CN109161507B - Corynebacterium glutamicum capable of producing L-ornithine at high yield and application thereof - Google Patents
Corynebacterium glutamicum capable of producing L-ornithine at high yield and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of biological fermentation, and discloses Corynebacterium glutamicum capable of producing L-ornithine with high yield and application thereof, wherein the Corynebacterium glutamicum is classified and named as Corynebacterium glutamicum (Corynebacterium glutamicum) jp-07092, which is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, the preservation number is CGMCC No. 15856. the strain is utilized to produce L-ornithine by fermentation, and the method has the advantages of low cost, short fermentation period, high yield in unit time, simple subsequent purification process and the like, and is more suitable for industrial popularization.
Description
Technical Field
The invention belongs to the technical field of biological fermentation, and particularly relates to corynebacterium glutamicum capable of producing L-ornithine at a high yield and application thereof.
Background
Corynebacterium glutamicum (Corynebacterium glutamicum) is a very important production strain in the fermentation industry at present, and has been widely applied to the production of various amino acids and related derivatives thereof, the main products include L-glutamic acid, L-lysine, L-ornithine, L-glutamine, etc.
L-ornithine mainly participates in urea circulation in organisms and plays an important role in discharging ammonia nitrogen, in recent years, L-ornithine has wide application in the medicine and food industries, in the medicine industry, L-ornithine can be used for protecting liver, protecting liver and treating hyperammonemia, and recovery of trauma and burn, in the food industry, L-ornithine has the effects of health care and weight reduction and body immunity improvement, and is an ideal nutritional supplement for bodybuilders and athletes, the multifunctional health care effect of L-ornithine continuously attracts people's attention, and the market demand of the ornithine is increased greatly, but L-ornithine is difficult to produce, at present, L-ornithine is mainly produced by an enzyme method and a fermentation method, arginine is converted into L-ornithine by the enzyme method under the action of arginase, but arginine and arginase are difficult to obtain, so that the production cost is high, and the fermentation method is simple and easy to implement and is particularly suitable for industrial production.
In the existing technology for producing L-ornithine by fermenting corynebacterium glutamicum, when the glucose addition amount of the corynebacterium glutamicum with the patent number of 2010102862368 and the strain number of CGMCC No.3663 is 60 g/L in the fermentation process, the yield of L-ornithine is 38.5 g/L, and when the glucose addition amount is 180 g/L, the yield of L-ornithine is 45 g/L.
Disclosure of Invention
The invention aims to provide corynebacterium glutamicum with high yield of L-ornithine, which has high yield of L-ornithine and can bring benefits for industrially improving the yield of L-ornithine.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
the Corynebacterium glutamicum strain with high yield of L-ornithine is classified and named as Corynebacterium glutamicum (Corynebacterium glutamicum) jp-07092, and is preserved in China general microbiological culture Collection center with the preservation number of CGMCC No. 15856.
The application of the corynebacterium glutamicum with high yield of L-ornithine in the fermentation production of L-ornithine.
The application comprises the steps of activating jp-07092 of Corynebacterium glutamicum (Corynebacterium glutamicum), and carrying out aerobic fermentation for 42-72 hours at 25-58 ℃, wherein an aerobic fermentation culture medium contains 80-120 g/L of carbon source, 50-90 g/L of nitrogen source, 0-10 g/L of inorganic salt, a proper amount of water and pH5.5-8.0.
Wherein the carbon source is glucose; the nitrogen source is ammonium sulfate; the inorganic salt is any one or combination of magnesium salt, potassium salt, manganese salt, calcium salt, sulfate, phosphate or hydrochloride.
Further, the culture medium may also contain vitamins and arginine.
The application comprises activating corynebacterium glutamicum jp-07092, carrying out aerobic fermentation at 30 ℃ for 65h, and stirring at 220r/min with the aeration ratio of 1.5v/vm in the fermentation stage;
wherein the aerobic fermentation medium contains 80-140 g/L (NH) of glucose4)2SO450～90g/L，K2HPO4·3H2O 1～3g/L，KH2PO41～4g/L，MgSO4·7H2O 0.2～2g/L,CaCO310-40 g/L, proper amount of water, pH 6-8, and stabilizing the pH by adding ammonia water in a flowing manner in the fermentation process.
The application is that the culture medium is optimized to be 120 g/L (NH) of glucose4)2SO480g/L，K2HPO4·3H2O 1g/L，KH2PO41g/L，MgSO4·7H2O 0.25g/L，MnSO4·H2O 200mg/L，FeSO4·7H2O200 mg/L, arginine 200 mg/L21mg/L，CuSO40.2 mg/L, biotin 0.1 g/L, vitamin B10.2mg/L330 g/L, appropriate amount of water, pH 7.0.
The culture medium for high yield L-ornithine of corynebacterium glutamicum is an aerobic fermentation culture medium containing 80-140 g/L of glucose,(NH4)2SO450～90g/L，K2HPO4·3H2O 1～3g/L，KH2PO41～4g/L，MgSO4·7H2O 0.2～2g/L,CaCO310-40 g/L, and proper amount of water, wherein the pH value is 6-8.
The culture medium is optimized to be 120 g/L (NH) of glucose4)2SO480g/L，K2HPO4·3H2O1g/L，KH2PO41g/L，MgSO4·7H2O 0.25g/L，MnSO4·H2O 200mg/L，FeSO4·7H2O200 mg/L, arginine 200 mg/L21mg/L，CuSO40.2 mg/L, biotin 0.1 g/L, vitamin B10.2mg/L330 g/L, appropriate amount of water, pH 7.0.
In particular, the first and second (c) substrates,
a Corynebacterium glutamicum for high yield of L-ornithine, which is a Corynebacterium glutamicum (Corynebacterium glutamicum) jp-07092, which has been preserved in the China general microbiological culture Collection center in 6.4.2018, wherein the address is No.3 of the Xilu No.1 of the Xingyang district of Beijing, the collection number is CGMCC NO. 15856.
The strain is obtained by the following steps of purchasing Corynebacterium glutamicum ATCC 13032 powder which is subjected to freeze-drying preservation from the national strain preservation center CGMCC, activating the strain, screening a Corynebacterium glutamicum strain with high yield of L-arginine through ion beam mutagenesis, then screening the Corynebacterium glutamicum strain with high yield of L-arginine to obtain a L-arginine defective Corynebacterium glutamicum strain through ion beam mutagenesis, screening the Corynebacterium glutamicum strain with high yield of L-arginine through ion beam mutagenesis, performing basic culture on the Corynebacterium glutamicum ATCC 13032 by using a liquid L B culture medium, culturing at 30 ℃ for 12 hours, and diluting the cultured Corynebacterium glutamicum ATCC 13032 bacterial liquid by a 10-fold dilution method until the cell number is 109M L, uniformly spreading the bacterial liquid 0.1m L in a sterile empty plate, and air-drying the plate and then carrying out N+Ion beam implantation, N+The ion beam implantation dosage is (95, 140, 185, 230, 275) × 2.6.6 2.6 × 1013N+/cm2，N+The ion beam injection energy is 15keV, after irradiation, cells are washed by 1m L sterile water, diluted by a 10-fold dilution method and coated into a plate culture medium, arginine methyl ester resistance is added into the culture medium, inverted culture is carried out for 1d at 30 ℃, after a single colony is picked, shake flask detection is carried out, a bacterial strain which is high in colony growth speed and produces L-arginine is screened out, under the condition of not optimizing fermentation, the yield of L-arginine is 18 g/L, and the bacterial strain is named as Best-1.
Using Best-1 as an original strain, and screening L-arginine deficient strain from the Best-1 by an ion beam mutagenesis method, wherein the mutagenesis method is the same as the above method, performing basic culture on the Best-1 by using a liquid L B culture medium, culturing at 30 ℃ for 12h, and diluting the cultured Best-1 bacterial liquid by a 10-fold dilution method until the cell number is 109M L, uniformly spreading the bacterial liquid 0.1m L in a sterile empty plate, and air-drying the plate and then carrying out N+Ion beam implantation, N+The ion beam implantation dosage is (95, 140, 185, 230, 275) × 2.6.6 2.6 × 1013N+/cm2，N+Ion beam injection energy is 15keV, after irradiation, cells are washed with 1m L sterile water, diluted by 10-fold dilution method, and applied to a plate-based medium and a L-arginine-containing medium, wherein the basic medium (g/L) includes glucose 5, (NH 26)4)2SO43，K2HPO40.2，KH2PO40.2，MgSO40.5，NaH2PO40.2, L-arginine medium was supplemented with L-arginine 150 mg/L in minimal medium to find out strains which did not grow in minimal medium and could grow in L-arginine medium, which were arginine-deficient strains, and ornithine-producing strains were selected from among the arginine-deficient strains, and the selection medium (g/L) was glucose 25, (NH)4)2SO415，K2HPO41，KH2PO40.5，MgSO42.5，NaH2PO40.5，CaCO310, pH7.0, separating and purifying the screened higher-yield strain to obtain a high-yield L-ornithine strain, optimizing fermentation, and adding 120 g/L of glucose (NH)4)2SO480g/L，K2HPO4·3H2O 1g/L，MgSO4·7H2O·0.25g/L，KH2PO41g/L，MnSO4·H2O 200mg/L，FeSO4·7H2O200 mg/L, arginine 200 mg/L21mg/L，CuSO40.2 mg/L, biotin 0.1 g/L, vitamin B10.2mg/L3The yield of 30 g/L-ornithine can reach 45 g/L, which is named as Corynebacterium glutamicum (Corynebacterium glutamicum) jp-07092.
The culture conditions of the corynebacterium glutamicum (Corynebacterium glutamicum) jp-07092 are as follows:
the strain is cultured aerobically, and the carbon source for culturing the strain can be glucose; the nitrogen source for culturing the strain may be ammonium sulfate. The optimal temperature range of the growth of the strain is 25-58 ℃, the pH range is 5-9, and the optimal pH range is 6-8. Inorganic salts such as dipotassium hydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate, manganese sulfate, ferrous sulfate, etc. can also be added in the process of culturing the strain.
The screened corynebacterium glutamicum with high yield of L-ornithine is subjected to subculture about 30 times, and the growth and yield of L-ornithine are measured by shake flask fermentation, and the strain still has good genetic stability after 30 passages.
The physiological morphological characteristics of the corynebacterium glutamicum (Corynebacterium glutamicum) jp-07092 are that the corynebacterium glutamicum grows vigorously on a L B plate, and obviously, a single colony is observed to be uniformly colored, is in a circular or spherical shape, and is in an off-white color.
The application of the corynebacterium glutamicum (corynebacterium glutamicum) jp-07092 in liquid fermentation production of L-ornithine comprises the following steps:
1) seed culture: inoculating a loopful of bacteria from the activated flat plate into a liquid seed culture medium, and culturing for 12h at 25-58 ℃;
2) inoculating the seed solution obtained in the step 1) into a liquid culture medium for fermentation culture, wherein the inoculation amount is 3.5%, the culture temperature is 25-58 ℃, and liquid fermentation is carried out.
The liquid seed culture medium in the step 1) comprises, by mass, 10-60 g/L of a carbon source, 15-35 g/L of a nitrogen source, 0-2 g/L of inorganic salt and a proper amount of water, wherein the carbon source is selected from glucose, the nitrogen source is ammonium sulfate, and the inorganic salt is at least one selected from dipotassium hydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate, manganese sulfate and ferrous sulfate.
The liquid culture medium in the step 2) comprises, by mass, 80-120 g/L parts of a carbon source, 50-90 g/L parts of a nitrogen source, 0-10 g/L parts of inorganic salt and a proper amount of water, wherein the pH value is 5.5-8.0.
The most preferable production mode is that after the strain is activated, aerobic fermentation is carried out for 30-78h at 25-58 ℃, the liquid loading amount is 30m L fermentation liquid/500 m L triangular flask, and the rotating speed is 220 r/min.
By optimizing the fermentation medium, the fermentation medium of the corynebacterium glutamicum jp-07092 is prepared by adding 120 g/L (NH) of glucose4)2SO480g/L，K2HPO4·3H2O 1g/L，MgSO4·7H2O 0.25g/L，KH2PO41g/L，MnSO4·H2O 200mg/L，FeSO4·7H2O200 mg/L, arginine 200 mg/L21mg/L，CuSO40.2 mg/L, biotin 0.1 g/L, vitamin B10.2mg/L3Sterilizing at 30 g/L, pH 7.0.110 deg.C for 15 min.
The invention has the beneficial effects that:
compared with the prior art, the invention has the following advantages:
compared with the existing Corynebacterium glutamicum CGMCC No.3663, the Corynebacterium glutamicum has high metabolic activity, the fermentation period is shortened to 65h by reducing the time of the fermentation period, and the yield of L-ornithine can reach 45 g/L when the addition amount of glucose is 120 g/L, so that the use of expensive glucose is greatly reduced industrially.
As can be seen from the above table, compared with other strains, the yield of 45 g/L can be achieved by using ammonium sulfate as the only nitrogen source for the Corynebacterium glutamicum jp-07092 in a low-sugar medium (120 g/L glucose), and the use of the nitrogen source is reduced compared with other strains, wherein the unit price of yeast extract is 30 yuan/kg, and the unit price of ammonium sulfate is 1.2 yuan/kg, so the production cost is greatly reduced by using ammonium sulfate as the nitrogen source, the fermentation period of the Corynebacterium glutamicum jp-07092 is shortened by nearly 10%, the fermentation batch is increased within a certain period of time, the total yield of L-ornithine is increased, and the carbon source and the like required by the Corynebacterium glutamicum are less in use amount under the condition of the same ornithine yield.
Compared with the traditional fermentation of corynebacterium glutamicum, the fermentation process of the strain adopts ammonium sulfate as a unique nitrogen source to replace the traditional organic nitrogen source such as yeast extract, beef extract and the like, so that the cost is reduced for industrial production, and a plurality of byproducts which are difficult to treat are avoided being generated by adopting the organic nitrogen source as a culture medium component at the later stage of the fermentation, and compared with the prior art, the problem of the type of the ammonium sulfate is avoided because the ammonium sulfate is single in component.
In general, under the specific optimized fermentation process, compared with the traditional corynebacterium glutamicum, the strain provided by the invention has the advantages of low cost, short fermentation period, high yield per unit time, simple subsequent purification process and the like, and is more suitable for industrial popularization.
Strain preservation information:
the strain adopted by the invention is classified and named as Corynebacterium glutamicum (Corynebacterium glutamicum) jp-07092, which is preserved in China general microbiological culture Collection center (CGMCC for short, the address: Beijing area Hongyang region No.1 Xilu No.3, China academy of sciences microbial research institute), the preservation number is CGMCC No.15856, and the preservation date is 2018, 6 months and 4 days.
Detailed Description
The invention will be better understood from the following examples. However, those skilled in the art will readily appreciate that the specific material ratios, process conditions and results described in the examples are merely illustrative of the invention and should not be construed to limit the invention as described in the claims. The culture medium which is mentioned in the invention and is not given a specific preparation process is prepared according to the culture medium which is commonly used in microbiological experiments.
EXAMPLE 1 mutagenic screening of Corynebacterium glutamicum (Corynebacterium glutamicum) Best-1
The culture medium of Corynebacterium glutamicum ATCC 13032 purchased from national culture Collection is subjected to basic culture by using liquid L B culture medium, after the culture is carried out for 1 day at 30 ℃, the biomass and the yields of L-arginine and L-ornithine are simultaneously measured, the culture is carried out in liquid L B seed culture medium, the cultured Corynebacterium glutamicum bacterial liquid is diluted by 10-fold dilution method until the cell number is 109M L, uniformly spreading the bacterial liquid 0.1m L in a sterile empty plate, and air-drying the plate and then carrying out N+Ion beam implantation, N+The ion beam implantation dosage is (95, 140, 185, 230, 275) × 2.6.6 2.6 × 1013N+/cm2，N+The ion beam injection energy is 15keV, after irradiation, cells are washed by 1m L sterile water, diluted by a 10-fold dilution method and coated into a plate culture medium, arginine methyl ester resistance is added into the culture medium, inverted culture is carried out for 1d at 30 ℃, after a single colony is picked, shake flask detection is carried out, a bacterial strain which is high in colony growth speed and produces L-arginine is screened out, under the condition of not optimizing fermentation, the yield of L-arginine is 18 g/L, and the bacterial strain is named as Best-1.
Example 2 mutagenesis screening of Corynebacterium glutamicum (Corynebacterium glutamicum) jp-07092
Basic culture is carried out by using Best-1 as starting strain and liquid L B culture medium, after 1 day of culture at 30 ℃, biomass and yields of L-arginine and L-ornithine are simultaneously measured, the culture is carried out in liquid L B seed culture medium, bacterial liquid of the cultured corynebacterium glutamicum is diluted to 10 cell number by 10-fold dilution method9M L, uniformly spreading the bacterial liquid 0.1m L in a sterile empty plate, and air-drying the plate and then carrying out N+Ion beam implantation, N+Ion beam implantationThe dosage is (95, 140, 185, 230, 275) × 2.6 × 1013N+/cm2，N+Ion beam injection energy is 15keV, after irradiation, cells are washed with 1m L sterile water, diluted by 10-fold dilution method, and applied to plate medium (g/L), glucose 5, (NH)4)2SO43，K2HPO40.2，KH2PO40.2，MgSO40.5，NaH2PO40.2, except the above-mentioned basic culture medium, it has need of setting blank combination control group, the blank group is cultured by above-mentioned basic culture medium, the control group is added with L-arginine 150 mg/L in the basic culture medium, and the strain which does not grow in the blank group and can grow in the control group is found out, and it is arginine deficient strain, and in the arginine deficient strain the ornithine high-producing strain is selected, and the screening culture medium is (g/L), glucose 25, (NH) and glucose 254)2SO415，K2HPO41，KH2PO40.5，MgSO42.5，NaH2PO40.5，CaCO310, pH 7.0. Finally, screening to obtain a high-yield ornithine bacterium named as Corynebacterium glutamicum (Corynebacterium glutamicum) jp-07092.
Example 3 culture and physiological characterization of Corynebacterium glutamicum (Corynebacterium glutamicum) jp-07092
The strain is cultured aerobically. The carbon source for culturing the strain may be glucose; the nitrogen source used for culturing the strain was ammonium sulfate. The optimal growth temperature of the strain is 25-50 ℃, the pH range is 5-9, and the optimal pH range is 6-8. Inorganic salts such as dipotassium hydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate, manganese sulfate, ferrous sulfate, etc. can also be added in the process of culturing the strain.
The physiological morphological characteristics of the corynebacterium glutamicum (corynebacterium glutamicum) jp-07092 are as follows:
corynebacterium glutamicum grew vigorously on L B plates, and it was clearly observed that single colonies were uniformly colored, and appeared round or spherical, beige in color.
EXAMPLE 4 fermentation of Corynebacterium glutamicum (Corynebacterium glutamicum) jp-07092 to L-ornithine
Fermentation of Corynebacterium glutamicum (Corynebacterium glutamicum) jp-07092
Fermentation medium is optimized by fermentation, glucose is 120 g/L, (NH)4)2SO480g/L，K2HPO4·3H2O 1g/L，MgSO4·7H2O 0.25g/L，KH2PO41g/L，MnSO4·H2O 200mg/L，FeSO4·7H2O200 mg/L, arginine 200 mg/L21mg/L，CuSO40.2 mg/L, biotin 0.1 g/L, vitamin B10.2mg/L330 g/L, pH 7.0. during the fermentation process, through feeding ammonia water to stabilize pH., culturing corynebacterium glutamicum jp-07092 with a corynebacterium glutamicum seed culture medium for 12 hours at 30 ℃ under the condition of 200r/min, and then inoculating into a sterilized 5L fermentation tank for culturing, wherein the liquid loading of the fermentation tank is 3L, the culture condition is that 30 ℃, the aeration ratio is 1.5v/vm, the stirring speed is 220r/min, ammonium sulfate is used as a unique nitrogen source during the fermentation process, the fermentation period is 65 hours, and the yield of L-ornithine can reach 45 g/L after the fermentation is finished.
The method comprises the steps of firstly subpackaging and centrifuging fermentation liquor after fermentation is finished, wherein the centrifugation condition is 7000r/min, centrifuging for 10min, then keeping supernatant, removing components such as glucose, nitrogen source, other impurities and residual thalli which are not completely consumed in the culture medium by using a membrane filtration method, selecting a ceramic membrane with the molecular weight of 500KD, carrying out filtration under the condition of pH 2.5 and the operating pressure difference of 0.24MPa and the operating temperature of 58 ℃, obtaining filtrate, then carrying out cation exchange resin adsorption, carrying out L-ornithine column loading concentration of 45 g/L, carrying out loading column flow of 10m 34/min and carrying out ammonia water with the eluent of 0.5 mol/L, carrying out decoloration and crystallization, wherein the concentration of decoloration liquid is 450 g/L, the crystallization temperature is 20 ℃, the stirring speed is 200rpm, and the purity of ornithine can be up to 397-L.7% after crystallization is 6 h.
Claims (8)
1. The Corynebacterium glutamicum strain with high yield of L-ornithine is classified and named as Corynebacterium glutamicum (Corynebacterium glutamicum) jp-07092, and is preserved in China general microbiological culture Collection center with the preservation number of CGMCC No. 15856.
2. Use of the highly L-ornithine producing corynebacterium glutamicum of claim 1 in the fermentative production of L-ornithine.
3. The application of claim 2, wherein Corynebacterium glutamicum (Corynebacterium glutamicum) jp-07092 is activated and aerobically fermented for 42-72 hours at 25-58 ℃, wherein the aerobically fermented culture medium comprises 80-120 g/L of carbon source, 50-90 g/L of nitrogen source, 0-10 g/L of inorganic salt and proper amount of water, and the pH value is 5.5-8.0.
4. The use of claim 3, wherein the carbon source is glucose; the nitrogen source is ammonium sulfate; the inorganic salt is any one or combination of magnesium salt, potassium salt, manganese salt, calcium salt, sulfate, phosphate or hydrochloride.
5. The use as claimed in claim 3, characterized in that, after activation of the Corynebacterium glutamicum jp-07092, it is aerobically fermented at 30 ℃ for 65h, with an aeration ratio of 1.5v/vm in the fermentation stage and stirring at 220 r/min;
wherein the aerobic fermentation medium contains 80-140 g/L (NH) of glucose4)2SO450～90g/L，K2HPO4·3H2O 1～3g/L，KH2PO41～4g/L，MgSO4·7H2O 0.2～2g/L,CaCO310-40 g/L, proper amount of water, pH 6-8, and stabilizing the pH by adding ammonia water in a flowing manner in the fermentation process.
6. The use of claim 5, wherein the culture medium is optimized to 120 g/L (NH) glucose4)2SO480g/L，K2HPO4·3H2O 1g/L，KH2PO41g/L，MgSO4·7H2O 0.25g/L，MnSO4·H2O 200mg/L，FeSO4·7H2O200 mg/L, arginine 200 mg/L21mg/L，CuSO40.2 mg/L, biotin 0.1 g/L, vitamin B10.2mg/L330 g/L, appropriate amount of water, pH 7.0.
7. The culture medium for high yield of L-ornithine from Corynebacterium glutamicum of claim 1, wherein the culture medium is an aerobic fermentation medium comprising glucose 80-140 g/L, (NH)4)2SO450～90g/L，K2HPO4·3H2O 1～3g/L，KH2PO41～4g/L，MgSO4·7H2O 0.2～2g/L,CaCO310-40 g/L, and proper amount of water, wherein the pH value is 6-8.
8. The culture medium of claim 7, wherein the culture medium is optimized to 120 g/L (NH) of glucose4)2SO480g/L，K2HPO4·3H2O 1g/L，KH2PO41g/L，MgSO4·7H2O 0.25g/L，MnSO4·H2O 200mg/L，FeSO4·7H2O200 mg/L, arginine 200 mg/L21mg/L，CuSO40.2 mg/L, biotin 0.1 g/L, vitamin B10.2mg/L330 g/L, appropriate amount of water, pH 7.0.
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