Extracellular carboxypeptidase was isolated from culture filtrates of Str. spheroides strain 35, using affinity chromatography on bacitracin-silochrome, bacitracin-Sepharose and CABS-Sepharose. The electrophoretically homogenous enzyme was obtained with a 44% yield and 4160-fold purification. The enzyme-molecular weight is 33,000 Da; pI is 4.7. The amino acid composition of carboxypeptidase is as follows: Asp43, Thr30, Ser35, Glu33, Pro30, Gly47-50, Ala38, 1/2 Cys5-6, Val16, Met2, Ile11, Leu15, Tyr8, Phe10, Lys10, His6, Arg9. The enzyme shows an activity optimum at pH 7.5 is stable at pH 6-8, is completely inhibited with EDTA and can be reactivated by Ca2+. The carboxypeptidase from Str. spheroides strain 35 has a dual substrate specificity, i. e., it splits N-substituted di-, three- and tetrapeptides having both neutral and basic amino acids at the C-ends similar to mammalian carboxypeptidases A and B. The enzyme belongs to the family of metallocarboxypeptidases; its properties are very similar to those of carboxypeptidase S from Str. griseus K-1 and of carboxypeptidase T from Thermoactinomyces sp.
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Int J Syst Evol Microbiol
December 2008
NITE Biological Resource Center, National Institute of Technology and Evaluation, Kisarazu, Chiba, Japan.
Previous studies have proposed that Streptomyces caeruleus is an earlier heterotypic synonym for Streptomyces niveus and Streptomyces spheroides. In this study, phylogenetic analysis of the almost complete 16S rRNA gene sequences of the Streptomyces caeruleus type strains NBRC 13344(T), JCM 4014(T) and NRRL B-2194(T) revealed that S. caeruleus was closely related to Actinoalloteichus cyanogriseus and not to S.
View Article and Find Full Text PDFMicrobiology (Reading)
June 2005
Pharmazeutische Biologie, Pharmazeutisches Institut, Eberhard-Karls-Universität Tübingen, Auf der Morgenstelle 8, 72076 Tübingen, Germany.
The biosynthetic gene cluster of the aminocoumarin antibiotic novobiocin contains two putative regulatory genes, i.e. novE and novG.
View Article and Find Full Text PDFChem Biol
November 2004
Pharmazeutische Biologie, Pharmazeutisches Institut, Eberhard-Karls-Universität Tübingen Auf der Morgenstelle 8, 72076 Tübingen, Germany.
In the present study, we produced a hybrid antibiotic, carrying a chlorine atom instead of a methyl group at position 8 of the aminocoumarin moiety of novobiocin. This compound was not accessible by conventional gene inactivation/gene expression experiments due to difficulties in the genetic manipulation of the novobiocin producer Streptomyces spheroides. However, the desired compound was obtained after modification of the novobiocin biosynthetic gene cluster by using lambda-Red-mediated recombination in Escherichia coli, followed by integration of the resulting modified cosmid into the phiC31 attachment site of Streptomyces coelicolor and coexpression of the halogenase Clo-hal of clorobiocin biosynthesis.
View Article and Find Full Text PDFAnal Biochem
December 2004
Department of Clinical Pharmacology, Institute of Pharmacology and Toxicology, University Hospital Tübingen, 72076 Tübingen, Germany.
Streptomyces spheroides, Streptomyces rishiriensis, and Streptomyces roseochromogenes are producers of the aminocoumarin-type antibiotics novobiocin, coumermycin A(1), and clorobiocin, respectively, all of which are bacterial gyrase inhibitors. In an attempt to develop a general analytical method for pathway monitoring of secondary metabolites from culture extracts of these strains, we used superior mass spectrometric methods. The aim was to develop and apply a technique for the rapid analysis of Streptomyces culture extracts with respect to those substances, thereby providing a method for screening extracts of genetically modified strains for new pharmaceutically active antibiotics with improved pharmacological effects.
View Article and Find Full Text PDFA collection of 93 Streptomyces reference strains were investigated using SDS-PAGE of whole-cell proteins. Computer-assisted numerical analysis revealed 24 clusters encompassing strains with very similar protein profiles. Five of them grouped several type strains with visually identical patterns.
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