Abstract
Flow microreactors are emergent engineering tools for the development of continuous biocatalytic transformations. Exploiting enzymes in continuous mode requires their retention for multiple rounds of conversions. To achieve this goal, immobilizing the enzymes on microchannel walls is a promising approach. However, protein immobilization within closed structures is difficult. Here, we describe a methodology based on the confluent design of enzyme and microreactor; fusion to the silica-binding module Zbasic2 is used to engineer enzymes for high-affinity-oriented attachment to the plain wall surface of glass microchannels. As a practical case, the methodology is described using a sucrose phosphorylase; the assayed reaction is synthesis of α-d-glucose 1-phosphate (αGlc 1-P) from sucrose and phosphate using the immobilized enzyme microreactor. Procedures of enzyme immobilization, reactor characterization, and operation are described. The methodology is applicable for any other enzymes fused to Zbasic2 and silica (glass)-based microfluidic reactors.
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References
Tamborini L, Fernandes P, Paradisi F, Molinari F (2018) Flow bioreactors as complementary tools for biocatalytic process intensification. Trends Biotechnol 36:73–88. https://doi.org/10.1016/j.tibtech.2017.09.005
Gutmann B, Cantillo D, Kappe CO (2015) Continuous-flow technology-a tool for the safe manufacturing of active pharmaceutical ingredients. Angew Chem Int Ed 54:6688–6728. https://doi.org/10.1002/anie.201409318
Wohlgemuth R, Plazl I, Žnidaršič-Plazl P et al (2015) Microscale technology and biocatalytic processes: opportunities and challenges for synthesis. Trends Biotechnol 33:302–314. https://doi.org/10.1016/j.tibtech.2015.02.010
Munirathinam R, Huskens J, Verboom W (2015) Supported catalysis in continuous-flow microreactors. Adv Synth Catal 357:1093–1123. https://doi.org/10.1002/adsc.201401081
Rossetti I (2018) Continuous flow (micro-)reactors for heterogeneously catalyzed reactions: main design and modelling issues. Catal Today 308:20–31. https://doi.org/10.1016/j.cattod.2017.09.040
Bolivar JM, Wiesbauer J, Nidetzky B (2011) Biotransformations in microstructured reactors: more than flowing with the stream? Trends Biotechnol 29:333–342
Bolivar JM, Tribulato MA, Petrasek Z, Nidetzky B (2016) Let the substrate flow, not the enzyme: practical immobilization of d-amino acid oxidase in a glass microreactor for effective biocatalytic conversions. Biotechnol Bioeng 113:2342–2349. https://doi.org/10.1002/bit.26011
Valikhani D, Bolivar JM, Pfeiffer M, Nidetzky B (2017) Multivalency effects on the immobilization of sucrose phosphorylase in flow microchannels and their use in the development of a high-performance biocatalytic microreactor. ChemCatChem 9:161–166. https://doi.org/10.1002/cctc.201601019
Miložič N, Lubej M, Lakner M et al (2017) Theoretical and experimental study of enzyme kinetics in a microreactor system with surface-immobilized biocatalyst. Chem Eng J 313:374–381. https://doi.org/10.1016/j.cej.2016.12.030
Bolivar JM, Nidetzky B (2013) Smart enzyme immobilization in microstructured reactors. Chimica Oggi/Chemistry Today 31:50–54
Hernandez K, Fernandez-Lafuente R (2011) Control of protein immobilization: coupling immobilization and site-directed mutagenesis to improve biocatalyst or biosensor performance. Enzym Microb Technol 48:107–122. https://doi.org/10.1016/j.enzmictec.2010.10.003
Bolivar JM, Nidetzky B (2012) Positively charged mini-protein Zbasic2 as a highly efficient silica binding module: opportunities for enzyme immobilization on unmodified silica supports. Langmuir 28:10040–10049. https://doi.org/10.1021/la3012348
Bolivar JM, Gascon V, Marquez-Alvarez C et al (2017) Oriented coimmobilization of oxidase and catalase on tailor-made ordered mesoporous silica. Langmuir 33:5065–5076. https://doi.org/10.1021/acs.langmuir.7b00441
Valikhani D, Bolivar JM, Viefhues M et al (2017) A spring in performance: silica nanosprings boost enzyme immobilization in microfluidic channels. ACS Appl Mater Interfaces 9:34641–34649. https://doi.org/10.1021/acsami.7b09875
Goedl C, Sawangwan T, Mueller M et al (2008) A high-yielding biocatalytic process for the production of 2-O-(alpha-D-glucopyranosyl)-sn-glycerol, a natural osmolyte and useful moisturizing ingredient. Angew Chem Int Ed Engl 47:10086–10089. https://doi.org/10.1002/anie.200803562
Goedl C, Schwarz A, Minani A, Nidetzky B (2007) Recombinant sucrose phosphorylase from Leuconostoc mesenteroides: characterization, kinetic studies of transglucosylation, and application of immobilised enzyme for production of alpha-D-glucose 1-phosphate. J Biotechnol 129:77–86. https://doi.org/10.1016/j.jbiotec.2006.11.019
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Valikhani, D., Bolivar, J.M., Nidetzky, B. (2020). Enzyme Immobilization in Wall-Coated Flow Microreactors. 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_16
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DOI: https://doi.org/10.1007/978-1-0716-0215-7_16
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