Isolation, Identification and Molecular Characterization of Phytase Producing Bacteria, Pseudomonas Sp. aazad

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Phytic acid is the major storage form of phosphorus and inostiol in plant seeds, comprising about 3-5% of the dry weight particularly in cereal grains and legumes (Zhang et al., 2010).Phytic acid can chelates divalent cations like calcium, magnesium, iron and zinc, decreasing their bioavailability.These minerals are essential in the diet of both humans and animals.Phytic acid has anti-nutritional effect since it reduce food intake and nutrient utilization in animals and humans (Urbano et al., 2000;Lei et al., 2003).Phytases are enzymes capable of hydrolyzing phytic acid to myo-inostiol and inorganic phosphorus.Phytases are mainly used as animal feed additive, as they enhance the nutritional quality of plant material in feed by increasing the bioavailability of minerals for monagastric animals.Moreover, animal feeds devoid of phytase causes immense release of undigested phytate into the environment.The phosphorus excreted in the environment enhances the growth of phosphorus assimilating microorganisms resulting in eutrophication (Schroder et al., 1996).
Phytases are found in plants, animals and microorganisms.However, researchers have shown that microbial phytases are most promising for biotechnological application (Angelis et al., 2003).Studies have shown that bacterial phytases offer several advantages over fungal phytases because of higher substrate specificity, greater resistance to proteolysis and better catalytic capability.There is an ongoing interest in microorganisms including bacteria for novel and efficient phytases as there is no single phytase that may be able to meet the diverse need for all commercial and environmental applications.The present study reports isolation, production and molecular identification of efficient phytate degrading bacteria, Pseudomonas sp.

Sample collection
Isolation of the phytase-producing bacteria was carried out by sampling 150 soil samples from different geographical regions.Samples were collected from 10 cm below soil surface aseptically with the help of sterile spatula in sterile polythene bags.Three serial dilutions of each sample were prepared (10 -1 , 10 -3 , and 10 -5 ) in normal saline.100 µl of each dilution were inoculated on freshly prepared nutrient agar plates having pH of (4.0, 7.0 and 10.0) and incubated at 10 ºC (24-96 h), 30 ºC (24-72 h) and 60 ºC (12-24 h).Isolated colonies of different morphological characteristics (variations in size, shape, color and regularity) were sub-cultured on fresh nutrient agar medium plates at neutral pH.

Phytase enzyme assay
Phytase producing bacterial isolates were inoculated in 50 ml PSM broth containing 0.5% calcium phytate and were cultured in a rotary shaker (200 rpm) at 30 ºC for 72 hr. 2 ml cultures were withdrawn aseptically from flasks periodically.The cell-free supernatant obtained after centrifugation at 10,000 rpm for 10 min, was used as the source of extracellular phytase enzyme.Phytase assay was done by measuring the amount of phosphate released using sodium phytate as a substrate.The reaction mixture comprised 0.5 ml of 0.1 M Mop's buffer (pH 7.0), 0.5 ml of sodium phytate and 1.0 ml crude enzyme.The reaction was carried out at 40 ºC for 10 min and stopped by adding 2.0 ml 10% (w/v) TCA.The phosphate released was determined spectrophotometrically according to Fiske and Subbarow (1925).A reference standard curve of KH 2 PO 4 was simultaneously assayed with the sample.'One unit of enzyme activity was defined as the amount of enzyme used to liberate one µmole inorganic phosphate/ml/minute under the assay conditions'.

pH and temperature optima of phytase enzyme
The pH optima of enzyme were determined by incubating reaction mixture on different pH ranging from pH 2.0-9.0 [0.2 M glycine/HCl buffer (pH 2.0 to 3.0), 0.2 M acetic acid/sodium acetate buffer (pH 4.0 to 6.0)], 0.2 M Tris/HCl (pH 7.0 to 8.0)].The temperature optima of enzyme were determined by incubating the reaction mixture at different temperature 30 0 C-70 0 C and the phytase activity was assayed

Identification of phytase producing bacterial isolate
The phytase producing bacterial isolates were identified by colony morphology and microscopic examination (gram staining).Metabolic fingerprinting is also done for the identification.The culture was sent to BTK biosciences, New Delhi, for the biochemical identification of bacterial species.

Molecular identification
Genomic DNA was extracted by a modified method of Bazzicalupo and Fani (1994).PCR amplification of 16S rDNA of bacterial isolate was carried out using eubacterial universal primers Gm3f and Gm4r.Primer sequences were as follow: Gm3f (5'-AGA GTT TGA TCM TGG -3') and Gm4r (5' -TAC CTT GTT ACG ACT T -3') (Eden et al., 1991).PCR amplification was done in 10 µl reaction mixture containing 5 µl taq master mix, 1.25 µl primers, 1.25 µl genomic DNA and 1.25 µl sterile deionised water.The reaction conditions were as follows: initial denaturation at 95 0 C for 7 min, 35 amplification cycles of denaturation at 94 0 C for 1 min, annealing at 51 0 C for 1 min and primer extension at 72 0 C for 1 min; followed by a final extension at 72 0 C for 10 min.The PCR product was run on 0.8% agarose gel and visualized under UV transilluminator.The Ltd., Bangalore, India.

Isolation of phytase producing bacteria
Phytase, much in demand, has potential application in food and feed industry for ameliorating digestibility and assimilation of nutrients by improving growth of poultry, pigs and fishes.On primary qualitative screening, 14 bacterial isolates were found to produce clear zone of hydrolysis.The formation of zone of hydrolysis around the bacterial colony was a positive indication of extracellular phytase production (Hill et al. 2009).Fig. 1 show zone of clearance around colonies of some isolate.

Phytase activity and optimization
Phytase activity was determined by measuring the amount of liberated inorganic phosphate and its reaction with colour reagent.Phytase production was done in PSM broth and five bacterial isolates 100A, 9H, 14A, 4D, and 2B showed significant phytase production in submerged fermentation.The isolate 14A was found to be the best among all with the maximum production of 89.09 U/ml at 72 h (Table .1),and was finally selected for further study.Most bacterial phytases studied show their optimum activity in acidic range.For instance, for Aerobacter aerogenes (Greaves et al., 1967), E. coli (Greiner et al., 1993), S. castellii (Segueilha et al., 1992), A. adeninivorans (Sano et al., 1999), Selenomonas ruminatium (Sreeramulu et al., 1996) and Pseudomonas sp.(Irving and Cosgrove, 1971), the optimum pH was between 4.0 and 5.5.In our study, the maximum enzyme activity (771 U/ml) was observed at pH 6, and comparable activity was observed at pH range (6)(7)(8).Activity of enzyme showed a decrement as the pH increases.The maximum activity (784 U/ml) was measured at 50 ºC, and the activity was also considerably good at temperature range 40-60 ºC.Activity decreases as the temperature increases above 50 ºC (Fig. 2).Phytase production at elevated temperature is desirable for feed application.

Identification of phytase producing bacterial isolate
Colony morphology and microscopic examination (Gram's staining) of maximal phytase producers suggested that the isolates 100A, and 14A are gram negative rod, and 9H, 4D, and 2B are gram positive rods and cocci (Fig. 3).Using the Biolog system the metabolic pattern of 14A was analyzed.The culture was positive for pH 6 , it can utilize D-Glactouronic acid, L-Aspartic acid, D-Glucuronic acid, D-Serine and was found sensitive towards Troleandomycin, Lincomycin, Vancomycin, Rifamycin, Tetrazolium violet, Tetrazolium blue, 1% Sodium lactate, Potassium tellurite and 1% NaCl.The result was recorded which identified it as a species of Pseudomonas florescence.Although, when strain 14A was treated under UV light (Brodsky and Nixon, 1974), it did not show any florescence.

Molecular identification
The molecular identification of bacterial isolate (14A) was done using 16S rDNA (Fig. 4).The 16S rDNA gene was amplified by PCR using eubacterial universal primers.The PCR product was estimated to be approximately 1.5 kb.By constructing the phylogenetic tree of the gene sequence the isolate showed maximum similarity with Pseudomonas sp.Phylogenetic relationship is shown in Fig. 5. 16S rDNA sequencing and sequence similarity search using BLAST showed that it matches 97 % with Pseudomonas pulluriniana, while 3% was found to be unique sequence (submitted at NCBI Gene bank with the accession number 157554).On the basis of these results, we concluded that the strain 14A belongs to genus Pseudomonas and were named as Pseudomonas sp.strain aazad.Phytase from Pseudomonas spp.were also reported by Richardson and  far with each producing a positive response to the plate-clearing as well as an enzyme activity.The phytase from the isolated Pseudomonas sp.aazad showed a good enzyme titre and an optimum pH and temperature of 6 and 60 ºC respectively that can be used for application as feed supplement for enhancing the utilization of phytate bound phosphorus and thus decreasing pollution caused by it.

Fig. 1 .
Fig. 1.Zone of hydrolysis shown by some isolates on phytase screening medium containing Ca phytate