A Spytag / SpyCatcher cyclized D-allulose-3-epimerase mutant and its applications

Abstract

The application belongs to the technical field of genetic engineering, and particularly relates to a Spytag / SpyCatcher cyclization modified D-allulose-3-epimerase mutant and application thereof. The mutant intermediate is obtained by simultaneously mutating an aspartic acid at the 109th position and a serine at the 160th position of a wild-type D-allulose-3-epimerase, and the mutant intermediate is cyclization modified by connecting a Spytag label and a SpyCatcher label at the N terminal and the C terminal of the mutant intermediate, respectively. When D-allulose is produced by using fructose as a raw material, the conversion rate reaches 35.7% when the host bacteria is Escherichia coli and the catalysis of 600 g / L fructose to produce D-allulose for 4 h, and the conversion rate reaches 36.5% when the host bacteria is Bacillus subtilis and the catalysis of 500 g / L fructose to produce D-allulose for 4 h, which is 13.5% higher than that of the wild-type D-allulose-3-epimerase strain of Bacillus subtilis.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a Spytag / SpyCatcher cyclized D-allulose-3-epimerase mutant and its applications. Background Technology

[0002] D-Allulose is a hexulose, structurally a C-3 epimer of D-fructose. It is a rare, functional sugar with high sweetness but extremely low energy content, containing only 10% of the calories of sucrose. It is an ideal natural sweetener and sucrose substitute, and has been classified as a "Generally Recognized as Safe" food by the U.S. Food and Drug Administration, allowing it to be added as an additive to candies, juices, soy sauces, and other dietary products.

[0003] The main methods for producing D-allulose include extraction, chemical synthesis, and biotransformation. Extraction methods have low yields and high costs; chemical synthesis involves complex reaction conditions, expensive raw materials, and safety risks; while biotransformation, due to its high specificity and environmental friendliness, has become the primary method for the industrial production of D-allulose. Biotransformation uses D-fructose as a substrate and ketose 3-epimerase as a catalyst, resulting in isomerization at the C-3 position to produce D-allulose in one step. Ketosose 3-epimerases can be classified into three types: D-allulose 3-epimerase (DAEase), D-tagatose 3-epimerase (DTEase), and L-ribulose 3-epimerase (LREase). Among these, D-allulose 3-epimerase is the most diverse and has been the most extensively studied.

[0004] However, wild-type D-allulose 3-epimerase has low catalytic activity for D-fructose and poor thermal stability, while industrial production requires reactions at high temperatures, which limits the practical industrial application of D-allulose production using enzymatic methods. Summary of the Invention

[0005] To overcome the above problems, the present invention provides a Spytag / SpyCatcher cyclized D-allulose-3-epimerase mutant and its application.

[0006] To achieve the above technical objectives, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides a Spytag / SpyCatcher cyclized D-allulose-3-epimerase mutant, which is obtained by mutating the aspartic acid at position 109 to cysteine ​​and the serine at position 160 to phenylalanine in the wild-type D-allulose-3-epimerase as shown in SEQ ID NO: 2, to obtain a mutant intermediate, and attaching a Spytag tag and a SpyCatcher tag to the N-terminus and C-terminus of the mutant intermediate, respectively, and then cyclizing the intermediate to obtain the mutant intermediate.

[0008] The amino acid sequence of the Spytag tag is shown in SEQ ID NO: 23, and the amino acid sequence of the SpyCatcher tag is shown in SEQ ID NO: 24.

[0009] In one or more embodiments, the N-terminus of the mutant intermediate is connected to the Spytag tag via linker1; the C-terminus of the mutant intermediate is connected to the SpyCatcher tag via linker2.

[0010] The amino acid sequence of linker1 is shown in SEQ ID NO: 25; the amino acid sequence of linker2 is shown in SEQ ID NO: 26.

[0011] A second aspect of the invention provides a gene encoding a Spytag / SpyCatcher cyclized D-allulose-3-epimerase mutant as described in the first aspect.

[0012] A third aspect of the invention provides an expression cassette comprising the gene described in the second aspect.

[0013] A fourth aspect of the present invention provides a recombinant expression vector comprising the gene described in the second aspect.

[0014] A fifth aspect of the present invention provides a recombinant bacterium comprising the genes described in the second aspect.

[0015] A sixth aspect of the present invention provides a transgenic cell line comprising the genes described in the second aspect.

[0016] A seventh aspect of the present invention provides the use of the D-allulose-3-epimerase mutant described in the first aspect, the encoding gene described in the second aspect, or the recombinant bacteria described in the fifth aspect in the catalytic synthesis of D-allulose.

[0017] An eighth aspect of the present invention provides a method for synthesizing D-allulose, comprising the following steps:

[0018] Using wet cells obtained by inducing culture of genetically engineered bacteria containing Spytag / SpyCatcher cyclized D-allulose-3-epimerase mutant, or crude enzyme solution extracted by ultrasonic disruption of wet cells, or immobilized enzyme as a catalyst, and fructose as a substrate, containing Co... 2+ An aqueous solution was used as the reaction medium to form the reaction system, and the reaction yielded D-allulose.

[0019] The genetically engineered bacteria are constructed by introducing the Spytag / SpyCatcher cyclized D-alokulose-3-epimerase mutant described in the first aspect into a host bacterium.

[0020] In one or more embodiments, the host bacterium is a bacterium; preferably, the bacterium is one or more of Escherichia coli or Bacillus subtilis.

[0021] In one or more embodiments, the amount of catalyst used is 5-25 g / L based on the total weight of wet cells or immobilized enzymes, and the final concentration of the substrate fructose is 450-550 g / L. 2+ The concentration is 0.8~1.2mM.

[0022] In one or more embodiments, the reaction temperature is 50-60 °C; the reaction time is 4-6 h.

[0023] The beneficial effects of this invention are as follows:

[0024] This invention utilizes the method of processing bacteria derived from rumenococcus CAG55 ( Ruminococcus sp. The wild-type D-allulose-3-epimerase (CAG55) was modified by simultaneous mutation of aspartic acid at position 109 and serine at position 160 to obtain a mutant intermediate. Subsequently, Spytag and SpyCatcher tags were attached to the N-terminus and C-terminus of the mutant intermediate, respectively, for cyclization modification, resulting in a Spytag / SpyCatcher cyclized D-allulose-3-epimerase mutant. This significantly improved the catalytic activity and stability of the D-allulose-3-epimerase. Testing showed that when fructose was used as a raw material to produce D-allulose, 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 h, achieving a conversion rate of 35.7%. With *Bacillus subtilis* as the host bacterium, intracellular expression of *Bacillus subtilis* catalyzed the production of D-allulose from 500 g / L fructose for 4 h, achieving a conversion rate of 36.5%. The cyclized D-alulose-3-epimerase mutant strain of *Bacillus subtilis* showed a 13.5% higher conversion rate compared to the wild-type D-alulose-3-epimerase strain of *Bacillus subtilis*. Attached Figure Description

[0025] 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.

[0026] Figure 1 In Embodiment 6 of the present invention E.coli Graph of the reaction process of fructose to D-allulose catalyzed by BL21(DE3) / / pET28a-Spytag-RsDAE-D109C-S160F-SpyCatcher.

[0027] Figure 2 The intracellular expression strain in Example 7 of this invention Bacillus subtilis Schematic diagram of the reaction process of fructose to D-allulose catalyzed by BS168 / PWB980-Spytag-RsDAE-D109C-S160F-SpyCatcher. Detailed Implementation

[0028] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0029] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0030] 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.

[0031] The culture medium formulations used in the following examples are as follows:

[0032] LB medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, water as solvent, pH 7.4.

[0033] 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.

[0034] The concentration of the product D-alokulose was determined by high performance liquid chromatography (HPLC). The analytical method was as follows: column type: cation calcium exchange column; mobile phase: water; injection volume: 10 μL; differential detector: detection time: 25 min; flow rate: 0.6 mL / min; column temperature: 80 ℃.

[0035] 0.1 mM Co was added to all catalytic reactions. 2+ This is due to the presence of rumenococcus CAG55 ( Ruminococcus sp. The D-allulose-3-epimerases of CAG:55 are all metal-dependent, and the addition of Co... 2+ It can stimulate the catalytic activity of D-allulose-3-epimerase. In the following examples, the divalent cobalt salt is cobalt chloride.

[0036] Example 1

[0037] (1) Construction of expression vector and engineered bacteria:

[0038] 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.

[0039] Primers F1 (nucleotide sequence as shown in SEQ ID NO: 3), R1 (nucleotide sequence as shown in SEQ ID NO: 4), F2 (nucleotide sequence as shown in SEQ ID NO: 5), and R2 (nucleotide sequence as shown in SEQ ID NO: 6) were designed based on the nucleotide sequence shown in SEQ ID NO: 1 and the pET-28a vector sequence.

[0040] (2) Construction of recombinant expression plasmids:

[0041] 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. Homologous recombination was then performed between the D-allulose-3-epimerase gene sequence with homologous arms and the linearized vector to construct the homologous recombination product pET28a-RSDAE.

[0042] (3) Construction of recombinant Escherichia coli:

[0043] Stored at -80℃ E.coli BL21(DE3) competent cells were incubated at 0 °C for 10 min on ice, then 5 µL of the homologous recombinant product (pET28a-RSDAE) was added in a clean bench, incubated at 0 °C for 30 min on ice, 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 on a shaker. The cultured cells were then plated on LB agar 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 recombinant expression plasmid pET28a-RSDAE were screened for these cells. E.coli BL21(DE3) / / pET28a-RsDAE.

[0044] Example 2

[0045] The D-allulose-3-epimerase gene was induced to express wet bacterial cells containing the D-allulose-3-epimerase gene.

[0046] The recombinant Escherichia coli obtained in Example 1 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 sample 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.

[0047] Example 3

[0048] Construction of a mutant library of D-allulose-3-epimerase gene:

[0049] The recombinant Escherichia coli expressing D-allulose-3-epimerase constructed in Example 2 E.coli BL21(DE3) / / pET28a-RsDAE is the originating strain.

[0050] Modification was performed using directed evolution theory, based on the crystal structure of D-allulose-3-epimerase obtained through homology modeling. Based on calculations, site-directed mutagenesis was performed at sites R22Y, D109C, I110Y, K145Y, S160F, V203A, I250V, and M285F.

[0051] 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.

[0052] The PCR conditions were as follows: pre-denaturation at 95 °C for 3 min, followed by 30 cycles: 95 °C for 15 s, 60 °C for 15 s, 72 °C for 7 min 20 s, and final extension at 72 °C for 10 min.

[0053] PCR results were verified by DNA agarose gel electrophoresis: PCR products were digested with DpnI enzyme, inactivated at 37 ℃ for 1 h, 200 rpm, 65 ℃ for 1 min, and then transformed by heat shock into E. coli. E.coli BL21(DE3) was activated, incubated at 37 ℃ and 200 rpm for 1 h, spread on LB plates containing 50 μg / mL kanamycin resistance, and incubated upside down at 37 ℃ overnight.

[0054] Table 1. Primer design for site-directed mutagenesis of D-allulose-3-epimerase

[0055]

[0056] DNA sequencing revealed that the site-directed mutations in R22Y, D109C, I110Y, K145Y, S160F, V203A, I250V, and M285F were completely consistent with the intended mutations.

[0057] Example 4

[0058] Screening of D-allulose-3-epimerase gene mutant libraries:

[0059] 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. 600When 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.

[0060] (1) Initial screening:

[0061] 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.

[0062] Reaction conditions: After reacting for 1 h 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.

[0063] Table 2 Initial screening reaction results

[0064]

[0065] Initial screening revealed that mutants D109C and S160F produced higher concentrations of D-allulose than the parent mutant when catalyzing the preparation of D-allulose from fructose, indicating enhanced catalytic activity in both mutants. Subsequent experiments were conducted using mutants D109C and S160F.

[0066] (2) Secondary screening:

[0067] The strains obtained from the initial screening were then subjected to a second screening.

[0068] The D109C+S160F mutant was constructed using the method described in Example 3. DNA sequencing showed that the mutant's DNA sequencing results were completely consistent with the intended design mutation.

[0069] Re-screening and reaction solution preparation (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.

[0070] Reaction conditions: After reacting for 1 h 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.

[0071] Table 3 Results of the secondary screening reaction

[0072]

[0073] After further screening, it was confirmed that the catalytic activities of all the mutants recorded in Table 3 were significantly improved. Among them, the mutant D109C+S160F showed the highest catalytic activity. Therefore, the mutant D109C+S160F9 (denoted as RsDAE-D109C-S160F) was selected for subsequent experiments.

[0074] Example 5

[0075] Construction of Spytag / SpyCatcher cyclized D-allulose-3-epimerase mutant:

[0076] Spytag / SpyCatcher cyclization modification can effectively improve enzyme stability. The plan is to attach a SpyTag (amino acid sequence SEQ ID NO: 23) and a SpyCatcher (amino acid sequence SEQ ID NO: 24) tag, respectively, to the N-terminus and C-terminus of the D-allulose-3-epimerase mutant RsDAE-D109C-S160F obtained in Example 4. Specifically: the N-terminus is linked to the Spytag tag via linker1 (amino acid sequence SEQ ID NO: 25); the C-terminus is linked to the SpyCatcher tag via linker2 (amino acid sequence SEQ ID NO: 26), thus constructing the SpyTag-RsDAE-D109C-S160F-SpyCatcher (sequence SEQ ID NO: 24) cyclized D-allulose-3-epimerase mutant. 27), and the whole gene was synthesized and constructed into the pET-28a plasmid to obtain the recombinant plasmid pET28a-SpyTag-RsDAE-D109C-S160F-SpyCatcher, strain E.coli For the specific transformation and induced expression process of BL21(DE3) / pET28a-SpyTag-RsDAE-D109C-S160F-SpyCatcher, please refer to Examples 1 and 2.

[0077] To compare enzyme activities between cyclized and uncyclized enzymes, the reaction solution (10 mL) was prepared with a final concentration of 300 g / L fructose substrate and a 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.

[0078] Reaction conditions: After reacting for 1 h 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 4.

[0079] Table 4 Reaction Results

[0080]

[0081] As can be seen from Table 4, the Spytag / SpyCatcher cyclization modification effectively improves the enzyme's reactivity. Therefore, the mutant SpyTag-RsDAE-D109C-S160F-SpyCatcher was selected for subsequent experiments.

[0082] Example 6

[0083] Application of D-allulose-3-epimerase isomer in the synthesis of D-allulose:

[0084] The Spytag / SpyCatcher cyclized D-allulose-3-epimerase mutant strain obtained in Example 4 was used. E.coli BL21(DE3) / pET28a-SpyTag-RsDAE-D109C-S160F-SpyCatcher was inoculated into LB liquid medium containing a final concentration of 50 μg / mL kanamycin and cultured at 37 ℃ for 9 h. 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 ℃ 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 was fitted with a sterilizing membrane). The outlet was inserted below the liquid level in the conical flask. When the autoclave temperature dropped to 37 ℃, a flame ring was placed over the inoculation port, and the cultured seed culture was inoculated into the fermenter. After incubation at 37 ℃ and 500 rpm for approximately 3-4 hours, the bacterial density OD... 600 To achieve the desired fermentation conditions (6-8), the fermenter temperature was lowered to 25 °C, and lactose at a final concentration of 16 g / L was added as an inducer. The fermentation was then incubated at 25 °C and 500 rpm for 12 h. The fermented broth was centrifuged at 8000 rpm for 10 min to obtain the Spytag / SpyCatcher cyclized D-allulose-3-epimerase mutant strain. E.coli Wet cells of BL21(DE3) / pET28a-SpyTag-RsDAE-D109C-S160F-SpyCatcher.

[0085] The fermentation tank 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.

[0086] The catalyst dosage was 15 g / L based on the total weight of wet bacterial cells, the final concentration of the substrate fructose was 600 g / L, and the final concentration of Co was 1 mM. 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.

[0087] The reaction process curve is as follows Figure 1 As shown, after the reaction, the concentration of D-allulose was 214.2 g / L, and the conversion rate was 35.7%.

[0088] Example 7

[0089] Construction of SpyTag-RsDAE-D109C-S160F-SpyCatcher Bacillus subtilis strain and its application in the synthesis of D-allulose:

[0090] The recombinant D-allulose-3-epimerase SpyTag-RsDAE-D109C-S160F-SpyCatcher, which had the highest activity obtained in Example 5, was codon-optimized for Bacillus subtilis and its entire genome was synthesized. The nucleotide sequence is shown in SEQ ID NO: 28. The plan is to construct SpyTag-RsDAE-D109C-S160F-SpyCatcher onto the secretory expression plasmid PWB980(XhoI-NheI), and simultaneously construct a recombinant plasmid of PWB980 with the secretory signal peptide knocked out, resulting in intracellular expression.

[0091] The intracellular expression recombinant plasmid PWB980 was used to design primers F3 (nucleotide sequence as shown in SEQ ID NO: 29), R3 (nucleotide sequence as shown in SEQ ID NO: 30), F4 (nucleotide sequence as shown in SEQ ID NO: 31), and R4 (nucleotide sequence as shown in SEQ ID NO: 32) based on the nucleotide sequence shown in SEQ ID NO: 28 and the PWB980 vector sequence.

[0092] Construction of recombinant expression plasmid: Under the initiation of primers F3 / R3 and F4 / R4, the target gene was amplified using high-fidelity Pfu DNA polymerase to obtain the D-alulose-3-epimerase gene sequence with homologous arms (Spytag / SpyCatcher circularized). Using PWB980 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 D-alulose-3-epimerase gene sequence with homologous arms (Spytag / SpyCatcher circularized) and the linearized vector to construct the homologous recombination product PWB980-SpyTag-RsDAE-D109C-S160F-SpyCatcher.

[0093] Construction of recombinant Bacillus subtilis: 200 µL of Bacillus subtilis stored at -80 °C was used... Bacillus subtilis BS168 competent cells were incubated on ice at 0 °C for 10 min, then 20 µL of homologous recombinant product (PWB980-SpyTag-RsDAE-D109C-S160F-SpyCatcher) 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 containing intracellular expression recombinant plasmids were screened. Bacillus subtilis BS168 / PWB980-SpyTag-RsDAE-D109C-S160F-SpyCatcher.

[0094] Recombinant Bacillus subtilis containing a recombinant plasmid expressing intracellular expression was used. Bacillus subtilis BS168 / PWB980-SpyTag-RsDAE-D109C-S160F-SpyCatcher was inoculated into LB liquid medium containing a final concentration of 25 μg / mL kanamycin and cultured at 37 ℃ for 10 h. 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 h of fermentation, the supernatant was collected by centrifugation to obtain the fermentation broth containing the target protein.

[0095] 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.

[0096] The supplemental culture medium (0.5 L) consists of: 15 g peptone, 30 g yeast extract, and 3 g dipotassium hydrogen phosphate.

[0097] Assay for expression strain activity: Add fructose substrate to a final concentration of 500 g / L, and Co to a final concentration of 1 mM. 2+ The catalyst dosage was 25 g / L based on the total weight of the wet bacterial cells. Pure water was used as the reaction medium to form the reaction solution, with a total volume of 1 L. Reaction conditions: 55 ℃, 500 rpm for 4 h. 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. The reaction progress curve is shown below. Figure 2 As shown, after the reaction, the concentration of D-allulose was 182.5 g / L, and the conversion rate was 36.5%.

[0098] Comparative Example 1

[0099] Catalytic effect of primitive wild-type D-allulose-3-epimerase:

[0100] Recombinant Bacillus subtilis Bacillus subtilis BS168 / PWB980-RsDAE was prepared using the same method as in Example 7. The catalyst dosage was 25 g / L based on the total weight of the wet cells before high-pressure homogenization. The final concentration of the substrate fructose was 500 g / L, and the final concentration of Co was 1 mM. 2+ The reaction solution, consisting of 1 L of purified water, was prepared using purified water as the reaction medium. Reaction conditions: 55 ℃, 500 rpm for 4 hours. After the reaction, 20 μL of the final sample was diluted 50-fold, filtered through a 0.22 μm filter, and analyzed by HPLC. The final D-allulose concentration was 115 g / L, with a conversion rate of 23%.

[0101] 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 Spytag / SpyCatcher cyclized modified D- Psicose-3-epimerase mutant, characterized in that, which is obtained by mutating aspartic acid at position 109 and serine at position 160 of wild-type D-allulose 3-epimerase with the amino acid sequence shown in SEQ ID NO: 2 into cysteine and phenylalanine respectively, obtaining a mutant intermediate, connecting a Spytag tag and a SpyCatcher tag at the N-terminus and C-terminus of the mutant intermediate respectively, and obtaining after cyclization; the amino acid sequence of the Spytag tag is shown in SEQ ID NO: 23, and the amino acid sequence of the SpyCatcher tag is shown in SEQ ID NO:

24.

2. The Spytag / SpyCatcher cyclized modified D-allulose-3-epimerase mutant of claim 1, wherein, the N-terminus of the mutant intermediate is connected with the Spytag tag through linker 1; and the C-terminus of the mutant intermediate is connected with the SpyCatcher tag through linker 2; the amino acid sequence of linker 1 is shown in SEQ ID NO: 25; and the amino acid sequence of linker 2 is shown in SEQ ID NO:

26.

3. A gene encoding the Spytag / SpyCatcher cyclization-modified D-allulose 3-epimerase mutant of claim 1 or 2.

4. An expression cassette comprising, comprising the gene of claim 3.

5. A recombinant expression vector, characterized in that, comprising the gene of claim 3.

6. A recombinant bacterium, characterized in that, comprising the gene of claim 3.

7. A transgenic cell line, characterized in that, comprising the gene of claim 3. comprising the gene of claim 3.

9. A method of synthesizing D-allulose, characterized by, 8. Use of the Spytag / SpyCatcher cyclization-modified D-allulose 3-epimerase mutant of claim 1 or 2 or the gene of claim 3 or the recombinant bacterium of claim 6 in catalyzing synthesis of D-allulose. The genetically engineered bacteria of the Spytag / SpyCatcher cyclization modified D- Psicose-3-epimerase mutant are induced to obtain wet bacteria or wet bacteria ultrasonic broken extraction of crude enzyme solution or immobilized enzyme as a catalyst, fructose as a substrate, and a Co 2+ containing aqueous solution as a reaction medium to form a reaction system, and D-psicose is obtained by reaction. comprising the following steps:

10. The method of claim 9, wherein, wherein the genetically engineered bacterium is constructed by introducing the Spytag / SpyCatcher cyclization-modified D-allulose 3-epimerase mutant of claim 1 or 2 into a host bacterium. The amount of catalyst is 5-25 g / L based on the total weight of wet bacteria or immobilized enzyme, the final concentration of substrate fructose is 450-550 g / L, the concentration of Co 2+ is 0.8-1.2 mM; The host bacterium is a bacterium; and the bacterium is one or more of Escherichia coli or Bacillus subtilis. The temperature of the reaction is 50-60 ℃; and the time of the reaction is 4-6 h.

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