Tagolose-6-phosphate phosphatase mutant and application thereof in production of D-tagatose

Abstract

The invention discloses a tagatose-6-phosphate phosphatase mutant and application of the tagatose-6-phosphate phosphatase mutant in production of D-tagatose, and belongs to the technical field of enzyme engineering. The tagatose-6-phosphate phosphatase mutant disclosed by the invention is obtained by mutating wild type tagatose-6-phosphate phosphatase of which the amino acid sequence is shown as SEQ ID NO.5, and the mutation site is selected from at least one of the 45th site, the 108th site, the 168th site and the 186th site. After the mutant is coupled with isoamylase, dextran phosphorylase, glucose phosphate mutase, glucose-6-phosphate isomerase, 4-alpha-glucosyltransferase and tagatose-6-phosphate isomerase, the yield of D-tagatose synthesized by substrate starch is increased, the accumulation amount of by-products glucose and fructose is low, and the yield of D-tagatose synthesized by substrate starch is increased. Therefore, the production efficiency is greatly improved, and the separation and purification cost is reduced. Therefore, the mutant disclosed by the invention is suitable for industrial production and has a good application prospect.

Description

Technical Field

[0001] This invention relates to the field of enzyme engineering technology, and more specifically, to a tagatose-6-phosphate phosphatase mutant and its application in the production of D-tagatose. Background Technology

[0002] D-tagatose is a rare hexanone sugar found in nature. It is an isomer of D-galactose and also an epimer of fructose. Its sweetness is similar to that of sucrose, but it provides only 30% of the calories of sucrose. It can be used as a low-calorie sweetener, and will not cause a rapid rise in blood sugar after consumption. It can be used as a special sweetener for diabetics and is widely used in low-sugar health foods and various weight-loss foods.

[0003] D-tagatose is extremely rare in nature, making its extraction via natural methods difficult, resulting in very low yields that cannot meet market demand, and the extraction costs are high. Chemical synthesis methods require harsh reaction conditions and generate numerous byproducts, increasing the difficulty of subsequent separation and purification processes, leading to low D-tagatose yields. Biotransformation methods, with their advantages of high specificity, environmental friendliness, mild reaction conditions, and elimination of the need for multi-step separation and purification, have attracted widespread attention.

[0004] Currently, scholars both domestically and internationally have reported various biological preparation routes for D-tagatose. Zheng Xu proposed a method derived from bacteria... Lactobacillus fermentum L-arabinose isomerase of CGMCC2921 and derived from Thermus thermophilus β-D-galactosidase of HB27 was co-expressed in Escherichia coli. The expression levels of the two enzymes were balanced by optimizing the SD sequence and its distance from the start codon. After 16 hours of reaction, the recombinant bacteria were able to convert lactose to obtain 101 g / L D-tagatose, with a yield and empty product rate of 20.2% and 6.3 g / L / h, respectively. Chinese patent CN103045575B constructed a recombinant L-arabinose isomerase Bacillus subtilis and investigated the expression of L-arabinose isomerase and the conditions for converting galactose to prepare D-tagatose. Using 100 g / L galactose as a substrate, the conversion rate reached about 40% after 10 hours of conversion.

[0005] Existing methods for preparing D-tagatose suffer from drawbacks such as high substrate costs, low conversion rates, and difficulties in separation and purification, which significantly increase the cost of D-tagatose preparation. Therefore, providing a low-cost, high-conversion-efficiency method for preparing D-tagatose is of great significance.

[0006] In view of this, the present invention is proposed. Summary of the Invention

[0007] The purpose of this invention is to provide a tagatose-6-phosphate phosphatase mutant and its application in the production of D-tagatose. This mutant can be coupled with isoamylase, dextran phosphorylase, glucose phosphate mutase, glucose-6-phosphate isomerase, 4-α-glucose transferase and tagatose-6-phosphate isomerase to efficiently convert the inexpensive substrate starch into D-tagatose.

[0008] This invention is implemented as follows: In a first aspect, the present invention provides a tagatose-6-phosphate phosphatase mutant obtained by mutating wild-type tagatose-6-phosphate phosphatase (T6PP) with the amino acid sequence shown in SEQ ID NO.5, wherein the mutation site is selected from at least one of the 45th, 108th, 168th and 186th positions.

[0009] To obtain T6PP with higher catalytic efficiency, this invention first screened different sources, among which those derived from... Mesobacterium hydrothermale The T6PP sequence (Genbank accession WP_326297441.1, amino acid sequence as shown in SEQ ID NO. 5, nucleotide sequence as shown in SEQ ID NO. 6) exhibited the highest activity in converting tagatose-6-phosphate to D-tagatose. Therefore, the inventors subsequently optimized this wild-type sequence by performing site-directed mutagenesis on the amino acid sequence shown in SEQ ID NO. 5. Specifically, the mutation sites for T6PP were selected from at least one of positions 45, 108, 168, and 186.

[0010] The mutation at position 45 is as follows: glycine is mutated to any one of serine, asparagine, or valine, namely G45S, G45N, and G45V.

[0011] The mutation at position 108 is as follows: threonine is mutated to any one of tyrosine, histidine, glutamine, or methionine, namely T108Y, T108H, T108Q, and T108M.

[0012] The mutation at position 168 is aspartic acid mutated to any one of glutamic acid, leucine, or isoleucine, namely D168E, D168L, and D168I.

[0013] The mutation at position 186 is as follows: tyrosine is mutated to any one of phenylalanine, lysine, or arginine, namely Y186F, Y186K, and Y186R.

[0014] The mutations at positions 45, 108, 168, and 186 can enhance the catalytic activity of T6PP. Coupling T6PP with isoamylase, glucan phosphorylase, glucose phosphate mutase, glucose-6-phosphate isomerase, 4-α-glucose transferase, and tagatose-6-phosphate isomerase can synthesize D-tagatose.

[0015] Furthermore, the mutation modes of the T6PP mutant include, but are not limited to, G45S, G45N, G45V, G45N / T108Y, G45N / T108H, G45N / T108Q, G45N / T108M, G45N / T108H / D168E, G45N / T108H / D168L, G45N / T108H / D168I, G45N / T108H / D168L / Y186F, G45N / T108H / D168L / Y186K, and G45N / T108H / D168L / Y186R.

[0016] Furthermore, among the many mutation modes, the mutant corresponding to G45N / T108H / D168L / Y186K has higher enzyme activity and higher efficiency in producing D-tagatose. Its amino acid sequence is shown in SEQ ID NO.7, and its nucleotide sequence is shown in SEQ ID NO.8.

[0017] In a second aspect, the present invention provides biological materials related to the above-mentioned tagatose-6-phosphate phosphatase mutant, which are any one of the following (1)-(4): (1) The nucleic acid molecule encoding the above tagatose-6-phosphate phosphatase mutant; (2) An expression cassette containing the nucleic acid molecule described in (1); (3) A recombinant vector containing the nucleic acid molecule described in (1) or the expression cassette described in (2); (4) Recombinant bacteria containing the nucleic acid molecule described in (1), the expression cassette described in (2), or the recombinant vector described in (3).

[0018] In some embodiments, the method for constructing recombinant bacteria expressing the T6PP mutant includes: ligating the nucleotide sequence of the T6PP mutant to an expression vector, transforming the obtained recombinant vector into host cells, culturing the host cells, and inducing the expression of the T6PP mutant.

[0019] The expression vector and host cell can be conventional choices in the art; in some embodiments, the expression vector is pET28a(+) and the host cell is Escherichia coli. E.coli BL21(DE3). By using the above method to induce protein expression and obtain crude enzyme solution through cell disruption, the catalytic activity of the obtained T6PP mutant was superior to that of the wild-type enzyme.

[0020] Thirdly, the present invention provides an enzyme preparation comprising the above-mentioned tagatose-6-phosphate phosphatase mutant or biological material.

[0021] Fourthly, the present invention provides the application of the above-mentioned tagatose-6-phosphate phosphatase mutant or biological material in the synthesis of D-tagatose.

[0022] Fifthly, the present invention provides a method for synthesizing D-tagatose, the synthetic route of which is as follows: Figure 1 As shown, the method includes: lysing recombinant bacteria expressing the above-mentioned tagatose-6-phosphate phosphatase mutant, and recombinant bacteria expressing isoamylase, glucan phosphorylase, glucose phosphate mutase, glucose-6-phosphate isomerase, 4-α-glucose transferase and tagatose-6-phosphate isomerase, and then adding their supernatant to the transformation system to react and obtain D-tagatose.

[0023] In some embodiments, the above-mentioned isoamylase, glucan phosphorylase, glucose phosphate mutase, glucose-6-phosphate isomerase, 4-α-glucose transferase and tagatose-6-phosphate isomerase are obtained by constructing recombinant bacteria for expression, and the construction method is the same as that of recombinant bacteria with T6PP mutant.

[0024] In some embodiments, the amino acid sequence of the isoamylase is shown in SEQ ID No. 9, and its nucleotide sequence is shown in SEQ ID No. 10.

[0025] In some embodiments, the amino acid sequence of the dextran phosphorylase is shown in SEQ ID No. 11, and its nucleotide sequence is shown in SEQ ID No. 12.

[0026] In some embodiments, the amino acid sequence of glucose phosphate mutase is shown in SEQ ID No. 13, and its nucleotide sequence is shown in SEQ ID No. 14.

[0027] In some embodiments, the amino acid sequence of glucose-6-phosphate isomerase is shown in SEQ ID No. 15, and its nucleotide sequence is shown in SEQ ID No. 16.

[0028] In some embodiments, the amino acid sequence of 4-α-glucose transferase is shown in SEQ ID No. 17, and its nucleotide sequence is shown in SEQ ID No. 18.

[0029] In some embodiments, the amino acid sequence of tagatose-6-phosphate isomerase is shown in SEQ ID No. 19, and its nucleotide sequence is shown in SEQ ID No. 20.

[0030] In some embodiments, the conversion system includes: starch, phosphate buffer, and supernatant of tagatose-6-phosphate phosphatase mutant, isoamylase, dextran phosphorylase, glucose phosphate mutase, glucose-6-phosphate isomerase, 4-α-glucose transferase, and tagatose-6-phosphate isomerase.

[0031] In some embodiments, the phosphate buffer is a sodium phosphate buffer or a potassium phosphate buffer, or a combination of sodium phosphate buffer and potassium phosphate buffer.

[0032] In some embodiments, the phosphate buffer is selected from at least one of K2HPO4-KH2PO4 buffer and Na2HPO4-NaH2PO4 buffer.

[0033] In some embodiments, the conversion system includes: 10-250 g / L starch, 20-100 mM phosphate buffer, and 1-10 g / L tagatose-6-phosphate phosphatase mutant, 1-5 g / L isoamylase, 1-5 g / L glucan phosphorylase, 1-5 g / L glucose phosphate mutase, 1-5 g / L glucose-6-phosphate isomerase, 1-5 g / L 4-α-glucose transferase, and 10-20 g / L tagatose-6-phosphate isomerase, based on cell weight.

[0034] In some embodiments, the reaction conditions are: pH 7.5-8.5, temperature 60-75°C, shaking speed 150-250 rpm, and reaction time 10-20 h.

[0035] The present invention has the following beneficial effects: This invention, through the source Mesobacterium hydrothermale Site-directed mutagenesis of four amino acids in the amino acid sequence of tagatose-6-phosphate phosphatase yielded a series of tagatose-6-phosphate phosphatase mutants with high substrate specificity. Compared with wild-type tagatose-6-phosphate phosphatase, these mutants, upon coupling with isoamylase, dextran phosphorylase, glucose phosphate mutase, glucose-6-phosphate isomerase, 4-α-glucose transferase, and tagatose-6-phosphate isomerase, showed increased yield of D-tagatose from starch substrate and lower accumulation of byproducts glucose and fructose, thus significantly improving production efficiency and reducing separation and purification costs. Therefore, the tagatose-6-phosphate phosphatase mutants of this invention are suitable for industrial production and have promising application prospects. Attached Figure Description

[0036] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0037] Figure 1 This is the synthetic route for D-tagatose in this invention. Detailed Implementation

[0038] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0039] This invention uses high-performance liquid chromatography (HPLC) to analyze product concentration. The specific analytical method is as follows: The conversion solution was analyzed using a Shimadzu LC-20AT high-performance liquid chromatograph (HPLC) with a RID-20A differential detector. The chromatographic conditions were as follows: mobile phase was 5 mM sulfuric acid aqueous solution, BioRad column (Aminex HPX-87H 7.8×300 mm, 5 μm), flow rate was 0.6 mL / min, column temperature was 60℃, and injection volume was 20 μL.

[0040] Example 1 This example demonstrates enzyme screening. 1. Enzyme source and construction of recombinant bacteria Tapatose-6-phosphate phosphatase was obtained from the NCBI database, derived from... Thermococcus indicus (Genbank code QDA30996.1) Methanococcoides orientis (Genbank number WP_233084546.1) and Mesobacterium hydrothermale (Genbank accession number WP_326297441.1), and named TiT6PP, MoT6PP, and MhT6PP respectively, three nucleotide sequences were synthesized using conventional genetic engineering methods based on the amino acid sequence and codon optimization according to the codon preference of E. coli, as shown in SEQ ID NO.2, SEQ ID NO.4, and SEQ ID NO.6 respectively; the amino acid sequences encoding the enzymes are shown in SEQ ID NO.1, SEQ ID NO.3, and SEQ ID NO.5 respectively.

[0041] A 6×His-tag was added to the end of the nucleotide sequence, and NdeI and XhoI restriction enzyme sites were added to both ends. The gene was cloned into the NdeI and XhoI sites corresponding to pET28a(+) to obtain recombinant expression plasmids pET28a-TiT6PP, pET28a-MoT6PP, and pET28a-MhT6PP. These three plasmids were then transformed into E. coli. Escherichia coli Recombinant bacteria were obtained from BL21(DE3) competent cells. Escherichia coli BL21(DE3) / pET28a-TiT6PP, Escherichia coli BL21(DE3) / pET28a-MoT6PP and Escherichia coli BL21(DE3) / pET28a-MhT6PP.

[0042] 2. Inducible expression of tagatose-6-phosphate phosphatase, isoamylase, glucan phosphorylase, glucose phosphate mutase, glucose-6-phosphate isomerase, 4-α-glucose transferase, and tagatose-6-phosphate isomerase from different sources. The isoamylase gene IA (nucleotide sequence shown in SEQ ID No. 10, amino acid sequence shown in SEQ ID No. 9) is synthesized from the whole gene and obtained from [source missing]. Thermosipho sp Recombinant isoamylase strain E.coli BL21(DE3) / pET28a-IA.

[0043] The dextran phosphorylase gene αGP (nucleotide sequence shown in SEQ ID No. 12, amino acid sequence shown in SEQ ID No. 11) was synthesized from a whole gene source. Thermus scotoductus Recombinant dextran phosphorylase strain E.coli BL21(DE3) / pET28a-αGP.

[0044] The glucose-phosphoryl mutase gene PGM (nucleotide sequence shown in SEQ ID No. 14, amino acid sequence shown in SEQ ID No. 13) is synthesized from the whole gene and obtained from [source missing]. Geobacillus zalihae Recombinant glucose phosphate mutase strain E.col i BL21(DE3) / pET28a-PGM.

[0045] The glucose-6-phosphate isomerase gene PGI (nucleotide sequence shown in SEQ ID No. 16, amino acid sequence shown in SEQ ID No. 15) is synthesized from the whole gene and obtained from [source missing]. Thermus altitudinis Recombinant glucose-6-phosphate isomerase strain E.coli BL21(DE3) / pET28a-PGI.

[0046] The 4-α-glucose transferase gene 4GT (nucleotide sequence shown in SEQ ID No. 18, amino acid sequence shown in SEQ ID No. 17) is synthesized from the whole gene and obtained from [source missing]. Pyrococcus kukulkanii Recombinant 4-α-glucose transferase strain E.coli BL21(DE3) / pET28a-4GT.

[0047] The tagatose-6-phosphate isomerase gene T6PE (nucleotide sequence shown in SEQ ID No. 20, amino acid sequence shown in SEQ ID No. 19) is synthesized from the whole gene and obtained from [source missing]. Caldilinea sp Recombinant tagatose-6-phosphate isomerase strain E.coli BL21(DE3) / pET28a-T6PE.

[0048] Recombinant *E. coli* was inoculated into LB medium containing 50 mg / L kanamycin and cultured at 37°C and 200 rpm for 12 h to obtain a seed culture. The seed culture was then inoculated into fresh LB medium at a rate of 2% and cultured at 37°C and 200 rpm until the bacterial concentration reached OD500. 600nm When the concentration reaches 0.7, add 0.5 mM IPTG, induce at 28℃ for 15 h, centrifuge at 8000 rpm for 10 min, discard the supernatant, wash the wet bacterial cells twice with 0.9% physiological saline, centrifuge, and set aside.

[0049] 3. Comparison of the activities of recombinant tagatose-6-phosphate phosphatase strains against glucose-6-phosphate, fructose-6-phosphate, and tagatose-6-phosphate. Enzyme activity assay method: Wet bacterial cells were ultrasonically disrupted. 1 g of the prepared wet bacterial cells were resuspended in 50 mL of 50 mM K₂HPO₄-KH₂PO₄ buffer (pH 8.0) and ultrasonically disrupted for 15 min at 35 W to obtain a sonicated suspension. The suspension was centrifuged, and the supernatant was collected. 1 mL of the supernatant was used for the reaction. Reaction system: 50 mM K₂HPO₄-KH₂PO₄ buffer (pH 8.0), 5 g / L glucose-6-phosphate, fructose-6-phosphate, or tagatose-6-phosphate, 0.5 g / L magnesium chloride, and 100 μL of T₆PP disruption supernatant, totaling 1 mL. Reaction conditions: The reaction was carried out at 65℃ for 10 min. Samples were taken, and the yield of glucose, fructose, or tagatose was determined by HPLC.

[0050] Enzyme activity is defined as the amount of enzyme required to produce 1 μmol of glucose, fructose, or tagatose per minute at 65°C and pH 8.0.

[0051] Table 1. Comparison of the activities of various recombinases for different substrates

[0052] As can be seen from Table 1, MhT6PP has the highest enzyme activity in the tagatose-6-phosphate reaction system, and relatively low activity for glucose-6-phosphate and fructose-6-phosphate. Therefore, T6PP from this source is more suitable for the production of D-tagatose.

[0053] Example 2 This example demonstrates the construction and screening of the MhT6PP unit point mutant. 1. Construction of mutants Site-directed mutagenesis primers were designed based on the parental sequence of MhT6PP (amino acid sequence SEQ ID NO. 5, nucleotide sequence SEQ ID NO. 6). Using rapid PCR technology, a single mutation was introduced at position 45 using recombinant pET28a-MhT6PP as a template. The primers are as follows: Forward primer 45G: TTATC NNN AACGGCGCCGCTGTGTTCGTCAAA- SEQ ID NO. 21; Reverse primer 45G: GGCGCCGTT NNN GATAAAGTCACGCGCCTGGG-SEQ ID NO. 22.

[0054] PCR reaction system: 25 μL of 2×FastPfu Fly Reaction Mix, 2 μL of 45G (10μM) forward primer, 2 μL of 45G (10μM) reverse primer, 1 μL of template DNA, 1 μL of FastPfu Fly DNA Polymerase, and ddH2O added to a final volume of 50 μL.

[0055] PCR amplification conditions: 95℃ for 5 min; (95℃ for 20 s, 60℃ for 15 s, 72℃ for 1.5 min) 30 cycles; 72℃ for 10 min.

[0056] 2. Mutant Transformation Expression The PCR results were verified by agarose gel electrophoresis. The PCR product was then digested with DpnI enzyme at 37°C for 1 h and inactivated at 65°C for 1 min. 10 μL of the PCR product was added to... E.coli In BL21(DE3) competent cells, heat shock transformation was performed, followed by incubation at 37℃ and 200 rpm for 1 h. The bacterial culture was then plated and incubated at 37℃ for 12 h.

[0057] 3. High-throughput screening of positive transformants Reaction mixture: 50 mM K₂HPO₄-KH₂PO₄ buffer (pH 8.0), 0.5 g / L magnesium chloride, 5 g / L glucose-6-phosphate or fructose-6-phosphate or tagatose-6-phosphate; Add 200 μL of LB medium containing 50 mg / L kanamycin to each well of a 96-well plate. Pick different single colonies and incubate at 37℃ and 200 rpm until the OD600 reaches 0.5-0.6. Add 0.5 mM IPTG to the culture medium and induce expression at 28℃ for 12 h. Centrifuge at 20℃ and 4200 rpm for 10 min and discard the supernatant. Add 100 μL of the reaction mixture to a 96-well plate containing bacterial cells, mix well, and react at 65℃ for 10 min. Terminate the reaction with dilute hydrochloric acid, take a sample, and determine the yield of the product by HPLC. (Using recombinant bacteria...) E.coli The reaction of BL21(DE3) / pET28a-MhT6PP was used as a control, and the tagatose yield ratio was taken. E.coli The BL21(DE3) / pET28a-MhT6PP reaction was high, and the glucose and fructose yields were higher than those of BL21(DE3) / pET28a-MhT6PP. E.coli The enzyme activity of mutant strains with low BL21(DE3) / pET28a-MhT6PP response was accurately measured.

[0058] 4. Precise determination of enzyme activity in positive transformants The procedure was the same as in Example 1, “Comparison of the activities of recombinant tagatose-6-phosphate phosphatase on glucose-6-phosphate, fructose-6-phosphate and tagatose-6-phosphate”.

[0059] The results of this embodiment are as follows: Of the 178 recombinant transformant strains initially screened, 3 mutant strains with increased enzyme activity were identified. Further precise enzyme activity determination was then performed on these mutants, and the specific results are shown in Table 2. Analysis determined that the reason the specificity of the remaining 175 recombinant strains remained unchanged or decreased was due to a mutation at position 45 (glycine (G) in which an amino acid other than S, N, and V was replaced.

[0060] Table 2 Enzyme activity assay of single-point mutant recombinant bacteria

[0061] The MhT6PP mutant-G45N with the highest enzyme activity was designated as MhT6PP-1, and recombinant bacteria were obtained. E.coli BL21(DE3) / pET28a-MhT6PP-1.

[0062] Example 3 This example demonstrates the construction and screening of MhT6PP dual-site mutants. Site-directed mutagenesis primers were designed based on the single mutant MhT6PP-1 sequence constructed in Example 2. Using rapid PCR technology, a single mutation was introduced at position 108 using recombinant pET28a-MhT6PP-1 as a template. The primers are as follows: Forward primer 108T: CCTGGGTATCTGC NNN AACAAACCTATTCGTCCGACC-SEQ ID NO.23; Reverse primer 108T: T NNN GCAGATACCCAGGATGTGACCTTCAGCT-SEQ ID NO. 24.

[0063] PCR reaction system: 25 μL of 2×FastPfu Fly Reaction Mix, 2 μL of forward primer 108T (10 μM), 2 μL of reverse primer 108T (10 μM), 1 μL of template DNA, 1 μL of FastPfu Fly DNA Polymerase, and ddH2O added to 50 μL.

[0064] PCR amplification conditions: 95℃ for 5 min; (95℃ for 20 s, 60℃ for 15 s, 72℃ for 1.5 min) 30 cycles; 72℃ for 10 min.

[0065] PCR results were verified by agarose gel electrophoresis. The PCR product was digested with DpnI enzyme at 37°C for 1 h and then inactivated at 65°C for 1 min. 10 μL of the PCR product was then added to... E.coli In BL21(DE3) competent cells, heat shock transformation was performed, followed by incubation at 37°C and 200 rpm for 1 h. The bacterial culture was then plated and incubated at 37°C for 12 h. The mutants were then subjected to initial screening (the procedure was the same as "High-throughput screening of positive transformants" in Example 2).

[0066] The wet bacterial cells were ultrasonically disrupted, and the enzyme activity was accurately measured (the procedure was the same as in Example 1, “Comparison of the activities of recombinant tagatose-6-phosphate phosphatase on glucose-6-phosphate, fructose-6-phosphate and tagatose-6-phosphate”).

[0067] The results of this embodiment are as follows: Of the 198 recombinant transformant strains initially screened, 4 mutant strains with increased enzyme activity were identified. Further precise enzyme activity determination was then performed on these mutants, and the specific results are shown in Table 3. Analysis determined that the reason the enzyme activity of the remaining 194 recombinant strains remained unchanged or decreased was due to a mutation at position 108 (threonine (T) in which an amino acid other than Y, H, Q, and M was replaced.

[0068] Table 3 Enzyme activity assay of recombinant bacteria with double-point mutations

[0069] The MhT6PP mutant with the highest increase in enzyme activity, -G45N-T108H, was designated as MhT6PP-2, and recombinant bacteria were obtained. E.coli BL21(DE3) / pET28a-MhT6PP-2.

[0070] Example 4 This embodiment describes the construction and screening of the MhT6PP three-point mutant. Based on the double mutant MhT6PP-2 sequence constructed in Example 3, site-directed mutagenesis primers were designed. Using rapid PCR technology, with recombinant pET28a-MhT6PP-2 as a template, a single mutation was introduced at position 168. The primers are as follows: Forward primer 168D: CTGATGTT NNN GCGGGTACCGCTGTAAACGCC-SEQ ID NO.25; Reverse primer 168D: ACCCGC NNN AACATCAGAGTCGCCCACGTACA-SEQ ID NO. 26.

[0071] PCR reaction system: 2×FastPfu Fly Reaction Mix 25 μL, forward primer 168D (10 μM) 2 μL, reverse primer 168D (10 μM) 2 μL, template DNA 1 μL, FastPfu Fly DNA Polymerase 1 μL, add ddH2O to 50 μL.

[0072] PCR amplification conditions: 95℃ for 5 min; (95℃ for 20 s, 60℃ for 15 s, 72℃ for 1.5 min) 30 cycles; 72℃ for 10 min.

[0073] PCR results were verified by agarose gel electrophoresis. The PCR product was digested with DpnI enzyme at 37°C for 1 h and then inactivated at 65°C for 1 min. 10 μL of the PCR product was then added to... E.coli In BL21(DE3) competent cells, heat shock transformation was performed, followed by incubation at 37°C and 200 rpm for 1 h. The bacterial culture was then plated and incubated at 37°C for 12 h. The mutants were then subjected to initial screening (the procedure was the same as "High-throughput screening of positive transformants" in Example 2).

[0074] The wet bacterial cells were ultrasonically disrupted, and the enzyme activity was accurately measured (the procedure was the same as in Example 1, “Comparison of the activities of recombinant tagatose-6-phosphate phosphatase on glucose-6-phosphate, fructose-6-phosphate and tagatose-6-phosphate”).

[0075] The results of this embodiment are as follows: Initial screening of 213 recombinant transformants revealed 3 mutant strains with increased enzyme activity. Further precise enzyme activity determination was performed on these strains, and the specific results are shown in Table 4. Analysis determined that the reason the enzyme activity of the remaining 210 recombinant strains remained unchanged or decreased was due to a mutation at position 168 (aspartic acid (D) in which an amino acid other than E, L, and I was replaced.

[0076] Table 4 Enzyme activity assay of the three-point mutant recombinant bacteria

[0077] The MhT6PP mutant with the highest increased enzyme activity, -G45N-T108H-D168L, was designated MhT6PP-3, and recombinant bacteria were obtained. E.coli BL21(DE3) / pET28a-MhT6PP-3.

[0078] Example 5 This example demonstrates the construction and screening of the MhT6PP four-site mutant. Based on the triple mutant MhT6PP-3 sequence constructed in Example 4, site-directed mutagenesis primers were designed. Using rapid PCR technology, with recombinant pET28a-MhT6PP-3 as a template, a single mutation was introduced at position 186. The primers are as follows: Forward primer 186Y: TGAGGGC NNN CGTAAACAACCGGTGGCCGAGA- SEQ ID NO.27; Reverse primer 186Y: GTTACG NNN GCCCTCAGTAAACAGCAGAAACG-SEQ ID NO. 28.

[0079] PCR reaction system: 2×FastPfu Fly Reaction Mix 25 μL, forward primer 186Y (10 μM) 2 μL, reverse primer 186Y (10 μM) 2 μL, template DNA 1 μL, FastPfu Fly DNA Polymerase 1 μL, add ddH2O to 50 μL.

[0080] PCR amplification conditions: 95℃ for 5 min; (95℃ for 20 s, 60℃ for 15 s, 72℃ for 1.5 min) 30 cycles; 72℃ for 10 min.

[0081] PCR results were verified by agarose gel electrophoresis. The PCR product was digested with DpnI enzyme at 37°C for 1 h and then inactivated at 65°C for 1 min. 10 μL of the PCR product was then added to... E.coli In BL21(DE3) competent cells, heat shock transformation was performed, followed by incubation at 37°C and 200 rpm for 1 h. The bacterial culture was then plated and incubated at 37°C for 12 h. The mutants were then subjected to initial screening (the procedure was the same as "High-throughput screening of positive transformants" in Example 2).

[0082] The wet bacterial cells were ultrasonically disrupted, and the enzyme activity was accurately measured (the procedure was the same as in Example 1, “Comparison of the activities of recombinant tagatose-6-phosphate phosphatase on glucose-6-phosphate, fructose-6-phosphate and tagatose-6-phosphate”).

[0083] The results of this embodiment are as follows: Of the 185 recombinant transformant strains initially screened, 3 mutant strains with increased enzyme activity were identified. Further precise enzyme activity determination was then performed on these mutants, and the specific results are shown in Table 5. Analysis determined that the reason the enzyme activity of the remaining 182 recombinant strains remained unchanged or decreased was due to a mutation at position 186 (tyrosine Y) into an amino acid other than F, K, and R.

[0084] Table 5 Enzyme activity assay of the four-point mutant recombinant bacteria

[0085] The MhT6PP mutant with the highest increased enzyme activity, -G45N-T108H-D168L-Y186K, was designated MhT6PP-4, and recombinant bacteria were obtained. E.coli BL21(DE3) / pET28a-MhT6PP-4.

[0086] Example 6 In this embodiment, D-tagatose is prepared by coupling tagatose-6-phosphate phosphatase MhT6PP, isoamylase IA, dextran phosphorylase αGP, glucose phosphate mutase PGM, glucose-6-phosphate isomerase PGI, 4-α-glucose transferase 4GT, and tagatose-6-phosphate isomerase T6PE with starch.

[0087] According to the induction expression method described in Example 1, E.coli BL21(DE3) / pET28a-MhT6PP, E.coli BL21(DE3) / pET28a-IA, E.coli BL21(DE3) / pET28a-αGP, E.coli BL21(DE3) / pET28a-PGM, E.coli BL21(DE3) / pET28a-PGI E.coliBL21(DE3) / pET28a-4GT and E.coli After BL21(DE3) / pET28a-T6PE induction expression was completed, the bacterial cells were collected. The two bacterial cells were ultrasonically disrupted according to the ultrasonic disruption method described in Example 1, and centrifuged to obtain the supernatant of each enzyme.

[0088] In a 50 mL system, the following were added: 5 g / L wet weight of MhT6PP (added as supernatant), 2 g / L wet weight of IA (added as supernatant), 2 g / L wet weight of αGP (added as supernatant), 2 g / L wet weight of PGM (added as supernatant), 2 g / L wet weight of PGI (added as supernatant), 2 g / L wet weight of 4GT (added as supernatant), 15 g / L wet weight of T6PE (added as supernatant), 200 g / L starch, 0.5 g / L magnesium chloride, 50 mL M K2HPO4-KH2PO4 buffer (pH 8.0), at 65℃, with a shaking speed of 200 rpm, for a conversion time of 15 h.

[0089] HPLC analysis showed that the yield of D-tagatose was 24.3 g / L, the glucose accumulation was 98.2 g / L, the fructose accumulation was 66.5 g / L, and the yield of D-tagatose was 12.2%.

[0090] Example 7 This embodiment describes the preparation of D-tagatose by coupling tagatose-6-phosphate phosphatase MhT6PP-1, isoamylase IA, dextran phosphorylase αGP, glucose phosphate mutase PGM, glucose-6-phosphate isomerase PGI, 4-α-glucose transferase 4GT, and tagatose-6-phosphate isomerase T6PE with starch.

[0091] According to the induction expression method described in Example 1, E.coli BL21(DE3) / pET28a-MhT6PP-1, E.coli BL21(DE3) / pET28a-IA, E.coli BL21(DE3) / pET28a-αGP, E.coli BL21(DE3) / pET28a-PGM, E.coli BL21(DE3) / pET28a-PGI E.coli BL21(DE3) / pET28a-4GT and E.coli After BL21(DE3) / pET28a-T6PE induction expression was completed, the bacterial cells were collected. The two bacterial cells were ultrasonically disrupted according to the ultrasonic disruption method described in Example 1, and centrifuged to obtain the supernatant of each enzyme.

[0092] In a 50 mL system, the following were added: 5 g / L wet weight of MhT6PP-1 (added as supernatant), 2 g / L wet weight of IA (added as supernatant), 2 g / L wet weight of αGP (added as supernatant), 2 g / L wet weight of PGM (added as supernatant), 2 g / L wet weight of PGI (added as supernatant), 2 g / L wet weight of 4GT (added as supernatant), 15 g / L wet weight of T6PE (added as supernatant), 200 g / L starch, 0.5 g / L magnesium chloride, 50 mL M K2HPO4-KH2PO4 buffer (pH 8.0), at 65℃, with a shaking speed of 200 rpm, for a conversion time of 15 h.

[0093] HPLC analysis showed that the yield of D-tagatose was 57.9 g / L, the glucose accumulation was 82.3 g / L, the fructose accumulation was 51.7 g / L, and the yield of D-tagatose reached 29%.

[0094] Example 8 This embodiment describes the preparation of D-tagatose by coupling tagatose-6-phosphate phosphatase MhT6PP-2, isoamylase IA, dextran phosphorylase αGP, glucose phosphate mutase PGM, glucose-6-phosphate isomerase PGI, 4-α-glucose transferase 4GT, and tagatose-6-phosphate isomerase T6PE with starch.

[0095] According to the induction expression method described in Example 1, E.coli BL21(DE3) / pET28a-MhT6PP-2, E.coli BL21(DE3) / pET28a-IA, E.coli BL21(DE3) / pET28a-αGP, E.coli BL21(DE3) / pET28a-PGM, E.coli BL21(DE3) / pET28a-PGI E.coli BL21(DE3) / pET28a-4GT and E.coli After BL21(DE3) / pET28a-T6PE induction expression was completed, the bacterial cells were collected. The two bacterial cells were ultrasonically disrupted according to the ultrasonic disruption method described in Example 1, and centrifuged to obtain the supernatant of each enzyme.

[0096] In a 50 mL system, the following were added: 5 g / L wet weight of MhT6PP-2 (added as supernatant), 2 g / L wet weight of IA (added as supernatant), 2 g / L wet weight of αGP (added as supernatant), 2 g / L wet weight of PGM (added as supernatant), 2 g / L wet weight of PGI (added as supernatant), 2 g / L wet weight of 4GT (added as supernatant), 15 g / L wet weight of T6PE (added as supernatant), 200 g / L starch, 0.5 g / L magnesium chloride, 50 mL M K2HPO4-KH2PO4 buffer (pH 8.0), temperature 65℃, shaker speed 200 rpm, conversion time 15 h. HPLC analysis showed that the yield of D-tagatose was 108.4 g / L, the glucose accumulation was 48.3 g / L, the fructose accumulation was 30.5 g / L, and the yield of D-tagatose was 54.2%.

[0097] Example 9 This embodiment describes the preparation of D-tagatose by coupling tagatose-6-phosphate phosphatase MhT6PP-3, isoamylase IA, dextran phosphorylase αGP, glucose phosphate mutase PGM, glucose-6-phosphate isomerase PGI, 4-α-glucose transferase 4GT, and tagatose-6-phosphate isomerase T6PE with starch.

[0098] According to the induction expression method described in Example 1, E.coli BL21(DE3) / pET28a-MhT6PP-3, E.coli BL21(DE3) / pET28a-IA, E.coli BL21(DE3) / pET28a-αGP, E.coli BL21(DE3) / pET28a-PGM, E.coli BL21(DE3) / pET28a-PGI E.coli BL21(DE3) / pET28a-4GT and E.coli After BL21(DE3) / pET28a-T6PE induction expression was completed, the bacterial cells were collected. The two bacterial cells were ultrasonically disrupted according to the ultrasonic disruption method described in Example 1, and centrifuged to obtain the supernatant of each enzyme.

[0099] In a 50 mL system, the following were added: 5 g / L wet weight of MhT6PP-3 (added as supernatant), 2 g / L wet weight of IA (added as supernatant), 2 g / L wet weight of αGP (added as supernatant), 2 g / L wet weight of PGM (added as supernatant), 2 g / L wet weight of PGI (added as supernatant), 2 g / L wet weight of 4GT (added as supernatant), 15 g / L wet weight of T6PE (added as supernatant), 200 g / L starch, 0.5 g / L magnesium chloride, 50 mL M K2HPO4-KH2PO4 buffer (pH 8.0), at 65 °C, with a shaking speed of 200 rpm, for a conversion time of 15 h. HPLC analysis showed that the yield of D-tagatose was 165.7 g / L, the glucose accumulation was 20.5 g / L, the fructose accumulation was 5.2 g / L, and the yield of D-tagatose reached 82.8%.

[0100] Example 10 This embodiment describes the preparation of D-tagatose by coupling tagatose-6-phosphate phosphatase MhT6PP-4, isoamylase IA, dextran phosphorylase αGP, glucose phosphate mutase PGM, glucose-6-phosphate isomerase PGI, 4-α-glucose transferase 4GT, and tagatose-6-phosphate isomerase T6PE with starch.

[0101] According to the induction expression method described in Example 1, E.coli BL21(DE3) / pET28a-MhT6PP-4, E.coli BL21(DE3) / pET28a-IA, E.coli BL21(DE3) / pET28a-αGP, E.coli BL21(DE3) / pET28a-PGM, E.coli BL21(DE3) / pET28a-PGI E.coli BL21(DE3) / pET28a-4GT and E.coli After BL21(DE3) / pET28a-T6PE induction expression was completed, the bacterial cells were collected. The two bacterial cells were ultrasonically disrupted according to the ultrasonic disruption method described in Example 1, and centrifuged to obtain the supernatant of each enzyme.

[0102] In a 50 mL system, the following were added: 5 g / L wet weight of MhT6PP-4 (added as supernatant), 2 g / L wet weight of IA (added as supernatant), 2 g / L wet weight of αGP (added as supernatant), 2 g / L wet weight of PGM (added as supernatant), 2 g / L wet weight of PGI (added as supernatant), 2 g / L wet weight of 4GT (added as supernatant), 15 g / L wet weight of T6PE (added as supernatant), 200 g / L starch, 0.5 g / L magnesium chloride, 50 mL M K2HPO4-KH2PO4 buffer (pH 8.0), at 65 °C, with a shaking speed of 200 rpm, for a conversion time of 15 h. HPLC analysis showed that the yield of D-tagatose was 186.4 g / L, the glucose accumulation was 4.1 g / L, the fructose accumulation was 0.8 g / L, and the yield of D-tagatose reached 93.2%.

[0103] 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 tagatose-6-phosphate phosphatase mutant, characterized in that, It is obtained by mutating wild-type tagatose-6-phosphate phosphatase with an amino acid sequence as shown in SEQ ID NO.5, with the mutation site selected from at least one of positions 45, 108, 168 and 186.

2. The tagatose-6-phosphate phosphatase mutant according to claim 1, characterized in that, The tagatose-6-phosphate phosphatase mutant is any one or a combination of the following (1)-(4): (1) Mutate the glycine at position 45 of the amino acid sequence shown in SEQ ID NO.5 to any one of serine, asparagine or valine; (2) Mutate the threonine at position 108 in the amino acid sequence shown in SEQ ID NO.5 to any one of tyrosine, histidine, glutamine or methionine; (3) Mutate the aspartic acid at position 168 in the amino acid sequence shown in SEQ ID NO.5 to any one of glutamic acid, leucine or isoleucine; (4) Mutate the tyrosine at position 186 in the amino acid sequence shown in SEQ ID NO.5 to any one of phenylalanine, lysine or arginine.

3. The tagatose-6-phosphate phosphatase mutant according to claim 2, characterized in that, The mutation mode of the tagatose-6-phosphate phosphatase mutant is any one or a combination of the following (1)-(4): (1) Mutate the glycine at position 45 of the amino acid sequence shown in SEQ ID NO.5 to asparagine; (2) Mutate the threonine at position 108 in the amino acid sequence shown in SEQ ID NO.5 to histidine; (3) Mutate the aspartic acid at position 168 of the amino acid sequence shown in SEQ ID NO.5 to leucine; (4) Mutate the tyrosine at position 186 in the amino acid sequence shown in SEQ ID NO.5 to lysine.

4. The tagatose-6-phosphate phosphatase mutant according to claim 3, characterized in that, The amino acid sequence of the tagatose-6-phosphate phosphatase mutant is shown in SEQ ID NO.

7.

5. A biomaterial relating to the tagatose-6-phosphate phosphatase mutant according to any one of claims 1-4, characterized in that, It can be any one of the following (1)-(4): (1) A nucleic acid molecule encoding the tagatose-6-phosphate phosphatase mutant according to any one of claims 1-4; (2) An expression cassette containing the nucleic acid molecule described in (1); (3) A recombinant vector containing the nucleic acid molecule described in (1) or the expression cassette described in (2); (4) Recombinant bacteria containing the nucleic acid molecule described in (1), the expression cassette described in (2), or the recombinant vector described in (3).

6. An enzyme preparation, characterized in that, The enzyme preparation comprises the tagatose-6-phosphate phosphatase mutant according to any one of claims 1-4 or / and the biological material according to claim 5.

7. The use of the tagatose-6-phosphate phosphatase mutant as described in any one of claims 1-4 or the biomaterial as described in claim 5 in the synthesis of D-tagatose.

8. A method for synthesizing D-tagatose, characterized in that, include: The recombinant bacteria expressing the tagatose-6-phosphate phosphatase mutant according to any one of claims 1-4, and the recombinant bacteria expressing isoamylase, glucan phosphorylase, glucose phosphate mutase, glucose-6-phosphate isomerase, 4-α-glucose transferase and tagatose-6-phosphate isomerase were lysed, and their supernatant was added to the transformation system to react and obtain D-tagatose. Preferably, the amino acid sequence of the isoamylase is shown in SEQ ID No. 9; Preferably, the amino acid sequence of the dextran phosphorylase is shown in SEQ ID No. 11; Preferably, the amino acid sequence of the glucose phosphate mutase is shown in SEQ ID No. 13; Preferably, the amino acid sequence of the glucose-6-phosphate isomerase is shown in SEQ ID No. 15; Preferably, the amino acid sequence of the 4-α-glucose transferase is shown in SEQ ID No. 17; Preferably, the amino acid sequence of the tagatose-6-phosphate isomerase is shown in SEQ ID No.

19.

9. The method according to claim 8, characterized in that, The host of the recombinant bacteria includes Escherichia coli.

10. The method according to claim 8, characterized in that, The conversion system includes: starch, phosphate buffer, and supernatant of tagatose-6-phosphate phosphatase mutant, isoamylase, dextran phosphorylase, glucose phosphate mutase, glucose-6-phosphate isomerase, 4-α-glucose transferase and tagatose-6-phosphate isomerase. Preferably, the transformation system comprises: 10-250 g / L starch, 20-100 mM phosphate buffer, and 1-10 g / L tagatose-6-phosphate phosphatase mutant, 1-5 g / L isoamylase, 1-5 g / L glucan phosphorylase, 1-5 g / L glucose phosphate mutase, 1-5 g / L glucose-6-phosphate isomerase, 1-5 g / L 4-α-glucose transferase, and 10-20 g / L tagatose-6-phosphate isomerase, based on cell weight. Preferably, the reaction conditions are: pH 7.5-8.5, temperature 60-75℃, shaking speed 150-250 rpm, and reaction time 10-20 h.

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