Refolding and Purification of Cephalosporium Acremonium Deacetoxycephalosporin C synthetase/hydroxylase From Granules of Recombinant Escherichia Coli

Biotechnol Appl Biochem. 1996 Oct;24(2):109-19.


The gene for bifunctional deacetoxycephalosporin C synthetase/hydroxylase of Cephalosporium acremonium was cloned and overexpressed as an insoluble and inactive enzyme in granules of recombinant Escherichia coli. About 40-60% of expected synthetase activity along with 50-80% protein purity could be recovered directly from granular extracts with only a single empirically optimized refolding step. Further purification to homogeneity was achieved by a single anion-exchange-chromatographic step in the presence of denaturing concentrations of urea. The main obstacle to converting the homogeneous unfolded protein into the active enzyme was a urea-dependent aggregation during refolding that led to irreversible enzyme inactivation. Information obtained from refolding studies using gel-filtration HPLC, fluorescence spectroscopy and disulphide analysis led to an optimal enzyme refolding scheme that resulted in a highly active (i.e. 65-75% of the expected activity) and moderately stable fungal synthetase/hydroxylase.

MeSH terms

  • Acremonium / enzymology*
  • Disulfides / chemistry
  • Enzyme Stability
  • Escherichia coli / chemistry
  • Escherichia coli / enzymology
  • Escherichia coli / genetics*
  • Intramolecular Transferases*
  • Isomerases / chemistry*
  • Isomerases / genetics*
  • Isomerases / metabolism
  • Models, Chemical
  • Penicillin-Binding Proteins*
  • Protein Folding
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Structure-Activity Relationship
  • Sulfhydryl Compounds / chemistry


  • Disulfides
  • Penicillin-Binding Proteins
  • Recombinant Proteins
  • Sulfhydryl Compounds
  • Isomerases
  • Intramolecular Transferases
  • deacetoxycephalosporin C synthetase