Zb l. Mikrobiol. 140 (1985), 325- 332 [Facult y of Agricult ure, Ain Shams University, an d N a t .i ona l Organization for D ru g Control and Research , E gypt]
Purificati on and I dentificati on of Streptomsjces aureofaciens ID13 Antibiotic E. A. SALEH,
S. A . Z.
EL- HADDAD ,
and M. K .
With :3 F igures
Summary Complete ex t ract ion of S . aureofaciens lD 13 antibiotic was achieved by ad ding n-butanol to the clarified cult ure f iltrate (v /2v) at p H 8.0. Using the column chroma t ography technique, 85.7% of t he init ial a m oun t of the antibiotic wa s obtained in a purified form. D ata of t he Rr value s of the a nt ibiot ic in d ifferent organic solve nts r eve aled that it be long s to t he tetracycli ne gro up . The a ntib iot ic w as chro mat ogr aph ically anal yzed, using t he thin-layer te ch nique. UV an d IR s pect ra , optica l rotation, melting point as well as 15 colour rea ctions were a lso determined.
Zusammenfassung Eine vo llstandige E xt rakti on des aus Streptom yces aur eofaciens ID13 gewonnenen Antibiot ik u ms gelang durch Zusat z von n -B utanol zum gek larten Kulturfiltr a t (v/2v) bei p H 8. D urch Anwen d ung del' Kolonnenchromatographie wurden 85,7 % des Antib iotikums in gerein igter F or m erhalten. Die R r-\Verte in verseh iedenen organisehe n Losungsrnitteln zeigten, daf3 es zur Tetrazyklin-Gruppe gehort DaB Antibiotikum wurde diinnBchi chtchromatographisch analy siert. UV u n d I R -Sp ek t ren, optisch e Drehung, Schmelzp unkt Bowie 15 F arbreaktionen wurden eb enfalls b estim mt.
Th ere are two ways for the chemical class ification of an tibiotics. T he fir st rests on t he description of the phys ico-chemical properti es of ant ibiotics, such as solvent ext ractability, adsorban cy on active charcoal or ion exchang e resins, ultraviolet abs orption an d R r va lues in pa per or t hin -layer chromat ography. Th e second way ma kes use of the chemi cal st ru ct ure of antibiotics (YONEHARA 1970). Th e presen t st udy was designed to ex tract S . aureofaci ens lD 13 antibiotic in a purified for m as a trial to st udy its ph ysico-chemical properties.
l\:laterials and Methods Organisms Streptomy ces aureofa ciens lD 13 , which was locall y isol ated and identified by the a u t hors et al. 1983 a ), wa s use d for antibi otic product ion. St ap hylococcus au reu.s ATCC 6 5 3 8P , kindl y s u p p li e d b y '1'he BaderioJ og ic al L abor a t or y o f the National Or ganization of Drug Cont r ol and R esearch, E gypt, was employ ed for antibioti c bio-
(MAHM OU D
assay. 22 ZhL -'li kr obiol. Ed . 140
E. A. SALEH et a!.
Media The extract of 2.24 % of corn meal was used according to EL-HADDAD et al. (1983) as a complete medium for antibiotic production. Nutrient agar medium (JACOBS and GERSTEIN 1960) was used for growing the test organisms.
Fermentation procedure Cultivation was carried out as described by the authors elsewhere (MAHMOUD et al, 1983b), except corn meal extract medium was used. At the end of fermentation the filtrates were gathered for extracting the antibiotic.
Extraction and purification of the antibiotic Different developing systems, namely, distilled water, 3 % (w/v) ammonium chloride, methanol acetone, ethyl acetate saturated with water, n-butanol saturated with water, ether saturated with water, chloroform, benzene, and petroleum ether were studied for the extraction of the antibiotic. To explore the most suitable pH for maximum separation of the antibiotic under investigation, different pH values, viz, 2.3, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, and 10.1 were studied. This was done, using the paper chromatography as described by BETINA (1958), applying the developing system, which proved to be the best one. The pH was measured, using Beckman pH-meter with a glass electrode. The recovered antibiotic on the chromatographic strips was determined, using the bioautographic method (BETINA 1965). Purification of the antibiotic was carried out, using the method recommended by MARTINet al. (1967). Purity of the antibiotic was checked by determining its melting point according to British Pharmacopoeia (1968).
Identification of the antibiotic The class of the antibiotic was determined by comparing its R r values in four solvents, namely, distilled water, n-butanol saturated with water, ethyl acetate saturated with water, and benzene saturated with water, with those obtained by BETINA (1967). To nominate the antibiotic, its Rr values were again determined according to MARTIN et al, (1967), using other three solvent systems, namely, n-butariol saturated with water, ethyl acetate saturated with water, and chloroform-2-chloro-ethan-water (2: 1: 1).
Thin-layer chromatography The R r value of the antibiotic under investigation was compared with those of the standard oxytetracycline, chlorotetracycline, and tetracycline, using the thin-layer chromatography technique (SELZER and WRIGHT 1957 and RAGAZZI and VERONESE 1977). Detection of spots was carried out bioautographically against Staphylococcus aureus according to BETINA (1973).
Characteristics of the antibiotic Optical rotation This was measured using a Carl-Zeiss JENA 11/0.05 Polarimeter at 20°C with the sodium light (British Pharmacopoeia 1968).
The infrared and ultraviolet spectra IR and UV spectra of the antibiotic under investigation were measured according to STEIN and BASSLER (1967).
Colour reactions The antibiotic produced by S. aureofaciens lD 13 was subjected to different selective tests (HAWK et al. 1954, KaHN 1961 and SMITH 1969) to obtain additional information about its structure and chemical properties. The same was done with a standard tetracycline.
Purification and Identification of Antibiotic
Results and Discussion Extraction of S. aureojaciens ID 1 3 antibiotic Ten solvents were tested to explore the most favourable one for antibiotic extraction. The results presented in Table 1 indicate that the antibiotic under investigation was found to be soluble in water, 3 % NH 4CI, methanol, and n-butanol, while its solubility in acetone and ethyl acetate was very low. The R r values, as shown in Table 1, reveal that a good separation of the antibiotic was achieved when methanol or n-butanol saturated with water was used. The latter is immiscible with water, therefore it was selected for extraction of the antibiotic. Table 1. R f values of S. oureofaciens ID13 antibiotic, using different developing systems Developing systems
R f values
1 2 3 4 5 6 7 8 9 10
0.32-0.59 0.42-0.62 0.94 0.0-0.15 0.04-0.06 0.52 0.0 0.0 0.0 0.0
Distilled water 3 % ammonium chloride Methanol Acetone Ethyl acetate saturated with water n-butanol saturated with water Chloroform Benzene saturated with water Petroleum ether Ethyl ether
Separation of the antibiotic by the organic solvents depends largely on the pH value (BETINA 1958), therefore it was found of importance to determine the most suitable pH for maximum separation, using n-butanol saturated with water. Results presented in Table 2 clearly show that the highest R r values (0.57-0.69) were obtained when the pH of the chromatogram was adjusted to 8.0. Increasing or decreasing the pH level of the chromatogram above or below pH 8.0 leads to a decrease in the R r values. In other words, at pH 8.0 the partition coefficient of the antibiotic was sufficiently high. It may be concluded that the n-butanol system and pH 8.0 proved to be the most suitable condition for the extraction of S. aureojaciene lD 13 antibiotic. Table 2. R f values of S. aureofaciens ID13 antibiotic in n-butanol saturated with water, as affected by different pH values pH values
R f values
To verify the most suitable concentration of n-butanol for maximum antibiotic extraction, different volumes of the solvent were added to a standard volume of the filtrate to give the following butanol: filtrate ratios, 0: 10, 1: 10, 2 : 10, 3: 10, 4: 10, and 5: 10. It is clear from the data presented in Table 3 that the recovery of the antibiotic increased gradually by increasing the ratio of n-butanol : filtrate. A complete recovery (100 %) was obtained nsing one volume of n-butanol per 2 volumes of filtrate. 22"
Tabl e 3. E ffect of different ratios of' n -bu tan ol and filtrat e (v/v ) on the ex traction of S . au reofuciens ID13 antibiot ic ml n-butan ol/IOOml filtrate
Antibiotic recovered (mg/IOO ml) R ecover y
In the ligh t of t he above-mentioned results, t he cult ure filtrate of S . aureojaciens lD 13 was subject ed to the following ste ps for extraction of th e antibiotic: The culture filtrate was adjusted to pH 2.0 to precipitate the inert protein (ABOU-ZEID et al. 1977) and t he precipitate was remov ed by filtration. The filt rate was re-adjusted to pH 8.0, and a complete extraction of the antibiot ic was attained by adding n-butanol to the filtrate at the ratio of vj2v in the respective order. Th e extract was evaporated under va cuum at 50 °0 to obtain a crude solid antibiotic. Purifi cation of the ant ibio ti c Purification of the antibioti c was carried out , using t he method recomm end ed by i\1ARTIN et al. (1967). The fr actions, containing the antibiotic, were gat hered, concentrate d under vacuum, and t hen lyophilized to give a yellow powd er. Applying t he scheme proposed by MARTIK et al. (1967), t he results showed that about 85.7 % of the ini tial amount of th e antibiotic was obtained in a purified form. The purity of the antibiotic was checked by measuring its melting point, as recommend ed by BRITISH PHARMACOPOEIA (1968). Th e purified antibiot ic exerted a clearcut melting point at 216 °0 . Thi s indi cates that the an tibioti c under investigation was obtained in a highly p urified for m. Id en t ifi cation of t he an t ibi o ti c The results presented in Table 4 reveal, according to BETINA, (1967), that the antibiotic under study belon gs to class lb. However, according to MARTIN et al. (1967) the data recorded in Table 4 indicate that the present antibioti c is tetracycline. Fig. 1 shows t hat the R r value of t he antibiotic under study was very near to that of the standa rd pure tetracyclin e. Table 4. R r values of S. aureojacie ns I D I3 antibiot ic, using differen t developing systems Developing sys te ms
R r values
a ) Sys te ms recommend ed b y BETINA ( 1967) Distill ed water n-Butano l saturated wit h water Et h yl acet at e saturated with water Ben zene saturated wit h water
0.58 0.52 0.05 0.00
b) Syst ems recommended b y M ARTI N et al, (1967) n-But anol sat ur ated with wate r E thyl acetate saturated with water Chloroform-2-chloroethan -water (2: 1: 1)
0.39 0.05 0.20
P urification and Identification of Antibioti c
-- - - -
o F ig. i . Thin-la yer ch romatography of l. ehlorotebraey cline , 2. tetracycl ine . 3. oxytetra cycline. 4. t he a ntibiotic of S . au reofaciens lD u'
C h a r a c t e r i s t i c s of th e S . aur eojaci en s l D 13 antib i o ti c Th e charact erist ics of th e tetracycline, pr od uced by S. oureojacien s ID13 • were st udied and compared with t hose of a standard t etracycline to confirm t he identification of t he form er result . Regarding the infrared and ultraviolet spectra, results presented in Fi gs. 2 and 3 clea rly show t hat t he UV and IR spectra of t he S. aureojaciens lD 13 an tibioti c and t he standa rd t etracyclin e were concomitant. The optical rot at ion of t he an tibiot ic at 25 °C was found t o be -260°C in 0.1 N H OI. This figure is close to t he optical rot ation of t he standa rd t et ra cycline, report ed by B OOTH E et al. (1958).
c: tJ -:: 0.3
// 0/ 0 P
\. o~ ,
\. / ~..... ~
300 320 3'tO Wave length t nm )
F ig. 2. UV a bs or p t ion spectra of t he a ntibiotic produced by S. aureoj acie ns (. - -- .) I D 13 a nd pure tetracycli ne (0 - - -- - - -0 ).
wavetenqt» LpmJ 3 t, 7 10 11 12 lJ II, 15 10 6 5 5 8 9 100F=:::::+:====t=============t======Ir=======I=======RIOO
l....-_ _..I.-_ _- ' -_ _- ' -_ _----'
Fig. 3. The IR spectra of the antibiotic, produced by S. aureofaciens 1D 13 and pure tetracycline antibiotic.
Table 5. Comparison of colour reaction of S. oureofaciens 1D 13 antibiotic with colour reaction of standard tetracycline Reaction
S. aureofaciens ID13 antibiotic
Molisch's Buiret Ehrlich's Fehling Meyer's Tollen's
2) Sakaguchi's Ninhydrin Nitro-prusside Phenol Potassium permanganate Ferric chloride Millon's 3) Schiff's 1) HAWK
2) SMITH 3) KOHN
+ + +
+ + +
reduced group no peptide linkage no indolic group reducing group no nitro group no aldehyde or aromatic amines unsaturated bonds no guanido group no amino acids no amino group no carbohydrate unsaturated bonds diketone compound phenol group no aldehyde group
et al. (1954). (1969). (1961).
Purification and Identification of Antibiotic
The results presented in Table 5 reveal that the antibiotic under investigation behaved similarly as the standard tetracycline with respect to colour reactions, which indicates that both antibiotics were found to be free from the peptide linkage, aromatic amines, amino acids and nitro, guanido, indole and aldehyde groups. On the other hand, they were characterized by the presence of reducing group, unsaturated bonds, diketone and phenol groups. In the light of the above-mentioned results the antibiotic, produced by S. aureo[aciens lD 13 , could by identified as tetracycline. This finding is in accordance with that by VECHER et al. (1978), who found that S. aureofaciens produces tetracycline in media of low chloride content.
References ABOU-ZEID, A. A., EISSA A. 1., EL-DEWANY', A. 1., ABD EL-HAMID, M. M., FouAD, M., FAHMI, M., and YASSEN, ]VI.: The fermentative production of oxytetracycline on industrial by-products by S. rimosus 12907. Folia Microbiol. 22 (1977), 47-54. BETINA, V.: A paper chromatography method for the determination of suitable pH values for the extraction of antibiotics. Nature 182 (1958), 769-797. Paper chromatography of antibiotics. In: Chromatographic Review, vol. 7. (LEDERER, M., ed.], Amsterdam 1965. Identification of antibiotic in crude materials. Antimicrobial Agents and Chemotherapy 1966 (1967), 637-643. Bioautography in paper and thin-layer chromatography and its scope in the antibiotic field. J. Chromatography 78 (1973), 41. BOOTHE, J. H., BONVICINO, G. E., WALLER, C. W., LETISI, J. P., SILKON R. W., and BROSCHARD, R.: Chemistry of the tetracycline antibiotics. 1. Quaternary derivatives. J. Amer. Chern, Soc. 80(1958),1634. British Pharmacopoeia: Biological assay of antibiotics. London 1968,1313 and 1317. EL-HADDAD, M. E., MAHMOUD, S. A. Z., SALEH, E. A., and ABDEL-FATAH, M. K.: A suggested medium for antibiotic production by S. aureofaciens ID I3 under different environmental conditions. Ann. Agric. Sci., Fac. Agric., Ain Shams Univ., Cairo, Egypt 28 (1) (1983), 109. HAWK, P. B., OSAR, B. 1., and SUMMERSON. W. H.: Practical Physiological Chemistry. Bth ed. New York 1954, 63-209. JACOBS, :VI. B., and GERSTEIN M. J.: Handbook of Microbiology. New York 1960, 139-207. KOHN. J.: A lipoprotein staining method for zone electrophoresis. Nature 189 (1961),312. MAHMOlJD, S. A. Z., EL-HADDAD, M. E., SALEH, E. A., and ABDEL-FATAH, M. K.: Evaluation of the antagonistic activity of some local isolates of streptomycetes. 1st Hon. Conf. Agric. Bot. Sci., EI-Mansoura Univ., Egypt (l98:3a), 64-81. Factors affecting the antibiotic production by Streptomyces aureofaciens ID I3. 1. Time of fermentation period and medium composition. Ist Hon. Conf. Agric. Bot Sci., EI-Mansoura Univ., Egypt (1983b), 82-101. MARTIN, J. H., MITSSCHER, L. A., MILLER, P. A., SHU, P., and BOHONOS, N.: 5-Hydroxy-7-chlortetracycline. 1. Preparation, isolation, and physicochemical properties. Antimicrobial Agents and Chemotherapy (1967), 56:3-567. RAGAZZI, E., and VERONESE, G.: Simple method for the quantitative analysis of tetracyclines by fluorimetry after thin-layer chromatography on cellulose plates. J. Chromatogr. 132 (1977), 105-114. SELZER, G. B., and "VRIGHT, W. W.: Paper chromatography of tetracycline antibiotics and their cpimers. Antibiotics and Chemotherapy 7 (1957),292. SILVERSTEIN R. M., and BASSLER, G. G.: Spectrometric Identification of Organic Compounds. 2nd ed. New York 1967, 64-109. SMITH, I : Chromatographic and Electrophoretic Techniques. Vol. 1. Chromatography. London 1969, 82 and 281.
E. A. SALEH et a l., Purification and Identification of An t ibi oti c
VECHER, A. S., P AROMCHIK, 1. 1., SHA CHKOV, E. N ., RE SHETNIKOV, V. N ., ZABORONK OL, V. U. , AKIMOVA, L. N., and TSARENKOVA, 1. S. : U se of protein-free p ot ato juice co nce n t rate in antibioti c production. Antib iotik i 23 (11) (19 78), 963-965. YONEHARA, H. : Tentati ve classificatio n of ant ibiot ics by chemica l st ruct ure. Progress in antimi cr obial an d anti cancer chemotherapy. Proc. 6th Int ern. Congres of Che mothe rapy. T ok y o 1970, 969. Authors ' a dd resses : D r. E. A. SALEH, Prof. Dr. S. A. Z. MAHMOUD, an d D r. ;\I.E . EI,-HADDAD, Agricultural Micr obi ology D epartmen t, Facul t y of Agri culture, Ain Shams U niversit y , Sho u bra El-Kheima, Ca iro, a n d Dr. M. K. ABDEL-F ATAH, N ati on al Organization for Drug Cont ro l a nd R esearch, Giza, E gypt.