Fermentation and purification method of recombinant glucose dehydrogenase in pichia pastoris

Abstract

The invention discloses a fermentation and purification method of recombinant glucose dehydrogenase in pichia pastoris. The method comprises the following steps: adding the pichia pastoris with the recombinant glucose dehydrogenase into a fermentation system to produce the glucose dehydrogenase; fermented fermentation liquor is subjected to centrifugation, ultrafiltration, affinity chromatography and elution, and the method specifically comprises a strain culture stage, a carbon source feeding stage, a starvation feeding stage, an induced expression stage, a centrifugation stage, an ultrafiltration stage, an affinity chromatography stage and an elution stage. On the basis of the traditional pichia pastoris fermentation process, the yield and purification yield of the FAD-dependent glucose dehydrogenase are improved by adding adenosine and using an NTA Ni + gel chromatographic column technology through experimental exploration of technical parameters. Meanwhile, the fermentation and purification process provided by the invention is simple and feasible, mild in condition, good in reproducibility, high in controllability and suitable for large-scale production.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a fermentation and purification method of recombinant glucose dehydrogenase in Pichia pastoris. Background Art

[0002] Glucose dehydrogenase (GDH) is an enzyme that catalyzes glucose oxidation reactions and is widely used in biosensors, clinical testing, and the food industry. It is present in the liver of higher animals and in certain microorganisms such as Acetobacter oxydans. The reaction catalyzed by glucose dehydrogenase is the conversion of D-glucose and NAD(P) to D-gluconolactone and NAD(P)H, where NAD(P) can be NAD+ or NADP+. This enzyme also has some activity on D-xylose, but has little effect on other natural hexoses and pentoses.

[0003] In the field of blood glucose monitoring, glucose dehydrogenase is used to develop second-generation electrochemical sensors to overcome the limitation of first-generation sensors (based on glucose oxidase) that rely on oxygen as an electron acceptor. Glucose dehydrogenase sensors are oxygen-independent and can therefore provide more stable blood glucose measurements in environments with varying oxygen concentrations. In addition, glucose dehydrogenase sensors can use different electron mediators to further improve the performance of the sensor.

[0004] Therefore, by optimizing the heterologous expression system, the production of glucose dehydrogenase can be significantly increased and the production cost can be reduced, which is of great significance for improving its performance in the field of blood glucose monitoring. Adenosine participates in almost all life activities of organisms, such as acting on cell energy transfer: adenosine helps cell energy transfer by forming molecules such as adenosine triphosphate (ATP) and adenosine diphosphate (ADP). Signal transduction pathway: adenosine affects protein synthesis and secretion by forming signal molecules such as cyclic adenosine monophosphate (cAMP). In theory, adenosine provides more ATP to affect the ratio of NADH / NAD+ in microbial cells, which will help improve the expression of FAD-dependent glucose dehydrogenase.

[0005] In metal chelation chromatography, Ni+ gel chromatography columns have two different chelation methods, resulting in two valent ligands that can bind to the His purification tag, namely nitrilotriacetic acid (IDA) and iminodiacetic acid (NTA), and two valent bound proteins that can be eluted by imidazole. As a trivalent ligand, IDA has an extremely high metal ion leakage rate, especially in the equilibration and elution steps. Although the elution components of NTA agarose also contain some leaked metal ions, they are significantly less than IDA. Due to changes in the properties of the target protein, the purity of histidine-tagged proteins purified using IDA agarose is usually lower than that purified using NTA agarose.

[0006] There are currently no reports on increasing protein expression by adding adenosine or using NTA Ni+ gel chromatography columns. Summary of the invention

[0007] The purpose of the present invention is to provide a fermentation purification method of recombinant glucose dehydrogenase in Pichia pastoris, zinc-loaded lactoferrin and application, so as to solve one or more technical problems existing in the prior art and at least provide a beneficial choice or create conditions.

[0008] The present invention adopts the following technical solutions to achieve the above-mentioned invention object: The present invention provides a method for fermenting and purifying recombinant glucose dehydrogenase in Pichia pastoris, comprising: Pichia pastoris containing recombinant glucose dehydrogenase is added into the fermentation system to produce glucose dehydrogenase; The fermentation broth after fermentation is subjected to centrifugation, ultrafiltration, affinity chromatography and elution, specifically including: Strain culture stage The inoculation amount is 10% to 15%, the fermentation medium is BS M medium containing glycerol, each liter of fermentation culture liquid contains 4 to 4.5 mL of PTM1, and the culture is carried out under the conditions of aeration and stirring at a temperature of 28° C. to 30° C., a stirring speed of 600 to 700 rpm, a pH of 4.8 to 5.2, and a ventilation volume of 1.2 to 2.0 vvm for 18 to 24 hours; Carbon source feeding stage When glycerol is exhausted during the fermentation process, 10 to 14 mL / LPTM1 of glycerol with a mass volume ratio of 50% is added, the addition amount is 12 to 16 mL / h / L, and the addition time is 4 to 6 hours; Starvation feeding stage When the OD600 reaches 160-180, stop adding glycerol until the glycerol in the culture medium is exhausted; Induced expression stage Methanol containing 10-14 mL / LPTM1 is added to maintain the final methanol concentration in the fermentation broth at 0.2%-0.3%, the pH is controlled at 5.9-6.1, the flow acceleration of the methanol solution is controlled to maintain the dissolved oxygen content in the fermentation broth at 0-10%, an adenosine aqueous solution is added to the culture broth during the induction process to achieve a final adenosine concentration of 0.5 mM-1 mM, and the fermentation is stopped when the enzyme activity no longer increases to obtain a glucose dehydrogenase fermentation broth; Centrifugal stage Collect the glucose dehydrogenase fermentation broth and remove bacterial cells by centrifugation; Ultrafiltration stage The fermentation supernatant is microfiltered using a hollow fiber microfiltration system, and the filtrate is collected; Affinity chromatography stage Purification was performed using a Ni metal chelate ion gel column, and the buffers used included: buffer A: 20 mM Tris-HCl, 0.5 M NaCl, 0 mM imidazole, pH 8.0; buffer B: 20 ​​mM Tris-HCl, 0.5 M NaCl, 250 mM imidazole, pH 8.0, loading flow rate 0.5 mL / min, re-equilibration flow rate 0.5 mL / min; Elution stage The ratio of buffer B to buffer A was adjusted, and imidazole concentrations of 25, 50, 100, 150, 250, and 500 mM were used for elution at a flow rate of 0.5 mL / min.

[0009] Furthermore, before the strain culture stage, an expansion culture stage is required, in which Pichia pastoris carrying a glucose dehydrogenase gene is prepared into a seed liquid.

[0010] Further, Pichia pastoris carrying the glucose dehydrogenase gene is prepared into a seed solution, specifically comprising: Pichia pastoris carrying the glucose dehydrogenase gene was inoculated into 5 mL YPD liquid medium; The primary seeds are prepared by shaking culture at a temperature of 28 to 30° C. and a shaking rate of 200 to 250 rpm for 12 to 16 hours; The first-level seeds are transferred to 80-120 mL of BMGY shake flask medium at an inoculum volume ratio of 3% to 5%; The culture was carried out at a temperature of 28°C to 30°C and a rotation speed of 200 to 250 rpm for 6 to 10 hours until the OD600 was 6 to 8 to prepare a seed solution.

[0011] Furthermore, the amount of the fermentation broth in the reactor is 45% to 56%.

[0012] Furthermore, concentrated ammonia water is used to adjust the pH in the strain culture stage and the induction expression stage.

[0013] Furthermore, during the induction expression stage, samples were taken every 12 hours to detect OD600 and wet weight of the bacteria, and the activity and protein content of the expressed glucose dehydrogenase were measured every 12 hours.

[0014] Furthermore, in the centrifugation stage, the centrifugation temperature is between 4°C and 25°C, and the rotation speed is between 8000 and 12000 rpm.

[0015] Furthermore, in the ultrafiltration stage, the pore size of the hollow fiber is 0.1 μm or 0.22 μm.

[0016] The beneficial effects of the present invention are as follows: The present invention improves the yield and purification yield of FAD-dependent glucose dehydrogenase by adding adenosine and using NTA Ni+ gel chromatography column technology on the basis of the traditional Pichia fermentation process, through experimental exploration of technical parameters. At the same time, the fermentation and purification process provided by the present invention is simple and feasible, with mild conditions, good reproducibility, strong controllability, and suitable for large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The protein electrophoresis diagram of glucose dehydrogenase in Examples 1-3 of the present invention; Figure 2 The protein electrophoresis diagram of glucose dehydrogenase in Example 4-5 of the present invention. DETAILED DESCRIPTION

[0018] The present invention will be further described below in conjunction with specific examples. The following examples are only used to more clearly illustrate the technical solutions of the present invention, and are not intended to limit the scope of protection of the present invention. Example 1

[0019] The method of using adenosine to increase the production of FAD-dependent glucose dehydrogenase is to inoculate Pichia pastoris with recombinant glucose dehydrogenase into a fermentation culture medium with glycerol as a carbon source for cultivation; the method includes the following process steps: A. Strain culture stage The inoculation volume was 15%, and the fermentation medium was BSM medium, with 4.5 mL PTM1 per liter of fermentation medium. The culture was carried out under the conditions of 30°C, 700 rpm, pH=5.2 and 2.0 vvm of ventilation and stirring for 24 hours; B. Carbon source feeding stage When glycerol was exhausted during the fermentation process, 12 mL / L PTM1 with a mass volume ratio of 50% glycerol was added at a rate of 16 mL / h / L for 6 hours. C. Starvation feeding stage When OD600 ≈ 180, stop adding glycerol until the glycerol in the culture medium is exhausted; D. Induction expression stage Methanol containing 14 mL / LPTM1 was fed to maintain the final methanol concentration in the fermentation broth at about 0.1%, the pH was controlled to be 6, the flow acceleration of the methanol solution was controlled to make the dissolved oxygen content in the fermentation broth ≈ 0, and the fermentation was stopped when the enzyme activity no longer increased.

[0020] Before the strain culture stage, an expansion culture stage is required, in which Pichia pastoris with recombinant glucose dehydrogenase is inoculated into 5 mL YPD liquid culture medium and cultured at a temperature of 30° C. and a shaking rate of 250 rpm for 16 hours to prepare a primary seed.

[0021] The primary seeds were transferred to 120 mL of BMGY shake flask medium at a volume ratio of 5% inoculation amount, and cultured at a temperature of 30° C. and a rotation speed of 250 rpm for 10 hours until OD600≈8 to prepare seed solution.

[0022] The loading amount of the fermentation broth in the reactor was 50%.

[0023] In the steps A and D, concentrated ammonia water is used to adjust the pH.

[0024] In step D, samples were taken every 12 hours to detect OD600 and wet weight of the bacteria, and the activity of the expressed glucose dehydrogenase was measured every 12 hours. Example 2

[0025] The difference between this embodiment and embodiment 1 is that: A. Strain culture stage The inoculation amount was 10%, the fermentation medium was BSM medium containing glycerol, each liter of fermentation culture medium contained 4.5 mL of PTM1, and the culture was carried out under aeration and stirring conditions of 30° C., 700 rpm, pH=5.0, and 2.0 vvm for 24 hours; B. Carbon source feeding stage When glycerol was exhausted during the fermentation process, 12 mL / L PTM1 with a mass volume ratio of 50% glycerol was added at a rate of 14 mL / h / L for 6 hours. C. Starvation feeding stage When OD600 ≈ 160, stop adding glycerol until the glycerol in the culture medium is exhausted; D. Induction expression stage Methanol containing 14 mL / L PTM1 was added to maintain the final methanol concentration in the fermentation broth at about 0.1%, the pH was controlled at 5, the flow rate of the methanol solution was controlled to make the dissolved oxygen content in the fermentation broth ≈ 0, and adenosine aqueous solution was added to the culture medium during the induction process. The final concentration of adenosine in the nutrient solution was 1 mM, and the fermentation was stopped when the enzyme activity no longer increased.

[0026] The rest of the contents are the same as in Example 1. Example 3

[0027] The difference between this embodiment and embodiment 1 is that: A. Strain culture stage The inoculation amount was 10%, the fermentation medium was BSM medium containing glycerol, each liter of fermentation culture medium contained 4.5 mL of PTM1, and the culture was carried out under aeration and stirring conditions of 30° C., 700 rpm, pH=5.5, and 2.0 vvm for 24 hours; B. Carbon source feeding stage When glycerol was exhausted during the fermentation process, 12 mL / L PTM1 with a mass volume ratio of 50% glycerol was added at a rate of 14 mL / h / L for 6 hours. C. Starvation feeding stage When OD600 ≈ 160, stop adding glycerol until the glycerol in the culture medium is exhausted; D. Induction expression stage Methanol containing 14 mL / L PTM1 was fed to maintain the final methanol concentration in the fermentation broth at about 0.1%, the pH was controlled at 5.5, and the flow acceleration of the methanol solution was controlled to make the dissolved oxygen content in the fermentation broth ≈ 0. During the induction process, adenosine aqueous solution was added to the culture medium to a final adenosine concentration of 0.1 mM. Fermentation was stopped when the enzyme activity no longer increased.

[0028] The rest of the contents are the same as in Example 1.

[0029] The enzymatic activity of the glucose dehydrogenase obtained in Example 1-3 was measured, and the protein expression level was measured. The results are as follows:

[0030] Reference Figure 1 As shown in the above experiments, it is not difficult to see that by adding adenosine, the specific enzyme activity and protein expression of glucose dehydrogenase are increased. The increase range is 13.8% to 35.8% and 32% to 44.8%. Example 4

[0031] Based on the above fermentation process, the purification process using Ni metal chelate ion gel column includes the following steps: A. Centrifugal stage The glucose dehydrogenase fermentation broth was collected and the bacterial cells were removed by centrifugation at 4°C, 8000 rpm.

[0032] B. Ultrafiltration stage The fermentation supernatant was microfiltered using a 0.22 μm hollow fiber microfiltration system, and the filtrate was collected.

[0033] C. Affinity chromatography stage Ni-IDA metal chelate ion gel column was used for purification, and the buffer used was: Buffer A: 20 mMTris-HCl, 0.5 M NaCl, 0 mM imidazole, pH 8.0; Buffer B: 20 ​​mM Tris-HCl, 0.5 M NaCl, 250 mM imidazole, pH 8.0. The loading flow rate was 0.5 mL / min, and the re-equilibration flow rate was 0.5 mL / min.

[0034] D. Elution stage The ratio of buffer B to buffer A was adjusted, and imidazole concentrations of 25, 50, 100, 150, 250, and 500 mM were used for elution at a flow rate of 0.5 mL / min. Example 5

[0035] The difference between this embodiment and embodiment 4 is that: A. Centrifugal stage The glucose dehydrogenase fermentation broth was collected and the bacterial cells were removed by centrifugation at 25°C and 12,000 rpm.

[0036] B. Ultrafiltration stage The fermentation supernatant was microfiltered using a 0.1 micron hollow fiber microfiltration system, and the filtrate was collected.

[0037] C. Affinity chromatography stage Purification was performed using a Ni-NTA metal chelate ion gel column.

[0038] The rest of the contents are the same as in Example 4.

[0039] The applicant measured the enzyme activity of the glucose dehydrogenase obtained in Example 4 and Example 5, and measured the protein expression level. The results are as follows:

[0040] Reference Figure 2 As shown, it is not difficult to see from the above experiments that by changing the NI+ metal chelate ions, the recovery rate of the target protein can be effectively improved.

[0041] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the technical principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A fermentation and purification method for recombinant glucose dehydrogenase in Pichia pastoris, characterized in that: include: Pichia pastoris containing recombinant glucose dehydrogenase is added into the fermentation system to produce glucose dehydrogenase; The fermentation broth after fermentation is subjected to centrifugation, ultrafiltration, affinity chromatography and elution, specifically including: Strain culture stage The inoculation amount is 10% to 15%, the fermentation medium is BS M medium containing glycerol, each liter of fermentation culture liquid contains 4 to 4.5 mL PTM1, and the culture is carried out under the conditions of a temperature of 28° C. to 30° C., a stirring speed of 600 to 700 rpm, a pH of 4.8 to 5.2, and a ventilation rate of 1.2 to 2.0 vvm for 18 to 24 hours; Carbon source feeding stage When glycerol is exhausted during the fermentation process, 10-14 mL / L PTM1 with a mass volume ratio of 50% glycerol is fed at a rate of 12-16 mL / h / L for 4-6 hours; Starvation feeding stage When the OD600 reaches 160-180, stop adding glycerol until the glycerol in the culture medium is exhausted; Induced expression stage Methanol containing 10-14 mL / LPTM1 is added to maintain the final methanol concentration in the fermentation broth at 0.2%-0.3%, the pH is controlled at 5.9-6.1, the flow acceleration of the methanol solution is controlled to maintain the dissolved oxygen content in the fermentation broth at 0-10%, an adenosine aqueous solution is added to the culture broth during the induction process to achieve a final adenosine concentration of 0.5 mM-1 mM, and the fermentation is stopped when the enzyme activity no longer increases to obtain a glucose dehydrogenase fermentation broth; Centrifugal stage Collect the glucose dehydrogenase fermentation broth and remove bacterial cells by centrifugation; Ultrafiltration stage The fermentation supernatant is microfiltered using a hollow fiber microfiltration system, and the filtrate is collected; Affinity chromatography stage Purification was performed using a Ni metal chelate ion gel column, and the buffers used included: buffer A: 20 mM Tris-HCl, 0.5 M NaCl, 0 mM imidazole, pH 8.0; buffer B: 20 ​​mM Tris-HCl, 0.5 M NaCl, 250 mM imidazole, pH 8.0, loading flow rate 0.5 mL / min, re-equilibration flow rate 0.5 mL / min; Elution stage The ratio of buffer B to buffer A was adjusted, and imidazole concentrations of 25, 50, 100, 150, 250, and 500 mM were used for elution at a flow rate of 0.5 mL / min.

2. The fermentation and purification method of a recombinant glucose dehydrogenase in Pichia pastoris according to claim 1, characterized in that: Before the strain culture stage, an expansion culture stage is required, in which Pichia pastoris carrying the glucose dehydrogenase gene is made into a seed liquid.

3. The fermentation and purification method of a recombinant glucose dehydrogenase in Pichia pastoris according to claim 2, characterized in that: The Pichia pastoris carrying the glucose dehydrogenase gene is prepared into a seed liquid, specifically comprising: Pichia pastoris carrying the glucose dehydrogenase gene was inoculated into 5 mL YPD liquid medium; The primary seeds are prepared by shaking culture at a temperature of 28 to 30° C. and a shaking rate of 200 to 250 rpm for 12 to 16 hours; The first-level seeds are transferred to 80-120 mL of BMGY shake flask medium at an inoculum volume ratio of 3% to 5%; The culture was carried out at a temperature of 28°C to 30°C and a rotation speed of 200 to 250 rpm for 6 to 10 hours until the OD600 was 6 to 8 to prepare a seed solution.

4. The fermentation and purification method of recombinant glucose dehydrogenase in Pichia pastoris according to claim 1, characterized in that: The loading amount of the fermentation culture liquid in the reaction kettle is 45% to 56%.

5. The fermentation and purification method of recombinant glucose dehydrogenase in Pichia pastoris according to claim 1, characterized in that: Concentrated ammonia water is used to adjust the pH in the strain culture stage and the induction expression stage.

6. The fermentation and purification method of recombinant glucose dehydrogenase in Pichia pastoris according to claim 1, characterized in that: During the induction expression stage, samples were taken every 12 hours to detect OD600 and wet weight of the bacteria, and the activity and protein content of the expressed glucose dehydrogenase were measured every 12 hours.

7. The fermentation and purification method of recombinant glucose dehydrogenase in Pichia pastoris according to claim 1, characterized in that: In the centrifugation stage, the centrifugation temperature is between 4°C and 25°C, and the rotation speed is between 8000 and 12000 rpm.

8. The fermentation and purification method of recombinant glucose dehydrogenase in Pichia pastoris according to claim 1, characterized in that: In the ultrafiltration stage, the pore size of the hollow fiber is 0.1 μm or 0.22 μm.

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