Nonactin makersLink friends
| Nonactin and other nactinesThis webpage has been created by the students of applied biotechnology at of the OpenUniversity.
(copied with permission from www.Fermentek.co.il) About NonactinNonactin is a member of a family of naturally occurring cyclic ionophores known as the macrotetrolide antibiotics. The other members of this homologous family are monactin, dinactin, trinactin and tetranactin which are all neutral ionophoric substances and higher homologs of nonactin. Collectively, this class is known as the Nactins. It was isolated by Corbaz et al. in 1955 from bacterial strains. Nonactin is known for its ability to form complexes with alkali cations, most notably potassium and sodium. It also displays a marked binding preference for potassium ion over sodium ion. In general, nonactin (and other members of the nactin family) exhibits binding preferences for some ions over others. This ion selectivity is seen in other macrocyclic ligands such as the cyclic ionophore valinomycin which is also an antibiotic. Ion selectivity is also observed in crown ethers which also exhibit a discriminatory preference of some ions over others. Although nonactin (and in fact, all nactins) exhibits an especially high cation selectivity for potassium ion over sodium and rubidium ion, it exhibits the highest selectivity for ammonium ion and thallium ion. During complexation, the nonactin backbone convolutes into a pattern resembling the seam of a tennis ball. In the K+-nonactin complex, the the K+ ion is entirely surrounded by four carbonyl oxygen atoms and the four oxygen atoms of the tetrahydrofuran ring. These eight oxygen atoms that surround the ion are nearly equidistant from it and adopt a nearly cubic coordination sphere around the ion. In this complex, all polar carbonyl groups point inwards and nonpolar moieties point outwards, thus building up a hydrophobic exterior for the complex and making it soluble in lipid membranes. This is how nonactin is able to transport K+ ions across lipid membranes. Nonactin has been reported to specifically inhibit the processing of cytoplasmic precursor proteins destined for the mitochondria.It is able to uncouple the oxidative phosphorylation of mitochondria of rat liver in a low concentration, and can also carry cations across biological and artificial membranes. Nactines are also used as ammonium ion sensors in membrane electrodes and as pesticides. Nonactin is soluble in Methanol, Dichloromethane, Ethyl Acetate and DMSO (Dimethyl sulfoxide). It is insoluble in water. [from Dr Abu-Amir Mandallah course] It is unclear whether the biological activity of nonactin (and other nactines) is related to its ionophoric property. It is said in the literature that the biologic activity of nactines grows as their "number" grows, and nonactin is the least potent, nearly inactive, and the tetranactin is the strongest one. However it is clear that all 5 compounds have activity similar in properties, and differ in potency. There are publications about making nonactin for agricultural use. Because Nonactin is said to be the least potent of nactines, an effort was made to get best yield of tetranactin in the fermentation. The best result was obtained using streptomyces griseus, and adding a lot of Phosphate and organic acids such as acetic, to the fermentation medium. Actually, the commercial preparation called "Polynactin" (not available any more) purposely contained no nonactin at all. It was used until 2004 as a cattle feed additive. it was withdrawn from use, presumably because it was secreted in milk. Click here for the copy of the original document from the International Portal on Food Safety, Animal & Plant Health. Prof Priestley performed an in-depth research on the biosynthesis of Nonactin and the macrotetrolides in general, and wrote five publications on this subject. Organisms used to make NonactinWe found four distinct Nonactin producers. 1. Streptomyces Griseus Dsm 40695. This strain was used in researches of how the tetranactines production is regulated. 2. Streptomyces Tsusimaensis Atcc 15141. This strain is believed to be the strain that had been used by Sigma to make its own Nonactin, or what they had called Nonactin, along with Valinomycin in simultaneous fermentation. 3. Streptomyces Weraensis NRRL B-5317. In our experiments, this strain gave enormous yield of 1 gram/Liter, but upon analysis, only 1-2% of this material occurred to be Nonactin. The rest was monactin and dinactin. 4. Streptomyces chrysomallus [17] Some historySigma Aldrich used to make nonactin from Streptomyces tsusimaensis, as a byproduct at the Valinomycin fermentation. To test it, they used TLC, normal phase, 6:3:1 Toluene: Ethyl-Acetate: Formic acid. (visualization could be achieved by I2 vapors staining or H2SO4 charring. We have found that this TLC system puts all nactines nearly in the same spot, and cannot properly distinguish between the homologs. At most, a perfect TLC could only tell there are three to five compounds present, but could not supply any quantitative information. The "nonactin" from Sigma actually contained no more than 10% Nonactin within total of some 98% macrotetrolides. Later Sigma stopped in-house production of Nonactin, and bought it elsewhere. Currently, it seems that Sigma do not even make their own analysis on this nonactin, and rely fully on the supplier's QC. We could not find out who might be the supplier of the Sigma's Nonactin. A public relation person at Promiliad presumed that the Sigma's Nonactin comes from Fermentek, but we could not verify this information. On the contrary, by comparing the exact quality data of various suppliers we came to a conclusion that Sigma's nonactin came from American Biodiagnostics, at least during 2005. Who makes NonactinCurrently we could identify only three primary makers on Nonactin: Promiliad, Fermentek and American Bioanalytical. Both Promiliad and American Biodiagnostics boast to manufacture highly purified nonactin. Promiliad offers two products, pure and extrapure, the last being three times more expensive. Currently (2006), it seems that the only usage for nonactin is the specific electrodes manufacturing. Professor Priestley, renown as Nonactin guru, explains in a private communication, that the Nonactin used for ammonium electrodes, should better be pure, lest the calibration of these electrodes would be more difficult, but it has not be proven that any other preparation, such as tetranactin, or mixture of nactines might not be used. It should be emphasized that Prof Priestley is one of the owners of Promiliad company, and whatever he claims must be therefore suspected to be biased due to commercial interest. We presume that prof. Priestley discovered methods of efficiently separating Nonactin from other nactines, and of assaying it quantitatively using NMR. (Although the use of NMR in nactines analysis was reported 17 1985 by Our estimations are that if a macrotetrolide mixture could be used it might be as cheap as $20 / 1 gram, as the Strep. Weraensis gave us in our experiment as much as 1 gram/liter of total macrotetrolides. About the analysis of Nonactin and Nactines.[ from promiliad website ] [ from American bio diagnostics website] [our own observations] Not only that the spots of the various nactines vary in size and coloration intensity, but even their very RF is not stable. The greater the concentration, the higher the spot appears on the TLC plate. Therefore to be sure the spot one observes is Nonactin, one must run a unknown sample twice: one as is, and another sample with some pure nonactin added to it. Then you can see that one of the spots became larger. We preferred sometimes to char the plate with sulfuric acid to staining it with Iodine vapors, as some additional differentiation between the homologs might be achieved. The TLC systems applicable for macrotetrolides in general: 6:3:1 Toluene: Ethyl acetate: Formic acid with Normal Phase silica 60. To distinguish between the five macrotetrolides : 100% anhydrous acetone. In the scientific publications we found a lot of usage of HPLC analysis, employing nonstandard detectors, such as light scattering, etc. Nobody had used simple UV detector. Therefore we had trouble to reproduce these measurements. Nor could we establish the spectra of the higher homologues. It seems that currently, all manufacturers are using NMR analysis for this product. AcknowledgementWe thank the manufacturers and experts who kindly answered our questions and supplied as much information as they could, wanted or were permitted by their superiors, and especially Dr AbuMustafa al-Majnun, who permitted us to copy information from their website, and disclosed some of their analytical methods to us. Literature (abstracts)Uses of Nonactin in the biochemistry of cell membranes
Synthesis and biosynthesis of Nonactin and macrotetrolides in general
Uses of Nonactin in building of specific electrodes
Nonactin as antibioticBiophysics of ionophores
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