Enzymatic reactions in liposomes using the detergent-induced liposome loading method

Abstract

Microcompartmentalization is a crucial step in the origin of life. More than 30 years ago, Oparin et al. proposed models based on biochemical reactions taking place in so-called coacervates. Their intention was to develop systems with which semipermeable microcompartments could be established. In the present work we follow their intuition, but we use well-characterized bilayer structures instead of the poorly characterized coacervates. Liposomes from phospholipids can be used as microreactors but they exhibit only a modest permeability and, therefore, chemical reactions occurring inside these structures are depleted after a relatively short period. Here it is shown that even highly stable liposomes from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) can be used as semipermeable microreactors when treated with sodium cholate. Using this kind of mixed liposomes, we describe a biochemical reaction occurring inside the liposomes while the same reaction is prevented in the external medium. In addition, we show that this cholate-induced permeability of POPC bilayers can even be used to load macromolecules such as enzymes from the outside.

Organic Nano-Compartments as Biomimetic Reactors and Protocells

“…Unfortunately, whatever methods are used to improve liposomal encapsulation, complete encapsulation is never reached. Thus, to prevent reactions in the external aqueous medium, an additional preparation step (compared to emulsion systems), such as enzymatic digestion or external medium exchange by chromatography or dialysis, will be required to neutralize non-encapsulated catalytic assemblies [11,76,77]. A few additional complications also exist.…”

“…During homogenization of liposome suspensions, some fraction of the encapsulated solutes will be released into the external medium. These "free" molecules need to be inactivated by removing them from the preparation (i.e., by gel exclusion chromatography or enzymatic digestion [11,74,77]) to prevent a residual catalytic activity in the external medium.…”

Catalysis in abiotic structured media: an approach to selective synthesis of biopolymers

“…Transient packing defects in the bilayers, more frequent at the lipid phase transition temperature (the temperature at which lipid molecules in the bilayer gradually pass from 529 a gel state to a liquid-crystalline state) [102,104], can allow the passive diffusion of ionic solutes, such as NDPs (nucleotide diphosphates) [101,105] and NTPs (nucleotide triphosphates) [102], which serve as substrates for RNA polymerases. The addition of detergents, which permeabilize PC bilayers at sublytic concentrations [106], can be used to supply encapsulated enzymes [106,107]. This method is rather difficult to implement because the threshold concentration of detergent which allows small solute to permeate but prevents protein crossing (e.g., DNase I [106]) is not well defined.…”

“…The addition of detergents, which permeabilize PC bilayers at sublytic concentrations [106], can be used to supply encapsulated enzymes [106,107]. This method is rather difficult to implement because the threshold concentration of detergent which allows small solute to permeate but prevents protein crossing (e.g., DNase I [106]) is not well defined. Oberholzer and colleagues used lipid-layer interdigitation by ethanol [2], but this effect only applies to GUVs because of their low curvature stress.…”

Chitosanase displayed on liposome can increase its activity and stability

“…The enzymatic reaction inside small and giant liposome vesicles have been dedicated by many researchers (Fischer et al, 2002;Yoshimoto et al, 2004;Oberholzer et al, 1995Oberholzer et al, , 1999Treyer et al, 2002) because of its merits for applications in medical and biomedical treatment (Walde and Ichikawa, 2001). Furthermore, the enzymes entrapped inside liposome have been proved to be more stable under high temperature (Wang et al, 2003;Han et al, 1998) and higher selectivity .…”