Novi aspekti upotrebe hidroksiapatita kao nosača za imobilizaciju industrijski značajnih lipaza i glikozidaza - dizajn, ispitivanje mehanizama vezivanja, stabilnost, primena i značaj

Abstract

U ovoj doktorskoj disertaciji je ispitan potencijal HAP za imobilizaciju industrijski značajnih enzima na primeru 4 lipaze i 4 glikozidaze različitog porekla, strukture i supstratne specifičnosti. Od testiranih enzima za HAP se najefikasnije vezuju lipaza iz Candida rugosa (CRL) i β-galaktozidaza (laktaza) iz Aspergillus oryzae (AOL). Oba enzima se imobilizuju na HAP jednostavnom, brzom i efikasnom fizičkom adsorpcijom po mehanizmu metal-koordinatne veze. Kombinovanjem bioinformatičkog pristupa i molekulskog modelovanja za oba enzima su identifikovani i opisani površinski HAP-vezujući motivi.Imobilizacija na HAP je uzrokovala promenu supstratne specifičnosti CRL ka supstratima sa kraćim alkil-nizom i drastično povećanje stabilnosti i aktivnosti enzima u metanolu. Ove karakteristike imobilizata CRL na HAP (CRL-HAP) su prvi put u literaturi primenjene za sintezu metil-acetata, estra sa mirisom jabuke, koga slobodna CRL sintetiše u zanemarljivom prinosu. Pažljivim odabirom rastvarača i supstrata je, takođe po prvi put pokazano kako ovaj popularni i jeftin enzim, u slobodnom ili imobilizovanom obliku, može da se primeni i za efikasnu sintezu kapsinoida direktnom esterifikacijom vanilil-alkohola (VA) i masnih kiselina (MK) i transesterifikacijom kokosovog ulja.Efikasnost imobilizovanog preparata AOL (AOL-HAP) je ispitana u sintezi bioaktivnih supstanci, vanilil-galaktozida (VG) i galakto-oligosaharida (GOS). Po efikasnosti u sintezi ovih proizvoda preparat AOL-HAP se našao u rangu sa kovalentno imobilizovanim preparatima AOL. Jedna ista šarža AOL-HAP može se iskoristiti u 10 uzastopnih reakcionih ciklusa sinteze VG, odnosno GOS, sa poluživotom od 15 h.

Potential of hydroxyapatite (HAP) as immobilization carrier for industrially important enzymes was examined in a study, comprising of 4 lipases and 4 glycosidases of different origin, structure and substrate specificity. Lipase from Candida rugosa (CRL) and β-galactosidase (lactase) from Aspergillus oryzae (AOL) exhibited the most efficient binding to HAP. Both of the enzymes were immobilized on HAP by simple, fast and efficient physical adsorption through formation of metal-coordinative bond. Furthermore, by the combining of bioinformatic approach and molecular modeling, HAP-speciffic superficial motif on both selected enzymes was identified and described for the first time in the literature.Immobilization on HAP has shifted substrate specificity of CRL towards shorter alkyl-chains, coupled with significant increase in stability and activity of CRL in methanol. These characteristics of immobilized Candida rugosa lipase preparation (CRL-HAP) were sucessfully implemented in synthesis of methyl-acetate, an apple flavour for the first time in the literature. Free CRL synthesized methyl-acetate in very low yield, because of methanol-induced inactivation. By a meticulous selection of organic solvent and susbtrates, it was shown also for the first time that this popular and cost-effective microbial lipase, free or immobilized on HAP, can be used for efficient synthesis of capsinoids in two distinctive reactions: direct esterification of vanillyl-alcohol (VA) with free fatty acids and transesterification of coconut oil.Efficiency of AOL immobilized on HAP (AOL-HAP), was evaluated in synthesis of bioactive compounds: vanillyl-galactoside (VG) and galacto-oligosaccharide (GOS) probiotics. Efficiency of AOL-HAP in synthesis of these compounds was in a good correlation with the procedures in which covalently immobilized AOL was employed. Immobilized AOL preparation can be used in maximum 10 consecutive reaction cycles during VG and GOS synthesis, with the half-life of 15 hours.