State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu, 214122, China.
State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu, 214122, China; Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan Universtiy, Wuxi, Jiangsu, 214122, China.
Int J Biol Macromol. 2016 Nov;92:723-730. doi: 10.1016/j.ijbiomac.2016.07.092. Epub 2016 Jul 28.
In this work, a novel gene encoding DFA I-forming inulin fructotransferase (IFTase) from Streptomyces davawensis SK39.001 was cloned and expressed in Escherichia coli. The enzyme was purified, identified, and characterized. The results showed that this IFTase (DFA I-forming) is a trimer (molecular weight of 125KDa) consisting of three identical subunits (the molecular weight as assayed by SDS-PAGE was approximately 40KDa). At pH 5.5 and 40°C, the maximum specific activity (approximately 100Umg) was achieved. Moreover, the enzyme was stable up to 70°C. K and V were 2.89±0.2mM and 1.94±0.9mMmin, respectively. For exploring putative active sites and probable catalytic mechanisms, homology modelling and molecular docking methods after site-directed mutagenesis were applied to IFTase (DFA I-forming). The results revealed that D183 and E194 were potential catalytic residues of the purified enzyme.
在这项工作中，从链霉菌 SK39.001 中克隆并在大肠杆菌中表达了一种新型的菊粉果糖基转移酶（IFTase）基因，该基因编码 DFA I 形成。对酶进行了纯化、鉴定和特性分析。结果表明，该 IFTase（DFA I 形成）是由三个相同亚基组成的三聚体（分子量为 125KDa）（通过 SDS-PAGE 测定的分子量约为 40KDa）。在 pH5.5 和 40°C 下，达到最大比活力（约 100Umg）。此外，该酶在 70°C 下稳定。K 和 V 分别为 2.89±0.2mM 和 1.94±0.9mMmin。为了探索可能的活性位点和可能的催化机制，对 IFTase（DFA I 形成）进行了定向突变后的同源建模和分子对接方法。结果表明，D183 和 E194 是纯化酶的潜在催化残基。
