A pyridoxal kinase mutant, a recombinant expression vector and a microbial cell and their applications

Abstract

The present application relates to a pyridoxal kinase mutant, a recombinant expression vector and a microbial cell and their applications, and belongs to the technical field of biological catalysis. In order to solve the problem of low product concentration in existing enzyme catalysis, a pyridoxal kinase mutant is provided, the amino acid sequence is selected from the amino acid sequence shown in SEQ ID NO. 1, the lysine at position 229 is mutated to alanine, phenylalanine, methionine, arginine, threonine, histidine, serine, tyrosine, valine, leucine, isoleucine, proline, asparagine, aspartic acid or glutamic acid; the pyridoxal kinase mutant is used for catalyzing pyridoxal to synthesize pyridoxal phosphate, and a recombinant expression vector and a microbial cell can be further formed. The present application has good enzyme activity, high product conversion rate, high concentration of pyridoxal phosphate obtained, and the concentration of the product catalyzed by the wild-type pyridoxal kinase to phosphorylate pyridoxal is obviously improved.

Description

TECHNICAL FIELD

[0001] The present application relates to a pyridoxal kinase mutant, a recombinant expression vector and a microbial cell and their applications, and belongs to the technical field of biological catalysis. BACKGROUND

[0002] Pyridoxal phosphate is a very important biochemical substance in the metabolic process of organisms, which is the active form of vitamin B6 and also the coenzyme of many enzyme reactions, especially the enzyme reactions involving amino groups. Pyridoxal phosphate has various medical uses, such as the treatment of anemia, atherosclerosis and other diseases, and the prevention of cardiovascular and cerebrovascular diseases. Therefore, it has a wide application prospect.

[0003] At present, the synthesis of pyridoxal phosphate (PLP) can be achieved by chemical method or enzymatic phosphorylation of pyridoxal. The chemical method of pyridoxal phosphate requires far excessive phosphorus pentoxide and phosphoric acid, resulting in a large amount of high-concentration phosphorus-containing wastewater and high production cost. The enzymatic process mainly uses ATP as the phosphate donor, which is catalyzed by pyridoxal kinase, and the reaction can be carried out at room temperature. The process has high potential advantages, but the main product pyridoxal phosphate has a low concentration, which is the main reason affecting the industrial application of enzyme method PLP.

[0004] The technical level of enzymatic synthesis of pyridoxal phosphate in existing literature is relatively low. For example, a Chinese patent application (publication number: CN107236770A) reports that approximately 100 g / L of pyridoxal hydrochloride can be enzymatically reacted to produce pyridoxal phosphate. However, the document does not disclose the specific enzyme raw material used for pyridoxalase, and is therefore not of reference value. For example, the mutants of pyridoxal kinase and their applications disclosed in the Chinese patent document (publication number: CN117467641A) are also based on the wild-type amino acid sequence and are formed by site-directed mutation at at least position 135. The mutants are further disclosed to contain at least one of the following amino acid mutations: mutations of amino acids 29, 52, 57, 195, 209, 210, 220, 237, 261, and 277. However, more importantly, although the mutant is mentioned in the document, it can be seen from the full text that although the document mentions that the pyridoxal kinase has enzymatic activity in pyridoxal catalysis, However, it has enzymatic activity, which does not mean that the conversion rate of the catalytic synthesis of pyridoxal phosphate is high, nor does it necessarily mean that the product yield is high. Further research is needed. In fact, the document discloses a method in which pyridoxine is enzymatically catalyzed by the pyridoxal stimulator to generate pyridoxine phosphate, which is then further oxidized to generate pyridoxal phosphate. In essence, pyridoxal phosphate is synthesized by a combined enzymatic and chemical synthesis method. The conversion ability of pyridoxal to pyridoxal phosphate and the product yield are not disclosed. In addition, the document discloses that the effect on enzyme activity is better through simultaneous mutation of multiple amino acids, which are all aimed at the synthesis of pyridoxine phosphate. Summary of the Invention

[0005] The present invention addresses the defects in the above-mentioned prior art and provides a pyridoxal kinase mutant, a recombinant expression vector, a microbial cell and their applications, and solves the problem of low conversion rate of pyridoxal phosphorylation products catalyzed by the existing pyridoxal kinase by providing a new mutant enzyme.

[0006] The object of the present invention is achieved by the following technical solution: a pyridoxal kinase mutant, characterized in that the amino acid sequence of the pyridoxal kinase mutant is selected from the amino acid sequence shown in SEQ ID NO.1, and the 229th lysine is mutated to alanine, phenylalanine, methionine, arginine, threonine, histidine, serine, tyrosine, valine, leucine, isoleucine, proline, asparagine, aspartic acid or glutamate; the pyridoxal kinase mutant is used to catalyze the synthesis of pyridoxal phosphate from pyridoxal.

[0007] The present invention is in the actual research and development process, find that although the catalytic efficiency may be reduced after the wild-type pyridoxal kinase amino acid mutation at position 229, the above-mentioned pyridoxal kinase mutant can greatly improve the product concentration of pyridoxal catalyzed synthesis of pyridoxal phosphate, with a high quality effect of product concentration, with high product yield. Specifically, the present invention is by selectively mutating position 229 in the amino acid sequence of wild-type pyridoxal kinase so that it is mutated into the above-mentioned amino acid, all with good enzymatic activity, more importantly, this mutant not only has good enzyme activity, and can directly carry out enzymatic phosphorylation of pyridoxal and convert it into pyridoxal phosphate, with high product conversion rate, the concentration of the obtained pyridoxal phosphate is high, compared to the concentration of the product of pyridoxal kinase catalyzed pyridoxal phosphorylation of wild-type is significantly improved; At the same time, the mutant of the present invention only needs to mutate position 229 of the above-mentioned amino acid sequence as shown in SEQ ID NO.1, without highlighting other positions, it is possible to effectively convert pyridoxal directly into pyridoxal phosphate by enzymatic catalysis, with a specific high selectivity effect.

[0008] The description of other amino acids after the above-mentioned lysine (K) variant at position 229 can be expressed in the following manner:

[0009] The mutation of lysine at position 229 to alanine can be described as K229A;

[0010] The mutation of lysine at position 229 to phenylalanine can be described as K229F;

[0011] The mutation of lysine at position 229 to methionine can be described as K229M;

[0012] The mutation of lysine at position 229 to arginine can be described as K229R;

[0013] The mutation of lysine at position 229 to threonine can be described as K229T;

[0014] The mutation of lysine at position 229 to histidine can be described as K229H;

[0015] The mutation of lysine at position 229 to serine can be described as K229S;

[0016] The mutation of lysine at position 229 to tyrosine can be described as K229Y;

[0017] The mutation of lysine at position 229 to valine can be described as K229V;

[0018] The mutation of lysine at position 229 to leucine can be described as K229L;

[0019] The mutation of lysine at position 229 to isoleucine can be described as K229I;

[0020] The mutation of lysine at position 229 to proline can be described as K229P;

[0021] The mutation of lysine at position 229 to asparagine can be described as K229N;

[0022] The mutation of lysine at position 229 to aspartic acid can be described as K229D;

[0023] The mutation of lysine at position 229 to glutamic acid can be described as K229E.

[0024] The preferred pyridoxal kinase mutant can further improve the concentration of the product of pyridoxal phosphorylation catalyzed by the wild-type pyridoxal kinase, thereby increasing the concentration of the product pyridoxal phosphate to 2 times or more.

[0025] In order to better improve the conversion ability of the selected pyridoxal kinase mutant to catalyze the phosphorylation of pyridoxal and achieve a high product concentration, as a further preferred embodiment, the amino acid sequence of the pyridoxal kinase mutant is such as the amino acid sequence shown in SEQ ID NO.1, and the 229th lysine is mutated to alanine, phenylalanine, histidine, serine or tyrosine.

[0026] That is, the corresponding mutation of lysine at position 229 to alanine can be described as K229A;

[0027] The mutation of lysine at position 229 to phenylalanine can be described as K229F;

[0028] The mutation of lysine at position 229 to histidine can be described as K229H;

[0029] The mutation of lysine at position 229 to serine can be described as K229S;

[0030] The mutation of lysine at position 229 to tyrosine can be described as K229Y.

[0031] In the above-mentioned pyridoxal kinase mutant, the pyridoxal kinase mutant is formed by mutating lysine at position 229 of the amino acid sequence derived from Escherichia coli, the amino acid sequence of which is shown in SEQ ID NO.1, and the nucleotide sequence of which is shown in SEQ ID NO.2. Using Escherichia coli as the basis for mutation is equivalent to mutating on the basis of wild-type pyridoxal kinase, which has better mutability, and mutating on the basis of the amino acid sequence, ultimately only a single mutation at position 229 is required to achieve the high enzymatic catalytic ability of the obtained mutant for pyridoxal, and specifically a high concentration of the product of pyridoxal phosphate. It can also more directly illustrate that the mutant of the present invention has a much better catalytic conversion ability for the product of pyridoxal phosphorylation than the wild-type enzyme, achieving better application value.

[0032] The second object of the present invention is achieved by the following technical solutions: a recombinant expression vector, characterized in that the recombinant expression vector carries the above-mentioned pyridoxal kinase mutant. Since the above-mentioned pyridoxal kinase mutant of the present invention has excellent enzymatic activity and the ability to catalyze pyridoxal to be directly converted into pyridoxal phosphate, and has the advantage of a high product yield of pyridoxal phosphate, the recombinant expression vector carrying the above-mentioned pyridoxal kinase mutant can also achieve the above-mentioned effect. Various expression vectors can be used for the selection of recombinant expression vectors, including but not limited to the following PET expression vectors, PUC expression vectors, etc., which can all be achieved. However, in order to better realize the above-mentioned expression vector, as further preferred, the recombinant expression vector uses PET-30a as an expression vector.

[0033] The third object of the present invention is achieved by the following technical solution: a microbial cell, wherein the microbial cell uses a microorganism as an expression host, characterized in that the microorganism carries a pyridoxal kinase mutant or carries a recombinant expression vector containing the pyridoxal kinase mutant, the amino acid sequence of the pyridoxal kinase mutant is selected from the amino acid sequence shown in SEQ ID NO.1, in which lysine at position 229 is mutated to alanine, phenylalanine, methionine, arginine, threonine, histidine, serine, tyrosine, valine, leucine, isoleucine, proline, asparagine, aspartic acid or glutamate; the pyridoxal kinase mutant is used to catalyze the synthesis of pyridoxal phosphate from pyridoxal.

[0034] Similarly, because the pyridoxal kinase mutant of the present invention has excellent enzymatic activity and the ability to catalyze the direct conversion of pyridoxal into pyridoxal phosphate, and has the advantage of a high concentration of the pyridoxal phosphate product, microbial cells carrying the pyridoxal kinase mutant or recombinant expression vector can also achieve the above-mentioned effects, with the effect of efficiently catalyzing pyridoxal to synthesize pyridoxal phosphate. The microorganisms of the above-mentioned expression host can be genetically engineered bacteria, such as microorganisms such as Escherichia coli and Bacillus subtilis. As a further preferred embodiment, the microorganism is Escherichia coli.

[0035] In the above-mentioned microbial cells, preferably, the pyridoxal kinase mutant is obtained by mutating lysine 229 of the amino acid sequence derived from Escherichia coli, the amino acid sequence of Escherichia coli is shown in SEQ ID NO.1, and the nucleotide sequence thereof is shown in SEQ ID NO.2.

[0036] The fourth object of the present invention is achieved by the following technical solution: a pyridoxal kinase mutant, a recombinant expression vector, and a microbial cell, wherein under the action of pyridoxal kinase, the substrate pyridoxal or pyridoxal acid salt is phosphorylated to prepare pyridoxal phosphate, characterized in that the pyridoxal kinase is selected from one or more of the above-mentioned pyridoxal kinase mutant, the recombinant expression vector, and the above-mentioned microbial cell. Since the pyridoxal kinase mutant of the present invention is efficient in enzymatically converting pyridoxal into pyridoxal phosphate, the expression vector or microorganism carrying the above-mentioned pyridoxal kinase mutant can also achieve the performance of efficiently phosphorylating pyridoxal, and also has the effect of high product concentration. Compared with the enzymatic conversion ability of the wild type, the concentration of pyridoxal phosphate can be increased to more than 2 times.

[0037] In the applications of the above-mentioned pyridoxal kinase mutant, recombinant expression vector and microbial cells, preferably, the specific preparation method of the pyridoxal phosphate is:

[0038] In the reaction system of pyridoxal hydrochloride, magnesium salt, ATP, pyridoxal kinase, acetate kinase and ACP, a reaction is carried out to prepare pyridoxal phosphate. The raw materials such as the above-mentioned acetate kinase, ATP (adenosine triphosphate disodium salt), and ACP (acetyl phosphate diammonium salt) can be directly purchased. Pyridoxal kinase, i.e., the above-mentioned pyridoxal kinase mutant, recombinant expression vector and microbial cells, are introduced into an ATP regeneration system and the regeneration of ADP to ATP is achieved with relatively cheap acetyl phosphate, so that the reaction is carried out more efficiently and the yield of the product is better guaranteed. The concentration of the obtained pyridoxal phosphate is high, which is more conducive to industrial production. Furthermore, although some mutants may be reduced in catalytic speed after the amino acid at position 229 of the wild-type pyridoxal kinase is mutated in the present invention, the inhibitory effect of high concentrations of substrate and product on the catalytic activity of the enzyme can be avoided; combined with the use of an ATP regeneration system to change the equilibrium state of the reaction from the reaction kinetics, the above-mentioned pyridoxal kinase mutant can be better enabled to break through the self-inhibition of pyridoxal and pyridoxal phosphate for the enzymatic reaction, and finally catalyze and synthesize the effect of obtaining a high concentration of pyridoxal phosphate product. The magnesium salt is preferably selected from magnesium sulfate or magnesium chloride, and magnesium sulfate may be, for example, magnesium sulfate heptahydrate. The reaction is preferably carried out at a pH of 5-6. The pH of the system may be adjusted using an alkali metal hydroxide, such as an aqueous hydroxide solution.

[0039] In the applications of the above-mentioned pyridoxal kinase mutant, recombinant expression vector and microbial cells, preferably, the molar ratio of pyridoxal:ATP:ACP:magnesium salt is 1:0.05-0.5:1.0-6.0:0.01-0.20.

[0040] Here, the corresponding amounts of pyridoxal kinase and acetate kinase can be added in catalytic amounts. For example, while ensuring that the reaction is carried out under enzyme activity conditions, it is best to make the weight ratio of pyridoxal: pyridoxal kinase enzyme solution: acetate kinase enzyme solution be 1:0.2-5.0:0.1-5.0.

[0041] In the applications of the above-mentioned pyridoxal kinase mutant, recombinant expression vector and microbial cells, preferably, the reaction temperature is controlled to be carried out at 30°C-37°C.

[0042] The above reaction equation of pyridoxal phosphate can be expressed as follows:

[0043]

[0044] In summary, the present invention has the following advantages compared with the prior art:

[0045] 1. The pyridoxal kinase mutant of the present invention can directly enzymatically phosphorylate pyridoxal to convert it into pyridoxal phosphate, with a high product conversion rate. Compared with the wild-type pyridoxal kinase-catalyzed pyridoxal phosphorylation, the product concentration is significantly improved.

[0046] 2. The present invention only requires mutation of position 229 of the amino acid sequence shown in SEQ ID NO. 1, without mutating other positions, to effectively convert pyridoxal directly into pyridoxal phosphate through enzymatic catalysis, with a high selectivity and a high concentration of the obtained pyridoxal phosphate. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 It is the SDS-PAGE image of protein expression of wild-type PdxK and pyridoxal kinase mutant K229F.

[0048] Figure 2 This is a reaction process curve diagram of the synthesis of PLP catalyzed by wild-type PdxK.

[0049] Figure 3 This is a reaction process curve diagram of the synthesis of PLP catalyzed by the pyridoxal kinase mutant K229F.

[0050] Figure 1 In the figure, 1. Total protein after induction with wild-type PdxK; 2. Supernatant after induction with wild-type PdxK; 3. Precipitate after induction with wild-type PdxK; 4. Total protein after induction with pyridoxal kinase mutant K229F; 5. Supernatant after induction with pyridoxal kinase mutant K229F; 6. Precipitate after induction with mutant K229F. DETAILED DESCRIPTION

[0051] The technical solution of the present invention will be further specifically described below through specific embodiments and drawings, but the present invention is not limited to these embodiments.

[0052] Example 1

[0053] 1L shake flask fermentation and enzyme activity determination method of wild-type pyridoxal kinase (PdxK) engineered bacteria

[0054] PdxK wild-type glycerol bacteria were inoculated into 5 mL LB liquid medium containing a final concentration of 50 μg / mL kanamycin at an inoculation amount of 0.1% v / v, and cultured at 37°C, 200 rpm for 12 h. Then, 5 mL of the liquid culture was inoculated into 200 mL TB liquid medium containing a final concentration of 50 μg / mL kanamycin at an inoculation amount of 1% v / v, and cultured at 37°C, 200 rpm for 4-6 h until the OD value reached about 0.8. Then, 1 mM IPTG was added for induction at 25°C for 12 h. The wet bacteria were collected by centrifugation at 4°C, 8000 rpm for 10 min, and the supernatant was discarded. The wet bacteria were further broken by an ultrasonic cell crusher to obtain a cell lysate, i.e., a PdxK crude enzyme solution.

[0055] The amino acid sequence of the wild-type PdxK derived from Escherichia coli is shown in SEQ ID NO. 1, and the nucleotide sequence is shown in SEQ ID NO. 2.

[0056] Example 2

[0057] PdxK mutant cloning process

[0058] Primers were designed, and the gene template pET-30a(+) of the expression vector pET30(a)+ was downloaded from the SnapGene website. The PdxK sequence was inserted into the vector through two enzyme cutting sites of "Hind III" and "Nde I". The 229th amino acid (K229) in the target sequence was mutated, and the primer sequence with a length of 30-36 bp and a Tm value of 55-65°C was selected. The designed primers were synthesized according to the conventional method, and the obtained mutant primers are shown in Table 1:

[0059] Table 1:

[0060]

[0061]

[0062] The amplification conditions were as follows: initial denaturation at 95°C for 3 min, denaturation at 95°C for 30 sec, annealing at 59°C for 30 sec, extension at 72°C for 45 s (short fragment) / 3 min (long fragment), for a total of 30 cycles, and finally extension at 72°C for 10 min.

[0063] After the reaction is complete, the PCR amplification product is detected by 1% agarose gel electrophoresis to obtain the target band that meets the expected results. According to the standard operation of the kit, the target fragment is recovered and purified. Subsequently, the target gene fragment and the vector are ligated using Exnase II ligase to obtain the ligation product recombinant expression vector. The resulting ligation product is transformed into E. coli BL21 (DE3) competent cells. The transformed cells are spread on LB solid plates containing 50μg / mL kanamycin. Several well-grown single colonies are picked and inoculated into LB liquid medium containing a final concentration of 50μg / mL kanamycin. They are cultured at 37°C, 200rpm for 12-16 hours, and glycerol strains are prepared and stored at -80°C for use. The remaining culture is used to extract the plasmid and send it for sequencing. The results show that the cloned K229 gene sequence is correct and has been correctly inserted into the pET30a plasmid, resulting in the recombinant plasmid pET30a-LysX (the corresponding recombinant expression vector). Plasmids with correct sequencing results are stored in a -20°C refrigerator for use.

[0064] If further preparation of microorganisms is required, the above-mentioned recombinant plasmid pET30a-LysX can be introduced into Escherichia coli through genetic engineering to obtain the corresponding recombinant microorganisms. The specific processing method of genetic engineering here can be conventional.

[0065] Example 3

[0066] In order to determine the enzymatic activity of the pyridoxal kinase mutant, the corresponding pyridoxal kinase mutant obtained by the method of Example 2 was selected, that is, the lysine at position 229 was substituted with the corresponding amino acid to obtain the pyridoxal kinase mutants shown below, specifically corresponding to K229A, K229C, K229D, K229E, K229F, K229H, K229I, K229L, K229M, K229N, K229P, K229R, K229S, K229T, K229V, K229G, K229W, and K229Y.

[0067] Enzyme activity assay for wild-type PdxK and pyridoxal kinase mutants:

[0068] Take 3 mL of a reaction solution containing 20 g / L pyridoxal hydrochloride, 10 g / L adenosine triphosphate disodium salt, 30 g / L acetyl phosphate diammonium salt, and 2 g / L magnesium sulfate heptahydrate. Stir until dissolved, adjust the pH to 5.0 with 10 M sodium hydroxide solution, add 0.2 mL of acetate kinase (the supernatant after disruption) and 0.2 mL of crude enzyme solution (the supernatant after disruption), and shake the reaction solution in a water bath at 35°C and 200 rpm. After 15 minutes, sample the solution and boil for 5 minutes to terminate the reaction. The concentration of pyridoxal phosphate in the reaction solution is determined by HPLC.

[0069] The crude enzyme solution corresponds to wild-type PdxK crude enzyme solution or pyridoxal kinase mutant crude enzyme solution respectively.

[0070] The enzyme activity is defined as the amount of enzyme required to catalyze the production of 1 μmol of pyridoxal phosphate per minute under the reaction condition of 35°C.

[0071] Comparison of enzyme activity of wild-type PdxK and pyridoxal kinase mutant

[0072] The genetically engineered bacteria of the pyridoxal kinase mutant (PL kinase mutant) gene successfully constructed in Example 2 were cultured by the method of Example 1 to obtain the bacterial bodies, and after being broken, the corresponding crude enzyme solution of the pyridoxal kinase mutant was obtained, and data analysis was performed. The enzyme activity data analysis results of each pyridoxal mutant are shown in Table 2.

[0073] Table 2:

[0074]

[0075]

[0076] The enzyme activity data results of the mutants in Table 1 above show that by mutating the lysine at position 229, the enzyme activity of not all mutants is better than that of the wild type, and the enzyme activity of some mutants is not as good as that of the wild type. The pyridoxal mutants of the present application can all achieve the performance of improving enzyme activity, and are also much better than the enzyme activity level of the wild type, but the specific enzyme activity cannot necessarily achieve the effect of converting to pyridoxal phosphate with high concentration.

[0077] At the same time, the wild-type PdxK and K229F were specifically selected for enzyme protein expression analysis and comparison, and the specific analysis results are shown in Table 3. Figure 1 The results show that the proteins of the mutant and the wild type are highly expressed, and the solubility of the proteins is good.

[0078] Example 4

[0079] The wild-type PdxK genetically engineered bacteria and acetic acid kinase engineered bacteria (existing acetic acid kinase) were fermented, and the obtained crude enzyme solution after ultrasonic disruption was used as a catalyst for the reaction process and result analysis of preparing pyridoxal phosphate.

[0080] Use 2.05g pyridoxal hydrochloride and 2.35g acetyl phosphate diammonium salt as substrates, add 0.2g magnesium sulfate heptahydrate and 1.4g adenosine triphosphate disodium salt, add 100mL water to dissolve, adjust the reaction pH to 5.0 with 10M NaOH, add 2g PdxK crude enzyme solution and 2g acetate kinase crude enzyme solution, and use 3M NaOH solution to control the reaction at pH 5.0 and 35°C. During the reaction, 0.5g acetyl phosphate diammonium salt (ACP) was added every 1h, and samples were taken every half hour for centrifugation and the concentration of the product was determined by HPLC. The reaction finally generated 22.18g / L pyridoxal phosphate in 4h. The concentration of the products during the reaction was analyzed as follows. Figure 2 shown.

[0081] Example 5

[0082] The reaction process and result analysis of the pyridoxal kinase mutant as an enzyme-catalyzed catalyst are shown in FIG. 1 . This example takes the reaction of the K229F mutant as an example.

[0083] The total volume of the reaction solution is 20 mL, and it contains 1.00 g of pyridoxal hydrochloride, 0.28 g of adenosine triphosphate disodium salt, 0.80 g of acetyl phosphate diammonium salt, and 0.04 g of magnesium sulfate heptahydrate. It is loaded into a 50 mL round-bottom reaction tube, and 15.80 mL of purified water is added. After stirring until dissolved, 10 M sodium hydroxide is used to adjust the pH to 5.0, and then 0.50 g of acetate kinase and 1.00 g of mutant K229F crude enzyme solution are added. The reaction is carried out at 35 ° C, pH = 5.0, and 200 rpm in a water bath shaker. Samples are taken every half hour to detect changes in the concentration of pyridoxal phosphate by HPLC, and 0.2 g of acetyl phosphate diammonium salt is added every half hour. The liquid phase detection results show that mutant K229F catalyzes the generation of 58.87 g / L of pyridoxal phosphate in 3.5 h. The concentration of the products in the reaction process is analyzed as follows. Figure 3 shown.

[0084] Example 6

[0085] Summary of reaction results of other pyridoxal kinase mutants

[0086] Other pyridoxal kinase mutant engineered bacteria were fermented and crushed in 1L shake flasks to obtain crude enzyme liquid, which was used as a catalyst together with acetate kinase.

[0087] The enzyme-catalyzed reaction was carried out using pyridoxal hydrochloride as the substrate and acetyl phosphate diammonium salt as the cosubstrate. The reaction solution had a pH of 5.0 and contained 20 mL of pyridoxal hydrochloride (1.00 g), adenosine triphosphate disodium salt (0.28 g), acetyl phosphate diammonium salt (0.80 g), and magnesium sulfate heptahydrate (0.04 g). The reaction was stirred in a 35°C water bath at 200 rpm. HPLC was used to monitor the concentration of the product, pyridoxal phosphate, during the reaction. The final product concentration was shown in Table 3 below.

[0088] Table 3:

[0089]

[0090] Combined with the corresponding reaction process tracking analysis in Examples 4 and 5 above, and the level of reaction product concentration reached in this example, Figure 2 and Figure 3 When the wild type and the pyridoxal kinase mutant of the present invention are used to catalyze the phosphorylation of pyridoxal to synthesize pyridoxal phosphate, the combination Figure 2 The concentration of pyridoxal phosphate obtained during the catalytic conversion reaction of the corresponding wild type can be clearly seen. It can be seen that the concentration of the product pyridoxal phosphate obtained by enzyme catalysis using the mutant of the present invention is much higher than the catalytic conversion ability of the wild type, and some can even be increased to a concentration level of more than 2 times. Combined with the data analysis in Table 3 above, it can be seen that among the mutants of the present invention, only K229C, K229W and K229G have low catalytic efficiency, and the remaining mutants all show good application value.

[0091] The specific embodiments described herein are merely illustrative of the spirit of the present invention. Persons skilled in the art may make various modifications, additions, or substitutions to the described embodiments without departing from the spirit of the present invention or exceeding the scope of the appended claims.

[0092] Although the present invention has been described in detail and certain specific embodiments have been cited, it will be apparent to those skilled in the art that various changes and modifications can be made without departing from the spirit and scope of the invention.

Claims

1. A pyridoxal kinase mutant, characterized in that The amino acid sequence of the pyridoxal kinase mutant is selected from the amino acid sequence shown in SEQ ID NO.1, in which the 229th lysine is mutated to alanine, phenylalanine, arginine, threonine, histidine, serine, tyrosine, valine, leucine, isoleucine, proline, aspartic acid or glutamate; the pyridoxal kinase mutant is used to catalyze the synthesis of pyridoxal phosphate from pyridoxal.

2. The pyridoxal kinase mutant according to claim 1, characterized in that The pyridoxal kinase mutant is obtained by mutating lysine at position 229 of the amino acid sequence derived from Escherichia coli. The amino acid sequence of the Escherichia coli is shown in SEQ ID NO.1, and the nucleotide sequence thereof is shown in SEQ ID NO.

2.

3. A recombinant expression vector, characterized in that: The recombinant expression vector carries the pyridoxal kinase mutant according to claim 1 or 2.

4. A microbial cell, wherein the microbial cell uses a microorganism as an expression host, characterized in that: The microorganism carries a pyridoxal kinase mutant or a recombinant expression vector containing the pyridoxal kinase mutant, wherein the amino acid sequence of the pyridoxal kinase mutant is selected from the amino acid sequence shown in SEQ ID NO.1, in which the 229th lysine is mutated to alanine, phenylalanine, arginine, threonine, histidine, serine, tyrosine, valine, leucine, isoleucine, proline, aspartic acid or glutamic acid; and the pyridoxal kinase mutant is used to catalyze the synthesis of pyridoxal phosphate from pyridoxal.

5. The microbial cell according to claim 4, characterized in that The microorganism is Escherichia coli.

6. The microbial cell according to claim 4 or 5, characterized in that The pyridoxal kinase mutant is obtained by mutating lysine at position 229 of the amino acid sequence derived from Escherichia coli. The amino acid sequence of the Escherichia coli is shown in SEQ ID NO.1, and the nucleotide sequence thereof is shown in SEQ ID NO.

2.

7. A use of a pyridoxal kinase mutant, a recombinant expression vector, and a microbial cell for phosphorylating pyridoxal hydrochloride to prepare pyridoxal phosphate under the action of pyridoxal kinase and acetate kinase, characterized in that: The pyridoxal kinase mutant is selected from the pyridoxal kinase mutant according to claim 1 or 2; the recombinant expression vector is selected from the recombinant expression vector according to claim 3; and the microbial cell is selected from the microbial cell according to any one of claims 4-6.

8. The use of the pyridoxal kinase mutant, recombinant expression vector and microbial cells according to claim 7, characterized in that: The specific preparation method of the pyridoxal phosphate is: Pyridoxal phosphate is prepared by reacting in a reaction system of pyridoxal hydrochloride, magnesium salt, ATP, pyridoxal kinase, acetate kinase and ACP.

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