CN117821489A - Method for degrading renewable plastic by using recombinant yeast whole cells - Google Patents
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W30/00—Technologies for solid waste management
- Y02W30/50—Reuse, recycling or recovery technologies
- Y02W30/62—Plastics recycling; Rubber recycling
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Abstract
The invention discloses a method for degrading renewable plastics by utilizing recombinant yeast whole cells, which comprises the following steps: obtaining recombinant yeast strain for intracellular expression of heat-resistant cutinase, inducing and fermenting the recombinant yeast strain, and taking thalli to directly perform strain-induced fermentation on degradable plasticsDegradation is carried out, wherein the thermostable cutinase is LCC ICCG Degradable plastics include PBAT, PHA, PBSA, PBS and PHBV. The method is suitable for treating the degradable plastic at high temperature and normal temperature in soil, does not need protein purification and concentration processes, has low production cost, is simple and convenient to operate and high in degradation speed, and provides a new idea for green degradation of the degradable plastic.
Description
Technical Field
The invention relates to the technical field of plastic biodegradation and genetic engineering, in particular to a method for degrading renewable plastics by utilizing recombinant yeast whole cells.
Background
The plastic is an artificial macromolecular polymer, and is widely applied to aspects of human life due to the advantages of light weight, good chemical stability, good water resistance, good insulativity, good wear resistance, low price and the like. However, the stability and the wear resistance of the plastic make the plastic products difficult to degrade, and the environmental pollution problem is increasingly serious. In recent years, more environment-friendly degradable plastics, such as biodegradable plastics, photodegradable plastics and the like, are receiving more and more attention, and have great market potential. The degradable plastic is a plastic which can meet the use requirement of various properties of the product, has unchanged properties in the storage period and can degrade substances harmless to the environment under the natural environment condition after being used. The degradable plastic has better ductility, elongation at break, heat resistance and impact resistance, and simultaneously has excellent degradability, and is widely applied to the production of agricultural mulching films, plastic packaging bags, garbage bags, shopping bags in markets, disposable tableware and the like at present. The currently commonly used degradable plastics include bio-based polylactic acid (PLA), copolymers of 3-hydroxybutyrate and 3-hydroxyvalerate (PHBV) and the like, fossil-based polybutylene terephthalate adipate (PBAT) and the like.
Although biodegradable plastics can be decomposed by microorganisms in the environment, the degradation conditions of the biodegradable plastics commonly used at present are severe, such as industrial composting, and due to lack of supporting facilities, a huge amount of biodegradable plastics are difficult to treat at present, and the decomposition of PBAT and the like still needs about 60-120 days in an ideal composting environment, so that the final lodging of most of the biodegradable plastics is still incineration and landfill, and cannot really play an environment-friendly function. In addition, PBAT is made from petrochemical resources (including coal and petroleum) and requires more non-renewable petrochemicals to be consumed for production than traditional non-degradable plastics, so the production costs are high. Therefore, the enzyme for efficiently degrading the degradable plastic is excavated, the recycling of TPA is realized, the use cost of PBAT is effectively reduced, the green recycling technology of the renewable plastic is developed, and the method has important significance for environmental protection and reduction of the production cost of the renewable plastic.
In 2005, muller et al realized hydrolysis of PET plastics for the first time using a Thermobifida fusca-derived cutinase TfCut, as a result of subsequent sequence alignment to find a number of enzymes with PET degradation capability, such as LCC (leaf-branch compost cutinase), isPETase (Ideonella sakaiensis-F6), etc., which are hydrolases capable of degrading PET to mono (2-hydroxyethyl) terephthalate (MHET), terephthalic acid (TPA), and ethylene glycol at normal or high temperature. Wherein, LCC is a cutinase found from a metagenome of compost, has an optimal reaction temperature of 65 ℃, and has better heat resistance. Scientists obtain LCC mutants ICCG and WCCG through rational design and disulfide bond introduction, the hydrolytic activity of the LCC mutants ICCG and WCCG on PET plastics is greatly improved, the thermal stability is also remarkably improved, and the LCC mutants ICCG and WCCG can efficiently decompose the PET plastics at 72 ℃ to generate TPA. No PET plastic hydrolase is reported to be used for degrading degradable plastics at present after searching.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a method for degrading degradable plastics by utilizing recombinant yeast with high-efficiency intracellular expression of heat-resistant cutinase through whole cells, which has the advantages of no need of protein purification and concentration processes, low production cost, simple and convenient operation and high degradation speed.
The technical scheme of the invention is as follows:
a method for degrading renewable plastics by using recombinant yeast whole cells, comprising the following steps:
s1, obtaining a recombinant yeast strain for intracellular expression of heat-resistant cutinase, wherein the heat-resistant cutinase has the activity of degrading degradable plastics at the temperature of more than or equal to 70 ℃;
s2, after the recombinant yeast strain is induced and fermented, the thalli are taken to directly degrade the degradable plastic.
In the method of the present invention, the degradable plastic includes, but is not limited to: PBAT, poly (3-hydroxyalkanoic acid) (PHA), polybutylene butyrate adipate (PBSA), polybutylene succinate (PBS), PHBV, and the like. The experimental data of the invention show that the degradation capability of the method of the invention to different degradable plastics is obviously different, for example, in the degradation system of an embodiment of the invention, the degradation efficiency of the adopted recombinant yeast engineering strain to PHBV and PBS is highest, and the degradation effect to PHA is poor.
Preferably, in the above method, the thermostable cutinase is a mutant ICCG of cutinase LCC (LCC for short) ICCG ). The prior art shows that LCC ICCG Compared with LCC, the hydrolysis activity of cutinase LCC and mutants thereof on other plastics is not explored in the prior study, and the hydrolysis activity of the cutinase LCC and mutants thereof on other plastics is better; the invention discovers that LCC expressed by yeast recombination for the first time ICCG Has excellent degradation capability for some degradable plastics.
Preferably, in the above method, the yeast is pichia pastoris.
Preferably, in the above method, step S1 includes the following process:
s11, constructing a recombinant plasmid carrying one or more target gene copies;
s12, transforming the recombinant plasmid into yeast cells.
In one embodiment of the invention, the gene of interest is a thermotolerant cutinase LCC ICCG The sequence of the coding gene is shown as SEQ ID NO. 1; and when the copy number of the target gene is 3, LCC can be realized ICCG High intracellular expression in yeast.
More preferably, in the above method, step S11 specifically includes: according to a T5 exonuclease mediated cloning method, PCR amplification is respectively carried out on a heat-resistant cutinase encoding gene (namely a target gene) and an expression vector pHBM905M, products are recovered and mixed, the products are added with T5 exonuclease and then are incubated in an ice bath, escherichia coli is transformed and identified to obtain recombinant plasmids, and the recombinant plasmids carrying two or more target gene copies are constructed by a biological brick method.
Preferably, in the above method, step S2 uses methanol to induce expression of the recombinant yeast.
Preferably, in the above method, step S2 is either of the following two cases:
after the recombinant yeast strain is induced and fermented, the thalli are taken to directly degrade the degradable plastic in a solution environment with the temperature of more than or equal to 70 ℃; or alternatively, the first and second heat exchangers may be,
after induced fermentation of the recombinant yeast strain, the bacterial suspension is taken out, and the bacterial suspension is added into soil embedded with degradable plastics to react at room temperature (15-30 ℃).
One embodiment of the present invention shows that LCC is expressed intracellularly ICCG The recombinant pichia pastoris engineering bacteria completely decompose the PBAT plastic bag in 12h at a high temperature (72 ℃), and simultaneously the engineering bacteria can completely degrade the PBAT plastic bag in soil in 10 days at room temperature.
Compared with the prior art, the invention has the beneficial effects that:
(1) The invention utilizes pichia pastoris cell to efficiently express the mutant ICCG of cutinase LCC for the first time, and the expression mode is intracellular expression; LCC expressed relative to E.coli ICCG The invention utilizes LCC produced by recombinant pichia pastoris ICCG Has good heat stability, no obvious denaturation and inactivation in 48h under the high temperature environment of 72 ℃, and LCC expressed by escherichia coli ICCG Will rapidly denature at the same temperature.
(2) The invention discovers LCC prepared by recombination of pichia pastoris cells ICCG Has the ability to degrade renewable plastics (e.g. PBAT, PHA, PBSA, PBS, PHBV, etc.).
(3) LCC based on constructed intracellular expression ICCG The invention specifically develops two modes for degrading the degradable plastics; the first way is: the yeast cell is utilized to thermally crack under high temperature environment to release intracellular protein, the endogenous protein of the yeast is denatured and deactivated under heating under high temperature environment, and the LCC expressed exogenously is utilized ICCG Can still keep activity, thus degrading the degradable plastic; the second mode is as follows: adding the recombinant yeast suspension directly into soil to make it match withThe degradable plastic in the soil reacts at room temperature. The method does not need protein purification and concentration processes, has low production cost, simple and convenient operation and high degradation speed, and provides a new idea for green degradation of degradable plastics.
Drawings
FIG. 1 is a SDS-PAGE detection of sampled thalli every 12 hours in the fermentation process of the recombinant Pichia pastoris prepared by the invention.
FIG. 2 shows the recombinant expression of LCC of Pichia and E.coli in example 1 of the present invention ICCG Is a comparison of the thermal stability of (2).
FIG. 3 is a photograph showing the whole cell degradation of different kinds of degradable plastics by recombinant Pichia pastoris in example 2 of the present invention.
FIG. 4 is a graph showing the variation trend of the plastic quality in the process of degrading the degradable plastic by the recombinant Pichia pastoris whole cell in the embodiment 2 of the invention.
FIG. 5 is a photograph showing the degradation of PBAT plastic bags by recombinant Pichia pastoris whole cells in a high temperature environment (72 ℃) in example 3 of the present invention.
FIG. 6 is a photograph of a recombinant Pichia pastoris whole cell and a room temperature soil environment degradation PBAT plastic bag according to example 4 of the present invention.
Detailed Description
The technical scheme of the present invention will be clearly and completely described in the following with reference to the embodiments and the accompanying drawings. It should be understood that the detailed description and specific examples, while indicating and illustrating the invention, are not intended to limit the invention.
Unless defined otherwise, 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. The terms "comprising" and "having" and any variations thereof in the description and claims are intended to cover a non-exclusive inclusion.
The following examples are not to be construed as limiting the specific techniques or conditions described in the literature in this field or as per the product specifications; the reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
EXAMPLE 1 preparation of recombinant Pichia pastoris
This example provides for the efficient intracellular expression of cutinase LCC by the following steps ICCG The Pichia pastoris engineering bacteria are as follows:
(1) Cutinase LCC ICCG Construction of recombinant expression vectors.
Synthesis of LCC from Jin Kairui Co ICCG Coding gene (the sequence of which is shown as SEQ ID NO.1, the amino acid sequence of which is shown as SEQ ID NO. 2) is used for carrying out LCC on plastic hydrolase according to a T5 exonuclease mediated cloning method ICCG The encoding gene and the expression vector pHBM905BDM are respectively amplified by PCR, the products are recovered by agarose gel, the nucleic acid concentration is measured by a Nanodrop 8000 spectrophotometer, the gene fragment and the vector fragment are mixed according to the molar ratio of 3:1, the T5 exonuclease is added and then reacted in an ice-water mixture for 5min, and then the escherichia coli DH5 alpha is transformed.
Single colonies are respectively picked to 3mL of LB liquid medium added with ampicillin, the culture is carried out for a plurality of hours at 37 ℃, the whole cell PCR identification is carried out, the positive clone is sent to company for sequencing identification, and the obtained recombinant vector is named pHBM905BDM-ICCG. Then constructing a recombinant vector carrying three target gene copies by a biological brick method, and the recombinant vector is named pHBM905BDM-ICCG-3.
(2) Cutinase LCC ICCG Is expressed by the gene.
The recombinant vector pHBM905BDM-ICCG-3 is transformed into Pichia pastoris GS115 strain, and after screening and identification, the recombinant yeast is inoculated into a 5L fermentation tank for high-density fermentation, and 1% methanol is used for continuous induction for 5 days. 5mL of the supernatant was collected every 12 hours, and the cells were collected by centrifugation at 5000rpm and resuspended in an equal volume of 100mM KH 2 PO 4 In NaOH buffer (pH 8.0) and in a water bath at 72℃for 3h, the supernatant was centrifuged, an equal volume of 2 Xloadingbuffer was added, incubated at 95℃for 5-10min, and 10. Mu.L of the resulting mixture was loaded onto a 15% polyacrylamide gel. SDS-PAGE detection (FIG. 1) shows that the target protein is successfully expressed.
(3) Thermotolerant cutinase LCC ICCG Is used for detecting the thermal stability of the steel sheet.
Referring to the step (2), constructing a recombinant expression vector pET23a-LCC ICCG And transforming into Escherichia coli BL21C43 to obtain LCC-containing strain ICCG E.coli engineering bacteria.
LCC-containing solutions are obtained separately by shake flask fermentation ICCG Pichia pastoris and E.coli cells were collected by centrifugation at 5000rpm and resuspended in an equal volume of 100mM KH 2 PO 4 In NaOH buffer (pH 8.0). Then treating the LCC-containing material in a water bath at 72 DEG C ICCG The cells of Pichia pastoris and E.coli are sampled every 3 hours and the supernatant is centrifuged, an equal volume of 2 Xloadingbuffer is added, incubated at 95℃for 5-10min, and then 10. Mu.L is taken for SDS-PAGE.
The results show LCC in E.coli ICCG Unstable and rapidly denatured; pichia endogenous proteins are denatured by heating, and LCC ICCG But can withstand high temperatures and remain soluble (fig. 2A). The reason for this may be that the optimum growth temperature of E.coli is around 37℃and its endogenous protease is relatively thermostable, while the optimum growth temperature of Pichia pastoris is around 28℃and its endogenous protease is not thermostable, so that during heating, the endogenous protease of Pichia pastoris is relatively thermostable to LCC ICCG The influence of (c) is relatively small.
Further prolonging the treatment time, LCC expressed by pichia pastoris ICCG Shows good thermal stability, no significant denaturation deactivation was seen over 48h (fig. 2B).
EXAMPLE 2 recombinant Pichia pastoris whole cell degradation Plastic granules
The recombinant yeast prepared in example 1 was inoculated into a 5L fermenter for high-density fermentation, and cells were collected after continuous induction with 1% methanol for 5 days to obtain recombinant cells.
10mL of 100mM KH was added to a 50mL centrifuge tube 2 PO 4 NaOH buffer (pH 8.0), 25mg recombinant cells and 5 particles PBAT, PHA, PBS, PHBV, PBSA (about 0.08-0.18 g) were incubated in a 72℃water bath shaker.
The results are shown in FIGS. 3 and 4, where the above degradable plastic particles are degraded to different extents.
EXAMPLE 3 recombinant Pichia pastoris whole cell degradation PBAT Plastic bag
1L100mM KH was added to a 1L capacity blue-capped bottle 2 PO 4 NaOH buffer (pH 8.0), 5g recombinant cells and 1 PBAT plastic bag (about 13.65 g) were incubated in a 72℃water bath shaker.
As shown in FIG. 5, the plastic bag was completely degraded after 12 hours, and LCC-free was added ICCG In the control group of GS115 engineering bacteria, the plastic bags have no obvious change.
EXAMPLE 4 recombinant Pichia pastoris whole cell degradation of PBAT Plastic in soil
Adding appropriate amount of thallus into 100mM KH 2 PO 4 NaOH buffer to bring the concentration of the bacterial suspension to 1g/L and 2g/L. 20mL of the bacterial suspension was mixed with 5-10g of vermiculite or garden soil, then the PBAT plastic bag was buried in the soil, placed in a dish, then placed in a sealed bag for sealing, placed on a laboratory desktop, photographed every 3 days and the soil was replaced. As a result, as shown in FIG. 6, the plastic bag had been substantially decomposed within 10 days, and no LCC was added ICCG In the control group of GS115 engineering bacteria, the plastic bags are not changed obviously.
Pichia pastoris used in the invention has no function of degrading plastics, but LCC expressed by the Pichia pastoris ICCG Has high activity, and has certain activity even at normal temperature, LCC ICCG Slowly releasing pichia pastoris into soil, and gradually decomposing the plastic bag; the amount of cells required for normal temperature degradation in the soil environment was relatively large and the time was relatively long, compared with example 2.
In conclusion, the invention efficiently recombining and expressing the heat-resistant cutinase LCC by the pichia pastoris ICCG The resulting LCC ICCG Compared with the expression of escherichia coli, the recombinant pichia pastoris constructed by the method has better thermal stability, not only realizes the efficient degradation of degradable plastics (such as PBAT, PHA, PBS and the like) under the high-temperature condition, but also can degrade the degradable plastics in the soil environment at room temperature, and provides a new idea for the degradation treatment of the renewable plastics.
It should be noted that the above-mentioned embodiments are only some embodiments of the present invention, but not all embodiments, and are only used for illustrating the technical scheme of the present invention, not limiting; all other embodiments, which can be made by one of ordinary skill in the art without undue burden on the person of ordinary skill in the art based on embodiments of the present invention, are within the scope of the present invention.
Claims (10)
1. A method for degrading renewable plastics by using recombinant yeast whole cells, which is characterized by comprising the following steps:
s1, obtaining a recombinant yeast strain for intracellular expression of heat-resistant cutinase, wherein the heat-resistant cutinase has the activity of degrading degradable plastics at the temperature of more than or equal to 70 ℃;
s2, after the recombinant yeast strain is induced and fermented, the thalli are taken to directly degrade the degradable plastic.
2. The method of claim 1, wherein the degradable plastic comprises polybutylene terephthalate, poly 3-hydroxyalkanoic acid, polybutylene butyrate adipate, polybutylene succinate, a copolymer of 3-hydroxybutyrate and 3-hydroxyvalerate.
3. The method of claim 1, wherein the thermostable cutinase is LCC ICCG 。
4. The method of claim 1, wherein the yeast is pichia pastoris.
5. The method according to claim 1, wherein step S1 comprises the following process:
s11, constructing a recombinant plasmid carrying one or more target gene copies;
s12, transforming the recombinant plasmid into yeast cells.
6. The method according to claim 5, wherein the sequence of the target gene is shown in SEQ ID NO.1, and the copy number of the target gene is 3.
7. The method according to claim 5, wherein step S11 is specifically: and respectively carrying out PCR amplification on the heat-resistant cutinase encoding gene and the expression vector pHBM905M, mixing after recovering the products, adding T5 exonuclease, incubating in an ice bath, then converting escherichia coli, identifying to obtain recombinant plasmids, and constructing the recombinant plasmids carrying two or more target gene copies by a method of biological bricks.
8. The method of claim 1, wherein the inducing is performed with methanol.
9. The method according to claim 1, wherein step S2 is specifically: after the recombinant yeast strain is induced and fermented, the thalli are taken to directly degrade the degradable plastic in a solution environment with the temperature of more than or equal to 70 ℃.
10. The method according to claim 1, wherein step S2 is specifically: after the recombinant yeast strain is induced and fermented, the bacterial suspension is taken out, and is added into soil embedded with degradable plastics to react at room temperature.
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