Purification of Cyclodextrin Glycosyltransferase and Amylomaltase for Rutin Glycoside Synthesis
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Abstract
Rutin, a bioflavonoid with beneficial properties, suffers from poor water solubility and low stability, leading to inadequate gastrointestinal absorption. To overcome these limitations, the synthesis of rutin glycosides with improved solubility and stability was investigated using cyclodextrin glycosyltransferase (CGTase) and amylomaltase (AMase). This study aimed to identify the most effective enzyme for catalyzing the transglycosylation reaction. CGTase, derived from recombinant p19bBC in Escherichia coli BL21 (DE3), was produced by shaking incubation at 250 rpm and 37°C for 24 hours, with an IPTG concentration of 0.2 mM. The CGTase was purified by starch adsorption, which increased the enzyme purity 5-fold with a specific activity of 4,530 U/mg, a 73% yield, and a molecular weight of 72 kDa. Similarly, AMase was produced from recombinant p19bAMY in E. coli BL21 (DE3) under the same incubation conditions, with an IPTG concentration of 0.8 mM. The enzyme was purified using HisTrap FF column, resulting in a 56-fold increase in purity, a specific activity of 391 U/mg, a 51% yield, and a molecular weight of 60 kDa. Thin-layer chromatography (TLC) analysis of the transglycosylation reactions revealed that CGTase catalyzed the formation of four distinct rutin glycosides (RG1-RG4) when beta-cyclodextrin was used as the glycosyl donor. In contrast, AMase did not catalyze the formation of any rutin derivatives. Therefore, CGTase is considered a promising enzyme for the synthesis of rutin glycosides.
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