Faculty of Food Technology and Biotechnology, University of Zagreb
Abstract
α-Pinene oxide lyase is able to catalyze the cleavage of both rings of α-pinene oxide to form cis-2-methyl-5-isopropylhexa-2,5-dienal (isonovalal) with no cofactor requirements. This bioconversion, when carried out under biphasic conditions (water/hexadecane) by Pseudomonas rhodesiae CIP 107491, allows the accumulation of a very high concentration of the product. Nevertheless, the reaction stopped due to the loss of enzyme activity, which has been demonstrated to be an irreversible phenomenon. The enzyme was purified by chromatographic methods to study the reasons of its instability. Bioconversion with pure enzyme showed that a protein precipitation occurred at the liquid-liquid interface, giving rise to a decrease in soluble protein and in residual enzymatic activity. The rate of decrease in soluble protein concentration was not related to the presence of the precursor, which indicated that the inactivation was interfacial, i.e. due to the direct contact of the enzyme with the hexadecane layer. This phenomenon was not present when crude enzymatic extract was used as a biocatalyst. Biomass components present in this extract thus had a protective effect against interfacial phenomena, demonstrating that α-pinene oxide lyase purification does not attract much practical interest for isonovalal production.Liaza α-pinen oksida katalizira cijepanje oba prstena α-pinen oksida bez prisutnosti kofaktora, pri čemu nastaje cis-2-metil-5-izopropilheksa-2,5-dienal (izonovalal). Provedbom takve biokonverzije na granici dviju faza (voda/heksadekan), s pomoću bakterije Pseudomonas rhodesiae CIP 107491, postignuto je nakupljanje velike koncentracije produkta. No, gubitkom aktivnosti enzima dolazi do ireverzibilnog prestanka reakcije. Stoga je enzim pročišćen kromatografijom i ispitani su uzroci njegove nestabilnosti. Utvrđeno je da biokonverzija pomoću pročišćenog enzima dovodi do taloženja proteina na granici faza, a time i smanjenja koncentracije topljivih proteina i enzimske aktivnosti. Smanjenje koncentracije topljivih proteina nije ovisilo o prisutnosti prekurzora, čime je dokazano da je izravni kontakt enzima s površinom heksadekana uzrokovao inaktivaciju međupovršine. Primjena sirovog ekstrakta enzima nije dovela do inaktivacije, pa je zaključeno da sastojci ekstrakta biomase imaju zaštitni učinak na međupovršinu, te da pročišćavanje liaze α-pinen oksida za proizvodnju izonovalala nema praktičnu primjenu
