CN113061596B - Immobilized enzyme catalyst, preparation method thereof and application thereof in synthesis of vitamin A palmitate - Google Patents

Immobilized enzyme catalyst, preparation method thereof and application thereof in synthesis of vitamin A palmitate Download PDF

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CN113061596B
CN113061596B CN202110332121.6A CN202110332121A CN113061596B CN 113061596 B CN113061596 B CN 113061596B CN 202110332121 A CN202110332121 A CN 202110332121A CN 113061596 B CN113061596 B CN 113061596B
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immobilized enzyme
enzyme catalyst
lipase
catalyst according
organic polymer
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CN113061596A (en
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罗朝辉
林龙
王嘉辉
吕英东
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Wanhua Chemical Group Nutrition Technology Co ltd
Wanhua Chemical Group Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • C12N9/20—Triglyceride splitting, e.g. by means of lipase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/14—Enzymes or microbial cells immobilised on or in an inorganic carrier
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P23/00—Preparation of compounds containing a cyclohexene ring having an unsaturated side chain containing at least ten carbon atoms bound by conjugated double bonds, e.g. carotenes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/01—Carboxylic ester hydrolases (3.1.1)
    • C12Y301/01003—Triacylglycerol lipase (3.1.1.3)

Abstract

The invention discloses an immobilized enzyme catalyst, a preparation method and application thereof in synthesizing vitamin A palmitate, wherein the method utilizes an electrostatic spinning technology to synthesize a metal oxide nanorod, adopts a hydrophobic natural organic polymer to modify the surface of the metal oxide nanorod, and utilizes an acoustic wave auxiliary method to fix lipase on the surface of a modified carrier, thereby effectively solving the problems of poor acid resistance, hydrophobicity and diffusivity of the traditional enzyme catalysis. The invention also provides an application of the immobilized enzyme in synthesizing vitamin A palmitate, the process does not need an organic solvent, and the immobilized enzyme is environment-friendly, can be used in batches for a long time and has extremely high physical and chemical stability.

Description

Immobilized enzyme catalyst, preparation method thereof and application thereof in synthesis of vitamin A palmitate
Technical Field
The invention belongs to the technical field of chemical synthesis, and particularly relates to an immobilized enzyme catalyst, a preparation method thereof and application thereof in synthesis of vitamin A palmitate.
Background
Compared with vitamin A acetate (VA acetate), vitamin A palmitate (VA palmitate) has the advantages of long carbon chain, low melting point, good oil solubility, good low-temperature stability and the like, is an indispensable substance for normal metabolism of human bodies and animals, and has been widely applied to cosmetics, medicines, feeds and the like.
Esterification or transesterification in the presence of lipase to give VA palmitate is one of the common methods for preparing VA palmitate. Patent JP62248495 discloses a method for synthesizing VA palmitate by lipase catalysis, wherein the reaction is carried out for 12 hours at the temperature of 30 ℃, the yield is only 80%, and the lipase is difficult to separate after the reaction. Patent CN101200740 discloses a method for synthesizing vitamin a fatty acid ester by catalyzing reaction of vitamin a and fatty acid with lipase, silica gel is added in the reaction, and the reaction is carried out for 8 hours at 30 ℃, so that the conversion rate is about 90%. Wherein, the preparation and fixation of the enzyme and the preparation of the vitamin A raw material are relatively complex, and are not suitable for continuous industrial production.
It can be found that the VA palmitate prepared by the enzymatic reaction in the above patent has the problems of long production period, high enzyme preparation cost, poor selectivity, extremely high enzyme chemical stability and the like, and the green and environment-friendly biocatalytic esterification reaction is found, so that the VA palmitate has good economic effect.
Disclosure of Invention
The invention aims to provide an immobilized enzyme catalyst, a preparation method thereof and application thereof in synthesizing vitamin A palmitate, wherein immobilized enzyme is used for synthesizing a polymer modified metal oxide nanorod by using an electrostatic spinning technology, and the problems of poor acid resistance, hydrophobicity and diffusivity in the traditional enzyme catalysis are effectively solved. The immobilized enzyme is used for synthesizing the vitamin A palmitate, an organic solvent is not needed in the process, and the immobilized enzyme is environment-friendly, can be used in batches for a long time and has extremely high physicochemical stability.
In order to achieve the above object, the present invention has the following technical scheme:
the invention provides an immobilized enzyme catalyst which comprises lipase and a carrier, wherein the carrier is an organic polymer modified metal oxide.
Wherein the organic polymer comprises one or more of chitin, lignin, cellulose, starch, natural rubber, pectin and the like, preferably cellulose, and the organic polymer can be added to serve as a spinning aid to better promote the metal oxide to form uniformly dispersed nano rods; and secondly, the organic polymer is wound on the surface of the metal oxide, so that the hydrophobicity of the metal oxide is enhanced.
The metal oxide is an oxide of a metal M, wherein the metal M is preferably one or more of the oxides of a metal Fe, co, ti, ni, cr, V, hf, ce, zn, ni, preferably an oxide of Ti;
the lipase comprises one or more of chirazyme-2 lipase, candida rugosa lipase, novo435 lipase, CALB lipase, RMIM lipase and Candida sp.99-125 lipase, preferably Candida sp.99-125 lipase;
preferably, the lipase is supported in an amount of 1 to 80.0wt%, preferably 10 to 40.0wt% based on the mass of the modified support.
Preferably, when preparing the organic polymer modified metal oxide, the mass of the added organic polymer is 1 to 80wt%, preferably 10 to 50wt% of the mass of the added metal precursor. The metal precursor is a salt or an organic matter of the metal oxide relative to the metal, and the metal oxide can be obtained through processes such as roasting.
The preparation method of the organic polymer modified metal oxide comprises the following steps:
(1) Adding a metal precursor into the first mixed solvent, stirring for a certain time, sealing with nitrogen for later use, adding an organic polymer into the second organic solvent to prepare an organic polymer spinning aid solution with a certain concentration, stirring and heating to a certain temperature, and sealing with nitrogen for later use.
(2) And (3) dropwise adding the metal precursor solution into the organic polymer spinning aid solution at a certain speed, uniformly stirring, transferring into an electrostatic spinning injector, electrospinning at a certain voltage, and roasting at a certain temperature to obtain the organic polymer modified metal oxide nanorod.
In the step (1) of the invention, the metal precursor is a salt or an organic matter of metal M, and the inorganic salt comprises chloride, sulfate, nitrate, phosphate and the like; organic compounds such as organometallic halides, organometallic phosphine complexes, organometallic framework MOF materials, and the like.
The metal M is preferably one or more of the metals Fe, co, ti, ni, cr, V, hf, ce, zn, ni, preferably Ti; the metal precursor is preferably a titanium-containing compound such as one or more of titanium tetrachloride, tetrabutyl titanate, potassium tetrachlorotitanate, titanium sulfate, titanium nitrate, and the like, preferably tetrabutyl titanate.
In the step (1), the first mixed solvent is a mixture of alcohol and acid, such as methanol/formic acid, ethanol/acetic acid, methanol/acetic acid, isopropanol/butyric acid, ethylene glycol/succinic acid, etc., preferably ethanol/acetic acid. The volume ratio of the alcohol to the acid is 0.1:1 to 2:1, preferably 0.5:1 to 1:1.
The volume of the mixed solvent I added per gram of metal precursor is 1-100 mL, preferably 10-50 mL.
In the step (1) of the present invention, the second organic solvent is an organic alcohol, such as methanol, ethanol, n-butanol, ethylene glycol, propylene glycol, isopropanol, and the like, preferably methanol.
The volume of the organic solvent II added into each gram of organic polymer is 10-1000 mL, preferably 50-250 mL. The organic polymer solution is heated to a temperature of 10 to 100 ℃, preferably 30 to 70 ℃.
In the step (2) of the invention, the time for adding the organic polymer spinning aid to the metal precursor solution is 0.5-5 h, preferably 1-2.5 h.
Preferably, the organic polymer is added in an amount of 1 to 80wt%, preferably 10 to 50wt% based on the mass of the metal precursor.
Preferably, the voltage of the electrospinning is 1-30 kV, preferably 10-20 kV. The electrospinning time is 1 to 20 hours, preferably 5 to 10 hours. The roasting temperature is 100-1000 ℃, preferably 300-700 ℃, and the roasting time is 1-20 h, preferably 4-8 h.
The preparation method of the immobilized enzyme catalyst comprises the following steps:
the free lipase is placed in a buffer solution, the nanorods of the organic polymer modified metal oxide are added after the pH is regulated, and the enzyme is uniformly fixed on the composite carrier under the condition of acoustic wave assistance.
The buffer solution is phosphate buffer solution, such as one or more of sodium hydrogen phosphate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium phosphate, potassium phosphate and the like, preferably sodium hydrogen phosphate; the buffer concentration is 0.01mol/L to 2.0mol/L, preferably 0.2mol/L to 0.8mol/L. The pH is 3 to 9, preferably 4.5 to 6.5.
The addition amount of the buffer solution is as follows: the buffer is added to the dry powder of the free enzyme in a volume of 10 to 200mL, preferably 50 to 100mL.
In the present invention, the immobilization conditions of the free lipase are as follows: the reaction temperature is 20-60 ℃, preferably 30-40 ℃, the lipase is immobilized under stirring, the rotating speed is 20-200 rpm, preferably 80-150 rpm, the lipase is uniformly stirred for 1-10 hours, preferably 2-5 hours, and the sound wave frequency is 10-60 HZ, preferably 20-40 HZ.
The immobilized enzyme catalyst is used for synthesizing vitamin A palmitate.
The invention provides a method for synthesizing vitamin A palmitate, which comprises the steps of adding VA acetate and palmitic acid serving as raw materials and an immobilized enzyme catalyst disclosed by the invention into a reactor, reacting for a certain time at a certain temperature and under a certain pressure, sampling, analyzing the reaction conversion rate by liquid chromatography, and calculating the reaction selectivity. The granular immobilized enzyme can be recycled after simple filtration and separation.
In the preparation method of VA palmitate, the VA acetate is firstly preheated to 30-100 ℃, preferably 50-60 ℃.
The molar ratio of the VA acetate to the palmitic acid is 1:0.7-1:2, preferably 1:1.05-1:1.5.
The mass ratio of the immobilized enzyme to the VA acetate is 1:100-1:10, preferably 1:50-1:20.
In the preparation method of VA palmitate, the reaction temperature in the reactor is 20-80 ℃, preferably 50-60 ℃; the reaction time is 1 to 10 hours, preferably 3 to 7 hours; the reaction pressure is micro positive pressure of 0.001-0.08 MPa, preferably 0.01-0.03 MPa; the stirring rotation speed is 50-700 rpm, preferably 100-400 rpm; the reaction atmosphere is inert gas atmosphere, preferably nitrogen, and the flow rate of the nitrogen is 1-5 mL/min, preferably 2-3.5 mL/min.
And filtering the reaction liquid obtained after the VA palmitate preparation reaction is finished to separate enzyme particles and the reaction liquid, and recycling the enzyme particles.
Adding a certain amount of crystallization reagent into the reaction solution, cooling and crystallizing at a certain temperature to obtain purified product crystals, and sampling for purity analysis by liquid chromatography.
In the invention, the crystallization reagent added into the reaction liquid after separation is organic alcohol substances such as methanol, ethanol, isopropanol, n-butanol, glycerol and the like, preferably ethanol. The addition amount of the crystallization reagent is 1 to 10g, preferably 2 to 5g, of the crystallization reagent per gram of the reaction solution. The temperature of the cooling crystallization is-10 ℃ to 10 ℃, preferably-2 ℃ to 5 ℃.
The invention has the beneficial effects that:
1. the metal oxide nanorods have a quasi-one-dimensional structure, and free enzyme can be completely dispersed on the outer layers of the nanorods, so that the loading capacity of the free enzyme is remarkably improved, and the problem of internal diffusion in the macromolecular reaction process is solved.
2. The natural organic polymer modified metal oxide nanorods are adopted, so that the problem of acid resistance of the traditional enzyme catalysis is effectively solved, and the structural stability of the immobilized enzyme is enhanced.
3. The lipophilic organic macromolecule adapter enzyme protein and the metal oxide surface solve the problem of hydrophobicity of immobilized enzyme in a solvent-free system, and improve the hydrophobicity of the catalyst, thereby improving the use efficiency of the catalyst.
4. The immobilized enzyme is used for synthesizing the vitamin A palmitate, an organic solvent is not needed in the process, and the immobilized enzyme is environment-friendly, can be used in batches for a long time and has extremely high physicochemical stability.
Detailed Description
The raw material sources are as follows:
VA acetate was purchased from zhejiang pharmaceutical stock, bio-enzyme was purchased from novelin biotechnology, alpha-cellulose, pectin, starch, metal precursors were purchased from shanghai taitan technology, and the remaining chemicals were purchased from ala Ding Shiji, inc.
Example 1:
preparation of immobilized enzyme catalyst:
5.0g of free enzyme (Candida sp.99-125 enzyme dry powder) was dissolved in 250mL of 0.2mol/L sodium hydrogen phosphate buffer, pH was adjusted to 4.5, and the mixture was kept under stirring in a nitrogen atmosphere for further use.
10.0g of tetrabutyl titanate was weighed and added to 100mL of a mixed solution of ethanol/acetic acid (volume ratio of ethanol to acetic acid: 0.5:1). 1g of alpha-cellulose is weighed and added into 50mL of methanol, and after the temperature is raised to 30 ℃, the mixture is stirred strongly to be dispersed uniformly. The mixed solution of tetrabutyl titanate is added into cellulose methanol liquid in a dropwise manner for 1h, the obtained mixture is transferred into an electrostatic spinning injector, and after being electrospun for 5h under 10kV, the obtained solid is baked for 4h under the nitrogen atmosphere at 300 ℃.
Accurately weighing 10.0g of baked solid, adding into sodium hydrogen phosphate buffer solution containing free enzyme, uniformly adsorbing for 2h under the assistance of sound waves with the temperature of 30 ℃ and the rotation speed of 80rpm and 20HZ, taking out, sealing and preserving in a refrigerator with the temperature of 4 ℃ for standby, and marking the prepared immobilized lipase as Candida sp.99-125@P/TiO 2 The mass of the loaded solid was 13.4g, and the loading of lipase was initially calculated to be 34%.
10.0g of the immobilized lipase catalyst is put into 0.1mol/L palmitic acid ethanol solution to be stirred for 12 hours, and then the immobilized lipase catalyst is taken out to be dried, and the mass of the immobilized lipase is 9.95g, and the mass loss rate is only 0.5%, which indicates that the immobilized lipase has good acid resistance. The average diameter of the granular enzyme was 10.5nm by using a transmission electron microscope, and the enzyme was almost entirely supported on the outer surface of the carrier.
Preparation reaction of VA palmitate:
the reaction vessel was purged with nitrogen before the reaction, and was continuously fed with an amount of 2.0 mL/min. After the crude 328.5. 328.5gVA acetate oil was preheated to 50℃it was added to a 5L reactor, followed by the addition of 6.57g of immobilized enzyme and 268.8g of palmitic acid. After reacting for 5 hours at 50 ℃ and 0.02MPa and stirring speed of 100rpm, the conversion rate of VA acetate obtained by liquid phase analysis is 99.1%, and the yield of VA palmitate is 90.3%.
In the reaction process, the reaction solution after the reaction is started for 6min is taken to analyze the conversion rate in liquid phase, and the initial reaction rate is calculated to be 30.44 mol/(g.h.m) 3 ). The reaction solution after complete reaction was taken out and weighed, 1200g of ethanol was immediately added thereto, cooled to-2℃and subjected to cooling crystallization, and the purity of VA palmitate obtained by liquid phase analysis was 98.5%. After the reaction is finished, the immobilized enzyme and the reaction solution are separated by simple filtration, the immobilized enzyme is recycled according to the same reaction process conditions, and after recycling for 5 times, the yield of the VA palmitate is sequentially 90.1%, 89.8%, 89.6%, 89.1% and 89.0%, which indicates that the immobilized lipase has good recycling rate and is suitable for industrial scale development.
Example 2:
preparation of immobilized enzyme catalyst:
5.0g of free enzyme (Candida sp.99-125 enzyme dry powder) was dissolved in 300mL of 0.3mol/L sodium hydrogen phosphate buffer, the pH was adjusted to 5.0, and the mixture was kept under stirring in a nitrogen atmosphere for further use.
10.0g of cobalt nitrate was weighed into 200mL of ethanol/acetic acid mixture (0.6:1 ethanol to acetic acid by volume). 2.0g of alpha-cellulose is weighed and added into 400mL of methanol, and after the temperature is raised to 40 ℃, the mixture is stirred strongly to be dispersed uniformly. The mixed solution of cobalt nitrate is dripped into a cellulose methanol solution for 1.2 hours, the obtained mixture is transferred into an electrostatic spinning injector, and after being electrospun for 6 hours under 15kV, the obtained solid is roasted for 4.5 hours under the nitrogen atmosphere at the temperature of 350 ℃.
Accurately weighing 10.0g of baked solid, adding into sodium hydrogen phosphate buffer solution containing free enzyme, uniformly adsorbing for 2.5h under the assistance of 25HZ sound wave at 35 ℃ and 90rpm, taking out, sealing in a refrigerator at 4 ℃ for standby, and marking the prepared immobilized lipase as Candida sp.99-125@P/Co 2 O 3 . The mass of the loaded solid was 12.4g, and the loading of lipase was initially calculated to be 24%.
10.0g of the immobilized lipase catalyst is put into 0.1mol/L palmitic acid ethanol solution and stirred for 12 hours, and then the immobilized lipase catalyst is taken out and dried, and the mass of the immobilized lipase is 9.98g, and the mass loss rate is only 0.2%, which indicates that the immobilized lipase has good acid resistance. The average diameter of the granzyme was counted to be 12.4nm by a transmission electron microscope.
Preparation reaction of VA palmitate:
the reaction vessel was purged with nitrogen before the reaction, and was continuously fed with an amount of 2.5 mL/min. After the crude 328.5. 328.5gVA acetate oil was preheated to 55℃it was added to a 5L reactor, followed by the addition of 8.56g of immobilized enzyme and 268.8g of palmitic acid. After reacting for 5 hours at 55 ℃ and 0.01MPa and stirring speed of 200rpm, the conversion rate of VA acetate obtained by liquid phase analysis is 98.5%, and the yield of VA palmitate is 89.4%.
In the reaction process, the reaction solution after the reaction is started for 6min is taken to analyze the conversion rate in liquid phase, and the initial reaction rate is calculated to be 29.84 mol/(g.h.m) 3 ). The reaction solution after complete reaction was taken out and weighed, 1500g of ethanol was added thereto immediately, and cooled to-1℃for cooling crystallization, and the purity of VA palmitate obtained by liquid phase analysis was 95.9%.
Example 3:
modification and immobilization preparation of free enzyme:
5.0g of dry powder of the free enzyme Candida sp.99-125 is dissolved in 300mL of 0.5mol/L sodium hydrogen phosphate buffer, the pH is adjusted to 5.5, and the mixture is stirred and preserved in a nitrogen atmosphere for standby. 10.0g of tetrabutyl titanate was weighed and added to 300mL of a mixed solution of ethanol/acetic acid (volume ratio of ethanol to acetic acid: 0.8:1). 3.0g of pectin is weighed and added into 600mL of methanol, and after the temperature is raised to 60 ℃, the pectin is stirred strongly to be dispersed uniformly.
The mixed solution of tetrabutyl titanate is dripped into pectin methanol solution for 1.8 hours, the obtained mixture is transferred into an electrostatic spinning injector, and after being electrospun for 8 hours under 18kV, the obtained solid is roasted for 6.5 hours under the nitrogen atmosphere at 450 ℃. Accurately weighing 10.0g of baked solid, adding into sodium hydrogen phosphate buffer solution containing free enzyme, uniformly adsorbing for 3.5h under the assistance of sound waves with the temperature of 35 ℃ and the rotation speed of 120rpm and 30HZ, taking out, sealing and preserving in a refrigerator with the temperature of 4 ℃ for standby, and marking the prepared immobilized lipase as Candida sp.99-125@P/TiO 2 The mass of the loaded solid was 13.7g, and the loading of lipase was initially calculated to be 37%.
10.0g of the immobilized lipase catalyst is weighed and put into 0.1mol/L palmitic acid ethanol solution to be stirred for 12 hours, and then the immobilized lipase catalyst is taken out and dried, and the mass of the immobilized lipase is 9.96g, and the mass loss rate is only 0.4%, which indicates that the immobilized lipase has good acid resistance. The average diameter of the granzyme was found to be 16.8nm by means of a transmission electron microscope.
Preparation reaction of VA palmitate:
the reaction vessel was purged with nitrogen before the reaction, and the reaction vessel was continuously purged with 3.5mL/min. After the crude 328.5. 328.5gVA acetate oil was preheated to 60 ℃, the crude oil was added to a 5L reaction vessel, and 8.65g of immobilized enzyme and 281.6g of palmitic acid were added in sequence. After reaction for 6 hours at 60℃and 0.02MPa and stirring speed of 250rpm, the conversion of VA acetate was 99.2% and the yield of VA palmitate was 90.1% by liquid phase analysis.
In the reaction process, the reaction solution after the reaction is started for 6min is taken to analyze the conversion rate in liquid phase, and the initial reaction rate is calculated to be 24.26 mol/(g.h.m) 3 ). The reaction solution after completion of the reaction was taken out and weighed, 1800g of ethanol was added thereto immediately, and cooled to-3℃for cooling crystallization, and the purity of VA palmitate obtained by liquid phase analysis was 97.5%.
Example 4:
modification and immobilization preparation of free enzyme:
5.0g of dry powder of free enzyme Candida sp.99-125 is dissolved in 400mL of 0.75mol/L sodium hydrogen phosphate buffer, the pH is adjusted to 6.0, and the mixture is stirred and preserved in a nitrogen atmosphere for standby. 10.0g of nickel nitrate was weighed into 400mL of ethanol/acetic acid mixture (volume ratio of ethanol to acetic acid 1:1). 4.0g of alpha-cellulose is weighed and added into 800mL of methanol, and after the temperature is raised to 70 ℃, the mixture is stirred strongly to be dispersed uniformly.
The mixed solution of nickel nitrate is dripped into cellulose methanol solution for 2.5 hours, the obtained mixture is transferred into an electrostatic spinning injector, and after being electrospun for 10 hours under 20kV, the obtained solid is roasted for 8.0 hours under the nitrogen atmosphere at 700 ℃. Accurately weighing 10.0g of roasted solid, adding the roasted solid into the sodium hydrogen phosphate containing free enzyme for 4.5 hours, taking out, sealing and preserving in a refrigerator at 4 ℃ for standby, marking the prepared immobilized lipase as Candida sp.99-125@P/NiO, weighing 13.2g of loaded solid, and preliminarily calculating the loading amount of the lipase to be 32%.
10.0g of the immobilized lipase catalyst is weighed and put into 0.1mol/L palmitic acid ethanol solution to be stirred for 12 hours, and then the immobilized lipase catalyst is taken out and dried, and the mass of the immobilized lipase is weighed to be 9.89g, the mass loss rate is only 1.1%, which indicates that the immobilized lipase has good acid resistance. The average diameter of the granzyme was 20.8nm by means of a transmission electron microscope.
Preparation reaction of VA palmitate:
the reaction vessel was purged with nitrogen before the reaction, and the reaction vessel was continuously purged with 3.5mL/min. After the crude 328.5. 328.5gVA acetate oil was preheated to 60 ℃, it was added to a 5L reactor, followed by the addition of 6.57g of immobilized enzyme and 332.8g of palmitic acid. After 7h reaction at 60℃and 0.02MPa and stirring at 250rpm, the conversion of VA acetate was 97.5% and the yield of VA palmitate was 85.6% by liquid phase analysis.
In the reaction process, the reaction solution after the reaction is started for 6min is taken to analyze the conversion rate in liquid phase, and the initial reaction rate is calculated to be 21.54 mol/(g.h.m) 3 ). Taking out the reaction solution after complete reaction, weighing, immediately adding 1800g of ethanol into the reaction solution, cooling to-3 ℃, cooling and crystallizing,the purity of the VA palmitate obtained by liquid phase analysis was 96.5%.
Example 5:
preparation of immobilized enzyme catalyst:
5.0g of dry powder of free enzyme Candida sp.99-125 is dissolved in 500mL of 0.8mol/L sodium hydrogen phosphate buffer solution, the pH is adjusted to 6.5, and the mixture is stirred and preserved in a nitrogen atmosphere for standby. 10.0g of tetrabutyl titanate was weighed and added to 500mL of a mixed solution of ethanol/acetic acid (volume ratio of ethanol to acetic acid: 0.75:1). 5.0g of alpha-cellulose starch is weighed and added into 1000mL of methanol, and after the temperature is raised to 60 ℃, the mixture is stirred strongly to be dispersed uniformly.
The mixed solution of tetrabutyl titanate is dripped into starch methanol solution for 2.0h, the obtained mixture is transferred into an electrostatic spinning injector, and after being electrospun for 8h under 20kV, the obtained solid is roasted for 6.5h under the nitrogen atmosphere at 400 ℃. Accurately weighing 10.0g of baked solid, adding into the sodium hydrogen phosphate containing free enzyme for 8.0h, taking out, sealing and preserving in a refrigerator at 4 ℃ for standby, and marking the prepared immobilized lipase as Candida sp.99-125@P/TiO 2 The mass of the loaded solid was 13.8g, and the loading of lipase was calculated initially to be 38%.
10.0g of the immobilized lipase catalyst is weighed and put into 0.1mol/L palmitic acid ethanol solution to be stirred for 12 hours, and then the immobilized lipase catalyst is taken out and dried, and the mass loss rate of the immobilized lipase is only 0.7% when the weight of the immobilized lipase is 9.93g, which indicates that the immobilized lipase has good acid resistance. The average diameter of the granzyme was found to be 14.3nm by means of a transmission electron microscope.
Preparation reaction of VA palmitate:
the reaction vessel was purged with nitrogen before the reaction, and the reaction vessel was continuously purged with 3.5mL/min. After the crude 328.5. 328.5gVA acetate oil was preheated to 40 ℃, the crude oil was added to a 5L reaction vessel, and 9.69g of immobilized enzyme and 384.0g of palmitic acid were added in sequence. After reaction for 4 hours at 40℃and 0.03MPa under a stirring speed of 400rpm, the conversion of VA acetate was 96.5% and the yield of VA palmitate was 87.6% by liquid phase analysis. And taking the reaction liquid after the reaction is started for 6min in the reaction process, analyzing the conversion rate by liquid phase, and calculating the initial reaction rate as29.54mol/(g·h·m 3 ). The reaction solution after completion of the reaction was taken out and weighed, 1800g of ethanol was added thereto immediately and cooled to 2℃for cooling crystallization, and the purity of VA palmitate obtained by liquid phase analysis was 97.8%.
Comparative example 1:
the other conditions of the immobilized preparation method of the free enzyme are the same as in example 1, except that: in this comparative example 1, the same mass of complex carrier was directly used in the reaction by the same immobilization preparation method without adding free enzyme dry powder. After the reaction, the conversion rate of VA acetate is 20.1%, the yield of VA palmitate is 10.6%, and the purity of VA palmitate is 7.8%.
As is clear from the results of comparative example 1, no biological enzyme is added as an active component for catalyzing the reaction in the reaction process, and the yield of the product obtained by the reaction is extremely high, which indicates that the biological enzyme is an active center for catalyzing the transesterification reaction.
Comparative example 2:
in this comparative example 2, the free enzyme was immobilized on a conventional titania support by a conventional adsorption method, and the specific steps were as follows:
5.0g of free enzyme (Candida sp.99-125 enzyme dry powder) was dissolved in 250mL of 0.2mol/L sodium hydrogen phosphate buffer, pH was adjusted to 4.5, and the mixture was kept under stirring in a nitrogen atmosphere for further use.
10.0g of titanium dioxide powder was weighed into 100mL of ethanol/acetic acid mixed solution (volume ratio of ethanol to acetic acid 0.5:1). 1g of alpha-cellulose is weighed and added into 50mL of methanol, and after the temperature is raised to 30 ℃, the mixture is stirred strongly to be dispersed uniformly. The mixed solution containing titanium dioxide is added into cellulose methanol liquid in a dropwise manner for 1h, and the obtained solid is roasted for 4h at 300 ℃ in a nitrogen atmosphere.
Accurately weighing 10.0g of baked solid, adding the solid into the sodium hydrogen phosphate buffer solution containing free enzyme, uniformly adsorbing for 2 hours under the assistance of sound waves with the temperature of 30 ℃ and the rotating speed of 80rpm and 20HZ, taking out the solid, sealing and preserving the solid in a refrigerator with the temperature of 4 ℃ for standby, wherein the mass of the loaded solid is 11.3g, and the loading amount of lipase is calculated to be 13 percent preliminarily.
10.0g of the immobilized lipase catalyst was put into a 0.1mol/L ethanol palmitate solution, stirred for 12 hours, taken out and dried, and the mass of the immobilized lipase was 9.45g, and the mass loss rate was 5.5%. The average diameter of the granzyme was counted to be 40.5nm by a transmission electron microscope.
VA palmitate was prepared under the same process conditions as in example 1, and after the reaction was completed, the conversion of VA acetate was 50.5%, the yield of VA palmitate was 35.6%, and the purity of the obtained VA palmitate was 80.8%.
As can be seen from the result of comparative example 2, the polymer modified quasi-one-dimensional nanorod carrier prepared by adopting the electrospinning method can remarkably improve the dispersity of active enzyme, further improve the mass transfer efficiency, strengthen the mass transfer and improve the catalytic activity.

Claims (49)

1. The immobilized enzyme catalyst is characterized by comprising lipase and a carrier, wherein the carrier is an organic polymer modified metal oxide;
the organic polymer comprises one or more of cellulose and pectin,
the metal oxide is the oxide of metal M, wherein the metal M is one or more of metal Co, ti, ni,
the preparation method of the organic polymer modified metal oxide comprises the following steps:
(1) Adding a metal precursor into the first mixed solvent, stirring for a certain time, sealing with nitrogen for standby, adding an organic polymer into the second organic solvent to prepare an organic polymer spinning aid solution with a certain concentration, stirring and heating to a certain temperature, and sealing with nitrogen for standby;
(2) Dropwise adding a metal precursor solution into an organic polymer spinning aid solution at a certain speed, uniformly stirring, transferring into an electrostatic spinning injector, electrospinning at a certain voltage, and roasting at a certain temperature to obtain an organic polymer modified metal oxide nanorod;
in the step (1), the first mixed solvent is a mixture of alcohol and acid, and the volume ratio of the alcohol to the acid is 0.1:1-2:1; the first mixed solvent is methanol/formic acid, ethanol/acetic acid, methanol/acetic acid, isopropanol/butyric acid or ethylene glycol/succinic acid; in the step (1), the second organic solvent is an organic alcohol substance.
2. The immobilized enzyme catalyst according to claim 1, wherein,
the organic polymer is cellulose; the metal oxide is an oxide of Ti.
3. The immobilized enzyme catalyst of claim 1, wherein the lipase comprises one or more of chirazyme-2 lipase, candida rugosa lipase, novo435 lipase, CALB lipase, RMIM lipase, candida sp.99-125 lipase.
4. The immobilized enzyme catalyst of claim 3, wherein the lipase is Candida sp.99-125 lipase.
5. The immobilized enzyme catalyst of claim 1, wherein the lipase loading is 1-80.0. 80.0wt% of the modified support mass.
6. The immobilized enzyme catalyst according to claim 5, wherein the loading of the lipase is 10-40.0. 40.0wt% of the modified support mass.
7. The immobilized enzyme catalyst according to claim 1, wherein the mass of the organic polymer added in the preparation of the organic polymer-modified metal oxide is 1 to 80% wt% of the mass of the metal precursor added.
8. The immobilized enzyme catalyst according to claim 7, wherein the mass of the organic polymer added in the preparation of the organic polymer-modified metal oxide is 10 to 50% wt% of the mass of the metal precursor added.
9. The immobilized enzyme catalyst according to claim 1, wherein the metal precursor in step (1) is a salt or an organic matter of metal M, and the inorganic salts include chloride, sulfate, nitrate and phosphate; the organic compounds include organometallic halides, organometallic phosphine complexes, and organometallic framework MOF materials.
10. The immobilized enzyme catalyst according to claim 1, wherein the volume ratio of alcohol to acid in the first mixed solvent of step (1) is 0.5:1 to 1:1.
11. The immobilized enzyme catalyst according to claim 1, wherein in step (1), the first mixed solvent is ethanol/acetic acid.
12. The immobilized enzyme catalyst according to claim 1, wherein in step (1), the volume of the first mixed solvent added per gram of the metal precursor is 1 to 100mL.
13. The immobilized enzyme catalyst according to claim 12, wherein in step (1), the volume of the first mixed solvent added per gram of the metal precursor is 10 to 50mL.
14. The immobilized enzyme catalyst according to claim 1, wherein in step (1), the organic solvent two is methanol, ethanol, n-butanol, ethylene glycol, propylene glycol or isopropanol.
15. The immobilized enzyme catalyst of claim 14, wherein in step (1), the organic solvent two is methanol.
16. The immobilized enzyme catalyst according to claim 1, wherein in the step (1), the volume of the organic solvent II added per gram of the organic polymer is 10 to 1000. 1000 mL.
17. The immobilized enzyme catalyst according to claim 16, wherein in step (1), the volume of the organic solvent two added per gram of the organic polymer is 50 to 250mL.
18. The immobilized enzyme catalyst according to claim 1, wherein in the step (1), the organic polymer solution is heated to a temperature of 10 to 100 ℃.
19. The immobilized enzyme catalyst according to claim 18, wherein in step (1), the organic polymer solution is heated to a temperature of 30 to 70 ℃.
20. The immobilized enzyme catalyst according to claim 1, wherein in the step (2), the metal precursor solution is added to the organic polymer co-spinning agent for a time of 0.5 to 5h.
21. The immobilized enzyme catalyst according to claim 20, wherein in step (2), the metal precursor solution is added to the organic polymer co-spinning agent for a period of time of 1 to 2.5 to h.
22. The immobilized enzyme catalyst according to claim 1, wherein,
the voltage of the electrospinning is 1-30 kV; the electrospinning time is 1-20 h.
23. The immobilized enzyme catalyst of claim 22, wherein the voltage of electrospinning is from 10 to 20 kV; the electrospinning time is 5-10 h.
24. The immobilized enzyme catalyst according to claim 1, wherein the calcination temperature is 100-1000 ℃ and the calcination time is 1-20 h.
25. The immobilized enzyme catalyst of claim 24, wherein the calcination temperature is 300-700 ℃ and the calcination time is 4-8 hours.
26. The method for preparing an immobilized enzyme catalyst according to claim 1, wherein,
placing free lipase in buffer solution, adjusting pH, adding organic polymer modified metal oxide nanorods, and uniformly fixing enzyme on a composite carrier under the condition of acoustic wave assistance.
27. The method for preparing an immobilized enzyme catalyst according to claim 26, wherein the buffer solution is phosphate buffer solution; the concentration of the buffer solution is 0.01 mol/L-2.0 mol/L.
28. The method for preparing an immobilized enzyme catalyst according to claim 27, wherein the buffer solution is one or more of sodium hydrogen phosphate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium phosphate, and potassium phosphate; the concentration of the buffer solution is 0.2 mol/L-0.8 mol/L.
29. The method for producing an immobilized enzyme catalyst according to claim 28, wherein the pH is adjusted to 3 to 9.
30. The method for producing an immobilized enzyme catalyst according to claim 29, wherein the pH is adjusted to 4.5 to 6.5.
31. The method for preparing an immobilized enzyme catalyst according to claim 26, wherein the buffer is added in an amount of: the volume of buffer solution added into each gram of free enzyme dry powder is 10-200 mL.
32. The method for preparing an immobilized enzyme catalyst according to claim 31, wherein the buffer is added in an amount of: the volume of buffer solution added into each gram of free enzyme dry powder is 50-100 mL.
33. The method for preparing an immobilized enzyme catalyst according to claim 26, wherein the immobilization conditions of the free lipase are: the reaction temperature is 20-60 ℃, the lipase is immobilized under the stirring condition, the rotating speed is 20-200 rpm, the lipase is uniformly stirred for 1-10 h, and the sound wave frequency is 10-60 HZ.
34. The method for preparing an immobilized enzyme catalyst according to claim 33, wherein the immobilization conditions of the free lipase are: the reaction temperature is 30-40 ℃, the lipase is immobilized under the stirring condition, the rotating speed is 80-150 rpm, the lipase is uniformly stirred for 2-5 h, and the sound wave frequency is 20-40 HZ.
35. An immobilized enzyme catalyst according to any one of claims 1-25 or prepared by the method of preparation of claims 26-34 for use in the synthesis of vitamin a palmitate.
36. A method for synthesizing vitamin a palmitate, which is characterized in that raw materials VA acetate and palmitic acid, the immobilized enzyme catalyst prepared by any one of claims 1-25 or the immobilized enzyme catalyst prepared by the preparation method of claims 26-34 are added into a reactor to react to prepare the vitamin a palmitate.
37. The method of claim 36, wherein the molar ratio of VA acetate to palmitic acid is 1:0.7 to 1:2.
38. The method of claim 37, wherein the molar ratio of VA acetate to palmitic acid is 1:1.05 to 1:1.5.
39. The method according to claim 36, wherein the mass ratio of the immobilized enzyme to the VA acetate is 1:100 to 1:10.
40. The method according to claim 39, wherein the mass ratio of the immobilized enzyme to the VA acetate is 1:50-1:20.
41. The synthetic method of claim 36 wherein the reaction temperature is 20 ℃ to 80 ℃; the reaction time is 1-10 h; the reaction pressure is micro positive pressure of 0.001-0.08 MPa.
42. The method of claim 41, wherein the reaction temperature is 50℃to 60 ℃; the reaction time is 3-7 h; the reaction pressure is 0.01-0.03 MPa.
43. The method according to claim 36, wherein the enzyme particles and the reaction liquid are separated by filtering the reaction liquid obtained after the completion of the reaction for preparing the vitamin a palmitate, and the reaction liquid is added with a certain amount of crystallization reagent and cooled and crystallized at a certain temperature.
44. The method of claim 43, wherein the crystallization reagent is an organic alcohol.
45. The method of claim 44, wherein the crystallization reagent is selected from the group consisting of methanol, ethanol, isopropanol, n-butanol, and glycerol.
46. The synthesis method according to claim 43, wherein the crystallization reagent is added in an amount of 1 to 10 g/g of crystallization reagent.
47. The method according to claim 46, wherein the crystallization reagent is added in an amount of 2 to 5 g/g of crystallization reagent per gram of reaction solution.
48. The synthesis method according to claim 43, wherein the temperature of the cooling crystallization is-10℃to 10 ℃.
49. The method of claim 48, wherein the temperature of the cooling crystallization is-2℃to 5 ℃.
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