CURRENT MICROBIOLOGY Vol. 47 (2003), pp. 290–294 DOI: 10.1007/s00284-002-3966-4 Current Microbiology An International Journal © Springer-Verlag New York Inc. 2003 Purification and Characterization of a Phytase from Pseudomonas syringae MOK1 Jaie Soon Cho,1 Chang Whan Lee,2 Seung Ha Kang,1 Jae Cheon Lee,1 Jin Duck Bok,2 Yang Soo Moon,1 Hong Gu Lee,1 Sung Chan Kim,1 Yun Jaie Choi1 1School of Agricultural Biotechnology, College of Agriculture and Life Science, Seoul National University, 441-744, Suweon, Korea 2Choong Ang Biotech Co., Ltd., 833-6 Wonsi-Dong, Ansan-City, Kyunggi-Do, Korea Received: 9 September 2002 / Accepted: 6 December 2002 Abstract. A phytase (EC 3.1.3.8) from Pseudomonas syringae MOK1 was purified to apparent homo- geneity in two steps employing cation and an anion exchange chromatography. The molecular weight of the purified enzyme was estimated to be 45 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The optimal activity occurred at pH 5.5 and 40°C. The Michaelis constant (Km) and maximum reaction rate (Vmax) for sodium phytate were 0.38 mM and 769 U/mg of protein, respectively. The enzyme was strongly inhibited by Cu2ϩ,Cd2ϩ,Mn2ϩ, and ethylenediaminetetraacetic acid (EDTA). It showed a high substrate specificity for sodium phytate with little or no activity on other phosphate conjugates. The enzyme efficiently released orthophosphate from wheat bran and soybean meal. Phytic acid (myo-inositol hexakis phosphate; phytate) is interest for biotechnological applications, as environ- the predominant storage form of phosphorus in cereals, ment-friendly feed additives in feed manufacturing in- oilseed meals, and legumes [16]. In terms of animal dustry. Because of their great industrial significance, nutrition, monogastric animals such as swine and poultry there is an ongoing interest in isolating new microbial are not capable of metabolizing phytate phosphorus ow- strains producing efficient phytases and characterizing ing to the lack of digestive enzymes hydrolyzing the these enzymes. substrate, and therefore inorganic phosphate is added to Until now, the majority of microbial phytase re- their diets to meet the phosphorus requirement, while search has been directed to fungal phytases such as undigested phytate phosphorus is excreted in manure and Aspergillus niger [17], Aspergillus ficuum [4], Aspergil- poses a serious phosphorus pollution problem, contrib- lus terreus [13, 21], Aspergillus fumigatus [15], Emeri- uting to the eutrophication of surface waters in areas of cella nidulans [14], Myceliophthora thermophila [13], intensive livestock production [16, 20]. In addition, Thermomyces lanuginosus [2], and Talaromyces ther- phytic acid also acts as an anti-nutritional agent by form- mophilus [14]. However, only a few reports are available ing complexes with proteins and various metal ions, on bacterial phytases, including Bacillus sp. [9, 11], thereby decreasing the dietary bioavailability of these Escherichia coli [5, 6], and Klebsiella terrigena [7]. nutrients [20]. In the present study, we describe the purification and Phytase (myo-inositol hexakisphosphate phosphohy- biochemical properties of a phytase from Pseudomonas drolase; EC 3.1.3.8) hydrolyzes phytic acid to myo- syringae MOK1. Inorganic phosphate release by this inositol and phosphoric acid. The supplementation of enzyme from major ingredients of animal feedstuff, such animal feedstuff with phytase increases the utilization of as corn meal, soybean meal, and wheat bran was also phosphate and diminishes the anti-nutritional effects of analyzed in vitro. high-phytate diets, ultimately improving the nutritional quality of the diets. Recently, phytases have been of Materials and Methods Organism. A bacterial strain, Pseudomonas syringae MOK1, showing Correspondence to: Y.J. Choi; email: [email protected] a high phytase activity in phytase differential staining assay [1], was J.S. Cho et al.: Phytase from Pseudomonas syringae MOK1 291 Table 1. The purification of MOK1 phytase Total Total Sp. activity Yield Purification Step protein (mg) activity (U)a (U/mg) (%) (fold) Crude enzyme 11.2 72 6.4 100 1 UNO-S6 0.096 52.6 548 73 85.2 Bio-scale Q2 0.049 31.8 649 44.2 101 aOne unit of phytase activity was defined as the amount of enzyme required to liberate 1 ␮mol of orthophosphate from sodium phytate per min at 40°C and pH 5.5. previously isolated from the soil of a Korean cattle farm (data not mM) was pre-incubated with the phytase for 15 min at 40°C before the shown). standard assay was performed, and the residual activity was measured. The kinetic parameters (Km and Vmax) were determined from the Enzyme purification. The strain was inoculated on 1-L of phytase- Lineweaver-Burk plot with different concentrations of sodium phytate specific medium [0.5% (NH ) SO , 0.5% KCl, 0.01% MgSO ⅐ 7H O, 4 2 4 4 2 (0.05–1mM). All experimental data were means of triplicate determi- 0.01% NaCl, 0.01% CaCl ⅐ 2H O, 0.001% FeSO , 0.001% MnSO ; 2 2 4 4 nations. pH 6.5] with 0.5% sodium phytate (phytic acid dodecasodium salt) (Sigma) as a sole source of carbon and phosphorus, and was cultivated General protein techniques. Sodium dodecyl sulfate polyacrylamide in a rotary shaker (200 rpm) at 30°C for 3 days. The cells were gel electrophoresis (SDS-PAGE) was performed as described by Lae- harvested by centrifugation at 10,000 g for 15 min at 4°C, resuspended mmli [12], using a 10% acrylamide gel. Proteins were stained with in 20 mL of buffer A [20 mM acetate buffer (pH 5.5)]. Ten g of glass Coomassie brilliant blue R-250. Protein concentration was measured by beads (425–600 microns; Sigma) was added and vortexed at maximum Bradford’s method [3], using a protein assay kit (Bio-Rad) with bovine speed for a total of 30 min with 2 min chilling on ice for every 2-min serum albumin as the standard. vortexing. Then, the homogenate was centrifuged at 10,000 g for 20 min at 4°C. After filtering the supernatant fluid with a 0.45-␮m filter N-terminal amino acid analysis. The purified enzyme was electro- unit (ADVANTEC), the crude extract was used for the enzyme puri- phoretically transferred to a sheet of polyvinylidene difluoride (PVDF) fication. Purification of MOK1 phytase was carried out through a membrane (Bio-Rad) from SDS-PAGE (10%). The phytase band was two-step process at 4°C by using a BioLogic HR LC System (Bio Rad). cut out, and its N-terminal amino acid sequence was analyzed by the In the first step, the crude extract was loaded onto a cation-exchange Edman degradation method with Procise 491 automatic protein se- UNO S6 column (6-mL bed volume; Bio-Rad) that had previously been quencer (Applied Biosystems Inc., Foster City, CA). equilibrated with buffer A. The column was washed with the same Phosphate liberation from feed substrates. Finely ground grains of buffer, and bound proteins were eluted in a linear salt gradient of 0–1 corn meal, soybean meal, and wheat bran were autoclaved for 20 min M NaCl in buffer A at a flow rate of 2.0 mL/min. The fractions showing at 120°C in order to inactivate endogenous phytases, and 5 g each of the phytase activity were pooled and diluted tenfold with buffer B [25 these autoclaved feed substrates was resuspended in 40 mL of 200 mM mM Tris buffer (pH 8.5)]. In the second step, these diluted active acetate buffer (pH 5.5). Each suspension was incubated with 250 U of fractions were applied to an anion-exchange Bio-scale Q2 column MOK1 phytase per kg of feed for 300 min at 37°C in a shaking water (2-mL bed volume; Bio-Rad) that had previously been equilibrated bath. From the incubation mixtures, 1-mL aliquots were removed with buffer B, and the phytase was eluted with a linear salt gradient of periodically and centrifuged at 10,000 g for 2 min at 4°C in a micro- 0–1 M NaCl in buffer B at a flow rate of 2.0 mL/min. The active centrifuge. The supernatant was collected, and the liberated phosphates fractions were pooled, dialyzed in 10 mM Tris buffer (pH 7.5), and were quantified as described above. stored at Ϫ20°C. Enzyme assays and characterization. Phytase assay was carried out in 1-mL volume at 40°C for 30 min in 50 mM acetate buffer (pH 5.5) Results and Discussion containing 1 mM sodium phytate. The liberated inorganic orthophos- Purification and properties of phytase. The purifica- phates were quantitated spectrophotometrically through a modified method of Heinonen and Lahti [8], with a freshly prepared acetone tion of MOK1 phytase is summarized in Table 1. The ammonium molybdate (AAM) reagent consisting of acetone, 5 N enzyme was purified 101-fold with 44.2% yield, starting sulfuric acid, and 10 mM ammonium molybdate (2:1:1, vol/vol). Two from the crude extract. The specific activity of the puri- ml of AAM solution was added per assay tube to terminate the phytase fied enzyme was 649 U/mg of protein at 40°C and pH assay. After 30 s, 0.2 mL of 1 M citric acid was added to each tube. 5.5. This value was much higher than 196 and 20 U/mg Absorbance was read at 405 nm after blanking the spectrophotometer with an appropriate control. One unit of phytase activity was defined as of Aspergillus sp. [19] and Bacillus sp. DS11 [11] phy- the amount of enzyme required to liberate 1 ␮mol of phosphate per min tases, respectively. The purified enzyme gave a single under the assay conditions. The assay for enzyme activity with other protein band on a sodium dodecyl sulfate (SDS)-poly- phosphorylated compounds was done as described above by using 1 acrylamide gel electrophoresis, and its molecular weight mM of each substrate.