Alginate-oil palm-derived nanocellulose-montmorillonite composite for lipase immobilization and its application in ethyl levulinate production

Abstract

The extension of large-scale oil palm plantations to meet global demands has led to a new environmental challenge. The issue rose from the passive dumping and the open burning of unwanted agriculture biomass i.e. matured oil palm fronds leaves (OPFL). Therefore, a future development toward further technological utilization of OPFL warrants attention of the scientific society. This study capitalized on developing a ternary support that combines nanocellulose (NC) derived from OPFL and montmorillonite (MMT) as reinforcing agents into alginate (ALG) polymer for covalent immobilization of Candida rugosa lipase (CRL). Successive treatments of OPFL with bleach, alkali, and acid hydrolysis produced NC in the form of whitish powder. Analysis of NC by attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR), thermal gravimetric analysis (TGA), X-ray diffraction (XRD), field emission scanning electron microscopy-energy dispersive X-ray (FESEM-EDX), transmission electron microscopy (TEM) and atomic force microscopy (AFM) confirmed the successful extraction of NC from OPFL. Surface area of NC obtained (5.11 m2/g) was corresponded to a crystallinity index of 45.0%. Meanwhile, the surface showed needle-like structures with diameters of 10-30 nm. Covalent immobilization of CRL onto the ALG/NC/MMT via epichlorohydrin attained a maximum enzyme loading and specific activity of 5.80 mg/g and 31.90 ± 0.80 U/mg, respectively. Data on surface topography and morphology of CRL-ALG/NC/MMT using the same analyses as mentioned above showed that CRL was present on the surface of the ALG/NC/MMT support. The suitability of protocol to immobilize CRL onto the ALG/NC/MMT supports was assessed for factors namely immobilization time, temperature, pH buffer and protein loading, to yield the highest conversion of ethyl levulinate (EL) within 2 h of reaction. Optimal conditions that gave the highest yield of EL (92.89%) using Taguchi design were 7.00 mg/mL protein loading, incubated for 7 h at 35? and buffer of pH 5, with factors of immobilization time and protein loading displayed the most prominent effect on the process. CRL-ALG/NC/MMT showed an extended operational stability, attaining approximately 50% of its initial activity after nine consecutive esterification cycles. Analyses on purified EL confirmed that the ester was successfully synthesized. Based on the results, it can be concluded that the newly developed ALG/NC/MMT can potentially be employed as a support for lipase immobilization.