Premium
Extracellular production of biologically active deacetoxycephalosporin C synthase from Streptomyces clavuligerus in Pichia pastoris
Author(s) -
Adrio José L.,
Velasco Javier,
Soler Gloria,
Saiz Marta Rodriguez,
Barredo José L.,
Moreno Miguel A.
Publication year - 2001
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.10028
Subject(s) - streptomyces clavuligerus , pichia pastoris , biochemistry , extracellular , fermentation , chemistry , biology , recombinant dna , microbiology and biotechnology , gene , clavulanic acid , antibiotics , amoxicillin
We have successfully expressed and observed secretion of the Streptomyces clavuligerus deacetoxycephalosporin C synthase (DAOCS) using the Pichia pastoris expression system. Two clones having multiple copies of the expression cassette were selected and used for protein‐expression analysis. SDS‐PAGE showed efficient expression and secretion of the bacterial recombinant DAOCS. The highest yield (120 μg/mL) was obtained when expression was induced with 2% methanol. Free and immobilized protein were assayed for biological activity and found to expand penicillin N (its natural substrate) and penicillin G to deacetoxycephalosporin C (DAOC) and deacetoxycephalosporin G (DAOG), respectively. © 2001 John Wiley & Sons, Inc. Biotechnol Bioeng 75: 485–491, 2001.