title: Purification and immobilization of recombinant variants of Brevundimonas diminuta glutaryl-7-aminocephalosporanic acid acylase expressed in Escherichia coli cells.
abstract: 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.
authors.affiliation: Centre Bioengineering, Russian Academy of Sciences, Prospekt 60-letiya Oktyabrya, 7/1, 117312 Moscow, Russia. svehat@yahoo.com
authors.forename: S A
authors.initials: SA
authors.lastname: Khatuntseva
authors.forename: M A
authors.initials: MA
authors.lastname: Eldarov
authors.forename: V A
authors.initials: VA
authors.lastname: Redo
authors.forename: K G
authors.initials: KG
authors.lastname: Skryabin
mesh_terms: D000581:Amidohydrolases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D004789:Enzyme Activation; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D014644:Genetic Variation / Q000235:genetics; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D004800:Enzymes, Immobilized; D011994:Recombinant Proteins; D000581:Amidohydrolases; C509512:glutaryl-7-aminocephalosporanic acid acylase, Bacillus laterosporus