Key Laboratory of Industrial Biotechnology of Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China.
Key Laboratory of Industrial Biotechnology of Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China.
N Biotechnol. 2016 Jan 25;33(1):164-70. doi: 10.1016/j.nbt.2015.04.005. Epub 2015 May 11.
In this work, the cDNA encoding fructosyltransferase (FTase) from Aspergillus niger YZ59 (CICIM F0901) was obtained and expressed in the methylotrophic yeast Pichia pastoris strain GS115. The yield of recombinant FTase in a 5-L fermentor reached 1020.0 U/mL after 96 h of induction, which was 1160.4 times higher that of native FTase from A. niger YZ59. The specific activity of recombinant FTase was 6.8×10(4) U/mg. The optimum temperature and pH of the recombinant FTase were 55 °C and 5.5, respectively. The recombinant FTase was stable below 40 °C and at pH from 3.0 to 10.0. Using sucrose as the substrate, the Km and Vmax values of recombinant FTase were 159.8 g/L and 0.66 g/(L min), respectively. The turnover number (kcat) and catalytic efficiency (kcat/Km) of recombinant FTase was 1.1×10(4) min(-1) and 68.8 L/(g min), respectively. The recombinant FTase was slightly activated by 5mM Ni(2+), Mg(2+), K(+), Fe(3+), or Mn(2+), but inhibited by all other metal ions (Na(+), Li(+), Ba(2+), Ca(2+), Zn(2+), and Cu(2+)). The highest yield of fructooligosaccharides for purified FTase reached approximately 343.3 g/L (w/v). This is the first study reporting the heterologous expression of FTases from A. niger in P. pastoris. This study plays an important role in the fructooligosaccharide synthesis industry by recombinant FTases.
在这项工作中，获得了来自黑曲霉 YZ59（CICIM F0901）的果糖基转移酶（FTase）的 cDNA，并在甲醇营养酵母毕赤酵母 GS115 中表达。在 5-L 发酵罐中诱导 96 小时后，重组 FTase 的产量达到 1020.0 U/mL，比来自黑曲霉 YZ59 的天然 FTase 高 1160.4 倍。重组 FTase 的比活为 6.8×10(4) U/mg。重组 FTase 的最适温度和 pH 分别为 55°C 和 5.5。重组 FTase 在低于 40°C 和 pH 3.0 至 10.0 的条件下稳定。以蔗糖为底物时，重组 FTase 的 Km 和 Vmax 值分别为 159.8 g/L 和 0.66 g/（L min）。重组 FTase 的周转数（kcat）和催化效率（kcat/Km）分别为 1.1×10(4) min(-1)和 68.8 L/（g min）。重组 FTase 被 5mM 的 Ni(2+)、Mg(2+)、K(+)、Fe(3+)或 Mn(2+)轻微激活，但被所有其他金属离子（Na(+)、Li(+)、Ba(2+)、Ca(2+)、Zn(2+)和 Cu(2+)）抑制。纯化 FTase 的果糖低聚糖最高产量达到约 343.3 g/L（w/v）。这是首次在毕赤酵母中异源表达黑曲霉 FTase 的研究。这项研究对重组 FTase 在果糖低聚糖合成工业中具有重要意义。
