Application of Bacillus subtilis in high-yield adenosine and acetoin co-production

Abstract

The invention discloses the application of Bacillus subtilis in high-production of adenosine and co-production of acetoin. The Bacillus subtilis (Bacillus subtilis) is CGMCC No. 4484. The strains screened in the present invention can utilize a variety of carbon sources and nitrogen sources to ferment to produce adenosine and co-produce acetoin. By adding sodium citrate to the fermentation medium, the production of adenosine and acetoin can be significantly increased, and the operation is easy. It is simple, has extensive culture conditions, can maintain a high level in a 5L fermentation tank, and has the potential for large-scale industrialization.

Description

technical field

[0001] The invention belongs to the technical field of microorganisms and fermentation engineering, and specifically relates to the application of Bacillus subtilis in high-yield adenosine and co-production of acetoin. Background technique

[0002] Adenosine, also known as adenosine nucleoside, is composed of adenine and ribose linked by glycosidic bonds. It is an endogenous nucleoside with important physiological functions in human cells. Adenosine is well known for its vasodilator effect, and has a wide range of cardiac effects and rapid and significant coronary artery dilation. A large number of studies have shown that adenosine can also trigger or mediate ischemic preconditioning, reduce reperfusion injury, etc. protective effect. In the United States, adenosine is an FDA-approved first-line drug to convert paroxysmal supraventricular tachycardia (PSVT) and one of two FDA-approved drugs for cardiac stress testing, which has become an emergency management...

Examples

Embodiment 1

[0034] Slant medium: glucose 15g / L, peptone 10g / L, yeast extract 5g / L, corn steep liquor 10ml / L, MgSO 4 ·7H 2 O 0.1g / L, NaCl 3g / L, guanosine 0.03g / L, agar 15g / L, the rest is water, pH7.0;

[0035] Seed medium: glucose 15g / L, peptone 10g / L, yeast extract 10g / L, corn steep liquor 10ml / L, MgSO 4 ·7H 2 O 0.1g / L, NaCl 4g / L, guanosine 0.03g / L, the rest is water, pH7.0;

[0036] Fermentation medium: glucose 80g / L, yeast extract 15g / L, (NH 4 ) 2 SO 4 16g / L, urea 3g / L, KH 2 PO 4 4g / L, KCl 4g / L, MgSO 4 ·7H 2 O 0.5g / L, MnSO 4 ·H 2 O 0.01g / L, soybean cake hydrolyzate 20ml / L, CaCO 3 30g / L, the rest is water, pH7.0;

[0037] The strain A409 was transferred from the slant to a 500mL shake flask containing 30mL liquid seed medium, and cultured on a reciprocating shaker at 200rpm at 32°C for 20 hours. Transfer 3mL to a 500mL shake flask containing 30mL liquid fermentation medium, and ferment and culture on a shaker at 250rpm at 33°C for 66 hours. The yield of adenosine reached...

Embodiment 2

[0039] Fermentation medium: glucose 100g / L, yeast extract 15g / L, (NH 4 ) 2 SO 4 16g / L, urea 3g / L, KH 2 PO 4 4g / L, KCl 4g / L, MgSO 4 ·7H 2 O 0.5g / L, MnSO 4 ·H 2 O 0.01g / L, soybean cake hydrolyzate 20ml / L, CaCO 3 30g / L, the rest is water, pH7.0;

[0040] The strain A409 was transferred from the slant to a 500mL shake flask filled with 30mL liquid seed medium (same as Example 1), and cultured on a reciprocating shaker at 200rpm at 32°C for 20 hours. Transfer 2 mL to a 500 mL shake flask containing 20 mL of liquid fermentation medium, and ferment and culture on a shaker at 250 rpm at 33°C for 66 hours. The yield of adenosine reached 21.6g / L, and that of acetoin reached 13.6g / L.

Embodiment 3

[0042] Synthesize adenosine with Bacillus subtilis A409 in a 5L fermenter, prepare seed culture medium, formula and culture method are the same as embodiment 1, prepare fermentation culture medium, formula and culture method are the same as embodiment 2, the seed liquid is pressed the volume of fermented liquid 10% (v / v) inoculum was added to the cooled fermentation medium, the volume of the fermentation medium was 3L, the ventilation volume was controlled at 2vvm, the stirring speed was 500rmp, and the pH was adjusted to 6.5 with 20% ammonia water, at 35°C Fermented for 66h, the content of adenosine in the fermented liquid obtained at last is 23g / L, and the content of acetoin is 15g / L;

technical field

[0001] The invention belongs to the technical field of microorganisms and fermentation engineering, and specifically relates to the application of Bacillus subtilis in high-yield adenosine and co-production of acetoin. Background technique

[0002] Adenosine, also known as adenosine nucleoside, is composed of adenine and ribose linked by glycosidic bonds. It is an endogenous nucleoside with important physiological functions in human cells. Adenosine is well known for its vasodilator effect, and has a wide range of cardiac effects and rapid and significant coronary artery dilation. A large number of studies have shown that adenosine can also trigger or mediate ischemic preconditioning, reduce reperfusion injury, etc. protective effect. In the United States, adenosine is an FDA-approved first-line drug to convert paroxysmal supraventricular tachycardia (PSVT) and one of two FDA-approved drugs for cardiac stress testing, which has become an emergency management...

Examples

Embodiment 1

[0034] Slant medium: glucose 15g / L, peptone 10g / L, yeast extract 5g / L, corn steep liquor 10ml / L, MgSO 4 ·7H 2 O 0.1g / L, NaCl 3g / L, guanosine 0.03g / L, agar 15g / L, the rest is water, pH7.0;

[0035] Seed medium: glucose 15g / L, peptone 10g / L, yeast extract 10g / L, corn steep liquor 10ml / L, MgSO 4 ·7H 2 O 0.1g / L, NaCl 4g / L, guanosine 0.03g / L, the rest is water, pH7.0;

[0036] Fermentation medium: glucose 80g / L, yeast extract 15g / L, (NH 4 ) 2 SO 4 16g / L, urea 3g / L, KH 2 PO 4 4g / L, KCl 4g / L, MgSO 4 ·7H 2 O 0.5g / L, MnSO 4 ·H 2 O 0.01g / L, soybean cake hydrolyzate 20ml / L, CaCO 3 30g / L, the rest is water, pH7.0;

[0037] The strain A409 was transferred from the slant to a 500mL shake flask containing 30mL liquid seed medium, and cultured on a reciprocating shaker at 200rpm at 32°C for 20 hours. Transfer 3mL to a 500mL shake flask containing 30mL liquid fermentation medium, and ferment and culture on a shaker at 250rpm at 33°C for 66 hours. The yield of adenosine reached...

Embodiment 2

[0039] Fermentation medium: glucose 100g / L, yeast extract 15g / L, (NH 4 ) 2 SO 4 16g / L, urea 3g / L, KH 2 PO 4 4g / L, KCl 4g / L, MgSO 4 ·7H 2 O 0.5g / L, MnSO 4 ·H 2 O 0.01g / L, soybean cake hydrolyzate 20ml / L, CaCO 3 30g / L, the rest is water, pH7.0;

[0040] The strain A409 was transferred from the slant to a 500mL shake flask filled with 30mL liquid seed medium (same as Example 1), and cultured on a reciprocating shaker at 200rpm at 32°C for 20 hours. Transfer 2 mL to a 500 mL shake flask containing 20 mL of liquid fermentation medium, and ferment and culture on a shaker at 250 rpm at 33°C for 66 hours. The yield of adenosine reached 21.6g / L, and that of acetoin reached 13.6g / L.

Embodiment 3

[0042] Synthesize adenosine with Bacillus subtilis A409 in a 5L fermenter, prepare seed culture medium, formula and culture method are the same as embodiment 1, prepare fermentation culture medium, formula and culture method are the same as embodiment 2, the seed liquid is pressed the volume of fermented liquid 10% (v / v) inoculum was added to the cooled fermentation medium, the volume of the fermentation medium was 3L, the ventilation volume was controlled at 2vvm, the stirring speed was 500rmp, and the pH was adjusted to 6.5 with 20% ammonia water, at 35°C Fermented for 66h, the content of adenosine in the fermented liquid obtained at last is 23g / L, and the content of acetoin is 15g / L;

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