Acid resistance and high succinic acid yield strain and preparation method thereof

A succinic acid and acid-resistant technology, applied in the field of bioengineering, can solve problems such as large demand for petrochemical materials and power consumption, cultivation conditions and yield limitations, and environmental pollution.

Active Publication Date: 2015-04-01
JILIN AGRICULTURAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] The purpose of the embodiments of the present invention is to provide an acid-resistant and high-yield succinic acid strain and its preparation method, aiming to solve the problem that the existing production methods of succinic acid mainly include chemical synthe

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  • Acid resistance and high succinic acid yield strain and preparation method thereof
  • Acid resistance and high succinic acid yield strain and preparation method thereof
  • Acid resistance and high succinic acid yield strain and preparation method thereof

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preparation example Construction

[0038] Such as figure 1 As shown, the method for preparing the acid-resistant and high-yield succinic acid strain of the embodiment of the present invention comprises the following steps:

[0039] S101: strain mutagenesis, ultraviolet (UV) mutagenesis and nitrosoguanidine (NTG) mutagenesis were performed on the Actinobacillus succinogenes strain AS-R2, respectively;

[0040] S102: Screening of mutagenized strains, screen out the most acid-resistant colony after ultraviolet (UV) mutagenesis and nitrosoguanidine (NTG) mutagenesis on an acid-resistant plate, scrape into normal saline, serially dilute, and spread Cultivate on bromocresol green plate at 37°C for 3-5 days in an anaerobic incubator; select colonies with larger color-changing circles, mark them with a marker pen, and pick a colony with larger color-changing circles to culture in the fermentation medium , and measured its succinic acid production; strains with high production continued to be used for genome shuffling;...

Embodiment 1

[0089] Protoplast preparation conditions:

[0090] Pick one loop of each mutant strain in the activation medium to activate the first generation (cultivate for 24 hours), activate it for the second time in the activation medium (cultivate for 24 hours) with 3% inoculum, then inoculate with 3% inoculum to contain 1.0% In the fermentation medium of glycine, cultivate for 6 hours, take 10ml of the culture solution in a sterilized centrifuge tube, centrifuge at a speed of 8000r / min for 15min, pour out the supernatant, and resuspend the bacterial cell pellet in 10ml of osmotic pressure stabilization solution. Centrifuge and wash the bacteria twice, continue to resuspend the bacteria pellet in 10ml osmotic pressure stabilization solution, shake well, add lysozyme solution with a final concentration of 0.08mg / ml, and enzymolyze in a water bath at 37°C for 40min at constant temperature; After the solution is completed, take it out from the water bath and centrifuge at a speed of 6000r...

Embodiment 2

[0092] Protoplast inactivation:

[0093] The protoplast suspension obtained by enzymatic hydrolysis of each mutant strain was mixed evenly in equal amounts, and then divided into two parts, one was inactivated by heat inactivation, and the other was inactivated by ultraviolet inactivation;

[0094] Heat inactivation: put the protoplast suspension used for heat inactivation in a 60°C water bath, and inactivate at a constant temperature in the dark for 30 minutes;

[0095] Ultraviolet inactivation: Divide the protoplast suspension used for ultraviolet inactivation equally, each portion contains 10ml of protoplast suspension, pour it into a sterilized petri dish, put it into a sterilized magnetic stirring bar, and place it under the ultraviolet light for 30cm UV irradiation on a magnetic stirrer for 35 minutes, when the time is up, turn off the magnetic stirrer;

[0096] The above-mentioned inactivation process must be carried out in the dark;

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Abstract

The invention discloses an acid resistance and high succinic acid yield strain and a preparation method thereof. According to the present invention, after the critical growth pH of an Actinobacillus succinogenes strain ATCC55618 is determined as 4.5, ultraviolet mutagenesis is performed on the strain, the strain M1 capable of growing on a solid flat plate with the pH value of 4.3 is screened, and two rounds of ultraviolet-nitrosoguanidine composite mutagenesis are performed on the M1 to screen the high succinic acid yield strain AS-R2, wherein the high succinic acid yield strain AS-R2 can grow and breed under the condition of pH4.1, and the acid yield is only 16.54 g/L under the condition of pH5.0; and the three rounds of genome re-organizations are performed on the AS-R2 by using the genome re-organization technology to obtain the acid resistance and high succinic acid yield strain AS-F3-2, wherein the acid resistance and high succinic acid yield strain AS-F3-2 can grow and breed under the condition of pH3.2, and the succinic acid yield is 25.2 g/L after fermentation for 36 h under the condition of pH5.0.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to an acid-resistant and high-yield succinic acid strain and a preparation method thereof. Background technique [0002] Succinic acid (Succinic acid) is a kind of dicarboxylic acid, which is a common natural organic acid, also known as succinic acid, and is one of the products of the tricarboxylic acid cycle, which widely exists in human body, animals, plants and microorganisms. As a C4 platform compound, succinic acid has a wide range of uses, and is currently widely used in food, agriculture, medicine, chemistry and other fields. [0003] The existing production methods of succinic acid mainly include chemical synthesis methods, but chemical synthesis methods have a large demand for petrochemical materials and electricity consumption, petrochemical materials are non-renewable, resources are limited and will cause serious environmental pollution; in recent years...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/03C12N15/01C12N13/00C12R1/01
CPCC12N13/00C12N15/01C12N15/03C12N1/205C12R2001/01
Inventor 王玉华夏飞飞姚曼于寒松刘俊梅朴春红代伟长刘彦隆
Owner JILIN AGRICULTURAL UNIV
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