Development of a Multi-Enzymatic Biocatalytic System through Immobilization on High Quality Few-Layer bio-Graphene

Christina Alatzoglou, Michaela Patila*, Archontoula Giannakopoulou, Konstantinos Spyrou, Feng Yan, Wenjian Li, Nikolaos Chalmpes, Angeliki C. Polydera, Petra Rudolf, Dimitrios Gournis*, Haralambos Stamatis*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

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In this work, we report the green production of few-layer bio-Graphene (bG) through liquid exfoliation of graphite in the presence of bovine serum albumin. Microscopic characterization evaluated the quality of the produced nanomaterial, showing the presence of 3–4-layer graphene. Moreover, spectroscopic techniques also confirmed the quality of the resulted bG, as well as the presence of bovine serum albumin on the graphene sheets. Next, for the first time, bG was used as support for the simultaneous covalent co-immobilization of three enzymes, namely β-glucosidase, glucose oxidase, and horseradish peroxidase. The three enzymes were efficiently co-immobilized on bG, demonstrating high immobilization yields and activity recoveries (up to 98.5 and 90%, respectively). Co-immobilization on bG led to an increase of apparent KM values and a decrease of apparent Vmax values, while the stability of the nanobiocatalysts prevailed compared to the free forms of the enzymes. Co-immobilized enzymes exhibited high reusability, preserving a significant part of their activity (up to 72%) after four successive catalytic cycles at 30 °C. Finally, the tri-enzymatic nanobiocatalytic system was applied in three-step cascade reactions, involving, as the first step, the hydrolysis of p-Nitrophenyl-β-D-Glucopyranoside and cellobiose.

Original languageEnglish
Article number127
Number of pages19
Issue number1
Publication statusPublished - Jan-2023


  • bio-Graphene
  • bovine serum albumin
  • cascade reactions
  • green synthesis
  • multi-enzymatic immobilization
  • nanobiocatalytic system

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