CN109321559B - Magnetic Fe3O4Method for immobilizing fructosyltransferase by taking polysaccharide microspheres as carrier - Google Patents
Magnetic Fe3O4Method for immobilizing fructosyltransferase by taking polysaccharide microspheres as carrier Download PDFInfo
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Abstract
The invention discloses a magnetic Fe3O4A method for immobilizing fructosyltransferase by taking polysaccharide microspheres as carriers, belonging to the technical field of biological engineering. Two functional polysaccharides inulin and fructan are used as adsorbents to synthesize magnetic Fe by chemical coprecipitation method3O4-polysaccharide microspheres. Magnetic Fe3O4The polysaccharide microspheres can effectively improve the stability of the carrier and the enzyme loading capacity, have the advantage of strong magnetic responsiveness, and are applied to immobilization of fructosyltransferase. After the immobilized enzyme is repeatedly reacted for 6 times, 81 percent of enzyme activity is remained.
Description
Technical Field
The invention relates to a magnetic Fe3O4A method for immobilizing fructosyltransferase by taking polysaccharide microspheres as carriers, belonging to the technical field of biological engineering.
Background
Fructooligosaccharide (FOS), also known as Fructooligosaccharide, has a molecular formula: G-F-Fn, n-1-3 (G is glucose, F is fructose), is a mixture of kestose, nystose and nystose, wherein 1-3 fructose groups are combined with the fructose group in sucrose through beta-2-1 glycosidic bonds.
Because fructo-oligosaccharide has the beneficial functions of effectively proliferating bifidobacteria, promoting gastrointestinal function, reducing calorie, preventing decayed teeth and the like, and is more favored by people, the industrial production of fructo-oligosaccharide has important significance for the health of people and the development of national economy. In general, fructo-oligosaccharide is obtained by transglycosylation of Fructosyltransferase (EC 2.4.1.9, FTS) with sucrose as a substrate in industry, and the reaction mechanism is as follows:
G-F (sucrose) → G (glucose) + G-F (sucrose) + G-F-F (kestose) + G-F-F (nystose) + G-F-F-F-F (nystose)
At present, the domestic technology for industrially producing fructo-oligosaccharide is mainly a liquid submerged fermentation method, which has long fermentation period and low utilization rate of fructosyltransferase. The immobilized enzyme method is a technique widely used in industrial enzymatic reactions. The immobilized enzyme method has many advantages, such as that the enzyme can be repeatedly utilized, the pollution by metabolites can be avoided, and the production cost is reduced, which becomes a research hotspot.
At present, the immobilization mainly comprises an adsorption method, an embedding method, covalent bonding and the like, the adopted carriers comprise macroporous resin, sodium alginate, novel organic materials and the like, and the use of a cross-linking agent and the covalent bonding method can cause partial inactivation of enzyme and change of an enzyme catalysis microenvironment and even cause change of reaction kinetics. Simple adsorption methods are not suitable for macromolecular substances, not for multienzyme reactions. The embedding method is not easy to be applied to industrial large-scale production, and can cause the phenomena of difficult separation of products and substrates and difficult control of enzyme reaction.
In 2005, Iraj Ghazi et al covalently immobilized fructosyltransferase on polymethacrylate polymer activated by epoxy group, the immobilized enzyme activity reached 25.9U/g carrier, and the concentration of fructo-oligosaccharide reached 61.5% at most. However, the method requires complex pretreatment of the carrier, low activity of the immobilized enzyme and limited recovery rate of the enzyme activity. In 2014, Ganaie et al used chitosan beads and alginate beads to immobilize fructosyltransferase, wherein the immobilized enzymes are 25U/g carrier and 43U/g carrier respectively, and the obtained fructo-oligosaccharide concentrations are 42.79% and 67.75% respectively. The method adopts a stable-structure matrix to embed the mycelium, the recycling efficiency of the immobilized enzyme taking chitosan beads as the carrier is low, and the thermal stability taking alginate beads as the carrier is still to be improved. Therefore, some methods are used for helping to improve the defects, and the improved immobilized enzyme method is used for producing beneficial substances with wide functions, such as fructo-oligosaccharide, and has important significance for further application of the immobilized enzyme.
Disclosure of Invention
One of the objectives of the present invention is to provide a magnetic Fe3O4The method for immobilizing fructosyltransferase by taking polysaccharide microspheres as carriers comprises the steps of firstly preparing the fructosyltransferase and magnetic Fe3O4Polysaccharide microspheres, with magnetic Fe3O4Polysaccharide microsphere immobilized fructosyltransferase.
In one embodiment of the invention, the polysaccharide comprises inulin or levan.
In one embodiment of the present invention, the specific steps for preparing the fructosyl transferase comprise:
(1) fermenting Aspergillus niger for 20-48h, and suction filtering to collect wet thallus;
(2) washing with 0.1-0.5mol/L, pH 5.0.0-7.0 citric acid-phosphate buffer solution, and making the obtained thallus into thallus suspension with the above buffer solution;
(3) crushing thallus, freezing and centrifuging, and taking supernatant;
(4) concentrating the supernatant to obtain crude enzyme solution.
In one embodiment of the present invention, the magnetic Fe3O4The preparation method of the polysaccharide microspheres comprises the following steps: preparing 90-100mL of 1-50mg/mL polysaccharide solution, and adding FeCl with the molar ratio of 1:22And FeCl3Vigorously stirring at room temperature, adjusting pH to 9-11, reacting for 0.5-2 hr, heating the solution at 40-80 deg.C for 20-40min, filtering, washing, and freeze vacuum drying to obtain magnetic Fe3O4-polysaccharide microspheres.
In one embodiment of the present invention, the magnetic Fe3O4The mass ratio of polysaccharide to iron ions in the polysaccharide microspheres is 1:5-1: 10. In one embodiment of the present invention, the magnetic Fe3O4The specific steps of immobilizing fructosyltransferase by polysaccharide microspheres comprise:
(1) taking magnetic Fe3O4Adding the polysaccharide microspheres into a buffer solution, fully soaking and swelling, and carrying out magnetic separation after 4-5 hours;
(2) adding fructosyltransferase enzyme solution for immobilization;
(3) standing at 0-5 deg.C for 10-14 h;
(4) separating by a magnetic field, and repeatedly washing the precipitate by using a buffer solution until the enzyme activity cannot be detected in the washing solution, thereby obtaining the immobilized enzyme.
In one embodiment of the present invention, the magnetic Fe3O4The addition amount of polysaccharide microspheres is 0.5 g; the addition amount of fructosyltransferase is 100-400U/g magnetic Fe3O4-polysaccharide microspheres.
Another object of the present invention is to provide a magnetic Fe3O4Polysaccharide microspheres, which are synthesized in one step by a chemical coprecipitation method, using polysaccharide inulin or levan as an adsorbent.
The fructosyl transferase is immobilized by taking polysaccharide such as inulin or fructan as a carrier and adsorbing or embedding a magnetic functional polysaccharide material formed by ferroferric oxide. Resulting magnetic Fe3O4The polysaccharide microspheres can effectively improve the stability of the carrier and the enzyme loading capacity, have the advantage of strong magnetic responsiveness, and are applied to immobilization of fructosyltransferase. After the immobilized enzyme is applied for repeated reaction for 6 times, 81 percent of enzyme activity remains, and the immobilized enzyme shows better adaptability under different temperature and pH conditions, and is suitable for industrial production.
Detailed Description
(ii) measurement of fructosyl transferase enzymatic Activity
Determination of free fructosyltransferase: preparing 10% (w/v) sucrose solution with 0.1mol/L, pH 5.0.0 citric acid-phosphate buffer solution, placing 50mL substrate solution in a constant temperature jacket enzyme reactor, adding 0.05mL fructosyltransferase enzyme solution, stirring at 50 deg.C for reaction for 60min, and boiling in boiling water for 10min to terminate the reaction.
Determination of immobilized fructosyl transferase: preparing 10% (w/v) sucrose solution by using 0.1mol/L, pH 5.0.0 citric acid-phosphoric acid buffer solution, putting 50mL substrate solution into a constant-temperature jacket enzyme reactor, adding 0.1g immobilized enzyme, stirring at a constant temperature of 50 ℃ for reaction for 60min, and quickly separating the product from the immobilized enzyme by using a magnetic field after the reaction is finished.
Definition of enzyme activity: under the above conditions, the amount of enzyme required to produce 1. mu. mol of kestose per minute.
(II) determination of sugar Components and content by HPLC method
An Agilent1100 series, equipped with Shodex RI-101 refractive index detector and M70 data processor; chromatography column, Shodex Asahipak (model); mobile phase, acetonitrile: water 75:25, flow rate 1 mL/min; the temperature is 30 ℃; the amount of the sample was 10. mu.L.
(III) recovery rate of immobilized enzyme activity (%) < activity of immobilized enzyme/total activity of added enzyme >. times.100%
The first embodiment is as follows: with magnetic Fe3O4Method 1 for immobilizing fructosyltransferase by taking polysaccharide microspheres as carriers
(1) Preparation of fructosyltransferase: fermenting Aspergillus niger in a 30L medium-sized fermentation tank for 48h, suction-filtering to collect wet thallus, washing with 0.1mol/L, pH 5.0.0 citric acid-phosphoric acid buffer solution, repeating for three times, making the thallus into thallus suspension with the buffer solution, crushing the thallus by a colloid mill, freezing, centrifuging, and collecting supernatant, i.e. crude enzyme solution of fructosyltransferase; after the crude enzyme solution is concentrated by ultrafiltration, the enzyme activity of the crude enzyme solution is measured to be 102U/mL.
(2) Magnetic Fe3O4-preparation of inulin microspheres: 100mL of 20mg/mL inulin was prepared, and 0.1mol FeCl was added2And 0.2mol FeCl3Vigorous stirring at room temperature followed by dropwise addition of NH4OH solution until pH rises to 10, reacting for 1h, heating the solution at 80 deg.C for 30min, filtering, repeatedly washing with ethanol and deionized water until the product is neutral, and freeze-drying under vacuum to obtain magnetic Fe3O4Inulin microspheres.
(3) Magnetic Fe3O4Inulin microsphere immobilized fructosyltransferase: 0.5g of magnetic Fe was taken3O4Adding the inulin microspheres into 0.1mol/L, pH 5.0.0 citric acid-phosphoric acid buffer solution for full soaking and swelling, carrying out magnetic separation after 5h, adding 1mL of fructosyltransferase with the total enzyme activity of 102U, and oscillating and fixing in a constant temperature shaking table at the rotating speed of 100-200rpm under the conditions of pH 5.0-7.0 and 20-25 ℃. Taking out after 4-8h reaction, putting into a refrigerator at 4 ℃ for standing overnight, separating by a magnetic field after immobilization is completed,and (3) repeatedly washing the precipitate with a buffer solution until the enzyme activity cannot be detected in the washing solution, thus obtaining the immobilized enzyme. The enzyme activity of the immobilized enzyme is measured to be 126.65U/g of carrier, and the recovery rate of the enzyme activity is 62.08%.
Example two: with magnetic Fe3O4Method 2 for immobilizing fructosyltransferase by taking polysaccharide microspheres as carriers
(1) Preparation of fructosyltransferase: fermenting Aspergillus niger in a 30L medium-sized fermentation tank for 24h, performing suction filtration to collect wet thalli, cleaning with 0.1mol/L, pH 5.0.0 citric acid-phosphoric acid buffer solution for three times, crushing the obtained thalli with the buffer solution to obtain thalli suspension, performing freeze centrifugation to obtain supernatant, and performing ultrafiltration concentration to obtain crude enzyme solution with fructosyltransferase activity of 188U/mL.
(2) Magnetic Fe3O4-preparation of fructan microspheres: preparing 100mL of 20mg/mL fructan solution, adding 0.1mol FeCl2And 0.2mol FeCl3Vigorous stirring at room temperature followed by dropwise addition of NH4OH solution until pH rises to 10, reacting for 1h, heating the solution at 80 deg.C for 30min, filtering, repeatedly washing with ethanol and deionized water until the product is neutral, and freeze-drying under vacuum to obtain magnetic Fe3O4-fructan microspheres.
(3) Magnetic Fe3O4-fructosan microsphere immobilized fructosyltransferase: 0.5g of magnetic Fe was taken3O4The fructan microspheres are added into 0.1mol/L, pH 5.0.0 citric acid-phosphoric acid buffer solution for full soaking and swelling, magnetic separation is carried out after 5h, 1mL of fructosyltransferase with 188U total enzyme activity is added, and the mixture is oscillated and fixed in a constant temperature shaking table at the rotating speed of 100-200rpm under the conditions of pH 5.0-7.0 and 20-25 ℃. Taking out after 4-8h reaction, putting the mixture into a refrigerator at 4 ℃ for standing overnight, separating the mixture through a magnetic field after immobilization is finished, and repeatedly washing the precipitate with a buffer solution until the enzyme activity cannot be detected in the washing solution, thus obtaining the immobilized enzyme. The enzyme activity of the immobilized enzyme is measured to be 297.36U/g carrier, and the recovery rate of the enzyme activity is 79.08 percent.
Example 3: magnetic Fe3O4Operating stability of the fructan microsphere immobilized enzyme:
taking 50mL of substrate solution into a constant-temperature jacket enzyme reactor, adding 0.1g of immobilized enzyme, stirring at a constant temperature of 50 ℃ for reaction for 60min, quickly separating a product from the immobilized enzyme by using a magnetic field after the reaction is finished, recovering the immobilized enzyme, washing with a buffer solution for three times, carrying out the next batch of reaction, repeating the operation for 6 times, and comparing the change of the enzyme activity. The 6 times of enzyme activities are 297.36U/g vector, 281.15U/g vector, 269.54U/g vector, 256.23U/g vector, 247.74U/g vector and 240.89U/g vector respectively.
Example 4 magnetic Fe3O4Tolerance of immobilized enzyme to low/high temperature by taking inulin microspheres as carrier
(1) Enzyme activity assay of fructosyltransferase at 30 ℃
Preparing 10% (w/v) sucrose solution with 0.1mol/L, pH 5.0.0 citric acid-phosphoric acid buffer solution, placing 50mL substrate solution in a constant temperature jacket enzyme reactor, adding 0.1g immobilized enzyme, stirring at constant temperature of 30 deg.C for reaction for 60min, boiling in boiling water for 10min to terminate the reaction. And (4) measuring enzyme activity. The enzyme activity is 253.27U/g vector.
(2) Enzyme activity assay of immobilized enzyme at 70 DEG C
Preparing 10% (w/v) sucrose solution by using 0.1mol/L, pH 5.0.0 citric acid-phosphoric acid buffer solution, putting 50mL substrate solution into a constant-temperature jacket enzyme reactor, adding 0.1g immobilized enzyme, stirring at a constant temperature of 70 ℃ for reaction for 60min, and quickly separating the product from the immobilized enzyme by using a magnetic field after the reaction is finished. And (4) measuring enzyme activity. The enzyme activity is 258.65U/g vector.
Example 5: with magnetic Fe3O4Tolerance of immobilized enzyme to low/high pH with inulin microspheres as carrier
(1) Enzyme activity determination of fructosyl transferase under pH 3
Preparing 10% (w/v) sucrose solution with 0.1mol/L, pH 3 citric acid-phosphoric acid buffer solution, placing 50mL substrate solution in a constant temperature jacket enzyme reactor, adding 0.1g immobilized enzyme, stirring at 50 deg.C for reaction for 60min, boiling in boiling water for 10min to terminate the reaction. And (4) measuring enzyme activity. The enzyme activity is 243.24U/g vector.
(2) Enzyme activity determination of immobilized enzyme under pH 8 condition
Preparing 10% (w/v) sucrose solution by using 0.1mol/L, pH 8 citric acid-phosphoric acid buffer solution, putting 50mL substrate solution into a constant-temperature jacket enzyme reactor, adding 0.1g immobilized enzyme, stirring at a constant temperature of 50 ℃ for reaction for 60min, and quickly separating the product from the immobilized enzyme by using a magnetic field after the reaction is finished. And (4) measuring enzyme activity. The enzyme activity is 241.09U/g carrier.
Comparative example 1: magnetic Fe without addition of polysaccharide such as fructan or inulin3O4Microsphere-carrier immobilized fructosyltransferase
(1) Preparation of fructosyltransferase: fermenting Aspergillus niger in a 30L medium-sized fermentation tank for 48h, suction-filtering to collect wet thallus, washing with 0.1mol/L, pH 5.0.0 citric acid-phosphoric acid buffer solution, repeating for three times, crushing the thallus with the above buffer solution to obtain thallus suspension, freeze-centrifuging to obtain supernatant, and ultrafiltering and concentrating to obtain crude enzyme solution.
(2) Magnetic Fe3O4Preparing microspheres: a100 mL portion of distilled water was prepared and 0.1mol FeCl was added2And 0.2mol FeCl3Vigorous stirring at room temperature followed by dropwise addition of NH4OH solution until pH rises to 10, reacting for 1h, heating the solution at 80 deg.C for 30min, filtering, repeatedly washing with ethanol and deionized water until the product is neutral, and freeze-drying under vacuum to obtain magnetic Fe3O4And (3) microspheres.
(3) Magnetic Fe3O4Microsphere immobilized fructosyltransferase: 0.5g of magnetic Fe was taken3O4Adding the microspheres into 0.1mol/L, pH 5.0.0 citric acid-phosphoric acid buffer solution, fully soaking and swelling, performing magnetic separation after 5h, adding 1mL of fructosyltransferase with the total enzyme activity of 102U, and oscillating and fixing in a constant temperature shaking table at the rotating speed of 100-200rpm under the conditions of pH 5.0-7.0 and 20-25 ℃. Taking out after 4-8h reaction, putting the mixture into a refrigerator at 4 ℃ for standing overnight, separating the mixture through a magnetic field after immobilization is finished, and repeatedly washing the precipitate with a buffer solution until the enzyme activity cannot be detected in the washing solution, thus obtaining the immobilized enzyme. The enzyme activity of the immobilized enzyme is measured to be 45.77U/g carrier, and the recovery rate of the enzyme activity is 24.37%.
Comparative example 2: changing only to magnetic Fe3O4Inulin microsphere as carrierThe amount of the immobilized fructosyltransferase to be added was otherwise the same as in example 1
(1) Preparation of fructosyltransferase: fermenting Aspergillus niger in a 30L medium-sized fermentation tank for 48h, performing suction filtration to collect wet thalli, cleaning with 0.1mol/L, pH 5.0.0 citric acid-phosphoric acid buffer solution for three times, crushing the obtained thalli with the buffer solution to obtain thalli suspension, performing freeze centrifugation to obtain supernatant, performing ultrafiltration concentration to obtain crude enzyme solution, and measuring the enzyme activity of the initial fructosyltransferase to be 102U/mL.
(2) Magnetic Fe3O4-preparation of inulin microspheres: 100mL of 20mg/mL inulin was prepared, and 0.1mol FeCl was added2And 0.2mol FeCl3Vigorous stirring at room temperature followed by dropwise addition of NH4OH solution until pH rises to 10, reacting for 1h, heating the solution at 80 deg.C for 30min, filtering, repeatedly washing with ethanol and deionized water until the product is neutral, and freeze-drying under vacuum to obtain magnetic Fe3O4Inulin microspheres.
(3) Magnetic Fe3O4Inulin microsphere immobilized fructosyltransferase: 0.5g of magnetic Fe was taken3O4Adding the inulin microspheres into 0.1mol/L, pH 5.0.0 citric acid-phosphoric acid buffer solution for full soaking and swelling, carrying out magnetic separation after 5h, adding fructosyltransferase with total enzyme activity of 51U, 102U, 153U, 204U, 255U and 306U respectively, and oscillating and fixing in a constant temperature shaking table at the rotation speed of 100-200rpm under the conditions of pH 5.0-7.0 and 20-25 ℃. Taking out after 4-8h reaction, putting the mixture into a refrigerator at 4 ℃ for standing overnight, separating the mixture through a magnetic field after immobilization is finished, and repeatedly washing the precipitate with a buffer solution until the enzyme activity cannot be detected in the washing solution, thus obtaining the immobilized enzyme. The enzyme activity of the immobilized enzyme is measured to be 56.97U/g carrier, 126.65U/g carrier, 210.04U/g carrier, 306.32U/g carrier, 354.28U/g carrier and 413.42U/g carrier, and the recovery rate of the enzyme activity is 55.85%, 62.08%, 68.64%, 75.08%, 69.48% and 67.5%.
Comparative example 3: changing only to magnetic Fe3O4Addition of immobilized fructosyltransferase on Fructosan microspheres as carrier, the other conditions being identical to those of example 2
(1) Preparation of fructosyltransferase: fermenting Aspergillus niger in a 30L medium-sized fermentation tank for 24h, performing suction filtration to collect wet thalli, cleaning with 0.1mol/L, pH 5.0.0 citric acid-phosphoric acid buffer solution for three times, crushing the obtained thalli with the buffer solution to obtain thalli suspension, performing freeze centrifugation to obtain supernatant, and performing ultrafiltration concentration to obtain crude enzyme solution with fructosyltransferase activity of 188U/mL.
(2) Magnetic Fe3O4-preparation of fructan microspheres: 100mL of 20mg/mL inulin was prepared, and 0.1mol FeCl was added2And 0.2mol FeCl3Vigorous stirring at room temperature followed by dropwise addition of NH4OH solution until pH rises to 10, reacting for 1h, heating the solution at 80 deg.C for 30min, filtering, repeatedly washing with ethanol and deionized water until the product is neutral, and freeze-drying under vacuum to obtain magnetic Fe3O4-fructan microspheres.
(3) Magnetic Fe3O4-fructosan microsphere immobilized fructosyltransferase: 0.5g of magnetic Fe was taken3O4The fructan microspheres are added into 0.1mol/L, pH 5.0.0 citric acid-phosphate buffer solution for full soaking and swelling, magnetic separation is carried out after 5h, 1mL of fructosyltransferase with total enzyme activity of 51U, 102U, 153U, 204U, 255U and 306U is added, and the mixture is shaken and fixed in a constant temperature shaking table at the rotating speed of 100-200rpm under the conditions of pH 5.0-7.0 and 20-25 ℃. Taking out after 4-8h reaction, putting the mixture into a refrigerator at 4 ℃ for standing overnight, separating the mixture through a magnetic field after immobilization is finished, and repeatedly washing the precipitate with a buffer solution until the enzyme activity cannot be detected in the washing solution, thus obtaining the immobilized enzyme. The enzyme activity of the immobilized enzyme is measured to be 55.11U/g carrier, 123.25U/g carrier, 210.23U/g carrier, 310.54U/g carrier, 350.75U/g carrier and 410.32U/g carrier, and the recovery rate of the enzyme activity is 54.03%, 60.42%, 68.7%, 76.11%, 68.77% and 67.05%.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (6)
1. Magnetic Fe3O4-a method for immobilizing fructosyltransferase using polysaccharide microspheres as a carrier, characterized in that it comprises the following steps:
(1) preparation of fructosyltransferase: fermenting Aspergillus niger for 20-48h, and suction filtering to collect wet thallus; washing with 0.1-0.5mol/L, pH 5.0.0-7.0 citric acid-phosphate buffer solution, and making the obtained thallus into thallus suspension with the above buffer solution; crushing thallus, freezing and centrifuging, and taking supernatant; concentrating the supernatant to obtain crude enzyme solution;
(2) magnetic Fe3O4-preparation of polysaccharide microspheres: preparing 90-100mL of 1-50mg/mL polysaccharide solution, and adding FeCl with the molar ratio of 1:22And FeCl3Vigorously stirring at room temperature, adjusting pH to 9-11, reacting for 0.5-2 hr, heating the solution at 40-80 deg.C for 20-40min, filtering, washing, and freeze vacuum drying to obtain magnetic Fe3O4-polysaccharide microspheres; the polysaccharide is inulin or levan;
(3) magnetic Fe3O4Polysaccharide microsphere immobilized fructosyltransferase: taking magnetic Fe3O4Adding the polysaccharide microspheres into citric acid-phosphoric acid buffer solution with the pH value of 5.0-7.0, fully soaking and swelling, and carrying out magnetic separation after 4-5 h; adding fructosyltransferase enzyme solution for immobilization; standing at 0-5 deg.C for 10-14 h; separating by a magnetic field, and repeatedly washing the precipitate by using a buffer solution until the enzyme activity cannot be detected in the washing solution, thereby obtaining the immobilized enzyme.
2. The method of claim 1, the magnetic Fe3O4The mass ratio of polysaccharide to iron ions in the polysaccharide microspheres is 1:5-1: 10.
3. Method according to claim 1 or 2, characterized in that magnetic Fe3O4The addition amount of polysaccharide microspheres is 0.5 g; the addition amount of fructosyltransferase is 100-400U/g magnetic Fe3O4-polysaccharide microspheres.
4. A method according to claim 3, characterized in that the fixed conditions are: oscillating in a constant temperature shaker at a rotation speed of 100-200rpm under the conditions of pH 5.0-7.0 and 20-25 ℃.
5. Magnetic Fe3O4-polysaccharide microspheres, characterized in that said magnetic Fe3O4Polysaccharide microspheres are prepared as follows: preparing 90-100mL of 1-50mg/mL polysaccharide solution, wherein the polysaccharide is inulin or levan; FeCl is added into the polysaccharide solution in a molar ratio of 1:22And FeCl3Vigorously stirring at room temperature, adjusting pH to 9-11, reacting for 0.5-2 hr, heating the solution at 40-80 deg.C for 20-40min, filtering, washing, and freeze vacuum drying to obtain magnetic Fe3O4-polysaccharide microspheres.
6. Magnetic Fe as claimed in claim 53O4Use of polysaccharide microspheres for immobilizing fructosyltransferase.
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| CN101560511B (en) * | 2009-05-19 | 2011-01-05 | 江南大学 | Method of immobilization of fructosyl transferase by macroporous anion resin |
| CN104774889B (en) * | 2015-03-25 | 2018-08-17 | 郑州轻工业学院 | A method of preparing cane sugar-6-acetic ester using transfructosylase |
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