The present invention relates to phytase, nucleic acids encoding phytase as well as methods for the production of phytase and its use.

Background of the Invention

Phosphorous is an essential element for growth. A substantial amount of the phosphorous found in many foods and animal feeds is present in the form of phosphate which is covalently bound in a molecule known as phytate (myo-inositol hexakisphosphate) . Since phytate itself is poorly digested and phosphate is to a large extent absorbed in the small intestine of an animal, phosphate sequestered in phytate and not made available to an animal in the small intestine is not absorbed, passes through the digestive tract and is excreted. This leads to an increased ecological phosphorus burden to land and water. In addition, since phytate chelates several essential minerals and prevents or inhibits their absorption in the digestive tract, phytate decreases the nutritional value of food and animal feeds.

Another problem associated with poor phytate digestability is that inorganic phosphates need to be added to animal feeds, thereby increasing their costs.

Phytate is converted by enzymes known as phytases which catalyse the hydrolysis of phytate to inositol and inorganic phosphate. Phytase is found in wheat bran and plant seeds and is known to be produced by various micro-organisms including yeast, fungi and bacteria.

Among known fungal phytases, Aspergillus terreus phytase was purified to homogeneity by Yamada et al.(Agr. Biol. Chem. , 32 (10) (1968) , 1275-1282) and shown to have a pH optimum of pH

4.5, a temperature optimum of about 70°C at pH 4.5 and a thermal stability over a temperature range from 30 to 60 °C at pH 4.5. However, said enzyme was shown to be completely inactive at neutral pH values, particularly at pH 7.0.

In addition, the Aspergillus ficuu phytase isolated and characterised by H.J. Ullah and D.M. Gibson (Preparative Biochemistry, 17 (1) (1987) , 63-91) was shown to have two pH optima, one at 2.2 and the other at 5.0-5.5, a temperature optimum of 58°C at pH 5.0 and a thermal stability up to 68°C at pH 5.0. However, as is the case with Aspergillus terreus phytase, Aspergillus ficuum phytase was shown to be inactive at pH 7.0.

DNA sequences encoding phytases from Aspergillus terreus (EP 684 313) and Aspergillus ficuum (EP 420 358) as well as Aspergillus niger var. awamori (Piddington et al. , (1993) Gene, 133, 55-62) have been characterised and recombinantly expressed.

Phytases are also known from bacterial sources such as Bacillus subtilis (V.K. Powar and V. Jagannathan, (1982) J. Bacteriology, 151 (3), 1102-1108) and Bacillus subtilis (natto) ( . Shimizu, (1992) Biosci. Biotech. Biochem., 56 (8), 1266-1269 and Japanese Patent Application 6-38745) .

Bacillus subtilis (natto) phytase described by Shimizu (supra) was purified to homogeneity by SDS-PAGE and was shown to have a molecular weight of between 36 and 38 kD. This enzyme was shown to have a pH optimum between pH 6.0 and 6.5 when measured in an assay solution at 37°C comprising 0.1 M maleic acid, 2 mM CaCl2 and 1.6 mM sodium phytate and a pH optimum of pH 7.0 when assayed in a solution comprising 0.1 M Tris-HCl buffer, 2 mM CaCl ₂ and 1.6 mM sodium phytate at 37°C. The temperature optimum for this phytase was shown to be 60°C and the enzyme is stable up to 50°C when incubated in the above mentioned assay solution containing Tris-HCl buffer for 15

min. The specific activity of this purified Bacillus subtilis (natto) phytase in said Tris-HCl containing solution was reported as 8.7 U/mg protein. One unit of phytase was defined as the amount of enzyme required to liberate one μmol of Pi per minute under tha assay conditions. This definition is used throughout.

Powar et al. (supra) described the isolation of a phytate specific phosphatase preparation from Bacillus subtilis which has a molecular weight of 36.5 kD. This enzyme preparation, which was purified by SDS-PAGE and found to comprise two phytase isozymes, was shown to have a pH optimum between 7.0 and 7.5 when measured in an assay solution comprising 0.1 M Tris-HCl buffer, 0.5 mM CaCl ₂ and 0.34 mM sodium phytate at 30°C. This phytase isozyme mixture exhibited a maximum activity at a temperature of 60°C and was stable up to a temperature of 70°C. The specific activity of the purified enzyme was reported as 8.5 to 9.0 U/mg protein when measured in the above assay solution. In addition, it was reported by Powar et al (supra) that the purified isozyme mixture contained proteolytic activity which resulted in the loss of activity.

The amino acid sequence of Bacillus phytase as well as nucleic acids which encode Bacillus phytases are not known to date.

The idea of supplementing foods and animal feed with naturally occurring or recombinant phytases in order to enzymatically convert phytate to digestible phosphate during food and animal feed processing has been described. JP-A-6-38745 describes the use of purified naturally occurring Bacillus subtilis (natto) phytase for use in processing feeds and foods. In addition, EP 420 358 and EP 684 313 describe the use of Aspergillus phytase in animal feeds.

Furthermore, it has also been suggested to add phytase to animal feeds which have already been processed in order to

allow the enzymatic action of said phytases to take place in the digestive tract of the animal.

However, the above mentioned Aspergillus phytases are either inactive or lose a substantial amount of their activity at the temperature and/or pH at which foods or animal feeds are processed (generally 65 to 95°C, pH 5.5 to 7.5) and at the pH of the small intestine of monogastric animals (generally 37- 41°C, pH 5.5 to 7.5) .

Furthermore, the specific activity, and therefore the relative activity, of the above mentioned Bacillus phytases is very low under the above conditions.

Summary of the invention

Due to the difference in the temperatures and/or pH used during processing of foodstuffs and in the digestive tract of animals, it is desirable to have available a phytase which has a high specific activity as well as a high relative activity both at the processing temperature and/or pH of foods and animal feeds and at the temperature and/or pH in the digestive tract of animals in order to both maximise the effects of phytase during food and feed processing, during digestion within the digestive tract and to reduce the phosphorous burden to the environment resulting from digestion of phytate containing animal feedstuffs.

Moreover, a method for the production of large quantities of phytase which fulfils the above criteria is also desirable for the economic production of said foods and animal feeds.

An object of the present invention is to provide phytase with a high specific activity which is capable of functioning with a high relative activity during the processing of foods and animal feeds and/or is capable of functioning with high relative activity in the digestive tract of farmed animals.

A further object of the present invention is to provide nucleic acid molecules which encode phytase of the present invention.

A further object of the present invention is to provide methods for the production of said phytase as well as means for delivering said phytase to said animals.

Other objects of the present invention will become apparent from the following detailed specification.

Subject matter of the invention is phytase or a functional derivative thereof, characterised in that said phytase has a specific activity of at least 20 U/mg protein, wherein said specific activity is determined by incubating said phytase in a solution containing 100 mM Tris-HCl, pH 7.5, 1 mM CaCl2, and 1.6 mM sodium phytate at 37°C for 30 minutes. Preferably, the phytase of the present invention has a specific activity of at least 29 U/mg protein, more preferably at least 80 U/mg protein, and most preferably at least 88 U/mg protein when assayed under the above conditions.

According to a preferred embodiment, said phytase has a pH optimum of at least pH 6.5, wherein said pH optimum is determined by incubating said phytase in a solution containing 100 mM maleic acid-Tris, 1 mM CaCl ₂ , and 1.6 mM sodium phytate at 37°C for 30 minutes or a pH optimum of at least pH 7.0, wherein said pH optimum is determined by incubating said phytase in a solution containing 100 mM Tris-HCl, 1 mM CaCl2; and 1.6 mM sodium phytate at 37°C for 30 minutes or by incubating said phytase in a solution containing wheat bran extract, 1 mM CaCl2, and 1.6 mM sodium phytate at 37°C for 30 minutes.

It is advantageous for phytase to have a relatively high activity both during food or feed processing and in the

i digestive tract of farmed animals such that the enzyme is capable of functioning well under both conditions. The activity of phytase of the present invention in feed or food during processing is preferably greater than or equal to 30%, more preferably greater than or equal to 35%, and most preferably greater than or equal to 37%, compared to the activity of said phytase in the digestive tract, preferably the crop and/or small intestine, of a farm animal.

In addition, said phytase is preferably capable of functioning in the presence of digestive enzymes found in the small intestine of animals. Enzymes which are found in the small intestine of animals include pancreatic enzymes such as trypsin, chymotrypsin and lipase.

The present invention relates to phytase with one or more of the above characteristics.

The phytase of the present invention is obtainable from a microbial source, preferably a strain of Bacillus, more preferably a Bacillus strain selected from the group comprising Bacillus subtilis and Bacillus amyloliquefaciens, and most preferably Bacillus subtilis strain B 13 deposited on August 1, 1996 at the National Collections of Industrial and Marine Bacteria, Ltd. (NCIMB) in Scotland under accession number NCIMB-40819.

In a preferred embodiment, phytase of the present invention comprises the amino acid sequence according to SEQ ID NO: 1 or a functional derivative thereof. The term "a functional derivative thereof" as it relates to phytase is used throughout the specification to indicate a derivative of phytase which has the functional characteristics of phytase of the present invention. Functional derivatives of phytase encompass naturally occurring, synthetically or recombinantly produced peptides or peptide fragments, mutants or variants which may have one or more amino acid deletions, substitutions

? or additions which have the general characteristics of the phytase of the present invention.

Further subject matter of the present invention is an isolated nucleic acid or a functional derivative thereof, which encodes a phytase having one or more of the above characteristics. Preferably, said nucleic acid comprises a DNA sequence according to SEQ ID NO: 1 or a functional derivative thereof, or hybridises to a DNA sequence according to SEQ ID NO: 1 or a functional derivative thereof.

Further subject matter is an isolated nucleic acid which encodes a phytase or a functional derivative thereof, characterized in that said nucleic acid hybridises to a DNA according to SEQ ID NO: 1 and encodes a phytase having a pH optimum of greater than or equal to pH 5.0 and a specific activity of at least 10 U/mg protein as determined in a solution containing 100 mM maleic acid-Tris, 1 mM CaCl2; and 1.6 mM sodium phytate at 37°C for 30 minutes.

Said nucleic acid is preferably a DNA molecule. The term "a functional derivative thereof" as it relates to nucleic acids encoding phytase is used throughout the specification to indicate a derivative of a nucleic acid which has the functional characteristics of a nucleic acid which encodes phytase. Functional derivatives of a nucleic acid which encode phytase of the present invention encompass naturally occurring, synthetically or recombinantly produced nucleic acids or fragments, mutants or variants thereof which may have one or more nucleic acid deletions, substitutions or additions and encode phytase characteristic of the present invention. Variants of nucleic acid encoding phytase according to the invention include alleles and variants based on the degeneracy of the genetic code known in the art. Mutants of nucleic acid encoding phytase according to the invention include mutants produced via site-directed mutagenesis techniques (see for example, Botstein, D. and Shortle, D., 1985, Science 229:

1193-1201 and Myers, R.M., Lerman, L.S., and Maniatis, T., 1985, Science 229: 242-247), error-prone PCR (see for example, Leung, D.W., Chen, E., and Goeddel, D.V., 1989, Technique 1: 11-15; Eckert, K.A. and Kunkel, T.A., 1991, PCR Methods Applic. 1: 17-24; and Cadwell, R.C. and Joyce, G.F., 1992, PCR Methods Applic. 2: 28-33) and/or chemical-induced mutagenesis techniques known in the art (see for example, Elander, R.P 'Microbial screening, Selection and Strain Improvement' in Basic Biotechnology, J. Bu'lock and B. Kristiansen Eds., Academic Press, New York, 1987, 217).

Subject matter of the present invention is also a method for the production of a nucleic acid of the invention, characterised in that a probe comprising a nucleic acid as described above is hybridised under standard conditions to a sample suspected of containing said nucleic acid and said nucleic acid is recovered. Standard techniques employing said probe for hybridisation include Southern blotting (see for example, Sambrook et al. , Molecular Cloning, a Laboratory Manual, 2nd. Edition, Cold Spring Harbor Laboratory Press, 1989), PCR and RT-PCR(see for example, PCR Protocols: A Guide to Methods and Applications, Innis, M.A. , Gelfand, D.H., Sninsky, J.J. and White, T.J. Eds., Academic Press New York, 1990) . Standard conditions for hybridization are preferably 6 x SSC, 0.5% SDS, 50°C overnight or functional equivalents thereof for Southern blotting and for PCR: 5 mM Mg ²⁺ , Taq enzyme, premelting, 94°C for 2 min and 30 cycles of melting at 92°C for 20 sec, annealing at 50°C for 30 sec and extension at 72 °C for 1 min, or functional equivalents thereof.

Subject matter of the present invention is also a vector comprising a DNA molecule of the present invention. Preferably, said vector is characterised in that said DNA molecule is functionally linked to regulatory sequences capable of expressing phytase from said DNA sequence. Preferably, said DNA molecule comprises a leader sequence capable of providing for the secretion of said phytase. Said

regulatory sequences can comprise prokaryotic or eukaryotic regulatory sequences.

Depending on whether the phytase of the invention is expressed intracellularly or is secreted, a DNA sequence or vector of the invention can be engineered such that the mature form of the phytase of the invention is expressed with or without a natural phytase signal sequence or a signal sequence which functions in Bacillus, other prokaryotes or eukaryotes. Expression can also be achieved by either removing or partially removing said signal sequence.

Subject matter of the present invention is also a prokaryotic host cell transformed by a nucleic acid or vector as described above. Preferably said host cell is selected from the group comprising E. coli, Bacillus sp., Lactobacillus and Lactococcus .

Subject matter of the present invention is also a eukaryotic host cell transformed by a nucleic acid or vector as described above. Preferably said host cell is selected from the group comprising Aspergillus sp., Humicola sp., Pichia sp. , Trichoderma sp. Saccharomyces sp. and plants such as soybean, maize and rapeseed.

Subject matter of the present invention is also a method for the recombinant production of phytase, characterised in that a prokaryotic or eukaryotic host cell as described above is cultured under suitable conditions and said phytase is recovered.

A preferred embodiment of the phytase of the present invention is a phytase obtainable according to the above method.

Further subject matter of the present invention is the use of bacterial cells or spores capable of producing phytase according to the invention as a probiotic or direct fed

to icrobial product. Preferred embodiments for said uses are phytase-producing Bacillus sp. and Lactobacillus sp. of the invention.

Further subject matter of the invention is also a use of phytase according to the present invention in food or animal feed.

Further subject matter is food or animal feed comprising phytase according to the invention. Preferably, said food or animal feed comprises phytase as an additive which is active in the digestive tract, preferably the crop and/or small intestine, of said animal, wherein said animal is preferably selected from the group comprising avians including poultry, ruminants including bovine and sheep, pig, and aquatic farm animals including fish and shrimp. Said additive is also preferably active in food or feed processing.

Further subject matter is food or animal feed comprising prokaryotic cells or spores capable of expressing phytase according to the present invention.

Subject matter of the present invention is also a method for the production of a food or animal feed, characterised in that phytase according to the invention is mixed with said food or animal feed. Said phytase is added as a dry product before processing or as a liquid before or after processing. If a dry powder is used, the enzyme would be diluted as a liquid onto a dry carrier such as milled grain.

Subject matter of the present invention is also a method for the production of a food or animal feed, characterised in that prokaryotic cells and/or spores capable of expressing phytase according to the inveniton are added to said food or animal feed.

i l Subject matter of the present invention is also a use of phytase according to the invention with or without accessory phosphataεes in the production of inositol and inorganic phosphate.

Further subject matter of the present invention is a method for the reduction of levels of phosphorous in animal manure, characterised in that an animal is fed an animal feed according to the invention in an amount effective in converting phytate contained in said animal feed.

Definitions

The term "phytase" is defined throughout the specification as a protein or polypeptide which is capable of catalysing the hydrolysis of phytate and releasing inorganic phosphate.

Specific activity of phytase is defined throughout specification as the number of units (U) / mg protein of a solution comprising phytase, wherein said phytase is detectable as a single band by SDS-PAGE. One unit is the amount of enzyme required to liberate one μmol of Pi per minute when said enzyme is incubated in a solution containing 100 mM Tris-HCl, pH 7.5, 1 mM CaCl ₂ , and 1.6 mM sodium phytate at 37°C for 30 minutes.

Relative activity of phytase is defined throughout the specification as the activity of the enzyme at a given temperature and/or pH compared to the activity of the enzyme at the optimal temperature and/or pH of said enzyme.

Brief description of the drawings

Figure 1: SDS-PAGE gel of phytase purification(procedure) ;

Figure 2: Isoelectric focusing gel of purified phytase,-

Figure 3 : Effect of pH on the activity of phytase at different temperatures;

Figure 4: Effect of pH on the temperature activity profile of phytase in defined buffers,-

Figure 5: Effect of pH on the activity of phytase in wheat bran extract at different temperatures;

Figure 6 : Effect of pH on the temperature activity profile of phytase in wheat bran extract;

Figure 7: Relative activity of phytase under pH and temperature corresponding to feed processing and digestion processes;

Figure 8: Results of PCR amplification of gene encoding B. subtilis phytase using primers derived from amino acid sequence;

Figure 9: Structure of B. subtilis phytase gene; and

Detailed description of the invention

The invention is more closely illustrated by the following examples.

Example 1

Bacillus subtilis B 13 deposited at the National Collections of Industrial and Marine Bacteria, Ltd. (NCIMB) in Scotland under accession number NCIMB-40819 was used throughout the study.

Media

Luria medium, containing 5 g of yeast extract, 10 g of tryptone and 10 g of NaCl per litre, was used to grow the inoculum for the production of phytase.

Wheat bran extract was used as the enzyme production medium and it was prepared as follows. 100 grams of wheat bran was extracted with 1000 ml of water by autoclaving at 121°C for 60 minutes. The extract was filtered through six layers of cheesecloth and then the volume of the extract was adjusted to one litre by addition of water. This extract was supplemented with: (NH ) Sθ ₄ 0.4 g, MgS0 ₄ ^• 7H 0 0.2 g, casitone 10 g, KH P0 ₄ 0.5 g and K ₂ HP0 ₄ 0.4 g. The final pH of the extract was 6.5. The extract base was autoclaved at 121°C for 15 minutes. Prior to inoculation, 5% CaCl2 (filter sterilised) was added to the final concentration of 0.2%.

Production of enzyme

Inoculum was grown up from the frozen stock in Luria medium supplemented with 0.2% CaCl2. The initial inoculum was grown for 24 hours at 30°C in a rotatory shaker. The cultivation was scaled up using successive 10% inoculations in wheat bran

"1 medium. For enzyme production the 5 litre batch was grown in wheat bran medium at 30°C for 91 hours with vigorous shaking.

Protein assay

Protein concentrations were determined by Bio-Rad Protein Microassay Procedure according to the recommendations of the manufacturer by using Bovine Serum Albumin as a standard.

Purification of phytase

All purification steps were carried out at 0 - 4°C unless otherwise stated. Bacteria were pelleted by centrifugation at 7000 x g for 30 minutes. The volume of the collected supernatant was determined and CaCl2 added to a final concentration of 1 mM. The enzyme was precipitated by adding three volumes of cold (-20°C) ethanol, which was added with constant stirring to the supernatant. Stirring was continued for 45 minutes and the precipitation was carried out overnight. The precipitate was collected by centrifugation at 1800 x g for 20 minutes. The collected precipitate was washed once with cold (-20°C) ethanol and once with cold (-20°C) acetone. Excess acetone was evaporated from the precipitate under nitrogen gas flow and then the drying was completed by lyophilisation.

The dried precipitate was dissolved in 300 ml of 100 mM Tris- HCl, pH 7.5, supplemented with 1 mM CaCl2 • Ammonium sulphate was added slowly to the solution under constant stirring until 65% saturation was reached. The solution was incubated at 4 °C overnight, cleared by centrifugation at 9000 x g for 60 minutes at 4°C and then ammonium sulphate added until 85% saturation was reached. The solution was again incubated over night at 4 °C. Precipitate was collected by centrifugation as before and then dissolved in 100 mM Tris-HCl, pH 7.5, supplemented with 1 mM CaCl2- Aliquots of enzyme preparation were stored at -20°C. When used for experiments the enzyme

IS preparations were gel filtered to a desired defined buffer by using PD-10 (Pharmacia) gel filtration columns. The purification scheme of phytase is shown in Table 1.

Table 1: Specific activity of purified phytase

Estimation of molecular weight and isoelectric point

The molecular weight of phytase as purified above was estimated in Pharmacia Phast electrophoresis equipment by using SDS 8-25% gradient polyacrylamide gel electrophoresis (PhastGel ^® SDS-page) and the Pharmacia Low Molecular Weight Electrophoresis Calibration Kit as a standard according to recommendations by the manufacturer. The isoelectric point was determined with the same system using PhastGel IEF 3-9 isoelectric focusing gel and the Pharmacia IEF Calibration Kit as a standard.

Molecular weight of the B 13 phytase was 43,000 as determined by SDS-PAGE (Figure 1) . Isoelectric pH of the B 13 phytase was 6.5 (Figure 2) .

ι

Substrate specificity

Substrate specificity of the phytase (in 0.1 M Tris-HCl, pH 7.5) was determined by using the standard activity assay of each enzyme. Besides phytic acid, β-glycerophosphate, D- glucose-6-phosphate, p-nitrophenylphosphate, ATP, ADP, AMP, fructose, 1, 6-diphosphate, 3-phosphoglyceric acid, bis- (p- nitrophenyl)phosphate and α,β-methyleneadenosine-5' - diphosphate were used as alternative substrates. The results of the analysis of substrate specificity are shown in Table 2.

Table 2 : Substrate specificity of phytase

Enzyme assay

Unless otherwise stated, the activity of phytase was measured by incubating 150 μl enzyme preparation with 600 μl of 2 mM sodium phytate in 100 mM Tris-HCl buffer pH 7.5, supplemented with 1 mM CaCl2 for 30 minutes at 37°C. After incubation the reaction was stopped by adding 750 μl of 5% trichloroacetic acid. Phosphate released was measured against phosphate

standard spectrophotometrically at 700 nm after adding 1500 μl of the colour reagent (4 volumes of 1.5% ammonium molybdate in 5.5% sulphuric acid and 1 volume of 2.7% ferrous sulphate; Shimizu, M., 1992; Biosci. Biotech. Biochem. , 56:1266-1269) . One unit of enzyme activity was defined as the amount of enzyme required to liberate one μmol Pi per min under assay conditions. The specific activity was expressed in units of enzyme activity per mg protein. The characteristics of the phytase purified in the above manner are summarised in Table 3.

Table 3: Characteristics of phytase

pH and temperature activity profiles

Temperature and pH activity profiles of phytase were analysed in defined buffers and in wheat bran extract. The enzyme concentrations used in the assays gave linear orthophosphate release for the 30 minute incubation period under optimum conditions at 37°C.

Defined buffers used were 100 mM Glycine pH 3.0, 100 mM Succinate pH 5.0, 100 mM Tris-maleate pH 5.0, 6.0, 7.0 and 8.0, 100 mM Tris-HCl pH 7.5, 8 and 9. All buffers were supplemented with 2 mM sodium phytate and 1 mM CaCl2- Enzyme assays were performed in these buffers at five different temperatures (37, 45, 55, 65 and 75°C) . 600 μl of a buffer was temperated at the relevant temperature and the enzyme reaction was started by adding 150 μl of an enzyme preparation. Reactions were stopped after 30 minutes incubation and liberated inorganic orthophosphate was

(3 measured as earlier described. Enzyme assays were run in duplicates. The true pH in the reaction mixture was measured in the beginning and at the end of each assay. Protein concentrations were measured as described earlier and the specific activities of enzymes were calculated at various pH and temperature.

Wheat bran extract was prepared by dissolving 50 g wheat bran in 500 ml of distilled water followed by autoclaving at 121°C for 60 minutes. The extract was filtered through cheese cloth, volume adjusted to 500 ml with distilled water and then the extract was centrifuged at 15,000 rpm for 15 minutes and the supernatant collected. The aliquots of the supernatant were adjusted to pH 3.0, 5.5, 7.0, 8.0 and 9.0, diluted 1:10 in distilled water and supplemented with 2 mM sodium phytate and 1 mM CaCl ₂ . 600 μl of a pH adjusted wheat bran extract was temperated to desired temperature (37, 55 and 75°C) and the enzyme reactions were started by adding 150 μl of enzyme preparation. Reactions were stopped after 30 minutes incubation and liberated inorganic orthophosphate was measured as described above. Enzyme assays were assayed in duplicates. The true pH of each reaction mixture was measured in the beginning and at the end of the enzyme assay.

Effect of pH on the phytase activity

Relative activity of phytase was determined over a pH ranging from 3.0 to 8.5 using both defined buffers and pH adjusted wheat bran extract. It was obvious that not only the pH of the buffer, but also acid composition of the buffer affected relative phytase activity. To cover the pH range, four different defined buffers or wheat bran extract, the pH of which was adjusted by HC1 or NaOH addition, were used. Since enzyme addition affected pH of the reaction mixture, the true pH of each assay mixture was measured in the beginning and in the end of the 30 minute incubation. During the reaction the changes of pH were insignificant.

True reaction pH was used in the determination of pH activity profiles.

Figures 3a to 3e show the pH activity profiles of B 13 phytase in defined buffers at five different temperatures between 37 and 75°C. Irrespective of the reaction temperature, phytase showed highest phytase activity at pH 7.5.

Animal compound feed typically has a pH ranging from pH 5.5 to 7.5.

Temperature optimum of phytase was 55°C. The effect of pH on the temperature activity profile of phytase in the above defined buffers is shown in Figure 4.

Wheat bran extract is likely to provide an environment that is closer to feed and animal digesta than any of the defined buffers. We determined the pH activity profiles of the phytases at 37, 55 and 75°C. Activity of the enzyme in wheat bran extract doubled as compared to its activity in defined buffers (Figures 5a to 5c) . The profiles did not differ from those found in the defined buffers (Figure 6) .

Figure 7 illustrates the relative activity of the two phytases under pH and temperature conditions relevant to feed manufacturing and the digestive process of the broiler chicken. The data for this presentation has been taken from the experiment described above (Figures 5a to 5c) .

Example 2 : Cloning of the gene encoding phytase

N-terminal sequencing

The N-terminal sequence of B. subtilis B 13 phytase purified by SDS-PAGE was sequenced with a Perkin-Elmer Procice Sequencing System using Edman degradation. A twenty five amino

acid long N-terminal sequence was obtained. To obtain more information about the amino acid sequence, the purified phytase was digested with lysC enzyme to obtain internal peptides and the digest was purified with RP-HPLC. LysC digestion was also performed to alcylated phytase following RP-HPLC purification. Non-alcylated RP-HPLC purified phytase peptides were sequenced with same system. Alcylation of phytase was done to determine whether possible sulphur bridges were present. There was no difference between alcylated and non-alcylated phytase lysC digestion RP-HPLC chromathograms showing that there were no sulphur bridges in the phytase.

Nineteen purified peptides were sequenced giving fourteen peptides which were different from each other (5 to 32 amino acids) and a total of 227 amino acids. All peptide sequences are shown in Table 4, including the sequence corresponding to the N-terminus of phytase. The molecular weight of the peptides was measured using mass spectrometer and compared with calculated molecular weights.

Table 4: Peptides obtained by N-terminal amino acid sequencing

* N-terminal sequence

Identification of phytase coding sequences by PCR

On the basis of these peptide sequences, primers for PCR were designed (see Table 5) . All PCR were performed using a PTC-255 DNA Engine and Perkin-Elmer Taq polymerase.

1%

Table 5 : PCR primers giving only one fragment each under optimal conditions

N = A, T, G or C; I = inosine;

PCR was performed with these primers using B. subtilis B 13 DNA isolated according to Sambrook el al . (supra) as the template at different annealing temperatures (45, 50, 55 and 60 C) and at different magnesium concentrations (1.25, 2.5, 5 and 10 mM) to optimize PCR conditions. The following PCR protocol was chosen: 94°C pre-melting for 2 min. before 30 cycles of 92°C melting for 20 sec, 50°C annealing for 30 sec, 72°C extension for 60 sec. in 5 mM magnesium concentration. The primers given in Table 5 amplified only one fragment each under optimal conditions. These amplified PCR fragments are shown in Figure 8.

The longest PCR fragment (amplified with primers 6465 and 6470) was cloned to pCR 2.1 vector (Invitrogen Corp., Inc., San Diego, USA) and sequenced using Sanger Dideoxy method. This resulted in determination of the partial DNA sequence (exact length 989 bp) of phytase of the present invention.

Restriction enzyme analysis of PCR products of phytase gene

To verify that these PCR fragments were phytase fragments, restriction enzyme Hinf I which cleaves the shortest PCR fragment into two approximately 100 bp long fragments was used. These fragments cut with Hinf I gave the same sized fragment from the N-terminal end. PCR fragments were also cut with EcoRI; two of the longest phytase PCR fragments cut with EcoR I which confirms the scheme presented in the Figure 9.

Southern blot analysis of phytase of the phytase gene

Genomic DNA was isolated from B . subtilis B 13, as described in Sambrook et. al. (supra, 1989). Restriction enzymes used were those of Boehringer-Mannheim. B. subtilis B 13 DNA was partially digested with EcoRI and the fragments were separated on agarose gel. Separated fragments were Southern-Blotted to nylon membrane. Nylon membrane was Southern-Hybridized with 32P-labelled N-terminal oligonucleotide probe, GA(C/T)CC(G/A/T)TA(C/T)CA(C/T)TT(C/T)AC(G/A/T)GTNAA(C/T)GC (G/A/T)GC(G/A/T)GC(G/A/T)GAAAC, in order to determine the approximate size of the fragment containing the putative phytase gene. Southern-Hybridisation showed two bands of approximately 1700 bp and 1000 bp consistant with the structure of the gene given in Figure 9.

Screening of a B. subtilis B 13 genomic library

Partially EcoR I digested genomic B. subtilis B 13 DNA was cloned into Lambda Zap II using a Stratagene Lambda Zap II/EcoRI/CIAP Cloning kit according to the recommendations of the manufacturer. Lambda Zap II library was screened with Boehringer-Mannheim EasyToHyb hybridisation kit according the recommendations provided by the manufacturer using the above mentioned longest PCR fragment (989 bp) labeled with digoxigenin as the hybridisation probe.

XL-1 Blue MRF' host cells were infected with 100 000 pfu's of lambda Zap II B . subtilis B 13 genomic library phages. Infected cells were plated with TOP agarose on LB agar plates. Formed plaques were transferred to nylon membranes and screened with the 989 bp digoxigenin labeled hybridisation probe. Several intense positive clones were found with practically no backround. These positive plaques were cored and used in a second round of hybridisation. Positive plaques remained positive in a second round of hybridisation and were cored and excised with helper phage to obtain pBluescript SK(-) phagemid. Obtained phagemids were transformed to E. coli host cells and DNA from minipreps were used in analysis of insert DNA and DNA sequencing.

Determination of the DNA sequence of the gene encoding phytase

The DNA sequence encoding for phytase as well as the deduced amino acid sequence are shown in SEQ ID NO: 1. The molecular weight of phytase as deduced from the amino acid sequence in SEQ ID NO: 1 is ca. 41,900 daltons for the pre-protein and ca. 39,000 for the mature protein (i.e. without the signal sequence) . This is in agreement with the molecular weight of phytase as determined from SDS-PAGE (Figure 1) .

The N- terminus of the mature protein corresponds to amino acid number 30 (Leu-30) of SEQ. ID. NO: 1.

Example 3: Expression of recombinant phytase in E. coli

DNA coding for the mature protein was amplified by PCR using primers which also contained restriction sites for cloning into vectors pQE-30 and pQE-60 (Qiagen, Chatsworth, CA, USA) . The 5' primer in each case encoded a Mfe I site (compatible with Eco RI) followed by a ribosome binding site and the amino terminus of the mature protein. The 3' primer for the pQE-30 construct hybridized downstream of the stop codon of the native protein followed by a Sal I site for cloning. The

resulting PCR product was cloned into pQE-30 digested with Eco Rl/Sal I. This construct should produce the same protein as the mature native product with an additional methionine residue on the amino terminus.

5' primer for both pQE-30 and pQE-60 constructs:

GTTTCTCAATTGAAGGAGGAATTTAAATGCTGTCCGATCCTTATCATTTTAC Mfe I RBS MetLeuSerAspProTyrHisPhe

3' primer for pQE-30 construct:

AATAAGTCGACGTACGACCGGATTCCGGCTGTGCT

Sal I

The 3' primer used for the pQE-60 construct encoded the C- terminus of the protein (without stop codon) followed by a Bgl II cloning site. The vector sequence provides the nucleotides encoding a histidine tag to facilitate purification of the expressed protein. The PCR product was cloned into pQE-60 digested with Eco RI/Bgl II. The enzyme expressed from this construct can be purified from the cell lysate using Ni-NTA resin according to the manufacturer's instructions (Qiagen) .

3' primer for pQE-60 construct:

AATAAAGATCTTTTTCCGCTTCTGTCGGTCAGTT Bgl II

Said constructs were then transformed into the expression host M15/pREP4 cell line (Quiagen) . The M15/pREP4 cell line was made competent and transformed using standard procedures (Sambrook, J., Fritsch, E.F. and Maniatis, T., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harboe, New York, 1989) . This cell line contains a plas id (pREP4) which constitutively expresses the lac repressor protein. This allows strong repression of the

expression constructs in pQE-30 and pQE-60 which have two lac repressor recognition sequences upstream of the open reading frame. The vectors use the phage T5 promoter which is efficiently recognized by the E. coli RNA polymerase. These constructs were grown overnight in LB medium supplimented with ampicillin, methicillin and kanamycin at 37°C. The overnight cultures were diluted 1:30 in fresh media and grown to OD ₆₀ o 0.8 at which point they were induced with 1.5 mM IPTG. After three additional hours of growth, the cells were havested, washed, and lysed by sonication. The lysates were cleared of debris by centrifugation. Aliquots of cleared lysates were also assayed for enzyme activity. The assays were performed in reaction buffer (100 mM Tris-100 mM maleate, pH 7, 1 mM CaCl2 and 2 mM sodium phytate) at 42°C for 30 minutes. The results are presented in Table 6.

Table 6

SEQUENCE LISTING ² 7

(1) GENERAL INFORMATION:

(i) APPLICANT:

(A) NAME: Finnfeeds International, Ltd.

(B) STREET: P.O. Box 777

(C) CITY: Marlborough

(D) STATE: Wiltshire

(E) COUNTRY: United Kingdom

(F) POSTAL CODE (ZIP) : SNΘ 1XN

(ii) TITLE OF INVENTION: Phytase, gene encoding said phytase, method for its production and use

(iii) NUMBER OF SEQUENCES; 2

(iv) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentln Release #1.0, Version #1.30 (EPO)

2S

(2) INFORMATION FOR SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1290 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Bacillus subtilis

(B) STRAIN: B13

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 91..1239

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

CACATTTGAC AATTTTCACA AAAACTTAAC ACTGACAATC ATGTATATAT GTTACAATTG 60

AAGTGCACGT TCATAAAAGG AGGAAGTAAA ATG AAT CAT TCA AAA ACA CTT TTG 114

Met Asn His Ser Lys Thr Leu Leu

1 5

TTA ACC GCG GCG GCC GGA CTG ATG CTC ACA TGC GGT GCG GTG TCT TCC 162 Leu Thr Ala Ala Ala Gly Leu Met Leu Thr Cys Gly Ala Val Ser Ser 10 15 20

CAG GCA AAG CAT AAG CTG TCC GAT CCT TAT CAT TTT ACC GTG AAT GCA 210 Gin Ala Lys His Lys Leu Ser Asp Pro Tyr His Phe Thr Val Asn Ala 25 30 35 40

GCG GCG GAA ACG GAA CCG GTT GAT ACG GCC GGT GAC GCG GCT GAT GAT 258 Ala Ala Glu Thr Glu Pro Val Asp Thr Ala Gly Asp Ala Ala Asp Asp 45 50 55

CCT GCG ATT TGG CTG GAC CCC AAG ACT CCT CAG AAC AGC AAA TTG ATT 306 Pro Ala lie Trp Leu Asp Pro Lys Thr Pro Gin Asn Ser Lys Leu lie 60 65 70

ACG ACC AAT AAA AAA TCA GGT TTA GTC GTT TAC AGC CTT GAT GGT AAG 354 Thr Thr Asn Lys Lys Ser Gly Leu Val Val Tyr Ser Leu Asp Gly Lys 75 80 85

ATG CTT CAT TCC TAT AAT ACC GGG AAG CTG AAC AAT GTC GAT ATC CGT 402 Met Leu His Ser Tyr Asn Thr Gly Lys Leu Asn Asn Val Asp lie Arg 90 95 100

TAT GAT TTT CCG TTG AAC GGC AAA AAA GTC GAT ATC GCG GCA GCA TCC 450 Tyr Asp Phe Pro Leu Asn Gly Lys Lys Val Asp lie Ala Ala Ala Ser 105 110 115 120

AAT CGG TCT GAA GGA AAA AAT ACC ATT GAG ATT TAC GCT ATT GAT GGA 498 Asn Arg Ser Glu Gly Lys Asn Thr lie Glu lie Tyr Ala lie Asp Gly 125 130 135

AAA AAC GGC ACA TTA CAA AGC ATG ACA GAT CCA GAC CAT CCG ATT GCA 546 Lys Asn Gly Thr Leu Gin Ser Met Thr Asp Pro Asp His Pro lie Ala 140 145 150

ACA GCA ATT AAT GAG GTA TAC GGT TTT ACC TTA TAC CAC AGT CAA AAA 594 Thr Ala lie Asn Glu Val Tyr Gly Phe Thr Leu Tyr His Ser Gin Lys 155 160 165

ACA GGA AAA TAT TAC GCG ATG GTG ACA GGA AAA GAG GGT GAA TTT GAA 642 Thr Gly Lys Tyr Tyr Ala Met Val Thr Gly Lys Glu Gly Glu Phe Glu 170 175 180

CAA TAC GAA TTA AAG GCG GAC AAA AAT GGA TAC ATA TCC GGC AAA AAG 690 Gin Tyr Glu Leu Lys Ala Asp Lys Asn Gly Tyr lie Ser Gly Lys Lys 185 190 195 200

GTA CGG GCG TTT AAA ATG AAT TCC CAG ACG GAA GGG ATG GCA GCA GAC 738 Val Arg Ala Phe Lys Met Asn Ser Gin Thr Glu Gly Met Ala Ala Asp 205 210 215

GAT GAA TAC GGC AGG CTT TAT ATC GCA GAA GAA GAT GAG GCC ATT TGG 786 Asp Glu Tyr Gly Arg Leu Tyr He Ala Glu Glu Asp Glu Ala He Trp 220 225 230

AAG TTC AGC GCC GAG CCG GAC GGC GGC AGT AAC GGA ACG GTT ATC GAC 834 Lys Phe Ser Ala Glu Pro Asp Gly Gly Ser Asn Gly Thr Val He Asp 235 240 245

CGT GCC GAC GGC AGG CAT TTA ACT CGT GAT ATT GAA GGA TTG ACG ATT 882 Arg Ala Asp Gly Arg His Leu Thr Arg Asp He Glu Gly Leu Thr He 250 255 260

TAC TAC GCT GCT GAC GGG AAA GGC TAT CTG ATG GCA TCA AGC CAG GGA 930 Tyr Tyr Ala Ala Asp Gly Lys Gly Tyr Leu Met Ala Ser Ser Gin Gly 265 270 275 280

AAC AGC AGC TAC GCC ATT TAT GAC AGA CAA GGA AAG AAC AAA TAT GTT 978 Asn Ser Ser Tyr Ala He Tyr Asp Arg Gin Gly Lys Asn Lys Tyr Val 285 290 295

GCG GAT TTT CGC ATA ACA GAC GGT CCT GAA ACA GAC GGG ACA AGC GAT 1026 Ala Asp Phe Arg He Thr Asp Gly Pro Glu Thr Asp Gly Thr Ser Asp 300 305 310

ACA GAC GGA ATT GAC GTT CTG GGT TTC GGA CTG GGG CCT GAA TAT CCG 1074 Thr Asp Gly He Asp Val Leu Gly Phe Gly Leu Gly Pro Glu Tyr Pro 315 320 325

TTC GGT ATT TTT GTC GCA CAG GAC GGT GAA AAT ATA GAT CAC GGC CAA 1122 Phe Gly He Phe Val Ala Gin Asp Gly Glu Asn He Asp His Gly Gin 330 335 340

AAG GCC AAT CAA AAT TTT AAA ATC GTG CCA TGG GAA AGA ATT GCT GAT 1170 Lys Ala Asn Gin Asn Phe Lys He Val Pro Trp Glu Arg He Ala Asp 345 350 355 360

CAA ATC GGT TTC CGC CCG CTG GCA AAT GAA CAG GTT GAC CCG AGA AAA 1218 Gin He Gly Phe Arg Pro Leu Ala Asn Glu Gin Val Asp Pro Arg Lys 365 370 375

CTG ACC GAC AGA AGC GGA AAA TAAACATGCA AAAAGCAGCT TATACAAGCT 1269

Leu Thr Asp Arg Ser Gly Lys 380

GCTTTTTGCA TGTGAAGAAC G 1290

(2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 383 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

Met Asn His Ser Lys Thr Leu Leu Leu Thr Ala Ala Ala Gly Leu Met 1 5 10 15

Leu Thr Cys Gly Ala Val Ser Ser Gin Ala Lys His Lys Leu Ser Asp 20 25 30

Pro Tyr His Phe Thr Val Asn Ala Ala Ala Glu Thr Glu Pro Val Asp 35 40 45

Thr Ala Gly Asp Ala Ala Asp Asp Pro Ala He Trp Leu Asp Pro Lys 50 55 60

Thr Pro Gin Asn Ser Lys Leu He Thr Thr Asn Lys Lys Ser Gly Leu 65 70 75 80

Val Val Tyr Ser Leu Asp Gly Lys Met Leu His Ser Tyr Asn Thr Gly 85 90 95

Lys Leu Asn Asn Val Asp He Arg Tyr Asp Phe Pro Leu Asn Gly Lys 100 105 110

Lys Val Asp He Ala Ala Ala Ser Asn Arg Ser Glu Gly Lys Asn Thr 115 120 125

He Glu He Tyr Ala He Asp Gly Lys Asn Gly Thr Leu Gin Ser Met 130 135 140

Thr Asp Pro Asp His Pro He Ala Thr Ala He Asn Glu Val Tyr Gly 145 150 155 160

Phe Thr Leu Tyr His Ser Gin Lys Thr Gly Lys Tyr Tyr Ala Met Val 165 170 175

Thr Gly Lys Glu Gly Glu Phe Glu Gin Tyr Glu Leu Lys Ala Asp Lys 180 185 190

Asn Gly Tyr He Ser Gly Lys Lys Val Arg Ala Phe Lys Met Asn Ser 195 200 205

Gin Thr Glu Gly Met Ala Ala Asp Asp Glu Tyr Gly Arg Leu Tyr He 210 215 220

Ala Glu Glu Asp Glu Ala He Trp Lys Phe Ser Ala Glu Pro Asp Gly 225 230 235 240

Gly Ser Asn Gly Thr Val He Asp Arg Ala Asp Gly Arg His Leu Thr 245 250 255

Arg Asp He Glu Gly Leu Thr He Tyr Tyr Ala Ala Asp Gly Lys Gly 260 265 270

Tyr Leu Met Ala Ser Ser Gin Gly Asn Ser Ser Tyr Ala He Tyr Asp

275 280 285

Arg Gin Gly Lys Asn Lys Tyr Val Ala Asp Phe Arg He Thr Asp Gly

290 295 300

Pro Glu Thr Asp Gly Thr Ser Asp Thr Asp Gly He Asp Val Leu Gly

305 310 315 320

Phe Gly Leu Gly Pro Glu Tyr Pro Phe Gly He Phe Val Ala Gin Asp

325 330 335

Gly Glu Asn He Asp His Gly Qln Lys Ala Asn Gin Asn Phe Lys He

340 345 350

Val Pro Trp Glu Arg He Ala Asp Gin He Gly Phe Arg Pro Leu Ala

355 360 365

Asn Glu Gin Val Asp Pro Arg Lys Leu Thr Asp Arg Ser Gly Lys 370 375 380