Purification and immobilization of recombinant variants of Brevundimonas diminuta glutaryl-7-aminocephalosporanic acid acylase expressed in Escherichia coli cells.

Modified chitin-binding domain (ChBD) from Bacillus circulans chitinase A1 with W42F mutation in chitin-binding site was genetically fused to different positions within alpha-subunit of glutaryl-7-aminocephalosporanic acid acylase (GLA) gene. Hybrid proteins were efficiently expressed in E. coli cells as soluble, enzymatically active and correctly processed holoenzymes. ChBD-GLA fusions were easily affinity purified on chitin column by changing the salt concentration of binding and elution buffer. The developed one-step affinity purification procedure is thus a promising approach for scaled-up isolation of GLA variants for preparation of industrial biocatalysts as well as for structure-functional studies.