CN113088544A - Method for synthesizing bio-based epoxy vegetable oil in deep eutectic solvent through enzyme method - Google Patents
Method for synthesizing bio-based epoxy vegetable oil in deep eutectic solvent through enzyme method Download PDFInfo
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Abstract
The invention relates to a method for synthesizing bio-based epoxy vegetable oil in a deep eutectic solvent by an enzyme method, which comprises the following steps: (1) mixing an oxidation source and choline chloride, heating and stirring to prepare a deep eutectic solvent; (2) mixing the deep eutectic solvent, the vegetable oil, the catalyst and the free fatty acid, stirring, and carrying out an epoxidation reaction to obtain an epoxidation crude product; (3) centrifuging the crude product of the epoxidation, taking an upper oil layer as a finished product of the epoxidized vegetable oil, and recycling a lower catalyst; the oxidizing source is carbamide peroxide. The method applies the deep eutectic solvent to the epoxidation reaction of vegetable oil, and the deep eutectic solvent serves as a solvent and a reaction substrate to participate in the reaction, so that a complete bifunctional epoxidation system is formed. The deep eutectic solvent is low in cost, non-toxic and harmless, the whole system does not need to be additionally added with a solvent and an oxidation source, reaction byproducts are few, the deep eutectic solvent is green and environment-friendly, no waste is generated, and the deep eutectic solvent has a good research prospect.
Description
Technical Field
The invention belongs to the technical field of biochemical engineering, and relates to a method for synthesizing bio-based epoxy vegetable oil in a deep eutectic solvent by an enzyme method.
Background
Vegetable oils containing epoxy groups are important oleochemicals. The main applications of these oils are in various areas as plasticizers and stabilizers for polyvinyl chloride (PVC), and epoxy vegetable oils are used as reactive diluents for coatings and intermediates in the production of polyurethane polyols.
Although the enzymatic catalytic epoxidation has high conversion rate, a hydrolysis side reaction exists in the reaction process, an oxidation source of epoxidation generally uses hydrogen peroxide, the hydrogen peroxide solution has great damage to the activity of a biocatalyst, the hydrolysis side reaction and the damage of hydrogen peroxide to the enzyme can be well reduced by adding a solvent, the epoxidation reaction can be better promoted by adding organic solvents such as toluene and the like, the cost is low, but the later process flow is complicated by adding the organic solvents such as toluene and the like, the later separation is not facilitated, and the organic solvents have great toxicity due to great volatility and great harm to human bodies and the environment. Toluene is a common enzymatic epoxidation solvent, and the addition amount of toluene is large, so that a reaction system is complex, and a later separation step is complicated.
Disclosure of Invention
The invention aims to solve the problem of the prior art and provides a method for synthesizing bio-based epoxy vegetable oil in a deep eutectic solvent by an enzyme method. The method uses a deep eutectic solvent formed by choline chloride and carbamide peroxide in the process of the oil epoxidation of the plant, plays a good role in protecting the activity of the biological enzyme, and the components of the solvent can be simultaneously used as a substrate and a solvent, thereby not only providing an oxidation source for a reaction system, but also playing a role in the solvent.
Therefore, the invention provides a method for enzymatically synthesizing bio-based epoxy vegetable oil in a deep eutectic solvent, which comprises the following steps:
b, mixing an oxidation source and choline chloride, heating and stirring to prepare a deep eutectic solvent;
step C, mixing the deep eutectic solvent, the vegetable oil, the catalyst and the free fatty acid, stirring, and carrying out an epoxidation reaction to obtain an epoxidation crude product;
and D, carrying out centrifugal treatment on the crude product of the epoxidation reaction to obtain a finished product of the epoxy vegetable oil.
According to the invention, in step B, the molar ratio of the oxidizing source to choline chloride is (1-3): 1.
In some embodiments of the present invention, in step B, the temperature for making the deep eutectic solvent is 60-80 ℃.
In other embodiments of the present invention, in step B, the time for preparing the deep eutectic solvent is 10-20 min.
In still other embodiments of the present invention, in step B, the rotation speed of the stirring is above 300 rpm.
In the invention, the oxidizing source can continuously and slowly release hydrogen peroxide in the reaction; preferably, the oxidizing source is carbamide peroxide.
In some embodiments of the invention, the molar ratio of hydrogen peroxide in the carbamide peroxide to double bonds contained in the vegetable oil is (0.5-3): 1.
In the invention, the catalyst is microbial lipase and mainly yeast lipase, and the yeast mainly comprises one or more of candida antarctica, candida rugosa, yarrowia lipolytica, candida citrina, candida tropicalis, candida parapsilosis and candida olivifolia.
In some embodiments of the invention, the catalyst is added in an amount of 5% to 30%, preferably 5% to 10% by mass of the vegetable oil.
In some embodiments of the present invention, the free fatty acid is added in an amount of 1% to 10% by molar mass of the double bonds contained in the vegetable oil.
In the invention, the free fatty acid comprises one or more of oleic acid, linoleic acid and linolenic acid.
According to the invention, in step C, the temperature of the epoxidation reaction is between 40 and 70 ℃.
In some embodiments of the present invention, in step C, the epoxidation reaction is carried out for a period of 12 to 24 hours, preferably 16 to 20 hours.
In other embodiments of the present invention, in step C, the stirring speed is above 300 rpm.
According to the invention, in the step D, the crude product of the epoxidized vegetable oil is subjected to centrifugal treatment, the upper layer is taken as a finished product of the epoxidized vegetable oil, and the lower layer catalyst is recycled.
In the invention, the vegetable oil comprises one or more of linseed oil, soybean oil, perilla oil, sunflower seed oil, peanut oil and rapeseed oil.
Compared with the prior art, the invention has the following characteristics:
(1) the oxidizing source is carbamide peroxide in the epoxidation process, and the carbamide peroxide can slowly release hydrogen peroxide in the reaction process, so that the concentration of the hydrogen peroxide in the reaction system is at a stable level, and the epoxidation can be stably and continuously carried out.
(2) In the epoxidation reaction process, the used solvent is a deep eutectic solvent, and the deep eutectic solvent is prepared by heating and stirring choline chloride and carbamide peroxide. The application of the carbamide peroxide not only provides a deep eutectic solvent for synthesizing the urea and the choline chloride, but also provides an oxidation source hydrogen peroxide required by epoxidation, and the carbamide peroxide is used as a substrate and a solvent to participate in a reaction system in the reaction process.
(3) The solvent is a deep eutectic solvent, and the existence of more chloride ions can strengthen the hydrogen bond interaction with the biological enzyme active center, thereby improving the enzyme activity and stability. In addition, the stability of the enzyme can be improved by hydrogen bond formation between surface amino acid residues and chloride ions, choline and urea.
(4) The invention has the advantages of high selectivity of target products, easy separation of products after the reaction, mild reaction conditions, few byproducts, no pollution of the products and low price, and the anhydrous hydrogen peroxide is safer to treat and minimally hydrolyzes epoxy products without water.
Drawings
The invention is described in further detail below with reference to the attached drawing figures:
FIG. 1 illustrates the epoxidation principle of vegetable oils; the biocatalyst is exemplified by Novozym 435.
FIG. 2 shows the process of epoxidation of vegetable oil after addition of free fatty acid; the biocatalyst is exemplified by Novozym 435.
Detailed Description
In order that the invention may be readily understood, reference will now be made in detail to the present invention as illustrated in the accompanying drawings. However, before the invention is described in detail, it is to be understood that this invention is not limited to particular embodiments described. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.
Where a range of values is provided, it is understood that each intervening value, to the extent that there is no stated or intervening value in that stated range, to the extent that there is no such intervening value, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where a specified range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.
Unless otherwise defined, all terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described.
Term (I)
The term "Deep eutectic solvent" (DES) as used herein has properties similar to those of ionic liquids ILs and is in a liquid state at a relatively low temperature; the deep eutectic solvent is a binary system and a ternary system which are mainly composed of hydrogen bond donors (polyhydric alcohols, urea and carboxylic acid) and hydrogen bond acceptors (quaternary ammonium salts, such as choline chloride and the like), and the most remarkable physical property is the reduction of the melting point of the solvent. As a novel green reagent which can replace ionic liquid, the method has wider application, and can be used for separating natural organic components, such as separating and extracting rutin and other substances.
The term "microbial lipase production" as used herein refers to lipase produced by a microorganism by fermentation, and is also referred to herein as microbial lipase.
The terms "about," "substantially," and "primarily," when used in conjunction with a range of concentrations, temperatures, or other physical or chemical properties or characteristics, as used herein, cover variations that may exist in the upper and/or lower limits of the range of properties or characteristics, including variations that may result, for example, from rounding, measurement, or other statistical variations. As used herein, numerical values associated with amounts, weights, and the like, are defined as all values for each particular value plus or minus 1%. For example, the term "about 10%" should be understood as "9% to 11%".
Embodiments II
As mentioned above, although the conversion rate of enzymatic catalytic epoxidation is high, a hydrolysis side reaction exists in the reaction process, and the oxidative source hydrogen peroxide used in the reaction process has great damage to the enzyme activity, a solvent needs to be added in the reaction process to reduce the hydrolysis rate and reduce the damage to the enzyme, but the addition of the solvent makes the later process flow complicated, which is not favorable for the separation of the later product, toluene is used as a commonly used enzymatic epoxidation solvent, the reaction addition amount is large, the reaction system is complicated and toxic and harmful, and the later separation step is complicated and is not favorable for the further utilization of the product. In view of this, the present inventors have studied a process for preparing an epoxy vegetable oil.
The inventors have found that "ideal" solvents in biocatalysis must meet a large number of requirements, such as high substrate solubility, high enzymatic activity and stability and a positive influence on the reaction balance. Over the past few decades, many enzymatic synthetic routes have been developed in aqueous-based and non-aqueous (organic solvent, ionic or supercritical fluid) reaction media. Ionic Liquids (ILS) have gained tremendous attention as reaction media. However, their technical applicability and "greenness" tend to be limited, mainly due to high cost, poor biodegradability, poor biocompatibility and low sustainability. Among the different biocatalytic reactions, the Deep Eutectic Solvent (DES) was evaluated as the most promising green solvent.
The Deep Eutectic Solvent (DES) is the prototype of the desired solvent, has versatility and biodegradability, has excellent solubilizing properties for acute and even opposite compounds, and is enzyme friendly. The deep eutectic solvent formed by choline chloride and urea is green and environment-friendly, low in cost, mature in research, still liquid at room temperature, and clear and transparent in solution.
Carbamide peroxide (UHP) has the ability to slowly release hydrogen peroxide, is less harmful to enzymes, and has the advantage of producing harmless by-products.
The inventor further researches and discovers that the deep eutectic solvent prepared by carbamide peroxide and choline chloride can provide an oxidation source and also play a role as a solvent in an epoxidation system of vegetable oil. The deep eutectic solvent is used as a substrate and a solvent simultaneously in the whole reaction system to participate in the whole reaction. So that the epoxidation reaction of the vegetable oil is carried out almost under solvent-free conditions. The reaction condition is mild, almost no by-product is formed, the product after the reaction is finished is obviously layered through centrifugal treatment, the product is easy to separate, the whole reaction process is green and pollution-free, and the cost is low. The present invention has been completed based on the above findings.
The traditional method for synthesizing bio-based epoxy vegetable oil by enzyme method is to mix vegetable oil with catalyst (such as Novozym 435) and free fatty acid, add hydrogen peroxide, mix, heat and stir, and perform epoxidation reaction. The main substances in the vegetable oil are triglyceride, unsaturated fatty acid, etc. The epoxidation principle of vegetable oil is as follows: the oxidation source reacts with unsaturated fatty acid in vegetable oil under the catalysis of biological enzyme to generate peroxy fatty acid, namely double bond is epoxidized. The resulting peroxyfatty acid reacts with the triglycerides in the vegetable oil and the resulting mixture contains epoxidized triglycerides, along with small amounts of epoxidized free fatty acids, by addition of free fatty acids (see figure 2).
In the process of the vegetable oil epoxidation reaction, the free fatty acid is added to have the following functions: the epoxidation of vegetable oils results in products which are not only epoxidized triglycerides but also epoxidized mono-and diglycerides (see fig. 1), it is almost impossible to separate these mono-and diglycerides from the reaction mixture, the addition of free fatty acids causes all hydroxyl groups to be immediately re-esterified with excess free fatty acids, whereby the end product only leaves epoxidized triglycerides (see fig. 2).
In the present invention, the double bond conversion means the double bond epoxidation conversion, as will be understood from the above.
The method for synthesizing the bio-based epoxy vegetable oil by the enzyme method in the deep eutectic solvent can be understood as a method for synthesizing the bio-based epoxy vegetable oil by the enzyme method by utilizing the deep eutectic solvent, and comprises the following steps:
b, mixing an oxidation source and choline chloride, heating and stirring to prepare a deep eutectic solvent;
step C, mixing vegetable oil with a catalyst and free fatty acid, adding the deep eutectic solvent prepared in the step B, mixing, heating and stirring, and carrying out an epoxidation reaction to obtain an epoxidation reaction crude product;
and D, carrying out centrifugal treatment on the crude product of the epoxidation reaction to obtain a finished product of the epoxy vegetable oil.
In the preparation of the deep eutectic solvent, the molar ratio of the oxidation source to the choline chloride is (1-3):1, more preferably (1-2):1, and still more preferably 2: 1.
In the step B, the temperature for preparing the deep eutectic solvent is 60-80 ℃, preferably 70-80 ℃, and more preferably 80 ℃; the rotation speed of the stirring is 300rpm or more, preferably 500rpm or more.
It will be appreciated by those skilled in the art that in step B above, the oxidizing source is mixed with the two solids choline chloride, and heated and stirred until a clear solution state is formed, i.e. a deep eutectic solvent is formed, which takes about 10-20min, preferably 10-15 min. This is understood to mean the time required for the oxidizing source to be mixed with the two solids choline chloride, heated and stirred until a clear solution is formed.
In the conventional epoxidation reaction, hydrogen peroxide is generally used as an oxidation source, but hydrogen peroxide solution is harmful to the activity of the biocatalyst, so that the present inventors consider adopting other oxidation sources instead of the hydrogen peroxide solution, and therefore, the oxidation source in the present invention is characterized in that it can continuously and slowly release hydrogen peroxide in the reaction.
The inventor of the invention considers that three problems mainly exist in the process of plant oil oxidation: firstly, the unsaturated fatty acid content in the vegetable oil is high, the amount of hydrogen peroxide required for oxidizing double bonds is high, and the hydrogen peroxide has great damage to the enzyme activity, so that the maintenance of the enzyme activity is important; secondly, the generation of side reaction, namely the hydrolysis of triglyceride is reduced, and the yield of the final product is influenced; and thirdly, after the reaction is finished, the product is separated and the later reaction is recovered, and the whole process needs to be green and environment-friendly.
The synthesis of Deep Eutectic Solvents (DESs) generally involves the mixing of quaternary ammonium salts with alcohols, amides, carboxylic acids and polyols as Hydrogen Bond Donors (HBDs), and the DES produced thereby is referred to as natural deep eutectic solvents (NADES) when both the Hydrogen Bond Acceptor (HBA) and the hydrogen bond donor HBD are naturally occurring primary metabolites (i.e., amino acids, choline, sugars, alcohols or amino acids and organic acids). The superior properties of DESs compared to volatile organic solvents and ILS have prompted their use in a range of biochemical applications.
UHP (Urea-Hydrogen peroxide Complex, molecular formula is CO (NH)2)2.H2O2) White crystal with melting point of 90-93 deg.c, and is easy to dissolve in water, ethanol, glycol, dichloromethane and other organic solvent. The urea hydrogen peroxide complex (UHP) is a complex resulting from strong hydrogen bonding between urea and hydrogen peroxide. It converts unstable hydrogen peroxide into solid which is easy to operate, safe and stable at room temperature, and has low price. UHP exhibits different oxidation capacities in different solvents and catalysts and is an important oxidizing agent in organic synthesis.
UHP has the properties of hydrogen peroxide, is commonly used for epoxidation of double bonds, and has a high regio-and stereoselectivity as well as a high conversion.
Based on the above description, carbamide peroxide is preferred as an oxidation source to synthesize a green and pollution-free deep eutectic solvent with choline chloride in the invention.
As described above, the inventors of the present invention have found that the deep eutectic solvent is used as a substrate and a solvent for a reaction in an epoxidation reaction of vegetable oil, and is a green, environmentally friendly and promising solvent. The low-cost raw materials of carbamide peroxide and choline chloride are synthesized into a deep eutectic solvent which serves as a solvent in an epoxidation reaction, so that the structural stability and the activity of the biocatalyst are fully protected, and an organic solvent is replaced; carbamide peroxide in the deep eutectic solvent serves as an oxidation source to slowly and continuously release hydrogen peroxide to replace the use of a hydrogen peroxide solution; specifically, during the reaction process, carbamide peroxide is decomposed into urea and hydrogen peroxide, the urea and choline chloride form a deep eutectic solvent, and the hydrogen peroxide provides an oxidation source for the epoxidation process of vegetable oil. So that the whole reaction system is reacted under the condition of almost no solvent. Therefore, in the invention, the solvent is a deep eutectic solvent synthesized by carbamide peroxide and choline chloride, and the deep eutectic solvent synthesized by carbamide peroxide and choline chloride is used in the oxidation reaction of the vegetable oil, so that the whole system forms a dual-function system, and the deep eutectic solvent serves as a reaction solvent and a reaction substrate in the reaction.
Therefore, the invention uses the green and environment-friendly deep eutectic solvent, and the deep eutectic solvent is synthesized by carbamide peroxide and choline chloride through heating and stirring, wherein the carbamide peroxide slowly releases hydrogen peroxide in the reaction process, and the damage to the enzyme activity is greatly reduced; the deep eutectic solvent can stabilize enzyme activity and reduce side reactions of a reaction system, before reaction, the deep eutectic solvent is a solvent formed by carbamide peroxide and choline chloride, and in the reaction process, the deep eutectic solvent is a deep eutectic solvent synthesized by carbamide and choline chloride; the deep eutectic solvent is easy to dissolve in water, the oil layer and the solvent layer are obviously layered through centrifugal treatment after the reaction is finished, and the product is easy to separate.
In the invention, no hydrogen peroxide solution is added in the reaction process, the oxidation source is provided by carbamide peroxide, the adding amount of the deep eutectic solvent is calculated according to the molar ratio of double bonds of the vegetable oil to the hydrogen peroxide, and then the adding amount of the deep eutectic solvent is calculated according to the molar ratio of the carbamide peroxide to the choline chloride. For example, in some instances, the molar ratio of hydrogen peroxide in the carbamide peroxide to the double bonds contained in the vegetable oil is (0.5-3):1, preferably (0.5-1): 1.
According to the method of the invention, in step C, the stirring speed is above 300rpm, preferably above 500rpm, and the temperature is 40-70 ℃, preferably 50-60 ℃; the epoxidation reaction time is 12-24h, preferably 16-20h, more preferably 16 h.
The inventor researches and discovers that the epoxidation reaction is carried out at the temperature of 40-70 ℃, the influence on the activity of enzyme is not great, a reaction solvent and an oxidation source are not required to be additionally added in the whole reaction system, the addition of a deep eutectic solvent serves as the reaction solvent and provides the oxidation source, the determination of the epoxy value of an epoxidation product shows that the epoxidation can be successfully carried out by the method, the epoxidation value can reach 4, the conversion rate of double bonds of epoxidation can reach 41.7 percent, and the full demonstration that the invention can be applied to the epoxidation of vegetable oil is provided.
It is easily understood that the free fatty acid plays a role of mediating the vegetable oil epoxidation reaction in the epoxidation reaction of step C, and the amount of the free fatty acid added is 1% to 10%, preferably 5% to 10%, and more preferably 5% of the molar mass of the double bonds contained in the vegetable oil.
In the invention, the free fatty acid comprises one or more of oleic acid, linoleic acid and linolenic acid, preferably oleic acid or a mixture of oleic acid, linoleic acid and linolenic acid, and the mass ratio of the oleic acid to the linoleic acid to the linolenic acid in the mixture of the oleic acid, the linoleic acid and the linolenic acid is (1-2): (1-5), preferably 2:2: 5.
In the present invention, the sources of the free fatty acid, carbamide peroxide and choline chloride are not particularly limited, and for example, the free fatty acid may be made by itself or may be commercially available.
In the invention, the catalyst is microbial lipase and mainly yeast lipase, and the yeast mainly comprises one or more of candida antarctica, candida rugosa, yarrowia lipolytica, candida citrina, candida tropicalis, candida parapsilosis and candida olivifolia.
In some embodiments of the present invention, the catalyst is added in a single step, and the amount of the catalyst added is 5% to 30%, preferably 5% to 10%, and more preferably 5% of the mass of the vegetable oil.
According to the method of the invention, in step D, after the reaction is finished, the reactant is centrifuged, and after centrifugation, the reactant is divided into two and three layers, wherein the two layers are an oil phase and a water phase, the three layers are an oil layer, an enzyme layer and a solvent layer, more specifically, the solvent layer is two layers, the upper layer is a transparent deep eutectic solvent synthesized by choline chloride and urea, and the lower layer is a milky deep eutectic solvent synthesized by choline chloride and carbamide peroxide. The whole process is very obvious in delamination.
In some preferred embodiments of the present invention, in step D, after the crude epoxidized vegetable oil product is centrifuged, the upper oil layer is taken as the finished epoxidized oil product, and the lower layer can be recycled. Experimental results show that the bio-enzyme catalyst used as the catalyst can be recycled after being recycled, and can be recycled for more than three times.
The vegetable oil is used in the epoxidation reaction, mainly in order to utilize unsaturated parts in the vegetable oil, and the unsaturated vegetable oil is epoxidized to obtain the required epoxy oil. In the invention, the vegetable oil comprises one or more of linseed oil, soybean oil, perilla oil, sunflower seed oil, peanut oil and rapeseed oil, and preferably linseed oil and soybean oil.
In some specific embodiments of the present invention, the preparation of epoxidized vegetable oil using a bio-enzyme catalyst comprises the steps of:
(1) preparing a deep eutectic solvent: choline chloride and carbamide peroxide are heated and stirred to obtain a solution state, namely a deep eutectic solvent is obtained, the deep eutectic solvent is green and environment-friendly and has low cost, and the deep eutectic solvent can be used as a solvent and a substrate in the epoxidation reaction of vegetable oil, so that the raw materials and the solvent form a complete and compact dual-function epoxidation system. Meanwhile, the performance of the enzyme is enhanced and the epoxidation side reaction is reduced.
(2) After reaction raw materials (vegetable oil and free fatty acid) and a catalyst are uniformly mixed with a deep eutectic solvent and a system is stable (the system is stable, namely the temperature reaches the set temperature of the epoxidation reaction, namely the epoxidation reaction temperature), reacting for 16-20h, and obtaining an epoxidation reaction crude product after the reaction is finished.
(3) And (4) centrifuging the crude epoxy vegetable oil product, taking out an upper oil layer to serve as a finished epoxy vegetable oil product, and recovering the enzyme for recycling.
The temperature of the deep eutectic solvent prepared in the step (1) is 70-80 ℃, the magnetic rotation speed is more than 300rpm, and the time is 10-15 min.
The temperature of the epoxidation reaction in the step (2) is 40-70 ℃, the stirring speed is more than 300rpm, and the reaction time is 12-24 h.
The oxidation source is provided by carbamide peroxide, and the dosage of the carbamide peroxide is as follows: the molar ratio of the hydrogen peroxide to the double bonds of the vegetable oil is (0.5-3): 1.
The addition mode of the biocatalyst is one-time addition, and the addition amount of the biocatalyst is 5% -10% of the vegetable oil.
The addition amount of the free fatty acid is 1-10% of the molar mass of double bonds contained in the vegetable oil.
The result shows that the epoxide prepared by the method has the epoxidation value of 3.5-4.0 and the double bond epoxidation conversion rate of 36.5-41.7 percent, and the bio-enzyme catalyst used as the catalyst can be recycled after being recycled, and can be recycled for more than 3 times.
The related parameter detection and calculation method in the invention is as follows:
(1) the method for measuring the double bond content in the vegetable oil comprises the following steps:
methyl-esterifying the vegetable oil, performing gas phase analysis by using a Saimerfi TRACE1300 gas chromatograph (RTX-WAX chromatographic column) to obtain the relative contents of different types of fatty acids in the vegetable oil, further obtaining the total double bond mole number in the vegetable oil, namely,
wherein W is the vegetable oil molecular weight; a oleic acid content; b is the linoleic acid content; c is the linolenic acid content; and 92 is glycerol molecular weight.
(2) The epoxide number is determined with reference to the determination of the epoxide number of the GB1677-2008 plasticizer.
Wherein V is the amount of the sodium hydroxide standard solution consumed in the blank test, and the unit is ml;
v1 is the amount of sodium hydroxide standard solution consumed in the sample test, and the unit is ml;
v2 is the amount of the sodium hydroxide standard solution used for determining the acid value in ml in the sample;
n is the equivalent concentration of the sodium hydroxide standard solution, and the unit is mol/L;
w sample weight in g;
g is the weight of the sample in G when the acid value is measured;
0.016 is the oxygen meq.
Note: the epoxidation value is determined by using a titration form, the color change in the titration process is clear, the solution color is changed from pink purple to yellow, and the titration is continued to be changed into grey blue, namely the end of the titration.
(3) The double bond conversion is theoretically calculated according to the following formula:
in the above formula:
the amount of material having a double bond content equal to the oxygen content of the epoxy number, in mol;
the theoretical double bond content, namely the total double bond content in the vegetable oil, is obtained by the related parameter detection and calculation method involved in the invention, namely the determination method of the double bond content in the vegetable oil (1), and the unit is mol.
III example
The present invention will be specifically described below with reference to specific examples. The experimental methods described below are, unless otherwise specified, all routine laboratory procedures. The experimental materials described below, unless otherwise specified, are commercially available.
Example 1:
(1) mixing choline chloride and carbamide peroxide at the temperature of 80 ℃ and the rotation speed of 500rpm to form a clear solution state (about 15 min) to obtain a deep eutectic solvent, and cooling the solution to the temperature of 60 ℃ required by epoxidation reaction for later use;
(2) mixing a deep eutectic solvent, 25g of linseed oil, free fatty acid (a mixture of oleic acid, linoleic acid and linolenic acid, wherein the mass ratio of the oleic acid to the linoleic acid to the linolenic acid is 2:2:5) and commercial lipase from candida antarctica in a 100ml round-bottom flask, wherein the addition amount of the enzyme is 5 percent, the oxidation source is carbamide peroxide, the molar ratio of choline chloride to carbamide peroxide in the deep eutectic solvent is 1:2, the molar ratio of hydrogen peroxide in the carbamide peroxide to double bonds contained in raw oil is 1:1, the addition amount of the free fatty acid is 5 percent of the molar amount of the double bonds in the raw oil, the reaction temperature is 60 ℃, the rotation speed of 500rpm, reacting for 16 hours, and obtaining an epoxidized crude product after the reaction is finished;
(3) the crude epoxidation product is centrifuged and separated into three layers, an oil phase, a water phase, an oil layer, a catalyst layer and a solvent layer. Centrifuging, and taking out the upper layer to obtain the epoxy linseed oil. The epoxy value was determined to be 4.0 and the double bond conversion was 41.7%.
Example 2:
(1) mixing choline chloride and carbamide peroxide at the temperature of 80 ℃ and the rotation speed of 500rpm to form a clear solution state (about 15 min) to obtain a deep eutectic solvent, and cooling the solution to the temperature of 60 ℃ required by epoxidation reaction for later use;
(2) mixing a deep eutectic solvent, 25g of linseed oil, free fatty acid (a mixture of oleic acid, linoleic acid and linolenic acid, wherein the mass ratio of the oleic acid to the linoleic acid to the linolenic acid is 2:2:5) and commercial lipase from candida antarctica in a 100ml round-bottom flask, wherein the addition amount of the enzyme is 5 percent, the oxidation source is carbamide peroxide, the molar ratio of choline chloride to carbamide peroxide in the deep eutectic solvent is 1:3, the molar ratio of hydrogen peroxide in the carbamide peroxide to double bonds contained in raw oil is 1:1, the addition amount of the free fatty acid is 5 percent of the molar amount of the double bonds in the raw oil, the reaction temperature is 60 ℃, the rotation speed of 500rpm, reacting for 16 hours, and obtaining an epoxidized crude product after the reaction is finished;
(3) the crude epoxidation product is centrifuged and separated into three layers, an oil phase, a water phase, an oil layer, a catalyst layer and a solvent layer. Centrifuging, and taking out the upper layer to obtain the epoxy linseed oil. The epoxy value was determined to be 3.5 and the double bond conversion was 36.5%.
Example 3:
(1) mixing choline chloride and carbamide peroxide at the temperature of 80 ℃ and the rotation speed of 500rpm to form a clear solution state (about 15 min) to obtain a deep eutectic solvent, and cooling the solution to the temperature of 60 ℃ required by epoxidation reaction for later use;
(2) mixing a deep eutectic solvent, 25g of linseed oil, free fatty acid (a mixture of oleic acid, linoleic acid and linolenic acid, wherein the mass ratio of the oleic acid to the linoleic acid to the linolenic acid is 2:2:5) and commercial lipase from candida antarctica in a 100ml round-bottom flask, wherein the addition amount of the enzyme is 5 percent, the oxidation source is carbamide peroxide, the molar ratio of choline chloride to carbamide peroxide in the deep eutectic solvent is 1:2, the molar ratio of hydrogen peroxide in the carbamide peroxide to double bonds contained in raw oil is 1:2, the addition amount of the free fatty acid is 5 percent of the molar amount of the double bonds of the raw oil, the reaction temperature is set to be 60 ℃, the rotating speed of 500rpm is rotated, the reaction is carried out for 16 hours, and after the reaction is finished, an epoxidized crude product is obtained;
(3) the crude epoxidation product is centrifuged and separated into three layers, an oil phase, a water phase, an oil layer, a catalyst layer and a solvent layer. Centrifuging, and taking out the upper layer to obtain the epoxy linseed oil. The epoxy value was determined to be 3.7 and the double bond conversion was 38.5%.
Example 4:
(1) mixing choline chloride and carbamide peroxide at the temperature of 80 ℃ and the rotation speed of 500rpm to form a clear solution state (about 15 min) to obtain a deep eutectic solvent, and cooling the solution to the temperature of 60 ℃ required by epoxidation reaction for later use;
(2) mixing a deep eutectic solvent, 25g of linseed oil, free fatty acid (a mixture of oleic acid, linoleic acid and linolenic acid, wherein the mass ratio of the oleic acid to the linoleic acid to the linolenic acid is 2:2:5) and thalli enzyme from yarrowia lipolytica in a 100ml round-bottom flask, wherein the addition amount of the enzyme is 5 percent, the oxidation source is carbamide peroxide, the molar ratio of choline chloride to carbamide peroxide in the deep eutectic solvent is 1:2, the molar ratio of hydrogen peroxide in the carbamide peroxide to double bonds contained in raw oil is 1:1, the addition amount of the free fatty acid is 5 percent of the molar amount of the double bonds of the raw oil, the reaction temperature is 60 ℃, the rotation speed of 500rpm, reacting for 16 hours, and obtaining an epoxidized crude product after the reaction is finished;
(3) the crude epoxidation product is centrifuged and separated into three layers, an oil phase, a water phase, an oil layer, a catalyst layer and a solvent layer. Centrifuging, and taking out the upper layer to obtain the epoxy linseed oil. The epoxy value was determined to be 3.8 and the double bond conversion was 39.6%.
Example 5:
(1) mixing choline chloride and carbamide peroxide at the temperature of 80 ℃ and the rotation speed of 500rpm to form a clear solution state (about 15 min) to obtain a deep eutectic solvent, and cooling the solution to the temperature of 60 ℃ required by epoxidation reaction for later use;
(2) mixing a deep eutectic solvent, 25g of soybean oil, free fatty acid (a mixture of oleic acid, linoleic acid and linolenic acid, wherein the mass ratio of the oleic acid to the linoleic acid to the linolenic acid is 2:2:5) and commercial lipase enzyme derived from candida antarctica A in a 100ml round-bottom flask, wherein the addition amount of the enzyme is 5 percent, the oxidation source is carbamide peroxide, the molar ratio of choline chloride to carbamide peroxide in the deep eutectic solvent is 1:2, the molar ratio of hydrogen peroxide in the carbamide peroxide to double bonds contained in raw oil is 1:1, the addition amount of the free fatty acid is 5 percent of the molar amount of the double bonds of the raw oil, the reaction temperature is 60 ℃, the rotation speed is 500rpm, reacting for 16 hours, and obtaining an epoxidized crude product after the reaction is finished;
(3) the crude epoxidation product is centrifuged and separated into three layers, an oil phase, a water phase, an oil layer, a catalyst layer and a solvent layer. Centrifuging, and taking out the upper layer to obtain the epoxy linseed oil. The epoxy value was determined to be 3.9 and the double bond conversion was 40.6%.
Test results show that the biological enzyme catalyst used as the catalyst can be recycled and reused for more than 3 times.
It should be noted that the above-mentioned embodiments are only for explaining the present invention, and do not constitute any limitation to the present invention. The invention has been described with reference to an exemplary embodiment, but the words which have been used herein are words of description and illustration, rather than words of limitation. The invention can be modified, as prescribed, within the scope of the claims and without departing from the scope and spirit of the invention. Although the invention has been described herein with reference to particular means, materials and embodiments, the invention is not intended to be limited to the particulars disclosed herein, but rather extends to all other methods and applications having the same functionality.
Claims (9)
1. A method for enzymatically synthesizing bio-based epoxy vegetable oil in a deep eutectic solvent, comprising:
b, mixing an oxidation source and choline chloride, heating and stirring to prepare a deep eutectic solvent;
step C, mixing the deep eutectic solvent, the vegetable oil, the catalyst and the free fatty acid, stirring, and carrying out an epoxidation reaction to obtain an epoxidation crude product;
and D, carrying out centrifugal treatment on the crude product of the epoxidation reaction to obtain a finished product of the epoxy vegetable oil.
2. The process of claim 1, wherein in step B, the molar ratio of oxidizing source to choline chloride is (1-3): 1; and/or the temperature for preparing the deep eutectic solvent is 60-80 ℃; and/or, the time for preparing the deep eutectic solvent is 10-20 min; and/or the rotation speed of the stirring is above 300 rpm.
3. The method according to claim 1 or 2, wherein the oxidizing source is capable of releasing hydrogen peroxide continuously and slowly in the reaction; preferably, the oxidizing source is carbamide peroxide; more preferably, the molar ratio of hydrogen peroxide in the carbamide peroxide to double bonds contained in the vegetable oil is (0.5-3): 1.
4. The method of any one of claims 1-3, wherein the catalyst is microbial lipase and is primarily yeast lipase, and the yeast primarily comprises one or more of Candida antarctica, Candida rugosa, yarrowia lipolytica, Candida citrifolia, Candida tropicalis, Candida parapsilosis, and Candida oliviformis.
5. The method according to any one of claims 1 to 4, wherein the catalyst is added in an amount of 5% to 30%, preferably 5% to 10% by mass of the vegetable oil.
6. The method according to any one of claims 1-5, wherein: the addition amount of the free fatty acid is 1-10% of the molar mass of double bonds contained in the vegetable oil; preferably, the free fatty acid comprises one or more of oleic acid, linoleic acid and linolenic acid.
7. The method according to any one of claims 1-6, wherein: in step C, the temperature of the epoxidation reaction is 40-70 ℃; and/or the time of epoxidation reaction is 12-24h, preferably 16-20 h; and/or the stirring speed is above 300 rpm.
8. The method according to any one of claims 1-7, wherein: and D, centrifuging the crude epoxy vegetable oil product, taking the upper layer as a finished epoxy vegetable oil product, and recycling the lower layer catalyst.
9. The method according to any one of claims 1-8, wherein: the vegetable oil comprises one or more of linseed oil, soybean oil, perilla oil, sunflower seed oil, peanut oil and rapeseed oil.
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| US20100136635A1 (en) * | 2008-11-28 | 2010-06-03 | Council Of Scientific & Industrial Research | Method of Inducing Chirality to Epoxides Using 2,3:4,6 di-O-isopropylidene-2-keto-L-gulonic Acid Monohydrate |
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| US20100136635A1 (en) * | 2008-11-28 | 2010-06-03 | Council Of Scientific & Industrial Research | Method of Inducing Chirality to Epoxides Using 2,3:4,6 di-O-isopropylidene-2-keto-L-gulonic Acid Monohydrate |
| CN107254494A (en) * | 2017-06-22 | 2017-10-17 | 华南理工大学 | A kind of glycerine ester type PUFA preparation method |
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