A method for synthesizing a novel bio-based polyester by combining enzyme cascade catalysis with microfluidic field coupling

Abstract

The present invention discloses a method for synthesizing a novel bio-based polyester by combining enzyme cascade catalysis with microfluidic field coupling, comprising the following steps: (1) in a first solvent, under the action of a catalyst, carrying out a first reaction between propyl-caprolactone and an initiator; (2) adding lactide and a second solvent to the first reaction system to carry out a second reaction to obtain a bio-based polyester, poly(propylcaprolactone)-b-poly(lactide); the number average molecular weight of the bio-based polyester is 1000-25000 g / mol. The present invention greatly improves the reaction efficiency by using a microreactor system, reduces the reaction time, and realizes continuous production, which has the advantages of safety, high efficiency, low energy consumption, fast reaction rate, mild reaction conditions, etc.

Description

Technical Field

[0001] The present invention relates to the fields of polymer materials, chemistry and chemical engineering, and particularly relates to a method for synthesizing a novel bio-based polyester by using two-enzyme cascade catalysis combined with microfluidic field coupling. Background Art

[0002] Thermoplastic elastomers have characteristics such as good elasticity and reprocessability, and are widely used in fields such as hot melt pressure-sensitive adhesives, automotive parts, and medical devices. Currently, common commercial thermoplastic elastomers include polystyrene-b-polybutadiene-b-polystyrene and polystyrene-b-polyisoprene-b-polystyrene, etc. Although these materials have wide applications, they rely on non-renewable raw materials and cannot be degraded in the natural environment after being discarded. Therefore, the preparation of sustainable bio-based thermoplastic elastomers using safe and non-toxic biomass-based raw materials has attracted increasing attention.

[0003] L-lactide is derived from plants such as sugarcane, and polylactic acid obtained by its ring-opening polymerization is a polyester material that can be completely biodegradable. Hillmyer et al. reported the synthesis of poly(lactic acid)-b-poly(β-methyl-δ-valerolactone)-b-poly(lactic acid) triblock copolymer, and the obtained triblock copolymer exhibited the properties of a thermoplastic elastomer and had relatively high mechanical strength and elongation at break. However, β-methyl-δ-valerolactone as a synthetic raw material is not a commercial monomer, and its synthesis steps are cumbersome and complex, making it difficult to carry out large-scale industrial production. Propyl-ε-caprolactone (ε-prCL) is a six-membered ring lactone with a propyl substitution at the ε position and is one of the depolymerization products of lignin bio-oil, which is composed of hundreds of phenolic and cyclic aromatic compounds, and its products vary due to different raw material types, depolymerization methods, heating rates, reaction temperatures, and catalysts, etc. The chemical route proposed by Roman-Leshkov and his colleagues: Cresol isolated from lignin pyrolysis oil is hydrogenated to form methyl-cyclohexanone, and then undergoes Baeyer-Villiger oxidation (BVO) to form methyl-ε-caprolactone. These combined reactions provide the possibility of downstream applications for lignin-derived alkylphenols, and at the same time can produce alkyl caprolactones with high net yield and high selectivity. Conventional linear-chain poly(ε-caprolactone) (PCL) is a semi-crystalline material, and the ester part in the polymer backbone endows biodegradability, and it can be mixed and copolymerized with other polymers to improve its properties. Branched poly(methyl-ε-caprolactone) (PMCL) and caprolactones containing other alkyl groups such as polypropyl-ε-caprolactone (PprCL) also benefit from this, but the addition of branched chains will lead to the formation of amorphous materials with a low glass transition temperature (Tg≈-60 °C), presenting a rubber state at room temperature and expected to be used as the soft segment of a thermoplastic elastomer. Therefore, the present invention provides a method for synthesizing a novel bio-based polyester by using enzyme cascade catalysis combined with microfluidic field coupling. Summary of the Invention

[0004] Objective of the Invention: Aiming at the deficiencies of the prior art, the technical problem to be solved by the present invention is to provide a method for synthesizing a novel bio-based polyester by using enzyme cascade catalysis combined with microfluidic field coupling.

[0005] Idea of the Invention: The present invention uses propylcyclohexanol derived from lignin pyrolysis oil as the starting material, synthesizes propyl-ε-caprolactone with chiral high selectivity through double enzyme cascade, and controllably synthesizes biodegradable block polymers based on organic catalysis in a microfluidic field. By changing the concentration ratio of the two monomers and the ratio of monomer to initiator, random / block copolymers PprCL-b-PLLA with different molecular weights can be controllably synthesized.

[0006] To solve the above technical problems, the present invention discloses a method for synthesizing a bio-based polyester (a novel pure bio-based degradable thermoplastic elastomer). The reaction mechanism is as follows: using p-propylcyclohexanol as the raw material, first using alcohol reductase and polycyclic ketone monooxygenase, and realizing the recycling of cofactors in the reaction at the same time. Propyl-caprolactone is synthesized through double enzyme cascade reaction. Subsequently, 1,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD) is used as a catalyst in a microreactor for catalysis. The obtained polyester is used as a macroinitiator for ring-opening polymerization of lactide, and a block copolymer can be synthesized in a short time under the same catalysis.

[0007] In some embodiments, the method for synthesizing the bio-based polyester comprises the following steps:

[0008] (1) In a first solvent, under the action of a catalyst, propyl-caprolactone and an initiator are subjected to a first reaction under an anhydrous inert gas atmosphere to obtain polypropyl-ε-caprolactone;

[0009] (2) Lactide and a second solvent are added to the first reaction system for a second reaction to obtain a bio-based polyester, polypropylcaprolactone-b-lactide PprCL-b-PLLA.

[0010]

[0011] PprCL-b-PLLA

[0012] In some embodiments, the number average molecular weight of the bio-based polyester is 1000 - 25000 g / mol, preferably 2000 - 24000 g / mol, and more preferably 6000 - 22000 g / mol.

[0013] In some embodiments, the preparation method of propyl-caprolactone is as follows: using p-propylcyclohexanol as the raw material, and using alcohol reductase and polycyclic ketone monooxygenase as biocatalysts, propyl-caprolactone is prepared through a cofactor regeneration and recycling one-pot system.

[0014] In some embodiments, the alcohol dehydrogenase (LK-ADH) is a free alcohol dehydrogenase obtained by cloning the ADH gene (GenBank: AY267012.1) encoded by Lactobacillus kefir DSM 20587 into pET22b(+) and transferring it into Escherichia coli for culture.

[0015] In some embodiments, the polycyclic ketone monooxygenase (PockMe) is a free polycyclic ketone monooxygenase obtained by cloning the BVMO gene encoded by Trypanosoma thermophila (ATCC42464) into pET22b(+) and transferring it into Escherichia coli for culture.

[0016] Among them, both the alcohol dehydrogenase and the polycyclic ketone monooxygenase are selected to participate in the reaction in a free state.

[0017] In some embodiments, the solvent for the reaction is a combination of an organic solvent and a buffer solution.

[0018] In some embodiments, the organic solvent accounts for 0.5% - 10% of the total volume of the solvent.

[0019] In some embodiments, the organic solvent is any one or a combination of methanol, acetonitrile, dimethyl sulfoxide, isopropanol, and dioxane, preferably dioxane.

[0020] In some embodiments, the buffer solution is a Tris-HCL buffer solution with a pH of 7 - 8, preferably a Tris-HCL buffer solution with a pH of 7.5.

[0021] In some embodiments, in the reaction system, the concentration of 4-propylcyclohexanol is 4 - 8 mM, preferably 4 mM.

[0022] In some embodiments, in the reaction system, the concentration of the alcohol dehydrogenase is 0.1 - 0.5 g / mL, preferably 0.2 g / mL.

[0023] In some embodiments, in the reaction system, the concentration of the polycyclic ketone monooxygenase is 0.1 - 0.5 g / mL, preferably 0.1 g / mL.

[0024] In some embodiments, in the reaction system, the concentration of the cofactor is 0.3 - 10 mM, preferably 0.3 mM.

[0025] In some embodiments, the temperature of the reaction is 25 - 35 °C.

[0026] In some embodiments, after the reaction is completed, the reaction solution is collected, the reaction solution is extracted with an organic solvent, the organic phase is retained, and after drying the organic phase, it is concentrated under reduced pressure to obtain propyl-ε-caprolactone; the organic solvent is any one or a combination of ethyl acetate, petroleum ether, dichloromethane, and n-hexane; preferably, the organic phase is dried with anhydrous sodium sulfate.

[0027] In step (1), the first solvent is an organic solvent, preferably any one or a combination of toluene, tetrahydrofuran, and dichloromethane, preferably toluene.

[0028] In step (1), the catalyst is an organic base catalyst, preferably 1,5,7-triazabicyclo(4.4.0)dec-5-ene.

[0029] In step (1), the initiator is an alcohol, preferably any one of n-hexanol, n-butanol, benzyl alcohol, and isopropanol, preferably benzyl alcohol.

[0030] In step (1), the concentration of propyl-caprolactone is 0.5 to 5 mol / L, preferably 0.5 to 3 mol / L, preferably 0.8 to 3 mol / L.

[0031] In step (1), the molar ratio of propyl-caprolactone to the initiator is 10 to 100:1, preferably 30 to 50:1.

[0032] In step (1), the temperature of the reaction is 40 to 80 °C, preferably 40 to 60 °C, preferably 40 to 50 °C.

[0033] In step (2), the second solvent is an organic solvent, preferably any one or a combination of toluene, tetrahydrofuran, and dichloromethane, preferably toluene.

[0034] In step (2), the molar ratio of lactide to propyl-caprolactone is 0.5 to 10:1, preferably 0.5 to 5:1, preferably 0.5 to 3:1, preferably 0.5 to 2.5:1.

[0035] In step (2), the temperature of the reaction is 25 to 80 °C, preferably 25 to 60 °C, preferably 30 to 50 °C, preferably 40 to 50 °C.

[0036] In some embodiments, the method for synthesizing the bio-based polyester is to synthesize the bio-based polyester in a microchannel reaction device, including the following steps:

[0037] S1: A first solution containing propyl-caprolactone, a catalyst, and a first solvent is subjected to a first reaction with a second solution containing an initiator in a first microreactor in the microchannel reaction device;

[0038] S2: The third solution containing lactide and the second solvent is subjected to a second reaction with the first reaction effluent in a second microreactor in the microchannel reaction device to obtain a bio-based polyester, polypropyl caprolactone-b-lactide.

[0039] Among them, the channel in the microchannel reaction device is a capillary or a polytetrafluoroethylene tube, preferably a polytetrafluoroethylene tube; the inner diameter of the microreactor in the microchannel reaction device is 1 to 3 mm, preferably 1 to 2 mm, preferably 2 mm; the length of the microreactor in the microchannel reaction device is 50 to 600 mm, preferably 150 to 300 mm.

[0040] Wherein, the volume ratio of the first solvent to the second solvent is 1:0.5-1.5, preferably 1:0.8-1.2, preferably 1:1.

[0041] The method is characterized in that the pumping rate of the first solution and the second solution is 0.01-0.03 mL / min, preferably 0.018 mL / min.

[0042] Wherein, the pumping rate of the third solution is 0.01-0.8 mL / min, preferably 0.01-0.5 mL / min, and preferably 0.01-0.3 mL / min.

[0043] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0044] (1) The present invention provides a method for synthesizing a novel block copolymer PprCL-b-PLLA by using a dual enzyme cascade and a microchannel reaction intensification device. Micro-reaction units are constructed for specific catalysts and corresponding monomers to achieve an increase in polymerization reaction rate and an optimization of molecular weight distribution; through the organic series connection of micro-reaction units, efficient copolymerization of different monomers is achieved in the same reaction process, and customized synthesis of fully bio-based degradable thermoplastic elastomers with different properties has potential prospects for industrial production.

[0045] (2) The raw materials used in the present invention are non-toxic and harmless and are derived from biomass, and the prepared block copolymer can be completely degraded under natural conditions.

[0046] (3) The catalytic system used in the present invention has high catalytic activity and high monomer conversion rate. The conversion rate of ε-caprolactone can reach about 99%, and the conversion rate of lactide is >90%.

[0047] (4) The reaction conditions of the present invention are mild and the reaction time is short, which greatly reduces energy consumption. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] The following further specifically describes the present invention in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become clearer.

[0049] Figure 1 It is a schematic diagram of the reaction device process.

[0050] Figure 2 It is the 1H NMR spectrum of the polymer.

[0051] Figure 3 It is the 13C NMR spectrum of the polymer.

[0052] Figure 4 It is the DSC analysis of copolymers with different components.

[0053] Figure 5 It is the Gel Permeation Chromatography (GPC) diagram of copolymers with different components. Specific Embodiments

[0054] In the following embodiments, the experimental methods are all conventional methods unless otherwise specified; the reagents and materials can all be obtained from commercial channels unless otherwise specified.

[0055] The microchannel reaction device described in the following embodiments is as Figure 1 shown, including a first raw material storage tank, a second raw material storage tank, a third raw material storage tank, a first microreactor, a second microreactor, and a product collector. The first raw material storage tank and the second raw material storage tank are connected in series with the first microreactor in parallel, the third raw material storage tank and the first microreactor are connected in series with the second microreactor in parallel, and the second microreactor is connected in series with the product collector; the connection is through a pipeline connection. The pipeline in the microchannel reaction device is a polytetrafluoroethylene tube; the inner diameter of the coiled tubes of the first microreactor and the second microreactor is 2 mm, and the length is 25 cm.

[0056] In the following examples, recombinant strains E. coli (LK-ADH) and E. coli (PockMe) were used to highly express alcohol dehydrogenase LK-ADH and polycyclic ketone monooxygenase. The construction methods of E. coli (LK-ADH) and E. coli (PockMe) are as follows: Alcohol reductase (LK-ADH) comes from the ADH gene (GenBank: AY267012.1) encoded by Lactobacillus kefir DSM 20587, and polycyclic ketone monooxygenase (PockMe) is derived from Thermothelomyces thermophila (Grogan classification: XP_003661890: PockeMO (Thermothelomyces thermophila ATCC 42464)). Then, single colonies of alcohol reductase and polycyclic ketone monooxygenase were separately picked from the plate and inoculated into LB medium containing 100 mg / L ampicillin resistance or 50 mg / L kanamycin resistance, and cultured with shaking at 37 °C and 200 rpm for 7 h. Then, inoculate into 50 mL TB medium containing 100 mg / L ampicillin resistance or 50 mg / L kanamycin resistance at an inoculation amount of 2%, and culture at 37 °C and 200 rpm. When the OD600 of the culture broth reaches 0.6, add 0.5 mM IPTG, and induce at 20 °C and 200 rpm for 12 h. Collect the bacteria, centrifuge at 8000 rpm for 5 min at 4 °C, remove the supernatant, suspend the obtained cells with deionized water, place on ice, and perform ultrasonic disruption 90 times at 400 W, with a working time of 4 s and an intermittent time of 4 s. Then, centrifuge the mixture at 1000 rpm and 4 °C for 30 minutes to remove cell debris, obtain the crude enzyme solution of the recombinant bacteria, and elute three times with 50 mM Tris / HCl pH 7.5 and 500 mM imidazole. Finally, the obtained light yellow enzyme solution was stored at -80 °C.

[0057] Example 1 Preparation of propyl-caprolactone

[0058] A water-organic two-phase system composed of Tris-HCl buffer (50 mM, pH 7.5) and 10% dioxane was used as the reaction medium. The enzyme solutions of thawed recombinant bacteria E. coli (LK-ADH) and E. coli (PockMe) were mixed as the biocatalyst according to a molar ratio of 2:1. The final concentrations of E. coli (LK-ADH) and E. coli (PockMe) were 0.2 g / mL and 0.1 g / mL, respectively. The substrate 4-propylcyclohexanol with a final concentration of 4 mM and NADP+ with a final concentration of 0.3 mM were added. Finally, the reaction system was made up to 2 mL with Tris-HCl buffer (50 mM, pH 7.5). The reaction was carried out at 200 rpm and 30 °C for 24 hours. 300 μL of the reaction solution was taken, extracted with an equal volume of ethyl acetate containing an internal standard (dodecane at 20 mM), centrifuged at 10000 rpm for 5 min, and then the supernatant was dried with anhydrous sodium sulfate. The changes in the substrate and the target product were monitored by gas chromatography.

[0059] 1H NMR (400 MHz, CDCl3): δ = 4.29–4.24 (m, 1H), 4.15 (dd, J = 12.4, 10.0 Hz, 1H), 2.71–2.65 (m, 1H), 2.64–2.57 (m, 1H), 2.01–1.89 (m, 2H), 1.67 - 1.59 (m, 1H), 1.52–1.43 (m, 1H), 1.37 - 1.23 (m, 5H), 0.90 (t, J = 7.2 Hz, 3H). 13C NMR (100 MHz, CDCl3): δ = 176.28, 68.24, 39.90, 38.66, 35.32, 33.20, 28.87, 19.86, 14.16 ppm.

[0060] Example 2 Preparation of Propyl-ε-caprolactone

[0061] In a water-organic two-phase system composed of Tris-HCl buffer (50 mM, pH 7.5) and 10% acetonitrile as the reaction medium, the enzyme solutions of thawed recombinant bacteria E. coli (LK-ADH) and E. coli (PockMe) were mixed as the biocatalyst according to a ratio of 2:1. The final concentrations of E. coli (LK-ADH) and E. coli (PockMe) were 0.2 g / mL and 0.1 g / mL, respectively. The 2 mL reaction system was ensured to contain 4 mM propylcyclohexanol and 0.3 mM NADP+ at the final concentrations. The reaction was carried out at 200 rpm and 30 °C. The substrate conversion rate was monitored by GC at regular intervals until the reaction was complete. Finally, it was extracted with ethyl acetate, centrifuged at 10000 rpm for 5 min, and the supernatant was dried with anhydrous sodium sulfate. The yield of the target product was determined by GC.

[0062] Preparation of Propyl-Caprolactone in Example 3

[0063] In a water-organic two-phase system composed of Tris-HCl buffer (50 mM, pH 7.5) and 10% dimethyl sulfoxide as the reaction medium, the enzyme solutions of thawed recombinant E. coli (LK-ADH) and E. coli (PockMe) were mixed in a ratio of 2:1 as the biocatalyst. The final concentrations of E. coli (LK-ADH) and E. coli (PockMe) were 0.2 g / mL and 0.1 g / mL, respectively. The 2 mL reaction system was ensured to contain a final concentration of 4 mM propylcyclohexanol and 0.3 mM NADP+. The reaction was carried out at 200 rpm and 30 °C. The substrate conversion rate was monitored by GC at regular intervals until the reaction was complete. Finally, extraction was performed with ethyl acetate, centrifugation was carried out at 10000 rpm for 5 min, the supernatant was dried with anhydrous sodium sulfate, and the yield of the target product was determined by GC.

[0064] Preparation of Propyl-Caprolactone in Example 4

[0065] Using a water-organic two-phase system composed of Tris-HCl buffer (50 mM, pH 8) and 10% dioxane as the reaction medium, the enzyme solutions of thawed recombinant E. coli (LK-ADH) and E. coli (PockMe) were mixed in a ratio of 2:1 as the biocatalyst. The final concentrations of E. coli (LK-ADH) and E. coli (PockMe) were 0.2 g / mL and 0.1 g / mL, respectively. The 2 mL reaction system was ensured to contain a final concentration of 4 mM propylcyclohexanol and 0.3 mM NADP+. The reaction was carried out at 200 rpm and 30 °C. The substrate conversion rate was monitored by GC at regular intervals until the reaction was complete. Finally, extraction was performed with ethyl acetate, centrifugation was carried out at 10000 rpm for 5 min, the supernatant was dried with anhydrous sodium sulfate, and the yield of the target product was determined by GC.

[0066] Preparation of Propyl-Caprolactone in Example 5

[0067] Using a water-organic two-phase system composed of Tris-HCl buffer (50 mM, pH 9) and 10% dioxane as the reaction medium, the enzyme solutions of thawed recombinant bacteria E. coli (LK-ADH) and E. coli (PockMe) were mixed in a ratio of 2:1 as the biocatalyst, and the final concentrations of E. coli (LK-ADH) and E. coli (PockMe) were 0.2 g / mL and 0.1 g / mL, respectively; the 2 mL reaction system was ensured to contain a final concentration of 4 mM propylcyclohexanol and 0.3 mM NADP⁺. The reaction was carried out at 200 rpm and 30 °C, and the substrate conversion rate was monitored by GC at regular intervals until the reaction was complete. Finally, extraction was performed with ethyl acetate, centrifugation was carried out at 10000 rpm for 5 min, the supernatant was dried with anhydrous sodium sulfate, and the yield of the target product was determined by GC.

[0068] Example 6 Preparation of Propyl-ε-caprolactone

[0069] Using a water-organic two-phase system composed of Tris-HCl buffer (50 mM, pH 7.5) and 10% dioxane as the reaction medium, the enzyme solutions of thawed recombinant bacteria E. coli (LK-ADH) and E. coli (PockMe) were mixed in a ratio of 1:1 as the biocatalyst, and the final concentrations of E. coli (LK-ADH) and E. coli (PockMe) were 0.1 g / mL and 0.1 g / mL, respectively; the 2 mL reaction system was ensured to contain a final concentration of 4 mM propylcyclohexanol and 0.3 mM NADP⁺. The reaction was carried out at 200 rpm and 30 °C, and the substrate conversion rate was monitored by GC at regular intervals until the reaction was complete. Finally, extraction was performed with ethyl acetate, centrifugation was carried out at 10000 rpm for 5 min, the supernatant was dried with anhydrous sodium sulfate, and the yield of the target product was determined by GC.

[0070] Example 7 Preparation of Propyl-ε-caprolactone

[0071] Using a water-organic two-phase system composed of Tris-HCl buffer (50 mM, pH 7.5) and 10% dioxane as the reaction medium, the enzyme solutions of thawed recombinant bacteria E. coli (LK-ADH) and E. coli (PockMe) were mixed in a ratio of 1:2 as the biocatalyst, and the final concentrations of E. coli (LK-ADH) and E. coli (PockMe) were 0.1 g / mL and 0.2 g / mL, respectively; the 2 mL reaction system was ensured to contain a final concentration of 4 mM propylcyclohexanol and 0.3 mM NADP⁺. The reaction was carried out at 200 rpm and 30 °C, and the substrate conversion rate was monitored by GC at regular intervals until the reaction was complete. Finally, extraction was performed with ethyl acetate, centrifugation was carried out at 10000 rpm for 5 min, the supernatant was dried with anhydrous sodium sulfate, and the yield of the target product was determined by GC.

[0072] Table 1 Results of double-enzyme catalysis in Examples 1-7

[0073] Example Solvent System pH ADH:PockMe Alcohol conversion rate % Ester yield % 1 Tris-HCl + dioxane 7.5 2：1 95 76 2 Tris-HCl + acetonitrile 7.5 2：1 85 45 3 Tris-HCl + dimethyl sulfoxide 7.5 2：1 63 33 4 Tris-HCl + dioxane 8 2：1 88 42 5 Tris-HCl + dioxane 9 2：1 74 20 6 Tris-HCl + dioxane 7.5 1：1 23 0 7 Tris-HCl + dioxane 7.5 1：2 10 0

[0074] Preparation of block copolymer PprCL-b-PLLA in Example 8

[0075] In the glove box, purified propyl-caprolactone (1000 mg, 6.41 mmol), initiator benzyl alcohol (13.845 mg, 0.1282 mmol), and catalyst TBD (53.45 mg, 0.3846 mmol) were dissolved in 6 mL of toluene. Subsequently, it was transferred to an oil bath at 45 °C to start the reaction. After 2.5 h of reaction, the first monomer reaction was completed. The process was monitored by size exclusion chromatography. Finally, it was transferred back to the glove box, and lactide (2153.75 mg, 14.956 mmol) and 9 mL of toluene were added to the reaction system. The temperature was maintained at 45 °C. After about 2 h of reaction, benzoic acid was added to terminate the reaction. It was precipitated at low temperature in cold methanol for 4 h, filtered and washed three times to collect the precipitate. After air drying, it was placed in a vacuum drying oven and dried for 48 h, and analyzed by size exclusion chromatography and proton nuclear magnetic resonance spectroscopy. The nuclear magnetic resonance of the obtained polymer is as Figure 2 and Figure 3 shown, and the GPC monitoring is as Figure 5 shown.

[0076] Preparation of block copolymer PprCL-b-PLLA in Example 9

[0077] In the glove box, purified propyl-caprolactone (1000 mg, 6.41 mmol), initiator benzyl alcohol (9.88 mg, 0.0915 mmol), and catalyst TBD (38.18 mg, 0.2747 mmol) were dissolved in 6 mL of toluene. Subsequently, it was transferred to an oil bath at 45 °C to start the reaction. After 2.7 h of reaction, the first monomer reaction was completed. It was transferred to the glove box, and lactide (1383.48 mg, 9.6075 mmol) and 4 mL of toluene were added to the reaction system. The temperature was maintained at 45 °C. After about 1.5 h of reaction, benzoic acid was added to terminate the reaction. It was precipitated at low temperature in cold methanol for 4 h, filtered and washed three times to collect the precipitate. After air drying, it was placed in a vacuum drying oven and dried for 48 h, and analyzed by size exclusion chromatography and proton nuclear magnetic resonance spectroscopy.

[0078] Preparation of block copolymer PprCL-b-PLLA in Example 10

[0079] In the glove box, purified propyl-caprolactone (1000 mg, 6.41 mmol), initiator benzyl alcohol (9.88 mg, 0.0915 mmol), and catalyst TBD (38.18 mg, 0.2747 mmol) were dissolved in 6 mL of toluene. Subsequently, it was transferred to an oil bath at 45 °C to start the reaction. After 2.7 h of reaction, the first monomer reaction was completed. It was transferred back to the glove box, and lactide (923.04 mg, 6.41 mmol) and 3 mL of toluene were added to the reaction system. The temperature was maintained at 45 °C. After about 1 h of reaction, benzoic acid was added to terminate the reaction. It was precipitated at low temperature in cold methanol for 4 h, filtered and washed three times to collect the precipitate. After air drying, it was placed in a vacuum drying oven and dried for 48 h, and analyzed by size exclusion chromatography and proton nuclear magnetic resonance spectroscopy.

[0080] Preparation of Block Copolymer PprCL-b-PLLA in Example 11

[0081] In the glove box, purified propyl-caprolactone (1000 mg, 6.41 mmol), initiator benzyl alcohol (6.92 mg, 0.0641 mmol), and catalyst TBD (38.18 mg, 0.2747 mmol) were dissolved in 6 mL of toluene. Subsequently, it was transferred to an oil bath at 45 °C to start the reaction. After 3 h of reaction, the first monomer reaction was completed. It was transferred back to the glove box, and lactide (609.20 mg, 4.23 mmol) and 3 mL of toluene were added to the reaction system. The temperature was maintained at 45 °C. After about 0.5 h of reaction, benzoic acid was added to terminate the reaction. It was precipitated at low temperature in cold methanol for 4 h, filtered and washed three times to collect the precipitate. After air drying, it was placed in a vacuum drying oven and dried for 48 h, and analyzed by size exclusion chromatography and proton nuclear magnetic resonance spectroscopy.

[0082] Preparation of Block Copolymer PprCL-b-PLLA in Example 12

[0083] The chemoselective polymerization platform consists of 3 50 mL airtight syringes / syringe pumps, 1 PTFE tube (diameter = 2.0 mm, retention volume = 0.56 mL), 1 PTFE tube (diameter = 3.8 mm, retention volume = 2.22 mL), and 1 T-shaped mixer.

[0084] In the glove box, prepare argon-filled stock solution A (PrCL (2000 mg, 12.82 mmol) and TBD (178.20 mg, 1.282 mmol) dissolved in 6.41 mL of dry toluene), stock solution B (benzyl alcohol (138.45 mg, 1.282 mmol) dissolved in 6.41 mL of dry toluene), stock solution C (lactide (1846.08 mg, 12.82 mmol) dissolved in 12 mL of dry toluene), and transfer them to 50 mL airtight syringes respectively. The assembled tubular reactor was flushed with nitrogen and dry toluene to remove moisture and air.

[0085] Stock solution A and stock solution B were pumped into a tubular reactor at a flow rate of 0.018 mL / min, and it was placed in a constant temperature water bath at 45 °C to control the experimental temperature. The retention time was 15 min. Stock solution C was pumped into the tubular reactor at a flow rate of 0.2405 mL / min, and the residence time was 8 min. After the reaction, cold methanol and benzoic acid (quenching agent) were added. After stirring, it was precipitated at low temperature for 4 h. The precipitate was collected by filtration, air-dried and then placed in a vacuum drying oven for drying for 48 h to obtain the block copolymer PprCL-b-PLLA, which was detected by size exclusion chromatography.

[0086] Preparation of Block Copolymer PprCL-b-PLLA in Example 13

[0087] The chemoselective polymerization platform consisted of three 50 mL airtight syringes / syringe pumps, one PTFE tube (diameter = 2.0 mm, retention volume = 0.56 mL), one PTFE tube (diameter = 3.8 mm, retention volume = 2.22 mL) and one T-shaped mixer.

[0088] Stock solution A (PrCL (2000 mg, 12.82 mmol) and TBD (178.20 mg, 1.282 mmol) dissolved in 6.41 mL of dry toluene), stock solution B (benzyl alcohol (46.188 mg, 0.427 mmol) dissolved in 6.41 mL of dry toluene), and stock solution C (lactide (1846.08 mg, 12.82 mmol) dissolved in 12 mL of dry toluene) were prepared in a glove box filled with argon and transferred to 50 mL airtight syringes respectively. The assembled tubular reactor was rinsed with nitrogen and dry toluene to remove moisture and air.

[0089] Stock solution A and stock solution B were pumped into a tubular reactor at a flow rate of 0.018 mL / min, and it was placed in a constant temperature water bath at 45 °C to control the experimental temperature. The retention time was 15 min. Stock solution C was pumped into the tubular reactor at a flow rate of 0.111 mL / min, and the residence time was 17 min. After the reaction, cold methanol and benzoic acid (quenching agent) were added. After stirring, it was precipitated at low temperature for 4 h. The precipitate was collected by filtration, air-dried and then placed in a vacuum drying oven for drying for 48 h to obtain the block copolymer PprCL-b-PLLA, which was detected by size exclusion chromatography.

[0090] Preparation of Block Copolymer PprCL-b-PLLA in Example 14

[0091] The chemoselective polymerization platform consists of three 50 mL airtight syringes / syringe pumps, one PTFE tube (diameter = 2.0 mm, retention volume = 0.56 mL), one PTFE tube (diameter = 3.8 mm, retention volume = 2.22 mL), and one T-shaped mixer.

[0092] In the glove box, prepare the argon-filled stock solution A (PrCL (2000 mg, 12.82 mmol) and TBD (178.20 mg, 1.282 mmol) dissolved in 2.31 mL of dry toluene), stock solution B (benzyl alcohol (46.188 mg, 0.427 mmol) dissolved in 2.31 mL of dry toluene), and stock solution C (lactide (1846.08 mg, 12.82 mmol) dissolved in 12 mL of dry toluene), and transfer them to 50 mL airtight syringes respectively. Flush the assembled tubular reactor with nitrogen and dry toluene to remove moisture and air.

[0093] Pump stock solution A and stock solution B into the tubular reactor at a flow rate of 0.018 mL / min, place it in a 45 °C constant temperature water bath to control the experimental temperature, and the retention time is 15 min; monitor the process by size exclusion chromatography. Finally, pump stock solution C into the tubular reactor at a flow rate of 0.111 mL / min, and the residence time is 17 min. After the reaction is completed, add cold methanol and benzoic acid (quencher), stir, precipitate at low temperature for 4 h, filter to collect the precipitate, air-dry it, and then place it in a vacuum drying oven to dry for 48 h to obtain the block copolymer PprCL-b-PLLA, which is detected by size exclusion chromatography as Figure 5 shown.

[0094] From Figure 5 the GPC monitoring of the molecular weight distribution time at each stage of the polymerization analysis, it is found that the addition of the second monomer prolongs the molecular weight of the first-stage polymerization and shows only a single peak, further proving the successful synthesis of the block copolymer and the little change in the molecular weight distribution.

[0095] Table 2 Ring-opening polymerization experiments of Examples 8 - 14

[0096]

[0097] Preparation of homopolymer PprCL in Example 15

[0098] In the glove box, the purified propyl - caprolactone (1000 mg, 6.41 mmol), initiator benzyl alcohol (6.92 mg, 0.0641 mmol), and catalyst TBD (8.99 mg, 0.0641 mmol) were dissolved in 3 mL of toluene. Subsequently, it was transferred to an oil bath at 45 °C to start the reaction. After reacting for about 3 h, benzoic acid was added to terminate the reaction. It was precipitated at low temperature in cold methanol for 4 h, the precipitate was collected after filtering and washing three times, air - dried, and then placed in a vacuum drying oven for drying for 48 h, and analyzed by size - exclusion chromatography and proton nuclear magnetic resonance spectroscopy.

[0099] Preparation of Homopolymer PLA in Example 16

[0100] In the glove box, the purified lactide (1000 mg, 6.94 mmol), initiator benzyl alcohol (7.50 mg, 0.0694 mmol), and catalyst TBD (9.65 mg, 0.0694 mmol) were dissolved in 8 mL of toluene. Subsequently, it was transferred to an oil bath at 45 °C to start the reaction. After reacting for about 1 h, benzoic acid was added to terminate the reaction. It was precipitated at low temperature in cold methanol for 4 h, the precipitate was collected after filtering and washing three times, air - dried, and then placed in a vacuum drying oven for drying for 48 h, and analyzed by size - exclusion chromatography and proton nuclear magnetic resonance spectroscopy.

[0101] Table 3: Thermodynamic Data of Copolymers

[0102]

[0103] Note: a is the monomer feed ratio; b is the composition ratio, obtained by nuclear magnetic calculation; c is obtained by nuclear magnetic calculation; d is measured by DSC.

[0104] DSC Thermogram of Copolymers ( Figure 4 , in the figure, PLA70 - PPRCL30 is Example 8, PLA40 - PPRCL60 is Example 9, PLA50 - PPRCL50 is Example 10, PLA60 - PPRCL40 is Example 11) shows the influence of the average block length of LA on the glass transition temperature (Tg) and crystallization temperature (Tc) of the copolymer. After adding the PLA segment, it can be observed that the Tg of the copolymer is higher than that of amorphous P(prCL). In addition, the change in the crystallization temperature is due to the fact that the less favorable supramolecular chain arrangement may form microcrystals with a higher melting point, making the LA unit sequence in the copolymer tend to grow imperfect crystals.

[0105] The present invention provides an idea and method for synthesizing a novel bio-based polyester by using enzyme cascade catalysis combined with microfluidic field coupling. There are many methods and ways to specifically implement this technical solution. The above description is only the preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention. Each component not clearly defined in this embodiment can be implemented by using the existing technology.

Claims

1. A method for synthesizing a bio-based polyester, characterized in that, Synthesizing a bio - based polyester in a microchannel reactor includes the following steps: (1) In a first solvent, under the action of a catalyst, reacting propyl - caprolactone with an initiator in a first reaction; (2) Adding lactide and a second solvent to the first reaction system for a second reaction to obtain a bio - based polyester, poly(propylcaprolactone - b - lactide); The number - average molecular weight of the bio - based polyester is 1000 - 25000 g / mol; The preparation method of the propyl - caprolactone is using p - propylcyclohexanol as a raw material, and using alcohol reductase and polycyclic ketone monooxygenase as biocatalysts to obtain propyl - caprolactone through a cofactor regeneration cycle system; The alcohol reductase is a free - state alcohol reductase obtained by cloning the ADH gene (GenBank: AY267012.1) encoded by Lactobacillus kefir DSM 20587 into pET22b + and transferring it into Escherichia coli for culture; The polycyclic ketone monooxygenase is a free - state polycyclic ketone monooxygenase obtained by cloning the BVMO gene encoded by Trypanosoma thermophila ATCC 42464 into pET22b + and transferring it into Escherichia coli for culture; The catalyst is 1,5,7 - triazabicyclo(4.4.0)dec - 5 - ene.

2. The method according to claim 1, wherein The number - average molecular weight of the bio - based polyester is 2000 - 24000 g / mol.

3. The method according to claim 1, wherein The number - average molecular weight of the bio - based polyester is 6000 - 22000 g / mol.

4. The method according to claim 1, characterized in that, The solvent for the reaction is a combination of an organic solvent and a buffer solution.

5. The method according to claim 4, wherein The organic solvent accounts for 0.5% - 10% of the total volume of the solvent.

6. The method according to claim 4, wherein The organic solvent is any one or a combination of methanol, acetonitrile, dimethyl sulfoxide, isopropanol, and dioxane.

7. The method according to claim 4, wherein The buffer solution is a Tris - HCL buffer solution with a pH of 7 - 8.

8. The method according to claim 1, wherein In the reaction system, the concentration of p - propylcyclohexanol is 4 - 8 mM, the concentration of alcohol reductase is 0.1 - 0.5 g / mL, the concentration of polycyclic ketone monooxygenase is 0.1 - 0.5 g / mL, and the concentration of cofactor is 0.3 - 10 mM.

9. The method according to claim 1, wherein The temperature of the reaction is 25 - 35 °C.

10. The method according to claim 1, wherein In step (1), the first solvent is an organic solvent.

11. The method according to claim 10, wherein The organic solvent is any one or a combination of toluene, tetrahydrofuran, and dichloromethane.

12. The method according to claim 1, wherein In step (1), the initiator is an alcohol.

13. The method according to claim 1, wherein In step (1), the initiator is any one of n - hexanol, n - butanol, benzyl alcohol, and isopropanol.

14. The method according to claim 1, wherein In step (1), the concentration of propyl - caprolactone is 0.5 - 5 mol / L.

15. The method according to claim 1, characterized in that In step (1), the molar ratio of propyl - caprolactone to the initiator is 10 - 100:

1.

16. The method according to claim 1, characterized in that In step (1), the temperature of the reaction is 40 - 80 °C.

17. The method according to claim 1, characterized in that, In step (2), the second solvent is an organic solvent.

18. The method according to claim 1, characterized in that, In step (2), the second solvent is any one or a combination of toluene, tetrahydrofuran, and dichloromethane.

19. The method according to claim 1, wherein In step (2), the molar ratio of lactide to propyl - caprolactone is 0.5 - 10:

1.

20. The method according to claim 1, wherein In step (2), the temperature of the reaction is 25 - 80 °C.

21. The method according to claim 1, wherein Includes the following steps: S1: Carry out a first reaction on a first solution containing propyl-caprolactone, a catalyst, and a first solvent and a second solution containing an initiator in a first microreactor in a microchannel reaction device; S2: Carry out a second reaction on a third solution containing lactide and a second solvent and the effluent of the first reaction in a second microreactor in the microchannel reaction device to obtain a bio-based polyester, polypropyl caprolactone-b-lactide.

22. The method according to claim 21, wherein The volume ratio of the first solvent to the second solvent is 1:0.5 - 1.

5.

23. The method according to claim 21, wherein The pumping rates of the first solution and the second solution are 0.01 - 0.03 mL / min; the pumping rate of the third solution is 0.01 - 0.8 mL / min.

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