Green synthesis of magnetic bio-Graphene nanohybrid for the immobilization of hydrolytic enzymes towards sustainable bioconversion of cellulose

In this work, we report a green and sustainable synthetic route for producing magnetic few-layer bio-Graphene (MbG) for the first time. Bio-Graphene (bG) was prepared <i>via</i> a green method using an aqueous olive leaf extract (OLE) as both the exfoliating and stabilizing agent, aiming to reduce the environmental impact of the traditional chemical methods. In the following step, iron oxide nanoparticles were created <i>in situ</i> on bG-OLE <i>via</i> co-precipitation using ferrous precursors. MbG was subsequently used to support the co-immobilization of cellulase (cel) and β-glucosidase (bgl), enabling the design of a recyclable, magnetically separable nanobiocatalyst. Various spectroscopic and microscopic techniques were employed to characterize the produced MbG and the resulting nanobiocatalysts. Both simultaneous and sequential immobilization strategies were applied to evaluate the synergy between cel and bgl. Several parameters were studied, such as the support-to-enzyme mass ratio, immobilization incubation time, and the order in which the enzymes were added. Although the 1-hour simultaneous co-immobilization resulted in low cel and bgl immobilization yields, the highest specific activity was observed (∼0.33 units mg^-1). Moreover, the bi-enzymatic nanobiocatalyst demonstrated better reusability for carboxymethyl (CMC) and microcrystalline cellulose (Avicel) hydrolysis compared to the mono-enzymatic nanobiocatalyst. Subsequently, the mono- and bi-enzymatic systems were employed in continuous-flow microreactors for the hydrolysis of CMC towards glucose production. The bi-enzymatic system exhibited significantly higher turnover frequency (TOF) (0.105 h^-1) and operational stability than the mono-enzymatic system (0.029 h^-1). The entire synthetic route is characterized by a minimal environmental footprint, offering a platform for sustainable bioprocessing.