Zhou Chaoqiang, Saravanan Thangavelu, Lorillière Marion, Wei Dongzhi, Charmantray Franck, Hecquet Laurence, Fessner Wolf-Dieter, Yi Dong
State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Meilong Road 130, 200237, Shanghai, P.R. China.
Institut für Organische Chemie und Biochemie, Technische Universität Darmstadt, Alarich-Weiss-Strasse 4, 64287, Darmstadt, Germany.
Chembiochem. 2017 Mar 2;18(5):455-459. doi: 10.1002/cbic.201600609. Epub 2017 Jan 27.
The transketolase from Geobacillus stearothermophilus (TK ) is a thermostable enzyme with notable high activity and stability at elevated temperatures, but it accepts non-α-hydroxylated aldehydes only with low efficiency. Here we report a protein engineering study of TK based on double-site saturation mutagenesis either at Leu191 or at Phe435 in combination with Asp470; these are the residues responsible for substrate binding in the active site. Screening of the mutagenesis libraries resulted in several positive variants with activity towards propanal up to 7.4 times higher than that of the wild type. Variants F435L/D470E and L191V/D470I exhibited improved (73 % ee, 3S) and inverted (74 % ee, 3R) stereoselectivity, respectively, for propanal. L191V, L382F/E, F435L, and D470/D470I were concluded to be positive mutations at Leu191, Leu382, Phe435, and Asp470 both for activity and for stereoselectivity improvement. These results should benefit further engineering of TK for various applications in asymmetric carboligation.
嗜热栖热放线菌转酮醇酶（TK）是一种热稳定酶，在高温下具有显著的高活性和稳定性，但它对非α-羟基化醛的接受效率很低。在此，我们报告了一项基于Leu191或Phe435与Asp470组合的双位点饱和诱变对TK进行的蛋白质工程研究；这些是负责活性位点底物结合的残基。对诱变文库的筛选产生了几个阳性变体，它们对丙醛的活性比野生型高7.4倍。变体F435L/D470E和L191V/D470I对丙醛分别表现出改善的（73% ee，3S）和反转的（74% ee，3R）立体选择性。L191V、L382F/E、F435L和D470/D470I被认为是Leu191、Leu382、Phe435和Asp470在活性和立体选择性改善方面的正向突变。这些结果将有助于进一步对TK进行工程改造，以用于不对称碳连接的各种应用。
