Variant lipolytic enzymes

US007465570B2
(12) United States Patent
Borch et a1.
(54)
VARIANT LIPOLYTIC ENZYMES
(75) Inventors: Kim Borch, Birkerod (DK); Luise
Erlandsen, CopenhagenV (DK); Jesper
Vind, Vaerlose (DK); Allan Svendsen,
Horsholm (DK); Christel Thea
(10) Patent N0.:
(45) Date of Patent:
(30)
J orgensen, Kgs Lyngby (DK)
(73) Assignee: Novozymes A/S, Bagsvaerd (DK)
(*)
Notice:
(21) Appl. No.:
Apr. 29, 2004
(86)
PCT No.:
PCT/DK2004/000292
(2), (4) Date:
Nov. 9, 2005
(87)
PCT Pub. No.: WO2004/099400
PCT Pub. Date: Nov. 18, 2004
(65)
Prior Publication Data
US 2006/0228446 A1
Oct. 12, 2006
Related US. Application Data
(60)
Provisional application No. 60/479,647, ?led on Jun.
3, 1920, provisional application No. 60/474,881, ?led
on May 30, 2003, provisional application No. 60/469,
228, ?led on May 9, 2003.
Int. Cl.
C12N 9/16
A21D 8/02
............................. .. 2003 00709
............................. .. 2003 00811
(2006.01)
(2006.01)
US. Cl. ....................................... .. 435/196; 426/20
(58)
Field of Classi?cation Search ............... .. 435/198,
435/196
See application ?le for complete search history.
(56)
10/556,511
(22) PCT Filed:
(DK)
(DK)
(52)
Subject to any disclaimer, the term of this
patent is extended or adjusted under 35
U.S.C. 154(b) by 153 days.
Dec. 16, 2008
Foreign Application Priority Data
May 9, 2003
May 30, 2003
(51)
US 7,465,570 B2
References Cited
FOREIGN PATENT DOCUMENTS
EP
W0
W0
W0
W0
W0
0 869 167
WO 98/26057
WO 00/32758
WO 01/83770
WO 02/00852
W0 02/055679
10/1998
6/1998
6/2000
11/2001
1/2002
7/2002
Primary ExamineriTekchand Saidha
(74) Attorney, Agent, or FirmiKristin J. McNamara
(57)
ABSTRACT
The inventors have developed improved polypeptides by sub
stituting or deleting speci?ed amino acids in fungal lipolytic
enzymes. More particularly, the polypeptides result in a
reduction of dough stickiness When they are added to a dough.
The polypeptides may particularly have activity on polar
lipids.
15 Claims, 3 Drawing Sheets
US. Patent
Dec. 16, 2008
Sheet 3 of3
US 7,465,570 B2
Fig. 1 cont.
301
350
SEQ
ID
NO:
1
QATDA.CNAG
GFS
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286
SEQ
ID
NO:
2
QATDA.CNAG
GFS .
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269
SEQ
ID
NO:
3
MNTGL.CL.
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SEQ
ID
NO:
4
MNTGL.CL.
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SEQ
ID
NO:
5
INTGL.CT
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.CL.
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SEQ
ID
NO:
6
INEGS
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SEQ
ID
NO:
7
GMTSGACTW.
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SEQ ID NO: 8
GMTSGHCTW
SEQ
ID
NO:
9
QSMAT.CAPI
AIPWKR.
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SEQ
ID
NO:
10
FIHADACKGP
GLPLR .
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LPFKR .
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SEQ
ID
NO:
11
VQVDAGKGPG
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SEQ
ID
NO:
12
FAISE.CLL.
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SEQ
ID
NO:
13
FAISE..CLL.
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SEQ
ID
NO:
14
IYISE.CS.
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SEQ
ID
NO:
15
GLIGT.CL.
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269
US 7,465,570 B2
1
2
VARIANT LIPOLYTIC ENZYMES
BRIEF DESCRIPTION OF DRAWINGS
CROSS-REFERENCE TO RELATED
APPLICATIONS
fungal lipolytic enZymes to identify corresponding amino
FIG. 1 shoWs an alignment of amino acid sequences of
acids in SEQ ID NO: 1 to 15. SEQ ID NO: 1 is the lipase/
phospholipase from Fusarium oxysporum (WO 9826057).
This application is a 35 U.S.C. 371 national application of
SEQ ID NO: 2 is a variant With phospholipase and galactoli
pase activity disclosed in WO 0032758. SEQ ID NO: 3 to 15
PCT/DK2004/000292 ?led Apr. 29, 2004, Which claims pri
ority or the bene?t under 35 USC 1 19 of Danish application
nos. PA 2003 00709 ?led May 9, 2003 and PA 2003 00811
are knoWn lipolytic enZymes from the folloWing organisms:
?led May 30, 2003 and US. Provisional application Nos.
Absidia re?exa, Absidia corymbefera, Rhizomucor miehei,
60/469,228, 60/474,881 and 60/479,647 ?led May 9, 2003,
Rhizopus delemar (oryzae), Aspergillus niger, Aspergillus
May 30, 2003, and Jun. 19, 2003, respectively, the contents of
Which are fully incorporated herein by reference.
Penicilium camemberli, Aspergillus foelidus, Aspergillus
Zubingensis, Fusarium helerosporum, Aspergillus oryzae,
niger, Aspergillus oryzae and Thermomyces lanuginosus.
FIELD OF INVENTION
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to variant polypeptides made
by altering the amino acid sequence of a fungal lipolytic
enzyme, particularly to such polypeptides With improved
properties for use in a dough, e. g. for making bread and other
Parent Polypeptide
20
baked products, and more particularly to such polypeptides
The parent polypeptide may have the sequence SEQ ID
NO: 1 or 2 or one Which can be aligned With SEQ ID NO: 1 or
2. It may have at least 50% amino acid identity to SEQ ID NO:
having hydrolytic activity toWards ester bonds in polar lipids.
1 or 2, eg at least 60%, at least 70% or at least 80%.
Examples are the polypeptides having the sequences SEQ ID
BACKGROUND OF THE INVENTION
NO: 1 to 14 or a variant disclosed in WO 0032758.
25
With hydrolytic activity toWards ester bonds in polar lipids
Variant Polypeptide
such as phospholipids and galactolipids. WO 0032758 dis
The amino acid at the position corresponding to A29 in
SEQ ID NO: 1 may be P. The amino acid at the position
corresponding to K33 in SEQ ID NO: 1 may be N. The amino
closes lipolytic enZyme variants having phospholipase and
galactolipase activity and their use in baking. WO 9826057
discloses a lipase/phospholipase from Fusarium oxysporum
and its use in baking. WO 0183770 describes variants of a
fungal lipase.
SUMMARY OF THE INVENTION
35
The inventors have developed variant polypeptides by
modifying the amino acid sequence of a parent polypeptide
Which is a fungal lipolytic enZymes. The variant polypeptides
result in a reduced dough stickiness, compared to the parent
polypeptide, When they are added to a dough.
Accordingly, the invention provides a method of producing
a polypeptide, comprising:
a) selecting an amino acid sequence for a parent polypep
tide Which is a fungal lipolytic enzyme,
b) selecting an amino acid residue in the sequence Which
corresponds to A29, K33, I83 or A255 of SEQ ID NO: 1
(corresponding to P29, N33, R84 or P256 of SEQ ID NO: 2),
c) modifying the amino acid sequence by substituting or
40
45
position N247, D at position N248, K/R at position Q249,
K/T at position P250, T at position N251, F at position I252,
M/R at position P253, S/Y/W at position D254, L at position
I255, G at positionA257, H/C at position W260, G at position
Q263, L at position A264, I at position T265, G/S/A at posi
50
tion D266, T at position A267, L at position N269 and/or
truncation after N269.
The polypeptide may additionally comprise amino acid
modi?cations such as insertions or deletions. Also, the N- or
amino acid sequence, and
55
DAELEKKLNSYVQMDKEYVKNNQARS. The C-termi
nal may be truncated after position 272, 273, 274 or 286 in
lipid, and
60
different amino acid or an amino acid deletion at a posi
tion corresponding to R84 or P256.
SEQ ID NO: 1 . The N-terminal may have a peptide extension,
eg as described in WO 0032758 or WO 9704079, such as the
addition of the amino acid residues SPIRR.
A similar amino acid substitution or deletion may be made
in other fungal lipolytic enZymes, e. g. SEQ ID NO: 3-14 at a
different amino acid or an amino acid deletion at a posi
tion corresponding to A29, K33, I83 or A255, or
ii) has at least 80% identity to SEQ ID NO: 2 and has a
C-terminus may be modi?ed, eg by truncating residues in
SEQ ID NO: 2 after position 269 or by extending the C-ter
minal of SEQ ID NO: 2 With WRRYRSAESVDKRATMT
stickiness.
The invention also provides a variant polypeptide Which:
a) has hydrolytic activity toWards an ester bonds in a polar
b) has an amino acid sequence Which
i) has at least 80% identity to SEQ ID NO: 1 and has a
acid at the position corresponding to I83 of SEQ ID NO: 1
may be A/R/N/D/C/Q/E/G/H/L/K/M/F/P/S/T/Y/V. The
amino acid at the position corresponding to A255 in SEQ ID
NO: 1 may be R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/V
The amino acid at the position corresponding to R84 of
SEQ ID NO: 2 may be A/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/T/
Y/V. The amino acid at the position corresponding to P256 in
SEQ ID NO: 2 may be A/R/N/D/C/Q/E/G/H/I/L/K/M/F/S/T/
W/Y/V. The polypeptide may comprise further modi?cations
compared to SEQ ID NO: 2., eg as disclosed in WO
0032758. Thus, it may have the amino acid A/T at position
D62, G/T at position A91, D/F/S/G at position W96, E at
position K99, G at position S158, D at position G240, S at
deleting the selected residue,
d) preparing a variant polypeptide having the modi?ed
e) adding the polypeptide to a dough and testing dough
The parent polypeptide has lipolytic enZyme activity, eg
hydrolytic activity toWards an ester bond in a polar lipid.
Phospholipases and galactolipases are knoWn as enZymes
corresponding position. The corresponding positions may be
65
found by aligning a given sequence With SEQ ID NO: 1 or 2,
eg as shoWn in FIG. 1. The alignment may be done by use of
the GAP program as described beloW.
US 7,465,570 B2
3
4
The variant polypeptide may have improved therrno stabil
ity compared to the parent polypeptide, particularly a variant
polypeptide having a substitution at a position corresponding
retention and volume during processing and baking, particu
larly in a stressed system, eg in the case of over-proo?ng or
over-mixing. It may also lead to a loWer initial ?rmness and/or
a more uniform and ?ne crumb, improved crumb structure
to A29 or K33 ofSEQ ID NO: 1, eg the substitutionA29P or
K33N.
(?ner crumb, thinner cell Walls, more rounded cells), of the
Sequence Identity
baked product, and it may further improve dough properties,
The variant polypeptide has at least 80% identity to SEQ
eg a less soft dough, higher elasticity and/or loWer extensi
ID NO: 1 or 2, particularly at least 85%, at least 90%, at least
95%, or at least 98%. The degree of identity betWeen tWo
sequences may be suitably determined by means of computer
programs knoWn in the art, such as GAP provided in the GCG
bility.
The process may be conducted in analogy With US. Pat.
No. 5,578,489 or US. Pat. No. 6,077,336. In the case of
program package (Program Manual for the Wisconsin Pack
age, Version 8, August 1994, Genetics Computer Group, 575
un-proofed froZen dough the polypeptides of the invention
Science Drive, Madison, Wis., USA 53711) (Needleman, S.
B. and Wunsch, C. D., (1970), Journal of Molecular Biology,
48, 443-45), using GAP With the folloWing settings for
polypeptide sequence comparison: GAP creation penalty of
of volume and crumb structure.
perform better than knoWn lipolytic enZyme variants in terms
The polypeptide can be used in a process for making bread,
comprising adding the polypeptide to the ingredients of a
dough, kneading the dough and baking the dough to make the
3.0 and GAP extension penalty of 0.1.
Dough Stickiness
bread. This can be done in analogy With US. Pat. No. 4,567,
20
The variant polypeptide may be tested by adding it to a
dough and evaluating the dough stickiness. The dough may be
generated according to a typical European straight dough
procedure, a typical American sponge & dough procedure or
any other bread making procedures. The polypeptide may be
added at a dosage of 0.01- 1 0 mg enZyme protein per kg ?our,
and the dough stickiness may be evaluated directly after mix
25
ing or at any point during processing. Of particular impor
tance is the dough stickiness of the ?nally mixed dough, i.e. at
the time Where the dough runs through processing equipment
such as divider, molder, sheeter and conveyor belts. The mix
ing time varies depending on procedure. For a typical Euro
pean straight dough procedure, the mixing time can eg be in
the range of 6-10 minutes. For a typical American Sponge &
dough procedure the mixing time can eg be in the range of
6-20 minutes (on ?nal dough). The dough may have a resting
30
described in W0 99/ 53769, e. g. derived from Aspergillus, in
particular of A. aculealus, A. niger (cf. WO 91/19782), A.
awamori (WO 91/18977), or A. Zubigensis (WO 92/01793),
The dough stickiness may be evaluated by hand by trained
from a strain of Trichoderma, e. g. T reesei, or from a strain of
bakers, by a sensory panel or by instrumental measurements
40
Humicula, e.g. H. insolens (WO 92/17573), a proteiase and/
or a glucose oxidase.
Stable Micro Systems TA-XT2 texture analyser, commer
The dough may further comprise an emulsi?er such as
cially available from Brook?eld Engineering Laboratories,
mono- or diglycerides, diacyl tartaric acid esters of mono- or
Inc.
Hydrolytic Activity toWards Ester Bonds in Polar Lipids
The parent and variant polypeptides have lipolytic enZyme
activity, i.e. they have hydrolytic activity toWards an ester
426211 (Unilever) or WO 99/53769 (NovoZymes).
The composition of a typical dough can be found in W0
99/ 53769.
The polypeptide of the invention may be added together
With an anti-staling amylase and optionally also a phospho
lipid as described in WO 9953769, particularly a maltogenic
alpha-amylase (eg from Bacillus sp., such as Novamyl®
from Novo Nordisk). Also, a fungal or bacterial ot-amylase
may be added, eg from Aspergillus or Bacillus, particularly
A. oryzae, B. lichenlformis or B. amyloliquefaciens. Option
ally an additional enZyme may be added, eg an amyloglu
cosidase, a beta-amylase, a pentosanase such as a xylanase as
35
period of 5-20 min before further processing, e. g. at 20-350 C.
eg by the Chen-Hoseney dough stickiness rig developed for
046 (KyoWa Hakko), JP-A 60-78529 (QP Corp.), JP-A
62-111629 (QP Corp.), JP-A 63-258528 (QP Corp.), EP
45
diglycerides, sugar esters of fatty acids, polyglycerol esters of
fatty acids, lactic esters of monoglycerides, acetic acid esters
of monoglycerides, polyoxyethylene stearates, polysorbates
bond and are classi?ed in EC 3.1.1 Carboxylic Ester Hydro
or lysolecithin.
lases according to EnZyme Nomenclature (available at
The dough may also comprise other conventional dough
ingredients, e.g.: proteins, such as milk poWder, gluten, and
WWW.chem.qmW.ac.uk/iubmb/enZyme). More speci?cally,
they have hydrolytic activity toWards ester bonds in polar
soy; eggs (either Whole eggs, egg yolks or egg Whites); an
lipids so as to split off acyl groups at the sn-1 and/or sn-2
oxidant such as ascorbic acid, potassium bromate, potassium
iodate, aZodicarbonamide (ADA) or ammonium persulfate;
position of polar lipids such as phospholipids and galactolip
ids. Accordingly, they may have phospholipase activity or
galactolipase activity (EC 3.1.1.26), e.g. phospholipase A1
activity (EC 3.1.1.32).
an amino acid such as L-cysteine; a sugar; a salt such as
55
sodium chloride, calcium acetate, sodium sulfate or calcium
sulfate.
Pho spholipase activity may be determined by knoWn meth
ods, eg the “monolayer phospholipase assay” or the plate
assay described in WO 0032758. Galactolipase activity may
be determined With digalactosyl diglyceride as substrate, eg
as described in WO 0032758.
EXAMPLES
60
The polypeptide may be added to a dough, and the dough
may be used to prepare a steamed bread, a baked product
(particularly bread), pasta or noodles. The addition of the
polypeptide may lead to improved dough stabiliZation, i.e. a
larger loaf volume of the baked product and/or a better shape
Baking Evaluation of Polypeptides With Phospholipase
Activity
Use of Polypeptide
65
In the examples, polypeptides according to the invention
Were tested together With the corresponding parent polypep
tide in a baking evaluation experiment by using conventional
baking protocols for European straight dough procedure and
US sponge & dough procedure, as folloWs:
US 7,465,570 B2
5
European Straight Dough Procedure:
-continued
A dough is prepared by mixing the below ingredients for 3
minutes sloW and 7 minutes fast.
5
Water
Ascorbicc acid
% (baker’s — by Weight)
Flour
Sponge
Dough
% (baker’s — by Weight)
% (baker’s — by Weight)
34.4
20.4
50
100
4
A liquid sponge is prepared by mixing a sponge consisting
Salt
1.5
Sugar
1.5
Water
Ascorbic acid
62
40 ppm
of the above listed sponge ingredients for 1 minute sloW and
4 minutes fast. The sponge is fermented for 3 hours at 27 C,
86% RH. The sponge is mixed With the dough ingredients
listed above and With enZymes for 1 minutes sloW and 18
minutes fast.
Compressed yeast
Dough stickiness is evaluated right after mixing and again
ter the dough is extruded on a rebuild pasta-machine to simu
late the dough extrusion used for dough dividing in US.
Dough stickiness is evaluated again after extrusion. Dough
evaluation by hand. Dough stickiness is a measure of hoW
sticky the dough feels and is expressed on a scale from 0 (little
stickiness) to 10 (very sticky). The dough With the variant is
compared to a reference dough, Which is alWays given the
Dough stickiness is evaluated right after mixing, Whereaf
15
after a resting period of 15 minutes. Dough stickiness is
evaluated by a trained and experienced bakers by sensory
stickiness is evaluated by a trained and experienced bakers by
20
sensory evaluation by hand. Dough stickiness is a measure of
hoW sticky the dough feels and is expressed on a scale from 0
score 5.
(little stickiness) to 10 (very sticky). The dough With the
Sponge & Dough Procedure:
variant polypeptide is compared to a reference dough made
With the parent polypeptide, Which is alWays given the score
25
Flour
Sponge
Dough
% (baker’s — by Weight)
% (baker’s — by Weight)
60
Compressed yeast
7.5
Oil
Salt
2.5
High fructose syrup
40
2
5.
Example 1
Construction of Polypeptides
30
Polypeptides according to the invention Were prepared as
described in WO 00/32758. The polypeptides Were derived
from SEQ ID NO: 15 by making the folloWing amino acid
modi?cations.
Polypeptide Amino acid alterations compared to SEQ ID NO: 15
US 7,465,570 B2
-continued
Polypeptide Amino acid alterations compared to SEQ ID NO: 15
10
11
12
14
16
17
18
19
50
Example 2
Baking Evaluation of a Polypeptide According to the
Invention
55
Polypeptide
Dough
stickiness after
mixing
5 variant polypeptides according to the invention Were
Dough
stickiness after
15 min table
time
compared to the parent polypeptide (SEQ ID NO: 2) in the
European straight dough procedure described above. 40 ppm
Fungamyl Super MA (a blend of fungal alpha-amylase and
6
5
5
5
5
5
Xylanase) Was added as background to all doughs. The parent
enzyme and the variants Were dosed at their optimal level, i.e.
the level giving best volume and dough stabilising effect. The
beloW results shoW that all 5 variants give reduced dough
stickiness compared to the parent polypeptide.
3 variant polypeptides according to the invention Were
65
compared to the parent polypeptide (SEQ ID NO: 1) in the
European straight dough procedure described above. 40 ppm
US 7,465,570 B2
9
10
Fungamyl Super MA (a blend of fungal alpha-amylase and
xylanase) Was added as background to all doughs. The parent
enzyme and the variants Were dosed at their optimal level, i.e.
the level giving best volume and dough stabilising effect. The
beloW results shoW that all 4 variants give reduced dough 5
stickiness compared to the parent enzyme
Polypeptide
Dough stickiness
A29P
K33N
Parent
A29P+K33N+I83T
A29P+K33N+I83H
A29P+K33N+I83Q
5
4
4
4
5
4
4
4
after mixing
Dough stickiness
after 15 min table
time
4 variant polypeptides according to the invention Were
compared to the parent enZyme (SEQ ID NO: 1) in the European straight dough procedure described above. IOFAU Fun- 20
-c0minued
A29P +I83T
gamyl/kg Was added as background to all doughs. The parent
enZyme and the variants Were dosed at their optimal level, i.e.
A291, +I83N
A291, +I83C
the level giving best volume and dough stabilising effect. The
beloW results shoW that all 4 variants give reduced dough
stickiness compared to the parent enZyme
Polypeptide
Dough stickiness
A29P +1331:
A29P +I83L
Parent
A29P + K33N
A29P + I83N
K33N + 183E
K33N + 183K
7
6
6
6
6
7
6
6
6
6
after mixing
Dough stickiness
after 15 min table
time
A variant polypeptide according to the invention Was com
pared to its parent enZyme (SEQ ID NO: 1) in the US sponge
& dough procedure described above. 40 ppm Fungamyl 40
Super MA (a blend of fungal alpha-amylase and xylanase)
K33N +133L
Was added as background to all doughs. The parent enZyme
and the variant Were dosed at their opt1mal level, 1.e. the level
K33N +I83Q
K33N +I83N
giving best volume and dough stabilising effect. The beloW
K33N +I83N
.
.
.
.
-continued
K33N +183“,
K33N +1838
results shoW that the variant gives reduced dough stickiness 45
+1235
+
Compared to the Parent enzyme
K33N +270VASLGDDTEAPRASTRGPP
A29P +I83N +A255V
Polypeptide
Dough Stickiness mm
Par?nt
6
A29P+I83N
5
mixing
Doug? Stickin?ss a?er
mmslon
6-5
5
50
unmodi?ed sequence of SEQ ID NO: 1.
Baking tests With each dough shoWed that all polypeptides
55 improved the crumb structure, the loaf volume and the dough
stability, both for the modi?ed and unmodi?ed sequences.
Example 3
Variant Polypeptides Derived from SEQ ID NO: 1
The results Were that With each of the above polypeptides,
dough stickiness Was better than With the polypeptide With the
Example 4
60
Variant Polypeptides Derived from SEQ ID NO: 2
Variant polypeptides With the folloWing amino acid alterVariant polypeptides With the folloWing amino acid alter
ations compared SEQ ID NO: 1 (lipase/phosapholipase from
ations compared SEQ ID NO: 2 (variant of T lanuginosus
F oxysporum) Were prepared and tested by adding each 65 lipase) Were prepared and tested by adding each polypeptide
polypeptide to a dough. The polypeptide With unmodi?ed
to a dough. The polypeptide With unmodi?ed SEQ ID NO: 2
SEQ ID NO: 1 Was also tested, for comparison.
Was also tested for comparison.
US 7,465,570 B2
11
12
-continued
R841)
5
P25 68
R841
P25 6V
R84M
R84Q
P25 6A
P256D
The results Were that With each of the above polypeptides,
dough stickiness Was better than With the polypeptide With the
10 unmodi?ed sequence of SEQ ID NO: 2.
P25 61
P25 6L
P25 6Q
Baking tests With each dough shoWed that all polypeptides
improved the crumb structure, the loaf Volume and the dough
stability, both for the modi?ed and unmodi?ed sequences.
SEQUENCE LISTING
<l60> NUMBER OF SEQ ID NOS :
15
<2ll> LENGTH: 286
<2l2> TYPE: PRT
<2l3> ORGANISM:
Fusarium oxysporum
<400> SEQUENCE:
1
Ala Val Gly Val Thr Thr Thr Asp Phe Ser Asn Phe Lys Phe Tyr Ile
1
5
1O
15
Gln His Gly Ala Ala Ala Tyr Cys Asn Ser Glu Ala Ala Ala Gly Ser
20
25
3O
Lys Ile Thr Cys Ser Asn Asn Gly Cys Pro Thr Val Gln Gly Asn Gly
35
4O
45
Ala Thr Ile Val Thr Ser Phe Val Gly Ser Lys Thr Gly Ile Gly Gly
50
55
6O
Tyr Val Ala Thr Asp Ser Ala Arg Lys Glu Ile Val Val Ser Phe Arg
65
7O
75
8O
Gly Ser Ile Asn Ile Arg Asn Trp Leu Thr Asn Leu Asp Phe Gly Gln
85
9O
95
Glu Asp Cys Ser Leu Val Ser Gly Cys Gly Val His Ser Gly Phe Gln
100
105
110
Arg Ala Trp Asn Glu Ile Ser Ser Gln Ala Thr Ala Ala Val Ala Ser
115
120
125
Ala Arg Lys Ala Asn Pro Ser Phe Asn Val Ile Ser Thr Gly His Ser
130
135
140
Leu Gly Gly Ala Val Ala Val Leu Ala Ala Ala Asn Leu Arg Val Gly
145
150
155
160
Gly Thr Pro Val Asp Ile Tyr Thr Tyr Gly Ser Pro Arg Val Gly Asn
165
1'70
175
Ala Gln Leu Ser Ala Phe Val Ser Asn Gln Ala Gly Gly Glu Tyr Arg
180
185
190
Val Thr His Ala Asp Asp Pro Val Pro Arg Leu Pro Pro Leu Ile Phe
195
200
205
Gly Tyr Arg His Thr Thr Pro Glu Phe Trp Leu Ser Gly Gly Gly Gly
210
215
220
Asp Lys Val Asp Tyr Thr Ile Ser Asp Val Lys Val Cys Glu Gly Ala
225
230
235
240
Ala Asn Leu Gly Cys Asn Gly Gly Thr Leu Gly Leu Asp Ile Ala Ala
245
250
255
US 7,465,570 B2
13
—cont inued
His Leu His Tyr Phe Gln Ala Thr Asp Ala Cys Asn Ala Gly Gly Phe
260
265
270
Ser Trp Arg Arg Tyr Arg Ser Ala Glu Ser Val Asp Lys Arg
275
<210>
<211>
<212>
<2l3>
<220>
<223>
280
285
SEQ ID NO 2
LENGTH: 2'74
TYPE: PRT
ORGANISM: Artificial
FEATURE:
OTHER INFORMATION: Variant disclosed in WO 0032758
<400> SEQUENCE: 2
Glu Val Ser Gln Asp Leu Phe Asn Gln Phe Asn Leu Phe Ala Gln Tyr
1
5
10
15
Ser Ala Ala Ala Tyr Cys Gly Lys Asn Asn Asp Ala Pro Ala Gly Thr
20
25
30
Asn Ile Thr Cys Thr Gly Asn Ala Cys Pro Glu Val Glu Lys Ala Asp
35
40
45
Ala Thr Phe Leu Tyr Ser Phe Glu Asp Ser Gly Val Gly Asp Val Thr
50
55
60
Gly Phe Leu Ala Leu Asp Asn Thr Asn Lys Leu Ile Val Leu Ser Phe
65
'70
75
80
Arg Gly Ser Arg Ser Ile Glu Asn Trp Ile Ala Asn Leu Asn Phe Trp
85
90
95
Leu Lys Lys Ile Asn Asp Ile Cys Ser Gly Cys Arg Gly His Asp Gly
100
105
110
Phe Thr Ser Ser Trp Arg Ser Val Ala Asp Thr Leu Arg Gln Lys Val
115
120
125
Glu Asp Ala Val Arg Glu His Pro Asp Tyr Arg Val Val Phe Thr Gly
130
135
140
His Ser Leu Gly Gly Ala Leu Ala Thr Val Ala Gly Ala Asp Leu Arg
145
150
155
160
Gly Asn Gly Tyr Asp Ile Asp Val Phe Ser Tyr Gly Ala Pro Arg Val
165
170
175
Gly Asn Arg Ala Phe Ala Glu Phe Leu Thr Val Gln Thr Gly Gly Thr
180
185
190
Leu Tyr Arg Ile Thr His Thr Asn Asp Ile Val Pro Arg Leu Pro Pro
195
200
205
Arg Glu Phe Gly Tyr Ser His Ser Ser Pro Glu Tyr Trp Ile Lys Ser
210
215
220
Gly Thr Leu Val Pro Val Thr Arg Asn Asp Ile Val Lys Ile Glu Gly
225
230
235
240
Ile Asp Ala Thr Gly Gly Asn Asn Gln Pro Asn Ile Pro Asp Ile Pro
245
250
255
Ala His Leu Trp Tyr Phe Gln Ala Thr Asp Ala Cys Asn Ala Gly Gly
260
265
270
Phe Ser
<210>
<211>
<212>
<2l3>
SEQ ID NO 3
LENGTH: 265
TYPE: PRT
ORGANISM: Absidia reflexa
<4 00> SEQUENCE: 3
Ser Ser Ser Ser Thr Gln Asp Tyr Arg Ile Ala Ser Glu Ala Glu Ile
US 7,465,570 B2
15
16
—cont inued
10
Lys
Ala His Thr Phe
Tyr
Thr Ala Leu Ser Ala Asn Ala
20
Thr Val Ile Pro
15
25
Gly Gly Arg Trp
Ser
Tyr Cys Arg
30
Cys
Pro His
Cys Gly
Val Ala
45
Ser Asn Leu Gln Ile Thr Lys Thr Phe Ser Thr Leu Ile Thr
50
55
60
Asn Val Leu Val Ala Val
65
Phe
Arg Gly
Tyr
100
Asp
Ser
Tyr
115
Lys
Ala Gln Leu
Glu
Lys
Arg
Gly
Asn Glu Val Gln
120
Asp Lys
Asp Arg His
Gly Gly
145
Pro
Gln
Ala Phe
His Ala Asn Ile Glu Ile
Tyr
Tyr
Arg Asp
Phe Leu His Ala
Arg
Val Ile
Val
<210>
<211>
<212>
<213>
Met Asn Thr
260
Gly
SEQ ID NO 4
LENGTH: 264
TYPE: PRT
ORGANISM: Absidia
Leu
Cys
Gly
Gln Pro
Gly
Thr
Lys
Arg
Ile Pro
Ile Val Pro His Leu Pro Pro
205
Gly
Glu Glu Phe
Trp
Ile Met
Lys
220
Pro Asn
Gly
Ile Glu Thr
Asp
Asn
235
Leu
Tyr
190
230
Asp
Asp
175
215
Ser Ser Leu
Gly
160
Thr Gln
Cys
Cys
240
Leu Ser
255
Tyr
Ile Ala Ser Glu Ala Glu Ile
Lys
Tyr Cys Arg
Thr
Ser Asn Ser Ile Val Pro Phe Thr Ser Val Ile
245
250
Leu
Ile Val Val Thr
200
225
Lys Gly
Leu Val Ala Glu Val
125
185
210
Asp
Val His
170
Leu Val His Glu
Gly
Ile Val Phe
155
195
Gly
Lys
Asp
Ala Thr Ala Val Leu Ser Ala Leu
Thr Pro Ala Phe Ala Asn
Arg
Val Val
140
180
Tyr
Ala
Gly Tyr Lys
165
Gly
Tyr
110
150
Gly
Thr Ile
Asn Ala Ile Ala
135
His Ser Leu
His
Lys
Pro Pro Val Asn
105
130
Ile
Glu
Thr
80
Thr Ser Ser Ile
Val Pro Val Asn
Phe Leu
Gly
Asp
Asp His
Leu
265
corymbifera
<400> SEQUENCE: 4
Ser Ser Ser Thr Gln
1
Ala His Thr Phe
Tyr
Asp Tyr Arg
Thr Ala Leu Ser Ala Asn Ala
25
Val Ile Pro
Gly Gly
Gln
Trp
35
Asn Leu Asn Ile Thr
50
Lys
Val Leu Val Ala Val
Gly
Ser
40
Cys
30
Pro His
Cys Asp
Val Ala Pro
45
Thr Phe Thr Thr Leu Ile Thr
Asp
Thr Asn
55
Glu Asn Glu
Lys
Thr Ile
Tyr
Val Val Phe
65
Arg Gly
80
Thr Ser Ser Ile
Arg
Asp
Asn Ala Ile
Ile Val Phe Val
85
Pro Val Asn
Tyr
100
Pro Pro Val Asn
95
Gly
105
Ala
Lys
Val His
Lys Gly
110
Phe
US 7,465,570 B2
17
18
continued
Leu
Asp
Ala
Ser
115
Tyr
Leu
Asp Arg His
130
Ser Leu
145
Gly
Gly
Gly Gly
Leu Val Ala Glu Val
125
Lys
120
Asp Lys
Gly Tyr Lys
Ile Val Val Thr
Gly
His
Asn Glu Val
Pro
135
140
Ala Thr Ala Val Leu Ser Ala Leu
150
His
Asp
Asn Ile Glu Ile
165
Thr Pro Glu Phe Ala Asn
Tyr
180
Gln
Arg
Ala Phe
Arg Asp
Tyr
Thr Gln
Arg
Val
Cys
Val Ile
Gly
<210>
<211>
<212>
<213>
Gly
Leu
Arg
Gly
Thr
Lys
Ile Pro
Ile Val Pro His Leu Pro Pro
Tyr
Gly
Glu Glu Phe
Trp
Ile Met
Lys Asp
Asp
Cys
Ser
240
Tyr
Leu
220
Pro Asn
Gly
Ile Glu Thr
Asn
235
Cys
Ile
205
230
Met Asn Thr
260
Gln Pro
190
Asn Ser Ile Val Pro Phe Thr Ser Val Ile
245
250
Asp
Tyr His
175
215
Ser Ser Leu
225
Gly
170
200
Phe Leu His Ala
210
Leu
160
185
Leu Val Asn Glu
195
Gly
Asp
155
Asp His
Leu Ser
255
Leu
SEQ ID NO 5
LENGTH: 269
TYPE: PRT
ORGANISM: Rhizomucor miehei
<4 00> SEQUENCE:
Ser
1
Ile Asp Gly Gly Ile Arg Ala Ala Thr
10
Leu Thr
Tyr Tyr
Thr Thr Leu Ser Ala Asn Ser
20
Ile Pro
Gly
Lys
Ala Thr
Trp Asp Cys
Ile His
Cys Asp
40
Ile Ile
Lys
Thr
Trp
Asp
Arg Gly Asp
Ser Thr Leu Ile
Tyr
Ser Glu
Arg
Asn
Pro Pro Val Ser
100
Tyr Gly
Lys
Tyr Asp
Thr Asn Ala
Gly Gly
Thr Ile
Tyr
Ile Val Phe
Trp
Ile Ala
Asp
Arg
Gly
Thr
Lys
Leu Thr Phe Val Pro
95
Val His
Phe Leu
11O
120
Gln
Lys Gly
105
Glu Val Gln Asn Glu Leu Val Ala Thr Val Leu
Tyr
130
Leu
145
Lys
90
115
Gln Phe
Asp
45
80
Ser Ser Ser Ile
85
Ser
Ala Thr Glu
60
Met Val Ala
65
Val Ser
Thr Val
30
50
Gly
Tyr Cys Arg
25
35
Leu
Ser Gln Glu Ile Asn Glu
15
Pro Ser
135
Asp
125
Tyr Lys
Val Ala Val Thr
Gly
His Ser
Tyr
Gln
140
Ala Thr Ala Leu Leu
Cys
Ala Leu
150
Asp
Leu
155
Arg
160
Glu Glu
Gly
Leu Ser Ser Ser Asn Leu Phe Leu
165
Tyr
Gly
Gln
Arg
Val
Gly Asp
Val Val Ser Thr
Gly
Pro
180
Ile Pro
Tyr Arg Arg
Pro Ala Phe Ala Asn
185
Thr Val Asn Glu
195
Pro Pro Ala Ala Phe
210
Tyr
190
Arg Asp
Ile Val Pro His Leu
200
Gly
Phe Leu His Ala
215
Thr Gln
205
Gly
Glu Glu
220
Tyr Trp
Ile
US 7,465,570 B2
19
20
continued
Thr Asp Asn Ser Pro Glu Thr Val Gln Val
Cys
Thr Ser Asp Leu Glu
225
235
240
230
Thr Ser Asp Cys Ser Asn Ser Ile Val Pro Phe Thr Ser Val Leu Asp
245
His Leu Ser Tyr Phe
250
Gly
255
Ile Asn Thr Gly Leu
260
Cys
Thr
265
SEQ ID NO 6
LENGTH:
TYPE: PRT
ORGANISM: Rhizopus oryzae
SEQUENCE: 6
Ser Ala Ser Asp
Gly Gly Lys
1
5
Gln Glu Phe Thr
Val Val Ala Ala Thr Thr Ala
10
Lys Tyr
Ala
Gly
20
Ser Val Val Pro
35
Val Pro
Gly
Asn
Ile
15
Ile Ala Ala Thr Ala
Tyr Cys Arg
25
30
Lys Trp Asp Cys
Val Gln
Cys
Gln
Lys Trp
45
Asp Gly Lys
Ile Ile Thr Thr Phe Thr Ser Leu Leu Ser
Asp
50
Thr Asn
Gly Tyr
Val Leu
Arg Gly
Thr Asn Ser Phe
Arg
Ser
Asp Lys
Gln
Lys
Thr Ile
Tyr
65
Val Phe
Arg
85
Phe Asn Phe Ser
Asp Tyr Lys
100
Gly
Phe Leu Ser Ser
Tyr
His Ser Leu
Gly Gly
Gln
Arg
Gly Gly
Ser Thr
Gly
Glu Pro
Pro
180
Arg
Tyr Lys
Asp
Leu
160
Gly
Gly
Lys Asp Cys
Leu Ser
260
Met
Val
245
Asp His
Gly
Leu Ser Pro Lys Asn Leu Ser Ile Phe Thr
170
Asn Pro Thr Phe Ala
185
Arg
Thr Val His
Tyr
Tyr Tyr
Val Glu
190
Lys Arg Asp
200
Ser
Val Ile Val Thr
Arg
Ile Pro Phe Gln
Lys
Phe Pro Val
155
Ile Val
205
Gly Phe Leu His Pro Gly Val Glu
220
Thr Ser Asn Val
230
Ile Glu Thr
Asp Tyr
140
Pro His Val Pro Pro Gln Ser Phe
210
215
Ile
Lys
11O
Ala Gln Ala Leu Leu Ala
195
Trp
Ala
Ile Val
95
125
150
165
Leu
Val His Ala
135
145
Ser
225
Lys Gly
120
130
Val
Asp
Glu Gln Leu Thr Ala His Pro Thr
Val
Tyr
Ser Ala Ile Thr
90
Glu Gln Val Val Asn
115
Gly
Pro Val
105
Leu
80
Ile
Cys
Thr Ser Glu
235
240
Ser Asn Ser Ile Val Pro Phe Thr Ser Ile
250
255
Phe
Asp
Ile Asn Glu
Gly
Ser
Leu
265
<211> LENGTH: 267
<2l2> TYPE: PRT
<2l3> ORGANISM: Aspergillus
niger
<400> SEQUENCE: 7
Thr Ala Gly His Ala Leu Ala Ala Ser Thr Gln Gly Ile Ser Glu Asp
1
5
15
US 7,465,570 B2
21
22
continued
Leu
Tyr
Ser
Arg
20
Ala
Asp
Tyr
Asn Ser Gln Thr
Leu
35
Cys
Leu Val Glu Met Ala Thr Ile Ser Gln Ala Ala
25
30
Asn Ile Pro Ser Thr Ile Ile
40
Asp
Ile Asn
Gly Trp
Ile Leu
55
Ser
65
Lys
Lys
Ile
Arg Asp Asp
Ser
Glu
60
Glu Ile Ile Thr Val Phe
Arg Gly
Thr
Gly
Ser
Asp
Thr Asn
80
Leu Gln Leu
Asp
Thr Asn
Tyr
Thr Leu Thr Pro Phe
85
Gln
Lys Gly
Tyr
Cys
Asn
Gly Cys
Asp
Thr Leu Pro
90
Glu Val His
95
Gly Gly Tyr Tyr
Ile
Gly Trp
Val
105
Ser Val Gln
115
Tyr
Pro
130
Asp
Gln Val Glu Ser Leu Val
Lys
Gln
120
Asp Tyr
Ala Leu Thr Val Thr
Gly
Leu
Tyr
Thr Phe
Gly
Tyr
Tyr
Phe
Met Asn
180
Asp
Glu Pro
Arg
Tyr
Cys Cys
Val Thr His Ala Asn
195
200
Phe
Gly
Asn Gln Ala Phe Ala
Asp
Gly Tyr
Ala His
205
Gly Gly
Val Glu
Gly Gly
Tyr Trp
Ser Val
Gln
Gly
Cys
Thr
Gly Asp
Glu Val Gln
235
240
Val Asn Asn Ala His Thr Thr
250
Met Thr Ser
260
Gly
Asp
220
Ser Ala Gln Asn Thr Phe Val
230
Glu Ala Gln
Thr Thr Gln
Ile Pro Asn Leu Pro Pro
215
Gly
Asn Ile
160
190
Asp Gly
245
Tyr
Tyr Asp
185
210
Pro
225
Ser
170
Ala Phe Gln Ala Ser Ser Pro
Arg
Val Glu Gln
Ala Ser
140
165
Ser
Gly
His Ser Leu
135
Leu Ala Ala Leu Thr Ala Ala Gln Leu Ser Ala Thr
145
150
155
Arg
Val Ser Gln
125
Ala
Cys
Thr
255
Trp
265
SEQ ID NO 8
LENGTH: 266
TYPE: PRT
ORGANISM: Aspergillus tubingensis
SEQUENCE: 8
Thr Ala Gly His Ala Leu Ala Ala Ser Thr Gln Gly Ile Ser Glu Asp
1
Leu
5
Tyr
Ser
Arg
20
Ala
Tyr
Asp
Leu
35
Cys
10
15
Leu Val Glu Met Ala Thr Ile Ser Gln Ala Ala
25
30
Asn Ile Pro Ser Thr Ile Ile
40
Asn Ser Gln Thr
Asp
Ile Asn
Gly Trp
Lys
Lys
Ile
Arg Asp Asp
Ser
Glu
45
Ile Leu
55
Ser
Lys Gly
Tyr
60
Glu Ile Ile Thr Val Phe
Arg Gly
Thr
Gly
Ser
Asp
Thr Asn
80
Leu Gln Leu
Asp
Thr Asn
Tyr
Thr Leu Thr Pro Phe
85
Gln
Cys
Asn Ser
100
Ser Val Gln
Asp
Cys
Asp
90
Glu Val His
Gly Gly Tyr Tyr
Thr Leu Pro
95
Ile
Gly Trp
Ile
105
Gln Val Glu Ser Leu Val Gln Gln Gln Val Ser Gln
US 7,465,570 B2
24
23
—cont inued
115
Phe Pro
120
Asp Tyr
125
Ala Leu Thr Val Thr
130
Gly
Leu
Tyr
Thr Phe
Gly
Glu Pro
Arg
165
Tyr
Met Asn
Asp
Asp
Arg
Glu
Asp Gly
195
200
Ala His
210
Tyr
Gly
Glu Ala Gln
Gly Gly
Gln
205
Gly
Cys
Tyr Trp
Ser Val
Thr
Gly Asp
Glu Val Gln
His
Pro
Cys
Val Asn Asn Ala His Thr Thr
Thr
Cys
240
250
Gly
Asp
235
245
Met Thr Ser
260
Tyr
220
Cys
<210>
<211>
<212>
<213>
Thr Thr Gln
Ile Pro Asn Leu Pro Pro Ala
Val Val Glu
Ser Ala Gln Asn Thr Phe Val
230
Gly
Asp
190
215
225
Phe
Asn Ile
160
185
Val Thr His Ala Asn
Gly Tyr
Tyr Asp
Ser Asn Gln Ala Phe Ala Ser
170
Ala Phe Gln Ala Ser Ser Pro
180
Phe
Ala Ser
140
Leu Ala Ala Leu Thr Ala Ala Gln Leu Ser Ala Thr
145
150
155
Arg
Gly
His Ser Leu
135
Tyr
255
Trp
265
SEQ ID NO 9
LENGTH: 275
TYPE: PRT
ORGANISM: Fusarium
heterosporum
<400> SEQUENCE: 9
Ala Val Thr Val Thr Thr Gln
Asp
1
Leu Ser Asn Phe
10
Arg
Phe
Asp
Ala Ala
Tyr Cys
Asn Phe Asn Thr Ala Val
25
30
Pro Val His
35
Cys
Ser Ala
Gly
Asn
40
Cys
Ser Val Val
55
Gly
Ala Ile Val Val
Tyr
Val Ala Thr
Gly
Asp
Asn Ala
Gly Lys
Lys Asp
Ala
Gly
Ala
Glu Ile Val Val Ser Val
Arg
Pro
Ile Glu
45
Thr
Lys
Thr
Gly
Ile
60
Arg Lys
65
Gly
Lys
80
Ser Ile Asn Val
85
Thr
Cys Asp
Arg
Asn
Trp
Ala
Trp
Leu Val Ala
Gly Cys Gly
Lys
Val His Thr
Lys
Gly Gly
Gly
Phe Pro Phe
Lys
Phe Val Val Thr
150
Leu
Gly
His Ser
140
Ala Val Ala Thr Ile Ala Ala Ala
Asp
Phe Leu
125
135
Leu
145
Gly
Ala Ala Val Ser Ala
120
Thr Ala Asn Pro Thr Phe
Tyr
Leu
Arg Lys Asp
155
Tyr
Gln
11O
Glu Glu Val Ala Ala Asn Val
130
Gly
95
105
115
Ala
Ile Thr Asn Phe Asn Phe
90
100
Asp
Leu
15
Gln His Ala
Asp
Tyr
Thr
Tyr Gly
160
Ser Pro
Arg
Val
Gly
Asn
165
Asp
Phe Phe Ala
Phe Val Thr
180
Val Thr His
Gly Asp Asp
Gln Thr
Pro Val Pro
210
His Thr Ser Pro Glu
215
Ala Glu
Tyr Arg
19O
Arg
195
Gly Tyr Arg
Gly
185
Tyr Trp
Leu Pro Pro Ile Val Phe
205
Leu Asn
220
Gly Gly
Pro Leu
US 7,465,570 B2
25
26
—cont inued
Asp Lys Asp Tyr
Thr Val Thr Glu Ile
225
Lys
230
Val Met
Ile Thr
Tyr
Cys
Asn
245
Val Cys Glu Gly Ile Ala
235
Gly Gly
Thr Ile
Phe Gln Ser Met Ala Thr
260
Gly
240
250
Leu Asp Ile Leu Ala His
255
Cys
Ala Pro Ile Ala Ile Pro
265
Trp Lys Arg
275
SEQ ID NO 10
LENGTH:
TYPE: PRT
ORGANISM: Aspergillus oryzae
SEQUENCE: 10
Asp
Ile Pro Thr Thr Gln Leu Glu Asp Phe
Lys Phe Trp
15
Ala Ala Ala Thr
Leu Asn
35
Cys
Ser Thr Val
50
Lys
Gly
Asp Gly
Glu
Val Glu Ala Ala
Gly
Tyr Cys
Pro Asn Asn
25
Tyr
Val Ala Lys
Ser Val
Gly
Pro
Asp
20
Lys
Tyr
Val
1
Cys
45
Leu Ser Phe Ser Asp
55
Phe Val Ala Val
Asp
Asn Thr Asn
Asp
Thr Ile Thr
Asp
Thr Ala
6O
Lys
Ala Ile Val Val Ala Phe
65
80
Arg Gly
Gln Thr
Trp
Ser
Asp
Thr Ala
Tyr
Pro
100
Ser Ile
85
Arg Asn Trp
Gly
Cys Asp Gly Cys Lys
Leu
90
Lys
130
Trp Lys
Val Val
Pro Glu His Ser
135
Gly
Lys
Tyr Asp
Lys
Asp Tyr Lys
Ile Val Val Val
His
Arg
Thr
155
Tyr
170
Asp Asp
Gly
140
165
160
Arg
Val Ala
Asn Asn
190
Tyr Arg
Ala Ala Pro
Gly
Pro Val Pro
200
Lys
Leu Pro Leu Leu Thr Met
205
Val His Ile Ser Pro Glu Tyr
Tyr
Ile Thr Ala Pro
215
Asp
225
Lys Gly
Asn Thr
245
Phe His Ser His Val
Gly
Leu Pro Leu
275
Asn
Gly
Asp Gly Tyr
Val Asn
240
Asp
Leu Leu Ala
255
Phe Ile His Ala Asp Ala
Lys Gly
Thr Ser Gly
Leu Pro
250
Trp Tyr
Asp
220
Asn Gln Val Thr Val Leu
230
235
260
<210>
<211>
<212>
<2l3>
Asp
Pro Leu Ala Glu Phe Ile Thr Asn Gln
180
185
Thr Thr Val Thr
Gly
Ile Ile Lys Thr Leu
125
Ala Ile Leu Tyr Ala
210
Pro
Arg Asp Arg
150
195
Phe
Phe
110
Ala Ala Ile Ala Ser Leu Ala Ala Ala Asp Leu
Phe Thr His Asn
Gly Tyr
Gly
Ala Glu Leu
120
Ser Leu
145
Asn
95
105
115
Glu Leu
Val Thr Asp Ala Thr Phe Pro
265
Arg
SEQ ID NO 11
LENGTH:
TYPE: PRT
ORGANISM: Penicillium camemberti
US 7,465,570 B2
27
28
continued
<400> SEQUENCE: 11
Asp Val Ser Thr Ser Glu Leu Asp Gln Phe Glu Phe
1
5
Trp
Val Gln
1O
Ala Ala Ala Ser Tyr Tyr Glu Ala Asp Tyr Thr Ala Gln Val
20
Tyr
15
25
Gly Asp
30
Lys Leu Ser Cys Ser Lys Gly Asn Cys Pro Glu Val Glu Ala Thr Gly
35
40
45
Ala Thr Val Ser Tyr Asp Phe Ser Asp Ser Thr Ile Thr Asp Thr Ala
5O
55
60
Gly Tyr Ile Ala Val Asp His Thr Asn Ser Ala Val Val Leu Ala Phe
80
Arg Gly Ser Tyr Ser Val Arg Asn Trp Val Ala Asp Ala Thr Phe Val
85
90
His Thr Asn Pro Gly Leu Cys
Asp Gly Cys
1OO
105
95
Leu Ala Glu Leu
110
Gly
Phe
Trp Ser Ser Trp Lys Leu Val Arg Asp Asp Ile Ile Lys Glu Leu Lys
115
120
125
Glu Val Val Ala Gln Asn Pro Asn Tyr Glu Leu Val Val Val
130
135
140
Ser Leu Gly Ala Ala Val Ala Thr Leu Ala Ala Thr
145
150
Asp
Leu
Gly
His
Arg Gly
155
160
Lys Gly Tyr Pro Ser Ala Lys Leu Tyr Ala Tyr Ala Ser Pro Arg Val
165
175
Gly Asn Ala Ala Leu Ala Lys Tyr Ile Thr Ala Gln
180
Gly
185
Asn Asn Phe
190
Arg Phe Thr His Thr Asn Asp Pro Val Pro Lys Leu Pro Leu Leu Ser
195
200
205
Met Gly Tyr Val His Val Ser Pro Glu Tyr Trp Ile Thr Ser Pro Asn
210
215
Asn Ala Thr Val Ser Thr Ser
225
230
220
Asp Ile Lys Val Ile Asp Gly Asp Val
235
240
Ser Phe Asp Gly Asn Thr Gly Thr Gly Leu Pro Leu Leu Thr Asp Phe
245
250
255
Glu Ala His Ile Trp Tyr Phe Val Gln Val Asp Ala
260
Gly Lys Gly
265
Pro
270
Gly Leu Pro Phe Lys Arg
275
<210> SEQ ID NO 12
<211> LENGTH: 2'70
<212> TYPE: PRT
<213> ORGANISM: Aspergillus foetidus
<400> SEQUENCE: 12
Ser Val Ser Thr Ser Thr Leu Asp Glu Leu Gln Leu Phe Ala Gln
1
5
10
15
Ser Ala Ala Ala Tyr Cys Ser Asn Asn Ile
20
Asp
Ser
Lys Asp
Trp
Ser Asn
25
Leu Thr Cys Thr Ala Asn Ala Cys Pro Ser Val Glu Glu Ala Ser Thr
35
40
Thr Met Leu Leu Glu Phe Asp Leu Thr Asn
5O
45
Asp
Phe
Gly Gly
Thr Ala
55
Gly Phe Leu Ala Ala Asp Asn Thr Asn Lys Arg Leu Val Val Ala Phe
65
7O
75
8O
US 7,465,570 B2
29
30
continued
Arg Gly
Leu Glu
Phe
Ser Ser Thr Ile Glu Asn
85
Trp
Asp
Thr
Trp Lys
Asn
100
Asp Asp
Ala
Trp
Leu
Glu Ser Ala Ala
His Ser Leu
Gly Gly
145
Asn
Gly
Asp Gly Tyr
Asn
Tyr
Met
Asp
Asn
Val Thr His Leu Asn
200
Phe Ser
Phe Thr
Gly
Ala Thr Val Leu
Arg
Tyr
140
Tyr
Thr
160
Tyr Gly Cys
Asp
Gly
Ile
Gly
Ser
190
Gly
Arg
Val Pro Pro
Ala
205
Pro Ser Pro Glu
Tyr Trp
Ile Thr Ser
220
Asn Ala
Gly
245
Trp Tyr
Arg
175
Ile Val Pro
Asp
230
Ile Asn Ser Thr Ala
Pro
170
Ala Ser Val Thr Ala Ser
225
Ala His Leu
Ile
Thr Leu
215
Gly
Lys
185
210
Gly
Gly Tyr
155
195
Gly
125
Ala Leu Ala Glu His Ile Thr Ser Gln
Gly
Val His Thr
Glu Leu Thr Ser
Gly
Arg
Phe
Asp
150
Ser Val Glu Leu
165
Phe Ile
110
Ala Leu Ala Thr Leu
180
Phe
Gly Cys Lys
120
Ser Ala Met Ser Thr Tyr Ser
130
135
Asp
95
105
115
Lys
Cys
Ile Ala Asn Leu
Ile Glu Val Ile Glu
Gly
235
240
Glu Ala Thr Val Ser Val Leu
250
255
Phe Phe Ala Ile Ser Glu
260
Cys
265
Leu Leu
270
SEQ ID NO 13
LENGTH:
TYPE: PRT
ORGANISM: Aspergillus niger
SEQUENCE: 13
Ser Val Ser Thr Ser Thr Leu
1
Ser Ala Ala Ala
20
Val Thr
Cys
Tyr Cys
Thr Ala
Asp
Asp
10
Ser Asn Asn Ile
25
Ala
35
Cys
40
15
Asp
Ser
Met Leu Leu Glu Phe Asp Leu Thr Asn Asn Phe
50
55
Gly
Phe Leu Ala Ala
Arg Gly
Leu Gln
Phe
Ser Ser Thr Ile
85
Lys
Ile Ala
Asp
Asp
Leu
Trp Lys
Asn
100
Asp Asp
Ala
Trp
Asn
Cys
Thr
Leu
Gly Cys Lys
Gly Gly
Asp
Phe Ile
Val His Thr
105
Glu Ala Ala Ala
Asp
Gly Tyr
Lys
Ile
Phe Thr
Gly
Ala Thr Val Leu
Arg
Asn Leu Thr Ser
125
Thr Leu
Tyr
140
Ala Leu Ala Thr Leu
Gly
150
155
Ser Val Glu Leu
165
Gly
110
120
Asp Gly Tyr
Tyr
Trp
95
Ser Ala Met Ser Thr Tyr Ser
130
135
Asn
Thr Ala
Leu Val Val Ala Phe
145
Gly
Gly Gly
Lys Arg
His Ser Leu
Asn
Ser Asn
Asn Thr Asn
115
Lys
Asp Asp
Pro Ser Val Glu Glu Ala Ser Thr
45
Lys
Asp
Trp
Glu Leu Gln Leu Phe Ser
Tyr
Thr
160
Tyr Gly Cys
Pro
170
Ala Leu Ala Glu His Ile Thr Ser Gln
Arg
Val
175
Gly
Ser
Gly
Ala
US 7,465,570 B2
31
32
—cont inued
180
185
Phe Pro Val Thr His Leu Asn
195
Met
Asp
Phe
Thr
Ile Val Pro
200
Gly
Phe Ser
210
Gly
Asp
190
Gly
Pro Ser Pro Glu
Asp
230
Gly
Asn Ala
Gly
245
Ala His Leu
Trp Tyr
Ile Thr Ser
Ile Glu Leu Ile Glu
Gly
235
240
Glu Ala Thr Val
Asp
250
Val Leu
255
Phe Phe Ala Ile Ser Glu
260
<210>
<211>
<2l2>
<213>
Tyr Trp
220
Ala Ser Val Thr Ala Ser
Ile Asn Ser Thr Ala
Val Pro Pro
205
215
225
Arg
Cys
265
Leu Leu
270
SEQ ID NO 14
LENGTH: 269
TYPE: PRT
ORGANISM: Aspergillus oryzae
<400> SEQUENCE: 14
Asp Val Ser Ser Ser Leu Leu Asn Asn Leu
1
Asp
Ser Ala Ala Ala
Tyr Cys Asp
Tyr
Leu Phe Ala
10
15
Glu Asn Leu Asn Ser Thr
Gly
Thr
Lys
25
Leu Thr
Cys
Ser Val
Gly
Asn
35
Gln Ser Leu
Asp
Pro Leu Val Glu Ala Ala Ser Thr
45
Glu Phe Asn Glu Ser Ser Ser
50
Gly Tyr
Cys
40
55
Leu Ala Ala
Asp
Ser Ala
Asp
Glu Thr Asn
Leu Ala Asn
Trp
85
Leu Glu
Asp
Ala Ser
Lys
Trp Lys
Ala
Trp
Asp
Leu
Cys
Ser
105
Gly
Ala Ala Leu Ala Ala Leu Ala Ala Thr Ala Leu
Arg
Ser
155
His Ser Val Glu Leu
Asn Glu Ala Leu Ala Thr
180
Tyr
Val Thr His Thr Asn
200
Asp
Asn
170
Ile Thr
Asp
Asn
Asp
Arg Trp Tyr
260
<210> SEQ ID NO 15
<211> LENGTH: 269
Gln Asn
Ile Val Pro
Lys
Tyr Tyr
Asp
Leu Pro Pro
Ile Ser Ser
Asp Gly
Thr
Val Thr Glu Val Thr
Gly
235
240
Asp Gly
Thr Ser Ile
250
Phe Ile
Lys Gly Gly
220
245
Ala His
Leu
175
His His Phe Ser Pro Glu
Gly Gly
Arg
205
Glu Ala Thr Val Thr Thr Thr
Ala Thr
Gln Pro
190
230
Asp
Tyr Gly
215
225
160
185
195
210
Ile
Tyr
Tyr Arg
Gly Tyr
Thr Ile Thr Ser
125
Asp Tyr
165
Thr Leu Leu
Gly
Gly
Asp His
Asp
150
Asn Ser
Glu Val His Ser
110
135
145
Ala
Gly Cys
Ser Leu Val Leu Thr
140
Ser Glu Ile Ala
120
130
Tyr Gly
Gly
95
Val
Glu Ser Ala Leu Ser
Gly
Val Ala Asn Leu Asn Phe
Lys
115
His Ser
Leu Leu Val Leu Ser Phe
75
90
100
Phe
Asn Pro Ala
60
65
Arg Gly
Tyr Gly
Tyr
Ile Ser Glu
265
255
Cys
Ser
Asp