Appl Microbiol Biotechnol DOI 10.1007/s00253-011-3554-2 MINI-REVIEW Characteristic features and biotechnological applications of cross-linked enzyme aggregates (CLEAs) Roger A. Sheldon Received: 11 July 2011 /Revised: 8 August 2011 /Accepted: 13 August 2011 # The Author(s) 2011. This article is published with open access at Springerlink.com Abstract Cross-linked enzyme aggregates (CLEAs) have reactors (no specialized equipment is needed) under mild many economic and environmental benefits in the context conditions (ambient temperature and pressure, physiological of industrial biocatalysis. They are easily prepared from pH) in an environmentally acceptable solvent (water) using a crude enzyme extracts, and the costs of (often expensive) biocompatible and biodegradable catalyst (an enzyme) that is carriers are circumvented. They generally exhibit improved itself derived from renewable resources. storage and operational stability towards denaturation by Enzymatic reactions proceed with high regio- and stereo- heat, organic solvents, and autoproteolysis and are stable selectivity and generally without the need for functional group towards leaching in aqueous media. Furthermore, they have activation and protection and deprotection steps required in high catalyst productivities (kilograms product per kilogram traditional syntheses. Hence, generally speaking, enzymatic biocatalyst) and are easy to recover and recycle. Yet another processes generate less waste than conventional synthetic advantage derives from the possibility to co-immobilize two routes, are more energy efficient, and provide products in or more enzymes to provide CLEAs that are capable of higher purity. The application of modern protein engineering catalyzing multiple biotransformations, independently or in techniques, such as directed evolution, has enabled the sequence as catalytic cascade processes. optimization of their properties to fit pre-defined process parameters (Luetz et al. 2008; Turner 2009). In short, Keywords Immobilization . Hydrolases . Oxidoreductases . enzymes can be “tailor made” and can be produced on a Lyases . Magnetic . Sustainable largescaleforanattractiveprice. Notwithstanding all these advantages, enzymes have some drawbacks that have limited their widespread appli- Introduction cation. They often lack operational and storage stability, for example. Enzymes are complex, highly sensitive molecules One of the great challenges that the pharmaceutical, with unique three-dimensional structures that are essential chemical, and allied industries face in the 21st century is for their activities. Exposure to certain conditions, such as the transition to a greener and more sustainable manufac- elevated temperatures or organic solvents, can lead to turing base that minimizes, or preferably avoids, the denaturation (unfolding) and concomitant loss of activity. generation of waste and the use of toxic and/or hazardous Furthermore, enzymes are generally used as aqueous materials. Biocatalysis has many benefits to offer in this solutions which makes recovery and reuse problematical respect. Reactions can be performed in conventional and can also result in contamination of the product. These obstacles can generally be overcome by immobilization of the enzyme, affording improved storage and operational R. A. Sheldon (*) stability and providing for its facile separation and reuse Department of Biotechnology, Delft University of Technology, (Sheldon 2007a). Moreover, immobilized enzymes, in Julianalaan 136, NL-2628 BL Delft, The Netherlands contrast to free enzymes which can penetrate the skin, are e-mail: [email protected] unlikely to cause allergic reactions. Appl Microbiol Biotechnol Enzyme immobilization: an enabling technology Several years ago, we reasoned that crystallization could perhaps be replaced by precipitation of the enzyme from Immobilization can enable economically viable applications aqueous buffer, a simpler and less expensive method not of enzymes that, for one or more of the reasons outlined requiring highly pure enzymes. It is well documented that above, would not have been viable using the free enzyme. the addition of salts, or water-miscible organic solvents or It typically involves binding the enzyme to a carrier non-ionic polymers, to aqueous solutions of proteins leads (support), such as an ion exchange resin or silica, or to their precipitation as physical aggregates that are held encapsulation in an inert carrier, affording a biocatalyst with together by non-covalent bonding (Burgess 2009). Addition superior operational performance compared to the free of water to this precipitate results in dissolution of the enzyme. Binding to the carrier can involve simple adsorp- enzyme. In contrast, we reasoned that cross-linking of these tion, e.g., via hydrophobic or ionic interactions or actual physical aggregates, to form what is essentially a cross- covalent bonding. A serious drawback of such carrier- linked polymer, would render them permanently insoluble bound enzymes (Cao et al. 2003) in general is their low while maintaining their pre-organized superstructure and productivities (kilograms product per kilogram enzyme) hence their catalytic activity. This led us to develop (Cao et owing to the large amount of non-catalytic ballast (often al. 2000;Sheldon2011) a new class of immobilized >95% of the total mass). They can also be costly as some of enzymes which we called cross-linked enzyme aggregates the carriers that are used are themselves expensive (CLEAs). Since selective precipitation with ammonium materials. In contrast, immobilization by cross-linking of sulfate is a commonly used method for enzyme purification enzyme molecules affords carrier-free immobilized (Englard and Seifter 1990), the CLEA methodology enzymes with high productivities and avoids the costs of essentially combines two unit processes, purification and the carrier. immobilization, into a single operation. In principle, one Cross-linked enzymes, produced by mixing an aqueous can even take the crude enzyme extract from fermentation solution of the enzyme with an aqueous solution of broth and produce the immobilized enzyme in one simple glutaraldehyde, were already known in the 1960s but were operation. Hence, one can envisage the direct immobilization generally difficult to handle, gelatinous materials exhibiting of an enzyme, at the production site, without any need for low activity, poor reproducibility, and low stability and shelf intermediate work-up or purification. life. Consequently, carrier-bound enzymes became the method of choice for the next three decades. In the early 1990s, Altus Biologics introduced the use of cross-linked Preparation of CLEAs enzyme crystals (CLECs) as industrial biocatalysts (Margolin and Navia 2001). The methodology was applicable to a wide A general scheme for the preparation of a CLEA is outlined variety of enzymes, and CLECs exhibited excellent opera- in Fig. 1. Glutaraldehyde is generally the cross-linking tional stability, controllable particle size coupled with high agent of choice as it is inexpensive and readily available in productivity, and facile recovery and reuse, making them commercial quantities. It has been used for decades for ideally suited to industrial biocatalysis. However, they had cross-linking proteins. However, the chemistry is complex one inherent limitation: the need to crystallize the enzyme, a and not fully understood. Cross-linking occurs via reaction laborious procedure requiring enzyme of high purity which of the free amino groups of lysine residues, on the surface meant relatively high costs. of neighboring enzyme molecules, with oligomers or Fig. 1 General scheme for CLEA preparation X-linking aggregation Classic CLEA X-linking e.g. (NH4)2SO4 or tert-butanol Dissolved Aggregates Copolymerization Enzyme e.g. with RSi(OR)3 Silica-CLEA composite Appl Microbiol Biotechnol polymers of glutaraldehyde resulting from inter- and high, too much cross-linking can result in a complete loss intramolecular aldol condensations. Cross-linking can of the enzyme's flexibility and hence its activity. Since involve both Schiff's base formation and Michael-type 1,4 every enzyme has a unique surface structure, containing addition to α,β-unsaturated aldehyde moieties, and the varying numbers of lysine residues, the optimum ratio has exact mode of cross-linking is pH dependent (Migneault et to be determined for each enzyme. With enzymes contain- al. 2004; Walt and Agayn 1994). Other dialdehydes, ing few or no accessible lysine residues, cross-linking may involving less complicated chemistry, can be used as be insufficient and lead to CLEAs that are unstable towards cross-linkers, e.g., dextran polyaldehyde, followed by leaching in aqueous media. One way to overcome this reduction of the Schiff's base moieties with sodium problem is to add polyamines, such as polyethyleneimine, cyanoborohydride to form irreversible amine linkages which are then co-immobilized with the enzyme (Lopez- (Mateo et al. 2004). Gallego et al. 2005). Problems can also be encountered in A variation on this theme involves performing the cross- CLEA formation when the protein concentration in the linking in the presence of a monomer that undergoes enzyme preparation is low. In such cases, CLEA formation polymerization under these conditions. This results in the can be promoted by the addition of a second protein, such formation of CLEA–polymer composites with tunable as bovine serum albumin, as a so-called proteic feeder physical properties.