Experiments on the preparation of yeast sucrase
Experiments on the preparation of yeast sucrase
Sucrase (inver tase) (β-D-fructofuranoside fructohydrolase) (EC. 3 . 2 . 1 . 26 ) catalyzes the hydrolysis of α-fructofuranoside bonds in non-reducing sugars with relative specificity. It can not only catalyze the hydrolysis of sucrose to produce glucose and fructose, but also catalyze the hydrolysis of raffinose to produce dystroglycan and fructose. The aim of this experiment was to extract sucrase from brewer's yeast.
Operation method
abrasive method
Principle
Sucrase (inver tase) (β-D-fructofuranoside fructohydrolase) ( fructofuranosidefructohydrolase) (EC. 3 . 2 . 1 . 26 ) catalyzes the hydrolysis of α-fructofuranoside bonds in non-reducing sugars with relative specificity. It can not only catalyze the hydrolysis of sucrose to produce glucose and fructose, but also catalyze the hydrolysis of raffinose to produce dystroglycan and fructose. Brewer's yeast contains a large number of sucrase, through grinding to break the cell wall, so that the enzyme free, with water to extract the enzyme, and then precipitate the enzyme protein with organic solvents to get the crude products, but also can be further purified by column chromatography to get the refined products.
Materials and Instruments
Brewer's yeast Move 1. Extraction For more product details, please visit Aladdin Scientific website.
Silicon dioxide Deionized water Ice salt Acetic acid Ethanol
Mortar and pestle Centrifuge tube Dropper tube Measuring cylinder Constant temperature water bath Beaker pH paper High-speed freezing centrifuge
(1) Prepare an ice bath and place the mortar securely in the ice bath.
(2) Weigh 10 g of wet brewer's yeast into the mortar together with an appropriate amount (approx. 5 g) of silicon dioxide. The silicon dioxide should be finely ground beforehand.(3) Slowly add 30 ml of pre-cooled deionized water, about 2 ml at a time, and grind for at least 30 minutes. Grind for at least 30 minutes until most of the yeast cells are crushed, so that sucrase can be fully transferred to the aqueous phase.(4) (Optional) Use a microscope to check the effect of grinding while grinding.
(5) Transfer the mixture into two centrifuge tubes, equilibrate, and centrifuge at 4 °C, 10 000 r/min, for 15 min in a high-speed refrigerated centrifuge.(6) Carefully remove the aqueous phase with a dropper and transfer to another clean centrifuge tube, 4 °C , 10 000 r/ min , centrifuge for 15 min.(7) Transfer the supernatant to a measuring cylinder and measure the volume. Check the pH of the supernatant with a wide range of pH paper and adjust the pH to 5.0 with 1 mol/ L acetic acid, which is called grade I. Set aside 1.5 ml for enzyme activity and protein content, and transfer the rest into a clean centrifuge tube.
2. Heat treatment and ethanol precipitation
(1) Set the thermostatic water bath to 50 ℃ in advance, put the centrifuge tube containing grade I into the water bath steadily, and keep warm at 50 ℃ for 30 minutes, and gently shake the tube during the warming process.(2) Remove the centrifuge tube, cool it quickly in an ice bath, and centrifuge it at 4 ℃, 10 000 r/min, for 10 min.(3) Transfer the supernatant into a small beaker, put it into an ice-salt bath (sprinkle a small amount of salt into the crushed ice without water), slowly add an equal volume of pre-cooled 95% ethanol to -20 ℃, while gently stirring, and then leave it in the ice-salt bath for 10 minutes to complete the precipitation. Centrifuge for 10 min at 10,000 r/min at 4 ℃, decant the supernatant, drip-dry, and store the precipitate in a centrifuge tube with a lid or film seal, then place it in the freezer for storage (referred to as Stage II).Before discarding the supernatant, check the enzyme activity with a urine glucose test strip (do this with the next experiment).