טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentMoscovici Alice Maayan
SubjectEnzymatic Formation of Copolymers and Block-Copolymers
based on Derivatized Polysaccharides
DepartmentDepartment of Biotechnology
Supervisor Dr. Eyal Shimoni
Full Thesis textFull thesis text - English Version


Abstract

Recent years have witnessed increased interest in functional foods as means of preventing diseases and improving well-being. However, the efficient delivery and controlled release of bioactives remains a significant challenge. The overall goal of this research was to employ glycosyltransferases, namely dextransucrase, to enzymatically synthesize food-grade copolymers and block-copolymers, which may be used in the future as delivery systems.

First, dextransucrase was evaluated for its ability to elongate amylopectin in an acceptor reaction, using waxy corn starch as a commercial source. High Performance Anion Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD) and enzymatic analyses demonstrated differences between dextransucrase reaction with and without waxy addition, which may indicate that amylopectin was enzymatically extended. Nevertheless, cryogenic (cryo-) and negative staining Transmission Electron Microscopy (TEM) showed that dextransucrase also synthesized dextran, at all sucrose:waxy weight ratios examined.

Next, dextransucrase was evaluated for its ability to elongate modified oligosaccharides obtained from the enzymatic cleavage of 2-hydroxypropyl-β-cyclodextrin (HP-β-CD) by Taka Amylase A (TAA). HPAEC-PAD, reducing ends assay and cryo-TEM confirmed that HP-β-CD was hydrolyzed by TAA. Products after a 24 hr cleavage reaction were taken for an acceptor reaction with dextransucrase. HPAEC-PAD and turbidity development analyses showed clear differences between dextransucrase reaction with and without the addition of cleaved HP-β-CD, which suggest that the hydroxypropylated oligosaccharides were in fact elongated by dextransucrase. Still, cryo-TEM images interestingly revealed round particles similar to dextran. It is suggested that other reaction conditions may diminish dextran formation.

This study has shown, for the first time, that dextransucrase can elongate polysaccharides other than dextran, specifically amylopectin, as well as modified oligosaccharides acquired from enzymatic cleavage of substituted cyclodextrins. It is the first step towards enzymatically designing tailored copolymers and block-copolymers, which may be used as novel bioactive delivery vehicles.