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Related Experiment Video

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Determination of Microbial Extracellular Enzyme Activity in Waters, Soils, and Sediments using High Throughput Microplate Assays
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Developing a fluorometric urease activity microplate assay suitable for automated microbioreactor experiments.

Frédéric M Lapierre1, Isabel Bolz1, Jochen Büchs2

  • 1Munich University of Applied Sciences HM, Munich, Germany.

Frontiers in Bioengineering and Biotechnology
|October 3, 2022
PubMed
Summary
This summary is machine-generated.

A new fluorescent assay accurately quantifies urease activity, crucial for Microbial Induced Calcite Precipitation research. This method enables efficient, automated measurements during microbial cultivation experiments.

Keywords:
MICPautomationenzymatic assaymicrobioreactorsporosarcina pasteuriiurease

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Area of Science:

  • Biochemistry
  • Microbiology
  • Environmental Science

Background:

  • Urease activity quantification is vital for Microbial Induced Calcite Precipitation (MICP) research.
  • Existing methods may require extensive sample preparation or lack automation capabilities.
  • Efficient and reliable urease activity measurement is needed to advance MICP studies.

Purpose of the Study:

  • To develop and validate a novel microplate assay for quantifying urease activity.
  • To enable automated and high-throughput measurements of urease activity.
  • To provide a straightforward, non-hazardous, and cost-effective method for MICP research.

Main Methods:

  • A microplate assay utilizing the fluorescent pH indicator fluorescein, urea, and a phosphate buffer.
  • Measurement of fluorescence increase resulting from urea hydrolysis by microbial urease.
  • Validation using microplate readers and the BioLector microbioreactor system for automated measurements.

Main Results:

  • The fluorescence signal slope strongly correlated with urease activity measured by standard offline methods.
  • Automated measurements were achieved without sample pre-treatment (e.g., centrifugation, OD adjustment).
  • The assay demonstrated robustness against sample turbidity, salinity, and buffer concentration variations.

Conclusions:

  • The developed fluorescent microplate assay provides a reliable, efficient, and automated method for quantifying urease activity.
  • This assay significantly enhances the efficiency of research on ureolytic bacteria for MICP.
  • The assay's principle is adaptable for measuring other pH-altering enzyme activities.