{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,4]],"date-time":"2026-03-04T17:57:41Z","timestamp":1772647061626,"version":"3.50.1"},"reference-count":52,"publisher":"MDPI AG","issue":"2","license":[{"start":{"date-parts":[[2023,1,14]],"date-time":"2023-01-14T00:00:00Z","timestamp":1673654400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001871","name":"Funda\u00e7\u00e3o para a Ci\u00eancia e Tecnologia (FCT)","doi-asserted-by":"publisher","award":["UID\/CTM\/50025\/2019"],"award-info":[{"award-number":["UID\/CTM\/50025\/2019"]}],"id":[{"id":"10.13039\/501100001871","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001871","name":"Funda\u00e7\u00e3o para a Ci\u00eancia e Tecnologia (FCT)","doi-asserted-by":"publisher","award":["CTTI-51\/18-IPC"],"award-info":[{"award-number":["CTTI-51\/18-IPC"]}],"id":[{"id":"10.13039\/501100001871","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001871","name":"Funda\u00e7\u00e3o para a Ci\u00eancia e Tecnologia (FCT)","doi-asserted-by":"publisher","award":["UI\/BD\/150854\/2021"],"award-info":[{"award-number":["UI\/BD\/150854\/2021"]}],"id":[{"id":"10.13039\/501100001871","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001871","name":"Funda\u00e7\u00e3o para a Ci\u00eancia e Tecnologia (FCT)","doi-asserted-by":"publisher","award":["2018\/022726"],"award-info":[{"award-number":["2018\/022726"]}],"id":[{"id":"10.13039\/501100001871","id-type":"DOI","asserted-by":"publisher"}]},{"name":"ALBA synchrotron governance","award":["UID\/CTM\/50025\/2019"],"award-info":[{"award-number":["UID\/CTM\/50025\/2019"]}]},{"name":"ALBA synchrotron governance","award":["CTTI-51\/18-IPC"],"award-info":[{"award-number":["CTTI-51\/18-IPC"]}]},{"name":"ALBA synchrotron governance","award":["UI\/BD\/150854\/2021"],"award-info":[{"award-number":["UI\/BD\/150854\/2021"]}]},{"name":"ALBA synchrotron governance","award":["2018\/022726"],"award-info":[{"award-number":["2018\/022726"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Molecules"],"abstract":"<jats:p>This study reports on the synthesis of novel bienzyme polymer-assisted nanoflower complexes (PANF), their morphological and structural characterization, and their effectiveness as cascade biocatalysts. First, highly porous polyamide 6 microparticles (PA6 MP) are synthesized by activated anionic polymerization in solution. Second, the PA6 MP are used as carriers for hybrid bienzyme assemblies comprising glucose oxidase (GOx) and horseradish peroxidase (HRP). Thus, four PANF complexes with different co-localization and compartmentalization of the two enzymes are prepared. In samples NF GH\/PA and NF GH@PA, both enzymes are localized within the same hybrid flowerlike organic-inorganic nanostructures (NF), the difference being in the way the PA6 MP are assembled with NF. In samples NF G\/PAiH and NF G@PAiH, only GOx is located in the NF, while HRP is preliminary immobilized on PA6 MP. The morphology and the structure of the four PANF complexes have been studied by microscopy, spectroscopy, and synchrotron X-ray techniques. The catalytic activity of the four PANF was assessed by a two-step cascade reaction of glucose oxidation. The PANF complexes are up to 2\u20133 times more active than the free GOx\/HRP dyad. They also display enhanced kinetic parameters, superior thermal stability in the 40\u201360 \u00b0C range, optimum performance at pH 4\u20136, and excellent storage stability. All PANF complexes are active for up to 6 consecutive operational cycles.<\/jats:p>","DOI":"10.3390\/molecules28020839","type":"journal-article","created":{"date-parts":[[2023,1,16]],"date-time":"2023-01-16T04:57:19Z","timestamp":1673845039000},"page":"839","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":7,"title":["Synthesis of Novel Polymer-Assisted Organic-Inorganic Hybrid Nanoflowers and Their Application in Cascade Biocatalysis"],"prefix":"10.3390","volume":"28","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-6342-1967","authenticated-orcid":false,"given":"Joana F.","family":"Braz","sequence":"first","affiliation":[{"name":"IPC\u2014Institute for Polymers and Composites, University of Minho, 4800-056 Guimar\u00e3es, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1698-553X","authenticated-orcid":false,"given":"Nadya V.","family":"Dencheva","sequence":"additional","affiliation":[{"name":"IPC\u2014Institute for Polymers and Composites, University of Minho, 4800-056 Guimar\u00e3es, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5231-1896","authenticated-orcid":false,"given":"Marc","family":"Malfois","sequence":"additional","affiliation":[{"name":"ALBA Synchrotron Facility, Cerdanyola del Val\u00e9s, 0890 Barcelona, Spain"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9057-9380","authenticated-orcid":false,"given":"Zlatan Z.","family":"Denchev","sequence":"additional","affiliation":[{"name":"IPC\u2014Institute for Polymers and Composites, University of Minho, 4800-056 Guimar\u00e3es, Portugal"}]}],"member":"1968","published-online":{"date-parts":[[2023,1,14]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1254","DOI":"10.1016\/j.cej.2019.05.141","article-title":"Recent progress in multienzymes co-immobilization and multienzyme system applications","volume":"373","author":"Ren","year":"2019","journal-title":"J. Chem. Eng."},{"key":"ref_2","unstructured":"Singh, S., Pandey, A., Singhania, R.R., Larroche, C., and Li, Z. (2020). Chapter 20\u2014Biocatalysis for cascade reactions to produce high-value chemicals. Biomass, Biofuels, Biochemicals, Elsevier. Advances in Enzyme Catalysis and Technology."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"270","DOI":"10.1021\/acs.chemrev.7b00033","article-title":"Artificial biocatalytic linear cascades for preparation of organic molecules","volume":"118","author":"Schrittwieser","year":"2018","journal-title":"Chem. Rev."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"100548","DOI":"10.1016\/j.cogsc.2021.100548","article-title":"Recent trends in synthetic enzymatic cascades promoted by alcohol dehydrogenases","volume":"32","author":"Gonzalo","year":"2021","journal-title":"Curr. Opin. Green Sustain. Chem."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"1359","DOI":"10.1002\/adsc.201801501","article-title":"Enzyme cascade reactions for the biosynthesis of long chain aliphatic amines from renewable fatty acids","volume":"361","author":"Lee","year":"2019","journal-title":"Adv. Synth. Catal."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"2164","DOI":"10.1002\/cctc.201701669","article-title":"Whole-cell cascade biotransformations for one-pot multistep organic synthesis","volume":"10","author":"Wu","year":"2018","journal-title":"ChemCatChem"},{"key":"ref_7","doi-asserted-by":"crossref","unstructured":"Nikulin, M., and \u0160vedas, V. (2021). Prospects of using biocatalysis for the synthesis and modification of polymers. Molecules, 26.","DOI":"10.3390\/molecules26092750"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"107584","DOI":"10.1016\/j.biotechadv.2020.107584","article-title":"Enzyme co-immobilization: Always the biocatalyst designers\u2019 choice or not?","volume":"51","author":"Carballares","year":"2021","journal-title":"Biotechnol. Adv."},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"Zdarta, J., Meyer, A.S., Jesionowski, T., and Pinelo, M. (2018). A general overview of support materials for enzyme immobilization: Characteristics, properties, practical utility. Catalysts, 8.","DOI":"10.3390\/catal8020092"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"660","DOI":"10.3389\/fbioe.2020.00660","article-title":"Immobilization of multienzymes on support materials for efficient biocatalysis","volume":"8","author":"Xu","year":"2020","journal-title":"Front. Bioeng. Biotechnol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"1332","DOI":"10.1021\/jz1002007","article-title":"Channeling by proximity: The catalytic advantages of active site colocalization using Brownian dynamics","volume":"1","author":"Bauler","year":"2010","journal-title":"J. Phys. Chem. Lett."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"299","DOI":"10.1038\/nchem.2459","article-title":"Substrate channeling as an approach to cascade reactions","volume":"8","author":"Wheeldon","year":"2016","journal-title":"Nat. Chem."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"531","DOI":"10.1038\/nnano.2014.100","article-title":"Multienzyme complexes on DNA scaffolds capable of substrate channeling with an artificial swinging arm","volume":"9","author":"Fu","year":"2014","journal-title":"Nat. Nanotechnol."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"8658","DOI":"10.1021\/nn402823k","article-title":"Origins of activity enhancement in enzyme cascades on scaffolds","volume":"7","author":"Idan","year":"2013","journal-title":"ACS Nano"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"13982","DOI":"10.1038\/ncomms13982","article-title":"Proximity does not contribute to activity enhancement in the glucose oxidase\u2013horseradish peroxidase cascade","volume":"7","author":"Zhang","year":"2016","journal-title":"Nat. Commun."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"5161","DOI":"10.1021\/acscatal.6b01302","article-title":"Role of dimension and spatial arrangement on the activity of biocatalytic cascade reactions on scaffolds","volume":"6","author":"Chado","year":"2016","journal-title":"ACS Catal."},{"key":"ref_17","unstructured":"Singh, S.P., Pandey, A., Singhania, R.R., Larroche, C., and Li, Z. (2020). Chapter 12\u2014Enzyme immobilization strategies and bioprocessing applications. Biomass, Biofuels, Biochemicals, Advances in Enzyme Catalysis and Technology, Elsevier."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"6223","DOI":"10.1039\/C3CS60075K","article-title":"Enzyme immobilization in biocatalysis: Why, what and how","volume":"42","author":"Sheldon","year":"2013","journal-title":"Chem. Soc. Rev."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"226","DOI":"10.1016\/0167-7799(91)90075-S","article-title":"Multienzyme systems obtained by gene fusion","volume":"9","author":"Mosbach","year":"1991","journal-title":"Trends Biotechnol."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"e00670","DOI":"10.1016\/j.btre.2021.e00670","article-title":"Protein scaffolds: A tool for multienzyme assembly","volume":"32","author":"Gad","year":"2021","journal-title":"Biotechnol. Rep."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"4402","DOI":"10.1021\/acscatal.8b04921","article-title":"Multienzymatic cascade reactions via enzyme complex by immobilization","volume":"9","author":"Hwang","year":"2019","journal-title":"ACS Catal."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"110607","DOI":"10.1016\/j.mcat.2019.110607","article-title":"Industrial applications of immobilized enzymes\u2014A review","volume":"479","author":"Basso","year":"2019","journal-title":"Mol. Catal."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"178","DOI":"10.1002\/bit.20131","article-title":"Enabling multienzyme biocatalysis using nanoporous materials","volume":"87","author":"Jia","year":"2004","journal-title":"Biotechnol. Bioeng."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"249","DOI":"10.1038\/nnano.2009.50","article-title":"Enzyme cascades activated on topologically programmed DNA scaffolds","volume":"4","author":"Wilner","year":"2009","journal-title":"Nat. Nanotechnol."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"5605","DOI":"10.1002\/anie.200701173","article-title":"Shell-in-shell microcapsules: A novel tool for integrated, spatially confined enzymatic reactions","volume":"46","author":"Kreft","year":"2007","journal-title":"Angew. Chem. Int. Ed."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"7378","DOI":"10.1002\/anie.200701125","article-title":"Positional assembly of enzymes in polymersome nanoreactors for cascade reactions","volume":"46","author":"Vriezema","year":"2007","journal-title":"Angew. Chem. Int. Ed."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"5305","DOI":"10.1038\/ncomms6305","article-title":"Vesicle-based artificial cells as chemical microreactors with spatially segregated reaction pathways","volume":"5","author":"Elani","year":"2014","journal-title":"Nat. Commun."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"9595","DOI":"10.1021\/la000942h","article-title":"Enzyme multilayers on colloid particles: Assembly, stability, and enzymatic activity","volume":"16","author":"Caruso","year":"2000","journal-title":"Langmuir"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"428","DOI":"10.1038\/nnano.2012.80","article-title":"Protein\u2013inorganic hybrid nanoflowers","volume":"7","author":"Ge","year":"2012","journal-title":"Nat. Nanotechnol."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"54","DOI":"10.1186\/s12951-015-0118-0","article-title":"Organic\u2013inorganic hybrid nanoflowers: Types, characteristics, and future prospects","volume":"13","author":"Lee","year":"2015","journal-title":"J. Nanobiotechnol."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"249","DOI":"10.1016\/j.ccr.2017.09.008","article-title":"Organic\u2013inorganic hybrid nanoflowers: A novel host platform for immobilizing biomolecules","volume":"352","author":"Cui","year":"2017","journal-title":"Coord. Chem. Rev."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"175","DOI":"10.1016\/j.bios.2018.08.058","article-title":"Recent progress in biosensors based on organic-inorganic hybrid nanoflowers","volume":"120","author":"Zhu","year":"2018","journal-title":"Biosens. Bioelectron."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"268","DOI":"10.1007\/s13206-018-2409-7","article-title":"Organic-inorganic hybrid nanoflowers as potent materials for biosensing and biocatalytic applications","volume":"12","author":"Tran","year":"2018","journal-title":"BioChip J."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"1488","DOI":"10.1080\/10408347.2021.1889962","article-title":"Organic-inorganic hybrid nanoflower production and analytical utilization: Fundamental to cutting-edge technologies","volume":"52","author":"Subramani","year":"2021","journal-title":"Crit. Rev. Anal. Chem."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"255","DOI":"10.1039\/C3NR04425D","article-title":"Multienzyme co-embedded organic-inorganic hybrid nanoflowers: Synthesis and application in colorimetric sensor","volume":"6","author":"Sun","year":"2014","journal-title":"Nanoscale"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"12465","DOI":"10.1039\/C4CC05478D","article-title":"Spatial co-localization of multienzymes by inorganic nanocrystal\u2013protein complexes","volume":"50","author":"Li","year":"2014","journal-title":"Chem. Commun."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"228","DOI":"10.1016\/j.apsusc.2017.06.018","article-title":"Feasible synthesis of protein-templated zinc phosphate-supported Pt nanoparticle with enhanced electrocatalysis for methanol oxidation","volume":"422","author":"Song","year":"2017","journal-title":"Appl. Surf. Sci."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"24","DOI":"10.1016\/j.enzmictec.2017.06.006","article-title":"Preparation of glutaraldehyde-treated lipase-inorganic hybrid nanoflowers and their catalytic performance as immobilized enzymes","volume":"105","author":"Lee","year":"2017","journal-title":"Enzym. Microb. Technol."},{"key":"ref_39","doi-asserted-by":"crossref","unstructured":"Guimar\u00e3es, J.R., Carballares, D., Tardioli, P.W., Rocha-Martin, J., and Fernandez-Lafuente, R. (2022). Tunning immobilized commercial lipase preparations features by simple treatment with phosphate salts. Molecules, 27.","DOI":"10.3390\/molecules27144486"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"442","DOI":"10.1016\/j.jhazmat.2018.01.003","article-title":"Synthesis of cross-linked protein-metal hybrid nanoflowers and its application in repeated batch decolorization of synthetic dyes","volume":"347","author":"Patel","year":"2018","journal-title":"J. Hazard. Mater."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"87","DOI":"10.1016\/j.procbio.2017.03.026","article-title":"Enzyme-inorganic nanoflowers\/alginate microbeads: An enzyme immobilization system and its potential application","volume":"57","author":"Zhao","year":"2017","journal-title":"Process Biochem."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"128419","DOI":"10.1016\/j.colsurfa.2022.128419","article-title":"In-situ growth of multienzyme-inorganic hybrid nanoflowers on PVA-co-PE nanofibrous strip for colorimetric biosensor","volume":"640","author":"Luo","year":"2022","journal-title":"Colloids Surf. A Physicochem. Eng. Asp."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"119","DOI":"10.1002\/mame.201500194","article-title":"One-step in situ synthesis of polyamide microcapsules with inorganic payload and their transformation into responsive thermoplastic composite materials","volume":"301","author":"Dencheva","year":"2016","journal-title":"Macromol. Mater. Eng."},{"key":"ref_44","unstructured":"Denchev, Z., and Dencheva, N. (2014). Polyamide Microcapsules and Method to Produce the Same. (Application Number PT107679), Patent."},{"key":"ref_45","doi-asserted-by":"crossref","unstructured":"Dencheva, N., Oliveira, S., Braz, J., Getya, D., Malfois, M., Denchev, Z., and Gitsov, I. (2021). Magnetically responsive PA6 microparticles with immobilized laccase show high catalytic efficiency in the enzymatic treatment of catechol. Catalysts, 11.","DOI":"10.3390\/catal11020239"},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"22412","DOI":"10.1038\/srep22412","article-title":"Amino acids-incorporated nanoflowers with an intrinsic peroxidase-like activity","volume":"6","author":"Wu","year":"2016","journal-title":"Sci. Rep."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"2154","DOI":"10.1002\/adfm.200800167","article-title":"Synthesis of Cu2PO4OH hierarchical superstructures with photocatalytic activity in visible light","volume":"18","author":"Cho","year":"2008","journal-title":"Adv. Funct. Mater."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"505","DOI":"10.1021\/cs401009z","article-title":"Design and analysis of enhanced catalysis in scaffolded multienzyme cascade reactions","volume":"4","author":"Lin","year":"2014","journal-title":"ACS Catal."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"2104884","DOI":"10.1002\/advs.202104884","article-title":"Multimodal enzyme-carrying suprastructures for rapid and sensitive biocatalytic cascade reactions","volume":"9","author":"Jo","year":"2022","journal-title":"Adv. Sci."},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"21205","DOI":"10.1039\/C7RA02291C","article-title":"Magnetic metal\u2013organic frameworks as scaffolds for spatial co-location and positional assembly of multi-enzyme systems enabling enhanced cascade biocatalysis","volume":"7","author":"Chen","year":"2017","journal-title":"RSC Adv."},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"4979","DOI":"10.1021\/acscatal.5b00958","article-title":"Bienzyme\u2212polymer\u2212graphene oxide quaternary hybrid biocatalysts: Efficient substrate channeling under chemically and thermally denaturing conditions","volume":"5","author":"Zore","year":"2015","journal-title":"ACS Catal."},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"510","DOI":"10.1107\/S1600576715004306","article-title":"The fast azimuthal integration Python library: pyFAI","volume":"48","author":"Ashiotis","year":"2015","journal-title":"J. Appl. Crystallogr."}],"container-title":["Molecules"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1420-3049\/28\/2\/839\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T18:05:57Z","timestamp":1760119557000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1420-3049\/28\/2\/839"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2023,1,14]]},"references-count":52,"journal-issue":{"issue":"2","published-online":{"date-parts":[[2023,1]]}},"alternative-id":["molecules28020839"],"URL":"https:\/\/doi.org\/10.3390\/molecules28020839","relation":{},"ISSN":["1420-3049"],"issn-type":[{"value":"1420-3049","type":"electronic"}],"subject":[],"published":{"date-parts":[[2023,1,14]]}}}