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Applied and Environmental Microbiology, October 2001, p. 4701-4707, Vol. 67, No. 10
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.10.4701-4707.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Expression, Gene Cloning, and Characterization of
Five Novel Phytases from Four Basidiomycete Fungi: Peniophora
lycii, Agrocybe pediades, a Ceriporia sp., and
Trametes pubescens
Søren F.
Lassen,1,*
Jens
Breinholt,2
Peter R.
Østergaard,1
Roland
Brugger,3
Andrea
Bischoff,3
Markus
Wyss,3 and
Claus C.
Fuglsang1
Novozymes A/S, DK-2880
Bagsværd,1 and Novo Nordisk A/S,
DK-2760 Måløv,2 Denmark, and F.
Hoffmann-La Roche, Ltd., CH-4070 Basel, Switzerland3
Received 30 April 2001/Accepted 1 August 2001
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ABSTRACT |
Phytases catalyze the hydrolysis of phosphomonoester bonds of
phytate (myo-inositol hexakisphosphate), thereby creating
lower forms of myo-inositol phosphates and inorganic
phosphate. In this study, cDNA expression libraries were constructed
from four basidiomycete fungi (Peniophora lycii, Agrocybe
pediades, a Ceriporia sp., and Trametes
pubescens) and screened for phytase activity in yeast. One
full-length phytase-encoding cDNA was isolated from each library, except for the Ceriporia sp. library where two different
phytase-encoding cDNAs were found. All five phytases were expressed in
Aspergillus oryzae, purified, and characterized. The
phytases revealed temperature optima between 40 and 60°C and pH
optima at 5.0 to 6.0, except for the P. lycii phytase,
which has a pH optimum at 4.0 to 5.0. They exhibited specific
activities in the range of 400 to 1,200 U · mg, of
protein
1 and were capable of hydrolyzing phytate down to
myo-inositol monophosphate. Surprisingly, 1H
nuclear magnetic resonance analysis of the hydrolysis of phytate by all
five basidiomycete phytases showed a preference for initial attack at
the 6-phosphate group of phytic acid, a characteristic that was
believed so far not to be seen with fungal phytases. Accordingly, the
basidiomycete phytases described here should be grouped as 6-phytases
(EC 3.1.3.26).
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INTRODUCTION |
Phytases
(myo-inositol hexakisphosphate phosphohydrolases) belong to
the family of histidine acid phosphatases sharing the sequence
consensus pattern
[LIVM]-X-X-[LIVMA]-X-X-[LIVM]-X-R-H-[GN]-X-R-X-[PAS] (27;
http://www.expasy.ch/cgi-bin/get-prodoc-entry?PDOC00538). They are
capable of catalyzing the hydrolysis of phosphomonoester bonds of
phytate (salts of myo-inositol hexakisphosphate or
myo-inositol 1,2,3,4,5,6-hexakis dihydrogen phosphate), thereby
creating lower forms of myo-inositol phosphates and
inorganic phosphate (22, 24). Phytases are grouped
according to the specific position of the phosphate ester group on the
phytate molecule at which hydrolysis is initiated, i.e., as 3-phytases
(EC 3.1.3.8) or as 6-phytases (EC 3.1.3.26).
Phytate is the primary source of inositol and the primary storage form
of phosphate in plant seeds (23). Seeds, cereal grains, and legumes are important components of food and, in particular, of
animal feed preparations. However, monogastric animals such as poultry
and swine are incapable of utilizing the phosphorus bound in phytate
due to low levels of phytase activity in the digestive tract.
Furthermore, phytate acts as an antinutrient by chelating divalent
cations and preventing the uptake of minerals, e.g., Zn
(9). Thus, phytases are used as a cereal feed additive that enhances the phosphorus and mineral uptake in monogastric animals
and reduces the level of phosphate output in their manure. Recently,
there have been several reports on the cloning of fungal PhyA phytases
from Aspergillus niger (16, 20, 28),
Aspergillus fumigatus (19), Aspergillus
terreus, Myceliophthora thermophila (14),
Emericella nidulans, Talaromyces thermophilus
(18), and Thermomyces lanuginosus
(2). Based on their characteristics and on their sequence
similarity, the PhyA phytases form a subclass within the histidine acid
phosphatase family (14). So far, all the reported cloned
filamentous fungal PhyA phytases were from the order
Eurotiales or Sordariales within the phylum
Ascomycota. Here, we report evidence for PhyA phytases being
more widely distributed in the fungal kingdom. This was done by
expression cloning, overexpression, and characterization of the first
five PhyA phytases from four orders within the phylum
Basidiomycota: Stereales, Agaricales, Phanerochaetales, and Coriolales.
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MATERIALS AND METHODS |
Materials.
All chemicals were obtained from Merck and were
of analytical grade unless stated otherwise.
Fungal strains and growth conditions.
Peniophora
lycii CBS 686.96, Agrocybe pediades CBS No. 900.96, Ceriporia sp. CBS 100231 (the CBS 100231 strain was
previously designated Paxillus involutus), and
Trametes pubescens CBS 100232 were cultivated in shake
flasks containing 100 ml of growth medium (soya flour, 30 g/liter;
maltodextrin, 15 g/liter; Bacto Peptone, 5 g/liter; and pluronic PE
6100, 0.2 g/liter). The P. lycii culture was
incubated without agitation at 26°C for 15 days, and the A. pediades, Ceriporia sp., and T. pubescens cultures were
incubated at 26°C for 5 days with agitation. Aspergillus
oryzae A1560 transformants were grown in YP medium containing
35 g of maltodextrin per liter as described in reference
4.
RNA isolation and construction of directional cDNA
libraries.
RNA isolation and the construction of the four
directional cDNA libraries in the yeast expression vector pYES2
(Invitrogene) were carried out essentially as described in reference
13.
Screening of the cDNA library.
The libraries were amplified
in Escherichia coli strain DH10B (Life Technologies) and
transformed into Saccharomyces cerevisiae W3124
(25) by electroporation. The S. cerevisiae
transformants were plated on synthetic complete agar containing
2% glucose and incubated at 30°C for 3 days. The transformants were
transferred to phytate replication plates (synthetic complete agar
containing 2% galactose, 0.5% threonine, 20 mM CaCl2, 20 mM MgCl2, 20 mM Na-phytate, and 0.1% trace element
solution [pH 6.5]) (trace element solution no. 141; Deutsche Sammlung
von Mikroorganismen und Zellkulturen GmbH Catalogue of Strains)
and incubated for 3 to 5 days at 30°C. LSB-agarose
(1%; BioWhittaker Molecular Applications) containing 0.2 M
CaCl2 was poured over the plates and after 1 to 4 days, the
phytase-positive colonies were identified by a surrounding clearing
zone. Isolation and rescuing of the phytase-encoding cDNA in pYES2 were
carried out as previously described (5).
Nucleotide sequence analysis.
The cDNA sequences from both
strands were determined with the Dye Terminator Cycle Sequencing kit
(Perkin-Elmer) and an Applied Biosystems ABI PRISM 377 DNA sequencer
according to the manufacturer's instructions.
Recombinant expression in A. oryzae.
The
phytase-encoding cDNA inserts were subcloned into
Aspergillus expression vector pHD414 (7) or,
for the A. pediades insert, into the pHD423 expression
vector (a pHD414 derivative with a KpnI site in the
polylinker). Plasmid DNA was isolated and cotransformed into A. oryzae A1560 with an amdS+ plasmid
(4). The amdS+ transformants were
screened for phytase activity in culture, and phytase-producing
transformants were isolated.
Purification.
The phytases were purified from the culture
supernatants of phytase-producing A. oryzae transformants.
The A. pediades, P. lycii, and T. pubescens
phytases were purified according to the following procedure. Filter aid
was added to the culture broth, and the broth was first filtered
through a filtration cloth and then through a Seitz depth filter plate. The filtrate was concentrated by ultrafiltration on 10-kDa cut-off polyethersulfone membranes, followed by diafiltration with distilled water to reduce the conductivity to less than 2 mS/cm. The concentrated enzyme was adjusted to a pH of 7.5 and applied to a Q-Sepharose FF
anion-exchange column (Amersham Pharmacia Biotech) equilibrated in 20 mM Tris-CH3COOH, pH 7.5. Bound proteins were eluted with a
linear NaCl gradient from 0 to 0.5 M. After addition of
(NH4)2SO4 to a final concentration
of 1.5 M, the phytase eluate was applied to a Phenyl Toyopearl 650S
column (TosoHaas) equilibrated in 1.5 M
(NH4)2SO4-10 mM succinic
acid-NaOH, pH 6.0. The column was washed with the equilibration
buffer, and bound protein was eluted with a linear
(NH4)2SO4 gradient from 1.5 to 0 M. The phytase eluate was buffer exchanged on a Sephadex G25 column
(Amersham Pharmacia Biotech) equilibrated in 20 mM HEPES-NaOH, pH 7.5, and applied to a SOURCE 30Q column (Amersham Pharmacia Biotech)
equilibrated in the same buffer. The column was washed thoroughly with
the equilibration buffer, and bound proteins were eluted with a linear NaCl gradient from 0 to 0.3 M. Fractions from the SOURCE 30Q column were evaluated by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE), pure phytase fractions were pooled, and the
pH was adjusted to 6.0.
The two phytases from the
Ceriporia sp., PhyA1 and PhyA2,
were purified as described above with the following modifications:
after concentration by ultrafiltration,
(NH
4)
2SO
4 was added to
a final
concentration of 2.0 M and the sample was applied to a
Phenyl Toyopearl
650S column equilibrated in 2.0 M
(NH
4)
2SO
4 and
10 mM succinic
acid-NaOH, pH 6.0, followed by the elution of bound
protein with a
linear (NH
4)
2SO
4 gradient from 2.0 to 0 M. The
phytase eluate was buffer exchanged on a Sephadex G25
column equilibrated
in 20 mM HEPES-NaOH, pH 7.5, and applied to a Q
Sepharose FF column
equilibrated in the same buffer, followed by the
elution of bound
protein as described above. The phytase eluate was
dialyzed overnight
against 20 mM HEPES-NaOH, pH 7.5, before being
applied to a SOURCE
30Q column. The remaining steps of the procedure
were performed
as described
above.
Tris-glycine SDS-PAGE, and IEF.
SDS-PAGE was
performed with 8 to 16% Tris-glycine gradient gels and isoelectric
focusing (IEF) on pH 3 to 7 or pH 3 to 10 IEF gels
(Novex/Invitrogen). The gels were stained with colloidal Coomassie blue
(Novex/Invitrogen) or semidry blotted onto Immobilon P polyvinylidene
difluoride membranes (Millipore), followed by amido black (naphthol
blue-black) staining.
Measurement of enzymatic activity.
Enzyme samples diluted in
0.1 M sodium acetate and 0.01% Tween-20, pH 5.5, were further diluted
26-fold into the substrate solution (5 mM sodium phytate [Sigma] in
0.1 M sodium acetate, and 0.01% Tween-20 [pH 5.5], preincubated at
37°C) to start the reaction. After 30 min at 37°C, the reaction was
stopped by adding an equal volume of 10% trichloroacetic acid. Free
inorganic phosphate was measured by the addition of an equal volume of
molybdate reagent containing, in 100 ml, 7.3 g of
FeSO4, 1.0 g of
(NH4)6Mo7O24 · 4H2O, and 3.2 ml of H2SO4.
Absorbance was measured at 750 nm (Vmax microtiter plate reader;
Molecular Devices). 1 unit equals the amount of enzyme that releases 1 µmol of phosphate per min. pH activity profiles were obtained by
running the assay with 0.1 M concentrations of the following buffers:
glycine-HCl at pH 3.0 to 3.5, sodium acetate at pH 4.0 to 5.5, morpholincethanesulfonic acid (MES) at pH 6.0 to 6.5, and
Tris-HCl at pH 7.0 to 9.0.
N-terminal sequencing.
Automated Edman degradation of
purified phytases was done with a Perkin-Elmer ABI 494HT sequencer with
online microbore phenylthiohydantoin-amino acid detection.
DSC.
The samples were desalted on a NAP-5 column (Amersham
Pharmacia Biotech) equilibrated with 0.1 M sodium acetate, pH 5.5. Differential scanning calorimetry (DSC) was performed with the VP-DSC
Micro Calorimeter (MicroCal) by applying a constant scan rate of
90°/h from 20 to 100°C.
Calculation of theoretical Mr and pI
values.
Theoretical Mr and pI values were
calculated from the cDNA deduced amino acid sequences corresponding to
the mature proteins with the programs in Vector NTI (Molecular Biology
software; InforMax, Inc.).
HPLC analysis of phytic acid degradation intermediates.
Samples of purified phytases were incubated at 37°C in 200 µl of an
assay mixture containing 200 mM sodium acetate, pH 5.0, with a final
concentration of 0.2 mM phytic acid which was radioactively labeled
with 1 µCi of
myo-[inositol-2-3H(N)]hexakisphosphate
(NEN Research Products, DuPont) per ml. The incubation was stopped
after 2.5, 5, 10, 15, 20, 25, 30, 40, 50, 60, or 90 min by the addition
of an equal volume of acetonitrile and subsequent heating at 95°C for
2 min. After centrifugation for 10 min at 10,000 × g,
a 150-µl aliquot of the supernatant was diluted with an equal volume
of H2O. A 200-µl sample was assayed by high-performance
liquid chromatography (HPLC) anion-exchange chromatography on a
4.6 by 250 mm Zorbax SAX (5-µm) column at a flow rate of 1 ml/min, according to reference 26.
1H NMR spectroscopic analysis of phytase-catalyzed
hydrolysis of phytic acid.
Nuclear magnoic resonance (NMR) spectra
were recorded at 27°C with a Bruker DRX400 instrument equipped with a
5-mm selective inverse probe head. A total of 16 scans preceded by 4 dummy scans were accumulated using a sweep width of 2,003 Hz (5 ppm)
covered by 8,000 data points. Attenuation of the residual
HOD resonance was achieved by a weak presaturation period of 3 seconds. The spectra were referenced to the HOD signal (
4.75).
Phytate samples for NMR analysis were prepared as follows: phytate (100 mg of phytic acid dipotassium salt; Sigma P-5681)
was dissolved in
deionized water (4.0 ml), and the pH was adjusted
to 5.5 or 3.5 by the
addition of NaOH (4 N). Deionized water was
added to a total volume of
5 ml, 1-ml portions were transferred
to screw-cap vials, and the
solvent was evaporated (vacuum centrifuge).
The dry samples were
dissolved in deuterium oxide (2 ml, 99.5%
D; Merck), evaporated to
dryness, and stored at
18°C until
use.
For NMR analysis, a phytate sample was dissolved in deuterium oxide
(1.0 ml), thus yielding a phytate concentration of 27
mM. The solution
was transferred to an NMR tube, and the
1H NMR spectrum was
recorded. One unit of the phytase in question
was added, followed by
mixing and immediate initiation of recording
of
1H NMR
spectra. Subsequent spectra were acquired after 5, 10, 15,
20, 25, 30, 45, 60, 75, 90, 105, 120, 135, 150, 165, 180, 195,
and 210 min (=
3.5 h) and 4.5, 5.5, 6.5, 7.5, 8.5, 9.5, 11.5,
13.5, 15.5, 17.5, 19.5, 21.5, and 23.5
h.
Nucleotide sequence accession numbers.
The cDNA nucleotide
sequences of P. lycii phyA, A. pediades phyA, Ceriporia sp.
phyA1 and phyA2, and T. pubescens phyA
have been deposited in the EMBL database under accession no. AJ310696, AJ310697, AJ310698, AJ310699, and AJ310700, respectively.
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RESULTS |
Isolation and characterization of phytase-encoding cDNA
clones.
Between 20,000 and 30,000 S. cerevisiae
colonies from each of the four cDNA libraries were screened for phytase
activity, and one to four phytase-producing S. cerevisiae
clones were isolated from each cDNA library. The positive colonies
corresponded to five different phytase genes, P. lycii phyA, A. pediades phyA, Ceriporia sp. phyA1 and
phyA2, and T. pubescens phyA. Where more than one
clone containing the same gene was isolated, the clone with the longest
cDNA insert was chosen for further analysis. For details of the cDNA
sequences, the reader is referred to the EMBL database (for accession
numbers, see Materials and Methods).
All five basidiomycete phytases contain the consensus pattern of
histidine acid phosphatases and display some sequence similarity
to the
known PhyA phytases of the phylum
Ascomycota, although
there
is a higher internal sequence similarity within the basidiomycete
phytases. This suggests that these basidiomycete phytases form
a
distinct class of phytases, which is further supported by the
observation of several amino acid sequence motifs conserved for
the
basidomycetes phytases (Fig.
1).
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FIG. 1.
Amino acid sequence alignment of three regions where
basidiomycete phytases share a high degree of amino acid conservation.
In P. lycii PhyA, consensus region I corresponds to residues
68 to 83, consensus region II corresponds to residues 162 to 171, and
consensus region III corresponds to residues 415 to 433. Identical
residues in at least 10 of the sequences are indicated by a black box;
a white box indicates identical residues for the basidiomycete
phytases.
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Enzyme characterization.
Molecular properties and
characteristics of the recombinantly expressed and purified
basidiomycete phytases are presented in Tables
1 and 2.
SDS-PAGE analysis showed the pure preparations for the recombinantly
expressed phytases as a single broad band, and furthermore the
Mrs determined were significantly higher than the Mrs calculated from the corresponding mature
amino acid sequences. This is probably due, as shown previously for
several ascomycete phytases (29), to glycosylation of the
secreted protein as all the phytase sequences contain several potential
N-glycosylation sites (Table 1).
The temperature optima obtained from temperature-activity profiles
fully agree with the thermal stabilities measured by DSC;
even at the
relatively high enzyme concentrations used in DSC,
the phytases showed
a significant degree of refolding after thermal
unfolding. This
confirms the findings in reference
30 that the
previously
reported heat stability of fungal PhyAs (
20) is due
to
reversible thermal unfolding rather than to intrinsic
thermostability.
The pattern of phytic acid degradation intermediates obtained with the
different phytases was studied by HPLC using a limited
amount of
phytate (0.2 mM) as the substrate at pH 5.0 (Fig.
2).
A rapid decline
of the
myo-inositol hexakisphosphate is observed
for all the
phytases, as is the rapid buildup and decline of the
myo-inositol pentakisphosphate. After prolonged incubation,
all
the phytases tended to accumulate relatively high levels of
myo-inositol
bisphosphate, whereas
myo-inositol
monophosphate was accumulated
only slowly. This indicates that the more
highly phosphorylated
inositols are better substrates for the phytases.
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FIG. 2.
HPLC analysis of phytate degradation patterns for
recombinant basidiomycete phytases. The fraction of the different
degrada tion intermediates out of the total amount of
myo-inositol compounds is plotted as the percentage versus
time.  , myo-Inositol hexakisphosphate;  ,
myo-inositol pentakisphosphate;  , myo-inositol
tetrakisphosphate;  , myo-inositol trisphosphate;  ,
myo-inositol bisphosphate;  , myo-inositol
monophosphate. Concentrations: P. lycii PhyA, 0.1 U/ml; A. pediades PhyA, 0.2 U/ml; Ceriporia sp.
PhyA1, 0.25 U/ml; Ceriporia sp. PhyA2, 0.1 U/ml; T. pubescens PhyA, 0.1 U/ml.
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Figure
3 displays stacked plots of
1H NMR spectra recorded over a period of 24 h during
the hydrolysis of 27 mM phytate by
the
A. niger, P. lycii,
and
A. pediades phytases (27°C, pH 5.5).
Concomitant with
the disappearance of the phytate-derived signals,
resonances shifted up
field as a result of dephosphorylation upgrowth.
Distinctive
differences, particularly with respect to the positional
preference for
the initial attack by the enzyme on the substrate,
are reflected in the
initial product profiles (Fig.
4). For
the
A. niger phytase, the dominant pentakisphosphate was
identified
as Ins(1,2,4,5,6)P
5, corresponding to removal of
the phosphate
group in the 3-position, as diagnosed by the intense
doublet signal
at
3.72 attributable to H-3. For the
P. lycii phytase, the triplet
signal at
3.92 was assigned to H-6
in Ins(1,2,3,4,5)P
5, formed
by hydrolysis of the
6-phosphate ester linkage. Accordingly, the
P. lycii phytase
was characterized as a 6-phytase. Finally, for
the
A. pediades phytase, Ins(1,2,3,4,5)P
5 was identified as
the
most abundant pentaphosphate, accompanied by significant amounts
of
Ins(1,2,4,5,6)P
5. Thus, the
A. pediades phytase
attacks both
the 3- and 6-positions, but with a preference for the
6-position.
A similar mixed 3- or 6-position specificity was
observed for
the
Ceriporia sp. and
T. pubescens
phytases (data not shown).
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FIG. 3.
1H NMR profiles of phytate degradation by
A. niger, P. lycii, and A. pediades phytases (27 mM phytate, pH 5.5, 27°C) recorded over a period of 24 h.
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FIG. 4.
The 4.1- to 3.5-ppm region of the 1H NMR
profiles recorded after 20 min of hydrolysis with A. niger, P. lycii, and A. pediades phytases (27 mM phytate, pH 5.5, 27°C) exhibiting the characteristic signals corresponding to H-3 in
Ins(1,2,4,5,6)P5 (A) and H-6 in
Ins(1,2,3,4,5)P5 (B).
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The positional specificities were likewise studied at pH 3.5. The
results were similar to those obtained at pH 5.5, except
for a slightly
decreased 6-position selectivity of the
P. lycii phytase.
H-5 in Ins(1,2)P
2 and Ins(2)P displays characteristic
triplet signals in the
1H NMR spectrum at
3.22 and
3.29, respectively. The transient
accumulation of
Ins(1,2)P
2 and its subsequent transformation into
Ins(2)P
for the
A. niger, P. lycii, and
A. pediades
phytases are
shown in Fig.
5. For the
P. lycii phytase, the Ins(1,2)P
2 level
rises
steeply and peaks after 5 h where it represents approximately
two-thirds of total inositol; whereas in the case of
A. niger phytase, the build-up of Ins(1,2)P
2 occurs more
slowly. In contrast,
the subsequent hydrolysis to the end product,
Ins(2)P, proceeded
more rapidly with the
A. niger phytase
than with the
P. lycii phytase. Except for the initial
specificity differences between
the
P. lycii and
A. pediades phytase, the overall picture, including
their Ins(2)P and
Ins(1,2)P
2 concentration profiles, appears very
similar and
distinctively different from that of
A. niger phytase.
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FIG. 5.
Concentration profiles of Ins(1,2)P2 and
Ins(2)P observed by 1H NMR spectroscopy during enzymatic
digestion of phytate by A. niger phytase [,
Ins(1,2)P2; , Ins(2)P], P. lycii phytase
[, Ins(1,2)P2; , Ins(2)P], and A. pediades phytase [, Ins(1,2)P2; , Ins(2)P] (27 mM phytate, pH 5.5, 27°C).
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DISCUSSION |
We have cloned and characterized five full-length cDNAs encoding
phytase, phyA, from four different basidiomycete fungi. The deduced
amino acid sequences show the characteristics of extracellular fungal
enzymes with a cleavable signal sequence (Table 1).
The N-terminal sequences determined for the mature proteins did not
correspond to the predicted signal peptide cleavage site (17) for any of the five recombinantly expressed
basidiomycete phytases, a phenomenon also seen with the recombinantly
expressed ascomycete phytases studied in reference 29. It
has been proposed that the difference of 10 amino acids between the
predicted and observed N-terminal amino acid sequences for the T. lanuginosus phytase is due to a propeptide, a notion that is
supported by the observation of a Kex2 site (2). It is not
possible to apply this hypothesis in general to the phytases here
reported, since a Kex2 site (Pro-Arg) (15) was found only
for Ceriporia sp. PhyA2. It is more likely that the N
terminus was exposed to aminopeptidase activity after the signal
sequence had been cleaved off. This notion is further supported by the
recent characterization of secreted aminopeptidase (3),
dipeptidyl peptidases (8), and tripeptidyl peptidase (K. Holm, G. Rasmussen, T. Halkaier, and J. Lehmbeck, 8 November
1995, world patent application WO 96/14404) in A. oryzae, of
which at least the aminopeptidase and the tripeptidyl peptidase are
non-specific.
Most of the known phytases are either 3- or 6-phytases (EC 3.1.3.8 or
EC 3.1.3.26, respectively), grouped according to the specific position
of the phosphomonoester group on the phytate molecule at which
hydrolysis is initiated. One exception is an alkaline phytase from lily
pollen that initially hydrolyzes the 5-phosphate group
(1). Previously, phytases of microbial origin such as
A. niger (11), Neurospora crassa
(12), and Pseudomonas phytase (6)
were generally considered to belong to the 3-phytases, while 6-phytases
such as wheat bran phytase (21) were believed to be
restricted mainly to the plant kingdom. Exceptions are the 6-phytases
from Paramecium (26) and E. coli
(10) and now the 6-phytases reported here. Thus, general
rules about the evolutionary distribution of 3- and 6-phytases appear
to be of limited relevance. Furthermore, the distinction between 3- and
6-specific phytases is not clear cut. The E. coli phytase
was shown not to be strictly 6-specific but also to initiate phytate
hydrolysis at the 3-phosphate group, and vice versa for the 3-phytase
from A. niger. A notable example of an enzyme displaying
such a mixed-type behavior is the A. pediades phytase.
The 3- and 6-phytase classification and the NMR experiments do not
address all aspects of stereoisomerism; namely, no distinction is made
between enantiomers. Therefore, the group of 3-phytases may comprise
enzymes with preference for the D-3 (= L-1) or L-3 (= D-1) positions,
and the group of 6-phytases enzymes may comprise those with preference
for the D-6 (= L-4) or L-6 (= D-4) positions. Since the
pentakisphosphates formed by cleaving the D-6 and L-6 phosphomonoester
bonds are enantiomers (mirror-image isomers), they are
indistinguishable by most techniques, including NMR spectroscopy, and
more elaborate procedures have to be applied to determine the exact stereospecificity.
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FOOTNOTES |
*
Corresponding author. Mailing address: Novozymes A/S,
Krogshoejvej 36, DK-2880 Bagsværd, Denmark. Phone: 45 4442 2556. Fax: 45 4442 7828. E-mail: sfl{at}novozymes.com.
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REFERENCES |
| 1.
|
Barrientos, L.,
J. J. Scott, and P. P. N. Murthy.
1994.
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Applied and Environmental Microbiology, October 2001, p. 4701-4707, Vol. 67, No. 10
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.10.4701-4707.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
