Gluconobacter oxydans containing specific membrane-bound glycerol dehydrogenase gene sequence and applications thereof
A technology of glycerol dehydrogenase and oxidizing glucose, which is applied in the field of catalyzing the conversion of glycerol into 1, oxidizing gluconic acid bacteria, can solve the problems of structural modification, improvement of enzymatic properties, and reduction of inhibitory effect, so as to accelerate electron transfer efficiency and improve performance. , The effect of improving the activity of glycerol dehydrogenase enzyme
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Embodiment 1
[0040] Embodiment 1: the comparison of the fermentative conversion glycerol of wild strain and mutant strain to produce DHA
[0041] Seed medium (g / L): glycerol 20, glucose 20, yeast extract 5
[0042] Solid plate medium (g / L): glycerol 20, glucose 20, yeast powder 5, CaCO 3 5. Agar powder 20
[0043] Slant medium (g / L): glycerol 20, glucose 20, yeast powder 5, agar powder 20, CaCO 3 10
[0044] Fermentation medium (g / L): glycerin 60-140, yeast powder 5, defoamer 0.1, MgSO 4 ·7H 2 O 0.1, CaCO 3 2
[0045] After recovering the screened Gluconobacter oxydans wild strain L-6 and mutant strain I-2-239 respectively, they were activated on the slope at 30°C, inoculated into the seed culture solution, cultivated to the middle and late stages of logarithmic growth, and then inoculated in the culture medium containing In the sterilized fermentation medium of 60g / L and 120g / L glycerol, the initial pH of the medium is 4.0-8.0, the fermentation temperature is 30-33°C, and the rotat...
Embodiment 2
[0046] Embodiment 2: the enzyme activity assay comparison of wild strain and mutant strain
[0047] The fermentation broth in Example 1 was centrifuged separately, and the obtained bacterial cells were washed 3 times with a phosphate buffer solution of pH 6.0, and the bacterial cells were resuspended according to the ratio of 100 mg cells: 1 mL phosphate buffer solution, and then the fermentation broth containing 100 g / L glycerin was added After 3 hours of biotransformation, the cells were centrifuged, and the DHA content of the supernatant was determined. The enzyme activity of glycerol dehydrogenase is defined as the amount of DHA produced per hour. The enzyme activities of glycerol dehydrogenase in wild strain and mutant strain were 0.19±0.05mM DHA / h and 0.34±0.05mM DHA / h, respectively. In parallel comparison, the glycerol dehydrogenase activity of the mutant strain was significantly higher than that of the wild strain.
Embodiment 3
[0048] Embodiment 3: analysis and comparison of the glycerol dehydrogenase gene transcription level of wild strain and mutant strain
[0049] The somatic cells of the wild strain and the mutant strain in the late logarithmic growth stage were selected respectively, and total RNA was extracted. After reverse transcription, cDNA was used as a template and the housekeeping gene 16SrDNA was used as an internal reference to perform fluorescence real-time quantitative PCR to determine the glycerol dehydrogenase gene sldA and Transcript levels of the glycerol dehydrogenase gene sldB. The transcription level of glycerol dehydrogenase gene sldA in the mutant strain was 4.8 times that of the wild strain, while the transcription level of sldB was 5.4 times that of the wild strain. The mutant glycerol dehydrogenase transcription level is significantly improved, which will help to realize the high-efficiency expression of the glycerol dehydrogenase gene, so that the catalytic activity of t...
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