Biochemical Characterization and Functional Analysis of Heat Stable High Potential Protease of Bacillus amyloliquefaciens Strain HM48 from Soils of Dachigam National Park in Kashmir Himalaya.

A novel temperature stable alkaline protease yielding bacteria was isolated from the soils of Dachigam National Park, which is known to be inhabited by a wide variety of endemic plant and animal species of Western Himalaya. This high-potential protease producing isolate was characterized and identified as Bacillus amyloliquefaciens strain HM48 by morphological, Gram's staining and biochemical techniques followed by molecular characterization using 16S rRNA approach. The extracellular protease of B. amyloliquefaciens HM48 was purified by precipitating with ammonium sulfate (80%), followed by dialysis and Gel filtration chromatography increasing its purity by 5.8-fold. The SDS-PAGE analysis of the purified enzyme confirmed a molecular weight of about ≈25 kDa. The enzyme displayed exceptional activity in a broad temperature range (10-90 °C) at pH 8.0, retaining its maximum at 70 °C, being the highest reported for this proteolytic Bacillus sp., with KM and Vmax of 11.71 mg/mL and 357.14 µmol/mL/min, respectively. The enzyme exhibited remarkable activity and stability against various metal ions, surfactants, oxidizing agent (H2O2), organic solvents and displayed outstanding compatibility with widely used detergents. This protease showed effective wash performance by exemplifying complete blood and egg-yolk stains removal at 70 °C and efficiently disintegrated chicken feathers making it of vital importance for laundry purpose and waste management. For functional analysis, protease gene amplification of strain HM48 yielded a nucleotide sequence of about 700 bp, which, when checked against the available sequences in NCBI, displayed similarity with subtilisin-like serine protease of B. amyloliquefaciens. The structure of this protease and its highest-priority substrate ß-casein was generated through protein modeling. These protein models were validated through futuristic algorithms following which protein-protein (protease from HM48 and ß-casein) docking was performed. The interaction profile of these proteins in the docked state with each other was also generated, shedding light on their finer details. Such attributes make this thermally stable protease novel and suitable for high-temperature industrial and environmental applications.

Bioprocessing of Biodiesel Industry Effluent by Immobilized Bacteria to Produce Value-Added Products.

Biodiesel industrial effluent rich in crude glycerol (CG) was processed to produce value-added product. Under continuous culture system, Bacillus amyloliquefaciens strain CD16 immobilized within its biofilm, produced 3.2 L H2/day/L feed, over a period of 60 days at a hydraulic retention time of 2 days. The effective H2 yield by B. amyloliquefaciens strain CD16 was 165 L/L CG. This H2 yield was 1.18-fold higher than that observed with non-biofilm forming Bacillus thuringiensis strain EGU45. Bioprocessing of the effluent released after this stage, by recycling it up to 25% did not have any adverse effect on H2 production by strain EGU45; however, a 25% reduction in yield was recorded with strain CD16. Biofilm forming H2 producers thus proved effective as self-immobilizing system leading to enhanced process efficiency.

The antibacterial activity and mechanism of ginkgolic acid C15:1.

BACKGROUND: The present study investigated the antibacterial activity and underlying mechanisms of ginkgolic acid (GA) C15:1 monomer using green fluorescent protein (GFP)-labeled bacteria strains. RESULTS: GA presented significant antibacterial activity against Gram-positive bacteria but generally did not affect the growth of Gram-negative bacteria. The studies of the antibacterial mechanism indicated that large amounts of GA (C15:1) could penetrate GFP-labeled Bacillus amyloliquefaciens in a short period of time, and as a result, led to the quenching of GFP in bacteria. In vitro results demonstrated that GA (C15:1) could inhibit the activity of multiple proteins including DNA polymerase. In vivo results showed that GA (C15:1) could significantly inhibit the biosynthesis of DNA, RNA and B. amyloliquefaciens proteins. CONCLUSION: We speculated that GA (C15:1) achieved its antibacterial effect through inhibiting the protein activity of B. amyloliquefaciens. GA (C15:1) could not penetrate Gram-negative bacteria in large amounts, and the lipid soluble components in the bacterial cell wall could intercept GA (C15:1), which was one of the primary reasons that GA (C15:1) did not have a significant antibacterial effect on Gram-negative bacteria.

Effects of MreB paralogs on poly-γ-glutamic acid synthesis and cell morphology in Bacillus amyloliquefaciens.

Actin-like MreB paralogs play important roles in cell shape maintenance, cell wall synthesis and the regulation of the D,L-endopeptidases, CwlO and LytE. The gram-positive bacteria, Bacillus amyloliquefaciens LL3, is a poly-γ-glutamic acid (γ-PGA) producing strain that contains three MreB paralogs: MreB, Mbl and MreBH. In B. amyloliquefaciens, CwlO and LytE can degrade γ-PGA. In this study, we aimed to test the hypothesis that modulating transcript levels of MreB paralogs would alter the synthesis and degradation of γ-PGA. The results showed that overexpression or inhibition of MreB, Mbl or MreBH had distinct effects on cell morphology and the molecular weight of the γ-PGA products. In fermentation medium, cells of mreB inhibition mutant were 50.2% longer than LL3, and the γ-PGA titer increased by 55.7%. However, changing the expression level of mbl showed only slight effects on the morphology, γ-PGA molecular weight and titer. In the mreBH inhibition mutant, γ-PGA production and its molecular weight increased by 56.7% and 19.4%, respectively. These results confirmed our hypothesis that suppressing the expression of MreB paralogs might reduce γ-PGA degradation, and that improving the cell size could strengthen γ-PGA synthesis. This is the first report of enhanced γ-PGA production via suppression of actin-like MreB paralogs.

Tolerance Induction of Temperature and Starvation with Tricalcium Phosphate on Preservation and Sporulation in Bacillus amyloliquefaciens Detected by Flow Cytometry.

The Bacillus species have many applications in the preparation of various enzymes, probiotic, biofertilizer, and biomarkers for which the survival of resting cells and spore formation under different conditions are important. In this study, water and saline along with different mineral substances such as calcium carbonate, calcium phosphate, and silica were used for the detection of survival and preservation of Bacillus amyloliquefaciens. The results showed intensive death of resting cells at 8 °C, but significant survival at 28 °C after one month. However, preservation by minerals significantly decreased the rate of death and induced sporulation at both the temperatures. The resting cells were maintained at room temperature (about 60 % of the initial population survived after a month) in the presence of tricalcium phosphate. The results showed that temperature has more effect on sporulation compare with starvation. The sporulation in normal saline at 28 °C was 70 times more than that at 8 °C; meanwhile, addition of tricalcium phosphate increases sporulation by 90 times. Also, the FTIR data showed the interaction of tricalcium phosphate with spores and resting cells. The discrimination of sporulation from non-sporulation state was performed by nucleic acid staining with thiazole orange and detected by flow cytometry. The flow cytometric studies confirmed that the rates of sporulation in pure water were significantly more at 28 °C. This is the first report on the detection of bacterial spore with thiazole orange by flow cytometry and also on the interaction of tricalcium phosphate with spores by FTIR analyses.

The smaller, the better? The size effect of alginate beads carrying plant growth-promoting bacteria for seed coating.

A range of lab-scale methods for encapsulation of plant growth-promoting bacteria in alginate beads intended for seed coating was evaluated: contact-spotting, extrusion through syringe with/without vibration, ejection by robotic liquid handler, extrusion by centrifugal force and commercial devices (nanodispenser, aerodynamically assisted jetting, encapsulator). Two methods were selected based on throughput (encapsulator: 1.5-5 mL/min; syringe with subsequent pulverisation: 5 mL/min). Four bead sizes (55 ± 39 µm, 104 ± 23 µm, 188 ± 16 µm and 336 ± 20 µm after lyophilisation) were produced. Bacterial viability, release, bead morphology, seed surface coverage and attrition were investigated. Release from the smallest bead size was approximately 10 times higher than from the largest. Seed surface coverage was highest (69 ± 3%) when alginate beads produced with nozzle size 80 µm were applied. Pulverised macro-beads are an alternative option, if high throughput is top priority.