One of the great challenges that the pharmaceutical, chemical and allied industries face in the 21st century is the transition to greener and more sustainable manufacturing processes that minimize, or preferably avoid, the generation of waste and the use of toxic and/or hazardous materials. Biocatalysis has many benefits to offer in this respect. Reactions can be performed in conventional reactors (no specialized equipment is needed) under mild conditions (ambient temperature and pressure, physiological pH) in an environmentally acceptable solvent (water) using a biocompatible and biodegradable catalyst (an enzyme) that is itself derived from renewable resources.

By using enzymes, reactions involving multifunctional molecules can proceed with high regio- and stereoselectivity and generally without the need for functional group activation and protection and deprotection steps required in traditional syntheses.

Hence, generally speaking, enzymatic processes generate less waste than conventional synthetic routes, are more energy efficient and provide products in higher purity. The use of enzymes also circumvents product contamination with traces of metal catalysts, which often necessitates expensive purification steps in pharmaceutical manufacture.

The time is ripe for the widespread application of enzymes in chemicals manufacture. Thanks to the sequencing of numerous bacterial and fungal genomes, many new enzymes have been identified in recent years. The application of modern protein engineering techniques, mostly developed in the last two decades, such as directed-evolution by gene shuffling, has enabled the optimization of their properties to fit pre-defined process parameters.

Advances in recombinant DNA technology have paved the way for their economically viable large-scale production. In short, more enzymes are available; they can be "tailor made" and can be produced on a large scale for an attractive price.

Enzyme Immobilization

Nothing is perfect, however, and enzymes do have some limitations. For example, they often lack operational and storage stability. Enzymes are complex, highly sensitive molecules with unique three- dimensional structures that are essential for their activities. Exposure to certain conditions, such as elevated temperatures or organic solvents, can lead to denaturation (unfolding) and concomitant loss of activity.

Furthermore, enzymes are generally used as aqueous solutions, which makes recovery and re-use problematical 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 stability and providing for its facile separation and re-use. Moreover, immobilized enzymes, in contrast to free enzymes, which can penetrate the skin, do not cause allergic reactions.

Immobilization is an enabling technology that typically involves binding the enzyme to a support, such as an ion exchange resin or silica, or encapsulation in an inert matrix. Such strategies can be costly and afford carrier-bound enzymes with low productivities (kilograms of product per kilograms of enzyme) owing to the large amount of non-catalytic ballast (often more than 95% of the total mass).

In contrast, immobilization by cross linking of enzyme molecules affords carrier-free immobilized enzymes with high productivities. Cross-linked enzymes, produced by mixing an aqueous solution of the enzyme with an aqueous solution of glutaraldehyde, were already known in the 1960s but generally had low activity, poor reproducibility, low stability and shelf life, and were difficult to handle and gelatinous materials. 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 enzyme crystals (CLECs) as industrial biocatalysts.

The methodology was applicable to a wide variety of enzymes and CLECs exhibited excellent operational stability, controllable particle size coupled with high productivity and facile recovery and re-use, making them ideally suited to industrial biocatalysis. However, they had one inherent limitation: the need to crystallize the enzyme, a laborious procedure requiring enzyme of high purity. In practice this translates to relatively high costs.

Enter Cross-Linked Enzyme Aggregates (CLEAs)

Several years ago, we reasoned that crystallization could perhaps be replaced by precipitation of the enzyme from aqueous buffer, a simpler and less expensive method not requiring highly pure enzymes. This led us to develop a new class of immobilized enzymes, which we called cross-linked enzyme aggregates (CLEAs). The CLEA methodology essentially combines two unit processes, purification and immobilization, into a single operation. In principle, one can even take the crude enzyme extract from fermentation broth and produce the immobilized enzyme in one simple operation. A variation on this theme involves performing the cross-linking in the presence of a monomer that undergoes polymerization under these conditions. This results in the formation of CLEA-polymer composites with tunable physical properties. For example, if the cross-linking is performed in the presence of a siloxane the latter undergoes polymerization to afford a CLEA-silica composite.

The hydrophobic/hydrophilic properties and particle size of the latter can be tailored by manipulating the structure of the siloxane used. More recently, we have made "smart" magnetic CLEAs by conducting the cross-linking in the presence of functionalized magnetic nanoparticles. These mCLEAs can be separated by magnetic decantation or can be used in a magnetically stabilized fluidized bed and we envisage that this will lead to novel combinations of bioconversions and downstream processing. A further elaboration of the CLEA methodology is the preparation of combi-CLEAs, from mixtures of two or more enzymes, for use in multi-enzyme cascade processes.

CLEAs have many economic and environmental benefits in the context of industrial biocatalysis. They are easily prepared from crude enzyme extracts and the costs of (often expensive) carriers are circumvented. They generally exhibit improved storage and operational stability towards denaturation by heat, organic solvents and autoproteolysis and are stable towards leaching in aqueous media. Furthermore, they have high catalyst productivities (kilograms of product per kilograms of biocatalyst) and are easy to recover and re-use. CLEAs are highly porous materials and diffusional limitations are generally not observed in typical bioconversions. Diffusional limitations have been observed in colorimetric assays which are usually very fast reactions. Obviously, the rate of diffusion is also influenced by the particle size which is amenable to tuning. Optimum rates are observed with smaller particles but practical considerations, e.g. ease of filtration, often dictate the use of larger particles.

The proprietary CLEA methodology has been commercialized by CLEA Technologies, which produces CLEAs from commercially available enzymes for sale on the open market as well as custom CLEAs from enzymes provided by clients on an exclusive basis. CLEAs are currently being developed for application in a wide variety of industrial settings, from peptide synthesis and chiral resolutions to cosmetic ingredients, oils and fats conversions, carbon capture and biofuels.