Insoluble Chromogenic Biomass-Based Enzyme Screening: An Assay to Evaluate the Degradation Activity of Plant Biomass-Degrading Enzymes

Abstract

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

This video demonstrates an assay to screen enzymes effective in degrading complex polysaccharides in plant biomass using insoluble chromogenic biomass or ICB. The enzymes hydrolyze ICB to release soluble dye-linked oligosaccharides, and the enzyme activity is assessed by colorimetric analysis of the reaction product.

Protocol

1. Chromogenic Assay with ICB Substrates in a 96-well Format

1. Enzyme reaction
   1. Add the 150 µl 100 mM sodium acetate buffer, pH 4.5 and 5 µl 31 U/ml endo-xylanase solution to each well of the assay kit plate containing red ICB-wheat straw (final enzyme concentration in the well: 1 U/ml).
      NOTE: The assay kit plates (96-well filter plates containing ICB substrates) are manufactured as described in literature (see List of Materials). The ICB substrates are stable in buffers with a pH range between pH 3.0 to 10.0. Always include buffer alone as a negative control, commercial enzymes as ....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Assay kit plates	Glycospot		Customized assay kit plates
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-β-1,4-xylanase M4 (Aspergillus niger)	Megazyme	E-XYAN4	Xylanase [xyl]