Extracellular production of biologically active deacetoxycephalosporin C synthase from Streptomyces clavuligerus in Pichia pastoris. |
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Authors: | J L Adrio J Velasco G Soler M Rodriguez-Saiz J L Barredo M A Moreno |
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Affiliation: | Laboratorio de Bioquímica, Departamento de I+D, Antibióticos, S.A.U., Avda. Antibioticos, 59-61, 24009-León, Spain. |
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Abstract: | 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 microg/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. |
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Keywords: | deacetoxycephalosporin C synthase expandase Pichia pastoris penicillin cephalosporin |
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