Self-assembly amphipathic peptides induce active enzyme aggregation that dramatically increases the operational stability of nitrilase

Abstract

Oligomeric nitrilase was fused with an amphipathic self-assembly peptide 18A at the C-terminus and expressed in Escherichia coli. The fusion enzyme spontaneously assembled into active aggregates with >90% native nitrilase activity. Much higher specific activities than the native nitrilase (Nit) were recorded for the fusion nitrilase (Nit-SEA) at higher temperatures. The enzyme aggregates were purified through cell lysis and centrifugation led to the facile preparation of immobilized particles (Nit-iSEA). Approximately 86% of the initial nitrilase activity was incorporated into the Ca-alginate entrapment beads. The thermostability of the four kinds of different nitrilase variants showed that the Nit-SEA and Nit-iSEA at 45 °C were about 6.7- and 10-fold more stable than the native nitrilase. The nitrile tolerance was also dramatically improved, no apparent substrate inhibition was observed for Nit-iSEA over the range of 30 to 120 mM mandelonitrile. Additionally, the Nit-iSEA could be recycled 20 times with ∼5% loss in activity.