubation at 37°C for 24 hours, the reaction is terminated with 500 μL of acetonitrile, and the product concentration is measured using HPLC. Among them, the PUR powder products are adipic acid, butanediol and 2,6-TDA; the PET oligomer powder and nanoparticle products are BHET, MHET, TPA and EG; the BHET powder products are MHET, TPA and EG; the PBAT powder products are adipic acid, butanediol and TPA; the PBS powder products are succinic acid and butanediol.

[0053] Esterase 8GL004290 can rapidly degrade polyurethane powder and produce a large amount of butanediol and adipic acid, while also producing a small amount of degradation product MDA.

[0054] Esterase 8GL004290 degraded 5 g / L PBS powder to produce degradation products succinic acid and butanediol. The concentration of succinic acid was about 0.31 g / L, and the concentration of butanediol was 0.26 g / L.

[0055] Esterase 8GL004290 degraded 5g / L PBAT powder to produce adipic acid, butanediol and a small amount of TPA. The concentration of product butanediol was about 0.1g / L, the concentration of adipic acid was about 0.28g / L, and the concentration of substrate TPA was 0.04g / L.

[0056] Esterase 8GL004290 degraded 5g / L BHET powder to produce a large amount of MHET and EG. The product MHET concentration was about 1.7g / L and EG concentration was about 0.35g / L. At the same time, the BHET concentration was much lower than the blank control and dropped to 3.0g / L.

[0057] Esterase 8GL004290 degraded PET oligomer powder to produce a large amount of MHET and EG. The product MHET concentration was about 1.2 g / L and EG concentration was about 0.35 g / L. At the same time, the BHET concentration was much lower than the blank control and dropped to 0.75 g / L.

[0058] In summary, esterase 8GL004290 can degrade polyester plastics PUR, PBS, PBAT and polyester substrates BHET and PET oligomers.

[0059] The amino acid sequence involved in the present invention

[0060] Esterase 8GL004290

[0061] MQMHKFTLGCLSATALVALSVSAIAAPLPDTPGAPFPAVSSFDNSG

[0062] PYAVTSQSEGPSCRVYRPRTLGQGGVRHPIILWGNGTGTGPTTYSG

[0063] LLTHWASHGFVVAAAETSNAGTGREMLACLDYLVQESNRTYGTY

[0064] VGVLNTGRVGTSGHSQGGGGSIMAGRDDRVRATAPIQPYTIGLGH

[0065] DSSSQRNQRGPMFLMSGGADTIAVPYLNAQPVYSRANVPIFWGER

[0066] CYVSHFEPVGNGGEYRGPSTAWFRYQLMDDQSARSTFYGRLCRLCTSLLCSVERKGIELE(SEQ IDNO.1)

[0067] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. An esterase, named 8GL004290, characterized in that: The esterase 8GL004290 has: a1) the amino acid sequence shown in SEQ ID NO.1; or, a2) an amino acid sequence having at least 90% sequence homology with SEQ ID NO.

1.

2. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the esterase according to claim 1.

3. A recombinant expression vector, characterized in that: The recombinant expression vector contains the nucleic acid molecule according to claim 2.

4. A host cell, characterized in that The host cell has the recombinant expression vector or chromosome described in claim 3 integrated with the nucleic acid molecule described in claim 2, or is capable of expressing the esterase described in claim 1.

5. The host cell according to claim 4, characterized in that The host cell is a prokaryotic cell or a eukaryotic cell.

6. The host cell according to claim 5, characterized in that The host cell is any one or more of a bacterial cell or a fungal cell; wherein the bacterial cell is any species within the genus Escherichia, Agrobacterium, Bacillus, Streptomyces, Pseudomonas or Staphylococcus; Further, the bacterial cell is Escherichia coli (including Escherichia coli BL21 (DE3)), Agrobacterium tumefaciens, Agrobacterium rhizogenes, Bacillus subtilis, Bacillus cereus or Pseudomonas; The fungal cells include yeast (including Pichia pastoris).

7. A method for preparing the esterase according to claim 1, characterized in that: The method comprises the steps of: culturing the host cell according to any one of claims 4 to 6 to express the esterase; and separating and purifying to obtain the esterase.

8. Use of the esterase according to claim 1, the nucleic acid molecule according to claim 2, the recombinant expression vector according to claim 3, and the host cell according to any one of claims 4 to 6 in the field of plastic hydrolysis, depolymerization, degradation and / or catalysis.

9. The use according to claim 8, characterized in that The plastic is polyester plastic, and the polyester plastic includes any one or more of PET plastic, polyurethane plastic, bis(hydroxyethyl terephthalate) plastic, PBAT plastic and PBS plastic.

10. A method for degrading polyester plastics, characterized in that: The method comprises: applying the esterase according to claim 1 or the host cell according to any one of claims 4 to 6 to a polyester plastic for reaction.

Images