Glutamic acid production strain and preparation method and application thereof
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A technology for the production of bacterial strains, Corynebacterium glutamicum, applied in the field of bioengineering, can solve problems such as gaps, and achieve the effect of promoting mutual penetration healing and strong glutamate dehydrogenase activity
Inactive Publication Date: 2020-11-13
HENAN UNIVERSITY OF TECHNOLOGY +1
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[0008] In recent years, although my country's glutamic acid fermentation has greatly improved...
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Embodiment 1
[0037] The preparation of glutamic acid high-yielding bacterial strain FM19-007 comprises the following steps:
[0038] (1) Preparation of protoplasts: Pretreat the seed liquid of Corynebacterium glutamicum A (preservation number CGMCC NO.1.10760) and Corynebacterium glutamicum B (preservation number CGMCC NO. 1.1735) with 1U / mL penicillin for 4 hours After that, add 1U / mL lysozyme to the seed liquid, and act at 34 °C for 12h, and add wheat germ agglutinin to the seed liquid at the fifth hour after adding lysozyme, and the amount of wheat germ agglutinin added is 0.1 g / L, that is, the amount of wheat germ agglutinin per liter of seed solution is 0.1g to obtain a protoplast suspension;
[0039] (2) Protoplast fusion: take 1 mL each of the protoplast suspensions of Corynebacterium glutamicum A (preservation number CGMCC NO. 1.10760) and Corynebacterium glutamicum B (preservation number CGMCC NO. 1.1735) in step (1) and mix , centrifuged for 15 min (10 000 × g), discarded the s...
Embodiment 2
[0047] Utilize the FM19-007 bacterial strain among the embodiment 1, ferment and produce glutamic acid, the steps are as follows:
[0049] (2) Inoculation: Inoculate the above-mentioned FM19-007 strain with a cell concentration of 1×10 8 Individual / mL or more, the inoculum volume is 1%;
[0050] (3) Fermentation: pH 7.3±0.05, temperature 33-38.5°C, time 34-36 hours, after the 30th hour of fermentation, add scopolamine at a concentration of 5ppm, 5 μL of scopolamine per liter of fermentation broth;
[0051] (4) Observation of obvious morphological changes in the fermentation process of the bacteria and at the same time changes in cell membrane permeability (forming cell leakage), such as figure 2 , image 3 shown;
[0052] (5) To measure the acid production rate of the fermentatio...
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Abstract
The invention discloses a glutamic acid production strain ands a preparation method and application thereof, and belongs to the field of bioengineering. The method comprises the following steps: fusing protoplast between corynebacterium glutamicum A (the preservation number is CGMCC NO.1. 10760) and corynebacterium glutamicum B (the preservation number is CGMCC NO.1. 1735), and performing mutationand regeneration so that the FM19-007 strain (CGMCC No. 18678) is obtained. The strain is characterized in that the strain is short-rod-shaped, spore-free and flagellate-free when observed under a microscope, gram staining is positive, the cell size is 0.7-1 * 1.2-3 [mu] m, and alpha-ketoglutarate dehydrogenase is lost; the glutamate dehydrogenase activity is high; glutamic acid is not used, glutamic acid feedback inhibition is relieved, and the capacity of secreting glutamic acid to the outside is achieved; in the fermentation process, obvious morphological change and cell membrane permeability change (forming cell leakage) occur at the decay stage. The mutant strain is fermented to produce glutamic acid, the yield of glutamic acid is 22%, the yield is increased by 51.72%, and the yieldof glutamic acid is remarkably increased.
Description
technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a glutamic acid production strain, a preparation method and application thereof. Background technique [0002] The fermentation of glutamic acid bacteria is divided into four periods: adaptation period, logarithmic growth period, acid production period (stable period), and decline period, and the decline period basically does not produce acid. The glutamic acid secreted by glutamicum is inside the cell and is secreted out of the cell through the cell membrane. In order to obtain intracellular glutamic acid, external interference is needed to secrete the extracellular. At present, the glutamic acid-producing bacteria used in industry include Corynebacterium glutamicum, Brevibacterium lactose fermenterum, Brevibacterium spirochete, Brevibacterium flavum, Brevibacterium ammoniaphagosus, etc. Commonly used strains in my country are Corynebacterium Beijing, Coryneb...
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