Investigation of Horseradish Peroxidase Kinetics in an “Organelle-Like” Environment

Abstract

In order to mimic cell organelles, artificial nanoreactors have been investigated based on polymeric vesicles with reconstituted channel proteins (outer membrane protein F) and coencapsulated enzymes horseradish peroxidase (HRP) along with a crowding agent (Ficoll or polyethylene glycol) inside the cavity. Importantly, the presence of macromolecules has a strong impact on the enzyme kinetics, but no influence on the integrity of vesicles up to certain concentrations. This particular design allows for the first time the determination of HRP kinetics inside nanoreactors with crowded milieu. The values of the Michaelis-Menten constant (K ) measured for HRP in a confined space (encapsulated in nanoreactors) in the absence of macromolecules are ≈50% lower than in free conditions, and the presence of a crowding agent results in a further pronounced decrease. These results clearly suggest that activities of enzymes in confined spaces can be tuned by varying the concentrations of crowding compounds. The present investigation represents an advance in nanoreactor design by considering the influence of environmental factors on enzymatic performance, and it demonstrates that both encapsulation and the presence of a crowding environment increase the enzyme-substrate affinity.

Bioinspired Molecular Factories with Architecture and In Vivo Functionalities as Cell Mimics

“…We used AOs based on PMOXA 6 ‐PDMS 44 ‐PMOXA 6 polymersomes combined with membrane proteins and enzymes, since they have already been reported to preserve their functionality in cells and in vivo . Spatial localization of enzymes within AOs increases the enzyme‐substrate affinity . As a model we loaded horseradish peroxidase (HRP) into the cavities of PMOXA 6 ‐PDMS 44 ‐PMOXA 6 polymersomes that contained the bacterial outer membrane protein F (OmpF) in the membrane to control passage of specific substrates and products.…”

“…[29,43] Spatial localization of enzymes within AOs increases the enzyme-substrate affinity. [28] As a model we loaded horseradish peroxidase (HRP) into the cavities of PMOXA 6 -PDMS 44 -PMOXA 6 polymersomes that contained the bacterial outer membrane protein F (OmpF) in the membrane to control passage of specific substrates and products. We selected HRP because of its biological role in detoxification of H 2 O 2 , known as toxic by-products of mitochondrial respiration in eukaryotic cells, and its role in modulating the protein function of by thiol oxidation.…”

“…AOs are formed by encapsulation of enzymes inside polymersomes equipped with channel porines, prior to their internalization by donor cells. AOs have various advantages, such as an increase in the in situ enzymatic activity by nanoscale confinement, a protection of the enzymes inside their cavity and the possibility to obtain separate reaction spaces by coencapsulation of different AOs when more complex reactions are studied . In our approach, donor cells are first preloaded with supplementary cargos (synthetic molecules, nanocompartments and AOs) and simultaneously engineered (genetically if necessary) to overexpress specific biomacromolecules.…”

“…AOs have various advantages, such as an increase in the in situ enzymatic activity by nanoscale confinement, a protection of the enzymes inside their cavity and the possibility to obtain separate reaction spaces by coencapsulation of different AOs when more complex reactions are studied. [28][29][30] In our approach, donor cells are first preloaded with supplementary cargos (synthetic molecules, nanocompartments and AOs) and simultaneously engineered (genetically if necessary) to overexpress specific biomacromolecules. Then a special "vesicular buffer" is added to the cells and induces formation of GPMVs that are equipped with the functional artificial cargoes, specifically implemented by prior internalization into the donor cells (E-GPMVs).…”

Recent Progress in Micro/Nanoreactors toward the Creation of Artificial Organelles

“…They incorporated the bacterial porin outer membrane protein F (OmpF), into polymersomes since porins allow for passive diffusion of small solutes . OmpF was incorporated within the hydrophobic domain of polymersomes prepared from the amphiphilic PMOXA‐ b ‐PDMS‐ b ‐PMOXA block copolymer and several enzymes including β‐lactamase, acid phosphatase, penicillin acylase, or HRP, have been successfully encapsulated demonstrating encapsulated enzymatic reactions …”