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The Enzymatic Properties of Rutin-α-rhamnosidase and Preparation of Isoquercitrin

Author: WuDi
Tutor: YuHongShan
School: Dalian University of Technology
Course: Fermentation Engineering
Keywords: rutin-α-rhamnosidase isoquercitrin enzymatic transformation sepration and purification
CLC: TQ28
Type: Master's thesis
Year: 2012
Downloads: 4
Quote: 0
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Abstract


In this paper, rutin-a-rhamnosidase from Absidia sp.R9g strain was used to transform rutin into higher active products isoquercitrin and rhamnose by hydrolyzing the rhamnoside of rutin. And macroporous resin used to separate the mixture of isoquercitrin and rhamnose. That will provide a theoretical basis to industrial production. Rutin-a-rhamnosidase was purified, and its molecular was determined. The enzymatic properties and the speciality of the enzyme were also investigated.The rutin-a-rhamnosidase is obtained by incubating the microorganism Absidia sp.R9strain. And the reaction condition of the rutin-a-rhamnosidase hydrolyzing rutin to isoquercitrin and rhamnose was optimized:the optimal substrate concentration was4%; the optimal reaction time was6hours; the optimal temperature was50℃; the optimal pH was5.0. The crude enzyme solution was prepared used to hydrolyze24.0g rutin. Isoquercitrin and rhamnose can be fully separated by macroporous resin SP207, crude product of isoquercitrin was obtained from the mixture.8.01g pure isoquercitrin was obtained and the yield was33.3%. The purity of isoquercitrin was98.3%based on HPLC.The enzyme was purified using DEAE-cellulose column. The pure enzyme purity and molecular weight of the rutin-a-rhamnosidase was determined by SDS-PAGE, and the molecular mass was66kDa. The optimum conditions of the enzyme were as follows:pH5.0, stable at pH4.0-8.0; temperature was50℃, stable below60℃; It was not effected by K+, Na+, Mg2+, Zn2+, Ca2+; it was inhibited by Cu2+, and effect was increase with the increasing of Cu2+concentration. The Vmax is56.1mmol/L·h, the Km is11.7mmol/L.

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