Preparation And Application Of Immobilized β-glucosidase On Cross-linked Chitosan Microsphere Modified By Lysine

The crosslinked chitosan (CCR) was synthesized by inverse suspension-crossinking method with glutaraldehyde as the crosslinking agent and CaCO3as theprogenic agent. In the presence of condensation agent DCC, crosslinked chitosan-lysineresin (LMCCR) was prepared through the condensation reaction.The immobilizedβ-glucosidase was prepared on LMCCR as the immobilizationcarrier. The optimal conditions for immobilization are as follows: the molar ratio amongβ-glucosidase, glutaraldehyde and amino group of LMCCR is1.8×10-4﹕1﹕1.Crosslinking for3h in buffer pH7.0at room temperature, with results of coupling ratio of62.02%,32.24U/mg of activity and80.9%of relative activity. Enzymatic propertiesofimmobilizd β-glucosidase are as follows: the optimal pH is5.5~6.5; the optimaltemperature is45~55℃; Kmand Vmaxare47.69mmol/L and34.60U/mg respectively;the half-life is15.7h holding at65℃; the half-life for repeated use at40℃is44times;the storage half-life at4℃is295days.The batch equilibrium adsorption of XAD-7HP for resveratrol and polydatin are37.63mg/g and25.47mg/g, respectively, under the condition of adsorption time2hours.The column equilibrium adsorption for resveratrol and polydatin are30.04mg/g and20.36mg/g, respectively. The column elution rate for resveratrol reaches92.58%using70%ethanol as eluent at the velocity of2mL/min, and the column elution rate for polydation reaches98.58%using40%ethanol eluent. The hydrosates are absobored by XAD-7HP resin andgradient eluted by40%and70%ethanol solution with elution rate of2mL/min. Theresults of high performance liquid chromatography of elution solution prove thatresveratrol can be separated and purified effectively.