title: Redox-initiated hydrogel system for detection and real-time imaging of cellulolytic enzyme activity.
abstract: Understanding the process of biomass degradation by cellulolytic enzymes is of urgent importance for biofuel and chemical production. Optimizing pretreatment conditions and improving enzyme formulations both require assays to quantify saccharification products on solid substrates. Typically, such assays are performed using freely diffusing fluorophores or dyes that measure reducing polysaccharide chain ends. These methods have thus far not allowed spatial localization of hydrolysis activity to specific substrate locations with identifiable morphological features. Here we describe a hydrogel reagent signaling (HyReS) system that amplifies saccharification products and initiates crosslinking of a hydrogel that localizes to locations of cellulose hydrolysis, allowing for imaging of the degradation process in real time. Optical detection of the gel in a rapid parallel format on synthetic and natural pretreated solid substrates was used to quantify activity of T. emersonii and T. reesei enzyme cocktails. When combined with total internal reflection fluorescence microscopy and AFM imaging, the reagent system provided a means to visualize enzyme activity in real-time with high spatial resolution (<2 μm). These results demonstrate the versatility of the HyReS system in detecting cellulolytic enzyme activity and suggest new opportunities in real-time chemical imaging of biomass depolymerization.
authors.affiliation: Lehrstuhl für Angewandte Physik and Center for Nanoscience, Ludwig-Maximilians-Universität, Amalienstrasse 54, 80799 Munich (Germany).
authors.forename: Klara H
authors.initials: KH
authors.lastname: Malinowska
authors.forename: Tobias
authors.initials: T
authors.lastname: Verdorfer
authors.forename: Aylin
authors.initials: A
authors.lastname: Meinhold
authors.forename: Lukas F
authors.initials: LF
authors.lastname: Milles
authors.forename: Victor
authors.initials: V
authors.lastname: Funk
authors.forename: Hermann E
authors.initials: HE
authors.lastname: Gaub
authors.forename: Michael A
authors.initials: MA
authors.lastname: Nash
mesh_terms: D018533:Biomass; D002480:Cellulase / Q000378:metabolism*; D002482:Cellulose / Q000378:metabolism*; D020136:Hydrogel, Polyethylene Glycol Dimethacrylate / Q000737:chemistry*; D006868:Hydrolysis; D018625:Microscopy, Atomic Force; D008856:Microscopy, Fluorescence; D010084:Oxidation-Reduction
chemical_list: D020136:Hydrogel, Polyethylene Glycol Dimethacrylate; D002482:Cellulose; D002480:Cellulase
keywords: TIRF imaging; Trichoderma reesei; biomass; cellulase enzymes; radical reactions