Phosphotransferase mutants and uses thereof

Abstract

The application discloses a phosphotransferase mutant and application thereof. The amino acid sequence of the phosphotransferase mutant is shown in SEQ ID No. 2, SEQ ID No. 4 or SEQ ID No. 6, and the nucleotide sequence for coding the phosphotransferase mutant is shown in SEQ ID No. 1, SEQ ID No. 3 or SEQ ID No. 5. The application also provides a method for producing 5'-nucleotide by using recombinant microorganism fermentation and preparing phosphotransferase with high enzyme activity. The phosphotransferase with high enzyme activity prepared by the application can be used for producing high-purity flavor nucleotide disodium product 5'-inosinic acid disodium and 5'-guanylic acid disodium, the purity of which can reach more than 97.5 %, reaching the requirement of food grade, and the yield is also obviously improved, reaching more than 125 %, so that the production cost can be greatly reduced in industrial production.

Description

Technical Field

[0001] This invention relates to the field of bioenzyme catalysis technology, specifically to phosphotransferase mutants and their application in catalyzing the reaction of nucleotides and phosphate donors to produce 5'-nucleotides. Background Technology

[0002] The phoC gene encodes an acid phosphatase, which has phosphotransferase activity and can catalyze the formation of nucleotides from nucleoside substrates. For example, it can catalyze the formation of guanosine or inosine from guanylic acid (GMP) or inosine (IMP). IMP and GMP have many applications in the food and pharmaceutical industries.

[0003] In the food industry, IMP and GMP are mainly used in the production of disodium inosinate (I+G), which, when mixed with monosodium glutamate (MSG), increases umami flavor several times to dozens of times. To achieve the same effect, the dosage is only 2-5% of MSG, significantly reducing costs. In the pharmaceutical field, IMP can be used as a drug to treat leukopenia and thrombocytopenia, and it has certain therapeutic effects on various acute and chronic hepatitis, hepatogenic heart disease, and ophthalmic diseases such as optic neuritis.

[0004] Currently, there are four main methods for preparing 5'-nucleotides: chemical synthesis, fermentation, enzymatic hydrolysis, and biocatalysis. Chemical synthesis primarily uses nucleosides as raw materials and involves phosphorylation. However, because appropriate protecting groups are required to protect the 2' and 3' hydroxyl groups of the ribose on the nucleoside before phosphorylation, the protection and deprotection steps reduce the overall yield of the reaction.

[0005] Fermentation methods include direct fermentation and fermentation-conversion methods. Direct fermentation typically uses naturally occurring biotin-containing substances such as corn steep liquor as a culture medium, utilizing Bacillus subtilis and ammonia-producing breviculobacillus to directly produce IMP and GMP. The process is relatively simple, but its yield is low, making industrialization difficult. The fermentation-conversion method utilizes inosine and guanosine-producing bacteria to first produce inosine or guanosine, which is then phosphorylated to IMP and GMP via chemical phosphorylation. This method offers high yield, short cycle time, and easily controllable fermentation conditions. It provides inexpensive nucleoside raw materials for phosphorylation, and the phosphorylation product is singular, with nucleoside conversion rates generally exceeding 98%, significantly reducing production costs.

[0006] Enzymatic hydrolysis methods include RNA hydrolysis and bacterial autolysis. RNA hydrolysis involves first extracting RNA from bacterial cells, preparing 5'-phosphodiesterase, and then performing RNA hydrolysis followed by the separation and purification of mononucleotides. However, while this method yields four nucleotides at once, and although the enzyme reaction yield is high, obtaining four high-purity products during subsequent extraction is difficult, leading to a long production cycle, cumbersome extraction process, and low product purity. Autolysis utilizes the abundant 5'-phosphodiesterase within bacterial cells to specifically act on their own ribonucleic acid, degrading it to generate 5'-mononucleotides, which are then exuded from the cell. The key to autolysis lies in determining the autolysis conditions; firstly, bacterial cells with enzyme activity must be selected.

[0007] Biocatalytic method: Inosine or guanosine reacts with a phosphate donor to synthesize 5'-inosinate disodium (IMP) and 5'-guanylate disodium (GMP) under the action of phosphorylase. The biocatalytic method has advantages such as high specificity, wide availability and low cost of phosphate donors, simple and easy-to-operate extraction process, and environmentally friendly raw materials. However, existing biocatalytic methods for preparing 5'-inosinate disodium (IMP) and 5'-guanylate disodium (GMP) suffer from low conversion efficiency. The activity of phosphotransferase is crucial to the technology, directly determining the conversion efficiency, as well as the subsequent extraction purity and yield. Summary of the Invention

[0008] The purpose of this invention is to provide a phosphotransferase mutant that can be used to produce 5'-nucleotides. The technical solution provided by this invention is as follows.

[0009] In a first aspect, the present invention provides a phosphotransferase mutant, the amino acid sequence of which is shown in SEQ ID No. 2, SEQ ID No. 4 or SEQ ID No. 6.

[0010] This invention uses the primers shown in SEQ ID No. 9-10 and the nucleotide sequence shown in SEQ ID No. 7 as a template for error-prone PCR amplification. The fragments obtained after PCR amplification are assembled using a linearized vector and transformed into the BL21 strain. Mutant strains are obtained through resistance and PCR verification screening. The mutant strains are then induced to express enzymes, and strains with higher enzyme activity are selected.

[0011] The synonymous mutations occurring in the amino acid sequence of the mutant described in this invention are all replaced with codons preferred by microorganisms, which greatly enhances the phosphotransferase activity of the enzyme.

[0012] Secondly, the present invention provides a DNA molecule encoding the above-mentioned phosphotransferase mutant. Specifically, the nucleotide sequence of the DNA molecule is shown in SEQ ID No. 1, SEQ ID No. 3, or SEQ ID No. 5.

[0013] Thirdly, the present invention seeks protection for biological materials containing the aforementioned DNA molecules, wherein the biological material is an expression cassette, a vector, or a host cell.

[0014] Based on the understanding of those skilled in the art, the present invention also claims protection for the above-described phosphotransferase mutant, the above-described DNA molecule, or the above-described biological material in any of the following applications:

[0015] (1) Preparation of highly active phosphotransferase;

[0016] (2) Increase 5'-nucleotide production;

[0017] (3) Improve the purity of disodium 5'-guanylate or disodium 5'-inosinate.

[0018] Fourthly, the present invention provides a recombinant microorganism that expresses the above-mentioned phosphotransferase mutant or whose genome contains the above-mentioned DNA molecule.

[0019] The recombinant microorganisms provided in this invention are Enterobacter aerogenes or Escherichia coli.

[0020] This invention also claims protection for the use of the above-mentioned recombinant microorganisms in increasing the yield of phosphotransferase or 5'-nucleotides, producing high-activity phosphotransferases, or producing high-purity 5'-nucleotides.

[0021] Fifthly, the present invention provides a method for preparing phosphotransferase, wherein the above-mentioned recombinant microorganism is activated and then inoculated into a fermentation medium. During the fermentation process, the pH is adjusted to 6.8-7.2, the dissolved oxygen to 30-60%, the initial fermentation temperature is 37°C, the OD reaches 20-25 and then the temperature is lowered to 28°C, the glucose concentration is reduced to 1-3 g / L and then sugar is added, the residual sugar is controlled to 0-1 g / L, and the fermentation is stopped when the OD reaches 75-80.

[0022] In the preparation method provided by the present invention, the fermentation culture medium formula is as follows: glucose 15-30 g / L, corn steep liquor 20-40 g / L, (NH4)2SO4 5-10 g / L, K2HPO4 1-5 g / L, MgSO4·7H2O 0.5-5 g / L, biotin 0.0001-0.0005 g / L and thiamine hydrochloride 0.0001-0.001 g / L;

[0023] The added sugar formulation is glucose and α-lactose in a mass ratio of (2-2.5):(1-1.5).

[0024] Specifically, as a specific embodiment of the present invention, a method for preparing a phosphotransferase comprises the following steps:

[0025] The mutant strain expressing the above phosphotransferase was cultured overnight in LB medium shake flasks. The LB medium formula was: yeast extract 5 g / L, peptone 10 g / L, NaCl 10 g / L, pH 7.2-7.4. 10% of the yeast extract was inoculated into the shake flask medium. The shake flask medium formula was: glucose 30 g / L, yeast extract 12 g / L, peptone 18 g / L, NaCl 20 g / L, NaH2PO4·2H2O 8 g / L, MgSO4·7H2O 5 g / L, (NH4)2SO4 8 g / L. The culture was carried out in a reciprocating shaker at 220 r / min and 37 ℃ until the OD reached 8-10. The shake flask seed was then prepared.

[0026] Take the prepared shake flask seeds and inoculate them into a 50L fermenter. The fermenter should contain 20L of liquid. The fermentation medium formula is as follows: glucose 30g / L, corn steep liquor 40g / L, (NH4)2SO4 10g / L, K2HPO4 5g / L, MgSO4·7H2O 5g / L, biotin 0.0005g / L, and thiamine hydrochloride 0.001g / L.

[0027] The fermentation process involves adjusting the pH to 6.8-7.2 by adding ammonia water, and alternately increasing the rotation speed, air volume, and tank pressure to maintain dissolved oxygen at 30-60%. The initial fermentation temperature is 37℃, and when the OD reaches 20-25, the temperature is lowered to 28℃. After the glucose concentration drops to 1-3 g / L, sugar is added. The added sugar formula is: 600 g / L glucose, 300 g / L α-lactose, with residual sugar controlled at 0-1 g / L. Fermentation is stopped when the OD reaches 75-80.

[0028] The fermentation broth was centrifuged at 5000 r / min for 5 minutes to collect the cells. The cells were then resuspended in an equal volume of phosphate buffer (1 g / L dipotassium hydrogen phosphate, 1 g / L potassium dihydrogen phosphate, pH 7.0) to prepare the phosphotransferase enzyme solution.

[0029] The beneficial effects of this invention are at least as follows:

[0030] (1) This invention uses error-prone PCR technology to obtain a new phoC gene mutation. The obtained phosphotransferase mutant is reported for the first time. The activity of the phosphotransferase mutant of this invention is significantly enhanced. Its application in the industrial production of nucleotides has the advantages of high yield, short cycle, easy control of fermentation conditions, and single phosphorylation product, which will greatly reduce production costs.

[0031] (2) This invention also provides a method for producing 5'-nucleotides and preparing phosphotransferases with high enzyme activity using recombinant microbial fermentation. The high-activity phosphotransferases prepared by this invention can be used to produce high-purity disodium 5'-inosinate and disodium 5'-guanylate products with a purity of over 97.5%, meeting food-grade requirements. The yield is also significantly improved, reaching over 125%, which can greatly reduce production costs in industrial production. Attached Figure Description

[0032] Figure 1 This is a graph showing the enzyme activity results of different mutant phosphotransferases in Example 2 of the present invention.

[0033] Figure 2 This is a graph showing the results of fermentation production of 5'-IMP or 5'-GMP by the mutant strain in Example 3 of this invention. Detailed Implementation

[0034] The following examples are for illustrative purposes only and are not intended to limit the scope of the invention. Where specific techniques or conditions are not specified in the examples, they should be performed according to the techniques or conditions described in the literature in this field, or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased from legitimate channels.

[0035] The phosphate donor referred to in this invention refers to a phosphate or phosphorus-containing compound capable of providing a phosphate group, including polyphosphates, phenyl phosphates, or carbamoyl phosphates. Specifically, polyphosphates include pyrophosphates, tripolyphosphates, tetrapolyphosphates, and hexametaphosphates.

[0036] This invention employs liquid chromatography to determine the purity of disodium 5'-guanylate or disodium 5'-inosinate. The specific method is as follows:

[0037] Mobile phase A: 0.02 mol / L potassium dihydrogen phosphate solution (pH = 4.5 ± 0.01). Weigh 2.7218 g of potassium dihydrogen phosphate and place it in a 1000 ml beaker. Add 1000 ml of ultrapure water and stir on a magnetic stirrer until the potassium dihydrogen phosphate is completely dissolved. Then adjust the pH to 4.5 with phosphoric acid, filter, and set aside for use.

[0038] Mobile phase B: Acetonitrile.

[0039] Detection method: Column: XDB-C18 (5μm, 4.6×250mm).

[0040] Mobile phase A: Mobile phase B = 99.5: 0.5 (v / v).

[0041] Column temperature: 40℃.

[0042] Flow rate: 1 ml / min.

[0043] Detector: Ultraviolet detector (λ = 254 nm).

[0044] Injection volume: 2 μL.

[0045] The culture medium involved in this invention has the following formulation:

[0046] LB medium formula (g / L): yeast extract 5g, peptone 10g, NaCl 10g, pH 7.2-7.4; steam sterilize at 121℃ for 20 minutes.

[0047] Shake flask culture medium formula (g / L): glucose 30, yeast extract 12, peptone 18, NaCl 20, NaH2PO4·2H2O 8, MgSO4·7H2O 5, (NH4)2SO4 8; steam sterilize at 121℃ for 20 minutes.

[0048] Fermentation medium formula (g / L): glucose 30, corn steep liquor 40, (NH4)2SO4 10, K2HPO4 5, MgSO4·7H2O 5, biotin 0.0005, thiamine hydrochloride 0.001; steam sterilize at 121℃ for 20 minutes.

[0049] Table 1. Primer sequences involved in this invention.

[0050] phoC-21f cagcaaatgggtcgcggatccATGAAAAAGCGCGTTCTCGC SEQ ID No. 9 phoC-21r ctcgagtgcggccgcaagcttTTACTTCTGCGTTTTGGCGAA SEQ ID No. 10

[0051] Example 1: Obtaining the mutant phoC gene

[0052] Error-prone PCR is currently the simplest and most effective in vitro random mutagenesis technique. PCR itself has a certain mismatch rate, typically 10-1. -6 -10 -5 Using low-fidelity Taq enzyme, Mg was added. 2+ Mn 2+ Methods such as these can be used to increase the mutation rate during mutant PCR amplification.

[0053] This embodiment provides a method for obtaining the mutated phoC gene, and the specific steps are as follows.

[0054] The phoC gene containing *Enterobacter aerogenes* (gene sequence see SEQ ID No. 7, corresponding amino acid sequence see SEQ ID No. 8) was synthesized and constructed into the PUC19 plasmid. Primers phoC-21f / phoC-21r (SEQ ID No. 9 / SEQ ID No. 10) were designed using this plasmid as a template for error-prone PCR amplification.

[0055] The amplification system consisted of: 5 μL of 10× error-prone PCR buffer (100 mmol / L Tris-HCl pH 8.3, 500 mmol / L KCl, 1% Triton, 20 mmol / L MgCl2, 5 μL of 10× dNTP mixture (1 mmol / L dGTP, 1 mmol / L dATP, 5 mmol / L dCTP, 5 mmol / L dTTP) per 50 μL system; 50 pmol each of primers phoC-20f / phoC-20r or phoC-28f / phoC-28r; 10 ng of plasmid DNA template; and 5 μL of 5 mmol / L MnO2. 2+ 2.5 μL of 15 U / μL Taq DNA polymerase, 5 μL of 25 mmol / L Mg 2+ Add sterile ultrapure water to a total volume of 50 μL. The error-prone PCR program is as follows: 95℃ for 5 min; 94℃ for 1 min, 55℃ for 1 min, 72℃ for 1 min, 30 cycles; 72℃ for 10 min. Use the recovered PCR gel product from the first run as a template for the next round of error-prone PCR. Repeat this process for a total of 5 rounds of error-prone PCR to finally obtain the phoC gene containing multiple different mutations.

[0056] Example 2: Construction of phoC mutant library and screening of highly active phosphotransferase mutants

[0057] This embodiment provides a method for screening highly active phosphotransferase mutants, the steps of which are as follows.

[0058] pET21a plasmid was extracted and linearized using BamHI / HindIII restriction endonuclease. The linearized vector and the PCR fragments containing various mutations obtained in Example 1 were assembled according to the assembly kit instructions and transformed into BL21 strain by chemical transformation.

[0059] Numerous mutant PCR fragments were obtained from Example 1. After successful assembly of the mutant PCR fragments with the linearized vector and transformation into the BL21 strain, 20 mutant strains were obtained through resistance and PCR verification, numbered 21a-1 to 20. These 20 strains were induced to express enzymes, and 5 strains showed increased enzyme activity compared to the control. The strain with the highest enzyme activity showed a 3.5-fold increase compared to the control. Figure 1 As shown.

[0060] The enzyme activity assay method is as follows: Phosphotransferase activity is determined in 1 ml of a standard reaction mixture (containing 100 μM sodium acetate buffer (pH 5.0), 40 μM inosine or guanosine, 100 μM tetrasodium pyrophosphate, and enzyme solution). The reaction is incubated at 30 °C for 10 minutes, and then terminated by adding 0.2 ml of 2N HCl. Inosine, guanosine, and 5′-IMP and 5′-GMP are quantitatively determined by HPLC. One unit of enzyme activity refers to the amount of enzyme that catalyzes the reaction of inosine with sodium tripolyphosphate to produce 1 μmol of inosine acid within 1 minute at 30 °C and pH 4.0, defined as 1 U.

[0061] Example 3: Production of 5'-nucleotides using shake flasks

[0062] This embodiment uses the strain with high enzyme activity obtained in Example 2 to produce 5'-nucleotides, and the steps are as follows.

[0063] Of the five strains with high enzyme activity obtained in Example 2, three strains, 21a-4, 21a-19, and 21a-20, were selected. The three strains were cultured overnight in LB medium via shake flasks. The LB medium formulation was: 5 g / L yeast extract, 10 g / L peptone, 10 g / L NaCl, pH 7.2-7.4. 10% of each strain was inoculated into shake flasks containing 30 g / L inosine or guanosine, and 150 g / L disodium pyrophosphate. The shake flask medium formulation was: 30 g / L glucose, 12 g / L yeast extract, 18 g / L peptone, 20 g / L NaCl, 8 g / L NaH2PO4·2H2O, 5 g / L MgSO4·7H2O, and 8 g / L (NH4)2SO4. 1 mM IPTG was added for induction. Fermentation was induced at 30°C for 18 h. After fermentation, the inosine or guanosine content in the fermentation medium was determined by liquid chromatography. The results showed that the inosinic acid or guanylic acid content of BL21 (pET21a) was 8-13 times higher than that of the control BL21 (pET21a). Fermentation results are shown in […]. Figure 2 .

[0064] Example 4: Fermentation preparation of phosphotransferase with high enzyme activity

[0065] This embodiment uses the strain with the highest enzyme activity to produce phosphotransferase, and the specific steps are as follows.

[0066] The strain 21a-20, which had the highest enzyme activity in Example 3, was selected and fermented in a 50L tank to prepare phosphotransferase culture.

[0067] Strains 21a-20 were cultured overnight in LB medium in shake flasks (LB medium formula (g / L): yeast extract 5, peptone 10, NaCl 10, pH 7.2-7.4). 10% of this culture was then inoculated into shake flask medium (shake flask medium formula (g / L): glucose 30, yeast extract 12, peptone 18, NaCl 20, NaH2PO4·2H2O 8, MgSO4·7H2O 5, (NH4)2SO4 8) and cultured in a reciprocating shaker at 220 r / min and 37℃ until the OD reached 8-10, thus preparing shake flask seed culture.

[0068] Take 50 mL of the prepared shake-flask seed culture and inoculate it into a 50 L fermenter. The fermenter should have a liquid volume of 20 L. The fermentation medium formula (g / L) is: glucose 30, corn steep liquor 40, (NH4)2SO4 10, K2HPO4 5, MgSO4·7H2O 5, biotin 0.0005, and thiamine hydrochloride 0.001. During fermentation, adjust the pH to 6.8-7.2 by adding ammonia water, and alternately increase the rotation speed, air volume, and tank pressure to maintain dissolved oxygen at 30-60%. The initial fermentation temperature is 37℃. When the OD reaches 20-25, cool down to 28℃. After the glucose concentration drops to 1-3 g / L, add sugar (sugar formula: glucose 600 g / L, α-lactose 300 g / L). Control the residual sugar to 0-1 g / L. Stop fermentation when the OD reaches 75-80. The fermentation broth was centrifuged at 5000 r / min for 5 minutes to collect the cells. The cells were then resuspended in an equal volume of phosphate buffer (1 g / L dipotassium hydrogen phosphate, 1 g / L potassium dihydrogen phosphate, pH 7.0) to prepare the phosphotransferase enzyme solution.

[0069] The phosphotransferase enzyme solution prepared above was detected by HPLC, and the enzyme activity reached 510±20 U / mL, which is much higher than the enzyme activity of phosphotransferase obtained in Patent Document 1 (CN96195770.0) and Patent Document 2 (CN201310574198.X).

[0070] Example 5: Bioenzyme-catalyzed production of 5'-nucleotides

[0071] This embodiment provides a method for producing 5'-nucleotides using the phosphotransferase prepared in Example 4, and the steps are as follows.

[0072] 240g of guanosine, 489g of sodium tripolyphosphate, and 111g of sodium pyrophosphate were suspended in 2280g of water. The pH was adjusted to 4.5±0.02 with 6mol / L hydrochloric acid. The solution temperature was maintained at 37±0.5℃. 684mL of the phosphotransferase enzyme solution prepared in Example 4 was added. The temperature was maintained at 37±0.5℃, and the reaction was completed after 8 hours to obtain the enzyme reaction solution.

[0073] The pH of the above reaction solution was adjusted to 10.2±0.02 using 40% sodium hydroxide, and the temperature was gradually decreased to 10°C for phosphorus precipitation. The temperature was decreased at a rate of 3°C per hour, and the solution was centrifuged to obtain a clear liquid.

[0074] The above clear liquid was heated to 60°C, 0.3% activated carbon was added, and after stirring for 30 minutes, it was centrifuged to obtain the decolorized clear liquid.

[0075] The above decolorized clear liquid was taken, and the pH was adjusted to 8.0±0.02 with 6 mol / L hydrochloric acid. Crystallization was then carried out by gradient cooling to 10℃ at a rate of 3℃ per hour. The mixture was centrifuged, and the solid product was collected and dried at 40℃ to obtain 301g of disodium 5'-guanylate with a purity of 98.3%, a yield of 125.4%. The yield was calculated using the formula: (disodium 5'-guanylate / guanosine) × 100%.

[0076] The 5'-inosinate disodium and 5'-guanylate disodium products obtained by the method provided in this embodiment have high purity, reaching over 97.5%, which meets food-grade requirements. At the same time, the yield also shows a significant leap, reaching over 125%, which can greatly reduce production costs for industrialization.

[0077] The preparation method described in this invention uses simple and readily available raw materials, has mild reaction conditions, low energy consumption, and can be rapidly scaled up for industrial production, while also realizing the concept of green production.

[0078] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention. sequence list <110> Feng Bo <120> A phosphotransferase mutant and its application <130> KHP211121301.2 <160> 10 <170> SIPOSequenceListing 1.0 <210> 1 <211> 747 <212> DNA <213> Artificial Sequence <400> 1 atgaaaaagc gcgttctcgc cctctgcctc gccagtttgt tttccgttaa cgctttcgcg 60 ctggtccctg ccggcaatga tgcaaccacc aaaccggatc tctattatct gaaaaatgca 120 caggccatcg atagtctggc gctgttgccg ccgccgccgg aagttggcag catcgcattt 180 ttaaacgatc aggcgatgta tgagaaagga cggctgctgc gcaataccga acgcggcaag 240 caggcgcagg aagatgctga cctggccaaa ggcgacgtcg cgaatgcctt ctccagcgct 300 tttggttcgc ccatcaccga aaaagacgcg ccggagttac ataagctgct gacaaatatg 360 attgaagatg ccggcgatct ggctacccgc agcgcgaaag aaaaatatat gcgcattcgc 420 ccgtttgcgt tctacggcgt ttcaacctgt aacactaaag agcaggacaa gctgtcgaaa 480 aacggatctt acccttccgg tcatgggtct tccggttggg caaccgcgct ggtactggcg 540 gagatcaatc cgcagcggca aaacgaaatt ctcaaacgcg gctatgagtt gggcgaaagc 600 cgggttatct gcggctatca ttggcagagc gacgtcgatg cggcgcggat agtcggctcg 660 gcggtggtgg cgaccctgca taccaacccg gccttccaac agcagttgca gaaagcaaag 720 gatgaattcg ccaaaacgca gaagtaa 747 <210> 2 <211> 248 <212> PRT <213> Artificial Sequence <400> 2 Met Lys Lys Arg Val Leu Ala Leu Cys Leu Ala Ser Leu Phe Ser Val 1 5 10 15 Asn Ala Phe Ala Leu Val Pro Ala Gly Asn Asp Ala Thr Thr Lys Pro 20 25 30 Asp Leu Tyr Tyr Leu Lys Asn Ala Gln Ala Ile Asp Ser Leu Ala Leu 35 40 45 Leu Pro Pro Pro Pro Glu Val Gly Ser Ile Ala Phe Leu Asn Asp Gln 50 55 60 Ala Met Tyr Glu Lys Gly Arg Leu Leu Arg Asn Thr Glu Arg Gly Lys 65 70 75 80 Gln Ala Gln Glu Asp Ala Asp Leu Ala Lys Gly Asp Val Ala Asn Ala 85 90 95 Phe Ser Ser Ala Phe Gly Ser Pro Ile Thr Glu Lys Asp Ala Pro Glu 100 105 110 Leu His Lys Leu Leu Thr Asn Met Ile Glu Asp Ala Gly Asp Leu Ala 115 120 125 Thr Arg Ser Ala Lys Glu Lys Tyr Met Arg Ile Arg Pro Phe Ala Phe 130 135 140 Tyr Gly Val Ser Thr Cys Asn Thr Lys Glu Gln Asp Lys Leu Ser Lys 145 150 155 160 Asn Gly Ser Tyr Pro Ser Gly His Gly Ser Ser Gly Trp Ala Thr Ala 165 170 175 Leu Val Leu Ala Glu Ile Asn Pro Gln Arg Gln Asn Glu Ile Leu Lys 180 185 190 Arg Gly Tyr Glu Leu Gly Glu Ser Arg Val Ile Cys Gly Tyr His Trp 195 200 205 Gln Ser Asp Val Asp Ala Ala Arg Ile Val Gly Ser Ala Val Val Ala 210 215 220 Thr Leu His Thr Asn Pro Ala Phe Gln Gln Gln Leu Gln Lys Ala Lys 225 230 235 240 Asp Glu Phe Ala Lys Thr Gln Lys 245 <210> 3 <211> 747 <212> DNA <213> Artificial Sequence <400> 3 atgaaaaagc gcgttctcgc cctctgcctc gccagtttgt tttccgttaa cgctttcgcg 60 ctggtccctg ccggcaatga tgcaaccacc aaaccggatc tctattatct gaaaaatgca 120 caggccatcg atagcctggc gctgttgccg ccgccgccgg aagttggcag catcgcattt 180 ttaaacgatc aggcgatgta tgagaaagga cgcctgctgc gcaataccga acgcggcaag 240 ctggcggctg aagatgctga cctgagcgcc ggcggcgtcg cgaatgcctt ctccagcgct 300 tttggttacc ccatcaccga aaaagacgcg ccggagttac ataagctgct gacaaatatg 360 attgaggatg ccggcgatct ggccacccgc agcgcgaaag agaaatatat gcgcattcgc 420 ccgtttgcgt tctacggcgt ttcaacctgt aacactaccg agcaggacaa gctgtcgaaa 480 aacggatctt acccttccgg ccatacctct atcggttggg catccgcgct ggttctggcg 540 gagatcaatc cgcagcggca aaacgaaatt ctcaaacgcg gctacgagtt gggcgaaagc 600 cgggttatct gcggctatca ttggcagagc gatgtcgatg cggcgcggat agtcggctcg 660 gcggtggtgg cgaccctgca taccaacccg gccttccaac agcagttgca gaaagcaaag 720 gatgaattcg ccaaaacgca gaagtaa 747 <210> 4 <211> 248 <212> PRT <213> Artificial Sequence <400> 4 Met Lys Lys Arg Val Leu Ala Leu Cys Leu Ala Ser Leu Phe Ser Val 1 5 10 15 Asn Ala Phe Ala Leu Val Pro Ala Gly Asn Asp Ala Thr Thr Lys Pro 20 25 30 Asp Leu Tyr Tyr Leu Lys Asn Ala Gln Ala Ile Asp Ser Leu Ala Leu 35 40 45 Leu Pro Pro Pro Pro Glu Val Gly Ser Ile Ala Phe Leu Asn Asp Gln 50 55 60 Ala Met Tyr Glu Lys Gly Arg Leu Leu Arg Asn Thr Glu Arg Gly Lys 65 70 75 80 Leu Ala Ala Glu Asp Ala Asp Leu Ser Ala Gly Gly Val Ala Asn Ala 85 90 95 Phe Ser Ser Ala Phe Gly Tyr Pro Ile Thr Glu Lys Asp Ala Pro Glu 100 105 110 Leu His Lys Leu Leu Thr Asn Met Ile Glu Asp Ala Gly Asp Leu Ala 115 120 125 Thr Arg Ser Ala Lys Glu Lys Tyr Met Arg Ile Arg Pro Phe Ala Phe 130 135 140 Tyr Gly Val Ser Thr Cys Asn Thr Thr Glu Gln Asp Lys Leu Ser Lys 145 150 155 160 Asn Gly Ser Tyr Pro Ser Gly His Thr Ser Ile Gly Trp Ala Ser Ala 165 170 175 Leu Val Leu Ala Glu Ile Asn Pro Gln Arg Gln Asn Glu Ile Leu Lys 180 185 190 Arg Gly Tyr Glu Leu Gly Glu Ser Arg Val Ile Cys Gly Tyr His Trp 195 200 205 Gln Ser Asp Val Asp Ala Ala Arg Ile Val Gly Ser Ala Val Val Ala 210 215 220 Thr Leu His Thr Asn Pro Ala Phe Gln Gln Gln Leu Gln Lys Ala Lys 225 230 235 240 Asp Glu Phe Ala Lys Thr Gln Lys 245 <210> 5 <211> 747 <212> DNA <213> Artificial Sequence <400> 5 atgaaaaagc gcgttctcgc cctctgcctc gccagcctgt tttccgttaa cgctttcgcg 60 ctggtccctg ccggcaatga tgcaaccacc aaaccggatc tctattatct gaaaaatgca 120 gaggccatcg atagcctggc cctgttgccg ccgccgccgg aagttggcag catcgccttt 180 ttaaacgatc aggcgatgta tgagaaagga cgcctgctgc gcaataccga acgcggcaag 240 ccggcggctg cagatgctga cctgagcgcc ggcgacgtcg cgaatgcctt ctccagcgct 300 tttggttacc ccatcaccga aaaagacgcg ccgcagttac ataagctgct gacaaatatg 360 attgaggatg ccggcgatct ggccacccgc agcgcgaaag agaaatatat gcgcattcgc 420 ccgtttgcgt tctacggcgt ttcaacctgt aacactaccg accaggacaa gctgtcgaaa 480 aacggatctt acccttccgg ccatacctct accggttggg caaccgcgct ggtactggcg 540 gagatcaatc cgcagcggca aaacgaaatt ctcaaacgcg gctatgagtt gggccaaagc 600 cgggttatct gcggctatca ttggcagagc gacgtcgatg cggcgcggat agtcggctcg 660 gcggtggtgg cgaccctgca ttccaacccg gccttccaac agcagttgca gaaagcaaag 720 gatgaattcg ccaaaacgca gaagtaa 747 <210> 6 <211> 248 <212> PRT <213> Artificial Sequence <400> 6 Met Lys Lys Arg Val Leu Ala Leu Cys Leu Ala Ser Leu Phe Ser Val 1 5 10 15 Asn Ala Phe Ala Leu Val Pro Ala Gly Asn Asp Ala Thr Thr Lys Pro 20 25 30 Asp Leu Tyr Tyr Leu Lys Asn Ala Glu Ala Ile Asp Ser Leu Ala Leu 35 40 45 Leu Pro Pro Pro Pro Glu Val Gly Ser Ile Ala Phe Leu Asn Asp Gln 50 55 60 Ala Met Tyr Glu Lys Gly Arg Leu Leu Arg Asn Thr Glu Arg Gly Lys 65 70 75 80 Pro Ala Ala Ala Asp Ala Asp Leu Ser Ala Gly Asp Val Ala Asn Ala 85 90 95 Phe Ser Ser Ala Phe Gly Tyr Pro Ile Thr Glu Lys Asp Ala Pro Gln 100 105 110 Leu His Lys Leu Leu Thr Asn Met Ile Glu Asp Ala Gly Asp Leu Ala 115 120 125 Thr Arg Ser Ala Lys Glu Lys Tyr Met Arg Ile Arg Pro Phe Ala Phe 130 135 140 Tyr Gly Val Ser Thr Cys Asn Thr Thr Asp Gln Asp Lys Leu Ser Lys 145 150 155 160 Asn Gly Ser Tyr Pro Ser Gly His Thr Ser Thr Gly Trp Ala Thr Ala 165 170 175 Leu Val Leu Ala Glu Ile Asn Pro Gln Arg Gln Asn Glu Ile Leu Lys 180 185 190 Arg Gly Tyr Glu Leu Gly Gln Ser Arg Val Ile Cys Gly Tyr His Trp 195 200 205 Gln Ser Asp Val Asp Ala Ala Arg Ile Val Gly Ser Ala Val Val Ala 210 215 220 Thr Leu His Ser Asn Pro Ala Phe Gln Gln Gln Leu Gln Lys Ala Lys 225 230 235 240 Asp Glu Phe Ala Lys Thr Gln Lys 245 <210> 7 <211> 747 <212> DNA <213> Artificial Sequence <400> 7 atgaaaaagc gcgttctcgc cctctgcctc gccagcctgt tttccgttaa cgctttcgcg 60 ctggtccctg ccggcaatga tgcaaccacc aaaccggatc tctattatct gaaaaatgca 120 caggccatcg atagcctggc gctgttgccg ccgccgccgg aagttggcag catcgcattt 180 ttaaacgatc aggcgatgta tgagaaagga cggctgctgc gcaataccga acgcggcaag 240 ctggcggctg aagatgctaa cctgagcgcc ggcggcgtcg cgaatgcctt ctccagcgct 300 tttggttcgc ccatcaccga aaaagacgcg ccgcagttac ataagctgct gacaaatatg 360 attgaggatg ccggcgatct ggctacccgc agcgcgaaag agaaatatat gcgcattcgc 420 ccgtttgcgt tctacggcgt ttcaacctgt aacactaccg agcaggacaa gctgtcgaaa 480 aacggatctt acccttccgg tcatacctct atcggttggg caaccgcgct ggtactggcg 540 gagatcaatc cgcagcggca aaacgaaatt ctcaaacgcg gctatgagtt gggcgaaagc 600 cgggttatct gcggctatca ttggcagagc gacgtcgatg cggcgcggat agtcggctcg 660 gcggtggtgg cgaccctgca taccaacccg gccttccaac agcagttgca gaaagcaaag 720 gatgaattcg ccaaaacgca gaagtaa 747 <210> 8 <211> 248 <212> PRT <213> Artificial Sequence <400> 8 Met Lys Lys Arg Val Leu Ala Leu Cys Leu Ala Ser Leu Phe Ser Val 1 5 10 15 Asn Ala Phe Ala Leu Val Pro Ala Gly Asn Asp Ala Thr Thr Lys Pro 20 25 30 Asp Leu Tyr Tyr Leu Lys Asn Ala Gln Ala Ile Asp Ser Leu Ala Leu 35 40 45 Leu Pro Pro Pro Pro Glu Val Gly Ser Ile Ala Phe Leu Asn Asp Gln 50 55 60 Ala Met Tyr Glu Lys Gly Arg Leu Leu Arg Asn Thr Glu Arg Gly Lys 65 70 75 80 Leu Ala Ala Glu Asp Ala Asn Leu Ser Ala Gly Gly Val Ala Asn Ala 85 90 95 Phe Ser Ser Ala Phe Gly Ser Pro Ile Thr Glu Lys Asp Ala Pro Gln 100 105 110 Leu His Lys Leu Leu Thr Asn Met Ile Glu Asp Ala Gly Asp Leu Ala 115 120 125 Thr Arg Ser Ala Lys Glu Lys Tyr Met Arg Ile Arg Pro Phe Ala Phe 130 135 140 Tyr Gly Val Ser Thr Cys Asn Thr Thr Glu Gln Asp Lys Leu Ser Lys 145 150 155 160 Asn Gly Ser Tyr Pro Ser Gly His Thr Ser Ile Gly Trp Ala Thr Ala 165 170 175 Leu Val Leu Ala Glu Ile Asn Pro Gln Arg Gln Asn Glu Ile Leu Lys 180 185 190 Arg Gly Tyr Glu Leu Gly Glu Ser Arg Val Ile Cys Gly Tyr His Trp 195 200 205 Gln Ser Asp Val Asp Ala Ala Arg Ile Val Gly Ser Ala Val Val Ala 210 215 220 Thr Leu His Thr Asn Pro Ala Phe Gln Gln Gln Leu Gln Lys Ala Lys 225 230 235 240 Asp Glu Phe Ala Lys Thr Gln Lys 245 <210> 9 <211> 41 <212> DNA <213> Artificial Sequence <400> 9 cagcaaatgg gtcgcggatc catgaaaaag cgcgttctcg c 41 <210> 10 <211> 42 <212> DNA <213> Artificial Sequence <400> 10 ctcgagtgcg gccgcaagct tttacttctg cgttttggcg aa 42

Claims

1. A phosphotransferase mutant, characterized in that, The amino acid sequence of the phosphotransferase mutant is shown in SEQ ID No. 2, SEQ ID No. 4 or SEQ ID No.

6.

2. A DNA molecule encoding the phosphotransferase mutant of claim 1.

3. The DNA molecule according to claim 2, characterized in that, The nucleotide sequence of the DNA molecule is shown in SEQ ID No. 1, SEQ ID No. 3 or SEQ ID No.

5.

4. A biomaterial containing the DNA molecule according to any one of claims 2-3, characterized in that, The biomaterial is an expression cassette, vector, or host cell.

5. The application of the phosphotransferase mutant of claim 1, the DNA molecule of any one of claims 2-3, or the biological material of claim 4 in any of the following: (1) Increase the yield of disodium 5'-guanylate or disodium 5'-inosinate; (2) Improve the purity of disodium 5'-guanylate or disodium 5'-inosinate.

6. The use of the DNA molecule according to any one of claims 2-3 or the biomaterial according to claim 4 in the preparation of highly active phosphotransferases.

7. A recombinant microorganism, characterized in that, The recombinant microorganism expresses the phosphotransferase mutant of claim 1 or the genome of the recombinant microorganism contains the DNA molecule of any one of claims 2-3.

8. The recombinant microorganism according to claim 7, characterized in that, The recombinant microorganism is Enterobacter aerogenes or Escherichia coli.

9. The use of the recombinant microorganism according to any one of claims 7-8 in increasing the yield of disodium 5'-guanylate or disodium 5'-inosinate, producing high-activity phosphotransferase or high-purity disodium 5'-guanylate or disodium 5'-inosinate.

10. A method for preparing a phosphotransferase, characterized in that, After activating the recombinant microorganisms described in any one of claims 7-8, they are inoculated into the fermentation medium. During the fermentation process, the pH is adjusted to 6.8-7.2, the dissolved oxygen to 30-60%, the initial fermentation temperature is 28-37℃, the OD reaches 20-25, the temperature is lowered to 28℃, and the glucose concentration is reduced to 1-3 g / L before adding sugar, controlling the residual sugar to 0-1 g / L.

11. The preparation method according to claim 10, characterized in that, The fermentation medium formula is as follows: glucose 15-30 g / L, corn steep liquor 20-40 g / L, (NH4)2SO4 5-10 g / L, K2HPO4 1-5 g / L, MgSO4·7H2O 0.5-5 g / L, biotin 0.0001-0.0005 g / L and thiamine hydrochloride 0.0001-0.001 g / L; The formulation of the added sugar is glucose and α-lactose in a mass ratio of (2-2.5):(1-1.5).

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