Overexpression Of The Endo-inulinase And Its Characteristics Research

Endo-inulinase,a enzyme which can hydrolyze the internal ?-2,1 fructofuranosidic linkages of inulin to inulooligosaccharide(IOS)as the main products specificity.The IOS has low calorific,can regulate blood lipid,anti-cancer effect,improve on human immunity,and many other regulative functions and health functions.The inulinases almost derived from fungi,but it is more complex of the fermentation enzymes from fungi,at the same time,with low content of inulinase and the extracellular enzyme mostly exo-inulinase,using inulin as substrate to product IOS was restricted.This study aims to screen endo-inulinase gene from the fungi microorganisms and overexpress the coding gene in Pichia pastoris to study its application in IOS production.The main research results were as follows.We queried the database of beta-D-fructofuranosidase gene from NCBI.by reconstruction of a phylogenetic tree to analyze these enzymes and available researchs.We choose the enzymes from Aspergillus niger and Penicillium as the research objects.The endo-inulinase gene inuA was amplified from the total DNA of A.niger(TCCC 41064)by PCR and subsequently cloned into pPIC9K.Sequencing results showed that the homology with A.niger CBS 513.88 was 97.68%.The recombinant plasmid was linearized by SacI and transformed into P.pastoris GS115 by electroporation,yielding the recombinant strain P.pastoris GS115/pPIC9K-inuA.Through shaking flask fermentation,the enzyme activity of culture supernatant reached to 7.81±0.23 U/mL.InuA was purified to homogeneity with the recycle rate was 9.78 and 55.5%.Its optimal pH and optimum temperature of the recombinant enzyme was 4.0 and 50?,moreover 83.2%of the original enzymatic activity remained after the incubation of recombinant enzyme at 55? for 9 hours.It showed that Cu2+?Ag+ strongly inhibited the recombinant enzyme activity while Ca2+ activated the enzyme.The hydrolytic process analyses showed that the recombinant endo-inulinase can hydrolyze 4%(w/v)inulin into DP3-6 of IOS in 8 hours.The endo-inulinase gene from Penicillium was optimized according to the usage of P.pastoris and synthesized.Two recombinant strains were constructed as pPIC9K-inuC?pPIC9K-inuC(P)with one contains its natural signal peptide.They were both transformed into P.pastoris GS115 by electroporation.Finally it showed that the enzymes activity were 11.23±0.79 U/mL?69.3± 1.17 U/mL respectively.The InuC was purified to homogeneity with the recycle rate was 4.61 and 43.5%with the enzyme activity 792.09 U/mg.Its optimal pH and optimum temperature of the recombinant enzyme was 5.0 and 60? respectively,75.4%of the original enzymatic activity remained after the incubation at 40? for 9 hours.The pH stability was 3.0?6.0.It was shown that Cu2+strongly inhibited the recombinant enzyme activity,too.The hydrolytic process analyses showed that the recombinant InuC can hydrolyze inulin into the most DP2(74.8%)of IOS in 8 hours.High density fermentation was performed in 5 L fermentor,and the recombinant enzyme activity reached 672 U/mL.On the IOS production application,InuC was choosed to study the hydrolytic process of inulin.Hydrolyzing inulin at 40??pH 4.0 showed that with 20 U/g enzyme added in ten percent concentration of inulin,81.4%of IOS gained in 9 hours;at the same time,we added 10 U/g enzyme to hydrolyze five percent of Jerusalem artichoke,yielding 20.21 g/L of IOS after 20 hours reaction.