A new generation of versatile chromogenic substrates for high-throughput analysis of biomass-degrading enzymes

Abstract

BackgroundEnzymes that degrade or modify polysaccharides are widespread in pro- and eukaryotes and have multiple biological roles and biotechnological applications. Recent advances in genome and secretome sequencing, together with associated bioinformatic tools, have enabled large numbers of carbohydrate-acting enzymes to be putatively identified. However, there is a paucity of methods for rapidly screening the biochemical activities of these enzymes, and this is a serious bottleneck in the development of enzyme-reliant bio-refining processes.ResultsWe have developed a new generation of multi-coloured chromogenic polysaccharide and protein substrates that can be used in cheap, convenient and high-throughput multiplexed assays. In addition, we have produced substrates of biomass materials in which the complexity of plant cell walls is partially maintained.ConclusionsWe show that these substrates can be used to screen the activities of glycosyl hydrolases, lytic polysaccharide monooxygenases and proteases and provide insight into substrate availability within biomass. We envisage that the assays we have developed will be used primarily for first-level screening of large numbers of putative carbohydrate-acting enzymes, and the assays have the potential to be incorporated into fully or semi-automated robotic enzyme screening systems.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-015-0250-y) contains supplementary material, which is available to authorized users.

High-throughput Screening of Carbohydrate-degrading Enzymes Using Novel Insoluble Chromogenic Substrate Assay Kits

“…The reproducibility of the assay is shown by the error bars (standard error of mean, SEM, of three replicas). More detailed experiments on the reproducibility of this assay are published elsewhere 8 .…”

“…The limitation of this assay kit lies in the detection of endo-enzyme activity, as CPH as well as the ICB substrates are not degradable by exo-enzymes most likely due to steric hindrance arising from the dye and crosslinker molecules 8 .…”

“…It has been shown, that this screening assay using CPH substrates can be also used in an agar plate format, where the soluble reaction products create a colored halo after overnight incubation. 8 The assay kits can be used to screen purified enzymes and their potential side-activities as demonstrated in Figure 2. Side-activities can arise from a single enzyme and its promiscuous specificity but also from a fact that the sample analyzed is a mixture of different enzymes and their synergic effect needs to be studied.…”

Pea Border Cell Maturation and Release Involve Complex Cell Wall Structural Dynamics

“…These observations suggested that cell wall functional fine-tuning associated with border cell release is achieved by coordinated cell wall remodeling rather than by extensive polysaccharide degradation. To test this assumption, we used a recently described set of chromogenic hydrogel substrates developed for screening hydrolytic cell walldegrading enzyme activities (Kra cun et al, 2015). We used a panel of seven chromogenic substrates (chromogenic polymer hydrogel [CPH]) representing major cell wall components and one insoluble chromogenic biomass substrate (pectin-rich orange [Citrus spp.…”

“…] peel as a tested substrate for HG-degrading enzymes). As with the CoMPP work, we separately analyzed homogenates of border cells, root tips, as well as washout material, and using this approach, no appreciable enzyme activity was observed, at least not at detection levels reported for CPH substrates (Kra cun et al, 2015) even after 24 h (Supplemental Fig. S5).…”