U.S. patent application number 13/232588 was filed with the patent office on 2013-05-30 for enzymes.
This patent application is currently assigned to DUPONT NUTRITION BIOSCIENCES APS. The applicant listed for this patent is Oliver Kensch, Ulrich Kettling, Andre Koltermann, Vijay Kumar, Birgitta Leuthner, Andrei Miasnikov, Klaus Pellangahr. Invention is credited to Oliver Kensch, Ulrich Kettling, Andre Koltermann, Vijay Kumar, Birgitta Leuthner, Andrei Miasnikov, Klaus Pellangahr.
Application Number | 20130136825 13/232588 |
Document ID | / |
Family ID | 33428071 |
Filed Date | 2013-05-30 |
United States Patent
Application |
20130136825 |
Kind Code |
A2 |
Miasnikov; Andrei ; et
al. |
May 30, 2013 |
ENZYMES
Abstract
The present invention relates to enzymes and processes. In
particular, there is described an isolated polypeptide comprising
the amino acid sequence corresponding to Citrobacter freundii
phytase or a homologue, a modified form, a functional equivalent or
an effective fragment thereof. There is also described a host cell
transformed or transfected with a nucleic acid encoding a bacterial
phytase enzyme or a modified form as well as the use of such a
phytase or modified form in food or animal feed.
Inventors: |
Miasnikov; Andrei; (Degerby,
FI) ; Kumar; Vijay; (Casnate, IT) ; Kensch;
Oliver; (Cologne, DE) ; Pellangahr; Klaus;
(Cologne, DE) ; Leuthner; Birgitta; (Cologne,
DE) ; Kettling; Ulrich; (Cologne, DE) ;
Koltermann; Andre; (Koln, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Miasnikov; Andrei
Kumar; Vijay
Kensch; Oliver
Pellangahr; Klaus
Leuthner; Birgitta
Kettling; Ulrich
Koltermann; Andre |
Degerby
Casnate
Cologne
Cologne
Cologne
Cologne
Koln |
|
FI
IT
DE
DE
DE
DE
DE |
|
|
Assignee: |
DUPONT NUTRITION BIOSCIENCES
APS
COPENHAGEN K
DK
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20120301578 A1 |
November 29, 2012 |
|
|
Family ID: |
33428071 |
Appl. No.: |
13/232588 |
Filed: |
September 14, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
11696162 |
Mar 27, 2012 |
8143045 |
|
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13232588 |
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PCTIB2005003660 |
Oct 4, 2005 |
|
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11696162 |
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Current U.S.
Class: |
426/61 ; 435/196;
435/252.1; 435/252.31; 435/252.33; 435/254.2; 435/254.21;
435/320.1; 536/23.2 |
Current CPC
Class: |
C12N 9/16 20130101 |
Class at
Publication: |
426/61 ; 435/196;
536/23.2; 435/320.1; 435/252.31; 435/252.33; 435/254.21; 435/254.2;
435/252.1 |
International
Class: |
C12N 9/16 20060101
C12N009/16; C12N 15/63 20060101 C12N015/63; A23K 1/165 20060101
A23K001/165; C12N 1/19 20060101 C12N001/19; C12N 1/20 20060101
C12N001/20; A23L 1/30 20060101 A23L001/30; C12N 15/55 20060101
C12N015/55; C12N 1/21 20060101 C12N001/21 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 4, 2004 |
GB |
0422052.1 |
Feb 15, 2005 |
IB |
PCTIB2005000598 |
Oct 4, 2005 |
IB |
PCTIB2005003660 |
Claims
1. An isolated polypeptide comprising the amino acid sequence
corresponding to Citrobacter freundii phytase or a homologue, a
modified form, a functional equivalent or an effective fragment
thereof.
2. An isolated polypeptide as claimed in claim 1 comprising the
amino acid sequence as shown in SEQ ID NO: 3 or a sequence having
at least 75% identity (homology) thereto or a functional fragment
thereof.
3. An isolated polypeptide having the amino acid sequence as set
out in SEQ ID NO: 3 or a sequence having at least 75% identity
(homology) thereto or a functional fragment thereof.
4. A phytase characterised in that it is derived from Citrobacter
freundii strain P3-42 deposited under accession number NCIMB
41247.
5. A phytase or functional equivalent thereof characterised in that
said phytase has a specific activity of at least 1100 U/mg wherein
said specific activity is determined by incubating said phytase in
a solution containing 2 mM phytate, 0.8 mM CaCl.sub.2 in 200 mM
sodium acetate buffer at pH 3.5.
6. A phytase or functional equivalent thereof characterised in that
said phytase has two activity maxima around pH 3 and pH 4-4.5
wherein said activity is determined by incubating said phytase in a
solution containing 2 mM phytate, 0.8 mM CaCl.sub.2 in 200 mM
sodium acetate buffer.
7. An isolated polypeptide or phytase as claimed claim 1 that
comprises one or more mutations at the following positions
(numbering according to the numbering in SEQ ID No. 3): 22, 23, 24,
28, 46, 53, 57, 67, 74, 75, 77, 78, 79, 82, 88, 95, 96, 97, 98,
101, 102, 103, 105, 109, 112, 122, 126, 136, 140, 142, 143, 148,
151, 152, 154, 156, 160, 161, 164, 168, 170, 176, 177, 195, 199,
203, 204, 205, 206, 207, 215, 224, 225, 229, 233, 235, 274, 279,
288, 301, 307, 308, 322, 343, 358, 360, 362, 365, 366, 367, 370,
383, 384, 385, 386, 391, 393, 395, 397, 408, 414.
8. An isolated polypeptide or phytase as claimed in claim 7 wherein
said phytase comprises one or more of the following mutations:
A22T, E23K, E23Q, E24D, M28L, K46E, K46R, D53K, D53N, D57Y, G67R,
G74R, E75K, E75V, V77I, S78T, E79V, Q82H, Q82K, Q82R, F88Y, N95D,
N95P, N96P, N96S, N96Y, Q97T, T98G, T98P, S101F, P102L, G103E,
V105I, A109T, D112V, D112Y, F122Y, L126I, Y136N, E140V, K142R,
T143I, T143P, N148D, K151G, M152K, M152V, T154I, S156T, L160F,
K161N, N164D, E168D, A170T, L176Q, L176V, Y177F, S195T, T199I,
T203I, T203L, T203S, T203W, E204A, E204G, E204H, E204I, E204N,
E204R, E204V, K205P, K205R, S206R, S206T, T207A, T207S, L215F,
D224H, N225D, N225E, P229S, S233C, S235A, Q274H, Q274L, Q279E,
R288M, L301S, E307Y, N308D, N308T, A322V, G343A, K358R, K360N,
T362A, T362I, N365D, T366S, D367N, Q370H, D383V, I384F, I384L,
I384M, Q385R, P386Q, K391N, A393P, K395T, D397N, S408I, L414I.
9. An isolated polypeptide or phytase as claimed in claim 7
comprising one mutation selected from the group consisting of:
P229S; D112V; Q82R; Q274H; D112Y; F88Y; K46E; S233C; R288M; I384L;
Q385R; Q274L; E307Y; T199I; Q82K and T203I.
10. An isolated polypeptide or phytase as claimed in claim 7
comprising a combination of mutations selected from the group
consisting of: K46E/Q82H; Q82K/V105I; N148D/T362I; K46E/L414I;
F88Y/Y136N; T154I/P386Q; N95P/N96S; N95P/N96P; Q97T/T98G;
D224H/N225E; Y177F/T199I; Q274L/Q370H; K46E/N96Y; N148D/L301S;
E24D/R288M; E140V/A322V; K46E/S195T; E75K/N365D; T98P/S235A;
L160F/L215F; Q274L/K395T; G67R/Q279E/N308T; K161N/P229S/R288M;
D53N/D57Y/M152V; F122Y/S156T/P229S; T199I/S206R/T207S;
E23K/K46E/Q82H; K46E/Q82H/Q385R; T203W/E204N/K205R;
T203W/E204H/K205R; T203W/E204R/K205R; T203W/E204A/K205R;
A22T/K151G/N308D; E23K/E75K/F88Y; M152K/N225D/L301S;
S78T/Q274L/S408I; L176Q/T199I/T366S; K46E/V77I/T203S;
K46R/T199I/D367N; G74R/E204G/R288M; A22T/T199I/S206T/T207A;
Q82R/F88Y/L126I/I384L; K46E/Q82H/E168D/Q274L;
Q82K/T154I/Q279E/N308T; Q82R/D112V/Q274H/T362A;
E24D/E79V/N95D/K360N; E23K/M28L/A109T/T143P/I384L;
D53N/D57Y/T199I/P229S/R288M; K46E/Q82H/N148D/T154I/T362I;
D53N/D57Y/P229S/R288M/K358R; D53N/D57Y/T154I/P229S/R288M;
Y136N/T199I/T203L/E204I/K205P; E23Q/S101F/Q274L/I384M/K391N;
K46E/Q82H/N95D/D112V/K142R/D383V;
D53N/D57Y/M152V/P229S/R288M/A393P;
D53K/D57Y/M152V/P229S/R288M/A393P;
D53N/D57Y/F88Y/M152V/P229S/Q279E/N308T;
D53N/D57Y/M152V/E204V/P229S/R288M/A393P;
D53N/D57Y/M152V/T154I/P229S/R288M/A393P;
D53N/D57Y/Q82H/G103E/M152V/P229S/R288M/A393P;
K46E/D53N/D57Y/T143I/M152V/L176V/P229S/R288M/A393P;
Q82K/F88Y/N96P/Q97T/T98G/V105I/Q274H/Q279E/A393P;
Q82R/F88Y/N95P/N96P/Q97T/Q279E/I384L/P386Q/A393P;
H18Q/D53N/D57Y/E75V/M152V/A170T/P229S/R288M/Q385R/A393P;
Q82K/F88Y/N96P/T98G/Y136N/M152V/Y177F/T362I/I384F/A393P/D397N;
D53N/D57Y/F88Y/N95P/N96P/V105I/D112V/Y136N/N148D/N164D/Q274H/T362I/I384L/-
A393P;
D53N/D57Y/Q82K/F88Y/N95P/P102L/V105I/Y136N/N148D/Y177F/Q274H/Q279E/-
T362I/A393P;
D53N/D57Y/Q82K/F88Y/N96P/T98G/V105I/D112V/Y177F/Q274L/G343A/T362I/I384L/A-
393P;
11. An isolated nucleic acid molecule coding for the enzyme of
Citrobacter freundii phytase, or a homologue thereof.
12. An isolated nucleic acid molecule as claimed in claim 11
encoding a polypeptide comprising the amino acid sequence as shown
in SEQ ID NO: 3 or a sequence having at least 75% identity
(homology) thereto or an effective fragment thereof.
13. An isolated nucleic acid molecule comprising a nucleotide
sequence that is the same as, or is complementary to, or contains
any suitable codon substitutions for any of those of SEQ ID NO: 2
or comprises a sequence which has at least 75%, 80%, 85%, 90%, 95%
or 99% sequence homology with SEQ ID NO: 2.
14. An isolated nucleic acid molecule coding for the isolated
polypeptide or phytase as claimed in claim 1.
15. An isolated nucleic acid molecule comprising the sequence as
set out in SEQ ID NO: 2.
16. A plasmid or vector system comprising an isolated polypeptide
or phytase as claimed in claim 1 or a homologue or derivative
thereof.
17. A plasmid or vector system as claimed in claim 16 which
comprises a nucleic acid sequence as claimed in claim 11.
18. A plasmid or vector system as claimed in claim 16 which
comprises a nucleic acid sequence as set out in SEQ ID No: 2 or a
sequence that is at least 75% homologous thereto or an effective
fragment thereof.
19. A plasmid or vector system as claimed in claim 16 wherein said
plasmid or vector system is an expression vector for the expression
of the respective phytase enzyme or homologue, modified form,
functional equivalent or effective fragment thereof, in a host cell
or a microorganism.
20. A host cell transformed or transfected with a plasmid or vector
system as claimed in claim 16.
21. A host cell as claimed in claim 20 which comprises a phytase
which comprises an amino acid sequence, or functional fragment
thereof, as set out in SEQ ID NO: 3, or a sequence that is at least
75% homologous thereto, or a variant thereof according to claims
7.
22. A host cell as claimed in claim 20 wherein said host cell is
derived from a microorganism including bacteria, such as B.
subtilis, E. coli and fungi, including yeast such as H. polymorpha,
S. pombe, S. cerevisiae.
23. A host cell as claimed in claim 22 wherein said microorganism
is a prokaryotic bacterial cell and, preferably, E. coli.
24. A bacterial cell strain Citrobacter freundii P3-42 deposited
under accession number NCIMB 41247.
25. A method of producing a phytase comprising expressing an amino
acid sequence as set out in SEQ ID NO: 3, or a sequence having at
least 75% homology thereto or a modified form or a variant or an
effective fragment thereof in a host cell and separating the
phytase from the host cell culture medium.
26. A food or animal feed composition comprising a phytase as
claimed in claim 1.
27. Use of a phytase as claimed in claim 1 in food or animal
feed.
28. A method for production of food or animal feed comprising a
step of spraying a phytase as claimed in claim 1 in liquid form
onto said food or animal feed.
29. A method for production of food or animal feed comprising a
step of mixing the phytase as claimed in claim 1 as a dry product
with said food or animal feed.
30. A method of preparing a phytase enzyme variant, which method
comprises: a) selecting a parent phytase enzyme, wherein the parent
phytase enzyme is selected from: i. a parent phytase enzyme with at
least 75% homology to SEQ ID NO 3 ii. a parent phytase enzyme
derived from Citrobacter spp. b) making at least one alteration
which is an insertion, a deletion or a substitution of an amino
acid residue in the parent phytase enzyme to obtain a phytase
enzyme variant c) screening for a phytase enzyme variant which
compared to the parent phytase enzyme has: i. higher thermal
stability and/or ii. specific activity and/or iii. proteolytic
stability d) preparing the phytase enzyme variant.
31. A method of preparing a phytase enzyme variant, which method
comprises: a) subjecting DNA sequence encoding a parent phytase
enzyme to mutagenesis, wherein the parent phytase is selected from
i. a parent phytase enzyme with at least 75% homology to SEQ ID NO
3 ii. a parent phytase enzyme derived from Citrobacter spp. b)
expressing the mutated DNA sequence obtained in strep (A) in a host
cell, and c) screening for host cells expressing a phytase enzyme
variant which compared to the parent phytase enzyme has: i. higher
thermal stability and/or ii. higher specific activity and/or iii.
higher proteolytic stability d) preparing the phytase enzyme
variant expressed by the host cell.
Description
REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 11/696,162, filed Apr. 3, 2007 which is a continuation-in-part
of International Patent Application PCT/IB2005/003660 filed Oct. 4,
2005 which published as WO 2006/038128 on Apr. 13, 2006, and which
claims priority to International Patent Application
PCT/IB2005/000598 filed Feb. 15, 2005, and Great Britain Patent
Application No. 0422052.1 filed Oct. 4, 2004.
[0002] Each of the above referenced applications, and each document
cited in this text ("application cited documents") and each
document cited or referenced in each of the application cited
documents, and any manufacturer's specifications or instructions
for any products mentions in this text and in any document
incorporated into this text, are hereby incorporated herein by
reference; and, technology in each of the documents incorporated
herein by reference can be used in the practice of this
invention.
[0003] It is noted that in this disclosure, terms such as
"comprises", "comprised", "comprising", "contains", "containing"
and the like can have the meaning attributed to them in U.S. Patent
law; e.g., they can mean "includes", "included", "including" and
the like. Terms such as "consisting essentially of" and "consists
essentially of" have the meaning attributed to them in U.S. Patent
law, e.g., they allow for the inclusion of additional ingredients
or steps that do not detract from the novel or basic
characteristics of the invention, i.e., they exclude additional
unrecited ingredients or steps that detract from novel or basic
characteristics of the inventions, and they exclude ingredients or
steps for the prior art, such as documents in the art that are
cited herein or are incorporated by reference herein, especially as
it is a goal of this document to define embodiments that are
patentable, e.g., novel, nonobvious, inventive, over the prior art,
e.g., over documents cited herein or incorporated by reference
herein. And, the terms "consists of" and "consisting of" have the
meaning ascribed to them in U.S. Patent law, namely, that these
terms are closed ended.
[0004] The present invention relates to phytases, nucleotide
sequences for same, methods of production of phytases and their
use.
FIELD OF THE INVENTION
[0005] The present invention relates to the field of enzymes for
additives to feedstuffs. More specifically, the present invention
relates to phytases which can be used for enhancing phosphate
digestion in foods and animal feeds.
TECHNICAL BACKGROUND AND PRIOR ART
[0006] Phytate is the major storage form of phosphorus in cereals
and legumes. However, monogastric animals such as pig, poultry and
fish are not able to metabolise or absorb phytate (or phytic acid)
and therefore it is excreted leading to phosphorous pollution in
areas of intense livestock production. Moreover phytic acid also
acts as an antinutritional agent in monogastric animals by
chelating metal agents such as calcium, copper and zinc.
[0007] In order to provide sufficient phosphates for growth and
health of these animals, inorganic phosphate is added to their
diets. Such addition can be costly and further increases pollution
problems.
[0008] Phytate is converted by phytases which generally catalyse
the hydrolysis of phytate to lower inositol-phosphates and
inorganic phosphate. Phytases are useful as additives to animal
feeds where they improve the availability of organic phosphorus to
the animal and decrease phosphate pollution of the environment
(Wodzinski R J, Ullah A H. Adv Appl Microbiol. 42, 263-302
(1996)).
[0009] A number of phytases of fungal (Wyss M. et al. Appl.
Environ. Microbiol. 65 (2), 367-373 (1999); Berka R. M. et al.
Appl. Environ. Microbiol. 64 (11), 4423-4427 (1998); Lassen S. et
al. Appl. Environ. Microbiol. 67 (10), 4701-4707 (2001)) and
bacterial (Greiner R. et al Arch. Biochem. Biophys. 303 (1),
107-113 (1993); Kerovuo et al. Appl. Environ. Microbiol. 64 (6),
2079-2085 (1998); Kim H. W. et al. Biotechnol. Lett. 25, 1231-1234
(2003); Greiner R. et al. Arch. Biochem. Biophys. 341 (2), 201-206
(1997); Yoon S. J. et al. Enzyme and microbial technol. 18, 449-454
(1996); Zinin N. V. et al. FEMS Microbiol. Lett. 236, 283-290
(2004))) origin have been described in the literature.
[0010] However, to date, none of these phytases display the
properties required for effective use as an animal feed supplement.
In particular, fungal phytases tend to be proteolytically unstable
(Igbasan F. A. et al. Arch. Anim. Nutr. 53, 353-373 (2000)) and
therefore susceptible to degradation, while most bacterial phytases
have a narrow substrate specificity for phytate alone and degrade
poorly inositol phosphates of intermediate degrees of
phosphorylation (Greiner R. et al., Arch. Biochem. Biophys. 303
(1), 107-113 (1993); Kerovuo J et al. Biochem. J. 352, 623-628
(2000)).
[0011] Accordingly, there is a need for improved phytases.
SUMMARY OF THE INVENTION
[0012] In a broad aspect, the present invention relates to phytases
derived from a bacterium and modified forms thereof. In particular
the invention relates to wild type phytases derived from the
bacterium, Citrobacter freundii, and variant/modified forms thereof
showing improved characteristics compared to the wild-type
enzyme.
[0013] The present invention is advantageous as it provides for
novel phytases that have properties making them particularly useful
and efficient as feed enzymes. In particular the invention relates
to isolated and/or purified novel phytase polypeptide as described
herein or functional fragments or variants or modified forms
thereof. The invention also provides the nucleic acid and amino
acid sequences encoding said phytases.
[0014] To be efficient as an enzyme additive to food or animal
feed, a phytase has to combine a number of different properties. In
order to be able to degrade phytic acid in the acidic environment
of an animal's stomach it has to be active at low pH, preferably
over a broad range of pH values. In addition, it has to have high
specific activity and preferably high thermostability to enable the
protein to withstand high temperatures commonly used in preparation
of feedstuffs such as feed pellets.
[0015] It is also important that the enzyme has broad substrate
specificity allowing it to hydrolyse not only phytate but also
intermediate products of phytate degradation such as inositol
pentaphosphates, tetraphosphates and triphosphates. Studies on
phytate degradation in pigs show that these inositol
oligophosphates otherwise remain largely insoluble in the small and
large intestine and thus inaccessible to alkaline phosphatases
produced by the animal and gut microflora (Schlemmer U. et al.
Arch. Anim. Nutr. 55, 255-280 (2001)). Variations in substrate
specificity profiles of different enzymes have been identified. For
example, inositol-triphosphates generated by the phytase from B.
subtilis are essentially resistant to further hydrolysis by this
enzyme (Kerovuo J. et al. Biochem J. (200) 352, 623-628).
[0016] In another aspect of the invention there is provided a
plasmid or a vector system or a transformed or a transgenic
organism comprising a novel phytase as described herein or a
modified form thereof.
[0017] In another aspect the present invention relates to
transgenic organisms modified to express a novel phytase as
described herein or a modified form thereof and therefore being
capable of producing a phytase. The present invention further
provides means and methods for the biotechnological production of
phytases and their use as feed supplements.
[0018] Aspects of the present invention are presented in the claims
and in the following commentary.
[0019] For ease of reference, these and further aspects of the
present invention are now discussed under appropriate section
headings. However, the teachings under each section are not
necessarily limited to each particular section.
[0020] As used with reference to the present invention, the terms
"produce", "producing", "produced", "produceable", "production" are
synonymous with the respective terms "prepare", "preparing",
"prepared", "preparation", "generated", "generation" and
"preparable".
[0021] As used with reference to the present invention, the terms
"expression", "expresses", "expressed" and "expressable" are
synonymous with the respective terms "transcription",
"transcribes", "transcribed" and "transcribable".
[0022] As used with reference to the present invention, the terms
"transformation" and "transfection" refer to a method of
introducing nucleic acid sequences into hosts, host cells, tissues
or organs.
[0023] Other aspects concerning the nucleotide sequences which can
be used in the present invention include: a construct comprising
the sequences of the present invention; a vector comprising the
sequences for use in the present invention; a plasmid comprising
the sequences for use in the present invention; a transformed cell
comprising the sequences for use in the present invention; a
transformed tissue comprising the sequences for use in the present
invention; a transformed organ comprising the sequences for use in
the present invention; a transformed host comprising the sequences
for use in the present invention; a transformed organism comprising
the sequences for use in the present invention. The present
invention also encompasses methods of expressing the nucleotide
sequence for use in the present invention using the same, such as
expression in a host cell; including methods for transferring same.
The present invention further encompasses methods of isolating the
nucleotide sequence, such as isolating from a host cell.
[0024] Other aspects concerning the amino acid sequences for use in
the present invention include: a construct encoding the amino acid
sequences for use in the present invention; a vector encoding the
amino acid sequences for use in the present invention; a plasmid
encoding the amino acid sequences for use in the present invention;
a transformed cell expressing the amino acid sequences for use in
the present invention; a transformed tissue expressing the amino
acid sequences for use in the present invention; a transformed
organ expressing the amino acid sequences for use in the present
invention; a transformed host expressing the amino acid sequences
for use in the present invention; a transformed organism expressing
the amino acid sequences for use in the present invention. The
present invention also encompasses methods of purifying the amino
acid sequences for use in the present invention using the same,
such as expression in a host cell; including methods of
transferring same, and then purifying said sequence.
BRIEF DESCRIPTION OF THE DRAWINGS
[0025] FIG. 1 shows SDS PAGE analysis of the recombinant phytase
from C. freundii P3-42 purified by DEAE-Sepharose chromatography.
The figure presents the scanning trace of a digital photographic
image of the lane containing a sample of the C. freundii
phytase.
[0026] FIG. 2 shows pH profile of the phytase from C. freundii
P3-42
[0027] FIG. 3 shows substrate specificity of the purified
recombinant phytase from C. freundii P3-42 with inositol phosphate
fractions of different degree of phosphorylation and model
substrates. Abbreviations: IP6--phytic acid, IP5, IP4 and
IP3--mixtures of isomeric inositol penta-, tetra- and triphosphates
respectively. Fru P2--fructose 1,6-diphosphate, Fru P1--fructose
6-phosphate.
[0028] SEQ ID NO: 1 lists the sequence obtained for identification
of the bacterial strain.
[0029] SEQ ID NO: 2 lists the sequence comprising the phytase gene
from C. freundii P3-42.
[0030] SEQ ID NO: 3 lists the amino acid sequence of the phytase
gene C. freundii P3-42.
[0031] SEQ ID NO:8 lists the amino acid sequence of the phytase
gene C. freundii P3-42 with possible amino acid substitutions
marked.
DETAILED DISCLOSURE OF INVENTION
[0032] The present invention features an enzyme comprising the
amino acid sequence corresponding to Citrobacter freundii phytase
or a modified form, a variant, a functional equivalent or an
effective fragment thereof.
[0033] The term "phytase" means a protein or polypeptide which is
capable of catalysing the hydrolysis of esters of phosphoric acid
including phytate and releasing inorganic phosphate. Phytases are
capable to hydrolyse, in addition to phytate, at least some of the
inositol-phosphates of intermediate degrees of phosphorylation.
[0034] The term "corresponding to Citrobacter freundii phytase"
means that the enzyme need not have been obtained from a source of
Citrobacter freundii. Instead, the enzyme has to have essentially
the same functional characteristics or sequence as that of
Citrobacter freundii phytase.
[0035] The term "functional equivalent thereof" means that the
enzyme has to have essentially the same functional characteristics
as that of wild-type Citrobacter freundii phytase. The term
"modified form" or "variant" means that the enzyme has been
modified from its original form but retains essentially the same
enzymatic functional characteristics as that of wild-type
Citrobacter freundii phytase. In particular, the terms "variant" or
"modified form" encompass phytase enzymes with an amino acid
sequence derived from the amino acid sequence of the
parent/wild-type phytase and having one or more amino acid
substitutions, insertions, and/or deletions, which together are
referred to as mutations. Modified forms or variants may be altered
in the enzyme characteristics compared to the parent enzyme.
Preferably, modified forms or variants have an increased
thermostability, an increased pepsin stability, an increased
specific activity, a broader substrate specificity, or other
modifications that are beneficial for the application of the
enzyme. The term "functional" or "effective" fragment means a
fragment or portion of the Citrobacter freundii phytase that
retains essentially the same enzymatic function or effect.
[0036] Preferably the enzyme of this aspect of the present
invention has the same sequence or a sequence that is at least 75%
identical (homologous) to that of Citrobacter freundii phytase.
[0037] Suitably, the enzyme comprises the amino acid sequence as
shown in SEQ ID NO: 3 or a sequence having at least 75% identity
(homology) thereto or a functional fragment thereof. In a preferred
embodiment, the invention provides an isolated and/or purified
polypeptide having the amino acid sequence as set out in SEQ ID NO:
3 or a sequence having at least 75% identity (homology) thereto or
an effective fragment thereof.
[0038] In another embodiment, the phytase is characterised in that
it is derived from Citrobacter freundii strain P3-42 deposited
under accession number NCIMB 41247 on Sep. 22, 2004, under the
terms of the Budapest Treaty, with the National Collections of
Industrial, Marine and Food Bacteria (NCIMB) in Bucksburn, Aberdeen
AB21 9YA, Scotland, UK. Deposited microorganism(s) will be
irrevocably and without restriction or condition released to the
public during the effective term of any patent issued from this
application.
[0039] In a preferred embodiment, the invention relates to a
phytase in accordance with any embodiment of the first aspect of
the invention that comprises one or more mutations at the following
positions (numbering according to the numbering in SEQ ID No. 3):
22, 23, 24, 28, 46, 53, 57, 67, 74, 75, 77, 78, 79, 82, 88, 95, 96,
97, 98, 101, 102, 103, 105, 109, 112, 122, 126, 136, 140, 142, 143,
148, 151, 152, 154, 156, 160, 161, 164, 168, 170, 176, 177, 195,
199, 203, 204, 205, 206, 207, 215, 224, 225, 229, 233, 235, 274,
279, 288, 301, 307, 308, 322, 343, 358, 360, 362, 365, 366, 367,
370, 383, 384, 385, 386, 391, 393, 395, 397, 408 and 414.
[0040] These positions are characterized in that mutagenesis of the
enzyme at these positions lead to an improvement in the desired
enzyme characteristics.
[0041] Preferred mutations include:
[0042] A22T, E23K, E23Q, E24D, M28L, K46E, K46R, D53K, D53N, D57Y,
G67R, G74R, E75K, E75V, V77I, S78T, E79V, Q82H, Q82K, Q82R, F88Y,
N95D, N95P, N96P, N96S, N96Y, Q97T, T98G, T98P, S101F, P102L,
G103E, V105I, A109T, D112V, D112Y, F122Y, L126I, Y136N, E140V,
K142R, T143I, T143P, N148D, K151G, M152K, M152V, T154I, S156T,
L160F, K161N, N164D, E168D, A170T, L176Q, L176V, Y177F, S195T,
T199I, T203I, T203L, T203S, T203W, E204A, E204G, E204H, E204I,
E204N, E204R, E204V, K205P, K205R, S206R, S206T, T207A, T207S,
L215F, D224H, N225D, N225E, P229S, S233C, S235A, Q274H, Q274L,
Q279E, R288M, L301S, E307Y, N308D, N308T, A322V, G343A, K358R,
K360N, T362A, T362I, N365D, T366S, D367N, Q370H, D383V, I384F,
I384L, I384M, Q385R, P386Q, K391N, A393P, K395T, D397N, S408I and
L414I or conservative mutations at each position.
[0043] By "conservative mutations" is meant mutations to amino acid
residues that are conservative in terms of the amino acid
characteristics compared to the amino acid residue indicated. Amino
acid characteristics include the size of the residue,
hydrophobicity, polarity, charge, pK-value, and other amino acid
characteristics known in the art and also described in more detail
below.
[0044] In a particularly preferred embodiment, the mutations are at
one or more of the following positions:
[0045] 23, 46, 53, 75, 82, 88, 95, 96, 98, 112, 143, 152, 176, 177,
199, 203, 204, 205, 225, 229, 233, 274, 288, 307, 308, 362, 370,
384 and 385
[0046] Preferred mutations at these specific positions include:
[0047] E23K, E23Q, K46E, K46R, D53K, D53N, E75K, E75V, Q82H, Q82K,
Q82R, F88Y, N95D, N95P, N96P, N96S, N96Y, T98G, D112V, D112Y,
T143I, T143P, M152K, M152V, L176Q, L176V, Y177F, T199I, T203I,
T203L, T203S, T203W, E204A, E204G, E204H, E204I, E204N, E204R,
E204V, K205P, K205R, N225D, N225E, P229S, S233C, Q274H, Q274L,
R288M, E307Y, N308D, N308T, T362A, T362I, Q370H, I384F, I384L,
I384M and Q385R or conservative mutations at each position.
[0048] In one embodiment, there is provided a phytase comprising
one mutation selected from the group consisting of:
[0049] P229S; D112V; Q82R; Q274H; D112Y; F88Y; K46E; S233C; R288M;
I384L; Q385R; Q274L; E307Y; T199I; Q82K and T203I.
[0050] In a further preferred embodiment, there is provided a
phytase comprising a combination of mutations selected from the
group consisting of:
[0051] K46E/Q82H; Q82K/V105I; N148D/T362I; K46E/L414I; F88Y/Y136N;
T154I/P386Q; N95P/N96S; N95P/N96P; Q97T/T98G; D224H/N225E;
Y177F/T199I; Q274L/Q370H; K46E/N96Y; N148D/L301S; E24D/R288M;
E140V/A322V; K46E/S195T; E75K/N365D; T98P/S235A; L160F/L215F;
Q274L/K395T; G67R/Q279E/N308T; K161N/P229S/R288M; D53N/D57Y/M152V;
F122Y/S156T/P229S; T199I/S206R/T207S; E23K/K46E/Q82H;
K46E/Q82H/Q385R; T203W/E204N/K205R; T203W/E204H/K205R;
T203W/E204R/K205R; T203W/E204A/K205R; A22T/K151G/N308D;
E23K/E75K/F88Y; M152K/N225D/L301S; S78T/Q274L/S408I;
L176Q/T199I/T366S; K46E/V77I/T203S; K46R/T199I/D367N;
G74R/E204G/R288M; A22T/T199I/S206T/T207A; Q82R/F88Y/L126I/I384L;
K46E/Q82H/E168D/Q274L; Q82K/T154I/Q279E/N308T;
Q82R/D112V/Q274H/T362A; E24D/E79V/N95D/K360N;
E23K/M28L/A109T/T143P/I384L; D53N/D57Y/T199I/P229S/R288M;
K46E/Q82H/N148D/T154I/T362I; D53N/D57Y/P229S/R288M/K358R;
D53N/D57Y/T154I/P229S/R288M; Y136N/T199I/T203L/E204I/K205P;
E23Q/S101F/Q274L/I384M/K391N; K46E/Q82H/N95D/D112V/K142R/D383V;
D53N/D57Y/M152V/P229S/R288M/A393P;
D53K/D57Y/M152V/P229S/R288M/A393P;
D53N/D57Y/F88Y/M152V/P229S/Q279E/N308T;
D53N/D57Y/M152V/E204V/P229S/R288M/A393P;
D53N/D57Y/M152V/T154I/P229S/R288M/A393P;
D53N/D57Y/Q82H/G103E/M152V/P229S/R288M/A393P;
K46E/D53N/D57Y/T143I/M152V/L176V/P229S/R288M/A393P;
Q82K/F88Y/N96P/Q97T/T98G/V105I/Q274H/Q279E/A393P;
Q82R/F88Y/N95P/N96P/Q97T/Q279E/I384L/P386Q/A393P;
H18Q/D53N/D57Y/E75V/M152V/A170T/P229S/R288M/Q385R/A393P;
Q82K/F88Y/N96P/T98G/Y136N/M152V/Y177F/T362I/I384F/A393P/D397N;
[0052]
D53N/D57Y/F88Y/N95P/N96P/V105I/D112V/Y136N/N148D/N164D/Q274H/T362I/-
I384L/A393P;
[0053]
D53N/D57Y/Q82K/F88Y/N95P/P102L/V105I/Y136N/N148D/Y177F/Q274H/Q279E/-
T362I/A393P, and;
[0054]
D53N/D57Y/Q82K/F88Y/N96P/T98G/V105I/D112V/Y177F/Q274L/G343A/T362I/I-
384L/A393P.
[0055] In a yet further preferred embodiment, there is provided a
phytase comprising a combination of mutations selected from the
group consisting of:
D57Y/F88Y/N95P/Q97T/N148D/M152V/T154I/Y177F/Q274H/I384L;
D53N/D57Y/F88Y/N95P/N96P/Q97T/M152V/Y177F/Q274H/Q279E/T362I/I384L;
D53N/Q82K/F88Y/N96P/T98G/V105I/N148D/T154I/Q274H/T362I/I384L/P386Q;
Q82R/F88Y/N96P/T98G/V105I/D112V/Y136N/N148D/T154I/Y177F/P386Q/A393P;
[0056]
D53N/Q82K/F88Y/N95P/N96P/T98G/Y136N/N148D/T154I/I384L/P386Q/A393P;
D57Y/Q82K/F88Y/N96P/Q97T/T98G/V105I/N148D/T154I/Y177F/Q274H/I384L/P386Q/A-
393P;
[0057]
D53N/Q82K/F88Y/N95P/Q97T/T98G/D112V/Y136N/N148D/T154I/Q274H/Q279E/I-
384L/P386Q/A393P
and
[0058]
D53N/D57Y/Q82R/F88Y/N95P/N96P/Q97T/T98G/V105I/Y136N/N148D/M152V/Y17-
7F/I384L/P386Q.
[0059] Accordingly, a preferred phytase in accordance with the
present invention is a variant consisting of the amino acid
sequence listed as SEQ ID NO: 3 and having one or more of the amino
acid mutations listed above or one of the combinations of mutations
listed above.
[0060] In these embodiments, the nomenclature indicates a phytase
comprising the amino acid sequence set out in SEQ ID NO: 3 with the
mutations indicated by reference to the positions of the amino
acids in SEQ ID NO: 3. The nomenclature is described in more detail
below.
[0061] Suitably these variants show improved characteristics with
respect to any one of the following: temperature stability, pH
range, pepsin stability, specific activity, substrate specificity.
Suitable methods for determining these characteristics are
disclosed herein.
[0062] In particular, the improvements in phytase characteristics
are directed to the enzyme stability under food and feed processing
conditions, to the enzyme stability during stomach transit, and to
the enzyme activity and stability in human or animal stomach and/or
intestinal tract making the improved variants particularly suitable
for use as feed supplements. Thus, such improvements comprise among
other parameters the increase in stability at elevated
temperatures, preferably at temperatures above 65.degree. C., the
increase in stability against proteolytic digestion, preferably
protease of the digestive tract, the increase in catalytic activity
at low pH, preferably catalytic activity below pH 5.5, and the
general efficiency of releasing phosphate groups from phytate.
[0063] Suitably, in one embodiment, the phytase or functional
equivalent of the present invention is characterised in that said
phytase has a specific activity of 1000 U/mg or higher wherein said
specific activity is determined by incubating said phytase in a
solution containing 2 mM phytate, 0.8 mM CaCl.sub.2 in 200 mM
sodium acetate buffer at pH 3.5. In another embodiment, the phytase
of the present invention or functional equivalent thereof may also
suitably be characterised in that said phytase has two activity
maxima around pH 3 and pH 4-4.5 wherein said activity is determined
by incubating said phytase in a solution containing 2 mM phytate,
0.8 mM CaCl.sub.2 in 200 mM sodium acetate buffer.
[0064] In a further embodiment the invention provides a method of
preparing a phytase enzyme variant, which method comprises:
a) Selecting a parent phytase enzyme, wherein the parent phytase
enzyme is selected from i. a parent phytase enzyme with at least
75% homology to SEQ ID No 3 ii. a parent phytase enzyme derived
from Citrobacter spp. b) Making at least one alteration which is an
insertion, a deletion or a substitution of an amino acid residue in
the parent phytase enzyme to obtain a phytase enzyme variant c)
Screening for a phytase enzyme variant which compared to the parent
phytase enzyme has: i. higher thermal stability and/or ii. specific
activity and/or iii. proteolytic stability and/or d) Preparing the
phytase enzyme variant
[0065] In a further embodiment the invention provides a method of
preparing a phytase enzyme variant, which method comprises:
a) Subjecting DNA sequence encoding a parent phytase enzyme to
mutagenesis, wherein the parent phytase enzyme is selected from i.
a parent phytase enzyme with at least 75% homology to SEQ ID No 3
ii. a parent phytase enzyme derived from Citrobacter spp. b)
Expressing the mutated DNA sequence obtained in step (A) in a host
cell, and c) Screening for host cells expressing a for a phytase
enzyme variant which compared to the parent phytase enzyme has: iv.
higher thermal stability and/or v. higher specific activity* and/or
vi. higher proteolytic stability and/or
Preparing the Phytase Enzyme Variant Expressed by the Host Cell
[0066] In the above embodiments of the invention, which relate to
methods of preparing phytase enzyme variant the phytase enzyme
variant is preferably screened for higher thermal stability.
[0067] In the above embodiments of the invention, which relate to
methods of preparing phytase enzyme variant the phytase enzyme
variant is preferably screened for higher thermal stability and
higher proteolytic stability.
[0068] In the above embodiments of the invention, which relate to
methods of preparing phytase enzyme variant the phytase enzyme
variant is preferably screened for higher thermal stability and
higher proteolytic stability and higher specific activity.
[0069] The parent phytase enzyme is preferably derived from
Citrobacter freundii, more preferably Citrobacter freundii
P3-42.
[0070] In methods of preparing a phytase enzyme variant, which
method comprises subjecting DNA sequence encoding a parent phytase
enzyme to mutagenesis, the DNA sequence encoding a parent phytase
enzyme is preferably subjected to random mutagenesis, more
preferably error prone PCR, even more preferably error threshold
PCR.
[0071] The preferred methods of mutagenesis of DNA sequence
encoding a parent phytase enzyme is error prone PCR, more
preferably error threshold PCR, other methods of mutagenesis may be
used either in place of error prone/threshold PCR or in conjunction
with error prone/threshold PCR. See Example 12 which provides
references for suitable error prone PCR and error threshold PCR
methods. Other methods are disclosed under the
[0072] The term `expression in a host cell` when used in the
context of the embodiments which refer to `a method of preparing a
phytase enzyme variant` is preferably defined as production of the
phytase enzyme variant in a living organism, organ or cell as
herein defined. However, it is considered that for the purpose of
selection the phytase enzyme variants may also be produced via in
vitro methods which utilise the transcription and translation
machinery isolated from of one or more cells isolated from one or
more living organism. Such in vitro production of variant phytases
on the invention can also be used for selecting preferred variant
phytases. In vitro expression can suitable performed using standard
techniques. For reference please see In vitro Expression Guide'
available from Promega Inc (Part# BR053).
DEFINITIONS OF VARIANT PHENOTYPES
[0073] Variants with higher thermal stability (thermal stability
difference) is preferably determined using the methods disclosed in
Example 12.
[0074] The variant phytase enzyme prepared by the method of
preparing phytase enzyme variants preferably has a thermal
stability difference of at least 1.5, more preferably 2, 2.5, 3,
3.5, 4, 5, 6, 7, 8, 9, most preferably at least 10.
[0075] Variants with higher proteolytic stability is preferably
determined by the methods disclosed in Example 12.
[0076] Preferably the phytase enzyme variant of the invention has a
proteolytic stability of at least 45%, preferably 50%, 55%, more
preferably at least 60%.
Further Variant Embodiments
[0077] In a further embodiment the invention provides methods for
the preparation of an animal feed comprising a phytase enzyme
variant.
a) Selecting a parent phytase enzyme, wherein the parent phytase
enzyme is selected from i. a parent phytase enzyme with at least
75% homology to SEQ ID No 3 ii. a parent phytase enzyme derived
from Citrobacter spp. b) Making at least one alteration which is an
insertion, a deletion or a substitution of an amino acid residue in
the parent phytase enzyme to obtain a phytase enzyme variant c)
Screening for a phytase enzyme variant which compared to the parent
phytase enzyme has: i. higher thermal stability and/or ii. specific
activity and/or iii. proteolytic stability and/or d) Preparing the
phytase enzyme variant e) Adding the prepared phytase enzyme
variant to an animal feed.
[0078] In a further embodiment the invention provides methods for
the preparation of an animal feed comprising a phytase enzyme
variant.
a) Subjecting DNA sequence encoding a parent phytase enzyme to
mutagenesis, wherein the parent phytase enzyme is selected from
iii. a parent phytase enzyme with at least 75% homology to SEQ ID
No 3 iv. a parent phytase enzyme derived from Citrobacter spp. b)
Expressing the mutated DNA sequence obtained in step (A) in a host
cell, and c) Screening for host cells expressing a for a phytase
enzyme variant which compared to the parent phytase enzyme has:
vii. higher thermal stability and/or viii. higher specific
activity* and/or ix. higher proteolytic stability and/or d)
Preparing the phytase enzyme variant expressed by the host cell f)
Adding the prepared phytase enzyme variant to an animal feed.
[0079] The preferred aspects of the method of preparing a phytase
enzyme variant also apply to the above methods of preparing an
animal feed comprising a phytase enzyme variant.
[0080] In another aspect, the invention provides an isolated and/or
purified nucleic acid molecule or nucleotide sequence coding for
the enzyme comprising the amino acid sequence corresponding to
Citrobacter freundii phytase, or a homologue thereof. Suitably said
isolated and/or purified nucleic acid molecule encodes a
polypeptide comprising the amino acid sequence as shown in SEQ ID
NO: 3 or a sequence having at least 75% identity (homology) thereto
or an effective fragment thereof. In one embodiment, the nucleic
acid molecule encodes a polypeptide comprising SEQ ID NO:3 and
including mutations at the preferred positions listed herein or any
of the specific mutations or combinations of mutations listed
herein. In another embodiment, the invention provides an isolated
and/or purified nucleic acid molecule comprising a nucleotide
sequence that is the same as, or is complementary to, or contains
any suitable codon substitutions for any of those of SEQ ID NO: 2
or comprises a sequence which has at least 75%, 80%, 85%, 90%, 95%,
96%, 97%, 98% or 99% sequence homology with SEQ ID NO: 2.
[0081] In a yet further aspect, the invention relates to a
nucleotide sequence and to the use of a nucleotide sequence shown
as:
(a) the nucleotide sequence presented as SEQ ID No. 2, (b) a
nucleotide sequence that is a variant, homologue, derivative or
fragment of the nucleotide sequence presented as SEQ ID No. 2; (c)
a nucleotide sequence that is the complement of the nucleotide
sequence set out in SEQ ID No. 2; (d) a nucleotide sequence that is
the complement of a variant, homologue, derivative or fragment of
the nucleotide sequence presented as SEQ ID No 2; (e) a nucleotide
sequence that is capable of hybridising to the nucleotide sequence
set out in SEQ ID No. 2; (f) a nucleotide sequence that is capable
of hybridising to a variant, homologue, derivative or fragment of
the nucleotide sequence presented as SEQ ID No. 2; (g) a nucleotide
sequence that is the complement of a nucleotide sequence that is
capable of hybridising to the nucleotide sequence set out in SEQ ID
No. 2; (h) a nucleotide sequence that is the complement of a
nucleotide sequence that is capable of hybridising to a variant,
homologue, derivative or fragment of the nucleotide sequence
presented as SEQ ID No. 2; (i) a nucleotide sequence that is
capable of hybridising to the complement of the nucleotide sequence
set out in SEQ ID No. 2; (j) a nucleotide sequence that is capable
of hybridising to the complement of a variant, homologue,
derivative or fragment of the nucleotide sequence presented as SEQ
ID No. 2.
[0082] The nucleotide sequence of the present invention may
comprise sequences that encode for SEQ ID No. 3 or a variant,
modified form, homologue or derivative thereof.
[0083] In particular, the invention provides a plasmid or vector
system comprising a phytase as described herein or a homologue or
derivative thereof. Preferably, the plasmid or vector system
comprises a nucleic acid sequence as set out in SEQ ID No: 2 or a
sequence that is at least 75% homologous thereto or an effective
fragment thereof. Suitably the plasmid or vector system is an
expression vector for the expression of any of the enzymes encoded
by a nucleic acid sequence as set out in any of SEQ ID No: 2 or a
sequence that is at least 75% homologous (identical) thereto in a
microorganism. In addition, the invention provides a plasmid or
vector system for expression of any of the modified enzymes or
variants described herein. Suitable expression vectors are
described herein.
[0084] In another aspect of the invention there is provided a host
cell transformed or 575 transfected with a nucleic acid encoding a
phytase as described herein.
[0085] Suitably, the host cell in accordance with this aspect of
the invention comprises a phytase which comprises an amino acid
sequence, or functional fragment thereof, as set out in SEQ ID NO:
3 or a sequence that is at least 75% homologous thereto.
[0086] In a preferred embodiment, said host cell produces a
phytase.
[0087] In a further aspect of the invention there is provided a
host cell transformed or transfected with a nucleic acid encoding a
phytase in accordance with the invention. Preferably, the phytase
is a Citrobacter freundii phytase as described herein or a
homologue or derivative thereof. Suitably, said phytase enzyme
comprises an amino acid sequence, or functional fragment thereof,
as set out in any of SEQ ID No: 3 or a sequence that is at least
75% homologous (identical) thereto. Preferably, said host cell
produces a phytase.
[0088] In one embodiment, the nucleotide sequence which can be used
in the present invention is obtainable from (though it does not
have to be actually obtained from) Citrobacter freundii, although
it will be recognised that enzymes isolated and/or purified from
equivalent strains may equally be used.
[0089] Suitably the host cell is derived from a microorganism
including bacteria and fungi, including yeast. In a particularly
preferred embodiment the host cell is a prokaryotic bacterial cell.
Suitable bacterial host cells include bacteria from different
prokaryotic taxonomic groups including proteobacteria, including
members of the alpha, beta, gamma, delta and epsilon subdivision,
gram-positive bacteria such as Actinomycetes, Firmicutes,
Clostridium and relatives, flavobacteria, cyanobacteria, green
sulfur bacteria, green non-sulfur bacteria, and archaea.
Particularly preferred are the Enterobacteriaceae such as
Escherichia coli proteobacteria belonging to the gamma subdivision
and low GC-Gram positive bacteria such as Bacillus.
[0090] Suitable fungal host cells include yeast selected from the
group consisting of Ascomycota including Saccharomycetes such as
Pichia, Hansenula, and Saccharomyces, Schizosaccharmycetes such as
Schizosaccharomyces pombe and anamorphic Ascomycota including
Aspergillus.
[0091] Other suitable eukaryotic host cells include insect cells
such as SF9, SF21, Trychplusiani and M121 cells. For example, the
polypeptides according to the invention can advantageously be
expressed in insect cell systems. As well as expression in insect
cells in culture, phytase genes can be expressed in whole insect
organisms. Virus vectors such as baculovirus allow infection of
entire insects. Large insects, such as silk moths, provide a high
yield of heterologous protein. The protein can be extracted from
the insects according to conventional extraction techniques.
Expression vectors suitable for use in the invention include all
vectors which are capable of expressing foreign proteins in insect
cell lines.
[0092] Other host cells include plant cells selected from the group
consisting of protoplasts, cells, calli, tissues, organs, seeds,
embryos, ovules, zygotes, etc. The invention also provides whole
plants that have been transformed and comprise the recombinant DNA
of the invention.
[0093] The term "plant" generally includes eukaryotic alga,
embryophytes including Bryophyta, Pteridophyta and Spermatophyta
such as Gymnospermae and Angiospermae.
[0094] Preferably, said host cell is a microorganism. Preferred
microorganisms include prokaryotic bacterial cells preferably, E.
coli, B. subtilis and other species of the genus Bacillus, yeast,
preferably, Hansenula polymorpha and Schizosaccharomyces pombe.
[0095] In another aspect of the invention there is provided a
bacterial cell strain Citrobacter freundii P3-42 deposited by
Danisco Global Innovation, Sokeritehtaantie 20, FIN-02460 Kantvik,
Finland under accession number NCIMB 41247. Such a cell can be
incorporated directly into feed.
[0096] In another aspect, there is provided a method for the
production of phytases comprising transfecting a host cell with an
expression vector or plasmid in accordance with the invention,
culturing said host cell under conditions for the expression of the
phytase and extracting said phytase from the host cell culture
media.
[0097] Suitably said method is for the production of a phytase
comprising expressing an amino acid sequence as set out in SEQ ID
NO: 3 or a sequence having at least 75% homology thereto or an
effective fragment thereof in a host cell and extracting the
secreted protein from the host cell culture medium.
[0098] Another aspect of the invention provides a feed composition
comprising a phytase in accordance with the invention. Preferably,
the feed composition comprises a phytase at a concentration of
10-10000 U/kg feed, preferably, 200-2000 U/kg feed, more
preferably, 500-1000 U/kg feed.
[0099] In one embodiment, the feed composition comprises a host
cell in accordance with the invention.
[0100] In a further aspect there is provided the use of a phytase
in accordance with the invention in food or animal feed.
Preferable Aspects
[0101] Preferable aspects are presented in the accompanying claims
and in the following description and Examples section.
Additional Advantages
[0102] The present invention is advantageous as it provides
phytases that have a number of properties that make them
particularly useful as additives to animal feeds.
[0103] In particular, the phytases of the present invention are
active at low pH and, preferably in the range pH 2 to 5.5 with
activity maxima around pH 3 and 4.5. Suitably the phytases of the
present invention are active at low pHs of the stomach
environment.
[0104] Furthermore, the phytases of the present invention are
efficiently secreted both in the native host and during
heterologous expression thus leading to more efficient production
and isolation for addition to feed.
[0105] Moreover, the phytases of the present invention have a broad
substrate specificity including penta- tetra, tri and di-phosphate
substrates thereby increasing the total available phosphate to the
animal. The phytases of the present invention also have a high
specific activity in the region of 1000 U/mg+/-approximately
10%.
[0106] The products of the present invention may be used as
additives/supplements to foods and feed. The products may also be
useful in the commercial production of various
inositol-phosphates.
Phytate/Phytic Acid/Phytases
[0107] Phytic acid (myo-inositol hexakisphosphate) is an important
constituent in cereals, legumes and oilseed crops. The salt form,
phytate, is the major storage form of phosphorous in these
plants.
[0108] Phytases catalyse phosphate monoester hydrolysis of phytic
acid which results in the step-wise formation of myo-inositol
pentakis-, tetrakis-, tris-, bis- and monophosphates, as well as
the liberation of inorganic phosphate.
[0109] The terms "wild type phytase" or "wild type" as used herein
refer to a phytase enzyme with an amino acid sequence found in
nature.
[0110] The terms "phytase variant" or "variant" or "modified form"
refer to a phytase enzyme with an amino acid sequence derived from
the amino acid sequence of a parent phytase having one or more
amino acid substitutions, insertions, and/or deletions, which
together are referred to as "mutations".
[0111] The terms "parent phytase" or "parent enzyme" refer to a
phytase enzyme from which a phytase variant is derived. A parent
phytase can be a wild type phytase or another phytase variant. In
particular, in the present invention, a "parent phytase" may be
derived from a Citrobacter freundii. Suitably, the "parent phytase"
is derived from Citrobacter freundii strain P3-42 as described
herein which, preferably has the amino acid sequence set out in SEQ
ID NO:3.
Isolated
[0112] In one aspect, preferably the nucleotide or amino acid
sequence is in an isolated form. The term "isolated" means that the
sequence is at least substantially free from at least one other
component with which the sequence is naturally associated in nature
and as found in nature.
Purified
[0113] In one aspect, preferably the nucleotide or amino acid
sequence is in a purified form. The term "purified" means that the
sequence is in a relatively pure state--e.g. at least about 90%
pure, or at least about 95% pure or at least about 98% pure.
Nucleotide Sequence
[0114] The scope of the present invention encompasses nucleotide
sequences encoding enzymes having the specific properties as
defined herein.
[0115] The term "nucleotide sequence" as used herein refers to an
oligonucleotide sequence, nucleic acid or polynucleotide sequence,
and variant, homologues, fragments and derivatives thereof (such as
portions thereof). The nucleotide sequence may be of genomic or
synthetic or recombinant origin, which may be double-stranded or
single-stranded whether representing the sense or anti-sense
strand.
[0116] The term "nucleotide sequence" or "nucleic acid molecule" in
relation to the present invention includes genomic DNA, cDNA,
synthetic DNA, and RNA. Preferably it means DNA, more preferably
cDNA sequence coding for the present invention.
[0117] In a preferred embodiment, the nucleotide sequence when
relating to and when encompassed by the per se scope of the present
invention does not include the native nucleotide sequence according
to the present invention when in its natural environment and when
it is linked to its naturally associated sequence(s) that is/are
also in its/their natural environment. For ease of reference, we
shall call this preferred embodiment the "non-native nucleotide
sequence". In this regard, the term "native nucleotide sequence"
means an entire nucleotide sequence that is in its native
environment and when operatively linked to an entire promoter with
which it is naturally associated, which promoter is also in its
native environment. However, the amino acid sequence encompassed by
scope the present invention can be isolated and/or purified post
expression of a nucleotide sequence in its native organism.
Preferably, however, the amino acid sequence encompassed by scope
of the present invention may be expressed by a nucleotide sequence
in its native organism but wherein the nucleotide sequence is not
under the control of the promoter with which it is naturally
associated within that organism.
Preparation of a Nucleotide Sequence
[0118] Typically, the nucleotide sequence encompassed by scope of
the present invention or the nucleotide sequences for use in the
present invention are prepared using recombinant DNA techniques
(i.e. recombinant DNA). However, in an alternative embodiment of
the invention, the nucleotide sequence could be synthesised, in
whole or in part, using chemical methods well known in the art (see
Caruthers M H et al., 765 (1980) Nuc Acids Res Symp Ser 215-23 and
Horn T et al., (1980) Nuc Acids Res Symp Ser 225-232).
[0119] A nucleotide sequence encoding either an enzyme which has
the specific properties as defined herein or an enzyme which is
suitable for modification may be identified and/or isolated and/or
purified from any cell or organism producing said enzyme. Various
methods are well known within the art for the identification and/or
isolation and/or purification of nucleotide sequences. By way of
example, PCR amplification techniques to prepare more of a sequence
may be used once a suitable sequence has been identified and/or
isolated and/or purified.
[0120] By way of further example, a genomic DNA and/or cDNA library
may be constructed using chromosomal DNA or messenger RNA from the
organism producing the enzyme. If the amino acid sequence of the
enzyme or a part of the amino acid sequence of the enzyme is known,
labelled oligonucleotide probes may be synthesised and used to
identify enzyme-encoding clones from the genomic library prepared
from the organism. Alternatively, a labelled oligonucleotide probe
containing sequences homologous to another known enzyme gene could
be used to identify enzyme-encoding clones. In the latter case,
hybridisation and washing conditions of lower stringency are
used.
[0121] Alternatively, enzyme-encoding clones could be identified by
inserting fragments of genomic DNA into an expression vector, such
as a plasmid, transforming enzyme-negative bacteria with the
resulting genomic DNA library, and then plating the transformed
bacteria onto agar plates containing a substrate for the enzyme
(e.g. maltose for a glucosidase (maltase) producing enzyme),
thereby allowing clones expressing the enzyme to be identified.
[0122] In a yet further alternative, the nucleotide sequence
encoding the enzyme may be prepared synthetically by established
standard methods, e.g. the phosphoroamidite method described by
Beucage S. L. et al., (1981) Tetrahedron Letters 22, p 1859-1869,
or the method described by Matthes et al., (1984) EMBO J. 3, p
801-805. In the phosphoroamidite method, oligonucleotides are
synthesised, e.g. in an automatic DNA synthesiser, purified,
annealed, ligated and cloned in appropriate vectors.
[0123] The nucleotide sequence may be of mixed genomic and
synthetic origin, mixed synthetic and cDNA origin, or mixed genomic
and cDNA origin, prepared by ligating fragments of synthetic,
genomic or cDNA origin (as appropriate) in accordance with standard
techniques. Each ligated fragment corresponds to various parts of
the entire nucleotide sequence. The DNA sequence may also be
prepared by polymerase chain reaction (PCR) using specific primers,
for instance as described in U.S. Pat. No. 4,683,202 or in Saiki R
K et al., (Science (1988) 239, pp 487-491).
[0124] Due to degeneracy in the genetic code, nucleotide sequences
may be readily produced in which the triplet codon usage, for some
or all of the amino acids encoded by the original nucleotide
sequence, has been changed thereby producing a nucleotide sequence
with low homology to the original nucleotide sequence but which
encodes the same, or a variant, amino acid sequence as encoded by
the original nucleotide sequence. For example, for most amino acids
the degeneracy of the genetic code is at the third position in the
triplet codon (wobble position) (for reference see Stryer, Lubert,
Biochemistry, Third Edition, Freeman Press, ISBN 0-7167-1920-7)
therefore, a nucleotide sequence in which all triplet codons have
been "wobbled" in the third position would be about 66% identical
to the original nucleotide sequence however, the amended nucleotide
sequence would encode for the same, or a variant, primary amino
acid sequence as the original nucleotide sequence.
[0125] Therefore, the present invention further relates to any
nucleotide sequence that has alternative triplet codon usage for at
least one amino acid encoding triplet codon, but which encodes the
same, or a variant, polypeptide sequence as the polypeptide
sequence encoded by the original nucleotide sequence.
[0126] Furthermore, specific organisms typically have a bias as to
which triplet codons are used to encode amino acids. Preferred
codon usage tables are widely available, and can be used to prepare
codon optimised genes. Such codon optimisation techniques are
routinely used to optimise expression of transgenes in a
heterologous host.
Molecular Evolution
[0127] Once an enzyme-encoding nucleotide sequence has been
isolated and/or purified, or a putative enzyme-encoding nucleotide
sequence has been identified, it may be desirable to modify the
selected nucleotide sequence, for example it may be desirable to
mutate the sequence in order to prepare an enzyme having improved
stability characteristics in accordance with the present
invention.
[0128] Mutations may be introduced using synthetic
oligonucleotides. These oligonucleotides contain nucleotide
sequences flanking the desired mutation sites.
[0129] A suitable method is disclosed in Morinaga et al
(Biotechnology (1984) 2, p 646-649). Another method of introducing
mutations into enzyme-encoding nucleotide sequences is described in
Nelson and Long (Analytical Biochemistry (1989), 180, p
147-151).
[0130] Instead of site directed mutagenesis, such as described
above, one can introduce mutations randomly for instance using a
commercial kit such as the GeneMorph PCR mutagenesis kit from
Stratagene, or the Diversify PCR random mutagenesis kit from
Clontech.
[0131] A third method to obtain novel sequences is to fragment
non-identical nucleotide sequences, either by using any number of
restriction enzymes or an enzyme such as Dnase I, and reassembling
full nucleotide sequences coding for functional proteins.
Alternatively one can use one or multiple non-identical nucleotide
sequences and introduce mutations during the reassembly of the full
nucleotide sequence.
[0132] Thus, it is possible to produce numerous site directed or
random mutations into a nucleotide sequence, either in vivo or in
vitro, and to subsequently screen for improved functionality of the
encoded polypeptide by various means.
[0133] As a non-limiting example, mutations or natural variants of
a polynucleotide sequence can be recombined with either the
wildtype or other mutations or natural variants to produce new
variants. Such new variants can also be screened for improved
functionality of the encoded polypeptide. The production of new
preferred variants may be achieved by various methods well
established in the art, for example the Error Threshold Mutagenesis
(WO 92/18645), oligonucleotide mediated random mutagenesis (U.S.
Pat. No. 5,723,323), DNA shuffling (U.S. Pat. No. 5,605,793),
exo-mediated gene assembly (WO 0058517), or RCR.RTM. Recombination
Chain Reaction (EP 1230390 and U.S. Pat. No. 6,821,758). Other
suitable methods are described, for example in WO 0134835, WO
02/097130, WO 03/012100, WO03/057247, WO 2004/018674, U.S. Pat. No.
6,303,344 and U.S. Pat. No. 6,132,970.
[0134] The application of the above-mentioned and similar molecular
evolution methods allows the identification and selection of
variants of the enzymes of the present invention which have
preferred characteristics without any prior knowledge of protein
structure or function, and allows the production of non-predictable
but beneficial mutations or variants. There are numerous examples
of the application of molecular evolution in the art for the
optimisation or alteration of enzyme activity, such examples
include, but are not limited to one or more of the following:
optimised expression and/or activity in a host cell or in vitro,
increased enzymatic activity, altered substrate and/or product
specificity, increased or decreased enzymatic or structural
stability, altered enzymatic activity/specificity in preferred
environmental conditions, e.g. temperature, pH, substrate
Amino Acid Sequences
[0135] The scope of the present invention also encompasses amino
acid sequences of enzymes having the specific properties as defined
herein.
[0136] As used herein, the term "amino acid sequence" is synonymous
with the term "polypeptide" and/or the term "protein". In some
instances, the term "amino acid sequence" is synonymous with the
term "peptide". In some instances, the term "amino acid sequence"
is synonymous with the term "enzyme".
[0137] The amino acid sequence may be prepared/isolated from a
suitable source, or it may be made synthetically or it may be
prepared by use of recombinant DNA techniques.
[0138] The enzyme encompassed in the present invention may be used
in conjunction with other enzymes. Thus the present invention also
covers a combination of enzymes wherein the combination comprises
the enzyme of the present invention and another enzyme, which may
be another enzyme according to the present invention. This aspect
is discussed in a later section.
[0139] Preferably the amino acid sequence when relating to and when
encompassed by the per se scope of the present invention is not a
native enzyme. In this regard, the term "native enzyme" means an
entire enzyme that is in its native environment and when it has
been expressed by its native nucleotide sequence.
Variants/Homologues/Derivatives
[0140] The present invention also encompasses the use of variants,
homologues and derivatives of any amino acid sequence of an enzyme
or of any nucleotide sequence encoding such an enzyme.
[0141] Here, the term "homologue" means an entity having a certain
homology with the amino acid sequences and the nucleotide
sequences. Here, the term "homology" can be equated with
"identity". Suitably, "homologous" in this context refers to the
percentage of sequence identity between two enzymes after aligning
their sequences using alignment algorithms as described in more
detail below.
[0142] In the present context, a homologous amino acid sequence is
taken to include an amino acid sequence which may be at least 75,
80, 81, 85 or 90% identical, preferably at least 95, 96, 97, 98 or
99% identical to the sequence. Typically, the homologues will
comprise the same active sites etc. --e.g. as the subject amino
acid sequence. Although homology can also be considered in terms of
similarity (i.e. amino acid residues having similar chemical
properties/functions), in the context of the present invention it
is preferred to express homology in terms of sequence identity.
[0143] By "functional fragment" is meant a fragment of the
polypeptide that retains that characteristic properties of that
polypeptide. In the context of the present invention, a functional
fragment of a phytase enzyme is a fragment that retains the
carotenoid cleavage capability of the whole protein.
[0144] In the present context, an homologous nucleotide sequence is
taken to include a nucleotide sequence which may be at least 75,
80, 81, 85 or 90% identical, preferably at least 95, 96, 97, 98 or
99% identical to a nucleotide sequence encoding an enzyme of the
present invention (the subject sequence). Typically, the homologues
will comprise the same sequences that code for the active sites
etc. as the subject sequence. Although homology can also be
considered in terms of similarity (i.e. amino acid residues having
similar chemical properties/functions), in the context of the
present invention it is preferred to express homology in terms of
sequence identity.
[0145] For the amino acid sequences and the nucleotide sequences,
homology comparisons can be conducted by eye, or more usually, with
the aid of readily available sequence comparison programs. These
commercially available computer programs can calculate % homology
between two or more sequences.
[0146] % homology may be calculated over contiguous sequences, i.e.
one sequence is aligned with the other sequence and each amino acid
in one sequence is directly compared with the corresponding amino
acid in the other sequence, one residue at a time. This is called
an "ungapped" alignment. Typically, such ungapped alignments are
performed only over a relatively short number of residues.
[0147] Although this is a very simple and consistent method, it
fails to take into consideration that, for example, in an otherwise
identical pair of sequences, one insertion or deletion will cause
the following amino acid residues to be put out of alignment, thus
potentially resulting in a large reduction in % homology when a
global alignment is performed. Consequently, most sequence
comparison methods are designed to produce optimal alignments that
take into consideration possible insertions and deletions without
penalising unduly the overall homology score. This is achieved by
inserting "gaps" in the sequence alignment to try to maximise local
homology.
[0148] However, these more complex methods assign "gap penalties"
to each gap that occurs in the alignment so that, for the same
number of identical amino acids, a sequence alignment with as few
gaps as possible--reflecting higher relatedness between the two
compared sequences--will achieve a higher score than one with many
gaps. "Affine gap costs" are typically used that charge a
relatively high cost for the existence of a gap and a smaller
penalty for each subsequent residue in the gap. This is the most
commonly used gap scoring system. High gap penalties will of course
produce optimised alignments with fewer gaps. Most alignment
programs allow the gap penalties to be modified. However, it is
preferred to use the default values when using such software for
sequence comparisons. For example when using the GCG Wisconsin
Bestfit package the default gap penalty for amino acid sequences is
-12 for a gap and -4 for each extension.
[0149] Calculation of maximum % homology therefore firstly requires
the production of an optimal alignment, taking into consideration
gap penalties. A suitable computer program for carrying out such an
alignment is the GCG Wisconsin Bestfit package (Devereux et al 1984
Nuc. Acids Research 12 p 387). Examples of other software than can
perform sequence comparisons include, but are not limited to, the
BLAST package (see Ausubel et al., 1999 Short Protocols in
Molecular Biology, 4.sup.th Ed--Chapter 18), FASTA (Altschul et
al., 1990 J. Mol. Biol. 403-410) and the GENEWORKS suite of
comparison tools. Both BLAST and FASTA are available for offline
and online searching (see Ausubel et al., 1999, Short Protocols in
Molecular Biology, pages 7-58 to 7-60).
[0150] However, for some applications, it is preferred to use the
GCG Bestfit program. A new tool, called BLAST 2 Sequences is also
available for comparing protein and nucleotide sequence (see FEMS
Microbiol Lett 1999 174(2): 247-50; FEMS Microbiol Lett 1999
177(1): 187-8 and tatiana@ncbi.nlm.nih.gov).
[0151] Although the final % homology can be measured in terms of
identity, the alignment process itself is typically not based on an
all-or-nothing pair comparison. Instead, a scaled similarity score
matrix is generally used that assigns scores to each pairwise
comparison based on chemical similarity or evolutionary distance.
An example of such a matrix commonly used is the BLOSUM62
matrix--the default matrix for the BLAST suite of programs. GCG
Wisconsin programs generally use either the public default values
or a custom symbol comparison table if supplied (see user manual
for further details). For some applications, it is preferred to use
the public default values for the GCG package, or in the case of
other software, the default matrix, such as BLOSUM62.
[0152] Alternatively, percentage homologies may be calculated using
the multiple alignment feature in DNASIS.TM. (Hitachi Software),
based on an algorithm, analogous to CLUSTAL (Higgins D G &
Sharp P M (1988), Gene 73(1), 237-244).
[0153] Once the software has produced an optimal alignment, it is
possible to calculate % homology, preferably % sequence identity.
The software typically does this as part of the sequence comparison
and generates a numerical result.
[0154] The sequences may also have deletions, insertions or
substitutions of amino acid residues which produce a silent change
and result in a functionally equivalent substance. Deliberate amino
acid substitutions may be made on the basis of similarity in amino
acid properties (such as polarity, charge, solubility,
hydrophobicity, hydrophilicity, and/or the amphipathic nature of
the residues) and it is therefore useful to group amino acids
together in functional groups Amino acids can be grouped together
based on the properties of their side chain alone. However it is
more useful to include mutation data as well. The sets of amino
acids thus derived are likely to be conserved for structural
reasons. These sets can be described in the form of a Venn diagram
(Livingstone C. D. and Barton G. J. (1993) "Protein sequence
alignments: a strategy for the hierarchical analysis of residue
conservation" Comput. Appl Biosci. 9: 745-756) (Taylor W. R. (1986)
"The classification of amino acid conservation" J. Theor. Biol.
119; 205-218). Conservative substitutions may be made, for example
according to the table below which describes a generally accepted
Venn diagram grouping of amino acids.
TABLE-US-00001 SET SUB-SET Hydrophobic FWYHKMILVAGC Aromatic FWYH
Aliphatic ILV Polar WYHKREDCSTNQ Charged HKRED Positively HKR
charged Negatively ED charged Small VCAGSPTND Tiny AGS
[0155] The present invention also encompasses conservative or
homologous substitutions or mutations (substitution and replacement
are both used herein to mean the interchange of an existing amino
acid residue, with an alternative residue) that may occur, i.e.
like-for-like substitution. Thus, the term "conservative mutation"
refers to an amino acid mutation that a person skilled in the art
would consider conservative to a first mutation. "Conservative" in
this context means conserving or invariable in terms of the amino
acid characteristics. If, for example, a mutation leads at a
specific position to a substitution of an aromatic amino acid
residue (e.g. Tyr) with an aliphatic amino acid residue (e.g. Leu)
then a substitution at the same position with a different aliphatic
amino acid (e.g. Ile or Val) is referred to as a conservative
mutation. Further amino acid characteristics include size of the
residue, hydrophobicity, polarity, charge, pK-value, and other
amino acid characteristics known in the art. Accordingly, a
conservative mutation may include substitution such as basic for
basic, acidic for acidic, polar for polar etc.
[0156] Non-conservative substitution may also occur i.e. from one
class of residue to another or alternatively involving the
inclusion of unnatural amino acids such as ornithine (hereinafter
referred to as Z), diaminobutyric acid ornithine (hereinafter
referred to as B), norleucine ornithine (hereinafter referred to as
O), pyriylalanine, thienylalanine, naphthylalanine and
phenylglycine.
[0157] Replacements may also be made by unnatural amino acids.
[0158] Variant amino acid sequences may include suitable spacer
groups that may be inserted between any two amino acid residues of
the sequence including alkyl groups such as methyl, ethyl or propyl
groups in addition to amino acid spacers such as glycine or
.beta.-alanine residues. A further form of variation, involves the
presence of one or more amino acid residues in peptoid form, will
be well understood by those skilled in the art. For the avoidance
of doubt, "the peptoid form" is used to refer to variant amino acid
residues wherein the .alpha.-carbon substituent group is on the
residue's nitrogen atom rather than the .alpha.-carbon. Processes
for preparing peptides in the peptoid form are known in the art,
for example Simon R J et al., PNAS (1992) 89(20), 9367-9371 and
Horwell D C, Trends Biotechnol. (1995) 13(4), 132-134.
Nomenclature
[0159] In the present invention, the conventional one-letter and
three-letter codes for amino acid residues are used. For ease of
reference, mutations in enzyme variants are described by use of the
following nomenclature: amino acid residue in the parent enzyme;
position; substituted amino acid residue(s). According to this
nomenclature, the substitution of, for instance, an alanine residue
for a glycine residue at position 20 is indicated as Ala20Gly or
A20G. The deletion of alanine in the same position is shown as
Ala20* or A20*. The insertion of an additional amino acid residue
(e.g. a glycine) is indicated as Ala20AlaGly or A20AG. The deletion
of a consecutive stretch of amino acid residues (e.g. between
alanine at position 20 and glycine at position 21) is indicated as
.DELTA.(Ala20-Gly21) or .DELTA.(A20-G21). When a parent enzyme
sequence contains a deletion in comparison to the enzyme sequence
used for numbering an insertion in such a position (e.g. an alanine
in the deleted position 20) is indicated as *20Ala or *20A.
Multiple mutations are separated by a plus sign or a slash. For
example, two mutations in positions 20 and 21 substituting alanine
and glutamic acid for glycine and serine, respectively, are
indicated as A20G+E21S or A20G/E21S. When an amino acid residue at
a given position is substituted with two or more alternative amino
acid residues these residues are separated by a comma or a slash.
For example, substitution of alanine at position 30 with either
glycine or glutamic acid is indicated as A20G,E or A20G/E, or A20G,
A20E. When a position suitable for modification is identified
herein without any specific modification being suggested, it is to
be understood that any amino acid residue may be substituted for
the amino acid residue present in the position. Thus, for instance,
when a modification of an alanine in position 20 is mentioned but
not specified, it is to be understood that the alanine may be
deleted or substituted for any other amino acid residue (i.e. any
one of R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V).
[0160] The nucleotide sequences for use in the present invention
may include within them synthetic or modified nucleotides. A number
of different types of modification to oligonucleotides are known in
the art. These include methylphosphonate and phosphorothioate
backbones and/or the addition of acridine or polylysine chains at
the 3' and/or 5' ends of the molecule. For the purposes of the
present invention, it is to be understood that the nucleotide
sequences described herein may be modified by any method available
in the art. Such modifications may be carried out in order to
enhance the in vivo activity or life span of nucleotide sequences
of the present invention.
[0161] The present invention also encompasses the use of nucleotide
sequences that are complementary to the sequences presented herein,
or any derivative, fragment or derivative thereof. If the sequence
is complementary to a fragment thereof then that sequence can be
used as a probe to identify similar coding sequences in other
organisms etc.
[0162] Polynucleotides which are not 100% homologous to the
sequences of the present invention but fall within the scope of the
invention can be obtained in a number of ways. Other variants of
the sequences described herein may be obtained for example by
probing DNA libraries made from a range of individuals, for example
individuals from different populations. In addition, other
homologues may be obtained and such homologues and fragments
thereof in general will be capable of selectively hybridising to
the sequences shown in the sequence listing herein. Such sequences
may be obtained by probing cDNA libraries made from or genomic DNA
libraries from other species, and probing such libraries with
probes comprising all or part of any one of the sequences in the
attached sequence listings under conditions of medium to high
stringency. Similar considerations apply to obtaining species
homologues and allelic variants of the polypeptide or nucleotide
sequences of the invention.
[0163] Variants and strain/species homologues may also be obtained
using degenerate PCR which will use primers designed to target
sequences within the variants and homologues encoding conserved
amino acid sequences within the sequences of the present invention.
Conserved sequences can be predicted, for example, by aligning the
amino acid sequences from several variants/homologues. Sequence
alignments can be performed using computer software known in the
art. For example the GCG Wisconsin PileUp program is widely
used.
[0164] The primers used in degenerate PCR will contain one or more
degenerate positions and will be used at stringency conditions
lower than those used for cloning sequences with single sequence
primers against known sequences.
[0165] Alternatively, such polynucleotides may be obtained by site
directed mutagenesis of characterised sequences. This may be useful
where for example silent codon sequence changes are required to
optimise codon preferences for a particular host cell in which the
polynucleotide sequences are being expressed. Other sequence
changes may be desired in order to introduce restriction enzyme
recognition sites, or to alter the property or function of the
polypeptides encoded by the polynucleotides.
[0166] Polynucleotides (nucleotide sequences) of the invention may
be used to produce a primer, e.g. a PCR primer, a primer for an
alternative amplification reaction, a probe e.g. labelled with a
revealing label by conventional means using radioactive or
non-radioactive labels, or the polynucleotides may be cloned into
vectors. Such primers, probes and other fragments will be at least
15, preferably at least 20, for example at least 25, 30 or 40
nucleotides in length, and are also encompassed by the term
polynucleotides of the invention as used herein.
[0167] Polynucleotides such as DNA polynucleotides and probes
according to the invention may be produced recombinantly,
synthetically, or by any means available to those of skill in the
art. They may also be cloned by standard techniques.
[0168] In general, primers will be produced by synthetic means,
involving a stepwise manufacture of the desired nucleic acid
sequence one nucleotide at a time. Techniques for accomplishing
this using automated techniques are readily available in the
art.
[0169] Longer polynucleotides will generally be produced using
recombinant means, for example using a PCR (polymerase chain
reaction) cloning techniques. The primers may be designed to
contain suitable restriction enzyme recognition sites so that the
amplified DNA can be cloned into a suitable cloning vector.
Biologically Active
[0170] Preferably, the variant sequences etc. are at least as
biologically active as the sequences presented herein.
[0171] As used herein "biologically active" refers to a sequence
having a similar structural function (but not necessarily to the
same degree), and/or similar regulatory function (but not
necessarily to the same degree), and/or similar biochemical
function (but not necessarily to the same degree) of the naturally
occurring sequence.
[0172] In particular, variant sequences or modified forms thereof
have a similar enzymatic profile to the profile of the phytase
identified herein. This profile includes characteristics such as
being a secreted protein, having a pH optimum in the range of pH 2
to 5.5, preferably 3.0 to 3.5, retaining at least 50% of the
maximum activity over the pH range 2.0-5.5 and/or having a specific
activity over 1000 U/mg.
Hybridisation
[0173] The present invention also encompasses sequences that are
complementary to the nucleic acid sequences of the present
invention or sequences that are capable of hybridising either to
the sequences of the present invention or to sequences that are
complementary thereto.
[0174] The term "hybridisation" as used herein shall include "the
process by which a strand of nucleic acid joins with a
complementary strand through base pairing" as well as the process
of amplification as carried out in polymerase chain reaction (PCR)
technologies.
[0175] The present invention also encompasses the use of nucleotide
sequences that are capable of hybridising to the sequences that are
complementary to the sequences presented herein, or any derivative,
fragment or derivative thereof.
[0176] The term "variant" also encompasses sequences that are
complementary to sequences that are capable of hybridising to the
nucleotide sequences presented herein.
[0177] Preferably, the term "variant" encompasses sequences that
are complementary to sequences that are capable of hybridising
under stringent conditions (e.g. 50.degree. C. and 0.2.times.SSC
{1.times.SSC=0.15 M NaCl, 0.015 M Na.sub.3citrate pH 7.0}) to the
nucleotide sequences presented herein.
[0178] More preferably, the term "variant" encompasses sequences
that are complementary to sequences that are capable of hybridising
under high stringent conditions (e.g. 65.degree. C. and
0.1.times.SSC {1.times.SSC=0.15 M NaCl, 0.015 M Na.sub.3citrate pH
7.0}) to the nucleotide sequences presented herein.
[0179] The present invention also relates to nucleotide sequences
that can hybridise to the nucleotide sequences of the present
invention (including complementary sequences of those presented
herein).
[0180] The present invention also relates to nucleotide sequences
that are complementary to sequences that can hybridise to the
nucleotide sequences of the present invention (including
complementary sequences of those presented herein).
[0181] Also included within the scope of the present invention are
polynucleotide sequences that are capable of hybridising to the
nucleotide sequences presented herein under conditions of
intermediate to maximal stringency.
[0182] In a preferred aspect, the present invention covers
nucleotide sequences that can hybridise to the nucleotide sequence
of the present invention, or the complement thereof, under
stringent conditions (e.g. 50.degree. C. and 0.2.times.SSC).
[0183] In a more preferred aspect, the present invention covers
nucleotide sequences that can hybridise to the nucleotide sequence
of the present invention, or the complement thereof, under high
stringent conditions (e.g. 65.degree. C. and 0.1.times.SSC).
Site-Directed Mutagenesis
[0184] Once an enzyme-encoding nucleotide sequence has been
isolated and/or purified, or a putative enzyme-encoding nucleotide
sequence has been identified, it may be desirable to mutate the
sequence in order to prepare an enzyme of the present
invention.
[0185] Mutations may be introduced using synthetic
oligonucleotides. These oligonucleotides contain nucleotide
sequences flanking the desired mutation sites.
[0186] A suitable method is disclosed in Morinaga et al.,
(Biotechnology (1984) 2, p 646-649). Another method of introducing
mutations into enzyme-encoding nucleotide sequences is described in
Nelson and Long (Analytical Biochemistry (1989), 180, p 147-151). A
further method is described in Sarkar and Sommer (Biotechniques
(1990), 8, p 404-407--"The megaprimer method of site directed
mutagenesis").
Recombinant
[0187] In one aspect the sequence for use in the present invention
is a recombinant sequence--i.e. a sequence that has been prepared
using recombinant DNA techniques.
[0188] These recombinant DNA techniques are within the capabilities
of a person of ordinary skill in the art. Such techniques are
explained in the literature, for example, J. Sambrook, E. F.
Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory
Manual, Second Edition, Books 1-3, Cold Spring Harbor Laboratory
Press.
Synthetic
[0189] In one aspect the sequence for use in the present invention
is a synthetic sequence--i.e. a sequence that has been prepared by
in vitro chemical or enzymatic synthesis. It includes, but is not
limited to, sequences made with optimal codon usage for host
organisms--such as the methylotrophic yeasts Pichia and
Hansenula.
Expression of Enzymes
[0190] The nucleotide sequence for use in the present invention may
be incorporated into a recombinant replicable vector. The vector
may be used to replicate and express the nucleotide sequence, in
enzyme form, in and/or from a compatible host cell.
[0191] Expression may be controlled using control sequences e.g.
regulatory sequences.
[0192] The enzyme produced by a host recombinant cell by expression
of the nucleotide sequence may be secreted or may be contained
intracellularly depending on the sequence and/or the vector used.
The coding sequences may be designed with signal sequences which
enhance direct secretion of the substance coding sequences through
a particular prokaryotic or eukaryotic cell membrane.
[0193] Advantageously, the enzymes of the present invention are
secreted.
Expression Vector
[0194] The terms "plasmid", "vector system" or "expression vector"
means a construct capable of in vivo or in vitro expression. In the
context of the present invention, these constructs may be used to
introduce genes encoding enzymes into host cells. Suitably, the
genes whose expression is introduced may be referred to as
"expressible transgenes".
[0195] Preferably, the expression vector is incorporated into the
genome of a suitable host organism. The term "incorporated"
preferably covers stable incorporation into the genome.
[0196] The nucleotide sequences described herein including the
nucleotide sequence of the present invention may be present in a
vector in which the nucleotide sequence is operably linked to
regulatory sequences capable of providing for the expression of the
nucleotide sequence by a suitable host organism.
[0197] The vectors for use in the present invention may be
transformed into a suitable host cell as described below to provide
for expression of a polypeptide of the present invention.
[0198] The choice of vector e.g. a plasmid, cosmid, or phage vector
will often depend on the host cell into which it is to be
introduced.
[0199] The vectors for use in the present invention may contain one
or more selectable marker genes--such as a gene, which confers
antibiotic resistance e.g. ampicillin, kanamycin, chloramphenicol
or tetracyclin resistance. Alternatively, the selection may be
accomplished by co-transformation (as described in WO91/17243).
[0200] Vectors may be used in vitro, for example for the production
of RNA or used to transfect, transform, transduce or infect a host
cell.
[0201] Thus, in a further embodiment, the invention provides a
method of making nucleotide sequences of the present invention by
introducing a nucleotide sequence of the present invention into a
replicable vector, introducing the vector into a compatible host
cell, and growing the host cell under conditions which bring about
replication of the vector.
[0202] The vector may further comprise a nucleotide sequence
enabling the vector to replicate in the host cell in question.
Examples of such sequences are the origins of replication of
plasmids pUC19, pACYC177, pUB110, pE194, pAMB1, pIJ702 and
pET11.
Regulatory Sequences
[0203] In some applications, the nucleotide sequence for use in the
present invention is operably linked to a regulatory sequence which
is capable of providing for the expression of the nucleotide
sequence, such as by the chosen host cell. By way of example, the
present invention covers a vector comprising the nucleotide
sequence of the present invention operably linked to such a
regulatory sequence, i.e. the vector is an expression vector.
[0204] The term "operably linked" refers to a juxtaposition wherein
the components described are in a relationship permitting them to
function in their intended manner A regulatory sequence "operably
linked" to a coding sequence is ligated in such a way that
expression of the coding sequence is achieved under condition
compatible with the control sequences.
[0205] The term "regulatory sequences" includes promoters and
enhancers and other expression regulation signals.
[0206] The term "promoter" is used in the normal sense of the art,
e.g. an RNA polymerase binding site.
[0207] Enhanced expression of the nucleotide sequence encoding the
enzyme of the present invention may also be achieved by the
selection of heterologous regulatory regions, e.g. promoter,
secretion leader and terminator regions.
[0208] Preferably, the nucleotide sequence according to the present
invention is operably linked to at least a promoter.
[0209] Examples of suitable promoters for directing the
transcription of the nucleotide sequence in a bacterial, fungal or
yeast host are well known in the art.
Constructs
[0210] The term "construct"--which is synonymous with terms such as
"conjugate", "cassette" and "hybrid"--includes a nucleotide
sequence for use according to the present invention directly or
indirectly attached to a promoter.
[0211] An example of an indirect attachment is the provision of a
suitable spacer group such as an intron sequence, such as the
Sh1-intron or the ADH intron, intermediate the promoter and the
nucleotide sequence of the present invention. The same is true for
the term "fused" in relation to the present invention which
includes direct or indirect attachment. In some cases, the terms do
not cover the natural combination of the nucleotide sequence coding
for the protein ordinarily associated with the wild type gene
promoter and when they are both in their natural environment.
[0212] The construct may even contain or express a marker, which
allows for the selection of the genetic construct.
[0213] For some applications, preferably the construct of the
present invention comprises at least the nucleotide sequence of the
present invention operably linked to a promoter.
Host Cells
[0214] The term "host cell"--in relation to the present invention
includes any cell that comprises either the nucleotide sequence or
an expression vector as described above and which is used in the
recombinant production of an enzyme having the specific properties
as defined herein or in the methods of the present invention.
[0215] Thus, a further embodiment of the present invention provides
host cells transformed or transfected with a nucleotide sequence
that expresses the enzymes described in the present invention. The
cells will be chosen to be compatible with the said vector and may
for example be prokaryotic (for example bacterial), fungal, yeast
or plant cells. Preferably, the host cells are not human cells.
[0216] Examples of suitable bacterial host organisms are gram
positive or gram negative bacterial species.
[0217] Depending on the nature of the nucleotide sequence encoding
the enzyme of the present invention, and/or the desirability for
further processing of the expressed protein, eukaryotic hosts such
as yeasts or other fungi may be preferred. In general, yeast cells
are preferred over fungal cells because they are easier to
manipulate. However, some proteins are either poorly secreted from
the yeast cell, or in some cases are not processed properly (e.g.
hyperglycosylation in yeast). In these instances, a different
fungal host organism should be selected.
[0218] The use of suitable host cells--such as yeast, fungal and
plant host cells--may provide for post-translational modifications
(e.g. myristoylation, glycosylation, truncation, lapidation and
tyrosine, serine or threonine phosphorylation) as may be needed to
confer optimal biological activity on recombinant expression
products of the present invention.
[0219] The host cell may be a protease deficient or protease minus
strain.
[0220] The genotype of the host cell may be modified to improve
expression.
[0221] Examples of host cell modifications include protease
deficiency, supplementation of rare tRNA's, and modification of the
reductive potential in the cytoplasm to enhance disulphide bond
formation.
[0222] For example, the host cell E. coli may overexpress rare
tRNA's to improve expression of heterologous proteins as
exemplified/described in Kane (Curr Opin Biotechnol (1995), 6,
494-500 "Effects of rare codon clusters on high-level expression of
heterologous proteins in E. coli"). The host cell may be deficient
in a number of reducing enzymes thus favouring formation of stable
disulphide bonds as exemplified/described in Bessette (Proc Natl
Acad Sci USA (1999), 96, 13703-13708 "Efficient folding of proteins
with multiple disulphide bonds in the Escherichia coli
cytoplasm").
[0223] In one embodiment, host cells in the context of the present
invention include those cells that can be added directly into
animal feed.
Organism
[0224] The term "organism" in relation to the present invention
includes any organism that could comprise the nucleotide sequence
coding for the enzymes as described in the present invention and/or
products obtained therefrom, and/or wherein a promoter can allow
expression of the nucleotide sequence according to the present
invention when present in the organism.
[0225] Suitable organisms may include a prokaryote, fungus, yeast
or a plant.
[0226] The term "transgenic organism" in relation to the present
invention includes any organism that comprises the nucleotide
sequence coding for the enzymes as described in the present
invention and/or the products obtained therefrom, and/or wherein a
promoter can allow expression of the nucleotide sequence according
to the present invention within the organism. Preferably the
nucleotide sequence is incorporated in the genome of the
organism.
[0227] The term "transgenic organism" does not cover native
nucleotide coding sequences in their natural environment when they
are under the control of their native promoter which is also in its
natural environment.
[0228] Therefore, the transgenic organism of the present invention
includes an organism comprising any one of, or combinations of, the
nucleotide sequence coding for the enzymes as described in the
present invention, constructs according to the present invention,
vectors according to the present invention, plasmids according to
the present invention, cells according to the present invention,
tissues according to the present invention, or the products
thereof.
[0229] For example the transgenic organism may also comprise the
nucleotide sequence coding for the enzyme of the present invention
under the control of a heterologous promoter.
Transformation of Host Cells/Organism
[0230] As indicated earlier, the host organism can be a prokaryotic
or a eukaryotic organism. Examples of suitable prokaryotic hosts
include E. coli and Bacillus subtilis.
[0231] Teachings on the transformation of prokaryotic hosts is well
documented in the art, for example see Sambrook et al (Molecular
Cloning: A Laboratory Manual, 2nd edition, 1989, Cold Spring Harbor
Laboratory Press). Other suitable methods are set out in the
Examples herein. If a prokaryotic host is used then the nucleotide
sequence may need to be suitably modified before
transformation--such as by removal of introns.
[0232] Filamentous fungi cells may be transformed using various
methods known in the art--such as a process involving protoplast
formation and transformation of the protoplasts followed by
regeneration of the cell wall in a manner known. The use of
Aspergillus as a host microorganism is described in EP 0 238
023.
[0233] Another host organism can be a plant. A review of the
general techniques used for transforming plants may be found in
articles by Potrykus (Annu Rev Plant Physiol Plant Mol Biol [1991]
42:205-225) and Christou (Agro-Food-Industry Hi-Tech March/April
1994 17-27). Further teachings on plant transformation may be found
in EP-A-0449375.
[0234] General teachings on the transformation of fungi, yeasts and
plants are presented in following sections.
Transformed Fungus
[0235] A host organism may be a fungus--such as a filamentous
fungus. Examples of suitable such hosts include any member
belonging to the genera Thermomyces, Acremonium, Aspergillus,
Penicillium, Mucor, Neurospora, Trichoderma and the like.
[0236] Teachings on transforming filamentous fungi are reviewed in
U.S. Pat. No. 5,741,665 which states that standard techniques for
transformation of filamentous fungi and culturing the fungi are
well known in the art. An extensive review of techniques as applied
to N. crassa is found, for example in Davis and de Serres, Methods
Enzymol (1971) 17A: 79-143.
[0237] Further teachings on transforming filamentous fungi are
reviewed in U.S. Pat. No. 5,674,707.
[0238] In one aspect, the host organism can be of the genus
Aspergillus, such as Aspergillus niger.
[0239] A transgenic Aspergillus according to the present invention
can also be prepared by following, for example, the teachings of
Turner G. 1994 (Vectors for genetic manipulation. In: Martinelli S.
D., Kinghorn J. R. (Editors) Aspergillus: 50 years on. Progress in
industrial microbiology vol 29. Elsevier Amsterdam 1994. pp.
641-666).
[0240] Gene expression in filamentous fungi has been reviewed in
Punt et al. (2002) Trends Biotechnol 2002 May; 20(5):200-6, Archer
& Peberdy Crit Rev Biotechnol (1997) 17(4):273-306.
Transformed Yeast
[0241] In another embodiment, the transgenic organism can be a
yeast.
[0242] A review of the principles of heterologous gene expression
in yeast are provided in, for example, Methods Mol Biol (1995),
49:341-54, and Curr Opin Biotechnol (1997) October; 8(5):554-60
[0243] In this regard, yeast--such as the species Saccharomyces
cerevisi or Pichia pastoris (see FEMS Microbiol Rev (2000
24(1):45-66), may be used as a vehicle for heterologous gene
expression.
[0244] A review of the principles of heterologous gene expression
in Saccharomyces cerevisiae and secretion of gene products is given
by E Hinchcliffe E Kenny (1993, "Yeast as a vehicle for the
expression of heterologous genes", Yeasts, Vol 5, Anthony H Rose
and J Stuart Harrison, eds, 2nd edition, Academic Press Ltd.).
[0245] For the transformation of yeast, several transformation
protocols have been developed. For example, a transgenic
Saccharomyces according to the present invention can be prepared by
following the teachings of Hinnen et al., (1978, Proceedings of the
National Academy of Sciences of the USA 75, 1929); Beggs, J D
(1978, Nature, London, 275, 104); and Ito, H et al (1983, J
Bacteriology 153, 163-168).
[0246] The transformed yeast cells may be selected using various
selective markers--such as auxotrophic markers dominant antibiotic
resistance markers.
Transformed Plants/Plant Cells
[0247] A host organism suitable for the present invention may be a
plant. A review of the general techniques may be found in articles
by Potrykus (Annu Rev Plant Physiol Plant Mol Biol [1991]
42:205-225) and Christou (Agro-Food-Industry Hi-Tech March/April
1994 17-27).
Culturing and Production
[0248] Host cells transformed with the nucleotide sequence of the
present invention may be cultured under conditions conducive to the
production of the encoded enzyme and which facilitate recovery of
the enzyme from the cells and/or culture medium.
[0249] The medium used to cultivate the cells may be any
conventional medium suitable for growing the host cell in questions
and obtaining expression of the enzyme.
[0250] The protein produced by a recombinant cell may be displayed
on the surface of the cell.
[0251] The enzyme may be secreted from the host cells and may
conveniently be recovered from the culture medium using well-known
procedures.
Secretion
[0252] It may be desirable for the enzyme to be secreted from the
expression host into the culture medium from where the enzyme may
be more easily recovered. According to the present invention, the
secretion leader sequence may be selected on the basis of the
desired expression host. Hybrid signal sequences may also be used
with the context of the present invention.
[0253] Typical examples of heterologous secretion leader sequences
are those originating from the fungal amyloglucosidase (AG) gene
(glaA--both 18 and 24 amino acid versions e.g. from Aspergillus),
the .alpha.-factor gene (yeasts e.g. Saccharomyces, Kluyveromyces
and Hansenula) or the .alpha.-amylase gene (Bacillus).
[0254] By way of example, the secretion of heterologous proteins in
E. coli is reviewed in Methods Enzymol (1990) 182:132-43.
Detection
[0255] A variety of protocols for detecting and measuring the
expression of the amino acid sequence are known in the art.
Examples include enzyme-linked immunosorbent assay (ELISA),
radioimmunoassay (RIA) and fluorescent activated cell sorting
(FACS).
[0256] A wide variety of labels and conjugation techniques are
known by those skilled in the art and can be used in various
nucleic and amino acid assays.
[0257] A number of companies such as Pharmacia Biotech (Piscataway,
N.J.), Promega (Madison, Wis.), and US Biochemical Corp (Cleveland,
Ohio) supply commercial kits and protocols for these
procedures.
[0258] Suitable reporter molecules or labels include those
radionuclides, enzymes, fluorescent, chemiluminescent, or
chromogenic agents as well as substrates, cofactors, inhibitors,
magnetic particles and the like. Patents teaching the use of such
labels include U.S. Pat. No. 3,817,837; U.S. Pat. No. 3,850,752;
U.S. Pat. No. 3,939,350; U.S. Pat. No. 3,996,345; U.S. Pat. No.
4,277,437; U.S. Pat. No. 4,275,149 and U.S. Pat. No. 4,366,241.
[0259] Also, recombinant immunoglobulins may be produced as shown
in U.S. Pat. No. 4,816,567.
[0260] Other suitable assays for detecting phytase activity are
known in the art and exemplified herein.
Fusion Proteins
[0261] The amino acid sequence for use according to the present
invention may be produced as a fusion protein, for example to aid
in extraction and purification. Examples of fusion protein partners
include glutathione-S-transferase (GST), 6.times.His, GAL4 (DNA
binding and/or transcriptional activation domains) and
(.beta.-galactosidase). It may also be convenient to include a
proteolytic cleavage site between the fusion protein partner and
the protein sequence of interest to allow removal of fusion protein
sequences.
[0262] Preferably, the fusion protein will not hinder the activity
of the protein sequence.
[0263] Gene fusion expression systems in E. coli have been reviewed
in Curr Opin Biotechnol (1995) 6(5):501-6.
[0264] In another embodiment of the invention, the amino acid
sequence may be ligated to a heterologous sequence to encode a
fusion protein. For example, for screening of peptide libraries for
agents capable of affecting the substance activity, it may be
useful to encode a chimeric substance expressing a heterologous
epitope that is recognised by a commercially available
antibody.
ADDITIONAL SEQUENCES
[0265] The sequences for use according to the present invention may
also be used in conjunction with one or more additional proteins of
interest (POIs) or nucleotide sequences of interest (NOIs).
[0266] Non-limiting examples of POIs include: Xylanase, lipases,
acid phosphatases and/or others. These include enzymes that, for
example, modulate the viscosity of the feed. The NOI may even be an
antisense sequence for any of those sequences.
[0267] The POI may even be a fusion protein, for example to aid in
extraction and purification or to enhance in vivo phytate
metabolism.
[0268] The POI may even be fused to a secretion sequence.
[0269] Other sequences can also facilitate secretion or increase
the yield of secreted POI. Such sequences could code for chaperone
proteins as for example the product of Aspergillus niger cyp B gene
described in UK patent application 9821198.0.
[0270] The NOI coding for POI may be engineered in order to alter
their activity for a number of reasons, including but not limited
to, alterations, which modify the processing and/or expression of
the expression product thereof. By way of further example, the NOI
may also be modified to optimise expression in a particular host
cell. Other sequence changes may be desired in order to introduce
restriction enzyme recognition sites.
[0271] The NOI coding for the POI may include within it synthetic
or modified nucleotides--such as methylphosphonate and
phosphorothioate backbones.
[0272] The NOI coding for the POI may be modified to increase
intracellular stability and half-life. Possible modifications
include, but are not limited to, the addition of flanking sequences
of the 5' and/or 3' ends of the molecule or the use of
phosphorothioate or 2' O-methyl rather than phosphodiesterase
linkages within the backbone of the molecule.
Antibodies
[0273] One aspect of the present invention relates to amino acids
that are immunologically reactive with the amino acid of SEQ ID No.
3.
[0274] Antibodies may be produced by standard techniques, such as
by immunisation with the substance of the invention or by using a
phage display library.
[0275] For the purposes of this invention, the term "antibody",
unless specified to the contrary, includes but is not limited to,
polyclonal, monoclonal, chimeric, single chain, Fab fragments,
fragments produced by a Fab expression library, as well as mimetics
thereof. Such fragments include fragments of whole antibodies which
retain their binding activity for a target substance, Fv, F(ab')
and F(ab').sub.2 fragments, as well as single chain antibodies
(scFv), fusion proteins and other synthetic proteins which comprise
the antigen-binding site of the antibody. Furthermore, the
antibodies and fragments thereof may be humanised antibodies.
Neutralising antibodies, i.e., those which inhibit biological
activity of the substance polypeptides, are especially preferred
for diagnostics and therapeutics.
[0276] If polyclonal antibodies are desired, a selected mammal
(e.g., mouse, rabbit, goat, horse, etc.) is immunised with the
sequence of the present invention (or a sequence comprising an
immunological epitope thereof). Depending on the host species,
various adjuvants may be used to increase immunological
response.
[0277] Serum from the immunised animal is collected and treated
according to known procedures. If serum containing polyclonal
antibodies to the sequence of the present invention (or a sequence
comprising an immunological epitope thereof) contains 1680
antibodies to other antigens, the polyclonal antibodies can be
purified by immunoaffinity chromatography. Techniques for producing
and processing polyclonal antisera are known in the art. In order
that such antibodies may be made, the invention also provides
polypeptides of the invention or fragments thereof haptenised to
another polypeptide for use as immunogens in animals or humans.
[0278] Monoclonal antibodies directed against the sequence of the
present invention (or a sequence comprising an immunological
epitope thereof) can also be readily produced by one skilled in the
art and include, but are not limited to, the hybridoma technique
Koehler and Milstein (1975 Nature 256:495-497), the human B-cell
hybridoma technique (Kosbor et al., (1983) Immunol Today 4:72; Cote
et al., (1983) Proc Natl Acad Sci 80:2026-2030) and the
EBV-hybridoma technique (Cole et al., (1985) Monoclonal Antibodies
and Cancer Therapy, Alan Rickman Liss Inc, pp 77-96).
[0279] In addition, techniques developed for the production of
"chimeric antibodies", the splicing of mouse antibody genes to
human antibody genes to obtain a molecule with appropriate antigen
specificity and biological activity may be used (Morrison et al.,
(1984) Proc Natl Acad Sci 81:6851-6855; Neuberger et al., (1984)
Nature 312:604-608; Takeda et al., (1985) Nature 314:452-454).
[0280] Alternatively, techniques described for the production of
single chain antibodies (U.S. Pat. No. 4,946,779) can be adapted to
produce the substance specific single chain antibodies.
[0281] Antibody fragments which contain specific binding sites for
the substance may also be generated. For example, such fragments
include, but are not limited to, the F(ab').sub.2 fragments which
can be produced by pepsin digestion of the antibody molecule and
the Fab fragments which can be generated by reducing the disulfide
bridges of the F(ab').sub.2 fragments. Alternatively, Fab
expression libraries may be constructed to allow rapid and easy
identification of monoclonal Fab fragments with the desired
specificity (Huse W D et al., (1989) Science 256:1275-128 1).
Large Scale Application
[0282] In one preferred embodiment of the present invention, the
amino acid sequence encoding a C. freundii-derived phytase or the
methods of the present invention are used for large scale
applications. In particular, the methods of the present invention
may be used for the large scale production of phytases for
industrial use as additives/supplements to food or feed
compositions.
[0283] Preferably the amino acid sequence is produced in a quantity
of from 5 g per litre to about 10 g per litre of the total cell
culture volume after cultivation of the host organism.
[0284] Preferably the amino acid sequence is produced in a quantity
of from 100 mg per litre to about 900 mg per litre of the total
cell culture volume after cultivation of the host organism.
[0285] Preferably the amino acid sequence is produced in a quantity
of from 250 mg per litre to about 500 mg per litre of the total
cell culture volume after cultivation of the host organism.
Use of Phytases
[0286] As stated above, the present invention also relates to the
production of phytases as described herein.
[0287] In particular, the present invention also relates to the use
of the amino acid sequences as disclosed herein in the production
of organic and inorganic phosphate compounds.
[0288] Thus, the present invention further relates to the use of
the nucleotide sequences encoding phytases in generating expression
vectors or systems for the expression of the phytases.
[0289] In addition, the present invention relates to the use of
such expression vectors or systems in the generation of host cells
which express phytases.
[0290] The invention further relates to the use of modified host
cells in the generation of precursors of organic and inorganic
phosphate compounds or in the generation of specific organic
phosphate compounds.
[0291] Suitable organic and inorganic phosphate compounds include
myo-inositol pentakis-, tetrakis-, tris-, bis- and
monophosphates.
[0292] Suitably, the invention therefore provides a method of
producing an organic phosphate compound comprising treating a
phytate with a phytase derived from Citrobacter freundii.
Preferably, the method is characterised in that the enzyme
comprises the amino acid sequences shown as SEQ ID NOs: 3 or a
sequence having at least 75% identity (homology) thereto or an
effective fragment, or modified form thereof. Suitably, the organic
phosphate is phytate or all possible stereoisomers of myo-inositol
di-, tri-, tetra, and pentaphosphates. Other suitable organic
phosphates include inositol-tetraphosphates and
inositol-oligophosphates. In a preferred embodiment, the method is
an in vivo biotechnological process.
[0293] Such methods for producing an organic phosphate compound may
suitably comprise the steps of: [0294] a) providing a host cell
that comprises expressible transgenes comprising C. freundii
phytase; [0295] b) culturing the transgenic organism under
conditions suitable for expression of the transgene; and [0296] c)
recovering the organic phosphate compound from the culture.
[0297] The compounds can be used for a number of applications
including in assays for the characterisation of phytases. Some
inositol phosphates are involved as signal molecules in
intracellular regulation and can be used research chemicals.
[0298] In another aspect there is provided a method for production
of food or animal feed Animal feed is typically produced in feed
mills in which raw materials are first ground to a suitable
particle size and then mixed with appropriate additives. The feed
may then be produced as a mash or pellets; the later typically
involves a method by which the temperature is raised to a target
level and then the feed is passed through a die to produce pellets
of a particular size. Subsequently liquid additives such as fat and
enzyme may be added. The pellets are allowed to cool prior to
transportation. Production of animal feed may also involve an
additional step that includes extrusion or expansion prior to
pelleting.
[0299] Accordingly, the invention further provides the use of an
amino acid sequence encoding a phytase or a host cell expressing a
phytase to produce a phytase for use in the manufacture of a food
or feed product. In one aspect, there is provided a use of an amino
acid sequence as described herein in the manufacture of a food or
feed product. In another aspect, there is provided a use of a host
cell in accordance with the invention in the manufacture of a food
or feed product. In another aspect, there is provided a use of an
expression vector or system in accordance with the invention in the
manufacture of a food or feed product.
[0300] The present invention also covers using the enzymes as a
component of feed combinations with other components to deliver to
animals.
Combination with Other Components
[0301] The enzymes of the present invention may be used in
combination with other components or carriers.
[0302] Suitable carriers for feed enzymes include wheat (coarsely
ground). In addition there are a number of encapsulation techniques
including those based on fat/wax coverage, adding plant gums
etc.
[0303] Examples of other components include one or more of:
thickeners, gelling agents, emulsifiers, binders, crystal
modifiers, sweetners (including artificial sweeteners), rheology
modifiers, stabilisers, anti-oxidants, dyes, enzymes, carriers,
vehicles, excipients, diluents, lubricating agents, flavouring
agents, colouring matter, suspending agents, disintegrants,
granulation binders etc. These other components may be natural.
These other components may be prepared by use of chemical and/or
enzymatic techniques.
[0304] As used herein the term "thickener or gelling agent" as used
herein refers to a product that prevents separation by slowing or
preventing the movement of particles, either droplets of immiscible
liquids, air or insoluble solids.
[0305] The term "stabiliser" as used here is defined as an
ingredient or combination of ingredients that keeps a product (e.g.
a food product) from changing over time.
[0306] The term "emulsifier" as used herein refers to an ingredient
(e.g. a food product ingredient) that prevents the separation of
emulsions.
[0307] As used herein the term "binder" refers to an ingredient
(e.g. a food ingredient) that binds the product together through a
physical or chemical reaction.
[0308] The term "crystal modifier" as used herein refers to an
ingredient (e.g. a food ingredient) that affects the
crystallisation of either fat or water.
[0309] "Carriers" or "vehicles" mean materials suitable for
compound administration and include any such material known in the
art such as, for example, any liquid, gel, solvent, liquid diluent,
solubiliser, or the like, which is non-toxic and which does not
interact with any components of the composition in a deleterious
manner.
[0310] Examples of nutritionally acceptable carriers include, for
example, grain, water, salt solutions, alcohol, silicone, waxes,
petroleum jelly, vegetable oils, and the like.
[0311] Examples of excipients include one or more of:
microcrystalline cellulose and other celluloses, lactose, sodium
citrate, calcium carbonate, dibasic calcium phosphate, glycine,
starch, milk sugar and high molecular weight polyethylene
glycols.
[0312] Examples of disintegrants include one or more of: starch
(preferably corn, potato or tapioca starch), sodium starch
glycollate, croscarmellose sodium and certain complex
silicates.
[0313] Examples of granulation binders include one or more of:
polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC),
hydroxypropylcellulose (HPC), sucrose, maltose, gelatin and
acacia.
[0314] Examples of lubricating agents include one or more of:
magnesium stearate, stearic acid, glyceryl behenate and talc.
[0315] Examples of diluents include one or more of: water, ethanol,
propylene glycol and glycerin, and combinations thereof.
[0316] The other components may be used simultaneously (e.g. when
they are in admixture together or even when they are delivered by
different routes) or sequentially (e.g. they may be delivered by
different routes).
[0317] As used herein the term "component suitable for animal or
human consumption" means a compound which is or can be added to the
composition of the present invention as a supplement which may be
of nutritional benefit, a fibre substitute or have a generally
beneficial effect to the consumer.
[0318] By way of example, the components may be prebiotics such as
alginate, xanthan, pectin, locust bean gum (LBG), inulin, guar gum,
galacto-oligosaccharide (GOS), fructo-oligosaccharide (FOS),
lactosucrose, soybean oligosaccharides, palatinose,
isomalto-oligosaccharides, gluco-oligosaccharides and
xylo-oligosaccharides.
Food or Feed Substance
[0319] The compounds may be used as--or in the preparation of--a
food or feed substance. Here, the term "food" is used in a broad
sense--and covers food and food products for humans as well as food
for animals (i.e. a feed). The term "feed" is used with reference
to products that are fed to animals in the rearing of livestock. In
a preferred aspect, the food or feed is for consumption by
monogastric animals such as pig, poultry and fish.
[0320] The food or feed may be in the form of a solution or as a
solid--depending on the use and/or the mode of application and/or
the mode of administration.
Food and Feed Ingredients and Supplements
[0321] The compounds may be used as a food or feed ingredient.
[0322] As used herein the term "food or feed ingredient" includes a
formulation, which is or can be added to foods or foodstuffs and
includes formulations which can be used at low levels in a wide
variety of products.
[0323] The food ingredient may be in the form of a solution or as a
solid--depending on the use and/or the mode of application and/or
the mode of administration.
[0324] The compounds may be--or may be added to--food
supplements.
Foods and Feed Compositions
[0325] Feed compositions for monogastric animals typically include
compositions comprising plant products which contain phytate. Such
compositions include cornmeal, soybean meal, rapeseed meal,
cottonseed meal, maize, wheat, barley and sorghum-based feeds.
[0326] The phytases described herein may be--or may be added
to--foods or feed compositions.
[0327] The present invention also provides a method of preparing a
food or a feed ingredient or supplement, the method comprising
admixing phytases produced by the process of the present invention
or the composition according to the present invention with another
food ingredient. The method for preparing or a food ingredient is
also another aspect of the present invention. Methods for preparing
animal feed are set out above. The enzyme can be added also in the
form of a solid formulation, or as a feed additive, such as a
pre-mix. A solid form is typically added before or during the
mixing step; and a liquid form is typically added after the
pelleting step.
Pharmaceutical
[0328] The phytases of the present invention may also be used in
pharmaceutical preparations or for combination into food stuffs in
order to provide some pharmaceutical effect. For example, EP
1,389,915 describes the use of a phytase in a food or drink for
increasing the availability of Calcium, Iron and/or Zinc of the
food or drink for humans
[0329] In addition, EP 1,392,353 describes a medicament or
nutritional supplement containing phytase, which is useful for
increasing bioavailability of bioelements, e.g., calcium and iron,
and for combating deficiency diseases.
[0330] Here, the term "pharmaceutical" is used in a broad
sense--and covers pharmaceuticals and/or nutraceuticals for humans
as well as pharmaceuticals and/or nutraceuticals for animals (i.e.
veterinary applications). In a preferred aspect, the pharmaceutical
is for human use and/or for animal husbandry.
[0331] The pharmaceutical can be for therapeutic purposes--which
may be curative or palliative or preventative in nature. The
pharmaceutical may even be for diagnostic purposes.
[0332] When used as--or in the preparation of--a pharmaceutical,
the product and/or the compounds of the present invention may be
used in conjunction with one or more of: a pharmaceutically
acceptable carrier, a pharmaceutically acceptable diluent, a
pharmaceutically acceptable excipient, a pharmaceutically
acceptable adjuvant, a pharmaceutically active ingredient.
[0333] The pharmaceutical may be in the form of a solution or as a
solid--depending on the use and/or the mode of application and/or
the mode of administration.
Pharmaceutical Ingredient
[0334] The product and/or the compounds of the present invention
may be used as pharmaceutical ingredients. Here, the product and/or
the composition of the present invention may be the sole active
component or it may be at least one of a number (i.e. 2 or more)
active components.
[0335] The pharmaceutical ingredient may be in the form of a
solution or as a solid--depending on the use and/or the mode of
application and/or the mode of administration.
[0336] The pharmaceutical ingredient may be in the form of an
effervescent product to improve the dissolving properties of the
pharmaceutical.
Forms
[0337] The product and/or the compounds of the present invention
may be used in any suitable form--whether when alone or when
present in a composition. Likewise, phytases produced in accordance
with the present invention (i.e. ingredients--such as food
ingredients, functional food ingredients or pharmaceutical
ingredients) may be used in any suitable form.
[0338] Suitable examples of forms include one or more of: tablets,
pills, capsules, ovules, solutions or suspensions, which may
contain flavouring or colouring agents, for immediate-, delayed-,
modified-, sustained-, pulsed- or controlled-release
applications.
[0339] By way of example, if the product and/or the composition are
used in a tablet form--such as for use as a functional
ingredient--the tablets may also contain one or more of:
excipients, disintegrants, granulation binders, or lubricating
agents.
[0340] Examples of nutritionally acceptable carriers for use in
preparing the forms include, for example, water, salt solutions,
alcohol, silicone, waxes, petroleum jelly and the like.
[0341] Preferred excipients for the forms include lactose, starch,
a cellulose, milk sugar or high molecular weight polyethylene
glycols.
[0342] For aqueous suspensions and/or elixirs, carotenoid cleavage
compounds may be combined with various sweetening or flavouring
agents, colouring matter or dyes, with emulsifying and/or
suspending agents and with diluents such as water, ethanol,
propylene glycol and glycerin, and combinations thereof.
[0343] The forms may also include gelatin capsules; fibre capsules,
fibre tablets etc.
General Recombinant DNA Methodology Techniques
[0344] The present invention employs, unless otherwise indicated,
conventional techniques of chemistry, molecular biology,
microbiology, recombinant DNA and immunology, which are within the
capabilities of a person of ordinary skill in the art. Such
techniques are explained in the literature. See, for example, J.
Sambrook, E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning:
A Laboratory Manual, Second Edition, Books 1-3, Cold Spring Harbor
Laboratory Press; Ausubel, F. M. et al. (1995 and periodic
supplements; Current Protocols in Molecular Biology, ch. 9, 13, and
16, John Wiley & Sons, New York, N.Y.); B. Roe, J. Crabtree,
and A. Kahn, 1996, DNA Isolation and Sequencing: Essential
Techniques, John Wiley & Sons; M. J. Gait (Editor), 1984,
Oligonucleotide Synthesis: A Practical Approach, Irl Press; and, D.
M. J. Lilley and J. E. Dahlberg, 1992, Methods of Enzymology: DNA
Structure Part A: Synthesis and Physical Analysis of DNA Methods in
Enzymology, Academic Press. Each of these general texts is herein
incorporated by reference.
EXAMPLES
[0345] The invention is now further illustrated in the following
non-limiting examples.
Example 1
Phytase Activity Assay
[0346] Phytase assays were carried out in microtitre plates. The
reaction mixture (100 .mu.l) contained: 2 mM phytate and 0.8 mM
CaCl.sub.2 in 200 mM sodium acetate buffer, pH 3.5. The reaction
was allowed to proceed for 1 h at 37.degree. C. after which time
the released phosphate was measured by a modification of a known
procedure (Heinonen J. K., Lahti R. J. Anal Biochem. 113 (2),
313-317 (1981)). Briefly, 200 .mu.l of a freshly prepared AMM
solution (7.5 N H.sub.2SO.sub.4, 15 mM ammonium molybdate and
acetone -1:1:2) was added to the 100 .mu.l reaction mixture in each
microtitre plate well. The absorbance at 390 nm was measured not
earlier than 10 min and not later than 30 min after addition of the
AMM reagent. The amount of phosphate was determined by building a
calibration curve with phosphate solutions of known concentrations.
For assaying phytase activity at different pH values the following
(all 200 mM) buffers were used: glycine/HCl between pH 2.0 and 3.0,
sodium acetate/acetic acid between pH 3.5 and 5.5, Tris/maleic acid
between pH 6.0 and 7.5.
Example 2
Phytase-Producing Strain P3-42
[0347] Bacterial strain P3-42 was originally isolated from a mass
of decaying birch leaves collected in a wet forest in southern
Finland. The strain can be aerobically cultivated at 30.degree. C.
on many simple culture media e.g. LB (1% peptome, 0.5% yeast
extract, 1% NaCl, pH 7.4) or low phosphate medium PP1 (1% peptone,
1%, beef extract, 0.5%, yeast extract, CaCl.sub.2-0.2M. The medium
is adjusted to pH 11 with NaOH and boiled for 10 min. The
precipitate is removed by filtration, pH re-adjusted to 5.5 and the
medium sterilised by autoclaving for 15 min at 121.degree. C.).
[0348] After growth in liquid PP1 medium the strain was found to
exhibit phytase activity both at pH 3.5 and 5.5 (assayed as
described in Example 1). The ratio of activities at 3.5 and 5.5 was
about 1.5. The activity was also measured separately in the cells
and culture supernatant of P3-42. According to these measurements
about 90% of all phytase activity was found in supernatant. The
strain was deposited with NCIMB on 22 Sep. 2004 under accession
number NCIMB 41247.
Example 3
Isolation of Chromosomal DNA from the Strain P3-42
[0349] Chromosomal DNA was prepared essentially by the standard
procedure (Ausubel et al., Current Protocols in Molecular Biology,
John Wiley & Sons, New York, 1996). A 250 ml culture grown
overnight at 30.degree. C. in LB medium was centrifuged at 10,000
rpm for 30 min, washed in 20 ml of 50 mM tris-HCl, 5 mM EDTA pH 8
and re-suspended in 10 ml of cold TES (50 mM tris-HCl, 5 mM EDTA,
15% glucose pH 8). Lysozyme was added to 10 mg/ml, and the cell
suspension was incubated at 37.degree. C. for 30-60 min until lysis
occurred, ascertained for by dilution of 100 .mu.l of the reaction
mixture into 1 ml of 1% SDS and checking for a "slimy" consistency.
At this time, SDS and Proteinase K (Sigma) were added to a final
concentration of 1% and 0.5 mg/ml respectively. The reaction
mixture was incubated for 30 min at 56.degree. C. followed by
addition of 2 ml of 5 M NaCl and 1.4 ml 10% cetyltrimethylammonium
bromide (Sigma). The incubation was continued for 15 min 65.degree.
C. The solution was extracted once with chloroform/isoamyl alcohol
(24:1) and once with phenol/chloroform. After the extractions, the
water phase was mixed with 0.6 vol of isopropanol, the DNA
precipitate collected by centrifugation (10,000 rpm, 15 min),
washed with 70% ethanol, vacuum dried and re-suspended in 2 ml of
10 mM tris-HCl, 1 mM EDTA pH 8, 5 .mu.g/ml. RNAse.
Example 4
Taxonomic Identification of the Bacterial Strain P3-42
[0350] A fragment of the 16S rRNA gene of the strain P3-42 was
amplified by the polymerase chain reaction (PCR) with Taq DNA
polymerase (Roche) using the primers; 536f (CAGCMGCCGCGGTAATWC)
(SEQ ID No. 4) and 1392r (ACGGGCGGTGTGTRC) (SEQ ID No. 5), (Lane,
D. J. In Nucleic acid techniques in bacterial systematics,
Stackbrandt, E. and Goodfellow, M. eds, John Wiley & Sons, New
York: pp 115-117 (1991)). The following program was used: 1)
initial DNA denaturation step of 5 min at 95.degree. C.; 2) 30
cycles of 1 min at 94.degree. C., 1 min at 55.degree. C., 1 min at
72.degree. C.; 3) a final extension step of 70.degree. C. for 10
min. The PCR products, approximately 900 base pairs in size, were
purified by electrophoresis in a 0.8% agarose gel and extracted
from the gel using a PCR Purification Kit (Qiagen) according to the
manufacturer's instructions. The purified PCR products were
sequenced by Medprobe (Norway) as a commercial service. The
sequenced area is listed as SEQ ID No 1. This sequence was compared
to DNA sequences in the GenBank database
(http://www.ncbi.nlm.nih.gov/blast/). The highest match (823 out of
824 nucleotides, 99.9%) was found with the sequence of 16S RNA gene
from Citrobacter freundii DSM 30039. Therefore, strain P3-42 can be
taxonomically classified as Citrobacter freundii.
Example 5
Cloning of the Phytase Gene from C. Freundii P3-42
[0351] Chromosomal DNA from the Citrobacter freundii strain P3-42
was partially digested with restriction endonuclease Sau3A and the
digest fractionated on 1% agarose gel. The DNA fragments of 3 to 5
kb were isolated from the gel using a gel purification kit (Qiagen)
and ligated with BamHI digested dephosphorylated .lamda.-ZAP arms
(Stratagene). Subsequent steps for library construction followed
the instructions of Stratagene's ZAP Express Predigested
Vector/Gigapack Cloning Kit. The phage form of the library was
converted into a plasmid form by the "mass excision" procedure as
described by the manufacturer (Stratagene). Screening of plasmid
library was done by similarly to the earlier published methods for
the detection of phytase activity on Petri plates (Howson and
Davis. Enzyme Microb. Technol. 5, 377-382 (1983); Chen J. C.
Biotechnology techniques 12 (10) 751-761 (1998); Riccio M. L. et
al, J. Appl. Microbiol. 82, 177-185 (1997)). Several
phytase-positive clones were isolated and purified by sub-cloning.
These isolates were grown in liquid culture (LB medium at
30.degree. C. and 200 rpm for about 24 h) and phytase activity was
measured (Example 1) in the resulting cell suspensions. One clone
that had the highest phytase activity (about 5 U/ml at pH 3.5) was
selected for subsequent characterisation. Plasmid DNA was isolated
this clone, named pBK(P3-42) and characterised by partial DNA
sequencing of the insert DNA (sequencing service was obtained from
Medprobe (Norway). This sequence comprising the phytase gene is
listed as SEQ ID No: 2. The deduced amino acid sequence of the C.
freundii phytase is listed as SEQ ID No: 3. Comparison of the SEQ
ID No: 3 with the sequences in GenBank using the BLAST service
provided by NCBI identifies the phytase from E. coli as the closest
known homologue of the C. freundii phytase. However, the level
homology is low--only about 62% of amino acid residues are
identical in both proteins.
Example 6
Amplification and Expression of Phytase Gene from C. Freundii
P3-42
[0352] Phytase gene was amplified by PCR. Chromosomal DNA of the
strain C. freundii P3-42 was used as template and oligonucleotides
o42-5 (GGAATTCATATGAGTACATTCATCATTCG) (SEQ ID No. 6) and o42-3
(GGAATTCGGATCCCTTATTCCGTAACTGCACAC) (SEQ ID No. 7) as primers. The
amplification was carried out using the Expand High Fidelity PCR
System kit (Roche). The following program was used: 1) initial DNA
denaturation for 3 min at 94.degree. C.; 2) 35 cycles of 45 sec at
94.degree. C., 45 sec at 55.degree. C., 1 min at 68.degree. C., 1
min at 72.degree. C., 1 min at 74.degree. C.; 3) a final extension
step of 10 min at 72.degree. C. The resulting PCR product was
purified by electrophoresis in a 0.8% agarose gel followed by DNA
extraction from the gel using a Gel Purification Kit (Qiagen). The
purified PCR product was digested with the restriction enzymes NdeI
and BamHI and isolated from the reaction mixture by the PCR
Purification Kit (Qiagen). The vector plasmid pET11a (Novagen) was
digested with the restriction endonucleases NdeI and BamHI,
de-phosphorylated using shrimp alkaline phosphatase (Roche) and
purified by electrophoresis in a 0.8% agarose gel. The linearised
plasmid DNA band was excised from the gel and purified using a Gel
Purification Kit (Qiagen). The two purified DNA fragments were
ligated using T4 DNA ligase (Roche). The ligation reaction was
precipitated with 70% ethanol, washed with ethanol and re-suspended
directly into 50 .mu.l of electrocompetent E. coli XL1-Blue MRF'
cells. The suspension was transferred to a 0.1 cm electroporation
cuvette (BioRad) and electroporated using a Gene Pulser Xcell
(BioRad) set at 1800 V, 25 .mu.F and 200.OMEGA.. Immediately after
electroporation 1 ml of LB medium was added, the cell suspension
was transferred to a 15 ml plastic tube (Falcon) and incubated at
37.degree. C. with shaking (200 rpm) for 1 hr. The transformed
cells were plated onto LB plates containing 100 .mu.g/ml ampicillin
and incubated overnight at 37.degree. C. 24 transformants were
grown in liquid culture and the cultures used for assaying phytase
activity and isolation of plasmid DNA. One clone producing highest
phytase activity and generating the expected restriction pattern of
the plasmid DNA was selected. The plasmid contained by this clone
named pET11(P3-42) was used to transform the expression host strain
BL21(DE3)pLysS (Novagen). The transformed cell suspension, was
shaken for 1 h at 37.degree. C. in LB containing 2% glucose and
inoculated into 50 ml of LB containing ampicillin (100 .mu.g/ml)
and glucose (2%) and grown overnight at 30.degree. C. with shaking
(200 rpm). The OD of the resulting culture was measured at 600 nm
and the culture was used to inoculate 1 l of LB+ampicillin (100
.mu.g/ml) to an OD.sub.600 of 0.04. Growth was continued overnight
at 30.degree. C. The phytase activity in such cultures was
typically 50-60 U/ml (measured at pH 3.5). Almost all of the
phytase was secreted into the culture medium. The fact that C.
freundii phytase is an efficiently secreted enzyme both in its
native host and in during heterologous expression in E. coli is in
contrast to the intracellular nature of a phytase from C. brakii
(Kim H. W. et al. Biotechnol. Lett. 25, 1231-1234 (2003)). The
activity in the culture of a control strain BL21(DE3)pLysS
transformed with pET11 grown under the same conditions was below
0.05 U/ml.
Example 7
Purification of the Recombinant Phytase from C. Freundii P3-42
[0353] The culture of BL21(DE3)pLysS transformed with pET11(P3-42)
was centrifuged to remove the bacterial cells, concentrated using a
rotary evaporator to about 1/10 of the original volume and dialysed
against water until the conductivity of the solution decreased
below 250 .mu.S/cm. The pH of the solution was adjusted to 8.0 with
tris base and it was applied to a column (3.times.20 cm) of DEAE
Sepharose Fast Flow (Amersham Biosciences) equilibrated with 25 mM
tris-HCl, pH 8.0. The column was washed with the equilibration
buffer at a flow rate of 3 ml/min for 30 min followed by elution
with three successive gradients of NaCl in 25 mM tris-HCl, pH 8.0:
0-50 mM, 50-150 mM and 150-500 mM. Each of the three gradients was
programmed for 1 h with a constant flow rate of 3 ml/min. 9 ml
fractions were collected and assayed for phytase activity. One
strong peak of activity was detected. The protein in the peak
fraction was concentrated using Centriplus concentrators (Amicon)
and analysed SDS PAGE using a 12% gel and the standard Laemmli
buffer system. The results of this analysis indicated that the
preparation of recombinant C. freundii P3-42 phytase obtained by
DEAE Sepharose contains a single prominent protein component.
Semi-quantitative analyses based on scanning of the digital image
of the gel (FIG. 1) indicate the purity of about 60-70%.
Example 8
pH Profile of the Recombinant Phytase from C. Freundii P3-42
[0354] Dependence of the activity of the C. freundii P3-42 phytase
from (purified according to the Example 7) on pH was studied in
buffers and under conditions described in Example 1 The enzyme was
active in a broad pH area (2-5.5) with two activity maxima around
pH 3 and 4-4.5 (FIG. 2).
Example 9
Substrate Specificity of the Recombinant Phytase from C. Freundii
P3-42
[0355] The fractions of inositol phosphates containing three, four
or five phosphates per inositol residue were isolated by
ion-exchange chromatography from a partial hydrolysate of phytic
acid treated with fungal phytase (Natuphos). Production and
purification of these preparations was a commercial service of
BioChemis Ltd (St. Petersburg, Russia). Contamination of each
fraction with inositol-phosphates having a different degree of
phosphorylation was less that 5% as judged by HPLC (Sandberg A. S.,
Ahderinne R. J. Food Sci. 51 (3), 547-550). Commercial fructose
1,6-diphosphate and fructose 6-phosphate (Sigma) were used as model
substrates used to estimate the specificity of the C. freundii
P3-42 phytase towards di- and monophosphate substrates. The
activity of the C. freundii phytase purified according to the
Example 7 with different substrates was measured by the standard
assay (Example 1) at pH 3.5 using 2 mM concentrations of substrates
in the final reaction mixture. The results (FIG. 3) indicate that
that the enzyme has maximum activity with inositol pentaphosphate.
Activities with inositol tri- and tetraphosphates as well as phytic
acid were rather similar while fructose 1,6-diphosphate was a
rather poor substrate. Hydrolysis of fructose 6-phosphate was below
reliable detection limit
Example 10
Specific Activity of the Recombinant Phytase from C. Freundii
P3-42
[0356] Specific activity of the C. freundii phytase was estimated
using the purified preparation according to the Example 7. The
phytase activity was measured at pH 3.5 according to the Example 1.
Phytase concentration was calculated by measuring total protein
concentration with BCA Protein Assay Kit (Pierce) and correcting it
by phytase content estimated by SDS PAGE (Example 7). According to
these measurements, the specific activity of the recombinant
phytase from C. freundii P3-42 is about 1100 U/mg.
Example 11
Comparison of C. Freundii P3-42 Phytase with the Phytase from C.
brakii YH-15
[0357] The only phytase from a bacterium belonging to the
Citrobacter family described earlier is the intracellular phytase
from C. brakii YH-15 (Kim H. W. et al. Biotechnol. Lett. 25,
1231-1234 (2003)). This enzyme shares some properties with the
secreted phytase of C. freundii of the present invention both
enzymes are acid phytases of high specific activity. Direct
comparison of the amino acid sequences of the two enzymes is
impossible because the sequence information regarding the C. brakii
enzyme is limited to a stretch of 10 amino acid residues. The
deduced amino acid sequence of C. freundii phytase contains a
fragment sharing 9 out of 10 residues with the sequence from C.
brakii enzymes. However, comparison of such short fragments of
sequence does not allow any conclusions about the overall homology
of the two enzymes to be made. The most striking difference between
the two enzymes is in cellular location: while the enzyme from C.
brakii is intracellular, the C. freundii phytase is clearly a
secreted enzyme. The enzyme is secreted in its native host, its
deduced amino acid sequence does contain a signal peptide (as
predicted by the Signal P algorithm:
http://www.cbs.dtu.dk/services/SignaIP/), the enzyme is also very
efficiently secreted from E. coli under its native signal peptide.
In addition to that, there are a number of significant differences
in biochemical properties of the two enzymes (Table 1). Table 1
Comparison of the Phytase from C. Freundii P3-42 with Phytase from
C. brakii YH-14
TABLE-US-00002 Property C. brakii YH-15 phytase C. freundii P3-42
phytase Localisation Intracellular Secreted Specific activity 3457
U/mg (pH 4) 1100 U/mg (pH 3.5) pH optimum 4.0 3.0, 5.0
Thermostability 20%.sup.(*.sup.) 58% .sup.(*.sup.)Measured under
conditions described by Kim et al. (Biotechnol. Lett. 25, 1231-1234
(2003)): heat treatment in 100 mM Na acetate, pH 4, 60.degree. C.,
30 min followed by standard assay at 37.degree. C.
Example 12
Generation and Characterisation of Phytase Variants
[0358] Phytase variants were constructed by mutagenesis of the
sequence SEQ ID No. 2 using mutagenesis methods as listed above
such as the methods disclosed in Morinaga et al (Biotechnology
(1984) 2, p 646-649), or in Nelson and Long (Analytical
Biochemistry (1989), 180, p 147-151), or the Error Threshold
Mutagenesis protocol described in WO 92/18645.
[0359] Phytase enzyme variants were characterized after
heterologous expression in one or more of the following expression
hosts: Escherichia coli K12; Bacillus subtilis; Saccharomyces
cerevisiae.
1. Thermostability
[0360] The thermostability of the variants was characterized by the
inactivation temperature of the enzyme. The inactivation
temperature was determined by measuring the residual activity of
the enzyme in an enzyme assay as described in Example 1 after
incubation for 10 min at different temperatures and subsequent
cooling to room temperature. The inactivation temperature is the
temperature at which the residual activity is 50% compared to the
residual activity after incubation for the same duration under the
same conditions at room temperature. Where appropriate
interpolations and extrapolations from the measured activity data
are done in order to determine the temperature corresponding to 50%
residual activity. Thermostability differences in .degree. C. were
calculated by subtracting the inactivation temperatures of two
enzymes from each other. (i.e. Thermostability difference (T.D.) is
compared to parent phytase (=inactivation temperature
(variant)-inactivation temperature (parent))
[0361] Table 2 lists the thermostability differences for different
variants:
TABLE-US-00003 TABLE 2 Thermostability differences for variants
derived from the parent phytase P3-42 having the sequence shown in
Seq ID No 3. Variant T.D. P229S 1.5 D112V 1.5 Q82R 1.5 Q274H 1.0
D112Y 2.5 F88Y 1.5 K46E 2.0 S233C 2.0 R288M 4.0 I384L 1.0 Q385R 1.5
Q274L 2.0 E307Y 1.0 T199I 2.0 Q82K 2.0 T203I 1.0 K46E/Q82H 2.5
Q82K/V105I 1.0 N148D/T362I 1.5 K46E/L414I 1.0 F88Y/Y136N 1.0
N95P/N96S 1.5 N95P/N96P 2.0 Q97T/T98G 1.0 Y177F/T199I 2.5
Q274L/Q370H 3.0 K46E/N96Y 2.5 N148D/L301S 1.5 E24D/R288M 1.5
E140V/A322V 2.0 K46E/S195T 2.0 E75K/N365D 1.5 T98P/S235A 2.0
L160F/L215F 1.0 Q274L/K395T 1.5 G67R/Q279E/N308T 2.0
K161N/P229S/R288M 2.0 D53N/D57Y/M152V 2.0 F122Y/S156T/P229S 1.5
E23K/K46E/Q82H 6.0 K46E/Q82H/Q385R 5.0 T203W/E204N/K205R 2.0
T203W/E204H/K205R 3.0 T203W/E204R/K205R 3.0 T203W/E204A/K205R 3.0
A22T/K151G/N308D 2.0 E23K/E75K/F88Y 2.0 M152K/N225D/L301S 2.0
S78T/Q274L/S408I 2.0 L176Q/T199I/T366S 1.5 K46E/V77I/T203S 3.0
K46R/T199I/D367N 1.5 G74R/E204G/R288M 1.5 A22T/T199I/S206T/T207A
1.5 Q82R/F88Y/L126I/I384L 3.0 K46E/Q82H/E168D/Q274L 5.0
Q82K/T154I/Q279E/N308T 5.5 Q82R/D112V/Q274H/T362A 5.0
E24D/E79V/N95D/K360N 1.0 E23K/M28L/A109T/T143P/I384L 2.0
D53N/D57Y/T199I/P229S/R288M 6.0 K46E/Q82H/N148D/T154I/T362I 7.0
D53N/D57Y/P229S/R288M/K358R 5.5 D53N/D57Y/T154I/P229S/R288M 7.0
Y136N/T199I/T203L/E204I/K205P 3.0 E23Q/S101F/Q274L/I384M/K391N 2.0
K46E/Q82H/N95D/D112V/K142R/D383V 5.5
D53N/D57Y/M152V/P229S/R288M/A393P 7.0
D53K/D57Y/M152V/P229S/R288M/A393P 8.0
D53N/D57Y/F88Y/M152V/P229S/Q279E/N308T 6.5
D53N/D57Y/M152V/E204V/P229S/R288M/A393P 8.0
D53N/D57Y/M152V/T154I/P229S/R288M/A393P 8.0
D53N/D57Y/Q82H/G103E/M152V/P229S/R288M/A393P 8.5
K46E/D53N/D57Y/T143I/M152V/L176V/P229S/R288M/A393P 8.0
Q82K/F88Y/N96P/Q97T/T98G/V105I/Q274H/Q279E/A393P 9.0
Q82R/F88Y/N95P/N96P/Q97T/Q279E/I384L/P386Q/A393P 9.0
D53N/D57Y/E75V/M152V/A170T/P229S/R288M/Q385R/A393P 7.5
Q82K/F88Y/N96P/T98G/Y136N/M152V/Y177F/T362I/I384F/ 10.0 A393P/D397N
D53N/D57Y/F88Y/N95P/N96P/V105I/D112V/Y136N/N148D/ 10.0
N164D/Q274H/T362I/I384L/A393P
D53N/D57Y/Q82K/F88Y/N95P/P102L/V105I/Y136N/N148D/ 10.0
Y177F/Q274H/Q279E/T362I/A393P
D53N/D57Y/Q82K/F88Y/N96P/T98G/V105I/D112V/Y177F/ 9.0
Q274L/G343A/T362I/I384L/A393P
2. Other Characteristics
[0362] Other characteristics were also improved.
[0363] Thermostability, specific activity, and pepsin stability of
selected variants were compared using assays as described above.
The pepsin stability of such variants was characterized by residual
activities measured at pH 3.5, 37.degree. C. after pepsin
incubation compared to control conditions (residual
activity=activity after pepsin incubation/activity after incubation
under control conditions). The pepsin incubation was performed for
2 hours at pH 2.0, 0.25 mg/ml pepsin, 1 mM CaCl2 and 5 mg/ml BSA at
37.degree. C. Control conditions were 2 hours at pH 5.0, 1 mM CaCl2
and 5 mg/ml BSA at 37.degree. C.
[0364] Table 3 shows properties of selected variants (derived from
and compared to wt phytase according to Seq ID No. 3).
TABLE-US-00004 Specific Pepsin stability activity [% [% residual
activity] Variant T.D. [.degree. C.] of wt activity] (wt = 41%)
K46E/ 2.2 96 65 Q82H
SEQUENCE INFORMATION
TABLE-US-00005 [0365] SEQ ID No: 1
CGATTACTAGCGATTCCGACTTCTGGAGTCGAGTTGCAGACTCCAATCCG
GACTACGACATACTTTATGAGGTCCGCTTGCTCTCGCGAGGTCGCTTCTC
TTTGTATATGCCATTGTAGCACGTGTGTAGCCCTACTCGTAAGGGCCATG
ATGACTTGACGTCATCCCCACCTTCCTCCAGTTTATCACTGGCAGTCTCC
TTTGAGTTCCCGGCCGAACCGCTGGCAACAAAGGATAAGGGTTGCGCTCG
TTGCGGGACTTAACCCAACATTTCACAACACGAGCTGACGACAGCCATGC
AGCACCTGTCTCAGAGTTCCCGAAGGCACCAAAGCATCTCTGCTAAGTTC
TCTGGATGTCAAGAGTAGGTAAGGTTCTTCGCGTTGCATCGAATTAAACC
ACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCATTTGAGTTTTAAC
CTTGCGGCCGTACTCCCCAGGCGGTCGACTTAACGCGTTAGCTCCGGAAG
CCACGCCTCAAGGGCACAACCTCCAAGTCGACATCGTTTACGGCGTGGAC
TACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGCACCTGAGCGT
CAGTCTTTGTCCAGGGGGCCGCCTTCGCCACCGGTATTCCTCCAGATCTC
TACGCATTTCACCGCTACACCTGGAATTCTACCCCCCTCTACAAGACTCT
AGCCTGCCAGTTTCGGATGCAGTTCCCAGGTTGAGCCCGGGGATTTCACA
TCCGACTTGACAGACCGCCTGCGTGCGCTTTACGCCCAGTAATTCCGATT
AACGCTTGCACCCTCCGTATTAC SEQ ID No 2:
AAAGGTGGTGCTGGTAATGAGTACATTCATCATTCGTTTATTATTTTTTT
CTCTCTTATGCGGTTCTTTCTCAATACATGCTGAAGAGCCGAACGGTATG
AAACTTGAGCGGGTTGTGATAGTGAGCCGTCATGGAGTAAGAGCACCTAC
GAAGTTCACTCCAATAATGAAAGATGTTACACCCGATCAATGGCCACAAT
GGGATGTGCCGTTAGGATGGCTAACGCCTCGTGGGGGAGAACTTGTTTCT
GAATTAGGTCAGTATCAACGTTTATGGTTCACAAGCAAAGGTCTGTTGAA
TAATCAAACGTGCCCATCTCCAGGGCAGGTTGCTGTTATTGCAGACACGG
ATCAACGCACCCGTAAAACGGGTGAGGCGTTTCTGGCTGGGTTAGCACCA
AAATGTCAAATTCAAGTGCATTATCAGAAGGATGAAGAAAAAACTGATCC
TCTTTTTAATCCAGTAAAAATGGGGACATGTTCGTTTAACACATTGAAGG
TTAAAAACGCTATTCTGGAACGGGCCGGAGGAAATATTGAACTGTATACC
CAACGCTATCAATCTTCATTTCGGACCCTGGAAAATGTTTTAAATTTCTC
ACAATCGGAGACATGTAAGACTACAGAAAAGTCTACGAAATGCACATTAC
CAGAGGCTTTACCGTCTGAACTTAAGGTAACTCCTGACAATGTATCATTA
CCTGGTGCCTGGAGTCTTTCTTCCACGCTGACTGAGATATTTCTGTTGCA
AGAGGCCCAGGGAATGCCACAGGTAGCCTGGGGGCGTATTACGGGAGAAA
AAGAATGGAGAGATTTGTTAAGTCTGCATAACGCTCAGTTTGATCTTTTG
CAAAGAACTCCAGAAGTTGCCCGTAGTAGGGCCACACCATTACTCGATAT
GATAGACACTGCATTATTGACAAATGGTACAACAGAAAACAGGTATGGCA
TAAAATTACCCGTATCTCTGTTGTTTATTGCTGGTCATGATACCAATCTT
GCAAATTTAAGCGGGGCTTTAGATCTTAACTGGTCGCTGCCCGGTCAACC
CGATAATACCCCTCCTGGTGGGGAGCTTGTATTCGAAAAGTGGAAAAGAA
CCAGTGATAATACGGATTGGGTTCAGGTTTCATTTGTTTATCAGACGCTG
AGAGATATGAGGGATATACAACCGTTGTCGTTAGAAAAACCTGCCGGCAA
AGTTGATTTAAAATTAATTGCATGTGAAGAGAAAAATAGTCAGGGAATGT
GTTCGTTAAAAAGTTTTTCCAGGCTCATTAAGGAAATTCGCGTGCCAGAG
TGTGCAGTTACGGAATAAGTAACTAATTACTATATATAGCGTATTAAAAA
ATAGAAACCCCCGGTTTGTAGTCGGGGGTATTCGTATTGTTCATAATTAC A SEQ ID No: 3
MSTFIIRLLFFSLLCGSFSIHAEEPNGMKLERVVIVSRHGVRAPTKFTPI
MKDVTPDQWPQWDVPLGWLTPRGGELVSELGQYQRLWFTSKGLLNNQTCP
SPGQVAVIADTDQRTRKTGEAFLAGLAPKCQIQVHYQKDEEKTDPLFNPV
KMGTCSFNTLKVKNAILERAGGNIELYTQRYQSSFRTLENVLNFSQSETC
KTTEKSTKCTLPEALPSELKVTPDNVSLPGAWSLSSTLTEIFLLQEAQGM
PQVAWGRITGEKEWRDLLSLHNAQFDLLQRTPEVARSRATPLLDMIDTAL
LTNGTTENRYGIKLPVSLLFIAGHDTNLANLSGALDLNWSLPGQPDNTPP
GGELVFEKWKRTSDNTDWVQVSFVYQTLRDMRDIQPLSLEKPAGKVDLKL
IACEEKNSQGMCSLKSFSRLIKEIRVPECAVTE
[0366] All publications mentioned in the above specification, and
references cited in said publications, are herein incorporated by
reference. Various modifications and variations of the described
methods and system of the present invention will be apparent to
those skilled in the art without departing from the scope and
spirit of the present invention. Although the invention has been
described in connection with specific preferred embodiments, it
should be understood that the invention as claimed should not be
unduly limited to such specific embodiments. Indeed, various
modifications of the described modes for carrying out the invention
which are obvious to those skilled in molecular biology or related
fields are intended to be within the scope of the following
claims.
[0367] The invention will now be further described by the following
numbered paragraphs:
1. An isolated polypeptide comprising the amino acid sequence
corresponding to Citrobacter freundii phytase or a homologue, a
modified form, a functional equivalent or an effective fragment
thereof. 2. An isolated polypeptide as paragraphed in paragraph 1
comprising the amino acid sequence as shown in SEQ ID NO: 3 or a
sequence having at least 75% identity (homology) thereto or a
functional fragment thereof. 3. An isolated polypeptide having the
amino acid sequence as set out in SEQ ID NO: 3 or a sequence having
at least 75% identity (homology) thereto or a functional fragment
thereof. 4. A phytase characterised in that it is derived from
Citrobacter freundii strain P3-42 deposited under accession number
NCIMB 41247. 5. A phytase or functional equivalent thereof
characterised in that said phytase has a specific activity of at
least 1100 U/mg wherein said specific activity is determined by
incubating said phytase in a solution containing 2 mM phytate, 0.8
mM CaCl.sub.2 in 200 mM sodium acetate buffer at pH 3.5. 6. A
phytase or functional equivalent thereof characterised in that said
phytase has two activity maxima around pH 3 and pH 4-4.5 wherein
said activity is determined by incubating said phytase in a
solution containing 2 mM phytate, 0.8 mM CaCl.sub.2 in 200 mM
sodium acetate buffer. 7. An isolated polypeptide or phytase as
paragraphed in any of paragraphs 1 to 6 that comprises one or more
mutations at the following positions (numbering according to the
numbering in SEQ ID No. 3): 22, 23, 24, 28, 46, 53, 57, 67, 74, 75,
77, 78, 79, 82, 88, 95, 96, 97, 98, 101, 102, 103, 105, 109, 112,
122, 126, 136, 140, 142, 143, 148, 151, 152, 154, 156, 160, 161,
164, 168, 170, 176, 177, 195, 199, 203, 204, 205, 206, 207, 215,
224, 225, 229, 233, 235, 274, 279, 288, 301, 307, 308, 322, 343,
358, 360, 362, 365, 366, 367, 370, 383, 384, 385, 386, 391, 393,
395, 397, 408, 414. 8. An isolated polypeptide or phytase as
paragraphed in paragraph 7 wherein said phytase comprises one or
more of the following mutations: A22T, E23K, E23Q, E24D, M28L,
K46E, K46R, D53K, D53N, D57Y, G67R, G74R, E75K, E75V, V77I, S78T,
E79V, Q82H, Q82K, Q82R, F88Y, N95D, N95P, N96P, N96S, N96Y, Q97T,
T98G, T98P, S101F, P102L, G103E, V105I, A109T, D112V, D112Y, F122Y,
L126I, Y136N, E140V, K142R, T143I, T143P, N148D, K151G, M152K,
M152V, T154I, S156T, L160F, K161N, N164D, E168D, A170T, L176Q,
L176V, Y177F, S195T, T199I, T203I, T203L, T203S, T203W, E204A,
E204G, E204H, E204I, E204N, E204R, E204V, K205P, K205R, S206R,
S206T, T207A, T207S, L215F, D224H, N225D, N225E, P229S, S233C,
S235A, Q274H, Q274L, Q279E, R288M, L301S, E307Y, N308D, N308T,
A322V, G343A, K358R, K360N, T362A, T362I, N365D, T366S, D367N,
Q370H, D383V, I384F, I384L, I384M, Q385R, P386Q, K391N, A393P,
K395T, D397N, S408I, L414I. 9. An isolated polypeptide or phytase
as paragraphed in paragraph 7 or paragraph 8 comprising one
mutation selected from the group consisting of:
P229S; D112V; Q82R; Q274H; D112Y; F88Y; K46E; S233C; R288M; I384L;
Q385R; Q274L; E307Y; T199I; Q82K and T203I.
[0368] 10. An isolated polypeptide or phytase as paragraphed in
paragraph 7 or 8 comprising a combination of mutations selected
from the group consisting of: K46E/Q82H; Q82K/V105I; N148D/T362I;
K46E/L414I; F88Y/Y136N; T154I/P386Q; N95P/N96S; N95P/N96P;
Q97T/T98G; D224H/N225E; Y177F/T199I; Q274L/Q370H; K46E/N96Y;
N148D/L301S; E24D/R288M; E140V/A322V; K46E/S195T; E75K/N365D;
T98P/S235A; L160F/L215F; Q274L/K395T; G67R/Q279E/N308T;
K161N/P229S/R288M; D53N/D57Y/M152V; F122Y/S156T/P229S;
T199I/S206R/T207S; E23K/K46E/Q82H; K46E/Q82H/Q385R;
T203W/E204N/K205R; T203W/E204H/K205R; T203W/E204R/K205R;
T203W/E204A/K205R; A22T/K151G/N308D; E23K/E75K/F88Y;
M152K/N225D/L301S; S78T/Q274L/S408I; L176Q/T199I/T366S;
K46E/V77I/T203S; K46R/T199I/D367N; G74R/E204G/R288M;
A22T/T199I/S206T/T207A; Q82R/F88Y/L126I/I384L;
K46E/Q82H/E168D/Q274L; Q82K/T154I/Q279E/N308T;
Q82R/D112V/Q274H/T362A; E24D/E79V/N95D/K360N;
E23K/M28L/A109T/T143P/I384L; D53N/D57Y/T199I/P229S/R288M;
K46E/Q82H/N148D/T154I/T362I; D53N/D57Y/P229S/R288M/K358R;
D53N/D57Y/T154I/P229S/R288M; Y136N/T199I/T203L/E204I/K205P;
E23Q/S101F/Q274L/I384M/K391N; K46E/Q82H/N95D/D112V/K142R/D383V;
D53N/D57Y/M152V/P229S/R288M/A393P;
D53K/D57Y/M152V/P229S/R288M/A393P;
D53N/D57Y/F88Y/M152V/P229S/Q279E/N308T;
D53N/D57Y/M152V/E204V/P229S/R288M/A393P;
D53N/D57Y/M152V/T154I/P229S/R288M/A393P;
D53N/D57Y/Q82H/G103E/M152V/P229S/R288M/A393P;
K46E/D53N/D57Y/T143I/M152V/L176V/P229S/R288M/A393P;
Q82K/F88Y/N96P/Q97T/T98G/V105I/Q274H/Q279E/A393P;
Q82R/F88Y/N95P/N96P/Q97T/Q279E/I384L/P386Q/A393P;
H18Q/D53N/D57Y/E75V/M152V/A170T/P229S/R288M/Q385R/A393P;
Q82K/F88Y/N96P/T98G/Y136N/M152V/Y177F/T362I/I384F/A393P/D397N;
D53N/D57Y/F88Y/N95P/N96P/V105I/D112V/Y136N/N148D/N164D/Q274H/T362I/I384L/A-
393P;
D53N/D57Y/Q82K/F88Y/N95P/P102L/V105I/Y136N/N148D/Y177F/Q274H/Q279E/T362I/A-
393P;
D53N/D57Y/Q82K/F88Y/N96P/T98G/V105I/D112V/Y177F/Q274L/G343A/T362I/I384L/A3-
93P;
[0369] 11. An isolated nucleic acid molecule coding for the enzyme
of Citrobacter freundii phytase, or a homologue thereof. 12. An
isolated nucleic acid molecule as paragraphed in paragraph 11
encoding a polypeptide comprising the amino acid sequence as shown
in SEQ ID NO: 3 or a sequence having at least 75% identity
(homology) thereto or an effective fragment thereof. 13. An
isolated nucleic acid molecule comprising a nucleotide sequence
that is the same as, or is complementary to, or contains any
suitable codon substitutions for any of those of SEQ ID NO: 2 or
comprises a sequence which has at least 75%, 80%, 85%, 90%, 95% or
99% sequence homology with SEQ ID NO: 2. 14. An isolated nucleic
acid molecule coding for the isolated polypeptide or phytase as
paragraphed in any of paragraphs 1 to 10. 15. An isolated nucleic
acid molecule comprising the sequence as set out in SEQ ID NO: 2.
16. A plasmid or vector system comprising an isolated polypeptide
or phytase as paragraphed in any of paragraphs 1 to 10 or a
homologue or derivative thereof. 17. A plasmid or vector system as
paragraphed in paragraph 16 which comprises a nucleic acid sequence
as paragraphed in any of paragraphs 11 to 15. 18. A plasmid or
vector system as paragraphed in paragraph 16 which comprises a
nucleic acid sequence as set out in SEQ ID No: 2 or a sequence that
is at least 75% homologous thereto or an effective fragment
thereof. 19. A plasmid or vector system as paragraphed in any of
paragraphs 16 to 18 wherein said plasmid or vector system is an
expression vector for the expression of the respective phytase
enzyme or homologue, modified form, functional equivalent or
effective fragment thereof, in a host cell or a microorganism. 20.
A host cell transformed or transfected with a plasmid or vector
system as paragraphed in any of paragraphs 16 to 19. 21. A host
cell as paragraphed in paragraph 20 which comprises a phytase which
comprises an amino acid sequence, or functional fragment thereof,
as set out in SEQ ID NO: 3, or a sequence that is at least 75%
homologous thereto, or a variant thereof according to any of
paragraphs 7-10. 22. A host cell as paragraphed in paragraph 20 or
21 wherein said host cell is derived from a microorganism including
bacteria, such as B. subtilis, E. coli and fungi, including yeast
such as H. polymorpha, S. pombe, S. cerevisiae. 23. A host cell as
paragraphed in paragraph 22 wherein said microorganism is a
prokaryotic bacterial cell and, preferably, E. coli. 24. A
bacterial cell strain Citrobacter freundii P3-42 deposited under
accession number NCIMB 41247. 25. A method of producing a phytase
comprising expressing an amino acid sequence as set out in SEQ ID
NO: 3, or a sequence having at least 75% homology thereto or a
modified form or a variant or an effective fragment thereof in a
host cell and separating the phytase from the host cell culture
medium. 26. A food or animal feed composition comprising a phytase
as paragraphed in any of paragraphs 1 to 25. 27. Use of a phytase
as paragraphed in any of paragraphs 1 to 10 in food or animal feed.
28. A method for production of food or animal feed comprising a
step of spraying a phytase as paragraphed in any of paragraphs 1 to
10 in liquid form onto said food or animal feed. 29. A method for
production of food or animal feed comprising a step of mixing the
phytase as paragraphed in any of paragraphs 1 to 10 as a dry
product with said food or animal feed. 30. A method of preparing a
phytase enzyme variant, which method comprises: [0370] a) selecting
a parent phytase enzyme, wherein the parent phytase enzyme is
selected from: [0371] i. a parent phytase enzyme with at least 75%
homology to SEQ ID NO 3 [0372] ii. a parent phytase enzyme derived
from Citrobacter spp. [0373] b) making at least one alteration
which is an insertion, a deletion or a substitution of an amino
acid residue in the parent phytase enzyme to obtain a phytase
enzyme variant [0374] c) screening for a phytase enzyme variant
which compared to the parent phytase enzyme has: [0375] i. higher
thermal stability and/or [0376] ii. specific activity and/or [0377]
iii. proteolytic stability [0378] d) preparing the phytase enzyme
variant. 31. A method of preparing a phytase enzyme variant, which
method comprises: [0379] a) subjecting DNA sequence encoding a
parent phytase enzyme to mutagenesis, wherein the parent phytase is
selected from [0380] i. a parent phytase enzyme with at least 75%
homology to SEQ ID NO 3 [0381] ii. a parent phytase enzyme derived
from Citrobacter spp. [0382] b) expressing the mutated DNA sequence
obtained in strep (A) in a host cell, and [0383] c) screening for
host cells expressing a phytase enzyme variant which compared to
the parent phytase enzyme has: [0384] i. higher thermal stability
and/or [0385] ii. higher specific activity and/or [0386] iii.
higher proteolytic stability [0387] d) preparing the phytase enzyme
variant expressed by the host cell.
Sequence CWU 1
1
81823DNACitrobacter freundii 1cgattactag cgattccgac ttctggagtc
gagttgcaga ctccaatccg gactacgaca 60tactttatga ggtccgcttg ctctcgcgag
gtcgcttctc tttgtatatg ccattgtagc 120acgtgtgtag ccctactcgt
aagggccatg atgacttgac gtcatcccca ccttcctcca 180gtttatcact
ggcagtctcc tttgagttcc cggccgaacc gctggcaaca aaggataagg
240gttgcgctcg ttgcgggact taacccaaca tttcacaaca cgagctgacg
acagccatgc 300agcacctgtc tcagagttcc cgaaggcacc aaagcatctc
tgctaagttc tctggatgtc 360aagagtaggt aaggttcttc gcgttgcatc
gaattaaacc acatgctcca ccgcttgtgc 420gggcccccgt caattcattt
gagttttaac cttgcggccg tactccccag gcggtcgact 480taacgcgtta
gctccggaag ccacgcctca agggcacaac ctccaagtcg acatcgttta
540cggcgtggac taccagggta tctaatcctg tttgctcccc acgctttcgc
acctgagcgt 600cagtctttgt ccagggggcc gccttcgcca ccggtattcc
tccagatctc tacgcatttc 660accgctacac ctggaattct acccccctct
acaagactct agcctgccag tttcggatgc 720agttcccagg ttgagcccgg
ggatttcaca tccgacttga cagaccgcct gcgtgcgctt 780tacgcccagt
aattccgatt aacgcttgca ccctccgtat tac 82321401DNACitrobacter
freundii 2aaaggtggtg ctggtaatga gtacattcat cattcgttta ttattttttt
ctctcttatg 60cggttctttc tcaatacatg ctgaagagcc gaacggtatg aaacttgagc
gggttgtgat 120agtgagccgt catggagtaa gagcacctac gaagttcact
ccaataatga aagatgttac 180acccgatcaa tggccacaat gggatgtgcc
gttaggatgg ctaacgcctc gtgggggaga 240acttgtttct gaattaggtc
agtatcaacg tttatggttc acaagcaaag gtctgttgaa 300taatcaaacg
tgcccatctc cagggcaggt tgctgttatt gcagacacgg atcaacgcac
360ccgtaaaacg ggtgaggcgt ttctggctgg gttagcacca aaatgtcaaa
ttcaagtgca 420ttatcagaag gatgaagaaa aaactgatcc tctttttaat
ccagtaaaaa tggggacatg 480ttcgtttaac acattgaagg ttaaaaacgc
tattctggaa cgggccggag gaaatattga 540actgtatacc caacgctatc
aatcttcatt tcggaccctg gaaaatgttt taaatttctc 600acaatcggag
acatgtaaga ctacagaaaa gtctacgaaa tgcacattac cagaggcttt
660accgtctgaa cttaaggtaa ctcctgacaa tgtatcatta cctggtgcct
ggagtctttc 720ttccacgctg actgagatat ttctgttgca agaggcccag
ggaatgccac aggtagcctg 780ggggcgtatt acgggagaaa aagaatggag
agatttgtta agtctgcata acgctcagtt 840tgatcttttg caaagaactc
cagaagttgc ccgtagtagg gccacaccat tactcgatat 900gatagacact
gcattattga caaatggtac aacagaaaac aggtatggca taaaattacc
960cgtatctctg ttgtttattg ctggtcatga taccaatctt gcaaatttaa
gcggggcttt 1020agatcttaac tggtcgctgc ccggtcaacc cgataatacc
cctcctggtg gggagcttgt 1080attcgaaaag tggaaaagaa ccagtgataa
tacggattgg gttcaggttt catttgttta 1140tcagacgctg agagatatga
gggatataca accgttgtcg ttagaaaaac ctgccggcaa 1200agttgattta
aaattaattg catgtgaaga gaaaaatagt cagggaatgt gttcgttaaa
1260aagtttttcc aggctcatta aggaaattcg cgtgccagag tgtgcagtta
cggaataagt 1320aactaattac tatatatagc gtattaaaaa atagaaaccc
ccggtttgta gtcgggggta 1380ttcgtattgt tcataattac a
14013433PRTCitrobacter freundii 3Met Ser Thr Phe Ile Ile Arg Leu
Leu Phe Phe Ser Leu Leu Cys Gly1 5 10 15Ser Phe Ser Ile His Ala Glu
Glu Pro Asn Gly Met Lys Leu Glu Arg 20 25 30Val Val Ile Val Ser Arg
His Gly Val Arg Ala Pro Thr Lys Phe Thr 35 40 45Pro Ile Met Lys Asp
Val Thr Pro Asp Gln Trp Pro Gln Trp Asp Val 50 55 60Pro Leu Gly Trp
Leu Thr Pro Arg Gly Gly Glu Leu Val Ser Glu Leu65 70 75 80Gly Gln
Tyr Gln Arg Leu Trp Phe Thr Ser Lys Gly Leu Leu Asn Asn 85 90 95Gln
Thr Cys Pro Ser Pro Gly Gln Val Ala Val Ile Ala Asp Thr Asp 100 105
110Gln Arg Thr Arg Lys Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro
115 120 125Lys Cys Gln Ile Gln Val His Tyr Gln Lys Asp Glu Glu Lys
Thr Asp 130 135 140Pro Leu Phe Asn Pro Val Lys Met Gly Thr Cys Ser
Phe Asn Thr Leu145 150 155 160Lys Val Lys Asn Ala Ile Leu Glu Arg
Ala Gly Gly Asn Ile Glu Leu 165 170 175Tyr Thr Gln Arg Tyr Gln Ser
Ser Phe Arg Thr Leu Glu Asn Val Leu 180 185 190Asn Phe Ser Gln Ser
Glu Thr Cys Lys Thr Thr Glu Lys Ser Thr Lys 195 200 205Cys Thr Leu
Pro Glu Ala Leu Pro Ser Glu Leu Lys Val Thr Pro Asp 210 215 220Asn
Val Ser Leu Pro Gly Ala Trp Ser Leu Ser Ser Thr Leu Thr Glu225 230
235 240Ile Phe Leu Leu Gln Glu Ala Gln Gly Met Pro Gln Val Ala Trp
Gly 245 250 255Arg Ile Thr Gly Glu Lys Glu Trp Arg Asp Leu Leu Ser
Leu His Asn 260 265 270Ala Gln Phe Asp Leu Leu Gln Arg Thr Pro Glu
Val Ala Arg Ser Arg 275 280 285Ala Thr Pro Leu Leu Asp Met Ile Asp
Thr Ala Leu Leu Thr Asn Gly 290 295 300Thr Thr Glu Asn Arg Tyr Gly
Ile Lys Leu Pro Val Ser Leu Leu Phe305 310 315 320Ile Ala Gly His
Asp Thr Asn Leu Ala Asn Leu Ser Gly Ala Leu Asp 325 330 335Leu Asn
Trp Ser Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly 340 345
350Glu Leu Val Phe Glu Lys Trp Lys Arg Thr Ser Asp Asn Thr Asp Trp
355 360 365Val Gln Val Ser Phe Val Tyr Gln Thr Leu Arg Asp Met Arg
Asp Ile 370 375 380Gln Pro Leu Ser Leu Glu Lys Pro Ala Gly Lys Val
Asp Leu Lys Leu385 390 395 400Ile Ala Cys Glu Glu Lys Asn Ser Gln
Gly Met Cys Ser Leu Lys Ser 405 410 415Phe Ser Arg Leu Ile Lys Glu
Ile Arg Val Pro Glu Cys Ala Val Thr 420 425 430Glu418DNAArtificial
SequenceOligonucleotide primer 536f 4cagcmgccgc ggtaatwc
18515DNAArtificial SequenceOligonucleotide primer 1392r 5acgggcggtg
tgtrc 15629DNAArtificial SequenceOligonucleotide primer o42-5
6ggaattcata tgagtacatt catcattcg 29733DNAArtificial
SequenceOligonucleotide primer o42-3 7ggaattcgga tcccttattc
cgtaactgca cac 338433PRTCitrobacter freundiiSITE(22)..(22)Xaa may
be Ala or Thr 8Met Ser Thr Phe Ile Ile Arg Leu Leu Phe Phe Ser Leu
Leu Cys Gly1 5 10 15Ser Phe Ser Ile His Xaa Xaa Xaa Pro Asn Gly Xaa
Lys Leu Glu Arg 20 25 30Val Val Ile Val Ser Arg His Gly Val Arg Ala
Pro Thr Xaa Phe Thr 35 40 45Pro Ile Met Lys Xaa Val Thr Pro Xaa Gln
Trp Pro Gln Trp Asp Val 50 55 60Pro Leu Xaa Trp Leu Thr Pro Arg Gly
Xaa Xaa Leu Xaa Xaa Xaa Leu65 70 75 80Gly Xaa Tyr Gln Arg Leu Trp
Xaa Thr Ser Lys Gly Leu Leu Xaa Xaa 85 90 95Xaa Xaa Cys Pro Xaa Xaa
Xaa Gln Xaa Ala Val Ile Xaa Asp Thr Xaa 100 105 110Gln Arg Thr Arg
Lys Thr Gly Glu Ala Xaa Leu Ala Gly Xaa Ala Pro 115 120 125Lys Cys
Gln Ile Gln Val His Xaa Gln Lys Asp Xaa Glu Xaa Xaa Asp 130 135
140Pro Leu Phe Xaa Pro Val Xaa Xaa Gly Xaa Cys Xaa Phe Asn Thr
Xaa145 150 155 160Xaa Val Lys Xaa Ala Ile Leu Xaa Arg Xaa Gly Gly
Asn Ile Glu Xaa 165 170 175Xaa Thr Gln Arg Tyr Gln Ser Ser Phe Arg
Thr Leu Glu Asn Val Leu 180 185 190Asn Phe Xaa Gln Ser Glu Xaa Cys
Lys Thr Xaa Xaa Xaa Xaa Xaa Lys 195 200 205Cys Thr Leu Pro Glu Ala
Xaa Pro Ser Glu Leu Lys Val Thr Pro Xaa 210 215 220Xaa Val Ser Leu
Xaa Gly Ala Trp Xaa Leu Xaa Ser Thr Leu Thr Glu225 230 235 240Ile
Phe Leu Leu Gln Glu Ala Gln Gly Met Pro Gln Val Ala Trp Gly 245 250
255Arg Ile Thr Gly Glu Lys Glu Trp Arg Asp Leu Leu Ser Leu His Asn
260 265 270Ala Xaa Phe Asp Leu Leu Xaa Arg Thr Pro Glu Val Ala Arg
Ser Xaa 275 280 285Ala Thr Pro Leu Leu Asp Met Ile Asp Thr Ala Leu
Xaa Thr Asn Gly 290 295 300Thr Thr Xaa Xaa Arg Tyr Gly Ile Lys Leu
Pro Val Ser Leu Leu Phe305 310 315 320Ile Xaa Gly His Asp Thr Asn
Leu Ala Asn Leu Ser Gly Ala Leu Asp 325 330 335Leu Asn Trp Ser Leu
Pro Xaa Gln Pro Asp Asn Thr Pro Pro Gly Gly 340 345 350Glu Leu Val
Phe Glu Xaa Trp Xaa Arg Xaa Ser Asp Xaa Xaa Xaa Trp 355 360 365Val
Xaa Val Ser Phe Val Tyr Gln Thr Leu Arg Asp Met Arg Xaa Xaa 370 375
380Xaa Xaa Leu Ser Leu Glu Xaa Pro Xaa Gly Xaa Val Xaa Leu Lys
Leu385 390 395 400Ile Ala Cys Glu Glu Lys Asn Xaa Gln Gly Met Cys
Ser Xaa Lys Ser 405 410 415Phe Ser Arg Leu Ile Lys Glu Ile Arg Val
Pro Glu Cys Ala Val Thr 420 425 430Glu
* * * * *
References