Charged Polypeptide Tail Boosts the Salt Resistance of Enzyme-Containing Complex Coacervate Micelles

Abstract

Encapsulation of proteins can have advantages for their protection, stability, and delivery purposes. One of the options to encapsulate proteins is to incorporate them in complex coacervate core micelles (C3Ms). This can easily be achieved by mixing aqueous solutions of the protein and an oppositely charged neutral-hydrophilic diblock copolymer. However, protein-containing C3Ms often suffer from salt-inducible disintegration due to the low charge density of proteins. The aim of this study is to improve the salt stability of protein-containing C3Ms by increasing the net charge of the protein by tagging it with a charged polypeptide. As a model protein, we used CotA laccase and generated variants with 10, 20, 30, and 40 glutamic acids attached at the C-terminus of CotA using genetic engineering. Micelles were obtained by mixing the five CotA variants with poly( N -methyl-2-vinyl-pyridinium)- block -poly(ethylene oxide) (PM2VP 128 - b -PEO 477 ) at pH 10.8. Hydrodynamic radii of the micelles of approximately 31, 27, and 23 nm for native CotA, CotA-E20, and CotA-E40, respectively, were determined using dynamic light scattering (DLS) and fluorescence correlation spectroscopy (FCS). The encapsulation efficiency was not affected using enzymes with a polyglutamic acid tail but resulted in more micelles with a smaller number of enzyme molecules per micelle. Furthermore, it was shown that the addition of a polyglutamic acid tail to CotA indeed resulted in improved salt stability of enzyme-containing C3Ms. Interestingly, the polyglutamic acid CotA variants showed an enhanced enzyme activity. This study demonstrates that increasing the net charge of enzymes through genetic engineering is a promising strategy to improve the practical applicability of C3Ms as enzyme delivery systems.

Biochemical Characteristics of Laccases and their Practical Application in the Removal of Xenobiotics from Waters

“…Currently, work is being undertaken to modify the enzymes, which are then immobilized. When a charged polypeptide tail was added at the C-terminus, the salt resistance of laccase placed in coacervate core micelles (C3M) by encapsulation was enhanced [330]. The use of CotA with a polyglutamic acid tail resulted in more micelles with fewer enzyme molecules per micelle, better stability and increased laccase activity.…”

pH-Dependent Solution Micellar Structure of Amphoteric Polypeptoid Block Copolymers with Positionally Controlled Ionizable Sites

“…Nonequilibrium assembly of ionic BCP micelles can afford a diverse range of intermediate morphologies that are kinetically trapped . Electrostatic interactions are known to play an important role in the micellization and micellar structure of ionic BCPs, giving rise to their structural dependence on the solution pH, − ionic strength, − and specific ions in aqueous media. , As a result, ionic BCP micelles are used in a wide range of technical applications including protein encapsulation and delivery, , ion transport media, template synthesis, etc. Early studies of ionic BCP micelles have mainly focused on those where the ionizable monomers are randomly distributed in the solvophilic segments, installed by postpolymerization functionalization approach .…”