Optimized D-psicose 3-epimerase immobilization process

Abstract

The invention relates to the technical field of biology, in particular to an optimized D-psicose 3-epimerase immobilization process. According to the method disclosed by the invention, the immobilization process of various immobilization carriers such as diatomite, polymer microspheres, epoxy group type carriers and epoxy group type carriers is optimized, so that the conversion rate of the D-psicose 3-epimerase in the prior art can reach the same or even higher under the conditions of shorter time and lowest cost, and the optimization strategy enables the production efficiency to be higher and the production cost to be lower. The method is more suitable for large-scale industrial production.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to an optimized process for immobilizing D-allulose 3-epimerase. Background Technology

[0002] D-Allulose is a naturally occurring but very rare sugar, found in trace amounts in figs, raisins, maple syrup, wheat, dates, or certain fruits. It has about 70% the sweetness of sucrose but is extremely low in calories, making it suitable as a low-calorie sweetener in various foods. It is especially suitable for people who need to control their calories, blood sugar, or who are looking for a healthy sugar substitute.

[0003] Because D-allulose is very rare in nature and difficult to obtain in large quantities from natural sources, it is mostly synthesized artificially. Currently, it is mainly produced on a large scale through enzymatic methods. The most common method is to use fructose as a substrate and convert it using specific enzymes, such as D-allulose 3-epimerase (DPEase).

[0004] However, the efficiency of preparing D-allulose using only D-allulose 3-epimerase is not high enough, and the existing immobilization process is also not effective enough. There is an urgent need for a more efficient production method. Summary of the Invention

[0005] In view of this, the present invention provides an optimized immobilization process for D-allulose 3-epimerase, which optimizes the immobilization process of various immobilization carriers such as diatomaceous earth, polymer microspheres, epoxy-based carriers, and epoxy-based carriers, enabling D-allulose 3-epimerase to achieve the same or even higher conversion rates as existing technologies in a shorter time and at the lowest cost. This optimization strategy results in higher production efficiency and is more suitable for large-scale industrial production.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides an immobilized enzyme comprising a carrier and an enzyme, said enzyme including D-allulose 3-epimerase derived from Dorea sp. of the actinomycetes phylum;

[0008] The carrier is:

[0009] (i) Diatomaceous earth; or

[0010] (ii) A carrier whose functional groups include amino groups; or

[0011] (iii) A carrier whose functional groups include polyamine groups; or

[0012] (iv) Supports with functional groups including epoxy groups.

[0013] In some specific embodiments of the present invention, the enzyme of the above-mentioned immobilized enzyme has:

[0014] (1) The amino acid sequence as shown in SEQ ID NO: 2; or

[0015] (2) An amino acid sequence obtained by substituting, deleting, or adding one or more residues as shown in (1), and whose function is the same as or similar to that of (1); or

[0016] (3) An amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the amino acid sequence shown in (1) or (2);

[0017] The "multiple" refers to 2, 3, 4, or 5.

[0018] In some specific embodiments of the present invention, the carrier for the functional group of the above-mentioned immobilized enzyme including amino groups is an amino-type carrier LX1000HA;

[0019] The carrier for the functional groups including polyamine groups is polymer microspheres AGZ-21S;

[0020] The carrier containing the functional group including the epoxy group is the epoxy-type carrier LX1000EP.

[0021] This invention also provides a method for preparing immobilized enzymes, comprising:

[0022] (A) Diatomaceous earth is mixed with an enzyme solution of 1.5 mg / mL, wherein the mass ratio of enzyme to carrier in the enzyme solution is 1:40. The mixture is inverted and mixed for 30 min, then mixed with polyethyleneimine for flocculation for 30 min, wherein the final concentration of polyethyleneimine is 0.2%. Finally, it is covalently cross-linked with glutaraldehyde for 30 min, wherein the final concentration of glutaraldehyde is 0.4%. The mixture is then washed with PBS buffer to obtain the immobilized enzyme; or

[0023] (B) Mix polymer microspheres AGZ-21S with an enzyme solution of 1.5 mg / mL, wherein the mass ratio of enzyme to carrier in the enzyme solution is 1.5:45, 1.5:46, 1.5:47, 1.5:48, 1.5:49, 1.5:50, 1.5:51, 1.5:52, 1.5:53, 1.5:54, 1.5:55, 1.5:56, 1.5:57, 1.5:58, 1.5:59, or 1.5:60. Mix thoroughly by inverting for 30 min, then mix with polyethyleneimine for flocculation for 30 min, the final concentration of polyethyleneimine being 0.2%. Then covalently crosslink with glutaraldehyde for 30 min, the final concentration of glutaraldehyde being 0.4%. Wash with PBS buffer to obtain the immobilized enzyme; or

[0024] (C) Equilibrate the amino-type carrier LX1000HA with 20 mM phosphate buffer (pH 8.0), then activate the carrier with 2% glutaraldehyde solution. Filter to obtain a wet carrier. Mix the enzyme solution with the wet carrier at a mass ratio of 50:1000, 55:1000, 60:1000, 65:1000, 70:1000, 75:1000, 80:1000, 85:1000, 90:1000, 95:1000, or 100:1000. React at 70-80 rpm, 20℃, 21℃, 22℃, 23℃, 24℃, 25℃, 26℃, 27℃, 28℃, 29℃, or 30℃ for 18 h. Filter and wash to obtain the immobilized enzyme; or

[0025] (D) Equilibrate the epoxy-type carrier LX1000EP with 20 mM phosphate buffer at pH 8.0, filter to obtain a wet carrier, mix the enzyme solution with the wet carrier, the mass ratio of enzyme in the enzyme solution to the wet carrier is 50:1000, 55:1000, 60:1000, 65:1000, 70:1000, 75:1000, 80:1000, 85:1000, 90:1000, 95:1000 or 100:1000, react at 70~80 rpm, 20℃, 21℃, 22℃, 23℃, 24℃, 25℃, 26℃, 27℃, 28℃, 29℃ or 30℃ for 18 h, let stand, filter, wash to obtain immobilized enzyme;

[0026] The enzyme in the enzyme solution is D-allulose 3-epimerase derived from Dorea sp. of the actinomycete phylum.

[0027] In some specific embodiments of the present invention, the enzyme prepared by the above method has the following characteristics:

[0028] (1) The amino acid sequence as shown in SEQ ID NO: 2; or

[0029] (2) An amino acid sequence obtained by substituting, deleting, or adding one or more residues as shown in (1), and whose function is the same as or similar to that of (1); or

[0030] (3) An amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the amino acid sequence shown in (1) or (2);

[0031] The "multiple" refers to 2, 3, 4, or 5.

[0032] The present invention also provides an immobilized enzyme prepared according to the above preparation method.

[0033] The present invention also provides the use of any of the following in the preparation of D-allulose:

[0034] (i) the above-mentioned immobilized enzymes;

[0035] (ii) Immobilized enzyme prepared according to the above preparation method.

[0036] This invention also provides a method for preparing D-allulose, comprising:

[0037] The immobilized enzyme was mixed with the reaction solution, reacted, and centrifuged at 13,000 rpm to discard the precipitate, yielding D-allulose.

[0038] The reaction solution includes a substrate and acceptable auxiliaries;

[0039] The immobilized enzyme is the immobilized enzyme described above or the immobilized enzyme prepared according to the above preparation method.

[0040] In some specific embodiments of the present invention, the reaction solution of the above preparation method is a phosphate buffer containing 400 g / L crystalline fructose, 140 μM CoCl2, and pH 7.5.

[0041] In some specific embodiments of the present invention, the reaction conditions of the above preparation method are 60°C and 300 rpm.

[0042] This invention optimizes the immobilization process of various immobilization carriers, such as diatomaceous earth, polymer microspheres, epoxy-based carriers, and epoxy-based carriers. Through this optimization strategy, D-allulose 3-epimerase can operate under more economical and efficient conditions, achieving or even surpassing the conversion level of existing technologies at a lower cost and in a shorter cycle, laying a solid foundation for large-scale commercial production. Attached Figure Description

[0043] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0044] Figure 1 Structural diagrams of amino-type and epoxy-type supports are shown;

[0045] Figure 2 This shows the Maillard reaction that occurs at a reaction temperature of 70°C. Detailed Implementation

[0046] This invention discloses an optimized process for immobilizing D-allulose 3-epimerase. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0047] It should be understood that the expression “one or more of…” individually includes each of the objects described after the expression, as well as various different combinations of two or more of the described objects, unless otherwise understood from the context and usage. The expression “and / or” combined with three or more described objects should be understood to have the same meaning, unless otherwise understood from the context.

[0048] The terms “including,” “having,” or “containing,” including the use of their grammatical synonyms, should generally be understood as open-ended and non-restrictive, for example, not excluding other unstated elements or steps, unless otherwise specifically stated or understood from the context.

[0049] It should be understood that the order of the steps or the order in which certain actions are performed is not important as long as the invention remains operational. Furthermore, two or more steps or actions can be performed simultaneously.

[0050] The use of any and all instances or exemplary language such as “e.g.” or “including” in this document is merely intended to better illustrate the invention and does not constitute a limitation on the scope of the invention. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of the invention.

[0051] Furthermore, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as precisely as possible. However, any value inevitably contains standard deviations due to individual test methods. Therefore, unless explicitly stated otherwise, it should be understood that all ranges, quantities, values, and percentages used in this disclosure are modified with the word "approximately". Here, "approximately" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a specific value or range.

[0052] The polyethyleneimine involved in this invention has CAS 9002-98-6 and a molecular weight of 600.

[0053] Unless otherwise specified, the raw materials, reagents, consumables and instruments involved in this invention are all commercially available products and can be purchased from the market.

[0054] The present invention will be further illustrated below with reference to the embodiments.

[0055] Example

[0056] 1. Plasmid transformation and seed culture preparation

[0057] The DPEase (Y21K) gene sequence is derived from a mutant of the Dorea sp. microorganism, a phylum of actinomycetes. Nucleotide sequence:

[0058] ATGAAGCATGGCATTTATTACGCATATTGGGAAAAAGAATGGGCAGCAGATTATCTGTATAAAGTGGAAAAAGTGGCACGCCTGGGTTTTGATCTGCTGGAAATTGGTGCCGCCCCGCTGCCGGAATATAGCACCGATCAGATTAAAGCACTGCGCGATTGTGCCAGTCAGAATGGCATTCAGCTGACCGCAGGCTATGGTCCGACCTATGATCATAATATGGGTAGTAGTGATGCAGGCATTCGCGCAGGTGCCCTGGAATGGTATAAACGTCTGTTTGATGTGATGGAACAGCTGGATATTCATCTGATTGGTGGCGCACTGTATGGTTATTGGCCGGTTGATTTTAGCAATATTAATAAAGAGGAGGACTGGAAACGTAGCGTTGAAGGTATGCATCTGCTGGCCCCGATTGCCAAAGAACATGATATTAATCTGGGCATGGAAGTGCTGAATCGCTTTGAAAGCCATATTCTGAATACCGCAGAAGAAGGTGTTGCATTTGTTAAAGAAGTGGGTCAGGAAAATGTTAAAGTGATGCTGGATACCTTTCATATGAATATTGAAGAAGAGAGTATCGGCGATGCAATTCGTACCGCAGGTAATCTGCTGGGTCATTTTCATACCGGTGAATGTAATCGCATGGTTCCGGGTAAAGGTCGTACCCCGTGGCGCGAAATTGGCAATGCACTGCGTGATATTGAATATGATGGTACAGTTGTTATGGAACCGTTTGTTAGTATGGGTGGTCAGGTTGGCCGTGATATTCATATTTGGCGCGATATTAGTCGCGGTGCAAGCGAAGCCGAACTGGATAAAGATGCAAAAAATGCAGTGGCATTTCAGAAATATATGCTGGATTGGAAA (SEQ ID NO: 1);

[0059] Amino acid sequence:

[0060] MKHGIYYAYWEKEWAADYLYKVEKVARLGFDLLEIGAAPLPEYSTDQIKALRDCASQNGIQLTAGYGPTYDHNMGSSDAGIRAGALEWYKRLFDVMEQLDIHLIGGALYGYWPVDFSNINKEEDWKRSVEGMHLLAPIAKEHDINL GMEVLNRFESHILNTAEEGVAFVKEVGQENVKVMLDTFHMNIEEESIGDAIRTAGNLLGHFHTGECNRMVPGKGRTPWREIGNALRDIEYDGTVVMEPFVSMGGQVGRDIHIWRDISRGASEAELDKDAKNAVAFQKYMLDWK (SEQ IDNO: 2).

[0061] 2. Shake-flask fermentation

[0062] The recombinant plasmid pET-21b-DPEase (Y21K) was transformed into *E. coli* BL21 (DE3). DPEase-E. coli BL21 (DE3) was plated on LB agar plates containing ampicillin and incubated overnight at 37°C. The next day, single clones were picked and inoculated into 3–5 mL of LB liquid medium containing antibiotics (formulation: 1.0% peptone, 0.5% yeast extract, 1.0% sodium chloride; sterilized at 121°C for 30 minutes). The medium was then incubated at 37°C with a shaker at 220 rpm for 5–8 hours. OD 600 At a growth rate of 0.8–1.2, transfer to 1 L LB liquid medium at an inoculum rate of 0.1% (v / v) and incubate until OD500. 600 Set the temperature to 3-5. Cool to 25°C, add 1 mL of 0.5 M IPTG inducer, and express overnight for 8-12 hours.

[0063] 3. Collecting and breaking down the bacteria

[0064] After expressing the target protein, the fermentation broth was centrifuged at 8000 rpm for 15 min, the supernatant was discarded, and the bacterial cells were collected. The cells were resuspended on ice at a ratio of 10 mL of Lysis Buffer (200 mM NaCl, 25 mM pH 8.0 Tris-HCl, 10 mM imidazole, 5% glycerol) per gram of cells. 0.1% PMSF protease inhibitor was added for lysis. The cells were homogenized using an autoclave (600-800 bar) 2-3 times until complete cell disruption and the liquid was no longer viscous. 0.1% PMSF was added again after lysis. The mixture was centrifuged at 12000 rpm at 4°C for 60 min, and the supernatant was collected. This supernatant, containing the target protein, was stored on ice.

[0065] 4. Affinity chromatography purification

[0066] Since the recombinant target protein contains a His-tag, it was purified using nickel column affinity chromatography.

[0067] 1) Column equilibration: Wash the nickel column with double-distilled water 3-5 times, then incubate with twice the column volume of affinity chromatography binding buffer. Resuspend the column with the crude enzyme solution obtained in the previous step, and flow through the column 2-3 times using a certain volume of nickel column.

[0068] 2) Washing: Wash the nickel column 10 times with twice the column volume of Wash Buffer (150~200 mM NaCl, 25 mM pH 8.0 Tris-HCl, 30 mM imidazole, 5% glycerol) each time to remove impurities and proteins.

[0069] 3) Elution: Elute 5 times with affinity chromatography Elution Buffer (150-200 mM NaCl, 25 mM pH 8.0 Tris-HCl, 150-200 mM imidazole, 5% glycerol), 1 column volume each time, and collect the eluent on ice;

[0070] 4) Column cleaning: Clean the column with double-distilled water 3-5 times, and then recycle the column material;

[0071] 5) Crude enzyme purification: Desalt the crude enzyme solution using an ultrafiltration tube. Gradually replace the crude enzyme solution with phosphate buffer (20 mM, pH 7.5, 5% glycerol) using a 30 kDa ultrafiltration tube. Add the crude enzyme solution in batches to a 30 kDa molecular sieve, centrifuge at 4000 rpm for 15 min, observe after multiple centrifugations, concentrate 15 mL of crude enzyme solution to 1.5 mL, replace with 20 mM phosphate buffer, and continue centrifugation for concentration. Aliquot the concentrated protein into 1.5 mL EP tubes and store at -80℃.

[0072] 5. DPEase Immobilization

[0073] 5.1 Enzyme Immobilization Materials

[0074] The diatomaceous earth was purchased from Maya Reagents, CAS No. 91053-39-3; 61790-53-2, Batch No. MAYA-CR-8606 (http: / / www.maya-r.com / goods-870725.html).

[0075] The polymer microspheres AGZ-21S were purchased from Shandong Junzhi Biotechnology Co., Ltd. It is a large network polymer microsphere with polyamine functional groups. The main raw materials for the synthesis of the material are toluene, divinylbenzene, chlorobenzene, and polyethylene polyamines.

[0076] The amino-type and epoxy-type carriers were purchased from Xi'an Lanxiao Technology Sepite. ® The 1LX-1000 series immobilized enzyme vectors, see the structural diagram below. Figure 1 .

[0077] 5.2 Immobilized enzyme activity assay

[0078] 5.2.1 Immobilized enzyme reaction with substrate

[0079] Weigh 20 mg of immobilized enzyme (diatomaceous earth / polymer microspheres AGZ-21S / amino-type carrier / epoxy-type carrier immobilized enzyme), add 1 mL of reaction solution (containing 400 g / L crystalline fructose, 140 μM CoCl2 or 10 mM MgCl2, pH 7.5 phosphate buffer), and react at 60℃ and 300 rpm. Transfer the supernatant after 30 min, 1 h, 2 h, and 3 h of reaction, and heat at 100℃ for 5 min to terminate the reaction. Centrifuge at 13000 rpm for 10 min, transfer the supernatant to an inner tube, and determine the contents of D-fructose and D-allulose by HPLC.

[0080] 5.2.2 HPLC determination of reaction products

[0081] Instrument parameters for HPLC determination of D-fructose and D-allulose:

[0082] Instrument Consumables: Chromatographic column model: Sugar-Pak Column, 10 µm, 6.5 mm × 300 mm, 1 / pk; LC / MS instrument model: Ultra-high performance liquid chromatograph (LC-40BXR, RID)

[0083] Mobile phase: 50 mg / L EDTA, column temperature: 85℃, mobile phase flow rate: 0.6 mL / min

[0084] Calculate the allulose conversion rate = D-allulose / (D-fructose + D-allulose) * 100%.

[0085] The conversion rates of D-allulose by 20 mg of enzymes immobilized on different carriers over 1 hour, as determined by HPLC, are shown in the table below.

[0086] 5.2 Enzyme Immobilization Method (Before Optimization)

[0087] 5.2.1 Enzyme Immobilization in Diatomite

[0088] Weigh 37.5 mg of diatomaceous earth into a 2 mL centrifuge tube. Add 1 mL of 1.5 mg / mL DPEase pure enzyme solution (phosphate buffer, pH 7.5) at a ratio of enzyme:diatomaceous earth = 1:25 (mg:mg). Mix by inversion for 30 min. Then add 0.2% glutaraldehyde for covalent cross-linking (rotation / stirring) for 30 min. Wash 2-3 times with PBS buffer to obtain the immobilized enzyme.

[0089] Reaction conditions: Reaction temperature 70℃, 20 mg immobilized DPEase diatomaceous earth reacted with 1 mL substrate solution (400 g / L fructose solution containing 140 μM CoCl2 (pH 7.5)). The finished product was a pink, pasty mass.

[0090] Reaction results: The conversion rate of D-allulose was 8.0% after 3 hours. It reacted with D-fructose substrate to form brown flocculent material, which was a Maillard reaction.

[0091] 5.2.2 Conventional methods for AGZ-21S enzyme immobilization

[0092] 16 mg of polymer microspheres AGZ-21S were appropriately swollen in water, the water was removed, and 0.2% glutaraldehyde aqueous solution was added. The mixture was stirred at 25℃ for 5 h. Excess glutaraldehyde was washed with distilled water to complete the cross-linking of glutaraldehyde with the carrier. 1 mL of 1.5 mg / mL DPEase pure enzyme was added, and the mixture was shaken at 60℃ for 8-10 h.

[0093] Table 1: Conversion rate of AGZ-21S without optimization

[0094]

[0095] 5.2.3 Activation and Immobilization of Amino-based Carrier (LX1000HA)

[0096] Add 400 mL of 0.1 M pH 8.0 phosphate buffer to 100 g of carrier, stir for 15 minutes, and monitor the pH to maintain it at 7.8–8.2. After 1 hour, filter and dry. Add the pretreated 100 g of carrier to 400 mL of 2% glutaraldehyde phosphate buffer, stir at 25°C for 1 hour, filter, and wash the carrier with deionized water until the water is clear. Carrier addition amount: Carrier amount = enzyme activity × volume × 25% / 50. Immobilization conditions: Immobilization temperature 25°C, pH 8.0, 18–20 hours.

[0097] 5.2.4 Activation and Enzyme Immobilization of Epoxy-Based Carrier (LX1000EP)

[0098] Add 400 mL of 0.1 M pH 8.0 phosphate buffer to 100 g of carrier, stir for 15 minutes, and monitor the pH to maintain it at 7.8–8.2. After 1 hour, filter and dry. Carrier quantity = enzyme activity × volume × 25% / 50. Immobilization conditions: immobilization temperature 25℃, pH 8.0, 24–36 hours.

[0099] 6. Optimization of enzyme immobilization scheme (after optimization)

[0100] 6.1 Enzyme Immobilization in Diatomite

[0101] Weigh 60 mg of diatomaceous earth into a 2 mL centrifuge tube. Add 1 mL of 1.5 mg / mL DPEase enzyme solution (dissolved in phosphate buffer, pH 7.5) at a ratio of enzyme:diatomaceous earth = 1:40 (mg:mg). Mix by inversion for 30 min. Then add 0.2% of the original volume of polyethyleneimine (rotate / stir) and flocculate for 30 min. Finally, add 0.4% of the original volume of glutaraldehyde and covalently crosslink (rotate / stir) for 30 min. Wash 2-3 times with PBS buffer to obtain the immobilized enzyme.

[0102] Table 2: Conversion rates of D-fructose and D-allulose determined by the optimized ratio of diatomaceous earth immobilization

[0103]

[0104] Table 3: Enzyme activity of DPEase immobilized in diatomaceous earth after 48 hours.

[0105]

[0106] Table 4: Enzyme activity of DPEase immobilized in diatomaceous earth after 14 days.

[0107]

[0108] 6.2 Enzyme Immobilization of AGZ-21S

[0109] Weigh 45-60 mg of polymer microspheres AGZ-21S into a 2 mL EP tube, add 1 mL of 1.5 mg / mL DPEase pure enzyme solution and mix by inversion for 30 min. Then add polyethyleneimine to a final concentration of 0.2% and flocculate for 30 min. Finally, add glutaraldehyde to a final concentration of 0.4% and covalently crosslink for 30 min. Wash with PBS buffer 3-4 times to obtain DPEase immobilized enzyme.

[0110] Temperature optimization conditions:

[0111] Table 5: Conversion rate of D-allulose at reaction temperatures of 70℃, 65℃, 60℃ and 55℃

[0112]

[0113] Table 6: Conversion rate of D-allulose determined after 2 days of storage

[0114]

[0115] Table 7: Conversion rate of D-allulose determined after 2 weeks of storage

[0116]

[0117] 6.3 Activation and Immobilization of Amino-based Carrier (LX1000HA)

[0118] a. Carrier equilibration: Wash repeatedly 2-4 times with 20 mM pH 8.0 phosphate buffer at a carrier / buffer ratio of 1:1 (g:mL), and filter dry after each wash;

[0119] b. Prepare a 2% glutaraldehyde solution using 20 mM pH 8.0 phosphate buffer;

[0120] c. Activation of the carrier: 2% glutaraldehyde was added to the carrier, and the ratio of carrier to glutaraldehyde buffer was 1:4 (g:mL). The mixture was stirred at 20~25℃ for 60 min. After filtration, the carrier was washed with immobilization buffer at a ratio of 1:4 (g:mL). After activation, the carrier turned orange to brownish-yellow.

[0121] d. The protein loading is 50-100 mg / g wet carrier, and the ratio of carrier to enzyme buffer is 1:4 (g:mL). This ratio can be optimized between 1:1 and 1:4.

[0122] e. Add the enzyme-containing buffer and carrier to the reactor and react at 70-80 rpm for 18 h. The immobilization temperature is 20-30℃ (generally 25℃).

[0123] f. After immobilization, collect the immobilized enzyme by filtration, wash with 20 mM phosphate buffer or deionized water, repeating 2–4 times, either by gentle stirring or in a column. Further deep washing with 0.5 M NaCl can be performed, followed by filtration to remove free water. Determine the enzyme activity and water content of the immobilized enzyme, and store at 2–8 °C.

[0124] 6.4 Activation and Enzyme Immobilization of Epoxy-Based Carrier (LX1000EP)

[0125] a. Carrier equilibration: Wash repeatedly 2-4 times with 20 mM pH 8.0 phosphate buffer at a carrier / buffer ratio of 1:1 (g:mL), and filter dry after each wash;

[0126] b. Protein loading is 50~100 mg / g wet carrier;

[0127] c. The ratio of carrier to enzyme buffer is 1:4 (g:mL), which can be optimized between 1:1 and 1:4;

[0128] d. Add the enzyme-containing buffer and carrier to the reactor and react at 70-80 rpm for 18 h, then let it stand for 20 h. The immobilization temperature can be selected at 20-30℃ (generally 25℃).

[0129] e. After immobilization, filter and collect the immobilized enzyme, wash with 20 mM phosphate buffer or deionized water, repeat 2 to 4 times, which can be done by gentle stirring or column washing. 0.5 M NaCl can be used for further deep washing. Filter dry to remove free water, determine the enzyme activity and water content of the immobilized enzyme, and store at 2 to 8 °C.

[0130] Table 8: Conversion rates of D-allulose by DPEase immobilized on different carriers within 30 days.

[0131]

[0132] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An immobilized enzyme, comprising a carrier and an enzyme, characterized in that, The enzyme includes D-allulose 3-epimerase derived from Dorea sp. of the actinomycetes phylum Dorea; The carrier is: (i) Diatomaceous earth; or (ii) A carrier whose functional groups include amino groups; or (iii) A carrier whose functional groups include polyamine groups; or (iv) Supports with functional groups including epoxy groups.

2. The immobilized enzyme as described in claim 1, characterized in that, The enzyme has the following characteristics: (1) The amino acid sequence as shown in SEQ ID NO: 2; or (2) An amino acid sequence obtained by substituting, deleting, or adding one or more residues as shown in (1), and whose function is the same as or similar to that of (1); or (3) An amino acid sequence that is at least 90% homologous to the amino acid sequence shown in (1) or (2); The number of items is 2 to 5.

3. The immobilized enzyme as described in claim 1, characterized in that, The carrier containing amino groups is an amino-type carrier LX1000HA. The carrier for the functional groups including polyamine groups is polymer microspheres AGZ-21S; The carrier containing the functional group including the epoxy group is the epoxy-type carrier LX1000EP.

4. A method for preparing immobilized enzymes, characterized in that, include: (A) Diatomaceous earth is mixed with an enzyme solution of 1.5 mg / mL, wherein the mass ratio of enzyme to carrier in the enzyme solution is 1:

40. The mixture is inverted and mixed for 30 min, then mixed with polyethyleneimine for flocculation for 30 min, wherein the final concentration of polyethyleneimine is 0.2%. Finally, it is covalently cross-linked with glutaraldehyde for 30 min, wherein the final concentration of glutaraldehyde is 0.4%. The mixture is then washed with PBS buffer to obtain the immobilized enzyme; or (B) Mix polymer microspheres AGZ-21S with an enzyme solution of 1.5 mg / mL, wherein the mass ratio of enzyme to carrier in the enzyme solution is 1.5:(45~60). Mix by inversion for 30 min, then mix with polyethyleneimine for flocculation for 30 min, wherein the final concentration of polyethyleneimine is 0.2%. Then covalently crosslink with glutaraldehyde for 30 min, wherein the final concentration of glutaraldehyde is 0.4%. Wash with PBS buffer to obtain immobilized enzyme; or (C) Equilibrate the amino-type carrier LX1000HA with 20 mM phosphate buffer (pH 8.0), then activate the carrier with 2% glutaraldehyde solution. Filter to obtain a wet carrier. Mix the enzyme solution with the wet carrier at a mass ratio of (50-100):1000. React at 70-80 rpm and 20-30°C for 18 h. Filter and wash to obtain the immobilized enzyme; or (D) Equilibrate the epoxy-type carrier LX1000EP with 20 mM phosphate buffer at pH 8.0, filter to obtain a wet carrier, mix the enzyme solution with the wet carrier, the mass ratio of enzyme in the enzyme solution to the wet carrier is (50~100):1000, react at 70~80 rpm and 20~30℃ for 18 h, let stand, filter, wash to obtain immobilized enzyme; The enzyme in the enzyme solution is D-allulose 3-epimerase derived from Dorea sp. of the actinomycete phylum.

5. The preparation method according to claim 4, characterized in that, The enzyme has the following characteristics: (1) The amino acid sequence as shown in SEQ ID NO: 2; or (2) An amino acid sequence obtained by substituting, deleting, or adding one or more residues as shown in (1), and whose function is the same as or similar to that of (1); or (3) An amino acid sequence that is at least 90% homologous to the amino acid sequence shown in (1) or (2); The number of items is 2 to 5.

6. The immobilized enzyme prepared by the method according to claim 4 or 5.

7. Any of the following applications in the preparation of D-allulose: (i) the immobilized enzyme according to any one of claims 1 to 3; (ii) The immobilized enzyme prepared by the method according to claim 4 or 5.

8. A method for preparing D-allulose, characterized in that, include: The immobilized enzyme was mixed with the reaction solution, reacted, and centrifuged at 13,000 rpm to discard the precipitate, yielding D-allulose. The reaction solution includes a substrate and acceptable auxiliaries; The immobilized enzyme is the immobilized enzyme according to any one of claims 1 to 3 or the immobilized enzyme prepared according to the preparation method according to claim 4 or 5.

9. The preparation method according to claim 8, characterized in that, The reaction solution is a phosphate buffer solution containing 400 g / L crystalline fructose, 140 μM CoCl2, and pH 7.

5.

10. The preparation method according to claim 8, characterized in that, The reaction conditions were 60°C and 300 rpm.

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