U.S. patent application number 10/797393 was filed with the patent office on 2004-11-04 for alcohol product processes.
This patent application is currently assigned to Novozymes A/S. Invention is credited to Festersen, Rikke Monica, Olsen, Hans Sejr, Pedersen, Sven.
Application Number | 20040219649 10/797393 |
Document ID | / |
Family ID | 32990756 |
Filed Date | 2004-11-04 |
United States Patent
Application |
20040219649 |
Kind Code |
A1 |
Olsen, Hans Sejr ; et
al. |
November 4, 2004 |
Alcohol product processes
Abstract
The present invention relates to processes for production of an
alcohol product from granular starch comprising a pre-treatment at
an elevated temperature below the initial gelatinization
temperature of said granular starch followed by simultaneous
saccharification and fermentation, and optionally recovery of
ethanol.
Inventors: |
Olsen, Hans Sejr; (Holte,
DK) ; Pedersen, Sven; (Gentofte, DK) ;
Festersen, Rikke Monica; (Copenhagen K, DK) |
Correspondence
Address: |
NOVOZYMES NORTH AMERICA, INC.
500 FIFTH AVENUE
SUITE 1600
NEW YORK
NY
10110
US
|
Assignee: |
Novozymes A/S
Bagsvaerd
DK
|
Family ID: |
32990756 |
Appl. No.: |
10/797393 |
Filed: |
March 10, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60453326 |
Mar 10, 2003 |
|
|
|
Current U.S.
Class: |
435/161 ;
435/170; 435/201 |
Current CPC
Class: |
C12C 7/04 20130101; C12N
9/2425 20130101; C12N 9/2411 20130101; C12N 9/2417 20130101; C12N
9/2414 20130101; C12P 19/14 20130101; C12P 7/06 20130101; Y02E
50/10 20130101; Y02E 50/17 20130101; C12P 7/10 20130101; C12Y
302/01001 20130101; C12N 9/242 20130101; C12P 19/02 20130101; C12N
9/2428 20130101; Y02E 50/16 20130101; C12Y 302/01003 20130101; C12C
5/004 20130101 |
Class at
Publication: |
435/161 ;
435/170; 435/201 |
International
Class: |
C12P 007/06; C12P
001/04; C12N 009/26 |
Claims
1-37 (Canceled)
38. A process for production of an alcohol product comprising the
sequential steps of: (a) providing a slurry comprising water and
granular starch, (b) holding said slurry in the presence of an acid
alpha-amylase and a glucoamylase at a temperature of 0.degree. C.
to 20.degree. C. below the initial gelatinization temperature of
said granular starch for a period of 5 minutes to 12 hours, (c)
holding said slurry in the presence of an acid alpha-amylase and a
glucoamylase and a yeast at a temperature between 10.degree. C. and
35.degree. C. to produce ethanol and, (d) optionally recovering the
ethanol.
39. The process of claim 1, wherein the product is fuel ethanol,
potable ethanol and/or industrial ethanol.
40. The process of claim 1, wherein the temperature under step (c)
is between 28.degree. C. and 36.degree. C.
41. The process of claim 1, wherein the temperature under step (c)
is between 29.degree. C. and 35.degree. C.
42. The process of claim 1, wherein the temperature under step (c)
is between 30.degree. C. and 34.degree. C.
43. The process of claim 1, wherein the temperature under step (c)
is between 11.degree. C. and 17.degree. C.
44. The process of claim 1, wherein the temperature under step (c)
is between 12.degree. C. and 16.degree. C.
45. The process of claim 1, wherein the temperature under step (c)
is between 13.degree. C. and 15.degree. C.
46. The process of claim 1, wherein the alcohol product is a
beer.
47. The process of claim 1, wherein the acid alpha-amylase and the
glucoamylase are added in step (b) in a ratio of between 0.30 and
5.00 AFAU/AGU.
48. The process of claim 1, wherein the acid alpha-amylase and the
glucoamylase are added in step (c) in a ratio of between 0.30 and
5.00 AFAU/AGU.
49. The process of claim 1, wherein the acid alpha-amylase is an
acid fungal alpha-amylase.
50. The process of claim 1, wherein the acid fungal alpha-amylase
is obtained from a strain of Aspergillus.
51. The process of claim 1, wherein the acid fungal alpha-amylase
is obtained from a strain of Aspergillus niger or a strain of
Aspergillus oryzae.
52. The process of claim 1, wherein the acid alpha-amylase is an
acid alpha-amylase having an amino acid sequence of SEQ ID
NO:1.
53. The process according to claim 1, wherein the glucoamylase is
obtained from a strain of Aspergillus, Talaromyces or
Clostridium.
54. The process according to claim 1, wherein the glucoamylase is
obtained from a strain of Aspergillus niger.
55. The process of claim 1, wherein the acid alpha-amylase is an
acid bacterial alpha-amylase.
56. The process of claim 55, wherein the acid alpha-amylase is
derived from a strain of B. licheniformis, B. amyloliquefaciens or
B. stearothermophilus alpha-amylase.
57. The process of claim 1, wherein the acid alpha-amylase activity
is present in an amount of 50-500 AFAU/kg of DS.
58. The process of claim 1, wherein the glucoamylase activity is
present in an amount of 20-200 AGU/kg of DS.
59. The process of claim 1, wherein the ratio between acid
alpha-amylase activity and glucoamylase activity is between 0.35
and 5.00 AFAU/AGU.
60. The process of claim 1, wherein step (b) is performed in the
presence of an enzyme activity selected from the group consisting
of xylanase, cellulase and phytase.
61. The process of claim 1, wherein step (c) is performed in the
presence of an enzyme activity selected from the group consisting
of xylanase, cellulase and phytase.
62. The process of claim 1, wherein the starch slurry has 5-60% DS
granular starch.
63. The process of claim 1, wherein the starch slurry has 10-50% DS
granular starch.
64. The process of claim 1, wherein the starch slurry has 20-40% DS
granular starch.
65. The process of claim 1, wherein the pH during step (b) is in
the range of 3.0 to 7.0.
66. The process of claim 1, wherein the pH during step (b) is in
the range of 3.5 to 6.0.
67. The process of claim 1, wherein the pH during step (b) is in
the range of 4.0-5.0.
68. The process of claim 1, wherein the pH during step (c) is in
the range of 3.0 to 7.0.
69. The process of claim 1, wherein the pH during step (c) is in
the range of 3.5 to 6.0.
70. The process of claim 1, wherein the pH during step (c) is in
the range of 4.0-5.0.
71. The process of claim 1, wherein the granular starch is obtained
from tubers, roots, stems, fruits, seeds or whole grain.
72. The process of claim 1, wherein the granular starch is obtained
from corn, cobs, wheat, barley, rye, milo, sago, cassava, manioc,
tapioca, sorghum, rice or potatoes.
73. The process of claim 1, wherein the granular starch is obtained
from cereals.
74. The process of claim 1, wherein the granular starch is obtained
from dry milling or wet milling of whole grain.
75. The process of claim 1, wherein the holding time under step (b)
is from 10 minutes to 6 hours.
76. The process of claim 1, wherein the holding time under step (b)
is from 15 minutes to 3 hours.
77. The process of claim 1, wherein the holding time under step (b)
is from 20 minutes to 11/2 hour.
78. The process of claim 1, wherein the holding time under step (b)
is from 30 minutes to 11/4 hour.
79. The process of claim 1, wherein the holding time under step (b)
is from 40 to 70 minutes.
80. The process of claim 1, wherein the holding time under step (b)
is from 50 to 60 minutes.
81. The process of claim 1, wherein the holding time under step (c)
for a period of 20 to 250 hours.
82. The process of claim 1, wherein the holding time under step (c)
for a period of 5 to 190 hours.
83. The process of claim 1, wherein the holding time under step (c)
for a period of 30 to 180 hours.
84. The process of claim 1, wherein the holding time under step (c)
for a period of 40 to 170 hours.
85. The process of claim 1, wherein the holding time under step (c)
for a period of 50 to 160 hours.
86. The process of claim 1, wherein the holding time under step (c)
for a period of 60 to 150 hours.
87. The process of claim 1, wherein the holding time under step (c)
for a period of 70 to 140 hours.
88. The process of claim 1, wherein the holding time under step (c)
for a period of 80 to 130 hours.
89. The process according to claim 1, wherein the temperature under
step (b) is from 45.degree. C. to 75.degree. C.
90. An enzyme composition comprising acid alpha-amylase activity
and glucoamylase activity in a ratio of between 0.30 and 5.00
AFAU/AGU and one or more additional enzymes selected from the group
consisting of cellulase, xylanase and phytase.
91. A mashing process comprising treating a mash with an acid
alpha-amylase.
92. The process of claim 91, wherein the acid alpha-amylase is
derived from a fungus.
93. The process of claim 91, wherein the acid alpha-amylase is
derived from Aspergillus.
94. The process of claim 91, wherein the acid alpha-amylase is
derived from A. niger.
95. The process of claim 91, wherein the acid alpha-amylase has the
amino acid sequence shown in SEQ ID NO:1
96. The process of claim 91, comprising; (a) forming a mash
comprising between 5% and 100% barley malt (w/w of the grist); (b)
prior to, during or after a) adding an acid alpha-amylase and at
least one enzyme selected from the list comprising: a protease,
cellulase and a maltose generating enzyme. (c) attaining within 15
minutes of a) an initial incubation temperature of at least
70.degree. C.; (d) following c) incubating the mash at a
temperature of at least 70.degree. C. for a period of time
sufficient to achieve an extract recovery of at least 80%.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims, under 35 U.S.C. 119, the benefit of
U.S. provisional application No. 60/453,326 filed on March 10, 2003
the contents of which are fully incorporated herein by
reference.
FIELD OF THE INVENTION
[0002] The present invention relates to processes for production of
an alcohol product from granular starch comprising a pre-treatment
at an elevated temperature below the initial gelatinization
temperature of the granular starch followed by simultaneous
saccharification and fermentation.
BACKGROUND OF THE INVENTION
[0003] Granular starch is found in grains, cereals or tubers of
plants. The granular starch is in the form of microscopic granules,
which are insoluble in water at room temperature. When an aqueous
starch slurry is heated, the granules swell and eventually burst,
dispersing the starch molecules into the solution. During this
"gelatinization" process, there is a dramatic increase in
viscosity. Because the solids level is 30-40% in a typical
industrial process, the starch has to be thinned or "liquefied" so
that it can be handled. This reduction in viscosity is generally
accomplished by enzymatic degradation in a process referred to as
liquefaction. During liquefaction, the long-chained starch is
degraded into smaller branched and linear chains of glucose units
(dextrins) by an alpha-amylase.
[0004] A conventional enzymatic liquefaction process may be carried
out as a three-step hot slurry process. The slurry is heated to
between 80-85.degree. and thermostable alpha-amylase added to
initiate liquefaction. The slurry is then jet-cooked at a
temperature between 105-125.degree. C. to complete gelatinization
of the slurry, cooled to 60-95.degree. C. and, generally,
additional alpha-amylase is added to finalize hydrolysis. The
liquefaction process is generally carried out at pH between 5 and
6. Milled and liquefied whole grains are known as mash.
[0005] During saccharification, the dextrins from the liquefaction
are further hydrolyzed to produce low molecular sugars DP1-3 that
can be metabolized by yeast. The hydrolysis is typically
accomplished using glucoamylases, alternatively or in addition to
glucoamylases, alpha-glucosidases and/or acid alpha-amylases can be
used. A full saccharification step typically last up to 72 hours,
however, it is common only to do a pre-saccharification of, e.g.,
40-90 minutes at a temperature above 50.degree. C., followed by a
complete saccharification during fermentation in a process known as
simultaneous saccharification and fermentation (SSF).
[0006] Fermentation, may be performed using a yeast, e.g., from
Saccharomyces spp., which added to the mash. When the alcohol
product is recovered ethanol, e.g. fuel, potable, or industrial
ethanol, the fermentation is carried out, for typically 35-60 hours
at a temperature of typically around 32.degree. C. When the alcohol
product is beer, the fermentation is carried out, for typically up
to 8 days at a temperature of typically around 14.degree. C.
[0007] Following fermentation, the mash may be used, e.g. as a
beer, or distilled to recover ethanol. The ethanol may be used as,
e.g., fuel ethanol, drinking ethanol, and/or industrial
ethanol.
[0008] It will be apparent from the above discussion that the
starch hydrolysis in a conventional alcohol product process is very
energy consuming due to the different temperature requirements
during the various steps. U.S. Pat. No. 4,316,956 provides a
fermentation process for conversion of granular starch into
ethanol. The European Patent EP0140410B2 provides an enzyme
composition for starch hydrolysis. The object of the present
invention is to provide improved processes for conversion of
granular starch into alcohol products.
SUMMARY OF THE INVENTION
[0009] The present invention provides methods for producing an
alcohol product from granular starch without prior gelatinization
of said starch. Accordingly in a first aspect, the invention
provides a process for production of an alcohol product comprising
the sequential steps of: (a) providing a slurry comprising water
and granular starch, (b) holding said slurry in the presence of an
acid alpha-amylase and a glucoamylase at a temperature of 0.degree.
C. to 20.degree. C. below the initial gelatinization temperature of
said granular starch for a period of 5 minutes to 12 hours, (c)
holding said slurry in the presence of an acid alpha-amylase, and a
glucoamylase, and a yeast at a temperature of between 10.degree. C.
and 35.degree. C. for a period of 20 to 250 hours to produce
ethanol and, (d) optionally recovering the ethanol.
[0010] Although not limited to any one theory of operation, the
present invention, in particular, process step (b), is believed to
result in swelling of starch granules enclosed in the plant cells
resulting in the disruption of cell walls and release of the starch
granules thereby rendering the starch granules more accessible to
further hydration and the action of the enzymes. As hydration
progresses through step (b), the acid alpha-amylase degrades the
starch granules to produce dextrins, which are degraded by the
glucoamylase into glucose. This process continues during step (c)
in which the glucose is continuously fermented to ethanol by the
yeast, thereby maintaining the concentration of fermentable sugar
at a relatively low concentration throughout the fermentation.
Without being limited to any one theory of operation, it is
believed that due to the low concentration of sugars present during
fermentation, the production of glycerol by the yeast is decreased
as there is a limited need for glycerol for osmoregulation. In this
regard, the present invention may be used to produce an alcohol
product which has a reduced glycerol and/or methanol content
compared to conventional processes.
[0011] The present invention provides a less energy consuming
alternative to conventional processes which must employ significant
amounts of energy to gelatinize the starch slurry. Other advantages
of the present invention include, without limitation, the ability
to employ a low pH throughout the process, thus reducing the risk
of unwanted microbial growth, and reducing or eliminating the need
for expensive equipment to gelatinize the starch, such as, jetting
installations and steam plant equipment.
[0012] In a second aspect the present invention relates to an
enzyme composition comprising an acid alpha-amylase and a
glucoamylase, wherein the ratio between the acid alpha-amylase
activity and glucoamylase activity is from 0.30 to 5.00 AFAU/AGU
wherein an additional enzyme activity is present; said enzyme
activity is selected from the list consisting of cellulase,
xylanase and phytase.
[0013] In a third aspect the present invention relates to a use of
the enzyme composition of the second aspect in an alcohol product
process or a starch hydrolysis process.
[0014] In a fourth aspect the present invention relates to a use of
an enzyme composition comprising an acid alpha-amylase and a
glucoamylase, wherein the ratio between the acid alpha-amylase
activity and glucoamylase activity is from 0.30 to 5.00 AFAU/AGU,
in an alcohol product process comprising hydrolysis of granular
starch.
[0015] In a sixth aspect the present invention relates to a mashing
process comprising application of an acid alpha-amylase.
DETAILED DESCRIPTION OF THE INVENTION
[0016] The term "alcohol product" means a product comprising
ethanol, e.g. fuel ethanol, potable and industrial ethanol.
However, the alcohol product may also be a beer, which beer may be
any type of beer. Preferred beer types comprise ales, stouts,
porters, lagers, bitters, malt liquors, happoushu, high-alcohol
beer, low-alcohol beer, low-calorie beer or light beer.
[0017] The term "granular starch" means raw uncooked starch, i.e.
starch in its natural form found in cereal, tubers or grains.
Starch is formed within plant cells as tiny granules insoluble in
water. When put in cold water, the starch granules may absorb a
small amount of the liquid and swell. At temperatures up to
50.degree. C. to 75.degree. C. the swelling may be reversible.
However, with higher temperatures an irreversible swelling called
gelatinization begins.
[0018] The term "initial gelatinization temperature" means the
lowest temperature at which gelatinization of the starch commences.
Starch heated in water begins to gelatinize between 50.degree. C.
and 75.degree. C.; the exact temperature of gelatinization depends
on the specific starch, and can readily be determined by the
skilled artisan. Thus, the initial gelatinization temperature may
vary according to the plant species, to the particular variety of
the plant species as well as with the growth conditions. In the
context of this invention the initial gelatinization temperature of
a given starch is the temperature at which birefringence is lost in
5% of the starch granules using the method described by Gorinstein.
S. and Lii. C., Starch/Strke, Vol. 44 (12) pp. 461-466 (1992).
[0019] The polypeptide "homology" means the degree of identity
between two amino acid sequences. The homology may suitably be
determined by computer programs known in the art, such as, GAP
provided in the GCG program package (Program Manual for the
Wisconsin Package, Version 8, August 1994, Genetics Computer Group,
575 Science Drive, Madison, Wis., USA 53711) (Needleman, S. B. and
Wunsch, C. D., (1970), Journal of Molecular Biology, 48, 443-453.
The following settings for polypeptide sequence comparison are
used: GAP creation penalty of 3.0 and GAP extension penalty of
0.1.
[0020] The term "acid alpha-amylase" means an alpha-amylase (E.C.
3.2.1.1) which added in an effective amount has activity at a pH in
the range of 3.0 to 7.0, preferably from 3.5 to 6.0, or more
preferably from 4.0-5.0. Any suitable acid alpha-amylase may be
used in the present invention.
[0021] In a preferred embodiment, the acid alpha-amylase is an acid
fungal alpha-amylase or an acid bacterial alpha-amylase. Preferably
the acid fungal alpha-amylase is obtained from a strain of
Aspergillus, preferably a strain of Aspergillus niger or a strain
of a strain of Aspergillus oryzae. More preferably the acid
alpha-amylase is an acid alpha-amylase having at least 70%
homology, such as at least 80% or even at least 90% homology to the
acid fungal alpha-amylase having the amino acid sequence set forth
in SEQ ID NO:1 or having at least at least at least 70% homology,
such as at least 80% or even at least 90% homology to the acid
fungal alpha-amylase having the amino acid in the sequence shown in
SWISPROT No: P10529.
[0022] Most preferably the acid alpha-amylase is an acid fungal
alpha-amylase having the amino acid sequence set forth in SEQ ID
NO:1 or variants thereof having one or more amino acid residues
which have been deleted, substituted and/or inserted compared to
the amino acid sequence of SEQ ID NO:1; which variants have
alpha-amylase activity.
[0023] Preferred acid alpha-amylase for use in the present
invention may be derived from a strain of B. licheniformis, B.
amyloliquefaciens, and B. stearothermophilus. Also preferred are
acid alpha-amylases having an amino acid sequence which has at
least 50% homology, preferably at least 60%, 70%, 80%, 85% or at
least 90%, e.g. at least 95%, 97%, 98%, or at least 99% homology to
the sequences set forth in SEQ ID NO:2 or SEQ ID NO:3. Preferably
the acid alpha-amylase used for the process of the invention is one
of the acid alpha-amylase variants and hybrids described in
WO96/23874, WO97/41213, and WO99/19467, such as the Bacillus
stearothermophilus alpha-amylase (BSG alpha-amylase) variant having
the following mutations delta(181-182)+N193F (also denoted
1181*+G182*+N193F) compared to the wild type amino acid sequence
set forth in SEQ ID NO:2. The acid bacterial alpha-amylase may also
be a hybrid alpha-amylase comprising the 445 C-terminal amino acid
residues of the Bacillus licheniformis alpha-amylase set forth in
SEQ ID NO:3 and the 37 N-terminal amino acid residues of the
alpha-amylase derived from Bacillus amyloliquefaciens set forth in
SEQ ID NO:4, which may further have the substitutions
G48A+T49I+G107A+H156Y+A181T+N190F+I201F+A209V+Q264S using the
numbering in SEQ ID NO:3. Also preferred are alpha-amylase variants
derived from Bacillus amyloliquefaciens and having at least 50%
homology, such as at least 60%, at least 70%, at least 80%, or even
90% homology to the sequence set forth in SEQ ID NO:4. Especially
preferred are variants having one or more of the mutations H154Y,
A181T, N190F, A209V and Q264S and/or deletion of two residues
between positions 176 and 179, preferably deletion of E178 and
G179.
[0024] Preferred commercial compositions comprising alpha-amylase
include Mycolase from DSM (Gist Brochades), BAN.TM., TERMAMYL.TM.
SC, FUNGAMYL.TM., LIQUOZYME.TM. X and SAN.TM. SUPER, SAN.TM. EXTRA
L (Novozymes A/S) and Clarase L-40,000, DEX-LO.TM., Spezyme FRED,
SPEZYME.TM. AA, and SPEZYME.TM. DELTA AA (Genencor Int.).
[0025] A glucoamylase (E.C.3.2.1.3) to be used in the processes of
the invention may be derived from a microorganism or a plant.
Preferred is glucoamylases of fungal origin such as Aspergillus
glucoamylases, in particular A. niger G1 or G2 glucoamylase (Boel
et al. (1984), EMBO J. 3 (5), p. 1097-1102). Also preferred are
variants thereof, such as disclosed in WO92/00381 and WO00/04136;
the A. awamori glucoamylase (WO84/02921), A. oryzae (Agric. Biol.
Chem. (1991), 55 (4), p. 941-949), or variants or fragments
thereof. Preferred glucoamylases include the glucoamylases derived
from Aspergillus niger, such as a glucoamylase having 50%, 55%,
60%, 65%, 70%, 75%, 80%, 85% or even 90% homology to the amino acid
sequence set forth in WO00/04136 and SEQ ID NO: 13. Also preferred
are the glucoamylases derived from Aspergillus oryzae, such as a
glucoamylase having 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or even
90% homology to the amino acid sequence set forth in WO0/04136 SEQ
ID NO:2.
[0026] Other preferred glucoamylases include Talaromyces
glucoamylases, in particular derived from Talaromyces emersonii
(WO99/28448), Talaromyces leycettanus (U.S. Pat. No. Re. 32, 153),
Talaromyces duponti, Talaromyces thermophilus (U.S. Pat. No.
4,587,215), Clostridium, in particular C. thermoamylolyticum
(EP135,138), and C. thermohydrosulfuricum (WO86/01831).
[0027] Commercially available compositions comprising glucoamylase
include AMG 200L; AMG 300 L; SAN.TM. SUPER, SAN EXTRA L and AMG.TM.
E (from Novozymes A/S); OPTIDEX.TM. 300 (from Genencor Int.);
AMIGASE.TM. and AMIGAS.TM. PLUS (from DSM); G-ZYME.TM. G900,
G-ZYME.TM. and G990 ZR (from Genencor Int.).
[0028] An additional enzyme that may be used in the processes of
the invention includes xylanases, cellulases and phytases.
[0029] A xylanase used according to the invention may be derived
from any suitable organism, including fungal and bacterial
organisms, such as Aspergillus, Disporotrichum, Penicillium,
Neurospora, Fusarium and Trichoderma.
[0030] Preferred commercially available preparations comprising
xylanase include SHEARZYME.RTM., BIOFEED WHEAT.RTM.,
CELLUCLAST.RTM., ULTRAFLO.RTM., VISCOZYME.RTM. (from Novozymes A/S)
and SPEZYME.RTM. CP (from Genencor Int.).
[0031] The cellulase activity (E.C. 3.2.1.4) may be a cellulase of
microbial origin, such as derivable from a strain of a filamentous
fungus (e.g., Aspergillus, Trichoderma, Humicola, Fusarium).
[0032] Commercially available preparations comprising cellulase
which may be used include CELLUCLAST.RTM., CELLUZYME.RTM.,
CEREFLO.RTM. and ULTRAFLO.RTM. (from Novozymes A/S), LAMINEX.TM.
and SPEZYME.RTM. CP (from Genencor Int.) and ROHAMENT.RTM. 7069 W
(from Rohm GmbH).
[0033] A phytase used according to the invention may be any enzyme
capable of effecting the liberation of inorganic phosphate from
phytic acid (myo-inositol hexakisphosphate) or from any salt
thereof (phytates). Phytases can be classified according to their
specificity in the initial hydrolysis step, viz. according to which
phosphate-ester group is hydrolyzed first. The phytase to be used
in the invention may have any specificity, e.g., be a 3-phytase
(E.C. 3.1.3.8), a 6-phytase (E.C. 3.1.3.26) or a 5-phytase (no E.C.
number).
[0034] Commercially available phytases preferred according to the
invention include BIO-FEED PHYTASE.TM., PHYTASE NOVO.TM. CT or L
(Novozymes A/S), or NATUPHOS.TM. NG 5000 (DSM).
[0035] Another enzyme used in the process may be a debranching
enzyme, such as an isoamylase (E.C. 3.2.1.68) or a pullulanases
(E.C. 3.2.1.41). Isoamylase hydrolyses alpha-1,6-D-glucosidic
branch linkages in amylopectin and beta-limit dextrins and can be
distinguished from pullulanases by the inability of isoamylase to
attack pullulan, and by the limited action on alpha-limit dextrins.
Debranching enzyme may be added in effective amounts well known to
the person skilled in the art.
[0036] In a first preferred embodiment of the first aspect, the
invention provides a process for production of ethanol, comprising
the steps of: (a) providing a slurry comprising water and granular
starch, (b) holding said slurry in the presence of an acid
alpha-amylase and a glucoamylase at a temperature of 0.degree. C.
to 20.degree. C. below the initial gelatinization temperature of
said granular starch for a period of 5 minutes to 12 hours, (c)
holding said slurry in the presence of an acid alpha-amylase, and a
glucoamylase, and a yeast at a temperature of between 30.degree. C.
and 35.degree. C. for a period of 20 to 200 hours to produce
ethanol, and, (d) recovering the ethanol. The steps (a), (b), (c)
and (d) are performed sequentially; however, the process may
comprise additional steps not specified in this description which
are performed prior to, between or after any of steps (a), (b), (c)
and (d).
[0037] In the first preferred embodiment of the first aspect the
temperature under step (c) is between 28.degree. C. and 36.degree.
C., preferably from 29.degree. C. and 35.degree. C., more
preferably from 30.degree. C. and 34.degree. C., such as around
32.degree. C. and the slurry is held in contact with the acid
alpha-amylase, the glucoamylase and the yeast for a period of time
sufficient to allow hydrolysis of the starch and fermentation of
the released sugars during step (c), preferably for a period of 25
to 190 hours, preferably from 30 to 180 hours, more preferably from
40 to 170 hours, even more preferably from 50 to 160 hours, yet
more preferably from 60 to 150 hours, even yet more preferably from
70 to 140 hours, and most preferably from 80 to 130 hours, such as
85 to 110 hours.
[0038] In a second preferred embodiment of the first aspect, the
invention provides a process for production of a beer, comprising
the steps of: (a) providing a slurry comprising water and granular
starch, (b) holding said slurry in the presence of an acid
alpha-amylase and a glucoamylase at a temperature of 0.degree. C.
to 20.degree. C. below the initial gelatinization temperature of
said granular starch for a period of 5 minutes to 12 hours, (c)
holding said slurry in the presence of an acid alpha-amylase, and a
glucoamylase, and a yeast at a temperature between 10.degree. C.
and 18.degree. C. for a period of 20 to 200 hours to produce
ethanol. The steps (a), (b), and (c) are performed sequentially;
however, the process may comprise additional steps not specified in
this description which are performed prior to, between or after any
of steps (a), (b), and (c).
[0039] In the second preferred embodiment of the first aspect the
temperature under step (c) is between 10.degree. C. and 18.degree.
C., preferably from 11.degree. C. and 17.degree. C., more
preferably from 12.degree. C. and 16.degree. C., such as between
13.degree. C. and 15.degree. C., e.g. around 14.degree. C. and the
slurry is held in contact with the acid alpha-amylase, the
glucoamylase and the yeast for a period of time sufficient to allow
hydrolysis of the starch and fermentation of the released sugars
during step (c), preferably for a period of 100 to 230 hours,
preferably from 150 to 210 hours, more preferably from 170 to 200
hours.
[0040] The enzyme activities may preferably be dosed in form of the
composition of the second aspect of the invention.
[0041] The acid alpha-amylase is preferably an acid bacterial
alpha-amylase and/or an acid fungal alpha-amylase and/or a variant
of an acid alpha-amylase derived from a bacterial and/or a fungal
source.
[0042] The acid alpha-amylase is added in an effective amount,
which is a concentration of acid alpha-amylase sufficient for its
intended purpose of converting the granular starch in the starch
slurry to dextrins. Preferably the acid alpha-amylase is present in
an amount of 10-10000 AFAU/kg of DS, in an amount of 500-2500
AFAU/kg of DS, or more preferably in an amount of 100-1000 AFAU/kg
of DS, such as approximately 500 AFAU/kg DS. When measured in AAU
units the acid alpha-amylase activity is preferably present in an
amount of 5-500000 AAU/kg of DS, in an amount of 500-50000 AAU/kg
of DS, or more preferably in an amount of 100-10000 AAU/kg of DS,
such as 500-1000 AAU/kg DS.
[0043] The glucoamylases is added in an effective amount, which is
a concentration of glucoamylase amylase sufficient for its intended
purpose of degrading the dextrins resulting from the acid
alpha-amylase treatment of the starch slurry. Preferably the
glucoamylase activity is present in an amount of 20-200 AGU/kg of
DS, preferably 100-1000 AGU/kg of DS, or more preferably in an
amount of 200-400 AGU/kg of DS, such as 250 AGU/kg DS. When
measured in AGI units the glucoamylase activity is preferably
present in an amount of 10-100000 AGI/kg of DS, 50-50000 AGI/kg of
DS, preferably 100-10000 AGI/kg of DS, or more preferably in an
amount of 200-5000 AGI/kg of DS.
[0044] Preferably the activities of acid alpha-amylase and
glucoamylase are present in a ratio of between 0.3 and 5.0
AFAU/AGU. More preferably the ratio between acid alpha-amylase
activity and glucoamylase activity is at least 0.35, at least 0.40,
at least 0.50, at least 0.60, at least 0.7, at least 0.8, at least
0.9, at least 1.0, at least 1.1, at least 1.2, at least 1.3, at
least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8,
at least 1.85, or even at least 1.9 AFAU/AGU. However, the ratio
between acid alpha-amylase activity and glucoamylase activity
should preferably be less than 4.5, less than 4.0, less than 3.5,
less than 3.0, less than 2.5, or even less than 2.25 AFAU/AGU. In
AUU/AGI the activities of acid alpha-amylase and glucoamylase are
preferably present in a ratio of between 0.4 and 6.5 AUU/AGI. More
preferably the ratio between acid alpha-amylase activity and
glucoamylase activity is at least 0.45, at least 0.50, at least
0.60, at least 0.7, at least 0.8, at least 0.9, at least 1.0, at
least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5,
at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least
2.0, at least 2.1, at least 2.2, at least 2.3, at least 2.4, or
even at least 2.5 AUU/AGI. However, the ratio between acid
alpha-amylase activity and glucoamylase activity is preferably less
than 6.0, less than 5.5, less than 4.5, less than 4.0, less than
3.5, or even less than 3.0 AUU/AGI.
[0045] In a preferred embodiment of the first aspect of the
invention the step (b) and/or step (c) is performed in the presence
of an additional enzyme activity selected from the list consisting
of xylanase, cellulase and phytase. The additional enzyme is
preferably added together with the acid alpha-amylase and the
glucoamylase. Xylanases may be added in amounts of 1-50000 FXU/kg
DS, preferably 5-5000 FXU/kg DS, or more preferably 10-500 FXU/kg
DS. Cellulases may be added in the amounts of 0.01-500000 EGU/kg
DS, preferably from 0.1-10000 EGU/kg DS, preferably from 1-5000
EGU/kg DS, more preferably from 10-500 EGU/kg DS and most
preferably from 100-250 EGU/kg DS. The dosage of the phytase may be
in the range 0.5-250000 FYT/kg DS, particularly 1-100000 FYT/kg DS,
preferably in the range from 5-25000 FYT/kg DS, preferably 10-10000
FYT/kg, such as 100-1000 FYT/kg DS.
[0046] In a preferred embodiment the starch slurry comprises water
and 5-60% DS (dry solids) granular starch, preferably 10-50% DS
granular starch, more preferably 15-40% DS, especially around
20-25% DS granular starch. The granular starch to be processed in
the processes of the invention may in particular be obtained from
tubers, roots, stems, cobs, legumes, cereals or whole grain. More
specifically the granular starch may be obtained from corns, cobs,
wheat, barley, rye, milo, sago, cassava, tapioca, sorghum, rice,
peas, bean, banana or potatoes. Preferred are both waxy and
non-waxy types of corn and barley. The granular starch to be
processed may preferably comprising milled whole grain or it may be
a more refined starch quality, preferably more than 90%, 95%, 97%
or 99.5% pure starch. The raw material comprising the starch is
preferably milled in order to open up the structure and allowing
for further processing. Dry milling as well as wet milling may be
used. When wet milling is applied it may be preceded by a soaking,
or steeping step. Both dry and wet milling is well known in the art
of alcohol manufacturing and is preferred for the processes of the
invention. In the second embodiment of the first aspect of the
invention wherein the alcohol product is a beer the granular starch
may preferably comprise at least 10%, 20%, 30%, 40%, 50%, 60%, 70%,
80% or even at least 90% granular starch derived from malted
cereals, e.g. barley malt.
[0047] The pH during step (b) and/or (c) is preferably in the range
of 3.0 to 7.0, more preferably from 3.5 to 6.0, or most preferably
from 4.0-5.0, such as from 4.3 to 4.6.
[0048] The slurry is held in contact with the acid alpha-amylase
and glucoamylase at an elevated temperature but below the initial
gelatinization temperature for a period of time effective to render
the starch granules susceptible for enzymatic degradation (step b),
preferably for a period of 5 minutes to 12 hours, preferably from
10 minutes to 6 hours, more preferably from 15 minutes to 3 hours,
even more preferably from 20 minutes to 11/2 hour, such as from 30
minutes to 11/4 hour, from 40 to 70 minutes, and even from 50 to 60
minutes. The temperature during step (b) should always be adjusted
to be below the initial gelatinization temperature of the
particular granular starch to be processed, and will typically be
between 45.degree. C. and 75.degree. C. According to the invention
step (b) is conducted at a temperature from 0.degree. C. to
20.degree. C., preferably to from 0.degree. C. 15.degree. C., more
preferably from 0.degree. C. to 10.degree. C., or even more
preferably from 0.degree. C. to 5.degree. C. below the initial
gelatinization temperature of the particular starch to be
processed. The actual temperature may be from 45.degree. C. to
75.degree. C., but is preferably from 55.degree. C. to 65.degree.
C. Preferably the temperature at which step (b) is conducted is at
least 45.degree. C., 46.degree. C., 47.degree. C., 48.degree. C.,
49.degree. C., 50.degree. C., 51.degree. C., 52.degree. C.,
53.degree. C., 54.degree. C., 55.degree. C., 56.degree. C.,
57.degree. C., 58.degree. C. or preferably at least 55.degree. C.,
and preferably the temperature is no more than 74.degree. C.,
73.degree. C., 72.degree. C., 71.degree. C., 70.degree. C.,
69.degree. C., 68.degree. C., 67.degree. C., 66.degree. C.,
65.degree. C., 64.degree. C., 63.degree. C. or preferably no more
than 62.degree. C.
[0049] After being subjected to the process of the first aspect of
the invention at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, or preferably 99% of the dry solids of the
granular starch is converted into ethanol.
[0050] The ethanol may optionally be recovered. The ethanol
recovery may be performed by any conventional manner such as e.g.
distillation and may be used as fuel ethanol and/or potable ethanol
and/or industrial ethanol.
[0051] In a particularly preferred embodiment the granular starch
to be processed is derived from dry or wet milled cereal, such as
wheat, barley, rye, and/or corn, the starch slurry has a DS of
20-40 percent, the temperature during step (b) is from 50.degree.
C. to 60.degree. C., such as 55.degree. C., the duration of step
(b) is from 30 minutes to 75 minutes, such as 60 minutes and step
(c) is carried out for 60 to 90 hours. The acid alpha amylase is
dosed at 300 to 700 AFAU/kg DS, such as 500 AFAU/kg DS and the
glucoamylase is dosed at 100 to 500 AGU/kg DS, such as 250 AGU/kg
DS. The ratio of acid alpha amylase to glucoamylase is from 1.0 to
3.0 AFAU/AGU or preferably from 1.5 to 2.5 AFAU/AGU, such as
approximately 2.0 AFAU/AGU. In AAU/AGI the ratio of acid alpha
amylase to glucoamylase is from 1.3 to 4.0 AAU/AGI or preferably
from 2.0 to 2.3 AAU/AGI, such as approximately 2.7 AAU/AGI.
[0052] In a second aspect the invention provides an enzyme
composition which may be used in any process, including the process
of the first aspect of the invention, said enzyme composition
having a ratio between acid alpha-amylase activity and glucoamylase
activity of at least 0.35, at least 0.40, at least 0.50, at least
0.60, at least 0.70, at least 0.80, at least 0.90, at least 1.00,
at least 1.20, at least 1.30, at least 1.40, at least 1.50, at
least 1.60, at least 1.70, at least 1.80, or even at least 1.85
AFAU/AGU. Preferably said enzyme composition has a ratio between
acid alpha-amylase activity and glucoamylase activity is less than
5.00, less than 4.50, less than 4.00, less than 3.00, less than
2.50, or even less than 2.25 AFAU/AGU. Measured in AAU/AGI the
ratio of acid alpha amylase to glucoamylase in said enzyme
composition is at least 0.45, at least 0.50, at least 0.60, at
least 0.70, at least 0.80, at least 0.90, at least 1.00, at least
1.20, at least 1.30, at least 1.40, at least 1.50, at least 1.60,
at least 1.70, at least 1.80, at least 1.90, at least 2.00, at
least 2.10, at least 2.20, at least 2.30, at least 2.40 or even at
least 2.50 AAU/AGI. Preferably said enzyme composition has a ratio
between acid alpha-amylase activity and glucoamylase activity is
less than 6.50, less than 5.00, less than 4.50, less than 4.00, or
even less than 3.50 AAU/AGI.
[0053] In a preferred embodiment the composition of the second
aspect of the invention further comprises an additional enzyme
activity is present; said enzyme activity is selected from the list
consisting of cellulase, xylanase and phytase.
Additional Applications
[0054] In a sixth aspect of the invention an acid alpha-amylase,
such as an acid alpha-amylase derived from a fungus, preferably of
the genus Aspergillus, preferably from the species A. niger, and
most preferably having at least 50%, at least 60%, at least 70%, at
least 80% or even at least 90% homology to the sequence shown in
SEQ ID No:1 is used in a brewing process.
[0055] Brewing processes are well-known in the art, and generally
involve the steps of malting, mashing, and fermentation. In the
traditional brewing process the malting serves the purpose of
converting insoluble starch to soluble starch, reducing complex
proteins, generating color and flavor compounds, generating
nutrients for yeast development, and the development of enzymes.
The three main steps of the malting process are steeping,
germination, and kilning.
[0056] Steeping includes mixing the barley kernels with water to
raise the moisture level and activate the metabolic processes of
the dormant kernel. In the next step, the wet barley is germinated
by maintaining it at a suitable temperature and humidity level
until adequate modification, i.e. such as degradation of starch and
activation of enzymes, has been achieved. The final step is to dry
the green malt in the kiln.
[0057] Mashing is the process of converting starch from the milled
barley malt and solid adjuncts into fermentable and unfermentable
sugars to produce wort of the desired composition. Traditional
mashing involves mixing milled barley malt and adjuncts with water
at a set temperature and volume to continue the biochemical changes
initiated during the malting process. The mashing process is
conducted over a period of time at various temperatures in order to
activate the endogenous enzymes responsible for the degradation of
proteins and carbohydrates. By far the most important change
brought about in mashing is the conversion of starch molecules into
fermentable sugars. The principal enzymes responsible for starch
conversion in a traditional mashing process are alpha- and
beta-amylases. Alpha-amylase very rapidly reduces insoluble and
soluble starch by splitting starch molecules into many shorter
chains that can be attacked by beta-amylase. The disaccharide
produced is maltose.
[0058] To day the double-mash infusion system is the most widely
used system for industrial production of beer, especially lager
type beer. This system prepares two separate mashes. It utilizes a
cereal cooker for boiling adjuncts and a mash tun for
well-modified, highly enzymatically active malts. As the
traditionally mashing processes utilize the endogenous enzymes of
the barley malt the temperature is maintained below 70.degree. C.
as inactivation of the enzymes would otherwise occur.
[0059] After mashing, when all the starch has been broken down, it
is necessary to separate the liquid extract (the wort) from the
solids (spent grains). Wort separation is important because the
solids contain large amounts of protein, poorly modified starch,
fatty material, silicates, and polyphenols (tannins). The
objectives of wort separation include the following:
[0060] to produce clear wort,
[0061] to obtain good extract recovery, and
[0062] to operate within the acceptable cycle time.
[0063] Wort clarity, extraction recovery, and overall cycle times
is greatly affected by the standard of the grist, e.g. the barley
malt and the types of adjunct, as well as the applied mashing
procedure.
[0064] Following the separation of the wort from the spent grains
the wort may be fermented with brewers yeast to produce a beer.
[0065] Further information on conventional brewing processes 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.
[0066] An acid alpha-amylase, such as an acid alpha-amylase derived
from a fungus, preferably of the genus Aspergillus, preferably from
the species A. niger, and most preferably having at least 50%, at
least 60%, at least 70%, at least 80% or even at least 90% homology
to the sequence shown in SEQ ID No:1 be applied in any brewing
process as a supplement to the enzymes comprised in the malted
and/or unmalted grain or in a higher temperature mashing (HTM)
process such as the one disclosed in WO 2004/011591.
[0067] In the HTM process the temperature regime applied in the
initial mashing phase ensures that the activity of the various
endogenous enzymes of the barley malt or of the adjunct is
significantly reduced or even eliminated. The high temperature
process preferably comprises, forming a mash comprising between 5%
and 100% barley malt, adding prior to, during or after forming the
mash a mashing enzyme composition, attaining within 15 minutes of
forming the mash an initial incubation temperature of at least
70.degree. C., followed by incubation of the mash at a temperature
of at least 70.degree. C. for a period of time, and separating the
wort from the spent grains. Preferably the period of time of
mashing is sufficient to achieve an extract recovery of at least
80%. The term "initial incubation temperature" is understood as the
temperature regime during the initial part of the incubation in
question.
[0068] Thus at temperatures in the interval 70.degree. C. to
78.degree. C. only the barley malt alpha- and beta-amylases will
exhibit notable activity, and at temperatures above 78.degree. C.
the endogenous enzymes activity will be negligible. In such a
mashing process the added mashing enzymes will thus constitute a
very essential part of or all enzyme activity. According to one
embodiment of the sixth aspect of the invention enzyme activities
needed for the mashing process to proceed are exogenously supplied
and may be added to the mash ingredients, e.g. the water or the
grist before forming the mash, or it may be added during or after
forming the mash. The enzymes are preferably supplied all at one
time at the start of the process; however, one or more of the
enzymes may be supplied at one or more times prior to, at the
start, or during the process of the sixth aspect of the invention.
In addition to an acid alpha-amylase (E.C. 3.2.1.1) the enzyme
activities added may comprise one or more of the following
activities; a protease (E.C. 3.4.), cellulase (E.C. 3.2.1.4) and a
maltose generating enzyme. The maltose generating enzyme is
preferably a beta-amylase (E.C. 3.2.1.2) or even more preferably a
maltogenic alpha-amylase (E.C. 3.2.1.133).
[0069] In yet a preferred embodiment a further enzyme is added,
said enzyme being selected from the group consisting of laccase,
lipase, glucoamylase, phospholipolase, phytase, phytin esterase,
pullulanase, and xylanase.
[0070] In accordance with the sixth aspect of the invention a
starch containing slurry, the mash, is obtained by mixing a grist
comprising at least 5%, or 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) barley malt with water.
Preferably at least 5%, preferably at least 10%, more preferably at
least 20%, even more preferably at least 50%, at least 75% or even
100% of the barley malt is well modified barley malt. In one
embodiment the grist comprises other malted grain than barley malt,
so that at least 10%, at least 25%, preferably at least 35%, more
preferably at least 50%, even more preferably at least 75%, most
preferably at least 90% (w/w) of the grist is other malted grain
than barley malt.
[0071] Prior to forming the mash the malted and/or unmalted grain
is preferably milled and most preferably dry milled. In a preferred
embodiment the malted and/or unmalted grain used is a husk free (or
hull free) variety or the husks are removed from the malted and/or
unmalted grain before forming the mash. Removal of husks is
preferably applied where the mashing programs comprising
temperatures above 75.degree. C., such as at temperatures above
76.degree. C., 77.degree. C., 78.degree. C., 79.degree. C.,
80.degree. C., 81.degree. C., 82.degree. C., 83.degree. C.,
84.degree. C., 85.degree. C. or even above 86.degree. C.
[0072] The water may preferably, before being added to the grist,
be preheated in order for the mash to attain the initial incubation
temperature at the moment of mash forming. If the temperature of
the formed mash is below the initial incubation temperature
additional heat is preferably supplied in order to attain the
initial incubation temperature. Preferably the initial incubation
temperature is attained within 15 minutes, or more preferably
within 10 minutes, such as within 9, 8, 7, 6, 5, 4, 2 minutes or
even more preferably within 1 minute after the mash forming, or
most preferably the initial incubation temperature is attained at
the mash forming.
[0073] The initial incubation temperature is preferably at least
70.degree. C., preferably at least 71.degree. C., more preferably
at least 72.degree. C., even more preferably at least 73.degree.
C., or most preferably at least 74.degree. C., such as at least
75.degree. C., at least 76.degree. C., at least 77.degree. C., at
least 78.degree. C., at least 79.degree. C., at least 80.degree.
C., at least 81.degree. C., such as at least 82.degree. C. A
preferred embodiment of the mashing process of the sixth aspect of
the invention includes incubating the mash at the initial
incubation temperature of at least 70.degree. C. and maintaining a
temperature of at least 70.degree. C., preferably at least
71.degree. C., more preferably at least 72.degree. C., even more
preferably at least 73.degree. C., or most preferably at least
74.degree. C., such as at least 75.degree. C., at least 76.degree.
C., at least 77.degree. C., at least 78.degree. C., at least
79.degree. C., at least 80.degree. C., at least 81.degree. C., at
least 82.degree. C., at least 83.degree. C., at least 84.degree.
C., or at least 85.degree. C. i.e. a temperature that never falls
below 70.degree. C. for the duration of the incubation period.
During the incubation period the temperature is preferably held
below 100.degree. C., such as below 99.degree. C., 98.degree. C.,
97.degree. C., 96.degree. C., 95.degree. C., 94.degree. C.,
93.degree. C., 92.degree. C., 91.degree. C., or even below
90.degree. C.
[0074] In the mashing process of the sixth aspect of the invention
the temperature may be held constant for the duration of the
incubation, or, following a period of an essentially constant
temperature (the initial incubation temperature) for the first part
of the incubation the temperature may be raised, either as a slow
continuously increase, or as one or more stepwise increment(s)
during the incubation. Alternatively the temperature may be
decreased during the incubation. In one embodiment the initial
incubation temperature is at least 70.degree. C. and during the
incubation the temperature is increased with at least 1.degree. C.,
2.degree. C., 3.degree. C., 4.degree. C., 5.degree. C., 6.degree.
C., 7.degree. C., 8.degree. C., 9.degree. C. or preferably with at
least 10.degree. C., or more preferably with at least 12.degree.
C., such as 15.degree. C. In another embodiment the initial
incubation temperature is at least 75.degree. C., or preferably at
least 80.degree. C., and the temperature is decreased during the
incubation with at least 5.degree. C., or preferably with at least
1.degree. C., 2.degree. C., 3.degree. C., 4.degree. C., 5.degree.
C., 6.degree. C., 7.degree. C., 8.degree. C., 9.degree. C. or
preferably with at least 10.degree. C., or more preferably with at
least 15.degree. C. In a particular embodiment the incubation
comprises maintaining the mash at a temperature of at least
75.degree. C., preferably at least 76.degree. C., more preferably
at least 77.degree. C., even more preferably at least 78.degree.
C., such as at least 79.degree. C., at least 80.degree. C., at
least 81.degree. C., 82.degree. C., 83.degree. C., 84.degree. C.,
85.degree. C., 86.degree. C., 87.degree. C., 88.degree. C.,
89.degree. C. or at least 90.degree. C. for a period of at least 1
minute, preferably for at least 5 minutes, more preferably for at
least 15 minutes, even more preferably for at least 20 minutes,
such as at least 30 minutes, at least 40 minutes, at least 50
minutes, at least 60 minutes, at least 90 minutes, or at least 120
minutes. In another particular embodiment the incubation comprises
maintaining the mash at a temperature of at least 75.degree. C.,
preferably at least 76.degree. C., more preferably at least
77.degree. C., even more preferably at least 78.degree. C., such as
at least 79.degree. C., at least 80.degree. C., such as at least
81.degree. C., 82.degree. C., 83.degree. C., 84.degree. C.,
85.degree. C., 86.degree. C., 87.degree. C., 88.degree. C.,
89.degree. C. or at least 90.degree. C. for at least 1% of the
total incubation time, preferably for at least 5%, more preferably
for at least 15%, even more preferably for at least 20%, or at
least 30%, at least 40%, at least 50%, at least 60%, at least 70%,
at least 80%, at least 90%, such as for 100% of the total
incubation time. The duration of the incubation is preferably at
least 15 minutes, typically between 30 minutes and 21/2 hours, e.g.
at least 45 minutes, at least 1 hour, at least 11/4 hour, at least
11/2 hour, at least 13/4 hour or at least 2 hours.
[0075] In the mashing process of the sixth aspect of the invention
the grist may in addition to barley malt preferably comprise
adjunct such as unmalted barley, or other malted or unmalted grain,
such as wheat, rye, oat, corn, rice, milo, millet and/or sorghum,
or raw and/or refined starch and/or sugar containing material
derived from plants like wheat, rye, oat, corn, rice, milo, millet,
sorghum, potato, sweet potato, cassava, tapioca, sago, banana,
sugar beet and/or sugar cane. For the invention adjuncts may be
obtained from tubers, roots, stems, leaves, legumes, cereals and/or
whole grain. Preferably the adjunct to be added to the mash of the
sixth aspect of the invention has gelatinization temperatures at or
below the process temperature. If adjuncts such as rice or corn, or
other adjuncts with similar high gelatinization temperature, are to
be used in the process of the sixth aspect of the invention, they
may preferably be cooked separately to ensure gelatinization before
being added to the mash of the sixth aspect of the invention, or
the gelatinized adjunct starch may be mashed separately from the
mash adding appropriate enzymes to ensure saccharification before
being added to the mash. Methods for gelatinization and
saccharification of brewing adjuncts are well known in the arts.
Adjunct comprising readily fermentable carbohydrates such as sugars
or syrups may be added to the barley malt mash before, during or
after mashing process of the sixth aspect of the invention but is
preferably added after the mashing process. Preferably a part of
the adjunct is treated with a protease and/or a beta-glucanase
before being added to the mash of the sixth aspect of the
invention. During the mashing process, starch extracted from the
grist is gradually hydrolyzed into fermentable sugars and smaller
dextrins. Preferably the mash is starch negative to iodine testing,
before extracting the wort.
[0076] Following the mashing step of the sixth aspect of the
invention obtaining the wort from the mash typically includes
straining the wort from the spent grains, i.e. the insoluble grain
and husk material forming part of grist. Hot water may be run
through the spent grains to rinse out, or sparge, any remaining
extract from the grist.
[0077] In the embodiment wherein the husks are removed from malted
and/or unmalted grain comprised in the grist the wort separation
may comprise a centrifugation step.
[0078] The wort produced by the mashing process of the sixth aspect
of the invention may be fermented to produce a beer. Fermentation
of the wort may include pitching the wort with a yeast slurry
comprising fresh yeast, i.e. yeast not previously used for the
invention or the yeast may be recycled yeast. The yeast applied may
be any yeast suitable for beer brewing, especially yeasts selected
from Saccharomyces spp. such as S. cerevisiae and S. uvarum,
including natural or artificially produced variants of these
organisms. The methods for fermentation of wort for production of
beer are well known to the person skilled in the arts.
[0079] Preferred beer types comprise ales, strong ales, stouts,
porters, lagers, bitters, export beers, malt liquors, happoushu,
high-alcohol beer, low-alcohol beer, low-calorie beer or light
beer.
[0080] The enzymes to be applied in the sixth aspect of present
invention should be selected for their ability to retain sufficient
activity at elevated temperatures, such as at the process
temperature of the processes, as well as for their ability to
retain sufficient activity under the moderately acid pH regime in
the mash and should be added in effective amounts. The enzymes may
be derived from any source, preferably from a plant or an algae,
and more preferably from a microorganism, such as from a bacteria
or a fungi.
[0081] Suitable proteases include microbial proteases, such as
fungal and bacterial proteases. Preferred proteases are acidic
proteases, i.e., proteases characterized by the ability to
hydrolyze proteins under acidic conditions below pH 7.
[0082] Contemplated acid fungal proteases include fungal proteases
derived from Aspergillus, Mucor, Rhizopus, Candida, Coriolus,
Endothia, Enthomophtra, Irpex, Penicillium, Sclerotiumand
Torulopsis. Especially contemplated are proteases derived from
Aspergillus niger (see, e.g., Koaze et al., (1964), Agr. Biol.
Chem. Japan, 28, 216), Aspergillus saitoi (see, e.g., Yoshida,
(1954) J. Agr. Chem. Soc. Japan, 28, 66), Aspergillus awamori
(Hayashida et al., (1977) Agric. Biol. Chem., 42(5), 927-933,
Aspergillus aculeatus (WO 95/02044), or Aspergillus oryzae, such as
the pepA protease; and acidic proteases from Mucor pusillus or
Mucor miehei.
[0083] Contemplated are also neutral or alkaline proteases, such as
a protease derived from a strain of Bacillus. A particular protease
contemplated for the invention is derived from Bacillus
amyloliquefaciens and has the sequence obtainable at Swissprot as
Accession No. P06832 (SEQ ID NO 5). Also contemplated are the
proteases having at least 90% homology to amino acid sequence
obtainable at Swissprot as Accession No. P06832 (SEQ ID NO 5) such
as at least 92%, at least 95%, at least 96%, at least 97%, at least
98%, or particularly at least 99%.
[0084] Further contemplated are the proteases having at least 90%
homology to amino acid sequence disclosed as SEQ.ID.NO:1 in the
Danish patent applications PA 2001 01821 and PA 2002 00005, such as
at 92%, at least 95%, at least 96%, at least 97%, at least 98%, or
particularly at least 99%.
[0085] Also contemplated are papain-like proteases such as
proteases within E.C. 3.4.22.* (cysteine protease), such as EC
3.4.22.2 (papain), EC 3.4.22.6 (chymopapain), EC 3.4.22.7
(asclepain), EC 3.4.22.14 (actinidain), EC 3.4.22.15 (cathepsin L),
EC 3.4.22.25 (glycyl endopeptidase) and EC 3.4.22.30
(caricain).
[0086] Proteases may be added in the amounts of 0.1-1000 AU/kg dm,
preferably 1-100 AU/kg dm and most preferably 5-25 AU/kg dm.
[0087] The cellulase (E.C. 3.2.1.4) may be of microbial origin,
such as derivable from a strain of a filamentous fungus (e.g.,
Aspergillus, Trichoderma, Humicola, Fusarium). Specific examples of
cellulases include the endo-glucanase (endo-glucanase I) obtainable
from H. insolens and further defined by the amino acid sequence of
FIG. 14 in WO 91/17244 and the 43 kD H. insolens endo-glucanase
described in WO 91/17243.
[0088] A particular cellulase to be used in the processes of the
sixth aspect of the invention may be an endo-glucanase, such as an
endo-1,4-beta-glucanase. Contemplated are beta-glucanases having at
least 90% homology to amino acid sequence disclosed as SEQ.ID.NO:1
in Danish patent application PA2002 00130, such as at least 92%, at
least 95%, at least 96%, at least 97%, at least 98%, or
particularly at least 99%.
[0089] Commercially available cellulase preparations which may be
used include CELLUCLAST.RTM., CELLUZYME.RTM.), CEREFLO.RTM. and
ULTRAFLO.RTM. (available from Novozymes A/S), LAMINEX.TM. and
SPEZYME.RTM. CP (available from Genencor Int.) and ROHAMENT.RTM.
7069 W (available from Rohm, Germany).
[0090] Beta-glucanases may be added in the amounts of 1.0-10000
BGU/kg dm, preferably from 10-5000 BGU/kg dm, preferably from
50-1000 BGU/kg dm and most preferably from 100-500 BGU/kg dm.
[0091] A particular alpha-amylase (EC 3.2.1.1) to be used in the
processes of the sixth aspect of the invention may be any fungal
alpha-amylase, preferably an acid alpha-amylase. Preferably the
acid alpha-amylase is derived from a fungus of the genus
Aspergillus, preferably from the species A. niger, and most
preferably having at least 50%, at least 60%, at least 70%, at
least 80% or even at least 90% homology to the sequence shown in
SEQ ID No:1 is used in a brewing process. Fungal alpha-amylases may
be added in an amount of 1-1000 AFAU/kg DM, preferably from 2-500
AFAU/kg DM, preferably 20-100 AFAU/kg DM.
[0092] Another acid alpha-amylase enzyme to be used in the
processes of the sixth aspect of the invention may be a Bacillus
alpha-amylase. Well-known Bacillus alpha-amylases include
alpha-amylase derived from a strain of B. licheniformis, B.
amyloliquefaciens, and B. stearothermophilus. Other Bacillus
alpha-amylases include alpha-amylase derived from a strain of the
Bacillus sp. NCIB 12289, NCIB 12512, NCIB 12513 or DSM 9375, all of
which are described in detail in WO95/26397, and the alpha-amylase
described by Tsukamoto et al., Biochemical and Biophysical Research
Communications, 151 (1988), pp. 25-31. In the context of the
present invention a contemplated Bacillus alpha-amylase is an
alpha-amylase as defined in WO99/19467 on page 3, line 18 to page
6, line 27. A preferred alpha-amylase has an amino acid sequence
having at least 90% homology to SEQ ID NO:4 in WO99/19467, such as
at least 92%, at least 95%, at least 96%, at least 97%, at least
98%, or particularly at least 99%. Most preferred variants of the
maltogenic alpha-amylase comprise the variants disclosed in
WO99/43794. Contemplated variants and hybrids are described in
WO96/23874, WO97/41213, and WO99/19467. Specifically contemplated
is a recombinant B.stearothermophilus alpha-amylase variant with
the mutations; I181*+G182*+N193F. Bacillus alpha-amylases may be
added in the amounts of 1.0-1000 NU/kg dm, preferably from 2.0-500
NU/kg dm, preferably 10-200 NU/kg dm.
[0093] Maltogenic Alpha-amylase
[0094] A particular enzyme to be used in the processes of the sixth
aspect of the invention is a maltogenic alpha-amylase (E.C.
3.2.1.133). Maltogenic alpha-amylases (glucan
1,4-alpha-maltohydrolase) are able to hydrolyse amylose and
amylopectin to maltose in the alpha-configuration. Furthermore, a
maltogenic alpha-amylase is able to hydrolyse maltotriose as well
as cyclodextrin. Specifically contemplated maltogenic
alpha-amylases may be derived from Bacillus sp., preferably from
Bacillus stearothermophilus, most preferably from Bacillus
stearothermophilus C599 such as the one described in EP 120.693.
This particular maltogenic alpha-amylase has the amino acid
sequence shown as amino acids 1-686 of SEQ ID NO:1 in U.S. Pat. No.
6,162,628. A preferred maltogenic alpha-amylase has an amino acid
sequence having at least 90% homology to amino acids 1-686 of SEQ
ID NO:1 in U.S. Pat. No. 6,162,628 preferably at least 92%, at
least 95%, at least 96%, at least 97%, at least 98%, or
particularly at least 99%. Most preferred variants of the
maltogenic alpha-amylase comprise the variants disclosed in
WO99/43794.
[0095] Maltogenic alpha-amylases may be added in amounts of
0.1-1000 MANU/kg dm, preferably from 1-100 MANU/kg dm, preferably
5-25 MANU/kg dm.
[0096] Another particular enzyme to be used in the processes of the
sixth aspect of the invention may be a beta-amylase (E.C
3.2.1.2).
[0097] Beta-amylases have been isolated from various plants and
microorganisms (W. M. Fogarty and C. T. Kelly, Progress in
Industrial Microbiology, vol. 15, pp. 112-115, 1979). These
beta-amylases are characterized by having optimum temperatures in
the range from 40.degree. C. to 65.degree. C. and optimum pH in the
range from 4.5 to 7.0. Specifically contemplated beta-amylase
include the beta-amylases SPEZYME.RTM. BBA 1500, SPEZYME.RTM. DBA
and OPTIMALT.TM. ME, OPTIMALT.TM. BBA from Genencor Int. as well as
the beta-amylases NOVOZYM.TM. WBA from Novozymes A/S. Beta-amylases
may be added in effective amounts well known to the person skilled
in the art.
[0098] A further particular enzyme to be used in the processes of
the sixth aspect of the invention may be a glucoamylase
(E.C.3.2.1.3) derived from a microorganism or a plant. Preferred
are glucoamylases of fungal or bacterial origin selected from the
group consisting of Aspergillus glucoamylases, in particular A.
niger G1 or G2 glucoamylase (Boel et al. (1984), EMBO J. 3 (5), p.
1097-1102), or variants thereof, such as disclosed in WO92/00381
and WO00/04136; the A. awamori glucoamylase (WO84/02921), A. oryzae
(Agric. Biol. Chem. (1991), 55 (4), p. 941-949), or variants or
fragments thereof. Glucoamylases may be added in effective amounts
well known to the person skilled in the art.
[0099] Another enzyme of the process of the sixth aspect of the
present invention may be a debranching enzyme, such as an
isoamylase (E.C. 3.2.1.68) or a pullulanases (E.C. 3.2.1.41).
Isoamylase hydrolyses alpha-1,6-D-glucosidic branch linkages in
amylopectin and beta-limit dextrins and can be distinguished from
pullulanases by the inability of isoamylase to attack pullulan, and
by the limited action on alpha-limit dextrins. Debranching enzyme
may be added in effective amounts well known to the person skilled
in the art.
Materials and Methods
[0100] Alpha-amylase Activity (KNU)
[0101] The amylolytic activity may be determined using potato
starch as substrate. This method is based on the break-down of
modified potato starch by the enzyme, and the reaction is followed
by mixing samples of the starch/enzyme solution with an iodine
solution. Initially, a blackish-blue color is formed, but during
the break-down of the starch the blue color gets weaker and
gradually turns into a reddish-brown, which is compared to a
colored glass standard.
[0102] One Kilo Novo alpha amylase Unit (KNU) is defined as the
amount of enzyme which, under standard conditions (i.e. at
37.degree. C.+/-0.05; 0.0003 M Ca.sup.2+; and pH 5.6) dextrinizes
5260 mg starch dry substance Merck Amylum solubile.
[0103] A folder EB-SM-0009.02/01 describing this analytical method
in more detail is available upon request to Novozymes A/S, Denmark,
which folder is hereby included by reference.
[0104] Acid Alpha-amylase Activity
[0105] When used according to the present invention the activity of
any acid alpha-amylase may be measured in AFAU (Acid Fungal
Alpha-amylase Units). Alternatively activity of acid alpha-amylase
may be measured in AAU (Acid Alpha-amylase Units).
[0106] Acid Alpha-amylase Units (AAU)
[0107] The acid alpha-amylase activity can be measured in AAU (Acid
Alpha-amylase Units), which is an absolute method. One Acid Amylase
Unit (AAU) is the quantity of enzyme converting 1 g of starch (100%
of dry matter) per hour under standardized conditions into a
product having a transmission at 620 nm after reaction with an
iodine solution of known strength equal to the one of a color
reference.
1 Standard conditions/reaction conditions: Substrate: Soluble
starch. Concentration approx. 20 g DS/L. Buffer: Citrate, approx.
0.13 M, pH = 4.2 Iodine solution: 40.176 g potassium iodide + 0.088
g iodine/L City water 15.degree.-20.degree. dH (German degree
hardness) pH: 4.2 Incubation temperature: 30.degree. C. Reaction
time: 11 minutes Wavelength: 620 nm Enzyme concentration: 0.13-0.19
AAU/mL Enzyme working range: 0.13-0.19 AAU/mL
[0108] The starch should be Lintner starch, which is a thin-boiling
starch used in the laboratory as calorimetric indicator. Lintner
starch is obtained by dilute hydrochloric acid treatment of native
starch so that it retains the ability to color blue with iodine.
Further details can be found in EP0140410B2, which disclosure is
hereby included by reference.
[0109] Acid Alpha-amylase Activity (AFAU)
[0110] Acid alpha-amylase activity may be measured in AFAU (Acid
Fungal Alpha-amylase Units), which are determined relative to an
enzyme standard. 1 FAU is defined as the amount of enzyme which
degrades 5.260 mg starch dry matter per hour under the below
mentioned standard conditions.
[0111] Acid alpha-amylase, an endo-alpha-amylase
(1,4-alpha-D-glucan-gluca- nohydrolase, E.C. 3.2.1.1) hydrolyzes
alpha-1,4-glucosidic bonds in the inner regions of the starch
molecule to form dextrins and oligosaccharides with different chain
lengths. The intensity of color formed with iodine is directly
proportional to the concentration of starch. Amylase activity is
determined using reverse colorimetry as a reduction in the
concentration of starch under the specified analytical conditions.
1
2 Standard conditions/reaction conditions: Substrate: Soluble
starch, approx. 0.17 g/L Buffer: Citrate, approx. 0.03 M Iodine
(I2): 0.03 g/L CaCl2: 1.85 mM pH: 2.50 .+-. 0.05 Incubation
temperature: 40.degree. C. Reaction time: 23 seconds Wavelength:
590 nm Enzyme concentration: 0.025 AFAU/mL Enzyme working range:
0.01-0.04 AFAU/mL
[0112] A folder EB-SM-0259.02/01 describing this analytical method
in more detail is available upon request to Novozymes A/S, Denmark,
which folder is hereby included by reference.
[0113] Glucoamylase Activity
[0114] Glucoamylase activity may be measured in AGI units or in
AmyloGlucosidase Units (AGU)
[0115] Glucoamylase Activity (AGI)
[0116] Glucoamylase (equivalent to amyloglucosidase) converts
starch into glucose. The amount of glucose is determined here by
the glucose oxidase method for the activity determination. The
method described in the section 76-11 Starch-Glucoamylase Method
with Subsequent Measurement of Glucose with Glucose Oxidase in
"Approved methods of the American Association of Cereal Chemists".
Vol. 1-2 AACC, from American Association of Cereal Chemists,
(2000); ISBN: 1-891127-12-8.
[0117] One glucoamylase unit (AGI) is the quantity of enzyme which
will form 1 micromol of glucose per minute under the standard
conditions of the method.
3 Standard conditions/reaction conditions: Substrate: Soluble
starch, concentration approx. 16 g dry matter/L. Buffer: Acetate,
approx. 0.04 M, pH = 4.3 pH: 4.3 Incubation temperature: 60.degree.
C. Reaction time: 15 minutes Termination of the reaction: NaOH to a
concentration of approximately 0.2 g/L (pH.about.9) Enzyme
concentration: 0.15-0.55 AAU/mL.
[0118] The starch should be Lintner starch, which is a thin-boiling
starch used in the laboratory as colorimetric indicator. Lintner
starch is obtained by dilute hydrochloric acid treatment of native
starch so that it retains the ability to color blue with
iodine.
[0119] Glucoamylase Activity (AGU)
[0120] The Novo Glucoamylase Unit (AGU) is defined as the amount of
enzyme, which hydrolyzes 1 micromole maltose per minute under the
standard conditions 37.degree. C., pH 4.3, substrate: maltose 23.2
mM, buffer: acetate 0.1 M, reaction time 5 minutes.
[0121] An autoanalyzer system may be used. Mutarotase is added to
the glucose dehydrogenase reagent so that any alpha-D-glucose
present is turned into beta-D-glucose. Glucose dehydrogenase reacts
specifically with beta-D-glucose in the reaction mentioned above,
forming NADH which is determined using a photometer at 340 nm as a
measure of the original glucose concentration.
4 AMG incubation: Substrate: maltose 23.2 mM Buffer: acetate 0.1 M
pH: 4.30 .+-. 0.05 Incubation temperature: 37.degree. C. .+-. 1
Reaction time: 5 minutes Enzyme working range: 0.5-4.0 AGU/mL Color
reaction: GlucDH: 430 U/L Mutarotase: 9 U/L NAD: 0.21 mM Buffer:
phosphate 0.12 M; 0.15 M NaCl pH: 7.60 .+-. 0.05 Incubation
temperature: 37.degree. C. .+-. 1 Reaction time: 5 minutes
Wavelength: 340 nm
[0122] A folder (EB-SM-0131.02/01) describing this analytical
method in more detail is available on request from Novozymes A/S,
Denmark, which folder is hereby included by reference.
[0123] Xylanolvtic Activity
[0124] The xylanolytic activity can be expressed in FXU-units,
determined at pH 6.0 with remazol-xylan
(4-O-methyl-D-glucurono-D-xylan dyed with Remazol Brilliant Blue R,
Fluka) as substrate.
[0125] A xylanase sample is incubated with the remazol-xylan
substrate. The background of non-degraded dyed substrate is
precipitated by ethanol. The remaining blue color in the
supernatant (as determined spectrophotometrically at 585 nm) is
proportional to the xylanase activity, and the xylanase units are
then determined relatively to an enzyme standard at standard
reaction conditions, i.e. at 50.0.degree. C., pH 6.0, and 30
minutes reaction time.
[0126] A folder EB-SM-352.02/01 describing this analytical method
in more detail is available upon request to Novozymes A/S, Denmark,
which folder is hereby included by reference.
[0127] Cellulytic Activity
[0128] The cellulytic activity may be measured in endo-glucanase
units (EGU), determined at pH 6.0 with carboxymethyl cellulose
(CMC) as substrate. A substrate solution is prepared, containing
34.0 g/l CMC (Hercules 7 LFD) in 0.1 M phosphate buffer at pH 6.0.
The enzyme sample to be analyzed is dissolved in the same buffer. 5
ml substrate solution and 0.15 ml enzyme solution are mixed and
transferred to a vibration viscosimeter (e.g. MIVI 3000 from
Sofraser, France), thermostated at 40.degree. C. for 30 minutes.
One EGU is defined as the amount of enzyme that reduces the
viscosity to one half under these conditions. The amount of enzyme
sample should be adjusted to provide 0.01-0.02 EGU/ml in the
reaction mixture. The arch standard is defined as 880 EGU/g.
[0129] A folder EB-SM-0275.02/01 describing this analytical method
in more detail is available upon request to Novozymes A/S, Denmark,
which folder is hereby included by reference.
[0130] Phytase Activity
[0131] The phytase activity is measured in FYT units, one FYT being
the amount of enzyme that liberates 1 micromole inorganic
ortho-phosphate per min. under the following conditions: pH 5.5;
temperature 37.degree. C.; substrate: sodium phytate
(C.sub.6H.sub.6O.sub.24P.sub.6Na.sub.12) at a concentration of
0.0050 mole/l.
[0132] Proteolytic Activity (AU)
[0133] The proteolytic activity may be determined with denatured
hemoglobin as substrate. In the Anson-Hemoglobin method for the
determination of proteolytic activity denatured hemoglobin is
digested, and the undigested hemoglobin is precipitated with
trichloroacetic acid (TCA). The amount of TCA soluble product is
determined with phenol reagent, which gives a blue color with
tyrosine and tryptophan.
[0134] One Anson Unit (AU) is defined as the amount of enzyme which
under standard conditions (i.e. 25.degree. C., pH 7.5 and 10 min.
reaction time) digests hemoglobin at an initial rate such that
there is liberated per minute an amount of TCA soluble product
which gives the same color with phenol reagent as one
milliequivalent of tyrosine.
[0135] A folder AF 4/5 describing the analytical method in more
detail is available upon request to Novozymes A/S, Denmark, which
folder is hereby included by reference.
[0136] Enzymes Preparation
[0137] The following enzyme preparations were used:
[0138] Bacterial alpha-amylase; An enzyme preparations comprising a
polypeptide with alpha-amylase activity (E.C. 3.2.1.1) derived from
B. stearothermophilus and having the amino acid sequence disclosed
as SEQ.NO:4 in WO99/19467. Activity: 120 KNU/g (density=1.20-1.25
g/mL).
[0139] A preferred acid fungal alpha-amylase; an enzyme
preparations derived from Aspergillus niger comprising acid fungal
alpha-amylase and some glucoamylase. Activities: 114 AFAU/g, 25
AGU/g (density=1.2 g/mL).
[0140] Glucoamylase; an enzyme preparations derived from
Aspergillus niger comprising glucoamylase and some acid fungal
alpha-amylase. Activity: 363 AGU/g, 86 AFAU/g (density=1.2
g/mL).
[0141] An enzyme preparation comprising xylanase and cellulase
activities derived from Trichoderma and Aspergillus. Activity: 140
FXU/g+350 EGU/g (density=1.2 g/mL).
EXAMPLE 1
[0142] A conventional ethanol process using a traditional
pre-liquefaction called the non-pressure cooking (NPC) is compared
with the process of the invention. Traditional non-pressure batch
cooking processes for production of potable alcohol is described in
the Novozymes publication No. 2001-10782-01 entitled "Use of
Novozymes enzymes in alcohol production".
[0143] A 20% D.S. slurry of the milled barley grain was made in
room temperature (RT) tap water.
[0144] The NPC pre-treatment of the conventional ethanol process
was performed in 6.times.1-litre tubs with stirring. Bacterial
alpha-amylase was added and the tubs were placed in water bath at
65.degree. C. When the temperature in the mash reached 55.degree.
C. the heating was increased to heat the mash to 90.degree. C. over
60 minutes. The temperature was then adjusted to 32.degree. C. and
3.times.250 g mash was portioned in 500 mL blue cap flasks with air
locks. To all flasks 0.25 g dry bakers yeast was added
(corresponding to 5-10 million vital cells/g mash). Enzyme
activities were added according to the table below and each flask
was weighed. The flasks were placed in a shaking water bath at
32.degree. C. for 72 hours. At 48 and 72 hours the flasks were
weighed and CO.sub.2 weight loss measured for monitoring of the
fermentation progress. The relationship used between amount of
CO.sub.2 loss and the weight of ethanol was: CO.sub.2 loss
(g).times.1.045=EtOH (g).
[0145] For the process of the invention 500 mL blue cap
fermentation flasks each with 250 g slurry was fitted with air
locks. Using 6.0 N HCl the pH was adjusted to 4.5 and glucoamylase
and acid alpha-amylase was dosed according to table 1. The flasks
were held at 55.degree. C. for 60 minutes. The temperature was then
adjusted to 32.degree. C. and fermentation was performed and
monitored as described above.
5TABLE 1 Barley, the weight loss (g) at 48 and 72 hours. Bacterial
alpha-amylase acid (KNU), acid alpha-amylase (AFAU) and
glucoamylase (AGU) activity was added according to the table.
Traditional non- Low Process of pressure cooking AFAU/AGU the
invention KNU/kg DS 36 0 0 AFAU/kg DS 39 39 540 AGU/kg DS 163 163
273 AFAU/AGU 0.24 0.24 1.98 Weight loss (g), 48 hours 9.0 11.0 14.7
Weight loss (g), 72 hours 12.2 13.0 16.4 Ethanol %, 48 Hrs 3.76
4.60 4.60 Ethanol %, 72 Hrs 5.10 5.43 6.86 *based on weight loss at
48 and 72 hours, CO.sub.2 loss (g) .times. 1.045 = EtOH (g).
EXAMPLE 2
[0146] This example illustrates the use of an enzyme composition of
the invention consisting of acid alpha-amylase, glucoamylase,
cellulase and xylanase activity.
[0147] A 20% D.S. slurry of the milled barley grain was made in RT
tap water. For each treatment 2.times.250 g was portioned in 500 mL
blue cap flasks. Using 6 N HCl the pH was adjusted to 4.5. Enzymes
activities were added according to table 2 and 3, and a
pre-treatment corresponding to step (b) of the invention was
carried out for one hour at 55.degree. C. in a shaking water bath.
The temperature was adjusted to 32.degree. C. and 0.25 g dry
baker's yeast Fermentation was performed and monitored as described
above.
6TABLE 2 Barley, the weight loss (g) at 48 and 72 hours. Acid
alpha-amylase (AFAU), glucoamylase (AGU), cellulase (EGU) and
xylanase (FXU) activity was added according to the table. Low
Process of AFAU/AGU the invention AFAU/AGU 0.24 0.24 1.98 1.98
AFAU/kg DS 39 39 540 540 AGU/kg DS 163 163 273 273 FXU/kg DS 0 70 0
70 EGU/kg DS 0 175 0 175 Weight loss (g), 48 hours 9.9 11.2 12.8
14.3 Weight loss (g), 72 hours 12.1 13.8 15.5 16.5 Ethanol %, 48
Hrs 4.14 4.68 5.35 5.98 Ethanol %, 72 Hrs 5.06 5.77 6.48 6.90
*based on weight loss at 48 and 72 hours, CO.sub.2 loss (g) .times.
1.045 = EtOH (g).
EXAMPLE 3
[0148] This example illustrates the process of the invention using
various raw materials. A 20% D.S. slurry of the milled grain or
corn meal was made in RT tab water. For each treatment 2.times.250
g was portioned in 500 mL blue cap flasks. Using 6 N HCl the pH was
adjusted to 4.5 Enzymes were dosed according to table 3, 4 and 5,
and a pre-treatment was carried out for one hour at 55.degree. C.
in a shaking water bath. The flasks were cooled to 32.degree. C.
and 0.25 g dry bakers yeast added. The flasks were placed in a
water bath at 32.degree. C. for 72 hours (90 hours for wheat).
7TABLE 3 Rye, the weight loss (g) at 48 hours and at 72 hours. Acid
alpha- amylase (AFAU), and glucoamylase (AGU) activity was added
according to the table. AFAU/kg DS 39 540 AGU/kg DS 163 273
AFAU/AGU 0.24 1.98 Weight loss (g), 48 hours 14.7 17.4 Weight loss
(g), 72 hours 16.2 19.2 Ethanol %, 48 Hrs 6.14 7.27 Ethanol %, 72
Hrs 6.77 8.03 *based on weight loss at 48 and 72 hours, CO.sub.2
loss (g) .times. 1.045 = EtOH (g).
[0149]
8TABLE 4 Yellow corn meal, the weight loss (g) at 48 hours and at
72 hours. Acid alpha-amylase (AFAU), and glucoamylase (AGU)
activity was added according to the table. AFAU/kg DS 39 540 AGU/kg
DS 163 273 AFAU/AGU 0.24 1.98 Weight loss (g), 48 hours 12.4 14.5
Weight loss (g), 72 hours 15.6 17.5 Ethanol %, 48 Hrs* 5.18 6.06
Ethanol %, 72 Hrs* 6.52 7.32 *based on weight loss at 48 and 72
hours, CO.sub.2 loss (g) .times. 1.045 = EtOH (g).
[0150]
9TABLE 5 Wheat, the weight losses (g) at 48 hours and at 90 hours.
Acid alpha-amylase (AFAU), and glucoamylase (AGU) activity was
added according to the table. AFAU/kg DS 39 540 AGU/kg DS 163 273
AFAU/AGU 0.24 1.98 Weight loss (g), 48 hours 13.8 16.3 Weight loss
(g), 90 hours 16.7 18.5 Ethanol %, 48 Hrs 5.77 6.81 Ethanol %, 72
Hrs 6.98 7.73 *based on weight loss at 48 and 72 hours, CO.sub.2
loss (g) .times. 1.045 = EtOH (g).
EXAMPLE 4
[0151] This example illustrates a process of the invention using
wheat. A 20% D.S. slurry of milled wheat was made in RT tab water.
For each treatment 2.times.250 g slurry was portioned in 500 mL
blue cap flasks. The pH was adjusted to 4.5 using 6 N HCl. Enzyme
activities were dosed according to table 6, and the flasks were
incubated for one hour at 55.degree. C. in a shaking water bath.
The flasks were cooled to 32.degree. C. and 0.25 g dry bakers yeast
added. The flasks were placed in a water bath at 32.degree. C. for
72 hours. Weight loss data was recorded. At 50 and 72.5 hours the
flasks were weighed and CO.sub.2 weight loss measured for
monitoring of the fermentation progress. The relationship used
between amount of CO.sub.2 loss and the weight of ethanol was:
CO.sub.2 loss (g).times.1.045=EtOH (g).
[0152] At 100 hours HPLC samples were drawn and the content of
ethanol, methanol and glycerol was recorded.
10TABLE 6 Wheat; the weight loss (g) at 50 hours and at 72.5 hours.
Glucoamylase (AGU), Acid alpha-amylase (AFAU) and bacterial
alpha-amylase (KNU) was added according to the table. AGU/kg DS
0.30 0.30 0.30 0.30 0.30 0.30 0.30 AFAU/kg DS 0.08 0.13 0.26 0.51
1.01 -- -- KNU/kg DS -- -- -- -- -- 0.05 0.15 Weight loss (g),
10.67 11.01 11.71 12.53 14.14 14.68 15.55 50 hours Weight loss (g),
12.53 12.90 13.78 14.80 16.90 16.62 17.52 72.5 hours Ethanol % w/w,
4.46 4.60 4.89 5.24 5.91 6.14 6.50 50 Hrs* Ethanol % w/w, 5.24 5.39
5.76 6.19 7.06 6.95 7.32 72.5 Hrs* Ethanol % w/w, 6.36 6.64 7.01
7.47 7.73 8.13 8.59 100 hrs** Methanol % w/w, 0.17 0.09 0.11 0.12
0.12 0.17 0.22 100 hrs** Glycerol % w/w, 0.52 0.54 0.49 0.54 0.53
0.65 0.72 100 hrs** 3-methyl-1-butanol, 0.01 0.01 0.01 0.04 0.01
0.01 0.01 100 hrs** *based on weight loss at 50 and 72.5 hrs,
CO.sub.2 loss (g) .times. 1.045 = EtOH (g), **based on HPLC at 100
hrs.
EXAMPLE 5
[0153] This example demonstrates the use of an acid alpha-amylase
in a brewing process. The enzymes used comprised an acid fungal
alpha-amylase derived from Aspergillus niger having the sequence
shown in SEQ ID NO:1. An alpha-amylase (E.C. 3.2.1.1) from B.
stearothermophilus having the amino acid sequence disclosed as
SEQ.NO:4 in WO99/19467 with the mutations: I181*+G182*+N193F. A
glucoamylase derived from Aspergillus niger. A protease having the
amino acid sequence shown as amino acids no. 1-177 of SEQ.ID.NO 2
in Danish patent applications WO 2003/048353. A cellulase (E.C.
3.2.1.4), a beta-glucanase having the amino acid sequence shown as
SEQ.ID.NO:1 in WO 2003/062409
[0154] A xylanase from Aspergillus aculeatus having the sequence
amino acid disclosed as SEQ ID NO:2 in WO 9421785.
[0155] The acid alpha-amylase from Aspergillus niger SP288 was
tested in a mashing set up using both Congress mashing and Higher
Temperature Mashing (HTM) conditions. The effect was evaluated on
formation of fermentable sugars in the wort, which is a key wort
quality parameter. A bacterial heat stable alpha-amylase from
Bacillus stearothermophilus was applied for comparison. All worts
were added a xylanase 5 mg EP/kg DS, betaglucanase 5 mg EP/kg DS
and protease 2.5 mg EP/kg DS.
[0156] Unless otherwise stated mashing was preformed according to
EBC: 4.5.1 using malt grounded according to EBC: 1.1. Mashing
trials were performed in 500 ml lidded vessels each containing a
mash with 50 g grist and adjusted to a total weight of 450.+-.0.2 g
with water preheated to the initial incubation
temperature+1.degree. C. During mashing the vessels were incubated
in water bath with stirring.
[0157] One treatment comprised using Congress mashing described in
EBC: 4.5.1. Thus 50.0 g malt is mixed with 200 ml water from
beginning, 100 ml water at 70.degree. C. is added when profile
reached 70.degree. C. All mashcups are standardized to 450.0 g at
the end of mashing, which gives approximately 8.6.degree. P.
[0158] The second treatment comprised using HTM as disclosed in WO
2004/011591 with the following temperature profile: an initial
incubation temperature of 70.degree. C. for 65 minutes, increasing
to 90.degree. C., with 1.0.degree. C./min for 20 minutes, followed
by 90.degree. C. for 15 minutes and finalized by cooling with
4.5.degree. C./min to 20.degree. C. The recipe applied was: 50.0 g
malt added 200 ml water from beginning, at the end of mashing the
mash-cups are standardized to 300.0 g, which gives app. 13.degree.
P.
[0159] Fermentable sugars were analysed at 8.6.degree. P using HPLC
method equivalent to EBC: 8.7
11TABLE 7 Extract E2, extract in dry malt, % (m/m) from Congress
mashing (8.6.degree. P) and HTM (13.degree. P). To all treatments
were added xylanase 5 mg EP/kg DS, betaglucanase 5 mg EP/kg DS and
Protease 2.5 mg EP/kg DS. No additional 75 AFAU/kg DS + enzymes 61
AGU/kg DS 75 KNU/kg DS Congress mashing 80.90 81.79 81.97 HTM 81.42
81.23 81.33 N.B. comparison only intended between enzyme
treatments, not between methods.
[0160]
12TABLE 8 Overview of fermentable sugar profile from Congress
mashing, 8.6.degree. P. To all treatments were added xylanase 5 mg
EP/kg DS, betaglucanase 5 mg EP/kg DS and Protease 2.5 mg EP/kg DS
No additional 75 AFAU/kg DS + enzymes 61 AGU/kg DS 75 KNU/kg DS
Glucose 7.65 10.40 7.82 Maltose 43.18 43.08 43.54 Maltotriose 8.25
7.08 9.08 Fructose 3.24 3.29 3.40 Sum of 62.32 63.83 63.83
fermentable sugars
[0161]
13TABLE 9 Fermentable sugar profile from HTM mashing, 13.degree. P.
To all treatments were added xylanase 5 mg EP/kg DS, betaglucanase
5 mg EP/kg DS and Protease 2.5 mg EP/kg DS. No additional 75
AFAU/kg DS + enzymes 61 AGU/kg DS 75 KNU/kg DS Glucose 10.19 14.12
10.43 Maltose 65.98 67.10 66.46 Maltotriose 12.61 11.07 12.89
Fructose 5.27 5.15 5.13 Sum of 94.05 97.44 94.91 fermentable
sugars
[0162] Congress trial (a): SP288 combined with AMG showed good
effect on formation of fermentable sugars, as the glucose
concentration was increased in wort by the addition of acid
alpha-amylase both compared to Termamyl SC and with out adding
amylase.
[0163] Acid alpha-amylase showed very good effect on formation of
fermentable sugars, predominantly the glucose concentration was
increased in wort by the addition of acid alpha-amylase both
compared to Termamyl SC and with out adding amylase. Overall the
sum of fermentable sugars is increased from 94.91 g/L to 97.44 g/L
from performance of acid alpha-amylase. This means that an
increased part of the extract is now fermentable, which will yield
higher alcohol amounts.
Sequence CWU 1
1
5 1 484 PRT Aspergillus niger 1 Leu Ser Ala Ala Ser Trp Arg Thr Gln
Ser Ile Tyr Phe Leu Leu Thr 1 5 10 15 Asp Arg Phe Gly Arg Thr Asp
Asn Ser Thr Thr Ala Thr Cys Asn Thr 20 25 30 Gly Asn Glu Ile Tyr
Cys Gly Gly Ser Trp Gln Gly Ile Ile Asp His 35 40 45 Leu Asp Tyr
Ile Glu Gly Met Gly Phe Thr Ala Ile Trp Ile Ser Pro 50 55 60 Ile
Thr Glu Gln Leu Pro Gln Asp Thr Ala Asp Gly Glu Ala Tyr His 65 70
75 80 Gly Tyr Trp Gln Gln Lys Ile Tyr Asp Val Asn Ser Asn Phe Gly
Thr 85 90 95 Ala Asp Asn Leu Lys Ser Leu Ser Asp Ala Leu His Ala
Arg Gly Met 100 105 110 Tyr Leu Met Val Asp Val Val Pro Asp His Met
Gly Tyr Ala Gly Asn 115 120 125 Gly Asn Asp Val Asp Tyr Ser Val Phe
Asp Pro Phe Asp Ser Ser Ser 130 135 140 Tyr Phe His Pro Tyr Cys Leu
Ile Thr Asp Trp Asp Asn Leu Thr Met 145 150 155 160 Val Glu Asp Cys
Trp Glu Gly Asp Thr Ile Val Ser Leu Pro Asp Leu 165 170 175 Asp Thr
Thr Glu Thr Ala Val Arg Thr Ile Trp Tyr Asp Trp Val Ala 180 185 190
Asp Leu Val Ser Asn Tyr Ser Val Asp Gly Leu Arg Ile Asp Ser Val 195
200 205 Leu Glu Val Gln Pro Asp Phe Phe Pro Gly Tyr Asn Lys Ala Ser
Gly 210 215 220 Val Tyr Cys Val Gly Glu Ile Asp Asn Gly Asn Pro Ala
Ser Asp Cys 225 230 235 240 Pro Tyr Gln Lys Val Leu Asp Gly Val Leu
Asn Tyr Pro Ile Tyr Trp 245 250 255 Gln Leu Leu Tyr Ala Phe Glu Ser
Ser Ser Gly Ser Ile Ser Asn Leu 260 265 270 Tyr Asn Met Ile Lys Ser
Val Ala Ser Asp Cys Ser Asp Pro Thr Leu 275 280 285 Leu Gly Asn Phe
Ile Glu Asn His Asp Asn Pro Arg Phe Ala Lys Tyr 290 295 300 Thr Ser
Asp Tyr Ser Gln Ala Lys Asn Val Leu Ser Tyr Ile Phe Leu 305 310 315
320 Ser Asp Gly Ile Pro Ile Val Tyr Ala Gly Glu Glu Gln His Tyr Ala
325 330 335 Gly Gly Lys Val Pro Tyr Asn Arg Glu Ala Thr Trp Leu Ser
Gly Tyr 340 345 350 Asp Thr Ser Ala Glu Leu Tyr Thr Trp Ile Ala Thr
Thr Asn Ala Ile 355 360 365 Arg Lys Leu Ala Ile Ala Ala Asp Ser Ala
Tyr Ile Thr Tyr Ala Asn 370 375 380 Asp Ala Phe Tyr Thr Asp Ser Asn
Thr Ile Ala Met Ala Lys Gly Thr 385 390 395 400 Ser Gly Ser Gln Val
Ile Thr Val Leu Ser Asn Lys Gly Ser Ser Gly 405 410 415 Ser Ser Tyr
Thr Leu Thr Leu Ser Gly Ser Gly Tyr Thr Ser Gly Thr 420 425 430 Lys
Leu Ile Glu Ala Tyr Thr Cys Thr Ser Val Thr Val Asp Ser Ser 435 440
445 Gly Asp Ile Pro Val Pro Met Ala Ser Gly Leu Pro Arg Val Leu Leu
450 455 460 Pro Ala Ser Val Val Asp Ser Ser Ser Leu Cys Gly Gly Ser
Gly Arg 465 470 475 480 Leu Tyr Val Glu 2 514 PRT Bacillus
stearothermophilus 2 Ala Ala Pro Phe Asn Gly Thr Met Met Gln Tyr
Phe Glu Trp Tyr Leu 1 5 10 15 Pro Asp Asp Gly Thr Leu Trp Thr Lys
Val Ala Asn Glu Ala Asn Asn 20 25 30 Leu Ser Ser Leu Gly Ile Thr
Ala Leu Trp Leu Pro Pro Ala Tyr Lys 35 40 45 Gly Thr Ser Arg Ser
Asp Val Gly Tyr Gly Val Tyr Asp Leu Tyr Asp 50 55 60 Leu Gly Glu
Phe Asn Gln Lys Gly Ala Val Arg Thr Lys Tyr Gly Thr 65 70 75 80 Lys
Ala Gln Tyr Leu Gln Ala Ile Gln Ala Ala His Ala Ala Gly Met 85 90
95 Gln Val Tyr Ala Asp Val Val Phe Asp His Lys Gly Gly Ala Asp Gly
100 105 110 Thr Glu Trp Val Asp Ala Val Glu Val Asn Pro Ser Asp Arg
Asn Gln 115 120 125 Glu Ile Ser Gly Thr Tyr Gln Ile Gln Ala Trp Thr
Lys Phe Asp Phe 130 135 140 Pro Gly Arg Gly Asn Thr Tyr Ser Ser Phe
Lys Trp Arg Trp Tyr His 145 150 155 160 Phe Asp Gly Val Asp Trp Asp
Glu Ser Arg Lys Leu Ser Arg Ile Tyr 165 170 175 Lys Phe Arg Gly Ile
Gly Lys Ala Trp Asp Trp Glu Val Asp Thr Glu 180 185 190 Asn Gly Asn
Tyr Asp Tyr Leu Met Tyr Ala Asp Leu Asp Met Asp His 195 200 205 Pro
Glu Val Val Thr Glu Leu Lys Ser Trp Gly Lys Trp Tyr Val Asn 210 215
220 Thr Thr Asn Ile Asp Gly Phe Arg Leu Asp Ala Val Lys His Ile Lys
225 230 235 240 Phe Ser Phe Phe Pro Asp Trp Leu Ser Asp Val Arg Ser
Gln Thr Gly 245 250 255 Lys Pro Leu Phe Thr Val Gly Glu Tyr Trp Ser
Tyr Asp Ile Asn Lys 260 265 270 Leu His Asn Tyr Ile Met Lys Thr Asn
Gly Thr Met Ser Leu Phe Asp 275 280 285 Ala Pro Leu His Asn Lys Phe
Tyr Thr Ala Ser Lys Ser Gly Gly Thr 290 295 300 Phe Asp Met Arg Thr
Leu Met Thr Asn Thr Leu Met Lys Asp Gln Pro 305 310 315 320 Thr Leu
Ala Val Thr Phe Val Asp Asn His Asp Thr Glu Pro Gly Gln 325 330 335
Ala Leu Gln Ser Trp Val Asp Pro Trp Phe Lys Pro Leu Ala Tyr Ala 340
345 350 Phe Ile Leu Thr Arg Gln Glu Gly Tyr Pro Cys Val Phe Tyr Gly
Asp 355 360 365 Tyr Tyr Gly Ile Pro Gln Tyr Asn Ile Pro Ser Leu Lys
Ser Lys Ile 370 375 380 Asp Pro Leu Leu Ile Ala Arg Arg Asp Tyr Ala
Tyr Gly Thr Gln His 385 390 395 400 Asp Tyr Leu Asp His Ser Asp Ile
Ile Gly Trp Thr Arg Glu Gly Val 405 410 415 Thr Glu Lys Pro Gly Ser
Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro 420 425 430 Gly Gly Ser Lys
Trp Met Tyr Val Gly Lys Gln His Ala Gly Lys Val 435 440 445 Phe Tyr
Asp Leu Thr Gly Asn Arg Ser Asp Thr Val Thr Ile Asn Ser 450 455 460
Asp Gly Trp Gly Glu Phe Lys Val Asn Gly Gly Ser Val Ser Val Trp 465
470 475 480 Val Pro Arg Lys Thr Thr Val Ser Thr Ile Ala Trp Ser Ile
Thr Thr 485 490 495 Arg Pro Trp Thr Asp Glu Phe Val Arg Trp Thr Glu
Pro Arg Leu Val 500 505 510 Ala Trp 3 483 PRT Bacillus
licheniformis 3 Ala Asn Leu Asn Gly Thr Leu Met Gln Tyr Phe Glu Trp
Tyr Met Pro 1 5 10 15 Asn Asp Gly Gln His Trp Arg Arg Leu Gln Asn
Asp Ser Ala Tyr Leu 20 25 30 Ala Glu His Gly Ile Thr Ala Val Trp
Ile Pro Pro Ala Tyr Lys Gly 35 40 45 Thr Ser Gln Ala Asp Val Gly
Tyr Gly Ala Tyr Asp Leu Tyr Asp Leu 50 55 60 Gly Glu Phe His Gln
Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr Lys 65 70 75 80 Gly Glu Leu
Gln Ser Ala Ile Lys Ser Leu His Ser Arg Asp Ile Asn 85 90 95 Val
Tyr Gly Asp Val Val Ile Asn His Lys Gly Gly Ala Asp Ala Thr 100 105
110 Glu Asp Val Thr Ala Val Glu Val Asp Pro Ala Asp Arg Asn Arg Val
115 120 125 Ile Ser Gly Glu His Leu Ile Lys Ala Trp Thr His Phe His
Phe Pro 130 135 140 Gly Arg Gly Ser Thr Tyr Ser Asp Phe Lys Trp His
Trp Tyr His Phe 145 150 155 160 Asp Gly Thr Asp Trp Asp Glu Ser Arg
Lys Leu Asn Arg Ile Tyr Lys 165 170 175 Phe Gln Gly Lys Ala Trp Asp
Trp Glu Val Ser Asn Glu Asn Gly Asn 180 185 190 Tyr Asp Tyr Leu Met
Tyr Ala Asp Ile Asp Tyr Asp His Pro Asp Val 195 200 205 Ala Ala Glu
Ile Lys Arg Trp Gly Thr Trp Tyr Ala Asn Glu Leu Gln 210 215 220 Leu
Asp Gly Phe Arg Leu Asp Ala Val Lys His Ile Lys Phe Ser Phe 225 230
235 240 Leu Arg Asp Trp Val Asn His Val Arg Glu Lys Thr Gly Lys Glu
Met 245 250 255 Phe Thr Val Ala Glu Tyr Trp Gln Asn Asp Leu Gly Ala
Leu Glu Asn 260 265 270 Tyr Leu Asn Lys Thr Asn Phe Asn His Ser Val
Phe Asp Val Pro Leu 275 280 285 His Tyr Gln Phe His Ala Ala Ser Thr
Gln Gly Gly Gly Tyr Asp Met 290 295 300 Arg Lys Leu Leu Asn Gly Thr
Val Val Ser Lys His Pro Leu Lys Ser 305 310 315 320 Val Thr Phe Val
Asp Asn His Asp Thr Gln Pro Gly Gln Ser Leu Glu 325 330 335 Ser Thr
Val Gln Thr Trp Phe Lys Pro Leu Ala Tyr Ala Phe Ile Leu 340 345 350
Thr Arg Glu Ser Gly Tyr Pro Gln Val Phe Tyr Gly Asp Met Tyr Gly 355
360 365 Thr Lys Gly Asp Ser Gln Arg Glu Ile Pro Ala Leu Lys His Lys
Ile 370 375 380 Glu Pro Ile Leu Lys Ala Arg Lys Gln Tyr Ala Tyr Gly
Ala Gln His 385 390 395 400 Asp Tyr Phe Asp His His Asp Ile Val Gly
Trp Thr Arg Glu Gly Asp 405 410 415 Ser Ser Val Ala Asn Ser Gly Leu
Ala Ala Leu Ile Thr Asp Gly Pro 420 425 430 Gly Gly Ala Lys Arg Met
Tyr Val Gly Arg Gln Asn Ala Gly Glu Thr 435 440 445 Trp His Asp Ile
Thr Gly Asn Arg Ser Glu Pro Val Val Ile Asn Ser 450 455 460 Glu Gly
Trp Gly Glu Phe His Val Asn Gly Gly Ser Val Ser Ile Tyr 465 470 475
480 Val Gln Arg 4 480 PRT Aspergillus amyloliquefaciens 4 Val Asn
Gly Thr Leu Met Gln Tyr Phe Glu Trp Tyr Thr Pro Asn Asp 1 5 10 15
Gly Gln His Trp Lys Arg Leu Gln Asn Asp Ala Glu His Leu Ser Asp 20
25 30 Ile Gly Ile Thr Ala Val Trp Ile Pro Pro Ala Tyr Lys Gly Leu
Ser 35 40 45 Gln Ser Asp Asn Gly Tyr Gly Pro Tyr Asp Leu Tyr Asp
Leu Gly Glu 50 55 60 Phe Gln Gln Lys Gly Thr Val Arg Thr Lys Tyr
Gly Thr Lys Ser Glu 65 70 75 80 Leu Gln Asp Ala Ile Gly Ser Leu His
Ser Arg Asn Val Gln Val Tyr 85 90 95 Gly Asp Val Val Leu Asn His
Lys Ala Gly Ala Asp Ala Thr Glu Asp 100 105 110 Val Thr Ala Val Glu
Val Asn Pro Ala Asn Arg Asn Gln Glu Thr Ser 115 120 125 Glu Glu Tyr
Gln Ile Lys Ala Trp Thr Asp Phe Arg Phe Pro Gly Arg 130 135 140 Gly
Asn Thr Tyr Ser Asp Phe Lys Trp His Trp Tyr His Phe Asp Gly 145 150
155 160 Ala Asp Trp Asp Glu Ser Arg Lys Ile Ser Arg Ile Phe Lys Phe
Arg 165 170 175 Gly Glu Gly Lys Ala Trp Asp Trp Glu Val Ser Ser Glu
Asn Gly Asn 180 185 190 Tyr Asp Tyr Leu Met Tyr Ala Asp Val Asp Tyr
Asp His Pro Asp Val 195 200 205 Val Ala Glu Thr Lys Lys Trp Gly Ile
Trp Tyr Ala Asn Glu Leu Ser 210 215 220 Leu Asp Gly Phe Arg Ile Asp
Ala Ala Lys His Ile Lys Phe Ser Phe 225 230 235 240 Leu Arg Asp Trp
Val Gln Ala Val Arg Gln Ala Thr Gly Lys Glu Met 245 250 255 Phe Thr
Val Ala Glu Tyr Trp Gln Asn Asn Ala Gly Lys Leu Glu Asn 260 265 270
Tyr Leu Asn Lys Thr Ser Phe Asn Gln Ser Val Phe Asp Val Pro Leu 275
280 285 His Phe Asn Leu Gln Ala Ala Ser Ser Gln Gly Gly Gly Tyr Asp
Met 290 295 300 Arg Arg Leu Leu Asp Gly Thr Val Val Ser Arg His Pro
Glu Lys Ala 305 310 315 320 Val Thr Phe Val Glu Asn His Asp Thr Gln
Pro Gly Gln Ser Leu Glu 325 330 335 Ser Thr Val Gln Thr Trp Phe Lys
Pro Leu Ala Tyr Ala Phe Ile Leu 340 345 350 Thr Arg Glu Ser Gly Tyr
Pro Gln Val Phe Tyr Gly Asp Met Tyr Gly 355 360 365 Thr Lys Gly Thr
Ser Pro Lys Glu Ile Pro Ser Leu Lys Asp Asn Ile 370 375 380 Glu Pro
Ile Leu Lys Ala Arg Lys Glu Tyr Ala Tyr Gly Pro Gln His 385 390 395
400 Asp Tyr Ile Asp His Pro Asp Val Ile Gly Trp Thr Arg Glu Gly Asp
405 410 415 Ser Ser Ala Ala Lys Ser Gly Leu Ala Ala Leu Ile Thr Asp
Gly Pro 420 425 430 Gly Gly Ser Lys Arg Met Tyr Ala Gly Leu Lys Asn
Ala Gly Glu Thr 435 440 445 Trp Tyr Asp Ile Thr Gly Asn Arg Ser Asp
Thr Val Lys Ile Gly Ser 450 455 460 Asp Gly Trp Gly Glu Phe His Val
Asn Asp Gly Ser Val Ser Ile Tyr 465 470 475 480 5 499 PRT
Aspergillus oryzae 5 Met Met Val Ala Trp Trp Ser Leu Phe Leu Tyr
Gly Leu Gln Val Ala 1 5 10 15 Ala Pro Ala Leu Ala Ala Thr Pro Ala
Asp Trp Arg Ser Gln Ser Ile 20 25 30 Tyr Phe Leu Leu Thr Asp Arg
Phe Ala Arg Thr Asp Gly Ser Thr Thr 35 40 45 Ala Thr Cys Asn Thr
Ala Asp Gln Lys Tyr Cys Gly Gly Thr Trp Gln 50 55 60 Gly Ile Ile
Asp Lys Leu Asp Tyr Ile Gln Gly Met Gly Phe Thr Ala 65 70 75 80 Ile
Trp Ile Thr Pro Val Thr Ala Gln Leu Pro Gln Thr Thr Ala Tyr 85 90
95 Gly Asp Ala Tyr His Gly Tyr Trp Gln Gln Asp Ile Tyr Ser Leu Asn
100 105 110 Glu Asn Tyr Gly Thr Ala Asp Asp Leu Lys Ala Leu Ser Ser
Ala Leu 115 120 125 His Glu Arg Gly Met Tyr Leu Met Val Asp Val Val
Ala Asn His Met 130 135 140 Gly Tyr Asp Gly Ala Gly Ser Ser Val Asp
Tyr Ser Val Phe Lys Pro 145 150 155 160 Phe Ser Ser Gln Asp Tyr Phe
His Pro Phe Cys Phe Ile Gln Asn Tyr 165 170 175 Glu Asp Gln Thr Gln
Val Glu Asp Cys Trp Leu Gly Asp Asn Thr Val 180 185 190 Ser Leu Pro
Asp Leu Asp Thr Thr Lys Asp Val Val Lys Asn Glu Trp 195 200 205 Tyr
Asp Trp Val Gly Ser Leu Val Ser Asn Tyr Ser Ile Asp Gly Leu 210 215
220 Arg Ile Asp Thr Val Lys His Val Gln Lys Asp Phe Trp Pro Gly Tyr
225 230 235 240 Asn Lys Ala Ala Gly Val Tyr Cys Ile Gly Glu Val Leu
Asp Gly Asp 245 250 255 Pro Ala Tyr Thr Cys Pro Tyr Gln Asn Val Met
Asp Gly Val Leu Asn 260 265 270 Tyr Pro Ile Tyr Tyr Pro Leu Leu Asn
Ala Phe Lys Ser Thr Ser Gly 275 280 285 Ser Met Asp Asp Leu Tyr Asn
Met Ile Asn Thr Val Lys Ser Asp Cys 290 295 300 Pro Asp Ser Thr Leu
Leu Gly Thr Phe Val Glu Asn His Asp Asn Pro 305 310 315 320 Arg Phe
Ala Ser Tyr Thr Asn Asp Ile Ala Leu Ala Lys Asn Val Ala 325 330 335
Ala Phe Ile Ile Leu Asn Asp Gly Ile Pro Ile Ile Tyr Ala Gly Gln 340
345 350 Glu Gln His Tyr Ala Gly Gly Asn Asp Pro Ala Asn Arg Glu Ala
Thr 355 360 365 Trp Leu Ser Gly Tyr Pro Thr Asp Ser Glu Leu Tyr Lys
Leu Ile Ala 370 375 380 Ser Ala Asn Ala Ile Arg Asn Tyr Ala Ile Ser
Lys Asp Thr Gly Phe 385 390 395 400 Val Thr Tyr Lys Asn Trp Pro Ile
Tyr Lys Asp Asp Thr Thr Ile Ala 405 410 415 Met Arg Lys Gly Thr Asp
Gly Ser Gln Ile Val Thr Ile Leu Ser Asn 420 425 430 Lys Gly Ala Ser
Gly Asp Ser Tyr Thr Leu Ser Leu Ser Gly Ala Gly 435 440 445 Tyr Thr
Ala Gly Gln Gln Leu Thr Glu Val Ile Gly Cys Thr Thr Val 450 455 460
Thr Val Gly Ser Asp
Gly Asn Val Pro Val Pro Met Ala Gly Gly Leu 465 470 475 480 Pro Arg
Val Leu Tyr Pro Thr Glu Lys Leu Ala Gly Ser Lys Ile Cys 485 490 495
Ser Ser Ser
* * * * *