Computational protocol: Efficient Degradation of Feather by Keratinase Producing Bacillus sp.

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Protocol publication

[…] Identification of the keratinolytic bacterial isolates was carried out by determining morphological, cultural, biochemical, and genetic characters. The colony morphology, pigment production, and diffusibility were tested on the nutrient agar. Microscopic morphology of the isolates was studied by Gram staining using bright field [, ] and by negative staining with 1% phosphotungstic acid for electron microscopic examination []. The optimum cultural characterization of the two isolates was determined by testing range of growth temperature (from 4°C to 65°C), pH (5.5 to 8.5), salinity (1–10%), oxygen requirement, and so forth. Further biochemical characterization was carried out adopting standard tests as described in Collee et al. [].16S ribosomal typing was carried out for characterization of the isolates to the species level and phylogenetic analysis. The genomic DNA was extracted by using bead beater (Omni model 8) using 2 mL polypropylene Beadbeater vial with 0.5 g each of 0.1 mm and 3.0 mm silica-zirconium beads at 4 m/s for 40 seconds []. 16S rDNA amplification was carried out using PB36 as forward primer (AGR GTT TGA TCM TGG CTC AG (R=G/M=A/C K=T/G A)) and PB38 as reverse primer (GKT ACC TTG TTA CGA CTT) []. The primer pair, dNTP, Taq polymerase, and other buffers were purchased from Invitrogen. DNA sample was purified from gel slices using PCR-DNA purification kit (Qualigens) followed by sequencing of the PCR product in a 313 OXL capillary DNA sequencer (Applied Biosystems). The gene sequence obtained was BLAST searched to fish out homologous sequences. The CLUSTAL W aligned sequences were analyzed using neighbor-joining method [] and phylogenetic analysis was carried out using Treecon package version 1.3b for Windows. The sequences are deposited in GenBank with accession numbers EU360724.1 (BF11) and EU360725.1 (BF21). […]

Pipeline specifications

Software tools Clustal W, TREECON
Application Phylogenetics
Organisms Bacillus subtilis, Bacillus cereus
Chemicals Carbon, Cysteine, Cystine, Methionine, Nitrogen