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Applied and Environmental Microbiology, November 1998, p. 4446-4451, Vol. 64, No. 11
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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
Markus
Wyss,1,*
Luis
Pasamontes,1
Roland
Rémy,1
Josiane
Kohler,2
Eric
Kusznir,2
Martin
Gadient,3
Francis
Müller,2 and
Adolphus P. G. M.
van
Loon1
Vitamins and Fine Chemicals Division,
Biotechnology Section,1
Product Form
Development,3 and
Pharma
Division,2 Preclinical Research, F. Hoffmann-La
Roche Ltd., CH-4070 Basel, Switzerland
Received 6 May 1998/Accepted 23 August 1998
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ABSTRACT |
Enzymes that are used as animal feed supplements should be able to
withstand temperatures of 60 to 90°C, which may be reached during the
feed pelleting process. The thermostability properties of three
histidine acid phosphatases, Aspergillus fumigatus phytase, Aspergillus niger phytase, and A. niger optimum
pH 2.5 acid phosphatase, were investigated by measuring circular
dichroism, fluorescence, and enzymatic activity. The phytases of
A. fumigatus and A. niger were both denatured
at temperatures between 50 and 70°C. After heat denaturation at
temperatures up to 90°C, A. fumigatus phytase refolded
completely into a nativelike, fully active conformation, while in the
case of A. niger phytase exposure to 55 to 90°C was associated with an irreversible conformational change and with losses
in enzymatic activity of 70 to 80%. In contrast to these two phytases,
A. niger pH 2.5 acid phosphatase displayed considerably higher thermostability; denaturation, conformational changes, and
irreversible inactivation were observed only at temperatures of
80°C. In feed pelleting experiments performed at 75°C, the recoveries of the enzymatic activities of the three acid phosphatases were similar (63 to 73%). At 85°C, however, the recovery of
enzymatic activity was considerably higher for A. fumigatus
phytase (51%) than for A. niger phytase (31%) or pH 2.5 acid phosphatase (14%). These findings confirm that A. niger pH 2.5 acid phosphatase is irreversibly inactivated at
temperatures above 80°C and that the capacity of A. fumigatus phytase to refold properly after heat denaturation may
favorably affect its pelleting stability.
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INTRODUCTION |
The industrial importance of
thermostable enzymes is increasing. Therefore, it comes as no surprise
that isolation, characterization, and engineering of thermostable
enzymes, as well as the search for the determinants of thermostability,
are hot spots of current research (2, 3, 9-11). Different
strategies can be used to obtain a thermostable enzyme with the desired
catalytic activity. These strategies include (i) screening of
thermophilic and hyperthermophilic organisms for the catalytic activity
and substrate specificity of interest; (ii) mutagenesis of a mesophilic
enzyme in order to increase its thermostability; and (iii) mutagenesis
of a known thermostable enzyme that catalyzes a closely related
reaction, with the aim of modifying its substrate specificity.
Phytases (EC 3.1.3.8) belong to the family of histidine acid
phosphatases (4, 7) and are found primarily in
microorganisms and plants. These enzymes catalyze the release of
phosphate from phytic acid (myo-inositol hexakisphosphate),
the major phosphorus storage form in plants. Since monogastric animals
like poultry and pigs have very low or no phytase activities in their
digestive tracts, inorganic phosphorus has to be added to the feed in
order to meet the phosphorus requirements of these animals.
Supplementation with inorganic phosphate, however, imposes ecological
problems (eutrophication), which makes partial substitution of
inorganic phosphate by phytase desirable. Because poultry and pig feed
commonly is pelleted, a commercially attractive phytase should be able to withstand the temperatures that may be reached temporarily during
the pelleting process (60 to 90°C).
As a first step towards the development of a thermostable phytase, a
number of phytases from various molds have been cloned and
overexpressed (4-6). In the present investigation, we
analyzed the thermostabilities of selected wild-type phytases and a pH 2.5 acid phosphatase by measuring circular dichroism (CD),
fluorescence, and activity and by performing feed pelleting
experiments. Aspergillus niger phytase is the only phytase
that is commercially available at present for feed application.
Aspergillus fumigatus phytase performed better in animal
experiments (16) and, at least in a purified form in test
tubes, resisted heat treatment at temperatures up to 100°C
(5). A. niger pH 2.5 acid phosphatase was
included since it was reported to have a higher intrinsic
thermostability than A. niger phytase (13, 14).
The results which we obtained may facilitate the design and development
of improved phytases.
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MATERIALS AND METHODS |
Materials.
Phytic acid (dodecasodium salt) was purchased
from Sigma, and 4-nitrophenyl phosphate (disodium salt) was purchased
from Merck. All of the other chemicals used were at least analytical
grade and were products of Fluka or Bio-Rad. The phytases of A. niger T213 and A. fumigatus ATCC 34625, as well as the
pH 2.5 acid phosphatase of A. niger T213, were overexpressed
in A. niger and purified to apparent homogeneity (17,
18).
The sequence of the A. niger T213 phytase used in the
present investigation differs from the sequence of the previously
described A. niger NRRL3135 phytase (15) in 12 amino acids: S14A, S30T, E66D, D89E, A106V, V155I, K171E, V236A, N292H,
Q297R, S345N, and V438I (4). In addition, it differs in two
amino acids (Q297R and S466F) from the sequence of the A. niger var. awamori ATCC 38854 phytase (7).
Fluorescence spectroscopy.
Fluorescence measurements were
performed with an SLM model 4048S instrument. The slit width was set at
4 nm, the gain was set at 200, and the excitation and emission
wavelengths were set at 280 and 340 nm, respectively. The effects of
temperature on the fluorescence properties of acid phosphatases were
investigated in the following experiment. Samples (in 10 mM sodium
acetate, pH 5.0) were diluted to an optical density at 280 nm of 0.1. Each acid phosphatase was measured in a thermostatted quartz cuvette consecutively at 30, 50, 70, 80, 90, 80, 70, 50, and 30°C. Individual measurements were obtained at 10-min intervals, the time needed to heat
or cool the sample and to allow for adequate preincubation at the
subsequent temperature. The enzymatic activities of the samples were
measured both at the beginning and at the end of the experiment.
CD spectroscopy.
CD spectra were recorded with a Jobin Yvon
model CD 6 dichrograph. The instrument was calibrated with
epiandrosterone (
= 3.3 at 304 nm [2a]) and
camphorsulfonic acid ( = 
4.72 at 188 nm and = 2.37 at
289 nm [1]). Far-UV CD spectra were acquired over a
wavelength range of 190 to 260 nm by using 1-nm increments. The
baseline obtained with solvent alone was subtracted from the sample
spectra. Temperature was controlled with a Haake model D8 thermostat
and with a thermostatted cell holder supplied by Jobin Yvon. Residual
molar ellipticities (in degrees per square centimeter per decimole)
were plotted against wavelength. The proportions of secondary-structure
elements (
-helix and 
-sheet) were calculated from the CD spectra
with the program Provencher (8).
The samples (in 10 mM sodium acetate, pH 5.0) used for the CD
measurements were filtered through 0.22-µm-pore-size membranes to
remove particles and aggregates; the protein concentrations in these
samples were 0.27 to 0.41 mg/ml. The samples were incubated for 20 min
at 30, 50, 70, 80, or 90°C. Subsequently, either the CD spectra were
measured directly at the same temperatures, or the samples were first
allowed to renature for 1 h at 30°C and then the CD spectra were
measured at 30°C.
Measurements of enzymatic activity.
Phytase activity was
measured in an assay mixture containing 0.5% phytic acid (~5 mM) and
200 mM sodium acetate (pH 5.0). The enzymatic activity of the pH 2.5 acid phosphatase was measured in an assay mixture containing
4-nitrophenyl phosphate (0.65 mg/ml) and 50 mM glycine (pH 2.5). In
feed samples (see Fig. 7), the phytase activity was measured in an
assay mixture containing 5 mM phytic acid and 200 mM sodium acetate (pH
5.5), and the acid phosphatase activity was measured in an assay
mixture containing 5 mM 4-nitrophenyl phosphate and 200 mM sodium
acetate (pH 5.0).
After 15 min of incubation at 37°C (or at 40, 45, 50, 55, 60, 65, 70, 80, and 90°C for the experiment shown in Fig.
6), the
reaction was
stopped by adding an equal volume of 15% trichloroacetic
acid. The
liberated phosphate ions were quantified by mixing 100
µl of the
assay mixture with 900 µl of H
2O and 1 ml of 0.6 M
H
2SO
4-2%
ascorbic acid-0.5% ammonium
molybdate. After incubation for 20
min at 50°C, the absorbance at 820 nm was measured. Standard solutions
of potassium phosphate were used as
references. In one experiment
(see Fig.
6), the enzyme and substrate
were preincubated separately
for 10 min at the appropriate temperatures
and then mixed to start
the
reaction.
Gel permeation chromatography.
The three acid phosphatases
(concentrations, 0.27 to 0.41 mg/ml in 10 mM sodium acetate [pH 5.0])
were incubated for 20 min at 30, 70, 80, or 90°C and then for 2 h on ice. Their molecular sizes were then determined at room
temperature by gel filtration on a Superdex 200 column (fast protein
liquid chromatography; Pharmacia). The elution buffer contained 50 mM
sodium phosphate, 150 mM NaCl, 0.2 mM disodium EDTA, 2 mM
2-mercaptoethanol, and 1 mM sodium azide (pH 7.2).
Feed pelleting.
A commercial broiler feed containing 55%
maize, 27% soya 50, 10% extruded soya, 3% fish meal, 1% soya oil,
and 4% mineral premix B4245 (Agrobase, Bourgen Bresse, France) was
extruded at 120°C (in order to inactivate acid phosphatases present
in the feed) and crumbled. For each test, approximately 15 kg of the treated feed and 500 to 800 U of phytase or acid phosphatase (as a
fermentation supernatant) per kg of feed were combined and mixed with a
Forberg type F-60 mixer. Subsequently, the mixed product was pelleted
with a Bühler model DFPL pellet mill at 75 or 85°C. For each
batch, one sample before pelleting and three samples after pelleting
were withdrawn and used to analyze the phytase or acid phosphatase
activity. The feed pellets were ground with a mortar and pestle,
aliquots (5 g) were suspended in 40 ml of 200 mM sodium acetate (pH
5.5), and each suspension was stirred for 30 min at 4°C. After
centrifugation for 10 min at 3,000 × g and extensive
dialysis of the supernatant against 200 mM sodium acetate (pH 5.5) at
4°C, phytase or acid phosphatase activity was determined as described above.
Other methods.
Protein concentrations were calculated from
the optical densities at 280 nm by using theoretical absorption values
calculated from the known protein sequences with the DNA* software
(DNASTAR, Inc.). An absorption value of 1 A at 280 nm corresponded to
0.63 mg of pH 2.5 acid phosphatase per ml, 0.94 mg of A. fumigatus phytase per ml, and 1.04 mg of A. niger T213
phytase per ml. Sodium dodecyl sulfate-polyacrylamide gel
electrophoresis was performed with 8 to 16% Tris-glycine gradient gels
supplied by Novex. The gels were stained with colloidal Coomassie stain (Novex).
| |
RESULTS |
Temperature-dependent changes in protein fluorescence.
Fluorescence measured at excitation and emission wavelengths of 280 and
340 nm, respectively, reveals the local environments of aromatic
residues in proteins. Fluorescence typically decreases as the
temperature increases and when aromatic residues become exposed to a
more hydrophilic environment.
As shown in Fig.
1, protein fluorescence
in fact decreased as the temperature was increased. In the case of
A. niger T213
phytase (Fig.
1B), at any temperature between
70 and 90°C, the
observed fluorescence was the same whether the
temperature was
increased from 70 to 90°C or decreased subsequently
from 90 to
70°C. In contrast, the values obtained at 30 and 50°C
were considerably
higher after heat treatment than before heat
treatment. At the
end of the experiment (i.e., when the preparation was
returned
to 30°C), the residual enzymatic activity of
A. niger T213 phytase
was only 22% of the initial activity. When in
a separate experiment
the enzyme was incubated consecutively at 20 and
50°C and then
again at 20°C, fluorescence returned to the control
value (0.226
versus 0.231 arbitrary unit). After incubation at 20 and
55°C
and then again at 20°C, however, a large increase in
fluorescence
was noticed (0.355 versus 0.226 arbitrary unit),
suggesting that
there was an irreversible change in the protein
conformation at
temperatures between 50 and 55°C that was associated
with inactivation
of the enzyme.
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FIG. 1.
Temperature-dependent changes in protein fluorescence.
(A) A. fumigatus phytase. (B) A. niger T213
phytase. (C) A. niger pH 2.5 acid phosphatase. Symbols:  ,
fluorescence changes associated with increases in temperature from 30 to 50, 70, 80, and 90°C;  , fluorescence changes associated with
decreases in temperature from 90 to 80, 70, 50, and 30°C. F,
fluorescence; a.u., arbitrary units. For experimental details see
Materials and Methods.
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In contrast to
A. niger T213 phytase, there was no evidence
that there were irreversible changes in the conformations of
A. fumigatus phytase (Fig.
1A) and
A. niger pH 2.5 acid
phosphatase
(Fig.
1C) after they were heated to 90°C and then cooled.
However,
pH 2.5 acid phosphatase was completely inactivated in this
experiment,
while
A. fumigatus phytase exhibited 87%
residual
activity.
Unfolding and refolding properties of acid phosphatases revealed by
CD spectroscopy.
CD spectroscopy was used to determine the effects
of temperature on the proportions of secondary-structure elements. When CD spectra are compared to a reference data set, they can be used to
predict the -helical and -sheet contents of a protein. Aliquots of the individual acid phosphatases were preincubated for 20 min at 30, 50, 70, 80, or 90°C and were then directly measured at the same
temperatures (Fig. 2A, C, and E).
Alternatively, after the initial 20-min incubation period, the proteins
were allowed to renature for 1 h at 30°C and then were measured
at 30°C (Fig. 2B, D, and F).
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FIG. 2.
Temperature-dependent unfolding and refolding of
phytases and pH 2.5 acid phosphatase as determined by CD measurements.
(A and B) A. fumigatus phytase. (C and D) A. niger T213 phytase. (E and F) A. niger pH 2.5 acid
phosphatase. The individual acid phosphatases were incubated for 20 min
at 30°C ( ), 50°C (----), 70°C
(······),
80°C
(-·-·-),
or 90°C
(-···-···-).
Then either the CD spectra were determined directly at the same
temperatures (A, C, and E), or the phosphatases were allowed to
renature for 1 h at 30°C, and then the spectra were determined
at 30°C (B, D, and F). The isodichroitic point observed for A. fumigatus phytase is indicated by an arrow in panel A.  ,
residual molar ellipticity.
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|
A. fumigatus phytase (Fig.
2A) and
A. niger T213
phytase (Fig.
2C) seemed to be equally sensitive to heat and were
denatured
at temperatures between 50 and 70°C. When heat-denatured
A. fumigatus phytase was returned to 30°C, it refolded
into a nativelike conformation
(Fig.
2B). Proper refolding of
A. niger T213 phytase, on the other
hand, was observed only after
exposure to temperatures up to 50°C
(Fig.
2D). Consistent with the
hypothesis that there was an irreversible
conformational change at
temperatures between 50 and 55°C (see
above), the CD spectra of
refolded
A. niger T213 phytase after
exposure to 70, 80, and
90°C were significantly different from
the control spectrum of the
native enzyme. The different behaviors
of the two phytases are also
reflected in the calculated
-helical
contents shown in Fig.
3 and by the fact that the CD curves for
A. fumigatus phytase have an isodichroitic point (Fig.
2A,
arrow),
while those of
A. niger T213 phytase do not (Fig.
2C). An isodichroitic
point is a strong indication that one defined
species is transformed
into another.
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FIG. 3.
Temperature-dependent changes in calculated -helical
contents. (A) A. fumigatus phytase. (B) A. niger
T213 phytase. (C) A. niger pH 2.5 acid phosphatase. The
-helical contents were calculated from the CD spectra shown in Fig.
2. Symbols: , -helical contents after 20 min of incubation at 30, 50, 70, 80, or 90°C; , -helical contents after a subsequent 1-h
renaturation period at 30°C.
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|
Almost no change in the CD spectral characteristics of pH 2.5 acid
phosphatase was observed at temperatures up to 80°C (Fig.
2E),
suggesting that this enzyme is in fact more thermostable
than the two
phytases which we investigated. Only at 90°C was
a clear shift in the
CD spectrum noticed, and this shift apparently
was irreversible (Fig.
2E and
F).
In order to correlate the CD spectral changes with enzyme inactivation,
aliquots of the individual incubation mixtures were
withdrawn at the
end of the 20-min heating period and immediately
put on ice. Since the
samples were on ice for at least 1 h prior
to analysis, the data
in Fig.
4 reflect the enzymatic
activities
of the refolded proteins. Evidently, there was a perfect
correlation
among activity, CD, and fluorescence measurements. Only two
points
deserve further attention. First, although
A. niger
pH 2.5 acid
phosphatase is more thermostable than the two phytases, it
is
in fact completely and irreversibly inactivated once the
conformational
transition occurs (at 90°C and, to a small extent, at
80°C). Second,
sodium dodecyl sulfate-polyacrylamide gel
electrophoresis control
experiments confirmed that interference by
protein degradation,
even at 90°C, could not account for any of the
findings presented
in this paper (data not shown).
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FIG. 4.
Temperature-dependent enzyme inactivation. (A) A. fumigatus phytase. (B) A. niger T213 phytase. (C)
A. niger pH 2.5 acid phosphatase. The individual acid
phosphatases were incubated for 20 min at 30, 50, 70, 80, or 90°C and
then immediately put on ice. The results represent means ± standard deviations (n = 3). Enzymatic activities were
measured as described in Materials and Methods.
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|
Heat-induced aggregation of pH 2.5 acid phosphatase.
The
phytases of A. niger T213 and A. fumigatus are
monomeric proteins, while A. niger pH 2.5 acid phosphatase
is an oligomer that is most likely composed of four identical protomers
(Fig. 5) (3a). It might be
anticipated that heat denaturation disrupts the tetrameric structure of
pH 2.5 acid phosphatase and that, once dissociated, the individual
protomers are unable to refold and reassociate properly into an active
tetramer. Gel permeation chromatography, however, demonstrated that
exposure to 90°C and, to a lesser extent, exposure to 80°C were
associated with the formation of high-Mr
aggregates (Fig. 5). No such aggregates were observed for the phytases
of A. niger T213 and A. fumigatus despite the
fact that A. niger T213 phytase is also inactivated
significantly at 90°C.
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FIG. 5.
Effect of heat denaturation on the molecular sizes of
phytases and pH 2.5 acid phosphatase. (A) A. fumigatus
phytase. (B) A. niger T213 phytase. (C) A. niger
pH 2.5 acid phosphatase. The individual acid phosphatases were
incubated for 20 min at 30°C (curve a in panels A and B), 70°C
(curve a in panel C), 80°C (curve b in panel C), and 90°C (curve b
in panels A and B and curve c in panel C), allowed to refold for 2 h on ice, and then analyzed by gel permeation chromatography. The
reference proteins blue dextran 2000 (Mr,
2,000,000), ferritin (Mr, 440,000), and
albumin (Mr, 67,000) eluted at 8.5, 11.3, and 14.4 ml, respectively. OD280, optical density at 280 nm.
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|
Temperature dependence of enzymatic activity.
To assess
specifically heat denaturation of the active site, activity was
measured directly at a series of temperatures between 37 and 90°C
(Fig. 6). Both phytases were inactivated
at temperatures above 55°C, while loss of activity of pH 2.5 acid
phosphatase was observed only at temperatures above 60 to 65°C. Since
pH 2.5 acid phosphatase activity had to be measured at pH 2.5 (this
enzyme exhibits virtually no activity at pH 5.0), this experiment
suffered from the limitation of not corresponding strictly with the
other investigations. Nevertheless, in qualitative terms, its results are consistent with the previous conclusions and confirm that pH 2.5 acid phosphatase is more thermostable than the A. niger T213
and A. fumigatus phytases.
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FIG. 6.
Temperature dependence of enzymatic activity. Symbols:
 , A. fumigatus phytase;  , A. niger T213
phytase; , A. niger pH 2.5 acid phosphatase. The
enzymatic activities were measured directly at the temperatures
indicated. The activity at 37°C was defined as 100%.
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Thermostability in feed pelleting experiments.
Experiments
performed in test tubes may not properly reflect the conditions in the
feed pelleting process. Therefore, the three acid phosphatases were
added in small volumes of liquid to extruded mash feed which
subsequently was pelleted at either 75 or 85°C. No significant
differences in the recovery of enzymatic activities were observed after
feed pelleting at 75°C (Fig. 7) despite
rather pronounced differences in the thermostability properties of the
purified proteins in dilute, buffered aqueous solutions (see above).
The low recovery of A. niger pH 2.5 acid phosphatase activity after pelleting at 85°C (14.1% ± 1.7%) may have been due
to the fact that this enzyme undergoes an irreversible conformational change that is associated with enzyme inactivation at temperatures above 80°C (see above). The recovery of more A. fumigatus
phytase activity than of A. niger phytase activity at 85°C
may be a reflection of the capacity of A. fumigatus phytase
to refold properly into an active conformation after heat denaturation.
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FIG. 7.
Recovery of enzymatic activity after feed pelleting. The
three acid phosphatases were added in liquid form to extruded mash feed
which was then pelleted at 75°C (solid bars) or 85°C (open bars).
Phytase and pH 2.5 acid phosphatase activities were measured in the
feed samples before and after pelleting.
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| |
DISCUSSION |
In the present investigation, we analyzed the temperature
sensitivity of acid phosphatases at three different levels of molecular organization, as follows: (i) fluorescence spectroscopy probed structural alterations in the immediate vicinity of aromatic residues; (ii) the temperature dependence of enzymatic activity revealed at which
temperature the active site was denatured or structurally affected in
such a way that catalysis was no longer possible; and (iii) far-UV CD
spectra revealed overall changes in the proportions of
secondary-structure elements. The last two approaches do not necessarily provide identical results, since it has been shown previously that the active sites of diverse enzymes are more
susceptible to denaturants than the global structures are (12,
19). Despite the different parameters that are measured by the
techniques which we used, all of the experiments described above
provided a consistent picture which showed that the three acid
phosphatases have distinct thermostability properties.
A. niger T213 phytase is not thermostable, nor does it have
the capacity to refold properly after heat denaturation. At
temperatures between 50 and 55°C, this enzyme undergoes an
irreversible conformational rearrangement that is associated with
losses in enzymatic activity of 70 to 80%.
A. fumigatus phytase, like A. niger T213 phytase,
is not thermostable but has the remarkable property of being able to
refold completely into a nativelike, fully active conformation after heat denaturation.
Compared to the two phytases, A. niger T213 pH 2.5 acid
phosphatase has higher intrinsic thermostability, a conclusion that is
consistent with previous findings (13, 14). At temperatures up to 80°C, only minor changes in CD spectral characteristics and
only slight (irreversible) enzyme inactivation were observed under the
experimental conditions used. Exposure to 90°C, however, resulted in
an irreversible conformational change that was associated with the
formation of high-Mr aggregates and with
complete inactivation of the enzyme. It is quite surprising that two
proteins from the same organism, the phytase and the pH 2.5 acid
phosphatase of A. niger T213, exhibit such a pronounced
difference in thermostability. Whether this finding has physiological
relevance remains to be determined.
A number of criteria must be fulfilled by a phytase if it is to be
attractive for widespread use in the animal feed industry. It should be
thermostable and protease resistant, and it should have broad substrate
specificity and a high specific activity. Even though the recovery of
enzymatic activity after feed pelleting of the nonformulated acid
phosphatases at 85°C ranged from 14 to 51%, the results presented
here are a step towards meeting these ambitious criteria.
| |
ACKNOWLEDGMENTS |
Kurt Vogel and Martin Lehmann are gratefully acknowledged for
stimulating discussions, and Alexandra Kronenberger and Stefan Jäggli are acknowledged for expert technical assistance.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: F. Hoffmann-La
Roche Ltd., Vitamins and Fine Chemicals Division, Biotechnology
Section, Bldg. 93/456, CH-4070 Basel, Switzerland. Phone:
41-61-688-2972. Fax: 41-61-688-1645. E-mail:
markus.wyss{at}roche.com.
| |
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Applied and Environmental Microbiology, November 1998, p. 4446-4451, Vol. 64, No. 11
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
