Intraparticle pH Sensing Within Immobilized Enzymes: Immobilized Yellow Fluorescent Protein as Optical Sensor for Spatiotemporal Mapping of pH Inside Porous Particles

Tanja Consolati, Juan M. Bolivar, Zdenek Petrasek, Jose Berenguer, Aurelio Hidalgo, Jose M. Guisan, Bernd Nidetzky

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pH is a fundamental variable in enzyme catalysis and its measurement therefore is crucial for understanding and optimizing enzyme-catalyzed reactions. Whereas measurements within homogeneous bulk liquid solution are prominently used, enzymes immobilized inside porous particles often suffer from pH gradients due to partition effects and heterogeneously catalyzed biochemical reactions. Unfortunately, the measurements of intraparticle pH are not available due to the lack of useful suitable methodologies; as a consequence the biocatalyst characterization is hampered. Here, a fully biocompatible methodology for real-time optical sensing of pH within porous materials is described. A genetically encoded ratiometric pH indicator, the superfolder yellow fluorescent protein (sYFP), is used to functionalize the internal surface of enzyme carrier supports. By using controlled, tailor-made immobilization, sYFP is homogeneously distributed within these materials, and so enables, via self-referenced imaging analysis, pH measurements in high accuracy and with useful spatiotemporal resolution. The hydrolysis of penicillin by a penicillin acylase, taking place in solution or confined to the solid surface of the porous matrix is used to show the monitoring of evolution of internal pH. Thus, pH sensing based on immobilized sYFP represents a broadly applicable technique to the study of the internally heterogeneous environment of immobilized enzymes into solid particles.

Original languageEnglish
Pages (from-to)319-333
Number of pages15
JournalMethods in molecular biology (Clifton, N.J.)
Publication statusPublished - 1 Jan 2020


  • Immobilization
  • Internal pH sensing
  • Porous materials
  • Spatiotemporal pH mapping
  • Yellow fluorescent protein

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

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