Method for preparing epsilon-caprolactone from 6-hydroxycaproic acid in biological catalytic liquid

Abstract

The invention discloses a method for preparing epsilon-caprolactone from 6-hydroxycaproic acid in a biological catalytic liquid. The specific process comprises the following steps: adding a certain proportion of isooctane in an enzyme catalysis process, and after the catalysis process is finished, carrying out microfiltration and ultrafiltration on a catalytic solution containing 6-hydroxyhexanoic acid to remove cells and proteins; if the concentration is relatively low, the 6-hydroxyhexanoic acid in the system is esterified into 6-hydroxyhexanoate under the action of a catalyst through nanofiltration and normal-temperature concentration, then alcohol is added, and after the temperature is raised, isooctane and water are azeotropic with water; 6-hydroxyhexanoate and excessive alcohol are in phase with water, and the upper layer is 6-hydroxyhexanoate and alcohol; and cyclizing the 6-hydroxyhexanoate at the upper layer into epsilon-caprolactone, and then separating out the epsilon-caprolactone through rectification.

Description

Technical Field

[0001] The present invention belongs to the technical field of biochemistry, and relates to a method for preparing ε-caprolactone from 6-hydroxyhexanoic acid in a biocatalytic solution. Background Art Caprolactone is a colorless and aromatic oily liquid with a boiling point of 235-236 °C and a melting point of -18 °C. Caprolactone is unstable and prone to polymerization. It is usually used as a novel polyester monomer and can be ring-opened and polymerized into polycaprolactone (PCL) under the conditions of a catalyst and an initiator. Polycaprolactone is a typical biodegradable polymer and has wide applications in the fields of biomedicine and environmental protection materials. Caprolactone is also often used to modify polymer materials to improve the performance of the materials.

[0002] Currently, the most commonly used method for industrial production of ε-caprolactone is the Baeyer-Villiger oxidation of cyclohexanone with peracids such as peracetic acid. There are not many domestic and foreign enterprises producing ε-caprolactone, mainly including BASF, Daicel, and Perstor. In China, there is Julong Chemical Industry, and some are still in the pilot stage.

[0003] A method for preparing ε-caprolactone disclosed in the US patent US6531615 applied by Solvay. This method is also a globally common industrial production method for ε-caprolactone. In this method, cyclohexanone and hydrogen peroxide are used as raw materials, and the molar ratio of cyclohexanone to hydrogen peroxide is 1:2 - 1:3. The catalyst is antimony trifluoride supported on mesoporous silica. The catalyst dosage per 100 ml of cyclohexanone does not exceed 2 g, and each gram of the catalyst contains 0.1 - 1.5 mmol SbF3. The catalyst and cyclohexanone are mixed at 70 °C for 5 - 120 minutes, and then hydrogen peroxide is added to ensure that the reaction occurs at 60 °C, 100 °C, and 50 - 150 mbar. In this method, hydrogen peroxide is an explosive substance, and the safety risk is high.

[0004] A method for preparing ε-caprolactone disclosed in US Patent No. US6156910 applied for by a Japanese enterprise in the road of the competition. This method uses cyclohexanone and peracetic acid as raw materials. The process includes reacting cyclohexanone, peracetic acid, and ethyl acetate (solvent / diluent, or it can be not used) at 50°C and then entering the first distillation column for separation. The boiling point of the solvent is preferably lower than that of caprolactone, which is beneficial to subsequent separation. The top temperature of the first distillation column is 30 - 40°C, the top pressure vacuum degree does not exceed 200 mmHg, the reflux ratio is 0.5 - 5, the bottom temperature is 120 - 180°C, the bottom of the kettle is mainly crude caprolactone, 0.5 - 3% of caprolactone and low-boiling substances are evaporated to the top, and the crude caprolactone enters the second distillation column. The theoretical number of plates in the rectifying section is 15 - 40 stages, the stripping section is 10 - 35, the top temperature is 110 - 130°C, the bottom temperature is 130 - 160°C, the top vacuum degree does not exceed 50 mmHg, the reflux ratio is 0.5 - 3. A third distillation column can also be added, with the same conditions as the second distillation column, but the number of plates is reduced and the residence time is shortened. The separation of the three distillation columns to obtain ε-caprolactone results in extremely high energy consumption.

[0005] US Patent No. 5,068,361 applied for by BASF discloses a method for preparing ε-caprolactone. In this method, in the presence of an oxidation catalyst (magnesium oxide, zinc oxide, cadmium oxide, aluminum oxide, and titanium dioxide), 6-hydroxycaproic acid ester is heated at 150°C to 450°C, and the vapor of 6-hydroxycaproic acid ester is passed through a fixed bed or a fluidized bed together with an inert carrier gas, and the amount of the inert carrier gas used is 10 - 30 times the amount of substance of 6-hydroxycaproic acid ester. The imperfection of this method is that it will consume a large amount of inert carrier gas.

[0006] Currently, in the large-scale production of caprolactone, both at home and abroad, the chemical method is adopted, using cyclohexanone as the raw material and oxidizing it with peroxyacid to produce high-purity ε-caprolactone. The biological method for preparing caprolactone still remains at the laboratory stage.

[0007] Currently, in the whole-cell catalysis constructed by the biological method in the laboratory, cyclohexanol is oxidized to cyclohexanone through ADH enzyme, and then cyclohexanone is oxidized to caprolactone by CHMO enzyme. However, the stability of CHMO is not high, and caprolactone will cause product inhibition to CHMO, resulting in a decrease in enzyme activity. Therefore, LAC enzyme is used to convert caprolactone into 6-hydroxycaproic acid. Summary of the Invention The present invention uses 6-hydroxycaproic acid in the catalytic solution prepared by the biological method as the raw material to prepare high-purity caprolactone. Low-molecular alcohols can be recycled, with low energy consumption and high yield. And the raw materials do not contain easily explosive peroxides, reducing potential safety hazards.

[0008] The technical solution adopted by the present invention is as follows: During the catalytic process, a certain proportion of isooctane is added, and the addition of isooctane makes the enzyme more active. After the catalytic reaction is completed, the fermentation broth containing 6-hydroxyhexanoic acid is first subjected to microfiltration and ultrafiltration, and then alcohols are added after low-temperature acidification to esterify 6-hydroxyhexanoic acid into 6-hydroxyhexanoic acid ester. During the heating process, isooctane and water form an azeotrope to remove water, and the water is removed from the system, shifting the esterification equilibrium to the right; the generated 6-hydroxyhexanoic acid ester, excessive alcohol, and water are phase-separated, and the upper layer is 6-hydroxyhexanoic acid ester and alcohol; the 6-hydroxyhexanoic acid ester in the upper layer is cyclized into ε-caprolactone; and then ε-caprolactone is separated by distillation.

[0009] A method for preparing ε-caprolactone from 6-hydroxyhexanoic acid in a biocatalytic solution, the method comprising the following steps: S1: Filtering the catalytic solution containing 6-hydroxyhexanoic acid; S2: Adding an acid for acidification, adding an alcohol to the acidified catalytic solution, removing water by azeotropy of isooctane and water during the heating process, oxidizing 6-hydroxyhexanoic acid into 6-hydroxyhexanoic acid ester under the action of a catalyst, phase-separating the 6-hydroxyhexanoic acid ester, excessive alcohol, and water, with the upper layer being 6-hydroxyhexanoic acid ester and alcohol, and the lower layer being an aqueous solution of hydrochloride, nitrate, or sulfate; S3: Separating the upper layer of 6-hydroxyhexanoic acid ester and alcohol from the lower phase, then cyclizing the 6-hydroxyhexanoic acid ester into ε-caprolactone, and purifying to prepare high-purity ε-caprolactone by distillation.

[0010] Preferably, the process for preparing the 6-hydroxyhexanoic acid catalytic solution comprises the following steps: 1) Adding a fermentation microorganism seed solution to a fermentation medium and performing fermentation to obtain a fermentation broth; 2) Enzymatic catalytic reaction: Using the fermentation broth after fermentation as a conversion system, continuously adding a substrate, with the substrate concentration being lower than 5 g / L, pH 6-9, and temperature 20-45 °C, and ending the conversion to obtain a catalytic solution when the substrate consumption rate < 0.5 g / L·h.

[0011] More preferably, during the enzymatic catalysis in step 2), an organic solvent isooctane is also added to form a two-phase fermentation enzymatic catalysis system; the added organic solvent is isooctane, and the addition ratio is 5-50 V / V% based on the volume of the fermentation broth. The addition of isooctane can increase the activity of the CHMO enzyme, and within a certain period of time, more cyclohexanol is converted into caprolactone.

[0012] More preferably, in step 2), the substrate is one or several of cyclohexanol, cyclohexane, and cyclohexanone; Using sodium hydroxide, ammonia water, potassium hydroxide, or others to control the pH at 6-9.

[0013] Further preferably, in the step 1), the fermentation microorganisms are yeast, Escherichia coli, coryneform bacteria, filamentous fungi or actinomycetes; the microorganisms used can be microorganisms isolated from natural environments or microorganisms obtained by mutation or gene recombination; the microorganisms can produce ADH, CHMO and LAC enzymes; glycerol tubes are streaked on solid medium to prepare single colonies, and the single colonies are added to the seed medium for cultivation to obtain seed liquid.

[0014] Further preferably, the solid medium is LB solid medium (1% peptone, 0.5% yeast powder, 1% sodium chloride, 1.5% agar, pH 7.0); the seed medium is prepared with LB medium (1% peptone, 0.5% yeast powder, 1% sodium chloride, pH 7.0) to obtain the seed solution. The fermentation medium is 15 - 25 g / L corn steep liquor dry powder, 0.8 - 1.2 g / L dipotassium hydrogen phosphate, 1.5 - 2.5 g / L potassium dihydrogen phosphate, 3 - 5 g / L ammonium sulfate, 10 - 12 g / L glucose monohydrate, and the feed supplement is 45 - 55% glucose monohydrate. The fermentation culture temperature is 35 - 38 °C, the induction temperature is 23 - 27 °C, and the fermentation period is 24 - 30 h to prepare the fermentation broth; preferably, the fermentation medium is 20 g / L corn steep liquor dry powder, 1 g / L dipotassium hydrogen phosphate, 2 g / L potassium dihydrogen phosphate, 4 g / L ammonium sulfate, 11 g / L glucose monohydrate, and the feed supplement is 50% glucose monohydrate), the fermentation culture temperature is 37 °C, the induction temperature is 25 °C, and the fermentation period is 24 - 30 h to prepare the fermentation broth.

[0015] Preferably, in the step S1, the filtration is microfiltration, ultrafiltration or nanofiltration to remove cells and proteins. Ultrafiltration can be carried out in one or multiple stages. In the first stage, 50KDa or 30KDa or 10KDa ultrafiltration membranes are used, and in the second stage, 3KDa or 1KDa ultrafiltration membranes are used. To remove bacteria and proteins, if necessary, a part of the bacteria can also be removed by centrifugation before this operation.

[0016] Preferably, in the step S2, when hydrochloric acid, nitric acid or sulfuric acid is added to acidify 6 - hydroxyhexanoic acid hydrochloride, the acidification temperature is 2 - 15 °C; the alcohol is methanol, ethanol, isopropanol, n - propanol, n - butanol or n - octanol. Alcohol is added to the acidified catalytic solution and heated. At a certain temperature, isooctane and water form an azeotrope, and water is taken out of the system. The low - molecular - weight alcohol esterifies 6 - hydroxyhexanoic acid in the catalytic solution to 6 - hydroxyhexanoic acid ester under the action of the catalyst.

[0017] Preferably, in step S2, the catalyst is an inorganic acid such as concentrated sulfuric acid, hydrochloric acid, phosphoric acid or an inorganic acid salt such as phthalate, zirconate or oxide. The inorganic acid is concentrated sulfuric acid, hydrochloric acid or phosphoric acid or others. The anhydrous acid salt is AlCl3, FeCl3, KHSO4, CH3COONa, ferric sulfate, ferrous sulfate, zinc sulfate, zinc chloride, copper sulfate, zirconium sulfate or cerium sulfate or others. The oxide is Al2O3, SiO2, ZnO, TiO2, tin oxide, stannous oxide, antimony trioxide or solid superacid or others. The oxidation temperature is 50 - 200 °C. The catalyst dosage is 0.1wt - 5wt% of 6-hydroxyhexanoic acid. The molar ratio of alcohol to acid is 1:(1 - 5).

[0018] In step S2, after the reaction, 6-hydroxyhexanoate, the excess alcohol and the water generated by esterification are separated into layers. The upper layer is 6-hydroxyhexanoate and alcohol, and the lower layer is the aqueous phase of hydrochloride or nitrate, sulfate.

[0019] Preferably, in step S3, the catalytic liquid after separation can be separated by overflow. The separated upper layer is heated to 200 - 350 °C, and 6-hydroxyhexanoate is cyclized to form caprolactone. The catalysts used in the cyclization process include alkaline earth metal oxides, alkaline earth metal carbonates, organic acids or inorganic acids. The catalyst dosage is 0.1%wt - 5wt% of 6-hydroxyhexanoic acid. The distillation of the excess alcohol during the cyclization process helps the reaction shift to the right, resulting in more ε-caprolactone production. More preferably, in step S3, the product after cyclization is rectified under reduced pressure to 0.001 - 0.05 Mpa, and the rectification temperature is controlled at 40 - 150 °C to separate ε-caprolactone from the polymer, and the distilled ε-caprolactone is condensed and collected. Before entering the rectification column, the pH of the product after cyclization should be controlled at 6.5 - 7.5, and it is rectified under reduced pressure to 0.001 - 0.05 Mpa, with the rectification temperature controlled at 40 - 150 °C. The temperature of the kettle should not exceed 150 °C. If it exceeds 150 °C, caprolactone will polymerize. ε-Caprolactone is separated from isooctane and low molecular weight alcohols. The low boiling point alcohols and isooctane are distilled out from the top of the rectification column, and high-purity ε-caprolactone is obtained from the side line or bottom, and the ε-caprolactone is condensed and collected.

[0020] The beneficial effects of the present invention are as follows: The production of 6-hydroxyhexanoic acid by biological method has mild conditions. However, directly extracting high-purity 6-hydroxyhexanoic acid from the catalytic solution usually requires a large amount of trioctylamine, a substance extremely destructive to natural water bodies. Moreover, high-purity 6-hydroxyhexanoic acid is chemically active and not easy to store and transport. Directly esterifying 6-hydroxyhexanoic acid in the fermentation broth to form stable 6-hydroxyhexanoic acid ester, and then cyclizing the 6-hydroxyhexanoic acid ester into the bulk chemical raw material ε-caprolactone effectively solves this problem. Moreover, compared with the production of ε-caprolactone using hydrogen peroxide or perpropionic acid (peracetic acid) as raw materials, this method has higher safety because hydrogen peroxide and peracids are both easy to explode; the process is simpler because the large-scale chemical production requires three distillation towers to separate the solvent dissolving the peracid, the acid, water, and ε-caprolactone, and the equipment required is cheaper. Not only that, frequent and long-term high-temperature operations will also cause the polymerization of ε-caprolactone, resulting in a decrease in the yield of ε-caprolactone. In the catalytic process of the present invention, isooctane is added, which can not only improve the activity of CHMO enzyme and the conversion efficiency of cyclohexanol, but also act as a water-carrying agent in the esterification reaction of 6-hydroxyhexanoic acid and low-molecular-weight alcohol, shifting the esterification reaction equilibrium to the right and obtaining a higher yield of 6-hydroxyhexanoic acid ester. Detailed implementation mode

[0021] The technical solution of the present invention will be further elaborated below in conjunction with the embodiments, but the scope of protection of the present invention is not limited thereto. The specific implementation modes described herein are only used to illustrate and explain the present disclosure, and are not used to limit the present disclosure. Equivalent substitutions or corresponding improvements made to the content of the present invention still fall within the scope of protection of the present invention.

[0022] The same conditions used in the following examples and comparative examples are as follows: The process for preparing the catalytic solution of 6-hydroxyhexanoic acid includes the following steps: 1) Add the fermentation microbial seed liquid to the fermentation medium and carry out fermentation to obtain the fermentation broth; 2) Enzymatic catalysis reaction: Using the fermentation broth after the end of fermentation as the conversion system, continuously add the substrate cyclohexanol, the substrate concentration is maintained at 4 g / L, pH 8, temperature 35 °C, and when the substrate consumption rate < 0.5 g / L*h, end the conversion to obtain the catalytic solution.

[0023] Further preferably, in the step 1), the fermentation microorganism is Escherichia coli; draw a line on the solid medium with a glycerol tube to prepare single colonies, and add the single colonies to the seed medium for cultivation to obtain the seed liquid. The solid medium is LB solid medium (1% peptone, 0.5% yeast powder, 1% sodium chloride, 1.5% agar, pH 7.0); the seed medium is LB medium (1% peptone, 0.5% yeast powder, 1% sodium chloride, pH 7.0) to prepare the seed liquid; The fermentation medium is 20 g / L corn steep liquor dry powder, 1 g / L dipotassium hydrogen phosphate, 2 g / L potassium dihydrogen phosphate, 4 g / L ammonium sulfate, 11 g / L glucose monohydrate, and the feeding is 50% glucose monohydrate). The fermentation temperature is 37 °C, the induction temperature is 25 °C, and the fermentation period is 26 h to prepare the fermentation broth. A method for preparing ε-caprolactone from 6-hydroxyhexanoic acid in a biocatalytic solution, the method comprising the following steps: S1: Filter the catalytic solution containing 6-hydroxyhexanoic acid; S2: Add acid for acidification, add alcohol to the acidified catalytic solution, remove water by azeotropic distillation of isooctane and water during heating, and esterify 6-hydroxyhexanoic acid into 6-hydroxyhexanoate under the action of an acid catalyst. The 6-hydroxyhexanoate and the excess alcohol are separated from the water phase. The upper layer is 6-hydroxyhexanoate and alcohol, and the lower layer is an aqueous solution of hydrochloride or nitrate, sulfate; S3: Separate the upper layer of 6-hydroxyhexanoate and alcohol from the lower phase, then cyclize 6-hydroxyhexanoate into ε-caprolactone, and purify to prepare high-purity ε-caprolactone by rectification.

[0024] Preferably, in the step S1, the filtration is microfiltration, ultrafiltration or nanofiltration to remove cells and proteins. The ultrafiltration is carried out in one or multiple stages. The first stage uses a 50 KDa or 30 KDa, 10 KDa ultrafiltration membrane, and the second stage uses a 3 KDa or 1 KDa ultrafiltration membrane. Remove bacteria and proteins.

[0025] Preferably, in the step S2, the catalyst is concentrated sulfuric acid, the esterification temperature is 50-200 °C, the catalyst dosage is 3 wt% of 6-hydroxyhexanoic acid, and the molar ratio of alcohol to acid is 1:3.

[0026] In the step S2, after the reaction, the 6-hydroxyhexanoate, the excess alcohol and the water generated by esterification are separated into layers. The upper layer is 6-hydroxyhexanoate and alcohol, and the lower layer is an aqueous phase of hydrochloride or nitrate, sulfate.

[0027] Preferably, in the step S3, the separated fermentation broth can be separated by overflow, and the separated upper layer is heated to 300 °C, and 6-hydroxyhexanoate is cyclized to form caprolactone; the catalyst used in the cyclization process is tetrabutyl titanate, and the catalyst dosage is 3 wt% of 6-hydroxyhexanoic acid. The distillation of the excess alcohol during the cyclization process helps the reaction to shift to the right and more ε-caprolactone is generated; Further preferably, in step S3, after cyclization, the product is depressurized to 0.004 Mpa for rectification, the rectification temperature is controlled at 100 °C, ε-caprolactone is separated from the polymer, and the distilled ε-caprolactone is condensed and collected. Before the cyclized product enters the rectification column, the pH should be controlled at 6.8, depressurized to 0.004 Mpa for rectification, the rectification temperature is controlled at 100 °C, and the kettle temperature should not exceed 150 °C. If it exceeds 150 °C, caprolactone will polymerize. ε-Caprolactone is separated from isooctane and low molecular weight alcohols. The low boiling point alcohols and isooctane are distilled out from the top of the rectification column, and high purity ε-caprolactone is obtained from the side line or bottom. The ε-caprolactone is condensed and collected. Preferably, in step S2, when hydrochloric acid, nitric acid or sulfuric acid is added to acidify 6-hydroxyhexanoic acid hydrochloride, the acidification temperature is 10 °C; the alcohol is methanol, ethanol, isopropanol, n-propanol, n-butanol or n-octanol. Alcohol is added to the acidified catalytic solution and heated. At a certain temperature, isooctane and water form an azeotrope, and water is taken out of the system. The low molecular weight alcohol esterifies 6-hydroxyhexanoic acid in the catalytic solution to 6-hydroxyhexanoic acid ester under the action of the catalyst. Example 1 On the basis of the above conditions, in step 2) during the enzymatic catalysis process, an organic solvent isooctane is also added to form a biphasic fermentation enzymatic catalysis system; the added organic solvent is isooctane, and the addition ratio is 10 V / V% of the fermentation broth by volume. The addition of isooctane can improve the activity of CHMO enzyme. Within a certain period of time, more cyclohexanol is converted into caprolactone.

[0028] Substrate cyclohexanol is continuously added dropwise, the substrate concentration is maintained at 4 g / L, pH 8, temperature 35 °C, dissolved oxygen content 40%. Cyclohexanol is continuously added. After 1 hour of detection, the conversion rate of cyclohexanol is 13%. After 2, 3, and 4 hours of detection, the conversion rates of cyclohexanol are 13%, 58%, and 82% respectively.

[0029] Comparative Example 1 On the basis of the above conditions, the difference is that: if organic solvent isooctane is not added in step 2) during the enzymatic catalysis process, and the others are the same as in Example 1. Substrate cyclohexanol is continuously added dropwise, the substrate concentration is maintained at 4 g / L, pH 8, temperature 35 °C, dissolved oxygen content 40%. Cyclohexanol is continuously added. After 1 hour of detection, the conversion rate of cyclohexanol is 8%. After 2, 3, and 4 hours of detection, the conversion rates of cyclohexanol are 20%, 34%, and 50% respectively.

[0030] It can be seen from Example 1 and Comparative Example 1 that adding isooctane during the enzymatic catalysis process helps to improve the enzyme activity, and the substrate conversion rate is thereby increased.

[0031] Example 2 The process for preparing the 6-hydroxyhexanoic acid catalytic solution includes the following steps: 1) Add the fermentation microorganism seed liquid to the fermentation medium and carry out fermentation to obtain the fermentation broth; 2) Enzymatic catalysis reaction: Using the post-fermentation broth after fermentation as the conversion system, continuously feed cyclohexanol as the substrate, maintaining the substrate concentration at 4 g / L, pH 8, and temperature 35 °C. When the substrate consumption rate < 0.5 g / L·h, the conversion is terminated to obtain the catalytic solution.

[0032] In step 1), the fermentation microorganism is Escherichia coli; streak the glycerol tube on the solid medium to prepare single colonies, and add the single colonies to the seed medium for cultivation to obtain the seed solution. The solid medium is LB solid medium (1% peptone, 0.5% yeast extract, 1% sodium chloride, 1.5% agar, pH 7.0); the seed medium is LB medium (1% peptone, 0.5% yeast extract, 1% sodium chloride, pH 7.0) to prepare the seed solution; The fermentation medium is 20 g / L corn steep liquor dry powder, 1 g / L dipotassium hydrogen phosphate, 2 g / L potassium dihydrogen phosphate, 4 g / L ammonium sulfate, 11 g / L glucose monohydrate, and the feeding supplement is 50% glucose monohydrate). The fermentation temperature is 37 °C, the induction temperature is 25 °C, and the fermentation period is 26 h to prepare the fermentation broth. The differences are as follows: Example 2-1: In step 2), take about 1.8 L of the fermentation broth after fermentation as the conversion system. During the catalysis process, continuously feed a total of 170 g of cyclohexanol, 120 g of 25% ammonia water, and 180 g of isooctane. After the catalysis is completed, obtain about 2.2 L of the catalytic solution containing 100 g / L of 6-hydroxyhexanoic acid.

[0033] S1: Filter through a 30 KDa ultrafiltration membrane in the first stage and a 1 KDa ultrafiltration membrane in the second stage at room temperature to remove a large amount of proteins and cells. Then concentrate the catalytic solution through a 100 Da nanofiltration membrane. After concentration, the content of 6-hydroxyhexanoic acid reaches 235 g / L.

[0034] S2: Take 220 mL of the concentrated catalytic solution in a beaker, place it in an ice-water bath, maintain the temperature at 5 °C, stir, slowly add sulfuric acid to adjust the pH to 1.0, add 50 g of n-butanol, connect a four-neck flask to an oil-water separator, heat, and azeotropically distill isooctane and a large amount of water out of the system. React for 2 hours, let it stand and separate layers. The upper layer is a mixture of butyl 6-hydroxyhexanoate, n-butanol, and isooctane. Collect the upper organic phase. Gas phase detection shows that it contains 55 g of butyl 6-hydroxyhexanoate, and the esterification rate can reach 74.7%.

[0035] Example 2-2: In step 2), take about 1.8 L of the fermentation broth after fermentation as the conversion system. During the catalysis process, continuously feed a total of 170 g of cyclohexanol, 120 g of 25% ammonia water, and 180 g of isooctane. After the catalysis is completed, obtain about 2.2 L of the catalytic solution containing 100 g / L of 6-hydroxyhexanoic acid.

[0036] S1 was first filtered through a 30KDa ultrafiltration membrane at room temperature and then through a 1KDa ultrafiltration membrane for secondary filtration, removing a large amount of proteins and cells. The catalytic solution was then concentrated through a 100Da nanofiltration membrane, and the 6-hydroxyhexanoic acid content reached 235g / L after concentration.

[0037] S2 Take 220 mL of the catalytic solution in a beaker, place it in an ice-water bath, maintain the temperature at 10°C, stir, slowly add sulfuric acid to adjust the pH to 1.0, add 50 g of n-butanol, connect a four-necked flask to an oil-water separator, heat, and azeotrope isooctane and a large amount of water out of the system. React for 2 hours, let it stand and then layer. The upper layer is a mixture of butyl 6-hydroxyhexanoate, n-butanol, and isooctane. Collect the upper organic phase. Gas phase detection shows that it contains 53 g of butyl 6-hydroxyhexanoate, and the esterification rate can reach 72.0%.

[0038] Example 2-3: In step 2), take about 1.8 L of the fermentation broth after fermentation as the conversion system. During the catalytic process, continuously feed 170 g of cyclohexanol, 120 g of 25% ammonia water, and 180 g of isooctane. After catalysis, about 2.2 L of the catalytic solution containing 100 g / L of 6-hydroxyhexanoic acid is obtained.

[0039] S1 was first filtered through a 30KDa ultrafiltration membrane at room temperature and then through a 1KDa ultrafiltration membrane for secondary filtration, removing a large amount of proteins and cells. The catalytic solution was then concentrated through a 100Da nanofiltration membrane, and the 6-hydroxyhexanoic acid content reached 235g / L after concentration.

[0040] S2 Take 220 mL of the catalytic solution in a beaker, place it in an ice-water bath, maintain the temperature at 15°C, stir, slowly add sulfuric acid to adjust the pH to 1.0, add 50 g of n-butanol, connect a four-necked flask to an oil-water separator, heat, and azeotrope isooctane and a large amount of water out of the system. React for 2 hours, let it stand and then layer. The upper layer is a mixture of butyl 6-hydroxyhexanoate, n-butanol, and isooctane. Collect the upper organic phase. Gas phase detection shows that it contains 50 g of butyl 6-hydroxyhexanoate, and the esterification rate can reach 67.9%.

[0041] Example 2-4: In step 2), take about 1.8 L of the fermentation broth after fermentation as the conversion system. During the catalytic process, continuously feed 170 g of cyclohexanol, 120 g of 25% ammonia water, and 180 g of isooctane. After catalysis, about 2.2 L of the catalytic solution containing 100 g / L of 6-hydroxyhexanoic acid is obtained.

[0042] S1 was first filtered through a 30KDa ultrafiltration membrane at room temperature and then through a 1KDa ultrafiltration membrane for secondary filtration, removing a large amount of proteins and cells. The catalytic solution was then concentrated through a 100Da nanofiltration membrane, and the 6-hydroxyhexanoic acid content reached 235g / L after concentration.

[0043] Take 220 mL of the catalytic solution in a beaker, place it in a cold water bath, maintain the temperature at 20 °C, stir, slowly add sulfuric acid to adjust the pH to 1.0, add 50 g of n-butanol. Connect a four-necked flask to an oil-water separator and heat. Isooctane and a large amount of water are carried out of the system by azeotropic distillation. React for 2 hours, let it stand and then separate the layers. The upper layer is a mixture of butyl 6-hydroxyhexanoate, n-butanol and isooctane. Collect the upper organic phase. Gas phase detection shows that it contains 30 g of butyl 6-hydroxyhexanoate, and the esterification rate can reach 40.7%. If there is a large amount of water in the esterification process system, it is not conducive to esterification. Isooctane can form an azeotrope with water and carry the water out of the system, which is conducive to the right shift of the esterification reaction equilibrium and the formation of more esters.

[0044] Comparative Example 2: Take about 1.8 L of the fermentation broth after fermentation as the conversion system. Continuously feed a total of 170 g of cyclohexanol and 120 g of 25% ammonia water during the catalytic process. After catalysis, about 2.0 L of the catalytic solution containing 90 g / L of 6-hydroxyhexanoic acid is obtained.

[0045] S1: Filter through a 30 KDa ultrafiltration membrane in the first stage and a 1 KDa ultrafiltration membrane in the second stage at room temperature to remove a large amount of proteins and cells. Then concentrate the fermentation broth through a 100 Da nanofiltration membrane. After concentration, the content of 6-hydroxyhexanoic acid reaches 235 g / L. S2: Take 220 mL of the fermentation broth in a beaker, place it in an ice water bath, keep it at 10 °C, stir, slowly add sulfuric acid to adjust the pH to 1.0, add 50 g of n-butanol. In the flask, reflux and react at 90 °C for 2 hours. Let it stand and then separate the layers. The upper layer is a mixture of butyl 6-hydroxyhexanoate and n-butanol. Collect the upper organic phase. Gas phase detection shows that it contains 36 g of butyl 6-hydroxyhexanoate, and the esterification rate is 48.9%.

[0046] Through Examples 2-1, 2-2, 2-3, and 2-4, maintaining a lower temperature during the acidification process of the catalytic solution is beneficial to ensuring the stability of 6-hydroxyhexanoic acid, thereby increasing the yield of 6-hydroxyhexanoate. From Example 2-2 and Comparative Example 2, it can be seen that without adding isooctane during the catalytic process, water cannot be carried out by the azeotropic distillation of isooctane and water in the subsequent separation and esterification stage, and the esterification effect is poor. Example 3 The process for preparing the 6-hydroxyhexanoic acid catalytic solution includes the following steps: 1) Add the fermentation microorganism seed liquid to the fermentation medium and carry out fermentation to obtain the fermentation broth; 2) Enzymatic catalytic reaction: Use the fermentation broth after fermentation as the conversion system, continuously flow-feed the substrate cyclohexanol, maintain the substrate concentration at 4 g / L, pH 8, and temperature 35 °C. When the substrate consumption rate < 0.5 g / L·h, end the conversion to obtain the catalytic solution.

[0047] In step 1), the fermentation microorganism is Escherichia coli. The glycerol stock is streaked on a solid medium to prepare single colonies, and the single colonies are added to a seed medium for cultivation to obtain a seed solution. The solid medium is LB solid medium (1% peptone, 0.5% yeast extract, 1% sodium chloride, 1.5% agar, pH 7.0); the seed medium is LB medium (1% peptone, 0.5% yeast extract, 1% sodium chloride, pH 7.0) to prepare the seed solution; The fermentation medium is 20 g / L corn steep liquor dry powder, 1 g / L dipotassium hydrogen phosphate, 2 g / L potassium dihydrogen phosphate, 4 g / L ammonium sulfate, 11 g / L glucose monohydrate, and the feeding is 50% glucose monohydrate). The fermentation temperature is 37 °C, the induction temperature is 25 °C, and the fermentation period is 26 h to prepare the fermentation broth. The difference lies in: Example 3-1: On the basis of the above experimental conditions, in step 2) of the enzymatic catalysis reaction: about 1.8 L of the fermentation broth after fermentation is taken as the conversion system, and a total of 170 g of cyclohexanol, 120 g of 25% ammonia water, and 180 g of isooctane are continuously fed during the catalysis process. After the catalysis, about 2.2 L of the catalytic solution containing 100 g / L of 6-hydroxyhexanoic acid is obtained.

[0048] S1 is first filtered through a 30KDa ultrafiltration membrane and then through a 1KDa ultrafiltration membrane at room temperature to remove a large amount of proteins and cells. Then, the catalytic solution is concentrated through a 100Da nanofiltration membrane, and the content of 6-hydroxyhexanoic acid after concentration reaches 235 g / L.

[0049] S2: Take 220 mL of the catalytic solution in a beaker, place it in an ice-water bath, maintain the temperature at 5 °C, stir, slowly add sulfuric acid to adjust the pH to 1.0, add 50 g of ethanol, connect a four-neck flask to an oil-water separator, heat, and isooctane and a large amount of water are taken out of the system by azeotropic distillation. React for 2 hours, let it stand and separate layers. The upper layer is a mixture of ethyl 6-hydroxyhexanoate, ethanol, and isooctane. Collect the upper organic phase, and gas phase detection shows that it contains 40 g of ethyl 6-hydroxyhexanoate, and the esterification rate can reach 63.8%.

[0050] Example 3-2, based on Example 3-1, the difference is that 60 g of ethanol is added after acidification in step S2, and the feeding and other processes are the same as those in Example 3-1. Gas phase detection shows that it contains 45 g of ethyl 6-hydroxyhexanoate, and the esterification rate can reach 71.8%.

[0051] Example 3-3, based on Example 3-1, the difference is that 70 g of ethanol is added after acidification in step S2, and the feeding and other processes are the same as those in Example 3-1. Gas phase detection shows that it contains 48 g of ethyl 6-hydroxyhexanoate, and the esterification rate can reach 76.6%. Comparative Example 3-1: Based on Example 3-1, the difference is that 10 g of ethanol is added after acidification in step S2, and the feeding and other processes are the same as those in Example 3-1. After detection, it contains 8 g of ethyl 6-hydroxyhexanoate, and the esterification rate can reach 12.8%.

[0052] Comparative Example 3-2: Based on Example 3-1, the difference is that 15 g of ethanol is added after acidification in step S2, and the feeding and other processes are the same as those in Example 3-1. After detection, it contains 9 g of ethyl 6-hydroxyhexanoate, and the esterification rate can reach 14.4%.

[0053] It can be seen from Example 3-1, Example 3-2, Example 3-3, Comparative Example 3-1, 3-2, and 3-3 that a higher content of alcohol during the esterification process is beneficial to maintaining the stability of 6-hydroxyhexanoic acid and preventing intermolecular polymerization of 6-hydroxyhexanoic acid at a higher temperature; at the same time, a high alcohol-acid ratio is also beneficial to shifting the esterification reaction equilibrium to the right, thereby increasing the esterification rate.

[0054] Example 4: A rectifying column filled with packing is connected above a four-necked flask. 0.5 g of tetrabutyl titanate is added as a catalyst to the flask, and 100 g of the mixture of ethyl 6-hydroxyhexanoate and ethanol in Example 3-3 is refluxed and heated at 180 °C for cyclization. While 61 g of a mixture of ethanol and a small amount of water is distilled off at the top, the reaction is stopped after 1 hour. The product remaining at the bottom of the flask is weighed as 39 g. After detection, the bottom of the flask contains 30 g of ε-caprolactone.

[0055] Example 5: Under 0.05 Mpa and a kettle temperature of 145 °C, the bottom product containing 30 g of ε-caprolactone in Example 4 is subjected to vacuum distillation, and 25 g of distillate is collected. After detection, the purity of ε-caprolactone is 99.5%.

[0056] The above embodiments are only the preferred technical solutions of the present invention and should not be regarded as limitations on the present invention. The embodiments and the features in the embodiments in this application can be arbitrarily combined with each other without conflict. The protection scope of the present invention should be the technical solutions recorded in the claims, including the equivalent replacement solutions of the technical features in the technical solutions recorded in the claims. That is, the equivalent replacement improvements within this scope are also within the protection scope of the present invention.

Claims

1. A method for preparing ε-caprolactone from 6-hydroxyhexanoic acid in a biocatalytic solution, characterized in that, The method includes the following steps: S1: Filter the catalytic solution containing 6-hydroxyhexanoic acid; S2: Add an acid for acidification, add an alcohol to the acidified catalytic solution, remove water by azeotropic distillation of isooctane and water during heating, oxidize 6-hydroxyhexanoic acid to 6-hydroxyhexanoate under the action of a catalyst, separate the 6-hydroxyhexanoate and the excessive alcohol from water, with the upper layer being 6-hydroxyhexanoate and alcohol, and the lower layer being an aqueous solution of hydrochloride or nitrate or sulfate; S3: Separate the upper layer of 6-hydroxyhexanoate and alcohol from the lower phase, then cyclize the 6-hydroxyhexanoate into ε-caprolactone, and purify it by rectification to prepare high-purity ε-caprolactone.

2. The method according to claim 1, wherein: The process for preparing the catalytic solution of 6-hydroxyhexanoic acid includes the following steps: 1) Add a fermentation microbial seed solution to a fermentation medium, and perform fermentation to obtain a fermentation broth; 2) Enzymatic catalytic reaction: Using the fermentation broth after fermentation as a conversion system, continuously add a substrate, with the substrate concentration being lower than 5 g / L, pH 6-9, temperature 20-45 °C, and end the conversion to obtain a catalytic solution when the substrate consumption rate < 0.5 g / L*h.

3. The method according to claim 2, characterized in that: During the enzymatic catalysis in step 2), an organic solvent isooctane is also added to form a two-phase fermentation enzymatic catalytic system; the added organic solvent is isooctane, and the addition ratio is 5-50 V / V% based on the volume of the fermentation broth.

4. The method according to claim 2, wherein: In step 2), the substrate is one or more of cyclohexanol, cyclohexane, and cyclohexanone; Use sodium hydroxide, ammonia water, potassium hydroxide or others to control the pH at 6-9.

5. The method according to claim 2, wherein: In step 1), the fermentation microorganism is yeast, Escherichia coli, coryneform bacteria, filamentous fungi or actinomycetes; the microorganisms used can be microorganisms isolated from the natural environment, or microorganisms obtained by mutation or gene recombination; the microorganisms can produce ADH, CHMO and LAC enzymes; streak a glycerol tube on a solid medium to prepare single colonies, and add the single colonies to a seed medium for culture to obtain a seed solution.

6. The method according to claim 5, characterized in that: The solid medium is an LB solid medium (peptone 1%, yeast powder 0.5%, sodium chloride 1%, agar 1.5%, pH 7.0); the seed medium is an LB medium (peptone 1%, yeast powder 0.5%, sodium chloride 1%, pH 7.0) for preparing the seed solution; The fermentation medium is 15-25 g / L of corn steep liquor dry powder, 0.8-1.2 g / L of dipotassium hydrogen phosphate, 1.5-2.5 g / L of potassium dihydrogen phosphate, 3-5 g / L of ammonium sulfate, 10-12 g / L of glucose monohydrate, the feeding is 45-55% of glucose monohydrate, the fermentation culture temperature is 35-38 °C, the induction temperature is 23-27 °C, and the fermentation period is 24-30 h to prepare a fermentation broth; preferably, the fermentation medium is 20 g / L of corn steep liquor dry powder, 1 g / L of dipotassium hydrogen phosphate, 2 g / L of potassium dihydrogen phosphate, 4 g / L of ammonium sulfate, 11 g / L of glucose monohydrate, the feeding is 50% of glucose monohydrate, the fermentation culture temperature is 37 °C, the induction temperature is 25 °C, and the fermentation period is 24-30 h to prepare a fermentation broth.

7. The method according to claim 1, wherein: In the step S1, the filtration is microfiltration, ultrafiltration or nanofiltration to remove cells and proteins. The ultrafiltration can be in one stage or multiple stages. In the first stage, a 50KDa, 30KDa or 10KDa ultrafiltration membrane is used, and in the second stage, a 3KDa or 1KDa ultrafiltration membrane is used.

8. The method according to claim 1, wherein: In the step S2, when hydrochloric acid, nitric acid or sulfuric acid is added to acidify 6-hydroxyhexanoic acid hydrochloride, the acidification temperature is 2-15°C; the alcohol is methanol, ethanol, isopropanol, n-propanol, n-butanol or n-octanol. Alcohol is added to the acidified catalytic solution and heated. At 70-75°C, isooctane and water form an azeotrope, and water is taken out of the system. Under the action of the catalyst, the low-molecular-weight alcohol esterifies 6-hydroxyhexanoic acid in the catalytic solution into 6-hydroxyhexanoic acid ester.

9. The method according to claim 1, wherein: In the step S2, the catalyst is an inorganic acid such as concentrated sulfuric acid, hydrochloric acid, phosphoric acid or an inorganic acid salt such as phthalate, zirconate or oxide. The inorganic acid is concentrated sulfuric acid, hydrochloric acid or phosphoric acid or others. The anhydrous acid salt is AlCl3, FeCl3, KHSO4, CH3COONa, iron sulfate, ferrous sulfate, zinc sulfate, zinc chloride, copper sulfate, zirconium sulfate or cerium sulfate or others. The oxide is Al2O3, SiO2, ZnO, TiO2, tin oxide, stannous oxide, antimony trioxide or solid superacid or others. The oxidation temperature is 50-200°C. The catalyst dosage is 0.1wt-5wt% of 6-hydroxyhexanoic acid, and the molar ratio of alcohol to acid is 1:(1-5).

10. In the step S2, after the reaction, the 6-hydroxyhexanoic acid ester, the excess alcohol and the water generated by esterification are separated into layers. The upper layer is the 6-hydroxyhexanoic acid ester and alcohol, and the lower layer is the aqueous phase of hydrochloride or nitrate, sulfate.

11. The method according to claim 1, wherein: In the step S3, the separated upper layer is heated to 200-350°C, and the 6-hydroxyhexanoic acid ester undergoes cyclization. The catalysts used in the cyclization process include alkaline earth metal oxides, alkaline earth metal carbonates, organic acids or inorganic acids. The catalyst dosage is 0.1%wt-5wt% of 6-hydroxyhexanoic acid. The distillation of the excess alcohol during the cyclization process helps the reaction shift to the right, generating more ε-caprolactone. In the step S3, the product after cyclization is rectified under reduced pressure to 0.001-0.05 Mpa, and the rectification temperature is controlled at 40-150°C to separate ε-caprolactone from the polymer, and the distilled ε-caprolactone is condensed and collected.