HK1058213A - Phytase enzymes, nucleic acids encoding phytase enzymes and vectors and host cells incorporation same - Google Patents

Description

Phytases, nucleic acids encoding phytases, and vectors and host cells comprising the nucleic acids

RELATED APPLICATIONS

This application claims priority from U.S. provisional patent application 60/148,960 filed on 13.8.1999, which is hereby incorporated by reference in its entirety.

Technical Field

The present invention relates to phytases, nucleic acids encoding phytases, as well as the preparation of phytases and the use thereof.

Reference to the literature

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Background

Phosphorus (P) is an essential element for growth. The phosphorus present in conventional livestock feed (e.g., grain, oilseed meal, and seed-derived by-products) is in considerable part in the form of phosphate covalently bound in a molecule called phytic acid (inositol hexaphosphoric acid). For non-ruminant animals such as poultry and swine, the bioavailability of this form of phosphorus is generally low due to their lack of digestive enzymes for separating the phosphorus from the phytate molecule.

Several important consequences of the inability of non-ruminant animals to utilize phytic acid may be noteworthy. For example, expenses are incurred when inorganic phosphorus (e.g., dicalcium phosphate, defluorinated phosphates) or animal products (e.g., meat and bone meal, fish meal) are added in order to meet the nutritional requirements for phosphorus for these animals. Furthermore, phytic acid also binds or sequesters many minerals (e.g., calcium, zinc, iron, magnesium, copper) in the gastrointestinal tract, thereby rendering them unabsorbed. Furthermore, most of the phytic acid present in the feed passes through the gastrointestinal tract, causing an increase in the amount of phosphorus in the feces. Which will increase the ecological phosphorus burden of the environment.

In contrast, ruminants such as cattle readily utilize phytate, thanks to the rumen microorganisms producing an enzyme called phytase. Phytases catalyze the hydrolysis of phytate to (1) inositol and/or (2) its mono-, di-, tri-, tetra-and/or pentaphosphates and (3) inorganic phosphoric acid. Two different types of phytases are known: (1) known as 3-phytase (phytase 3-phosphohydrolase, EC3.1.3.8) and (2) known as 6-phytase (phytase 6-phosphohydrolase, EC3.1.3.26). The 3-phytase first hydrolyzes the ester bond at the 3-position, while the 6-phytase first hydrolyzes the ester bond at the 6-position.

It has been found that the bioavailability of phytases in foods typical of non-ruminant animals can be increased by using microbial phytases as feed additives (see, e.g., Cromwell et al, 1993). The result is a reduction in the need for inorganic phosphorus to be added to animal feed and a reduction in the phosphorus level in the excreted manure (see, e.g., Kornegay et al, 1996).

Despite these advantages, few known phytases have gained wide acceptance in the feed industry. The reason for this varies from enzyme to enzyme. Typical related problems relate to high production costs, and/or low stability/activity of the enzyme in the desired application environment (e.g. pH/temperature encountered in feed processing, or in the animal gut).

Therefore, it is generally desirable to discover and develop new enzymes with good stability and phytase activity for animal feeding related applications and to apply advances in fermentation technology to the production of these enzymes in order to make them commercially viable. It is also desirable to identify nucleotide sequences that can be used to generate more efficient genetically engineered organisms that are capable of expressing these phytases in large quantities to be suitable for industrial production. Furthermore, it is desirable to develop phytase expression systems by genetic engineering that make it possible to purify and utilize relatively pure enzymes in a working amount.

Summary of The Invention

The present invention provides purified enzymes having phytase activity from microbial sources, preferably from fungal sources such as species of the genus Penicillium, such as P.hordei (formerly Penicillium hirsutum; ATCC No. 22053), Penicillium juniperi (ATCC No. 10519), or Penicillium brevi-compactum (ATCC No. 48944).

The invention also provides a polynucleotide sequence encoding the enzyme and comprising the DNA shown in any one of FIGS. 1A-1C; a polynucleotide encoding the amino acid sequence shown in figure 2; a polynucleotide encoding a phytase comprising an amino acid segment other than the sequence in figure 2, provided that the polynucleotide encodes a derivative of the phytase specifically described herein; and a polynucleotide encoding a phytase comprising an amino acid segment other than the sequence in figure 2, provided that the polynucleotide hybridizes to DNA comprising all or part of the DNA in any one of figures 1A-1C under medium to high stringency conditions.

The present invention also provides a polynucleotide encoding an enzyme having phytate hydrolyzing activity and comprising the nucleotide sequence set forth in figure 17; a polynucleotide encoding the amino acid sequence set forth in figure 17; a polynucleotide encoding a phytase comprising an amino acid segment that differs from the sequence in figure 17, provided that the polynucleotide encodes a derivative of the phytase specifically described herein; and a polynucleotide encoding a phytase comprising an amino acid segment that differs from the sequence in figure 17, provided that the polynucleotide hybridizes to the nucleotide sequence shown in figure 17 under medium to high stringency conditions.

In addition, the present invention includes vectors comprising the polynucleotide sequences, host cells transformed with the polynucleotides or vectors, fermentation broths containing the host cells, and phytase proteins encoded by the polynucleotides expressed by the host cells. Preferably, the polynucleotides of the present invention are in purified or isolated form and used to prepare transformed host cells capable of producing the protein products encoded thereby. In addition, polypeptides that are the expression products of the above-described polynucleotide sequences are also included in the scope of the present invention.

In one embodiment, the invention provides an isolated or purified polynucleotide from a fungal source of the genus penicillium, the polynucleotide comprising a nucleotide sequence encoding an enzyme having phytase activity. The fungal source may be selected from, for example, Penicillium juniperum (Penicillium piceum) and Penicillium hordei.

According to one embodiment, the polynucleotide encodes a phytate hydrolase comprising an amino acid sequence which is substantially identical to the amino acid sequence of SEQ ID NO: 4, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, and/or about 100% identical.

One embodiment of the present invention provides an isolated polynucleotide comprising a nucleotide sequence which (i) hybridizes with SEQ ID NO: 1. SEQ ID NO: 2. or SEQ ID NO: 3, or (ii) is capable of at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, and/or about 100% identity to a nucleotide sequence disclosed in SEQ ID NO: 1. SEQ ID NO: 2. or SEQ ID NO: 3 under medium to high stringency conditions, or (iii) hybridizes to SEQ ID NO: 1. SEQ ID NO: 2. or SEQ ID NO: 3 is complementary to the nucleotide sequence disclosed in 3.

Another aspect of the invention provides an isolated polynucleotide encoding an enzyme having phytase activity, wherein the enzyme is from a Penicillium (Penicillium) source. The source may be selected from, for example, Penicillium juniperi and Penicillium hordei.

In one embodiment, the polynucleotide encodes a phytate hydrolase comprising a nucleotide sequence substantially similar to the nucleotide sequence set forth in SEQ ID NO: 4, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, and/or about 100% identical.

In another embodiment, the polynucleotide is identical to SEQ ID NO: 1. SEQ ID NO: 2. or SEQ ID NO: 3, or (ii) is capable of at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, and/or about 100% identity to a nucleotide sequence disclosed in SEQ ID NO: 1. SEQ ID NO: 2. or SEQ ID NO: 3 under medium to high stringency conditions, or (iii) hybridizes to SEQ ID NO: 1. SEQ ID NO: 2. or SEQ ID NO: 3 is complementary to the nucleotide sequence disclosed in 3.

In a further aspect the present invention provides an expression construct comprising a polynucleotide sequence which (i) hybridizes with SEQ ID NO: 1. SEQ ID NO: 2. or SEQ ID NO: 3, or (ii) is capable of at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, and/or about 100% identity to a nucleotide sequence disclosed in SEQ ID NO: 1. SEQ ID NO: 2. or SEQ ID NO: 3 under moderate to high stringency conditions, or (iii) hybridizes to a nucleic acid sequence disclosed in SEQ id no: 1. SEQ ID NO: 2. or SEQ ID NO: 3 is complementary to the nucleotide sequence disclosed in 3. Also provided are vectors (e.g., plasmids) containing the expression constructs, and host cells (e.g., Aspergillus, such as Aspergillus niger or Aspergillus nidulans) transformed with the vectors.

In a further aspect, the present invention provides a probe for detecting a nucleic acid sequence encoding an enzyme having phytase activity from a microbial source, the probe comprising: (i) and SEQ ID NO: 1. SEQ ID NO: 2 or SEQ ID NO: 3, or (ii) a nucleotide sequence that is at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, and/or about 100% identical to a nucleotide sequence disclosed in SEQ ID NO: 1. SEQ ID NO: 2 or SEQ ID NO: 3 under medium to high stringency conditions, or (iii) a nucleotide sequence that hybridizes to a polynucleotide of a sequence disclosed in SEQ ID NO: 1. SEQ ID NO: 2 or SEQ ID NO: 3, or a nucleotide sequence complementary to the nucleotide sequence disclosed in 3.

In one embodiment, the microbial source is a fungal source, for example a species of the genus penicillium, such as penicillium hordei or penicillium juniperi.

In addition, the invention provides a food or animal feed comprising an enzyme having phytase activity, wherein the enzyme comprises an amino acid sequence substantially identical to SEQ ID NO: 4, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, and/or about 100% identical.

The present invention provides food or animal feed comprising an enzyme having phytase activity, wherein the enzyme is from a fungal source selected from Penicillium hordei and Penicillium juniperi.

One aspect of the present invention provides an isolated phytase enzyme, wherein the enzyme is obtained from a fungus selected from the group consisting of penicillium juniperi and p.hordei, and has the following physicochemical properties: (1) molecular weight: about 45-55kDa (unglycosylated); and (2) specificity: phytic acid.

In one embodiment, the invention provides a fungal species (e.g. penicillium, such as penicillium juniperi and p.hordei) derived from a fungal species or a fungal species consisting of a fungal strain capable of hybridizing to SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 3. or an enzyme encoded by a nucleotide sequence in which the polynucleotide sequence of figure 17 hybridizes under medium to high stringency conditions, having one or more of the following physicochemical properties:

(1) molecular weight: about 45-60kDa (not glycosylated) (based on a protein with 489 amino acids);

(2) activity specific to phytate, phytic acid or phytate, and/or lower phosphate derivatives thereof;

(3) theoretical pI of about 7-7.6; such as 7.3;

(4) the pH optimum is in the range of about 4.5-5.5, e.g., about 5; and/or

(5) The optimum ambient temperature is 40-45 deg.C, for example 42-44 deg.C.

Another aspect of the present invention provides a method of preparing an enzyme having phytase activity, comprising:

(a) providing a host cell transformed with an expression vector comprising a polynucleotide described herein;

(b) culturing the transformed host cell under conditions suitable for the production of the phytase by the transformed host cell; and

(c) recovering the phytase.

According to one embodiment, the host cell is an Aspergillus species, such as Aspergillus niger or Aspergillus nidulans.

In another aspect, the present invention provides a method for separating phosphorus from phytic acid comprising the steps of: using a nucleic acid comprising a nucleotide sequence identical to SEQ ID NO: 4, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, and/or about 100% identical in amino acid sequence.

The present invention also provides a process for separating phosphorus from phytic acid comprising the steps of: treating the phytate with an enzyme as defined above.

Another aspect of the invention provides a phytate hydrolase comprising an amino acid sequence having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, and/or about 100% identity to the amino acid sequence disclosed in figure 17.

Yet another aspect of the invention provides an isolated polynucleotide comprising a nucleotide sequence that is (i) at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, and/or about 100% identical to the nucleotide sequence disclosed in figure 17, or (ii) capable of hybridizing to a probe derived from the nucleotide sequence disclosed in figure 17 under medium to high stringency conditions, or (iii) complementary to the nucleotide sequence disclosed in figure 17.

In one embodiment, the isolated polynucleotide encodes a phytate hydrolase derived from Penicillium juniperi or Penicillium hordei. According to one embodiment, the enzyme comprises an amino acid sequence which is at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, and/or about 100% identical to the amino acid sequence disclosed in figure 17.

In another embodiment, the polynucleotide comprises a nucleotide sequence that is (i) at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, and/or about 100% identical to the nucleotide sequence disclosed in fig. 17, or (ii) capable of hybridizing to a probe derived from the nucleotide sequence disclosed in fig. 17 under medium to high stringency conditions, or (iii) complementary to the nucleotide sequence disclosed in fig. 17.

Another aspect of the invention provides an expression construct comprising a polynucleotide comprising: (i) a nucleotide sequence at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, and/or about 100% identical to the nucleotide sequence disclosed in figure 17, or (ii) a nucleotide sequence capable of hybridizing to a probe derived from the nucleotide sequence disclosed in figure 17 under medium to high stringency conditions, or (iii) a nucleotide sequence complementary to the nucleotide sequence disclosed in figure 17. The invention also provides vectors (e.g., plasmids) comprising the expression constructs, and host cells (e.g., Aspergillus niger or Aspergillus nidulans) transformed with the vectors.

Furthermore, the present invention provides a probe for detecting a nucleic acid sequence encoding an enzyme having phytase activity, derived from a microbial source, the probe comprising: (i) a nucleotide sequence at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, and/or about 100% identical to the nucleotide sequence disclosed in figure 17, or (ii) a nucleotide sequence capable of hybridizing to a polynucleotide comprising the sequence disclosed in figure 17 under medium to high stringency conditions, or (iii) a nucleotide sequence complementary to the nucleotide sequence disclosed in figure 17.

In one embodiment, the microbial source is a fungal source, for example a penicillium species, such as p.

The invention also provides food or animal feed comprising an enzyme having phytase activity, wherein the enzyme comprises an amino acid sequence that is at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, and/or about 100% identical to the amino acid sequence disclosed in figure 17.

Further, the present invention provides a method for separating phosphorus from phytic acid comprising the steps of: treating the phytate with (i) an enzyme having phytate hydrolysis activity and (ii) an enzyme comprising an amino acid sequence at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, and/or about 100% identical to the amino acid sequence disclosed in figure 17.

It will be appreciated that an advantage of the present invention is that the polynucleotide has been isolated, which enables the isolation of other polynucleotides encoding proteins having phytase activity.

Another advantage of the present invention is that by providing a polynucleotide encoding a protein having phytase activity, host cells capable of producing the protein having phytase activity in relatively large amounts can be prepared by recombinant means.

Yet another advantage of the present invention is that it enables commercial application of proteins having phytase activity. For example, the invention provides animal feed incorporating a phytase as described herein.

It is a further advantage of the present invention to provide an enzyme having phytate hydrolase activity and which activity is optimal at temperatures of about 40-45 ℃, which makes the enzyme well suited for use in animal feed (i.e., the enzyme has high activity at the site of action (in the stomach of the animal)).

Other objects and advantages of the present invention will be apparent from the following detailed description.

Brief Description of Drawings

FIG. 1A depicts the nucleic acid sequence (SEQ ID NO: 1) corresponding to the gene encoding the phytate hydrolase from Penicillium hordei, with the following features highlighted: the phytase ATG start codon encoding Met (bold); introns (lowercase letters); and a TAG stop codon (bold). Notably, the figure shows two exons (120bp and 1347bp) separated by a 120bp long 5' intron.

FIG. 1B shows the continuous region (SEQ ID NO: 2) between the start codon (ATG) and the stop codon (TAG) of the sequence in FIG. 1A.

FIG. 1C shows, in the form of a continuous sequence, the region of the sequence of FIG. 1A between the start codon and the stop codon, excluding the 120bp intron (SEQ ID NO: 3).

FIG. 2 depicts the amino acid sequence (SEQ ID NO: 4) encoded by the nucleic acid sequence of FIG. 1.

Fig. 3 and 4 are growth curves for penicillium juniperi and p.hordei, respectively, showing the effect of available P in the medium on growth over time.

FIG. 5A shows an alignment of 4 published fungal phytase amino acid sequences and indicates the conserved regions (highlighted in black boxes) used to design degenerate PCR primers.

FIG. 5B shows an alignment of the 4 published fungal phytase amino acid sequences of FIG. 5A and the sequences of the P.hordei and P.juniperi sequences of the present invention (SEQ ID NO: and SEQ ID NO: respectively).

FIG. 6 shows a published alignment between the amino acid sequences of A.niger phyA and phyB phytases, whereby degenerate primers CS1 and CS2 were designed. The conserved sequences used for designing the primers CS1, CS2 are shown.

FIG. 7 shows 4 published fungal phytase amino acid sequences aligned together with the following amino acid sequences: (i) an amino acid sequence translated from the PCR product obtained with primers CS1 and CS2 (line 2, denoted "p.hordei3d"), and (ii) an amino acid sequence translated from approximately 80% (i.e., lacking the N-terminal portion) of the p.hordei phytase gene obtained from the first p.hordei genomic library (line 1, denoted "p.hordei").

FIG. 8 shows a Southern blot gel showing the hybridization between probes containing PCR products obtained with degenerate primers CS1 and CS2 and various fungal genomic DNA digests; lane 1-size standard reference; lane 2-Aspergillus niger-EcoRI; lane 3-Penicillium juniperi-EcoRI; lane 4-Penicillium hordei-EcoRI; lane 5-Peniciliumhorrei-BamHI; lane 6-Penicillium hordei-SalI; lane 7-Penicillium hordi-KpnI; and lane 8-Penicillium hordei-SacI.

FIG. 9 depicts the nucleic acid sequence of a clone designated CS101 (SEQ ID NO: _) obtained by construction and screening of a P.hordei genomic library. CS101 contained a 4.8kb fragment from P.hordei in the commercial cloning vector pSKII + Bluescript (Stratagene; La Jolla, California). The 1.7kb of this insert is the phyA phytase sequence of P.hordei, representing 80% of the coding region (including the 3' end) and downstream regulatory regions of the gene.

FIG. 10 shows the 5 '/N-terminal nucleic acid sequence (SEQ ID NO: A)) and the 3'/C-terminal sequence (SEQ ID NO: A)) of a clone designated CS 158. The deduced amino acid sequences corresponding to each nucleic acid sequence are also shown (SEQ ID NO: and SEQ ID NO: respectively). Clone CS158 was prepared by PCR from a combination of primers CS 201-204. Bands of appropriate size were generated (FIG. 12) and cloned and sequenced. The deduced amino acid sequence is listed below. The primarily deduced phytase amino acid sequence is shown in bold. Sequencing of CS158 was incomplete because of the lack of about 70 amino acids in the middle of the gene. Reamplification with the redesigned primers (FIG. 13) yielded the complete sequence, which was analyzed to be phytase. The C-terminus of CS158 shows 100% homology to the C-terminus of CS 101.

Figure 11 provides a diagram of the pGAPT-PG vector that can be used for expressing p.

Fig. 12 shows PCR amplification of putative phytase genes using primers designed from the sequence of a genomic phytase clone isolated from p.

FIG. 13 shows PCR amplification of a putative phytase gene using primers designed from the phytase gene sequence of clone CS 158.

Fig. 14 shows Southern blot analysis of aspergillus nidulans transformed with the p. hordei phytase gene.

Fig. 15 shows the pH profile of phytase from p.

Fig. 16 shows the temperature-activity curves of phytase from p.

FIG. 17 shows the complete sequence (5 '-3') (SEQ ID NO: (R)) of the 853bp fragment of P.juniperi phytase from a clone designated CS142, together with the deduced amino acid sequence (283 amino acids) (SEQ ID NO: (R)). Motifs of the sequence that are important for phytase structure and function, and Cys residues that are important for structure are shown in bold.

Detailed Description1. Definition of

Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton et al, DICTIONARY OF MICROBIOLOGY AND molecular biology (DICTIONARY OF MICROBIOLOGY AND MOLECULARBIOLOGY), 2 nd edition, John Wiley AND Sons, New York (1994); and Hale and Marham, Harper Collins biology DICTIONARY (THE HARPER Collins DICTIONARY OFBIOLOGY), Harper Perennial, NY (1991), provide the skilled artisan with a general explanation of many of the terms used in the present invention. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described. Numerical ranges include the numbers per se that define the range. Unless otherwise indicated, nucleic acids are written from left to right in the 5 'to 3' direction, respectively; amino acid sequences are written from left to right in the amino to carboxy direction. The headings provided herein are not to be construed as limitations of various aspects or embodiments of the invention which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below may be more fully defined by reference to this specification in its entirety.

The term "phytase" or "phytase activity" as used herein refers to a protein or polypeptide capable of catalyzing the hydrolysis of phytate to (1) inositol and/or (2) its mono-, di-, tri-, tetra-and/or pentaphosphates and (3) inorganic phosphate. For example, enzymes having the catalytic activity defined in enzyme commission EC No. 3.1.3.8 or EC No. 3.1.3.26.

The term "identical" with respect to two nucleic acid or polypeptide sequences refers to the identical residues in the two sequences when aligned for maximum correspondence, as can be measured using one of the following sequence comparison or analysis algorithms.

The "best alignment" is defined as the alignment that gives the highest percent identity score. The alignment can be performed using a variety of commercially available sequence analysis programs, such as the local alignment program LALIGN using ktup of 1, default parameters, and default PAM. One preferred alignment is a pairwise alignment using the CLUSTAL-W program in MACVECTOR, which runs in a "slow" alignment mode using default parameters including an open gap penalty of 10.0, an extended gap penalty of 0.1, and the BLOSUM30 similarity matrix (similarity). If a deletion region (gap) needs to be inserted in the first sequence in order to optimally align the first sequence with the second sequence, then only residues that pair with the corresponding amino acid residues are used to calculate percent identity (i.e., the calculation does not take into account the residues in the second sequence that are located within the "deletion region" of the first sequence).

For comparison, optimal alignment of sequences can also be performed by, for example, the local homology algorithm of Smith and Waterman (adv. Appl. Math.2: 482(1981)), the homology alignment algorithm of Needleman and Wunsch (J.mol. biol.48: 443(1970)), the similarity search method of Pearson and Lipman (Proc. Natl. Acad. Sci.USA 85: 2444(1988)), the Computer-implemented programs for these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics software package), or visual inspection.

"percent sequence identity," with respect to two amino acid or polynucleotide sequences, refers to the percentage of residues in the two sequences that are identical when the two sequences are optimally aligned. Thus, 80% amino acid sequence identity means that 80% of the amino acids in the two optimally aligned polypeptide sequences are identical.

For example, the percent identity may be determined by: the sequence information between two molecules is directly compared by aligning the sequences, counting the exact number of matching residues between the two aligned sequences, dividing by the length of the shorter sequence, and multiplying the result by 100. Analysis can be aided by readily available computer programs such as ALIGN (Dayhoff, M.O., "Protein Sequence and Structure Association" M.O.Dayhoff eds.,. 5 suppl.3: 353-358, National biological Research Foundation, Washington, D.C.) that have been adapted to the local homology algorithm of Smith and Waterman (1981) (Advances in appl.Math.2: 482-489). There are programs for determining nucleotide sequence identity, such as BESTFIT, FASTA and GAP programs, which also rely on Smith and Waterman algorithms, in the Wisconsin sequence analysis software package, version 8 (available from Genetics Computer Group, Madison, Wis.). These programs can be readily utilized with the manufacturer's recommendations and default parameters described in the Wisconsin sequence analysis software package mentioned above.

One example of an algorithm suitable for determining sequence similarity is the BLAST algorithm described in Altschul et al, j.mol.biol.215: 403-410(1990). Software for performing BLAST analysis is available from the national center for bioinformatics (http：//www.ncbi.nlm.nih.gov/) Is obtained by disclosure. The algorithm involves first identifying high scoring sequence pairs (HSPs) by determining in the query sequence the W long and short words that meet or satisfy some positive threshold score T when aligned with words of the same length in a database sequence. These initial neighboring matched words are used as starting points to find longer HSPs containing them. The matches are extended toward the ends along each of the two sequences being compared, as long as the cumulative alignment score can be increased. When: cumulative ratio of bisectionThe amount of decrease X from the obtained maximum value; cumulative scores of zero or less; or the end of either sequence, the extension of the match word is terminated. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLAST program uses a word length (W) of 11, a BLOSUM62 scoring matrix (see Henikoff)&Henikoff, proc.natl.acad.sci.usa89: 10915(1989)), alignment positions of 50 show (B), expected values of 10, M '5, N' -4, and double-strand comparisons as default values.

The BLAST algorithm then performs a statistical analysis of the similarity between the two sequences (see, e.g., Karlin & Altschul, Proc. Natl. Acad. Sci. USA 90: 5873-. One measure of similarity provided by the BLAST algorithm is the minimum sum probability (P (N)), which indicates the probability at which a match between two nucleotide or amino acid sequences occurs randomly. For example, a test nucleic acid is considered similar to a phytase nucleic acid of the invention when the smallest sum probability of a nucleic acid compared to a phytase nucleic acid of the invention is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001. In the case where the test nucleic acid encodes a phytase polypeptide, the test nucleic acid is considered similar to the specified phytase nucleic acid if the comparison results in a minimum sum probability of less than about 0.5, more preferably less than about 0.2.

Thus, the phrase "substantially identical" with respect to two nucleic acids or polypeptides typically means that one polynucleotide or polypeptide contains a sequence that has at least 60% sequence identity, preferably at least 80%, more preferably at least 90%, and most preferably at least 95%, when compared to a reference sequence in the programs described above (e.g., BLAST, ALIGN, CLUSTAL) using standard parameters. One indication that two polypeptides are substantially identical is that the first polypeptide is immunologically cross-reactive with the second polypeptide. Typically, there is immunological cross-reactivity between polypeptides that differ by conservative amino acid substitutions. Thus, for example, a polypeptide is substantially identical to another polypeptide when the two polypeptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules can hybridize to each other under stringent conditions (e.g., conditions in the range of medium to high stringency).

"hybridization" includes any process by which a strand of nucleic acid is joined to a complementary nucleic acid strand by base pairing. Thus, strictly speaking, the term refers to the ability of the complement of the target sequence to bind to the test sequence and vice versa.

Typically, "hybridization conditions" are classified according to the degree of "stringency" of the conditions used to measure hybridization. The degree of stringency can be based, for example, on the melting temperature (Tm) of the nucleic acid binding complex or probe. For example, "maximum stringency" typically occurs at about Tm-5 ℃ (below probe Tm5 ℃); "high stringency" occurs at about 5-10 ℃ below Tm; "moderate stringency" occurs about 10-20 ℃ below the Tm of the probe; "Low stringency" occurs at about 20-25 ℃ below the Tm. Alternatively, or in addition, hybridization conditions may be based on salt or ionic strength conditions for hybridization and/or one or more stringency washes. For example, 6 × SSC is extremely low stringency; 3 × SSC — low to medium stringency; 1 × SSC to medium stringency; 0.5 × SSC indicates high stringency. Functionally, conditions of maximum stringency can be used to determine nucleic acid sequences that are strictly or near-strictly identical to the hybridization probes; and high stringency conditions are used to determine nucleic acid sequences that have about 80% or more sequence identity with the probe.

For applications requiring high selectivity, it is typically desirable to employ relatively stringent conditions to form the hybrid, e.g., relatively low salt and/or high temperature conditions are selected. Hybridization conditions, including medium stringency and high stringency, are provided in the book by Sambrook et al, incorporated herein by reference.

The term "isolated" or "purified" means that a material is separated from its original environment (e.g., its natural environment when it is a naturally occurring material). For example, a material is considered "purified" when it is present in a composition at a concentration that is higher or lower than the concentration at which it is present in a native or wild-type organism, or in combination with a component that is not normally expressed by a native or wild-type organism. For example, a native polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide is isolated when it is separated from some or all of the coexisting materials in the natural system. These polynucleotides may be part of a vector, and/or these polynucleotides or polypeptides may be part of a composition, but also isolated in that the vector or composition is not part of its natural environment. For example, a nucleic acid or protein is considered to be purified if it gives essentially only one band in an electrophoretic gel.

The term "derived from" as used herein when referring to a phytase (phytase) is intended to refer not only to a phytase produced or producible by a strain of the organism in question, but also to a phytase encoded by a DNA sequence isolated from the strain, produced in a host organism containing the DNA sequence. Furthermore, the term is also intended to refer to phytases encoded by DNA sequences of synthetic and/or cDNA origin having the distinguishing characteristics of the phytase in question. For example, "phytases from the genus Penicillium" refers to those enzymes naturally produced by the genus Penicillium that have phytase activity, as well as phytases similar to those produced by the genus Penicillium, but produced by non-Penicillium organisms transformed with nucleic acids encoding the phytases by employing genetic engineering techniques.

The present invention includes phytate hydrolases equivalent to those from the particular microbial strains mentioned. By "equivalent" herein is meant that the phytate hydrolase is encoded by a polynucleotide capable of hybridizing to a polynucleotide having a sequence as set forth in any of figures 1A-1C under conditions of moderate to high stringency. Equivalent means that the phytate hydrolase has at least 55%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the phytate hydrolase having the amino acid sequence disclosed in FIG. 2 (SEQ ID NO: 4).

The present invention also includes mutants, variants and derivatives of the phytate hydrolase of the invention, provided that the mutation, variant or derivative phytate hydrolase is capable of retaining at least one of the characteristic activities of the naturally occurring phytate hydrolase.

The term "mutant and variant" as used herein when referring to a phytate hydrolase refers to a phytate hydrolase obtained by altering the native amino acid sequence and or structure of the phytate hydrolase, for example by altering the DNA nucleotide sequence of the structural gene and/or by direct substitution and/or alteration of the amino acid sequence and/or structure of the phytate hydrolase.

The term "derivative" or "functional derivative" when referring to a phytase is used herein to refer to a phytase derivative having the functional characteristics of the phytase of the invention. Functional derivatives of phytases include naturally occurring, synthetically or recombinantly produced peptides or peptide fragments, mutants or variants which may contain one or more amino acid deletions, substitutions or additions and which possess the general characteristics of the phytases of the invention.

The term "functional derivative" when referring to a phytase-encoding nucleic acid is used throughout the specification to refer to a nucleic acid derivative having the functional characteristics of a phytase-encoding nucleic acid. Functional derivatives of nucleic acids encoding phytases of the invention include naturally occurring, synthetically or recombinantly produced nucleic acids or fragments, mutants or variants thereof which may contain one or more nucleic acid deletions, substitutions or additions and encode the phytases characteristic of the invention. Nucleic acid variants encoding phytases of the invention include alleles and variants based on the degeneracy of the genetic code known in the art. Nucleic acid mutants encoding phytases of the invention include those obtained by site-directed mutagenesis techniques (see, e.g., Botstein, D. and Shortle, D., 1985, Science 229: 1193-, error-prone PCR (error-prone PCR) (see, e.g., Leung, D.W., Chen, E., and Goeddel, D.V., 1989, techniques (Technique) 1: 11-15; Eckert, K.A., and Kunkel, T.A., 1991, application of PCR methods (PCRmethods application) 1: 17-24; and Cadwell, R.C., and Joyce, G.F., 1992, application of PCR methods 2: 28-33) and/or chemically-induced mutagenesis techniques known in the art (see, e.g., Elander, R.P., microbial identification, screening and strain improvement, Basic Biotechnology, J.Biotechnology and B.Kristiansen, academic Press, New York, 1987, 217).

An "expression vector" refers to a DNA construct comprising a DNA sequence operably linked to suitable control sequences capable of effecting the expression of the DNA in a suitable host. These control sequences may include a promoter to effect transcription, an operator sequence to selectively control such transcription, a sequence encoding a suitable ribosome binding site on the mRNA, and sequences which control termination of transcription and translation. It is preferred for different expression vectors to be used with different cell types. For vectors used in Bacillus subtilis, the preferred promoter is the aprE promoter; coli (e.coli) and glaA in a. niger. The vector may be a plasmid, a phage particle, or just one potential genomic insert. Once transformed into a suitable host, the vector may replicate and function independently of the host genome, or may integrate into the genome itself under the appropriate conditions. In the present specification, a plasmid and a vector are sometimes used interchangeably. However, the present invention is intended to include such other forms of expression vectors which are, or are, known in the art as performing equivalent functions. Thus, a variety of host/expression vector combinations may be employed to express the DNA sequences of the present invention. For example, useful expression vectors can be composed of chromosomal, nonchromosomal, and synthetic DNA sequence fragments, such as various known derivatives of SV40 and known bacterial plasmids (e.g., plasmids from E.coli, including col E1, pCR1, pBR332, pMb9, pUC19, and derivatives thereof), plasmids of a broader host range (e.g., RP4), phage DNA (e.g., various derivatives of lambda phage such as NM989, and other DNA phages such as M13 and filamentous single stranded DNA phages), yeast plasmids (e.g., 2 μ plasmids or derivatives thereof), vectors used in eukaryotic cells (e.g., vectors used in animal cells), and vectors derived from combinations of plasmids and phage DNA (e.g., plasmids modified to employ phage DNA or other expression control sequences). Expression techniques using the expression vectors of the invention are known in the art and are generally described, for example, in Sambrook et al, molecular cloning: a laboratory Manual (Molecular Cloning: laboratory Manual), 2 nd edition, Cold Spring Harbor Press (1989). Typically, these expression vectors containing the DNA sequences of the present invention are transformed into unicellular hosts by direct insertion into the genome of a particular species via an integration event (see, e.g., Bennett and Lasure, More fungal Gene manipulations in Fungi, academic Press, San Diego, pp.70-76 (1991), and the articles cited therein describing targeted genomic insertion in fungal hosts, all of which are incorporated herein by reference).

"host strain" or "host cell" refers to a host suitable for an expression vector containing the DNA of the present invention. Host cells useful in the present invention are generally prokaryotic or eukaryotic hosts in which expression can be achieved, including any transformable microorganism. For example, the host strain may be Bacillus subtilis, Escherichia coli (Escherichia coli), Trichoderma longibrachiatum, Saccharomyces cerevisiae (Saccharomyces cerevisiae), Aspergillus niger and Aspergillus nidulans. The vectors constructed using recombinant DNA techniques are used to transform or transfect host cells. These transformed host cells are capable of not only replicating vectors encoding phytases and variants (mutants) thereof, but also expressing the peptide product of interest.

Examples of suitable expression hosts include: bacterial cells, such as E.coli, Streptomyces (Streptomyces), Salmonella typhimurium (Salmonella typhimurium); fungal cells, such as Aspergillus and Penicillium; insect cells such as Drosophila (Drosophila) and armyworm (Spodoptera) Sf 9; animal cells, such as CHO, COS, HEK 293 or Bowes melanoma; plant cells, etc. Selection of an appropriate host is considered to be within the purview of one skilled in the art from the description herein. It should be noted that the present invention is not limited by the particular host cell used. Phytases and nucleic acids encoding phytases

One aspect of the invention provides a protein or polypeptide capable of catalyzing the hydrolysis of phytate and releasing inorganic phosphate; for example enzymes having a catalytic activity as defined in enzyme commission EC3.1.3.8 or EC3.1.3.26. In a preferred embodiment, the present invention provides what is known as a 3-phytase. In addition, the present invention also includes polynucleotides (e.g., DNA) encoding these phytate hydrolyzed proteins or polypeptides.

Preferably, the phytase according to the invention and/or the polynucleotide encoding the phytase is derived from a fungus, more preferably from an anaerobic fungus, more preferably from a species of the genus Penicillium, such as Penicillium hordei or Penicillium juniperi. Thus, it is contemplated that the phytases of the invention and/or the DNA encoding the phytases may be from species of the genus absidia (Absidiaspp.); species of Acremonium (Acremonium spp.); species of the genus actinomycete (Actinomycetes spp.); species of the genus Agaricus (Agaricus spp.); anaeromyces spp.; species of the genus Aspergillus (Aspergillus spp.) including a.auricularia, Aspergillus awamori (a.awamori), Aspergillus flavus (a.flavus), Aspergillus foetidus (a.foetidus), Aspergillus butenedioic (a.fumaricus), Aspergillus fumigatus (a.fumigatus), Aspergillus nidulans (a.nidulans), Aspergillus niger (a.niger), Aspergillus oryzae (a.oryzae), Aspergillus terreus (a.terreus) and Aspergillus versicolor (a.versicolor); aeurobasidium spp; species of cephalosporium (cephalosporium spp.); species of Chaetomium spp; coprinus spp (Coprinus spp.); dactyllum spp.; fusarium spp including f.conglomeratans, Fusarium polydatium (f.decelcellulare), Fusarium javanicum (f.javanium), Fusarium linonense (f.lini), Fusarium oxysporum (f.oxysporum), and Fusarium solani (f.solani); species of the genus gloomycopa (Gliocladium spp.); species of the genus humicola (humicola spp.), including h.insolens and h.lanuginosa; mucor spp); species of the genus Neurospora (Neurospora spp.) including Neurospora crassa (n.crassa) and Neurospora acidophilus (n.sitophila); neocallimastix spp.; orpinomyes spp.; species of the genus Penicillium (Penicillium spp.); phanerochaete spp.; species of the genus Phlebia (Phlebia spp.); piromyces spp.; species of the genus Pseudomonas (Pseudomonas spp.); rhizopus species (Rhizopus spp.); schizophyllum spp (Schizophyllum spp.); species of Streptomyces (Streptomyces spp.); trametes spp (Trametes spp.); and Trichoderma species (Trichoderma spp.) including t.reesei, t.longibrachiatum and Trichoderma viride (t.viride); and species of the genus A (Zygorhynchus spp.). Similarly, we contemplate that the phytases and/or DNA encoding the phytases described herein may be derived from bacteria such as Streptomyces spp, including Streptomyces olivochromogenes(s); especially fiber degrading ruminal bacteria (fiber degrading ruminal bacteria) such as filamentous bacillus succinogenes (fibrobacter succinogenes); and yeasts include Candida torreliii, Candida parapsilosis (C.parapsilosis), Candida sake (C.sake), Candida salivarius (C.zearaloides), Pichia minuta, Rhodotorula mucilaginosa (Rhodotorula glutinis), Rhodotorula glutinis (R.mucopolysaccharides), and Sporobolomyces castinosus (Sporobolomyces castanosarcina).

In a preferred embodiment, the phytase of the invention and/or the polynucleotide encoding the phytase is derived from (i) a grain-spoiling fungus (grain-fungi fungal), such as Penicillium hordei, Penicillium juniperi or Penicillium brevicaulis; or (ii) a root-associated ectomycorrhizal fungus (ectomycorrhizal fungi), such as a true Lacquertree (Laccialaccata), Laccaracia rufus, Paxillus (Paxillus involuteus), Gliocladium giganteum (Hebeloma crustiniforme), Amanita ruditapes (Amanita rubescens), or a toadstool (Amania muscaria).

According to a preferred embodiment, the phytase according to the invention and/or the polynucleotide encoding the phytase is present in purified form, i.e. in a composition at a concentration higher or lower than its concentration in the natural or wild-type organism, or in combination with components which are not normally produced by expression in the natural or wild-type organism.

The present invention includes phytate hydrolyzed proteins and peptides having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity as compared to a phytate hydrolase having the amino acid sequence disclosed in FIG. 2 (SEQ ID NO: 4).

The present invention also includes polynucleotides, e.g., DNA, encoding a phytate hydrolase from a fungal source, e.g., a Penicillium species, the polynucleotides comprising a sequence at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, and at least 95% identical to the polynucleotide sequence disclosed in any one of FIGS. 1A-1C, provided that the polynucleotide encodes an enzyme capable of catalyzing the hydrolysis of phytate and the release of inorganic phosphate. In a preferred embodiment, the polynucleotide encoding a phytate hydrolase has the polynucleotide sequence shown in any one of figures 1A-1C, or is capable of hybridising to, or is complementary to, the polynucleotide sequence shown in any one of figures 1A-1C. It will be appreciated by those skilled in the art that, due to the degeneracy of the genetic code, a variety of polynucleotides can encode the phytate hydrolase disclosed in FIG. 2 (SEQ ID NO: 4). The present invention includes all such polynucleotides. Obtaining a polynucleotide encoding a phytate hydrolase

Nucleic acids encoding phytate hydrolase can be obtained, for example, from cloned DNA (e.g., DNA "libraries") by standard procedures known in the art, or by chemical synthesis, cDNA Cloning, PCR, or Cloning of genomic DNA or fragments thereof, or purified from desired cells, such as fungal species (see, e.g., Sambrook et al, 1989, molecular Cloning: A Laboratory Manual, 2 nd edition, Cold Spring harbor Laboratory Press, Cold Spring harbor, N.Y.; Glover, DM and Hames, BD (eds.), (DNA Cloning: practice methods) (DNA Cloning: A practical Aprrroach, volumes 1 and 2, 2 nd edition). Nucleic acid sequences derived from genomic DNA may contain coding regions as well as regulatory regions.

When the gene is cloned from a genomic DNA molecule, DNA fragments are prepared some of which will contain at least a portion of the gene of interest. Various restriction enzymes can be used to cleave DNA at specific sites. Alternatively, DNA may be fragmented in the presence of manganese using dnase, or may be sheared by physical methods such as ultrasound. These linear DNA fragments are then separated by size by standard methods including, but not limited to, agarose and polyacrylamide gel electrophoresis, PCR, and column chromatography.

Once the nucleic acid fragment is generated, the particular DNA fragment encoding the phytate hydrolase can be determined in a variety of ways. For example, the phytate hydrolase encoding gene of the invention or a specific RNA thereof, or fragments thereof such as probes or primers, can be isolated and labeled and then used in hybridization assays to identify the resulting gene. (Benton, W. and Davis, R., 1977, science 196: 180; Grunstein, M. and Hogness, D.1975, Proc. Natl. Acad. Sci. USA 72: 3961). Those DNA fragments that have substantial sequence similarity to the probe will hybridize under moderate to high stringency conditions.

The invention comprises the use of SEQ ID NO: 1. SEQ ID NO: 2 or SEQ ID NO: 3. or a suitable portion or fragment thereof (e.g., at least about 10-15 contiguous nucleotides) as a probe or primer for screening nucleic acids of genomic or cDNA origin for a phytase enzyme derived from a fungal species, particularly a Penicillium species. A polypeptide encoding a phytase from a penicillium species and having the amino acid sequence shown in SEQ ID NO: 1. SEQ ID NO: 2 or SEQ ID NO: 3. or a portion or fragment thereof, can be identified by using a probe, i.e., seq id NO: 1. SEQ ID NO: 2 or SEQ ID NO: 3, and performing DNA-DNA or DNA-RNA hybridization or amplification. Accordingly, the present invention provides a method for detecting a nucleic acid encoding a phytate hydrolase of the invention, which method comprises contacting a genomic or cDNA derived penicillium nucleic acid with a nucleic acid sequence as set forth in SEQ ID NO: 1. SEQ ID NO: 2 or SEQ ID NO: 3, or a part or all of the nucleic acid sequence of 3.

Is capable of hybridizing to SEQ ID NO: 1. SEQ ID NO: 2 or SEQ ID NO: 3 under moderate to high stringency conditions are also included within the scope of the present invention. In one embodiment, hybridization conditions are based on the melting temperature (Tm) of the nucleic acid binding complex taught by Berger and Kimmel (1987, molecular cloning techniques, Methods in Enzymology, Vol.152, Academic Press, san Diego CA), which is incorporated herein by reference, and are given a defined stringency. In this embodiment, typically, "maximum stringency" occurs at about Tm-5 ℃ (5 ℃ below the probe Tm); "high stringency" occurs at about 5-10 ℃ below Tm; "moderate" or "intermediate stringency" occurs about 10-20 ℃ below Tm; "Low stringency" occurs at about 20-25 ℃ below the Tm. Maximum stringency hybridization can be used to determine or detect identical or near identical polynucleotide sequences, while intermediate or low stringency hybridization can be used to determine or detect polynucleotide sequence homologs.

In another embodiment, stringency is determined by the wash conditions employed after hybridization. For this embodiment, "low stringency" conditions comprise washing with 0.2 XSSC/0.1% SDS solution at 20 ℃ for 15 minutes. "Standard stringency" conditions comprise a further washing step: the cells were washed with 0.2 XSSC/0.1% SDS solution at 37 ℃ for 30 minutes.

Amplification methods employed in Polymerase Chain Reaction (PCR) technology are described in DieffenbachCW and GS Dveksler (1995, PCR primers, A laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y.). Has the sequence shown in SEQ ID NO: 1. SEQ ID NO: 2. or SEQ ID NO: 3 and up to about 60 nucleotides, preferably about 12-30 nucleotides, more preferably about 25 nucleotides, can be used as probes or PCR primers.

One preferred method for isolating the nucleic acid construct of the invention from a cDNA or genomic library is to use a nucleic acid construct according to the invention having the sequence given in SEQ ID NO: 4 of the amino acid sequence of the protein is subjected to Polymerase Chain Reaction (PCR). This PCR can be performed, for example, using the technique described in U.S. Pat. No. 4,683,202.

In view of the above, it will be appreciated that the polynucleotide sequences provided in FIGS. 1A-1C are useful for obtaining identical or homologous fragments of polynucleotides encoding enzymes having phytase activity from other species, particularly from fungi (e.g., grain-spoiling fungi, or ectomycorrhizal fungi). Expression and recovery of Phytase

The polynucleotide sequences of the present invention may be expressed by operably linking to expression control sequences in an appropriate expression vector and may be used in such expression vectors to transform appropriate hosts according to techniques well established in the art. The polypeptide produced by expression of the DNA sequence of the invention may be isolated from the fermentation cell culture and purified in a variety of ways according to techniques well established in the art. The skilled person will be able to select the most appropriate separation and purification technique.

More specifically, the present invention provides host cells, expression methods and systems for producing microbial (e.g., penicillium species) derived phytate hydrolase. Once a nucleic acid encoding a phytate hydrolase of the invention has been obtained, recombinant host cells containing the nucleic acid can be constructed using techniques well known in the art. Molecular biology techniques are disclosed in Sambrook et al, molecular biology cloning: a Laboratory Manual, 2 nd edition (1989), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989).

In one embodiment, a nucleic acid encoding a phytase from a penicillium species that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and at least 95% identical to a nucleic acid of any one of figures 1A-1C, or capable of hybridizing to a nucleic acid of any one of figures 1A-1C under intermediate to high stringency conditions, or complementary to a nucleic acid of any one of figures 1A-1C, is obtained and transformed into a host cell with an appropriate vector.

The nucleic acid encoding a phytase may contain a leader sequence which enables the encoded phytase to gain secretion. Depending on whether the phytase is expressed or secreted in cells, the DNA sequences or expression vectors of the invention may be engineered to express the mature form of phytase with or without a signal sequence that is native to the phytase or that functions in fungi (e.g., Aspergillus niger), other prokaryotic or eukaryotic organisms. Expression can also be effected by removing or partially removing the signal sequence.

A wide variety of vectors and transformation and expression cassettes suitable for cloning, transformation and expression in fungal, yeast, bacterial, insect and plant cells are known to those skilled in the art. Typically, the vector or cassette contains sequences that direct the transcription and translation of the nucleic acid, a selectable marker, and sequences that allow for autonomous replication or chromosomal integration. Suitable vectors contain a 5 'region of the gene that contains the transcriptional initiation control, and a 3' region of the DNA segment that controls transcriptional termination. These control regions may be derived from genes homologous or heterologous to the host, so long as the selected control region is capable of functioning in the host cell.

Initiation control regions or promoters useful for driving expression of phytate hydrolase in a host cell are known to those skilled in the art. A nucleic acid encoding a phytate hydrolase is operably linked via a start codon to an expression control region selected for efficient expression of the enzyme. Once the appropriate cassettes have been constructed, they can be used to transform host cells.

When plant expression vectors are used, the phytase encoding sequence may be expressed from a variety of promoters. For example, viral promoters such as the 35S and 19S promoters of CaMV (Brisson et al (1984) Nature 310: 511-. Alternatively, plant promoters such as the promoter of the small subunit of rubisco can be used (Coruzzi et al (1984) EMBO J3: 1671-; or heat shock promoters (Winter J and Sinibaldi RM (1991) Results System Cell Differ 17: 85-105). These constructs can be introduced into plant cells by direct DNA transformation or pathogen-mediated transfection. For a review of these techniques, see Hobbs S or Murry LE (1992) McGrawHill scientific and technical Ann (McGraw Hill Yeast of Science and technology), McGraw Hill, New York, N.Y., pp.191-196; or Weissbach and Weissbach (1988) Methods in plant molecular Biology (Methods for plant molecular Biology), Academic Press, New York, N.Y., pp.421-463.

General transformation methods are described in Current protocols Molecular Biology (Current protocols Biology) (Vol.1, eds. Ausubel et al, John Wiley & Sons, Inc.1987, Chapter 9), which include calcium phosphate methods, transformation with PEG and electroporation. For Aspergillus and Trichoderma (Trichoderma), PEG and calcium mediated protoplast transformation (Finkelstein, DB1992 transformation, Biotechnology, technology and products of Filamentous fungi (Biotechnology of Filamentous fungi and technologies and products) (edited by Finkelstein and Bill) 113-. Electroporation of protoplasts is disclosed in Finkelestein, DB1992 transformation, Biotechnology, technology and products of filamentous fungi (Finkelstein and Bill eds.) 113-156. Microprojectile bombardment of conidia is disclosed in Fungaro et al (1995) transformation of Aspergillus nidulans, FEMSMICmicrobiology Letters125293-298 by microprojectile bombardment of intact conidia. Agrobacterium-mediated transformation is disclosed in Groot et al (1998) Agrobacterium tumefaciens-mediated transformation of filamentous fungi, Nature Biotechnology 16839-842. For the transformation of Saccharomyces, lithium acetate mediated transformation and PEG and calcium mediated protoplast transformation and electroporation techniques are known to those skilled in the art.

Host cells containing the coding sequence for the phytate hydrolase of the invention and expressing the protein can be identified by a variety of methods known to those skilled in the art. These methods include, but are not limited to, DNA-DNA or DNA-RNA hybridization and protein bioassay or immunoassay techniques, including membrane-based, solution-based, or chip-based techniques for detecting and/or quantifying nucleic acids or proteins.

It should also be noted that the present invention contemplates in vitro expression of the phytases described herein.

In one embodiment of the invention, the polynucleotide sequence encoding a phytate hydrolase from Penicillium hordei (ATCC22053) is isolated and expressed in A.niger, while in another embodiment the polynucleotide sequence is expressed in A.nidulans. The expressed phytase can then be recovered, for example, as described below.

The phytases of the invention can be recovered from the culture medium or from host cell lysates. If in membrane bound form, it may be released from the membrane by use of a suitable detergent solution (e.g.Triton-X100) or by enzymatic cleavage. Cells used to express phytase may be disrupted by a variety of physical or chemical means, such as freeze-thaw cycles, sonication, mechanical disruption, or cell lysis reagents. It may be desirable to purify the phytase from a protein or polypeptide of a recombinant cell. The following methods are examples of suitable purification methods: carrying out fractionation on an ion exchange column; ethanol precipitation; reverse phase HPLC; chromatography on silica or cation exchange resins such as DEAE; carrying out chromatographic focusing; SDS-PAGE; ammonium sulfate precipitation; gel filtration using, for example, Sephadex G-75; protein A Sepharose column for removing impurities; and a metal chelating column that binds to the epitope tagged phytase form. A variety of protein purification methods may be employed, and are known in the art and described, for example, in Deutscher, methods in enzymology, 182 (1990); scopes, protein purification: principles and practices (Protein Purification: Principles and Practice), Springer-Verlag, New York (1982). The purification steps used will depend, for example, on the nature of the preparation method used and the particular form of phytase produced. Use of phytate hydrolase

The phytases and derivatives thereof described herein can be used in a variety of applications where it is desirable to separate phosphorus from phytic acid. Several examples of applications are given below.

For example, the invention provides the use of bacterial cells or spores producing the phytase of the invention as probiotic or direct fed microbial products. A preferred embodiment of said use is the phytase producing Aspergillus species of the invention.

In addition, the present invention also contemplates the use of the phytases described herein in food or animal feed.

The present invention provides food or animal feed containing the phytases described herein. Preferably, the food or animal feed contains as an additive a phytase enzyme active in the alimentary canal, preferably the crop and/or the small intestine, of livestock, such as poultry and pigs, and of farmed aquatic animals, including fish and shrimp. Preferably, the additive is also active in the processing of food or feed.

In addition, the invention provides food or animal feed containing cells or spores capable of expressing the phytases described herein.

Furthermore, the present invention contemplates a method for preparing a food or animal feed, characterized in that a phytase according to the invention is mixed with said food or animal feed. The phytase is added before processing in dry form, or before or after processing in liquid form. According to one embodiment using a dry powder, the enzyme is diluted in liquid form on a dry carrier such as ground particles.

The invention also provides a method for preparing a food or animal feed, characterized in that cells and/or spores capable of expressing a phytase according to the invention are added to said food or animal feed.

Furthermore, the present invention provides the use of the phytases described herein, with or without an additional phosphatase, for the production of myo-inositol and inorganic phosphate, as well as intermediates for phytic acid.

The invention also provides a method for reducing the level of phosphorus in animal manure, characterized in that an animal feed according to the invention is fed to the animal in an amount effective to convert phytic acid contained in the animal feed.

The phytase and phytate derived intermediates of the invention can also be used in grain wet milling, cleaning and personal care, and textile processing.

The following examples are provided for illustration only and are not intended to limit the scope of the present invention in any way. All patents and publications referred to in this specification are hereby incorporated by reference in their entirety.

Example (b):

example 1

Evidence of phytate hydrolytic Activity in liquid cultures

Penicillium juniperi (ATCC10519) and p.hordei (ATCC22053) were cultured in designated media containing various concentrations of inorganic phosphate, and then growth characteristics and phytase production were determined and compared. Using a spore suspension (2X 10)⁶Final concentration of spores/ml) was inoculated into minimal medium (Vogels) and the concentration of phosphate in the medium was varied to see how this would affect growth and phytase production. Cultures were grown by shake flask culture in 50ml medium at 25 ℃ (P.hordei) or 30 ℃ (Penicillium juniperi). Cultures were harvested at 24, 48, 72 and 96 hours. Culture supernatants were assayed for phytase activity using the method of Fiske and Subbrow. Growth was determined by dry weight (p. hordei) or OD reading (penicillium juniperi). Effect of different Medium conditions on growth and morphology

A series of penicillium growth curves, such as the sabina growth curve of fig. 3 and the p.hordei growth curve of fig. 4, were prepared and the effect of available P on growth and phytase production in the medium was observed. When the P level was reduced to 0.57mM (1/64P of the growth curve [ FIG. 3]), morphological changes associated with stress conditions in fungal growth (e.g.fragmentation of the mycelium, formation of precipitates, heterogeneous growth (heterosis) and overall pale yellow color) were observed, especially for P.juniperi. This physiological strain is associated with the appearance of phytase activity near the late exponential phase of the growth curve (48 hours; see Table 1 below). Morphological evidence of phytic acid utilization was also observed in the same low P (0.57mM) cultures supplemented with 1mM phytic acid as a source of phosphorus after 24 hours of culture. The morphological changes observed in the absence of added phytic acid were not significant, and in fact these phytate supplemented samples were similar to cultures in higher P medium where P was not limiting. This clearly shows that phytate specific hydrolytic activity is produced so as to be able to provide P to the growing fungus. However, when supplemented with 5mM phytate, the culture did not grow, suggesting that this level of phytate in the medium will chelate with essential minerals resulting in a medium that is unable to support fungal growth and nutrition.

Hordei study, culture media for fungi contained:

high phosphate (1.14mM)

Low phosphate (0.57mM)

Low phosphate plus 1mM supplemental Phytic acid

Growth was monitored by gravity measurements over 0, 24, 48, 72 and 96 hours and observed for morphological features that appeared in response to different media conditions. The following are the main observations and conclusions:

1. there was expected good growth in the high phosphate, consistent fungal morphology indicating healthy cultures.

2. Growth was markedly poor under low phosphate conditions, the fungus morphology was heterogeneous with evidence of aggregation and fragmentation of the mycelium. The cultures exhibited a morbid yellow color.

3. Cultures similar to (2) no longer appear to be under the same physiological stress when supplemented with phytic acid (substrate). The growth of biomass was similar to condition (1) and the morphology of the fungus was the same as the fungus under high phosphate conditions.

4. Growth curves and photographic evidence of these cultures support these observations, the main conclusion overall being that we observed phytate hydrolysis activity in condition (3) that allows the fungus to obtain phosphate from phytate and thus prevent the phosphate starvation stress to which the culture is being subjected. Phytase Activity in culture supernatants

Table 1 below shows the phytase activity in the supernatant of the culture of Penicillium juniperi. Table 1 also shows data for Aspergillus niger cultures grown under the same conditions in Vogels 1/64P (0.57mM) medium as a control for phytase activity. We can observe that the aspergillus niger culture shows similar levels to the penicillium juniperi culture. TABLE 1 Phytase Activity of fungal culture supernatants cultured in media containing varying levels of inorganic P

Fungal species	Type of culture medium	P content (mM)	Phytase Activity (μmolsPmin)-1ml-1)
				Penicillium juniperi	1/2×MEB	8-12	0±
Penicillium juniperi	Vogels (full P)	37.0	0±
				Penicillium juniperi	Vogels(1/4P)	9.20	0±
Penicillium juniperi*	Vogels(1/64P)	0.57	0.121±0.077
				Penicillium juniperi**	Vogels(1/64P)	0.57	0.028±0.027
Aspergillus niger	Vogels(1/64P)	0.57	0.036±0.027

The activity detected in these samples at 48 hours post inoculation was compared. The phytase activity is expressed as the amount of μmol P released per minute per ml culture supernatant. The activity of the samples was calculated from triplicate identical flasks and the supernatants were repeatedTwo phytase assays. The activity is shown as mean ± SD, n ═ 6. Penicillium juniperi^*Penicillium juniperi (Roxb.) kummer^**Refers to data from two independent time course experiments.

Thus, significant physiological stress is associated with phosphate-limited cultures, which adversely affects growth, and is linked to the occurrence of phytase activity. Concentration of culture supernatant

Another evidence of phytase activity could be expected from concentrated supernatants (concentrated proteins). For example, a concentrated protein sample can be obtained from:

1. a Penicillium culture (here expected to express phytase) under stress and low phosphate conditions,

2. a culture of Penicillium under high phosphate and non-stressed conditions, where no phytase production is expected, and

3. cultures supplemented with low phosphate and supplemented phytic acid.

Silver stained SDS-PAGE gels of these concentrated protein samples are expected to show a protein profile in which a protein band (the putative phytase band) is present in the concentrated protein from condition 1 (above) and is absent in condition 2. A similar, albeit lower, level band is expected to occur in condition 3. Based on the amino acid sequence of p.hordei phytase, and on the fact that it appears to be an extracellular enzyme, the expected size of the protein is about 50 kDa. However, it should be noted that glycosylation modifications on the extracellular enzyme can increase the MW to 60-90 kDa.

Example 2

PCR amplification of Phytase Gene fragments

Degenerate primer design

Based on published alignments of phytase amino acid sequences, a number of degenerate primers were designed to conserved structural and catalytic regions. These regions include those that are highly conserved in phytases, as well as those known to be important for the structure and function of enzymes.

In one study, 4 published phytase amino acid sequences were aligned. These phytase sequences are derived from: (i) aspergillus niger (definition: Aspergillus niger phyA gene; ACCESSION: Z16414; van Hartingsveldt, W. et al, 1992); (ii) aspergillus fumigatus (definition: Aspergillus fumigatus phytase gene, complete cds.; ACCESSION: U59804; Pasamontes, L. et al 1997); (iii) aspergillus terreus (definition: Aspergillus terreus 9A1 phytase gene, complete cds.; ACCESSION: U59805; Mitchell, D.B. 1997); and (iv) Myceliophtora thermophilus (definition: Myceliophtatothermophila phytase gene, complete cds.; ACCESSION: U59806; Mitchell, D.B. 1997). It should be noted that all of these sequences are publicly available from GENbank, each of which is incorporated herein by reference.

5 specific regions that meet the above criteria were selected, and then forward and reverse primers were designed from these amino acid sequences. The specific amino acid regions used to design these primers are indicated in the protein sequence alignment of FIG. 5 in black boxes. Degenerate nucleotide PCR primers were synthesized by MWG-Biotech, Inc. using the genetic code according to codon usage.

In another study, a pair of primers, designated CS1 and CS2, was designed from only the published amino acid sequences of a. niger phyA and phyB phytases. The design of these primers is as follows:

primer CS 1: the forward (5 '-3') primer, derived from the RHGARYPT region (see FIG. 5, amino acids at positions 110-120), which is the phosphate binding domain of phyA phytase, is required for catalytic activity.

Primer CS 2: the reverse primer, derived from the FT (H/Q) DEW (I/V) region (see FIG. 5, amino acids 335 and 345), which is the central region of the phytase, appears to be relatively well conserved.

The sequences of these primers are given below:

CS1：5’CGI CAT/C GGI GCI CGI TAT/C CC3’

CS2：3’AAA/G TGI GTI CTA/G CTT/C ACC T/CAI5’

CS2：5’AC/TC CAC/T TCG/A TCI TGI GTG/A AA3’

since all primers were synthesized in the 5 '-3' direction, the reverse primer was prepared as CS2 shown in bold. The amino acids were changed to triplet codons using the standard genetic code and the standard IUB code was used for mixed base positions (e.g., A/C/T/G was named I).

For additional details on the design of primers CS1 and CS2, we will now describe with reference to FIG. 6. FIG. 6 shows published amino acid sequences of the A.niger phyA and phyB phytases from which the primers CS1 and CS2 were designed. Specifically, the sequences aligned are from 1: piddington et al, 1993 (Phya and Phyb of a. niger var. awamori), 2: van Hartingsveldt et al, 1993 (Phya. niger), 3: erlich et al, 1993 (Phy B from Aspergillus niger). The alignment of FIG. 1 was performed using CLUSTAL V (Higgins D.G., et al, 1992) from PHYLIP software package version 3.5. The conserved sequences used to design degenerate primers CS1 and CS2 are shown.

We can observe from the alignment of FIG. 6 that the phosphate binding domains of PhyA and PhyB are well conserved in PhyA (RHG)ARYP, respectively; van Hartingsveldt et al, 1993) and PhyB (RHG)ERYP, respectively; piddington et al, 1993) differ by only a single amino acid. Degenerate primer CS1 was designed to be complementary only to this region in the phyA version of the sequence, i.e., using RHGARYPT as the basis for primer design. In this case, the primer would thus be biased towards the phyA-type phosphate binding domain. The second conserved region underlying primer CS2 is located in the middle of the PhyA and PhyB amino acid sequences. This conservation is a measure ofCorresponding to amino acids 285-291 in PhyA (FTHDEWI). In PhyB, the amino acid sequence (FTQDEWV) corresponds to amino acids 280-286.

Degenerate primers CS1 and CS2 successfully amplified a 650bp region from p.hordei by PCR, as described below.

PCR amplification of Phytase Gene fragments

The putative phytase gene fragment was PCR amplified using genomic DNA from Penicillium species as template and the combination of primers described above. PCR was performed using PCR Ready-to-go Beads from Amersham Pharmacia. Conditions were determined by a separate experiment, but typically run in a Techne thermocycler for 30 cycles. Successful amplification was verified by running the PCR reaction products on a 1% agarose gel. PCR products of the correct expected size (650bp) amplified from p.hordei by primers CS1 and CS2 were purified by gel extraction using Qiaquick Spin gel extraction kit from Qiagen. The purified PCR product was ligated into a commercial pGEM-T Easy vector system (Promega Co.) for cloning. The ligation reaction was incubated overnight at 4 ℃ with a total of 10. mu.l volumes of 10 Xligase buffer and 1. mu.l (1U. mu.l)^-1) The T4DNA ligase of (1). The insert DNA fragment is typically used in this reaction in a molar ratio of 1-4: 1 between insert and vector DNA. A100. mu.l aliquot of CaCl was removed from the-80 ℃ storage₂Competent E.coli XL-1Blue cells were used for transformation after thawing on ice. To the cells add 3 u l connection mixture, and in ice incubated the mixture for 20 minutes. The cells were then heat shocked at 42 ℃ for 1 minute before being placed back on ice for 5 minutes. To the transformation mixture was added 0.9mL L-broth and the cells were incubated without selection with shaking in order to allow the ampicillin resistance gene product to be expressed before selection was applied (37 ℃, 1 h). Samples of 200, 300 and 400. mu.l of this culture were then spread directly onto selective agar plates. The plates were incubated overnight at 37 ℃. Colonies containing the recombinant plasmid were observed by blue/white screening. To rapidly screen for recombinant transformants, positivity was inferred (white)Color) colonies plasmid DNA was prepared. DNA was isolated according to the procedures of Sambrook et al (1989) using the method of Birnboim and Doly. The presence of the correct insert (650bp) in the recombinant plasmid was confirmed by restriction analysis. The DNA was digested with Not1-pPstI restriction enzyme overnight at 37 ℃ and the digestion products were visualized by agarose gel electrophoresis. Many clones contained the correct size insert, and some were selected for manual sequencing to see if the insert was a phytase gene fragment. Sequencing of the insert was performed using the dideoxy chain termination method of Sanger et al (1977) and a modified form of T7DNA polymerase (sequencer enzyme version 2.0). The reaction was carried out using the reagents provided in the Sequenase kit version 2.0 (Amersham Life Science-United States Biochemical Co., Ltd.) according to the manufacturer's instructions. Partial sequences from the ends of two clones (designated 3D and 3G) indicated that phytase gene fragments have been cloned. Plasmid DNA from these two clones was sent to MWG-Biotech for full length sequencing of the double stranded insert. 2C sequence analysis

These sequences were analyzed by BLAST and protein translation sequence tools. BLAST comparisons at the nucleotide level showed various levels of homology to published phyA phytase sequences. Initially, the nucleotide sequences were submitted to BLAST (basic BLAST version 2.0) by accessing the BLAST database on the world wide web. The net value used ishttp：//ncbi.nlm.nih.gov/cgi-bin/ BLAST. The selected program is blastn and the selected database is nr. Standard/default parameter values are used. The sequence data for the putative p.hordei gene fragment is entered as a sequence in FASTA format, and the query is submitted to BLAST to compare the sequences of the invention to sequences already in the database. For this 650bp fragment and the EcoRI gene fragment from the first library screen (discussed below), the results returned show a large number of matching positions with the phytase genes of aspergillus niger, e.nidulans, aspergillus fumigatus and actinomyces thermophilus (t.thermophilus).

These sequences are then processed using a DNA-to-protein translation tool called the protein machinery. The tool may also be online (http:// medkey. gu. se/edu/trans. ht. httml) was obtained. Another suitable translation tool is used as a translation machine, which can be accessed from the webhttp：//www2.ebi.ac.uk/translate/obtain. Hordei was inserted into the analysis block with the DNA sequence of the putative phytase gene fragment from p.hordei, using the standard genetic code as the basis for translation. The forward and reverse strands are translated in all three reading frames. The analysis tool transmits the translated amino acid sequence as a one-letter code amino acid sequence to the screen. When translated into an amino acid sequence, these clones appeared to contain 212 amino acids (636bp is the actual size of the gene fragment) with no stop codon. Analysis of the amino acid sequence showed that this fragment contained two correct ends (for the design of primers CS1 and CS 2), contained the necessary P-binding motif (RHGARYP) and 3 cysteines that were also present in the published phyA phytase sequence. We reasoned that this 636bp fragment cloned was the phyA phytase gene fragment of p.

Sequence alignments and analysis of these alignments were performed at the nucleotide and amino acid levels using the ALIGN program (Alignment Editor Version 4/97; dominikHepperine, Fontanenster.9c, D016775, Neuglobsow, Germany). In the analysis, the subject sequence is pasted and in PHYLIP interleaving format. Homology analysis was performed using the "analysis" section of the program, specifically an option entitled "distance analysis". Using a minimum of two amino acid sequences (i.e., two "species"), this allows the calculation of% homology and the number of distinct sites between the species. Minimum and maximum homology are calculated as%. The basis for homology analysis is% identity, i.e., the calculation yields a percentage value based on the number of identical amino acids (or bases) divided by the total number of amino acids (or bases) and then multiplied by 100 ". The amino acid sequence of p.hordei and published phytase sequence were placed in ALIGN program and manually aligned at amino acid level. Exemplary results are shown in fig. 7. In FIG. 7, the sequence designated "P.hordei3D" (SEQ ID NO:) is the deduced translation of the PCR product obtained with the degenerate primers CS1 and CS 2.

Example 3

Southern analysis for library preparation

Genomic DNA from P.hordei, P.juniperi and A.niger was digested with a panel of restriction enzymes at 37 ℃ overnight. Successfully digested DNA was run on a 1% agarose gel for transfer to a nylon membrane. After the electrophoresis was completed, the agarose gel was soaked in 0.2M HCl for 10 minutes to depurinate the DNA, and then in ddH₂And O, short-time washing. DNA transfer to Hybond by alkaline capillary blotting^TM-N + membrane (Amersham International PLC). The blotting system was installed so that the nylon filter was sandwiched between the gel and a stack of absorbent paper towels. A core of Whatman3MM paper (Schleicher and Schuell, Dassel, Germany) was prepared on a glass plate across a bath of transfer buffer (0.4M NaOH). The gel was inverted on the core, taking care to avoid bubble formation, and a Nescofilm band was used around the gel edge to prevent the blotting action of the paper towel from bypassing the gel. Using an equal size Hybond^TMthe-N + film covers the gel, which was previously cut off a corner to match the gel and pre-wetted in 3 x SSC. Next, 3-5 sheets of 3MM paper were placed on top of the filter membrane, a stack of 10cm blotting paper was added, followed by a 0.5kg weight to complete the blotting system installation. The blotting system was maintained for 8-24 hours to transfer the DNA. The membrane was then washed briefly in 2 XSSC at RT and baked in a vacuum oven at 80 ℃ to fix the DNA on the membrane. This Southern blot was probed with a 636bp fragment from p.hordei. First of all by³²The P isotope labels the fragment with the aid of the High Prime DNA labelling kit (Boehringer Mannheim). Denatured fragments were added to the random priming labeling reaction to incorporate radiolabeled adenine. Southern blots were prehybridized in hybridization tubes at 42 ℃ for 1 hour in 12ml Easy-Hyb buffer (Boehringer Mannheim). The radiolabeled probe was denatured, added to 5ml Easy-Hyb hybridization buffer, and then hybridized overnight at 42 ℃. After hybridization, the blot was washed by incubation in 40ml3 XSSC, 0.1% SDS for 15 min at 42 ℃. This low stringency wash was repeated with fresh wash solution. After stringent washing, the blot was rinsed in 3 XSSC or less,then sealed in clean plastic and exposed to an x-ray film. After exposure for 2 hours, the x-ray film was developed.

As shown in fig. 8, strong hybridization bands were observed in p. Specifically, FIG. 8 depicts a Southern blot gel showing hybridization between probes containing PCR products obtained using degenerate primers CS1 and CS2 and various fungal genomic DNA digests; lane 1-size standard reference; lane 2-Aspergillus niger-EcoRI; lane 3-Penicillium juniperi-EcoRI; lane 4-Penicillium hordei-EcoRI; lane 5-Peniciliumhorrei-BamHI; lane 6-Penicillium hordei-Sall; lane 7-Penicillium hordei-KpnI; lane 8-Penicillium hordei-SacI. These results indicate that the 636bp fragment can be used as a probe for library screening.

Example 4

Isolation of a polynucleotide sequence encoding a phytase from the P.hordei genome4A. Preparation and screening of Hordei genomic library

After Southern hybridization analysis, we decided to prepare a partial genomic library of p.hordei in order to attempt to clone the full-length phytase gene. A size-restricted plasmid library targeted to the 4.4Kb EcoRI fragment (estimated from Southern analysis) was prepared. EcoRI digested p.hordei genomic DNA was run on a 1.25% agarose gel. About 4.4Kb of the digested fragment was extracted from the gel and purified by Glass-Max (Gibco-BRL, Scotland). The purified genomic fragment and EcoRI linearized pSKII Bluescript vector (Stratagene) were used in the shotgun ligation reaction. The vector was first dephosphorylated prior to ligation, and then the ligation reaction was performed overnight at 14 ℃. The library was generated by transforming E.coli XL-10Gold super competent cells (Stratagene). A100. mu.l portion of the cells was removed from the-80 ℃ storage and thawed on ice for transformation. Add 4. mu.l of beta-mercaptoethanol to the cells on ice. To this mixture was added 3. mu.l of the ligation mixture and incubated on ice for 20 minutes. The cells were then heat shocked at 42 ℃ for 30 seconds and then reweighedPlace freshly on ice for 2 minutes. To this transformation mixture, 0.9ml of NZY liquid medium was added, and the cells were incubated without selection with shaking to express the ampicillin resistance gene. These transformed cells were plated on LB agar plates with blue/white spot selection and incubated overnight at 37 ℃. A total of 728 colonies were observed on the plates, 450 of which were white. Colonies were transferred to nylon filters as per Maniatis (10% SDS-lysis, 3 min; 1.5M NaOH-denaturation, 5 min; 1.5M TricHCl-neutralization, 5 min; 3 XSSC-wash, 5 min). The filters were then vacuum-dried at 80 ℃ for 2 hours to immobilize the DNA. The library was screened using a 32P radiolabeled 636bp probe in the same manner as Southern hybridization. After hybridization, the filters were washed in 3 XSSC, 0.1% SDS at 42 ℃ for 15 minutes. The filters were then rinsed in 3 XSSC, sealed in plastic and exposed to x-ray film overnight at-80 ℃.5 positive cross-over points were observed on this sheet. These spots were compared to agar plates containing transformants. These hybridization spots were paired with more than one single colony on an agar plate. All colonies located in the radius of the intersection were picked with a sterile loop and inoculated with 2ml Luria broth. The culture was incubated at 37 ℃ for 2 hours. The culture was diluted from 10^-1To 10^-5Then, 100. mu.l of each sample was spread on LB-amp agar plates and incubated overnight at 37 ℃. Plates with 10-150 colonies on top were selected for secondary screening. Colonies were transferred as described above and filters were processed using the same procedure. Preparation of fresh³²The probe is P-labeled and the filters are then screened in the same manner as described above. Stringent washes were performed using 2 XSSC, 0.1% SDS at 42 ℃ for 15 minutes. The filters were then rinsed in 2 XSSC, sealed in plastic and exposed to x-ray film for 2 hours. The developed discs showed a very high number of strong cross-over points, consistent with the amplification of positive colonies from the first screening. The plate was then compared to the plate and the spots were adjusted to see if they corresponded to a single isolated colony. The best 12 positive spots matching a single colony were picked for inoculation with Luria broth for plasmid DNA preparation. Plasmid DNA was purified using the Qiapin Mini-Prep kit (Qiagen) and subjected to restriction analysis to estimate the size of the insert.All 12 clones gave the same restriction map, suggesting that the insert size was 3-4 Kb. Of these 6 clones were sent to MWG-Biotech for partial sequencing to determine if they were the correct gene/gene fragment. Sequence analysis showed that 3 of these clones contained a fragment of the phyA phytase gene, but only to one end. These 3 clones were then sent out to completely sequence the insert.

Sequence analysis of the complete sequence of these clones revealed that all three clones were identical and that they encoded 355 amino acids corresponding to approximately 80% of the phyA phytase gene (see FIG. 7, line 1, referred to as "P hordei"). Obviously, there is an internal EcoRI site in the gene, located at a position 300 and 400bp downstream of the start site of the gene. 4B.Comparison of percent identity between fungal phytases

The deduced polypeptide product using the cloned phytase gene fragment was compared for homology with published phytases. Analysis showed about 42-56% identity (see Table 2 below), which in combination with analysis of the translated sequence demonstrated that the cloned gene fragment was phyA phytase. TABLE 2 comparison of percent identity between fungal phytases (ALIGN editor)

Species (II)	P.hordei	Aspergillus niger	Aspergillus fumigatus	Aspergillus terreus	M.thermophilus
						P.hordei		55.6	56.2	52.5	42.4
Aspergillus niger			67.4	62.1	44.9
						Aspergillus fumigatus				61.7	47.6
Aspergillus terreus					43.7
						M.thermophilus

Note that: hordei phytase amino acid sequence based on approximately 80% of the deduced p.hordei phytase amino acid sequence (i.e., lacking the N-terminal portion [ about 140 amino acids ]]) Comparative example 4℃ preparation and screening of SalI-based size-restricted genomic library to isolate the remainder of the Phytase Gene

To isolate the estimated remaining 20% of the p.hordei phytase gene, we decided to prepare a second partial genomic library using a second restriction enzyme to isolate the 5' end of the gene and then attempt to subclone the two fragments together. The restriction endonuclease recognition sites present in the cloned phytase sequence were determined using Webcutter. Of particular interest are the sites of enzymes used in the Southern analysis shown in FIG. 8. We found that all of these enzymes (KpnI, SacI, BamHI and SalI) have a cut point within the phytase sequence, 48bp, 75bp, 486bp and 660bp from the 5' end of the cloned fragment, respectively. Southern analysis of the hordei genomic DNA digest (fig. 8) showed that the BamHI fragment was very large (about 8Kb) and would be difficult to clone in a plasmid-based library. The extent of hybridization with the KpnI band is not strong enough for library screening, and the presence of two bands on the SacI lane may complicate the screening process. We decided to prepare a SalI-based size-restricted library in the same manner as described above to isolate a 1.8kb SalI band. Since a known SalI site exists at a position about 120bp downstream of the end of the probe sequence, it is possible to have a SaII site at a position upstream of the starting position of the phytase gene. Libraries were prepared in pBluescript SKII as described above and screened using the same 636bp probe. Selected positively hybridizing colonies were selected and compared to colonies on the plate. All 12 colonies were paired with a single isolated colony, and these colonies were extracted for plasmid DNA preparation. Restriction analysis showed that only 3 clones contained inserts and all were about 1.8 kb. Both clones (CS112, CS114) were then sent to MWG-Biotech for complete sequencing. The deduced amino acid sequences showed that these clones contained sequences with motifs belonging to phyA phytase, but CS112 had a large number of sequencing errors. Analysis of clone CS114 showed a 450bp overlap between the two genomic fragments of the phytase gene. In addition to the putative initiation codon, the 5' intron and upstream regulatory elements are defined on CS 114. 4D. amplification of continuous Phytase genes for heterologous expression

A pooled phytase sequence was generated from two genomic clones CS114 and CS101 (FIG. 9), and we used this sequence to design a number of gene sequences that could be used to amplify a continuous phytase geneThe upstream and downstream primers of (1). We also designed PCR amplification to facilitate the cloning and expression of the complete phytase gene in the heterologous expression vector pGAPT-PG, a 5.1kb construct supplied by Genencor International (see FIG. 11). There are two restriction enzyme sites (EcoRV and Agel) in the multiple cloning site of pGAPT-PG, which are not present in this phytase gene sequence. We used this phytase gene sequence to design a number of 5 'and 3' flanking primers and modified them to include the restriction enzyme recognition sites for these enzymes (Table 3). TABLE 3 sequences of phytase-specific primers designed from the phyA gene sequences of the cloned CS101 and CS114 combinations

Primer and method for producing the same	Region(s)	Sequence of
			CS201(F)	5' upstream flanking region, including EcoRV site	5’CGG C GA TAT CAG TAT CCC TGC GGT C3’
CS202(F)	5' upstream flanking region, including EcoRV site	5’CGG C GA TAT CCC GGT GAC GTC GGG T3’
			CS203(R)	3' downstream flanking region including AgeI site	5’CGG C AC CGG TGG AAG AGG ACC AAC C3’
CS204(R)	3' downstream flanking region including AgeI site	5’CGG C AC CGG TGC ATT AAT ATT GGC C3’

F means forward primer on the forward strand (5 '→ 3'); r is a reverse primer on the minus strand. Restriction enzyme recognition sites designed into the primer sequences to facilitate cloning into the expression vector pGAPT-PG are underlined and in bold. The upstream and downstream flanking regions used to design the primers were subjectively selected and located approximately 100bp upstream of the ATG (start) codon and downstream of the TAG (stop) codon, respectively. The gene sequence used is also chosen to contain as many bases as possible in equilibrium.

Using p.hordei genomic DNA and these primer combinations, we attempted to amplify the phytase gene by PCR. PCR should amplify a region of approximately 1.7kb corresponding to the full-length phytase gene. FIG. 12 shows the results of PCR using the primers CS201 or CS202 and CS 203. One band of the correct size can be observed for the CS201-CS203 combination, but multiple lower molecular weight bands can also be seen. No amplification was observed with CS202-CS203 under these conditions, but a very faint band at about 1.7kb was observed when the annealing temperature was 50 ℃ (not shown). A strong band (not shown) is generated at about 700bp for CS201/CS202 and CS 204. The expected product resulting from the CS201-CS203 amplification observed in fig. 12 was cloned into the vector pTT and several clones containing inserts of the correct size were selected for sequencing (CS158 and CS 167).

FIG. 10 depicts the 5 '/N-terminal nucleic acid sequence and the 3'/C-terminal sequence of clone CS 158. The primarily deduced phytase amino acid sequence is shown in bold.

The sequence of clone CS167 was not considered a phytase after analysis. Analysis of clone CS158 revealed some interesting properties. The CS158 amino acid sequence deduced from the 3' end of the CS158 clone shows a high homology to CS101 phytase. However, the sequence generated from the 5' end is substantially different from that of CS 114. This is particularly true for sequences upstream of the phosphate binding domain motif (RHGARY). Notably, the sequence from the 5' end of CS 158closely resembles the published phytase sequence and contains a number of key structural motifs and conserved amino acids that are not present in the N-terminal amino acid sequence of CS 114. We reasoned that clones CS101 and CS114 were derived from 2 different genes, while CS158 was the correct full-length phytase.

There are many apparent sequencing errors in the CS158 sequence. This may be caused by the fact that the 1.7kb band shown in FIG. 12 was amplified using Taq polymerase. From this new sequence two new 5' primers were designed to re-amplify the phytase gene for expression. Two regions upstream of the putative initiation codon in this new phytase sequence from CS158 were selected and modified to include an EcoRv recognition site for cloning into pGAPT-PG (table 4). TABLE 4 Phytase specific primer sequences designed from the sequence of the phyA gene generated by Taq polymerase amplification from clone CS158

Primer and method for producing the same	Region(s)	Sequence of
			CS22(F)	5' upstream flanking region, including EcoRV site	5’GTT GAT ATC ACT TGT CGT GAT ACC C3’
CS23(F)	5' upstream flanking region, including EcoRV site	5’TCT GAT ATC TCG ATA TCC TTG CAG G3’

F refers to the forward primer on the forward strand (5 '→ 3'). Restriction enzyme recognition sites designed into the primer sequences to facilitate cloning into the expression vector pGAPT-PG are underlined and in bold. The upstream flanking region used to design the primers was selected subjectively and was located approximately 50-70bp upstream of the putative ATG (start) codon. The gene sequence used is also chosen to contain as many bases as possible in equilibrium.

P. hordei genomic DNA was PCR amplified using high fidelity DNA polymerase Pfu using these 5 'primers in combination with 3' primers designed from CS101 (table 3) to minimize expression errors of the phytase gene (fig. 13). This polymerase is Pfu DNA polymerase (Stratagene) and is part of a Pfu DNA polymerase kit for PCR. For these reactions, reaction buffer, dNTPs, target DNA and primers were mixed together and 2.5 units of Pfu polymerase were added to a final reaction volume of 50. mu.l. After amplification, 5. mu.l of the reaction mixture was analyzed by gel electrophoresis. The combination of the primer designed from the new sequence and primer CS204 produced a single strong product at the expected size position (1.7 kb). This fragment was cloned directly into the vector pCR-Blunt II TOPO (Invitrogen) and the selected number of clones was analyzed to confirm the presence of the correct insert. (cloning of blunt-ended PCR product by Pfu DNA polymerase to ZeroBlunt^TM TOPO^TMPCR cloning kit (Invitrogen). The vector contains an MCS site and a kanamycin gene that generates antibiotic resistance, and also allows screening based on disruption of the lethal E.coli gene ccdb, which is different from blue/white spot screening. To the purified PCR product (50-200ng) was added 1. mu.l of pCR-BluntII-TOPO vector, and the reaction volume was supplemented to 5. mu.l with sterile water. After gentle mixing, incubate for 5min at room temperature. Add 1. mu.l of 6 XPTOPO clone to stop lysisThe reaction was either placed on ice or frozen at-20 ℃ for 24 hours to effect transformation. ) The integrity of the engineered EcoRV and AgeI sites was also verified by this analysis. Several clones, CS212 and CS213, were prepared and sequenced. Sequence analysis confirmed the presence of the entire phyA phytase gene. The gene is further used for expression in a heterologous system and the enzyme is subsequently biochemically analysed. Analysis of 4e.p.hordei phytase sequence

CS213 represents the full-length phytase sequence of p. The genomic sequence of this clone can be seen in FIG. 1A. The deduced amino acid sequence of p.hordei phytase can be seen in fig. 2.

Hordei sequences and published phytases were aligned and homology analysis was performed based on% identity. The results can be seen in table 4 below. TABLE 4 comparison of% identity between phytases

	P.hordei	Aspergillus niger phyA	Aspergillus fumigatus phyA	Aspergillus terreus 9A-1	M.thermo
						P.hordei	-	59.1	58.9	55.4	44.0
Aspergillus niger	212/518	-	68.3	63.7	46.3
						Aspergillus fumigatus	213/518	164/518	-	62.2	49.2
Aspergillus terreus 9A-1	231/518	188/518	196/518	-	44.6
						M.thermo	290/518	278/518	263/518	287/518	-

The numbers in the left/lower region indicate the number of different sites between the two species. Minimum homology: 44.0%, maximum homology: 68.3 percent of

Example 5

Cloning, expression and characterization of P.hordei Phytase

We decided to try to overexpress the phytase gene in a heterologous host to produce enough protein for the enzyme's characterization. Cloning of Phytase Gene in expression vector and transformation of Aspergillus nidulans

Full-length phytase genes amplified with high-fidelity DNA polymerases were generated with primers engineered to contain two restriction enzyme sites (EcoRV, AgeI). These sites were used to facilitate cloning in the expression vector pGAPT-PG (FIG. 11). The phytase clones CS212 and CS213 were digested with these enzymes to generate a 1.7kb single insert. pGAPT-PG was also digested with these enzymes and linearized. The phytase gene fragments were ligated to this expression vector to generate a number of transformants. Selected clones were analyzed to verify the presence of the insert. These phytase clones were then used to transform the swollen spores of aspergillus nidulans by electroporation (swellensporones).

The methods for transformation of aspergillus niger strain FGSC a767 and aspergillus nidulans FGSC a1032 by electroporation are variations of the experimental procedures developed by o.sanchez and j.agiurre for aspergillus nidulans. Using a suitable spore suspension as 10⁷Spores/ml were inoculated with 50ml YG medium (0.5% yeast extract, 2% glucose, supplemented with 10mM uridine and 10mM uracil). Cultures were grown at 25 ℃ for 4 hours on a rotary shaker at 300 rp. The swollen spores were collected by centrifugation at 4000rpm for 5 minutes at 4 ℃. The spores were resuspended in 200ml ice-cold sterile water and centrifuged at 4000rpm for 5 minutes at 4 ℃. The supernatant was decanted, and the spores were resuspended in 12.5ml pH8.0YED medium (1% yeast extract, 1% glucose, 20mM HEPES), and incubated at 30 ℃ for 60 minutes at 100rpm on a rotary shaker. The spores were collected by centrifugation at 4000rpm for 5 minutes and then 10⁹Conidia. ml^-1Concentration of (2)Suspended in 1ml ice-cold EB buffer (10mM tris-HCl, pH7,5, 270mM sucrose, 1mM lithium acetate) and placed on ice. 50. mu.l of the swollen spore suspension was mixed with 1-2. mu.g of DNA in a sterile Eppendorf tube in a total volume of 60. mu.l and then placed on ice for 15 minutes. This suspension was transferred to a 0.2cm electroporation cuvette. Electroporation was performed in a BioRad electroporation apparatus (set at 1kv, 400W, 25. mu.F). After electroporation 1ml ice cold YED was added to the suspension and the combined mixture was transferred to a pre-cooled 15ml sterile Falcon tube and placed on ice for 15 minutes. The tube was then placed in a horizontal position on a rotary shaker and incubated at 30 ℃ at 100rpm for 90 minutes. Spores were plated on plates and transformants were observed after 36-48 hours.

In all cases with circular plasmid DNA, 15 transformants were generated for clones derived from CS213, and only 2 transformants were generated for those derived from CS 212. Aspergillus niger strain FGSC A767 and Aspergillus nidulans strain FGSC A1032 were obtained from the fungal genetics storage Center, University of Kansas Medical Center, 3901Rainbow boularard, city of Kansas, USA. Preliminary characterization of Aspergillus nidulans transformants

A total of 10 A.nidulans transformants were selected for further analysis (CS212-1, CS213-1 to 9). Spores from these transformants, respectively, were used to inoculate selective media and spore suspensions of individual clones were prepared. These spore suspensions were used to inoculate liquid media for transformants, which were then screened for phytase activity. After 72 hours of culture growth, the supernatant was collected. Samples were desalted on a PD-10 column and these protein samples were then diluted in 0.25M sodium acetate. The phytase assay was carried out under standard conditions (pH5.5, 37 ℃, 30 min). Two of the clones (CS213-2, CS213-4) showed phytase activity in the culture supernatants. The phytase activity of CS231-2 was recorded to release 0.12mmoles Pi per ml culture supernatant per minute, whereas transformant CS213-4 had an activity of 0.08mmoles Pi per ml per minute. These transformants were used for further analysis. Time to maximum expression of Phytase in liquid culture

To assess when the phytase production levels peak for subsequent biochemical profiling, a series of liquid cultures of CS213-2 and CS213-4 were prepared over a 2-7 day period. The medium is inoculated with a spore suspension of the appropriate transformant and harvested daily during this period. The culture supernatant was processed according to standard methods and the desalted culture supernatant was assayed under standard phytase conditions. Table 5 summarizes the results of these measurements. Phytase activity was observed over time, but its expression reached a maximum on day three. TABLE 5 Phytase Activity in the transformant culture supernatants over time

Time point (sky)	Activity of CS213-2	Activity of CS213-4
			2	35	36
3	76	75
			4	36	33
5	35	35
			6	34	33
7	33	32

At each time point, liquid cultures were harvested and eluted after desalting in 0.25mM sodium acetate (pH 5.5). The phytase assay was repeated twice under standard conditions (pH5.5, 37 ℃, 30 min). Activity is expressed as units of phytase per ml culture supernatant (released Pi. mu. moles. min.)^-1.ml^-1)。

Failure to transform A.nidulans at these time points was also determined as a control. No phytase activity was detected. Selected supernatant samples (day 4 and day 6) were analyzed by SDS-PAGE for protein samples before and after desalting. None of the coomassie stained gels using the samples showed bands. This suggests that the total level of protein secreted into the medium is low. Silver stained gel also showed no signs of any protein bands. Southern analysis of transformants

Although there is evidence that the p. hordei phytase gene has been successfully cloned into the expression vector pGAPT-PG and that expression of the active enzyme is achieved, there is still a lack of molecular evidence to date. Genomic DNA was prepared from transformed A.nidulans CS213-2 and CS213-4, as well as the original untransformed host. Since there is no internal EcoRV site in the p.hordei phytase gene, DNA was digested with EcoRV and then subjected to Southern hybridization analysis of transformants. These Southern blots were analyzed using a 650bp phytase probe from p.hordei (fig. 14). Under medium to high stringency conditions (3 XSSC), a single strong hybridizing band was observed for both transformants CS213-2 and CS 213-4. There was no evidence of any other bands of hybridization, and it was concluded that this indicated a single copy of the phytase gene in transformed A.nidulans. No hybridization bands could be observed in the untransformed samples, indicating that there is no homology between the p. Biochemical characterization of 5e.p.hordei phytase

To confirm that the cloned gene has specific phytase activity and characterize this activity, a set of biochemical assays was performed on the overexpressed enzyme. Preliminary qualitative analysis showed that the gene produced phytic acid hydrolysis activity. The assay was extended to detect activity at different pH, temperature and against different substrates.

The transformant CS213-4 was used for these analyses. Cultures were harvested on day 3. When phytic acid was used as a substrate, the enzyme exhibited activity between pH3.0 and pH7.0 (FIG. 15). The purified enzyme sample from the culture supernatant was desalted and eluted in 0.025mM sodium acetate (pH 5.0). This was then added to a substrate prepared in the following buffer: glycine hydrochloride of pH 3.0: 0.4M, sodium acetate of pH 4.0: 0.4M, sodium acetate of pH 5.0: 0.4M, imidazole hydrochloride of pH 6.0: 0.4M, Tris-HCl of pH 7.0: 0.4M, Tris-HCl of pH 8.0: 0.4M, Tris-HCl of pH 9.0: 0.4M. There was a clear optimum at pH5.0 (240 units/ml supernatant) with a broad range of flanking activity from 5.0 up to 6.0. When 4-nitrophenyl phosphate was used as substrate, little activity was observed, indicating a high level of specificity of the enzyme for phytic acid substrates. The highest activity against this substrate occurs at pH3.0, but even this is only 25% of the activity of the enzyme at this pH against phytic acid. The temperature profile of the enzyme was characterized for a range of temperatures using a pH5.0 buffer, this time using only phytic acid as the substrate (FIG. 16). Hordei phytase showed activity at 30 ℃ -85 ℃, but had a clear temperature optimum at 44 ℃. The enzyme activity is higher closer to ambient temperature, losing only 22% of the relative activity between 44 ℃ and 37 ℃. Since the total level of protein produced is low and these assays are performed on all supernatant proteins, it is difficult to determine the specific activity of the phytase. The average total protein amount of these samples was 8. mu.g/ml, which would be expected to give a specific activity of 240000 units per mg protein.

We also performed preliminary stability studies on this phytase. Samples of day 3 protein were placed at-20 ℃, 4 ℃ and 37 ℃ overnight and then assayed under standard conditions. No residual activity was measured. We also exposed the samples to 85 ℃ for 20 minutes, and 100 ℃ for 10 minutes to determine the thermostability of the phytase activity. There was a considerable reduction in residual enzyme activity when measured at 37 ℃ pH5.0 after exposure to these temperatures (Table 6). TABLE 6

Stability conditions	Residual Activity (%)
		85℃20min	20％
100℃10min	21％
		24 hours at-20 DEG C	ND
24 hours at 4 DEG C	ND
		24 hours at 37 DEG C	ND

Residual activity was based on comparison with phytase activity measurements obtained from samples before exposure to each condition (100% activity corresponds to 112 units per ml culture supernatant). Although the samples were assayed in duplicate only, these were standard phytase assay conditions, i.e., pH5.0, 37 ℃, 30 minutes. The samples were then all tested under the same test conditions.

Example 6

Cloning and analysis of Phytase Gene fragment from Penicillium Sabinae

We designed two primers to amplify phytase gene fragments from this fungal target (table 7). We designed CS11 based on the DNA sequence of the cloned phytase genes (aspergillus niger, aspergillus fumigatus, e.nidulans and thermoactinomyces thermophilus), the gene sequence of the cloned p.hordei phytase, and the sequences of the designed primers CS1 and N1. For this primer, the first two bases of each codon are well conserved. The third base was selected based on the bias shown in published DNA sequences, in particular based on the base at the third position in the p. CS12 was designed based on the conserved amino acid motif YADF/SHDN in the published phytase sequence, and the deduced amino acid sequence of the p. TABLE 7 sequences of degenerate primers designed based on conserved sequences in fungal phytases

Primer and method for producing the same	Region(s)	Sequence of
			CS11(F)	5' redesigned CS1 primer from the phytase motif RHGARYP	5，CG(T/G/C)CA(C/T)GG(A/G/C)GC(G/T)CG(G/C)TA(C/T)CC(A/G/T)(A/T)C3’
CS12(R)	3' reverse primer designed from the phytase motif YADFTHDN	5’(A/G)TT(A/G)TC(A/G)TG(A/G/C/T)(G/C)(A/G)(A/G)AA(A/G)TC(A/C/G/T)(A/G)C(A/G)TA3′

F means forward primer on the forward strand (5 '→ 3'); r is a reverse primer on the minus strand.

A genomic sample of DNA from Penicillium juniperi was subjected to PCR and amplified to produce a fragment of the expected size, i.e., about 800 bp. This fragment was cloned into the pTT vector and E.coli XL1-blue cells were transformed according to standard methods. The selected transformants were screened and plasmid DNA was isolated. Selected clones with the correct size insert were sent to MWG-Biotech for sequencing. One clone, CS142, contains a sequence with high homology to phytase. The deduced amino acid sequence showed that this fragment (853bp) cloned was from the phytase gene. FIG. 17 depicts the genomic sequence and deduced amino acid sequence of the P.juniperi fragment. It has the expected number of Cys residues in this region (3), and it also contains all motifs critical to the structure and function of the phytase, notably:

·RHGARYP

·3×Cys

HD (in YADFTHDN)

There are many other regions that are well conserved (see the alignment in fig. 5), and a% identity analysis using this alignment shows about 51% identity between penicillium juniperi and p.hordei phytase. The pencillium sabdariffae phytase fragment (283 amino acids) makes up about 65% of the total phytase.

Southern analysis of the digest of Aspergillus niger, P.hordei, Penicillium juniperi and Penicillium brevicaulis with this fragment under medium stringency conditions (3 XSSC) showed hybridization only to the digest of Penicillium juniperi.

It will, of course, be understood that various modifications and changes may be made to the preferred embodiments described above. It is, therefore, intended that the foregoing detailed description be understood in the following claims (including all equivalents) that are intended to define the scope of this invention.

Claims (38)

1. An isolated polynucleotide from a fungal source of the genus penicillium, the polynucleotide comprising a nucleotide sequence encoding an enzyme having phytase activity.

2. The polynucleotide of claim 1, wherein the fungal source is selected from the group consisting of Penicillium juniperi and Penicillium hordei.

3. The polynucleotide of claim 1, wherein the enzyme comprises a nucleotide sequence identical to SEQ ID NO: 4, optionally at least 80% identical to the amino acid sequence disclosed in claim 4.

4. An isolated polynucleotide comprising a nucleotide sequence wherein said nucleotide sequence (i) differs from the nucleotide sequence set forth in SEQ ID NO: 1. SEQ ID NO: 2. or SEQ ID NO: 3, or (ii) is capable of hybridizing to a nucleic acid molecule derived from seq id NO: 1. SEQ ID NO: 2. or SEQ ID NO: 3 under medium to high stringency conditions, or (iii) hybridizes to SEQ ID NO: 1. SEQ ID NO: 2. or SEQ ID NO: 3 is complementary to the nucleotide sequence disclosed in 3.

5. The polynucleotide of claim 4, wherein the nucleotide sequence is identical to SEQ ID NO: 1. SEQ ID NO: 2. or SEQ ID NO: 3, or a pharmaceutically acceptable salt thereof, and optionally at least 85% identity thereto.

6. An isolated polynucleotide encoding an enzyme having phytase activity, wherein the enzyme is from a penicillium source.

7. The polynucleotide of claim 6, wherein the enzyme comprises a nucleotide sequence identical to SEQ ID NO: 4, optionally at least 80% identical to the amino acid sequence disclosed in claim 4.

8. The polynucleotide of claim 6, wherein the polynucleotide (i) hybridizes to SEQ ID NO: 1. SEQ ID NO: 2. or SEQ ID NO: 3, or (ii) is capable of at least 55% identity, optionally at least 65% identity, to a nucleotide sequence disclosed in SEQ ID NO: 1. SEQ ID NO: 2. or SEQ ID NO: 3 under medium to high stringency conditions, or (iii) hybridizes to SEQ ID NO: 1. SEQ ID NO: 2. or SEQ ID NO: 3 is complementary to the nucleotide sequence disclosed in 3.

9. An expression construct comprising a polynucleotide sequence wherein said polynucleotide sequence (i) differs from the polynucleotide sequence of SEQ ID NO: 1. SEQ ID NO: 2. or SEQ ID NO: 3, or (ii) is capable of hybridizing to a nucleotide sequence disclosed in SEQ id no: 1. SEQ ID NO: 2. or SEQ ID NO: 3 under medium to high stringency conditions, or (iii) hybridizes to SEQ ID NO: 1. SEQ ID NO: 2. or SEQ ID NO: 3 is complementary to the nucleotide sequence disclosed in 3.

10. A vector comprising the expression construct of claim 9.

11. A host cell transformed with the vector of claim 10.

12. A probe for detecting a nucleic acid sequence encoding an enzyme having phytase activity from a microbial source, comprising: (i) and SEQ ID NO: 1. SEQ ID NO: 2. or SEQ ID NO: 3, or (ii) a nucleotide sequence that is at least 55% identical, optionally at least 65% identical to a nucleotide sequence disclosed in SEQ ID NO: 1. SEQ ID NO: 2. or SEQ ID NO: 3 under medium to high stringency conditions, or (iii) a nucleotide sequence that hybridizes to a polynucleotide of a sequence disclosed in SEQ ID NO: 1. SEQ ID NO: 2. or SEQ ID NO: 3, or a nucleotide sequence complementary to the nucleotide sequence disclosed in 3.

13. The probe of claim 12, wherein the microbial source is a fungal source.

14. The probe of claim 13, wherein the fungal source is a penicillium species.

15. A food or animal feed comprising an enzyme having phytase activity, wherein the enzyme comprises an amino acid sequence substantially identical to SEQ ID NO: 4, optionally at least 80% identical to the amino acid sequence disclosed in claim 4.

16. Food or animal feed comprising an enzyme having phytase activity, wherein the enzyme is from a fungal source selected from Penicillium hordei and Penicillium juniperi.

17. An isolated phytase, wherein the enzyme is obtained from a fungus selected from the group consisting of Penicillium juniperi and P.hordei, and has the following physicochemical properties: (1) molecular weight: about 45-55kDa (unglycosylated); and (2) specificity: phytic acid.

18. A method of preparing an enzyme having phytase activity, comprising:

(a) providing a host cell transformed with an expression vector comprising a polynucleotide as defined in claim 4;

(b) culturing said transformed host cell under conditions suitable for production of said phytase by said transformed host cell; and

(c) recovering the phytase.

19. The method of claim 18, wherein the host cell is an aspergillus species.

20. A method for separating phosphorus from phytic acid comprising:

using a nucleic acid comprising a nucleotide sequence identical to SEQ ID NO: 4, optionally at least 80% identical to the amino acid sequence disclosed in claim 4.

21. A method for separating phosphorus from phytic acid comprising:

treating said phytate with an enzyme as defined in claim 17.

22. The polynucleotide of claim 1, wherein said enzyme comprises an amino acid sequence which is at least 70% identical, optionally at least 80% identical, to the amino acid sequence disclosed in figure 17.

23. An isolated polynucleotide comprising a nucleotide sequence which (i) is at least 55% identical, optionally at least 65% identical to the nucleotide sequence disclosed in figure 17, or (ii) is capable of hybridizing to a probe derived from the nucleotide sequence disclosed in figure 17 under conditions of moderate to high stringency, or (iii) is complementary to the nucleotide sequence disclosed in figure 17.

24. The polynucleotide of claim 23 wherein said nucleotide sequence has at least 85% identity to the nucleotide sequence disclosed in figure 17.

25. The isolated polynucleotide of claim 6, wherein the enzyme is derived from Penicillium juniperi or Penicillium hordei.

26. The polynucleotide of claim 25, wherein the enzyme comprises an amino acid sequence which is at least 70% identical, optionally at least 80% identical, to the amino acid sequence disclosed in figure 17.

27. The polynucleotide of claim 25, wherein the polynucleotide comprises (i) a nucleotide sequence at least 55% identical, optionally at least 65% identical to the nucleotide sequence disclosed in figure 17, or (ii) a nucleotide sequence capable of hybridizing to a probe derived from the nucleotide sequence disclosed in figure 17 under conditions of medium to high stringency, or (iii) a nucleotide sequence complementary to the nucleotide sequence disclosed in figure 17.

28. An expression construct comprising a polynucleotide, wherein said polynucleotide comprises (i) a nucleotide sequence at least 55% identical, optionally at least 65% identical to the nucleotide sequence disclosed in figure 17, or (ii) a nucleotide sequence capable of hybridizing to a probe derived from the nucleotide sequence disclosed in figure 17 under conditions of moderate to high stringency, or (iii) a nucleotide sequence complementary to the nucleotide sequence disclosed in figure 17.

29. A vector comprising the expression construct of claim 28.

30. A host cell transformed with the vector of claim 29.

31. A probe for detecting a nucleic acid sequence encoding an enzyme having phytase activity from a microbial source, comprising: (i) a nucleotide sequence at least 55% identical, optionally at least 65% identical to the nucleotide sequence disclosed in figure 17, or (ii) a nucleotide sequence capable of hybridizing to a polynucleotide comprising the sequence disclosed in figure 17 under conditions of medium to high stringency, or (iii) a nucleotide sequence complementary to the nucleotide sequence disclosed in figure 17.

32. The probe of claim 31, wherein the microbial source is a fungal source.

33. The probe of claim 32, wherein the fungal source is a penicillium species.

34. A food or animal feed comprising an enzyme having phytase activity, wherein the enzyme comprises an amino acid sequence which is at least 70% identical, optionally at least 80% identical, to the amino acid sequence disclosed in figure 17.

35. A method of preparing an enzyme having phytase activity, comprising:

(a) providing a host cell transformed with an expression vector comprising a polynucleotide as defined in claim 23;

(b) culturing said transformed host cell under conditions suitable for production of said phytase by said transformed host cell; and

(c) recovering the phytase.

36. The method of claim 35, wherein said host cell is an aspergillus species.

37. A method for separating phosphorus from phytic acid comprising:

treating the phytate with (i) an enzyme having phytate hydrolase activity and (ii) an enzyme comprising an amino acid sequence having at least 65% identity, optionally at least 70% identity, to the amino acid sequence disclosed in figure 17.

38. An enzyme derived from a penicillium species, optionally penicillium juniperi or p.hordei; the enzyme consists of a polypeptide capable of hybridizing to SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 3. or a nucleotide sequence encoding a polynucleotide sequence as set forth in figure 17 that hybridizes under medium to high stringency conditions; the enzyme has one or more of the following physicochemical properties:

(1) molecular weight: about 45-60kDa (unglycosylated);

(2) activity specific to phytate, phytic acid or phytate, and/or lower phosphate derivatives thereof;

(3) theoretical pI of about 7-7.6; optionally, about 7.3;

(4) the pH optimum is in the range of about 4.5-5.5, optionally about 5; and/or

(5) The optimum ambient temperature is in the range of about 40 to about 45 ℃; optionally, 42-44 ℃.