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

• Published in: Journal of biotechnology Vol. 133; no. 1; pp. 123 - 126
• Main Authors: Khatuntseva, S.A., Eldarov, M.A., Redo, V.A., Skryabin, K.G.
• Format: Journal Article
• Language: English
• Published: Lausanne Elsevier B.V 2008; Amsterdam [New York, NY]: Elsevier; New York, NY Elsevier
• Subjects: Affinity tag; Amidohydrolases - chemistry; Amidohydrolases - genetics; Amidohydrolases - isolation & purification; Amidohydrolases - metabolism; Bacillus - enzymology; Bacillus - genetics; Bacillus circulans; Brevundimonas diminuta; Chitin-binding domain; Enzyme Activation; Enzyme Stability; Enzymes, Immobilized - chemistry; Escherichia coli; Escherichia coli - enzymology; Escherichia coli - genetics; Fusion protein; Genetic Variation - genetics; Glutaryl-7-aminocephalosporanic acid acylase; Protein Engineering - methods; Recombinant Proteins - chemistry; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Glutaryl-7-aminocephalosporanic acid acylase; Affinity tag; Fusion protein; Chitin-binding domain
• ISSN: 0168-1656; 1873-4863
• DOI: 10.1016/j.jbiotec.2007.08.044

Modified chitin-binding domain (ChBD) from Bacillus circulans chitinase A1 with W42F mutation in chitin-binding site was genetically fused to different positions within α-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.