Advances in Microbiology, 2012, 2, 395-398
http://dx.doi.org/10.4236/aim.2012.23050 Published Online September 2012 (http://www.SciRP.org/journal/aim)
Ethanol Tolerance in Aspergillus niger and
Escherichia coli Phytase
Edward J. Mullaney, Kandan Sethumadhavan, Stephanie Boone, Abul H. J. Ullah*
Commodity Utilization, Southern Regional Research Center, Agricultural Research Service,
United States Department of Agriculture, New Orleans, USA
Received July 9, 2012; revised August 10, 2012; accepted August 18, 2012
ABSTRACT
Despite yeast having its own native phytase, the high levels of phytate found in DDGS, a byproduct of ethanol (ETOH)
fermentation, suggest that its activity is diminished in the presence of ETOH. Ethanol, a product of grain fermentation,
is known to inactivate several hydrolytic enzymes but its effect on phytases is relatively unknown. In this study, two
phytases, Aspergillus niger (PhyA) and Escherichia coli (AppA2), were tested for ETOH tolerance. The E. coli phytase
displayed greater ethanol tolerance over fungal phytase in the 5 to 10% range. However, ETOH inactivation was found
to be reversible for both the enzymes. These differences in ETOH tolerance do suggest that there is a potential to
achieve higher ETOH tolerance in phytases by “structure-function” studies to lower phytic acid levels in DDGS and for
other applications.
Keywords: Phytase; Ethanol; DDGS; Fermentation; Aspergillus niger
1. Introduction
Phytate is the principal storage compound for phosphorus
in plants. With the increased utilization of high phytate
containing plant meals over the last several decades,
extensive research has focused on the deployment of
phytases as an animal feed additive. This is to allow
monogastric animals (swine, poultry, etc.) which lack a
digestive phytase to obtain the phytate’s ortho-phosphate
groups, which otherwise will be unavailable.
More recently, demands for additional bio-based fuels
have spurred enhanced fermentation of corn and other
grains such as sweet sorghum to produce ethanol in re-
sponse to the growing demand. This has resulted in in-
creased amounts of dried distillers grains with solubles
(DDGS) emanating from this process. The DDGS is rich
in nutrients and has much potential as an animal feed.
However, recent studies have reported that DDGS con-
tains high levels of phytate [1]. Moreover, ethanol is
known to inhibit the activity of several hydrolytic en-
zymes [2,3] and these results suggest that the phytase
produced by the native yeast, Saccharomyces cerevisiae,
[4] would be inhibited by increased concentration of
ethanol. In this study, the ethanol tolerances of two phy-
tases that are marketed as animal feed additive are de-
termined. While both are histidine acid phosphatases
(HAPs) and share the same active site geometry and
catalytic mechanism, one is from Aspergillus niger and
the other is produced by Escherichia coli, each has its
own unique catalytic properties. While no information
exist on ethanol tolerance in phytases, any differences in
ethanol tolerance in the two enzymes can enhance our
understanding of how ethanol interacts with this class of
enzymes and this may contribute to the designing of
phytases that retains more activity during fermentation
and thus lower the phytic acid content of DDGS. In addi-
tion, the achievement of a molecular modification to en-
hance ethanol tolerance in phytase may also have further
applications in enhancing the ethanol tolerance of other
hydrolytic enzymes.
2. Materials and Methods
2.1. Source of Phytase
Fungal phytase was obtained from the cloned Aspergillus
niger phyA gene that was overexpressed in Pichia pas-
toris. The recombinant phytase was purified using se-
quential ion-exchange column chromatographies.
E.coli phytase, the AppA2 gene product, was a gift
from Phytex LLC, Portland, Maine, which was over-
expressed in Pichia pastoris. The crude culture filtrate
was dialyzed against 25 mM glycine, pH 2.8 buffer and
loaded onto a MacroPrep™ S column and eluted as a
single activity component in a linear salt (0 - 0.5 M
sodium chloride) gradient. The final specific activity of
the phytase was about 15,000 nkat/mg of protein at 55˚C.
*Corresponding author.
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opyright © 2012 SciRes. AiM

E. J. MULLANEY ET AL. 396
2.2. Phytase Assay
Phytase assays were carried out in 1.0 mL 50 mM acetate
buffer, pH 5.0˚C at 55˚C similar to A. niger phytase as-
say [5]. Liberated inorganic ortho-phosphates were quan-
titated spectrophotometrically using a freshly prepared
AMA reagent consisting of acetone, 10 mM ammonium
molybdate, and 5.0 N sulfuric acid, (2:1:1, v/v) [6].
Adding 2.0 mL AMA solution per assay tube terminated
phytase assay. After 30 seconds, 0.1 mL of 1.0 M citric
acid was added to each tube to fix the color generated by
AAM reagent. Absorbance was read at 355 nm after
blanking the spectrophotometer with appropriate control.
Values were expressed as nkat/mL, where kat is defined
as moles of substrate converted per second.
2.3. Ethanol in the Inorganic Phosphate
Measurement
A 50 mM acetate buffer containing 0% - 10% ethanol
and 200 µM aliquot of potassium phosphate (K2HPO4) in
1 ml volume was mixed with AMA reagent followed by
citrate as in the phytase assay to measure the inorganic
ortho-phosphate. This is to rule out any inference of
ethanol in the detection and quantification of inorganic
orthophosphates.
2.4. Effect of Ethanol on Phytase Activity
Phytases (8 µL PhyA and 15 µL AppA2) were incubated
with 0 to 10% ethanol in 1 mL volume at room tem-
perature for 10 min. Then they were transferred to a 55˚C
water bath for 2 min before phytase assay.
2.5. Stability of Phytases after Exposure to
Ethanol
A 100 μL aliquot of PhyA and 4 µL of AppA2 were
incubated at room temperature for 30 min in 0% - 10%
ethanol in 1 mL 50 mM acetate buffer, pH 5.0. After
incubation, an aliquot of PhyA (10 µL) and AppA2 (15
µL) were incubated with 75 µL of 10 mM phytate for 1
min at 55˚C. The liberated inorganic ortho-phosphates
were measured as above.
3. Results
3.1. Ethanol Tolerance of Microbial Phytase
Samples of both PhyA and AppA2 phytase were in-
cubated for 10 minutes in 0% to 10% ethanol to deter-
mine their respective ethanol tolerance. The results are
shown in Figure 1. The PhyA phytase had activity (566
nkat/mL) at 0% ethanol to no activity at 10% denaturant
concentration. However, E. coli phytase, AppA2, which
had a comparably lower activity at 0% ethanol (275 nkat/
mL), still had some activity (55 nkat/mL) at 10% dena-
turant concentration. This means a loss of 80% activity
for AppA2 due to 10% ethanol.
3.2. Phytase Activity at Varying Concentration
of Ethanol
The activity of the two phytases as a function of various
concentration of ethanol is shown in Figure 2. The loss
of activity in both the phytases were very similar in 0.5
to 3% range of ethanol concentration. While AppA2
phytase retained nearly 20% of its activity at 10% etha-
nol, the A. niger phytase was completely inactivated at
10% ethanol.
3.3. Ethanol Does Not Effect Phytase Assay
The possibility of ethanol interference with the phytase
assay was shown not to be a significant factor (Figure 3).
3.4. Ethanol Effect on Phytase Is Reversible
Since inactivation of the enzyme by ethanol does not
result in permanent denaturation of either of the enzyme
(Figure 4), it indicates that the inhibition of phytases by
Figure 1. Effect of ethanol on phytase enzyme activity.
Phytases (8 µL phytase A and 15 µL AppA2) were incu-
bated with 0 to 10% ethanol in 1 mL volume at room tem-
perature for 10 min. Then they were tranferred to a 55˚C
water bath for 2 min before phytase assay.
Figure 2. Effects of various ethanol concentration on phy-
tase activity over the 0% - 10% range.

E. J. MULLANEY ET AL. 397
Figure 3. Ethanol in the inorganic phosphate measurement.
200 µM potassium orthophosphate (K2HPO4) was mixed
with 0 to 10% ethanol in 1 mL 50 mM acetate buffer, pH
5.0 followed by AMA reagent and citrate. This is to rule out
any interference of ethanol in the phytase assay.
Figure 4. Recovery of phytases after exposure to ethanol.
100 µL phytase PhyA and 4 µL of AppA2 were incubated at
room temperature for 30 min in presence and absence
(control) of 10% ethanol in 1 mL 50 mM acetate buffer, pH
5.0. After incubation, an aliquot of Phy A (10 µL) and
AppA2 (15 µL) were incubated with 75 µL of 10 mM phy-
tate for 1 min at 55˚C. The liberated inorganic ortho-
phosphates were me asur e d as above.
ethanol is a reversible process.
4. Discussion
Both A. niger and E. coli phytase displayed activity inhi-
bition with increasing amounts of ethanol. However, at
concentrations of ethanol above 3% the E. coli phytase
retained significantly more activity than A. niger PhyA
phytase. Both of these enzymes not only share a common
catalytic mechanism, but they also display considerable
amino acids divergence in their molecular structure.
Previous studies have shown that a change of just a
single amino acid can alter physical properties of these
enzymes [7,8]. In addition, researchers have shown that
significant differences exist in these two enzymes in their
response to sodium chloride [9]. The addition of sodium
chloride increases activity of the fungal phytase in the pH
range 1.5 - 6.0. No increase in activity was achieved
when AppA2 was tested with the same sodium chloride
solution. Ullah and coworkers [9] attributed the differ-
ences in the response to sodium chloride to divergence in
the electrostatic environment in the active site for the two
enzymes.
While no information exist on the ethanol tolerance of
any phytase, ethanol inhibition of other hydrolytic en-
zymes such as, cellulase, has been cited [2,10,11] and
this has hindered the development of simultaneous sac-
charification and fermentation techniques in bio-ethanol
production to maximize yields and lower both the cost
and energy requirements.
A number of benefits have been proposed for the addi-
tion of phytase during fermentation. First, the hydrolysis
of phytic acid results in more free minerals e.g., calcium,
magnesium, zinc, iron, etc., that are needed for yeast
metabolism and whose availability results in a higher
fermentation rate [12]. Another is that starch hydrolysis
with a α-Amylase has also been shown to be benefitted
by the addition of a phytase to relieve phytic acid inhibi-
tion ofα-Amylase during fermentation [13].
In the present study, a significant difference in ethanol
tolerance has been observed in two commercially mar-
keted phytases. Inactivation of both enzymes by ethanol
is reversed by the removal of ethanol. The fact that dif-
ferences in ethanol tolerance do exist supports the thesis
that further tolerance can be achieved by structural modi-
fication of the enzyme. Such molecular modification may
have application in elevating the ethanol tolerance in
other hydrolytic enzymes.
5. Conclusion
The results presented in this study clearly demonstrated
that ethanol, the main product of starch fermentation,
severely inactivates two commercially important phyta-
ses' catalytic activity even at a low concentration of 10%.
The differences in ethanol tolerance in the two phosphor-
hydrolases can enhance our understanding of how etha-
nol interacts with this class of enzymes and this may
contribute to the design of phytases that retain more ac-
tivity during fermentation and thus lower the phytic acid
content of dried distillers grains with solubles (DDGS).
The achievement of a molecular modification to enhance
ethanol tolerance in phytase may find applications in
other hydrolytic enzymes.
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