Bacterial strain and medium
The microorganism used in this study was Bacillus sp. C4; CCUG 66887. It has earlier been isolated from the compost and identified using biochemical tests and 16S rDNA technique (GenBank accession: FJ214667) (Fellahi et al. 2014). Before the strain was used for protease production it was grown at 37 °C for 24 h on peptone yeast extract medium containing Bactopeptone, 10 g/l; Yeast extract, 5 g/l and NaCl, 5 g/l.
Keratinase gene sequence determination
Multiple sequence alignment with CLUSTALW2 (http://www.ebi.ac.uk/Tools/msa/clustalw2/) (Chenna et al. 2003) was used to align keratinase genes from different Bacillus pumilus strains to investigate the resemblance among the strains to be able to choose one strain for designing the first sequencing primer set (F: TTAGAAGCCGCTTGAACGTTA, R: ATGTGCGTGAAAAAGAAAAATGTG). Genomic DNA was isolated from strain C4 using MasterPure™ Gram Positive DNA Purification Kit (Epicentre), and the DNA was sent together with the sequence for the first primer set to Eurofins Genomics, Germany where the primers were synthesized and both DNA strands sequenced by Sanger method. From the retrieved two DNA sequences, a new primer set (F: AAGTATTAGATCGTTACGGCGATGGAC, R: CCAAGAACACCAATCGTGTTATCAAGG) was designed and once again sent to Eurofins Genomics together with genomic DNA. This procedure was repeated a third time with primer (F: TTGCCAACGTGAACAGCAAC) to determine the open reading frame (ORF) of the gene.
De novo sequencing and genome annotation
To be able to search the genome of strain C4 for additional putative keratinase genes, de novo sequencing of the whole genome using the instrument MiSeq and the MiSeq Control Software 188.8.131.52 was performed by Eurofins Genomics, Germany. The sequence assembly and scaffolding was done using the Newbler assembler software v2.9. The genome sequence was annotated using the prokaryotic annotation pipeline at the National Center for Biotechnology Information, Bethesda, USA (NCBI).
Partial purification of proteases for nanoHPLC–ESI–MS/MS
The protease production from Bacillus C4 strain was done according to Fellahi and coworkers. (Fellahi et al. 2014). In short: the C4 strain was grown in 50 ml of modified Schaeffer’s medium (Leighton and Doi 1971) containing Beef extract, 3 g/l; Bactopeptone, 5 g/l; KCl, 2 g/l; Yeast extract, 2 g/l; pH 7, and supplemented with 2 mM MgSO4.7H2O; 1 mM CaCl2; 0.1 mM MnCl2; 1 mM FeSO4; and 0.1 % (w/v) glucose. The production was done using a 250-ml E-flask with a 2 % inoculum size in a shaker incubator (Excella 24, New Brunswick Scientific) at 37 °C, 160 rpm. After 24 h, the cell-free supernatant was received by centrifugation at 8000 rpm for 15 min at 4 °C (Optima Max-XP, Beckman Coulter).
For the identification of the enzyme by nanoHPLC–ESI–MS/MS the cell-free supernatant was precipitated by adding NH3SO4 to 65 % saturation at 4 °C and slowly mixing in a shaker incubator (Excella 24, New Brunswick Scientific) for 1 h. The precipitate was collected by centrifugation at 12,000 rpm for 30 min at 4 °C (Optima Max-XP, Beckman Coulter). The pellet was re-suspended in 500 µl of 20 mM Tris–HCl buffer, pH 8, followed by dialysis overnight against the same buffer. Proteolytic activity was measured as described by Cliffe and Law (1982), using Hide Powder Azure (HPA, Sigma) as substrate. Approximately 20 µg of the purified enzyme was run on a 12 % SDS-PAGE-gel, according to Laemmli (1970) along with a molecular weight protein marker (All Blue Protein Precision Standard, Bio-Rad).
Protein identification by nanoHPLC–ESI–MS/MS and data base search
Protein identification of the crude enzyme was performed by Proteome Factory AG, Germany. Two protein spots from a 12 % SDS-PAGE-gel were cut out and digested in-gel by trypsin (Promega, Mannheim, Germany) and analyzed by nanoHPLC–ESI–MS/MS. The LCMS system consisted of an Agilent 1100 nanoHPLC system (Agilent, Waldbronn, Germany), PicoTip electrospray emitter (New Objective, Woburn, MA, USA), and an Orbitrap XL or LTQFT. The retrieved peptides were analyzed using an ultra-mass spectrometer (ThermoFisher Scientific, Bremen, Germany). Peptides were first trapped and desalted on the enrichment column Zorbax 300SB-C18, 0.3 mm × 5 mm (Agilent) for 5 min (solvent: 2.5 % acetonitrile/0.5 % formic acid), then separated on a Zorbax 300SB-C18, 75 μm × 150 mm column (Agilent) using a linear gradient from 10 to 32 % B (Solvent A: 5 % acetonitrile in water, Solvent B: acetonitrile. Both solvents contained 0.1 % formic acid). Ions of interest were data-dependently subjected to MS/MS according to the expected charge state distribution of the peptide ions. MS/MS ion search of the Mascot search engine (Matrix Science, London, England) was performed, and only peptide matches with a score of 20 or above were accepted. Proteins were identified against the B. pumilus entries from the RefSeq protein database available at NCBI, which was appended to an existing bacterial database. The search results were also run against the amino acid sequences retrieved from the Ker1 and Ker2 genes.
Amino acid sequence alignment
The amino acid sequences of the retrieved enzymes from the ultra-mass spectrometer analysis were compared to other proteases produced by the B. pumilus strains using the Basic Local Alignment Search Tool Blastp 184.108.40.206 available at NCBI.