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High-adenosine-yield Bacillus subtilis

A Bacillus subtilis, adenosine technology, applied in the field of microorganisms, can solve the problems of low adenosine yield, reverse mutation, and unstable passage, and achieve the effects of genetic stability, high mutation rate, and wide mutation spectrum.

Inactive Publication Date: 2012-06-27
NANJING TECH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The technical problem to be solved by the present invention is to provide a high-yield adenosine-producing Bacillus subtilis to overcome the problems of low adenosine production, unstable passage, and prone to reverse mutation

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] The low-yield adenosine-producing Bacillus subtilis (Bacillus subtilis) 301 preserved in the laboratory was used as the starting strain, and the + Ion beam mutagenesis (the vacuum degree in the target chamber of the low-energy ion implanter is 2×10 -4 , the injection energy is 20kev, and the dose is 5×10 13 ions cm -2 .s -1 ) after the bacterial suspension was diluted 100 times with sterile water, took 0.1ml and spread it on a complete medium plate and cultured it at 32°C for 3 days, from which a single colony with a large and full circle was selected, and the corresponding points were in the basic medium, bird On the purine-supplemented medium plate, select colonies at corresponding positions that do not grow on the basic medium but grow on the supplemented medium containing guanine. The detected auxotrophs were then slanted, and transferred to a 30mL liquid seed medium (glucose 20g / L, peptone 10g / L, yeast extract 12g / L, corn steep liquor 14g / L, NaCl 3g / L , guanosi...

Embodiment 2

[0059] Transfer a loop of bacterial strain A409 from the beef extract slant to 30mL liquid seed medium (glucose 25g / L, peptone 10g / L, yeast extract 12g / L, corn steep liquor 15g / L, NaCl 3g / L, guanosine 0.025 g / L) in a 500 mL shaker flask, cultured on a reciprocating shaker at 100 rpm at 32°C for 20 hours. Transfer 3mL to 30mL liquid fermentation medium (glucose 90g / L, K 2 HPO 4 4g / L, (NH 4 ) 2 SO 4 15g / L, MgSO 4 ·7H 2 O 5g / L, MnSO 4 ·H 2 O 0.02g / L, KCl 3g / L, urea 5g / L, yeast extract 16g / L, guanine 0.1g / L, bean cake hydrolyzate 15g / L, CaCO 3 15g / L) in a 500mL shake flask, fermented on a shaker at 270rpm at 33°C for 66 hours. The yield of adenosine reached 24.9g / L.

Embodiment 3

[0061] The strain A409 was transferred from the beef extract slant to a 500mL shake flask equipped with 30mL liquid seed medium (same as Example 1), and cultivated on a reciprocating shaker at 100rpm at 32°C for 20 hours. Transfer 2mL to 20mL liquid fermentation medium (glucose 95g / L, K 2 HPO 4 4g / L, (NH 4 ) 2 SO 4 15g / L, MgSO 4 ·7H 2 O 5g / L, MnSO 4 ·H 2 O 0.02g / L, KCl 2g / L, urea 5g / L, yeast extract 18g / L, guanine 0.1g / L, bean cake hydrolyzate 15g / L, CaCO 3 15g / L) in a 500mL shake flask, fermented and cultured on a shaker at 270rpm at 33°C for 68 hours. The yield of adenosine reached 25.3g / L.

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Abstract

The invention discloses high-adenosine-yield Bacillus subtilis. The class name of the high-adenosine-yield Bacillus subtilis is Bacillus subtilis A409; the genetic markers of the high-adenosine-yield Bacillus subtilis include: guanine auxotroph, xanthylate ammoniation enzyme deletion and 8-nitroxanthine resistance. The collection number of the Bacillus subtilis is CGMCC No.4484, and the date of collection is December 17th, 2010. The adenosine yield of the Bacillus subtilis strain is stabilized at 25g / L, which is over 5 times higher than that of a starting strain. The adenosine producing performance of the strain is stable after being passes for more than 10 generations. And the strain can keep a high level on a 5-liter fermentation tank and has a large-scale industrialization potential.

Description

technical field [0001] The invention belongs to the technical field of microorganisms and relates to a bacillus subtilis with high adenosine production. Background technique [0002] Adenosine (Adenosine), also known as adenosine, is an endogenous nucleoside with important physiological functions in human cells. It can directly enter the myocardium and be phosphorylated to generate adenosine to participate in the energy metabolism of the myocardium. It has antiepileptic and strong Effective expansion of the role of coronary arteries, clinically used in the treatment of coronary vascular disorders, myocardial infarction, arteriosclerosis, stroke sequelae, essential hypertension, angina pectoris, etc. At the same time, adenosine is also an important pharmaceutical intermediate, which can be used to synthesize 8-chloroadenosine, purine toxin, homocitrulamino adenosine, lysylamino adenosine, s-methylthio adenosine, vidarabine adenosine, and cyclic adenosine monophosphate , aden...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12P19/40C12R1/125
Inventor 应汉杰曹家明柏建新陈晓春任华景陈勇熊健吴菁岚谢婧婧
Owner NANJING TECH UNIV
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