Immobilization of Thermomyces lanuginosus Lipase by Different Techniques on Immobead 150 Support: Characterization and Applications

Abstract

Thermomyces lanuginosus lipase (TLL) was immobilized on native and modified Immobead 150, with epoxy groups removed by hydrolysis and oxidized to add aldehyde on its surface. Immobilizations on both supports were performed by adsorption, adsorption and cross-linking, covalent attachment, multipoint covalent attachment, and, for the modified support, multipoint covalent attachment using ethylenediamine. Biocatalysts were evaluated for thermal and solvent stabilities, and the best biocatalyst was also tested after incubation in ionic liquids and used in the synthesis of butyl butyrate and isoamyl butyrate. Multipoint covalent immobilized TLL on the native Immobead 150 (Emulti) showed a half-life of 5.32 h at 70 °C, being approximately 30 times more stable than its soluble form; it showed high stability in acetone, hexane, and isooctane. Its enzymatic activity was up to 40% when incubated in ionic liquids. Ester synthesis produced yields of esterification above 60% in 24 h. Of all immobilization protocols, the Emulti performed best concerning the thermal, solvent, and ionic liquids stabilities. Emulti was successfully applied to the synthesis of butyl butyrate and isoamyl butyrate, which are very important products for the food and beverage industries.

Physical-Chemical Properties of the Support Immobead 150 Before and After the Immobilization Process of Lipase

“…These results correlates well with previous reports on the literature, 29 in which, using the same protocol but 1 M immobilization buffer and protein loads varying from 5 to 20 mg g -1 , it was observed a decrease in the efficiency inversely proportional to protein load. This loss of efficiency can be attributed to high ionic strength of the immobilization buffer, possibly causing the aggregation of the protein.…”

“…The fact that this support has a small associated amount of water is remarkable in the sense that enzyme stability can be greatly enhanced when exposed to organic solvents and ionic liquids, as it was observed in a previous work. 29 The control of water content on enzymatic synthesis reactions is important because this fact can affect the reaction either positively or negatively. Water acts as a 'lubricant', maintaining the enzyme in the active conformation.…”

Immobilization of Amano Lipase A onto Stöber silica surface: process characterization and kinetic studies

“…The hydrolytic activity of the immobilized enzymes was estimated according to Matte et al [28]. Briefly, the spectrophotometric measurements were based on the ability of the enzymes to transform the p-NPP (paranitrophenyl palmitate) into pNP (para-nitrophenol).…”

Utilization of spent coffee grounds for isolation and stabilization of Paenibacillus chitinolyticus CKS1 cellulase by immobilization

“…The immobilization yield (%) was the term used to determine the success of the cellulase immobilization process onto the coffee carrier, and was calculated by the following equation [19]:$Yield(\%)=100·(\frac{immobilizedactivity}{strartinga\mathrm{c}tivity})$…”