Enhanced Heat Stability of α‐Chymotrypsin through Single‐Enzyme Confinement in Attoliter Liposomes

Abstract

The entrapment of α-chymotrypsin (α-CT) within 70-140 nm liposomes formed from POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) leads to an unexpected and remarkable increase in the thermal stability of the enzyme. This finding is based on the observation that heating aqueous suspensions of α-CT-containing POPC liposomes to 80 °C for 30 minutes resulted in partial enzyme inactivation, whereas the same treatment of aqueous solutions of free α-CT inactivated the enzyme completely. The stabilizing effect of enzyme confinement in the attoliter volumes of the liposomes was found to increase with decreasing numbers of α-CT molecules per liposome. Single-enzyme confinement was particularly effective, as intermolecular interactions between heat-denatured α-CT molecules (causing irreversible inactivation) are not possible.

Spectrophotometric Quantification of Peroxidase with p-Phenylene-diamine for Analyzing Peroxidase-Encapsulating Lipid Vesicles

“…This calculated value is lower than the HRP concentration used for dispersing the POPC molecules (62 μ M or 31 μ M after dilution, see the Experimental Section). We did not attempt to increase the entrapment efficiency, for example, by using freeze–thawing cycles53 or by applying another method 54…”

“…In the case of the reducing substrate ABTS 2− , the situation is a bit different, since the rate of reaction is determined by analyzing the formation of ABTS •− , i.e., no nonenzymatic follow-up reactions are involved in the spectrophotometric analysis, as long as the disproportionation of two ABTS •− species into ABTS 2− and ABTS can be ignored. 8 For the sake of a direct comparison, we also determined the apparent K M and k cat values for ABTS The general methodology used for the preparation of HRPcontaining POPC vesicles is basically the same as described previously for encapsulating HRP in vesicles formed from a mixture of egg yolk phosphatidylcholine (egg PC), 1,2dimyristoyl-sn-glycero-3-phospho-(1′-rac-glycerol sodium salt (DMPG), and cholesterol (4:1:5, molar ratios), 40 or for encapsulating α-chymotrypsin in POPC vesicles, 41,53 see the Supporting Information (Figure S-2). In short, a thin film of POPC was first formed inside a round-bottom flask made from silicate glass.…”

“…The general methodology used for the preparation of HRP-containing POPC vesicles is basically the same as described previously for encapsulating HRP in vesicles formed from a mixture of egg yolk phosphatidylcholine (egg PC), 1,2-dimyristoyl- sn -glycero-3-phospho-(1′- rac -glycerol sodium salt (DMPG), and cholesterol (4:1:5, molar ratios), or for encapsulating α-chymotrypsin in POPC vesicles, , see the Supporting Information (Figure S-2). In short, a thin film of POPC was first formed inside a round-bottom flask made from silicate glass.…”

High Permeability of Polyunsaturated Lipid Bilayers As Applied to Attoliter Enzyme Reactors

“…1−4 Polymersomes and phospholipid vesicles or liposomes are well-characterized assemblies which offer the opportunity to spatially confine catalytically different enzymes for a cascade reaction 5−10 or to isolate single enzyme molecules for their thermal stabilization. 11 These systems are also applicable to fabricate multicompartmentalized soft materials like vesosomes 12 for achieving spatially controlled encapsulation of different compounds and chemical reactions. 13 Among molecular assemblies, phospholipid vesicles with high biocompatibility are suitable for developing functional drug carriers and materials mimicking biological membranes.…”

Fabrication of Shape‐Controlled Palladium Nanoparticle‐Decorated Electrospun Polypyrrole/Polyacrylonitrile Nanofibers for Hydrogen Peroxide Coalescing Detection

“…Nevertheless, the nature of enzymes makes this strategy sensitive to pH, salt concentration, humidity, and temperature. This intrinsic thermal and chemical instability of enzyme‐based systems results in short sensor lifetimes and poor storage characteristics …”