Phosphatase mutant and application thereof

Abstract

The invention belongs to the field of biological catalysis, and particularly relates to a D-psicose 6-phosphate phosphatase mutant derived from Defluviitoga tonsiensis in sewage and application of the D-psicose 6-phosphate phosphatase mutant derived from Defluviitoga tonsiensis in the sewage and the application of the D-psicose 6-phosphate phosphatase mutant derived from Defluviitoga tonsiensis in the sewage. The D-psicose 6-phosphate phosphatase mutant is obtained by taking D-psicose 6-phosphate phosphatase of which the amino acid sequence is shown as SEQ ID NO.2 as a template and carrying out site-directed saturation mutation on the D-psicose 6-phosphate phosphatase; the mutation site of the D-psicose 6-phosphate phosphatase mutant is one or more than two of the following: (1) K at the 160th site is mutated into C; (2) S at the 228th site is mutated into A; and (3) C at the 48th site is mutated into P. When the D-psicose 6-phosphate phosphatase mutant provided by the invention is used for preparing D-psicose, the yield reaches 60%, and the ratio of the D-psicose to the byproduct fructose in the product is increased to 6: 1 from 1: 1 of a wild type. The phosphatase mutant subjected to substrate specificity regulation and control has important industrial application potential in production of D-psicose by an in-vitro multi-enzyme synthesis system.

Description

Technical Field

[0001] The invention relates to the technical field of bioengineering, and in particular to a phosphatase mutant derived from Defluviitogatunisiensis with improved reaction specificity to D-psicose 6-phosphate and application thereof. Background Art

[0002] D-psicose is a low-energy sweetener with a sweetness of 70% of sucrose. It is considered a potential sucrose substitute and is widely used in food, medicine, health care and other fields. Its efficient production has received widespread attention. The commonly used method for producing psicose is to use fructose as a substrate and use isomerase to isomerize D-psicose in one step. However, this reaction is subject to thermodynamic limitations, resulting in a low yield of D-psicose, which requires 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 the starting substrate, constructing a multi-enzyme synthesis system consisting of α-glucan phosphorylase, phosphoglucose mutase, phosphoglucose isomerase, 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] The phosphatases reported so far have a broad substrate spectrum and can catalyze the dephosphorylation of a variety of phosphate monosaccharides, resulting in the accumulation of subsequent by-products and difficulties in separation and purification. Therefore, the regulation of phosphatase substrate specificity is of great significance for the synthesis of high-purity D-psicose. Tang et al. semi-rationally designed the phosphatase of Thermosipho atlanticus to improve its substrate specificity, and the ratio of the target product fructose to the by-product glucose was increased from 2:1 to 19:1. (Biotechnol Bioeng. 2022, 119, 3462–3473).

[0005] Molecular modification technology is an important means to improve the application properties of enzymes, such as catalytic efficiency, substrate specificity and thermal stability. The present invention screened and mined phosphatase from Defluviitoga tunisiensis, modified the enzyme through molecular modification technology, and regulated it to obtain mutants with improved substrate specificity, which were applied to the efficient synthesis of D-psicose by multi-enzymes in vitro. Summary of the invention

[0006] In order to solve the problem of low substrate specificity of phosphatases, the present invention provides a D-psicose 6-phosphate phosphatase mutant derived from Tunisian sewage sheath bacillus and its application through mutation screening. The mutant changes the enzymatic characteristics of the phosphatase derived from thermophilic bacteria with poor substrate specificity to a certain extent. The phosphatase mutant can be used in the in vitro multi-enzyme directed synthesis reaction of D-psicose, and has good application prospects. To achieve the above-mentioned purpose, the present invention is implemented by the following technical solutions:

[0007] In a first aspect, the present invention first provides a D-psicose 6-phosphate phosphatase mutant, using the D-psicose 6-phosphate phosphatase with an amino acid sequence as shown in SEQ ID NO.2 as a template, performing site-directed saturation mutagenesis on it, and obtaining 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 mutates to C; (2) S at position 228 mutates to A; (3) C at position 48 mutates to P.

[0008] The present invention uses D-psicose 6-phosphate phosphatase (DtA6PP) from Tunisian sewage sheath bacillus (Defluviitogatunisiensis) constructed and preserved in the laboratory in the early stage as a template (the amino acid sequence of which is shown in SEQ ID NO.2, and the nucleotide sequence is shown in SEQ ID NO.1), performs site-directed saturation mutagenesis on it, and obtains the D-psicose 6-phosphate phosphatase mutant.

[0009] 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.

[0010] 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; the amino acid sequence of DtA6PP / K160C / S228A / C48P (K at position 160 mutated to C, S at position 180 mutated to A, C at position 48 mutated to P) is shown in SEQ ID NO.12. Compared with the wild-type DtA6PP, the substrate specificity of the above 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 in the product to the by-product fructose is 3:1; for the D-psicose 6-phosphate phosphatase mutant DtA6PP / K160C / S228A, the ratio of D-psicose in the product to the by-product fructose is 4:1; for the D-psicose 6-phosphate phosphatase mutant DtA6PP / K160C / S228A / C48P, the ratio of D-psicose in the product to the by-product fructose is 6:1.

[0011] In a second aspect, the present invention provides a gene encoding the D-psicose 6-phosphate phosphatase mutant.

[0012] 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.

[0013] In a third aspect, the present invention provides a recombinant vector containing a coding gene.

[0014] Preferably, the original vector of the present invention is pET28b.

[0015] In a fourth aspect, the present invention provides a recombinant genetically engineered bacterium containing a recombinant vector.

[0016] 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.

[0017] In a fifth aspect, the present invention provides a method for constructing the D-psicose 6-phosphate phosphatase mutant, the preparation method comprising the following steps: (S.1) designing site-directed mutagenesis primers, using the recombinant plasmid containing the D-psicose 6-phosphate phosphatase gene as a template, and performing overlap extension PCR to obtain single-site mutation products in which the K at position 160 is mutated to C or the S at position 228 is mutated to A in the parental D-psicose 6-phosphate phosphatase; (S.2) using the single-site mutation product as a template, performing overlap extension PCR using the site-directed mutation primers to obtain a double-site mutation product; (S.3) using the double-site mutation product as a template, performing overlap extension PCR using the site-directed mutation primers to obtain a triple-site mutation product; (S.4) The single-site mutation product, double-site mutation product, and triple-site mutation product are respectively transformed into host bacteria, 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.

[0018] 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 D-psicose 6-phosphate 3-epimerase to catalyze the preparation of D-psicose from D-fructose 6-phosphate.

[0019] The application of the present invention is: using wet bacteria or enzyme-containing supernatant obtained by centrifugation after fermentation and culture of engineered bacteria containing a D-psicose 6-phosphate phosphatase mutant encoding gene as a catalyst, coupling psicose 6-phosphate 3-epimerase, using D-fructose 6-phosphate as a substrate, and using HEPES buffer as a reaction medium for reaction. After the reaction is completed, the reaction solution is separated and purified to obtain D-psicose.

[0020] 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 bacteria, resuspending with a buffer solution, ultrasonically disrupting, and centrifuging; heat-treating the supernatant in a water bath and centrifuging again, using the supernatant as a catalyst, coupling psicose 6-phosphate 3-epimerase, reacting with D-fructose 6-phosphate as a substrate and HEPES buffer as a reaction medium, placing the bacteria in an ice bath and adding HCl to terminate the reaction; after the reaction, separating and purifying the reaction solution to obtain D-psicose.

[0021] In the reaction system, the substrate concentration is 5 mM, the catalyst is an engineered bacterium containing a D-psicose 6-phosphate phosphatase mutant encoding gene, which is fermented and cultured, centrifuged, resuspended in a buffer solution, ultrasonically broken, and centrifuged. The supernatant is heat-treated in a 60°C water bath for 30 minutes and then centrifuged again to obtain the supernatant.

[0022] Preferably, the fermentation culture method of the engineered bacteria is as follows: the recombinant engineered bacteria are inoculated into a LB liquid medium containing kanamycin to obtain a seed solution; the seed solution is inoculated into a fresh LB liquid medium containing kanamycin, and the culture is shaken until the bacterial OD 600 The pH value was 0.6-0.8, isopropyl-β-D-thiogalactopyranoside (IPTG) was added to induce the culture, and the bacterial cells were collected by centrifugation.

[0023] 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.

[0024] 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 under shaking conditions of 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 under shaking conditions of 37°C and 150 rpm until the bacterial OD 600 The pH value was 0.6-0.8, and isopropyl-β-D-thiogalactopyranoside (IPTG) was added at a final concentration of 0.1 mM. The cells were induced at 18°C ​​and 180 rpm for 20 h, and the bacterial cells were collected by centrifugation at 4°C and 8000 rpm for 10 min. The cells were washed twice with physiological saline, and the cells obtained by centrifugation were stored in a -20°C refrigerator.

[0025] Preferably, the reaction medium is a HEPES buffer with a pH value of 6.5-7.5, and the catalytic reaction temperature is 45°C-55°C.

[0026] The beneficial effects of the present invention are mainly reflected in: (1) The D-psicose 6-phosphate phosphatase mutants provided by the present invention have significantly improved substrate specificity compared to the wild-type DtA6PP. In particular, the triple mutant DtA6PP / K160C / S228A / C48P is coupled with psicose 6-phosphate 3-epimerase to prepare D-psicose. Compared with the wild-type DtA6PP product, the ratio of D-psicose to byproduct fructose in the product is 1:1. The yield of psicose in the triple mutant can reach 60%, and the ratio of D-psicose to byproduct fructose in the product is 6:1, and the amount of byproduct fructose is greatly reduced. (2) The D-psicose 6-phosphate phosphatase mutant has, to a certain extent, changed the enzymatic properties of the phosphatase derived from thermophilic bacteria, which has poor substrate specificity. The 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

[0027] Figure 1 The relative enzyme activities of each D-psicose 6-phosphate phosphatase mutant and the wild-type DtA6PP in catalyzing D-fructose 6-phosphate to produce D-fructose 6-phosphate (F6P) and D-psicose 6-phosphatase (A6P), respectively; Figure 2 This is a reaction process diagram of the synthesis of D-psicose from D-fructose 6-phosphate catalyzed by various D-psicose 6-phosphate phosphatase mutants and wild-type DtA6PP. DETAILED DESCRIPTION

[0028] The present invention is further described below in conjunction with specific embodiments. Those of ordinary skill in the art will be able to implement the present invention based on these descriptions. In addition, the embodiments of the present invention involved in the following description are generally only embodiments of a part of the present invention, rather than all embodiments. Therefore, based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work should fall within the scope of protection of the present invention. Example 1: Construction of phosphatase gene

[0029] The D-psicose 6-phosphate phosphatase from Defluviitogatunisiensis was obtained by mining databases such as NCBI, the amino acid sequence of which 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. Example 2: Site-directed saturation mutagenesis of sites 160 and 228 of D-psicose 6-phosphate phosphatase

[0030] In order to perform saturation mutation 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. Table 1: Primer design table Note: N=A / G / C / T, K=G / T, M=A / C.

[0031] The recombinant plasmid pET28b-DtA6PP containing the target gene fragment was used as a template, and the whole plasmid of the template was amplified according to the overlap extension PCR method.

[0032] The PCR amplification system was (50 μL): template DNA 0.1 ng-1 ng, 2×Phanta Max Buffer 25 μL, dNTPs (10 mM each) 1 μL, upstream and downstream mutation primers 1 μL each, Phanta Max Super-Fidelity DNA Polymerase 1U, and the rest ddH2O to make up the total volume.

[0033] PCR reaction parameters: (1) pre-denaturation at 95°C for 30 s; (2) denaturation at 95°C for 15 s; (3) annealing at 58°C for 15 s; (4) extension at 72°C for 6 min, and steps (2) to (4) were cycled 30 times; (5) complete extension at 72°C for 5 min, and stored at 4°C.

[0034] After the PCR product was positive after 0.9% agarose gel electrophoresis analysis, 20 μL of the PCR reaction solution was taken, and 1 μL of endonuclease Dpn I was added to remove the template plasmid DNA at 37°C for 3h, and inactivated at 65°C for 10min. Heat shock transformation was performed into E. coli BL21 (DE3) competent cells, and after recovery, it was spread on LB plates containing kanamycin and cultured overnight. A mutant library of about 300 clones was obtained on each plate.

[0035] 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 reaches 600 When the pH is about 0.6-0.8, add IPTG (final concentration is 0.1mM) to the above LB liquid medium, and induce culture at 18°C, 150rpm for 20h. Centrifuge in a 96-well plate centrifuge at 4°C, 4000rpm for 20min, discard the supernatant, add 600μL HEPES buffer (50mM, pH 7.0) to the collected bacteria, mix evenly, freeze-thaw and break (-80°C 30min, 37°C 30min), cycle 4 times, centrifuge at 4000rpm for 10min, and take the supernatant as the phosphatase enzyme solution.

[0036] Take 200 μL of phosphatase solution and mix it with the pre-prepared reaction system (1 mM F6P, 5 mM Mg 2+, 100mM HEPES buffer, excess allulose-6-phosphate 3-epimerase) and mix well, shake at 50℃ for 0.5h, and add 30μL 6MHCl to each well to terminate the reaction. Pipette 50μL of the reaction solution and 150μL of the molybdenum blue method phosphate working solution (solution A: a mixture of 20mM ammonium molybdate and 100mM zinc acetate (pH=5.0), solution B: 10% ascorbic acid ultrapure aqueous solution) and mix well, incubate in a 37℃ incubator for 30min; detect the fluorescence intensity of each sample at a wavelength of 850nm under an ELISA instrument. According to the change in fluorescence intensity, the catalytic activity of the mutant is judged, and then the clones with a change intensity far greater than that of the control strain (starting strain) are screened.

[0037] After rescreening and verification of the screened positive clones, the whole plasmid of the mutant strain was extracted, and the introduced point mutations were determined by DNA sequencing. The DNA sequencing results of the mutant strain with the highest substrate specificity at each site showed that Lys at position 160 mutated to Cys (K160C), Ser at position 228 mutated to Ala (S228A), and Cys at position 48 mutated to Pro (C48P). Thus, the D-psicose 6-phosphate phosphatase mutant engineering 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). Example 3: Construction of D-psicose 6-phosphate phosphatase combination mutants

[0038] Using the expression plasmid pET28b-DtA6PP / K160C or pET28b-DtA6PP / S228A as a template, a double mutant pET28b-DtA6PP / K160C / S228A was obtained by site-directed mutagenesis through full plasmid amplification; and then using the expression plasmid pET28b-DtA6PP / K160C / S228A as a template, a triple mutant pET28b-DtA6PP / K160C / S228A / C48P was obtained by site-directed mutagenesis through full plasmid amplification. The corresponding primers were designed as shown in Table 2. Table 2: Primer design table

[0039] The PCR amplification system was (50 μL): template DNA 0.1 ng-1 ng, 2×Phanta Max Buffer 25 μL, dNTPs (10 mM each) 1 μL, upstream and downstream mutation primers 1 μL each, Phanta Max Super-Fidelity DNA Polymerase 1 μL, and the rest ddH2O to make up the total volume.

[0040] PCR reaction parameters: (1) pre-denaturation at 95°C for 30 s; (2) denaturation at 95°C for 15 s; (3) annealing at 60°C for 15 s; (4) extension at 58°C for 5 min, and steps (2) to (4) were cycled 30 times; (5) complete extension at 72°C for 5 min, and stored at 4°C.

[0041] After the PCR product was positive after 0.9% agarose gel electrophoresis analysis, 20 μL of the PCR reaction solution was taken, and 1 μL of endonuclease Dpn I was added to remove the template plasmid DNA at 37°C for 3h, and inactivated at 65°C for 10min. Heat shock transformation was performed into E. coli BL21 (DE3) competent cells, and after recovery, it was spread on LB plates containing kanamycin and cultured overnight. A single colony was picked and cultured in LB liquid medium containing kanamycin resistance (final concentration of 50 mg / L), and the plasmid was extracted for sequencing.

[0042] The correct sequencing results are D-psicose 6-phosphate phosphatase combined mutant engineering strains E. coli BL21 (DE3) / pET28b-DtA6PP / K160C / S228A, E. coli BL21 (DE3) / pET28b-DtA6PP / K160C / S228A / C48P. The corresponding nucleotide sequences of the combined mutant engineering strains are SEQ ID NO.9 and SEQ ID NO.11 (the corresponding amino acid sequences are SEQ ID NO.10 and SEQ ID NO.12). Example 4: Expression and purification of D-psicose 6-phosphate phosphatase mutants

[0043] 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), cultured at 37°C for 6-8 h, and transferred to fresh LB liquid medium containing kanamycin (final concentration of 50 mg / L) at a 2% (v / v) inoculum for expansion culture, and cultured at 37°C, 150 rpm until the bacterial OD 600When the pH 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, and 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 broken (60W, lasting 2s, intermittent 4s, continuous breaking for 15min) in a 4°C ice bath to obtain a cell lysis solution, centrifuged at 12000×g for 10min, and 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 a phosphatase enzyme solution, and the target protein content is detected using a BCA protein content detection kit. Example 5: Determination of the Enzyme Activity of the Parent D-psicose 6-phosphate Phosphatase and Its Mutants on Structurally Similar Substrates The recombinant Escherichia coli obtained in Example 4 was subjected to enzyme activity assays on other structurally similar substrates. 1 mL of the reaction system consisted of: HEPES buffer solution (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 phosphatase enzyme solution prepared by the method of Example 4. The reaction solution was preheated at 50 ° C for 2 min and reacted at 1000 rpm for 60 min. After the reaction was completed, ice bath was added and HCl was added to terminate the reaction. 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. Table 3: Enzyme activity ratio of wild-type phosphatase and its mutants on different structurally similar substrates Example 6: In vitro synthesis of D-psicose by multienzyme catalysis of D-fructose 6-phosphate (I)

[0044] The substrate specificity of the recombinant Escherichia coli obtained in Example 4 was determined. The composition of the 1 mL reaction system was: HEPES buffer solution (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 by the method of Example 4. The reaction solution was preheated at 50° C. for 2 min and reacted at 1000 rpm for 60 min. After the reaction was completed, the reaction was terminated by adding HCl in an ice bath, and the reaction solution was separated and purified. The content of D-psicose and byproduct fructose in the product was determined by liquid chromatography.

[0045] The product D-psicose and the by-product fructose were determined by liquid chromatography. The liquid chromatography model was 1260 Infinity RID (Agilent), and the capillary column model was Aminex HPX-87H Column. The chromatographic conditions were: injection volume 10 μL, injection port and detector temperature were both 40°C, column temperature was 60°C, flow rate was 0.6 mL / min, and detection time was 15 min.

[0046] According to the results of liquid phase detection, after 60 minutes of reaction, the psicose yield of wild-type DtA6PP was 19.7%, and the ratio of product D-psicose to byproduct fructose was about 1:1. The psicose yields of mutants DtA6PP / K160C, DtA6PP / K160C / S228A and DtA6PP / K160C / S228A / C48P reached 21%, 29.8% and 31.2%, respectively, and the ratios of product D-psicose to byproduct fructose were 2:1, 3:1 and 4:1, respectively. Example 6: In vitro multienzyme catalysis of D-fructose 6-phosphate to synthesize D-psicose (II)

[0047] The reaction progress of the recombinant Escherichia coli obtained in Example 4 was determined. The 10 mL reaction system consisted of: HEPES buffer solution (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 by the method of Example 4. After the reaction solution was preheated at 50° C. for 2 min, the reaction solution was directly sampled after reacting at 50° C. for 0.5, 1, 2, 3, 4, 5, 6, 7, 8, and 9 h, and the reaction was terminated by adding HCl in an ice bath. The reaction solution was separated and purified, and the content of D-psicose and byproduct fructose in the product was determined by liquid chromatography.

[0048] The effects of wild-type phosphatase and its mutants on the in vitro multi-enzyme synthesis of D-psicose are shown in Table 2. The liquid phase detection results after 9 hours of reaction showed that the yield of wild-type DtA6PP psicose was 30%, and the ratio of psicose to by-product fructose in the product was 1:1; the yield of single-point mutant DtA6PP / K160C psicose was 32%, and the ratio of psicose to by-product fructose in the product was 3:1; the yield of double mutant DtA6PP / K160C / S228A psicose was 47%, and the ratio of psicose to by-product fructose in the product was 4:1; the yield of triple mutant DtA6PP / K160C / S228A / C48P psicose was 60%, and the ratio of psicose to by-product fructose in the product was 6:1. Table 2: Effects of wild-type phosphatase and its mutants on in vitro multienzyme synthesis of D-psicose The present invention is not limited by the above specific textual description. The present invention can make various changes within the scope summarized 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: Using the D-psicose 6-phosphate phosphatase with an amino acid sequence as shown in SEQ ID NO.2 as a template, site-directed saturation mutagenesis is performed to obtain a 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.

2. A D-psicose 6-phosphate phosphatase mutant as described in claim 1, characterized in that: Its amino acid sequence is shown in SEQ ID NO.4, SEQ ID NO.8 or SEQ ID NO.

12.

3. A gene encoding the D-psicose 6-phosphate phosphatase mutant according to claim 1 or 2, characterized in that: The nucleotide sequence thereof is shown in SEQ ID NO.3, SEQ ID NO.7 or SEQ ID NO.

11.

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

5. A recombinant genetically engineered bacterium containing the recombinant vector as claimed in claim 4.

6. Use of the D-psicose 6-phosphate phosphatase mutant according to claim 1 or 2 in catalyzing the preparation of D-psicose from D-psicose 6-phosphate.

7. Use of the D-psicose 6-phosphate phosphatase mutant according to claim 1 or 2 in coupling with psicose 6-phosphate 3-epimerase to catalyze D-fructose 6-phosphate to prepare D-psicose.

8. A method for preparing D-psicose, characterized in that: The method comprises the following steps: fermenting and culturing an engineered bacterium containing a gene encoding a D-psicose 6-phosphate phosphatase mutant as claimed in claim 1 or 2, centrifuging to obtain wet bacterial cells, resuspending the cells with a buffer solution, ultrasonically breaking the cells, and centrifuging the cells; heat-treating the supernatant in a water bath and centrifuging the supernatant again as a catalyst; coupling 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 separating and purifying the reaction solution after the reaction is completed to obtain D-psicose.

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

10. The method for preparing D-psicose according to claim 8 or 9, wherein: The reaction medium is a HEPES buffer solution with a pH value of 6.5 to 7.5, and the catalytic reaction temperature is 45° C. to 55° C.

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