Streptomycin purification

As Streptomyces contain high proportions of proteases, a protease inhibitor, diphenylcarbamoyl chloride, was added to the buffer during purification of the dTDP-dihydrostreptose synthase. A partially purified, enzyme preparation from S. griseus could be obtained by removal of nucleic acids with streptomycin and fractionation with ammonium sulfate.40 However, when this enzyme preparation was subjected to gel filtration on a column of Sephadex G-100, enzyme activity was completely lost. By combining certain fractions of the column eluate, enzyme activity could be partially restored. 

It became evident that not all strains of Streptomyces griaeus were capable of producing streptomycin and that streptomycin-producing strains formed active and inactive variants. On the basis that those strains which can actively produce streptomycin would be more resistant to the antibiotic, it was proposed to utilize streptomycin-enriched media for the purification of active cultures of Streptomyces griseus from inactive variants. 

Waksman and Reilly have summarized the factors which have a bearing upon the choice of the method to be employed in measuring quantitatively the activity or potency of an antibiotic substance. They have cited literature references to the more commonly used methods. Loo and coworkers have described a suitable method for the quantitative determination of streptomycin by the filter paper disc, agar plate diffusion technique using Bacillus subtilis as the test organism. The procedure proved satisfactory in the assay of surface and submerged culture beers and of preparations obtained in isolation and purification... 

Wolfrom and coworkers had found that the mercaptolysis of the crystalline calcium chloride double salt of streptomycin trihydrochloride yielded, after chromatographic purification, two anomeric forms of ethyl tetraacetylthiostreptobiosaminide diethyl mercaptal. Mercaptolysis of dihydrostreptomycin yielded an amorphous ethyl dihydrothio-streptobiosaminide hydrochloride which formed crystalline N-acetyl and pentaacetyl derivatives. The ability of the ring in this latter compound to resist hydrolysis and to form a thioglycoside instead of a thio-... 

Sterilized mash is inoculated with a pure culture of Streptomyces griseus and aerated under aseptic conditions for several days at 25° to 30° C. The broth is filtered and activated carbon is added to the clear filtrate to adsorb the streptomycin. The carbon dosage must be closely controlled as an insufficient amount results in incomplete adsorption, and an excess of carbon reduces the yield by elution. The carbon is separated by filtration and washed with alcohol to remove loosely held impurities, after which the streptomycin is eluted from the carbon with acidified methanol. The eluate is neutralized and evaporated at reduced pressure under carefully controlled conditions to prevent decomposition of the streptomycin. At this point, the streptomycin is 25 to 30% pure and a number of subsequent steps are required to obtain a final product free from all toxic impurities. In some operations, a subsequent purification with activated carbon is provided under conditions in which the impurities are adsorbed and not the streptomycin. 

There is always a need for streamlining the initial purification steps of products that are derived from fermentation. Recent advances in a technique called expanded-bed adsoiption have shown the ability to elimmate the solid-4iquid separation step and thereby increase product yield and reduce processing time. Expanded-bed adsorption is not a new concept, since there are reported applications for streptomycin (23) in the 1950s and novobiocin (24) in the 1970s. However, these applications used adsorbents that were not specifically designed for expanded-bed-adsorption procedures and often required compli-... 

Sugiyama, M. Sakamoto, M. Mochizuki, H. Nimi, O. Nomi, R. Purification and characterization of streptomycin 6-kinase, an enzyme implicated in selfprotection of a streptomycin-producing micro-organism. J. Gen. Microbiol., 129, 1683-1687 (1983)... 

Mitsuhashi and coworkers found streptomycin phosphate transferase in Pseudomonas aeruginosa TI-13 they isolated the inactivated compound, which was shown to have the empirical formula of a mono-0-phosphonodihydrostreptomycin monocarbonate trihydrate by elementary analysis. They also found the same enzyme in other strains, namely, 99, 137, 138, 351, 10126, Cape 18, and TI-11. The enzyme in the supernatant liquor (600 ml) from centrifugation of disrupted eells of strain TI-13 at 105,000g was precipitated by ammonium sulfate at 33-66% saturation. Further purification by successive, column chromatography on Sephadex G-75, DEAE-Sephadex A-50 (with a gradient of potassium... 

Aminobemoylhydrazone of Streptomycin (7). Streptomycin sulfate (Boehringer, Germany) and 4-aminobenzoylhydrazine (Fluka-AG, Switzerland) are mixed in water in a molar ratio of 1 2. After 2 hr at room temperature, the hydrazone is precipitated by the addition of 10 volumes of ethanol and is maintained for at least 2 hr at —20 . The precipitate is collected by centrifugation and is then dissolved in water. It is reprecipitated twice with cold ethanol. Final purification is carried out by descending paper chromatography with butanol(l)/ethanol/water (3 2 5) as solvent system. Rf relative to dihydrostreptomycin, 1.77 absorption spectrum (pH 6), Xmax 298 nm, Amin 255 nm, Azas/A.ss 2.36. The reaction is quantitative. 

Wutz et al. (2011) developed for the first time a multianalyte immunoassay based on an automated flow-through CL microarray technique for identification and quantification of antibiotic derivatives in honey samples using regenerable antigen microarrays, an indirect competitive immunoassay format using horseradish peroxidase (HRP)-labeled antibodies and CL read-out with a CCD camera. The method allows the analysis of four analytes (enrofloxacin, sulfadiazine, sulfamethazine, and streptomycin) simultaneously in 8 min with adequate recoveries and without purification or extraction. Due to the regenerability of the microarray each chip could be individually calibrated before the analysis and allowed more than 40 assays, which reduces the costs per analysis and permits an automated work flow in routine laboratories. 

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