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Preparation method of hyaluronic acid

A technology of hyaluronic acid and JM109 is applied in the field of engineering Escherichia coli to produce hyaluronic acid, which can solve the problems of resource shortage and low safety of hyaluronic acid.

Active Publication Date: 2011-08-17
MICROBIAL FERMENTATION ENG RES CENT CO LTD OF YUNNAN PROVINCE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Overcoming resource shortages, safety issues, and low hyaluronic acid synthesis in engineered E. coli in prior art production of hyaluronic acid

Method used

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  • Preparation method of hyaluronic acid
  • Preparation method of hyaluronic acid
  • Preparation method of hyaluronic acid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Shake flask culture synthesis of hyaluronic acid in pHK / JM109:

[0039] In a 500ml Erlenmeyer flask, contain 50 mM K in a total volume of 100ml 2 HPO 4 ·3H 2 O, (pH7.0) 4mM g / L MgSO4 7H2O, 20mM / L citric acid, 1mM MnCl2, 300mM glucose, 50 mM nitrogen acetylglucosamine (GlcNAc), 10mM lactose, 10g / L glycerol, 5g / LCasamino acid (hydrolyzed Casamino acids) added to the system expressed pmHas and kfi The pHK / JM109 of D makes the bacterial cell concentration reach 5% (W / V), the fermentation condition is 30°C, 250rpm, and the pH is adjusted to neutral with 28% (V / V) ammonia water, and the fermentation reaction time is 36 hours . The determined HA content can reach about 0.75-1 g / L (see figure 2 ).

Embodiment 2

[0041] In a 2500l Erlenmeyer flask, with a total volume of 500ml containing 25 mM K 2 HPO 4 ·3H 2 O, (pH6.5) 2mM g / L MgSO4 7H2O, 40mM / L citric acid, 2mM MnCl2, 400mM glucose, 75 mM nitrogen acetylglucosamine (GlcNAc), 5mM lactose, 30g / L glycerol, 3g / L hydrolyzed casein Amino acid (Casamino acid) system added to the expressed pmHas and kfi The pHK / JM109 of D makes the bacterial cell concentration reach 4% (W / V), the fermentation condition is 33°C, 250rpm, and the pH is adjusted to neutral with 28% (V / V) ammonia water, and the fermentation reaction time is 30 hours .

[0042] It has been determined that the HA content can reach about 0.7-8.5 g / L. Because the synthesis of HA requires oxygen and relatively stable pH, in order to further improve the output of engineering bacteria, the present invention has carried out the cultivation of HA synthesis in a fermenter.

Embodiment 3

[0044] Fermentation tank synthesis of hyaluronic acid in pHK / JM109:

[0045] Pick up 3-5 streaked and overnight cultured pHK / JM109 single colonies from the LB plate of Amp100ug / ml+Km100 ug / ml, and place them in 100ml TB medium (Terrific Broth): 12 g / L tryptone (Tryptone), 24 g / L Yeast extract, 9.4 g / L K 2 HPO 4 and 2.2 g / L KH 2 PO 4 (pH6.8), cultivated overnight. The culture was collected by centrifugation, suspended in 20ml TB, and transferred to a 1L fermenter (Bioreactor, BIOSTAT Bplus Sartoris BBI system German product). The fermenter medium is TB containing ampicillin (Amp) 100ug / ml+kanamycin (Km) 100ug / ml, the total volume is 600ml. The temperature is controlled at 37°C, the rotation speed is 1200rpm, and the mixed gas (oxygen:air 1:10 v / v) is introduced at a flow rate of 60ml / min, and the pH is automatically adjusted to 6.8 with 28% ammonia water and 4 mol / L HCl. Incubate for about 4 hours, when the culture OD 600 When it reaches 5, add IPTG to make the final c...

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Abstract

The invention provides a preparation method of hyaluronic acid. The method comprises the following steps: transforming a recombinant expression vector into the colibacillus to obtain engineering colibacillus by constructing the recombinant expression vector of which hyaluronic acid synzyme gene Has and uridine diphosphate glucose dehydrogenase gene are jointly expressed in the colibacillus; and fermenting the engineering colibacillus to obtain the hyaluronic acid. After the method is used, the production yield of the hyaluronic acid achieves 2.5g / L. compared with the prior art, the yield of the highest HA (hyaluronic acid) obtained by gram positive chained coccus HA synthetic gene spHas expressed in the colibacillus is approximately improved by 10 times.

Description

[0001] technical field [0002] The invention relates to the technical field of genetic engineering of microorganisms, in particular to a method for preparing hyaluronic acid produced by engineered Escherichia coli constructed in the form of a plasmid or in a genome integration manner. Background technique [0003] Escherichia coli K12 strain is widely used in the field of metabolic engineering to produce a variety of high value-added products due to its clear genetic background, numerous efficient genetic transformation systems and the ease of genome manipulation. No natural strain of E. coli has been found to have the ability to produce hyaluronic acid. [0004] Hyaluronic acid (Hyaluronic acid, HA) is a high-viscosity amino polysaccharide that was first isolated from bull eyes. Hyaluronic acid widely exists in human and animal tissues, and its molecular weight is 5×10 4 to 8×10 6 Between Daltons, it is glucuronic acid linked by β-1,4-glycosidic bonds and nitrogen acety...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/04C12R1/19
Inventor 毛自朝杨发祥尚海丽程倩
Owner MICROBIAL FERMENTATION ENG RES CENT CO LTD OF YUNNAN PROVINCE
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