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Strain for producing 3-hydroxyl propionic acid at high yield and application thereof

A technology for high-yield and bacterial strains of hydroxypropionic acid, applied in fungi, microorganism-based methods, microorganisms, etc., can solve the problems of insecurity, high production cost, and difficult separation and purification of products, and achieve the effects of stable genetic traits and high yield.

Inactive Publication Date: 2012-03-21
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it is very difficult to introduce functional groups at the carbon end, and its products are not easy to separate and purify, the production cost is high, and there are unsafe factors in the production process. The production of 3-HP by microbial fermentation can effectively avoid these unfavorable factors

Method used

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  • Strain for producing 3-hydroxyl propionic acid at high yield and application thereof
  • Strain for producing 3-hydroxyl propionic acid at high yield and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0011] The acquisition of embodiment 1 new bacterial strain CGMCC No.5344

[0012] (a) domesticate the wild fungus Candida sp.CCTCC NO: M 93018 with a medium containing propionic acid, and select the starting strain a;

[0013] (b) subjecting the starting strain a to ultraviolet-nitrosoguanidine-cobalt 60 compound mutagenesis to obtain mixed seed solution b;

[0014] (c) B is coated and cultivated completely to obtain a single bacterial colony screening medium;

[0015] (d) Cultivate and screen a single colony, and verify by fermentation detection, to obtain the mutagenized high-yielding strain CGMCC No.5344.

[0016] The acclimation concentration of propionic acid in step (a) is 0.5%-3%.

[0017] In step (b), irradiate with a 15W ultraviolet lamp for 70s, add nitrosoguanidine solution to make the final concentration 25mg / mL, and cobalt 60 dose is 1000Gy for the best mutagenesis effect.

[0018] In step (c), the complete medium formula is (g / L): glucose 20, peptone 20, yeas...

Embodiment 2

[0020] Example 2 Fermentation of mutant strain CGMCC No.5344 to produce 3-HP

[0021] (1) Seed liquid culture: inoculate the seed medium with the activated colonies, and cultivate at 220 r / min at 30° C. for 20 h. Seed medium (g / L): glucose / glycerol 20, yeast extract 10, (NH 4 ) 2 SO 4 15, KH 2 PO 4 9,K 2 HPO 4 3. MgSO 4 ·7H 2 O 1.5, FeSO 4 ·7H 2 O 0.03, pH 6.8±0.2.

[0022] (2) Fermentation culture

[0023] Fermentation medium (g / L): yeast extract 10, (NH 4 ) 2 SO 4 15, KH 2 PO 4 9,K 2 HPO 4 3. MgSO 4 ·7H 2 O 1.5, FeSO 4 ·7H 2 O 0.03, CaCl 2 0.04, propionic acid 15, TES 5mL, with KOH / H 2 SO 4 Adjust the pH to 6.0±0.2, sterilize at 115°C for 15 minutes, and mix with 50g / L glucose / glycerol.

[0024] Inoculate with 4% inoculum, culture at 220r / min, 30°C, ferment for 96h, centrifuge the fermentation broth, dilute, and filter through a microporous membrane, then measure the 3-hydroxypropionic acid production of wild bacteria and mutant strain CGMCC No.5344...

Embodiment 3

[0025] The influence of embodiment 3 quiescent cells on the production of 3-hydroxypropionic acid

[0026] In the absence of exogenous carbon sources, 3-HP will be consumed by the bacteria to supply energy to maintain the normal metabolism of the bacteria. In comparison, the degradation rate of 3-hydroxypropionic acid in wild bacteria Candida sp. is higher than that of CGMCC No.5344 (as shown in Table 1).

[0027] Preparation and cultivation of resting cells: After culturing in the fermentation medium for 72 hours, the cells were collected by centrifugation, and the cells were washed twice with 0.2M phosphate buffer. Transfer to phosphate buffer solution containing 1% 3-hydroxypropionic acid, and culture on a rotary shaker at 220r / min at 30°C for 48h. Samples were taken every 12 hours to detect the bacterial concentration and the content of 3-HP.

[0028] Table 1 Degradation of 3-HP by resting cells of Candida sp. and CGMCC No.5344

[0029]

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Abstract

The invention discloses a strain for producing 3-hydroxyl propionic acid at high yield, which is a compound mutant strain of Candida sp., is stable in hereditary features and yield, is applicable to industrial popularization, and is collected in the China general microbiological culture collection center with the collection number of CGMCC No.5344. The invention further discloses a method for converting propionic acid into 3-hydroxyl propionic acid. The method comprises the following steps of when the growth of the strain reaches a stable stage, supplementing glucose in a limited way till theconcentration of the strain is 15-25g / L when the growth of the strain reaches a stable stage; controlling the concentration OD600 of the strain is 18-20; adding propionic acid, potassium hydroxide, strong sodium oxide and ammonia water (according to a ratio of 8: 1: 1: 1) in a flowing way, so as to keep the concentration of the propionic acid at 10-20g / L; and fermenting and culturing for 96 hoursto obtain a fermentation liquid of 3-hydroxyl propionic acid. After optimization, the yield of 3-hydroxyl propionic acid on the culture medium is increased to 30g / L.

Description

Technical field: [0001] The invention relates to a 3-hydroxypropionic acid high-yield bacterial strain and its application, in particular to a yeast Candida sp. obtained through physicochemical compound mutagenesis and its application to transform propionic acid into 3-hydroxypropionic acid. Background technique: [0002] 3-Hydroxypropionic acid (3-Hydroxypropionic acid; also known as β-Hydroxypropionic acid; abbreviated as 3-HP; molecular formula C 3 h 6 o 3 ; CAS No. 503-66-2; pK4.51; molecular weight 90; density 1.08) is a three-carbon achiral organic acid, which is isomers with lactic acid. 3-HP has two functional groups, hydroxyl and carboxyl, and is the precursor of many optically active substances. It can be dehydrated to generate acrylic acid, oxidized to generate malonic acid, reduced to generate 1,3-propanediol, and polymerized to generate polymer materials. It is an important chemical platform product. In 2004, the U.S. Department of Energy listed it as one of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/16C12P7/42C12R1/72
Inventor 诸葛斌范俊英方慧英诸葛健
Owner JIANGNAN UNIV
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