Phosphopyridoxine oxidase mutant and application thereof in synthesis of phosphopyridoxal

Abstract

The invention provides a pyridoxine phosphate oxidase mutant and application of the pyridoxine phosphate oxidase mutant in synthesis of pyridoxal phosphate. The mutant is obtained by performing one or more mutations on an amino acid sequence as shown in SEQ: ID: NO: 4, wherein the amino acid sequence contains the 24th site (D24N), the 141 site (Q141S), the 190 site (R190G) and the 195 site (H195A) as a mutant 1; the mutant 2 contains a 66th site (R66K), a 141 site (Q141S), a 180 site (Q180T) and a 193 site (R193H); and the mutant 3 contains the 141 site (Q141S) and the 190 site (R190G). Compared with wild type pyridoxine phosphate oxidase, the pyridoxine phosphate oxidase mutant disclosed by the invention has relatively high catalytic efficiency, and when the pyridoxine phosphate oxidase mutant is used for synthesizing pyridoxal phosphate, the conversion rate of 99.76% or above can be realized within 5 hours.

Description

Technical Field

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

[0002] Vitamin B6 (VB6) 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 forms are pyridoxine-5'-phosphate (PNP), pyridoxamine-5'-phosphate (PMP), and pyridoxal-5'-phosphate (PLP).

[0003] VB6 has various physiological functions such as anti-inflammatory, antioxidant, neuromodulatory, and anti-tumor, and is an important nutritional chemical. VB6 has great potential in the treatment of diseases. For example, in the treatment of cardiovascular diseases, VB6 is an essential cofactor in the metabolism of homocystine (Hcy), which can convert the Hcy cycle into methionine or cysteine to reduce the content of Hcy in the blood, thereby reducing the incidence of coronary atherosclerotic heart disease, hypertension, and stroke.

[0004] PLP is the biologically active form of VB6 and is a cofactor for more than 180 enzymes. For example, PLP is a cofactor for dopamine (vasopressin) synthase, and 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 the small gas molecule hydrogen sulfide (H2S) to stabilize the physiological level of H2S and relieve the imbalance of endogenous H2S metabolism in the human body, thereby preventing damage to pancreatic islet β-cell function. In the treatment of cancer, the active form of PLP can regulate two key metabolic enzymes required for the growth of acute myeloblastic leukemia (AML) cells, thereby slowing down the deterioration of AML. Therefore, a large intake of VB6 may inhibit the development of pancreatic cancer, cancer cell metastasis, and tumor growth.

[0005] Currently, the production of PLP mostly adopts chemical methods. Chinese Patent CN 110016049A discloses a method for preparing pyridoxal phosphate by a chemical method, which not only consumes time and energy, causes pollution, has a high cost, but also has a low yield. Preparing PLP by the biological enzyme method has milder conditions and shorter consumption time; it is environmentally friendly, efficient, has good selectivity, high yield, convenient operation, low cost, and has good industrial application value.

[0006] PLP is oxidized and generated under the action of pyridoxine 5’-phosphate oxidase (PdxH). Among them, PdxH encodes a classical PNP / PMP flavoprotein oxidase, which requires flavin mononucleotide (FMN) as a cofactor, and oxygen is reduced to hydrogen peroxide (H2O2) as an electron acceptor. However, at present, the pyridoxine phosphate oxidase derived from wild type has poor thermal stability and low enzyme activity. Summary of the Invention

[0007] The purpose of the present invention is to provide a pyridoxine phosphate oxidase mutant to solve the problems of poor thermal stability and low enzyme activity of wild-type pyridoxine phosphate oxidase. At the same time, the present invention also provides the application of the pyridoxine phosphate oxidase mutant in the synthesis of pyridoxal phosphate to improve the production efficiency of pyridoxal phosphate and reduce the reaction cost.

[0008] The purpose of the present invention is achieved by the following technical solutions: A pyridoxine phosphate oxidase mutant, which is any of the following mutants: Mutant 1, the Mutant 1 is based on the wild-type pyridoxine phosphate oxidase with the amino acid sequence shown in SEQ: ID: NO:4, in which Asp at the 24th position is mutated to Asn, Gln at the 141st position is mutated to Ser, Arg at the 190th position is mutated to Gly, and His at the 195th position is mutated to Ala, and its amino acid sequence is shown in SEQ: ID: NO:6; Mutant 2, the Mutant 2 is based on the wild-type pyridoxine phosphate oxidase with the amino acid sequence shown in SEQ: ID: NO:4, in which Arg at the 66th position is mutated to Lys, Gln at the 141st position is mutated to Ser, Gln at the 180th position is mutated to Thr, and Arg at the 193rd position is mutated to His, and its amino acid sequence is shown in SEQ: ID: NO:8; Mutant 3, the Mutant 3 is based on the wild-type pyridoxine phosphate oxidase with the amino acid sequence shown in SEQ: ID: NO:4, in which Gln at the 141st position is mutated to Ser, and Arg at the 190th position is mutated to Gly, and its amino acid sequence is shown in SEQ: ID: NO:2.

[0009] The present invention also provides nucleotide sequences encoding the above pyridoxine oxidase mutants and wild-type pyridoxine oxidase: The nucleotide sequence encoding Mutant 1 is shown as SEQ: ID: NO:5, the nucleotide sequence encoding Mutant 2 is shown as SEQ: ID: NO:7, and the nucleotide sequence encoding Mutant 3 is shown as SEQ: ID: NO:1; the nucleotide sequence encoding wild-type pyridoxine oxidase is shown as SEQ: ID: NO:3.

[0010] The method for obtaining the above pyridoxine oxidase mutants and the mutation positions are shown in Table 1.

[0011] Table 1 Mutation sites and obtaining methods of wild-type and mutants .

[0012] Furthermore, the present invention provides a recombinant expression vector containing nucleic acid encoding any of the above pyridoxine oxidase mutants.

[0013] Preferably, the recombinant expression vector uses pET-29a(+) as the original expression vector.

[0014] Even further, the present invention also provides a recombinant bacterium containing the above recombinant expression vector.

[0015] Preferably, the host bacterium of the recombinant bacterium is Escherichia coli, preferably Escherichia coli BL21(DE3).

[0016] Among them, the construction method of the recombinant bacterium is as follows: 1) Insert the coding gene fragment of any of the above pyridoxine oxidase mutants between the NdeI and EcoRI restriction enzyme sites of the original expression vector pET-29a(+) to obtain a recombinant expression vector; 2) Transform the recombinant expression vector into Escherichia coli BL21(DE3) by heat shock method to obtain the recombinant bacterium.

[0017] The application of the above pyridoxine oxidase mutants in the synthesis of pyridoxal.

[0018] Specifically, using pyridoxine as a substrate, FMN, catalase, and the enzyme solution of pyridoxine oxidase mutant are added in sequence for a catalytic reaction to generate pyridoxal.

[0019] Preferably, the mass ratio of the pyridoxine substrate to the coenzyme FMN is (2000~3000):1.

[0020] Preferably, the reaction pH is 8~10, and the catalytic reaction temperature is 36~40°C.

[0021] Preferably, in every 1 liter of the system, 400 U of the pyridoxine oxidase mutant enzyme solution and 40,000 U of the catalase enzyme solution are added.

[0022] The present invention also provides a preparation for catalyzing the synthesis of pyridoxal phosphate from pyridoxine, and the preparation contains the pyridoxine oxidase mutant described in the present invention.

[0023] Advantages of the present invention: By using molecular biology techniques, the present invention conducts molecular modification on wild-type-derived pyridoxine oxidase and designs a pyridoxine oxidase mutant. The pyridoxine oxidase mutant described in the present invention uses pyridoxine as a substrate and can be used for synthesizing pyridoxal phosphate through a specific catalytic process. The conversion rate can reach more than 99.76% within 5 h. Its thermal stability and enzyme activity are significantly improved compared with wild-type pyridoxine oxidase, which improves the efficiency of biocatalytic preparation of pyridoxal phosphate and reduces the reaction cost. Compared with the previous chemical synthesis method and biocatalytic method, the reaction time is shorter and the cost is lower. It has good industrial application prospects and promotes the process of producing pyridoxal phosphate by biological methods. Description of the drawings

[0024] Figure 1 It is the plasmid map of pet29a-PNPO-Q141S-R190G.

[0025] Figure 2 It is the agarose gel electrophoresis diagram of site-directed mutagenesis PCR for transforming wild-type pyridoxine oxidase into mutant 3; where M is DNA Marker; lane 1 is the recombinant plasmid pET29a-Q141S-R190G.

[0026] Figure 3 It is the liquid chromatography diagram of PNP and PLP. Detailed implementation manners

[0027] 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 objectives of the present invention.

[0028] Sequence description SEQ: ID: NO:1 is the nucleotide sequence encoding pyridoxine oxidase mutant 3: with a full length of 645 bases; SEQ: ID: NO:2 is the amino acid sequence of pyridoxine oxidase mutant 3: with a full length of 214 amino acids; SEQ: ID: NO: 3 is the nucleotide sequence encoding wild-type pyridoxine oxidase: 645 bases in full length; SEQ: ID: NO: 4 is the amino acid sequence of wild-type pyridoxine oxidase: 214 amino acids in full length; SEQ: ID: NO: 5 is the nucleotide sequence encoding pyridoxine oxidase mutant 1: 645 bases in full length; SEQ: ID: NO: 6 is the amino acid sequence of pyridoxine oxidase mutant 1: 214 amino acids in full length; SEQ: ID: NO: 7 is the nucleotide sequence encoding pyridoxine oxidase mutant 2: 645 bases in full length; SEQ: ID: NO: 8 is the amino acid sequence of pyridoxine oxidase mutant 2: 214 amino acids in full length.

[0029] Enzyme activity definition Under specific temperature, pH and stirring speed conditions, the amount of enzyme required to convert 1 micromole of pyridoxine or generate 1 micromole of pyridoxal within 1 minute is defined as one activity unit (U).

[0030] Enzyme activity assay method Add the reaction solution to the Erlenmeyer flask, where the reaction solution includes pyridoxine, FMN, catalase and the enzyme solution of pyridoxal mutant. Place it in a shaker for reaction, immediately inactivate it in a metal bath after sampling, dilute it, and analyze it by HPLC.

[0031] Example 1 Construction of recombinant vector of wild-type pyridoxine oxidase and acquisition of corresponding strain The nucleotide sequence shown in SEQ: ID: NO: 3 (this sequence was originally derived from Deinococcus radiodurans the strain, and the protein sequence number in NCBI is WP_010887140.1) was artificially synthesized and cloned between the Nde I and Eco RI restriction enzyme sites of the pET-29a(+) expression vector to obtain the recombinant vector pET29a-PNPO-18; transform the recombinant vector pET29a-PNPO-18 into Escherichia coli E. coli BL21(DE3), send it for sequencing, and the successfully sequenced one is the wild-type pyridoxine oxidase strain PNPO-WT.

[0032] Example 2 Construction of recombinant vectors of mutant 1 and mutant 2 and acquisition of corresponding strains Using the above pET29a-PNPO-18 as a template, random mutations were introduced through a random mutation PCR kit (product number BTN1010055). After the PCR products were purified and recovered, they were transformed into Escherichia coli E. coli BL21(DE3). Two mutants with increased activity and enhanced stability were obtained through a method of batch screening by color development in 96-well plates. By comparing the sequencing results, it was found that four amino acid site mutations were introduced on the basis of the wild-type sequence. The significantly changed sequences were selected and named mutant 1 and mutant 2 respectively. Among them, mutant 1 contained mutations at positions 24 (D24N), 141 (Q141S), 190 (R190G), and 195 (H195A), and the resulting recombinant plasmid was named pET29a-PNPO-M1; mutant 2 contained mutations at positions 66 (R66K), 141 (Q141S), 180 (Q180T), and 193 (R193H), and the resulting recombinant plasmid was named pET29a-PNPO-M2. The above two recombinant vectors were respectively transformed into E. coli BL21(DE3), and sent for sequencing. The successfully sequenced strains were named PNPO-M1 and PNPO-M2 respectively.

[0033] Example 3 Construction of the recombinant vector of mutant 3 and obtaining of the strain Based on the fact that the enzyme activities of the above mutants 1 and 2 were higher than those of the normal wild-type enzyme, the inventors speculated through bioinformatics analysis that the double-site mutations at positions 141 (Q141S) and 190 (R190G) had an important impact on the enzyme activity, and further site-directed mutagenesis was carried out to construct mutant 3 with double-site mutations of Q141S and R190G.

[0034] Using the pET-29a recombinant plasmid pET29a-PNPO-18 containing the nucleotide sequence shown in SEQ: ID: NO:3 as a template, two pairs of primers, F1primer / R1primer and F2primer / R2primer, were respectively designed (the primer sequences are shown in Table 2). The recombinant plasmid was constructed by the whole plasmid PCR iteration method (the reaction system is shown in Table 3, and the reaction conditions are shown in Table 4), and the recombinant expression vector pET29a-PNPO-Q141S-R190G ( Figure 1 ).

[0035] Table 2 Primers designed for site-directed mutagenesis PCR

[0036] Table 3 PCR reaction system

[0037] Table 4 PCR reaction conditions

[0038] First, the PCR products were examined by gel electrophoresis, and the results were as Figure 2 shown. The target band was consistent with the theory. Then, 1 μL of Dpn I restriction endonuclease was added to 20 μL of the PCR product to digest the template plasmid, and it was incubated at 37 °C for 3 h. 10 μL of the digested product was taken and transformed into Escherichia coli BL21(DE3) to obtain the corresponding recombinant Escherichia coli, which was spread on an LB plate containing kanamycin (100 mg / L) and cultured overnight at 37 °C. Monoclonal colonies were randomly selected for colony PCR identification and sequencing verification. The results showed that the recombinant expression vector containing the pyridoxine oxidase mutant was successfully transformed into the expression host Escherichia coli BL21(DE3). The strain with successful mutation verified by sequencing was named PNPO-M3. Glycerol was added to the PNPO-M3 bacterial solution and stored in a refrigerator at -70 °C.

[0039] Example 4 Obtaining of wild-type pyridoxine oxidase and mutant crude enzyme solutions The above wild-type pyridoxine oxidase and mutants 1, 2, and 3 were respectively inoculated into LB medium. When the OD600 value of the bacteria reached 0.6, IPTG with an appropriate concentration (0.1 mM) was added to induce the expression of wild-type pyridoxine oxidase and its mutants. The induction conditions were 20 °C and the induction time was 16 h. The amount of bacteria collected at the shake-flask level was 5 g / L. The bacteria were ultrasonically disrupted to prepare crude enzyme solutions.

[0040] Example 5 Method for determining the enzyme activity of pyridoxine oxidase Activity determination: The crude enzyme solutions of wild-type and mutant pyridoxine oxidases were prepared using shake flasks. Equal amounts of pyridoxine oxidase and various mutant pyridoxine oxidases (5 mg / mL) were diluted 100 times, and 100 μL was taken and added to 1 mL of PBS buffer (800 μL of 0.65 g / L PNP, 50 μL of 0.5 g / L catalase, 50 μL of 0.48 g / L FMN). The pH was adjusted to 9, and after reacting at 37 °C for 30 min, inactivation was carried out.

[0041] The production amount of pyridoxal and the reduction amount of the substrate pyridoxine were respectively determined by liquid chromatography, and the activities of wild-type and three mutant pyridoxine oxidases were calculated. Taking the wild-type sequence as 100%, the activity changes of the three mutant pyridoxine oxidases were evaluated. The specific enzyme activity results of the three strains are shown in Table 5. It can be seen that the enzyme activities of the three mutant strains are all higher than that of the wild-type, and the enzyme activity of the further rationally designed mutant 3 (Q141S, R190G) is higher than that of mutant 1 and mutant 2.

[0042] Table 5 Influence of amino acid sequence mutation positions on enzyme activity 。

[0043] Example 6 Evaluation of the Thermal Stability of Pyridoxine Oxidase Equal amounts of wild-type pyridoxine oxidase and three pyridoxine oxidase mutants (5 mg / mL) were separately incubated at different temperatures (40 °C, 45 °C, 50 °C, 55 °C) for 0.5 h. After returning to room temperature, their enzyme activities were measured using the above method respectively.

[0044] The enzyme activity results are shown in Table 6. It can be seen that during the heating process from 40 °C to 55 °C, the enzyme activities of both wild-type and mutant pyridoxine oxidases gradually decreased. When incubated at 55 °C for 0.5 h, the enzyme activity of wild-type pyridoxine oxidase basically disappeared, while the mutant pyridoxine oxidase still had a relatively low enzyme activity. When incubated at 50 °C for 0.5 h, more than 50% of the enzyme activities of pyridoxine oxidase mutants 2 and 3 remained.

[0045] Since pyridoxine oxidase mutant 3 has high enzyme activity and thermal stability, mutant 3 was selected for subsequent biocatalytic reactions.

[0046] Table 6 Changes in Temperature Tolerance of Different Mutants 。

[0047] Example 7 Catalytic Synthesis of Pyridoxal by Pyridoxine Oxidase Mutant 3 1. Catalytic Reactions in a 50 mL System under Different Temperature Conditions In a 50 mL system, 0.63 g of pyridoxine, 0.2 mg of coenzyme FMN, 2000 U of catalase, and 20 U of the enzyme solution of pyridoxine oxidase mutant 3 were sequentially added. The pH was adjusted to 9, and the volume was fixed to 50 mL. The reaction was started under stirring at 200 rpm at different temperatures. After 5 h of reaction, the conversion rates and formation rates under different reaction conditions were measured as shown in Table 7 below.

[0048] Table 7 Effects of Temperature in Different Reaction Systems on Conversion Rates and Formation Rates

[0049] From the results in the above table, it can be seen that under the condition of a reaction temperature of 37 °C, the catalytic efficiency of this enzyme is the highest and the effect is the best, with a conversion rate of 99.82% and a formation rate of 95.31%.

[0050] 2. Catalytic Reactions in a 50 mL System under Different pH Conditions In a 50-ml system, 0.63 g of pyridoxine phosphate, 0.2 mg of coenzyme FMN, 2000 U of catalase, and 20 U of the enzyme solution of pyridoxine phosphate oxidase mutant 3 were added successively. The pH value was adjusted with 5 M NaOH, and the volume was made up to 50 ml. The reaction was started under stirring at 37°C and 200 rpm. After reacting for 5 h, the conversion rates and production rates under different reaction conditions were measured as shown in Table 8 below.

[0051] Table 8 Effects of pH in different reaction systems on the conversion rate and production rate

[0052] It can be seen from the results in the above table that under the condition of a reaction pH of 9, the catalytic efficiency of this enzyme is the highest and the effect is the best, with a conversion rate of 99.78% and a production rate of 94.85.

[0053] 3. Catalytic reaction of different enzyme dosages of pyridoxine phosphate oxidase mutant in a 50-mL system In a 50-ml system, 0.63 g of pyridoxine phosphate, 0.2 mg of coenzyme FMN, 2000 U of catalase, and 20 - 50 U of the enzyme solution of pyridoxine phosphate oxidase mutant 3 were added successively. The pH was adjusted to 9, and the volume was made up to 50 ml. The reaction was started under stirring at 37°C and 200 rpm. After reacting for 5 h, the conversion rates and production rates under different reaction conditions were measured as shown in Table 9 below.

[0054] Table 9 Effects of different enzyme dosages of pyridoxine phosphate oxidase mutant on the conversion rate and production rate

[0055] It can be seen from the results in the above table that when the enzyme solution dosage of the mutant is 20 U - 50 U, there are relatively high conversion rates and production rates. Considering the cost, an enzyme dosage of 20 U can be selected.

[0056] Example 8 Catalytic synthesis of pyridoxal phosphate by pyridoxine phosphate oxidase mutant 3 (catalytic reaction in a 1-L system) In a 1-L system, 12.5 g of pyridoxine phosphate, 4 mg of coenzyme FMN, 40000 U of catalase, and 400 U of the enzyme solution of pyridoxine phosphate oxidase mutant 3 were added successively. The pH was adjusted to 9, and the volume was made up to 1 L. The reaction was started under stirring at 37°C and 200 rpm. After reacting for 5 h, the concentration of pyridoxine phosphate was measured to be 0.03 g / L, and the conversion rate was 99.76%.

[0057] Example 9 Catalytic synthesis of pyridoxal phosphate by pyridoxine phosphate oxidase mutant 3 (catalytic reaction in a 10-L system) In a 10 L system, 125 g of pyridoxine phosphate, 40 mg of coenzyme FMN, 400,000 U of catalase, and 4000 U of the enzyme solution of pyridoxine phosphate oxidase mutant 3 were added successively. The pH was adjusted to 9, and the volume was made up to 10 L. The reaction was started at 37 °C with stirring at 200 rpm. After reacting for 5 h, the concentration of pyridoxine phosphate was measured to be 0.05 g / L, and the conversion rate was 99.60%, indicating that the scale-up of this reaction was okay and could guide industrial production.

Claims

1. A pyridoxal phosphate oxidase mutant, characterized in that, It is any of the following mutants: Mutant 1, which is obtained by mutating Asp at position 24 to Asn, Gln at position 141 to Ser, Arg at position 190 to Gly, and His at position 195 to Ala on the basis of the wild-type pyridoxine oxidase with the amino acid sequence shown in SEQ: ID: NO:4, and its amino acid sequence is shown in SEQ: ID: NO:6; Mutant 2, which is obtained by mutating Arg at position 66 to Lys, Gln at position 141 to Ser, Gln at position 180 to Thr, and Arg at position 193 to His on the basis of the wild-type pyridoxine oxidase with the amino acid sequence shown in SEQ: ID: NO:4, and its amino acid sequence is shown in SEQ: ID: NO:8; Mutant 3, which is obtained by mutating Gln at position 141 to Ser and Arg at position 190 to Gly on the basis of the wild-type pyridoxine oxidase with the amino acid sequence shown in SEQ: ID: NO:4, and its amino acid sequence is shown in SEQ: ID: NO:

2.

2. A nucleic acid encoding the pyridoxal phosphate oxidase mutant according to claim 1, characterized in that, The nucleotide sequence encoding Mutant 1 is shown in SEQ: ID: NO:5, the nucleotide sequence encoding Mutant 2 is shown in SEQ: ID: NO:7, and the nucleotide sequence encoding Mutant 3 is shown in SEQ: ID: NO:

1.

3. A recombinant expression vector containing the nucleic acid according to claim 2.

4. A recombinant bacterium containing the recombinant expression vector according to claim 3.

5. Use of the pyridoxine oxidase mutant according to claim 1 in the synthesis of pyridoxal.

6. The application according to claim 5, wherein Using pyridoxine as a substrate, flavin mononucleotide, catalase, and the enzyme solution of the pyridoxine oxidase mutant are added in sequence to carry out a catalytic reaction to generate pyridoxal.

7. The application according to claim 6, characterized in that, The mass ratio of pyridoxine to flavin mononucleotide is (2000-3000):

1.

8. The application according to claim 6, characterized in that, In each 1 liter of the synthesis system, 40000 U of catalase enzyme solution and 400 U of the pyridoxine oxidase mutant enzyme solution are added.

9. The application according to claim 6, characterized in that, The pH of the catalytic reaction is 8-10, and the catalytic reaction temperature is 36-40°C.

10. A preparation for catalyzing the synthesis of pyridoxal phosphate from pyridoxine phosphate, characterized in that, The preparation contains the pyridoxine oxidase mutant according to claim 1.

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