Liposomes Carrying Surface-Conjugated Trypsin for Controlled Proteolysis Reactions

Control of the proteolytic activity of trypsin is an important challenge in proteomics and food engineering. In this work, trypsin was covalently conjugated to lipid membranes possessing poly-(ethylene glycol) in a 0.1 M sodium phosphate buffer solution (pH = 7.0) to control the enzyme functions using a liposomal environment. The amount of trypsin conjugated per liposome membrane area was maximized based on the size of the polymer chain of liposomes and the type of cross-linker (glutaraldehyde or tris-succinimidyl aminotriacetate). The secondary structure of trypsin was altered through the conjugation reaction, as revealed with circular dichroism measurements and spectral analyses. Stability of trypsin at 40-60 °C significantly increased through being conjugated to liposomes, whereas free trypsin was clearly deactivated even at 40 °C in an enzyme concentration-dependent manner, indicating the involvement of autolysis mechanism. Liposome-conjugated trypsin was applied to catalyze the digestion of bovine serum albumin at 37 °C and a substrate/trypsin weight ratio of 100. The proteolysis rate observed with liposome-conjugated trypsin was smaller than that of the free enzyme, while the rate clearly depended on the characteristics of liposomes. The results obtained demonstrate that liposomes carrying surface-conjugated trypsin are biocompatible, heat stable, and reactivity-controllable catalysts for proteolysis reactions.