Abstract
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.
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References
Bolivar JM, Consolati T, Mayr T, Nidetzky B (2013) Shine a light on immobilized enzymes: real-time sensing in solid supported biocatalysts. Trends Biotechnol 31:194–203. https://doi.org/10.1016/j.tibtech.2013.01.004
Benítez-Mateos AI, Nidetzky B, Bolivar JM, López-Gallego F (2018) Single-particle studies to advance the characterization of heterogeneous biocatalysts. ChemCatChem 10:654–665. https://doi.org/10.1002/cctc.201701590
Bolivar JM, Eisl I, Nidetzky B (2016) Advanced characterization of immobilized enzymes as heterogeneous biocatalysts. Catal Today 259:66–80. https://doi.org/10.1016/j.cattod.2015.05.004
Casey JR, Grinstein S, Orlowski J (2010) Sensors and regulators of intracellular pH. Nat Rev Mol Cell Biol 11:50–61. https://doi.org/10.1038/nrm2820
Yang Z, Cao J, He Y et al (2014) Macro−/micro-environment-sensitive chemosensing and biological imaging. Chem Soc Rev 43:4563–4601. https://doi.org/10.1039/c4cs00051j
Yamaguchi A, Namekawa M, Kamijo T et al (2011) Acid−base equilibria inside amine-functionalized mesoporous silica. Anal Chem 83:2939–2946. https://doi.org/10.1021/ac102935q
Sun X, Xie J, Xu J et al (2015) Single-molecule studies of acidity distributions in mesoporous aluminosilicate thin films. Langmuir 31:5667–5675. https://doi.org/10.1021/acs.langmuir.5b01628
Begemann J, Spiess AC (2015) Dual lifetime referencing enables pH-control for oxidoreductions in hydrogel-stabilized biphasic reaction systems. Biotechnol J 10:1822–1829. https://doi.org/10.1002/biot.201500198
Huang HY, Shaw J, Yip C, Wu XY (2008) Microdomain pH gradient and kinetics inside composite polymeric membranes of pH and glucose sensitivity. Pharm Res 25:1150–1157
Boniello C, Mayr T, Klimant I et al (2010) Intraparticle concentration gradients for substrate and acidic product in immobilized cephalosporin C amidase and their dependencies on carrier characteristics and reaction parameters. Biotechnol Bioeng 106:528–540. https://doi.org/10.1002/bit.22694
Zahel T, Boniello C, Nidetzky B (2013) Real-time measurement and modeling of intraparticle pH gradient formation in immobilized cephalosporin C amidase. Process Biochem 48:593–604
Spiess A, Schlothauer R, Hinrichs J et al (1999) pH gradients in immobilized amidases and their influence on rates and yields of beta-lactam hydrolysis. Biotechnol Bioeng 62:267–277
Spiess AC, Kasche V (2001) Direct measurement of pH profiles in immobilized enzyme carriers during kinetically controlled synthesis using CLSM. Biotechnol Prog 17:294–303. https://doi.org/10.1021/bp000149e
Wencel D, Abel T, McDonagh C (2014) Optical chemical pH sensors. Anal Chem 86:15–29. https://doi.org/10.1021/ac4035168
Barczak M, McDonagh C, Wencel D (2016) Micro- and nanostructured sol-gel-based materials for optical chemical sensing (2005–2015). Microchim Acta 183:2085–2109. https://doi.org/10.1007/s00604-016-1863-y
Luo H, Zhu L, Chang Y et al (2017) Microenvironmental pH changes in immobilized cephalosporin C acylase during a proton-producing reaction and regulation by a two-stage catalytic process. Bioresour Technol 223:157–165. https://doi.org/10.1016/j.biortech.2016.10.038
Boniello C, Mayr T, Bolivar JM, Nidetzky B (2012) Dual-lifetime referencing (DLR): a powerful method for on-line measurement of internal pH in carrier-bound immobilized biocatalysts. BMC Biotechnol 12:11. https://doi.org/10.1186/1472-6750-12-11
Kuwana E, Liang F, Sevick-Muraca EM (2004) Fluorescence lifetime spectroscopy of a pH-sensitive dye encapsulated in hydrogel beads. Biotechnol Prog 20:1561–1566
Schwendt T, Michalik C, Zavrel M et al (2010) Determination of temporal and spatial concentration gradients in hydrogel beads using multiphoton microscopy techniques. Appl Spectrosc 64:720–726
Zavrel M, Michalik C, Schwendt T et al (2010) Systematic determination of intrinsic reaction parameters in enzyme immobilizates. Chem Eng Sci 65:2491–2499. https://doi.org/10.1016/j.ces.2009.12.026
Consolati T, Bolivar JM, Petrasek Z et al (2018) Biobased, internally pH-sensitive materials: immobilized yellow fluorescent protein as an optical sensor for spatiotemporal mapping of pH inside porous matrices. ACS Appl Mater Interfaces 10:6858–6868. https://doi.org/10.1021/acsami.7b16639
Pédelacq J-D, Cabantous S, Tran T et al (2006) Engineering and characterization of a superfolder green fluorescent protein. Nat Biotechnol 24:79–88. https://doi.org/10.1038/nbt1172
Aliye N, Fabbretti A, Lupidi G et al (2015) Engineering color variants of green fluorescent protein (GFP) for thermostability, pH-sensitivity, and improved folding kinetics. Appl Microbiol Biotechnol 99:1205–1216. https://doi.org/10.1007/s00253-014-5975-1
Mateo C, Grazu V, Guisan JM (2013) Immobilization of enzymes on monofunctional and heterofunctional epoxy-activated supports. In: Guisan JM (ed) Immobilization of enzymes and cells. Humana Press, Totowa, NJ, pp 43–57
López-Gallego F, Fernandez-Lorente G, Rocha-Martin J et al (2013) Stabilization of enzymes by multipoint covalent immobilization on supports activated with glyoxyl groups. In: Guisan JM (ed) Immobilization of enzymes and cells. Humana Press, Totowa, NJ, pp 59–71
Valencia P, Wilson L, Aguirre C, Illanes A (2010) Evaluation of the incidence of diffusional restrictions on the enzymatic reactions of hydrolysis of penicillin G and synthesis of cephalexin. Enzym Microb Technol 47:268–276. https://doi.org/10.1016/j.enzmictec.2010.07.010
Alvaro G, Fernandez-Lafuente R, Blanco RM, Guisán JM (1990) Immobilization-stabilization of penicillin G acylase from Escherichia coli. Appl Biochem Biotechnol 26:181–195
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Consolati, T. et al. (2020). Intraparticle pH Sensing Within Immobilized Enzymes: Immobilized Yellow Fluorescent Protein as Optical Sensor for Spatiotemporal Mapping of pH Inside Porous Particles. In: Guisan, J., Bolivar, J., López-Gallego, F., Rocha-Martín, J. (eds) Immobilization of Enzymes and Cells. Methods in Molecular Biology, vol 2100. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0215-7_21
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DOI: https://doi.org/10.1007/978-1-0716-0215-7_21
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