Unesco Chinese Center of Marine Biotechnology, Institute of Marine Biodiversity and Evolution, Ocean University of China, Yushan Road, No. 5, Qingdao, China.
Metab Eng. 2010 Sep;12(5):469-76. doi: 10.1016/j.ymben.2010.04.004. Epub 2010 May 6.
The INU1 gene encoding exo-inulinase cloned from Kluyveromyces marxianus CBS 6556 was ligated into the surface display plasmid and expressed in the cells of the marine-derived yeast Yarrowia lipolytica which can produce citric acid. The expressed inulinase was immobilized on the yeast cells. The activity of the immobilized inulinase with 6 x His tag was found to be 22.6 U mg(-1) of cell dry weight after cell growth for 96 h. The optimal pH and temperature of the displayed inulinase were 4.5 and 50 degrees C, respectively and the inulinase was stable in the pH range of 3-8 and in the temperature range of 0-50 degrees C. During the inulin hydrolysis, the optimal inulin concentration was 12.0% and the optimal amount of added inulinase was 181.6 U g(-1) of inulin. Under such conditions, over 77.9% of inulin was hydrolyzed within 10h and the hydrolysate contained main monosaccharides and disaccharides, and minor trisaccharides. During the citric acid production in the flask level, the recombinant yeast could produce 77.9 g L(-1) citric acid and 5.3 g L(-1) iso-citric acid from inulin while 68.9 g L(-1) of citric acid and 4.1 g L(-1) iso-citric acid in the fermented medium were attained within 312 h of the 2-L fermentation, respectively.
从马克斯克鲁维酵母 CBS 6556 中克隆的 INU1 基因编码外切菊粉酶，被连接到表面展示质粒中，并在能产生柠檬酸的海洋酵母解脂耶氏酵母的细胞中表达。表达的菊粉酶被固定在酵母细胞上。经过 96 小时的细胞生长，带有 6xHis 标签的固定化菊粉酶的活性为 22.6 U mg(-1)细胞干重。展示菊粉酶的最适 pH 和温度分别为 4.5 和 50°C，并且该酶在 pH 3-8 和 0-50°C 的温度范围内稳定。在菊粉水解过程中，菊粉的最适浓度为 12.0%，添加的菊粉酶最适量为 181.6 U g(-1)菊粉。在这种条件下，超过 77.9%的菊粉在 10 小时内被水解，水解产物含有主要的单糖和二糖，以及少量的三糖。在摇瓶水平的柠檬酸生产过程中，重组酵母可以从菊粉中生产 77.9 g L(-1)柠檬酸和 5.3 g L(-1)异柠檬酸，而在 2-L 发酵的 312 小时内，发酵培养基中分别可以获得 68.9 g L(-1)柠檬酸和 4.1 g L(-1)异柠檬酸。
