Primena agro-industrijskog otpada za dobijanje enzima destransaharaze i proizvodnja dekstrana i ologosaharida pomoću imobilisanih sistema

Predmet istraživanja u okviru ove doktorske disertacije je optimizacija proizvodnje enzima dekstransaharaze, dobijene iz novog soja Leuconostoc mesenteroides T3, na komercijalnoj podlozi i na otpadnom supstratu, kao i imobilizacija ovog enzima na nanočestice TiO2 i unutar agar-agar filmova u cilju proizvodnje dekstrana i glukooligosaharida (GLOS). Novi soj Lc. mesenteroides T3, izolovan iz zrna vodenog kefira korišćen je kao mikroorganizam za proizvodnju i izolaciju dekstransaharaze. Optimalna podloga za maksimalnu proizvodnju enzima dobijena je variranjem uslova gajenja (kao što su temperatura i aeracija) i komponenata podloge (koncentracija saharoze i izvora azota). Dekstransaharaza dobijena u optimalnoj podlozi je zatim izolovana i prečišćena taloženjem uz pomoć polietilen glikola 400 (PEG). Karakterizacijom delimično prečišćene dekstransaharaze utvrđeno je da pokazuje najveću aktivnost na 30 0C pri pH vrednosti 5,4 i da dodatak jona Mn2+ ima najveći pozitivan uticaj na povećanje aktivnosti od čak 73 %. Zimogramom je pokazano da dekstransahraza ima molekulsku masu od oko 180 kDa. Karakterizacija proizvoda reakcije katalizovane dekstransaharazom iz radnog mikroorganizma, kada u reakcionoj smeši nisu bili prisutni akceptorski molekuli, izvršena je FTIR spektroskopijom. Potvrđeno je da je molekul glukana koji je sintetisan dekstransaharazom iz Lc. mesenteroides T3 dekstran u kome su molekuli glukoze dominantno povezani α-(1→6) glikozidnim vezama. Za proizvodnju dekstransaharaze na otpadnom supstratu, izluženi repin rezanac (SBP, od engl. sugar beet pulp), veličina zrna 500 μm‒800 μm, korišćen je kao nosač za imobilizaciju ćelija Lc. mesenteroides T3 dok je melasa bila izvor ugljenika, vitamina i mineralnih materija. Ispitivanjem uticaja ne-tretiranog (SBP-N) i izluženog repinog rezanca nakon tretmana sa NaOH (SBP-NaOH) na proizvodnju dekstransaharaze, bolji se pokazao repin rezanac nakon alkalnog pretretmana. Optimalan sastav podloge za maksimalnu proizvodnju dekstr
The scope of this doctoral dissertation is the optimization of the production of dextransaccharase (DS) enzyme, obtained from a new strain Leuconostoc mesenteroides T3, using a commercial medium and a waste substrate, as well as the immobilization of this enzyme on TiO2 nanoparticles and within agar-agar and application of these imobilised systems for the production of dextran and glucooligosaccharides (GLOS). A new strain Lc. mesenteroides T3, isolated from water kefir grains, was used as a microorganism for production and isolation of enzyme dextransucrase. The optimal medium for maximum enzyme production was defined by varying the growth conditions (such as temperature and aeration) and the broth/medium components (sucrose concentration and nitrogen source). The enzyme, purified by polyethylene glycol 400 (PEG) fractionation, displayed the maximum activity at 30 0C and pH 5.4. The addition of Mn2+ cation caused a signficant change of DS activity, with 73% increase. Zymogram analysis showed the presence of DS of approximately 180 kDa. Characterization of the dextransucrase catalyzed reaction product, when acceptor molecules were absesent from the reaction mixture, was performed by FTIR spectroscopy. The results indicated that a glucan molecule synthesized by dextransucrase from Lc. mesenteroides T3 was dextran in which glucose molecules were dominantly linked by α- (1 → 6) glycosidic bonds. For the dextransaccharase production on waste substrated, sugar beet pulp (SBP) of grain size 500 μm ‒ 800 μm, was used as a carrier in order to immobilize Lc. mesenteroides T3 cells, while molasses was a source of carbon, vitamins, and minerals. The influence of SBP in native form and after treatment with NaOH (SBP-NaOH) on dextransucrase production was investigated. The optimal substrate composition for maximum dextransucrase production was determined by varying the concentration of molasses, SBP-NaOH and sucrose. The maximum enzyme activity of 2,02 IU/ml was obtained on the