TY - JOUR
T1 - Intraparticle pH Sensing Within Immobilized Enzymes
T2 - Immobilized Yellow Fluorescent Protein as Optical Sensor for Spatiotemporal Mapping of pH Inside Porous Particles
AU - Consolati, Tanja
AU - Bolivar, Juan M.
AU - Petrasek, Zdenek
AU - Berenguer, Jose
AU - Hidalgo, Aurelio
AU - Guisan, Jose M.
AU - Nidetzky, Bernd
PY - 2020/1/1
Y1 - 2020/1/1
N2 - 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.
AB - 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.
KW - Immobilization
KW - Internal pH sensing
KW - Porous materials
KW - Spatiotemporal pH mapping
KW - Yellow fluorescent protein
UR - http://www.scopus.com/inward/record.url?scp=85077843074&partnerID=8YFLogxK
U2 - 10.1007/978-1-0716-0215-7_21
DO - 10.1007/978-1-0716-0215-7_21
M3 - Article
C2 - 31939133
AN - SCOPUS:85077843074
SN - 1064-3745
VL - 2100
SP - 319
EP - 333
JO - Methods in molecular biology (Clifton, N.J.)
JF - Methods in molecular biology (Clifton, N.J.)
ER -