Preparation method of ketose 3-epimerase immobilized enzyme and application thereof

Abstract

This invention discloses a method for preparing an immobilized ketose 3-epimerase and its application, belonging to the field of immobilized enzyme preparation technology. The invention comprises the following steps: first, pretreatment of anion exchange resin; second, adsorption of the protease onto the ion exchange resin; and third, cross-linking with glutaraldehyde. The immobilized enzyme prepared by this invention is simple to prepare, inexpensive, and requires simple and mild reaction conditions. Immobilization improves the relative activity and stability of the enzyme, and the prepared immobilized enzyme is firmly bound and can undergo multiple enzyme-catalyzed reactions. This discovery has significant research value for the industrial preparation of D-allulose.

Description

Technical Field

[0001] This invention relates to a method for preparing an immobilized ketose 3-epimerase and its application, belonging to the field of immobilized enzyme preparation technology. Background Technology

[0002] D-allulose, classified as a hexulose, is a naturally occurring but extremely rare low-energy functional sugar. It exhibits a certain sweetness without bitterness, and its taste is similar to sucrose. When sucrose is considered a sweetness value of 100, D-allulose has a sweetness value of approximately 70. According to the guidance document from the U.S. Food and Drug Administration (FDA) (FDA-2019-D-0725), D-allulose provides only 0.4 kcal / g of dietary energy. Due to its low-energy, high-sweetness characteristics, D-allulose shows great potential as a novel sweetener in the production of candies, desserts, bread, and other food processing products. Furthermore, D-allulose can be used in the Maillard process to obtain caramel-like products and maintains relatively stable performance under various pH conditions. Studies have found that the final concentration of D-allulose remained unchanged when the initial pH of the reaction mixture was 4.0 and 6.0; however, when the initial pH was set to 7.5, the retention rate of D-allulose was 91.3%, indicating its stability under normal cooking and production conditions. Although D-allulose cannot be metabolized for energy in the human body, it exhibits many unique physiological functions. Hossain et al. found that D-allulose can partially inhibit the uptake of D-glucose and D-fructose by competitively inhibiting them, which also require glucose transporters. Braunstein et al. found that D-allulose can significantly improve postprandial glucose levels by inhibiting the activity of α-glucosidase and α-amylase in the intestine and promoting the conversion of glucose to glycogen by glucokinase in the liver. Pongkan et al. found that D-allulose can protect mice by enhancing insulin regulation and provides cardioprotection by reducing cardiac mitochondrial dysfunction. Studies by Moon et al. have found that D-allulose can effectively inhibit adipocyte differentiation and hepatic lipase activity, reducing fat accumulation and lowering the probability of obesity. Research by Kimura et al. has reported that D-allulose can regulate the expression of genes related to lipid metabolism and lipogenesis, enhancing energy metabolism by increasing fatty acid oxidation. Furthermore, D-allulose also exhibits anti-inflammatory effects by reducing the levels of inflammatory cytokines. Kanasaki et al. found that D-allulose can regulate cholesterol metabolism by reducing PCSK9 protein in hamster serum and increase the uptake of high-density lipoprotein cholesterol by hepatocytes. The diverse functional properties exhibited by D-allulose in physiological studies lay a theoretical foundation for its further application research.

[0003] According to Izumoring's rare sugar conversion strategy, ketose 3-epimerase (KEase) plays an irreplaceable role in the bioconversion of D-allulose, catalyzing a reversible epimerization reaction of D-fructose at the C-3 position to generate D-allulose. Currently, research on ketose 3-epimerase is quite extensive, including microbial screening and identification, enzyme isolation and purification, heterologous recombinant expression, enzyme immobilization, food-grade expression, molecular modification, and crystal structure determination. In downstream research on D-allulose production, controlling production costs is crucial; therefore, the immobilization of ketose 3-epimerase, the isolation and purification of D-allulose, and the exploration of low-cost raw materials are key technical focuses.

[0004] Free enzymes suffer from drawbacks such as inability to be reused and poor stability, leading to the increasing adoption of immobilized enzyme and cell immobilization technologies. Current reports on the immobilization of ketose 3-epimerases are hampered by issues such as complex procedures, low enzyme activity, and difficulty in separating them from the reaction system, hindering their application in practical industrial production. Therefore, developing an immobilized ketose 3-epimerase that is simple to operate, highly stable, easily separable, and has a high reusability rate for industrial production is a problem that needs to be solved. Summary of the Invention

[0005] In downstream research on D-allulose production, cost control is crucial. The immobilization of ketose 3-epimerase, the isolation and purification of D-allulose, and the exploration of low-cost raw materials are key technical focuses. Free enzymes suffer from drawbacks such as inability to be reused and poor stability, leading to the increasing attention given to immobilized enzymes and immobilized cell technologies. Current reports on KEase immobilization suffer from problems such as complex operation, low enzyme activity, and difficulty in separating from the reaction system, hindering their application in actual industrial production. Therefore, developing an immobilized ketose 3-epimerase that is simple to operate, highly stable, easily separable, and has a high reusability rate for industrial production is a problem that needs to be solved.

[0006] This invention provides a method for immobilizing ketose 3-epimerase using anion exchange resin as a carrier. This discovery has important practical significance for the industrial application of D-allulose and ketose 3-epimerase in industrial production.

[0007] To address the aforementioned technical problems, this invention provides a method for immobilizing ketose 3-epimerase using anion exchange resin as a carrier.

[0008] The purpose of this invention is to provide a method for immobilizing ketose 3-epimerase using anion exchange resin as a carrier, and further to provide the application of immobilized ketose 3-epimerase in the production of D-allulose.

[0009] This invention provides a method for immobilizing ketose 3-epomerase, characterized in that the method comprises the following steps:

[0010] a. Ketosaccharide 3-epimerase from Labedella endophytica was added to a reaction system containing pretreated ion exchange resin for adsorption.

[0011] The pretreated ion exchange resin is treated as follows: (1) Soak the resin in ethanol for 12-24 hours, discard the impurities, and soak it in a sodium chloride solution with a volume fraction of 10-15% for 10-15 hours. Then wash away the impurities and floating color with deionized water until the solution pH is neutral. (2) Soak the resin treated in step (1) in a sodium hydroxide solution with a volume fraction of 2-5% for 10-15 hours, and wash it with deionized water until the solution pH is neutral. (3) Soak the resin treated in step (2) in a hydrochloric acid solution with a volume fraction of 2-5% for 10-15 hours, and wash it with deionized water until the solution pH is neutral.

[0012] b. After cross-linking the enzyme with glutaraldehyde cross-linking agent, immobilized ketose 3-epimerase is obtained.

[0013] In one embodiment of the present invention, the ion exchange resin is: D301 macroporous weakly basic styrene-based anion exchange resin.

[0014] In one embodiment of the present invention, the treatment method for the pretreated ion exchange resin is as follows:

[0015] (1) Soak in 95% ethanol with twice the volume of resin for 20 hours, discard the impurities, and soak in 10% sodium chloride solution for 12 hours. Then wash away the impurities and floating color with deionized water until the pH of the solution is neutral.

[0016] (2) Soak the resin treated in step (1) in a 5% sodium hydroxide solution for 12 hours, and wash with deionized water until the solution pH is neutral.

[0017] (3) Soak the resin in a 2% hydrochloric acid solution for 12 hours, and wash it with deionized water until the pH of the solution is neutral.

[0018] The pretreated resin is soaked in deionized water at twice its volume and stored in a refrigerator at 4°C for later use; when using, the resin is filtered and wiped dry.

[0019] In one embodiment of the present invention, the total volume of the reaction system is 2-4 mL. The reaction system is adsorbed on a shaker at 15-37°C for 2-9 h, and then placed at 2-8°C for 10-30 min. Glutaraldehyde with a final concentration of 0.005-0.1% is added for cross-linking, and the cross-linking is carried out at 2-8°C for 1-8 h. The supernatant is discarded, the mixture is washed with deionized water, and vacuum filtered to obtain immobilized ketose 3-epimerase, which is stored at 4°C for later use.

[0020] In one embodiment of the present invention, the enzyme immobilization step is as follows: (1) Take 0.5g of resin, add 0.5mL of crude enzyme solution of the source microorganism Labedella endophytica, the amount of enzyme added is 0.4mg protein / g resin, and the final concentration is 50mM Tris-HCl buffer (pH 8.0).

[0021] In one embodiment of the present invention, the total volume of the immobilization system is 2 mL. Adsorption is performed on a shaker at 18°C ​​for 2 h, followed by standing at 4°C for 30 min. Then, glutaraldehyde with a final concentration of 0.01% is added for crosslinking, and the mixture is placed at 4°C for 1 h for crosslinking.

[0022] In one embodiment of the present invention, after immobilization, the supernatant is discarded, the resin is washed with deionized water, and stored in a refrigerator at 4°C for later use.

[0023] In one embodiment of the present invention, the reaction system further contains a Tris-HCl buffer solution (pH 8.0) with a final concentration of 50 mM.

[0024] In one embodiment of the present invention, the amount of ketose 3-epimerase added to the reaction system is 0.2 to 6.0 mg protein / g resin.

[0025] In one embodiment of the present invention, the amino acid sequence of the ketose 3-epimerase is numbered WP_127049469.1 on NCBI.

[0026] In one embodiment of the present invention, the nucleotide sequence encoding the ketose 3-epimerase is numbered NZ_RZGZ01000002.1 (1271830..1272699) on NCBI.

[0027] The present invention also provides an immobilized ketose 3-epomerase prepared by the above-described method for immobilizing ketose 3-epomerase.

[0028] The present invention also provides the above-described method for immobilizing ketose 3-epimerase, or the application of the above-described immobilized ketose 3-epimerase in the preparation of products containing D-allulose or containing D-allulose.

[0029] The present invention also provides the application of the immobilized ketose 3-epimerase in the pharmaceutical and food industries.

[0030] Beneficial effects

[0031] (1) The immobilized ketose 3-epimerase provided by the present invention has a simple processing technology, low cost, small activity loss, high efficiency, stable properties and can be reused.

[0032] (2) The immobilization method provided by the present invention improves the heat resistance and enhances the pH stability of the immobilized ketose 3-epimerase;

[0033] (3) The immobilized ketose 3-epimerase of the present invention can be reused, avoiding the waste of the carrier;

[0034] (4) Experiments show that the immobilized enzyme obtained in this invention can be converted into D-allulose using fructose as a substrate. Attached Figure Description

[0035] Figure 1 pH optimization diagram for immobilized ketose 3-epimerase adsorption.

[0036] Figure 2 : Optimization of adsorption temperature for immobilized ketose 3-epimerase.

[0037] Figure 3 Optimization of adsorption time for immobilized ketose 3-epimerase.

[0038] Figure 4 Optimization of glutaraldehyde concentration for immobilized ketose 3-epimerase.

[0039] Figure 5 Optimization of cross-linking time for immobilized ketose 3-epimerase.

[0040] Figure 6 : Optimal pH of immobilized ketose 3-epimerase and crude enzyme solution.

[0041] Figure 7 Thermostability diagram of immobilized ketose 3-epimerase and crude enzyme solution.

[0042] Figure 8 : Reuse count of immobilized ketose 3-epimerase. Detailed Implementation

[0043] The culture media involved in the following examples are as follows:

[0044] LB medium: 1% (w / v) sodium chloride, 1% (w / v) tryptone, 0.5% (w / v) yeast extract, sterilized at 121°C for 20 min. Add ampicillin (Amp) to a final concentration of 50 μg / mL before use.

[0045] The detection methods involved in the following embodiments are as follows:

[0046] Enzyme activity assay of crude enzyme solution:

[0047] Using 50 g / L D-fructose as a substrate, 0.5 μmol / L crude enzyme and 1 mmol / L CoCl2 were added. The enzyme reaction was carried out at 80 °C and pH 6.0 for 5 min, followed by boiling for 10 min to inactivate the enzyme. After the reaction, the product was centrifuged, filtered through a membrane, diluted to a certain concentration, and then detected by HPLC.

[0048] Enzyme activity assay of immobilized enzymes:

[0049] Using 50 g / L D-fructose as a substrate, 0.25 g of immobilized enzyme and 1 mmol / L CoCl2 were added. The enzyme reaction was carried out at 80 °C and pH 6.0 for 5 min. The supernatant was then boiled for 10 min to inactivate the enzyme. After the reaction, the product was centrifuged, filtered through a membrane, diluted to a certain concentration, and detected by HPLC.

[0050] Enzyme activity (U): Under standard reaction conditions, the amount of enzyme required to catalyze the synthesis of 1 μmol of D-allulose per unit time (min). Enzyme activity recovery rate = (Immobilized enzyme specific activity / Crude enzyme solution specific activity) * Immobilized enzyme dosage * 100%.

[0051] Example 1: Method for preparing KEase expression from Labedella endophytica

[0052] (1) Preparation of recombinant vector pET-22b(+)-Laen

[0053] A gene fragment of KEase derived from L. endophytica was synthesized, thereby obtaining a recombinant plasmid. pET-22b(+) was used as the expression vector for the KEase target gene, and Xho I and Nde I were used as restriction enzyme sites. The target gene fragment was inserted into the vector to obtain the pET-22b(+)-Laen recombinant plasmid.

[0054] (2) Construction of recombinant bacteria

[0055] The recombinant vector pET-22b(+)-Laen was transformed into E. coli BL21(DE3) cells to prepare E. coli BL21(DE3) / pET-22b(+)-Laen.

[0056] (3) Enzyme production by recombinant bacteria fermentation

[0057] Select the positive transformants from step (2) and incubate them in LB medium at 37°C and 200 rpm for 12 h to prepare seed culture;

[0058] The prepared seed culture was inoculated into LB medium at an inoculation rate of 0.01% and cultured at 37℃ for 3-4 hours until the OD value reached 0.6-0.8. The temperature was then lowered to 30℃, and IPTG was added at a final concentration of 1.0 mM to induce fermentation for 6 hours to obtain the fermentation broth.

[0059] (4) Preparation of crude enzyme solution

[0060] The fermentation broth obtained in step (3) was centrifuged at 4℃ and 8000 rpm for 20 min to collect the bacterial cells. 20 mL of buffer (50 mM Tris, 200 mM NaCl, pH adjusted to 7.5) was added to fully resuspend the bacterial cells. The centrifuge tube was then placed in an ice bath and placed in an ultrasonic cell disruptor. The ultrasonic disruption conditions were: working time 1 ls, stop time 2 s, for a total of 15 min. The obtained disrupted solution was centrifuged at low temperature and high speed at 4℃ and 8000 rpm for 10 min to obtain the crude enzyme solution. Filtered through a 0.45 μm microporous membrane for later use.

[0061] The enzyme activity of the crude enzyme solution of ketose 3-epimerase derived from Labedella endophytica was measured to be 25.4 U / g resin.

[0062] Example 2: Immobilization of ketose 3-epimerase using resin as a carrier.

[0063] The specific steps are as follows:

[0064] (1) Resin pretreatment:

[0065] 1) Soak in 95% ethanol with twice the volume of resin for 20 hours, discard the impurities, soak in 10% sodium chloride solution for 12 hours, and then wash away impurities and floating color with deionized water until the pH of the solution is neutral.

[0066] 2) Soak the resin treated in step 1) above in a 2% hydrochloric acid solution for 12 hours, and wash with deionized water until the solution pH is neutral.

[0067] 3) Soak the resin treated in step 2) for 12 hours in a 5% sodium hydroxide solution, then wash with deionized water until the solution pH is neutral.

[0068] The pretreated resin is soaked in twice its volume of deionized water and stored in a refrigerator at 4°C for later use. Before use, the resin is filtered and dried.

[0069] (2) Enzyme immobilization:

[0070] Take 0.5 g of resin and add 0.5 mL of crude enzyme solution of the source microorganism Labedella endophytica prepared in Example 1. The amount of enzyme added is 0.4 mg protein / g resin, and the final concentration is 50 mM Tris-HCl buffer (pH 8.0).

[0071] The total volume of the immobilization system was 2 mL; the immobilization conditions were as follows: adsorption on a shaker at 18 °C for 2 h, standing at 4 °C for 30 min, cross-linking with glutaraldehyde at a final concentration of 0.01%, and cross-linking at 4 °C for 1 h.

[0072] After immobilization, discard the supernatant, wash the resin with deionized water, and store it in a refrigerator at 4°C for later use.

[0073] Following the above method, Dowex 1x8 ion exchange resin and D301 macroporous weakly basic styrene-based anion exchange resin were used respectively. FPA53 anion exchange resin, D152 macroporous acrylic weakly acidic cation exchange resin, and D311 macroporous weakly basic acrylic anion exchange resin. 15. Ion exchange resin, strong acid cation exchange resin (NA) IRA-410(Cl) ion exchange resin, IRA-900 anion exchange resin, Amberlite TM XAD761 ion exchange macroporous adsorption resin, Amberlite XAD7HP ion exchange resin The enzyme activity of immobilized enzymes was detected using IRC-748 chelating ion exchange resin, and the results were 0, 18.2, 1.5, 0, 2.3, 0, 0, 0, 3.5, 0, 0, and 0.9, respectively.

[0074] The results showed that the immobilized enzyme prepared using D301 macroporous weakly basic styrene-based anion exchange resin had the best effect.

[0075] Example 3: pH optimization.

[0076] The specific implementation method is the same as in Examples 1 and 2, except that the resin in Example 2 is changed to D301 resin, and the enzyme immobilization step is changed as follows:

[0077] (1) Preparation of enzyme solution:

[0078] The crude enzyme solution was diluted to 0.4 mg / mL using enzyme solutions prepared at 50 mM with pH values ​​of 6.0, 6.5, 7.0 (acetate buffer), 7.5, 8.0, 8.5, and 9.0 (Tris-HCl buffer).

[0079] (2) Weigh 0.5 g of resin and add 2 mL of the enzyme solution obtained in step (1). The enzyme concentration is 0.4 mg protein / g resin. Adsorb the solution in a constant temperature shaker at 18℃ and 200 r / min for 2 h. Place the solution in a refrigerator at 4℃ and let it stand for 30 min. Add 10 μL of 1% glutaraldehyde to make the final concentration 0.01%. Let the solution stand at 4℃ for 1 h for crosslinking. Discard the supernatant and wash the immobilized enzyme three times with buffer. Detect the specific enzyme activity and recovery rate of the immobilized enzyme.

[0080] The results are as follows Figure 1 As shown in the figure, the optimal adsorption pH is 8.0.

[0081] Example 4: Temperature optimization.

[0082] The specific implementation method is the same as in Examples 1 and 2, except that the resin in Example 2 is changed to XX resin, and the enzyme immobilization step is changed as follows:

[0083] (1) Preparation of enzyme solution:

[0084] The crude enzyme solution was diluted to 0.4 mg / mL using an enzyme solution prepared with 50 mM, pH 8.0 Tris-HCl buffer.

[0085] (2) Weigh 0.5 g of resin and add 2 mL of the enzyme solution obtained in step (1). The enzyme concentration is 0.4 mg protein / g resin. Adsorb the enzyme in a shaker at 15, 18, 25, 28, 30, 37 °C and 200 r / min for 2 h. Place the mixture in a refrigerator at 4 °C and let it stand for 30 min. Add 10 μL of 1% glutaraldehyde to make the final concentration 0.01%. Let the mixture stand at 4 °C for 1 h for crosslinking. Discard the supernatant and wash the immobilized enzyme three times with buffer. Detect the specific enzyme activity and recovery rate of the immobilized enzyme.

[0086] The results are as follows Figure 2 As shown in the figure, the results indicate that the optimal adsorption temperature is 18℃.

[0087] Example 5: Optimization of adsorption time.

[0088] The specific implementation method is the same as in Examples 1 and 2, except that the resin in Example 2 is changed to D301 resin, and the enzyme immobilization step is changed as follows:

[0089] (1) Preparation of enzyme solution:

[0090] The crude enzyme solution was diluted to 0.4 mg / mL using an enzyme solution prepared with 50 mM, pH 8.0 Tris-HCl buffer.

[0091] (2) Weigh 0.5 g of resin and add 2 mL of the enzyme solution obtained in step (1). The enzyme concentration is 0.4 mg protein / g resin. Adsorb the enzyme in a shaker at 18℃ and 200 r / min for 0, 2, 4, 6, 8, and 9 h, respectively. Place the mixture in a refrigerator at 4℃ and let it stand for 30 min. Add 10 μL of 1% glutaraldehyde to make the final concentration 0.01%. Let the mixture stand at 4℃ for 1 h for crosslinking. Discard the supernatant and wash the immobilized enzyme three times with buffer. Detect the specific enzyme activity and recovery rate of the obtained immobilized enzyme.

[0092] The results are as follows Figure 3 As shown in the figure, the optimal adsorption time is 2 hours.

[0093] Example 6: Optimization of crosslinking agent concentration.

[0094] The specific implementation method is the same as in Examples 1 and 2, except that the resin in Example 2 is changed to D301 resin, and the enzyme immobilization step is changed as follows:

[0095] (1) Preparation of enzyme solution:

[0096] The crude enzyme solution was diluted to 0.4 mg / mL using an enzyme solution prepared with 50 mM, pH 8.0 Tris-HCl buffer.

[0097] (2) Weigh 0.5 g of resin, add 2 mL of the enzyme solution obtained in step (1), and add enzyme at a rate of 0.4 mg protein / g resin. Adsorb the enzyme in a shaker at 18℃ and 200 r / min for 2 h. Place the mixture in a refrigerator at 4℃ and let it stand for 30 min. Add 10 μL of glutaraldehyde with volume fractions of 0, 0.5, 1, 2, 4, 8, and 10% to make the final concentrations 0, 0.005, 0.01, 0.02, 0.04, 0.08, and 0.1%, respectively. Let the mixture stand at 4℃ for 1 h for crosslinking. Discard the supernatant and wash the immobilized enzyme three times with buffer. Detect the specific enzyme activity and recovery rate of the obtained immobilized enzyme.

[0098] The results are as follows Figure 4 As shown in the figure, the optimal crosslinking agent concentration is 0.01%.

[0099] Example 7: Optimization of crosslinking time.

[0100] The specific implementation method is the same as in Examples 1 and 2, except that the resin in Example 2 is changed to D301 resin, and the enzyme immobilization steps are adjusted as follows:

[0101] (1) Preparation of enzyme solution:

[0102] The crude enzyme solution was diluted to 0.4 mg / mL using an enzyme solution prepared with 50 mM, pH 8.0 Tris-HCl buffer.

[0103] (2) Weigh 0.5 g of resin, add 2 mL of the enzyme solution obtained in step (1), and add enzyme at a concentration of 0.4 mg protein / g resin. Adsorb the enzyme in a shaker at 18℃ and 200 r / min for 2 h. Place the mixture in a refrigerator at 4℃ and let it stand for 30 min. Add 10 μL of 1% glutaraldehyde to bring the final concentration to 0.01%. Allow the mixture to stand at 4℃ for crosslinking for 0, 1, 2, 4, 6, and 8 h respectively. Discard the supernatant and wash the immobilized enzyme three times with buffer. Detect the specific enzyme activity and recovery rate of the immobilized enzyme.

[0104] The results are as follows Figure 5 As shown in the figure, the optimal crosslinking time is 1 hour.

[0105] Example 8: Determination of the optimal pH of the enzyme before and after immobilization.

[0106] The enzyme reaction was carried out using acetate buffer (50 mM, pH range 4.0-6.0), PBS buffer (50 mM, pH range 6.0-7.5), and Tris-HCl buffer (50 mM, pH range 7.5-9.0) as buffer systems, respectively. Except for the change in the buffer solutions, the other reaction and detection conditions were the same as described in Example 3.

[0107] The optimal reaction pH is the pH corresponding to the highest enzyme activity. To compare the effect of different reaction pH on recombinant enzyme activity, the enzyme activity at the optimal reaction pH was set as 100% relative enzyme activity, and the relative enzyme activity at other pH values ​​was calculated.

[0108] The relative enzyme activity of the immobilized enzyme prepared according to the method of the optimal resin D301 in Example 2 and the crude enzyme solution before immobilization prepared in Example 1 were tested according to the enzyme activity detection method.

[0109] Results are attached to the instruction manual. Figure 6 The optimal pH for immobilized enzymes is 5.5. It is noteworthy that at pH 4.0, the free enzyme completely loses its activity, but the immobilized enzyme retains 80% of its activity. This indicates that the immobilized enzyme has strong pH tolerance and will not become inactive in acidic environments, which suggests promising industrial applications.

[0110] Example 9: Test of enzyme thermostability before and after immobilization.

[0111] The crude enzyme solution of Laen prepared in Example 2 and the immobilized enzyme prepared by the method of the optimal resin D301 in Example 2 were placed at 60 and 65°C for constant temperature incubation, respectively. At certain intervals, the crude enzyme and immobilized enzyme incubated under different conditions were taken out, and the residual enzyme activity was determined according to the enzyme activity detection method.

[0112] The results are as follows Figure 7 As shown, the results are as follows:

[0113] After incubation at 60 and 65℃ for 6 hours, the residual enzyme activity of the immobilized enzyme reached over 80%, while the residual enzyme activity of the crude enzyme solution was 60% (60℃) and less than 20% (65℃), respectively. The residual enzyme activity of the immobilized enzyme was higher than that of the free enzyme, indicating that the immobilized enzyme has better thermal stability and is more tolerant to high temperatures.

[0114] Example 10: Reusability test of immobilized enzyme.

[0115] Weigh 1g of the immobilized enzyme prepared by the optimal resin D301 method in Example 2, and add it to 1mL of the reaction system: 100g / L fructose, 50mM PBS 6.0 solution, 1mM Ni 2+ The reaction was carried out continuously at 70℃. 100 μL of the reaction solution after 5 min was taken to determine the D-allulose yield. The reaction time was 2 h. After the reaction, the immobilized enzyme was washed three times with deionized water, and fresh reaction solution was added before the next batch was reacted. The operation was repeated, and the enzyme activity of each batch was calculated by HPLC to analyze the reusability of the immobilized enzyme.

[0116] The results are as follows Figure 8 As shown, the results indicate that after 20 repeated uses, the residual enzyme activity was still 60%. After 20 repeated uses, the residual enzyme activity decreased rapidly, and after 30 repeated uses, the residual enzyme activity was only 27%.

[0117] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A method for immobilizing ketose 3-epomerase, characterized in that, The method includes: Labedella endophytica Ketosaccharide 3-epomerase was added to a reaction system containing pretreated D301 macroporous weakly basic styrene-based anion exchange resin for adsorption; after cross-linking the enzyme with glutaraldehyde cross-linking agent, immobilized ketose 3-epomerase was obtained; the amino acid sequence of the ketose 3-epomerase is numbered WP_127049469.1 on NCBI. The pretreated ion exchange resin is treated as follows: (1) Soak the resin in 95% ethanol (twice the volume of the resin) for 20 hours, discard the impurities, and soak it in 10% sodium chloride solution for 12 hours. Then wash away the impurities and floating color with deionized water until the solution pH is neutral. (2) Soak the resin treated in step (1) in 2% hydrochloric acid solution for 12 hours, and wash it with deionized water until the solution pH is neutral. (3) Soak the resin in 5% sodium hydroxide solution for 12 hours, and wash it with deionized water until the solution pH is neutral. The pretreated resin is soaked in deionized water (twice the volume of the resin) and stored in a refrigerator at 4°C for later use. When using the resin, filter it and wipe it dry. The total volume of the reaction system was 2 mL. The reaction system contained Tris-HCl buffer at pH 8.0 with a final concentration of 50 mM. The amount of resin added was 0.5 g. The amount of ketose 3-epimerase added to the reaction system was 0.4 mg protein / g resin. The reaction system was adsorbed on a shaker at 18 °C for 2 h, and then placed at 4 °C for 30 min. 0.01% glutaraldehyde was added for cross-linking, and the cross-linking was carried out at 4 °C for 1 h. The supernatant was discarded, and the mixture was washed with deionized water. Vacuum filtration was performed to obtain immobilized D-allulose 3-epimerase, which was stored at 4 °C for later use.

2. The immobilized ketose 3-epimerase prepared by the method of claim 1.

3. The method of claim 1, or the immobilized ketose 3-epimerase of claim 2, in the preparation of products containing D-allulose or D-allulose.

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