In 2001 Sheldon and co-workers patented the method for the production of Cross-Linked Enzyme aggregates. The preparation of CLEAs involved the precipitation of the enzyme followed by crosslinking of the precipitated enzyme aggregates with a bifunctional crosslinking agent such as glutaraldehyde. The cross-linking occurs between amino groups of the lysine residues on the surface of the enzyme and the aldehyde group, the formation of covalent bonds, makes the CLEAs permanently insoluble. This method is robust and straightforward, and the CLEAs are significantly more stable to denaturation by proteolysis, heat and organic solvents than the corresponding soluble enzyme.
A commercial variant of Thermomyces lanouginosa lipase (Lipex 16L) has been used to develop an improved method for CLEAs production, with applications in detergent products. Lipex 16L differs from the wild-type T. lanouginosa lipase due to two mutations (T231R, N233R) which improve wash performance and show beneficial synergistic results when combined with bleach catalysts. Lipex 16L is widely used in the laundry industry where it is considered one of the benchmark enzymes.
A new improved immobilization method has been developed and compared to a commercially available CLEA. It has been shown to be able to prepare homogenuous particles which are more active than the free enzyme and that these immobilised enzymes are suitable for HPC applications.