Austrian Centre of Industrial Biotechnology, ACIB , Konrad Lorenz Strasse 20, 3430 Tulln, Austria.
Department of Environmental Systems Science, Institute of Biogeochemistry and Pollutant Dynamics, Swiss Federal Institute of Technology Zurich (ETHZ) , Universitätstrasse 16, 8092 Zurich, Switzerland.
Biomacromolecules. 2015 Dec 14;16(12):3889-96. doi: 10.1021/acs.biomac.5b01219. Epub 2015 Nov 24.
Mimicking a concept of nature for the hydrolysis of biopolymers, the Thermobifida cellulosilytica cutinase 1 (Thc_Cut1) was fused to a polymer binding module (PBM) to enhance the hydrolysis of the polyester poly(1,4-butylene adipate) (PBA). Namely, the binding module of a polyhydroxyalkanoate depolymerase from Alcaligenes faecalis (Thc_Cut1_PBM) was attached to the cutinase via two different linker sequences varying in length. In order to investigate the adsorption behavior, catalytically inactive mutants both of Thc_Cut1 and Thc_Cut1_PBM were successfully constructed by site-directed mutagenesis of serine 131 to alanine. Quartz crystal microbalance with dissipation monitoring (QCM-D) analysis revealed that the initial mass increase during enzyme adsorption was larger for the inactive enzymes linked with the PBM as compared to the enzyme without the PBM. The hydrolysis rates of PBA were significantly enhanced when incubated with the active, engineered Thc_Cut1_PBM as compared to the native Thc_Cut1. Thc_Cut1_PBM completely hydrolyzed PBA thin films on QCM-D sensors within approximately 40 min, whereas twice as much time was required for the complete hydrolysis by the native Thc_Cut1.
受生物聚合物水解仿生概念的启发，将嗜热纤维梭菌角质酶 1（Thc_Cut1）与聚合物结合模块（PBM）融合，以增强聚酯聚（1,4-丁二酸丁二醇酯）（PBA）的水解。也就是说，来自粪产碱杆菌的聚羟基烷酸酯解聚酶的结合模块（Thc_Cut1_PBM）通过两种不同长度的连接序列附着在角质酶上。为了研究吸附行为，通过定点突变将丝氨酸 131突变为丙氨酸，成功构建了具有催化活性的突变体 Thc_Cut1 和 Thc_Cut1_PBM。石英晶体微天平耗散监测（QCM-D）分析表明，与没有 PBM 的酶相比，与 PBM 连接的无活性酶的初始酶吸附时的质量增加更大。与天然的 Thc_Cut1 相比，当与活性工程化的 Thc_Cut1_PBM 孵育时，PBA 的水解速率显著提高。Thc_Cut1_PBM 在 QCM-D 传感器上完全水解 PBA 薄膜大约需要 40 分钟，而天然 Thc_Cut1 则需要两倍的时间才能完全水解。
