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Purification and Properties of Alkaline Phosphatase from Bacillus Cereus

Article  in  Biotechnology & Biotechnological Equipment · April 2014

DOI: 10.1080/13102818.2010.10817906

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Sonya Kostadinova Mariana Marhova

Plovdiv University "Paisii Hilendarski" Plovdiv University "Paisii Hilendarski"
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PURIFICATION AND PROPERTIES OF ALKALINE PHOSPHATASE
FROM BACILLUS CEREUS

S. Kostadinova and M. Marhova

University of Plovdiv, Department of Biochemistry and Microbiology, Plovdiv, Bulgaria
Correspondence to: Sonya Kostadinova
E-mail: skosta@uni-plovdiv.bg

ABSTRACT
Extracellular and membrane-bound alkaline phosphatases were produced at the middle stationary phase of growth by a strain
Bacillus cereus. Twenty two percent of the enzyme activity was secreted into the culture media. An extracellular alkaline
phosphatase (AP I) and a membrane-bound alkaline phosphatase (AP II) were purified 282-fold and 70-fold, respectively by a
combination of chromatographic methods. Enzyme activity of alkaline phosphatase preparations was maximal at pH 9.5. Both
enzymes were inhibited by EDTA and were reactivated by addition of Ca 2+. The molecular weight of AP I was estimated to be
43 ± 1 kDa, and that of AP II was estimated to be 44 ± 1 kDa. Alkaline phosphatase activity of both enzyme preparations was
completely lost by heating at 80°C.

Keywords: alkaline phosphatase, APSE-cellulose
chromatography, Bacillus cereus, enzyme purification
Introduction
Alkaline phosphatase (AP) (EC 3.1.3.1.) is a hydrolase
enzyme responsible for removing phosphate groups in the 5-
and 3- positions from many types of molecules, including
nucleotides, proteins, and alkaloids.
Alkaline phosphatases have been identified in a wide
variety of organisms, including bacteria (Escherichia coli,
Bacillus species, Mycobacterium smegmatis, Thermotoga
maritime, Haloarcula marismortui) and humans (3, 15, 12,
13, 17, 21, 22). Although the actual purpose of the enzyme is
still not fully understood, the simple hypothesis, that it is a
means for the bacteria to generate free phosphate groups for
uptake and use (20). In bacteria, alkaline phosphatase is
located in the periplasmic space.
Bacillus species produce alkaline phosphatase when
phosphate becomes growth limiting as well as during
sporulation, when phosphate supplies are abundant (1).
Alkaline phosphatase of Bacillus licheniformis and Bacillus
subtilis is located intracellularly and extracellularly (9, 23). It
has been shown that culturing conditions significantly affect
both the distribution and the amount of synthesis of alkaline
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phosphatase (8). It is difficult to interpret the localization
data (active dimer associated with inner leaflet of
cytoplasmic membrane versus active dimer secreted based on
any of the current models for protein secretion or insertion of
proteins into membranes (8, 19).
In this study we reported the purification of extracellular
and membrane-bound forms of alkaline phosphatase from
Bacillus cereus strain and determined some of their
molecular and enzymatic properties.
Materials and Methods
Culture method
Bacillus cereus strain No 15 was used as a producer of alka-
line phosphatase. Bacillus cereus was cultured in modified
low phosphate medium (14). Volume of 30 ml of this me-
dium in 300-ml conical flasks was inoculated directly from a
plate colony. The mixture was incubated at 33°C with shak-
ing by a rotary shaker (100 rev/min) and after that was cen-
trifuged for 20 min at 10000 g and 0-4C to remove the cells.
Enzyme purification
Salt extraction
Membrane preparation was suspended in a solution
containing 1 M Tris-hydrochloride, 3 M MgCl2, and Ca-Co,
SECOND BALKAN CONFERENCE ON BIOLOGY
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5O YEARS UNIVERSITY OF PLOVDIV
pH 7.0. The suspension was shaken at 5°C overnight and
then centrifuged at 60 000 g for 2 h.
2-Propanol precipitation
The culture supernatant was saturated to 75% (by volume)
with 2-propanol at a flow rate of 3 ml/min (kept at 0 to -4°C)
under continuous stirring. The mixture was allowed to stand
at –20°C for 2 h and centrifuged for 15 min at 13 000 g and
0°C. The precipitate was then dissolved in 0.1 M Borax-HCl,
pH 7.2 and dialysed twice against 0.03 M Tris-HCl buffer,
pH 7.8.
APSE-cellulose column chromatography
Enzyme preparation (after salt extraction or 2-propanol
precipitation) was dialysed and applied to a 2-(4-
aminophenylsulphonyl)-ethyl (APSE)-cellulose column (5 x
2.5 cm, i.d.) equilibrated with 0.03 M Tris-HCl (pH 7.8). The
column was washed with three column volumes of the same
buffer. The proteins were eluted with linear gradient (0-1 M)
of NaCl with a flow rate of 18 ml/h. Fractions of 5 ml were
collected throughout. All the procedures were carried out at 0
to 4°C.
Gel-filtration on Sephadex G-200
The concentrated eluate from the APSE-cellulose column
was placed on a column (95 x 2.1 cm, i.d.) of Sephadex G-
200. Fraction of 6 ml was collected throughout with a flow
rate of 15 ml/h.
Assay of alkaline phosphatase activity
p-NPP (Sigma) was used as a substrate. The reaction mixture
contained: 0.5 ml of 6 mM p-NPP (containing 3 mM MgCl2,
0.2 M Tris-HCl buffer, рН 9.5) and 0.1 ml of enzyme
solution. After incubation at 37°C the reaction was stopped
by adding 0.4 ml of 0.4 N NaOH. The absorbance at 405 nm
was measured and the enzyme activity was estimated by the
method of Ikezawa et al. (11).
One unit of AP was defined as the amount that would
catalyse the hydrolysis 1 µmol of substrate per minute at pH
10.0 and 37°C.
Estimation of protein
Protein contents were estimated by the method of Hartree (6)
or Bradford (2) with bovine serum albumin (Serva) as a
standard.
Results and Discussion
Purification of alkaline phosphatase
Bacillus cereus strain No 15 was selected as a producer of
extracellular (AP I) and membrane-bound alkaline
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phosphatase (AP II). Enzyme synthesis was maximal when
Bacillus cereus was cultured in modified low phosphate
medium (14). In the middle stationary phase of growth
enzyme activity in the culture media was 0.31 U/mg protein
and a membrane-bound activity was 1.4 U/mg (Fig. 1).
Fig. 1. Production of alkaline phosphatase by B. cereus during growth
The gram-positive species Bacillus are well known for its
high capacity to secrete proteins, both in its natural habitat
and in biotechnological applications (4, 16, 18).
Bacillus cereus produced alkaline phosphatase during late
logarithmic and middle stationary phase of growth. We ob-
served high level of activity of AP II for a long time period
(6 h). Maximum of AP II activity correlated with release of
AP I in the culture broth.
Similar alkaline phosphatase production pattern was
reported for Bacillus subtilis enzyme (17).
In our previous study we describe purification of an
extracellular alkaline phosphatase from B.cereus strain (14).
The purification scheme was optimised by adding
precipitation with 2-propanol and replacing the column
chromatography on DEAE-cellulose with APSE-cellulose
chromatography (Table 1).
A commercially obtained 2-(4-aminophenylsulphonyl)-
ethyl-cellulose containing 23 µmol/ml amino groups was
used (Ostsorb AV, Spolchemie, Czech Republic). Bacillus
cereus extracellular alkaline phosphatase was absorbed to the
sorbent and was eluted with 0.2 M NaCl. The APSE-
cellulose chromatography decreased protein content in the
enzyme preparation, which resulted in a significant increase
of specific activity - from 4.8 U.mg-1 for 2-propanol
precipitation to 40.9 U.mg-1 (Fig. 2).
A summary of the purification procedure is given in
Table 1. Bacillus cereus alkaline phosphatase was purified
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21-23 MAY 2010, PLOVDIV
5O YEARS UNIVERSITY OF PLOVDIV
282-fold over the supernatant with a recovery of 11.2%. column. The column was equilibrated with 0.03 M Tris-HCl
(pH 7.8) and eluted with the same buffer. Alkaline
TABLE 1 phosphatise C activity appeared in the minor protein
Purification of an extracellular alkaline phosphatase from fractions randing from 400 and 456 ml (Fig. 3).
Bacillus cereus The purification scheme resulted in a recovery of 19%
Pro- Ac- Specific Purifica- Re- with a 70-fold increase in specific activity (Table 2).
tein tivity activity tion covery
Step
(mg) (U) (U/mg) (fold) (%)
TABLE 2
Culture
Purification of membrane-bound alkaline phosphatase from
supernatant 800 250 0.31 - 100
Bacillus cereus
2-propanol
precipitate 80 121 1.51 4.8 48.47 Reco-
Pro- Activ- Specific Purifica-
APSE - very
tein ity activity tion
cellulose 4 51 12.7 40.9 20.4 Step (%)
(mg) (U) (U/mg) (fold)
Sephadex
Membrane
G-200 0.32 28 87.5 282 11.2
preparation 480 672 1.4 - 100

Salt extrac-
98 537 5.4 3.8 80
tion
APSE-
cellulose 6.5 233 35.8 25.6 34.7
Sephadex
G-200 1.3 129 99.2 70.8 19.2

2
1,8
1,6
1,4
1,2
(■) alkaline phosphatase activity x 10; (♦) protein Activity U/ml
1
Protein A595
Fig. 2. APSE-cellulose chromatography of Bacillus cereus alkaline phospha- 0,8
tase 0,6
0,4
A membrane-bound alkaline phosphatase was purified by 0,2

a combination of salt extraction and chromatography on 0

11
13
15
17
19
21
23
25
27
29
31
1
3
5
7
9

APSE-cellulose and Sephadex G-200 (Table 2).

Fractions No
Membrane preparation was suspended and subjected to
salt extraction. After five sequential extractions about 80% of Fig. 3. Sephadex G 200 chromatography of Bacillus cereus alkaline
the AP activity was solubilized from the membrane phosphatase

preparation.
The solubilized material, containing AP activity was
dialyzed against 0.03 M Tris-HCl (pH 7.8) and then
separated with column chromatography. Alkaline
phosphatase from B.cereus was adsorbed onto APSE-
cellulose and was eluted with 0.2 M NaCl. The specific
activity increased 6.6-fold and the recovery of activity was
43% (compared to previous step).
Fractions containing phosphatase activity were collected,
concentrated, dialysed and applied to a Sephadex G-75
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Properties of alkaline phosphatases
Molecular weight. The molecular sizes of the two purified
enzymes were estimated by comparing their behavior in
Sephadex G-200 gel filtration to that of proteins of known
molecular weight (RNAse II – 13.7 kDa; PC–PLC from
B.cereus – 23 kDa; peroxydase – 40 kDa, bovine serum
albumin – 65 kDa, and aldolose – 158 kDa). From the linear
relationship of the logarithm of molecular weight of the
elution volume in the gel filtration we estimated the
molecular weight of API as 43 ± 1 kDa and that of AP II as
SECOND BALKAN CONFERENCE ON BIOLOGY
21-23 MAY 2010, PLOVDIV
5O YEARS UNIVERSITY OF PLOVDIV
44 ± 1 kDa. These results suggest that both API and APII are determined (Fig. 4). Enzyme activity was completely lost by
composed of single polypeptide chains. heating at 80°C. AP II was more stable than AP I at higher
Yamane and Maruo (23) estimated the molecular weight temperatures.
of Bacillus subtilis extracellular and membrane-bound
alkaline phosphatases as 45 and 46 kDa, respectively.
Alkaline phosphatase of B. licheniformis is 60 kDa protein
and those of E.coli – 84 kDa (5, 8).
рН optimum and pH stability
The pH optimum of phosphatase activity against p-NPP of
AP I and AP II were determined in 0.2 M Tris-hydrochloride
buffer and 0.1 M glycine-sodium hydroxide buffer of varying
pH. The enzyme activities of both alkaline phosphatases
were greatest at pH 9.5.
To examine the effect of pH on AP I and on AP II, each
enzyme preparation was mixed with a buffer solution ranging
Fig. 4. Thermostability of Bacillus cereus alkaline phosphatases
from pH 3.2 to 11.0, and allowed to stand at 37°C overnight.
The mixtures were then adjusted to pH 9.5 and AP activity Conclusions
was assayed. Both enzyme preparations were stable at pH 7.0 We found production of extracellular and membrane-bound
to 9.5. alkaline phospatases by a strain Bacillus cereus. Enzyme
Effect of metal ions and EDTA preparations were purified by column chromatographic
The effect of metal ions on API and APII activity of the two methods. Molecular and enzymatic properties of both AP I
enzyme preparations was examined by assaying the activity and AP II were similar. This proposed the release of a part of
in the presence of various metal ions at a 1 mM alkaline phospatase into the culture media, which provide
concentration of each. Only Ca2+ was found to activate inorganic phosphate for Bacillus cereus.
enzyme activity of the two preparations. Na+, K+, Mg2+,
Co2+, and Zn2+ had no effect. Mn2+ and Cu2+ had an
Acknowledgement
inhibitory effect on the enzyme activity of the preparations.
The effect of ethylenediaminetetraacetate (EDTA) was This work was supported by Grant RSFB 044 from the NSF
examined in 10 mM Tris-hydrochloride, pH 7.5. After University of Plovdiv, Bulgaria.
incubation with various concentrations of EDTA (0.1 – 2.0
mM) for 3 h at 37°C, the remaining activity was determined.
The enzyme activitie of AP I was inactivated by the
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