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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 50C. 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]
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
563
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
ESCOBIN-MOPERA ET AL.
J. BIOSCI. BIOENG.,
10.00
Growth (OD660)
4.5
25
1.00
0.10
0.01
10
15
20
25
4
20
3.5
3
15
2.5
2
10
1.5
1
0.5
1.5
3
1.0
2
0.5
0.0
12 16 20 24 28 32 36 40 44 48
Liberated inositol
(mM/mg protein)
Growth (OD660)
564
0
0 mM 12.8 mM 64 mM 128 mM 256 mM MM9
FIG. 2. Phytase specific activity during the growth of K. pneumoniae 9-3B in a medium
with phytate as the sole carbon and phosphate source at 37C for 48 h. Symbols: circles,
phytase specific activity; squares, cell growth (OD600).
30
25
20
15
10
5
2
0
45 kda,Phytase 9-3B
Log MW
565
1.5
44 kda
13.7 kda
6.5 kda
0.4
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 37C to
measure the liberated phosphate using standard procedure. Symbols: closed circles,
phytate; open circles, myo-inositol-2-monophosphate.
0.3
Substrate (mM)
29 kda
0.5
0.2
0.5
0.6
0.7
Kav
DISCUSSION
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 anhydrase), 13.7 kDa (ribonuclease A) and 6.5 kDa (aprotinin).
TABLE 1. Purification of phytase and liberated inositol from Klebsiella pneumoniae 9-B.
Purification step
Protein
(mg)
Phytase
Phytase
(U) a
Specific activity
(U/mg)
596
547
502
153
10.8
5.64
6.89
1078
1346
1360
% yield
100
91.8
84.2
25.7
1.81
Inositol liberation
Purification fold
1.00
1.22
191
239
241
Liberated inositol
(U) b
7.17
6.83
5.88
5.37
1.13
% yield
100
95.2
82.0
74.9
15.8
Purification fold
1.00
1.26
184
658
2077
566
ESCOBIN-MOPERA ET AL.
J. BIOSCI. BIOENG.,
TABLE 3. Substrate specificity of phytase from K. pneumoniae 9-3B.
180
Substrate
160
Phytate
-Nitrophenyl phosphate
1-Naphtyl phosphate
Glucose-6-phosphate
2-Glycerolphosphate
NADP
ATP
ADP
AMP
140
120
100
80
60
40
Km
100.00
27.66
11.39
0.84
5.04
55.68
4.45
8.16
3.49
0.04
2.12
1.18
4.56
140.00
2.62
0.86
0.47
0.41
20
0
10
pH
120
100
80
60
40
20
0
30
40
50
60
70
Temperature ( )
FIG. 5. The effect of pH (A) and temperature (B) on the phytase activity of K.
pneumoniae 9-3B. Optimum pH and stability was determined at pH 2.0 and 3.0
(GlycineHCl), pH 4.0, 5.0 and 6.0 (Sodium acetate), pH 7.0, 8.0 and 9.0 (TrisHCl) and
pH 10.0 (GlycineNaOH). Symbols: closed squares, stability; open squares, optima.
Ca2 +
Zn2 +
Mg2 +
Mn2 +
Fe2 +
Co2 +
EDTA
None
5 mM
103.21 0.16
46.07 0.04
79.49 0.01
86.89 0.01
47.57 0.06
78.77 0.01
102.79 0.03
100.00 0.01
78.90 0.01
37.68 0.05
67.10 0.04
59.17 0.08
29.45 0.03
54.52 0.03
101.85 0.01
100.00 0.01
567
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