DEAE-cellulose immobilized D-psicose-3-epimerase as well as preparation method and application thereof

Abstract

The invention relates to the technical field of immobilized enzymes, and provides DEAE (diethylaminoethanol)-cellulose immobilized D-psicose-3-epimerase as well as a preparation method and application thereof. The preparation method comprises the following steps: soaking DEAE-cellulose in water, then carrying out acid-alkali treatment, and then soaking the DEAE-cellulose in a phosphate buffer solution until the DEAE-cellulose is balanced to obtain activated DEAE-cellulose; the D-psicose-3-epimerase solution and a cross-linking agent are mixed for a cross-linking reaction, and a cross-linked enzyme solution is obtained; and mixing the cross-linked enzyme solution with the activated DEAE-cellulose, and carrying out immobilization, so as to obtain the DEAE-cellulose immobilized D-psicose-3-epimerase. The DEAE-cellulose immobilized D-psicose-3-epimerase prepared by the preparation method disclosed by the invention is good in stability and reusability, high in enzymatic activity, relatively high in conversion rate when catalyzing fructose isomerization to prepare psicose, and suitable for continuously converting fructose to generate psicose.

Description

Technical Field

[0001] The invention relates to the technical field of immobilized enzymes, and in particular to a DEAE-cellulose immobilized D-psicose-3-epimerase and a preparation method and application thereof. Background Art

[0002] Allulose is a naturally occurring sugar that has attracted attention for its low calorie and sucrose-like sweetness. It has multiple health and medicinal functions, such as hypoglycemic, anti-oxidant, and neuroprotective. Allulose has broad application prospects in the food industry and can be used in non-alcoholic beverages, chewing gum, candy, frosting, frozen dairy desserts, salad dressings, soft and hard candies, and other products. In recent years, with the rise of healthy diets and sugar-free foods, the market size of allulose has grown year by year. At present, the industrial production method of allulose is mainly based on the whole-cell enzyme catalysis method, that is, the mutual conversion between fructose and allulose is achieved by sugar isomerase, and the commonly used sugar isomerase is D-psicose-3-epimerase. However, when the whole-cell enzyme catalysis method is used for production, the entire reaction process needs to be carried out under high temperature and high osmotic pressure conditions, which is a great challenge to the activity of the enzyme and the stability of its expression host.

[0003] Immobilized enzyme is a method of combining or embedding enzymes on a carrier by physical or chemical methods to form an insoluble enzyme preparation. This technology can improve the stability and reusability of the enzyme, simplify the product purification process, and improve the efficiency of enzyme use. Common methods for preparing immobilized enzymes mainly include adsorption, embedding, covalent bonding, and cross-linking. However, although the current method can combine free enzymes with carriers, the activity of the obtained immobilized enzymes is poor, and the conversion rate when catalyzing fructose isomerization to prepare psicose is low. Summary of the invention

[0004] In view of this, the present invention provides a DEAE-cellulose immobilized D-psicose-3-epimerase and a preparation method and application thereof. The present invention uses DEAE-cellulose as a carrier to immobilize D-psicose-3-epimerase, and the obtained immobilized enzyme has good stability and reusability, high enzyme activity, high conversion rate when catalyzing fructose isomerization to prepare psicose, and is suitable for continuous conversion of fructose to produce psicose.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions: A method for preparing DEAE-cellulose immobilized D-psicose-3-epimerase comprises the following steps: The DEAE-cellulose is soaked in water, treated with acid and alkali, and then soaked in phosphate buffer until equilibrium is reached to obtain activated DEAE-cellulose; The D-psicose-3-epimerase solution and the cross-linking agent are mixed to perform a cross-linking reaction to obtain a cross-linked enzyme solution; The cross-linked enzyme solution and the activated DEAE-cellulose are mixed and immobilized to obtain the DEAE-cellulose immobilized D-psicose-3-epimerase.

[0006] Preferably, the material-liquid ratio of the water immersion is 1 g:5-8 mL; and the time of the water immersion is 2-5 h.

[0007] Preferably, the acid-base treatment comprises acid treatment and alkali treatment in sequence, or comprises alkali treatment and acid treatment in sequence.

[0008] Preferably, the alkaline reagent used in the alkali treatment is an alkali metal hydroxide solution or an alkali metal carbonate solution; the concentration of the alkaline reagent is 0.3~0.8mol / L; the material-liquid ratio of the alkali treatment is 1g:3~8mL; the alkali treatment includes oscillation and static soaking in sequence, the oscillation time is 10~25min, and the static soaking time is 3~8h.

[0009] Preferably, the acidic reagent used in the acid treatment is hydrochloric acid solution, phosphoric acid solution or acetic acid solution; the concentration of the acidic reagent is 0.3~0.8mol / L; the material-liquid ratio of the acid treatment is 1g:3~8mL; the acid treatment includes oscillation and static soaking in sequence, the oscillation time is 10~25min, and the static soaking time is 3~8h.

[0010] Preferably, after the acid treatment or alkali treatment is completed, the obtained treatment liquid is filtered and the obtained solid is washed with deionized water until the pH value is 6-7.

[0011] Preferably, the concentration of the D-psicose-3-epimerase solution is 8-24 mg / mL; the cross-linking agent comprises one or more of glutaraldehyde, ethylenediamine, maleic anhydride and isocyanate derivatives; the mass of the cross-linking agent is 0.05-1% of the mass of the D-psicose-3-epimerase solution; The cross-linking reaction is carried out at a temperature of 20-35° C. and a time of 10-25 min. The cross-linking reaction is carried out under oscillation conditions, and the oscillation speed is 150-250 rpm.

[0012] Preferably, the dosage ratio of the cross-linking enzyme solution and the activated DEAE-cellulose is 0.5~2mL:1g; the immobilization temperature is 20~35°C, and the time is 2~5h; the immobilization is carried out under oscillation conditions, and the oscillation rate is 150~250rpm.

[0013] The present invention also provides DEAE-cellulose immobilized D-psicose-3-epimerase prepared by the preparation method described in the above scheme, including DEAE-cellulose and D-psicose-3-epimerase immobilized on the DEAE-cellulose.

[0014] The present invention also provides the use of the DEAE-cellulose immobilized D-psicose-3-epimerase described in the above scheme in the preparation of psicose by fructose isomerization reaction.

[0015] The present invention provides a preparation method of DEAE-cellulose immobilized D-psicose-3-epimerase, comprising the following steps: soaking DEAE-cellulose in water, performing acid-base treatment, and then soaking in phosphate buffer until equilibrium to obtain activated DEAE-cellulose; mixing D-psicose-3-epimerase solution and cross-linking agent for cross-linking reaction to obtain cross-linked enzyme solution; mixing the cross-linked enzyme solution and the activated DEAE-cellulose for immobilization to obtain the DEAE-cellulose immobilized D-psicose-3-epimerase. The present invention first removes impurities in DEAE-cellulose by soaking in water, and then adjusts the pH value of DEAE-cellulose by acid-base treatment to achieve an optimal working state, which is beneficial to the activation of the carrier and the subsequent ion exchange process; and the acid treatment can enhance the mechanical strength of DEAE-cellulose, making it more stable and not easy to break during the reaction process, and can also help remove impurities in DEAE-cellulose, such as metal ions, organic matter, etc., thereby improving the purity and efficiency of the carrier, improving the stability of the carrier at different pH values, and allowing the immobilized enzyme to maintain activity in a wide pH range. In addition, the present invention cross-links D-psicose-3-epimerase before immobilization, which can form a stable covalent bond for D-psicose-3-epimerase, improve the rigidity, thermal stability and pH tolerance of the enzyme, maintain the structure and function of the enzyme, promote the formation of multi-point cross-linking between enzyme molecules or between the enzyme and the carrier, create a stable three-dimensional network structure, promote the integration of the enzyme and the carrier, increase the active sites on the carrier surface, increase the loading amount of the immobilized enzyme, and improve the binding performance of the immobilized carrier and the enzyme. The results of the embodiment show that the DEAE-cellulose immobilized D-psicose-3-epimerase of the present invention is applied to the reaction of preparing psicose by fructose isomerization, and the conversion rate of fructose reaches 26.6%. DETAILED DESCRIPTION

[0016] The present invention provides a method for preparing DEAE-cellulose immobilized D-psicose-3-epimerase, comprising the following steps: The DEAE-cellulose is soaked in water, treated with acid and alkali, and then soaked in phosphate buffer until equilibrium is reached to obtain activated DEAE-cellulose; The D-psicose-3-epimerase solution and the cross-linking agent are mixed to perform a cross-linking reaction to obtain a cross-linked enzyme solution; The cross-linked enzyme solution and the activated DEAE-cellulose are mixed and immobilized to obtain the DEAE-cellulose immobilized D-psicose-3-epimerase.

[0017] The present invention soaks DEAE-cellulose in water, performs acid-base treatment, and then soaks it in phosphate buffer until equilibrium to obtain activated DEAE-cellulose. In the present invention, the water soaking specifically uses deionized water to soak the DEAE cellulose; the material-liquid ratio of the water soaking is preferably 1g:5~8mL, specifically 1g:5mL, 1g:6mL, 1g:7mL or 1g:8mL; the time of the water soaking is preferably 2~5h, specifically 2h, 3h, 4h or 5h; the temperature of the water soaking is preferably room temperature. After the water soaking is completed, the water soaking liquid is preferably drained and filtered, and repeatedly washed with deionized water. The present invention removes water-soluble impurities in DEAE-cellulose by water soaking.

[0018] In the present invention, the acid-base treatment preferably includes sequentially performing an acid treatment and an alkali treatment, or sequentially performing an alkali treatment and an acid treatment.

[0019] In the present invention, the alkaline agent used in the alkali treatment is preferably an alkali metal hydroxide solution or an alkali metal carbonate solution; the alkali metal hydroxide is preferably potassium hydroxide and / or sodium hydroxide; the alkali metal carbonate is preferably sodium carbonate; the concentration of the alkaline agent is preferably 0.3-0.8 mol / L, specifically 0.5 mol / L; the material-liquid ratio of the alkali treatment is preferably 1g:3-8mL, specifically 1g:3mL, 1g:4mL, 1g:5mL, 1g:6mL, 1g:7mL or 1g:8mL; the alkali treatment includes shaking and standing soaking in sequence, the shaking time is preferably 10-25min, specifically 15min, the standing soaking time is preferably 3-8h, specifically 6h. The alkali treatment is preferably carried out at room temperature.

[0020] In the present invention, the acidic reagent used in the acid treatment is preferably a hydrochloric acid solution, a phosphoric acid solution or an acetic acid solution; the concentration of the acidic reagent is preferably 0.3-0.8 mol / L, specifically 0.5 mol / L; the solid-liquid ratio of the acid treatment is preferably 1g:3-8mL, specifically 1g:3mL, 1g:4mL, 1g:5mL, 1g:6mL, 1g:7mL or 1g:8mL; the acid treatment preferably includes oscillation and standing soaking in sequence, the oscillation time is preferably 10-25min, specifically 15min, and the standing soaking time is preferably 3-8h, specifically 6h. The acid treatment is preferably carried out at room temperature. The present invention adjusts the pH value of DEAE-cellulose through acid-base treatment to achieve the best working state, improve the mechanical strength and stability of the carrier, and also helps to further remove impurities in the carrier.

[0021] In the present invention, after the acid treatment or alkali treatment is completed, it is preferred that the obtained treatment solution is filtered and the obtained solid is washed with deionized water until the pH value is 6-7.

[0022] In the present invention, the pH value of the phosphate buffer is preferably 7-7.5; the soaking time of the phosphate buffer is preferably more than 2 hours, specifically 2-3 hours. In a specific embodiment of the present invention, the pH value of the slurry is soaked until it stabilizes at the pH value of the phosphate buffer.

[0023] The present invention mixes a D-psicose-3-epimerase solution and a cross-linking agent for cross-linking reaction to obtain a cross-linked enzyme solution. In the present invention, the concentration of the D-psicose-3-epimerase solution is preferably 8-24 mg / mL, specifically 12 mg / mL; the solvent of the D-psicose-3-epimerase solution is preferably PBS buffer, and the pH value of the PBS buffer is preferably 7; the cross-linking agent preferably includes one or more of glutaraldehyde, ethylenediamine, maleic anhydride and isocyanic acid derivatives, and the isocyanic acid derivative is preferably one or two of monoisocyanate and diisocyanate; in a specific embodiment of the present invention, the cross-linking agent is preferably glutaraldehyde; the mass of the cross-linking agent is preferably 0.05-1% of the mass of the D-psicose-3-epimerase solution, specifically 0.1%.

[0024] In the present invention, the temperature of the cross-linking reaction is preferably 20-35°C, specifically 25°C, the time is preferably 10-25min, specifically 15min, the cross-linking reaction is preferably carried out under oscillation conditions, and the oscillation speed is preferably 150-250rpm, specifically 200rpm. The present invention cross-links the D-psicose-3-epimerase solution and the cross-linking agent to form a covalent bond, increase the rigidity of the enzyme, improve the thermal stability and pH tolerance of the enzyme, and the cross-linked enzyme has good reusability.

[0025] After obtaining the cross-linked enzyme solution and activated DEAE-cellulose, the present invention mixes the cross-linked enzyme solution and the activated DEAE-cellulose for immobilization to obtain the DEAE-cellulose immobilized D-psicose-3-epimerase. In the present invention, the dosage ratio of the cross-linked enzyme solution and the activated DEAE-cellulose is preferably 0.5~2mL:1g, specifically 1mL:1g; the immobilization temperature is preferably 20~35°C, specifically 30°C, and the time is preferably 2~5h, specifically 4h; the immobilization is preferably carried out under oscillation conditions, and the oscillation rate is preferably 150~250rpm, specifically 200rpm. After the immobilization is completed, the obtained immobilized solution is preferably filtered to obtain the DEAE-cellulose immobilized D-psicose-3-epimerase.

[0026] The present invention also provides a DEAE-cellulose immobilized D-psicose-3-epimerase prepared by the preparation method described in the above scheme, comprising DEAE-cellulose and D-psicose-3-epimerase immobilized on the DEAE-cellulose. In the present invention, the enzyme activity of the DEAE-cellulose immobilized D-psicose-3-epimerase is 70-120 u / g.

[0027] The present invention also provides the use of the DEAE-cellulose immobilized D-psicose-3-epimerase described in the above scheme in the preparation of psicose by fructose isomerization reaction. In a specific embodiment of the present invention, the DEAE-cellulose immobilized D-psicose-3-epimerase and a fructose solution are preferably mixed for isomerization reaction to obtain psicose; the temperature of the isomerization reaction is preferably 35-75°C, more preferably 55°C, and the pH value of the isomerization reaction is 5.5-9.5, preferably 7-8; the concentration of the fructose solution is preferably 40-60wt%, more preferably 50wt%; the ratio of the mass of the DEAE-cellulose immobilized D-psicose-3-epimerase to the volume of the fructose solution is preferably 1g:3-10mL, more preferably 1g:4mL.

[0028] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0029] Example 1 (1) Take 1 g of DEAE-cellulose and add 5 mL of deionized water to soak for 3 h. After soaking, drain and filter the water.

[0030] (2) Add 5 mL of 0.5 mol / L NaOH solution to 1 g of DEAE-cellulose after soaking, shake for 15 min, and soak for 6 h; wash repeatedly with deionized water, drain and filter.

[0031] (3) Add 0.5 mol / L HCl solution to the alkali-treated DEAE-cellulose at a solid-liquid ratio of 1 g:5 mL, shake for 15 min, and soak for 6 h; use a Buchner funnel to drain and filter, and add deionized water to wash until the pH value is 6-7.

[0032] (4) The acid-treated DEAE-cellulose is soaked in a phosphate buffer solution with a pH value of 7.5 for 2 h until equilibrium is reached to obtain activated DEAE-cellulose.

[0033] (5) Take 0.12 g of D-psicose-3-epimerase, add 10 mL of PBS buffer with a pH value of 7.5 to dissolve it to obtain an enzyme solution, then add 0.1% glutaraldehyde by mass fraction of the enzyme solution, and shake at 25°C and 200 rpm for 15 min for cross-linking to obtain a cross-linked enzyme solution.

[0034] (6) Take 1 g of activated DEAE-cellulose and add 1 mL of cross-linking enzyme solution. Oscillating and adsorbing at 30 °C and 200 rpm for 4 h. After the oscillation is completed, filtering is performed to obtain DEAE-cellulose immobilized D-psicose-3-isomerase (referred to as immobilized enzyme) with an enzyme activity of 120 u / g.

[0035] (7) 1 g of DEAE-cellulose-immobilized D-psicose-3-isomerase was mixed with 4 mL of 50 wt% fructose solution and reacted at 55°C and pH 7.5. After the reaction was completed (the reaction was considered to be completed when it reached equilibrium), the conversion rate was detected to be 26.6%.

[0036] Example 2 Other conditions were the same as those in Example 1, except that during the fructose isomerization reaction, the pH value of the reaction was adjusted to 5, 5.5, 6, 7, 8 or 9 using hydrochloric acid solution or sodium hydroxide solution. After the reaction, the fructose conversion rate was detected. The results are shown in Table 1.

[0037] Table 1 Fructose conversion rate under different pH conditions

[0038] According to the data in Table 1, it can be seen that the DEAE-cellulose immobilized D-psicose-3-epimerase prepared in the present invention has good pH adaptability and can catalyze the isomerization of fructose to produce psicose within the pH range of 5 to 9.

[0039] Example 3 After the isomerization reaction in Example 1 was completed, the DEAE-cellulose immobilized D-psicose-3-epimerase was separated, washed and filtered, and the recovered immobilized enzyme was used to repeat the fructose isomerization experiment. The experimental conditions were the same as in Example 1. A total of 6 isomerization experiments were performed, and the fructose conversion rate of each experiment is shown in Table 2.

[0040] Table 2 Results of repeatability test of immobilized enzyme

[0041] According to the data in Table 2, it can be seen that when the immobilized enzyme was used 6 times, the conversion rate of fructose could still reach 22.4%, indicating that the DEAE-cellulose immobilized D-psicose-3-epimerase prepared by the present invention has good reusability.

[0042] Example 4 The other conditions were the same as those in Example 1, except that the reaction temperature was changed to 35° C., 45° C., 65° C. or 75° C. during the fructose isomerization reaction. After the reaction, the fructose conversion rate was tested, and the results are shown in Table 3.

[0043] Table 3 Thermal stability test results of immobilized enzyme

[0044] According to the data in Table 3, it can be seen that within the range of 35-75° C., the DEAE-cellulose immobilized D-psicose-3-epimerase of the present invention can catalyze the isomerization of fructose to produce psicose with a high conversion rate, indicating that it has good thermal stability.

[0045] Example 5 The DEAE-cellulose-immobilized D-psicose-3-epimerase after immobilization in Example 1 was rinsed with deionized water and filtered, and the rinsing was repeated five times, each time with 25 mL of water, and the enzyme adsorption rate was tested after each rinse. The test results are shown in Table 4.

[0046] Table 4 Immobilized enzyme elution test

[0047] According to the data in Table 4, it can be seen that after repeated elution for five times, the enzyme adsorption rate of the immobilized enzyme prepared by the present invention is still maintained above 70%, indicating that the immobilized carrier and the enzyme are stably combined and not easily eluted.

[0048] Example 6 The other conditions were the same as those in Example 1, except that the alkali treatment and the acid treatment were reversed, that is, the acid treatment was performed first and then the alkali treatment. The obtained DEAE-cellulose immobilized D-psicose-3-epimerase was used to catalyze the isomerization reaction of the fructose solution, and the reaction conditions were the same as those in Example 1. After the reaction was completed, the conversion rate was detected to be 25.8%.

[0049] Example 7 The other conditions were the same as those in Example 1, except that the solution used for the alkali treatment was replaced with a KOH solution. The obtained DEAE-cellulose immobilized D-psicose-3-epimerase was used to catalyze the isomerization reaction of the fructose solution. The reaction conditions were the same as those in Example 1. After the reaction was completed, the conversion rate was detected to be 26.2%.

[0050] Example 8 The other conditions were the same as those in Example 1, except that the solution used for the alkali treatment was replaced with a sodium carbonate solution. The obtained DEAE-cellulose immobilized D-psicose-3-epimerase was used to catalyze the isomerization reaction of the fructose solution. The reaction conditions were the same as those in Example 1. After the reaction was completed, the conversion rate was detected to be 25.8%.

[0051] Example 9 The other conditions were the same as those in Example 1, except that the solution used for the acid treatment was replaced with an acetic acid solution having a concentration of 0.5 mol / L. The obtained DEAE-cellulose immobilized D-psicose-3-epimerase was used to catalyze the isomerization reaction of the fructose solution. The reaction conditions were the same as those in Example 1. After the reaction was completed, the conversion rate was detected to be 25.7%.

[0052] Example 10 The other conditions were the same as those in Example 1, except that the solution used for the acid treatment was replaced with a 0.5 mol / L phosphoric acid solution. The obtained DEAE-cellulose immobilized D-psicose-3-epimerase was used to catalyze the isomerization reaction of the fructose solution. The reaction conditions were the same as those in Example 1. After the reaction, the conversion rate was detected to be 25.6%.

[0053] Comparative Example 1: Omitting the crosslinking agent (1) Take 1 g of DEAE-cellulose and add 5 mL of deionized water to soak for 3 h. After soaking, drain and filter the water.

[0054] (2) Add 5 mL of 0.5 mol / L NaOH solution to 1 g of DEAE-cellulose after soaking, shake for 15 min, and soak for 6 h; then wash repeatedly with deionized water, dry and filter.

[0055] (3) Add 0.5 mol / L HCl solution to the alkali-treated DEAE-cellulose at a solid-liquid ratio of 1 g:5 mL, shake for 15 min, and soak for 6 h; use a Buchner funnel to drain and filter, and add deionized water to wash until the pH value is 6-7.

[0056] (4) The acid-treated DEAE-cellulose was soaked in pH 7.5 phosphate buffer for 2 h to reach equilibrium, thereby obtaining activated DEAE-cellulose.

[0057] (5) Take 0.12 g of D-psicose-3-epimerase, add 10 mL of PBS buffer (pH 7.5) to dissolve it, and shake at 25°C and 200 rpm for 15 min.

[0058] (6) Take 1 g of activated DEAE-cellulose and add 1 mL of enzyme solution. Oscillating adsorption was carried out at 30 °C and 200 rpm for 4 h to obtain DEAE-cellulose immobilized D-psicose-3-isomerase with an enzyme activity of 75 u / g.

[0059] (7) 1 g of DEAE-cellulose-immobilized D-psicose-3-isomerase was mixed with 4 mL of 50 wt % fructose solution and reacted at 55 °C and pH 7.5. After the reaction, the conversion rate was detected to be 20.68%.

[0060] (8) The repeatability, stability and pH stability of the immobilized enzyme prepared in Comparative Example 1 were tested as follows: After the isomerization reaction was completed, the immobilized enzyme was separated, washed and filtered. The fructose isomerization experiment was repeated under the same conditions using the recovered immobilized enzyme. The results are shown in Table 5.

[0061] Table 5 Repeatability test results

[0062] The other conditions were the same as step (7), except that the reaction temperature was changed to 25°C, 45°C, 65°C or 75°C during the fructose isomerization reaction. After the reaction, the fructose conversion rate was measured, and the results are shown in Table 6.

[0063] Table 6 Temperature stability test results

[0064] The other conditions were the same as step (7), except that during the fructose isomerization reaction, the pH value of the reaction was adjusted to 5, 5.5, 6, 7, 8 or 9 using hydrochloric acid solution or sodium hydroxide solution. After the reaction, the fructose conversion rate was measured. The results are shown in Table 7.

[0065] Table 7 pH stability test results

[0066] According to the results in Table 5, it can be seen that the fructose conversion rate of the immobilized enzyme prepared in Comparative Example 1 dropped to 13.54% after repeated use for 3 times, and the repeatability was poor. According to Table 6, the fructose conversion rate decreased under low or high temperature conditions, especially at 75°C, the fructose conversion rate was only 11.7%, indicating that its temperature stability was poor. According to Table 7, the fructose conversion rate decreased significantly under low or high pH conditions, indicating that its pH adaptability was poor.

[0067] Comparative Example 2 Omitting acid treatment (1) Take 1 g of DEAE-cellulose and add 5 mL of deionized water to soak for 3 h. After soaking, drain and filter the water.

[0068] (2) Add 5 mL of 0.5 mol / L NaOH solution to 1 g of DEAE-cellulose after soaking, shake for 15 min, and soak for 6 h; wash repeatedly with deionized water, drain and filter.

[0069] (3) The alkali-treated DEAE-cellulose was soaked in pH 7.5 phosphate buffer for 2 h until equilibrium was reached to obtain activated DEAE-cellulose.

[0070] (4) Take 0.12 g of D-psicose-3-epimerase, add 10 mL of PBS buffer with a pH value of 7.5 to dissolve it to obtain an enzyme solution, and then add 0.1% glutaraldehyde by mass fraction of the enzyme solution to perform cross-linking, and shake at 25°C and 200 rpm for 15 min to obtain a cross-linked enzyme solution.

[0071] (5) Take 1 g of activated DEAE-cellulose and add 1 mL of cross-linking enzyme solution. Oscillating adsorption was carried out at 30 °C and 200 rpm for 4 h to obtain DEAE-cellulose immobilized D-psicose-3-isomerase with an enzyme activity of 86 u / g.

[0072] (6) 1 g of DEAE-cellulose-immobilized D-psicose-3-isomerase was mixed with 4 mL of 50 wt % fructose solution and reacted at 55 °C and pH 7.5. After the reaction, the conversion rate was detected to be 20.07%.

[0073] (7) The repeatability, stability and pH stability of the immobilized enzyme prepared in Comparative Example 2 were tested as follows: After the isomerization reaction was completed, the immobilized enzyme was separated, washed and filtered. The fructose isomerization experiment was repeated under the same conditions using the recovered immobilized enzyme. The results are shown in Table 8.

[0074] Table 8 Repeatability test results

[0075] The other conditions were the same as step (6), except that the reaction temperature was changed to 35°C, 45°C, 65°C or 75°C during the fructose isomerization reaction. After the reaction, the fructose conversion rate was measured, and the results are shown in Table 9.

[0076] Table 9 Temperature stability test results

[0077] The other conditions were the same as step (6), except that during the fructose isomerization reaction, the pH value of the reaction was adjusted to 5, 5.5, 6, 7, 8 or 9 using hydrochloric acid solution or sodium hydroxide solution. After the reaction, the fructose conversion rate was measured. The results are shown in Table 10.

[0078] Table 10 pH stability test results

[0079] According to the results in Tables 8 to 10, it can be seen that the immobilized enzyme prepared in Comparative Example 2 has poor repeatability, stability and pH stability.

[0080] Comparative Example 3: Omitting alkali treatment (1) Take 1g DEAE-cellulose and add 5mL deionized water to soak for 3h. After soaking, drain and filter the water; (2) Add the soaked DEAE cellulose into 0.5 mol / L HCl solution with a solid-liquid ratio of 1 g:5 mL, shake for 15 min and soak for 6 h; use a Buchner funnel to drain and filter, and add deionized water to wash until the pH value is 6-7.

[0081] (3) The acid-treated DEAE cellulose is soaked in a phosphate buffer solution with a pH value of 7.5 for 2 h until equilibrium is reached to obtain activated DEAE cellulose.

[0082] (4) Take 0.12 g of D-psicose-3-epimerase, add 10 mL of PBS buffer with a pH value of 7.5 to dissolve it to obtain an enzyme solution, then add 0.1% glutaraldehyde by mass fraction of the enzyme solution, and shake at 25°C and 200 rpm for 15 min for cross-linking to obtain a cross-linked enzyme solution.

[0083] (5) Take 1 g of activated DEAE-cellulose and add 1 mL of cross-linking enzyme solution. Oscillate and adsorb at 30 °C and 200 rpm for 4 h to obtain DEAE-cellulose immobilized D-psicose-3-isomerase with an enzyme activity of 83 u / g.

[0084] (6) 1 g of DEAE-cellulose-immobilized D-psicose-3-isomerase was mixed with 4 mL of 50 wt% fructose solution and reacted at 55°C and pH 7.5. After the reaction, the conversion rate was detected to be 19.14%.

[0085] (7) The repeatability, stability and pH stability of the immobilized enzyme prepared in Comparative Example 3 were tested as follows: After the isomerization reaction was completed, the immobilized enzyme was separated, washed and filtered, and the fructose isomerization experiment was repeated under the same conditions using the recovered immobilized enzyme. The results are shown in Table 11.

[0086] Table 11 Repeatability test results

[0087] The other conditions were the same as step (6), except that the reaction temperature was changed to 35°C, 45°C, 65°C or 75°C during the fructose isomerization reaction. After the reaction, the fructose conversion rate was measured, and the results are shown in Table 12.

[0088] Table 12 Temperature stability test results

[0089] The other conditions were the same as step (6), except that during the fructose isomerization reaction, the pH value of the reaction was adjusted to 5, 5.5, 6, 7, 8 or 9 using hydrochloric acid solution or sodium hydroxide solution. After the reaction, the fructose conversion rate was measured. The results are shown in Table 13.

[0090] Table 13 pH stability test results

[0091] According to the results in Tables 11 to 13, it can be seen that the immobilized enzyme prepared in Comparative Example 3 has poor repeatability, stability and pH stability.

[0092] It can be seen from Comparative Examples 1 to 3 that after omitting the cross-linking agent, acid treatment or alkali treatment steps, the activity of the obtained immobilized enzyme decreases, the conversion rate when catalyzing the fructose isomerization reaction decreases, and the repeatability, temperature stability and pH stability of the immobilized enzyme all decrease.

[0093] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing DEAE-cellulose immobilized D-psicose-3-epimerase, characterized in that: The following steps are involved: The DEAE-cellulose is soaked in water, treated with acid and alkali, and then soaked in phosphate buffer until equilibrium is reached to obtain activated DEAE-cellulose; The D-psicose-3-epimerase solution and the cross-linking agent are mixed to perform a cross-linking reaction to obtain a cross-linked enzyme solution; The cross-linked enzyme solution and the activated DEAE-cellulose are mixed and immobilized to obtain the DEAE-cellulose immobilized D-psicose-3-epimerase.

2. The preparation method according to claim 1, characterized in that: The material-liquid ratio of the water immersion is 1 g:5-8 mL; and the time of the water immersion is 2-5 h.

3. The preparation method according to claim 1, characterized in that: The acid-base treatment includes sequentially performing an acid treatment and an alkali treatment, or sequentially performing an alkali treatment and an acid treatment.

4. The preparation method according to claim 3, characterized in that: The alkaline reagent used in the alkali treatment is an alkali metal hydroxide solution or an alkali metal carbonate solution; the concentration of the alkaline reagent is 0.3-0.8 mol / L; the material-liquid ratio of the alkali treatment is 1 g:3-8 mL; the alkali treatment includes oscillation and static soaking in sequence, the oscillation time is 10-25 min, and the static soaking time is 3-8 h.

5. The preparation method according to claim 3, characterized in that: The acidic reagent used in the acid treatment is hydrochloric acid solution, phosphoric acid solution or acetic acid solution; the concentration of the acidic reagent is 0.3~0.8mol / L; the material-liquid ratio of the acid treatment is 1g:3~8mL; the acid treatment includes oscillation and static soaking in sequence, the oscillation time is 10~25min, and the static soaking time is 3~8h.

6. The preparation method according to claim 3, characterized in that: After the acid treatment or the alkali treatment is completed, the obtained treatment liquid is filtered and the obtained solid is washed with deionized water until the pH value is 6-7.

7. The preparation method according to claim 1, characterized in that: The concentration of the D-psicose-3-epimerase solution is 8-24 mg / mL; the cross-linking agent includes one or more of glutaraldehyde, ethylenediamine, maleic anhydride and isocyanate derivatives; the mass of the cross-linking agent is 0.05-1% of the mass of the D-psicose-3-epimerase solution; The cross-linking reaction is carried out at a temperature of 20-35° C. and a time of 10-25 min. The cross-linking reaction is carried out under oscillation conditions, and the oscillation speed is 150-250 rpm.

8. The preparation method according to claim 1, characterized in that: The dosage ratio of the cross-linking enzyme solution and the activated DEAE-cellulose is 0.5~2mL:1g; the immobilization temperature is 20~35°C and the time is 2~5h; the immobilization is carried out under oscillation conditions, and the oscillation rate is 150~250rpm.

9. The DEAE-cellulose immobilized D-psicose-3-epimerase prepared by the preparation method according to any one of claims 1 to 8, comprising DEAE-cellulose and D-psicose-3-epimerase immobilized on the DEAE-cellulose.

10. Use of the DEAE-cellulose immobilized D-psicose-3-epimerase according to claim 9 in the preparation of psicose by fructose isomerization reaction.

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