Bacilysin biosynthesis by a partially-purified enzyme fraction from Bacillus subtilis

Yazgan, A
Özcengiz, Gülay
Ozcengiz, E
Kilinc, K
Marahiel, MA
Alaeddinoglu, NG
Biosynthesis of dipeptide antibiotic bacilysin by a partially purified enzyme prepared from Bacillus subtilis PY79 was studied. Cell material was desintegrated by treatment with lysozyme and sonication and the extract was subjected to ammonium sulfate fractionation. Bacilysin-synthesizing enzyme activity was precipitated between 40% to 70% ammonium sulfate saturation. In vitro enzymatical synthesis of bacilysin was confirmed by performing thin layer chromatographic comparison of the antibiotic formed with the authentic bacilysin. An enzyme fraction (ca. 125 kDa) was prepared by fast flow gel permeation chromatography which was further purified by anion exchange FPLC. The enzymatic synthesis of bacilysin required either ATP or 2'-deoxy ATP and was entirely dependent on the presence of constituting amino acids. Although anticapsin, at the concentration used in enzyme assay, did not produce an inhibition zone when assayed against Staphylococcus aureus ATCC 9144, it exhibited a slight inhibition zone after incubation with the enzyme fraction in the absence of alanine under the standard assay conditions. To determine the mechanism of amino acid activation, ATP-PPi and ATP-P-i exchange reactions were performed with component amino acids L-alanine and L-anticapsin. The enzyme catalyzed ATP-PPi exchange reaction dependent on L-alanine, but did not activate L-anticapsin in this way. There was also no evidence for activation of this amino acid as an amino acid phosphate. Pantothenic acid was liberated from the enzyme fraction as determined microbiologically. Consistently, covalent binding as thioester was shown for L-alanine. These results indicated that the mechanism of bacilysin biosynthesis is not typical of the general multicarrier thiotemplate model. (C) 2001 Elsevier Science Inc. All rights reserved.


Killer toxin of Pichia anomala NCYC 432; purification, characterization and its exo-beta-1,3-glucanase activity
Izgu, Fatih; Altinbay, Demet; Acun, Tolga (Elsevier BV, 2006-08-02)
Pichia anomala NCYC 432 secretes a killer toxin which is inhibitory to a variety of yeasts including pathogenic Candida spp. The killer toxin in the culture supernatant was concentrated by ultratiltration and purified to homogenity by two successive gel filtration chromatographies with a TSK G2000SW column. Biochemical characterization of the toxin showed that it is a glycosylated protein with a molecular mass of 47 kDa and pI values of 3.4 and 3.7. The toxin showed high stability at pH values between 3 and...
Bacillus subtilis overproduces industrially important extracellular enzymes upon the targeted deletion of bacilysin biosynthetic operon
Özcengiz, Gülay; Islerel, E. Tekin; Aktas, C. (Elsevier BV, 2018-10-10)
Bacilysin being produced by Bacillus subtilis is the smallest peptide antibiotic ever known. It is composed of an N-terminal l-alanine and a modified amino acid at its C-terminal, namely anticapsin. bacABCDEF operon and a monocistronic gene bacG are functional for bacilysin production in the organism, bacABCDFG being needed for the flux from prephenate to anticapsin and then to mature bacilysin while bacE gene within the operon is involved in resistance of the producer by pumping bacilysin out of the cell. ...
Analysis of a bac operon-silenced strain suggests pleiotropic effects of bacilysin in Bacillus subtilis
Ertekin, Ozan; Taskin, Ash Aras; Demir, Mustafa; Karataş, Ayten; Özcengiz, Gülay (Springer Science and Business Media LLC, 2020-04-01)
Bacilysin, as the simplest peptide antibiotic made up of only L-alanine and L-anticapsin, is produced and excreted by Bacillus subtilis under the control of quorum sensing. We analyzed bacilysin-nonproducing strain OGU1 which was obtained by bacA-targeted pMutin T3 insertion into the parental strain genome resulting in a genomic organization (bacA '::lacZ::erm::bacABCDEF) to form an IPTG-inducible bac operon. Although IPTG induction provided 3- to 5-fold increment in the transcription of bac operon genes, n...
Preparation of cross-linked tyrosinase aggregates
Aytar, Burcu Selin; Bakir, Ufuk (Elsevier BV, 2008-02-01)
Tyrosinase from mushroom was immobilized as a cross-linked enzyme aggregate (CLEA) via precipitation with ammonium sulfate and cross-linking with glutaraldehyde. The effects of precipitation and cross-linking on CLEA activity were investigated and the immobilized tyrosinase was characterized. Sixty percent ammonium sulfate saturation and 2% glutaraldehyde were used; a 3-h cross-linking reaction at room temperature, at pH 7.0 was performed; particle sizes of the aggregates were reduced; consequently, 100% ac...
Kinetics of riboflavin production by Brewers' yeasts
Tamer, I.M.; Özilgen , Mustafa; Ungan, Suat (Elsevier BV, 1988-12)
The kinetics of riboflavin production by Saccharomyces cerevisiae and Saccharomyces carlsbergensis in synthetic media and wort were studied. The results indicated that riboflavin was produced by growing cells only. Riboflavin production rate was proportional to growth rate of the yeasts in the exponential phase. Riboflavin was depleted in the stationary phase. The depletion rate was expressed with a first-order kinetic expression in yeast concentration. The kinetics of substrate utilization and ethanol prod...
Citation Formats
A. Yazgan, G. Özcengiz, E. Ozcengiz, K. Kilinc, M. Marahiel, and N. Alaeddinoglu, “Bacilysin biosynthesis by a partially-purified enzyme fraction from Bacillus subtilis,” ENZYME AND MICROBIAL TECHNOLOGY, pp. 400–406, 2001, Accessed: 00, 2020. [Online]. Available: