Chromogenic Polymer Hydrogel-Based Enzyme Screening Assay: A High Throughput Method to Screen the Carbohydrate-Active Enzymes Using Synthetic Substrates

Abstract

Source: Schückel, J., et al. High-throughput Screening of Carbohydrate-degrading Enzymes Using Novel Insoluble Chromogenic Substrate Assay Kits. J. Vis. Exp. (2016).

In this video, we demonstrate a high throughput assay for carbohydrate-active enzyme screening using insoluble chromogenic polymer hydrogel substrates. The enzyme degrades these substrates into a soluble colored product that can be quantified by measuring the color intensity of the product.

Protocol

1. Activation of the assay kit plate

1. Activate the 96-well filter (containing blue CPH-xylan) assay kit plate (List of Materials) by adding 200 µl activation solution (obtained with the kit) into each well, followed by 10 min incubation at room temperature without agitation.
2. Apply vacuum using a vacuum manifold (with the spacer block inside and any standard, transparent 96-well plate as a collection plate) to remove the extant activation solution. It is also possible to use a centrifuge at 2,700 x g for 10 min for this step instead of the vacuum manifold.
3. Wash the CPH substrates by adding 100 µl steril....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Assay kit plates	Glycospot		customized assay kit plates
Activation solution	Glycospot		for activating CPH substrates
350 ml receiver plate spacer block for vacuum manifold	Pall Corporation	5015	spacer block
96-well MultiScreen HV filter plate,  0.45 µm, clear, non-sterile	Millipore	MSHVN4510	assay plate
96-Well Microplates, Polypropylene	Greiner Bio-One	651201	collection plate after washing the substrates
Nunc MicroWell 96-Well Microplates	Thermo Scientific	269620	product plate
Diaphragm pump MZ 2 NT	Vacuubrand	732000	vacuum pump used with the vacuum manifold
Infors HT Ecotron	Infors HT	4950132 (Buch & Holm)	horizontal shaker
SpectraMax M5	Molecular Devices	10067-750 (VWR)	96-well plate absorbance reader
Vacuum manifold	Pall Corporation	5017	vacuum manifold
Endo-cellulase (EGII) (Trichoderma longibrachiatum)	Megazyme	E-CELTR	cellulase [cel]
α-amylase (Bacillus licheniformis)	Megazyme	E-BLAAM	amylase [amy]