Polyelectrolyte Complex Beads by Novel Two-Step Process for Improved Performance of Viable Whole-Cell Baeyer-Villiger Monoxygenase by Immobilization

Abstract

A novel immobilization matrix for the entrapment of viable whole-cell Baeyer-Villiger monooxygenase was developed. Viable recombinant Escherichia coli cells overexpressing cyclohexanone monooxygenase were entrapped in polyelectrolyte complex beads prepared by a two-step reaction of oppositely-charged polymers including highly defined cellulose sulphate. Immobilized cells exhibited higher operational stability than free cells during 10 repeated cycles of Baeyer-Villiger biooxidations of rac-bicyclo[3.2.0]hept-2-en-6-one to the corresponding lactones (1R,5S)-3-oxabicyclo-[3.3.0]oct-6-en-3-one and (1S,5R)-2-oxabicyclo-[3.3.0]oct-6-en-3-one. The morphology of polyelectrolyte complex beads was characterised by environmental scanning electron microscopy; the spatial distribution of polymers in the beads and cell viability were examined using confocal laser scanning microscopy, and the texture was characterised by the mechanical resistance measurements.

Core–Shell Spheroidal Hydrogels Produced via Charge-Driven Interfacial Complexation

“…Similar core–shell structures have been previously reported, for instance, by coextruding an alginate solution around a second liquid phase and precipitating the core–shell droplet into a divalent cation bath (sometimes termed ionotropic gelation), leading to the gelation of the alginate around a liquid core. , Although these structures have long-term stability in water, they have a limited life span in the range of just a few minutes when stored in a monovalent electrolyte solution, even at concentrations as low as 10 mM, due to alginate dissolution . The use of charge-driven complexation for the production of core–shell structures at the micro- and nanoscale has been broadly reported, while to our knowledge, stable macroscopic core–shell porous hydrogels have only been reported using a combination of ionotropic gelation and charge-driven complexation of oppositely charged species. − Macroscopic core–shell hydrogels offer applications in tissue engineering, cell culture, and controlled delivery of active excipients. Core–shell hydrogels are potential microreactors for biocatalysis from which substrate and product are freely able to migrate through the shell while avoiding enzyme leakage.…”

Baeyer–Villiger Monooxygenases: Tunable Oxidative Biocatalysts

“…Over the past decades, some progress was made in optimizing large-scale reactions, employing strategies such as biphasic systems, whole cell conversions, and enzyme immobilization. ,, Reviews focusing on biocatalysis with BVMOs from prior years are referred to for a broader overview. ,− Alongside these developments, several groups have explored different reactions and combinations of reactions with BVMOs, of which we present an overview, focused on studies from recent years. In particular, these combinations of reactions include cascades, as well as chemoenzymatic routes.…”

Internal Structure of Matrix-Type Multilayer Capsules Templated on Porous Vaterite CaCO3 Crystals as Probed by Staining with a Fluorescence Dye

“…Modern methods used for the investigation of the internal structure of the capsules are usually based on imaging technologies and include characterization of capsule mechanical properties using atomic force microscopy (AFM) [ 24 ]; capsule structure using scanning electron microscopy (SEM) including cryo-SEM [ 25 ] and environmental SEM [ 26 ] to analyze fully hydrated and chemically unmodified state of the capsules. However, these methods can be destructive or require drying of the capsules.…”