D-allulose 3-epimerase immobilized enzyme, and preparation method and application thereof

Abstract

The application provides a D-allulose 3-epimerase immobilized enzyme and a preparation method and application thereof. The D-allulose 3-epimerase immobilized enzyme comprises an enzyme carrier and D-allulose 3-epimerase immobilized on the enzyme carrier. The enzyme carrier is a short-chain amino resin with a carbon atom number of 2-4. The D-allulose 3-epimerase is from Rhizobium freirei. The D-allulose 3-epimerase immobilized enzyme has the advantages of high enzyme activity under weak acid conditions, short preparation period of D-allulose and high reusability.

Description

Technical Field

[0001] This invention relates to a D-allulose 3-epimerase immobilized enzyme, its preparation method, and its application. Background Technology

[0002] D-Allulose (D-Psicose), also known as allulose, boasts significant advantages in taste, physicochemical properties, physiological functions, and food safety, making it a promising new generation of natural low-intensity sweetener. Its sweetness and texture are very similar to sucrose, with approximately 70% of the sweetness of sucrose, but its calorie content is far lower (approximately 0.2–0.4 calories per gram of D-Allulose, compared to 3.89 calories per gram of sucrose). For obese individuals and those with diabetes, D-Allulose is an ideal sucrose substitute. As a weak inhibitor of α-glucosidase, α-amylase, maltase, and sucrase, D-Allulose can inhibit the metabolism of starch and disaccharides in the gastrointestinal tract and can suppress blood glucose spikes by protecting pancreatic β-cells, exhibiting potential anti-hyperglycemic effects. Therefore, D-Allulose may help increase insulin sensitivity and reduce the risk of type 2 diabetes. Toxicological studies have confirmed that the median lethal dose (LD50) of D-allulose is 16.3 g / kg, the LD50 of fructose is 14.7 g / kg, and the LD50 of erythritol is 15.3 g / kg. Based on toxicity ratings, D-allulose falls into the "relatively harmless" category (the lowest toxicity rating). In 2014, the U.S. Food and Drug Administration (FDA) officially approved D-allulose as Generally Recognized as Safe (GRAS), allowing its use in food, dietary supplements, and pharmaceutical preparations.

[0003] Currently, the industrial production of D-allulose generally employs a biological method, specifically the preparation of D-allulose under the catalysis of D-allulose-3-epimerase (DPE). However, non-enzymatic browning, also known as the Maillard reaction, easily occurs during the preparation process. Weakly acidic conditions can effectively inhibit the Maillard reaction, but the activity of D-allulose-3-epimerase is low under these conditions. Therefore, finding a way to maintain high enzyme activity under weakly acidic conditions for efficient and rapid production of D-allulose is of significant value in industrial production. Summary of the Invention

[0004] The purpose of this invention is to provide an immobilized D-allulose 3-epimerase, its preparation method, and its applications. The immobilized D-allulose 3-epimerase provided by this invention has advantages such as high enzyme activity under weakly acidic conditions, a short preparation cycle for D-allulose, and high reusability.

[0005] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0006] The first aspect of the present invention is to provide an immobilized D-allulose-3-epimerase, comprising an enzyme carrier and a D-allulose-3-epimerase immobilized on the enzyme carrier, wherein the enzyme carrier is a short-chain amino resin having 2 to 4 carbon atoms, and the D-allulose-3-epimerase is derived from Rhizobium freirei.

[0007] Preferably, the amino acid sequence of the D-allulose 3-epimerase is as shown in SEQ ID NO.1.

[0008] Preferably, the enzyme carrier is LX-1000EA.

[0009] A second aspect of the present invention is to provide a method for preparing D-allulose-3-epimerase immobilized enzyme, the method comprising the following steps:

[0010] (1) Synthesize the nucleotide sequence shown in SEQ ID NO.2;

[0011] (2) Construct an expression vector and genetically engineered bacteria containing the nucleotide sequence described in step (1);

[0012] (3) Cultivate the genetically engineered bacteria obtained in step (2) to obtain D-allulose 3-epimerase;

[0013] (4) The enzyme carrier is mixed with the D-allulose 3-epimerase obtained in step (3) to obtain the D-allulose 3-epimerase immobilized enzyme, wherein the enzyme carrier is a short-chain amino resin with 2 to 4 carbon atoms.

[0014] Preferably, the genetically engineered bacteria is *Escherichia coli* comprising the nucleotide sequence shown in SEQ ID NO.2.

[0015] Preferably, between steps (3) and (4), there is an additional step of purifying the D-allulose 3-epimerase to obtain a pure D-allulose 3-epimerase solution, wherein the pure D-allulose 3-epimerase solution is subjected to step (4).

[0016] More preferably, the purification step includes: collecting the genetically engineered bacteria in step (3) and taking the supernatant after ultrasonic disruption and centrifugation to obtain crude D-allulose 3-epimerase solution; adding the crude D-allulose 3-epimerase solution to a gravity purification column, eluting with imidazole solution, and collecting the eluent to obtain pure D-allulose 3-epimerase solution.

[0017] More preferably, the ultrasonic crushing is performed on ice for 12 to 18 minutes, for example, 13 minutes, 14 minutes, 16 minutes, 17 minutes, etc., with a power of 200W to 500W, an on-time of 1 to 5 seconds, and an off-time of 4 to 8 seconds.

[0018] More preferably, the centrifugation is carried out at 2℃~6℃ and 4000rpm~7000rpm.

[0019] Preferably, the pure enzyme solution of D-allulose 3-epimerase described in step (4) is covalently loaded onto the enzyme carrier.

[0020] Preferably, in step (4), the ratio of the D-allulose 3-epimerase pure enzyme solution to the enzyme carrier is 1g of the enzyme carrier added for every 3mL to 8mL of the D-allulose 3-epimerase pure enzyme solution, for example, 3.5mL, 4.2mL, 4.8mL, 5.4mL, 6.0mL, 6.5mL, etc.

[0021] More preferably, in step (4), the ratio of the D-allulose 3-epimerase pure enzyme solution to the enzyme carrier is 1g of the enzyme carrier added for every 3.5mL to 6mL of the D-allulose 3-epimerase pure enzyme solution.

[0022] Preferably, in step (3), IPTG is added to induce the culture of the genetically engineered bacteria. The concentration of IPTG is 0.15 mmol / L to 0.5 mmol / L, for example, 0.16 mmol / L, 0.22 mmol / L, 0.26 mmol / L, 0.30 mmol / L, 0.35 mmol / L, 0.40 mmol / L, etc.

[0023] More preferably, in step (3), IPTG is added to induce the culture of the genetically engineered bacteria, and the concentration of IPTG is 0.18 mmol / L to 0.3 mmol / L.

[0024] Preferably, the enzyme carrier in step (4) is activated with 3% to 6% glutaraldehyde at 22°C to 26°C and 150 r / min to 200 r / min for 0.5 h to 2 h.

[0025] More preferably, the enzyme carrier in step (4) is activated with 4% to 5.5% glutaraldehyde at 24°C to 26°C and 160 r / min to 190 r / min for 0.8 h to 1.5 h.

[0026] A third aspect of the present invention is to provide a method for preparing D-allulose using D-allulose-3-epimerase immobilized enzyme, the method being as follows: adding the above-mentioned D-allulose-3-epimerase immobilized enzyme or the D-allulose-3-epimerase immobilized enzyme prepared by the above method to a fructose solution, and reacting at a pH of 6.0 to 9.5 and at 40°C to 65°C to obtain the D-allulose.

[0027] Preferably, the reaction is carried out at a pH of 6.0 to 6.5, such as 6.1, 6.2, 6.3, 6.4, etc.

[0028] Preferably, the pH of the reaction is controlled using a HEPES buffer solution with a concentration of 18 mmol / L to 22 mmol / L, wherein the HEPES buffer solution contains 0.05 mmol / L to 0.2 mmol / L Co. 2+ .

[0029] More preferably, the concentration of the HEPES buffer is 19 mmol / L to 21 mmol / L, for example, 19.2 mmol / L, 19.6 mmol / L, 20.2 mmol / L, 20.8 mmol / L, etc.

[0030] More preferably, the HEPES buffer contains Co 2+ The concentration is 0.08 mmol / L to 0.15 mmol / L, for example 0.12 mmol / L, 0.13 mmol / L, 0.14 mmol / L, etc.

[0031] Preferably, the reaction is controlled to be carried out at 55°C to 60°C, for example, 56°C, 57°C, 58°C, 59°C, etc.

[0032] Compared with the prior art, the present invention has the following advantages:

[0033] The D-allulose-3 epimerase immobilized enzyme provided by this invention has high enzyme activity under weakly acidic conditions. The preparation cycle of D-allulose using it is short, requiring only 30 minutes to obtain a high concentration of D-allulose. Furthermore, the enzyme has a high reusability and can be used to continuously react multiple batches under weakly acidic conditions. Attached Figure Description

[0034] Figure 1SDS-PAGE analysis of D-allulose-3-epimerase in Example 1: Lane M: Marker; Lane 1: Crude D-allulose-3-epimerase; Lane 2: Flow-through sample; Lanes 3-10: D-allulose-3-epimerases eluted from the 1st to 8th elutions, respectively.

[0035] Figure 2 The relative enzyme activities of free DPE enzyme and DPE immobilized enzyme of LX-1000EA under different pH conditions;

[0036] Figure 3 The relative enzyme activities of free DPE enzyme and DPE immobilized enzyme of LX-1000EA under different temperature conditions. Detailed Implementation

[0037] The present invention will be further described below with reference to embodiments. However, the present invention is not limited to the following embodiments. The implementation conditions used in the embodiments can be further adjusted according to different requirements of specific applications, and the implementation conditions not specified are conventional conditions in the industry. The technical features involved in the various embodiments of the present invention can be combined with each other as long as they do not conflict with each other.

[0038] This invention provides an immobilized D-allulose 3-epimerase, comprising an enzyme carrier and a D-allulose 3-epimerase immobilized on the enzyme carrier. The enzyme carrier is a short-chain amino resin with 2-4 carbon atoms, and the D-allulose 3-epimerase is derived from Rhizobium freirei. Specifically, the enzyme carrier is LX-1000EA amino resin, and the amino acid sequence of the D-allulose 3-epimerase is shown in SEQ ID NO.1. The DPE immobilized enzyme provided by this invention exhibits high enzyme activity under weakly acidic conditions and also possesses advantages such as a short preparation cycle for D-allulose and high reusability.

[0039] SEQ ID NO.1

[0040] MKHGIYYSYWEHEWSAKFGPYVEKVAKLGFDVIEVAAHHINEYSDAELAEIRRTAKDNNIILTAGIGPSKTKNLSSPDIAVRQAGKAFFEQTLTNVAKLDIKTIGGALHSYWPVDYSKPVDKEGDRARGVEGIHGIADFAGNLG INLCIEVLNRFENHVLNTAAEGVAFVKDVGKPNVKVMLDTFHMNIEEDSFGEAIRTAGPLLGHFHTGESNRRVPGKGRMPWHEIGLALRDISYAGAVVMEPFVKTGGTIGSDIRVWRDLTDGADETKMDEDARNALAFSRFVLGG

[0041] DPE enzyme activity unit: The amount of enzyme required to catalyze the production of 1 μmol of D-allulose within 1 minute is defined as one enzyme activity unit.

[0042] Equilibrium conversion rate: refers to the percentage of D-fructose converted to D-allulose when the reversible chemical reaction of D-fructose to D-allulose reaches chemical equilibrium.

[0043] HPLC detection conditions for D-allulose: Carbomix Ca-NP column, water as mobile phase, column temperature 80℃, flow rate 0.5 mL / min, and detection using a differential detector.

[0044] The present invention will now be described in further detail with reference to specific embodiments.

[0045] Unless otherwise specified, the raw materials and reagents used in the following examples are all commercially available products.

[0046] Example 1

[0047] DPE preparation

[0048] (1) Constructing engineered strains expressing DPE

[0049] A D-allulose-3-epimerase gene sequence containing Rhizobium freirei was synthesized, as shown in SEQ ID NO.2. This gene sequence was ligated into a pET28a(+) plasmid with BamHI and XhoI restriction sites at both ends, respectively, to construct the recombinant expression vector pET28a(+)-His6-3Ep-His6. The recombinant expression vector was transformed into the BL21 expression strain using a heat shock method, and resistance screening and verification were performed.

[0050] SEQ ID NO.2

[0051] ATGAAACACGGTATCTACTACAGCTACTGGGAGCACGAATGGTCCGCAAAATTCGGCCCGTACGTTGAAAAAGTGGCTAAACTGGGCTTTGATGTAATCGAGGTAGCAGCTCACCACATCAACGAGTACTCTGACGCCGAACTGGCAGAAATTCGTCGTACCGCTAAAGACAATAACATCATCCTGACCGCTGGCATCGGCCCTAGCAAAACTAAAAACCTGTCCAGCCCGGATATTGCAGTACGCCAAGCAGGTAAAGCTTTTTTTGAACAGACCCTGACCAATGTTGCTAAACTGGATATTAAGACTATCGGTGGTGCTCTGCACTCCTACTGGCCGGTCGATTACAGCAAACCGGTTGACAAAGAAGGCGACCGTGCGCGTGGTGTCGAAGGCATTCACGGTATCGCGGATTTCGCGGGTAACCTGGGTATTAACCTGTGTATCGAGGTTCTGAACCGTTTCGAAAACCACGTGCTGAACACTGCGGCGGAAGGTGTGGCTTTCGTTAAAGACGTTGGTAAACCAAACGTTAAGGTTATGCTGGACACCTTCCACATGAACATCGAAGAAGATTCCTTCGGCGAAGCGATCCGTACTGCTGGTCCGCTGCTGGGCCATTTCCATACCGGTGAGTCTAACCGCCGTGTGCCGGGCAAAGGTCGCATGCCGTGGCACGAAATCGGTCTGGCGCTGCGTGATATCAGCTACGCCGGTGCTGTAGTCATGGAACCGTTCGTTAAGACCGGCGGTACCATCGGTTCCGATATTCGCGTTTGGCGCGACCTGACTGATGGTGCAGATGAAACCAAAATGGACGAGGATGCGCGTAATGCGCTGGCATTCTCCCGCTTCGTGCTGGGCGGC

[0052] (2) Preparation of crude DPE enzyme solution

[0053] Solid culture: The above strains were inoculated into solid LB medium by streaking and activated, and cultured overnight at 37°C; then a single colony was picked from the LB medium.

[0054] The formula for LB solid medium (g / L) is: 5 g / L yeast extract, 10 g / L peptone, 10 g / L sodium chloride, and 10 g / L agar powder.

[0055] Seed culture: The above single colonies were inoculated into LB liquid medium and cultured at 37℃ and 200r / min for 20h to obtain primary seed culture.

[0056] Liquid culture: The primary seed culture was transferred to 3L of LB liquid medium at an inoculation rate of 1%, and cultured at 37℃ for 5h. Then, 0.2mmol / L IPTG was added, and expression was induced at 16℃ for 16h.

[0057] The formula for LB liquid medium (g / L) is: 5 g / L yeast extract, 10 g / L peptone, and 10 g / L sodium chloride.

[0058] After centrifugation, the expressed strain was collected and centrifuged at 6000 r / min for 15 min at 4 °C. The supernatant was discarded to obtain wet cells. The wet cells were weighed and resuspended in 100 mmol / L potassium phosphate buffer (pH 8.0) to make the final cell concentration 100 g / L. The cells were ultrasonically disrupted on ice for 15 min at 400 W with a 3 s on and 5 s off program. Finally, the cells were centrifuged at 11000 r / min for 10 min at 4 °C. The supernatant was collected, which is the crude DPE enzyme solution.

[0059] (3) Preparation of DPE pure enzyme solution

[0060] The above DPE crude enzyme solution was mixed with BeyoGold TM His-tag purification resin was mixed and placed in a gravity purification column; it was then slowly shaken for 60 min at 4°C on a side-shaking or horizontal shaking table; the cap at the bottom of the purification column was then opened, and the liquid inside the column was allowed to flow out under gravity to wash the column 5 times. D-allulose-3-epimerase was eluted 6–10 times with 0.5 mL of potassium phosphate buffer (pH 8.0) containing 100 mmol / L imidazole to obtain pure DPE enzyme solution. The obtained DPE was then subjected to SDS-PAGE electrophoresis, and the electrophoresis image is shown below. Figure 1 As shown.

[0061] Example 2

[0062] DPE Immobilized Enzyme Preparation

[0063] (1) Resin activation

[0064] Prepare a 5% glutaraldehyde solution using 0.1 mol / L potassium phosphate buffer (pH 8.0).

[0065] Weigh 5g of LX-1000EA amino resin, add 20mL of 0.1mol / L potassium phosphate buffer (pH 8.0) and mix well. Shake at 25℃ and 170r / min for 1h. After washing with distilled water, add 20mL of prepared 5% glutaraldehyde solution and activate at 25℃ and 170r / min for 1h. Then wash with 20mmol / L potassium phosphate buffer (pH 8.0) until no glutaraldehyde residue remains.

[0066] (2) Preparation of DPE immobilized enzyme

[0067] Weigh 4g of the activated amino resin and place it in a 250mL Erlenmeyer flask. Add 20mL of 1.0mol / L potassium phosphate buffer (pH 8.0) and 20mL of DPE pure enzyme solution to the flask and place it in a shaker at 25℃ for 24h to immobilize. Vacuum filter the enzyme, wash the immobilized enzyme particles five times with 20mmol / L potassium phosphate buffer (pH 8.0), dry them, and store them at 4℃ to obtain the DPE immobilized enzyme LX-1000EA.

[0068] Example 3

[0069] Enzymatic Characteristics Study of DPE Immobilized Enzymes

[0070] The enzymatic properties of the DPE immobilized enzyme obtained in Example 2 were studied, including the determination of the optimal pH and optimal temperature.

[0071] (1) Determination of the optimal pH for DPE free enzyme and DPE immobilized enzyme

[0072] Prepare HEPES buffer solutions (20 mmol / L, 0.1 mmol / L Co) at different pH values ​​(pH 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, and 9.5). 2+ ).

[0073] DPE free enzyme activity assay: 200 μL of appropriately diluted DPE pure enzyme solution was added to 800 μL of HEPES buffer containing fructose at different pH values ​​to achieve a final fructose concentration of 80 g / L. After vortexing and mixing, the mixture was placed in a 55℃ water bath for 10 min, and the reaction was terminated by boiling for 10 min to inactivate the enzyme. After centrifugation and membrane filtration, the D-allulose content was analyzed by high-performance liquid chromatography (HPLC). The relative enzyme activity of DPE free enzyme under different pH conditions was calculated based on the D-allulose content measured under each pH reaction condition.

[0074] DPE immobilized enzyme activity assay: Weigh 0.5 g of LX-1000EA DPE immobilized enzyme and add it to 10 mL of HEPES buffer containing fructose at different pH values ​​to achieve a final fructose concentration of 80 g / L. After vortexing and mixing, incubate in a 55°C water bath for 10 min. Take the supernatant and boil for 10 min to inactivate the enzyme and terminate the reaction. After centrifugation and membrane filtration, analyze the D-allulose content by high-performance liquid chromatography (HPLC). Calculate the relative enzyme activity of LX-1000EA DPE immobilized enzyme under different pH conditions based on the D-allulose content measured at each pH reaction condition.

[0075] The relative enzyme activities mentioned above are defined as follows: the enzyme activity with the highest D-allulose yield is defined as 100% relative enzyme activity. The relative enzyme activities of free DPE enzyme and LX-1000EA immobilized enzyme under different pH conditions are shown below. Figure 2 .

[0076] Depend on Figure 2 It can be seen that in the optimal pH determination, the enzyme activity of the DPE immobilized enzyme of LX-1000EA is significantly higher than that of the DPE free enzyme, and the optimal pH increases from 8.5 to 9.0. However, under the conditions of pH 6.0 to 9.5, the relative enzyme activity of the DPE immobilized enzyme of LX-1000EA can still be maintained at more than 70%, which is higher than that of the DPE free enzyme.

[0077] (2) Determination of the optimal temperature for DPE free enzyme and DPE immobilized enzyme

[0078] The enzyme activities of free DPE enzyme and DPE immobilized enzyme of LX-1000EA were measured at different temperatures. The steps were roughly the same as in (1) of this embodiment. The difference was that the pH of HEPES buffer was 9.0 and the incubation temperature was set to 40℃, 45℃, 50℃, 55℃, 60℃ and 65℃ respectively.

[0079] The relative enzyme activities of free DPE enzyme and DPE immobilized enzyme of LX-1000EA under different temperature conditions are as follows: Figure 3 As shown.

[0080] Depend on Figure 3 It can be seen that, in the optimal temperature determination, the DPE immobilized enzyme of LX-1000EA has a temperature 5℃ higher than that of the DPE free enzyme, and its activity is increased by more than 10% under the reaction conditions of 60℃.

[0081] Through experimentation and exploration, the inventors have found that DPE immobilized enzymes obtained using LX-1000EA amino resin have significant advantages under weakly acidic conditions compared to other types of resin materials, such as epoxy resin.

[0082] Example 4

[0083] Stability determination of DPE immobilized enzyme at different temperatures

[0084] The preparation of DPE-immobilized enzymes using two other types of amino resin materials follows the same steps as in Example 2, except that the enzyme carriers bound to DPE are LX-1000HA and LX-1000HFA, respectively.

[0085] The DPE free enzyme and the three types of immobilized enzymes mentioned above were mixed with HEPES buffer containing fructose and incubated at 50℃, 55℃ and 60℃ respectively. Samples were taken at fixed time intervals to determine the enzyme activity of each enzyme. The enzyme activity measured in the enzyme solution without incubation was recorded as the relative enzyme activity of 100%. The half-life of each enzyme at the above three temperatures was measured and is shown in Table 1.

[0086] Table 1

[0087]

[0088] As shown in Table 1, among the three types of immobilized enzymes, the DPE immobilized enzyme with amino resin material LX-1000EA has a longer half-life than the other two types of DPE immobilized enzymes. Under standard reaction conditions of 55℃, the half-life of the DPE immobilized enzyme with LX-1000EA can reach 14 times that of the free enzyme, up to 56 hours; at 60℃, the half-life can reach 5 times that of the free DPE enzyme, up to 11 hours. Compared with other materials, the DPE immobilized enzyme with amino resin material LX-1000EA has great potential for industrial application.

[0089] Example 5

[0090] Stability determination of DPE immobilized enzyme at different pH levels

[0091] The three types of immobilized enzymes were mixed with HEPES buffer containing fructose and reacted in multiple batches at 55°C and pH 6.5 and 6.0, respectively. The equilibrium conversion rate and enzyme activity of each batch were measured, and the results are shown in Table 2.

[0092] Table 2

[0093]

[0094] The DPE immobilized enzyme of LX-1000EA maintained an equilibrium conversion rate above 28% at pH 6.5, with enzyme activity decreasing from the 32nd batch to half its original value by the 36th batch (see Table 2). At pH 6, the DPE immobilized enzyme of amino resin material LX-1000EA maintained an equilibrium conversion rate above 28%, with enzyme activity decreasing from the 17th batch to half its original value by the 20th batch (see Table 2). The other two immobilized enzymes did not perform well at pH 6.5 and 6.0. Therefore, the DPE immobilized enzyme of LX-1000EA has excellent industrial application potential under weakly acidic conditions where Maillard reactions are unlikely, and can even produce multiple batches under the more stringent pH 6 conditions, representing a significant improvement over current production levels.

[0095] Example 6

[0096] DPE immobilized enzyme continuous transformation

[0097] Based on the equilibrium conversion characteristics of DPE, 50g of LX-1000EA DPE immobilized enzyme and a chromatographic column were used to prepare a continuous conversion reaction tube, which was then placed in a 55℃ water bath to maintain the reaction temperature. The DPE immobilized enzyme was continuously converted at a fructose concentration of 750g / L and a flow rate of 0.5mL / min, reacting in fructose solutions at pH 6, 6.5, and 7, respectively. The product was analyzed every 10 minutes. After the reaction reached equilibrium, samples were taken periodically to detect the D-allulose concentration and conversion rate, and the continuous conversion reaction time was calculated. Before the conversion rate fell below 25%, the reaction could continue for 15 days at pH 6, 17 days at pH 6.5, and up to 23 days at pH 7.

[0098] Example 7

[0099] Application of DPE immobilized enzyme in the conversion of D-fructose to D-allulose

[0100] Add 1.5L of HEPES buffer (20mmol / L, pH 6.0, 0.1mmol / L) containing fructose to a 3L fermenter. 2+ In the reaction, the final fructose concentration was 750 g / L, and 6 g of the DPE immobilized enzyme of LX-1000EA prepared according to the method in Example 2 was added. The reaction temperature was 55°C. Every 5 minutes, 1 mL of the reaction solution was taken out, and the content of D-fructose and D-allulose in the sample was detected using a differential detector. After catalytic reaction for 30 min, the concentration of D-allulose was measured to be 212.1 g / L, and the conversion rate was 28.2%. According to the definition of DPE enzyme activity unit, the enzyme activity of the DPE immobilized enzyme of LX-1000EA under this pH condition was calculated to be 1478.1 U / mg.

[0101] The above results show that:

[0102] (1) Enzymatic characteristics studies of three DPE immobilized enzymes showed that the enzyme activity and reusability of the DPE immobilized enzyme of LX-1000EA were the best, and it has better prospects for industrial application.

[0103] (2) The enzyme activity of DPE immobilized enzyme in LX-1000EA was 1478.1 U / mg under weakly acidic conditions (pH 6.0), which is more than twice that of free DPE enzyme.

[0104] (3) The half-life of the DPE immobilized enzyme of LX-1000EA is 56 hours under the reaction conditions of 55℃, which is 14 times that of the free DPE enzyme. Under the higher temperature of 60℃, the half-life is 11 hours, which is 5 times that of the free DPE enzyme.

[0105] (4) The DPE immobilized enzyme with amino resin LX-1000EA can be continuously and repeatedly produced for at least 36 batches under conditions of 55℃ and pH 6.0.

[0106] (5) The DPE immobilized enzyme with amino resin LX-1000EA can be continuously transformed for 15 days at 55℃ and pH 6.0.

[0107] (6) The preparation of D-allulose using the DPE immobilized enzyme of LX-1000EA is a short process, requiring only 30 minutes to obtain a high concentration of D-allulose.

[0108] In summary, the LX-1000EA DPE immobilized enzyme provided by this invention has the advantages of high enzyme activity under weakly acidic conditions, short preparation cycle for D-allulose, and high reusability.

[0109] The present invention has been described in detail above, with the aim of enabling those skilled in the art to understand and implement the invention. However, this description should not be construed as limiting the scope of protection of the invention. All equivalent changes or modifications made in accordance with the spirit and essence of the invention should be included within the scope of protection of the invention.

Claims

1. A method for preparing D-allulose, characterized in that: D-allulose 3-epimerase immobilized enzyme was added to a fructose solution and reacted at pH 6.0–6.4 and 40–65°C to obtain the D-allulose; wherein the D-allulose 3-epimerase immobilized enzyme comprises an enzyme carrier and D-allulose 3-epimerase immobilized on the enzyme carrier; the enzyme carrier is LX-1000EA; the D-allulose 3-epimerase is derived from Rhizobium freirei, and its amino acid sequence is shown in SEQ ID NO.

1.

2. The method for preparing D-allulose according to claim 1, characterized in that, The preparation method of the D-allulose 3-epimerase immobilized enzyme includes the following steps: (1) Synthesize the nucleotide sequence shown in SEQ ID NO.2; (2) Construct a genetically engineered bacterium containing the nucleotide sequence described in step (1); (3) Cultivate the genetically engineered bacteria obtained in step (2) to obtain D-allulose 3-epimerase; (4) The enzyme carrier is mixed with the D-allulose 3-epimerase obtained in step (3) to obtain the D-allulose 3-epimerase immobilized enzyme.

3. The method for preparing D-allulose as described in claim 2, characterized in that, The genetically engineered bacteria is Escherichia coli containing the nucleotide sequence shown in SEQ ID NO.

2.

4. The method for preparing D-allulose as described in claim 2, characterized in that, Between steps (3) and (4), there is also a step of purifying the D-allulose 3-epimerase to obtain a pure D-allulose 3-epimerase solution, wherein the pure D-allulose 3-epimerase solution is subjected to step (4).

5. The method for preparing D-allulose as described in claim 4, characterized in that, In step (4), the D-allulose 3-epimerase pure enzyme solution is covalently loaded onto the enzyme carrier; the ratio of the D-allulose 3-epimerase pure enzyme solution to the enzyme carrier is 1g of the enzyme carrier for every 3mL to 8mL of the D-allulose 3-epimerase pure enzyme solution.

6. The method for preparing D-allulose as described in claim 2, characterized in that, In step (3), IPTG is added to induce the culture of the genetically engineered bacteria, and the concentration of IPTG is 0.15 mmol / L to 0.5 mmol / L.

7. The method for preparing D-allulose as described in claim 2, characterized in that, The enzyme carrier described in step (4) is activated with 3% to 6% glutaraldehyde at 22°C to 26°C and 150 to 200 r / min for 0.5 to 2 hours.

8. The method for preparing D-allulose according to claim 1, characterized in that: The reaction was carried out at a pH of 6.0–6.

2.

9. The method for preparing D-allulose according to claim 1, characterized in that: The pH of the reaction was controlled using a HEPES buffer solution with a concentration of 18 mmol / L to 22 mmol / L, the HEPES buffer solution containing 0.05 mmol / L to 0.2 mmol / L Co. 2+ .

10. The method for preparing D-allulose according to claim 1, characterized in that: The reaction was carried out at 55°C to 60°C.

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