Exogenous Expression Of Endoinulinase From Aspergillus Niger And Its Application In Inulooligosaccharide Production

Inulooligosaccharide（IOS） consists of linear chains of β-2,1-linked fructose residues attached to a terminal sucrose molecule, with a degree of polymerization（DP） 3-10. Endoinulinase（EC 3.2.1.7）, derived from fungi, yeasts and bacteria, is specific for inulin and hydrolyze the internal β-2,1-glucopyranosoyl bonds to yield IOS as the main products. It may provide an important application value for large-scale industrial producuction of IOS using endoinulinase.A strain of A. niger DSM 2466 which could secrete endoinulinase was stored in our laboratory. In order to improve the activity of endolinase, to realize the application of endoinulinase in industrial production, we cloned and expressed the endoinulinase gene. On this basis, we studied the endoinulinase in IOS production. The results of the study are as following:（1） A 1551 bp endoinulinase genes of A. niger DSM 2466 was cloned and constructed the recombinant expression plasmid on the carrier of pETDuet-1, and it was expressed in Escherichia coli BL21（DE3）. The recombinant E. coli BL21（DE3）-pETDuet-Inu was incubated and induced with 1 mmol/L IPTG in 23°C for 6 h. The recombinant endoinulinase successfully expressed and the SDS-PAGE analysis showed that an about 66 kDa protein strip was obtained. The optimum temperature and pH of endoinulinase were 50 oC and 6.0. And the specific activities of endoinulinase was reach to 4.91 U/mg. Al³⁺, Cu²⁺, Ag+, Mn²⁺ and Fe³⁺ cound inhibit activity intensively while Ca²⁺ and Ni²⁺ could activate the endoinulinase. In addition, K+ and Mg²⁺ had no significant effect on the enzyme activity.（2） The codon optimized gene fragment encoding endoinulinase from A. niger DSM 2466 was cloned into pPIC9 K vector, linearized by Sac I and was transformed into P. pastoris KM71. After 120 h of methanol induction in the shaking flask, the endoinulinase activity of recombinant P. pastoris KM71/pPIC9K-Inuop culture supernatant was 480 U/mL. Whereafter, the high cell density and high expression level of recombinant enzyme were achieved in 3-L fermenter. When the initial cell density OD600 nm was 180, inducing temperature was 28 oC, pH was 5 and the dissolved oxygen concentration was maintained about 20%, after 120 h of methanol induction the activity of endoinulinase reached 858 U/mL and the protein concentration in the culture media was 3024.3 μg/m L.（3） The conditions for IOS production from inulin employing endoinulinases were optimized, including temperature, pH, substrate concentration, and enzyme dosage. IOS were harvested with a high concentration of 365.1 g/L and high yield up to 91.3% after 4 h of hydrolysis. IOS with different degree of polymerization（DP, mainly DP 3-6） were distributed in the final reaction products. And the major IOS were 8.84% GF2, 18.05% GF3, 30.18% F3, 15.67% GF4, 10.58% F4, and 7.97% GF5 oligomer, respectively.The endoinulinase from A. niger DSM 2466 was displayed on the cell wall of S. cerevisiae and P. pastoris. The endoinulinase displayed on the cell wall of S. cerevisiae EBY100 was using a-agglutinin as anchor protein. The activity of displayed endoinulinase was 1.89 U/mg dry cell after induction at 20°C for 72 h. Moreover, the endoinulinase was displayed on the cell wall of P.pastoris KM71 using Pir1 from S. cerevisiae as anchor protein for the first time. After electroporation, the recombinant strain KM71/ pPIC9K-Inuop-Pir1 was obtained. The activity of displayed endoinulinase was 1.13 U/mg dry cell by flaskfermentation.