Novel method for extracting and purifying beer malt root phospholipase D
A technology of phospholipase and a new method, applied in biochemical equipment and methods, hydrolytic enzymes, pharmaceutical formulations, etc., can solve unseen problems and achieve the effect of high enzyme activity
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[0045] : Crushing treatment of beer malt roots
[0046] Crush an appropriate amount of malt root through an 80-mesh sieve, then add a small amount of quartz sand in a mortar to mechanically grind for 15 minutes, and then add complex enzymes (1.0% cellulase and 0.6% pectinase) at pH 5.5, 40 Under the condition of ℃, the cell wall was broken by enzymolysis for 4 hours to obtain the sample to be separated and purified.
Example Embodiment
[0047] : Extraction and separation of phospholipase D
[0048] The sample after the wall-breaking treatment was added with a material-to-water ratio of 1:11, and it was extracted in water for 24 hours at 20°C and pH 5.5. After centrifugation, the supernatant was taken, and 1:4 alcohol was added for precipitation. Leave it for 3 hours, centrifuge to remove the supernatant, and harvest the extract-like precipitate.
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[0049] : Purification of Phospholipase D
[0050] The invention uses epichlorohydrin to activate the carrier and the coupling ligand. Take 10mL of the precipitation volume of Sepharose 4B in a G2 funnel, suction and filter it into a semi-dry, rinse with about 100mL of 0.5mol / L NaCl solution, and then wash with 100mL of distilled water to remove the protective agent and preservative. Drain about 6g semi-dry product, put it in a 50mL Erlenmeyer flask, add 6.5mL 2mol / L NaOH, 2mL epichlorohydrin and 15mL 56% dimethyl sulfoxide, mix well, and shake gently at 40℃2 Hours, then transfer the glue to the G2 funnel, rinse with distilled water to remove excess reagents, and finally use about 100mL 0.2mol / L Na 2 CO 3 Wash with pH 9.5 buffer. Then carry out the coupling experiment as soon as possible. Transfer the activated Sepharose 4B to the Erlenmeyer flask. Use 10mL 0.2mol / L Na 2 CO 3 , PH9.5 buffer solution to dissolve 2g of phosphatidylcholine, then transfer to a triangular flask to coupl...
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