A process for obtaining a consensus protein from a group of amino acid sequences of a defined protein family, proteins and polynucleotides so obtained, and compositions containing such proteins.
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3. A purified polynucleotide which encodes a consensus protein having the amino acid sequence of SEQ ID NO:2 SEQ ID NO:17 except that Q at position 50 has been replaced by L, T, or G.
5. A purified polynucleotide which encodes a consensus protein having the amino acid sequence of SEQ ID NO:2 SEQ ID NO:17 except that Q at position 50 has been replaced by L and Y at position 51 has been replaced by N.
4. A purified polynucleotide which encodes a consensus protein having the amino acid sequence of SEQ ID NO:2 SEQ ID NO: 17 except that Q at position 50 has been replaced by T and Y at position 51 has been replaced by N.
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This is a divisional of U.S. application Ser. No. 09/121,425, filed Jul. 23, 1998, now U.S. Pat. No. 6,153,418.
Phytases (myo-inositol hexakisphosphate phosphohydrolases; EC3.1.3.8) are enzymes that hydrolyze phytate (myo-inositol hexakisphosphate) to myo-inositol and inorganic phosphate and are known to be valuable feed additives.
A phytase was first described in rice bran in 1907 [Suzuki et al., Bull. Coll. Agr. Tokio Imp. Univ. 7, 495 (1907)] and phytases from Aspergillus species in 1911 [Dox and Golden, J. Biol. Chem. 10, 183-186 (1911)]. Phytases have also been found in wheat bran, plant seeds, animal intestines and in microorganisms [Howsen and Davis, Enzyme Microb. Technol. 5, 377-382 (1983), Lambrechts et al., Biotech. Lett. 14, 61-66 (1992), Shieh and Ware, Appl. Microbiol. 16, 1348-1351 (1968)].
The cloning and expression of the phytase from Aspergillus niger (ficuum) has been described by Van Hartingsveldt et al., in Gene, 127, 87-94 (1993) and in European Patent Application, Publication No. (EP) 420 358 and from Aspergillus niger var. awamori by Piddington et al., in Gene 133, 55-62 (1993).
Cloning, expression and purification of phytases with improved properties have been disclosed in EP 684 313. However, since there is a still ongoing need for further improved phytases, especially with respect to the thermostability, it is an object of the present invention to provide the following process which is, however, not only applicable to phytases.
The invention herein is a process for the preparation of a consensus protein, especially a phytase. The invention is also directed to a consensus phytase and to a DNA sequence encoding the consensus phytase. As is well known, a consensus protein is a new protein whose sequence is created from sequence information obtained from at least three other proteins having a similar biological activity. The object in preparing a consensus protein is to obtain a single protein which combines the advantageous properties of the original proteins.
The process is characterized by the following steps:
In a preferred embodiment of this process step b) can also be defined as follows: b) amino acids at the same position according to such an alignment are compared regarding their evolutionary similarity by any standard program known in the art, whereas the degree of similarity provided by such program is set at the lowest possible value and the amino acid which is the most similar for at least half of the sequences used for the comparison is selected for the corresponding position in the amino acid sequence of the consensus protein.
Thus the claimed invention is a process for obtaining a consensus protein from a group of amino acid sequences of a defined protein family, which comprises:
The present invention is also directed to new phytases, preferably phytases having the amino acid sequence depicted in FIG. 2 and variants and muteins thereof. In addition, the invention includes polynucleotides which encode such new phytases.
The PCR products (670 and 905 bp) were purified by an agarose gel electrophoresis (Q.9% agarose) and a following gel extraction (QIAEX II Gel Extraction Kit, Qiagen, Hilden, Germany). The purified DNA fragments were used for the PCR reaction c.
PCR reaction c: 6 μl PCR product of reaction a (≈50 ng)
The resulting PCR product (1.4 kb) was purified as mentioned above, digested with Eco RI, and ligated in an Eco RI-digested and dephosphorylated pBsk(−)-vector (Stratagene, La Jolla, Calif., USA). 1 μl of the ligation mixture was used to transform E. coli XL-1 competent cells (Stratagene, La Jolla, Calif., USA). All standard procedures were carried out as described by Sambrook et al. (1987). The constructed fungal consensus phytase gene (fcp) was verified by sequencing (plasmid pBsk−-fcp).
A fungal consensus phytase gene was isolated from the plasmid pBsk−fcp ligated into the Eco RI sites of the expression cassette of the Saccharomyces cerevisiae expression vector pYES2 (Invitrogen, San Diego, Calif., USA) or subcloned between the shortened GAPFL (glyceraldhyde-3-phosphate dehydrogenase) promoter and the pho5 terminator as described by Janes et al. (1990). The correct orientation of the gene was checked by PCR. Transformation of S. cerevisiae strains. e.g. INVSc1 (Invitrogen, San Diego, Calif., USA) was done according to Hinnen et al. (1978). Single colonies harboring the phytase gene under the control of the GAPFL promoter were picked and cultivated in 5 ml selection medium (SD-uracil, Sherman et al., 1986) at 30° C. under vigorous shaking (250 rpm) for one day. The preculture was then added to 500 ml YPD medium (Sherman et al., 1986) and grown under the same conditions. Induction of the gall promoter was done according to manufacturer's instruction. After four days of incubation cell broth was centrifuged (7000 rpm, GS3 rotor, 15 min, 5° C.) to remove the cells and the supernatant was concentrated by way of ultrafiltration in Amicon 8400 cells (PM30 membranes) and ultrafree-15 centrifugal filter device (Biomax-30K, Millipore, Bedford, Mass., USA). The concentrate (10 ml) was desalted on a 40 ml Sephadex G25 Superfine column (Pharmacia Biotech, Freiburg, Germany), with 10 mM sodium acetate, pH 5.0, serving as elution buffer. The desalted sample was brought to 2 M (NH4)2SO4 and directly loaded onto a 1 ml Butyl Sepharose 4 Fast Flow hydrophobic interaction chromatography column (Pharmacia Biotech, Feiburg, Germany) which was eluted with a linear gradient from 2 M to 0 M (NH4)2SO4 in 10 mM sodium acetate, pH 5.0. Phytase was eluted in the break-through concentrated and loaded on a 120 ml Sephacryl S-300 gel permeation chromatography column. (Pharmacia Biotech, Freiburg, Germany). Fungal consensus phytase and fungal consensus phytase 7 eluted as a homogeneous symmetrical peak and was shown by SDS-PAGE to be approx. 95% pure.
The phytase expression vectors, used to transform H. polymorpha, was constructed by inserting the Eco RI fragment of pBsk−fcp encoding the consensus phytase or a variant into the multiple cloning site of the H. polymorpha expression vector pFPMT121, which is based on an ura3 selection marker and the FMD promoter. The 5′ end of the fcp gene is fused to the FMD promoter, the 3′ end to the MOX terminator (Gellissen et al., 1996; EP 0299 108 B). The resulting expression vector are designated pFPMTfcp and pBsk−fcp7.
The constructed plasmids were propagated in E. coli. Plasmid DNA was purified using standard state of the art procedures. The expression plasmids were transformed into the H. polymorpha strain RP11 deficient in orotidine-5′-phosphate decarboxylase (ura3) using the procedure for preparation of competent cells and for transformation of yeast as described in Gelissen et al. (1996). Each transformation mixture was plated on YNB (0.14% w/v Difco YNB and 0.5% ammonium sulfate) containing 2% glucose and 1.8% agar and incubated at 37° C. After 4 to 5 days individual transformant colonies were picked and grown in the liquid medium described above for 2 days at 37° C. Subsequently, an aliquot of this culture was used to inoculate fresh vials with YNB-medium containing 2% glucose. After seven further passages in selective medium, the expression vector integrates into the yeast genome in multimeric form. Subsequently, mitotically stable transformants were obtained by two additional cultivation steps in 3 ml non-selective liquid medium (YPD, 2% glucose, 10 g yeast extract, and 20 g peptone). In order to obtain genetically homogeneous recombinant strains an aliquot from the last stabilization culture was plated on a selective plate. Single colonies were isolated for analysis of phytase expression in YNB containing 2% glycerol instead of glucose to derepress the find promoter. Purification of the fungal consensus phytases was done as described in Example 5.
Plasmid pBsk−fcp or the corresponding plasmid of a variant of the fcp gene were used as template for the introduction of a Bsp HI-site upstream of the start codon of the genes and an Eco RV-site downstream of the stop codon. The Expand™ High Fidelity PCR Kit (Boehringer Mannheim, Mannheim, Germany) was used with the following primers:
Primer Asp-1 (SEQ ID NO:22):
Bsp HI
5′-TAT ATC ATG AGC GTG TTC GTC GTG CTA CTG TTC-3′
Primer Asp-2 for cloning of fcp and fcp7
(SEQ ID NO:23):
3′-ACC CGA CTT ACA AAG CGA ATT CTA TAG ATA TAT-5′
Eco RV
The reaction was performed as described by the supplier. The PCR-amplified fcp gene had a new Bsp HI site at the start codon, introduced by primer Asp-1, which resulted in a replacement of the second amino acid residue glycine by serine. Subsequently, the DNA-fragment was digested with Bsp HI and Eco RV and ligated into the Nco I site downstream of the glucoamylase promoter of Aspergillus niger (glaA) and the Eco RV site upstream of the Aspergillus nidulans tryptophan C terminator (trpC) (Mullaney et al., 1985). After this cloning step, the genes were sequenced to detect possible failures introduced by PCR. The resulting expression plasmids which basically corresponds to the pGLAC vector as described in Example 9 of EP 684 313, contained the orotidine-5′-phosphate decarboxylase gene (pyr4) of Neurospora crassa as a selection marker. Transformation of Aspergillus tiger and expression of the consensus phytase genes was done as described in EP 684 313. The fungal consensus phytases were purified as described in Example 5.
To construct muteins for expression in A. niger, S. cerevisiae, or H. polymorpha, the corresponding expression plasmid containing the fungal consensus phytase gene was used as template for site-directed mutagenesis. Mutations were introduced using the “quick exchange site-directed mutagenesis kit” from Stratagene (La Jolla, Calif., USA) following the manufacturer's protocol and using the corresponding primers. All mutations made and the corresponding primers are summarized in Table 4. Clones harboring the desired mutation were identified by DNA sequence analysis as known in the art. The mutated phytase were verified by sequencing of the complete gene.
TABLE 4
mutation Primer set
t1,1
Q50L
Ssp BI
(SEQ ID NO:24)
5′-CAC TTG TGG GGT TTG TAC AGT CCA TAC TTC TC-3′
(SEQ ID NO:25)
5′-GAG AAG TAT GGA CTG TAC AAA CCC CAC AAG TG-3′
Q50T
Kpn I
(SEQ ID NO:26)
5′-CAC TTG TGG GGT ACC TAC TCT CCA TAC TTC TC-3′
(SEQ ID NO:27)
5′-GA GAA GTA TGG AGA GTA GGT ACC CCA CAA GTG-3′
Q50G
(SEQ ID NO:28)
5′-CAC TTG TGG GGT GGT TAC TCT CCA TAC TTC TC-3′
(SEQ ID NO:29)
5′-GA GAA GTA TGG AGA GTA ACC ACC CCA CAA GTG-3′
Q50T-Y51N
Kpn I
(SEQ ID NO:30)
5′-CAC TTG TGG GGT ACCAAC TCT CCA TAC TTC TC-3′
(SEQ ID NO:31)
5′-GA GAA GTA TGG AGA GTT GGT ACC CCA CAA GTG-3′
Q50L-Y51N
Bsa I
(SEQ ID NO:32)
5′-CAC TTG TGG GGT CTCAAC TCT CAA TAC TTC TC-3′
(SEQ ID NO:33)
5′-GA GAA GTA TGG AGA GTT GAG ACC CCA CAA GTG-3′
Table 4: Primers used for the introduction of single mutations into fungal consensus phytase. For the introduction of each mutation, two primers containing the desired mutation were required (see Example 8). The changed triplets are highlighted in bold letters.
Phytase activity was determined basically as described by Mitchell et al. (1997). The activity was measured in a assay mixture containing 0.5% phytic acid (≈5 mM), 200 mM sodium acetate, pH 5.0. After 15 min incubation at 37° C., the reaction was stopped by addition of an equal volume of 15% trichloroacetic acid. The liberated phosphate was quantified by mixing 100 μl of the assay mixture with 900 μl H2O and 1 ml of 0.6 M H2SO4, 2% ascorbic acid and 0.5% ammonium molybdate. Standard solutions of potassium phosphate were used as reference. One unit of enzyme activity was defined as the amount of enzyme that releases 1 μmol phosphate per minute at 37° C. The protein concentration was determined using the enzyme extinction coefficient at 280 nm calculated according to Pace et al. (1995): fungal consensus phytase, 1.101; fungal consensus phytase 7, 1.068. In case of pH-optimum curves, purified enzymes were diluted in 10 mM sodium acetate, pH 5.0. Incubations were started by mixing aliquots of the diluted protein with an equal volume of 1% phytic acid (≈10 mM) in a series of different buffers; 0.4 M glycine/HCl, pH 2.5; 0.4 M acetate/NaOH, pH 3.0, 3.5, 4.0, 4.5, 5.0, 5.5; 0.4 M imidazole/HCl, pH 6.0, 6.5; 0.4 M Tris/HCl pH 7.0, 7.5, 8.0, 8.5, 9.0. Control experiments showed that pH was only slightly affected by the mixing step. Incubations were performed for 15 min at 37° C. as described above.
For determination of the substrate specificities of the phytases, phytic acid in the assay mixture was replaced by 5 mM concentrations of the respective phosphate compounds. The activity tests were performed as described above.
For determination of the temperature optimum, enzyme (100 μl) and substrate solution (100 μl) were pre-incubated for 5 min at the given temperature. The reaction was started by addition of the substrate solution to the enzyme. After 15 min incubation, the reaction was stopped with trichloroacetic acid and the amount of phosphate released was determined.
The pH-optimum of the original fungal consensus phytase was around pH 6.0-6.5 (70 U/mg). By introduction of the Q50T mutation, the pH-optimum shifted, to pH 6.0 (130 U/mg), while the replacement by a leucine at the same position resulted in a maximum activity around pH 5.5 (212 U/mg). The exchange Q50G resulted in a pH-optimum of the activity above pH 6.0 (see FIG. 4). The exchange of tyrosine at position 51 with asparagine resulted in a relative increase of the activity below pH 5.0 (see FIG. 5). Especially by the Q50L mutation, the specificity for phytate of fungal consensus phytase was drastically increased (see FIG. 6).
The temperature optimum of fungal consensus phytase (70° C.) was 15-25° C. higher than the temperature optimum of the wild-type phytases (45-55° C.) which were used to calculate the consensus sequence (see Table 5 and FIG. 3).
TABLE 5
temperature
phytase
optimum
Tm
Consensus phytase
70° C.
78.0° C.
A. niger NRRL3135
55° C.
63.3° C.
A. fumigatus 13073
55° C.
62.5° C.
A. terreus 9A-1
49° C.
57.5° C.
A. terreus cbs
45° C.
58.5° C.
A. nidulans
45° C.
55.7° C.
M. thermophila
55° C.
—
Table 5: Temperature optimum and Tm-value of fungal consensus phytase and of the phytases from A. fumigatus, A. niger, A. nidulans, and M. thermophila. The temperature optima were taken from
In order to determine the unfolding temperature of the fungal consensus phytases, differential scanning calorimetry was applied as previously published by Brugger et al. (1997). Solutions of 50-60 mg/ml homogeneous phytase were used for the tests. A constant heating rate of 10° C./min was applied up to 90° C.
The determined melting points clearly show the strongly improved thermostability of the fungal consensus phytase in comparison to the wild-type phytases (see Table 5 and FIG. 7).
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