{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,6]],"date-time":"2026-05-06T11:07:51Z","timestamp":1778065671008,"version":"3.51.4"},"reference-count":55,"publisher":"MDPI AG","issue":"12","license":[{"start":{"date-parts":[[2019,11,27]],"date-time":"2019-11-27T00:00:00Z","timestamp":1574812800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Project \u201cSynthetic Biology: from omics technologies to genomic engineering (OMIC-ENGINE)\u201d , which is implemented under the Action \u201cReinforcement of the Research and Innovation Infrastructure\u201d, funded by the Operational Program \u201cCompetitiveness, Entreprene","award":["MIS 5002636"],"award-info":[{"award-number":["MIS 5002636"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Catalysts"],"abstract":"<jats:p>We report the preparation, characterization and application of a novel magnetic four-enzyme nanobiocatalyst prepared by the simultaneous covalent co-immobilization of cellulase (CelDZ1), \u03b2-glucosidase (bgl), glucose oxidase (GOx) and horseradish peroxidase (HRP) onto the surface of amino-functionalized magnetic nanoparticles (MNPs). This nanobiocatalyst was characterized by various spectroscopic techniques. The co-immobilization process yielded maximum recovered enzymatic activity (CelDZ1: 42%, bgl: 66%, GOx: 94% and HRP: 78%) at a 10% v\/v cross-linker concentration, after 2 h incubation time and at 1:1 mass ratio of MNPs to total enzyme content. The immobilization process leads to an increase of Km and a decrease of Vmax values of co-immobilized enzymes. The thermal stability studies of the co-immobilized enzymes indicated up to 2-fold increase in half-life time constants and up to 1.5-fold increase in their deactivation energies compared to the native enzymes. The enhanced thermodynamic parameters of the four-enzyme co-immobilized MNPs also suggested increment in their thermal stability. Furthermore, the co-immobilized enzymes retained a significant part of their activity (up to 50%) after 5 reaction cycles at 50 \u00b0C and remained active even after 24 d of incubation at 5 \u00b0C. The nanobiocatalyst was successfully applied in a four-step cascade reaction involving the hydrolysis of cellulose.<\/jats:p>","DOI":"10.3390\/catal9120995","type":"journal-article","created":{"date-parts":[[2019,11,27]],"date-time":"2019-11-27T11:07:00Z","timestamp":1574852820000},"page":"995","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":33,"title":["Development of a Four-Enzyme Magnetic Nanobiocatalyst for Multi-Step Cascade Reactions"],"prefix":"10.3390","volume":"9","author":[{"given":"Archontoula","family":"Giannakopoulou","sequence":"first","affiliation":[{"name":"Biotechnology Laboratory, Department of Biological Applications and Technologies, University of Ioannina, 45110 Ioannina, Greece"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Michaela","family":"Patila","sequence":"additional","affiliation":[{"name":"Biotechnology Laboratory, Department of Biological Applications and Technologies, University of Ioannina, 45110 Ioannina, Greece"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Konstantinos","family":"Spyrou","sequence":"additional","affiliation":[{"name":"Department of Materials Science &amp; Engineering, University of Ioannina, 45110 Ioannina, Greece"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2744-5934","authenticated-orcid":false,"given":"Nikolaos","family":"Chalmpes","sequence":"additional","affiliation":[{"name":"Department of Materials Science &amp; Engineering, University of Ioannina, 45110 Ioannina, Greece"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Dimitra","family":"Zarafeta","sequence":"additional","affiliation":[{"name":"Institute of Biology, Medicinal Chemistry &amp; Biotechnology, National Hellenic Research Foundation, 11635 Athens, Greece"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Georgios","family":"Skretas","sequence":"additional","affiliation":[{"name":"Institute of Biology, Medicinal Chemistry &amp; Biotechnology, National Hellenic Research Foundation, 11635 Athens, Greece"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4256-8190","authenticated-orcid":false,"given":"Dimitrios","family":"Gournis","sequence":"additional","affiliation":[{"name":"Department of Materials Science &amp; Engineering, University of Ioannina, 45110 Ioannina, Greece"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8196-4885","authenticated-orcid":false,"given":"Haralambos","family":"Stamatis","sequence":"additional","affiliation":[{"name":"Biotechnology Laboratory, Department of Biological Applications and Technologies, University of Ioannina, 45110 Ioannina, Greece"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2019,11,27]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"553","DOI":"10.1007\/s12257-014-0173-7","article-title":"Recent progress in nanobiocatalysis for enzyme immobilization and its application","volume":"19","author":"Min","year":"2014","journal-title":"Biotechnol. 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