表面活性剂调控疏水性污染物跨膜过程及分子机制

Transmembrane process of hydrophobic organic compounds (HOCs) is the rate-limited step for their biodegradation. The outer membrane of Gram-negative bacteria provides an efficient barrier for the transmembrane process of HOC molecules owing to the presence of polar lipopolysaccharide, which limiting the efficiency and application of bioremediation. .This project is to perform a basic research for the enhanced-transport of HOCs. In this research, we focus on the interfaces in the hydrophobic channel through outer membrane of Gram-negative bacteria. We will investigate the interactions among surfactant, outer membrane and the HOCs in a molecular level. The solubilization and replacement of C8E4 component in the hydrophobic channel by surfactants will be also conducted. Then, we will investigate the selective binding of surfactant to high- and low-affinity sites inside the channel, as well as the surfactant properties. Besides, the conformation change of hydrophobic channel protein under surfactants will also be noticed. Finally, we will particularly focus on the effects and molecular mechanisms of surfactants on the effective concentration of HOCs outside the channel, on the directed force due to hydrophobic gradient, on the opening of lateral exit of the hydrophobic channel. These information is necessary for the understanding on transport route of HOCs into bacteria periplasm, surfactant-enhanced transmembrane process and underlying mechanisms. These information is also useful for solving the rate-limited biodegradation process and even for the development of new techniques to remediate contaminated soils.

疏水性有机污染物的跨膜传输过程是细菌降解有机物的限速步骤，通常会受到脂多糖层等亲水结构的阻碍，严重制约了生物修复有机污染土壤的效率和应用。本项目以强化疏水性污染物的跨膜传输过程为目标，以革兰氏阴性菌细胞膜疏水性孔道的蛋白质分子界面为研究对象，从分子水平研究表面活性剂-细胞外膜-HOCs的相互作用，表面活性剂对疏水性孔道内C8E4组分的增溶、替换作用，研究表面活性剂与疏水性孔道中高、低疏水基团的选择性结合及其性质的影响，探讨表面活性剂对疏水孔道构象的影响。重点研究表面活性剂对增大疏水孔道外部有效底物浓度、增强内部疏水梯度引导力以及提高横向出口触发敏感性的影响及机制，试图阐明疏水性有机污染物跨膜进入细菌胞内的途径、表面活性剂强化污染物的跨膜传输过程及其分子机制，解决疏水性有机污染物跨膜进入细菌胞内量少速度慢而导致土壤细菌降解有机污染物效率低的问题，为拟定经济安全高效的土壤修复技术提供科学依据。

疏水性有机污染物（HOCs）的跨膜传输过程是细菌降解有机物的限速步骤，是影响生物修复有机污染土壤效率和应用的重要过程，通常会受到菌表吸附的可参与跨膜传输的疏水底物浓度、细胞膜透性等因素的影响。本项目旨在通过表面活性剂对细菌迁移活性、菌表吸附调控菌表可参与跨膜的底物浓度，通过与细菌细胞内/外膜的相互作用影响细菌的膜透性，从而促进疏水性污染物的跨膜传输。研究发现：（1）表面活性剂可加速细菌在土柱中的迁移，且可降低细菌在土柱中的残留率，从而提高细菌与污染物接触可能性。表面活性剂对细菌迁移的促进作用与其浓度和性质相关，1.0和4.0 g/L Tween 80最终平衡洗出细菌量占细菌总量的比例分别为0.74和1.00，均远高于纯水体系的0.412；对细菌迁移活性影响程度顺序为Tween 80 > Tween 40 > Tween 20，与其HLB值正相关。（2）得到两株PAHs降解菌Klebsiella oxytoca和Pseudomonas aeruginosa，均可高效降解、去除菲、苊等典型HOCs。Triton X系列表面活性剂对细菌吸附PAHs表现为浓度依赖，低浓度对吸附系数影响不大， 而高浓度（CMC以上）则显著降低Kd值；且对Kd的影响与表面活性剂HLB呈负相关。共存污染物（碘、铜、镉）均会促进细菌对HOC的吸附，重金属（铜和镉）主要是通过n-π键合起作用，而碘则通过I-C特殊结合其作用。（3）表面活性剂可增加P. aeruginosa的内膜、外膜透性，如8 CMC Triton X-114存在时，P. aeruginosa内膜渗透性是无表面活性剂对照的2.20倍。但对细菌相对电导率的影响不明显。利用脂质体模拟细胞膜研究HOCs的跨膜扩散，发现表面活性剂可加速和提高脂质体包裹菲的扩散，如Tween 80-皂角苷（1:9）可以协助将脂质体中87%左右的菲扩散出来，远高于无表面活性剂对照。