A bacteriocin from
Bacillus subtilis
(MK733983) originated from ethnomedicinal plant was purified using Preparative RP-HPLC. The HPLC fraction eluted with 65% acetonitrile showed the highest antimicrobial activity with
Mycobacterium smegmatis
as an indicator. Its specific activity and purification fold increased by 70.5% and 44%, respectively, compared to the crude bacteriocin. The bacteriocin showed stability over a wide range of pH (3.0–8.0) and preservation (− 20 °C and 4 °C), also thermal stability up to 80 °C for 20 min. Its proteinaceous nature was confirmed with complete loss of activity on its treatment with Trypsin, Proteinase K, and α-Chymotrypsin. Nevertheless, the bacteriocin retained up to 45% activity with Papainase treatment and was unaffected by salivary Amylase. It maintained ~ 95% activity on UV exposure up to 3 h and its activity was augmented by ethyl alcohol and metal ions like Fe
2+
and Mn
2+
. Most of the common organic solvents, general surfactants, preservatives, and detergents like Sulfobetaine-14, Deoxy-cholic-acid did not affect the bacteriocin’s action. Its molecular weight was estimated to be 3.4KDa by LC-ESI-MS/MS analysis. The bacteriocin is non-hemolytic and exhibited a broad inhibition spectrum with standard strains of
Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli and Chromobacterium violaceum
with MICs ranging 0.225 ± 0.02–0.55 ± 0.05 mg/mL. Scanning Electron Microscopy showed cell annihilation with pores in cell membranes of
S. aureus
and
P. aeruginosa
treated with the bacteriocin, implicating bactericidal mode of action. These promising results suggest that the bacteriocin is significant and has wide-ranging application prospects.
Supplementary Information
The online version contains supplementary material available at 10.1007/s00203-020-02173-7.
Pseudomonas oleovorans PAMD_1 produced an intracellular azoreductase as the more prominent enzyme that reduces the azo bridge during the azo dye decolorization process. In order to optimize the expression of azoreductase, statistically based experiments were applied. Eleven significant factors were screened on decolorization activity using Plackett-Burman design. Dye, NADH, glucose, and peptone were identified as having highest positive influence on the decolorization activity. Central composite design of response surface methodology was employed for the concerted effect of these four factors on decolorization activity. This method showed that the optimum medium containing dye (200 mg L(-1)), NADH (1.14 mM), glucose (2.07 g L(-1)), and peptone (6.44 g L(-1)) for the decolorization of Orange II up to 87% in 48 hr. The applied methodology was validated through the adequacy and accuracy of the overall experiments, and the results proved that the applied methods were most effective. Further, the enzyme was purified ninefold with 16% yield by anion-exchange chromatography and a specific activity of 26 U mg(-1). The purified enzyme with a molecular mass of 29,000 Da gave a single band on sodium dodecyl sulfate (SDS) gel, and the degradation products sulfanilic acid and 1-amino-2-napthol of Orange II by azoreductase were analyzed by using an ultraviolet-visible (UV-Vis) spectrophotometer and hish-performance liquid chromatography (HPLC).
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