Pyridoxal kinase mutant, recombinant expression vector, microbial cell and application of pyridoxal kinase mutant, recombinant expression vector and microbial cell

Abstract

The invention relates to a pyridoxal kinase mutant, a recombinant expression vector, a microbial cell and application thereof, and belongs to the technical field of biological catalysis. In order to solve the problem of low concentration of the existing enzyme catalysis product, the invention provides the pyridoxal kinase mutant. The amino acid sequence is selected from lysine at the 229 site of the amino acid sequence as shown in SEQ ID NO.1, and the lysine at the 229 site of the amino acid sequence as shown in SEQ ID NO.1 is mutated into alanine, phenylalanine, methionine, arginine, threonine, histidine, serine, tyrosine, valine, leucine, isoleucine, proline, asparagine, asparaginic acid or glutamic acid; the pyridoxal kinase mutant is used for catalyzing pyridoxal to synthesize pyridoxal phosphate, and recombinant expression vectors, microbial cells and the like can be further formed. The pyridoxal phosphate has good enzyme activity and high product conversion rate, the concentration of the obtained pyridoxal phosphate is high, and the concentration of a product obtained by catalyzing phosphorylation of pyridoxal through the pyridoxal phosphate kinase is obviously improved compared with that of a product obtained by catalyzing phosphorylation of pyridoxal through wild type pyridoxal kinase.

Description

Technical Field

[0001] The invention relates to a pyridoxal kinase mutant, a recombinant expression vector and a microbial cell and applications thereof, belonging to the technical field of biocatalysis. Background Art

[0002] Pyridoxal phosphate is a very important biochemical substance in the metabolic process of organisms. It is the active form of vitamin B6 and a coenzyme for many enzymatic reactions, especially those involving amino groups. Pyridoxal phosphate also has many uses in medicine. It can be used to treat anemia, atherosclerosis and other diseases, as well as prevent cardiovascular and cerebrovascular diseases. Therefore, it has broad application prospects.

[0003] At present, the synthesis of pyridoxal phosphate (PLP) can be carried out by phosphorylating pyridoxal by chemical method or enzymatic phosphorylation. The chemical method of pyridoxal phosphate requires a far excess of phosphorus pentoxide and phosphoric acid, which produces a large amount of highly concentrated phosphorus-containing wastewater, resulting in high production costs. The enzyme-catalyzed process mainly uses ATP as a phosphate donor and is catalyzed by pyridoxal kinase. The reaction can be carried out at room temperature and the process has high potential advantages. However, the concentration of the main product pyridoxal phosphate is low, which is the main reason affecting the industrial application of enzymatic 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 about 100 g / L of pyridoxal hydrochloride can be enzymatically reacted to form pyridoxal phosphate. However, the document does not disclose the specific enzyme raw material of the pyridoxal enzyme used, and is not of reference value. For example, in the mutants of pyridoxal kinase and their applications disclosed in the Chinese patent document (publication number: CN117467641A), the mutants of pyridoxal kinase mentioned are also based on the wild-type amino acid sequence at least 135 position for site-directed mutation, and further disclosed to contain at least one of the following amino acid mutations: 29, 52, 57, 195, 209, 210, 220, 237, 261, 277 amino acid mutations, but more importantly, although the document mentions the mutants, 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 enzyme activity, which does not mean that the conversion rate of catalyzing the synthesis of pyridoxal phosphate is high, nor does it necessarily mean that the product yield is high. Instead, further research is needed. In fact, the document discloses that pyridoxine is 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, it is disclosed that the simultaneous mutation of multiple amino acids has a better effect on the enzyme activity, which is also aimed at the synthesis of pyridoxine phosphate. Summary of the invention

[0005] The present invention aims at the defects existing in the above prior art, provides a pyridoxal kinase mutant, a recombinant expression vector and a microbial cell and their applications, and solves the problem of low conversion rate of pyridoxal phosphorylation product 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 scheme: 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, 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] In the actual research and development process, the present invention found that although the catalytic efficiency may be reduced after the 229th amino acid mutation of the wild-type pyridoxal kinase, the above-mentioned pyridoxal kinase mutant can greatly improve the product concentration of pyridoxal catalyzed synthesis of pyridoxal phosphate, with a high product concentration quality effect, with a high product yield. Specifically, the present invention selectively mutates the 229th position in the amino acid sequence of the wild-type pyridoxal kinase to the above-mentioned amino acid, and has good enzyme activity. More importantly, this mutant not only has good enzyme activity, but also can directly carry out enzyme-catalyzed phosphorylation of pyridoxal and convert it into pyridoxal phosphate, with a high product conversion rate, and the concentration of the obtained pyridoxal phosphate is high, which is significantly improved compared to the concentration of the product of the wild-type pyridoxal kinase catalyzed pyridoxal phosphorylation; At the same time, the mutant of the present invention only needs to mutate the 229th position of the amino acid sequence shown in SEQ ID NO.1, and no other positions need to be highlighted, so that pyridoxal can be effectively directly converted into pyridoxal phosphate by enzyme 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 lysine at position 229 is mutated to alanine, which can be described as K229A;

[0010] The lysine at position 229 is mutated to phenylalanine, which can be described as K229F;

[0011] The lysine at position 229 is mutated to methionine, which can be described as K229M;

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

[0013] The lysine at position 229 is mutated to threonine, which can be described as K229T;

[0014] The lysine at position 229 is mutated to histidine, which can be described as K229H;

[0015] The lysine at position 229 is mutated to serine, which can be described as K229S;

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

[0017] The lysine at position 229 is mutated to valine, which can be described as K229V;

[0018] The lysine at position 229 is mutated to leucine, which 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 lysine at position 229 is mutated to asparagine, which 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] By using the above preferred pyridoxal kinase mutant, the concentration of the product of pyridoxal phosphorylation catalyzed by the wild-type pyridoxal kinase can be further improved, so that the concentration of the product pyridoxal phosphate is increased to a level of 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 preference, 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 lysine at position 229 is mutated to phenylalanine, which can be described as K229F;

[0028] The lysine at position 229 is mutated to histidine, which can be described as K229H;

[0029] The lysine at position 229 is mutated to serine, which 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 the 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 its nucleotide sequence is shown in SEQ ID NO.2. Using Escherichia coli as the basis for mutation is equivalent to mutating on the basis of the wild-type pyridoxal kinase, which has better mutability, and mutating on the basis of the amino acid sequence, and finally only needing to mutate a single site at position 229, the obtained mutant can achieve the high enzyme catalytic ability of pyridoxal, and the specific product concentration of pyridoxal phosphate is high. It can also be more directly explained that the mutant of the present invention has a far superior catalytic conversion ability of the wild-type enzyme to the product conversion rate of phosphorylating pyridoxal, and achieves 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. Because the above-mentioned pyridoxal kinase mutant of the present invention has excellent enzyme activity and the ability of catalyzing pyridoxal to be directly converted into pyridoxal phosphate, and has the advantage of 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 vector, including but not limited to the following PET expression vector, PUC expression vector and other expression vectors can be realized. But in order to better realize the above-mentioned expression vector, as further preferred, the recombinant expression vector is with PET-30a as expression vector.

[0033] The third object of the present invention is achieved by the following technical scheme: 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, and 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, 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 above-mentioned pyridoxal kinase mutant of the present invention has excellent enzyme activity and the ability of catalyzing pyridoxal to directly convert into pyridoxal phosphate, and has the advantage of high product concentration of pyridoxal phosphate, the microbial cells carrying the above-mentioned pyridoxal kinase mutant or recombinant expression vector can also achieve the above-mentioned effect, and have the effect of efficiently catalyzing pyridoxal to synthesize pyridoxal phosphate. Genetically engineered bacteria, such as microorganisms such as Escherichia coli and Bacillus subtilis, can be used for the microorganism of the above-mentioned expression host. As further preferred, the microorganism is Escherichia coli.

[0035] In the above-mentioned microbial cells, preferably, 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 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 scheme, a pyridoxal kinase mutant, a recombinant expression vector and a microbial cell application, 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, therefore, 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 enzyme catalytic conversion ability of the wild type, the concentration of pyridoxal phosphate can be increased to more than 2 times.

[0037] In the application of the above-mentioned pyridoxal kinase mutant, recombinant expression vector and microbial cell, 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, pyridoxal phosphate is reacted to prepare pyridoxal. The above-mentioned acetate kinase, ATP (adenosine triphosphate disodium salt), ACP (acetyl phosphate diammonium salt) and other raw materials can be directly purchased, and pyridoxal kinase, i.e., the above-mentioned pyridoxal kinase mutant, recombinant expression vector and microbial cells, can be introduced into the ATP regeneration system and realize the regeneration of ADP to ATP with relatively cheap acetyl phosphate, so that the reaction can be carried out more efficiently, and the yield of the product can be better guaranteed, and the concentration of the obtained pyridoxal phosphate is high, which is more conducive to industrial production. Further, although some mutants may be reduced in catalytic speed after the amino acid mutation of the wild-type pyridoxal kinase at position 229 in the present invention, the inhibitory effect of high-concentration substrates and products on the catalytic activity of the enzyme can be avoided; combined with the use of the ATP regeneration system, the equilibrium state of the reaction is changed from the reaction kinetics, so that the above-mentioned pyridoxal kinase mutant can better 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 pyridoxal phosphate product. It is preferred that the magnesium salt be selected from magnesium sulfate or magnesium chloride, and magnesium sulfate may be magnesium sulfate heptahydrate, etc. It is preferred that the reaction be carried out at a pH of 5-6, and the pH of the system may be adjusted by using an alkali metal hydroxide, such as an aqueous solution of a hydroxide.

[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 at 30°C-37°C.

[0042] The above reaction equation of pyridoxal phosphate can be expressed in the following way:

[0043]

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

[0045] 1. The pyridoxal kinase mutant of the present invention can directly perform enzymatic phosphorylation on pyridoxal to convert it into pyridoxal phosphate, and has the effect of high product conversion rate. Compared with the wild-type pyridoxal kinase, the product concentration of pyridoxal phosphorylation catalyzed by pyridoxal kinase is significantly improved.

[0046] 2. The present invention only needs to mutate the 229th position of the amino acid sequence shown in SEQ ID NO.1, without mutating other positions, so that pyridoxal can be directly converted into pyridoxal phosphate by enzyme catalysis, with a specific high selectivity effect, and the concentration of the obtained pyridoxal phosphate is high. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0048] Figure 2 It 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 1. Total protein after induction of wild-type PdxK; 2. Supernatant after induction of wild-type PdxK; 3. Precipitate after induction of wild-type PdxK; 4. Total protein after induction of pyridoxal kinase mutant K229F; 5. Supernatant after induction of pyridoxal kinase mutant K229F; 6. Precipitate after induction of mutant K229F. DETAILED DESCRIPTION

[0051] The technical solution of the present invention is 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 engineered bacteria of pyridoxal kinase (PdxK)

[0054] The wild-type glycerol bacteria of PdxK were inoculated into 5 mL of LB liquid culture medium containing a final concentration of 50 μg / mL kanamycin at a 0.1% v / v inoculation amount, and cultured at 37°C and 200 rpm for 12 hours. Then, 5 mL of the liquid culture was inoculated into 200 mL of TB liquid culture medium containing a final concentration of 50 μg / mL kanamycin at a 1% v / v inoculation amount, and cultured at 37°C and 200 rpm for 4-6 hours until the OD value reached about 0.8, 1 mM IPTG was added for induction at 25°C for 12 hours, and centrifuged at 4°C and 8000 rpm for 10 minutes, and the supernatant was discarded to collect the wet cells. The collected wet cells were further broken by an ultrasonic cell disruptor to obtain a cell lysate, which was the 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 thereof is shown in SEQ ID NO.2.

[0056] Example 2

[0057] Pyridoxal kinase mutant cloning process

[0058] Design primers, download the gene template pET-30a(+) of the expression vector pET30(a)+ from the SnapGene official website, and insert the PdxK sequence into the vector through the two restriction sites "HindⅢ" and "NdeⅠ". Mutate the 229th amino acid (K229) in the target sequence, and select primer sequences with a length of 30-36bp and a Tm value of 55-65℃. The designed primers were synthesized separately according to conventional methods, and the mutant primers obtained are shown in Table 1:

[0059] Table 1:

[0060]

[0061]

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

[0063] After the reaction is completed, 1% agarose gel electrophoresis is used to detect the PCR amplification product 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, and then the target gene fragment is connected to the vector using ExnaseⅡ ligase to obtain a recombinant expression vector of the connection product, and the obtained connection product is transformed into Escherichia coli E.coliBL21 (DE3) competent cells, and the transformed cells are spread on LB solid plates containing 50μg / mL kanamycin, and multiple well-grown single colonies are picked and inoculated in LB liquid culture medium containing a final concentration of 50μg / mL kanamycin, 37℃, 200rpm culture for 12-16h, and prepared into glycerol bacteria and stored at -80℃ for standby use. The remaining culture extracts the plasmid and sends it for sequencing. The results show that the cloned K229 gene sequence is correct and has been correctly connected to the pET30a plasmid to obtain the recombinant plasmid pET30a-LysX (corresponding recombinant expression vector). The plasmid with the correct sequencing results is stored in a -20℃ refrigerator for standby use.

[0064] If it is further used to make microorganisms, 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 enzyme activity of the pyridoxal kinase mutant, the corresponding pyridoxal kinase mutant obtained by the method of Example 2 was selected, that is, the lysine mutant at position 229 was replaced by 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 3mL of reaction solution, which contains 20g / L pyridoxal hydrochloride, 10g / L adenosine triphosphate disodium salt, 30g / L acetyl phosphate diammonium salt, and 2g / L magnesium sulfate heptahydrate, stir until dissolved, adjust pH to 5.0 with 10M sodium hydroxide solution, add 0.2mL acetate kinase (supernatant after crushing), 0.2mL crude enzyme solution (supernatant after crushing), and shake the reaction solution at 35°C and 200rpm in a water bath shaker. After 15min, take a sample and heat and boil for 5min to terminate the reaction, and determine the change in the concentration of pyridoxal phosphate in the reaction solution by HPLC.

[0069] The above crude enzyme solutions correspond to the wild-type PdxK crude enzyme solution or the pyridoxal kinase mutant crude enzyme solution, respectively.

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

[0071] Comparison of enzyme activities between wild-type PdxK and pyridoxal kinase mutants

[0072] The genetically engineered bacteria of the pyridoxal kinase mutant (PL kinase mutant) successfully constructed in Example 2 were cultured using the method of Example 1 to obtain bacterial cells, and after crushing, the crude enzyme solution of the corresponding 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 enzymatic activity data of the mutants in Table 1 above show that by mutating the lysine at position 229, not all mutants have better enzymatic activity than the wild type, and some mutants have less enzymatic activity than the wild type. The pyridoxal mutants of the present invention can achieve the performance of improving enzymatic activity, which is also far better than the enzymatic activity level of the wild type, but the specific enzymatic activity does not necessarily enable the conversion into pyridoxal phosphate with a high concentration effect.

[0077] At the same time, the wild-type PdxK and K229F were specifically selected for analysis of enzyme protein expression for comparison. The specific analysis results are as follows: Figure 1 As shown, the results showed that both the mutant and wild-type proteins were highly expressed and had good solubility.

[0078] Example 4

[0079] The crude enzyme solution obtained by fermentation and ultrasonic crushing of wild-type PdxK genetic engineering bacteria and acetate kinase engineering bacteria (existing acetate kinase) was used as a catalyst for the reaction process and result analysis of preparing pyridoxal phosphate.

[0080] Take 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, add 0.5g acetyl phosphate diammonium salt (ACP) every 1h, take samples every half hour, centrifuge and determine the concentration of the product by HPLC. The reaction finally generates 22.18g / L pyridoxal phosphate in 4h. The concentration of the product during the reaction is 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, this example takes the K229F mutant reaction as an example.

[0083] The total volume of the reaction solution is 20 mL, including 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. They are 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 an hour to detect changes in the concentration of pyridoxal phosphate by HPLC, and 0.2 g of acetyl phosphate diammonium salt is supplemented every half an hour. The liquid phase detection results show that mutant K229F catalyzes the generation of 58.87 g / L of pyridoxal phosphate in 3.5 hours. 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 in 1L shake flasks and crushed to obtain crude enzyme liquid, which was used as a catalyst together with acetate kinase.

[0087] The enzyme catalyzed reaction was carried out with pyridoxal hydrochloride as substrate and acetyl phosphate diammonium salt as cosubstrate. The pH value of the reaction solution was 5.0, with a total of 20 mL, including 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, and the reaction was shaken at 200 rpm in a 35 ° C water bath. HPLC was used to monitor the concentration change of the product pyridoxal phosphate during the reaction process, and the concentration of the final product of the reaction could reach the following Table 3.

[0088] Table 3:

[0089]

[0090] Combined with the corresponding reaction process tracking analysis in the above Examples 4 and 5, 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, and the concentration of the product pyridoxal phosphate obtained by using the mutant of the present invention for enzyme catalysis 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 only K229C, K229W and K229G have low catalytic efficiency in the mutant of the present invention, and the remaining mutants all show good application value.

[0091] The specific embodiments described in the present invention are merely examples of the spirit of the present invention. Those skilled in the art may make various modifications or additions to the specific embodiments described or replace them in similar ways, but they will not deviate from the spirit of the present invention or exceed the scope defined by the attached claims.

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

2. The pyridoxal kinase mutant according to claim 1, characterized in that The pyridoxal kinase mutant is obtained by mutating the 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, methionine, arginine, threonine, histidine, serine, tyrosine, valine, leucine, isoleucine, proline, asparagine, aspartic acid or glutamic acid; and the pyridoxal kinase mutant is used for catalyzing pyridoxal to synthesize pyridoxal phosphate.

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 the 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. An application of a pyridoxal kinase mutant, a recombinant expression vector and a microbial cell, wherein under the action of pyridoxal kinase, a 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 pyridoxal kinase mutant of claim 1 or 2, the recombinant expression vector of claim 3, and the microbial cell of 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: The 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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