Research Information System
V. International Enzyme and Bioprocess Days EBDays 2024 , İzmir, Turkey, 27 - 29 August 2024, pp.22
α-Glucosidases (EC 3.2.1.20) are typically exo-hydrolase enzymes. They liberates α-glucose from the
unreduced end of substrates such as oligos and polysaccharides. They are usually associated with other
amylolytic enzymes that accompany the complete degradation of starch. They are found intracellularly,
extracellularly or cell-bound in the microorganism. α-amylase (EC 3.2.1.1) is one of the best-known
glycoside hydrolases. Amylases hydrolyze α-(1-4) glycosidic bonds and are thus also known as
glycoside hydrolases. Amylases can be divided into endo-amylases and exo-amylases. Endo-amylases
catalyze the random hydrolysis of starch molecules. This action results in the formation of linear and
branched oligosaccharides of various chain lengths. Cross-linking as an enzyme immobilization
technique involves directly binding enzymes together, eliminating the need for external support
materials. It is usually produced by direct cross-linking of dissolved enzymes.
In this study, Anoxybacillus flavithermus DSM 2641T α-amylase and α-glucosidase genes were cloned
into the pET28a(+) expression vector and expressed in E.coli (DE3)pLysS. After the partial
purification, α-amylase and α-glucosidase were then used in combi-CLEA immobilization studies. For
cross-linking, different crosslinking time, and precipitation temperature were tested. Then, the
optimum pH and optimum temperature of combi-CLEA were observed. Enzymes were successfully
immobilized in the presence of cross-linking agent (5 mM Glutaraldehyde) after 3 hours. After
immobilization, optimum temperature and pH were found to be 55°C and 7.4, respectively.