生化试验（Biochemical test）.docVIP

生化试验（Biochemical test） 1. Sucrose Enzyme Extraction and preliminary purification: containing yeast invertase, sucrose and enzyme belongs to the endoenzyme, so often the cell wall broken extraction, this method adopts the autolysis method of fungus to break the cell wall after cell extraction and separation of sucrose enzyme solution, followed by heat extraction, ethanol precipitation extraction, sucrose the preliminary purification of enzyme. Note: before each centrifugation, balance, balance for the first time, second water balance, third water balance, fourth ethanol balance, fifth tris-Hcl balance. Water bath insulation temperature is not higher than 50 degrees Celsius, and constantly shake conical bottle. In the ice bath, the alcohol should be stirred and added slowly, and the alcohol will be frozen. The white solids should be retained and ready for final consolidation. Initial extraction process: after the yeast was added to distilled water, the solution was centrifuged, and the solution was divided into three layers: organic layer, protein layer and solution layer. Centrifugal principle: the powerful centrifugal force produced by the high-speed rotation of the rotor of the ion machine can accelerate the settling velocity of particles in the liquid and separate the material with different sedimentation coefficient and buoyancy density in the sample. Heat extraction process: according to the thermostable property of sucrase, the impurity protein of thermal instability was denatured and removed, while the sucrase remained alive in the supernatant. Ethanol precipitation extraction process: according to the molecular weight of invertase and hydrophilicity, adding a certain amount of 95% ethanol solution, the dehydration, destruction of protein surface membrane hydration, sucrose enzyme aggregation precipitation down, and separated from other impurities. The purified -Q Sepharose column chromatography 2. Sucrose Enzyme: ion exchange chromatography is a common method