Primena imobilizovanog ćelijskog zida kvasca Saccharomyces cerevisiae u proizvodnji invertnog šećera

Abstract

Zidna invertaza je izolovana iz ćelija kvasca Saccharomyces cerevisiae. Izolovan enzimski preparat je detaljno okarakterisan i dobijeni su pH optimum na 4,0, temperaturni optimum na 60°C, KM 30,93 mM, Vmax 11,64 mM/min i energija aktivacije od 38 kJ/mol. Stabilnost zidne invertaze detaljno je ispitana i upoređena sa stabilnošću rastvorne invertaze izolovane iz ćelija kvasca S. cerevisiae na povišenoj temperaturi i u prisustvu različitih koncentracija raznih enzimskih denaturanata i inhibitora. Dokazana je veća stabilnost zidne invertaze u odnosu na rastvornu. Zidna invertaza je uspešno imobilizovana u Ca–alginatu sa prinosom imobilizacije od 100%. pH optimum imobilizata je na 5,0, temperaturni optimum 50–80°C, KM 72 mM, Vmax 0,42 mM/min i energija aktivacije 25 kJ/mol. Imobilizovana zidna invertaza pokazala je značajno većutemperaturnu stabilnost u poređenju sa slobodnim enzimom. Osim dobre termalne stabilnosti pokazano je da imobilizat ima i dobru operativnu stabilnost, pa se njegova aktivnost nakon 40 ponavljajućih ciklusa rada u šaržnom reaktoru minimalno smanjila (8%). Zidna invertaza hemijski je modifikovana različitim modifikatorima. Pokazano je da hemijskom modifikacijom zidne invertaze ne dolazi do promena u pH stabilnosti i optimumu enzima, kao ni temperaturnom optimumu, ali da se hemijskom modifikacijom zidne invertaze dobija termalno stabilniji enzim u odnosu na nemodifikovan. Zidna invertaza modifikovana glutaraldehidom je uspešno imobilizovana u Ca–alginatu. Aktivnost imobilizata je bila 91 U/g. Stabilnost i produktivnost dobijenog imobilizata izmerene su korišćenjem visoko koncentrovanih rastvora saharoze u reaktoru sa napakovanim slojem tokom 30 dana kontinualnog rada reaktora. Ukupna produktivnost reaktora bila je 3844 kg invertnog šećera po kg imobilizata. Dobijeni proizvod (invertni šećer) je bio bez boje, imao je provodljivost od 16 μS/cm, pH 5,44, količinu suve mase 70,3°Bx i bez štetnih sastojaka.

Cell wall invertase (CWI) was isolated from yeast Saccharomyces cerevisiae. Isolated enzyme was characterized in detail; the optimum pH value was 4.0, temperature optimum 60°C, KM 30.93 mM, Vmax 11.64 mM/min and the activation energy was 38 kJ/mol. Stability of CWI was examined in detail and compared with the stability of soluble invertase isolated from yeast cells S. cerevisiae at high temperatures and in the presence of different concentrations of some enzyme inhibitors and denaturants. It was shown that cell wall invertase had better stability than soluble enzyme. Cell wall invertase was successfully immobilized in Ca–alginate beads, with immobilization yield of 100%. The pH optimum of immobilized CWI was 5.0; optimum temperature 50–80ºC, KM 72 mM, Vmax 0.42 mM/min and the activation energy was 25 kJ/mol. Obtained biocatalysts had enhanced thermal stability compared with the free enzyme. Immobilized CWI was tested in a batch reactor. After 40 consecutive cycles it retained more than 90% of its activity. CWI was modified with different chemically reactive substances. Chemically modified and native CWIs showed similar pH stability (pH 3–11), pH optimum and temperature optimum values, while modified CWIs showed higher thermostability than the native one. CWI modified with glutaraldehyde was successfully immobilized in Ca–alginate beads, with enzyme activity of 91 U/g. Stability and productivity of obtained biocatalyst were measured using the highly concentrated substrate solution in the packed bead reactor. One month productivity of 3844 kg of inverted sugar per kg of the immobilized biocatalyst was obtained. Produced invert sugar was colourless, pH neutral (5.44), with low conductivity (16 μS/cm), with 70.3°Bx dry weight and without unhealthy byproducts.