Esterase and its application in plastic degradation

Abstract

The present application belongs to the technical field of enzyme engineering and plastic degradation, and particularly relates to an esterase, a preparation method thereof and application thereof in plastic degradation. Genome sequencing analysis is performed on a screened polyester plastic degrading strain, and an esterase 8GL004290 is successfully screened, which belongs to the alpha / beta hydrolase family. It is verified through experiments that the polyester degrading enzyme provided by the present application can not only degrade polyester model small molecule substrates, but also exhibits good degradation capacity for different polyester plastics. The esterase 8GL004290 has the highest degradation activity on a polyurethane substrate, and the substrate types are widely covered. Therefore, the present application has extremely important application value for the biodegradation treatment of mixed plastic waste.

Description

TECHNICAL FIELD

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

[0002] The information disclosed in this Background section is for the purpose of generally presenting the context of the application. The information disclosed in this Background section is not to be necessarily taken as an acknowledgement or any form of suggestion that this information forms part of 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 class of high molecular materials polymerized by ester bonds, mainly including polyethylene terephthalate (PET), polyurethane (PU), polylactic acid (PLA), polybutylene adipate / terephthalate (PBAT), polybutylene succinate (PBS) and polyhydroxyalkanoate (PHA) and the like. They have excellent mechanical properties, transparency and chemical resistance, and are widely used in packaging, textiles, agriculture and medical treatment and the like. However, most of the polyester plastics are difficult to degrade quickly under natural conditions at present, and there are problems of environmental pollution, resource waste and the like in recycling treatment by landfill, incineration and the like, and the use of enzymatic degradation of polyester plastics has great application potential.

[0004] Esterases widely exist in nature, belong to the α / β hydrolase superfamily, and play an important role in the process of cell lipid metabolism and protein biosynthesis. They have high similarity in core structural domains and catalytic mechanisms, and form a catalytic triad composed of serine, histidine and negatively charged amino acids (usually aspartic acid or glutamic acid) to catalyze the hydrolysis of ester bonds. At present, some esterases can degrade the ester bonds in polyester plastics. Leaf-branch compost cutinase LCC has degradation activity to PET and PBAT. In 2017, Potocki Veronese isolated 26 strains showing esterase activity from the rumen microbiome, and isolated a new esterase CE_Ubrb with polyurethane degradation activity from strain 44I12. In 2019, Avérous selected esterase E3576 from 50 hydrolytic enzymes to degrade polyester polyurethane. However, the inventors found that the esterases reported above have single type of polyester plastic degradation, and do not have broad-spectrum degradation characteristics to polyester plastics. SUMMARY

[0005] In view of the problems in the prior art, the present application provides an esterase and its application in plastic degradation. Specifically, the present application analyzes the genome sequencing of the screened polyester plastic degrading strain, successfully screens an esterase 8GL004290, which belongs to the alpha / beta hydrolase family and has broad-spectrum degradation activity of polyester plastic. Based on the above research, the present application is completed.

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

[0007] In a first aspect, the present application 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] In a second aspect, the present application provides a nucleic acid molecule encoding the esterase of the first aspect.

[0011] In a third aspect, the present application provides a recombinant expression vector containing the nucleic acid molecule of the second aspect.

[0012] In a fourth aspect, the present application provides a host cell containing the vector of the third aspect or having the nucleic acid molecule of the second aspect integrated into the chromosome or expressing the esterase of the first aspect.

[0013] In a fifth aspect, the present application provides a method for preparing the esterase, comprising the steps of: culturing the host cell of the fourth aspect to express the esterase; and isolating and purifying the esterase.

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

[0015] 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] In a seventh aspect, the present application provides a method for degrading a polyester plastic, which comprises: applying the esterase of the first aspect or the host cell of the fourth aspect to the polyester plastic for reaction.

[0017] The above one or more technical solutions have the beneficial technical effects that:

[0018] The above technical solutions provide an esterase, a preparation method thereof and application in plastic degradation. Specifically, the polyester-degrading enzyme provided by the above technical solutions can not only degrade polyester model small molecule substrates, but also has good degradation capacity for different polyester plastics. The esterase 8GL004290 has the highest polyurethane substrate degradation activity, and the substrate types are widely covered. Therefore, the application has extremely important application value for the biodegradation treatment of mixed plastic waste. BRIEF DESCRIPTION OF DRAWINGS

[0019] The accompanying drawings, which form a part of the present description, illustrate the present application and, together with the written description, serve to explain the principles of the present application. The drawings are illustrative only and are not intended to limit the present application.

[0020] Figure 1 A phylogenetic tree of the esterase 8GL004290 of the present application.

[0021] Figure 2 SDS-PAGE results of the esterase 8GL004290 of the present application.

[0022] Figure 3 Substrate specificity of the esterase 8GL004290 of the present application.

[0023] Figure 4 Decomposition of polyurethane film by the esterase 8GL004290 of the present application to produce adipic acid and butanediol.

[0024] Figure 5 Product concentration of different polyester plastics by the esterase 8GL004290 of the present application; wherein (a) PUR; (b) PBS; (c) PBAT; (d) BHET; (e) PET oligomer. DETAILED DESCRIPTION

[0025] It should be noted that the following detailed description is illustrative only and is not intended to limit the present application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0026] It is to be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms "comprises" and / or "comprising," when used in this specification, specify the presence of stated features, steps, operations, elements, components, and / or groups thereof, but do not preclude the presence or addition of one or more other features, steps, operations, elements, components, and / or groups thereof. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the application pertains. Methods and materials are described herein for use in the present application; other methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present application. Throughout the specification, any and all references to a method comprising a step should be interpreted as including the possibility of additionally taking that step in an alternative order, and / or omitting that step.

[0027] In one exemplary embodiment of the present application, an esterase is provided, which is named as 8GL004290, the esterase 8GL004290 has:

[0028] a1) an amino acid sequence as 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 exemplary embodiment of the present application, the amino acid sequence of the esterase has at least 95% homology compared with SEQ ID NO. 1; such as having at least 95%, 96%, 97%, 98%, 99% homology.

[0031] In another exemplary embodiment of the present application, a nucleic acid molecule is provided, which encodes the above-mentioned esterase.

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

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

[0034] In another exemplary embodiment of the present application, a host cell is provided, which contains the vector or chromosome of the third aspect of the present application, or has the above-mentioned nucleic acid molecule integrated or is capable of expressing the above-mentioned esterase.

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

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

[0037] The bacterial cell is any one of Escherichia, Agrobacterium, Bacillus, Streptomyces, Pseudomonas or Staphylococcus.

[0038] In another specific embodiment of the present application, 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 cell includes yeast (such as Pichia pastoris), etc.

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

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

[0042] 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 application, a method for degrading a 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 application is further explained by the following examples, which do not limit the present application. It should be understood that these examples are used to illustrate the present application but not to limit the scope of the present application.

[0046] Example 1

[0047] The screened polyester plastic degrading strain was subjected to genome sequencing analysis, and esterase 8 GL004290 (the amino acid sequence of which is shown as SEQ ID NO. 1) was screened. Esterase 8 GL004290 belongs to the alpha / beta hydrolase family together with polyurethane degrading enzyme Tcur_1278, Tcur0390, and PET degrading enzymes LCC, TfCut2 and IsPETase, and has a polyester plastic degrading activity, and its phylogenetic tree is shown as Figure 1 .

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

[0049] The substrate specificity of esterase 8 GL004290 for acyl chains of different lengths was tested using the p-nitrophenol substrate pNPEs (C2-C16). The results are shown in Figure 3 , esterase 8 GL004290 showed a preference for short acyl chains (C2-C6), with the highest activity for p-nitrophenyl hexanoate (C6) and the lowest esterase detection activity for p-nitrophenyl palmitate (C16). This indicates that 8 GL004290 as an esterase is more inclined to degrade low molecular weight ester bond-containing substrates.

[0050] After IPTG-induced expression of the E. coli BL21 (DE3) expressing the esterase 8 GL004290 gene, the E. coli BL21 (DE3) was able to completely degrade the polyurethane film within 14 hours, and accumulated the degradation products adipic acid and butanediol. The results are shown in Figure 4 .

[0051] Example 2

[0052] The esterase 8 GL004290 was concentrated to 1 mL using the BCA kit to measure the protein concentration, and the esterase 8 GL004290 purification concentration was 2.124 g / L. 500 μL of the reaction system (enzyme + buffer) was taken, the buffer was 20 mM Tris-HCl buffer with pH = 8, and the final concentration of the enzyme was 400 nmol / L. The substrates were 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 substrate addition amount of PET oligomer powder, polyurethane powder, BHET powder, PBAT powder and PBS powder was 5 g / L, that is, 5 mg of substrate powder was added per 1 mL of reaction system. After incubation at 37°C for 24 h, the reaction was terminated using 500 μL of acetonitrile, and the product concentration was measured using HPLC. Among them, the PUR powder products were adipic acid, butanediol and 2,6-TDA, respectively; the PET oligomer powder and nanoparticle products were BHET, MHET, TPA and EG, respectively; the BHET powder products were MHET, TPA and EG, respectively; the PBAT powder products were adipic acid, butanediol and TPA, respectively; and the PBS powder products were butanedioic acid and butanediol, respectively.

[0053] The esterase 8 GL004290 can quickly degrade the polyurethane powder and produce a large amount of butanediol and adipic acid, while producing a small amount of degradation product MDA.

[0054] The esterase 8 GL004290 degrades 5 g / L PBS powder to produce degradation products butanedioic acid and butanediol, with a product butanedioic acid concentration of about 0.31 g / L and a butanediol concentration of 0.26 g / L.

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

[0056] The esterase 8 GL004290 degrades 5 g / L BHET powder to produce a large amount of MHET and EG, with a product MHET concentration of about 1.7 g / L and an EG concentration of about 0.35 g / L, while the BHET concentration is much smaller than the blank control and decreases to 3.0 g / L.

[0057] The esterase 8 GL004290 degrades the PET oligomer powder to produce a large amount of MHET and EG, with a product MHET concentration of about 1.2 g / L and an EG concentration of about 0.35 g / L, while the BHET concentration is much smaller than the blank control and decreases to 0.75 g / L.

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

[0059] Amino acid sequence involved in the present application

[0060] Esterase 8 GL004290

[0061] MQMHKFTLGCLSATALVALSVSAIAAPLPDTPGAPFPAVSSFDNSG

[0062] PYAVTSQSEGPSCRVYRPRTLGQGGVRHPIILWGNGTGTGPTTYSG

[0063] LLTHWASHGFVVAAAETSNAGTGREMLACLDYLVQESNRTYGTY

[0064] VGVLNTGRVGTSGHSQGGGGSIMAGRDDRVRATAPIQPYTIGLGH

[0065] DSSSQRNQRGPMFLMSGGADTIAVPYLNAQPVYSRANVPIFWGER

[0066] CYVSHFEPVGNGGEYRGPSTAWFRYQLMDDQSARSTFYGRLCRLCTSLLCSVERKGIELE (SEQ ID NO. 1)

[0067] Finally, it should be noted that: the above only for the preferred embodiments of the present application, and not for limiting the present application, although the foregoing detailed description of the present application is made with reference to the foregoing embodiments, for those skilled in the art, it still can be modified, or part of the technical features of the equivalent replacement of the technical solutions described in the foregoing embodiments. Any modification, equivalent replacement, improvement, etc. within the spirit and principles of the present application, shall be included within the scope of protection of the present application.

Claims

1. An esterase designated 8GL004290, characterized in that, The amino acid sequence of the esterase 8GL004290 is shown as SEQ ID NO.

1.

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

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

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

5. The host cell of claim 4, wherein the nucleic acid molecule is integrated into the host cell genome. The host cell is a prokaryotic cell or a eukaryotic cell.

6. The host cell of claim 5, wherein The host cell is any one or more of a bacterial cell, a fungal cell.

7. The host cell of claim 6, wherein The bacterial cell is any species within the genus Escherichia, Agrobacterium, Bacillus, Streptomyces, Pseudomonas or Staphylococcus.

8. The host cell of claim 7, wherein The bacterial cell is Escherichia coli, Agrobacterium tumefaciens, Agrobacterium rhizogenes, Bacillus subtilis, Bacillus cereus or Pseudomonas.

9. The host cell of claim 8, wherein The Escherichia coli is Escherichia coli BL21 (DE3).

10. The host cell of claim 6, wherein The fungal cell is a yeast.

11. The host cell of claim 10, wherein The yeast is Pichia pastoris.

12. A method for preparing the esterase of claim 1, characterized by, The method comprises culturing the host cell of any one of claims 4-11, thereby expressing the esterase; and isolating and purifying the esterase.

13. Use of the esterase of claim 1, the nucleic acid molecule of claim 2, the recombinant expression vector of claim 3, or the host cell of any one of claims 4-11 in the field of plastic degradation and / or catalysis. The plastic is a polyester plastic, and the polyester plastic is any one or more of PET plastic, polyurethane plastic, polyethylene terephthalate glycol plastic, PBAT plastic and PBS plastic.

14. Use of the esterase of claim 1, the nucleic acid molecule of claim 2, the recombinant expression vector of claim 3, or the host cell of any one of claims 4-11 in the field of plastic hydrolysis and / or catalysis. The plastic is a polyester plastic, and the polyester plastic is any one or more of PET plastic, polyurethane plastic, polyethylene terephthalate glycol plastic, PBAT plastic and PBS plastic.

15. Use of the esterase of claim 1, the nucleic acid molecule of claim 2, the recombinant expression vector of claim 3, or the host cell of any one of claims 4-11 in the field of plastic depolymerization and / or catalysis. The plastic is a polyester plastic, and the polyester plastic is any one or more of PET plastic, polyurethane plastic, polyethylene terephthalate glycol plastic, PBAT plastic and PBS plastic.

16. A method of degrading a polyester-type plastic, characterized by, The method comprises applying the esterase of claim 1 or the host cell of any one of claims 4-11 to a polyester plastic for reaction. The plastic is a polyester plastic, and the polyester plastic is any one or more of PET plastic, polyurethane plastic, polyethylene terephthalate glycol plastic, PBAT plastic and PBS plastic.

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