Strategies to immobilize the individual enzymes are crucial for enhancing catalytic applicability and require a controlled immobilization process. Herein, protocol for immobilizing Candida rugosa lipase (CRL) onto modified magnetic silica derived from oil palm leaves ash (OPLA) was optimized for the effects of concentration of CRL, immobilization time, and temperature, monitored by titrimetric and spectrometric methods. XRD and TGA-DTG spectrometric observations indicated that OPLA-silica was well coated over magnetite (SiO2-MNPs) and CRLs were uniformly bound by covalent bonds to SiO2-MNPs (CRL/Gl-A-SiO2-MNPs). The optimized immobilization protocol showed that in the preparation of CRL/Gl-A-SiO2-MNPs, CRL with 68.3 mg/g protein loading and 74.6 U/g specific activity was achieved using 5 mg/mL of CRL, with an immobilization time of 12 h at 25 °C. The present work also demonstrated that acid-pretreated OPLA is a potential source of renewable silica, envisioning its applicability for practical use in enzymatic catalysis on solid support.
The study reports the preparation of a composite consisting of magnetite coated with nanosilica extracted from oil palm leaves (OPL) ash as nanosupports for immobilization of Candida rugosa lipase (CRL) and its application for the synthesis of butyl butyrate. Results of immobilization parameters showed that ∼ 80% of CRL (84.5 mg) initially offered was immobilized onto the surface of the nanosupports to yield a maximum protein loading and specific activity of 67.5 ± 0.72 mg/g and 320.8 ± 0.42 U/g of support, respectively. Surface topography, morphology as well as information on surface composition obtained by Raman spectroscopy, atomic force microscopy, field emission scanning electron microscopy and transmission electron microscopy showed that CRL was successfully immobilized onto the nanosupports, affirming its biocompatibility. Under optimal conditions (3.5 mg/mL protein loading, at 45 ℃, 3 h and molar ratio 2:1 (1-butanol:n-butyric acid) the CRL/Gl-A-SiO2-MNPs gave a maximum yield of 94 ± 0.24% butyl butyrate as compared to 84 ± 0.32% in the lyophilized CRL. CRL/Gl-A-SiO2-MNPs showed an extended operational stability, retaining 50% of its initial activity after 17 consecutive esterification cycles. The results indicated that OPL derived nanosilica coated on magnetite can potentially be employed as carrier for lipase immobilization in replacement of the non-renewable conventionalsilica sources.