Method for improving growth density and enzyme yield of glucose-6-phosphate isomerase strain produced by escherichia coli

Abstract

The invention discloses a method for improving the growth density and enzyme yield of a glucose-6-phosphate isomerase strain produced by escherichia coli, and belongs to the technical field of biology. According to the method for improving the growth density and the enzyme yield of the glucose-6-phosphate isomerase strain produced by escherichia coli, a fermentation mechanism is controlled by establishing bacterium concentration-pH-dissolved oxygen joint control and continuous feeding, corn steep liquor is used as a main raw material, a key cofactor Co < 2 + > is added, and the bacterium concentration and the enzyme activity are improved. The method has the advantages of simple operation, cheap raw materials, low cost, short period and significantly improved enzyme yield, and meets the requirements of industrial production.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and more particularly to a method for improving the growth density and enzyme production of Escherichia coli strains producing glucose-6-phosphate isomerase. Background Art

[0002] Glucose-6-phosphate isomerase (GPI) is an enzyme with great industrial application value. Glucose-6-phosphate isomerase participates in sugar metabolism and catalyzes the isomerization of D-glucose-6-phosphate into D-fructose-6-phosphate. Industrially, it is widely used in the production of fructose, high fructose corn syrup (HFCS), and bioethanol. The production method of fructose usually involves liquefying starch and then converting it into glucose by glucoamylase, and obtaining fructose through isomerization by GPI. The fructose is obtained by separating and crystallizing the fructose syrup; or sucrose can be hydrolyzed to produce fructose syrup, and the fructose is obtained by separating and crystallizing the fructose syrup.

[0003] The application of fructose instead of sucrose in food has great advantages. The metabolism of fructose does not depend on insulin and is easily absorbed and utilized by the human body. It is more suitable for the supply of energy and body fluids for diabetic and liver disease patients; fructose has health care functions such as promoting the absorption of trace elements, protecting liver cells, assisting in the treatment of alcoholism, preventing dental caries, and enhancing endurance; crystalline fructose has been widely used in the food, pharmaceutical and other industries due to its high purity and stable properties.

[0004] Corn steep liquor is a liquid obtained by clarifying the corn steep water extracted from corn after wet milling and hot sulfurous acid impregnation, and then sending it to triple-effect evaporation for concentration. Its main components include water, starch, protein, fat, ash, total nitrogen, and total sugar, etc. It is inexpensive and rich in nutrients. Using corn steep liquor as the main fermentation raw material is beneficial to reducing costs. The total sugar content in corn steep liquor is between 0.74% and 4.39%, and the main component is glucose. It contains a certain amount of protein and is rich in various amino acids, such as arginine, glutamic acid, histidine, phenylalanine, alanine, and β-phenethylamine, etc., which are beneficial to the growth requirements of bacteria. The total nitrogen content is between 3.33% and 3.67%, providing a nitrogen source for the growth of bacteria and contributing to the growth and reproduction of microorganisms. In addition, corn steep liquor also contains various vitamins and minerals, such as elements like potassium, magnesium, phosphorus, and iron, which are helpful for bacteria to produce enzymes.

[0005] The glucose-6-phosphate isomerase gene is ubiquitous in prokaryotes, and the glucose-6-phosphate isomerase gene has been found in many microorganisms such as Escherichia coli, Streptomyces, and Lactobacillus. In order to improve the expression of glucose-6-phosphate isomerase, methods such as optimizing the culture medium and gene recombination are mainly used. At present, there are problems such as low conversion rate in the direct production of high fructose syrup (containing 55% fructose) by biocatalytic conversion method; the existing glucose-6-phosphate isomerase has low enzyme activity, and the highest conversion rate can only reach about 40%, which increases the production and application costs; it is urgent to develop a glucose-6-phosphate isomerase with low cost and good stability.

[0006] Therefore, it is an urgent problem for those skilled in the art to provide a method for improving the growth density and enzyme production of Escherichia coli strains producing glucose-6-phosphate isomerase. Summary of the Invention

[0007] In view of this, the present invention provides a method for improving the growth density and enzyme production of Escherichia coli strains producing glucose-6-phosphate isomerase.

[0008] In order to achieve the above object, the present invention adopts the following technical scheme:

[0009] A method for improving the growth density and enzyme production of Escherichia coli strains producing glucose-6-phosphate isomerase, through the joint control of cell density-pH-dissolved oxygen, continuous feeding control fermentation mechanism, using corn steep liquor as the main raw material, adding the key cofactor Co 2+ , to improve cell density and enzyme activity, the specific steps are as follows:

[0010] (1) Inoculate the seed liquid of 10% (v / v) E. coli BL21(DE3) / pET21-PGI (see Patent 202111512164.9, obtained by transferring the pET21-PGI plasmid into E. coli BL21(DE3)) into the fermentation medium for culture and fermentation, and control the fermentation parameters: the initial temperature is 37±2°C, the pH value is 6.9-7.1, the stirring speed is 200-500 rpm, the tank pressure is 0.02-0.05 Mpa, the air volume is 300-400 m 3 / h, and regulate the dissolved oxygen DO of fermentation to 7-15% to make the OD increase rapidly to the cell density OD of Escherichia coli 600nm =10-15;

[0011] (2) When the pH is greater than 7.1 and the dissolved oxygen is greater than 15%, start to feed and supplement, and the feeding rate is 2.52-3.80 g / L / h;

[0012] (3) The cell density OD of E. coli BL21(DE3) / pET21-PGI 600nmWhen it is 30 - 45, cool down to 30°C and add 0.05 - 0.1 mmol / L inducer IPTG (isopropyl-beta-D-thiogalactopyranoside) to enter the stage of fermenting to produce glucose-6-phosphate isomerase; regulate the fermentation process parameters as follows: stirring speed 400 - 800 rpm, tank pressure 0.02 - 0.05 Mpa, air volume in the reaction tank 500 - 1000 m 3 / h, pH 6.9 - 7.1, dissolved oxygen DO 10 - 20%; feeding rate 3.10 - 4.50 g / L / h; control the dissolved oxygen at 24 - 25% 8 - 12 h after induction. When the cell OD 600nm > 120, end the fermentation to obtain the fermentation broth.

[0013] Furthermore, the preparation method of the E. coli BL21(DE3) / pET21-PGI seed solution in step (1) is as follows:

[0014] Inoculate E. coli BL21(DE3) / pET21-PGI in the first-stage seed medium and culture it in a shaking flask at 37°C with a rotation speed of 200 r / min. When the OD 600nm is 3 - 4, inoculate it into the second-stage seed medium at an inoculation amount of 1%, control the reaction temperature at 37°C, and culture it at a rotation speed of 200 r / min until the OD 600nm is 2 - 4.

[0015] Furthermore, the compositions of the first-stage seed medium and the second-stage seed medium are: yeast powder 5 g / L, peptone 10 g / L, sodium chloride 10 g / L, pH 7.0, 100 μg / mL ampicillin.

[0016] Furthermore, the composition of the fermentation medium in step (1) is as follows: potassium dihydrogen phosphate 1 - 10 g / L, dipotassium hydrogen phosphate 1 - 8 g / L, cobalt chloride 0.01 - 0.05 g / L, corn steep liquor 5 - 10 g / L, peptone 2 - 6 g / L, magnesium sulfate heptahydrate 1 - 1.2 g / L, glucose with a final concentration of 2%, 100 μg / mL ampicillin.

[0017] Furthermore, the feeding formula in step (2) is as follows: corn steep liquor 550 g, cobalt chloride 0.5 g, magnesium sulfate heptahydrate 10 g, add water to make up the volume to 3 L.

[0018] Through the above technical solutions, compared with the prior art, the present invention discloses a method for improving the growth density and enzyme production of Escherichia coli strains producing glucose-6-phosphate isomerase. Using Escherichia coli as the fermentation strain, by controlling the culture conditions and fermentation conditions, the cell OD 600nm>120, the enzyme activity of Escherichia coli producing glucose-6-phosphate isomerase is increased to 900-1300 U / mL. The method of the present invention is simple to operate, low in cost, short in cycle, and the enzyme production is significantly increased, which is suitable for the industrial production requirements of fructose and high fructose syrup. Detailed implementation manners

[0019] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0020] One enzyme activity unit (U / mL) of glucose-6-phosphate isomerase is defined as: the amount of enzyme required for 1 mL of glucose-6-phosphate isomerase to catalyze the formation of 1 μmol of fructose-6-phosphate per minute.

[0021] The method for measuring enzyme activity is as follows: Centrifuge the fermentation broth, remove the supernatant, dilute the bacterial sludge, break the bacteria by ultrasonic wave (ice bath, power 20%, ultrasonic wave for 3 s, interval 10 s, repeat 30 times) to prepare a crude enzyme solution, and place it on ice for measurement.

[0022] Take 0.5 mL of 100 mM Tris-HCl buffer (pH 7.8), 0.1 mL of 20 mM glucose-6-phosphate, 10 μL of 30 mM NADP + , 40 μL of 20 mM magnesium chloride solution and 10 μL of 100 U glucose-6-phosphate dehydrogenase solution, mix them, preheat at 37 °C for 5 min, add 10 μL of the crude enzyme solution at 37 °C, make up the volume to 1 mL with water, mix immediately and start timing. Use an ultraviolet-visible spectrophotometer to record the absorbance every 30 s at a wavelength of 340 nm for 3 min, and calculate the enzyme activity through formula (1).

[0023] Enzyme activity (U / mL) = (△A 340 / min × V 总 ) / (6.22 × d × V 酶 ) × bacterial sludge dilution factor (1)

[0024] Where 6.22 is the molar extinction coefficient of NADPH (mM -1 cm -1 );△A 340 / min is the absorbance change rate after deducting the blank; d is the optical path of the cuvette (cm); V 总 is the total reaction volume (L); V 酶 is the total volume of the crude enzyme solution (L).

[0025] The composition of the primary seed culture medium and the secondary seed culture medium is: 5 g / L yeast powder, 10 g / L peptone, 10 g / L sodium chloride, pH 7.0, and 100 μg / mL ampicillin.

[0026] The feed formula is as follows: 550 g corn syrup, 0.5 g cobalt chloride, 10 g magnesium sulfate heptahydrate, and add water to make up to 3 L.

[0027] Corn syrup was purchased from Shandong Fuyang Biotechnology Co., Ltd.

[0028] Example 1

[0029] A method for increasing the growth density and enzyme production of an Escherichia coli glucose-6-phosphate isomerase producing strain, comprising the following specific steps:

[0030] (1) 30 mL primary shake flask seed preparation.

[0031] Prepare the primary seed culture medium and inoculate E.coliBL21(DE3) / pET21-PGI. Cultivate at 37℃, 200rpm in a shaking incubator until OD 600nm The value is 3.25, and the first-grade seed solution is obtained.

[0032] (2) 1L secondary shake flask seed preparation.

[0033] Transfer the primary seed solution to a secondary shake flask containing 1 L of secondary culture medium at a 1% inoculation rate, control the reaction temperature at 37°C, and culture at 200 rpm until the OD 600nm is 2.15, and the secondary seed liquid is obtained.

[0034] (3) Cultivation in 15 L fermenter.

[0035] A 15L fermenter (using a 10L amount) was weighed separately: 5g / L potassium dihydrogen phosphate, 4g / L potassium hydrogen phosphate, 0.03g / L cobalt chloride, 8g / L corn steep liquor, 4g / L peptone, 1.1g / L magnesium sulfate heptahydrate, and glucose with a final concentration of 2%. The mixture was dissolved in water and added to a volume of 9L in the fermenter, and sterilized at 115°C for 20min. The sterilized fermenter was cooled to 37°C at room temperature, and 10mL of ampicillin with a concentration of 100mg / mL was sterilized separately and placed in the fermenter together before inoculation.

[0036] (4) The secondary seed liquid was inoculated into the fermentation tank sterilized in advance in step (3) with an inoculation amount of 10%. After inoculation, the temperature was controlled at 37° C., the pH was adjusted to 6.9 with ammonia water (17%), the initial stirring speed was 300 rpm, the tank pressure was 0.03 MPa, and the air volume was 400 m 3 / h.

[0037] (5) Establish a joint control mechanism for mycelial concentration - pH - dissolved oxygen, and a continuous feeding control fermentation mechanism.

[0038] ① At 8 h of fermentation, the dissolved oxygen rapidly decreases. During the fermentation process, improve the fermentation conditions, regulate the dissolved oxygen DO to 10 - 11%, and make the OD increase rapidly; at 11.5 h of fermentation, when OD 600nm = 14.72, pH is greater than 7.1 and dissolved oxygen is greater than 15%, start to feed supplementary materials, and the feeding rate is 3.15 g / L / h.

[0039] ② At 22 h of fermentation, when the cell density OD 600nm = 38.3, cool down to 30 °C and add 0.075 mmol / L inducer IPTG to enter the fermentation stage for producing glucose - 6 - phosphate isomerase; regulate the fermentation process parameters as follows: stirring speed 600 rpm, tank pressure 0.035 Mpa, air volume in the reaction tank 750 m 3 / h, pH is 6.9 - 7.1, dissolved oxygen DO is 14 - 15%; the feeding rate is 3.80 g / L / h.

[0040] ③ At 30 h of fermentation, in the middle and late stage of the fermentation process, the mycelial concentration increases slowly. Control the dissolved oxygen at 24 - 25%. At 36 h of fermentation, the cell OD 600nm = 121, end the fermentation, and obtain the fermentation broth.

[0041] (6) Enzyme activity determination

[0042] Centrifuge the obtained fermentation broth, remove the supernatant, dilute the mycelial sludge, and break the bacteria by ultrasonic wave; the measured enzyme activity is 1258.4 U / mL.

[0043] Example 2

[0044] A method for improving the growth density and enzyme production of Escherichia coli strains producing glucose - 6 - phosphate isomerase, and the specific steps are as follows:

[0045] (1) Preparation of 30 mL of first - stage shake - flask seeds.

[0046] Prepare the first - stage seed medium and inoculate E.coli BL21(DE3) / pET21 - PGI, culture at 37 °C and 200 rpm on a shaker until OD 600nm reaches 3.02 to obtain the first - stage seed solution.

[0047] (2) Preparation of 1 L of second - stage shake - flask seeds.

[0048] Transfer the first - stage seed solution to a second - stage shake - flask containing 1 L of second - stage medium according to an inoculation amount of 1%, control the reaction temperature at 37 °C, and the rotation speed at 200 rpm and culture until OD 600nm reaches 2.23 to obtain the second - stage seed solution.

[0049] (3) Culturing in a 15L fermenter.

[0050] Weigh the following components for a 15L fermenter (with a usage volume of 10L) respectively: potassium dihydrogen phosphate 10g / L, dipotassium hydrogen phosphate 8g / L, cobalt chloride 0.05g / L, corn steep liquor 10g / L, peptone 6g / L, magnesium sulfate heptahydrate 1.2g / L, glucose with a final concentration of 2%. Dissolve them in water and add to the fermenter to a volume of 9L, then sterilize at 115°C for 20 min. Place the sterilized fermenter at room temperature and cool it to 37°C. Sterilize 10 mL of ampicillin with a concentration of 100mg / mL separately and add it to the fermenter together before inoculation.

[0051] (4) Inoculate the secondary seed liquid into the fermenter sterilized in step (3) with an inoculation amount of 10%. After inoculation, control the temperature at 37°C, adjust and control the pH to 7.0 with ammonia water (17%), with an initial stirring speed of 200 rpm, a tank pressure of 0.02 Mpa, and an air volume of 300m 3 / h.

[0052] (5) Establish a control mechanism for cell density - pH - dissolved oxygen and a continuous feeding control fermentation mechanism.

[0053] ① At 6 h of fermentation, the dissolved oxygen drops rapidly. Improve the fermentation conditions during the fermentation process, regulate the dissolved oxygen DO to 14 - 15% to make the OD increase rapidly. At 10 h of fermentation, when OD 600nm = 14.58, pH is greater than 7.1 and dissolved oxygen is greater than 15%, start feeding at a feeding rate of 3.8 g / L / h.

[0054] ② At 20.5 h of fermentation, when the cell density OD 600nm = 45, cool down to 30°C and add 0.1 mmol / L inducer IPTG to enter the stage of fermenting to produce glucose - 6 - phosphate isomerase. Regulate the fermentation process parameters as follows: stirring speed 700 rpm, tank pressure 0.05 Mpa, air volume in the reaction tank 1000m 3 / h, pH is 6.9 - 7.1, dissolved oxygen DO is 19 - 20%; feeding rate is 4.50 g / L / h.

[0055] ③ At 29 h of fermentation, in the stationary phase after the logarithmic growth phase of Escherichia coli, the increase in cell concentration is slow. Control the dissolved oxygen at 24 - 25%. At 32 h of fermentation, the cell density OD 600nm = 122.4, end the fermentation to obtain the fermentation broth.

[0056] (6) Enzyme activity determination

[0057] Centrifuge the obtained fermentation broth, remove the supernatant, dilute the cell sludge, break the bacteria by ultrasonic wave, and measure the enzyme activity to be 1343.2 U / mL.

[0058] Example 3

[0059] A method for improving the growth density and enzyme production of Escherichia coli strains producing glucose-6-phosphate isomerase, the specific steps are as follows:

[0060] (1) Preparation of 30 mL of first-stage shake flask seeds.

[0061] Prepare the first-stage seed medium and inoculate E. coli BL21(DE3) / pET21-PGI, incubate at 37 °C and 200 rpm on a shaker until OD 600nm reaches 4.02 to obtain the first-stage seed solution.

[0062] (2) Preparation of 1 L of second-stage shake flask seeds.

[0063] Transfer the first-stage seed solution to a second-stage shake flask containing 1 L of second-stage medium at an inoculation amount of 1%, control the reaction temperature at 37 °C and the rotation speed at 200 rpm, and incubate until OD 600nm reaches 4.15 to obtain the second-stage seed solution.

[0064] (3) Cultivation in a 15 L fermenter.

[0065] Weigh the following components for a 15 L fermenter (usage amount is 10 L): potassium dihydrogen phosphate 1 g / L, dipotassium hydrogen phosphate 1 g / L, cobalt chloride 0.01 g / L, corn steep liquor 10 g / L, peptone 2 g / L, magnesium sulfate heptahydrate 1 g / L, glucose with a final concentration of 2%. Dissolve with water and add to the fermenter to a volume of 9 L, sterilize at 115 °C for 20 min; cool the sterilized fermenter to 37 °C at room temperature, sterilize 10 mL of ampicillin with a concentration of 100 mg / mL separately, and put it into the fermenter together before inoculation.

[0066] (4) Inoculate the second-stage seed solution into the fermenter sterilized in step (3) at an inoculation amount of 10%. After inoculation, control the temperature at 37 °C, adjust and control the pH to 7.0 with ammonia water (17%), the initial stirring speed is 500 rpm, the tank pressure is 0.05 Mpa, and the air volume is 400 m 3 / h.

[0067] (5) Establish a joint control of cell density - pH - dissolved oxygen and a continuous feeding control fermentation mechanism.

[0068] ① At 4 h of fermentation, the dissolved oxygen drops rapidly. During the fermentation process, improve the fermentation conditions, regulate the dissolved oxygen DO to 14 - 15% to make the OD increase rapidly; at 8 h of fermentation, OD 600nm = 10.21, pH is greater than 7.1 and dissolved oxygen is greater than 15%, start to feed continuously, and the feeding rate is 3.8 g / L / h.

[0069] ② At 18 h of fermentation, the cell density OD 600nmWhen it reaches 30.5, cool down to 30°C and add 0.1 mmol / L inducer IPTG to enter the stage of fermenting to produce glucose-6-phosphate isomerase; regulate the fermentation process parameters as follows: stirring speed 600 rpm, tank pressure 0.05 Mpa, air volume in the reaction tank 500 m 3 / h, pH is 6.9 - 7.1, dissolved oxygen DO is 9 - 10%; the feeding rate is 4.50 g / L / h.

[0070] ③ When fermenting for 30 h, which is in the middle and late stage of the fermentation process, the bacterial concentration increases slowly. Control the dissolved oxygen at 24 - 25%. After fermenting for 34 h, the OD of the bacterial cells 600nm is 120.5, end the fermentation to obtain the fermentation broth.

[0071] (6) Enzyme activity determination

[0072] Centrifuge the obtained fermentation broth, remove the supernatant, dilute the bacterial sludge, break the bacteria by ultrasonic wave, and measure the enzyme activity to be 953.4 U / mL.

[0073] Example 4

[0074] A method for improving the growth density and enzyme production of Escherichia coli strains producing glucose-6-phosphate isomerase, the specific steps are as follows:

[0075] (1) Preparation of 30 mL of first-stage shake flask seeds.

[0076] Prepare the first-stage seed medium and inoculate E.coli BL21(DE3) / pET21-PGI, culture at 37°C, 200 rpm on a shaker until OD 600nm reaches 3.52 to obtain the first-stage seed liquid.

[0077] (2) Preparation of 1 L of second-stage shake flask seeds.

[0078] Transfer the first-stage seed liquid to a second-stage shake flask containing 1 L of second-stage medium at an inoculation amount of 1%, control the reaction temperature at 37°C, and culture at a rotation speed of 200 rpm until OD 600nm reaches 3.16 to obtain the second-stage seed liquid.

[0079] (3) Culture in a 15 L fermenter.

[0080] Weigh respectively for a 15 L fermenter (usage amount is 10 L): potassium dihydrogen phosphate 8 g / L, dipotassium hydrogen phosphate 6 g / L, cobalt chloride 0.04 g / L, corn steep liquor 5 g / L, peptone 6 g / L, magnesium sulfate heptahydrate 1.2 g / L, glucose with a final concentration of 2%. Dissolve with water and add 9 L to the fermenter, sterilize at 115°C for 20 min; cool the sterilized fermenter to 37°C at room temperature. Sterilize 10 mL of ampicillin with a concentration of 100 mg / mL separately and put it into the fermenter together before inoculation.

[0081] (4) Inoculate the secondary seeds into the fermenter that has been sterilized in advance in step (3) with an inoculation amount of 10%. After inoculation, control the temperature at 37°C, adjust and control the pH to 7.0 with ammonia water (17%), set the initial stirring speed at 350 rpm, the tank pressure at 0.035 Mpa, and the air volume at 350 m 3 / h.

[0082] (5) Establish a joint control mechanism for cell concentration - pH - dissolved oxygen and a continuous feeding control fermentation mechanism.

[0083] ① At 6 h of fermentation, the dissolved oxygen drops rapidly. Improve the fermentation conditions during the fermentation process, regulate the dissolved oxygen DO to 7 - 8% to make the OD increase rapidly; at 12 h of fermentation, OD 600nm = 12.76, pH > 7.1 and dissolved oxygen > 15%, start to feed supplementary materials at a feeding rate of 2.52 g / L / h.

[0084] ② At 24 h of fermentation, when the cell density OD 600nm = 40.2, cool down to 30°C and add 0.05 mmol / L inducer IPTG to enter the stage of fermenting to produce glucose - 6 - phosphate isomerase; regulate the fermentation process parameters as follows: stirring speed 400 rpm, tank pressure 0.035 Mpa, air volume in the reaction tank 500 m 3 / h, pH 6.9 - 7.1, dissolved oxygen DO 14 - 15%; feeding rate 3.10 g / L / h.

[0085] ③ At 34 h of fermentation, in the middle and late stage of the fermentation process, the increase in cell concentration is slow. Control the dissolved oxygen at 24 - 25%. After 40 h of fermentation, the cell OD 600nm = 123.1, end the fermentation to obtain the fermentation broth.

[0086] (6) Enzyme activity assay

[0087] Centrifuge the obtained fermentation broth, remove the supernatant, dilute the cell sludge, break the bacteria by ultrasonic wave, and measure the enzyme activity to be 1172.0 U / mL.

[0088] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be obvious to those skilled in the art. The general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to these embodiments shown herein, but rather will be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for improving the growth density and enzyme production of Escherichia coli strains producing glucose-6-phosphate isomerase, characterized in that, Through the joint control of cell concentration - pH - dissolved oxygen and continuous feeding to control the fermentation mechanism, using corn steep liquor as the main raw material and adding the key cofactor Co 2+ , to increase cell concentration and enzyme activity. The specific steps are as follows: (1) Inoculate the seed liquid of 10% (volume fraction) E. coli BL21(DE3) / pET21-PGI into the fermentation medium for fermentation, and control the fermentation parameters: the initial temperature is 37 ± 2 °C, the pH value is 6.9 - 7.1, the stirring speed is 200 - 500 rpm, the tank pressure is 0.02 - 0.05 Mpa, the air volume is 300 - 400 m 3 / h, regulate the dissolved oxygen DO during fermentation to 7 - 15%, and make the OD increase rapidly to the E. coli cell density OD 600nm = 10 - 15; (2) When the pH is greater than 7.1 and the dissolved oxygen is greater than 15%, start feeding, and the feeding rate is 2.52 - 3.80 g / L / h; (3) When the cell density OD of E. coli BL21(DE3) / pET21-PGI 600nm reaches 30 - 45, cool down to 30 °C and add 0.05 - 0.1 mmol / L inducer IPTG to enter the stage of fermenting glucose-6-phosphate isomerase; regulate the fermentation process parameters as follows: stirring speed 400 - 800 rpm, tank pressure 0.02 - 0.05 Mpa, air volume in the reaction tank 500 - 1000 m 3 / h, pH 6.9 - 7.1, dissolved oxygen DO 10 - 20%; feeding rate 3.10 - 4.50 g / L / h; control the dissolved oxygen at 24 - 25% 8 - 12 h after induction. When the cell OD 600nm > 120, end the fermentation to obtain the fermentation broth.

2. A method for improving the growth density and enzyme production of Escherichia coli strains producing glucose-6-phosphate isomerase according to claim 1, characterized in that, The method for preparing the E. coli BL21(DE3) / pET21-PGI seed solution described in step (1) is as follows: Inoculate E. coli BL21(DE3) / pET21-PGI into the first-stage seed culture medium and culture it in a shaking flask at 37°C with a rotation speed of 200 r / min. When the OD 600nm reaches 3 - 4, inoculate it into the second-stage seed culture medium at an inoculation amount of 1%, control the reaction temperature at 37°C, and culture it at a rotation speed of 200 r / min until the OD 600nm reaches 2 - 4.

3. A method for increasing the growth density and enzyme production of Escherichia coli strains producing glucose-6-phosphate isomerase according to claim 2, characterized in that, The compositions of the primary seed medium and the secondary seed medium are as follows: yeast powder 5 g / L, peptone 10 g / L, sodium chloride 10 g / L, pH 7.0, 100 μg / mL ampicillin.

4. A method for improving the growth density and enzyme production of Escherichia coli strains producing glucose-6-phosphate isomerase according to claim 1, characterized in that The composition of the fermentation medium described in step (1) is as follows: potassium dihydrogen phosphate 1 - 10 g / L, dipotassium hydrogen phosphate 1 - 8 g / L, cobalt chloride 0.01 - 0.05 g / L, corn steep liquor 5 - 10 g / L, peptone 2 - 6 g / L, magnesium sulfate heptahydrate 1 - 1.2 g / L, glucose with a final concentration of 2%, 100 μg / mL ampicillin.

5. A method for increasing the growth density and enzyme production of Escherichia coli strains producing glucose-6-phosphate isomerase according to claim 1, characterized in that, The feeding formula described in step (2) is as follows: corn steep liquor 550 g, cobalt chloride 0.5 g, magnesium sulfate heptahydrate 10 g, add water to make up to 3 L.