Chaize Barnabé, Colletier Jacques-Philippe, Winterhalter Mathias, Fournier Didier
Groupe de Biophysique et de Biotechnologie des Protéines, IPBS-UMR 5089, Toulouse, France.
Artif Cells Blood Substit Immobil Biotechnol. 2004 Feb;32(1):67-75. doi: 10.1081/bio-120028669.
Enzyme encapsulation into liposomes is a promising technique to stabilize and prevent them from denaturation and proteolysis. We demonstrate this using acetylcholinesterase which is the main target for pesticides. In order to achieve a reasonable encapsulation yield, we analyzed the parameters involved in each step of various encapsulation procedures. The only encapsulation method which did not denature the protein was the lipid film hydration technique, however the encapsulation efficiency was usually low. The efficiency could be increased up to more than 40% by induction of a specific interaction between the enzyme and the lipid surface. Once encapsulated, the enzyme encountered another problem: the permeability barrier of the lipid membrane drastically diminished the activity of the enzyme entrapped in the liposome by reducing the entrance rate of the substrate molecules and then reducing the substrate concentration inside the liposome. To solve this problem, we controlled the permeability of the liposome wall by reconstituting a porin from Escherichia coli. We succeeded to recover the full functionality of the enzyme, while retaining the protection against denaturation and proteolytic enzymes.
将酶包裹于脂质体中是一种很有前景的技术，可使其稳定并防止其变性和被蛋白水解。我们以农药的主要靶标乙酰胆碱酯酶为例来证明这一点。为了获得合理的包裹产率，我们分析了各种包裹程序每个步骤中涉及的参数。唯一不会使蛋白质变性的包裹方法是脂质膜水化技术，然而其包裹效率通常较低。通过诱导酶与脂质表面之间的特异性相互作用，效率可提高至40%以上。一旦被包裹，酶又遇到了另一个问题：脂质膜的通透性屏障通过降低底物分子的进入速率进而降低脂质体内的底物浓度，极大地降低了包裹于脂质体中的酶的活性。为了解决这个问题，我们通过重组来自大肠杆菌的孔蛋白来控制脂质体壁的通透性。我们成功恢复了酶的全部功能，同时保留了对变性和蛋白水解酶的保护作用。
