U.S. patent application number 16/006521 was filed with the patent office on 2018-09-27 for detoxifying pre-treated lignocellulose-containing materials.
This patent application is currently assigned to Novozymes A/S. The applicant listed for this patent is Novozymes A/S, Novozymes Inc., Novozymes North America, Inc.. Invention is credited to Bjorn Lennart Pierre Alexander Cassland, Donald L. Higgins, Qiming Jin, Jiyin Liu.
Application Number | 20180273984 16/006521 |
Document ID | / |
Family ID | 39545105 |
Filed Date | 2018-09-27 |
United States Patent
Application |
20180273984 |
Kind Code |
A1 |
Jin; Qiming ; et
al. |
September 27, 2018 |
DETOXIFYING PRE-TREATED LIGNOCELLULOSE-CONTAINING MATERIALS
Abstract
The invention relates to a process of detoxifying pre-treated
lignocellulose-containing material comprising subjecting the
pre-treated lignocellulose-containing material to one or more
phenolic compound oxidizing enzymes.
Inventors: |
Jin; Qiming; (Sacremento,
CA) ; Liu; Jiyin; (Raleigh, NC) ; Alexander
Cassland; Bjorn Lennart Pierre; (Malmo, SE) ;
Higgins; Donald L.; (Franklinton, NC) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Novozymes A/S
Novozymes Inc.
Novozymes North America, Inc. |
Bagsvaerd
Davis
Franklinton |
CA
NC |
DK
US
US |
|
|
Assignee: |
Novozymes A/S
Bagsvaerd
CA
Novozymes Inc.
Davis
NC
Novozymes North America, Inc.
Franklinton
|
Family ID: |
39545105 |
Appl. No.: |
16/006521 |
Filed: |
June 12, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12597834 |
Nov 24, 2009 |
10023881 |
|
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PCT/US08/60766 |
Apr 18, 2008 |
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16006521 |
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60988949 |
Nov 19, 2007 |
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60946272 |
Jun 26, 2007 |
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60913581 |
Apr 24, 2007 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12P 7/10 20130101; Y02E
50/16 20130101; Y02E 50/10 20130101 |
International
Class: |
C12P 7/10 20060101
C12P007/10 |
Claims
1-20. (canceled)
21. A process for producing a fermentation product from
lignocellulose-containing material, comprising the steps of: (a)
pre-treating lignocellulose-containing material; (b) detoxifying
comprising subjecting the pre-treated lignocellulose-containing
material to one or more phenolic compound oxidizing enzymes and/or
one or more enzymes exhibiting peroxidase activity; (c)
hydrolyzing; and (d) fermenting using a fermenting organism,
wherein the detoxification is carried out simultaneously with
hydrolysis or fermentation.
22. The process of claim 21, wherein the one or more phenolic
compound oxidizing enzymes are selected from the group consisting
of a catechol oxidase (EC 1.10.3.1), laccase (EC 1.10.3.2),
o-aminophenol oxidase (EC 1.10.3.4); and monophenol monooxygenase
(EC 1.14.18.1).
23. The process of claim 21, wherein the enzyme exhibiting
peroxidase activity is selected from the group consisting of a
peroxidase (EC 1.11.1.7), haloperoxidase (EC1.11.1.8 and EC
1.11.1.10); lignin peroxidase (EC 1.11.1.14); manganese peroxidase
(EC 1.11.1.13); and lipoxygenase (EC.1.13.11.12).
24. The process of claim 21, wherein the solids after pre-treating
the lignocellulose-containing material in step (a) are
removed/separated from the liquor before detoxification.
25. The process of claim 21, wherein the lignocellulose-containing
material is chemically, mechanically and/or biologically
pre-treated in step (a).
26. The process of claim 21, wherein hydrolysis and/or fermentation
is carried out using one or more carbohydrases selected from the
group consisting of cellulase, hemicellulase, amylase, protease,
esterase, endoglucanase, beta-glucosidase, cellobiohydrolase,
xylanase, alpha-amylase, alpha-glucosidase, glucoamylase, protease
and lipase.
27. The process of claim 21, wherein the fermenting organism is a
yeast strain of the genus Saccharomyces.
28. The process of claim 1, wherein the fermentation product is an
alcohol.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a 35 U.S.C. 371 national application of
international application no. PCT/US2008/60766 filed Apr. 18, 2008,
which claims priority or the benefit under 35 U.S.C. 119 of U.S.
provisional application Nos. 60/988,949, 60/946,272 and 60/913,581
filed Nov. 9, 2007, Jun. 26, 2007 and Apr. 24, 2007 the contents of
which are fully incorporated herein by reference.
TECHNICAL FIELD
[0002] The present invention relates to processes of detoxifying
pre-treated lignocellulose-containing material. The invention also
relates to processes of producing a fermentation product from
lignocellulose-containing material using a fermenting organism
including a detoxification process of the invention.
BACKGROUND OF THE INVENTION
[0003] Due to the limited reserves of fossil fuels and worries
about emission of greenhouse gases there is an increasing focus on
using renewable energy sources. Production of fermentation products
from lignocellulose-containing material is known in the art and
conventionally includes pretreatment, hydrolysis, and fermentation
of the lignocellulose-containing material. Pre-treatment results in
the release of, e.g., phenolics and furans, from the
lignocellulose-containing material that may irreversibly bind
enzymes added during hydrolysis and fermentation. These compounds
may also be toxic to the fermenting organism's metabolism and
inhibit the performance of the fermenting organism.
[0004] Detoxification by steam stripping has been suggested but it
is a cumbersome and a costly additional process step. It has also
been suggested to wash the pre-treated lignocellulose-containing
material before hydrolysis. This requires huge amounts of water,
that needs to be removed again, and is therefore also costly.
[0005] Consequently, there is a need for providing processes for
detoxifying pre-treated lignocellulose-containing material suitable
for fermentation product production processes.
SUMMARY OF THE INVENTION
[0006] The present invention relates to processes of detoxifying
pre-treated lignocellulose-containing material. The invention also
relates to processes of producing a fermentation product from
lignocellulose-containing material using a fermenting organism
including a detoxification process of the invention.
[0007] In the first aspect the invention relates to processes for
detoxifying pre-treated lignocellulose-containing material
comprising subjecting the pre-treated lignocellulose-containing
material to one or more phenolic compound oxidizing enzymes and/or
one or more enzymes exhibiting peroxidase activity.
[0008] In the second aspect the invention relates to processes for
producing a fermentation product from lignocellulose-containing
material comprising the steps of:
[0009] (a) pre-treating lignocellulose-containing material;
[0010] (b) hydrolyzing;
[0011] (c) detoxifying in accordance with a detoxification process
of the invention; and
[0012] (d) fermenting using a fermenting organism.
[0013] The invention also relates to processes for producing a
fermentation product from lignocellulose-containing material
comprising the steps of:
[0014] (i) pre-treating lignocellulose-containing material;
[0015] (ii) detoxifying in accordance with the fermentation process
of the invention;
[0016] (iii) hydrolyzing; and
[0017] (iv) fermenting using a fermenting organism.
BRIEF DESCRIPTION OF THE FIGURES
[0018] FIG. 1 shows the effect of laccase treatment on cellulose
conversion.
DETAILED DESCRIPTION OF THE INVENTION
[0019] In the first aspect the invention relates to processes of
detoxifying pre-treated lignocellulose-containing material suitable
for producing a fermentation product.
Lignocellulose-Containing Material
[0020] The term "lignocellulose-containing materials" used herein
refers to material that primarily consists of cellulose,
hemicellulose, and lignin. Such material is often referred to as
"biomass".
[0021] The structure of lignocellulose is not directly accessible
to enzymatic hydrolysis. Therefore, the lignocellulose has to be
pre-treated, e.g., by acid hydrolysis under adequate conditions of
pressure and temperature, in order to break the lignin seal and
disrupt the crystalline structure of cellulose. This causes
solubilization and saccharification of the hemicellulose fraction.
The cellulose fraction can then be hydrolyzed enzymatically, e.g.,
by cellulase enzymes (or cellulolytic enzymes), to convert the
carbohydrate polymers into fermentable sugars which may be
fermented into a desired fermentation product, such as ethanol.
Optionally the fermentation product is recovered after
fermentation, e.g., by distillation.
[0022] Any lignocellulose-containing material is contemplated
according to the present invention. The lignocellulose-containing
material may be any material containing lignocellulose. In a
preferred embodiment the lignocellulose-containing material
contains at least 30 wt. %, preferably at least 50 wt. %, more
preferably at least 70 wt. %, even more preferably at least 90 wt.
% lignocellulose. It is to be understood that the
lignocellulose-containing material may also comprise other
constituents such as cellulosic material, including cellulose and
hemicellulose, and may also comprise other constituents such as
proteinaceous material, starch, sugars, such as fermentable sugars
and/or un-fermentable sugars.
[0023] Lignocellulose-containing material is generally found, for
example, in the stems, leaves, hulls, husks, and cobs of plants or
leaves, branches, and wood of trees. Lignocellulose-containing
material can also be, but is not limited to, herbaceous material,
agricultural residues, forestry residues, municipal solid wastes,
waste paper, and pulp and paper mill residues. It is understood
herein that lignocellulose-containing material may be in the form
of plant cell wall material containing lignin, cellulose, and
hemi-cellulose in a mixed matrix.
[0024] In a preferred embodiment the lignocellulose-containing
material is corn fiber, rice straw, pine wood, wood chips, poplar,
bagasse, paper and pulp processing waste.
[0025] Other examples include corn stover, hardwood, such as poplar
and birch, softwood, cereal straw, such as wheat straw,
switchgrass, municipal solid waste (MSW), industrial organic waste,
office paper, or mixtures thereof.
[0026] In a preferred embodiment the lignocellulose-containing
material is corn stover. In another preferred embodiment the
material is corn fiber.
Process of Detoxifying Pre-Treated Lignocellulose-Containing
Material
[0027] When lignocellulose-containing material is pre-treated,
degradation products that may inhibit enzymes and/or may be toxic
to fermenting organisms are produced. These degradation products
severely decrease both the hydrolysis and fermentation rate.
[0028] Methods for pre-treating lignocellulose-containing material
are well known in the art. Examples of contemplated methods are
described below in the section "Pre-treatment".
[0029] The present inventors have found that phenolic compound
oxidizing enzymes can be used to detoxify pre-treated
lignocellulose-containing material. The fermentation time can be
reduced as a result of improved performance of the fermenting
organism during fermentation. In other words, detoxification in
accordance with the invention may result in a shorter
"lignocellulose-containing material-to-fermentation product"
process time. Furthermore, the need for a washing step after
pre-treatment of the lignocellulose-containing material, to remove
toxic compounds, and/or adaption of the fermentation organism to
the medium/broth can be eliminated. Also, the dosing of the
fermentation organism may be reduced.
[0030] In a preferred embodiment the pre-treated
lignocellulose-containing material may be treated with cellulase
(cellulolytic enzymes) and/or hemicellulase (hemicellulolytic
enzymes).
[0031] Specific examples of detoxifying compounds can be found in
the "Detoxifying Compounds"-section below.
[0032] In the first aspect the invention relates to processes for
detoxifying pre-treated lignocellulose-containing material
comprising subjecting the pre-treated lignocellulose-containing
material to one or more phenolic compound oxidizing enzymes and/or
one or more enzymes exhibiting peroxidase activity.
[0033] The pre-treated lignocellulose degradation products include
lignin degradation products, cellulose degradation products and
hemicellulose degradation products. The pre-treated lignin
degradation products may be phenolics in nature.
[0034] The hemicellulose degradation products include furans from
sugars (such as hexoses and/or pentoses), including xylose,
mannose, galactose, rhamanose, and arabinose. Examples of
hemicelluloses include xylan, galactoglucomannan, arabinogalactan,
arabinoglucuronoxylan, glucuronoxylan, and derivatives and
combinations thereof.
[0035] Examples of inhibitory compounds, i.e., pre-treated
lignocellulose degradation products, include 4-OH benzyl alcohol,
4-OH benzaldehyde, 4-OH benzoic acid, trimethyl benzaldehyde,
2-furoic acid, coumaric acid, ferulic acid, phenol, guaiacol,
veratrole, pyrogallollol, pyrogallol mono methyl ether, vanillyl
alcohol, vanillin, isovanillin, vanillic acid, isovanillic acid,
homovanillic acid, veratryl alcohol, veratraldehyde, veratric acid,
2-O-methyl gallic acid, syringyl alcohol, syringaldehyde, syringic
acid, trimethyl gallic acid, homocatechol, ethyl vanillin, creosol,
p-methyl anisol, anisaldehyde, anisic acid, furfural,
hydroxymethylfurfural, 5-hydroxymethylfurfural, formic acid, acetic
acid, levulinic acid, cinnamic acid, coniferyl aldehyde,
isoeugenol, hydroquinone, eugenol or combinations thereof. Other
inhibitory compounds can be found in, e.g., Luo et al., 2002,
Biomass and Bioenergy 22: 125-138.
[0036] The detoxification process of the invention may preferably
be carried out at a pH that is suitable of the phenolic compound
oxidizing enzymes and hydrolyzing enzyme(s) and/or fermenting
organism if detoxification is carried out simultaneously with
hydrolysis or simultaneously with hydrolysis and fermentation. In
one embodiment the pH is between 2 and 7, preferably between 3 and
6, especially between 4 and 5. In a preferred embodiment the
temperature during detoxification is a temperature suitable for the
phenolic compound oxidizing enzyme(s) and/or enzyme exhibiting
peroxidase activity and hydrolyzing enzyme(s) and/or fermenting
organism if detoxification is carried out a simultaneous with
hydrolysis or simultaneously with hydrolysis and fermentation. In
one embodiment the temperature during detoxification is between
25.degree. C. and 70.degree. C., preferably between 30.degree. C.
and 60.degree. C. In cases where detoxification is carried out
simultaneously with fermentation the temperature will depend on the
fermenting organism. For ethanol fermentations with yeast the
temperature would be between 26-38.degree. C., such as between
26-34.degree. C. or between 30-36.degree. C., such as around
32.degree. C.
[0037] Suitable pHs, temperatures and other process conditions can
easily be determined by one skilled in the art.
Detoxifying Enzymes
[0038] The detoxifying enzyme(s) may be of any origin including of
mammal, plant and microbial origin, such as of bacteria and fungal
origin.
[0039] Phenolic compound oxidizing enzymes may in preferred
embodiments belong to any of the following EC classes including:
Catechol oxidase (EC 1.10.3.1), Laccase (EC 1.10.3.2),
o-Aminophenol oxidase (1.10.3.4); and Monophenol monooxygenase
(1.14.18.1).
[0040] The enzyme exhibiting peroxidase activity may in a preferred
embodiment belong to any of the following EC classes including
those selected from the group consisting of a peroxidase (EC
1.11.1.7), Haloperoxidase (EC1.11.1.8 and EC 1.11.1.10); Lignin
peroxidase (EC 1.11.1.14); manganese peroxidase (EC 1.11.1.13); and
Lipoxygenase (EC.1.13.11.12).
[0041] Examples of detoxifying enzymes contemplated according to
the invention can be found in the "Enzymes"-section below.
Production of Fermentation Products from Lignocellulose-Containing
Material
[0042] In the second aspect the invention relates to processes of
producing fermentation products from lignocellulose-containing
material. Conversion of lignocellulose-containing material into
fermentation products, such as ethanol, has the advantages of the
ready availability of large amounts of feedstock, including wood,
agricultural residues, herbaceous crops, municipal solid wastes,
etc.
[0043] The structure of lignocellulose is not directly accessible
to enzymatic hydrolysis. Therefore, the lignocellulose-containing
material has to be pre-treated, e.g., by acid hydrolysis under
adequate conditions of pressure and temperature, in order to break
the lignin seal and disrupt the crystalline structure of cellulose.
This causes solubilization of the hemicellulose and cellulose
fractions. The cellulose and hemicellulose can then be hydrolyzed
enzymatically, e.g., by cellulase enzymes (cellulolytic enzymes),
to convert the carbohydrate polymers into fermentable sugars which
may be fermented into a desired fermentation product, such as
ethanol. Optionally the fermentation product may be recovered,
e.g., by distillation.
[0044] More precisely the invention relates in this embodiment to
processes for producing a fermentation product from
lignocellulose-containing material comprising the steps of:
[0045] (a) pre-treating lignocellulose-containing material;
[0046] (b) hydrolyzing;
[0047] (c) detoxifying; and
[0048] (d) fermenting using a fermenting organism;
wherein detoxification is carried out in accordance with a
detoxification process of the invention. More details on the steps
are described below in the sections "Pre-treatment", "Hydrolysis"
and "Fermentation".
[0049] In another embodiment the invention relates to processes for
producing a fermentation product from lignocellulose-containing
material comprising the steps of:
[0050] (i) pre-treating lignocellulose-containing material;
[0051] (ii) detoxifying;
[0052] (iii) hydrolyzing; and
[0053] (iv) fermenting using a fermenting organism;
wherein detoxification is carried out in accordance with a
detoxification process of the invention.
[0054] One or more detoxifying enzymes may be added after
pre-treatment step (i), but before hydrolysis step (iii).
Detoxifying the pre-treated material in step (ii) may be carried
out before hydrolysis, but detoxification and hydrolysis may also
be carried out simultaneously. The detoxification step (ii) may be
carried out separately from hydrolysis. Further hydrolysis step
(iii) and fermentation step (iv) may be carried out simultaneously
or sequentially. In one embodiment the solids (comprising mainly
lignin and unconverted polysaccharides) may, after pre-treating the
lignocellulose-containing material in step (i), be
removed/separated from the liquor before detoxification. The
removed solids and the detoxified liquor may be combined before
hydrolysis in step (iii) or simultaneous hydrolysis and
fermentation.
[0055] The solids may be removed/separated in any suitable way know
in the art. In suitable embodiments the solids are removed by
filtration, or by using a filter press and/or centrifuge, or the
like. The reduced inhibitory effect of the hydrolyzing enzymes is
tested in Example 4.
[0056] Examples of pre-treatment methods, hydrolysis and
fermentation conditions for both of above embodiment is described
below.
Pre-Treatment
[0057] The lignocellulose-containing material may be pre-treated in
any suitable way. Pre-treatment may be carried out before and/or
during hydrolysis and/or fermentation. In a preferred embodiment
the pre-treated material is hydrolyzed, preferably enzymatically,
before and/or during fermentation and/or before and/or during
detoxification. The goal of pre-treatment is to separate and/or
release cellulose; hemicellulose and/or lignin and this way improve
the rate of hydrolysis. Pre-treatment methods such as wet-oxidation
and alkaline pre-treatment targets lignin, while dilute acid and
auto-hydrolysis targets hemicellulose. Steam explosion is an
example of a pre-treatment that targets cellulose.
[0058] According to the invention pre-treatment in step (a) or (i)
may be a conventional pre-treatment step using techniques well
known in the art. Examples of suitable pre-treatments are disclosed
below. In a preferred embodiment pre-treatment takes place in
aqueous slurry.
[0059] The lignocellulose-containing material may during
pre-treatment be present in an amount between 10-80 wt. %,
preferably between 20-70 wt. %, especially between 30-60 wt. %,
such as around 50 wt. %.
Chemical, Mechanical and/or Biological Pre-Treatment
[0060] The lignocellulose-containing material may according to the
invention be chemically, mechanically and/or biologically
pre-treated before hydrolysis and/or fermentation. Mechanical
treatment (often referred to as physical treatment) may be used
alone or in combination with subsequent or simultaneous hydrolysis,
especially enzymatic hydrolysis.
[0061] Preferably, chemical, mechanical and/or biological
pre-treatment is carried out prior to the hydrolysis and/or
fermentation. Alternatively, the chemical, mechanical and/or
biological pre-treatment may be carried out simultaneously with
hydrolysis, such as simultaneously with addition of one or more
cellulase enzymes (cellulolytic enzymes), or other enzyme
activities mentioned below, to release, e.g., fermentable sugars,
such as glucose and/or maltose.
[0062] In an embodiment of the invention the pre-treated
lignocellulose-containing material may be washed before and/or
after detoxification. However, washing is not mandatory and is in a
preferred embodiment eliminated.
[0063] According to one embodiment of the invention one or more
detoxifying enzymes may be added to the pre-treated
lignocellulose-containing material in step (c) or (ii).
Detoxification step (c) or (ii) and hydrolysis step (b) or (iii)
may be carried out either simultaneously or sequentially. The
reduced toxic effect on the fermenting organism is shown in
Examples 2 and 3.
[0064] The steps may in one embodiment be done in one treating
solution (ie., one bath). In one embodiment the hydrolyzing
enzyme(s) and the detoxifying enzyme(s) are added simultaneously to
the treating solution. In another embodiment the hydrolyzing
enzyme(s) are added before the detoxifying enzyme(s). It may be
advantageous to complete above 50% of hydrolysis, preferably above
70% of hydrolysis, especially above 90% of hydrolysis before adding
the detoxifying enzyme(s) to the treating solution. If the
pre-treated lignocellulose-containing material is hydrolyzed
enzymatically, it is advantageous to do detoxification before
and/or simultaneous with hydrolysis. However, if hydrolysis is
carried out using one or more acids, i.e., acid hydrolysis,
detoxification is preferably carried out after and/or
simultaneously with acid hydrolysis.
[0065] In another embodiment detoxification step (c) or (ii) may be
carried out separately from hydrolysis step (b) or (iii) and
fermentation step (d) or (iv), respectively, which in one
embodiment may be carried out simultaneously. In a further
embodiment all of steps (b), (c) and (d) or (i), (ii), (iii) and
(iv), respectively, are carried out simultaneously or sequentially.
When detoxification is done as a separate step, it typically is
carried out for between 1-24 hours.
[0066] In a preferred embodiment the pre-treated
lignocellulose-containing material is unwashed.
[0067] In an embodiment the phenolic compound oxidizing enzyme(s)
is(are) dosed in the range from above 0, such as 0.01 to 1 mg/g DS
or in the range from above 0 to 100 LACU/g DS. In an embodiment the
enzyme(s) exhibiting peroxidase activity is(are) dosed in the range
from above 0, such as 0.01 to 10 mg/g DS or above 0, such as 0.01
to 100 PODU/g DS.
Chemical Pre-Treatment
[0068] The term "chemical treatment" refers to any chemical
pre-treatment which promotes the separation and/or release of
cellulose, hemicellulose and/or lignin. Examples of suitable
chemical pre-treatments include treatment with; for example, dilute
acid, lime, alkaline, organic solvent, ammonia, sulfur dioxide,
carbon dioxide. Further, wet oxidation and pH-controlled
hydrothermolysis are also considered chemical pre-treatment.
[0069] In a preferred embodiment the chemical pre-treatment is acid
treatment, more preferably, a continuous dilute and/or mild acid
treatment, such as, treatment with sulfuric acid, or another
organic acid, such as acetic acid, citric acid, tartaric acid,
succinic acid, hydrogen chloride or mixtures thereof. Other acids
may also be used. Mild acid treatment means that the treatment pH
lies in the range from 1-5, preferably 1-3. In a specific
embodiment the acid concentration is in the range from 0.1 to 2.0
wt. % acid, preferably sulphuric acid. The acid may be contacted
with the lignocellulose-containing material and the mixture may be
held at a temperature in the range of 160-220.degree. C., such as
165-195.degree. C., for periods ranging from minutes to seconds,
e.g., 1-60 minutes, such as 2-30 minutes or 3-12 minutes. Addition
of strong acids, such as sulphuric acid, may be applied to remove
hemicellulose. This enhances the digestibility of cellulose.
[0070] Other techniques are also contemplated. Cellulose solvent
treatment has been shown to convert about 90% of cellulose to
glucose. It has also been shown that enzymatic hydrolysis could be
greatly enhanced when the lignocellulose structure is disrupted.
Alkaline H.sub.2O.sub.2, ozone, organosolv (uses Lewis acids,
FeCl.sub.3, (Al).sub.2SO.sub.4 in aqueous alcohols), glycerol,
dioxane, phenol, or ethylene glycol are among solvents known to
disrupt cellulose structure and promote hydrolysis (Mosier et al.,
2005, Bioresource Technology 96: 673-686).
[0071] Alkaline chemical pre-treatment with base, e.g., NaOH,
Na.sub.2CO.sub.3 and/or ammonia or the like, is also contemplated
according to the invention. Pre-treatment methods using ammonia are
described in, e.g., WO 2006/110891, WO 2006/110899, WO 2006/110900,
and WO 2006/110901 (which are hereby incorporated by
reference).
[0072] Wet oxidation techniques involve use of oxidizing agents,
such as: sulphite based oxidizing agents or the like. Examples of
solvent pre-treatments include treatment with DMSO (Dimethyl
Sulfoxide) or the like. Chemical pre-treatment is generally carried
out for 1 to 60 minutes, such as from 5 to 30 minutes, but may be
carried out for shorter or longer periods of time dependent on the
material to be pre-treated.
[0073] Other examples of suitable pre-treatment methods are
described by Schell et al., 2003, Appl. Biochem and Biotechn. Vol.
105-108: 69-85, and Mosier et al., 2005, Bioresource Technology 96:
673-686, and U.S. Publication No. 2002/0164730, which references
are hereby all incorporated by reference.
Mechanical Pre-Treatment
[0074] The term "mechanical pre-treatment" refers to any mechanical
(or physical) treatment which promotes the separation and/or
release of cellulose, hemicellulose and/or lignin from
lignocellulose-containing material. For example, mechanical
pre-treatment includes various types of milling, irradiation,
steaming/steam explosion, and hydrothermolysis.
[0075] Mechanical pre-treatment includes comminution (mechanical
reduction of the size). Comminution includes dry milling, wet
milling and vibratory ball milling. Mechanical pre-treatment may
involve high pressure and/or high temperature (steam explosion). In
an embodiment of the invention high pressure means pressure in the
range from 300 to 600 psi, preferably 400 to 500 psi, such as
around 450 psi. In an embodiment of the invention high temperature
means temperatures in the range from about 100 to 300.degree. C.,
preferably from about 140 to 235.degree. C. In a preferred
embodiment mechanical pre-treatment is a batch-process, steam gun
hydrolyzer system which uses high pressure and high temperature as
defined above. A Sunds Hydrolyzer (available from Sunds Defibrator
AB (Sweden) may be used for this.
Combined Chemical and Mechanical Pre-Treatment
[0076] In a preferred embodiment both chemical and mechanical
pre-treatments are carried out. For instance, the pre-treatment
step may involve dilute or mild acid treatment and high temperature
and/or pressure treatment. The chemical and mechanical
pre-treatment may be carried out sequentially or simultaneously, as
desired.
[0077] Accordingly, in a preferred embodiment, the
lignocellulose-containing material is subjected to both chemical
and mechanical pre-treatment to promote the separation and/or
release of cellulose, hemicellulose and/or lignin.
[0078] In a preferred embodiment the pre-treatment is carried out
as a dilute and/or mild acid steam explosion step. In another
preferred embodiment pre-treatment is carried out as an ammonia
fiber explosion step (or AFEX pre-treatment step).
Biological Pre-Treatment
[0079] As used in the present invention the term "biological
pre-treatment" refers to any biological pre-treatment which
promotes the separation and/or release of cellulose, hemicellulose,
and/or lignin from the lignocellulose-containing material.
Biological pre-treatment techniques can involve applying
lignin-solubilizing microorganisms (see, for example, Hsu, 1996,
Pretreatment of biomass, in Handbook on Bioethanol: Production and
Utilization, Wyman, ed., Taylor & Francis, Washington, D.C.,
179-212; Ghosh and Singh, 1993, Physicochemical and biological
treatments for enzymatic/microbial conversion of lignocellulosic
biomass, Adv. Appl. Microbiol. 39: 295-333; McMillan, 1994,
Pretreating lignocellulosic biomass: a review, in Enzymatic
Conversion of Biomass for Fuels Production, Himmel, Baker, and
Overend, eds., ACS Symposium Series 566, American Chemical Society,
Washington, D.C., chapter 15; Gong, Cao, Du, and Tsao, 1999,
Ethanol production from renewable resources, in Advances in
Biochemical Engineering/Biotechnology, Scheper, ed.,
Springer-Verlag Berlin Heidelberg, Germany, 65: 207-241; Olsson and
Hahn-Hagerdal, 1996, Fermentation of lignocellulosic hydrolysates
for ethanol production, Enz. Microb. Tech. 18: 312-331; and
Vallander and Eriksson, 1990, Production of ethanol from
lignocellulosic materials: State of the art, Adv. Biochem.
Eng./Biotechnol. 42: 63-95).
Hydrolysis
[0080] Before and/or simultaneously with fermentation the
pre-treated lignocellulose-containing material may be hydrolyzed to
break down cellulose and hemicellulose.
[0081] The dry solids content during hydrolysis may be in the range
from 5-50 wt. %, preferably 10-40 wt. %, preferably 20-30 wt. %.
Hydrolysis may in a preferred embodiment be carried out as a fed
batch process where the pre-treated lignocellulose-containing
material (substrate) is fed gradually to an, e.g., enzyme
containing hydrolysis solution.
[0082] In an embodiment of the invention detoxification takes place
before, during and/or after hydrolysis.
[0083] In a preferred embodiment hydrolysis is carried out
enzymatically. According to the invention the pre-treated
lignocellulose-containing material may be hydrolyzed by one or more
hydrolases (class EC 3 according to Enzyme Nomenclature),
preferably one or more carbohydrases selected from the group
consisting of cellulase, hemicellulase, amylase, such as
alpha-amylase, protease, carbohydrate-generating enzyme, such as
glucoamylase, esterase, such as lipase. Alpha-amylase, glucoamylase
and/or the like may be present during hydrolysis and/or
fermentation as the lignocellulose-containing material may include
some starch.
[0084] The enzyme(s) used for hydrolysis is(are) capable of
directly or indirectly converting carbohydrate polymers into
fermentable sugars which can be fermented into a desired
fermentation product, such as ethanol.
[0085] In a preferred embodiment the carbohydrase has cellulase
enzyme activity. Suitable carbohydrases are described in the
"Enzymes"-section below.
[0086] Hemicellulose polymers can be broken down by hemicellulases
and/or acid hydrolysis to release its five and six carbon sugar
components. The six carbon sugars (hexoses), such as glucose,
galactose, arabinose, and mannose, can readily be fermented to,
e.g., ethanol, acetone, butanol, glycerol, citric acid, fumaric
acid, etc. by suitable fermenting organisms including yeast.
Preferred for ethanol fermentation is yeast of the species
Saccharomyces cerevisiae, preferably strains which are resistant
towards high levels of ethanol, i.e., up to, e.g., about 10, 12 or
15 vol. % ethanol or more, such as 20 vol. % ethanol.
[0087] In a preferred embodiment the pre-treated
lignocellulose-containing material is hydrolyzed using a
hemicellulase, preferably a xylanase, esterase, cellobiase, or
combination thereof.
[0088] Hydrolysis may also be carried out in the presence of a
combination of hemicellulases and/or cellulases, and optionally one
or more of the other enzyme activities mentioned in the "Enzyme"
section below.
[0089] In a preferred embodiment hydrolysis and fermentation is
carried out as a simultaneous hydrolysis and fermentation step
(SSF). In general this means that combined/simultaneous hydrolysis
and fermentation are carried out at conditions (e.g., temperature
and/or pH) suitable, preferably optimal, for the fermenting
organism(s) in question.
[0090] In another preferred embodiment hydrolysis and fermentation
are carried out as hybrid hydrolysis and fermentation (HHF). HHF
typically begins with a separate partial hydrolysis step and ends
with a simultaneous hydrolysis and fermentation step. The separate
partial hydrolysis step is an enzymatic cellulose saccharification
step typically carried out at conditions (e.g., at higher
temperatures) suitable, preferably optimal, for the hydrolyzing
enzyme(s) in question. The subsequent simultaneous hydrolysis and
fermentation step is typically carried out at conditions suitable
for the fermenting organism(s) (often at lower temperatures than
the separate hydrolysis step). Finally, hydrolysis and fermentation
may also be carried out a separate hydrolysis and fermentation,
where the hydrolysis is taken to completion before initiation of
fermentation. This often referred to as "SHF".
[0091] Enzymatic treatments may be carried out in a suitable
aqueous environment under conditions which can readily be
determined by one skilled in the art.
[0092] In a preferred embodiment hydrolysis is carried out at
suitable, preferably optimal conditions for the enzyme(s) in
question.
[0093] Suitable process time, temperature and pH conditions can
readily be determined by one skilled in the art present invention.
Preferably, hydrolysis is carried out at a temperature between 25
and 70.degree. C., preferably between 40 and 60.degree. C.,
especially around 50.degree. C. The process is preferably carried
out at a pH in the range from 3-8, preferably pH 4-6, especially
around pH 5.
[0094] Preferably, hydrolysis is carried out for between 12 and 96
hours, preferable 16 to 72 hours, more preferably between 24 and 48
hours.
[0095] According to the invention hydrolysis in step (b) or (iii)
and fermentation in step (d) or (iv) may be carried out
simultaneously (SSF process) or sequentially (SHF process) or as a
hybrid hydrolysis and fermentation (HHF).
Fermentation
[0096] According to the invention the pre-treated (and hydrolyzed)
lignocellulose-containing material is fermented by at least one
fermenting organism capable of fermenting fermentable sugars, such
as glucose, xylose, mannose, and galactose directly or indirectly
into a desired fermentation product.
[0097] The fermentation is preferably ongoing for between 8 to 96
hours, preferably 12 to 72 hours, more preferable from 24 to 48
hours.
[0098] In an embodiment the fermentation is carried out at a
temperature between 20 to 40.degree. C., preferably 26 to
34.degree. C., in particular around 32.degree. C. In an embodiment
the pH is from pH 3 to 6, preferably around pH 4 to 5.
[0099] Contemplated according to the invention is simultaneous
hydrolysis and fermentation (SSF). In an embodiment there is no
separate holding stage for the hydrolysis, meaning that the
hydrolyzing enzyme(s) and the fermenting organism are added
together. When the fermentation (e.g., ethanol fermentation using
Saccharomyces yeast) is performed simultaneous with hydrolysis the
temperature is preferably between 26.degree. C. and 35.degree. C.,
more preferably between 30.degree. C. and 34.degree. C., such as
around 32.degree. C. A temperature program comprising at least two
holding stages at different temperatures may be applied according
to the invention.
[0100] The process of the invention may be performed as a batch,
fed-batch or as a continuous process.
Recovery
[0101] Subsequent to fermentation the fermentation product may be
separated from the fermentation medium/broth. The medium/broth may
be distilled to extract the fermentation product or the
fermentation product may be extracted from the fermentation
medium/broth by micro or membrane filtration techniques.
Alternatively the fermentation product may be recovered by
stripping. Recovery methods are well known in the art.
Fermentation Products
[0102] Processes of the invention may be used for producing any
fermentation product. Especially contemplated fermentation products
include alcohols (e.g., ethanol, methanol, butanol); organic acids
(e.g., citric acid, acetic acid, itaconic acid, lactic acid,
gluconic acid); ketones (e.g., acetone); amino acids (e.g.,
glutamic acid); gases (e.g., H.sub.2 and CO.sub.2); antibiotics
(e.g., penicillin and tetracycline); enzymes; vitamins (e.g.,
riboflavin, B12, beta-carotene); and hormones.
[0103] Also contemplated products include consumable alcohol
industry products, e.g., beer and wine; dairy industry products,
e.g., fermented dairy products; leather industry products and
tobacco industry products. In a preferred embodiment the
fermentation product is an alcohol, especially ethanol. The
fermentation product, such as ethanol, obtained according to the
invention, may preferably be fuel alcohol/ethanol. However, in the
case of ethanol it may also be used as potable ethanol.
Fermenting Organism
[0104] The term "fermenting organism" refers to any organism,
including bacterial and fungal organisms, suitable for producing a
desired fermentation product. Especially suitable fermenting
organisms according to the invention are able to ferment, i.e.,
convert, sugars, such as glucose, directly or indirectly into the
desired fermentation product. Examples of fermenting organisms
include fungal organisms, such as yeast. Preferred yeast includes
strains of the genus Saccharomyces, in particular a strain of
Saccharomyces cerevisiae or Saccharomyces uvarum; a strain of
Pichia, in particular Pichia stipitis or Pichia pastoris; a strain
of the genus Candida, in particular a strain of Candida utilis,
Candida arabinofermentans, Candida diddensii, or Candida boidinii.
Other contemplated yeast includes strains of Hansenula, in
particular Hansenula polymorpha or Hansenula anomala; strains of
Kluyveromyces, in particular Kluyveromyces marxianus or
Kluyveromyces fagilis, and strains of Schizosaccharomyces, in
particular Schizosaccharomyces pombe.
[0105] Preferred bacterial fermenting organisms include strains of
Escherichia, in particular Escherichia coli, strains of Zymomonas,
in particular Zymomonas mobilis, strains of Zymobacter in
particular Zymobactor palmae, strains of Klebsiella, in particular
Klebsiella oxytoca, strains of Leuconostoc, in particular
Leuconostoc mesenteroides, strains of Clostridium, in particular
Clostridium butyricum, strains of Enterobacter in particular
Enterobacter aerogenes and strains of Thermoanaerobacter, in
particular Thermoanaerobacter BG1L1 (Appl. Micrbiol. Biotech. 77:
61-86) and Thermoanarobacter ethanolicus.
[0106] Commercially available yeast includes, e.g., RED STAR.TM. or
ETHANOL RED.TM. yeast (available from Fermentis/Lesaffre, USA),
FALI (available from Fleischmann's Yeast, USA), SUPERSTART and
THERMOSACC.TM. fresh yeast (available from Ethanol Technology, WI,
USA), BIOFERM AFT and XR (available from NABC--North American
Bioproducts Corporation, GA, USA), GERT STRAND (available from Gert
Strand AB, Sweden), and FERMIOL (available from DSM
Specialties).
Enzymes
[0107] Even though not specifically mentioned in context of
processes of the invention, it is to be understood that the enzymes
(as well as other compounds) are used in an "effective amount". For
instance, "effective amount" means, in context of phenolic compound
oxidizing enzyme(s) that it has an improving effect compared to a
corresponding process where no phenolic compound oxidizing
enzyme(s) was (were) added.
Phenolic Compound Oxidizing Enzymes
[0108] Preferred phenolic compound oxidizing enzymes belong to any
of the following EC classes: Catechol oxidase (EC 1.10.3.1),
Laccase (EC 1.10.3.2), o-Aminophenol oxidase (1.10.3.4); and
Monophenol monooxygenase (1.14.18.1).
Laccase
[0109] Laccases (EC 1.10.3.2) are multi-copper-containing enzymes
that catalyze the oxidation of phenolic compounds. Laccases are
produced by plants, bacteria and also a wide variety of fungi,
including Ascomycetes such as Aspergillus, Neurospora, and
Podospora; Deuteromycete including Botrytis, and Basidiomycetes
such as Collybia, Fomes, Lentinus, Pleurotus, Trametes, and perfect
forms of Rhizoctonia. A number of fungal laccases have been
isolated. For example, Choi et al. (Mol. Plant-Microbe Interactions
5: 119-128, 1992) describe the molecular characterization and
cloning of the gene encoding the laccase of the chestnut blight
fungus, Cryphonectria parasitica. Kojima et al. (J. Biol. Chem.
265: 15224-15230, 1990; JP 2-238885) provide a description of two
allelic forms of the laccase of the white-rot basidiomycete
Coriolus hirsutus. Germann and Lerch (Experientia 41: 801, 1985;
PNAS USA 83: 8854-8858, 1986) have reported the cloning and partial
sequencing of the Neurospora crassa laccase gene. Saloheimo et al.
(J. Gen. Microbiol. 137: 1537-1544, 1985; WO 92/01046) have
disclosed a structural analysis of the laccase gene from the fungus
Phlebia radiata.
[0110] Especially contemplated laccases include those derived from
a strain of Polyporus, preferably Polyporus pinsitus; Melanocarpus,
preferably Melanocarpus albomyces; Myceliophtora, preferably
Myceliophtora thermophila; Coprinus, preferably Coprinus cinereus;
Rhizoctonia, preferably Rhizoctonia solani or Rhizoctonia
praticola; Scytalidium, preferably Scytalidium thermophilum;
Pyricularia, preferably Pyricularia oryzae.
[0111] In an embodiment the laccase is derived from the tree Rhus
vernicifera (Yoshida, 1983, Chemistry of Lacquer (Urushi) part 1.
J. Chem. Soc. 43: 472-486).
[0112] In another embodiment the laccase is derived from
Myceliopthora thermophila, e.g., the one described in WO 95/33836
(Novozymes).
[0113] In another embodiment the laccase is derived from Polyporus
pinsitus, e.g., the one described in WO 96/00290 (Novozymes).
[0114] Jonsson et al., 1998, Appl. Microbiol. Biotechnol. 49:
691-697, also discloses a suitable laccase derived from Polyporus
versicolar.
[0115] Other laccases include the one derived from Pyricularia
oryzae concerned in, e.g., Muralikrishna et al., 1995, Appl.
Environ. Microbiol. 61(12): 4374-4377, or the laccase derived from
Scytalidium thermophilum, which is disclosed in Abstract of Papers
American Chemical Society vol. 209, no. 1-2, 1995.
[0116] The laccase may also be one derived from Coprinus cinereus,
e.g., the one concerned in Schneider et al., 1999, Enzyme and
Microbial Technology 25: 502-508.
[0117] Other suitable laccases include those derived from
Rhizoctonia solani concerned in Waleithner et al., Curr. Genet.,
1996, 29: 395-403, or derived from Melanocarpus albomyces concerned
in Kiiskinen et al., 2004, Microbiology 150: 3065-3074.
[0118] Suitable bacterial laccase include those derived from
Streptomyces coelicolor, e.g., disclosed by Machczynski et al. in
Protein Science, 2004, 13: 2388-2397.
Enzymes Exhibiting Peroxidase Activity
[0119] According to the invention any enzyme exhibiting peroxidase
activity may be used.
[0120] The enzyme exhibiting peroxidase activity may be selected
from the group consisting of a peroxidase (EC 1.11.1.7),
haloperoxidase (EC1.11.1.8 and EC 1.11.1.10), lignin perocidase (EC
1.11.1.14), manganese peroxidase (EC 1.11.1.13); and lipoxygenase
(EC.1.13.11.12).
Peroxidase
[0121] The enzyme exhibiting peroxidase activity may be any
peroxidase classified as EC 1.11.1.7.
[0122] Peroxidases suitable in processes of the invention may be of
plant (e.g., horseradish or soybean peroxidase), or microbial
origin, such as of fungal or bacteria origin. Examples include
peroxidases derived from fungi of the subdivision Deuteromycotina,
class Hyphomycetes, e.g., Fusarium, Humicola, Tricoderma,
Myrothecium, Verticillum, Arthromyces, Caldariomyces, Ulocladium,
Embellisia, Cladosporium or Dreschlera, in particular Fusarium
oxysporum (DSM 2672), Humicola insolens, Trichoderma reesii,
Myrothecium verrucana (IFO 6113), Verticillum alboatrum,
Verticillum dahlia, Arthromyces ramosus (FERM P-7754),
Caldariomyces fumago, Ulocladium chartarum, Embellisia alli or
Dreschlera halodes.
[0123] Other suitable peroxidases are derived from fungi including
strains of the subdivision Basidiomycotina, class Basidiomycetes,
e.g., Coprinus, Phanerochaete, Coriolus or Trametes, in particular
Coprinus cinereus f. microsporus (IFO 8371), Coprinus macrorhizus,
Phanerochaete chrysosporium (e.g., NA-12) or Trametes (previously
called Polyporus), e.g., T. versicolor (e.g., PR4 28-A).
[0124] Other peroxidases may be derived from fungi including
strains belonging to the subdivision Zygomycotina, class
Mycoraceae, e.g., Rhizopus or Mucor, in particular Mucor
hiemalis.
[0125] Bacterial peroxidases may be derived from strains of the
order Actinomycetales, e.g., Streptomyces spheroides (ATTC 23965),
Streptomyces thermoviolaceus (IFO 12382) or Streptoverticillum
verticillium ssp. Verticillium; Bacillus pumilus (ATCC 12905),
Bacillus stearothermophilus, Rhodobacter sphaeroides, Rhodomonas
palustri, Streptococcus lactis, Pseudomonas purrocinia (ATCC 15958)
or Pseudomonas fluorescens (NRRL B-11); Myxococcus, e.g., M.
virescens.
[0126] Recombinantly produced peroxidases derived from Coprinus
sp., in particular C. macrorhizus or C. cinereus are described in
WO 92/16634. Variants thereof are described in WO 94/12621.
Haloperoxidase
[0127] The enzyme exhibiting peroxidase activity may be any
haloperoxidase. Haloperoxidases are widespread in nature and are
known to be produced by mammals, plants, algae, lichen, bacteria,
and fungi. There are three types of haloperoxidases, classified
according to their specificity for halide ions: Chloroperoxidases
(E.C. 1.11.1.10) which catalyze the chlorination, bromination and
iodination of compounds; bromoperoxidases which show specificity
for bromide and iodide ions; and iodoperoxidases (E.C. 1.11.1.8)
which solely catalyze the oxidation of iodide ions.
[0128] Haloperoxidases include the haloperoxidase from Curvularia,
in particular, C. verruculosa, such as, C. verruculosa CBS 147.63
or C. verruculosa CBS 444.70. Curvularia haloperoxidase and
recombinant production hereof is described in WO97/04102.
[0129] Bromide peroxidase has been isolated from algae (see U.S.
Pat. No. 4,937,192). Haloperoxidases are also described in U.S.
Pat. No. 6,372,465 (Novozymes A/S).
[0130] In a preferred embodiment, the haloperoxidase is a
chloroperoxidase (E.C.1.11.1.10). Chloroperoxidases are known in
the art and may be obtained from Streptomyces aureofaciens,
Streptomyces lividans, Pseudomonas fluorescens, Caldariomyces
fumago, Curvularia inaequalis, and Corallina officinalis. A
preferred chloroperoxidase is the chloroperoxidase from
Caldariomyces fumago (available from SIGMA, C-0278).
[0131] Haloperoxidases containing a vanadium prosthetic group are
known to include at least two types of fungal chloroperoxidases
from Curvularia inaequalis (van Schijndel et al., 1993, Biochimica
Biophysica Acta 1161:249-256; Simons et al., 1995, European Journal
of Biochemistry 229: 566-574; WO 95/27046) and Curvularia
verruculosa (WO 97/04102) or Phaeotrichoconis crotalariae
haloperoxidase (WO 2001/079461).
Lipoxygenase (LOX)
[0132] The enzyme exhibiting peroxidase activity may be any
lipoxygenase (LOX). Lipoxygenases are classified as EC 1.13.11.12,
which is an enzyme that catalyzes the oxygenation of
polyunsaturated fatty acids, especially cis,cis-1,4-dienes, e.g.,
linoleic acid and produces a hydroperoxide. But also other
substrates may be oxidized, e.g., monounsaturated fatty acids.
Microbial lipoxygenases may be derived from, e.g., Saccharomyces
cerevisiae, Thermoactinomyces vulgaris, Fusarium oxysporum,
Fusarium proliferatum, Thermomyces lanuginosus, Pyricularia oryzae,
and strains of Geotrichum. The preparation of a lipoxygenase
derived from Gaeumannomyces graminis is described in Examples 3-4
of WO 02/20730. The expression in Aspergillus oryzae of a
lipoxygenase derived from Magnaporthe salvinii is described in
Example 2 of WO 02/086114, and this enzyme can be purified using
standard methods, e.g., as described in Example 4 of WO
02/20730.
[0133] Lipoxygenase (LOX) may also be extracted from plant seeds,
such as soybean, pea, chickpea, and kidney bean. Alternatively,
lipoxygenase may be obtained from mammalian cells, e.g., rabbit
reticulocytes.
Cellulases or Cellulolytic Enzymes
[0134] The term "cellulases" or "cellulolytic enzymes" as used
herein are understood as comprising the cellobiohydrolases (EC
3.2.1.91), e.g., cellobiohydrolase I and cellobiohydrolase II, as
well as the endo-glucanases (EC 3.2.1.4), and beta-glucosidases (EC
3.2.1.21).
[0135] In order to be efficient, the digestion of cellulose and
hemicellulose requires several types of enzymes acting
cooperatively. At least three categories of enzymes are important
to convert cellulose into fermentable sugars: endo-glucanases (EC
3.2.1.4) cut cellulose chains at random; cellobiohydrolases (EC
3.2.1.91) cleave cellobiosyl units from the cellulose chain ends
and beta-glucosidases (EC 3.2.1.21) convert cellobiose and soluble
cellodextrins into glucose. Among these three categories of enzymes
involved in the biodegradation of cellulose, cellobiohydrolases are
the key enzymes for the degradation of native crystalline
cellulose. The term "cellobiohydrolase I" is defined herein as a
cellulose 1,4-beta-cellobiosidase (also referred to as
exo-glucanase, exo-cellobiohydrolase or 1,4-beta-cellobiohydrolase)
activity, as defined in the enzyme class EC 3.2.1.91, which
catalyzes the hydrolysis of 1,4-beta-D-glucosidic linkages in
cellulose and cellotetraose, by the release of cellobiose from the
non-reducing ends of the chains. The definition of the term
"cellobiohydrolase II activity" is identical, except that
cellobiohydrolase II attacks from the reducing ends of the
chains.
[0136] Endoglucanases (EC No. 3.2.1.4) catalyze endo hydrolysis of
1,4-beta-D-glycosidic linkages in cellulose, cellulose derivatives
(such as carboxy methyl cellulose and hydroxy ethyl cellulose),
lichenin, beta-1,4 bonds in mixed beta-1,3 glucans such as cereal
beta-D-glucans or xyloglucans and other plant material containing
cellulosic parts. The authorized name is endo-1,4-beta-D-glucan
4-glucano hydrolase, but the abbreviated term endoglucanase is used
in the present specification.
[0137] The cellulases or cellulolytic enzymes may comprise a
carbohydrate-binding module (CBM) which enhances the binding of the
enzyme to a cellulose-containing fiber and increases the efficacy
of the catalytic active part of the enzyme. A CBM is defined as
contiguous amino acid sequence within a carbohydrate-active enzyme
with a discreet fold having carbohydrate-binding activity. For
further information on CBMs see, e.g., the CAZy internet server
(Supra) or Tomme et al., 1995, in Enzymatic Degradation of
Insoluble Polysaccharides (Saddler & Penner, eds.),
Cellulose-binding domains: classification and properties. pp.
142-163, American Chemical Society, Washington.
[0138] The cellulase activity may, in a preferred embodiment, be
derived from a fungal source, such as a strain of the genus
Trichoderma, preferably a strain of Trichoderma reesei; a strain of
the genus Humicola, such as a strain of Humicola insolens; or a
strain of Chrysosporium, preferably a strain of Chrysosporium
lucknowense.
[0139] In a preferred embodiment cellulase or cellulolytic enzyme
preparation is a composition concerned in co-pending application
U.S. provisional application No. 60/941,251, which is hereby
incorporated by reference. In a preferred embodiment the cellulase
or cellulolytic enzyme preparation comprising a polypeptide having
cellulolytic enhancing activity, preferably a family GH61A
polypeptide, preferably one disclosed in WO 2005/074656
(Novozymes). The cellulolytic enzyme preparation may further
comprise a beta-glucosidase, such as a beta-glucosidase derived
from a strain of the genus Trichoderma, Aspergillus, or
Penicillium, including the fusion protein having beta-glucosidase
activity disclosed in U.S. provisional application No. 60/832,511
(PCT/US2007/074038) (Novozymes). In a preferred embodiment the
cellulolytic enzyme preparation may also comprises a CBH II enzyme,
preferably Thielavia terrestris cellobiohydrolase II (CEL6A). In
another preferred embodiment the cellulolytic enzyme preparation
may also comprise cellulolytic enzymes, preferably one derived from
Trichoderma reesei or Humicola insolens.
[0140] In a specific embodiment the cellulolytic enzyme preparation
may also comprise a polypeptide having cellulolytic enhancing
activity (GH61A) disclosed in WO 2005/074656; a CBH II from
Thielavia terrestris cellobiohydrolase II (CEL6A); and a
beta-glucosidase (fusion protein disclosed in U.S. provisional
application No. 60/832,511 (or PCT/US2007/074038)), and
cellulolytic enzymes derived from Trichoderma reesei.
[0141] In another specific embodiment the cellulolytic enzyme
preparation may also comprise a polypeptide having cellulolytic
enhancing activity (GH61A) disclosed in WO 2005/074656; a
beta-glucosidase (fusion protein disclosed in U.S. provisional
application No. 60/832,511 (or PCT/US2007/074038)), and
cellulolytic enzymes derived from Trichoderma reesei.
[0142] In preferred embodiments the cellulase or cellulolytic
preparations are Cellulolytic preparations A and B used in Examples
1 and 4, respectively, disclosed in U.S. provisional application
No. 60/941,251.
[0143] In an embodiment the cellulase is the commercially available
product CELLUCLAST.RTM. 1.5 L or CELLUZYME.TM. (Novozymes A/S,
Denmark) or ACCELERASE.TM. 1000 (from Genencor Inc., USA).
[0144] A cellulase or cellulolytic enzyme may be added for
hydrolyzing the pre-treated lignocellulose-containing material. The
cellulase may be dosed in the range from 0.1-100 FPU per gram total
solids (TS), preferably 0.5-50 FPU per gram TS, especially 1-20 FPU
per gram TS.
Hemicellulases
[0145] Hemicellulose can be broken down by hemicellulases and/or
acid hydrolysis to release its five and six carbon sugar
components. In an embodiment of the invention the lignocellulose
derived material may be treated with one or more hemicellulase.
[0146] Any hemicellulase suitable for use in hydrolyzing
hemicellulose may be used. Preferred hemicellulases include
xylanases, arabinofuranosidases, acetyl xylan esterase, feruloyl
esterase, glucuronidases, endo-galactanase, mannases, endo or exo
arabinases, exo-galactanases, and mixtures of two or more thereof.
Preferably, the hemicellulase for use in the present invention is
an exo-acting hemicellulase, and more preferably, the hemicellulase
is an exo-acting hemicellulase which has the ability to hydrolyze
hemicellulose under acidic conditions of below pH 7, preferably pH
3-7. An example of hemicellulase suitable for use in the present
invention includes VISCOZYME.TM. (available from Novozymes A/S,
Denmark).
[0147] Arabinofuranosidase (EC 3.2.1.55) catalyzes the hydrolysis
of terminal non-reducing alpha-L-arabinofuranoside residues in
alpha-L-arabinosides.
[0148] Galactanase (EC 3.2.1.89), arabinogalactan
endo-1,4-beta-galactosidase, catalyzes the endohydrolysis of
1,4-D-galactosidic linkages in arabinogalactans.
[0149] Pectinase (EC 3.2.1.15) catalyzes the hydrolysis of
1,4-alpha-D-galactosiduronic linkages in pectate and other
galacturonans.
[0150] Xyloglucanase catalyzes the hydrolysis of xyloglucan.
[0151] The hemicellulase may be added in an amount effective to
hydrolyze hemicellulose, such as, in amounts from about 0.001 to
0.5 wt. % of total solids (TS), more preferably from about 0.05 to
0.5 wt. % of TS.
Alpha-Amylase
[0152] According to the invention an alpha-amylase may be used. In
a preferred embodiment the alpha-amylase is an acid alpha-amylase,
e.g., fungal acid alpha-amylase or bacterial acid alpha-amylase.
The term "acid alpha-amylase" means an alpha-amylase (E.C. 3.2.1.1)
which added in an effective amount has activity optimum at a pH in
the range of 3 to 7, preferably from 3.5 to 6, or more preferably
from 4-5.
Bacterial Alpha-Amylase
[0153] According to the invention the bacterial alpha-amylase is
preferably derived from the genus Bacillus.
[0154] In a preferred embodiment the Bacillus alpha-amylase is
derived from a strain of B. licheniformis, B. amyloliquefaciens, B.
subtilis or B. stearothermophilus, but may also be derived from
other Bacillus sp. Specific examples of contemplated alpha-amylases
include the Bacillus licheniformis alpha-amylase shown in SEQ ID
NO: 4 in WO 99/19467, the Bacillus amyloliquefaciens alpha-amylase
SEQ ID NO: 5 in WO 99/19467 and the Bacillus stearothermophilus
alpha-amylase shown in SEQ ID NO: 3 in WO 99/19467 (all sequences
hereby incorporated by reference). In an embodiment of the
invention the alpha-amylase may be an enzyme having a degree of
identity of at least 60%, preferably at least 70%, more preferred
at least 80%, even more preferred at least 90%, such as at least
95%, at least 96%, at least 97%, at least 98% or at least 99% to
any of the sequences shown in SEQ ID NOS: 1, 2 or 3, respectively,
in WO 99/19467.
[0155] The Bacillus alpha-amylase may also be a variant and/or
hybrid, especially one described in any of WO 96/23873, WO
96/23874, WO 97/41213, WO 99/19467, WO 00/60059, and WO 02/10355
(all documents hereby incorporated by reference). Specifically
contemplated alpha-amylase variants are disclosed in U.S. Pat. No.
6,093,562, 6,297,038 or 6,187,576 (hereby incorporated by
reference) and include Bacillus stearothermophilus alpha-amylase
(BSG alpha-amylase) variants having a deletion of one or two amino
acid in positions R179 to G182, preferably a double deletion
disclosed in WO 1996/023873--see e.g., page 20, lines 1-10 (hereby
incorporated by reference), preferably corresponding to
delta(181-182) compared to the wild-type BSG alpha-amylase amino
acid sequence set forth in SEQ ID NO:3 disclosed in WO 99/19467 or
deletion of amino acids R179 and G180 using SEQ ID NO:3 in WO
99/19467 for numbering (which reference is hereby incorporated by
reference). Even more preferred are Bacillus alpha-amylases,
especially Bacillus stearothermophilus alpha-amylase, which have a
double deletion corresponding to delta(181-182) and further
comprise a N193F substitution (also denoted 1181*+G182*+N193F)
compared to the wild-type BSG alpha-amylase amino acid sequence set
forth in SEQ ID NO:3 disclosed in WO 99/19467.
Bacterial Hybrid Alpha-Amylase
[0156] A hybrid alpha-amylase specifically contemplated comprises
445 C-terminal amino acid residues of the Bacillus licheniformis
alpha-amylase (shown in SEQ ID NO: 4 of WO 99/19467) and the 37
N-terminal amino acid residues of the alpha-amylase derived from
Bacillus amyloliquefaciens (shown in SEQ ID NO: 5 of WO 99/19467),
with one or more, especially all, of the following substitution:
G48A+T49I+G107A+H156Y+A181T+N190F+I201F+A209V+Q264S (using the
Bacillus licheniformis numbering in SEQ ID NO: 4 of WO 99/19467).
Also preferred are variants having one or more of the following
mutations (or corresponding mutations in other Bacillus
alpha-amylase backbones): H154Y, A181T, N190F, A209V and Q264S
and/or deletion of two residues between positions 176 and 179,
preferably deletion of E178 and G179 (using the SEQ ID NO: 5
numbering of WO 99/19467).
Fungal Alpha-Amylase
[0157] Fungal alpha-amylases include alpha-amylases derived from a
strain of the genus Aspergillus, such as, Aspergillus oryzae,
Aspergillus niger and Aspergillis kawachii alpha-amylases.
[0158] A preferred acidic fungal alpha-amylase is a Fungamyl-like
alpha-amylase which is derived from a strain of Aspergillus oryzae.
According to the present invention, the term "Fungamyl-like
alpha-amylase" indicates an alpha-amylase which exhibits a high
identity, i.e., more than 70%, more than 75%, more than 80%, more
than 85% more than 90%, more than 95%, more than 96%, more than
97%, more than 98%, more than 99% or even 100% identity to the
mature part of the amino acid sequence shown in SEQ ID NO: 10 in WO
96/23874.
[0159] Another preferred acidic alpha-amylase is derived from a
strain Aspergillus niger. In a preferred embodiment the acid fungal
alpha-amylase is the one from A. niger disclosed as "AMYA_ASPNG" in
the Swiss-prot/TeEMBL database under the primary accession no.
P56271 and described in WO 89/01969 (Example 3). A commercially
available acid fungal alpha-amylase derived from Aspergillus niger
is SP288 (available from Novozymes A/S, Denmark).
[0160] Other contemplated wild-type alpha-amylases include those
derived from a strain of the genera Rhizomucor and Meripilus,
preferably a strain of Rhizomucor pusillus (WO 2004/055178
incorporated by reference) or Meripilus giganteus.
[0161] In a preferred embodiment the alpha-amylase is derived from
Aspergillus kawachii and disclosed by Kaneko et al., 1996, J.
Ferment. Bioeng. 81: 292-298, "Molecular-cloning and determination
of the nucleotide-sequence of a gene encoding an acid-stable
alpha-amylase from Aspergillus kawachii; and further as EMBL:
#AB008370.
[0162] The fungal alpha-amylase may also be a wild-type enzyme
comprising a starch-binding domain (SBD) and an alpha-amylase
catalytic domain (i.e., non-hybrid), or a variant thereof. In an
embodiment the wild-type alpha-amylase is derived from a strain of
Aspergillus kawachii.
Fungal Hybrid Alpha-Amylase
[0163] In a preferred embodiment the fungal acid alpha-amylase is a
hybrid alpha-amylase. Preferred examples of fungal hybrid
alpha-amylases include the ones disclosed in WO 2005/003311 or U.S.
Publication No. 2005/0054071 (Novozymes) or U.S. provisional
application No. 60/638,614 (Novozymes) which is hereby incorporated
by reference. A hybrid alpha-amylase may comprise an alpha-amylase
catalytic domain (CD) and a carbohydrate-binding domain/module
(CBM), such as a starch binding domain, and optional a linker.
[0164] Specific examples of contemplated hybrid alpha-amylases
include those disclosed in Table 1 to 5 of the examples in U.S.
provisional application No. 60/638,614, including Fungamyl variant
with catalytic domain JA118 and Athelia rolfsii SBD (SEQ ID NO:100
in U.S. provisional application No. 60/638,614), Rhizomucor
pusillus alpha-amylase with Athelia rolfsii AMG linker and SBD (SEQ
ID NO:101 in U.S. provisional application No. 60/638,614),
Rhizomucor pusillus alpha-amylase with Aspergillus niger
glucoamylase linker and SBD (which is disclosed in Table 5 as a
combination of amino acid sequences SEQ ID NO:20, SEQ ID NO:72 and
SEQ ID NO:96 in U.S. application Ser. No. 11/316,535 and further as
SEQ ID NO: 13 herein) or as V039 in Table 5 in WO 2006/069290, and
Meripilus giganteus alpha-amylase with Athelia rolfsii glucoamylase
linker and SBD (SEQ ID NO: 102 in U.S. provisional application No.
60/638,614). Other specifically contemplated hybrid alpha-amylases
are any of the ones listed in Tables 3, 4, 5, and 6 in Example 4 in
U.S. application Ser. No. 11/316,535 and WO 2006/069290 (hereby
incorporated by reference).
[0165] Other specific examples of contemplated hybrid
alpha-amylases include those disclosed in U.S. Publication no.
2005/0054071, including those disclosed in Table 3 on page 15, such
as Aspergillus niger alpha-amylase with Aspergillus kawachii linker
and starch binding domain.
[0166] Contemplated are also alpha-amylases which exhibit a high
identity to any of above mention alpha-amylases, i.e., more than
70%, more than 75%, more than 80%, more than 85% more than 90%,
more than 95%, more than 96%, more than 97%, more than 98%, more
than 99% or even 100% identity to the mature enzyme sequences.
[0167] An acid alpha-amylases may according to the invention be
added in an amount of 0.1 to 10 AFAU/g DS, preferably 0.10 to 5
AFAU/g DS, especially 0.3 to 2 AFAU/g DS.
Commercial Alpha-Amylase Products
[0168] Preferred commercial compositions comprising alpha-amylase
include MYCOLASE from DSM, BAN.TM., TERMAMYL.TM. SC, FUNGAMYL.TM.,
LIQUOZYME.TM. X and SAN.TM. SUPER, SAN.TM. EXTRA L (Novozymes A/S)
and CLARASE.TM. L-40,000, DEX-LO.TM., SPEZYME.TM. FRED, SPEZYME.TM.
AA, and SPEZYME.TM. DELTA AA (Genencor Int.), and the acid fungal
alpha-amylase sold under the trade name SP288 (available from
Novozymes A/S, Denmark).
Carbohydrate-Source Generating Enzyme
[0169] The term "carbohydrate-source generating enzyme" includes
glucoamylase (being glucose generators), beta-amylase and
maltogenic amylase (being maltose generators). A
carbohydrate-source generating enzyme is capable of producing a
carbohydrate that can be used as an energy-source by the fermenting
organism(s) in question, for instance, when used in a process of
the invention for producing a fermentation product, such as
ethanol. The generated carbohydrate may be converted directly or
indirectly to the desired fermentation product, preferably ethanol.
According to the invention a mixture of carbohydrate-source
generating enzymes may be used. Especially contemplated mixtures
are mixtures of at least a glucoamylase and an alpha-amylase,
especially an acid amylase, even more preferred an acid fungal
alpha-amylase. The ratio between acidic fungal alpha-amylase
activity (AFAU) per glucoamylase activity (AGU) (AFAU per AGU) may
in an embodiment of the invention be at least 0.1, in particular at
least 0.16, such as in the range from 0.12 to 0.50 or more.
Alternatively the ratio between acid fungal alpha-amylase activity
(FAU-F) and glucoamylase activity (AGU) (i.e., FAU-F per AGU) may
in an embodiment of the invention be between 0.1 and 100 AGU/FAU-F,
in particular between 2 and 50 AGU/FAU-F, such as in the range from
10-40 AGU/FAU-F.
Glucoamylase
[0170] A glucoamylase used according to the invention may be
derived from any suitable source, e.g., derived from a
microorganism or a plant. Preferred glucoamylases are 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): 1097-1102), or variants thereof, such
as those disclosed in WO 92/00381, WO 00/04136 and WO 01/04273
(from Novozymes, Denmark); the A. awamori glucoamylase disclosed in
WO 84/02921, A. oryzae glucoamylase (Agric. Biol. Chem., 1991,
55(4): 941-949), or variants or fragments thereof. Other
Aspergillus glucoamylase variants include variants with enhanced
thermal stability: G137A and G139A (Chen et al., 1996, Prot. Eng.
9: 499-505); D257E and D293E/Q (Chen et al., 1995, Prot. Eng. 8:
575-582); N182 (Chen et al., 1994, Biochem. J. 301: 275-281);
disulphide bonds, A246C (Fierobe et al., 1996, Biochemistry 35:
8698-8704; and introduction of Pro residues in position A435 and
S436 (Li et al., 1997, Protein Eng. 10: 1199-1204.
[0171] Other glucoamylases include Athelia rolfsii (previously
denoted Corticium rolfsi) glucoamylase (see U.S. Pat. No. 4,727,026
and Nagasaka et al., 1998, "Purification and properties of the
raw-starch-degrading glucoamylases from Corticium rolfsii, Appl
Microbiol Biotechnol 50: 323-330), Talaromyces glucoamylases, in
particular derived from Talaromyces emersonii (WO 99/28448),
Talaromyces leycettanus (U.S. Pat. No. Re. 32,153), Talaromyces
duponti, Talaromyces thermophilus (U.S. Pat. No. 4,587,215).
[0172] Bacterial glucoamylases contemplated include glucoamylases
from the genus Clostridium, in particular C. thermoamylolyticum (EP
135,138), and C. thermohydrosulfuricum (WO 86/01831) and Trametes
cingulata disclosed in WO 2006/069289 (which is hereby incorporated
by reference).
[0173] Also hybrid glucoamylase are contemplated according to the
invention. Examples the hybrid glucoamylases disclosed in WO
2005/045018. Specific examples include the hybrid glucoamylase
disclosed in Table 1 and 4 of Example 1 (which hybrids are hereby
incorporated by reference.).
[0174] Contemplated are also glucoamylases which exhibit a high
identity to any of above mention glucoamylases, i.e., more than
70%, more than 75%, more than 80%, more than 85% more than 90%,
more than 95%, more than 96%, more than 97%, more than 98%, more
than 99% or even 100% identity to the mature enzymes sequences.
[0175] Commercially available compositions comprising glucoamylase
include AMG 200L; AMG 300 L; SAN.TM. SUPER, SAN.TM. EXTRA L,
SPIRIZYME.TM. PLUS, SPIRIZYME.TM. FUEL, SPIRIZYME.TM. B4U and
AMG.TM. E (from Novozymes A/S); OPTIDEX.TM. 300 (from Genencor
Int.); AMIGASE.TM. and AMIGASE.TM. PLUS (from DSM); G-ZYME.TM.
G900, G-ZYME.TM. and G990 ZR (from Genencor Int.).
[0176] Glucoamylases may in an embodiment be added in an amount of
0.02-20 AGU/g DS, preferably 0.1-10 AGU/g DS, especially between
1-5 AGU/g DS, such as 0.5 AGU/g DS.
Beta-Amylase
[0177] At least according to the invention beta-amylase (E.C
3.2.1.2) is the name traditionally given to exo-acting maltogenic
amylases, which catalyze the hydrolysis of 1,4-alpha-glucosidic
linkages in amylose, amylopectin and related glucose polymers.
Maltose units are successively removed from the non-reducing chain
ends in a step-wise manner until the molecule is degraded or, in
the case of amylopectin, until a branch point is reached. The
maltose released has the beta anomeric configuration, hence the
name beta-amylase.
[0178] Beta-amylases have been isolated from various plants and
microorganisms (Fogarty and Kelly, 1979, Progress in Industrial
Microbiology 15: 112-115). 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. A
commercially available beta-amylase from barley is NOVOZYM.TM. WBA
from Novozymes A/S, Denmark and SPEZYME.TM. BBA 1500 from Genencor
Int., USA.
Maltogenic Amylase
[0179] The amylase may also be a maltogenic alpha-amylase. A
"maltogenic alpha-amylase" (glucan 1,4-alpha-maltohydrolase, E.C.
3.2.1.133) is able to hydrolyze amylose and amylopectin to maltose
in the alpha-configuration. A maltogenic amylase from Bacillus
stearothermophilus strain NCIB 11837 is commercially available from
Novozymes A/S. Maltogenic alpha-amylases are described in U.S. Pat.
Nos. 4,598,048, 4,604,355 and 6,162,628, which are hereby
incorporated by reference.
[0180] The maltogenic amylase may in a preferred embodiment be
added in an amount of 0.05-5 mg total protein/gram DS or 0.05-5
MANU/g DS.
Proteases
[0181] The protease may according to the invention be any protease.
In a preferred embodiment the protease is an acid protease of
microbial origin, preferably of fungal or bacterial origin.
[0182] 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.
[0183] Contemplated acid fungal proteases include fungal proteases
derived from Aspergillus, Mucor, Rhizopus, Candida, Coriolus,
Endothia, Enthomophtra, Irpex, Penicillium, Scierotiumand, and
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.
[0184] 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. Also contemplated are the proteases having at
least 90% identity to amino acid sequence obtainable at Swissprot
as Accession No. P06832 such as at least 92%, at least 95%, at
least 96%, at least 97%, at least 98%, or particularly at least 99%
identity.
[0185] Further contemplated are the proteases having at least 90%
identity to amino acid sequence disclosed as SEQ.ID.NO:1 in the WO
2003/048353 such as at 92%, at least 95%, at least 96%, at least
97%, at least 98%, or particularly at least 99% identity.
[0186] 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).
[0187] Proteases may be added in the amounts of 0.1-1000 AU/kg dm,
preferably 1-100 AU/kg DS and most preferably 5-25 AU/kg DS.
Use
[0188] In the third aspect the invention relates to the use of one
or more phenolic compound oxidizing enzymes and/or enzymes
exhibiting peroxidase activity for detoxifying pre-treated
lignocellulose-containing material.
[0189] In a preferably embodiment the phenolic compound oxidizing
enzyme may be selected from the group comprising catechol oxidase
(EC 1.10.3.1), laccase (EC 1.10.3.2), o-aminophenol oxidase (EC
1.10.3.4); and monophenol monooxygenase (EC 1.14.18.1) for
detoxifying pre-treated lignocellulose-containing material.
[0190] In another preferred embodiment the enzyme exhibiting
peroxidase activity may be selected from the group comprising
peroxidase (EC 1.11.1.7), haloperoxidase (EC1.11.1.8 and EC
1.11.1.10); lignin peroxidase (EC 1.11.1.14); manganese peroxidase
(EC 1.11.1.13); and lipoxygenase (EC.1.13.11.12) for detoxifying
pre-treated lignocellulose-containing material.
[0191] The detoxification may be part of a fermentation product
production process of the invention.
Materials & Methods
Enzymes:
Laccase PpL:
[0192] Laccase derived from Polyporus pinsitus disclosed in WO
1996/000290 (Novozymes).
Laccase MtL:
[0193] Laccase derived from Myceliopthora thermophila disclosed in
WO 1995/033836 (Novozymes).
Laccase CcL:
[0194] Laccase derived from Coprinus cinereus disclosed in WO
97/08325 (Novozymes)
Peroxidase CcP:
[0195] Peroxidase is derived from Coprinus cinereus disclosed in
Petersen et al., 1994, FEBS Letters 339: 291-296.
Cellulolutic Preparation A:
[0196] Cellulolytic composition comprising a polypeptide having
cellulolytic enhancing activity (GH61A) disclosed in WO
2005/074656; a beta-glucosidase (fusion protein disclosed in U.S.
provisional application No. 60/832,511), Thielavia terrestris
cellobiohydrolase II (CEL6A), and cellulolytic enzymes preparation
derived from Trichoderma reesei. Cellulase preparation A is
disclosed in U.S. provisional application No. 60/941,251.
Cellulolytic Preparation B:
[0197] Cellulolytic composition comprising a polypeptide having
cellulolytic enhancing activity (GH61A) disclosed in WO
2005/074656; a beta-glucosidase (fusion protein disclosed in U.S.
provisional application No. 60/832,511); and cellulolytic enzymes
preparation derived from Trichoderma reesei. Cellulase preparation
A is disclosed in U.S. provisional application No. 60/941,251.
Yeast:
[0198] RED STAR.TM. available from Red Star/Lesaffre, USA
Pre-Treated Corn Stover:
[0199] dilute acid-catalyzed steam explosion corn stover (28.6% DS)
was obtained from NREL (National Renewable Research Laboratory,
USA).
Determination of Identity
[0200] The relatedness between two amino acid sequences or between
two nucleotide sequences is described by the parameter
"identity".
[0201] The degree of identity between two amino acid sequences may
be determined by the Clustal method (Higgins, 1989, CABIOS 5:
151-153) using the LASERGENE.TM. MEGALIGN.TM. software (DNASTAR,
Inc., Madison, Wis.) with an identity table and the following
multiple alignment parameters: Gap penalty of 10 and gap length
penalty of 10. Pairwise alignment parameters are Ktuple=1, gap
penalty=3, windows=5, and diagonals=5.
[0202] The degree of identity between two nucleotide sequences may
be determined by the Wilbur-Lipman method (Wilbur and Lipman, 1983,
Proceedings of the National Academy of Science USA 80: 726-730)
using the LASERGENE.TM. MEGALIGN.TM. software (DNASTAR, Inc.,
Madison, Wis.) with an identity table and the following multiple
alignment parameters: Gap penalty of 10 and gap length penalty of
10. Pairwise alignment parameters are Ktuple=3, gap penalty=3, and
windows=20.
Determination of Laccase Activity (LACU)
[0203] Laccase activity is determined from the oxidation of
syringaldazin under aerobic conditions. The violet color produced
is photometered at 530 nm. The analytical conditions are 19 mM
syringaldazin, 23.2 mM acetate buffer, pH 5.5, 30.degree. C., 1
min. reaction time.
[0204] 1 laccase unit (LACU) is the amount of enzyme that catalyzes
the conversion of 1.0 micromole syringaldazin per minute at these
conditions.
Determination of Peroxidase Activity (PODU)
[0205] One peroxidase unit (PODU) is defined as the amount of
enzyme which, under standard conditions (i.e., pH 7.0; temperature
30.degree. C.; reaction time 3 minutes) catalyzes the conversion of
1 micromole hydrogen peroxide per minute. The activity is
determined using an assay based on ABTS.RTM.
(2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonate)) as the
chromophore, the greenish-blue colour produced being photometered
at 418 nm. A folder AF 279/2 describing this analytical method in
more detail is available upon request to Novozymes A/S, Denmark,
which folder is hereby included by reference.
Glucoamylase Activity
[0206] Glucoamylase activity may be measured in Glucoamylase Units
(AGU).
Glucoamylase Activity (AGU)
[0207] 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.
[0208] 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.
TABLE-US-00001 AMG incubation: Substrate: maltose 23.2 mM Buffer:
acetate 0.1M pH: 4.30 .+-. 0.05 Incubation temperature: 37.degree.
C. .+-. 1 Reaction time: 5 minutes Enzyme working range: 0.5-4.0
AGU/mL
TABLE-US-00002 Color reaction: GlucDH: 430 U/L Mutarotase: 9 U/L
NAD: 0.21 mM Buffer: phosphate 0.12M; 0.15M NaCl pH: 7.60 .+-. 0.05
Incubation temperature: 37.degree. C. .+-. 1 Reaction time: 5
minutes Wavelength: 340 nm
[0209] 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.
Alpha-Amylase Activity (KNU)
[0210] The alpha-amylase 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.
[0211] 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.
[0212] 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.
Acid Alpha-Amylase Activity
[0213] When used according to the present invention the activity of
an acid alpha-amylase may be measured in AFAU (Acid Fungal
Alpha-amylase Units) or FAU-F (Fungal Alpha-Amylase Units).
Acid Alpha-Amylase Activity (AFAU)
[0214] Acid alpha-amylase activity may be measured in AFAU (Acid
Fungal Alpha-amylase Units), which are determined relative to an
enzyme standard. 1 AFAU is defined as the amount of enzyme which
degrades 5.260 mg starch dry matter per hour under the below
mentioned standard conditions.
[0215] Acid alpha-amylase, an endo-alpha-amylase
(1,4-alpha-D-glucan-glucanohydrolase, 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.
##STR00001##
[0216] Standard Conditions/Reaction Conditions: [0217] Substrate:
Soluble starch, approx. 0.17 g/L [0218] Buffer: Citrate, approx.
0.03 M [0219] Iodine (12): 0.03 g/L [0220] CaC.sub.2: 1.85 mM
[0221] pH: 2.50.+-.0.05 [0222] Incubation temperature: 40.degree.
C. [0223] Reaction time: 23 seconds [0224] Wavelength: 590 nm
[0225] Enzyme concentration: 0.025 AFAU/mL [0226] Enzyme working
range: 0.01-0.04 AFAU/mL
[0227] 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.
Determination of FAU-F
[0228] FAU-F Fungal Alpha-Amylase Units (Fungamyl) is measured
relative to an enzyme standard of a declared strength.
TABLE-US-00003 Reaction conditions Temperature 37.degree. C. pH
7.15 Wavelength 405 nm Reaction time 5 min Measuring time 2 min
[0229] A folder (EB-SM-0216.02) describing this standard method in
more detail is available on request from Novozymes A/S, Denmark,
which folder is hereby included by reference.
Measurement of Cellulase Activity Using Filter Paper Assay (FPU
Assay)
1. Source of Method
[0230] 1.1 The method is disclosed in a document entitled
"Measurement of Cellulase Activities" by Adney and Baker, 1996,
Laboratory Analytical Procedure, LAP-006, National Renewable Energy
Laboratory (NREL). It is based on the IUPAC method for measuring
cellulase activity (Ghose, 1987, Measurement of Cellulase
Activities, Pure & Appl. Chem. 59: 257-268.
2. Procedure
[0231] 2.1 The method is carried out as described by Adney and
Baker, 1996, supra, except for the use of a 96 well plates to read
the absorbance values after color development, as described
below.
2.2 Enzyme Assay Tubes:
[0232] 2.2.1 A rolled filter paper strip (#1 Whatman; 1.times.6 cm;
50 mg) is added to the bottom of a test tube (13.times.100 mm).
[0233] 2.2.2 To the tube is added 1.0 mL of 0.05 M Na-citrate
buffer (pH 4.80). [0234] 2.2.3 The tubes containing filter paper
and buffer are incubated 5 min. at 50.degree. C. (.+-.0.1.degree.
C.) in a circulating water bath. [0235] 2.2.4 Following incubation,
0.5 mL of enzyme dilution in citrate buffer is added to the tube.
[0236] Enzyme dilutions are designed to produce values slightly
above and below the target value of 2.0 mg glucose. [0237] 2.2.5
The tube contents are mixed by gently vortexing for 3 seconds.
[0238] 2.2.6 After vortexing, the tubes are incubated for 60 mins.
at 50.degree. C. (.+-.0.1.degree. C.) in a circulating water bath.
[0239] 2.2.7 Immediately following the 60 min. incubation, the
tubes are removed from the water bath, and 3.0 mL of DNS reagent is
added to each tube to stop the reaction. The tubes are vortexed 3
seconds to mix.
2.3 Blank and Controls
[0239] [0240] 2.3.1 A reagent blank is prepared by adding 1.5 mL of
citrate buffer to a test tube. [0241] 2.3.2 A substrate control is
prepared by placing a rolled filter paper strip into the bottom of
a test tube, and adding 1.5 mL of citrate buffer. [0242] 2.3.3
Enzyme controls are prepared for each enzyme dilution by mixing 1.0
mL of citrate buffer with 0.5 mL of the appropriate enzyme
dilution. [0243] 2.3.4 The reagent blank, substrate control, and
enzyme controls are assayed in the same manner as the enzyme assay
tubes, and done along with them.
[0244] 2.4 Glucose Standards [0245] 2.4.1 A 100 mL stock solution
of glucose (10.0 mg/mL) is prepared, and 5 mL aliquots are frozen.
Prior to use, aliquots are thawed and vortexed to mix. [0246] 2.4.2
Dilutions of the stock solution are made in citrate buffer as
follows: G1=1.0 mL stock+0.5 mL buffer=6.7 mg/mL=3.3 mg/0.5 mL
G2=0.75 mL stock+0.75 mL buffer=5.0 mg/mL=2.5 mg/0.5 mL G3=0.5 mL
stock+1.0 mL buffer=3.3 mg/mL=1.7 mg/0.5 mL G4=0.2 mL stock+0.8 mL
buffer=2.0 mg/mL=1.0 mg/0.5 mL [0247] 2.4.3 Glucose standard tubes
are prepared by adding 0.5 mL of each dilution to 1.0 mL of citrate
buffer. [0248] 2.4.4 The glucose standard tubes are assayed in the
same manner as the enzyme assay tubes, and done along with
them.
[0249] 2.5 Color Development [0250] 2.5.1 Following the 60 min.
incubation and addition of DNS, the tubes are all boiled together
for 5 mins. in a water bath. [0251] 2.5.2 After boiling, they are
immediately cooled in an ice/water bath. [0252] 2.5.3 When cool,
the tubes are briefly vortexed, and the pulp is allowed to settle.
Then each tube is diluted by adding 50 microL from the tube to 200
microL of ddH.sub.2O in a 96-well plate. Each well is mixed, and
the absorbance is read at 540 nm.
[0253] 2.6 Calculations (Examples are Given in the NREL Document)
[0254] 2.6.1 A glucose standard curve is prepared by graphing
glucose concentration (mg/0.5 mL) for the four standards (G1-G4)
vs. A.sub.540. This is fitted using a linear regression (Prism
Software), and the equation for the line is used to determine the
glucose produced for each of the enzyme assay tubes. [0255] 2.6.2 A
plot of glucose produced (mg/0.5 mL) vs. total enzyme dilution is
prepared, with the Y-axis (enzyme dilution) being on a log scale.
[0256] 2.6.3 A line is drawn between the enzyme dilution that
produced just above 2.0 mg glucose and the dilution that produced
just below that. From this line, it is determined the enzyme
dilution that would have produced exactly 2.0 mg of glucose. [0257]
2.6.4 The Filter Paper Units/mL (FPU/mL) are calculated as follows:
FPU/mL=0.37/enzyme dilution producing 2.0 mg glucose
Protease Assay Method--AU(RH)
[0258] 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.
[0259] One Anson Unit (AU-RH) is defined as the amount of enzyme
which under standard conditions (i.e., 25.degree. C., pH 5.5 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.
[0260] The AU(RH) method is described in EAL-SM-0350 and is
available from Novozymes A/S Denmark on request.
EXAMPLES
Example 1
Effect of Laccase on Ethanol Yield During Enzymatic Hydrolysis
[0261] Dilute acid-catalyzed steam exploded pre-treated corn stover
was obtained from NREL. The pre-treated corn stover (15% DS) was
hydrolyzed at pH 4.5, 50.degree. C. for 75 hours with Cellulolytic
preparation A (5 FPU/g DS) with and without Laccase PpL (20-30
LACU/g DS). The enzymatic treatment was carried out with open lid
so that consistent air flow was provided during the treatment.
Evaporation was control by daily water supplement based on weight
loss. The enzyme treated samples were used for ethanol fermentation
at 32.degree. C. for up to 88 hours in a closed vessel where a
needle was punched in the cap, with yeast (RED STAR.TM.) at initial
dosage of 0.2 g/L. The fermentation mixture also contains YPU (5
g/L yeast extract, 5 g/L peptone and 10 g/L Urea). After 25 hours
fermentation, the laccase treated sample resulted in 10 g/L ethanol
production, whereas the non-laccase treated sample resulted in no
ethanol yield. With initial yeast dosage of 1.6 g/L, about 10-fold
higher ethanol production was observed with laccase treated sample
after 25 hours fermentation (18.87 g/L for laccase treated samples
vs 1.83 for non-laccase treated sample).
Example 2
Effect of Laccase on Ethanol Yield During Fermentation
[0262] The pre-treated corn stover (28.6% DS) used was the same as
in Example 1. The pre-treated corn stover (15% DS) was hydrolyzed
with Cellulolytic preparation A (5 FPU/g DS) at pH 4.5, 50.degree.
C. for 75 hours in the absence of laccase with covered lid. After
the enzymatic hydrolysis, material was fermented at 32.degree. C.
for up to 88 hours in a closed vessel where a needle was punched in
the cap, with yeast (RED STAR.TM.) at dosage of 1.6 g/L with and
without laccase PpL (20-30 LACU/g DS). The fermentation mixture
also contains YPU (5 g/L yeast extract, 5 g/L peptone and 10 g/L
Urea). After 25 hours, the ethanol production was doubled in the
sample where laccase was present during fermentation (4.59 g/L
laccase treated vs 1.83 non-laccase treated).
Example 3
Effect of Oxidoreductases Treatment in Between Hydrolysis and Yeast
Fermentation
[0263] The pre-treated corn stover (28.6% DS) used was the same as
in Example 1. Enzymatic hydrolysis was carried out with pre-treated
corn stover (15% DS) at pH 4.5, 50.degree. C. for 72 hours in the
absence of laccase with covered lid. Oxidoreductase treatment was
conducted at 50.degree. C. for one or two hours before fermentation
at 32.degree. C. (up to 48 hours) with yeast (RED STAR.TM.) at
dosage of 0.5 g/L in a closed vessel where a needle was punched in
the cap. During oxidoreductase treatment, lids were opened every 20
minutes. The fermentation mixture also contains YPU (5 g/L yeast
extract, 5 g/l peptone and 10 g/L Urea). Up to 40% increase in
ethanol production was observed with Laccase PpL (4.5-30 LACU/g
DS). Up to 27% increase in ethanol production was observed with
Laccase MtL (0.5-30 LACU/g DS). About 27% increase in ethanol
production was observed with Laccase CcL (36 LACU/g DS). About 40%
increase in ethanol production was observed with peroxidase CcP in
the presence of 5% H.sub.2O.sub.2.
Example 4
Laccase-Mediated Improvement of PCS Enzymatic Hydrolysis
Collection of Pretreatment Liquor:
[0264] Liquor was collected from both neutral, steam exploded corn
stover and from acid, steam exploded corn stover. Each PCS was
slurried in deionized water to a final total solids (TS) level of
15 wt. % with mixing at ambient temperature for 1 hour. Each slurry
was stored at 4.degree. C. for 16-20 hours. Slurries were then
mixed at ambient temperature for 1 hour, and liquor was collected
by vacuum filtration through a glass fiber filter (Whatman GF/D).
Sodium azide was added to a final concentration of 0.02% w/w. The
pH of each was adjusted to 5.0. After mixing for 1 hour at ambient
temperature, liquor was vacuum filtered through a 0.2 micro m
membrane and stored at 4.degree. C.
Laccase Treatment:
[0265] All PCS liquors were adjusted to pH 5.0. Laccase MtL was
dosed into PCS liquor to a final concentration of 100 ppm. The
liquor containing laccase was incubated alongside a negative
control liquor for 18 hours at 50.degree. C. with 150 rpm of
agitation. Any precipitated material was removed by centrifugation
at 3000 rpm for 10 minutes prior to further characterization.
Folin-Ciocalteu (FC) Method for Phenolics:
[0266] The method was modified from a published procedure
(Singleton, Orthofer, and Lamuela-Raventos, 1999, Methods Enzymol.
299: 152-178). Catechol (Sigma #135011) calibration standards and
sample dilutions were prepared in deionized water. Fifty microL of
diluted sample or catechol calibration standard were transferred to
the wells of a microtiter plate. Deionized water (50 microL) was
added to each well followed by 50 microL/well of FC reagent (Sigma
#F9252). The plate was incubated for 5 minutes at ambient
temperature. Sodium carbonate (15% w/v, 100 microL) was then added
to each well and the plate was incubated for 30 minutes at ambient
temperature in the dark. The absorbance at 770 nm of each well was
collected. Unknown total phenolic concentrations were calculated
from the catechol standard curve by linear regression analysis in
Microsoft Excel.
PCS Hydrolysis and Glucose Monitoring.
[0267] Washed PCS solids were slurried in the appropriate PCS
liquor at a final concentration of 4% total solids and dosed with
Cellulolytic preparation B (3 mg protein/g cellulose). Hydrolysis
reactions were incubated at 50.degree. C. with shaking (150 rpm)
for 48 hours. Glucose concentrations were monitored over time using
an enzyme-coupled glucose assay.
TABLE-US-00004 TABLE 1 Effect of laccase treatment on soluble
phenolics as measured by FC method Phenolics, mg/mL Liquor -laccase
+laccase % Decrease Neutral PCS 2.30 .+-. 0.05 1.63 .+-. 0.01 29.1
Acid PCS 1.96 .+-. 0.02 1.34 .+-. 0.02 31.8
CONCLUSIONS
[0268] Enzymatic hydrolysis of PCS was improved by treating PCS
liquor with a laccase. Laccase treatment of either neutral or acid
PCS liquors resulted in an 8-10% improvement in cellulose
conversion.
[0269] The invention described and claimed herein is not to be
limited in scope by the specific embodiments herein disclosed,
since these embodiments are intended as illustrations of several
aspects of the invention. Any equivalent embodiments are intended
to be within the scope of this invention. Indeed, various
modifications of the invention in addition to those shown and
described herein will become apparent to those skilled in the art
from the foregoing description. Such modifications are also
intended to fall within the scope of the appended claims. In the
case of conflict, the present disclosure, including definitions
will be controlling.
[0270] Various references are cited herein, the disclosures of
which are incorporated by reference in their entireties.
Sequence CWU 1
1
131483PRTB. licheniformis 1Ala 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 2480PRTB. amyloliquefaciens 2Val 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 3514PRTB.
stearothermophilus 3Ala 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 4485PRTBacillus sp. 4His His Asn
Gly Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr 1 5 10 15 Leu
Pro Asn Asp Gly Asn His Trp Asn Arg Leu Arg Asp Asp Ala Ala 20 25
30 Asn Leu Lys Ser Lys Gly Ile Thr Ala Val Trp Ile Pro Pro Ala Trp
35 40 45 Lys Gly Thr Ser Gln Asn Asp Val Gly Tyr Gly Ala Tyr Asp
Leu Tyr 50 55 60 Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg
Thr Lys Tyr Gly 65 70 75 80 Thr Arg Asn Gln Leu Gln Ala Ala Val Thr
Ser Leu Lys Asn Asn Gly 85 90 95 Ile Gln Val Tyr Gly Asp Val Val
Met Asn His Lys Gly Gly Ala Asp 100 105 110 Gly Thr Glu Ile Val Asn
Ala Val Glu Val Asn Arg Ser Asn Arg Asn 115 120 125 Gln Glu Thr Ser
Gly Glu Tyr Ala Ile Glu Ala Trp Thr Lys Phe Asp 130 135 140 Phe Pro
Gly Arg Gly Asn Asn His Ser Ser Phe Lys Trp Arg Trp Tyr 145 150 155
160 His Phe Asp Gly Thr Asp Trp Asp Gln Ser Arg Gln Leu Gln Asn Lys
165 170 175 Ile Tyr Lys Phe Arg Gly Thr Gly Lys Ala Trp Asp Trp Glu
Val Asp 180 185 190 Thr Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala
Asp Val Asp Met 195 200 205 Asp His Pro Glu Val Ile His Glu Leu Arg
Asn Trp Gly Val Trp Tyr 210 215 220 Thr Asn Thr Leu Asn Leu Asp Gly
Phe Arg Ile Asp Ala Val Lys His 225 230 235 240 Ile Lys Tyr Ser Phe
Thr Arg Asp Trp Leu Thr His Val Arg Asn Thr 245 250 255 Thr Gly Lys
Pro Met Phe Ala Val Ala Glu Phe Trp Lys Asn Asp Leu 260 265 270 Gly
Ala Ile Glu Asn Tyr Leu Asn Lys Thr Ser Trp Asn His Ser Val 275 280
285 Phe Asp Val Pro Leu His Tyr Asn Leu Tyr Asn Ala Ser Asn Ser Gly
290 295 300 Gly Tyr Tyr Asp Met Arg Asn Ile Leu Asn Gly Ser Val Val
Gln Lys 305 310 315 320 His Pro Thr His Ala Val Thr Phe Val Asp Asn
His Asp Ser Gln Pro 325 330 335 Gly Glu Ala Leu Glu Ser Phe Val Gln
Gln Trp Phe Lys Pro Leu Ala 340 345 350 Tyr Ala Leu Val Leu Thr Arg
Glu Gln Gly Tyr Pro Ser Val Phe Tyr 355 360 365 Gly Asp Tyr Tyr Gly
Ile Pro Thr His Gly Val Pro Ala Met Lys Ser 370 375 380 Lys Ile Asp
Pro Leu Leu Gln Ala Arg Gln Thr Phe Ala Tyr Gly Thr 385 390 395 400
Gln His Asp Tyr Phe Asp His His Asp Ile Ile Gly Trp Thr Arg Glu 405
410 415 Gly Asn Ser Ser His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser
Asp 420 425 430 Gly Pro Gly Gly Asn Lys Trp Met Tyr Val Gly Lys Asn
Lys Ala Gly 435 440 445 Gln Val Trp Arg Asp Ile Thr Gly Asn Arg Thr
Gly Thr Val Thr Ile 450 455 460 Asn Ala Asp Gly Trp Gly Asn Phe Ser
Val Asn Gly Gly Ser Val Ser 465 470 475 480 Val Trp Val Lys Gln 485
5485PRTBacillus sp. 5His His Asn Gly Thr Asn Gly Thr Met Met Gln
Tyr Phe Glu Trp His 1 5 10 15 Leu Pro Asn Asp Gly Asn His Trp Asn
Arg Leu Arg Asp Asp Ala Ser 20 25 30 Asn Leu Arg Asn Arg Gly Ile
Thr Ala Ile Trp Ile Pro Pro Ala Trp 35 40 45 Lys Gly Thr Ser Gln
Asn Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr 50 55 60 Asp Leu Gly
Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly 65 70 75 80 Thr
Arg Ser Gln Leu Glu Ser Ala Ile His Ala Leu Lys Asn Asn Gly 85 90
95 Val Gln Val Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp
100 105 110 Ala Thr Glu Asn Val Leu Ala Val Glu Val Asn Pro Asn Asn
Arg Asn 115 120 125 Gln Glu Ile Ser Gly Asp Tyr Thr Ile Glu Ala Trp
Thr Lys Phe Asp 130 135 140 Phe Pro Gly Arg Gly Asn Thr Tyr Ser Asp
Phe Lys Trp Arg Trp Tyr 145 150 155 160 His Phe Asp Gly Val Asp Trp
Asp Gln Ser Arg Gln Phe Gln Asn Arg 165 170 175 Ile Tyr Lys Phe Arg
Gly Asp Gly Lys Ala Trp Asp Trp Glu Val Asp 180 185 190 Ser Glu Asn
Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Val Asp Met 195 200 205 Asp
His Pro Glu Val Val Asn Glu Leu Arg Arg Trp Gly Glu Trp Tyr 210 215
220 Thr Asn Thr Leu Asn Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His
225 230 235 240 Ile Lys Tyr Ser Phe Thr Arg Asp Trp Leu Thr His Val
Arg Asn Ala 245 250 255 Thr Gly Lys Glu Met Phe Ala Val Ala Glu Phe
Trp Lys Asn Asp Leu 260 265 270 Gly Ala Leu Glu Asn Tyr Leu Asn Lys
Thr Asn Trp Asn His Ser Val 275 280 285 Phe Asp Val Pro Leu His Tyr
Asn Leu Tyr Asn Ala Ser Asn Ser Gly 290 295 300 Gly Asn Tyr Asp Met
Ala Lys Leu Leu Asn Gly Thr Val Val Gln Lys 305 310 315 320 His Pro
Met His Ala Val Thr Phe Val Asp Asn His Asp Ser Gln Pro 325 330
335 Gly Glu Ser Leu Glu Ser Phe Val Gln Glu Trp Phe Lys Pro Leu Ala
340 345 350 Tyr Ala Leu Ile Leu Thr Arg Glu Gln Gly Tyr Pro Ser Val
Phe Tyr 355 360 365 Gly Asp Tyr Tyr Gly Ile Pro Thr His Ser Val Pro
Ala Met Lys Ala 370 375 380 Lys Ile Asp Pro Ile Leu Glu Ala Arg Gln
Asn Phe Ala Tyr Gly Thr 385 390 395 400 Gln His Asp Tyr Phe Asp His
His Asn Ile Ile Gly Trp Thr Arg Glu 405 410 415 Gly Asn Thr Thr His
Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp 420 425 430 Gly Pro Gly
Gly Glu Lys Trp Met Tyr Val Gly Gln Asn Lys Ala Gly 435 440 445 Gln
Val Trp His Asp Ile Thr Gly Asn Lys Pro Gly Thr Val Thr Ile 450 455
460 Asn Ala Asp Gly Trp Ala Asn Phe Ser Val Asn Gly Gly Ser Val Ser
465 470 475 480 Ile Trp Val Lys Arg 485 6478PRTArtificial
SequenceSynthetic construct 6Ala Thr Pro Ala Asp Trp Arg Ser Gln
Ser Ile Tyr Phe Leu Leu Thr 1 5 10 15 Asp Arg Phe Ala Arg Thr Asp
Gly Ser Thr Thr Ala Thr Cys Asn Thr 20 25 30 Ala Asp Gln Lys Tyr
Cys Gly Gly Thr Trp Gln Gly Ile Ile Asp Lys 35 40 45 Leu Asp Tyr
Ile Gln Gly Met Gly Phe Thr Ala Ile Trp Ile Thr Pro 50 55 60 Val
Thr Ala Gln Leu Pro Gln Thr Thr Ala Tyr Gly Asp Ala Tyr His 65 70
75 80 Gly Tyr Trp Gln Gln Asp Ile Tyr Ser Leu Asn Glu Asn Tyr Gly
Thr 85 90 95 Ala Asp Asp Leu Lys Ala Leu Ser Ser Ala Leu His Glu
Arg Gly Met 100 105 110 Tyr Leu Met Val Asp Val Val Ala Asn His Met
Gly Tyr Asp Gly Ala 115 120 125 Gly Ser Ser Val Asp Tyr Ser Val Phe
Lys Pro Phe Ser Ser Gln Asp 130 135 140 Tyr Phe His Pro Phe Cys Phe
Ile Gln Asn Tyr Glu Asp Gln Thr Gln 145 150 155 160 Val Glu Asp Cys
Trp Leu Gly Asp Asn Thr Val Ser Leu Pro Asp Leu 165 170 175 Asp Thr
Thr Lys Asp Val Val Lys Asn Glu Trp Tyr Asp Trp Val Gly 180 185 190
Ser Leu Val Ser Asn Tyr Ser Ile Asp Gly Leu Arg Ile Asp Thr Val 195
200 205 Lys His Val Gln Lys Asp Phe Trp Pro Gly Tyr Asn Lys Ala Ala
Gly 210 215 220 Val Tyr Cys Ile Gly Glu Val Leu Asp Gly Asp Pro Ala
Tyr Thr Cys 225 230 235 240 Pro Tyr Gln Asn Val Met Asp Gly Val Leu
Asn Tyr Pro Ile Tyr Tyr 245 250 255 Pro Leu Leu Asn Ala Phe Lys Ser
Thr Ser Gly Ser Met Asp Asp Leu 260 265 270 Tyr Asn Met Ile Asn Thr
Val Lys Ser Asp Cys Pro Asp Ser Thr Leu 275 280 285 Leu Gly Thr Phe
Val Glu Asn His Asp Asn Pro Arg Phe Ala Ser Tyr 290 295 300 Thr Asn
Asp Ile Ala Leu Ala Lys Asn Val Ala Ala Phe Ile Ile Leu 305 310 315
320 Asn Asp Gly Ile Pro Ile Ile Tyr Ala Gly Gln Glu Gln His Tyr Ala
325 330 335 Gly Gly Asn Asp Pro Ala Asn Arg Glu Ala Thr Trp Leu Ser
Gly Tyr 340 345 350 Pro Thr Asp Ser Glu Leu Tyr Lys Leu Ile Ala Ser
Ala Asn Ala Ile 355 360 365 Arg Asn Tyr Ala Ile Ser Lys Asp Thr Gly
Phe Val Thr Tyr Lys Asn 370 375 380 Trp Pro Ile Tyr Lys Asp Asp Ile
Thr Ile Ala Met Arg Lys Gly Thr 385 390 395 400 Asp Gly Ser Gln Ile
Val Thr Ile Leu Ser Asn Lys Gly Ala Ser Gly 405 410 415 Asp Ser Tyr
Thr Leu Ser Leu Ser Gly Ala Gly Tyr Thr Ala Gly Gln 420 425 430 Gln
Leu Thr Glu Val Ile Gly Cys Thr Thr Val Thr Val Gly Ser Asp 435 440
445 Gly Asn Val Pro Val Pro Met Ala Gly Gly Leu Pro Arg Val Leu Tyr
450 455 460 Pro Thr Glu Lys Leu Ala Gly Ser Lys Ile Cys Ser Ser Ser
465 470 475 7586PRTArtificial SequenceSynthetic construct 7Ala Thr
Pro Ala Asp Trp Arg Ser Gln Ser Ile Tyr Phe Leu Leu Thr 1 5 10 15
Asp Arg Phe Ala Arg Thr Asp Gly Ser Thr Thr Ala Thr Cys Asn Thr 20
25 30 Ala Asp Gln Lys Tyr Cys Gly Gly Thr Trp Gln Gly Ile Ile Asp
Lys 35 40 45 Leu Asp Tyr Ile Gln Gly Met Gly Phe Thr Ala Ile Trp
Ile Thr Pro 50 55 60 Val Thr Ala Gln Leu Pro Gln Thr Thr Ala Tyr
Gly Asp Ala Tyr His 65 70 75 80 Gly Tyr Trp Gln Gln Asp Ile Tyr Ser
Leu Asn Glu Asn Tyr Gly Thr 85 90 95 Ala Asp Asp Leu Lys Ala Leu
Ser Ser Ala Leu His Glu Arg Gly Met 100 105 110 Tyr Leu Met Val Asp
Val Val Ala Asn His Met Gly Tyr Asp Gly Pro 115 120 125 Gly Ser Ser
Val Asp Tyr Ser Val Phe Val Pro Phe Asn Ser Ala Ser 130 135 140 Tyr
Phe His Pro Phe Cys Phe Ile Gln Asn Trp Asn Asp Gln Thr Gln 145 150
155 160 Val Glu Asp Cys Trp Leu Gly Asp Asn Thr Val Ser Leu Pro Asp
Leu 165 170 175 Asp Thr Thr Lys Asp Val Val Lys Asn Glu Trp Tyr Asp
Trp Val Gly 180 185 190 Ser Leu Val Ser Asn Tyr Ser Ile Asp Gly Leu
Arg Ile Asp Thr Val 195 200 205 Lys His Val Gln Lys Asp Phe Trp Pro
Gly Tyr Asn Lys Ala Ala Gly 210 215 220 Val Tyr Cys Ile Gly Glu Val
Leu Asp Gly Asp Pro Ala Tyr Thr Cys 225 230 235 240 Pro Tyr Gln Glu
Val Leu Asp Gly Val Leu Asn Tyr Pro Ile Tyr Tyr 245 250 255 Pro Leu
Leu Asn Ala Phe Lys Ser Thr Ser Gly Ser Met Asp Asp Leu 260 265 270
Tyr Asn Met Ile Asn Thr Val Lys Ser Asp Cys Pro Asp Ser Thr Leu 275
280 285 Leu Gly Thr Phe Val Glu Asn His Asp Asn Pro Arg Phe Ala Ser
Tyr 290 295 300 Thr Asn Asp Ile Ala Leu Ala Lys Asn Val Ala Ala Phe
Ile Ile Leu 305 310 315 320 Asn Asp Gly Ile Pro Ile Ile Tyr Ala Gly
Gln Glu Gln His Tyr Ala 325 330 335 Gly Gly Asn Asp Pro Ala Asn Arg
Glu Ala Thr Trp Leu Ser Gly Tyr 340 345 350 Pro Thr Asp Ser Glu Leu
Tyr Lys Leu Ile Ala Ser Ala Asn Ala Ile 355 360 365 Arg Asn Tyr Ala
Ile Ser Lys Asp Thr Gly Phe Val Thr Tyr Lys Asn 370 375 380 Trp Pro
Ile Tyr Lys Asp Asp Thr Thr Ile Ala Met Arg Lys Gly Thr 385 390 395
400 Asp Gly Ser Gln Ile Val Thr Ile Leu Ser Asn Lys Gly Ala Ser Gly
405 410 415 Asp Ser Tyr Thr Leu Ser Leu Ser Gly Ala Gly Tyr Thr Ala
Gly Gln 420 425 430 Gln Leu Thr Glu Val Ile Gly Cys Thr Thr Val Thr
Val Asp Ser Ser 435 440 445 Gly Asp Val Pro Val Pro Met Ala Gly Gly
Leu Pro Arg Val Leu Tyr 450 455 460 Pro Thr Glu Lys Leu Ala Gly Ser
Lys Ile Cys Ser Ser Ser Gly Ala 465 470 475 480 Thr Ser Pro Gly Gly
Ser Ser Gly Ser Val Glu Val Thr Phe Asp Val 485 490 495 Tyr Ala Thr
Thr Val Tyr Gly Gln Asn Ile Tyr Ile Thr Gly Asp Val 500 505 510 Ser
Glu Leu Gly Asn Trp Thr Pro Ala Asn Gly Val Ala Leu Ser Ser 515 520
525 Ala Asn Tyr Pro Thr Trp Ser Ala Thr Ile Ala Leu Pro Ala Asp Thr
530 535 540 Thr Ile Gln Tyr Lys Tyr Val Asn Ile Asp Gly Ser Thr Val
Ile Trp 545 550 555 560 Glu Asp Ala Ile Ser Asn Arg Glu Ile Thr Thr
Pro Ala Ser Gly Thr 565 570 575 Tyr Thr Glu Lys Asp Thr Trp Asp Glu
Ser 580 585 8558PRTArtificial SequenceRhizomucor pusillus amylase
with linker and SBD from A. rolfsii 8Ser Pro Leu Pro Gln Gln Gln
Arg Tyr Gly Lys Arg Ala Thr Ser Asp 1 5 10 15 Asp Trp Lys Ser Lys
Ala Ile Tyr Gln Leu Leu Thr Asp Arg Phe Gly 20 25 30 Arg Ala Asp
Asp Ser Thr Ser Asn Cys Ser Asn Leu Ser Asn Tyr Cys 35 40 45 Gly
Gly Thr Tyr Glu Gly Ile Thr Lys His Leu Asp Tyr Ile Ser Gly 50 55
60 Met Gly Phe Asp Ala Ile Trp Ile Ser Pro Ile Pro Lys Asn Ser Asp
65 70 75 80 Gly Gly Tyr His Gly Tyr Trp Ala Thr Asp Phe Tyr Gln Leu
Asn Ser 85 90 95 Asn Phe Gly Asp Glu Ser Gln Leu Lys Ala Leu Ile
Gln Ala Ala His 100 105 110 Glu Arg Asp Met Tyr Val Met Leu Asp Val
Val Ala Asn His Ala Gly 115 120 125 Pro Thr Ser Asn Gly Tyr Ser Gly
Tyr Thr Phe Gly Asp Ala Ser Leu 130 135 140 Tyr His Pro Lys Cys Thr
Ile Asp Tyr Asn Asp Gln Thr Ser Ile Glu 145 150 155 160 Gln Cys Trp
Val Ala Asp Glu Leu Pro Asp Ile Asp Thr Glu Asn Ser 165 170 175 Asp
Asn Val Ala Ile Leu Asn Asp Ile Val Ser Gly Trp Val Gly Asn 180 185
190 Tyr Ser Phe Asp Gly Ile Arg Ile Asp Thr Val Lys His Ile Arg Lys
195 200 205 Asp Phe Trp Thr Gly Tyr Ala Glu Ala Ala Gly Val Phe Ala
Thr Gly 210 215 220 Glu Val Phe Asn Gly Asp Pro Ala Tyr Val Gly Pro
Tyr Gln Lys Tyr 225 230 235 240 Leu Pro Ser Leu Ile Asn Tyr Pro Met
Tyr Tyr Ala Leu Asn Asp Val 245 250 255 Phe Val Ser Lys Ser Lys Gly
Phe Ser Arg Ile Ser Glu Met Leu Gly 260 265 270 Ser Asn Arg Asn Ala
Phe Glu Asp Thr Ser Val Leu Thr Thr Phe Val 275 280 285 Asp Asn His
Asp Asn Pro Arg Phe Leu Asn Ser Gln Ser Asp Lys Ala 290 295 300 Leu
Phe Lys Asn Ala Leu Thr Tyr Val Leu Leu Gly Glu Gly Ile Pro 305 310
315 320 Ile Val Tyr Tyr Gly Ser Glu Gln Gly Phe Ser Gly Gly Ala Asp
Pro 325 330 335 Ala Asn Arg Glu Val Leu Trp Thr Thr Asn Tyr Asp Thr
Ser Ser Asp 340 345 350 Leu Tyr Gln Phe Ile Lys Thr Val Asn Ser Val
Arg Met Lys Ser Asn 355 360 365 Lys Ala Val Tyr Met Asp Ile Tyr Val
Gly Asp Asn Ala Tyr Ala Phe 370 375 380 Lys His Gly Asp Ala Leu Val
Val Leu Asn Asn Tyr Gly Ser Gly Ser 385 390 395 400 Thr Asn Gln Val
Ser Phe Ser Val Ser Gly Lys Phe Asp Ser Gly Ala 405 410 415 Ser Leu
Met Asp Ile Val Ser Asn Ile Thr Thr Thr Val Ser Ser Asp 420 425 430
Gly Thr Val Thr Phe Asn Leu Lys Asp Gly Leu Pro Ala Ile Phe Thr 435
440 445 Ser Ala Gly Ala Thr Ser Pro Gly Gly Ser Ser Gly Ser Val Glu
Val 450 455 460 Thr Phe Asp Val Tyr Ala Thr Thr Val Tyr Gly Gln Asn
Ile Tyr Ile 465 470 475 480 Thr Gly Asp Val Ser Glu Leu Gly Asn Trp
Thr Pro Ala Asn Gly Val 485 490 495 Ala Leu Ser Ser Ala Asn Tyr Pro
Thr Trp Ser Ala Thr Ile Ala Leu 500 505 510 Pro Ala Asp Thr Thr Ile
Gln Tyr Lys Tyr Val Asn Ile Asp Gly Ser 515 520 525 Thr Val Ile Trp
Glu Asp Ala Ile Ser Asn Arg Glu Ile Thr Thr Pro 530 535 540 Ala Ser
Gly Thr Tyr Thr Glu Lys Asp Thr Trp Asp Glu Ser 545 550 555
9450PRTRhizomucor pusillus 9Ser Pro Leu Pro Gln Gln Gln Arg Tyr Gly
Lys Arg Ala Thr Ser Asp 1 5 10 15 Asp Trp Lys Gly Lys Ala Ile Tyr
Gln Leu Leu Thr Asp Arg Phe Gly 20 25 30 Arg Ala Asp Asp Ser Thr
Ser Asn Cys Ser Asn Leu Ser Asn Tyr Cys 35 40 45 Gly Gly Thr Tyr
Glu Gly Ile Thr Lys His Leu Asp Tyr Ile Ser Gly 50 55 60 Met Gly
Phe Asp Ala Ile Trp Ile Ser Pro Ile Pro Lys Asn Ser Asp 65 70 75 80
Gly Gly Tyr His Gly Tyr Trp Ala Thr Asp Phe Tyr Gln Leu Asn Ser 85
90 95 Asn Phe Gly Asp Glu Ser Gln Leu Lys Ala Leu Ile Gln Ala Ala
His 100 105 110 Glu Arg Asp Met Tyr Val Met Leu Asp Val Val Ala Asn
His Ala Gly 115 120 125 Pro Thr Ser Asn Gly Tyr Ser Gly Tyr Thr Phe
Gly Asp Ala Ser Leu 130 135 140 Tyr His Pro Lys Cys Thr Ile Asp Tyr
Asn Asp Gln Thr Ser Ile Glu 145 150 155 160 Gln Cys Trp Val Ala Asp
Glu Leu Pro Asp Ile Asp Thr Glu Asn Ser 165 170 175 Asp Asn Val Ala
Ile Leu Asn Asp Ile Val Ser Gly Trp Val Gly Asn 180 185 190 Tyr Ser
Phe Asp Gly Ile Arg Ile Asp Thr Val Lys His Ile Arg Lys 195 200 205
Asp Phe Trp Thr Gly Tyr Ala Glu Ala Ala Gly Val Phe Ala Thr Gly 210
215 220 Glu Val Phe Asn Gly Asp Pro Ala Tyr Val Gly Pro Tyr Gln Lys
Tyr 225 230 235 240 Leu Pro Ser Leu Ile Asn Tyr Pro Met Tyr Tyr Ala
Leu Asn Asp Val 245 250 255 Phe Val Ser Lys Ser Lys Gly Phe Ser Arg
Ile Ser Glu Met Leu Gly 260 265 270 Ser Asn Arg Asn Ala Phe Glu Asp
Thr Ser Val Leu Thr Thr Phe Val 275 280 285 Asp Asn His Asp Asn Pro
Arg Phe Leu Asn Ser Gln Ser Asp Lys Ala 290 295 300 Leu Phe Lys Asn
Ala Leu Thr Tyr Val Leu Leu Gly Glu Gly Ile Pro 305 310 315 320 Ile
Val Tyr Tyr Gly Ser Glu Gln Gly Phe Ser Gly Gly Ala Asp Pro 325 330
335 Ala Asn Arg Glu Val Leu Trp Thr Thr Asn Tyr Asp Thr Ser Ser Asp
340 345 350 Leu Tyr Gln Phe Ile Lys Thr Val Asn Ser Val Arg Met Lys
Ser Asn 355 360 365 Lys Ala Val Tyr Met Asp Ile Tyr Val Gly Asp Asn
Ala Tyr Ala Phe 370 375 380 Lys His Gly Asp Ala Leu Val Val Leu Asn
Asn Tyr Gly Ser Gly Ser 385 390 395 400 Thr Asn Gln Val Ser Phe Ser
Val Ser Gly Lys Phe Asp Ser Gly Ala 405 410 415 Ser Leu Met Asp Ile
Val Ser Asn Ile Thr Thr Thr Val Ser Ser Asp 420 425 430 Gly Thr Val
Thr Phe Asn Leu Lys Asp Gly Leu Pro Ala Ile Phe Thr 435 440 445 Ser
Ala 450 1037PRTAspergillus niger 10Thr Gly Gly Thr Thr Thr Thr Ala
Thr Pro Thr Gly Ser Gly Ser Val 1 5 10 15 Thr Ser Thr Ser Lys Thr
Thr Ala Thr Ala Ser Lys Thr Ser Thr Ser 20 25 30 Thr Ser Ser Thr
Ser 35 11108PRTAspergillus niger 11Cys Thr Thr Pro Thr Ala Val Ala
Val Thr Phe Asp Leu Thr Ala Thr 1 5 10
15 Thr Thr Tyr Gly Glu Asn Ile Tyr Leu Val Gly Ser Ile Ser Gln Leu
20 25 30 Gly Asp Trp Glu Thr Ser Asp Gly Ile Ala Leu Ser Ala Asp
Lys Tyr 35 40 45 Thr Ser Ser Asp Pro Leu Trp Tyr Val Thr Val Thr
Leu Pro Ala Gly 50 55 60 Glu Ser Phe Glu Tyr Lys Phe Ile Arg Ile
Glu Ser Asp Asp Ser Val 65 70 75 80 Glu Trp Glu Ser Asp Pro Asn Arg
Glu Tyr Thr Val Pro Gln Ala Cys 85 90 95 Gly Thr Ser Thr Ala Thr
Val Thr Asp Thr Trp Arg 100 105 12574PRTArtificial SequenceHybrid
of Meripilus giganteus amylase with A.rolfsii SBD 12Arg Pro Thr Val
Phe Asp Ala Gly Ala Asp Ala His Ser Leu His Ala 1 5 10 15 Arg Ala
Pro Ser Gly Ser Lys Asp Val Ile Ile Gln Met Phe Glu Trp 20 25 30
Asn Trp Asp Ser Val Ala Ala Glu Cys Thr Asn Phe Ile Gly Pro Ala 35
40 45 Gly Tyr Gly Phe Val Gln Val Ser Pro Pro Gln Glu Thr Ile Gln
Gly 50 55 60 Ala Gln Trp Trp Thr Asp Tyr Gln Pro Val Ser Tyr Thr
Leu Thr Gly 65 70 75 80 Lys Arg Gly Asp Arg Ser Gln Phe Ala Asn Met
Ile Thr Thr Cys His 85 90 95 Ala Ala Gly Val Gly Val Ile Val Asp
Thr Ile Trp Asn His Met Ala 100 105 110 Gly Val Asp Ser Gly Thr Gly
Thr Ala Gly Ser Ser Phe Thr His Tyr 115 120 125 Asn Tyr Pro Gly Ile
Tyr Gln Asn Gln Asp Phe His His Cys Gly Leu 130 135 140 Glu Pro Gly
Asp Asp Ile Val Asn Tyr Asp Asn Ala Val Glu Val Gln 145 150 155 160
Thr Cys Glu Leu Val Asn Leu Ala Asp Leu Ala Thr Asp Thr Glu Tyr 165
170 175 Val Arg Gly Arg Leu Ala Gln Tyr Gly Asn Asp Leu Leu Ser Leu
Gly 180 185 190 Ala Asp Gly Leu Arg Leu Asp Ala Ser Lys His Ile Pro
Val Gly Asp 195 200 205 Ile Ala Asn Ile Leu Ser Arg Leu Ser Arg Ser
Val Tyr Ile Thr Gln 210 215 220 Glu Val Ile Phe Gly Ala Gly Glu Pro
Ile Thr Pro Asn Gln Tyr Thr 225 230 235 240 Gly Asn Gly Asp Val Gln
Glu Phe Arg Tyr Thr Ser Ala Leu Lys Asp 245 250 255 Ala Phe Leu Ser
Ser Gly Ile Ser Asn Leu Gln Asp Phe Glu Asn Arg 260 265 270 Gly Trp
Val Pro Gly Ser Gly Ala Asn Val Phe Val Val Asn His Asp 275 280 285
Thr Glu Arg Asn Gly Ala Ser Leu Asn Asn Asn Ser Pro Ser Asn Thr 290
295 300 Tyr Val Thr Ala Thr Ile Phe Ser Leu Ala His Pro Tyr Gly Thr
Pro 305 310 315 320 Thr Ile Leu Ser Ser Tyr Asp Gly Phe Thr Asn Thr
Asp Ala Gly Ala 325 330 335 Pro Asn Asn Asn Val Gly Thr Cys Ser Thr
Ser Gly Gly Ala Asn Gly 340 345 350 Trp Leu Cys Gln His Arg Trp Thr
Ala Ile Ala Gly Met Val Gly Phe 355 360 365 Arg Asn Asn Val Gly Ser
Ala Ala Leu Asn Asn Trp Gln Ala Pro Gln 370 375 380 Ser Gln Gln Ile
Ala Phe Gly Arg Gly Ala Leu Gly Phe Val Ala Ile 385 390 395 400 Asn
Asn Ala Asp Ser Ala Trp Ser Thr Thr Phe Thr Thr Ser Leu Pro 405 410
415 Asp Gly Ser Tyr Cys Asp Val Ile Ser Gly Lys Ala Ser Gly Ser Ser
420 425 430 Cys Thr Gly Ser Ser Phe Thr Val Ser Gly Gly Lys Leu Thr
Ala Thr 435 440 445 Val Pro Ala Arg Ser Ala Ile Ala Val His Thr Gly
Gln Lys Gly Ser 450 455 460 Gly Gly Gly Ala Thr Ser Pro Gly Gly Ser
Ser Gly Ser Val Glu Val 465 470 475 480 Thr Phe Asp Val Tyr Ala Thr
Thr Val Tyr Gly Gln Asn Ile Tyr Ile 485 490 495 Thr Gly Asp Val Ser
Glu Leu Gly Asn Trp Thr Pro Ala Asn Gly Val 500 505 510 Ala Leu Ser
Ser Ala Asn Tyr Pro Thr Trp Ser Ala Thr Ile Ala Leu 515 520 525 Pro
Ala Asp Thr Thr Ile Gln Tyr Lys Tyr Val Asn Ile Asp Gly Ser 530 535
540 Thr Val Ile Trp Glu Asp Ala Ile Ser Asn Arg Glu Ile Thr Thr Pro
545 550 555 560 Ala Ser Gly Thr Tyr Thr Glu Lys Asp Thr Trp Asp Glu
Ser 565 570 131344DNAThermoascus aurantiacus 13aagtctaccc
agtatcctgt caacatgcgg ctcgttgctt ccctaacggc cttggtggcc 60ttgtccgtac
ctgtctttcc cgctgctgtc aacgtgaagc gtgcttcgtc ctacctggag
120atcactctga gccaggtcag caacactctg atcaaggccg tggtccagaa
cactggtagc 180gacgagttgt ccttcgttca cctgaacttc ttcaaggacc
ccgctcctgt caaaaaggta 240tcggtctatc gcgatgggtc tgaagtgcag
ttcgagggca ttttgagccg ctacaaatcg 300actggcctct ctcgtgacgc
ctttacttat ctggctcccg gagagtccgt cgaggacgtt 360tttgatattg
cttcgactta cgatctgacc agcggcggcc ctgtaactat ccgtactgag
420ggagttgttc cctacgccac ggctaacagc actgatattg ccggctacat
ctcatactcg 480tctaatgtgt tgaccattga tgtcgatggc gccgctgctg
ccactgtctc caaggcaatc 540actcctttgg accgccgcac taggatcagt
tcctgctccg gcagcagaca gagcgctctt 600actacggctc tcagaaacgc
tgcttctctt gccaacgcag ctgccgacgc ggctcagtct 660ggatcagctt
caaagttcag cgagtacttc aagactactt ctagctctac ccgccagacc
720gtggctgcgc gtcttcgggc tgttgcgcgg gaggcatctt cgtcttcttc
gggagccacc 780acgtactact gcgacgatcc ctacggctac tgttcctcca
acgtcctggc ttacaccctg 840ccttcataca acataatcgc caactgtgac
attttctata cttacctgcc ggctctgacc 900agtacctgtc acgctcagga
tcaagcgacc actgcccttc acgagttcac ccatgcgcct 960ggcgtctaca
gccctggcac ggacgacctg gcgtatggct accaggctgc gatgggtctc
1020agcagcagcc aggctgtcat gaacgctgac acctacgctc tctatgcgaa
tgccatatac 1080cttggttgct aagcgcagag cggtccattg gcgagttggt
cgcggtccag ctctagctgg 1140gatcggccat ggatggtttg agctctgtaa
atgacggtcc cgatcttgca gctttgattc 1200catctaaacg cgcaggaagg
aatattagga tgaggatgtt tctatgagac ggctgtgcgc 1260agagttccga
cgagtgacgg taactatttt tgccatagct acataatgca tctacaagtt
1320atctaaaaaa aaaaaaaaaa aaaa 1344
* * * * *