Purification and characterization of phytase from two enteric bacteria isolated from cow dung

Phytase-producing Klebsiella and Shigella sp. were isolated from environmental samples based on their ability to degrade phytate and the enzymes were purified and characterized. The enzymes from both sources were intracellular monomeric proteins with the molecular mass of about 40kDa. The enzymes were active in the pH range of 2.0 to 7.5 with pH optima at 5.5. The optimum in vitro temperatures of the enzymes from Shigella and Klebsiella were 60°C and 55°C respectively. The enzyme from Shigella retained about 80% activity up to 75°C. Due to their relatively high specific activity, substrate specificity, good pH profile and thermostability, the enzymes could be interesting candidate for agricultural and feed application. INTRODUCTION Phytic acid (myo-inositol 1,2,3,4,5,6 hexakisphosphate) is the main storage form of phosphorus in cereals, legumes, and oilseed crops [13]. Although phytic acid serves as the major source of energy and phosphorus for seed germination, the bound phosphorus is poorly available to monogastric animals. Phytic acid also acts as an antinutritional agent in several ways as it interacts with other food ingredients [4]. Phytase (myo-inositol hexakisphosphate phosphohydrolase) is the enzyme that catalyses the hydrolysis of phytic acid to inorganic monophosphate and lower myo-inositol phosphates, and in some cases to free myo-inositol [5, 16]. The ruminants digest phytic acid through the action of phytases produced by the anaerobic gut fungi and bacteria present in their ruminal microflora. However, monogastric animals such as pigs, poultry and fishes utilize phytate phosphorus poorly because they are deficient in gastrointestinal tractphytases and therefore an inorganic, non-renewable and expensive mineral supplement is used in diets for swine, poultry and fishes to meet their nutritional requirement of phosphorus. The unutilized phytate phosphorus from plant feeds is excreted as an environmental pollutant in areas of intensive live-stock production [10]. Excessive phosphorus in soil runs off to lakes and the sea, causing eutrophication and also stimulating growth ofvplanktonic vegetation such as algal blooms and aquatic blooms [19, 11] that may produce neurotoxins, injurious to human beings [9, 10]. Therefore, the enzymatic hydrolysis of phytic acid into less-phosphorylated myo-inositol derivatives in the intestine of the monogastric animals is desirable. Attempts to enzymatically hydrolyze phytic acid have been made to improve the nutritional value of feed and to decrease the amount of phosphorus excreted by animals [9, 6]. When used as additives in feed of monogastric animals, phytases not only enhance utilization of phosphate and reduce phosphate output in manures but also increase mineral uptake [3, 19]. Studies are also going on the utilization of phytases in food and industrial purposes [11]. In recent years, bacterial phytases have been isolated, characterized and proposed as potential tools in biotechnology. The highest frequency of phytase-like proteins has been reported in members of the gamma-proteobacteria group. Pure culture studies of ruminal bacteria have demonstrated phytate-degrading activity in various strains belonging to the Enterobacteriaeceae family, such as in Enterobacter, E.coli, Klebsiella, Citrobacter, Yersinia etc. Here, we report a comparative study on isolation, purification and characterization of phytases from two enteric bacteria (Klebsiella sp. RS4 and Shigella sp. W3). MATERIALS AND METHODS A. Isolation and selection of phytase-producing bacteria Bacterial strains were isolated from cow dung. Isolation of pure cultures from different collected samples was done by the simple serial dilution method followed by plating onto phytase screening medium (PSM) at 37°C. Phytase-producing bacteria were screened on the phytase-producing medium (PPM) as described by Kerovuo et al. [5]. On the basis of qualitative and quantitative enzymatic analysis, two potent phytaseproducing strains were selected for further studies. Those strains were characterized on the morphological, biochemical, cultural and molecular level. B. Optimization of phytase production under shake flask cultures Pure cultures were isolated and screened for phytase production in the PPM at 37°C under shake flask culture and analyzed for intracellular and extracellular phytase activity. The effect of different production parameters, i.e. inoculum age, pH, temperature, simple and complex carbon sources, nitrogen sources, on enzyme production was studied. C. Phytase activity assays Enzyme assays were preformed as described by Shimizu [10]. One unit of enzyme activity was defined as the amount of enzyme hydrolyzing 1 μmol of Pi per min under assay conditions. The specific activity was expressed in units of enzyme activity per milligram of protein. D. Enzyme purification Phytase enzyme was purified from the phytase-producing bacterial strains by the methods based on solubility as well as standard chromatographic techniques. A combination of two-step ammonium sulfate precipitation, gel filtration and ion exchange chromatographic methods was found to be the best purification method. Unless otherwise indicated, all operations during extraction, separation and purification of the enzyme were carried out at 4°C. E. Protein estimation and SDS-PAGE analysis The amount of protein was determined by the method of Bradford [12] using Bovine Serum Albumin as standard. The enzyme fractions from various steps of purification were separated by Sodium dodecyle sulphate polyacrylamide gel electrophoresis (SDS-PAGE), by the method of Laemmli [17]. F. Characterization of the enzyme The kinetic properties such as Km, pH optima, temperature optima, thermostability and substrate specificity of the purified enzymes were also compared. RESULTS AND DISCUSSION Six strains that can solubilize sodium phytate and form peripheral halo zone on phytase screening agar medium around colonies were isolated form cow dung. By plate screening, RS4 and W3 were found to be the most potent strains as phytase producers. Quantitative screening methods done by phytase assay also supported the same fact. The phytase activity assay was carried out using media supernatant as well as sonicated fractions showing that phytase production in both the isolated strains is intracellular. RS4 produced maximum amount of phytase after 24h of incubation, while W3 showed maximum Proceedings of 5th International Conference on Environmental Aspects of Bangladesh [ICEAB 2014] Page | 58 phytase activity at 72h (Fig.1a). 16S ribosomal DNA analysis revealed that RS4 was a representative of Klebsiella sp. showing close sequence homology to Klebsiella pneumoniae and W3 was a representative of Shigella sp. (GenBank accession number FR745402 and FR682761 respectively). Morphological and biochemical characterization were also done for both the strains. For optimization of culture condition, the parameters tested were carbon sources such as 1% glucose, maltose, sucrose, mannose, lactose, galactose or starch and 0.5% glucose + 0.5% mannose and nitrogen sources, both organic and inorganic, such as ammonium sulphate, sodium nitrate, ammonium nitrate, peptone, tryptone, casein, beef extract and yeast extract. Result of the experiments showed the best carbon source for both the strains was glucose while in presence of ammonium sulphate they showed highest phytase activity (Fig.1b & c). Fig. 1. Optimization of phytase production for both Klebsiella sp. RS4 and Shigella sp W3 with respect to (a) Incubation time (b) Carbon sources (c) Nitrogen sources The Klebsiella phytase was purified 101 fold from the cell extract with specific activity of 598 U mg-1 (Table 2) for sodium phytate hydrolysis and the 133 fold purified enzyme from Shigella sp. W3 had specific activity of 780 U mg-1 protein (Table 1). Table 1. Purification of phytase from Shigella sp.W3