Nancy L. St. Clair

Crosslinked enzyme crystals

A protein such as an enzyme of antibody is immobilized by crosslinking crystals of the protein with a multifunctional crosslinking agent. The crosslinked protein crystals may be lyophilized for storage. A preferred protein is an enzyme such as thermolysin, elastase, asparaginase, lysozyme, lipase or urease. Crosslinked enzyme crystals preferably retain at least 91% activity after incubation for three hours in the presence of a concentration of Pronase™ that causes the soluble uncrosslinked form of the enzyme to lose at least 94% of its initial activity under the same conditions. A preferred enzyme:Pronase™ ratio is 1:40. Enzyme crystals that are crosslinked may be microcrystals having a cross-section of 10-1 mm or less. Crosslinked enzyme or antibody crystals may be used in an assay, diagnostic kit or biosensor for detecting an analyte, in an extracorporeal device for altering a component of a fluid, in producing a product such as using crosslinked thermolysin crystals to produce aspartame, in separating a substance from a mixture, and in therapy.

Cofactor-Bound Cross-Linked Enzyme Crystals (CLEC) of Alcohol Dehydrogenase

Horse liver alcohol dehydrogenase was crystallized in the presence of its cofactor. The cross-linked enzyme crystals (CLECs) produced showed good catalytic activity without the addition of extra cofactor (see scheme). The enantioselectivity and stereochemical preference of the CLEC were the same as with the soluble enzyme, and both cofactor and enzyme were considerably more stable in CLEC form. Cofactor regeneration studies on the reduction of cinnemaldehyde indicate the potential for a high level of catalyst productivity.