Fermentation preparation of feruloyl esterase and application of feruloyl esterase in polyester plastic degradation

Abstract

The invention discloses feruloyl esterase fermentation preparation and application of feruloyl esterase in polyester plastic degradation, and belongs to the field of environmental science. According to the method, feruloyl esterase FAE4 derived from Anerocephalum thermophilum is prepared through fermentation, and is compounded with polyester degrading enzymes which are different in source and are used for degrading polyester plastic oligomers, so that a compound reaction system which remarkably improves the degradation efficiency of poly (butylene adipate-co-terephthalate) or poly (butylene adipate-co-terephthalate) (PBAT) and the content proportion of a final product is obtained; good industrial prospects are realized.

Description

Technical Field

[0001] The invention relates to the fermentation preparation of ferulic acid esterase and its application in the degradation of polyester plastics, and belongs to the field of environmental science. Background Art

[0002] Only 9% of plastic waste can be recycled, and most of it is directly discarded, incinerated or landfilled, causing irreversible and serious damage to the environment. Therefore, the problem of plastic "white pollution" is becoming increasingly serious. With people's attention to the problem of plastic pollution and the promulgation of the national plastic restriction order, the use of degradable plastics to replace traditional plastics has gradually become the future development trend of plastic products. Among them, the bio-based degradable plastic polybutylene adipate / terephthalate (PBAT) has been widely used because of its structure containing flexible fatty chains and rigid aromatic segments, and has some excellent properties of polybutylene adipate (PBA) and polybutylene terephthalate (PBT). However, it often takes several months or even longer for PBAT to degrade in the natural environment. The microplastics produced in this process will still enter the human body through the food chain and threaten the health of the ecosystem. Therefore, it is of great significance to explore a feasible technology that can efficiently and thoroughly degrade PBAT. Summary of the invention

[0003] The purpose of the present invention is to further improve the efficiency of polyesterase degradation of PBAT by using ferulic acid esterase and plastic degradation polyesterase, and provide new enzymes for degrading PBAT. The present invention synthesizes FAE4 from Anaerocellumthermophilum and expresses and purifies it, and also expresses and purifies polyesterases from different sources, and compound the two in a certain ratio to degrade PBAT, so as to increase the complete degradation efficiency of PBAT.

[0004] The present invention provides a recombinant microorganism expressing ferulic acid esterase FAE4.

[0005] In one embodiment, the recombinant microorganism expresses the nucleotide sequence shown in SEQ ID NO.2.

[0006] In one implementation method, the vector includes but is not limited to pET series, Duet series, pGEX series, pHY300, pHY300PLK, pPIC3K or pPIC9K series vectors.

[0007] In one implementation method, the vector is pET-24a, and the polynucleotide encoding the ferulic acid esterase is inserted into the multiple cloning restriction site of the pET24a vector.

[0008] In one embodiment, the cells include but are not limited to Escherichia coli, Pichia pastoris, Bacillus subtilis, and Saccharomyces cerevisiae.

[0009] The invention provides an enzyme preparation, which contains ferulic acid esterase FAE4 and plastic-degrading polyesterase; the ferulic acid esterase FAE4 is derived from Anaerocellum thermophilum, and the plastic-degrading polyesterase includes but is not limited to: Tcur0390 derived from Thermomonospora curvata, Est119 from Thermobifida alba AHK119, DuraPETase from Ideonellasakaiensis, cutinase ICCG from Leaf-branch compost, cutinase TfC from Thermobifidafusca, and hydrolase BhrPETase from Bacterium HR29.

[0010] In one implementation method, the amino acid sequence of Tcur0390 derived from Thermomonospora curvata is shown in SEQ ID NO.4, and the nucleotide sequence is shown in SEQ ID NO.5; the amino acid sequence of Est119 derived from Thermobifida albaAHK119 is shown in SEQ ID NO.6, and the nucleotide sequence is shown in SEQ ID NO.7; the amino acid sequence of DuraPETase derived from Ideonella sakaiensis is shown in SEQ ID NO.8, and the nucleotide sequence is shown in SEQ ID NO.9; the amino acid sequence of cutinase ICCG derived from Leaf-branch compost is shown in SEQ ID NO.10, and the nucleotide sequence is shown in SEQ ID NO.11; the amino acid sequence of cutinase TfC derived from Thermobifida fusca is shown in SEQ ID NO.12, and the nucleotide sequence is shown in SEQ ID NO.13; the hydrolase BhrPETase derived from BacteriumHR29 has an amino acid sequence as shown in SEQ ID NO.14, and the nucleotide sequence is shown in SEQ ID NO.15.

[0011] In one implementation method, the amino acid sequence of the ferulic acid esterase FAE4 derived from Anaerocellum thermophilum is shown as SEQ ID NO.1.

[0012] In one implementation method, the enzyme preparation or composition contains an enzyme protectant; the protectant includes an acid-base regulator and a freeze-drying protectant.

[0013] The present invention provides a method for preparing the enzyme preparation.

[0014] In one implementation, the method includes:

[0015] a) cultivating a recombinant cell expressing the plastic polyesterase and / or ferulic acid esterase FAEE; and

[0016] b) optionally recovering the plastic polyesterase and / or ferulic acid esterase.

[0017] In one embodiment, the step a) is culturing the host cell in a culture medium containing a carbon source, a nitrogen source and an inorganic salt.

[0018] In one embodiment, the a) is to culture the host cells in TB medium at 35-40°C and 150-250 rpm until the OD 600 =0.8±0.5, then add IPTG with a final concentration of 0.1 mM, and culture at 20-30°C and 150-250 rpm for 24 h to obtain the fermentation broth.

[0019] In one implementation method, the fermentation broth expressing TfC, ICCG, BhrPETase or DuraPETase is centrifuged and the supernatant is collected as the crude enzyme solution; the fermentation broth expressing FAE4, Tcur0390 or Est119 is homogenized under high pressure and then centrifuged to break the cell wall, and the supernatant is collected as the crude enzyme solution.

[0020] The present invention also provides a method for degrading poly(butylene adipate / terephthalate) or a substance containing poly(butylene adipate / terephthalate), wherein the method comprises compounding the ferulic acid esterase FAE4 and a plastic-degrading esterase in a system containing poly(butylene adipate / terephthalate) to carry out a catalytic reaction.

[0021] In one implementation method, the added amount of the plastic degrading esterase and FAE4 is ≥ 0.3 mg / g substrate.

[0022] In one implementation method, the added amount of the plastic degrading esterase and FAE4 is 0.3 mg / g substrate.

[0023] In one implementation method, the reaction is carried out at pH 8.0±0.5, 70-75° C., and 150-250 rpm.

[0024] In one embodiment, the reaction time is not less than 24 hours.

[0025] In one embodiment, the reaction time is not less than 48 hours.

[0026] The present invention provides the use of the plastic degrading enzyme and FAE4 in degrading poly(butylene adipate / terephthalate) or a material containing poly(butylene adipate / terephthalate).

[0027] In one implementation method, the material containing polybutylene adipate / terephthalate includes agricultural mulch films, straws, plastic bags and other materials made of polybutylene adipate / terephthalate.

[0028] Beneficial effects: The present invention recombinantly expresses ferulic acid esterase FAE4 from Anaerocellum thermophilum and combines it with esterase with plastic degradation ability to degrade PBAT. Compared with using different types of esterase with plastic degradation ability to degrade PBAT alone, the dual enzyme combination can improve the PBAT degradation efficiency to varying degrees. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The fermentation process curves of ferulic acid esterase FAE4 in 3L and 6-ton fermenters.

[0030] Figure 2 The degradation of polyester plastic oligomers by ferulic acid esterase; among them, (a): degradation of MHET by FAE; (b): degradation of BHET by FAE; (c): degradation of MHBT by FAE; (d): degradation of BHBT by FAE.

[0031] Figure 3 The degradation rates of TPA and MHBT by enzyme reactions of different groups; (a) the degradation rates of ICCG enzyme, FAE4 enzyme, and ICCG and FAE4 combined enzyme after reaction at 75°C for different time periods; (b) the degradation rates of Tcur0390 enzyme, FAE4 enzyme, and Tcur0390 and FAE4 combined enzyme after reaction at 55°C for different time periods; (c) the degradation rates of TfC enzyme, FAE4 enzyme, and TfC and FAE4 combined enzyme after reaction at 55°C for different time periods; (d) the degradation rates of Est119 enzyme, FAE4, Est119 and FAE4 combined enzyme after reaction at 50°C for different time periods; (e) the degradation rates of DuraPETase enzyme, FAE4, DuraPETase and FAE4 combined enzyme after reaction at 50°C for different time periods; (f) the degradation rates of BhrPETase enzyme, FAE4, BhrPETase and FAE4 combined enzyme after reaction at 65°C for different time periods. DETAILED DESCRIPTION

[0032] The present invention will be further described below in conjunction with specific embodiments.

[0033] The polybutylene adipate / terephthalate (PBAT) plastics involved in the following examples were purchased from Shanghai McLean Biochemical Technology Co., Ltd. 4-(4-hydroxybutoxycarbonyl)benzoic acid (MHBT) and terephthalic acid (2-hydroxyethyl) ester (MHET) were purchased from Shanghai Bid Pharmaceutical Technology Co., Ltd. Bis(2-hydroxyethyl) terephthalate (BHET) was purchased from Sinopharm Chemical Reagent Co., Ltd.

[0034] The detection methods involved in the following embodiments are as follows:

[0035] The E. coli JM109 and E. coli BL21 (DE3) involved in the following examples were purchased from Takara Biotech (Beijing) Co., Ltd.

[0036] The culture media involved are as follows:

[0037] LB solid medium (g / L): peptone 10, yeast powder 5, sodium chloride 10, agar 13, pH 7.0.

[0038] LB liquid medium (g / L): peptone 10, yeast powder 5, sodium chloride 10, pH 7.0.

[0039] Fermentation medium: (NH4)2HPO4 4.0, KH2PO4 13.5, MgSO4·7H2O 1.4, citric acid 1.7, industrial grade peptone 1.0, industrial grade yeast powder 2.0, glycerol 8.0, trace element solution 10.0 mL.

[0040] Trace element solution (g / L): FeSO4·7H2O 10.0, ZnSO4·7H2O 2.2, CaCl2 2.0, CuSO4·5H2O1.0, MnSO4·4H2O 0.5, Na2B4O7·10H2O 0.2, (NH4)6Mo7O 24 0.1.

[0041] Feed medium: glycerol 500.0, MgSO4·7H2O 15.0, industrial grade peptone 2.0, industrial grade yeast powder 4.0.

[0042] Protein concentration determination method:

[0043] The protein concentration was determined by the Coomassie Brilliant Blue method (Analytical Biochemistry 1976 72(1-2):248-54).

[0044] Detection method of degradation products and their content:

[0045] Standard treatment: Weigh TPA and MHBT standards and dissolve them in dimethyl sulfoxide (DMSO) to prepare a mother solution. Dilute the mother solution with sterile water to a 0.1 mg / mL standard solution, filter with a 0.22 μM filter head, and inject into a liquid phase bottle with a syringe for HPLC detection;

[0046] Sample treatment: After the reaction is completed, take 0.5 mL of the reaction solution, centrifuge to obtain the supernatant, add methanol to dilute, filter with a 0.22 μM filter head, inject into a liquid phase bottle with a syringe, and perform HPLC detection.

[0047] HPLC determination method:

[0048] Chromatographic conditions: Agilent 1200HPLC chromatograph, Agilent autosampler, Athena C18-WP, 4.6 mm × 250 mm, 5 μm column, Agilent UV detector, detection wavelength of 254 nm, mobile phase of acetonitrile and 1‰ formic acid, running program of 25%-85% acetonitrile elution within 40 min, column temperature set at 30 °C, flow rate 1 mL min -1 .

[0049] Calculation method of PBAT degradation rate:

[0050] Degradation rate (%) = A / (B×C)×100+D*0.69 / (B×C)×100;

[0051] A is the mass (mg) of TPA in the reaction system; B is the percentage of TPA determined when PBAT is fully degraded by 20% NaOH; C is the mass (mg) of PBAT in the reaction system; D is the mass (mg) of MHBT in the reaction system.

[0052] Uses of ferulic acid esterase

[0053] The ferulic acid esterase of the present invention is a carboxylic acid ester hydrolase, which mainly hydrolyzes and breaks the ester bonds between methyl ferulate, oligosaccharide ferulic acid ester and polysaccharide ferulic acid ester, thereby releasing ferulic acid and its dimer, and promoting the depolymerization of covalent cross-linking between cell wall polysaccharides and hydroxycinnamic acid. At the same time, the ferulic acid esterase also has the ability to synergistically degrade crude fiber with other crude fiber degrading enzymes such as cellulase, xylanase and the like.

[0054] The reaction substrate structure of ferulic acid esterase is similar to 4-(4-hydroxybutoxycarbonyl)benzoic acid (MHBT) and monohydroxyethyl terephthalate (MHET), and the overall structure of ferulic acid esterase is similar to MHETase which has the ability to degrade the above intermediates. Therefore, applying ferulic acid esterase to decompose the intermediates of degradation of different types of PBAT products can obtain more final hydrolysis products TPA, thereby promoting the green recycling of plastics.

[0055] Example 1: Construction of FAE4 recombinant Escherichia coli

[0056] The synthetic plasmid containing the FAE4 gene (nucleotide sequence as shown in SEQ ID NO:2) and having a 6×his tag was used as a template to obtain the gene by PCR, and the PCR product was recovered by a nucleic acid gel recovery kit to obtain the target gene fragment. The target gene fragment was connected to the pET-24a expression vector by Megawhop PCR to obtain a recombinant plasmid. The constructed recombinant plasmid was transformed into Escherichia coli JM109 to obtain a transformation product; the transformation product was spread on LB solid culture medium (containing 30μg / mL kanamycin), and inverted in a 37°C constant temperature incubator for 8-12h to obtain a transformant; the transformant was picked and inoculated into LB liquid culture medium, and the plasmid was extracted for sequencing verification after shaking the flask at 37°C and 120-180rpm for 8-12h to obtain a recombinant plasmid expressing different ICCG mutants.

[0057] The correct recombinant plasmid obtained was transformed into Escherichia coli BL21 to obtain a transformation product; the transformation product was spread on LB solid culture medium (containing 30 μg / mL kanamycin), and inverted cultured in a 37°C constant temperature incubator for 8 to 12 hours to obtain transformants; the transformants were picked and inoculated into LB liquid culture medium, and after shaking flask culture at 37°C and 120 to 180 rpm for 8 to 12 hours, the plasmid was extracted for enzyme digestion verification and sequencing verification. If the verification was correct, the recombinant Escherichia coli: E. coli BL21 / pET-24(+)-FAE4 was obtained.

[0058] Example 2: Recombinant expression of FAE4 and preparation by 3L tank fermentation

[0059] The recombinant E. coli BL21 / pET-24(+)-M14 constructed in Example 1 was transferred to 50 mL of industrial-grade LB medium at a volume fraction of 2%, and a final concentration of 0.1 g·L -1 of ampicillin, 37°C, 200 r·min -1After 8 hours of cultivation, seed solution was obtained. The above seeds were transferred to a 3L fermenter at a volume fraction of 10%, and the initial liquid volume was 1L (including 900mL of fermentation medium and 100mL of seed solution). The growth temperature was maintained at 37°C through the instrument's own control, the dissolved oxygen was maintained at around 30% by adjusting the rotation speed and changing the oxygen introduction ratio, and the pH was maintained at 7.0 by adding ammonia water. After 5-7 hours of cultivation, the carbon source in the culture medium of the upper tank was exhausted, and the dissolved oxygen rebounded. A final concentration of 0.1mM IPTG was added to start induced fermentation. Thereafter, feed medium was added to the fermenter in an exponential manner until the end of fermentation, and the specific growth rate was controlled at μ=0.2h by adjusting the feed rate and dissolved oxygen dO2. -1 After induction, samples were taken at regular intervals to measure the bacterial biomass and crude enzyme activity of the samples. The fermentation was terminated when the enzyme activity showed a significant downward trend. The enzyme activity in the fermentation broth at the end of the fermentation was measured, such as Figure 1 As shown, the enzyme activity reached 124.6 U / mL after 60 h of fermentation.

[0060] Example 3: Fermentation of FAE4 enzyme in a 6-ton fermenter

[0061] Specific implementation method is the same as Example 2, except that, compared with the 3L tank, the 6-ton tank is manually controlled to increase or decrease the speed to ensure that the dissolved oxygen dO2 is maintained at 30% (20-40%), and the exponential feed is changed every 0.5h to control the specific growth rate at μ = 0.2h -1 Due to the limitation of the rotation speed of the large tank, the dissolved oxygen level during the fermentation process was guaranteed by appropriately limiting the glycerol feed.

[0062] The crude enzyme activity in the fermentation broth was measured and the results were as follows: Figure 1 As shown, the enzyme activity reached 123.2 U / mL after 60 h of fermentation.

[0063] Example 4: Degradation performance of FAE4 on MHET, MHBT, BHET, and BHBT

[0064] The enzyme solutions of MHETase (SEQ ID NO.16), FAE1 (SEQ ID NO.17), TtFAE (SEQ ID NO.18) and TsFAE (SEQ ID NO.19) were prepared according to the same strategy as in Examples 1-2.

[0065] MHET, MHBT, BHET, and BHBT were dissolved in dimethyl sulfoxide (DMSO) and added to the reaction system to a final concentration of 4.0 mmol·L -1 , at 0.1 mg·mL -1The enzyme solutions of FAE4, MHETase, FAE1, TtFAE, and TsFAE were added to the reaction sample tubes of different groups; -1 The total reaction volume was 5 mL in a potassium phosphate buffer solution of pH 8.0, and the mixture was stirred in a constant temperature water bath shaker at 70 °C and 200 rpm for 2 h. Since PBAT continuously produces acid during the degradation process, 6 mol·L -1 The pH was maintained stable by adding NaOH. After centrifugation, methanol was added to dilute the reaction solution, and finally the degradation rate was calculated by HPLC analysis.

[0066] The results are as follows Figure 2 As shown, FAE4 had the highest degradation rate compared with other enzymes with the same degradation function, and the degradation rates of MHET, MHBT, BHET and BHBT were 94.3%, 93.4%, 87.2% and 65.7%, respectively.

[0067] Example 5: PBAT degradation performance of FAE4 combined with different plastic degrading polyester enzymes

[0068] The enzyme solutions of Tcur0390 (SEQ ID NO.4), Est119 (SEQ ID NO.6), DuraPETase (SEQ ID NO.8), ICCG (SEQ ID NO.10), BhrPETase (SEQ ID NO.14), and TfC (SEQ ID NO.12) were prepared according to the same strategy as in Examples 1 and 2. The FAE4 enzyme was prepared according to the method in Example 2.

[0069] FAE4 enzyme was used with Tcur0390, Est119, DuraPETase, ICCG, BhrPETase, and TfC to degrade PBAT. The specific steps are:

[0070] 100 mg of PBAT particles that had been crushed and passed through a 60-mesh sieve were added to the reaction flask, and each enzyme was added to the reaction system at an addition rate of 0.3 mg enzyme protein / g substrate; -1 The total reaction system was 5 mL in potassium phosphate buffer solution of pH 8.0. The group with ICCG enzyme solution was shaken in a constant temperature water bath at 75°C and 200 rpm for 24 h; the group with BhrPETase enzyme solution was shaken in a constant temperature water bath at 65°C and 200 rpm for 24 h; the group with DuraPETase and Est119 enzyme solution was shaken in a constant temperature water bath at 50°C and 200 rpm for 24 h; the group with Tcur0390 and TfC enzyme solution was shaken in a constant temperature water bath at 55°C and 200 rpm for 24 h. Since PBAT continuously produces acid during the degradation process, 6 mol·L-1 The pH was maintained stable by adding NaOH. After centrifugation, methanol was added to dilute the reaction solution, and finally the degradation rate was calculated by HPLC analysis.

[0071] The results are as follows Figure 3 As shown, the degradation of PBAT by the combination of FAE4 and different plastic-degrading polyesterases can increase the total degradation rate and the proportion of the final product TPA in the degradation solution. The degradation rate of PBAT by FAE4 and TfC polyesterase can reach up to 83.2%.

[0072] Example 6: Composition or enzyme preparation containing FAE4

[0073] FAE4 was prepared according to the method of Example 2, and the enzyme protein was mixed with an enzyme protective agent; the protective agent included an acid-base regulator and a lyophilization protective agent.

[0074] The acid-base regulator is a conventional buffer capable of maintaining pH, such as malic acid-sodium malate buffer, acetic acid-sodium acetate buffer, citric acid-sodium citrate buffer, citric acid-sodium hydroxide-hydrochloric acid buffer, Tris-hydrochloric acid buffer. The buffer is used to maintain the ferulic acid esterase protein at its optimal pH condition to ensure its maximum catalytic activity.

[0075] The lyoprotectant is used to reduce the chemical and / or physical instability of the ferulic acid esterase protein during freeze-drying and / or subsequent storage, including but not limited to sugars and their corresponding sugar alcohols, such as sucrose, lactose, trehalose, dextran, erythritol, arabitol, xylitol, sorbitol, mannitol; amino acids, such as arginine or histidine; soluble salts, such as magnesium sulfate; polyols, such as propylene glycol, glycerol, poly (ethylene glycol) or poly (propylene glycol); and any combination of the above.

[0076] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A recombinant microorganism, characterized in that The ferulic acid esterase gene FAE4 shown in SEQ ID NO.1 was expressed.

2. The recombinant microorganism according to claim 1, characterized in that Escherichia coli is used as a host to express the nucleotide sequence shown in SEQ ID NO.2; the expression vector includes but is not limited to pET series, Duet series, pGEX series, pHY300, pHY300PLK, pPIC3K or pPIC9K series vectors.

3. The recombinant microorganism according to claim 1, characterized in that The cells include, but are not limited to, Escherichia coli, Pichia pastoris, Bacillus subtilis or Saccharomyces cerevisiae.

4. An enzyme preparation, characterized in that Contains ferulic acid esterase FAE4 and plastic degrading polyesterase; the amino acid sequence of the ferulic acid esterase FAE is shown in SEQ ID NO.1, and the plastic degrading polyesterase includes but is not limited to: Tcur0390 from Thermomonospora curvata, Est119 from Thermobifida alba AHK119, DuraPETase from Ideonellasakaiensis, cutinase ICCG from Leaf-branch compost, cutinase TfC from Thermobifidafusca, and hydrolase BhrPETase from Bacterium HR29.

5. The enzyme preparation according to claim 4, characterized in that The enzyme preparation contains enzyme protectants; the protectants include acid-base regulators and freeze-drying protectants.

6. A method for preparing the enzyme preparation according to claim 4 or 5, characterized in that: The method comprises: a) cultivating a recombinant microorganism expressing a plastic polyesterase and / or a ferulic acid esterase; and b) optionally recovering the plastic polyesterase and / or ferulic acid esterase.

7. The method according to claim 6, characterized in that The fermentation broth expressing TfC, ICCG, BhrPETase or DuraPETase was centrifuged, and the supernatant was collected as the crude enzyme solution; the fermentation broth expressing FAE4, Tcur0390 or Est119 was homogenized under high pressure and then centrifuged to break the cell wall, and the supernatant was collected as the crude enzyme solution.

8. A method for degrading polybutylene adipate / terephthalate or a substance containing polybutylene adipate / terephthalate, characterized in that: The ferulic acid esterase FAE4 and the plastic-degrading esterase are compounded in a system containing poly(adipate) / butylene terephthalate to carry out a catalytic reaction; the amino acid sequence of the ferulic acid esterase FAE4 is shown in SEQ ID NO.1; the plastic-degrading esterase includes but is not limited to Tcur0390 derived from Thermomonospora curvata, Est119 from Thermobifida alba AHK119, DuraPETase from Ideonella sakaiensis, cutinase ICCG from Leaf-branchcompost, cutinase TfC from Thermobifida fusca, and hydrolase BhrPETase from Bacterium HR29.

9. The method according to claim 8, characterized in that The added amount of the plastic-degrading esterase and FAE4 is ≥0.3 mg / g substrate.

10. Use of ferulic acid esterase FAE4 in the degradation of poly(butylene adipate / terephthalate) or substances containing poly(butylene adipate / terephthalate).

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