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RSS FeedsMolecules, Vol. 24, Pages 3042: Synthesis of Isorhamnetin-3-O-Rhamnoside by a Three-Enzyme (Rhamnosyltransferase, Glycine Max Sucrose Synthase, UDP-Rhamnose Synthase) Cascade Using a UDP-Rhamnose Regeneration System (Molecules)

 
 

22 august 2019 10:03:35

 
Molecules, Vol. 24, Pages 3042: Synthesis of Isorhamnetin-3-O-Rhamnoside by a Three-Enzyme (Rhamnosyltransferase, Glycine Max Sucrose Synthase, UDP-Rhamnose Synthase) Cascade Using a UDP-Rhamnose Regeneration System (Molecules)
 


Isorhamnetin-3-O-rhamnoside was synthesized by a highly efficient three-enzyme (rhamnosyltransferase, glycine max sucrose synthase and uridine diphosphate (UDP)-rhamnose synthase) cascade using a UDP-rhamnose regeneration system. The rhamnosyltransferase gene (78D1) from Arabidopsis thaliana was cloned, expressed, and characterized in Escherichia coli. The optimal activity was at pH 7.0 and 45 °C. The enzyme was stable over the pH range of 6.5 to 8.5 and had a 1.5-h half-life at 45 °C. The Vmax and Km for isorhamnetin were 0.646 U/mg and 181 μM, respectively. The optimal pH and temperature for synergistic catalysis were 7.5 and 25 °C, and the optimal concentration of substrates were assayed, respectively. The highest titer of isorhamnetin-3-O-rhamnoside production reached 231 mg/L with a corresponding molar conversion of 100%. Isorhamnetin-3-O-rhamnoside was purified and the cytotoxicity against HepG2, MCF-7, and A549 cells were evaluated. Therefore, an efficient method for isorhamnetin-3-O-rhamnoside production described herein could be widely used for the rhamnosylation of flavonoids.


 
222 viewsCategory: Biochemistry, Chemistry, Molecular Biology
 
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