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Calcium Alginate Bead-mediated Enhancement of the Selective Recovery of a Lead Novel Antifungal Bacillomycin Variant

Ramachandran, Ramya, Ramesh, Swetha, Ramkumar, Srinath, Chakrabarti, Arunaloke, Roy, Utpal
Applied biochemistry and biotechnology 2018 v.186 no.4 pp. 917-936
Bacillus subtilis, Candida, antifungal agents, calcium alginate, cytotoxicity, fermentation, fungi, glucose, lead, lipopeptides, malt extract, metal ions, minimum inhibitory concentration, mycelium, peptones, sodium alginate, sodium chloride, temperature
In the pursuit of new antifungal compounds, five coproduced lipopeptide variants (AF₁ to AF₅) from wild-type Bacillus subtilis RLID 12.1 were identified in our previous study. Out of five, AF₄ was identified as a novel lead molecule belonging to the bacillomycin family showing less cytotoxicity at its respective minimum inhibitory concentrations (MIC) evaluated against 81 strains of Candida and Cryptococcus species (including clinical isolates); besides this, AF₄ purified in the present study exhibited encouraging MIC values against 10 clinical mycelial fungi. Aiming for a selective production augmentation of AF₄ lipopeptide variant, a new fermentation media comprising malt extract (1.01%), dextrose (0.55%), peptone (1.79%), MnSO₄ (2 mM), and NaCl (0.5%) was formulated. Maximum production of 954.8 ± 10.8 mg/L was achieved with 44% selectivity at 30 °C compared to unoptimized conditions (186.4 ± 6.1 mg/L). Use of calcium alginate beads in the formulated media during the onset of lipopeptide production resulted in an augmentation in the selectivity of the most efficacious AF₄ variant to about 72% presumably due to attenuation of other coproduced lipopeptide variants AF₁ and AF₂. Difference in yield of lipopeptides varied with bead size, bead preparation ratios, and sodium alginate concentrations. Use of Ca-alginate beads in the upstream production process of the lead AF₄ variant may be considered as a novel strategy to address the potential challenge that may arise during the scale-up and downstream processing steps. Another significant finding derived from the study is that the proportion of bacillomycin variants of B. subtilis RLID 12.1 could be controlled by temperature and metal ions under static and shaking conditions.