Folia Microbiol. 51 (6), 541–545 (2006) http://www.biomed.cas.cz/mbu/folia/
Aspergillus niger pH 2.1 Optimum Acid Phosphatase
with High Affinity for Phytate
S. GARGOVA, M. SARIYSKA, A. ANGELOV, I. STOILOVA
Department of Biotechnology, University of Food Technologies, 4002 Plovdiv, Bulgaria
Received 12 September 2005
Revised version 30 January 2006
ABSTRACT. An extracellular acid phosphatase isolated from the culture of a wild strain Aspergillus niger,
producing the dephosphorylating 3-phytase, was obtained in a homogeneous form by sequential application
of ultrafiltration through PS 50 membrane, gel filtration on Sephadex G-100 and ion exchange chromatogra-
phy on DEAE-Sepharose CL 6B and CM-Sepharose CL 6B. The enzyme showed a maximum catalytic value
in a strongly acidic range (pH 2.0–2.4) with pHopt 2.1 and topt 66 °C. The acid phosphatase showed a wide
substrate specificity and a high affinity for sodium phytate, 2.5× higher than with 4-nitrophenyl phosphate.
This property of the acid phosphatase demonstrated that it is a potent 3-phytase at pH 2.1 and is of great sig-
nificance for a practical application of the dephosphorylating complex – its addition to the diets of mono-
gastric animals in view of the low pH values in the digestive tract.
Acid phosphatases (EC 188.8.131.52) catalyze the hydrolysis of various phosphorylated substrates and
are common in nature. These enzymes normally accompany the 3-phytase of mold origin (Żyła 1990; Shi-
mizu 1993; Ullah and Phillippy 1994; Gargova et al. 1997; Dvořáková 1998; Voříšek and Kalachová 2003).
They can be used to increase the amount of free phosphorus in cereal and legume forage, as well as in pro-
tein soybean isolates. In combination with 3-phytase (EC 184.108.40.206) their action is much more efficient than
with a separate application (Żyła 1993; Wyss et al. 1998; Näsi et al. 1999).
Depending on their pH optimum these enzymes are divided into 3 groups: (a) strongly acid with pH
optimum 2.2–2.5, (b) moderately acid with pH optimum 5.0–5.5, and (c) weakly acid with pH optimum ≈6.0
(Ullah et al. 1994).
We examined the strain A. niger 307, selected for the biosynthesis of 3-phytase, which we have
reported to be accompanied by acid phosphatase (Gargova et al. 1997). This 3-phytase was obtained in
a homogeneous form, had 2 pH optima (at 2.6 and 5.0), temperature optimum at 55–58 °C, and a narrow
substrate specificity with a maximum enzyme activity with dodecasodium phytate, while with regard to other
phosphorylated substrates its activity was only 2–3 % of the maximum (Sariyska et al. 2005).
Here we present the results of the purification and report on the properties of acid phosphatase
isolated from the culture of a natural strain A. niger 307.
MATERIALS AND METHODS
Medium and cultivation. Acid phosphatase was produced in a culture medium containing (in g/L):
corn starch 50, glucose 50, NaNO3 8.6, MgSO4·7H2O 0.5, KCl 0.5, FeSO4·7H2O 0.1, K2HPO4 0.1 g P; pH 5.
Erlenmeyer flasks (300 mL), with 30 mL medium were inoculated with 50-d conidia (final concentration
20–30/nL, i.e. 2–3 × 107/mL) and shaken (30 °C, 3.7 Hz, 7 d; Gargova and Sariyska 2003).
Steps of purification. The filtrate of the culture medium was concentrated by using a PS 50 mem-
brane (Membrane Technologies Ltd., Bulgaria) at room temperature and pressure of 0.3 MPa (achieved with
nitrogen). Portions of the concentrate containing 8 mg protein per mL were run through 12/600 Sephadex
G-100 column (Pharmacia), equilibrated with 200 mmol/L sodium acetate buffer (pH 5.0). The active fract-
ions were eluted with the same buffer (0.15 mL/min), pooled and applied on a 14/180 DEAE-Sepharose CL
6B (Fluka) column, equilibrated with 50 mmol/L Tris-HCl buffer (pH 8.0). The active fractions were pooled
and chromatographed on a 14/180 CM-Sepharose CL 6B (Fluka) column, equilibrated with 200 mmol/L
glycine (Merck) buffer (pH 2.6). From both ion exchange columns the proteins were eluted with a stepwise
gradient of pH.
542 S. GARGOVA et al. Vol. 51
SDS-PAGE (15 %) was performed (Laemmli 1970) at room temperature and 20–30 mA. Standards
(Merck) were applied at 40 mg per well, samples up to 100 µg per well. The slab gels were stained with
Comassie Brilliant Blue R-250.
Properties of the enzyme. The effect of pH was investigated within pH 1.8–5.8 at intervals of 0.2 and
around the pH optimum at an interval of 0.1 pH. Glycine-HCl (50 mmol/L) and sodium acetate (200 mmol/L)
buffers were used for pH 1.8–3.6 and 3.6–5.8, respectively. The effect of the temperature was determined in
the range of 20–80 °C at 10 and 1 °C around the optimum. The kinetic parameters Km and v lim for the hydro-
lysis of 4-nitrophenyl phosphate (4-NPP) were determined at pH 2.1 and 30 °C. To investigate the substrate
specificity, each of the substrates (1.25 mmol/L) was assayed at pH 2.1 and 30 °C.
Enzyme assay. The activity of acid phosphatase was determined according to Ullah and Cummins
(1987) with 1.25 mmol/L 4-NPP at pH 2.5 and 30 °C and was expressed in nkat (nmol of 4-nitrophenol re-
leased per s).
The 3-phytase activity was determined according to Ullah and Gibson (1987) with 0.5 mmol do-
decasodium phytate and released monophosphate was measured according to Heinonen and Lahti (1981), the
activity being expressed in nkat (nmol monophosphate released per s).
Protein concentration was determined according to Lowry using bovine serum albumin (Sigma) as
RESULTS AND DISCUSSION
Under the conditions of limiting the biosynthesis of acid phosphatase by phosphorus, A. niger strain
307 formed a dephosphorylating complex of 3-phytase and acid phosphatase with an activity of 324 and
254 nkat/mL culture liquid, respectively. When culture liquid was run through PS 50 membrane and the
volume was reduced 10×, 37 % of the total protein passed into the permeate, but the acid phosphatase acti-
vity was almost completely (94.3 %) preserved in the concentrate (Table I). Obviously, ultrafiltration was an
appropriate method for a considerable concentration of the enzyme and release of low-molar-mass proteins
in an initial step of the purification.
Table I. Purification of A. niger 307 acid phosphatase
Total activity Total protein Purification
Purification step activity Yield, %
nkat mg fold
Culture liquid 12 540 78 161 100 1.0
Ultrafiltration 11 820 49 242 94.3 1.51
Chromatography: Sephadex G-100 1 105 0.86 1284 8.81 7.99
DEAE-Sepharose CL 6B 509 0.11 4549 4.06 28.3
CM-Sepharose CL 6B 163 0.03 6023 1.29 37.5
The acid phosphatase flowed out of the column with Sephadex G-100 in the first fractions which is
a convincing argument that the enzyme has a molar mass higher than the bulk of the accompanying proteins
(data not shown). In the next step, the acid phosphatase did not bind to DEAE-Sepharose CL 6B, but was
separated from the 3-phytase (after the elution with a pH 6.0 buffer; Fig. 1). An ion exchange chromato-
graphy with CM-Sepharose CL 6B was applied, as the acid phosphatase was determined only at the 1st elu-
tion peak (Fig. 2). The monitoring of the purification of the product after each step is shown on Fig. 3. The
single band obtained suggested that the acid phosphatase was in a homogeneous form. Molar mass determi-
ned by SDS-PAGE was ≈70 kDa. Since the enzyme is eluted with the 1st heavy protein fractions during gel
filtration, we could suggest that the native form is an oligomer, similar to other acid phosphatases (Kostrewa
et al. 1999).
The enzyme showed a maximum activity at pH 2.0–2.4 with pHopt 2.1 (Fig. 4A). Too et al. (1997)
reported a similar pHopt of 2.0–2.5, but for an intracellular acid mold phosphatase. The temperature optimum
was observed at 66 °C (Fig. 4B). Using a Lineweaver–Burk plot, the Km was calculated to be 950 nmol/L
for 4-NPP, which was a lower value than that reported for acid phosphatase from A. oryzae – 800 µmol/L
(Shimizu 1993) or for that enzyme from A. ficuum – 270 µmol/L (Ullah and Cummins 1987). The value of
v lim was 232 nkat/mL.
2006 A. niger ACID PHOSPHATASE WITH HIGH AFFINITY FOR PHYTATE 543
Fig. 1. Elution profile of dephosphorylating enzymes on DEAE-Sepharose CL 6B; elution buffers (mmol/L, open diamonds):
Tris-HCl 50 (pH 8.0), sodium acetate 200 (pH 6.0), sodium acetate 200 (pH 4.0), glycine 200 (pH 2.5); A280 – absorbance
(closed diamonds), nkat/mL – activity (3-phytase – closed circles, acid phosphatase – closed triangles), n – fraction
Fig. 2. Elution profile of acid phosphatase on CM-Sepharose CL 6B; elution buffers (mmol/L, open diamonds): glycine 200
(pH 2.5), sodium acetate 200 (pH 4.0), sodium acetate 200 (pH 6.0), Tris-HCl 50 (pH 8.0); A280 – absorbance (closed
diamonds), nkat/mL – activity (closed triangles), n – fraction number.
Fig. 3. Purity monitoring of 3-phytase and acid phos-
phatase by SDS-PAGE: A – standards, B – culture filtrate,
C – ultraconcentrate, D – fractions after Sephadex G-100,
E – 3-phytase after DEAE-Sepharose CL 6B, F – acid
phosphatase after CM-Sepharose CL 6B.
544 S. GARGOVA et al. Vol. 51
The acid phosphatase hydrolyzed various phosphorylated substrates (Table II). With AMP and hexa-
sodium phytate the enzyme activity was 2.5× higher than for 4-NPP. Similar to other acid phosphatases
(Ullah and Cummins 1987; Żyła 1990; Shimizu 1993; Kostrewa et al. 1999) the enzyme showed a wide
Fig. 4. Effect of pH (A) and temperature (°C, B) on acid phosphatase activity (relative %).
substrate specificity to various phosphomonoesters. The acid phosphatase from A. niger strain KU-8 also
showed a broad substrate specificity, but with a strict affinity to 6-phosphorylated oligosaccharides, prepared
from a potato starch hydrolysate (Too et al. 1997). Our 3-phytase manifested a distinct affinity only for hexa-
sodium phytate (see above) (Sariyska et al. 2005). The results obtained for the substrate specificity as well
as the electrophoretic data suggested that this strain produced 2 proteins which have a catalytic effect on phos-
phorylated substrates, but differ in a number of properties, e.g., temperature and pH optima, molar mass and
affinity to their substrates. However, both acid phosphatase and 3-phytase showed the maximum activity
towards hexasodium phytate. Their property demonstrated that our enzyme is a potent 3-phytase at this pH.
Table II. Substrate specificity of acid phosphatase from A. niger 307a
Substrate Activity, % Substrate Activity, %
AMP 255 NADP+ 129
Dodecasodium phytate 245 Glucose 6-phosphate 85.2
1-Naphthyl phosphate 163 2-O-Phosphoglycerate 80.9
Phenyl phosphate 134 2-Phosphoenolpyruvate 75.6
aActivity of 4-NPP = 100 % (control).
It is of great significance for the practical application of the dephosphorylating complex from A. niger strain
307 in stock breeding (direct addition to the diets of monogastric animals). On the other hand, in view of the
low values of pH in the digestive tract, the low pH optimum of the acid phosphatase is beneficial. The pre-
sence of the 2 activities will increase the total conversion of phytates to myo-inositol and inorganic phosphate
at low pH.
In conclusion, a 4-step scheme was used for the purification of acid phosphatase derived from A. niger
strain 307 culture. The enzyme, which was obtained in a homogeneous form, showed the maximum catalytic
effect in a strongly acidic region and demonstrated a wide substrate specificity with high affinity for hexa-
DVOŘÁKOVÁ J.: Phytase sources, preparation and exploitation. Folia Microbiol. 48, 323–338 (1998).
GARGOVA S., ROSHKOVA Z., VANCHEVA G.: Screening of fungi for phytase production. Biotech.Techn. 11, 221–224 (1997).
GARGOVA S., SARIYSKA M.: Effect of culture conditions on the biosynthesis of Aspergillus niger phytase and acid phosphatase. Enzyme
Microb.Technol. 32, 231–235 (2003).
HEINONEN J.K., LAHTI R.J.: A new and convenient colorimetric determination of inorganic orthophosphate and its application to the
assay of inorganic pyrophosphatase. Anal.Biochem. 113, 313–317 (1981).
KOSTREWA D., WYSS M., D’ARCY A., VAN LOON A.P.G.M.: Crystal structure of Aspergillus niger pH 2.5 acid phosphatase at 2.4 Å re-
solution. J.Mol.Biol. 288, 965–974 (1999).
LAEMMLI U.K.: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685 (1970).
2006 A. niger ACID PHOSPHATASE WITH HIGH AFFINITY FOR PHYTATE 545
NÄSI M., PARTANEN K., PIIRONEN J.: Comparison of Aspergillus niger phytase and Trichoderma reesei phytase and acid phosphatase
on phytate phosphorus availability in pigs fed on maize-soybean meal or barley-soybean meal diets. Arch.Anim.Nutr. 52, 15–
SARIYSKA M., GARGOVA S., KOLEVA L., ANGELOV A.: Aspergillus niger phytase: purification and characterization. Biotechnol.Bio-
technol.Eq. 19, 98–106 (2005).
SHIMIZU M.: Purification and characterization of phytase and acid phosphatase produced by Aspergillus oryzae K1. Biosci.Biotech.
Biochem. 57, 1364–1365 (1993).
TOO K., KAMASAKA H., KUSAKA K., KURIKI T., KOMETANI T., OKADA S.: A novel acid phosphatase from Aspergillus niger KU-8 that
specifically hydrolyzes C-6 phosphate groups of phosphoryl oligosaccharides. Biosci.Biotechnol.Biochem. 61, 1512–1517
ULLAH A.N.J., CUMMINS B.J.: Purification, N-terminal amino acid sequence and characterization of pH 2.5 optimum acid phosphatase
(EC 220.127.116.11) from Aspergillus ficuum. Prepar.Biochem. 17, 397–422 (1987).
ULLAH A.N.J., GIBSON D.M.: Extracellular phytase (18.104.22.168) from Aspergillus ficuum NRRL 3135: purification and characterization.
Prepar.Biochem. 17, 63–91 (1987).
ULLAH A.N.J., PHILLIPPY B.Q.: Substrate selectivity in Aspergillus ficuum phytase and acid phosphatases using myo-inositol phospha-
tes. Agric.Food Chem. 42, 423–425 (1994).
ULLAH A.H.J., MULLANEY E.M., DISCHINGER N.C.: The complete primary structure elucidation of Aspergillus ficuum (niger), pH 6.0,
optimum acid phosphatase by Edman degradation. Biochem.Biophys.Res.Comm. 203, 182–189 (1994).
VOŘÍŠEK J., KALACHOVÁ L.: Secretion of acid phosphatase in Claviceps purpurea – an ultracytochemical study. Folia Microbiol. 48,
WYSS M., PASAMONTES L., RÉMY R., KOHLER J., KUSZNIR E., GADIENT M., MÜLLER F., VAN LOON A.P.G.M.: Comparison of the
thermostability properties of three acid phosphatases from molds: Aspergillus fumigatus phytase, A. niger phytase and
A. niger pH 2.5 acid phosphatase. Appl.Environ.Microbiol. 64, 4446–4451 (1998).
ŻYŁA K.: Acid phosphatases purified from industrial waste mycelium of Aspergillus niger used to produce citric acid. Acta Biotechnol.
10, 319–327 (1990).
ŻYŁA K.: The role of acid phosphatase activity during enzymic dephosphorylation of phytates by Aspergillus niger phytase. World
J.Microbiol.Biotechnol. 9, 117–119 (1993).