Biotechnology Techniques, Vol 11, No 4, April 1997, pp. 221–224
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Screening of fungi for phytase
production
S. Gargova*, Z. Roshkova and G. Vancheva
Department of Biotechnology, Higher Institute of Food and Flavour Industries, 26 Maritza Blvd., 4002 Plovdiv,
Bulgaria
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Over 200 fungal strains were screened for phytase production using a two-step procedure. The best strain, an
Aspergillus sp., produced phytase almost equally active at both pH 5.0 and 2.5. Synthesis of the enzyme was limited
by content of inorganic phosphorus above 20 mg/dm3.
24 pts min base to base from Key words to line 1 of text
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Introduction
Phytase (E.C. 3.1.3.8) catalyses the de-esterification of
phytic acid (myo-inositol hexakisphosphate, IP6) to
myo-inositol and free ortho-phosphoric acid. The phytic
acid and its salts, the phytates, contain the major part
(up to 90%) of the total phosphorus in the seeds of
many plants (cereals, leguminous, oil-producing). This
form of phosphorus however is not easily assimilated by
monogastric animals probably because of lack of the
necessary enzymes in them, which requires addition to
the forage of sources of this element, for example, as
Ca3(PO4)2. The phytic acid excreted by the animals is
degraded by microorganisms in the soil and the released
phosphorus at high concentrations gets to the rivers
where it causes eutrophication. In addition, the phytic
acid, having six reactive phosphatic groups, acts as a
strong chelating agent binding Ca2+, Mg2+, Fe3+ and
Zn2+ and decreasing their assimilation.
The importance of phytic acid as a source of phosphorus,
its ability to cause undesirable ecological effects and its
antinutritive properties has stimulated research into
ways for its dephosphorylation.
Aspergillus ficuum NRRL 3135 phytase which produces
a high activity (Shieh and Ware, 1968) has been studied
by many investigators (Howson and Davis, 1983;
Gibson, 1987; Ullah and Gibson, 1987; Chelius and
Wodzinski, 1994). Other known fungi that produce
phytase: Rhizopus oligosporus (Howson and Davis, 1983),
A. oryzae (Shimizu, 1993), A. carbonarius (Al-Asheh and
Duvnjak, 1994), A. niger (Volfova et al., 1994), have
less activity, that is why the selection and the study
of fungi with high production of enzymes dephosphorylating phytates are of scientific and practical
interest.
© 1997 Chapman & Hall
In this work the results of a study of fungi producing
extracellular phytase are reported.
Materials and methods
Microorganisms and screening of strains
203 fungal strains belonging to the genera Aspergillus,
Penicillium, Mucor and Rhizopus were used. The initial
screening was made on synthetic agar phytase screening
medium, containing (g/dm3): D-glucose – 15.0; calcium
phytate – 5.0; (NH4)NO3 – 5.0; MgSO4?7H2O – 0.5;
KCl – 0.5; FeSO4?7H2O – 0.01; MnSO4?4H2O – 0.01.
The pH was adjusted to 5.5 using 1M HCl before autoclaving at 120°C for 20 min (Howson and Davis, 1983).
For the second screening was used a liquid corn
starch medium of the following composition (g/dm3):
corn starch – 80.0; glucose – 30.0; NaNO3 – 8,6;
MgSO4?7H2O – 0.5; KCl – 0.5; FeSO4?7H20 – 0.1; the
phosphorus was supplied as K2HPO4; pH 5.0 (Shieh
and Ware, 1968). The cultivation in the liquid medium
was performed in 500 cm3 Erlenmeyer flasks containing
50 cm3 nutrient medium inoculated with 107/cm3
spores of the appropriate strain on a shaker (220 rpm)
at 30°C. At the end of the process the cultures were
successively filtered and centrifuged at 12 000 g for 20
min and the enzyme activity of the supernatants was
determined.
Study of Aspergillus sp. 307 for phytase
production
These studies were performed in the liquid corn starch
medium at the already described cultivation conditions.
In order to investigate the effect of the phosphorus
content on the phytase production K2HPO4 was added
supplying a concentration of phosphorus from 2 to 100
mg/dm3 . The influence of the initial pH value of the
medium was studied in the interval between 3.0 and
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6.0 (before sterilization). The development of the culture
and the enzyme activity were analyzed after 144 hours.
around them for two Aspergillus strains are shown in
Fig. 1.
Analytical methods
The extracellular phytase activity (PhA) was determined
according to Ullah and Gibson (1987) with substrate
sodium phytate at concentration 500 nmoles in the final
volume of 1.0 cm3 at two pH values in the reaction
mixture – pH 5.0 and pH 2.5 at temperature of hydrolysis 58°C. The released inorganic orthophosphate was
measured according to Heinonen and Lahti (1981). The
phytase activity (nkat) was expressed as nmoles inorganic orthophosphate released per second.
The visual estimation of the phytate-hydrolyzing effect
does not allow to distinguish between the phytase
activity at pH 5.0 (PhA5.0), the phytase activity at pH
2.5 (PhA2.5) and the concomitant acid phosphatase
which also participates in the total dephosphorylating
action of this enzyme group. A significant number of
the microorganisms produces acids which decrease the
pH near the colonies. As a result sufficient phosphorus
may be released for growth (Chelius and Wodzinski,
1994). That is why 60 of our strains with clear zones
were subjected to a second screening.
The acid phosphatase activity (APhA) was determined
by the method of Ullah and Cummins (1987) with
substrate p-nitrophenylphosphate at concentration 1.25
mmoles in the final volume of 1.0 cm3. The incubation
was at pH 2.5 and temperature was 30°C. The enzyme
activity (nkat) was expressed as nmoles p-nitrophenol
released per second.
The content of the phosphorus in the starch was determined after the mineralization of the sample and the
measuring of the phosphorus according to Heinonen and
Lahti (1981). The biomass was measured after drying
at 105°C.
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Results and discussion
During the initial screening of fungi for synthesis of
dephosphorylating enzymes we found that almost all
studied strains grew on the medium with Ca-phytate
as a selective agent. Clear zones were formed around the
colonies of 91.6% of the strains, the zones of six of them
being rather large. The colonies and the clear zones
The data from the quantitative determination of the
phytase activity during the second screening show that
86.6% of the strains produce phytase activity at pH 5.0
up to 10 nkat/cm3. Only two strains – Aspergillus sp.
307 and Aspergillus sp. 7/5 showed activity above 10
nkat/cm3. Similar results were obtained for the phytase
activity at pH 2.5. Aspergillus sp 307 was chosen for
further studies.
The phosphorus in the nutrient medium affects phytase
production by Aspergillus sp. 307 (Fig. 2). The biomass
increases with the increase of the concentration of phosphorus supplied by K2HPO4 and only at 80–100
mg/dm3 remains at an almost constant level (data not
shown). As to enzyme activities, however, a very strict
dependence on the concentration of the phosphorus
was observed. The strain shows a maximum phytase
production at 20 mg/dm3 phosphorus from K2HPO4
(26.6 mg/dm3 the control). A maximal value of the
acid phosphatase activity was found at even lower
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Figure 1 Growth and clear zones of Aspergillus sp. 307 (a) and Aspergillus sp. 7/5 (b) on solid medium selective for
phytase producers
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Screening of fungi for phytase production
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Figure 2 Effect of concentration of phosphorus from
K2HPO4 on the growth of Aspergillus sp. 307 and biosynthesis of dephosphorylating enzymes on nutrient medium at
an initial pH of 5.0.
Figure 3 Effect of the initial pH of the nutrient medium on
the growth of Aspergillus sp. 307 and biosynthesis of
dephosphorylating enzymes.
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concentration of phosphorus – 10 mg/dm3. Having in
mind the phosphorus content in the starch, the optimal
total phosphorus in the nutrient medium for the phytase
production is 45.6 mg/dm3, for the acid phosphatase it
is 35.6 mg/dm3. Obviously the content of phosphorus
in the medium is one of the basic factors which determine to a great degree the biosynthetic process of the
studied enzymes. Our conclusion about the role of the
inorganic phosphorus as a regulator of the phytase and
acid phosphatase production corresponds with that of
other authors (Shieh and Ware, 1968; Howson and
Davis, 1983; Chelius and Wodzinski, 1994).
When the initial pH value of the nutrient medium is
higher than 3.0 the development of the fungus is stimulated (Fig. 3). The production of the acid phosphatase
is favoured by low pH values. A maximum phytase
activity was found at initial pH of the medium of 5.0.
Acid pH values (3.0–5.0) are more favourable for
phytase production than those above 5.0.
Aspergillus sp. 307 develops most intensly up to the
72 d h, but retains its freshness and vitality up to
the 192 d h, which correlates with the constant increase
of the biomass (Fig. 4). Meanwhile the pH of the
medium gradually decreases. Obviously the slow change
of pH around 3.0 favours the production of phytase and
acid phosphatase. The maximum phytase activity found
here (52.1 nkat/cm3 at pH 5.0 and 62.5 nkat/cm3 at pH
2.5) is higher than the activity of A. ficuum (Ullah and
Gibson, 1987) and A. oryzae (Shimizu, 1993) and lower
than the activity of A.niger (Volfova( et al., 1994). Our
assumption that a phytase with two pH optimums of
Figure 4 Dynamics of growth and production of dephosphorylating enzymes by Aspergillus sp. 307.
action is produced needs further investigations on the
purification and characterization of the enzymes in this
complex. The value of the acid phosphatase activity
(400 nkat/cm3) is also higher than that of A. ficuum
(Ullah and Cummins, 1987; Chelius and Wodzinski,
1994).
Conclusion
The screening of 203 fungal strains resulted in selection of Aspergillus sp. 307, which synthetizes both acid
phosphatase and phytase acting equally well at pH 5.0
and 2.5.
Aspergillus sp. 307 is of interest for further studies
because it produces high levels of extracellular dephosphorylating enzymes.
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Received as Revised 29 January 1997
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