Method for accelerating bacillus to synthesize gamma-polyglutamic acid by using gas signal molecules
A gas signal, polyglutamic acid technology, applied in the field of microorganisms, to achieve the effect of strong genetic stability
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Embodiment 1
[0038] Example 1: Effect of Nitric Oxide Synthase on Nitric Oxide Synthesis in Bacillus amyloliquefaciens SQR9
[0039] Through information comparison, it was found that there is a gene yflM (NCBI ID: AHZ14748.1) encoding nitric oxide synthase in the strain SQR9, which is located at V529_07220 in the SQR9 genome, and the gene was knocked out by homologous recombination to obtain a mutation Body ΔyflM. The ability of the two strains to produce nitric oxide was measured by a nitric oxide detection kit.
[0040] The results of this experiment are as Figure 1As shown, the nitric oxide synthesis level decreased by 5 times after knocking out nitric oxide synthase (YflM) in strain SQR9, which proved that nitric oxide synthase involved in the synthesis of nitric oxide was the main source of nitric oxide in strain SQR9 , this mutant strain ΔyflM was used to measure the synthesis ability of γ-polyglutamic acid in the strain SQR9 at a low level.
Embodiment 2
[0041] Embodiment 2: Fermentation and extraction of gamma-polyglutamic acid
[0042] This embodiment is divided into SQR9 group and ΔyflM group.
[0043] The strains SQR9 and ΔyflM activated overnight were respectively connected to Landy medium (Landy medium, the recipe for 1L medium: 1. Glucose 20g, sterilized separately, 115°C, 30min. 2. MgSO 4 ·7H 2 O 0.5g; KH 2 PO 4 1g; KCl0.2g; MnSO 4 ·H 2 O 10mg; FeSO 4 ·7H 2 O 5mg; CuSO 4 ·5H 2 O 0.2mg; Yeast powder 1g; L-alanine 2mg; L-glutamic acid 5g, adjusted to pH=6.7 with 5M NaOH, sterilized at 115°C for 30min. 1 and 2 were sterilized separately and mixed together), cultivated at 37°C and 170rpm for 48h.
[0044] Centrifuge the two groups of fermentation broths at 4°C and 8000r / min for 15min to remove bacteria, add 3 times the volume of absolute ethanol, let stand overnight at 4°C, centrifuge at 8000r / min for 15min to get the precipitate, add the same volume of fermentation broth Distilled water was dissolved to obtain...
Embodiment 3
[0045] Embodiment 3: the mensuration of gamma-polyglutamic acid content
[0046] Configuration of γ-polyglutamic acid standard solution: Accurately weigh 0.1g of γ-polyglutamic acid, dissolve it in distilled water and set the volume to 100ml to obtain 1mg / ml γ-polyglutamic acid standard solution, and serially dilute the standard solution A γ-polyglutamic acid standard solution of 10 μg-1 mg / ml was obtained. Preparation of CTAB solution: prepare 2% NaOH solution, and use it as a solvent, add CTAB to make the final concentration 25g / L, and heat appropriately to accelerate the dissolution.
[0047] Take respectively 2ml of the fermentation product samples of the SQR9 group and ΔyflM group prepared in Example 2 and 2ml of the standard solution in a test tube, accurately add 2ml of CTAB test solution, fully shake and let stand for 3min, measure the absorbance value at a wavelength of 250nm .
[0048] The results of this experiment are as Figure II It was shown that after Bacill...
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