RESUMO
Considering the advantages of bioimprinting and carrier free immobilization, cross-linked enzyme aggregates (CLEA) were prepared by using bioimprinted Candida rugosa lipase (CRL) with Bovine serum albumin (BSA), Polyethyleneimine and glutaraldehyde. Effect of various factors such as CRL-Oleic acid ratio, CRL-BSA ratio, CRL- Polyethyleneimine ratio, glutaraldehyde loading, cross-linking time etc., on lipase activity recovery and aggregate yield were studied and optimized. This immobilized lipase (CRL-CLEA) was used for the selective hydrolysis of ester linkages of non-PUFA glycerides, with an aim to concentrate EPA and DHA glycerides in the Sardine oil. Imprinting with oleic acid in the presence of ethanol and Tween 60, and further immobilization with co-aggregates and cross-linking agent showed 10.4 times higher degree of hydrolysis compared to free enzyme. As result, 2.83-fold increase of n-3 PUFA content in deacidified oil was obtained by using CRL-CLEA. The resultant oil had negligible di- and triglycerides content, proving higher efficiency in hydrolysing ester bonds of fatty acids, other than n-3 PUFA. Reusability studies showed CRL-CLEA could be reused up to 5 runs without a substantial reduction in its performance. Improvement in degree of hydrolysis, thermostability, efficiency of hydrolysis and reusability were achieved due to bioimprinting and subsequent immobilization of CRL in the form of CLEA.