Department of Chemical Engineering, National Institute of Technology Karnataka, Surathkal, Srinivasnagar, Mangaluru 575 025, India.
Department of Chemical Engineering, National Institute of Technology Karnataka, Surathkal, Srinivasnagar, Mangaluru 575 025, India.
Enzyme Microb Technol. 2018 Mar;110:20-29. doi: 10.1016/j.enzmictec.2017.12.003. Epub 2017 Dec 19.
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.
考虑到印迹技术和无载体固定化的优势，用生物印迹法制备了交联酶聚集体（CLEA），印迹模板为固定化脂肪酶（CRL），印迹分子为牛血清白蛋白（BSA）、聚乙烯亚胺和戊二醛。研究并优化了各种因素，如酶与油的摩尔比、酶与 BSA 的摩尔比、酶与聚乙烯亚胺的摩尔比、戊二醛的用量、交联时间等，对脂肪酶活性回收率和聚集体产率的影响。将这种固定化脂肪酶（CRL-CLEA）用于非多不饱和脂肪酸甘油酯酯键的选择性水解，目的是在沙丁鱼油中浓缩 EPA 和 DHA 甘油酯。在乙醇和吐温 60 的存在下用油酸印迹，然后用共聚集体和交联剂进一步固定化，与游离酶相比，水解度提高了 10.4 倍。结果，使用 CRL-CLEA 可使脱酸油中 n-3PUFA 的含量增加 2.83 倍。所得油中二酰基和三酰基甘油的含量可忽略不计，证明其在水解脂肪酸酯键方面的效率更高，而不仅仅是 n-3PUFA。重复使用研究表明，CRL-CLEA 可重复使用 5 次以上，而其性能没有明显下降。由于印迹技术和随后以 CLEA 形式对 CRL 的固定化，水解度、热稳定性、水解效率和重复使用性能得到了提高。
