Direct Conversion Of Inulin Into Single Cell Protein By The Engineered Yarrowia Lipolytica Carrying Inulinase Gene

In recent years,the demand of protein in food and feed industries is being increased. SCP production technologies arose as a promising way to solve the problem of worldwide protein shortage. They evolved as bioconversion processes which turned low value by-products, into products with added nutritional and market value. So it is crucial to choose cheap raw materials for controlling the cost of producing SCP.Inulin is present as a reserve carbohydrate in the roots and tubers of plants such as Jerusalem artichoke, chicory, dahlia, and yacon. Inulin sources have recently received increasing attention as they represent a renewable, inexpensive and abundant raw material. These advantages make inulin and inulin-containing materials good potential candidates for the economic production of SCP.The INU1 gene encoding exo-inulinase cloned from Kluyveromyces marxianus CBS 6556 was ligated into the expression plasmid pINA1317 and expressed in the cells of the marine-derived yeast Yarrowia lipolytica with high content of protein. The activity of the secreted inulinase with 6×His tag was found to be 43.1±0.9 U/ml after cell growth for 80 h. When the engineered yeast cells were grown in the medium containing 4.0% inulin in 2-l fermentor, crude protein in the cells, cell mass, reducing sugar and total sugar utilization could reach 47.5%, 20.1 g/l, 0.6±0.1 g/l and 96.4%, respectively within 72 h. Furthermore, when the engineered yeast cells were grown in the medium containing 8.0% the meal of Jerusalem artichoke tuber in 2-l fermentor, crude protein in the cells, cell mass,reducing sugar and total sugar utilization could reach 53.7%, 20.8 g/l, 1.3±0.2 g/l and 94.2%, respectively within 80 h. Therefore, the engineered Y. lipolytica could be used to produce single cell protein from inulin and inulin-containing materials.The recombinant inulinase secreted into the medium was purified with affinity chromatography using a Ni SepharoseTM 6 Fast Flow column. The results of SDS-PAGE reveal that the molecular mass was approximatly 84 kDa. The optimal pH and temperature of the purified recombinant inulinase were 4.5 and 50°C, respectively. The enzyme was stable below 50℃and between pH 4.0 and 5.5, respectively. Na⁺ and Zn²⁺ had an activating effect on the recombinant inulinase activity while Hg²⁺ and Ag⁺ acted as inhibitors in decreasing activity of the purified inulinase. The recombinant inulinase activity was also intensively inhibited by SDS. The K_m and V_max values of the purified recombinant inulinase for inulin were 16.7mg/ml and 0.0052mg/min, respectively. A large amount of monosaccharides were detected after the hydrolysis, indicating the purified inulinase had a high exoinulinase activity.