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A strain of anti-phage aspartase variant production bacteria and its selection method and application

An aspartase and anti-bacteriophage technology, applied in the field of bioengineering, can solve the problems of prolonged fermentation period, loss, and reduced fermentation enzyme production, so as to solve the problems of susceptibility to bacteriophage infection, good genetic stability, and improve economic benefits Effect

Active Publication Date: 2022-05-24
QINHUANGDAO HUAHENG BIOENG CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, in actual production, bacteriophage contamination is easy to occur in the process of fermentation and enzyme production, which may prolong the fermentation cycle and reduce the fermentation enzyme production; To great waste, the loss is immeasurable

Method used

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  • A strain of anti-phage aspartase variant production bacteria and its selection method and application
  • A strain of anti-phage aspartase variant production bacteria and its selection method and application
  • A strain of anti-phage aspartase variant production bacteria and its selection method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] The source of the strain: the aspartase variant producing strain pL181, which can produce enzymes and catalyze the ammoniation of acrylic acid to produce β-alanine.

[0030] The aspartase variant producing strain pL181 corresponds to the aspartase variant AHB032, which is described in the following patent: Wu Bian et al. Aspartase variants and their preparation methods and applications [P].ZL201710659654. 9. The aspartase variant is modified on the basis of the wild-type aspartase to improve its catalytic activity for promoting the hydrogenation of acrylic acid to generate β-alanine.

[0031] Separation and purification of bacteriophage by double-layer plate method: take 10ml of aspartase variant production bacteria pL181 fermentation broth contaminated by phage in the workshop, centrifuge at 8000rpm for 15min, take the supernatant and filter it through a 0.22μm sterile microporous membrane to sterilize. Take 100μl of the filtrate and mix it with 300μl of pL181 bacteria...

Embodiment 2

[0034] Starting strain: aspartase variant producer pL181.

[0035] Phage: Escherichia coli phage CGMCC NO.17999, isolated through Example 1.

[0036] Medium: LB medium (yeast powder 5.0g / L, peptone 10.0g / L, sodium chloride 10.0g / L, pH 7.0-7.2, solid medium plus agar 2%).

[0037] Continuous spontaneous breeding method: Pick a single colony of pL181 from a fresh plate and insert it into LB liquid medium (3ml / 10ml test tube), culture overnight at 37°C and 200rpm for 16-18h, and insert 0.1ml of titer to 10 10pfu / ml of phage lysate, and detect the initial OD value. After continuing to culture with shaking for 6-24 hours, check the OD value again, select a diluted LB plate with a smaller decrease in OD value, and place it in a 37°C incubator for overnight culture. Pick a single colony from the plate and repeat the above operation for 10 consecutive times.

Embodiment 3

[0039] Starting strain: the strain obtained by screening in Example 2.

[0040] Low-energy ion implantation mutagenesis method: Take out the fresh plate of the bacterial strain, add 15ml of sterile water, scrape and wash the bacteria and transfer them into a 250ml conical flask with glass beads, add sterile water to the conical flask to make a bacterial suspension, Adjust the bacterial concentration at 10 8 pieces / ml. Take 200 μl of the above-mentioned bacterial suspension and evenly spread it on a sterile blank LB culture dish, air-dry it in a sterile state, inject the energy at 20keV, and inject the dose at 10×10 14 , 15×10 14 , 20×10 14 ions / cm 2 , the target chamber vacuum is 10 -3 pa, injected in a 5s pulse, with an interval of 15s for nitrogen ion implantation, after the ion injection was completed, the petri dish was eluted with 1ml sterile physiological saline under aseptic conditions, and 10 4 、10 5 、10 6 The diluted samples were spread on the LB plate medium ...

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Abstract

The invention discloses an anti-bacteriophage aspartase variant production bacterium, a breeding method and an application thereof. The present invention carries out continuous spontaneous mutation breeding on the aspartase variant producing bacteria pL181 which is easily polluted by phages, and then utilizes the low-energy ion implantation mutagenesis method to efficiently breed a strain of aspartase with phage resistance Acidase variant producing bacteria Escherichia coli CGMCC NO.18005. The strain not only has the resistance to bacteriophage (CGMCC NO.17999), but also has 31% higher enzyme activity than the original strain pL181. While improving the enzyme-producing ability of the producing bacteria, the present invention fundamentally solves the difficult problem that β-alanine is easily infected by phages in the production, and improves the economic benefits of β-alanine production enterprises.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a bacteriophage-resistant aspartase variant-producing bacterium, a breeding method and its application. Background technique [0002] β-alanine, also known as 3-alanine, is the only β-type amino acid that exists in nature. The physiological function of β-alanine is mainly as an intermediate product of metabolism, which widely exists in the metabolic process of animals, plants and microorganisms, and plays an essential role in the growth and development of organisms. It is mainly used in the synthesis of pantothenic acid and calcium pantothenate, carnosine, sodium pamidronate, balsalazide, etc., and is widely used in medicine, feed, food and other fields. In recent years, foreign reports on β-alanine have gradually increased, most of which are studies on β-alanine derivatives. The role of derivatives of β-alanine in medical treatment, environment and daily che...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N7/00C12N13/00C12P13/06C12Q1/18C12R1/19
CPCC12N13/00C12Q1/18C12P13/06C12N7/00C12N2795/10121C12R2001/19C12N1/205
Inventor 李慧荣刘洋赵晶晶李美华高雄伟陈金花夏丹丹张聪
Owner QINHUANGDAO HUAHENG BIOENG CO LTD
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