Method for producing psicose 3-epimerase by high cell density fermentation

Abstract

To provide, in the technical field of microbial fermentation engineering, particularly a method for producing psicose 3-epimerase by high-density fermentation, and solve problems existing in conventional fermentation, such as a low expression level of psicose 3-epimerase.SOLUTION: By controlling the feeding rate in a fermentation process, by improving the culture temperature in middle and late stages of the fermentation and by other measures, an OD value during the fermentation with recombinant Bacillus subtilis and the total enzyme activity of psicose 3-epimerase in a fermentation broth are significantly increased, the expression of psicose 3-epimerase is markedly improved, and the production cost of allulose is reduced. Therefore, the present invention has a very broad prospect of application in industry.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] This application claims the priority of Chinese Patent Application No. 202210288790.2, filed on March 22, 2022, which is incorporated herein by reference in its entirety for all purposes as if fully set forth herein.

[0002] The present invention relates to the technical field of microbial fermentation engineering, and particularly to a method for producing psicose 3-epimerase by high-density fermentation.

Background Art

[0003] D-Psicose is a rare sugar that has 70% of the sweetness of sucrose while having low calories and can be a good substitute for sucrose. Furthermore, D-psicose has a lower absorption rate compared to other sweeteners, can suppress the absorption of fructose and glucose in the body, and reduce fat accumulation, thus reducing the risk of diseases such as type 2 diabetes and obesity. Currently, it has been found in research that D-psicose also has the function of lowering blood lipids and blood glucose levels. Since the US Food and Drug Administration (FDA) has recognized D-psicose as Generally Recognized as Safe (GRAS), D-psicose can be used as a component of food additives. D-Psicose is expected to have a wide range of applications in food and healthcare products.

[0004] D-Psicose 3-epimerase can epimerize D-fructose to D-psicose and is an important enzyme in the production of psicose. The production of this enzyme greatly affects the production cost of psicose.

[0005] Currently, research on D-psicose mainly focuses on the construction of genetically recombinant strains, site-directed mutagenesis and directed evolution of enzymes, and enzyme immobilization. There is little research on methods to increase the fermentation density of genetically recombinant strains expressing psicose 3-epimerase and significantly increase the expression level.

[0006] High-density fermentation refers to a highly efficient fermentation technology in liquid fermentation by microorganisms, which increases the cell density to more than 10 times that of the prior art through the control of fermentation parameters and feed control. High-density fermentation can increase the cell density in the fermentation tank and the expression of the product. Accordingly, the volume of the fermentation tank is reduced, the production capacity of the device per unit volume is increased, the cost of biomass separation is reduced, and the manufacturing period is shortened. As a result, the manufacturing cost is reduced and the manufacturing efficiency is improved. Therefore, high-density fermentation has great significance in industrial applications.

[0007] Chinese Patent Application Publication No. 112852795 (CN112852795A) provides a method for producing psicose 3-epimerase by fermentation. In this patent, by optimizing the SR medium and fermentation conditions, the activity of psicose 3-epimerase finally reaches 1436 U / mL. However, in this method, the feeding control strategy during fermentation to achieve high-density fermentation is not adopted, and the expression level during fermentation still needs to be improved.

Prior Art Documents

Patent Documents

[0008]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0009] In view of the low expression level, low purity, high cost and other defects and drawbacks of psicose 3-epimerase in fermentation and production in the prior art, the object of the present invention is to provide a method for producing psicose 3-epimerase by high-density fermentation. This method can significantly improve the cell density during fermentation, increase the expression level and purity of psicose 3-epimerase, reduce the manufacturing cost, and has great significance in industrial applications.

Means for Solving the Problems

[0010] The object of the present invention is achieved by the following technical solutions.

[0011] A method for producing psicose 3-epimerase by high-density fermentation includes the following steps: (1) Activation of the strain Inoculating and activating a recombinant Bacillus subtilis strain in an activation medium to obtain an activated seed cell suspension; and (2) Fermentation culture Fermentation culture: Adding 1-10% by volume of the activated seed cell suspension cultured in step (1) to a fermentation medium and fermenting.

[0012] Initial setting of the fermentation conditions: temperature 37.0 °C, air flow rate 1-6 L / min, oxygen flow rate 0-3 L / min, rotation speed 200-1000 rpm. Control of the fermentation process: During fermentation, the dissolved oxygen is controlled in the range of 10%-20%, and when OD 600 rises to 80-120, the fermentation temperature is raised to 40-42 °C. During the fermentation process, when OD 600 rises to 5-20, the fed-batch medium is added continuously at a flow rate of 10-30 mL / hour. When OD 600 rises to 30-50, the fed-batch medium is added continuously at a flow rate of 30-60 mL / hour. When OD 600 rises to 50-80, the fed-batch medium is added continuously at a flow rate of 60-90 mL / hour. When OD 600 rises to 80-100, the fed-batch medium is added continuously at a flow rate of 90-120 mL / hour.

[0013] The recombinant Bacillus subtilis described in step (1) is preferably a genetically engineered recombinant Bacillus subtilis B-3-1 strain (the name "Psicose 3-epimerase variant, genetically engineered strain expressing the variant, and its use" disclosed in Application No. 202010496928.9).

[0014] The activation medium in step (1) contains 5 - 15 g / L of peptone, 1 - 5 g / L of yeast powder, 8 - 12 g / L of sodium chloride, and 25 - 50 mg / L of kanamycin.

[0015] The activation medium in step (1) preferably contains 10 g / L of peptone, 5 g / L of yeast powder, 10 g / L of sodium chloride, and 25 - 50 mg / L of kanamycin.

[0016] The activation of the recombinant Bacillus subtilis in step (1) preferably specifically includes the following: Primary strain activation: Inoculate 0.1 - 1% by volume of the frozen recombinant Bacillus subtilis cell suspension into the activation medium, and culture it with shaking at a certain temperature to obtain a primary activated seed cell suspension. Here, the culture temperature is 37.0 °C, the rotation speed is 180 - 220 rpm, and the culture time is 18 - 24 hours; and Secondary strain activation: Inoculate 1 - 10% by volume of the cultured primary activated seed cell suspension into the activation medium, and culture it with shaking at a certain temperature to obtain a secondary activated seed cell suspension. Here, the culture temperature is 37.0 °C, the rotation speed is 180 - 220 rpm, and the culture time is 6 - 12 hours.

[0017] The primary activated seed cell suspension is preferably prepared as follows: Inoculate 0.2% by volume of the frozen recombinant Bacillus subtilis cell suspension into the activation medium, and culture the cells with shaking at a certain temperature until the OD 600 value of the cell culture reaches 1.0 - 3.0 to obtain a primary activated seed cell suspension. Here, the culture temperature is 37.0 °C, the rotation speed is 200 rpm, and the culture time is 18 - 24 hours.

[0018] The secondary activated seed cell suspension is preferably prepared as follows: Inoculate 10% by volume of the cultured primary activated seed cell suspension into the activation medium, and the OD of the cell culture 600Cultivate while shaking at a constant temperature until the value reaches 4.0 - 6.0 to obtain a secondary activated cell suspension. Here, the culture temperature is 37.0 °C, the rotation speed is 200 rpm, and the culture time is 6 - 12 hours.

[0019] The fermentation medium in step (2) contains 5 - 15 g / L of peptone, 1 - 5 g / L of yeast powder, 0.5 - 5 g / L of potassium dihydrogen phosphate, 5 - 20 g / L of dipotassium hydrogen phosphate, 0.02 - 0.2 g / L of manganese chloride tetrahydrate, and 2 - 10 g / L of glucose.

[0020] The fermentation medium in step (2) preferably contains 10 g / L of peptone, 5 g / L of yeast powder, 2.5 g / L of potassium dihydrogen phosphate, 15 g / L of dipotassium hydrogen phosphate, 0.1 g / L of manganese chloride tetrahydrate, and 6 g / L of glucose.

[0021] The fed-batch medium in step (2) preferably contains 10 - 30 wt% of glucose and 10 - 30 wt% of yeast powder. The specific concentrations, feeding rate, and feeding amount of glucose and yeast powder depend on the growth of the strain.

[0022] The activation medium, fermentation medium, and fed-batch medium in steps (1) and (2) are preferably sterilized at high temperature. The components other than kanamycin in the activation medium are sterilized at high temperature, and then kanamycin is added.

[0023] The high-temperature sterilization is preferably sterilization at 121 °C for 20 minutes.

[0024] The fermentation culture in step (2) is preferably Fermentation culture: adding 10% by volume of the activated cell suspension cultured in step (1) to the fermentation medium and fermenting, which is included.

[0025] The initial setting of the fermentation conditions is a temperature of 37.0 °C, an air flow rate of 5 L / min, an oxygen flow rate of 1 L / min, and a rotation speed of 800 rpm. In order to control the fermentation process, during fermentation, the dissolved oxygen is controlled at 15%, OD 600When it rises to 100, the fermentation temperature rises to 40°C. During the fermentation process, when OD 600 rises to 10, the feeding medium is added continuously at a flow rate of 15 mL / hour. When OD 600 rises to 35, the feeding medium is added continuously at a flow rate of 40 mL / hour. When OD 600 rises to 60, the feeding medium is added continuously at a flow rate of 80 mL / hour. When OD 600 rises to 90, the feeding medium is added continuously at a flow rate of 110 mL / hour.

[0026] The method for producing psicose 3-epimerase by high-density fermentation of the present invention further comprises the following steps: When the enzyme activity no longer increases, stop the fermentation, perform solid-liquid separation to obtain a crude enzyme solution, and further purify it to obtain psicose 3-epimerase. It includes.

[0027] As the method for solid-liquid separation, centrifugation is preferred.

[0028] The present invention further provides the use of the method for producing psicose 3-epimerase by high-density fermentation in the production of D - psicose.

[0029] The principle of the present invention: (1) For the growth and metabolism of microorganisms, inorganic nitrogen (such as ammonium chloride, ammonium sulfate, etc.) or organic nitrogen (such as peptone, yeast powder, etc.) can be used. When using inorganic nitrogen, it is required that the microorganisms convert the inorganic nitrogen into amino acids by their own metabolism to complete the expression of proteins. Therefore, the metabolic load of the cells increases, which affects the expression of proteins. In addition, excessive ammonia concentration causes ammonia poisoning of the cells. The present invention provides a fermentation medium and a feeding medium added with organic nitrogen. The amino acids contained are directly absorbed and utilized by the cells, which can reduce the metabolic load of the cells and increase the expression of psicose 3-epimerase.

[0030] (2) The presence of metal ions is often important for the activity of enzymes in the following aspects: Metal ions can form a complex with the enzyme as a coenzyme and become a component of the active center of the enzyme; they can stabilize the conformation of the enzyme molecule; and they can function as a bridge between the enzyme and the substrate. It has been found through research that manganese ions can promote the enzyme activity of psicose 3-epimerase expressed in the present invention, while the presence of other metal ions such as magnesium ions, calcium ions, iron ions, copper ions, and zinc ions has an inhibitory effect on the enzyme activity. Therefore, in order to improve the enzyme activity of psicose 3-epimerase, an appropriate amount of manganese chloride is added to the fermentation medium of the present invention.

[0031] (3) Enzymes are generally secreted through transport proteins on the cell membrane, and their secretion rate is limited by the permeability of the cell membrane and the metabolic rate of the cell. In addition, cell death and lysis promote the release of intracellular enzymes, and at the same time, impurity proteins in the cell are also released, resulting in a decrease in the purity of the target protein and an increase in the difficulty and cost of subsequent separation and purification. In the present invention, a method of increasing the culture temperature in the middle and late stages of fermentation is adopted to improve the permeability of the cell membrane and increase the metabolic rate of the cell, thereby promoting the secretion of the target enzyme, avoiding the decrease in enzyme purity caused by cell death and lysis in the late stage of fermentation, facilitating subsequent separation and purification, and promoting cost reduction.

[0032] (4) In the fed-batch process of fermentation, the cell density increases during the feeding. When the cell density is high, the expression of the enzyme is likely to be improved. However, if the glucose concentration in the medium is too high due to inappropriate fed-batch control, the expression of the CcpA protein (carbon catabolite repression regulator) in the cell is induced, and the transcription and translation of the protein are suppressed. In addition, the plasmid may disappear because the growth rate of the cell is too high due to the high concentration of the carbon source. Therefore, in the present invention, according to the fact that the amount of nutrients required varies depending on the difference in cell density at different times, different OD 600A policy is established to adjust the feeding amount according to the value, and in the fermentation process, the carbon source and nitrogen source are maintained at a relatively balanced and low level, thereby increasing the expression of psicose 3-epimerase and reducing the production cost of psicose.

[0033] The present invention has the following advantages and effects compared with the prior art.

[0034] (1) In the present invention, by controlling the organic nitrogen content in the fermentation medium and the feeding medium during the fermentation process, the metabolic load of the cells is reduced, and the expression of psicose 3-epimerase is improved.

[0035] (2) In the present invention, by controlling the content of manganese chloride in the fermentation medium during the fermentation process, the enzyme activity of psicose 3-epimerase is improved.

[0036] (3) In the present invention, the policy of high-density fermentation is adopted, and by controlling the supply rate at different cell densities during the fermentation process, the cell density of recombinant Bacillus subtilis during fermentation is significantly increased, and the OD 600 value increases to 150 or more.

[0037] (4) In the present invention, by raising the culture temperature in the middle and late stages of fermentation to 40-42 °C, the permeability of the cell membrane is improved, the metabolic rate of the cells is increased, thereby promoting the secretion of the target enzyme, avoiding the decrease in enzyme purity due to cell death and lysis in the late stage of fermentation, facilitating subsequent separation and purification, and promoting cost reduction.

[0038] (5) By the method for producing psicose 3-epimerase provided by the present invention, the total enzyme activity of psicose 3-epimerase in the fermentation broth can be increased to 4783 U / mL. By this method, the expression of psicose 3-epimerase during fermentation is significantly improved.

[0039] (6) In the present invention, the production cost of psicose is reduced. Therefore, the present invention is expected to be very widely applied in the industry.

Brief Description of the Drawings

[0040]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Modes for Carrying Out the Invention

[0041] (Detailed Description of the Preferred Embodiment) Hereinafter, the present invention will be described in more detail with reference to Examples and the accompanying drawings. However, the present invention is not limited thereto.

[0042] The recombinant Bacillus subtilis in step (1) in the examples is the genetically engineered recombinant Bacillus subtilis strain B-3-1 disclosed in Chinese Patent Application No. 202010496928.9, titled "Psicose 3-epimerase variant, genetically engineered strain expressing the variant, and its use".

[0043] Unless otherwise specified, commercially available reagents are used in the following examples.

Examples

[0044] Example 1: High-density fermentation (using peptone and yeast powder as nitrogen sources in the fermentation medium and the feeding medium)

[0045] (1) Preparation of the medium A. Activation medium: Composition of the activation medium: peptone 10 g / L, yeast powder 5 g / L, sodium chloride 10 g / L (sterilized at 121 °C for 20 minutes, and 50 mg / L of kanamycin was added after sterilization).

[0046] B. Fermentation medium: Composition of the fermentation medium: peptone 10 g / L, yeast powder 5 g / L, potassium dihydrogen phosphate 2.5 g / L, dipotassium hydrogen phosphate 15 g / L, manganese chloride tetrahydrate 0.1 g / L, glucose 6 g / L (sterilized at 121 °C for 20 minutes).

[0047] C. Feeding medium: Composition of the feeding medium: glucose 15 wt%, yeast powder 20 wt% (sterilized at 121 °C for 20 minutes).

[0048] (2) Activation of the strain Activation of the primary strain: 100 μL of the preserved cell suspension of the genetically engineered recombinant Bacillus subtilis strain B-3-1 was inoculated into a 250 mL Erlenmeyer flask containing 50 mL of the activation medium, and the cells were cultured with shaking at a constant temperature until the OD 600 value reached 2.0 to obtain a primary activated seed cell suspension. The culture temperature was 37.0 °C, the rotation speed was 200 rpm, and the culture time was 24 hours.

[0049] Activation of secondary strain: 50 mL of the cultured primary activated cell suspension was inoculated into a 3000 mL Erlenmeyer flask containing 500 mL of activation medium, and the OD of the cell suspension 600 was shake-cultured at a constant temperature until the value reached 5.0 to obtain a secondary activated cell suspension. The culture temperature was 37.0 °C, the rotation speed was 200 rpm, and the culture time was 8 hours.

[0050] (3) Fermentation culture Fermentation culture: 3150 mL of fermentation medium was added to a 5 L fermentation tank and sterilized at 121 °C for 20 minutes. 350 mL of the secondary activated cell suspension obtained in step (2) was added to the fermentation medium and fermented. Initial setting of fermentation conditions: temperature 37.0 °C, air flow rate 5 L / min, oxygen flow rate 1 L / min, rotation speed 800 rpm. Control of fermentation process: During the fermentation process, the dissolved oxygen was controlled at 15%, and when the OD 600 rose to 100, the fermentation temperature was raised to 40 °C. During the fermentation process, when the OD 600 rose to 10, the feeding medium was added continuously at a flow rate of 15 mL / hour. When the OD 600 rose to 35, the feeding medium was added continuously at a flow rate of 40 mL / hour. When the OD 600 rose to 60, the feeding medium was added continuously at a flow rate of 80 mL / hour. When the OD 600 rose to 90, the feeding medium was added continuously at a flow rate of 110 mL / hour.

[0051] (4) Discharge from the fermentation tank: Fermentation was stopped when the enzyme activity no longer increased, and the fermentation broth was centrifuged at 4000 rpm for 30 minutes to obtain a crude enzyme solution.

[0052] Example 2: High-density fermentation (using peptone and yeast powder as nitrogen sources for the fermentation medium and feeding medium)

[0053] (1) Preparation of medium A. Activation medium: Composition of activation medium: peptone 5 g / L, yeast powder 1 g / L, sodium chloride 8 g / L (sterilized at 121 °C for 20 minutes, and 25 mg / L of kanamycin was added after sterilization).

[0054] B. Fermentation Medium: Composition of the fermentation medium: 5 g / L of peptone, 1 g / L of yeast powder, 0.5 g / L of potassium dihydrogen phosphate, 5 g / L of dipotassium hydrogen phosphate, 0.02 g / L of manganese chloride tetrahydrate, 2 g / L of glucose (sterilized at 121 °C for 20 minutes).

[0055] C. Feed Medium: Composition of the feed medium: 10 wt% of glucose, 10 wt% of yeast powder (sterilized at 121 °C for 20 minutes).

[0056] (2) Activation of the Strain Activation of the primary strain: 50 μL of the preserved cell suspension of the genetically engineered recombinant Bacillus subtilis B-3-1 strain was inoculated into a 250 mL Erlenmeyer flask containing 50 mL of the activation medium, and the OD of the cell suspension 600 was cultured with shaking at a constant temperature until the value reached 1.0 to obtain a primary activated seed cell suspension. The culture temperature was 37.0 °C, the rotation speed was 180 rpm, and the culture time was 24 hours.

[0057] Activation of the secondary strain: 5 mL of the cultured primary activated seed cell suspension was inoculated into a 3000 mL Erlenmeyer flask containing 500 mL of the activation medium, and the OD of the cell suspension 600 was cultured with shaking at a constant temperature until the value reached 4.0 to obtain a secondary activated seed cell suspension. The culture temperature was 37.0 °C, the rotation speed was 180 rpm, and the culture time was 12 hours.

[0058] (3) Fermentation Culture Fermentation culture: 3150 mL of the fermentation medium was added to a 5 L fermentation tank and sterilized at 121 °C for 20 minutes. 350 mL of the secondary activated seed cell suspension obtained in step (2) was added to the fermentation medium and fermented. Initial setting of the fermentation conditions: temperature 37.0 °C, air flow rate 1 L / min, oxygen flow rate 0 L / min, and rotation speed 200 rpm. Control of the fermentation process: During the fermentation process, the dissolved oxygen was controlled at 10%, and when the OD 600 rose to 80, the fermentation temperature was raised to 40 °C. During the fermentation process, when the OD 600When it increased to 5, the feeding medium was added continuously at a flow rate of 10 mL / hour, and the OD 600 When it increased to 30, the feeding medium was added continuously at a flow rate of 30 mL / hour, and the OD 600 When it increased to 50, the feeding medium was added continuously at a flow rate of 60 mL / hour, and the OD 600 When it increased to 80, the feeding medium was added continuously at a flow rate of 90 mL / hour.

[0059] (4) Discharge from the fermentation tank: When the enzyme activity no longer increased, the fermentation was stopped, and the fermentation broth was centrifuged at 4000 rpm for 30 minutes to obtain a crude enzyme solution.

[0060] Example 3: High-density fermentation (using peptone and yeast powder as nitrogen sources for the fermentation medium and the feeding medium)

[0061] (1) Preparation of the medium A. Activation medium: Composition of the activation medium: peptone 15 g / L, yeast powder 5 g / L, sodium chloride 10 g / L (sterilized at 121 °C for 20 minutes, and 50 mg / L of kanamycin was added after sterilization).

[0062] B. Fermentation medium: Composition of the fermentation medium: peptone 15 g / L, yeast powder 5 g / L, potassium dihydrogen phosphate 5 g / L, dipotassium hydrogen phosphate 20 g / L, manganese chloride tetrahydrate 0.2 g / L, glucose 10 g / L (sterilized at 121 °C for 20 minutes).

[0063] C. Feeding medium: Composition of the feeding medium: glucose 30 wt%, yeast powder 30 wt% (sterilized at 121 °C for 20 minutes).

[0064] (2) Activation of the strain Activation of the primary strain: 500 μL of the preserved cell suspension of the genetically engineered recombinant Bacillus subtilis B-3-1 strain was inoculated into a 250 mL Erlenmeyer flask containing 50 mL of the activation medium, and the OD of the cell suspension 600It was cultured while shaking at a constant temperature until the value reached 3.0 to obtain a primary activated cell suspension. The culture temperature was 37.0 °C, the rotation speed was 220 rpm, and the culture time was 18 hours.

[0065] Activation of the secondary strain: 50 mL of the cultured primary activated cell suspension was inoculated into a 3000 mL Erlenmeyer flask containing 500 mL of the activation medium, and the OD of the cell suspension 600 was cultured with shaking at a constant temperature until the value reached 6.0 to obtain a secondary activated cell suspension. The culture temperature was 37.0 °C, the rotation speed was 220 rpm, and the culture time was 6 hours.

[0066] (3) Fermentation culture Fermentation culture: 3150 mL of the fermentation medium was added to a 5 L fermentation tank and sterilized at 121 °C for 20 minutes. 350 mL of the secondary activated cell suspension obtained in step (2) was added to the fermentation medium and fermented. Initial setting of fermentation conditions: temperature 37.0 °C, air flow rate 6 L / min, oxygen flow rate 3 L / min, rotation speed 1000 rpm. Control of the fermentation process: During the fermentation process, the dissolved oxygen was controlled at 20%, and when the OD 600 rose to 120, the fermentation temperature was raised to 42 °C. During the fermentation process, when the OD 600 rose to 20, the feeding medium was added continuously at a flow rate of 30 mL / hour. When the OD 600 rose to 50, the feeding medium was added continuously at a flow rate of 60 mL / hour. When the OD 600 rose to 80, the feeding medium was added continuously at a flow rate of 90 mL / hour. When the OD 600 rose to 100, the feeding medium was added continuously at a flow rate of 120 mL / hour.

[0067] (4) Discharge from the fermentation tank: Fermentation was stopped when the enzyme activity no longer increased, and the fermentation broth was centrifuged at 4000 rpm for 30 minutes to obtain a crude enzyme solution.

[0068] Comparative Example 1: Isothermal fermentation at 37 °C without feeding

[0069] (1) Preparation of the medium A. Activation medium: The same as in Example 1. B. Fermentation medium: The same as in Example 1.

[0070] (2) Activation of the strain Activation of the primary strain: The same as in Example 1. Activation of the secondary strain: The same as in Example 1.

[0071] (3) Fermentation culture Fermentation culture: 3150 mL of the fermentation medium was added to a 5 L fermentation tank and sterilized at 121 °C for 20 minutes. 350 mL of the secondary activated seed cell suspension obtained in step (2) was added to the fermentation medium and fermented. Initial setting of fermentation conditions: temperature 37.0 °C, air flow rate 5 L / min, oxygen flow rate 1 L / min, rotation speed 800 rpm. Control of the fermentation process: During the fermentation process, the dissolved oxygen was controlled at 15% by controlling the rotation speed and the introduction rate of pure oxygen.

[0072] (4) Discharge from the fermentation tank: The same as in Example 1.

[0073] Comparative Example 2: Isothermal fermentation at 37 °C with feeding

[0074] (1) Preparation of the medium A. Activation medium: The same as in Example 1. B. Fermentation medium: The same as in Example 1. C. Feeding medium: The same as in Example 1.

[0075] (2) Activation of the strain Activation of the primary strain: The same as in Example 1. Activation of the secondary strain: The same as in Example 1.

[0076] (3) Fermentation culture: 3150 mL of fermentation medium was added to a 5 L fermentation tank and sterilized at 121 °C for 20 minutes. 350 mL of the secondary activated seed cell suspension obtained in step (2) was added to the fermentation medium and fermented. Initial setting of fermentation conditions: temperature 37.0 °C, air flow rate 5 L / min, oxygen flow rate 1 L / min, rotation speed 800 rpm. Control of the fermentation process: During the fermentation process, the dissolved oxygen was controlled at 15%. During the fermentation process, when OD 600 rose to 10, the feeding medium was added continuously at a flow rate of 15 mL / h. When OD 600 rose to 35, the feeding medium was added continuously at a flow rate of 40 mL / h. When OD 600 rose to 60, the feeding medium was added continuously at a flow rate of 80 mL / h. When OD 600 rose to 90, the feeding medium was added continuously at a flow rate of 110 mL / h.

[0077] (4) Discharge from the fermentation tank: The same as in Example 1.

[0078] Comparative Example 3: Fermentation without feeding, raising the temperature to 40 °C in the middle and late stages of fermentation

[0079] (1) Preparation of the medium A. Activation medium: The same as in Example 1. B. Fermentation medium: The same as in Example 1.

[0080] (2) Activation of the strain Activation of the primary strain: The same as in Example 1. Activation of the secondary strain: The same as in Example 1.

[0081] (3) Fermentation culture Fermentation culture: 3150 mL of fermentation medium was added to a 5 L fermentation tank and sterilized at 121 °C for 20 minutes. 350 mL of the secondary activated seed cell suspension obtained in step (2) was added to the fermentation medium and fermented. Initial setting of fermentation conditions: temperature 37.0 °C, air flow rate 5 L / min, oxygen flow rate 1 L / min, rotation speed 800 rpm. Control of the fermentation process: During the fermentation process, the dissolved oxygen was controlled at 15% by controlling the rotation speed and the introduction rate of pure oxygen. OD 600 When it increased to 20, the fermentation temperature was raised to 40 °C.

[0082] (4) Discharge from the fermentation tank: The same as in Example 1.

[0083] Comparative Example 4: Fermentation with a fermentation medium and a feeding medium using ammonium chloride as a nitrogen source

[0084] (1) Preparation of the medium A. Activation medium: The same as in Example 1. B. Fermentation medium: Composition of the fermentation medium: 5 g / L of ammonium chloride, 2.5 g / L of potassium dihydrogen phosphate, 15 g / L of dipotassium hydrogen phosphate, 0.1 g / L of manganese chloride tetrahydrate, 6 g / L of glucose (sterilized at 121 °C for 20 minutes). C. Feeding medium: Composition of the feeding medium: 20 wt% glucose, 10 wt% ammonium chloride (sterilized at 121 °C for 20 minutes).

[0085] (2) Activation of the strain Activation of the primary strain: The same as in Example 1. Activation of the secondary strain: The same as in Example 1.

[0086] (3) Fermentation culture Fermentation culture: 3150 mL of fermentation medium was added to a 5 L fermentation tank and sterilized at 121 °C for 20 minutes. 350 mL of the secondary activated seed cell suspension obtained in step (2) was added to the fermentation medium and fermented. Initial setting of fermentation conditions: temperature 37.0 °C, air flow rate 5 L / min, oxygen flow rate 1 L / min, rotation speed 800 rpm. Control of the fermentation process: During the fermentation process, the dissolved oxygen was controlled at 15% and OD600 When it rises to 100, raise the fermentation temperature to 40 °C. During the fermentation process, when OD 600 rises to 10, add the fed-batch medium continuously at a flow rate of 15 mL / hour. When OD 600 rises to 35, add the fed-batch medium continuously at a flow rate of 40 mL / hour. When OD 600 rises to 60, add the fed-batch medium continuously at a flow rate of 80 mL / hour. When OD 600 rises to 90, add the fed-batch medium continuously at a flow rate of 110 mL / hour.

[0087] (4) Discharge from the fermentation tank: The same as in Example 1.

[0088] Effect Example (1) Detection of samples during fermentation 1. Detection of absorbance: In Example 1 and Comparative Examples 1 to 4, the fermentation broth was sampled every 4 hours after the start of fermentation, and the OD 600 value of the fermentation broth was detected and recorded with an ultraviolet spectrophotometer. A growth curve was plotted.

[0089] 2. Detection of enzyme activity: Using a 10 wt% fructose solution (containing potassium phosphate buffer pH 8.0, 1 mM Mn 2+ ) as a substrate, an appropriate amount of the fermentation broth of Comparative Examples 1 to 4 was added and reacted at 60 °C for 20 minutes. After the reaction, the reaction system was treated in a boiling water bath for 5 minutes, and the content of the product D-psicose was measured by HPLC, and the enzyme activity was calculated therefrom.

[0090] (2) Detection by SDS-PAGE: The crude enzyme solutions prepared in Example 1, Comparative Example 1, and Comparative Example 4 were diluted to the same enzyme activity (100 U / mL), loading buffer was added and mixed uniformly, and boiled at 100 °C for 10 minutes. The samples were loaded in a volume of 10 μL, and electrophoresis was performed under conditions such as an extraction gel of 12%, a stacking gel of 5%, and a constant voltage of 100 V.

Table 1

[0091] The present inventors unexpectedly found that the enzyme secretion rate by the genetically engineered recombinant Bacillus subtilis B-3-1 strain was limited during the fermentation culture process. In Comparative Example 1, high enzyme activity was also detected in the cells in the middle and late stages of fermentation, indicating that some enzymes were not secreted outside the cells. Further culturing for a certain period of time resulted in a decrease in cell density. At this time, since the enzyme activity was the highest (Table 1, Figure 3), it was suggested that the cells were lysed and the intracellular enzymes were released. However, since intracellular impure proteins were also released by cell lysis, the amount of impure proteins in the crude enzyme solution increased, and the difficulty and cost of purification increased (Figure 6).

[0092] Comparative Example 2 differed from Example 1 in the isothermal fermentation at 37°C and from Comparative Example 1 in the fed-batch fermentation. As can be seen from Figure 3, compared with Comparative Example 1, the fed-batch fermentation in Comparative Example 2 could improve the cell density and enzyme activity in the fermentation tank. However, the enzyme activity in Example 1 was significantly higher than that in Comparative Example 2, indicating that the secretion of the target enzyme could be promoted by raising the culture temperature in the middle and late stages of fermentation to the appropriate temperature.

[0093] Comparative Example 3 differed from Example 1 in the fed-batch-free fermentation with a temperature increase to 40°C in the middle and late stages of fermentation. As can be seen from Figure 4, simply raising the culture temperature in the middle and late stages of fermentation could not effectively improve the cell density and enzyme activity. However, compared with Comparative Example 1, the fermentation period could be shortened from 48 hours to 40 hours, and the enzyme activity during fermentation was not significantly different from that in Comparative Example 1. Furthermore, it was shown that the secretion rate of the enzyme could be accelerated by raising the temperature in the late stage of fermentation.

[0094] Comparative Example 4 is different from Example 1 in that ammonium chloride, an inorganic nitrogen source, is used in the fermentation medium and the fed-batch medium. As can be seen from FIG. 5, when an inorganic nitrogen source is used, the cell density decreases and the enzyme activity decreases by at least one-fold. The organic nitrogen source used in Example 1 reduces the metabolic load of cells and increases the cell density and the expression level of psicose 3-epimerase. For the growth and metabolism of microorganisms, it is preferable to use organic nitrogen (such as peptone and yeast powder). When using inorganic nitrogen, microorganisms need to convert inorganic nitrogen into amino acids by their own metabolism to complete the expression of proteins. Therefore, the metabolic load of cells increases, affecting the expression of proteins. As can be seen from FIGS. 1 and 5, although both Example 1 and Comparative Example 4 employ high-density fermentation, the protein expression levels are more than one-fold different. This may be due to the type of nitrogen source used in the fermentation process. Inorganic nitrogen promotes cell growth, while organic nitrogen promotes protein expression.

[0095] From FIGS. 1 and 6, it can be seen that due to the fed-batch control during the fermentation of Example 1, the cell density continues to increase continuously. By increasing the culture temperature in the late fermentation stage, the permeability of the cell membrane is improved, and it can be seen that the enzyme secretion rate is significantly improved. It is not necessary to lyse the cells to release intracellular enzymes and impure proteins, the fermentation period is shortened, and accordingly, the purity of the crude enzyme solution is also improved. Using the method of Example 1, the cell density at the time of discharge from the tank increases by about 6-fold, and the enzyme activity increases by about 2-fold.

[0096] As described above, the preferred embodiments of the present invention have been explained, but the present invention is not limited thereto. All other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention are all equivalent alternatives and are included in the protection scope of the present invention.

Claims

【Claim 1】 A method for producing psicose 3-epimerase by high-density fermentation, comprising: (1) Activation of the strain Inoculating 0.1 to 1% by volume of a frozen recombinant Bacillus subtilis strain cell suspension into an activation medium containing peptone and yeast powder as nitrogen sources, and culturing with shaking at 37 °C and a rotational speed of 200 rpm for 24 hours until the OD ６００ value reaches 2.0 to activate the primary strain and obtain a primary activated seed cell suspension. Inoculating 1 to 10% by volume of the cultured primary activated seed cell suspension into the activation medium, and then culturing with shaking at 37 °C and a rotational speed of 200 rpm for 8 hours until the OD ６００ value reaches 5.0 to activate the secondary strain and obtain a secondary activated seed cell suspension; and (2) Fermentation culture Adding 10% by volume (350 mL) of the secondary activated seed cell suspension cultured in step (1) to a fermentation medium containing peptone and yeast powder as nitrogen sources in a 5 L tank and fermenting, wherein the initial setting of the fermentation conditions includes a temperature of 37.0 °C, an air flow rate of 5 L / min, an oxygen flow rate of 1 L / min, and a rotational speed of 800 rpm. To control the fermentation process, during fermentation, the dissolved oxygen is controlled within a range of 15%. During the fermentation process, when the OD ６００ rises to 10, a feeding medium containing yeast powder as a nitrogen source is added continuously at a flow rate of 15 mL / hour. When the OD ６００ rises to 35, the feeding medium is added continuously at a flow rate of 40 mL / hour. When the OD ６００ rises to 60, the feeding medium is added continuously at a flow rate of 80 mL / hour. When the OD ６００ rises to 90, the feeding medium is added continuously at a flow rate of 110 mL / hour. OD ６００ When it rises to 100, raising the fermentation temperature to 40 °C, including the step. Here, the method wherein the recombinant Bacillus subtilis in step (1) is the genetically engineered recombinant Bacillus subtilis strain B-3-1. **Claim 2** The method for producing psicose 3-epimerase by high-density fermentation according to claim 1, wherein the activation medium in step (1) contains 5-15 g / L of peptone, 1-5 g / L of yeast powder, 8-12 g / L of sodium chloride, and 25-50 mg / L of kanamycin. **Claim 3** The fermentation medium in step (2) contains 10 g / L of peptone, 5 g / L of yeast powder, 2.5 g / L of potassium dihydrogen phosphate, 15 g / L of dipotassium hydrogen phosphate, 0.1 g / L of manganese chloride tetrahydrate, and 6 g / L of glucose; The method for producing psicose 3-epimerase by high-density fermentation according to claim 1, wherein the fed-batch medium in step (2) contains 10-30% by weight of glucose and 10-30% by weight of yeast powder. **Claim 4** The method for producing psicose 3-epimerase by high-density fermentation according to claim 1, further comprising the steps of stopping the fermentation when the enzyme activity no longer increases, performing solid-liquid separation to obtain a crude enzyme solution, and further purifying to obtain psicose 3-epimerase.

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