Quantifying Synergy, Thermostability, and Targeting of Cellulolytic Enzymes and Cellulosomes with Polymerization-Based Amplification

Abstract

We present a polymerization-based assay for determining the potency of cellulolytic enzyme formulations on pretreated biomass substrates. Our system relies on monitoring the autofluorescence of cellulose and measuring the attenuation of this fluorescent signal as a hydrogel consisting of poly(ethylene glycol) (PEG) polymerizes on top of the cellulose in response to glucose produced during saccharification. The one-pot method we present is label-free, rapid, highly sensitive, and requires only a single pipetting step. Using model enzyme formulations derived from Trichoderma reesei, Trichoderma longibrachiatum, Talaromyces emersonii and recombinant bacterial minicellulosomes from Clostridium thermocellum, we demonstrate the ability to differentiate enzyme performance based on differences in thermostability, cellulose-binding domain targeting, and endo/exoglucanase synergy. On the basis of its ease of use, we expect this cellulase assay platform to be applicable to enzyme screening for improved bioconversion of lignocellulosic biomass.

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

“…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] .…”

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

“…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.…”

Hydrophobic Coatings by Thiol-Ene Click Functionalization of Silsesquioxanes with Tunable Architecture

“…The brightest emission of raw cotton is produced by the cotton autofluorescence, which lies in the same spectral region of CPM emission. As it is common for natural fibers, cotton fluorescence has its origin in residual nucleotides, such as nicotinamide adenine dinucleotide phosphate (NADPH), and in the structure of cellulose, though the molecular origin of the latter phenomenon is still poorly understood [ 28 , 29 ]. Therefore, while it is not possible to determine the extent of CPM binding to SH-NBBs, the lower emission of Cotton-NBB80h can be reasonably ascribed to the quenching of cotton self-fluorescence resulting from the surface functionalization with SH-NBBs.…”