A kind of Bacillus subtilis FJ-3-16 and its application
A technology of Bacillus subtilis and strains, applied in the field of microorganisms, can solve the problems of single action site, low enzyme activity, high price, etc., and achieve good degradation effect
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Embodiment 1
[0016] Example 1 Separation and purification of B.subtilis FJ-3-16
[0017] Milk plate: skimmed milk powder with a mass fraction of 10% was sterilized at 116°C for 30 minutes, then mixed with beef extract peptone medium at a ratio of 1:9, mixed evenly and poured onto the plate.
[0018] Feathers, wool, and sheepskin are purchased from slaughterhouses, washed with soapy water, rinsed with clean water, and dried for later use.
[0019] FM1 feather liquid medium (g / L): NaCl 0.5, K 2 HPO 4 1.0, KH 2 PO 4 0.4, MgCl 2 ·7H 2 O 0.1, CaCl 2 0.06, adjust the pH to 7.5 with NaOH. Pack into 30mL / 100mL Erlenmeyer flasks. After autoclaving, about 20 mg of feather substrate was added.
[0020] Weigh 1 g of soil sample, put it into a conical flask containing 10 mL of sterilized saline and glass beads, and shake it to fully mix the soil sample with water. Take 1mL soil suspension and add it into a test tube filled with 9mL sterilized normal saline and mix well, this is 10 -1 Dilu...
Embodiment 2
[0028] Example 2 B. subtilis FJ-3-16 strain identification
[0029] LB liquid medium (g / L): peptone 10.0, yeast powder 5.0, NaCl 10.0, pH 7.5. For transformant screening, add ampicillin (Amp) at a final concentration of 50 μg / mL to the culture medium.
[0030] LB solid medium (g / L): peptone 10.0, yeast powder 5.0, NaCl 10.0, agar 15.0, pH 7.5.
[0031] A single colony of the FJ-3-16 strain was inoculated in 5 mL of liquid LB medium and cultured overnight at 37°C. Take 1mL of the cultured bacterial liquid, and extract the total bacterial DNA according to the instructions of "Bitec Bacterial Genome Extraction Kit".
[0032] 16S universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-ACGGCTACCTTGTTACGACTT-3') were designed. Using the bacterial genome as a template, according to the PCR system and procedures in the instruction manual of Taq DNA polymerase, PCR was used to obtain DNA fragments. The PCR system is shown in Table 1, and the PCR program is shown in Table 2: ...
Embodiment 3
[0040] Example 3 B.subtilis FJ-3-16 enzyme production condition optimization
[0041] (1) Selection of medium
[0042] experimental method:
[0043] FM1 feather liquid medium (g / L): NaCl 0.5, K 2 HPO 4 1.0, KH 2 PO 4 0.4, MgCl 2 ·7H 2 O 0.1, CaCl 2 0.06, adjust the pH to 7.5 with NaOH. Pack into 30mL / 100mL Erlenmeyer flasks. After autoclaving, about 20 mg of feather substrate was added.
[0044] FM2 feather fermentation medium (g / L): peptone 5.0, glucose 10, K 2 HPO 4 3.0, KH 2 PO 4 1.0, adjust the pH to 7.5 with NaOH, divide into 100mL / 500mL Erlenmeyer flasks, then add Feather 5, and sterilize by high pressure steam for later use (Tiwary E, GuptaR. Bioresource Technol. 2010, 101: 6103-6110).
[0045] FM3 fermentation medium (g / L): corn flour 20, soybean flour 20, bran 10, KH 2 PO 4 1.0,K 2 HPO 4 3.0, CaCl 2 1.0, adjust the pH to 7.5 with NaOH, pack into 50mL / 500mL Erlenmeyer flasks, and sterilize by high pressure steam for later use.
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