Phytic acid sodium salt was purchased from Sigma Chemical, USA. Sephacryl S-200HR was obtained from Amersham Pharmacia Biotech, Sweden. The enzymes and kits for molecular biology experiments were purchased from standard suppliers and used as per manufactures’ instructions. All the general chemicals used for this report were of analytical grade and purchased from either Sisco Research Laboratories or HiMedia Laboratories, Pvt. Ltd, Mumbai, India.
Fungal strain and culture condition
The spore suspension (1.8 × 108 spores/mL) of A. foetidus MTCC 11682 was immobilized on poly urethane foam (PUF) cubes and cultured on an optimized media as per Ajith et al. (2018b). After each cycle of 10 days, the spent culture media were harvested, filtered and filtrates were subjected to assay for phytase activity.
Assay for phytase activity
Phytase activity was determined colorimetrically by monitoring the release of inorganic phosphorous from phytic acid using Phytex method as described by Kim and Lei (2005).
Partial purification of phytase
From immobilized fungal fermentation culture, spent media was collected, filtered through Whatman No. 1 filter paper and centrifuged at 10,000 rpm for 10 min at 4 °C. Clear supernatant was adjusted to 90% ammonium sulfate [(NH4)2SO4] saturation for precipitation of proteins. The precipitated proteins was centrifuged at 10,000 rpm for 45 min at 4 °C, solubilized in small volume of 0.01 M sodium acetate buffer (pH 5.0) and desalted by dialysis against the same buffer in refrigerated temperature using 10–14 kDa cut off membrane (HiMedia, Mumbai, India) for 48 h with four buffer changes. The sample was clarified again by centrifugation at 10,000 rpm for 10 min and then filtered through 0.45 μM membrane filter (Uniflow TM, GE Healthcare Life Sciences) and concentrated using Maxidry Lyo® (M/s Heto-Holten, Denmark). After desalting, total protein was quantified by Lowry’s method (Lowry et al. 1951). About 20 mg total protein was further fractionated in Sephacryl S-200 HR column (95 cm × 1.6 cm) using an Automated Biologic Duo-flow system (BioRad, USA) at a flow rate of 1 mL/min in 0.1 M sodium acetate buffer (pH 5.0). The eluted 3 mL per tube fractions were collected for the entire run. Each tube of column eluents was screened at 280 nm for the protein. Phytase activity in each tubes were also screened as per the method described earlier. The column void volume (V0) was determined by loading 20 mg dextran blue 2000 (2000 kDa) in 2 mL volume and was used for calibration with standard protein BSA dimer (~ 136 kDa) and monomer (~ 68 kDa).
Determination of proteins in phytase active column fractions
Elutes in multiple tubes showing phytase activity were pooled, dialyzed in 0.1 M sodium acetate buffer (pH 5.0), concentrated in Maxidry Lyo®. Samples were further tested for purity by separating the protein under reducing and non-reducing SDS-PAGE using 10% separating and 4% stacking gel (Laemmli 1970). About 80 μg of column eluted proteins and the standard molecular weight marker was loaded on each well for electrophoresis separation. Following separation, protein bands in gels were stained with Coomassie brilliant blue R-250 and phytase activity was detected by staining procedure (Bae et al. 1999). Gel image was taken in LAS-3000 gel documentation system (M/s Fuji Film, Japan) and the molecular weights of the unknown major and minor protein bands of each lane were determined by comparing the relative front (Rf) of the known MW protein markers (Bench Mark unstained protein ladder, Life technologies, USA).
Effects of temperature, pH, reducing agents and detergents on phytase activity
Phytase activity profile for the temperature optima and thermal stability of the partially purified phytase were determined over a temperature range between 4 and 80 °C for 30 min incubation.
Effect of pH on enzyme activity was determined by pre-incubating the purified enzyme in buffers with pH ranges of 2.5–3.5 (0.2 M glycine), 4.5–5.5 (0.2 M sodium acetate) and 6.5–7.5 (0.2 M Tris HCl) for 6 h followed by phytase assay at 37 °C.
To determine the effect on enzymatic activity, purified phytase was first incubated at 0.05% concentration each of 2-mercaptoethenol (reducing agent), SDS, Triton X-100, Tween-20 and Tween-80 (all detergents) for 15 min at 37 °C and then phytase activity was assayed following the methodology by Kim and Lei (2005).
Mass spectroscopy analysis of the protein bands
Major protein bands (151.4, 138.5, 121.3, 99.7 and 90.5 kDa) from the gel were excised and washed in 50% acetonitrile solution containing 100 mM ammonium bicarbonate (NH4HCO3). Samples were reduced using 10 mM dithiothreitol (DTT) in 100 mM NH4HCO3 solution for 45 min at 56 °C, followed by alkylation using 55 mM iodoacetamide solution in 100 mM NH4HCO3 for 30 min at room temperature in dark. Finally in-gel digestion was carried out using 20 μL (10 ng/μL) of sequencing grade trypsin in 50 mM NH4HCO3 overnight at 37 °C. Peptides generated after digestion were extracted in NH4HCO3 buffer with 5% formic acid. Samples were vacuum-dried and reconstituted in buffer with 5% formic acid. The protein digest spectrum was acquired on M/s Bruker Daltonics Autoflex TOF/TOF mass spectrometer equipped with ion source and FLEX PC instrument. The generated mass (m) to charge (z) ratio peaks were analyzed with the setting of 10% threshold, precursor and fragment mass tolerances of 0.15 Da, cysteine carbamido-methylation as fixed modification and methionine oxidation as variable modification in the Masscot Ion search engine (http://www.matrixscience.com/search_form_select.html).
Isolation of total RNA and cDNA conversion
About 300 mg mycelium from 18 to 24 h old biomass of A. foetidus MTCC 11682 was collected and macerated in liquid nitrogen using a sterile mortar and pestle. To each sample, 2 mL of TriReagent (Sigma Aldrich, USA) was added and then passed through a 20 g needle repeatedly to make it a homogeneous solution. Total RNA from the solution was isolated following Sigma Technical bulletin protocol (https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Bulletin/t942bul.pdf). About 2 µg total RNA was reverse transcribed using Reverse Aid H minus first stand cDNA synthesis kit (M/s Fermentas USA).
Polymerase chain reaction (PCR) amplification, cloning and analysis of sequence
Partial coding region of phyA gene was amplified by PCR using gene specific primers (Forward 5′ GTATCAATGCTTCTCCGAGACTTCG and reverse 5′ CGATCATTAACCAAGACACGGACC) designed from the consensus region of phytase gene coding sequences of four different Aspergillus species—A. niger (M94550), A. fumigates (U59804), A. terreus (U59805) and A. terreus (U60412). A typical 20 µL PCR reaction was performed in volume that contained 20 ng total RNA equivalent cDNA, 0.5 µL (5 µM) forward and reverse primers, 10 µL 2XGoTaq® Green master mix. PCR was performed with an initial denaturation at 94 °C for 2 min, followed by 35 cycles each of denaturation at 94 °C for 1 min, annealing at 57 °C for 30 s and extension at 65 °C for 1 min 30 s, and finally an extension at 72 °C for 5 min. A total of 120 µL PCR reaction mix was prepared for the purpose of cloning the fragment. Amplified product was electrophoresed on 1% agarose gel in 1X TAE buffer and visualized with ethidium bromide staining. The desired band was excised and purified using QIAquick gel extraction kit (Qiagen, Germany). About 7 µL of gel purified PCR product was ligated to pJET 1.2 vector as per manufacturer’s protocol, transformed to chemically competent Top10 E. coli cells by heat shock treatment and cultured in Luria–Bertani (LB) plate containing Ampicillin (50 μg/mL media). Individual bacterial colonies were picked from the LB plate and were grown overnight in LB broth with Ampicillin (100 μg/mL media). Plasmids were isolated from the grown E. coli using QIAprep Spin mini prep kit (Qiagen, Germany). Presence of inserts in plasmids was confirmed by gene specific PCR. Plasmids isolated from three different clones positive for gene specific PCR were further sequenced for both strands by Sanger sequencing reactions with Big Dye Terminator v3.1 Cycle Sequencing method (Applied Biosystems® Inc, USA). Output sequences were derived, confirmed and annotated by homology search using NCBI-BLAST database (https://www.ncbi.nlm.nih.gov/BLAST/). Based on open reading frame, amino acid sequences were deduced and locations of disulphide bonds in the amino acid chain were predicted using online software (http://clavius.bc.edu/~clotelab/DiANNA/). The N- and O-glycosylation sites were predicted by (http://www.cbs.dtu.dk/services/NetNGlyc) and (http://www.cbs.dtu.dk/services/NetOGlyc) online tools, respectively. The expected amino acid (aa) sequence was deduced. Both nucleic acid and derived aa sequences of nearby species were aligned by CLUSTAL-W program and phylogenetic tree was constructed by neighborhood joining method (http://www.genome.jp/tools/clustalw/). The tree was drawn to scale with branch length in the same units as those of the evolutionary distance of other similar species and synthetic sequences.