Cross-linked enzyme aggregates (CLEAs) are considered as an effective tool for the immobilization
of enzyme. In this study, Pencillium notatum lipase (PNL) was immobilized as carrier free
cross-linked enzyme aggregates using glutaraldehyde (GLA) and Ethylene glycol-bis [succinic acid Nhydroxysuccinimide]
(EG-NHS) as cross-linking agents. The optimal conditions for the synthesis of an
efficient lipase CLEAs such as precipitant type, the nature and amount of cross-linking reagent, and
cross-linking time were optimized. The recovered activities of CLEAs were considerably dependent on
the concentration of GLA; however, the activity recovery was not severely affected by EG-NHS as a
mild cross-linker. The EG-NHS aggregates displayed superior hydrolytic (52.08 ± 2.52%) and esterification
(64.42%) activities as compared to GLA aggregates which showed 23.8 ± 1.86 and 34.54% of hydrolytic
and esterification activity, respectively. Morphological analysis by fluorescence and scanning electron
microscope revealed that EG-NHS aggregates were smaller in size with larger surface area compared to
GLA aggregates. The pH optima of both types of CLEAs were displaced to slightly alkaline region and
higher temperature as compared to native enzyme. Highest enzyme activity of CLEAs was achieved at
the pH of 9.0 and 42 ◦C temperature. Moreover, a significant improvement in the thermal resistance was
also recorded after immobilization. After ten reusability cycles in aqueous medium, GLA and EG-NHS
cross-linked lipase CLEAs preserved 63.62% and 70.9% of their original activities, respectively. The results
suggest that this novel CLEA-lipase is potentially usable in many industrial applications.