U.S. patent application number 15/590867 was filed with the patent office on 2017-11-02 for enzymes.
This patent application is currently assigned to DuPont Nutrition Biosciences APS. The applicant listed for this patent is DuPont Nutrition Biosciences APS. Invention is credited to Lone Brond MILLER, Jens Frisb.ae butted.k SORENSEN.
Application Number | 20170314004 15/590867 |
Document ID | / |
Family ID | 47883847 |
Filed Date | 2017-11-02 |
United States Patent
Application |
20170314004 |
Kind Code |
A1 |
SORENSEN; Jens Frisb.ae butted.k ;
et al. |
November 2, 2017 |
ENZYMES
Abstract
The present invention relates to new enzymes with improved
properties and to compositions comprising these enzymes suitable
for use in the production of a food, feed, or malt beverage
product, such as in a brewing process.
Inventors: |
SORENSEN; Jens Frisb.ae
butted.k; (Arhus N, DK) ; MILLER; Lone Brond;
(Viby J, DK) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
DuPont Nutrition Biosciences APS |
Copenhagen |
|
DK |
|
|
Assignee: |
DuPont Nutrition Biosciences
APS
Copenhagen
DK
|
Family ID: |
47883847 |
Appl. No.: |
15/590867 |
Filed: |
May 9, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14344508 |
Mar 12, 2014 |
9683224 |
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PCT/EP2012/068041 |
Sep 14, 2012 |
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15590867 |
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61534574 |
Sep 14, 2011 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
Y02E 50/10 20130101;
C12N 9/2482 20130101; Y02E 50/17 20130101; C12P 7/06 20130101; C12N
9/24 20130101; C12C 12/002 20130101; D21H 17/005 20130101; C12N
9/2408 20130101; C12Y 302/01006 20130101; A23L 5/25 20160801; C12C
11/003 20130101; C12Y 302/01014 20130101; C12N 9/244 20130101; C12N
9/242 20130101; C12Y 302/01008 20130101 |
International
Class: |
C12N 9/24 20060101
C12N009/24; D21H 17/00 20060101 D21H017/00; C12P 7/06 20060101
C12P007/06; C12C 11/00 20060101 C12C011/00; C12N 9/30 20060101
C12N009/30; C12N 9/26 20060101 C12N009/26; C12N 9/24 20060101
C12N009/24; C12C 12/00 20060101 C12C012/00; C12N 9/42 20060101
C12N009/42; A23L 5/20 20060101 A23L005/20 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 14, 2011 |
EP |
11181241.8 |
Claims
1. An enzyme exhibiting endo-1,4-.beta.-xylanase activity, which
enzyme comprises an amino acid sequence having at least 80%
identity with any one selected from SEQ ID NO:1, SEQ ID NO:2, SEQ
ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:17, and
SEQ ID NO:18, or any functional fragment thereof.
2. The enzyme according to claim 1, which enzyme has a ratio in
activity on soluble arabinoxylan substrate (WE-AX) to insoluble
arabinoxylan substrate (WU-AX) arabinoxylan substrate of less than
about 7.0, such as less than about 6.5, such as less than about
6.0, such as less than about 5.5, such as less than about 5.0, such
as less than about 4.5.
3. An enzyme exhibiting endo-1,3(4)-.beta.-glucanase activity,
which enzyme comprises an amino acid sequence having at least 80%
identity with any one selected from SEQ ID NO:7, SEQ ID NO:8, SEQ
ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, SEQ ID NO:12, SEQ ID
NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, or any functional
fragment thereof.
4. The enzyme according to claim 1, which enzyme has a temperature
optimum in the range of 40-70.degree. C., such as in the range of
45-65.degree. C., such as in the range of 50-65.degree. C., such as
in the range of 55-65.degree. C.
5. The enzyme according to claim 4, wherein said enzyme has at
least 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,
96, 97, 98 or 99% identity with any one amino acid sequence
selected from SEQ ID NO: 1-18, or any functional fragment
thereof.
6. The enzyme according to claim 5 having a total number of amino
acids of less than 350, such as less than 340, such as less than
330, such as less than 320, such as less than 310, such as less
than 300 amino acids, such as in the range of 200 to 350, such as
in the range of 220 to 345 amino acids.
7. The enzyme according to claim 6, wherein the amino acid sequence
of said enzyme has at least one, two, three, four, five, six,
seven, eight, nine or ten amino acid substitutions as compared to
any one amino acid sequence selected from SEQ ID NO: 1-18, or any
functional fragment thereof.
8. The enzyme according to claim 7, wherein the amino acid sequence
of said enzyme has a maximum of one, two, three, four, five, six,
seven, eight, nine or ten amino acid substitutions compared to any
one amino acid sequence selected from SEQ ID NO: 1-18, or any
functional fragment thereof.
9. The enzyme according to claim 8, which enzyme comprises the
amino acid sequence identified by any one of SEQ ID NO: 1-18, or
any functional fragment thereof.
10. The enzyme according to claim 3, which enzyme consists of the
amino acid sequence identified by any one of SEQ ID NO: 1-18, or
any functional fragment thereof.
11. A DNA construct comprising a DNA sequence encoding an enzyme
according to claim 1.
12. A recombinant expression vector comprising a DNA construct
according to claim 11.
13. A cell that has been transformed with a DNA construct of claim
11.
14. A preparation comprising an enzyme according to claim 10.
15. A composition comprising an enzyme exhibiting
endo-1,4-.beta.-xylanase activity according to claim 1 in
combination with any one or more .beta.-glucanase.
16. The composition according to claim 15, wherein said one or more
.beta.-glucanase is an enzyme exhibiting
endo-1,3(4)-.beta.-glucanase activity according to any one of
claims 3-10.
17. A composition comprising an enzyme exhibiting
endo-1,3(4)-.beta.-glucanase activity according to claim 3 in
combination with any one or more xylanase.
18. The composition according to claim 17, wherein said one or more
xylanase is an enzyme exhibiting endo-1,4-.beta.-xylanase
activity.
19. The composition according to claim 18, wherein said
endo-1,3(4)-.beta.-glucanase activity and said
endo-1,4-.beta.-xylanase activity are derived from at least two
different enzymes, such as at least two different enzymes from two
different species.
20. The composition according to claim 19, comprising a combination
of at least two enzymes, said two enzymes, or two enzymes with an
amino acid sequence having at least 80% sequence identity with the
respective SEQ ID, or any functional fragment thereof, being
selected from the list consisting of SEQ ID NO:1 and SEQ ID NO:7;
SEQ ID NO:2 and SEQ ID NO:7; SEQ ID NO:3 and SEQ ID NO:7; SEQ ID
NO:4 and SEQ ID NO:7; SEQ ID NO:5 and SEQ ID NO:7; SEQ ID NO:6 and
SEQ ID NO:7; SEQ ID NO:17 and SEQ ID NO:7; SEQ ID NO:18 and SEQ ID
NO:7; SEQ ID NO:1 and SEQ ID NO:8; SEQ ID NO:2 and SEQ ID NO:8; SEQ
ID NO:3 and SEQ ID NO:8; SEQ ID NO:4 and SEQ ID NO:8; SEQ ID NO:5
and SEQ ID NO:8; SEQ ID NO:6 and SEQ ID NO:8; SEQ ID NO:17 and SEQ
ID NO:8; SEQ ID NO:18 and SEQ ID NO:8; SEQ ID NO:1 and SEQ ID NO:9;
SEQ ID NO:2 and SEQ ID NO:9; SEQ ID NO:3 and SEQ ID NO:9; SEQ ID
NO:4 and SEQ ID NO:9; SEQ ID NO:5 and SEQ ID NO:9; SEQ ID NO:6 and
SEQ ID NO:9; SEQ ID NO:17 and SEQ ID NO:9; SEQ ID NO:18 and SEQ ID
NO:9; SEQ ID NO:1 and SEQ ID NO:10; SEQ ID NO:2 and SEQ ID NO:10;
SEQ ID NO:3 and SEQ ID NO:10; SEQ ID NO:4 and SEQ ID NO:10; SEQ ID
NO:5 and SEQ ID NO:10; SEQ ID NO:6 and SEQ ID NO:10; SEQ ID NO:17
and SEQ ID NO:10; SEQ ID NO:18 and SEQ ID NO:10; SEQ ID NO:1 and
SEQ ID NO:11; SEQ ID NO:2 and SEQ ID NO:11; SEQ ID NO:3 and SEQ ID
NO:11; SEQ ID NO:4 and SEQ ID NO:11; SEQ ID NO:5 and SEQ ID NO:11;
SEQ ID NO:6 and SEQ ID NO:11; SEQ ID NO:17 and SEQ ID NO:11; SEQ ID
NO:18 and SEQ ID NO:11; SEQ ID NO:1 and SEQ ID NO:12; SEQ ID NO:2
and SEQ ID NO:12; SEQ ID NO:3 and SEQ ID NO:12; SEQ ID NO:4 and SEQ
ID NO:12; SEQ ID NO:5 and SEQ ID NO:12; SEQ ID NO:6 and SEQ ID
NO:12; SEQ ID NO:17 and SEQ ID NO:12; SEQ ID NO:18 and SEQ ID
NO:12; SEQ ID NO:1 and SEQ ID NO:13; SEQ ID NO:2 and SEQ ID NO:13;
SEQ ID NO:3 and SEQ ID NO:13; SEQ ID NO:4 and SEQ ID NO:13; SEQ ID
NO:5 and SEQ ID NO:13; SEQ ID NO:6 and SEQ ID NO:13; SEQ ID NO:17
and SEQ ID NO:13; SEQ ID NO:18 and SEQ ID NO:13; SEQ ID NO:1 and
SEQ ID NO:14; SEQ ID NO:2 and SEQ ID NO:14; SEQ ID NO:3 and SEQ ID
NO:14; SEQ ID NO:4 and SEQ ID NO:14; SEQ ID NO:5 and SEQ ID NO:14;
SEQ ID NO:6 and SEQ ID NO:14; SEQ ID NO:17 and SEQ ID NO:14; SEQ ID
NO:18 and SEQ ID NO:14; SEQ ID NO:1 and SEQ ID NO:15; SEQ ID NO:2
and SEQ ID NO:15; SEQ ID NO:3 and SEQ ID NO:15; SEQ ID NO:4 and SEQ
ID NO:15; SEQ ID NO:5 and SEQ ID NO:15; SEQ ID NO:6 and SEQ ID
NO:15; SEQ ID NO:17 and SEQ ID NO:15; SEQ ID NO:18 and SEQ ID
NO:15; SEQ ID NO:1 and SEQ ID NO:16; SEQ ID NO:2 and SEQ ID NO:16;
SEQ ID NO:3 and SEQ ID NO:16; SEQ ID NO:4 and SEQ ID NO:16; SEQ ID
NO:5 and SEQ ID NO:16; SEQ ID NO:6 and SEQ ID NO:16; SEQ ID NO:17
and SEQ ID NO:16; and SEQ ID NO:18 and SEQ ID NO:16.
21. The composition according to claim 20, wherein when used prior
to the lautering in a brewing application the total pressure built
up is reduced to a value of less than 470 mm WC, such as less than
450 mm WC, such as less than 430 mm WC, such as less than 410 mm
WC, such as less than 390 mm WC, such as less than 370 mm WC, such
as less than 350 mm WC, such as less than 330 mm WC, such as less
than 310 mm WC, such as less than 300 mm WC, such as less than 290
mm WC.
22. The composition according to claim 21, wherein when used prior
to the lautering in a brewing application the total pressure built
up is reduced by at least 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25,
27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59,
61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93
or 95% compared to the use of a negative control without said
composition.
23. The composition according to claim 22, wherein when used in a
brewing application prior to the wort separation, the wort
filterability as measured by volume wort collected after 5 min of
filtration relative to a control without enzymes is increased to
above 1.5, such as above 1.6, such as above 1.7, such as above 1.8,
such as above 1.9, such as above 2.0, such as above 2.1, such as
above 2.2, such as above 2.3, such as above 2.4, such as above
2.5.
24. The composition according to claim 23, when used in a brewing
application prior to the wort separation, the wort filterability as
measured by volume wort collected after 5 min of filtration is
increased by at least 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27,
29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61,
63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95,
97, 99, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220,
230, 240, 250, 260, 270, 280, 290, or 300% as compared to the use
of a negative control without said composition.
25. The composition according to claim 24, comprising any one or
more further enzyme.
26. The composition according to claim 25, wherein said one or more
further enzyme is selected from list consisting of a xylanase
classified in EC 3.2.1.32, EC 3.2.1.136, or EC 3.2.1.156, a
cellulase, a laminarinase, an endo-1,5-.alpha.-L-arabinanase, a
beta-D-glucoside glucohydrolase, a .beta.-Xylosidase, a
cellobiohydrolase, a glucan 1,4-beta-glucosidase, a
xyloglucan-specific exo-beta-1,4-glucanase and an
.alpha.-N-Arabinofuranosidase.
27. Use of an enzyme according to claim 1 in the production of a
food, feed, or malt beverage product.
28. Use of an enzyme according to claim 1 in the production of
dough or baked products.
29. Use of an enzyme according to claim 1 in the preparation of
pulp or paper.
30. Use of an enzyme according to claim 1 for the preparation of
cereal components.
31. The use according to claim 30, in which the cereal is rye,
wheat, or barley.
32. Use of an enzyme according to claim 1 in the production of beer
or modification of by-products from a brewing process.
33. Use of an enzyme according to claim 1 in the production of wine
or juice.
34. Use of an enzyme according to claim 1 in the production of a
first- or second-generation biofuel, such as bioethanol.
35. Method of altering filterability of a starch comprising
material, said method comprising the step of treating said starch
comprising material with enzyme according to claim 1.
36. Method of reducing pressure built up during lautering in a
brewing application, said method comprising the step of treating a
brewing mash with enzyme according to claim 1.
37. Method for the production of a food, feed, or beverage product,
such as an alcoholic or non-alcoholic beverage, such as a cereal-
or malt-based beverage like beer or whiskey, said method comprising
the step of treating a starch comprising material with enzyme
according to claim 1.
38. Method for the production of a brewing mash, said method
comprising the step of treating a starch comprising material with
enzyme according to claim 1.
39. Method for the production of a first- or second-generation
biofuel, such as bioethanol, said method comprising the step of
treating a starch comprising material with an enzyme according to
claim 1.
40. Product obtained by the method according to claim 39.
41. A composition comprising the product according to claim 40,
such as wherein the product is in a range of 0.1%-99.9%.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to enzymes with improved
properties and to compositions comprising these enzymes suitable
for use in the production of a food, beverage (e.g. beer), feed, or
biofuel, such as in a brewing process.
BACKGROUND OF THE INVENTION
[0002] The use of enzymes in beer production is well known.
Application of enzymes to the mashing step to improve mash
filterability and increase extract yield is described in WO
97/42302.
[0003] WO2005118769 and WO2005059084 relates to a mashing and
filtration step in a process for the production of beer, and to
enzymatic compositions for use in such a process.
[0004] WO1999057325 relates to strains of Penicillium funiculosum,
to new enzyme mixtures obtained from it and nucleic sequences
thereto.
[0005] However, there is a need for improved enzymes as well as
combination of enzymes useful in the productions of food and
beverage products, such as in the mashing, cooking and filtration
steps in the production of an alcoholic beverage, such as beer or
whiskey.
OBJECT OF THE INVENTION
[0006] It is an object of embodiments of the invention to provide
enzymes suitable for the production of food and beverage products,
such as in the production of an alcoholic or non-alcoholic
beverage, such as a cereal- or malt-based beverage like beer or
whiskey. The enzymes provided may have improved properties in
relation to the use in brewing. These wide varieties of improved
properties comprise e.g. improved temperature optimums, improved
ratio in activity on soluble (WE-AX) to insoluble (WU-AX)
arabinoxylan substrates, reduced total pressure built up during
lautering and/or filtration steps of a brewing process, as well as
increased filterability of enzyme treated material.
SUMMARY OF THE INVENTION
[0007] It has been found by the present inventor(s) that one or
more enzyme as well as certain combinations of enzymes have
improved properties relative to known enzymes and enzyme
combinations, particularly in relation to the use in a process of
brewing, wherein starch containing material is treated with the one
or more enzyme to produce a brewing mash.
[0008] So, in a first aspect the present invention relates to an
enzyme exhibiting endo-1,4-.beta.-xylanase activity, which enzyme
comprises an amino acid sequence having at least 80% identity with
any one selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID
NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:17, and SEQ ID NO:18, or
any functional fragment thereof.
[0009] As used herein "functional fragment" refers to a truncated
version of an enzyme with essentially the same or at least a
significant degree of enzyme activity as the non-truncated
reference enzyme.
[0010] In a second aspect, the present invention relates to an
enzyme exhibiting endo-1,3(4)-.beta.-glucanase activity, which
enzyme comprises an amino acid sequence having at least 80%
identity with any one selected from SEQ ID NO:7, SEQ ID NO:8, SEQ
ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, SEQ ID NO:12, SEQ ID
NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, or any functional
fragment thereof.
[0011] In a third aspect the present invention relates to a DNA
construct comprising a DNA sequence encoding an enzyme according to
the invention.
[0012] In a further aspect the present invention relates to a
recombinant expression vector comprising a DNA construct comprising
a DNA sequence encoding an enzyme according to the invention.
[0013] In a further aspect the present invention relates to a cell
that has been transformed with a DNA construct comprising a DNA
sequence encoding an enzyme according to the invention.
[0014] In a further aspect the present invention relates to
preparation comprising an enzyme, or a DNA construct, or a vector,
or a cell according to the invention.
[0015] In a further aspect the present invention relates to
composition comprising an enzyme exhibiting
endo-1,4-.beta.-xylanase activity according to the invention in
combination with any one or more .beta.-glucanase.
[0016] In a further aspect the present invention relates to a
composition comprising an enzyme exhibiting
endo-1,3(4)-.beta.-glucanase activity according to the invention in
combination with any one or more xylanase.
[0017] In a further aspect the present invention relates to the use
of an enzyme according to the invention, or a preparation according
to the invention, or a composition according to the invention in
the production of a food, feed, or malt beverage product, such as
beer or whiskey.
[0018] In a further aspect the present invention relates to the use
of an enzyme according to the invention, or a preparation according
to the invention, or a composition according to the invention, in
the production of dough or baked products.
[0019] In a further aspect the present invention relates to the use
of an enzyme according to the invention, or a preparation according
to the invention, or a composition according to the invention, in
the preparation of pulp or paper.
[0020] In a further aspect the present invention relates to the use
of an enzyme according to the invention, or a preparation according
to the invention, or a composition according to the invention, for
the preparation of cereal components. In some embodiments the
cereal is rye, wheat, or barley.
[0021] In a further aspect the present invention relates to the use
of enzyme according to the invention, or a preparation according to
the invention, or a composition according to the invention, in the
production of beer or modification of by-products from a brewing
process.
[0022] In a further aspect the present invention relates to the use
of enzyme according to the invention, or a preparation according to
the invention, or a composition according to the invention, in the
production of wine or juice.
[0023] In a further aspect the present invention relates to the use
of enzyme according to the invention, or a preparation according to
the invention, or a composition according to the invention, in the
production of a first- or second-generation biofuel, such as
bioethanol.
[0024] In a further aspect the present invention relates to a
method of altering filterability of a starch comprising material,
said method comprising the step of treating said starch comprising
material with enzyme, or a preparation, or a composition according
to the invention.
[0025] In a further aspect the present invention relates to a
method of reducing pressure built up during lautering in a brewing
application, said method comprising the step of treating a brewing
mash with enzyme, or a preparation, or a composition according to
the invention.
[0026] In a further aspect the present invention relates to a
method for the production of a food, feed, or beverage product,
such as an alcoholic or non-alcoholic beverage, such as a cereal-
or malt-based beverage like beer or whiskey, said method comprising
the step of treating a starch comprising material with enzyme, or a
preparation, or a composition according to the invention.
[0027] In a further aspect the present invention relates to a
method for the production of a brewing mash, said method comprising
the step of treating a starch comprising material with an enzyme,
or a preparation, or a composition according to the invention.
[0028] In a further aspect the present invention relates to a
method for the production of a first- or second-generation biofuel,
such as bioethanol, said method comprising the step of treating a
starch comprising material with an enzyme, or a preparation, or a
composition according to the invention.
[0029] In a further aspect the present invention relates to a
product obtained by a method according to the invention.
[0030] In a further aspect the present invention relates to a
composition comprising the product obtained by a method according
to the invention, such as wherein the product is in a range of
0.1%-99.9%.
LEGENDS TO THE FIGURE
[0031] FIG. 1: Mashing profile used in lab scale and pilot scale
brewing. Mashing was initiated by a 10 min mashing-in period after
which the enzyme was added.
[0032] FIG. 2: Pilot scale Brewing application results from
verification of the glucanase and xylanases screening. The B. sub
glucanase S combined with the A. tub xylanases was tested against a
blank and UltraFlo max. Data collected was the average flow (L/h),
the total pressure build up over the lautering (mm WC, where 1 mm
WC=9.80665 Pa) and the max pressure recorded during the lautering
(mm WC).
[0033] FIG. 3: Xylanase functionality in brewing
[0034] FIG. 4: Flow--lautering applying various xylanase
candidates
[0035] FIG. 5: Beer filtration--average of repeated filtrations
[0036] FIG. 6: Beer filtration--average of repeated filtrations
[0037] FIG. 7: Mashing diagram of example 3.
DETAILED DISCLOSURE OF THE INVENTION
[0038] Beer is traditionally referred to as an alcoholic beverage
derived from malt, such as malt derived from barley grain, and
optionally adjunct, such as starch containing plant material (e.g.
cereal grains) and optionally flavoured, e.g. with hops.
[0039] In the context of the present invention, the term "beer" is
meant to comprise any fermented wort, produced by
fermentation/brewing of a starch-containing plant material, thus in
particular also beer produced exclusively from adjunct, or any
combination of malt and adjunct.
[0040] The term "fermentation" means in the present context
production of a substance such as ethanol by growing microorganisms
in a culture. Commonly, microorganisms such as yeast are used for
fermentation.
[0041] As used herein the term "malt" is understood as any malted
cereal grain, such as malted barley. "Adjunct" can be defined as
any starch-containing plant material which is not malt or barley
malt.
[0042] "Starch-containing plant material" can e.g. be one or more
cereal, such as barley, wheat, maize, rye, sorghum, millet, or
rice, and any combination thereof. The starch-containing plant
material can be processed, e.g. milled, malted, partially malted or
unmalted. Unmalted cereal is also called "raw grain". Examples of
non-cereal starch-containing plant material comprise e.g. tubers,
such as potatoes and cassava.
[0043] As used herein, the terms "beverage" and "beverage(s)
product" includes such foam forming fermented beverages as full
malted beer, beer brewed under the "Reinheitsgebot", ale, dry beer,
near beer, light beer, low alcohol beer, low calorie beer, porter,
bock beer, stout, malt liquor, non-alcoholic beer, non-alcoholic
malt liquor and the like. The term "beverages" or "beverages
product" also includes non-foaming beer and alternative malt
beverages such as fruit flavoured malt beverages, e. g., citrus
flavoured, such as lemon-, orange-, lime-, or berry-flavoured malt
beverages, liquor flavoured malt beverages, e. g., vodka-, rum-, or
tequila-flavoured malt liquor, or coffee flavoured malt beverages,
such as caffeine-flavoured malt liquor, and the like.
[0044] Beer can be made from a variety of starch-containing plant
material by essentially the same process, where the starch is
consists mainly of glucose homopolymers in which the glucose
residues are linked by either alpha-1, 4- or alpha-1,6-bonds, with
the former predominating.
[0045] The process of making fermented beverages such as beer is
commonly referred to as brewing. The traditional raw materials used
in making these beverages are water, hops and malt. In addition or
instead of malt, adjuncts such as common corn grits, refined corn
grits, brewer's milled yeast, rice, sorghum, refined corn starch,
barley, barley starch, dehusked barley, wheat, wheat starch,
torrified cereal, cereal flakes, rye, oats, potato, tapioca, and
syrups, such as corn syrup, sugar cane syrup, inverted sugar syrup,
barley and/or wheat syrups, and the like may be used as a source of
starch. The starch will eventually be converted enzymatically into
fermentable sugars.
[0046] Concerning beers made predominantly from malt (e.g. up to
15-20% adjunct), for a number of reasons, the malt, which is
produced principally from selected varieties of barley, has the
greatest effect on the overall character and quality of the beer.
First, the malt is the primary flavouring agent in beer. Second,
the malt provides the major portion of the fermentable sugar.
Third, the malt provides the proteins, which will contribute to the
body and foam character of the beer. Fourth, the malt provides the
necessary enzymatic activity during mashing.
[0047] Hops also contribute significantly to beer quality,
including flavouring. In particular, hops (or hops constituents)
add desirable bittering substances to the beer. In addition, the
hops act as protein precipitants, establish preservative agents and
aid in foam formation and stabilization. Not all beers are produced
using hops. Other stabilizing agents, such as proteases (e.g.
papain) may also be used.
[0048] Without Wanting to be Construed as Limiting for the Present
Invention, a Conventional Brewing Process can be Described as
Follows:
[0049] The process for making beer is well known in the art, but
briefly, it involves five steps: (a) mashing and/or adjunct cooking
(b) wort separation and extraction (c) boiling and hopping of wort
(d) cooling, fermentation and storage, and (e) maturation,
processing and packaging. Typically, in the first step, milled or
crushed malt is mixed with water and held for a period of time
under controlled temperatures to permit the enzymes present in the
malt to convert the starch present in the malt into fermentable
sugars.
[0050] In the second step, the mash is transferred to a "lauter
tun" or mash filter where the liquid is separated from the grain
residue. This sweet liquid is called "wort" and the left over grain
residue is called "spent grain". The mash is typically subjected to
an extraction, which involves adding water to the mash in order to
recover the residual soluble extract from the spent grain.
[0051] In the third step, the wort is boiled vigorously. This
sterilizes the wort and helps to develop the colour, flavour and
odour. Hops are added at some point during the boiling.
[0052] In the fourth step, the wort is cooled and transferred to a
fermentor, which either contains the yeast or to which yeast is
added. The yeast converts the sugars by fermentation into alcohol
and carbon dioxide gas; at the end of fermentation the fermentor is
chilled or the fermentor may be chilled to stop fermentation. The
yeast flocculates and is removed.
[0053] In the last step, the beer is cooled and stored for a period
of time, during which the beer clarifies and its flavour develops,
and any material that might impair the appearance, flavour and
shelf life of the beer settles out. Prior to packaging, the beer is
carbonated and, optionally, filtered and pasteurized.
[0054] After fermentation, a beverage is obtained which usually
contains from about 2% to about 10% alcohol by weight. The
non-fermentable carbohydrates are not converted during fermentation
and form the majority of the dissolved solids in the final
beer.
[0055] This residue remains because of the inability of malt
amylases to hydrolyze the alpha-1,6-linkages of the starch. The
non-fermentable carbohydrates contribute about 50 calories per 12
ounces of beer.
[0056] Recently, there has been a widespread popularization of
brewed beverages called light beers, reduced calorie beers or low
calorie beers, particularly in the U. S. market. As defined in the
U. S., these beers have approximately 30% fewer calories than a
manufacturer's "normal" beer.
[0057] Further information on conventional brewing processes, as
well as definitions for terms used in the field of brewing
technology to be applied for the present invention, may be found in
"Technology Brewing and Malting" by Wolfgang Kunze of the Research
and Teaching Institute of Brewing, Berlin (VLB), 2nd revised
Edition 1999, ISBN 3-921690-39-0, 3rd edition (2004): ISBN
3-921690-49-8, 4.sup.th updated edition, 2010 (ISBN
978-3-921690-64-2).
[0058] Xylanases are classified in EC 3.2.1.8, EC 3.2.1.32, EC
3.2.1.136 and EC 3.2.1.156; their activity may be measured e.g. as
described in the examples. Suitable xylanases to be used in
combination with an enzyme exhibiting endo-1,3(4)-.beta.-glucanase
activity according to the invention includes any xylanase
classified in EC 3.2.1.8, EC 3.2.1.32, EC 3.2.1.136 and EC
3.2.1.156, such as any one disclosed in WO 2010072226, WO
2010072225, WO 2010072224, WO 2005059084, WO2007056321,
WO2008023060A, WO9421785, WO2006114095, WO2006066582, US
2008233175, and WO10059424.
[0059] Endo-1,4-beta xylanase is classified as EC 3.2.1.8. The
enzyme causes endohydrolysis of 1,4-beta-D-xylosidic linkages in
xylans.
[0060] The terms "family 11 xylanase", "Glycoside hydrolase (GH)
family 11" or simply "GH 11 xylanase" as used herein refers to an
endo-1,4-beta xylanase classified as EC 3.2.1.8, which causes
endohydrolysis of 1,4-beta-D-xylosidic linkages in xylans and which
is classified as a family 11 xylanase according to B. Henrissat, A
classification of glycosyl hydrolases based on amino acid sequence
similarities. Biochem. J. 280 (1991), pp. 309-316.
[0061] The terms "Family 10 xylanase", "Glycoside hydrolase (GH)
family 10", or simply "GH 10 xylanase" comprises enzymes with a
number of known activities, such as xylanase (EC:3.2.1.8);
endo-1,3-beta-xylanase (EC:3.2.1.32); cellobiohydrolase
(EC:3.2.1.91). These enzymes were formerly known as cellulase
family F.
[0062] In some embodiments the enzyme exhibiting
endo-1,4-.beta.-xylanase activity is a family 11 xylanase. In some
embodiments the enzyme exhibiting endo-1,4-.beta.-xylanase activity
is a family 10 xylanase.
[0063] In one aspect, the enzyme composition according to the
invention has endo-1,4-beta xylanase activity as measured by the
assay described in the examples.
[0064] An assay for measuring xylanase activity may be carried out
at pH 3.5 or pH 5 and 50.degree. C. using xylan as substrate, or it
can be performed at different pH and temperature values for the
additional characterisation and specification of enzymes. Enzyme
activity is calculated from the increase in absorbance caused by
xylose at 540 nm per unit time.
[0065] In some embodiments the enzyme composition according to the
invention comprises a xylanase activity of at least about 5000 U/g,
such as at least about 6000 U/g, such as at least about 7000 U/g,
such as at least about 8000 U/g, such as at least about 8500 U/g,
as measured by in the assay described in the examples.
[0066] The enzyme composition according to the invention may have
cellulolytic activity. The systematic name of cellulose is
4-(1,3;1,4)-.beta.-D-glucan 4-glucanohydrolase and cellulolytic
enzymes or cellulases are classified in EC 3.2.1.4. Cellulase
endohydrolyse (1.fwdarw.4)-.beta.-D-glucosidic linkages in e.g.
cellulose, lichenin and cereal .beta.-D-glucans and will also
hydrolyse 1,4-linkages in .beta.-D-glucans also containing
1,3-linkages. Cellulase also have other names such as
endo-1,4-.beta.-D-glucanase, .beta.-1,4-glucanase,
.beta.-1,4-endoglucan hydrolase, cellulase A, cellulosin AP,
endoglucanase D, alkali cellulose, cellulase A 3, celludextrinase,
9.5 cellulase, avicelase, pancellase SS and
1,4-(1,3;1,4)-.beta.-D-glucan 4-glucanohydrolase.
[0067] In one aspect of the invention, the cellulase activity of
the enzyme composition according to the invention is measured by
the "Cellulase activity method" as described in the following under
the heading "Assays".
[0068] In further aspects, the present invention relates to enzymes
having endo-1,3(4)-.beta.-glucanase activity is determined by the
assay described in the examples.
[0069] ".beta.-glucanase" or "beta-glucanase" as used herein refers
to an endo-1,3(4)-beta-glucanase of EC 3.2.1.6. Catalyze the
endohydrolysis of (1->3)- or (1->4)-linkages in
beta-D-glucans when the glucose residue whose reducing group is
involved in the linkage to be hydrolyzed is itself substituted at
C-3. Suitable beta-glucanases to be used in combination with an
enzyme exhibiting endo-1,4-.beta.-xylanase activity according to
the invention includes any one beta-glucanase disclosed in
WO2004087889, WO2005059084, WO9414953, WO2007056321, WO9531533,
WO08023060, WO2005100582, WO9828410, WO9742301, WO2006066582,
WO05118769, WO2005003319, and WO10059424.
[0070] The standard assay is carried out at pH 5.0, and it can be
performed at different pH values for the additional
characterisation and specification of enzymes.
[0071] One unit of endo-1,3(4)-.beta.-glucanase activity is defined
as the amount of enzyme which produces 1 .mu.mole glucose
equivalents per minute under the conditions of the assay (pH 5.0
(or as specified) and 50.degree. C.).
[0072] In some embodiments the enzyme composition according to the
invention comprises a .beta.-glucanase activity of at least about
10000 U/g, such as at least about 12000 U/g, such as at least about
14000 U/g, such as at least about 15000 U/g, such as at least about
18000 U/g as measured by the assay described in the examples.
[0073] In further aspects, the enzyme composition according to the
invention has laminarinase activity or comprises any one or more
further enzyme having laminarinase activity. The laminarinase
activity is determined as described in the laminarase assay
described in the Assay section.
[0074] Laminarinase may be an endo-1,3(4)-beta-glucanase classified
in E.C. 3.2.1.6 or glucan endo-1,3-beta-D-glucosidase classified in
E.C. 3.2.1.39. Endo-1,3(4)-beta-glucanase with the alternative
names, laminarinase, endo-1,3-beta-glucanase,
Endo-1,4-beta-glucanase is classified in E.C. 3.2.1.6. The
substrates include laminarin, lichenin and cereal D-glucans and the
enzyme catalyse endohydrolysis of (1->3)- or (1->4)-linkages
in beta-D-glucans when the glucose residue whose reducing group is
involved in the linkage to be hydrolyzed is itself substituted at
C-3. Glucan endo-1,3-beta-D-glucosidase with the alternative names
(1->3)-beta-glucan endohydrolase, Endo-1,3-beta-glucanase and
laminarinase is classified in E.C. 3.2.1.39 and hydrolyse
(1->3)-beta-D-glucosidic linkages in (1->3)-beta-D-glucans in
substrates as eg. laminarin, paramylon and pachyman.
[0075] In some aspects, the enzyme composition according to the
invention has arabinanase activity or comprises a further enzyme
having arabinanase activity. Arabinanase is classified as EC
3.2.1.99. The systematic name is 5-.alpha.-L-arabinan
5-.alpha.-L-arabinanohydrolase but it has several other names such
as arabinan endo-1,5-.alpha.-L-arabinosidase, and
endo-1,5-.alpha.-L-arabinanase, endo-.alpha.-1,5-arabanase,
endo-arabanase, 1,5-.alpha.-L-arabinan and
1,5-.alpha.-L-arabinanohydrolase. Arabinase endohydrolyses
(1.fwdarw.5)-.alpha.-arabinofuranosidic linkages in
(1.fwdarw.5)-arabinans. Arabinanase also acts on arabinan.
[0076] In one aspect of the invention, the arabinase activity of
the enzyme composition according to the invention is measured by
arabinase assay as described in the following under the heading
"Assays". The assay can be carried out at pH 3.5 and 50.degree. C.
using sugar beet arabinan as substrate, and it can be performed at
different pH and temperature values for the additional
characterisation and specification of enzymes. Enzyme activity is
calculated from the increase in absorbance at 540 nm per unit
time.
[0077] One unit of arabinase activity is defined as the amount of
enzyme (normalised for total assay volume) that gives an increase
in .DELTA.OD.sub.540 nmmin.sup.-1 under the conditions of the assay
(pH 3.5 and 50.degree. C.).
[0078] In some aspects, the enzyme composition according to the
invention has beta-D-glucoside glucohydrolase activity or comprises
a further enzyme having beta-D-glucoside glucohydrolase activity.
Beta-D-glucoside glucohydrolase refers to enzymes of E.C
3.2.1.21.
[0079] In some aspects, the enzyme composition according to the
invention has .beta.-Xylosidase activity or comprises a further
enzyme having .beta.-Xylosidase activity. ".beta.-Xylosidase" or
"Xylan 1,4-beta-xylosidase" refers to enzymes of E.C 3.2.1.37.
.beta.-Xylosidase catalyze the hydrolysis of
(1->4)-beta-D-xylans, to remove successive D-xylose residues
from the non-reducing termini.
[0080] In some aspects of the invention, the enzyme composition
according to the invention has cellobiohydrolase activity or
comprises a further enzyme having cellobiohydrolase activity.
"Cellobiohydrolase" or "Cellulose 1,4-beta-cellobiosidase" refers
to enzymes of EC 3.2.1.91. Cellulose 1,4-beta-cellobiosidase
catalyze hydrolysis of 1,4-beta-D-glucosidic linkages in cellulose
and cellotetraose, releasing cellobiose from the non-reducing ends
of the chains.
[0081] The cellobiohydrolase activity of the enzyme composition
according to the invention is measured by the cellobiohydrolase
assay as described in the following under the heading "Assays". The
standard assay is carried out at pH 5.0, and it can be performed at
different pH values for the additional characterisation and
specification of enzymes.
[0082] One unit of cellobiohydrolase activity is defined as the
amount of enzyme which produces 1 .mu.mole p-nitrophenol from
p-nitrophenyl .beta.-D-cellobiopyranoside per minute under the
conditions of the assay (pH 5.0 (or as specified) and 50.degree.
C.).
[0083] In some aspects, the enzyme composition according to the
invention has .alpha.-N-arabinofuranosidase activity or comprises a
further enzyme having arabinofuranosidase activity.
".alpha.-N-arabinofuranosidase" or "Alpha-N-arabinofuranosidase"
refers to enzymes of EC 3.2.1.55. .alpha.-N-arabinofuranosidase
catalyze the hydrolysis of terminal non-reducing
alpha-L-arabinofuranoside residues in alpha-L-arabinosides.
[0084] In one aspect of the invention, the arabinofuranosidase
activity of the enzyme composition according to the invention is
measured by the arabinofuranosidase assay as described in the
following under the heading "Assays". The standard assay can be
carried out at pH 5.0 and 50.degree. C. and it can be performed at
different values of pH and temperature for the additional
characterisation and specification of enzymes.
[0085] One unit of .alpha.-N-arabinofuranosidase activity is
defined as the amount of enzyme which produces 1 .mu.mole
p-nitrophenol from p-nitrophenyl .alpha.-L-arabinofuranoside per
minute under the conditions of the assay (pH 5.0 and 50.degree. C.
(or as specified)).
[0086] In some aspects, the enzyme composition according to the
invention has glucan 1,4-beta-glucosidase activity or comprises a
further enzyme having glucan 1,4-beta-glucosidase activity. "Glucan
1,4-beta-glucosidase" or "glucan 1,4-beta-glucosidase" refers to
enzymes of E.C3.2.1.74. Glucan 1,4-beta-glucosidase catalyze the
hydrolysis of (1->4)-linkages in (1->4)-beta-D-glucans, to
remove successive glucose units.
[0087] In some aspects, the enzyme composition according to the
invention has xyloglucan-specific exo-beta-1,4-glucanase activity
or comprises a further enzyme having xyloglucan-specific
exo-beta-1,4-glucanase activity. "xyloglucan-specific
exo-beta-1,4-glucanase" refers to enzymes of E.C3.2.1.155.
Xyloglucan-specific exo-beta-1,4-glucanase catalyze the
exohydrolysis of (1->4)-beta-D-glucosidic linkages in
xyloglucan.
[0088] The enzymes and enzyme compositions according to the
proceeding aspects may be used in a process comprising reducing the
viscosity of an aqueous solution comprising a starch
hydrolysate.
[0089] The enzymes and enzyme compositions may also be used in a
process comprising filtering of an aqueous solution comprising a
starch hydrolysate. In some embodiments the aqueous solution
comprising a starch hydrolysate is a mash for beer making, and in
other embodiments the aqueous solution comprising a starch
hydrolysate is a food composition.
[0090] Alternatively, the enzyme composition according to the
present invention may be used in the production of fruit juice,
wine, grain processing, fuel alcohol, first- or second-generation
biofuel, such as bioethanol, and potable alcohol.
[0091] In some embodiments the first- or second-generation biofuel,
such as bioethanol is produced from agricultural feed stocks such
as sugar cane, potato, corn, wheat sorghum etc. or from cellulosic
material such as corn stover, switchgrass or other plant material.
In both cases fermentable sugars are extracted from the raw
material and fermented by microorganisms into alcohol, which is
distilled and may be used as transportation fuel. The enzyme
composition according to the present invention may be used in this
production of biofuel. The enzymes complex may be added to enhance
extraction of polysaccharides from the raw material, help degrade
polysaccharides down into fermentable sugars and/or to enhance
processing parameters such as separation of liquids from solids,
flow characteristics and pumpability.
[0092] The process of the invention may be applied in the mashing
of any grist. According to the invention the grist may comprise any
starch and/or sugar containing plant material derivable from any
plant and plant part, including tubers, roots, stems, leaves and
seeds.
[0093] In some embodiments the grist comprises grain, such as grain
from barley, wheat, rye, oat, corn, rice, milo, millet and sorghum,
and more preferably, at least 10%, or more preferably at least 15%,
even more preferably at least 25%, or most preferably at least 35%,
such as at least 50%, at least 75%, at least 90% or even 100% (w/w)
of the grist of the wort is derived from grain.
[0094] In some embodiments the grist comprises malted grain, such
as barley malt. Preferably, at least 10%, or more preferably at
least 15%, even more preferably at least 25%, or most preferably at
least 35%, such as at least 50%, at least 75%, at least 90% or even
100% (w/w) of the grist of the wort is derived from malted
grain.
[0095] The term "mash" is understood as aqueous starch slurry, e.
g. comprising crushed barley malt, crushed barley, and/or other
adjunct or a combination hereof, mixed with water later to be
separated into wort+spent grains.
[0096] The term "mash separation" is understood as the separation
of wort from spent grains, such as by lautering or mash
filtration.
[0097] The term "beer filtration" is understood as a separation
process in which the yeast cells and other turbidity-causing
materials still present in the beer are removed, such as by
microfiltration or membrane processes.
[0098] The enzyme preparation, such as in the form of a food
ingredient prepared according to the present invention, may be in
the form of a solution or as a solid--depending on the use and/or
the mode of application and/or the mode of administration. The
solid form can be either as a dried enzyme powder or as a
granulated enzyme.
[0099] In one aspect the invention provides an enzyme composition
preparation comprising the enzyme or enzyme composition according
to the invention, an enzyme carrier and optionally a stabilizer
and/or a preservative.
[0100] In yet a further aspect of the invention, the enzyme carrier
is selected from the group consisting of glycerol or water.
[0101] In a further aspect, the preparation comprises a stabilizer.
In one aspect, the stabilizer is selected from the group consisting
of inorganic salts, polyols, sugars and combinations thereof. In
one aspect, the stabilizer is an inorganic salt such as potassium
chloride. In another aspect, the polyol is glycerol, propylene
glycol, or sorbitol. In yet another aspect, the sugar is a
small-molecule carbohydrate, in particular any of several
sweet-tasting ones such as glucose, fructose and saccharose.
[0102] In yet at further aspect, the preparation comprises a
preservative. In one aspect, the preservative is methyl paraben,
propyl paraben, benzoate, sorbate or other food approved
preservatives or a mixture thereof.
Specific Embodiments of the Invention
[0103] In some embodiments the enzyme exhibiting
endo-1,4-.beta.-xylanase activity, optionally in combination with
any one or more .beta.-glucanase according to the present invention
provides for a significantly reduced viscosity in brewing
applications facilitating improved mash and beer separation.
[0104] Desired xylanase characteristics for brewing applications
may include one or more of the following aspects: [0105] a) Enzyme
substrate specificity [0106] WE-AX/WU-AX ratio has an impact on
viscosity. In some embodiments this ratio is less than about 7.0,
such as less than about 6.5, such as less than about 6.0, such as
less than about 5.5, such as less than about 5.0, such as less than
about 4.5. [0107] b) Enzyme substrate selectivity [0108] How close
to branch points the enzyme(s) cuts is believed to have an impact
on the functionality. [0109] c) Enzyme thermostability [0110]
Continuous solubilisation of AX during mashing--thermostability a
key feature. Accordingly, in some embodiments, the enzyme
exhibiting endo-1,4-.beta.-xylanase activity according to the
present invention is thermostable within a temperature range of
65-78.degree. C. [0111] d) Enzyme pH optimum. Accordingly, in some
embodiments, the enzyme exhibiting endo-1,4-.beta.-xylanase
activity has a pH optimum in the range of pH 5.4-5.6. [0112] e)
Enzyme inhibition (e.g. known key factor for xylanases)
[0113] Said significantly reduced viscosity in brewing applications
may be measured as a reduced viscosity in the brewing application
as compared to a control with a known enzyme or combination of
enzyme activities, such as Ultraflo.RTM. Max used under same
conditions and amounts.
[0114] In some embodiments, the enzyme exhibiting
endo-1,4-.beta.-xylanase activity according to the present
invention, optionally in combination with any one or more
.beta.-glucanase according to the present invention provides for an
improved mash and beer separation in brewing applications.
[0115] In some embodiments, the enzyme exhibiting
endo-1,4-.beta.-xylanase activity according to the present
invention, optionally in combination with any one or more
.beta.-glucanase according to the present invention provides for a
low potential for off flavour formation, such as off flavour
formation related to arabinoxylan breakdown.
[0116] In some embodiments, the enzyme exhibiting
endo-1,4-.beta.-xylanase activity according to the present
invention, optionally in combination with any one or more
.beta.-glucanase according to the present invention provides for a
decreased risk of filter bed collapse, such as at lautering.
[0117] In some embodiments, the enzyme exhibiting
endo-1,4-.beta.-xylanase activity according to the present
invention, optionally in combination with any one or more
.beta.-glucanase according to the present invention provides for a
reduction in off flavour potential and/or reduction in off flavor
formation. One aspect of the invention relates to an enzyme
exhibiting endo-1,4-.beta.-xylanase activity, which enzyme
comprises an amino acid sequence having at least 80% identity with
any one selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID
NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:17, and SEQ ID NO:18, or
any functional fragment thereof.
[0118] Another aspect relates to an enzyme exhibiting
endo-1,3(4)-.beta.-glucanase activity, which enzyme comprises an
amino acid sequence having at least 80% identity with any one
selected from SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10,
and SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID
NO:15, SEQ ID NO:16, or any functional fragment thereof.
[0119] In some embodiments of the invention the enzyme exhibiting
endo-1,4-.beta.-xylanase activity has a ratio in activity on
soluble arabinoxylan substrate (WE-AX) to insoluble arabinoxylan
substrate (WU-AX) arabinoxylan substrate of less than about 7.0,
such as less than about 6.5, such as less than about 6.0, such as
less than about 5.5, such as less than about 5.0, such as less than
about 4.5.
[0120] In some embodiments the enzyme according to the invention
has a temperature optimum in the range of 40-70.degree. C., such as
in the range of 45-65.degree. C., such as in the range of
50-65.degree. C., such as in the range of 55-65.degree. C.
[0121] In some embodiments the enzyme according to the invention
has at least 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,
94, 95, 96, 97, 98 or 99% identity with any one amino acid sequence
selected from SEQ ID NO: 1-18, or any functional fragment
thereof.
[0122] In some embodiments the enzyme according to the invention
has a total number of amino acids of less than 350, such as less
than 340, such as less than 330, such as less than 320, such as
less than 310, such as less than 300 amino acids, such as in the
range of 200 to 350, such as in the range of 220 to 345 amino
acids.
[0123] In some embodiments the amino acid sequence of said enzyme
according to the invention has at least one, two, three, four,
five, six, seven, eight, nine or ten amino acid substitutions as
compared to any one amino acid sequence selected from SEQ ID NO:
1-18, or any functional fragment thereof.
[0124] In some embodiments the amino acid sequence of said enzyme
according to the invention has a maximum of one, two, three, four,
five, six, seven, eight, nine or ten amino acid substitutions
compared to any one amino acid sequence selected from SEQ ID NO:
1-18, or any functional fragment thereof.
[0125] In some embodiments the enzyme according to the invention
comprises the amino acid sequence identified by any one of SEQ ID
NO: 1-18, or any functional fragment thereof.
[0126] In some embodiments the enzyme according to the invention
consists of the amino acid sequence identified by any one of SEQ ID
NO: 1-18, or any functional fragment thereof.
[0127] A further important aspect of the invention relates to a
composition comprising an enzyme exhibiting
endo-1,4-.beta.-xylanase activity according to the invention in
combination with any one or more .beta.-glucanase. In some
embodiments this one or more .beta.-glucanase is according to the
invention.
[0128] A further important aspect of the invention is a composition
comprising an enzyme exhibiting endo-1,3(4)-.beta.-glucanase
activity according to the invention in combination with any one or
more xylanase. In some embodiments this one or more xylanase is an
enzyme exhibiting endo-1,4-.beta.-xylanase activity according to
the invention. In some embodiments this one or more xylanase is an
enzyme according to SEQ ID NO:17 and/or SEQ ID NO:18, or any
functional fragment thereof.
[0129] In some embodiments the combination of an enzyme exhibiting
endo-1,4-.beta.-xylanase activity with an enzyme exhibiting
endo-1,3(4)-.beta.-glucanase activity is according to the following
table:
TABLE-US-00001 1.sup.st enzyme (Xylanase) 2.sup.nd enzyme SEQ ID
SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID (Glucanase) NO: 1
NO: 2 NO: 3 NO: 4 NO: 5 NO: 6 NO: 17 NO: 18 SEQ ID NO: 7 X X X X X
X X X SEQ ID NO: 8 X X X X X X X X SEQ ID NO: 9 X X X X X X X X SEQ
ID NO: 10 X X X X X X X X SEQ ID NO: 11 X X X X X X X X SEQ ID NO:
12 X X X X X X X X SEQ ID NO: 13 X X X X X X X X SEQ ID NO: 14 X X
X X X X X X SEQ ID NO: 15 X X X X X X X X SEQ ID NO: 16 X X X X X X
X X
[0130] It is to be understood that any one of the above combination
of a 1.sup.st enzyme being an enzyme exhibiting
endo-1,4-.beta.-xylanase activity may be combined with one one
enzyme exhibiting endo-1,3(4)-.beta.-glucanase activity with a
ratio between the two enzymes of 1:10, 2:10, 3:10, 4:10, 5:10,
6:10, 7:10, 8:10, 9:10, 10:10, 10:9, 10:8, 10:7, 10:6, 10:5, 10:4,
10:3, 10:2, or 10:1, such as within a range of 1:10-10:1, such as
2:10-10:2, such as 3:10-10:3, such as 4:10-10:4, such as 5:10-10:5,
such as 6:10-10:6, such as 7:10-10:7, such as 8:10-10:8, or within
9:10-10:9.
[0131] In some embodiments the composition according to the
invention comprises a combination of at least two enzymes, said two
enzymes, or two enzymes with an amino acid sequence having at least
80% sequence identity with the respective SEQ ID, or any functional
fragment thereof, being selected from the list consisting of
SEQ ID NO:1 and SEQ ID NO:7;
SEQ ID NO:2 and SEQ ID NO:7;
SEQ ID NO:3 and SEQ ID NO:7;
SEQ ID NO:4 and SEQ ID NO:7;
SEQ ID NO:5 and SEQ ID NO:7;
SEQ ID NO:6 and SEQ ID NO:7;
SEQ ID NO:17 and SEQ ID NO:7;
SEQ ID NO:18 and SEQ ID NO:7;
SEQ ID NO:1 and SEQ ID NO:8;
SEQ ID NO:2 and SEQ ID NO:8;
SEQ ID NO:3 and SEQ ID NO:8;
SEQ ID NO:4 and SEQ ID NO:8;
SEQ ID NO:5 and SEQ ID NO:8;
SEQ ID NO:6 and SEQ ID NO:8;
SEQ ID NO:17 and SEQ ID NO:8;
SEQ ID NO:18 and SEQ ID NO:8;
SEQ ID NO:1 and SEQ ID NO:9;
SEQ ID NO:2 and SEQ ID NO:9;
SEQ ID NO:3 and SEQ ID NO:9;
SEQ ID NO:4 and SEQ ID NO:9;
SEQ ID NO:5 and SEQ ID NO:9;
SEQ ID NO:6 and SEQ ID NO:9;
SEQ ID NO:17 and SEQ ID NO:9;
SEQ ID NO:18 and SEQ ID NO:9;
SEQ ID NO:1 and SEQ ID NO:10;
SEQ ID NO:2 and SEQ ID NO:10;
SEQ ID NO:3 and SEQ ID NO:10;
SEQ ID NO:4 and SEQ ID NO:10;
SEQ ID NO:5 and SEQ ID NO:10;
SEQ ID NO:6 and SEQ ID NO:10;
SEQ ID NO:17 and SEQ ID NO:10;
SEQ ID NO:18 and SEQ ID NO:10;
SEQ ID NO:1 and SEQ ID NO:11;
SEQ ID NO:2 and SEQ ID NO:11;
SEQ ID NO:3 and SEQ ID NO:11;
SEQ ID NO:4 and SEQ ID NO:11;
SEQ ID NO:5 and SEQ ID NO:11;
SEQ ID NO:6 and SEQ ID NO:11;
SEQ ID NO:17 and SEQ ID NO:11;
SEQ ID NO:18 and SEQ ID NO:11;
SEQ ID NO:1 and SEQ ID NO:12;
SEQ ID NO:2 and SEQ ID NO:12;
SEQ ID NO:3 and SEQ ID NO:12;
SEQ ID NO:4 and SEQ ID NO:12;
SEQ ID NO:5 and SEQ ID NO:12;
SEQ ID NO:6 and SEQ ID NO:12;
SEQ ID NO:17 and SEQ ID NO:12;
SEQ ID NO:18 and SEQ ID NO:12;
SEQ ID NO:1 and SEQ ID NO:13;
SEQ ID NO:2 and SEQ ID NO:13;
SEQ ID NO:3 and SEQ ID NO:13;
SEQ ID NO:4 and SEQ ID NO:13;
SEQ ID NO:5 and SEQ ID NO:13;
SEQ ID NO:6 and SEQ ID NO:13;
SEQ ID NO:17 and SEQ ID NO:13;
SEQ ID NO:18 and SEQ ID NO:13;
SEQ ID NO:1 and SEQ ID NO:14;
SEQ ID NO:2 and SEQ ID NO:14;
SEQ ID NO:3 and SEQ ID NO:14;
SEQ ID NO:4 and SEQ ID NO:14;
SEQ ID NO:5 and SEQ ID NO:14;
SEQ ID NO:6 and SEQ ID NO:14;
SEQ ID NO:17 and SEQ ID NO:14;
SEQ ID NO:18 and SEQ ID NO:14;
SEQ ID NO:1 and SEQ ID NO:15;
SEQ ID NO:2 and SEQ ID NO:15;
SEQ ID NO:3 and SEQ ID NO:15;
SEQ ID NO:4 and SEQ ID NO:15;
SEQ ID NO:5 and SEQ ID NO:15;
SEQ ID NO:6 and SEQ ID NO:15;
SEQ ID NO:17 and SEQ ID NO:15;
SEQ ID NO:18 and SEQ ID NO:15;
SEQ ID NO:1 and SEQ ID NO:16;
SEQ ID NO:2 and SEQ ID NO:16;
SEQ ID NO:3 and SEQ ID NO:16;
SEQ ID NO:4 and SEQ ID NO:16;
SEQ ID NO:5 and SEQ ID NO:16;
SEQ ID NO:6 and SEQ ID NO:16;
SEQ ID NO:17 and SEQ ID NO:16; and
SEQ ID NO:18 and SEQ ID NO:16.
[0132] In some embodiments the endo-1,3(4)-.beta.-glucanase
activity and the endo-1,4-.beta.-xylanase activity are derived from
at least two different enzymes, such as at least two different
enzymes from two different species.
[0133] In some embodiments the total pressure built up is reduced
to a value of less than 470 mm WC, such as less than 450 mm WC,
such as less than 430 mm WC, such as less than 410 mm WC, such as
less than 390 mm WC, such as less than 370 mm WC, such as less than
350 mm WC, such as less than 330 mm WC, such as less than 310 mm
WC, such as less than 300 mm WC, such as less than 290 mm WC, when
the composition according to the present invention is used prior to
the lautering in a brewing application.
[0134] In some embodiments the total pressure built up is reduced
by at least 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31,
33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65,
67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93 or 95%
compared to the use of a negative control without said composition;
when used prior to the lautering in a brewing application.
[0135] In some embodiments the wort filterability as measured by
volume wort collected after 5 min of filtration relative to a
control without enzymes is increased to above 1.5, such as above
1.6, such as above 1.7, such as above 1.8, such as above 1.9, such
as above 2.0, such as above 2.1, such as above 2.2, such as above
2.3, such as above 2.4, such as above 2.5, when the composition
according to invention is used in a brewing application prior to
the wort separation.
[0136] In some embodiments the wort filterability as measured by
volume wort collected after 5 min of filtration is increased at
least 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35,
37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69,
71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 110,
120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240,
250, 260, 270, 280, 290, or 300% as compared to the use of a
negative control without said composition.
[0137] In some embodiments the composition according to the
invention comprises any one or more further enzyme. In some
embodiments the one or more further enzyme is selected from list
consisting of a xylanase classified in EC 3.2.1.32, EC 3.2.1.136,
or EC 3.2.1.156, a cellulase, a laminarinase, an
endo-1,5-.alpha.-L-arabinanase, a beta-D-glucoside glucohydrolase,
a .beta.-Xylosidase, a cellobiohydrolase, a glucan
1,4-beta-glucosidase, a xyloglucan-specific exo-beta-1,4-glucanase
and an .alpha.-N-Arabinofuranosidase.
[0138] Sequences and enzymes identified by a sequence as mentioned
herein and used according to the present invention alone or in
combinations with other enzymes or compounds may be with or without
signal peptide.
[0139] Assays
[0140] DNS Cellulase Activity Method (DNS CMC Method)
[0141] Systematic Name: 1,4-(1,3;1,4)-.beta.-D-glucan
4-glucanohydrolase
[0142] IUB Number: EC 3.2.1.4
[0143] Principle
[0144] The assay of cellulase is based on the enzymatic
endo-hydrolysis of the 1,4-.beta.-D-glucosidic bonds in
carboxymethylcellulose (CMC), a .beta.-1,4-glucan. The products of
the reaction (.beta.-1,4 glucan oligosaccharides) was determined
colorimetrically by measuring the resulting increase in reducing
groups using a 3,5-dinitrosalicylic acid reagent. Enzyme activity
was calculated from the relationship between the concentration of
reducing groups, as glucose equivalents, and absorbance at 540
nm.
[0145] The assay was carried out at pH 5.0, but it can be performed
at different pH values for the additional characterisation and
specification of enzymes.
[0146] Unit Definition
[0147] One unit of cellulase activity is defined as the amount of
enzyme which produces 1 .mu.mole glucose equivalents per minute
under the conditions of the assay (pH 5.0 (or as specified) and
50.degree. C.).
[0148] Materials
[0149] Carboxymethylcellulose. Supplier: Megazyme Ltd. Product no.:
CM-Cellulose 4M
[0150] D-Glucose `AnalaR`. Supplier: Merck Ltd (BDH). Product no.:
10117. M.W.: 180.16
[0151] Sodium acetate anhydrous `AnalaR`. Supplier: Merck Ltd
(BDH). Product no.: 10236. M.W.: 82.03
[0152] Acetic acid ("glacial") `AnalaR`. Supplier: Merck Ltd (BDH).
Product no.: 10001. M.W.: 60.05
[0153] 3,5-Dinitrosalicylic acid GPR (3,5-dinitro-2-hydroxybenzoic
acid). Supplier: Merck Ltd (BDH). Product no.: 28235
[0154] Sodium hydroxide pellets `AnalaR`. Supplier: Merck Ltd
(BDH). Product no.: 10252. M.W.: 40.00
[0155] Potassium sodium (+)-tartrate `AnalaR`. Supplier: Merck Ltd
(BDH). Product no.: 10219. M.W.: 282.22
[0156] 1.5% (w/v solution) Carboxymethylcellulose (CMC) solution in
0.1M sodium acetate buffer, pH 5.0 (substrate solution).
[0157] 3,5-Dinitrosalicylic acid (DNS) solution. 20 g/L of DNS in
buffer containing 32 g/L sodium hydroxide pellets, and 600 g/L
potassium sodium (+)-tartrate.
[0158] Glucose standard solution (0.50 mg/ml)
[0159] Procedure
[0160] The enzyme composition was diluted into samples and a
glucose standard curve as shown in FIG. 2 was made using glucose
concentrations of 0, 0.125, 0.25, 0.375, and 0.5 mg/ml.
[0161] 0.25 ml of enzyme solution was mixed with 1.75 ml of the
substrate solution (1.5% w/v) at 50.degree. C. and the reaction was
stopped after 10 min by addition of DNS solution. This is followed
by heating to 95.degree. C. for 5 minutes.
[0162] The optical density was measured at 540 nm (OD.sub.540 nm)
of the different samples.
[0163] Calculation
[0164] The enzyme activity is determined from the standard curve as
shown in FIG. 2.
[0165] The activity is calculated as follows:
Activity ( u ml - 1 or u g - 1 ) = T - c m .times. A .times. 1
180.16 .times. 10 3 1 V .times. 1 t .times. D ##EQU00001##
where:
T=.DELTA.OD.sub.540 nm TEST
[0166] =OD.sub.540 nm TEST-OD.sub.540 nm BLANK m=gradient of the
standard curve (approximately 1.0) c=y axis intercept of the
standard curve (always negative and approximately -0.02)
180.16.ident.molecular weight of glucose 10.sup.3.ident.to convert
to .mu.moles A.ident.assay volume in ml V.ident.enzyme volume in ml
t.ident.assay time in minutes D=actual enzyme dilution factor (e.g.
for 1.000 g diluted to 1 litre D=1000)
[0167] Laminarinase (DNS Laminarin Method)
[0168] Principle
[0169] The reaction, catalysed by laminarinase, involves the
endohydrolysis of 1,3-glucosidic bonds in 1,3-.beta.-D-glucans.
Substrates include laminarin, paramylon and pachyman. The products
of the reaction (.beta.-1,3-glucan oligosaccharides) are determined
colourimetrically by measuring the resulting increase in reducing
groups using a 3,5-dintrosalicylic acid reagent. Enzyme activity is
calculated from the relationship between the concentration of
reducing groups, as glucose equivalents, and absorbance at 540
nm.
[0170] The assay was carried out at pH 5.0 and 50.degree. C., but
it can be performed at different values of pH and temperature for
the additional characterisation and specification of enzymes.
[0171] Unit Definition
[0172] One unit of laminarinase activity is defined as the amount
of enzyme which produces 1 .mu.mole glucose equivalents per minute
under the conditions of the assay (pH 5.0 and 50.degree. C. (or as
specified)).
[0173] Materials
[0174] See materials given above for the Cellulase activity
assay.
[0175] Laminarin (from Laminaria digitata). Supplier: Sigma-Aldrich
Co. Ltd. Product no.: L 9634
[0176] 1.00% (w/v solution) Laminarin solution (substrate solution
0.1M sodium acetate buffer, pH 5.0)
[0177] 1.75 ml laminarin solution is mixed with 0.25 ml diluted
enzyme solution at 50.degree. C. for 10 minutes and the reaction
stopped by addition of 2 ml DNS solution.
[0178] Standard curve was made using 0, 0.125, 0.25, 0.5 and 0.75
mg/ml glucose solution.
[0179] Optical density was measured at 540 nm (OD.sub.540 nm).
[0180] Calculation
[0181] The activity is calculated as follows:
Activity ( u ml - 1 or u g - 1 ) = T - c m .times. A .times. 1
180.16 .times. 10 3 .times. 1 V .times. 1 t .times. D ##EQU00002##
[0182] where:
T=.DELTA.OD.sub.540 nm TEST
[0182] [0183] =OD.sub.540 nm TEST-OD.sub.540 nm BLANK m=gradient of
the standard curve (approximately 1.0) c=y axis intercept of the
standard curve (always negative and approximately -0.03)
180.16.ident.molecular weight of glucose 10.sup.3.ident.to convert
to .mu.moles A.ident.assay volume in ml V.ident.enzyme volume in ml
t.ident.assay time in minutes D=enzyme dilution factor (e.g. for 1
g diluted to 1 litre D=1000)
[0184] Arabinase Assay.
[0185] Principle
[0186] The assay of Arabinase activity is based on colorimetrically
determination by measuring the resulting increase in reducing
groups using a 3,5-dinitrosalicylic acid reagent. Enzyme activity
was calculated from the relationship between the concentration of
reducing groups, as arabinose equivalents, and absorbance at 540
nm.
[0187] The assay was carried out at pH 3.5, but it can be performed
at different pH values for the additional characterisation and
specification of enzymes.
[0188] Unit Definition
[0189] One unit of arabinase (Arabinanase
(endo-1,5-alpha-L-arabinanase)) activity is defined as the amount
of enzyme which produces 1 .mu.mole arabinose equivalents per
minute under the conditions of the assay (pH 3.5 (or as specified)
and 50.degree. C.).
[0190] Materials
[0191] Megazyme Sugar Beet Arabinan
[0192] Arabinose Sigma A3131 M.W.: 150.1
[0193] Sodium acetate anhydrous `AnalaR`. Supplier: Merck Ltd
(BDH). Product no.: 10236. M.W.: 82.03
[0194] Acetic acid ("glacial") `AnalaR`. Supplier: Merck Ltd (BDH).
Product no.: 10001. M.W.: 60.05
[0195] 3,5-Dinitrosalicylic acid GPR (3,5-dinitro-2-hydroxybenzoic
acid). Supplier: Merck Ltd (BDH). Product no.: 28235
[0196] Sodium hydroxide pellets `AnalaR`. Supplier: Merck Ltd
(BDH). Product no.: 10252. M.W.: 40.00
[0197] Potassium sodium (+)-tartrate `AnalaR`. Supplier: Merck Ltd
(BDH). Product no.: 10219. M.W.: 282.22
[0198] 1.5% (w/v solution) Arabinan solution in 0.1M sodium acetate
buffer, pH 3.5 (substrate solution).
[0199] 3,5-Dinitrosalicylic acid (DNS) solution. 20 g/L of DNS in
buffer containing 32 g/L sodium hydroxide pellets, and 600 g/L
potassium sodium (+)-tartrate.
[0200] Arabinase standard solution (0.50 mg/ml)
[0201] Procedure
[0202] The enzyme composition was diluted into samples and a
glucose standard curve was made using arabinase concentrations of
0, 0.125, 0.25, 0.375, and 0.5 mg/ml.
[0203] 0.25 ml of enzyme solution was mixed with 1.75 ml of the
substrate solution (1.5% w/v) at 50.degree. C. and the reaction was
stopped after 10 min by addition of DNS solution. Followed by
heating to 95.degree. C. for 5 minutes.
[0204] The optical density was measured at 540 nm (OD.sub.540 nm)
of the different samples.
[0205] Calculation
[0206] The enzyme activity is determined from the standard
curve.
[0207] The activity is calculated as follows:
Activity ( u ml - 1 or u g - 1 ) = T - c m .times. A .times. 1
150.13 .times. 10 3 .times. 1 V .times. 1 t .times. D
##EQU00003##
where:
T=.DELTA.OD.sub.540 nm TEST
[0208] =OD.sub.540 nm TEST-OD.sub.540 nm BLANK m=gradient of the
standard curve (approximately 1.0) c=y axis intercept of the
standard curve (always negative and approximately -0.02)
150.13.ident.molecular weight of arabinase 10.sup.3.ident.to
convert to .mu.moles A.ident.assay volume in ml V.ident.enzyme
volume in ml t.ident.assay time in minutes D=actual enzyme dilution
factor (e.g. for 1.000 g diluted to 1 litre D=1000)
[0209] Arabinofuranosidase Assay.
[0210] The reaction, catalysed by .alpha.-N-arabinofuranosidase,
involves the hydrolysis of the terminal bond, at the non-reducing
.alpha.-L-arabinofuranoside residue, of .alpha.-L-arabinosides. The
enzyme acts on .alpha.-L-arabinofuranosides, .alpha.-L-arabinans
containing (1,3)- and/or (1,5)-linkages, arabinoxylans and
arabinogalactans.
[0211] The assay of .alpha.-N-arabinofuranosidase is based upon the
enzymatic hydrolysis of p-nitrophenyl .alpha.-L-arabinofuranoside.
The assay is a "two-point", rather than a "continuous monitoring",
method. The calculation of enzyme activity is based on measurements
taken only at the beginning and end of the incubation period. A
product of the reaction, p-nitrophenol is determined
colourimetrically (after pH adjustment). Enzyme activity is
calculated from the relationship between the concentration of
p-nitrophenol and absorbance at 400 nm.
[0212] Preparation of Diluted Enzyme Solution:
[0213] Prepare all enzyme solutions, from powder or liquid enzyme
preparations, with glass distilled water. Minimise assay dilution
errors by avoiding large dilution steps involving small volumes or
weights. In making enzyme dilutions it is more accurate, even for a
liquid sample, to weigh out the initial enzyme sample. If this is
done, in the case of liquid samples it is therefore necessary to
measure the specific gravity of the liquid at 20.degree. C.
[0214] As the assay is a "two-point", rather than a "continuous
monitoring", method it is important to ensure the linearity within
the incubation period with different enzyme systems and conditions.
Under the standard assay conditions of substrate concentration, pH,
temperature and assay time the assay has been demonstrated to be
linear in the range .DELTA.OD.sub.540 nm TEST (T)=0.20-1.50.
However, for good practice, the assay is operated within a defined
range of .DELTA.OD.sub.540 nm TEST (T)=0.400-0.800.
[0215] Procedure
[0216] Each enzyme sample assay involves three analyses: duplicate
test (TEST) analyses and a blank (BLANK) analysis. The procedure
given describes the analysis of a single enzyme sample.
TABLE-US-00002 TEST BLANK 0.2M Sodium acetate buffer, pH 5.0 1.00
ml 1.00 ml Glass distilled water 1.00 ml 1.00 ml
p-Nitrophenyl-.alpha.-L-arabinofuranoside solution 1.00 ml 1.00
ml
[0217] 0.25 ml diluted enzyme solution was added to the solutions
at 50.degree. C., the reaction was stopped after 10 minutes by
addition of 4 ml of 0.4M glycine solution, pH 10.8 (stop
reagent).
[0218] Absorbance was measured at 400 nm at 25.degree. C. against a
water blank. [0219] determine OD.sub.400 nm TEST for the duplicate
TESTS measured; [0220] determine OD.sub.400 nm BLANK.
[0221] Calculation
.DELTA. OD 400 nm TEST ( T ) = OD 400 nm TEST - OD 400 nm BLANK
##EQU00004## Units ( mol min - 1 ) = T 18300 .times. V 1000 .times.
10 6 .times. 1 t Activity ( u ml - 1 or u g - 1 ) = Units .times. 1
E .times. D ##EQU00004.2##
where: T=OD400 nm TEST-OD400 nm BLANK 18300=Molar extinction
coefficient for p-nitrophenol (1 cm path length) V=7.25 (total
liquid volume in test in ml) t=10 (minutes) 1 u=1 .mu.molmin-1
E=0.25 (volume of diluted enzyme sample in ml) D=Enzyme dilution
factor e.g. for 1 ml diluted to 1 litre D=1000)
[0222] Cellobiohydrolase Assay.
[0223] Principle
[0224] The reaction, catalysed by cellobiohydrolase, involves the
hydrolysis of 1,4-.beta.-D-glucosidic linkages in cellulose and
cellotetraose, releasing cellobiose from the non-reducing ends of
the chains.
[0225] The assay of cellobiohydrolase is based on the enzymatic
hydrolysis of p-nitrophenyl .beta.-D-cellobiopyranoside. The
product of the reaction, p-nitrophenol is determined
colorimetrically (after pH adjustment). Enzyme activity is
calculated from the relationship between the concentration of
p-nitrophenol and absorbance at 400 nm.
[0226] The assay is operated within the linear defined range of
.DELTA.OD.sub.540 nm TEST (T)=0.400-0.800.
[0227] Procedure
[0228] Each enzyme sample assay involves three analyses: duplicate
test (TEST) analyses and a blank (BLANK) analysis. The procedure
given describes the analysis of a single enzyme sample.
TABLE-US-00003 TEST BLANK 0.2M Sodium acetate buffer, pH 5.0 1.00
ml 1.00 ml Glass distilled water 1.00 ml 1.00 ml p-Nitrophenyl
.beta.-D-cellobiopyranoside solution 1.00 ml 1.00 ml
[0229] 0.25 ml diluted enzyme solution was added to the test
solution at 50.degree. C., after 30 minutes 4 ml of 0.4M glycine
solution, pH 10.8 (stop reagent) was added to each tube. [0230]
Absorbance was measured at 20.degree. C. at 400 nm in a 1 cm glass
cuvette against a water blank. [0231] determine OD400 nm TEST for
the duplicate TESTS measured; [0232] determine OD400 nm BLANK.
[0233] Calculation
.DELTA. OD 400 nm TEST ( T ) = OD 400 nm TEST - OD 400 nm BLANK
##EQU00005## Units ( mol min - 1 ) = T 18300 .times. V 1000 .times.
10 6 .times. 1 t Activity ( u ml - 1 or u g - 1 ) = Units .times. 1
E .times. D ##EQU00005.2##
where: T=OD.sub.400 nm TEST-OD.sub.400 nm BLANK 18300=Molar
extinction coefficient for p-nitrophenol (1 cm path length) V=7.25
(total liquid volume in test in ml) 1000=to convert to litres
10.sub.6=to convert to .mu.moles t=30 (minutes) 1 u=1
.mu.molmin.sup.-1 E=0.25 (volume of diluted enzyme sample in ml)
D=Enzyme dilution factor e.g. for 1 ml diluted to 1 litre
D=1000)
[0234] Numbered embodiments according to the invention:
[0235] 1. An enzyme exhibiting endo-1,4-.beta.-xylanase activity,
which enzyme comprises an amino acid sequence having at least 80%
identity with any one selected from SEQ ID NO:1, SEQ ID NO:2, SEQ
ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:17, and
SEQ ID NO:18, or any functional fragment thereof.
[0236] 2. The enzyme according to embodiment 1, which enzyme has a
ratio in activity on soluble arabinoxylan substrate (WE-AX) to
insoluble arabinoxylan substrate (WU-AX) arabinoxylan substrate of
less than about 7.0, such as less than about 6.5, such as less than
about 6.0, such as less than about 5.5, such as less than about
5.0, such as less than about 4.5.
[0237] 3. An enzyme exhibiting endo-1,3(4)-.beta.-glucanase
activity, which enzyme comprises an amino acid sequence having at
least 80% identity with any one selected from SEQ ID NO:7, SEQ ID
NO:8, SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, SEQ ID NO:12,
SEQ ID NO: 13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, or any
functional fragment thereof.
[0238] 4. The enzyme according to any one of embodiment 1-3, which
enzyme has a temperature optimum in the range of 40-70.degree. C.,
such as in the range of 45-65.degree. C., such as in the range of
50-65.degree. C., such as in the range of 55-65.degree. C.
[0239] 5. The enzyme according to any one of embodiments 1-4,
wherein said enzyme has at least 81, 82, 83, 84, 85, 86, 87, 88,
89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity with any one
amino acid sequence selected from SEQ ID NO: 1-18, or any
functional fragment thereof.
[0240] 6. The enzyme according to any one of embodiments 1-5 having
a total number of amino acids of less than 350, such as less than
340, such as less than 330, such as less than 320, such as less
than 310, such as less than 300 amino acids, such as in the range
of 200 to 350, such as in the range of 220 to 345 amino acids.
[0241] 7. The enzyme according to any one of embodiments 1-6,
wherein the amino acid sequence of said enzyme has at least one,
two, three, four, five, six, seven, eight, nine or ten amino acid
substitutions as compared to any one amino acid sequence selected
from SEQ ID NO: 1-18, or any functional fragment thereof.
[0242] 8. The enzyme according to any one of embodiments 1-7,
wherein the amino acid sequence of said enzyme has a maximum of
one, two, three, four, five, six, seven, eight, nine or ten amino
acid substitutions compared to any one amino acid sequence selected
from SEQ ID NO: 1-18, or any functional fragment thereof.
[0243] 9. The enzyme according to any one of embodiments 1-8, which
enzyme comprises the amino acid sequence identified by any one of
SEQ ID NO: 1-18, or any functional fragment thereof.
[0244] 10. The enzyme according to any one of embodiments 1 or 3,
which enzyme consists of the amino acid sequence identified by any
one of SEQ ID NO: 1-18, or any functional fragment thereof.
[0245] 11. A DNA construct comprising a DNA sequence encoding an
enzyme according to any of embodiments 1-10.
[0246] 12. A recombinant expression vector comprising a DNA
construct according to embodiment 11.
[0247] 13. A cell that has been transformed with a DNA construct of
embodiment 11 or the vector of embodiment 12.
[0248] 14. A preparation comprising an enzyme according to any one
of embodiments 1-10, or a DNA construct according to embodiment 11,
or a vector according to embodiment 12, or a cell according to
embodiment 13.
[0249] 15. A composition comprising an enzyme exhibiting
endo-1,4-.beta.-xylanase activity according to any one of
embodiments 1, 2, 4-10 in combination with any one or more
.beta.-glucanase.
[0250] 16. The composition according to embodiment 15, wherein said
one or more .beta.-glucanase is an enzyme exhibiting
endo-1,3(4)-.beta.-glucanase activity according to any one of
embodiments 3-10.
[0251] 17. A composition comprising an enzyme exhibiting
endo-1,3(4)-.beta.-glucanase activity according to any one of
embodiments 3-10 in combination with any one or more xylanase.
[0252] 18. The composition according to embodiment 17, wherein said
one or more xylanase is an enzyme exhibiting
endo-1,4-.beta.-xylanase activity according to any one of
embodiments 1, 2, 4-10.
[0253] 19. The composition according to any one of embodiments
15-18, wherein said endo-1,3(4)-.beta.-glucanase activity and said
endo-1,4-.beta.-xylanase activity are derived from at least two
different enzymes, such as at least two different enzymes from two
different species.
[0254] 20. The composition according to any one of embodiments
15-19, comprising a combination of at least two enzymes, said two
enzymes, or two enzymes with an amino acid sequence having at least
80% sequence identity with the respective SEQ ID, or any functional
fragment thereof, being selected from the list consisting of
SEQ ID NO:1 and SEQ ID NO:7;
SEQ ID NO:2 and SEQ ID NO:7;
SEQ ID NO:3 and SEQ ID NO:7;
SEQ ID NO:4 and SEQ ID NO:7;
SEQ ID NO:5 and SEQ ID NO:7;
SEQ ID NO:6 and SEQ ID NO:7;
SEQ ID NO:17 and SEQ ID NO:7;
SEQ ID NO:18 and SEQ ID NO:7;
SEQ ID NO:1 and SEQ ID NO:8;
SEQ ID NO:2 and SEQ ID NO:8;
SEQ ID NO:3 and SEQ ID NO:8;
SEQ ID NO:4 and SEQ ID NO:8;
SEQ ID NO:5 and SEQ ID NO:8;
SEQ ID NO:6 and SEQ ID NO:8;
SEQ ID NO:17 and SEQ ID NO:8;
SEQ ID NO:18 and SEQ ID NO:8;
SEQ ID NO:1 and SEQ ID NO:9;
SEQ ID NO:2 and SEQ ID NO:9;
SEQ ID NO:3 and SEQ ID NO:9;
SEQ ID NO:4 and SEQ ID NO:9;
SEQ ID NO:5 and SEQ ID NO:9;
SEQ ID NO:6 and SEQ ID NO:9;
SEQ ID NO:17 and SEQ ID NO:9;
SEQ ID NO:18 and SEQ ID NO:9;
SEQ ID NO:1 and SEQ ID NO:10;
SEQ ID NO:2 and SEQ ID NO:10;
SEQ ID NO:3 and SEQ ID NO:10;
SEQ ID NO:4 and SEQ ID NO:10;
SEQ ID NO:5 and SEQ ID NO:10;
SEQ ID NO:6 and SEQ ID NO:10;
SEQ ID NO:17 and SEQ ID NO:10;
SEQ ID NO:18 and SEQ ID NO:10;
SEQ ID NO:1 and SEQ ID NO:11;
SEQ ID NO:2 and SEQ ID NO:11;
SEQ ID NO:3 and SEQ ID NO:11;
SEQ ID NO:4 and SEQ ID NO:11;
SEQ ID NO:5 and SEQ ID NO:11;
SEQ ID NO:6 and SEQ ID NO:11;
SEQ ID NO:17 and SEQ ID NO:11;
SEQ ID NO:18 and SEQ ID NO:11;
SEQ ID NO:1 and SEQ ID NO:12;
SEQ ID NO:2 and SEQ ID NO:12;
SEQ ID NO:3 and SEQ ID NO:12;
SEQ ID NO:4 and SEQ ID NO:12;
SEQ ID NO:5 and SEQ ID NO:12;
SEQ ID NO:6 and SEQ ID NO:12;
SEQ ID NO:17 and SEQ ID NO:12;
SEQ ID NO:18 and SEQ ID NO:12;
SEQ ID NO:1 and SEQ ID NO:13;
SEQ ID NO:2 and SEQ ID NO:13;
SEQ ID NO:3 and SEQ ID NO:13;
SEQ ID NO:4 and SEQ ID NO:13;
SEQ ID NO:5 and SEQ ID NO:13;
SEQ ID NO:6 and SEQ ID NO:13;
SEQ ID NO:17 and SEQ ID NO:13;
SEQ ID NO:18 and SEQ ID NO:13;
SEQ ID NO:1 and SEQ ID NO:14;
SEQ ID NO:2 and SEQ ID NO:14;
SEQ ID NO:3 and SEQ ID NO:14;
SEQ ID NO:4 and SEQ ID NO:14;
SEQ ID NO:5 and SEQ ID NO:14;
SEQ ID NO:6 and SEQ ID NO:14;
SEQ ID NO:17 and SEQ ID NO:14;
SEQ ID NO:18 and SEQ ID NO:14;
SEQ ID NO:1 and SEQ ID NO:15;
SEQ ID NO:2 and SEQ ID NO:15;
SEQ ID NO:3 and SEQ ID NO:15;
SEQ ID NO:4 and SEQ ID NO:15;
SEQ ID NO:5 and SEQ ID NO:15;
SEQ ID NO:6 and SEQ ID NO:15;
SEQ ID NO:17 and SEQ ID NO:15;
SEQ ID NO:18 and SEQ ID NO:15;
SEQ ID NO:1 and SEQ ID NO:16;
SEQ ID NO:2 and SEQ ID NO:16;
SEQ ID NO:3 and SEQ ID NO:16;
SEQ ID NO:4 and SEQ ID NO:16;
SEQ ID NO:5 and SEQ ID NO:16;
SEQ ID NO:6 and SEQ ID NO:16;
SEQ ID NO:17 and SEQ ID NO:16; and
SEQ ID NO:18 and SEQ ID NO:16.
[0255] 21. The composition according to any one of embodiments
15-20, wherein when used prior to the lautering in a brewing
application the total pressure built up is reduced to a value of
less than 470 mm WC, such as less than 450 mm WC, such as less than
430 mm WC, such as less than 410 mm WC, such as less than 390 mm
WC, such as less than 370 mm WC, such as less than 350 mm WC, such
as less than 330 mm WC, such as less than 310 mm WC, such as less
than 300 mm WC, such as less than 290 mm WC.
[0256] 22. The composition according to any one of embodiments
15-21, wherein when used prior to the lautering in a brewing
application the total pressure built up is reduced by at least 5,
7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39,
41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73,
75, 77, 79, 81, 83, 85, 87, 89, 91, 93 or 95% compared to the use
of a negative control without said composition.
[0257] 23. The composition according to any one of embodiments
15-22, wherein when used in a brewing application prior to the wort
separation, the wort filterability as measured by volume wort
collected after 5 min of filtration relative to a control without
enzymes is increased to above 1.5, such as above 1.6, such as above
1.7, such as above 1.8, such as above 1.9, such as above 2.0, such
as above 2.1, such as above 2.2, such as above 2.3, such as above
2.4, such as above 2.5.
[0258] 24. The composition according to any one of embodiments
15-23, when used in a brewing application prior to the wort
separation, the wort filterability as measured by volume wort
collected after 5 min of filtration is increased by at least 5, 7,
9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41,
43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75,
77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 110, 120, 130, 140,
150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270,
280, 290, or 300% as compared to the use of a negative control
without said composition.
[0259] 25. The composition according to any one of embodiments
15-24, comprising any one or more further enzyme.
[0260] 26. The composition according to embodiment 25, wherein said
one or more further enzyme is selected from list consisting of a
xylanase classified in EC 3.2.1.32, EC 3.2.1.136, or EC 3.2.1.156,
a cellulase, a laminarinase, an endo-1,5-.alpha.-L-arabinanase, a
beta-D-glucoside glucohydrolase, a .beta.-Xylosidase, a
cellobiohydrolase, a glucan 1,4-beta-glucosidase, a
xyloglucan-specific exo-beta-1,4-glucanase and an
.alpha.-N-Arabinofuranosidase.
[0261] 27. Use of an enzyme according to embodiments 1-10, or a
preparation according to embodiment 14, or a composition according
to any one of embodiments 15-26 in the production of a food, feed,
or malt beverage product.
[0262] 28. Use of an enzyme according to embodiments 1-10, or a
preparation according to embodiment 14, or a composition according
to any one of embodiments 15-26, in the production of dough or
baked products.
[0263] 29. Use of an enzyme according to embodiments 1-10, or a
preparation according to embodiment 14, or a composition according
to any one of embodiments 15-26, in the preparation of pulp or
paper.
[0264] 30. Use of an enzyme according to embodiments 1-10, or a
preparation according to embodiment 14, or a composition according
to any one of embodiments 15-26, for the preparation of cereal
components.
[0265] 31. The use according to embodiment 29, in which the cereal
is rye, wheat, or barley.
[0266] 32. Use of enzyme according to embodiments 1-10, or a
preparation according to embodiment 14, or a composition according
to any one of embodiments 15-26, in the production of beer or
modification of by-products from a brewing process.
[0267] 33. Use of enzyme according to embodiments 1-10, or a
preparation according to embodiment 14, or a composition according
to any one of embodiments 15-26, in the production of wine or
juice.
[0268] 34. Use of enzyme according to embodiments 1-10, or a
preparation according to embodiment 14, or a composition according
to any one of embodiments 15-26, in the production of a first- or
second-generation biofuel, such as bioethanol.
[0269] 35. Method of altering filterability of a starch comprising
material, said method comprising the step of treating said starch
comprising material with enzyme according to embodiments 1-10, or a
preparation according to embodiment 14, or a composition according
to any one of embodiments 15-26.
[0270] 36. Method of reducing pressure built up during lautering in
a brewing application, said method comprising the step of treating
a brewing mash with enzyme according to embodiments 1-10, or a
preparation according to embodiment 14, or a composition according
to any one of embodiments 15-26.
[0271] 37. Method for the production of a food, feed, or beverage
product, such as an alcoholic or non-alcoholic beverage, such as a
cereal- or malt-based beverage like beer or whiskey, said method
comprising the step of treating a starch comprising material with
enzyme according to embodiments 1-10, or a preparation according to
embodiment 14, or a composition according to any one of embodiments
15-26.
[0272] 38. Method for the production of a brewing mash, said method
comprising the step of treating a starch comprising material with
enzyme according to embodiments 1-10, or a preparation according to
embodiment 14, or a composition according to any one of embodiments
15-26.
[0273] 39. Method for the production of a first- or
second-generation biofuel, such as bioethanol, said method
comprising the step of treating a starch comprising material with
an enzyme according to embodiments 1-10, or a preparation according
to embodiment 14, or a composition according to any one of
embodiments 15-26.
[0274] 40. Product obtained by the method according to any one of
embodiments 38-39.
[0275] 41. A composition comprising the product according to
embodiment 40, such as wherein the product is in a range of
0.1%-99.9%.
EXAMPLES
Example 1
[0276] Methods and results in relation to xylanases/glucanase
filing for brew application.
[0277] The below methods have been used to screen for xylanases and
glucanases with application in brewing:
[0278] Methods:
[0279] Water Extractable Arabinoxylan (WE-AX) Xylanase Method:
[0280] Samples, to obtain approx. OD540=0.25-0.30 in this assay and
xylose standards (0, 0.125, 0.250, 0.375 and 0.500 mg/ml distilled
water) are prepared in distilled water. At time t=0 minutes, 1.75
ml soluble wheat arabinoxylan (0.5% wheat arabinoxylan (PWAXYH,
Megazyme, Bray, Ireland)) in 0.1M sodium acetate/acetic acid, pH 5)
is placed in a test tube at 50.degree. C. At time t=5 minutes, 250
.mu.l enzyme solution is added to the substrate at 50.degree. C.
followed by mixing. Distilled water is used as blank. At time t=15
minutes, 2 ml DNS solution (1% 3,5-Dinitrosalicylic acid (DNS),
1.6% sodium hydroxide, 30% potassium sodium tartrate in distilled
water) is added to the enzyme-substrate solution and 2.0 ml
standard solution. Samples, blanks and standards added DNS are
placed in a boiling water bath (95.degree. C.) for 5 minutes.
Hereafter samples, blanks and standards are cooled by placing them
in a 25.degree. C. water bath for 20 minutes. The Optical density
of all samples are read at OD540 using a spectrophotometer. Based
on the dilution of the samples, the amount of sample taking into
work and the standards, the xylanases activity of the sample can be
calculated.
[0281] One Unit of endo-1,4-beta-xylanase WE-AX activity is defined
as the amount of enzyme which produces 1 .mu.mole xylose
equivalents per minute under the conditions mentioned above (Water
extractable arabinoxylan (WE-AX) xylanase method).
[0282] Water Un-Extractable Arabinoxylan (WU-AX) Xylanase
Method:
[0283] Samples are prepared in distilled water. At time t=0
minutes, 1.75 ml Insoluble wheat (0.5% wheat arabinoxylan (PWAXYI,
Megazyme, Bray, Ireland)) in 0.1M sodium acetate/acetic acid, pH 5)
is placed in a test tube at 50.degree. C. At time t=5 minutes, 250
.mu.l enzyme solution is added to the substrate at 50.degree. C.
followed by mixing. Distilled water is used as blank. At time t=15
minutes, samples and blanks are placed in a boiling water bath
(95.degree. C.) for 5 minutes.
[0284] Hereafter samples and blanks are centrifuged to precipitate
residual insoluble substrate. The amount of arabinoxylan brough
into solution is determined using the method described by Rouau, X.
and Surget, A. (1994), Carbohydrate Polymers, 24, 123-132.
[0285] WU-AX endo-1,4-beta-xylanase activity is defined as the
amount of pentoses solubilised (.mu.g pentoses) under the
conditions described above giving a unit definition of .mu.g
pentose/gram of xylanase sample.
[0286] Xylanase Activity Assay
[0287] Samples, to obtain approx. OD540=0.25-0.30 in this assay and
xylose standards (0, 0.125, 0.250, 0.375 and 0.500 mg/ml distilled
water) are prepared in distilled water. At time t=0 minutes, 1.75
ml soluble wheat arabinoxylan (0.5% wheat arabinoxylan (PWAXYH,
Megazyme, Bray, Ireland)) in 0.1M sodium acetate/acetic acid, pH 5)
is placed in a test tube at 50.degree. C. At time t=5 minutes, 250
.mu.l enzyme solution is added to the substrate at 50.degree. C.
followed by mixing. Distilled water is used as blank. At time t=15
minutes, 2 ml DNS solution (1% 3,5-Dinitrosalicylic acid (DNS),
1.6% sodium hydroxide, 30% potassium sodium tartrate in distilled
water) is added to the enzyme-substrate solution and 2.0 ml
standard solution. Samples, blanks and standards added DNS are
placed in a boiling water bath (95.degree. C.) for 5 minutes.
Hereafter samples, blanks and standards are cooled by placing them
in a 25.degree. C. water bath for 20 minutes. The Optical density
of all samples are read at OD540 using a spectrophotometer. Based
on the dilution of the samples, the amount of sample taking into
work and the standards, the xylanases activity of the sample can be
calculated.
[0288] One Unit of endo-1,4-beta-xylanase WE-AX activity is defined
as the amount of enzyme which produces 1 .mu.mole xylose
equivalents per minute under the conditions mentioned above
[0289] Glucanase Activity Assay
[0290] Samples, to obtain OD.sub.540 within the standard curve in
this assay and glucose standards (0; 0.125; 0.250; 0.500; and 0.750
mg/ml distilled water) are prepared in distilled water. At time t=0
minutes, 1.75 ml barley beta-glucan (1.5% barley beta-glucan
(P-BGBM, Megazyme, Bray, Ireland)) in 1M sodium acetate/acetic
acid, pH 5) is placed in a test tube at 50.degree. C. At time t=5
minutes, 250 .mu.l enzyme solution is added to the substrate at
50.degree. C. followed by mixing. Distilled water is used as blank.
At time t=15 minutes, 2 ml DNS solution (1% 3,5-Dinitrosalicylic
acid (DNS), 1,6% sodium hydroxide, 30% potassium sodium tartrate in
distilled water) is added to the enzyme-substrate solution and 2.0
ml standard solution. Samples, blanks and standards added DNS are
placed in a boiling water bath (95.degree. C.) for 15 minutes.
Hereafter samples, blanks and standards are cooled by placing them
in a 25.degree. C. water bath for 20 minutes. The Optical density
of all samples are read at OD.sub.540 using a spectrophotometer.
Based on the dilution of the samples, the amount of sample taking
into work and the standards, the glucanase activity of the sample
can be calculated.
[0291] One unit of endo-1,3(4)-.beta.-glucanase activity is defined
as the amount of enzyme which produces 1 .mu.mole glucose
equivalents per minute under the conditions of the assay (pH 5.0
(or as specified) and 50.degree. C.).
[0292] Lab Scale Brewing Application Method:
[0293] Lab scale brewing application studies were conducted using
Pilsner malt:Barley in a 75:25 ratio at a water:grist ratio of 3:1
(150 ml:50 g grist). Initially water was preheated to 53.degree. C.
before mashing in and pH adjustment (5.4, 2 M H2SO4). After
regaining initial temperature (10 min period) the mashing profile
(see FIG. 1) is initiated and enzymes are added. After mashing off
wort separation is conducted using a conventional plastic funnel
and filter paper (paper filter No 1, 24 cm diameter, Whatman,
England). Filtration performance was evaluated as well as several
other wort parameters, such as i.e. viscosity, .beta.-glucan and
pentosan.
[0294] Wort filtration was measured for 30 min after which
filtration was terminated. Collected wort was cooled before any
further analysis.
[0295] Filtration
[0296] Filtration data are presented as volume wort collected after
5, 10, 15 and 30 minutes relative to a blank (brewing without added
exogenous enzymes).
[0297] Pilot Scale Brewing
[0298] Trials were conducted in a pilot scale brewing facility (2
HL capacity). Wort separation was conducted by lautering and beer
filtration by horizontal kiselguhr filtration.
[0299] To elucidate filtration optimization by combination of
glucanase and xylanase under "challenging" brewing conditions,
pilot scale brewing trails were conducted using a mixed grist
comprising of 75% malt and 25% barley. Initially, the water:grist
ratio was set at 2.8:1 (mash start) increasing to 3.1:1 at the
start of lautering. In comparison water:grist ratios around 3.2-3.8
are typical in full scale brew house lautering. Thus the current
pilot trial settings of a 3.1:1 water:grist ratio are believed to
be in the challenging end of the scale.
[0300] Malt and barley was ground dry using a two-roller mill. Both
barley and malt was milled twice using a roller distance of
.about.0.7 mm.
[0301] Mashing-in was conducted aiming at an initial mash
temperature of 53.degree. C. After mashing-in small adjustments
were conducted such as: mash volume adjustment for water:grist
ratio of 2.8:1 and pH adjustment to .about.5.56 (Lactic acid).
After fine tuning the mash, enzyme was added and the mashing
profile given in FIG. 1 was followed. Saccharification rest at
70.degree. C. was programmed to 15 min, however rest period was
extended by 5 min until an iodine test showed that no starch was
present. (Ludwig Narziss and Werner Back, Technische Universitaet
Muenchen (Fakultaet fuer Brauwesen, Weihenstephan), Abriss der
Bierbrauerei. WILEY-VCH Verlags GmbH Weinheim Germany, 2005).
[0302] Mashing-off was initiated after a 5 min rest at 78.degree.
C. Mash was transferred to the Lauter Tun, which was beforehand
prefilled with water to a height just below the "false bottom". The
mash was left to rest for 5 min for settling of filter cake. This
was followed by a 15 min recirculation (140 L/h) ensuring filter
cake settling and wort clarification. Typically in full scale
brewing, filtration will be initiated when a given wort turbidity
is obtained, however in the current trials recirculation was kept
constant at 15 min enabling comparison of trials. During lautering
the following data were collected, including time (min), wort
volume collected (L), filtration pressure difference across filter
cake (mmWC, mm Water Column), pump capacity (%), wort turbidity
(EBC) and mash temperature (.degree. C.).
[0303] The pressure build up across the filter cake during
filtration is believed to be a factor contributing to setting the
standard of the wort lautering performance. Reaching very high
pressure differences--e.g. 250 mmWC during first wort collection
and e.g. 450 mmWC for the reminder of the lautering--a filter cake
racking (also known as deep cut) is induced. Racking is a process
where a filter cake collapses or a filtration channel formation is
relieved by slowing cutting the filter cake with special designed
knives. Following filter cake racking a 6 min wort recirculation
(flow rate: 120 l/h) was introduced priming the filter cake for
continued filtration. Filter cake racking relieves an otherwise
compromised filtration performance which would otherwise also
result in poor wort quality. If no pressure induced racking has
been introduced by the beginning of the 3rd sparging, automatic
rackings were conducted at the beginning of the 3rd and 4th
spargings to ensure that no full filtration block would occur just
before finishing wort separation.
[0304] Lautering was conducted with the settings illustrated in
table 1.
TABLE-US-00004 TABLE 1 Lautering settings. Volumes collected (L),
filtration flow (L/h) and Sparging volumes (L). Volume Filtration
Sparging Wort collected, L flow, L/h volume, L First wort 0-60 130
1 st sparging 60-78 140 18 2nd sparging 78-96 160 18 3rd sparging
96-114 180 18 4th sparging 114-140 180 26
[0305] After end lautering, sweet wort was returned to the Mash
Tun, heated to boiling and hops were added. Hopping was continued
for 80 min and at the end of hopping pH is adjusting to
5.10.+-.0.05. Hops were cleared from the bitter wort by use of
whirlpool and following wort was cooled to .about.8.degree. C. For
fermentation, a bottom fermenting dried yeast (Saccharomyces
cerevisiae) W34/70 from Fermentis was chosen. Yeast was rehydrated
for 30 min and pitched at 100 g/HL. Main fermentation was hold for
5-6 days at 10.degree. C., followed by maturation at 15.degree. C.
until attenuated and Diacetyl below 80 ppb. Beer was stored for
another 2-3 weeks at 1.degree. C. and 0.7 bar before filtering.
[0306] Beer was filtered horizontally by use of 1.2 .mu.m PP-candle
plates and kieselguhr. Up to 8 plates could be included in the
filtration unit, resulting in a total filtration area of .about.0.5
m2. In the current studies 3 plates were included and filtration
was conducted at a flow rate of 130 L/h, resulting in a speed of
filtration of 6.9 HL/(hm2). In full scale breweries, speed of
filtration is usually set between 5-7 HL/(hm2). It is thus obvious
that the current settings are in the high end--a deliberate choice
challenging the beer filtration conditions to verify potential
benefits from the choice of using an enzyme in the brewing process.
During beer filtration, flow rates (L/h) as well as pressure values
(P-in and P-out) were monitored to verify beer filtration
performance. Also a number of beer analyses, such as Original
Gravity (OG), Apparent Extract (AE), Alcohol By Volume (ABV),
Apparent Degree of Fermentation (ADF), Reel Degree of Fermentation
(RDF), pH, colour and bitterness were conducted for evaluation of
beer quality.
[0307] Results:
[0308] Xylanases:
[0309] Xylanases were screened for their activity on soluble
substrate and insoluble substrate, their pH and temperature
characteristics.
[0310] Results are shown in table 2.
TABLE-US-00005 TABLE 2 Xylanases screened, their activity on
soluble (WE-AX) and insoluble (WU-AX) arabinoxylan substrate and
their biochemical characteristics in regard to temp and pH. Temp
T1/2 WU-AX/ opt, temp, pH Name Origin GH WE-AX WU-AX WE-AX .degree.
C. .degree. C. opt. AfuXyn2 Aspergillus 11 7798 68790526 8822 65 59
5.5 fumingatus AfuXyn3 Aspergillus 11 26283 99716865 3794 60 62 5
fumingatus AfuXyn5 Aspergillus 11 90005 714363158 7937 60 50 4
fumingatus BsuXyn3 Bacillus 11 82 1095357 13388 50 n.d. 6 subtilis,
BS3 BsuXyn4 Bacillus 11 54 1005400 18619 50 n.d. 6 subtilis, BS4
#160 TerXyn1 Geosmithia 10 1467 6208786 4232 78 >78 3 emersonii
AtuXyn3 Aspergillus 10 1220 7760982 6361 65 67 4.5 tubigensis
AtuXyn4 Aspergillus 11 1600 12934971 8084 45 58 5 tubigensis
AacXyn2 Aspergillus 10 777 3880491 4994 70 73 4 aculeatus TreXyn2
Trichoderma 11 2244 16015846 7137 55 n.d. 5 reesei TreXyn3
Trichoderma 10 21487 141108772 6567 60 64 5.5 reesei TreXyn5
Trichoderma 11 1410 8842816 6272 70 68 5 reesei n.d. = Not
determined
[0311] WE-AX and WU-AX enzyme activities (U) were measured as
described in sections "water extractable arabinoxylan (WE-AX)
xylanase method" and "water un-extractable arabinoxylan (WU-AX)
xylanase method".
[0312] Based on the results from the biochemical screening,
xylanases having an appropriate activity ratio on soluble vs.
insoluble arabinoxylan were chosen for further testing in
application trials. The results are shown in table 3.
TABLE-US-00006 TABLE 3 Xylanases screened and the relative extract
yield obtained using the xylanases versus a blank (without
xylanases). Finally the xylanases substrate specificity is
illustrated as a ration of their activity on insoluble vs. soluble
arabinoxylan (WU-AX/WE-AX). Filtration Performance 5 10 15 30
WU-AX/ Name Origin min min min min WE-AX Blank 1.00 1.00 1.00 1.00
BsuXyn3 Bacillus 0.93 0.95 0.96 0.95 13388 subtilis, BS3 BsuXyn4
Bacillus n.d. n.d. n.d. n.d. 18619 subtilis, BS4 #160 TerXyn1
Geosmithia 2.19 1.92 1.70 1.44 4232 emersonii (Taleromyces
emersonii) AtuXyn3 Aspergillus 2.06 1.75 1.59 1.37 6361 tubigensis
AtuXyn4 Aspergillus 1.02 1.01 1.01 1.01 8084 tubigensis AacXyn2
Aspergillus 2.07 1.86 1.67 1.43 4994 aculeatus TreXyn3 Trichoderma
2.41 2.02 1.81 1.55 6567 reesei TreXyn5 Trichoderma 2.06 1.75 1.59
1.37 6272 reesei
[0313] Filtration performance was measured as described earlier
("filtration"), and is presented as volume filtrate at the
different time points relative to the negative control (blank).
[0314] WE-AX and WU-AX enzyme activities (U) were measured as
described in sections "water extractable arabinoxylan (WE-AX)
xylanase method" and "water un-extractable arabinoxylan (WU-AX)
xylanase method".
[0315] Glucanases:
[0316] Glucanases were screened for their activity and temperature
characteristics, and the results are shown in table 4.
TABLE-US-00007 TABLE 4 Glucanases screened, their activity and
their biochemical characteristics in regard to temperature Temp
T1/2 T1/2 opt, temp_buffer, temp_wort, pH Name Origin U/ml .degree.
C. .degree. C. .degree. C. opt. TerGlu1 Talaromyces 7338 70 78 78 3
emersonii/ Geosmithia emersonii BsuGluS Bacillus 208 55-65 60 68
.sup. 5-6 subtilis BsuGlu103FULL Bacillus 391 50-60 53 58 .sup. 5-6
subtilis TreGlu2 Trichoderma 13 40-50 70 74 4.5-6 reesei TreGlu3
Trichoderma 9215 40-51 58 62 4.5-6 reesei TreGlu4 Trichoderma n.d.
40-52 62 62 4.5-6 reesei TreGlu6 Trichoderma n.d. 40-53 62 64 4.5-6
reesei TreGlu7 Trichoderma n.d. 40-54 62 62 4.5-6 reesei TreGlu8
Trichoderma n.d. 40-55 61 63 4.5-6 reesei BsuGluC CBD Bacillus 10
50-60 60 67 .sup. 5-6 subtilis n.d. = Not determined
[0317] Glucanase activity/units was/were determined as described in
the glucanase activity assay as described above.
[0318] Based on the results from the biochemical screening,
glucanases having suitable characteristics were chosen for further
testing in application trials. The results are shown in table
5.
TABLE-US-00008 TABLE 5 Name and origin of glucanases screened and
the relative extract yield obtained using the glucanases versus a
blank (without enzyme). Filtration performance Name Origin 5 min 10
min 15 min 30 min Blank Neg control 1.00 1.00 1.00 1.00 TerGlu1
Geosmithia 1.36 1.43 1.46 1.36 emersonii BsuGluS Bacillus 1.48 1.49
1.48 1.35 subtilis BsuGlu103FULL Bacillus 1.29 1.28 1.30 1.22
subtilis TreGlu2 Trichoderma 1.15 1.18 1.20 1.15 reesei TreGlu3
Trichoderma 1.29 1.32 1.30 1.22 reesei TreGlu4 Trichoderma 1.11
1.11 1.11 1.09 reesei TreGlu6 Trichoderma 1.13 1.15 1.13 1.10
reesei TreGlu7 Trichoderma 1.06 n.d. 1.01 1.02 reesei TreGlu8
Trichoderma 1.12 1.11 1.13 1.09 reesei BsuGluC CBD Bacillus 1.33
1.37 1.37 1.32 subtilis
[0319] Filtration performance was measured as described earlier
("filtration"), and is presented as volume filtrate at the
different time points relative to the negative control (blank).
[0320] Based on the individual screening of xylanases and
glucanases, combinatorial experiments were conducted, and results
are illustrated in table 6.
TABLE-US-00009 TABLE 6 Brewing application results from
combinatorial experiments of xylanases and glucanases versus a
blank and versus UltraFlo .RTM. Max. 250 Fungal Xylanase Units FXU-
S/g; 700 Cellulase Units EGU/g (Novozymes, Denmark) results are
illustrated as relative extract yield obtained. Filtration
Performance Name Origin 5 min 10 min 15 min 30 min Control 1.00
1.00 1.00 1.00 UFmax 0.1 A. aculeatus 2.29 2.13 2.00 1.77 BsuGluS/
B. sub/ 1.70 1.69 1.00 1.47 TauXyn1 T. aurantiacus BsuGluS/ B. sub/
2.57 2.14 1.96 1.75 AtuXyn3 A. tubingensis
[0321] (Origin of UltraFlo.RTM. Max may include other
microorganisms than A. aculeatus, such as described in
WO05059084)
[0322] Filtration performance was measured as described earlier
("filtration"), and is presented as volume filtrate at the
different time points relative to the negative control (blank).
[0323] Suitable combinations were further tested in a 2HL pilot
scale facility for verification, and results are shown in table 7
and FIG. 3.
TABLE-US-00010 TABLE 7 Pilot scale Brewing application results from
verification of the glucanase and xylanases screening. The B. sub
glucanase S combined with the A. tub xylanases were tested against
a blank and UltraFlo .RTM. Max. Data collected was the average flow
(L/h), the total pressure build up over the lautering (mm WC) and
the max pressure recorded during the lautering (mm WC). Avg Flow
Total pressure Max pressure ID (L/h) build up (mm WC) (mm WC) Blank
148 556 356 UltraFlo max 149 478 280 BsuGluS/ 147 263 163
AtuXyn3
Example 2
[0324] In this example it was attempted to show that xylanases for
brewing applications may have a very high selectivity for High
Molecular Weight Soluble-arabinoxylan (HMWS-AX) and water
extractable arabinoxylan (WE-AX). It is believed that hereby only
limited amounts of arabinoxylan need to be solubilised.
Consequently, the related off flavour potential is highly
reduced.
[0325] A significantly reduced viscosity is facilitating mash and
beer separation. Desired xylanase characteristics for brewing
applications may include one or more of the following aspects of
table 8:
TABLE-US-00011 TABLE 8 Screening criterias for xylanase selection
Enzyme substrate specificity WE-AX/WU-AX ratio has an impact on
viscosity Enzyme substrate selectivity How close to branch points
the enzyme will cut has an impact on the functionality Enzyme
thermostability Continuous solubilisation of AX during mashing -
thermostability is a key feature Enzyme pH optimum (pH 5.4-5.6)
Enzyme inhibition (e.g. known key factor for xylanases)
TABLE-US-00012 TABLE 9 Xylanases - biochem characteristics
Inhibition by endogenous cereal xylanase inhibitors occur in both
xylanase GH's Xylanase GH GH10 GH11 Mw +30 kDa 20 kDa Substrate
Hydrolyse close Need more specificity to Arabinose unsubstituted
substitutions Xylose to hydrolyse AX Substrate WE-AX/WU-AX
WE-AX/WU-AX selectivity typically >1 typically <1 SBD Often
separate No classical SBD SBD, but secondary BD on surface
Technological Viscosity Solubilizer/ effect reducers viscosity
reducers
[0326] Water-Unsoluble ArabinoXylan (WU-AX) in cereals as shown in
FIG. 3 is linked to filter cake stability in the brew house.
[0327] The concentration of ferulic acid (FA) in cereals very much
depends on the tissue. The highest concentration is found in the
pericarp material, whereas the concentration in the endosperm is
much lower. Different concentrations are reported. A concentration
of 2700 .mu.g/g insoluble fiber, 185 .mu.g/g soluble fiber is
likely (Bunzel et al. 2001, Journal of Sc. of food and agriculture,
vol. 81, p. 653-60).
[0328] To put this into perspective it means that FA is only found
for every 200th xylose molecules in arabinoxylan in insoluble fiber
(WU-AX) and for every 2500 xylose in soluble fiber (WE-AX).
[0329] It is a well-known fact that xylanases may lead to
off-flavor formation in beer such as free ferulic acid and
4-VG.
[0330] Methods:
[0331] Based on the criteria mentioned in table 8+9 more than 15
xylanases from DuPont Industrial Biosciences were found as
potential candidates. The xylanases were screened in laboratory
mashing application applying up to 30% barley in combination with
malt. Among others, mash separation speed, pentosan/arabinoxylan
level and wort viscosities were monitored. Top candidates where
tested at several pilot brewery plant studies to test our
hypothesis and link xylanase characteristics to functionality in
brewing. The optimal dosage of the selected xylanase candidate was
tested in combination with a .beta.-glucanase.
[0332] Results and Discussion:
TABLE-US-00013 TABLE 10 Sample ID Ref Control (X + B). X1 X2 X3
Dyn. 1.798 1.670 1.801 1.746 1.794 Viscosity (12.degree. Plato) mPa
s Extract 15.1 15.7 15.6 15.2 15.1 (.degree. Plato) Total 1610 1910
2440 2020 1710 pentosan (mg/l)
[0333] Pilot plant brews where enzyme dosage is the only variable.
Applying a WU-AX selective xylanase (X1) results in filter bed
collapse. WE-AX selective xylanase candidates (reference, X2, X3)
results in low pressure buildup. The reference is a blend of
xylanase+beta-glucanase.
TABLE-US-00014 TABLE 11 Wort analyses - pilot plant studies Sample
ID Ref. X + B Xh + B Extract (.degree. Plato) 15.70 16.00 15.95
Betaglucan in wort (mg/l) 44 35 25 Dyn. Viscosity at 1.65 1.68 1.68
12.degree. Plato (mPa s) Total pentosan (mg/l) 3540 2970 3010
TABLE-US-00015 TABLE 12 Strecker Aldehyd analysis of aged beer
Aging markers (forced aged beer) Unit Ref. X + B Xh + B 2-Me--Pr
ppb 25 24 22 2-Me--Bu ppb 3 2 3 3-Me--Bu ppb 9 7 8 Furfural ppb 113
85 93 Methional ppb 6 5 6 PheAcal ppb 10 9 10 T2N ppb 0.022 0.022
0.022
[0334] Optimized blends of a WE-AX selective xylanase applied at a
medium (X) and a high dosage (Xh) in combination with
.beta.-glucanase (B) on 20% barley/80% malt. The results indicate a
good mash and beer separation performance with a low risk of
off-flavor formation and filter bed collapse.
[0335] Conclusion
[0336] The study has proven the importance of applying xylanases
for brewing which are highly selective for the WE-AX during
mashing. The following benefits are achieved: [0337] Good mash
separation and beer filtration performance [0338] Minimized risk of
filter bed collapse at lautering [0339] Reduced potential for
off-flavor formation related to arabinoxylan breakdown [0340]
Tolerance towards xylanase overdose
[0341] Xylanases can often be applied with a high beneficial effect
in combination with beta-glucanases for separation control.
Example 3
[0342] Evaluation of X3/BglS (also referred to as AtuXyn3/BsuGluS)
combinations in 2 HL Pilot brewing trials
[0343] Material and Methods:
[0344] Experiments: Enzymes:
[0345] AtuXyn3 (X3)/BsuGluS (Bgls) (a): Combination of BglS
(Bacillus glucanase) and X3 (Aspergillus xylanase; BgLS: 0.50 mg
protein/kg grist and X3: 1.50 mg protein/kg grist).
[0346] AtuXyn3 (X3)/BsuGluS (Bgls) (b): As AtuXyn3 (X3)/BsuGluS
(Bgls) (a), but with 20% increase X3 dose to test robustness.
[0347] Reference: Benchmark enzyme product (Ultraflo.RTM. Max)
dosed at 0.20 kg/T grist.
[0348] Raw Material:
[0349] Adjunct material: barley 22% w/w.
[0350] Malt: Pilsner malt Chiraz 42.6% w/w, Pilsner malt Quench DMG
35.4% weight pr. weight (ww).
[0351] All material used for acid adjustment of pH, Calcium, Zink
and bitterness levels are food grade and considered as standard
brewing materials.
[0352] The recipe for the brew was aiming at a beer style as an
international lager beer.
[0353] Milling:
[0354] Kunzel 2 roller pilot mill. The milled material was passing
the rollers twice simulating a 4 roller mill.
[0355] Malt grist: the mill was running at 1.5 mm at the first pass
and 0.7 mm at the second pass of the rollers.
[0356] Barley grist: the mill was running at 1.5 mm at the first
pass and 0.4 mm at the second pass of the rollers.
[0357] Brewhouse 2 HL:
[0358] All brews were based on HGB (High Gravity Brewing) infusion
mashing and standard lautering of 190 L wort aiming at 16.degree.
Plato. During lautering which was performed at fixed flow, the
differential pressure was recorded (used as parameter for
evaluating lautering performance). All brew materials were milled
ahead of time (24 h) and kept in closed buckets prior to water
contact. All material was dumped in the mash kettle within the
first 3 minutes after start of mashing. Calcium and pH adjustment
were done prior to enzyme addition. pH (20.degree. C.) was
rechecked at the 52.degree. C. break. Iodine normality was
confirmed after 10 minutes at 72.degree. C. Lautering was performed
at 78.degree. C.
[0359] Lautering performance was evaluated on fixed flow at 90 l/H
during first wort collection. Flow was increased to 110 L/hour and
130 L/hour during sparging and weak wort collection. Chemical
analysis was performed on cold wort.
[0360] Wort Boiling:
[0361] Boiling was performed using an external boiler with 4-5%
evaporation. Hop extracts were added from the beginning of the wort
boiling aiming at 20 BU in the final beer.
[0362] Fermentation 50 L:
[0363] All fermentations were performed in 50 L cylindriconical
tanks. Fermentation was made according to standard operation
procedures. Pitching was done with 15.times.10.sup.6 live yeast
cells/ml. Yeast counts and viability was calculated using a Nucleo
counter.
[0364] Beer Processing:
[0365] Plate and frame filter operated at constant pressure. Flow
evaluation was done by weight.
[0366] Data was collected from 1 and 3 filter plates.
[0367] Debrewing:
[0368] All beers were de-brewed to 5.0% ABV (Alcohol By Volume),
considered as international lager beer standard.
[0369] Bottling:
[0370] CO.sub.2 was adjusted to 5.0 g/L. All beer samples were
bottled in 33 cl standard bottles on a McLennon automatic filling
machine using single evacuation.
[0371] Beer Analysis:
[0372] Fresh beers were analysed using GC-MS
[0373] Chemical aging profile was determined using GC-MS.
[0374] Results and observations: Mashing.
[0375] Mashing was performed with the following condition:
[0376] 52.degree. C. for 10 minutes simulating a 15-20 minutes
mashing using a running mill.
[0377] 65.degree. C. for 40 minutes.
[0378] 72.degree. C. for 30 minutes.
[0379] 78.degree. C. for 10 minutes.
[0380] All ramping steps were executed at 1.degree. C. A graphic
representation is given in FIG. 7.
[0381] All trials were made with this mashing regime aiming at a
16.degree. Plato brew. There were no remarks to this process
step.
[0382] Results and observations: Lautering.
[0383] The lautering was performed in the 2 hl brewery with a load
of 150 kg/m.sup.2. This is representative for a standard brew house
operation. Control of the lautering process was made as a fixed
flow at an average of 100 litre/hour. Initial flow rate is 90
litre/hour, increasing to 130 litre/hour during weak wort
collection. Differential pressure and in-line measurement of haze
was recorded for the four brews. Total lautering and wort
collection was done over approximately 2 hours.
[0384] Trial X3/BglS (b) and X3/BglS (a) are suggested to be the
trials that had the best lautering performance followed by trial
X3/BglS (a) and trial UF max with the worst performance.
TABLE-US-00016 TABLE 13 Data collected during lautering of the mash
from the four trials. UF X3/BglS X3/BglS X3/BglS max (a) (b) (a)
Lauter tun load (kg/m3) 153 153 153 153 Lauter tun time (min) 154
164 170 154 Diff. Pressure (cm) 40 30 30 30 Racking (# deep cuts) 1
1 1 1 Haze (EBC) 10 15 10 10 First wort pressure 40 33 31 30 build
up (cm/h) Time to first deep cut 45 60 120 115 (min) "Diff.
Pressure" and "First wort pressure build up" in the table was
measured as cmWC (cm Water column) and not as (cm) and (cm/h)
respectively.
[0385] Results and observations: Wort analysis after boiling.
[0386] Analysis of the cold wort shows similar results. The
beta-glucan analysis indicates a slight difference between the
samples.
TABLE-US-00017 TABLE 14 Chemical analysis of the cold wort. UF
X3/BglS X3/BglS X3/BglS Wort max (a) (b) (a) Extract 16.09 16.05
15.99 16.1 (% plato) Color (EBC) 9.7 9.3 9.3 9.3 pH 5 5.2 5.2 5.2
Iodine (Y/N) N N N N Bitterness 52 51 46 50 (BU, EBC)
TABLE-US-00018 TABLE 15 Analytical data on cold wort. UF X3/BglS
X3/BglS X3/BglS max (a) (b) (a) Beta-glucan in wort 49 40 25 30
(mg/L) Dyn. Viscosity at 1,888 1,685 1,679 1,686 12.degree. C. (mPa
s) Pentosan (mg/l) 3365 2975 3014 2964 Ferulic acid (ug/ml) 4.3 3.9
3.8 3.9 4-VG (ug/ml) <0.49 <0.49 <0.49 <0.49
(12.degree. C. is 12.degree. Plato); (% Plato may be used
interchangeably with .degree. Plato)
[0387] Results and observations: Fermentation.
[0388] Analysis of the green beer is given in table 16.
TABLE-US-00019 TABLE 16 Green beer analysis. UF X3/BglS X3/BglS
X3/BglS Green beer max (a) (b) (a) Alcohol (% vol) 6.79 6.79 6.7
6.86 Real extract (% P) 6.28 6 6 6 RDF (%) 63.5 63.7 63.5 64.4
Original extract 16.29 16.25 16.09 16.24 (% P) Color (EBC) 8.3 8.5
-- -- pH 4.4 4.4 4.4 4.4 SO2 (ppm) 7 9 11 10 Bitterness (BU, EBC)
29 28 27 27
[0389] The green beer analysis shows a high degree of similarity
between the trials. All trials have relative low RDF, but this is
normally seen with the inclusion of 22% barley calculated on the
basis of weight per weight (ww).
[0390] Results and observations: Beer filtration.
[0391] Beer samples were filtered using a plate and frame filter
using a fixed pressure. Two kegs of approximately 15 kg were
filtered and the individual keg filtration data are presented in
table 17. The first keg was filtered using 1 filter sheet and the
second keg was filtered using 3 filter sheets. The differential
pressure was always 0.5 bar. The filter plates are KD7 (20
cm.times.20 cm) from Begerow.
[0392] The overall picture of the filtrations curves from either 1
or 3 filter plate filtrations is the same. We believe that the 1
filter plate record may be too sensitive to show the real ratio
difference.
TABLE-US-00020 TABLE 17 Keg filtration data from the four trials UF
X3/BglS X3/BglS X3/BglS Filtration max (a) (b) (a) Filtration speed
- 4.8 5.6 9.9 11.8 1 filter sheet (L/h) Filtration speed - 77.2
59.6 70.6 105.4 3 filter sheet (L/h) Results and observations:
Final beer analysis.
[0393] Trial beers were analysed according to standard operation
procedures (EBC) and presented in table 18.
TABLE-US-00021 TABLE 18 Final beer analysis. UF X3/BglS X3/BglS
X3/BglS Finished beer max (a) (b) (a) alcohol (%) 4.82 4.89 5.01
4.92 Real extract (% P) 4.5 4.6 4.6 4.4 RDF (%) 63.2 63.4 63.8 64.3
Original extract 11.85 11.99 12.19 11.89 (% P) Color (EBC) 4.8 4.9
5 5 pH 4.4 4.4 4.4 4.4 SO2 (ppm) 13 13 10 6 Bitterness (BU, 22 22
20 18 EBC) Haze (EEC) 0.43 0.4 0.38 0.4 Total haze - 8.6 12.9 7.3
6.2 5 d-60 dg C. (EBC) CO2 (g/L) 4.9 5.3 5.1 5.2 Diacetyl (ppb) 12
11 8 10 Head retention (S) 107 111 119 108 Foam volume (ml) 452 476
460 470
[0394] Results and observations: Strecker aldehydes and "age
markers" in final beer.
[0395] Analysis was performed both on fresh and aged beer. Strecker
aldehydes and the "age and heat markers" (2-Me-Pr (2-methyl
Propanal), 2-Me-Bu (2-methyl Butanal), 3-Me-Bu (3-methyl Butanal),
Furfural, Methional, PheAcal (phenyl Acetaldehyde) and T2N
(trans-2-nonenal)) were analysed by GC-MS on both fresh and aged
beer. The data from analysis of fresh beer is presented in table
19.
TABLE-US-00022 TABLE 19 Strecker aldehyde analysis of fresh beer.
Markers for heat and aging (Furfural and trans-2-Nonenal) are used
as sample control. Aging markers UF X3/BglS X3/BglS X3/BglS (fresh
beer) max (a) (b) (a) 2-ME--Pr (ppb) 5 5 5 6 2-ME--Bu (ppb) 2 2 2 2
3-ME--Bu (ppb) 6 6 6 6 Furfural (ppb) 10 11 11 10 Methional (ppb) 4
4 4 4 PheAcal (ppb) 6 6 6 7 T2N (ppb) 0.0011 0.005 0.004 0.006
[0396] The trial beers were incubated at 37.degree. C. for 2 weeks
prior to the Strecker aldehyde analysis. The data for aged beer
samples are presented in table 20.
TABLE-US-00023 TABLE 20 Strecker aldehyde analysis of aged beer.
Markers for heat and aging (furfural and trans-2-Nonenal) is used
as a sample control. Aging markers UF X3/BglS X3/BglS X3/BglS
(forced aged beer) max (a) (b) (a) 2-ME--Pr (ppb) 25 23 22 26
2-ME--Bu (ppb) 3 3 3 2 3-ME--Bu (ppb) 9 7 7 8 Furfural (ppb) 111 92
93 78 Methional (ppb) 6 5 6 6 PheAcal (ppb) 10 9 9 10 T2N (ppb)
0.017 0.022 0.022 0.022
[0397] The data presented in table 20 show an expected increase in
Strecker aldehyde level. The increase in furfural and
trans-2-Nonenal reach an expected level.
[0398] Conclusion:
[0399] Based on the pilot scale experiments, we can conclude that
the ratios of the BglS and X3 tested in this Experiment performs as
good or even better than the reference UltraFlo Max in pilot scale
brewing.
[0400] The results are surprising, seen in the light of the
challenging raw material used, 22% barley inclusion in combination
with the 300 mg/I 6-glucan containing malt. The performance is not
only seen in the mash separation results, also in the beer
filtration. Due to the low solubilisation of cell wall material
when using the BREW2 (pentosan data), a lower degree of cell wall
material that might cause quality issues in relation to off-taste
and stability, can be recorded.
[0401] Finally it can be concluded that a 20% increase in the dose
of the xylanase component in X3/Bgls (b) appears not to have any
impact on any of the evaluated parameters, indicating that X3/Bgls
(a) is a robust enzyme combination.
TABLE-US-00024 Sequences: AtuXyn3, Aspergillus tubigensis,302 aa
(SEQ ID NO: 1)
QASVSIDTKFKAHGKKYLGNIGDQYTLTKNSKTPAIIKADFGALTPENSMKWDATEPSRGQFSFSGS
DYLVNFAQSNNKLIRGHTLVWHSQLPSWVQAITDKNTLIEVMKNHITTVMQHYKGKIYAWDVVNEIF
NEDGSLRDSVFYQVIGEDYVRIAFETARAADPNAKLYINDYNLDSASYPKLTGMVSHVKKWIEAGIP
IDGIGSQTHLSAGGGAGISGALNALAGAGTKEIAVTELDIAGASSTDYVEVVEACLDQPKCIGITVW
GVADPDSWRSSSTPLLFDSNYNPKPAYTAIANAL TerXynl, Geosmithia emersonii
(Taleromyces emersonii) (SEQ ID NO: 2)
AGLNTAAKAIGLKYFGTATDNPELSDTAYETQLNNTQDFGQLTPANSMKWDATEPEQNVFTFSAGDQ
IANLAKANGQMLRCHNLVWYNQLPSWVTSGSWTNETLLAAMKNHITNVVTHYKGQCYAWDVVNEALN
DDGTYRSNVFYQYIGEAYIPIAFATAAAADPNAKLYYNDYNIEYPGAKATAAQNLVKLVQSYGARID
GVGLQSHFIVGETPSTSSQQQNMAAFTALGVEVAITELDIRMQLPETEALLTQQATDYQSTVQACAN
TKGCVGITVWDWTDKYSWVPSTFSGYGDACPWDANYQKKPAYEGILTGLGQTVISTTYIISPTTSVG
IGTTTSSGGSGGTTGVAQHWEQCGGLGWTGPTVCASGYTCTVINEYYSQCL AtuXyn4,
Aspergillus tubigensis (SEQ ID NO: 3)
EPIEPRQASVSIDTKFKAHGKKYLGNIGDQYTLTKNSKTPAIIKADFGALTPENSMKWDATEPSRGQF
SFSGSDYLVNFAQSNNKLIRGHTLVWHSQLPSWVQSITDKNTLIEVMKNHITTVMQHYKGKIYAWDVV
NEIFNEDGSLRDSVFYKVIGEDYVRIAFETARAADPNAKLYINDYNLDSASYPKLTGMVSHVKKWIAA
GIPIDGIGSQTHLSAGGGAGISGALNALAGAGTKEIAVTELDIAGASSTDYVEVVEACLNQPKCIGIT
VWGVADPDSWRSSSTPLLFDSNYNPKPAYTAIANAL AacXyn2, Aspergillus aculeatus
(SEQ ID NO: 4)
MVGLLSITAALAATVLPNIVSAVGLDQAAVAKGLQYFGTATDNPELTDIPYVTQLNNTADFGQITPGN
SMKWDATEPSQGTFTFTKGDVIADLAEGNGQYLRCHTLVWYNQLPSWVTSGTWTNATLTAALKNHITN
VVSHYKGKCLHWDVVNEALNDDGTYRTNIFYTTIGEAYIPIAFAAAAAADPDAKLFYNDYNLEYGGAK
AASARAIVQLVKNAGAKIDGVGLQAHFSVGTVPSTSSLVSVLQSFTALGVEVAYTEADVRILLPTTAT
TLAQQSSDFQALVQSCVQTTGCVGFTIWDWTDKYSWVPSTFSGYGAALPWDENLVKKPAYNGLLAGMG
VTVTTTTTTTTATATGKTFTTFTGATSTGTTAAHWGQCGGLNWSGPTACATGYTCTYVNDYYSQCL
TreXyn3, Trichoderma reesei (SEQ ID NO: 5)
MKANVILCLLAPLVAALPTETIHLDPELAALRANLTERTADLWDRQASQSIDQLIKRKGKLYFGT
ATDRGLLQREKNAAIIQADLGQVTPENSMKWQSLENNQGQLNWGDADYLVNFAQQNGKSIRGHTL
IWHSQLPAWVNNINNADTLRQVIRTHVSTVVGRYKGKIRAWDVVNEIFNEDGILRSSVFSRLLGE
EFVSIAFRAARDADPSARLYINDYNLDRANYGKVNGLKTYVSKWISQGVPIDGIGSQSHLSGGGG
SGTLGALQQLATVPVTELAITELDIQGAPTTDYTQVVQACLSVSKCVGITVWGISDKDSWRASTN
PLLFDANFNPKPAYNSIVGILQ TreXyn5, Trichoderma reesei (SEQ ID NO: 6)
QCIQPGTGYNNGYFYSYWNDGHGGVTYCNGPGGQFSVNWSNSGNFVGGKGWQPGTKNRVINFSGSY
NPNGNSYLSVYGWSRNPLIEYYIVENFGTYNPSTGATKLGEVTSDGSVYDIYRTQRVNQPSIIGTA
TFYQYWSVRRNHRSSGSVNTANHFNAWAQQGLTLGTMDYQIVAVEGYFSSGSASITVSD
BsuGluS, Bacillus subtilis, 214 aa (SEQ ID NO: 7)
QTGGSFFDPFNGYNSGFWQKADGYSNGNMFNCTWRANNVSMTSLGEMRLALTSPAYNKFDCGENRSV
QTYGYGLYEVRMKPAKNTGIVSSFFTYTGPTDGTPWDEIDIEFLGKDTTKVQFNYYTNGAGNHEKIV
DLGFDAANAYHTYAFDWQPNSIKWYVDGQLKHTATNQIPTTPGKIMMNLWNGTGVDEWLGSYNGVNP
LYAHYDWVRYTKK TerGlu1, Geosmithia emersonii (Taleromyces emersonii)
(SEQ ID NO : 8)
APVKEKGIKKRASPFQWFGSNESGAEFGNNNIPGVEGTDYTFPNTSAIQILIDQGMNIFRVPFLMER
MVPNQMTGPVDSAYFQGYSQVINYITSHGASAVIDPHNFGRYYNNIISSPSDFQTFWHTIASNFADN
DNVIFDTNNEYHDMDESLVVQLNQAAIDGIRAAGATSQYIFVEGNSWTGAWTWTQVNDAMANLTDPQ
NKIVYEMHQYLDSDGSGTSDQCVNSTIGQDRVESATAWLKQNGKKAILGEYAGGANSVCETAVTGML
DYLANNTDVWTGAIWWAAGPWWGDYIFSMEPPSGIAYEQVLPLLQPYL BsuGlu103FULL,
Bacillus subtilis (SEQ ID NO: 9)
DDYSVVEEHGQLSISNGELVNERGEQVQLKGMSSHGLQWYGQFVNYESMKWLRDDWGITVFRAAMYT
SSGGYIDDPSVKEKVKETVEAAIDLGIYVIIDWHILSDNDPNIYKEEAKDFFDEMSELYGDYPNVIY
EIANEPNGSDVTWDNQIKPYAEEVIPVIRDNDPNNIVIVGTGTWSQDVHHAADNQLADPNVMYAFHF
YAGTHGQNLRDQVDYALDQGAAIFVSEWGTSAATGDGGVFLDEAQVWIDFMDERNLSWANWSLTHKD
ESSAALMPGANPTGGWTEAELSPSGTFVREKIRESASIPPSDPTPPSDPGEPDPGEPDPTPPSDPGE
YPAWDSNQIYTNEIVYHNGQLWQAKWWTQNQEPGDPYGPWEPLKSDPDSGEPDPTPPSDPGEYPAWD
SNQIYTNEIVYHNGQLWQAKWWTQNQEPGDPYGPWEPLN TreGlu2, Trichoderma reesei
(SEQ ID NO: 10)
QQTVWGQCGGIGWSGPTNCAPGSACSTLNPYYAQCIPGATTITTSTRPPSGPTTTTRATSTSSSTPP
TSSGVRFAGVNIAGFDFGCTTDGTCVTSKVYPPLKNFTGSNNYPDGIGQMQHFVNDDGMTIFRLPVG
WQYLVNNNLGGNLDSTSISKYDQLVQGCLSLGAYCIVDIHNYARWNGGIIGQGGPTNAQFTSLWSQL
ASKYASQSRVWFGIMNEPHDVNINTWAATVQEVVTAIRNAGATSQFISLPGNDWQSAGAFISDGSAA
ALSQVTNPDGSTTNLIFDVHKYLDSDNSGTHAECTTAINIDGAFSPLATWLRQNNRQAILTETGGGN
VQSCIQDMCQQIQYLNQNSDVYLGYVGWGAGSFDSTYVLTETPTGSGNSWTDTSLVSSCLARK
TreGlu3, Trichoderma reesei (SEQ ID NO: 11)
QTSCDQWATFTGNGYTVSNNLWGASAGSGFGCVTAVSLSGGASWHADWQWSGGQNNVKSYQNSQI
AIPQKRTVNSISSMPTTASWSYSGSNIRANVAYDLFTAANPNHVTYSGDYELMIWLGKYGDIGPI
GSSQGTVNVGGQSWILYYGYNGAMQVYSFVAQINTTNYSGDVKNFFNYLRDNKGYNAAGQYVLSY
QFGTEPFTGSGTLNVASWTASIN TreGlu4, Trichoderma reesei (SEQ ID NO: 12)
HGHINDIVINGVWYQAYDPTTFPYESNPPIVVGWTAADLDNGFVSPDAYQNPDIICHKNATNAKGHA
SVKAGDTILFQWVPVPWPHPGPIVDYLANCNGDCETVDKTTLEFFKIDGVGLLSGGDPGTWASDVLI
SNNNTWVVKIPDNLAPGNYVLRHEIIALHSAGQANGAQNYPQCFNIAVSGSGSLQPSGVLGTDLYHA
TDPGVLINIYTSPLNYIIPGPTVVSGLPTSVAQGSSAATATASATVPGGGSGPTSRTFTTARTTQAS
SRPSSTPPATTSAPAGGPTQTLYGQCGGSGYSGPTRCAPPATCSTNPYYAQCLN TreGlu6,
Trichoderma reesei (SEQ ID NO: 13)
AFSWKNVKLGGGGGFVPGIIFHPKTKGVAYARTDIGGLYRLNADDSWTAVTDGIADNAGWHNWGIDAV
ALDPQDDQKVYAAVGMYTNSWDPSNGAIIRSSDRGATWSFTNLPFKVGGNMPGRGAGERLAVDPANSN
HYFGARSGNGLWKSTDGGVTFSKVSSFTATGTYIPDPSDSNGYNSDKQGLMWVTFDSTSSTTGGATSR
IFVGTADNITASVYVSTNAGSTWSAVPGQPGKYFPHKAKLQPAEKALYLTYSWWPDAQLFRSTDSGTF
WSPIWAWASYPTETYYYSISTPKAPWIKNNFIDVTSESPSDGLIKRLGWMIESLEIDPTDSNHWLYGT
GMTIFGGHDLTNWDTRHNVSIQSLADGIEEFSVQDLASAPGGSELLAAVGDDNGFTFASRNDLGTSPQ
TVWATPTWATSTSVDYAGNSVKSVVRVGNTAGTQQVAISSDGGATWSIDYAADTSMNGGTVAYSADGD
TILWSTASSGVQRSQFQGSFASVSSLPAGAVIASDKKTNSVFYAGSGSTFYVSKDTGSSFTRGPKLGS
AGTIRDIAAHPTTAGTLYVSTDVGIFRSTDSGTTFGQVSTALTNTYQIALGVGSGSNWNLYAFGTGPS
GARLYASGDSGASWTDIQGSQGFGSIDSTKVAGSGSTAGQVYVGTNGRGVFYAQGTVGGGTGGTSSST
KQSSSSTSSASSSTFLRSSVVSTTRASTVTSSRTSSAAGPTGSGVAGHYAQCGGIGWTGPTQCVAPYV
CQKQNDYYYQCV TreGlu7, Trichoderma reesei (SEQ ID NO: 14)
HGQVQNFTINGQYNQGFILDYYYQKQNTGHFPNVAGWYAEDLDLGFISPDQYTTPDIVCHKNAAPGAI
SATAAAGSNIVFQWGPGVWPHPYGPIVTYVVECSGSCTTVNKNNLRWVKIQEAGINYNTQVWAQQDLI
NQGNKWTVKIPSSLRPGNYVFRHELLAAHGASSANGMQNYPQCVNIAVTGSGTKALPAGTPATQLYKP
TDPGILFNPYTTITSYTIPGPALWQG TreGlu8, Trichoderma reesei (SEQ ID NO:
15)
GKIKYLGVAIPGIDFGCDIDGSCPTDTSSVPLLSYKGGDGAGQMKHFAEDDGLNVFRISATWQF
VLNNTVDGKLDELNWGSYNKVVNACLETGAYCMIDMHNFARYNGGIIGQGGVSDDIFVDLWVQI
AKYYEDNDKIIFGLMNEPHDLDIEIWAQTCQKVVTAIRKAGATSQMILLPGTNFASVETYVSTG
SAEALGKITNPDGSTDLLYFDVHKYLDINNSGSHAECTFDNVDAFNDFADWLRQNKRQAIISET
GASMEPSCMTAFCAQNKAISENSDVYIGFVGWGAGSFDTSYILTLTPLGKPGNYTDNKLMNECI
LDQFTLDEKYRPTPTSISTAAEETATATATSDGDAPSTTKPIFREETASPTPNAVTKPSPDTSD
SSDDDKDSAASMSAQGLTGTVLFTVAALGYMLVAF BsuGluC CBD, Bacillus subtilis
(SEQ ID NO: 16)
MKRSISIFITCLLITLLTMGGMIASPASAAGTKTPVAKNGQLSIKGTQLVNRDGKAVQLKGISS
HGLQWYGEYVNKDSLKWLRDDWGITVFRAAMYTADGGYIDNPSVKNKVKEAVEAAKELGIYVII
DWHILNDGNPNQNKEKAKEFFKEMSSLYGNTPNVIYEIANEPNGDVNWKRDIKPYAEEVISVIR
KNDPDNIIIVGTGTWSQDVNDAADDQLKDANVMYALHFYAGTHGQFLRDKANYALSKGAPIFVT
EWGTSDASGNGGVFLDQSREWLKYLDSKTISWVNWNLSDKQESSSALKPGASKTGGWRLSDLSA
SGTFVRENILGTKDSTKDIPETPSKDKPTQENGISVQYRAGDGSMNSNQIRPQLQIKNNGNTTV
DLKDVTARYWYKAKNKGQNFDCDYAQIGCGNVTHKFVTLHKPKQGADTYLELGFKNGTLAPGAS
TGNIQLRLHNDDWSNYAQSGDYSFFKSNTFKTFKKITLYDQGKLIWGTEPN BsuXyn3,
Bacillus subtilis xylanase variant (SEQ ID NO: 17)
ASTDYWQNWTFGGGIVNAVNGSGGNYSVNWSNTGNFVVGKGWTTGSPFRTINYNAGVWAPNGNGYL
TLYGWTRSPLIEYYVVDSWGTYRPTGTYKGTVKSDGGTYDIYTTTRYNAPSIDGDDTTFTQYWSVR
QSKRPTGSNATITFSNHVNAWKSHGMNLGSNWAYQVMATEGYQSSGSSNVTVW BsuXyn4,
Bacillus subtilis xylanase variant (SEQ ID NO: 18)
ASTDYWQNWTDGYGIVNAVNGSGGNYSVNWSNTGNFVVGKGWTTGSPFRTINYNAGVWAPNGNGYL
TLYGWTRSPLIEYYVVDSWGTYRPTGTYKGTVYSDGGWYDIYTATRDNAPSIDGDFTTFTQYWSVR
QSKRPTGSNATITFSNHVNAWRSHGMDLGSNWAYQVMATEGYLSSGSSNVTVW
Sequence CWU 1
1
181302PRTAspergillus tubigensis 1Gln Ala Ser Val Ser Ile Asp Thr
Lys Phe Lys Ala His Gly Lys Lys 1 5 10 15 Tyr Leu Gly Asn Ile Gly
Asp Gln Tyr Thr Leu Thr Lys Asn Ser Lys 20 25 30 Thr Pro Ala Ile
Ile Lys Ala Asp Phe Gly Ala Leu Thr Pro Glu Asn 35 40 45 Ser Met
Lys Trp Asp Ala Thr Glu Pro Ser Arg Gly Gln Phe Ser Phe 50 55 60
Ser Gly Ser Asp Tyr Leu Val Asn Phe Ala Gln Ser Asn Asn Lys Leu 65
70 75 80 Ile Arg Gly His Thr Leu Val Trp His Ser Gln Leu Pro Ser
Trp Val 85 90 95 Gln Ala Ile Thr Asp Lys Asn Thr Leu Ile Glu Val
Met Lys Asn His 100 105 110 Ile Thr Thr Val Met Gln His Tyr Lys Gly
Lys Ile Tyr Ala Trp Asp 115 120 125 Val Val Asn Glu Ile Phe Asn Glu
Asp Gly Ser Leu Arg Asp Ser Val 130 135 140 Phe Tyr Gln Val Ile Gly
Glu Asp Tyr Val Arg Ile Ala Phe Glu Thr 145 150 155 160 Ala Arg Ala
Ala Asp Pro Asn Ala Lys Leu Tyr Ile Asn Asp Tyr Asn 165 170 175 Leu
Asp Ser Ala Ser Tyr Pro Lys Leu Thr Gly Met Val Ser His Val 180 185
190 Lys Lys Trp Ile Glu Ala Gly Ile Pro Ile Asp Gly Ile Gly Ser Gln
195 200 205 Thr His Leu Ser Ala Gly Gly Gly Ala Gly Ile Ser Gly Ala
Leu Asn 210 215 220 Ala Leu Ala Gly Ala Gly Thr Lys Glu Ile Ala Val
Thr Glu Leu Asp 225 230 235 240 Ile Ala Gly Ala Ser Ser Thr Asp Tyr
Val Glu Val Val Glu Ala Cys 245 250 255 Leu Asp Gln Pro Lys Cys Ile
Gly Ile Thr Val Trp Gly Val Ala Asp 260 265 270 Pro Asp Ser Trp Arg
Ser Ser Ser Thr Pro Leu Leu Phe Asp Ser Asn 275 280 285 Tyr Asn Pro
Lys Pro Ala Tyr Thr Ala Ile Ala Asn Ala Leu 290 295 300
2386PRTGeosmithia emersonii 2Ala Gly Leu Asn Thr Ala Ala Lys Ala
Ile Gly Leu Lys Tyr Phe Gly 1 5 10 15 Thr Ala Thr Asp Asn Pro Glu
Leu Ser Asp Thr Ala Tyr Glu Thr Gln 20 25 30 Leu Asn Asn Thr Gln
Asp Phe Gly Gln Leu Thr Pro Ala Asn Ser Met 35 40 45 Lys Trp Asp
Ala Thr Glu Pro Glu Gln Asn Val Phe Thr Phe Ser Ala 50 55 60 Gly
Asp Gln Ile Ala Asn Leu Ala Lys Ala Asn Gly Gln Met Leu Arg 65 70
75 80 Cys His Asn Leu Val Trp Tyr Asn Gln Leu Pro Ser Trp Val Thr
Ser 85 90 95 Gly Ser Trp Thr Asn Glu Thr Leu Leu Ala Ala Met Lys
Asn His Ile 100 105 110 Thr Asn Val Val Thr His Tyr Lys Gly Gln Cys
Tyr Ala Trp Asp Val 115 120 125 Val Asn Glu Ala Leu Asn Asp Asp Gly
Thr Tyr Arg Ser Asn Val Phe 130 135 140 Tyr Gln Tyr Ile Gly Glu Ala
Tyr Ile Pro Ile Ala Phe Ala Thr Ala 145 150 155 160 Ala Ala Ala Asp
Pro Asn Ala Lys Leu Tyr Tyr Asn Asp Tyr Asn Ile 165 170 175 Glu Tyr
Pro Gly Ala Lys Ala Thr Ala Ala Gln Asn Leu Val Lys Leu 180 185 190
Val Gln Ser Tyr Gly Ala Arg Ile Asp Gly Val Gly Leu Gln Ser His 195
200 205 Phe Ile Val Gly Glu Thr Pro Ser Thr Ser Ser Gln Gln Gln Asn
Met 210 215 220 Ala Ala Phe Thr Ala Leu Gly Val Glu Val Ala Ile Thr
Glu Leu Asp 225 230 235 240 Ile Arg Met Gln Leu Pro Glu Thr Glu Ala
Leu Leu Thr Gln Gln Ala 245 250 255 Thr Asp Tyr Gln Ser Thr Val Gln
Ala Cys Ala Asn Thr Lys Gly Cys 260 265 270 Val Gly Ile Thr Val Trp
Asp Trp Thr Asp Lys Tyr Ser Trp Val Pro 275 280 285 Ser Thr Phe Ser
Gly Tyr Gly Asp Ala Cys Pro Trp Asp Ala Asn Tyr 290 295 300 Gln Lys
Lys Pro Ala Tyr Glu Gly Ile Leu Thr Gly Leu Gly Gln Thr 305 310 315
320 Val Thr Ser Thr Thr Tyr Ile Ile Ser Pro Thr Thr Ser Val Gly Thr
325 330 335 Gly Thr Thr Thr Ser Ser Gly Gly Ser Gly Gly Thr Thr Gly
Val Ala 340 345 350 Gln His Trp Glu Gln Cys Gly Gly Leu Gly Trp Thr
Gly Pro Thr Val 355 360 365 Cys Ala Ser Gly Tyr Thr Cys Thr Val Ile
Asn Glu Tyr Tyr Ser Gln 370 375 380 Cys Leu 385 3308PRTAspergillus
tubigensis 3Glu Pro Ile Glu Pro Arg Gln Ala Ser Val Ser Ile Asp Thr
Lys Phe 1 5 10 15 Lys Ala His Gly Lys Lys Tyr Leu Gly Asn Ile Gly
Asp Gln Tyr Thr 20 25 30 Leu Thr Lys Asn Ser Lys Thr Pro Ala Ile
Ile Lys Ala Asp Phe Gly 35 40 45 Ala Leu Thr Pro Glu Asn Ser Met
Lys Trp Asp Ala Thr Glu Pro Ser 50 55 60 Arg Gly Gln Phe Ser Phe
Ser Gly Ser Asp Tyr Leu Val Asn Phe Ala 65 70 75 80 Gln Ser Asn Asn
Lys Leu Ile Arg Gly His Thr Leu Val Trp His Ser 85 90 95 Gln Leu
Pro Ser Trp Val Gln Ser Ile Thr Asp Lys Asn Thr Leu Ile 100 105 110
Glu Val Met Lys Asn His Ile Thr Thr Val Met Gln His Tyr Lys Gly 115
120 125 Lys Ile Tyr Ala Trp Asp Val Val Asn Glu Ile Phe Asn Glu Asp
Gly 130 135 140 Ser Leu Arg Asp Ser Val Phe Tyr Lys Val Ile Gly Glu
Asp Tyr Val 145 150 155 160 Arg Ile Ala Phe Glu Thr Ala Arg Ala Ala
Asp Pro Asn Ala Lys Leu 165 170 175 Tyr Ile Asn Asp Tyr Asn Leu Asp
Ser Ala Ser Tyr Pro Lys Leu Thr 180 185 190 Gly Met Val Ser His Val
Lys Lys Trp Ile Ala Ala Gly Ile Pro Ile 195 200 205 Asp Gly Ile Gly
Ser Gln Thr His Leu Ser Ala Gly Gly Gly Ala Gly 210 215 220 Ile Ser
Gly Ala Leu Asn Ala Leu Ala Gly Ala Gly Thr Lys Glu Ile 225 230 235
240 Ala Val Thr Glu Leu Asp Ile Ala Gly Ala Ser Ser Thr Asp Tyr Val
245 250 255 Glu Val Val Glu Ala Cys Leu Asn Gln Pro Lys Cys Ile Gly
Ile Thr 260 265 270 Val Trp Gly Val Ala Asp Pro Asp Ser Trp Arg Ser
Ser Ser Thr Pro 275 280 285 Leu Leu Phe Asp Ser Asn Tyr Asn Pro Lys
Pro Ala Tyr Thr Ala Ile 290 295 300 Ala Asn Ala Leu 305
4406PRTAspergillus aculeatus 4Met Val Gly Leu Leu Ser Ile Thr Ala
Ala Leu Ala Ala Thr Val Leu 1 5 10 15 Pro Asn Ile Val Ser Ala Val
Gly Leu Asp Gln Ala Ala Val Ala Lys 20 25 30 Gly Leu Gln Tyr Phe
Gly Thr Ala Thr Asp Asn Pro Glu Leu Thr Asp 35 40 45 Ile Pro Tyr
Val Thr Gln Leu Asn Asn Thr Ala Asp Phe Gly Gln Ile 50 55 60 Thr
Pro Gly Asn Ser Met Lys Trp Asp Ala Thr Glu Pro Ser Gln Gly 65 70
75 80 Thr Phe Thr Phe Thr Lys Gly Asp Val Ile Ala Asp Leu Ala Glu
Gly 85 90 95 Asn Gly Gln Tyr Leu Arg Cys His Thr Leu Val Trp Tyr
Asn Gln Leu 100 105 110 Pro Ser Trp Val Thr Ser Gly Thr Trp Thr Asn
Ala Thr Leu Thr Ala 115 120 125 Ala Leu Lys Asn His Ile Thr Asn Val
Val Ser His Tyr Lys Gly Lys 130 135 140 Cys Leu His Trp Asp Val Val
Asn Glu Ala Leu Asn Asp Asp Gly Thr 145 150 155 160 Tyr Arg Thr Asn
Ile Phe Tyr Thr Thr Ile Gly Glu Ala Tyr Ile Pro 165 170 175 Ile Ala
Phe Ala Ala Ala Ala Ala Ala Asp Pro Asp Ala Lys Leu Phe 180 185 190
Tyr Asn Asp Tyr Asn Leu Glu Tyr Gly Gly Ala Lys Ala Ala Ser Ala 195
200 205 Arg Ala Ile Val Gln Leu Val Lys Asn Ala Gly Ala Lys Ile Asp
Gly 210 215 220 Val Gly Leu Gln Ala His Phe Ser Val Gly Thr Val Pro
Ser Thr Ser 225 230 235 240 Ser Leu Val Ser Val Leu Gln Ser Phe Thr
Ala Leu Gly Val Glu Val 245 250 255 Ala Tyr Thr Glu Ala Asp Val Arg
Ile Leu Leu Pro Thr Thr Ala Thr 260 265 270 Thr Leu Ala Gln Gln Ser
Ser Asp Phe Gln Ala Leu Val Gln Ser Cys 275 280 285 Val Gln Thr Thr
Gly Cys Val Gly Phe Thr Ile Trp Asp Trp Thr Asp 290 295 300 Lys Tyr
Ser Trp Val Pro Ser Thr Phe Ser Gly Tyr Gly Ala Ala Leu 305 310 315
320 Pro Trp Asp Glu Asn Leu Val Lys Lys Pro Ala Tyr Asn Gly Leu Leu
325 330 335 Ala Gly Met Gly Val Thr Val Thr Thr Thr Thr Thr Thr Thr
Thr Ala 340 345 350 Thr Ala Thr Gly Lys Thr Thr Thr Thr Thr Thr Gly
Ala Thr Ser Thr 355 360 365 Gly Thr Thr Ala Ala His Trp Gly Gln Cys
Gly Gly Leu Asn Trp Ser 370 375 380 Gly Pro Thr Ala Cys Ala Thr Gly
Tyr Thr Cys Thr Tyr Val Asn Asp 385 390 395 400 Tyr Tyr Ser Gln Cys
Leu 405 5347PRTTrichoderma reesei 5Met Lys Ala Asn Val Ile Leu Cys
Leu Leu Ala Pro Leu Val Ala Ala 1 5 10 15 Leu Pro Thr Glu Thr Ile
His Leu Asp Pro Glu Leu Ala Ala Leu Arg 20 25 30 Ala Asn Leu Thr
Glu Arg Thr Ala Asp Leu Trp Asp Arg Gln Ala Ser 35 40 45 Gln Ser
Ile Asp Gln Leu Ile Lys Arg Lys Gly Lys Leu Tyr Phe Gly 50 55 60
Thr Ala Thr Asp Arg Gly Leu Leu Gln Arg Glu Lys Asn Ala Ala Ile 65
70 75 80 Ile Gln Ala Asp Leu Gly Gln Val Thr Pro Glu Asn Ser Met
Lys Trp 85 90 95 Gln Ser Leu Glu Asn Asn Gln Gly Gln Leu Asn Trp
Gly Asp Ala Asp 100 105 110 Tyr Leu Val Asn Phe Ala Gln Gln Asn Gly
Lys Ser Ile Arg Gly His 115 120 125 Thr Leu Ile Trp His Ser Gln Leu
Pro Ala Trp Val Asn Asn Ile Asn 130 135 140 Asn Ala Asp Thr Leu Arg
Gln Val Ile Arg Thr His Val Ser Thr Val 145 150 155 160 Val Gly Arg
Tyr Lys Gly Lys Ile Arg Ala Trp Asp Val Val Asn Glu 165 170 175 Ile
Phe Asn Glu Asp Gly Thr Leu Arg Ser Ser Val Phe Ser Arg Leu 180 185
190 Leu Gly Glu Glu Phe Val Ser Ile Ala Phe Arg Ala Ala Arg Asp Ala
195 200 205 Asp Pro Ser Ala Arg Leu Tyr Ile Asn Asp Tyr Asn Leu Asp
Arg Ala 210 215 220 Asn Tyr Gly Lys Val Asn Gly Leu Lys Thr Tyr Val
Ser Lys Trp Ile 225 230 235 240 Ser Gln Gly Val Pro Ile Asp Gly Ile
Gly Ser Gln Ser His Leu Ser 245 250 255 Gly Gly Gly Gly Ser Gly Thr
Leu Gly Ala Leu Gln Gln Leu Ala Thr 260 265 270 Val Pro Val Thr Glu
Leu Ala Ile Thr Glu Leu Asp Ile Gln Gly Ala 275 280 285 Pro Thr Thr
Asp Tyr Thr Gln Val Val Gln Ala Cys Leu Ser Val Ser 290 295 300 Lys
Cys Val Gly Ile Thr Val Trp Gly Ile Ser Asp Lys Asp Ser Trp 305 310
315 320 Arg Ala Ser Thr Asn Pro Leu Leu Phe Asp Ala Asn Phe Asn Pro
Lys 325 330 335 Pro Ala Tyr Asn Ser Ile Val Gly Ile Leu Gln 340 345
6191PRTTrichoderma reesei 6Gln Cys Ile Gln Pro Gly Thr Gly Tyr Asn
Asn Gly Tyr Phe Tyr Ser 1 5 10 15 Tyr Trp Asn Asp Gly His Gly Gly
Val Thr Tyr Cys Asn Gly Pro Gly 20 25 30 Gly Gln Phe Ser Val Asn
Trp Ser Asn Ser Gly Asn Phe Val Gly Gly 35 40 45 Lys Gly Trp Gln
Pro Gly Thr Lys Asn Arg Val Ile Asn Phe Ser Gly 50 55 60 Ser Tyr
Asn Pro Asn Gly Asn Ser Tyr Leu Ser Val Tyr Gly Trp Ser 65 70 75 80
Arg Asn Pro Leu Ile Glu Tyr Tyr Ile Val Glu Asn Phe Gly Thr Tyr 85
90 95 Asn Pro Ser Thr Gly Ala Thr Lys Leu Gly Glu Val Thr Ser Asp
Gly 100 105 110 Ser Val Tyr Asp Ile Tyr Arg Thr Gln Arg Val Asn Gln
Pro Ser Ile 115 120 125 Ile Gly Thr Ala Thr Phe Tyr Gln Tyr Trp Ser
Val Arg Arg Asn His 130 135 140 Arg Ser Ser Gly Ser Val Asn Thr Ala
Asn His Phe Asn Ala Trp Ala 145 150 155 160 Gln Gln Gly Leu Thr Leu
Gly Thr Met Asp Tyr Gln Ile Val Ala Val 165 170 175 Glu Gly Tyr Phe
Ser Ser Gly Ser Ala Ser Ile Thr Val Ser Asp 180 185 190
7214PRTBacillus subtilis 7Gln Thr Gly Gly Ser Phe Phe Asp Pro Phe
Asn Gly Tyr Asn Ser Gly 1 5 10 15 Phe Trp Gln Lys Ala Asp Gly Tyr
Ser Asn Gly Asn Met Phe Asn Cys 20 25 30 Thr Trp Arg Ala Asn Asn
Val Ser Met Thr Ser Leu Gly Glu Met Arg 35 40 45 Leu Ala Leu Thr
Ser Pro Ala Tyr Asn Lys Phe Asp Cys Gly Glu Asn 50 55 60 Arg Ser
Val Gln Thr Tyr Gly Tyr Gly Leu Tyr Glu Val Arg Met Lys 65 70 75 80
Pro Ala Lys Asn Thr Gly Ile Val Ser Ser Phe Phe Thr Tyr Thr Gly 85
90 95 Pro Thr Asp Gly Thr Pro Trp Asp Glu Ile Asp Ile Glu Phe Leu
Gly 100 105 110 Lys Asp Thr Thr Lys Val Gln Phe Asn Tyr Tyr Thr Asn
Gly Ala Gly 115 120 125 Asn His Glu Lys Ile Val Asp Leu Gly Phe Asp
Ala Ala Asn Ala Tyr 130 135 140 His Thr Tyr Ala Phe Asp Trp Gln Pro
Asn Ser Ile Lys Trp Tyr Val 145 150 155 160 Asp Gly Gln Leu Lys His
Thr Ala Thr Asn Gln Ile Pro Thr Thr Pro 165 170 175 Gly Lys Ile Met
Met Asn Leu Trp Asn Gly Thr Gly Val Asp Glu Trp 180 185 190 Leu Gly
Ser Tyr Asn Gly Val Asn Pro Leu Tyr Ala His Tyr Asp Trp 195 200 205
Val Arg Tyr Thr Lys Lys 210 8316PRTGeosmithia emersonii 8Ala Pro
Val Lys Glu Lys Gly Ile Lys Lys Arg Ala Ser Pro Phe Gln 1 5 10 15
Trp Phe Gly Ser Asn Glu Ser Gly Ala Glu Phe Gly Asn Asn Asn Ile 20
25 30 Pro Gly Val Glu Gly Thr Asp Tyr Thr Phe Pro Asn Thr Ser Ala
Ile 35 40 45 Gln Ile Leu Ile Asp Gln Gly Met Asn Ile Phe Arg Val
Pro Phe Leu 50 55 60 Met Glu Arg Met Val Pro Asn Gln Met Thr Gly
Pro Val Asp Ser Ala 65 70 75 80 Tyr Phe Gln Gly Tyr Ser Gln Val Ile
Asn Tyr Ile Thr Ser His Gly 85 90 95 Ala Ser Ala Val Ile Asp Pro
His Asn Phe Gly Arg Tyr Tyr Asn Asn 100 105 110 Ile Ile Ser Ser Pro
Ser Asp Phe Gln Thr Phe Trp His Thr Ile
Ala 115 120 125 Ser Asn Phe Ala Asp Asn Asp Asn Val Ile Phe Asp Thr
Asn Asn Glu 130 135 140 Tyr His Asp Met Asp Glu Ser Leu Val Val Gln
Leu Asn Gln Ala Ala 145 150 155 160 Ile Asp Gly Ile Arg Ala Ala Gly
Ala Thr Ser Gln Tyr Ile Phe Val 165 170 175 Glu Gly Asn Ser Trp Thr
Gly Ala Trp Thr Trp Thr Gln Val Asn Asp 180 185 190 Ala Met Ala Asn
Leu Thr Asp Pro Gln Asn Lys Ile Val Tyr Glu Met 195 200 205 His Gln
Tyr Leu Asp Ser Asp Gly Ser Gly Thr Ser Asp Gln Cys Val 210 215 220
Asn Ser Thr Ile Gly Gln Asp Arg Val Glu Ser Ala Thr Ala Trp Leu 225
230 235 240 Lys Gln Asn Gly Lys Lys Ala Ile Leu Gly Glu Tyr Ala Gly
Gly Ala 245 250 255 Asn Ser Val Cys Glu Thr Ala Val Thr Gly Met Leu
Asp Tyr Leu Ala 260 265 270 Asn Asn Thr Asp Val Trp Thr Gly Ala Ile
Trp Trp Ala Ala Gly Pro 275 280 285 Trp Trp Gly Asp Tyr Ile Phe Ser
Met Glu Pro Pro Ser Gly Ile Ala 290 295 300 Tyr Glu Gln Val Leu Pro
Leu Leu Gln Pro Tyr Leu 305 310 315 9441PRTBacillus subtilis 9Asp
Asp Tyr Ser Val Val Glu Glu His Gly Gln Leu Ser Ile Ser Asn 1 5 10
15 Gly Glu Leu Val Asn Glu Arg Gly Glu Gln Val Gln Leu Lys Gly Met
20 25 30 Ser Ser His Gly Leu Gln Trp Tyr Gly Gln Phe Val Asn Tyr
Glu Ser 35 40 45 Met Lys Trp Leu Arg Asp Asp Trp Gly Ile Thr Val
Phe Arg Ala Ala 50 55 60 Met Tyr Thr Ser Ser Gly Gly Tyr Ile Asp
Asp Pro Ser Val Lys Glu 65 70 75 80 Lys Val Lys Glu Thr Val Glu Ala
Ala Ile Asp Leu Gly Ile Tyr Val 85 90 95 Ile Ile Asp Trp His Ile
Leu Ser Asp Asn Asp Pro Asn Ile Tyr Lys 100 105 110 Glu Glu Ala Lys
Asp Phe Phe Asp Glu Met Ser Glu Leu Tyr Gly Asp 115 120 125 Tyr Pro
Asn Val Ile Tyr Glu Ile Ala Asn Glu Pro Asn Gly Ser Asp 130 135 140
Val Thr Trp Asp Asn Gln Ile Lys Pro Tyr Ala Glu Glu Val Ile Pro 145
150 155 160 Val Ile Arg Asp Asn Asp Pro Asn Asn Ile Val Ile Val Gly
Thr Gly 165 170 175 Thr Trp Ser Gln Asp Val His His Ala Ala Asp Asn
Gln Leu Ala Asp 180 185 190 Pro Asn Val Met Tyr Ala Phe His Phe Tyr
Ala Gly Thr His Gly Gln 195 200 205 Asn Leu Arg Asp Gln Val Asp Tyr
Ala Leu Asp Gln Gly Ala Ala Ile 210 215 220 Phe Val Ser Glu Trp Gly
Thr Ser Ala Ala Thr Gly Asp Gly Gly Val 225 230 235 240 Phe Leu Asp
Glu Ala Gln Val Trp Ile Asp Phe Met Asp Glu Arg Asn 245 250 255 Leu
Ser Trp Ala Asn Trp Ser Leu Thr His Lys Asp Glu Ser Ser Ala 260 265
270 Ala Leu Met Pro Gly Ala Asn Pro Thr Gly Gly Trp Thr Glu Ala Glu
275 280 285 Leu Ser Pro Ser Gly Thr Phe Val Arg Glu Lys Ile Arg Glu
Ser Ala 290 295 300 Ser Ile Pro Pro Ser Asp Pro Thr Pro Pro Ser Asp
Pro Gly Glu Pro 305 310 315 320 Asp Pro Gly Glu Pro Asp Pro Thr Pro
Pro Ser Asp Pro Gly Glu Tyr 325 330 335 Pro Ala Trp Asp Ser Asn Gln
Ile Tyr Thr Asn Glu Ile Val Tyr His 340 345 350 Asn Gly Gln Leu Trp
Gln Ala Lys Trp Trp Thr Gln Asn Gln Glu Pro 355 360 365 Gly Asp Pro
Tyr Gly Pro Trp Glu Pro Leu Lys Ser Asp Pro Asp Ser 370 375 380 Gly
Glu Pro Asp Pro Thr Pro Pro Ser Asp Pro Gly Glu Tyr Pro Ala 385 390
395 400 Trp Asp Ser Asn Gln Ile Tyr Thr Asn Glu Ile Val Tyr His Asn
Gly 405 410 415 Gln Leu Trp Gln Ala Lys Trp Trp Thr Gln Asn Gln Glu
Pro Gly Asp 420 425 430 Pro Tyr Gly Pro Trp Glu Pro Leu Asn 435 440
10397PRTTrichoderma reesei 10Gln Gln Thr Val Trp Gly Gln Cys Gly
Gly Ile Gly Trp Ser Gly Pro 1 5 10 15 Thr Asn Cys Ala Pro Gly Ser
Ala Cys Ser Thr Leu Asn Pro Tyr Tyr 20 25 30 Ala Gln Cys Ile Pro
Gly Ala Thr Thr Ile Thr Thr Ser Thr Arg Pro 35 40 45 Pro Ser Gly
Pro Thr Thr Thr Thr Arg Ala Thr Ser Thr Ser Ser Ser 50 55 60 Thr
Pro Pro Thr Ser Ser Gly Val Arg Phe Ala Gly Val Asn Ile Ala 65 70
75 80 Gly Phe Asp Phe Gly Cys Thr Thr Asp Gly Thr Cys Val Thr Ser
Lys 85 90 95 Val Tyr Pro Pro Leu Lys Asn Phe Thr Gly Ser Asn Asn
Tyr Pro Asp 100 105 110 Gly Ile Gly Gln Met Gln His Phe Val Asn Asp
Asp Gly Met Thr Ile 115 120 125 Phe Arg Leu Pro Val Gly Trp Gln Tyr
Leu Val Asn Asn Asn Leu Gly 130 135 140 Gly Asn Leu Asp Ser Thr Ser
Ile Ser Lys Tyr Asp Gln Leu Val Gln 145 150 155 160 Gly Cys Leu Ser
Leu Gly Ala Tyr Cys Ile Val Asp Ile His Asn Tyr 165 170 175 Ala Arg
Trp Asn Gly Gly Ile Ile Gly Gln Gly Gly Pro Thr Asn Ala 180 185 190
Gln Phe Thr Ser Leu Trp Ser Gln Leu Ala Ser Lys Tyr Ala Ser Gln 195
200 205 Ser Arg Val Trp Phe Gly Ile Met Asn Glu Pro His Asp Val Asn
Ile 210 215 220 Asn Thr Trp Ala Ala Thr Val Gln Glu Val Val Thr Ala
Ile Arg Asn 225 230 235 240 Ala Gly Ala Thr Ser Gln Phe Ile Ser Leu
Pro Gly Asn Asp Trp Gln 245 250 255 Ser Ala Gly Ala Phe Ile Ser Asp
Gly Ser Ala Ala Ala Leu Ser Gln 260 265 270 Val Thr Asn Pro Asp Gly
Ser Thr Thr Asn Leu Ile Phe Asp Val His 275 280 285 Lys Tyr Leu Asp
Ser Asp Asn Ser Gly Thr His Ala Glu Cys Thr Thr 290 295 300 Asn Asn
Ile Asp Gly Ala Phe Ser Pro Leu Ala Thr Trp Leu Arg Gln 305 310 315
320 Asn Asn Arg Gln Ala Ile Leu Thr Glu Thr Gly Gly Gly Asn Val Gln
325 330 335 Ser Cys Ile Gln Asp Met Cys Gln Gln Ile Gln Tyr Leu Asn
Gln Asn 340 345 350 Ser Asp Val Tyr Leu Gly Tyr Val Gly Trp Gly Ala
Gly Ser Phe Asp 355 360 365 Ser Thr Tyr Val Leu Thr Glu Thr Pro Thr
Gly Ser Gly Asn Ser Trp 370 375 380 Thr Asp Thr Ser Leu Val Ser Ser
Cys Leu Ala Arg Lys 385 390 395 11218PRTTrichoderma reesei 11Gln
Thr Ser Cys Asp Gln Trp Ala Thr Phe Thr Gly Asn Gly Tyr Thr 1 5 10
15 Val Ser Asn Asn Leu Trp Gly Ala Ser Ala Gly Ser Gly Phe Gly Cys
20 25 30 Val Thr Ala Val Ser Leu Ser Gly Gly Ala Ser Trp His Ala
Asp Trp 35 40 45 Gln Trp Ser Gly Gly Gln Asn Asn Val Lys Ser Tyr
Gln Asn Ser Gln 50 55 60 Ile Ala Ile Pro Gln Lys Arg Thr Val Asn
Ser Ile Ser Ser Met Pro 65 70 75 80 Thr Thr Ala Ser Trp Ser Tyr Ser
Gly Ser Asn Ile Arg Ala Asn Val 85 90 95 Ala Tyr Asp Leu Phe Thr
Ala Ala Asn Pro Asn His Val Thr Tyr Ser 100 105 110 Gly Asp Tyr Glu
Leu Met Ile Trp Leu Gly Lys Tyr Gly Asp Ile Gly 115 120 125 Pro Ile
Gly Ser Ser Gln Gly Thr Val Asn Val Gly Gly Gln Ser Trp 130 135 140
Thr Leu Tyr Tyr Gly Tyr Asn Gly Ala Met Gln Val Tyr Ser Phe Val 145
150 155 160 Ala Gln Thr Asn Thr Thr Asn Tyr Ser Gly Asp Val Lys Asn
Phe Phe 165 170 175 Asn Tyr Leu Arg Asp Asn Lys Gly Tyr Asn Ala Ala
Gly Gln Tyr Val 180 185 190 Leu Ser Tyr Gln Phe Gly Thr Glu Pro Phe
Thr Gly Ser Gly Thr Leu 195 200 205 Asn Val Ala Ser Trp Thr Ala Ser
Ile Asn 210 215 12322PRTTrichoderma reesei 12His Gly His Ile Asn
Asp Ile Val Ile Asn Gly Val Trp Tyr Gln Ala 1 5 10 15 Tyr Asp Pro
Thr Thr Phe Pro Tyr Glu Ser Asn Pro Pro Ile Val Val 20 25 30 Gly
Trp Thr Ala Ala Asp Leu Asp Asn Gly Phe Val Ser Pro Asp Ala 35 40
45 Tyr Gln Asn Pro Asp Ile Ile Cys His Lys Asn Ala Thr Asn Ala Lys
50 55 60 Gly His Ala Ser Val Lys Ala Gly Asp Thr Ile Leu Phe Gln
Trp Val 65 70 75 80 Pro Val Pro Trp Pro His Pro Gly Pro Ile Val Asp
Tyr Leu Ala Asn 85 90 95 Cys Asn Gly Asp Cys Glu Thr Val Asp Lys
Thr Thr Leu Glu Phe Phe 100 105 110 Lys Ile Asp Gly Val Gly Leu Leu
Ser Gly Gly Asp Pro Gly Thr Trp 115 120 125 Ala Ser Asp Val Leu Ile
Ser Asn Asn Asn Thr Trp Val Val Lys Ile 130 135 140 Pro Asp Asn Leu
Ala Pro Gly Asn Tyr Val Leu Arg His Glu Ile Ile 145 150 155 160 Ala
Leu His Ser Ala Gly Gln Ala Asn Gly Ala Gln Asn Tyr Pro Gln 165 170
175 Cys Phe Asn Ile Ala Val Ser Gly Ser Gly Ser Leu Gln Pro Ser Gly
180 185 190 Val Leu Gly Thr Asp Leu Tyr His Ala Thr Asp Pro Gly Val
Leu Ile 195 200 205 Asn Ile Tyr Thr Ser Pro Leu Asn Tyr Ile Ile Pro
Gly Pro Thr Val 210 215 220 Val Ser Gly Leu Pro Thr Ser Val Ala Gln
Gly Ser Ser Ala Ala Thr 225 230 235 240 Ala Thr Ala Ser Ala Thr Val
Pro Gly Gly Gly Ser Gly Pro Thr Ser 245 250 255 Arg Thr Thr Thr Thr
Ala Arg Thr Thr Gln Ala Ser Ser Arg Pro Ser 260 265 270 Ser Thr Pro
Pro Ala Thr Thr Ser Ala Pro Ala Gly Gly Pro Thr Gln 275 280 285 Thr
Leu Tyr Gly Gln Cys Gly Gly Ser Gly Tyr Ser Gly Pro Thr Arg 290 295
300 Cys Ala Pro Pro Ala Thr Cys Ser Thr Asn Pro Tyr Tyr Ala Gln Cys
305 310 315 320 Leu Asn 13761PRTTrichoderma reesei 13Ala Phe Ser
Trp Lys Asn Val Lys Leu Gly Gly Gly Gly Gly Phe Val 1 5 10 15 Pro
Gly Ile Ile Phe His Pro Lys Thr Lys Gly Val Ala Tyr Ala Arg 20 25
30 Thr Asp Ile Gly Gly Leu Tyr Arg Leu Asn Ala Asp Asp Ser Trp Thr
35 40 45 Ala Val Thr Asp Gly Ile Ala Asp Asn Ala Gly Trp His Asn
Trp Gly 50 55 60 Ile Asp Ala Val Ala Leu Asp Pro Gln Asp Asp Gln
Lys Val Tyr Ala 65 70 75 80 Ala Val Gly Met Tyr Thr Asn Ser Trp Asp
Pro Ser Asn Gly Ala Ile 85 90 95 Ile Arg Ser Ser Asp Arg Gly Ala
Thr Trp Ser Phe Thr Asn Leu Pro 100 105 110 Phe Lys Val Gly Gly Asn
Met Pro Gly Arg Gly Ala Gly Glu Arg Leu 115 120 125 Ala Val Asp Pro
Ala Asn Ser Asn Ile Ile Tyr Phe Gly Ala Arg Ser 130 135 140 Gly Asn
Gly Leu Trp Lys Ser Thr Asp Gly Gly Val Thr Phe Ser Lys 145 150 155
160 Val Ser Ser Phe Thr Ala Thr Gly Thr Tyr Ile Pro Asp Pro Ser Asp
165 170 175 Ser Asn Gly Tyr Asn Ser Asp Lys Gln Gly Leu Met Trp Val
Thr Phe 180 185 190 Asp Ser Thr Ser Ser Thr Thr Gly Gly Ala Thr Ser
Arg Ile Phe Val 195 200 205 Gly Thr Ala Asp Asn Ile Thr Ala Ser Val
Tyr Val Ser Thr Asn Ala 210 215 220 Gly Ser Thr Trp Ser Ala Val Pro
Gly Gln Pro Gly Lys Tyr Phe Pro 225 230 235 240 His Lys Ala Lys Leu
Gln Pro Ala Glu Lys Ala Leu Tyr Leu Thr Tyr 245 250 255 Ser Trp Trp
Pro Asp Ala Gln Leu Phe Arg Ser Thr Asp Ser Gly Thr 260 265 270 Thr
Trp Ser Pro Ile Trp Ala Trp Ala Ser Tyr Pro Thr Glu Thr Tyr 275 280
285 Tyr Tyr Ser Ile Ser Thr Pro Lys Ala Pro Trp Ile Lys Asn Asn Phe
290 295 300 Ile Asp Val Thr Ser Glu Ser Pro Ser Asp Gly Leu Ile Lys
Arg Leu 305 310 315 320 Gly Trp Met Ile Glu Ser Leu Glu Ile Asp Pro
Thr Asp Ser Asn His 325 330 335 Trp Leu Tyr Gly Thr Gly Met Thr Ile
Phe Gly Gly His Asp Leu Thr 340 345 350 Asn Trp Asp Thr Arg His Asn
Val Ser Ile Gln Ser Leu Ala Asp Gly 355 360 365 Ile Glu Glu Phe Ser
Val Gln Asp Leu Ala Ser Ala Pro Gly Gly Ser 370 375 380 Glu Leu Leu
Ala Ala Val Gly Asp Asp Asn Gly Phe Thr Phe Ala Ser 385 390 395 400
Arg Asn Asp Leu Gly Thr Ser Pro Gln Thr Val Trp Ala Thr Pro Thr 405
410 415 Trp Ala Thr Ser Thr Ser Val Asp Tyr Ala Gly Asn Ser Val Lys
Ser 420 425 430 Val Val Arg Val Gly Asn Thr Ala Gly Thr Gln Gln Val
Ala Ile Ser 435 440 445 Ser Asp Gly Gly Ala Thr Trp Ser Ile Asp Tyr
Ala Ala Asp Thr Ser 450 455 460 Met Asn Gly Gly Thr Val Ala Tyr Ser
Ala Asp Gly Asp Thr Ile Leu 465 470 475 480 Trp Ser Thr Ala Ser Ser
Gly Val Gln Arg Ser Gln Phe Gln Gly Ser 485 490 495 Phe Ala Ser Val
Ser Ser Leu Pro Ala Gly Ala Val Ile Ala Ser Asp 500 505 510 Lys Lys
Thr Asn Ser Val Phe Tyr Ala Gly Ser Gly Ser Thr Phe Tyr 515 520 525
Val Ser Lys Asp Thr Gly Ser Ser Phe Thr Arg Gly Pro Lys Leu Gly 530
535 540 Ser Ala Gly Thr Ile Arg Asp Ile Ala Ala His Pro Thr Thr Ala
Gly 545 550 555 560 Thr Leu Tyr Val Ser Thr Asp Val Gly Ile Phe Arg
Ser Thr Asp Ser 565 570 575 Gly Thr Thr Phe Gly Gln Val Ser Thr Ala
Leu Thr Asn Thr Tyr Gln 580 585 590 Ile Ala Leu Gly Val Gly Ser Gly
Ser Asn Trp Asn Leu Tyr Ala Phe 595 600 605 Gly Thr Gly Pro Ser Gly
Ala Arg Leu Tyr Ala Ser Gly Asp Ser Gly 610 615 620 Ala Ser Trp Thr
Asp Ile Gln Gly Ser Gln Gly Phe Gly Ser Ile Asp 625 630 635 640 Ser
Thr Lys Val Ala Gly Ser Gly Ser Thr Ala Gly Gln Val Tyr Val 645 650
655 Gly Thr Asn Gly Arg Gly Val Phe Tyr Ala Gln Gly Thr Val Gly Gly
660 665 670 Gly Thr Gly Gly Thr Ser Ser Ser Thr Lys Gln Ser Ser Ser
Ser Thr 675 680 685 Ser Ser Ala Ser Ser Ser Thr Thr Leu Arg Ser Ser
Val Val Ser Thr 690 695 700 Thr Arg Ala Ser Thr Val Thr Ser Ser Arg
Thr Ser Ser Ala Ala Gly 705 710
715 720 Pro Thr Gly Ser Gly Val Ala Gly His Tyr Ala Gln Cys Gly Gly
Ile 725 730 735 Gly Trp Thr Gly Pro Thr Gln Cys Val Ala Pro Tyr Val
Cys Gln Lys 740 745 750 Gln Asn Asp Tyr Tyr Tyr Gln Cys Val 755 760
14230PRTTrichoderma reesei 14His Gly Gln Val Gln Asn Phe Thr Ile
Asn Gly Gln Tyr Asn Gln Gly 1 5 10 15 Phe Ile Leu Asp Tyr Tyr Tyr
Gln Lys Gln Asn Thr Gly His Phe Pro 20 25 30 Asn Val Ala Gly Trp
Tyr Ala Glu Asp Leu Asp Leu Gly Phe Ile Ser 35 40 45 Pro Asp Gln
Tyr Thr Thr Pro Asp Ile Val Cys His Lys Asn Ala Ala 50 55 60 Pro
Gly Ala Ile Ser Ala Thr Ala Ala Ala Gly Ser Asn Ile Val Phe 65 70
75 80 Gln Trp Gly Pro Gly Val Trp Pro His Pro Tyr Gly Pro Ile Val
Thr 85 90 95 Tyr Val Val Glu Cys Ser Gly Ser Cys Thr Thr Val Asn
Lys Asn Asn 100 105 110 Leu Arg Trp Val Lys Ile Gln Glu Ala Gly Ile
Asn Tyr Asn Thr Gln 115 120 125 Val Trp Ala Gln Gln Asp Leu Ile Asn
Gln Gly Asn Lys Trp Thr Val 130 135 140 Lys Ile Pro Ser Ser Leu Arg
Pro Gly Asn Tyr Val Phe Arg His Glu 145 150 155 160 Leu Leu Ala Ala
His Gly Ala Ser Ser Ala Asn Gly Met Gln Asn Tyr 165 170 175 Pro Gln
Cys Val Asn Ile Ala Val Thr Gly Ser Gly Thr Lys Ala Leu 180 185 190
Pro Ala Gly Thr Pro Ala Thr Gln Leu Tyr Lys Pro Thr Asp Pro Gly 195
200 205 Ile Leu Phe Asn Pro Tyr Thr Thr Ile Thr Ser Tyr Thr Ile Pro
Gly 210 215 220 Pro Ala Leu Trp Gln Gly 225 230 15419PRTTrichoderma
reesei 15Gly Lys Ile Lys Tyr Leu Gly Val Ala Ile Pro Gly Ile Asp
Phe Gly 1 5 10 15 Cys Asp Ile Asp Gly Ser Cys Pro Thr Asp Thr Ser
Ser Val Pro Leu 20 25 30 Leu Ser Tyr Lys Gly Gly Asp Gly Ala Gly
Gln Met Lys His Phe Ala 35 40 45 Glu Asp Asp Gly Leu Asn Val Phe
Arg Ile Ser Ala Thr Trp Gln Phe 50 55 60 Val Leu Asn Asn Thr Val
Asp Gly Lys Leu Asp Glu Leu Asn Trp Gly 65 70 75 80 Ser Tyr Asn Lys
Val Val Asn Ala Cys Leu Glu Thr Gly Ala Tyr Cys 85 90 95 Met Ile
Asp Met His Asn Phe Ala Arg Tyr Asn Gly Gly Ile Ile Gly 100 105 110
Gln Gly Gly Val Ser Asp Asp Ile Phe Val Asp Leu Trp Val Gln Ile 115
120 125 Ala Lys Tyr Tyr Glu Asp Asn Asp Lys Ile Ile Phe Gly Leu Met
Asn 130 135 140 Glu Pro His Asp Leu Asp Ile Glu Ile Trp Ala Gln Thr
Cys Gln Lys 145 150 155 160 Val Val Thr Ala Ile Arg Lys Ala Gly Ala
Thr Ser Gln Met Ile Leu 165 170 175 Leu Pro Gly Thr Asn Phe Ala Ser
Val Glu Thr Tyr Val Ser Thr Gly 180 185 190 Ser Ala Glu Ala Leu Gly
Lys Ile Thr Asn Pro Asp Gly Ser Thr Asp 195 200 205 Leu Leu Tyr Phe
Asp Val His Lys Tyr Leu Asp Ile Asn Asn Ser Gly 210 215 220 Ser His
Ala Glu Cys Thr Thr Asp Asn Val Asp Ala Phe Asn Asp Phe 225 230 235
240 Ala Asp Trp Leu Arg Gln Asn Lys Arg Gln Ala Ile Ile Ser Glu Thr
245 250 255 Gly Ala Ser Met Glu Pro Ser Cys Met Thr Ala Phe Cys Ala
Gln Asn 260 265 270 Lys Ala Ile Ser Glu Asn Ser Asp Val Tyr Ile Gly
Phe Val Gly Trp 275 280 285 Gly Ala Gly Ser Phe Asp Thr Ser Tyr Ile
Leu Thr Leu Thr Pro Leu 290 295 300 Gly Lys Pro Gly Asn Tyr Thr Asp
Asn Lys Leu Met Asn Glu Cys Ile 305 310 315 320 Leu Asp Gln Phe Thr
Leu Asp Glu Lys Tyr Arg Pro Thr Pro Thr Ser 325 330 335 Ile Ser Thr
Ala Ala Glu Glu Thr Ala Thr Ala Thr Ala Thr Ser Asp 340 345 350 Gly
Asp Ala Pro Ser Thr Thr Lys Pro Ile Phe Arg Glu Glu Thr Ala 355 360
365 Ser Pro Thr Pro Asn Ala Val Thr Lys Pro Ser Pro Asp Thr Ser Asp
370 375 380 Ser Ser Asp Asp Asp Lys Asp Ser Ala Ala Ser Met Ser Ala
Gln Gly 385 390 395 400 Leu Thr Gly Thr Val Leu Phe Thr Val Ala Ala
Leu Gly Tyr Met Leu 405 410 415 Val Ala Phe 16499PRTBacillus
subtilis 16Met Lys Arg Ser Ile Ser Ile Phe Ile Thr Cys Leu Leu Ile
Thr Leu 1 5 10 15 Leu Thr Met Gly Gly Met Ile Ala Ser Pro Ala Ser
Ala Ala Gly Thr 20 25 30 Lys Thr Pro Val Ala Lys Asn Gly Gln Leu
Ser Ile Lys Gly Thr Gln 35 40 45 Leu Val Asn Arg Asp Gly Lys Ala
Val Gln Leu Lys Gly Ile Ser Ser 50 55 60 His Gly Leu Gln Trp Tyr
Gly Glu Tyr Val Asn Lys Asp Ser Leu Lys 65 70 75 80 Trp Leu Arg Asp
Asp Trp Gly Ile Thr Val Phe Arg Ala Ala Met Tyr 85 90 95 Thr Ala
Asp Gly Gly Tyr Ile Asp Asn Pro Ser Val Lys Asn Lys Val 100 105 110
Lys Glu Ala Val Glu Ala Ala Lys Glu Leu Gly Ile Tyr Val Ile Ile 115
120 125 Asp Trp His Ile Leu Asn Asp Gly Asn Pro Asn Gln Asn Lys Glu
Lys 130 135 140 Ala Lys Glu Phe Phe Lys Glu Met Ser Ser Leu Tyr Gly
Asn Thr Pro 145 150 155 160 Asn Val Ile Tyr Glu Ile Ala Asn Glu Pro
Asn Gly Asp Val Asn Trp 165 170 175 Lys Arg Asp Ile Lys Pro Tyr Ala
Glu Glu Val Ile Ser Val Ile Arg 180 185 190 Lys Asn Asp Pro Asp Asn
Ile Ile Ile Val Gly Thr Gly Thr Trp Ser 195 200 205 Gln Asp Val Asn
Asp Ala Ala Asp Asp Gln Leu Lys Asp Ala Asn Val 210 215 220 Met Tyr
Ala Leu His Phe Tyr Ala Gly Thr His Gly Gln Phe Leu Arg 225 230 235
240 Asp Lys Ala Asn Tyr Ala Leu Ser Lys Gly Ala Pro Ile Phe Val Thr
245 250 255 Glu Trp Gly Thr Ser Asp Ala Ser Gly Asn Gly Gly Val Phe
Leu Asp 260 265 270 Gln Ser Arg Glu Trp Leu Lys Tyr Leu Asp Ser Lys
Thr Ile Ser Trp 275 280 285 Val Asn Trp Asn Leu Ser Asp Lys Gln Glu
Ser Ser Ser Ala Leu Lys 290 295 300 Pro Gly Ala Ser Lys Thr Gly Gly
Trp Arg Leu Ser Asp Leu Ser Ala 305 310 315 320 Ser Gly Thr Phe Val
Arg Glu Asn Ile Leu Gly Thr Lys Asp Ser Thr 325 330 335 Lys Asp Ile
Pro Glu Thr Pro Ser Lys Asp Lys Pro Thr Gln Glu Asn 340 345 350 Gly
Ile Ser Val Gln Tyr Arg Ala Gly Asp Gly Ser Met Asn Ser Asn 355 360
365 Gln Ile Arg Pro Gln Leu Gln Ile Lys Asn Asn Gly Asn Thr Thr Val
370 375 380 Asp Leu Lys Asp Val Thr Ala Arg Tyr Trp Tyr Lys Ala Lys
Asn Lys 385 390 395 400 Gly Gln Asn Phe Asp Cys Asp Tyr Ala Gln Ile
Gly Cys Gly Asn Val 405 410 415 Thr His Lys Phe Val Thr Leu His Lys
Pro Lys Gln Gly Ala Asp Thr 420 425 430 Tyr Leu Glu Leu Gly Phe Lys
Asn Gly Thr Leu Ala Pro Gly Ala Ser 435 440 445 Thr Gly Asn Ile Gln
Leu Arg Leu His Asn Asp Asp Trp Ser Asn Tyr 450 455 460 Ala Gln Ser
Gly Asp Tyr Ser Phe Phe Lys Ser Asn Thr Phe Lys Thr 465 470 475 480
Thr Lys Lys Ile Thr Leu Tyr Asp Gln Gly Lys Leu Ile Trp Gly Thr 485
490 495 Glu Pro Asn 17185PRTArtificial sequenceBacillus subtilis
xylanase variant 17Ala Ser Thr Asp Tyr Trp Gln Asn Trp Thr Phe Gly
Gly Gly Ile Val 1 5 10 15 Asn Ala Val Asn Gly Ser Gly Gly Asn Tyr
Ser Val Asn Trp Ser Asn 20 25 30 Thr Gly Asn Phe Val Val Gly Lys
Gly Trp Thr Thr Gly Ser Pro Phe 35 40 45 Arg Thr Ile Asn Tyr Asn
Ala Gly Val Trp Ala Pro Asn Gly Asn Gly 50 55 60 Tyr Leu Thr Leu
Tyr Gly Trp Thr Arg Ser Pro Leu Ile Glu Tyr Tyr 65 70 75 80 Val Val
Asp Ser Trp Gly Thr Tyr Arg Pro Thr Gly Thr Tyr Lys Gly 85 90 95
Thr Val Lys Ser Asp Gly Gly Thr Tyr Asp Ile Tyr Thr Thr Thr Arg 100
105 110 Tyr Asn Ala Pro Ser Ile Asp Gly Asp Asp Thr Thr Phe Thr Gln
Tyr 115 120 125 Trp Ser Val Arg Gln Ser Lys Arg Pro Thr Gly Ser Asn
Ala Thr Ile 130 135 140 Thr Phe Ser Asn His Val Asn Ala Trp Lys Ser
His Gly Met Asn Leu 145 150 155 160 Gly Ser Asn Trp Ala Tyr Gln Val
Met Ala Thr Glu Gly Tyr Gln Ser 165 170 175 Ser Gly Ser Ser Asn Val
Thr Val Trp 180 185 18185PRTArtificial sequenceBacillus subtilis
xylanase variant 18Ala Ser Thr Asp Tyr Trp Gln Asn Trp Thr Asp Gly
Tyr Gly Ile Val 1 5 10 15 Asn Ala Val Asn Gly Ser Gly Gly Asn Tyr
Ser Val Asn Trp Ser Asn 20 25 30 Thr Gly Asn Phe Val Val Gly Lys
Gly Trp Thr Thr Gly Ser Pro Phe 35 40 45 Arg Thr Ile Asn Tyr Asn
Ala Gly Val Trp Ala Pro Asn Gly Asn Gly 50 55 60 Tyr Leu Thr Leu
Tyr Gly Trp Thr Arg Ser Pro Leu Ile Glu Tyr Tyr 65 70 75 80 Val Val
Asp Ser Trp Gly Thr Tyr Arg Pro Thr Gly Thr Tyr Lys Gly 85 90 95
Thr Val Tyr Ser Asp Gly Gly Trp Tyr Asp Ile Tyr Thr Ala Thr Arg 100
105 110 Asp Asn Ala Pro Ser Ile Asp Gly Asp Phe Thr Thr Phe Thr Gln
Tyr 115 120 125 Trp Ser Val Arg Gln Ser Lys Arg Pro Thr Gly Ser Asn
Ala Thr Ile 130 135 140 Thr Phe Ser Asn His Val Asn Ala Trp Arg Ser
His Gly Met Asp Leu 145 150 155 160 Gly Ser Asn Trp Ala Tyr Gln Val
Met Ala Thr Glu Gly Tyr Leu Ser 165 170 175 Ser Gly Ser Ser Asn Val
Thr Val Trp 180 185
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