Studies On The Bioconversion Of Puerarin And Enzyme Immobilization

Puerarin (8-C-β-D-Glucopyranosyl-7,4'-hydroxy-Isoflavone), the most abundant isoflavone in Puerariae radix, is prescribed to treat coronary heart diseases, diabetes, sudden alcoholism and cancer. But there are two major drawbacks limit its wide use in clinical practice, which are low water solubility and low bioavailability. Modification on puerarin with the purpose of improving its water-soluble, increasing their bioavailability and selective, and ultimately enhancing its pharmacological activity, is the focus of the puerarin study in future.Doctor Ding Juanfang and Jiang Jierong in our laboratory screened a Bacillus M. oxydans CGMCC 1788. The bacteria cells can transform puerarin into puerarin-7-O-glucoside. Doctor Ding Juanfang in our laboratory purified the enzyme, and studied its properties. Yu Cigang of our laboratory found the permeabilized cells with ethanol can convert puerarin into puerarin-7-O-fructoside.Industry media were optimized by orthogonal test L9(34). M.oxydans CGMCC 1788 was cultured in 5-litre fermenter to convert puerarin into puerarin-7-O-fructoside. Puerarin-7-O-fructoside was eluted from the column with 10% ethanol at 10 mL/min in a two column chromatography. We gained puerarin-7-O-fructoside 3.84 g at 97% purity.Compared with two celluloses and glutaraldehyde-crosslinked method, it was found that the activity of immobilized enzyme on DEAE-cellulose was 3.6 times more than on CM-cellulose. At the same time, the activity loss of the enzyme immobilized on glutaraldehyde-crosslinked DEAE-cellulose was more than 85% after eluted by 0.5mol/L Nacl.The enzyme from M.oxydans CGMCC 1788 was immobilized onto DEAE-Cellulose via ion adsorption method. The optimum conditions for the immobilization were as follows:12mg enzyme per g DEAE-Cellulose, pH 8.0,4℃. The activity of the immobilized enzyme was 1.2U and the activity yield was about 54.8%.In this study, the properties of the immobilized enzyme was obtained. The optimum reaction temperature,30℃, was consistent with the free enzyme. Compared with the free enzyme, the optimal reaction pH6.5 was shifting to acidity, and the pH stability were improved in the range of pH6.5～pH8.0. The immobilized enzyme showed good operation stability, its enzyme activity loss was less than 50% after 10 times reaction. After storing at 4℃for 55 days, its remnant enzyme activity was 45%.