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 α-subunit of glutaryl-7-aminocephalosporanic acid acylase (GLA) gene. Hybrid proteins were efficiently expressed in E. coli cells a...

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Bibliographic Details
Published in:Journal of biotechnology 2008, Vol.133 (1), p.123-126
Main Authors: Khatuntseva, S.A., Eldarov, M.A., Redo, V.A., Skryabin, K.G.
Format: Article
Language:eng ; rus
Subjects:
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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
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Summary: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.
ISSN:0168-1656
1873-4863
DOI:10.1016/j.jbiotec.2007.08.044