Candida rugosa lipase supported on silica-coated magnetite nanoparticles for hydrolysis of olive oil

• Main Author: Wong, Winny Kai Li
• Format: Thesis
• Language: English
• Published: 2019
• Subjects: Q Science (General)
• Online Access: http://eprints.utm.my/id/eprint/81163/1/WinnyWongKaiMFS2019.pdf; http://eprints.utm.my/id/eprint/81163/; http://dms.library.utm.my:8080/vital/access/manager/Repository/vital:125022

Oil palm leaves (OPL) has high content of silica (SiO2). SiO2 has a high surface area and large pore volume which could reduce the aggregation of magnetite (Fe3O4). The coating of the superparamagnetic Fe3O4 was to enable easy separation from the reaction mixture. SiO2 extracted from OPL was coated on Fe3O4 followed by functionalization of 3-aminopropyltriethoxysilane (APTES) and activation of glutaraldehyde to prepare a nanosupport (G-AP-SiO2-Fe3O4) for immobilisation of Candida rugosa lipase (CRL). The feasibility of the biocatalyst (CRL/G-AP-SiO2- Fe3O4) has yet to be tested in aqueous environment. In this research, the CRL/G-APSiO2- Fe3O4 was used to determine the optimum condition for hydrolysis of olive oil. The kinetic and thermodynamic properties of the CRL/G-AP-SiO2-Fe3O4 was investigated for the hydrolysis of olive oil. The study first characterised the components and the treated OPL, whereby data of the thermal gravimetric analysis (TGA) indicated that the hemicellulose and lignin components in OPL were successfully reduced by acid treatment and calcination. The morphological and physiochemical facets of the extracted SiO2 were investigated by fourier transform infrared (FTIR) spectroscopy, X-ray diffraction (XRD) and thermogravimetry analysis-differential scanning calorimetry (TGA-DTG). The results revealed that SiO2 was successfully extracted from OPL and coated on the Fe3O4. Subsequently, it was activated by APTES and glutaraldehyde to yield CRL/G-AP-SiO2-Fe3O4. FTIR, XRD and TGA-DTG data showed that CRL was successfully immobilised on G-APSiO2- Fe3O4, as seen with the band arising at 1639 cm-1 by C=O and C=N stretching in FTIR. Specifically, immobilisation of CRL onto the G-AP-SiO2-Fe3O4 yielded an enzyme loading and specific activity of 14.7 mg/g and 183 U/g. The CRL/G-APSiO2- Fe3O4 was then tested to establish the optimal conditions for catalysing hydrolysis of olive oil. It was found that the optimal conditions for the reaction that yielded the best activity were enzyme loading of 1.00 mg/mL, incubation temperature of 40 ??C, pH 8.0, ratio of olive oil: water of 2.5:1, and an agitation speed of 200 rpm. Assessments of thermal stability showed that CRL/G-AP-SiO2-Fe3O4 was more resistant to thermally-induced denaturation, than free CRL. The CRL/GAP- SiO2-Fe3O4 was kinetically shown to have higher affinity towards the substrate (Michaelis-Menten constant, Km = 0.583 g/mL) but catalysed at a lower maximum rate of reaction (Vmax = 833.3 µmol/ml.min) as compared to free CRL (Km = 6.00 g/mL, Vmax = 3330 µmol/ml.min), respectively. The thermodynamic parameters based on values of half-life (t1/2 = 38.94 min), D-value (129.4 min), thermal deactivation energy (Ed = 112.90 kJ/mol), standard enthalpy of deactivation (ΔHd° = 110.10 kJ/mol) and Gibbs free energy (ΔGd° = 11.32 kJ/mol) for CRL/G-AP-SiO2- Fe3O4 conclusively showed that the lipase was appreciably more thermostable than free CRL (t1/2 = 23.89 min, D-value = 79.67 min, Ed= 93.3 kJ/mol, ΔHd° = 87.5 kJ/mol and ΔGd° = 9.8111 kJ/mol) at 60'C. The finding shows that SiO2 extracted from OPL could be coated on Fe3O4 to be used as an inorganic support for enzyme immobilisation. The results thus demonstrated that the CRL/G-AP-SiO2-Fe3O4 biocatalyst was a potential candidate for catalysing hydrolytic reactions with good reaction rates, thus envisaging its prospective application as a commercially relevant biocatalyst.