The growing need for technologies that are safe, eco-friendly, and capable of handling high volumes has resulted in a greater focus on utilizing enzymes as biochemical catalysts in the industrial processes (Jafarian et al., 2020). This enzyme-based approach provides several benefits, such as biodegradability, mild reaction conditions, and significantly higher catalytic efficiency than purely chemical methods (Mohamad et al., 2015). Nonetheless, effectively incorporating enzymes in the industrial processes necessitates the resolution of certain limitations, including difficulties or impossibilities in retrieving enzymes following catalytic reactions, the low stability of enzymes in harsh conditions, and their subpar performance at hydrophobic/aqueous interfaces (Brady and Jordaan, 2009, Hanefeld et al., 2009).

In order to overcome these obstacles, enzyme immobilization has been identified as an effective approach to improve the stability, reusability, and efficiency of enzymes (Bilal et al., 2018). The success of an industrial process depends heavily on selecting an appropriate immobilization technique and optimizing the reaction conditions (Chapman et al., 2018). Of the various strategies available, the immobilization of enzymes onto solid supports through reversible physical methods or irreversible covalent attachment is widely considered and utilized (Rodrigues et al., 2021). While the physical method is relatively simple and cost-effective, its main drawback is the easy detachment of enzyme. Conversely, covalent bonding is robust but requires considerably higher enzyme concentrations due to enzymes' occasional inactivation (leakage) (Nisha et al., 2012).

Lipases have garnered significant scientific consideration due to their unique properties, including the broad substrate specificities, regioselectivity, enantioselectivity, and the ability to hydrolyze oils, esterify, and undergo acidolysis. Lipases are found in various kingdoms of animals, plants, and molds, making them highly versatile (Asmat and Husain, 2018). Consequently, lipases have found numerous potential applications in diverse industries, including but not limited to laundry soap, food, leather, cosmetics, biofuel production, textiles, paper, and pharmacology (Silva and Guidini, 2019). Given the two different closed and open conformations reported for lipases, which are mainly affected by the hydrophobicity of the surrounding medium, the immobilization employed is anticipated to affect the final conformation and, consequently, the enzyme's activity.

Our team recently developed a membrane reactor through the surface modification of polyethersulfone (PES) membranes with graphene oxide nanosheets (GON) followed by the physical immobilization of Candida rugosa lipase (CRL). The enzyme's activity was enhanced by surface engineering of the PES using various GON concentrations. The resulting membrane reactors were successfully used to hydrolyze p-nitrophenyl palmitate (p-NPP) in pure water flux. However, the relatively wide hydrophobic area of lipase led to the domination of the closed form of CRL in the aqueous environment. As a result, we investigated the possibility of further improving enzyme activity through covalent immobilization in this study. We first developed GON-modified PES hybrid membranes through the phase inversion method followed by the surface functionalization with APTES. We then utilized glutaraldehyde (GLU), as a crosslinker, to covalently immobilize CRL on the hybrid membranes. After characterizing the EHMR using XRD, FE-SEM, FT-IR, ATR-FTIR, and EDX, its catalytic activity was evaluated for the hydrolysis of p-NPP, as a precursor, to produce p-nitrophenol (p-NP). Additionally, we evaluated and compared the activity, reusability, and storage stability of immobilized lipase with free CRL under various conditions (pH, temperature, etc.).