Enhancement of apparent substrate selectivity of proteinase K encapsulated in liposomes through a cholate‐induced alteration of the bilayer permeability

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Abstract
Proteinase K‐containing liposomes with highly selective membrane permeability properties were prepared. The selectivity obtained was with respect to the two substrate molecules added to the external aqueous phase of the liposomes: acetyl‐L‐Ala‐Ala‐Ala‐p‐nitroanilide (Ac‐AAA‐pNA) and succinyl‐L‐Ala‐Ala‐Ala‐p‐nitroanilide (Suc‐AAA‐pNA). The liposome‐forming lipid used was POPC (1‐palmitoyl‐2‐oleoyl‐sn‐glycero‐3‐phosphocholine) and modulation of the membrane permeability was achieved using the detergent cholate. Proteinase K‐containing mixed liposomes (PKCL) were prepared by adding cholate to preformed proteinase K‐containing POPC liposomes (PKL) at a defined effective cholate/POPC molar ratio in the liposomal bilayer membrane Re. Proteinase K was kept inside PKCL with a negligible amount of leakage into the bulk aqueous phase at Re ≤ 0.30. At higher Re, leakage of proteinase K was pronounced, even under conditions where POPC/cholate mixed liposomes seemed to be still intact (0.30 < Re ≤ 0.39). At Re ≤ 0.30, the reactivity of proteinase K in the PKCL measured with the externally added substrate Ac‐AAA‐pNA increased with increasing Re, while the reactivity measured with Suc‐AAA‐pNA remained low, regardless of the Re value. This showed that externally added Ac‐AAA‐pNA molecules permeated the liposomal membrane more easily than Suc‐AAA‐pNA by modulating the membrane with cholate. Consequently, Ac‐AAA‐pNA was hydrolyzed in PKCL with considerably higher apparent substrate selectivity in comparison with the cases of proteinase K in PKL and free proteinase K (without liposomal encapsulation). The results obtained clearly demonstrate that the prepared PKCL can be utilized as a kind of nano‐scaled bioreactor system which can take up a particular target substrate with high apparent substrate selectively from the external phase of the liposomes. Inside the liposomes, the target substrate is then converted into the corresponding products.

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