Het is echter wel schadelijk om overmatig te alcohol te drinken, zeker als u een infectie heeft. Dit komt de infectie eerder verergerd door het gebruik van alcohol amoxicilline kopen zonder recept Doorgaans worden ze voorgeschreven bij bacteriële infecties die om de één of andere reden niet uit zichzelf over gaan; ze doden dan de bacteriën.
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Appl Microbiol Biotechnol (2004) 65: 110–118
A P P L I E D M I C R O B I A L A N D C E L L P H Y S I O L O G Y
A. Sajidan . A. Farouk . R. Greiner . P. Jungblut . E.-C. Müller . R. Borriss
Molecular and physiological characterisation of a 3-phytasefrom soil bacterium Klebsiella sp. ASR1
Received: 11 September 2003 / Revised: 10 November 2003 / Accepted: 21 November 2003 / Published online: 16 January 2004
Abstract Klebsiella sp. strain ASR1 isolated from an
Indonesian rice field is able to hydrolyse myo-inositolhexakis phosphate (phytate). The phytase protein was
Phytate (myo-inositol 1,2,3,4,5,6-hexakisphosphate) is the
purified and characterised as a 42 kDa protein accepting
main storage form of phosphorous in plants and accounts
phytate, NADP and sugar phosphates as substrates. The
for 20–50% of total soil organic phosphorous (Selle et al.
corresponding gene (phyK) was cloned from chromosomal
). Due to limitation of digestible phosphorous in plant
DNA using a combined approach of protein and genome
and in animal nutrition, it is still common practice to add
analysis, and expressed in Escherichia coli. The recom-
inorganic phosphorous as plant fertiliser and as an animal
binant enzyme was identified as a 3-phytase yielding myo-
feed supplement. Especially in areas of intensive crop and
inositol monophosphate, Ins(2)P, as the final product of
livestock production, this can lead to environmental
enzymatic phytate hydrolysis. Based on its amino acid
pollution when phytase-producing soil microorganisms
sequence, PhyK appears to be a member of a hitherto
hydrolyse phytate to release inorganic orthophosphate into
unknown subfamily of histidine acid phytate-degrading
enzymes with the active site RHGXRXP and HD sequence
Improved phosphorous nutrition is achievable by
motifs, and is different from other general phosphatases
mobilisation of phytate phosphorous by phytate-degrading
and phytases. Due to its ability to degrade sodium phytate
enzymes (phytases). Aspergillus niger phytase is currently
to the mono phosphate ester, the phyK gene product is an
in use as a supplement of animal diet in order to improve
interesting candidate for industrial and agricultural
phosphorus utilisation. Based on sequence homology,
applications to make phytate phosphorous available for
phytases (EC 3.1.3.8 for 3-phytase and EC 3.1.8.26 for 6-
phytase) can be divided into histidine acid phosphatases,plant purple acid phosphatases and Bacillus beta-propellerphytases. Besides their ability to make phytate phospho-rous available, elimination of chelate-forming phytate,which is known to bind nutritionally important minerals is
Electronic Supplementary Material Supplementary material is
another beneficial effect of phytase activity (Reddy et al.
available in the online version of this article at http://dx.doi.
). Most of the known microbial phytases are encoded
by genes that have evolved from histidine acid phospha-
A. Sajidan . A. Farouk . R. Borriss (*)
tases containing the RHGXRXP sequence motif (Mitchell
Department of Bacterial Genetics, Institute of Biology,
et al. With the exception of the E. coli appA gene
(Dassa et al. ), and despite the high number of cloned
fungal phytase genes, there is little information about
10115 Berlin, Germanye-mail: [email protected]
bacterial phytase sequences. In fact, the only phytase
genes known to date from soil bacteria are derived from
Bacillus spp. (Tye et al. These do not contain theRHGXRXP sequence motif and may have evolved from a
R. GreinerCentre for Molecular Biology, Federal Research Centre for
Bacillus alkaline phosphatase ancestor (Idriss et al.
The native phytase produced by Klebsiella terrigena has
been isolated and characterised as a 3-phytase (EC 3.1.3.8;Greiner et al. ); however, the genes encoding
Klebsiella spp. phytase are still unknown.
Max Delbrück Centre for Molecular Medicine,13125 Berlin, Germany
To extend our present knowledge about bacterial
Isolation, PCR amplification, sequencing and cloning of DNA
phytases we performed a survey of phytase-producing
bacteria sampled from soil of an Indonesian rice field.
Genomic DNA from bacteria was isolated from logarithmic growing
Here we report the gene sequence encoding a phytate-
culture by conventional phenol/chloroform extraction (Sambrook et
degrading enzyme from Klebsiella sp. ASR1 and the
al. ). Amplification of the 16S rDNA with sequence-specific
functional characterisation of its product, which degrades
primers 27f 5′ GAGAGTTTGATCCTGGCTCAG 3′ and 765r 5′
phytate to myo-inositol monophosphate [Ins(2)P]. The
CTGTTTGCTCCCCACGCTTTC 3′ yielding a 738 bp fragment,
was carried out as described previously (Damiani et al. For
deduced amino acid sequence of the 3-phytase-encoding
cloning the phytase gene, a 1,263 bp coding region fragment was
gene phyK was found to be distinct from that of other
amplified from the genomic DNA of Klebsiella sp. ASR1 using
known bacterial phytase genes, but it contains sequence
primers AS23 [forward: 1–27]: 5′ atgcaagacatcaggggctgttacgcc 3′
motifs generally conserved in histidine acid phosphatases.
and AS22 [reverse: 1,257–1,233]: 5′ cggcaggaccatggctaccgccgg 3′.
The initial denaturation step was performed for 4 min at 94°C, andwas followed by 30 cycles as follows: denaturation at 94°C for1 min, annealing at 54°C for 1 min, and extension at 72°C for 2 min.
A final extension step at 72°C for 8 min was carried out.
For expression cloning of phosphatase genes, genomic DNA was
partially degraded by digestion with Sau3AI. Following electropho-resis, fragments of 3–15 kb were eluted from agarose gels using a
Qiaex system (Qiagen, Hilden, Germany). Fragments were ligated
Most of the enzyme substrates were purchased from Merck
into dephosphorylated BamHI-linearised pUC18 vector. After
(Darmstadt, Germany). Phytic acid dodecasodium salt was from
transformation into E. coli DH5α, collected plasmid DNA was
Sigma (Steinheim, Germany). All other chemicals such as restriction
retransformed into the phoA− E. coli GE334 in order to avoid
enzymes, salt alkaline phosphatase (SAP), T4 DNA ligase and Taq
endogenous background phosphatase activity due to the host cells.
polymerase were purchased from Appligene (Illkirch, France),
Phosphatase-expressing clones were detected on LB plates by their
Promega (Heidelberg, Germany), and USB Biochemicals (Cleve-
brown-red colour after developing with a reagent containing 1-
land, Ohio), and were used according to the instructions of the
naphthyl phosphate (0.1%, w/v), Fast Garnet salt (Sigma, 0.1% w/v)
manufacturers. The oligonucleotides were products from Genset
and 0.5 M sodium acetate buffer pH 5.
Oligos (France). S-Sepharose, Q-Sepharose, Blue Sepharose and
DNA sequences were determined with an automatic sequencing
Superdex 2000 were obtained from Pharmacia (Freiburg, Germany).
system (ALF, Pharmacia). Sequence analysis was performed withOmiga (Oxfords Molecular, Oxford, UK), ClustalW (Thompson etal. ), and PAUP (phylogenetic analysis using parsimony;
The isolated gene fragments were inserted between the NdeI and
E. coli strains DH5α (Sambrook et al. GE334 (leuB6 lac-
HindIII sites of pET 22b(+) vector (Novagen, Madison, Wis.) and
290, tsx-96 recA1 rpsE2018 aroE24 rpsL86 cysG132 malT1 gal-290
transformed into E. coli C41(DE3) in order to express enzyme
ilv591 ΔphoA, kindly supplied by P. Belin, Gif-sur-Yvette, France)
and C41(DE3), a derivative of BL21(DE3) (Miroux and Walker) were grown in Luria-Bertani broth (LB; Sambrook et al. ).
Purification of native Klebsiella phytase
Cells were disrupted by sonication. Cell debris and proteins
Isolation and characterisation of phytase-producing strains
precipitated at 30% ammonium sulfate saturation were removedby centrifugation and the cleared supernatant was subjected to
Bacterial strains were isolated from a soil sample taken from an
ammonium sulfate precipitation at 70% saturation. The protein
Indonesian rice (Oryza sativa var. IR64) field in the direct vicinity of
material obtained was dialysed against 25 mM sodium acetate,
the plant roots. The diluted sample was plated on MA agar (see
pH 5.4. Purification of phytase was carried out by FPLC chroma-
below) containing 1% pancreatic peptone; 0.5% soya peptone; 0.2%
tography with S-Sepharose, Blue-Sepharose, Q-Sepharose and
phytic acid dodecasodium salt, and 0.25% calcium chloride. The
Superdex TM 2000 (see Electronic Supplementary Material).
plates were incubated at 37°C for 3 days. Five hundred isolates fromsoil sampled from an Indonesian rice field were tested for phytaseactivity using the disappearance of precipitated calcium phytate as
an indication of enzyme activity (Bae et al. ). Bacterial strainshydrolysing phytate agar were cultivated in liquid MAS medium
Phytase activity was assayed in 0.1 M sodium acetate pH 5.4 with
[MA medium supplemented with 0.5% MgSO4, 0.5% MgCl2, 0.2%
phytic acid dodecasodium salt as previously described (Greiner et al.
NaCl, 0.05% KCl, 0.05% CaCl2, and 1% glucose (w/v)] and tested
One unit of activity was defined as the amount of enzyme
for cellular-bound phytase activity. Finally, four strains were
that liberated 1 µmol phosphate in 1 min at 37°C. Phosphatase
confirmed as potent producer strains with activities ranging between
activities were measured with the substrate p-nitrophenyl phosphate
10 and 20 mU (mg protein)−1 (ASR3, ASR4, and ASR5) and 100–
(0.4% w/v) in 0.1 M sodium acetate buffer pH 5 or Tris-HCl buffer
200 mU (mg protein)−1 (ASR1). Strain ASR1 was chosen for further
pH 8. One unit of activity was defined as described for phytase
studies. ASR1 was characterised as a Gram-negative Klebsiella sp.-
like bacterium: not motile, rod-shaped, Gram-negative, negative in
To detect phosphatase activity in SDS PAGE gels, the gel was
Voges Proskauer reaction and in indole and Methyl red test, positive
incubated for 1 h in 1% Triton X100 and then incubated for a further
in Simmon citrate agar, resistant against ampicillin and producing β-
hour in 25 mM sodium acetate, pH 5.4. Finally, the gel was
incubated in a solution of 1-naphthyl phosphate and Fast Garnet salt
16S ribosomal DNA (rDNA) analysis (see below) revealed that
(Sigma), 0.1 and 0.2% (w/v), in 25 mM sodium acetate, pH 5.4,
ASR1 was a representative of Klebsiella sp. displaying close
sequence homology to Klebsiella pneumoniae (Sajidan ).
Protein concentrations were determined by the method of
ASR1 was deposited in the DSMZ culture collection as Klebsiella
Bradford using bovine serum albumin as standard. Specific
enzyme activities are defined as milliunits per milligram cell protein.
Protein identification by matrix assisted laser desorption/ionisation
mass spectroscopy (MALDI-MS) was performed as describedpreviously (Jungblut et al. In brief, the excised 42 kDa
The Klebsiella sp. ASR1 nucleotide sequence data reported has been
protein band resolved by SDS-PAGE was subjected to in-gel tryptic
deposited in the GenBank nucleotide sequence database under
digestion. The resulting peptide mixture was desalted using ZipTips
accession numbers AF453251 (16S rDNA), AF453252 (aphA),
(Millipore, Bedford, Mass.). Mass spectra were acquired using
MALDI-MS (Voyager Elite spectrometer; Perseptive, Framingham,Miss.). The amino acid sequences of the proteolytic peptides wereused in subsequent database searches with the program MS-FIT(http://www.prospector.ucsf.edu/ucsfhtml/msfit.hm) in the NCBIdatabase
MGH78578). Partial enzymatic cleavages leaving two cleavagesites, oxidation of methionine, pyroglutamic acid formation at the N-
Production and purification of native phytase from
terminal glutamine, and modification by acrylamide were consid-ered in these searches.
Direct identification of peptide sequences from the tryptic digest
of the 42 kDa protein was performed with the aid of the highly
Soil bacterium Klebsiella sp. ASR1 was characterised as a
sensitive nanoflow-electrospray mass spectrometry technique em-
potent producer strain for cell bound phytase (see
ploying a hybrid quadruple time of flight mass spectrometer (Q-Tof;Micromass, Manchester, UK) with a nanoflow electrospray ion
Materials and methods). Phytase was prepared from a
20 l culture of stationary phase cells grown for 16 h inMAS medium. Following ammonium sulfate precipitationand several steps of ion exchange column chromatography
Expression and purification of recombinant phytase
and a final gel filtration step, phytase activity was morethan 1,200 times enriched from cell extract (see Electronic
Recombinant E. coli strain C41(DE3) was cultured at 30°C in LB
Supplementary Material). Analytical SDS-PAGE revealed
containing ampicillin (50 µg/ml). At OD600=0.6, phytase expressionwas induced by addition of isopropyl-β-
a protein with an apparent molecular mass of 42 kDa with
(IPTG, final concentration 1 mM) and the cultures were further
activity against 1-naphthyl phosphate in direct gel staining
incubated at 30°C for 6 h. The cells were collected by centrifuga-
(see Electronic Supplementary Material). Pooled fractions
tion, suspended in 20 mM Tris-HCl/500 mM NaCl pH 7.9, and
containing the 42 kDa protein displayed specific activities
sonicated. After centrifugation, phytase was purified from the
of 224 U mg−1 using phytate and 49 U mg−1 using p-
supernatant by affinity chromatography with Ni-NT agarose(Qiagen).
nitrophenyl phosphate as substrate. Apparent molecularmass and specific activities of the enzyme prepared fromASR1 are similar to those described for phytase from K.
Identification of enzymatically formed hydrolysis products
Enzyme and sodium phytate were incubated in 0.1 M sodium
Cloning and expression of alkaline and acid
acetate buffer pH 5.4 as described for activity determination(Greiner et al. ). From the incubation mixture, samples
(200 μl) were removed periodically and the reaction was stopped
by heat treatment (90°C, 5 min); 50 μl of the heat-treated samples
Our first strategy to clone the phytase-encoding gene from
was resolved on a high performance ion chromatography (HPIC)
Kl. pneumoniae was based on the unspecific phosphatase
system using a Carbo Pac PA-100 (4×250 mm) analytical columnand a gradient of 5–98% HCl (0.5 M, 0.8 ml min−1) as described
activity of the purified 42 kDa phytase (see above). A
(Skoglund et al. ). The eluants were mixed in a post-column
plasmid library prepared from a Sau3A partial digest of
reactor with 0.1% Fe(NO3)3·9H2O in a 2% HClO4 solution
chromosomal DNA was used to clone genes encoding
(0.4 ml min−1) (Phillippy and Bland ). The combined flow
enzymes with phosphatase activity. Only transformants
that were able to hydrolyse 1-naphthyl phosphate butunable to hydrolyse phytate were selected and used forDNA sequencing. Clones with more than 90% sequence
Identification of the myo-inositol monophosphate isomer
identity to E. coli alkaline phosphatase (phoA, AF 453253)
Myo-inositol monophosphate was produced by incubation of 1.0 U
and acid phosphatase (aphA, AF 453252) genes were
legume phytase (Greiner et al. with a limiting amount of myo-
obtained. Expression cloning of the phoA- and aphA-like
inositol hexakisphosphate (0.1 µmol) in a final volume of 500 µl
genes in pET22b(+) with subsequent purification of the
50 mM NH4-formate. After lyophilisation, the residues were
His-tagged proteins bound on Ni NT agarose columns
dissolved in 500 µl of a solution of pyridine:bis (trimethylsilyl)trifluoroacetamide (1:1 v/v) and incubated at room temperature for
confirmed that we had cloned an alkaline phosphatase
24 h. The silylated products were injected at 270°C into a gas
exhibiting a pH maximum at 8.5–9.0 and an acid
chromatograph coupled with a mass spectrometer (GC-MS). The
phosphatase with a pH optimum at 4.0–5.0.
stationary phase was methyl silicon in a fused silica column(0.25 mm ×15 m). Helium was used as the carrier gas at a flow rateof 0.5 m s−1. The following heating program was used for thecolumn: 100–340°C, rate increase: 4°C min−1. Ionisation was
performed by electron impact at 70 eV and 250°C.
Cloning of the phytase gene using sequence
information obtained by nanoflow-electrospray massspectrometry of the trypsinised 42 kDa protein
The deduced amino acid sequence of the phyK gene is a421 amino acid protein with a molecular mass of
The tryptic digest of the 42 kDa protein band excised from
46,239 Da. The first 27 N-terminal amino acids form a
SDS PAGE was subjected to MALDI MS. Since this
cleavable signal peptide with a putative processing site
peptide mass fingerprinting revealed that no protein
AAA↓ADWQ (Nielsen et al. The amino acid
homologous to ASR1 phytase has been deposited within
sequence of the mature protein contains the active site
the protein databases, the highly sensitive nanoflow-
motif RHGXRXP that is shared by other histidine acid
electrospray mass spectrometry technique was applied in
phosphatases and phytases (Mitchell et al.
order to obtain specific sequence information of the
An extensive tBLASTN search in available databases,
Klebsiella sp. ASR1 phytase. This method allows direct
including unfinished and finished genomes, was per-
mass spectrometric sequencing of the peptides (Müller et
formed in order to detect proteins similar to PhyK. The
al. ). The resulting peptide sequences, together with
highest similarity was detected with two putative phytases
sequences already obtained for K. terrigena phytase (R.
from Pseudomonas syringae. Similarity scores signifi-
Greiner, unpublished), were used for similarity searches
cantly higher than with AppA (Dassa et al. were
(tBLASTN) within unfinished and finished bacterial
also found in ORFs in the genomes of two Xanthomonas
genomes. We were able to detect significant homology
species, Yersinia pestis, and Caulobacter crescentus.
to an open reading frame (ORF) located between nucle-
Pairwise alignment (Needleman and Wunsch ) of
otides 22,323 and 21,061 in contig118 of the unfinished
the whole sequences identified as similar to PhyK
genome of the human pathogenic K. pneumoniae
confirmed the close relatedness of the Klebsiella phytase
MGH78578 (McClelland et al. ). Using this sequence
with the putative phytases detected in the genomes of P.
information, a fragment of 1,263 bp was amplified from
syringae strains. Moreover, PhyK also displays homology
chromosomal DNA isolated from soil strain ASR1. The
to members of the yeast histidine acid phosphatase
deduced amino acid sequence of the fragment exhibited
superfamily with 3- or 6-phytase activity, including
MGH78578. The identity of the amplified sequence with
An amino acid sequence alignment was performed
the protein isolated from Klebsiella sp. ASR1 was
using ClustalW (Thompson et al. and the data were
confirmed by mass spectrometric measurements. PhyK
used to generate a phylogenetic tree. The tree obtained was
sequence was covered to 39.6% by the MALDI-MS
used as starting point for parsimony heuristic search with
peptide spectrum of the trypsinised 42 kDa protein
bootstrap support. The topology of the resulting phyloge-
isolated from Klebsiella sp. ASR1, which is well above
netic tree was very similar to the tree obtained by the
the cut-off for correct protein identification of 30%
neighbour joining (genetic-distance) method. Klebsiella
sp. ASR1 acid phosphatase forms a separate branch toother members of the histidine acid phosphatase familycharacterised by the RHGXRXP active site motif. Withinthis family, PhyK and two predicted phytases from P. syringae cluster on a separate branch, which is clearlydistinct from E. coli AppA, glucose-1-phosphatase and
Sequence comparison of microbial histidine acid phytases
pneumoniae was used for comparison and displays no significant
by EMBOSS Align (http://www.ebi.ac.uk/emboss/align/). The
homology to PhyK. Percentages of identical (% identity) and similar
deduced amino acid sequence of acid phosphatase, aphK from K.
(% similarity) amino acid residues are presented
other unknown proteins predicted to be histidine acid
phytase was purified from the culture supernatant by
affinity chromatography on Ni NT agarose yielding asingle homogeneous band in SDS-PAGE with a specificactivity of 169 U (mg protein)−1 when assayed at the
Expression, purification and properties of recombinant
The host E. coli C41 (DE3) was used for over-expression
of the phyK gene. Part of the phyK gene encoding themature phytase was fused in-frame with the pelB signal
Recombinant Klebsiella sp. ASR1 phytase has a single pH
peptide under the control of the strong IPTG-inducible T7
optimum at pH 5.0. The enzyme is virtually inactive at
RNA polymerase promoter present in vector pET22b(+).
values less than pH 4.0 and above pH 7.0. No shift in pH
Transformed C41 cells started to express phytase 2 h after
optimum was detected with p-nitrophenyl phosphate as an
IPTG induction. Initially, some cell-bound activity was
alternative substrate for the recombinant phytase.
detected, but the majority of the activity was found in theculture filtrate 8 h after induction. Since the pelB signalpeptide enables only SecA-dependent export into the
Temperature optimum and thermal stability
periplasmic space, the extracellular phytase activitydetected might be attributed to unspecific lysis of IPTG-
The temperature profile of the purified recombinant
induced recombinant E. coli cells. His-tagged recombinant
phytase was determined from 4°C to 70°C using thestandard phytase assay at the given temperature. Enzymeactivity increased with increasing temperature up to 45°Cand declined above 50°C. Thermal stability was testedfrom 0°C to 95°C. The phytase was fairly stable for15 min when incubated in temperatures from 0°C to 45°C. However, between 55°C and 60°C, enzyme activitydropped significantly. If incubated at 65°C, no phytaseactivity was detectable. In summary, the pH and temper-ature behaviour of the recombinant ASR1 enzyme weresimilar to the biochemical properties of the native phytaseof Klebsiella sp. ASR1 (Sajidan ) and those reportedfor K. terrigena phytase (Greiner et al. ).
The actions of native and recombinant phytase PhyK, andof the acid phosphatase AphK from Klebsiella sp. ASR1on several phosphorylated compounds were comparedwith data reported for the 3-phytase of K. terrigena(Greiner et al. the 6-phytase AppA from E. coli(Golovan et al. ) and Bacillus amyloliquefaciensphytase (Greiner et al. The relative rates ofenzymatic hydrolysis performed at 37°C are summarisedin Table Like E. coli AppA and K. terrigena phytase,PhyK is specific for phytate, displaying activity towardsphytate over four times higher than that towards p-nitrophenyl phosphate, and 20- to 40-fold higher than
Phylogenetic tree of bacterial histidine acid phosphatases
towards 2-naphthyl phosphate and 1-naphthyl phosphate.
genes on deduced protein level constructed by random stepwiseparsimony using the PAUP program package (Swofford 2002),
In contrast, no activity of recombinant ASR1 acid and
supported by 1,000 bootstrap repetitions. phyK_AS Klebsiella sp.
alkaline phosphatases AphK and PhoK towards sodium
ASR1 3-phytase, phyP_MOK1 Pseudomonas syringae MOK1
putative phytase, phyP_B728a Pseudomonas syringae B728A
Differences in specific activity determined at pH 5 and
putative phytase, phyX_Xa Xanthomonas axonopodis putativephytase, phyX_Xc Xanthomonas campestris putative phytase,
37°C of the native phytase (224 U mg protein−1) and the
phyC_Cc Caulobacter crescentus putative phytase, appA_Yp Yer-
recombinant phytase (99 U mg protein−1) were observed.
sinia pestis_KIM acid phosphatase, appA_Eco, Escherichia coli
These differences might be due to the presence of the His-
phytase, agp_Eco E. coli glucose-1-phosphatase, aphK Klebsiella
tagged C-terminus and/or the presence of some denatured
sp. ASR1 acid phosphatase, phoK Klebsiella sp. ASR1 alkaline
material in the preparation of the recombinant enzyme.
phosphatase. Members of the phyK family of acid histidinephosphatase are boxed. For accession numbers see Table 1
Km value obtained for recombinant phytase,
Substrate specificities of selected phytases and of the acid phosphatase AphK from Klebsiella sp. ASR1. All enzyme activities
were assayed at 37°C. Relative activities compared to phytate (100%) are shown
aNative phytase purified from Klebsiella pneumoniae ASR1bRecombinant phytase from K. pneumoniae ASR1 expressed in E. colicNative phytase, purified from Klebsiella terrigena. Data from Greiner et al. 1997dNative phytase, purified from E. coli. Data from Golovan et al. 2000eRecombinant phytase from Bacillus amyloliquefaciens FZB45. Data from Greiner et al. 2002fRecombinant acid phosphatase from Klebsiella pneumoniae nPhyK, native phytase purified from Klebsiella sp. ASR1
280 µmol l−1 phytate, is similar to that reported for K.
terrigena (Greiner et al. ). The kcat/Km value of therecombinant
Cloning of the phyK gene from a soil isolate ASR1
23.57 s−1 µmol l−1 exceeds by far the value of 0.65
identified as Klebsiella sp. was achieved by successful
s−1 µmol l−1 determined for the substrate p-nitrophenyl
purification and partial amino acid sequencing of the
phosphate, again suggesting that phytate is the preferred
protein revealing similarity to an ORF identified in the
unfinished genome of the human pathogenic K. pneumo-niae strain MGH78578. Phytases seem to be common inKlebsiella spp. since an ORF homologous to phyK is
present in strain MGH78578, and a phytase of K. terrigena with properties similar to the ASR1 enzyme
The hydrolysis products of the recombinant phytase were
has been reported (Greiner et al. ). Until now, the E.
separated by HPLC, ion pair chromatography, and ion
coli periplasmic phospho-anhydride phosphohydrolase
exchange chromatography (Fig. The results suggested
AppA (Dassa et al. ), which has been characterised
possible myo-inositol hexakisphosphate degradation path-
as 6-phytase (EC 3.1.3.26, Greiner et al. ), is the only
ways by the Klebsiella sp. ASR1 PhoK phytase as
characterised bacterial representative of histidine acid
outlined in Fig. Stepwise dephosphorylation occurs
phosphatases possessing the RHGXRXP and the HD
via (1) myo-inositol pentakisphosphate, D/L-Ins(1,2,4,5,6)
motifs. The mature PhyK displayed only a weak sequence
P5; (2) myo-inositol tetrakisphosphates, D/L-Ins(1,2,5,6)P4
similarity of 25% identical residues to E. coli phytase
or Ins(2,4,5,6)P4; (3) myo-inositol trisphosphates, D/L-Ins
AppA. However, active site residues H17 (nucleophilic
(1,2,6)P3 or Ins(1,2,3)P3 or D/L-Ins(1,4,5)P3; and (4) myo-
acceptor) and H303/D304 (proton donor) in the sequence
inositol bisphosphates, D/L-Ins(1,2)P2 or Ins(2,5)P2 or D/L-
A/G-H-D-T-X-I/L, and the residues R16, R20, and R92,
Ins(4,5)P2 and D/L-Ins(2,4)P2. myo-Inositol monophos-
which together with H303 and D304 are probably
phate, Ins(2)P was identified as the final product of
involved in coordinating the scissile 3-phosphate (Lim et
al. are well conserved in both enzymes. Alignmentto conserved domains (CD alignment) of the mature PhyKsequence by RPS-Blast with the conserved domaindatabase (CDD) revealed a similarity score of 80 bits (e-value: 4e-16) to the histidine acid phosphatase domain
High performance ion chromatography (HPIC) analysis
on sodium phytate. Enzyme and substrate were incubated at pH 5.0
of hydrolysis products of myo-inositol hexakisphosphate by the
and reaction products were analysed by HPIC (see Materials and
purified recombinant phytate-degrading enzyme PhyK from Kleb-
methods): 1D/L-Ins (1,2,3,4,5,6)P6, 3D/L-Ins (1,2,4,5,6)P5, 7D/L-Ins
siella sp. ASR1. A Profile of the reference myo-inositol phosphates.
(2,4,5,6)P4, 8D/L-Ins (1,2,5,6)P4, 17D/L-Ins (1,2,5)P3, 18D/L-Ins
The source of the reference myo-inositol phosphates is as indicated
(1,2,6)P3, 21D/L-Ins (2,4) P2, 22 Ins (1,2)P2
in Skoglund et al. (1998). B Action of purified recombinant phytase
pfam00328 (gn1│CDD │7564, http://www.ncbi.nlm.nih.
phosphatases have been classified either as six-bladed
propeller alkaline phosphatases (Shin et al. or as
The deduced amino acid sequence of the phyK gene,
purple acid phosphatases (Hegemann and Grabau ).
although containing the functional residues of histidine
The specific activity of the native Klebsiella sp. ASR1
acid phosphatases, displayed only 25% overall homology
phytase (224 U mg−1) exceeds the activity of phosphatase
to AppA and 15–17% to fungal histidine acid phytases,
measured against p-nitrophenyl phosphate (49 U mg−1)
suggesting that PhyK represents a novel subfamily of
4.56 times. In general, the presence of substantial amounts
histidine acid phytate-degrading enzymes that is clearly
of unspecific phosphatase activity is typical of phytases
distinct from the other previously characterised members
belonging to the histidine acid phosphatase family (Wyss
of this family. Other bacterial and plant phytases not
et al. but the ratio, together with a significant lower
containing the signature sequences of histidine acid
Km, characterises PhyK as a true phytase. With the
D/L-Ins(1,2,6)P3 and D/L-Ins(1,2)P2 or D/L-Ins(2,4)P2
D/L-Ins(1,2,5)P3 and D/L-Ins(1,2)P2 or Ins(2,5)P2
D/L-Ins(2,5,6)P3 and Ins(2,5)P2 or D/L-Ins(2,4)P2
Ins(2,4,5,6)P4 can only be degraded via Ins(2,4,6)P3
and D/L-Ins(2,4)P2 to, finally, Ins(2)P. Since pure Ins(2,4,6)P3 is not available, it was impossible to prove, or toexclude, the generation of Ins(2,4,6)P3. If in factgenerated, it possibly eluted together with Ins(1,4,5)P3. The experimentally supported pathway of phytate degra-dation is clearly different from that reported for otherbacterial phytate-degrading enzymes, but is similar to thatof the phytase of K. terrigena (R. Greiner, unpublishedobservation). The E. coli phytate degrading enzyme P2,which is identical to the appA gene product (Golovan etal. ), initially dephosphorylates myo-inositol hexaki-sphosphate at the 6-position followed by sequentialremoval of phosphate groups at the 1- and 3-position. The resulting myo-inositol trisphosphate is degradedfurther to Ins(2,5)P2 and Ins(2)P as final product ofhydrolysis following the notation 6/1/3/4/5 (Greiner et al. ). Recently, the 3-phytase from Bacillus was charac-terised as using two independent routes of degradation ofD-Ins(1,2,3,4,5,6)P6 via Ins(2,4,5,6)P4 and D-Ins(1,2,5,6)
Suggested degradation pathways of phytate by phytase
P4. However, in Bacillus the main end products of
PhyK from Klebsiella sp. ASR1. Ins(2,4,6)P3 is unavailable as a
enzymatic phytate hydrolysis are myo-inositol trispho-
reference compound in HPIC experiments (see Discussion), there-
sphates. The final monophosphate Ins(2)P, which is
fore it could not be excluded as a degradation intermediate
generated via D-Ins(2,6)P2, is detectable only afterprolonged incubation of Ins(1,2,6)P
exception of E. coli AppA, specific activities reported for
enzyme concentrations (Greiner et al. Therefore,
other bacterial and fungal phytases are in the same range
different phytases generate different products of enzymatic
(Greiner et al. ; Lassen et al. as found for
hydrolysis, which might be desirable in specific applica-
PhyK. E. coli phytase is reported to possess a specific
tions. Moreover, the action of several phytate-degrading
activity for hydrolysis of myo-inositol hexakisphosphate of
enzymes could lead to synergistic effects in phosphate
1,800 U mg−1, i.e. exceeding by 8-fold the specific activity
mobilisation in animal feed and under environmental
of the commercially used A. niger phytase (Golovan et al.
conditions such as in the plant rhizosphere (Richardson et
al. which is colonised by different phytase-
Our data suggest that Klebsiella phytase dephosphor-
producing beneficial microorganisms, e.g. Bacillus spp
ylates myo-inositol hexakisphosphate by sequential re-
(Idriss et al. Pseudomonas spp (Irving and
moval of phosphate groups via two independent routes. In
Cosgrove ) and Klebsiella spp (Chelius and Triplett
contrast to E. coli AppA (EC 3.1.26), but similar to fungal
). The potential of microbial phytases for stimulating
phytases, PhyK was characterised as a 3-phytase (EC
plant growth under conditions of limited access to
3.1.3.8), since the phosphoester bond at position 1 or 3 of
phosphate in complex environmental systems such as the
the myo inositol residue is preferentially hydrolysed
soil micro-cosmos remains to be further elucidated.
yielding D/L-Ins(1,2,4,5,6)P5 as the first degradationproduct. The two independent pathways proceed eithervia Ins (2,4,5,6)P
4 or D/L-Ins(1,2,5,6)P4 (Fig. ). Since all
theoretically existing myo-inositol pentakis- and tetraki-
gratefully acknowledged. We are especially grateful to MonikaSchmid for analysis of peptide masses and Thomas Leya for his help
sphosphate isomers are well resolved on the HPIC system
in using the PAUP package for construction of evolutionary trees.
used, the identity of the myo-inositol pentakis- and
We thank Romy Scholz and Kristin Rosner for their support in DNA
tetrakisphosphate isomer produced by the Klebsiella sp.
sequence analysis. Dr. Steffen Porwollik is thanked for critical
reading of the manuscript. The technical assistance of Christiane
Müller and Sybille Striegl is gratefully acknowledged.
4 is well established. A clear identifica-
tion of the formed myo-inositol tris-, bis- and monophos-phate isomers by HPIC cannot be achieved since not all
theoretically possible isomers are available. D/L-Ins(1,2,5,6)P4 may be degraded to Ins(2)P via the following
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