Heterologous Expression Of Aspergillus Niger Endoinulinase In Pichia Pastoris And Its Application

Endoinulinases are specific for inulin and hydrolyze the internal β-2,1fructofuranosidiclinkages to yield inulooligosaccharide (IOS) as the main products. The enzyme derived fromfungi, yeasts and bacteria and the Aspergillus niger endoinulinase had various advantagesincluding good specificity, thermostability and wide pH stability. IOS belongs to functionaloligosaccharide, which has low calories, can inhibit starch retrogradation and restrain thegrowth of facial pathogen etc. Therefore, IOS can be widely used in the industries of food,nutrition, cosmetics etc.In this study, the endoinulinase gene from A. niger CICIM F0620was expressed in Pichiapastoris KM71. Then the fermentation process was optimized, followed by the characterizationof the recombinant enzyme. Afterwards, the effects of different conditions on the IOSconversion yield was studied. The results of the study are as following:(1)The endoinulinase DNA (EnInu) was amplified from the total DNA of A. niger byPCR and was ligated into pPIC9K. The recombinant plasmid was linearized by Sac I andtransformed into P. pastoris KM71by electroporation, yielding the recombinant strainP. pastoris KM71/pPIC9K-EnInu. After120h of methanol induction in the shaking flask, theinulin hydrolase activity of recombinant P. pastoris KM71/pPIC9K-EnInu culture supernatantwas16.7U·mL-1.(2)The endoinulinase gene (EnInu) from Aspergillus niger CICIM F0620was optimizedaccording to the codon usage of Pichia pastoris, named EnInuop. Then EnInuop was ligatedinto pPIC9K, linearized by Sac I and transformed into P. pastoris KM71by electroporation,yielding the recombinant strain P. pastoris KM71/pPIC9K-EnInuop. After120h of methanolinduction in the shaking flask, the endoinulinase activity of recombinant P. pastorisKM71/pPIC9K-EnInuop culture supernatant was26.5U·mL-1,1.59-fold of the natural gene.Fed-batch cultivation process of the recombinant strain in3L fermenter was optimized. Whenthe initial cell density (OD600) was160, the methanol concentration was1%(v/v), inducingtemperature was26℃, pH was5and the dissolved oxygen concentration was maintainedabout20%, after120h of methanol induction the endoinulinase activity of the culturesupernatant was1349U·mL-1, which was the highest activity of endoinulinase reported so far.(3)The recombinant endoinulinase was purified through70%ammonium sulphateprecipitation, DEAE-cellulose column chromatography and Mono Q column chromatography.The temperature for optimal activity of the endoinulinase was determined to be60℃and therecombinant EnInuop showed good thermostability. The enzyme exhibited the highest activityat pH6.0, and was stable over a pH range of4.0-9.0. Ca2+can activate the EnInuop, while Co2+can inhibit the EnInuop. (4) The hydrolysis products of the recombinant EnInuop on inulin were mainly IOS withDP3-6. The conversion conditions were optimized. After8h under optimal conditions, whichincluded400g·L-1inulin, an enzyme concentration of40U·g-1,50℃and pH5.0, the IOS yieldwas89%. Thus, this study may provide the basis for the industrial use of this recombinantendoinulinase for the production of IOS.