Candida rugosa lipase imobilization on modified mesoporous kit-6 from rice husk as biocatalyst in esterification for butyl butyrate

Abstract

Lipases as are widely exploited in various industries, especially in the food processing industries for their ability to act on a variety of substrates, pH and thermal stability, resistance to solvents, and they are also highly specific as chemo-, regio-, and enantioselective biocatalysts. However, enzymes have serious drawbacks such as, often unstable in the long term operation, high cost and non-reusability. Enzyme immobilization is a creative solution to alleviate these problems, whereas the mesoporous silica KIT-6 has great potential as catalyst support due to the high surface area, porosity and stability. This research aimed to explore the potential of APTES-functionalized KIT-6 silica as immobilization matrix for lipase from Candida rugosa (CRL). In particular, the immobilized CRL onto KIT-6 silica nanoparticles derived from rice husk ash, is reported here for the first time as a catalyst in the esterification reaction. Additionally, a comparative study of the effectiveness of immobilization methods (physical adsorption and covalent binding) on the biophysical properties of the immobilized CRL, and the hydrolytic activities of free and immobilized enzymes in the esterification of butyric acid with butanol were carried out. The different techniques used for the biophysical characterization of the immobilized CRL onto KIT-6 were SAXS, TEM, FESEM, FTIR, XPS, fluorescence spectra, and TGA. The results showed that both functionalization and immobilization procedures did not disrupt the structural integrity of the immobilized CRL, but found significant differences in the morphology of each of the resultant catalysts. The KIT-6/a/g/Crl catalyst displayed highly dense and more compact morphology than the KIT-6/Crl with a higher enzyme loading. The findings suggest that some of the attached APTES-glutaraldehyde compounds including CRL molecules may be physically adsorbed inside the pores of the KIT-6/a/g/Crl, which resulted in the obvious reduction in surface area and total pore volume when the CRL loading increased. Further, the structure of immobilized CRL in KIT-6 was verified by FTIR spectra and correlated with the XPS analysis. The values of binding constant, Kass, determined by fluorescence quenching studies, confirmed that the direct physical interaction of KIT-6 with CRL was much weaker (5.20 × 102 M-1), compared to the covalent binding (1.20 × 104 M-1). The optimal assay conditions of the CRL catalyzed esterification were identified to be pH 7.0 at 35 °C with 0.10 mg/mL concentration of the CRL in n-hexane as the solvent. The CRL immobilized onto KIT-6 through physical adsorption and covalent binding method is more advantageous compared to the free CRL, as it could withstand extreme reaction conditions and capable to achieve a high activity. Besides, the conformational changes in the CRL were restricted due to entrapment in the porous silica matrix. Overall, Crl immobilized onto silica KIT-6 demonstrated improved properties compared with the free CRL giving excellent results in the thermal stability, storage and reusability studies. Both the immobilized CRL onto KIT-6 by covalent binding method (KIT-6/a/g/Crl) and physical adsorption (KIT-6/Crl) were able to retain more than 50% of the activity after the heat treatment. Moreover, KIT- 6/a/g/Crl retained 67.3% of the activity after 85 days of storage, indicating enhanced thermal stability and shelf-life through the immobilization process. Recycling studies showed that the KIT-6/a/g/Crl, which has less loading amount of CRL (2.57 mg/g support versus 3.10 mg/g support in KIT-6/Crl) was reusable up to 20 cycles, with only 75.3% of its activity lost during the first 10 cycles; as compared to KIT-6/Crl which rendered inactive over 7 cycles. Under an optimized reaction condition, even using low CRL loadings, the specific activity of KIT-6/a/g/Crl was shown higher (2144.7 U/mg) than the KIT-6/Crl (1847.6 U/mg) and free CRL (1604.3 U/mg). Optimal yields of the esterification of butyric acid with butanol in hexane using the KIT-6/Crl and KIT-6/a/g/Crl catalysts were 94% and 90% respectively, with 100% selectivity. The kinetic studies revealed that Km of immobilized CRL was higher than that of free CRL, implying a high affinity towards the substrate. Also, the Vmax/Km (catalytic efficiency) value of KIT-6/a/g/Crl was comparable with the free CRL, whereas the value determined for the KIT-6/Crl was lower compared to the KIT-6/a/g/Crl.