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A microplate assay for extracellular hydrolase detection.

Athayde Neves Junior1, Veronica da Silva Cardoso1, Felipe Raposo Passos Mansoldo1

  • 1Bioinovar Laboratory, Institute of Microbiology Paulo de Góes, Centro de Ciências da Saúde, Federal University of Rio de Janeiro (UFRJ), Rio de Janeiro, Brazil.

Journal of Microbiological Methods
|May 25, 2020
PubMed
Summary

A new 24-well microplate method offers a rapid, low-cost alternative for detecting enzyme activity, simplifying microbial bioprospecting and industrial hydrolase screening.

Keywords:
AmylasesEndoglucanasesEnzymatic activity detectionEnzymatic agar-platesMicroplate qualitative testsPolygalacturonases

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Area of Science:

  • Biotechnology
  • Microbiology
  • Enzymology

Background:

  • Conventional methods for detecting microbial hydrolases are time-consuming and costly.
  • Qualitative detection of enzyme activity often requires large quantities of microbial strains or extracts.
  • Bioprospecting for enzyme-producing microorganisms necessitates efficient screening techniques.

Purpose of the Study:

  • To introduce a novel, simplified qualitative method for detecting enzyme activity using 24-well microplates.
  • To establish optimal substrate concentrations for enzyme detection in microplates.
  • To validate the new method against traditional techniques and assess its efficiency for industrial hydrolase screening.

Main Methods:

  • Utilized 24-well microplates for qualitative enzyme activity detection.
  • Tested various substrate concentrations (0-20 g/L) in agar for enzymatic hydrolysis analysis.
  • Validated the method using quantitative enzymatic assays and thin-layer chromatography.

Main Results:

  • The new microplate method demonstrated statistically relevant results for amylase, endoglucanase, and polygalacturonase.
  • Optimal substrate concentrations were identified: 4 g/L carboxymethylcellulose for endoglucanase and 8 g/L for amylase and polygalacturonase.
  • High halo diameters were observed even at low substrate concentrations, indicating method sensitivity.

Conclusions:

  • The 24-well microplate method is a faster, less costly alternative for detecting enzyme activity.
  • This technique is suitable for general screening of industrial hydrolases and studying microbial enzyme production.
  • The method offers a simplified approach for bioprospecting enzyme-producing microorganisms.