Preparation of cross-linked enzyme aggregates of lipase from Aspergillus niger: process optimization, characterization, stability, and application for epoxidation of lemongrass oil.

Abstract

Cross-linked enzyme aggregates (CLEAs) of lipase were prepared after fractional precipitation with 40-50% ammonium sulfate and then cross-linking with glutaraldehyde. The process variables for the preparation of lipase-CLEAs such as glutaraldehyde concentration, cross-linking period, and initial pH of medium were optimized. The optimized conditions for the preparation of lipase-CLEAs were 25mM/80min/pH 7.0, and 31.62mM/90min/pH 6.0 with one factor at a time approach and numerical optimization with central composite design, respectively. Lipase-CLEAs were characterized by particle size analysis, SEM, and FTIR. Cross-linking not only shifted the optimal pH and temperature from 7.0 to 7.5 and 40-45 to 45-50°C, but also altered the secondary structure. Lipase-CLEAs showed an increase in Km by 7.70%,and a decrease in Vmax by 16.63%. Lipase-CLEAs presented better thermostability than free lipase as evident from thermal inactivation constants (t1/2, D and Ed value), and thermodynamic parameters (Ed, ΔH°, ΔG°, and ΔS°) in the range of 50-70°C. Lipase-CLEAs retained more than 65% activity up to four cycles and showed good storage stability for 12days when stored at 4 ± 2°C. They were successfully utilized for the epoxidation of lemongrass oil which was confirmed by changes in iodine value, epoxide value, and FTIR spectra.