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Results 3.1. Isolation and Screening of Marine Actinomycetes Few actinomycetes colonies isolated from marine invertebrate sample (Sample ID: NIO_SA_An_15) were found to grow on ASW-AIA plate. The white sporulating slow growing colony showing black diffusible pig- ment on the agar was picked up and referred to as PM0525875. The extracts of PM0525875 showed exclusive antifungal activity, specific towards Candida albicans in Table 1. The medium ASW-36P(1) supported better activity than other two media. During the secondary screening, the antifungal activity against Candida albicans was determined and results are shown in Table 2. The culture filtrate obtained from all the three media did not show any active zone. The antifungal activity was found to be confined to mycelia, indicating its intracellular concentration. The medium ASW-36P(1) was shortlisted for large scale cultivation in fermenter. Based on the UV profile displayed the extract we ruled out presence of any polyene type of compound in it. Prior to it, total four of the morphological variants were selected (shown in Figure 1) and their antifungal activity was compared with the parent strain and results are showed in Table 3. The variant 4 (PM0525875-V4) displayed best anti- fungal activity as it showed the antifungal activity at the lowest dilution of broth (1:64). So PM0525875-V4 was selected for further studies. 3.2. Large Scale Fermentation of PM0525875-V4 Large scale fermentation was carried out using PM0525875-V4 and fermentation medium ASW-36P(1). The fermentation was carried out till 96 h. The time course of fermentation batch is shown in Table 4 and expressed in graphical form as shown in Figure 2. During fermentation, the pH was dropped with the time and later main- tained to 6.5 till end of the fermentation. The rise in biomass (PCV) was slow initially and was picked up after 24 h. After 48 h, the growth rate was steady and the stationary phase was achieved. Dissolve oxygen (DO) dropped significantly till 24 h and was maintained at around 40% till end of the process. The total sugar con- sumption was slow at beginning and it was reduced to half of the concentration at 48 h. Based on the bioactivity pattern obtained by the broth dilution method (as mentioned in 2.4), the maximum activity was attended at 72 h and maintained steady till end of the fermentation process. Hence, the fermentation cycle was terminated at 96h. 3.3. Isolation and Characterization of Bioactive Compound The active compound was found to be intracellular, hence mycelial extract was subjected to bioactivity guided isolation and purification as mentioned in 2.7. The active compound was able to adsorb on HP-20 resins. Further fractionation of crude material using petroleum ether helped removing the impurities and subsequent dichloro- methane fraction was found to be active. The pure compound was finally isolated by silica gel chromatography followed by preparative HPLC using RP-18 resin. The characterization of isolated compound was carried out based on data obtained from mass, IR and 1 H NMR spectra (Figures 3-5). In 1 H NMR spectra, the signals at δ 7.8 and 7.35 were assigned for protons attached at C-3 and C-5 respectively. Proton at unsaturated C-7 appeared at δ 8.13 due to nitrogen and OH group being in proximity. The aromatic protons associated with other pyridine ring were assigned in the range of δ 7.4 - 8.3. The O-methyl group showed a singlet for 3 H at δ 3.9. Structural confirmation was done by comparing the spectral values with the published data [19]. Based on the data, the ac- tive compound was characterized as Caerulomycin A. Figure 6 and its chemical properties are shown in Table 5. From the final yield of the isolated compound (20 mg from 8.5 L broth), the back calculated titer in the har- vested broth was estimated as 2.3 mg/L. In order to simplify the extraction method for scale-up purification, the whole broth extraction with equal quantity of ethyl acetate was attempted. Caerulomycin A was found to be completely extractable in ethyl acetate hence this method could be used for large scale extraction batches. 3.4. Phylogenetic Analysis of PM0525875 The 16S rRNA gene from PM0525875 was sequenced. The partial sequence containing 1377 nucleotide base pairs showed close similarity with Actinoalloteichus cyanogriseus. The phylogenic tree constructed using ten matching sequences of 16S rRNA from NCBI data-base is shown in Figure 7. The evolutionary history was in- ferred using the Neighbor-Joining method [20]. The bootstrap consensus tree inferred from 500 replicates was represented in the evolutionary history of the taxa [21] and the branches corresponding to partitions reproduced in less than 50% bootstrap replicates were collapsed. The evolutionary distances were computed using the Ki- mura 2-parameter method [22]. The analysis involved 11 nucleotide sequences and the codon positions included were 1st + 2nd + 3rd + Noncoding. There were a total of 1381 positions in the final datasheet. Evolutionary analyses were conducted on computer software MEGA5 [23]. Based on this results the isolated actinomycetes strain was identified as Actinoalloteichus cyanogriseus. The gene sequence was deposited in NCBI GenBank under accession no KF861694.1. 3.5. MIC of Caerulomycin A MIC of the isolated compound Caerulomycin A and two standard antifungal compounds was determined using NCCLS Macrobroth dilution method and results are shown in Table 6. Caerulomycin A showed potent activity against four Candida strains, including two fluconazole resistant strains. The MIC of the compound was in the range of 0.39 - 1.256 μg/ml. The MIC values obtained of Caerulomycin A against fluconazole resistant Candida glabrata were comparable with the MIC values obtained for Amphotericin B. This result indicates the potential of Caerulomycin A as a potent and broad spectrum antifungal agent. |
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