An Exo-inulinase From Aspergillus Ficuum SK004 And HFCS Production By Its Hydrolysis On Inulin

Inulin has been defined as a polydisperse carbohydrate material consisting mainly of β-2,1fructosyl-fructose links and end in a glucose unit. Inulin is the main carbohydrate in a variety ofplants. Jerusalem Artichoke contains 70%⁸0% inulin (dry weight). Because of its ease ofcultivation and harvesting, Jerusalem Artichoke has become the principal source of producinghigh fructose syrup and oligofructose today. Exo-inulinase is 2,1-β-D-fructan fructanohydrolaseswhich convert inulin to fructose by hydrolase the β-2,1 linkage in its unreducing end. Because ofthe hygienical character of high fructose syrup, it is very important to screen outinuinase-producing strain with high enzyme activity in industry.A strain of Aspergillus ficuum SK004, with a ratio of inuluinase activity to invertase activity(I /S ratio) of 0.43, was stored in our laboratory. The effects of nutrients and other culturalconditions on the exo-inulinase was studied. The optimal medium compositions and culturalconditions were (g /L): inulin 25 , peptone 25, NaCl 5, NH4H2PO4 4, MgSO4·7H2O 0.1,ZnSO4·7H2O 0.1, pH 6.5, temperature 30℃, shaken at 200 rpm with 30 ml medium per flask(250 mL) for 7 d. The maximum enzyme activity was 56.7 U /mL.The crude inulinase preparation was purified in a sequence of operations includingdecoloration by active carbon, ammonium sulfate fractionation, dialysis, ion exchangechromatography on DEAE-Sepharose CL-6B and column chromatography with Sephadex G-75.One exo-inulinase was obtained. The single band on SDS-PAGE showed that the emzyme washighly purified and its molecular weight was 53665Da.Properties of the purified exo-inulinase were also studied. The optimal reaction temperatureand pH of exo-inulinase were 60℃ and 4.5 respectively. The enzyme was stable within pH4.0⁶.0 and below the temperature 50℃. With inulin as substrate, Km =115.8 mg /mL,Vmax=7.80 mg /ml·min;with sucrose as substrate, Km=60.06 mg / mL,Vmax=7.69 mg /ml·min. Themetal ions (2 mM), with inulin as substrate, the effects on exo-inulinase activity of Urea(10 mM),K+, Al³⁺ were negligible, whereas Mn²⁺, Li⁺ and Fe²⁺ exhibited positive effects on exo-inulinaseactivity;Zn²⁺, Mg²⁺, EDTA, Ca²⁺, Cu²⁺ and SDS(40 mM) inhibited activity to different degree.With sucrose as substrate, Al3+ had slight positive effects on inulinase activity;other metals allhad negative effects.Effects of temperature, pH, enzyme and substrate concentration on the inulin-hydrolyzingdegree were determined. When hydrolyzed in system of 2% inulin solution added crude inulinaseof 25 U/g inulin at pH 4.0, 55℃ for 4 h, the inulin-hydrolyzing degree was 88% ,if hydrolyzedfor 10h, the degree up to 96%. After 10 h, the hydrolyzing degree increased slowly and the totalreducing sugar yield can get to 97.55% at 30h.A technological line was proposed according to the hydrolysis research. Over 90% fructosesyrup could be obtained by this line.