Method for preparing neohesperidin by enzymic method
A technology of neohesperidin and hesperidin, applied in the field of enzymatic preparation of neohesperidin, which can solve the problems of high cost of rhamnosidase and rhamnosyltransferase, increased difficulty in product separation and purification, and complex hydrolyzed products , to achieve the effect of saving separation and purification cost, low cost and wide source of raw materials
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Embodiment 1
[0020] Take 0.25 g of Sepabeads EC-EP carrier (80-120 mesh), soak in water for 24 hours, filter out, and set aside. Add to 1.25 ml rhamnosidase solution, add 1 ml of 1.4 M potassium dihydrogen phosphate buffer solution with a pH of 8, add the above-mentioned treated carrier, and shake at a constant temperature of 26±1°C for 72 hours. The carrier after the immobilized enzyme was filtered, washed with distilled water until the eluate was free of protein, and then washed three times with 0.02 M phosphate buffer at pH 8, and the enzyme activity was 150 U / g.
Embodiment 2
[0022] Take 0.25 g of 80-120 mesh Sepabeads EC-HA carrier filtered out after soaking in water for 24 hours, add 1.25 ml of 4% glutaraldehyde solution, shake for 30 min, filter out the carrier and add it to 1.25 ml of rhamnosyltransferase solution, Add 1 ml of 0.02 M potassium dihydrogen phosphate buffer solution with a pH of 8, and shake the reaction system at a constant temperature of 26±1°C for 12 hours. Filter, wash with distilled water until the eluate is free of protein, then wash three times with 0.02 M phosphate buffer with pH 8, and the enzyme activity is 250 U / g.
Embodiment 3
[0024] In Reactor 1, prepare 5×10 with 80% methanol -3 M Hesperidin and 3×10 -3 M CuCl 2 Solution, heated and reacted in a water bath at 40 °C for 1 h, distilled off methanol under reduced pressure to obtain an aqueous solution of hesperidin copper complex with a concentration of 1.5 g / L. Add the immobilized rhamnosidase prepared in Example 1, 45 °C, pH 4.0, react for 1 h, the raw materials are completely converted into hesperetin-7-glucoside copper complex and rhamnose. Separation through the 400-mesh sieve at the bottom of Reactor 1, the immobilized enzyme remained in Reactor 1; the reaction solution entered into Reactor 2, added the immobilized rhamnosyltransferase prepared in Example 2, and reacted at 35°C for 2h, orange Cortex-7-glucoside and rhamnose were completely converted to neohesperidin copper complex. Separation through the 400-mesh screen at the bottom of the reactor, the immobilized rhamnosyltransferase remains in reactor 2, the solution enters reactor 3, a...
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