Crosslinked aggregates of Rhizopus oryzae lipase as industrial biocatalysts: Preparation, optimization, characterization, and application for enantioselective resolution reactions

Abstract

Lipase from Rhizopus oryzae (ROL) was immobilized as crosslinked enzyme aggregate (CLEA) via precipitation with ammonium sulfate and simultaneous crosslinking with glutaraldehyde. The optimum conditions of the immobilization process were determined. Lipase CLEAs showed a twofold increase in activity when Tween 80-pretreated lipase was used for CLEA preparation. CLEAs were shown to have several advantages compared to free lipase. CLEAs were more stable at 50°C and 60°C as well as for a wide range of pH. After incubation at 50°C, CLEA showed 74% of initial activity whereas free enzyme was totally inactivated. Reduction of Schiff bases has been performed for the first time in the CLEA preparation process significantly improving the chemically modified CLEAs' reusability, thus providing an enzyme with high potential for recycling even under aqueous reaction conditions where enzyme leakage is, in general, one of the major problems. The CLEA retained 91% activity after 10 cycles in aqueous medium. The immobilized enzyme was used for kinetic resolution reactions. Results showed that immobilization had an enhancing effect on the conversion (c) as well as on the enantiomeric ratio (E). ROL CLEA displayed five times higher enantioselectivity for the hydrolysis of (R,S)-1-phenylethyl acetate and likewise 1.5 times higher enantioselectivity for the transesterification of racemic (R-S)-1-phenylethanol with vinylacetate.

Stabilization of a Lipolytic Enzyme for Commercial Application

“…Kartal and Kilinc [27] showed that increasing the particle size of the CLEA resulted in lower activity because of the loss of enzyme flexibility caused by excessive cross-linking and/or diffusion limitations [27]. The CLEA particle size gradually increased as a result of the cross-linking times and the process needed to be fine-tuned in order to obtain a positive effect.…”

“…Final The phenomenon of interfacial activation of lipases by additives such as surfactants, crown ethers or amines is well documented [27,35] and generally ascribed to the lipase adopting a more active conformation under these conditions. On preparing Lipex CLEAs, most of the tested compounds produced a negative effect with the exception of Tween 80, which had 129.7% retained activity as compared to the free (native) enzyme, and 150.3% retained activity as compared to the Lipex CLEAs prepared without surfactant.…”

“…Regardless of the choice, both immobilization strategies have a common feature of not requiring a support since the biocatalyst is predominantly the enzyme [32]. Hyperactive lipase CLEAs have been made in the presence of surfactants and detergents [27,33]. These compounds are added to the enzyme prior to cross-linking and initiate the process of interfacial activation, opening the lipase "lid", by binding to the hydrophobic active site of the enzyme [10].…”

“…Hyperactive lipase CLEAs have been made in the presence of surfactants and detergents [27,33]. These compounds are added to the enzyme prior to cross-linking and initiate the process of interfacial activation, opening the lipase "lid", by binding to the hydrophobic active site of the enzyme [10].…”

Recent Trends in Biodiesel and Biogas Production

“…Therefore, they are more stable, active and enantioselective than their corresponding solubilized form (58). Several reports on successful application of these two immobilization methods for biodiesel production could be found in literature (48,(59)(60)(61)(62)(63). However, immobilized enzymes are much more expensive than free enzymes (50).…”