Method for generating phospholipase D with streptomyces cinnamonensis and method for determining activity of phospholipase D
A technology of Streptomyces cinnamon and Streptomyces cinnamomi, applied in the field of phospholipase D activity determination and Streptomyces cinnamon producing phospholipase D, can solve the problems of limitation, long cycle, low yield, etc., and achieve the effect of high phosphoryl transfer activity
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Embodiment 1
[0017] Embodiment 1 strain screening
[0018] 1. Plate initial screening: Treat the soil samples near the soybean oil factory, use lecithin as the substrate and inducer, use bromocresol violet as the color reagent, culture at a constant temperature of 30°C, and the ones that can produce white transparent circles around the colonies are selected. For the strains produced by phospholipase, pick a single colony with a transparent circle, culture it on a slant for 72 hours, and carry out the next step of re-screening. Slant culture conditions are: glucose 12g / L, yeast extract 15g / L, peptone 10g / L, agar 18g / L, K 2 HPO 4 1g / L, MgSO 4 ·7H 2 O 1.5g / L, sterilized at 121°C for 30min.
[0019] 2. Re-screening by shaking flask bacterial viability detection
[0020] Shake flask re-screening: culture the strains obtained by the plate primary screening in shake flasks, using glucose 12g / L, yeast extract 15g / L, peptone 10g / L, K 2 HPO 4 1g / L, MgSO 4 ·7H 2 O 1.5g / L, sterilized at 121°...
Embodiment 2
[0022] Embodiment 2 strain fermentation
[0023] Streptomyces cinnamon was fermented under the following conditions:
[0024] (1) Incline culture: use glucose 10g / L, yeast extract 20g / L, peptone 5g / L, agar 15g / L, K 2 HPO 4 0.5g / L, MgSO 4 ·7H 2 O 0.5g / L, sterilized at 121°C for 30min to obtain a slant culture medium, inoculated Streptomyces cinnamomi on the slant medium, and cultivated for 72h to obtain Streptomyces cinnamon cultivated on a slant.
[0025] (2) Seed culture: such as figure 2 It can be seen that the Streptomyces cinnamona was in the growth phase and the number of viable bacteria increased linearly at 48h, and entered a stable phase after 84h. Suitable for fermentation culture. Glucose 10g / L, yeast extract 20g / L, peptone 5g / L, K 2 HPO 4 0.5g / L, MgSO 4 ·7H 2O 0.5g / L, sterilized at 121 DEG C for 30min to obtain a seed culture medium, and the Streptomyces cinnamona cultivated on the slant that step (1) obtained was inoculated onto the seed culture medium ...
Embodiment 3
[0029] (1) Incline culture: use glucose 15g / L, yeast extract 10g / L, peptone 20g / L, agar 20g / L, K 2 HPO 4 2g / L, MgSO 4 ·7H 2 O 2.5g / L, sterilized at 121°C for 30min to obtain a slant medium, inoculated Streptomyces cinnamon on the slant medium, and cultivated for 72h to obtain Streptomyces cinnamon cultured on the slant.
[0030] (2) Seed culture: use glucose 15g / L, yeast extract 10g / L, peptone 15g / L, K 2 HPO 4 2g / L, MgSO 4 ·7H 2 02.5g / L, sterilized at 121 DEG C for 30min to obtain a seed culture medium, and the Streptomyces cinnamona cultured on the slant that step (1) obtained was inoculated onto the seed culture medium and cultivated. The culture temperature was 32° C., the rotation speed of the shaker was 220 rpm, and the culture time was 5 days to obtain a seed culture solution.
[0031] (3) Basic fermentation culture: use glucose 25g / L, yeast extract 5g / L, peptone 20g / L, K 2 HPO 4 2g / L, MgSO 4 ·7H 2 O2.5g / L, antifoaming agent polyoxyethylene oxypropylene trig...
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