Bioactivity guided enzymatic hydrolysis of saponin molecules in microfluidic systems

Microfluidics systems which enable the manipulation of liquids in small volumes have various advantages over conventional systems such as enhanced heat and mass transfer, lower reagent consumption and efficient mixing. In this thesis, development of an enzymatic microreactor which combines the advantages of microfluidic systems and enzymatic reactions and the hydrolysis of saponin molecules in the developed system were aimed. In accordance with this purpose, β-glucosidase enzyme was immobilized in silica and alginate hydrogels, these hydrogels were characterized in terms of morpholgy, surface area and shrinkage/swelling properties and the kinetic parameters of free and immobilized enzyme such as Vmax ve KM were determined. After the characterization studies, enzymatic microreactors were prepared and hydrolysis of pNPG (model substrate), ginsenoside Rb1, soyasaponin Bb and soyasaponin Aa were carried out. The characterization studies indicated that the high surface area (∼304 m2/g) and porosity properties of alginate hydrogels made them suitable matrices for the enzyme immobilization. The studies carried out in enzymatic microreactors showed that immobilization method and the residence time had significant effect on the reaction yield and increasing residence time enhanced the conversion yield. Performance of the enzymatic microreactor and free and immobilized enzymes in batch conditions were compared and in microreactor experiments higher conversion yield (37.52%) was obtained than the conventional reaction systems (27% and 16% for free and immobilized enzyme, respectively). Saponin molecules were hydrolyzed by three different approaches. By the acidic hydrolysis, not only the intermediate products but also undesired side products were obtained while no side product formation occured during enzymatic hydrolysis which indicated the main advantage of enzymes over chemical catalysts. The hydrolysis of ginsenoside Rb1 by free enzyme resulted in the formation of ginsenoside Rd, the molecule obtained by the removal of a glucose from ginsenoside Rb1. For the same residence time, ginsenoside Rd and also ginsenoside F2 (the follow up product of ginsenoside Rd) were determined in enzymatic microreactor experiments. In a similar way, only intermediate products (soyasaponin III and soyasapogenol B monoglucuronide) were obtained by the hydrolysis of soyasaponin Bb with free enzyme while enzymatic microreactor was able to produce the aglycone, soyasapogenol B. These results clearly showed the high substrate specifity of enzymes and the efficacy of enzymatic microreactors over conventional systems. In addition, MTT analyses were carried out to determine the cytotoxicity of the saponins and aglycones on different cancer cell lines. Soyasapogenols showed higher cytotoxicity than soyasaponins. The most effective molecule on all tested cell lines was compound K and it was found that compound K induced cell death by apoptosis. Keywords: Enzymatic microreactor, β-glucosidase, hydrogel, ginsenoside Rb1, soyasaponins, cytotoxicity.