Identification of the Streptomyces isolate
16S rRNA gene was sequenced and the sequence data was stored in GenBank (accession number: MZ159946). Homology search identified the isolate to be Streptomyces decoyicus (Fig. 1). This organism optimally grew at pH7 at 37°C. It formed yellowish colonies on agar medium. Gram staining was used for morphological identification under the light microscope.
Resolution of the crude extract
Crude n-butanol extract was first run in thin layer chromatography sheets and two visible bands were obtained. The Rf values of these bands were 0.30 and the 0.23 (Fig. 2). These wo bands were also shown in SDS-PAGE gels (Fig. 3). The bands were cut out from the gel and their contents were eluted overnight in n-butanol. Dry extracts were obtained by evaporating the organic solvent and were then analysed by FT-IR and GC-MS.
Metabolite identification and characterization
The elution samples were identified by GC-MS analysis (Fig. 4). One of the bands, M1, contained 61.12% tromethamine, 17.77% 1-dodecanol, 7.71% 13-docosenamide (Table 1). The band M2 had 54.06% 1-dodecanol and 41.16% tromethamine (Table 2). The presence of these compounds was also verified by FTIR (Fig. 5): ѵ max. (cm-1): 3347, 3327 (aliphatic NH-stretch bonds - 3288 shoulder bond), 3182 (OH-stretch bond), 2900 (aliphatic CH-asymmetric stretch bond), 2850 (aliphatic CH-symmetric stretch bond), 1586 (NH-bending), 1287 (CN-stretch bond), and 1206 (C-O stretch bond). N-H asymmetric and symmetric tensile bonds expected around 3550-3420 cm-1 and 3450-3320 cm-1of the aliphatic NH2 group (Erdik, 2007, p:118) were observed in the 3347-3327 cm-1 region. Since the primary amine showed the characteristic doublet structure, it was thought that the metabolites mostly contained the primary NH2 group. O-H stretch band at the end of aliphatic chains was observed in the region of 3183 cm-1. In addition, the specific diffuse band of the O-H stretch band (Besson et al, 1997) was also observed.
The results of the GC-MS analysis suggested that mostly tromethamine and 1-dodecanol, and some 13-docosenamide were present in the metabolite samples. Accordingly, the asymmetric and symmetric tension and bending vibration bands of CH2 and CH aliphatic groups, 2900 cm-1 and 2850 cm-1, common to the three compounds, were observed in the FT-IR spectra.
In general, when the C=O group is attached to an atom carrying an unshared electron or is conjugated with a double bond, the effect of unshared electron pairs and double bonds in a structure creates the mesomeric effect, while the electron-withdrawing or electron-donating creates the inductive effect. Any factor that increases the dipole character of the C=O group, decreases the value of the force constant, and thus the absorption shifts to the lower frequency (Erdik, 2007, p:151-152). Although the amide C=O stretch band in the 13-docosenamide compound was expected to be observed within the 1650-1700 cm-1 region, no band was obvious in this region. In addition, there was also no C=C alkene bands available. These two findings might argue against the presence of 13-docosenamide in the metabolite samples, and it could be considered as the GC-MS artefact, resulting from the compendium of compounds specified by its software. Hence, the FT-IR results confirmed the abundance of two compounds, tromethamine and 1-dodecanol.
The FT-IR spectra of the M2 sample almost overlapped with the M1, except for the CO-stretch bond at 1206 (Fig. 6): ѵ max. (cm-1): 3347, 3326 (aliphatic NH-stretch bonds - 3288 shoulder bond), 3186 (OH-stretch bond), 2920 (aliphatic CH-asymmetric stretch bond), 2851(aliphatic CH-symmetric stretch bond), 1587 (C=O stretch bond), 1535 (NH-bending), and 1287 (CN-stretch bond). Here, theoretically, the NH2 group (Erdik, 2007, p:118), which is expected to be observed around 3550-3420 cm-1 and 3450-3320 cm-1, was observed in the 3347-3326 cm-1 region with N-H asymmetric and symmetric tension bands. The characteristic doublet structure of the primary amine suggested that the metabolites mostly contain the primary NH2 group. From these data, it could be said that the metabolite content consisted mostly of tromethamine and 1-dodecanol (Besson et al, 1997).