WO2020181779A1 - 一种基于低共熔溶剂的脂氨基酸类化合物的制备方法 - Google Patents

一种基于低共熔溶剂的脂氨基酸类化合物的制备方法 Download PDF

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WO2020181779A1
WO2020181779A1 PCT/CN2019/113559 CN2019113559W WO2020181779A1 WO 2020181779 A1 WO2020181779 A1 WO 2020181779A1 CN 2019113559 W CN2019113559 W CN 2019113559W WO 2020181779 A1 WO2020181779 A1 WO 2020181779A1
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acid
lipoamino
eutectic solvent
hydrogen bond
prepare
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PCT/CN2019/113559
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English (en)
French (fr)
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刘元法
年彬彬
曹晨
李进伟
徐勇将
翟颖红
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江南大学
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Priority claimed from CN201910193954.1A external-priority patent/CN109776348B/zh
Priority claimed from CN201910447896.0A external-priority patent/CN110042130B/zh
Application filed by 江南大学 filed Critical 江南大学
Publication of WO2020181779A1 publication Critical patent/WO2020181779A1/zh
Priority to US17/139,067 priority Critical patent/US11319565B2/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/02—Amides, e.g. chloramphenicol or polyamides; Imides or polyimides; Urethanes, i.e. compounds comprising N-C=O structural element or polyurethanes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
    • A23L29/10—Foods or foodstuffs containing additives; Preparation or treatment thereof containing emulsifiers
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C231/00—Preparation of carboxylic acid amides
    • C07C231/02—Preparation of carboxylic acid amides from carboxylic acids or from esters, anhydrides, or halides thereof by reaction with ammonia or amines
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C233/00—Carboxylic acid amides
    • C07C233/01—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms
    • C07C233/45—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by carboxyl groups
    • C07C233/46—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by carboxyl groups with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by an acyclic carbon atom
    • C07C233/47—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by carboxyl groups with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by an acyclic carbon atom having the carbon atom of the carboxamide group bound to a hydrogen atom or to a carbon atom of an acyclic saturated carbon skeleton
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/01—Carboxylic ester hydrolases (3.1.1)
    • C12Y301/01003—Triacylglycerol lipase (3.1.1.3)

Definitions

  • the invention relates to a method for preparing a lipoamino acid compound based on a eutectic solvent, and belongs to the field of organic synthesis.
  • surfactants are often used as emulsifiers, thickeners, defoamers and many other aspects in the food industry due to their unique properties. They are called "industrial monosodium glutamate" and are in great demand. In recent years, the research on lipoamino acid surfactants has become a hot spot. Traditional lipoamino acid surfactants are mostly quaternary ammonium salts. Although they have good surface activity and antibacterial properties, they also have a certain degree of strong cytotoxicity, which limits their large-scale application in the food field. Compared with traditional surfactants, lipoamino acid surfactants have higher safety and have better application prospects in the food field.
  • Natural eutectic solvents have been widely used in esterification reactions. Unlike esterification reactions, the reaction of fatty acids and amino acids usually produces quaternary ammonium salts instead of fatty amino acids. In order to increase the yield of fatty amino acids At present, a two-step method is often used, that is, firstly activate the fatty acid or the nucleophilic reaction of the amino acid. Therefore, there are many by-products and low product synthesis efficiency.
  • Gemini-type lipoamino acids are composed of two hydrophilic or two hydrophobic groups and can be considered as a dimer of lipoamino acids.
  • CMC value of Gemini-type lipid amino acids can be one or even several orders of magnitude lower than that of single-chain surfactants; there are also reports that a series of new L-lysine-based Gemini surfactants have been synthesized and compared with traditional lipoamino acids. In comparison, better antibacterial activity was found in a broad spectrum of Gram-positive bacteria and Gram-negative bacteria.
  • Gemini-type lipoamino acids have the above-mentioned many advantages, they have not been used industrially, because currently Gemini-type lipoamino acids can only be prepared by complex multi-step organic synthesis.
  • the existing synthesis of Gemini-type fatty amino acids is mostly accomplished by the Schotten-Bauman reaction, which uses fatty acid chlorides instead of fatty acids as raw materials.
  • the product yield is improved to a certain extent, there are still great potential safety hazards and environmental problems.
  • the present invention provides an enzymatic preparation method of lipoamino acid surfactants based on eutectic solvents.
  • the technical scheme adopted in the present invention includes: using fatty acids and amino acids as raw materials, lipase CALB as catalyst, eutectic solvent as medium, direct synthesis of fatty amino acids in one step, and directly obtained by simple and efficient water washing-methanol recrystallization method Products with higher purity
  • the method of the invention is simple, green, safe and efficient.
  • the present invention provides a eutectic solvent design method and a method for synthesizing Gemini-type lipoamino acid based on the eutectic solvent/enzyme synergistic catalysis Efficient preparation method.
  • the technical scheme adopted by the present invention includes: using lipase and amino acids containing two amino groups as raw materials, enzymes as catalysts, hydrophobic natural eutectic solvents as media, and one-step method of directly synthesizing fatty amino acids, and adopting simple and efficient
  • the water washing-methanol recrystallization method directly obtains products with higher purity, and the method of the invention is simple, green, safe and efficient.
  • the first objective of the present invention is to provide a method for preparing a lipoamino acid compound, the method comprising:
  • fatty acids and amino acids are directly prepared under the action of a catalyst to obtain lipoamino acid compounds.
  • the molar ratio of fatty acid to amino acid is 1:1 to 2:1.
  • the amino acids include glycine, lysine, arginine, and alanine.
  • the fatty acid is a C2-C20 fatty acid.
  • the fatty acid includes lauric acid and palmitic acid.
  • the catalyst includes lipase CALB.
  • eutectic solvents are screened.
  • the eutectic solvents include betaine-glycerin and betaine-xylitol. , Choline Chloride-Glycerin, Choline Chloride-Xylitol.
  • the eutectic solvent is obtained by rotary evaporation from a hydrogen bond donor and a hydrogen bond acceptor at a certain stoichiometric ratio at 60-100°C.
  • the stoichiometric ratio of hydrogen bond donor and hydrogen bond acceptor is 2:1 to 1:2.
  • the method is carried out at 40-60°C, pH 7.0-8.0, and stirring speed 200-300rpm.
  • the amount of water used is 50%-100% (V/V) of the reaction system.
  • the second object of the present invention is to provide a method for preparing a surfactant.
  • the method is to prepare a lipoamino acid compound in advance, and then prepare the surfactant.
  • the preparation method of the lipoamino acid compound is the above method .
  • the third object of the present invention is to provide an emulsifier.
  • the method is to prepare a lipoamino acid compound in advance and then prepare the emulsifier.
  • the preparation method of the lipoamino acid compound is the above-mentioned method.
  • the fourth object of the present invention is to provide a thickening agent.
  • the method is to prepare a lipoamino acid compound in advance, and then prepare the thickening agent.
  • the preparation method of the lipoamino acid compound is the above method.
  • the fifth object of the present invention is to provide an antifoaming agent.
  • the method is to prepare a lipoamino acid compound in advance and then prepare the antifoaming agent.
  • the preparation method of the lipoamino acid compound is the above method.
  • the sixth objective of the present invention is to provide a method for preparing Gemini-type amino acids.
  • the method is to prepare Gemini-type amino acids by reacting amino acids and fatty acids in a eutectic solvent with Amano lipase M as a catalyst;
  • the hydrogen bond donor in the eutectic solvent includes oleic acid and/or linoleic acid, and the hydrogen bond acceptor is choline chloride; the number of amino groups in the amino acid is not less than 2.
  • the amino acids include lysine, cystine, and arginine.
  • the fatty acid is a C2-C20 fatty acid.
  • the fatty acid includes lauric acid and palmitic acid.
  • the eutectic solvent is preferably choline chloride-oleic acid.
  • the eutectic solvent is obtained by rotary evaporation from a hydrogen bond donor and a hydrogen bond acceptor at a certain stoichiometric ratio at 60-100°C.
  • the stoichiometric ratio of the hydrogen bond donor to the hydrogen bond acceptor in the eutectic solvent is 2:1 to 1:2.
  • the temperature of the reaction is 40-80°C.
  • the temperature of the reaction is preferably 60°C.
  • the amount of Amano lipase M added is 50-100 mg per mole of amino acid.
  • the amount of Amano lipase M added is preferably 60-75 mg per mole of amino acid.
  • the Amano lipase M is from Mucor (MJL), CAS NO: 9001-62-1.
  • the molar ratio of fatty acid to amino acid is 2.5:1 to 1:1.
  • the product obtained from the reaction undergoes the following purification process: extraction with ethyl acetate and water, and recrystallization with methanol to obtain a pure product.
  • 50-100 mL of ethyl acetate and 250-500 mL of water are used for extraction per 20 mL of the reaction system.
  • the seventh object of the present invention is to apply the above method to the preparation of food emulsifiers.
  • the present invention uses a green and safe natural eutectic solvent to replace traditional organic reagents as the reaction medium, and does not require water as an auxiliary mixed solvent, which not only greatly reduces environmental pollution, but also because of the natural eutectic solvent
  • the good substrate solubility and the ability to better maintain the stability and activity of the enzyme have greatly changed the current situation of low substrate solvent and poor lipase activity in the traditional organic solvent enzymatic synthesis of lipoamino acids.
  • the reaction yield is significantly higher than the enzymatic synthesis of lipoamino acids that have been reported by other methods.
  • the current natural eutectic for enzymatic synthesis is mostly characterized by substrate conversion rate, which does not indicate the level of product yield, and basically does not involve the purification process of the product.
  • the present invention uses a simple and efficient water washing-methanol weight The crystallization method obtains products with higher purity, which greatly simplifies the purification process of enzymatic synthesis of lipoamino acids.
  • the present invention also recovers and recycles the natural eutectic solvent through simple processing. After 10 cycles, the synthesis yield can reach 52.4% and the initial effect can be maintained at 88.48%, which has excellent effects. Greatly reduce production costs.
  • Figure 1 is a synthetic route diagram of lipoamino acids
  • Figure 2 shows the solubility of glycine acid (a) and lauric acid (b) in different natural eutectic solvents.
  • the solvents from left to right at each temperature are betaine-glycerol (B-Gly) and betaine-xylose.
  • Alcohol (BX) Choline Chloride-Glycerin (C-Gly), Choline Chloride-Xylitol (CX), Betaine-Glycerin-Water (B-Gly-W), Choline Chloride-Glycerin- Water (C-Gly-W);
  • Figure 3 shows the activity of lipase in different natural eutectic solvents.
  • the solvent from left to right at each time is Tris buffer (50mM, PH 8.0) (control group), betaine-glycerol (B-Gly), betaine-xylitol (BX), choline chloride-glycerol (C-Gly), choline chloride-xylitol (CX ), betaine-glycerol-water (B-Gly-W), choline chloride-glycerol-water (C-Gly-W);
  • Figure 4 shows the thermal stability of lipase in different natural eutectic solvents.
  • the solvents from left to right at each time are betaine-glycerol (B-Gly), betaine-xylitol (BX), chlorine Choline-glycerol (C-Gly), choline chloride-xylitol (CX), betaine-glycerol-water (B-Gly-W), choline chloride-glycerol-water (C-Gly- W);
  • Figure 5 shows the infrared spectrum of the product
  • Figure 6 is a mass spectrum of the product
  • Figure 7 is a hydrogen nuclear magnetic resonance spectrum and its corresponding data chart
  • Figure 8 is a mass spectrum of Gemini type lauroyl lysine (GLL);
  • Figure 9 is a mass spectrum of single-chain lauroyl glycine (LAA).
  • Figure 10 shows the recycling efficiency of eutectic solvents.
  • the main idea of the present invention is:
  • the present invention first investigates the synthesis of Gemini-type lipid amino acids in traditional organic solvents, and secondly investigates the synthesis of lipid amino acids in self-designed natural eutectic solvents.
  • the process includes: enzyme screening, solvent screening, reaction conditions optimization, and solvent Recycling, product purification program.
  • the yield of Gemini lipoamino acids actual value of Gemini lipoamino acids/theoretical value of Gemini lipoamino acids;
  • the conductivity method is used to determine the cmc value: using the relationship between the conductivity of the ionic surfactant aqueous solution and the concentration, a ⁇ m -C 1/2 curve is drawn, and the CMC value is obtained from the turning point of the curve.
  • the molar conductivity ⁇ m of the strong electrolyte solution has a linear relationship with the concentration of the solution c 1/2 .
  • the conductivity in the dilute solution the change in the molar conductivity
  • the law is the same as that of a strong electrolyte, but with the formation of micelles in the solution, the electrical conductivity and molar conductivity change significantly, which is the basis for determining CMC.
  • Lauroylglycine is 0.0044mol/L, which is significantly lower than Tween sodium dodecyl sulfonate (0.009mol/L), which is a common surfactant in the market. Better surfactant.
  • Example 5 Investigate the effect of eutectic solvent on the reaction substrate
  • S is the solvent degree
  • m t is the total amount of substrate added.
  • the final lauric acid solubility is 24.98 (g/100g).
  • Lauric acid is slightly soluble in commonly used enzyme-friendly solvent acetone (0.01-0.1g/100g), and its solubility in water is 0.
  • the solubility of glycine in the eutectic solvent is 22.78 (g/100g), the solubility in water is 25g/100g, and it is almost insoluble in acetone.
  • Example 6 Investigate the influence of different eutectic solvents on lipase catalyst
  • lipase CALB can still maintain a high activity after being treated in NADES at 60°C for 360 minutes, reaching 6400 U/mL.
  • the balance between structural rigidity and flexibility is the basis for the high stability and activity of lipase.
  • NADES can interact with enzymes to increase its structural rigidity, thereby improving its stability.
  • the lipase still maintains a high activity after 360 minutes; while in the participating system, the enzyme activity is gradually inhibited .
  • the inventor believes that it may be because the surface of the enzyme can be bonded with NADES, such as glycerol, xylitol, etc. These small organic molecules can form a complex hydrogen bond network with the enzyme.
  • Example 1 the eutectic solvent was replaced with acetone, and other conditions were unchanged, and the product yield was basically negligible.
  • the cmc value of several products was determined by the conductivity method, and the cmc of Gemini-type lauroyl amino acid was 0.0044
  • the mol/Lmol/L is significantly lower than Tween sodium dodecyl sulfonate (0.009mol/L), a surfactant commonly used in the market. It is a surfactant with better performance.
  • Example 8 The effect of different eutectic solvents on the preparation of Gemini lipoamino acids
  • N.R. means no product is obtained.
  • Example 7 the enzyme catalysts were replaced with the enzymes shown in Table 2, and other conditions were unchanged, and Gemini-type lipid amino acids were prepared by reaction. The results obtained are shown in Table 2.
  • DA D-aminoacylase from E. coli
  • PPL lipase from porcine pancreas
  • CALB Candida antarctica (Candida Antarctica) lipase B
  • BSAP Bacillus subtilis (Bacillus subtilis) alkaline protease.
  • Example 7 With reference to Example 7, the single factor test method was used to optimize the substrate molar ratio, reaction time, reaction temperature, enzyme addition and other indicators. Table 3 shows the specific results of generating Gemini lipoamino acids.
  • Lauric acid lysine molar ratio Response time (h) Enzyme addition amount (g) Reaction temperature (°C) Yield(%) 2:1 72 60 60 44.3 2:1 72 60 80 45.6 2:1 72 60 40 41.3 2:1 72 60 60 50. 4 2:1 72 75 60 57.6 2:1 72 50 60 51.4 2:1 72 30 60 30.3 2:1 96 60 60 48.0 2.5:1 72 60 60 59.6 1:1 72 60 60 43.9 1:2 72 60 60 28.8
  • Example 11 Investigate the recovery efficiency of the eutectic solvent after the reaction
  • the recovered eutectic solvent was used in the synthesis of lauroyl lysine to test its performance. Refer to Example 1 for specific reaction conditions. After 10 cycles of the solvent, the synthesis yield of Gemini lipoamino acids decreased from 59.6% to 52.4 %, it can still maintain 88.48% of the initial effect.

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Abstract

一种基于低共熔溶剂的脂氨基酸类化合物的制备方法，属于有机合成领域。以脂肪酸、氨基酸为底物，以低共熔溶剂为介质，在脂肪酶CALB的催化作用下直接高效制得单链型脂氨基酸类表面活性剂；或者，以Amano脂肪酶M作为催化剂，高效催化含多个氨基的氨基酸与脂肪酸反应制得Gemini型氨基酸。所述方法无需经过氨基酸的保护以及脱保护，不涉及常用的脂肪酰氯，相对于传统化学合成法具有绿色、安全、选择性高、产率高等优势。

Description

一种基于低共熔溶剂的脂氨基酸类化合物的制备方法 技术领域
本发明涉及一种基于低共熔溶剂的脂氨基酸类化合物的制备方法，属于有机合成领域。
 背景技术
表面活性剂由于其独特的性质在食品领域内常被用于乳化剂、增稠剂、消泡剂等诸多方面，被称为“工业味精”，具有很大的需求量。近年来关于脂氨基酸类表面活性剂的研究成为热点。传统脂氨基酸类表面活性剂多为季铵盐类，虽具有良好的表面活性以及抑菌性，但也存在一定的细胞毒性强，限制了其在食品领域内的大量应用。相对于传统表面活性剂，脂氨基酸类表面活性剂具有更高的安全性，在食品领域内具有较好的应用前景。
目前，关于脂氨基酸类表面活性剂的合成研究多集中于化学法，对环境产生极大污染。酶法合成过程多采用有机溶剂作为反应体系，不仅不利于环境，且很容易导致酶的变性失活。目前的制备方法有：在无溶剂体系中以脂肪酶Novozyme435为催化剂合成了甘油酰精氨酸表面活性剂，其合成得率仅为15%。所以，如何寻找一种合适的绿色溶剂已经成为了限制其合成以及应用的一大难题。
天然低共熔溶剂在酯化反应中已经得到了较多的应用，与酯化反应不同的是，脂肪酸与氨基酸的反应通常会生成季铵盐、而非脂氨基酸，为了提高脂氨基酸的得率，目前多采用两步法进行，即首先对脂肪酸或进行活化，而后进行氨基酸的亲核反应。因此，存在副产物多、产物合成效率低等问题。
此外，作为新一代脂氨基酸，Gemini型脂氨基酸由两个亲水或两个疏水基团组成，可以认为是脂氨基酸的二聚体。目前有报道认为Gemini型脂氨基酸的CMC值可以比单链表面活性剂低一个甚至几个数量级；也有报道合成了一系列新的基于L-赖氨酸的Gemini表面活性剂，与传统型脂氨基酸相比，在广谱的革兰氏阳性菌和革兰氏阴性菌中发现了更好的抗菌活性。尽管Gemini型脂氨基酸具有上述诸多优势，其仍没有被工业化应用，这是因为目前Gemini型脂氨基酸只能通过复杂的多步有机合成来制备。现有合成Gemini型脂氨基酸多采用肖顿-鲍曼反应来完成，该反应利用脂肪酰氯代替脂肪酸作为原料，虽一定程度提高了产物得率，但是仍存在极大的安全隐患以及环境问题。
为了缓解Gemini型脂氨基酸生产过程中的污染以及安全性问题，酶法合成被逐渐重视。但是目前酶法合成仍存在一系列问题难以解决，主要集中于：很难找到一种溶剂在具有良好的底物溶解性的同时还能保持较高的酶活性。这些问题都导致，酶法合成Gemini型脂氨基酸产率较低。
发明内容
为了克服上述现有技术中脂氨基酸类表面活性剂合成方法的不足，本发明提供了一种基于低共熔溶剂的脂氨基酸类表面活性剂酶法制备方法。本发明所采用的技术方案包括：以脂肪酸、氨基酸为原料，脂肪酶CALB作为催化剂，低共熔溶剂为介质，一步法直接合成脂氨基酸，并采用简单、高效的水洗-甲醇重结晶法直接得到纯度较高的产物本发明方法简便、绿色、安全、高效。进一步地，为了克服上述现有技术中Gemini型脂氨基酸合成方法存在的不足，本发明提供了一种低共熔溶剂的设计方法以及基于此低共熔溶剂/酶协同催化合成Gemini型脂氨基酸的高效制备方法。本发明所采用的技术方案包括：以脂肪酶、含有两个氨基的氨基酸为原料，酶作为催化剂，以疏水性天然低共熔溶剂为介质，一步法直接合成脂氨基酸，并采用简单、高效的水洗-甲醇重结晶法直接得到纯度较高的产物，本发明方法简便、绿色、安全、高效。
本发明的第一个发明目的是提供一种脂氨基酸类化合物的制备方法，所述方法包括：
以低共熔溶剂为介质，脂肪酸、氨基酸在催化剂作用下直接制备得到脂氨基酸类化合物。
在本发明的一种实施方式中，脂肪酸与氨基酸的摩尔比为1：1~2:1。
在本发明的一种实施方式中，所述氨基酸包括甘氨酸、赖氨酸、精氨酸、丙氨酸。
在本发明的一种实施方式中，所述脂肪酸为C2-C20脂肪酸。
在本发明的一种实施方式中，所述脂肪酸包括月桂酸、棕榈酸。
在本发明的一种实施方式中，所述催化剂包括脂肪酶CALB。
在本发明的一种实施方式中，针对酰胺化反应的特点以及氨基酸、脂肪酸的溶解度对目前常用的低共熔溶剂进行了筛选，低共熔溶剂包括甜菜碱-甘油、甜菜碱-木糖醇、氯化胆碱-甘油、氯化胆碱-木糖醇中的任意一种。
在本发明的一种实施方式中，所述的低共熔溶剂是由氢键供体和氢键受体在60-100℃条件下按照一定化学计量比，经过旋转蒸发获得。
在本发明的一种实施方式中，氢键供体以及氢键受体的化学计量比为2:1~1:2。
在本发明的一种实施方式中，所述方法是在40-60℃，pH值7.0-8.0，搅拌速度200-300rpm下进行。
在本发明的一种实施例中，水的用量为反应体系的50%~100%（V/V）。
本发明的第二个目的是提供一种表面活性剂的制备方法，所述方法是预先制备脂氨基酸类化合物，然后在进行表面活性剂的制备，所述脂氨基酸类化合物的制备方法为上述方法。
本发明的第三个目的是提供一种乳化剂，所述方法是预先制备脂氨基酸类化合物，然后在进行乳化剂的制备，所述脂氨基酸类化合物的制备方法为上述方法。
本发明的第四个目的是提供一种增稠剂，所述方法是预先制备脂氨基酸类化合物，然后在进行增稠剂的制备，所述脂氨基酸类化合物的制备方法为上述方法。
本发明的第五个目的是提供一种消泡剂，所述方法是预先制备脂氨基酸类化合物，然后在进行消泡剂的制备，所述脂氨基酸类化合物的制备方法为上述方法。
本发明的第六个发明目的是提供一种制备Gemini型氨基酸的方法，所述方法是在低共熔溶剂中，以Amano脂肪酶M作为催化剂，氨基酸与脂肪酸反应制得Gemini型氨基酸；所述低共熔溶剂中的氢键供体包括油酸和/或亚油酸，氢键受体为氯化胆碱；所述氨基酸中氨基的数量不低于2。
在本发明的一种实施方式中，所述氨基酸包括赖氨酸、胱氨酸、精氨酸。
在本发明的一种实施方式中，所述脂肪酸为C2-C20脂肪酸。
在本发明的一种实施方式中，所述脂肪酸包括月桂酸、棕榈酸。
在本发明的一种实施方式中，所述低共熔溶剂优选氯化胆碱-油酸。
在本发明的一种实施方式中，低共熔溶剂是由氢键供体和氢键受体在60-100℃条件下按照一定化学计量比，经过旋转蒸发获得。
在本发明的一种实施方式中，低共熔溶剂中氢键供体与氢键受体的化学计量比为2:1~1:2。
在本发明的一种实施方式中，所述反应的温度为40-80℃。
在本发明的一种实施方式中，所述反应的温度优选60℃。
在本发明的一种实施方式中，所述Amano脂肪酶M的添加量为每摩尔氨基酸添加50-100mg。
在本发明的一种实施方式中，所述Amano脂肪酶M的添加量优选每摩尔氨基酸添加60-75mg。
在本发明的一种实施方式中，所述Amano脂肪酶M来自毛霉（MJL），CAS NO：9001-62-1。
在本发明的一种实施方式中，脂肪酸与氨基酸的摩尔比为2.5:1~1:1。
本发明的一种实施例中，反应所得产物进过如下纯化过程：采用乙酸乙酯、水萃取，并利用甲醇重结晶，得到纯品。
本发明的一种实施例中，每20mL反应体系利用50-100mL乙酸乙酯和250-500mL水进行萃取。
本发明的第七个目的是将上述方法应用于食品乳化剂制备中。
与现有技术相比，本发明的有益效果是：
（1）本发明利用绿色安全的天然低共熔溶剂替代传统的有机试剂为反应介质，无需水作为辅助混合溶剂，不仅极大程度的减小了对环境的污染，且由于天然低共熔溶剂良好的底物溶解性，以及能够较好的保持酶的稳定性以及活性，极大程度的改变了传统有机溶剂酶法合成脂氨基酸过程中底物溶剂性低，脂肪酶活性差的现状。反应产率显著高于目前已报道其他方法的脂氨基酸的酶法合成。此外，目前天然低共熔用于酶法合成多采用底物转化率进行表征，并不能说明产物得率的高低，且基本没有涉及产物的纯化过程，本发明采用了简单高效的水洗-甲醇重结晶法得到纯度较高的产物，极大程度简化了酶法合成脂氨基酸的纯化过程。
（2）采用天然低共熔溶剂代替传统的有机溶剂作为反应介质，不仅仅解决了底物溶解性的问题，还极好的保持了较高的酶活性。此外，本发明首次报道了天然低共熔溶剂与酶之间存在协同催化效应，并在此基础上以较高的产物产率成功合成了Gemini型脂氨基酸类食品乳化剂（产率可达59.6%），打破了国内Gemini型脂氨基酸合成的瓶颈。此外，本发明还设计对产物提纯过程的简化，采用了乙酸乙酯、水提取，甲醇重结晶的方式获得了纯度较高的产物。为了进一步降低生产成本，本发明还通过简单的处理对天然低共熔溶剂进行了回收循环利用，10次循环后还能够合成产率达到52.4%，保持初始效果的88.48%，具有优异的效果，极大的降低了生产成本。
 附图说明
图1 为脂氨基酸的合成路线图；
图2 为不同天然低共熔溶剂中甘氨酸酸（a）、月桂酸（b）溶解度，每种温度下从左向右的溶剂分别为甜菜碱-甘油（B-Gly）、甜菜碱-木糖醇（B-X）、氯化胆碱-甘油（C-Gly）、氯化胆碱-木糖醇（C-X）、甜菜碱-甘油-水（B-Gly-W）、氯化胆碱-甘油-水（C-Gly-W）；
图3 为不同天然低共熔溶剂中脂肪酶的活性，每种时间下从左向右的溶剂分别为Tris缓冲液(50mM, PH 8.0)（对照组）、甜菜碱-甘油（B-Gly）、甜菜碱-木糖醇（B-X）、氯化胆碱-甘油（C-Gly）、氯化胆碱-木糖醇（C-X）、甜菜碱-甘油-水（B-Gly-W）、氯化胆碱-甘油-水（C-Gly-W）；
图4 为不同天然低共熔溶剂中脂肪酶的热稳定性，每种时间下从左向右的溶剂分别为甜菜碱-甘油（B-Gly）、甜菜碱-木糖醇（B-X）、氯化胆碱-甘油（C-Gly）、氯化胆碱-木糖醇（C-X）、甜菜碱-甘油-水（B-Gly-W）、氯化胆碱-甘油-水（C-Gly-W）；
图5为产物的红外光谱图；
图6为产物的质谱图；
图7为核磁共振氢谱图及其相应图谱数据图表；
图8为Gemini型月桂酰赖氨酸（GLL）的质谱图；
图9为单链型月桂酰甘氨酸（LAA）的质谱图；
图10为低共熔溶剂的循环使用效率。
具体实施方式
本发明的主要思路在于：
（1）为脂氨基酸的酶法合成提供了新的思路，即采用了一系列具有良好的底物溶解性的酶友好型溶剂。本发明首先考察了天然低共熔溶剂对底物的溶解性，其次，又考察了溶剂对酶的活性以及稳定性的影响及其机制；最后，进行了脂氨基酸的合成实验，合成过程包括两个步骤：1）脂肪酶与脂肪酸结合，完成对脂肪酸的活化；2）氨基酸的氨基对活化后的脂肪酸中间体亲核进攻，形成酰胺。具体步骤如图1所示。
（2）为Gemini型脂氨基酸的酶法合成提供了新的思路，即采用了一系列与酶存在协同催化效应的绿色溶剂作为反应介质。
本发明首先考察了传统有机溶剂中Gemini型脂氨基酸的合成，其次考察了自行设计的天然低共熔溶剂中脂氨基酸的合成，该过程包括：酶的筛选，溶剂的筛选，反应条件优化，溶剂的回收利用，产物的提纯方案。
下面结合具体的实施例对本发明做进一步详细的说明。
Gemini型脂氨基酸的产率=Gemini型脂氨基酸的实际值/Gemini型脂氨基酸的的理论值；
电导率法测定cmc值：利用离子型表面活性剂水溶液的电导率随浓度的变化关系，作Λ m-C 1/2曲线，由曲线的转折点求出CMC值。
Figure dest_path_image001
，
若温度恒定，在极稀的浓度范围内，强电解质溶液的摩尔电导率Λ m与其溶液浓度的c 1/2成线形关系，对于胶体电解质，在稀溶液时的电导率，摩尔电导率的变化规律与强电解质一样，但是随着溶液中胶团的生成，电导率和摩尔电导率发生明显变化，这就是确定CMC的依据。
实施例1单链型月桂酰甘氨酸的制备
分别称取25mM的月桂酸以及25mM的甘氨酸，于25ml反应瓶中.向其中加入10ml天然低共熔溶剂（氯化胆碱-甘油），以及12.5mg脂肪酶CALB。然后将反应瓶密封并置于恒温水浴摇床，60℃，2000rpm下持续反应48h。
反应结束后，取出反应瓶，加入大量的水，剧烈震荡，然后10000rpm离心10min，离心后，真空抽滤得到白色固体，加入甲醇，对粗产物重结晶两次，得到纯度为98%的纯产物。产物得率为50.49%。
通过图5中的质谱可以看出，产物中没有出现脂氨基酸合成过程中常见的副产物---二肽，具有较高的产物得率且同时副产物少。
采用电导率法测定了几种产物的cmc值，月桂酰甘氨酸0.0044mol/L，显著低于目前市面常用表面活性剂吐温十二烷基磺酸钠（0.009mol/L），是一种性能较好的表面活性剂。
实施例2单链型月桂酰甘氨酸的制备
分别称取25mM的月桂酸以及25mM的甘氨酸，于25ml反应瓶中.向其中加入10ml天然低共熔溶剂（甜菜碱-甘油），以及12.5mg脂肪酶CALB。然后将反应瓶密封并置于恒温水浴摇床，60℃，2000rpm下持续反应48h。
反应结束后，取出反应瓶，加入大量的水，剧烈震荡，然后10000rpm离心10min，离心后，真空抽滤得到白色固体，加入甲醇，对粗产物重结晶两次，得到纯度为98%的纯产物。产物最终得率为43.98%。
实施例3单链型月桂酰甘氨酸的制备
分别称取25mM的月桂酸以及25mM的甘氨酸，于25ml反应瓶中.向其中加入10ml天然低共熔溶剂（甜菜碱-木糖醇），以及12.5mg脂肪酶CALB。然后将反应瓶密封并置于恒温水浴摇床，60℃，2000rpm下持续反应48h。
反应结束后，取出反应瓶，加入大量的水，剧烈震荡，然后10000rpm离心10min，离心后，真空抽滤得到白色固体，加入甲醇，对粗产物重结晶两次，得到纯度为98%的纯产物。产物最终得率为38.97%。
实施例4单链型月桂酰甘氨酸的制备
分别称取25mM的月桂酸以及25mM的甘氨酸，于25ml反应瓶中.向其中加入10ml天然低共熔溶剂（氯化胆碱-木糖醇），以及12.5mg脂肪酶CALB。然后将反应瓶密封并置于恒温水浴摇床，60℃，2000rpm下持续反应48h。
反应结束后，取出反应瓶，加入大量的水，剧烈震荡，然后10000rpm离心10min，离心后，真空抽滤得到白色固体，加入甲醇，对粗产物重结晶两次，得到纯度为98%的纯产物。产物最终得率为41.40%。
实施例5：考察低共熔溶剂对反应底物的影响
准确称取10g天然低共熔溶剂（C-Gly），于60℃恒温震荡水浴中，待温度恒定，称取10mg月桂酸加入其中，200rpm下震荡30min，然后用毛细管取出一滴在偏光显微镜下检测，如不出现偏振光，则说明溶解完全，则再次加入10mg月桂酸。直至不再继续溶解（出现偏振光）。最终底物溶解度按照如下公式计算：
S=m t×10/1000                     (1)，
这里S为溶剂度，m t为总的底物添加量。最终月桂酸溶解度为24.98（g/100g）。
月桂酸在常用酶友好型溶剂丙酮中为微溶（0.01-0.1g/100g），在水中的溶解度为0。
甘氨酸在低共熔溶剂中的溶解度为22.78（g/100g），水中溶解度为25g/100g，丙酮中几乎不溶。
从图2可知，不同低共熔溶剂对两种底物的溶解度影响不一，其中在C-Gly-W的溶解性最好，底物普适性也较好。
实施例6：考察不同低共熔溶剂对脂肪酶催化剂的影响
通过pNPP测定法测量脂肪酶活性以及热稳定性：将10μL酶溶液，加入到3990μL NADES（PBS缓冲液，PH8.0，作为对照）中，在60℃（CALB的最佳温度）下以200rpm的速度反应120min，240min和360min。立即从中取出10μL，并加入80μLPBS缓冲液（50mM，pH8.0）和10μLpNPP溶液中。将混合物在60℃，200rpm下反应5min，加入100μL乙醇（95％）立即终止反应。用Spectramax 190酶标仪（Molecular Devices，German）在405nm处测量吸光度。根据pNP计算酶活性以及热稳定性。
结合图3可知，脂肪酶CALB在NADES中经过60℃，360min处理后依然能够保持较高的活性，可达6400U/mL。结构刚性和柔韧性之间的平衡是脂肪酶的高稳定性和活性的基础。NADES可与酶相互作用以增加其结构刚性，从而提高其稳定性。此外，令人意想不到的是，如图3所示，在没有水的四种NADES中，脂肪酶在360分钟后仍保持较高的活性；而在有参与的体系中，酶活逐渐收到抑制。发明人认为可能是因为酶表面可以与NADES键合，如甘油，木糖醇等，这些有机小分子可以与酶形成复杂的氢键网络。
如图4所示，水分的存在削弱了NADES与酶的相互作用，降低了酶的结构刚性和抵抗外部干扰的能力，导致脂肪酶的热稳定性降低，与酶活的效果趋势一致。
对照例1：
参照实施例1，将溶剂有低共熔溶剂替换为丙酮，其他条件不变，产物得率基本可以忽略不计。
实施例7 Gemini型月桂酰氨基酸的制备
取2.5mmol月桂酸、1mmol L-赖氨酸和60mgAmano脂肪酶M加入到20mL氯化胆碱-油酸（1：2）中，500rpm，60℃下进行72小时。反应后的混合物中加入200mL超纯水，并涡旋震荡1分钟；将稀释的反应溶液以10000rpm离心20分钟，保留沉淀物，同时收集水相用于溶剂回收；用25mL乙酸乙酯稀释沉淀物，用20ml饱和碳酸氢钠溶液中和脂肪酸。将混合物以10000rpm离心20分钟，在313K下真空干燥24小时；将干燥的产物加入到5mL甲醇中，涡旋1分钟，并以10000rpm离心20分钟，体系分层，去除上层（甲醇和单链型脂氨基酸），得到Gemini型脂氨基酸产品。产品纯度为98%的纯产物，产率为59.6 %。现有报道有利用低共熔溶剂（氯化胆碱-甘油）配合CALB脂肪酶合成N,N-二月桂酰赖氨酸，产率相为26.3%，不到本实施例效果一半。
通过图6中的质谱可以看出，本实施例制备得到了一种由两个亲水或两个疏水基团组成的Gemini型月桂酰甘氨酸，即脂氨基酸的二聚体。
采用电导率法测定了几种产物的cmc值，Gemini型月桂酰氨基酸的cmc为0.0044 mol/Lmol/L，显著低于目前市面常用表面活性剂吐温十二烷基磺酸钠（0.009mol/L），是一种性能较好的表面活性剂。
实施例8 不同低共熔溶剂对Gemini型脂氨基酸制备的影响
参照实施例7，将溶剂分别替换为表1中所示的溶剂，其他条件不变，反应制备Gemini型脂氨基酸。所得结果如表1所示。
表1 不同低共熔溶剂制备Gemini型脂氨基酸的结果
溶剂 Gemini型脂氨基酸产率（%）
氯化胆碱-亚油酸 29.3
甜菜碱-油酸 22.1
氯化胆碱-甘油 18.6
DMSO NR
丙酮 NR
[C4mim]Cl NR
其中，N.R. 表示没有产物得到。
实施例9 不同酶对Gemini型脂氨基酸制备的影响
参照实施例7，将酶催化剂分别替换为表2中所示的酶，其他条件不变，反应制备Gemini型脂氨基酸。所得结果如表2所示。
表2 不同低共熔溶剂制备Gemini型脂氨基酸的结果
酶催化剂 Gemini型脂氨基酸产率（%）
DA 8.9
PPL 14.6
CALB 19.1
BSAP 6.7
其中，DA为来自大肠杆菌的D-氨基酰化酶；PPL为来自猪胰腺的脂肪酶；CALB为南极假丝酵母（Candida Antarctica）的脂肪酶B；BSAP为来自枯草芽孢杆菌（Bacillus subtilis）的碱性蛋白酶。
实施例10：反应条件参数的优化
参照实施例7，采用单因素试验法，分别对底物摩尔比、反应时间、反应温度、酶添加量等指标进行优化。具体生成Gemini型脂氨基酸的结果如表3所示。
表3 反应条件参数的优化结果
月桂酸：赖氨酸摩尔比 反应时间（h） 酶添加量（g） 反应温度（℃） 产率（%）
2:1 72 60 60 44.3
2:1 72 60 80 45.6
2:1 72 60 40 41.3
2:1 72 60 60 50. 4
2:1 72 75 60 57.6
2:1 72 50 60 51.4
2:1 72 30 60 30.3
2:1 96 60 60 48.0
2.5:1 72 60 60 59.6
1:1 72 60 60 43.9
1:2 72 60 60 28.8
结果显示，反应过程中，酶添加量和反应温度是两种最为关键的影响因素。在60℃下，60mg酶添加量配合体系得到较好的反应效果。
实施例11：考察反应后低共熔溶剂的回收效率
采用在反应后处理过程中收集的水相来回收天然低共熔溶剂：向混合物中加入10质量当量的乙酸乙酯，涡旋1分钟，然后转移到分液漏斗中；弃去有机相，同时收集水相；通过在333K下真空干燥48小时除去过量的水和乙酸乙酯，回收得到低共熔溶剂。
回收后的低共熔溶剂用于月桂酰赖氨酸的合成，以检验其性能，具体反应条件参照实施例1，溶剂经过10次循环，Gemini型脂氨基酸的合成得率由59.6%降低到52.4%，还能够保持初始效果的88.48%。
上述实施例只为说明本发明的技术构思及特点。其目的在于让熟悉此项技术的人能够了解本发明的内容并据此加以实施。并不能以此限制本发明的保护范围，凡根据本发明精神实质所做的等效变化或修饰，都应该涵盖在本发明的保护范围之内。

Claims (20)

  1. 一种制备Gemini型氨基酸的方法，其特征在于，所述方法是在低共熔溶剂中，以Amano脂肪酶M作为催化剂，氨基酸与脂肪酸反应制得Gemini型氨基酸；所述低共熔溶剂中的氢键供体包括油酸和/或亚油酸，氢键受体为氯化胆碱；所述氨基酸中氨基的数量不低于2。
  2. 根据权利要求1所述的方法，其特征在于，所述Amano脂肪酶M的添加量为每摩尔氨基酸添加50-100mg。
  3. 根据权利要求1所述的方法，其特征在于，所述反应的温度为40-80℃。
  4. 根据权利要求1所述的方法，其特征在于，所述低共熔溶剂为氯化胆碱-油酸。
  5. 根据权利要求1所述的方法，其特征在于，低共熔溶剂中氢键供体与氢键受体的化学计量比为2:1~1:2。
  6. 根据权利要求1所述的方法，其特征在于，低共熔溶剂是由氢键供体和氢键受体在60-100℃条件下按照一定化学计量比，经过旋转蒸发获得。
  7. 根据权利要求1所述的方法，其特征在于，所述氨基酸包括赖氨酸、胱氨酸、精氨酸。
  8. 根据权利要求1所述的方法，其特征在于，所述脂肪酸为C2-C20脂肪酸。
  9. 根据权利要求1所述的方法，其特征在于，脂肪酸与氨基酸的摩尔比为2.5:1~1:1。
  10. 一种脂氨基酸类化合物的制备方法，其特征在于，所述方法包括：
    以低共熔溶剂为介质，脂肪酸、氨基酸在催化剂作用下直接制备得到脂氨基酸类表面活性。
  11. 根据权利要求10所述的方法，其特征在于，脂肪酸与氨基酸的摩尔比为1：1~2:1。
  12. 根据权利要求10所述的方法，其特征在于，所述低共熔溶剂包括甜菜碱-甘油、甜菜碱-木糖醇、氯化胆碱-甘油、氯化胆碱-木糖醇中的任意一种。
  13. 根据权利要求10所述的方法，其特征在于，所述的低共熔溶剂中的氢键供体与氢键受体的体积比为2:1~1:2。
  14. 根据权利要求10所述的方法，其特征在于，所述氨基酸包括甘氨酸、赖氨酸、精氨酸、丙氨酸。
  15. 根据权利要求10所述的方法，其特征在于，所述催化剂包括脂肪酶CALB。
  16. 一种表面活性剂的制备方法，其特征在于，所述方法是利用权利要求10-15任一所述方法制备脂氨基酸类化合物，然后进行表面活性剂的制备。
  17. 一种乳化剂的制备方法，其特征在于，所述方法是利用权利要求10-15任一所述方法制备脂氨基酸类化合物，然后在进行乳化剂的制备。
  18. 一种增稠剂的制备方法，其特征在于，所述方法是利用权利要求10-15任一所述方法制备脂氨基酸类化合物，然后在进行增稠剂的制备。
  19. 一种消泡剂的制备方法，其特征在于，所述方法是利用权利要求10-15任一所述方法制备脂氨基酸类化合物，然后在进行消泡剂的制备。
  20. 权利要求1-9任一所述方法在食品乳化剂制备中的应用。
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