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

Overview

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 a positive control and use a statistically appropriate number of replicas.
      1. Do not activate the ICB substrates like the CPH substrate plate, but remove the stabilizer by washing three times with 100 µl water followed by vacuum filtration or centrifugation.
   2. Put the product plate underneath the substrate plate to collect any potential leakage from the substrate plate during shaking.
   3. Incubate the reaction at 25 °C shaking at 150 rpm for 2 hr.
      NOTE: An active enzyme is degrading the insoluble chromogenic polysaccharides in the ICB substrate into soluble oligosaccharides, which are visible as colored supernatant. ICB substrates are stable up to 90 °C. The incubation time should be increased up to 24 hr if non-purified enzymes such as culture broths are used.
   4. Place the product plate inside the vacuum manifold with the spacer block inside.
   5. Place the assay kit plate on top and apply vacuum (maximum negative pressure of -60 kPa) or use a centrifuge to filtrate the product from the assay kit plate into the well of the product plate.
      NOTE: The filtrate containing the colored oligosaccharides as reaction product is now in the collection plate for further analysis.

2. Detection and quantification

1. Check that the volume of liquid in each well of the collection plate is approximately the same by visual inspection.
2. Read the absorbance of the collection plate at 517 nm for red ICB-wheat straw using a plate reader.
3. When doing data analysis — subtract the buffer — only negative control values from the values from the wells where an enzyme was added. Calculate the mean value and the standard error of means (SEM) from the replicate wells.
   NOTE: In case of screening unknown enzymes, we suggest making a dilution series in order to obtain more detailed data about the dynamic range of enzyme's activity.

Materials

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]