Over-expression Of Endo-inulinase And Invertase Gene And Their Application In Inulin Hydrolysis

Inulinases are fructofuranosyl hydrolases that target on the β-2,1linkage ofinulin and hydrolyze it into fructose and glucose. They can be divided intoexo-inulinase and endo-inulinase. The exo-inulinase catalyzes the removal of theterminal fructose residues from the non-reducing end of the inulin molecule while theendo-inulinase hydrolyzes the internal linkages in inulin to yield inulotriose,inulotetraose, and inulopentaose. Invertase is the beta-fructofuranosidase thatcatalyzes the irreversible hydrolysis of sucrose into glucose and frucose. It is widelypresent in bacteria, yeasts, fungi, higher plants and in some animal cells. Especially,invertase from Saccharomyces cerevisiae has been intensively investigated andapplied to food and fermentation industries. The SUC2gene encodes the maininvertase activity.In the study, the endo-inulinase gene (EnIA) from Arthrobacter sp. S37wasligated into the expression vector pINA1317and over-expressed in Yarrowialipolytica Po1h. It was found that the endo-inulinase activity and specificendo-inulinase activity produced by the transformant1317-EnIA were16.7U/mL and93.4U/mg, respectively. The recombinant EnIA was purified and characterized. Themolecular weight of the purified rEnIA was78.9kDa. The optimal pH andtemperature of the purified rEnIA were4.0and50℃, respectively. The purifiedrEnIA was stable in the temperature range of4-40℃and in the pH range of2.0-8.0.The activity of rEnIA was greatly stimulated in the presence of Li+. The purifiedrEnIA could actively convert inulin into disaccharides. Moreover, the mixture ofrEnIA and exo-inulinase has a higher inulinase activity than the sum of rEnIA andexo-inulinase activity. It suggested that expressing both the endo-inulinase andexo-inulinase gene in the same host would get the recombinants with higher inulinaseactivity. The high ethanol producing yeast Saccharomyces sp. strain W0, which ispreserved in our lab could ferment inulin and produce low concentration of ethanolfrom inulin hydrolysate. After the endo-inulinase gene from Arthrobacter sp. S37wasligated the expression vectors pMIDSC31and pMIRSC31, the endo-inulinase genewas inserted into the chromosomal DNA of Saccharomyces sp. W0. It was found thatthe inulinase activity of the recombinant yeast D5in which the endo-inulinase genewas inserted into the delta sequence was higher than that of the recombinant yeast R1in which the endo-inulinase gene was inserted into18S rDNA sequence. More ethanolfrom inulin was produced by the recombinant yeast D5than by the recombinant yeastR1. But Saccharomyces sp. W0produced the lowest inulinase activity andconcentration of ethanol. During the5-L fermentation, the recombinant yeast D5could produce13.6mL of ethanol per100mL of the fermented medium from30%(w/v) inulin. The recombinant yeast D5could actively convert the unsterilized mealof Jerusalem artichoke tubers, yielding10.1mL of ethanol per100mL of thefermented medium.The Saccharomyces sp. W0has the low inulinase activity. However, little isknown about relationship between the invertase gene (SUC2) and inulinase activityand ethanol production from inulin in strain W0. Therefore, in this study, the SUC2gene was disrupted in Saccharomyces sp. W0and the native SUC2gene wasover-expressed in the disruptant. Then, the relationship between the invertase geneand inulinase activity and ethanol production was examined. After the SUC2gene inSaccharomyces sp. W0was removed, the disruptant W4obtained could not produceany extracellular invertase and inulinase activities, could not grow in the mediacontaining sucrose and inulin, but could grow in the medium containing fructose.However, after the SUC2gene was over-expressed in the disruptant W4, therecombinant yeast strain SUC2-1obtained could produce much higher extracellularinvertase and inulinase activities than Saccharomyces sp. W0and the transcriptionallevel of the SUC2gene in the recombinant yeast strain SUC2-1was also much higherthan that in Saccharomyces sp. W0. This is the first time to report that the SUC2genein Saccharomyces sp. W0is closely related to both extracellular invertase and inulinase activities. The invertase over-produced by the recombinant yeast strainSUC2-1could actively convert inulin into monosaccharides. The recombinant yeaststrain SUC2-1over-expressing the SUC2gene could produce over13.4%(v/v)ethanol from30%(w/v) inulin, suggesting that Saccharomyces sp. W0over-producing invertase also can be applied to ethanol fermentation industry.