Insoluble Chromogenic Biomass-Based Enzyme Screening: An Assay to Evaluate the Degradation Activity of Plant Biomass-Degrading Enzymes
Published: April 30, 2023
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 Enzyme reaction 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 …
Disclosures
The authors have nothing to disclose.
Materials
| 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] |
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Cite This Article
Insoluble Chromogenic Biomass-Based Enzyme Screening: An Assay to Evaluate the Degradation Activity of Plant Biomass-Degrading Enzymes. J. Vis. Exp. (Pending Publication), e21177, doi: (2023).