Beta-lactam biosynthesis in a gram-negative eubacterium: purification and characterization of isopenicillin N synthase from Flavobacterium sp. strain SC 12.154
| Autor: | J. E. Baldwin, H. Palissa, Horst Kleinkauf, H. Von Döhren, Hong-Hoi Ting |
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| Rok vydání: | 1989 |
| Předmět: |
Time Factors
Blotting Western Size-exclusion chromatography Isopenicillin N synthase Polyethylene glycol Flavobacterium Microbiology beta-Lactamases chemistry.chemical_compound Biosynthesis Disulfides Sulfhydryl Compounds Molecular Biology Chromatography High Pressure Liquid chemistry.chemical_classification biology Molecular mass Temperature Hydrogen-Ion Concentration biology.organism_classification Cephalosporins Enzymes Amino acid Molecular Weight Kinetics Enzyme chemistry Biochemistry Fermentation biology.protein Oxidoreductases Research Article |
| Zdroj: | Journal of Bacteriology. 171:5720-5728 |
| ISSN: | 1098-5530 0021-9193 |
| DOI: | 10.1128/jb.171.10.5720-5728.1989 |
| Popis: | The occurrence, localization, and extraction of isopenicillin N-synthase (IPNS) were investigated in the gram-negative low-level beta-lactam producer Flavobacterium sp. strain SC 12.154, which forms deacetoxycephalosporin and excretes the cephabacin 7-formamidocephalosporin. IPNS was detected with anti-IPNS antibodies raised against the Cephalosporium acremonium enzyme. The flavobacterium enzyme, whose molecular mass (38 kilodaltons) and cofactor requirements resemble those of the fungal and Streptomyces enzymes, is formed at the transition from growth to the stationary phase. It was extracted into the polyethylene glycol phase of a polyethylene glycol-Ficoll-dextran three-phase system and was purified by quaternary aminoethyl ion-exchange chromatography, gel filtration, covalent chromatography on cystamine-Sepharose, and fast-protein liquid chromatography on Mono Q. The enzyme was characterized with respect to sulfhydryl requirement, inhibition by disulfides and metal ions, pH and temperature dependence, and stimulation by polyethylene glycol and low Triton X-100 concentrations, as well as by several amino acids, including alpha-aminoadipic acid and cysteine. The Km for alpha-aminoadipyl-cysteinyl-D-valine was 0.08 mM. An inactive membrane-associated form of IPNS was detected together with a beta-lactamase active on isopenicillin N. The system has been suggested as a model for the study of endogenous functions of beta-lactams in bacteria. |
| Databáze: | OpenAIRE |
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