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Brevibacterium and method for separating and purifying fumarase from Brevibacterium fermentation broth

A fumaric acid enzyme, Brevibacterium technology, applied in the directions of microorganism-based methods, biochemical equipment and methods, enzymes, etc., can solve the problems of low recovery rate, low purity of fumaric acid enzyme, etc., achieve high recovery rate, easy to realize industrialization, cost low effect

Inactive Publication Date: 2016-06-08
NANJING UNIV OF TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The purpose of the present invention is to provide a kind of Brevibacterium, and another purpose of the present invention is to solve the problem of low purity and low recovery rate of extracting fumarase in the prior art, and to provide a simple, low-cost, low-purity fumarase , a method for isolating and purifying fumarase from Brevibacterium fermentation broth, which is easy for large-scale production

Method used

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  • Brevibacterium and method for separating and purifying fumarase from Brevibacterium fermentation broth

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] (1) Brevibacterium was inoculated on the broth agar medium and cultured, picked a ring of single colony, put it into the Erlenmeyer flask containing the seed culture solution, controlled the volume of 15%, shaken at 30°C and 180r / min Cultivate in the bed for 24 hours to obtain seed liquid; inoculate into the fermentation medium according to the volume inoculum of 3%, the volume of the fermenter is 20%, the temperature is controlled at 25°C, the stirring rate is 220rpm, the pH is controlled at 7.0, and the fermentation is cultivated for 30h Get the fermentation broth. Among them: the seed medium components are: glucose 20g / L, yeast extract powder 1.0g / L, corn steep liquor 25g / L, MgSO 4 ·7H 2 O0.5g / L, control pH7. The medium used for fermentation includes the following components: glucose 15g / L, yeast extract powder 4g / L, corn steep liquor 35g / L, MgSO 4 ·7H 2 O0.7g / L, sodium chloride 2g / L, KH 2 PO 4 2g / L.

[0037] (2) The fermentation broth was centrifuged at 8000 ...

Embodiment 2

[0044](1) Brevibacteria were inoculated on broth agar medium and cultivated, picked 1 ring of single colony, inserted into the Erlenmeyer flask containing the seed culture solution, controlled the volume of 20% solution, and shaken at 35°C and 200r / min Cultivate in the bed for 28 hours to obtain seed liquid; inoculate into the fermentation medium according to the volume inoculum of 8%, the liquid volume of the fermenter is 30%, the control temperature is 30°C, the stirring rate is 300rpm, the pH is controlled at 7.0, and the fermentation is cultivated for 26 hours Get the fermentation broth. Among them: the seed medium components are: glucose 15g / L, yeast extract powder 5.0g / L, corn steep liquor 30g / L, MgSO 4 ·7H 2 O0.2g / L, control pH7.5. The medium used for fermentation includes the following components: glucose 17g / L, yeast extract powder 3g / L, corn steep liquor 21g / L, MgSO4 7H2O0.5g / L, sodium chloride 5g / L, KH 2 PO 4 2g / L.

[0045] (2) The fermentation broth was centri...

Embodiment 3

[0052] (1) Brevibacterium was inoculated on the broth agar medium and cultured, picked 1 ring of single colony, put it into the Erlenmeyer flask containing the seed culture solution, controlled the filling volume of 25%, 37°C, 220r / min Cultivate in a shaker for 25 hours to obtain a seed solution; inoculate into the fermentation medium at a volume inoculum of 10%, the volume of the fermenter is 40%, the temperature is controlled at 37°C, the stirring rate is 400rpm, and the pH is controlled at 7.5. 32h to get the fermentation broth. Among them: the seed medium components are: glucose 17g / L, yeast extract powder 4.0g / L, corn steep liquor 27g / L, MgSO 4 ·7H 2 O0.6g / L, control pH7.5. Fermentation medium components: glucose 20g / L, yeast extract powder 1.0g / L, corn steep liquor 30g / L, MgSO 4 ·7H 2 O0.6g / L, sodium chloride 5g / L, KH 2 PO 4 4g / L. (2) The fermentation broth was centrifuged at 8000 r / min for 25 minutes, filtered to obtain mycelia, and the cells of the bacteria were...

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Abstract

The invention relates to Brevibacterium and a method for separating and purifying fumarase from Brevibacterium fermentation broth. Latin name of this bacterium is Brevibacterium sp., reference is made to a microorganism: NJWGY2133, and Brevibacterium sp. is collected on 26th May, 2008 under CGMCC No. 2520. The method for separating and purifying fumarase comprises the specific steps: preparing crude enzyme liquid through Brevibacterium sp. fermentation broth culturing and ultrasonic crushing, and preparing a polyethylene glycol-inorganic salt two aqueous phase extraction system; carrying out two aqueous phase extraction; preparing fumarase through dialytic desalting and freeze drying. The process of the invention is simple, production cycle is short, operation conditions are mild, cost is low, high-purity enzymes may be acquired from microorganisms, and enzyme activity yield is higher than 90%.

Description

Technical field: [0001] The invention relates to a Brevibacterium and a method for separating and purifying fumarase from Brevibacterium fermentation liquid. Background technique: [0002] Fumarase, also known as malate lyase, fumarate hydratase or fumarase, oxidoreductase (E.C.4.2.1.2), is a key enzyme in the tricarboxylic acid cycle (TCA cycle). Catalyzing fumaric acid to generate L-malic acid with very high yield and optical purity by adding water, and the reaction mechanism is relatively simple, which is a very commercially valuable enzyme. Enzymes in industrial production are generally in the form of crude enzymes. However, enzymes with a certain purity can not only improve catalytic efficiency and reduce energy consumption, but also reduce by-products caused by the influence of heterozymes, affecting product purity. [0003] Common fumarase protein purification methods include salting out precipitation, hydrophobic chromatography, ion exchange chromatography and gel f...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N9/88C12R1/13
CPCC12N9/88C12Y402/01002C12R2001/13C12N1/205
Inventor 胡永红曹翠翠杨文革曹洋吴刚王春晓章泳
Owner NANJING UNIV OF TECH
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