CN114425136A

CN114425136A - Method for treating polyethylene by combining peroxide and cutinase

Info

Publication number: CN114425136A
Application number: CN202210027522.5A
Authority: CN
Inventors: 吴敬; 王蕾; 孔德民; 宿玲恰; 颜正飞; 陈晓倩
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2022-01-11
Filing date: 2022-01-11
Publication date: 2022-05-03
Anticipated expiration: 2042-01-11
Also published as: CN114425136B

Abstract

The invention discloses a method for treating polyethylene by combining peroxide and cutinase, belonging to the fields of environmental science, materials science and enzyme engineering. The invention provides a novel method for treating polyethylene by combining m-chloroperoxybenzoic acid and cutinase and optimizing LDPE (low-density polyethylene) and cutinase^mThe proportion of CPBA and the optimized enzymolysis reaction time can reduce the weight-average molecular weight of the treated polyethylene from the initial 210000Da to 3862Da, thereby providing a new idea for the biodegradation of the polyethylene.

Description

Method for treating polyethylene by combining peroxide and cutinase

Technical Field

The invention relates to a method for treating polyethylene by combining peroxide and cutinase, belonging to the fields of environmental science, materials science and enzyme engineering.

Background

Polyethylene is a high molecular polymer formed by connecting carbon-carbon single bonds as a skeleton, is the simplest polyolefin high polymer, and is widely used in daily life. However, the attendant consequences are the widespread distribution of polyolefin wastes and their enormous pressure on the ecosystem and biological health. Therefore, the degradation of polyethylene wastes has been a problem to the world. Compared with physical or chemical methods, the biological method has the advantages of low energy consumption and environmental protection in the aspect of degrading polyolefin.

Due to the higher bond energy of the carbon-carbon single bond (330-^-1) The bio-enzyme catalyzes the degradation of the polyethylene and needs to be activated firstly. At present, the oxidation treatment means mainly comprises the treatment of UV, strong acid and strong base or inorganic oxidant, and the generated functional groups mainly comprise hydroxyl, carbonyl, carbon-carbon double bond and the like. Based on the above pretreatment, it has been reported that a part of oxidoreductase is capable of degrading polyethylene. Laccase (Lac) can degrade UV oxidized polyethylene, and the weight average molecular weight loss is about 20%; lignin peroxidase (Lip) and manganese peroxidase (Mnp) can degrade K in black liquor medium₂Cr₂O₇Oxidized polyethylene; the weight average molecular weight loss after UV oxidized polyethylene catalyzed by latex scavenger protein (Lcp) was about 42.02%. Although the above pretreatment method can effectively improve the degradation action of the biological enzymes, no study has been made to clarify the mechanism of degrading polyethylene with oxygen-containing groups by these enzymes.

Disclosure of Invention

Aiming at the problems existing at present, the invention provides a method for treating polyethylene by combining peroxide and cutinase, wherein m-chloroperoxybenzoic acid (I), (II), (III), (IV), (V), (^mCPBA) to catalyze the Baeyer-Villiger reaction, ester bonds are added to the carbon-carbon single bonds of the main chain of the polyethylene, and the ester bonds are degraded by using a keratinase catalytic hydrolysis reaction from Thermobifida fusca, so that the aim of degrading the polyethylene is fulfilled.

In one embodiment, the mass ratio of the m-chloroperoxybenzoic acid to the polyethylene (LDPE) is (1-5): 1.

in one embodiment, the Thermobifida fusca-derived cutinase has an amino acid sequence shown in SEQ ID No. 1.

In one embodiment, the cutinase is catalyzed for a period of greater than or equal to 1 day, 5 days, or 7 days.

In one embodiment, the method comprises the steps of:

(1) dissolving LDPE in a 1,3, 5-trichlorobenzene-dichloroethane mixed solution, and stirring and dissolving at 80-120 ℃ for 10-14 h; adding^mCatalyzing CPBA at 80-120 ℃ for 6-10 h; adding methanol to precipitate LDPE, filtering, washing with methanol for 3-4 times to remove solvent, and drying to constant weight to obtain oxidized polyethylene containing ester bond;

(2) and (3) adding the oxidized polyethylene containing ester bonds prepared in the step (2) into the cutinase enzyme solution, and reacting at 55-65 ℃ for at least 1 day.

In one embodiment, the 1,3, 5-trichlorobenzene-dichloroethane mixed solution is prepared by mixing 1,3, 5-trichlorobenzene and dichloroethane in an equal volume ratio.

In one embodiment, the LDPE is dissolved in a 1,3, 5-trichlorobenzene-dichloroethane mixed solution in a ratio of 1 (10-20).

In one embodiment, the cutinase in step (2) has the amino acid sequence shown as SEQ ID No. 1.

In one embodiment, the cutinase obtained in step (2) is reacted at 60 ℃ and 150rpm for 1-7 days.

In one embodiment, the steps of the method are:

(1) adding LDPE into a 1,3, 5-trichlorobenzene-dichloroethane mixed solution mixed in a volume ratio of 1:1, stirring and dissolving for 12 hours at 100 ℃ to ensure that the concentration of the dissolved LDPE is 50 g/L; adding^mCPBA, order^mThe proportion of the CPBA to the LDPE is (1-5): 1, catalyzing at 100 ℃ for 8 hours; adding methanol with the same volume to precipitate LDPE, filtering, washing with methanol for 3-4 times to remove the solvent, and subsequently placing the sample in an oven to be dried to constant weight to obtain oxidized polyethylene containing ester bonds;

(2) and (2) taking the oxidized polyethylene obtained in the step (1) as a substrate, adding enzyme of T.fusca cutinase 4200U/g of oxidized polyethylene, and reacting for 1-7 d at 60 ℃ and 150 rpm.

In one embodiment, the polyethylene-containing product includes, but is not limited to, low density polyethylene, including cling film, mulching film, or disposable plastic bags.

The invention also provides application of the method in waste treatment or garbage recycling.

Has the beneficial effects that:

the invention provides a novel method for treating polyethylene by combining m-chloroperoxybenzoic acid and cutinase and optimizing LDPE (low-density polyethylene) and cutinase^mThe proportion of CPBA and the optimized enzymolysis reaction time can reduce the weight-average molecular weight of the treated polyethylene from the initial 210000Da to 3862Da, thereby providing a new idea for the biodegradation of the polyethylene.

Drawings

FIG. 1 shows FT-IR measurements of samples (top to bottom in sequence) after treatment with polyethylene, oxidized polyethylene and cutinase; wherein the curves are polyethylene, oxidized polyethylene and cutinase from top to bottom in sequence.

Detailed Description

The experimental reagent comprises polyethylene, 1,2, 4-trichlorobenzene (1,2,4-TCB), Dichloroethane (DCE), m-chloroperoxybenzoic acid ()^mCPBA), Sodium Dodecyl Sulfate (SDS).

LB culture medium: tryptone 10 g.L^-1Yeast powder 5 g.L^-1Sodium chloride 10 g.L^-1

TB culture medium: tryptone 12 g.L^-1Yeast powder 24 g.L^-1Glycerol 5 g.L^-1，KH₂PO₄ 2.31g·L^-1，K₂HPO₄·3H₂O 16.43g·L^-1。

The samples after the oxidation treatment and the hydrolysis treatment were analyzed by Fourier transform infrared spectroscopy (FT-IR) and hydrogen nuclear magnetic resonance spectroscopy (F)¹HNMR) and high temperature gel permeation chromatography (HT-GPC). The specific parameters are as follows: (1) functional groups on the surface of polyethylene samples were detected using a NEXUS Fourier transform Infrared spectrometer (Nicolet, USA) to establish their infrared spectra. The resolution is better than 0.5cm < -1 >, the signal-to-noise ratio is better than 44000: 1, spectral range set to 4000cm^-1-400cm^-1The number of scans was 32. In a transmittance (% T) mode,The same scale shows the case of the absorption peak of the sample analyzed in the mode. The integral information of different absorption peaks was collected in absorbance mode. (2) The proportion of oxygenated functions and the conversion factor (TF) in the sample were examined using an AVANCE NEO 700MHz nuclear magnetic resonance spectrometer (Bruker, Germany). The specific method comprises the following steps: sample (final concentration 5-15mg mL)^-1) Dissolving in deuterated o-dichlorobenzene at 100 ℃, testing at 100 ℃, scanning for 16 times, and scanning for-1-13 ppm. Wherein the conversion factor (TF) is defined as the ratio of functional groups of hydroxyl group (A), carbonyl group (K) and ester bond (E) contained per 100 degrees of polymerization. (3) Polyethylene samples were tested for weight average molecular weight (M) using HLC-8321 high temperature gel permeation chromatography (Eco-SEC, Japan)_w) Number average molecular weight (M)_n) Polydispersity (PDI) and dW/d (LogM) -Log (M) differential distribution curves. The specific method comprises the following steps: polyethylene samples were dissolved in 1,2, 4-trichlorobenzene (final concentration 2mg/mL), using TSK gel GMH (S) HT2, TSKgel GMH (S) HT2 and TSKgel H HFRC tandem chromatography columns and TSK guard column chromatography H (S) HT2(21489), at a detection temperature of 145 ℃, at a sample loading of 300. mu.L, eluting the solution in 1,2, 4-dichlorobenzene for a time of 50min, at an elution flow rate of 1.0 mL/min.

The detection method of enzyme activity comprises the following steps:

Tris-HCl buffer (10mM pH 7.0): 1.210g of Tris and 0.584g of NaCl are accurately weighed, about 800mL of deionized water is added, the mixture is fully stirred and dissolved, the pH value is adjusted to 8.0 by HCl, and the volume is adjusted to 1000 mL.

Substrate (50mmol/L p-nitrobenzoate solution): accurately weighing 0.1046g of p-nitrobenzyl butyrate, metering to 10mL by using acetonitrile, and storing at-20 ℃.

Preparing a p-nitrophenol standard curve: 13.9mg of p-nitrophenol is weighed, the solution is diluted to 1000mL by 10mM of Tris-HCl buffer solution with the pH value of 7.0 to prepare 100 mu mol/L p-nitrophenol mother solution, and the solution is diluted to 0, 20, 40, 60, 80 and 100 mu mol/L by 10mM of Tris-HCl buffer solution with the pH value of 7.0. The absorbance at a wavelength of 405nm was measured on a spectrophotometer using a 0.5cm glass cuvette, and a standard curve a ═ a × C + b was plotted using the p-nitrophenol concentration C as the abscissa and the absorbance a as the ordinate.

1.5mL of Tris-HCl buffer (preincubated at 37 ℃ for 10min) was accurately pipetted into a 0.5cm glass cuvette and zeroed at the absorption wavelength of 405 nm. Taking 1.44mL of Tris-HCl buffer solution to a 0.5cm quartz cuvette, taking 30 mu L of diluted enzyme solution to be detected, adding the diluted enzyme solution to be detected to the quartz cuvette, taking 30 mu L of substrate solution, adding the substrate solution to the quartz cuvette, shaking uniformly, immediately putting the substrate solution into a visible spectrophotometer, and detecting an A value at an absorption wavelength of 405 nm. The A value was recorded every 5 seconds with a reaction time of 1 minute.

And (3) calculating:

in the formula: k: the slope of a curve formed by different time A values and time (min) measured by enzyme reaction;

V₁: reaction volume (mL);

V₂: enzyme addition (mL);

n: dilution factor.

The enzyme activity is defined as the enzyme amount of the p-nitrophenol which catalyzes the hydrolysis of the p-nitrobenzoate to generate 1 mu mol per min under the environment of 65 ℃ and pH8.0 as one enzyme activity unit (U).

Example 1: preparation of a T.fusca cutinase

(1) Inserting the synthesized cutinase nucleotide sequence shown as SEQ ID NO.2 between Nde I site and Xho I site of pET24a through restriction enzyme to obtain recombinant plasmid pET24 a-TfC; then the constructed recombinant plasmid pET24a-TfC is transformed into Escherichia coli (Escherichia coli) JM109 to obtain a transformation product; coating the transformation product on an LB solid culture medium (containing 40 mu g/mL kanamycin), and carrying out inverted culture in a constant-temperature incubator at 37 ℃ for 8-12h to obtain a transformant; and (3) selecting a transformant, inoculating the transformant into an LB liquid culture medium, performing shake-flask culture for 8-12h at 37 ℃ and 120-180 rpm, extracting plasmids, performing sequencing verification, and preserving strains with correct verification in 30% glycerol.

(2) Inoculating the recombinant Escherichia coli expressing T.fusca cutinase obtained in the step (1) into 10mL of LB liquid medium according to the inoculation amount of 0.1%, and adding 40ng mu L^-1Kanamycin (1). At 37 ℃ and 200rpmAfter the culture for 8-12h, the cells were inoculated into 50mL TB medium in a 5% inoculum size, and 40 ng. mu.L of the medium was added^-1Kanamycin. Culturing at 37 deg.C and 200rpm for 2h to OD₆₀₀After changing to 0.8, IPTG was added to a final concentration of 0.4mM, and the mixture was incubated at 25 ℃ for 48 hours. Centrifuging at 8000rpm for 15min, and collecting the supernatant as crude enzyme solution with enzyme activity of 110.1U/mL.

EXAMPLE 2 Oxidation treatment of polyethylene with m-Chloroperoxybenzoic acid

(1) System optimization

By using^mCPBA pretreats polyethylene and carries out oxidation treatment on LDPE dissolved in a mixed solution of 1,2,4-TCB and DCE. The method comprises the following specific steps: (1) dissolving 50g/L LDPE in a 1,3, 5-trichlorobenzene-dichloroethane mixed solution in a volume ratio of 1:1, and stirring and dissolving at 100 ℃ for 12 hours; (2) adding of^mCPBA and catalyzing for 8 hours at 100 ℃; (3) adding methanol with the same volume to precipitate LDPE, filtering, washing with methanol for 3-4 times to remove the solvent, and then placing the sample in an oven to be dried to constant weight to obtain the sample, namely the oxidized polyethylene containing ester bonds.

Wherein, step (2) designs LDPE respectively:^mthe mass ratio of CPBA is 1:1, 1:4 and 1: 5.

The molecular weights of the oxidized polyethylene samples after the reaction were measured, respectively, and the results showed that the weight average molecular weights of the oxidized polyethylene samples prepared at catalyst ratios of 1:1, 1:4, and 1:5 were 26936Da, 11053Da, and 7121Da, respectively, and the number average molecular weights were 2033Da, 581Da, and 214Da, respectively.

(2) For LDPE:^mdetection of product with mass ratio of CPBA of 1:5

Oxidized polyethylene containing ester bonds was prepared as follows: dissolving 50g/L LDPE in a 1,3, 5-trichlorobenzene-dichloroethane mixed solution in a volume ratio of 1:1, and stirring and dissolving at 100 ℃ for 12 hours; (2) adding^mCPBA, order^mThe ratio of CPBA to LDPE added in step (1) is 5: 1, catalyzing at 100 ℃ for 8 hours; (3) adding methanol with the same volume to precipitate LDPE, filtering, washing with methanol for 3-4 times to remove the solvent, and then placing the sample in an oven to be dried to constant weight to obtain the oxidized polyethylene containing ester bonds and catalyzed into light yellow powder. The FT-IR results show that, compared to polyethylene,an-C ═ O stretching peak of 1770-^-1And 1711cm^-1Corresponding to the ester carbonyl and ketone carbonyl groups, respectively.¹H NMR results showed a ratio of different functional groups in the sample of a: E: K: 22:36:42, TF: 4.36. The HT-GPC results showed that the weight average molecular weight and the number average molecular weight of the oxidized polyethylene were significantly reduced compared to polyethylene (Mw 7121; Mn 214). The above results show that it is possible to obtain,^mthe CPBA catalyst system can activate a carbon-carbon single bond of polyethylene in a dissolved state, and add an oxygen-containing functional group (hydroxyl group, carbonyl group, and ester bond).

Example 3: thermobifida fusca-derived cutinase-treated oxidized polyethylene

(1) System optimization

Using the oxidized polyethylene obtained in the step (2) of example 1 as a substrate, and the amount of the T.fusca cutinase added was 4200U/g oxidized polyethylene, 1d, 3d or 7d were reacted at 60 ℃ and 150rpm, respectively. And after the catalysis is finished, filtering the mixture in a 500-mesh nylon net to obtain a product sample, sequentially performing ultrasonic treatment on the product sample by using 2% (w/v) SDS, deionized water and 75% ethanol for 30min, and placing the product sample in an oven to dry the product sample to obtain the oxidized polyethylene after the cutinase treatment. The weight average molecular weights of the oxidized polyethylene samples after the completion of the reaction were measured, respectively, and the results showed that the weight average molecular weights of the prepared oxidized polyethylene samples were 4862Da, 4257Da, and 3779Da, respectively, and the number average molecular weights were 29Da, 27Da, and 33Da, respectively, for reaction times of 1d, 3d, and 7d, respectively.

(2) Product detection

And detecting the degradation products after 7d of treatment. Compared to oxidized polyethylene, the color of the degradation products is darker than oxidized polyethylene. The FT-IR results show that 1650-^-1And 3700 to 3000cm^-1In the range of the absorption peaks of carboxylate and hydroxyl, 1770-1640cm in the cutinase-treated oxidized polyethylene^-1the-C ═ O stretching peak of (a) is lower than that of the ketone carbonyl group, and the contents of the two are close to each other in the oxidized polyethylene. At the same time, the user can select the desired position,¹h NMR showed the ratio of different functional groups A, E, K in the oxidized polyethylene after cutinase treatment was 28:28:44 and TF 3.95. Cutinase treated oxygen compared to oxidized polyethyleneThe ester bond content in the polyethylene is reduced.¹The results of HNMR further demonstrate that_CPBAPart of the ester bonds in PE are hydrolyzed by cutinase to produce new species. The HT-GPC results showed that the weight average molecular weight of the cutinase-treated oxidized polyethylene was reduced to 47.4% compared to the oxidized polyethylene. Overall, the t.fusca cutinase is able to undergo hydrolysis reactions with ester bonds in oxidized polyethylene and effectively reduce its weight average molecular weight. Nuclear magnetic results, however, show that there is still ester bond undegraded, probably because t.fusca cutinase cannot contact with the ester bond present inside oxidized polyethylene.

TABLE 1 HT-GPC and of samples after polyethylene, oxidized polyethylene and cutinase treatment¹H NMR measurement results

Example 4: treatment of polyethylene with m-chloroperoxybenzoic acid in combination with a Thermobifida fusca-derived cutinase

The method comprises the following specific steps:

(1) dissolving 50g/L LDPE in a 1,3, 5-trichlorobenzene-dichloroethane mixed solution in a volume ratio of 1:1, and stirring and dissolving at 100 ℃ for 12 hours; adding^mCPBA of^mThe proportion of the CPBA to the added LDPE is (1-5): 1, catalyzing at 100 ℃ for 8 hours; adding methanol with the same volume to precipitate LDPE, filtering, washing with methanol for 3-4 times to remove the solvent, and then placing the sample in an oven to be dried to constant weight to obtain oxidized polyethylene containing ester bonds.

(2) And (2) taking the oxidized polyethylene obtained in the step (1) as a substrate, adding enzyme of T.fusca cutinase 4200U/g of oxidized polyethylene, and reacting for 1-7 d at 60 ℃ and 150 rpm. And after the catalysis is finished, filtering the mixture in a 500-mesh nylon net to obtain a product sample, sequentially performing ultrasonic treatment on the product sample by using 2% (w/v) SDS, deionized water and 75% ethanol for 30min, and placing the product sample in an oven to dry the product sample to obtain the oxidized polyethylene after the cutinase treatment. The results show a weight average molecular weight reduction of the cutinase treated oxidized polyethylene of about 33.8% to 47.4% compared to oxidized polyethylene.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

SEQUENCE LISTING

<110> university of south of the Yangtze river

<120> method for treating polyethylene by combining peroxide and cutinase

<130> BAA211677A

<160> 2

<170> PatentIn version 3.3

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<211> 261

<212> PRT

<213> Thermobifida fusca

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Asn Thr Tyr Gly Ala Val Ala Ile Ser Pro Gly Tyr Thr Gly Thr Glu

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Ala Ser Ile Ala Trp Leu Gly Glu Arg Ile Ala Ser His Gly Phe Val

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Val Ile Thr Ile Asp Thr Ile Thr Thr Leu Asp Gly Pro Asp Ser Arg

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Ala Glu Gln Leu Asn Ala Ala Leu Asn His Met Ile Asn Arg Ala Ser

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Ser Thr Cys Pro Phe

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<213> Thermobifida fusca

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ttt 783

Claims

1. A process for the treatment of polyethylene or a product comprising polyethylene, characterized in that the polyethylene or the product comprising polyethylene is treated with a combination of peroxide and cutinase; the method comprises the steps of firstly, catalyzing a Baeyer-Villiger reaction by using m-chloroperoxybenzoic acid to add ester bonds to main chain carbon-carbon single bonds of polyethylene, and then catalyzing a hydrolysis reaction by using cutinase from Thermobifida fusca to degrade the ester bonds.

2. The method according to claim 1, wherein the mass ratio of m-chloroperoxybenzoic acid to polyethylene is (1-5): 1.

3. the method according to claim 1 or 2, characterized in that the cutinase has the amino acid sequence shown in SEQ ID No. 1.

4. A process according to any one of claims 1 to 3, wherein the cutinase is catalysed for a period of time of at least 1 day, 5 days or 7 days.

5. A method according to claim 1 or 3, characterized in that the method comprises the steps of:

6. The method as claimed in claim 5, wherein the 1,3, 5-trichlorobenzene-dichloroethane mixed solution in step (1) is prepared by mixing 1,3, 5-trichlorobenzene and dichloroethane in an equal volume ratio.

7. The method according to claim 5, wherein the LDPE in the step (1) is dissolved in a 1,3, 5-trichlorobenzene-dichloroethane mixed solution in a ratio of 1 (10-20).

8. The method according to claim 5, wherein the cutinase in the step (2) is reacted at 60 ℃ for 1-7 days.

9. The method of any one of claims 1 to 8, wherein the polyethylene-containing product includes, but is not limited to, low density polyethylene, including cling film, mulching film, or disposable plastic bags.

10. Use of the method of any one of claims 1 to 9 in waste treatment or waste recycling.