CURRENT MICROBIOLOGY Vol. 47 (2003), pp. 290Ð294 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 7H O, 4 2 4 4 2 (0.05Ð1mM). All experimental data were means of triplicate determi- 0.01% NaCl, 0.01% CaCl 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 (425Ð600 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 0Ð1 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- 0Ð1 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. Acid phosphatase activity was assayed by using was approximately 45 kDa (Fig. 1). The well-known p-nitrophenylphosphate as described by Greiner et al. [6]. The release of p-nitrophenol was measured at the absorbance of 410 nm. fungal phytases had larger molecular weights, for exam- The effects of metal ions and inhibitors on the enzyme activity ple, 214 kDa (a homohexamer) for A. terreus [21] and 85 were examined with sodium phytate as a substrate. Each additive (5 kDa for A. ficuum [18]. On the other hand, Bacillus sp. 292 CURRENT MICROBIOLOGY Vol. 47 (2003)

Fig. 2. The pH activity profile of MOK1 phytase. The activity was measured at 40¡C and expressed as a percentage of the maximum activity taken as 100%. Buffers used (final concentration, 50 mM) were glycine-HCl (pH 2.5Ð3.5); sodium acetate (pH 4.5Ð5.5); bis-Tris (pH 6.5); and TrisÐHCl (pH 7.5Ð8.5).

Fig. 1. SDS-polyacrylamide gel analysis of MOK1 phytase. Electro- phoresis was performed on a 10% polyacrylamide slab gel in 25 mM Tris-HClÐ0.192 M glycine containing 0.1% SDS. The proteins were stained with Coomassie brilliant blue R-250. Lane M, molecular weight standards including myosin (200 kDa), ␤-galactosidase (116.25 kDa), phosphorylase b (97.4 kDa), serum albumin (66.2 kDa), ovalbumin (45 kDa), carbonic anhydrase (31 kDa), trypsin inhibitor (21.5 kDa), ly- sozyme (14.4 kDa), and aprotinin (6.5 kDa). Lane 1, the purified MOK1 phytase; Lane 2, crude protein.

Table 2. N-terminal sequence of MOK1 phytase and comparison with sequences of other bacterial phytases

Origin N-terminal amino acid sequences Fig. 3. Effect of temperature on MOK1 phytase activity. The enzyme MOK1 phytase Ala-Ser-Ala-Asp-Gly-Tyr-Val-Leu-Asp- was incubated in 50 mM acetate buffer (pH 5.5) containing 1 mM Lys-Val-Val-Gln sodium phytate at various temperatures for 30 min, and the released Bacillus sp. DS11 phytase Ser-Asp-Pro-Tyr-His-Phe-Thr-Val-Asn- orthophosphate was measured. The value obtained at 40¡C was taken Ala-Ala-Ala-Glu-Thr as 100%. E. coli phytase Ser-Glu-Pro-Glu-Leu-Lys-Leu-Glu-Ala- Val-Val 6.5Ð7.0. The optimum temperature of MOK1 phytase was 40¡C, and no activity was observed above 70¡C (Fig. 3). The reported optimum temperatures of A. niger [9, 11] and E. coli [6] phytases had molecular weights of [16] and Bacillus sp. DS11 [11] phytases are 58¡C and 43Ð44 and 42 kDa (a monomer), respectively, similar to 70¡C, respectively. that of MOK1 phytase. However, since the N-terminal Substrate specificity. The hydrolysis activity of MOK1 amino acid sequence of this enzyme is quite different phytase on several phosphate conjugates was tested in 50 from other bacterial phytases (Table 2), the enzyme is mM acetate buffer (pH 5.5). The substrates included in considered to be a novel phytase. the test were as follows: sodium phytate, p-nitrophe- Effects of temperature and pH on phytase activity. nylphosphate, D-glucose-1-phosphate, fructose-6-phos- The purified MOK1 phytase showed a weakly acidic pH phate, glycerophosphate, sodium pyrophosphate, optimum of 5.5, similar to that of A. niger phytase [16], ␣-naphthylphosphate, and ATP. The enzyme was very and was virtually inactive above pH 7.0 (Fig. 2), whereas specific for phytic acid. It showed a maximum of about Bacillus sp. phytase [10, 11] had neutral optimum pH of 5% of relative activity on the other phosphate conjugates J.S. Cho et al.: Phytase from Pseudomonas syringae MOK1 293

acid salt form. Our preliminary results clearly indicate that MOK1 phytase may improve the nutritional quality of some grains such as wheat bran and soybean meal.

ACKNOWLEDGMENTS This research was funded by the TS Corporation, Republic of Korea. We are thankful for the graduate fellowship provided by the Ministry of Education through the Brain Korea 21 project.

Literature Cited 1. Bae HD, Yanke LJ, Cheng KJ, Selinger LB (1999) A novel staining method for detecting phytase activity. J Microbiol Meth- ods 39:17Ð22 Fig. 4. In vitro phosphate liberation from feedstuffs by MOK1 phytase. 2. Berka RM, Rey MW, Brown KM, Byun T, Klotz AV (1998) ● f Œ Symbols: , corn meal; , soybean meal; , wheat bran. Molecular characterization and expression of a phytase gene from the thermophilic fungus Thermomyces lanuginosus. Appl Environ Microbiol 64:4423Ð4427 compared with the phytic acid value (100%) (data not 3. Bradford MM (1976) A rapid and sensitive method of the quanti- shown). On the other hand, the substrate specificity of tation of microgram quantities of protein utilizing the principle of fungal phytases is broad, including p-nitrophenylphos- protein dye binding. Anal Biochem 72:248Ð254 phate (the general substrate of acid phosphatase), fruc- 4. Gibson DM (1987) Production of extracellular phytase from As- pergillus ficuum on starch media. Biotechnol Lett 9:305Ð310 tose 1,6-bisphosphate, and glucose-6-phosphate [19]. 5. Golovan S, Wang G, Zhang J, Forsberg CW (2000) Characteriza- The kinetic parameters on phytic acid were deter- tion and overproduction of the Escherichia coli appA encoded mined at pH 5.5¡C and 40¡C. The Michaelis constant bifunctional enzyme that exhibits both phytase and acid phospha- tase activities. Can J Microbiol 46:59Ð71 (Km) and the maximum reaction velocity (Vmax)of MOK1 phytase on phytic acid calculated from the Lin- 6. Greiner R, Konietzny U, Jany KD (1993) Purification and charac- terization of two phytases from Escherichia coli. Arch Biochem eweaver-Burk plot were 0.38 mM of sodium phytate and Biophys 303:107Ð113 769 U/mg of protein, respectively. This Km value is much 7. Greiner R, Haller E, Konietzny U, Jany KD (1997) Purification and higher than that of A. ficuum phytase (27 ␮M) [20], but characterization of a phytase from Klebsiella terrigena. Arch Bio- lower than that of Bacillus sp. DS11 phytase (0.55 mM) chem Biophys 341:201Ð206 8. Heinonen JK, Lahti RJ (1980) A new and convenient colori- [11] and E. coli phytase (0.78 mM) [5]. metric determination of inorganic orthophosphate and its appli- Effects of reagents and metal ions. The effects of cation to the assay of inorganic pyrophosphate. Anal Biochem inhibitors and metal ions on the enzyme activity were 72:248Ð254 9. Kerovuo J, Lauraeus M, Nurminen P, Kalkkinen N, Apajalahti J examined with sodium phytate as a substrate. The en- (1998) Isolation, characterization, molecular gene cloning, and zyme activity was greatly diminished by 70% with the sequencing of a novel phytase from Bacillus subtilis. Appl Environ 2ϩ 2ϩ 2ϩ addition of 5 mM each of Cu ,Cd ,Mn , or EDTA. Microbiol 64:2079Ð2085 These metal ions were found to form poorly soluble 10. Kerovuo J, Lappalainen I, Reinikainen T (2000) The metal depen- complexes with phytic acid and may, therefore, decrease dence of Bacillus subtilis phytase. Biochem Biophys Res Commun 268:365Ð369 the active concentration of phytic acid in an activity 11. Kim YO, Kim HK, Bae KS, Yu JH, Oh TK (1998) Purification and assay [19]. Similar to Bacillus sp. phytases [10, 11], properties of a thermostable phytase from Bacillus sp. DS11. chelating agents such as EDTA readily inactivated Enzyme Microb Technol 22:2Ð7 MOK1 phytase. Most of the fungal phytase activities 12. Laemmli UK (1970) Cleavage of structural protein during the were unaffected by EDTA, except A. fumigatus phytase, assembly of the head of bacteriophage T4. Nature 227:680Ð 685 13. Mitchell DB, Vogel K, Weimann BJ, Passamontes L, van Loon whose activity was increased up to 50% by the addition APGM (1997) The phytase subfamily of histidine acid phospha- of1mM EDTA [19]. tase: isolation of genes for two novel phytases from the fungi Aspergillus terreus and Myceliophthora thermophila. Microbiol In vitro phosphate liberation from feed samples. As 143:245Ð252 shown in Fig. 4, the net amounts of phosphate released 14. Pasamontes L, Haiker M, Henriquezhuecas M, Mitchel DB, van from corn meal, soybean meal, and wheat bran at pH 5.5 Loon APGM (1997a) Cloning of the phytase from Emericella for 300 min were 1.72, 3.27, and 4.02 mmol/g of grain, nidulans and the thermophilc fungus Talaromyces thermophilus. respectively. The ability of MOK1 phytase to hydrolyze Biochim Biophys Acta 1353:217Ð223 15. Pasamontes L, Haiker M, Wyss M, Tessier M, van Loon APGM not only chemically pure soluble sodium phytate, but (1997b.) Gene cloning, purification, and characterization of a heat- also natural phytate in feeds, is practically important, stable phytase from the fungus Aspergillus fumigatus. Appl Envi- because phytate in plants exists as an insoluble phytic ron Microbiol 63:1696Ð1700 294 CURRENT MICROBIOLOGY Vol. 47 (2003)

16. Reddy NR, Sathe SK, Salunkhe DK (1982) Phytates in legumes acterization of fungal phytases (myo-inositol hexakisphosphate and cereals. Adv Food Res 28:1Ð92 phosphohydrolases): catalytic properties. Appl Environ Microbiol 17. Shieh TR, Ware JH (1968) Survey of microorganisms for the 65:367Ð373 production of extracellular phytase. Appl Microbiol 16:1348Ð1351 20. Wodzinski RJ, Ullah AHJ (1996) Phytase. Adv Appl Microbiol 18. Ullah AHJ, Gibson DM (1987) Extracellular phytase (E. C. 3. 1. 3. 42:263Ð302 8) from Aspergillus ficuum NRRL 3135: purification and charac- 21. Yamada K, Minoda Y, Yamamoto S (1968). Phytase from terization. Prep Biochem 17:63Ð91 Aspergillus terreus. Part I. Production, purification, and some 19. Wiss M, Brugger R, Kronenberger A, Remy R, Fimbel R, Oster- general properties of the enzyme. Agric Biol Chem 32:1275Ð helt G, Lehmann M, van Loon APGM (1999) Biochemical char- 1283