Studies On The Screening Of Filamentous Fungi And The Production Of Inulinase

Inulinase is a hydrolase for natural Fructans, and many microbes can produce inulinase. Inulinase can catalyze the hydrolysis of the inulin to fructose or fructooligosaccharide. Therefore, it has a great potential in industrial production.From the soil around Jerusalem artichoke roots, 46 strains of filamentous fungi were isolated, and 3 of them with higher enzymatic activities were obtained after further screening. Strain A24 that had the highest enzymatic activities was selected for further research. By morphological methods strain A24 was identified as Aspergillus niger. After both single factor and multi-factor orthogonal design test, the optimal conditions for Inulinase enzyme activity in strain A24 were found: 3% Jerusalem artichoke juice, 0.5% beef extract, 2%(NH₄)₂ 2HPO₄, 1% CaCO₃, initial pH 6.5, flask shaking at 160r/min at 28℃.We studied the fermentative dynamics of inulinase by flask shaking under the optimal condition mentioned above. The results demonstrated that when Aspergillus niger entered into stationary phase (about 48 to 120 hours), and the reductive sugars dropped to the range from 0.11% to 0.32%, and the pH of culture below 5.0, the synthesis of enzyme was exuberant. At the end of stationary phase, the highest yield of enzyme was obtained with the enzyme activity 30.2U/ml.Regulation on Inulinase metabolism in Aspergillus niger A24 showed that inulinase was an inducible enzyme, with Inulin being the inducing factor. Glucose and relatively high-concentration fructose exercised less influence on the induction, though they contributed to the growth of Aspergillus niger. Reductive single sugar such as glucose and fructose could hinder the producing of inulinase. The possible metabolism might be the hindrance of catabolism.The optimal temperature for inulinase's hydrolytic activity was 60℃, andinulinase could be stable at or below 60 "C.The reaction rate varied under different pH. The optimal pH was 5.0. From pH 3.6 to 7.0, the enzyme was stable and maintained high enzyme activity.When inulinase was used to enzymolyze inulin and Jerusalem artichoke juice, (under the conditions: substrate concentration 10%, inulinase 3U/g, temperature 50'C, pH5.0 reaction time 14h), the substrates were nearly all hydrolyzed. (inulin and Jerusalem artichoke juice 90.3% and 94.2% respectively). When inulinase was used to produce high fructose syrups, the optimal conditions were: temperature 50'C, substrate concentration 10%²0%, enzyme 9U/g Jerusalem artichoke juice and reaction time 8 10 hours.The thin-layer chromatography showed that the major resultants from enzymatic hydrolysis were fructose with some fructooligosaccharide. The inulinase activity and glucose hydrolytic activity of inulin was 30U/ml and 70.5U/ml respectively, and its VS ratio was 0.426. The resultant analysis showed that the enzyme mixture mainly consisted of exterior contact enzyme with a little interior contact enzyme.