Effect of the Degree of Polymerization of Inulin on the Rate of Hydrolysis Using Immobilized Inulinase

U ovom su radu istražene dvije glavne značajke važne za razvoj industrijske proizvodnje fruktoze enzimskom hidrolizom inulina: uporaba imobiliziranog biokatalizatora za hidrolizu sirovog ekstrakta korijena cikorije, te procjena utjecaja stupnja polimerizacije inulina na ukupnu brzinu reakcije. Kao imobilizirani biokatalizator upotrijebljena je inulinaza kovalentno vezana na podlogu Sepabeads®. Aktivnost je katalizatora bila znatno veća na sirovom ekstraktu inulina (realni uzorak) nego na čistom inulinu (sintetska otopina). Eksperimentalno je dokazano da je pri praktičnoj primjeni stvarnog supstrata aktivnost imobiliziranog enzima bila čak 63 % aktivnosti ukupnog slobodnog enzima u homogenoj otopini, što može biti poticaj za industrijsku primjenu ovog imobiliziranog enzima. Stoga je ispitan utjecaj čistog i sirovog supstrata na kinetiku reakcije katalizirane imobiliziranim enzimom, te je utvrđeno da se ovisnost brzine reakcije o koncentraciji supstrata, tj. čistog (velike molekularne mase) i sirovog (male molekularne mase) inulina, može opisati Michaelis-Mentenovom jednadžbom. Zanimljivi su podaci dobiveni usporedbom vrijednosti Km i vmax. Povećanjem stupnja polimerizacije supstrata smanjila se vrijednost vmax, a povećala vrijednost Km. Nakon procjene koeficijenta prijenosa tvari zaključeno je da je uzrok tome različit stupanj polimerizacije inulina, jer je afinitet enzima bio veći prema inulinu manje molekularne mase.The present paper addresses two crucial features in the industrial development of fructose production by enzymatic hydrolysis of inulin: the use of immobilized biocatalyst in the hydrolysis of crude extracts of chicory roots and the evaluation of the effect of degree of polymerization of inulin on the overall reaction rate. The immobilized biocatalyst consisted of inulinase covalently bound to Sepabeads® supports. It was demonstrated that its catalytic activity towards crude inulin extract (real substrate) was much higher than that exhibited towards pure inulin (synthetic solution). Experiments revealed that, in applications of practical interest with real substrate, the activity of immobilized enzyme was as high as 63 % of that of free enzyme in homogeneous solution. This certainly was a driving force to potential industrial application of this immobilized enzyme preparation. Therefore, the effect of pure and crude substrates on the kinetics of the reaction catalysed by the immobilized enzyme was investigated. The kinetic analysis revealed a Michaelis-Menten dependence of the reaction rate on substrate concentration for both pure (high molecular mass) and crude (low molecular mass) inulin. Interesting results were derived from the comparison of Km and vmax values in the two cases. In particular, it was found that increasing degree of polymerization of the substrate caused vmax decrease and Km increase. After evaluation of mass transport effects, this was mainly associated with a different substrate/ enzyme affinity when exploiting inulin characterized by different (low or high) degree of polymerization