A new, mild cross-linking methodology to prepare cross-linked enzyme aggregates

Cross‐linked enzyme aggregates (CLEAs) were prepared from several enzymes (penicillin G acylase, hydroxynitrile lyase, alcohol dehydrogenase, and two different nitrilases) by precipitation and subsequent cross‐linking using dextran polyaldehyde. In most cases, higher immobilization yields were obtained using the latter cross‐linker as compared with the commonly used glutaraldehyde. Active site titration of penicillin acylase CLEAs showed that the higher activity originated from a significantly lower loss in active sites using dextran polyaldehyde as a cross‐linking agent. It is proposed that macromolecular cross‐linkers are too large to penetrate the protein active site and react with catalytically essential amino acid residues. © 2004 Wiley Periodicals, Inc.