Home › Biochemistry › High-throughput Screening of Carbohydrate-degrading Enzymes Using Novel Insoluble Chromogenic Substrate Assay Kits
Biochemistry JoVE (Open Access) Citable · DOI

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

DOI: 10.3791/54286-v
What you'll learn
  • ✓ Set up and execute high-throughput chromogenic enzyme assays using CPH substrates
  • ✓ Perform detection and quantification of polysaccharide-degrading enzyme activity
  • ✓ Interpret enzymatic degradation results from insoluble chromogenic biomass substrates
Protocol

Biopharma Insights A high-throughput assay for enzyme screening is described. This multiplexed ready-to-use assay kit comprises of pre-chosen Chromogenic Polymer Hydrogel (CPH) substrates and complex Insoluble Chromogenic Biomass (ICB) substrates. Target enzymes are polysaccharide degrading endo-enzymes and proteases.

Difficulty
intermediate
Total time
~2–4 hours per assay batch (including substrate preparation, incubation, and detection)
Biosafety
BSL-1

Steps

1
Perform chromogenic assay with CPH substrates

Prepare and incubate target enzymes with Chromogenic Polymer Hydrogel substrates in the ready-to-use assay kit, following the multiplexed protocol design.

▶ 00:40
2
Detect and quantify CPH substrate degradation

Measure enzymatic activity through chromogenic detection of CPH substrate breakdown, collecting quantitative readouts for enzyme screening.

▶ 00:40
3
Perform assay with insoluble chromogenic biomass

Incubate target enzymes with complex Insoluble Chromogenic Biomass substrates using the same high-throughput multiplexed approach.

▶ 02:41
4
Detect and quantify ICB substrate degradation

Measure enzymatic activity on ICB substrates through chromogenic detection and collect quantitative data for enzyme comparison.

▶ 02:41
5
Analyze enzymatic degradation results

Review and interpret degradation profiles of polysaccharide-degrading endo-enzymes and proteases across chromogenic substrate conditions.

▶ 04:15
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