粘土矿物-蛋白质超薄膜的设计与生物催化性能

Clay-protein ultrathin films:Design and bio-catalytic performance study

随着生物芯片及生物大分子有序自组装技术的发展,蛋白质-粘土矿物的超薄复合膜(clay-protein ultrathin films,CPUFs)的制备在生物催化领域引起人们的广泛关注.本文详述了蛋白质(溶解酵素、牛血清白蛋白、木瓜蛋白酶与精蛋白)与单片粘土(elementary sheet)矿物(钠化皂石)形成单层或多层纳米薄膜复合物的吸附过程及二维分子自组装的制备工艺,对交替层吸附(layer-by-layer,LbL)和Langmuir-Blodgett(LB)方法应用于CPUFs的构筑分别进行了介绍,并阐述了紫外可见分光光度计(UV-vis),衰减全反射红外光谱(ATR-FTIR),X射线衍射(XRD),原子力显微镜(AFM)和界面化学技术等手段对薄膜定性定量表征的研究方法及结果.研究表明,在CPUFs的形成过程中,静电相互作用是一个突出因素,但不是唯一驱动力.在利用LB技术构筑CPUFs的研究中,我们发现即使在无表面活性剂分子的协助下,水溶性蛋白质也能够在粘土矿物稀溶液的界面上形成较为稳定的蛋白质-粘土矿物Langmuir复合膜.通过研究表面压力与时间(?-t)的动力学曲线和表面压力对面积(?-A)的等温线,实现了粘土矿物与蛋白分子吸附过程即CPUFs形成过程的实时监测,并测定出CPUFs中蛋白质含量(NS)、蛋白质分子堆积密度(??)、单个蛋白质分子在粘土片层上的平均占位面积(??)及蛋白质在粘土层的饱和吸附量等物理化学参数.通过将界面化学测定结果、光谱学方法测定结果与粘土矿物体相溶液吸附实验结果相比较,发现LB技术实现了单层粘土片(厚度约1.3nm)的吸附与自组装,证明界面化学方法是定量化研究CPUFs的一种有效手段.通过将含有溶解酵素的CPUFs膜用于流水池反应器上进行生物活性检测,考察了其在睾丸酮丛毛单胞菌生长发育过程中的催化性能,结果表明LZ分子被锚固在CPUFs上以后,未发生明显的生物活性失活现象.

In recent years,immense interest has been paid to the biomolecular architecture with the aim of protein assembly in 2-dimensions on solid substrates,and the constructions of clay-protein ultrathin films（CPUFs） are particularly concerned.This paper gives an overview of the recent research concerning the protein molecules（lysozyme,papain,protamine,bovine serum albumin） immobilized on clay mineral（Na-saponite） platelets and assembled in monolayered or multilayered hybrid ultrafilms or nanofilms.Two techniques including alternate layer-by-layer（LbL） assembly and the Langmuir-Blodgett（LB） are described in detail.A variety of means,including UV-vis absorption,attenuated total reflectance Fourier transform infrared（ATR-FTIR） spectroscopy,XRD,AFM and surface chemistry techniques,have been described for characterization of the films in terms of quantification of protein and clay.The result reveals that electrostatic interaction is a prominent but not the only driving force in CPUF construction.In the case of LB technique,we managed to manipulate the elementary clay mineral platelets（1.3 nm in thickness） and assemble proteins into CPUFs with the aid of surfactants,and the formation of CPUFs was monitored via surface pressure vs.time（？-t） kinetics curves and surface pressure vs.area（？-A） isotherms.The factors that influence protein adsorption on the clay layer,such as surfactants,the concentration of clay,equilibrium time,categories of protein,and injection methods,were investigated.The parameters such as protein amount（nS）,packing density（？？）,and average surface area per molecule（？？） of deposited CPUFs were measured via method of surface chemistry and spectroscopy.By comparing the results of surface chemistry with those of adsorption experiments,we demonstrate that the surface chemistry method is a useful tool in investigating CPUFs.We also found that the water soluble protein molecules could form protein-clay hybrid monolayer over the dilute clay dispersions without addition of surfactants,and CPUFs containing elementary clay sheets and protein with great homogeneity were easily prepared by controlling certain surface pressure.To investigate the bio-catalytic performance of the immobilized lysozyme in CPUFs,we deposited CPUFs onto a cover glass,and installed the cover glass in a flow cell-grown reactor for Comamonas testosteroni（WDL7-GFP） incubation.The results show that the proliferation of WDL7-GFP is greatly suppressed by lysozyme,which demonstrates that lysozyme still retains its bioactivity after it is immobilized in the CPUFs.