Zhan Rongrong, Mu Wanmeng, Jiang Bo, Li Yungao, Zhou Liuming, Zhang Tao
State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, 214122, China; Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, Wuxi, 214122, China.
J Sci Food Agric. 2015 May;95(7):1408-13. doi: 10.1002/jsfa.6931. Epub 2014 Nov 3.
Inulin fructotransferase (IFTase) catalyzes inulin conversion to difructose anhydride (DFA III), which is a natural low-calorie sweetener. Although heterologous expression of IFTase was achieved in Escherichia coli, the extracellular enzyme activity was very low, which limited the commercialization of IFTase.
Active IFTase of about 43 kDa molecular mass of subunit was extracellularly expressed by Pichia pastoris and was greatly regulated by the IFTase gene copy number integrated into the P. pastoris genome and by the methanol concentration in the induction phase. Under optimized culture conditions, multicopy P. pastoris exhibited a maximum extracellular IFTase activity of 105.4 U mL(-1) in a 5 L fermenter, which was 8.9-fold the activity in shake flasks and 5.3-fold that obtained from wild-type strain.
IFTase was expressed in a eukaryotic P. pastoris system for the first time and achieved high-level extracellular expression using a high-cell-density fed-batch cultivation strategy. This demonstrated that P. pastoris was a good candidate for potential DFA III production as a novel IFTase expression system.
菊粉果糖转移酶（IFTase）催化菊粉转化为二果糖酐（DFA III），DFA III是一种天然低热量甜味剂。尽管已在大肠杆菌中实现了IFTase的异源表达，但胞外酶活性非常低，这限制了IFTase的商业化。
约43 kDa亚基分子量的活性IFTase由巴斯德毕赤酵母在胞外表达，并且受到整合到毕赤酵母基因组中的IFTase基因拷贝数以及诱导阶段甲醇浓度的显著调控。在优化的培养条件下，多拷贝毕赤酵母在5 L发酵罐中表现出最大胞外IFTase活性为105.4 U mL(-1)，这是摇瓶中活性的8.9倍，是野生型菌株活性的5.3倍。
首次在真核巴斯德毕赤酵母系统中表达了IFTase，并使用高细胞密度补料分批培养策略实现了高水平的胞外表达。这表明毕赤酵母作为一种新型IFTase表达系统，是潜在生产DFA III的良好候选者。
