Japan Agency for Marine-Earth Science and Technology (JAMSTEC), 2-15 Natsushima, Yokosuka, Kanagawa, 237-0061, Japan.
Appl Microbiol Biotechnol. 2014 Aug;98(15):6667-77. doi: 10.1007/s00253-014-5645-3. Epub 2014 Mar 15.
A β-fructofuranosidase from Microbacterium saccharophilum K-1 (formerly known as Arthrobacter sp. K-1) is useful for producing the sweetener lactosucrose (4(G)-β-D-galactosylsucrose). Thermostability of the β-fructofuranosidase was enhanced by random mutagenesis and saturation mutagenesis. Clones with enhanced thermostability included mutations at residues Thr47, Ser200, Phe447, Phe470, and Pro500. In the highest stability mutant, T47S/S200T/F447P/F470Y/P500S, the half-life at 60 °C was 182 min, 16.5-fold longer than the wild-type enzyme. A comparison of the crystal structures of the full-length wild-type enzyme and three mutants showed that various mechanisms appear to be involved in thermostability enhancement. In particular, the replacement of Phe447 with Val or Pro induced a conformational change in an adjacent residue His477, which results in the formation of a new hydrogen bond in the enzyme. Although the thermostabilization mechanisms of the five residue mutations were explicable on the basis of the crystal structures, it appears to be difficult to predict which amino acid residues should be modified to obtain thermostabilized enzymes.
一株嗜糖微杆菌（原称节杆菌 sp. K-1）β-呋喃果糖苷酶可用于生产甜味剂乳蔗糖（4(G)-β-D-半乳糖基蔗糖）。通过随机诱变和饱和诱变提高了β-呋喃果糖苷酶的热稳定性。热稳定性提高的克隆包括残基 Thr47、Ser200、Phe447、Phe470 和 Pro500 的突变。在热稳定性最高的突变体 T47S/S200T/F447P/F470Y/P500S 中，60°C 时的半衰期为 182 分钟，比野生型酶长 16.5 倍。全长野生型酶和三种突变体的晶体结构比较表明，各种机制似乎都参与了热稳定性的提高。特别是用缬氨酸或脯氨酸替代 Phe447 诱导了相邻残基 His477 的构象变化，导致酶中形成新的氢键。尽管基于晶体结构可以解释这 5 个残基突变的热稳定化机制，但似乎难以预测应该修饰哪些氨基酸残基以获得热稳定的酶。
