Horseradish Peroxidase as a Catalyst for Atom Transfer Radical Polymerization

Sigg, Severin J.; Seidi, Farzad; Renggli, Kasper; Silva, Tilana B.; Kali, Gergely; Bruns, Nico

doi:10.1002/marc.201100579

Cited by 19 publications

(24 citation statements)

References 2 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…During its catalytic cycle, GOx utilizes molecular oxygen as an electron acceptor, oxidizing β‐ d ‐glucose to d ‐gluconolactone and producing hydrogen peroxide as a byproduct (Swoboda & Massey, ; Figure a). We developed the current system relying on GOx and HRP working in tandem as a bienzymatic initiation system for polymerization of phenolated fluorescent macromonomers (Sakai, Komatani, & Taya, ; Sakai et al, ; Shoda, Uyama, Kadokawa, Kimura, & Kobayashi, ; Sigg et al, ; Uyama, Kurioka, & Kobayashi, ; Vanella et al, ; Zavada, Battsengel, & Scott, ). To produce the macromonomers, we modified sodium alginate (M W = 70,000 g/mol) with tyramine and aminofluorescein (Figure b; Liu, Sakai, Kawa, & Taya, ; Sakai & Kawakami, ) using carbodiimide activation chemistry.…”

Section: Resultsmentioning

confidence: 99%

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

Vanella

Nash

2019

Biotech & Bioengineering

View full text Add to dashboard Cite

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

show abstract

Section: Resultsmentioning

confidence: 99%

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

Vanella

Nash

2019

Biotech & Bioengineering

View full text Add to dashboard Cite

show abstract

“…Recently we demonstrated the use of GOx in combination with Fenton's reagent for quantifying enzyme activity based on fluorescent hydrogel formation 37,38 . In the current system, GOx and HRP worked in tandem as a bi-enzymatic initiation system for polymerization of phenolated macromonomers [39][40][41][42] .…”

Section: Resultsmentioning

confidence: 99%

Genetically Encoded Stimuli-Responsive Cytoprotective Hydrogel Capsules for Single Cells Provide Novel Genotype–Phenotype Linkage

Vanella

Bazin

et al. 2019

Chem. Mater.

View full text Add to dashboard Cite

Modification of cell surfaces with synthetic polymers is a promising approach for regulating cellular behavior. Here we describe a genetically controlled strategy for selectively encapsulating single yeast cells in synthetic microniches comprised of cross-linked phenol-modified alginate and chitosan hydrogel capsules. Our system links inducible gene expression with enzyme-mediated hydrogel polymerization and provides a novel genotype-phenotype linkage whereby only cells carrying a requisite gene encoding a flavin adenine dinucleotide (FAD)-dependent oxidoreductase undergo autonomous enzyme-mediated surface polymerization resulting in formation of hydrogel capsules. The composition of the hydrogel capsules is highly tunable and the capsule sizes are pH-responsive, allowing for control of capsule porosity and shell diameters over a range of 15-80 µm. The hydrogel capsules prevent extracellular proteins from reaching the cell surface, thereby conferring cellular immunity to lytic enzyme cocktails and rendering the hydrogel capsules cytoprotective against osmotic shock. We demonstrate the utility of this genetically controlled artificial hydrogel-encapsulated cell phenotype by isolating and enriching uniform eukaryotic cell lineages from genetically heterogeneous cell mixtures at 95-100% efficiency. The encapsulated cells remained viable and were capable of dividing and breaking free from their hydrogel capsules, allowing further propagation of selected cells. Our bottom-up approach to cellular compartmentalization links inducible intracellular genetic components with an artificial extracellular matrix that resists enzymatic lysis and mediates communication with the surrounding environment through a size-tunable and permeable hydrogel capsule.

show abstract

“…Very recently the groups around Bruns [39] and di Lena [40,41] independently reported on biocatalytic atom transfer radical polymerizations.…”

Section: Other Enzyme Systems/miscellaneousmentioning

confidence: 99%

Enzyme Initiated Radical Polymerizations

2012

View full text Add to dashboard Cite

show abstract

Horseradish Peroxidase as a Catalyst for Atom Transfer Radical Polymerization

Cited by 19 publications

References 2 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

Genetically Encoded Stimuli-Responsive Cytoprotective Hydrogel Capsules for Single Cells Provide Novel Genotype–Phenotype Linkage

Enzyme Initiated Radical Polymerizations

Contact Info

Product

Resources

About