Journal of Bioscience and Bioengineering

VOL. 113 No. 5, 562 – 567, 2012

www.elsevier.com/locate/jbiosc

Purification and characterization of phytase from Klebsiella pneumoniae 9-3B

Lotis Escobin-Mopera, 1, 2 Midori Ohtani, 1 Sachie Sekiguchi, 1 Teruo Sone, 1,⁎ Ayumi Abe, 1 Michiko Tanaka, 1
Vithaya Meevootisom, 3 and Kozo Asano 1

Laboratory of Applied Microbiology, Graduate Faculty of Agriculture, Hokkaido University, N9 W9, Kita-ku, Sapporo, Hokkaido 060-8589, Japan, 1 Institute
of Food Science and Technology, College of Agriculture, University of the Philippines Los Baños, College 4031, Los Baños, Laguna, Philippines, 2 and
Department of Microbiology, Faculty of Science, Mahidol University, Rama VI Rd., Bangkok 10400, Thailand 3

Received 24 October 2011; accepted 15 December 2011

Available online 13 January 2012

Phytase, an enzyme that catalyzes the hydrolysis of phytate, was purified from Klebsiella pneumoniae 9-3B. The isolate was
preferentially selected in a medium which contains phytate as a sole carbon and phosphate source. Phytic acid was utilized for
growth and consequently stimulated phytase production. Phytase production was detected throughout growth and the
highest phytase production was observed at the onset of stationary phase. The purification scheme including ion exchange
chromatography and gel filtration resulted in a 240 and 2077 fold purification of the enzyme with 2% and 15% recovery of the
total activity for liberation of inorganic phosphate and inositol, respectively. The purified phytase was a monomeric protein
with an estimated molecular weight of 45 kDa based on size exclusion chromatography and SDS-PAGE analyses. The phytase
has an optimum pH of 4.0 and optimum temperature of 50°C. The phytase activity was slightly stimulated by Ca2 + and EDTA
and inhibited by Zn2 + and Fe2 +. The phytase exhibited broad substrate specificity and the Km value for phytate was 0.04 mM.
The enzyme completely hydrolyzed myo-inositol hexakisphosphate (phytate) to myo-inositol and inorganic phosphate. The
properties of the enzyme prove that it is a good candidate for the hydrolysis of phytate for industrial applications.
© 2011, The Society for Biotechnology, Japan. All rights reserved.

[Key words: Phytase; Klebsiella pneumoniae; Phytate degradation; myo-Inositol; Complete hydrolysis]

Phytic acid is the major storage form of phosphorus and inositol in
plant seeds. It comprises about 3–5% of the dry weight particularly in
cereal grains and legumes (1). Phytic acid can chelate divalent cations
like calcium, magnesium, iron and zinc, decreasing their bioavailability
(2). These minerals are essential in the diet of both humans and animals.
Substances like phytic acid are commonly referred to as anti-nutritional
factor since they reduce food intake and nutrient utilization in animals
and humans.
Phytases are enzymes capable of catalyzing the sequential release of
the inorganic phosphorus from phytic acid. These enzymes have attracted
a lot of attention from both scientists and entrepreneurs in the areas of
nutrition, environmental protection and biotechnology (3). Originally
intended as animal feed additive, phytases enhance the nutritional quality
of plant material in feed by increasing the bioavailability of minerals for
monogastric animals(4). Phytase fortification improves the mineral
deficiency associated with the insolubility of phytic acid in animal feeds.
Moreover, animal feeds devoid of phytase causes immense release of
undigested phytates into the environment. The phosphorus excreted into
the environment enhances the growth of phosphorus assimilating
microorganisms resulting in eutrophication or algal bloom.
In the area of human nutrition, phytase is being used as a functional
food ingredient. Further applications are being explored on the
⁎ Corresponding author. Tel.: +81 11 706 2502; fax: +81 11 706 4961.
E-mail address: sonet@chem.agr.hokudai.ac.jp (T. Sone).
1389-1723/$ - see front matter © 2011, The Society for Biotechnology, Japan. All rights reserved.
doi:10.1016/j.jbiosc.2011.12.010
utilization of phytase in food processing (5). The potential applications
of phytases mentioned above emphasize the demand for the enzyme in
the field of agriculture and biotechnology. The annual sales value of
phytase was estimated at USD 500 million (6) and about 70% of the
global monogastric feed contains phytase (International Phytase
Summit 2010, http://www.ips2010.com). These enzymes are found in
plants, animals, and microorganisms. However, researches have shown
that microbial phytases are most promising for biotechnological
applications (7). Commercially produced phytases from Aspergillus
spp. and E. coli have been sold in the market for over a decade. Studies
have shown, however, that bacterial phytases offer several advantages
over fungal phytases because of higher substrate specificity, greater
resistance to proteolysis and better catalytic capability (3).
Recently, phosphatidylinositol received considerable interest due
to their applications in animal and human nutrition (3). These
compounds are derived only by enzymatic hydrolysis since it is
difficult to produce chemically. Thus, phytases are in demand in terms
of producing these compounds for various applications. Currently,
most acid phytases produced inositol-2-monophosphate as the final
product of hydrolysis. This indicates that there is a preferential
hydrolysis for phosphate group located at the equatorial position.
Often, the axial position is not acted upon by most phytases. On the
other hand, the final product of alkaline phytases is inositol-
triphosphate. Although these phytases can be used for some food
and environmental applications there is a need to find more phytases
that has the ability to efficiently hydrolyze the substrate for the above
VOL. 113, 2012
mentioned applications. No single phytase may be able to meet the
diverse need for all commercial and environmental applications (3).
Therefore, there is an ongoing interest in microorganisms including
bacteria for novel and efficient phytases.
To date, complete hydrolysis of phytic acid is accomplished by
employing enzyme systems of commercially available recombinant
phytase from Aspergillus and E. coli (8). A combined use of phytase
and phosphatase as feed supplement is known to work synergistically
for the hydrolysis of the phytate into phosphoric acid and myo-
inositol (9). Recent studies reported only two yeasts species capable
of complete hydrolysis of phytic acid namely: Debaryomyces castellii
CBS 2923 (10) and Schwanniomyces castellii CBS 2863 (11). In this
study, a new phytate degrading microorganism was reported.
Notably, to our knowledge, Klebsiella pneumoniae 9-3B phytase, is
the first bacterial phytase reported with the capability to completely
hydrolyze phytate. Myo-inositol was formed and detected in the
reaction mixture after enzyme treatment. This enzyme which also
exhibited high activity was purified and characterized.
MATERIALS AND METHODS
Screening and isolation of phytase producing strains Soil samples were
collected from a spinach field in Takaoka, Toyama, and a lawn in Sapporo, Hokkaido,
Japan. Enrichment culture containing phytic acid as sole carbon and phosphate source,
were prepared by inoculation of 1 g soil sample into 10 ml MM9 Medium. The medium
was prepared by mixing the following: 37.5 ml M9 Basal medium (30.6 g KCl, 20 g
NH4Cl, 100 g NaCl in 1 l distilled water); 7.5 ml Mg and Ca solution (12 g MgSO4, 1.1 g
CaCl2 and 1 l distilled water); 85.8 ml phytic acid solution [48 ml 50% Phytic acid
solution (Wako Pure Chemicals, Osaka, Japan), 40 ml 10 N NaOH and 8 ml distilled
water] and 869.2 ml distilled water. Each solution with the exception of phytic acid
solution was sterilized separately at 121°C for 15 min. Phytic acid solution was filter
sterilized (0.22 μm filter). After sterilization, the solutions were mixed as indicated. In
the case of solid medium, 1.5% agar was added to distilled water before sterilization.
Phytate degradation was further tested by measuring the zone of clearing produced in
phytase screening medium (PSM) (12). Isolates with apparent phytase activity were
selected and inoculated into MM9. The cultures were then incubated at 37°C and
agitated at 150 rpm in a reciprocal bioshaker. After 24–48 h incubation, the cells were
separated from the supernatant by centrifugation at 12,000 × g for 30 min.
Cell growth and cultivation Single colony of K. pneumoniae 9-3B was taken from
24 h incubated MM9 agar plates. The colony was transferred to 5 ml MM9 and was allowed
to grow for 24 h at 37°C with agitation at 140 rpm on a reciprocal shaker until a cell density
of OD600 =1.0 was attained. This culture was transferred to 30 ml MM9 following the same
culture condition. Thereafter, 1% (30 ml) inoculum was transferred to 5 l jar fermentor
containing 3 l of MM9. The initial pH was controlled at 6.4, then, the culture was incubated
at 37°C, 400 rpm agitation and 1.0 vvm aeration for 24 h. Optical density and pH was
monitored every 2 h post-inoculation.
Preparation of the cell extract Cells were harvested by centrifugation at
12,000 × g for 30 min. The cells were washed 3 times with 0.05 M sodium acetate buffer,
pH 5.5. Wet weight of the cells was measured after centrifugation and 200 ml sodium
acetate buffer was added. Cells (about 25 g/tube) were disrupted with a Multi beads
shocker (Yasui Kikai, Osaka, Japan) to release the enzyme. Glass beads (0.1 mm) were
added at a rate twice as much as the cell wet weight. Beads shocker condition was set at
2500 rpm for 10 cycles with intermittent stop and start for 30 s. Then, cell extract was
separated from the cell debris by centrifugation at 18,000 × g for 5 min. The cell extract
was subjected to 0–40% ammonium sulfate precipitation. Further separation was done
by centrifugation at 9000 × g for 45 min. The supernatant was collected and was again
subjected to ammonium sulfate precipitation at 60–80%. The pellet was collected by
centrifugation at 9000 × g for 45 min and the supernatant was discarded. The pellet was
suspended in 0.05 M sodium acetate buffer and filtered using a 0.45 μm filter. The crude
enzyme solution was passed through desalting column, 10DG column (Bio-Rad
Laboratories, USA). The filtrate was collected and frozen at − 80°C then lyophilized.
Purification of phytase All purification steps were conducted at 4°C using
ÄKTA Explorer system (GE Healthcare Lifesciences AB, Uppsala, Sweden). Freeze-dried
crude enzyme was dissolved in 50 mM sodium acetate buffer (pH 5.5) and was
separated by cation exchange chromatography by passing through 20 ml HiPrep SP FF
column (GE Healthcare Lifesciences AB). The fractions were eluted by a linear gradient
of 1 N NaCl in 50 mM sodium acetate buffer, pH 5.5. The fractions were collected,
desalted with 10DG column (Bio-Rad Laboratories) in 50 mM Tris–HCl, pH 8.0 and
further purified by passing through a 20 ml Hi-Trap Q XL (GE Healthcare Life sciences
AB) anionic column. The desired fractions were eluted through a linear gradient of 1 N
NaCl in 50 mM Tris–HCl, pH 8.0. Finally, active fractions were collected, desalted with
10DG column in 50 mM sodium acetate buffer with 150 mM NaCl, pH 6.4 and passed
through Superdex 75 Prep grade (GE Healthcare Life Sciences AB) XK 16 column in
50 mM sodium acetate buffer with 150 mM NaCl, pH 6.4. All separation steps were
conducted at a flow rate of 4 ml min− 1 at 4°C.
PHYTASE FROM K. PNEUMONIAE 9-3B 563
Measurement of phytase activity Samples collected from each purification
step were analyzed for phytase activity. Samples were diluted accordingly before the
analysis. Diluted 0.5 ml samples and 25 mM sodium phytate in 0.2 M acetate buffer, pH
5.5 were separately incubated at 37°C for 10 min. Then, 0.5 ml of substrate was added
to the sample and the mixture was incubated for another 10 min. Thereafter, 2 ml of
10 mM NH4Mo7O24·4H2O:5 N H2SO4:Acetone (1:1:2) was added. Reaction was
allowed to proceed for 30 s. The reaction was halted by adding 0.1 ml of 1 M citric
acid. The color reaction of the Pi (inorganic phosphate)–Mo complex was read at A380. A
reference standard (KH2PO4, 0.1–0.4 μM) was simultaneously assayed with the
samples. A unit (U) of phytase activity was defined as the amount of enzyme required
to release 1 μM of Pi per minute at 37°C.
Inositol liberation assay Samples (150 μl) were mixed with 25 mM sodium
phytate in 0.2 M acetate buffer (350 μl) make up to 1 ml with the same buffer. The
mixture was incubated for 24 h at 37°C. The reaction was stopped by boiling for 10 min.
The solution was immediately cooled in an ice bath, then, 375 μl 2 N HCl was added to
the reaction mixture. The hydrolysis products were detected by HPLC (JASCO System)
using an RI detector. The samples were eluted on a Shodex RSPak KC-811 column with
an RSPak KC-LG guard column (Showa Denko, Japan) using 3 mM HClO4 in 5% CH3CN as
mobile phase. Column oven was set at 60°C at a flow rate of 1.0 ml min− 1. Inositol
standard and chemically hydrolyzed sodium phytate samples were also analyzed as
external standard. For this assay, 1 U was defined as 1 μM of inositol liberated per
minute at 37°C.
Protein concentration and molecular weight estimation Protein concentra-
tion was determined by using Bradford's method (Bio-Rad Laboratories) with bovine
serum albumin as standard. The molecular weight of the purified phytase was
estimated by gel filtration on a Superdex 75 prep grade column and by SDS-PAGE. Gel
filtration was performed using ÄKTA system as mentioned above. The column was
calibrated by ovalbumin (44 kDa), carbonic anhydrase (29 kDa), ribonuclease A
(13.7 kDa) and aprotinin (6.5 kDa) and blue dextran (2000 Da) (all calibration proteins
were purchased from GE Healthcare, Buckinghamshire, UK). SDS-PAGE was performed
according to previously described procedure with 12% gel concentration (13).
Characterization of the purified phytase The temperature stability of phytase
was tested by subjecting the enzyme solution to different temperatures from 20 to 70°C
for 1 h. The remaining enzyme activity was analyzed according to standard procedure.
On the other hand, the effect of temperature on the phytase activity was determined at
20°C, 30°C, 37°C, 40°C, 50°C, 60°C and 70°C. Similarly, the optimum pH for the activity
was determined by mixing equal volumes of buffers at different pH values ranging from
2.0 to 10.0 at 37°C while the stability was examined at different pH values and
incubation for 6 h at 37°C. The residual phytase activity was determined after
incubation. The buffers used for the pH experiment were: 100 mM Glycine–HCl for
pH 2.0 and 3.0, 100 mM sodium acetate buffer for pH 4.0, 5.0 and 6.0, 100 mM Tris–HCl
for pH 7.0, 8.0 and 9.0, and 100 mM Glycine–NaOH for pH 10.0.
Equal volumes of metal ions or EDTA (to a final concentration of 1 mM and 5 mM)
were mixed with enzyme solution in 0.05 M sodium acetate buffer. The effect of metal
ions and EDTA were analyzed after incubation by proceeding with the usual phytase
assay stated above.
The substrate specificity of the purified enzyme was evaluated by following
standard assay procedure but the substrate was replaced with different phosphorylated
compounds at increasing concentrations.
RESULTS
Screening and isolation A screening method was developed to
isolate phytate-degrading enzyme producer from soil. Both solid and
liquid evaluation systems were employed to determine the ability of
the isolated strains to produce phytase. However, in contrast to
previously reported liquid medium (14,15), phytate was used as the
sole carbon and phosphorus source in this study. Initial enrichment
screening in liquid medium for phytate degrading microorganisms
yielded 81 strains. After three screening steps, the authors obtained
only 4 strains that exhibited high phytase activity. Consequently,
strain 9-3B was chosen because it exhibited the highest intracellular
phytase activity.
Stimulation of phytase production and utilization of phytic acid as
sole carbon and phosphate source was demonstrated by inoculating
isolate 9-3B into MM9 or MMPI medium. MMPI is modified MM9
medium which lacks phytic acid but contains myo-inositol, and
phosphate at different concentrations (Figs. 1A and B). The growth of
isolate 9-3B was inhibited in MMPI medium without additional
phosphate. Growth gradually resumed as the phosphate concentration
in the medium increase. In the MMPI medium with 64 mM or more
phosphate, growth was comparable to that in MM9 medium. Cells were
harvested from these culture and cell extracts were assayed for phytase
activity and inositol liberation. The extract of MM9-cultured cells
564 ESCOBIN-MOPERA ET AL.
A
10.00
Growth (OD660)
1.00
0.10
0.01
0 5 10 15
Culture Time (h)
B Cuture time (h)
Phytase specific activity (U/mg)
2.0
1.5
1.0
0.5
0.0
0 mM 12.8 mM 64 mM 128 mM 256 mM MM9
Additional Phosphate in MMPI
FIG. 1. The effect of additional phosphate in the MMPI medium on the growth and
phytase activity of K. pneumoniae 9-3B. (A) Growth of K. pneumoniae 9-3B in MMPI
liquid medium, a modified MM9 which lacks phytate and contain myo-inositol as
carbon source. Growth in MMPI with additional phosphate of 256 mM (open squares),
128 mM(open diamonds), 64 mM (closed triangles), 12.8 mM (closed squares), and no
addition (closed diamonds) are shown. Growth in MM9 is represented by closed circles.
(B) Phytase activity and inositol liberation activity in cell-free extracts. Open bars,
phytase activity; solid bars, liberated inositol.
showed detectable phytase activity and inositol in the reaction mixture
after 24 h (Figs. 1A and B). However, the cells harvested from MMPI
media did not yield cell extracts with phytase activity. These results
confirmed that isolate 9-3B produced phytate degrading enzyme which
liberated myo-inositol as final product, and was able to utilize
phosphate and myo-inositol for its growth. Phytase production was
stimulated by phytate in the medium. Phylogenetic studies using the
16S rDNA sequencing revealed that isolate 9-3B was identified as K.
pneumoniae with a sequence homology of 99.7%. The sequence
(1421 bp) has been deposited to DDBJ/EMBL/GenBank database under
the accession number AB675600. Glycerol stocks of K. pneumoniae 9-3B
were prepared and revived in MM9 for succeeding experiments.
Phytase production Phytase was reported to be intracellularly
produced in some organisms (12,16–20). As stated above, K. pneumoniae
9-3B was likewise proven to elicit the same property. Therefore, large
scale culture of K. pneumoniae 9-3B was employed to acquire more cells
for the recovery of the enzyme. Batch culture of the strain was carried out
in a 5 l bioreactor with 3 l working volume. K. pneumoniae 9-3B produced
significant amount of phytase paralleled with cell growth when the
culture medium was augmented with phytate (Fig. 2). Phytase
production was immediately observed in growing cultures continuing
throughout the stationary phase. However, the amount of cells as well as
the phytase produced after 24 h was not significantly different from that
of a 48 h culture so the phytase production was decided to be halted at
24 h (Fig. 2). The medium was highly buffered because highly acidic or
alkaline pH affected growth. The cell yield was 26 g/l (wet weight basis)
and was twice the amount produced in a 1 l jar fermentor (about 15 g/l,
data not shown).
J. BIOSCI. BIOENG.,
25 4.5
4
Phytase specific activity (U/mg)
20 3.5
Growth (OD660)
3
15
2.5
2
10
1.5
5 1
20 25 0.5
0 0
0 4 8 12 16 20 24 28 32 36 40 44 48
5
FIG. 2. Phytase specific activity during the growth of K. pneumoniae 9-3B in a medium
4 Liberated inositol
with phytate as the sole carbon and phosphate source at 37°C for 48 h. Symbols: circles,
(mM/mg protein) phytase specific activity; squares, cell growth (OD600).
3
Purification of phytase from K. pneumonia 9-3B Cells
2
harvested in the stationary phase were used as enzyme source.
Phytase were extracted from the cell by disrupting the cell membrane
1
followed by ammonium sulfate precipitation. The freeze dried crude
protein (2.5 g) was dissolved in 40 ml of 0.05 M sodium acetate
0
buffer, desalted and was passed through HiPrep SP FF column.
Desalted active fractions (20 ml) were then further purified by HiPrep
Q XL. Then, 10 ml of the desalted active fractions were subjected to gel
filtration chromatography and finally purified as a single band by
SDS-PAGE (Fig. 3A). Column chromatography purified the phytase to
a specific activity of 1360 U/mg (Table 1). Phytase activity from
K. pneumoniae 9-3B was purified to nearly 240 fold from cell extract
and recovery was nearly 2% (Table 1). The specific activity is the highest
reported among phytases produced by Klebsiella spp. (12,16,17,21).
Moreover, the purified enzyme was proven to exhibit complete
hydrolysis of phytate using the stipulated experimental conditions.
HPLC analysis of the assay mixture revealed the presence of inositol as
one of the products of hydrolysis (Table 1). There was a considerable
increase in the amount of inositol released from phytate as the enzyme
becomes homogenous. The specific inositol liberation activity was
recorded at 141.25 U mg− 1 protein. This activity was also able to
register a very high purification fold and yield which reached at least
2000 fold and 15%, respectively. In addition, the molar ratio of produced
phosphate to phytate in standard assay condition was estimated to be
6:1, indicating complete hydrolysis of phytate by this single phytase
(Fig. 4). Further, the enzyme was able to hydrolyzed myo-inositol-2-
monophosphate as a substrate exhibiting an equal released of inorganic
phosphorus per molecule of the substrate (Fig. 4).
The molecular mass of the purified enzyme exhibited by one single
band on SDS-PAGE was estimated to be 45 kDa (Fig. 3A). Similarly, the
apparent molecular weight was confirmed by gel permeation
chromatography (Fig. 3B). Consequently, the enzyme was deduced
to be a monomeric protein based on these results.
Effect of pH and temperature on enzymatic stability of
phytase The optimum pH of this enzyme was pH 4.0 (Fig. 5A).
The phytase did not show any decline in activity from pH 2.0 to 7.0 for
6 h at 37°C. However, 80% of the activity was lost at pH 8.0. The
temperature profiles of the purified enzyme were demonstrated from
20°C to 70°C using the standard assay procedure. The optimum
temperature was at 50°C. Thereafter, a remarkable 80% decrease in
activity was observed at 60°C and was completely lost at 70°C. On the
other hand, the enzyme activity remained stable for 1 h at tempera-
tures from 30°C to 50°C (Fig. 5B).
Effect of divalent metal ions and EDTA The effect of different
metal ions and EDTA on the phytase activity (Table 2) showed that
VOL. 113, 2012 PHYTASE FROM K. PNEUMONIAE 9-3B 565

35
A
30

Liberated Phosphate (mM)

25

20

15

10

B 2
5

0
0 1 2 3 4 5 6
45 kda,Phytase 9-3B Substrate (mM)
44 kda
1.5
Log MW

29 kda FIG. 4. Molar ratio of phytate (substrate) and liberated phosphate (product). Phytate
was solubilized in 0.2 M sodium acetate buffer, pH 5.5. Assay was conducted at 37°C to
measure the liberated phosphate using standard procedure. Symbols: closed circles,
13.7 kda phytate; open circles, myo-inositol-2-monophosphate.
1

6.5 kda
0.5
0.2 0.3 0.4 0.5 0.6 0.7
Kav
FIG. 3. Purification and molecular weight estimation of the phytase. (A) SDS-PAGE
protein profile of K. pneumoniae 9-3B phytase. Lane 1, molecular weight marker; 2, cell
extract; 3, ammonium sulfate precipitate; 4, HiPrep SPFF; 5, HiPrep QXL; and 6, Gel
filtration. (B) Estimation of the molecular weight by gel permeation chromatography
on a XK 16 Superdex 75 column. Symbols: open circles, native phytase; closed
diamonds, molecular weight standard, 44 kDa (ovalbumin), 29 kDa (carbonic anhy-
drase), 13.7 kDa (ribonuclease A) and 6.5 kDa (aprotinin).
Ca2 + at 1 mM concentration did not alter the activity of the phytase.
However, increasing the concentration to 5 mM affected the activity
to decrease at least 20%. Mg2 +, Mn2 + and Co2 + moderately inhibited
phytase activity at 14 to 20% and 33% to 45% at 1 mM and 5 mM,
respectively. Interestingly, Co2 + ions reduced the enzyme activity by
as much as 23% at 1 mM and about 45% at 5 mM. On the other hand,
Zn2 + and Fe2 + affected a marked decline in the enzyme activity. All
metal ions inhibited the activity at 5 mM concentration. EDTA, a
chelating agent, did not elicit any inhibitory effect on the enzyme
activity.
Substrate specificity and enzyme kinetics The relative rate of
hydrolysis of different phosphorylated compounds demonstrated that
purified phytase exhibited broad substrate specificity (Table 3). The
enzyme hydrolyzed phytate at a very high rate. This property
confirmed that this enzyme is recognized as a phytase. Moreover,
ρ-nitrophenyl phosphate (PNPP), a known substrate for testing
phosphatase activity, registered only 27.66%. On the other hand, the
other phosphorylated compounds exhibited very low rate of hydrolysis
ranging from 0.8% to 27% with the exception of NADP which registered
55%. The lowest turnover rate was observed for glucose-6-phosphate at
0.84% at pH 5.5 in 0.2 M sodium acetate buffer. The Km value for phytate
was 0.04 mM as derived from the Lineweaver–Burk plots.
DISCUSSION
Phytase is a special type of phosphatase capable of releasing the
phosphate moieties from phytate (17). Previous screening methods
for the isolation of phytate degrading microorganisms employed
sodium phytate as the sole phosphorus source (14,15). In this study,
however, phytate served as sole carbon and phosphorus source in the
screening medium. This screening yielded a unique microorganism K.
pneumoniae 9-3B, which is capable of utilizing phytate as a sole carbon
and phosphate source. In the absence of a readily utilizable energy
source, phytase was induced by phytate and both phosphate and
inositol was used by the organism for growth (Fig. 1). This indicates
that inositol was released after phytate degradation and served as
carbon source for the growth of the bacteria. Klebsiella terrigena, on
the other hand, produced significant amount of phytase in the
presence of phytate but did not utilize phytate as sole carbon source.
In this case, phytase is not necessary for the energy but rather a
response to phosphorus limitation (17).
The purified phytase from this study exhibited many characteris-
tics similar to other bacterial phytases, particularly phytases from
Klebsiella spp. (12,17). On the other hand, the specific activity
reported herein is the highest so far for all the Klebsiella spp. reported
to exhibit phytase activity (12,16,17,22). The phytase produced by
K. pneumoniae 9-3B is the only bacterial phytase that can cleave the
phosphate moiety at the axial position yielding inositol as the final
product. This was exemplified by the exact molar ratio of 1:6 (phytate:
phosphate) detected during measurement of liberated phosphate after

TABLE 1. Purification of phytase and liberated inositol from Klebsiella pneumoniae 9-B.
Purification step Protein Phytase Inositol liberation
(mg)
Phytase Specific activity % yield Purification fold Liberated inositol Specific inositol activity % yield Purification fold
(U) a (U/mg) (U) b (U/mg)

Cell extract (CE) 106 596 5.64 100 1.00 7.17 0.068 100 1.00
(NH4)2SO4 precipitation (ASP) 79.4 547 6.89 91.8 1.22 6.83 0.086 95.2 1.26
HiPrepSPFF 0.470 502 1078 84.2 191 5.88 12.5 82.0 184
HiPrepQXL 0.120 153 1346 25.7 239 5.37 44.8 74.9 658
Superdex 75 0.008 10.8 1360 1.81 241 1.13 141 15.8 2077
a
1 U is defined as 1 μM of Pi liberated per min.
b
1 U is defined as 1 μM of inositol liberated per min.
566 ESCOBIN-MOPERA ET AL. J. BIOSCI. BIOENG.,

A 180
TABLE 3. Substrate specificity of phytase from K. pneumoniae 9-3B.
Substrate Relative activity (%) Km
160
Phytate 100.00 0.04
Relative activity (%)

140 ρ-Nitrophenyl phosphate 27.66 2.12

120 1-Naphtyl phosphate 11.39 1.18
Glucose-6-phosphate 0.84 4.56
100
2-Glycerolphosphate 5.04 140.00
80 NADP 55.68 2.62
ATP 4.45 0.86
60
ADP 8.16 0.47
40 AMP 3.49 0.41
20
0
2 3 4 5 6 7 8 9 10
pH intracellular phytase exhibited pH optima at pH 4.0 and a broad pH
stability from 2.0 to 7.0. This characteristic will allow the enzyme to
B elicit activity in the stomach (pH 2.0 to 4.5) as well as the small
120 intestine (pH 6.5 to 7.5). This is a notable characteristic for application
as animal feed additive. The stability at pH 2.0 is a known
100
characteristic among acid phosphatases in microorganisms especially
Relative activity (%)

for Aspergilli, but not in E. coli (23). The high activity of K. pneumoniae
80
9-3B phytase can be used more effectively at acidic pH similar to the
60
stomach of monogastric animals. The optimum temperature and
stability was well within the typical temperature for phytase activity
40 (10,11). The enzyme activity was the highest at 50°C and was stable
even after 1 h of exposure. On the other hand, there was about 15%
20 reduction in the enzyme activity at 60°C for only 10 min. The activity
at this temperature is relatively higher than that observed for Klebsiella
0
30 40 50 60 70
sp. no. PG-2 (22). A phytase from Enterobacter had an optimum
temperature of 50°C but it is only active at neutral pH (27).
Temperature ( )
The enzymatic activity of the K. pneumoniae 9-3B was not
FIG. 5. The effect of pH (A) and temperature (B) on the phytase activity of K.
significantly affected by Ca2 + ions. Rather Ca2 + ions were found to
pneumoniae 9-3B. Optimum pH and stability was determined at pH 2.0 and 3.0 slightly stimulate phytase activity at 1 mM concentration. This
(Glycine–HCl), pH 4.0, 5.0 and 6.0 (Sodium acetate), pH 7.0, 8.0 and 9.0 (Tris–HCl) and indicates that even in the presence of Ca2 + ions or calcium–phytic
pH 10.0 (Glycine–NaOH). Symbols: closed squares, stability; open squares, optima. acid complexes, the enzyme still retains much of its activity. Often,
Ca2 + ions forms complex with phytate that results in precipitation
treatment with the purified enzyme and detection of inositol by HPLC thus decreasing enzyme efficiency. However, 5 mM Ca 2 + ions
analysis. HPLC analysis also revealed the presence of myo-inositol decreased the relative activity. This is in contrast to that observed in
phosphate intermediates before the complete hydrolysis of phytate Bacillus wherein calcium ions are required for phytase activity (28).
(data not shown). To our knowledge, there are very few reports of In addition, it was strongly inhibited by Zn2 + and Fe2 + but not by
microbial phytases capable of complete hydrolysis of phytate (10,11). chelating agent like EDTA. Similar characteristics were observed in K.
Generally, bacterial phytases release either inositol monophosphate oxytoca (21). On the other hand, EDTA even slightly stimulated
(18,19,23) or inositol triphosphate (24). Most phytases cleaved the phytase activity at 1 and 5 mM concentration. This implies that a
phosphate group at the equatorial position of the phytic acid. The metal chelator like EDTA can be added with this phytase as feed
phosphate group at position 2 located at the axial position is never additive to increase efficacy. Like most divalent cations, Co2 +, Mg2 +
hydrolyzed by those enzymes (25). Further, this phytase also and Mn2 + ions reduced the enzyme activity. On the contrary, Co2 +
demonstrated broad substrate specificity. Only a few phytases have was reported to enhance the phytase activity in K. pneumoniae subsp.
been reported to display broad substrate specificity (12,23,26). Phytases pneumoniae XY-5 (12). Cobalt together with aluminum was postulated
with broad substrate specificity are better suited for animal nutrition to interact with the enzyme in a way that it change the conformation of
purposes than those with narrow substrate specificity. Broad substrate the enzyme to be more active. Generally, the inhibitory effect of the
specificity is often associated with low specific activity (3). However, the metal ions can be attributed to the strong chelating property of the
phytase produced by K. pneumoniae 9-3B still exhibited a very high substrate which results in a metal–phytate complex that effectively
specific activity compared with other phytases (8,12). reduces the availability of the phytate for the enzyme.
The biochemical properties of the 9-3B phytase are very much In conclusion, the purified phytase in this study exemplified the
comparable with other fungal and bacterial phytases as well. This characteristics of a novel bacterial phytase. In other phytases, the
enzyme is normally produced at the stationary phase as a response to
TABLE 2. Effects of metal cations and EDTA on phytase activity. nutrient and energy limitation (3). In this study, phytase was
Reagents Relative activity (%)
produced as a mechanism of survival for K. pneumoniae 9-3B in a
medium with phytate as sole carbon and phosphate source. The
1 mM 5 mM
assimilation of phytate by K. pneumoniae 9-3B might explain why the
Ca2 + 103.21 ± 0.16 78.90 ± 0.01 phytase of this bacterium has the ability to completely hydrolyze
Zn2 + 46.07 ± 0.04 37.68 ± 0.05
phytate. Hitherto, no bacterial phytase have demonstrated this
Mg2 + 79.49 ± 0.01 67.10 ± 0.04
Mn2 + 86.89 ± 0.01 59.17 ± 0.08
characteristic. Further, this phytase retained its activity in broad pH
Fe2 + 47.57 ± 0.06 29.45 ± 0.03 range at an optimum temperature relatively higher than most
Co2 + 78.77 ± 0.01 54.52 ± 0.03 phytases. The enzyme also elicited activity against various types of
EDTA 102.79 ± 0.03 101.85 ± 0.01 substrates and a very high specific activity that allowed the hydrolysis
None 100.00 ± 0.01 100.00 ± 0.01
of the substrate even at a very low protein concentration. The
VOL. 113, 2012
mechanism behind phosphate utilization and acquisition for growth in
bacteria incapable of producing extracellular phytase is still unknown
(18). Further elucidation of the characteristics and properties of this
phytase is needed to obtain detailed information about the enzyme and
take advantage of its potential for industrial use.
ACKNOWLEDGMENTS
This work was supported by JSPS Asian CORE Program.
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