Preparation, Purification And Application Of Endoinulinase

Inulin is a natural fructopolysaccharide which was found in several plants such as Helianthus tuberosus. Inulinase, a multienzyme complex composed of endoinulinase and exoinulinase, cleaves theβ-2,1- linkage of inulin for preparation of fructo-oligosaccharides (FOS) and fructose. The dissertation investigated extraction of inulin from Helianthus tuberosus; preparation, purification and characterization of endoinulinase from Aspergillus niger, and application of endoinulinase in FOS preparation was also studied. The main results are as follows:The Helianthus tuberosus was extracted using hot water at the ratio of solid to liquid 1:25, 80℃for 35min, the concentration of total sugars in extractive was 38.1 g/L and the recovery of inulin was 83.89%.The optimal carbon and nitrogen sources for endoinulinase biosynthesis by Aspergillus niger submerged fermentation were Helianthus tuberosus and NH4Cl, respectively, and the optiml C/N and initial pH value were 30 and 5.0, respectively. The maximum endoinulinase activity 22.26U/ml was obtained under the optimal conditions.The homogeneity endoinulinase was purified from Aspergillus niger inulinase, the specific activity and the estimated molecular mass of endoinulinase were 12958U/mg and 64.2kDa, respectively. The optimal temperature and pH value of endoinulinase were 60 oC and pH 4.4, respectively. The endoinulinase kept stable at 30oC and lose majority activity when temperature was obove 60oC. The Km value of endoinulinase was 7.71mg/ml using inulin as substrate.Inulin was hydrolyzed by inulinase and endoinulinase at enzyme dosage 40U/g linulin, 55 oC and pH 4.5 for 4 h, the main products hydrolyzed by inuilinase were fructose and difructose, the fraction of difructose was 27.70 %; and the main products hydrolyzed by endoinuilinase were fructose and FOS, the fraction of FOS was 56.72%.The endoinulinase could be used for FOS preparation by selective hydrolysis of inulin, inulin was hydrolyzed by endoinulinase at enzyme dosage 40U/g inulin, 55 oC, pH4.5 for 24h, the hydrolysis yield of inulin and the yield of FOS were 86.94% and 52.29%, respectively. The concentrations of fructose and FOS in the hydrolysate were 5.23 and 3.70 g/L, respectively, and the ratio of FOS to total sugars in hydrolysate was 57.91%.