Combined cross-linking of Rhizomucor miehei lipase and Candida antarctica lipase B for the effective enrichment of omega-3 fatty acids in fish oil.

The incorporation of a non-specific lipase and a sn-1,3 specific one in a single immobilized system can be a promising approach for the exploitation of both lipases. A one-step immobilization platform mediated by an isocyanide-based multi-component reaction was applied to create co-cross-linked enzymes (co-CLEs) of lipases from Rhizomucor miehei (sn-1,3 specific) and Candida antarctica (non-specific). Glutaraldehyde was found to be effective cross-linker by producing specific activity of 16.9 U/mg and immobilization yield of 99 %. High activity recovery of up to 404 % was obtained for immobilized derivatives. Leaking experiment showed covalent nature of the cross-linking processes. BSA had considerable effect on the immobilization process, providing 87-100 % immobilization yields and up to 10 times improvement in the specific activity of the immobilized derivatives. Scanning electron microscopy images showed flower-like and rod-like structures for the CLEs prepared by glutaraldehyde and undecanedicarboxylic acid, respectively. The prepared co-CLEs were examined in non-selective enrichment of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) from fish oil, showing capability of releasing up to 100 % of both omega-3 fatty acids within 8 h of the reaction. The reusability of co-CLEs in five successive cycles presented retaining 63-72 % of their initial activities after the fifth reuse cycle in the hydrolysis reaction.

The effect of carbon chain length of cross-linking agent on the functionality of carrier- free immobilized Thermomyces lanuginosa lipase particles.

The cross-linked enzyme (CLEs) of Thermomyces lanuginosa lipase (TLL) was prepared in an isocyanide-based multi-component reactions (ICMRs) platform by applying three di-acidic cross-linkers to unveil more factors contributing to the functional properties of CLEs. The linkers were 1,11-undecanedicarboxylic acid, azelaic acid, and adipic acid with 11, 7, and 4 carbon lengths, respectively, providing a proper tool to investigate the effect of linker length on the activity, stability, and selectivity of the resulting CLEs. The immobilization yields of 60-90 % and the specific activities of 168, 88.4 and 49 U/mg were obtained for the CLEs of 1,11-undecanedicarboxylic acid, azelaic acid, adipic acid, respectively. The lower activity of azelaic and adipic acid-mediated CLEs compared to the soluble TLL (110 U/mg) was explained by in silico calculations. The results revealed that as opposed to 1,11-undecanedicarboxylic acid, both linkers tended to penetrate the enzyme active site, thus resulting in a major inhibitory effect on the enzyme functionality. The thermal and co-solvent stability of the immobilized derivatives improved compared to those of free TLL. The selectivity of CLEs was also examined by catalytic release of main omega-3 fatty acids from fish oil, presenting the highest selectivity of 22 for the CLEs of azelaic acid.

Isocyanide-based multi-component reactions for carrier-free and carrier-bound covalent immobilization of enzymes.

Strategies for the covalent immobilization of enzymes depend on the type of functional group selected to perform the coupling reaction, and on the relative importance of selectivity, loading capacity, immobilization time and activity/stability of the resulting immobilized preparation. However, no single strategy is applicable for all covalent immobilization methods or can meet all these criteria, exemplifying the challenge of introducing a versatile process broadly compatible with the residues on the surface of proteins and the functional groups of common linkers. Here, we describe the use of isocyanide-based multi-component reactions for the carrier-bound and carrier-free covalent immobilization of enzymes. Isocyanide-based multi-component reactions can accept a wide variety of functional groups such as epoxy, acid, amine and aldehyde, as well as many commercially available bi-functional linkers, and are therefore suitable for either covalent coupling of enzymes on a solid support or intermolecular cross-linking of enzymes. In this strategy, an isocyanide is directly added to the reaction medium, the enzyme supplies either the exposed amine or carboxylic acid groups, and the support (in carrier-bound immobilization) or the bi-functional cross-linking agent (in carrier-free immobilization) provides another reactive functional group. The protocol offers operational simplicity, high efficiency and a notable reduction in time over alternative strategies, and can be performed by users with expertise in chemistry or biology. The immobilization reactions typically require 1-24 h.

Sortase-mediated immobilization of Candida antarctica lipase B (CalB) on graphene oxide; comparison with chemical approach.

In this study, Candida antarctica lipase B (CalB) was covalently immobilized on the surface of graphene oxide (GO) nanoparticles by sortase-mediated immobilization as well as a chemical attachment approach. Sortase is a transpeptidase that provides one-step purification and targeted immobilization of CalB from one specific site, presenting oriented attachment of the enzyme to a solid support. Chemical immobilization, on the other hand, is considered as a random immobilization, in which the protein can bind to the support from different regions of the protein surface. In this approach, amine-functionalized GO was further modified with glutaraldehyde to facilitate the covalent binding of CalB via its amine residues. The applied methods produced 60% and 100% immobilization yields and presented 0.106 U/mg and 0.085 U/mg of specific activities for the oriented and random immobilization, respectively. The stabilized enzyme with the sortase-mediated approach retained approximately 80% of its initial activity at 50°C.

Soluble enzyme cross-linking via multi-component reactions: a new generation of cross-linked enzymes.

By using a isocyanide-based multi-component reaction for the immobilization of the soluble forms of Rhizomucor miehei lipase (RML) and Thermomyces lanuginosa lipase (TLL), the first step of enzyme aggregation or crystallization in the traditional methods of cross-linking was bypassed. High immobilization yields and specific activities were achieved for both lipases.