Modified general affinity adsorbent for large-scale purification of penicillinases

L. Kiss, Andrea Tar, Susanne Gál, Bela L. Toth-Martinez, Ferenc J. Hernádi

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

N-Acetyl-d-(-)-penicillamine as a stable second-generation biospecific affinity ligand has previously been suggested for purification of Bacillus cereus 569/H β-lactamase I. A complex spacer arm is coupled with the matrix by using epichlorohydrin and phloroglucinol doubly activated with divinyl sulphone in the meta position. Coupling of d-(-)-penicillamine ligand resulted in an active affigel. However, we found that two affinity ligands in close proximity prevents simultaneous binding of two penicillinase molecules, therefore one ligand is superfluous. Our results show that: (1) shortening the spacer arm by direct activation of the matrix with divinyl sulphone is satisfactory to produce the affinity material with N-acetyl-d-(-)-penicillamine; (2) incorporation of 15 μmol of N-acetyl-d-(-)-penicillamine per ml of wet Sepharose 4B satisfies the maximum binding capacity requirements of the affigel (about half of the originally incorporated amount of ligand); (3) our simplified affinity adsorbent is generally applicable for large-scale purification of penicillinases to homogeneity from various bacterial sources by the convenient batch method without prior concentration of these enzymes; (4) reacetylation for four/five times can regenerate the original binding capacity of the affigel.

Original languageEnglish
Pages (from-to)109-116
Number of pages8
JournalJournal of Chromatography A
Volume448
Issue numberC
DOIs
Publication statusPublished - 1988

Fingerprint

Penicillinase
Penicillamine
Adsorbents
Purification
Ligands
Epichlorohydrin
Phloroglucinol
Bacillus cereus
Sepharose
Chemical activation
Molecules
Enzymes

ASJC Scopus subject areas

  • Analytical Chemistry
  • Clinical Biochemistry
  • Molecular Medicine

Cite this

Modified general affinity adsorbent for large-scale purification of penicillinases. / Kiss, L.; Tar, Andrea; Gál, Susanne; Toth-Martinez, Bela L.; Hernádi, Ferenc J.

In: Journal of Chromatography A, Vol. 448, No. C, 1988, p. 109-116.

Research output: Contribution to journalArticle

Kiss, L. ; Tar, Andrea ; Gál, Susanne ; Toth-Martinez, Bela L. ; Hernádi, Ferenc J. / Modified general affinity adsorbent for large-scale purification of penicillinases. In: Journal of Chromatography A. 1988 ; Vol. 448, No. C. pp. 109-116.
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AB - N-Acetyl-d-(-)-penicillamine as a stable second-generation biospecific affinity ligand has previously been suggested for purification of Bacillus cereus 569/H β-lactamase I. A complex spacer arm is coupled with the matrix by using epichlorohydrin and phloroglucinol doubly activated with divinyl sulphone in the meta position. Coupling of d-(-)-penicillamine ligand resulted in an active affigel. However, we found that two affinity ligands in close proximity prevents simultaneous binding of two penicillinase molecules, therefore one ligand is superfluous. Our results show that: (1) shortening the spacer arm by direct activation of the matrix with divinyl sulphone is satisfactory to produce the affinity material with N-acetyl-d-(-)-penicillamine; (2) incorporation of 15 μmol of N-acetyl-d-(-)-penicillamine per ml of wet Sepharose 4B satisfies the maximum binding capacity requirements of the affigel (about half of the originally incorporated amount of ligand); (3) our simplified affinity adsorbent is generally applicable for large-scale purification of penicillinases to homogeneity from various bacterial sources by the convenient batch method without prior concentration of these enzymes; (4) reacetylation for four/five times can regenerate the original binding capacity of the affigel.

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