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Method for improving nitrile hydratase activity of bacillus subtilis

A technology of Bacillus subtilis and nitrile hydratase, which is applied in the field of improving the activity of Bacillus subtilis nitrile hydratase, can solve the problem of low activity of Bacillus subtilis nitrile hydratase, and achieve the effect of improving enzyme activity

Inactive Publication Date: 2017-03-22
ANHUI RUIBANG BIOLOGICAL SCI & TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The purpose of the present invention is to provide a kind of method that can improve the activity of Bacillus subtilis nitrile hydratase for the low activity of existing Bacillus subtilis nitrile hydratase, make the fermented liquid per unit volume have higher catalytic ability, strengthen nitrile hydratase activity, effectively reducing fermentation costs and increasing the yield of nicotinamide

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] A method for improving the activity of Bacillus subtilis nitrile hydratase, comprising the steps of: (1) slant culture: the bacteria were connected to the sterilized slant solid medium at 25°C and cultivated for 6 days, and the amount of liquid added was 50ml In a 500ml Erlenmeyer shaker flask with notches, culture on a shaker at 25°C for 72h, with a shaker speed of 300rpm, and use it as a first-grade seed for later use; (2) Seed cultivation: insert 10% of the cultivated first-grade seed into 50ml In the second-stage seed culture shake flask, 25 ° C shaker culture 72h, shaker speed 300rpm; (3) Fermentation culture: add 2L fermentation medium (quality is 2.05 tons) in 5L fermentation tank, 121 °C real tank disinfection 30min , transfer 400ml of seed liquid into the fermenter with 10% inoculation amount, under the conditions of ventilation rate 3vvm, tank pressure 0.05Mpa, stirring speed 300rpm, temperature 25°C, the fermentation period is 72h; during the cultivation proce...

Embodiment 2

[0027] A method for improving the activity of Bacillus subtilis nitrile hydratase, comprising the steps of: (1) slant culture: the bacterial classification was connected to the sterilized slant solid culture medium at 30°C and cultivated for 5 days, and the amount of liquid added was 50ml In a 500ml Erlenmeyer shaker flask with notches, cultivated on a shaking table at 30°C for 72 hours, and the rotating speed of the shaking table was 100rpm, which was used as the primary seed for standby; (2) Seed cultivation: insert the cultivated primary seed into 50m L secondary seed culture shake flask, 30 ℃ shaker culture 72hh, shaker speed 200rpm; (3) fermentation culture: add 3L fermentation medium (mass is 3.08 tons) in 5L fermentation tank, 121 ℃ real tank disinfection 30min, transfer 400ml seed liquid into the fermenter with 15% inoculation amount, under the condition of ventilation rate 2vvm, tank pressure 0.05Mpa, stirring speed 300rpm, temperature 25 ℃, the fermentation period is ...

Embodiment 3

[0030] A method for improving the activity of Bacillus subtilis nitrile hydratase, comprising the steps of: (1) slant culture: the bacteria were connected to the sterilized slant solid medium at 35° C. and cultivated for 4 days, and the amount of liquid added was 50 ml In a 500ml Erlenmeyer shaker flask with notches, cultivated on a shaking table at 35°C for 72h, the rotating speed of the shaking table was 200rpm, and it was used as the primary seed for standby; (2) Seed cultivation: insert the cultivated primary seed into 50ml according to the inoculation amount of 8%. In the second-stage seed culture shake flasks, culture on a shaking table at 35°C for 72 hours, and the rotating speed of the shaking table is 200rpm; (3) Fermentation culture: add 4L fermentation medium (quality is 4.1 tons) to a 5L fermenter, and sterilize the real tank at 121°C for 30min , transfer 400ml seed solution into the fermenter with 8% inoculum amount, under the conditions of ventilation rate 2vvm, t...

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Abstract

The invention discloses a method for improving the nitrile hydratase activity of bacillus subtilis. The method comprises the following steps: (1) carrying out slant culture; (2) carrying out seed culture; and (3) carrying out fermentation culture. The method has the advantages that on one hand, the nutrition components in the fermentation medium are changed, namely, the fermentation medium contains Tween-80 0.1-1 g / ml%, NH4Cl 0.1-1 g / ml%, and urea 0.01-1 g / ml%, a glucose-Co<2+> coupling fed batch process is adopted, and a supplementary material formed by mixing glucose and inducer IND is added; on the other hand, the growth environment of bacteria can be controlled, namely, in the fermentation process, by adding ammonium hydroxide, the pH is controlled to be 6.8-7.5, the temperature is controlled to be 25-35 DEG C, the oxygen quantity is maintained to be 1-3 vvm, and thus the purpose of greatly improving the enzymatic activity during the nitrile hydratase fermentation production is achieved.

Description

technical field [0001] The invention belongs to the technical field of biochemical industry, in particular to a method for improving the activity of bacillus subtilis nitrile hydratase. Background technique [0002] Nitrile hydratase is an important biocatalytic agent for the production of nicotinamide. At present, there are many reports on the use of nitrile hydratase in microorganisms to biocatalyze nicotinamide: for example, the invention patent with the publication number CN1424402A reports that 3-cyanopyridine is converted into nicotinamide by using the nitrile hydratase produced by Corynebacterium propionici. During the conversion process, when the product concentration is ≥ 5%, use the ultra flow membrane filtration system to discharge the product, and at the same time start to add 3-cyanopyridine and water, and wait for the cumulative concentration of the product to be ≥ 10%, and then discharge the tank at one time; as disclosed The invention patent No. CN1154364A r...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/88C12N1/20C12R1/125
CPCC12N9/88C12N1/20C12Y402/01084
Inventor 韦永飞胡其钊周寿芳汪中伟李文兵张全宝
Owner ANHUI RUIBANG BIOLOGICAL SCI & TECH CO LTD
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