Production, Purification, Characterization And Application Of Inulinase From A Novel Species Of P. Expansum SK16Grown On Dahlia Spoiled Tuber

Dahlia pinnata belongs to the family asteraceae and is a perennial growing to1m. It is inflower from June to October. The root is rich in the starch inulin. Whilst not absorbed bybody, this starch can be converted into fructose a sweetening substance suitable fordiabetes to use.The production of fructose has been increasing steadily in recent years due to itssweetness, favorable properties and the drastic increase in sugar prices. Fructose iscommercially produced from corn starch into glucose followed by isomerization ofglucose to fructose. This process produces syrups containing about42%fructose. Thefructose concentration in the syrup can be enhanced (to55%and90%High FructoseCorn Syrups, HFCS) by selective removal of glucose or by applying multistagechromatographic separation methods. These techniques are costly, which reflect on theprice of the product thus limiting its use as a common sweetener. A potentially promisingprocess for fructose production is based on hydrolyzing the fructan-rich plants to fructose.Microorganisms are the best sources for inulinases production, as they are easy to becultivated and produce high yields of enzymes. Meanwhile, the study of useful microbialand their biocatalytic applications is crucial to future developments in microbialbiotechnology.Inulinase offers interesting perspectives in view of the growing need for the production ofpure fructose syrups and may present an alternative way to produce the so-called UltraHigh Fructose-Glucose syrups not from starch, but from inulin.Inulinases have a molecular weight over50.0kDa, an optimal pH between4.5-7.0and anoptimal temperature between30°C and60°C, depending of their producers, moulds,bacteria or yeasts. A potentially promising process for fructose production is based on hydrolyzing thefructan-rich plants to fructose. The main purpose of this study was screening andisolation of the new strain with the purpose to produce FOS or FRUCTOSE.According to its morphological and biochemical characteristics, as well18S rRNA, it wasidentified as Penicillium expansum and deposit at China Center for Type CultureCollection (CCTCC) under accession number GU561988. Furthermore, the optimalconditions such as inoculum size, pH, Temperature, carbon sources, nitrogen sources onthe formation of inulinase were investigated, and inulin was used as unique sole carbon.In addition the enzyme was purified to electrophoresis homogeneity by a combination ofammonium sulphate precipitation, DEAE Fast Flow column and superdex200column.The optimization for inulinase production by Penicillium expansum SK16was achievedusing one-factor-at-a-time method. Results showed that the maximum yield of inulinasewas noticed when the inulin (4%) was used as carbon source and peptone from fish (0.7%)as nitrogen source. In addition, adding5%of inoculum to medium (pH5.0) andfermenting it for4days at28°C showed a high inulinase production.Inulinase activity varies markedly according to the composition or type of buffer, withsodium acetate buffer giving the best results.The pH profile of inulinase displayed an optimum pH around5.0. Concerning the pHstability, inulinase activity was found to be quite stable for2h in pH value ranging from4.5to6.5at room temperature. The temperature profile of inulinase displayed anoptimum temperature around50°C. In terms of thermal stability, inulinase activityremained stable after2h of exposure at50°C, with no significant loss of its initialactivity. Furthermore, inulinase revealed itself to be among the most thermostable. In fact,after2hours of heat exposure, only20%of inulinase activity was lost. These propertiesare favorable in view of large-scale inulinase application for pure fructose production. In fact, high operation temperatures would avoid microbial contamination of reactors andwould allow the use of high inulin substrate concentration, a factor which is limiting inobtaining high conversion ratios. In addition, the remarkably low pH optimum preventscolour formation and undesirable chemical side reactions.By using High Performance Liquid Chromatography (HPLC), the hydrolysis products ofinulin were analyzed. Fructose was observed as the main product of inulin afterhydrolysis. This result suggests that inulinase from decomposed Dahlia tubers extract wasan exo-type inulinase.Inulinase was found to be a homogeneous enzyme by SDS-PAGE, the molecular weightof the purified inulinase were estimated to be (approximately66kDa). Nevertheless, theP. expansum SK16strain is another kind of mould capable of producing inulinase.