CN108642036B - Preparation method of nickel-doped ordered mesoporous alumina and immobilized fructosyltransferase - Google Patents
Preparation method of nickel-doped ordered mesoporous alumina and immobilized fructosyltransferase Download PDFInfo
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- CN108642036B CN108642036B CN201810463193.2A CN201810463193A CN108642036B CN 108642036 B CN108642036 B CN 108642036B CN 201810463193 A CN201810463193 A CN 201810463193A CN 108642036 B CN108642036 B CN 108642036B
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- 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
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- C01F7/02—Aluminium oxide; Aluminium hydroxide; Aluminates
Abstract
The invention discloses a preparation method of nickel-doped ordered mesoporous alumina and immobilized fructosyltransferase, belonging to the technical field of bioengineering. The preparation method of the immobilized fructosyl transferase comprises the following steps: (1) synthesizing a nickel-doped ordered mesoporous alumina carrier; (2) immobilizing the fructosyltransferase on a carrier by coordination of aluminum and an amino acid; (3) the activation of nickel improves the enzymatic activity of the immobilized enzyme. The method has mild preparation conditions, the acting force between the enzyme and the carrier is a coordination bond, the acting force is strong, the enzyme is not easy to fall off, meanwhile, the enzyme can keep higher activity due to the activation effect of nickel, the carrier with a mesoporous structure is beneficial to the substrate to quickly reach a reaction site, and the balance between high activity and firm immobilization of the fructosyltransferase is realized.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a nickel-doped ordered mesoporous alumina body and a preparation method of immobilized fructosyltransferase.
Background
Fructooligosaccharides, also known as fructotriose oligosaccharides, are fructooligosaccharides formed by the linkage of 1-3 fructosyl groups to the fructose residue of a sucrose molecule via β 1-2-glycosidic bonds: kestose, nystose and other mixtures. The fructo-oligosaccharide has the effect of water-soluble dietary fiber, is a prebiotic, can promote the growth of bifidobacterium, reduce harmful bacteria, promote the absorption of mineral substances, enhance the body immunity, resist cancer, reduce blood sugar, prevent decayed teeth and the like, and has no toxic or side effect. The fructo-oligosaccharide is widely present in plants such as barley, tomato, banana and the like, but has low content and great development difficulty.
The fructosyltransferase can catalyze sucrose to be converted into the fructo-oligosaccharide, but the fructosyltransferase is easy to dissolve in water, is expensive, is difficult to separate from a substrate in the production process, can be utilized only once, causes extremely high production cost, and cannot realize industrial application. Therefore, the fructosyltransferase is immobilized on a water-insoluble carrier, so that the immobilized enzyme is extremely easy to separate from a substrate and a product, no enzyme residue exists in a product solution, the purification process is simplified, continuous reaction can be realized, and the automatic production is convenient, therefore, the immobilization of the fructosyltransferase becomes a hotspot of research. The reported immobilized carriers comprise high molecular materials such as macroporous resin, chitosan and the like, and inorganic materials such as hydroxyapatite, magnetic iron oxide, active carbon, molecular sieves, silicon oxide and the like, wherein the materials are mainly combined with enzymes through the acting force of hydrogen bonds, ionic bonds and covalent bonds, the enzymes are easy to fall off due to weak acting force of the hydrogen bonds and the ionic bonds, the covalent bonds need the action of the carriers and the enzymes although the acting force is strong, and the conditions in the synthesis process are harsh, so that the enzymes are easy to inactivate. Various immobilized enzyme methods have respective advantages and disadvantages, and how to balance the activity and stability of the enzyme is a key scientific problem to be solved for the application of the immobilized enzyme. Therefore, designing and synthesizing a carrier which can be mildly immobilized, has high reactivity and stable property becomes the key of industrial application of the immobilized enzyme.
Disclosure of Invention
The invention aims to provide a preparation method of nickel-doped ordered mesoporous alumina for mildly immobilizing fructosyltransferase by utilizing a coordinate bond, and solves the problem that high activity and stable immobilization of enzyme in a fructosyltransferase immobilization system are difficult to obtain simultaneously.
In order to achieve the purpose, the invention adopts the technical scheme that:
a preparation method of nickel-doped ordered mesoporous alumina comprises the following steps:
1) dissolving P123 (polyvinyl ether-polypropylene ether-polyvinyl ether block copolymer) in absolute ethyl alcohol at room temperature to obtain an absolute ethyl alcohol solution of P123;
2) adding aluminum isopropoxide and nickel nitrate into an absolute ethyl alcohol solution of P123, adding concentrated hydrochloric acid, fully stirring and dissolving, and stirring for 5-12h to form sol;
3) the sol is volatilized at the temperature of 55-65 ℃ for self-assembly for 45-50h to obtain dry gel, the temperature of the dry gel is raised to 480-500 ℃ at the speed of 1.8-2.2 ℃/min, and the temperature is kept for 3.5-4.5h to obtain the nickel-doped ordered mesoporous alumina carrier.
Further, the mass ratio of the polyvinyl ether-polypropylene ether-polyvinyl ether segmented copolymer to the aluminum isopropoxide to the nickel nitrate is 1: 1-4: 0.01-1, preferably 1: 1-4: 0.01-0.25.
The preparation method of the immobilized fructosyltransferase by using nickel-doped ordered mesoporous alumina as a carrier comprises the following steps: dissolving fructosyltransferase in disodium hydrogen phosphate-potassium dihydrogen phosphate buffer solution with pH of 6-8 to prepare enzyme solution with concentration of 10mg/mL, adding ordered mesoporous alumina doped with nickel of 10-90mg into 10mL of water, performing ultrasonic treatment for 15-25 min, adding 0.1-0.9mL of enzyme solution, placing into a shaking table for reaction for 22-26h, discarding supernatant, and washing solid particles with 0.05mol/L citric acid-phosphoric acid buffer solution with pH of 4.5-6.0 for 2-3 times to obtain immobilized fructosyltransferase.
According to the technical scheme, firstly, a nickel-doped ordered mesoporous alumina carrier is prepared, fructosyltransferase is fixed on the carrier through coordination of aluminum and amino acid, and the enzymatic activity of the immobilized fructosyltransferase is improved through activation of nickel.
According to the invention, aluminum is easy to chelate with amino acid in the enzyme to form a complex, the reaction condition is mild, and the structure and activity of the enzyme are not easily influenced; on the other hand, nickel ions are activators of the fructosyl transferase, so that the nickel ions are combined together, and the nickel ions are doped in the ordered mesoporous alumina, so that the activation capability of the nickel ions and the acting force of the coordination bond of the aluminum and the enzyme are utilized, meanwhile, the large specific surface area of the alumina is fully utilized to facilitate the contact of the immobilized enzyme and the substrate, and the high-activity and high-stability fructosyl transferase carrier is synthesized by combining the nickel ions, the aluminum ions and the substrate.
The invention has the following advantages: 1. the acting force between the enzyme and the carrier is a coordination bond, the acting force is strong, and the enzyme is not easy to fall off. 2. The enzyme can maintain high activity due to activation of nickel. 3. The carrier with the mesoporous structure is beneficial to the substrate to quickly reach the reaction site, and realizes the balance between high activity and firm immobilization of the fructosyltransferase.
Detailed Description
A preparation method of nickel-doped ordered mesoporous alumina comprises the following steps:
1) dissolving P123 (polyvinyl ether-polypropylene ether-polyvinyl ether block copolymer) in absolute ethyl alcohol at room temperature to obtain an absolute ethyl alcohol solution of P123;
2) adding aluminum isopropoxide and nickel nitrate into an absolute ethyl alcohol solution of P123, adding concentrated hydrochloric acid, fully stirring and dissolving, and stirring for 5-12h to form sol;
3) the sol is volatilized at the temperature of 55-65 ℃ for self-assembly for 45-50h to obtain dry gel, the temperature of the dry gel is raised to 480-500 ℃ at the speed of 1.8-2.2 ℃/min, and the temperature is kept for 3.5-4.5h to obtain the nickel-doped ordered mesoporous alumina carrier.
Wherein the mass ratio of the polyvinyl ether-polypropylene ether-polyvinyl ether segmented copolymer to the aluminum isopropoxide to the nickel nitrate is 1: 1-4: 0.01-1. The immobilization stability is affected by different nickel contents in the raw materials, particularly when the nickel content is high, the enzyme is easy to fall off, the stability is poor, and the mass ratio is preferably 1: 1-4: 0.01-0.25.
The preparation method of the immobilized fructosyltransferase by using nickel-doped ordered mesoporous alumina as a carrier comprises the following steps: dissolving fructosyltransferase in disodium hydrogen phosphate-potassium dihydrogen phosphate buffer solution with pH of 6-8 to prepare enzyme solution with concentration of 10mg/mL, adding ordered mesoporous alumina doped with nickel of 10-90mg into 10mL of water, performing ultrasonic treatment for 15-25 min, adding 0.1-0.9mL of enzyme solution, placing into a shaking table for reaction for 22-26h, discarding supernatant, and washing solid particles with 0.05mol/L citric acid-phosphoric acid buffer solution with pH of 4.5-6.0 for 2-3 times to obtain immobilized fructosyltransferase.
Example 1
(1) Preparation of nickel-doped ordered mesoporous alumina
At room temperature, 1g P123 (polyvinyl ether-polypropylene ether-polyvinyl ether block copolymer) is dissolved in 20mL of absolute ethyl alcohol, 2.07g of aluminum isopropoxide and 0.25g of nickel nitrate are added into the absolute ethyl alcohol solution of P123, 1.6mL of concentrated hydrochloric acid is added, after full stirring and dissolution, stirring is continued for 5h to form sol, the sol is subjected to solvent volatilization self-assembly at 60 ℃ for 48h to obtain dry gel, the temperature of the dry gel is raised to 500 ℃ at 2 ℃/min, and the temperature is maintained for 4h to obtain the nickel-doped ordered mesoporous alumina.
(2) Preparation method of immobilized fructosyl transferase
The nickel-doped ordered mesoporous alumina is adopted as a carrier to immobilize the fructosyltransferase: preparing 0.1mol/L disodium hydrogen phosphate and 0.1mol/L potassium dihydrogen phosphate solution, and preparing a buffer solution with the pH value of 6.8 by using the two solutions; dissolving fructosyltransferase in the buffer solution to prepare enzyme solution with the concentration of 10mg/mL, weighing 30mg of nickel-doped ordered mesoporous alumina carrier, adding the mixture into 10mL of water, carrying out ultrasonic treatment for 20min, adding 0.5mL of enzyme solution, placing the mixture into a shaking table for reaction for 24h, pouring out supernatant, and washing solid particles for 3 times by using 0.05mol/L citric acid-phosphoric acid buffer solution with the pH value of 5.0 to obtain the immobilized fructosyltransferase.
(3) Enzyme Activity assay
Adding immobilized fructosyltransferase into a triangular flask, adding 15mL of 50% sucrose and 5mL of citric acid-phosphoric acid buffer solution with pH =5.0, reacting for 1h in a 200r/min constant-temperature rotary shaking table at 50 ℃, taking out, putting into a boiling water bath, boiling for 10min, inactivating, cooling to room temperature, centrifuging for 2min at 5000r/min, taking supernatant, detecting the content of fructo-oligosaccharide by using HPLC, and calculating enzyme activity. The activity of the immobilized fructosyltransferase reached 78% of the free enzyme activity.
(4) Stability testing of immobilized fructosyltransferases
Adding immobilized fructosyltransferase into a triangular flask, adding 15mL of 50% sucrose and 5mL of citric acid-phosphate buffer solution with pH =5.0, reacting in a 200r/min constant-temperature rotary shaking table at 50 ℃ for 1h, after the reaction is finished, centrifuging the system at 10000r/min, removing the reaction liquid, continuously adding 15mL of 50% sucrose and 5mL of citric acid-phosphate buffer solution with pH =5.0 into the immobilized fructosyltransferase solid particles, and circulating for 8 times, wherein the activity of the immobilized fructosyltransferase is reduced to 65% of the activity of the free enzyme, the activity is slightly reduced, and the stability of the immobilized fructosyltransferase is good.
Example 2
(1) Preparation of nickel-doped ordered mesoporous alumina
Dissolving 1g P123 (polyvinyl ether-polypropylene ether-polyvinyl ether block copolymer) in 20mL of absolute ethyl alcohol at room temperature, adding 2.07g of aluminum isopropoxide and 0.75g of nickel nitrate into the absolute ethyl alcohol solution of P123, adding 1.6mL of concentrated hydrochloric acid, fully stirring and dissolving, continuing stirring for 5h to form sol, volatilizing the solvent at 60 ℃ for self-assembly for 48h to obtain dry gel, heating the dry gel to 500 ℃ at the temperature of 2 ℃/min, and keeping for 4h to obtain the nickel-doped ordered mesoporous alumina.
(2) Preparation method of immobilized fructosyl transferase
The nickel-doped ordered mesoporous alumina is adopted as a carrier to immobilize the fructosyltransferase: : preparing 0.1mol/L disodium hydrogen phosphate and 0.1mol/L potassium dihydrogen phosphate solution, and preparing a buffer solution with the pH value of 6.8 by using the two solutions; dissolving fructosyltransferase in the buffer solution to prepare enzyme solution with the concentration of 10mg/mL, weighing 30mg of nickel-doped ordered mesoporous alumina carrier, adding the mixture into 10mL of water, carrying out ultrasonic treatment for 20min, adding 0.5mL of enzyme solution, placing the mixture into a shaking table for reaction for 24h, pouring out supernatant, and washing solid particles for 2 times by using 0.05mol/L citric acid-phosphoric acid buffer solution with the pH value of 5.0 to obtain the domestic fructosyltransferase.
(3) Enzyme Activity assay
Adding immobilized fructosyltransferase into a triangular flask, adding 15mL of 50% sucrose and 5mL of citric acid-phosphate buffer solution with pH =5.0, reacting in a 200r/min constant-temperature rotary shaking table at 50 ℃ for 1h, taking out, putting into a boiling water bath, boiling for 10min, inactivating, cooling to room temperature, centrifuging at 5000r/min for 2min, taking supernatant, detecting the content of fructo-oligosaccharide by using HPLC, and calculating enzyme activity. The activity of the immobilized fructosyltransferase reached 42% of the free enzyme activity.
(4) Stability testing of immobilized fructosyltransferases
Adding immobilized fructosyltransferase into a triangular flask, adding 15mL of 50% sucrose and 5mL of citric acid-phosphate buffer solution with pH =5.0, reacting for 1h in a 200r/min constant-temperature rotary shaking table at 50 ℃, removing reaction liquid after the reaction is finished, continuously adding 15mL of 50% sucrose and 5mL of citric acid-phosphate buffer solution with pH =5.0 into immobilized enzyme solid particles, circulating for 8 times, and finding that the activity of the immobilized enzyme is reduced to 18% of the activity of free enzyme and the stability of the activity of the enzyme is poor. This is mainly because the nickel content of the raw material in this example is high, and the enzyme easily falls off, resulting in poor stability.
Example 3
(1) Preparation of nickel-doped ordered mesoporous alumina
At room temperature, 1g P123 (polyvinyl ether-polypropylene ether-polyvinyl ether block copolymer) is dissolved in 20mL of absolute ethyl alcohol, 2.07g of aluminum isopropoxide and 0.25g of nickel nitrate are added into the absolute ethyl alcohol solution of P123, 1.6mL of concentrated hydrochloric acid is added, after full stirring and dissolution, stirring is continued for 5h to form sol, the sol is volatilized at 60 ℃ for self-assembly for 48h to obtain dry gel, the temperature of the dry gel is raised to 500 ℃ at the rate of 2 ℃/min, and the temperature is maintained for 4h to obtain the nickel-doped ordered mesoporous alumina.
(2) Preparation method of immobilized fructosyl transferase
The method for immobilizing the fructosyltransferase by using the nickel-doped ordered mesoporous alumina carrier comprises the following steps: preparing 0.1mol/L disodium hydrogen phosphate and 0.1mol/L potassium dihydrogen phosphate solution, and preparing a buffer solution with the pH value of 6.8 by using the two solutions; dissolving fructosyltransferase in the buffer solution to prepare 10mg/mL enzyme solution, weighing 30mg carrier, adding 10mL water, performing ultrasonic treatment for 20min, adding 10mL enzyme solution, putting into a shaking table for reaction for 24h, pouring out supernatant, and washing solid particles with 0.05mol/L citric acid-phosphoric acid buffer solution with pH of 5.0 for 3 times to obtain the immobilized fructosyltransferase.
(3) Enzyme Activity assay
Adding immobilized fructosyltransferase into a triangular flask, adding 15mL of 50% sucrose and 5mL of citric acid-phosphate buffer solution with pH =5.0, reacting in a 200r/min constant-temperature rotary shaking table at 50 ℃ for 1h, taking out, putting into a boiling water bath, boiling for 10min, inactivating, cooling to room temperature, centrifuging at 5000r/min for 2min, taking supernatant, detecting the content of fructo-oligosaccharide by using HPLC, and calculating enzyme activity. The activity of the immobilized fructosyltransferase reached 65% of the activity of the free enzyme.
(4) Stability testing of immobilized fructosyltransferases
Adding immobilized fructosyltransferase into a triangular flask, adding 15mL of 50% sucrose and 5mL of citric acid-phosphate buffer solution with pH =5.0, reacting in a 200r/min constant-temperature rotary shaking table at 50 ℃ for 1h, after the reaction is finished, centrifuging the system at 10000r/min, removing the reaction liquid, continuously adding 15mL of 50% sucrose and 5mL of citric acid-phosphate buffer solution with pH =5.0 into immobilized enzyme solid particles, and circulating for 8 times, wherein the activity of the immobilized enzyme is reduced to 56% of the activity of the free enzyme, the activity of the enzyme is slightly reduced, and the stability of the immobilized enzyme is good.
Example 4
(1) Preparation of nickel-doped ordered mesoporous alumina
At room temperature, 1g P123 (polyvinyl ether-polypropylene ether-polyvinyl ether block copolymer) is dissolved in 20mL of absolute ethyl alcohol, 2.07g of aluminum isopropoxide and 0.25g of nickel nitrate are added into the absolute ethyl alcohol solution of P123, 1.6mL of concentrated hydrochloric acid is added, after full stirring and dissolution, stirring is continued for 12h to form sol, the sol volatilizes from self-assembly for 50h at 55 ℃ to obtain dry gel, the temperature of the dry gel is raised to 480 ℃ at 1.8 ℃/min, and the temperature is maintained for 4.5h to obtain the nickel-doped ordered mesoporous alumina.
(2) Preparation method of immobilized fructosyl transferase
The nickel-doped ordered mesoporous alumina is adopted as a carrier to immobilize the fructosyltransferase: preparing 0.1mol/L disodium hydrogen phosphate and 0.1mol/L potassium dihydrogen phosphate solution, and preparing a buffer solution with the pH value of 6 by using the two solutions; dissolving fructosyltransferase in the buffer solution to prepare enzyme solution with the concentration of 10mg/mL, weighing 10mg of nickel-doped ordered mesoporous alumina carrier, adding the mixture into 10mL of water, carrying out ultrasonic treatment for 15min, adding 0.1mL of enzyme solution, putting the mixture into a shaking table to react for 22h, pouring out supernatant, and washing solid particles for 2 times by using 0.05mol/L citric acid-phosphoric acid buffer solution with the pH value of 4.5 to obtain the immobilized fructosyltransferase.
(3) Enzyme Activity assay
Enzyme Activity test methods As in example 1, the activity of the immobilized fructosyltransferase reached 75% of the activity of the free enzyme.
(4) Stability testing of immobilized fructosyltransferases
The stability test method is the same as that of the example 1, and the activity of the immobilized enzyme is reduced to 63 percent of that of the free enzyme by 8 times of circulation, the activity is slightly reduced, and the stability of the immobilized enzyme is good.
Example 5
(1) Preparation of nickel-doped ordered mesoporous alumina
At room temperature, 1g P123 (polyvinyl ether-polypropylene ether-polyvinyl ether block copolymer) is dissolved in 20mL of absolute ethyl alcohol, 2.07g of aluminum isopropoxide and 0.25g of nickel nitrate are added into the absolute ethyl alcohol solution of P123, 1.6mL of concentrated hydrochloric acid is added, after full stirring and dissolution, stirring is continued for 10h to form sol, the sol volatilizes from the solvent at 65 ℃ for self-assembly for 50h to obtain dry gel, the temperature of the dry gel is raised to 500 ℃ at 2.2 ℃/min, and the temperature is maintained for 3.5h to obtain the nickel-doped ordered mesoporous alumina.
(2) Preparation method of immobilized fructosyl transferase
The nickel-doped ordered mesoporous alumina is adopted as a carrier to immobilize the fructosyltransferase: preparing 0.1mol/L disodium hydrogen phosphate and 0.1mol/L potassium dihydrogen phosphate solution, and preparing a buffer solution with the pH value of 6.8 by using the two solutions; dissolving fructosyltransferase in the buffer solution to prepare enzyme solution with the concentration of 10mg/mL, weighing 90mg of nickel-doped ordered mesoporous alumina carrier, adding the nickel-doped ordered mesoporous alumina carrier into 10mL of water, carrying out ultrasonic treatment for 25min, adding 0.9mL of enzyme solution, putting the mixture into a shaking table for reaction for 24h, pouring out supernatant, and washing solid particles for 3 times by using 0.05mol/L citric acid-phosphoric acid buffer solution with the pH value of 5.0 to obtain the immobilized fructosyltransferase.
(3) Enzyme Activity assay
Enzyme Activity assay As in example 1, the activity of the immobilized fructosyltransferase reached 73% of the free enzyme activity.
(4) Stability testing of immobilized fructosyltransferases
The stability test method is the same as that of the example 1, and the activity of the immobilized enzyme is reduced to 60 percent of that of the free enzyme by 8 times of circulation, the activity is slightly reduced, and the stability of the immobilized enzyme is good.
Claims (2)
1. A preparation method of immobilized fructosyltransferase by using nickel-doped ordered mesoporous alumina as a carrier is characterized by comprising the following steps: dissolving fructosyltransferase in disodium hydrogen phosphate-potassium dihydrogen phosphate buffer solution with pH of 6-8 to prepare enzyme solution with concentration of 10mg/mL, adding nickel-doped ordered mesoporous alumina into 10mL of water per 10-90mg, performing ultrasonic treatment for 15-25 min, adding 0.1-0.9mL of enzyme solution, placing into a shaking table to react for 22-26h, removing supernatant, and washing solid particles with citric acid-phosphoric acid buffer solution for 2-3 times to obtain immobilized fructosyltransferase;
the preparation method of the nickel-doped ordered mesoporous alumina comprises the following steps:
1) dissolving a polyvinyl ether-polypropylene ether-polyvinyl ether segmented copolymer in absolute ethyl alcohol at room temperature to obtain an absolute ethyl alcohol solution of P123;
2) adding aluminum isopropoxide and nickel nitrate into an absolute ethyl alcohol solution of P123, adding concentrated hydrochloric acid, and stirring for 5-12h to form sol; the mass ratio of the polyvinyl ether-polypropylene ether-polyvinyl ether segmented copolymer to the aluminum isopropoxide to the nickel nitrate is 1: 1-4: 0.01-0.25;
3) the sol is volatilized at the temperature of 55-65 ℃ for self-assembly for 45-50h to obtain dry gel, the temperature of the dry gel is raised to 480-500 ℃ at the speed of 1.8-2.2 ℃/min, and the temperature is kept for 3.5-4.5h to obtain the nickel-doped ordered mesoporous alumina carrier.
2. The method for preparing immobilized fructosyltransferase by using nickel-doped ordered mesoporous alumina as a carrier according to claim 1, wherein the immobilized fructosyltransferase is prepared by the following steps: the pH value of the citric acid-phosphoric acid buffer solution is 4.5-6.0, and the molar concentration is 0.05 mol/L.
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