| Inulin widely exists in root of many plants such as Jerusalem artichoke,chicory,dahlia,and so on.With many good properties such as cold tolerance,drought resistance,rapid propagating and resistance to salt-alkaline land,Jerusalem artichoke is widely grown in various areas in our country even in the desert and the saline and alkaline land.Inulin is accounted for 40%?70%of the Jerusalem artichoke tubers dry weight.As a kind of water soluble dietary fiber,fructooligosaccharides(FOS)is a bifidobacterium factor meets the prebiotics standard and its function of health caring was researched in detail.In the near decade,FOS is becoming more and more popular in the international food ingredient market because of its good properties such as difficult to be digested and low calorific value,improve lipid metabolism,regulating intestinal microbial flora,promoting mineral absorption.The method of produce FOS from inulin with Endo-inulinase is easy to be carried out,and with less other carbohydrate in the production.This process has tremendous application potential in industrial production.In this study,tag-fusion technology was applied to add a sequence encoding acidic/basic amino acid tag on the inu2 gene.The recombined Endo-inulinase INU2 were successfully expressed and the inulinase activities of recombined and no-labelized INU2 were compared after flask fermentation.The activity of INU2 labelized with acidic amino acid tag was better than the INU2 recombined with basic amino acid tag.The INU2 fused with 12 acidic amino acid sharing the similar activity with original INU2,suggesting that this strategy was feasible in transforming the INU2.This study constructed an engineered yeast strain with co-expressing INU2 boxes which were tagged,detected the enzyme activity of INU2 by shake-flask fermenting,and compared the enzyme activity of pPIC9K-INU2aa.When cultured in shake-flask,the microbe amount of the strain with two copies decreased nearly one quarter compared to the one with one copy.Meanwhile,compared to the one-copy strain,enzyme activity of INU2-6A had some increase,the strain including INU2-6B and INU2-12 had the same enzyme activity,and the strain with two INU2-12B copies had an 14.34%decrease.This study also immobilizated the tagged Endo-inulinase and wild Endo-inulinase with anion exchange resin and cation exchange resin,respectively.It was found that alkaline tag could enhance the immobilization efficiency of cation exchange resin.However,the efficiency was quite low with a value under 20%.When immobilized with amino resin,the enzyme activity of the non-tagged Endo-inulinase could reach to 115.23 U/g,with the immobilization efficiency reaching to 79.17%.This study determined the optimal conditions for Endo-inulinase to hydrolyze inulin into FOS as follow,70U/g for concentration of the free enzyme,5 h for the reaction time,EA resin treated by 2%as the braces.And the one immobilized with glucose oxidase had the highest enzyme activity,up to 52.78U/g,which remained 41.1U/g when used for ten times.This study has compared the products when different Endo-inulinase reacted with inulin.Meanwhile,several methods for remove sugars have been compared to verify the feasibility for combined-utilizing of glucose isomerase and glucose oxidase used for removing sugars. |
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