Carrier-Free Immobilization of Lipase from Candida rugosa with Polyethyleneimines by Carboxyl-Activated Cross-Linking

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• 作者：Susana Velasco-Lozano ; Fernando L贸pez-Gallego ; Rafael V谩zquez-Duhalt ; Juan C. Mateos-D铆az ; Jos茅 M. Guis谩n ; Ernesto Favela-Torres
• 刊名：Biomacromolecules
• 出版年：2014
• 出版时间：May 12, 2014
• 年：2014
• 卷：15
• 期：5
• 页码：1896-1903
• 全文大小：394K
• 年卷期：v.15,no.5(May 12, 2014)
• ISSN：1526-4602

文摘

Carrier-free immobilization of Candida rugosa lipase (CRL) and polymers containing primary amino groups were cross-linked using carbodiimide. To accomplish this, the free carboxyl groups of the enzyme were activated with carbodiimide-succinimide in organic medium, and then the activated proteins were cross-linked with different polyethylenimines (PEIs). The effect of the cross-linker chain length, the amount of added bovine serum albumin (BSA), and carbodiimide concentration on the catalytic properties of resulting cross-linked enzyme aggregates (CLEAs) was investigated. The CLEAs鈥?size, shape, specific activity, activity recovery, thermostability and enantioselectivity significantly varied according to the preparation procedure. The highest thermostable CRL-CLEA preparation was obtained with 1.3 kDa polyethyleneimine as cross-linker, 10 mg of BSA and 28 mM of carbodiimide. This preparation is 1.3-fold more active and thermostable than CLEAs prepared by the traditional method of amino cross-linking with glutaraldehyde, and retains 60% of residual activity after 22 h at 50 掳C. Additionally, the CRL-CLEA preparation showed an enantioselectivity of 91% enantiomeric excess (ee). This immobilization procedure provides an alternative strategy for CLEA production, particularly for enzymes where the traditional method of cross-linking via lysine residues leads to enzyme inactivation.