Abstract
In this study, Candida antarctica lipase A, which has a unique applicability for the conversion of highly branched and bulky substrates, was subjected to immobilization on the flexible nanoporous MIL-53(Fe) by two approaches: covalent coupling and in situ immobilization method. The pre-synthesized support under ultrasound irradiation was incubated with N,N-dicyclohexylcarbodiimide to mediate the covalent attachment between the carboxylic groups on the support surface and amino groups of enzyme molecules. The in situ immobilization in which the enzyme molecules directly were embedded into the metal-organic framework was performed under mild operating conditions in a facile one-step manner. Both immobilized derivatives of the enzyme were characterized by scanning electron microscopy, X-ray diffraction, thermogravimetric analysis, FT-IR spectra, and energy-dispersive X-ray spectroscopy. In the in situ immobilization method, the enzyme molecules were efficiently encapsulated within the support with a high loading capacity (220 ± 5 mg/g support). On the other hand, the covalent attachment resulted in immobilizing much lower concentrations of the enzyme (20 ± 2.2 mg/g support). Although both immobilized derivatives of lipase showed broader pH and temperature tolerance relative to the soluble enzyme, the biocatalyst, which was prepared through in situ method, was more stable at elevated temperatures than the covalently immobilized lipase. Furthermore, in situ immobilized derivatives of Candida antarctica lipase A could be efficiently reused for at least eight cycles (> 70% of retained activity). In contrast, its covalently immobilized counterpart showed a drastic decrease in activity after five cycles (less than 10% of retained activity at the end of 6 rounds).