Poly-gamma-glutamic acid generation bacteria and method for producing poly-gamma-glutamic acid

A glutamic acid and strain technology, applied in the field of applied microorganisms, can solve the problems of high fermentation cost, long fermentation period, limited performance application range, etc., and achieve the effects of stable genetic traits, reduced production costs, and fast growth speed.

Inactive Publication Date: 2004-10-13
HUAZHONG AGRI UNIV
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Problems solved by technology

[0009] During the synthesis of poly-γ-glutamic acid by Bacillus licheniformis ATCC 9945a and Bacillus subtilis (natto), which are commonly used at present, there is a phenomenon that the properties of poly-γ-glutamic acid synthesized by the strain are unstable. After preservation or transfer , most of the colonies lost the ability to synthesize poly-γ-glutamate (Ashiuchi et al., 2001, Isolation of Bacillus subtilis (chungkookjang), a poly-γ-glutamate producer with high genetic competence.Appl.Microbiol.Biotechnol., 57 : 764-769.), this situation requires repeated purification of strains in the experimental process; at the same time, adopt Bacillus licheniformis ATCC 9945a to synthesize poly-γ-glutamic acid, and need to add high concentration of glycerol (to 80g / L) in the medium , thereby causing the fermentation cost to be too high, in addition there are also problems such as long fermentation cycle (2 days to 5 days) (Soon et al., 2000, Biotech.Lett., 22:585-588.; Borbely, 2001, Polymeric product, United States Patent 6,326,511); These problems have seriously restricted the industrialization process of poly-γ-glutamic acid
In addition, the molecular weight of poly-γ-glutamic acid biosynthesized by strains such as Bacillus licheniformis ATCC 9945a and Bacillus subtilis (natto) is below 3000KD ((Cromwick, etc., 1995a.Effect of magnese (II) on Bacillus licheniformis ATCC9945Aphysiology and γ- poly(glutamic acid) formation.Int.J.Biol.Macromol.17: 259-267.)), when using poly-γ-glutamic acid to synthesize polymer biomaterials, it has received great attention in terms of performance and application range. big limit
At present, it is only reported that the molecular weight of poly-γ-glutamic acid biosynthesized by Bacillus subtilis (chungkookjang) can reach 10,000KD. Whether it can be screened to produce strains with higher molecular weight of poly-γ-glutamic acid is urgently to be developed

Method used

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Embodiment 1

[0047] Example 1: Isolation and identification of poly-γ-glutamic acid producing bacteria X-003:

[0048] Take the soil 5cm below the soil surface of the paddy field in the experimental farm of Huazhong Agricultural University, add an appropriate amount of sterile water, shake well, and dilute it by 100 times. Heat-treat the suspension at 80°C for 15 minutes, and apply LB (LB medium should be added per 1 liter of distilled water. The formula is as follows: peptone 10 g / L, NaCl 10 g / L, yeast extract 5 g / L , agar 20 g / liter, pH7.0) solid plate, through overnight cultivation, pick sticky colonies in 5 milliliters of poly-γ-glutamic acid screening medium E (E medium formula and production: see literature: Cromwick etc., Effect of magnese (II) on Bacillus licheniformis ATCC9945A physiology and poly-(glutamic acid) formation. Int. J. Biol. Macromol. 17: 259-267) cultured for 2-3 days. Centrifuge the culture solution to get the supernatant, precipitate with 3 times the volume of eth...

Embodiment 2

[0050] Embodiment 2: the HPLC analysis method of poly-γ-glutamic acid

[0051] The quantitative analysis and molecular weight determination of poly-γ-glutamic acid were completed by HPLC, chromatographic column: G6000PWXL, mobile phase was 25mM Na 2 SO 4 solution and acetonitrile in a ratio of 4:1. Purified poly-γ-glutamic acid was used as a quantitative standard, and the molecular weight standard was TSK standard Poly (ETHYLENE OXIDE).

[0052] The steps for determining the yield and molecular weight of poly-γ-glutamic acid are as follows: After quantitatively diluting the fermentation broth, adjust the pH value to 3.0, centrifuge at 10,000 rpm for 30 minutes, and collect the supernatant. The diluted fermentation broth was used to detect the concentration and molecular weight. The retention time of the fermentation product poly-γ-glutamic acid is 10min, and the molecular weight is above 10000KD (see figure 2 , 3 ) shown.

Embodiment 3

[0053] Embodiment 3: the liquid batch fermentation production of poly-γ-glutamic acid producing bacteria

[0054] 1. Use the strains preserved in cryovials as the original strains.

[0055] 2. Inclined bacteria

[0056] Inoculate the bacterial strain Bacillus subtilis x-003 preserved in the frozen tube onto the LB solid slant with an inoculation loop (add peptone 10 g / L, NaCl 10 g / L, and yeast extract 5 g to 1 liter of distilled water medium) / L, agar 20 g / L, pH7.0.), cultured at 37°C.

[0057] 3. Seed liquid culture

[0058] Inoculate the activated colony on the solid slant surface of LB (the medium composition is the same as above), inoculate 10 g / L of peptone, 10 g / L of NaCl, and 5 g / L of yeast extract into the medium of 1 liter of distilled water , pH 7.0 in 250 ml Erlenmeyer flasks, the filling volume of each bottle of culture solution is 20-40 ml, culture at 37° C. and 200 rpm for 5-6 hours to the mid-logarithmic growth phase.

[0059] 4. Fermentation tank culture: ...

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Abstract

The present invention discloses a strain capable of producing poly-gamma-glutamic acid and a method for producing poly-gamma-glutamic acid by utilizing said strain. The described strain is X-003, a Bacillussubilis, CCTCC, NO:M202048. It adopts seed liquor preparation and liquid submerged fermentation process to produce and obtain the poly-gamma-glutamic acid product. The hereditary feature of said strain is stable, and its capacity for synthesizing poly-gamma-glutamic acid is strong, and the molecular weight of synthetic poly-gamma-glutamic acid can be up to above 10000 KD. The shake-flask fermentation level of poly-gamma-glutamic acid by utilizing said invented strain and its fermentation method can be up to 10 mg/ml, and the fermentation level of the fermentation tank with 80L is 30 mg/ml.

Description

technical field [0001] The invention belongs to the technical field of applied microorganisms, and in particular relates to a poly-gamma-glutamic acid producing bacterium selected and a method for producing poly-gamma-glutamic acid using the strain. Background technique [0002] Poly-γ-glutamic acid (poly-γ-glutamic acid) is a biodegradable polymer composed of D-type and L-type glutamic acid linked by peptide bonds, with an average molecular weight ranging from 10,000 to 2,000,000 Daltons (Matsukawa et al.1997 Hydrophilic surface treating aqueous solution and hydrophilic surfacetreating method, U.S.Patents 5.616,585.Ito, Y., Tamaka et al., 1996.Glutamic acid Independent Production of poly(glutamic acid) by Bacillus 4nol-subtilisBiotech. .Biochen.60:1239-1242; Cromwick et al., Effect of magnese (II) on Bacilluslicheniformis ATCC9945A physiology and poly-(glutamic acid)formation.Int.J.Biol.Macromol.17:259-267; Cromwick et al., 1995 b .Investigati...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12P13/14
Inventor 陈守文喻子牛江昊孙明何进蔡皓李林
Owner HUAZHONG AGRI UNIV
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