Yu Shuhuai, Zhang Yanmin, Zhu Yingying, Zhang Tao, Jiang Bo, Mu Wanmeng
State Key Laboratory of Food Science and Technology, Jiangnan University , Wuxi, Jiangsu 214122, China.
Laboratory of Molecular Design and Drug Discovery, School of Science, China Pharmaceutical University , 639 Longmian Avenue, Nanjing 211198, China.
J Agric Food Chem. 2017 Aug 30;65(34):7579-7587. doi: 10.1021/acs.jafc.7b02897. Epub 2017 Aug 17.
Previously, a α-d-fructofuranose-β-d-fructofuranose 1,2':2,1'-dianhydride (DFA I)-forming inulin fructotransferase (IFTase), namely, SdIFTase, was identified. The enzyme does not show high performances. In this work, to improve catalytic behavior including activity and thermostability, the enzyme was modified using site-directed mutagenesis on the basis of structure. The mutated residues were divided into three groups. Those in group I are located at central tunnel including G236, A257, G281, T313, and A314S. The group II contains residues at the inner edge of substrate binding pocket including I80, while group III at the outer edge includes G121 and T122. The thermostability was reflected by the melting temperature (T) determined by Nano DSC. Finally, the T values of G236S/G281S/A257S/T313S/A314S in group I and G121A/T122L in group III were enhanced by 3.2 and 4.5 °C, and the relative activities were enhanced to 140.5% and 148.7%, respectively. The method in this work may be applicable to other DFA I-forming IFTases.
此前，已鉴定出一种能形成α-d-呋喃果糖-β-d-呋喃果糖1,2':2,1'-二酐（DFA I）的菊粉果糖转移酶（IFTase），即SdIFTase。该酶表现不佳。在本研究中，为改善其包括活性和热稳定性在内的催化行为，基于结构通过定点突变对该酶进行了修饰。突变残基分为三组。第一组位于中心通道，包括G236、A257、G281、T313和A314S。第二组包含位于底物结合口袋内边缘的残基，如I80，而位于外边缘的第三组包括G121和T122。热稳定性通过纳米差示扫描量热法（Nano DSC）测定的解链温度（T）来反映。最后，第一组中的G236S/G281S/A257S/T313S/A314S和第三组中的G121A/T122L的T值分别提高了3.2℃和4.5℃，相对活性分别提高到了140.5%和148.7%。本研究中的方法可能适用于其他能形成DFA I的IFTases。
