Method for fixing alpha-amylase
A technology of amylase and starch, which is applied in the field of bioengineering, can solve the problems of high cost and complicated preparation process, and achieve the effects of increased particle hardness and activity, simple operation and low cost
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Embodiment 1
[0018] Add the crude enzyme of α-amylase to the phosphate buffer (100mM, PH7.0) of 1 / 3 relative to the volume of the enzyme solution to dissolve 300rpm and stir at room temperature for 15min to prepare a crude α-amylase solution (or centrifuge the fermentation broth at 10000rpm 15 minutes, remove the bacteria, get the supernatant);
[0019] Slowly add solid ammonium sulfate to the enzyme solution so that the mass percentage of ammonium sulfate in the solution is 80%, stir at room temperature at 300rpm for 30min, take the supernatant and centrifuge to measure the protein content every 10 minutes while stirring, and after more than 95% of the enzyme is precipitated, Centrifuge at 10,000 rpm at 4°C for 15 min to discard the supernatant to obtain a crude enzyme product, and dissolve the crude enzyme with an equal volume of phosphate buffer (pH 7.0, 100 mM) to form a purified crude enzyme solution.
[0020] Get crude enzyme solution 1.6ml and add 1 / 4 volume of phosphate buffer (pH ...
Embodiment 2
[0024] Add the crude enzyme of α-amylase to the phosphate buffer (100mM, PH7.0) of 1 / 3 relative to the volume of the enzyme solution to dissolve 300rpm and stir at room temperature for 15min to prepare a crude α-amylase solution (or centrifuge the fermentation broth at 10000rpm 15 minutes, remove the bacteria, get the supernatant);
[0025] Slowly add solid ammonium sulfate to the enzyme solution so that the mass percentage of ammonium sulfate in the solution is 80%, stir at room temperature at 300rpm for 30min, stir while adding, take the supernatant and centrifuge to determine the protein content every 10 minutes, and make more than 95% of the enzyme After precipitation, centrifuge at 10,000 rpm at 4°C for 15 min to discard the supernatant to obtain a crude enzyme product, and dissolve the crude enzyme with a phosphate buffer solution (pH 7.0, 100 mM) equal to the volume of the original enzyme solution to form a purified crude enzyme solution.
[0026] Get crude enzyme liqui...
Embodiment 3
[0030] Add the crude enzyme of α-amylase to the phosphate buffer (100mM, PH7.0) of 1 / 3 relative to the volume of the enzyme solution to dissolve 300rpm and stir at room temperature for 15min to prepare a crude α-amylase solution (or centrifuge the fermentation broth at 10000rpm 15 minutes, remove the bacteria, get the supernatant);
[0031] Slowly add solid ammonium sulfate to the enzyme solution so that the mass percentage of ammonium sulfate in the solution is 80%, stir at room temperature at 300rpm for 30min, stir while adding, take the supernatant and centrifuge to determine the protein content every 10 minutes, and make more than 95% of the enzyme After precipitation, centrifuge at 10,000 rpm at 4°C for 15 min to discard the supernatant to obtain a crude enzyme product, and dissolve the crude enzyme with a phosphate buffer solution (pH 7.0, 100 mM) equal to the volume of the original enzyme solution to form a purified crude enzyme solution.
[0032] Get 1.6ml of crude enz...
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