Study On Immobilized Fructan Exohydrolases From Jerusalem Artichoke And Chicory For The Fructose Production

Jerusalem artichoke(Helianthus tuberosus L.)and chicory(Cichorium intybus L.)are typical plants which accumulate fructans in their tubers or roots.These fructans can not only provide nutrients for plant growth,but also play vital roles in resistant to salt and drought.1-Fructanexohydrolase(1-FEH)in plant plays a key role in the hydrolysis of fructan to fructose.With the improvement of people's living standard,fructose,as a kind of healthy food,has gradually entered people's life,especially in the field of food processing and medical treatrnent.Fructose is generally obtained by hydrolysis of sucrose in practise,while there are few studies on fructan hydrolysis by fructan exohydrolase.So in this paper,we cloned and expressed Ht1-FEH ? from Jerusalem artichoke,then compared the activities of six fructan exohydrolases from Jerusalem artichoke and chicory.The fructan exohydrolase owning the highest activity,chicory Cil-FEH ?A,was chosen to be immobilized.After immobilization,the fructan exohydrolase were used to hydrolyze inulin or tuber extract from Jerusalem artichoke to produce fructose.Fructose was further to be crystallizated.The main findings are as follows:1.Htl-FEH ? of Jerusalem artichoke was cloned and it had higher sequence simility with the prediction sequence in Jerusalem artichoke databases.The translated amino acid sequence of Ht1-FEH ? contained three conserved domains NDPNG,FRDP,WECPD,which are the symbols of FEH family.Ht1-FEH ? was then expressed in Pichia pastoris which displayed activity of 1-FEH enzyme.Even through the sequences of three 1-FEHs of chicory were previously known but their general properties of the enzyme were not clear.In this study we expressed and purified the enzyme of Cil-FEH I,Cil-FEH IIA and Cil-FEH ?B in Pichia pastoris also.Our results demonstrated that the optimal temperature of Ht1-FEH ? is 55? and the optimal pH is 5.5.The optimal temperature of Cil-FEH IIA was 40?,and the optimal pH was 5.The optimal temperature for Cil-FEH I and Cil-FEH IIB is 30? and the optimal pH is 6.As the substrate specificities,Htl-FEH ? showed the differences with typical fructan hydrolases.It could not only hydrolyze fructans with ?(2,1)and ?(2,6)bond,but also has a hydrolysis activity to sucrose.2.In the comparision of the activities of six fuctan exohydrolases from Jerusalem artichoke and chicory,Cil-FEH IIA showed the highest activity.We further took use of absorption-crosslinking method to immobilize this enzyme.The following are the optimization conditions of the immobilization process:the adsorption temperature was 30?;the adsorption pH was 4.5;the adsorption time was 3h;the concentration of glutaraldehyde was 0.125%;the crosslinking temperature was 10? and the crosslinking time was 2h respectively.Besides,our results identified that the optimal temperature of the immobilized Cil-FEH IIA was increased from 40? to 50?.In the process of hydrolyzing inulin and tuber extract of Jerusalem artichoke,it showed good hydrolysis ability.In the experiment of hydrolyzing the tuber extract of Jerusalem artichoke,the high degrees of polymerization of fructans could be converted to fructose in 48h.3.As the fructose is not easy to be transported or stored in liquid,we explored the conditions of fructose crystallization in this study.The optimal conditions for obtaining fructose crystallization were 1:10.The temperature gradient preferred 55? to 4? and the amount of seed crystal was 3%respectively.The tuber extract solution hydrolyzed by Cil-FEH IIA was first concentrated and the fructose in the solution was crystallized twice.The recovery of the fructose was reach to 21.51%.