Cross-Linked Enzyme Aggregates (CLEAs)
The use of cross-linked enzyme crystals (CLECs) as industrial biocatalysts was pioneered by Altus Biologics in the 1990s. CLECs proved to be significantly more stable to denaturation by heat, organic solvents and proteolysis than the corresponding soluble enzyme or lyophilized (freeze-dried) powder. CLECs are robust, highly active immobilized enzymes of controllable particle size, varying from 1 to 100 micrometer. Their operational stability and ease of recycling, coupled with their high catalyst and volumetric productivities, renders them ideally suited for industrial biotransformations.
However, CLECs have an inherent disadvantage: enzyme crystallization is a laborious procedure requiring enzyme of high purity, which translates to prohibitively high costs. The more recently developed cross-linked enzyme aggregates (CLEAs), on the other hand, are produced by simple precipitation of the enzyme from aqueous solution, as physical aggregates of protein molecules, by the addition of salts, or water miscible organic solvents or non-ionic polymers. The physical aggregates are held together by non-covalent bonding without perturbation of their tertiary structure, that is without denaturation. Subsequent cross-linking of these physical aggregates renders them permanently insoluble while maintaining their pre-organized superstructure, and, hence their catalytic activity. This discovery led to the development of a new family of immobilized enzymes: cross-linked enzyme aggregates (CLEAs). Since precipitation from an aqueous medium, by addition of ammonium sulfate or polyethylene glycol, is often used to purify enzymes, the CLEA methodology essentially combines purification and immobilization into a single unit operation that does not require a highly pure enzyme. It could be used, for example, for the direct isolation of an enzyme, in a purified and immobilized form suitable for performing biotransformations, from a crude fermentation broth.
CLEAs are very attractive biocatalysts, owing to their facile, inexpensive and effective production method. They can readily be reused and exhibit improved stability and performance. The methodology is applicable to essentially any enzyme, including cofactor dependent oxidoreductases. Application to penicillin acylase used in antibiotic synthesis showed large improvements over other type of biocatalysts.
The potential applications of CLEAs are numerous and include:
1. Synthesis of pharmaceuticals, flavors and fragrances, agrochemicals, nutraceuticals, fine chemicals, bulk monomers and biofuels.
2. Animal feed, e.g. phytase for utilization of organically bound phosphate by pigs and poultry.
3. Food and beverage processing, e.g. lipases in cheese manufacture and laccase in wine clarification.
4. Cosmetics, e.g. in skin care products
5. Oils and fats processing, e.g. in biolubricants, bioemulsifiers, bioemollients.
6. Carbohydrate processing, e.g. laccase in carbohydrate oxidations.
7. Pulp and paper, e.g. in pulp bleaching.
8. Detergents, e.g. proteases, amylases and lipases for removal of protein, carbohydrate and fat stains.
9. Waste water treatment, e.g. for removal of phenols, dyes, and endocrine disrupters.
10. Biosensors/diagnostics, e.g. glucose oxidase and cholesterol oxidase biosensors.
11. Delivery of proteins as therapeutic agents or nutritional/digestive supplements e.g. beta-galactosidase for digestive hydrolysis of lactose in dairy products to alleviate the symptoms of lactose intolerance.
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