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Corynebacterium glutamicum and method for preparing L-ornithine and salts thereof by using same

A technology of Corynebacterium glutamicum and ornithine, applied in the field of fermentation engineering, can solve the problems of low yield, many reaction steps, difficulty in large-scale production, etc., and achieves simple fermentation process, extensive culture conditions, and low production cost. Effect

Active Publication Date: 2011-01-26
NANJING UNIV OF TECH
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  • Abstract
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Problems solved by technology

The disadvantages of this method are: the raw material hydrogen cyanide is a highly toxic carcinogen, which will bring great safety hazards to production; there are many reaction steps, each step is not completely reacted, and the yield is not high; the reaction will get D-bird A racemic compound where amino acid and L-ornithine coexist, the chiral resolution of the product adds difficulty and cost to the separation and purification process
However, the disadvantage of preparing L-ornithine by hydrolyzing arginine is that its economic benefits are subject to the market price difference between arginine and ornithine, and the conversion rate of L-arginine is 90% of the theoretical value- 97%, and the extraction rate is 85%. Based on this calculation, at least 1.6-1.7 tons of L-arginine is required to produce 1 ton of L-ornithine hydrochloride. Amino acid application

Method used

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  • Corynebacterium glutamicum and method for preparing L-ornithine and salts thereof by using same
  • Corynebacterium glutamicum and method for preparing L-ornithine and salts thereof by using same
  • Corynebacterium glutamicum and method for preparing L-ornithine and salts thereof by using same

Examples

Experimental program
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Effect test

Embodiment 1

[0035] Slant medium: 1g glucose, 10g peptone, 5g beef extract, 5g NaCl, 20g agar, add water to 1000ml, pH 7.0-7.2, sterilize at 0.1Mpa at 121°C for 20min.

[0036] Shake flask medium: glucose 25g, yeast extract 10g, (NH 4 ) 2 SO 4 15g, MgSO 4 ·7H 2 O 2.5g, KH 2 PO 4 1g, K 2 HPO 4 ·3H 2 O 0.5g, NaH 2 PO 4 2H 2 O 0.5g, CaCO 3 10g, add water to 1000ml, pH7.6-7.8, sterilize at 0.1Mpa at 121℃ for 10min.

[0037] Fermentation medium: glucose 60g, yeast extract 30g, (NH 4 ) 2 SO 4 50g, MgSO 4 ·7H 2 O 2.50g, KH 2 PO 4 1g, K 2 HPO 4 ·3H 2 O 0.5g, Na 2 HPO 4 2H 2 O 0.50g, CaCO 3 10g, FeSO 4 ·7H 2 O 36.6mg / L, MnSO 4 ·H 2 O 22.38 mg, ZnSO 4 ·7H 2 O 17.8mg, Biotin (biotin) 0.05mg, V B1 0.05mg, add water to 1000ml, pH7.6-7.8, sterilize at 0.1Mpa at 121℃ for 10min.

[0038] Corynebacterium glutamicum 1006 with the preservation number CGMCC No.3663 was cultured statically on a slant medium at 30°C for 18-20 hours, and then a ring of well-grown activated strai...

Embodiment 2

[0040] Glucose 180g, yeast extract 90g, (NH 4 ) 2 SO 4 150g, MgSO 4 ·7H 2 O 7.5g, KH 2 PO 4 3g,K 2 HPO 4 ·3H 2 O 1.5g, Na 2 HPO 4 2H 2 O 1.5g, CaCO 3 30g, FeSO 4 ·7H 2 O 0.1g, MnSO 4 ·H 2 O0.067g, ZnSO 4 ·7H 2 O 0.053g, Biotin 0.15mg, V B1 0.15mg, adjust the pH to 7.6-7.8, dilute to 3L with water, put into a 5L automatic glass stirring fermenter, and steam sterilize at 121°C for 10min.

[0041] The activated Corynebacterium glutamicum 1006 with the preservation number of CGMCC No.3663 was cultivated at 30° C. for 11 hours with a seed medium to obtain a seed liquid, and 120 mL of the seed liquid was inserted into a cooled 5 L fermenter and cultivated at 28° C. ( Ventilation ratio 1: 0.7vvm, stirring speed is 220r / min), along with the increase of culture time, L-ornithine constantly increases, and to 72h, containing L-ornithine in the fermented liquid is 45g / L.

Embodiment 3

[0043] Same as Example 2, the process control pH value (ceramic membrane operating pH) is at 6.8 ± 0.05, and the final fermentation broth adopts a titanium dioxide ceramic membrane with a molecular weight cut-off of 50,000 Daltons, with an operating pressure difference of 0.22MPa, a temperature of 58°C, and a flux of 192L·m -2 h -1 , effectively remove the bacteria; the clear liquid is adsorbed by JK006 cation exchange resin, 0.5mol / L ammonia water is eluted, pure water is washed, the collected liquid is concentrated to remove ammonia, and activated carbon is decolorized; the decolorized liquid is adjusted to pH 4.8 with concentrated hydrochloric acid, and the temperature is 20 ℃, the stirring speed is 200r / min, crystallization with ethanol solution, the crystallization time is 6h, and the crystallization of L-ornithine hydrochloride is obtained, the yield is 88.1%, and the purity is 98.7%. (Calculation of yield: before and after extraction and separation, use HPLC-ELSD to de...

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Abstract

The invention belongs to the field of fermentation engineering and discloses corynebacterium glutamicum and a method for preparing L-ornithine and salts thereof by using the same. The corynebacterium glutamicum is corynebacterium glutamicum 1006 with the collection number of CGMCCNo.3663. The method comprises the following steps of: culturing the corynebacterium glutamicum in a culture medium containing carbon sources, nitrogen sources and inorganic salts at the temperature of between 28 and 30 DEG C to generate L-ornithine fermentation liquor; removing the thalli of the fermentation liquor by using ceramic membranes of 1 to 15 ten thousand Dalton; separating the fermentation liquor by using cation exchange resins to obtain L-ornithine solution; and decoloring and crystallizing the L-ornithine solution by a hydrochloric acid and ethanol method to obtain L-ornithine hydrochloride. The method has the advantages of simple thallus cultivation and contribution to industrialized mass production. The accumulation of the L-ornithine prepared from the corynebacterium glutamicum reaches 35 to 45g / L.

Description

technical field [0001] The invention belongs to the field of fermentation engineering, and relates to a high-yield L-ornithine Corynebacterium glutamicum and a method for preparing L-ornithine and its salt by using the bacteria. Background technique [0002] L-Ornithine is one of the ubiquitous amino acids in living organisms. It was discovered by Jeffrey in 1877 in the hydrolyzate of bird urine fed with benzoic acid. L-ornithine is an important metabolic compound in living cells. It is a component of bacterial cell membranes and polypeptide antibiotics. It mainly participates in the urea cycle in organisms and is used for citrulline, arginine, proline, The biosynthesis of polyamines plays an important role in the excretion of ammoniacal nitrogen in the body. In recent years, the development of L-ornithine products at home and abroad has gradually heated up, and the market prospect has become increasingly broad. [0003] L-ornithine hydrochloride is an important metabolic ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12P13/10C12R1/15
Inventor 万红贵蔡恒袁建锋陆彬
Owner NANJING UNIV OF TECH
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