| abstract |
A protein such as an enzyme or antibody is immobilized by crosslinking crystals of the protein with a multifunctional crosslinking agent such as glutaraldehyde, and if desired lyophilizing the crosslinked crystals for storage. Crosslinking of the protein crystals provides stabilization for use under harsh conditions and for lyophilizing. The crystals crosslinked may be microcrystals having a cross-section of 10-1 mm or less. Crosslinked thermolysin, esterase, elastase, asparaginase and lysozyme crystals and crosslinked crystals of lipase from Geotrichum candidum and Candida cylindracea and of porcine origin can be used to convert a substrate to a product. Crosslinked thermolysin crystals are prepared that retain at least 96% of their initial activity after incubation for 4 days in the presence of a concentration of Pronase TM such as a thermolysin:Pronase TM ratio of 1:40 that causes the soluble uncrosslinked form of thermolysin that is crystallized to form the crystals that are crosslinked to lose at least 99% of its initial activity after incubation for 90 minutes under the same conditions. Crosslinked thermolysin crystals can be used to produce aspartame by combining the crystals with N-(benzyloxycarbonyl)-L-aspartic acid and L-phenylalanine methyl ester in a mixed aqueous/organic solvent such as a water-ethyl acetate mixture, and maintaining the combination under conditions to cause a condensation reaction to produce N-(benzyloxycarbonyl)-L-aspartyl-L-phenylalanine methyl ester, and removing the benzyloxycarbonyl group to obtain aspartame. Crosslinked antibody crystals have uses as an immunospecific reagent such as for detection of a substance in a sample, and for therapeutic purposes. |