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Method for producing DPE through high-density fermentation of recombinant bacillus subtilis

A Bacillus subtilis, high-density fermentation technology, applied in the fields of bioengineering and fermentation engineering, can solve the problems of low bacterial OD, long fermentation cycle, and low protein expression, and achieve high cell density, simple operation, and high enzyme activity high price effect

Active Publication Date: 2022-01-21
山东福洋生物科技股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Recombinant Bacillus subtilis producing D-psicose 3-epimerase, strain OD 600 =10-15, the enzyme activity is 15-20U / mL; and the recombinant Bacillus subtilis has a long fermentation cycle, low bacterial OD, and low protein expression

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] A method for producing DPE by recombinant Bacillus subtilis high-density fermentation, the specific steps are as follows:

[0025] (1) 150mL shake flask seed preparation

[0026] Prepare primary seed medium (primary seed medium formula: peptone 1.5g, yeast powder 0.75g, sodium chloride 1.5g), 50μg / ml kanamycin (final concentration), pH7.0, 121°C, incinerated Bacteria 30min.

[0027] Inoculate the recombinant Bacillus subtilis with 1% inoculum amount, culture at 35° C., 220 rpm, on a shaking table for 5 hours, and obtain a first-grade seed liquid.

[0028] (2) Measure the OD of the primary seed fermentation liquid with an absorbance photometer 600 When =2.5, transfer to the 5L secondary seed culture medium that is equipped with 1L secondary seed medium (secondary seed medium formula: yeast powder 5g / L, peptone 10g / L, sodium chloride 10g / L) by 1% inoculum size In the seed tank; in the secondary seed tank, the culture temperature is controlled at 35°C, DO 40-50%, the ro...

Embodiment 2

[0038] A method for producing DPE by recombinant Bacillus subtilis high-density fermentation, the specific steps are as follows:

[0039] (1) 150mL shake flask seed preparation

[0040] Prepare primary seed medium (primary seed medium formula: peptone 1.5g, yeast powder 0.75g, sodium chloride 1.5g), 50μg / ml kanamycin (final concentration), pH7.0, 121°C, incinerated Bacteria 30min.

[0041] Inoculate the recombinant Bacillus subtilis with 1% inoculum amount, culture at 35° C., 220 rpm, on a shaking table for 5 hours, and obtain a first-grade seed liquid.

[0042] (2) Measure the OD of the primary seed fermentation liquid with an absorbance photometer 600 When =2.67, transfer to the 5L secondary seed medium that 1L secondary seed medium (secondary seed medium formula: yeast powder 5g / L, peptone 10g / L, sodium chloride 10g / L) is housed by 1% inoculum size In the seed tank; in the secondary seed tank, the culture temperature is controlled at 35°C, DO 40-50%, the rotation speed i...

Embodiment 3

[0052] A method for producing DPE by recombinant Bacillus subtilis high-density fermentation, the specific steps are as follows:

[0053] (1) 150mL shake flask seed preparation

[0054] Prepare primary seed medium (primary seed medium formula: peptone 1.5g, yeast powder 0.75g, sodium chloride 1.5g), 50μg / ml kanamycin (final concentration), pH7.0, 121°C, incinerated Bacteria 30min.

[0055] Inoculate the recombinant Bacillus subtilis with 1% inoculum amount, culture at 35° C., 220 rpm, on a shaking table for 5 hours, and obtain a first-grade seed liquid.

[0056] (2) Measure the OD of the primary seed fermentation liquid with an absorbance photometer 600 When =3, transfer to the 5L secondary seed culture medium that is equipped with 1L secondary seed medium (secondary seed medium formula: yeast powder 5g / L, peptone 10g / L, sodium chloride 10g / L) by 1% inoculum size. In the seed tank; in the secondary seed tank, the culture temperature is controlled at 35°C, DO 40-50%, the rot...

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PUM

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Abstract

The invention discloses a method for producing DPE through high-density fermentation of recombinant bacillus subtilis, and belongs to the technical field of bioengineering and fermentation engineering. According to the method for producing DPE through high-density fermentation of the recombinant bacillus subtilis, the recombinant bacillus subtilis is used as an original strain, an OD and enzyme activity feedback regulation fermentation mechanism is established, and after seed culture, the strain is transferred into a fermentation tank for high-density fermentation of the bacillus subtilis to produce D-psicose 3-epimerase. In the fermentation period, the air volume, the rotating speed and the material supplementing speed in the fermentation process are adjusted and controlled through OD change and enzyme activity increment feedback regulation, the preparation method of the D-psicose 3-epimerase which is simple to operate, high in thallus density and high in enzyme activity titer is obtained, the OD600 of fermentation finishing bacteria is equal to 100-110, and the enzyme activity is 200-210 U / mL.

Description

technical field [0001] The invention relates to the technical fields of bioengineering and fermentation engineering, and more specifically relates to a method for producing DPE by high-density fermentation of recombinant bacillus subtilis. Background technique [0002] D-psicose 3-epimerase (D-psicose3-epimerase, DPE for short) is currently the most effective enzyme for converting D-fructose into D-psicose by biological methods. [0003] In the prior art, the production of D-psicose 3-epimerase mainly comes from the exogenous expression of DPE gene in Escherichia coli, and the research on D-psicose 3-epimerase family enzymes Most of them use Escherichia coli as the expression host; Bacillus subtilis, as a traditional industrial production strain, has high protein secretion ability and is a good system for studying exogenous protein expression and secretion, and it is also a food-grade microorganism . Recombinant Bacillus subtilis producing D-psicose 3-epimerase, strain OD ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/90C12R1/125
CPCC12N9/90C12Y501/03Y02P60/87
Inventor 不公告发明人
Owner 山东福洋生物科技股份有限公司