A fermentation method for recombinant uricase from genetically engineered bacteria

Abstract

The present invention belongs to the field of recombinant DNA technology for producing genetically engineered drugs, and specifically discloses a fermentation medium and a fermentation method for recombinant uricase produced by genetically engineered bacteria. The present invention optimizes the basal medium and the feed medium and improves the fermentation process feed method, so that the fermentation density OD of recombinant uricase produced by Escherichia coli fermentation is 0.1%. 600nm The fermentation temperature reaches 110-130, and the bacterial density is increased by more than 4 times compared with low-density fermentation; the recombinant uricase production is as high as 5.5 g / L, the recombinant uricase protein expression accounts for 57% of the total bacterial protein, and the plasmid loss rate is low. The high-density fermentation of the recombinant uricase of the present invention improves production efficiency and has better application prospects.

Description

Technical Field

[0001] The invention belongs to the field of producing genetic engineering drugs by recombinant DNA technology, and specifically relates to a fermentation method of recombinant uricase from genetic engineering bacteria. Background Art

[0002] Uricase (EC 1.7.3.3, urate oxidase) is an enzyme in the purine degradation metabolic pathway in organisms, which can oxidize uric acid into allantoin and hydrogen peroxide with higher concentrations. Uricase is found in many species, but humans and some primates lack this enzyme. Due to mutations in the uricase gene, a premature termination codon is introduced into the coding sequence. Therefore, uric acid is the end product of purine metabolism. Uric acid and its salts have very low solubility in water. The uric acid saturation in plasma at 37°C pH7.4 is 380-420 μmol / L (6.4-7.1 mg / dL). When the blood uric acid concentration in the human body exceeds 7.0 mg / dL, it can cause hyperuricemia.

[0003] Hyperuricemia in the human body can cause or aggravate many diseases. Acute inflammation and pain caused by the deposition of uric acid crystals in peripheral joints during hyperuricemia are the main causes of gout, and severe gout can cause joint dysfunction. Uric acid can stimulate the proliferation of vascular smooth muscle cells and lead to endothelial cell dysfunction. Plasma hyperuricemia is an important risk factor for cardiovascular diseases such as atherosclerosis. Uric acid deposition in the renal tubules during hyperuricemia can cause renal dysfunction. High levels of plasma uric acid are also the main risk factor for acute renal failure, tubular injury, and IgA nephropathy. During chemotherapy for hematological tumors such as leukemia, lymphoma, and myeloma, tumor cells are lysed in large quantities, intracellular nucleic acids are degraded in large quantities, and plasma uric acid rises sharply, causing metabolic disorders mainly caused by hyperuricemia and causing renal failure and death in patients. This phenomenon is called tumor lysis syndrome. Therefore, serum uric acid levels have important reference value for the experimental diagnosis of diseases such as renal function, gout, hyperuricemia, and tumor lysis syndrome, and it is necessary to take measures to reduce plasma uric acid levels to treat these diseases.

[0004] With the rapid development of biotechnology, uricases from various mammalian and microbial sources have been widely used in genetic engineering to express and produce recombinant uricases. Currently, two recombinant uricase products have been launched in Europe and the United States: rasburicase and pegololac. Rasburicase is a recombinant uricase developed by Sanofi Synthelabo, France. The enzyme is derived from Aspergillus flavus and produced by a yeast expression system. It was approved for marketing by the US FDA in July 2002. The first approved indication is the prevention and treatment of hyperuricemia caused by high-risk tumor lysis syndrome after chemotherapy in patients with hematological malignancies. It is also used to treat hyperuricemia in patients with gout and rheumatoid arthritis. Clinically, rasburicase can quickly reduce the level of uric acid in serum, has a fast onset time, and is significantly more effective than allopurinol. It can also dissolve tophi and is more suitable for patients who are intolerant to conventional therapy. However, the drug is quite expensive, has a short half-life (only 18 hours), requires frequent injections, and is prone to hypersensitivity reactions. Therefore, the uricase inhibitor cannot be used for the long-term control and treatment of hyperuricemia. It is currently mainly used to control the transient increase of uric acid in tumor lysis syndrome, and conventional medication is controlled within seven days.

[0005] Another drug is Pegloticase (Krystexxa), developed by Savient in the United States. It was launched in the United States in 2010 and was subsequently approved by the European Union for the treatment of refractory gout that is ineffective in traditional uric acid lowering. Pegloticase is a pegylated recombinant uricase, the gene of which comes from the mammalian pig and is produced by an Escherichia coli expression system. Compared with rasburicase, the pegylated pegloticase effectively prolongs the half-life to 10 to 12 days, so it only needs to be administered once every two weeks. The drug can be used for treatment for up to six months, can reduce uric acid levels, reduce the size of tophi, improve the functional status and quality of life of patients, and achieve long-term control of serum uric acid levels. Although the immunogenicity of pegoloticase is significantly lower than that of other recombinant uricases, it is reported that about 40% of patients produce high titers of anti-uricase antibodies in their bodies as the medication time increases, causing serious adverse reactions such as allergic reactions, infusion reactions and gout attacks.

[0006] The sources of natural uricase oxidase are relatively limited, which greatly limits its research and application. At present, there are many methods for producing uricase by genetic engineering. Ma Xuefeng disclosed a human-dog chimeric uricase in "Construction, Expression, Purification and Property Research of Human-Dog Chimeric Uricase" (Master's Degree Thesis of Chongqing University of Technology, 2010), which modified the gene of dog uricase, introduced part of the amino acid sequence of human uricase into the C-terminus of the inactive center of mammalian dog uricase, expressed human-dog chimeric uricase by prokaryotic expression system, screened out high-expression uricase strains, and disclosed the fermentation and purification method of human-dog chimeric uricase, but the fermentation method is low-density fermentation, and the expression amount of recombinant uricase is low, only 30%.

[0007] Zhao Ming, He Zhongjie, and others disclosed a method for fermentation and cultivation of recombinant uricase in "Process conditions for fermentation and preparation of recombinant uricase", which specifically used a recombinant E. coli JM109 (DE3) strain containing a PEG-Urate Oxidase expression plasmid for large-scale fermentation and cultivation, optimized its separation and purification conditions according to the fermentation tank growth curve, and determined that 5h of fermentation was the best time for induction in the middle and late logarithmic growth of the bacteria, and 7h of induction was the best. This method uses lactose instead of IPTG as an inducer. Although the induction effect is better, the fermentation density in this process is low and the fermentation yield is still not high.

[0008] Yang Xiuxu disclosed a high-density fermentation process of recombinant urate oxidase (rUOX) genetically engineered Escherichia coli BL (DE3 / pET-32a-uox) in "Optimization of Fermentation Process of Recombinant Urate Oxidase" (Biotechnology Newsletter, 2012, Vol. 23 No. 2). The final OD of the bacteria in high-density fermentation was 600nm The value is 50, the recombinant protein accounts for 45.1% of the total bacterial protein, the yield reaches 3.45g / L, and the active protein is 27U / mg. The fermentation density in this process is low, and the fermentation yield needs to be improved. Therefore, the prior art needs to further develop the fermentation process of recombinant uricase, and it is urgent to provide a high-density, high-expression recombinant uricase fermentation process. Summary of the invention

[0009] Based on the deficiencies in the prior art, the object of the present invention is to provide a fermentation method for high-density and high-expression recombinant uricase.

[0010] The two most important issues in genetically engineered bacterial fermentation are high-density fermentation and the determination of induction conditions. High-density fermentation technology is a method of changing the culture method or culture conditions under a specific system to increase the fermentation density of the cultured bacterial community, thereby increasing the production of the corresponding product. This culture method is based on the current advanced fermentation technology and uses a variety of methods to increase the yield of the product. When applied to the fermentation and culture process of Escherichia coli recombinant uricase, it can effectively increase the synthesis of related recombinant proteins, reduce the culture volume, and improve the purity of separation and extraction, greatly shortening the production cycle, which also reduces production costs and improves production efficiency from another perspective.

[0011] Based on the successful construction of recombinant uricase genetically engineered bacteria, the present invention explores culture medium, culture conditions and induction conditions suitable for high-density fermentation of genetically engineered bacteria; in addition, a feeding strategy is optimized to achieve high-density fermentation of genetically engineered bacteria in a fermenter while ensuring a low plasmid loss rate.

[0012] The technical solution of the present invention is as follows:

[0013] A culture medium for producing recombinant uricase by genetically engineered bacteria comprises the following components: 2-4 g / L peptone, 6-8 g / L yeast extract, 8-12 g / L glucose, 3-5 g / L lactose, 17-30 g / L sodium dihydrogen phosphate, 4-6 g / L dipotassium hydrogen phosphate, 0.5-1.5 g / L magnesium sulfate and 0.5-1.5 g / L ammonium chloride.

[0014] Preferably, the culture medium consists of: 3 g / L peptone, 7 g / L yeast extract, 10 g / L glucose, 4 g / L lactose, 25 g / L sodium dihydrogen phosphate, 5 g / L dipotassium hydrogen phosphate, 1 g / L magnesium sulfate, and 1 g / L ammonium chloride.

[0015] A fermentation method for recombinant uricase from genetically engineered bacteria comprises the following steps:

[0016] (1) inoculating the genetically engineered bacteria into a seed culture medium and gradually amplifying the culture to obtain a seed solution;

[0017] (2) The seed liquid is inoculated into the basic culture medium of the fermentation tank for high-density fermentation, an inducer is added for induction, and a feed medium is added in a restrictive flow until the end of the culture.

[0018] Preferably, the basic culture medium consists of: 2-4 g / L peptone, 6-8 g / L yeast extract, 8-12 g / L glucose, 3-5 g / L lactose, 17-30 g / L sodium dihydrogen phosphate, 4-6 g / L dipotassium hydrogen phosphate, 0.5-1.5 g / L magnesium sulfate, and 0.5-1.5 g / L ammonium chloride.

[0019] Further preferably, the basic culture medium consists of: 3 g / L peptone, 7 g / L yeast extract, 10 g / L glucose, 4 g / L lactose, 25 g / L sodium dihydrogen phosphate, 5 g / L dipotassium hydrogen phosphate, 1 g / L magnesium sulfate, and 1 g / L ammonium chloride.

[0020] Preferably, the feed medium consists of: 50-70 g / L peptone, 70-90 g / L yeast extract, 100-300 g / L glucose, 50-70 g / L lactose, and 15-25 g / L magnesium sulfate.

[0021] Further preferably, the feed medium consists of: 60 g / L peptone, 80 g / L yeast extract, 200 g / L glucose, 60 g / L lactose, and 20 g / L magnesium sulfate.

[0022] Preferably, the genetically engineered bacteria is PET-UHC / BL21DE3PlysS, and the construction method is based on Ma Xuefeng, "Construction, Expression, Purification and Property Study of Human-Dog Chimeric Uricase", Master's thesis of Chongqing University of Technology, 2010.

[0023] Preferably, the inoculation amount of the seed liquid inoculated into the basal culture medium of the fermentation tank is 5-10%, that is, the volume of the seed liquid is 5-10% of the volume of the basal culture medium.

[0024] Preferably, during the fermentation process, the pH is 6.8-7.2; and the fermentation temperature is 35-37°C.

[0025] Preferably, the inducer is IPTG, and the amount added is a final concentration of 0.2 to 0.4 mmol / L; and the induction culture time is 9 to 12 hours.

[0026] Preferably, during the feeding process, the amount of feed medium required is: the total feed amount is 25-35% of the volume of the basal medium, more preferably, the total feed amount is 30-33% of the volume of the basal medium.

[0027] Preferably, the limiting feeding process controls the cumulative amount of glucose to be no more than 0.01%, and the flow acceleration is 0.20-0.50 mL / (L·min); further preferably, the feeding rate can be adjusted in time according to the detected OD value. In one embodiment, when the culture reaches OD 600nm When OD is 10-13, the flow acceleration is 0.20 mL / (L·min); 600nm When OD is 14-17, the flow acceleration is 0.25mL / (L·min); 600nm When the flow rate is 17-28 hours, the flow acceleration is 0.30mL / (L·min); OD 600nm When the flow rate is 28-39, the flow acceleration is 0.40mL / (L·min); OD600nm The flow acceleration was maintained at 0.50 mL / (L·min) when the flow rate was above 39 until the end of the culture.

[0028] The following further details a fermentation method of recombinant uricase from genetically engineered bacteria, comprising the following steps:

[0029] (1) inoculating the genetically engineered bacteria into a seed culture medium and gradually amplifying the culture to obtain a seed solution;

[0030] Specifically, a single colony is picked from the re-streaked plate and inoculated into a seed culture medium, and cultured at 35.0-37.0°C and 150rpm for 6-8h to obtain a primary seed solution; the cultured primary seed solution is inoculated into a seed culture medium, and cultured at 35.0-37.0°C and 150rpm for 10-12h to obtain a secondary seed solution, i.e., a seed solution for recombinant uricase fermentation.

[0031] Preferably, the genetically engineered bacteria is PET-UHC / BL21DE3PlysS, and the construction method is based on Ma Xuefeng, "Construction, Expression, Purification and Property Study of Human-Dog Chimeric Uricase", Master's thesis of Chongqing University of Technology, 2010.

[0032] Preferably, the seed culture medium formula is 5 g / L peptone, 5 g / L yeast extract, 10 g / L glucose, and 10 g / L sodium chloride.

[0033] Preferably, the primary seed solution is cultured to OD 600nm 0.6~0.8, secondary seed solution culture to OD 600nm 1.8~2.0.

[0034] (2) The seed solution is inoculated into the basic culture medium of the fermentation tank for high-density culture, and the feed medium is added restrictively until the end of the culture.

[0035] Specifically, a basic culture medium is prepared and sterilized for use; the secondary seed liquid is inoculated into the basic culture medium of the fermentation tank at an inoculation rate of 5-10%, and the dissolved oxygen in the fermentation tank is controlled to be not less than 35%, the temperature is 35-37°C, and the pH is 6.8-7.2 during the fermentation process; when the OD of the fermentation system is 600nm Start feeding the feed medium at 10-13 o'clock, and feed until OD 600nm At 14-17 pm, the inducer IPTG was added for induction culture, and then the remaining feed medium was added and the culture was continued until the end.

[0036] Preferably, the basic culture medium consists of: 2-4 g / L peptone, 6-8 g / L yeast extract, 8-12 g / L glucose, 3-5 g / L lactose, 17-30 g / L sodium dihydrogen phosphate, 4-6 g / L dipotassium hydrogen phosphate, 0.5-1.5 g / L magnesium sulfate, and 0.5-1.5 g / L ammonium chloride.

[0037] Further preferably, the basic culture medium consists of: 3 g / L peptone, 7 g / L yeast extract, 10 g / L glucose, 4 g / L lactose, 25 g / L sodium dihydrogen phosphate, 5 g / L dipotassium hydrogen phosphate, 1 g / L magnesium sulfate, and 1 g / L ammonium chloride.

[0038] Preferably, the feed medium consists of:

[0039] Peptone 50-70g / L, yeast extract 70-90g / L, glucose 100-300g / L, lactose 50-70g / L, magnesium sulfate 15-25g / L.

[0040] Further preferably, the feed medium consists of: 60 g / L peptone, 80 g / L yeast extract, 200 g / L glucose, 60 g / L lactose, and 20 g / L magnesium sulfate.

[0041] Preferably, during the induction culture stage, an inducer IPTG is added to a final concentration of 0.2 to 0.4 mmol / L for induction culture.

[0042] Preferably, during the induction culture process, the dissolved oxygen is controlled at 55-70%, the temperature is 35-37° C., and the pH is 7.0-7.4.

[0043] Preferably, the feed medium is fed in a restricted manner, with a flow rate of 0.20 to 0.50 mL / (L·min); further preferably, the feed rate can be increased in due course. In one embodiment, OD 600nm When the OD was 10-13, feed medium was added at a flow rate of 0.20 mL / (L·min); 600nm When the flow rate is 14-17, the flow acceleration is 0.25mL / (L·min); when the culture reaches OD 600nm When the flow rate is 17-28, the flow acceleration is 0.30mL / (L·min); when the culture reaches OD 600nm When the flow rate is 28-39, the flow acceleration is 0.40mL / (L·min); when the culture reaches OD 600nm The flow acceleration was maintained at 0.50 mL / (L·min) when the flow rate was above 39 until the end of the culture.

[0044] Preferably, the induction culture time is 9 to 12 hours; the cell density OD 600nm The fermentation process ends at 110-130 ℃.

[0045] The present invention has the following advantages:

[0046] By optimizing the seed culture medium, fermentation culture medium and feed culture medium and improving the feed method of the fermentation process, after the genetic engineering bacteria are fermented and cultured, the fermentation density OD of Escherichia coli for producing recombinant uricase is 0.0447 W / cm3. 600nm The fermentation temperature reaches 110-130, and the bacterial density is increased by more than 4 times compared with low-density fermentation; the recombinant uricase production is as high as 5.5 g / L, the recombinant uricase protein expression accounts for 57% of the total bacterial protein, and the plasmid loss rate is low. The high-density fermentation of the recombinant uricase of the present invention improves production efficiency and has better application prospects. DETAILED DESCRIPTION

[0047] The present invention is further described below in conjunction with specific examples. The following examples are only for illustrative purposes and are not intended to limit the present invention. Anyone familiar with this technology can make various deductions and substitutions without departing from the spirit and scope of the present invention. The protection scope of the present invention should be the technical solution recorded in the claims, including the equivalent replacement scheme of the technical features in the technical solution recorded in the claims. That is, equivalent replacement improvements within this scope, and the same proportion of enlargement or reduction experiments are also within the protection scope of the present invention.

[0048] The strain used in the following embodiments is PET-UHC / BL21DE3PlysS, and the construction method is based on Ma Xuefeng's "Construction, Expression, Purification and Properties of Human-Dog Chimeric Uricase" (Master's Thesis of Chongqing University of Technology, 2010). The reagents required in the fermentation process can be obtained from commercial sources unless otherwise specified.

[0049] Example 1

[0050] 1. Seed liquid culture

[0051] The components of the primary and secondary seed liquid culture medium are: peptone 5g / L, yeast extract 5g / L, glucose 10g / L, and sodium chloride 10g / L.

[0052] (1) Activation of bacteria

[0053] Take the stored PET-UHC / BL21DE3PlysS strain from the -70°C freezer, streak it onto a plate containing ampicillin sodium, and culture it at 37°C overnight;

[0054] (2) Cultivation of primary seeds

[0055] Pick a single colony from the cultured plate and inoculate it into a test tube containing 8 mL of seed culture medium containing 100 mg / L ampicillin. Cultivate at 35-37°C, 150 rpm for 7 h. Detect OD600nm is 0.75, used as the first-level seed liquid.

[0056] (3) Cultivation of secondary seeds

[0057] Inoculate the first-grade seed solution into 2L seed culture medium at 1% inoculation volume, culture at 35-37°C, 150rpm for 12h, and measure the OD 600nm It is 1.93 as the seed liquid for fermentation.

[0058] 2. 30L fermentation tank culture

[0059] Basic culture medium: peptone 3g / L, yeast extract 7g / L, glucose 10g / L, lactose 4g / L, sodium dihydrogen phosphate 25g / L, dipotassium hydrogen phosphate 5g / L, magnesium sulfate 1g / L, ammonium chloride 1g / L.

[0060] Feed medium: peptone 60 g / L, yeast extract 80 g / L, glucose 200 g / L, lactose 60 g / L, magnesium sulfate 20 g / L.

[0061] (1) Prepare 18 L of basal culture medium and transfer it to a 30 L fermenter. Sterilize at 115° C. for 20 minutes. After sterilization, the temperature of the fermentation medium is reduced to 35-37° C. The seed liquid is inoculated at a volume ratio of 10% into the fermenter. The fermentation process is controlled at a temperature of 35-37° C. and a ventilation volume of 1.2-1.5 m 3 / h, pressure 0.04~0.06MPa, speed 200rpm, pH 6.8~7.2, calibrated dissolved oxygen 100%, controlled dissolved oxygen to be maintained above 35% by adjusting the speed, cultured for 4 hours, and detected OD 600nm It is 11.82.

[0062] (2) Prepare sterilized feed medium in advance at a flow rate of 0.2 mL / (L·min). Stop feeding after 30 min. After the dissolved oxygen rises rapidly to above 95% and the pH rises simultaneously, measure the OD 600nm The final concentration of IPTG was 0.2 mmol / L, and the culture medium was added for induction. After 20 min of induction, the feed medium was added at a flow rate of 0.25 mL / (L·min). The OD was detected in due course during the addition process. 600nm , and monitor the glucose content not to exceed 0.01%; when the OD 600nm When the flow rate reaches 17-28 hours, the flow acceleration is 0.3 mL / (L·min); when the culture reaches OD 600nm When the flow rate is 28-39, the flow acceleration is 0.4mL / (L·min); when the culture reaches OD 600nmThe flow rate was maintained at 0.5 mL / (L·min) and above 39. During the fed-batch culture, the dissolved oxygen was maintained at 55% to 70%, and the pH was 7.0 to 7.4. The OD of the fermentation liquid was detected after about 12 hours of induction. 600nm When the pH value was 130.5, the fermentation culture was terminated and 6 L of feed medium was added. The samples were centrifuged and subjected to SDS-PAGE electrophoresis to detect the expression of the target protein and the plasmid loss rate.

[0063] Example 2

[0064] 1. Seed liquid culture

[0065] The components of the primary and secondary seed liquid culture medium are: peptone 5g / L, yeast extract 5g / L, glucose 10g / L, and sodium chloride 10g / L.

[0066] (1) Activation of bacteria

[0067] Take the stored PET-UHC / BL21DE3PlysS strain from the -70°C freezer, streak it onto a plate containing ampicillin sodium, and culture it at 37°C overnight;

[0068] (2) Cultivation of primary seeds

[0069] Pick a single colony from the cultured plate and inoculate it into a test tube containing 8 mL of seed culture medium containing 100 mg / L ampicillin. Cultivate at 35-37°C, 150 rpm for 6 h. Detect OD 600nm The value of 0.62 is used as the primary seed solution.

[0070] (3) Cultivation of secondary seeds

[0071] Inoculate the first-grade seed solution into 2L seed culture medium at 1% inoculation volume, culture at 35-37°C, 150rpm for 10h, and measure the OD 600nm It is 1.82 as the seed liquid for fermentation.

[0072] 2. 30L fermentation tank culture

[0073] Basic culture medium: peptone 2g / L, yeast extract 6g / L, glucose 8g / L, lactose 3g / L, sodium dihydrogen phosphate 17g / L, dipotassium hydrogen phosphate 4g / L, magnesium sulfate 0.5g / L, ammonium chloride 0.5g / L.

[0074] Feed medium: peptone 70 g / L, yeast extract 90 g / L, glucose 300 g / L, lactose 70 g / L, magnesium sulfate 25 g / L.

[0075] (1) Prepare 18 L of basal culture medium and transfer it to a 30 L fermenter. Sterilize at 115° C. for 20 minutes. After sterilization, the temperature of the fermentation medium is reduced to 35-37° C. The seed liquid is inoculated at a volume ratio of 10% into the fermenter. The fermentation process is controlled at a temperature of 35-37° C. and a ventilation volume of 1.2-1.5 m 3 / h, pressure 0.04~0.06MPa, speed 200rpm, pH 6.8~7.2, calibrated dissolved oxygen 100%, controlled dissolved oxygen to be maintained above 35% by adjusting the speed, cultured for 5 hours, and detected OD 600nm It is 10.72.

[0076] (2) Prepare sterilized feed medium in advance at a flow rate of 0.2 mL / (L·min). Stop feeding after 30 min. After the dissolved oxygen rises rapidly to above 95% and the pH rises simultaneously, measure the OD 600nm The final concentration of IPTG was 0.4 mmol / L, and the induction was carried out. After 20 min of induction, the feed medium was continued to be fed at a flow rate of 0.25 mL / (L·min). The OD was detected in time during the feeding process. 600nm , and monitor the glucose content not to exceed 0.01%; when the OD 600nm When the flow rate reaches 17-28 hours, the flow acceleration is 0.3 mL / (L·min); when the culture reaches OD 600nm When the flow rate is 28-39, the flow acceleration is 0.4mL / (L·min); when the culture reaches OD 600nm The flow rate was maintained at 0.5 mL / (L·min) and above 39. During the fed-batch culture, the dissolved oxygen was maintained at 55% to 70%, and the pH was 7.0 to 7.4. The OD of the fermentation liquid was detected after about 10 hours of induction. 600nm When the pH value was 110.7, the fermentation culture was terminated and 5.5 L of feed medium was added. The fermentation broth was centrifuged, the bacterial cells were collected, and the expression amount of the target protein and the plasmid loss rate were detected.

[0077] Example 3

[0078] 1. Seed liquid culture

[0079] The components of the primary and secondary seed liquid culture medium are: peptone 5g / L, yeast extract 5g / L, glucose 10g / L, and sodium chloride 10g / L.

[0080] (1) Activation of bacteria

[0081] Take the stored PET-UHC / BL21DE3PlysS strain from the -70°C freezer, streak it onto a plate containing ampicillin sodium, and culture it at 37°C overnight;

[0082] (2) Cultivation of primary seeds

[0083] Pick a single colony from the cultured plate and inoculate it into a test tube containing 8 mL of seed culture medium containing 100 mg / L ampicillin. Cultivate at 35-37°C, 150 rpm for 8 h. Detect OD 600nm The value of 0.81 is used as the primary seed solution.

[0084] (3) Cultivation of secondary seeds

[0085] Inoculate the first-grade seed solution into 2L seed culture medium at 1% inoculation volume, culture at 35-37°C, 150rpm for 10h, and measure the OD 600nm 1.95 as the seed liquid for fermentation.

[0086] 2. 30L fermentation tank culture

[0087] Basic culture medium: peptone 4g / L, yeast extract 8g / L, glucose 12g / L, lactose 5g / L, sodium dihydrogen phosphate 25g / L, dipotassium hydrogen phosphate 6g / L, magnesium sulfate 1.5g / L, ammonium chloride 1.5g / L.

[0088] Feed medium: peptone 50 g / L, yeast extract 70 g / L, glucose 100 g / L, lactose 50 g / L, magnesium sulfate 15 g / L.

[0089] (1) Prepare 18 L of basal culture medium and transfer it to a 30 L fermenter. Sterilize at 115° C. for 20 minutes. After sterilization, the temperature of the fermentation medium is reduced to 35-37° C. The seed liquid is inoculated at a volume ratio of 10% into the fermenter. The fermentation process is controlled at a temperature of 35-37° C. and a ventilation volume of 1.2-1.5 m 3 / h, pressure 0.04~0.06MPa, speed 200rpm, pH 6.8~7.2, calibrated dissolved oxygen 100%, controlled dissolved oxygen to be maintained above 35% by adjusting the speed, cultured for 4 hours, and detected OD 600nm It is 13.86.

[0090] (2) Prepare sterilized feed medium in advance at a flow rate of 0.2 mL / (L·min). Stop feeding after 30 min. After the dissolved oxygen rises rapidly to above 95% and the pH rises simultaneously, measure the OD 600nm The final concentration of IPTG was 0.3 mmol / L, and the culture medium was added for induction. After 20 min of induction, the feed medium was added at a flow rate of 0.12 mL / (L·min). The OD was detected in time during the addition process. 600nm , and monitor the glucose content not to exceed 0.01%; when the OD 600nm When the flow rate reaches 17-28 hours, the flow acceleration is 0.3 mL / (L·min); when the culture reaches OD 600nmWhen the flow rate is 28-39, the flow acceleration is 0.4mL / (L·min); when the culture reaches OD 600nm The flow rate was maintained at 0.5 mL / (L·min) and above 39. During the fed-batch culture, the dissolved oxygen was maintained at 55% to 70%, and the pH was 7.0 to 7.4. The OD of the fermentation liquid was detected after about 10 hours of induction. 600nm When the pH value was 120.2, the fermentation culture was terminated and 6.2 L of feed medium was added. The fermentation broth was centrifuged, the bacterial cells were collected, and the expression amount of the target protein and the plasmid loss rate were detected.

[0091] Example 4

[0092] 1. Seed liquid culture

[0093] The components of the primary and secondary seed liquid culture medium are: peptone 5g / L, yeast extract 5g / L, glucose 10g / L, and sodium chloride 10g / L.

[0094] (1) Activation of bacteria

[0095] Take the stored PET-UHC / BL21DE3PlysS strain from the -70°C freezer, streak it onto a plate containing ampicillin sodium, and culture it at 37°C overnight;

[0096] (2) Cultivation of primary seeds

[0097] Pick a single colony from the cultured plate and inoculate it into a test tube containing 8 mL of seed culture medium containing 100 mg / L ampicillin. Cultivate at 35-37°C, 150 rpm for 7 h. Detect OD 600nm The value of 0.78 is used as the primary seed solution.

[0098] (3) Cultivation of secondary seeds

[0099] Inoculate the first-grade seed solution into 2L seed culture medium at 1% inoculation volume, culture at 35-37°C, 150rpm for 12h, and measure the OD 600nm It is 1.97 as the seed liquid for fermentation.

[0100] 2. 30L fermentation tank culture

[0101] Basic culture medium: peptone 3g / L, yeast extract 7g / L, glucose 10g / L, lactose 4g / L, sodium dihydrogen phosphate 25g / L, dipotassium hydrogen phosphate 5g / L, magnesium sulfate 1g / L, ammonium chloride 1g / L.

[0102] Feed medium: peptone 60 g / L, yeast extract 80 g / L, glucose 200 g / L, lactose 60 g / L, magnesium sulfate 20 g / L.

[0103] (1) Prepare 18 L of basal culture medium and transfer it to a 30 L fermenter. Sterilize at 115° C. for 20 minutes. After sterilization, the temperature of the fermentation medium is reduced to 35-37° C. The seed liquid is inoculated at a volume ratio of 10% into the fermenter. The fermentation process is controlled at a temperature of 35-37° C. and a ventilation volume of 1.2-1.5 m 3 / h, pressure 0.04~0.06MPa, speed 200rpm, pH 6.8~7.2, calibrated dissolved oxygen 100%, controlled dissolved oxygen to be maintained above 35% by adjusting the speed, cultured for 4 hours, and detected OD 600nm It is 12.85.

[0104] (2) Prepare sterilized feed medium in advance at a flow rate of 0.5 mL / (L·min). Stop feeding after 30 min. After the dissolved oxygen rises rapidly to above 95% and the pH rises simultaneously, measure the OD 600nm The final concentration of IPTG was 16.62, and IPTG with a final concentration of 0.2 mmol / L was added for induction. After 20 minutes of induction, the feed medium was continued to be fed at a flow rate of 0.5 mL / (L·min). During the fed-batch culture, the dissolved oxygen was maintained at 55% to 70%, and the pH was 7.0 to 7.4. The OD of the fermentation liquid was detected after about 9 hours of induction. 600nm When the pH value was 136.7, the fermentation culture was terminated and 5.5 L of feed medium was added. The fermentation broth was centrifuged, the bacterial cells were collected, and the expression amount of the target protein and the plasmid loss rate were detected.

[0105] Example 5

[0106] 1. Seed liquid culture

[0107] The components of the primary and secondary seed liquid culture medium are: peptone 5g / L, yeast extract 5g / L, glucose 10g / L, and sodium chloride 10g / L.

[0108] (1) Activation of bacteria

[0109] Take the stored PET-UHC / BL21DE3PlysS strain from the -70°C freezer, streak it onto a plate containing ampicillin sodium, and culture it at 37°C overnight;

[0110] (2) Cultivation of primary seeds

[0111] Pick a single colony from the cultured plate and inoculate it into a test tube containing 8 mL of seed culture medium containing 100 mg / L ampicillin. Cultivate at 35-37°C, 150 rpm for 7 h. Detect OD 600nm The value of 0.76 is used as the primary seed solution.

[0112] (3) Cultivation of secondary seeds

[0113] Inoculate the first-grade seed solution into 2L seed culture medium at 1% inoculation volume, culture at 35-37°C, 150rpm for 12h, and measure the OD 600nm It is 1.89 as the seed liquid for fermentation.

[0114] 2. 30L fermentation tank culture

[0115] Basic culture medium: peptone 3g / L, yeast extract 7g / L, glucose 10g / L, lactose 4g / L, sodium dihydrogen phosphate 25g / L, dipotassium hydrogen phosphate 5g / L, magnesium sulfate 1g / L, ammonium chloride 1g / L.

[0116] Feed medium: peptone 60 g / L, yeast extract 80 g / L, glucose 200 g / L, lactose 60 g / L, magnesium sulfate 20 g / L.

[0117] (1) Prepare 18 L of basal culture medium and transfer it to a 30 L fermenter. Sterilize at 115° C. for 20 minutes. After sterilization, the temperature of the fermentation medium is reduced to 35-37° C. The seed liquid is inoculated at a volume ratio of 10% into the fermenter. The fermentation process is controlled at a temperature of 35-37° C. and a ventilation volume of 1.2-1.5 m 3 / h, pressure 0.04~0.06MPa, speed 200rpm, pH 6.8~7.2, calibrated dissolved oxygen 100%, controlled dissolved oxygen to be maintained above 35% by adjusting the speed, cultured for 4 hours, and detected OD 600nm It is 12.69.

[0118] (2) Prepare sterilized feed medium in advance at a flow rate of 0.3 mL / (L·min). Stop feeding after 30 min. After the dissolved oxygen rises rapidly to above 95% and the pH rises simultaneously, measure the OD 600nm The final concentration of IPTG was 17.63, and IPTG with a final concentration of 0.2 mmol / L was added for induction. After 20 minutes of induction, the feed medium was continued to be fed at a flow rate of 0.3 mL / (L·min). During the fed-batch culture, the dissolved oxygen was maintained at 55% to 70%, and the pH was 7.0 to 7.4. The OD of the fermentation liquid was detected after about 15 hours of induction. 600nm The pH value was 115.26, and the fermentation culture was terminated. 5.4 L of feed medium was added. The fermentation broth was centrifuged, and the bacterial cells were collected to detect the expression amount of the target protein and the plasmid loss rate.

[0119] Comparative Example 1

[0120] 1. Cultivation of seed solution

[0121] LB medium: yeast extract 5g / L, peptone 10g / L, sodium chloride 10g / L

[0122] (1) Activation of seed bacteria

[0123] The PET-UHC / BL21DE3PlysS strain was taken from the -70℃ freezer, streaked onto a LB slant containing ampicillin (100 μg / mL), cultured at 37℃ overnight, and then the bacterial moss was placed in a 100mL LB tube and cultured at 37℃ with shaking at 250rpm until the OD 600nm It is 0.4-0.5 and stored at 4°C as activated seeds.

[0124] (2) Seed solution preparation

[0125] The activated seeds were taken and inoculated into four 1000 mL Erlenmeyer flasks containing 500 mL LB medium at a ratio of 1:10. The culture was incubated at 30°C, 200 rpm, for 15 hours to serve as the fermentation tank seed solution.

[0126] 2. Fermentation culture

[0127] Basic culture medium: yeast extract 3g / L, peptone 5g / L, glucose 10g / L, disodium hydrogen phosphate 30g / L, potassium dihydrogen phosphate 5g / L, ammonium chloride 1g / L, sodium chloride 0.6g / L, magnesium sulfate 1g / L.

[0128] Feed medium: 40% glucose, 5% peptone, 3% yeast extract, total volume 2L

[0129] Prepare 18L of basal culture medium, sterilize the culture medium at 121℃ for 30min, and then cool it to 37℃. Take 2000mL of seed solution and add it to the fermentation tank aseptically to a total volume of 20L. Adjust the process parameters to set the temperature to 37℃, the speed to 300rpm, the tank pressure to 0.01Mpa, and the pH to 7.0 automatically with 50% ammonia water, and calibrate the dissolved oxygen to 100%. When the OD of the fermented bacteria is 600nm When the cell density OD reaches 8-10, start feeding the feed medium. 600nm When the temperature reaches 14-18 o'clock, add 0.2mMIPTG to induce for 3h, sample and collect the fermentation liquid. Monitor the growth of bacteria and glucose content during the fermentation process. Centrifuge the sample and perform SDS-PAGE electrophoresis to detect the target protein content and plasmid loss rate.

[0130] Comparative Example 2

[0131] 1. Cultivation of seed solution

[0132] LB medium: yeast extract 5g / L, peptone 10g / L, sodium chloride 10g / L

[0133] (1) Activation of seed bacteria

[0134] The preserved PET-UHC / BL21DE3PlysS strain was taken out from the -70°C refrigerator, streaked onto a plate, and cultured at 37°C and 220 r / min for 16 h. A single clone was picked and inoculated into a test tube containing 5 mL of LB medium (containing 100 μg / mL of ampicillin), and cultured at 37°C for 10 h.

[0135] (2) Seed solution preparation

[0136] Take the first-level seeds cultured in test tubes and transfer them to a 1000mL shake flask containing 200mL 2×YT medium at a 2% inoculation rate. Culture them at 37°C and 220r / min for about 6h for inoculation of the fermenter.

[0137] 2. Fermentation culture

[0138] Semi-synthetic medium for fed-batch culture in fermenters (g / L): 5 tryptone, 10 yeast extract, 5 glycerol, 3 potassium dihydrogen phosphate, 5 potassium dihydrogen phosphate trihydrate, 2 sodium dihydrogen phosphate dihydrate, 1 magnesium sulfate heptahydrate, 2 mL trace element solution

[0139] Composition of trace element solution (g / L): 3 manganese sulfate monohydrate, 0.5 cobalt chloride, 0.2 sodium molybdate dihydrate, 20 zinc chloride, 6 copper sulfate pentahydrate, 0.02 boric acid, 65 ferrous sulfate heptahydrate, 2 calcium chloride dihydrate, 0.08 sodium iodide, 5 mL sulfuric acid.

[0140] Feed matrix (g / L): 400 glycerol, 50 yeast extract, 100 tryptone, 5 magnesium sulfate heptahydrate.

[0141] Prepare 18L of basal culture medium, sterilize the culture medium at 121℃ for 30min, and then cool it to 37℃. Take 2000mL of seed solution and add it to the fermentation tank aseptically to a total volume of 20L. Adjust the process parameters to set the temperature to 37℃, the speed to 300rpm, the tank pressure to 0.01Mpa, and the pH to 7.0 automatically with 50% ammonia water, and calibrate the dissolved oxygen to 100%. When the fermentation culture reaches 5h, the dissolved oxygen and pH value rise sharply, and the feed medium is started. When the bacterial density OD 600nm When the pH reaches 35, add 0.5 mM IPTG to induce for 5 h, sample and collect the fermentation broth. Monitor the bacterial growth and glucose content during the fermentation process. Centrifuge the sample and perform SDS-PAGE electrophoresis to detect the target protein content and plasmid loss rate.

[0142] Verification Example

[0143] Determination of OD of the fermentation broth of Examples 1-5 and Comparative Examples 1-2 600nm , the yield of recombinant uricase, plasmid loss rate and electrophoresis expression level; the results are shown in Table 1.

[0144] Table 1: Test results of fermentation broth of Examples 1-5 and Comparative Examples 1-2

[0145] Fermentation Broth <![CDATA[OD 600nm ]]> Recombinant uricase production (g / L) Plasmid loss rate (%) Electrophoresis expression level (%) Example 1 130.5 5.5 1 57.1 Example 2 110.7 4.9 2 51.6 Example 3 120.2 5.1 1 53.8 Example 4 136.7 4.6 5 46.4 Example 5 115.2 4.2 6 43.2 Comparative Example 1 23.1 1.1 1 30.3 Comparative Example 2 70.5 3.4 11 42.7

Claims

1. A fermentation method for recombinant uricase from genetically engineered bacteria, characterized in that: The steps include: (1) Inoculating the genetically engineered bacteria PET-UHC / BL21DE3PlysS into a seed culture medium and amplifying the culture step by step to obtain a seed solution; (2) inoculating the seed solution into the fermentation tank medium for high-density fermentation, adding an inducer 0.2-0.4 mmol / L IPTG to induce the culture, and limiting the flow of feed medium until the end of the culture; The fermentation medium is 2-4 g / L peptone, 6-8 g / L yeast extract, 8-12 g / L glucose, 3-5 g / L lactose, 17-30 g / L sodium dihydrogen phosphate, 4-6 g / L dipotassium hydrogen phosphate, 0.5-1.5 g / L magnesium sulfate, and 0.5-1.5 g / L ammonium chloride. The feed medium components are peptone 50-70 g / L, yeast extract 70-90 g / L, glucose 100-300 g / L, lactose 50-70 g / L, magnesium sulfate 15-25 g / L, and the flow acceleration of the feed medium is 0.2-0.5 mL / (L·min); The pH value of the fermentation culture process is 6.8-7.2; the fermentation temperature is 35-37°C.

2. The method according to claim 1, characterized in that The feeding rate was adjusted in time according to the OD value of the culture system. When the OD600nm was 10-13, the flow acceleration was 0.20mL / (L·min); when the OD600nm was 14-17, the flow acceleration was 0.25mL / (L·min); when the OD600nm was 17-28, the flow acceleration was 0.30mL / (L·min); when the OD600nm was 28-39, the flow acceleration was 0.40mL / (L·min); when the OD600nm was above 39, the flow acceleration was maintained at 0.50mL / (L·min) until the end of the culture.

3. The method according to claim 1, characterized in that The amount of feed medium added is 25-35% of the volume of the basal medium.

4. The method according to claim 1, characterized in that The induction culture time is 9 to 12 hours.