Abstract
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.