U.S. patent application number 10/536101 was filed with the patent office on 2007-11-15 for fungal alpha-amylase variants.
This patent application is currently assigned to Novozymes A/S. Invention is credited to Lars Beier, Morten Tovborg Jensen, Tina Spendler, Allan Svendsen, Jesper Vind.
Application Number | 20070264700 10/536101 |
Document ID | / |
Family ID | 34203112 |
Filed Date | 2007-11-15 |
United States Patent
Application |
20070264700 |
Kind Code |
A1 |
Svendsen; Allan ; et
al. |
November 15, 2007 |
Fungal Alpha-Amylase Variants
Abstract
The inventors have developed a method of altering the amino acid
sequence of a fungal alpha-amylase to obtain variants, and they
have used the method to construct such variants. The variants may
be useful for anti-staling in baked products. Accordingly, the
invention provides a method of constructing fungal alpha-amylase
variants based on a comparison of three-dimensional (3D) structures
of the fungal alpha-amylase and a maltogenic alpha-amylase. One or
both models includes a substrate. The invention also provides novel
fungal alpha-amylase variants.
Inventors: |
Svendsen; Allan; (Horsholm,
DK) ; Beier; Lars; (Lyngby, DK) ; Vind;
Jesper; (Vaerlose, DK) ; Spendler; Tina;
(Malov, DK) ; Jensen; Morten Tovborg; (Vaerlose,
DK) |
Correspondence
Address: |
NOVOZYMES NORTH AMERICA, INC.
500 FIFTH AVENUE
SUITE 1600
NEW YORK
NY
10110
US
|
Assignee: |
Novozymes A/S
kROGSHOEJVEJ 36
bAGSVAERD
DK
DK-2880
|
Family ID: |
34203112 |
Appl. No.: |
10/536101 |
Filed: |
August 23, 2004 |
PCT Filed: |
August 23, 2004 |
PCT NO: |
PCT/DK04/00558 |
371 Date: |
February 22, 2005 |
Current U.S.
Class: |
435/203 |
Current CPC
Class: |
A21D 2/267 20130101;
C12N 9/2417 20130101; C12N 9/242 20130101; A21D 8/042 20130101 |
Class at
Publication: |
435/203 |
International
Class: |
C12N 9/30 20060101
C12N009/30 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 22, 2003 |
DK |
PA2003 01201 |
Claims
1-13. (canceled)
14. A method of producing a variant polypeptide, which method
comprises: a) providing an amino acid sequence and a
three-dimensional model for a fungal alpha-amylase and for a
maltogenic alpha-amylase wherein one or both models includes a
substrate, b) superimposing the two three-dimensional models, c)
selecting an amino acid residue in the fungal amylase which has a
C-alpha atom located >0.8 .ANG. from the C-alpha atom of any
amino acid residue in the maltogenic alpha-amylase and <11 .ANG.
from an atom of a substrate, d) altering the fungal amylase
sequence wherein the alteration comprises substitution or deletion
of the selected residue or by insertion of a residue adjacent to
the selected residue, and e) producing the polypeptide having the
resulting amino acid sequence.
15. The method of claim 14 wherein the substitution or insertion is
made with an amino acid residue of the same type as the
corresponding residue in the maltogenic alpha-amylase sequence,
wherein the type is positively charged, negatively charged,
hydrophilic or hydrophobic.
16. The method of claim 14 wherein the substitution or insertion is
made with a larger or smaller amino acid residue depending on
whether the corresponding residue in the maltogenic alpha-amylase
sequence is larger or smaller.
17. The method of claim 14 wherein, the alteration of the amino
acid sequence further comprises substitution of a fungal
alpha-amylase residue which has a C-alpha atom located less than 11
.ANG. from an atom of a substrate and <0.8 .ANG. from the
C-alpha atom of a maltogenic alpha-amylase residue.
18. The method of claim 17 wherein the substitution is made with an
amino acid residue of the same type as the corresponding maltogenic
alpha-amylase residue, wherein the type is positive, negative,
hydrophilic or hydrophobic.
19. A polypeptide which: a) has an amino acid sequence having at
least 70% identity to SEQ ID NO: 2; and b) compared to SEQ ID NO: 2
comprises an amino acid alteration which is a deletion,
substitution or insertion at a position corresponding to 15, 32-36,
63-64, 73-77, 119-120, 125-126, 151-152, 155-156, 167-172, 211 or
233-239, c) has the ability to hydrolyze starch.
20. The polypeptide of claim 19 wherein said polypeptide: a) has an
amino acid sequence having at least 80% identity to SEQ ID NO: 2;
and b) compared to SEQ ID NO: 2 comprises an amino acid alteration
which is a deletion, substitution or insertion at a position
corresponding to 15, 32-36, 63-64, 73-77, 119-120, 125-126,
151-152, 155-156, 167-172, 211 or 233-239, c) has the ability to
hydrolyze starch.
21. The polypeptide of claim 19 wherein said polypeptide; a) has an
amino acid sequence having at least 90% identity to SEQ ID NO: 2;
and b) compared to SEQ ID NO: 2 comprises an amino acid alteration
which is a deletion, substitution or insertion at a position
corresponding to 15, 32-36, 63-64, 73-77, 119-120, 125-126,
151-152, 155-156, 167-172, 211 or 233-239, c) has the ability to
hydrolyze starch.
22. A polypeptide of claim 19 wherein said polypeptide: a) has an
amino acid sequence having at least 95% identity to SEQ ID NO: 2;
and b) compared to SEQ ID NO; 2 comprises an amino acid alteration
which is a deletion, substitution or insertion at a position
corresponding to 15, 32-36, 63-64, 73-77, 119-120, 125-126,
151-152, 155-156, 167-172, 211 or 233-239, c) has the ability to
hydrolyze starch.
23. The polypeptide of claim 19 wherein the alteration comprises
substitution or insertion with an amino acid residue of the same
type as the corresponding residue in the maltogenic alpha-amylase
sequence, wherein the type is positively charged, negatively
charged, hydrophilic or hydrophobic.
24. The polypeptide of claim 19 wherein the alteration comprises
substitution or insertion with a larger or smaller amino acid
residue depending on whether the corresponding residue in the
maltogenic alpha-amylase sequence is larger or smaller.
25. The polypeptide of claim 19 comprising alteration corresponding
to Q35K/R, Y75A/F, Y155W, L166F, G167T, N169P, T170A, L232Y, D233G,
G234D, Y252F, Y256T, 166LGDNTV171 to FTDPAGF, 168-171 (DNTV)
substituted with DPAGF, 168-171 (DNTV) substituted with DPAGL,
168-171 (DNTV) substituted with DPAGC.
26. The polypeptide of claim 19 which has the amino acid sequence
of SEQ ID NO: 2 comprising one or more of the following
alterations: Q35K/R Y75A/F Y155W L166F G167T N169P T170A L232Y
D233G G234D Y252F Y256T 166LGDNTV171 to FTDPAGF 168-171 (DNTV)
substituted with DPAGF 168-171 (DNTV) substituted with DPAGL
168-171 (DNTV) substituted with DPAGC D233G+G234D Q35K+Y75F+D168Y
Q35R+Y75F Q35R+Y75F+D168Y 168-171 (DNTV) substituted with
DPAGF+Y75A 168-171 (DNTV) substituted with DPAGF+Q35K+Y75A 168-171
(DNTV) substituted with DPAGF+Q35K+Y75A+D233G+G234D 168-171 (DNTV)
substituted with DPAGF+Y75A+G234D 168-171 (DNTV) substituted with
DPAGF+Y75A+D233G+G234D 166-171 (LGDNTV) substituted with
FTDPAGF+Y75A 166-171 (LGDNTV) substituted with FTDPAGF+Q35K+Y75A
166-171 (LGDNTV) substituted with FTDPAGF+Q35K+Y75A+D233G+G234D
27. A polypeptide which: a) has an amino acid sequence having at
least 70% identity to SEQ ID NO: 3; b) compared to SEQ ID NO: 3
comprises an amino acid alteration which comprises Q35K, Q35R,
P70K, L151F, L151D, N233G+G234D, D75G, D75A or 166-171 (EGDTIV)
substituted with FTDPAGF, and c) has the ability to hydrolyze
starch.
28. The polypeptide of claim 27 wherein said polypeptide: a) has an
amino acid sequence having at least 80% identity to SEQ ID NO: 3;
b) compared to SEQ ID NO: 3 comprises an amino acid alteration
which comprises Q35K, Q35R, P70K, L151F, L151D, N233G+G234D, D75G,
D75A or 166-171 (EGDTIV) substituted with FTDPAGF, and c) has the
ability to hydrolyze starch.
29. The polypeptide of claim 27 wherein said polypeptide: a) has an
amino acid sequence having at least 90% identity to SEQ ID NO: 3;
b) compared to SEQ ID NO: 3 comprises an amino acid alteration
which comprises Q35K, Q35R, P70K, L151F, L151D, N233G+G234D, D75G,
D75A or 166-171 (EGDTIV) substituted with FTDPAGF, and c) has the
ability to hydrolyze starch.
30. The polypeptide of claim 27 wherein said polypeptide: a) has an
amino acid sequence having at least 95% identity to SEQ ID NO: 3;
b) compared to SEQ ID NO: 3 comprises an amino acid alteration
which comprises Q35K, Q35R, P70K, L151F, L151D, N233G+G234D, D75G,
D75A or 166-171 (EGDTIV) substituted with FTDPAGF, and c) has the
ability to hydrolyze starch.
31. A polypeptide which: a) has an amino acid sequence having at
least 70% identity to SEQ ID NO: 4; b) compared to SEQ ID NO: 4
comprises an amino acid alteration which comprises G35K, G35R,
A76del+D77del, D74del+A78del, D74A, D74G, D77A, D77G, Y157W or
L168F+A169T+T171P+P172A+T173G, and c) has the ability to hydrolyze
starch.
32. The polypeptide of claim 31 wherein said polypeptide: a) has an
amino acid sequence having at least 80% identity to SEQ ID NO: 4;
b) compared to SEQ ID NO: 4 comprises an amino acid alteration
which comprises G35K, G35R, A76del+D77del, D74del+A78del, D74A,
D74G, D77A, D77G, Y157W or L168F+A169T+T171P+P172A+T173G, and c)
has the ability to hydrolyze starch.
33. The polypeptide of claim 31 wherein said polypeptide: a) has an
amino acid sequence having at least 90% identity to SEQ ID NO: 4;
b) compared to SEQ ID NO: 4 comprises an amino acid alteration
which comprises G35K, G35R, A76del+D77del, D74del+A78del, D74A,
D74G, D77A, D77G, Y157W or L168F+A169T+T171P+P172A+T173G, and c)
has the ability to hydrolyze starch.
34. The polypeptide of claim 31 wherein said polypeptide: a) has an
amino acid sequence having at least 95% identity to SEQ ID NO: 4;
b) compared to SEQ ID NO: 4 comprises an amino acid alteration
which comprises G35K, G35R, A76del+D77del, D74del+A78del, D74A,
D74G, D77A, D77G, Y157W or L168F+A169T+T171P+P172A+T173G, and c)
has the ability to hydrolyze starch.
35. A process for preparing a dough or a baked from dough product
which comprises adding the polypeptide of claim 19.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to the construction of
variants of fungal alpha-amylases.
BACKGROUND OF THE INVENTION
[0002] WO 0134784 discloses variants of a fungal alpha-amylase. Pdb
files 2AAA, 6taa and 7taa (available at www.rcsb.org) show the
amino acid sequences and three-dimensional structures of fungal
alpha-amylases. WO 9943794 discloses the amino acid sequence and
three-dimensional structure of a maltogenic alpha-amylase from
Bacillus stearothermophilus, known as Novamyl.RTM..
SUMMARY OF THE INVENTION
[0003] The inventors have developed a method of altering the amino
acid sequence of a fungal alpha-amylase to obtain variants with
improved anti-staling effect and a higher degree of exo-amylase
activity (increased ratio of exo-amylase to endo-amylase), and they
have used the method to construct such variants. The variants may
be useful for anti-staling in baked products.
[0004] Accordingly, the invention provides a method of constructing
fungal alpha-amylase variants based on a comparison of
three-dimensional (3D) structures of the fungal alpha-amylase and a
maltogenic alpha-amylase. One or both models includes a substrate.
The invention also provides novel fungal alpha-amylase variants and
use of the variants in the preparation of dough and baked
products.
BRIEF DESCRIPTION OF DRAWINGS
[0005] FIG. 1 shows an alignment of fungal amylases SEQ ID NO: 2, 3
and 4.
[0006] FIG. 2 shows an alignment of the 3D structures 1QHO for the
maltogenic alpha-amylase Novamyl (SEQ ID NO: 1) at top and 6taa for
a fungal alpha-amylase (SEQ ID NO: 2) below. Details are described
in Example 1.
DETAILED DESCRIPTION OF THE INVENTION
Fungal Alpha-Amylase
[0007] The method of the invention uses an amino acid sequence of a
fungal alpha-amylase and a three-dimensional model for the fungal
alpha-amylase. The model may include a substrate.
[0008] The fungal alpha-amylase may be one of the following having
the indicated amino acid sequence and a three-dimensional structure
found under the indicated identifier in the Protein Data Bank
(www.rcsb.org): acid alpha-amylase from Aspergillus niger (2AAA,
SEQ ID NO: 3), Alpha-amylase (Taka amylase) from Aspergillus oryzae
(6taa or 7taa, SEQ ID NO: 2) or alpha-amylase from Thermomyces
lanuginosus (SEQ ID NO: 4, WO 9601323). Alternatively, the fungal
alpha-amylase may be a variant having at least 70% amino acid
identity with SEQ ID NO: 2, e.g. a variant described in WO
0134784.
[0009] 3D structures for other fungal alpha-amylases may be
constructed as described in Example 1 of WO 9623874. To develop
variants of a fungal alpha-amylase without a known 3D structure,
the sequence may be aligned with a fungal alpha-amylase having a
known 3D structure. The sequence alignment may be done by
conventional methods, e.g. by use the software GAP from UWGCG
Version 8. FIG. 1 shows an alignment of SEQ ID NO: 4 (without a
known 3D structure) with SEQ ID NO: 2 and 3 (with known
structures).
Maltogenic Alpha-Amylase
[0010] The method also uses an amino acid sequence of a maltogenic
alpha-amylase (EC 3.2.1.133) and a three-dimensional model of the
maltogenic alpha-amylase. The model may include a substrate. The
maltogenic alpha-amylase may have the amino acid sequence have the
amino acid sequence shown in SEQ ID NO: 1 (in the following
referred to as Novamyl). A 3D model for Novamyl with a substrate is
described in U.S. Pat. No. 6,162,628 and is found in the Protein
Data Bank with the identifier 1QHO. Alternatively, the maltogenic
alpha-amylase may be a Novamyl variant described in U.S. Pat. No.
6,162,628. A 3D structure of such a variant may be developed from
the Novamyl structure by known methods, e.g. as described in T. L.
Blundell et al., Nature, vol. 326, p. 347 ff (26 Mar. 1987); J.
Greer, Proteins: Structure, Function and Genetics, 7:317-334
(1990); or Example 1 of WO 9623874.
Superimposition of 3D Models
[0011] The two 3D models may be superimposed by aligning the amino
acid residues of each catalytic triad by methods known in the art.
This may be based on the deviations of heavy atoms (i.e.
non-hydrogen atoms) in the active sites, e.g. by minimizing the sum
of squares of deviations. Alternatively, the superimposition may be
based on the deviations of the three pairs of C-alpha atoms, e.g.
by minimizing the sum of squares of the three deviations or by
aligning so as to keep each deviation below 0.8 .ANG., e.g. below
0.6 .ANG., below 0.4 .ANG., below 0.3 .ANG. or below 0.2 .ANG..
[0012] The structural alignment may be done by use of known
software. In the structurally aligned models, pairs of residues
from different sequences are considered to be aligned when they are
located close to each other. The following software may be
used:
[0013] DALI software, available at http://www.ebi.ac.uk/dali/
[0014] CE software available at http://cl.sdsc.edu/
[0015] STAMP software available at
http://www.compbio.dundee.ac.uk/Software/Stamp/stamp.html
[0016] Protein 3Dhome at
http://www-lecb.ncifcrf.gov/.about.tsai/
[0017] Yale Gemstein Lab--spare parts at
http://bioinfo.mbb.yale.edu/align/
[0018] Structural alignment server at
http://www.molmovdb.org/align/
Substrate
[0019] A 3D structure of the enzyme(s) having a substrate or
substrate analog in the active site binding cleft. A "substrate"
could be a substrate bound in an inactive or active enzyme, or a
substrate inhibitor like acarbose bound in the active site, or a
modelled substrate in the active site, a docked substrate in the
active site, or a substrate superimposed into the enzyme of
interest and taken from a homologous 3D structure having such
substrate or substrate analog bound in the active site.
Selection of Amino Acid Residues
[0020] In the superimposed 3D models, amino acid residues in the
fungal alpha-amylase sequence are selected by two criteria:
Firstly, fungal alpha-amylase residues <11 .ANG. from a
substrate (i.e. residues having a C-alpha atom located <11 .ANG.
from an atom of a substrate) are selected. Secondly, fungal
alpha-amylase residues >0.8 .ANG. from any maltogenic
alpha-amylase residue (i.e. fungal alpha-amylase residues having a
C-alpha atom >0.8 .ANG. from the C-alpha atom of any maltogenic
alpha-amylase residue) are selected.
Alteration of Fungal Alpha-Amylase Amino Acid Sequence
[0021] One or more of the following alterations are made to the
fungal alpha-amylase sequence:
Deletion or Substitution
[0022] A fungal alpha-amylase residue <11 .ANG. from a substrate
and >0.8 .ANG. from any maltogenic alpha-amylase residue may be
deleted or may be substituted with a different residue.
[0023] The substitution may be made with the same amino acid
residue as found at a corresponding position in the maltogenic
alpha-amylase sequence or with a residue of the same type. The type
indicates a positively charged, negatively charged, hydrophilic or
hydrophobic residue, understood as follows (Tyr may be hydrophilic
or hydrophobic):
[0024] Hydrophobic amino acids: Ala, Val, Leu, Ile, Pro, Phe, Trp,
Gly, Met, Tyr
[0025] Hydrophilic amino acids: Thr, Ser, Gin, Asn, Tyr, Cys
[0026] Positively charged amino acids: Lys, Arg, His
[0027] Negatively charged amino acids: Glu, Asp
[0028] The fungal alpha-amylase residue may be substituted with a
larger or smaller residue depending on whether a larger or smaller
residue is found at a corresponding position in the maltogenic
alpha-amylase sequence. In this connection, the residues are ranked
as follows from smallest to largest: (an equal sign indicates
residues with sizes that are practically indistinguishable):
[0029]
G<A=S=C<V=T<P<L=I=N=D=M<E=Q<K<H<R=F.ltoreq-
.Y.ltoreq.W
Insertion
[0030] One or more amino acid residues may be inserted at a
position in the fungal alpha-amylase sequence corresponding to one
or more residues in the maltogenic alpha-amylase sequence which are
<11 .ANG. from a substrate and which are >0.8 .ANG. from any
fungal alpha-amylase residue. The insertion may be made with the
same residue as in the maltogenic alpha-amylase sequence or with
another amino acid residue of the same type. The type indicates a
positively charged, negatively charged, hydrophilic or hydrophobic
residue, as above.
[0031] Where the maltogenic alpha-amylase sequence contains a
consecutive stretch (a peptide loop) of residues which are >0.8
.ANG. from any fungal alpha-amylase residue and of which some are
<11 .ANG. from a substrate, the insertion at the corresponding
position in the fungal alpha-amylase sequence may consist of an
equal number of residues, or the insertion may have one or two
fewer or more residues. Thus, in the case of a stretch of 5 such
residues in the maltogenic alpha-amylase sequence, the insertion
may be made with 3-7 residues, e.g. 3, 4, 5, 6 or 7 residues. Each
inserted residue may be the same as one of the maltogenic
alpha-amylase residues or of the same type.
Optional Further Alterations of the Fungal Alpha-Amylase
Sequence
[0032] Optionally, one or more other residues in the fungal
alpha-amylase sequence may be substituted. The substitution may be
made as described in WO 0134784 and may improve the thermostability
of the variant.
[0033] A fungal alpha-amylase residue <11 .ANG. of a substrate
and <0.8 .ANG. of a maltogenic alpha-amylase residue may be
substituted with a residue identical to or of the same type as the
corresponding maltogenic alpha-amylase residue, or with a larger or
smaller residue depending on whether the corresponding maltogenic
alpha-amylase residue is larger or smaller.
Degree of Exo-Activity
[0034] The degree of exo amylase activity is given as a relative
activity compared to the endo amylase activity. The endo activity
can be measured by a number of well known assays e.g. starch
iodine, Phadebas (Amersham now GE Healthcare), or AZCL-amylose
(Megazyme). The exo activity is preferably a measure of the small
malto-oligomers released from starch at initial phases of
hydrolysis. It is preferably measured by total carbohydrate after
removal of the remaining starch, by the exo activity assay
described below or similar method, but could be measured by other
means e.g. the sum of oligomers by HPAEC-PAD (Dionex) or sum of
oligomers after size exclusion chromatography.
Endo-Amylase Activity Assay:
[0035] 1 mL resuspended Phadebas substrate (0.25 tablets/mL 50 mM
sodium acetate, 1 mM CaCl.sub.2, adjusted to pH 5.7) is incubated
with 25 micro-L enzyme for 15 min at 40.degree. C. with agitation.
The reaction is stopped by addition of 0.5 mL 1 M NaOH and the
mixture is centrifuged in a table centrifuge at 14,000 RPM. The
absorbance of the supernatant at 620 nm is measured. The activity
is determined by comparing to a standard with declared activity
(BAN 480 L, 480 KNU/g)
Exo-Amylase Activity Assay:
[0036] 900 .mu.L 3.3% solubilized waxy maize starch (3.3% starch is
boiled in 50 mM sodium acetate, 1 mM CaCl.sub.2, pH 5.7 for 5 min
and cooled to 40.degree. C.) is incubated with 100 micro-L enzyme
at 40.degree. C. with stirring. After appropriate reaction time the
remaining starch is precipitated by addition of 450 micro-L
4.degree. C. 96% ethanol. The precipitate is immediately removed by
centrifugation at 3000 G for 20 min. The total carbohydrate in the
supernatant is determined by mixing 200 micro-L supernatant with 50
micro-L 2% tryptophan and 900 micro-L 64% sulfuric acid. The
mixture is heated for 15 min at 95.degree. C. and the absorbance at
630 nm is measured after cooling to room temperature. The activity
is determined by comparing with the absorbance of glucose standards
in the same assay. One unit is defined as the amount of enzyme that
at initial rates liberates 1 mg oligomeric products (products that
are not precipitated by ethanol) per min.
Fungal Alpha-Amylase Variants
[0037] A fungal alpha-amylase variant may be a polypeptide
which:
[0038] a) has an amino acid sequence having at least 70% identity
to SEQ ID NO: 2, 3 or 4; and
[0039] b) comprises an amino acid alteration which is deletion,
substitution or insertion as described below, and
[0040] c) has the ability to hydrolyze starch.
[0041] The identity may be at least 80%, at least 90% or at least
95%. Amino acid identity may be determined as described in U.S.
Pat. No. 6,162,628.
Production of Fungal Alpha-Amylase Variants
[0042] A polypeptide having the resulting amino acid sequence may
be produced by conventional methods, generally involving producing
DNA with a sequence encoding the polypeptide together with control
sequences, transforming a suitable host organism with the DNA,
cultivating the transformed organism at suitable conditions for
expressing and optionally secreting the polypeptide, and optionally
recovering the expressed polypeptide.
[0043] DNA encoding any of the above fungal alpha-amylase variants
may be prepared, e.g. by point-specific mutation of DNA encoding
the parent fungal alpha-amylase. This may be followed by
transformation of a suitable host organism with the DNA, and
cultivation of the transformed host organism under suitable
conditions to express the encoded polypeptide (fungal alpha-amylase
variant). This may be done by known methods.
Optional Screening of Fungal Alpha-Amylase Variants
[0044] Optionally, one or more expressed polypeptides may be tested
for useful properties. This may include testing for the ability to
hydrolyze starch or a starch derivative by a conventional method,
e.g. a plate assay, use of Phadebas tablets or DSC on amylopectin.
Also, the polypeptide may be tested for thermostability, and a more
thermostable one may be preferred. Finally, the polypeptide may be
tested by adding it to a dough, baking it and testing the firmness
of the baked product during storage; a polypeptide with
anti-staling effect may be selected as described in WO 9104669 or
U.S. Pat. No. 6,162,628.
Optional Gene Recombination
[0045] Optionally, DNA encoding a plurality of the above fungal
alpha-amylase variants may be prepared and recombined, followed by
transformation of a suitable host organism with the recombined DNA,
and cultivation of the transformed host organism under suitable
conditions to express the encoded polypeptides (fungal
alpha-amylase variants). The gene recombination may be done by
known methods.
Dough and Baked Product
[0046] The variants are useful in the preparation of dough and
baked products from dough. Particularly, the variant may be added
in an amount which is effective to retard the staling of the baked
product.
[0047] The dough may be leavened e.g. by adding chemical leavening
agents or yeast, usually Saccharomyces cerevisiae (baker's yeast).
The dough generally comprises flour, particularly wheat flour.
Examples of baked products are bread and rolls.
[0048] The dough may comprise an additional enzyme, e.g. a second
amylase, a protease or peptidase, a transglutaminase, a lipolytic
enzyme, a cellulase, a xylanase or an oxidoreductase, e.g. a
carbohydrate oxidase with activity on glucose and/or maltose. The
lipolytic enzyme may have triacyl glycerol lipase activity,
phospholipase activity and/or galactolipase activity, e.g. as
described in WO 9953769, WO 9826057 or WO 0032758.
EXAMPLES
Example 1
Construction of Variants of Fungal Alpha-Amylase from A. oryzae
[0049] Two 3D structures with substrates were used: 6taa for a
fungal alpha-amylase (SEQ ID NO: 2) and 1QHO for a maltogenic
alpha-amylase (Novamyl, SEQ ID NO: 1), wherein the substrates are
indicated as ABC for 6taa and as ABD for 1QHO. The two structures
were superimposed using the heavy atoms of the three C-alpha atoms
at the catalytic triad: D206, E230 and D297 for 6taa, and D228,
E256 and D329 for Novamyl. The superimposed structures were
analyzed, and the result is shown in FIG. 2 with the Novamyl
sequence at the top and the fungal alpha-amylase sequence
below.
[0050] The following fungal alpha-amylase residues were found to
have a C-alpha atom <11
[0051] A from an atom of either substrate: 13, 14, 15, 18, 31, 32,
33, 34, 35, 36, 61, 62, 63, 64, 66, 68, 69, 71, 72, 73, 74, 75, 76,
77, 78, 79, 80, 81, 82, 83, 84, 94, 117, 118, 119, 120, 121, 122,
123, 124, 125, 126, 127, 151, 152, 153, 154, 155, 156, 157, 158,
160, 161, 162, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173,
174, 175, 204, 205, 206, 207, 208, 209, 210, 211, 216, 228, 229,
230, 231, 232, 233, 234, 235, 236, 237, 238, 239,242, 250, 251,
252, 253, 254, 255, 256, 257, 258, 259, 260, 275, 292, 294,295,
296, 297, 298, 299, 304, 328, 338, 239, 340, 341, 342, 343, 344.
They are indicated by the first underlining in FIG. 2.
[0052] The following fungal alpha-amylase residues were found to be
included in either of the above subsets (<11 .ANG. from a
substrate or in a loop) and to have a C-alpha atom >0.8 .ANG.
from the C-alpha atom of any Novamyl residue: 15, 32, 33, 34, 35,
36, 63, 64, 73, 74, 75, 76, 77, 119, 120, 125, 126, 151, 152, 155,
156, 167, 168, 169, 170, 171, 172, 211, 233, 234, 235, 236, 237,
238, 239. They are indicated by the second underlining in FIG.
2.
[0053] Variants were constructed by substituting a selected residue
in SEQ ID NO: 2 (fungal amylase) as indicated below: TABLE-US-00001
Selected Corresponding Particular residue in residue in
substitution SEQ ID NO: 2 SEQ ID NO: 1 Criteria for In SEQ ID
(fungal amylase) (Novamyl) substitution NO: 2 Q35 K44 larger and/or
Q35K/R positive Y75 T84 smaller Y75A/F Y155 W177 larger and/or
Y155W hydrophobic L166 F188 larger and/or L166F hydrophobic G167
T189 larger and/or G167T hydrophilic N169 P191 smaller and/or N169P
hydrophobic T170 A192 smaller and/or T170A hydrophobic L232 Y258
larger L232Y D233 G259 smaller and/or D233G hydrophobic G234 D260
larger and/or G234D negative Y252 F284 smaller and/or Y252F
hydrophobic Y256 T288 smaller and/or Y256T hydrophilic
[0054] Variants were constructed by altering a subsequence with
insertion of an additional residue in SEQ ID NO: 2 (fungal amylase)
to match the number of residues in SEQ ID NO: 1, as indicated
below: TABLE-US-00002 Alteration In SEQ ID NO: 2 (fungal amylase)
166LGDNTV171 to FTDPAGF (Novamyl loop (long)) 168-171 (DNTV)
substituted with DPAGF (Novamyl loop) 168-171 (DNTV) substituted
with DPAGL (Novamyl loop with adjustments in last part) 168-171
(DNTV) substituted with DPAGC (Novamyl loop with adjustments in
last part)
[0055] Further, amino acid alterations were combined as follows:
TABLE-US-00003 Alteration with insertion Substitutions in SEQ ID
NO: 2 in SEQ ID NO: 2 D233G + G234D Q35K + Y75F + D168Y Q35R + Y75F
Q35R + Y75F + D168Y 168-171 (DNTV) Y75A substituted with DPAGF
168-171 (DNTV) Q35K + Y75A substituted with DPAGF 168-171 (DNTV)
Q35K + Y75A + D233G + G234D substituted with DPAGF 168-171 (DNTV)
Y75A + G234D substituted with DPAGF 168-171 (DNTV) Y75A + D233G +
G234D substituted with DPAGF 166-171 (LGDNTV) Y75A substituted with
FTDPAGF 166-171 (LGDNTV) Q35K + Y75A substituted with FTDPAGF
166-171 (LGDNTV) Q35K + Y75A + D233G + G234D substituted with
FTDPAGF
Example 2
Construction of Variants of Acid Amylase from A. niger
[0056] The three-dimensional structure 2AAA for the acid
alpha-amylase from Aspergillus niger (SEQ ID NO: 3) was compared
with the structure of Novamyl 1QHO, and variants were constructed
by altering the sequence SEQ ID NO: 3 as follows:
[0057] Q35K
[0058] Q35R
[0059] P70K
[0060] L151F
[0061] L151D
[0062] N233G+G234D
[0063] D75G
[0064] D75A
[0065] 166-171 (EGDTIV) substituted with FTDPAGF (Novamyl loop
(long))
Example 3
Construction of Variants of Fungal Amylase from T. lanuginosus
[0066] A three-dimensional model of SEQ ID NO: 4 (fungal amylase
from T. lanuginosus) was constructed from a model of SEQ ID NO: 2
(fungal amylase from A. oryzae) using the alignment shown in FIG.
1. Residues were selected, and variants were constructed with amino
acid alterations to substitute or delete selected residues as
follows:
[0067] G35K
[0068] G35R
[0069] A76del+D77del
[0070] D74del+A78del
[0071] D74A
[0072] D74G
[0073] D77A
[0074] D77G
[0075] Y157W
[0076] L168F+A169T+T171P+P172A+T173G
Example 4
Anti-Staling Effect of Variants (Straight-Dough Method)
[0077] Baking tests were made with the following variants of SEQ ID
NO: 2 (fungal amylase from A. oryzae): TABLE-US-00004 Alteration in
SEQ ID NO: 2 (fungal amylase) 168-171 (DNTV) substituted with DPAGF
Y75A Q35R Q35R + Y75F 168-171 (DNTV) substituted with DPAGC L232Y
168-171 (DNTV) substituted with DPAGF + Y75A D233G + G234D 168-171
(DNTV) substituted with DPAGF + Q35K + Y75A
[0078] Doughs were made according to the straight dough method.
Bread was baked in lidded pans, and the bread was stored at ambient
temperature. Firmness and elasticity were evaluated after 1, 4 and
6 days. Each variant was added at a dosage of 1 mg per kg flour.
Controls were made without enzyme, with the parent fungal amylase
of SEQ ID NO: 2 and with Novamyl (maltogenic alpha-amylase of SEQ
ID NO: 1).
[0079] The results showed that the fungal alpha-amylase variants
and Novamyl improved the elasticity after storage compared to the
control without enzyme, whereas the fungal alpha-amylase gave a
slightly lower elasticity. All the enzymes tested (variants, fungal
amylase and Novamyl) improved the firmness after storage. In
conclusion, the amino acid alterations succeeded in changing the
functional properties of the fungal amylase to make it more
Novamyl-like.
Example 5
Anti-Staling Effect of Variants (Sponge-and-Dough Method)
[0080] Baking tests were made with the following variants of SEQ ID
NO: 2 (fungal amylase from A. oryzae): TABLE-US-00005 Alteration in
SEQ ID NO: 2 (fungal amylase) 168-171 (DNTV) substituted with DPAGF
Y75A
[0081] Doughs were made by the sponge & dough method, and the
variants were tested as in the preceding example. Controls were
made without enzyme, with the parent fungal amylase of SEQ ID NO: 2
and with Novamyl (maltogenic alpha-amylase of SEQ ID NO: 1).
[0082] The variants show comparable softness and improved
elasticity relative to the parent amylase, when dosed at optimal
dosage in this trial.
[0083] A sensory evaluation by a small panel agrees with NMR data
on mobility of free water and shows that the variants improve the
moistness of bread crumb to the same level or slightly better than
the parent amylase.
[0084] In conclusion, the variants showed improved effect (a more
Novamyl-like effect) compared to the parent amylase.
Example 6
Exo/Endo Ratio of A. oryzae Amylase Variants
[0085] The following variants of SEQ ID NO: 2 (fungal amylase from
A. oryzae) were tested: TABLE-US-00006 Alteration in SEQ ID NO: 2
168-171 (DNTV) substituted with DPAGF Y75A 168-171 (DNTV)
substituted with DPAGC Q35R Q35R + Y75F
[0086] The exo- and endo-amylase activities were determined for
each variant by the assays described in the specification, and the
parent amylase was tested for comparison. The results showed that
each variant had a higher degree of exo-amylase activity (higher
exo/endo-amylase ratio) that the parent fungal amylase.
Example 7
Exo/Endo Ratio of A. niger Amylase Variants
[0087] The following variants of SEQ ID NO: 3 (acid amylase from A.
niger) were tested: [0088] Alteration in SEQ ID NO: 3 [0089] D75G
[0090] Q35K [0091] L151F [0092] L151D [0093] N233G+G234D
[0094] The exo- and endo-amylase activities were determined for
each variant by the assays described in the specification, and the
parent amylase was tested for comparison. The results showed that
each variant had a higher degree of exo-amylase activity (higher
exo/endo-amylase ratio) that the parent fungal amylase.
Sequence CWU 1
1
4 1 686 PRT Bacillus stearothermophilus 1 Ser Ser Ser Ala Ser Val
Lys Gly Asp Val Ile Tyr Gln Ile Ile Ile 1 5 10 15 Asp Arg Phe Tyr
Asp Gly Asp Thr Thr Asn Asn Asn Pro Ala Lys Ser 20 25 30 Tyr Gly
Leu Tyr Asp Pro Thr Lys Ser Lys Trp Lys Met Tyr Trp Gly 35 40 45
Gly Asp Leu Glu Gly Val Arg Gln Lys Leu Pro Tyr Leu Lys Gln Leu 50
55 60 Gly Val Thr Thr Ile Trp Leu Ser Pro Val Leu Asp Asn Leu Asp
Thr 65 70 75 80 Leu Ala Gly Thr Asp Asn Thr Gly Tyr His Gly Tyr Trp
Thr Arg Asp 85 90 95 Phe Lys Gln Ile Glu Glu His Phe Gly Asn Trp
Thr Thr Phe Asp Thr 100 105 110 Leu Val Asn Asp Ala His Gln Asn Gly
Ile Lys Val Ile Val Asp Phe 115 120 125 Val Pro Asn His Ser Thr Pro
Phe Lys Ala Asn Asp Ser Thr Phe Ala 130 135 140 Glu Gly Gly Ala Leu
Tyr Asn Asn Gly Thr Tyr Met Gly Asn Tyr Phe 145 150 155 160 Asp Asp
Ala Thr Lys Gly Tyr Phe His His Asn Gly Asp Ile Ser Asn 165 170 175
Trp Asp Asp Arg Tyr Glu Ala Gln Trp Lys Asn Phe Thr Asp Pro Ala 180
185 190 Gly Phe Ser Leu Ala Asp Leu Ser Gln Glu Asn Gly Thr Ile Ala
Gln 195 200 205 Tyr Leu Thr Asp Ala Ala Val Gln Leu Val Ala His Gly
Ala Asp Gly 210 215 220 Leu Arg Ile Asp Ala Val Lys His Phe Asn Ser
Gly Phe Ser Lys Ser 225 230 235 240 Leu Ala Asp Lys Leu Tyr Gln Lys
Lys Asp Ile Phe Leu Val Gly Glu 245 250 255 Trp Tyr Gly Asp Asp Pro
Gly Thr Ala Asn His Leu Glu Lys Val Arg 260 265 270 Tyr Ala Asn Asn
Ser Gly Val Asn Val Leu Asp Phe Asp Leu Asn Thr 275 280 285 Val Ile
Arg Asn Val Phe Gly Thr Phe Thr Gln Thr Met Tyr Asp Leu 290 295 300
Asn Asn Met Val Asn Gln Thr Gly Asn Glu Tyr Lys Tyr Lys Glu Asn 305
310 315 320 Leu Ile Thr Phe Ile Asp Asn His Asp Met Ser Arg Phe Leu
Ser Val 325 330 335 Asn Ser Asn Lys Ala Asn Leu His Gln Ala Leu Ala
Phe Ile Leu Thr 340 345 350 Ser Arg Gly Thr Pro Ser Ile Tyr Tyr Gly
Thr Glu Gln Tyr Met Ala 355 360 365 Gly Gly Asn Asp Pro Tyr Asn Arg
Gly Met Met Pro Ala Phe Asp Thr 370 375 380 Thr Thr Thr Ala Phe Lys
Glu Val Ser Thr Leu Ala Gly Leu Arg Arg 385 390 395 400 Asn Asn Ala
Ala Ile Gln Tyr Gly Thr Thr Thr Gln Arg Trp Ile Asn 405 410 415 Asn
Asp Val Tyr Ile Tyr Glu Arg Lys Phe Phe Asn Asp Val Val Leu 420 425
430 Val Ala Ile Asn Arg Asn Thr Gln Ser Ser Tyr Ser Ile Ser Gly Leu
435 440 445 Gln Thr Ala Leu Pro Asn Gly Ser Tyr Ala Asp Tyr Leu Ser
Gly Leu 450 455 460 Leu Gly Gly Asn Gly Ile Ser Val Ser Asn Gly Ser
Val Ala Ser Phe 465 470 475 480 Thr Leu Ala Pro Gly Ala Val Ser Val
Trp Gln Tyr Ser Thr Ser Ala 485 490 495 Ser Ala Pro Gln Ile Gly Ser
Val Ala Pro Asn Met Gly Ile Pro Gly 500 505 510 Asn Val Val Thr Ile
Asp Gly Lys Gly Phe Gly Thr Thr Gln Gly Thr 515 520 525 Val Thr Phe
Gly Gly Val Thr Ala Thr Val Lys Ser Trp Thr Ser Asn 530 535 540 Arg
Ile Glu Val Tyr Val Pro Asn Met Ala Ala Gly Leu Thr Asp Val 545 550
555 560 Lys Val Thr Ala Gly Gly Val Ser Ser Asn Leu Tyr Ser Tyr Asn
Ile 565 570 575 Leu Ser Gly Thr Gln Thr Ser Val Val Phe Thr Val Lys
Ser Ala Pro 580 585 590 Pro Thr Asn Leu Gly Asp Lys Ile Tyr Leu Thr
Gly Asn Ile Pro Glu 595 600 605 Leu Gly Asn Trp Ser Thr Asp Thr Ser
Gly Ala Val Asn Asn Ala Gln 610 615 620 Gly Pro Leu Leu Ala Pro Asn
Tyr Pro Asp Trp Phe Tyr Val Phe Ser 625 630 635 640 Val Pro Ala Gly
Lys Thr Ile Gln Phe Lys Phe Phe Ile Lys Arg Ala 645 650 655 Asp Gly
Thr Ile Gln Trp Glu Asn Gly Ser Asn His Val Ala Thr Thr 660 665 670
Pro Thr Gly Ala Thr Gly Asn Ile Thr Val Thr Trp Gln Asn 675 680 685
2 478 PRT Aspergillus oryzae 2 Ala Thr Pro Ala Asp Trp Arg Ser Gln
Ser Ile Tyr Phe Leu Leu Thr 1 5 10 15 Asp Arg Phe Ala Arg Thr Asp
Gly Ser Thr Thr Ala Thr Cys Asn Thr 20 25 30 Ala Asp Gln Lys Tyr
Cys Gly Gly Thr Trp Gln Gly Ile Ile Asp Lys 35 40 45 Leu Asp Tyr
Ile Gln Gly Met Gly Phe Thr Ala Ile Trp Ile Thr Pro 50 55 60 Val
Thr Ala Gln Leu Pro Gln Thr Thr Ala Tyr Gly Asp Ala Tyr His 65 70
75 80 Gly Tyr Trp Gln Gln Asp Ile Tyr Ser Leu Asn Glu Asn Tyr Gly
Thr 85 90 95 Ala Asp Asp Leu Lys Ala Leu Ser Ser Ala Leu His Glu
Arg Gly Met 100 105 110 Tyr Leu Met Val Asp Val Val Ala Asn His Met
Gly Tyr Asp Gly Ala 115 120 125 Gly Ser Ser Val Asp Tyr Ser Val Phe
Lys Pro Phe Ser Ser Gln Asp 130 135 140 Tyr Phe His Pro Phe Cys Phe
Ile Gln Asn Tyr Glu Asp Gln Thr Gln 145 150 155 160 Val Glu Asp Cys
Trp Leu Gly Asp Asn Thr Val Ser Leu Pro Asp Leu 165 170 175 Asp Thr
Thr Lys Asp Val Val Lys Asn Glu Trp Tyr Asp Trp Val Gly 180 185 190
Ser Leu Val Ser Asn Tyr Ser Ile Asp Gly Leu Arg Ile Asp Thr Val 195
200 205 Lys His Val Gln Lys Asp Phe Trp Pro Gly Tyr Asn Lys Ala Ala
Gly 210 215 220 Val Tyr Cys Ile Gly Glu Val Leu Asp Gly Asp Pro Ala
Tyr Thr Cys 225 230 235 240 Pro Tyr Gln Asn Val Met Asp Gly Val Leu
Asn Tyr Pro Ile Tyr Tyr 245 250 255 Pro Leu Leu Asn Ala Phe Lys Ser
Thr Ser Gly Ser Met Asp Asp Leu 260 265 270 Tyr Asn Met Ile Asn Thr
Val Lys Ser Asp Cys Pro Asp Ser Thr Leu 275 280 285 Leu Gly Thr Phe
Val Glu Asn His Asp Asn Pro Arg Phe Ala Ser Tyr 290 295 300 Thr Asn
Asp Ile Ala Leu Ala Lys Asn Val Ala Ala Phe Ile Ile Leu 305 310 315
320 Asn Asp Gly Ile Pro Ile Ile Tyr Ala Gly Gln Glu Gln His Tyr Ala
325 330 335 Gly Gly Asn Asp Pro Ala Asn Arg Glu Ala Thr Trp Leu Ser
Gly Tyr 340 345 350 Pro Thr Asp Ser Glu Leu Tyr Lys Leu Ile Ala Ser
Ala Asn Ala Ile 355 360 365 Arg Asn Tyr Ala Ile Ser Lys Asp Thr Gly
Phe Val Thr Tyr Lys Asn 370 375 380 Trp Pro Ile Tyr Lys Asp Asp Thr
Thr Ile Ala Met Arg Lys Gly Thr 385 390 395 400 Asp Gly Ser Gln Ile
Val Thr Ile Leu Ser Asn Lys Gly Ala Ser Gly 405 410 415 Asp Ser Tyr
Thr Leu Ser Leu Ser Gly Ala Gly Tyr Thr Ala Gly Gln 420 425 430 Gln
Leu Thr Glu Val Ile Gly Cys Thr Thr Val Thr Val Gly Ser Asp 435 440
445 Gly Asn Val Pro Val Pro Met Ala Gly Gly Leu Pro Arg Val Leu Tyr
450 455 460 Pro Thr Glu Lys Leu Ala Gly Ser Lys Ile Cys Ser Ser Ser
465 470 475 3 476 PRT Aspergillus niger 3 Leu Ser Ala Ala Ser Trp
Arg Thr Gln Ser Ile Tyr Phe Leu Leu Thr 1 5 10 15 Asp Arg Phe Gly
Arg Thr Asp Asn Ser Thr Thr Ala Thr Cys Asn Thr 20 25 30 Gly Asn
Glu Ile Tyr Cys Gly Gly Ser Trp Gln Gly Ile Ile Asp His 35 40 45
Leu Asp Tyr Ile Glu Gly Met Gly Phe Thr Ala Ile Trp Ile Ser Pro 50
55 60 Ile Thr Glu Gln Leu Pro Gln Asp Thr Ala Asp Gly Glu Ala Tyr
His 65 70 75 80 Gly Tyr Trp Gln Gln Lys Ile Tyr Asp Val Asn Ser Asn
Phe Gly Thr 85 90 95 Ala Asp Asn Leu Lys Ser Leu Ser Asp Ala Leu
His Ala Arg Gly Met 100 105 110 Tyr Leu Met Val Asp Val Val Pro Asp
His Met Gly Tyr Ala Gly Asn 115 120 125 Gly Asn Asp Val Asp Tyr Ser
Val Phe Asp Pro Phe Asp Ser Ser Ser 130 135 140 Tyr Phe His Pro Tyr
Cys Leu Ile Thr Asp Trp Asp Asn Leu Thr Met 145 150 155 160 Val Glu
Asp Cys Trp Glu Gly Asp Thr Ile Val Ser Leu Pro Asp Leu 165 170 175
Asp Thr Thr Glu Thr Ala Val Arg Thr Ile Trp Tyr Asp Trp Val Ala 180
185 190 Asp Leu Val Ser Asn Tyr Ser Val Asp Gly Leu Arg Ile Asp Ser
Val 195 200 205 Leu Glu Val Gln Pro Asp Phe Phe Pro Gly Tyr Asn Lys
Ala Ser Gly 210 215 220 Val Tyr Cys Val Gly Glu Ile Asp Asn Gly Asn
Pro Ala Ser Asp Cys 225 230 235 240 Pro Tyr Gln Lys Val Leu Asp Gly
Val Leu Asn Tyr Pro Ile Tyr Trp 245 250 255 Gln Leu Leu Tyr Ala Phe
Glu Ser Ser Ser Gly Ser Ile Ser Asn Leu 260 265 270 Tyr Asn Met Ile
Lys Ser Val Ala Ser Asp Cys Ser Asp Pro Thr Leu 275 280 285 Leu Gly
Asn Phe Ile Glu Asn His Asp Asn Pro Arg Phe Ala Lys Tyr 290 295 300
Thr Ser Asp Tyr Ser Gln Ala Lys Asn Val Leu Ser Tyr Ile Phe Leu 305
310 315 320 Ser Asp Gly Ile Pro Ile Val Tyr Ala Gly Glu Glu Gln His
Tyr Ala 325 330 335 Gly Gly Lys Val Pro Tyr Asn Arg Glu Ala Thr Trp
Leu Ser Gly Tyr 340 345 350 Asp Thr Ser Ala Glu Leu Tyr Thr Trp Ile
Ala Thr Thr Asn Ala Ile 355 360 365 Arg Lys Leu Ala Ile Ala Ala Asp
Ser Ala Tyr Ile Thr Tyr Ala Asn 370 375 380 Asp Ala Phe Tyr Thr Asp
Ser Asn Thr Ile Ala Met Ala Lys Gly Thr 385 390 395 400 Ser Gly Ser
Gln Val Ile Thr Val Leu Ser Asn Lys Gly Ser Ser Gly 405 410 415 Ser
Ser Tyr Thr Leu Thr Leu Ser Gly Ser Gly Tyr Thr Ser Gly Thr 420 425
430 Lys Leu Ile Glu Ala Tyr Thr Cys Thr Ser Val Thr Val Asp Ser Ser
435 440 445 Gly Asp Ile Pro Val Pro Met Ala Ser Gly Leu Pro Arg Val
Leu Leu 450 455 460 Pro Ala Ser Val Val Asp Ser Ser Ser Leu Cys Gly
465 470 475 4 475 PRT Thermomyces lanuginosus 4 Ala Thr Pro Asp Glu
Trp Lys Ala Gln Ser Ile Tyr Phe Met Leu Thr 1 5 10 15 Asp Arg Phe
Ala Arg Thr Asp Asn Ser Thr Thr Ala Pro Cys Asp Thr 20 25 30 Thr
Ala Gly Lys Tyr Cys Gly Gly Thr Trp Arg Gly Ile Ile Asn Asn 35 40
45 Leu Asp Tyr Ile Gln Asp Met Gly Phe Thr Ala Ile Trp Ile Thr Pro
50 55 60 Val Thr Ala Gln Trp Asp Asp Asp Val Asp Ala Ala Asp Ala
Thr Ser 65 70 75 80 Tyr His Gly Tyr Trp Gln Lys Asp Leu Tyr Ser Leu
Asn Ser Lys Phe 85 90 95 Gly Thr Ala Asp Asp Leu Lys Ala Leu Ala
Asp Ala Leu His Ala Arg 100 105 110 Gly Met Leu Leu Met Val Asp Val
Val Ala Asn His Phe Gly Tyr Gly 115 120 125 Gly Ser His Ser Glu Val
Asp Tyr Ser Ile Phe Asn Pro Leu Asn Ser 130 135 140 Gln Asp Tyr Phe
His Pro Phe Cys Leu Ile Glu Asp Tyr Asp Asn Gln 145 150 155 160 Glu
Glu Val Glu Gln Cys Trp Leu Ala Asp Thr Pro Thr Thr Leu Pro 165 170
175 Asp Val Asp Thr Thr Asn Pro Gln Val Arg Thr Phe Phe Asn Asp Trp
180 185 190 Ile Lys Ser Leu Val Ala Asn Tyr Ser Ile Asp Gly Leu Arg
Val Asp 195 200 205 Thr Val Lys His Val Glu Lys Asp Phe Trp Pro Asp
Phe Asn Glu Ala 210 215 220 Ala Ala Cys Thr Val Gly Glu Val Phe Asn
Gly Asp Pro Ala Tyr Thr 225 230 235 240 Cys Pro Tyr Gln Glu Val Leu
Asp Gly Val Leu Asn Tyr Pro Ile Tyr 245 250 255 Tyr Pro Ala Leu Asp
Ala Phe Lys Ser Val Gly Gly Asn Leu Gly Gly 260 265 270 Leu Ala Gln
Ala Ile Thr Thr Val Gln Glu Ser Cys Lys Asp Ser Asn 275 280 285 Leu
Leu Gly Asn Phe Leu Glu Asn His Asp Ile Ala Arg Phe Ala Ser 290 295
300 Tyr Thr Asp Asp Leu Ala Leu Ala Lys Asn Gly Leu Ala Phe Ile Ile
305 310 315 320 Leu Ser Asp Gly Ile Pro Ile Ile Tyr Thr Gly Gln Glu
Gln His Tyr 325 330 335 Ala Gly Asp His Asp Pro Thr Asn Arg Glu Ala
Val Trp Leu Ser Gly 340 345 350 Tyr Asn Thr Asp Ala Glu Leu Tyr Gln
Phe Ile Lys Lys Ala Asn Gly 355 360 365 Ile Arg Asn Leu Ala Ile Ser
Gln Asn Pro Glu Phe Thr Ser Ser Lys 370 375 380 Thr Lys Val Ile Tyr
Gln Asp Asp Ser Thr Leu Ala Ile Asn Arg Gly 385 390 395 400 Gly Val
Val Thr Val Leu Ser Asn Glu Gly Ala Ser Gly Glu Thr Gly 405 410 415
Thr Val Ser Ile Pro Gly Thr Gly Phe Glu Ala Gly Thr Glu Leu Thr 420
425 430 Asp Val Ile Ser Cys Lys Thr Val Thr Ala Gly Asp Ser Gly Ala
Val 435 440 445 Asp Val Pro Leu Ser Gly Gly Leu Pro Ser Val Leu Tyr
Pro Ser Ser 450 455 460 Gln Leu Ala Lys Ser Gly Leu Cys Ala Ser Ala
465 470 475
* * * * *
References