Glucose feeding control method for fermentation production of glutamic acid
阅读说明：本技术 一种发酵生产谷氨酸的葡萄糖补料控制方法 (Glucose feeding control method for fermentation production of glutamic acid ) 是由 吕奎 霍星宇 戴京京 许慧 丁健 陈鹏程 于 2020-03-30 设计创作，主要内容包括：本发明公开了一种发酵生产谷氨酸的葡萄糖补料控制方法,属于生物技术领域。本发明提供了一种发酵生产谷氨酸的方法,此方法通过控制发酵产酸期发酵培养基中葡萄糖的流加大大提高了谷氨酸的产量,利用此方法将嗜乙酰乙酸棒杆菌基因工程菌接种至10L发酵罐中发酵35h,即可使发酵液中谷氨酸的产量高达127g/L。(The invention discloses a glucose feeding control method for producing glutamic acid by fermentation, belonging to the technical field of biology, and providing a method for producing glutamic acid by fermentation, which greatly improves the yield of glutamic acid by controlling the fed-batch of glucose in a fermentation medium in a fermentation acid production period of the fermentation, and by utilizing the method, corynebacterium acetoacidophilum genetic engineering bacteria are inoculated into a 10L fermentation tank for fermentation for 35 hours, so that the yield of glutamic acid in fermentation liquor can reach 127 g/L.)
1. A method for producing glutamic acid by fermentation is characterized in that glutamic acid producing strains are inoculated into a fermentation culture medium for fermentation, after the acid production period of the fermentation is started, the sugar consumption and the oxygen consumption are related, and glucose is fed into the fermentation culture medium according to the oxygen consumption.
2. The method for producing glutamic acid by fermentation as claimed in claim 1, wherein the glutamic acid-producing strain is inoculated into a fermentation medium for fermentation, and after the acid production period of the fermentation starts, the fermentation is performed for 4-5 min as a control period, and each control period isCalculating the consumption of glucose in the control period according to formula Y-2.65671X-7.74394, and calculating the consumption of glucose in the control period according to formulaAnd S (k +1) V ═ S (k) V + SFL (k) -G (k) calculating the theoretical dosage of glucose in the control period, and finally adding glucose in the fermentation medium;
in the formula Y-2.65671X-7.74394, Y is the glucose consumption in the acid production period of normal fermentation, and X is the oxygen consumption in the acid production period of normal fermentation;
formula (II)And S (k +1) V ═ S (k) V + SFL (k) -G (k), where S (k) is the glucose concentration at the time of fermentation k in g/L, S (k +1) is the glucose concentration at the time of fermentation k +1 in g/L, V is the volume of the fermentation broth in L, and S (k) is the glucose concentration at the time of fermentation k +1 in g/LFIs the glucose fed solution concentration in g/L (k) in k to k +1 intervals, the actual glucose fed in L, G (k) in k to k +1 intervals, the actual glucose consumed in g,is the theoretical flux of glucose in the interval k to k +1, in units of L;
taking the fermentation time k to the fermentation time k +1 as a fermentation period, when calculating the theoretical dosage of glucose in the control period, firstly assuming that S (k +1) at the time k +1 is to reach a set valueThenThen substituting S (k +1) into the glucose material balance relational expression S (k +1) V ═ S (k) V + SFL (k) -G (k), then can be solved to obtain
3. The method for fermentative production of glutamic acid according to claim 1 or 2, wherein the glucose is fed to the fermentation medium as a glucose solution.
4. The method for producing glutamic acid by fermentation according to claim 3, wherein the concentration of the glucose solution is 300 to 900 g/L.
5. The method for fermentative production of glutamic acid according to claim 3 or 4, wherein the concentration of the glucose solution is 500 g/L.
6. The method for producing glutamic acid by fermentation according to any one of claims 1 to 5, wherein the glutamic acid-producing strain is Corynebacterium glutamicum (Corynebacterium glutamicum) or a genetically engineered strain obtained by engineering Corynebacterium glutamicum (Corynebacterium glutamicum).
7. The method for producing glutamic acid by fermentation according to claim 6, wherein the genetically engineered bacterium is a Corynebacterium acetoacidophilum genetically engineered bacterium.
8. The method for the fermentative production of glutamic acid according to any one of claims 1 to 7, wherein the fermentation medium comprises C6H12O6、K2HPO4Corn steep liquor and MgSO4Urea, MnSO4、FeSO4Thiamine, and biotin.
9. The method for the fermentative production of glutamic acid according to any one of claims 1 to 8, wherein the fermentation medium comprises C6H12O680g/L、K2HPO41.5 g/L, corn steep liquor 5 g/L, MgSO40.8 g/L, urine5.5 g/L g of element and MnSO42×10-3g/L、FeSO42×10-3g/L, thiamine 5 × 10-5g/L and biotin 3 × 10-6g/L。
10. Use of the method of any one of claims 1-9 for the production of glutamic acid.
Technical Field
The invention relates to a glucose feeding control method for producing glutamic acid by fermentation, belonging to the technical field of biology.
Background
Glutamic acid is an important amino acid and has wide application in a plurality of fields. For example, glutamic acid has a strong umami taste and can be used as a flavoring agent in the food industry; glutamic acid can be used for synthesizing a surfactant and has important application in the field of cosmetics; glutamic acid is easy to form glutamine with blood ammonia after being absorbed by a human body, can relieve the toxic action of ammonia in the metabolic process, can be used as an auxiliary medicament for patients with liver diseases and has important application in the field of medicines, and the glutamic acid is used as a tonic for nerve center and cerebral cortex and has certain curative effect on treating concussion or nerve injury, epilepsy and children with mental retardation.
The production method of glutamic acid mainly comprises three methods, namely a protein hydrolysis method, a chemical synthesis method and a microbial fermentation method, wherein the protein hydrolysis method has the defects of complex operation, time and labor waste, high cost, low product yield and the like, and the chemical synthesis method has serious environmental pollution problem, so that the methods are not suitable for large-scale production. Compared with a protein hydrolysis method and a chemical synthesis method, the method for producing glutamic acid by using a microbial fermentation method has the advantages of environmental friendliness, mild conditions, low cost, easiness in industrial production and the like, so that the microbial fermentation method is commonly used for producing glutamic acid at present in the industry.
However, the existing microbial fermentation method still has certain defects, wherein the low yield is one of the defects which limit the industrial production process of the glutamic acid, for example, the recombinant strain F343 is inoculated into a fermentation medium by the Liu seedling for fermentation for 40h, and the yield of the glutamic acid in the fermentation liquid can only reach 78 g/L (particularly, the reference can be seen in the patent: Liu seedling, the high-temperature resistant glutamic acid producing strain [ D ]. Liu seedling is bred by applying genome shuffling technology, Jiangnan university, 2009(05)), the yield of the glutamic acid in the shake flask fermentation liquid can only reach 82 g/L by inoculating the Corynebacterium glutamicum S9114 into the fermentation medium through optimizing the concentration of the biotin in the medium for fermentation for 30h by the Yahui (particularly, the reference can be seen in the patent: fermentation optimization of the Corynebacterium glutamicum S9114 and regulation of the biotin on the fermentation of the Corynebacterium glutamicum [ D ]. Yahui.
Disclosure of Invention
[ problem ] to
The invention aims to solve the technical problem of providing a method for producing glutamic acid by fermentation with high yield.
[ solution ]
In order to solve the technical problem, the invention provides a method for producing glutamic acid by fermentation, which comprises the steps of inoculating a glutamic acid production strain into a fermentation culture medium for fermentation, correlating sugar consumption and oxygen consumption after the acid production period of the fermentation begins, and adding glucose into the fermentation culture medium according to the oxygen consumption.
In one embodiment of the invention, glutamic acid producing strain is inoculated into a fermentation medium for fermentation, after the acid production period of the fermentation is started, the fermentation is carried out for 4-5 min to serve as a control period, the consumption of glucose in the control period is calculated according to the formula Y-2.65671X-7.74394 in each control period, and then the consumption of glucose in the control period is calculated according to the formulaAnd S (k +1) V ═ S (k) V + SFL (k) -G (k) calculating the theoretical dosage of glucose in the control period, and finally adding glucose in the fermentation medium;
in the formula Y-2.65671X-7.74394, Y is the glucose consumption in the acid production period of normal fermentation, and X is the oxygen consumption in the acid production period of normal fermentation;
formula (II)And S (k +1) V ═ S (k) V + SFL (k) -G (k), where S (k) is the glucose concentration at the time of fermentation k in g/L, S (k +1) is the glucose concentration at the time of fermentation k +1 in g/L, V is the volume of the fermentation broth in L, and S (k) is the glucose concentration at the time of fermentation k +1 in g/LFIs the glucose fed solution concentration in g/L (k) in k to k +1 intervals, the actual glucose fed in L, G (k) in k to k +1 intervals, the actual glucose consumed in g,is the theoretical flux of glucose in the interval k to k +1, in units of L;
taking the time from the fermentation k to the fermentation k +1 as a fermentation period, and assuming k + first when calculating the theoretical dosage of glucose in the control periodS (k +1) at time 1 is to reach the set valueThenThen substituting S (k +1) into the glucose material balance relational expression S (k +1) V ═ S (k) V + SFL (k) -G (k), then can be solved to obtain
In one embodiment of the invention, the glucose is fed to the fermentation medium in the form of a glucose solution.
In one embodiment of the present invention, the concentration of the glucose solution is 300 to 900 g/L.
In one embodiment of the invention, the glucose solution has a concentration of 500 g/L.
In one embodiment of the present invention, the glutamic acid-producing strain is Corynebacterium glutamicum (Corynebacterium glutamicum) and the glutamic acid-producing strain is Corynebacterium glutamicum (Corynebacterium glutamicum) or a genetically engineered bacterium obtained by engineering the strain with Corynebacterium glutamicum (Corynebacterium glutamicum).
In one embodiment of the present invention, the genetically engineered bacterium is a genetically engineered bacterium of corynebacterium acetoacidophilum. The corynebacterium acetoacidophilum genetically engineered bacterium is described in patent application publication No. CN 104371961A.
In one embodiment of the invention, the composition of the fermentation medium comprises C6H12O6、K2HPO4Corn steep liquor and MgSO4Urea, MnSO4、FeSO4Thiamine, and biotin.
In one embodiment of the invention, the composition of the fermentation medium comprises C6H12O680g/L、K2HPO41.5 g/L, corn steep liquor 5 g/L, MgSO40.8 g/L, 5.5 g/L of urea,MnSO42×10-3g/L、FeSO42×10-3g/L, thiamine 5 × 10-5g/L and biotin 3 × 10-6g/L。
In one embodiment of the present invention, the pH of the fermentation medium is 7.0 to 7.2.
The invention provides the application of the method in the production of glutamic acid.
[ advantageous effects ]
The invention provides a method for producing glutamic acid by fermentation, which greatly improves the yield of the glutamic acid by controlling the fed-batch of glucose in a fermentation medium in the acid production period of the fermentation, and by utilizing the method, corynebacterium acetoacidophilum genetic engineering bacteria are inoculated into a 10L fermentation tank for fermentation for 35 hours, so that the yield of the glutamic acid in the fermentation liquid can reach 127 g/L.
Drawings
FIG. 1: linear relationship between sugar consumption and oxygen consumption.
Detailed Description
The genetically engineered bacteria of Corynebacterium acetoacidophilum mentioned in the following examples are described in the patent application publication No. CN 104371961A.
The media involved in the following examples are as follows:
seed culture medium: c6H12O625g/L、K2HPO41.5 g/L, corn steep liquor 40 g/L and FeSO45×10-3g/L、MnSO45×10-3g/L and 2.5 g/L of urea, and the pH value is 7.0-7.2.
Fermentation medium: c6H12O680g/L、K2HPO41.5 g/L, corn steep liquor 5 g/L, MgSO40.8 g/L, 5.5 g/L urea and MnSO42×10-3g/L、FeSO42×10-3g/L, thiamine 5 × 10-5g/L and biotin 3 × 10-6g/L, and the pH value is 7.0-7.2.
The detection methods referred to in the following examples are as follows:
and (3) measuring the content of glucose and the content of glutamic acid, namely analyzing the content of glucose and glutamic acid in the fermentation liquor by adopting a Bio-SBA bioanalyzer, sucking 25 mu L of standard solution SBA for calibration, diluting 1m L of fermentation liquor after the calibration is finished, sucking 25 mu L of diluted fermentation liquor for measurement, and recording data.
Measurement of cell concentration: the absorbance at 620nm was measured using a UV-2000Z UV-Vis spectrophotometer.
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