Inulin is a widespread polysaccharide, and it can be bio-transferred to α-d-fructofuranose-β-d-fructofuranose 1,2':2,1'-dianhydride (DFA I), a potential low-calorie sweetener, via inulin fructotransferase (IFTase, EC 4.2.2.17). In this study, a novel DFA I-forming IFTase was identified and characterized from Streptomyces peucetius subsp. caesius ATCC 27952 (SpIFTase). The specific activity of SpIFTase was determined to be 420.84 ± 6.21 U mg-1 at pH 6.5 and 45 °C. The enzyme exhibited a prominent thermostability with a half-life of 70 min at 70 °C and a structural melting temperature (Tm) of 75.48 °C. The values of Km and kcat/Km of SpIFTase against inulin substrate were 3.08 mM and 199.09 mM-1 s-1, respectively. Furthermore, the catalytic residues were investigated by site-directed mutagenesis with an alanine scanning method. Interestingly, the critical catalytic residues of SpIFTase were speculated to be residues D162 and E231, which were firstly non-conserved with those of previously reported IFTases. This work proposes a potential for industrial DFA I production and new insights into the catalytic mechanism of DFA I-forming IFTases.

中文翻译：

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来自 Streptomyces peucetius subsp. 的新型热稳定 DFA I 形成菊粉果糖转移酶的生化表征。凯西斯 ATCC 27952

菊粉是一种广泛使用的多糖，它可以通过菊粉生物转移为 α-d-呋喃果糖-β-d-呋喃果糖 1,2':2,1'-二酐 (DFA I)，一种潜在的低热量甜味剂果糖转移酶（IFTase，EC 4.2.2.17）。在这项研究中，从 Streptomyces peucetius subsp. 中鉴定并表征了一种新的 DFA I 形成 IFTase。caesius ATCC 27952 (SpIFTase)。在 pH 6.5 和 45 °C 下，SpIFTase 的比活性测定为 420.84 ± 6.21 U mg-1。该酶表现出突出的热稳定性，在 70°C 下的半衰期为 70 分钟，结构解链温度 (Tm) 为 75.48°C。SpIFTase 对菊粉底物的 Km 和 kcat/Km 值分别为 3.08 mM 和 199.09 mM-1 s-1。此外，通过定点诱变和丙氨酸扫描方法研究了催化残基。有趣的是，SpIFTase 的关键催化残基被推测为残基 D162 和 E231，它们首先与先前报道的 IFTase 的残基不保守。这项工作提出了工业 DFA I 生产的潜力和对 DFA I 形成 IFTases 催化机制的新见解。