Studies On Inulinase Production By Aspergillus Usamii And Enzyme Properties

The fuel ethanol is becoming the research focus today. However, due to the high price, fuel ethanol has no competition with the mineral fuel at present, so reducing the production cost is the key of the fuel ethanol research. Jerusalem artichoke is a new alternative to grain crop which can reduce the production cost of the fuel ethanol research. Jerusalem Artichoke mainly contains inulin that is composed of linear chains of D-fructose units which can be easily hydrolyzed by microbial inulinase into fructose. It is very important to screen out inulinase-producing strain with high enzyme activity in researching fuel ethanol production.A strain of aspergillus usamii with the highest enzymatic activities was obtained from five strains after further screening. Aspergillus usamii was selected for further reseach. The main results were as follows.1.The effects of nutrients and other cultural conditionas on inulinase activity from Aspergillus usamii was studies. To optimize the optimal conditions, experiment design employed single factor, Plackett-Burman design(PB) and response surface methodology(RSM). Three factors, Jerusalem artichoke juice, Peptone and (NH₄)₂HPO₄ significantly improving the inulinase activities, were screened for further investigation. By response surface methodology (RSM), a mathematical model was developed to show the significant effect of each factor and their interactions on the inulinase activities, and the optimum media composition for the inulinase activities by Aspergillus usamii in shaking flask culture was (g/L): Jerusalem artichoke juice117.28, Peptone22.14, (NH₄)₂HPO₄ 3.94, NaCl 5.0, MgSO₄ 0.50, MnSO₄ 0.10, initial pH 6.5, 50mL at 28℃. Under above conditions, the highest inulinase activities by Aspergillus usamii was achieved. 2. Properties of the crude inulinase were also studied. The optimal reaction temperature and pH of inulinase were 60℃and 6.0 respectively. Within pH3.0-7.0 and at 40-60℃, the enzyme was stable and maintained high enzyme. With inulin as substrate, Km=83.77mg/mL, Vmax=5.52mg/mL·min.The metal ions have different effects on the inulinase activity. The effects on inulinase of Cu²⁺ was negligible, whereas Na⁺ and Mn²⁺ exhibited positive effects on the inulinase activity; Fe²⁺ and Mg²⁺ inhibited activity to different degree.3 The crude inulinase preparation was purified in a sequence of operation including decoloration by ammonium sulfate fractionation, dialysis, ion exchange chromatography on DEAE-cellulose and column chromatography with Sephadex G-100. On SDS-PAGE the purified enzyme has five protein bands. The first protein band had not the enzyme activity, so it was not inuliase. Other protein bands had the enzyme activities. The result was showed that they might be inulinase isozymes.4. The reducing sugar's production from Jerusalem Artichoke by Aspergillus usamii was studied. After both single factor and multi-factor orthogonal design test, the optimal conditions were found: 110% Jerusalem artichoke juice, 0.3% KH₂PO₄ 0.4% MgSO₄.