Enzyme catalyst for preparing dicarboxylic acid and method for preparing dicarboxylic acid by using enzyme catalyst

Abstract

The present invention relates to an enzyme catalyst for preparing a dicarboxylic acid and a method for preparing a dicarboxylic acid using the same. The enzyme catalyst is prepared by a method comprising the following steps: (1) carrying out fermentation culture on enzyme-producing microorganisms in a culture medium to obtain a crude enzyme solution, and (2) immobilizing the crude enzyme solution on a molecular sieve to obtain the enzyme catalyst, wherein the enzyme producing microorganisms are selected from candida tropicalis, candida sake, pichia pastoris, saccharomyces cerevisiae, lactobacillus brevis, corynebacterium glutamicum, bacillus subtilis, bacillus cereus, escherichia coli and pseudomonas aeruginosa. The method comprises the following steps: in a PBS buffer solution, mixing triglyceride with the enzyme catalyst, and carrying out catalytic reaction under stirring at 20-50 DEG C; and then carrying out hydrolysis reaction at the temperature of 250-300 DEG C under the pressure of 30-100 bar. According to the method, the enzyme catalyst and the molecular sieve are immobilized, so that other oxidants or catalysts are not required to be added while the dicarboxylic acid with higher yield is obtained.

Description

Technical Field

[0001] The present invention relates to a method for preparing dicarboxylic acid, and more particularly to an enzyme catalyst for preparing dicarboxylic acid and a method for preparing dicarboxylic acid using the enzyme catalyst, in particular to a method for preparing and purifying dicarboxylic acid, which uses oil as raw material, obtains a mixture of triglycerides having more than one acid functional group through esterification and hydrolysis under enzyme catalysis, and performs a series of extraction and crystallization operations. Background Art

[0002] There are many methods for preparing dicarboxylic acids. For example, high-yield carboxylic acids are obtained by oxidative dissociation of renewable resources (animal oils, vegetable oils, unsaturated fatty acids and their derivatives). Through these methods, a mixture of monocarboxylic acids and dicarboxylic acids or their derivatives is obtained.

[0003] Azelaic acid is a medium-chain organic dibasic fatty acid, which is widely used in plastics, plasticizers, lubricants, coatings, resins and other industries. For example, nylon materials such as nylon 9 and nylon 69 synthesized based on azelaic acid, plasticizers such as dioctyl azelate and isobutyl azelate, and lubricants such as dioctyl azelate and ditridecyl azelate. In addition, it also has great application potential in medical and aesthetic materials and electrical materials.

[0004] The preparation methods of azelaic acid at home and abroad can be divided into unsaturated fatty acid oxidation method, ricinoleic acid oxidation method, wax oil thermal cracking method and microbial oxidation method. Among them, the most commonly used method in industry is unsaturated fatty acid oxidation method, and the raw material is mainly oleic acid. Unsaturated fatty acids react with glycerol at high temperature to form triglycerides. When triglycerides are subjected to oxidative dissociation process, a mixture of monocarboxylic acid and triglycerides containing saturated carboxylic acid with more than one acid functional group is obtained.

[0005] Chinese patent application CN101200735A discloses a method for preparing azelaic acid by enzyme-catalyzed hydrogen peroxide oxidation of oleic acid, but it requires the addition of H2O2 as an oxidant while using lipase as the catalyzer. Summary of the invention

[0006] The purpose of the present invention is to overcome the shortcomings of the prior art, avoid the use of strong oxidants, and prepare and separate dicarboxylic acids through enzyme catalytic reactions after the raw oil is glycerol esterified. The method does not require high temperature and high pressure, and does not require the addition of other oxidants or catalysts.

[0007] According to one aspect of the present invention, there is provided an enzyme catalyst for preparing dicarboxylic acid, wherein the enzyme catalyst is prepared by a method comprising the following steps: (1) fermenting an enzyme-producing microorganism in a culture medium to obtain a crude enzyme solution, (2) immobilizing the crude enzyme solution on a molecular sieve to obtain an enzyme catalyst,

[0008] Wherein, the enzyme-producing microorganism is selected from Candida tropicalis, Candida sake, Pichia sp., Saccharomyces cerevisiae, Lactobacillus brevis, Corynebacterium glutamicum, Bacillus subtilis, Bacillus cereus, Escherichia coli, and Pseudomonas aeruginosa.

[0009] According to another aspect of the present invention, there is provided a method for preparing an enzyme catalyst for preparing dicarboxylic acid, the method comprising the following steps: (1) fermenting an enzyme-producing microorganism in a culture medium to obtain a crude enzyme solution, (2) immobilizing the crude enzyme solution on a molecular sieve to obtain an enzyme catalyst,

[0010] Wherein, the enzyme-producing microorganism is selected from Candida tropicalis, Candida sakei, Pichia pastoris, Saccharomyces cerevisiae, Lactobacillus brevis, Corynebacterium glutamicum, Bacillus subtilis, Bacillus cereus, Escherichia coli, and Pseudomonas aeruginosa.

[0011] In some embodiments, the enzyme-producing microorganism is preferably selected from Candida tropicalis, Escherichia coli, Saccharomyces cerevisiae, and Bacillus subtilis.

[0012] According to the present invention, the enzyme-producing microorganisms can be purchased commercially.

[0013] In some embodiments, the method includes: inoculating the enzyme-producing microorganism into a culture medium, culturing at 20-40°C and 150-350rpm for 15-30 hours; when the bacterial concentration OD600 reaches 0.6-0.8, adding isopropyl-β-D-thiogalactopyranoside, continuing to culture at 20-30°C for 8-20 hours, collecting the bacterial liquid, centrifuging to obtain a bacterial cell precipitate, and then resuspending the collected cell precipitate in potassium dihydrogen phosphate buffer, after ultrasonic disruption, centrifuging to remove cell debris and unbroken cells, and the supernatant is the crude enzyme solution.

[0014] In some embodiments, the crude enzyme solution is immobilized on the molecular sieve at a mass ratio of the molecular sieve to the crude enzyme solution of 5:1-1:1.

[0015] In some embodiments, the crude enzyme solution is immobilized on the molecular sieve in the following manner: the molecular sieve is added to the crude enzyme solution at a mass ratio of the molecular sieve to the crude enzyme solution of 5:1-1:1, and the solution is vigorously stirred, for example, with a glass rod, and after the addition is completed, the solution is stirred magnetically for 10-15 hours at room temperature to obtain an initial mixed gel system of the synthetic crude enzyme solution; PBS buffer is then added to the mixed gel system obtained above, and the solution is stirred magnetically for 20-36 hours until the pH value of the solution is between 7 and 8 to obtain a molecular sieve-immobilized enzyme catalyst mixed system.

[0016] In one embodiment, the culture medium is selected from LB medium, YPD medium, and M9 medium.

[0017] In one embodiment, the culture medium contains antibiotics to prevent contamination.

[0018] In one embodiment, the culture medium is LB medium containing 10-1500 μg / mL kanamycin sulfate.

[0019] In one embodiment, during the culture process in the culture medium, when the bacterial cell concentration OD600 reaches 0.6-0.8, isopropyl-β-D-thiogalactopyranoside is added at a final concentration of 0.01-0.2 mM (eg, 0.05, 0.1, 0.15 mM).

[0020] In one embodiment, the enzyme catalyst is prepared by a method comprising the following steps: inoculating an enzyme-producing microorganism selected from tropical Candida, Escherichia coli, Saccharomyces cerevisiae, and Bacillus subtilis into a culture medium containing 30-100 μg / mL kanamycin sulfate, the culture medium also containing 0.5-2.0 g / L pancreatin, 0.5-1.5 g / L yeast extract and 1-3 g / L glucose, 0.5-2 g / L yeast nitrogen base, culturing at 20-40°C and 150-350 rpm for 15-30 hours; when the bacterial cell concentration OD600 reaches 0.6-0 .8, add isopropyl-β-D-thiogalactopyranoside at a final concentration of 0.15mM, continue to culture at 20-30℃ for 8-20h, collect the bacterial solution, centrifuge at 4℃, 6793×g for 1-5min to obtain the bacterial cell precipitate, and then resuspend the collected cell precipitate in 100-200mM, pH7.0-8.0 potassium dihydrogen phosphate buffer at 4℃, after ultrasonic disruption, centrifuge at 4℃, 10012×g for 20-40min to remove cell debris and unbroken cells, and the supernatant is the crude enzyme solution.

[0021] In one embodiment, the molecular sieve type may be MTW molecular sieve, MFI molecular sieve, ZSM molecular sieve, etc., preferably MTW zeolite molecular sieve.

[0022] In one embodiment, the molecular sieve is a hierarchical pore zeolite synthesized by using a structure directing agent.

[0023] In one embodiment, the BET specific surface area of ​​the molecular sieve is 350-400 m 2 g -1 .

[0024] In one embodiment, the surface area S of the molecular sieve obtained by the t-plot method is ext 50-150m 2 g -1 In one embodiment, the micropore volume of the molecular sieve obtained by the t-plot method is 0.10-0.15 cm 3 g -1 .

[0025] In one embodiment, the mesoporous volume of the molecular sieve obtained by BJH desorption is 0.1-0.5 cm 3 g -1 .

[0026] In one embodiment, the molecular sieve is synthesized by a method comprising the following steps: adding a structure directing agent to deionized water, stirring, adding NaAlO2 thereto, stirring until completely dissolved, and then adding a silica sol aqueous solution dropwise; stirring and aging the obtained sol at room temperature and then transferring it to a polytetrafluoroethylene reactor liner for sealing; then placing the reactor in an oven for static crystallization, then cooling, and when the temperature of the reactor drops to room temperature, separating, washing, drying, and calcining.

[0027] In one embodiment, the morphology of the molecular sieve is mainly adjusted by adjusting the synthesis parameters (such as sol ratio, aging time, crystallization temperature, etc.).

[0028] In one embodiment, the structure directing agent of the molecular sieve is selected from 1,8-octanediamine, methyltriethylammonium chloride, hexamethyleneimine, tetraethylammonium hydroxide, preferably tetraethylammonium hydroxide.

[0029] In one embodiment, the amount of the structure directing agent of the molecular sieve is 40-70 wt % of the amount of the silica sol.

[0030] In one embodiment, the molecular sieve may have a H2O / SiO2 ratio of 4-50, preferably 10-40.

[0031] In one embodiment, the Si / Al ratio of the molecular sieve may be 20-50, preferably 30-40.

[0032] In one embodiment, the stirring aging time may be 2-5 hours, preferably 3 hours.

[0033] In one embodiment, the crystallization temperature range of the molecular sieve is 150-180°C, such as 160, 170°C, etc., and the crystallization time is 40-150h, such as 60h, 80h, 100h, 120h, etc.

[0034] In one embodiment, the molecular sieve is calcined at a temperature ranging from 400 to 600° C., such as 450, 500, 550° C., etc., and the calcination time is 5 to 10 h, such as 6 h, 8 h, etc.

[0035] In one embodiment, the enzyme content in the enzyme catalyst is 0.5-2 mg / mL, such as 1 mg / mL, 1.5 mg / mL, 2 mg / mL.

[0036] According to another aspect of the present invention, there is provided a method for preparing a dicarboxylic acid, the method comprising:

[0037] In PBS buffer, triglyceride is mixed with the above enzyme catalyst, and catalytic reaction is carried out under stirring at 20-50°C; then, hydrolysis reaction is carried out at 250-300°C and 30-100 bar pressure.

[0038] In one embodiment, the mass ratio of triglyceride to enzyme is 1:0.0001-0.005, preferably 1:0.0002-0.002

[0039] In one embodiment, the enzyme catalyzed reaction is carried out at a pH of 6.5 to 10.0, preferably 7.0 to 9.5.

[0040] In one embodiment, the enzyme catalyzed reaction temperature is 25-40°C and the reaction time is 30-60h.

[0041] In one embodiment, after the hydrolysis reaction, the monoacid (e.g., propionic acid, hexanoic acid, nonanoic acid) is removed by extraction, and then the reaction mixture after the removal of the monoacid is cooled to 70-90° C., an extractant (e.g., an alkane, such as heptane, octane, decane) is added, the organic phase and the aqueous phase are separated, and the organic phase is treated to obtain a dicarboxylic acid. In particular, after the organic phase is filtered to remove the enzyme catalyst, the dicarboxylic acid is obtained by evaporating the solvent, acidifying, extracting, separating, recrystallizing, and drying. In particular, the above-mentioned acidification can be adjusted by adding hydrochloric acid to the system so that the pH of the reaction system is 3.5-5.5.

[0042] According to one embodiment of the present invention, the triglyceride is a triglyceride containing unsaturated carboxylic acid prepared by esterification reaction of fatty acids or fatty acids in oils and glycerol. In particular, the reaction conditions are as follows: normal pressure, reaction temperature of 180-350°C, preferably 200-280°C; molar ratio of glycerol to fatty acid of 0.2:1-4:1, preferably 0.6:1-3:1; nitrogen purge, reaction time of 1-6 hours, preferably 2-5 hours.

[0043] According to an embodiment of the present invention, the oils and fats include various waste oils and fats, such as waste cooking oil, soybean acidified oil and palmitic acidified oil, croton oil, sunflower oil, castor oil, peanut oil, and palm oil.

[0044] The present invention allows triglyceride containing unsaturated carboxylic acid to react with an enzyme catalyst to first obtain an intermediate product in which the terminal alkyl group is oxidized to a carboxyl group, and then continues the enzyme catalytic reaction to obtain triglyceride of carboxylic acid having more than one acid functional group and a small amount of monocarboxylic acid; hydrolyzes the mixture containing triglyceride to obtain a reaction product containing dicarboxylic acid and glycerol; separates an aqueous phase containing at least a portion of glycerol from an organic phase containing dicarboxylic acid; evaporates and / or distills the organic phase to separate the residue; and obtains dicarboxylic acid through crystallization and drying.

[0045] According to the present invention, the triglyceride can be a mixture of carboxylic acid triglycerides containing more than one acid functional group. It can be a mixture containing a single triglyceride or a mixture containing multiple triglycerides, that is, the triglycerides are the same or different and contain at least one dicarboxylic acid acyl group.

[0046] According to the present invention, suitable dicarboxylic acids are aliphatic diacids, preferably saturated C6-C24 chain aliphatic diacids, and preferably α, ω type, which are selected from, for example, oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, pimelic acid, suberic acid, azelaic acid, sebacic acid, undecane dioic acid, dodecanedioic acid, tridecanedioic acid, tetradecanedioic acid or pentadecanedioic acid. Preferably, the dicarboxylic acid is selected from adipic acid, suberic acid, azelaic acid, sebacic acid, undecane dioic acid, dodecanedioic acid, tridecanedioic acid and mixtures thereof. More preferably, the starting material mainly contains azelaic acid triglyceride.

[0047] In the present invention, the enzyme catalyst and the zeolite molecular sieve are immobilized, and while a higher yield of dicarboxylic acid such as azelaic acid is obtained, no other oxidant or catalyst needs to be added. After the reaction is completed, the enzyme catalyst can be recycled and reused, and the dicarboxylic acid can be obtained through simple separation and purification. The oil conversion rate is high, the application range is wide, the production cost is low, the implementation conditions are simple, and it is easy to carry out large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1The graph shows the change of the overall morphology over time during the immobilization process of the crude enzyme solution prepared with tropical yeast and MTW zeolite molecular sieve in the preparation example.

[0049] Figure 2 The transmission electron micrograph of the Candida tropicalis MTW zeolite-immobilized enzyme catalyst in the preparation example is shown. DETAILED DESCRIPTION

[0050] The following examples are only listed as examples of embodiments of the present invention and do not constitute any limitation to the present invention. It can be understood by those skilled in the art that modifications within the scope of the essence and concept of the present invention fall within the scope of protection of the present invention. Unless otherwise specified, the reagents and instruments used in the following examples are all commercially available products.

[0051] As mentioned above, in view of the many defects of the prior art, the inventor of this case has proposed the technical solution of the present invention after long-term in-depth research and extensive practice, which is described in detail below.

[0052] Preparation Example: Preparation of Catalyst

[0053] Preparation of crude enzyme solution: Inoculate enzyme-producing microorganisms, such as Candida tropicalis, Escherichia coli, Saccharomyces cerevisiae, and Bacillus subtilis, into a culture medium (1.0 g / L pancreatin, 0.5 g / L yeast extract, 1.5 g / L glucose, and 1 g / L yeast nitrogen base) containing 60 μg / mL kanamycin sulfate, and culture at 25°C and 200 rpm for 20 hours. When the bacterial concentration OD600 reaches 0.6-0.8, add isopropyl-β-D-thiogalactopyranoside (final concentration is 0.15 mM), continue to culture at 25°C for 12 hours, collect the bacterial solution, and centrifuge at 4°C and 6793×g for 1-5 minutes to obtain the bacterial cell precipitate. Then, at 4°C, the collected cell pellet was resuspended in potassium dihydrogen phosphate buffer (150 mM, pH 7.0-8.0), and after ultrasonic disruption, centrifuged at 4°C, 10012×g for 20-40 min to remove cell debris and unbroken cells. The supernatant was the crude enzyme solution (i.e., a mixed solution of enzyme and buffer salt, in which the enzyme content was about 1 mg / mL).

[0054] Preparation of MTW zeolite molecular sieve: Add 7.1g of tetraethylammonium hydroxide to 1.5g of deionized water, stir for 10min, add 0.17g of NaAlO2 to the above solution, stir until completely dissolved, and then add 11.5g of silica sol (40wt% aqueous solution) dropwise. After stirring and aging the obtained sol at room temperature for 3h, transfer it to a 25mL polytetrafluoroethylene reactor liner and seal it. Then put the reactor into a 160℃ oven for static crystallization for 80h. When the temperature of the reactor drops to room temperature, filter and separate, wash with deionized water several times, and dry the obtained sample in an 80℃ oven overnight. Finally, calcine the obtained white powder at 500℃ for 6h.

[0055] The specific surface area and pore data of the obtained MTW zeolite molecular sieve are shown in the following table:

[0056]

[0057] a By t-plot method; b Using BJH desorption data

[0058] Enzyme immobilization: Weigh an appropriate amount of the MTW zeolite molecular sieve prepared above and add it to the crude enzyme solution so that the mass ratio of molecular sieve: crude enzyme solution = 3:1, and stir vigorously with a glass rod while adding. After the addition is completed, magnetic stirring is performed at room temperature for 12 hours to obtain an initial mixed gel system of the synthetic crude enzyme solution; then weigh an appropriate amount of PBS buffer, add it to the mixed gel system obtained above, and magnetic stirring is performed for 36 hours until the pH value of the solution is between 7 and 8 to obtain a molecular sieve-immobilized enzyme catalyst mixed system.

[0059] Reagent sources and detection methods

[0060] High oleic peanut oil was purchased from Luhua Group; sunflower oil was purchased from Fulinmen; castor oil was purchased from Jiangsu Hai'an Petrochemical Plant; croton oil was purchased from Jiangxi Global Natural Flavor Co., Ltd.; glycerin was purchased from Sinopharm Group; culture media such as kanamycin sulfate and yeast paste were purchased from Maclean; tropical Candida was purchased from Ningbo Mingzhou Biotechnology Co., Ltd. with a product number of B89745; sake Candida CICC 33015 was purchased from Beijing Yuwei Technology Co., Ltd.; brewer's yeast was purchased from Ningbo Mingzhou Biotechnology Co., Ltd. with a product number of B312024; Bacillus subtilis was purchased from Ningbo Mingzhou Biotechnology Co., Ltd. with a product number of B98052; Escherichia coli was purchased from Ningbo Mingzhou Biotechnology Co., Ltd. with a product number of B81036; columnar Candida was purchased from Ningbo Testo Biotechnology Co., Ltd. with a product number of TS286496; octane and other organic solvents were purchased from Inokane; NaAlO2, tetraethylammonium hydroxide, and silica sol were all purchased from Aladdin. The products in the following examples were detected by gas chromatography-mass spectrometry (GC-MS) (Agilent, Santa Clara, CA, USA) to analyze the TMS derivatives of fatty acids. The gas chromatography-mass spectrometry (GC-MS) was equipped with a flame ionization detector and a split injection system and equipped with an SPB-1 capillary column (15 m x 0.32 mm inner diameter, 0.25 μm thickness) (Supelco, Bellfonte, PA, USA). The peak positions of the samples were: intermediate products of terminal carboxylic acids 14.426 min; azelaic acid 8.403 min; propionic acid 5.032 min; caproic acid 7.910 min; succinic acid 2.494 min; adipic acid 4.321 min; sebacic acid 9.867 min.

[0061] Inlet temperature: 250℃, column flow: 0.6mL / min, split ratio: 40:1, electron ionization (EI) source, ion source temperature: 250℃, interface temperature: 220℃, injection volume: 0.8uL, full scan mode, mass-to-charge ratio range: 35-500, total time 41min. Column temperature: 80℃, keep for 2min, then increase to 125℃ at a rate of 20℃ / min and keep for 1min. Then increase to 220℃ at a rate of 3℃ / min and keep for 5min.

[0062] The concentration of diacids during the conversion process was measured by high performance liquid chromatography (HPLC) with the purified product as the standard. High performance liquid chromatography analysis and quantification (Shimadazu LC-20A HPLC) was performed on a C18 reverse phase column with a size of 150×4.6mm and a particle size of 5μm. The mobile phase composition was: 10% H2O, 5% acetonitrile, 5% formic acid solution (≥98%, AR) and 80% methanol, v / v. Sample elution positions: intermediate products of terminal carboxylic acids 13.07min; azelaic acid 7.06min; propionic acid 4.331min; hexanoic acid 6.745min; succinic acid 1.862min; adipic acid 3.752min; sebacic acid 8.394min.

[0063] The grain morphology of the samples was measured using a Hitachi S-4800 cold-field high-resolution emission scanning electron microscope.

[0064] Example 1 Using high oleic acid peanut oil as raw material

[0065] Step (1) Esterification reaction of oil and glycerol to prepare triglyceride containing unsaturated carboxylic acid: glycerol and peanut oil with a molar ratio of glycerol to fatty acid of about 1:3 are added to a reactor, reacted at 200° C., and reacted for 3 hours under nitrogen purge to obtain peanut oleic acid triglyceride.

[0066] Step (2) 300 mL of PBS buffer solution with a pH of 7.0, 20 g of arachidonic acid triglyceride obtained in step (1) and 5 ml of tropical Candida MTW zeolite-immobilized enzyme catalyst solution are added to the reaction container in sequence, stirred evenly at 30° C. and 300 rpm with a stirring paddle, reacted for 20 hours, and the reaction conversion rate was about 60% by HPLC detection, and the presence of an intermediate product in which the terminal alkyl group was oxidized to a carboxyl group was detected by GC-MS in the reaction solution. The reaction was stirred for 20 hours, and the conversion rate of the arachidonic acid triglyceride by HPLC detection was about 91%.

[0067] Step (3) hydrolyzing the mixture containing triglycerides: hydrolyzing the mixture at 280°C and 50 bar for 4 hours to obtain a reaction product containing a small amount of monoacid (such as propionic acid, caproic acid, etc.), azelaic acid and glycerol; separating the monoacid by vacuum rotary evaporation; cooling the hydrolyzed reaction mixture to 80°C, adding octane in an amount equal to 4 wt%, and centrifuging the mixture to separate the aqueous phase containing glycerol from the organic phase. The glycerol can be recovered by purification and concentration.

[0068] Step (4) Filter the organic phase to recover the enzyme catalyst, rotary evaporate and recover the solvent, add hydrochloric acid to adjust the pH of the reaction system to 4.0, extract with hot water, recrystallize, wash with water, filter and dry to obtain 14.58g of the target product, azelaic acid. After testing, the total yield of azelaic acid is 81% (based on oleic acid content) and the purity is 95%.

[0069] Example 2 Using castor oil with low oleic acid content as raw material

[0070] Except that peanut oil is replaced with castor oil, the other process steps are the same as those in Example 1. After reacting for 20 hours, the conversion rate of castor oil detected by HPLC is about 66%, and the intermediate product of terminal alkyl being oxidized to carboxyl is detected by GC-MS in the reaction solution. The reaction is continued with stirring for 20 hours, and the conversion rate of ricinoleic acid triglyceride detected by HPLC is about 88%. After subsequent treatment, 10.5 g of sebacic acid is obtained, with a total yield of 75% (based on ricinoleic acid content) and a purity of 92%; 0.4 g of azelaic acid, a total yield of 13% (based on oleic acid content), and a purity of 90%.

[0071] Example 3

[0072] Step (1) Esterification reaction of oil and glycerol to prepare triglyceride containing unsaturated carboxylic acid: Glycerol with a molar ratio of 1:3 and the high oleic acid content peanut oil in Example 1 are added to a reactor, reacted at 200° C., and purged with nitrogen for 3 hours to obtain peanut oleic acid triglyceride.

[0073] Step (2) 300 mL of PBS buffer solution with a pH of 7.0, 20 g of arachidonic acid triglyceride obtained in step (1) and 5 ml of Escherichia coli MTW zeolite immobilized enzyme catalyst solution are added to the reaction container in sequence, stirred evenly at 30° C. and 300 rpm with a stirring paddle, reacted for 20 hours, and the reaction conversion rate was about 53% by HPLC detection, and the presence of an intermediate product in which the terminal alkyl group was oxidized to a carboxyl group was detected by GC-MS in the reaction solution. The reaction was stirred for 20 hours, and the conversion rate of the arachidonic acid triglyceride by HPLC detection was about 85%.

[0074] Step (3) hydrolyzing the mixture containing triglycerides: hydrolyzing the mixture at 280°C and 50 bar for 4 hours to obtain a reaction product containing a small amount of monoacid (such as propionic acid, caproic acid, etc.), azelaic acid and glycerol; separating the monoacid by hot water extraction; cooling the hydrolyzed reaction mixture to 80°C, adding octane in an amount equal to 4 wt%, and centrifuging the mixture to separate the aqueous phase containing glycerol from the organic phase. The glycerol can be recovered by purification and concentration.

[0075] Step (4) Filter the organic phase to recover the enzyme catalyst, rotary evaporate and recover the solvent, add hydrochloric acid to adjust the pH of the reaction system to 4.0, extract with hot water, recrystallize, wash with water, filter and dry to obtain 7.16 g of the target product, azelaic acid. After testing, the total yield of azelaic acid is 42% (based on oleic acid content) and the purity is 86%.

[0076] Example 4

[0077] Step (1) Esterification reaction of oil and glycerol to prepare triglyceride containing unsaturated carboxylic acid: Glycerol with a molar ratio of 1:3 and the high oleic acid content peanut oil in Example 1 are added to a reactor, reacted at 200° C., and purged with nitrogen for 3 hours to obtain peanut oleic acid triglyceride.

[0078] Step (2) 300 mL of PBS buffer solution with a pH of 7.0, 20 g of arachidonic acid triglyceride obtained in step (1) and 5 ml of MTW zeolite-immobilized enzyme catalyst solution of Saccharomyces cerevisiae were added to the reaction container in sequence, stirred evenly at 30° C. and 300 rpm with a stirring paddle, and reacted for 20 hours. The reaction conversion rate was about 57% by HPLC, and the presence of an intermediate product in which the terminal alkyl group was oxidized to a carboxyl group was detected by GC-MS. The reaction was continued to stir for 20 hours, and the conversion rate of arachidonic acid triglyceride by HPLC was about 82%.

[0079] Step (3) hydrolyzing the mixture containing triglycerides: hydrolyzing the mixture at 280°C and 50 bar for 4 hours to obtain a reaction product containing a small amount of monoacid (such as propionic acid, caproic acid, etc.), azelaic acid and glycerol; separating the monoacid by hot water extraction; cooling the hydrolyzed reaction mixture to 80°C, adding octane in an amount equal to 4 wt%, and centrifuging the mixture to separate the aqueous phase containing glycerol from the organic phase. The glycerol can be recovered by purification and concentration.

[0080] Step (4) Filter the organic phase to recover the enzyme catalyst, rotary evaporate and recover the solvent, add hydrochloric acid to adjust the pH of the reaction system to 4.0, extract with hot water, recrystallize, wash with water, filter and dry to obtain 11.32 g of the target product, azelaic acid. After testing, the total yield of azelaic acid is 64% (based on oleic acid content) and the purity is 84%.

[0081] Example 5

[0082] Except for the use of 5 ml of Bacillus subtilis MTW zeolite immobilized enzyme catalyst solution in step (2), the other process steps are the same as those in Example 1. The reaction is carried out for 20 hours. The reaction conversion rate is about 48% by HPLC detection, and the presence of an intermediate product in which the terminal alkyl is oxidized to a carboxyl group is detected by GC-MS. The reaction is continued to be stirred for 20 hours. The conversion rate of peanut oleic acid triglyceride by HPLC detection is about 73%. After subsequent treatment, 10.22 g of the target product azelaic acid is obtained, with a total yield of 57% (based on oleic acid content) and a purity of 90%.

[0083] Example 6

[0084] Step (1) Esterification reaction of oil and glycerol to prepare triglyceride containing unsaturated carboxylic acid: Glycerol and croton oil in a molar ratio of 1:3 are added to a reactor, reacted at 200° C., and purged with nitrogen for 3 hours to obtain croton oleic acid triglyceride.

[0085] Step (2) 300 mL of PBS buffer solution with a pH of 7.0, 20 g of croton oleic acid triglyceride obtained in step (1) and 5 ml of tropical Candida MTW zeolite immobilized enzyme catalyst solution are added to the reaction container in sequence, stirred evenly at 30° C. and 300 rpm with a stirring paddle, reacted for 20 hours, and the reaction conversion rate was about 53% by HPLC detection, and the presence of an intermediate product in which the terminal alkyl group was oxidized to a carboxyl group was detected by GC-MS in the reaction solution. The reaction was stirred for 20 hours, and the conversion rate of the croton oleic acid triglyceride by HPLC detection was about 85%.

[0086] Step (3) hydrolyzing the mixture containing triglycerides: hydrolyzing the mixture at 280° C. and 50 bar for 4 hours to obtain a reaction product containing a small amount of monoacid (such as propionic acid, caproic acid, etc.) and glycerol; separating the monoacid by hot water extraction; cooling the hydrolyzed reaction mixture to 80° C., adding octane in an amount equal to 4 wt %, and centrifuging the mixture to separate the aqueous phase containing glycerol from the organic phase. The glycerol can be recovered by purification and concentration.

[0087] Step (4) Filter the organic phase to recover the enzyme catalyst, rotary evaporate and recover the solvent, add hydrochloric acid to adjust the pH of the reaction system to 4.0, extract with hot water, recrystallize, wash with water, filter and dry to obtain 8.54 g of the target product adipic acid. After testing, the total yield of adipic acid is 58% (based on the content of croton oleic acid) and the purity is 93%.

[0088] Example 7

[0089] Step (1) Esterification reaction of oil and glycerol to prepare triglyceride containing unsaturated carboxylic acid: Glycerol and sunflower oil with a molar ratio of 1:3 are added to a reactor, reacted at 200° C., and purged with nitrogen for 3 hours to obtain vegetable oil triglyceride.

[0090] Step (2) 300 mL of PBS buffer solution with a pH of 7.0, 20 g of the vegetable oleic acid triglyceride obtained in step (1) and 5 ml of the Escherichia coli MTW immobilized enzyme catalyst solution were added to the reaction container in sequence, stirred evenly at 30° C. and 300 rpm with a stirring paddle, and reacted for 20 hours. The reaction conversion rate was about 53% by HPLC, and the presence of an intermediate product in which the terminal alkyl group was oxidized to a carboxyl group was detected by GC-MS in the reaction solution. The reaction was stirred for 20 hours, and the conversion rate of the vegetable oleic acid triglyceride by HPLC was about 85%.

[0091] Step (3) hydrolyzing the mixture containing triglycerides: hydrolyzing the mixture at 280° C. and 50 bar for 4 hours to obtain a reaction product containing a small amount of monoacid (such as propionic acid, caproic acid, etc.) and glycerol; separating the monoacid by hot water extraction; cooling the hydrolyzed reaction mixture to 80° C., adding octane in an amount equal to 4 wt %, and centrifuging the mixture to separate the aqueous phase containing glycerol from the organic phase. The glycerol can be recovered by purification and concentration.

[0092] Step (4) Filter the organic phase to recover the enzyme catalyst, rotary evaporate and recover the solvent, add hydrochloric acid to adjust the pH of the reaction system to 4.0, extract with hot water, recrystallize, wash with water, filter and dry to obtain 11.03 g of the target product suberic acid. After testing, the total yield of suberic acid is 65% (based on oleic acid content) and the purity is 86%.

[0093] The bacterial solutions in the following comparative examples were all directly used from the commercially available bacterial solutions mentioned above.

[0094] Comparative Example 1

[0095] Step (1) Esterification reaction of oil and glycerol to prepare triglyceride containing unsaturated carboxylic acid: Glycerol with a molar ratio of 1:3 and the high oleic acid content peanut oil in Example 1 or the low oleic acid content castor oil in Example 2 are added to a reactor, reacted at 200° C., and purged with nitrogen for 3 hours to obtain triglyceride containing unsaturated carboxylic acid.

[0096] Step (2) 300 mL of PBS buffer solution with a pH of 7.0 and 20 g of triglyceride obtained in step (1) were added to the reaction container in sequence, stirred evenly at 30°C and 300 rpm with a stirring paddle, and reacted for 20 hours. HPLC detected that there was no conversion. The reaction was stirred for 20 hours, and the reaction conversion rate was still 0 when detected by HPLC. This indicates that the reaction in Example 1 or 2 cannot proceed without adding an enzyme catalyst.

[0097] Comparative Example 2

[0098] Step (1) Esterification reaction of oil and glycerol to prepare triglyceride containing unsaturated carboxylic acid: Glycerol with a molar ratio of 1:3 and the high oleic acid peanut oil in Example 1 or the low oleic acid castor oil fatty acid in Example 2 are added to a reactor, reacted at 200° C., and purged with nitrogen for 3 hours to obtain triglyceride containing unsaturated carboxylic acid.

[0099] Step (2) 300 mL of PBS buffer solution with a pH of 7.0, 20 g of triglyceride obtained in step (1) and 5 ml of tropical Candida solution were added to the reaction container in sequence, stirred evenly at 30°C and 300 rpm with a stirring paddle, and reacted for 20 hours. HPLC detected no conversion. The reaction was continued to stir for 20 hours, and the reaction conversion rate was still 0 when detected by HPLC. This indicates that the reaction in Example 1 or 2 cannot be carried out if the tropical Candida solution is directly added.

[0100] Comparative Example 3

[0101] Step (1) Esterification reaction of oil and glycerol to prepare triglyceride containing unsaturated carboxylic acid: Glycerol with a molar ratio of 1:3 and the high oleic acid content peanut oil in Example 1 are added to a reactor, reacted at 200° C., and purged with nitrogen for 3 hours to obtain triglyceride containing unsaturated carboxylic acid.

[0102] Step (2) 300 mL of PBS buffer solution with a pH of 7.0, 20 g of triglyceride obtained in step (1) and 5 ml of E. coli liquid were added to the reaction container in sequence, stirred evenly at 30°C and 300 rpm with a stirring paddle, and reacted for 20 hours. HPLC detected that there was no conversion. The reaction was stirred for 20 hours, and the reaction conversion rate was still 0 when detected by HPLC. This indicates that the reaction in Example 3 cannot be carried out if the E. coli liquid is directly added.

[0103] Comparative Example 4

[0104] Step (1) Esterification reaction of oil and glycerol to prepare triglyceride containing unsaturated carboxylic acid: Glycerol with a molar ratio of 1:3 and the high oleic acid content peanut oil in Example 1 are added to a reactor, reacted at 200° C., and purged with nitrogen for 3 hours to obtain triglyceride containing unsaturated carboxylic acid.

[0105] Step (2) 300 mL of PBS buffer solution with a pH of 7.0, 20 g of triglyceride obtained in step (1) and 5 ml of saccharomyces cerevisiae liquid were added to the reaction container in sequence, and stirred evenly at 30°C and 300 rpm with a stirring paddle, and reacted for 20 hours. HPLC detected that there was no conversion. The reaction was stirred for 20 hours, and the reaction conversion rate was still 0 when detected by HPLC. This indicates that the reaction in Example 4 cannot be carried out if the saccharomyces cerevisiae liquid is directly added.

[0106] Comparative Example 5

[0107] Step (1) Esterification reaction of oil and glycerol to prepare triglyceride containing unsaturated carboxylic acid: Glycerol with a molar ratio of 1:3 and the high oleic acid content peanut oil in Example 1 are added to a reactor, reacted at 200° C., and purged with nitrogen for 3 hours to obtain triglyceride containing unsaturated carboxylic acid.

[0108] Step (2) 300 mL of PBS buffer solution with a pH of 7.0, 20 g of triglyceride obtained in step (1) and 5 ml of Bacillus subtilis liquid were added to the reaction container in sequence, stirred evenly at 30°C and 300 rpm with a stirring paddle, and reacted for 20 hours. HPLC detected no conversion. The reaction was stirred for 20 hours, and the reaction conversion rate was still 0 when detected by HPLC. This indicates that the reaction in Example 5 cannot be carried out if the Bacillus subtilis liquid is directly added.

[0109] Comparative Example 6

[0110] Step (1) Esterification reaction of oil and glycerol to prepare triglyceride containing unsaturated carboxylic acid: Glycerol with a molar ratio of 1:3 and the high oleic acid peanut oil in Example 1 or the low oleic acid castor oil fatty acid in Example 2 are added to a reactor, reacted at 200° C., and purged with nitrogen for 3 hours to obtain triglyceride containing unsaturated carboxylic acid.

[0111] Step (2) 300 mL of PBS buffer solution with a pH of 7.0, 20 g of triglyceride obtained in step (1) and 5 ml of columnar Candida liquid were added to the reaction container in sequence, stirred evenly at 30°C and 300 rpm with a stirring paddle, and reacted for 20 hours. HPLC detected no conversion. The reaction was continued to stir for 20 hours, and the reaction conversion rate detected by HPLC was still 0. This indicates that the reaction in Example 1 or 2 cannot be carried out by directly adding the columnar Candida liquid.

[0112] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An enzyme catalyst for preparing a dicarboxylic acid, wherein: The enzyme catalyst is prepared by a method comprising the following steps: (1) fermenting an enzyme-producing microorganism in a culture medium to obtain a crude enzyme solution; (2) immobilizing the crude enzyme solution on a molecular sieve to obtain an enzyme catalyst; Wherein, the enzyme-producing microorganism is selected from Candida tropicalis, Candida sakei, Pichia pastoris, Saccharomyces cerevisiae, Lactobacillus brevis, Corynebacterium glutamicum, Bacillus subtilis, Bacillus cereus, Escherichia coli, and Pseudomonas aeruginosa.

2. The enzyme catalyst according to claim 1, wherein The culture medium is selected from LB culture medium, YPD culture medium, M9 culture medium; and / or The culture medium is LB culture medium containing 10-1500 μg / mL kanamycin sulfate; and / or During the culture process of the culture medium, when the bacterial concentration OD600 reaches 0.6-0.8, isopropyl-β-D-thiogalactopyranoside is added at a final concentration of 0.01-0.2 mM; and / or The molecular sieve is selected from MTW molecular sieve, MFI molecular sieve, and ZSM molecular sieve; in particular, the molecular sieve is a hierarchical pore zeolite synthesized by a structure directing agent; more particularly, the molecular sieve is synthesized by a method comprising the following steps: adding a structure directing agent to deionized water, stirring, adding NaAlO2 thereto, stirring until completely dissolved, and then adding a silica sol aqueous solution dropwise; stirring and aging the obtained sol at room temperature, and then transferring it to a polytetrafluoroethylene reactor liner for sealing; then placing the reactor in an oven for static crystallization, and then cooling, and when the temperature of the reactor drops to room temperature, separating, washing, drying, and calcining; and / or The BET specific surface area of ​​the molecular sieve is 350-400m 2 g -1 ; and / or The surface area of ​​the molecular sieve obtained by the t-plot method is 50-150m 2 g -1 ; and / or The micropore volume of the molecular sieve obtained by the t-plot method is 0.10-0.15 cm 3 g -1 ; and / or The mesoporous volume of the molecular sieve obtained by BJH desorption is 0.1-0.5 cm 3 g -1 ; and / or The enzyme content in the enzyme catalyst is 0.5-2 mg / mL.

3. The enzyme catalyst according to claim 1 or 2, wherein The method comprises: inoculating enzyme-producing microorganisms into a culture medium, culturing at 20-40°C and 150-350rpm for 15-30 hours; when the bacterial concentration OD600 reaches 0.6-0.8, adding isopropyl-β-D-thiogalactopyranoside, continuing to culture at 20-30°C for 8-20 hours, collecting bacterial liquid, centrifuging to obtain bacterial cell sediment, then resuspending the collected cell sediment in potassium dihydrogen phosphate buffer, ultrasonically disrupting, and centrifuging to remove cell debris and unbroken cells, and the supernatant is the crude enzyme liquid.

4. The enzyme catalyst according to any one of claims 1 to 3, wherein The crude enzyme solution is immobilized on the molecular sieve in the following manner: the molecular sieve is added to the crude enzyme solution according to a mass ratio of the molecular sieve to the crude enzyme solution of 5:1-1:1, and the mixture is vigorously stirred while being added. After the addition is completed, the mixture is stirred at room temperature for, for example, 10-15 hours to obtain an initial mixed gel system of the synthetic crude enzyme solution; and PBS buffer is added to the mixed gel system obtained above, and the mixture is stirred for, for example, 20-36 hours until the pH value of the solution is between 7 and 8 to obtain a molecular sieve-immobilized enzyme catalyst mixed system.

5. The enzyme catalyst according to any one of claims 1 to 4, wherein The enzyme catalyst is prepared by a method comprising the following steps: inoculating an enzyme-producing microorganism selected from tropical Candida, Escherichia coli, Saccharomyces cerevisiae, and Bacillus subtilis into a culture medium containing 30-100 μg / mL kanamycin sulfate, wherein the culture medium further contains 0.5-2.0 g / L pancreatin, 0.5-1.5 g / L yeast extract, 1-3 g / L glucose, and 0.5-2 g / L yeast nitrogen base, and culturing at 20-40° C. and 150-350 rpm for 15-30 hours; when the bacterial cell concentration OD600 reaches 0.6-0.8, Add isopropyl-β-D-thiogalactopyranoside at a final concentration of 0.15 mM, continue to culture at 20-30°C for 8-20 h, collect the bacterial solution, centrifuge at 4°C, 6793×g for 1-5 min to obtain the bacterial cell precipitate, and then resuspend the collected cell precipitate in 100-200 mM, pH 7.0-8.0 potassium dihydrogen phosphate buffer at 4°C, after ultrasonic disruption, centrifuge at 4°C, 10012×g for 20-40 min to remove cell debris and unbroken cells, and the supernatant is the crude enzyme solution.

6. A method for preparing a dicarboxylic acid, the method comprising: In a PBS buffer, triglyceride is mixed with the enzyme catalyst according to any one of claims 1 to 5, and a catalytic reaction is carried out at 20-50° C. under stirring; then a hydrolysis reaction is carried out at 250-300° C. and a pressure of 30-100 bar.

7. The method according to claim 6, wherein: The mass ratio of triglyceride:enzyme is 1:0.0001-0.005; and / or The enzyme catalyzed reaction is carried out at a pH of 6.5 to 10.0; and / or The enzyme catalytic reaction temperature is 25-40°C, and the reaction time is 30-60h.

8. The method according to claim 6 or 7, wherein: After the hydrolysis reaction, the monoacid (e.g., propionic acid, hexanoic acid, nonanoic acid) is removed by extraction, and then the reaction mixture after the removal of the monoacid is cooled to 70-90°C, an extractant (e.g., an alkane, such as heptane, octane, decane) is added, the organic phase and the aqueous phase are separated, and the organic phase is treated to obtain the dicarboxylic acid. In particular, the organic phase is filtered to remove the enzyme catalyst, and then the dicarboxylic acid is obtained by evaporating the solvent, acidifying, extracting, separating, recrystallizing, and drying. In particular, the above-mentioned acidification can be adjusted by adding hydrochloric acid to the system so that the pH of the reaction system is 3.5-5.

5.

9. The method according to any one of claims 6 to 8, wherein: The triglyceride is a triglyceride containing unsaturated carboxylic acid prepared by esterification of fatty acids or fatty acids in oils and glycerol. In particular, the reaction conditions are as follows: normal pressure, reaction temperature of 180-350° C., preferably 200-280° C.; molar ratio of glycerol to fatty acid of 0.2:1-4:1; nitrogen purge, reaction time of 1-6 hours, In particular, the oils and fats include various waste oils and fats, such as waste cooking oil, soybean acidified oil and palmitic acidified oil, sunflower seed oil, castor oil, peanut oil, and palm oil.

10. The method according to any one of claims 6 to 9, wherein: The dicarboxylic acid is selected from saturated C6-C24 chain aliphatic diacids, and preferably α,ω type, selected from oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, pimelic acid, suberic acid, azelaic acid, sebacic acid, undecanedioic acid, dodecanedioic acid, tridecanedioic acid, tetradecanedioic acid or pentadecanedioic acid.

Images