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Applied and Environmental Microbiology, November 1998, p. 4423-4427, Vol. 64, No. 11
Novo Nordisk Biotech, Davis, California
95616-4880
Received 17 February 1998/Accepted 10 September 1998
The phyA gene encoding an extracellular phytase from
the thermophilic fungus Thermomyces lanuginosus was cloned
and heterologously expressed, and the recombinant gene product was
biochemically characterized. The phyA gene encodes a
primary translation product (PhyA) of 475 amino acids (aa) which
includes a putative signal peptide (23 aa) and propeptide (10 aa). The
deduced amino acid sequence of PhyA has limited sequence identity (ca.
47%) with Aspergillus niger phytase. The phyA
gene was inserted into an expression vector under transcriptional
control of the Fusarium oxysporum trypsin gene promoter and
used to transform a Fusarium venenatum recipient strain.
The secreted recombinant phytase protein was enzymatically active
between pHs 3 and 7.5, with a specific activity of 110 µmol of
inorganic phosphate released per min per mg of protein at pH 6 and
37°C. The Thermomyces phytase retained activity at assay
temperatures up to 75°C and demonstrated superior catalytic
efficiency to any known fungal phytase at 65°C (the temperature
optimum). Comparison of this new Thermomyces catalyst with
the well-known Aspergillus niger phytase reveals other
favorable properties for the enzyme derived from the thermophilic gene
donor, including catalytic activity over an expanded pH range.
Phytases (myo-inositol
hexakisphosphate phosphohydrolases; EC 3.1.3.8) catalyze the hydrolysis
of phytic acid (myo-inositol hexakisphosphate) to the mono-,
di-, tri-, tetra-, and pentaphosphates of myo-inositol and
inorganic phosphate. A broad range of microorganisms, including
bacteria (20), yeasts (2), and filamentous fungi (10, 19, 27), produce phytases.
Phytic acid is the primary storage form of phosphate in cereal grains,
legumes, and oilseeds, such as soy, which are the principal components
of animal feeds. However, monogastric animals are unable to metabolize
phytic acid and largely excrete it in their manure. Therefore, the
presence of phytic acid in animal feeds for chickens and pigs is
undesirable, because the phosphate moieties of phytic acid chelate
essential minerals and possibly proteins, rendering the nutrients
unavailable. Since phosphorus is an essential element for the growth of
all organisms, livestock feed must be supplemented with inorganic
phosphate. There are a number of published reports (12, 16, 18,
26) describing the use of phytases in the feeds of monogastric
animals and in human food.
When phytic acid is not metabolized by monogastric animals the
phosphate level in the manure can also create disposal problems. The
amount of manure produced worldwide has increased significantly as a
result of increased livestock production. Environmental pollution with
high-phosphate manure has caused problems in various locations around
the world due to the accumulation of phosphate, particularly in bodies
of water. Consequently, animal feed distributors in Europe have begun
to formulate feed products with supplemental phytase in order to
improve feedlot productivity and decrease phosphate waste. Thus,
phytases are also useful for reducing the amount of phytate in manure
(13, 18). The current commercial feed supplement is a
recombinant Aspergillus niger (previously Aspergillus
ficuum) phytase produced in Aspergillus niger (27) or
Aspergillus oryzae (i.e., Phytase Novo
[13]).
There is a definite commercial need for second-generation phytases with
improved properties (e.g., higher thermostability and catalytic
efficiency) that can be produced in commercially significant
quantities. Our objectives were to identify, clone, and characterize a
phytase from a thermophilic fungus in anticipation that this enzyme
would offer superior biochemical properties.
DNA extraction and hybridization analysis.
Total cellular
DNA was extracted from Thermomyces lanuginosus CBS 586.94 by
the procedure described by Timberlake and Bernard (21).
Genomic DNA samples were analyzed by Southern hybridization (6) under conditions of low stringency (i.e., 5× SSPE [1×
SSPE is 0.18 M NaCl, 10 mM NaH2PO4, and 1 mM
EDTA {pH 7.7}], 25% formamide, 0.3% sodium dodecyl sulfate
[SDS]). A phytase-specific probe fragment comprising the
Aspergillus niger phyA coding region (approximately 1.6 kb)
was radiolabeled by nick translation (11) with
[ DNA libraries and identification of phytase clones.
Genomic
DNA libraries were constructed with the bacteriophage cloning vector
Molecular analysis of the T. lanuginosus phytase
gene.
Restriction mapping of pMWR46 was performed by standard
methods (11). DNA sequencing of the phytase clones was
performed with model 373A automated DNA sequencer (Applied Biosystems,
Inc., Foster City, Calif.) by the primer-walking technique with
dye-terminator chemistry (7). In addition to the
lac forward and lac reverse primers, specific
oligonucleotide sequencing primers were synthesized on an Applied
Biosystems model 394 DNA-RNA synthesizer according to the
manufacturer's instructions.
Construction of the phytase expression vector pMWR48.
The
coding region of the T. lanuginosus phyA gene was amplified
by PCR with the forward primer 5'-ATTTAAATGGCGGGGATAGGTTTGG-3' and the reverse primer 5'-CTTAATTAATCAAAAGCAGCGATCCC-3'.
The sense primer incorporated the first in-frame ATG and extends
16 bp downstream. The antisense primer incorporated a region 14 bp
upstream of the translational stop codon and extends through the stop
codon. To facilitate the cloning of the amplified fragment, the sense
and antisense primers contain a SwaI and a PacI
restriction site, respectively. The amplified product was digested with
SwaI and PacI and ligated with pDM181 (also
digested with SwaI and PacI), a plasmid which
provides the Fusarium oxysporum trypsin gene promoter and
terminator and the bar resistance cassette (3).
The resulting expression vector was designated pMWR48.
Transformation of Fusarium venenatum and analysis of
transformants.
Transformation protocols and methods for
purification of F. venenatum (28) transformants
are described by Royer et al. (15). Mycelia from primary
transformants were used to inoculate shake flasks containing 25 ml of
M400 Da medium (50 g of maltodextrin, 2 g of
MgSO4 · 7H2O, 2 g of
KH2PO4, 4 g of citric acid, 8 g of yeast extract, 2 g of urea, and 0.5 ml of trace metal solution per
liter [15]) and incubated with shaking at 30°C. One
milliliter of culture supernatant was harvested at 4, 5, and 7 days and
stored at 4°C. Phytase activity was assayed as described below.
Spores from the primary transformants producing the highest phytase
activity were generated by inoculating 20 ml of R medium (12.1 g of
NaNO3/liter, 50 g of succinic acid/liter, 20 ml of
50× Vogel's salts, 25 mM NaNO3 [pH 6.0]
[15]) with mycelia and incubating it at 30°C with
shaking for 2 to 3 days. Single spores were isolated by spreading 150 ml of spore culture onto manipulator plates (1X Vogel's salts, 25 ml
of NaNO3, 2.5% sucrose, 2% Noble agar) containing 5 mg of Basta [phosphinothricin or
2-amino-4-(hydroxymethyl-phosphinyl)butanoic acid; Hoechst-Schering,
Rodovre, Denmark] per ml and using a micromanipulator to transfer
single spores to a clear region of the plate. After 3 days of growth at
room temperature, the germinated spores were transferred to individual
Vogel plates containing 5 mg of Basta/ml. Shake flasks containing 25 ml
of M400Da medium plus 5 mg of Basta/ml were inoculated in duplicate
with mycelial plugs from each single-spore isolate and incubated at
30°C. The best single-spore isolate was selected based on assay of
the secreted enzymatic activity, where the transformants produced
>150-fold more phytase activity than an untransformed control.
Protein purification.
The best F. venenatum
transformant was run in two 2-liter fermentors with a standard protocol
(3). The frozen cell-free broth (1,700 ml) was thawed,
clarified by centrifugation, and concentrated on a hollow-fiber Amicon
filtration unit with an S1Y10 filter to a volume of 350 ml. The sample
was adjusted to pH 7, diluted to a conductivity of 2 mS, and
chromatographed at room temperature on a 75-ml-bed-volume Q-Sepharose
Big Beads column (Pharmacia), which had been equilibrated in 20 mM
Tris-Cl, pH 7. The column was developed at 5 ml/min with the
equilibration buffer until the effluent A280 had
decreased to near baseline. The column was then developed at 5 ml/min
with a 600-ml gradient of 0 to 0.6 M NaCl in the same buffer. The bound
enzyme activity was found to elute in fractions corresponding to ca.
0.2 M NaCl.
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Molecular Characterization and Expression of a
Phytase Gene from the Thermophilic Fungus Thermomyces
lanuginosus
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
-32P]dCTP (Amersham, Arlington Heights, Ill.) and
added to the hybridization buffer at an activity of approximately
106 cpm per ml. The hybridization and washing conditions
have been described previously (4).
ZipLox (Life Technologies, Gaithersburg, Md.) with Escherichia
coli Y1090ZL cells (Life Technologies) as a host for plating and
purification of recombinant bacteriophages and E. coli
DH10Bzip (Life Technologies) for excision of individual pZL1-phytase
clones. Total cellular DNA was partially digested with
Tsp509I and size fractionated on 1% agarose gels. DNA
fragments migrating in the range of 3 to 7 kb were excised and eluted
from the gel with Prep-a-Gene reagents (Bio-Rad Laboratories, Hercules, Calif.). The eluted DNA fragments were ligated with
EcoRI-cleaved and dephosphorylated ZipLox vector arms
(Life Technologies), and the ligation mixtures were packaged with
commercial packaging extracts (Stratagene, La Jolla, Calif.). The
packaged DNA libraries were plated and amplified in E. coli
Y1090ZL cells (Life Technologies). Approximately 30,000 plaques from
the library were screened by plaque hybridization with the radiolabeled
phytase probe. One positive clone which hybridizes strongly to the
probe was picked and purified twice in E. coli Y1090ZL
cells. The phytase clone was subsequently excised from the ZipLox
vector as a pZL1-phytase clone (5) and designated pMWR46.
Physicochemical characterization. Isoelectric focusing (IEF) was performed with a Novex pH 3 to 7 IEF gel according to the instructions of the manufacturer. IEF standards from both Pharmacia and Bio-Rad were used to calibrate the gel.
The protein extinction coefficient was determined experimentally by quantitative amino acid analysis with a Hewlett-Packard AminoQuant system. The analysis assumed 49,700 for the protein molecular weight, based on the translated gene sequence for the mature protein. Amino-terminal sequence analysis was performed on an Applied Biosystems 476A sequencer.Enzyme assays. Phytase activity was measured by two different methods. During purification, fractions were rapidly evaluated by measuring the rate of p-nitrophenyl phosphate hydrolysis at 405 nm with 10 mM substrate in 0.2 M sodium citrate, pH 5.5, at 30°C with a plate reader (Thermomax; Molecular Devices).
Enzyme kinetics studies performed on purified enzyme samples were accomplished by the assay of inorganic phosphate liberated from corn phytic acid (Sigma catalog no. P 8810). Exhaustive phytate hydrolysis was accomplished by incubating 0.5 or 0.1% phytic acid with enzyme (1 U/ml) in 0.2 M sodium citrate, pH 5.5, at 37°C. Aliquots were removed over a period of 10 h and analyzed (see below) for kinetics of phosphorus release. Ten hours was found to be sufficient for the completion of product formation. Standard enzyme kinetics reactions were carried out for 30 min at 37°C in 0.5% (wt/wt) phytic acid. The reaction was quenched by the addition of an equal volume of 15% (wt/wt) trichloroacetic acid. After cooling, 100 µl of the resulting mixture was diluted in 1 ml of water. The sample was incubated at 50°C for 5 min. Color reagent (1 ml) was added, and the 50°C incubation was continued for 15 min. The absorbance of a 200-µl aliquot was measured at 690 nm with a microplate reader. The color reagent was composed of 6 N sulfuric acid-water-2.5% (wt/vol) hepta-ammonium molybdate-10% ascorbate (aqueous) in a ratio of 1:2:1:1 and was prepared fresh daily. Quantitation was based on a standard curve generated with a 10 mM sodium monobasic phosphate standard. One unit is defined as 1 µmol of inorganic phosphate released per min with 0.5% phytic acid in 0.2 M sodium citrate, pH 5.5, at 37°C. Steady-state kinetics measurements were made by substrate titration. Phytate concentrations were 2.16, 1.08, 0.541, 0.216, 0.108, and 0.0758 mM for Km determination. Phytate concentrations of 1.08, 0.541, 0.216, and 0.108 mM in the presence or absence of 1 mM sodium monobasic phosphate were used to evaluate product inhibition.Thermostability measurement. Phytase samples were dissolved at 100 U per ml in 0.2 M sodium citrate, pH 5.5. One hundred-microliter aliquots of each enzyme solution were incubated for 20 min in a water bath at 37, 45, 50, 55, 60, 65, 70, and 75°C. After the heat treatment, the samples were stored at 0°C until activity assays were performed. Each sample was diluted 1:80 in 0.2 M sodium citrate, pH 5.5, containing 0.01% (wt/wt) Tween 20, and the standard activity assay was performed.
pH-activity measurement. To attain a buffering range between pHs 2 and 7, a three-component 125 mM glycine-acetate-citrate buffer was employed. The buffer components were combined at final concentrations of 42 mM per component, and phytic acid was added as a solid to 1% (wt/wt). This mixture was adjusted to pH 7 with concentrated HCl, and a 10-ml aliquot was taken. This process was repeated for every 0.5 pH units through pH 2.
Enzyme stock solutions of 20 U per ml were prepared in 20 mM MES buffer, pH 5.5. Substrate (1% [wt/wt]; 850 µl) in buffer at a given pH was combined with 100 µl of water and 50 µl of enzyme stock solution and incubated for 30 min at 37°C. Subsequently, the enzyme reaction was quenched with 1 ml of 15% trichloroacetic acid and quantitated by the standard method.Temperature-activity measurement. Enzyme stock solutions of 12.5 U per ml were prepared in 0.2 M sodium citrate buffer, pH 5.5. Two hundred fifty microliters of 1% phytic acid substrate was added to a 1.7-ml Eppendorf tube followed by 240 µl of 0.2 M sodium citrate buffer, pH 5.5. This solution was vortexed and placed in a water bath at the designated temperature. After 20 min of equilibration in the water bath, the mixture was vortexed and 10 µl of phytase solution was added. The sample was vortexed and incubated in the water bath for an additional 30 min, and then the reaction was quenched with 1 ml of 15% trichloroacetic acid and quantitated by the standard method.
Nucleotide sequence accession number. The complete phyA gene sequence has been deposited in GENESEQN as accession no. T90070.
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RESULTS |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Cloning of phytase gene sequences from T. lanuginosus.
Southern blotting experiments indicated that an Aspergillus
phytase gene fragment could be used as a probe to identify phytase gene-specific fragments in T. lanuginosus genomic DNA (Fig.
1). We screened 30,000 plaques from a
genomic library of T. lanuginosus DNA constructed in
ZipLox for hybridization with the Aspergillus phytase
gene probe. Several positive clones were picked and excised by an in
vivo-excision protocol (5).
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Analysis of the T. lanuginosus phyA gene. DNA sequencing of one T. lanuginosus phytase clone (pMWR46) showed an open reading frame similar to the A. niger phytase gene. The positions of introns and exons within the phyA gene were assigned based on comparison of the deduced amino acid sequence with the deduced amino acid sequence of the corresponding A. niger phytase gene product. On the basis of this analysis, the T. lanuginosus phytase gene is comprised of two exons (47 and 1,377 bp), which are separated by a small intron (56 bp). The size and composition of the intron is consistent with those of other fungal genes (9) in that all contain consensus splice donor and acceptor sequences as well as a near approximation of the consensus lariat sequence (RCTRAC) near the 3' end of each intervening sequence.
The deduced amino acid sequence of the T. lanuginosus gene product shows the characteristics of an extracellular fungal enzyme with a cleavable signal sequence. Based on the rules of von Heijne (25), the first 22 amino acids of PhyA likely comprise a secretory signal peptide which directs the nascent polypeptide into the endoplasmic reticulum. Amino-terminal amino acid sequencing suggests that the next 10 amino acids constitute a propeptide which terminates with a dibasic cleavage site (LysLys). The mature PhyA is an acidic protein (predicted isoelectric point, 5.4) composed of 452 amino acids (molecular mass, 51 kDa). The amino acid sequence also contains the active-site motif RHGXRXP, which is shared by other known phytases and acid phosphatases (Fig. 2) (23, 27). Lastly, the deduced amino acid sequence of the mature PhyA has approximately 47.5% identity with the phytase from A. niger (GenBank accession no. M94550).
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Analysis of F. venenatum transformants expressing T. lanuginosus phytase. F. venenatum has recently been developed as an efficient fungal host for the production of heterologous proteins (15). Culture supernatants from 14 of the 17 primary transformants of pMWR48 were positive when assayed for phytase activity. Two primary transformants with the highest phytase activity were selected for single-spore isolation, and nine single-spore isolates were obtained.
Physicochemical characterization of the recombinant phytase. The purified T. lanuginosus phytase was apparently homogeneous in SDS-polyacrylamide gel electrophoresis, with a single component corresponding to a molecular weight of 60,000. The protein sample contained numerous components in IEF analysis ranging from pH 4.7 to 5.2. In contrast to the T. lanuginosus phytase, recombinant A. niger phytase is composed of a single major component with a pI near 4.9 and two minor bands around pI 4.7.
Amino-terminal sequence analysis of the purified T. lanuginosus enzyme identified three components: the major component (ca. 60%) is H2N-His-Pro-Asn-Val-Asp-Ile-Ala-Arg-His-Trp-Gly-Gln..., which corresponds to a Kex2 cleavage site at position 34 in the primary translation product. Two minor sequences, H2N-Gly-Glu-Asp-Glu-Pro-Phe-Val-Arg-Val-Leu-Val-Asn...(ca. 30%) and H2N-Ser-Glu-Glu-Glu-Glu-Glu-Gly-Glu-Asp-Glu-Pro-Phe...(ca. 10%), correspond to internal cleavage sites near the COOH terminal of the protein at positions 428 and 435 in the primary translation product. The observation that our protein sequence data exactly match the predicted translation product of the T. lanuginosus gene and the finding that untransformed Fusarium host strains produce 2 orders of magnitude less enzyme activity both argue strongly that we have isolated a heterologous gene product. The specific activities for the two recombinant phytases (i.e., those of T. lanuginosus and A. niger) were 91 and 180 U/mg, respectively, under standard assay conditions at pH 5.5. At its pH 6 optimum T. lanuginosus phytase had a specific activity of 110 µmol of inorganic phosphate released per min per mg of protein at 37°C. Exhaustive enzymatic hydrolysis of phytic acid revealed that A. niger and T. lanuginosus phytases released identical amounts (70%) of the total theoretically available phosphorus. Steady-state kinetic measurements disclosed that the apparent Km of T. lanuginosus phytase is approximately 110 µM with respect to phytate while A. niger has an apparent Km of 200 µM. There was a faint indication of excess substrate inhibition at the 2.16 mM substrate concentration, perhaps congruent with the report of inhibition above 2 mM for A. niger phytase (22). Steady-state kinetics measurements with 1 mM phosphate present failed to reveal any type of inhibition with this product. We estimate that the Ki for phosphate must exceed 3 mM to be undetectable in our experiments. In contrast Ullah (22) has reported that phosphate is a competitive inhibitor, with a Ki of 1.9 mM. A comparison of enzyme thermostability profiles (Fig. 3) suggests that differences between the stabilities of the two enzymes are small. Neither enzyme is fully inactivated by a high-temperature incubation, and the residual activity profiles are consistent with partially reversible thermal denaturation (24). Differential scanning calorimetry (DSC) experiments reveal that the A. niger enzyme has a transition at 60°C while T. lanuginosus phytase unfolds at 69°C. Others have reported an Aspergillus fumigatus phytase which has an apparently greater propensity for reversible thermal denaturation (14), as measured by residual enzyme activity. However, there are no published data on thermal denaturation points for the A. fumigatus phytase or other phytase species.
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Enzyme activity at elevated temperatures may be relevant in applications such as saccharification (a high-temperature industrial process to generate high-fructose corn syrup), where others have reported that the addition of phytase improves carbohydrate yields (1). Figure 5 demonstrates that at 55°C, the optimal temperature for A. niger phytase, the Thermomyces phytase performs at 79% of the A. niger phytase turnover number (despite lower specific activity for Thermomyces phytase at 37°C) and at 60°C the Thermomyces phytase is operating at 67%-greater catalytic efficiency than the A. niger enzyme. The A. niger phytase is inactivated at 65°C, where Thermomyces phytase activity is maximal.
Enzyme thermal stability is also relevant in animal feed applications, where the enzyme is normally incorporated into the grains prior to pelletization and the feed briefly reaches processing temperatures of 85 to 90°C. In this circumstance a commercial phytase product must be able to withstand brief heating prior to encountering an animal's digestive tract at 37°C. Our physicochemical data demonstrate an improvement of approximately 9°C in denaturation temperature for Thermomyces phytase versus the present A. niger product.
Animal-feeding trials with formulated phytase supplementation would involve testing a total of 300 broilers or piglets at two enzyme dosages plus a control without enzyme addition. Typically the apparent total-tract digestibility of dissolved matter, organic matter, nitrogen, calcium, and total phosphorus would be monitored at one or two points during an animal's growth to determine the effect of enzyme dosage on feed intake and conversion. Such animal-feeding trials and the level of analysis required to present and evaluate the data are beyond the scope of this paper.
It is tempting to speculate about the structural origins of thermal stability in phytases. However, there is no obvious pattern to the sequence differences between phytases from thermophiles (represented by Myceliophthora, Talaromyces, and Thermomyces) and mesophiles (represented by A. niger and A. fumigatus). For example, there are no gross differences in protein structure, such as addition or deletion of secondary structure elements. Nor is there a systematic pattern to the sequence differences between the two representative enzymes; i.e., hydrophobic replacements, addition of salt bridges, addition of potential disulfide bonding sites, and deletion of asparagine or aspartate residues are not readily apparent. The most striking difference is the additional consensus N-linked glycosylation site present in the two Aspergillus enzymes (sequence position 231 in reference 27) but missing in the three thermophile examples. We believe that the most likely explanation which can be deduced for the sequence differences is derived from evolutionary rather than functional factors.
Recently the discovery of new industrial enzymes has focused on novel microbial sources representing extreme conditions (extremophiles). In many cases the genes encoding these interesting enzymes can be cloned without prior isolation of the catalyst or culturing of the donor microbe. However, heterologous production of the novel enzyme often results in extremely low yields of secreted product or accumulation of inactive material as inclusion bodies. Either of these outcomes is incompatible with the production economics required for commercialization. We have searched for new industrial catalysts from a constellation of thermophilic fungi that are more closely related than the extremophiles to the industrial fungal production strains which are available. We have successfully isolated enzymes with both improved thermal stability characteristics and the potential for high-level commercial production (4).
T. lanuginosus phytase is an alternative enzyme with performance advantages over the conventional A. niger enzyme in the form of stable enzyme activity at elevated temperatures and superior substrate saturation kinetics at physiological pH. A second-generation commercial enzyme may also benefit from protein engineering when a three-dimensional protein structure is available, as is the case for the A. fumigatus enzyme (8).
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ACKNOWLEDGMENTS |
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We thank Carin Morris (Novo Nordisk Biotech) for performing phytase enzyme activity assays, Sam Johnstone (Novo Nordisk Biotech) for performing Fusarium fermentations, and Claus Crone Fuglsang (Novo Nordisk A/S) for measuring phytase stability by DSC.
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FOOTNOTES |
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* Corresponding author. Mailing address: Novo Nordisk Biotech, Inc., 1445 Drew Ave., Davis, CA 95616-4880. Phone: (530) 757-0822. Fax: (530) 758-0317. E-mail: magi{at}nnbt.com.
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REFERENCES |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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| 1. | Antrim, R. L., C. Mitchinson, and L. P. Solheim. 1996. Method for liquefying starch. PCT patent application WO 96/28567. |
| 2. |
Barbaric, S.,
B. Kozulic,
B. Reis, and P. Mildner.
1984.
Physicochemical and kinetic properties of acid phosphatase from Saccharomyces cerevisiae.
J. Biol. Chem.
259:878-883 |
| 3. | Berka, R. M., M. W. Rey, and A. V. Klotz. 1997. Polypeptides having phytase activity and nucleic acids encoding same. PCT patent application WO 97/35017. |
| 4. | Berka, R. M., P. Schneider, E. J. Golightly, S. H. Brown, M. Madden, K. M. Brown, T. Halkier, K. Mondorf, and F. Xu. 1997. Characterization of the gene encoding an extracellular laccase of Myceliophthora thermophila and analysis of the recombinant enzyme produced in Aspergillus oryzae. Appl. Environ. Microbiol. 63:3151-3157[Abstract]. |
| 5. | D'Alessio, J. M., R. Bebee, J. L. Hartley, M. C. Noon, and D. Polayes. 1992. Lambda ZipLoxTM: automatic subcloning of cDNA. Focus (Life Technologies, Gaithersburg, Md.) 14:76-79. |
| 6. | Davis, R. W., D. Botstein, and J. R. Roth. 1980. Advanced bacterial genetics, a manual for genetic engineering. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. |
| 7. | Giesecke, H., B. Obermaier, H. Domedy, and W. J. Neubert. 1992. Rapid sequencing of the Sendai virus 6.8-kb large L gene through primer walking with an automated DNA sequencer. J. Virol. Methods 38:47-60[Medline]. |
| 8. | Grueninger-Leitch, F., A. D'Arcy, B. D'Arcy, and C. Chene. 1996. Deglycosylation of proteins for crystallization using recombinant fusion protein glycosidases. Protein Sci. 5:2617-2622[Medline]. |
| 9. | Gurr, S. J., S. E. Unkles, and J. R. Kinghorn. 1987. The structure and organization of nuclear genes in filamentous fungi, p. 93-139. In J. R. Kinghorn (ed.), Gene structure in eukaryotic microbes. IRL Press, Oxford, England. |
| 10. | Howson, S. J., and R. P. Davis. 1983. Production of phytate-hydrolyzing enzyme by some fungi. Enzyme Microb. Technol. 5:377-382. |
| 11. | Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. |
| 12. | Nelson, T. S., T. R. Shieh, R. J. Wodzinski, and J. H. Ware. 1971. Effect of supplemental phytase on the utilization of phytate phosphorus by chicks. J. Nutr. 101:1289-1294. |
| 13. | Novo Nordisk A/S. 1995. Phytase Novo cuts phosphorus in manure by 30%. BioTimes 10:8-9. |
| 14. | Pasamontes, L., M. Haiker, M. Wyss, M. Tessier, and A. P. G. H. van Loon. 1997. Gene cloning, purification, and characterization of a heat-stable phytase from the fungus Aspergillus fumigatus. Appl. Environ. Microbiol. 63:1696-1700[Abstract]. |
| 15. | Royer, J. C., D. M. Moyer, S. G. Reiwitch, M. S. Madden, E. B. Jensen, S. H. Brown, C. C. Yonker, J. A. Johnstone, E. J. Golightly, W. T. Yoder, and J. R. Shuster. 1995. Fusarium graminearum A 3/5 as a novel host for heterologous protein production. Bio/Technology 13:1479-1483[Medline]. |
| 16. | Sandberg, A.-S., and H. Andersson. 1988. Effect of dietary phytase on the digestion of phytate in stomach and small intestine of humans. J. Nutr. 118:469-473. |
| 17. | Sandberg, A.-S., L. R. Hulthen, and M. Türk. 1996. Dietary Aspergillus niger phytase increases iron absorption in humans. J. Nutr. 126:476-480. |
| 18. | Schwarz, G., and P. P. Hoppe. 1992. Phytase enzyme to curb pollution from pigs and poultry. Feed Magazine 1992:22-26. |
| 19. | Shieh, T. R., and J. H. Ware. 1968. Survey of microorganisms for the production of extracellular phytase. Appl. Microbiol. 6:1348-1351. |
| 20. | Shimizu, M. 1992. Purification and characterization of phytase from Bacillus subtilis (natto) N-77. Biosci. Biotechnol. Biochem. 56:1266-1269. |
| 21. | Timberlake, W. E., and E. C. Bernard. 1981. Organization of a gene cluster expressed specifically in the asexual spores of Aspergillus nidulans. Cell 26:29-37[Medline]. |
| 22. | Ullah, A. H. J. 1988. Production, rapid purification and catalytic characterization of extracellular phytase from Aspergillus ficuum. Prep. Biochem. 18:443-458[Medline]. |
| 23. | Ullah, A. H. J., and H. C. Dischinger, Jr. 1993. Identification of active-site residues in Aspergillus ficuum extracellular pH 2.5 optimum acid phosphatase. Biochem. Biophys. Res. Commun. 192:754-759[Medline]. |
| 24. | Ullah, A. H. J., and E. J. Mullaney. 1996. Disulfide bonds are necessary for structure and activity in Aspergillus ficuum phytase. Biochem. Biophys. Res. Commun. 227:311-317[Medline]. |
| 25. | von Heijne, G. 1984. How signal sequences maintain cleavage specificity. J. Mol. Biol. 173:243-251[Medline]. |
| 26. | Wang, H. L., E. W. Swain, and C. W. Hasseltine. 1980. Phytase of moulds used in oriental food fermentation. J. Food Sci. 45:1262-1266. |
| 27. | Wodzinski, R. J., and A. H. J. Ullah. 1996. Phytase, p. 263-302. In S. L. Neidleman, and A. I. Laskin (ed.), Advances in applied microbiology, vol. 42. Academic Press, Inc., New York, N.Y. |
| 28. | Yoder, W. T., and L. M. Christianson. 1998. Species-specific primers resolve members of Fusarium section Fusarium: taxonomic status of the edible "Quorn" fungus reevaluated. Fungal Genet. Biol. 23:68-80[Medline]. |
| 29. | Zyla, K. 1993. The role of acid phosphatase activity during enzymic dephosphorylation of phytates by Aspergillus niger phytase. World J. Microbiol. Biotechnol. 9:117-119. |
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