Pyridoxal kinase mutant and application thereof in synthesis of pyridoxine phosphate

Abstract

The invention provides a pyridoxal kinase mutant and application thereof in synthesis of pyridoxine phosphate, and belongs to the field of enzyme engineering. The mutant disclosed by the invention is obtained by mutating an amino acid sequence of wild type pyridoxal kinase, and mutation sites comprise at least one of the 23rd amino acid, the 25th amino acid, the 30th amino acid, the 31st amino acid, the 52nd amino acid, the 54th amino acid, the 57th amino acid, the 57th amino acid, the 96th amino acid, the 125th amino acid, the 127th amino acid, the 130th amino acid, the 136th amino acid, the 157th amino acid, the 158th amino acid, the 159th amino acid, the 230th amino acid, the 233th amino acid, the 235th amino acid, the 236th amino acid, the 257th amino acid, the 260th amino acid and the 264th amino acid; wherein the amino acid sequence of the wild type pyridoxal kinase is shown as SEQ: ID: NO: 1. Compared with wild type pyridoxal kinase and other mutants, the pyridoxal kinase mutant provided by the invention has higher catalytic efficiency, has the advantages of simple process, high conversion rate and low production cost when being applied to a pyridoxine phosphate synthesis process, and has a good industrial application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and specifically relates to a pyridoxal kinase mutant and its application in the synthesis of pyridoxine phosphate. Background Art

[0002] Vitamin B6 belongs to water-soluble vitamins and is a general term for a class of pyridine compounds that can be converted into each other, including pyridoxine (PN), pyridoxamine (PM), and pyridoxal (PL). Their corresponding phosphate ester forms are pyridoxine-5'-phosphate (PNP), pyridoxamine-5'-phosphate (PMP), and pyridoxal-5'-phosphate (PLP). Among them, PLP is the bioactive form of VB6 and has extremely important physiological functions. As a cofactor for more than 180 enzymes, it participates in a variety of key biochemical reactions.

[0003] In terms of cardiovascular health, PLP is a cofactor for dopamine (vasopressin) synthase. Dopamine can stimulate the contraction of muscle tissues in capillaries and arteries, thereby reducing hypertension. In the treatment of diabetes, PLP can catalyze the decomposition of cysteine to generate a small gas molecule hydrogen sulfide (H2S) to stabilize the physiological level of H2S, relieve the imbalance of endogenous H2S metabolism in the human body, and thus prevent damage to pancreatic islet β cell function. In the treatment of cancer, PLP can regulate two key metabolic enzymes required for the growth of acute myeloblastic leukemia (AML) cells, thereby slowing down the deterioration of AML; a large intake of VB6 may inhibit the development of pancreatic cancer, cancer cell metastasis, and tumor growth, etc.

[0004] Currently, the production of PLP mostly uses chemical methods. For example, patent application CN 110016049A discloses a method for preparing pyridoxal phosphate by a chemical method, which is not only time-consuming, energy-consuming, seriously polluting, and costly, but also has a low yield. In contrast, the biosynthetic pathway has become a more promising alternative due to its environmental friendliness and high efficiency. As a precursor of PLP, PNP is an important source of pyridoxal phosphate. Oxidizing PNP to PLP in one step is an important way for the biological preparation of PLP. Therefore, it is of great significance to improve the production rate of PNP and further increase the yield of PLP. However, the enzyme activity of pyridoxal kinase used in the current PNP synthesis process is low, which limits the production rate of PNP. Therefore, it is particularly urgent and necessary to develop a pyridoxal kinase with high catalytic efficiency and an efficient process route for the production of PNP. Summary of the Invention

[0005] The object of the present invention is to provide a pyridoxal kinase mutant and its application in the synthesis of pyridoxine phosphate, so as to solve the problems of low activity, low catalytic efficiency and long reaction time of existing pyridoxal kinases.

[0006] The present invention is implemented as follows:

[0007] A pyridoxal kinase mutant, which is obtained by mutating the amino acid sequence of wild-type pyridoxal kinase, and the mutation sites include at least one of the 23rd, 25th, 30th, 31st, 52nd, 54th, 57th, 58th, 59th, 96th, 125th, 127th, 130th, 136th, 157th, 158th, 159th, 230th, 233rd, 235th, 236th, 257th, 260th, 264th amino acids.

[0008] Furthermore, the mutant includes at least one of the following mutations:

[0009] a1: Based on the amino acid sequence shown in SEQ: ID: NO: 1, mutate the Ser at its 23rd position to Thr;

[0010] a2: Based on the amino acid sequence shown in SEQ: ID: NO: 1, mutate the Ser at its 23rd position to Gly;

[0011] a3: Based on the amino acid sequence shown in SEQ: ID: NO: 1, mutate the Val at its 25th position to Asn;

[0012] a4: Based on the amino acid sequence shown in SEQ: ID: NO: 1, mutate the Val at its 25th position to Leu;

[0013] a5: Based on the amino acid sequence shown in SEQ: ID: NO: 1, mutate the Val at its 30th position to Gly;

[0014] a6: Based on the amino acid sequence shown in SEQ: ID: NO: 1, mutate the Gly at its 31st position to Ser;

[0015] a7: Based on the amino acid sequence shown in SEQ: ID: NO: 1, mutate the Val at its 52nd position to Gly;

[0016] a8: Based on the amino acid sequence shown in SEQ: ID: NO: 1, mutate the Val at its 52nd position to Ser;

[0017] a9: Based on the amino acid sequence shown in SEQ:ID:NO:1, mutate Leu at position 54 to Ile;

[0018] a10: Based on the amino acid sequence shown in SEQ:ID:NO:1, mutate Leu at position 54 to Thr;

[0019] a11: Based on the amino acid sequence shown in SEQ:ID:NO:1, mutate Thr at position 57 to Ser;

[0020] a12: Based on the amino acid sequence shown in SEQ:ID:NO:1, mutate Thr at position 57 to Pro;

[0021] a13: Based on the amino acid sequence shown in SEQ:ID:NO:1, mutate Pro at position 58 to His;

[0022] a14: Based on the amino acid sequence shown in SEQ:ID:NO:1, mutate Pro at position 58 to Arg;

[0023] a15: Based on the amino acid sequence shown in SEQ:ID:NO:1, mutate His at position 59 to Arg;

[0024] a16: Based on the amino acid sequence shown in SEQ:ID:NO:1, mutate Tyr at position 96 to Phe;

[0025] a17: Based on the amino acid sequence shown in SEQ:ID:NO:1, mutate Asp at position 125 to Leu;

[0026] a18: Based on the amino acid sequence shown in SEQ:ID:NO:1, mutate Val at position 127 to Gly;

[0027] a19: Based on the amino acid sequence shown in SEQ:ID:NO:1, mutate Asp at position 130 to Asn;

[0028] a20: Based on the amino acid sequence shown in SEQ:ID:NO:1, mutate Tyr at position 136 to Phe;

[0029] a21: Based on the amino acid sequence shown in SEQ:ID:NO:1, mutate Thr at position 157 to Ser;

[0030] a22: Based on the amino acid sequence shown in SEQ:ID:NO:1, mutate Pro at position 158 to His;

[0031] a23: Based on the amino acid sequence shown in SEQ: ID: NO: 1, Asn at position 159 is mutated to Arg;

[0032] a24: Based on the amino acid sequence shown in SEQ: ID: NO: 1, Gly at position 230 is mutated to Val;

[0033] a25: Based on the amino acid sequence shown in SEQ: ID: NO: 1, Asp at position 233 is mutated to Asn;

[0034] a26: Based on the amino acid sequence shown in SEQ: ID: NO: 1, Phe at position 235 is mutated to Tyr;

[0035] a27: Based on the amino acid sequence shown in SEQ: ID: NO: 1, Cys at position 236 is mutated to Ser;

[0036] a28: Based on the amino acid sequence shown in SEQ: ID: NO: 1, Gly at position 257 is mutated to Val;

[0037] a29: Based on the amino acid sequence shown in SEQ: ID: NO: 1, Val at position 260 is mutated to Gly;

[0038] a30: Based on the amino acid sequence shown in SEQ: ID: NO: 1, Met at position 264 is mutated to Thr;

[0039] a31: Based on the amino acid sequence shown in SEQ: ID: NO: 1, Leu at position 54 is mutated to Ile, and Thr at position 57 is mutated to Ser simultaneously, resulting in a double - combination mutation.

[0040] Furthermore, the pyridoxal kinase mutant is any one of the following mutants:

[0041] Mutant pdxK - L54I, the mutant pdxK - L54I is obtained by mutating Leu at position 54 to Ile based on the amino acid sequence shown in SEQ: ID: NO: 1, and its amino acid sequence is shown in SEQ: ID: NO: 3;

[0042] Mutant pdxK - L54T, the mutant pdxK - L54T is obtained by mutating Leu at position 54 to Thr based on the amino acid sequence shown in SEQ: ID: NO: 1, and its amino acid sequence is shown in SEQ: ID: NO: 5;

[0043] Mutant pdxK-T57S, which is obtained by mutating Thr at position 57 to Ser based on the amino acid sequence shown in SEQ: ID: NO: 1, and its amino acid sequence is shown in SEQ: ID: NO: 7;

[0044] Mutant pdxK-T57P, which is obtained by mutating Thr at position 57 to Pro based on the amino acid sequence shown in SEQ: ID: NO: 1, and its amino acid sequence is shown in SEQ: ID: NO: 9;

[0045] Mutant pdxK-L54I / T57S, which is obtained by mutating Leu at position 54 to Ile and then mutating Thr at position 57 to Ser based on the amino acid sequence shown in SEQ: ID: NO: 1, and its nucleotide sequence is shown in SEQ: ID: NO: 11.

[0046] The present invention also provides biological materials related to the above pyridoxal kinase mutants, and the biological materials are any one of B1) to B4):

[0047] B1) A nucleic acid molecule encoding the above pyridoxal kinase mutant;

[0048] B2) An expression cassette containing the nucleic acid molecule described in B1);

[0049] B3) A recombinant expression vector containing the nucleic acid molecule described in B1), or a recombinant expression vector containing the expression cassette described in B2);

[0050] B4) A recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3).

[0051] Furthermore, the nucleotide sequence encoding mutant PDXKpdxK-L54I is shown in SEQ: ID: NO: 4; the nucleotide sequence encoding mutant pdxK-L54T is shown in SEQ: ID: NO: 6; the nucleotide sequence encoding mutant pdxK-T57S is shown in SEQ: ID: NO: 8; the nucleotide sequence encoding mutant pdxK-T57P is shown in SEQ: ID: NO: 10; the nucleotide sequence encoding mutant pdxK-L54I / T57 is shown in SEQ: ID: NO: 12.

[0052] Preferably, the recombinant expression vector uses pET-29a(+) as the original expression vector.

[0053] The recombinant microorganism is a recombinant bacterium, and the host bacterium of the recombinant bacterium is Escherichia coli or filamentous fungi such as Bacillus subtilis, Corynebacterium glutamicum, yeast, Streptomyces, Penicillium or Acremonium chrysogenum; preferably, the host bacterium of the recombinant bacterium is Escherichia coli BL21(DE3).

[0054] The construction method of the recombinant bacterium is as follows:

[0055] 1) Insert the coding gene fragment of pyridoxal kinase from the above-mentioned Escherichia coli species between the NdeI and EcoRI restriction sites of the original expression vector pET-29a to obtain a recombinant vector named pET-29a-ZTWT;

[0056] 2) Transform the recombinant vector into Escherichia coli BL21(DE3) by heat shock method to obtain the recombinant bacterium.

[0057] The present invention also provides the application of any of the above pyridoxal kinase mutants in the synthesis of pyridoxine phosphate.

[0058] Specifically, it includes the following steps: using VB6 as a substrate, successively adding magnesium salt, sodium ATP and sodium hexametaphosphate, stirring and dissolving, adjusting the pH to 5-6, adding the pyridoxal kinase mutant enzyme solution and polyphosphate kinase enzyme solution, making up the volume, and carrying out a catalytic reaction at 36-40 °C to generate pyridoxine phosphate.

[0059] Preferably, the mass ratio of VB6, magnesium salt, sodium ATP and sodium hexametaphosphate is 5:1:5:5; the magnesium salt is magnesium chloride hexahydrate; the pH is 5, and the catalytic reaction temperature is 37 °C; in every 1 liter of the synthesis system, 1400-5600 U of the pyridoxal kinase mutant enzyme solution and 800-1340 U of the polyphosphate kinase enzyme solution are added;

[0060] More preferably, in every 1 liter of the synthesis system, 1400 U of the pyridoxal kinase mutant enzyme solution and 800 U of the polyphosphate kinase enzyme solution are added.

[0061] The preparation method of the crude enzyme solution of pyridoxal kinase and its mutants is as follows: transform the recombinant plasmid into Escherichia coli BL21(DE3) strain to obtain a recombinant strain; after activating the recombinant strain, inoculate it into LB medium, and when the OD 600 value reaches 0.6, add IPTG to induce the expression of pyridoxal kinase or its mutants; the final concentration of IPTG in the medium is 0.1 mM, the induction condition is 20 °C, and the induction time is 20 h; centrifuge and break to obtain the crude enzyme solution.

[0062] The present invention also provides a method for preparing pyridoxine phosphate, which comprises the following steps: using VB6 as a substrate and performing a catalytic reaction with the pyridoxal kinase mutant or the biological material as described above to obtain pyridoxine phosphate. Preferably, the temperature of the catalytic reaction is 36-40 °C and the pH is 5-6.

[0063] The present invention also provides a preparation for catalyzing the synthesis of pyridoxine phosphate from VB6, and the preparation contains the pyridoxal kinase mutant or its related biological material as described in the present invention.

[0064] The present invention has the following beneficial effects:

[0065] Based on the traditional chemical synthesis method and the biological enzyme method, the present invention analyzes the active site of pyridoxal kinase, finds potential target site residues, combines the saturation mutagenesis technology, modifies the enzyme molecule, and screens out a variety of pyridoxal kinase mutants. Compared with the wild-type pyridoxal kinase, the mutated enzyme has a higher catalytic efficiency. Using VB6 as a substrate and through a specific catalytic process, it can be used for the synthesis of pyridoxine phosphate, and a conversion rate of more than 99.8% can be achieved within 2 hours. Compared with the previous chemical synthesis method and biological enzyme method, the reaction time is shorter and the cost is lower, and it has good industrial application prospects. Description of the Drawings

[0066] Figure 1 is the liquid chromatography diagram of PNP.

[0067] Figure 2 is the plasmid map of pET-29a-pdxK-L54I / T57S in Example 3.

[0068] Figure 3 is the reaction route diagram from pyridoxine to pyridoxine phosphate. Detailed Embodiments

[0069] The following examples are used to further illustrate the present invention in detail, but do not limit the present invention in any form. The processes and methods not described in detail in the following examples are conventional methods well known in the art, and the reagents used in the examples can be purchased commercially or prepared by methods well known to those of ordinary skill in the art. The following examples have all achieved the purpose of the present invention.

[0070] Definition of enzyme activity: Under specific temperature, pH and stirring speed conditions, the amount of enzyme required to convert 1 micromole of pyridoxine or generate 1 micromole of pyridoxine phosphate within 1 minute is defined as one activity unit (U).

[0071] Method for measuring enzyme activity: Add the reaction solution to an Erlenmeyer flask, and the reaction solution includes VB6, sodium ATP, magnesium chloride hexahydrate, sodium hexametaphosphate, and the enzyme solution of the pyridoxal kinase mutant. Place it in a shaker for reaction, immediately perform metal bath inactivation, dilution, and HPLC analysis after sampling, and calculate the enzyme activity through the change in the peak area of PNP.

[0072] The present invention obtains a pyridoxal kinase pdxK mutant protein with the function of catalyzing the synthesis of PNP from PN by modifying the wild-type pyridoxal kinase pdxK. Specifically, the wild-type gene sequence of pyridoxal kinase pdxK is SEQ:ID:No.2 in the sequence listing, and the amino acid sequence of pyridoxal kinase pdxK encoded by it is SEQ:ID:No.1. The nucleotide sequence shown in SEQ:ID:NO:2 (this sequence was originally derived from the Escherichia coli strain, and the protein sequence number in UniProt is B1IX53) was artificially synthesized and cloned between the NdeI and EcoRI restriction sites of the pET-29a(+) expression vector to obtain a recombinant vector named pET-29a-ZTWT. The modified amino acid residue sites and the corresponding amino acid residue names are shown in Table 1 (where S23 represents serine at the 23rd position of the wild-type sequence SEQ:ID:No:2 of pyridoxal kinase), and the primers used to modify pyridoxal kinase pdxK are shown in Table 2.

[0073] Table 1 Amino acid residue sites modified in pyridoxal kinase pdxK

[0074] S23 V25 V30 G31 V52 L54 T57 P58 H59 Y96 D125 V127 D130 Y136 T157 P158 N159 G230 D233 F235 C236 G257 V260 M264

[0075] Table 2 Primer sequences used to modify pyridoxal kinase pdxK

[0076]

[0077]

[0078]

[0079] Example 1 Construction of a pyridoxal kinase pdxK mutant protein library

[0080] In this example, a pyridoxal kinase pdxK mutant protein was obtained by modifying pyridoxal kinase pdxK. Specifically, single-site saturation mutations were performed on the 24 amino acid residues shown in Table 1.

[0081] Construction of the S23 mutant library:

[0082] Using plasmid pET-29a-ZTWT as a template, the primers JM-S23-NDT-F1, JM-S23-VMA-F2, JM-S23-ATG-F3, and JM-S23-TGG-F4 in Table 2 were mixed in a molar ratio of 12∶6∶1∶1. 2 μL of the above primer mixture was taken as the upstream primer, and 2 μL of JMR was taken as the downstream primer for the first-round PCR to obtain the first-round PCR product of S23. The reaction system for the first-round PCR is shown in Table 3, and the reaction conditions for the first-round PCR are shown in Table 4.

[0083] Table 3 Reaction System for the First-Round PCR

[0084] Component Volume (μL) Forward primer 2 Reverse primer 2 Template pET-29a-ZTWT 1 5×PS GXL Buffer 10 dNTPs (2.5 mmol / L) 4 PrimeStar DNA polymerase (2.5 U / μL) 1 <![CDATA[ddH2O]]> 30 Total volume 50

[0085] Table 4 Reaction Conditions for the First-Round PCR

[0086]

[0087] Using 2 μL of the PCR product from the first round as a primer and plasmid pET-29a-ZTWT as a template for the second-round PCR. The reaction system for the second-round PCR is shown in Table 5, and the reaction conditions for the second-round PCR are shown in Table 6.

[0088] Table 5 Reaction System for the Second-Round PCR

[0089] Component Volume (μL) Primer (first-round PCR product) 1 Template pET-29a-ZTWT 1 5×PS GXL Buffer 10 dNTPs (2.5 mmol / L) 4 PrimeStar DNA polymerase (2.5 U / μL) 1 <![CDATA[ddH2O]]> 33 Total volume 50

[0090] Table 6 Reaction Conditions for the Second-Round PCR

[0091]

[0092] The second-round PCR product for constructing the S23 mutant library was obtained.

[0093] The following operations were performed on the above S23 second-round PCR product:

[0094] 1 μL of Dpn I enzyme was added to each of the products of the S23 second-round PCR to digest the plasmid template, and the treatment was carried out at a constant temperature of 37°C for 3 h. 2 μL of the enzyme-digested S23 second-round PCR product was electrotransformed into Escherichia coli BL21(DE3). The bacterial solution of Escherichia coli BL21(DE3) after electrotransformation of the S23 second-round PCR product was evenly spread on an LB plate with kanamycin resistance (concentration 50 μg / mL), and single colonies grew after culturing at a constant temperature of 37°C for 14 h, thus obtaining the S23 mutant library.

[0095] Construction of mutant libraries at the other 23 sites: According to the construction steps of the S23 mutant library, the mutant libraries at the other 23 sites were constructed, and the primer sequences used are shown in Table 2.

[0096] Scrape all the single colonies on the above 24 mutant library plates and send them for sequencing. After verifying the diversity of the 24 mutant libraries, use sterile toothpicks to pick the negative control bacterium BL21(DE3) / pET-29a (pick 3 single colonies), the positive control bacterium BL21(DE3) / pET-29a-JZWT (pick 3 single colonies), and the single colonies grown on the mutant library LB plate (pick a total of 90 single colonies) and transfer them to a 96-well deep-well culture plate containing 300 μL of TB medium. Add kanamycin to make its final concentration 50 μg / mL, and culture with shaking at 37 °C and 800 rpm for 12 h. Take 120 μL of the bacterial liquid from each well for bacteria preservation, then add 800 μL of TB medium to each well of the 96-well deep-well culture plate, and add kanamycin and isopropyl-β-D-thiogalactoside (IPTG) to make their final concentrations 50 μg / mL and 0.1 mM respectively, and culture with shaking at 20 °C and 800 rpm for 12 h. Then centrifuge at 4000 rpm and 4 °C for 10 min to collect the bacterial cells. Wash the bacterial cells once with 1000 μL of 50 mM phosphate buffer (pH 7.4), discard the supernatant after centrifugation, and collect the bacterial cells. For the next step of whole-cell catalysis and screening of the enzyme activity of the strains.

[0097] Among them, BL21(DE3) / pET-29a is a recombinant bacterium obtained by introducing pET-29a into Escherichia coli BL21(DE3), and BL21(DE3) / pET-29a-JZWT is a recombinant bacterium obtained by introducing pET-29a-ZTWT into Escherichia coli BL21(DE3).

[0098] Prepare the catalytic reaction system: Add 10 μL of 6 mM MgCl2, 15 μL of 10 mM ATP, 15 μL of 20 mM PN, 10 μL of 30 μM FMN, and 10 μL of pyridoxine oxidase enzyme solution to 240 μL of 50 mM Tris hydrochloride buffer (pH 7.4).

[0099] Add the above-obtained reaction solution to the 96-well plate for collecting the bacterial cells of each mutant library above, react at 37 °C for 2 h, centrifuge, aspirate 200 μL and transfer it to an enzyme-labeled plate, detect the absorbance of the 24 mutant libraries at a wavelength of 414 nm, obtain the monoclonal colonies with the absorbance measured at each mutation site higher than that of the wild type, and sequence the obtained monoclonal colonies. The sequencing results are shown in Table 7.

[0100] Table 7 Sequencing results of monoclonal colonies with higher absorbance values at the saturated mutation sites of pyridoxal kinase pdxK

[0101]

[0102]

[0103] The pyridoxal kinase pdxK mutants shown in Table 7 were prepared into crude enzyme solutions to further verify the enzyme activity after mutation.

[0104] Example 2 Preparation of Mutant Crude Enzyme Solution and Verification of Enzyme Activity

[0105] The crude enzyme solutions of the mutants screened as shown in Table 7 were used to further verify the activity of the mutants. The specific operation was as follows:

[0106] The negative control bacterium BL21(DE3) / pET-29a, the positive control bacterium BL21(DE3) / pET-29a-JZWT, and the monoclonal mutants of pyridoxal kinase pdxK shown in Table 7 were respectively picked. Among them, the mutant strain name was named BL21(DE3) / pET-29a-pdxK-X (X was the amino acids before and after the mutation site). For example, the strain name corresponding to pdxK-S23T was BL21(DE3) / pET-29a-pdxK-S23T.

[0107] The crude enzyme solution was prepared according to the following steps: The negative control bacterium BL21(DE3) / pET-29a, the positive control bacterium BL21(DE3) / pET-29a-JZWT, and the monoclonal mutants were transferred into test tubes containing 5 mL of LB medium (kanamycin concentration was 50 μg / mL), and cultured overnight at 37 °C and 220 rpm; then inoculated into 100 mL of TB medium (kanamycin concentration was 50 μg / mL) at an inoculation amount of 1%, and cultured at 37 °C and 220 rpm for about 3 h. When the OD600 value of the bacterial solution reached 0.6, IPTG with a final concentration of 0.1 mM was added to the system, and the culture was continued at 25 °C and 220 rpm for 18 h; then the cells were collected by centrifugation at 4000 rpm and 4 °C for 10 min, and the cells were ultrasonically disrupted and the supernatant was collected by centrifugation to obtain the crude enzyme solutions of the above strains respectively.

[0108] The activity of the crude enzyme solution was measured by high performance liquid chromatography. The specific steps were as follows:

[0109] Weighed 9.145 g / L of PN-HCl (VB6), 30.84 g / L of disodium ATP, and 10.17 g / L of MgCl2·6H2O into a container, and dissolved them with 30 mL of purified water. After complete dissolution, the pH of the solution was adjusted to 6.0 with 10 M NaOH, and the volume was made up to 40 mL with water to obtain the mother liquor.

[0110] Add 100 μL of the crude enzyme solution to 900 μL of the mother liquor prepared above, place it in a shaker, react at 37 °C and 220 rpm for 30 min, then take it out, immediately place it in a metal bath at 100 °C to inactivate for 2 min, let it stand at room temperature, dilute it 100 times, filter it, and perform high-performance liquid chromatography determination. Calculate the enzyme activity based on the peak area of PNP. The liquid chromatography diagram is as Figure 1 shown.

[0111] The enzyme activity results of the wild-type pyridoxal kinase pdxK and each mutant strain are shown in Table 8.

[0112] Table 8 Enzyme activity results of the wild-type pyridoxal kinase pdxK and the crude enzyme solution of each mutant

[0113]

[0114]

[0115] Example 3 Construction of the expression vector of the combinatorial mutant pyridoxal kinase

[0116] Based on BL21(DE3) / pET-29a-pdxK-L54I, mutate the threonine residue (T57) at its 57th position to obtain a mutant protein with a double mutation of L54I / T57S. The plasmid map of pET-29a-pdxK-L54I / T57S is as Figure 2 shown. The specific operation is as follows:

[0117] Extract the plasmid of BL21(DE3) / pET-29a-pdxK-L54I to obtain the plasmid pET29a-pdxK-L54I. Using the plasmid pET-29a-pdxK-L54I as a template, perform PCR with the upstream and downstream primers T57S-F and T57S-R. The PCR program is: pre-denaturation at 98 °C for 2 min, denaturation at 98 °C for 15 s, annealing at 55 °C for 15 s, extension at 68 °C for 6 min, and finally extension at 72 °C for 10 min. The denaturation to extension program is set for 30 cycles.

[0118] The PCR reaction system is shown in Table 9.

[0119] Table 9 PCR reaction system for combinatorial mutation

[0120] Component Volume (μL) T57S-F primer 2 T57S-R primer 2 Template pET-29a-ZTWT 1 5×PS GXL Buffer 10 dNTPs (2.5 mmol / L) 4 PrimeStar DNA polymerase (2.5 U / μL) 1 <![CDATA[ddH2O]]> 30 Total volume 50

[0121] After the PCR reaction, 1 μL of Dpn I enzyme was added to the PCR product, and the mixture was incubated at 37 °C for 3 h to remove the template plasmid. 2 μL of the enzyme-digested PCR product was electrotransformed into Escherichia coli BL21(DE3). The electrotransformed Escherichia coli BL21(DE3) bacterial solution was evenly spread on an LB plate containing 50 μg / mL kanamycin resistance, and single colonies grew after culturing at 37 °C for 14 h. Single colonies were picked to extract plasmids for sequencing. The correctly sequenced plasmid was pET-29a-pdxK-L54I / T57S containing the double mutant, and the Escherichia coli BL21(DE3) host containing this plasmid was named BL21(DE3)pET-29a-pdxK-L54I / T57S.

[0122] According to the steps in Example 2, crude enzyme solution preparation and enzyme activity determination were carried out for BL21(DE3)pET29a-pdxK-L54T / T57P. The enzyme activity determination results of five strains in the same batch are shown in Table 10.

[0123] Table 10 Enzyme activity results of crude enzyme solutions of five strains

[0124] Sample name Enzyme activity (U / mL) Fold increase relative to wild type BL21(DE3) / pET-29a \ \ BL21(DE3) / pET-29a-JZWT 1.2 1 BL21(DE3) / pET-29a-pdxK-L54I 31.5 26.3 BL21(DE3) / pET-29a-pdxK-T57S 30.4 25.3 BL21(DE3)pET-29a-pdxK-L54I / T57S 37.9 31.6

[0125] As can be seen from the above table, the enzyme activities of the crude enzyme solutions of the three mutants are all higher than that of the wild type. The enzyme activity of the double mutant BL21(DE3)pET-29a-pdxK-L54I / T57S is slightly higher than that of the two single mutants. The three pyridoxal kinase mutants were applied to the following catalytic synthesis of pyridoxine phosphate, and the catalytic reaction conditions refer to the optimized conditions of patent application CN116814586A.

[0126] The reaction route diagram of pyridoxine to pyridoxine phosphate is as Figure 3 shown.

[0127] Example 4 Catalytic synthesis of pyridoxine phosphate by pyridoxal kinase mutant BL21(DE3) / pET-29a-pdxK-L54I (catalytic reaction in a 1L system)

[0128] In a 1L system, 10 g of VB6, 2 g of sodium ATP, 10 g of magnesium chloride hexahydrate, and 10 g of sodium hexametaphosphate were added. The pH was adjusted to 5.0 with 5M NaOH. 1400 U of the enzyme solution of pyridoxal kinase pdxK mutant BL21(DE3) / pET29a-pdxK-L54I and 800 U of PpK kinase enzyme solution were added to make the volume up to 1L, and the reaction was started at 37 °C with stirring at 200 rpm. After 2 h of reaction, the concentration of VB6 was measured to be 0.19 g / L, and the conversion rate was 98.1%.

[0129] Example 5 Pyridoxal Kinase Mutant BL21(DE3) / pET-29a-pdxK-T57S Catalyzes the Synthesis of Pyridoxine Phosphate (Catalytic Reaction in 1L System)

[0130] In a 1L system, 10 g of VB6, 2 g of sodium ATP, 10 g of magnesium chloride hexahydrate, and 10 g of sodium metaphosphate were added. The pH was adjusted to 5.0 with 5M NaOH. 1400 U of the enzyme solution of pyridoxal kinase mutant BL21(DE3) / pET29a-pdxK-T57S and 800 U of the PpK kinase enzyme solution were added to make up to 1L, and the reaction was started with stirring at 37 °C and 200 rpm. After 2 h of reaction, the concentration of VB6 was measured to be 0.12 g / L, and the conversion rate was 98.8%.

[0131] Example 6 Pyridoxal Kinase Mutant BL21(DE3)pET29a-pdxK-L54I / T57S Catalyzes the Synthesis of Pyridoxine Phosphate (Catalytic Reaction in 1L System)

[0132] In a 1L system, 10 g of VB6, 2 g of sodium ATP, 10 g of magnesium chloride hexahydrate, and 10 g of sodium metaphosphate were added. The pH was adjusted to 5.0 with 5M NaOH. 1400 U of the enzyme solution of pyridoxal kinase mutant BL21(DE3)pET-29a-pdxK-L54I / T57S and 800 U of the PpK kinase enzyme solution were added to make up to 1L, and the reaction was started with stirring at 37 °C and 200 rpm. After 2 h of reaction, the concentration of VB6 was measured to be 0.02 g / L, and the conversion rate was 99.8%.

[0133] Since the double mutant pdxK-L54I / T57S has a higher conversion rate compared to the pdxK-L54I mutant and the pdxK-T57S mutant, the mutant pdxK-L54I / T57S was further selected for the catalytic reaction in a 10L system.

[0134] Example 7 Pyridoxal Kinase Mutant BL21(DE3)pET29a-pdxK-L54I / T57S Catalyzes the Synthesis of Pyridoxine Phosphate (Catalytic Reaction in 10L System)

[0135] In a 10L system, 100 g of VB6, 20 g of sodium ATP, 100 g of magnesium chloride hexahydrate, and 100 g of sodium metaphosphate were added. The pH was adjusted to 5.0 with 5M NaOH. 14000 U of the enzyme solution of the pyridoxal kinase mutant pdxK-L54I / T57S and 8000 U of the PpK kinase enzyme solution were added to make up to 10L, and the reaction was started with stirring at 37 °C and 200 rpm. After 2 h of reaction, the concentration of VB6 was measured to be 0.01 g / L, and the conversion rate was 99.9%. It shows that the reaction can be scaled up and can guide industrial production.

Claims

1. A pyridoxal kinase mutant, characterized in that, The mutant is obtained by mutating the amino acid sequence of wild-type pyridoxal kinase, and the mutation sites include at least one of the 23rd, 25th, 30th, 31st, 52nd, 54th, 57th, 58th, 59th, 96th, 125th, 127th, 130th, 136th, 157th, 158th, 159th, 230th, 233rd, 235th, 236th, 257th, 260th, and 264th amino acids. The amino acid sequence of the wild-type pyridoxal kinase is shown as SEQ:ID:NO:

1.

2. The pyridoxal kinase mutant according to claim 1, wherein The mutant includes at least one of the following mutations: a1: Based on the amino acid sequence shown in SEQ:ID:NO:1, mutate the Ser at its 23rd position to Thr; a2: Based on the amino acid sequence shown in SEQ:ID:NO:1, mutate the Ser at its 23rd position to Gly; a3: Based on the amino acid sequence shown in SEQ:ID:NO:1, mutate the Val at its 25th position to Asn; a4: Based on the amino acid sequence shown in SEQ:ID:NO:1, mutate the Val at its 25th position to Leu; a5: Based on the amino acid sequence shown in SEQ:ID:NO:1, mutate the Val at its 30th position to Gly; a6: Based on the amino acid sequence shown in SEQ:ID:NO:1, mutate the Gly at its 31st position to Ser; a7: Based on the amino acid sequence shown in SEQ:ID:NO:1, mutate the Val at its 52nd position to Gly; a8: Based on the amino acid sequence shown in SEQ:ID:NO:1, mutate the Val at its 52nd position to Ser; a9: Based on the amino acid sequence shown in SEQ:ID:NO:1, mutate the Leu at its 54th position to Ile; a10: Based on the amino acid sequence shown in SEQ:ID:NO:1, mutate the Leu at its 54th position to Thr; a11: Based on the amino acid sequence shown in SEQ:ID:NO:1, mutate the Thr at its 57th position to Ser; a12: Based on the amino acid sequence shown in SEQ:ID:NO:1, mutate the Thr at its 57th position to Pro; a13: Based on the amino acid sequence shown in SEQ:ID:NO:1, mutate the Pro at its 58th position to His; a14: Based on the amino acid sequence shown in SEQ:ID:NO:1, mutate the Pro at its 58th position to Arg; a15: Based on the amino acid sequence shown in SEQ:ID:NO:1, mutate the His at its 59th position to Arg; a16: Based on the amino acid sequence shown in SEQ:ID:NO:1, mutate the Tyr at its 96th position to Phe; a17: Based on the amino acid sequence shown in SEQ:ID:NO:1, Asp at position 125 is mutated to Leu; a18: Based on the amino acid sequence shown in SEQ:ID:NO:1, Val at position 127 is mutated to Gly; a19: Based on the amino acid sequence shown in SEQ:ID:NO:1, Asp at position 130 is mutated to Asn; a20: Based on the amino acid sequence shown in SEQ:ID:NO:1, Tyr at position 136 is mutated to Phe; a21: Based on the amino acid sequence shown in SEQ:ID:NO:1, Thr at position 157 is mutated to Ser; a22: Based on the amino acid sequence shown in SEQ:ID:NO:1, Pro at position 158 is mutated to His; a23: Based on the amino acid sequence shown in SEQ:ID:NO:1, Asn at position 159 is mutated to Arg; a24: Based on the amino acid sequence shown in SEQ:ID:NO:1, Gly at position 230 is mutated to Val; a25: Based on the amino acid sequence shown in SEQ:ID:NO:1, Asp at position 233 is mutated to Asn; a26: Based on the amino acid sequence shown in SEQ:ID:NO:1, Phe at position 235 is mutated to Tyr; a27: Based on the amino acid sequence shown in SEQ:ID:NO:1, Cys at position 236 is mutated to Ser; a28: Based on the amino acid sequence shown in SEQ:ID:NO:1, Gly at position 257 is mutated to Val; a29: Based on the amino acid sequence shown in SEQ:ID:NO:1, Val at position 260 is mutated to Gly; a30: Based on the amino acid sequence shown in SEQ:ID:NO:1, Met at position 264 is mutated to Thr.

3. The pyridoxal kinase mutant according to claim 1, wherein It is any of the following mutants: Mutant pdxK-L54I, which is obtained by mutating Leu at position 54 to Ile based on the amino acid sequence shown in SEQ:ID:NO:1, and its amino acid sequence is shown in SEQ:ID:NO:3; Mutant pdxK-L54T, which is obtained by mutating Leu at position 54 to Thr based on the amino acid sequence shown in SEQ:ID:NO:1, and its amino acid sequence is shown in SEQ:ID:NO:5; Mutant pdxK-T57S, which is obtained by mutating Thr at position 57 to Ser based on the amino acid sequence shown in SEQ:ID:NO:1, and its amino acid sequence is shown in SEQ:ID:NO:7; Mutant pdxK-T57P, which is obtained by mutating Thr at position 57 to Pro on the basis of the amino acid sequence shown in SEQ: ID: NO: 1, and its amino acid sequence is shown in SEQ: ID: NO: 9; Mutant pdxK-L54I / T57S, which is obtained by mutating Leu at position 54 to Ile and then mutating Thr at position 57 to Ser on the basis of the amino acid sequence shown in SEQ: ID: NO: 1, and its amino acid sequence is shown in SEQ: ID: NO:

11.

4. A biological material related to the pyridoxal kinase mutant according to any one of claims 1-3, characterized in that, The biological material is any one of B1) to B4): B1) A nucleic acid molecule encoding the pyridoxal kinase mutant according to any one of claims 1-3; B2) An expression cassette containing the nucleic acid molecule described in B1); B3) A recombinant expression vector containing the nucleic acid molecule described in B1), or a recombinant expression vector containing the expression cassette described in B2); B4) A recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3).

5. Use of the pyridoxal kinase mutant according to any one of claims 1-3 or the biological material according to claim 4 in the synthesis of pyridoxine phosphate.

6. The application according to claim 5, wherein Using VB6 as a substrate, successively adding magnesium salt, sodium ATP and sodium hexametaphosphate, stirring to dissolve, adjusting the pH to 5-6, adding the pyridoxal kinase mutant or its related biological material, adding polyphosphate kinase enzyme solution, making up the volume, and carrying out a catalytic reaction at 36-40 °C to generate pyridoxine phosphate.

7. The application according to claim 6, characterized in that, The mass ratio of VB6, magnesium salt, sodium ATP and sodium hexametaphosphate is 5:1:5:5; in each 1 liter of the synthesis system, 1400-5600 U of the pyridoxal kinase mutant enzyme solution and 800-1340 U of the polyphosphate kinase enzyme solution are added.

8. A method for preparing pyridoxine phosphate, characterized in that, It includes the following steps: using VB6 as a substrate and carrying out a catalytic reaction with the pyridoxal kinase mutant according to any one of claims 1-3 or the biological material according to claim 4 to obtain pyridoxine phosphate.

9. The method for preparing pyridoxine phosphate according to claim 8, characterized in that, The temperature of the catalytic reaction is 36-40 °C and the pH is 5-6.

10. A preparation for catalyzing the synthesis of pyridoxine phosphate from VB6, characterized in that, The preparation contains the pyridoxal kinase mutant according to any one of claims 1-3 or the biological material according to claim 4.

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