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Glutamic acid capable of having high-yield glutamine

A technology of Corynebacterium glutamicum and glutamine, applied in the direction of bacteria, etc.

Inactive Publication Date: 2005-12-21
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, at present, the high-yield glutamine Corynebacterium glutamicum that can reach 80g / l capacity has not been reported yet.

Method used

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  • Glutamic acid capable of having high-yield glutamine
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  • Glutamic acid capable of having high-yield glutamine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1: Screening of Corynebacterium glutamicum G32CCTCC NO.M205028 strain

[0037] Add a nitrosoguanidine (NTG) solution with a final concentration of 300ug / ml to the seed medium that has been cultivated to the mid-logarithmic growth phase (pre-added glass beads), treat it with constant temperature shaking at 30°C for 30 minutes, and then take 5ml and add it to a In a sterile test tube, immediately add 0.5ml of 25% sodium thiosulfate to terminate the effect. Take an appropriate dilution (preferably 10 5 bacterium / ml) coating screening plate, the screening plate is LB medium plus sulfaguanidine (SG) and 0.5% LiCl, wrap it with paper to avoid light, and culture it upside down at 30°C for 2 to 3 days , pick a single colony for shaking tube culture, shake tube culture is carried out on a reciprocating shaker at 30°C, speed 100r / min, stroke 8cm, after 72 hours of culture, use YSI2700 biochemical analyzer to detect glutamine production, and select high-yielding strains ...

Embodiment 2

[0041] Example 2: Extraction of the 16S rDNA gene of the above Corynebacterium glutamicum G32 CCTCCNO.M205028 strain

[0042] Cultivate 5ml of bacterial culture to saturation, take 1.5ml of culture, and centrifuge at 6000g for 2 minutes; add 565μl of TE buffer to the precipitate, and the formula of TE buffer is as follows: 10mmol / L Tris (Tris), 1mmol / L ethylenediaminetetraacetic acid (EDTA), adjust the pH to 8.0 with hydrochloric acid, resuspend it by repeatedly blowing with a pipette, add 30μl of sodium dodecylsulfonate (SDS) with a mass volume ratio of 10% and 5μl 20mg / mL proteinase K, mix well, incubate at 37°C for 1 hour; add 100μl, 5mol / L NaCl, mix thoroughly, then add 80μl CTAB / NaCl solution, the formula of CTAB / NaCl solution is as follows: the mass volume ratio is Dissolve 10% cetyltriethylammonium bromide (CTAB) in 0.7mol / L NaCl, mix well, and incubate at 65°C for 10 minutes; add an equal volume of chloroform / isoamyl alcohol, mix well, Centrifuge at 12000g for 5 minut...

Embodiment 3

[0070] Embodiment 3: above-mentioned Corynebacterium glutamicum (Corynebacterium glutamicum) G32 CCTCCN0.M205028 bacterial strain fermentation produces glutamine

[0071]After culturing Corynebacterium glutamicum (Cornyebacterium glutamicum) G32 CCTCC NO.M205028 on the LB slope for 12 hours, connect 1 to 2 rings of bacterial lawn to a Erlenmeyer flask containing 20ml of liquid medium, at 30°C, at a speed of 100r / min, Liquid seeds were obtained after culturing for 9 hours under a reciprocating shaker with a stroke of 8 cm.

[0072] The above-mentioned liquid seeds are inserted into the fermentation medium at a volume percentage of 5% for ventilated and stirred culture, and high-concentration glutamine is produced by fermentation. Control the degree of saturation at 20% to 40%, and the fermentation time is 35-60 hours. Control the pH at the initial stage of fermentation to be 6.6. When the bacteria grow to the middle and late stages of the index (17-18 hours), the pH is reduced...

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Abstract

This invention discloses the bacillus of glutaminic acid, this fungus by name glutaminic acid bacillus (Corynebacteriumglutamicum ) G32, own to preserve in ' China model culturation preservation center ' it is CCTCC NO.M205028 of preserve number. The bacterial strain is in short bar shape when it is young, exist individually or in pairs, ' V ' shape arranged, not forming mycelium, leather blue dye is positive but easy to fade, do not move, does not give birth to the spore , does not resist the acid , facultative anaero which needs nutrition-rich medium, its colony is protruding, semitransparent, the glassy surface. The ammonia amount of Gln accumulated in this fermented liquid of fungus reaches 75-80g / L, and this fungus tolerance is up to the salt concentration of 8%- 10% of ammonium sulfate.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a Corynebacterium glutamicum capable of high glutamine production. Background technique [0002] Glutamine is a special nutrient that has attracted widespread attention and has become a research hotspot in recent years. [0003] There are three main methods for the production of glutamine: chemical synthesis, enzymatic method, and fermentation method. [0004] The chemical synthesis method uses glutamic acid as the starting material, and generates glutamine through three steps of acidification, ammonolysis and acidolysis. The yield of chemical synthesis method can reach 70%-78%. However, due to the high price of required raw materials and reagents, the cost is also high, and the raw material products have a slight special smell, which is not suitable for acceptance by the food and pharmaceutical industries, so the chemical method is used to produce Glutamine is still di...

Claims

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Application Information

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IPC IPC(8): C12N1/20
Inventor 许平马翠卿杨春玉孙际宾李金山邱建华王霞
Owner SHANDONG UNIV
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