Acid protease
A technology of acid protease and acidic conditions, applied in the field of acid protease, can solve the problems of low hydrolysis rate and achieve the effects of high hydrolysis efficiency, high degree of hydrolysis and strong hydrolysis ability
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Embodiment 1
[0023] The fermentation of embodiment 1 hair enzyme and the extraction of crude enzyme
[0024] 1.1 Strains
[0025] Mucor actinosa AS3.2778 was purchased from the General Microbiology Center of China Committee for the Collection of Microorganisms.
[0026] 1.2 Seed medium and seed activation
[0027] Add 10g of bran and 10ml of water into a 250ml triangular flask, stir and mix evenly with a glass rod, and smash larger agglomerates (try to make the particles uniform). Sterilize at 121°C for 30 minutes, take it out from the heat, shake it up, and cool it down for later use.
[0028] The sterilized seed medium was inoculated with a ring of slant spores in a test tube per bottle, mixed evenly, and the surface of the medium was made as smooth as possible, and the thickness of the medium layer was consistent, and then placed in a biochemical incubator at 28°C for 3 days.
[0029] 1.3 Preparation of spore suspension
[0030] Under sterile conditions, add 100 ml of sterile water ...
Embodiment 2
[0039] The separation and purification of embodiment 2 acid protease
[0040] 2.1 Ammonium sulfate segmental salting out
[0041] Take a certain volume of crude enzyme solution, slowly add solid ammonium sulfate to 40% saturation on an ice-water bath, fully dissolve and let stand in a refrigerator at 4°C for 3 hours, centrifuge at 12000g / min for 20min at 4°C, take the supernatant and continue Add solid ammonium sulfate to 85% saturation, let it stand overnight in a refrigerator at 4°C, then centrifuge at 12000g / min for 20min at 4°C, dissolve the protein precipitate with 0.02M, pH7.5 Tris-HCl buffer, and dialyze for desalting.
[0042] 2.2CM-Sepharose cation exchange
[0043] Equilibrate the CM-Sepharose cation exchange column with 0.05M, pH 5.0 acetate buffer, and load the concentrated enzyme solution collected in the previous step into the cation exchange column. After fully washing the column with equilibration buffer, gradient elution was carried out with 0.5M NaCl soluti...
Embodiment 3
[0059] The mensuration of embodiment 3 mucor acid protease Pa2 catalytic properties
[0060] 3.1 Optimum action temperature of acid protease
[0061] According to the enzyme activity assay method, the activity of Mucor acid protease was measured at different temperatures (range of 30-70°C), and the effect of temperature on the activity of Mucor acid protease was investigated. According to the experimental results, the temperature-activity curve was drawn, and the optimum action temperature of Mucor acid protease was determined accordingly.
[0062] 3.2 Optimal pH of Mucor acid protease
[0063] According to the enzyme activity assay method, under different pH buffer conditions (pH 3.0-5.0, 0.05M acetic acid buffered saline; pH6.0-7.0, 0.05M phosphate buffered saline; pH 8.0-9.0, 0.02M Tris-HCl; pH 9.5- 10.5, 0.02M glycine-NaOH) to measure the activity of Mucor acid protease, and investigate the effect of pH on the activity of Mucor acid protease. According to the experiment...
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