Bacterial isolation, their screening and enzyme assay
Totally 20 fishes were collected from the Cauvery River at the Stanley Reservoir, Salem District, India (21°43.232′N, 87°48.884′E). The collected fish was identified as Systomus sarana by following the keys provided in the Manual of Jayaram et al. (2010). The fish gut contents were homogenized, and serial diluted up to 10-5. The spread plate technique was performed on sterilized tryptone soya agar-medium that composed of Sodium chloride-5.0 g/L, Dextrose-2.5 g/L, Dipotassium hydrogen phosphate-2.5 g/L and Agar-15.0 g/L (Hi Media, Mumbai, India). The isolate’s growth condition was 37°C, on nutrient agar media plate and finally 40% glycerol stock was made for the long-time storage at-20°C. For the primary screening of pectinase enzyme production, the isolates were grown on pectin-agar media (5 g/L pectin, 5 g/L tryptone, 2.5 g/L yeast extract, 5 g/L NaCl, 15 g/L agar; pH 7). By following the well diffusion method, 100 µl of cell-free supernatant was loaded on a pectin agar well plate
(of diameter 5 mm) and the plates were incubated at 37°C for 24 h. The amount of the enzyme produced was then calculated, and expressed as enzyme intensity by following protocol of Hankin et al. (1971) (EI) = (colony diameter + halo zone diameter) / colony diameter. The pectinase enzyme activity was determined using 3, 5-dinitrosalicylic acid method of Miller et al. (1959). A unit of pectinase activity is defined as 1 µmol of galacturonic acid released per minute.
Bacterial genomic DNA (gDNA) was isolated according to the phenol: chloroform method (Sambrook et al. 2001). The purified DNA was subjected to PCR amplification using a pair of 5'-AGAGTTTGATCMTGGCTCAG-3' and 5´-AAGGAGGTGATCCAGCCGCA-3´ forward and reverse primers for 16S rRNA gene amplification and sequencing. The phenotypic level identified gyrB F (5′-GAAGTCATCATGACCGTTCTGCAYGCNGGNGGNAARTTYGA-3′) and gryB R (5′-AGCAGGGTACGGATGTGCGAGCCRTCNACRTCNGCRTCNGTCAT-3′). A total volume of 25 µL (2x dream taq master mixed) was used for PCR reaction. The PCR program consisted of an initial denaturation step at 95°C for 5 min followed by 30 rounds of temperature cycling comprising denaturation at 95°C for 1 min, annealing at 55°C for 1 min, and extension at 72°C for 1.30 min, followed by a final extension at 72°C for 7 min and then cooling at 4°C. The sequences have been deposited in the National Center for Biotechnology Information (NCBI) Data Bank Accession number; MF800949. The phylogenetic and molecular evolutionary analyses were conducted by using the software MEGA by the neighbor-joining method (Tamura et al. 2013).
Response Surface Methodology (RSM)
Response surface methodology was performed as per the protocol of Ramesh et al. (2013), with slight modification. A five-factor Box-Behnken Design contained 46 experimental runs with three replications (Table 1a). It was also used to optimize the independent variables, like, pH, temperature, carbon sources, nitrogen sources, inoculum size and incubation day. The modeling and statistical analysis was used Stat-Ease Inc., Minneapolis, software. Plackett-Burman (PB) design: Totally, 12 factors viz; glucose, maltose, sucrose, galactose, lactose, mannitol, beef extract, peptone, yeast extract, NaNO3, NaCl and KH2PO4 were considered (Plackett et al. 1946).
Effect of various parameters on pectinase production
The effective isolate was inoculated into the pectinase production medium at the optimum ranges of distinct parameters viz; pH (3-10), temperatures (10-45°C) and incubation time (0-60 h) and incubated under rotary shaking at 150 rpm. The effect of six carbon sources each to 1% and six nitrogen sources each to 1% on the production of pectinase was carried out (Kumar et al. 2011).
Extraction and purification of protein
The isolated overnight culture broth (at the optimum condition) was centrifuged (10,000 rpm; 4°C; 20 min), the cell-free supernatant was collected and an 15% Ammonium per sulfate was added to precipitate the protein. And then the protein was purified using gel chromatography, where 20 ml of Sephadex G-100 solution was packed into a glass column (35×1.5 cm, 60 ml of bed volume). The 10 ml of precipitated protein-loaded column was eluted using the Tris-HCl buffer (10 mM, pH 7.5). Then, 1.5 ml volume of fractions was collected separately and stored at −80°C for further analysis.
Two-Dimensional (2-D) PAGE analysis
The purified protein (20 µl) was loaded onto 12% SDS-PAGE with a standard marker (GENEI, Bangalore, India) and electrophoresis was performed as per the protocol of Laemmli et al. (1970). Further 2D analyses, the protein samples were sent to Sandor life science Pvt. Ltd. (Hyderabad). The 2-D gel was analyzed by an in-silico method based on the isoelectric point (pI) and the molecular weight of separated proteins. The 2-D gel protein spot was analyzed by using the ExPASy tool.
FT-IR and HPLC analyses
ART model FT-IR spectrophotometer was used at the wavelength of 4000- 400 cm-1 and the resolutions of 4 cm-1 were averaged according to Cakmak et al. (2006). The enzyme fraction sample was investigated by HPLC using Shimadzu LC solution 20 AD, Japan and SPD 20 A, having a reverse phase water guard Column: Symmetry LCGC C18 (5 µm,4.6×250 mm) and Hamilton microliter syringe using an injection volume of 20 µL and detection was made at 254 nm by UV detector at 28ºC. The HPLC mobile phase comprised of methanol and water (50:50). Empower2 software was used to analyses the data.
Charaterization of the Mass Spectroscopy of extracted protein and homology modeling
Mass spectra of the digestion mixtures were acquired in a 4800 MALDI-TOF-MS instrument (Applied Biosystem) in reflector mode and were externally calibrated using a mixture of peptide standards by following the protocols of Patterson et al. (1995). Collision-induced dissociation MS/MS experiments of selected peptides were performed. A NCBI database search with peptide m/z values was made by using the MASCOT search tool (URL http://www.matrixscience.com) for the identification of tryptic maps of protein identification. Homology modeling was performed with SWISS-MODEL a homology-modeling server as described by Bordoli et al. (2009).
PCR amplification of exo-pelA gene
PCR using the exo-pelA gene primer pelA f (5' GATAAGGATCCGATGCAAGACAGCGACGTG 3') and pelA r (5' GCTTCGAATTCCGCACAAAGAGAAAGGAAT 3') in a 30-μL reaction volume that contained 15 μL of 2x dream taq master mix (Thermo), 1 μL genomic DNA (50 ng/μL), 1 μL of each primer (20 pM) and water was used. The PCR program consisted of an initial denaturation step at 94°C for 3 min followed by 35 rounds of temperature cycling comprising 1 min at 94ºC for denaturation, 30 s at 57ºC for annealing, 40 s at 74ºC for extension, and 7 min final extension.
DNA/pREST vector digestion and ligation
The pREST plasmid (~2.7 Kb) E. coli strain: For the isolation of plasmid genomic DNA, the protocol of Sambrook et al. (2001) was followed. An aliquot of DNA sample was mixed with 2 μl of 1 x restriction buffer (5 μl) and the volume was made up to 30 μl with ddH2O. The reaction was started with the addition of the enzyme EcoRI (1 μl), BamHI (1 μl) and incubated at 37°C for 2 h. Incubation at 70°C for 3 min terminated the reaction. The cocktail mixtures (30 µl reaction) contained 10x T4 DNA ligase buffer (4 µl), vector (4 µl), insert gene (3 µl), T4 DNA ligase (5 µl), ATP (1 µl) and ddH2O 13 µl. The mixtures were added into the microtubes and placed on ice, and then the samples were incubated at 4ºC for 16 h.
Preparation of competent cells and transformation
A single colony of E. coli (DH5α) culture was inoculated in 5 ml of LB medium (with AMP) and incubated at 37°C for 16 h. And 1.0 ml of 16 h grown culture was added to 100 ml of LB medium in a 250 ml conical flask and incubated at 37°C for 16 h. For the competent cells preparation (CaCl2-treated) and transformation (heat shock treatment), the method of Sambrook et al. (2001) was followed.
Gene expression analysis and recombinant protein purification
An LB medium containing AMP was used (pH 7.0) for pectinases production. A modified basal synthetic (BS) medium was used for recombinant exo-pelA production in a 250 ml fermenter. The glycerol feeding solution was used for high-cell-density cultivation (HCDC) of E. coli (BL21). Isopropyl β-D-1-thiogalactopyranoside (IPTG) was filter-sterilized and added to the bioreactor as an inducer. The recombinant protein was purified by Sephadex G 100.
Effect of metal ions on pectate lyases
20 ml of sterile production medium was prepared in different conical flasks. Each flask was amended with effective metal ions such as Ca2+, Mg2+, Co2+, Cu2+, Fe3+, and Mn2+, individually. The final concentration of the enzyme substrate system was 5 mM. The reaction mixture was terminated by adding 2 ml of 3, 5-dinitrosalicylicacid reagent and then the enzyme activity was assayed.