Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2000-08-08
2003-06-17
Prouty, Rebecca E. (Department: 1652)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C435S069100, C435S320100, C435S325000, C435S252300, C435S195000, C435S196000
Reexamination Certificate
active
06579975
ABSTRACT:
BACKGROUND OF THE INVENTION
Phytases (myo-inositol hexakisphosphate phosphohydrolases; EC 3.1.3.8) 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.
SUMMARY OF THE INVENTION
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:
a) at least three preferably four amino acid sequences of a defined protein family are aligned by any standard alignment program known in the art;
b) amino acids at the same position according to such alignment are compared regarding their evolutionary similarity by any standard program known in the art, whereas the degree of similarity provided by such a program which defines the least similarity of the amino acids that is used for the determination of an amino acid of corresponding positions is set to a less stringent number and the parameters are set in such a way that it is possible for the program to determine from only 2 identical amino acids at a corresponding position an amino acid for the consensus protein; however, if among the compared amino acid sequences are sequences that show a much higher degree of similarity to each other than to the residual sequences, the sequences are represented by their consensus sequence determined as defined in the same way as in the present process for the consensus sequence of the consensus protein or a vote weight of 1 divided by the number of such sequences is assigned to every of those sequences.
c) in case no common amino acid at a defined position can be identified by the program, any of the amino acids of all sequences used for the comparison, preferably the most frequent amino acid of all such sequences is selected or an amino acid is selected on the basis of the consideration given in Example 2.
d) once the consensus sequence has been defined, such sequence is back-translated into a DNA sequence, preferably using a codon frequency table of the organism in which expression should take place;
e) the DNA sequence is synthesized by methods known in the art and used either integrated into a suitable expression vector or by itself to transform an appropriate host cell;
f) the transformed host cell is grown under suitable culture conditions and the consensus protein is isolated from the host cell or its culture medium by methods known in the art.
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 inthe 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:
a) aligning a group consisting of three to one hundred, but preferably three or four amino acid sequences from a defined protein family;
b) comparing the evolutionary similarity of amino acids which occupy a position in the aligned sequences to select a consensus amino acid for said position using a system which is so organized that if two amino acids which occupy said position are identical, then the identical amino acid is selected as the consensus amino acid for said position, unless three or more other amino acids at said position have a higher degree of structural similarity to each other than to the identical amino acid, in which case the amino acid which has the highest degree of evolutionary similarity to the other amino acids is selected as the consensus amino acid for said position, with the proviso that if a set of amino acid sequences exists within the group of step a) such that the amino acid sequences within the set have more evolutionary similarity to each other than to any of the amino acid sequences of the group which are not part of the set, then the amino acids which occupy said position in members of the set will have a vote weight of one divided by the number of amino acid sequences in the set where the amino acids which occupy said position in amino acid sequences which are not in the set will have a vote weight of one, and repeating the procedure for each position in the aligned group of amino acid sequences;
c) if no consensus amino acid for said position is obtained by the method of step b), then any amino acid at said position is selected as the consensus sequence, preferably the most frequent amino acid;
d) combining the consensus amino acids obtained in steps b) and c) obtain a consensus amino acid sequence;
e) translating the consensus amino acid sequence into a DNA sequence, preferably using a codon frequency table specific to whichever host organism has been selected for expressing the DNA sequence;
f) obtaining the DNA sequence and using said DNA sequence to express a protein which is the consensus protein of the defined protein family.
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.
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Pasamontes et al. , Appl. Environ. Microbiol. 63
Bryan Cave LLP
Prouty Rebecca E.
Ramirez Delia
Roche Vitamins Inc.
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