20.500.12556/DKUM-13933 IMOBILIZACIJA ENCIMA [alfa] - AMILAZE V ZAMREŽENE ENCIMSKE SKUPKE (CLEA) IMOBILIZATION OF ENZYME [alpha] - AMYLASE AS CROSS-LINKED ENZYME AGGREGATES (CLEA) Delo opisuje pripravo katalitično aktivnih zamreženih encimskih skupkov iz encima α — amilaze ali na kratko CLEA. Postopek priprave zamreženih encimskih skupkov je bil razdeljen na dva ključna dela; na obarjanje topnega oziroma nativnega encima z ustreznim organskim topilom in zamreženje tako izoborjenega encima s pomočjo mrežnega povezovalca. Sinteza zamreženih encimskih skupkov je potekala pri stalni volumski koncentraciji mrežnega povezovalca (glutaraldehida, GA) 1 % (v/v) in obarjalnega reagenta (metanola) 90 % (v/v).V našem primeru smo aktivnost CLEA iz α - amilaze določili z reakcijo hidrolize škroba topnega v vodi. Optimalni reakcijski pogoji so bili doseženi pri koncentraciji proteinov iz encimskega preparata α — amilaze: γα - amilaza = 6 mg/mL in pri koncentraciji inertnega proteina albumina (EA): γEA = 2,5 mg/mL. Reakcija hidrolize škroba je potekala 3 h. Temperaturno stabilnost zamreženih encimskih skupkov smo določili po 30 minutni izpostavitvi CLEA pri različnih temperaturah. Vpliv temperature na reakcijo hidrolize škroba imobilizirane z α — amilazo smo določili v temperaturnem razponu od 45 °C do 110 °C. Stabilnost proste α — amilaze in zamreženih encimskih skupkov iz encima α — amilaze smo preučili tudi v superkritičnem ogljikovem dioksidu. Testirali smo tudi vpliv večkratne uporabe zamreženih encimskih skupkov na njihovo stabilnost. This work describes the preparation of catalytically active enzyme aggregates of the enzyme α – amylase or briefly CLEA. The procedure to prepare cross-linked enzyme aggregates was devided into two major steps: precipitation of soluble or native enzyme with a suitable precipitant such as organic solvent and consequently cross-linking of enzyme aggregates with the cross-linker. Synthesis of cross-linked enzyme aggregates was conducted at a constant volume concentration of the cross-linker (glutaraldehyde, GA) 1 % (v/v) and precipitant (methanol) 90 % (v/v).In the present study, the activity of CLEA from α - amylase was determined by the reaction of hydrolysis of soluble starch. The optimal reaction conditions were found to be: concentration of proteins from α – amylase enzyme preparation γα - amylase = 6 mg/mL and concentration of protein albumin (EA) γEA = 2.5 mg/mL. The hydrolysis of starch was carrier out for 3 h. Temperature stability of cross-linked enzyme aggregates was determined after exposure of the immobilized enzyme at different temperatures for 30 min. Effect of temperature on the reaction of hydrolysis of starch with immobilized α - amylase was determined in the temperature range from 45 °C to 110 °C. The stability of free α – amylase and cross – linked enzyme aggregates was finally tested in supercritical carbon dioxide. We also tested the reusability of cross – linked enzyme aggregates in consecutive cycle of hydrolysis of starch. imobilizacija α – amilaza zamreženi encimski skupki CLEA glutaraldehid immobilization α – amylase cross-linked enzyme aggregates CLEA glutaraldehyde true true false [N. Čurič] Slovenski jezik Angleški jezik Diplomsko delo 2010-05-11 18:16:31 2010-05-25 11:18:39 2022-04-12 11:36:10 2023-12-17 03:07:01 2010 0 Maribor 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 547.914+577.17(043.2) 14147094 URN:SI:UM:DK:OJ4XEWSQ UNI_Curic_Natalija_2010.pdf UNI_Curic_Natalija_2010.pdf 1 BE989ADAE3910730F6B24527174C65F1 193296e6683515fe588c363e13a17617173d7e4a8d14ab4c83cfc134c03140a6 077e1c77-7c04-11eb-bb7a-00155d0001ca 20.500.12556/dkum/177a2ca0-ea0b-4a7b-a2e8-4c3d4b7d6cbf https://dk.um.si/Dokument.php?lang=slv&id=14488 Fakulteta za kemijo in kemijsko tehnologijo 0 0 0