Purification and characterization of two distinct acidic phytases with broad pH stability from Aspergillus niger NCIM 563

S. K. Soni 0 A. Magdum 0 J. M. Khire 0 0 S. K. Soni A. Magdum J. M. Khire (&) Division of Biochemical Sciences, National Chemical Laboratory , Pune 411 008, India Aspergillus niger NCIM 563 produced two different extracellular phytases (Phy I and Phy II) under submerged fermentation conditions at 30 C in medium containing dextrin-glucose-sodium nitrate-salts. Both the enzymes were purified to homogeneity using Rotavapor concentration, Phenyl-Sepharose column chromatography and Sephacryl S-200 gel filtration. The molecular mass of Phy I and II as determined by SDS-PAGE and gel filtration were 66, 264, 150 and 148 kDa respectively, indicating that Phy I consists of four identical subunits and Phy II is a monomer. The pI values of Phy I and II were 3.55 and 3.91, respectively. Phy I was highly acidic with optimum pH of 2.5 and was stable over a broad pH range (1.5-9.0) while Phy II showed a pH optimum of 5.0 with stability in the range of pH 3.5-9.0. Phy I exhibited very broad substrate specificity while Phy II was more specific for sodium phytate. Similarly Phy II was strongly inhibited by Ag?, Hg2? (1 mM) metal ions and Phy I was partially inhibited. Peptide analysis by Mass Spectrometry (MS) MALDI-TOF also indicated that both the proteins were totally different. The Km for Phy I and II for sodium phytate was 2.01 and 0.145 mM while Vmax was 5,018 and 1,671 lmol min-1 mg-1, respectively. The N-terminal amino acid sequences of Phy I and Phy II were FSYGAAIPQQ and GVDERFPYTG, respectively. Phy II showed no homology with Phy I and any other known phytases from the literature suggesting its unique nature. This, according to us, is the first report of two distinct novel phytases from Aspergillus niger. - Phytase, a phosphomonoesterase, is an enzyme capable of hydrolysing phytate [myo-inositol (1,2,3,4,5,6) hexakisphosphate], the major storage form of phosphorus in cereals and legumes, representing 1888% of total phosphorus content (Konietzny and Greiner 2002). Phytases (EC 3.1.3.8 and EC 3.1.3.26) belong to the family of histidine acid phosphatases which hydrolyses phytate to liberate inositol and inorganic phosphate (Mullaney et al. 2000). Phytate phosphorus is biologically unavailable to non-ruminants as they lack or secrete a very low level of phytase activity in the intestine. Consequently, the phytate in animal feeds is discharged in the feces of these animals into rivers and seas, resulting in severe pollution of water resources (Mullaney et al. 2000). Similarly, phytic acid is an anti-nutrient, which due to its strong chelating potential can bind essential minerals such as calcium, zinc and copper, rendering them unavailable or poorly available for absorption (Singh 2008). To overcome this difficulty the feed has to be supplemented with inorganic phosphate to meet the nutritional requirements of the animals (Selle and Ravindran 2007). Supplementation of feed with phytases in contrast increases the bioavailability of phytic acid-bound phosphate, facilitating a reduction in the addition of inorganic phosphate to the feed and a decrease in phosphorus excretion in areas of intensive animal husbandry. Although a large number of bacteria, yeasts and fungi are reported to produce phytase (Vohra and Satyanarayana 2003; Vats and Banerjee 2004; Kaur et al. 2007) fungal phytases are preferred in animal feed due to their high yield and more acid tolerance as compared to phytases from bacteria (Kim et al. 1998). Similarly the pH in poultry gut varies from 2.5 to 6.0, thus phytase active and stable in acidic environment is highly preferred (Radcliffe et al. 1998). Among fungi many Aspergilli (Ullah 1988; Vohra and Satyanarayana 2003; Vats and Banerjee 2004; Vats et al. 2004) are known to be active phytase producers. As Aspergillus niger is Generally Recognised as Safe (GRAS) it is frequently used in food and feed applications. Earlier we have reported phytase production by Aspergillus niger NCIM 563 under submerged fermentation (Soni and Khire 2007; Bhavsar et al. 2008; Shah et al. 2009) which includes production and partial characterization of two types of phytase from Aspergillus niger NCIM 563. The present communication reports purification and characterization of two novel phytases (Phy I and Phy II), which according to us, is the first report of two distinct forms of extracellular acidic phytases produced simultaneously under submerged fermentation. Materials and methods Phytic acid sodium salt was purchased from Sigma Chemical Company, St Louise, MO, USA. All other chemicals used were of analytical grade and obtained from leading manufacturers including BDH, Sigma and Glaxo. SDSPAGE and gel filtration markers, Coomassie Brilliant Blue R-250 and Bromophenol Blue were purchased from Sigma Chemical Company, USA Sephacryl S-300, Phenyl-Sepharose CL-4B were obtained from Sigma. Organism and culture conditions The strain used throughout the present work was Aspergillus niger NCIM 563 (Soni and Khire 2007). It was maintained on Potato Dextrose Agar (PDA) slants. The fungus was grown in modified fermentation medium containing (per 100 ml): Dextrin 5 g; Glucose 2.5 g; NaNO3 0.86 g; KH2PO4 0.004 g; KCl 0.05 g; MgSO4 7H2O 0.05 g; FeSO4 7H2O 0.01 g. pH 5.5 before sterilization. Fermentation medium (100 ml in 250-ml Erlenmeyer flasks) was inoculated with 1% (v/v) of spore suspension (5 9 107 spores per ml) prepared by suspending the spores from 7-day-old sporulated slants of Aspergillus niger NCIM 563 grown on PDA in 10 ml of sterile distilled water containing 0.01% (v/v) Tween 80 and incubated at 30 C at 200 rev/min. Samples were removed after every 24 h and checked for pH, growth, total residual reducing sugar, extracellular protein and phytase activity. Assay of phytase and protein Phytase activity was measured at 55 C as described earlier (Soni and Khire 2007). The reaction for Phy I and Phy II was carried out at pH 2.5 (100 mM Glycine-HCl buffer) and pH 5.0 (100 mM acetate buffer) at 55 C for 30 min, respectively. The liberated inorganic phosphate was measured by a modification of the ammonium molybdate method (Heinohen and Lathi 1981). A freshly prepared 4 ml solution of acetone:2.5 M H2SO4:10 mM ammonium molybdate (2:1:1 v/v/v) and 400 ll of 1 M citric acid were added to the assay mixture. Absorbance was measured at 370 nm. One unit of phytase activity (IU) was expressed as the amount of enzyme that liberates 1 lmol phosphorus per minute under standard assay conditions. Each experiment was carried out in triplicate and the values reported are the mean of three such experiments in which a maximum of 35% variability was observed. Protein concentration in the fermentation broth and in the purified enzyme preparation was determined by the Lowry method as well as measurement of absorbance at 280 nm using BSA as a standard. Purification of phytase After fermentation, mycelium was separated by filtration followed by centrifugation at 10,0009g for 30 min and the clear supernata (...truncated)