CN114058650A - Method for preparing epoxy vegetable oil by double-enzyme coupling reaction system - Google Patents
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Abstract
The invention belongs to the field of biochemical engineering, and relates to a method for preparing epoxy vegetable oil by using a double-enzyme coupling reaction system. The method comprises the following steps: b, preparing buffer solution with corresponding concentration and pH value, and dissolving glucose; step C, adding vegetable oil, free fatty acid and an organic solvent into the prepared glucose solution, fully stirring, and simultaneously adding two biocatalysts when the reaction system is stabilized at 40 ℃ to perform epoxidation reaction; step D, centrifuging the solution after the reaction is finished, removing the organic solvent from the upper layer to obtain epoxy vegetable oil, and obtaining a gluconic acid solution from the lower layer, and simultaneously recycling the catalyst; the method utilizes the byproduct hydrogen peroxide generated by the oxidation reaction of glucose as an oxidation source to participate in the oil epoxidation reaction of plants. The system can simultaneously obtain the gluconic acid and the epoxy vegetable oil through reaction, and has good development prospect.
Description
Technical Field
The invention belongs to the technical field of biochemical engineering, and relates to a method for preparing epoxy vegetable oil by using a double-enzyme coupling reaction system.
Background
Polyvinyl chloride (PVC) is one of the most widely used thermoplastic materials at present and plays an important role in the plastics industry. By adding the plasticizer, the polyvinyl chloride can be widely applied to the fields of military affairs, infant care, building, biomedicine, food packaging and the like.
Plasticizers are one of the largest plastics additives worldwide in terms of production and consumption, and are widely used in industry. The primary function of the plasticizer is to improve the flexibility and processability of the polymer by lowering the secondary transition temperature. Plasticizers reduce secondary bonding between the polymer and the polymer chain, allowing the macromolecular species to have greater fluidity, resulting in a more flexible, more deformable product. However, it has been found through extensive research that the quality of the plasticizer is affected by other side reactions during the reaction or storage problems, and substances harmful to the environment and animals are generated. Therefore, the selection of a suitable plasticizer requires a lot of experimentation and great innovation and can be practically applied to industrial production, which is of great significance to the environmental problems which are now increasingly noticed.
Vegetable oils containing epoxy groups are important oleochemicals. The main application of these oils is their use as PVC plasticizers and stabilizers, and the current industrial epoxidized oils are made by epoxidizing unsaturated vegetable oils as raw materials. In the epoxidation reaction, a short-chain peroxy acid is preferably prepared by reacting acetic acid with hydrogen peroxide (H)2O2) The in situ reaction of (a) produces peroxyacetic acid and is used as a catalyst. However, such epoxidation results in highly corrosive waste products and also leads to ring opening of the epoxidation and polymerization under acidic conditions.
There has been some research effort to catalyze the epoxidation of vegetable oils using biocatalysts, using hydrogen peroxide (H)2O2) As an oxidation source, under the catalysis of biological enzyme and the condition of adding a small amount of free fatty acid, the vegetable oil is subjected to self-epoxidation, and hydrogen peroxide reacts with unsaturated fatty acid in the vegetable oil to generate peroxy fatty acid, namely double bonds are epoxidized. The resulting peroxyfatty acid reacts with the triglycerides in the vegetable oil and, under the conditions of addition of free fatty acid, the resulting mixture contains epoxidized triglycerides, as well as small amounts of epoxidized free fatty acid.
Addition of free fatty acids: the epoxidation of vegetable oils results in products which are not only epoxidized triglycerides but also epoxidized mono-and diglycerides (see fig. 2), separation from the reaction mixture is almost impossible, and the addition of free fatty acids allows all hydroxyl groups to be immediately re-esterified with excess free fatty acids, thus leaving the final product with only epoxidized triglycerides and a small amount of epoxidized free fatty acids.
But hydrogen peroxide (H)2O2) Is a strong oxidant, and is decomposed into water (H) by light2O) and oxygen (O)2) The biological enzyme is a biological catalyst with activity and specificity, is a protein and can be biodegraded, the condition of the enzyme catalysis is quite mild, and the H is converted into the protein2O2Directly applied to related biological enzyme method epoxidation reaction as an oxidant, the biological enzyme catalyst is easily deactivated, and H2O2Easy photolysis, low hydrogen peroxide utilization rate, and poor reutilization of biocatalyst due to its damaged activity.
Disclosure of Invention
The invention aims to solve the problem of the prior art and provides a method for preparing epoxidized vegetable oil by using a double-enzyme coupling reaction system. The method utilizes glucose to generate gluconic acid and hydrogen peroxide under the catalysis of glucose oxidase, and the generated byproduct hydrogen peroxide can be utilized to participate in the enzymatic plant oil epoxidation reaction. The hydrogen peroxide generated by the method hardly influences the activity of the biocatalyst, so that the biocatalyst can be effectively recycled for many times, and the enzyme activity is hardly influenced. The whole reaction condition is mild, the byproducts are few, the products are easy to separate, and a brand new research direction is provided for the preparation of the bio-based epoxy oil.
Therefore, the invention provides a method for preparing epoxidized vegetable oil by using a double-enzyme coupling reaction system, which comprises the following steps:
step B, dissolving glucose in a phosphate buffer solution to prepare a glucose solution;
step C, mixing and heating the glucose solution, the vegetable oil, the free fatty acid and the organic solvent, and adding the glucose oxidase and the biological lipase at the same time when the temperature is stabilized at the reaction temperature to form a double-enzyme coupling reaction system, so that the double-enzyme coupling reaction system performs an epoxidation reaction to obtain an epoxidation reaction crude product;
and D, carrying out centrifugal treatment on the crude epoxidation product, taking an oil phase for rotary evaporation, and removing the organic solvent to obtain a finished epoxy vegetable oil product.
According to the invention, in step C, the vegetable oil is subjected to an epoxidation reaction in the presence of an oxidation source, wherein the oxidation source is hydrogen peroxide generated by oxidation reaction of glucose under the catalysis of glucose oxidase; preferably, the glucose dosage is calculated by hydrogen peroxide required by the theory of the vegetable oil epoxidation reaction; further preferably, the molar ratio of hydrogen peroxide generated by oxidation of glucose to double bonds contained in the vegetable oil is (0.5-3): 1.
in some embodiments of the invention, the glucose oxidase is added in an amount of 5% to 10% of the amount of glucose added.
In other embodiments of the present invention, the amount of bio-lipase added is 5% to 10% of the amount of vegetable oil added.
Preferably, the addition ratio of the glucose oxidase to the biological lipase is (1-2) to (1-2).
In the invention, the main sources of the biological lipase comprise one or more of candida antarctica, candida rugosa, yarrowia lipolytica, candida citrinopilea, candida tropicalis, candida parapsilosis and candida olivaceus.
According to the invention, the volume ratio of vegetable oil to organic solvent is 1: 3.
in some embodiments of the invention: the addition amount of the free fatty acid is 1-10% of the vegetable oil, and preferably 5-10%.
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 B, the phosphate buffer has a pH value of 6.5 to 7.0 and a concentration of 50 to 150mmol/L, preferably 50 to 100 mmol/L.
The concentration of the glucose solution is 20-40% (g/mL), preferably 20-37% (g/mL)
In some particularly preferred embodiments of the present invention, the concentration of the glucose solution is 20% to 40% (g/mL), preferably 20% to 37% (g/mL).
According to the invention, in step C, the temperature of the epoxidation reaction is between 30 and 50 ℃.
In some embodiments of the invention, in step C, the epoxidation reaction is carried out for a period of time ranging from 10 to 20 hours.
In some embodiments of the invention, in step C, the speed of the agitation is above 150 rpm.
According to the invention: and D, centrifuging the crude epoxy vegetable oil product, taking oil phase substances for rotary evaporation, removing the organic solvent to obtain epoxy vegetable oil, and recycling the lower-layer catalyst.
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 oxidation source in the vegetable oil oxidation is a byproduct hydrogen peroxide generated by using glucose under the catalysis condition of glucose oxidase. Two problems are solved simultaneously: removing hydrogen peroxide which is a byproduct generated in the process of oxidizing glucose to generate gluconic acid; the influence of over high hydrogen peroxide concentration on the reaction activity of the biocatalyst in the process of enzyme-catalyzed epoxidation reaction is reduced.
(2) The double-enzyme catalytic epoxidation reaction process reacts in an oil-water two-phase to simultaneously generate gluconic acid and epoxy vegetable oil. The water phase is subjected to oxidation reaction of glucose under the catalytic condition of glucose oxidase to generate gluconic acid and hydrogen peroxide; the oil phase directly utilizes the hydrogen peroxide generated in the water phase reaction under the catalysis of biological lipase, participates in the epoxidation reaction of vegetable oil, and generates epoxy triglyceride and a small amount of epoxidized free fatty acid.
(3) The invention has the advantages of high selectivity of target products, easy treatment of the products after the reaction, mild reaction conditions, few byproducts, almost no influence on the activity of the biological lipase, and capability of ensuring the original catalytic activity of the biocatalyst to the maximum extent, so that the biocatalyst can be better recycled.
Furthermore, after the reaction of the method is finished, the oil phase and the water phase are obviously layered, the oil phase is treated to obtain the epoxidized vegetable oil, the water phase is treated to obtain the gluconic acid, and the gluconic acid is further treated to be utilized in other aspects.
Drawings
The invention is described in further detail below with reference to the attached drawing figures:
FIG. 1 shows the basic principle of a two-enzyme coupled reaction system.
FIG. 2 shows the epoxidation of vegetable oils under lipase catalyzed conditions.
Figure 3 shows the reaction in figure 1 with the addition of free fatty acid.
FIG. 4 shows the basic principle of the Glucose Oxidase (GOX) catalyzing the oxidation reaction of β -D-glucose.
FIG. 5 is a high performance liquid chromatogram of a gluconic acid standard.
Fig. 6 is a standard concentration curve of gluconic acid.
FIG. 7 shows a high performance liquid chromatogram of the product of the invention.
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)
Glucose oxidase: glucose Oxidase (GOX) from aspergillus niger is naturally produced in some fungi and insects, catalyzing the product hydrogen peroxide as an antibacterial and antifungal agent. GOX is generally recognized as safe, and the reaction catalyzed by GOX can remove oxygen and produce hydrogen peroxide, a characteristic used for food preservation. GOX is also used in baking, dry egg powder production, wine production, gluconic acid production, and the like. Its electrochemical activity makes it an important component of glucose sensors and possible for application in fuel cells. In addition, GOX preparations are relatively inexpensive and widely available, and potential uses remain to be developed.
Catalytic properties of glucose oxidase: glucose oxidase contains two units of FAD (flavin adenine dinucleotide) per enzyme molecule, and as a hydrogen acceptor, it catalyzes the oxidation of glucose to gluconic acid and produces hydrogen peroxide.
The catalytic reaction of glucose oxidase has the following three forms according to different conditions:
in the absence of catalase, 1 mole of oxygen is consumed per mole of glucose oxidase oxidation.
C6H12O6+O2→C6H12O7+H2O2
In the presence of catalase, 0.5 moles of oxygen are consumed per mole of glucose oxidase oxidation.
C6H12O6+1/2O2→C6H12O7
In the presence of ethanol and catalase, hydrogen peroxide is simultaneously used for the oxidation of ethanol, at this point. Every mole of glucose oxidase is oxidized consuming 1 mole of oxygen.
C6H12O6+C2H5OH+O2→C6H12O7+CH3CHO+H2O
Biological lipase: lipase EC 3.1.1.3 refers to ester bond hydrolase mainly used for hydrolyzing triglyceride, is a polymer taking glycoprotein as a core, consists of hydrophilic and hydrophobic parts, and has a molecular weight of about 20-360 kDa. The catalytic characteristics and effects, amino acid sequences and molecular sizes of the compounds can be greatly different due to different sources, but the active centers have more similar triad structures.
The study of enzymes has been long-standing so far, and lipase is widely distributed in animal and plant tissues and various microorganisms and is one of the earliest studied enzymes. Compared with animal and plant lipases, the microbial lipase has wider application and more varieties. Compared with lipases from other sources, the lipase has wide range of action temperature and pH value, wider sources, more advantages in substrate characteristics, stability and activity and stronger substrate specificity, can be better applied to industries, and becomes an important industrial enzyme in recent years.
Enzymatic vegetable oil epoxidation: 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 with small amounts of epoxidized free fatty acids by addition of free fatty acids (see figure 3).
Words and phrases of the invention"coupling" refers to the phenomenon that when one reaction occurs, other reactions proceed concomitantly in a stoichiometric relationship, and specifically in the present invention, the vegetable oil epoxidation reaction is coupled with the glucose oxidation reaction; wherein the vegetable oil oxidation reaction and the glucose oxidation reaction respectively use two different biological enzymes, the water phase has the glucose oxidation reaction under the action of the glucose oxidase, and the oil phase and the water phase generate H under the catalysis of the lipase2O2The epoxidation reaction of vegetable oil occurs as shown in fig. 1. The entire reaction system is therefore referred to as a two-enzyme coupled reaction system in the present invention.
The terms "two-enzyme coupled reaction system" and "two-enzyme coupled system" are used interchangeably herein.
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 described above, although hydrogen peroxide is an oxidation source for enzymatic vegetable oil epoxidation, although hydrogen peroxide is environmentally friendly, hydrogen peroxide has strong oxidation property and has a great influence on the activity of a biological enzyme, so that the recycling value of a biological catalyst is greatly reduced, and the catalytic effect is also influenced, at present, research is conducted on using other oxidation sources instead of high-concentration hydrogen peroxide, such as carbamide peroxide, and the hydrogen peroxide is correspondingly applied to epoxidation reaction because the hydrogen peroxide slowly releases in the reaction, but the epoxidation is limited to the epoxidation of compounds containing double bonds, such as terpenes, and the like, and no report is made on the oxidation source for vegetable oil epoxidation.
The glucose is oxidized to generate gluconic acid, which is widely applied in many fields, and in the research of the reaction formula, catalase is often added to remove the generated byproduct hydrogen peroxide, so that the production cost is increased.
In view of this, the present inventors have studied a process for preparing epoxidized vegetable oil using a two-enzyme coupling system.
Glucose Oxidase (GOX) catalyzes the oxidation of beta-D-glucose to produce D-gluconolactone and hydrogen peroxide. The generated hydrogen peroxide is partially decomposed, and part of the hydrogen peroxide and D-gluconolactone spontaneously react to generate gluconic acid (figure 4), and meanwhile, the application route of the gluconic acid is more. However, when hydrogen peroxide accumulates and inactivates the enzyme, the enzymatic activity of GOX decreases and hydrogen peroxide can lead to product inhibition of the enzyme. Therefore, catalase is often used industrially to remove hydrogen peroxide produced as a by-product in the oxidation reaction of glucose.
In the epoxidation reaction of vegetable oil catalyzed by a biological enzyme method, hydrogen peroxide is pollution-free and environment-friendly, and is often used as an oxygen donor to participate in the epoxidation reaction, but for a biological catalyst, the strong oxidizing property of the hydrogen peroxide has certain damage to the activity of the biological catalyst, and the hydrogen peroxide is easy to photolyze, so that the reaction utilization rate is low.
The inventor researches and discovers that hydrogen peroxide generated as a byproduct of glucose under the catalysis condition of glucose oxidase is used as an oxidation source for the oil epoxidation of the plant oil by the biological enzyme method, the problem of the hydrogen peroxide generated as the byproduct of the glucose oxidation reaction and the problem of the damage of the hydrogen peroxide concentration in the oil epoxidation of the plant oil by the enzyme method to the activity of the biocatalyst are solved, the hydrogen peroxide generated by the reaction can be timely applied by the oil epoxidation reaction of the plant oil, and the damage of the hydrogen peroxide to the activity of the biocatalyst is reduced.
The inventor further researches and finds that the epoxy vegetable oil prepared by coupling the glucose oxidase and the biological lipase is used as a brand new research direction of the bio-based plasticizer, and the research significance is greater. The present invention has been completed based on the above findings.
Therefore, the method for preparing the epoxidized vegetable oil by using the double-enzyme coupling reaction system comprises the following steps:
step B, preparing a phosphate buffer, and adding glucose into the phosphate buffer for dissolving to prepare a glucose solution;
step C, mixing and heating the glucose solution, the vegetable oil, the free fatty acid and the organic solvent, and adding the glucose oxidase and the biological lipase at the same time when the temperature is stabilized at the reaction temperature to form a double-enzyme coupling reaction system, so that the double-enzyme coupling reaction system performs an epoxidation reaction to obtain an epoxidation reaction crude product;
step D, carrying out centrifugal treatment on the crude epoxidation reaction product, carrying out rotary evaporation treatment on the upper oil phase, removing the organic solvent to obtain a finished product of the epoxidized vegetable oil, and carrying out recovery treatment on the biological lipase (intermediate phase); the lower aqueous phase was similarly centrifuged, and the aqueous phase was treated to recover gluconic acid and glucose oxidase.
According to the invention, in step C, the vegetable oil is subjected to an epoxidation reaction in the presence of an oxidizing source. As described above, the inventor researches and discovers that hydrogen peroxide, which is a byproduct generated by the catalytic action of glucose oxidase, can be used as an oxidation source to participate in the epoxidation reaction of vegetable oil, and the hydrogen peroxide solution used as the oxidation source in the enzymatic vegetable oil epoxidation has the advantages that the damage to the activity of a biocatalyst is minimized, and the hydrogen peroxide is better utilized. A double-enzyme coupling mode is commonly applied to industrial preparation of gluconic acid, the hydrogen peroxide generated as a byproduct in the reaction is usually removed by coupling glucose oxidase and catalase, and the hydrogen peroxide is accumulated in the process of glucose oxidation reaction, so that not only is the catalytic activity of the oxidase damaged, but also the reaction process is damaged; the technology for preparing the epoxy vegetable oil by the biological enzyme method is nearly mature, but has some problems that an oxidation source in the reaction is a hydrogen peroxide solution, the hydrogen peroxide has certain damage to the activity of a biological catalyst in the reaction process and is easy to carry out photolysis, and certain safety problems are also faced during operation.
Based on the above description, the present inventors propose a new research direction for preparing epoxidized vegetable oil by using a two-enzyme coupling reaction system.
The biological lipase applied in the oil epoxidation of the plant oil is commercial biological lipase, and the main source of the biological lipase is one or more of candida antarctica, candida rugosa, yarrowia lipolytica, candida citrinopileata, candida tropicalis, candida parapsilosis and candida olivifolia.
In some embodiments of the invention, the amount of glucose oxidase added is 5% -10% of the amount of glucose added, and the amount of bio-lipase added is 5% -10% of the amount of vegetable oil added; preferably, the addition ratio of the glucose oxidase to the biological lipase is (1-2) to (1-2).
The glucose used in the present invention is beta-D glucose, and the glucose on the market is not particularly specified to be beta-D glucose.
According to the present invention, the amount of glucose added is calculated by the molar ratio of the double bonds of the vegetable oil to hydrogen peroxide in the enzymatic vegetable oil catalytic epoxidation reaction, which is usually (0.5-3): 1.
The free fatty acid plays a role in mediating the vegetable oil epoxidation reaction in the epoxidation reaction of the step C, and the addition amount of the free fatty acid is 1-10%, preferably 5-10%, and more preferably 5% of 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 present invention, in step B, glucose is added to phosphate buffer solution to be dissolved, and a glucose solution is prepared, preferably, the concentration of the glucose solution is 20% to 40% (g/mL), preferably 20% to 37% (g/mL), and more preferably 37% (g/mL).
In some embodiments of the invention, the phosphate buffer dissolves glucose at a concentration of 20% to 40%.
In some embodiments, the phosphate buffer is fixed at a pH in the range of 6.5 to 7.0 and at a concentration in the range of 50 to 150mmol/L, preferably 50 to 100 mmol/L.
According to the invention, in step C, the epoxidation reaction is carried out at a temperature of from 30 to 70 ℃, preferably from 40 to 60 ℃.
In some embodiments of the present invention, in step C, the time for the epoxidation reaction is 8 to 20 hours, preferably 8 to 12 hours, and more preferably 10 hours.
In some embodiments of the invention, in step C, the stirring speed is above 150rpm, preferably 200-400 rpm.
In the step C, a glucose solution formed by dissolving glucose in a phosphate buffer solution forms a water phase, vegetable oil, free fatty acid and an organic solvent form an oil phase, and the water phase, the oil phase, glucose oxidase and biological lipase form a double-enzyme coupling reaction system, wherein in the reaction system, the glucose oxidase plays a catalytic role in the water phase, and the biological lipase plays a catalytic role in the oil phase; preferably, the adding amount ratio of the water phase phosphate buffer solution to the oil phase in the reaction system is (1-3): (3-1), preferably 1: 1.
In the present invention, the sources of the reaction raw materials, such as vegetable oil, biocatalyst, glucose oxidase, and phosphate buffer, are not particularly limited, and may be those commercially available or others.
The organic solvent in the present invention includes, but is not limited to, toluene.
In step D of the invention, after the reaction is finished, the reactant is centrifuged and divided into three layers, namely an organic phase, an enzyme phase and a water phase from top to bottom, so that the whole process is very obvious in layering and easy to sample.
In some preferred embodiments of the present invention, in step D, the reaction crude product after the reaction is finished is centrifuged, the upper layer is an organic phase, and the organic solvent is removed by rotary evaporation or the like; the middle layer biological catalyst layer can be recycled for more than three times; and recovering gluconic acid from the lower aqueous phase.
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.
In some embodiments of the present invention, the preparation of epoxidized vegetable oil with Novozym435 as the biocatalyst and glucose oxidase as the dual catalyst comprises the following steps:
(1) preparing phosphate buffer solution with corresponding concentration and pH (pH is 6.5-7.0, concentration is 50-150mmol/L, preferably 50-100mmol/L), wherein the phosphate buffer solution with certain concentration and pH value has certain stability for an epoxidation reaction system and certain benefit for the stability of the activity of the biological enzyme, and the phosphate buffer solution is used for dissolving a certain amount of glucose to obtain glucose solution;
(2) after the reaction raw materials and the phosphate buffer solution are uniformly mixed and the system is stable (the stable system means that the temperature reaches the set temperature of the epoxidation reaction, namely the epoxidation reaction temperature), the reaction is carried out for 8-20h, and after the reaction is finished, an epoxidation crude product and a gluconic acid product are obtained;
(3) and (3) when the reaction is finished, centrifuging the product after the reaction is finished, performing rotary evaporation treatment on the upper oil phase to remove the organic solvent, recovering the gluconic acid in the lower water phase, and recycling the two enzymes after the recovery treatment.
The concentration and the pH value (pH value) of the phosphate buffer solution in the step (1) are used for dissolving solid glucose on one hand and are used for the activity condition of the double-enzyme catalyst on the other hand, and the epoxidation reaction environment is suitable for two enzymes to carry out respective reactions by adjusting the concentration and the pH value of the buffer solution: oxidation reaction of glucose and epoxidation reaction of vegetable oil.
The glucose concentration in the step (1) is maintained at 20-40%.
The system temperature in the step (2) is simultaneously adapted to the activity conditions of two enzymes, which is beneficial to the oxidation reaction of glucose catalyzed by glucose oxidase and the epoxidation reaction of vegetable oil catalyzed by biological enzyme, the temperature range is 40-60 ℃, the rotating speed is 200-400rpm, and the reaction time is 8-12 h.
In the reaction system, for the glucose oxidation reaction: the byproduct hydrogen peroxide produced by the reaction is removed by using a biocatalyst lipase, and for the epoxidation reaction of vegetable oil: the oxidation source is hydrogen peroxide generated by the oxidation reaction of glucose. The addition amount of glucose is calculated by the theory of hydrogen peroxide needed by the oil oxidation of the plant oil.
The two enzymes are added at one time in a ratio of (1-2) to (1-2).
The addition amount of the free fatty acid is 5-10% of the addition amount of the vegetable oil.
III, the related parameter detection and calculation method in the invention is as follows:
(1) method for measuring double bond content in vegetable oil
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 is the oleic acid content; b is the linoleic acid content; c is the linolenic acid content; 92 is glycerol molecular weight;
the molar amount of the double bonds is calculated and multiplied by 0.016X 100, the molar equivalent of oxygen, to give the double bond content.
(2) Determining the epoxidation value by referring to the epoxy value of GB1677-81 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 by the test sample; the unit is mL;
v2 is the amount of the sodium hydroxide standard solution used for determining the acid value in mL;
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.
(3) Conversion of double bond
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.
(4) Detection method of gluconic acid
Centrifuging a product obtained by the reaction of the double-enzyme coupling reaction system, taking a lower-layer solution for dilution, using a Saimerfin high performance liquid chromatograph, using an ultraviolet detector, using an organic acid column, wherein a mobile phase is concentrated sulfuric acid with a certain concentration, a high performance liquid chromatogram of a gluconic acid standard substance is shown in figure 5, and gluconic acid solutions with different concentrations obtain different peak areas, and drawing a gluconic acid concentration standard curve (see figure 6) according to the principle, the high performance liquid chromatogram of the product is shown in figure 7, and as can be seen from figure 7, the peak position of the product spectrogram is almost the same as that of the gluconic acid standard substance spectrogram (figure 5), so that the generation of gluconic acid in the reaction can be verified; meanwhile, the content of gluconic acid in the reaction system can be calculated based on the standard curve of the concentration of the gluconic acid (see fig. 7), and the concentration unit of the gluconic acid is g/L, so that the conversion rate of glucose and the generation amount (g/L) of hydrogen peroxide are calculated.
Glucose oxidation equation: c6H12O6+O2+H2O→C6H12O7+H2O2
Wherein the glucose has a relative molecular weight of 180.16
Gluconic acid has a relative molecular weight of 196.16
Hydrogen peroxide has a relative molecular weight of 34.01
Remarking: as shown in the reaction formula, the molar ratio of the gluconic acid to the glucose and the hydrogen peroxide is 1:1:1,
then:
the glucose reaction amount and the hydrogen peroxide generation amount can be calculated by measuring the gluconic acid content.
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:
preparing phosphate buffer solution with pH of 6.5 and ionic strength of 100mmol/L, dissolving 14.8165g of monohydrate glucose in 40mL of phosphate buffer solution, adding 10mL of linseed oil and 30mL of toluene into a reaction system (oil-water ratio) when the temperature of the system is stabilized at 40 ℃, wherein the addition amount of glucose oxidase is 10% of the addition amount of glucose, the addition amount of Novozym435 is 10% of the addition amount of linseed oil, the addition amount of free fatty acid is 5% of raw oil, the reaction temperature is 40 ℃, the rotating speed is 300rpm, reacting for 12h, centrifuging after the reaction is finished, and dividing the reaction into two phases and three layers: water phase, catalyst and oil phase. Taking out the upper layer after centrifugation, and removing the organic solvent to obtain the epoxy linseed oil. The epoxy value is determined to be 7.87, the double bond conversion rate is 72.36%, and the content of the gluconic acid is determined to be 16.88 g/L.
Example 2:
preparing phosphate buffer solution with pH of 7.0 and ionic strength of 100mmol/L, dissolving 14.8165g of monohydrate glucose in 40mL of phosphate buffer solution, adding 10mL of linseed oil and 30mL of toluene into a reaction system (oil-water ratio) when the temperature of the system is stabilized at 40 ℃, wherein the addition amount of glucose oxidase is 10% of the addition amount of glucose, the addition amount of Novozym435 is 10% of the addition amount of linseed oil, the addition amount of free fatty acid is 5% of raw oil, the reaction temperature is 40 ℃, the rotating speed is 300rpm, reacting for 12h, centrifuging after the reaction is finished, and dividing the reaction into two phases and three layers: water phase, catalyst and oil phase. Taking out the upper layer after centrifugation, and removing the organic solvent to obtain the epoxy linseed oil. The epoxy value is determined to be 7.5, the double bond conversion rate is 68.9 percent, and the content of the gluconic acid is determined to be 13.09 g/L.
Example 3:
preparing phosphate buffer solution with pH of 6.5 and ionic strength of 100mmol/L, dissolving 14.8165g of monohydrate glucose in 40mL of phosphate buffer solution, adding 10mL of linseed oil and 30mL of toluene into a reaction system (oil-water ratio) when the temperature of the system is stabilized at 50 ℃, wherein the addition amount of glucose oxidase is 10% of the addition amount of glucose, the addition amount of Novozym435 is 10% of the addition amount of linseed oil, the addition amount of free fatty acid is 5% of raw oil, the reaction temperature is 40 ℃, the rotating speed is 300rpm, reacting for 12h, centrifuging after the reaction is finished, and dividing the reaction into two phases and three layers: water phase, catalyst and oil phase. Taking out the upper layer after centrifugation, and removing the organic solvent to obtain the epoxy linseed oil. The epoxy value of the product is 7.8, the conversion rate of double bonds is 71.72%, and the content of gluconic acid is 13.10 g/L.
Example 4:
preparing phosphate buffer solution with pH of 6.5 and ionic strength of 50mmol/L, dissolving 14.8165g of monohydrate glucose in 40mL of phosphate buffer solution, adding 10mL of linseed oil and 30mL of toluene into a reaction system (oil-water ratio) when the temperature of the system is stabilized at 40 ℃, wherein the addition amount of glucose oxidase is 10% of the addition amount of glucose, the addition amount of Novozym435 is 10% of the addition amount of linseed oil, the addition amount of free fatty acid is 5% of raw oil, the reaction temperature is 40 ℃, the rotating speed is 300rpm, reacting for 12h, centrifuging after the reaction is finished, and dividing the reaction into two phases and three layers: water phase, catalyst and oil phase. Taking out the upper layer after centrifugation, and removing the organic solvent to obtain the epoxy linseed oil. The epoxy value of the product is determined to be 6.7, the double bond conversion rate is 61.63%, and the content of the gluconic acid is determined to be 13.09 g/L.
The experimental result shows that the glucose oxidase serving as the catalyst and the Novozym435 in the invention can be recycled, the key point of the invention is that the byproduct hydrogen peroxide generated in the oxidation reaction of glucose is utilized in the oil epoxidation reaction of plants, the two reactions of oil and water phases are coordinated by using the reaction conditions, the reaction system is uniformly mixed under the stirring condition, so that the hydrogen peroxide directly participates in the reaction as the oxidation source of the oil epoxidation reaction of plants, and the reaction result shows that the oxidation effect is uniform. Fully illustrates the feasibility of preparing the epoxy vegetable oil by the double-enzyme coupling reaction system.
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 (10)
1. A method for preparing epoxidized vegetable oil by a two-enzyme coupling reaction system comprises the following steps:
step B, dissolving glucose in a phosphate buffer solution to prepare a glucose solution;
step C, mixing and heating the glucose solution, the vegetable oil, the free fatty acid and the organic solvent, and adding the glucose oxidase and the biological lipase at the same time when the temperature is stabilized at the reaction temperature to form a double-enzyme coupling reaction system, so that the double-enzyme coupling reaction system performs an epoxidation reaction to obtain an epoxidation reaction crude product;
and D, carrying out centrifugal treatment on the crude epoxidation product, taking an oil phase for rotary evaporation, and removing the organic solvent to obtain a finished epoxy vegetable oil product.
2. The method according to claim 1, wherein in step C, the vegetable oil is subjected to epoxidation reaction in the presence of an oxidizing source, the oxidizing source being hydrogen peroxide generated by oxidation of glucose catalyzed by glucose oxidase; preferably, the glucose dosage is calculated by hydrogen peroxide required by the theory of the vegetable oil epoxidation reaction; further preferably, the molar ratio of hydrogen peroxide generated by oxidation of glucose to double bonds contained in the vegetable oil is (0.5-3): 1.
3. the method according to claim 2, wherein the amount of glucose oxidase added is 5% -10% of the amount of glucose added; and/or the addition amount of the biological lipase is 5% -10% of the addition amount of the vegetable oil; preferably, the addition ratio of the glucose oxidase to the biological lipase is (1-2) to (1-2).
4. The method of claim 3, wherein the major sources of biological lipase include one or more of Candida antarctica, Candida rugosa, yarrowia lipolytica, Candida citrifolia, Candida tropicalis, Candida parapsilosis, and Candida olivaceus.
5. The method of claim 2, wherein the volume ratio of vegetable oil to organic solvent is 1: 3.
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 vegetable oil, and preferably 5-10%; further 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 to 6, wherein in step B, the phosphate buffer has a pH value of 6.5 to 7.0 and a concentration of 50 to 150mmol/L, preferably 50 to 100 mmol/L; and/or the concentration of the glucose solution is 20-40% (g/mL), preferably 20-37% (g/mL).
8. The method according to any one of claims 1-7, wherein: in step C, the temperature of the epoxidation reaction is 30-50 ℃; and/or the time of epoxidation reaction is 10-20 h; and/or the stirring speed is above 150 rpm.
9. The method according to any one of claims 1-8, wherein: and D, centrifuging the crude epoxy vegetable oil product, taking oil phase substances for rotary evaporation, removing the organic solvent to obtain epoxy vegetable oil, and recycling the lower-layer catalyst.
10. The method of any one of claims 1-9, wherein the vegetable oil comprises one or more of linseed oil, soybean oil, perilla oil, sunflower oil, peanut oil and rapeseed oil.
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