A fermentation method for producing thiamine pyrophosphokinase

Abstract

The application discloses a fermentation method for producing thiamine pyrophosphokinase and belongs to the technical field of bioengineering. The BL21 recombinant engineering bacteria containing pET28a-TPK, pET28a-ProS2-TPK or pET28a-SUMO-TPK are constructed, the seed liquid is prepared, the isopropyl-beta-D-thiogalactoside is used for inducing expression, the bacteria are collected, the cells are broken and the supernatant is collected to obtain the TPK crude enzyme liquid. In the fermentation, the LB / TB culture medium containing 50 μg / mL kanamycin or 100 μg / mL ampicillin is used. The SUMO label is introduced to guide the correct folding of the TPK, so that the soluble expression ratio of the TPK is more than 85%, the ratio is improved by 2 times compared with the common system, the inclusion body ratio is greatly reduced, the process is stable and has good repeatability, and an effective scheme is provided for the industrialized large-scale preparation of the TPK.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to a fermentation method for the efficient production of thiamine pyrophosphate kinase (TPK). Background Technology

[0002] Thiamine pyrophosphate kinase (TPK) is a key enzyme in the metabolism of vitamin B1. It can specifically catalyze the phosphorylation of thiamine (vitamin B1) to produce its active form, thiamine pyrophosphate (TPP). As an important coenzyme, TPP is widely involved in various physiological processes such as carbohydrate metabolism and the tricarboxylic acid cycle. Therefore, TPK has broad application prospects in biocatalysis, pharmaceutical research and development, food additives, and clinical diagnostics.

[0003] Currently, TPK preparation mainly relies on two technical routes: extraction from natural organisms and fermentation preparation using engineered strains constructed through conventional recombinant expression technology. However, both routes have significant technical drawbacks: natural extraction methods are limited by raw material sources, resulting in low extraction efficiency, high costs, and low TPK purity, making it difficult to meet the needs of large-scale industrial applications; while conventional recombinant expression technology can achieve large-scale TPK preparation, TPK in heterologous expression systems is prone to problems such as folding errors, low soluble expression levels, and inclusion body formation, which not only reduce the activity of the target protein but also significantly increase the difficulty and cost of subsequent purification processes, limiting its industrial application.

[0004] SUMO (small ubiquitin-like modified protein) tags, as highly efficient solubilization tags, can not only guide the correct folding of target proteins, increasing the soluble expression ratio and reducing inclusion body formation, but also be specifically cleaved by SUMO proteases (such as ULP1 protease), thereby obtaining target proteins with native conformation and complete activity. They have shown good application results in the heterologous expression of various poorly soluble proteins. However, to date, no reports have been published on the application of SUMO tags in the fermentation preparation of TPK to solve the technical challenges of its low solubility and easy inclusion body formation. Summary of the Invention

[0005] To overcome the above-mentioned technical defects, this invention provides a fermentation method for the efficient production of thiamine pyrophosphate kinase (TPK), thereby solving the problems of low expression levels and poor solubility in existing TPK preparation methods.

[0006] This invention provides a fermentation method for the efficient production of thiamine pyrokinase, comprising the following steps: (1) Seed culture preparation: 100 μL of glycerol bacteria containing recombinant genetically engineered bacteria was inoculated into 100 mL of antibiotic-containing culture medium and cultured with shaking at 37℃ and 180 rpm until the bacterial culture OD 600 When the value reaches 0.6-0.8, seed solution is obtained; (2) Induction of expression: Isopropyl-β-D-thiogalactoside was added to the seed culture obtained in step (1) to make the final concentration of isopropyl-β-D-thiogalactoside in the system reach 0.5mM. Then, the culture was induced by shaking at 18℃ and 180rpm for 12-16 hours to obtain the induction culture medium. (3) Collection of bacterial cells: Centrifuge the induction culture medium obtained in step (2) at 4℃ and 4000rpm for 20 minutes, discard the supernatant, and collect the bacterial cells; (4) Cell disruption: Add 20 ml of 50 mM Tris-HCl buffer solution to the cells collected in step (3) and add 20 μL of benzyl sulfonyl fluoride. Use a cell disruptor to disrupt the cells. The disruption parameters are set as follows: disruption for 3 seconds, pause for 3 seconds, power 14%, disruption time 30 minutes to obtain the cell disruption solution. (5) Collection of supernatant: Centrifuge the bacterial cell lysate obtained in step (4) at 12000 rpm for 30 minutes and collect the supernatant to obtain TPK crude enzyme solution.

[0007] Further, the antibiotic mentioned in step (1) is kanamycin or ampicillin, wherein the concentration of kanamycin is 50 μg / mL and the concentration of ampicillin is 100 μg / mL.

[0008] Further, the culture medium mentioned in step (1) is LB medium or TB medium.

[0009] Further, the recombinant genetically engineered bacteria described in step (1) are constructed by transforming the pET28a-TPK, pET28a-ProS2-TPK or pET28a-SUMO-TPK expression vector into the BL21(DE3) strain, and then screening to obtain the recombinant genetically engineered bacteria.

[0010] Compared with the prior art, the present invention has the following beneficial effects: 1. The method provided by this invention significantly improves soluble expression. By introducing the SUMO tag, its molecular chaperone-like function guides TPK to fold correctly, and the soluble expression rate reaches more than 85%, which is more than twice that of ordinary expression systems without the SUMO tag.

[0011] 2. The method and process provided by this invention are stable and have good repeatability.

[0012] 3. The method provided by this invention significantly reduces the proportion of inclusion bodies, thereby reducing protein loss. Attached Figure Description

[0013] Figure 1 This is the SDS-PAGE electrophoresis pattern of TPK obtained in Example 3. Number M is the protein molecular weight standard marker, number 1 is the whole bacterial lysate before TPK induction, number 2 is the whole bacterial lysate after induction, number 3 is the TPK supernatant, and number 4 is the TPK precipitate. Figure 2 These are SDS-PAGE electrophoresis patterns of SUMO-TPK obtained in Examples 4-5. M is the protein molecular weight standard marker; 1 is the whole-cell lysate before SUMO-TPK induction; 2 is the whole-cell lysate after SUMO-TPK induction; 3 is the SUMO-TPK supernatant; 4 is the SUMO-TPK precipitate; 5 is the whole-cell lysate before ProS2-TPK induction; 6 is the whole-cell lysate after ProS2-TPK induction; 7 is the ProS2-TPK supernatant; and 8 is the ProS2-TPK precipitate. Detailed Implementation

[0014] The present invention is illustrated by the following examples.

[0015] Example 1

[0016] TB culture medium is prepared using a two-step method. The specific preparation steps are as follows (for preparing 1L of TB culture medium): 1. Preparation of TB-A solution: Weigh 24g of yeast extract, 12g of tryptone, and 4g of glycerol, dissolve them in 900mL of ultrapure water, stir until completely dissolved, autoclave at 121℃ for 20 minutes, and cool to room temperature for later use; 2. Preparation of TB-B solution: Weigh 2.3g of potassium dihydrogen phosphate and 16.4g of dipotassium hydrogen phosphate, dissolve them in 100mL of ultrapure water, stir until completely dissolved, autoclave at 121℃ for 15min, and cool to room temperature for later use. 3. Mix and adjust volume: Gently mix 900 mL of cooled TB-A solution with 100 mL of TB-B solution to obtain 1 L of complete TB culture medium.

[0017] LB medium preparation: Weigh 10 g of tryptone, 5 g of yeast extract, and 10 g of NaCl, add distilled water to a final volume of 1 L, and autoclave at 121°C for 20 minutes.

[0018] TPK fermentation preparation: Take 100 mL of LB medium, add 100 μl of the glycerol bacteria and 50 μl of kanamycin at a concentration of 50 μg / mL, and incubate at 37℃ and 180 rpm with shaking for about 5 h, until the bacterial culture OD... 600 The value reached 0.6; then 100 μl of 0.5 mM IPTG was added to 100 ml of bacterial culture, and expression was induced overnight with shaking at 18℃ and 180 rpm for 12 h; after induction, the bacterial culture was aliquoted into 45 ml tubes, centrifuged at 4℃ and 4000 rpm for 20 min, and the supernatant was discarded to collect the bacterial cells; 20 ml of 50 mM Tris-HCl buffer and 20 μl of PMSF were added to the collected bacterial cells, mixed well, and the bacterial cells were disrupted using a cell disruptor with the following parameters: disruption for 3 seconds, pause for 3 seconds, disruption power of 14%, and total disruption time of 30 min; after all disruptions were completed, the disruption solution was centrifuged at 4℃ and 12000 rpm for 30 min, and the supernatant was collected to obtain the crude enzyme solution containing TPK. Protein expression was detected by SDS-PAGE electrophoresis.

[0019] SUMO-TPK fermentation preparation: Take 100 mL of TB medium, add 100 μl of the glycerol bacteria and 50 μl of kanamycin at a concentration of 50 μg / mL, and incubate at 37℃ and 180 rpm with shaking for about 3 hours until the bacterial culture OD reaches 100 μg / mL. 600 The value reached 0.6; then 100 μl of 0.5 mM IPTG was added to 100 ml of bacterial culture, and expression was induced overnight at 18℃ and 180 rpm with shaking, for a total induction time of 16 h; after induction, the bacterial culture was aliquoted into 45 ml tubes, centrifuged at 4℃ and 4000 rpm for 20 min, and the supernatant was discarded to collect the bacterial cells; 20 ml of 50 mM Tris-HCl buffer and 20 μl of PMSF were added to the collected bacterial cells, mixed well, and the bacterial cells were disrupted using a cell disruptor with the following parameters: disruption for 3 seconds, pause for 3 seconds, disruption power of 14%, and total disruption time of 30 min; after all disruptions were completed, the disruption solution was centrifuged at 4℃ and 12000 rpm for 30 min, and the supernatant was collected to obtain the crude enzyme solution containing SUMO-TPK. Protein expression was detected by SDS-PAGE electrophoresis.

[0020] Fermentation preparation of ProS2-TPK: Take 100 mL of TB medium, add 100 μl of the glycerol bacteria and 100 μl of ampicillin at a concentration of 100 μg / mL, and incubate at 37℃ and 180 rpm with shaking for about 3 hours, until the bacterial culture reaches OD. 600The value reached 0.6; then 100 μl of 0.5 mM IPTG was added to 100 ml of bacterial culture, and expression was induced overnight at 18℃ and 180 rpm with shaking, for a total induction time of 16 h; after induction, the bacterial culture was aliquoted into 45 ml tubes, centrifuged at 4℃ and 4000 rpm for 20 min, and the supernatant was discarded to collect the bacterial cells; 20 ml of 50 mM Tris-HCl buffer and 20 μl of PMSF were added to the collected bacterial cells, mixed well, and the bacterial cells were disrupted using a cell disruptor with the following parameters: disruption for 3 seconds, pause for 3 seconds, disruption power of 14%, and total disruption time of 30 min; after all disruptions were completed, the disruption solution was centrifuged at 4℃ and 12000 rpm for 30 min, and the supernatant was collected to obtain the crude enzyme solution containing ProS2-TPK. Protein expression was detected by SDS-PAGE electrophoresis.

[0021] SDS-PAGE electrophoresis analysis method: Add 2x SDS Loading Buffer to the prepared sample, heat-treat at 98℃ for 3 min, then load the sample onto a 12% SDS-PAGE precast gel. Electrophoresis is then performed at a constant voltage of 160V for 35-40 min. Coomassie brilliant blue staining is applied, followed by destaining and observation / analysis.

Claims

1. A fermentation method for the efficient production of thiamine pyrophosphate kinase, characterized in that, Includes the following steps: (1) Seed culture preparation: 100 μL of glycerol bacteria containing recombinant genetically engineered bacteria was inoculated into 100 mL of antibiotic-containing culture medium and cultured with shaking at 37℃ and 180 rpm until the bacterial culture OD 600 When the value reaches 0.6-0.8, seed solution is obtained; (2) Induction of expression: Isopropyl-β-D-thiogalactoside was added to the seed culture obtained in step (1) to make the final concentration of isopropyl-β-D-thiogalactoside in the system reach 0.5mM. Then, the culture was induced by shaking at 18℃ and 180rpm for 12-16 hours to obtain the induction culture medium. (3) Collection of bacterial cells: Centrifuge the induction culture medium obtained in step (2) at 4℃ and 4000rpm for 20 minutes, discard the supernatant, and collect the bacterial cells; (4) Cell disruption: Add 20 ml of 50 mM Tris-HCl buffer solution to the cells collected in step (3) and add 20 μL of benzyl sulfonyl fluoride. Use a cell disruptor to disrupt the cells. The disruption parameters are set as follows: disruption for 3 seconds, pause for 3 seconds, power 14%, disruption time 30 minutes to obtain the cell disruption solution. (5) Collection of supernatant: Centrifuge the bacterial cell lysate obtained in step (4) at 12000 rpm for 30 minutes and collect the supernatant to obtain TPK crude enzyme solution.

2. The fermentation method according to claim 1, characterized in that, The antibiotics mentioned in step (1) are kanamycin or ampicillin, wherein the concentration of kanamycin is 50 μg / mL and the concentration of ampicillin is 100 μg / mL.

3. The fermentation method according to claim 1, characterized in that, The culture medium mentioned in step (1) is LB medium or TB medium.

4. The fermentation method according to claim 1, characterized in that, The recombinant genetically engineered bacteria described in step (1) are constructed by transforming the pET28a-TPK, pET28a-ProS2-TPK or pET28a-SUMO-TPK expression vector into the BL21(DE3) strain, and then screening to obtain the recombinant genetically engineered bacteria.

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