D-allulose-3-epimerase mutant, host cell, and its application in allulose synthesis.

Abstract

This invention discloses a D-allulose-3-epimerase mutant, its host cell, and its application in the synthesis of allulose, belonging to the field of genetic engineering technology. This invention utilizes a mutant derived from Ruminococcus CAG55 (… Ruminococcus sp. Simultaneous mutations at positions 47, 72, 114, and 221 of wild-type D-allulose-3-epimerase (CAG55) yielded a series of D-allulose-3-epimerase mutants, significantly improving their catalytic activity and stability. The mutants obtained by simultaneous mutations at positions 47, 72, 114, and 221 exhibited the highest catalytic activity. The D-allulose-3-epimerase mutants provided by this invention have significant application value in the production of D-allulose.

Description

[0001] This application is a divisional application of the patent application filed on August 5, 2025, with application number 2025110860765, entitled "D-allulose-3-epimerase mutant, host cell and its application in the synthesis of allulose". Technical Field

[0002] This invention belongs to the field of genetic engineering technology, specifically involving D-allulose-3-epimerase mutants, host cells, and their application in the synthesis of allulose. Background Technology

[0003] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0004] D-Allulose, a low-calorie (approximately 0.2 kcal / g, compared to 4 kcal / g for sucrose) and high-sweetness (up to 70% of sucrose), is a rare functional ketose with significant applications in the food, health supplement, and pharmaceutical industries. Its unique physiological activities, including blood glucose and lipid regulation, antioxidant effects, and neuroprotective properties, make it an ideal choice for diabetic dietary supplements and healthy sweeteners.

[0005] Currently, industrial production mainly relies on D-allulose-3-epimerase (DAEase) to catalyze the conversion of D-fructose. However, existing natural DAEases suffer from several drawbacks: low catalytic efficiency and insufficient activity for converting the substrate D-fructose; and poor stability, making them unsuitable for the high-temperature, high-sugar, and high-ionic-strength production environments encountered in large-scale industrial production. Therefore, developing DAEase mutants with both high activity and high stability is an urgent need for industry. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a series of D-allulose-3-epimerase mutants, which solve the problems of limited catalytic activity, low conversion efficiency, poor stability, and unfavorable conditions for industrial production of existing D-allulose-3-epimerases.

[0007] To achieve the above objectives, the technical solution of the present invention is as follows:

[0008] In a first aspect, this invention provides a D-allulose-3-epimerase mutant, the NCBI accession number for D-allulose-3-epimerase being CDC15199.1, with the mutation site being:

[0009] F47A; or

[0010] N72E; or

[0011] N114G; or

[0012] C221S; or

[0013] F47A and N72E; or

[0014] F47A, N72E, and N114G; or

[0015] F47A, N72E, N114G, and C221S.

[0016] In a second aspect, the present invention provides a nucleic acid molecule having a nucleotide sequence encoding the above-described D-allulose-3-epimerase mutant.

[0017] A third aspect of the present invention provides an expression vector having the above-described nucleic acid molecule.

[0018] In some specific embodiments of the present invention, the expression vector comprises pET-28a plasmid or PWB980 plasmid.

[0019] A fourth aspect of the present invention provides a host cell having any of the following:

[0020] (a1) The aforementioned nucleic acid molecules;

[0021] (a2) The aforementioned expression carriers;

[0022] The host cell is bacteria.

[0023] In some specific embodiments of the present invention, the host cell is Escherichia coli or Bacillus subtilis.

[0024] A fifth aspect of the present invention provides a method for preparing a D-allulose-3-epimerase mutant, which is prepared based on any of the following:

[0025] (b1) The aforementioned nucleic acid molecules;

[0026] (b2) The aforementioned expression carriers;

[0027] (b3) The host cells mentioned above.

[0028] A sixth aspect of the present invention provides the application of the above-described D-allulose-3-epimerase mutant in the preparation of D-allulose.

[0029] A seventh aspect of the present invention provides a method for preparing D-allulose, comprising: preparing D-allulose based on the above-described host cells;

[0030] The prepared reaction system comprises: 450-650 g / L fructose, 1 mM Co 2+ 5-25 g / L of wet bacterial cells containing the host cells described above;

[0031] The preparation temperature is 50-60℃;

[0032] The preparation time is 4-6 hours.

[0033] It should be noted that the term "wet cell mass" refers to a collection of microbial cells containing a certain amount of moisture, collected from a liquid culture medium (such as fermentation broth) through centrifugation, filtration, or other separation methods.

[0034] In some specific embodiments of the present invention, the concentration of fructose is 450 g / L, 460 g / L, 470 g / L, 480 g / L, 490 g / L, 500 g / L, 510 g / L, 520 g / L, 530 g / L, 540 g / L, 550 g / L, 560 g / L, 570 g / L, 580 g / L, 590 g / L, 600 g / L, 610 g / L, 620 g / L, 630 g / L, 640 g / L, or 650 g / L.

[0035] In some specific embodiments of the present invention, the concentration of the wet bacterial cells is 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, 11 g / L, 12 g / L, 13 g / L, 14 g / L, 15 g / L, 16 g / L, 17 g / L, 18 g / L, 19 g / L, 20 g / L, 21 g / L, 22 g / L, 23 g / L, 24 g / L, or 25 g / L.

[0036] In some specific embodiments of the present invention, the prepared reaction system comprises: 500 g / L fructose, 1 mMCo 2+ 15 g / L of wet bacterial cells containing the host cells.

[0037] In some specific embodiments of the present invention, the prepared reaction system comprises: 600 g / L fructose, 1 mMCo 2+ 15 g / L of wet bacterial cells containing the host cells.

[0038] In some specific embodiments of the present invention, the preparation temperature is 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, or 60°C.

[0039] In some specific embodiments of the present invention, the preparation time is 4 hours, 4.5 hours, 5 hours, 5.5 hours or 6 hours.

[0040] In some specific embodiments of the present invention, the preparation temperature is 55°C and the time is 6 hours.

[0041] An eighth aspect of the present invention provides a method for preparing D-allulose, comprising: preparing D-allulose based on the above-described D-allulose-3-epimerase mutant;

[0042] The prepared reaction system comprises: 450-650 g / L fructose, 1 mM Co 2+ 5-25 g / L of the D-allulose-3-epimerase mutant;

[0043] The preparation temperature is 50-60℃;

[0044] The preparation time is 4-6 hours.

[0045] In some specific embodiments of the present invention, the concentration of fructose is 450 g / L, 460 g / L, 470 g / L, 480 g / L, 490 g / L, 500 g / L, 510 g / L, 520 g / L, 530 g / L, 540 g / L, 550 g / L, 560 g / L, 570 g / L, 580 g / L, 590 g / L, 600 g / L, 610 g / L, 620 g / L, 630 g / L, 640 g / L, or 650 g / L.

[0046] In some specific embodiments of the present invention, the concentration of the D-allulose-3-epimerase mutant is 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, 11 g / L, 12 g / L, 13 g / L, 14 g / L, 15 g / L, 16 g / L, 17 g / L, 18 g / L, 19 g / L, 20 g / L, 21 g / L, 22 g / L, 23 g / L, 24 g / L, or 25 g / L.

[0047] In some specific embodiments of the present invention, the prepared reaction system comprises: 500 g / L fructose, 1 mMCo 2+ ,20 g / L of the D-allulose-3-epimerase mutant.

[0048] In some specific embodiments of the present invention, the preparation temperature is 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, or 60°C.

[0049] In some specific embodiments of the present invention, the preparation time is 4 hours, 4.5 hours, 5 hours, 5.5 hours or 6 hours.

[0050] In some specific embodiments of the present invention, the preparation temperature is 55°C and the time is 4 hours.

[0051] In some specific embodiments of the present invention, the preparation temperature is 55°C and the time is 6 hours.

[0052] The beneficial effects of this invention are as follows:

[0053] This invention utilizes the method of processing bacteria derived from rumenococcus CAG55 ( Ruminococcus sp. Simultaneous mutations at positions 47, 72, 114, and 221 of wild-type D-allulose-3-epimerase (CAG55) yielded a series of D-allulose-3-epimerase mutants, significantly improving their catalytic activity and stability. The mutants obtained by simultaneous mutations at positions 47, 72, 114, and 221 exhibited the highest catalytic activity. Testing revealed that when producing D-allulose from fructose, with *E. coli* as the host bacterium, the engineered *E. coli* strain catalyzed the production of D-allulose from 600 g / L fructose for 4 hours, achieving a conversion rate of 36.3%. With *Bacillus subtilis* as the host bacterium, the supernatant of *B. subtilis* fermentation broth expressing *B. subtilis* catalyzed the production of D-allulose from 500 g / L fructose for 5 hours, achieving a conversion rate of 35.5%. Intracellular expression of *B. subtilis* fermentation broth supernatant catalyzed the production of D-allulose from 500 g / L fructose for 4 hours, achieving a conversion rate of 35.8%. Furthermore, after immobilization, the enzyme retained its activity at 80% even after 30 repeated batches of use. Therefore, the D-allulose-3-epimerase mutant provided by this invention has significant application value in the field of D-allulose production. Attached Figure Description

[0054] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0055] Figure 1 In Embodiment 5 of the present invention E.coli Graph of the reaction process of fructose to D-allulose catalyzed by BL21(DE3) / / pET28a-RsDAE-F47A-N72E-N114G-C221S.

[0056] Figure 2 The secretory expression strain in Example 6 of this invention Bacillus subtilisSchematic diagram of the reaction process for the synthesis of D-allulose from fructose catalyzed by BS168 / PWB980-RsDAE-F47A-N72E-N114G-C221S.

[0057] Figure 3 The intracellular expression strain in Example 6 of this invention Bacillus subtilis Schematic diagram of the reaction process for the synthesis of D-allulose from fructose catalyzed by BS168 / PWB980-RsDAE-F47A-N72E-N114G-C221S. Detailed Implementation

[0058] This invention discloses a D-allulose-3-epimerase mutant, a host cell, and its application in the synthesis of allulose. 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 is particularly important to note 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.

[0059] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0060] 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.

[0061] The culture medium formulations used in the following examples are as follows:

[0062] LB medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, water as solvent, pH 7.4.

[0063] LB plates: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, 18 g / L agar, water as solvent, pH 7.4.

[0064] The concentration of the product D-alokulose was determined by high performance liquid chromatography (HPLC). The analytical method was as follows: column type: calcium cation exchange column; mobile phase: water; injection volume: 10 μL; differential detector: detection time: 25 min; flow rate: 0.6 mL / min; column temperature: 80℃.

[0065] Sample preparation: Take 100 μL of the sample after the reaction is completed, dilute it 10 times with aqueous solution, filter it through a 0.22 μm filter membrane, and perform HPLC detection.

[0066] Example 1

[0067] 1. Construction of expression vectors and engineered bacteria:

[0068] Through library mining, a sample derived from Ruminococcus CAG55 was screened. Ruminococcus sp. The D-allulose-3-epimerase (CAG:55), NCBI accession number CDC15199.1, was synthesized in its entirety by Nanjing Genscript Biotech Co., Ltd. The nucleotide sequence is shown in SEQ ID NO.1 and the amino acid sequence is shown in SEQ ID NO.2.

[0069] The nucleotide sequence of D-allulose-3-epimerase (SEQ ID NO.1):

[0070] ATGAATAAAATAGGGGTACACTTTGGATATTTCAACCGTGACTGGAATACGGACTTCATCAAGCGCATTGAGCAAGTGAAAAAGATTGGTCTGGATATTTTGGAAGTCGCACCGGCACCGCTCTTAGCCTTGACCAAATTCCAACGCGACGAGATCGCCGCTGCGGCGAAAGCTAATGATATCGAGCTGACTTTCAGCGTTGGCTTGTCTGCGAACCAGGATCTGGCGAGCGAGGACGAAGAGATCCGTAAGAACGGCATTAAGTTTACCACCGATACCTTTCAAATTATGAGCGAGATGGGTGGTAAGACGTACAGTGGTGTTGATATCGCGGCTTGGAACAAAACCTTCATGGAAGGGATCACTGACAAAAGCGCTACCTGGGAACGTAGCATTAGCGCTGTGAAGGAGATTATGAAAGTGGCCGAAGACAAAGGTATCACCTTCGCCGTTGAAGTTGTCAACCGCTACGAATCGTCCTTGGTTAACACCGCGGAAGAAGCAGTGAAGTATGTGGACGAAGTGGGCAGCCCGAACTGCAAGATCCTGCTTGACACGTATCACATGAATATTGAGGAGGATAGCTTTGCGGGTGCGATCAAGCTGGTAGGCAATCGGCTGGGTCATTTTCACGTGGGCGAGTCCAACCGCCGTCCACCGTGTGAAAACGGTAAAATGCCGTGGAACGAAATCACCAATGCACTGAAAGAAATCGATTACCAGGGCGCGATTGTTATGGAGCCGTTTATTAAGATGGGCGGTGAAGTTGGTCGTGACATCAAGGTGTGGCGTGACATCTCTGAAGGTGCGTCGGAGTCCGAGATGGAGCAGCTGCTGGCGGATGCAGCGATGATGCTGCGTAAAAAAATGCAGCGTTAA

[0071] Amino acid sequence of D-allulose-3-epimerase (SEQ ID NO.2):

[0072] MNKIGVHFGYFNRDWNTDFIKRIEQVKKIGLDILEVAPAPLLALTKFQRDEIAAAAKANDIELTFSVGLSANQDLASEDEEIRKNGIKFTTDTFQIMSEMGGKTYSGVDIAAWNKTFMEGITDKSATWERSISAVKEIMKVAEDKG ITFAVEVVNRYESSLVNTAEEAVKYVDEVGSPNCKILLDTYHMNIEEDSFAGAIKLVGNRLGHFHVGESNRRPPCENGKMPWNEITNALKEIDYQGAIVMEPFIKMGGEVGRDIKVWRDISEGASESEMEQLLADAAMMLRKKMQR

[0073] 1. Design primers F1, R1, F2 and R2 based on the nucleotide sequence shown in SEQ ID NO.1 and the pET-28a vector sequence.

[0074] F1: 5'-CTTTAAGAAGGAGATATACCATGAATAAAATAGGGGTACACTTTGG-3' (SEQ ID NO.3)

[0075] R1: 5'-TGGTGGTGGTGGGTGCTCGAGTTAACGCTGCATTTTTTTACGC-3' (SEQ ID NO.4)

[0076] F2: 5'-CTCGAGCACCACCACCACC-3' (SEQ ID NO.5)

[0077] R2: 5'-GGTATATCTCCTTCTTAAAGTTAAACAAAAT-3' (SEQ ID NO.6)

[0078] 2. Using pET-28a plasmid as an expression vector, construct Escherichia coli... E.coli BL21(DE3) / / pET28a-RsDAE

[0079] 2.1 Construction of expression plasmids:

[0080] Initiated by primers F1 / R1 and F2 / R2, the target gene was amplified using high-fidelity Pfu DNA polymerase to obtain the D-allulose-3-epimerase gene sequence with homologous arms. Using pET-28a plasmid as a template, the linearized vector sequence was amplified using high-fidelity Pfu DNA polymerase to obtain the linearized vector sequence. Homologous recombination was then performed between the target gene and the linearized vector using homologous recombination enzyme to construct the expression plasmid pET28a-RSDAE.

[0081] 2.2 Construction of recombinant Escherichia coli:

[0082] Stored at -80℃ E.coli BL21(DE3) competent cells were incubated at 0°C on ice for 10 min, then 5 µL of the homologous recombinant product (expression plasmid pET28a-RSDAE) was added in a clean bench, incubated at 0°C on ice for 30 min, heat-shocked in a 42°C water bath for 90 s, incubated at 0°C on ice for 2 min, and then 600 µL of LB medium was added. The cells were cultured at 37°C and 200 rpm for 1 h. The culture was then spread on LB plates containing 50 μg / mL kanamycin and cultured at 37°C for 8–12 h. Clones were randomly selected, plasmids were extracted, and sequenced for identification. Recombinant *E. coli* containing the expression plasmid pET28a-RSDAE were screened for these cells. E.coli BL21(DE3) / / pET28a-RsDAE.

[0083] Example 2

[0084] The expression of D-allulose-3-epimerase was induced to obtain wet bacterial cells containing the D-allulose-3-epimerase gene:

[0085] The recombinant Escherichia coli obtained in Example 1 were respectively... E.coli BL21(DE3) / / pET28a-RsDAE was inoculated into LB liquid medium containing 50 μg / mL kanamycin resistance and cultured at 37°C and 200 rpm for 12 h. Then, it was inoculated at a 1% (v / v) inoculation rate into fresh LB liquid medium containing 50 μg / mL kanamycin resistance and cultured at 37°C and 200 rpm until the bacterial OD reached the target cell count. 600 When the concentration reached 0.6-0.8, isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 0.1 mM. After induction culture at 25°C for 16 h, the mixture was centrifuged at 4°C and 8000 rpm for 20 min. The supernatant was discarded, and the precipitate was collected to obtain recombinant Escherichia coli containing D-allulose-3-epimerase. E.coli Wet cells of BL21(DE3) / / pET28a-RsDAE.

[0086] Example 3

[0087] Construction of a mutant library of D-allulose-3-epimerase gene:

[0088] The recombinant Escherichia coli expressing D-allulose-3-epimerase constructed in Example 2 E.coli BL21(DE3) / / pET28a-RsDAE is the originating strain.

[0089] Modification was performed using directed evolutionary theory, based on the crystal structure of D-allulose-3-epimerase obtained through homology modeling. Based on protein folding free energy calculations, site-directed mutagenesis was performed on stability-enhancing sites T17G, P38A, F47A, V67G, S70P, N72E, T90L, Q95D, D109L, N114G, M118A, S160Y, C221S, and M284A.

[0090] The mutant PCR system (100 μL) consisted of: 25 μL of 2*PhantaMax buffer, 1 μL of dNTPs, 1 μL each of the upper and lower mutant primers (Table 1), 1 μL of template (starting strain), 0.5 μL of Pfu DNA polymerase, and ddH2O added to a final volume of 50 μL.

[0091] The PCR conditions were: 95℃ pre-denaturation for 3 min, followed by 30 cycles: 95℃ for 15 s, 60℃ for 15 s, 72℃ for 7 min 20 s, and finally 72℃ for 10 min final extension.

[0092] PCR results were verified positive by DNA agarose gel electrophoresis: PCR products were digested with DpnI enzyme, inactivated at 37°C for 1 hour, then at 200 rpm for 1 minute at 65°C. The PCR products were then transformed by heat shock into E. coli. E.coli BL21(DE3) was activated, incubated at 37°C and 200 rpm for 1 hour, and then spread onto LB plates containing 50 μg / mL kanamycin resistance. The plates were incubated upside down at 37°C overnight.

[0093] Table 1. Primer design for site-directed mutagenesis of D-allulose-3-epimerase

[0094]

[0095] DNA sequencing revealed that the site-directed mutations in T17G, P38A, F47A, V67G, S70P, N72E, T90L, Q95D, D109L, N114G, M118A, S160Y, C221S, and M284A were completely consistent with the intended mutations.

[0096] Example 4

[0097] Screening of D-allulose-3-epimerase gene mutant libraries:

[0098] Single colonies were picked from the plates obtained in Example 3 and inoculated into LB liquid medium containing 50 μg / mL kanamycin resistance. The culture was carried out at 37°C and 200 rpm for 12 h. Then, a 1% (v / v) inoculum was added to fresh LB liquid medium containing 50 μg / mL kanamycin resistance and cultured at 37°C and 200 rpm until the bacterial OD reached the target cell count. 600 When the concentration reaches 0.6~0.8, add isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 0.1 mM. After induction culture at 25℃ for 16 h, centrifuge at 4℃ and 8000 rpm for 20 min, discard the supernatant, collect the precipitate, and obtain wet bacterial cells containing the D-allulose-3-epimerase gene mutant library.

[0099] 1. Initial screening:

[0100] Preparation of reaction solution (200 μL): final concentration of 300 g / L substrate fructose, final concentration of 1 mM Co 2+ The catalyst dosage is 5 g / L based on the total weight of wet bacterial cells, and the reaction solution is prepared using purified water as the reaction medium.

[0101] Reaction conditions: After reacting for 1 hour in a reactor at 55℃ and 500 rpm, 20 μL of the reaction sample was taken after the reaction was completed, diluted 20 times, filtered through a 0.22 μm filter membrane, and analyzed by HPLC. The results are shown in Table 2.

[0102] Table 2 Initial screening reaction results

[0103]

[0104] Initial screening revealed that mutants F47A, N72E, N114G, and C221S produced higher concentrations of D-allulose than the parent mutant when catalyzing the preparation of D-allulose from fructose, indicating enhanced catalytic activity in these four mutants. Subsequent experiments were conducted using mutants F47A, N72E, N114G, and C221S.

[0105] 2. Secondary screening:

[0106] The strains obtained from the initial screening were then subjected to a second screening.

[0107] The mutants F47A+N72E, F47A+N72E+N114G, and F47A+N72E+N114G+C221S were constructed using the method described in Example 3. DNA sequencing of these three mutants showed that the results were completely consistent with the intended design mutations.

[0108] Re-screening reaction solution (10 mL): final concentration of 300 g / L substrate fructose, final concentration of 1 mM Co 2+ The catalyst dosage is 5 g / L based on the total weight of wet bacterial cells, and the reaction solution is prepared using purified water as the reaction medium.

[0109] Reaction conditions: After reacting for 1 hour in a reactor at 55℃ and 500 rpm, 20 μL of the reaction sample was taken after the reaction was completed, diluted 20 times, filtered through a 0.22 μm filter membrane, and analyzed by HPLC. The results are shown in Table 3.

[0110] The test results are shown in Table 3.

[0111] Table 3 Results of the secondary screening reaction

[0112]

[0113] After further screening, it was confirmed that the catalytic activity of all seven mutants recorded in Table 3 was significantly improved. Among them, mutant F47A+N72E+N114G+C221S exhibited the highest catalytic activity. Therefore, mutant F47A+N72E+N114G+C221S was selected for the following experiments.

[0114] Example 5

[0115] Application of D-allulose-3-epimerase isomer in the synthesis of D-allulose:

[0116] The recombinant D-allulose-3-epimerase mutant with the highest activity obtained in Example 4 was used. E.coliBL21(DE3) / / pET28a-RsDAE-F47A-N72E-N114G-C221S was inoculated into LB liquid medium containing a final concentration of 50 μg / mL kanamycin and cultured at 37°C for 9 hours. This seed culture was then inoculated into a 5 L fermenter containing 3 L of fermentation medium at a volume concentration of 3.5%. The prepared medium was added to the fermenter, and the inlet and outlet were sealed tightly. The inoculation port was left open, and the fermenter, along with the prepared lactose inducer, was placed in an autoclave at 115°C for 30 min for sterilization. The sterilized fermenter was then fitted with the inoculation port and attached to the operating system. Cooling water and air were introduced (the inlet pipe should be fitted with a sterilizing membrane). The outlet was inserted below the liquid level in the conical flask. When the autoclave temperature dropped to 37°C, a flame ring was placed over the inoculation port, and the cultured seed culture was inoculated into the fermenter. Incubate at 37℃ and 500 rpm for approximately 3-4 hours to achieve the desired bacterial density (OD). 600 To achieve the desired 6-8 results, the fermenter temperature was lowered to 25℃, and lactose at a final concentration of 16 g / L was added as an inducer. The fermentation was then incubated at 25℃ and 500 rpm for 12 h. The fermented broth was centrifuged at 8000 rpm for 10 min to obtain the D-allulose-3-epimerase mutant. E.coli Wet cells of BL21(DE3) / / pET28a-RsDAE-F47A-N72E-N114G-C221S.

[0117] The fermenter culture medium consists of: 45 g tryptone, 36 g yeast extract, 30 g sodium chloride, 4.08 g potassium dihydrogen phosphate, 45 g glycerol, 6.84 g dipotassium hydrogen phosphate trihydrate, 15 g ammonium sulfate, 1.125 g magnesium sulfate, and 4 g defoamer, which are dissolved in distilled water to a final volume of 3 L.

[0118] The catalyst dosage was 15 g / L based on the total weight of wet bacterial cells, with a final concentration of 600 g / L for the substrate fructose and a final concentration of 1 mM Co. 2+ The reaction solution was prepared using purified water as the reaction medium, with a total volume of 1 L. Reaction conditions: 55℃, 500 rpm for 4 hours. After the reaction, 20 μL of the final sample was taken, diluted 50 times, filtered through a 0.22 μm filter membrane, and analyzed by HPLC.

[0119] The reaction process curve is as follows Figure 1 As shown, after the reaction, the concentration of D-allulose was 217.8 g / L, and the conversion rate was 36.3%.

[0120] Example 6

[0121] Construction of Bacillus subtilis strain RsDAE-F47A-N72E-N114G-C221S and its application in the synthesis of D-allulose:

[0122] The recombinant D-allulose-3-epimerase RsDAE-F47A-N72E-N114G-C221S, which had the highest activity obtained in Example 4, was codon-optimized for Bacillus subtilis and its entire genome was synthesized. The nucleotide sequence is shown in SEQ ID NO. 35, and the amino acid sequence is shown in SEQ ID NO. 36. The plan is to construct RsDAE-F47A-N72E-N114G-C221S into the secretory expression plasmid PWB980(XhoI-NheI), and simultaneously construct a PWB980 recombinant plasmid with the secretory signal peptide knocked out and expressed intracellularly.

[0123] The nucleic acid sequence of the recombinant D-allulose-3-epimerase RsDAE-F47A-N72E-N114G-C221S after codon optimization of Bacillus subtilis (SEQ ID NO.35):

[0124] ATGAACAAAATCGGCGTTCATTTTGGCTATTTTAACCGTGATTGGAACACCGATTTTATTAAGCGGATTGAGCAAGTGAAAAAGATCGGGCTGGATATTTTAGAAGTGGCTCCCGCGCCGCTGCTGGCCTTGACAAAAGCCCAAAGAGACGAAATTGCCGCCGCAGCGAAAGCGAATGATATCGAGCTTACCTTCTCTGTAGGGCTTAGTGCAGAACAGGATTTAGCTTCAGAAGATGAAGAAATCAGAAAGAACGGCATCAAGTTTACGACTGATACATTCCAAATCATGAGCGAGATGGGAGGTAAAACATACTCTGGTGTGGACATTGCTGCTTGGGGTAAAACGTTTATGGAAGGCATCACAGATAAAAGCGCGACATGGGAAAGATCGATTTCCGCCGTTAAAGAAATTATGAAAGTGGCAGAAGACAAAGGAATTACATTCGCAGTTGAAGTTGTTAACCGCTATGAAAGCTCACTCGTCAATACTGCTGAAGAAGCAGTAAAATACGTAGATGAGGTCGGATCACCAAACTGCAAAATTTTGCTTGATACGTATCATATGAATATAGAAGAGGACAGCTTTGCGGGAGCTATTAAGCTTGTCGGCAACAGGCTCGGCCACTTTCATGTCGGTGAATCAAATCGCCGGCCGCCTAGCGAAAATGGGAAAATGCCTTGGAATGAAATCACGAATGCCTTAAAAGAGATTGATTATCAAGGAGCCATTGTCATGGAGCCGTTTATCAAAATGGGCGGTGAAGTAGGACGTGACATAAAGGTGTGGCGCGATATTTCTGAGGGAGCGAGTGAGTCCGAAATGGAACAGTTACTAGCAGACGCAGCGATGATGCTGCGAAAAAAGATGCAGCGTTAA

[0125] Nucleic acid sequence of recombinant D-allulose-3-epimerase RsDAE-F47A-N72E-N114G-C221S after codon optimization in Bacillus subtilis (SEQ ID NO.36):

[0126] MNKIGVHFGYFNRDWNTDFIKRIEQVKKIGLDILEVAPAPLLALTKAQRDEIAAAAKANDIELTFSVGLSAEQDLASEDEEIRKNGIKFTTDTFQIMSEMGGKTYSGVDIAAWGKTFMEGITDKSATWERSISAVKEIMKVAEDKG ITFAVEVVNRYESSLVNTAEEAVKYVDEVGSPNCKILLDTYHMNIEEDSFAGAIKLVGNRLGHFHVGESNRRPPSENGKMPWNEITNALKEIDYQGAIVMEPFIKMGGEVGRDIKVWRDISEGASESEMEQLLADAAMMLRKKMQR

[0127] Primers F3, R3, F4, and R4 were designed based on the nucleotide sequence shown in SEQ ID NO.35 and the PWB980 vector sequence to secrete the recombinant plasmid expressing PWB980.

[0128] Primers F5, R5, F6, and R6 were designed based on the nucleotide sequence shown in SEQ ID NO.35 and the PWB980 vector sequence to express the recombinant plasmid PWB980 intracellularly.

[0129] F3: 5'-ggcgcaactcaagcttttgcATGAACAAAATCGGCGTTCATTTTG-3'; (SEQ ID NO. 37)

[0130] R3: 5'-ggaattgtgctgaagctagcTTAACGCTGCATCTTTTTTCGCAGCATC-3'; (SEQ ID NO. 38)

[0131] F4: 5'-GCTAGCTTCAGCACAATTCCAA-3'; (SEQ ID NO. 39)

[0132] R4: 5'-GCAAAAGCTTGAGTTGCGCC-3'; (SEQ ID NO. 40)

[0133] F5: 5'-taaaaaaggagacatgaacgATGAACAAAATCGGGCTTTCATT-3'; (SEQ ID NO. 41)

[0134] R5: 5'-tccccgggtaccgagctcgaTTAACGCTGCATCTTTTTTCGC-3'; (SEQ ID NO. 42)

[0135] F6: 5'-TCGAGCTCGGTACCCGGG-3'; (SEQ ID NO. 43)

[0136] R6: 5'-CGTTCATGTCTCCTTTTTTATGTACTG-3'; (SEQ ID NO. 44)

[0137] Using PWB980 plasmid as an expression vector, a Bacillus subtilis secretory expression strain was constructed. Bacillus subtle BS168 / PWB980-RsDAE-F47A-N72E-N114G-C221S and Bacillus subtilis intracellular expression strain Bacillus subtilis BS168 / PWB980-RsDAE-F47A-N72E-N114G-C221S.

[0138] Construction of the secretory expression plasmid: Using primers F3 / R3 and F4 / R4 as the initiation point, the target gene was amplified using high-fidelity Pfu DNA polymerase to obtain the D-allulose-3-epimerase gene sequence with homologous arms. Using PWB980 plasmid as a template, high-fidelity Pfu DNA polymerase was used to amplify the linearized vector sequence. Homologous recombination of the target gene with the linearized vector was then performed using homologous recombination enzyme to construct the secretory expression plasmid PWB980-RsDAE-F47A-N72E-N114G-C221S. Then, the intracellular expression plasmid was constructed using the same method as the secretory expression plasmid.

[0139] Construction of recombinant Bacillus subtilis: 200 µL of Bacillus subtilis stored at -80℃ was used... Bacillus subtilis BS168 competent cells were incubated at 0°C on ice for 10 min, then 20 µL of homologous recombinant product (i.e., secretory expression plasmid or intracellular expression plasmid) was added in a clean bench and cultured at 37°C and 200 rpm for 2 h. 800 µL of LB medium was added, and the cells were cultured at 37°C and 200 rpm for another 1 h. The culture was then plated on LB agar plates containing 25 μg / mL kanamycin resistance and cultured at 37°C for 15 h. Clones were randomly selected, plasmids were extracted, and sequenced for identification. Recombinant Bacillus subtilis cells containing both secretory and intracellular expression of the recombinant product were screened. Bacillus subtilis BS168 / PWB980-RsDAE-F47A-N72E-N114G-C221S.

[0140] Recombinant Bacillus subtilis containing recombinant secretory expression and intracellular expression were respectively... Bacillus subtleBS168 / PWB980-RsDAE-F47A-N72E-N114G-C221S was inoculated into LB liquid medium containing a final concentration of 25 μg / mL kanamycin and cultured at 37°C for 10 hours. This seed culture was then inoculated into a 5 L fermenter containing 3 L of fermentation medium at a volume concentration of 5%. The pH of the fermentation process was controlled at approximately 7.0 using ammonia solution and 30% (v / v) phosphate solution. The fermentation speed was coupled with dissolved oxygen (DO), and the DO curve was closely monitored. When a DO rebound occurred, feed medium was added promptly to maintain the DO value at approximately 30% throughout the fermentation process. After 48 hours of fermentation, the supernatant was collected by centrifugation to obtain the fermentation broth containing the target protein.

[0141] The culture medium for the 3L fermenter consists of: 75 g yeast powder, 36 g glucose, 30 g corn steep liquor, 9 g dipotassium hydrogen phosphate, 3 g sodium chloride, 3 g magnesium sulfate, and 0.75 g calcium chloride.

[0142] The supplemental culture medium (0.5 L) consists of: 15 g peptone, 30 g yeast extract, and 3 g dipotassium hydrogen phosphate.

[0143] Assay for the activity of secretory expression strains: Add fructose substrate to a final concentration of 500 g / L and Co to a final concentration of 1 mM. 2+ 200 mL of fermentation broth supernatant was used to form the reaction solution, with a total volume of 1 L. Reaction conditions: 55℃, 500 rpm for 5 hours. After the reaction, 20 μL of the final sample was taken, diluted 50-fold, filtered through a 0.22 μm filter membrane, and analyzed by HPLC. The reaction progress curve is shown below. Figure 2 As shown, after the reaction, the concentration of D-allulose was 177.5 g / L, and the conversion rate was 35.5%.

[0144] Intracellular expression strain activity assay: Add substrate fructose to a final concentration of 500 g / L, and Co to a final concentration of 1 mM. 2+ The amount of bacterial sludge used was 25 g / L, constituting the reaction solution with a total volume of 1 L. Reaction conditions: 55℃, 500 rpm for 4 hours. After the reaction, 20 μL of the final sample was taken, diluted 50-fold, filtered through a 0.22 μm filter membrane, and analyzed by HPLC. The reaction progress curve is shown below. Figure 3 As shown, after the reaction, the concentration of D-allulose was 179 g / L, and the conversion rate was 35.8%.

[0145] Example 7

[0146] Immobilization of D-allulose-3-epimerase and its application in the synthesis of D-allulose:

[0147] Collect secretion expression Bacillus subtilis Add twice the volume of purified water to the supernatant of the BS168 / PWB980-RsDAE-F47A-N72E-N114G-C221S fermentation broth, weigh out an appropriate amount of diatomaceous earth (the addition ratio to the diluted fermentation broth is 1:8 by mass), stir and mix for 10-15 min, then add 0.3% polyethyleneimine by volume for flocculation for 20-30 min, and finally add 0.8% glutaraldehyde by volume for covalent cross-linking for 1 h. Wash with purified water 3-5 times to obtain immobilized RsDAE-F47A-N72E-N114G-C221S.

[0148] The catalyst dosage was 20 g / L based on the total weight of the immobilized enzyme, with a final concentration of 500 g / L substrate fructose and a final concentration of 1 mM Co. 2+ The reaction medium consisted of purified water, with a total volume of 1 L. Reaction conditions: 55℃, 500 rpm for 4–6 hours. After the reaction, 20 μL of the final sample was taken, diluted 50-fold, filtered through a 0.22 μm filter membrane, and analyzed by HPLC. The immobilized RsDAE-F47A-N72E-N114G-C221 achieved a conversion rate of 36.0% after 4 hours of reaction, and the enzyme activity remained above 80% even after 30 batches of reaction.

[0149] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A D-allulose-3-epimerase mutant, characterized in that, The amino acid sequence of D-allulose-3-epimerase is shown in SEQ ID NO.2, with the mutation site being N72E.

2. A nucleic acid molecule, characterized in that, It has a nucleotide sequence encoding the D-allulose-3-epimerase mutant of claim 1.

3. An expression carrier, characterized in that, It has the nucleic acid molecule as described in claim 2.

4. The expression vector as described in claim 3, characterized in that, The expression vector contains either the pET-28a plasmid or the PWB980 plasmid.

5. A host cell, characterized in that, It has any of the following: (a1) The nucleic acid molecule as described in claim 2; (a2) The expression vector as described in claim 3 or 4; The host cell is bacteria.

6. The host cell as described in claim 5, characterized in that, The host cell is either Escherichia coli or Bacillus subtilis.

7. A method for preparing a D-allulose-3-epimerase mutant, characterized in that, It is prepared based on any of the following: (b1) The nucleic acid molecule as described in claim 2; (b2) The expression vector as described in claim 3 or 4; (b3) The host cell as described in claim 5 or 6.

8. The use of the D-allulose-3-epimerase mutant of claim 1 in the preparation of D-allulose.

9. A method for preparing D-allulose, characterized in that, include: D-allulose was prepared based on the host cells described in claim 5 or 6; The prepared reaction system comprises: 450-650 g / L fructose, 1 mM Co 2+ 5-25 g / L of wet bacterial cells containing the host cells described above; The preparation temperature is 50-60℃; The preparation time is 4-6 hours.

10. A method for preparing D-allulose, characterized in that, include: D-allulose was prepared based on the D-allulose-3-epimerase mutant according to claim 1; The prepared reaction system comprises: 450-650 g / L fructose, 1 mM Co 2+ 5-25 g / L of the D-allulose-3-epimerase mutant; The preparation temperature is 50-60℃; The preparation time is 4-6 hours.

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