U.S. patent application number 10/495597 was filed with the patent office on 2005-11-17 for lipolytic enzyme variants and method for their production.
This patent application is currently assigned to Novozymes A/S. Invention is credited to Erlandsen, Luise, Heldt-Hansen, Hans Peter, Svendsen, Allan, Vind, Jesper.
Application Number | 20050255544 10/495597 |
Document ID | / |
Family ID | 8161014 |
Filed Date | 2005-11-17 |
United States Patent
Application |
20050255544 |
Kind Code |
A1 |
Svendsen, Allan ; et
al. |
November 17, 2005 |
Lipolytic enzyme variants and method for their production
Abstract
The inventors have developed a method using protein engineering
to produce lipolytic enzymes having a relatively high activity for
one ester bond in an amphiphilic substrate with two lipophilic
groups) and a relatively low activity for the ester bond in an
amphiphilic substrate with one lipophilic group, e.g. a relatively
high phospholipase activity and a relatively low lysophospholipase
activity.
Inventors: |
Svendsen, Allan; (Horsholm,
DK) ; Vind, Jesper; (Vaerlose, DK) ;
Heldt-Hansen, Hans Peter; (Virum, DK) ; Erlandsen,
Luise; (Copenhagen, 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: |
8161014 |
Appl. No.: |
10/495597 |
Filed: |
May 14, 2004 |
PCT Filed: |
January 16, 2003 |
PCT NO: |
PCT/DK03/00028 |
Current U.S.
Class: |
435/69.1 ;
435/198; 435/254.1; 435/483 |
Current CPC
Class: |
C12Y 301/01003 20130101;
C12N 9/20 20130101 |
Class at
Publication: |
435/069.1 ;
435/198; 435/254.1; 435/483 |
International
Class: |
C12P 021/06; C12N
009/20; C12N 001/14; C12N 015/74 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 16, 2002 |
DK |
PA 2002 00074 |
Claims
1. A method of producing a lipolytic enzyme variant comprising: a)
selecting a parent fungal lipolytic enzyme, b) selecting at least
one amino acid residue which comprises an atom (excluding H atoms)
which in a three-dimensional model lies within 10 .ANG. of an atom
(excluding H atoms) of a phospholipid docked with the parent
lipolytic enzyme, or which is the C-terminal amino acid, c)
altering the selected amino acid, d) optionally, altering one or
more amino acids other than those selected, e) preparing the
variant resulting from the preceding steps, f) testing hydrolytic
activities of the variant towards the bond B-C of a first substrate
having the general formula A-B-C and towards the bond B'-C of a
second substrate having the general formula A'-B'-(A"-B"-) C where:
i) A and A' are fatty acyl groups, ii) B and B' are oxygen or
sulfur iii) C is a polyol with B, B' and B" attached to OH groups,
optionally having other functional groups, and optionally having a
hydrophilic group attached to an OH group, g) selecting a variant
having a ratio of activity on the first substrate to activity on
the second substrate which is lower than the parent lipolytic
enzyme, and h) producing the selected variant.
2. A method of producing a lipolytic enzyme variant comprising: i)
selecting a parent fungal lipolytic enzyme, j) in the parent
lipolytic enzyme selecting at least one amino acid residue
corresponding to any of residues 17-18, 20-23, 26, 37, 39, 62, 64,
80-96, 110-113, 144-151, 171-177, 200-211, 213, 215, 227, 253-261
and 263-269 of the T. lanuginosus lipase (SEQ ID NO: 14), k)
altering the selected amino acid, l) optionally, altering one or
more amino acids other than those selected, m) preparing the
variant resulting from the preceding steps, n) testing hydrolytic
activities of the variant towards the bond B-C of a first substrate
having the general formula A-B-C and towards the bond B'-C of a
second substrate having the general formula A'-B'-(A"-B"-) C where:
i) A and A' are fatty acyl groups, ii) B and B' are oxygen or
sulfur iii) C is a polyol with B, B' and B" attached to OH groups,
optionally having other functional groups, and optionally having a
hydrophilic group attached to an OH group, o) selecting a variant
having a ratio of activity on the first substrate to activity on
the second substrate which is lower than the parent lipolytic
enzyme, and p) producing the selected variant.
3. The method of claim 1 wherein the first substrate is a
lyso-phospholipid and the second substrate is a phospholipid.
4. The method of claim 3 wherein the parent lipolytic enzyme has
phospholipase activity, particularly phospholipase A1 activity.
5. The method of claim 4 wherein the parent lipolytic enzyme also
has digalactosyl diglyceride hydrolyzing activity and optionally
triacylglycerol lipase activity.
6. The method of claim 1 wherein the parent lipolytic enzyme has an
amino acid sequence which is at least 50% (particularly at least
90%) identical to that of the Thermomyces lanuginosus lipase (SEQ
ID NO: 14).
7. The method of claim 6 wherein the alterations comprise
substitution of at least one of L93, F95, V203 and L206 with an
amino acid residue which is smaller and/or more hydrophilic.
8. The method of claim 7 wherein the alterations comprise at least
one of the substitutions L206V/S/T/A, V203T/S/A, F95I/L/Y and
L93V/I/A/T.
9. The method of claim 5 wherein the alterations comprise
substitution of at least one of P253, I255 and P256 with an amino
acid which is larger.
10. The method of claim 5 wherein the alterations comprise an amino
acid alteration in the region 247-260 or 265-269.
11. The method of claim 10 wherein the alterations comprise
substitution of at least one of P253 and P256 with a different
amino acid residue.
12. The method of claim 5 wherein the alterations comprise a
substitution L227D, P253T/G/L, D254S/L, I255, P256L/A, A257D/A,
L259 or W260H.
13. The method of claim 5 wherein the alterations comprise an amino
acid alteration in the region 247-260 and an alteration of amino
acid L93, F95, V203 and L206.
14. The method of claim 5 wherein the parent lipolytic enzyme
compared to the T. lanuginosus lipase (SEQ ID NO: 14) comprises an
amino acid alteration at a position corresponding to R81, R84, S85,
G263, L264, 1265, G266, T267 or L269 and/or a peptide extension of
1-10 amino acids art the C-terminal.
15. A method of preparing a dough or a baked product made from
dough, comprising preparing a lipolytic enzyme variant by the
method of any preceding claim 1 and adding the lipolytic enzyme
variant to the dough.
16. A variant of a parent fungal lipolytic enzyme which variant
comprises an amino acid alteration corresponding to R84G/A/Y/S or
L206F of SEQ ID NO: 14, has phospholipase activity and has a
lysophospholipase to phospholipase ratio corresponding to PLARN
below 500 or RLPLA below 1.0.
17-19. (canceled)
Description
FIELD OF THE INVENTION
[0001] The present invention relates to a lipolytic enzyme variant
and a method of producing such a variant. More particularly, the
variant has a relatively high activity for one ester bond in an
amphiphilic substrate with two lipophilic groups and a relatively
low activity for the ester bond in an amphiphilic substrate with
one lipophilic group, e.g. a relatively high phospholipase activity
and a relatively low lysophospholipase activity.
BACKGROUND OF THE INVENTION
[0002] EP 870840, JP-A 10-42884, JP-A 4-135456 or JP-A 249593
describe the use of a phospholipase to hydrolyze a phospholipid to
produce lyso-phospholipid.
[0003] .vertline.U.S. Pat. No. 4,567,046.vertline., WO 94/04035, EP
109244, .vertline.EP 585988.vertline., WO 98/26057, .vertline.WO
98/45453.vertline., WO 99/53769, WO 00/32758, .vertline.WO
0139602.vertline. and EP 575133 describe the addition of various
lipolytic enzymes to dough in the preparation of baked products and
the preparation of lipolytic enzyme variants.
[0004] .vertline.WO 00/32758.vertline. discloses that the substrate
specificity of a lipolytic enzyme can be modified by making
alterations to the amino acid sequence.
SUMMARY OF THE INVENTION
[0005] The inventors have developed a method using protein
engineering to produce lipolytic enzymes having a relatively high
activity for one ester bond in an amphiphilic substrate with two
lipophilic groups and a relatively low activity for the ester bond
in an amphiphilic substrate with one lipophilic group, e.g. a
relatively high phospholipase activity and a relatively low
lysophospholipase activity.
[0006] Accordingly, the invention provides a method of producing a
lipolytic enzyme variant comprising:
[0007] a) selecting a parent fungal lipolytic enzyme,
[0008] b) in the parent lipolytic enzyme altering at least one
specified amino acid residue,
[0009] c) optionally, altering one or more amino acid residues
other than b),
[0010] d) preparing the variant resulting from steps a)-c),
[0011] e) testing hydrolytic activities of the variant towards a
first substrate and a second substrate,
[0012] f) selecting a variant having a ratio of hydrolytic
activities towards the first substrate and the second substrate
which is lower than the parent lipolytic enzyme, and
[0013] g) producing the selected variant.
[0014] The first substrate is a molecule comprising one fatty acyl
group linked through an ester or thioester bond to a hydrophilic
group. The second substrate is a molecule comprising a first
lipophilic group which is a fatty acyl group linked through an
ester or thioester bond to a hydrophilic group, and a second
lipophilic group linked to the hydrophilic group, where the second
lipolhilic group may be a second fatty acyl group linked through an
ester, thioester or amide bond, or it may be a fatty alcohol linked
through an ether or thioether bond.
[0015] Each amino acid alteration may be an amino acid
substitution, deletion or insertion. The amino acid residue to be
altered may be determined from a three-dimensional model of a
phospholipid docked with the parent lipolytic enzyme as a residue
which comprises an atom (excluding H atoms) which lies within 10
.ANG. (particularly 7 .ANG. or 5 .ANG.) of an atom (excluding H
atoms) of the lyso-phospholipid, or it may be the C-terminal amino
acid.
[0016] Alternatively, the amino acid residue to be altered may be
determined by aligning the amino acid sequence of the parent
lipolytic enzyme with the T. lanuginosus lipase and selecting a
residue corresponding to any of residues 17-18, 20-23, 26, 37, 39,
62, 64, 80-96, 110-113, 144-151, 171-177, 200-211, 213, 215, 227,
253-261 or 263-269.
[0017] The invention also provides a 1. lipolytic enzyme having an
amino acid sequence derived from the T. lanuginosus lipase (SEQ ID
NO: 14) comprising the following amino acid alterations:
[0018] a)
R84W+G91A+D96F+E99K+G263Q+L264A+1265T+G266D+T267A+L269N,
[0019] b)
G91A+D96W+E99K+L227G+G263Q+L264A+1265T+G266D+T267A+L269N,
[0020] c) R84W+G91A:D96F+E99K+G263Q+L264A+1265T+G266S+T267A
25+L269N+270A+271G+272G+273F+274S,
[0021] d) SPPCGRRP(-E)+Y21K+E99N+N101S+E239C+Q249R,
[0022] e) G91A+D96K+E99K+G263Q+L264A+1265T+G266D+T267A+L269N,
[0023] f)
Y21V+R84G+G91A:D96F+E99K+G263Q+L264A+1265T+G266D+T267A+L269N+270-
A+271G+272G+273F+274S,
[0024] g)
V60G+D62W+R84W+G91A+D96F+E99K+G263Q+L264A+1265T+G266D+T267A+L269-
N,
[0025] h)
V60A+D62S+G91A+D96W+E99K+W221R+G263Q+L264A+1265T+G266D+T267A+L26-
9N+270A+271G+272G+273F+274S,
[0026] i) R84A+S85D+E87A+G91A+D96G+K98E+E99D,
[0027] j)
G91A+D96W+E99K+P250N+G263Q+L264A+1265T+G266D+T267A+L269N+270A+27-
1G+272G+273F+274S,
[0028] k)
G91A+D96W+E99K+P256N+G263Q+L264A+1265T+G266D+T267A+L269N+270A+27-
1G+272G+273F+274S,
[0029] l)
R84W+G91A+D96W+E99K+Y261Q+G263Q+L264A+1265T+G266D+T267A+L269N+27-
0A+271G+272G+273F+274S,
[0030] m)
G91A+D96W+E99K+P250L+P253Q+D254DEL+P257S+G263Q+L264A+1265T+G266D-
+T267A+L269N+270A+271G+272G+273F+274S,
[0031] n) R84Y+G91A+D96F+E99K+G263Q+L264A+1265T+G266D+T267A+L269N,
or
[0032] o)
R84S+G91A+D96F+E99K+E129A+V203i+L206F+G263Q+L264A+1265T+G266D+T2-
67A+L269N.
[0033] The invention also provides a lipolytic enzyme having an
amino acid sequence derived from a Fusarium lipase comprising at
least one amino acid alteration corresponding to the following in
the Fusarium oxysporum lipase(phospholipase (SEQ ID NO: 7):
[0034] a) H257W,
[0035] b) S142A,
[0036] c) V157D,
[0037] d) S271P,
[0038] e) S80T,
[0039] f) A127T,
[0040] g) D263G,
[0041] h) Y21W,
[0042] i) S80T,
[0043] j) R274A,
[0044] k) 275YRSAESVDKR or
[0045] l) 275YRSAESVDKMTMTDAELEKKLNSYVQMDKEYVKNNQARS.
[0046] Further, the invention provides a lipolytic enzyme
which:
[0047] a) has an amino acid sequence having at least 90% identity
to that of the Thermomyces lanuginosus lipase or the Fusarium
oxysporum lipase/phospholipase,
[0048] b) has phospholipase activity, and
[0049] c) has a lysophospholipase to phospholipase ratio below the
limit indicated below.
DETAILED DESCRIPTION OF THE INVENTION
[0050] Test Substrates
[0051] The invention uses two different polar lipids as test
substrates. Both are amphiphilic (amphipolaric), having a
hydrophilic part and one or two lipophilic groups,
respectively.
[0052] First Substrate
[0053] The first substrate has the general formula A-B-C where:
[0054] A is an acyl group, particularly straight-chain and
unsubstituted. It may be saturated or may have one or more double
bonds. It may have an even number of carbon atoms, e.g. from 12 to
24
[0055] B is O (oxygen) or S (sulfur) forming an ester or thioester
bond between A and C.
[0056] C is a polyol having B attached to an OH group, optionally
having other functional groups and/or a hydrophilic group attached
to an OH group. The polyol may be a sugar alcohol such as glycerol,
e.g. having B attached in the sn1 or sn2 position of glycerol. The
other functional groups may be one or more aldehyde, keto or
carboxyl groups; thus, the polyol may be a monosaccharide or a
corresponding uronic acid. The hydrophilic group linked to an OH
group, e.g. in the sn3 position of glycerol, may be:
[0057] A phosphate group, optionally linked to an alcohol such as
choline, ethanolamine, serine or inositol.
[0058] Mono- or digalactosyl link to C through a glycosidic
bond.
[0059] The first substrate may be a lysophospholipid such as
lyso-lecithin or a lyso-galactolipid such as digalactosyl
monoglyceride (DGMG) or monogalactosyl monoglyceride (MGMG). The
lyso-phospholipid may be a 1-lyso-phospholipid with an acyl group
at the sn1-position or a 2-lyso-phospholipid with an acyl group at
the sn2-position.
[0060] The activity of interest is a hydrolytic activity towards
the bond B-C.
[0061] Second Substrate
[0062] The second substrate has the general formula A'-B'-(A"-B"-)C
where:
[0063] A' is an acyl group defined as for A above.
[0064] B' is O (oxygen) or S (sulfur) forming an ester or thioester
bond between A' and C.
[0065] A" is an acyl or alkyl group particularly straight-chain and
unsubstituted. It may be saturated or may have one or more double
bonds. It may have an even number of carbon atoms, e.g. from 12 to
24
[0066] B" is O (oxygen), S (sulfur) or NH forming an ester,
thioester, amide, ether or thioether bond between A" and C.
[0067] C is the same as for the first substrate.
[0068] A" and B" of the second substrate may be chosen identical to
A and B of the second substrate and attached in the same position
of C, or they may be chosen independently.
[0069] The second substrate may be a phospholipid such as lecithin
or a galactolipid such as digalactosyl diglyceride (DGDG) or
monogalactosyl diglyceride (MGDG), or it may be prepared
synthetically by attaching a fatty alcohol through an ether bond or
thioether bond to a lysophospholipid or a lyso-galactolipid.
[0070] The activity of interest is a hydrolytic activity towards
the B'-C bond.
[0071] Lipolytic Enzyme Activities
[0072] The lipolytic enzyme of the invention has a low ratio of
activity for the first substrate compared to activity for the
second substrate. Thus, it has a relatively low hydrolytic activity
towards the B-C (thio)ester bond of the first substrate and a
relatively high hydrolytic activity towards the B'-C (thio)ester
bond of the second substrate. The activity towards the second
substrate may be phospholipase A1 (EC 3.1.1.32) or A2 (EC 3.1.1.4),
or it may be a galactolipase activity.
[0073] The activity ratio may be found by contacting the lipolytic
enzyme with each substrate separately or by contacting it with a
mixture including both substrates. The activity ratio may be
measured by the PLARN assay, the RLPLA assay or a plate assay
described below. The lipolytic enzyme may have a ratio of
lysophospholipase activity to phospholipase activity corresponding
to PLARN below 1000 (particularly 500, below 200 or below 50) or
RLPLA (0.1/2.5) below 2 (particularly below 1 or below 0.5).
[0074] The lipolytic enzyme of the invention may have phospholipase
(PL) activity with a relatively low lysophospholipase (LPL)
activity. The lyso-phospholipid may be a 1-lyso-phospholipid with
an acyl group at the sn1-position or a 2-lyso-phospholipid with an
acyl group at the sn2-position. The lipolytic enzyme may in
particular have phospholipase A1 activity with low
1-lysophospholipase activity.
[0075] The lipolytic enzyme of the invention may have hydrolytic
activity towards a carboxylic ester bond in DGDG (digalactosyl
diglyceride) with a relatively low hydrolytic activity towards the
ester bond in DGMG (digalactosyl monoglyceride).
[0076] Optionally, the lipolytic enzyme may also have
triacylglycerol lipase activity (EC 3.1.1.3), i.e. hydrolytic
activity for carboxylic ester bonds in triglycerides, e.g.
1,3-specific activity, particularly on long-chain triglycerides
such as olive oil. The enzyme may have a substrate specificity for
hydrolyzing long-chain fatty acyl groups rather than short-chain
groups, e.g. expressed as a high ratio of activities on olive oil
and tributyrin, e.g. a ratio SLU/LU>3 as described in WO
0032758.
[0077] LPUPL Ratio (PLARN)
[0078] Phospholipase activity is determined at 30.degree. C. using
4% (w/v) lecithin (phosphatidyl choline) in 50 mM sodium acetate, 5
mM CaCl2, pH 5.0. One unit of phospholipase activity is defined as
1 mmol free fatty acids released per minute per mg enzyme using the
above conditions.
[0079] Lysophospholipase activity is determined at 30.degree. C.
using 1% (w/v) lysolechitin in 50 mM sodium acetate, 5 mM CaCl2, pH
5.0. One unit of lysophospholipase activity is defined as 1 mmol
free fatty acids released per minute per mg enzyme using the above
conditions. The lysolechitin may be an equilibrium mixture or pure
1-lysolechitin in the case of a phospholipase A1 and pure
2-lysolechitin in the case of a phospholipase A2.
[0080] The PLARN ratio is defined as the lysophospholipase activity
divided by the phospholipase activity, both determined by the above
methods.
[0081] Relative Activity on Lysopholipids and Phospholipids
(RLPLA)
[0082] This activity measurement expresses the relative activity on
lysophosphatidyl choline and phosphatidyl choline in an equimolar
mixture.
[0083] More specifically the assay is carried out as follows: The
activity at different concentrations of phospholipase is determined
at 30.degree. C. blending 1:1 solutions of phosphatidyl choline (25
mM) and lysophosphatidyl choline (25 mM) in 50 mM NaOAc buffer (pH
5). 50 .mu.l enzyme solution (e.g. having 0.1 or 2.5 mg enzyme
protein per ml) is added to the substrate solution and allowed to
react for 30 minutes. 100 .mu.l of the sample is inactivated at
95.degree. C. for 5 minutes and dissolved in 900 .mu.l CHCl3/MeOH
50%/50%. The sample is centrifuged at 14000 rpm for 2 minutes. The
supernatant is analyzed by HPLC after filtering through a 0.45
.mu.m filter. Column: Microsorb-MV 100Si 250 mm column (analytical
instruments). Mobile phases: A: 80% CHCl3, 19.5% MeOH, 0.5% NH4OH;
B: 60% CHCl3, 34% MeOH, 0.5% NH4OH, 5.5% H.sub.2O. Gradient: 0-3
minutes 100% A, 3-23 minutes 100% B, 2345 minutes 100% A. Injection
volume 20 .mu.l Detector: Sedere, Sedex 75 light scattering, Temp
40.degree. C., pressure 3.5 Bar. The RLPLA is then measured as the
depletion of phosphatidyl choline relative to lysophosphatidyl
choline. A variant with a lower RLPLA value indicates a higher
accumulation of lysophospholipid under the conditions of
analysis.
[0084] The relative activity can be expressed as "RLPLA ratio" by
measuring lysolecithin hydrolysis at an enzyme dosage of 0.1 mg/ml
and lecithin hydrolysis at a dosage of 2.5 mg/ml, and taking the
ratio of the two.
[0085] Plate Assay
[0086] Plates including each of the substrates may be prepared in
analogy with WO 0032758 using suitable pH and substrate
concentration. Optionally, other ingredients such as flour may be
included. A suitably diluted enzyme solution is applied to holes in
the plates, and clearing zones are read after incubation for a
suitable time at a suitable temperature.
[0087] Preparation of Lipolytic Enzyme
[0088] The lipolytic enzyme may be obtained by preparing variants
of a parent lipolytic enzyme by altering its amino acid sequence
and screening for a variant with an improved activity ratio. The
parent lipolytic enzyme may have phospholipase activity, DGDG
hydrolytic activity and/or triacylglycerol lipase activity.
Variants may be prepared from the parent lipolytic enzyme by known
methods, e.g. by subjecting a DNA sequence encoding the parent
lipolytic enzyme to site-directed mutagenesis, localized random
mutagenesis or site-saturation mutagenesis, e.g. using methods
described in WO 0032758. Resulting DNA sequences may be further
modified by gene shuffling and directed evolution.
[0089] Parent Lipolytic Enzyme
[0090] The lipolytic enzyme to be used in the present invention is
one that can hydrolyze ester bonds. Such enzymes include, for
example, lipases, such as triacylglycerol lipase (EC 3.1.1.3),
lipoprotein lipase (EC 3.1.1.34), monoglyceride lipase (EC
3.1.1.23), phospholipase A1 or A2 (EC 3.1.1.26, 3.1.1.4),
lysophospholipase (EC 3.1.1.5), galactolipase (EC 3.1.1.26),
ferulic acid esterase and esterase (EC 3.1.1.1, EC 3.1.1.2).
[0091] The parent lipolytic enzyme may be the Thermomyces
lanuginosus lipase (Humicola lanuginosa lipase) (EP 305216) or it
may have an amino acid sequence with at least 50% identity (e.g. at
least 90% identity). The amino acid sequence of the T. lanuginosus
lipase is shown in U.S. Pat. No. 5,869,438 and as seq 14 in FIG. 2
of this application.
[0092] Thus, the parent lipolytic enzyme may be a naturally
occurring enzyme as described at pages 5-6 of WO 0032758. Examples
are the lipolytic enzymes from the following organisms. They are
known in the prior art; and their amino acid sequences are given in
the attached sequence listing.
[0093] 1. Absidia reflexa
[0094] 2. Absidia corymbefera
[0095] 3. Rhizmucor miehei
[0096] 4. Rhizopus delemar (oryzae)
[0097] 5. Aspergillus niger
[0098] 6. Aspergillus tubingensis
[0099] 7. Fusarium oxysporum
[0100] 8. Fusarium heterosporum
[0101] 9. Aspergillus oryzae
[0102] 10. Penicilium camembertii
[0103] 11. Aspergillus foetidus
[0104] 12. Aspergillus niger
[0105] 13. Aspergillus oryzae
[0106] 14. Thermomyces lanuginosus (Humicola lanuginosa)
[0107] As indicated above, the amino acid to be altered may be
determined on the basis of an alignment of the parent lipolytic
enzyme with the T. lanuginosus lipase. FIG. 2 shows an alignment of
the amino acid sequences of the above fungal lipolytic enzymes,
based on a comparison of the available 3-dimensional
structures:
[0108] Other amino acid sequences may be aligned with those shown
in FIG. 2 by using the GAP alignment to the most homologous
sequence found by the GAP program. GAP is provided in the GCG
program package (Program Manual for the Wisconsin Package, Version
8, August 1994, Genetics Computer Group, 575 Science Drive,
Madison, Wis., USA 53711) (Needleman, S. B. and Wunsch, C. D.,
(1970), Journal of Molecular Biology, 48, 443-45). The following
settings are used for polypeptide sequence comparison: GAP creation
penalty of 3.0 and GAP extension penalty of 0.1.
[0109] Alternatively, the parent lipolytic used in the present
invention may be a variant of the above, e.g. a variant of the T.
lanuginosus lipase (SEQ ID NO: 14) or the F. oxysporum
lipase/phospholipase (SEQ ID NO: 7). A variant with phospholipase
activity or galactolipase activity may be used, e.g. as described
in Example 5, 6 or 13 of WO 0032758. Particular examples are
variants of SEQ ID NO: 14 with the following amino acid
alterations:
1 L259S G266D G91A + D96W + E99K + G263Q + L264A + I265T + G266D +
T267A + L269N + 270A + 271G + 272G + 273F + 274S G266E G263A +
G266A E1SPCRPRP + E239C + Q249R + G266A E1SPCRPRP + E239C + Q249R +
G266S D96S + G266A D96S + G266S D96S + G266W E1SPPCGRRP + D96S +
E239C + Q249R + G263D + L264I + I265N + G266E + T267GS
[0110] Three-Dimensional Model
[0111] FIG. 1 gives the coordinates of a three-dimensional model of
the T. lanuginosus lipase docked with a phospholipid as substrate:
1-palmetoyl-2-oleylglycero-sn-3-phosphocholine (POPC). This may be
used as a starting point for building a similar model for any given
fungal lipolytic enzyme. Using this model, the following amino acid
residues are found to be within 10 .ANG., 7 .ANG. and 5 .ANG. of an
atom of the substrate (LIP1 in the pdb structure shown in FIG.
1):
[0112] 10 .ANG.: 17-18, 20-23, 26, 37, 39, 62, 64, 80-96, 110-113,
144-151, 171-177, 200-211, 213, 215, 227, 253-261, 263-269.
[0113] 7 .ANG.: 21, 81-95, 110, 113, 145-148, 150, 172-175,
201-208, 213, 254-256, 258-259, 264-269.
[0114] 5 .ANG.: 21, 82-86, 89-90, 92-93, 95, 110, 113, 145-147,
174, 202-203, 206-208, 255, 258-259, 265-268.
[0115] More particularly, amino acid alterations may be made at one
or more positions corresponding to the following amino acids in the
T. lanuginosus lipase (SEQ ID NO: 14): Y212, R84, S85, E87, D96,
V203, L206, L227, P253, D254, P256, P257 and/or Y261, particularly
one or more alterations corresponding to Y21V/K, R84W/A/G/Y/S,
S85D, E87A, D96F/K/G, V203I, L206F, P253Q, D254*, P256N, P257S
and/or Y261Q.
[0116] Further, amino acid alterations may be made at one or more
positions corresponding to H257, S142, S80, D263 and/or Y21 of the
F. oxysporum lipase/phospholipase (SEQ ID NO: 7), particularly one
or more corresponding to H257W, S142A, S80T, D263G and/or Y21W.
[0117] Also, amino acid alterations may be made at the C-terminal
or at any position downstream of L269 of SEQ ID NO: 14. Such
alteration may be addition or deletion of a peptide extension or
deletion of a peptide extension of one or more amino acids (e.g.
1-50 amino acids such as 2-15) at the C-terminal.
[0118] Amino Acid Alteration
[0119] The amino acid alteration may be substitution with a larger
amino acid. The amino acid residues are ranked by size as follows
from smallest to largest:
[0120] G, A, S, C, V, T, P, L, I, N, D, M, E, Q, K, H, R, F, Y,
W
[0121] An amino acid residue within 10 .ANG. (or 7 .ANG. or 5
.ANG.) of a C atom in the alkyl group R of R--COO attached to sn1
of the lyso-phospholipid may be substituted with an amino acid
residue which is larger or more hydrophilic.
[0122] An amino acid residue within 10 .ANG. (or 7 .ANG. or 5
.ANG.) of an atom (other than H) of the phosphate group attached to
sn3 (i.e. the O atom at sn3 or any atom beyond that) may be
substituted with an amino acid which is larger or more
hydrophobic.
[0123] Amino acid residues are ranked as follows from most
hydrophilic to most hydrophobic:
[0124] R, K, E, D, N, Q, H, S, T, Y, C, M, G, A, V, P, L, I, F,
W
[0125] Lipolytic Enzyme Variant
[0126] Starting from a variant having phospholipase activity
derived from the T. lanuginosus lipase, and improvement regarding
increased ratio of lecithin/lysolecithin(sn1) activity and lowered
activity against lysolecithin (sn1) of lysolecithin in general,
or/and with improved sn1 lecithase activity may be achieved by use
of the following concepts.
[0127] One concept is to lower the binding energy of sn1 acyl chain
and in this way increase the ratio of activity
lecithin/lysolecithin(sn1). Secondary to increase sn2 binding to
favour lecithin rather than lysolecithin.
[0128] Thus, 206, 95, 203 and 93 may be made smaller and more
hydrophilic. Also, positions 253, 255 and 256 may be made bigger.
Some particular examples of such variants are:
[0129] L206V/S/T/A
[0130] V203T/S/A
[0131] F951/LY
[0132] L93V/I/A1T
[0133] A doped library 206/203 and 95/93. appr. 90% wt and appr 10%
variant may be used.
[0134] A second concept is to make libraries in the contacts to
both acyl chains found docked structures, and in the regions close
to the substrate found by comparison of good and bad lysolecithase
active homologous enzymes. The regions will be doped according to
the homologous enzymes sequence.
[0135] Thus, region 265-269 is of interest, e.g. P253. Some
particular examples of such variants are:
[0136] A doped library 247-260 may be used, optionally together
with doped 1202P and L206V. The doping may be appr 90% wt and appr.
10% variants.
[0137] P253TG/L
[0138] D254S/L
[0139] 1255
[0140] P256LUA
[0141] A257D/A
[0142] L259
[0143] W260H
[0144] Proline removal(s):
[0145] P256X
[0146] P253X
[0147] together with 1202P
[0148] L227D
[0149] Library 247-260 and 206, 95, 203 and 93 may be combined.
[0150] Amino Acid Identity
[0151] The lipolytic enzyme of the invention and the parent
lipolytic enzyme may have an amino acid identity of at least 50%
(particularly at least 90%, e.g. more than 95% or more than 98%)
with the T. lanuginosus lipase (SEQ ID NO: 14).
[0152] The degree of identity may be suitably determined by means
of computer programs known in the art, such as GAP provided in the
GCG program package (Program Manual for the Wisconsin Package,
Version 8, August 1994, Genetics Computer Group, 575 Science Drive,
Madison, Wis., USA 53711) (Needleman, S. B. and Wunsch, C. D.,
(1970), Journal of Molecular Biology, 48, 44345), using GAP with
the following settings for polypeptide sequence comparison: GAP
creation penalty of 3.0 and GAP extension penalty of 0.1.
[0153] Use of Lipolytic Enzyme Variant
[0154] Hydrolysis of Phospholipid
[0155] A variant with phospholipase activity can be used to prepare
lysophospholipid (e.g. lyso-lecithin) by treating the corresponding
phospholipid with the variant, e.g. as described in EP 870840, JP-A
10-42884, JP-A 4-135456 or JP-A 2-49593. The variant can also be
used to make mayonnaise, e.g. as described in EP 628256, EP 398666
or EP 319064.
[0156] Advantageously, a low ratio of
lysophospholipase/phospholipase activity can lead to a high degree
of phospholipid hydrolysis with a low degree of lysophospholipid
hydrolysis. This may allow the use of long reaction time and the
use of phospholipid with a high lyso-phospholipid content, e.g.
from cereals such as oats.
[0157] Baking
[0158] Lipolytic enzymes according to the invention have improved
baking performance, e.g. a lower dough stickiness, a better dough
extensibility and elasticity, a better dough stability, a better
crumb structure of the baked product, a larger loaf volume and/or
improved resistance to over-proofing or other abuse.
[0159] The invention provides a baking additive in the form of a
granulate, an agglomerated powder or a stabilized liquid,
comprising a lipolytic enzyme which:
[0160] a) has phospholipase activity, and
[0161] b) has a lysophospholipase to phospholipase ratio
corresponding to PLARN below 500 or RLPLA (0.1/2,5) below 1.0.
[0162] The baking additive may have a narrow particle size
distribution with more than 95% (by weight) of the particles in the
range from 25 to 500 .mu.m.
[0163] Granulates and agglomerated powders may be prepared by
conventional methods, e.g. by spraying the lipolytic enzyme onto a
carrier in a fluid-bed granulator. The carrier may consist of
particulate cores having a suitable particle size. The carrier may
be soluble or insoluble, e.g. a salt (such as NaCl or sodium
sulfate), a sugar (such as sucrose or lactose), a sugar alcohol
(such as sorbitol), starch, rice, corn grits, or soy. Liquid enzyme
preparations may, for instance, be stabilized by adding a polyol
such as propylene glycol, a sugar or sugar alcohol, lactic acid or
boric acid according to established methods.
[0164] The invention further relates to a pre-mix comprising flour
and the lipolytic enzyme described above. The pre-mix may contain
other dough-improving and/or bread-improving additives, e.g. any of
the additives, including enzymes, mentioned above.
[0165] The invention also provides a method of preparing a dough or
a baked product prepared from dough. The method may comprise
preparing a variant by the above method and adding it to the dough.
Alternatively, the method may comprise:
[0166] a) testing at least one lipolytic enzyme for its hydrolytic
activities towards intact phospholipid (PL) and lyso-phospholipid
(LPL),
[0167] b) selecting a lipolytic enzyme having a hydrolytic activity
ratio for LPL/PL corresponding to PLARN below 500, and
[0168] c) adding the selected lipolytic enzyme to the dough.
[0169] Dough
[0170] The dough generally comprises wheat meal or wheat flour
and/or other types of meal, flour or starch such as corn flour,
corn starch, rye meal, rye flour, oat flour, oat meal, soy flour,
sorghum meal, sorghum flour, rice starch, rice flour, potato meal,
potato flour or potato starch. The dough may be fresh, frozen or
par-baked. It may particularly be a leavened dough.
[0171] 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 oxidant such
as ascorbic acid, potassium bromate, potassium iodate,
azodicarbonamide (ADA) or ammonium persulfate; an amino acid such
as L-cysteine; a sugar; a salt such as sodium chloride, calcium
acetate, sodium sulfate or calcium sulfate.
[0172] The dough may comprise fat (triglyceride) such as granulated
fat or shortening, but the invention is particularly applicable to
a dough where less than 1% by weight of fat (triglyceride) is
added, and particularly to a dough which is made without addition
of fat.
[0173] The dough may further comprise an emulsifier such as mono-
or diglycerides, diacetyl tartaric acid esters of mono- or
diglycerides, sugar esters of fatty acids, polyglycerol esters of
fatty acids, lactic acid esters of monoglycerides, acetic acid
esters of monoglycerides, polyoxyethylene stearates, or
lysolecithin, but the invention is particularly applicable to a
dough which is made without addition of emulsifiers (other than
optionally phospholipid).
[0174] Baked Product
[0175] The process of the invention may be used for any kind of
baked product prepared from dough, either of a soft or a crisp
character, either of a white, light or dark type. Examples are
bread (in particular white, whole-meal or rye bread), typically in
the form of loaves or rolls, French baguette-type bread, pita
bread, tortillas, cakes, pancakes, biscuits, cookies, muffins, pie
crusts, crisp bread, steamed bread, pizza and the like.
[0176] Additional Enzyme
[0177] Optionally, an additional enzyme may be used together with
the lipolytic enzyme. The additional enzyme may be a second
lipolytic enzyme (e.g. as described in PCT/DK01/00472), an amylase,
particularly an anti-staling amylase, an amyloglucosidase, a
cyclodextrin glucanotransferase, or the additional enzyme may be a
peptidase, in particular an exopeptidase, a transglutaminase, a
cellulase, a hemicellulase, in particular a pentosanase such as
xylanase, a protease, a protein disulfide isomerase, e.g., a
protein disulfide isomerase as disclosed in WO 95/00636, a
glycosyltransferase, a branching enzyme (1,4-.alpha.-glucan
branching enzyme), a 4-.alpha.-glucanotransferase (dextrin
glycosyltransferase), a lactase (galactosidase), or an
oxidoreductase, e.g., a peroxidase, a laccase, a glucose oxidase, a
pyranose oxidase, a lipoxygenase, an L-amino acid oxidase or a
carbohydrate oxidase.
[0178] The amylase may be a fungal or bacterial alpha-amylase, e.g.
from Bacillus, particularly B. licheniformis or B.
amyloliquefaciens, or from Aspergillus, particularly A. oryzae, a
betaamylase, e.g. from plant (e.g. soy bean) or from microbial
sources (e.g. Bacillus). The amylase may be an anti-staling
amylase, as described in WO 99/53769, i.e. an amylase that is
effective in retarding the staling (crumb firming) of baked
products, particularly a maltogenic alpha-amylase, e.g. from
Bacillus stearothermophilus strain NCIB 11837.
[0179] Other Uses
[0180] The lipolytic enzyme variant may also be used in the
production of pasta and noodles in analogy with EP 1057415.
[0181] A lipolytic enzyme variant with phospholipase activity may
be used in cheese production as described in WO 00/54601.
EXAMPLES
Example 1
Variants Based on a T. Lanuginosus Lipase Variant
[0182] A prior-art variant of the T. lanuginosus lipase with
phospholipase activity was chosen as the starting point (parent
lipolytic enzyme), and variants were prepared by introducing
further amino acid alterations into the prior-art variant.
[0183] Experiment A
[0184] Activities of the new variants were determined with lecithin
and lysolecithin (pure 1-phosphatidyl choline, 1-lysolecithin) as
substrates by the methods described above. More specifically, 1.7
mL of the reaction mixture was shaken for between 15 and 90 min in
an Eppendorf tube shaken at 1300 rpm by an "Eppendorf Thermomixer
comfort". The enzyme was inactivated at 95 C for 5 min, and
centrifuged at 14000 rpm by Eppendorf centrifuge 5417R. The
liberated fatty acids were determined (relative to a control sample
where the enzyme was inactivated before it was added to the
substrate) by NEFA C test from Wako following the ACS-ACOD method
described for the NEFA-C test.
[0185] Three variants were found to have phospholipase activity and
a to have a lower ratio of lysophospholipase to phospholipase than
the prior-art variant.
[0186] R84W+G91A+D96F+E99K+G263Q+L264A+1265T+G266D+T267A+L269N
G91A+D96W+E99K+L227G+G263Q+L264A+1265T+G266D+T267A+L269N
R84W+G91A+D96F+E99K+G263Q+L264A+1265T+G266S+T267A+L269N
+270A+271G+272G+273F+274S
[0187] Experiment B
[0188] Activities of further variants were determined with lecithin
and lysolecithin (mixture of 1- and 2-lysolecithin) as substrates
at 0.1 and 2.5 mg/ml by the RLPLA method described above. All
variants were found to have a high activity on lysolecithin
compared to lecithin.
[0189] Variants having the following amino acid alterations
compared to SEQ ID NO: 14 were found to have phospholipase activity
and a to have a lower ratio of lysophospholipase to phospholipase
than the prior-art variant.
2 SPPCGRRP(-E) + Y21K + E99N + N101S + E239C + Q249R G91A + D96K +
E99K + G263Q + L264A + I265T + G266D + T267A + L269N Y21V + R84G +
G91A + D96F + E99K + G263Q + L264A + I265T + G266D + T267A + L269N
+ 270A + 271G + 272G + 273F + 274S V60G + D62W + R84W + G91A + D96F
+ E99K + G263Q + L264A + I265T + G266D + T267A + L269N V60A + D62S
+ G91A + D96W + E99K + W221R + G263Q + L264A + I265T + G266D +
T267A + L269N + 270A + 271G + 272G + 273F + 274S R84A + S85D + E87A
+ G91A + D96G + K98E + E99D G91A + D96W + E99K + P250N + G263Q +
L264A + I265T + G266D + T267A + L269N + 270A + 271G + 272G + 273F +
274S G91A + D96W + E99K + P256N + G263Q + L264A + I265T + G266D +
T267A + L269N + 270A + 271G + 272G + 273F + 274S R84W + G91A + D96W
+ E99K + Y261Q + G263Q + L264A + I265T + G266D + T267A + L269N +
270A + 271G + 272G + 273F + 274S G91A + D96W + E99K + P250L + P253Q
+ D254DEL + P257S + G263Q + L264A + I265T + G266D + T267A + L269N +
270A + 271G + 272G + 273F + 274S R84Y + G91A + D96F + E99K + G263Q
+ L264A + I265T + G266D + T267A + L269N R84S + G91A + D96F + E99K +
E129A + V203i + L206F + G263Q + L264A + I265T + G266D + T267A +
L269N
Example 2
Variants of F. Oxysporum Lipase/Phospholipase
[0190] Variants of the F. oxysporum lipase/phospholipase were
prepared, having the following amino acid alterations compared to
SEQ ID NO: 7:
[0191] H257W
[0192] S142A
[0193] V157D+S271 P
[0194] S80T+A127T
[0195] D263G
[0196] Y21W
[0197] S80T
[0198] The following variants with modified C-terminal sequences
were prepared by making the substitutions R274A and/or R284A to
remove one or two cleavage points for the Kex-2 protease:
[0199] R274A+275YRSAESVDKR
[0200] R274A+275YRSAESVDKAATMTDAELEKKLNSYVQMDKEYVKNNQARS
[0201] Activities of the variants and the parent enzyme were
determined as in Example 2. Compared to the parent enzyme, the
variants were found to have lower activity on lysolecithin and a
lower ratio of lysolecithin activity to lecithin activity.
Example 3
Preparation of Dough
[0202] Doughs were prepared from Meneba flour according to the
European straight dough method (ABF-SP 1201.1) with 40 ppm Fungamyl
Super MA (Novozymes), 40 ppm ascorbic acid, and different dosages
of lipolytic enzyme.
[0203] The stickiness of the dough was evaluated an a 1-10 scale by
the baker as the degree to which a dough adheres to ones hands or
other surfaces, where 5 is identical to a control without addition
of lipolytic enzyme, 1 is the lowest degree of stickiness and 10 is
the highest degree of stickiness.
[0204] The extensibility of the dough was evaluated an a 1-10 scale
by the baker as the degree to which a dough can be stretched
without tearing, where 5 is identical to a control without addition
of lipolytic enzyme, 1 indicates the lowest (shortest)
extensibility and 10 indicates the highest (longest)
extensibility
[0205] The elasticity of the dough was evaluated an a 1-10 scale by
the baker as the degree to which a dough tends to recover its
original shape after release from a deforming force, where 5 is
identical to a control without addition of lipolytic enzyme, 1
indicates the lowest (weakest) elasticity and 10 indicates the
highest (strongest) elasticity.
[0206] A lipolytic enzymes prepared in Example 1 was tested, and
the prior-art variant was tested for comparison. The results were
as follows
3 Experiment A Lipolytic PLARN Sticki- Extensi- Elasti- enzyme
Dosage ratio ness bility city Invention 0.2 mg/kg dough 690 5 6 4
Prior art 250 LU/kg dough 5800 5 7 3
[0207]
4 Experiment B Lipolytic RLPLA Sticki- Extensi- Elasti- enzyme
Dosage ratio ness bility city Invention 0.2 mg/kg flour 1.06 4 4 6
Prior art 250 LU/kg dough 1.58 5 5 5
[0208] The results show that lipolytic enzymes with a lower ratio
of lysolecithin activity to lecithin activity make doughs with a
desirable combination of lower extensibility and higher elasticity
than the prior-art lipolytic enzymes, and they furthermore tend to
make a less sticky dough.
Sequence CWU 1
1
14 1 265 PRT Absidia reflexa 1 Ser Ser Ser Ser Thr Gln Asp Tyr Arg
Ile Ala Ser Glu Ala Glu Ile 1 5 10 15 Lys Ala His Thr Phe Tyr Thr
Ala Leu Ser Ala Asn Ala Tyr Cys Arg 20 25 30 Thr Val Ile Pro Gly
Gly Arg Trp Ser Cys Pro His Cys Gly Val Ala 35 40 45 Ser Asn Leu
Gln Ile Thr Lys Thr Phe Ser Thr Leu Ile Thr Asp Thr 50 55 60 Asn
Val Leu Val Ala Val Gly Glu Lys Glu Lys Thr Ile Tyr Val Val 65 70
75 80 Phe Arg Gly Thr Ser Ser Ile Arg Asn Ala Ile Ala Asp Ile Val
Phe 85 90 95 Val Pro Val Asn Tyr Pro Pro Val Asn Gly Ala Lys Val
His Lys Gly 100 105 110 Phe Leu Asp Ser Tyr Asn Glu Val Gln Asp Lys
Leu Val Ala Glu Val 115 120 125 Lys Ala Gln Leu Asp Arg His Pro Gly
Tyr Lys Ile Val Val Thr Gly 130 135 140 His Ser Leu Gly Gly Ala Thr
Ala Val Leu Ser Ala Leu Asp Leu Tyr 145 150 155 160 His His Gly His
Ala Asn Ile Glu Ile Tyr Thr Gln Gly Gln Pro Arg 165 170 175 Ile Gly
Thr Pro Ala Phe Ala Asn Tyr Val Ile Gly Thr Lys Ile Pro 180 185 190
Tyr Gln Arg Leu Val His Glu Arg Asp Ile Val Pro His Leu Pro Pro 195
200 205 Gly Ala Phe Gly Phe Leu His Ala Gly Glu Glu Phe Trp Ile Met
Lys 210 215 220 Asp Ser Ser Leu Arg Val Cys Pro Asn Gly Ile Glu Thr
Asp Asn Cys 225 230 235 240 Ser Asn Ser Ile Val Pro Phe Thr Ser Val
Ile Asp His Leu Ser Tyr 245 250 255 Leu Asp Met Asn Thr Gly Leu Cys
Leu 260 265 2 264 PRT Absidia corymbifera 2 Ser Ser Ser Thr Gln Asp
Tyr Arg Ile Ala Ser Glu Ala Glu Ile Lys 1 5 10 15 Ala His Thr Phe
Tyr Thr Ala Leu Ser Ala Asn Ala Tyr Cys Arg Thr 20 25 30 Val Ile
Pro Gly Gly Gln Trp Ser Cys Pro His Cys Asp Val Ala Pro 35 40 45
Asn Leu Asn Ile Thr Lys Thr Phe Thr Thr Leu Ile Thr Asp Thr Asn 50
55 60 Val Leu Val Ala Val Gly Glu Asn Glu Lys Thr Ile Tyr Val Val
Phe 65 70 75 80 Arg Gly Thr Ser Ser Ile Arg Asn Ala Ile Ala Asp Ile
Val Phe Val 85 90 95 Pro Val Asn Tyr Pro Pro Val Asn Gly Ala Lys
Val His Lys Gly Phe 100 105 110 Leu Asp Ser Tyr Asn Glu Val Gln Asp
Lys Leu Val Ala Glu Val Lys 115 120 125 Ala Gln Leu Asp Arg His Pro
Gly Tyr Lys Ile Val Val Thr Gly His 130 135 140 Ser Leu Gly Gly Ala
Thr Ala Val Leu Ser Ala Leu Asp Leu Tyr His 145 150 155 160 His Gly
His Asp Asn Ile Glu Ile Tyr Thr Gln Gly Gln Pro Arg Ile 165 170 175
Gly Thr Pro Glu Phe Ala Asn Tyr Val Ile Gly Thr Lys Ile Pro Tyr 180
185 190 Gln Arg Leu Val Asn Glu Arg Asp Ile Val Pro His Leu Pro Pro
Gly 195 200 205 Ala Phe Gly Phe Leu His Ala Gly Glu Glu Phe Trp Ile
Met Lys Asp 210 215 220 Ser Ser Leu Arg Val Cys Pro Asn Gly Ile Glu
Thr Asp Asn Cys Ser 225 230 235 240 Asn Ser Ile Val Pro Phe Thr Ser
Val Ile Asp His Leu Ser Tyr Leu 245 250 255 Asp Met Asn Thr Gly Leu
Cys Leu 260 3 269 PRT Rhizomucor miehei 3 Ser Ile Asp Gly Gly Ile
Arg Ala Ala Thr Ser Gln Glu Ile Asn Glu 1 5 10 15 Leu Thr Tyr Tyr
Thr Thr Leu Ser Ala Asn Ser Tyr Cys Arg Thr Val 20 25 30 Ile Pro
Gly Ala Thr Trp Asp Cys Ile His Cys Asp Ala Thr Glu Asp 35 40 45
Leu Lys Ile Ile Lys Thr Trp Ser Thr Leu Ile Tyr Asp Thr Asn Ala 50
55 60 Met Val Ala Arg Gly Asp Ser Glu Lys Thr Ile Tyr Ile Val Phe
Arg 65 70 75 80 Gly Ser Ser Ser Ile Arg Asn Trp Ile Ala Asp Leu Thr
Phe Val Pro 85 90 95 Val Ser Tyr Pro Pro Val Ser Gly Thr Lys Val
His Lys Gly Phe Leu 100 105 110 Asp Ser Tyr Gly Glu Val Gln Asn Glu
Leu Val Ala Thr Val Leu Asp 115 120 125 Gln Phe Lys Gln Tyr Pro Ser
Tyr Lys Val Ala Val Thr Gly His Ser 130 135 140 Leu Gly Gly Ala Thr
Ala Leu Leu Cys Ala Leu Asp Leu Tyr Gln Arg 145 150 155 160 Glu Glu
Gly Leu Ser Ser Ser Asn Leu Phe Leu Tyr Thr Gln Gly Gln 165 170 175
Pro Arg Val Gly Asp Pro Ala Phe Ala Asn Tyr Val Val Ser Thr Gly 180
185 190 Ile Pro Tyr Arg Arg Thr Val Asn Glu Arg Asp Ile Val Pro His
Leu 195 200 205 Pro Pro Ala Ala Phe Gly Phe Leu His Ala Gly Glu Glu
Tyr Trp Ile 210 215 220 Thr Asp Asn Ser Pro Glu Thr Val Gln Val Cys
Thr Ser Asp Leu Glu 225 230 235 240 Thr Ser Asp Cys Ser Asn Ser Ile
Val Pro Phe Thr Ser Val Leu Asp 245 250 255 His Leu Ser Tyr Phe Gly
Ile Asn Thr Gly Leu Cys Thr 260 265 4 270 PRT Rhizopus oryzae 4 Ser
Ala Ser Asp Gly Gly Lys Val Val Ala Ala Thr Thr Ala Gln Ile 1 5 10
15 Gln Glu Phe Thr Lys Tyr Ala Gly Ile Ala Ala Thr Ala Tyr Cys Arg
20 25 30 Ser Val Val Pro Gly Asn Lys Trp Asp Cys Val Gln Cys Gln
Lys Trp 35 40 45 Val Pro Asp Gly Lys Ile Ile Thr Thr Phe Thr Ser
Leu Leu Ser Asp 50 55 60 Thr Asn Gly Tyr Val Leu Arg Asp Lys Gln
Lys Thr Ile Tyr Leu Val 65 70 75 80 Phe Arg Gly Thr Asn Ser Phe Arg
Ser Ala Ile Thr Asp Ile Val Phe 85 90 95 Asn Phe Ser Asp Tyr Lys
Pro Val Lys Gly Ala Lys Val His Ala Gly 100 105 110 Phe Leu Ser Ser
Tyr Glu Gln Val Val Asn Asp Tyr Phe Pro Val Val 115 120 125 Gln Glu
Gln Leu Thr Ala His Pro Thr Tyr Lys Val Ile Val Thr Gly 130 135 140
His Ser Leu Gly Gly Ala Gln Ala Leu Leu Ala Gly Met Asp Leu Tyr 145
150 155 160 Gln Arg Glu Pro Arg Leu Ser Pro Lys Asn Leu Ser Ile Phe
Thr Val 165 170 175 Gly Gly Pro Arg Val Gly Asn Pro Thr Phe Ala Tyr
Tyr Val Glu Ser 180 185 190 Thr Gly Ile Pro Phe Gln Arg Thr Val His
Lys Arg Asp Ile Val Pro 195 200 205 His Val Pro Pro Gln Ser Phe Gly
Phe Leu His Pro Gly Val Glu Ser 210 215 220 Trp Ile Lys Ser Gly Thr
Ser Asn Val Gln Ile Cys Thr Ser Glu Ile 225 230 235 240 Glu Thr Lys
Asp Cys Ser Asn Ser Ile Val Pro Phe Thr Ser Ile Leu 245 250 255 Asp
His Leu Ser Tyr Phe Asp Ile Asn Glu Gly Ser Cys Leu 260 265 270 5
267 PRT Aspergillus niger 5 Thr Ala Gly His Ala Leu Ala Ala Ser Thr
Gln Gly Ile Ser Glu Asp 1 5 10 15 Leu Tyr Ser Arg Leu Val Glu Met
Ala Thr Ile Ser Gln Ala Ala Tyr 20 25 30 Ala Asp Leu Cys Asn Ile
Pro Ser Thr Ile Ile Lys Gly Glu Lys Ile 35 40 45 Tyr Asn Ser Gln
Thr Asp Ile Asn Gly Trp Ile Leu Arg Asp Asp Ser 50 55 60 Ser Lys
Glu Ile Ile Thr Val Phe Arg Gly Thr Gly Ser Asp Thr Asn 65 70 75 80
Leu Gln Leu Asp Thr Asn Tyr Thr Leu Thr Pro Phe Asp Thr Leu Pro 85
90 95 Gln Cys Asn Gly Cys Glu Val His Gly Gly Tyr Tyr Ile Gly Trp
Val 100 105 110 Ser Val Gln Asp Gln Val Glu Ser Leu Val Lys Gln Gln
Val Ser Gln 115 120 125 Tyr Pro Asp Tyr Ala Leu Thr Val Thr Gly His
Ser Leu Gly Ala Ser 130 135 140 Leu Ala Ala Leu Thr Ala Ala Gln Leu
Ser Ala Thr Tyr Asp Asn Ile 145 150 155 160 Arg Leu Tyr Thr Phe Gly
Glu Pro Arg Ser Gly Asn Gln Ala Phe Ala 165 170 175 Ser Tyr Met Asn
Asp Ala Phe Gln Ala Ser Ser Pro Asp Thr Thr Gln 180 185 190 Tyr Phe
Arg Val Thr His Ala Asn Asp Gly Ile Pro Asn Leu Pro Pro 195 200 205
Val Glu Gln Gly Tyr Ala His Gly Gly Val Glu Tyr Trp Ser Val Asp 210
215 220 Pro Tyr Ser Ala Gln Asn Thr Phe Val Cys Thr Gly Asp Glu Val
Gln 225 230 235 240 Cys Cys Glu Ala Gln Gly Gly Gln Gly Val Asn Asn
Ala His Thr Thr 245 250 255 Tyr Phe Gly Met Thr Ser Gly Ala Cys Thr
Trp 260 265 6 312 PRT Aspergillus tubingensis 6 Thr Ala Gly His Ala
Leu Ala Ala Ser Thr Gln Gly Ile Ser Glu Asp 1 5 10 15 Leu Tyr Ser
Arg Leu Val Glu Met Ala Thr Ile Ser Gln Ala Ala Tyr 20 25 30 Ala
Asp Leu Cys Asn Ile Pro Ser Thr Ile Ile Lys Gly Glu Lys Ile 35 40
45 Tyr Asn Ser Gln Thr Asp Ile Asn Gly Trp Ile Leu Arg Tyr Cys Asn
50 55 60 Ser Glu Ala Ala Ala Gly Ser Lys Ile Thr Cys Ser Asn Asn
Gly Cys 65 70 75 80 Pro Thr Val Gln Gly Asn Gly Ala Thr Ile Val Thr
Ser Phe Val Gly 85 90 95 Ser Lys Thr Gly Ile Gly Gly Tyr Val Ala
Thr Asp Asp Ser Ser Lys 100 105 110 Glu Ile Ile Thr Val Phe Arg Gly
Thr Gly Ser Asp Thr Asn Leu Gln 115 120 125 Leu Asp Thr Asn Tyr Thr
Leu Thr Pro Phe Asp Thr Leu Pro Gln Cys 130 135 140 Asn Ser Cys Glu
Val His Gly Gly Tyr Tyr Ile Gly Trp Ile Ser Val 145 150 155 160 Gln
Asp Gln Val Glu Ser Leu Val Gln Gln Gln Val Ser Gln Phe Pro 165 170
175 Asp Tyr Ala Leu Thr Val Thr Gly His Ser Leu Gly Ala Ser Leu Ala
180 185 190 Ala Leu Thr Ala Ala Gln Leu Ser Ala Thr Tyr Asp Asn Ile
Arg Leu 195 200 205 Tyr Thr Phe Gly Glu Pro Arg Ser Asn Gln Ala Phe
Ala Ser Tyr Met 210 215 220 Asn Asp Ala Phe Gln Ala Ser Ser Pro Asp
Thr Thr Gln Tyr Phe Arg 225 230 235 240 Val Thr His Ala Asn Asp Gly
Ile Pro Asn Leu Pro Pro Ala Asp Glu 245 250 255 Gly Tyr Ala His Gly
Val Val Glu Tyr Trp Ser Val Asp Pro Tyr Ser 260 265 270 Ala Gln Asn
Thr Phe Val Cys Thr Gly Asp Glu Val Gln Cys Cys Glu 275 280 285 Ala
Gln Gly Gly Gln Gly Val Asn Asn Ala His Thr Thr Tyr Phe Gly 290 295
300 Met Thr Ser Gly His Cys Thr Trp 305 310 7 274 PRT Fusarium
oxysporum 7 Gly Val Thr Thr Thr Asp Phe Ser Asn Phe Lys Phe Tyr Ile
Gln His 1 5 10 15 Gly Ala Ala Ala Tyr Cys Asn Ser Glu Ala Ala Ala
Gly Ser Lys Ile 20 25 30 Thr Cys Ser Asn Asn Gly Cys Pro Thr Val
Gln Gly Asn Gly Ala Thr 35 40 45 Ile Val Thr Ser Phe Val Gly Ser
Lys Thr Gly Ile Gly Gly Tyr Val 50 55 60 Ala Thr Asp Ser Ala Arg
Lys Glu Ile Val Val Ser Phe Arg Gly Ser 65 70 75 80 Ile Asn Ile Arg
Asn Trp Leu Thr Asn Leu Asp Phe Gly Gln Glu Asp 85 90 95 Cys Ser
Leu Val Ser Gly Cys Gly Val His Ser Gly Phe Gln Arg Ala 100 105 110
Trp Asn Glu Ile Ser Ser Gln Ala Thr Ala Ala Val Ala Ser Ala Arg 115
120 125 Lys Ala Asn Pro Ser Phe Asn Val Ile Ser Thr Gly His Ser Leu
Gly 130 135 140 Gly Ala Val Ala Val Leu Ala Ala Ala Asn Leu Arg Val
Gly Gly Thr 145 150 155 160 Pro Val Asp Ile Tyr Thr Tyr Gly Ser Pro
Arg Val Gly Asn Ala Gln 165 170 175 Leu Ser Ala Phe Val Ser Asn Gln
Ala Gly Gly Glu Tyr Arg Val Thr 180 185 190 His Ala Asp Asp Pro Val
Pro Arg Leu Pro Pro Leu Ile Phe Gly Tyr 195 200 205 Arg His Thr Thr
Pro Glu Phe Trp Leu Ser Gly Gly Gly Gly Asp Lys 210 215 220 Val Asp
Tyr Thr Ile Ser Asp Val Lys Val Cys Glu Gly Ala Ala Asn 225 230 235
240 Leu Gly Cys Asn Gly Gly Thr Leu Gly Leu Asp Ile Ala Ala His Leu
245 250 255 His Tyr Phe Gln Ala Thr Asp Ala Cys Asn Ala Gly Gly Phe
Ser Trp 260 265 270 Arg Arg 8 273 PRT Fusarium heterosporum 8 Thr
Val Thr Thr Gln Asp Leu Ser Asn Phe Arg Phe Tyr Leu Gln His 1 5 10
15 Ala Asp Ala Ala Tyr Cys Asn Phe Asn Thr Ala Val Gly Lys Pro Val
20 25 30 His Cys Ser Ala Gly Asn Cys Pro Asp Ile Glu Lys Asp Ala
Ala Ile 35 40 45 Val Val Gly Ser Val Val Gly Thr Lys Thr Gly Ile
Gly Ala Tyr Val 50 55 60 Ala Thr Asp Asn Ala Arg Lys Glu Ile Val
Val Ser Val Arg Gly Ser 65 70 75 80 Ile Asn Val Arg Asn Trp Ile Thr
Asn Phe Asn Phe Gly Gln Lys Thr 85 90 95 Cys Asp Leu Val Ala Gly
Cys Gly Val His Thr Gly Phe Leu Asp Ala 100 105 110 Trp Glu Glu Val
Ala Ala Asn Val Lys Ala Ala Val Ser Ala Ala Lys 115 120 125 Thr Ala
Asn Pro Thr Phe Lys Phe Val Val Thr Gly His Ser Leu Gly 130 135 140
Gly Ala Val Ala Thr Ile Ala Ala Ala Tyr Leu Arg Lys Asp Gly Phe 145
150 155 160 Pro Phe Asp Leu Tyr Thr Tyr Gly Ser Pro Arg Val Gly Asn
Asp Phe 165 170 175 Phe Ala Asn Phe Val Thr Gln Gln Thr Gly Ala Glu
Tyr Arg Val Thr 180 185 190 His Gly Asp Asp Pro Val Pro Arg Leu Pro
Pro Ile Val Phe Gly Tyr 195 200 205 Arg His Thr Ser Pro Glu Tyr Trp
Leu Asn Gly Gly Pro Leu Asp Lys 210 215 220 Asp Tyr Thr Val Thr Glu
Ile Lys Val Cys Glu Gly Ile Ala Asn Val 225 230 235 240 Met Cys Asn
Gly Gly Thr Ile Gly Leu Asp Ile Leu Ala His Ile Thr 245 250 255 Tyr
Phe Gln Ser Met Ala Thr Cys Ala Pro Ile Ala Ile Pro Trp Lys 260 265
270 Arg 9 278 PRT Aspergillus oryzae 9 Asp Ile Pro Thr Thr Gln Leu
Glu Asp Phe Lys Phe Trp Val Gln Tyr 1 5 10 15 Ala Ala Ala Thr Tyr
Cys Pro Asn Asn Tyr Val Ala Lys Asp Gly Glu 20 25 30 Lys Leu Asn
Cys Ser Val Gly Asn Cys Pro Asp Val Glu Ala Ala Gly 35 40 45 Ser
Thr Val Lys Leu Ser Phe Ser Asp Asp Thr Ile Thr Asp Thr Ala 50 55
60 Gly Phe Val Ala Val Asp Asn Thr Asn Lys Ala Ile Val Val Ala Phe
65 70 75 80 Arg Gly Ser Tyr Ser Ile Arg Asn Trp Val Thr Asp Ala Thr
Phe Pro 85 90 95 Gln Thr Asp Pro Gly Leu Cys Asp Gly Cys Lys Ala
Glu Leu Gly Phe 100 105 110 Trp Thr Ala Trp Lys Val Val Arg Asp Arg
Ile Ile Lys Thr Leu Asp 115 120 125 Glu Leu Lys Pro Glu His Ser Asp
Tyr Lys Ile Val Val Val Gly His 130 135 140 Ser Leu Gly Ala Ala Ile
Ala Ser Leu Ala Ala Ala Asp Leu Arg Thr 145 150 155 160 Lys Asn Tyr
Asp Ala Ile Leu Tyr Ala Tyr Ala Ala Pro Arg Val Ala 165 170 175 Asn
Lys Pro Leu Ala Glu Phe Ile Thr Asn Gln Gly Asn Asn Tyr Arg 180 185
190 Phe Thr His Asn Asp Asp Pro Val Pro Lys Leu Pro Leu Leu Thr Met
195 200 205 Gly Tyr Val His Ile Ser Pro Glu Tyr Tyr Ile Thr Ala Pro
Asp Asn 210
215 220 Thr Thr Val Thr Asp Asn Gln Val Thr Val Leu Asp Gly Tyr Val
Asn 225 230 235 240 Phe Lys Gly Asn Thr Gly Thr Ser Gly Gly Leu Pro
Asp Leu Leu Ala 245 250 255 Phe His Ser His Val Trp Tyr Phe Ile His
Ala Asp Ala Cys Lys Gly 260 265 270 Pro Gly Leu Pro Leu Arg 275 10
278 PRT Penicillium camemberti 10 Asp Val Ser Thr Ser Glu Leu Asp
Gln Phe Glu Phe Trp Val Gln Tyr 1 5 10 15 Ala Ala Ala Ser Tyr Tyr
Glu Ala Asp Tyr Thr Ala Gln Val Gly Asp 20 25 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 50 55 60
Gly Tyr Ile Ala Val Asp His Thr Asn Ser Ala Val Val Leu Ala Phe 65
70 75 80 Arg Gly Ser Tyr Ser Val Arg Asn Trp Val Ala Asp Ala Thr
Phe Val 85 90 95 His Thr Asn Pro Gly Leu Cys Asp Gly Cys Leu Ala
Glu Leu Gly Phe 100 105 110 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 Gly His 130 135 140 Ser Leu Gly Ala Ala Val
Ala Thr Leu Ala Ala Thr Asp Leu Arg Gly 145 150 155 160 Lys Gly Tyr
Pro Ser Ala Lys Leu Tyr Ala Tyr Ala Ser Pro Arg Val 165 170 175 Gly
Asn Ala Ala Leu Ala Lys Tyr Ile Thr Ala Gln Gly Asn Asn Phe 180 185
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 210 215 220 Asn Ala Thr Val Ser Thr Ser Asp Ile Lys Val Ile
Asp Gly Asp Val 225 230 235 240 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 Gly Lys Gly Pro 260 265 270 Gly Leu Pro Phe Lys
Arg 275 11 270 PRT Aspergillus foetidus 11 Ser Val Ser Thr Ser Thr
Leu Asp Glu Leu Gln Leu Phe Ala Gln Trp 1 5 10 15 Ser Ala Ala Ala
Tyr Cys Ser Asn Asn Ile Asp Ser Lys Asp Ser Asn 20 25 30 Leu Thr
Cys Thr Ala Asn Ala Cys Pro Ser Val Glu Glu Ala Ser Thr 35 40 45
Thr Met Leu Leu Glu Phe Asp Leu Thr Asn Asp Phe Gly Gly Thr Ala 50
55 60 Gly Phe Leu Ala Ala Asp Asn Thr Asn Lys Arg Leu Val Val Ala
Phe 65 70 75 80 Arg Gly Ser Ser Thr Ile Glu Asn Trp Ile Ala Asn Leu
Asp Phe Ile 85 90 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 12 270 PRT Aspergillus
niger 12 Ser Val Ser Thr Ser Thr Leu Asp Glu Leu Gln Leu Phe Ser
Gln Trp 1 5 10 15 Ser Ala Ala Ala Tyr Cys Ser Asn Asn Ile Asp Ser
Asp Asp Ser Asn 20 25 30 Val Thr Cys Thr Ala Asp Ala Cys Pro Ser
Val Glu Glu Ala Ser Thr 35 40 45 Lys Met Leu Leu Glu Phe Asp Leu
Thr Asn Asn Phe Gly Gly Thr Ala 50 55 60 Gly Phe Leu Ala Ala Asp
Asn Thr Asn Lys Arg Leu Val Val Ala Phe 65 70 75 80 Arg Gly Ser Ser
Thr Ile Lys Asn Trp Ile Ala Asp Leu Asp Phe Ile 85 90 95 Leu Gln
Asp Asn Asp Asp Leu Cys Thr Gly Cys Lys Val His Thr Gly 100 105 110
Phe Trp Lys Ala Trp Glu Ala Ala Ala Asp Asn 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 Val 165 170 175 Gly Asn Tyr Ala Leu Ala Glu His
Ile Thr Ser Gln Gly Ser Gly Ala 180 185 190 Asn Phe Pro 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 Thr
Gly Ala Ser Val Thr Ala Ser Asp Ile Glu Leu Ile Glu Gly 225 230 235
240 Ile Asn Ser Thr Ala Gly Asn Ala Gly Glu Ala Thr Val Asp Val Leu
245 250 255 Ala His Leu Trp Tyr Phe Phe Ala Ile Ser Glu Cys Leu Leu
260 265 270 13 269 PRT Aspergillus oryzae 13 Asp Val Ser Ser Ser
Leu Leu Asn Asn Leu Asp Leu Phe Ala Gln Tyr 1 5 10 15 Ser Ala Ala
Ala Tyr Cys Asp Glu Asn Leu Asn Ser Thr Gly Thr Lys 20 25 30 Leu
Thr Cys Ser Val Gly Asn Cys Pro Leu Val Glu Ala Ala Ser Thr 35 40
45 Gln Ser Leu Asp Glu Phe Asn Glu Ser Ser Ser Tyr Gly Asn Pro Ala
50 55 60 Gly Tyr Leu Ala Ala Asp Glu Thr Asn Lys Leu Leu Val Leu
Ser Phe 65 70 75 80 Arg Gly Ser Ala Asp Leu Ala Asn Trp Val Ala Asn
Leu Asn Phe Gly 85 90 95 Leu Glu Asp Ala Ser Asp Leu Cys Ser Gly
Cys Glu Val His Ser Gly 100 105 110 Phe Trp Lys Ala Trp Ser Glu Ile
Ala Asp Thr Ile Thr Ser Lys Val 115 120 125 Glu Ser Ala Leu Ser Asp
His Ser Asp Tyr Ser Leu Val Leu Thr Gly 130 135 140 His Ser Tyr Gly
Ala Ala Leu Ala Ala Leu Ala Ala Thr Ala Leu Arg 145 150 155 160 Asn
Ser Gly His Ser Val Glu Leu Tyr Asn Tyr Gly Gln Pro Arg Leu 165 170
175 Gly Asn Glu Ala Leu Ala Thr Tyr Ile Thr Asp Gln Asn Lys Gly Gly
180 185 190 Asn Tyr Arg Val Thr His Thr Asn Asp Ile Val Pro Lys Leu
Pro Pro 195 200 205 Thr Leu Leu Gly Tyr His His Phe Ser Pro Glu Tyr
Tyr Ile Ser Ser 210 215 220 Ala Asp Glu Ala Thr Val Thr Thr Thr Asp
Val Thr Glu Val Thr Gly 225 230 235 240 Ile Asp Ala Thr Gly Gly Asn
Asp Gly Thr Asp Gly Thr Ser Ile Asp 245 250 255 Ala His Arg Trp Tyr
Phe Ile Tyr Ile Ser Glu Cys Ser 260 265 14 269 PRT Thermomyces
lanuginosus 14 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 Gly Asn Leu Asn Phe Asp 85 90 95 Leu
Lys Glu 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 Gly Leu Ile Gly Thr Cys Leu
260 265
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