A phosphatase mutant and its application

Abstract

The present invention belongs to the field of biocatalysis, and specifically relates to a D-psicose 6-phosphate phosphatase mutant derived from Tunisian sewage bacterium (Defluviitoga tunisiensis) and its application. The D-psicose 6-phosphate phosphatase mutant is obtained by subjecting the D-psicose 6-phosphate phosphatase with an amino acid sequence as shown in SEQ ID NO.2 as a template to site-directed saturation mutagenesis; the mutation sites of the D-psicose 6-phosphate phosphatase mutant are one or more of the following: (1) K at position 160 is mutated to C; (2) S at position 228 is mutated to A; (3) C at position 48 is mutated to P. The D-psicose 6-phosphate phosphatase mutant provided by the present invention has a yield of 60% when preparing D-psicose, and the ratio of D-psicose to byproduct fructose in the product is increased from 1:1 of the wild type to 6:1. Phosphatase mutants with regulated substrate specificity have important industrial application potential in the production of D-psicose in an in vitro multi-enzyme synthesis system.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and in particular to a phosphatase mutant derived from Tunisian sewage bacterium (Defluviitogatunisiensis) with improved reaction specificity to D-psicose 6-phosphate and its application. Background Art

[0002] D-psicose is a low-energy sweetener with 70% the sweetness of sucrose. It is considered a potential sucrose substitute and is widely used in food, medicine, healthcare, and other fields. Its efficient production has attracted widespread attention. Currently, the most common method for producing psicose is to use fructose as a substrate and use an isomerase to isomerize it into D-psicose in a single step. However, this reaction is subject to thermodynamic limitations, resulting in low D-psicose yields, necessitating further separation and preparation using a simulated moving bed.

[0003] Li et al. constructed an in vitro multi-enzyme method for synthesizing D-psicose (ACS Catal. 2021, 11, 5088-5099). Using cheap starch as a starting substrate, constructing a multi-enzyme synthesis system consisting of α-glucan phosphorylase, phosphoglucomutase, phosphoglucomutase, psicose 6-phosphate 3-epimerase and psicose 6-phosphate phosphatase is an important method for the efficient synthesis of D-psicose. Among them, phosphatase is the key enzyme that catalyzes the dephosphorylation of D-psicose-6-phosphate (A6P) to synthesize D-psicose, and its substrate specificity determines the synthesis efficiency and purity of D-psicose.

[0004] Currently reported phosphatases have a broad substrate spectrum and can catalyze the dephosphorylation of a variety of monosaccharide phosphates, leading to the accumulation of subsequent byproducts and making their separation and purification difficult. Therefore, regulating the substrate specificity of phosphatases is crucial for the synthesis of high-purity D-psicose. Tang et al. semi-rationally engineered a phosphatase from Thermosipho atlanticus to improve its substrate specificity, increasing the ratio of the target product fructose to the byproduct glucose from 2:1 to 19:1. (Biotechnol Bioeng. 2022, 119, 3462–3473).

[0005] Molecular engineering is an important means of improving enzyme properties, including catalytic efficiency, substrate specificity, and thermal stability. This study screened and identified a phosphatase from Defluviitoga tunisiensis, a Tunisian sewage bacterium. Using molecular engineering techniques, the enzyme was modified and regulated to yield mutants with enhanced substrate specificity, which were then applied to the efficient in vitro multi-enzyme synthesis of D-psicose. Summary of the Invention

[0006] To address the low substrate specificity of current phosphatases, the present invention provides a D-psicose 6-phosphate phosphatase mutant derived from Tunisian sewage bacteria through mutation screening and its application. This mutant modifies the enzymatic properties of thermophilic bacteria, which have a relatively poor substrate specificity. This phosphatase mutant can be used in in vitro multi-enzyme directed synthesis reactions of D-psicose, demonstrating promising application prospects.

[0007] To achieve the above-mentioned purpose, the present invention is implemented through the following technical solutions:

[0008] In a first aspect, the present invention first provides a D-psicose 6-phosphate phosphatase mutant. The D-psicose 6-phosphate phosphatase with an amino acid sequence as shown in SEQ ID NO. 2 is used as a template and site-directed saturation mutagenesis is performed on it to obtain the D-psicose 6-phosphate phosphatase mutant. The mutation sites of the D-psicose 6-phosphate phosphatase mutant are one or more of the following: (1) K at position 160 is mutated to C; (2) S at position 228 is mutated to A; (3) C at position 48 is mutated to P.

[0009] The present invention uses D-psicose 6-phosphate phosphatase (DtA6PP) derived from Tunisian sewage bacterium (Defluviitogatunisiensis) constructed and preserved in the laboratory in the early stage as a template (its amino acid sequence is shown in SEQ ID NO. 2, and its nucleotide sequence is shown in SEQ ID NO. 1), and performs site-directed saturation mutagenesis on it to obtain the D-psicose 6-phosphate phosphatase mutant.

[0010] Preferably, a D-psicose 6-phosphate phosphatase mutant has an amino acid sequence as shown in SEQ ID NO. 4, SEQ ID NO. 8 or SEQ ID NO. 12.

[0011] The amino acid sequence of the mutant DtA6PP / K160C (K at position 160 mutated to C) of the present invention is shown in SEQ ID NO. 4; the amino acid sequence of the mutant DtA6PP / K160C / S228A (K at position 160 mutated to C, S at position 180 mutated to A) is shown in SEQ ID NO. 8; and the amino acid sequence of DtA6PP / K160C / S228A / C48P (K at position 160 mutated to C, S at position 180 mutated to A, and C at position 48 mutated to P) is shown in SEQ ID NO. 12. Compared to wild-type DtA6PP, the substrate specificity of these three D-psicose 6-phosphate phosphatase mutants is significantly improved. Specifically: for the D-psicose 6-phosphate phosphatase mutant DtA6PP / K160C, the ratio of D-psicose to by-product fructose in the product is 3:1; for the D-psicose 6-phosphate phosphatase mutant DtA6PP / K160C / S228A, the ratio of D-psicose to by-product fructose in the product is 4:1; and for the D-psicose 6-phosphate phosphatase mutant DtA6PP / K160C / S228A / C48P, the ratio of D-psicose to by-product fructose in the product is 6:1.

[0012] In a second aspect, the present invention provides a gene encoding the D-psicose 6-phosphate phosphatase mutant.

[0013] Preferably, the nucleotide sequence of the encoding gene is shown as SEQ ID NO.1 or SEQ ID NO.3 or SEQ ID NO.7 or SEQ ID NO.11.

[0014] In a third aspect, the present invention provides a recombinant vector containing a coding gene.

[0015] Preferably, the original vector of the present invention is pET28b.

[0016] In a fourth aspect, the present invention provides a recombinant genetically engineered bacterium containing a recombinant vector.

[0017] The recombinant vector is used to transform a host cell to obtain a recombinant genetically engineered bacterium. The host cell may be any conventional host cell in the art. Preferably, the host cell is Escherichia coli BL21.

[0018] In a fifth aspect, the present invention provides a method for constructing the D-psicose 6-phosphate phosphatase mutant, the method comprising the following steps:

[0019] (S.1) Design site-directed mutagenesis primers and perform overlap extension PCR using a recombinant plasmid containing the D-psicose 6-phosphate phosphatase gene as a template to obtain single-site mutation products in the parental D-psicose 6-phosphate phosphatase, where K at position 160 is mutated to C or S at position 228 is mutated to A.

[0020] (S.2) using the single-site mutation product as a template, performing overlap extension PCR with the site-directed mutagenesis primers to obtain a double-site mutation product;

[0021] (S.3) Using the double-site mutation product as a template, overlap extension PCR was performed using the site-directed mutagenesis primers to obtain a triple-site mutation product;

[0022] (S.4) The single-site mutation product, double-site mutation product, and triple-site mutation product are respectively transformed into a host bacterium, and a D-psicose 6-phosphate phosphatase mutant expression strain is obtained by screening, and the expression is induced to obtain the D-psicose 6-phosphate phosphatase mutant.

[0023] In a sixth aspect, the present invention also includes the use of a D-psicose 6-phosphate phosphatase mutant in catalyzing the preparation of D-psicose, especially in coupling with a psicose 6-phosphate 3-epimerase to catalyze the preparation of D-psicose from D-fructose 6-phosphate.

[0024] The application of the present invention involves using wet cells or enzyme-containing supernatant obtained by centrifugation of an engineered bacterium containing a gene encoding a mutant of D-psicose 6-phosphate phosphatase as a catalyst, coupled with psicose 6-phosphate 3-epimerase, and reacting with D-fructose 6-phosphate as a substrate and a HEPES buffer as a reaction medium. After the reaction, the reaction liquid is separated and purified to obtain D-psicose.

[0025] In a seventh aspect, the present invention provides a method for preparing D-psicose, comprising the following steps: fermenting and culturing an engineered bacterium containing the gene encoding the D-psicose 6-phosphate phosphatase mutant, centrifuging to obtain wet cells, resuspending the cells in a buffer solution, ultrasonically disrupting the cells, and centrifuging the cells; heat-treating the supernatant in a water bath, and re-centrifuging the supernatant as a catalyst; coupling the supernatant with psicose 6-phosphate 3-epimerase; reacting with D-fructose 6-phosphate as a substrate and HEPES buffer as a reaction medium; placing the cells in an ice bath and adding HCl to terminate the reaction; and after completion of the reaction, separating and purifying the reaction solution to obtain D-psicose.

[0026] In the reaction system, the substrate concentration is 5 mM, and the catalyst is a supernatant obtained by centrifuging the fermented culture of an engineered bacterium containing a gene encoding a mutant of D-psicose 6-phosphate phosphatase, resuspending the culture in a buffer solution, ultrasonically disrupting the culture, and centrifuging the supernatant. The supernatant is heat-treated in a 60°C water bath for 30 minutes and then centrifuged again.

[0027] As a preferred method, the fermentation culture method of the engineered bacteria is as follows: the recombinant engineered bacteria are inoculated into LB liquid medium containing kanamycin to obtain seed liquid; the seed liquid is inoculated into fresh LB liquid medium containing kanamycin, and shaken to culture until the bacterial OD 600 The concentration of β-D-thiogalactopyranoside (IPTG) was 0.6-0.8, and the culture was induced. The bacterial cells were collected by centrifugation.

[0028] The bacterial cell described in the present invention is a recombinant engineered bacterium E. coli BL21 (DE3) / pET28b-DtA6PP / K160C, E. coli BL21 (DE3) / pET28b-DtA6PP / K160C / S228A, or E. coli BL21 (DE3) / pET28b-DtA6PP / K160C / S228A / C48P containing a gene encoding a D-psicose 6-phosphate phosphatase mutant.

[0029] Specifically, the fermentation culture method of the present invention is as follows: the recombinant engineered bacteria are inoculated into LB liquid medium containing kanamycin (final concentration of 50 mg / L), and cultured at 37°C and 200 rpm for 8 h; the seed liquid is inoculated into fresh LB liquid medium containing kanamycin (final concentration of 50 mg / L) at a volume ratio of 2%, and cultured at 37°C and 150 rpm until the bacterial OD 600 When the pH is 0.6-0.8, add isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 0.1 mM, and culture at 18°C ​​and 180 rpm for 20 hours. Collect the cells by centrifugation at 4°C and 8000 rpm for 10 minutes. Wash twice with physiological saline and store the centrifuged cells in a -20°C refrigerator.

[0030] Preferably, the reaction medium is a HEPES buffer solution with a pH value of 6.5-7.5, and the catalytic reaction temperature is 45°C-55°C.

[0031] The beneficial effects of the present invention are mainly reflected in:

[0032] (1) The D-psicose 6-phosphate phosphatase mutants provided by the present invention exhibit significantly improved substrate specificity compared to wild-type DtA6PP. In particular, the triple mutant DtA6PP / K160C / S228A / C48P, coupled with psicose 6-phosphate 3-epimerase to produce D-psicose, achieves a 60% yield of psicose, with a D-psicose to fructose byproduct ratio of 6:1, compared to the wild-type DtA6PP product, which has a 1:1 ratio of D-psicose to fructose byproduct. This significantly reduces the amount of fructose byproduct.

[0033] (2) The D-psicose 6-phosphate phosphatase mutant has, to a certain extent, changed the enzymatic properties of the thermophilic bacteria-derived phosphatase, which has poor substrate specificity. This phosphatase mutant can be used in the in vitro multi-enzyme directed synthesis reaction of D-psicose, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The relative enzyme activities of each D-psicose 6-phosphate phosphatase mutant and wild-type DtA6PP in catalyzing D-fructose 6-phosphate to produce D-fructose 6-phosphate (F6P) and D-psicose 6-phosphatase (A6P), respectively;

[0035] Figure 2 Diagram showing the reaction process of synthesizing D-psicose from D-fructose 6-phosphate catalyzed by various D-psicose 6-phosphate phosphatase mutants and wild-type DtA6PP. DETAILED DESCRIPTION

[0036] The present invention will be further described below with reference to specific embodiments. Those skilled in the art will be able to implement the present invention based on these descriptions. Furthermore, the embodiments of the present invention described below generally represent only a portion of the present invention, rather than all of the embodiments. Therefore, all other embodiments derived by those skilled in the art based on the embodiments of the present invention without inventive effort should fall within the scope of protection of the present invention.

[0037] Example 1: Construction of phosphatase gene

[0038] D-psicose 6-phosphate phosphatase from Defluviitogatunisiensis was obtained by mining databases such as NCBI. The amino acid sequence is shown in SEQ ID NO. 2, and the nucleotide sequence is shown in SEQ ID NO. 1. The GenBank accession number is WP_045087739.1.

[0039] Example 2: Site-Directed Saturation Mutagenesis of Sites 160 and 228 of D-psicose 6-Phosphate Phosphatase

[0040] In order to perform saturation mutagenesis on Lys (K) at position 160, Ser (S) at position 228, and Cys (C) at position 48 in the parent amino acid sequence, corresponding primers were designed, as shown in Table 1.

[0041] Table 1: Primer design table

[0042]

[0043] Note: N=A / G / C / T, K=G / T, M=A / C.

[0044] The recombinant plasmid pET28b-DtA6PP containing the target gene fragment was used as a template and the whole plasmid was amplified according to the overlap extension PCR method.

[0045] The PCR amplification system was as follows (50 μL): template DNA 0.1 ng-1 ng, 2× Phanta Max Buffer 25 μL, dNTPs (10 mM each) 1 μL, 1 μL each of upstream and downstream mutation primers, Phanta Max Super-Fidelity DNA Polymerase 1 U, and the remaining ddH2O was added to the total volume.

[0046] PCR reaction parameters: (1) pre-denaturation at 95°C for 30 seconds; (2) denaturation at 95°C for 15 seconds; (3) annealing at 58°C for 15 seconds; (4) extension at 72°C for 6 minutes, and steps (2) to (4) were repeated 30 times; (5) complete extension at 72°C for 5 minutes, and storage at 4°C.

[0047] After the PCR product was positive by 0.9% agarose gel electrophoresis, 20 μL of the PCR reaction mixture was digested with 1 μL of the endonuclease Dpn I at 37°C for 3 hours to remove the template plasmid DNA, followed by inactivation at 65°C for 10 minutes. Heat-shock transformation was then performed into E. coli BL21(DE3) competent cells. After recovery, the cells were plated on LB plates containing kanamycin and cultured overnight. A mutant library of approximately 300 clones was obtained on each plate.

[0048] Pick a single colony and place it in a 96-well culture plate filled with LB medium and culture at 37°C until the bacterial OD 600 When the pH is approximately 0.6-0.8, IPTG (final concentration 0.1 mM) is added to the above LB liquid medium and cultured at 18°C, 150 rpm for 20 h. Centrifuge in a 96-well plate centrifuge at 4°C, 4000 rpm for 20 min, discard the supernatant, add 600 μL of HEPES buffer (50 mM, pH 7.0) to the collected cells, mix well, and freeze-thaw (-80°C for 30 min, 37°C for 30 min) for four cycles. Centrifuge at 4000 rpm for 10 min, and the supernatant is the phosphatase enzyme solution.

[0049] Take 200 μL of phosphatase solution and the reaction system prepared in advance (1mM F6P, 5mM Mg 2+, 100mM HEPES buffer, and excess psicose-6-phosphate 3-epimerase) were mixed and incubated at 50°C with shaking for 0.5 hours. The reaction was terminated by adding 30μL of 6M HCl to each well. 50μL of the reaction solution was aspirated and mixed with 150μL of molybdenum blue phosphate working solution (Solution A: a mixture of 20mM ammonium molybdate and 100mM zinc acetate (pH 5.0); Solution B: 10% ascorbic acid ultrapure water). The mixture was incubated in a 37°C incubator for 30 minutes. The fluorescence intensity of each sample was measured at a wavelength of 850nm using a microplate reader. The catalytic activity of the mutant was determined based on the change in fluorescence intensity, and clones with a significantly greater change than the control strain (starting strain) were selected.

[0050] After rescreening and verification of the positive clones, the full plasmids of the mutants were extracted, and the introduced point mutations were confirmed by DNA sequencing. The DNA sequencing results of the mutants with the highest substrate specificity at each site showed that Lys at position 160 was mutated to Cys (K160C), Ser at position 228 was mutated to Ala (S228A), and Cys at position 48 was mutated to Pro (C48P). Thus, the D-psicose 6-phosphate phosphatase mutant engineered bacteria E. coli BL21(DE3) / pET28b-DtA6PP / K160C, E. coli BL21(DE3) / pET28b-DtA6PP / S228A, and E. coli BL21(DE3) / pET28b-DtA6PP / C48P were obtained. The nucleotide sequences of mutants K160C, S228A and C48P are SEQ ID NO.3, SEQ ID NO.5 and SEQ ID NO.7, respectively (the corresponding amino acid sequences are SEQ ID NO.4, SEQ ID NO.6 and SEQ ID NO.8).

[0051] Example 3: Construction of D-psicose 6-phosphate phosphatase combination mutants

[0052] Using the expression plasmids pET28b-DtA6PP / K160C or pET28b-DtA6PP / S228A as templates, site-directed mutagenesis was performed by whole-plasmid amplification to obtain the double mutant pET28b-DtA6PP / K160C / S228A. Furthermore, using the expression plasmids pET28b-DtA6PP / K160C / S228A as templates, site-directed mutagenesis was performed by whole-plasmid amplification to obtain the triple mutant pET28b-DtA6PP / K160C / S228A / C48P. The corresponding primers were designed as shown in Table 2.

[0053] Table 2: Primer design table

[0054]

[0055] The PCR amplification system was as follows (50 μL): template DNA 0.1 ng-1 ng, 2× Phanta Max Buffer 25 μL, dNTPs (10 mM each) 1 μL, 1 μL each of upstream and downstream mutation primers, Phanta Max Super-Fidelity DNA Polymerase 1 μL, and the remaining ddH2O was added to the total volume.

[0056] PCR reaction parameters: (1) pre-denaturation at 95°C for 30 seconds; (2) denaturation at 95°C for 15 seconds; (3) annealing at 60°C for 15 seconds; (4) extension at 58°C for 5 minutes, and steps (2) to (4) were repeated 30 times; (5) complete extension at 72°C for 5 minutes, and storage at 4°C.

[0057] If the PCR product was positive by 0.9% agarose gel electrophoresis, 20 μL of the PCR reaction mixture was digested with 1 μL of the endonuclease Dpn I at 37°C for 3 hours to remove the template plasmid DNA, followed by inactivation at 65°C for 10 minutes. Heat-shock transformation was then performed into E. coli BL21(DE3) competent cells. After recovery, the cells were plated on LB plates containing kanamycin and cultured overnight. Single colonies were selected and cultured in LB liquid medium containing kanamycin (final concentration 50 mg / L), and the plasmids were extracted and sequenced.

[0058] Correct sequencing results identified the D-psicose 6-phosphate phosphatase combination mutant engineered strains: E. coli BL21(DE3) / pET28b-DtA6PP / K160C / S228A and E. coli BL21(DE3) / pET28b-DtA6PP / K160C / S228A / C48P. The corresponding nucleotide sequences for these combination mutant engineered strains are SEQ ID NOs. 9 and 11, respectively (and the corresponding amino acid sequences are SEQ ID NOs. 10 and 12).

[0059] Example 4: Expression and purification of D-psicose 6-phosphate phosphatase mutants

[0060] The wild-type DtA6PP and the mutants DtA6PP / K160C, DtA6PP / K160C / S228A, and DtA6PP / K160C / S228A / C48P obtained in Example 2 and Example 3 were inoculated into LB medium containing kanamycin (final concentration of 50 mg / L) and cultured at 37°C for 6-8 h. The inoculum was transferred to fresh LB liquid medium containing kanamycin (final concentration of 50 mg / L) at a 2% (v / v) inoculum size for expansion and cultured at 37°C at 150 rpm until the bacterial OD reached 0. 600When the p-value is about 0.6-0.8, IPTG (final concentration is 0.1mM) is added to the above LB liquid medium, and the culture is induced at 18°C ​​and 180rpm for 20h. The bacterial cells are collected by centrifugation at 4°C and 8000rpm for 10min, and the precipitate is resuspended in HEPES buffer (pH 7.0) to obtain a bacterial suspension. The bacterial suspension is ultrasonically disrupted (60W, continuous 2s, intermittent 4s, continuous disruption for 15min) in a 4°C ice bath to obtain a cell disruption solution, which is centrifuged at 12000×g for 10min. The supernatant is heat-treated in a 60°C water bath for 30min, and centrifuged at 4°C and 12000×g for 10min. The supernatant is taken as the phosphatase enzyme solution, and the target protein content is detected using a BCA protein content detection kit.

[0061] Example 5: Determination of Enzyme Activity of Parent D-psicose 6-phosphate Phosphatase and Its Mutants on Structurally Similar Substrates

[0062] The recombinant E. coli obtained in Example 4 was subjected to enzyme activity assays against other structurally similar substrates. A 1 mL reaction system consisted of: HEPES buffer (100 mM, pH 7.0), 5 mM D-fructose 6-phosphate, D-glucose 6-phosphate, D-glucose 1-phosphate, D-psicose 6-phosphate, 5 mM MgCl2, and the phosphatase enzyme solution prepared according to the method of Example 4. The reaction solution was preheated at 50°C for 2 minutes and then reacted at 1000 rpm for 60 minutes. After completion of the reaction, the reaction was terminated by adding HCl in an ice bath. The reaction solution was separated and purified, and the yield of the target product was determined by liquid chromatography. The enzyme activity data are shown in Table 3.

[0063] Table 3: Enzyme activity ratios of wild-type phosphatase and its mutants on different structurally similar substrates

[0064]

[0065] Example 6: In vitro multienzyme-catalyzed synthesis of D-psicose from D-fructose 6-phosphate (I)

[0066] The substrate specificity of the recombinant E. coli obtained in Example 4 was determined. A 1 mL reaction system consisted of: HEPES buffer (100 mM, pH 7.0), 5 mM D-fructose 6-phosphate, 5 mM MgCl2, the previously prepared psicose 6-phosphate 3-epimerase enzyme solution, and the phosphatase enzyme solution prepared according to the method of Example 4. The reaction solution was preheated at 50°C for 2 minutes and then reacted at 1000 rpm for 60 minutes. After completion of the reaction, the reaction was incubated on ice and terminated by the addition of HCl. The reaction solution was separated and purified, and the content of D-psicose and the byproduct fructose in the product was determined by liquid chromatography.

[0067] The product D-psicose and the byproduct fructose were determined by liquid chromatography using an Agilent 1260 InfinityRID and an Aminex HPX-87H capillary column. Chromatographic conditions included an injection volume of 10 μL, an injection port and detector temperature of 40°C, a column temperature of 60°C, a flow rate of 0.6 mL / min, and a detection time of 15 minutes.

[0068] Liquid chromatography analysis revealed that after 60 minutes of reaction, the wild-type DtA6PP produced a 19.7% psicose yield, with a D-psicose to fructose ratio of approximately 1:1. The mutants DtA6PP / K160C, DtA6PP / K160C / S228A, and DtA6PP / K160C / S228A / C48P achieved psicose yields of 21%, 29.8%, and 31.2%, respectively, with D-psicose to fructose ratios of 2:1, 3:1, and 4:1, respectively.

[0069] Example 7: In vitro multienzyme-catalyzed synthesis of D-psicose from D-fructose 6-phosphate (II)

[0070] The recombinant E. coli obtained in Example 4 was subjected to a reaction progress assay. A 10 mL reaction system consisted of: HEPES buffer (100 mM, pH 7.0), 5 mM D-fructose 6-phosphate, 5 mM MgCl2, previously prepared psicose 6-phosphate 3-epimerase enzyme solution, and phosphatase enzyme solution prepared according to the method of Example 4. The reaction solution was preheated at 50°C for 2 minutes. The reaction solution was reacted at 50°C for 0.5, 1, 2, 3, 4, 5, 6, 7, 8, and 9 hours. Samples were then taken directly after the reaction, ice-bathed, and HCl was added to terminate the reaction. The reaction solution was separated and purified, and the content of D-psicose and the byproduct fructose in the product was determined by liquid chromatography.

[0071] The effects of wild-type phosphatase and its mutants on the in vitro multi-enzyme synthesis of D-psicose are shown in Table 4. The liquid chromatography-mass spectrometry results after 9 h of reaction showed that the yield of allulose from the wild-type DtA6PP DtA6PP was 30%, and the ratio of allulose to byproduct fructose in the product was 1:1; the yield of allulose from the single-point mutant DtA6PP / K160C DtA6PP / K160C DtA6PP / K160C DtA6PP / S228A DtA6PP / K160C DtA6PP / S228A DtA6PP / K160C DtA6PP / S228A DtA6PP / K160C DtA6PP / S228A DtA6PP / K160C DtA6PP / S228A DtA6PP / C48P DtA6PP / K160C DtA6PP / S228A ...

[0072] Table 4: Effects of wild-type phosphatase and its mutants on in vitro multienzyme synthesis of D-psicose

[0073]

[0074] The present invention is not limited by the above specific description. Various changes can be made to the present invention within the scope outlined by the claims, and these changes are all within the scope of the present invention.

Claims

1. A D-psicose 6-phosphate phosphatase mutant, characterized in that: Its amino acid sequence is shown in SEQ ID NO.10 or SEQ ID NO.

12.

2. A gene encoding the D-psicose 6-phosphate phosphatase mutant according to claim 1, characterized in that: The nucleotide sequence thereof is shown in SEQ ID NO.9 or SEQ ID NO.

11.

3. A recombinant vector containing the coding gene as claimed in claim 2.

4. A recombinant genetically engineered bacterium containing the recombinant vector as claimed in claim 3. Use of the D-psicose 6-phosphate phosphatase mutant according to claim 1 in catalyzing the production of D-psicose from D-psicose 6-phosphate. Use of the D-psicose 6-phosphate phosphatase mutant according to claim 1 in preparing D-psicose by coupling D-fructose 6-phosphate catalyzed by psicose 6-phosphate 3-epimerase.

7. A method for preparing D-psicose, characterized in that: The method comprises the following steps: fermenting and culturing an engineered bacterium containing the gene encoding the D-psicose 6-phosphate phosphatase mutant according to claim 1, centrifuging to obtain wet bacterial cells, resuspending the cells in a buffer solution, ultrasonically disrupting the cells, and centrifuging the cells; heat-treating the supernatant in a water bath, and re-centrifuging the supernatant as a catalyst; coupling the supernatant with psicose 6-phosphate 3-epimerase; reacting the cells with D-fructose 6-phosphate as a substrate and HEPES buffer as a reaction medium; placing the cells in an ice bath and adding HCl to terminate the reaction; and separating and purifying the reaction solution after the reaction to obtain D-psicose.

8. The method for preparing D-psicose according to claim 7, wherein: The fermentation culture method of the engineered bacteria is as follows: the recombinant engineered bacteria are inoculated into LB liquid culture medium containing kanamycin to obtain seed liquid; the seed liquid is inoculated into fresh LB liquid culture medium containing kanamycin, and the culture is shaken until the bacterial OD 600 The concentration of isopropyl-β-D-thiogalactopyranoside (IPTG) was set to 0.6~0.8, and the culture was induced. The bacterial cells were collected by centrifugation.

9. The method for preparing D-psicose according to claim 7, wherein: The fermentation culture method of the engineered bacteria is as follows: the recombinant engineered bacteria are inoculated into LB liquid culture medium containing kanamycin to obtain seed liquid; the seed liquid is inoculated into fresh LB liquid culture medium containing kanamycin, and the culture is shaken until the bacterial OD 600 The concentration of isopropyl-β-D-thiogalactopyranoside (IPTG) was set to 0.6~0.8, and the culture was induced. The bacterial cells were collected by centrifugation.

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