Phytases

Details Phytases Phytases

2000-01-20

2004-04-13

Riuz, Rebecca (Department: 1652)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Hydrolase

C435S018000, C435S195000, C530S350000, C536S023200, C426S006000

Reexamination Certificate

active

06720174

ABSTRACT:

Phytases are enzymes that hydrolyze phytate (myo-inositol hexakisphosphate) to myo-inositol and inorganic phosphate. They are known to be valuable feed additives.
The present invention relates to improved phytases, viz. phytases of amended characteristics, e.g. amended activity characteristics, reference being made to e.g. the phytase(s) it has been derived from, or to known phytases. Amended activity characteristics means amended in at least one phytase activity related respect, such as (non-exclusive list): pH stability, temperature stability, pH profile, temperature profile, specific activity (in particular in relation to pH and temperature), substrate specificity, substrate cleavage pattern, substrate binding, position specificity, the velocity and level of release of phosphate from corn, reaction rate, phytate degradation rate), end level of released phosphate reached.
Examples of amended activity characteristics are amended specific activity (e.g. increased, e.g. increased at a pH of 3, 4, 5, or 6); amended pH or temperature profile; and/or amended (e.g. increased) thermostability, e.g. of an increased melting temperature as measured using Differential Scanning Calorimetry (DSC).
The present invention also relates to a process for the preparation of a modified protein, wherein in a first step a consensus sequence is determined from a number of highly homologous sequences according to steps a), b) and c) below:
a) at least three, preferably at least four amino acid sequences are aligned by any standard alignment program known in the art;
b) at every position of the amino acid sequence alignment, the amino acids are evaluated for their evolutionary similarity and a consensus residue is chosen by any standard program known in the art, whereby the minimal requirements for calculation of a consensus residue are set in such a way that the program is already able to determine a consensus residue if a given residue occurs in only two of the aligned sequences. However, if there is a subgroup of sequences among the compared amino acid sequences that shows a much higher degree of similarity with each other than with the remaining sequences of the alignment, the subgroup may be represented in the calculation only with its consensus sequence determined in the same way as outlined in EP 897985, or alternatively, to each sequence of the subgroup, a vote weight of 1 divided by the number of sequences in the subgroup will be assigned;
c) in case no consensus amino acid at a defined position is identified by the program, any of the amino acids, preferably the most frequently occurring amino acid at this position is selected.
In a second aspect of the invention, a homologous sequence is compared with the consensus sequence, and one or more non-consensus residues in this homologous sequence are replaced by the corresponding consensus residues.
Preferably, only such amino acid residues are replaced in the homologous amino acid sequence where a consensus residue can clearly be defined by the program under moderately stringent conditions whereas at all positions of the alignment where no preferred consensus amino acid can be determined under moderately stringent conditions, the amino acids of the homologous protein remain unchanged.
In a third aspect of the invention, the active center of the protein of interest is determined, comprising all amino acid residues that are involved in forming the active center, both in the consensus sequence, and in the sequence of a homologous protein; subsequently, some or all of the divergent amino acid residues of the homologous protein are inserted in the backbone of the consensus sequence.
In one embodiment of this process, the program used for the comparison of amino acids at a defined position regarding their evolutionary similarity is the program “PRETTY”.
The active center of the protein can be determined by using an analysis of the three-dimensional structure of the protein.
An example of a homologous protein is an enzyme family, an example of a defined protein family is the family of phytases, e.g. of fungal origin.
For example, the amino acid sequence of the phytase can be changed by the introduction of at least one mutation or substitution chosen from
E58A
F54Y
D69K
I73V
D197N
K94A
T214L
R101A
E222T
N153K
E267D
V158I
R291I
A203G
R329H
S205G
S364T
V217A
A379K
A227V
G404A
V234L
P238A
Q277E
A287H
A292Q
V366I
A396S
E415Q
G437A
R451E
For interpreting these abbreviations, as an example, the mutation E58A is to be interpreted as follows: When subtracting 26 from the number, you get the position or residue number in the consensus phytase sequence or another phytase sequence aligned as shown in
FIG. 1
(corresponding to the addition of a 26 amino acid signal sequence to the sequences shown in FIG.
1
). For example, in E58A, number 58 means position number 32 (58−26=32). And the letter before the number, i.e. E, represents the amino acid in the phytase to be modified which is replaced by the amino acid behind the number, i.e. A.
The above-mentioned amino acid replacements, alone and/or in combination, have a positive effect on the protein stability.
The following sub-groups of mutations are also interesting (i.e. phytases comprising at least one mutation selected from either one of the groups of):
E58A, D69K, D197N, T214L, E222T, E267D, R291I, R329H, S364T, A379K, G404A;
F54Y, I73V, K94A, R101A, N153K, V158I, A203G, S205G, V217A, A227V, V234L, P238A, Q277E, A287H, A292Q, V366I, A396S, E415Q, G437A, R451E;
E58A, D69K, D197N, F54Y, I73V, K94A;
T214L, E222T, E267DR101A, N153K, V158I;
R291I, R329H, S364TA203G, S205G, V217A;
A379K, G404AA227V, V234L, P238A, Q277E;
A287H, A292Q, V366I, A396S, E415Q, G437A, R451E;
T214L, E222T, S364T, V158I, A203G, G404A, A227V, P238A, A396S, G437A, R451E.
Examples of host cells are plant cells, animal cells, and microbial cells, e.g. prokaryotic or eukaryotic cells, such as bacterial, fungal or yeast cells. An example of a fungal host is a strain of the genus Aspergillus, and examples of yeast hosts are strains of Saccharomyces, and strains of Hansenula.
The invention also relates to a modified protein obtainable or obtained by any of the processes described above.
The invention also relates to a variant or mutein of a phytase such as (but not limited to) the consensus phytase-1, wherein, in the amino acid sequence in
FIG. 2
, at least one of the following replacements have been effected: Q50L, Q50T, Q50G, Q50T-Y51N, Q50L-Y51N or Q50T-K91A.
In the third aspect mentioned above, a consensus sequence is determined from homologous sequences as described above; in a second step the active center of the protein comprising all amino acid residues that are involved in forming the active center is determined in the consensus sequence and in the sequence of a single homologous protein as well. The single homologous protein may have preferred properties like high specific activity or different pH dependency of enzymatic activity. In a third step some or all amino acid residues that are involved in forming the active center of the homologous protein are inserted into the backbone of the consensus sequence. The result thereof is a chimeric protein having the active center derived from a single protein and the backbone of the consensus sequence.
The active center of the protein can be determined e.g. by using any analysis of the three-dimensional structure of the protein, e.g. by homology modelling on the basis of a known 3D-structure of a known protein.
The present invention also provides consensus proteins obtainable or obtained by such processes, in particular proteins comprising at least one of the amino acid sequences shown in
FIGS. 2-6
,
10
or
21
, or variants or muteins thereof. Examples of such variants are shown in
FIGS. 7-9
.
Such variants or muteins can be defined and prepared on the basis of the teachings given in European Patent Application number 0897010, e.g. Q50L, Q50T, Q50G, Q50L-Y51N, or Q50T-Y51N.
These mutations are defined as above, or, alternatively, by reference to FIG.
2
. When referring to
FIG. 2

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