CN113061596A - 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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CN113061596A
CN113061596A CN202110332121.6A CN202110332121A CN113061596A CN 113061596 A CN113061596 A CN 113061596A CN 202110332121 A CN202110332121 A CN 202110332121A CN 113061596 A CN113061596 A CN 113061596A
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lipase
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immobilized enzyme
organic polymer
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CN113061596B (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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    • 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
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    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
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    • 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
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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)

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Abstract

The invention discloses an immobilized enzyme catalyst, a preparation method thereof and application thereof in synthesizing vitamin A palmitate. The invention also provides the application of the immobilized enzyme in the synthesis of vitamin A palmitate, the process does not need an organic solvent, is green and environment-friendly, and the immobilized enzyme 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 is widely applied to cosmetics, medicines, feeds and the like.
One of the common methods for preparing VA palmitate is esterification or transesterification in the presence of lipase to obtain VA palmitate. Patent JP62248495 discloses a method for synthesizing VA palmitate under lipase catalysis, wherein the reaction is carried out at 30 ℃ for 12h, the yield is only 80%, and the separation of lipase after the reaction is difficult. Patent CN101200740 discloses a method for synthesizing vitamin a fatty acid ester by using lipase to catalyze the reaction of vitamin a and fatty acid, wherein silica gel is added in the reaction, the reaction is carried out at 30 ℃ for 8 hours, and the conversion rate is about 90%. Wherein, the preparation and fixation of the enzyme and the preparation of the raw material of the vitamin A are relatively complex and are not suitable for continuous industrial production.
It can be found that the preparation of VA palmitate by the enzymatic reaction in the patent has the problems of long production period, high preparation cost of enzyme, poor selectivity, extremely high chemical stability of enzyme and the like, and a green and environment-friendly biocatalytic esterification reaction is found, so that the method has a 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. The immobilized enzyme is used for synthesizing the vitamin A palmitate, the process does not need an organic solvent, and the method is green and environment-friendly, can be applied in batches for a long time, and has extremely high physicochemical stability.
In order to achieve the purpose, the technical scheme of the invention is as follows:
the invention provides an immobilized enzyme catalyst which comprises lipase and a carrier, wherein the carrier is a metal oxide modified by organic polymers.
The organic polymer comprises one or more of chitin, lignin, cellulose, starch, natural rubber, pectin and the like, preferably cellulose, and can be used as a spinning aid to better promote metal oxides to form uniformly dispersed nano rods; and secondly, organic polymers are 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 metal M, wherein the metal M is preferably one or more of oxides of metals Fe, Co, Ti, Ni, Cr, V, Hf, Ce, Zn and Ni, and is preferably an oxide of Ti;
the lipase comprises one or more of Chirazymel-2 lipase, Candida rugosa lipase, Novo435 lipase, CALB lipase, RMIM lipase, Candida sp.99-125 lipase, preferably Candida sp.99-125 lipase;
preferably, the loading amount of the lipase is 1-80.0 wt%, preferably 10-40.0 wt% of the mass of the modified carrier.
Preferably, when the organic polymer modified metal oxide is prepared, the mass of the added organic polymer is 1 to 80 wt%, preferably 10 to 50 wt%, of the mass of the added metal precursor. The metal precursor is metal oxide relative to metal salts or organic matters, and the metal oxide can be obtained by roasting and other processes.
The preparation method of the organic polymer modified metal oxide comprises the following steps:
(1) adding the metal precursor into the first mixed solvent, stirring for a certain time, then carrying out nitrogen sealing for standby application, adding the organic polymer into the second organic solvent to prepare an organic polymer spinning aid solution with a certain concentration, stirring, heating to a certain temperature, and then carrying out nitrogen sealing for standby application.
(2) Dripping the metal precursor solution into the organic polymer spinning aid solution at a certain speed, uniformly stirring, transferring into an injector for electrostatic spinning, electrospinning at a certain voltage, and then roasting at a certain temperature to obtain the organic polymer modified metal oxide nanorod.
In the step (1), the metal precursor is a salt or an organic matter of metal M, and inorganic salts include chloride, sulfate, nitrate, phosphate and the like; organic compounds such as organometallic halides, organometallic phosphine complexes, and organometallic framework MOF materials, among others.
The metal M is preferably one or more of metals Fe, Co, Ti, Ni, Cr, V, Hf, Ce, Zn and Ni, and is 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, etc., and tetrabutyl titanate is preferred.
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, and the like, 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 the metal precursor is 1-100 mL, preferably 10-50 mL.
In the step (1) of the present invention, the organic solvent II is an organic alcohol substance, such as methanol, ethanol, n-butanol, ethylene glycol, propylene glycol, isopropanol, etc., preferably methanol.
The volume of the organic solvent II added into each gram of organic polymer is 10-1000 mL, preferably 50-250 mL. Heating the organic polymer solution to a temperature of 10-100 ℃, preferably 30-70 ℃.
In the step (2), the time for adding the organic polymer spinning aid into the metal precursor solution is 0.5-5 h, preferably 1-2.5 h.
Preferably, the amount of the organic polymer added is 1 to 80 wt%, preferably 10 to 50 wt%, based on the mass of the metal precursor.
Preferably, the voltage of the electrospinning is 1-30 kV, and preferably 10-20 kV. The electrospinning time is 1-20 h, preferably 5-10 h. The roasting temperature is 100-1000 ℃, preferably 300-700 ℃, and the roasting time is 1-20 hours, preferably 4-8 hours.
The preparation method of the immobilized enzyme catalyst comprises the following steps:
and (2) placing the free lipase in a buffer solution, adjusting the pH, adding the organic polymer modified metal oxide nanorod into the buffer solution, and uniformly fixing the enzyme on the composite carrier under the condition of sound 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, and sodium hydrogen phosphate is preferred; the concentration of the buffer solution is 0.01 mol/L-2.0 mol/L, preferably 0.2 mol/L-0.8 mol/L. The pH is 3-9, preferably 4.5-6.5.
The adding amount of the buffer solution is as follows: the volume of the buffer solution added into each gram of the free enzyme dry powder is 10-200 mL, preferably 50-100 mL.
In the present invention, the immobilization conditions of the free lipase are: the reaction temperature is 20-60 ℃, preferably 30-40 ℃, the lipase is immobilized under the stirring condition, 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 raw materials of VA acetate and palmitic acid and an immobilized enzyme catalyst 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 the VA palmitate, the VA acetate is preheated to 30-100 ℃ at first, and 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 1-10 h, preferably 3-7 h; the reaction pressure is micro-positive pressure of 0.001-0.08 MPa, preferably 0.01-0.03 MPa; the stirring speed is 50-700 rpm, preferably 100-400 rpm; the reaction atmosphere is an inert gas atmosphere, preferably nitrogen, and the flow rate of the nitrogen is 1-5 mL/min, preferably 2-3.5 mL/min.
After the VA palmitate preparation reaction is finished, the obtained reaction liquid is filtered to separate enzyme particles and the reaction liquid, and the enzyme particles can be recycled.
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 to perform purity analysis by liquid chromatography.
In the present invention, the crystallization reagent added to the separated reaction solution is an organic alcohol substance, such as methanol, ethanol, isopropanol, n-butanol, glycerol, etc., preferably ethanol. The addition amount of the crystallization reagent is 1-10 g, preferably 2-5 g, of the crystallization reagent per gram of the reaction solution. The cooling crystallization temperature is-10 ℃ to 10 ℃, preferably-2 ℃ to 5 ℃.
The invention has the beneficial effects that:
firstly, the metal oxide nano-rod has a quasi-one-dimensional structure, and the free enzyme can be completely dispersed on the outer layer of the nano-rod, so that the loading capacity of the free enzyme is obviously improved, and the problem of internal diffusion in the macromolecular reaction process is solved.
And secondly, the acid resistance problem of the traditional enzyme catalysis is effectively solved by adopting the natural organic polymer modified metal oxide nanorod, and the structural stability of the immobilized enzyme is enhanced.
And thirdly, the lipophilic organic polymer is linked with the surfaces of the enzyme protein and the metal oxide, so that the problem of hydrophobicity of the immobilized enzyme in a solvent-free system is solved, and the hydrophobicity of the catalyst is improved, thereby improving the use efficiency of the catalyst.
The immobilized enzyme is used for synthesizing the vitamin A palmitate, the process does not need an organic solvent, and the method is green and environment-friendly, can be used in batches for a long time, and has extremely high physical and chemical stability.
Detailed Description
The raw material sources are as follows:
VA acetate is purchased from Zhejiang medical science, Inc., bio-enzyme is purchased from Novixin Biotechnology, Inc., alpha-cellulose, pectin, starch, and metal precursors are purchased from Shanghai Tantake, Inc., and the rest of chemical reagents are purchased from Aladdin reagents, Inc.
Example 1:
preparation of an 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 solution, the pH was adjusted to 4.5, and the solution was kept under stirring in a nitrogen atmosphere for further use.
10.0g of tetrabutyl titanate was weighed and added to 100mL of an ethanol/acetic acid mixed solution (volume ratio of ethanol to acetic acid was 0.5: 1). 1g of alpha-cellulose is weighed and added into 50mL of methanol, heated to 30 ℃ and stirred intensively to be dispersed evenly. And (2) dropwise adding the mixed solution of tetrabutyl titanate into a cellulose methanol solution, dropwise adding for 1h, transferring the obtained mixture into an electrostatic spinning injector, electrospinning at 10kV for 5h, and roasting the obtained solid at 300 ℃ for 4h in a nitrogen atmosphere.
Accurately weighing 10.0g of calcined solid, adding the solid into the sodium hydrogen phosphate buffer solution containing the free enzyme, uniformly adsorbing for 2h at the temperature of 30 ℃, the rotating speed of 80rpm and the assistance of 20HZ sound waves, taking out, sealing and storing in a refrigerator at the temperature of 4 ℃ for later use, and recording the immobilized lipase obtained by preparation as Candida sp.99-125@ P/TiO2The mass of the solid after loading was 13.4g, and the loading of lipase was preliminarily 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, then the immobilized lipase catalyst is taken out and dried, the weight of the immobilized lipase is weighed to be 9.95g, the mass loss rate is only 0.5 percent, and the immobilized lipase has good acid resistance. The average diameter of the granular enzyme was 10.5nm as counted by transmission electron microscopy, and the enzyme was almost completely supported on the outer surface of the carrier.
Preparation reaction of VA palmitate:
the reaction vessel was purged with nitrogen before the reaction, and the nitrogen was continuously introduced into the reaction vessel at a rate of 2.0 mL/min. 328.5gVA acetic ester crude oil is preheated to 50 ℃ and then added into a 5L reaction kettle, and 6.57g immobilized enzyme and 268.8g palmitic acid are sequentially added. After reacting for 5 hours at 50 ℃, the pressure of 0.02MPa and the stirring speed of 100rpm, the conversion rate of VA acetate obtained by liquid phase analysis is 99.1 percent, and the yield of VA palmitate is 90.3 percent.
In the reaction process, the reaction liquid after the reaction is started for 6min is taken to analyze the conversion rate by a liquid phase, and the initial reaction rate is calculated to be 30.44 mol/(g.h.m)3). Taking out the reaction solution after complete reaction, weighing, immediately adding 1200g ethanol, cooling to-2 deg.C for cooling crystallization, and analyzing the liquid phase to obtain VA palmitate with high purityThe content was 98.5%. After the reaction is finished, the immobilized enzyme and the reaction liquid are separated by simple filtration, the immobilized enzyme is recycled according to the same reaction process conditions, and after 5 times of recycling, the yield of the VA palmitate is 90.1%, 89.8%, 89.6%, 89.1% and 89.0% in sequence, which indicates that the immobilized lipase has good recycling rate and is suitable for industrial scale development.
Example 2:
preparation of an 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 solution, the pH was adjusted to 5.0, and the solution was kept under stirring in a nitrogen atmosphere for further use.
10.0g of cobalt nitrate was weighed and added to 200mL of a mixed solution of ethanol/acetic acid (volume ratio of ethanol to acetic acid was 0.6: 1). 2.0g of alpha-cellulose is weighed into 400mL of methanol, heated to 40 ℃ and stirred vigorously to disperse the alpha-cellulose uniformly. And (3) dropwise adding the mixed solution of the cobalt nitrate into a cellulose methanol solution, dropwise adding for 1.2h, transferring the obtained mixture into an electrostatic spinning injector, electrospinning at 15kV for 6h, and roasting the obtained solid at 350 ℃ for 4.5h in a nitrogen atmosphere.
Accurately weighing 10.0g of calcined solid, adding the solid into the sodium hydrogen phosphate buffer solution containing the free enzyme, uniformly adsorbing for 2.5h at the temperature of 35 ℃, the rotation speed of 90rpm and the assistance of 25HZ sound waves, taking out, sealing and storing in a refrigerator at 4 ℃ for later use, and recording the immobilized lipase obtained by preparation as Candida sp.99-125@ P/Co2O3. The mass of the solid after loading was 12.4g, and the loading of lipase was preliminarily calculated to be 24%.
10.0g of the immobilized lipase catalyst is put into 0.1mol/L palmitic acid ethanol solution to be stirred for 12 hours, then the immobilized lipase catalyst is taken out and dried, the weight of the immobilized lipase is weighed to be 9.98g, the mass loss rate is only 0.2 percent, and the immobilized lipase has good acid resistance. The mean diameter of the granzyme was 12.4nm as counted by a transmission electron microscope.
Preparation reaction of VA palmitate:
the reaction vessel was purged with nitrogen before the reaction, and the nitrogen was continuously introduced into the reaction vessel at a rate of 2.5 mL/min. The 328.5gVA acetic ester crude oil is preheated to 55 ℃, then added into a 5L reaction kettle, and sequentially added with 8.56g of immobilized enzyme and 268.8g of palmitic acid. After reacting for 5 hours at 55 ℃, the pressure of 0.01MPa and the stirring speed of 200rpm, the conversion rate of VA acetate obtained by liquid phase analysis is 98.5 percent, and the yield of VA palmitate is 89.4 percent.
In the reaction process, the reaction liquid after the reaction is started for 6min is taken to analyze the conversion rate by a 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 immediately added thereto, 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 immobilized preparation of free enzyme:
5.0g of a dry powder of the free enzyme Candida sp.99-125 was dissolved in 300mL of 0.5mol/L sodium hydrogen phosphate buffer, the pH was adjusted to 5.5, and the solution was kept under stirring in a nitrogen atmosphere for further use. 10.0g of tetrabutyl titanate was weighed and added to 300mL of an ethanol/acetic acid mixed solution (volume ratio of ethanol to acetic acid was 0.8: 1). 3.0g of pectin is weighed into 600mL of methanol, heated to 60 ℃ and stirred vigorously to disperse the pectin uniformly.
And (3) dropwise adding the mixed solution of tetrabutyl titanate into pectin methanol solution, dropwise adding for 1.8h, transferring the obtained mixture into an electrostatic spinning injector, electrospinning at 18kV for 8h, and roasting the obtained solid at 450 ℃ for 6.5h in a nitrogen atmosphere. Accurately weighing 10.0g of calcined solid, adding the solid into the sodium hydrogen phosphate buffer solution containing the free enzyme, uniformly adsorbing for 3.5h at the temperature of 35 ℃, the rotating speed of 120rpm and the assistance of 30HZ sound wave, taking out, sealing and storing in a refrigerator at 4 ℃ for later use, and recording the immobilized lipase obtained by preparation as Candida sp.99-125@ P/TiO2And the mass of the solid after loading was taken to be 13.7g, and the loading amount of lipase was preliminarily calculated to be 37%.
Weighing 10.0g of the obtained immobilized lipase catalyst, putting the immobilized lipase catalyst into 0.1mol/L palmitic acid ethanol solution, stirring for 12h, taking out and drying, weighing the immobilized lipase with the mass of 9.96g, and ensuring that the mass loss rate is only 0.4%, which indicates that the immobilized lipase has good acid resistance. The mean diameter of the granular enzyme was 16.8nm as counted by transmission electron microscopy.
Preparation reaction of VA palmitate:
the reaction vessel was purged with nitrogen before the reaction, and the nitrogen was continuously introduced into the reaction vessel at an amount of 3.5 mL/min. 328.5gVA acetic ester crude oil is preheated to 60 ℃ and then added into a 5L reaction kettle, and 8.65g immobilized enzyme and 281.6g palmitic acid are sequentially added. After reacting for 6 hours at 60 ℃, the pressure of 0.02MPa and the stirring speed of 250rpm, the conversion rate of VA acetate obtained by liquid phase analysis is 99.2 percent, and the yield of VA palmitate is 90.1 percent.
In the reaction process, the reaction liquid after the reaction is started for 6min is taken to analyze the conversion rate by a liquid phase, and the initial reaction rate is calculated to be 24.26 mol/(g.h.m)3). The reaction solution after complete reaction was taken out and weighed, and 1800g of ethanol was immediately added thereto, 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 immobilized preparation of free enzyme:
5.0g of a dry powder of the free enzyme Candida sp.99-125 was dissolved in 400mL of 0.75mol/L sodium hydrogen phosphate buffer, the pH was adjusted to 6.0, and the solution was stored under stirring in a nitrogen atmosphere for further use. 10.0g of nickel nitrate was weighed and added to 400mL of a mixed solution of ethanol/acetic acid (volume ratio of ethanol to acetic acid was 1: 1). 4.0g of alpha-cellulose is weighed into 800mL of methanol, heated to 70 ℃ and stirred vigorously to disperse the alpha-cellulose uniformly.
And (3) dropwise adding the mixed solution of the nickel nitrate into a cellulose methanol solution, dropwise adding for 2.5h, transferring the obtained mixture into an electrostatic spinning injector, performing electrostatic spinning for 10h at 20kV, and roasting the obtained solid for 8.0h at 700 ℃ in a nitrogen atmosphere. Accurately weighing 10.0g of calcined solid, adding the calcined solid into the sodium hydrogen phosphate containing the free enzyme, slowing for 4.5h, taking out, sealing and storing in a refrigerator at 4 ℃ for later use, marking the immobilized lipase obtained by preparation as Candida sp.99-125@ P/NiO, weighing the mass of the loaded solid as 13.2g, and preliminarily calculating the load of the lipase as 32%.
Weighing 10.0g of the obtained immobilized lipase catalyst, putting the immobilized lipase catalyst into 0.1mol/L palmitic acid ethanol solution, stirring for 12h, taking out and drying, weighing 9.89g of the immobilized lipase, and ensuring that the mass loss rate is only 1.1%, which indicates that the immobilized lipase has good acid resistance. The mean diameter of the granular enzyme was 20.8nm as counted by transmission electron microscopy.
Preparation reaction of VA palmitate:
the reaction vessel was purged with nitrogen before the reaction, and the nitrogen was continuously introduced into the reaction vessel at an amount of 3.5 mL/min. 328.5gVA acetic ester crude oil is preheated to 60 ℃ and then added into a 5L reaction kettle, and 6.57g immobilized enzyme and 332.8g palmitic acid are sequentially added. After reacting for 7 hours at 60 ℃, the pressure of 0.02MPa and the stirring speed of 250rpm, the conversion rate of VA acetate obtained by liquid phase analysis is 97.5 percent, and the yield of VA palmitate is 85.6 percent.
In the reaction process, the reaction liquid after the reaction is started for 6min is taken to analyze the conversion rate by a liquid phase, and the initial reaction rate is calculated to be 21.54 mol/(g.h.m)3). The reaction solution after complete reaction was taken out and weighed, and 1800g of ethanol was immediately added thereto, and cooled to-3 ℃ for cooling crystallization, and the purity of VA palmitate obtained by liquid phase analysis was 96.5%.
Example 5:
preparation of an immobilized enzyme catalyst:
5.0g of the enzyme dry powder of the free enzyme Candida sp.99-125 was dissolved in 500mL of 0.8mol/L sodium hydrogen phosphate buffer, the pH was adjusted to 6.5, and the solution was kept under stirring in a nitrogen atmosphere for further use. 10.0g of tetrabutyl titanate was weighed and added to 500mL of an ethanol/acetic acid mixed solution (volume ratio of ethanol to acetic acid was 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 intensively stirred to be uniformly dispersed.
And (3) dropwise adding the mixed solution of tetrabutyl titanate into a starch methanol solution, dropwise adding for 2.0h, transferring the obtained mixture into an electrostatic spinning injector, electrospinning at 20kV for 8h, and roasting the obtained solid at 400 ℃ for 6.5h in a nitrogen atmosphere. Accurately weighing 10.0g of calcined solid, adding into the above sodium hydrogen phosphate containing free enzyme, slowing for 8.0h, taking out, sealing and storing in a refrigerator at 4 deg.C for use, and recording the immobilized lipase obtained as Candida sp.99-125@P/TiO2And the mass of the solid after loading was taken to be 13.8g, and the loading amount of lipase was preliminarily calculated to be 38%.
Weighing 10.0g of the obtained immobilized lipase catalyst, putting the immobilized lipase catalyst into 0.1mol/L palmitic acid ethanol solution, stirring for 12h, taking out and drying, weighing 9.93g of the immobilized lipase, and ensuring that the mass loss rate is only 0.7%, which indicates that the immobilized lipase has good acid resistance. The mean diameter of the granular enzyme was 14.3nm as counted by transmission electron microscopy.
Preparation reaction of VA palmitate:
the reaction vessel was purged with nitrogen before the reaction, and the nitrogen was continuously introduced into the reaction vessel at an amount of 3.5 mL/min. 328.5gVA acetic ester crude oil is preheated to 40 ℃ and then added into a 5L reaction kettle, and 9.69g of immobilized enzyme and 384.0g of palmitic acid are sequentially added. After reacting for 4 hours at 40 ℃, the pressure of 0.03MPa and the stirring speed of 400rpm, the conversion rate of VA acetate obtained by liquid phase analysis is 96.5 percent, and the yield of VA palmitate is 87.6 percent. And the initial reaction rate is calculated as 29.54 mol/(g.h.m) by analyzing the conversion rate of the reaction liquid which is taken from the reaction process and reacts for 6min in a liquid phase3). The reaction solution after the complete reaction was taken out and weighed, and 1800g of ethanol was immediately added thereto and cooled to 2 ℃ to perform cooling crystallization, and the purity of VA palmitate obtained by liquid phase analysis was 97.8%.
Comparative example 1:
other conditions for the preparation of immobilized free enzyme were the same as in example 1 except that: in this comparative example 1, without adding the free enzyme dry powder, the same mass of the composite carrier was directly used in the reaction by the same immobilization preparation method. After the reaction, the conversion of VA acetate was 20.1%, the yield of VA palmitate was 10.6%, and the purity of VA palmitate was 7.8% as determined by analysis.
From the results of comparative example 1, it can be seen that the bio-enzyme is not added as an active component for catalyzing the reaction during the reaction, and the yield of the product obtained by the reaction is very high, indicating that the bio-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 carrier by a conventional adsorption method, which specifically comprises the following steps:
5.0g of free enzyme (Candida sp.99-125 enzyme dry powder) was dissolved in 250mL of 0.2mol/L sodium hydrogen phosphate buffer solution, the pH was adjusted to 4.5, and the solution was kept under stirring in a nitrogen atmosphere for further use.
10.0g of titanium dioxide powder was weighed and added to 100mL of an ethanol/acetic acid mixed solution (volume ratio of ethanol to acetic acid was 0.5: 1). 1g of alpha-cellulose is weighed and added into 50mL of methanol, heated to 30 ℃ and stirred intensively to be dispersed evenly. And (3) dropwise adding the mixed solution containing titanium dioxide into a cellulose methanol solution, dropwise adding for 1h, and roasting the obtained solid at 300 ℃ for 4h in a nitrogen atmosphere.
Accurately weighing 10.0g of roasted solid, adding the solid into the sodium hydrogen phosphate buffer solution containing the free enzyme, uniformly adsorbing for 2h under the assistance of 20HZ sound wave at the temperature of 30 ℃, the rotating speed of 80rpm, and taking out, sealing and storing for later use in a refrigerator at the temperature of 4 ℃, wherein the mass of the loaded solid is 11.3g, and the loading capacity of the lipase is 13% by preliminary calculation.
10.0g of the obtained immobilized lipase catalyst is put into 0.1mol/L palmitic acid ethanol solution to be stirred for 12 hours, then the immobilized lipase catalyst is taken out and dried, the mass of the immobilized lipase is weighed to be 9.45g, and the mass loss rate is 5.5%. The average diameter of the granular enzyme was counted by transmission electron microscopy to be 40.5 nm.
VA palmitate was prepared under the same process conditions as in example 1, and after the reaction, the conversion of VA acetate was 50.5%, the yield of VA palmitate was 35.6%, and the purity of VA palmitate was 80.8%.
From the results of comparative example 2, it can be seen that the polymer modified quasi-one-dimensional nanorod carrier prepared by the electrospinning method can significantly improve the dispersity of active enzymes, thereby improving the mass transfer efficiency, strengthening the mass transfer and improving the catalytic activity.

Claims (10)

1. An immobilized enzyme catalyst is characterized by comprising lipase and a carrier, wherein the carrier is a metal oxide modified by organic macromolecules;
preferably, the organic polymer comprises one or more of chitin, lignin, cellulose, starch, natural rubber and pectin, preferably cellulose;
preferably, the metal oxide is an oxide of metal M, wherein the metal M is preferably one or more of metals Fe, Co, Ti, Ni, Cr, V, Hf, Ce, Zn, Ni, preferably an oxide of Ti.
2. The immobilized enzyme catalyst according to claim 1, wherein the lipase comprises one or more of chiralymel-2 lipase, Candida rugosa lipase, Novo435 lipase, CALB lipase, RMIM lipase, Candida sp.99-125 lipase, preferably Candida sp.99-125 lipase;
preferably, the loading amount of the lipase is 1-80.0 wt%, preferably 10-40.0 wt% of the mass of the modified carrier;
preferably, when the organic polymer modified metal oxide is prepared, the mass of the added organic polymer is 1 to 80 wt%, preferably 10 to 50 wt%, of the mass of the added metal precursor.
3. The immobilized enzyme catalyst according to claim 1, wherein the preparation method of the organic polymer-modified metal oxide comprises:
(1) adding a metal precursor into the mixed solvent I, stirring for a certain time, then carrying out nitrogen sealing for standby application, adding an organic polymer into the organic solvent II to prepare an organic polymer spinning aid solution with a certain concentration, stirring, heating to a certain temperature, and then carrying out nitrogen sealing for standby application;
(2) dripping the metal precursor solution into the organic polymer spinning aid solution at a certain speed, uniformly stirring, transferring into an injector for electrostatic spinning, electrospinning at a certain voltage, and then roasting at a certain temperature to obtain the organic polymer modified metal oxide nanorod.
4. The immobilized enzyme catalyst according to claim 3, wherein the metal precursor in step (1) is a salt of metal M or an organic substance, and the inorganic salt includes chloride, sulfate, nitrate and phosphate; the organic matter comprises organic metal halide, organic metal phosphine coordination compound and organic metal framework MOF material;
the metal M is preferably one or more of metals Fe, Co, Ti, Ni, Cr, V, Hf, Ce, Zn and Ni, and is preferably Ti; preferably, in the step (1), the first mixed solvent is a mixture of alcohol and acid, preferably methanol/formic acid, ethanol/acetic acid, methanol/acetic acid, isopropanol/butyric acid, ethylene glycol/succinic acid, and more preferably ethanol/acetic acid; the volume ratio of the alcohol to the acid is 0.1: 1-2: 1, preferably 0.5: 1-1: 1;
preferably, in the step (1), the volume of the first mixed solvent added per gram of the metal precursor is 1-100 mL, and preferably 10-50 mL.
5. The immobilized enzyme catalyst according to claim 3, wherein in step (1), the organic solvent II is an organic alcohol substance, preferably methanol, ethanol, n-butanol, ethylene glycol, propylene glycol, isopropanol, preferably methanol;
preferably, in the step (1), the volume of the organic solvent II added in each gram of organic polymer is 10-1000 mL, preferably 50-250 mL;
preferably, in the step (1), the organic polymer solution is heated to a temperature of 10 to 100 ℃, preferably 30 to 70 ℃.
6. The immobilized enzyme catalyst according to any one of claims 3 to 5, wherein in the step (2), the organic polymer spinning aid is added into the metal precursor solution for 0.5 to 5 hours, preferably 1 to 2.5 hours;
preferably, the adding amount of the organic polymer is 1-80 wt% of the mass of the metal precursor, and preferably 10-50 wt%;
preferably, the voltage of the electrospinning is 1-30 kV, and preferably 10-20 kV. The electrospinning time is 1-20 h, preferably 5-10 h;
preferably, the roasting temperature is 100-1000 ℃, preferably 300-700 ℃, and the roasting time is 1-20 hours, preferably 4-8 hours.
7. The method for producing an immobilized enzyme catalyst according to any one of claims 1 to 6,
placing free lipase in a buffer solution, adjusting the pH, adding the organic polymer modified metal oxide nanorod into the buffer solution, and uniformly fixing the enzyme on the composite carrier under the auxiliary condition of sound waves;
preferably, the buffer solution is a phosphate buffer solution, preferably one or more of sodium hydrogen phosphate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium phosphate and potassium phosphate, preferably sodium hydrogen phosphate; the concentration of the buffer solution is 0.01-2.0 mol/L, preferably 0.2-0.8 mol/L;
preferably, the pH is adjusted to 3-9, preferably 4.5-6.5;
preferably, the addition amount of the buffer is as follows: the volume of the buffer solution added into the free enzyme dry powder per gram is 10-200 mL, preferably 50-100 mL;
preferably, the preparation conditions of the free lipase are as follows: the reaction temperature is 20-60 ℃, preferably 30-40 ℃, the lipase is immobilized under the stirring condition, 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.
8. The immobilized enzyme catalyst according to any one of claims 1 to 6 or the immobilized enzyme catalyst prepared by the preparation method according to claim 7 is used for synthesizing vitamin A palmitate.
9. A synthesis method of vitamin A palmitate is characterized in that raw materials of VA acetate and palmitic acid, the immobilized enzyme catalyst of any one of claims 1 to 6 or the immobilized enzyme catalyst prepared by the preparation method of claim 7 are added into a reactor to react to prepare the vitamin A palmitate;
preferably, the molar ratio of the VA acetate to the palmitic acid is 1: 0.7-1: 2, preferably 1: 1.05-1: 1.5;
preferably, the mass ratio of the immobilized enzyme to the VA acetate is 1: 100-1: 10, preferably 1: 50-1: 20;
the preferable reaction temperature is 20-80 ℃, and the preferable reaction temperature is 50-60 ℃; the reaction time is 1-10 h, preferably 3-7 h; the reaction pressure is micro-positive pressure of 0.001-0.08 MPa, preferably 0.01-0.03 MPa.
10. The synthesis method of claim 9, wherein the reaction solution obtained after the reaction for preparing vitamin a palmitate is filtered to separate enzyme particles and the reaction solution, and a certain amount of crystallization reagent is added into the reaction solution, and the reaction solution is cooled and crystallized at a certain temperature;
preferably, the crystallization reagent is an organic alcohol substance, preferably methanol, ethanol, isopropanol, n-butanol and glycerol, more preferably ethanol;
preferably, the addition amount of the crystallization reagent is 1-10 g, preferably 2-5 g, of the crystallization reagent added in each gram of reaction liquid;
preferably, the temperature of the cooling crystallization is-10 ℃ to 10 ℃, preferably-2 ℃ to 5 ℃.
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