Bacillus subtilis mutant strain for highly producing NK (nattokinase) and application of Bacillus subtilis mutant strain
A Bacillus subtilis, nattokinase-producing technology, applied in the application, enzyme, peptidase and other directions, can solve the problems of lack of good strains and high production cost of nattokinase, achieve high unit activity, strong nattokinase ability, The effect of high safety
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Embodiment 1
[0041] A subspecies of Bacillus subtilis subsp.UJS-18, the screening process is as follows:
[0042]1) Take 0.2 g of nattokinase bacteria powder purchased in the market, add 1 ml of sterile saline, shake and let stand, dip the supernatant with an inoculation loop and draw a line on the LB plate to separate a single colony. Use an inoculation loop to pick a single colony, refer to the eighth edition of "Berger's Bacterial Identification Manual" to observe whether it can be drawn, select a few single colonies of Gram-positive spores that can be drawn, and put them on a new LB plate for division and marking , separated and purified again, picked a single colony and inoculated it into the seed medium, 37°C, 200rpm, cultured for 6 hours, took 0.2ml and inoculated it into 40ml of new seed medium, cultivated for 6 hours to reach the logarithmic growth phase.
[0043] 2) Take 5ml of bacterial solution in the logarithmic growth phase, centrifuge at 5000rpm for 5min, discard the superna...
Embodiment 2
[0046] The PCR amplification of 16S rRNA, its steps are as follows:
[0047] Genomic DNA of UJS-18 strain was extracted using a bacterial genomic DNA extraction kit (Tiangen Biochemical Technology Co., Ltd.), diluted to 200 ng / μl with TE buffer, and used as a template for PCR amplification. Prepare the following reaction system: 10 μl of 2×PCR mix, 2 μl of genomic DNA, 1 μl of upstream primer (5'-AGAGTTTGATCCTGGCTCA-3'), 1 μl of downstream primer (5'-GGTTACCTTGTTACGACTT-3'), and 6 μl of pure water. Amplification was performed according to the following procedure: pre-denaturation at 94°C for 3 min, 35 cycles (pre-denaturation at 94°C for 20 s, annealing at 55°C for 20 s, extension for 1 min), extension at 72°C for 5 min, and storage at 4°C. After the PCR amplification, 3 μl of the reaction solution was taken for 1.5% agarose electrophoresis detection. Ultraviolet detection showed that the size of the amplified 16S rRNA band was about 1500bp.
[0048] Physiological and bioche...
Embodiment 3
[0055] Nattokinase fibrinolytic activity assay, its steps are as follows:
[0056] 1) Preparation of agarose-fibrin plate:
[0057] Use a serum bottle to measure 25ml of 1% agarose solution prepared with PBS buffer solution, cool down to 50-55°C, draw 200ul of 0.5% fibrinogen solution, and quickly pour the fibrinogen solution three times into the bottom, middle and upper parts of the bottle Pour in and stir vigorously for 5-8 times, pour 60ul of 20U / ml thrombin solution into the fibrinogen solution and stir vigorously, and immediately pour the solution in the serum bottle into the petri dish from the wall of the petri dish After shaking for a few times, let it stand still for 1 hour, and punch a hole on the plate with a glass tube with a diameter of 2 mm.
[0058] 2) The making of urokinase standard curve:
[0059] Prepare urokinase standard products (10, 20, 40, 60, 80 IU / ml) with different activities, take 10 μl each and add them to the sample wells of the agarose-fibrin p...
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