Lipolytic enzyme variants

US008298800B2
(12) Ulllted States Patent
(10) Patent N0.:
Borch et al.
(54)
US 8,298,800 B2
(45) Date of Patent:
LIPOLYTIC ENZYME VARIANTS
(51)
Int. Cl.
C12N 9/16
A 21D 8/02
(75) Inventors: Kim Borch, Birkerod (DK); Luise
*Oct. 30, 2012
(2006.01)
(2006.01)
glans/“Hi copeDngaféglilv (13K); gesPer
(52) us. Cl. ........................................ .. 435/196; 426/20
50255013110353Ch);iste:'¥h§n sen’
(58)
Field of Classi?cation Search ................ .. 435/196,
’
J orgensen, Kgs Lyngby (DK)
_
435/252.3, 320.1;
426/20
_
_
See application ?le for complete search history.
(73) Assignee: Novozymes A/S, Bagsvaerd (DK)
(*)
Notice:
Subject to any disclaimer, the term of this
patent is extended or adjusted under 35
(56)
References Clted
U S PATENT DOCUMENTS
U.S.C. 154(b) by 232 days.
' '
7,465,570 B2 *
This patent is subject to a terminal dis-
Clalmer'
(21)
Flled:
EP
N0“ 4’ 2009
(65)
Prior Publication Data
US 2010/0047388 A1
Borch et a1.
................ .. 435/196
FOREIGN PATENT DOCUMENTS
Appl. N0.: 12/612,398
.
(22)
12/2008
7,638,317 B2* 12/2009 Borch et a1. ................ .. 435/196
Feb. 25, 2010
869167
10/1998
W0
WO 98/26057
6/1998
W0
WO 00/32758
6/2000
11/2001
W0
WO 01/83770
W0
WO 02/00852
l/2002
W0
W0 02/055679
7/2002
WQ
‘NO/93060112
7/2003
* cited by examiner
Related US. Application Data
(60)
Continuation of application No. 12/ 169,679, ?led on
Jul. 9, 2008, noW Pat. No. 7,638,317, Which is a
Primary Examiner * Tekchand Saidha
(74) Attorney, Agent, or Firm * Kristin J. McNamara
division of application No. 10/556,511, ?led as
application No. PCT/DK2004/000292 on Apr. 29,
2004, noW Pat. No. 7,465,570.
(57)
(60) Provisional application No. 60/469,228, ?led on May
9, 2003, provisional application No. 60/474,881, ?led
on May 30, 2003, provisional application No.
60/ 479,647, ?led on Jun. 19, 2003.
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
(30)
Foreign Application Priority Data
May 9, 2003
(DK) ............................... .. 2003 00709
May 30, 2003
(DK) ............................... .. 2003 00811
lipids,
14 Claims, 3 Drawing Sheets
US. Patent
0a. 30, 2012
Sheet 3 of3
US 8,298,800 B2
Flgure 1C
301
350
SEQ
10
NO:
1
QA‘L'DA.CNAG
GE'S
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SEQ
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QATDA.CNAG
GES
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MNTGTHCL.
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MNTGL.CL.
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INTGL.C'I.
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INEGS.CL.
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SEQ
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SMwSGAc'm.
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SEQ 10 NO:
8
SM‘l‘SGHC‘lW
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SEQ
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NO:
9
QSMAT .CAPT
ATPWKR.
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FTHADACKGP
SLPLR .
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VQVDAGKGPG
LPFKR .
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SEQ
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FAISE
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SEQ
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FAISE
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10
NO:
14
iYlSE.CS
11>
NO:
15
GLIGT
SEQ
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269
US 8,298,800 B2
1
2
The invention also provides a variant polypeptide Which:
a) has hydrolytic activity toWards an ester bonds in a polar
LIPOLYTIC ENZYME VARIANTS
lipid, and
CROSS-REFERENCE TO RELATED
APPLICATIONS
b) has an amino acid sequence Which
i) has at least 80% identity to SEQ ID NO: 1 and has a
This application is a continuation of US. application Ser.
No. 12/169,679 ?led on Jul. 9, 2008, now US. Pat. No.
7,638,317, Which is a divisional ofU.S. application Ser. No.
different amino acid or an amino acid deletion at a
position corresponding to A29, K33, I83 or A255, or
ii) has at least 80% identity to SEQ ID NO: 2 and has a
10/556,511 ?led Nov. 9, 2005, now US. Pat. No. 7,465,570
different amino acid or an amino acid deletion at a
issued on Dec. 16, 2008, Which is a 35 USC 371 national
position corresponding to R84 or P256.
application of PCT/DK2004/000292 ?led Apr. 29, 2004,
BRIEF DESCRIPTION OF DRAWINGS
Which claims priority or the bene?t under 35 U.S.C. 119 of
Danish application nos. PA 2003 00709 and PA 2003 00811
FIG. 1 shoWs an alignment of amino acid sequences of
?led May 9, 2003 and May 30, 2003, respectively, and US.
provisional application Nos. 60/469,228, 60/474,881 and
60/479,647 ?led May 9, 2003, May 30, 2003, and Jun. 19,
fungal lipolytic enZymes to identify corresponding amino
acids in SEQ ID NOs: 1 to 15. SEQ ID NO: 1 is the lipase/
phospholipase from Fusarium oxysporum (WO 98/26057).
2003, respectively, the contents of Which are fully incorpo
rated herein by reference.
CROSS-REFERENCE TO SEQUENCE LISTING
SEQ ID NO: 2 is a variant With phospholipase and galactoli
pase activity disclosed in WO 00/32758. SEQ ID NOs: 3 to 15
20
are knoWn lipolytic enZymes from the folloWing organisms:
Absidia re?exa, Absidia corymbefera, Rhizomucor miehei,
The present application contains information in the form of
delemar (oryzae), Aspergillus niger, Aspergillus Zubingensis,
a sequence listing, Which is submitted on a data carrier
Fusarium helerosporum, Aspergillus oryzae, Penicilium
camemberlii, Aspergillus foelidus, Aspergillus niger,
accompanying this application. The contents of the data car
rier are fully incorporated herein by reference.
25
DETAILED DESCRIPTION OF THE INVENTION
FIELD 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
Aspergillus oryzae and Thermomyces lanuginosus.
Parent Polypeptide
30
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
properties for use in a dough, e. g., for making bread and other
baked products, and more particularly to such polypeptides
2. It may have at least 50% amino acid identity to SEQ ID NO:
having hydrolytic activity toWards ester bonds in polar lipids.
Examples are the polypeptides having the sequences of SEQ
1 or 2, e.g., at least 60%, at least 70% or at least 80%.
35
Variant Polypeptide
Phospholipases and galactolipases are knoWn as enZymes
With hydrolytic activity toWards ester bonds in polar lipids
such as phospholipids and galactolipids. WO 00/32758 dis
40
closes lipolytic enZyme variants having phospholipase and
galactolipase activity and their use in baking. WO 98/ 26057
discloses a lipase/phospholipase from Fusarium oxysporum
and its use in baking. WO 01/83770 describes variants of a
fungal lipase.
45
SUMMARY OF THE INVENTION
The inventors have developed variant polypeptides by
modifying the amino acid sequence of a parent polypeptide
Which is a fungal lipolytic enZyme. 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 orA255 of SEQ ID NO: 1
(corresponding to P29, N33, R84 or P256 of SEQ ID
50
e) adding the polypeptide to a dough and testing dough
stickiness.
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
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, e.g., as disclosed in WO
00/32758. Thus, it may have the amino acid A/T at position
55
60
NO: 2),
c) modifying the amino acid sequence by substituting or
deleting the selected residue,
d) preparing a variant polypeptide having the modi?ed
amino acid sequence, and
ID NOs: 1 to 14 or a variant disclosed in WO 00/32758.
The parent polypeptide has lipolytic enZyme activity, e. g.,
hydrolytic activity toWards an ester bond in a polar lipid.
BACKGROUND OF THE INVENTION
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
position N247, D at position N248, K/R at position Q249,
K/T at position P250, T at position N251, F at position 1252,
lVI/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
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
65
C-terminus may be modi?ed, e.g., 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
DAELEKKLNSYVQMDKEYVKNNQARS. The C-termi
US 8,298,800 B2
3
4
nal may be truncated after position 272, 273, 274 or 286 in
be determined With digalactosyl diglyceride as substrate, e. g.,
SEQ ID NO: 1 . The N-terminal may have a peptide extension,
e.g., as described in WO 00/32758 or WO 97/04079, such as
Use of Polypeptide
as described in WO 00/32758.
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
The polypeptide may be added to a dough, and the dough
may be used to prepare a steamed bread, a baked product
found by aligning a given sequence With SEQ ID NO: 1 or 2,
(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
e.g., as shoWn in FIG. 1. The alignment may be done by use of
retention and volume during processing and baking, particu
the GAP program as described beloW.
larly in a stressed system, e. g., 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
corresponding position. The corresponding positions may be
The variant polypeptide may have improved thermo stabil
ity compared to the parent polypeptide, particularly a variant
polypeptide having a substitution at a position corresponding
(?ner crumb, thinner cell Walls, more rounded cells), of the
to A29 or K33 of SEQ ID NO: 1, e.g., the substitution A29P
baked product, and it may further improve dough properties,
or K33N.
e.g., a less soft dough, higher elasticity and/or loWer extensi
Sequence Identity
bility.
The variant polypeptide has at least 80% identity to SEQ
The process may be conducted in analogy With US. Pat.
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
No. 5,578,489 or US. Pat. No. 6,077,336. In the case of
un-proofed froZen dough the polypeptides of the invention
20
of volume and crumb structure.
The polypeptide can be used in a process for making bread,
program package (Program Manual for the Wisconsin Pack
age, Version 8, August 1994, Genetics Computer Group, 575
comprising adding the polypeptide to the ingredients of a
dough, kneading the dough and baking the dough to make the
Science Drive, Madison, Wis., USA 53711) (Needleman, S.
B. and Wunsch, C. D., 1970, Journal of Molecular Biology,
perform better than knoWn lipolytic enZyme variants in terms
25
bread. This can be done in analogy With US. Pat. No. 4,567,
48, 443-45), using GAP With the folloWing settings for
polypeptide sequence comparison: GAP creation penalty of
046 (KyoWa Hakko), JP-A 60-78529 (QP Corp.), JP-A
62-111629 (QP Corp.), JP-A 63-258528 (QP Corp.), EP
3.0 and GAP extension penalty of 0.1.
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
Dough Stickiness
The variant polypeptide may be tested by adding it to a
30
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
35
lase may be added, e. g., from Aspergillus or Bacillus, particu
larly A. oryzae, B. lichenlformis or B. amyloliquefaciens.
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 equip
ment such as divider, molder, sheeter and conveyer belts. The
With an anti-staling amylase and optionally also a phospho
lipid as described in W0 99/ 53769, particularly a maltogenic
alpha-amylase (e.g., from Bacillus sp., such as Novamyl®
from Novo Nordisk). Also, a fungal or bacterial alpha-amy
40
mixing time varies depending on procedure. For a typical
European straight dough procedure, the mixing time can, e. g.,
Optionally an additional enZyme may be added, e. g., an amy
loglucosidase, a beta-amylase, a pentosanase such as a xyla
nase as described in W0 99/ 53769, e.g., derived from
Aspergillus, in particular A. aculealus, A. niger (cf. WO
91/19782), A. awamori (WO 91/18977), or A. Zubigensis
be in the range of 6-10 minutes. For a typical American
(WO 92/01793), from a strain of Trichoderma, e.g., T reesei,
Sponge & dough procedure the mixing time can, e.g., be in
the range of 6-20 minutes (on ?nal dough). The dough may
have a resting period of 5-20 min before further processing,
or from a strain of Humicola, e.g., H. insolens (WO
92/17573), a protease and/or a glucose oxidase.
The dough may further comprise an emulsi?er such as
45
e.g., at 20-350 C. The dough stickiness may be evaluated by
hand by trained bakers, by a sensory panel or by instrumental
measurements, e.g., by the Chen-Hoseney dough stickiness
rig developed for Stable Micro Systems TA-XT2 texture
analyser, commercially available from Brook?eld Engineer
ing Laboratories, 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
mono- or diglycerides, diacyl tartaric acid esters of mono- or
diglycerides, sugar esters of fatty acids, polyglycerol esters of
fatty acids, lactic esters of monoglycerides, acetic acid esters
50
of monoglycerides, polyoxyethylene stearates, polysorbates
or lysolecithin.
The dough may also comprise other conventional dough
ingredients, e.g., proteins, such as milk poWder, gluten, and
soy; eggs (either Whole eggs, egg yolks or egg Whites); an
bond and are classi?ed in EC 3.1.1 Carboxylic Ester Hydro
oxidant such as ascorbic acid, potassium bromate, potassium
iodate, aZodicarbonamide (ADA) or ammonium persulfate;
lases according to EnZyme Nomenclature (available at
an amino acid such as L-cysteine; a sugar; a salt such as
WWW.chem.qmW.ac.uk/iubmb/enZyme). More speci?cally,
sodium chloride, calcium acetate, sodium sulfate or calcium
sulfate.
55
they have hydrolytic activity toWards ester bonds in polar
lipids so as to split off acyl groups at the sn-1 and/or sn-2
60
position of polar lipids such as phospholipids and galactolip
ids. Accordingly, they may have phospholipase activity or
EXAMPLES
galactolipase activity (EC 3.1.1.26), e.g., phospholipase A1
Baking Evaluation of Polypeptides With
Phospholipase Activity
activity (EC 3.1.1.32).
Pho spholipase activity may be determined by knoWn meth
ods, e.g., the “monolayer phospholipase assay” or the plate
assay described in WO 00/32758. Galactolipase activity may
65
In the examples, polypeptides according to the invention
Were tested together With the corresponding parent polypep
US 8,298,800 B2
5
6
tide in a baking evaluation experiment by using conventional
-continued
baking protocols for European straight dough procedure and
US sponge & dough procedure, as folloWs:
European Straight Dough Procedure:
A dough is prepared by mixing the beloW ingredients for 3 5 Oil
minutes sloW and 7 minutes fast.
S?“
Sponge
Dough
% (baker’s — by Weight)
% (baker’s — by Weight)
2-5
2
High fructose syrup
12
Water
Ascorbicc acid
% (baker’s —
10
by Weight)
34.4
20.4
50
A liquid sponge is prepared by mixing a sponge consisting
of the above listed sponge ingredients for 1 minute sloW and
it? ressed
Salt p
sugar
t
yeas
102
4 minutes fast. The sponge is fermented for 3 hours at 270 C.,
L5
1_5
86% RH. The sponge is mixed With the dough ingredients
15 listed above and With enZymes for 1 minute sloW and 18
Water
62
minutes fast.
Ascorbic acid
40 ppm
Dough stickiness is evaluated right after mixing, Whereaf
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
Dough stickiness is evaluated right after mixing and again
after a resting period of 15 minutes. Dough stickiness is
evaluated by a trained and experienced baker by sensory
20
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
stickiness is evaluated by a trained and experienced baker by
sensory 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
25
score 5.
variant polypeptide is compared to a reference dough made
With the parent polypeptide, Which is alWays given the score
5.
Sponge & Dough Procedure:
Example 1
Flour
Compressed yeast
Sponge
Dough
% (baker’s — by Weight)
% (baker’s — by Weight)
60
40
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
7.5
modi?cations.
Polypeptide Amino acid alterations compared to SEQ ID NO: 15
1
10
ll
12
US 8,298,800 B2
-continued
Polypeptide Amino acid alterations compared to SEQ ID NO: 15
Example 2
~
~
-continued
~
~
25
A29P +
Baking Evaluat1on of a Polypeptlde According to the
Invent1on
Polypeptide
Dough stickiness
5 variant polypeptides according to the invention Were
K33N +
A29P +
A29P +
Parent
183T
K33N + I83H
K33N + I83Q
5
4
4
4
mm 15 min table
compared to the parent polypeptide (SEQ ID NO: 2) in the 30 time
European straight dough procedure described above. 40 ppm
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 stabilizing effect. The
beloW results shoW that all 5 variants give reduced dough
stickiness compared to the parent polypeptide.
35
4 variant polypeptides according to the invention Were
compared to the parent enzyme (SEQ ID NO: 1) in the Euro
pean straight dough procedure described above. 10 FAU Fun
gamyl/kg 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 stabilizing effect. The
Polypeptide
Dough stickiness after mixing
Dough stickiness
after 15 min table time
Parent
P25 6V
P256A
P256Q
P25 6L
P25 6W
6
6
5
5
5
5
5
5
5
5
5
5
3 variant polypeptides according to the invention Were
beloW results shoW that all 4 variants give reduced dough
compared to the parent polypeptide (SEQ ID NO: 1) in the 50 stickiness compared to the parent enzyme.
European straight dough procedure described above. 40 ppm
Fungamyl Super MA (a blend of fungal alpha-amylase and
Xylanase) Was added as background to all doughs. The parent
Parent
K3 3N
I83N
183E
183K
the level giving best volume and dough stabilizing effect. The
beloW results shoW that all 4 variants g1ve reduced dough
enzyme and the variants Were dosed at their optimal level, i.e., 55 Polypeptide
D Ough Stickiness
after mixing
7
6
6
6
6
stickiness compared to the parent enzyme.
Dough 5661611655
7
6
6
6
6
after 15 mln
60 table time
A29P +
Polypeptide
Dough Stickimss
a?er mixing
Parent
5
A variant polypeptide according to the invention Was com
K33N +
A29P +
A29P +
183T
4
K33N + I83H
4
K33N + I83Q
4
_
_
pared I0 115 Pawnt enzyme (SEQ ID NO? 1) 111 the Us Sponge
65 & dough procedure described above. 40 ppm Fungamyl
Super MA (a blend of fungal alpha-amylase and Xylanase)
Was added as background to all doughs. The parent enzyme
US 8,298,800 B2
10
and the variant Were dosed at their optimal level, i.e., the level
-continued
giving best volume and dough stabilizing effect. The below
results shoW that the variant gives reduced dough stickiness
compared to the parent enzyme
K33N + 270VASLGDDTEAPRASTRGPP
A29P + I83N + A255V
The results Were that With each of the above polypeptides,
dough stickiness Was better than With the polypeptide With the
unmodi?ed sequence of SEQ ID NO: 1.
O yp?p l 6
mm
Dough stickiness after mixing
Doug}? Stlckm?ss all“
Bakmg tests W1th each dough shoWed that all polypept1des
6
6-5
5
5
10 improved the crumb structure, the loaf volume and the dough
stability, both for the modi?ed and unmodi?ed sequences.
extrusion
Example 4
Example 3
_
_
15
Variant Polypeptides Derived from SEQ ID NO: 2
_
Variant polypeptides With the folloWing amino acid alter
Varlant Polypeptldes Derived frOm SEQ ID NO1 1
ations compared SEQ ID NO: 2 (variant of T lanuginosus
lipase) Were prepared and tested by adding each polypeptide
to a dough The polypapn‘l6 W1th unmodl?ed SEQ ID NO? 2
Variant polypeptides With the folloWing amino acid alter-
ations compared SEQ ID NO: 1 (lipase/phospholipase from 20 Was also tested for Compansoll
F oxysporum) Were prepared and tested by adding each
polypeptide to a dough. The polypeptide With unmodi?ed
SEQ ID NO: 1 Was also tested, for compar1son.
R841)
R841
R84M
25
R84Q
A29P
K33N
P256A
P256D
A29P + 183T
A29P + I83N
P2561
P256L
A29P + 183C
30
P256Q
A29P + I83F
P2568
A29P + I83L
P256V
K33N + 183W
K33N + I83L
_
K33N + I83Q
K33N + 1835
KEN +1831‘
K33N + HEN
K33N + 183R
K33N + I83L
_
The results Were that With each of the above polypept1des,
35 dough stickiness Was better than With the polypeptide With the
d'? d
f SEQ ID No-. 2 .
unmo '1 e sequence 0
'
. Bakmg tests W1th each dough shoWed that all polypept1des
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:
<21o>
<211>
<212>
<2l3>
15
SEQ ID NO 1
LENGTH: 286
TYPE: PRT
ORGANISM: Fusarium oxysporum
<4oo> 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
2o
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
5o
55
60
Tyr Val Ala Thr Asp Ser Ala Arg Lys Glu Ile Val Val Ser Phe Arg
65
7o
75
80
Gly Ser Ile Asn Ile Arg Asn Trp Leu Thr Asn Leu Asp Phe Gly Gln
85
9o
95
US 8,298,800 B2
11
12
—cont inued
Glu
Arg
Asp Cys
Ala
Trp
115
Ala
Arg Lys
Ser Leu Val Ser
100
Gly Cys Gly
Val His Ser
105
Gly
Phe Gln
11O
Asn Glu Ile Ser Ser Gln Ala Thr Ala Ala Val Ala Ser
120
125
Ala Asn Pro Ser Phe Asn Val Ile Ser Thr
130
135
Leu
145
Gly Gly
Gly
Thr Pro Val
Ala Val Ala Val Leu Ala Ala Ala Asn Leu
150
Asp
Ile
Gly
His Ser
Arg
Val
140
155
Tyr
Thr
Tyr Gly
Gly
160
Arg
Val
Gly
Gly Gly
Glu
Tyr Arg
Ser Pro
Asn
165
Ala Gln Leu Ser Ala Phe Val Ser Asn Gln Ala
180
185
Pro Val Pro
200
Arg
195
Leu Pro Pro Leu Ile Phe
205
Gly Tyr Arg
His Thr Thr Pro Glu Phe
Trp
Leu Ser
220
Gly Gly Gly Gly
Lys
Cys
Val Thr His Ala
Asp Asp
190
210
Asp Lys
215
Val
Asp Tyr
225
Thr Ile Ser
Asp
Ala Asn Leu
Gly Cys
Asn
Leu His
Tyr
Val
Glu
Gly Gly
Thr Leu
Gly
Phe Gln Ala Thr
Asp
Ala
Ala
240
Leu
Asp
Ile Ala Ala
250
255
Cys
260
265
Trp Arg Arg Tyr Arg
Ser Ala Glu Ser Val
280
275
Gly
235
245
Ser
Val
230
Asn Ala
Gly Gly
Phe
Asp Lys Arg
285
SEQ ID NO 2
LENGTH: 274
TYPE: PRT
ORGANISM: Artificial sequence
FEATURE:
OTHER INFORMATION: Variant disclosed in WO 0032 758
SEQUENCE: 2
Glu Val Ser Gln
Asp
1
5
Ser Ala Ala Ala
20
Tyr Cys Gly Lys
Leu Phe Asn Gln Phe Asn Leu Phe Ala Gln
15
Asn Asn
25
Asp
Ala Pro Ala
Thr
Gly
Asn Ala
40
Cys
Pro Glu Val Glu
45
Ala Thr Phe Leu
Tyr
Ser Phe Glu
55
Asp
Ser
50
Phe Leu Ala Leu
Asp
Gly
Gly
Thr
Ala
Asp
30
Cys
Asn Ile Thr
35
Tyr
Gly
Val
Lys
Gly Asp
Val Thr
60
Asn Thr Asn
Lys
Leu Ile Val Leu Ser Phe
65
Arg Gly
Leu
Ser
Lys Lys
Arg
Ser Ile Glu Asn
85
Ile Asn
Phe Thr Ser Ser
Asp
Trp Arg
115
Glu
Asp
Ala Val
Arg
Cys
Ile Ala Asn Leu Asn Phe
Ser
105
Gly Cys Arg Gly
Gly Gly
145
Asp
Gly Tyr Asp
165
Lys
Val
Val Val Phe Thr
Gly
Thr Leu
Asp Tyr Arg
Arg
Gln
140
Ala Leu Ala Thr Val Ala
Ile
Asp Gly
125
150
Asn
His
110
135
His Ser Leu
Gly
Ala
Asp
Leu
155
Asp
Trp
95
Ser Val Ala
120
Glu His Pro
130
Gly
Ile
Trp
Val Phe Ser
170
Tyr Gly
Arg
160
Ala Pro
Arg
175
Val
US 8,298,800 B2
14
13
—cont inued
Gly
Leu
Arg
Asn
Arg
Ala Phe Ala Glu Phe Leu Thr Val Gln Thr
Gly Gly
180
190
185
Tyr Arg
Ile Thr His Thr Asn
195
200
Glu Phe
Gly Tyr
210
Gly
Ile
Ile Val Pro
Arg
Asn
Asp
230
Asp
Ala Thr
Gly Gly
245
Ala His Leu
Trp Tyr
Arg
Leu Pro Pro
205
Ser His Ser Ser Pro Glu Tyr
215
220
Thr Leu Val Pro Val Thr
225
Asp
Ile Val
Trp
Ile
Lys
Ser
Lys
Ile Glu
Gly
235
240
Asn Asn Gln Pro Asn Ile Pro
250
Phe Gln Ala Thr
260
Thr
Asp
Ala
Cys
265
Asp
Ile Pro
255
Asn Ala
270
Gly Gly
Phe Ser
<210>
<211>
<212>
<2l3>
SEQ ID NO 3
LENGTH: 265
TYPE: PRT
ORGANISM: Absidia reflexa
<400> SEQUENCE: 3
Ser Ser Ser Ser Thr Gln
1
Asp Tyr Arg
Lys
Thr Ala Leu Ser Ala Asn Ala
Ala His Thr Phe
Tyr
15
20
Thr Val Ile Pro
Ile Ala Ser Glu Ala Glu Ile
25
Gly Gly Arg Trp
Ser
Tyr Cys Arg
30
Cys
Pro His
35
Cys Gly
Ser Asn Leu Gln Ile Thr Lys Thr Phe Ser Thr Leu Ile Thr
50
55
60
Gly
Glu
Lys
Thr Ser Ser Ile
Arg
Asn Ala Ile Ala
90
Asn Val Leu Val Ala Val
65
Phe
Arg Gly
85
Val Pro Val Asn
Tyr
100
Phe Leu
Asp
Ser
Tyr
115
Lys
Ala Gln Leu
Asn Glu Val Gln
120
Asp Lys
Pro
Gly Tyr Lys
135
Gly Gly
Gly
Gln
His Ala Asn Ile Glu Ile
Ala Phe
Asp
Tyr
Arg Asp
Phe Leu His Ala
Tyr
Ser Ser Leu
Arg
Val
Cys
Gly
Leu
Gly
Gln Pro
Val Ile
Gly
Thr
Lys
Arg
Ile Pro
190
Ile Val Pro His Leu Pro Pro
Gly
Glu Glu Phe
Trp
Ile Met
Lys
220
Pro Asn
Gly
Ile Glu Thr
Asp
Asn
235
Leu
Tyr
175
Ser Asn Ser Ile Val Pro Phe Thr Ser Val Ile
245
250
Met Asn Thr
260
Asp
205
230
Asp
Gly
160
Thr Gln
215
225
Leu
Ile Val Val Thr
200
210
Lys Gly
Leu Val Ala Glu Val
125
185
Leu Val His Glu
Gly
Val His
170
195
Gly
Ile Val Phe
155
Thr Pro Ala Phe Ala Asn
Arg
Val Val
95
Ala Thr Ala Val Leu Ser Ala Leu
180
Tyr
Lys
Asp
Tyr
Thr
140
165
Gly
Ala
Thr Ile
Asp
110
150
His
Lys
Gly
Asp Arg His
145
Ile
Glu
Pro Pro Val Asn
105
130
His Ser Leu
Val Ala
45
Cys
Leu
265
Cys
240
Asp His
Leu Ser
255
Tyr
US 8,298,800 B2
15
16
—cont inued
<210>
<211>
<212>
<213>
SEQ ID NO 4
LENGTH: 264
TYPE: PRT
ORGANISM: Absidia
corymbifera
<400> SEQUENCE:
Ser Ser Ser Thr Gln
1
Ala His Thr Phe
Tyr
Asp Tyr Arg
Ile Ala Ser Glu Ala Glu Ile
10
Thr Ala Leu Ser Ala Asn Ala
Tyr Cys Arg
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
Lys
15
Thr
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
80
Arg Gly
Thr Ser Ser Ile
Arg
Asn Ala Ile Ala
Asp
Ile Val Phe Val
95
Pro Val Asn
Tyr
Pro Pro Val Asn
100
Leu
Asp
Ala
Ser
115
Tyr
Leu
Asp Arg His
Gly
Gly
Gly Gly
Asn Glu Val Gln
120
Pro
135
Asp
Ala Phe
Gly
His
Ile Val Val Thr
Tyr
Tyr
Thr Gln
Arg
Val Ile
Val
Cys
Arg Asp
Leu
Tyr His
160
Gly
Gln Pro
Gly
Arg
Gly
Thr
Lys
Ile Pro
Tyr
Ile Val Pro His Leu Pro Pro
Gly
205
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
175
200
Asn Ser Ile Val Pro Phe Thr Ser Val Ile
245
250
Gly
Leu
190
230
Met Asn Thr
260
Asp
170
215
Ser Ser Leu
225
Asp
Lys
155
Phe Leu His Ala
Phe
Leu Val Ala Glu Val
125
185
210
Lys Gly
140
Asn Ile Glu Ile
165
Leu Val Asn Glu
195
Gly
Val His
Ala Thr Ala Val Leu Ser Ala Leu
Thr Pro Glu Phe Ala Asn
Arg
Asp Lys
150
His
Lys
110
Gly Tyr Lys
180
Gln
Ala
105
130
Ser Leu
145
Gly
Asp His
Leu Ser
255
Leu
SEQ ID NO 5
LENGTH: 269
TYPE: PRT
ORGANISM: Rhizomucor miehei
SEQUENCE: 5
Ser Ile Asp Gly Gly Ile Arg Ala Ala Thr Ser Gln Glu Ile Asn Glu
1
5
Leu Thr
10
Tyr Tyr Thr Thr
20
Ile Pro Gly Ala Thr
Leu Ser Ala Asn Ser
25
Trp Asp Cys
35
Leu
Lys Ile Ile Lys
15
Ile His
Thr
Trp
Thr Val
30
Cys Asp
40
Ala Thr Glu
Asp
45
Ser Thr Leu Ile
50
Met Val
65
Tyr Cys Arg
Tyr Asp
Thr Asn Ala
60
Ala Arg Gly Asp
7O
Ser Glu
Lys
Thr Ile
'75
Tyr
Ile Val Phe
Arg
80
US 8,298,800 B2
17
18
—cont inued
Gly
Ser Ser Ser Ile
85
Val Ser
Asp
Ser
Tyr
Arg
Asn
Lys
Gly Gly
Gly
Thr
Lys
Val His
Gln
Lys Gly
Phe Leu
105
120
Tyr
130
Leu
145
Leu Thr Phe Val Pro
95
Glu Val Gln Asn Glu Leu Val Ala Thr Val Leu
115
Gln Phe
Ile Ala
90
Pro Pro Val Ser
100
Tyr Gly
Trp
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
190
Arg Asp
Ile Val Pro His Leu
200
Pro Pro Ala Ala Phe
210
Gly
205
Phe Leu His Ala
Gly
215
Glu Glu
Asn Ser Pro Glu Thr Val Gln Val Cys Thr Ser
230
235
Thr Ser
Asp Cys
Leu Ser
Tyr
Asp
Phe
Gly
Ile
Leu Glu
240
Ser Asn Ser Ile Val Pro Phe Thr Ser Val Leu
245
250
255
Ile Asn Thr
260
<210>
<211>
<212>
<213>
Tyr Trp
220
225
Thr
Asp
Tyr
Thr Gln
Gly
Leu
Cys
Asp
Thr
265
SEQ ID NO 6
LENGTH: 271
TYPE: PRT
ORGANISM: Rhizopus
oryzae
<400> SEQUENCE: 6
Ser Ala Ser Asp
1
Gly Gly Lys
Gln Glu Phe Thr
Lys Tyr
Val Val Ala Ala Thr Thr Ala
10
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
Lys Gly
Val His Ala
His Ser Leu
Gly Gly
Tyr Lys
Glu Pro
Arg
Pro
180
Arg
Val Ile Val Thr
Gly
Met
155
Leu Ser Pro
Lys
170
Val
Phe Pro Val
140
Ala Gln Ala Leu Leu Ala
165
Gly Gly
Asp Tyr
125
150
Arg
Lys
Gly
Ile Val
95
11O
135
145
Gln
Ala
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
Asp
Leu
160
Asn Leu Ser Ile Phe Thr
1'75
Asn Pro Thr Phe Ala
185
Tyr Tyr
190
Val Glu
US 8,298,800 B2
19
20
—cont inued
Ser Thr
Gly
Ile Pro Phe Gln
195
Arg
Trp
Ile
Lys
Ser
Gly
Lys Asp Cys
245
Leu
Asp His
Leu Ser
260
Tyr
Ile Val
205
Gly
Phe Leu His Pro
Gly
Val Glu
220
Thr Ser Asn Val
230
Ile Glu Thr
Lys Arg Asp
200
Pro His Val Pro Pro Gln Ser Phe
210
215
Ser
225
Thr Val His
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
Gly
Ile Ser Glu
Leu
265
SEQ ID NO '7
LENGTH: 267
TYPE: PRT
ORGANISM: Aspergillus niger
SEQUENCE: 7
Thr Ala Gly His Ala Leu Ala Ala Ser Thr Gln
1
Leu
5
Tyr
Ser
Arg
20
Ala
Tyr
Asp
Leu
35
Cys
10
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
Asp
15
Lys Gly
Tyr
Lys
Ile
Arg Asp Asp
Ser
Glu
45
Ile Leu
60
Ser
65
Lys
Glu Ile Ile Thr Val Phe
Arg Gly
Gly
Ser
Asp
Thr Asn
80
Leu Gln Leu
Asp
Thr Asn
Tyr
Thr Leu Thr Pro Phe
85
Gln
Thr
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
Met Thr Ser
260
Gln
Gly
Cys
Asp
Thr
Ala
Cys
Gly Asp
Glu Val Gln
240
Val Asn Asn Ala His Thr Thr
Thr
265
<210> SEQ ID NO 8
<211> LENGTH: 266
<2l2> TYPE: PRT
<2l3> ORGANISM: Aspergillus
Ser Val
235
250
Gly
Tyr Trp
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
tubingensis
255
Trp
US 8,298,800 B2
21
22
—cont inued
<400> SEQUENCE: 8
Thr Ala Gly His Ala Leu Ala Ala Ser Thr Gln
1
Leu
Tyr
Ser
Arg
20
Asp
Tyr
Asn Ser Gln Thr
Leu
35
Cys
Asn Ile Pro Ser Thr Ile Ile
40
Asp
Ile Asn
Gly Trp
Lys
Lys Gly
Ile Leu
Arg Gly
Cys
Asp
Asn Ser
100
Ser Val Gln
115
Phe Pro
Thr Asn
Thr
Gly
Tyr
Thr Leu Thr Pro Phe
Asp
Cys
Glu Val His
Gly Gly Tyr Tyr
Ser
120
Asp Tyr
Leu
Asp
Thr Asn
Met Asn
Ala Leu Thr Val Thr
Thr Phe
Asp
Arg
Glu
Gly
Gly
Gly Trp
Ile
Val Ser Gln
Gly
Ala Ser
Tyr Asp
Asn Ile
160
His Ser Leu
Glu Pro
Arg
Ser Asn Gln Ala Phe Ala Ser
170
Ala Phe Gln Ala Ser Ser Pro
Asp
Thr Thr Gln
185
Asp Gly
195
200
Ala His
210
Gly
Ile Pro Asn Leu Pro Pro Ala
205
Val Val Glu
Tyr Trp
Cys
Glu Ala Gln
Gln
Gly
Cys
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
Ser Val
220
Ser Ala Gln Asn Thr Phe Val
230
Gly Gly
Tyr
190
215
225
Gly
Ile
140
Val Thr His Ala Asn
Gly Tyr
Thr Leu Pro
125
135
Tyr
Asp
Gln Val Glu Ser Leu Val Gln Gln
180
Phe
Ser
105
165
Tyr
Arg Asp Asp
95
Leu Ala Ala Leu Thr Ala Ala Gln Leu Ser Ala Thr
145
150
155
Asp
Ile
90
130
Phe
Lys
Glu
80
85
Tyr
Tyr
60
Glu Ile Ile Thr Val Phe
Leu Gln Leu
Arg
Asp
15
55
Gln
Ile Ser Glu
Leu Val Glu Met Ala Thr Ile Ser Gln Ala Ala
25
30
Ala
Ser
65
Gly
10
Tyr
255
Trp
265
SEQ ID NO 9
LENGTH:
TYPE: PRT
ORGANISM: Fusarium heterosporum
SEQUENCE:
Ala Val Thr Val Thr Thr Gln
1
Asp
5
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
Gly
50
Tyr
Val Ala Thr
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
Leu
15
Gln His Ala
Asp
Tyr
80
Ser Ile Asn Val
85
Arg
Asn
Trp
Ile Thr Asn Phe Asn Phe
90
Gly
95
Gln
US 8,298,800 B2
24
23
—cont inued
Lys
Thr
Cys Asp
Leu Val Ala
Gly Cys Gly
100
Asp
Ala
Trp
Lys
Glu Glu Val Ala Ala Asn Val
Lys
Thr Ala Asn Pro Thr Phe
130
125
Lys
Phe Val Val Thr
135
Gly Gly
Gly
Phe Pro Phe
Phe Leu
Ala Ala Val Ser Ala
120
Leu
145
Gly
105
115
Ala
Val His Thr
Ala Val Ala Thr Ile Ala Ala Ala
150
Asp
Leu
Gly
His Ser
140
Tyr
Leu
Arg Lys Asp
155
Tyr
Thr
Tyr Gly
160
Ser Pro
Arg
Val
Gly
Asn
165
Asp
Phe Phe Ala Asn Phe Val Thr
180
Val Thr His
Gly Asp Asp
195
Gly Tyr Arg
Gln Thr
Pro Val Pro
200
His Thr Ser Pro Glu
210
Arg
Tyr Trp
Thr Val Thr Glu Ile
225
Lys
230
Asn Val Met
Ile Thr
Tyr
Cys
Ala Glu
Asn
245
Tyr Arg
190
215
Asp Lys Asp Tyr
Gly
185
Leu Pro Pro Ile Val Phe
205
Leu Asn
220
Val
Cys
Gly Gly
Pro Leu
Gly
Ile Ala
Glu
235
Gly Gly
Thr Ile
Gly
Leu
240
Asp
Ile Leu Ala His
250
Phe Gln Ser Met Ala Thr
260
Cys
255
Ala Pro Ile Ala Ile Pro
265
2'70
Trp Lys Arg
275
<210>
<211>
<212>
<213>
SEQ ID NO 10
LENGTH: 278
TYPE: PRT
ORGANISM: Aspergillus oryzae
<400> SEQUENCE:
Asp Ile Pro Thr Thr Gln Leu Glu
Asp
Phe
Lys
Phe
Trp
Val Gln
1
Ala Ala Ala Thr
Leu Asn
35
Cys
Ser Thr Val
50
Lys
Gly
Lys Asp Gly
Glu
Val Glu Ala Ala
Gly
Tyr Cys
Pro Asn Asn
25
Tyr
Val Ala
Ser Val
Gly
Pro
Asp
20
Lys
Asn
Cys
45
Leu Ser Phe Ser
55
Phe Val Ala Val
Asp
Asp Asp
Asn Thr Asn
Lys
Thr Ile Thr
Asp
Thr Ala
Ala Ile Val Val Ala Phe
65
80
Arg Gly
Gln Thr
Trp
Ser
Asp
Thr Ala
Tyr
Pro
100
Ser Ile
85
Arg
Gly
Cys Asp Gly Cys Lys
Leu
Glu Leu
Lys
130
Val Thr
Asp
Val Val
Pro Glu His Ser
135
Lys
Tyr Asp
Ala Thr Phe Pro
95
Arg Asp Arg
Ile Ile
Lys
Thr Leu
Asp
Gly
His
Arg
Thr
125
Asp Tyr Lys
Ile Val Val Val
140
150
165
Phe
110
Ala Ala Ile Ala Ser Leu Ala Ala Ala
Ala Ile Leu
Gly
Ala Glu Leu
120
Gly
Lys
Trp
105
Trp Lys
Ser Leu
145
Asn
Asn
90
115
Asn
Tyr
15
Asp
Leu
155
Tyr
Ala
Tyr
160
Ala Ala Pro
Arg
170
Pro Leu Ala Glu Phe Ile Thr Asn Gln
180
185
Val Ala
1'75
Gly
Asn Asn
190
Tyr Arg
US 8,298,800 B2
25
26
—cont inued
Phe Thr His Asn
Asp Asp
195
Gly Tyr
Val His Ile Ser Pro Glu
210
Asp
225
Lys Gly
Asn Thr
245
Phe His Ser His Val
Gly
<210>
<211>
<212>
<213>
Tyr Tyr
Leu Pro Leu Leu Thr Met
205
Ile Thr Ala Pro
Asn Gln Val Thr Val Leu
230
235
Gly
Thr Ser
Gly Gly
Asp Gly Tyr
Asn
Val Asn
240
Leu Pro
Asp
Leu Leu Ala
255
Asp
Ala
Lys Gly
Gln Phe Glu Phe
Trp
250
Trp Tyr
Asp
220
Phe Ile His Ala
260
Pro
Lys
215
Thr Thr Val Thr
Phe
Pro Val Pro
200
265
Leu Pro Leu
275
Arg
SEQ ID NO 11
LENGTH:
TYPE: PRT
ORGANISM: Penicillium camemberti
<400> SEQUENCE: 11
Asp Val Ser Thr Ser Glu Leu
Asp
Val Gln
1
Ala Ala Ala Ser
Tyr Tyr
Glu Ala
20
Lys
Tyr
15
Leu Ser
35
Cys
Ala Thr Val Ser
Thr Ala Gln Val
25
Ser
Lys Gly
Tyr Asp
50
Gly Tyr
Asp Tyr
Asn
Cys
Pro Glu Val Glu Ala Thr
45
Phe Ser
Asp
Ser Thr Ile Thr
60
55
Ile Ala Val
Gly Asp
30
Asp His
Asp
Gly
Thr Ala
Thr Asn Ser Ala Val Val Leu Ala Phe
65
80
Arg Gly
Ser
Tyr
Ser Val
Arg
Asn
Trp
Val Ala
Asp
Ala Thr Phe Val
95
Thr Asn Pro
Gly
Leu
Cys Asp Gly Cys
100
Trp
Ser Ser
115
105
Trp Lys
Leu Val
Arg Asp Asp
130
Ser Leu
145
Gly
Arg
Lys
Leu
Asp
Tyr
Ala
Tyr
Pro Val Pro
200
Lys
Tyr Trp
Asp
Ile
Lys
Arg Gly
Arg
Gly
Val
Asn Asn Phe
190
Leu Pro Leu Leu Ser
205
Ile Thr Ser Pro Asn
Val Ile
Asp Gly Asp
235
Asn Thr
245
Gly
Glu Ala His Ile
Trp Tyr
Phe Val Gln Val
Thr
Gly
265
Lys Arg
Leu
220
Asp Gly
Leu Pro Phe
275
Asp
Ala Ser Pro
Ile Thr Ala Gln
215
Asn Ala Thr Val Ser Thr Ser
225
230
Gly
His
160
185
Val His Val Ser Pro Glu
260
Gly
175
Phe Thr His Thr Asn
Ser Phe
Lys
155
Lys Tyr
210
Glu Leu
140
Asn Ala Ala Leu Ala
180
Gly Tyr
Lys
Glu Leu Val Val Val
150
195
Met
Tyr
Ala Ala Val Ala Thr Leu Ala Ala Thr
Pro Ser Ala
165
Phe
125
135
Lys Gly Tyr
Gly
Ile Ile
120
Glu Val Val Ala Gln Asn Pro Asn
Gly
Leu Ala Glu Leu
110
Val
240
Leu Pro Leu Leu Thr Asp Phe
250
255
Asp
Ala
Gly Lys Gly
270
Pro
US 8,298,800 B2
27
—cont inued
<2lO> SEQ ID NO 12
<211> LENGTH: 2'70
<2l2> TYPE: PRT
<2l3> ORGANISM: Aspergillus foetidus
<400> SEQUENCE: 12
Ser Val Ser Thr Ser Thr Leu Asp Glu Leu Gln Leu Phe Ala Gln Trp
1
5
1O
15
Ser Ala Ala Ala Tyr Cys Ser Asn Asn Ile Asp Ser Lys Asp Ser Asn
Leu Thr Cys Thr Ala Asn Ala Cys Pro Ser Val Glu Glu Ala Ser Thr
35
4O
45
Thr Met Leu Leu Glu Phe Asp Leu Thr Asn Asp Phe Gly Gly Thr Ala
50
55
6O
Gly Phe Leu Ala Ala Asp Asn Thr Asn Lys Arg Leu Val Val Ala Phe
65
7O
75
8O
Arg Gly Ser Ser Thr Ile Glu Asn Trp Ile Ala Asn Leu Asp Phe Ile
85
9O
95
Leu Glu Asp Asn Asp Asp Leu Cys Thr Gly Cys Lys Val His Thr Gly
100
105
110
Phe Trp Lys Ala Trp Glu Ser Ala Ala Asp Glu Leu Thr Ser Lys Ile
115
120
125
Lys Ser Ala Met Ser Thr Tyr Ser Gly Tyr Thr Leu Tyr Phe Thr Gly
130
135
140
His Ser Leu Gly Gly Ala Leu Ala Thr Leu Gly Ala Thr Val Leu Arg
145
150
155
160
Asn Asp Gly Tyr Ser Val Glu Leu Tyr Thr Tyr Gly Cys Pro Arg Ile
165
170
175
Gly Asn Tyr Ala Leu Ala Glu His Ile Thr Ser Gln Gly Ser Gly Ala
180
185
190
Asn Phe Arg Val Thr His Leu Asn Asp Ile Val Pro Arg Val Pro Pro
195
200
205
Met Asp Phe Gly Phe Ser Gln Pro Ser Pro Glu Tyr Trp Ile Thr Ser
210
215
220
Gly Asn Gly Ala Ser Val Thr Ala Ser Asp Ile Glu Val Ile Glu Gly
225
230
235
240
Ile Asn Ser Thr Ala Gly Asn Ala Gly Glu Ala Thr Val Ser Val Leu
245
250
255
Ala His Leu Trp Tyr Phe Phe Ala Ile Ser Glu Cys Leu Leu
260
265
270
<2lO> SEQ ID NO 13
<211> LENGTH: 2'70
<2l2> TYPE: PRT
<2l3> ORGANISM: Aspergillus niger
<400> SEQUENCE: 13
Ser Val Ser Thr Ser Thr Leu Asp Glu Leu Gln Leu Phe Ser Gln Trp
1
5
1O
15
Ser Ala Ala Ala Tyr Cys Ser Asn Asn Ile Asp Ser Asp Asp Ser Asn
2O
25
30
Val Thr Cys Thr Ala Asp Ala Cys Pro Ser Val Glu Glu Ala Ser Thr
35
4O
45
Lys Met Leu Leu Glu Phe Asp Leu Thr Asn Asn Phe Gly Gly Thr Ala
50
55
6O
Gly Phe Leu Ala Ala Asp Asn Thr Asn Lys Arg Leu Val Val Ala Phe
65
7O
75
8O
US 8,298,800 B2
29
30
—cont inued
Arg Gly
Leu Gln
Phe
Ser Ser Thr Ile
85
Lys
Asp
Leu
Trp Lys
Asn
100
Asp Asp
Ala
Trp
Gly
Tyr
Glu Ala Ala Ala
Gly Gly
Ser Val Glu Leu
165
Thr
Gly Tyr
Tyr
Thr
Asp
Ala Thr Val Leu
Arg
160
Tyr Gly Cys
Gly
Arg
Val
175
Ile Val Pro
Pro Ser Pro Glu
Gly
Ser
190
Gly
Arg
Val Pro Pro
Ala
Tyr Trp
Ile Thr Ser
220
Asn Ala
Gly
245
Trp Tyr
Pro
205
Ala Ser Val Thr Ala Ser
Ile Asn Ser Thr Ala
Asp
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>
<212>
<213>
Gly
170
230
Ala His Leu
Phe Thr
Tyr
140
200
Phe Ser
Gly
Ile
Thr Leu
215
Gly
Val His Thr
185
225
Phe Ile
Lys
Ala Leu Ala Glu His Ile Thr Ser Gln
Gly
Asp
Asn Leu Thr Ser
125
155
210
Gly
Asp
Gly
195
Phe
Leu
110
150
Phe Pro Val Thr His Leu Asn
Asp
Asp
Gly Cys Lys
Ala Leu Ala Thr Leu
180
Met
Thr
120
Asp Gly Tyr
Asn
Ile Ala
105
145
Asn
Cys
Ser Ala Met Ser Thr Tyr Ser
130
135
His Ser Leu
Trp
95
115
Lys
Asn
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
20
Leu Thr
Cys
Tyr Cys Asp
Gln Ser Leu
Ser Val
Gly
Asn
Cys
Asp
Thr
Lys
Pro Leu Val Glu Ala Ala Ser Thr
45
55
Leu Ala Ala
Gly
30
Glu Phe Asn Glu Ser Ser Ser
50
Gly Tyr
Glu Asn Leu Asn Ser Thr
40
Asp
15
25
35
Tyr
Leu Phe Ala
10
Tyr Gly
Asn Pro Ala
60
Glu Thr Asn
Lys
Leu Leu Val Leu Ser Phe
65
Arg Gly
Ser Ala
Asp
Leu Ala Asn
Trp
85
Leu Glu
Asp
Ala Ser
90
Asp
Leu
Cys
100
Phe
Trp Lys
Ala
Trp
Ser
105
Gly
Ala Ala Leu Ala Ala Leu Ala Ala Thr Ala Leu
Arg
Asp His
Ser
Asp
Asp Tyr
155
His Ser Val Glu Leu
Tyr
165
Asn Glu Ala Leu Ala Thr
180
Thr Ile Thr Ser
125
150
Gly
Gly
110
135
145
Asn Ser
Glu Val His Ser
Ser Leu Val Leu Thr
140
Ser Glu Ile Ala
120
130
Gly
Gly Cys
Val
Glu Ser Ala Leu Ser
Tyr Gly
Gly
95
Lys
115
His Ser
Val Ala Asn Leu Asn Phe
Tyr
Asn
170
Ile Thr
185
160
Tyr Gly
Gln Pro
Arg
Leu
175
Asp
Gln Asn
Lys Gly Gly
190
US 8,298,800 B2
31
32
—cont inued
Asn
Tyr Arg
Val Thr His Thr Asn
195
200
Thr Leu Leu
Gly Tyr
Asp
Glu Ala Thr Val Thr Thr Thr
Ala Thr
Gly Gly
Asn
Asp Gly
245
Ala His
Arg Trp Tyr
Leu Pro Pro
Tyr Tyr
Ile Ser Ser
220
Asp
230
Asp
Lys
205
215
225
Ile
Ile Val Pro
His His Phe Ser Pro Glu
210
Ala
Asp
Thr
Val Thr Glu Val Thr
Gly
235
240
Asp Gly
Thr Ser Ile
250
Phe Ile
Tyr
260
Asp
255
Ile Ser Glu
Cys
Ser
265
SEQ ID NO 15
LENGTH: 269
TYPE: PRT
ORGANISM: Thermomyces
lanuginosus
SEQUENCE: 15
Glu Val Ser Gln
1
Asp
Ser Ala Ala Ala
20
Tyr Cys Gly Lys
15
Asn Asn
25
Asp
Ala Pro Ala
Thr
Gly
Asn Ala
40
Cys
Pro Glu Val Glu
45
Ala Thr Phe Leu
Tyr
Ser Phe Glu
55
Asp
Ser
50
Phe Leu Ala Leu
Asp
Gly
Gly
Thr
Ala
Asp
30
Cys
Asn Ile Thr
35
Tyr
Leu Phe Asn Gln Phe Asn Leu Phe Ala
Gly
Val
Lys
Gly Asp
Val Thr
60
Asn Thr Asn
Lys
Leu Ile Val Leu Ser Phe
Trp
Ile
Gly
Ser
105
Gly Cys Arg Gly
65
Arg Gly
Leu
Lys
Ser
Arg
Ser Ile Glu Asn
85
Glu Ile Asn
Phe Thr Ser Ser
Asp
Trp Arg
115
Glu
Asp
Ala Val
Arg
Cys
Ser Val Ala
120
Glu His Pro
130
Gly Gly
145
Gly Tyr Asp
Leu
Arg
Asn
Arg
Asp
Val Phe Ser
Ala
Asp
Leu
Tyr Gly
Ala Pro
Arg
Gly Gly
19O
Gly Tyr
Asp
Ile Val Pro
Arg
Asn
Asp
230
Ala Thr
Gly Gly
245
Trp Tyr
260
Phe
Arg
Ile Val
Trp
Ile
Lys
Ser
Lys
Ile Glu
Gly
240
Asn Asn Gln Pro Asn Ile Pro
250
Leu Ile
265
Gly
Thr
Cys
Asp
Ile Pro
255
Leu
The invention claimed is:
1. A Variant polypeptide Which:
(a) has hydrolytic activity towards an ester bond in a polar
lipid, and
Leu Pro Pro
235
Gly
Thr
205
Ser His Ser Ser Pro Glu Tyr
215
220
Thr Leu Val Pro Val Thr
Val
175
185
200
Arg
160
170
Ile Thr His Thr Asn
Ala His Leu
Gly
155
195
Asp
Gly
180
225
Ile
Val Val Phe Thr
Ala Phe Ala Glu Phe Leu Thr Val Gln Thr
210
Gly
Val
Gln
140
Tyr Arg
Glu Phe
Arg
125
Ala Leu Ala Thr Val Ala
Ile
Asp Gly
Lys
Thr Leu
Asp Tyr Arg
165
Gly
Asp
150
Asn
His
Asp
110
135
His Ser Leu
Gly
Ile
Asn Leu Asn Phe
95
(b) has an amino acid sequence Which
(i) has at least 80% identity to SEQ ID NO: 1 and has a
different amino acid or an amino acid deletion at a posi
tion corresponding to A29, K33, 183 or A255, or