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Assaying for Inorganic Polyphosphate in Bacteria
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A sensitive pH indicator-based spectrophotometric assay for PHB depolymerase activity on microtiter plates.

Maria Angeles Camacho-Ruiz1, Marcelo Müller-Santos, Xitlalli D Hernández-Mancillas

  • 1Biotecnología Industrial, Centro de Investigación y Asistencia en Tecnología y Diseño del Estado de Jalisco (CIATEJ), Zapopan, Jalisco 45019, Mexico. jrodriguez@ciatej.mx.

Analytical Methods : Advancing Methods and Applications
|August 7, 2020
PubMed
Summary
This summary is machine-generated.

A new spectrophotometric assay effectively screens for polyhydroxyalkanoate (PHA) depolymerase activity. This method enhances sensitivity and aids in discovering novel PHA-degrading bacterial strains.

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Area of Science:

  • Biochemistry
  • Microbiology
  • Enzyme Assays

Background:

  • Polyhydroxyalkanoates (PHAs) are biopolyesters with diverse applications.
  • Identifying enzymes like polyhydroxyalkanoate (PHA) depolymerases is crucial for PHA biodegradation and recycling.
  • Existing methods for screening PHA depolymerase activity can lack sensitivity or require specialized equipment.

Purpose of the Study:

  • To develop a continuous, sensitive spectrophotometric assay for screening polyhydroxyalkanoate (PHA) depolymerase activity.
  • To optimize the assay for use in microtiter plates for high-throughput screening.
  • To apply the assay to identify novel bacterial strains with PHA depolymerase activity.

Main Methods:

  • A continuous spectrophotometric assay was developed using crystalline polyhydroxyalkanoate (PHB) and a pH indicator.
  • The assay detects ester bond breakage via proton titration.
  • The method was optimized for microtiter plates and compared to a turbidimetric approach.

Main Results:

  • The assay demonstrated a linear correlation between reaction rate and enzyme concentration.
  • The spectrophotometric method showed a 5-fold increase in signal-to-noise ratio compared to the turbidimetric method.
  • Screening 140 bacterial strains identified eleven positive for PHA depolymerase activity, including novel strains with high enzyme activity.

Conclusions:

  • The developed assay is a sensitive and efficient tool for screening polyhydroxyalkanoate (PHA) depolymerase activity.
  • The method facilitates the discovery and characterization of new PHA-degrading microorganisms.
  • This assay is valuable for both qualitative screening and quantitative analysis of PHA depolymerase enzymes.