A method for preparing N-acyl amino acid compounds based on deep eutectic solvent

Abstract

The present invention discloses a one-step method for directly synthesizing N-acyl amino acid compounds based on deep eutectic solution and using a deep eutectic solvent as a medium. The preparation method of the deep eutectic solvent provided by the present invention uses an amino acid surfactant to produce a substrate as a component, reduces the addition of additional compounds in the reaction system, reduces production costs, and simplifies the post-processing impurity removal process. The deep eutectic solvent used therein has the advantages of maintaining enzymatic properties and good substrate solubility, and can be used to catalyze the esterification reaction of fatty acids and amino acids to prepare N-acyl amino acid compounds. The preparation method of N-acyl amino acid compounds provided by the present invention has a simple preparation process, low production cost, and is green and safe.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to a method for preparing N-acyl amino acid compounds based on a deep eutectic solvent, including its own application and a production method. Background Art

[0002] Amino acid surfactants are a type of renewable, green and environmentally friendly surfactant based on biomass. N-acyl amino acid surfactants not only have a wide range of raw material sources, low toxicity and side effects, mild performance, low irritation, good biodegradability, and green production process, but also have good emulsification, wetting, solubilization, dispersion, foaming and other properties that have attracted much attention and are gradually being used in many fields such as washing, personal care and food industry.

[0003] N-acyl amino acid compounds are an important type of amino acid surfactant. At present, the synthesis of N-acyl amino acids has disadvantages such as high difficulty, low yield, and non-green synthesis process. The main reason is that the synthesis of N-acyl amino acids that can be used for commercialization is mainly chemical. The most commonly used method is the Schotten-Bowman reaction, which is a type of reaction between fatty acyl chlorides and amino acids in an organic solvent-water mixture as a reaction medium and a strong base as a catalyst. The problems of reaction byproducts and environmental pollution are difficult to solve.

[0004] In 1989, Nagao of Novo-Nordisk proposed the enzyme-catalyzed synthesis of N-acyl amino acids. Compared with the chemical method, the enzymatic synthesis of N-acyl amino acids has milder reaction conditions and fewer toxic byproducts, so it has attracted widespread attention. However, there are many problems in the enzymatic synthesis of N-acyl amino acids. For example, the types of N-acyl amino acids synthesized by enzymatic methods are relatively small; in organic solvents, most enzymes cannot maintain high activity, but the substrate solubility in the aqueous phase is relatively poor, so the yield of enzymatic synthesis of N-acyl amino acids is low. Using lipase and protease as catalysts, lauroyl glycine was synthesized in an acetone-water solvent system with a conversion rate of 40% (Claudia Bernal ab, Fanny Guzman c, Andres Illanes d, Lorena Wilson d. Selective and eco-friendly synthesis of lipoaminoacid-based surfactants for food, using immobilized lipase and protease biocatalysts. Food chemistry 2018(239):189-195.). Therefore, the enzymatic synthesis of N-acylamino acids has not yet achieved large-scale industrial production.

[0005] Compared with chemical methods, enzymatic synthesis of N-acyl amino acids has great advantages. 2020, Mochamed ChafikBourkaib found that S. ambofaciens ATCC23877 The acylase in aqueous media has the ability to catalyze the production of N-acyl amino acids (Mochamed Chafik Bourkaib, Stephane Delaunay, Xavier Framboisier, Laurence Hotel, Bertrand Aigle, Catherine Humeau, Yann Guiavarch, Isabelle Chevalot. N-acylation of L-amino acids in aqueous media: Evaluation of the catalytic performance of Streptomyces ambofaciens aminoacylases. Enzyme and microbial technology 137 (2020) 109536.). Since the acylase has both the ability to synthesize N-acyl amino acids and the ability to hydrolyze, its conversion rate for synthesizing N-acyl amino acids in an aqueous environment is low. In order to maximize its synthetic ability and minimize its hydrolysis ability, its reaction environment should be a micro-aqueous environment or even an anhydrous environment. However, most biological enzymes have low tolerance to organic solvents. Excessive organic solvents will cause the biological enzymes to denature and inactivate. How to find a green solvent that has little effect on enzyme activity has become an important factor restricting the synthesis of N-acyl amino acid compounds.

[0006] Deep eutectic solvent is a low eutectic mixture with the advantages of being cheap, easy to obtain, low vapor pressure, non-toxic, non-flammable, biodegradable, controllable and recyclable. It is a new type of green solvent that can be widely used. It has a wide range of applications in organic reactions, biocatalysis, electrochemistry, material preparation and extraction and separation engineering. So far, deep eutectic solvents have been proven to play an important role in enzymatic reactions such as oxidation, hydrolysis, separation, transesterification, acylation and decarboxylation. C8-C18 fatty acids have poor hydrophilicity and are basically insoluble in water; while amino acids such as glycine, arginine, glutamic acid, lysine and alanine are easily soluble in water but difficult to dissolve in organic solvents. Therefore, finding a green solvent that is friendly to both fatty acids and amino acids is an important breakthrough in solving the low conversion rate of N-acyl amino acid compounds. Summary of the invention

[0007] In order to overcome the above shortcomings, the present invention provides an enzymatic preparation method of N-acyl amino acid compounds based on deep eutectic solvents, which directly synthesizes N-acyl amino acid compounds in one step. The deep eutectic solvent uses fatty acids as hydrogen bond acceptors and organic alcohols as hydrogen bond donors, thereby achieving high solubility of fatty acids and amino acids and efficient green synthesis of N-acyl amino acid compounds.

[0008] The present invention provides a method for preparing N-acyl amino acid compounds, which uses amino acids as raw materials, acylase as a catalyst, and a deep eutectic solvent as a medium to react and synthesize N-acyl amino acid compounds;

[0009] Preferably, the amino acid is selected from glycine, arginine, glutamic acid, lysine or alanine;

[0010] The deep eutectic solvent is obtained by mixing a hydrogen bond acceptor and a hydrogen bond donor.

[0011] In a specific embodiment, the deep eutectic solvent is a hydrogen bond donor that is an organic alcohol, and a hydrogen bond acceptor that is a fatty acid; more preferably, the organic alcohol is selected from glycerol, ethylene glycol, propylene glycol, methanol, cyclohexanol or butanol;

[0012] Preferably, the fatty acid is a C8-C18 fatty acid, such as caprylic acid, capric acid, lauric acid or stearic acid.

[0013] In a specific embodiment, the deep eutectic solvent is obtained by reacting a hydrogen bond donor and a hydrogen bond acceptor at 60-120°C for 2-4h; more specifically, the specific preparation steps of the deep eutectic solvent include: adding a hydrogen bond acceptor and a hydrogen bond donor in a magnetic stirrer at 60-120°C; stirring at 150-250r / min until a clear and uniform liquid is formed.

[0014] In a specific embodiment, the molar ratio of ethylene glycol to octanoic acid in the deep eutectic solvent is 2:1-5:1, preferably 3:1; the molar ratio of 1,3-propylene glycol to octanoic acid in the deep eutectic solvent is 2:1-8:1, preferably 5:1; the molar ratio of 1,2-propylene glycol to octanoic acid in the deep eutectic solvent is 2:1-8:1, preferably 3:1; the molar ratio of glycerol to octanoic acid in the deep eutectic solvent is 2:1-8:1, preferably 5:1.

[0015] In a specific embodiment, the reaction temperature is 25-65°C, preferably 45°C; the reaction time is 12-24h.

[0016] In a specific embodiment, the raw material, the acylase is added in an amount of 1 mg / mL-10 mg / mL, and the deep eutectic solvent is 20%-100% (V / V) of the reaction system.

[0017] Zn was also added to the reaction system 2+ 、Co 2+ , Mn 2+ , Fe 2+ 、Na + or K + , preferably adding Zn 2+ 、Co 2+ After that, its ion concentration is 50-150 μmol;

[0018] Water is further added to the reaction system, specifically 0-80% (V / V), preferably 20-50% (V / V).

[0019] The present invention also provides a method for preparing a surfactant, which uses the method for preparing N-acyl amino acid compounds to pre-prepare N-acyl amino acid compounds, and then prepares the surfactant. The method for preparing N-acyl amino acid compounds is the above method.

[0020] The present invention uses a green deep eutectic solvent as a reaction medium, which not only greatly reduces the environmental pollution caused by the production process of amino acid surfactants, but also has good substrate solubility, can better maintain the stability of enzyme activity, and can provide an anhydrous reaction environment for acylase, which can greatly exert the synthetic activity of amino acid surfactants of acylase. The reaction enzyme activity is significantly higher than the enzyme activity in aqueous or organic phase reactions. In the present invention, the deep eutectic solvent is composed of an amino acid surfactant production substrate, which reduces the addition of additional compounds in the reaction system, that is, reduces the production cost, and simplifies the post-processing impurity removal process. Therefore, the preparation method of the deep eutectic solvent provided by the present invention has a simple preparation process, low production cost, complete reaction, full utilization of raw materials, and no by-products. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 is the solubility of alanine in different deep eutectic solvents of Example 7. DETAILED DESCRIPTION

[0022] The present invention is further described below through specific examples in order to better understand the present invention, but does not constitute a limitation of the present invention.

[0023] The product detection method used in the embodiment is as follows:

[0024] The product prepared by the present invention was detected by high performance liquid chromatography, chromatographic column: C18, 4.6 mm × 250 mm, 5 μm, detection wavelength: ultraviolet (UV) = 200 nm; mobile phase: 85% acetonitrile and 15% 0.05% trifluoroacetic acid aqueous solution; column temperature: 30 ° C; flow rate: 1 mL / min; detection time: 15 min.

[0025] Example 1. Preparation of Lyophilized Acylase Powder

[0026] 1. Fermentation culture of acylase

[0027] Will S. ambofaciens ATCC23877 The strain was plated on an agar plate (4 g yeast extract, 10 g malt extract, 4 g glucose, 20 g agar, 1 L water, pH 7.3) at 30°C for 7 days. The strain was then cultured in 30 mL pre-culture medium (10 g glucose, 10 g dextrin, 5 g tryptone, 5 g yeast extract, 1 g CaCO3, 1 L water, pH 6.5) at 30°C, 200 rpm for 48 h. 1.8 mL of the pre-culture was added to 50 mL fermentation medium (40 g beef extract, 40 g soluble starch, 20 g peptone, 2 g K2HPO4, 20 g MgSO4, 1 L water, pH 7.0) at 30°C, 120 rpm for 8 days.

[0028] 2. Preparation of acylase lyophilized powder

[0029] 8000rpm, 4℃, centrifuged for 20min, and the fermentation broth of Example 1 was collected. The precipitate was dissolved in 25mM Tris-HCl, 50mMNaCl, pH 8. The bacteria were broken by homogenizer, and then centrifuged at 8000rpm, 4℃ for 20min, and the supernatant was collected to obtain a crude enzyme solution. Then 0.1% PEI was added, 4℃, stirred for 1h, and then centrifuged at 8000rpm, 4℃ for 20min, and the supernatant was collected and put into a freeze dryer to prepare acylase freeze-dried powder.

[0030] Example 2: Reactions in different solvents

[0031] In a 50 mL reaction bottle, 25 mL of water, PBS buffer (0.1 M, pH 7.0), methanol, ethylene glycol, glycerol, isopropanol, and 1,2-propylene glycol were added as solvents. 360 mg of octanoic acid, 1113.6 mg of alanine, and 100 mg of acylase freeze-dried powder were added respectively. After the reaction bottle was sealed, the reaction was carried out at 45 ° C and 200 rpm for 24 hours. After the reaction was completed, the reaction solution was analyzed by HPLC to obtain the enzyme activity of the acylase in the reaction solution, and its enzyme activity relative to the water solvent was recorded in Table 1. The reaction and analysis were carried out more than 3 times for each reaction system, and the data deviation caused by the operation was corrected.

[0032] Table 1. Enzyme activity of acylase in reaction solutions of different solvents relative to water solvent

[0033]

[0034] The above results show that the activity of acylase decreases sharply in organic solvents such as methanol, ethylene glycol, glycerol, isopropanol, and 1,2-propylene glycol.

[0035] Example 3

[0036] Ethylene glycol and octanoic acid were mixed in a molar ratio of 3:1 and stirred at 80°C for 2 hours to obtain a homogeneous transparent liquid, which was a deep eutectic solvent of ethylene glycol and octanoic acid. Subsequently, the synthesized deep eutectic solvent was cooled to room temperature.

[0037] In a 50 mL reaction bottle, 1113.6 mg of alanine, 25 mL of a deep eutectic solvent of ethylene glycol and octanoic acid, and 100 mg of the acylase freeze-dried powder prepared in Example 1 were added. The reaction bottle was sealed and reacted at 45° C. and 200 rpm for 24 h.

[0038] After the reaction was completed, the reaction solution was analyzed by HPLC to obtain the enzyme activity of the acylase in the reaction solution. The relative enzyme activity with respect to the water solvent was 4.15.

[0039] Example 4

[0040] 1,3-propylene glycol and octanoic acid were mixed in a molar ratio of 5:1 and stirred at 60°C for 2 hours to obtain a homogeneous transparent liquid, which was a deep eutectic solvent of 1,3-propylene glycol and octanoic acid. Subsequently, the synthesized deep eutectic solvent was cooled to room temperature.

[0041] In a 50 mL reaction bottle, 1113.6 mg of alanine, 25 mL of a deep eutectic solvent of 1,3-propanediol and octanoic acid, and 100 mg of the acylase freeze-dried powder prepared in Example 1 were added, the reaction bottle was sealed, and the reaction was carried out at 45° C. and 200 rpm for 24 h.

[0042] After the reaction was completed, the reaction solution was analyzed by HPLC to obtain the enzyme activity of the acylase in the reaction solution. The relative enzyme activity with respect to the water solvent was 2.76.

[0043] Example 5

[0044] 1,2-propylene glycol and octanoic acid were mixed in a molar ratio of 3:1 and stirred at 80°C for 2 hours to obtain a homogeneous transparent liquid, which was a deep eutectic solvent of 1,2-propylene glycol and octanoic acid. Subsequently, the synthesized deep eutectic solvent was cooled to room temperature.

[0045] In a 50 mL reaction bottle, 1113.6 mg of alanine, 25 mL of a deep eutectic solvent of 1,2-propylene glycol and octanoic acid, and 100 mg of the acylase lyophilized powder prepared in Example 1 were added, the reaction bottle was sealed, and the reaction was carried out at 45° C. and 200 rpm for 24 h.

[0046] After the reaction was completed, the reaction solution was analyzed by HPLC to obtain the enzyme activity of the acylase in the reaction solution. The relative enzyme activity with respect to the water solvent was 7.41.

[0047] Example 6

[0048] Glycerol and octanoic acid were mixed in a molar ratio of 5:1 and stirred at 120° C. for 2 h to obtain a homogeneous transparent liquid, which was a deep eutectic solvent of glycerol and octanoic acid. Subsequently, the synthesized deep eutectic solvent was cooled to room temperature.

[0049] In a 50 mL reaction bottle, 1113.6 mg of alanine, 25 mL of a deep eutectic solvent of glycine and octanoic acid, and 100 mg of the acylase lyophilized powder prepared in Example 1 were added, the reaction bottle was sealed, and the reaction was carried out at 45° C. and 200 rpm for 24 h.

[0050] After the reaction was completed, the reaction solution was analyzed by HPLC to obtain the enzyme activity of the acylase in the reaction solution. The relative enzyme activity with respect to the water solvent was 12.39.

[0051] Example 7. Solubility of deep eutectic solvents for alanine

[0052] Accurately weigh 10g of deep eutectic solvent, place it in a constant temperature shaking water bath at 45℃, wait until the temperature is constant, weigh 10mg of alanine and add it, shake at 200rpm for 30min. Then take out a drop with a capillary and test it under a polarizing microscope. If no polarized light appears, it means that the solution is completely dissolved; then add another 10mg of alanine. Until it no longer dissolves (polarized light appears). The final substrate solubility is calculated according to the following formula:

[0053] S=m×10 / 100

[0054] Where S is the solubility and m is the total amount of substrate added.

[0055] The solubility of alanine in water is 16.65 g / 100 g, and it is insoluble in ether or acetone.

[0056] from Figure 1 It can be seen that different deep eutectic solvents have different effects on the solubility of alanine. Among them, the solubility is highest in glycerol:octanoic acid = 5:1, which is 15.7g / 100g.

[0057] Example 8: The promoting effect of metal ions on the reaction

[0058] 1,3-propylene glycol and octanoic acid were mixed in a molar ratio of 5:1 and stirred at 60°C for 2 hours to obtain a homogeneous transparent liquid, which was a deep eutectic solvent of 1,3-propylene glycol and octanoic acid. Subsequently, the synthesized deep eutectic solvent was cooled to room temperature.

[0059] Take 6 50 mL reaction bottles, add 1113.6 mg of alanine, 25 mL of deep eutectic solvent (1,3-propylene glycol and octanoic acid), and 100 mg of the acylase lyophilized powder prepared in Example 1, and then add ZnSO4, CoCl2, MnCl2, FeCl2, NaCl, and KCl with a final concentration of 100 μmol / L, respectively. After sealing the reaction bottle, react at 45°C and 200 rpm for 24 hours.

[0060] After the reaction was completed, the reaction solution was analyzed by HPLC to obtain the enzyme activity of the acylase in different reaction solutions. The relative enzyme activity relative to Example 4 is recorded in Table 2.

[0061] Table 2. Relative enzyme activity of acylase in different reaction solutions relative to Example 4

[0062]

[0063] From the above results, it can be seen that adding Zn 2+ 、Co 2+ After that, it has a certain promoting effect on the reaction.

[0064] Example 9: Effect of water on the reaction

[0065] Glycerol and octanoic acid were mixed in a molar ratio of 5:1 and stirred at 60°C for 2 hours to obtain a homogeneous transparent liquid, which was a deep eutectic solvent of glycerol and octanoic acid. Subsequently, the synthesized deep eutectic solvent was cooled to room temperature.

[0066] 1113.6 mg of alanine, a solvent consisting of a deep eutectic solvent and water, and 100 mg of the acylase lyophilized powder prepared in Example 1 were added to four 50 mL reaction bottles, respectively. The reaction bottles were sealed and reacted at 45° C. and 200 rpm for 24 h.

[0067] After the reaction was completed, the reaction solution was analyzed by HPLC to obtain the enzyme activity of the acylase in different reaction solutions. The relative enzyme activity relative to Example 6 is recorded in Table 3.

[0068] Table 3. Relative enzyme activity of acylase in different reaction solutions relative to Example 6

[0069]

[0070] From the above results, it can be seen that adding a small amount of water and deep eutectic solvent to form a mixed solvent has a certain promoting effect on the reaction.

Claims

1. A method for preparing an N-acylamino acid compound, characterized in that: The method uses alanine as a raw material, acylase as a catalyst, and a deep eutectic solvent as a medium to react and synthesize N-acyl amino acid compounds; The deep eutectic solvent is obtained by mixing a hydrogen bond acceptor and a hydrogen bond donor, wherein the hydrogen bond donor is an organic alcohol and the hydrogen bond acceptor is a fatty acid; The organic alcohol is ethylene glycol, and the fatty acid is octanoic acid; or the organic alcohol is 1,3-propylene glycol, and the fatty acid is octanoic acid; or the organic alcohol is 1,2-propylene glycol, and the fatty acid is octanoic acid; or the organic alcohol is glycerol, and the fatty acid is octanoic acid.

2. The method for preparing N-acylamino acid compounds according to claim 1, characterized in that: The deep eutectic solvent is obtained by reacting a hydrogen bond donor and a hydrogen bond acceptor at 60-120° C. for 2-4 hours.

3. The method for preparing N-acylamino acid compounds according to claim 1, characterized in that: The specific preparation steps of the deep eutectic solvent include: adding a hydrogen bond acceptor and a hydrogen bond donor in a magnetic stirrer at 60-120° C.; and stirring at 150-250 r / min until a clear and uniform liquid is formed.

4. The method for preparing N-acylamino acid compounds according to claim 1, characterized in that: The molar ratio of ethylene glycol to octanoic acid in the deep eutectic solvent is 2:1-5:1; the molar ratio of 1,3-propylene glycol to octanoic acid in the deep eutectic solvent is 2:1-8:1; the molar ratio of 1,2-propylene glycol to octanoic acid in the deep eutectic solvent is 2:1-8:1; the molar ratio of glycerol to octanoic acid in the deep eutectic solvent is 2:1-8:

1.

5. The method for preparing N-acylamino acid compounds according to claim 4, characterized in that: The molar ratio of ethylene glycol to octanoic acid in the deep eutectic solvent is 3:1; the molar ratio of 1,3-propylene glycol to octanoic acid in the deep eutectic solvent is 5:1; the molar ratio of 1,2-propylene glycol to octanoic acid in the deep eutectic solvent is 3:1; and the molar ratio of glycerol to octanoic acid in the deep eutectic solvent is 5:

1.

6. The method for preparing N-acylamino acid compounds according to claim 1, characterized in that: The reaction temperature is 25-65°C.

7. The method for preparing N-acylamino acid compounds according to claim 6, characterized in that: The reaction temperature is 40-50°C, and the reaction time is 12-24h.

8. The method for preparing N-acylamino acid compounds according to claim 1, characterized in that: The raw material and the acylase are added in an amount of 1 mg / mL-10 mg / mL, and the deep eutectic solvent is 20%-100% (V / V) of the reaction system; Zn is also added to the reaction system 2+ or Co 2+ Ions, the ion concentration is 50-150 μmol.

9. The method for preparing N-acylamino acid compounds according to claim 8, characterized in that: Water is also added to the reaction system in an amount of 20-50% (V / V) of the total volume of the reaction system.

10. A method for preparing a surfactant, characterized in that: The method comprises the steps of preparing N-acyl amino acid compounds by the method for preparing N-acyl amino acid compounds according to any one of claims 1 to 9, and then preparing the N-acyl amino acid compounds into surfactants.

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