Redox‐Initiated Hydrogel System for Detection and Real‐Time Imaging of Cellulolytic Enzyme Activity

Malinowska, Klara H.; Verdorfer, Tobias; Meinhold, Aylin; Milles, Lukas F.; Funk, Victor; Gaub, Hermann E.; Nash, M.

doi:10.1002/cssc.201402428

Cited by 13 publications

(19 citation statements)

References 44 publications

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“…The system we reported was made possible by GOx initiator enzymes that were synthesized by the cells from single‐copy plasmids carrying the GOx gene, and surface‐displayed on the outer cell wall. A competing approach has also been reported to produce hydrogels using a GOx cascade with Fenton's reagent, which can produce hydroxyl radicals for polymerization of vinylated compounds and fluorophores (Malinowska & Nash, ; Malinowska et al, ; Malinowska, Rind, Verdorfer, Gaub, & Nash, ; Pitzler et al, ). Here, we report the utilization of the GOx‐mediated cell encapsulation system for directed evolution of GOx.…”

Section: Introductionmentioning

confidence: 99%

Enzyme‐mediated hydrogel encapsulation of single cells for high‐throughput screening and directed evolution of oxidoreductases

Vanella

Nash

2019

Biotech & Bioengineering

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Directed evolution of oxidoreductases to improve their catalytic properties is being ardently pursued in the industrial, biotechnological, and biopharma sectors.Hampering this pursuit are current enzyme screening methods that are limited in terms of throughput, cost, time, and complexity. We present a directed evolution strategy that allows for large-scale one-pot screening of glucose oxidase (GOx) enzyme libraries in well-mixed homogeneous solution. We used GOx variants displayed on the outer cell wall of yeasts to initiate a cascade reaction with horseradish peroxidase (HRP), resulting in peroxidase-mediated phenol crosscoupling and encapsulation of individual cells in well-defined fluorescent alginate hydrogel shells within~10 min in mixed cell suspensions. Following application of denaturing stress to whole-cell GOx libraries, only cells displaying GOx variants with enhanced stability or catalytic activity were able to carry out the hydrogel encapsulation reaction. Fluorescence-activated cell sorting was then used to isolate the enhanced variants. We characterized three of the newly evolved Aspergillus niger GOx enzyme sequences and found up to~5-fold higher specific activity, enhanced thermal stability, and differentiable glycosylation patterns. By coupling intracellular gene expression with the rapid formation of an extracellular hydrogel capsule, our system improves high-throughput screening for directed evolution of H 2 O 2producing enzymes many folds. K E Y W O R D Scell encapsulation, directed evolution, glucose oxidase, hydrogels, yeast display

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Section: Introductionmentioning

confidence: 99%

Enzyme‐mediated hydrogel encapsulation of single cells for high‐throughput screening and directed evolution of oxidoreductases

Vanella

Nash

2019

Biotech & Bioengineering

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“…Such fur‐shell technology represented a simple and rapid approach of identifying the most active variants from vast populations, as well as a platform for fabrication of polymer‐hybrid cells for interactive biomaterials . Introduced by Nash and co‐workers, fluorescent hydrogels synthesized through the Fenton chemistry–mediated FRP process, were also employed for detecting and imaging the degradation (or hydrolysis) of cellulosic substrates.…”

Section: Applications Of Fenton Reaction‐initiated Frpmentioning

confidence: 99%

Fenton‐Chemistry‐Mediated Radical Polymerization

Reyhani

McKenzie

et al. 2019

Macromol. Rapid Commun.

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have been developed for the synthesis of polymers with diverse structures (linear, star, cyclic, etc.), compositions ( • mopolymer, gradient, alternating, block, etc.), and functionalities (thiol, amine, etc.). [11] The main mechanism of RDRP which results in controllable polymerization is the establishment of a dynamic equilibrium between the propagating radicals (active polymers) and the dormant species via a reversible activation-deactivation process with rate constants k act. and k deact. , respectively. [7,12] With the emergence of RDRP methods such as atom transfer radical polymerization (ATRP), [13][14][15] nitroxide-mediated polymerization (NMP), [16] and reversible addition-fragmentation transfer (RAFT) [11,17,18] there has been significant effort to control these techniques via different physical and chemical stimuli. [19][20][21][22][23][24][25][26][27] Of these, RAFT holds particular promise due to its compatibility with a diverse range of solvents, monomers, and operating conditions. [11,24,[28][29][30][31][32][33][34][35] Among RDRP methods, one might argue that RAFT is the most similar to traditional FRP, with the simple addition of a thiocarbonylthio-containing compound chain transfer agent (CTA, termed as RAFT agent) [36] that can mediate the polymerization, allowing the synthesis of well-defined polymers (Scheme 1). In fact, RAFT CTAs aid in achieving control of the radical polymerization via reversible addition of the thiocarbonylthio group and rapid fragmentation reactions. [37,38] Therefore, relatively analogous activation/initiation systems such as thermal, [4,11,[28][29][30][31][39][40][41] photo, [2,24,[32][33][34][42][43][44][45][46][47][48][49] enzyme, [5,23,[50][51][52][53][54] and redox [3,[55][56][57][58][59] based radical formation can be employed to initiate both FRP and RAFT processes. Despite the huge advances made in this field, new techniques that can overcome some of the drawbacks of these systems are continuously sought. For example, using a thermal initiator requires a high operating temperature, limiting the possibility for in situ FRP/RAFT methods in biological systems, where temperature control is limited. The rate of visible light-activated RAFT polymerization without using photocatalysts is relatively slow, [34,60] while photocatalysts, e.g. iridium and rutheniumbased ones, [24,61] employed in photo-activated processes are expensive. Moreover, photo-mediated polymerization faces difficulties in terms of the equipment requisite to perform photoinitiation at scale. [29] Redox-responsive polymerization systems have many advantages, including a low activation energy (40-85 kJ mol −1 ), easy and facile control over the polymerization at ambient temperatures, very short induction periods, etc. [56,62] Polymerizations initiated by a redox reaction between an oxidizing and a reducing agent are referred to as "redox polymerizations." [63] Use of Radical PolymerizationIn this review, the power of a classical chemical reaction, the Fenton reaction for initiating radical polymeriza...

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“…For this particularc ase, AFM imagingh as offered many possibilities to researchers. [60] In this regard, AFM has delivered important information about cellulose fibers. Borrell et al reported on differences in the shift of the meltingt ransitionf or liposomes (measured in bulk through differentials canning calorimetry) and lipid bilayers (observed throughA FM).…”

Section: Membranes Fibers and Cellsmentioning

confidence: 99%

“…AFM, in combination with total internal reflection fluorescence microscopy,w as used to image, in real-time,c ellulose hydrolysis on nanometer-scalef ibers. [60] In this regard, AFM has delivered important information about cellulose fibers. Lahiji and co-workers investigated the topography and elastic and adhesivep roperties of wood-derived cellulose nanocrystals under different humidity conditions.…”

Section: Membranes Fibers and Cellsmentioning

confidence: 99%

Atomic Force Microscopy Meets Biophysics, Bioengineering, Chemistry, and Materials Science

Toca-Herrera

2019

ChemSusChem

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Briefly, herein the use of atomic force microscopy (AFM) in the characterization of molecules and (bioengineered) materials related to chemistry, materials science, chemical engineering, and environmental science and biotechnology is reviewed. First, the basic operations of standard AFM, Kelvin probe force microscopy, electrochemical AFM, and tip‐enhanced Raman microscopy are described. Second, several applications of these techniques to the characterization of single molecules, polymers, biological membranes, films, cells, hydrogels, catalytic processes, and semiconductors are provided and discussed.

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Redox‐Initiated Hydrogel System for Detection and Real‐Time Imaging of Cellulolytic Enzyme Activity

Cited by 13 publications

References 44 publications

Enzyme‐mediated hydrogel encapsulation of single cells for high‐throughput screening and directed evolution of oxidoreductases

Enzyme‐mediated hydrogel encapsulation of single cells for high‐throughput screening and directed evolution of oxidoreductases

Fenton‐Chemistry‐Mediated Radical Polymerization

Atomic Force Microscopy Meets Biophysics, Bioengineering, Chemistry, and Materials Science

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