Production, Purification, Characterization, And Application Of An Endoinulinase From Penicillium Purpurogenum Stoll To Produce Fructooligosaccharides Via Inulin Endo-Hydrolysis

The aim of this work was to produce a selective endoinulinase, free from invertase or exoinulinase activity through microbial fermentation process.Cultured in media of various combinations of different sources of sugar, nitrogen, salt and metal ions, Penicillium purpurogenum Stoll BCRC31690 released an extra-cellular endoinulinase in the culture medium.DNS, HPLC and The inulinase to invertase activity ratio (I/ S) were used for the detection and characterization of the reducing sugars and fructo-oligosaccharides released from the reaction of the enzyme with inulin and sucrose as substrates.The three-level full factorial design of experiments was used for the optimization of endoinulinase production and statistical treatment of the results was done using the SAS Software.Purification of the produced enzyme was done using methods of ammonium sulfate fractionation, DEAE- Sepharose CL-6B ion exchange chromatography, and Sepharose CL-6B gel filtration. The purified endoinulinase molecular weight was estimated by using SDS PAGE gel electrophoresis.The results showed that optimized conditions for the highest endoinulinase production were achieved in the culture mixture composition of Inulin (20g/l), Ammonium sulfate (5g/l), Potassium phosphate, monobasic (2.5g/l) and magnesium sulfate heptahydrate (0.5g/l); fermentation time, pH and temperature being respectively 3days, 5.5 and 24℃. The highest inulinase to invertase activity ratio I/S was 21.8. The optimized inulinase activity was 2.65U/ml. Its optimum pH and temperature were respectively 5 and 55°C. By SDS PAGE, this enzyme was revealed to have a molecular weight of 62.88kDa. Analyzis by HPLC of reaction products between this enzyme and inuline as substrate showed no sugar monomers but products with degrees of polymerization varying from 2 to 13, namely called fructooligosaccharides.