A kind of method for fermenting and producing vitamin C precursor 2-keto-l-gulonic acid

Abstract

The invention discloses a method for fermenting and producing vitamin C precursor 2-keto-L-gulonic acid, which comprises Bacillus licheniformis (associated bacteria) overexpressing D-sorbitol dehydrogenase (SLDH) Mixed culture with acid-producing bacteria Ketogulonigenium vulgare, fermented to produce 2‑keto‑L‑gulgare. The method of the invention realizes the "one-pot method" conversion from D-sorbitol to 2-keto-L-gulonic acid, simplifies the production process, shortens the fermentation period, and effectively reduces the production cost.

Description

technical field

[0001] The invention relates to the technical field of fermentation, in particular to a method for producing 2-keto-L-gulonic acid by fermentation involving only two strains of bacillus licheniformis and ketogenic cologne. Background technique

[0002] Vitamin C (Vitamin C, referred to as Vc), also known as L-ascorbic acid, is a water-soluble vitamin that maintains normal physiological functions of the body and is widely used in food, medicine, feed, cosmetics and other industries. At present, the production methods of vitamin C in the industry are mainly the Reyes method and the two-step fermentation method, and the two-step fermentation method is a method commonly used by vitamin C production enterprises in my country. The first step of the two-step fermentation method is to use Gluconobacter oxyans to convert D-sorbitol into L-sorbose; The mixed bacteria system composed of Bacillus megaterium (commonly known as big bacteria) converts L-sorbose into 2-keto...

Examples

Embodiment 1

[0035] Example 1 Screening of Bacillus licheniformis with high activity of D-sorbitol dehydrogenase (SLDH)

[0036] Medium: sorbitol 2%, yeast extract powder 0.5%, K 2 HPO 4 0.3%, MgSO 4 ·7H 2 O 0.1%, Sodium Glutamate 0.1%. Sterilization of various media was carried out at 121°C for 20 minutes in a high-pressure steam sterilizer.

[0037] Screening: Wash and scrape the activated slant bacteria with sterile physiological saline to prepare a bacterial suspension, dilute it appropriately, spread it on a plate, and place it in an incubator at 28°C for 72 hours. Pick a single colony on a solid slant medium, and then place it in an incubator at 28°C for 72 hours. Shake flask culture, detect bacterial biomass and sorbitol dehydrogenase activity, thereby select the strain with the highest biomass and sorbitol dehydrogenase activity.

Embodiment 2

[0038] Embodiment 2 mutagenesis improves bacillus licheniformis D-sorbitol dehydrogenase (SLDH) activity

[0039] According to the measured bacterial cell growth curve, the logarithmic phase of bacterial cell growth is reached when the OD600 value is 0.6-0.8. Use a suction gun on the ultra-clean workbench to draw an appropriate amount of bacterial suspension and dilute it with normal saline to an appropriate multiple, then draw 10 μL of the diluted bacterial suspension onto the special carrier sheet for ARTP equipment mutagenesis, and put it into the ARTP equipment for mutagenesis treatment. The mutagenesis time was set as 50s, 60s, 70s, 80s, 90s and 100s. After the mutagenesis treatment is completed, take out the carrier sheet, put it into a 2mL sterile PE tube prepared in advance and add 1mL of normal saline, and then place the PE tube on a rotary shaker for 2-3 minutes to make the PE tube The bacteria in the medium were mixed evenly; at the same time, 10 μL of the diluted ...

Embodiment 3

[0040] The mensuration of embodiment 3 sorbitol dehydrogenase enzymatic activity

[0041] The enzyme reaction system includes 100mM Tris-HCl buffer solution (pH 9.0), 1mM NAD+50mM sorbitol, an appropriate amount of mutagenized Bacillus licheniformis cells, and measures the rise of absorbance at 340nm at 30°C. Enzyme activity is defined as the amount of enzyme needed to generate 1 μmol NADH per minute as one enzyme activity unit U.

technical field

[0001] The invention relates to the technical field of fermentation, in particular to a method for producing 2-keto-L-gulonic acid by fermentation involving only two strains of bacillus licheniformis and ketogenic cologne. Background technique

[0002] Vitamin C (Vitamin C, referred to as Vc), also known as L-ascorbic acid, is a water-soluble vitamin that maintains normal physiological functions of the body and is widely used in food, medicine, feed, cosmetics and other industries. At present, the production methods of vitamin C in the industry are mainly the Reyes method and the two-step fermentation method, and the two-step fermentation method is a method commonly used by vitamin C production enterprises in my country. The first step of the two-step fermentation method is to use Gluconobacter oxyans to convert D-sorbitol into L-sorbose; The mixed bacteria system composed of Bacillus megaterium (commonly known as big bacteria) converts L-sorbose into 2-keto...

Examples

Embodiment 1

[0035] Example 1 Screening of Bacillus licheniformis with high activity of D-sorbitol dehydrogenase (SLDH)

[0036] Medium: sorbitol 2%, yeast extract powder 0.5%, K 2 HPO 4 0.3%, MgSO 4 ·7H 2 O 0.1%, Sodium Glutamate 0.1%. Sterilization of various media was carried out at 121°C for 20 minutes in a high-pressure steam sterilizer.

[0037] Screening: Wash and scrape the activated slant bacteria with sterile physiological saline to prepare a bacterial suspension, dilute it appropriately, spread it on a plate, and place it in an incubator at 28°C for 72 hours. Pick a single colony on a solid slant medium, and then place it in an incubator at 28°C for 72 hours. Shake flask culture, detect bacterial biomass and sorbitol dehydrogenase activity, thereby select the strain with the highest biomass and sorbitol dehydrogenase activity.

Embodiment 2

[0038] Embodiment 2 mutagenesis improves bacillus licheniformis D-sorbitol dehydrogenase (SLDH) activity

[0039] According to the measured bacterial cell growth curve, the logarithmic phase of bacterial cell growth is reached when the OD600 value is 0.6-0.8. Use a suction gun on the ultra-clean workbench to draw an appropriate amount of bacterial suspension and dilute it with normal saline to an appropriate multiple, then draw 10 μL of the diluted bacterial suspension onto the special carrier sheet for ARTP equipment mutagenesis, and put it into the ARTP equipment for mutagenesis treatment. The mutagenesis time was set as 50s, 60s, 70s, 80s, 90s and 100s. After the mutagenesis treatment is completed, take out the carrier sheet, put it into a 2mL sterile PE tube prepared in advance and add 1mL of normal saline, and then place the PE tube on a rotary shaker for 2-3 minutes to make the PE tube The bacteria in the medium were mixed evenly; at the same time, 10 μL of the diluted ...

Embodiment 3

[0040] The mensuration of embodiment 3 sorbitol dehydrogenase enzymatic activity

[0041] The enzyme reaction system includes 100mM Tris-HCl buffer solution (pH 9.0), 1mM NAD+50mM sorbitol, an appropriate amount of mutagenized Bacillus licheniformis cells, and measures the rise of absorbance at 340nm at 30°C. Enzyme activity is defined as the amount of enzyme needed to generate 1 μmol NADH per minute as one enzyme activity unit U.

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