CN112888782A - Liquid lipase immobilization method and sucrose-6-acetate preparation method - Google Patents
Liquid lipase immobilization method and sucrose-6-acetate preparation method Download PDFInfo
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- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
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- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
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Abstract
The application provides a method for immobilizing liquid lipase, which comprises the following steps of: a dissolving step: adding alginate into deionized water for complete dissolution, and adding liquid lipase at a preset temperature to obtain a mixed solution; a fixing step: mixing the mixed solution with a metal cross-linking agent solution, and keeping for a preset time to obtain spherical gel so as to fix the liquid lipase; and, a chelating step: and washing the spherical gel, adding a buffer solution and an organic complexing agent solution to carry out chelation reaction, and washing by adopting the buffer solution to obtain the immobilized liquid lipase. The gel obtained by the method has extremely high physical strength, can exist in a reaction system more stably, and basically cannot be broken and decomposed; can be repeatedly used for many times, and is more suitable for producing the sucrose-6-acetate by industrial large-scale continuous esterification; the manufacturing method is simple and the cost is low.
Description
Technical Field
The invention belongs to the technical field of fine chemical engineering, and particularly relates to an immobilization method of liquid lipase and a preparation method of sucrose-6-acetate.
Background
Sucralose is a fresh sweetener, which is a novel sweetener developed by the company of England and university of London and applied for patent in 1976, and is the only functional sweetener using sucrose as raw material. The sucralose has the effect of desalting sour and salty tastes, has the effect of covering up unpleasant tastes such as astringent taste, bitter taste, wine taste and the like, has the synergistic effect on spicy taste and milk taste, and has a very wide application range. Therefore, the product is the most ideal intense sweetener at present, can be eaten by children, teenagers, adolescents, middle-aged people, old people and patients with various diseases, and has no any nutritional doubt.
Sucrose-6-acetate is an important intermediate for synthesizing sucralose, the method for synthesizing sucrose-6-acetate is mainly a traditional chemical method, such as acetic anhydride esterification, organic metal synthesis, orthoester method, etc., the chemical synthesis reaction conditions of sucrose ester are generally harsh, the synthesized product has dark color, and by-products are easily generated due to poor selectivity.
In the prior art, there are reports about the enzymatic synthesis of sucrose-6-ethyl ester, such as chinese patent CN102618601A, but the reuse rate of the currently available liquid enzyme catalyst for preparing solidified sucrose-6-ethyl ester is very poor, and after the liquid enzyme catalyst is reused for about three times, most of the solidified liquid enzyme catalyst is broken, which results in substantial inactivation of the liquid enzyme, serious loss of the liquid enzyme, and high synthesis cost of sucrose-6-ethyl ester.
Disclosure of Invention
In view of the above problems, the present application has been made to provide a method for immobilizing a liquid lipase and a method for producing sucrose-6-acetate that overcome the above problems or at least partially solve the above problems.
According to an aspect of the present application, there is provided a method for immobilizing liquid lipase, comprising the steps of:
a dissolving step: adding alginate into deionized water for complete dissolution, and adding liquid lipase at a preset temperature to obtain a mixed solution;
a fixing step: mixing the mixed solution with a metal cross-linking agent solution, and keeping for a preset time to obtain spherical gel so as to fix the liquid lipase; and the number of the first and second groups,
a chelation step: and washing the spherical gel, adding a buffer solution and an organic complexing agent solution to carry out chelation reaction, and washing by adopting the buffer solution to obtain the immobilized liquid lipase.
According to another aspect of the present application, there is provided a method for preparing sucrose-6-acetate, comprising: and (3) carrying out dehydration esterification reaction on sucrose and fatty acid vinyl ester in a mixed organic solution under the catalytic action of the immobilized liquid lipase prepared by the method to obtain a sucrose-6-acetate solution.
To sum up, the beneficial effect of this application lies in:
1. the alginate is used as a carrier, and is subjected to secondary crosslinking with a metal crosslinking agent and an organic complexing agent to fix the liquid lipase, so that the obtained gel has extremely high physical strength, can exist in a reaction system more stably, and is basically not broken and decomposed;
2. because the gel strength is high, the solidified enzyme can be repeatedly used for many times, which is much higher than the repeated utilization rate of the solidified enzyme reported in the prior art, and the industrial large-scale continuous esterification is utilized to produce the sucrose-6-ethyl ester;
3. the preparation method is simple, the raw materials needed are wide in source, the cost of liquid lipase immobilization is greatly reduced, and further, the preparation cost of sucrose-6-ethyl ester is reduced.
The foregoing description is only an overview of the technical solutions of the present application, and the present application can be implemented according to the content of the description in order to make the technical means of the present application more clearly understood, and the following detailed description of the present application is given in order to make the above and other objects, features, and advantages of the present application more clearly understandable.
Drawings
Various other advantages and benefits will become apparent to those of ordinary skill in the art upon reading the following detailed description of the preferred embodiments. The drawings are only for purposes of illustrating the preferred embodiments and are not to be construed as limiting the application. Also, like reference numerals are used to refer to like parts throughout the drawings. In the drawings:
FIG. 1 shows a schematic flow diagram of a method for immobilizing liquid lipase according to one embodiment of the present application;
fig. 2 shows a schematic structure of a gel formed by sodium alginate and calcium lactate according to one embodiment of the present application.
Detailed Description
Exemplary embodiments of the present application will be described in more detail below with reference to the accompanying drawings. While exemplary embodiments of the present application are shown in the drawings, it should be understood that the present application may be embodied in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art.
The idea of the application is that aiming at the current situation that the immobilized liquid lipase (hereinafter called immobilized enzyme) in the prior art is low in strength and poor in reusability, alginate is used as a carrier, and secondary crosslinking is carried out on the alginate, a metal crosslinking agent and an organic complexing agent to prepare the gel containing the liquid lipase, and the gel is extremely high in strength and extremely high in reusability.
In this application, the raw materials and apparatus used are commercially available products.
Fig. 1 shows a method for immobilizing liquid lipase according to one embodiment of the present application, comprising the following steps in sequence:
a dissolving step S110: adding alginate into deionized water to be completely dissolved, and adding liquid lipase at a preset temperature to obtain a mixed solution.
Alginate is used as a carrier, and in the application, the alginate used as the carrier is soluble salt, such as sodium alginate, and insoluble salt such as calcium alginate is not included.
The following sodium alginate is exemplified by sodium alginate (C)6H7O6Na) n is mainly composed of sodium salt of alginic acid, a copolymer of β -D-mannuronic acid (M units) and α -L-guluronic acid (G units) linked by β -1, 4-glycosidic bonds and composed of different ratios of GM, MM and GG fragments. The molecular formula is formula (1), and the carboxyl group can be used as a crosslinking point to crosslink with a metal crosslinking agent and an organic complexing agent to form an irreversible gel.
Formula (1)
The alginate is completely dissolved in the deionized water, and a high-speed stirring device is adopted for stirring and heating to promote the dissolution of the alginate. After the alginate is completely dissolved, dissolving liquid lipase in the obtained alginate solution at a preset temperature, wherein the liquid lipase can keep activity at a certain temperature, and if the temperature is too high or too low, the inactivation of the lipase can be caused, so that the preset temperature can be determined according to the type of the selected liquid lipase, and the preset temperature is not the temperature for inactivating the liquid lipase.
Fixing step S120: and mixing the mixed solution with a metal cross-linking agent solution, and keeping for a preset time to obtain spherical gel so as to fix the liquid lipase.
The metal cross-linking agent is a soluble metal salt, which can be used only as a solution, in this application, it is preferable to use a compound containing divalent or trivalent metal ions as the metal cross-linking agent, taking calcium lactate and sodium alginate as an example, after adding a sodium alginate solution to a calcium lactate solution, sodium alginate will undergo ion exchange with calcium ions in calcium lactate to form calcium alginate, and calcium ions in calcium alginate will form ionic bonds with carboxylate groups on different polymer chains to link different polymer chains together, i.e. a cross-linking reaction will occur, similar to adding more branched chains on a straight chain, so that different polymer chains form a network structure, the structure of which is shown in fig. 2, so that the viscosity of the solution is further increased, and the solubility of calcium alginate is lower than that of sodium alginate, so as to form a gel. The hardness of the gel can be influenced by adding a proper amount of calcium ions into the sodium alginate solution, and generally, the more calcium ions are added, the firmer the colloid is. To obtain a spherical gel, the gel may be extruded using an extrusion machine, or using an injection with a needle.
And, a chelating step S130: and washing the spherical gel, adding a buffer solution and an organic complexing agent solution to carry out chelation reaction, and washing by adopting the buffer solution to obtain the immobilized liquid lipase.
Washing the obtained spherical gel to remove unreacted metal cross-linking agent, wherein the washing can adopt buffer solutions such as deionized water or normal saline, and a decompression suction filtration mode can be adopted to accelerate the washing process and promote the washing degree.
And (3) washing the spherical gel, adding a buffer solution and an organic complexing agent, and carrying out secondary crosslinking with the obtained spherical gel, namely carrying out chelation reaction. The buffer solution is used for providing a reaction environment for the spherical gel and the organic complexing agent, so that the use amount of the buffer solution is not limited, and the spherical gel can be submerged.
In the fixing step, all crosslinking groups, i.e. carboxyl groups, available for crosslinking of the alginate are not crosslinked with the metal crosslinking agent, and the remaining uncrosslinked carboxyl groups can be secondarily crosslinked with the organic complexing agent. Through the two crosslinking processes, the crosslinking degree of the alginate is further deepened, and the formed gel has strong strength and is not easy to damage.
In summary, the alginate is used as a carrier, and secondary crosslinking is carried out on the alginate, the metal crosslinking agent and the organic complexing agent so as to fix the liquid lipase, so that the obtained gel has extremely high physical strength, can exist in a reaction system more stably, and is basically not broken and decomposed; the method can be repeatedly used for many times, is much higher than the repeated utilization rate of the solid enzyme reported in the prior art, and more utilizes industrial large-scale continuous esterification to produce sucrose-6-acetate; the preparation method is simple, the raw materials needed are wide in source, the cost of liquid lipase immobilization is greatly reduced, and further, the preparation cost of sucrose-6-acetate is reduced.
Classes of liquid lipases
In some embodiments of the present application, in the above-mentioned method for immobilizing liquid lipase, the type of liquid lipase is not limited, and the lipase is a type of enzyme having multiple catalytic capabilities, and can be used for catalyzing hydrolysis, alcoholysis, esterification, transesterification, reverse synthesis reaction of esters, and the like, such as enzymatic synthesis and transesterification in an organic phase. In other embodiments of the present application, the liquid lipase can be any one of Lipozyme TL IM, Lipozyme TL100L, Lipozyme RM IM.
Kind of alginate
In some embodiments of the present application, in the above-mentioned method for immobilizing liquid lipase, the kind of alginate is not limited, and in other embodiments, the alginate is selected from sodium alginate.
The dosage of alginate, deionized water and liquid lipase
In some embodiments of the present application, in the method for immobilizing liquid lipase, the amounts of alginate, deionized water and liquid lipase are not limited, wherein the minimum amount of deionized water is determined to be able to completely dissolve alginate; in other embodiments, the total mass ratio of alginate to deionized water to liquid lipase is 1:5 to 1: 20; wherein the mass ratio of the deionized water to the liquid lipase is 0.1-10: 1, and in some embodiments, the mass ratio of the deionized water to the liquid lipase is 3: 1. If the mass consumption of the alginate is less than one twentieth of the total mass consumption of the deionized water and the liquid lipase, the consumption is too low, so that the formed gel cannot completely embed the liquid lipase; if the mass consumption of the alginate is more than one fifth of the total mass consumption of the deionized water and the liquid lipase, the consumption is excessive, the polymer in the formed gel is excessive, the liquid lipase is too little, and the subsequent utilization is not utilized.
Temperature of lipase addition
In some embodiments of the present application, in the method for immobilizing liquid lipase, the preset temperature in the dissolving step is not limited, and may be determined according to the type of the liquid lipase selected, so that the liquid lipase is not inactivated. In other embodiments, the predetermined temperature may be 10-37 deg.C, and in still other embodiments, the predetermined temperature may be 28 deg.C.
Class of Metal crosslinkers
In some embodiments of the present application, in the method for immobilizing liquid lipase, the type of the metal cross-linking agent is not limited, and the metal-soluble metal compound can be any metal-soluble metal compound that can form an irreversible gel with alginate; in other embodiments, the metal crosslinking agent is an inorganic salt of calcium, zinc, copper, magnesium, aluminum, iron, and the action mechanism of zinc, copper, magnesium, aluminum, and iron is the same as that of calcium, and is not repeated herein; in other embodiments, the metal crosslinker is an inorganic salt of calcium, and in still other embodiments, the metal crosslinker is calcium chloride or calcium lactate.
Amount of metal crosslinking agent
In some embodiments of the present application, in the above-mentioned liquid lipase immobilization method, the amount and concentration of the metal cross-linking agent are not limited, and in other embodiments, the ratio of the mass amount of the metal cross-linking agent to the mass amount of the alginate is 1: 1-1: 20, in still other embodiments, the mass ratio of the metal cross-linking agent to the alginate is 1:5, and the concentration of the metal cross-linking agent solution is 1 wt% to 5 wt%. If the mass consumption of the metal cross-linking agent is less than one twentieth of the mass consumption of the alginate, the consumption is too low, the strength of the formed gel is too low, and even the gel cannot be effectively formed; if the mass consumption of the metal cross-linking agent is more than one time of the mass consumption of the alginate, the consumption is too much, the cross-linking process is too deep, and the secondary cross-linking is not facilitated. If the concentration of the metal cross-linking agent solution is less than 1 wt%, after the metal cross-linking agent solution is mixed with the sodium alginate solution, the volume of the formed mixed solution is too large, so that metal ions are not easy to form gel with the sodium alginate; if the concentration of the metal cross-linking agent solution is more than 5 wt%, the concentration of metal ions is too high, so that the sodium alginate is excessively cross-linked locally, and the strength of the formed gel is not uniform.
Immobilization time and temperature
In some embodiments of the present application, in the method for immobilizing liquid lipase, the preset time in the immobilizing step is not limited, and in other embodiments, the preset time is 0.5h to 3 h. If the preset time is less than 0.5h, the crosslinking time of the sodium alginate and the metal crosslinking agent is too short, so that the aim of full crosslinking cannot be fulfilled; if the preset time is longer than 3h, the crosslinking time of the sodium alginate and the metal crosslinking agent is too long, the crosslinking can be completed within 3h under the condition that the use amounts of the sodium alginate and the metal crosslinking agent are determined, the crosslinking degree cannot be deepened after the time is prolonged, and other beneficial effects cannot be brought. The fixing temperature in the fixing step may be room temperature without special heating or cooling.
Buffer type
In some embodiments of the present application, in the liquid lipase immobilization method, the buffer solution serves to provide a reaction environment for immobilization, the buffer solution is not limited to a specific type, and in other embodiments, the buffer solution is any one of normal saline, phosphoric acid, citric acid, carbonic acid, acetic acid, barbituric acid, and tris, preferably normal saline.
Kind of organic complexing agent
In some embodiments of the present application, in the above method for immobilizing liquid lipase, the type of the organic complexing agent is not limited, and may be a substance that forms a chemical bond with a carboxyl group, and in other embodiments, the organic complexing agent is any one of formaldehyde, C2-C8 linear or branched dialdehyde, preferably glutaraldehyde.
Amount and solubility of organic complexing agent
In some embodiments of the present application, in the above method for immobilizing liquid lipase, the mass ratio of the alginate to the organic complexing agent is 1: 1-1: 20; in other embodiments, the mass ratio of the alginate to the organic complexing agent is 1:3 to 1: 5; the mass solubility of the organic complexing agent solution is 0.1 wt% -2 wt%. If the mass consumption of the organic complexing agent is less than one twentieth of the mass consumption of the alginate, the dosage is too small, so that the formed gel strength is too low; if the mass consumption of the organic complexing agent is more than one time of the mass consumption of the alginate, the consumption is too much, and the workload of the subsequent washing procedure is large. If the concentration of the organic complexing agent solution is less than 0.1 wt%, after the organic complexing agent solution is mixed with the sodium alginate solution, the formed mixed solution has large volume, so that the organic complexing agent is not easy to form gel with the sodium alginate; if the concentration of the organic complexing agent solution is more than 2 wt%, the concentration of the organic complexing agent is too high, which causes the problems of excessive local crosslinking of sodium alginate, uneven strength of formed gel and the like.
Chelating reaction conditions
In some embodiments of the present application, in the above-mentioned method for immobilizing liquid lipase, the temperature and time of the chelation reaction in the chelation step are not limited, and in other embodiments, the chelation reaction is performed at a temperature of 1 ℃ to 10 ℃, preferably 2 ℃ to 4 ℃; the time is 8-24 h. If the temperature is lower than 1 ℃, the temperature is too low, and the chelation reaction is difficult to carry out; if the temperature is higher than 10 ℃, the temperature is too high, which results in too fast chelation reaction and too deep crosslinking.
In still other embodiments of the present application, there is provided a method of preparing sucrose-6-acetate, the method comprising: and (3) carrying out dehydration esterification reaction on sucrose and fatty acid vinyl ester in a mixed organic solution under the action of the immobilized liquid lipase prepared by any one of the methods as a catalyst to obtain a sucrose-6-acetate solution. The prepared solidified enzyme is used for catalyzing sucrose and fatty acid vinyl ester to carry out dehydration esterification reaction in a mixed organic solvent to prepare sucrose-6-acetate, and the solidified enzyme has extremely high gel strength and can be recycled for multiple times; and the yield of the sucrose-6-acetate is high, the sucrose-6-acetate is generated by catalyzing esterification reaction with solidified enzyme for the first time, and the yield can reach more than 70%, and in some embodiments reaches 74.2-78.5%.
In some embodiments of the present application, the above method for preparing sucrose-6-acetate further comprises: the immobilized liquid lipase is recovered and used for dehydration esterification reaction of sucrose and fatty acid vinyl ester, and after the immobilized liquid lipase is repeatedly used for 7 times, the yield can still reach 42.4-45.8%.
Since immobilized enzyme has extremely high gel strength and is hardly broken in one reaction, it can be repeatedly recovered and used, at least 7 times or more, and since immobilized enzyme gel is not broken, liquid lipase can still maintain high enzyme activity.
Kind of vinyl ester of fatty acid
In some embodiments of the present application, in the above-described method for preparing sucrose-6-acetate, the kind of the fatty acid vinyl ester is not limited, and in other embodiments, the fatty acid vinyl ester is any one of vinyl acetate, vinyl butyrate, vinyl caprylate, vinyl caprate, vinyl palmitate, vinyl stearate, or vinyl oleate.
Mixed organic solvent
In some embodiments of the present application, in the above method for preparing sucrose-6-acetate, the kind of the mixed organic solvent is not limited, and in other embodiments, the mixed organic solvent is composed of DMF and t-butanol and/or t-amyl alcohol in a volume ratio of 1:1 to 10.
Means or conditions of testing
High performance liquid chromatography:agilent Poroshell 120 EC-18 column, mobile phase acetonitrile: water (95; 5) at a flow rate of1.0ml/min, the sample injection volume is 20uL, the column temperature is 40 ℃, and the detection wavelength of the ultraviolet detector is set to be 300 nm.
Method for calculating the conversion of sucrose:
the concentration is calculated from the data detected by high performance liquid chromatography, specifically, the reaction ending concentration of sucrose is compared with the initial concentration of sucrose.
Example 1: preparation of immobilized enzyme 1
(1) 500kg of sodium alginate is added with 4.4m of 3 ionized water, steam heating is carried out at 60 ℃, and stirring is started until the sodium alginate is completely dissolved.
(2) After cooling to room temperature (below 30 ℃), 600kg of lipase liquid is added, and the mixture is stirred uniformly and kept stand until bubbles disappear completely.
(3) 0.9 wt% physiological saline solution 5m is prepared3，10m33% of CaCl2And (3) solution.
(4) Pumping the mixed solution in the step (2) at a constant speed until CaCl is formed2Fixing in the solution, wherein the fixing process is about 2 hours.
(5) After the fixation is finished, washing the spherical gel twice by using 0.9 wt% of normal saline, then adding the normal saline which submerges the liquid level of the spherical gel and 0.5 wt% of glutaraldehyde 20L, and introducing cold air to control the temperature to be crosslinked for 5 hours in an environment of 2-4 ℃.
(6) After the crosslinking, the immobilized enzyme is obtained by washing with normal saline and is stored for later use.
Example 2: preparation of sucrose-6-ethyl ester Using immobilized enzyme 1
a. Preparing a mixed organic solution, DMF: the volume ratio of the tertiary amyl alcohol is 1:4, and the total volume is 6m3Pumping 4.8m3The tertiary amyl alcohol is pumped into the reaction kettle for 1.2m3DMF, start stirring.
b. Adding 123kg (0.06mol/L) of sucrose into the reaction kettle, stirring to dissolve completely, pumping 310kg (0.6mol/L) of vinyl acetate into the reaction kettle to be 6m3In the reaction kettle.
c. Then, 480kg of the immobilized enzyme 1 prepared in example 1 was charged into a reaction vessel, and the reaction was carried out while maintaining the temperature at 30 ℃ and carrying out the reaction at a stirring speed of 200rpm for 9 hours.
d. After the reaction is finished, filtering and filtering the unbroken immobilized enzyme 1 in the reaction system by a filter press, observing by naked eyes that most of spherical gel is unbroken, and soaking and placing in normal saline at room temperature after primary cleaning by using the normal saline.
e. The reacted mother liquor was filtered through a 0.22um filter membrane into a small liquid phase bottle, and the conversion rate of sucrose was quantitatively analyzed by High Performance Liquid Chromatography (HPLC), and the results are shown in Table 1.
f. Repeating the steps a to d7 times by using the immobilized enzyme 1 recovered in the step d (certain loss is caused in each reaction process, the recovered immobilized enzyme 1 is repeatedly used each time without additional addition) as a catalyst, weighing the mass of the immobilized enzyme 1 after each recovery, and measuring the conversion rate of the sucrose by using high performance liquid chromatography, wherein the results are shown in Table 1.
TABLE 1 results of preparation of sucrose-6-ethyl ester using immobilized enzyme 1
Example 3: preparation of immobilized enzyme 2
(1) Adding sodium alginate 400kg into the mixture of 3.8m3Ionized water, steam heating at 55 deg.C, and stirring to dissolve completely.
(2) After cooling to room temperature (below 30 ℃), 500kg of lipase liquid is added, and the mixture is stirred uniformly and kept stand until bubbles disappear completely.
(3) 0.9 wt% physiological saline solution 6m is prepared3，12m33% of CaCl2And (3) solution.
(4) Pumping the mixed solution in the step (2) at a constant speed until CaCl is formed2Fixing in the solution for about 3 h.
(5) After the fixation is finished, washing the spherical gel twice by using 0.9 wt% of normal saline, then adding the normal saline which submerges the liquid level of the spherical gel and 0.5 wt% of glutaraldehyde 50L, and introducing cold air to control the temperature to be crosslinked for 8 hours in an environment of 2-4 ℃.
(6) After the crosslinking, the immobilized enzyme 2 is obtained by washing with normal saline and stored for later use.
Example 4: preparation of sucrose-6-ethyl ester with immobilized enzyme 2
a. Preparing a mixed organic solution, DMF: the volume ratio of the tertiary amyl alcohol is 1:4, and the total volume is 6m3Pumping 4.8m3The tertiary amyl alcohol is pumped into the reaction kettle for 1.2m3DMF, start stirring.
b. Adding 123kg (0.06mol/L) of sucrose into the reaction kettle, stirring to dissolve completely, pumping 310kg (0.6mol/L) of vinyl acetate into the reaction kettle to be 6m3In the reaction kettle.
c. Subsequently, 450kg of immobilized enzyme 2 prepared in example 2 was charged into a reaction vessel, and the reaction was carried out while maintaining the temperature at 30 ℃ and the stirring speed at 200rpm for 10 hours.
d. After the reaction is finished, filtering out the unbroken immobilized enzyme 2 in the reaction system by a filter press, washing the immobilized enzyme by normal saline for the first time, and soaking the immobilized enzyme in the normal saline at room temperature for standing.
e. The reacted mother liquor was filtered through a 0.22um filter into a liquid phase vial, and the conversion of sucrose was quantitatively analyzed by HPLC, and the results are shown in Table 2.
f. Repeating the steps a to d7 times by using the immobilized enzyme 2 recovered in the step d (certain loss is caused in each reaction process, the recovered immobilized enzyme 2 is repeatedly used each time, and no additional addition is required), weighing the mass of the immobilized enzyme 2 after each recovery, and measuring the conversion rate of the sucrose by using high performance liquid chromatography, wherein the results are shown in Table 2.
TABLE 2 results of preparation of sucrose-6-ethyl ester using immobilized enzyme 2
As can be seen from tables 1 and 2, the immobilized enzyme provided by the application can be repeatedly used for at least 7 times, and the loss of the immobilized enzyme is very small each time; the conversion rate of the sucrose is very high and can reach over 74 percent in the first reaction, and still reach over 40 percent in the 7 th reaction.
To sum up, the beneficial effect of this application lies in:
1. the alginate is used as a carrier, and is subjected to secondary crosslinking with a metal crosslinking agent and an organic complexing agent to fix the liquid lipase, so that the obtained gel has extremely high physical strength, can exist in a reaction system more stably, and is basically not broken and decomposed;
2. because the gel strength is high, the solidified enzyme can be repeatedly used for many times, which is much higher than the repeated utilization rate of the solidified enzyme reported in the prior art, and the industrial large-scale continuous esterification is utilized to produce the sucrose-6-ethyl ester;
3. the preparation method is simple, the raw materials needed are wide in source, the cost of liquid lipase immobilization is greatly reduced, and further, the preparation cost of sucrose-6-ethyl ester is reduced.
The foregoing is merely a preferred embodiment of the invention and is not intended to limit the invention in any manner; those skilled in the art can make numerous possible variations and modifications to the present teachings, or modify equivalent embodiments to equivalent variations, without departing from the scope of the present teachings, using the methods and techniques disclosed above. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments according to the technical essence of the present invention are still within the scope of the protection of the technical solution of the present invention.
Claims (10)
1. A method for immobilizing liquid lipase, characterized by sequentially carrying out the following steps:
a dissolving step: adding alginate into deionized water for complete dissolution, and adding liquid lipase at a preset temperature to obtain a mixed solution;
a fixing step: mixing the mixed solution with a metal cross-linking agent solution, and keeping for a preset time to obtain spherical gel so as to fix the liquid lipase; and the number of the first and second groups,
a chelation step: and washing the spherical gel, adding a buffer solution and an organic complexing agent solution to carry out chelation reaction, and washing by adopting the buffer solution to obtain the immobilized liquid lipase.
2. The method of immobilizing a liquid lipase according to claim 1, wherein in the dissolving step, the liquid lipase is any one of Lipozyme TL IM, Lipozyme TL100L, and Lipozyme RM IM;
the alginate is sodium alginate;
the total mass and dosage ratio of the alginate to the deionized water to the liquid lipase is 1: 5-1: 20; wherein the mass usage ratio of the deionized water to the liquid lipase is 0.1-10: 1, preferably 3: 1;
the preset temperature is 10-37 ℃.
3. The method for immobilizing a liquid lipase according to claim 1, wherein the metal crosslinking agent is an inorganic salt of calcium, an inorganic salt of zinc, an inorganic salt of copper, an inorganic salt of magnesium, an inorganic salt of aluminum, an inorganic salt of iron, preferably an inorganic salt of calcium, more preferably calcium chloride;
the mass ratio of the metal cross-linking agent to the alginate is 1: 1-1: 20, preferably 1: 5;
the concentration of the metal cross-linking agent solution is 1 wt% -5 wt%.
4. The method of claim 1, wherein the predetermined time is 0.5 to 3 hours in the immobilizing step.
5. The method for immobilizing a liquid lipase according to claim 1, wherein the buffer is any one of physiological saline, phosphoric acid, citric acid, carbonic acid, acetic acid, barbituric acid, and tris, preferably physiological saline;
the organic complexing agent is any one of formaldehyde and C2-C8 straight chain or branched chain dialdehyde, and preferably is pentanediol.
6. The method for immobilizing liquid lipase according to claim 5, wherein the mass-to-use ratio of the alginate to the organic complexing agent is 1: 1-1: 20, preferably 1: 3-1: 5; the mass solubility of the organic complexing agent solution is 0.1 wt% -2 wt%.
7. The method for immobilizing a liquid lipase according to claim 1, wherein in the chelating step, the chelating reaction is performed at a temperature of 1 to 10 ℃, preferably 2 to 4 ℃; the time is 8-24 h.
8. A method for preparing sucrose-6-acetate, comprising: a step of subjecting sucrose and vinyl ester of fatty acid to a dehydration esterification reaction in a mixed organic solution under the catalytic action of an immobilized liquid lipase obtained by the method for immobilizing a liquid lipase according to any one of claims 1 to 7 to obtain a sucrose-6-acetate solution.
9. The method of making sucrose-6-acetate as in claim 8, further comprising: the immobilized liquid lipase is recovered and used for the dehydroesterification reaction of sucrose with vinyl esters of fatty acids.
10. The method for producing sucrose-6-acetate according to claim 9, wherein the fatty acid vinyl ester is any one of vinyl acetate, vinyl butyrate, vinyl caprylate, vinyl caprate, vinyl palmitate, vinyl stearate, or vinyl oleate;
the mixed organic solution is composed of DMF and tert-butyl alcohol and/or tert-amyl alcohol according to a volume ratio of 1: 1-10.
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| PCT/CN2021/071430 WO2022151024A1 (en) | 2021-01-13 | 2021-01-13 | Immobilization method of liquid lipase and preparation method of sucrose-6-acetate |
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| WO2022151024A1 (en) * | 2021-01-13 | 2022-07-21 | 安徽金禾实业股份有限公司 | Immobilization method of liquid lipase and preparation method of sucrose-6-acetate |
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| WO2022151024A1 (en) * | 2021-01-13 | 2022-07-21 | 安徽金禾实业股份有限公司 | Immobilization method of liquid lipase and preparation method of sucrose-6-acetate |
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