U.S. patent application number 14/649167 was filed with the patent office on 2016-04-28 for trichoderma reesei host cells expressing a glucoamylase from aspergillus fumigatus and methods of use thereof.
The applicant listed for this patent is DANISCO US INC.. Invention is credited to Jing Ge, Ling Hua, Sung Ho Lee, Jalsen Li, Jayarama K. Shetty, Zhongmei Tang, Bo Zhang, Kun Zhong.
Application Number | 20160115509 14/649167 |
Document ID | / |
Family ID | 49876974 |
Filed Date | 2016-04-28 |
United States Patent
Application |
20160115509 |
Kind Code |
A1 |
Ge; Jing ; et al. |
April 28, 2016 |
TRICHODERMA REESEI HOST CELLS EXPRESSING A GLUCOAMYLASE FROM
ASPERGILLUS FUMIGATUS AND METHODS OF USE THEREOF
Abstract
Fungal glucoamylases from Aspergillus fumigatus--expressed in
Trichoderma reesei host cells (AfGATR) are provided. Trichoderma
reesei host cells express AfGATRs at higher, or at least
comparable, levels to natively expressed AfGA Aspergillus
fumigatus. AfGATRs, including AfGA1TR and AfGA2TR, exhibit high
activity at elevated temperatures and at low pH, so AfGATRs can be
used efficiently in a process of saccharification in the presence
of alpha-amylase, such as Aspergillus kawachii alpha-amylase
(AkAA). AfGATRs advantageously catalyze starch saccharification to
an oligosaccharide composition significantly enriched in DP1 (i.e.,
glucose) compared to the products of saccharification catalyzed by
Aspergillus niger glucoamylase (AnGA) or native AfGA expressed in
Aspergillus fumigatus. AfGATRs such as AfGA1TR, AfGA2TR or a
variant thereof can be used at a lower dosage than AnGA and
natively expressed AfGAs to produce comparable levels of
glucose.
Inventors: |
Ge; Jing; (Shanghai, CN)
; Hua; Ling; (Hockessin, DE) ; Lee; Sung Ho;
(North Liberty, IA) ; Li; Jalsen; (Shanghai,
CN) ; Shetty; Jayarama K.; (Pleasanton, CA) ;
Tang; Zhongmei; (Shanghai, CN) ; Zhang; Bo;
(Shanghai, CN) ; Zhong; Kun; (Shanghai,
CN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
DANISCO US INC. |
Palo Alto |
CA |
US |
|
|
Family ID: |
49876974 |
Appl. No.: |
14/649167 |
Filed: |
November 21, 2013 |
PCT Filed: |
November 21, 2013 |
PCT NO: |
PCT/US13/71154 |
371 Date: |
June 2, 2015 |
Current U.S.
Class: |
435/99 ; 435/110;
435/115; 435/126; 435/134; 435/137; 435/139; 435/144; 435/145;
435/158; 435/160; 435/162; 435/167; 435/205; 435/254.6 |
Current CPC
Class: |
C12N 9/2428 20130101;
C12Y 302/01003 20130101; C12P 19/02 20130101; C12P 19/14 20130101;
Y02E 50/10 20130101 |
International
Class: |
C12P 19/14 20060101
C12P019/14; C12P 19/02 20060101 C12P019/02; C12N 9/34 20060101
C12N009/34 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 11, 2012 |
CN |
PCT/CN2012/086349 |
Claims
1. A recombinant Trichoderma reesei host cell expressing an AfGATR,
or variant thereof, having at least 80% sequence identity to SEQ ID
NO: 12 or 13, wherein said Trichoderma reesei host cell expresses
the AfGATR or variant at a comparable level to a Aspergillus
fumigatus host cell that expresses an AfGA, or variant thereof,
having the same amino acid sequence of the AfGATR, or variant
thereof, under identical conditions.
2. A recombinant Trichoderma reesei host cell expressing an AfGATR,
or variant thereof, having at least 80% sequence identity to SEQ ID
NO: 12 or 13, wherein the AfGATR, or variant thereof, is more
thermostable than an AfGA, or variant thereof, having the same
amino acid sequence of AfGATR, or variant thereof, and wherein the
AfGA, or variant thereof, is expressed in an A. fumigatus host
cell.
3. A recombinant AfGATR, or variant thereof, produced by the host
cell of claim 2.
4. A recombinant AfGATR, or variant thereof, produced by the host
cell of claim 1.
5. The recombinant AfGATR, or variant thereof, of claim 4, wherein
said AfGATR, or variant thereof, has at least 70% activity at
74.degree. C. at pH 5.0 over 10 min.
6. The recombinant AfGATR, or variant thereof, of claim 5, wherein
said AfGATR, or variant thereof, is an AfGA1TR, or variant
thereof.
7. The recombinant AfGA1TR, or variant thereof, of claim 6, wherein
said AfGA1TR, or variant, thereof has at least 70% activity over a
temperature range of 55.degree. to 74.degree. C. at pH 5.0 over 10
min.
8. The recombinant AfGA1TR, or variant thereof, of claim 7, wherein
said AfGATR, or variant thereof, has an optimum temperature of
about 68.degree. C.
9. The recombinant AfGATR, or variant, thereof of claim 5, wherein
said AfGATR, or variant thereof, is an AfGA2TR, or variant
thereof.
10. The recombinant AfGA2TR, or variant thereof, of claim 9,
wherein said AfGA2TR, or variant thereof, has at least 70% activity
over a temperature range of 61.degree. to 74.degree. C. at pH 5.0
over 10 min.
11. The recombinant AfGA2TR, or variant thereof, of claim 10,
wherein said AfGA2TR, or variant thereof, has an optimum
temperature of about 69.degree. C.
12. The recombinant AfGATR, or variant thereof, of claim 4, wherein
said AfGATR, or variant thereof, comprises an amino acid sequence
with at least 90%, 95%, or 99% amino acid sequence identity to SEQ
ID NO: 12.
13. The recombinant AfGATR, or variant thereof, of claim 12,
wherein said AfGATR, or variant, thereof comprises SEQ ID NO:
12.
14. The recombinant AfGATR, or variant thereof, of claim 4, wherein
said AfGATR, or variant thereof, consists of an amino acid sequence
with at least 80%, 90%, 95%, or 99% amino acid sequence identity to
SEQ ID NO: 12.
15. (canceled)
16. The recombinant AfGATR, or variant thereof, of claim 4, wherein
said AfGATR, or variant thereof, comprises an amino acid sequence
with at least 90%, 95%, or 99% amino acid sequence identity to SEQ
ID NO: 13.
17. The recombinant AfGATR, or variant thereof, of claim 16,
wherein said AfGATR, or variant, thereof comprises SEQ ID NO:
13.
18. The recombinant AfGATR, or variant thereof, of claim 4, wherein
said AfGATR, or variant thereof, consists of an amino acid sequence
with at least 80%, 90%, 95%, or 99% amino acid sequence identity to
SEQ ID NO: 13.
19. (canceled)
20. A method of saccharifying a composition comprising starch to
produce a composition comprising glucose, wherein said method
comprises: (i) contacting a starch composition with the isolated
AfGATR, or variant thereof, of claim 4; and (ii) saccharifying the
starch composition to produce said glucose composition; wherein
said AfGA1TR, or variant, thereof catalyzes the saccharification of
the composition comprising starch to a composition comprising
glucose.
21. The method of claim 20, wherein said composition comprising
glucose enriched in DP1 compared to a second composition comprising
DP1 produced by AnGA after 24 hours of saccharification under the
same conditions.
22. The method of claim 20, wherein said composition comprising
glucose is enriched in DP1 compared to a second composition
comprising DP1 produced by a wild-type AfGA under the same
conditions.
23. The method of claim 20, wherein said AfGATR, or variant
thereof, is AfGATR2 and wherein said composition comprising glucose
is enriched in DP1 compared to a second composition comprising DP1
produced by AfGA1TR under the same conditions.
24. The method of claim 20, wherein the AfGA1TR, or variant
thereof, is dosed at about 40%-50% the dose of AnGA, to produce the
same DP1 yield after 24 hours of saccharification under the same
conditions.
25-30. (canceled)
31. The method of claim 20, wherein the method further comprises
contacting a starch composition with an alpha-amylase.
32. The method of claim 31, wherein the alpha-amylase is AkAA.
33. The method of claim 20, wherein the method further comprises
contacting a starch composition with a pullulanase.
34. The method of claim 20, further comprising fermenting the
glucose composition to produce an End of Fermentation (EOF)
product.
35-40. (canceled)
41. The method of claim 34, wherein the EOF product comprises a
metabolite.
42-45. (canceled)
46. The method of claim 20, wherein said isolated AfGATR, or
variant thereof, is secreted by said Trichoderma reesei host
cell.
47. The method of claim 46, wherein said host cell further
expresses and secretes an alpha-amylase.
48. The method of claim 47, wherein said host cell further
expresses and secretes a pullulanase.
49. The method of claim 46, wherein said composition comprising
starch is contacted with said host cell.
50-74. (canceled)
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims benefit of priority from
international patent application no. PCT/CN2012/086349 filed on 11
Dec. 2012, and is incorporated herein by reference in its
entirety.
[0002] A sequence listing comprising SEQ ID NO: 1-14 is attached
herein and incorporated by reference in its entirety.
FIELD OF THE INVENTION
[0003] Trichoderma reesei host cells expressing a glucoamylase from
Aspergillus fumigatus, (AfGATR) or a variant thereof, and methods
of use thereof.
BACKGROUND
[0004] Starch consists of a mixture of amylose (15-30% w/w) and
amylopectin (70-85% w/w). Amylose consists of linear chains of
.alpha.-1,4-linked glucose units having a molecular weight (MW)
from about 60,000 to about 800,000. Amylopectin is a branched
polymer containing .alpha.-1,6 branch points every 24-30 glucose
units; its MW may be as high as 100 million.
[0005] Sugars from starch, in the form of concentrated dextrose
syrups, are currently produced by an enzyme catalyzed process
involving: (1) liquefaction (or viscosity reduction) of solid
starch with an .alpha.-amylase into dextrins having an average
degree of polymerization of about 7-10, and (2) saccharification of
the resulting liquefied starch (i.e. starch hydrolysate) with
glucoamylase (also called amyloglucosidase or GA). The resulting
syrup has a high glucose content. Much of the glucose syrup that is
commercially produced is subsequently enzymatically isomerized to a
dextrose/fructose mixture known as isosyrup. The resulting syrup
also may be fermented with microorganisms, such as yeast, to
produce commercial products including ethanol, citric acid, lactic
acid, succinic acid, itaconic acid, monosodium glutamate,
gluconates, lysine, other organic acids, other amino acids, and
other biochemicals, for example. Fermentation and saccharification
can be conducted simultaneously (i.e., an SSF process) to achieve
greater economy and efficiency.
[0006] Glucoamylases (glucan 1,4-.alpha.-glucohydrolases, EC
3.2.1.3) are starch hydrolyzing exo-acting carbohydrases, which
catalyze the removal of successive glucose units from the
non-reducing ends of starch or related oligo and polysaccharide
molecules. Glucoamylases can hydrolyze both the linear and branched
glucosidic linkages of starch (e.g., amylose and amylopectin).
.alpha.-Amylases, on the other hand, hydrolyze starch, glycogen,
and related polysaccharides by cleaving internal
.alpha.-1,4-glucosidic bonds at random. Glucoamylases have been
used for a variety of different purposes, including starch
saccharification, brewing, baking, production of syrups for the
food industry, production of feedstocks for fermentation processes,
and in animal feed to increase digestibility.
[0007] Glucoamylases are produced by numerous strains of bacteria,
fungi, and plants. For example, a glucoamylase is produced by
strains of Aspergillus fumigatus. Luo et al. (2008) "Production of
acid proof raw starch-digesting glucoamylase from a newly isolated
strain of Aspergillus fumigatus MS-09," Sci. Tech. Food Indus.
29(5): 151-154; Sellars et al. (1976) "Degradation of barley by
Aspergillus fumigatus Fres," Proc. Int. Biodegradation Symp., 3rd,
S. J. Miles et al., eds., Appl. Sci., Barking, UK, pp. 635-43;
Domingues et al. (1993) "Production of amylase by soil fungi and
partial biochemical characterization of amylase of a selected
strain (Aspergillus fumigatus Fresenius)," Can. J. Microbiol.
39(7): 681-85; Cherry et al. (2004) "Extracellular glucoamylase
from the isolate Aspergillus fumigatus," Pakistan J. Biol. Sci.
7(11): 1988-92. However, Aspergillus fumigatus is highly allergenic
and pathogenic to humans and plants. Thus, Aspergillus fumigatus is
not a viable production host for glucoamylases used in industrial
processes for manufacturing products for human consumption. There
is a need to produce A. fumigatus glucoamylases from a suitable
host.
SUMMARY
[0008] Glucoamylases from Aspergillus fumigatus that are expressed
in Trichoderma reesei (AfGATRs) catalyze saccharification for
extended periods at high temperatures and an acidic pH. Examples of
known glucoamylases from Aspergillus fumigatus (SEQ ID NO: 1 and
2), encoding nucleic acids, and Trichoderma reesei host cells that
express the polynucleotides are provided. Trichoderma reesei host
cells express AfGATRs at higher, or at least comparable, levels to
natively expressed AfGA Aspergillus fumigatus. AfGATRs, including
AfGA1TR and AfGA2TR, exhibit high activity at elevated temperatures
and at low pH, so AfGATRs can be used efficiently in a process of
saccharification in the presence of .alpha.-amylase, such as
Aspergillus kawachii .alpha.-amylase (AkAA). AfGATRs advantageously
catalyze starch saccharification to an oligosaccharide composition
significantly enriched in DP1 (i.e., glucose) compared to the
products of saccharification catalyzed by Aspergillus niger
glucoamylase (AnGA) or native AfGA expressed in Aspergillus
fumigatus. AfGATRs such as AfGA1TR, AfGA2TR or a variant thereof
can be used at a lower dosage than AnGA and natively expressed
AfGAs to produce comparable levels of glucose. AfGATRs or variants
thereof can be used in combination with enzymes derived from plants
(e.g., cereals and grains). AfGATRs or variants thereof also can be
used in combination with enzymes secreted by, or endogenous to, a
host cell. For example, an AfGATR or a variant thereof can be added
to a saccharification reaction, or SSF process during which one or
more amylases, additional glucoamylases, proteases, lipases,
phytases, esterases, redox enzymes, transferases, or other enzymes
are secreted by the production host. An AfGATR or a variant thereof
may also work in combination with endogenous non-secreted
production host enzymes. In another example, an AfGATR or a variant
thereof can be secreted by a production host cell with other
enzymes during saccharification or SSF. The AfGATR glucoamylase, or
a variant thereof, may be used in a process involving direct
hydrolysis of starch for syrup and/or biochemicals (e.g., alcohols,
organic acids, amino acids, other biochemicals and biomaterials)
where the reaction temperature is below the gelatinization
temperature of substrate. An AfGATR or a variant thereof can be
secreted by a Trichoderma reesei host cell with other enzymes
during saccharification or SSF.
[0009] Accordingly, provided is a recombinant Trichoderma reesei
host cell expressing an AfGATR or variant thereof having at least
80% sequence identity to SEQ ID NO: 12 or 13, wherein said
Trichoderma reesei host cell expresses the AfGATR or variant at a
comparable level to a Aspergillus fumigatus host cell, which
expresses an AfGA or variant thereof having the same amino acid
sequence of the AfGATR or variant thereof, under identical
conditions.
[0010] Also provided is a recombinant Trichoderma reesei host cell
expressing an AfGATR or variant thereof having at least 80%
sequence identity to SEQ ID NO: 12 or 13, wherein the AfGATR or
variant thereof is more thermostable than an AfGA or variant
thereof having the same amino acid sequence of AfGATR or variant
thereof, and wherein the AfGA or variant thereof is expressed in an
A. fumigatus host cell.
[0011] Also provided is a method for producing a recombinant AfGATR
or variant thereof, comprising: (a) providing a T. reesei host cell
that expresses a recombinant AfGATR or variant thereof having at
least 80% sequence identity to SEQ ID NO: 12 or 13; (b) culturing
said host cell under conditions which permit the production of said
recombinant AfGATR or variant thereof; and (c) isolating said
recombinant AfGATR, or variant thereof, wherein the AfGATR or
variant thereof is more thermostable than an AfGA or variant
thereof having the same amino acid sequence of AfGATR or variant
thereof, and wherein the AfGA or variant thereof is expressed in an
A. fumigatus host cell.
[0012] Also provided is a recombinant AfGATR, or variant thereof,
produced by the disclosed host cells. The recombinant AfGATR or
variant thereof may have at least 70% activity at 74.degree. C. at
pH 5.0 over 10 min. The recombinant AfGATR or variant thereof may
be AfGA1TR. The AfGA1TR may have at least 70% activity over a
temperature range of 55.degree.-74.degree. C. at pH 5.0 over 10
min. The AfGA1TR may have an optimum temperature of about
68.degree. C. The recombinant AfGATR or variant thereof may also be
AfGA2TR. The AfGA2TR may have at least 70% activity over a
temperature range of 61.degree. to 74.degree. C. at pH 5.0 over 10
min. The AfGA2TR may have an optimum temperature of about
69.degree. C. The recombinant AfGATR or variant thereof may
comprise an amino acid sequence with at least 90%, 95%, or 99%
amino acid sequence identity to sequence identity to SEQ ID NO: 12.
The recombinant AfGATR or variant thereof may comprise SEQ ID NO:
12. The recombinant AfGATR or variant thereof may also consist of
an amino acid sequence with at least 90%, 95%, or 99% amino acid
sequence identity to SEQ ID NO: 13. The recombinant AfGATR or
variant thereof may consist of SEQ ID NO: 12. The recombinant
AfGATR or variant thereof may also comprise an amino acid sequence
with at least 90%, 95%, or 99% amino acid sequence identity to
sequence identity to SEQ ID NO: 13. The recombinant AfGATR or
variant thereof may comprise SEQ ID NO: 13. The recombinant AfGATR
or variant thereof may also consist of an amino acid sequence with
at least 90%, 95%, or 99% amino acid sequence identity to SEQ ID
NO: 13. The recombinant AfGATR or variant thereof may consist of
SEQ ID NO: 13.
[0013] Also provided is a method of saccharifying a composition
comprising starch to produce a composition comprising glucose,
wherein said method comprises: (i) contacting a starch composition
with the isolated AfGATR or variant thereof of any of claims 4-15;
and (ii) saccharifying the starch composition to produce said
glucose composition; wherein said AfGA1TR or variant thereof
catalyzes the saccharification of the composition comprising starch
to a composition comprising glucose. The composition comprising
glucose may be enriched in DP1 compared to a second composition
comprising DP1 produced by AnGA under the same conditions. The
composition comprising glucose may also be enriched in DP1 compared
to a second composition comprising DP1 produced by a wild-type AfGA
under the same conditions. The AfGATR or variant thereof may be an
AfGATR2, and the composition comprising glucose may be enriched in
DP1 compared to a second composition comprising DP1 produced by
AfGA1TR under the same conditions. The AfGA1TR or variant thereof
may be dosed at about 40%-50% the dose of AnGA, to produce the same
DP1 yield under the same conditions.
[0014] It is also provided that the composition comprising starch
comprises liquefied starch, gelatinized starch, or granular starch.
Saccharification may be conducted at a temperature range of about
30.degree. C. to about 65.degree. C. The temperature range may be
47.degree. C.-60.degree. C. Saccharification may be conducted over
a pH range of pH 2.0-pH 6.0. The pH range may be pH 3.5-pH 5.5. The
pH range may also be pH 4.0-pH 5.0.
[0015] It is also provided that the method of saccharification may
further comprise contacting a starch composition with an
alpha-amylase. The alpha-amylase may be AkAA. The method of
saccharification may further comprise contacting a starch
composition with a pullulanase.
[0016] It is also provided that the method of saccharification may
further comprise fermenting the glucose composition to produce an
End of Fermentation (EOF) product. The fermentation may also be a
simultaneous saccharification and fermentation (SSF) reaction. The
fermentation may be conducted for 24-70 hours at pH 2-8 and in a
temperature range of 25.degree. C.-70.degree. C. The EOF product
may comprise ethanol. The EOF product may comprise 8%-18% (v/v)
ethanol. The method may further comprise contacting a mash and/or a
wort with a pullulanase, an alpha-amylase and the AfGA1TR or
variant thereof. The method may also comprise (a) preparing a mash;
(b) filtering the mash to obtain a wort; and (c) fermenting the
wort to obtain a fermented beverage, wherein the pullulanase, the
alpha-amylase and the AfGA1TR or variant thereof are added to: (i)
the mash of step (a) and/or (ii) the wort of step (b) and/or (iii)
the fermented wort of step (c). The EOF product may comprise a
metabolite. The metabolite may be citric acid, lactic acid,
succinic acid, monosodium glutamate, gluconic acid, sodium
gluconate, calcium gluconate, potassium gluconate, glucono
delta-lactone, sodium erythorbate, omega 3 fatty acid, butanol, an
amino acid, lysine, itaconic acid, 1,3-propanediol, or
isoprene.
[0017] It is also provided that the method of saccharification may
further comprise adding an additional glucoamylase, hexokinase,
xylanase, glucose isomerase, xylose isomerase, phosphatase,
phytase, protease, pullulanase, .beta.-amylase, an additional
.alpha.-amylase, protease, cellulase, hemicellulase, lipase,
cutinase, trehalase, isoamylase, redox enzyme, esterase,
transferase, pectinase, alpha-glucosidase, beta-glucosidase, lyase,
hydrolase, or a combination thereof, to said starch composition.
The AfGATR, or variant thereof, may be added at a dosage of 0.1 to
2 glucoamylase units (GAU)/g ds. The AfGATR, or variant thereof,
may be added at a dosage of about 49.5 .mu.g prot/g solid. The
pullulanase may also be added. The isolated AfGATR or a variant
thereof may be secreted by said Trichoderma reesei host cell. The
host cell may further express and secrete an alpha-amylase. The
host cell may further express and secrete a pullulanase.
[0018] It is also provided that the method of saccharification may
further contacting said composition comprising starch with said
host cell. The host cell is capable of fermenting the glucose
composition.
[0019] Also contemplated is a composition comprising glucose
produced by the disclosed methods of saccharification. Also
contemplated is a liquefied starch produced by the disclosed
methods of saccharification. Also contemplated is a fermented
beverage produced by the disclosed methods of saccharification.
[0020] Also contemplated is the use of saccharifying a composition
comprising starch, comprising an isolated AfGA1TR or variant
thereof. The composition may be a cultured cell material. The
composition may further comprise a glucoamylase. The AfGA1TR or
variant thereof may be purified. The AfGA1TR or variant thereof may
be secreted by the host cell.
[0021] Also contemplated is the use of an AfGA1TR or variant
thereof in the production of a composition comprising glucose. Also
contemplated is the use of an AfGA1TR or variant thereof in the
production of a liquefied starch. Also contemplated is the use of
an AfGA1TR or variant thereof in the production of a fermented
beverage. Also contemplated are methods of saccharification the
disclosed fermented beverage, or the disclosed uses of the end of
fermentation product, wherein the fermented beverage or end
fermentation product is selected from the group consisting of i) a
beer selected from the group consisting of full malted beer, beer
brewed under the "Reinheitsgebot", ale, IPA, lager, bitter,
Happoshu (second beer), third beer, dry beer, near beer, light
beer, low alcohol beer, low calorie beer, porter, bock beer, stout,
malt liquor, non-alcoholic beer, and non-alcoholic malt liquor; and
ii) cereal or malt beverages selected from the group consisting of
fruit flavoured malt beverages, liquor flavoured malt beverages,
and coffee flavoured malt beverages.
[0022] Also contemplated is a method of producing a food
composition, comprising combining (i) one or more food ingredients,
and (ii) an isolated AfGA1TR or variant thereof of claims 4-15,
wherein said pullulanase and said isolated AfGA1TR or variant
thereof catalyze the hydrolysis of starch components present in the
food ingredients to produce glucose. The food composition may be
selected from the group consisting of a food product, a baking
composition, a food additive, an animal food product, a feed
product, a feed additive, an oil, a meat, and a lard. The food
ingredients may comprise a baking ingredient or an additive. The
one or more food ingredients may be selected from the group
consisting of flour; an anti-staling amylase; a phospholipase; a
phospholipid; a maltogenic alpha-amylase or a variant, homologue,
or mutants thereof which has maltogenic alpha-amylase activity; a
bakery xylanase (EC 3.2.1.8); and a lipase. The one or more food
ingredients may be selected from the group consisting of (i) a
maltogenic alpha-amylase from Bacillus stearothermophilus, (ii) a
bakery xylanase is from Bacillus, Aspergillus, Thermomyces or
Trichoderma, (iii) a glycolipase from Fusarium heterosporum. The
food composition may comprise a dough or a dough product,
preferably a processed dough product. The method may comprise
baking the food composition to produce a baked good. The method may
further comprise (i) providing a starch medium; (ii) adding to the
starch medium the pullulanase and the AfGA1TR or variant thereof;
and (iii) applying heat to the starch medium during or after step
(b) to produce a baked good.
[0023] Also contemplated is a composition for use in producing a
food composition, comprising an AfGA1TR or variant thereof. Also
contemplated is a use of AfGA1TR or variant thereof in preparing a
food composition. The food composition may comprise a dough or a
dough product, preferably a processed dough product. The food
composition may be a bakery composition. Also contemplated is use
of AfGA1TR or variant thereof in a dough product to retard or
reduce staling, preferably detrimental retrogradation, of the dough
product.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] The accompanying drawings are incorporated in, and
constitute a part of, this specification and illustrate various
methods and compositions disclosed herein. In the drawings:
[0025] FIGS. 1A-B depict a ClustalW alignment of the AfGA1
catalytic core and carbohydrate binding domain (residues 27-476 and
524-631 of SEQ ID NO: 1, respectively or the full length, with the
corresponding residues of glucoamylases from: Aspergillus fumigatus
A1163 (AfGA2)(residues 27-476 and 524-631 of SEQ ID NO: 2,
respectively); Neosartorya fisheri NRRL 181 (residues 28-476 and
520-627 of SEQ ID NO: 3, respectively); Talaromyces stipitatus ATCC
10500 (residues 28-478 and 530-637 of SEQ ID NO: 4, respectively);
Penicillium marneffei ATCC 18224 (residues 31-481 and 534-641 of
SEQ ID NO: 5, respectively); and Aspergillus nidulans FGSC A4
(residues 55-493 and 544-661 of SEQ ID NO: 6, respectively).
Residues designated by an asterisk in FIG. 1 are AfGA1 residues
corresponding to conserved residues in SEQ ID NOS: 1-6.
[0026] FIG. 2 depicts a map of a pJG222 expression vector
comprising a polynucleotide that encodes an AfGA1 polypeptide,
pJG222 (Trex3gM-AfGA1).
[0027] FIG. 3 depicts the dependence of glucoamylase activity
(relative units) on pH. The glucoamylases include (1) wild-type
AfGA expressed in Aspergillus fumigatus, (2) AfGA1TR expressed in
Trichoderma reesei, and (2) AnGA expressed in Aspergillus niger.
Glucoamylase activity was assayed by the release of glucose from
soluble starch at 50.degree. C.
[0028] FIG. 4 depicts the dependence of glucoamylase activity
(relative units) on temperature. The glucoamylases include (1)
wild-type AfGA expressed in Aspergillus fumigatus, (2) AfGA1TR
expressed in Trichoderma reesei, and (3) and AnGA. Glucoamylase
activity was assayed by the release of glucose from soluble starch
at pH 5.0.
[0029] FIGS. 5A-B depict AfGA1TR and AnGA glucoamylase activity
assayed by the release of glucose from 35% dry solid starch at pH
4.5 and 5.0.
[0030] FIGS. 6A-B depict the hydrolysis of 35% dry solid starch to
DP1 and reversion of DP1 to DP2 by a composition containing
AfGATR1, pullulanase and AkAA.
[0031] FIGS. 7A-B depict the hydrolysis of 35% dry solid starch to
DP1 and reversion of DP1 to DP2 by a composition containing
AfGATR1, pullulanase and varying doses of AkAA.
[0032] FIG. 8 depicts the amount of DP2 found in a high glucose
composition containing 96% DP1 after the release of reducing sugar
from a 35% dry solid starch by compositions containing AfGA1TR or
AnGA, and further containing an alpha-amylase (OPTIMAX L-100) and
PU (GC636).
[0033] FIG. 9 depicts a map of a pJG313 expression vector
comprising a polynucleotide that encodes an AfGA2 polypeptide,
pJG313(Trex3gM-AfGA2).
[0034] FIG. 10 depicts the dependence of glucoamylase (relative
units) AfGA2TR expressed in Trichoderma reesei on pH. Glucoamylase
activity was assayed by the release of glucose from soluble starch
substrate at 50.degree. C.
[0035] FIG. 11 depicts the dependence of glucoamylase activity
(relative units) of AfGA2TR expressed in Trichoderma reesei on
temperature. Glucoamylase activity was assayed by the release of
glucose from soluble starch substrate at pH 5.0.
[0036] FIG. 12 depicts the thermostability of AfGA2TR in 50 mM
sodium acetate buffer at pH 5.0. The enzyme was incubated at
desired temperature for 2 hours in a thermocycler prior to addition
to soluble starch substrate.
[0037] FIG. 13 depicts an SDS gel of AfGA1TR expressed in T.
reesei. Column M contains a protein molecular weight (MW) ladder in
kDa. Columns 1-4 represent samples from T. reesei fermentation
producing AfGATR with elapsed fermentation times of 40.5 hours,
64.5 hours, 88.3 hours and 112 hours, respectively. The bands
labeled with an arrow at 75 kDa are AfGA1TR.
DETAILED DESCRIPTION
[0038] Fungal glucoamylases from Aspergillus fumigatus (AfGA1TR or
AfGA2TR) and variants thereof are provided. AfGA1TR or a variant
thereof has a pH optimum of pH 5.0 and at least 70% activity over a
range of pH 3.5 to pH 7.5. The enzyme has an optimum temperature of
68.degree. C. and at least 70% activity over a temperature range of
55.degree.-74.degree. C., when tested at pH 5.0. AfGA2TR or a
variant thereof has a pH optimum of pH 5.3 and at least 70%
activity over a range of pH 3.3 to pH 7.3. The enzyme has an
optimum temperature of 69.degree. C. and at least 70% activity over
a temperature range of 61.degree.-74.degree. C., when tested at pH
5.0. These properties allow these enzymes to be used in combination
with a .alpha.-amylase under the same reaction conditions. This
obviates the necessity of running a saccharification reaction as a
batch process, where the pH and temperature should be adjusted for
optimal use of the .alpha.-amylase or glucoamylase.
[0039] Exemplary applications for glucoamylases such as AfGATRs
(including AfGA1TR and AfGA2TR) or variants thereof can be used in
a process of starch saccharification, e.g., SSF, the preparation of
food compositions, the preparation of cleaning compositions, such
as detergent compositions for cleaning laundry, dishes, and other
surfaces, for textile processing (e.g., desizing). AfGATRs
advantageously catalyze starch saccharification to an
oligosaccharide composition significantly enriched in DP1 (i.e.,
glucose) compared to the products of saccharification catalyzed by
Aspergillus niger glucoamylase (AnGA). AfGATRs can be secreted by a
host cell with other enzymes during fermentation or SSF. For
example, AfGATRs demonstrate a greater rate of saccharification
over AnGA, producing more than 96% glucose in 24 hours. AfGATRs can
also be used at a lower dosage than AnGA to produce comparable
levels of DP1. At least a 50% dose saving can be expected. AfGATRs
are also statistically significantly more thermostable than AnGA
during saccharification. AfGATRs can be used in combination with
enzymes derived from plants (e.g., cereals and grains). AfGATRs
also can be used in combination with enzymes secreted by, or
endogenous to, a host cell such as T. reesei. For example, AfGATRs
can be added to a saccharification, fermentation or SSF process
during which one or more amylases, glucoamylases, proteases,
lipases, phytases, esterases, redox enzymes, transferases, or other
enzymes that are secreted by the production host. AfGATRs may be
combined with an accessory alpha-amylase to further improve the
rate of saccharification. For example, the addition of 0.1 SSU/gds
of AkAA improves the rate of saccharification. When combined with
AkAA and a pullulanase, AfGATRs were found to have lower DP3 by
0.1% than AnGA at the same glucose yield in a single pH process.
AfGATRs may also work in combination with endogenous non-secreted
production host enzymes. In another example, AfGATRs can be
secreted by a production host cell with other enzymes during
fermentation or SSF. The AfGATRs may also be effective in direct
hydrolysis of starch for syrup and/or biochemicals (e.g., alcohols,
organic acids, amino acids, other biochemicals and biomaterials)
where the reaction temperature is below the gelatinization
temperature of substrate.
1. Definitions & Abbreviations
[0040] In accordance with this detailed description, the following
abbreviations and definitions apply. Note that the singular forms
"a," "an," and "the" include plural referents unless the context
clearly dictates otherwise. Thus, for example, reference to "an
enzyme" includes a plurality of such enzymes, and reference to "the
dosage" includes reference to one or more dosages and equivalents
thereof known to those skilled in the art, and so forth.
[0041] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art. The following terms are provided
below.
1.1. Abbreviations and Acronyms
[0042] The following abbreviations/acronyms have the following
meanings unless otherwise specified:
TABLE-US-00001 ABTS 2,2-azino-bis-3-ethylbenzothiazoline-6-sulfonic
acid AcAmyl Aspergillus clavatus .alpha.-amylase AE alcohol
ethoxylate AEO alcohol ethoxylate AEOS alcohol ethoxysulfate AES
alcohol ethoxysulfate AfGA Aspergillus fumigatus glucoamylase AfGA1
Aspergillus fumigatus glucoamylase 1 AfGA2 Aspergillus fumigatus
glucoamylase 2 AfGATR Aspergillus fumigatus glucoamylase expressed
in Trichoderma reesei AfGA1TR Aspergillus fumigatus glucoamylase 1
expressed in Trichoderma reesei AfGA2TR Aspergillus fumigatus
glucoamylase 2 expressed in Trichoderma reesei AkAA Aspergillus
kawachii .alpha.-amylase AnGA Aspergillus niger glucoamylase AOS
.alpha.-olefinsulfonate AS alkyl sulfate cDNA complementary DNA CMC
carboxymethylcellulose DE dextrose equivalent DNA deoxyribonucleic
acid DPn degree of saccharide polymerization having n subunits ds
or DS dry solids DTMPA diethylenetriaminepentaacetic acid EC Enzyme
Commission EDTA ethylenediaminetetraacetic acid EO ethylene oxide
(polymer fragment) EOF End of Fermentation FGSC Fungal Genetics
Stock Center GA glucoamylase GAU/g ds glucoamylase activity
unit/gram dry solids HFCS high fructose corn syrup HgGA Humicola
grisea glucoamylase HS higher sugar IPTG isopropyl
.beta.-D-thiogalactoside IRS insoluble residual starch kDa
kiloDalton LAS linear alkylbenzenesulfonate MW molecular weight MWU
modified Wohlgemuth unit; 1.6 .times.10.sup.-5 mg/MWU = unit of
activity NCBI National Center for Biotechnology Information NOBS
nonanoyloxybenzenesulfonate NTA nitriloacetic acid OxAm Purastar
HPAM 5000 L (Danisco US Inc.) PAHBAH p-hydroxybenzoic acid
hydrazide PEG polyethyleneglycol pI isoelectric point ppm parts per
million PVA poly(vinyl alcohol) PVP poly(vinylpyrrolidone) RNA
ribonucleic acid SAS alkanesulfonate SDS-PAGE sodium dodecyl
sulfate polyacrylamide gel electrophoresis SSF simultaneous
saccharification and fermentation SSU/g solid soluble starch
unit/gram dry solids sp. species TAED tetraacetylethylenediamine
TrGA Trichoderma reesei glucoamylase w/v weight/volume w/w
weight/weight v/v volume/volume wt % weight percent .degree. C.
degrees Centigrade H.sub.2O water dH.sub.2O or DI deionized water
dIH.sub.2O deionized water, Milli-Q filtration g or gm grams .mu.g
micrograms mg milligrams kg kilograms .mu.L and .mu.l microliters
mL and ml milliliters mm millimeters .mu.m micrometer M molar mM
millimolar .mu.M micromolar U units sec seconds min(s)
minute/minutes hr(s) hour/hours DO dissolved oxygen Ncm Newton
centimeter ETOH ethanol eq. equivalents N normal
1.2. Definitions
[0043] The terms "amylase" or "amylolytic enzyme" refer to an
enzyme that is, among other things, capable of catalyzing the
degradation of starch. .alpha.-Amylases are hydrolases that cleave
the .alpha.-D-(1.fwdarw.4) O-glycosidic linkages in starch.
Generally, .alpha.-amylases (EC 3.2.1.1;
.alpha.-D-(1.fwdarw.4)-glucan glucanohydrolase) are described as
endo-acting enzymes cleaving .alpha.-D-(1.fwdarw.4) O-glycosidic
linkages within the starch molecule in a random fashion yielding
polysaccharides containing three or more (1-4)-.alpha.-linked
D-glucose units. In contrast, the exo-acting amylolytic enzymes,
such as .beta.-amylases (EC 3.2.1.2; .alpha.-D-(1.fwdarw.4)-glucan
maltohydrolase) and some product-specific amylases like maltogenic
.alpha.-amylase (EC 3.2.1.133) cleave the polysaccharide molecule
from the non-reducing end of the substrate. .beta.-amylases,
.alpha.-glucosidases (EC 3.2.1.20; .alpha.-D-glucoside
glucohydrolase), glucoamylase (EC 3.2.1.3;
.alpha.-D-(1.fwdarw.4)-glucan glucohydrolase), and product-specific
amylases like the maltotetraosidases (EC 3.2.1.60) and the
maltohexaosidases (EC 3.2.1.98) can produce malto-oligosaccharides
of a specific length or enriched syrups of specific
maltooligosaccharides.
[0044] As used herein, the term "glucoamylase" (EC 3.2.1.3)
(otherwise known as glucan 1,4-.alpha.-glucosidase; glucoamylase;
amyloglucosidase; .gamma.-amylase; lysosomal .alpha.-glucosidase;
acid maltase; exo-1,4-.alpha.-glucosidase; glucose amylase;
.gamma.-1,4-glucan glucohydrolase; acid maltase;
1,4-.alpha.-D-glucan glucohydrolase; or 4-.alpha.-D-glucan
glucohydrolase) refers to a class of enzymes that catalyze the
release of D-glucose from the non-reducing ends of starch and
related oligo- and polysaccharides. These are exo-acting enzymes,
which release glucosyl residues from the non-reducing ends of
amylose and amylopectin molecules. The enzymes also hydrolyze
alpha-1, 6 and alpha-1, 3 linkages although at much slower rates
than alpha-1, 4 linkages. The term "hydrolysis of starch" refers to
the cleavage of glucosidic bonds with the addition of water
molecules.
[0045] The term "pullulanase" (E.C. 3.2.1.41, pullulan
6-glucanohydrolase) refers to a class of enzymes that are capable
of hydrolyzing alpha 1-6 glucosidic linkages in an amylopectin
molecule.
[0046] "Enzyme units" herein refer to the amount of product formed
per time under the specified conditions of the assay. For example,
a "glucoamylase activity unit" (GAU) is defined as the amount of
enzyme that produces 1 g of glucose per hour from soluble starch
substrate (4% DS) at 60.degree. C., pH 4.2. A "soluble starch unit"
(SSU) is the amount of enzyme that produces 1 mg of glucose per
minute from soluble starch substrate (4% DS) at pH 4.5, 50.degree.
C. DS refers to "dry solids."
[0047] As used herein "dry solids" content refers to the total
solids of a slurry in a dry weight percent basis. The term "slurry"
refers to an aqueous mixture containing insoluble solids. The term
"high ds" refers to an aqueous starch slurry containing dry solids
greater than 38%.
[0048] The term "Brix" refers to a well-known hydrometer scale for
measuring the sugar content of a solution at a given temperature.
The Brix scale measures the number of grams of sucrose present per
100 grams of aqueous sugar solution (the total solubilized solid
content). Brix measurements are frequently performed using a
hydrometer or refractometer.
[0049] The term "degree of polymerization" (DP) refers to the
number (n) of anhydro-glucopyranose units in a given saccharide.
Examples of DP1 are monosaccharides, such as glucose and fructose.
Examples of DP2 are disaccharides, such as maltose and sucrose. HS
or DP4+(>DP3) denotes polymers with a degree of polymerization
of greater than 3. The term "DE," or "dextrose equivalent," is
defined as the percentage of reducing sugar, i.e., D-glucose, as a
fraction of total carbohydrate in a syrup. It is an industry
standard for the concentration of total reducing sugars, and is
expressed as % D-glucose on a dry weight basis. Unhydrolyzed
granular starch has a DE that is essentially 0 and D-glucose has a
DE of 100.
[0050] As used herein the term "starch" refers to any material
comprised of the complex polysaccharide carbohydrates of plants,
comprised of amylose and amylopectin with the formula
(C.sub.6H.sub.10O.sub.5).sub.x, wherein X can be any number. The
term includes plant-based materials such as grains, grasses, tubers
and roots, and more specifically materials obtained from wheat,
barley, corn, rye, rice, sorghum, brans, cassava, millet, potato,
sweet potato, and tapioca. The term "starch" includes granular
starch. The term "granular starch" refers to raw, i.e., uncooked
starch, e.g., starch that has not been subject to
gelatinization.
[0051] The term "glucose syrup" refers to an aqueous composition
containing glucose solids. Glucose syrup will have a DE of at least
20. In some embodiments, glucose syrup will not contain more than
21% water and will not contain less than 25% reducing sugar
calculated as dextrose. The glucose syrup will include at least 90%
D-glucose, perhaps at least 95% D-glucose. In some embodiments, the
terms glucose and glucose syrup are used interchangeably.
[0052] The term "total sugar content" refers to the total sugar
content present in a starch composition.
[0053] The term "Refractive Index Dry Substance" (RIDS) is defined
as the determination of the refractive index of a starch solution
at a known DE at a controlled temperature then converting the RI to
dry substance using an appropriate relationship, such as the
Critical Data Tables of the Corn Refiners Association.
[0054] The term "contacting" refers to the placing of the
respective enzymes in sufficiently close proximity to the
respective substrate to enable the enzymes to convert the substrate
to the end-product. Those skilled in the art will recognize that
mixing solutions of the enzyme with the respective substrates can
effect contacting.
[0055] The terms, "wild-type," "parental," or "reference," with
respect to a polypeptide, refer to a naturally-occurring
polypeptide that does not include a man-made substitution,
insertion, or deletion at one or more amino acid positions.
Similarly, the terms "wild-type," "parental," or "reference," with
respect to a polynucleotide, refer to a naturally-occurring
polynucleotide that does not include a man-made nucleoside change.
However, note that a polynucleotide encoding a wild-type, parental,
or reference polypeptide is not limited to a naturally-occurring
polynucleotide, and encompasses any polynucleotide encoding the
wild-type, parental, or reference polypeptide. Further, as used
herein and as will be clear from the context, it will be
appreciated that referring to a particular sequence as "wild-type"
is not meant to imply that other sequences in the example that are
not affixed with the pre-fix "wild-type" aren't wild type as
well.
[0056] As used herein, the term "comparable" in reference to
expression level refers to no more than 20% variance between the
samples of interest, unless the context clearly dictates
otherwise.
[0057] Reference to the wild-type protein is understood to include
the mature form of the protein. A "mature" polypeptide means a
polypeptide or variant thereof from which a signal sequence is
absent. For example, the signal sequence may be cleaved during
expression of the polypeptide. The mature AfGA1 or AfGA2 is 612
amino acids in length covering positions 1-612 of SEQ ID NO: 1 and
SEQ ID NO: 2 respectively, where positions are counted from the
N-terminus. The signal sequence of the wild-type AfGA1 or AfGA2 is
19 amino acids in length and has the sequence set forth in SEQ ID
NO: 11. Mature AfGA1, AfGA2, or variant thereof may comprise a
signal sequence taken from different proteins. The mature protein
can be a fusion protein between the mature polypeptide and a signal
sequence polypeptide.
[0058] The putative "catalytic core" of AfGA1, AfGA2 or a variant
thereof spans residues 41-453 of SEQ ID NO: 1. Amino acid residues
534-630 constitute the putative "carbohydrate binding domain" of
AfGA1, AfGA2 or a variant thereof. The "linker" or "linker region"
of AfGA1, AfGA2, or a variant thereof spans a region between the
"catalytic core" and "carbohydrate binding domain."
[0059] The term "variant," with respect to a polypeptide, refers to
a polypeptide that differs from a specified wild-type, parental, or
reference polypeptide in that it includes one or more
naturally-occurring or man-made substitutions, insertions, or
deletions of an amino acid. Similarly, the term "variant," with
respect to a polynucleotide, refers to a polynucleotide that
differs in nucleotide sequence from a specified wild-type,
parental, or reference polynucleotide. The identity of the
wild-type, parental, or reference polypeptide or polynucleotide
will be apparent from context.
[0060] In the case of the present enzymes, such as a glucoamylase,
"activity" refers to enzymatic activity, which can be measured as
described, herein.
[0061] The term "recombinant," when used in reference to a subject
cell, nucleic acid, protein or vector, indicates that the subject
has been modified from its native state. Thus, for example,
recombinant cells express genes that are not found within the
native (non-recombinant) form of the cell, or express native genes
at different levels or under different conditions than found in
nature. Recombinant nucleic acids differ from a native sequence by
one or more nucleotides and/or are operably-linked to heterologous
sequences, e.g., a heterologous promoter in an expression vector.
Recombinant proteins may differ from a native sequence by one or
more amino acids and/or are fused with heterologous sequences. A
vector comprising a nucleic acid encoding an AfGA1, AfGA2 or
variant thereof is a recombinant vector.
[0062] The terms "recovered," "isolated," and "separated," refer to
a compound, protein (polypeptides), cell, nucleic acid, amino acid,
or other specified material or component that is removed from at
least one other material or component with which it is naturally
associated as found in nature, e.g., an AfGATR isolated from a
recombinant host cell. An "isolated" AfGATR, or variant thereof
includes, but is not limited to, a culture broth containing
secreted AfGATR expressed in a heterologous host cell (i.e., a host
cell this not A. fumigatus).
[0063] As used herein, the term "purified" refers to material
(e.g., an isolated polypeptide or polynucleotide) that is in a
relatively pure state, e.g., at least about 90% pure, at least
about 95% pure, at least about 98% pure, or even at least about 99%
pure.
[0064] The terms "thermostable" and "thermostability," with
reference to an enzyme, refer to the ability of the enzyme to
retain activity after exposure to an elevated temperature. The
thermostability of an enzyme, such as an amylase enzyme, can be
measured by its T.sub.m, at which half the enzyme activity is lost
under defined conditions. The T.sub.m may be calculated by
measuring residual glucoamylase activity following exposure to
(i.e., challenge by) an elevated temperature.
[0065] A "pH range," with reference to an enzyme, refers to the
range of pH values under which the enzyme exhibits catalytic
activity.
[0066] As used herein, the terms "pH stable" and "pH stability,"
with reference to an enzyme, relate to the ability of the enzyme to
retain activity over a wide range of pH values for a predetermined
period of time (e.g., 15 min., 30 min., and 1 hour).
[0067] As used herein, the term "amino acid sequence" is synonymous
with the terms "polypeptide," "protein," and "peptide," and are
used interchangeably. Where such amino acid sequences exhibit
activity, they may be referred to as an "enzyme." The conventional
one-letter or three-letter codes for amino acid residues are used,
with amino acid sequences being presented in the standard
amino-to-carboxy terminal orientation (i.e., N.fwdarw.C).
[0068] The term "nucleic acid" encompasses DNA, RNA,
heteroduplexes, and synthetic molecules capable of encoding a
polypeptide. Nucleic acids may be single stranded or double
stranded, and may be chemical modifications. The terms "nucleic
acid" and "polynucleotide" are used interchangeably. Because the
genetic code is degenerate, more than one codon may be used to
encode a particular amino acid, and the present compositions and
methods encompass nucleotide sequences that encode a particular
amino acid sequence. Unless otherwise indicated, nucleic acid
sequences are presented in 5'-to-3' orientation.
[0069] As used herein, "hybridization" refers to the process by
which one strand of nucleic acid forms a duplex with, i.e., base
pairs with, a complementary strand, as occurs during blot
hybridization techniques and PCR techniques. Stringent
hybridization conditions are exemplified by hybridization under the
following conditions: 65.degree. C. and 0.1.times.SSC (where
1.times.SSC=0.15 M NaCl, 0.015 M Na.sub.3 citrate, pH 7.0).
Hybridized, duplex nucleic acids are characterized by a melting
temperature (T.sub.m), where one-half of the hybridized nucleic
acids are unpaired with the complementary strand. Mismatched
nucleotides within the duplex lower the T.sub.m. A nucleic acid
encoding a variant glucoamylase may have a T.sub.m reduced by
1.degree. C. to 3.degree. C. or more compared to a duplex formed
between the nucleotide of SEQ ID NO: 8 and its identical
complement.
[0070] As used herein, a "synthetic" molecule is produced by in
vitro chemical or enzymatic synthesis rather than by an
organism.
[0071] As used herein, the terms "transformed," "stably
transformed," and "transgenic," used with reference to a cell means
that the cell contains a non-native (e.g., heterologous) nucleic
acid sequence integrated into its genome or carried as an episome
that is maintained through multiple generations.
[0072] The term "introduced" in the context of inserting a nucleic
acid sequence into a cell, means "transfection," "transformation"
or "transduction," as known in the art.
[0073] A "host strain" or "host cell" is an organism into which an
expression vector, phage, virus, or other DNA construct, including
a polynucleotide encoding a polypeptide of interest (e.g., an
AfGATR or variant thereof) has been introduced. Exemplary host
strains are microorganism cells (e.g., bacteria, filamentous fungi,
and yeast, such as T. reesei) capable of expressing the polypeptide
of interest and/or fermenting saccharides. The term "host cell"
includes protoplasts created from cells.
[0074] The term "heterologous" with reference to a polynucleotide
or protein refers to a polynucleotide or protein that does not
naturally occur in a host cell.
[0075] The term "endogenous" with reference to a polynucleotide or
protein refers to a polynucleotide or protein that occurs naturally
in the host cell.
[0076] As used herein, the term "expression" refers to the process
by which a polypeptide is produced based on a nucleic acid
sequence. The process includes both transcription and
translation.
[0077] A "selective marker" or "selectable marker" refers to a gene
capable of being expressed in a host to facilitate selection of
host cells carrying the gene. Examples of selectable markers
include but are not limited to antimicrobials (e.g., hygromycin,
bleomycin, or chloramphenicol) and/or genes that confer a metabolic
advantage, such as a nutritional advantage on the host cell.
[0078] A "vector" refers to a polynucleotide sequence designed to
introduce nucleic acids into one or more cell types. Vectors
include cloning vectors, expression vectors, shuttle vectors,
plasmids, phage particles, cassettes, and the like.
[0079] An "expression vector" refers to a DNA construct comprising
a DNA sequence encoding a polypeptide of interest, which coding
sequence is operably linked to a suitable control sequence capable
of effecting expression of the DNA in a suitable host. Such control
sequences may include a promoter to effect transcription, an
optional operator sequence to control transcription, a sequence
encoding suitable ribosome binding sites on the mRNA, enhancers,
and sequences that control termination of transcription and
translation.
[0080] The term "operably linked" means that specified components
are in a relationship (including but not limited to juxtaposition)
permitting them to function in an intended manner. For example, a
regulatory sequence is operably linked to a coding sequence such
that expression of the coding sequence is under control of the
regulatory sequences.
[0081] A "signal sequence" is a sequence of amino acids attached to
the N-terminal portion of a protein, which facilitates the
secretion of the protein outside the cell. The mature form of an
extracellular protein lacks the signal sequence, which is cleaved
off during the secretion process.
[0082] As used herein, "biologically active" refer to a sequence
having a specified biological activity, such an enzymatic
activity.
[0083] As used herein, a "swatch" is a piece of material such as a
fabric that has a stain applied thereto. The material can be, for
example, fabrics made of cotton, polyester, or mixtures of natural
and synthetic fibers. The swatch can further be paper, such as
filter paper or nitrocellulose, or a piece of a hard material such
as ceramic, metal, or glass. For amylases, the stain is starch
based, but can include blood, milk, ink, grass, tea, wine, spinach,
gravy, chocolate, egg, cheese, clay, pigment, oil, or mixtures of
these compounds.
[0084] As used herein, a "smaller swatch" is a section of the
swatch that has been cut with a single-hole punch device, or has
been cut with a custom manufactured 96-hole punch device, where the
pattern of the multi-hole punch is matched to standard 96-well
microtiter plates, or the section has been otherwise removed from
the swatch. The swatch can be of textile, paper, metal, or other
suitable material. The smaller swatch can have the stain affixed
either before or after it is placed into the well of a 24-, 48- or
96-well microtiter plate. The smaller swatch can also be made by
applying a stain to a small piece of material. For example, the
smaller swatch can be a stained piece of fabric 5/8'' or 0.25'' in
diameter. The custom manufactured punch is designed in such a
manner that it delivers 96 swatches simultaneously to all wells of
a 96-well plate. The device allows delivery of more than one swatch
per well by simply loading the same 96-well plate multiple times.
Multi-hole punch devices can be conceived of to deliver
simultaneously swatches to any format plate, including but not
limited to 24-well, 48-well, and 96-well plates. In another
conceivable method, the soiled test platform can be a bead made of
metal, plastic, glass, ceramic, or another suitable material that
is coated with the soil substrate. The one or more coated beads are
then placed into wells of 96-, 48-, or 24-well plates or larger
formats, containing suitable buffer and enzyme.
[0085] As used herein, "a cultured cell material comprising an
AfGATR," or similar language, refers to a cell lysate or
supernatant (including media) that includes an AfGATR or a variant
thereof as a component. The cell material may be from a
heterologous host that is grown in culture for the purpose of
producing the AfGATR or variant thereof.
[0086] "Percent sequence identity" means that a variant has at
least a certain percentage of amino acid residues identical to a
wild-type AfGA1 or AfGA2, when aligned using the CLUSTAL W
algorithm with default parameters. See Thompson et al. (1994)
Nucleic Acids Res. 22:4673-4680. Default parameters for the CLUSTAL
W algorithm are:
TABLE-US-00002 Gap opening penalty: 10.0 Gap extension penalty:
0.05 Protein weight matrix: BLOSUM series DNA weight matrix: IUB
Delay divergent sequences %: 40 Gap separation distance: 8 DNA
transitions weight: 0.50 List hydrophilic residues: GPSNDQEKR Use
negative matrix: OFF Toggle Residue specific penalties: ON Toggle
hydrophilic penalties: ON Toggle end gap separation penalty
OFF.
[0087] Deletions are counted as non-identical residues, compared to
a reference sequence. Deletions occurring at either terminus are
included. For example, a variant with six amino acid deletions of
the C-terminus of the mature AfGA1 polypeptide of SEQ ID NO: 12
would have a percent sequence identity of 99% (606/612 identical
residues.times.100, rounded to the nearest whole number) relative
to the mature polypeptide. Such a variant would be encompassed by a
variant having "at least 99% sequence identity" to a mature AfGA1
polypeptide.
[0088] "Fused" polypeptide sequences are connected, i.e., operably
linked, via a peptide bond between the two polypeptide
sequences.
[0089] The term "filamentous fungi" refers to all filamentous forms
of the subdivision Eumycotina.
[0090] The phrase "simultaneous saccharification and fermentation
(SSF)" refers to a process in the production of biochemicals in
which a microbial organism, such as an ethanologenic microorganism,
and at least one enzyme, such as AfGA or a variant thereof, are
present during the same process step. SSF includes the
contemporaneous hydrolysis of starch substrates (granular,
liquefied, or solubilized) to saccharides, including glucose, and
the fermentation of the saccharides into alcohol or other
biochemical or biomaterial in the same reactor vessel.
[0091] As used herein "ethanologenic microorganism" refers to a
microorganism with the ability to convert a sugar or
oligosaccharide to ethanol.
[0092] The term "fermented beverage" refers to any beverage
produced by a method comprising a fermentation process, such as a
microbial fermentation, e.g., a bacterial and/or yeast
fermentation.
[0093] "Beer" is an example of such a fermented beverage, and the
term "beer" is meant to comprise any fermented wort produced by
fermentation/brewing of a starch-containing plant material. Often,
beer is produced exclusively from malt or adjunct, or any
combination of malt and adjunct. Examples of beers include: full
malted beer, beer brewed under the "Reinheitsgebot," ale, IPA,
lager, bitter, Happoshu (second beer), third beer, dry beer, near
beer, light beer, low alcohol beer, low calorie beer, porter, bock
beer, stout, malt liquor, non-alcoholic beer, non-alcoholic malt
liquor and the like, but also alternative cereal and malt beverages
such as fruit flavored malt beverages, e.g., citrus flavored, such
as lemon-, orange-, lime-, or berry-flavored malt beverages, liquor
flavored malt beverages, e.g., vodka-, rum-, or tequila-flavored
malt liquor, or coffee flavored malt beverages, such as
caffeine-flavored malt liquor, and the like.
[0094] The term "malt" refers to any malted cereal grain, such as
malted barley or wheat.
[0095] The term "adjunct" refers to any starch and/or sugar
containing plant material which is not malt, such as barley or
wheat malt. Examples of adjuncts include common corn grits, refined
corn grits, brewer's milled yeast, rice, sorghum, refined corn
starch, barley, barley starch, dehusked barley, wheat, wheat
starch, torrified cereal, cereal flakes, rye, oats, potato,
tapioca, cassava and syrups, such as corn syrup, sugar cane syrup,
inverted sugar syrup, barley and/or wheat syrups, and the like.
[0096] The term "mash" refers to an aqueous slurry of any starch
and/or sugar containing plant material, such as grist, e.g.,
comprising crushed barley malt, crushed barley, and/or other
adjunct or a combination thereof, mixed with water later to be
separated into wort and spent grains.
[0097] The term "wort" refers to the unfermented liquor run-off
following extracting the grist during mashing.
[0098] "Iodine-positive starch" or "IPS" refers to (1) amylose that
is not hydrolyzed after liquefaction and saccharification, or (2) a
retrograded starch polymer. When saccharified starch or saccharide
liquor is tested with iodine, the high DPn amylose or the
retrograded starch polymer binds iodine and produces a
characteristic blue color. The saccharide liquor is thus termed
"iodine-positive saccharide," "blue saccharide," or "blue sac."
[0099] The terms "retrograded starch" or "starch retrogradation"
refer to changes that occur spontaneously in a starch paste or gel
on ageing.
[0100] The term "about" refers to .+-.15% to the referenced
value.
2. Aspergillus fumigatus Glucoamylases (AfGA1 and AfGA2)
[0101] An isolated and/or purified AfGA1, or a variant thereof,
polypeptide from A. fumigatus sp., which has glucoamylase activity
is provided. The glucoamylase consists of three distinct structural
domains, including a catalytic domain, followed by a linker region,
that are in turn connected to a starch binding domain. The AfGA1
polypeptide can be the mature AfGA1 polypeptide depicted in SEQ ID
NO: 12. The polypeptides may be fused to additional amino acid
sequences at the N-terminus and/or C-terminus. Additional
N-terminal sequences can be a signal peptide, which may have the
sequence shown in SEQ ID NO:11, for example. Other amino acid
sequences fused at either termini include fusion partner
polypeptides useful for labeling or purifying the protein.
[0102] For example, the AfGA1 precursor includes the sequence below
(SEQ ID NO: 1):
TABLE-US-00003 MPRLSYALCALSLGHAAIAAPQLSARATGSLDSWLGTETTVALNGILANI
GADGAYAKSAKPGIIIASPSTSEPDYYYTWTRDAALVTKVLVDLFRNGNL
GLQKVITEYVNSQAYLQTVSNPSGGLASGGLAEPKYNVDMTAFTGAWGRP
QRDGPALRATALIDFGNWLIDNGYSSYAVNNIWPIVRNDLSYVSQYWSQS
GFDLWEEVNSMSFFTVAVQHRALVEGSTFAKRVGASCSWCDSQAPQILCY
MQSFWTGSYINANTGGGRSGKDANTVLASIHTFDPEAGCDDTTFQPCSPR
ALANHKVYTDSFRSVYAINSGIPQGAAVSAGRYPEDVYYNGNPWFLTTLA
AAEQLYDAIYQWKKIGSISITSTSLAFFKDIYSSAAVGTYASSTSTFTDI
INAVKTYADGYVSIVQAHAMNNGSLSEQFDKSSGLSLSARDLTWSYAAFL
TANMRRNGVVPAPWGAASANSVPSSCSMGSATGTYSTATATSWPSTLTSG
SPGSTTTVGTTTSTTSGTAAETACATPTAVAVTFNEIATTTYGENVYIVG
SISELGNWDTSKAVALSASKYTSSNNLWYVSVTLPAGTTFEYKYIRKESD
GSIVWESDPNRSYTVPAACGVSTATENDTWQ
[0103] An isolated and/or purified AfGA2, or a variant thereof,
polypeptide from A. fumigatus sp., which has glucoamylase activity
is also provided. The glucoamylase consists of three distinct
structural domains, including a catalytic domain, followed by a
linker region, that are in turn connected to a starch binding
domain. The AfGA2 polypeptide can be the mature AfGA2 polypeptide
depicted in SEQ ID NO: 13. The polypeptides may be fused to
additional amino acid sequences at the N-terminus and/or
C-terminus. Additional N-terminal sequences can be a signal
peptide, which may have the sequence shown in SEQ ID NO: 11, for
example. Other amino acid sequences fused at either termini include
fusion protein polypeptides useful for labeling or purifying the
protein.
[0104] For example, the AfGA2 precursor includes the sequence below
(SEQ ID NO: 2):
TABLE-US-00004 MPRLSYALCALSLGHAAIAAPQLSARATGSLDSWLGTETTVALNGILANI
GADGAYAKSAKPGIIIASPSTSEPDYYYTWTRDAALVTKVLVDLFRNGNL
GLQKVITEYVNSQAYLQTVSNPSGGLASGGLAEPKYNVDMTAFTGAWGRP
QRDGPALRATALIDFGNWLIDNGYSSYAVNNIWPIVRNDLSYVSQYWSQS
GFDLWEEVNSMSFFTVAVQHRALVEGSTFAKRVGASCSWCDSQAPQILCY
MQSFWTGSYINANTGGGRSGKDANTVLASIHTFDPEAGCDDTTFQPCSPR
ALANHKVYTDSFRSVYAINSGIPQGAAVSAGRYPEDVYYNGNPWFLTTLA
AAEQLYDAIYQWKKIGSISITSTSLAFFKDIYSSAAVGTYASSTSTFTDI
INAVKTYADGYVSIVQAHAMNNGSLSEQFDKSSGLSLSARDLTWSYAAFL
TANMRRNGVVPAPWGAASANSVPSSCSMGSATGTYSTATATSWPSTLTSG
SPGSTTTVGTTTSTTSGTATETACATPTAVAVTFNEIATTTYGENVYIVG
SISELGNWDTSKAVALSASKYTSSNNLWYVSVTLPAGTTFEYKYIRKESD
GSIVWESDPNRSYTVPAACGVSTATENDTWR
[0105] The bolded amino acids above constitute a C-terminal
carbohydrate binding (CBM) domain (SEQ ID NO: 7) for both AfGA1 and
AfGA2. A glycosylated linker region connects the N-terminal
catalytic core with the CBM domain. The CBM domain in AfGA1 and
AfGA2 is conserved with a CBM20 domain found in a large number of
starch degrading enzymes, including alpha-amylases, beta-amylases,
glucoamylases, and cyclodextrin glucanotransferases. CBM20 folds as
an antiparallel beta-barrel structure with two starch-binding sites
1 and 2. These two sites are thought to differ functionally: site 1
may act as the initial starch recognition site, whereas site 2 may
be involved in specific recognition of appropriate regions of
starch. See Sorimachi et al. (1997) "Solution structure of the
granular starch binding domain of Aspergillus niger glucoamylase
bound to beta-cyclodextrin," Structure 5(5): 647-61. Residues in
the AfGA1 and AfGA2 CBM domain that are conserved with starch
binding sites 1 and 2 indicated in the sequence below by the
numbers 1 and 2, respectively:
TABLE-US-00005 (SEQ ID NO: 7)
FNEIATTTYGENVYIVGSISELGNWDTSKAVALSASKYTSSNNLWYVSVTL 222222 1 1 1111
2 2222 22 PAGTTFEYKYIRKESDGSIVWESDPNRSYTVPAACGVSTATENDTW. 1
[0106] A variant AfGA1 or AfGA2 may comprise some or no amino acid
residues of the CBM domain of SEQ ID NO: 7. A variant alternatively
may comprise a CBM domain with at least 80%, 85%, 90%, 95%, or 98%
sequence identity to the CBM domain of SEQ ID NO: 7. A variant may
comprise a heterologous or an engineered CBM20 domain.
[0107] The AfGA or variant thereof may be expressed in a eukaryotic
host cell, e.g., a filamentous fungal cell that allows proper
glycosylation of the linker sequence, for example.
[0108] A representative polynucleotide encoding AfGA1 is the
polynucleotide sequence set forth in SEQ ID NO: 8. A representative
polynucleotide encoding AfGA2 is the polynucleotide sequence set
forth in SEQ ID NO: 14. (NCBI Reference Sequence NC_007195, the A.
fumigatus genome.) The polypeptide sequence, MPRLSYALCALSLGHAAIA
(SEQ ID NO: 11), shown in italics in the AfGA1 and AfGA2 precursor
sequences above, is an N-terminal signal peptide that is cleaved
when the protein is expressed in an appropriate host cell.
[0109] The polypeptide sequence of AfGA1 is similar to other fungal
glucoamylases, including AfGA2. For example, AfGA1 has the high
sequence identity to the following fungal glucoamylases: [0110] 99%
sequence identity to the glycosyl hydrolase from Aspergillus
fumigatus A1163 (SEQ ID NO: 2)(AfGA2); [0111] 92% sequence identity
to the glycosyl hydrolase from Neosartorya fisheri NRRL 181 (SEQ ID
NO: 3); and [0112] 82% sequence identity to the putative
glucoamylase from Talaromyces stipitatus ATCC 10500 (SEQ ID NO: 4);
[0113] 81% sequence identity to the putative glucoamylase from
Penicillium marneffei ATCC 18224 (SEQ ID NO: 5); [0114] 81%
sequence identity to the hypothetical glucoamylase from Aspergillus
nidulans FGSC A4 (SEQ ID NO: 6); Sequence identity was determined
by a BLAST alignment, using the precursor form of the AfGA1 of SEQ
ID NO: 1 as the query sequence. See Altschul et al. (1990) J. Mol.
Biol. 215: 403-410. Sequence identity may also optionally be based
on the mature form of the enzyme.
[0115] A variant of an AfGA1 polypeptide is provided. The variant
can consist of or comprise a polypeptide with at least 80%, at
least 90%, at least 95%, at least 98%, or at least 99% amino acid
sequence identity to the polypeptide of residues 1-631 of SEQ ID
NO: 1, wherein the variant comprises one or more amino acid
modifications selected from a substitution, insertion, or deletion
of one or more corresponding amino acids in SEQ ID NO: 2-6. A
variant of an AfGA2 polypeptide is also provided. The variant can
consist of, or comprise, a polypeptide with at least 80%, at least
90%, at least 95%, at least 98%, or at least 99% amino acid
sequence identity to the polypeptide of residues 1-631 of SEQ ID
NO: 2, wherein the variant comprises one or more amino acid
modifications selected from a substitution, insertion, or deletion
of one or more corresponding amino acids in SEQ ID NO: 1 and/or
3-6. For example, a variant consisting of a polypeptide with at
least 99% sequence identity to the polypeptide of residues 1-612 of
SEQ ID NO:1 may have one to six amino acid substitutions,
insertions, or deletions, compared to the AfGA1 of SEQ ID NO: 1.
The insertions or deletions may be may at either termini of the
polypeptide, for example. Alternatively, the variant can "comprise"
a polypeptide consisting of a polypeptide with at least 80%, at
least 90%, at least 95%, at least 98%, or at least 99% amino acid
sequence identity to the polypeptide of 1-631 of SEQ ID NO: 1 or 2.
In a variant, additional amino acid residues may be fused to either
termini of the polypeptide. The variant may be glycosylated,
regardless of whether the variant "comprises" or "consists" of a
given amino acid sequence.
[0116] A ClustalW alignment between AfGA1 (SEQ ID NO: 1); AfGA2
(SEQ ID NO: 2); the glucoamylase from Neosartorya fisheri NRRL 181
(SEQ ID NO: 3); the glucoamylase from Talaromyces stipitatus ATCC
10500 (SEQ ID NO: 4); the glucoamylase from Penicillium marneffei
ATCC 18224 (SEQ ID NO: 5); and the glucoamylase Aspergillus
nidulans FGSC A4 (SEQ ID NO: 6) is shown in FIG. 1. See Thompson et
al. (1994) Nucleic Acids Res. 22:4673-4680. As a general rule, the
degree to which an amino acid is conserved in an alignment of
related protein sequences is proportional to the relative
importance of the amino acid position to the function of the
protein. That is, amino acids that are common in all related
sequences likely play an important functional role, and cannot be
easily substituted. Likewise, positions that vary between the
sequences likely can be substituted with other amino acids or
otherwise modified, while maintaining the activity of the
protein.
[0117] The alignments shown in FIG. 1, for example, can guide the
construction of variant AfGA polypeptides having glucoamylase
activity. Variants of the AfGA1 polypeptide of SEQ ID NO: 1 can
include, but are not limited to, those with an amino acid
modification selected from a substitution, insertion, or deletion
of a corresponding amino acid in a polypeptide selected from the
group consisting of SEQ ID NOs: 2 (AfGA2), 3, 4, 5, and 6.
Correspondence between positions in the AfGA1 of SEQ ID NO: 1 and
the glucoamylases of SEQ ID NOs: 2, 3, 4, 5 and 6 is determined
with reference to the alignment shown in FIG. 1. For example, a
variant AfGA1 polypeptide can have the substitution D23N, where Asn
is the corresponding amino acid in SEQ ID NO: 6, referring to the
alignment in FIG. 1. Variant AfGA1 polypeptides also include, but
are not limited to, those with 1, 2, 3, or 4 randomly selected
amino acid modifications. Amino acid modifications can be made
using well-known methodologies, such as oligo-directed mutagenesis.
Similarly, variants of the AfGA2 polypeptide of SEQ ID NO: 2 can
include, but are not limited to, those with an amino acid
modification selected from a substitution, insertion, or deletion
of a corresponding amino acid in a polypeptide selected from the
group consisting of SEQ ID NOS: 1 (AfGA1), 3, 4, 5, and 6.
[0118] Nucleic acids encoding the AfGA1 polypeptide or variant
thereof also are provided. A nucleic acid encoding AfGA1 can be
genomic DNA. Or, the nucleic acid can be a cDNA comprising SEQ ID
NO: 8. Similarly, nucleic acids encoding the AfGA2 polypeptide or
variant thereof also are provided. A nucleic acid encoding AfGA2
can also be genomic DNA. Or, the nucleic acid can be a cDNA
comprising SEQ ID NO: 14. As is well understood by one skilled in
the art, the genetic code is degenerate, meaning that multiple
codons in some cases may encode the same amino acid. Nucleic acids
include all genomic DNA, mRNA, and cDNA sequences that encode an
AfGA1, AfGA2 or variant thereof.
[0119] The AfGA1, AfGA2 or variants thereof may be "precursor,"
"immature," or "full-length," in which case they include a signal
sequence, or "mature," in which case they lack a signal sequence.
The variant glucoamylases may also be truncated at the N- or
C-termini, so long as the resulting polypeptides retain
glucoamylases activity
2.1. AfGA Variant Characterization
[0120] Variant AfGA polypeptides retain glucoamylase activity. They
may have a specific activity higher or lower than the wild-type
AfGA polypeptide. Additional characteristics of the AfGA variant
include stability, pH range, temperature profile, oxidation
stability, and thermostability, for example. For example, the
variant may be pH stable for 24-60 hours from pH 3 to about pH 8,
e.g., pH 3.0-7.8; e.g., pH 3.0-7.5; pH 3.5-7.0; pH 4.0-6.7; or pH
5.0. An AfGA variant can be expressed at higher levels than the
wild-type AfGA, while retaining the performance characteristics of
the wild-type AfGA. AfGA variants also may have altered oxidation
stability in comparison to the parent glucoamylase. For example,
decreased oxidation stability may be advantageous in compositions
for starch liquefaction. The variant AfGA, have altered temperature
profile compared to the wild-type glucoamylase. Such AfGA variants
are advantageous for use in baking or other processes that require
elevated temperatures. Levels of expression and enzyme activity can
be assessed using standard assays known to the artisan skilled in
this field, including those disclosed below
3. Production of AfGA and Variants Thereof
[0121] The AfGA or variant thereof can be isolated from a host
cell, for example by secretion of the AfGA or variant from the host
cell. A cultured cell material comprising AfGA, or variant thereof,
can be obtained following secretion of the AfGA or variant from the
host cell. The AfGA, or variant thereof, is optionally purified
prior to use. The AfGA gene can be cloned and expressed according
to methods well known in the art. Suitable host cells include
bacterial, plant, or yeast cells, e.g., filamentous fungal cells.
Particularly useful host cells include Trichoderma reesei.
Trichoderma reesei host cells express AfGATRs at higher, or at
least comparable, levels to natively expressed AfGA Aspergillus
fumigatus.
[0122] In some embodiments, the AfGA is heterologously expressed in
a host at at least 10 g/liter. In some embodiments, the AfGA is
heterologously expressed at at least 15, 20, 25, 30, 35, 40, 45,
50, 55, 60, 65, 70, 75, 80, 90, 100, or 110 g/liter. In some
embodiments, the AfGA is heterologously expressed in a Trichoderma
reesei host, wherein the expression is at least 15, 20, 25, 30, 35,
40, 45, 50, 55, 60, 65, 70, 75, 80, 90, 100, or 110 g/liter. In
some embodiments, the AfGA is heterologously expressed in an
Aspergillus host, wherein the expression is at least 15, 20, 25,
30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, 100, or 110
g/liter.
[0123] The host cell may further express a nucleic acid encoding a
homologous or heterologous amylase, i.e., an amylase that is not
the same species as the host cell, or one or more other enzymes.
The amylase may be a variant amylase. Additionally, the host may
express one or more accessory enzymes, proteins, peptides. These
may benefit liquefaction, saccharification, fermentation, SSF, etc.
processes. Furthermore, the host cell may produce biochemicals in
addition to enzymes used to digest the carbon feedstock(s). Such
host cells may be useful for fermentation or simultaneous
saccharification and fermentation processes to reduce or eliminate
the need to add enzymes.
3.1. Vectors
[0124] A DNA construct comprising a nucleic acid encoding an AfGA
or variant thereof can be constructed to be expressed in a host
cell. Representative nucleic acids that encode AfGA1 include SEQ ID
NO: 8. Representative nucleic acids that encode AfGA2 include SEQ
ID NO: 14. Because of the well-known degeneracy in the genetic
code, variant polynucleotides that encode an identical amino acid
sequence can be designed and made with routine skill. It is also
well-known in the art to optimize codon use for a particular host
cell. Nucleic acids encoding an AfGA or variant thereof can be
incorporated into a vector. Vectors can be transferred to a host
cell using well-known transformation techniques, such as those
disclosed below.
[0125] The vector may be any vector that can be transformed into
and replicated within a host cell. For example, a vector comprising
a nucleic acid encoding an AfGA or variant thereof can be
transformed and replicated in a bacterial host cell as a means of
propagating and amplifying the vector. The vector also may be
transformed into an expression host, so that the encoding nucleic
acids can be expressed as a functional AfGA or variant thereof.
Host cells that serve as expression hosts can include filamentous
fungi, for example. The Fungal Genetics Stock Center (FGSC)
Catalogue of Strains lists suitable vectors for expression in
fungal host cells. See FGSC, Catalogue of Strains, University of
Missouri, at www.fgsc.net (last modified Jan. 17, 2007).
Representative vectors include pJG222 (Trex3gM-AfGA1) (FIG. 2) and
pJG313 (Trex3gM-AfGA2) (FIG. 10), each of which comprises a
pTrex3gM expression vector (U.S. Published Application No.
2011/0136197 A1), and allows expression a nucleic acid encoding
AfGA under the control of the cbh1 promoter in a fungal host. Both
pJG222 and pJG313 can be modified with routine skill to comprise
and express a nucleic acid encoding an AfGA variant.
[0126] A nucleic acid encoding an AfGA or a variant thereof can be
operably linked to a suitable promoter, which allows transcription
in the host cell. The promoter may be any DNA sequence that shows
transcriptional activity in the host cell of choice and may be
derived from genes encoding proteins either homologous or
heterologous to the host cell. For transcription in a fungal host,
examples of useful promoters are those derived from the gene
encoding Aspergillus oryzae TAKA amylase, Rhizomucor miehei
aspartic proteinase, Aspergillus niger neutral .alpha.-amylase, A.
niger acid stable .alpha.-amylase, A. niger glucoamylase,
Rhizomucor miehei lipase, A. oryzae alkaline protease, A. oryzae
triose phosphate isomerase, or A. nidulans acetamidase. When a gene
encoding an AfGA or variant thereof is expressed in a bacterial
species such as E. coli, a suitable promoter can be selected, for
example, from a bacteriophage promoter including a T7 promoter and
a phage lambda promoter. Examples of suitable promoters for the
expression in a yeast species include but are not limited to the
Gal 1 and Gal 10 promoters of Saccharomyces cerevisiae and the
Pichia pastoris AOX1 or AOX2 promoters. The pJG222 vector depicted
in FIG. 2, for example, contains a cbh1 promoter operably linked to
AfGA1. The pJG313 vector depicted in FIG. 10, contains a cbh1
promoter operably linked to AfGA2. cbh1 is an endogenous, inducible
promoter from T. reesei. See Liu et al. (2008) "Improved
heterologous gene expression in Trichoderma reesei by
cellobiohydrolase I gene (cbh1) promoter optimization," Acta
Biochim. Biophys. Sin (Shanghai) 40(2): 158-65.
[0127] The coding sequence can be operably linked to a signal
sequence. The DNA encoding the signal sequence may be the DNA
sequence naturally associated with the AfGA gene to be expressed.
For example, the DNA may encode the AfGA1 and AfGA2 signal sequence
of SEQ ID NO: 11 operably linked to a nucleic acid encoding an AfGA
or a variant thereof. The DNA encodes a signal sequence from a
species other than A. fumigatus. A signal sequence and a promoter
sequence comprising a DNA construct or vector can be introduced
into a fungal host cell and can be derived from the same source.
For example, the signal sequence is the cbh1 signal sequence that
is operably linked to a cbh1 promoter.
[0128] An expression vector may also comprise a suitable
transcription terminator and, in eukaryotes, polyadenylation
sequences operably linked to the DNA sequence encoding an AfGA or
variant thereof. Termination and polyadenylation sequences may
suitably be derived from the same sources as the promoter.
[0129] The vector may further comprise a DNA sequence enabling the
vector to replicate in the host cell. Examples of such sequences
are the origins of replication of plasmids pUC19, pACYC177, pUB110,
pE194, pAMB1, and pIJ702.
[0130] The vector may also comprise a selectable marker, e.g., a
gene the product of which complements a defect in the isolated host
cell, such as the dal genes from B. subtilis or B. licheniformis,
or a gene that confers antibiotic resistance such as, e.g.,
ampicillin, kanamycin, chloramphenicol or tetracycline resistance.
Furthermore, the vector may comprise Aspergillus selection markers
such as amdS, argB, niaD, and sC, a marker giving rise to
hygromycin resistance, or the selection may be accomplished by
co-transformation, such as known in the art. See e.g.,
International PCT Application WO 91/17243.
[0131] Intracellular expression may be advantageous in some
respects, e.g., when using certain bacteria or fungi as host cells
to produce large amounts of an AfGA or variant thereof for
subsequent purification. Extracellular secretion of the AfGA or
variant thereof into the culture medium can also be used to make a
cultured cell material comprising the isolated AfGA or variant
thereof.
[0132] The expression vector typically includes the components of a
cloning vector, such as, for example, an element that permits
autonomous replication of the vector in the selected host organism
and one or more phenotypically detectable markers for selection
purposes. The expression vector normally comprises control
nucleotide sequences such as a promoter, operator, ribosome binding
site, translation initiation signal and optionally, a repressor
gene or one or more activator genes. Additionally, the expression
vector may comprise a sequence coding for an amino acid sequence
capable of targeting the AfGA or variant thereof to a host cell
organelle such as a peroxisome, or to a particular host cell
compartment. Such a targeting sequence includes but is not limited
to the sequence serine-lysine-leucine (SKL), which is a known
peroxisome target signal. For expression under the direction of
control sequences, the nucleic acid sequence of the AfGA or variant
thereof is operably linked to the control sequences in proper
manner with respect to expression.
[0133] The procedures used to ligate the DNA construct encoding an
AfGA or variant thereof, the promoter, terminator and other
elements, respectively, and to insert them into suitable vectors
containing the information necessary for replication, are well
known to persons skilled in the art (see, e.g., Sambrook et al.,
MOLECULAR CLONING: A LABORATORY MANUAL, 2.sup.nd ed., Cold Spring
Harbor, 1989, and 3.sup.rd ed., 2001).
3.2. Transformation and Culture of Host Cells
[0134] A Trichoderma reesei host cell, comprising either a DNA
construct or an expression vector, is advantageously used as a host
cell in the recombinant production of an AfGATR or variant thereof.
The cell may be transformed with the DNA construct encoding the
enzyme, conveniently by integrating the DNA construct (in one or
more copies) in the host chromosome. This integration is generally
considered to be an advantage, as the DNA sequence is more likely
to be stably maintained in the cell. Integration of the DNA
constructs into the host chromosome may be performed according to
conventional methods, e.g., by homologous or heterologous
recombination. Alternatively, the cell may be transformed with an
expression vector as described above in connection with the
different types of host cells.
[0135] It is advantageous to delete genes from expression hosts,
where the gene deficiency can be cured by the transformed
expression vector. Known methods may be used to obtain a fungal
host cell having one or more inactivated genes. Gene inactivation
may be accomplished by complete or partial deletion, by insertional
inactivation or by any other means that renders a gene
nonfunctional for its intended purpose, such that the gene is
prevented from expression of a functional protein. Any gene from a
Trichoderma sp. or other filamentous fungal host that has been
cloned can be deleted, for example, cbh1, cbh2, egl1, and egl2
genes. Gene deletion may be accomplished by inserting a form of the
desired gene to be inactivated into a plasmid by methods known in
the art.
[0136] Introduction of a DNA construct or vector into a host cell
includes techniques such as transformation; electroporation;
nuclear microinjection; transduction; transfection, e.g.,
lipofection mediated and DEAE-Dextrin mediated transfection;
incubation with calcium phosphate DNA precipitate; high velocity
bombardment with DNA-coated microprojectiles; and protoplast
fusion. General transformation techniques are known in the art.
See, e.g., Sambrook et al. (2001), supra. The expression of
heterologous protein in Trichoderma is described, for example, in
U.S. Pat. No. 6,022,725. Reference is also made to Cao et al.
(2000) Science9:991-1001 for transformation of Aspergillus strains.
Genetically stable transformants can be constructed with vector
systems whereby the nucleic acid encoding an AfGA or variant
thereof is stably integrated into a host cell chromosome.
Transformants are then selected and purified by known
techniques.
[0137] The preparation of Trichoderma sp. for transformation, for
example, may involve the preparation of protoplasts from fungal
mycelia. See Campbell et al. (1989) Curr. Genet. 16: 53-56. The
mycelia can be obtained from germinated vegetative spores. The
mycelia are treated with an enzyme that digests the cell wall,
resulting in protoplasts. The protoplasts are protected by the
presence of an osmotic stabilizer in the suspending medium. These
stabilizers include sorbitol, mannitol, potassium chloride,
magnesium sulfate, and the like. Usually the concentration of these
stabilizers varies between 0.8 M and 1.2 M, e.g., a 1.2 M solution
of sorbitol can be used in the suspension medium.
[0138] Uptake of DNA into the host Trichoderma sp. strain depends
upon the calcium ion concentration. Generally, between about 10-50
mM CaCl.sub.2 is used in an uptake solution. Additional suitable
compounds include a buffering system, such as TE buffer (10 mM
Tris, pH 7.4; 1 mM EDTA) or 10 mM MOPS, pH 6.0 and polyethylene
glycol. The polyethylene glycol is believed to fuse the cell
membranes, thus permitting the contents of the medium to be
delivered into the cytoplasm of the Trichoderma sp. strain. This
fusion frequently leaves multiple copies of the plasmid DNA
integrated into the host chromosome.
[0139] Usually transformation of Trichoderma sp. uses protoplasts
or cells that have been subjected to a permeability treatment,
typically at a density of 10.sup.5 to 10.sup.7/mL, particularly
2.times.10.sup.6/mL. A volume of 100 .mu.L of these protoplasts or
cells in an appropriate solution (e.g., 1.2 M sorbitol and 50 mM
CaCl.sub.2) may be mixed with the desired DNA. Generally, a high
concentration of PEG is added to the uptake solution. From 0.1 to 1
volume of 25% PEG 4000 can be added to the protoplast suspension;
however, it is useful to add about 0.25 volumes to the protoplast
suspension. Additives, such as dimethyl sulfoxide, heparin,
spermidine, potassium chloride and the like, may also be added to
the uptake solution to facilitate transformation. Similar
procedures are available for other fungal host cells. See, e.g.,
U.S. Pat. No. 6,022,725.
3.3. Expression
[0140] A method of producing an AfGATR or variant thereof may
comprise cultivating a Trichoderma reesei host cell as described
above under conditions conducive to the production of the enzyme
and recovering the enzyme from the cells and/or culture medium.
Trichoderma reesei host cells express AfGATRs at higher, or at
least comparable, levels to natively expressed AfGA Aspergillus
fumigatus.
[0141] The medium used to cultivate the cells may be any
conventional medium suitable for growing the host cell in question
and obtaining expression of an AfGATR or variant thereof. Suitable
media and media components are available from commercial suppliers
or may be prepared according to published recipes (e.g., as
described in catalogues of the American Type Culture
Collection).
[0142] An enzyme secreted from the host cells can be used in a
whole broth preparation. In the present methods, the preparation of
a spent whole fermentation broth of a recombinant microorganism can
be achieved using any cultivation method known in the art resulting
in the expression of a glucoamylase. Fermentation may, therefore,
be understood as comprising shake flask cultivation, small- or
large-scale fermentation (including continuous, batch, fed-batch,
or solid state fermentations) in laboratory or industrial
fermenters performed in a suitable medium and under conditions
allowing the glucoamylase to be expressed or isolated. The term
"spent whole fermentation broth" is defined herein as the
unfractionated contents of fermentation material that includes
culture medium, extracellular proteins (e.g., enzymes), and
cellular biomass. It is understood that the term "spent whole
fermentation broth" also encompasses cellular biomass that has been
lysed or permeabilized using methods well known in the art.
[0143] An enzyme secreted from the host cells may conveniently be
recovered from the culture medium by well-known procedures,
including separating the cells from the medium by centrifugation or
filtration, and precipitating proteinaceous components of the
medium by means of a salt such as ammonium sulfate, followed by the
use of chromatographic procedures such as ion exchange
chromatography, affinity chromatography, or the like.
[0144] The polynucleotide encoding AfGA or a variant thereof in a
vector can be operably linked to a control sequence that is capable
of providing for the expression of the coding sequence by the host
cell, i.e. the vector is an expression vector. The control
sequences may be modified, for example by the addition of further
transcriptional regulatory elements to make the level of
transcription directed by the control sequences more responsive to
transcriptional modulators. The control sequences may in particular
comprise promoters.
[0145] Host cells may be cultured under suitable conditions that
allow expression of the AfGATR or variant thereof. Expression of
the enzymes may be constitutive such that they are continually
produced, or inducible, requiring a stimulus to initiate
expression. In the case of inducible expression, protein production
can be initiated when required by, for example, addition of an
inducer substance to the culture medium, for example dexamethasone
or IPTG or Sepharose. Polypeptides can also be produced
recombinantly in an in vitro cell-free system, such as the TNT.TM.
(Promega) rabbit reticulocyte system.
[0146] An expression host also can be cultured in the appropriate
medium for the host, under aerobic conditions. Shaking or a
combination of agitation and aeration can be provided, with
production occurring at the appropriate temperature for that host,
e.g., from about 25.degree. C. to about 78.degree. C. (e.g.,
30.degree. C. to 45.degree. C.), depending on the needs of the host
and production of the desired AfGATR or variant thereof. Culturing
can occur from about 12 to about 100 hours or greater (and any hour
value there between, e.g., from 24 to 72 hours). Typically, the
culture broth is at a pH of about 4.0 to about 8.0, again depending
on the culture conditions needed for the host relative to
production of an AfGATR or variant thereof.
3.4. Identification of AfGATR Activity
[0147] To evaluate the expression of an AfGATR or variant thereof
in a host cell, assays can measure the expressed protein,
corresponding mRNA, or glucoamylase activity. For example, suitable
assays include Northern blotting, reverse transcriptase polymerase
chain reaction, and in situ hybridization, using an appropriately
labeled hybridizing probe. Suitable assays also include measuring
AfGATR activity in a sample, for example, by assays directly
measuring reducing sugars such as glucose in the culture media. For
example, glucose concentration may be determined using glucose
reagent kit No. 15-UV (Sigma Chemical Co.) or an instrument, such
as Technicon Autoanalyzer. Glucoamylase activity also may be
measured by any known method, such as the PAHBAH or ABTS assays,
described below.
3.5. Methods for Purifying an AfGATR and Variants Thereof
[0148] Fermentation, separation, and concentration techniques are
well known in the art and conventional methods can be used in order
to prepare a concentrated AfGATR or variant glucoamylase
polypeptide-containing solution.
[0149] After fermentation, a fermentation broth is obtained, the
microbial cells and various suspended solids, including residual
raw fermentation materials, are removed by conventional separation
techniques in order to obtain an amylase solution. Filtration,
centrifugation, microfiltration, rotary vacuum drum filtration,
ultrafiltration, centrifugation followed by ultrafiltration,
extraction, or chromatography, or the like, are generally used.
[0150] It is desirable to concentrate an AfGATR or variant
glucoamylase polypeptide-containing solution in order to optimize
recovery. Use of unconcentrated solutions can require increased
incubation time in order to collect the purified enzyme
precipitate.
[0151] The enzyme containing solution is concentrated using
conventional concentration techniques until the desired enzyme
level is obtained. Concentration of the enzyme containing solution
may be achieved by any of the techniques discussed herein.
Exemplary methods of purification include but are not limited to
rotary vacuum filtration and/or ultrafiltration.
[0152] The enzyme solution is concentrated into a concentrated
enzyme solution until the enzyme activity of the concentrated
AfGATR or variant glucoamylase polypeptide-containing solution is
at a desired level.
[0153] Concentration may be performed using, e.g., a precipitation
agent, such as a metal halide precipitation agent. Metal halide
precipitation agents include but are not limited to alkali metal
chlorides, alkali metal bromides, and blends of two or more of
these metal halides. Exemplary metal halides include sodium
chloride, potassium chloride, sodium bromide, potassium bromide,
and blends of two or more of these metal halides. The metal halide
precipitation agent, sodium chloride, can also be used as a
preservative.
[0154] The metal halide precipitation agent is used in an amount
effective to precipitate the AfGATR or variant thereof. The
selection of at least an effective amount and an optimum amount of
metal halide effective to cause precipitation of the enzyme, as
well as the conditions of the precipitation for maximum recovery
including incubation time, pH, temperature and concentration of
enzyme, will be readily apparent to one of ordinary skill in the
art, after routine testing.
[0155] Generally, at least about 5% w/v (weight/volume) to about
25% w/v of metal halide is added to the concentrated enzyme
solution, and usually at least 8% w/v. Generally, no more than
about 25% w/v of metal halide is added to the concentrated enzyme
solution and usually no more than about 20% w/v. The optimal
concentration of the metal halide precipitation agent will depend,
among others, on the nature of the specific AfGATR or variant
glucoamylase polypeptide and on its concentration in the
concentrated enzyme solution.
[0156] Another alternative way to precipitate the enzyme is to use
organic compounds. Exemplary organic compound precipitating agents
include: 4-hydroxybenzoic acid, alkali metal salts of
4-hydroxybenzoic acid, alkyl esters of 4-hydroxybenzoic acid, and
blends of two or more of these organic compounds. The addition of
said organic compound precipitation agents can take place prior to,
simultaneously with or subsequent to the addition of the metal
halide precipitation agent, and the addition of both precipitation
agents, organic compound and metal halide, may be carried out
sequentially or simultaneously.
[0157] Generally, the organic precipitation agents are selected
from the group consisting of alkali metal salts of 4-hydroxybenzoic
acid, such as sodium or potassium salts, and linear or branched
alkyl esters of 4-hydroxybenzoic acid, wherein the alkyl group
contains from 1 to 12 carbon atoms, and blends of two or more of
these organic compounds. The organic compound precipitation agents
can be, for example, linear or branched alkyl esters of
4-hydroxybenzoic acid, wherein the alkyl group contains from 1 to
10 carbon atoms, and blends of two or more of these organic
compounds. Exemplary organic compounds are linear alkyl esters of
4-hydroxybenzoic acid, wherein the alkyl group contains from 1 to 6
carbon atoms, and blends of two or more of these organic compounds.
Methyl esters of 4-hydroxybenzoic acid, propyl esters of
4-hydroxybenzoic acid, butyl ester of 4-hydroxybenzoic acid, ethyl
ester of 4-hydroxybenzoic acid and blends of two or more of these
organic compounds can also be used. Additional organic compounds
also include but are not limited to 4-hydroxybenzoic acid methyl
ester (named methyl PARABEN), 4-hydroxybenzoic acid propyl ester
(named propyl PARABEN), which also are both amylase preservative
agents. For further descriptions, see, e.g., U.S. Pat. No.
5,281,526.
[0158] Addition of the organic compound precipitation agent
provides the advantage of high flexibility of the precipitation
conditions with respect to pH, temperature, AfGATR or variant
glucoamylase polypeptide concentration, precipitation agent
concentration, and time of incubation.
[0159] The organic compound precipitation agent is used in an
amount effective to improve precipitation of the enzyme by means of
the metal halide precipitation agent. The selection of at least an
effective amount and an optimum amount of organic compound
precipitation agent, as well as the conditions of the precipitation
for maximum recovery including incubation time, pH, temperature and
concentration of enzyme, will be readily apparent to one of
ordinary skill in the art, in light of the present disclosure,
after routine testing.
[0160] Generally, at least about 0.01% w/v of organic compound
precipitation agent is added to the concentrated enzyme solution
and usually at least about 0.02% w/v. Generally, no more than about
0.3% w/v of organic compound precipitation agent is added to the
concentrated enzyme solution and usually no more than about 0.2%
w/v.
[0161] The concentrated polypeptide solution, containing the metal
halide precipitation agent, and the organic compound precipitation
agent, can be adjusted to a pH, which will, of necessity, depend on
the enzyme to be purified. Generally, the pH is adjusted at a level
near the isoelectric point of the glucoamylase. The pH can be
adjusted at a pH in a range from about 2.5 pH units below the
isoelectric point (pI) up to about 2.5 pH units above the
isoelectric point.
[0162] The incubation time necessary to obtain a purified enzyme
precipitate depends on the nature of the specific enzyme, the
concentration of enzyme, and the specific precipitation agent(s)
and its (their) concentration. Generally, the time effective to
precipitate the enzyme is between about 1 to about 30 hours;
usually it does not exceed about 25 hours. In the presence of the
organic compound precipitation agent, the time of incubation can
still be reduced to less than about 10 hours and in most cases even
about 6 hours.
[0163] Generally, the temperature during incubation is between
about 4.degree. C. and about 50.degree. C. Usually, the method is
carried out at a temperature between about 10.degree. C. and about
45.degree. C. (e.g., between about 20.degree. C. and about
40.degree. C.). The optimal temperature for inducing precipitation
varies according to the solution conditions and the enzyme or
precipitation agent(s) used.
[0164] The overall recovery of purified enzyme precipitate, and the
efficiency with which the process is conducted, is improved by
agitating the solution comprising the enzyme, the added metal
halide and the added organic compound. The agitation step is done
both during addition of the metal halide and the organic compound,
and during the subsequent incubation period. Suitable agitation
methods include mechanical stirring or shaking, vigorous aeration,
or any similar technique.
[0165] After the incubation period, the purified enzyme can be then
separated from the dissociated pigment and other impurities and
collected by conventional separation techniques, such as
filtration, centrifugation, microfiltration, rotary vacuum
filtration, ultrafiltration, press filtration, cross membrane
microfiltration, cross flow membrane microfiltration, or the like.
Further purification of the purified enzyme precipitate can be
obtained by washing the precipitate with water. For example, the
purified enzyme precipitate is washed with water containing the
metal halide precipitation agent, or with water containing the
metal halide and the organic compound precipitation agents.
[0166] During fermentation, an AfGATR or variant glucoamylase
polypeptide accumulates in the culture broth. For the isolation and
purification of the desired AfGATR or variant glucoamylase, the
culture broth can be centrifuged or filtered to eliminate cells,
and the resulting cell-free liquid is used for enzyme purification.
In one embodiment, the cell-free broth is subjected to salting out
using ammonium sulfate at about 70% saturation; the 70%
saturation-precipitation fraction is then dissolved in a buffer and
applied to a column such as a Sephadex G-100 column, and eluted to
recover the enzyme-active fraction. For further purification, a
conventional procedure such as ion exchange chromatography may be
used.
[0167] Purified enzymes are useful for laundry and cleaning
applications. For example, they can be used in laundry detergents
and spot removers. They can be made into a final product that is
either liquid (solution, slurry) or solid (granular, powder).
[0168] A more specific example of purification, is described in
Sumitani et al. (2000) "New type of starch-binding domain: the
direct repeat motif in the C-terminal region of Bacillus sp. 195
glucoamylase contributes to starch binding and raw starch
degrading," Biochem. J. 350: 477-484, and is briefly summarized
here. The enzyme obtained from 4 liters of a Streptomyces lividans
TK24 culture supernatant was treated with (NH.sub.4).sub.2SO.sub.4
at 80% saturation. The precipitate was recovered by centrifugation
at 10,000.times.g (20 min. and 4.degree. C.) and re-dissolved in 20
mM Tris/HCl buffer (pH 7.0) containing 5 mM CaCl.sub.2. The
solubilized precipitate was then dialyzed against the same buffer.
The dialyzed sample was then applied to a Sephacryl S-200 column,
which had previously been equilibrated with 20 mM Tris/HCl buffer,
(pH 7.0), 5 mM CaCl.sub.2, and eluted at a linear flow rate of 7
mL/hr with the same buffer. Fractions from the column were
collected and assessed for activity as judged by enzyme assay and
SDS-PAGE. The protein was further purified as follows. A Toyopearl
HW55 column (Tosoh Bioscience, Montgomeryville, Pa.; Cat. No.
19812) was equilibrated with 20 mM Tris/HCl buffer (pH 7.0)
containing 5 mM CaCl.sub.2 and 1.5 M (NH.sub.4).sub.2SO.sub.4. The
enzyme was eluted with a linear gradient of 1.5 to 0 M
(NH.sub.4).sub.2SO.sub.4 in 20 mM Tris/HCL buffer, pH 7.0
containing 5 mM CaCl.sub.2. The active fractions were collected,
and the enzyme precipitated with (NH.sub.4).sub.2SO.sub.4 at 80%
saturation. The precipitate was recovered, re-dissolved, and
dialyzed as described above. The dialyzed sample was then applied
to a Mono Q HR5/5 column (Amersham Pharmacia; Cat. No. 17-5167-01)
previously equilibrated with 20 mM Tris/HCl buffer (pH 7.0)
containing 5 mM CaCl.sub.2, at a flow rate of 60 mL/hour. The
active fractions are collected and added to a 1.5 M
(NH.sub.4).sub.2SO.sub.4 solution. The active enzyme fractions were
re-chromatographed on a Toyopearl HW55 column, as before, to yield
a homogeneous enzyme as determined by SDS-PAGE. See Sumitani et al.
(2000) Biochem. J. 350: 477-484, for general discussion of the
method and variations thereon.
[0169] For production scale recovery, an AfGATR or variant
glucoamylase polypeptide can be partially purified as generally
described above by removing cells via flocculation with polymers.
Alternatively, the enzyme can be purified by microfiltration
followed by concentration by ultrafiltration using available
membranes and equipment. However, for some applications, the enzyme
does not need to be purified, and whole broth culture can be lysed
and used without further treatment. The enzyme can then be
processed, for example, into granules.
4. Compositions and Uses of AfGATR and Variants Thereof
[0170] AfGATR and its variants are useful for a variety of
industrial applications. For example, AfGATR and its variants are
useful in a starch conversion process, particularly in a
saccharification process of a starch that has undergone
liquefaction. The desired end-product may be any product that may
be produced by the enzymatic conversion of the starch substrate.
For example, the desired product may be a syrup rich in glucose,
which can be used in other processes, such as the preparation of
HFCS, or which can be converted into a number of other useful
products, such as ascorbic acid intermediates (e.g., gluconate;
2-keto-L-gulonic acid; 5-keto-gluconate; and 2,5-diketogluconate);
1,3-propanediol; aromatic amino acids (e.g., tyrosine,
phenylalanine and tryptophan); organic acids (e.g., lactate,
pyruvate, succinate, isocitrate, and oxaloacetate); amino acids
(e.g., serine and glycine); antibiotics; antimicrobials; enzymes;
vitamins; and hormones.
[0171] The starch conversion process may be a precursor to, or
simultaneous with, a fermentation process designed to produce
alcohol for fuel or drinking (i.e., potable alcohol). One skilled
in the art is aware of various fermentation conditions that may be
used in the production of these end-products. AfGATR and variants
thereof also are useful in compositions and methods of food
preparation. These various uses of AfGATR and its variants are
described in more detail below.
4.1. Preparation of Starch Substrates
[0172] Those of general skill in the art are well aware of
available methods that may be used to prepare starch substrates for
use in the processes disclosed herein. For example, a useful starch
substrate may be obtained from tubers, roots, stems, legumes,
cereals or whole grain. More specifically, the granular starch may
be obtained from corn, cobs, wheat, barley, rye, triticale, milo,
sago, millet, cassava, tapioca, sorghum, rice, peas, bean, banana,
or potatoes. Corn contains about 60-68% starch; barley contains
about 55-65% starch; millet contains about 75-80% starch; wheat
contains about 60-65% starch; and polished rice contains 70-72%
starch. Specifically contemplated starch substrates are cornstarch
and wheat starch. The starch from a grain may be ground or whole
and includes corn solids, such as kernels, bran and/or cobs. The
starch may be highly refined raw starch or feedstock from starch
refinery processes. Various starches also are commercially
available. For example, corn starch is available from Cerestar,
Sigma, and Katayama Chemical Industry Co. (Japan); wheat starch is
available from Sigma; sweet potato starch is available from Wako
Pure Chemical Industry Co. (Japan); and potato starch is available
from Nakaari Chemical Pharmaceutical Co. (Japan).
[0173] The starch substrate can be a crude starch from milled whole
grain, which contains non-starch fractions, e.g., germ residues and
fibers. Milling may comprise either wet milling or dry milling or
grinding. In wet milling, whole grain is soaked in water or dilute
acid to separate the grain into its component parts, e.g., starch,
protein, germ, oil, kernel fibers. Wet milling efficiently
separates the germ and meal (i.e., starch granules and protein) and
is especially suitable for production of syrups. In dry milling or
grinding, whole kernels are ground into a fine powder, and often
processed without fractionating the grain into its component parts.
In some cases, oils from the kernels are recovered. Dry ground
grain generally will comprise significant amounts of non-starch
carbohydrate compounds, in addition to starch. Dry grinding of the
starch substrate can be used for production of ethanol and other
biochemicals. The starch to be processed may be a highly refined
starch quality, for example, at least 90%, at least 95%, at least
97%, or at least 99.5% pure.
4.2. Gelatinization and Liquefaction of Starch
[0174] As used herein, the term "liquefaction" or "liquefy" means a
process by which starch is converted to less viscous and shorter
chain dextrins. Generally, this process involves gelatinization of
starch simultaneously with or followed by the addition of an
.alpha.-amylase, although additional liquefaction-inducing enzymes
optionally may be added. In some embodiments, the starch substrate
prepared as described above is slurried with water. The starch
slurry may contain starch as a weight percent of dry solids of
about 10-55%, about 20-45%, about 30-45%, about 30-40%, or about
30-35%. .alpha.-Amylase (EC 3.2.1.1) may be added to the slurry,
with a metering pump, for example. The .alpha.-amylase typically
used for this application is a thermally stable, bacterial
.alpha.-amylase, such as a Geobacillus stearothermophilus
.alpha.-amylase. The .alpha.-amylase is usually supplied, for
example, at about 1500 units per kg dry matter of starch. To
optimize .alpha.-amylase stability and activity, the pH of the
slurry typically is adjusted to about pH 5.5-6.5 and about 1 mM of
calcium (about 40 ppm free calcium ions) typically is added.
Geobacillus stearothermophilus variants or other .alpha.-amylases
may require different conditions. Bacterial .alpha.-amylase
remaining in the slurry following liquefaction may be deactivated
via a number of methods, including lowering the pH in a subsequent
reaction step or by removing calcium from the slurry in cases where
the enzyme is dependent upon calcium.
[0175] The slurry of starch plus the .alpha.-amylases may be pumped
continuously through a jet cooker, which is steam heated to
105.degree. C. Gelatinization occurs rapidly under these
conditions, and the enzymatic activity, combined with the shear
forces, begins the hydrolysis of the starch substrate. The
residence time in the jet cooker is brief. The partly gelatinized
starch may be passed into a series of holding tubes maintained at
105-110.degree. C. and held for 5-8 min. to complete the
gelatinization process ("primary liquefaction"). Hydrolysis to the
required DE is completed in holding tanks at 85-95.degree. C. or
higher temperatures for about 1 to 2 hours ("secondary
liquefaction"). These tanks may contain baffles to discourage back
mixing. As used herein, the term "minutes of secondary
liquefaction" refers to the time that has elapsed from the start of
secondary liquefaction to the time that the Dextrose Equivalent
(DE) is measured. The slurry is then allowed to cool to room
temperature. This cooling step can be 30 minutes to 180 minutes,
e.g. 90 minutes to 120 minutes.
[0176] The liquefied starch resulting from the process above
typically contains about 98% oligosaccharides and about 2% maltose
and 0.3% D-glucose. The liquefied starch typically is in the form
of a slurry having a dry solids content (w/w) of about 10-50%;
about 10-45%; about 15-40%; about 20-40%; about 25-40%; or about
25-35%.
[0177] AkAA, AtAmyl, AfAmyl, and AcAmyl and variants thereof can be
used in a process of liquefaction instead of bacterial
.alpha.-amylases. Liquefaction with these .alpha.-amylase and
variants thereof advantageously can be conducted at low pH,
eliminating the requirement to adjust the pH to about pH 5.5-6.5.
Theses .alpha.-amylases variants thereof can be used for
liquefaction at a pH range of 2 to 7, e.g., pH 3.0-7.5, pH 4.0-6.0,
or pH 4.5-5.8. They can maintain liquefying activity at a
temperature range of about 85.degree. C.-95.degree. C., e.g.,
85.degree. C., 90.degree. C., or 95.degree. C. For example,
liquefaction can be conducted with 800 .mu.g AcAmyl or a variant
thereof in a solution of 25% DS corn starch for 10 min at pH 5.8
and 85.degree. C., or pH 4.5 and 95.degree. C., for example.
Liquefying activity can be assayed using any of a number of known
viscosity assays in the art.
4.3. Saccharification
[0178] The liquefied starch can be saccharified into a syrup rich
in lower DP, especially DP1 saccharides, using the AfGATR and
variants thereof, optionally in the presence of another enzyme(s).
The exact composition of the products of saccharification depends
on the combination of enzymes used, as well as the type of granular
starch processed. Advantageously, the syrup obtainable using the
provided AfGATR and variants thereof may contain a weight percent
of DP1 of the total oligosaccharides in the saccharified starch
exceeding about 65%, e.g., 70%, 80%, 85%, 90%, 95%, or 96%.
[0179] Whereas liquefaction is generally run as a continuous
process, saccharification is often conducted as a batch process.
Saccharification typically is most effective at temperatures of
about 55.degree.-75.degree. C. and a pH of about 4.0-6.7, e.g., pH
5.0, necessitating cooling and adjusting the pH of the liquefied
starch. Saccharification may be performed, for example, at a
temperature between about 40.degree. C., about 55.degree. C., or
about 65.degree. C. to about 70.degree. C., about 75.degree. C., or
about 80.degree. C. Saccharification is normally conducted in
stirred tanks, which may take several hours to fill or empty.
Enzymes typically are added either at a fixed ratio to dried solids
as the tanks are filled or added as a single dose at the
commencement of the filling stage. A saccharification reaction to
make a syrup typically is run over about 24-72 hours, for example,
24-48 hours. When a maximum or desired DE has been attained, the
reaction is stopped by heating to 85.degree. C. for 5 min., for
example. Further incubation will result in a lower DE, eventually
to about 90 DE, as accumulated glucose re-polymerizes to isomaltose
and/or other reversion products via an enzymatic reversion reaction
and/or with the approach of thermodynamic equilibrium. When using
an AfGATR polypeptide or variants thereof, saccharification
optimally is conducted at a temperature range of about 40.degree.
C. to about 80.degree. C., e.g., about 55.degree. C. to about
75.degree. C. or about 65.degree. C. to about 70.degree. C. The
saccharifying may be conducted over a pH range of about pH 3.0 to
about pH 7.5, e.g., pH 3.5-pH 7.0, pH 4.0-pH 6.7, or pH 5.0.
[0180] AfGATR or a variant thereof may be added to the slurry in
the form of a composition. AfGATR or a variant thereof can be added
to a slurry of a granular starch substrate in an amount of about
0.6-10 ppm ds, e.g., 2 ppm ds. The AfGATR or variant thereof can be
added as a whole broth, clarified, partially purified, or purified
enzyme. The specific activity of the purified AfGA1TR or variant
thereof may be about 187.7 U/mg, for example, measured with the
ABTS assay. The specific activity of the purified AfGA2TR or
variant thereof may be about 213.7 U/mg, for example, measured with
the ABTS assay. The AfGATR or variant thereof also can be added as
a whole broth product.
[0181] AfGATR or a variant thereof may be added to the slurry as an
isolated enzyme solution. For example, AfGATR or a variant thereof
can be added in the form of a cultured cell material produced by
host cells expressing the AfGATR or variant thereof. AfGATR or a
variant thereof also may be secreted by a host cell into the
reaction medium during the fermentation or SSF process, such that
the enzyme is provided continuously into the reaction. The host
cell producing and secreting the AfGATR or a variant may also
express an additional enzyme, such as a glucoamylase. For example,
U.S. Pat. No. 5,422,267 discloses the use of a glucoamylase in
yeast for production of alcoholic beverages. For example, a host
cell, e.g., Trichoderma reesei may be engineered to co-express
AfGATR or a variant thereof and an .alpha.-amylase, including, but
not limited to AkAA, AcAmyl, native Trichoderma reesei
.alpha.-amylase, or variants thereof during saccharification. The
host cell can be genetically modified so as not to express its
endogenous glucoamylase and/or other enzymes, proteins or other
materials. The host cell can be engineered to express a broad
spectrum of various saccharolytic enzymes. For example, the
recombinant yeast host cell can comprise nucleic acids encoding a
glucoamylase, an alpha-glucosidase, an enzyme that utilizes pentose
sugar, an .alpha.-amylase, a pullulanase, an isoamylase, and/or an
isopullulanase. See, e.g., WO 2011/153516 A2.
4.4. Isomerization
[0182] The soluble starch hydrolysate produced by treatment with
AfGATR or variants thereof can be converted into high fructose
starch-based syrup (HFSS), such as high fructose corn syrup (HFCS).
This conversion can be achieved using a glucose isomerase,
particularly a glucose isomerase immobilized on a solid support.
The pH is increased to about 6.0 to about 8.0, e.g., pH 7.5, and
Ca.sup.2+ is removed by ion exchange. Suitable isomerases include
Sweetzyme.RTM., IT (Novozymes A/S); G-Zyme.RTM. IMGI, and
G-Zyme.RTM. G993, Ketomax.RTM., G-Zyme.RTM.G993, G-Zyme.RTM. G993
liquid, and GenSweet.RTM. IGI. Following isomerization, the mixture
typically contains about 40-45% fructose, e.g., 42% fructose.
4.5. Fermentation
[0183] The soluble starch hydrolysate, particularly a glucose rich
syrup, can be fermented by contacting the starch hydrolysate with a
fermenting organism typically at a temperature around 32.degree.
C., such as from 30.degree. C. to 35.degree. C. EOF products
include metabolites, such as citric acid, lactic acid, succinic
acid, monosodium glutamate, gluconic acid, sodium gluconate,
calcium gluconate, potassium gluconate, itaconic acid and other
carboxylic acids, glucono delta-lactone, sodium erythorbate, lysine
and other amino acids, omega 3 fatty acid, butanol, isoprene,
1,3-propanediol and other biomaterials.
[0184] Ethanologenic microorganisms include yeast, such as
Saccharomyces cerevisiae and bacteria, e.g., Zymomonas moblis,
expressing alcohol dehydrogenase and pyruvate decarboxylase. The
ethanologenic microorganism can express xylose reductase and
xylitol dehydrogenase, which convert xylose to xylulose. Improved
strains of ethanologenic microorganisms, which can withstand higher
temperatures, for example, are known in the art and can be used.
See Liu et al. (2011) Sheng Wu Gong Cheng Xue Bao 27(7): 1049-56.
Commercial sources of yeast include ETHANOL RED.RTM. (LeSaffre);
Thermosacc.RTM. (Lallemand); RED STAR.RTM. (Red Star); FERMIOL.RTM.
(DSM Specialties); and SUPERSTART.RTM. (Alltech). Microorganisms
that produce other metabolites, such as citric acid and lactic
acid, by fermentation are also known in the art. See, e.g.,
Papagianni (2007) "Advances in citric acid fermentation by
Aspergillus niger: biochemical aspects, membrane transport and
modeling," Biotechnol. Adv. 25(3): 244-63; John et al. (2009)
"Direct lactic acid fermentation: focus on simultaneous
saccharification and lactic acid production," Biotechnol. Adv.
27(2): 145-52.
[0185] The saccharification and fermentation processes may be
carried out as an SSF process. Fermentation may comprise subsequent
purification and recovery of ethanol, for example. During the
fermentation, the ethanol content of the broth or "beer" may reach
about 8-18% v/v, e.g., 14-15% v/v. The broth may be distilled to
produce enriched, e.g., 96% pure, solutions of ethanol. Further,
CO.sub.2 generated by fermentation may be collected with a CO.sub.2
scrubber, compressed, and marketed for other uses, e.g.,
carbonating beverage or dry ice production. Solid waste from the
fermentation process may be used as protein-rich products, e.g.,
livestock feed.
[0186] As mentioned above, an SSF process can be conducted with
fungal cells, such as Trichoderma reesei, that express and secrete
AfGATR or its variants continuously throughout SSF. The fungal
cells expressing AfGATR or its variants also can be the fermenting
microorganism, e.g., an ethanologenic microorganism. Ethanol
production thus can be carried out using a fungal cell that
expresses sufficient AfGATR or its variants so that less or no
enzyme has to be added exogenously. The fungal host cell can be
from an appropriately engineered fungal strain. Fungal host cells
that express and secrete other enzymes, in addition to AfGATR or
its variants, also can be used. Such cells may express
.alpha.-amylase and/or a pullulanase, phytase, alpha-glucosidase,
isoamylase, beta-amylase cellulase, xylanase, other hemicellulases,
protease, beta-glucosidase, pectinase, esterase, redox enzymes,
transferase, a glucoamylase other than AfGATR or other enzyme.
[0187] A variation on this process is a "fed-batch fermentation"
system, where the substrate is added in increments as the
fermentation progresses. Fed-batch systems are useful when
catabolite repression may inhibit the metabolism of the cells and
where it is desirable to have limited amounts of substrate in the
medium. The actual substrate concentration in fed-batch systems is
estimated by the changes of measurable factors such as pH,
dissolved oxygen and the partial pressure of waste gases, such as
CO.sub.2. Batch and fed-batch fermentations are common and well
known in the art.
[0188] Continuous fermentation is an open system where a defined
fermentation medium is added continuously to a bioreactor, and an
equal amount of conditioned medium is removed simultaneously for
processing. Continuous fermentation generally maintains the
cultures at a constant high density where cells are primarily in
log phase growth. Continuous fermentation permits modulation of
cell growth and/or product concentration. For example, a limiting
nutrient such as the carbon source or nitrogen source is maintained
at a fixed rate and all other parameters are allowed to moderate.
Because growth is maintained at a steady state, cell loss due to
medium being drawn off should be balanced against the cell growth
rate in the fermentation. Methods of optimizing continuous
fermentation processes and maximizing the rate of product formation
are well known in the art of industrial microbiology.
4.6. Compositions Comprising AfGATR or Variants Thereof
[0189] AfGATR or variants thereof may be combined with an
.alpha.-amylase (EC 3.2.1.1). In some embodiments, the
.alpha.-amylase is an acid stable alpha amylase which when added in
an effective amount has activity in the pH range of 3.0 to 7.0 and
preferably from 3.5 to 6.5. Alpha amylases may be a fungal
.alpha.-amylase or a bacterial .alpha.-amylase. Further, the
.alpha.-amylase may be a wild-type .alpha.-amylase or a variant
thereof.
[0190] Preferred examples of fungal alpha amylases include those
obtained from filamentous fungal strains including but not limited
to strains of Aspergillus (e.g., A. niger, A. kawachii, and A.
oryzae); Trichoderma sp., Rhizopus sp., Mucor sp., and Penicillium
sp. Lactobacilli sp. and Streptomuces sp. The acid stable
.alpha.-amylase may be derived from a bacterial strain. Preferred
bacterial strains include Bacillus sp., such as B. licheniformis,
B. stearothermophilus, B. amyloliquefaciens, B. subtilis, B.
lentus, and B. coagulans. Particularly preferred are B.
licheniformis, B. stearothermophilus, and B. amyloliquefaciens. One
of the bacterial alpha amylases used in the compositions and
processes of the invention may include one of the .alpha.-amylases
described in U.S. Pat. Nos. 5,093,257; 5,763,385; 5,824,532;
5,958,739; 6,008,026; 6,093,563; 6,187,576; 6,361,809; 6,867,031;
U.S. Publication No. 2006/0014265; and International PCT Nos. WO
96/23874, WO 96/39528; WO 97/141213, WO 99/19467; and WO
05/001064.
[0191] Exemplary .alpha.-amylases include is AkAA or AcAmyl and
variants thereof that possess superior specific activity and
thermal stability. Suitable variants of AkAA include those with
.alpha.-amylase activity and at least 80%, 90%, 95%, 98% or at
least 99% sequence identity to wild-type AkAA. Suitable variants of
AcAmyl include those with .alpha.-amylase activity and at least
80%, at least 90%, or at least 95% sequence identity to wild-type
AcAmyl. AfGATR and its variants advantageously increase the yield
of glucose produced in a saccharification process catalyzed by AnGA
or Tr-GA.
[0192] Commercially available alpha amylases contemplated for use
in the compositions and method include: SPEZYME.TM. AA; SPEZYME.TM.
FRED; SPEZYME.TM. XTRA; GZYME.TM. 997; and CLARASE.TM. L (Genencor
International Inc.); TERMAMYL.TM. 120-L, LC and SC and SUPRA
(Novozymes Biotech); LIQUOZYME.TM. X and SAN.TM. SUPER (Novozymes
A/S) and Fuelzyme.TM. LF (Diversa). In some embodiments, the alpha
amylase will include an alpha amylase derived from Bacillus
stearothermophilus such as SPEZYME.TM. AA, SPEZYME.TM. FRED or
SPEZYME.TM. XTRA. In some embodiments, the enzyme compositions will
include BP-WT, SPEZYME.TM. XTRA and optionally SPEZYME.TM. FRED. In
other embodiments, the compositions will include BP-17, SPEZYME.TM.
XTRA and optionally SPEZYME.TM. FRED.
[0193] Other suitable enzymes that can be used with AfGATR or its
variants include a glucoamylase that is not AfGATR, phytase,
protease, pullulanase, .beta.-amylase, isoamylase, .alpha.-amylase,
alpha-glucosidase, cellulase, xylanase, other hemicellulases,
beta-glucosidase, transferase, pectinase, lipase, cutinase,
esterase, redox enzymes, or a combination thereof.
[0194] For example, a debranching enzyme, such as a pullulanase (EC
3.2.1.41), e.g., Promozyme.RTM., may be added in effective amounts
well known to the person skilled in the art. Pullulanase typically
is added at 100 U/kg ds. Pullulanases are generally secreted by a
Bacillus species. Exemplary pullanases are described for Bacillus
deramificans (U.S. Pat. No. 5,817,498; 1998), Bacillus
acidopullulyticus (European Patent #0 063 909 and Bacillus
naganoensis (U.S. Pat. No. 5,055,403). Enzymes having pullulanase
activity used commercially are produced for examples, from Bacillus
species (trade name OPTIMAX.TM. L-1000 from Danisco-Genencor and
Promozyme.TM. from Novozymes).
[0195] Bacillus megaterium amylase/transferase (BMA): Bacillus
megaterium amylase has the ability to convert the branched
saccharides to a form that is easily hydrolysed by glucoamylase.
(Habeda R. E., Styrlund C. R and Teague, W. M.; 1988 Starch/Starke,
40, 33-36) The enzyme exhibits maximum activity at pH 5.5 and
temperature at 75 C. (David, M. H., Gunther H and Vilvoorde, H. R.;
1987, Starch/Starke, 39 436-440) The enzyme has been cloned,
expressed in a genetically engineered Bacillus subtilis and
produced on a commercial scale (Brumm, P. J., Habeda R. E, and
Teague W. M., 1991 Starch/Starke, 43 315-329). The enzyme is sold
under a trade name MEGADEX.TM. for enhancing the glucose yield
during the saccharification of enzyme liquefied starch by
Aspergillus niger glucoamylase.
[0196] An isoamylase (EC 3.2.1.68), may also be added in effective
amounts well known to the person skilled in the art. A pullulanase
(EC 3.2.1.41), e.g., Promozyme.RTM., is also suitable. Pullulanase
typically is added at 100 U/kg ds. Further suitable enzymes include
proteases, such as fungal and bacterial proteases. Fungal proteases
include those obtained from Aspergillus, such as A. niger, A.
awamori, A. oryzae; Mucor (e.g., M. miehei); Rhizopus; and
Trichoderma.
[0197] .beta.-Amylases (EC 3.2.1.2) are exo-acting maltogenic
amylases, which catalyze the hydrolysis of 1,4-.alpha.-glucosidic
linkages into amylopectin and related glucose polymers, thereby
releasing maltose. .beta.-Amylases have been isolated from various
plants and microorganisms. See Fogarty et al. (1979) in PROGRESS IN
INDUSTRIAL MICROBIOLOGY, Vol. 15, pp. 112-115. These
.beta.-Amylases have optimum temperatures in the range from
40.degree. C. to 65.degree. C. and optimum pH in the range from
about 4.5 to about 7.0. Contemplated .beta.-amylases include, but
are not limited to, .beta.-amylases from barley Spezyme.RTM. BBA
1500, Spezyme.RTM. DBA, Optimalt.TM. ME, Optimalt.TM. BBA (Danisco
US Inc.); and Novozym.TM. WBA (Novozymes A/S).
5. Compositions and Methods for Baking and Food Preparation
[0198] The present invention also relates to a "food composition,"
including but not limited to a food product, animal feed and/or
food/feed additives, comprising an AfGATR or variant thereof, and
methods for preparing such a food composition comprising mixing
AfGATR or variant thereof with one or more food ingredients, or
uses thereof.
[0199] The AfGATR or variant thereof can be used in the preparation
of a food composition, wherein the food composition is baked
subsequent to the addition of the polypeptide. As used herein the
term "baking composition" means any composition and/or additive
prepared in the process of providing a baked food product,
including but not limited to bakers flour, a dough, a baking
additive and/or a baked product. The food composition or additive
may be liquid or solid.
[0200] As used herein, the term "flour" means milled or ground
cereal grain. The term "flour" also may mean Sago or tuber products
that have been ground or mashed. In some embodiments, flour may
also contain components in addition to the milled or mashed cereal
or plant matter. An example of an additional component, although
not intended to be limiting, is a leavening agent. Cereal grains
include wheat, oat, rye, and barley. Tuber products include tapioca
flour, cassava flour, and custard powder. The term "flour" also
includes ground corn flour, maize-meal, rice flour, whole-meal
flour, self-rising flour, tapioca flour, cassava flour, ground
rice, enriched flower, and custard powder.
[0201] For the commercial and home use of flour for baking and food
production, it is important to maintain an appropriate level of
glucoamylase activity in the flour. A level of activity that is too
high may result in a product that is sticky and/or doughy and
therefore unmarketable. Flour with insufficient glucoamylase
activity may not contain enough sugar for proper yeast function,
resulting in dry, crumbly bread, or baked products. Accordingly, an
AfGATR or variant thereof, by itself or in combination with an
.alpha.-amylase(s), may be added to the flour to augment the level
of endogenous glucoamylase activity in flour.
[0202] An amylase can be added alone or in a combination with other
amylases to prevent or retard staling, i.e., crumb firming of baked
products. The amount of anti-staling amylase will typically be in
the range of 0.01-10 mg of enzyme protein per kg of flour, e.g.,
0.5 mg/kg ds. Additional anti-staling amylases that can be used in
combination with an AfGATR or variant thereof include an
endo-amylase, e.g., a bacterial endo-amylase from Bacillus. The
additional amylase can be another maltogenic .alpha.-amylase (EC
3.2.1.133), e.g., from Bacillus. Novamyl.RTM. is an exemplary
maltogenic .alpha.-amylase from B. stearothermophilus strain NCIB
11837 and is described, for example, in Christophersen et al.
(1997) Starch 50: 39-45. Other examples of anti-staling
endo-amylases include bacterial .alpha.-amylases derived from
Bacillus, such as B. licheniformis or B. amyloliquefaciens. The
anti-staling amylase may be an exo-amylase, such as .beta.-amylase,
e.g., from plant sources, such as soybean, or from microbial
sources, such as Bacillus.
[0203] The baking composition comprising an AfGATR or variant
thereof further can comprise a phospholipase or enzyme with
phospholipase activity. An enzyme with phospholipase activity has
an activity that can be measured in Lipase Units (LU). The
phospholipase may have A.sub.1 or A.sub.2 activity to remove fatty
acid from the phospholipids, forming a lysophospholipid. It may or
may not have lipase activity, i.e., activity on triglyceride
substrates. The phospholipase typically has a temperature optimum
in the range of 30-90.degree. C., e.g., 30-70.degree. C. The added
phospholipases can be of animal origin, for example, from pancreas,
e.g., bovine or porcine pancreas, snake venom, or bee venom.
Alternatively, the phospholipase may be of microbial origin, e.g.,
from filamentous fungi, yeast or bacteria, for example.
[0204] The phospholipase is added in an amount that improves the
softness of the bread during the initial period after baking,
particularly the first 24 hours. The amount of phospholipase will
typically be in the range of 0.01-10 mg of enzyme protein per kg of
flour, e.g., 0.1-5 mg/kg. That is, phospholipase activity generally
will be in the range of 20-1000 LU/kg of flour, where a Lipase Unit
is defined as the amount of enzyme required to release 1 .mu.mol
butyric acid per minute at 30.degree. C., pH 7.0, with gum arabic
as emulsifier and tributyrin as substrate.
[0205] Compositions of dough generally comprise wheat meal or wheat
flour and/or other types of meal, flour or starch such as corn
flour, cornstarch, rye meal, rye flour, oat flour, oatmeal, soy
flour, sorghum meal, sorghum flour, potato meal, potato flour or
potato starch. The dough may be fresh, frozen, or par-baked. The
dough can be a leavened dough or a dough to be subjected to
leavening. The dough may be leavened in various ways, such as by
adding chemical leavening agents, e.g., sodium bicarbonate or by
adding a leaven, i.e., fermenting dough. Dough also may be leavened
by adding a suitable yeast culture, such as a culture of
Saccharomyces cerevisiae (baker's yeast), e.g., a commercially
available strain of S. cerevisiae.
[0206] The dough may also comprise other conventional dough
ingredients, e.g., proteins, such as milk powder, gluten, and soy;
eggs (e.g., whole eggs, egg yolks or egg whites); an oxidant, such
as ascorbic acid, potassium bromate, potassium iodate,
azodicarbonamide (ADA) or ammonium persulfate; an amino acid such
as L-cysteine; a sugar; or a salt, such as sodium chloride, calcium
acetate, sodium sulfate, or calcium sulfate. The dough further may
comprise fat, e.g., triglyceride, such as granulated fat or
shortening. The dough further may comprise an emulsifier such as
mono- or diglycerides, diacetyl tartaric acid esters of mono- or
diglycerides, sugar esters of fatty acids, polyglycerol esters of
fatty acids, lactic acid esters of monoglycerides, acetic acid
esters of monoglycerides, polyoxyethylene stearates, or
lysolecithin. For example, the dough can be made without addition
of emulsifiers.
[0207] The dough product may be any processed dough product,
including fried, deep fried, roasted, baked, steamed and boiled
doughs, such as steamed bread and rice cakes. In one embodiment,
the food product is a bakery product. Typical bakery (baked)
products include bread--such as loaves, rolls, buns, bagels, pizza
bases etc. pastry, pretzels, tortillas, cakes, cookies, biscuits,
crackers etc.
[0208] Optionally, an additional enzyme may be used together with
the anti-staling amylase and the phospholipase. The additional
enzyme may be a second amylase, such as an amyloglucosidase, a
.beta.-amylase, a cyclodextrin glucanotransferase, or the
additional enzyme may be a peptidase, in particular an
exopeptidase, a transglutaminase, a lipase, a cellulase, a
xylanase, a protease, a protein disulfide isomerase, e.g., a
protein disulfide isomerase as disclosed in WO 95/00636, for
example, a glycosyltransferase, a branching enzyme
(1,4-.alpha.-glucan branching enzyme), a
4-.alpha.-glucanotransferase (dextrin glycosyltransferase) or an
oxidoreductase, e.g., a peroxidase, a laccase, a glucose oxidase, a
pyranose oxidase, a lipooxygenase, an L-amino acid oxidase or a
carbohydrate oxidase. The additional enzyme(s) may be of any
origin, including mammalian and plant, and particularly of
microbial (bacterial, yeast or fungal) origin and may be obtained
by techniques conventionally used in the art.
[0209] The xylanase is typically of microbial origin, e.g., derived
from a bacterium or fungus, such as a strain of Aspergillus.
Xylanases include Pentopan.RTM. and Novozym 384.RTM., for example,
which are commercially available xylanase preparations produced
from Trichoderma reesei. The amyloglucosidase may be an A. niger
amyloglucosidase (such as AMG.RTM.). Other useful amylase products
include Grindamyl.RTM. A 1000 or A 5000 (Grindsted Products,
Denmark) and Amylase.RTM. H or Amylase.RTM. P (DSM). The glucose
oxidase may be a fungal glucose oxidase, in particular an
Aspergillus niger glucose oxidase (such as Gluzyme.RTM.). An
exemplary protease is Neutrase.RTM..
[0210] The process may be used for any kind of baked product
prepared from dough, either of a soft or a crisp character, either
of a white, light or dark type. Examples are bread, particularly
white, whole-meal or rye bread, typically in the form of loaves or
rolls, such as, but not limited to, French baguette-type bread,
pita bread, tortillas, cakes, pancakes, biscuits, cookies,
piecrusts, crisp bread, steamed bread, pizza and the like.
[0211] The AfGATR or variant thereof may be used in a pre-mix,
comprising flour together with an anti-staling amylase, a
phospholipase, and/or a phospholipid. The pre-mix may contain other
dough-improving and/or bread-improving additives, e.g., any of the
additives, including enzymes, mentioned above. The AfGATR or
variant thereof can be a component of an enzyme preparation
comprising an anti-staling amylase and a phospholipase, for use as
a baking additive.
[0212] The enzyme preparation is optionally in the form of a
granulate or agglomerated powder. The preparation can have a narrow
particle size distribution with more than 95% (by weight) of the
particles in the range from 25 to 500 .mu.m. Granulates and
agglomerated powders may be prepared by conventional methods, e.g.,
by spraying the AfGATR or variant thereof onto a carrier in a
fluid-bed granulator. The carrier may consist of particulate cores
having a suitable particle size. The carrier may be soluble or
insoluble, e.g., a salt (such as NaCl or sodium sulfate), a sugar
(such as sucrose or lactose), a sugar alcohol (such as sorbitol),
starch, rice, corn grits, or soy.
[0213] Enveloped particles, i.e., glucoamylase particles, can
comprise an AfGATR or variants thereof. To prepare enveloped
glucoamylase particles, the enzyme is contacted with a food grade
lipid in sufficient quantity to suspend all of the glucoamylase
particles. Food grade lipids, as used herein, may be any naturally
organic compound that is insoluble in water but is soluble in
non-polar organic solvents such as hydrocarbon or diethyl ether.
Suitable food grade lipids include, but are not limited to,
triglycerides either in the form of fats or oils that are either
saturated or unsaturated. Examples of fatty acids and combinations
thereof which make up the saturated triglycerides include, but are
not limited to, butyric (derived from milk fat), palmitic (derived
from animal and plant fat), and/or stearic (derived from animal and
plant fat). Examples of fatty acids and combinations thereof which
make up the unsaturated triglycerides include, but are not limited
to, palmitoleic (derived from animal and plant fat), oleic (derived
from animal and plant fat), linoleic (derived from plant oils),
and/or linolenic (derived from linseed oil). Other suitable food
grade lipids include, but are not limited to, monoglycerides and
diglycerides derived from the triglycerides discussed above,
phospholipids and glycolipids.
[0214] The food grade lipid, particularly in the liquid form, is
contacted with a powdered form of the glucoamylase particles in
such a fashion that the lipid material covers at least a portion of
the surface of at least a majority, e.g., 100% of the glucoamylase
particles. Thus, each glucoamylase particle is individually
enveloped in a lipid. For example, all or substantially all of the
glucoamylase particles are provided with a thin, continuous,
enveloping film of lipid. This can be accomplished by first pouring
a quantity of lipid into a container, and then slurrying the
glucoamylase particles so that the lipid thoroughly wets the
surface of a glucoamylase particle. After a short period of
stirring, the enveloped glucoamylase particles, carrying a
substantial amount of the lipids on their surfaces, are recovered.
The thickness of the coating so applied to the particles of
glucoamylase can be controlled by selection of the type of lipid
used and by repeating the operation in order to build up a thicker
film, when desired.
[0215] The storing, handling and incorporation of the loaded
delivery vehicle can be accomplished by means of a packaged mix.
The packaged mix can comprise the enveloped glucoamylase. However,
the packaged mix may further contain additional ingredients as
required by the manufacturer or baker. After the enveloped
glucoamylase has been incorporated into the dough, the baker
continues through the normal production process for that
product.
[0216] The advantages of enveloping the glucoamylase particles are
two-fold. First, the food grade lipid protects the enzyme from
thermal denaturation during the baking process for those enzymes
that are heat labile. Consequently, while the glucoamylase is
stabilized and protected during the proving and baking stages, it
is released from the protective coating in the final baked good
product, where it hydrolyzes the glucosidic linkages in
polyglucans. The loaded delivery vehicle also provides a sustained
release of the active enzyme into the baked good. That is,
following the baking process, active glucoamylase is continually
released from the protective coating at a rate that counteracts,
and therefore reduces the rate of, staling mechanisms.
[0217] In general, the amount of lipid applied to the glucoamylase
particles can vary from a few percent of the total weight of the
glucoamylase to many times that weight, depending upon the nature
of the lipid, the manner in which it is applied to the glucoamylase
particles, the composition of the dough mixture to be treated, and
the severity of the dough-mixing operation involved.
[0218] The loaded delivery vehicle, i.e., the lipid-enveloped
enzyme, is added to the ingredients used to prepare a baked good in
an effective amount to extend the shelf-life of the baked good. The
baker computes the amount of enveloped .alpha.-amylase, prepared as
discussed above, that will be required to achieve the desired
anti-staling effect. The amount of the enveloped glucoamylase
required is calculated based on the concentration of enzyme
enveloped and on the proportion of glucoamylase to flour specified.
A wide range of concentrations has been found to be effective,
although, as has been discussed, observable improvements in
anti-staling do not correspond linearly with the glucoamylase
concentration, but above certain minimal levels, large increases in
glucoamylase concentration produce little additional improvement.
The glucoamylase concentration actually used in a particular bakery
production could be much higher than the minimum necessary to
provide the baker with some insurance against inadvertent
under-measurement errors by the baker. The lower limit of enzyme
concentration is determined by the minimum anti-staling effect the
baker wishes to achieve.
[0219] A method of preparing a baked good may comprise: a)
preparing lipid-coated glucoamylase particles, where substantially
all of the glucoamylase particles are coated; b) mixing a dough
containing flour; c) adding the lipid-coated glucoamylase to the
dough before the mixing is complete and terminating the mixing
before the lipid coating is removed from the .alpha.-amylase; d)
proofing the dough; and e) baking the dough to provide the baked
good, where the glucoamylase is inactive during the mixing,
proofing and baking stages and is active in the baked good.
[0220] The enveloped glucoamylase can be added to the dough during
the mix cycle, e.g., near the end of the mix cycle. The enveloped
glucoamylase is added at a point in the mixing stage that allows
sufficient distribution of the enveloped glucoamylase throughout
the dough; however, the mixing stage is terminated before the
protective coating becomes stripped from the glucoamylase
particle(s). Depending on the type and volume of dough, and mixer
action and speed, anywhere from one to six minutes or more might be
required to mix the enveloped glucoamylase into the dough, but two
to four minutes is average. Thus, several variables may determine
the precise procedure. First, the quantity of enveloped
glucoamylase should have a total volume sufficient to allow the
enveloped glucoamylase to be spread throughout the dough mix. If
the preparation of enveloped glucoamylase is highly concentrated,
additional oil may need to be added to the pre-mix before the
enveloped glucoamylase is added to the dough. Recipes and
production processes may require specific modifications; however,
good results generally can be achieved when 25% of the oil
specified in a bread dough formula is held out of the dough and is
used as a carrier for a concentrated enveloped glucoamylase when
added near the end of the mix cycle. In bread or other baked goods,
particularly those having a low fat content, e.g., French-style
breads, an enveloped glucoamylase mixture of approximately 1% of
the dry flour weight is sufficient to admix the enveloped
glucoamylase properly with the dough. The range of suitable
percentages is wide and depends on the formula, finished product,
and production methodology requirements of the individual baker.
Second, the enveloped glucoamylase suspension should be added to
the mix with sufficient time for complete mixture into the dough,
but not for such a time that excessive mechanical action strips the
protective lipid coating from the enveloped glucoamylase
particles.
[0221] A food composition is contemplated where the food is an oil,
meat, lard, composition comprising an AfGATR or a variant thereof.
In this context the term "[oil/meat/lard] composition" means any
composition, based on, made from and/or containing oil, meat or
lard, respectively. A method is contemplated for preparing an oil
or meat or lard composition and/or additive comprising an AfGATR or
a variant thereof, comprising mixing the polypeptide of the
invention with a oil/meat/lard composition and/or additive
ingredients.
[0222] The food composition can be an animal feed composition,
animal feed additive, and/or pet food comprising an AfGATR and
variants thereof. A method is contemplated for preparing such an
animal feed composition, animal feed additive composition and/or
pet food comprising mixing an AfGATR and variants thereof with one
or more animal feed ingredients and/or animal feed additive
ingredients and/or pet food ingredients. An AfGATR and variants
thereof can be used in the preparation of an animal feed
composition and/or animal feed additive composition and/or pet
food.
[0223] The term "animal" includes all non-ruminant and ruminant
animals. In a particular embodiment, the animal is a non-ruminant
animal, such as a horse and a mono-gastric animal. Examples of
mono-gastric animals include, but are not limited to, pigs and
swine, such as piglets, growing pigs, sows; poultry such as
turkeys, ducks, chicken, broiler chicks, layers; fish such as
salmon, trout, tilapia, catfish and carps; and crustaceans such as
shrimps and prawns. In a further embodiment the animal is a
ruminant animal including, but not limited to, cattle, young
calves, goats, sheep, giraffes, bison, moose, elk, yaks, water
buffalo, deer, camels, alpacas, llamas, antelope, pronghorn and
nilgai.
[0224] In the present context, it is intended that the term "pet
food" is understood to mean a food for a household animal such as,
but not limited to dogs, cats, gerbils, hamsters, chinchillas,
fancy rats, guinea pigs; avian pets, such as canaries, parakeets,
and parrots; reptile pets, such as turtles, lizards and snakes; and
aquatic pets, such as tropical fish and frogs.
[0225] The terms "animal feed composition," "feedstuff" and
"fodder" are used interchangeably and may comprise one or more feed
materials selected from the group comprising a) cereals, such as
small grains (e.g., wheat, barley, rye, oats and combinations
thereof) and/or large grains such as maize or sorghum; b) by
products from cereals, such as corn gluten meal, Distillers Dried
Grain Solubles (DDGS) (particularly corn based Distillers Dried
Grain Solubles (cDDGS), wheat bran, wheat middlings, wheat shorts,
rice bran, rice hulls, oat hulls, palm kernel, and citrus pulp; c)
protein obtained from sources such as soya, sunflower, peanut,
lupin, peas, fava beans, cotton, canola, fish meal, dried plasma
protein, meat and bone meal, potato protein, whey, copra, sesame;
d) oils and fats obtained from vegetable and animal sources; e)
minerals and vitamins.
6. Textile Desizing Compositions and Use
[0226] Also contemplated are compositions and methods of treating
fabrics (e.g., to desize a textile) using an AfGATR.
Fabric-treating methods are well known in the art (see, e.g., U.S.
Pat. No. 6,077,316). For example, the feel and appearance of a
fabric can be improved by a method comprising contacting the fabric
with an AfGATR in a solution. The fabric can be treated with the
solution under pressure.
[0227] An AfGATR can be applied during or after the weaving of a
textile, or during the desizing stage, or one or more additional
fabric processing steps. During the weaving of textiles, the
threads are exposed to considerable mechanical strain. Prior to
weaving on mechanical looms, warp yarns are often coated with
sizing starch or starch derivatives to increase their tensile
strength and to prevent breaking. An AfGATR can be applied during
or after the weaving to remove these sizing starches or starch
derivatives. After weaving, an AfGATR can be used to remove the
size coating before further processing the fabric to ensure a
homogeneous and wash-proof result.
[0228] An AfGATR can be used alone or with other desizing chemical
reagents and/or desizing enzymes to desize fabrics, including
cotton-containing fabrics, as detergent additives, e.g., in aqueous
compositions. An AfGATR also can be used in compositions and
methods for producing a stonewashed look on indigo-dyed denim
fabric and garments. For the manufacture of clothes, the fabric can
be cut and sewn into clothes or garments, which are afterwards
finished. In particular, for the manufacture of denim jeans,
different enzymatic finishing methods have been developed. The
finishing of denim garment normally is initiated with an enzymatic
desizing step, during which garments are subjected to the action of
amylolytic enzymes to provide softness to the fabric and make the
cotton more accessible to the subsequent enzymatic finishing steps.
An AfGATR can be used in methods of finishing denim garments (e.g.,
a "bio-stoning process"), enzymatic desizing and providing softness
to fabrics, and/or finishing process.
7. Cleaning Compositions
[0229] An aspect of the present compositions and methods is a
cleaning composition that includes an AfGATR or variant thereof as
a component. An amylase polypeptide can be used as a component in
detergent compositions for hand washing, laundry washing,
dishwashing, and other hard-surface cleaning.
7.1. Overview
[0230] Preferably, the AfGATR or variant thereof is incorporated
into detergents at or near a concentration conventionally used for
amylase in detergents. For example, an glucoamylase polypeptide may
be added in amount corresponding to 0.00001-1 mg (calculated as
pure enzyme protein) of amylase per liter of wash/dishwash liquor.
Exemplary formulations are provided herein, as exemplified by the
following:
[0231] A glucoamylase polypeptide may be a component of a detergent
composition, as the only enzyme or with other enzymes including
other amylolytic enzymes. As such, it may be included in the
detergent composition in the form of a non-dusting granulate, a
stabilized liquid, or a protected enzyme. Non-dusting granulates
may be produced, e.g., as disclosed in U.S. Pat. Nos. 4,106,991 and
4,661,452 and may optionally be coated by methods known in the art.
Examples of waxy coating materials are poly(ethylene oxide)
products (polyethyleneglycol, PEG) with mean molar weights of 1,000
to 20,000; ethoxylated nonylphenols having from 16 to 50 ethylene
oxide units; ethoxylated fatty alcohols in which the alcohol
contains from 12 to 20 carbon atoms and in which there are 15 to 80
ethylene oxide units; fatty alcohols; fatty acids; and mono- and
di- and triglycerides of fatty acids. Examples of film-forming
coating materials suitable for application by fluid bed techniques
are given in, for example, GB 1483591. Liquid enzyme preparations
may, for instance, be stabilized by adding a polyol such as
propylene glycol, a sugar or sugar alcohol, lactic acid or boric
acid according to established methods. Other enzyme stabilizers are
known in the art. Protected enzymes may be prepared according to
the method disclosed in for example EP 238 216. Polyols have long
been recognized as stabilizers of proteins, as well as improving
protein solubility.
[0232] The detergent composition may be in any useful form, e.g.,
as powders, granules, pastes, or liquid. A liquid detergent may be
aqueous, typically containing up to about 70% of water and 0% to
about 30% of organic solvent. It may also be in the form of a
compact gel type containing about 30% water.
[0233] The detergent composition comprises one or more surfactants,
each of which may be anionic, nonionic, cationic, or zwitterionic.
The detergent will usually contain 0% to about 50% of anionic
surfactant, such as linear alkylbenzenesulfonate (LAS);
.alpha.-olefinsulfonate (AOS); alkyl sulfate (fatty alcohol
sulfate) (AS); alcohol ethoxysulfate (AEOS or AES); secondary
alkanesulfonates (SAS); .alpha.-sulfo fatty acid methyl esters;
alkyl- or alkenylsuccinic acid; or soap. The composition may also
contain 0% to about 40% of nonionic surfactant such as alcohol
ethoxylate (AEO or AE), carboxylated alcohol ethoxylates,
nonylphenol ethoxylate, alkylpolyglycoside,
alkyldimethylamineoxide, ethoxylated fatty acid monoethanolamide,
fatty acid monoethanolamide, or polyhydroxy alkyl fatty acid amide
(as described for example in WO 92/06154).
[0234] The detergent composition may additionally comprise one or
more other enzymes, such as proteases, another amylolytic enzyme,
cutinase, lipase, cellulase, pectate lyase, perhydrolase, xylanase,
peroxidase, and/or laccase in any combination.
[0235] The detergent may contain about 1% to about 65% of a
detergent builder or complexing agent such as zeolite, diphosphate,
triphosphate, phosphonate, citrate, nitrilotriacetic acid (NTA),
ethylenediaminetetraacetic acid (EDTA),
diethylenetriaminepentaacetic acid (DTMPA), alkyl- or
alkenylsuccinic acid, soluble silicates or layered silicates (e.g.,
SKS-6 from Hoechst). The detergent may also be unbuilt, i.e.
essentially free of detergent builder. The enzymes can be used in
any composition compatible with the stability of the enzyme.
Enzymes generally can be protected against deleterious components
by known forms of encapsulation, for example, by granulation or
sequestration in hydro gels. Enzymes, and specifically amylases,
either with or without starch binding domains, can be used in a
variety of compositions including laundry and dishwashing
applications, surface cleaners, as well as in compositions for
ethanol production from starch or biomass.
[0236] The detergent may comprise one or more polymers. Examples
include carboxymethylcellulose (CMC), poly(vinylpyrrolidone) (PVP),
polyethyleneglycol (PEG), poly(vinyl alcohol) (PVA),
polycarboxylates such as polyacrylates, maleic/acrylic acid
copolymers and lauryl methacrylate/acrylic acid copolymers.
[0237] The detergent may contain a bleaching system, which may
comprise a H.sub.2O.sub.2 source such as perborate or percarbonate,
which may be combined with a peracid-forming bleach activator such
as tetraacetylethylenediamine (TAED) or nonanoyloxybenzenesulfonate
(NOBS). Alternatively, the bleaching system may comprise
peroxyacids (e.g., the amide, imide, or sulfone type peroxyacids).
The bleaching system can also be an enzymatic bleaching system, for
example, perhydrolase, such as that described in International PCT
Application WO 2005/056783.
[0238] The enzymes of the detergent composition may be stabilized
using conventional stabilizing agents, e.g., a polyol such as
propylene glycol or glycerol; a sugar or sugar alcohol; lactic
acid; boric acid or a boric acid derivative such as, e.g., an
aromatic borate ester; and the composition may be formulated as
described in, e.g., WO 92/19709 and WO 92/19708.
[0239] The detergent may also contain other conventional detergent
ingredients such as e.g., fabric conditioners including clays, foam
boosters, suds suppressors, anti-corrosion agents, soil-suspending
agents, anti-soil redeposition agents, dyes, bactericides, tarnish
inhibitors, optical brighteners, or perfumes.
[0240] The pH (measured in aqueous solution at use concentration)
is usually neutral or alkaline, e.g., pH about 7.0 to about
11.0.
[0241] Particular forms of detergent compositions for inclusion of
the present glucoamylase are described, below.
7.2. Heavy Duty Liquid (HDL) Laundry Detergent Composition
[0242] Exemplary HDL laundry detergent compositions includes a
detersive surfactant (10%-40% wt/wt), including an anionic
detersive surfactant (selected from a group of linear or branched
or random chain, substituted or unsubstituted alkyl sulphates,
alkyl sulphonates, alkyl alkoxylated sulphate, alkyl phosphates,
alkyl phosphonates, alkyl carboxylates, and/or mixtures thereof),
and optionally non-ionic surfactant (selected from a group of
linear or branched or random chain, substituted or unsubstituted
alkyl alkoxylated alcohol, for example a C.sub.8-C.sub.18 alkyl
ethoxylated alcohol and/or C.sub.6-C.sub.12 alkyl phenol
alkoxylates), wherein the weight ratio of anionic detersive
surfactant (with a hydrophilic index (HIc) of from 6.0 to 9) to
non-ionic detersive surfactant is greater than 1:1. Suitable
detersive surfactants also include cationic detersive surfactants
(selected from a group of alkyl pyridinium compounds, alkyl
quaternary ammonium compounds, alkyl quaternary phosphonium
compounds, alkyl ternary sulphonium compounds, and/or mixtures
thereof); zwitterionic and/or amphoteric detersive surfactants
(selected from a group of alkanolamine sulpho-betaines); ampholytic
surfactants; semi-polar non-ionic surfactants and mixtures
thereof.
[0243] The composition may optionally include, a surfactancy
boosting polymer consisting of amphiphilic alkoxylated grease
cleaning polymers (selected from a group of alkoxylated polymers
having branched hydrophilic and hydrophobic properties, such as
alkoxylated polyalkylenimines in the range of 0.05 wt %-10 wt %)
and/or random graft polymers typically comprising of hydrophilic
backbone comprising monomers selected from the group consisting of:
unsaturated C.sub.1-C.sub.6 carboxylic acids, ethers, alcohols,
aldehydes, ketones, esters, sugar units, alkoxy units, maleic
anhydride, saturated polyalcohols such as glycerol, and mixtures
thereof; and hydrophobic side chain(s) selected from the group
consisting of: C.sub.4-C.sub.25 alkyl group, polypropylene,
polybutylene, vinyl ester of a saturated C.sub.1-C.sub.6
mono-carboxylic acid, C.sub.1-C.sub.6 alkyl ester of acrylic or
methacrylic acid, and mixtures thereof.
[0244] The composition may include additional polymers such as soil
release polymers (include anionically end-capped polyesters, for
example SRP1, polymers comprising at least one monomer unit
selected from saccharide, dicarboxylic acid, polyol and
combinations thereof, in random or block configuration, ethylene
terephthalate-based polymers and co-polymers thereof in random or
block configuration, for example Repel-o-tex SF, SF-2 and SRP6,
Texcare SRA100, SRA300, SRN100, SRN170, SRN240, SRN300 and SRN325,
Marloquest SL), anti-redeposition polymers (0.1 wt % to 10 wt %,
include carboxylate polymers, such as polymers comprising at least
one monomer selected from acrylic acid, maleic acid (or maleic
anhydride), fumaric acid, itaconic acid, aconitic acid, mesaconic
acid, citraconic acid, methylenemalonic acid, and any mixture
thereof, vinylpyrrolidone homopolymer, and/or polyethylene glycol,
molecular weight in the range of from 500 to 100,000 Da);
cellulosic polymer (including those selected from alkyl cellulose,
alkyl alkoxyalkyl cellulose, carboxyalkyl cellulose, alkyl
carboxyalkyl cellulose examples of which include carboxymethyl
cellulose, methyl cellulose, methyl hydroxyethyl cellulose, methyl
carboxymethyl cellulose, and mixtures thereof) and polymeric
carboxylate (such as maleate/acrylate random copolymer or
polyacrylate homopolymer).
[0245] The composition may further include saturated or unsaturated
fatty acid, preferably saturated or unsaturated C.sub.12-C.sub.24
fatty acid (0 wt % to 10 wt %); deposition aids examples for which
include polysaccharides, preferably cellulosic polymers, poly
diallyl dimethyl ammonium halides (DADMAC), and co-polymers of DAD
MAC with vinyl pyrrolidone, acrylamides, imidazoles, imidazolinium
halides, and mixtures thereof, in random or block configuration,
cationic guar gum, cationic cellulose such as cationic hydroxyethyl
cellulose, cationic starch, cationic polyacrylamides, and mixtures
thereof.
[0246] The composition may further include dye transfer inhibiting
agents, examples of which include manganese phthalocyanine,
peroxidases, polyvinylpyrrolidone polymers, polyamine N-oxide
polymers, copolymers of N-vinylpyrrolidone and N-vinylimidazole,
polyvinyloxazolidones and polyvinylimidazoles and/or mixtures
thereof; chelating agents, examples of which include
ethylene-diamine-tetraacetic acid (EDTA), diethylene triamine penta
methylene phosphonic acid (DTPMP), hydroxy-ethane diphosphonic acid
(HEDP), ethylenediamine N,N'-disuccinic acid (EDDS), methyl glycine
diacetic acid (MGDA), diethylene triamine penta acetic acid (DTPA),
propylene diamine tetracetic acid (PDT A),
2-hydroxypyridine-N-oxide (HPNO), or methyl glycine diacetic acid
(MGDA), glutamic acid N,N-diacetic acid (N,N-dicarboxymethyl
glutamic acid tetrasodium salt (GLDA), nitrilotriacetic acid (NTA),
4,5-dihydroxy-m-benzenedisulfonic acid, citric acid and any salts
thereof, N-hydroxyethylethylenediaminetri-acetic acid (HEDTA),
triethylenetetraaminehexaacetic acid (TTHA),
N-hydroxyethyliminodiacetic acid (HEIDA), dihydroxyethylglycine
(DHEG), ethylenediaminetetrapropionic acid (EDTP), and derivatives
thereof.
[0247] The composition preferably included enzymes (generally about
0.01 wt % active enzyme to 0.03 wt % active enzyme) selected from
proteases, amylases, lipases, cellulases, choline oxidases,
peroxidases/oxidases, pectate lyases, mannanases, cutinases,
laccases, phospholipases, lysophospholipases, acyltransferases,
perhydrolases, arylesterases, and any mixture thereof. The
composition may include an enzyme stabilizer (examples of which
include polyols such as propylene glycol or glycerol, sugar or
sugar alcohol, lactic acid, reversible protease inhibitor, boric
acid, or a boric acid derivative, e.g., an aromatic borate ester,
or a phenyl boronic acid derivative such as 4-formylphenyl boronic
acid).
[0248] The composition optionally include silicone or fatty-acid
based suds suppressors; heuing dyes, calcium and magnesium cations,
visual signaling ingredients, anti-foam (0.001 wt % to about 4.0 wt
%), and/or structurant/thickener (0.01 wt % to 5 wt %, selected
from the group consisting of diglycerides and triglycerides,
ethylene glycol distearate, microcrystalline cellulose, cellulose
based materials, microfiber cellulose, biopolymers, xanthan gum,
gellan gum, and mixtures thereof).
[0249] The composition can be any liquid form, for example a liquid
or gel form, or any combination thereof. The composition may be in
any unit dose form, for example a pouch.
7.3. Heavy Duty Dry/Solid (HDD) Laundry Detergent Composition
[0250] Exemplary HDD laundry detergent compositions includes a
detersive surfactant, including anionic detersive surfactants
(e.g., linear or branched or random chain, substituted or
unsubstituted alkyl sulphates, alkyl sulphonates, alkyl alkoxylated
sulphate, alkyl phosphates, alkyl phosphonates, alkyl carboxylates
and/or mixtures thereof), non-ionic detersive surfactant (e.g.,
linear or branched or random chain, substituted or unsubstituted
C.sub.8-C.sub.18 alkyl ethoxylates, and/or C.sub.6-C.sub.12 alkyl
phenol alkoxylates), cationic detersive surfactants (e.g., alkyl
pyridinium compounds, alkyl quaternary ammonium compounds, alkyl
quaternary phosphonium compounds, alkyl ternary sulphonium
compounds, and mixtures thereof), zwitterionic and/or amphoteric
detersive surfactants (e.g., alkanolamine sulpho-betaines),
ampholytic surfactants, semi-polar non-ionic surfactants, and
mixtures thereof; builders including phosphate free builders (for
example zeolite builders examples which include zeolite A, zeolite
X, zeolite P and zeolite MAP in the range of 0 wt % to less than 10
wt %), phosphate builders (for example sodium tri-polyphosphate in
the range of 0 wt % to less than 10 wt %), citric acid, citrate
salts and nitrilotriacetic acid, silicate salt (e.g., sodium or
potassium silicate or sodium meta-silicate in the range of 0 wt %
to less than 10 wt %, or layered silicate (SKS-6)); carbonate salt
(e.g., sodium carbonate and/or sodium bicarbonate in the range of 0
wt % to less than 80 wt %); and bleaching agents including
photobleaches (e.g., sulfonated zinc phthalocyanines, sulfonated
aluminum phthalocyanines, xanthenes dyes, and mixtures thereof)
hydrophobic or hydrophilic bleach activators (e.g., dodecanoyl
oxybenzene sulfonate, decanoyl oxybenzene sulfonate, decanoyl
oxybenzoic acid or salts thereof, 3,5,5-trimethy hexanoyl
oxybenzene sulfonate, tetraacetyl ethylene diamine-TAED,
nonanoyloxybenzene sulfonate-NOBS, nitrile quats, and mixtures
thereof), sources of hydrogen peroxide (e.g., inorganic perhydrate
salts examples of which include mono or tetra hydrate sodium salt
of perborate, percarbonate, persulfate, perphosphate, or
persilicate), preformed hydrophilic and/or hydrophobic peracids
(e.g., percarboxylic acids and salts, percarbonic acids and salts,
perimidic acids and salts, peroxymonosulfuric acids and salts, and
mixtures thereof), and/or bleach catalysts (e.g., imine bleach
boosters (examples of which include iminium cations and polyions),
iminium zwitterions, modified amines, modified amine oxides,
N-sulphonyl imines, N-phosphonyl imines, N-acyl imines, thiadiazole
dioxides, perfluoroimines, cyclic sugar ketones, and mixtures
thereof, and metal-containing bleach catalysts (e.g., copper, iron,
titanium, ruthenium, tungsten, molybdenum, or manganese cations
along with an auxiliary metal cations such as zinc or aluminum and
a sequestrate such as ethylenediaminetetraacetic acid,
ethylenediaminetetra(methylenephosphonic acid), and water-soluble
salts thereof).
[0251] The composition preferably includes enzymes, e.g.,
proteases, amylases, lipases, cellulases, choline oxidases,
peroxidases/oxidases, pectate lyases, mannanases, cutinases,
laccases, phospholipases, lysophospholipases, acyltransferase,
perhydrolase, arylesterase, and any mixture thereof.
[0252] The composition may optionally include additional detergent
ingredients including perfume microcapsules, starch encapsulated
perfume accord, hueing agents, additional polymers, including
fabric integrity and cationic polymers, dye-lock ingredients,
fabric-softening agents, brighteners (for example C.I. Fluorescent
brighteners), flocculating agents, chelating agents, alkoxylated
polyamines, fabric deposition aids, and/or cyclodextrin.
7.4. Automatic Dishwashing (ADW) Detergent Composition
[0253] Exemplary ADW detergent composition includes non-ionic
surfactants, including ethoxylated non-ionic surfactants, alcohol
alkoxylated surfactants, epoxy-capped poly(oxyalkylated) alcohols,
or amine oxide surfactants present in amounts from 0 to 10% by
weight; builders in the range of 5-60% including phosphate builders
(e.g., mono-phosphates, di-phosphates, tri-polyphosphates, other
oligomeric-polyphosphates, sodium tripolyphosphate-STPP) and
phosphate-free builders (e.g., amino acid-based compounds including
methyl-glycine-diacetic acid (MGDA) and salts and derivatives
thereof, glutamic-N,N-diacetic acid (GLDA) and salts and
derivatives thereof, iminodisuccinic acid (IDS) and salts and
derivatives thereof, carboxy methyl inulin and salts and
derivatives thereof, nitrilotriacetic acid (NTA), diethylene
triamine penta acetic acid (DTPA), B-alaninediacetic acid (B-ADA)
and their salts, homopolymers and copolymers of poly-carboxylic
acids and their partially or completely neutralized salts,
monomeric polycarboxylic acids and hydroxycarboxylic acids and
their salts in the range of 0.5% to 50% by weight;
sulfonated/carboxylated polymers in the range of about 0.1% to
about 50% by weight to provide dimensional stability; drying aids
in the range of about 0.1% to about 10% by weight (e.g.,
polyesters, especially anionic polyesters, optionally together with
further monomers with 3 to 6 functionalities--typically acid,
alcohol or ester functionalities which are conducive to
polycondensation, polycarbonate-, polyurethane- and/or
polyurea-polyorganosiloxane compounds or precursor compounds,
thereof, particularly of the reactive cyclic carbonate and urea
type); silicates in the range from about 1% to about 20% by weight
(including sodium or potassium silicates for example sodium
disilicate, sodium meta-silicate and crystalline phyllosilicates);
inorganic bleach (e.g., perhydrate salts such as perborate,
percarbonate, perphosphate, persulfate and persilicate salts) and
organic bleach (e.g., organic peroxyacids, including diacyl and
tetraacylperoxides, such as diperoxydodecanedioc acid,
diperoxytetradecanedioc acid, and diperoxyhexadecanedioc acid);
bleach activators (i.e., organic peracid precursors in the range
from about 0.1% to about 10% by weight); bleach catalysts (e.g.,
manganese triazacyclononane and related complexes, Co, Cu, Mn, and
Fe bispyridylamine and related complexes, and pentamine acetate
cobalt(III) and related complexes); metal care agents in the range
from about 0.1% to 5% by weight (e.g., benzatriazoles, metal salts
and complexes, and/or silicates); enzymes in the range from about
0.01 to 5.0 mg of active enzyme per gram of automatic dishwashing
detergent composition (e.g., proteases, amylases, lipases,
cellulases, choline oxidases, peroxidases/oxidases, pectate lyases,
mannanases, cutinases, laccases, phospholipases,
lysophospholipases, acyltransferase, perhydrolase, arylesterase,
and mixtures thereof); and enzyme stabilizer components (e.g.,
oligosaccharides, polysaccharides, and inorganic divalent metal
salts).
7.5. Additional Detergent Compositions
[0254] Additional exemplary detergent formulations to which the
present amylase can be added are described, below, in the numbered
paragraphs.
[0255] 1) A detergent composition formulated as a granulate having
a bulk density of at least 600 g/L comprising linear
alkylbenzenesulfonate (calculated as acid) about 7% to about 12%;
alcohol ethoxysulfate (e.g., C.sub.12-18 alcohol, 1-2 ethylene
oxide (EO)) or alkyl sulfate (e.g., C.sub.16-18) about 1% to about
4%; alcohol ethoxylate (e.g., C.sub.14-15 alcohol, 7 EO) about 5%
to about 9%; sodium carbonate (e.g., Na.sub.2CO.sub.3) about 14% to
about 20%; soluble silicate (e.g., Na.sub.2O, 2SiO.sub.2) about 2
to about 6%; zeolite (e.g., NaAlSiO.sub.4) about 15% to about 22%;
sodium sulfate (e.g., Na.sub.2SO.sub.4) 0% to about 6%; sodium
citrate/citric acid (e.g.,
C.sub.6H.sub.5Na.sub.3O.sub.7/C.sub.6H.sub.8O.sub.7) about 0% to
about 15%; sodium perborate (e.g., NaBO.sub.3H.sub.2O) about 11% to
about 18%; TAED about 2% to about 6%; carboxymethylcellulose (CMC)
and 0% to about 2%; polymers (e.g., maleic/acrylic acid, copolymer,
PVP, PEG) 0-3%; enzymes (calculated as pure enzyme) 0.0001-0.1%
protein; and minor ingredients (e.g., suds suppressors, perfumes,
optical brightener, photobleach) 0-5%.
[0256] 2) A detergent composition formulated as a granulate having
a bulk density of at least 600 g/L comprising linear
alkylbenzenesulfonate (calculated as acid) about 6% to about 11%;
alcohol ethoxysulfate (e.g., C.sub.12-18 alcohol, 1-2 EO) or alkyl
sulfate (e.g., C.sub.16-18) about 1% to about 3%; alcohol
ethoxylate (e.g., C.sub.14-15 alcohol, 7 EO) about 5% to about 9%;
sodium carbonate (e.g., Na.sub.2CO.sub.3) about 15% to about 21%;
soluble silicate (e.g., Na.sub.2O, 2SiO.sub.2) about 1% to about
4%; zeolite (e.g., NaAlSiO.sub.4) about 24% to about 34%; sodium
sulfate (e.g,. Na.sub.2SO.sub.4) about 4% to about 10%; sodium
citrate/citric acid (e.g.,
C.sub.6H.sub.5Na.sub.3O.sub.7/C.sub.6H.sub.8O.sub.7) 0% to about
15%; carboxymethylcellulose (CMC) 0% to about 2%; polymers (e.g.,
maleic/acrylic acid copolymer, PVP, PEG) 1-6%; enzymes (calculated
as pure enzyme protein) 0.0001-0.1%; minor ingredients (e.g., suds
suppressors, perfume) 0-5%.
[0257] 3) A detergent composition formulated as a granulate having
a bulk density of at least 600 g/L comprising linear
alkylbenzenesulfonate (calculated as acid) about 5% to about 9%;
alcohol ethoxylate (e.g., C.sub.12-15 alcohol, 7 EO) about 7% to
about 14%; Soap as fatty acid (e.g., C.sub.16-22 fatty acid) about
1 to about 3%; sodium carbonate (as Na.sub.2CO.sub.3) about 10% to
about 17%; soluble silicate (e.g., Na.sub.2O, 2SiO.sub.2) about 3%
to about 9%; zeolite (as NaAlSiO.sub.4) about 23% to about 33%;
sodium sulfate (e.g., Na.sub.2SO.sub.4) 0% to about 4%; sodium
perborate (e.g., NaBO.sub.3H.sub.2O) about 8% to about 16%; TAED
about 2% to about 8%; phosphonate (e.g., EDTMPA) 0% to about 1%;
carboxymethylcellulose (CMC) 0% to about 2%; polymers (e.g.,
maleic/acrylic acid copolymer, PVP, PEG) 0-3%; enzymes (calculated
as pure enzyme protein) 0.0001-0.1%; minor ingredients (e.g., suds
suppressors, perfume, optical brightener) 0-5%.
[0258] 4) A detergent composition formulated as a granulate having
a bulk density of at least 600 g/L comprising linear
alkylbenzenesulfonate (calculated as acid) about 8% to about 12%;
alcohol ethoxylate (e.g., C.sub.12-15 alcohol, 7 EO) about 10% to
about 25%; sodium carbonate (as Na.sub.2CO.sub.3) about 14% to
about 22%; soluble silicate (e.g., Na.sub.2O, 2SiO.sub.2) about 1%
to about 5%; zeolite (e.g., NaAlSiO.sub.4) about 25% to about 35%;
sodium sulfate (e.g., Na.sub.2SO.sub.4) 0% to about 10%;
carboxymethylcellulose (CMC) 0% to about 2%; polymers (e.g.,
maleic/acrylic acid copolymer, PVP, PEG) 1-3%; enzymes (calculated
as pure enzyme protein) 0.0001-0.1%; and minor ingredients (e.g.,
suds suppressors, perfume) 0-5%.
[0259] 5) An aqueous liquid detergent composition comprising linear
alkylbenzenesulfonate (calculated as acid) about 15% to about 21%;
alcohol ethoxylate (e.g., C.sub.12-15 alcohol, 7 EO or C.sub.12-15
alcohol, 5 EO) about 12% to about 18%; soap as fatty acid (e.g.,
oleic acid) about 3% to about 13%; alkenylsuccinic acid
(C.sub.12-14) 0% to about 13%; aminoethanol about 8% to about 18%;
citric acid about 2% to about 8%; phosphonate 0% to about 3%;
polymers (e.g., PVP, PEG) 0% to about 3%; borate (e.g.,
B.sub.4O.sub.7) 0% to about 2%; ethanol 0% to about 3%; propylene
glycol about 8% to about 14%; enzymes (calculated as pure enzyme
protein) 0.0001-0.1%; and minor ingredients (e.g., dispersants,
suds suppressors, perfume, optical brightener) 0-5%.
[0260] 6) An aqueous structured liquid detergent composition
comprising linear alkylbenzenesulfonate (calculated as acid) about
15% to about 21%; alcohol ethoxylate (e.g., C.sub.12-15 alcohol, 7
EO, or C.sub.12-15 alcohol, 5 EO) 3-9%; soap as fatty acid (e.g.,
oleic acid) about 3% to about 10%; zeolite (as NaAlSiO.sub.4) about
14% to about 22%; potassium citrate about 9% to about 18%; borate
(e.g., B.sub.4O.sub.7) 0% to about 2%; carboxymethylcellulose (CMC)
0% to about 2%; polymers (e.g., PEG, PVP) 0% to about 3%; anchoring
polymers such as, e.g., lauryl methacrylate/acrylic acid copolymer;
molar ratio 25:1, MW 3800) 0% to about 3%; glycerol 0% to about 5%;
enzymes (calculated as pure enzyme protein) 0.0001-0.1%; and minor
ingredients (e.g., dispersants, suds suppressors, perfume, optical
brighteners) 0-5%.
[0261] 7) A detergent composition formulated as a granulate having
a bulk density of at least 600 g/L comprising fatty alcohol sulfate
about 5% to about 10%; ethoxylated fatty acid monoethanolamide
about 3% to about 9%; soap as fatty acid 0-3%; sodium carbonate
(e.g., Na.sub.2CO.sub.3) about 5% to about 10%; Soluble silicate
(e.g., Na.sub.2O, 2SiO.sub.2) about 1% to about 4%; zeolite (e.g.,
NaAlSiO.sub.4) about 20% to about 40%; Sodium sulfate (e.g.,
Na.sub.2SO.sub.4) about 2% to about 8%; sodium perborate (e.g.,
NaBO.sub.3H.sub.2O) about 12% to about 18%; TAED about 2% to about
7%; polymers (e.g., maleic/acrylic acid copolymer, PEG) about 1% to
about 5%; enzymes (calculated as pure enzyme protein) 0.0001-0.1%;
and minor ingredients (e.g., optical brightener, suds suppressors,
perfume) 0-5%.
[0262] 8) A detergent composition formulated as a granulate
comprising linear alkylbenzenesulfonate (calculated as acid) about
8% to about 14%; ethoxylated fatty acid monoethanolamide about 5%
to about 11%; soap as fatty acid 0% to about 3%; sodium carbonate
(e.g., Na.sub.2CO.sub.3) about 4% to about 10%; soluble silicate
(Na.sub.2O, 2SiO.sub.2) about 1% to about 4%; zeolite (e.g.,
NaAlSiO.sub.4) about 30% to about 50%; sodium sulfate (e.g.,
Na.sub.2SO.sub.4) about 3% to about 11%; sodium citrate (e.g.,
C.sub.6H.sub.5Na.sub.3O.sub.7) about 5% to about 12%; polymers
(e.g., PVP, maleic/acrylic acid copolymer, PEG) about 1% to about
5%; enzymes (calculated as pure enzyme protein) 0.0001-0.1%; and
minor ingredients (e.g., suds suppressors, perfume) 0-5%.
[0263] 9) A detergent composition formulated as a granulate
comprising linear alkylbenzenesulfonate (calculated as acid) about
6% to about 12%; nonionic surfactant about 1% to about 4%; soap as
fatty acid about 2% to about 6%; sodium carbonate (e.g.,
Na.sub.2CO.sub.3) about 14% to about 22%; zeolite (e.g.,
NaAlSiO.sub.4) about 18% to about 32%; sodium sulfate (e.g.,
Na.sub.2SO.sub.4) about 5% to about 20%; sodium citrate (e.g.,
C.sub.6H.sub.5Na.sub.3O.sub.7) about 3% to about 8%; sodium
perborate (e.g., NaBO.sub.3H.sub.2O) about 4% to about 9%; bleach
activator (e.g., NOBS or TAED) about 1% to about 5%;
carboxymethylcellulose (CMC) 0% to about 2%; polymers (e.g.,
polycarboxylate or PEG) about 1% to about 5%; enzymes (calculated
as pure enzyme protein) 0.0001-0.1%; and minor ingredients (e.g.,
optical brightener, perfume) 0-5%.
[0264] 10) An aqueous liquid detergent composition comprising
linear alkylbenzenesulfonate (calculated as acid) about 15% to
about 23%; alcohol ethoxysulfate (e.g., C.sub.12-15 alcohol, 2-3
EO) about 8% to about 15%; alcohol ethoxylate (e.g., C.sub.12-15
alcohol, 7 EO, or C.sub.12-15 alcohol, 5 EO) about 3% to about 9%;
soap as fatty acid (e.g., lauric acid) 0% to about 3%; aminoethanol
about 1% to about 5%; sodium citrate about 5% to about 10%;
hydrotrope (e.g., sodium toluensulfonate) about 2% to about 6%;
borate (e.g., B.sub.4O.sub.7) 0% to about 2%;
carboxymethylcellulose 0% to about 1%; ethanol about 1% to about
3%; propylene glycol about 2% to about 5%; enzymes (calculated as
pure enzyme protein) 0.0001-0.1%; and minor ingredients (e.g.,
polymers, dispersants, perfume, optical brighteners) 0-5%.
[0265] 11) An aqueous liquid detergent composition comprising
linear alkylbenzenesulfonate (calculated as acid) about 20% to
about 32%; alcohol ethoxylate (e.g., C.sub.12-15 alcohol, 7 EO, or
C.sub.12-15 alcohol, 5 EO) 6-12%; aminoethanol about 2% to about
6%; citric acid about 8% to about 14%; borate (e.g.,
B.sub.4O.sub.7) about 1% to about 3%; polymer (e.g., maleic/acrylic
acid copolymer, anchoring polymer such as, e.g., lauryl
methacrylate/acrylic acid copolymer) 0% to about 3%; glycerol about
3% to about 8%; enzymes (calculated as pure enzyme protein)
0.0001-0.1%; and minor ingredients (e.g., hydrotropes, dispersants,
perfume, optical brighteners) 0-5%.
[0266] 12) A detergent composition formulated as a granulate having
a bulk density of at least 600 g/L comprising anionic surfactant
(linear alkylbenzenesulfonate, alkyl sulfate,
.alpha.-olefinsulfonate, .alpha.-sulfo fatty acid methyl esters,
alkanesulfonates, soap) about 25% to about 40%; nonionic surfactant
(e.g., alcohol ethoxylate) about 1% to about 10%; sodium carbonate
(e.g., Na.sub.2CO.sub.3) about 8% to about 25%; soluble silicates
(e.g., Na.sub.2O, 2SiO.sub.2) about 5% to about 15%; sodium sulfate
(e.g., Na.sub.2SO.sub.4) 0% to about 5%; zeolite (NaAlSiO.sub.4)
about 15% to about 28%; sodium perborate (e.g.,
NaBO.sub.3.4H.sub.2O) 0% to about 20%; bleach activator (TAED or
NOBS) about 0% to about 5%; enzymes (calculated as pure enzyme
protein) 0.0001-0.1%; minor ingredients (e.g., perfume, optical
brighteners) 0-3%.
[0267] 13) Detergent compositions as described in compositions
1)-12) supra, wherein all or part of the linear
alkylbenzenesulfonate is replaced by (C.sub.12-C.sub.18) alkyl
sulfate.
[0268] 14) A detergent composition formulated as a granulate having
a bulk density of at least 600 g/L comprising (C.sub.12-C.sub.18)
alkyl sulfate about 9% to about 15%; alcohol ethoxylate about 3% to
about 6%; polyhydroxy alkyl fatty acid amide about 1% to about 5%;
zeolite (e.g., NaAlSiO.sub.4) about 10% to about 20%; layered
disilicate (e.g., SK56 from Hoechst) about 10% to about 20%; sodium
carbonate (e.g., Na.sub.2CO.sub.3) about 3% to about 12%; soluble
silicate (e.g., Na.sub.2O, 2SiO.sub.2) 0% to about 6%; sodium
citrate about 4% to about 8%; sodium percarbonate about 13% to
about 22%; TAED about 3% to about 8%; polymers (e.g.,
polycarboxylates and PVP) 0% to about 5%; enzymes (calculated as
pure enzyme protein) 0.0001-0.1%; and minor ingredients (e.g.,
optical brightener, photobleach, perfume, suds suppressors)
0-5%.
[0269] 15) A detergent composition formulated as a granulate having
a bulk density of at least 600 g/L comprising (C.sub.12-C.sub.18)
alkyl sulfate about 4% to about 8%; alcohol ethoxylate about 11% to
about 15%; soap about 1% to about 4%; zeolite MAP or zeolite A
about 35% to about 45%; sodium carbonate (as Na.sub.2CO.sub.3)
about 2% to about 8%; soluble silicate (e.g., Na.sub.2O,
2SiO.sub.2) 0% to about 4%; sodium percarbonate about 13% to about
22%; TAED 1-8%; carboxymethylcellulose (CMC) 0% to about 3%;
polymers (e.g., polycarboxylates and PVP) 0% to about 3%; enzymes
(calculated as pure enzyme protein) 0.0001-0.1%; and minor
ingredients (e.g., optical brightener, phosphonate, perfume)
0-3%.
[0270] 16) Detergent formulations as described in 1)-15) supra,
which contain a stabilized or encapsulated peracid, either as an
additional component or as a substitute for already specified
bleach systems.
[0271] 17) Detergent compositions as described supra in 1), 3), 7),
9), and 12), wherein perborate is replaced by percarbonate.
[0272] 18) Detergent compositions as described supra in 1), 3), 7),
9), 12), 14), and 15), which additionally contain a manganese
catalyst. The manganese catalyst for example is one of the
compounds described in "Efficient manganese catalysts for
low-temperature bleaching," Nature 369: 637-639 (1994).
[0273] 19) Detergent composition formulated as a non-aqueous
detergent liquid comprising a liquid nonionic surfactant such as,
e.g., linear alkoxylated primary alcohol, a builder system (e.g.,
phosphate), an enzyme(s), and alkali. The detergent may also
comprise anionic surfactant and/or a bleach system.
[0274] As above, the present amylase polypeptide may be
incorporated at a concentration conventionally employed in
detergents. It is at present contemplated that, in the detergent
composition, the enzyme may be added in an amount corresponding to
0.00001-1.0 mg (calculated as pure enzyme protein) of amylase
polypeptide per liter of wash liquor.
[0275] The detergent composition may also contain other
conventional detergent ingredients, e.g., deflocculant material,
filler material, foam depressors, anti-corrosion agents,
soil-suspending agents, sequestering agents, anti-soil redeposition
agents, dehydrating agents, dyes, bactericides, fluorescers,
thickeners, and perfumes.
[0276] The detergent composition may be formulated as a hand
(manual) or machine (automatic) laundry detergent composition,
including a laundry additive composition suitable for pre-treatment
of stained fabrics and a rinse added fabric softener composition,
or be formulated as a detergent composition for use in general
household hard surface cleaning operations, or be formulated for
manual or automatic dishwashing operations.
[0277] Any of the cleaning compositions described, herein, may
include any number of additional enzymes. In general the enzyme(s)
should be compatible with the selected detergent, (e.g., with
respect to pH-optimum, compatibility with other enzymatic and
non-enzymatic ingredients, and the like), and the enzyme(s) should
be present in effective amounts. The following enzymes are provided
as examples.
[0278] Proteases:
[0279] Suitable proteases include those of animal, vegetable or
microbial origin. Chemically modified or protein engineered mutants
are included, as well as naturally processed proteins. The protease
may be a serine protease or a metalloprotease, an alkaline
microbial protease, a trypsin-like protease, or a chymotrypsin-like
protease. Examples of alkaline proteases are subtilisins, for
example those derived from Bacillus, e.g., subtilisin Novo,
subtilisin Carlsberg, subtilisin 309, subtilisin 147, and
subtilisin 168 (see, e.g., WO 89/06279). Examples of trypsin-like
proteases are trypsin (e.g., of porcine or bovine origin), and
Fusarium proteases (see, e.g., WO 89/06270 and WO 94/25583).
Examples of useful proteases also include but are not limited to
the variants described in WO 92/19729, WO 98/20115, WO 98/20116,
and WO 98/34946. Commercially available protease enzymes include
but are not limited to: ALCALASE.RTM., SAVINASE.RTM., PRIMASE.TM.,
DURALASE.TM., ESPERASE.RTM., KANNASE.TM., and BLAZE.TM. (Novo
Nordisk A/S and Novozymes A/S); MAXATASE.RTM., MAXACAL.TM.,
MAXAPEM.TM., PROPERASE.RTM., PURAFECT.RTM., PURAFECT OXP.TM.,
FN2.TM., and FN3.TM. (Danisco US Inc.). Other exemplary proteases
include NprE from Bacillus amyloliquefaciens and ASP from
Cellulomonas sp. strain 69B4.
[0280] Lipases:
[0281] Suitable lipases include those of bacterial or fungal
origin. Chemically modified, proteolytically modified, or protein
engineered mutants are included. Examples of useful lipases include
but are not limited to lipases from Humicola (synonym Thermomyces),
e.g., from H. lanuginosa (T. lanuginosus) (see e.g., EP 258068 and
EP 305216), from H. insolens (see e.g., WO 96/13580); a Pseudomonas
lipase (e.g., from P. alcaligenes or P. pseudoalcaligenes; see,
e.g., EP 218 272), P. cepacia (see e.g., EP 331 376), P. stutzeri
(see e.g., GB 1,372,034), P. fluorescens, Pseudomonas sp. strain SD
705 (see e.g., WO 95/06720 and WO 96/27002), P. wisconsinensis (see
e.g., WO 96/12012); a Bacillus lipase (e.g., from B. subtilis; see
e.g., Dartois et al. Biochemica et Biophysica Acta, 1131: 253-360
(1993)), B. stearothermophilus (see e.g., JP 64/744992), or B.
pumilus (see e.g., WO 91/16422). Additional lipase variants
contemplated for use in the formulations include those described
for example in: WO 92/05249, WO 94/01541, WO 95/35381, WO 96/00292,
WO 95/30744, WO 94/25578, WO 95/14783, WO 95/22615, WO 97/04079, WO
97/07202, EP 407225, and EP 260105. Some commercially available
lipase enzymes include LIPOLASE.RTM. and LIPOLASE ULTRA.TM. (Novo
Nordisk A/S and Novozymes A/S).
[0282] Polyesterases:
[0283] Suitable polyesterases can be included in the composition,
such as those described in, for example, WO 01/34899, WO 01/14629,
and U.S. Pat. No. 6,933,140.
[0284] Amylases:
[0285] The compositions can be combined with amylases, such as
non-production enhanced amylase. These can include commercially
available amylases, such as but not limited to STAINZYME.RTM.,
NATALASE.RTM., DURAMYL.RTM., TERMAMYL.RTM., FUNGAMYL.RTM. and
BAN.TM. (Novo Nordisk A/S and Novozymes A/S); RAPIDASE.RTM.,
POWERASE.RTM., and PURASTAR.RTM. (from Danisco US Inc.).
[0286] Cellulases:
[0287] Cellulases can be added to the compositions. Suitable
cellulases include those of bacterial or fungal origin. Chemically
modified or protein engineered mutants are included. Suitable
cellulases include cellulases from the genera Bacillus,
Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium, e.g., the
fungal cellulases produced from Humicola insolens, Myceliophthora
thermophila and Fusarium oxysporum disclosed for example in U.S.
Pat. Nos. 4,435,307; 5,648,263; 5,691,178; 5,776,757; and WO
89/09259. Exemplary cellulases contemplated for use are those
having color care benefit for the textile. Examples of such
cellulases are cellulases described in for example EP 0495257, EP
0531372, WO 96/11262, WO 96/29397, and WO 98/08940. Other examples
are cellulase variants, such as those described in WO 94/07998; WO
98/12307; WO 95/24471; PCT/DK98/00299; EP 531315; U.S. Pat. Nos.
5,457,046; 5,686,593; and 5,763,254. Commercially available
cellulases include CELLUZYME.RTM. and CAREZYME.RTM. (Novo Nordisk
A/S and Novozymes A/S); CLAZINASE.RTM. and PURADAX HA.RTM. (Danisco
US Inc.); and KAC-500(B).TM. (Kao Corporation).
[0288] Peroxidases/Oxidases:
[0289] Suitable peroxidases/oxidases contemplated for use in the
compositions include those of plant, bacterial or fungal origin.
Chemically modified or protein engineered mutants are included.
Examples of useful peroxidases include peroxidases from Coprinus,
e.g., from C. cinereus, and variants thereof as those described in
WO 93/24618, WO 95/10602, and WO 98/15257. Commercially available
peroxidases include for example GUARDZYME.TM. (Novo Nordisk A/S and
Novozymes A/S).
[0290] The detergent composition can also comprise
2,6-.beta.-D-fructan hydrolase, which is effective for
removal/cleaning of biofilm present on household and/or industrial
textile/laundry.
[0291] The detergent enzyme(s) may be included in a detergent
composition by adding separate additives containing one or more
enzymes, or by adding a combined additive comprising all of these
enzymes. A detergent additive, i.e. a separate additive or a
combined additive, can be formulated e.g., as a granulate, a
liquid, a slurry, and the like. Exemplary detergent additive
formulations include but are not limited to granulates, in
particular non-dusting granulates, liquids, in particular
stabilized liquids or slurries.
[0292] Non-dusting granulates may be produced, e.g., as disclosed
in U.S. Pat. Nos. 4,106,991 and 4,661,452 and may optionally be
coated by methods known in the art. Examples of waxy coating
materials are poly(ethylene oxide) products (e.g.,
polyethyleneglycol, PEG) with mean molar weights of 1,000 to
20,000; ethoxylated nonylphenols having from 16 to 50 ethylene
oxide units; ethoxylated fatty alcohols in which the alcohol
contains from 12 to 20 carbon atoms and in which there are 15 to 80
ethylene oxide units; fatty alcohols; fatty acids; and mono- and
di- and triglycerides of fatty acids. Examples of film-forming
coating materials suitable for application by fluid bed techniques
are given in, for example, GB 1483591. Liquid enzyme preparations
may, for instance, be stabilized by adding a polyol such as
propylene glycol, a sugar or sugar alcohol, lactic acid or boric
acid according to established methods. Protected enzymes may be
prepared according to the method disclosed in EP 238,216.
[0293] The detergent composition may be in any convenient form,
e.g., a bar, a tablet, a powder, a granule, a paste, or a liquid. A
liquid detergent may be aqueous, typically containing up to about
70% water, and 0% to about 30% organic solvent. Compact detergent
gels containing about 30% or less water are also contemplated. The
detergent composition can optionally comprise one or more
surfactants, which may be non-ionic, including semi-polar and/or
anionic and/or cationic and/or zwitterionic. The surfactants can be
present in a wide range, from about 0.1% to about 60% by
weight.
[0294] When included therein the detergent will typically contain
from about 1% to about 40% of an anionic surfactant, such as linear
alkylbenzenesulfonate, .alpha.-olefinsulfonate, alkyl sulfate
(fatty alcohol sulfate), alcohol ethoxysulfate, secondary
alkanesulfonate, .alpha.-sulfo fatty acid methyl ester, alkyl- or
alkenylsuccinic acid, or soap.
[0295] When included therein, the detergent will usually contain
from about 0.2% to about 40% of a non-ionic surfactant such as
alcohol ethoxylate, nonylphenol ethoxylate, alkylpolyglycoside,
alkyldimethylamineoxide, ethoxylated fatty acid monoethanolamide,
fatty acid monoethanolamide, polyhydroxy alkyl fatty acid amide, or
N-acyl-N-alkyl derivatives of glucosamine ("glucamides").
[0296] The detergent may contain 0% to about 65% of a detergent
builder or complexing agent such as zeolite, diphosphate,
triphosphate, phosphonate, carbonate, citrate, nitrilotriacetic
acid, ethylenediaminetetraacetic acid (EDTA),
diethylenetriaminepentaacetic acid, alkyl- or alkenylsuccinic acid,
soluble silicates or layered silicates (e.g., SKS-6 from
Hoechst).
[0297] The detergent may comprise one or more polymers. Exemplary
polymers include carboxymethylcellulose (CMC),
poly(vinylpyrrolidone) (PVP), poly(ethylene glycol) (PEG),
poly(vinyl alcohol) (PVA), poly(vinylpyridine-N-oxide),
poly(vinylimidazole), polycarboxylates e.g., polyacrylates,
maleic/acrylic acid copolymers), and lauryl methacrylate/acrylic
acid copolymers.
[0298] The enzyme(s) of the detergent composition may be stabilized
using conventional stabilizing agents, e.g., as polyol (e.g.,
propylene glycol or glycerol), a sugar or sugar alcohol, lactic
acid, boric acid, or a boric acid derivative (e.g., an aromatic
borate ester), or a phenyl boronic acid derivative (e.g.,
4-formylphenyl boronic acid). The composition may be formulated as
described in WO 92/19709 and WO 92/19708.
[0299] It is contemplated that in the detergent compositions, in
particular the enzyme variants, may be added in an amount
corresponding to about 0.01 to about 100 mg of enzyme protein per
liter of wash liquor (e.g., about 0.05 to about 5.0 mg of enzyme
protein per liter of wash liquor or 0.1 to about 1.0 mg of enzyme
protein per liter of wash liquor).
[0300] Although the present compositions and methods have been
described with reference to the details below, it would be
understood that various modifications can be made.
7.6. Methods of Assessing Amylase Activity in Detergent
Compositions
[0301] Numerous glucoamylase cleaning assays are known in the art,
including swatch and micro-swatch assays. The appended Examples
describe only a few such assays.
8. Brewing Compositions
[0302] An AfGATR or variant thereof may be a component of a brewing
composition used in a process of providing a fermented beverage,
such as brewing. It is believed that non-fermentable carbohydrates
form the majority of the dissolved solids in the final beer. This
residue remains because of the inability of malt amylases to
hydrolyze the alpha-1,6-linkages of the starch. The non-fermentable
carbohydrates contribute about 50 calories per 12 ounces (about 340
grams) of beer. The AfGATR or variant thereof, usually in
combination with a glucoamylase and optionally a pullulanase and/or
isoamylase, assist in converting the starch into dextrins and
fermentable sugars, lowering the residual non-fermentable
carbohydrates in the final beer.
[0303] The principal raw materials used in making these beverages
are water, hops and malt. In addition, but also exclusively,
adjuncts such as common corn grits, refined corn grits, brewer's
milled yeast, rice, sorghum, refined corn starch, barley, barley
starch, dehusked barley, wheat, wheat starch, torrified cereal,
cereal flakes, rye, oats, potato, tapioca, and syrups, such as corn
syrup, sugar cane syrup, inverted sugar syrup, barley and/or wheat
syrups, and the like may be used as a source of starch.
[0304] For a number of reasons, the malt, which is produced
principally from selected varieties of barley, has an important
effect on the overall character and quality of the beer. First, the
malt is the primary flavoring agent in beer. Second, the malt
provides the major portion of the fermentable sugar. Third, the
malt provides the proteins, which will contribute to the body and
foam character of the beer. Fourth, the malt provides the necessary
enzymatic activity during mashing. Hops also contribute
significantly to beer quality, including flavoring. In particular,
hops (or hops constituents) add desirable bittering substances to
the beer. In addition, the hops can act as protein precipitants,
establish preservative agents and aid in foam formation and
stabilization.
[0305] Cereals (grains), such as barley, oats, wheat, but also corn
and rice are often used for brewing, both in industry and for home
brewing, but also other plant components, such as hops are often
added. The components used in brewing may be unmalted or may be
malted, i.e., partially germinated, resulting in an increase in the
levels of enzymes, including .alpha.-amylase. For successful
brewing, adequate levels of .alpha.-amylase enzyme activity are
necessary to ensure the appropriate levels of sugars for
fermentation. An AfGATR or variant thereof may also be added to the
components used for brewing.
[0306] As used herein, the term "stock" means grains and plant
components that are crushed or broken. For example, barley used in
beer production is a grain that has been coarsely ground or crushed
to yield a consistency appropriate for producing a mash for
fermentation. As used herein, the term "stock" includes any of the
aforementioned types of plants and grains in crushed or coarsely
ground forms. The methods described herein may be used to determine
.alpha.-amylase activity levels in both flours and stock.
[0307] Processes for making beer are well known in the art. See,
e.g., Wolfgang Kunze (2004) "Technology Brewing and Malting,"
Research and Teaching Institute of Brewing, Berlin (VLB), 3rd
edition. Briefly, the process involves: (a) preparing a mash, (b)
filtering the mash to prepare a wort, and (c) fermenting the wort
to obtain a fermented beverage, such as beer. Typically, milled or
crushed malt, malt and adjunct, or adjunct is mixed with water and
held for a period of time under controlled temperatures to permit
the enzymes present in the malt and/or adjunct to convert the
starch present in the malt into fermentable sugars. The mash is
then transferred to a mash filter where the liquid is separated
from the grain residue. This sweet liquid is called "wort," and the
left over grain residue is called "spent grain." The mash is
typically subjected to an extraction, which involves adding water
to the mash in order to recover the residual soluble extract from
the spent grain. The wort is then boiled vigorously to sterilizes
the wort and help develop the color, flavor and odor. Hops are
added at some point during the boiling. The wort is cooled and
transferred to a fermentor.
[0308] The wort is then contacted in a fermentor with yeast. The
fermentor may be chilled to stop fermentation. The yeast that may
flocculate is removed. Finally, the beer is cooled and stored for a
period of time, during which the beer clarifies and its flavor
develops, and any material that might impair the appearance,
flavor, and shelf life of the beer settles out. The beer usually
contains from about 2% to about 10% v/v alcohol, although beer with
a higher alcohol content, e.g., 18% v/v, may be obtained. Prior to
packaging, the beer is carbonated and, optionally, filtered, and
pasteurized.
[0309] The brewing composition comprising an alpha-amylase, often,
but not necessarily in combination with one or more exogenous
enzymes, such as glucoamylase(s) (e.g. AfGATR or variant thereof),
pullulanase(s) and/or isoamylase(s), and any combination thereof,
may be added to the mash of step (a) above, i.e., during the
preparation of the mash. Alternatively, or in addition, the brewing
composition may be added to the mash of step (b) above, such as
during the filtration of the mash. Alternatively, or in addition,
the brewing composition may be added to the wort of step (c) above,
such as during the fermenting of the wort.
[0310] Particular embodiments pertains to any of the above uses,
methods or fermented beverages, wherein said fermented beverage is
a beer, such as full malted beer, beer brewed under the
"Reinheitsgebot," ale, IPA, lager, bitter, Happoshu (second beer),
third beer, dry beer, near beer, light beer, low alcohol beer, low
calorie beer, porter, bock beer, stout, malt liquor, non-alcoholic
beer, non-alcoholic malt liquor and the like, but also alternative
cereal and malt beverages such as fruit flavoured malt beverages,
e.g., citrus flavoured, such as lemon-, orange-, lime-, or
berry-flavoured malt beverages, liquor flavoured malt beverages,
e.g., vodka-, rum-, or tequila-flavoured malt liquor, or coffee
flavoured malt beverages, such as caffeine-flavoured malt liquor,
and the like.
9. Reduction of Iodine-Positive Starch
[0311] AfGATR and variants thereof may reduce the iodine-positive
starch (IPS), when used in a method of liquefaction and/or
saccharification. One source of IPS is from amylose that escapes
hydrolysis and/or from retrograded starch polymer. Starch
retrogradation occurs spontaneously in a starch paste, or gel on
ageing, because of the tendency of starch molecules to bind to one
another followed by an increase in crystallinity. Solutions of low
concentration become increasingly cloudy due to the progressive
association of starch molecules into larger articles. Spontaneous
precipitation takes place and the precipitated starch appears to be
reverting to its original condition of cold-water insolubility.
Pastes of higher concentration on cooling set to a gel, which on
ageing becomes steadily firmer due to the increasing association of
the starch molecules. This arises because of the strong tendency
for hydrogen bond formation between hydroxy groups on adjacent
starch molecules. See J. A. Radley, ed., STARCH AND ITS DERIVATIVES
194-201 (Chapman and Hall, London (1968)).
[0312] The presence of IPS in saccharide liquor negatively affects
final product quality and represents a major issue with downstream
processing. IPS plugs or slows filtration system, and fouls the
carbon columns used for purification. When IPS reaches sufficiently
high levels, it may leak through the carbon columns and decrease
production efficiency. Additionally, it may results in hazy final
product upon storage, which is unacceptable for final product
quality. The amount of IPS can be reduced by isolating the
saccharification tank and blending the contents back. IPS
nevertheless will accumulate in carbon columns and filter systems,
among other things. The use of AfGATR or variants thereof thus is
expected to improve overall process performance by reducing the
amount of IPS.
[0313] In order to further illustrate the compositions and methods,
and advantages thereof, the following specific examples are given
with the understanding that they are illustrative rather than
limiting.
EXAMPLES
Example 1
Cloning of AfGA1
[0314] Genomic DNA of Aspergillus fumigatus Af293 was purchased
from Fungal Genetics Stock Center, Kansas City, Mo. (FGSC A1100).
The genome of Aspergillus fumigatus is sequenced. The nucleic acid
sequence for the AfGA1 gene (within the disclosed genome in NCBI
Reference Sequence NC_007195), and the amino acid sequence of the
predicted glucan 1,4-alpha-glucosidase (NCBI Accession No.
XP_749206) encoded by the AfGA1 gene were obtained in the NCBI
Databases. AfGA1 is homologous to other fungal glucoamylases as
determined from a BLAST search. See FIG. 1. The nucleotide sequence
of the AfGA1 gene, which comprises three introns, is set forth in
SEQ ID NO: 8.
[0315] The AfGA1 gene was amplified from genomic DNA of Aspergillus
fumigatus using the following primers: Primer 1: AfGA1-Fw 5'-GCG
GCGGCCGC ACC atgcctcgcctttcctacgc-3' (SEQ ID NO: 9), and Primer 2:
AfGA1-Rv 5'-cc ggcgcgccc TTA tcactgccaagtatcattctcg-3' (SEQ ID NO:
10). The forward primer contains a NotI restriction site, and the
reverse primer contains an AscI restriction site. After digestion
with NotI and AscI, the PCR product was cloned into the pTrex3gM
expression vector (described in U.S. Published Application
2011/0136197 A1) digested with the same restriction enzymes, and
the resulting plasmid was labeled pJG222. A plasmid map of pJG222
is provided in FIG. 2. The sequence of the AfGA1 gene was confirmed
by DNA sequencing.
Example 2
Expression and Purification of AfGA1TR
[0316] The plasmid pJG222(Trex3gM-AfGA1) was transformed into a
quad-deleted Trichoderma reesei strain (described in WO 05/001036)
using biolistic method (Te'o et al., J. Microbiol. Methods 51:
393-99, 2002). Transformed colonies (about 50) appeared in about 1
week. After growth on acetamide plates for 5 days, the colonies
were inoculated in 250 ml shake flasks with 30 ml Glucose/Sepharose
defined medium for protein expression. The protein, AfGA1TR, was
secreted into the extracellular medium, and the filtered culture
medium was used to perform SDS-PAGE and a glucoamylase activity on
DP7 assay to confirm the enzyme expression.
[0317] The stable strain was subsequently grown in a 7 L fermenter
in a defined medium. Fermentation broth was harvested by
centrifugation. Following centrifugation, filtration and
concentration, 450 ml of the concentrated sample was obtained. The
concentration of total protein in the sample was determined to be
83.70 g/L by using BCA method (protein quantification kit, Shanghai
Generay Biotech CO., Ltd). SDS-PAGE analysis suggested that 80% of
the total protein was the target protein. Thus, the concentration
of target protein in the concentrated sample was estimated to be
66.96 g/L.
[0318] AfGA1TR was purified by affinity chromatography using an
AKTA Explorer 100 FPLC system (GE Healthcare). .beta.-Cyclodextrin
(Sigma-Aldrich, 856088) was coupled to epoxy-activated Sepharose
beads (GE Healthcare, 17-0480-01) and employed for purification.
The pH of 40 ml concentrated fermentation broth from the 7 L
fermenter was adjusted to 4.3 and the solution was loaded onto 30
ml .beta.-CD-Sepharose column pre-equilibrated with 25 mM, pH 4.3
sodium acetate (buffer A). The column was washed with a 2 column
volume of buffer A. The target protein was eluted with three column
volume of buffer B which containing buffer A and 10 mM
.alpha.-cyclodextrin (Sigma-Aldrich, C4642). Fractions were
analyzed by SDS-PAGE gel, and assayed for glucoamylase activity.
The fractions containing target protein were pooled and run through
a Hiprep 26.times.10 desalting column to remove
.beta.-cyclodextrin. The resulting sample was more than 95% pure,
the solution was concentrated using 10K Amicon Ultra-15 devices and
stored in 40% glycerol at -80.degree. C.
Example 3
Determination of AfGA1TR Substrate Specificity
[0319] Glucoamylase activity was assayed based on the release of
glucose by glucoamylases, AfGA1TR, AnGA or wild-type AfGA, from
different substrates, including maltose, isomaltose, maltoheptaose
(DP7), maltodextrin (DE4-DE10), potato amylopectin, and soluble
starch. The rate of glucose release was measured using a coupled
glucose oxidase/peroxidase (GOX/HRP) method (Anal. Biochem. 105
(1980), 389-397). Glucose was quantified as the rate of oxidation
of 2,2'-Azino-bis 3-ethylbenzothiazoline-6-sulfonic acid (ABTS) by
peroxide which was generated from coupled GOX/HRP enzymes reacted
with glucose.
[0320] Substrate solutions were prepared by mixing 9 mL of each
substrate (1% in water, w/w) and 1 mL of 0.5 M pH 5.0 sodium
acetate buffer in a 15-mL conical tube. Coupled enzyme (GOX/HRP)
solution with ABTS was prepared by dissolving GOX/HRP in 50 mM
sodium acetate buffer (pH 5.0), with the final concentrations of
2.74 mg/mL ABTS, 0.1 U/mL HRP, and 1 U/mL GOX.
[0321] Serial dilutions of glucoamylase samples, the benchmark AnGA
(Genencor product, Optidex L-400), wild-type AfGA, and glucose
standard were also prepared in 50 mM sodium acetate buffer (pH
5.0). Each glucoamylase sample (10 .mu.l) was transferred into a
new microtiter plate (Corning 3641) containing 90 .mu.l of
substrate solution preincubated at 50.degree. C. for 5 min at 600
rpm. The reactions were carried out at 50.degree. C. for 10 min
with shaking (600 rpm) in a thermomixer (Eppendorf), 10 .mu.l of
reaction mixtures as well as 10 .mu.l of serial dilutions of
glucose standard were quickly transferred to new microtiter plates
(Corning 9017), respectively, followed by the addition of 100 .mu.l
of ABTS/GOX/HRP solution. The microtiter plates containing the
reaction mixture were immediately measured at 405 nm at 11 seconds
intervals for 5 min on SoftMax Pro plate reader (Molecular Device).
The output was the reaction rate, Vo, for each enzyme
concentration. Linear regression was used to determine the slope of
the plot Vo vs. enzyme dose. The specific activity of glucoamylase
activity was calculated based on the glucose standard curve using
Equation 1:
Specific Activity (Unit/mg)=Slope (enzyme)/slope (std).times.100
(1),
where 1 Unit=1 .mu.mol glucose/min.
[0322] Representative specific activities of AfGA1TR and the
benchmark glucoamylases AnGA and wild-type AfGA are shown in Table
1.
TABLE-US-00006 TABLE 1 Specific activity of purified glucoamylases
on various substrates. Specific activity (U/mg) Wild-type Substrate
AnGA AfGA AfGA1TR Maltose (DP2) 29.2 29.2 42.7 Isomaltose 0 0.6 0.9
Maltoheptaose (DP7) 159.9 180.3 254.8 Maltodextrin 128.8 127.8
211.5 (DE4-10DE) Amylopectin from 142.5 146.5 197.8 potato Soluble
starch 137.5 128.0 213.4 Pullulan 29.2 25.6 31.1
Example 4
Effect of pH on AfGA1TR Glucoamylase Activity
[0323] The effect of pH (from 3.0 to 10.0) on AfGA1TR activity was
monitored using the ABTS assay protocol as described in Example 3.
Buffer working solutions consisted of the combination of
glycine/sodium acetate/HEPES (250 mM), with pH varying from 3.0 to
10.0. Substrate solutions were prepared by mixing soluble starch
(1% in water, w/w) with 250 mM buffer solution at a ratio of 9:1.
Enzyme working solutions were prepared in water at a certain dose
(showing signal within linear range as per dose response curve).
All the incubations were carried out at 50.degree. C. for 10 min
following the same protocol as described for glucoamylase activity
assay in Example 3. Enzyme activity at each pH was reported as
relative activity compared to enzyme activity at optimum pH. The pH
profile of AfGA1TR is shown in Table 2 and FIG. 3. AfGA1TR was
found to have an optimum pH at about 5.0, and retain greater than
70% of maximum activity between pH 3.5 and 7.5.
TABLE-US-00007 TABLE 2 pH profiles for purified glucoamylases
Relative activity (%) Native pH AnGA AfGA AfGA1TR 3 73.5 54.9 52.6
4 94.9 92.5 88.3 5 100 97.6 100 6 95.2 100 99.3 7 66.5 79.2 87.9 8
23.8 43 42.1 9 9.9 11 11.7 10 5.3 8.4 5.3
Example 5
Effect of Temperature on AfGA1TR Glucoamylase Activity
[0324] The effect of temperature (from 40.degree. C. to 84.degree.
C.) on AfGA1TR activity was monitored using the ABTS assay protocol
as described in Example 3. Substrate solutions were prepared by
mixing 3.6 mL of soluble starch (1% in water, w/w) and 0.4 mL of
0.5 M buffer (pH 5.0 sodium acetate) into a 15-mL conical tube.
Enzyme working solutions were prepared in water at a certain dose
(showing signal within linear range as per dose response curve).
Incubations were done at temperatures from 40.degree. C. to
84.degree. C., respectively, for 10 min following the same protocol
as described for glucoamylase activity assay in Example 3. Enzyme
activity at each temperature was reported as relative activity
compared to enzyme activity at optimum temperature. The temperature
profile of AfGA1TR is shown in Table 3 and FIG. 4. AfGA1TR was
found to have an optimum temperature of 68.degree. C., and retain
greater than 70% of maximum activity between 56.degree. C. and
74.degree. C.
TABLE-US-00008 TABLE 3 Temperature profiles for glucoamylases.
Relative activity (%) Temp Native (.degree. C.) AnGA AfGA AfGA1TR
40 33.9 36.6 32.7 42.1 36 38.2 37.6 46.5 46.1 49.4 45 54 57.2 66.3
61.9 60 85.1 91.9 87.1 66.6 100 100 100 74.1 31.1 39.4 70.4 80.2
11.8 10.9 11.2 83.5 8.8 9.6 8.8
Example 6
AfGA1TR Product Profile Analysis
[0325] To assay the products of fungal glucoamylase catalysis of
polysaccharides, the glucoamylases, AnGA (0.118 mg/gds starch) and
AfGA1TR (0.118 mg/gds or 0.059 mg/gds), were incubated with 34% DS
LIQUOZYME.RTM. Supra liquefied starch (CPI, Stockton, Calif.), at
60.degree. C., pH 4.2 to 4.5 for 2 days. Pullulanase (PU) and
acid-stable alpha-amylase from Aspergillus kawachii, GC626.RTM.
(AkAA) were dosed along with purified AfGA1TR at 0.256 ASPU/gds and
0.35 SSU/gds, respectively. Samples were taken at different
intervals of time and analyzed for sugar composition by HPLC.
[0326] Table 4 shows the profile of oligosaccharides saccharified
by AnGA/PU/AkAA and AfGA1TR/PU/AkAA at 100% and 50% the
concentration of AnGA. (FIG. 6 depicts the profile of
oligosaccharides saccharified by AnGA and AfGA1TR at 100%, 50%, and
40% the concentration of AnGA, with and without PU and AkAA). Only
oligosaccharides with DP1, DP2, DP3, and HS are shown. The numbers
in Table 4 reflect the weight percentage of each DPn as a fraction
of the total DP1, DP2, DP3, and HS.
TABLE-US-00009 TABLE 4 Product profile of fungal glucoamylases on
liquefied starch. % % % % Flask Enzymes Dose: /gds Hr DP1 DP2 DP3
HS 1 An-GA + 0.118 mg + 8 69.75 9.29 0.66 20.30 PU + 0.256 ASPU +
24 93.40 2.21 0.71 3.68 GC626 0.35 SSU 31 95.40 2.15 0.67 1.79 37
95.90 2.22 0.60 1.27 48.5 96.10 2.48 0.53 0.89 55 96.09 2.64 0.49
0.78 70 95.95 2.99 0.45 0.62 2 AfGA1TR + 0.118 mg + 8 86.34 2.35
0.47 10.84 PU + 0.256 ASPU + 24 96.01 2.29 0.49 1.21 GC626 0.35 SSU
31 96.11 2.58 0.44 0.88 37 96.03 2.84 0.42 0.71 48.5 95.70 3.33
0.43 0.54 55 95.52 3.56 0.44 0.48 70 94.97 4.12 0.49 0.42 3 AfGA2TR
+ 0.059 mg + 8 71.27 9.07 0.61 19.05 PU + 0.256 ASPU + 24 93.80
2.19 0.72 3.29 GC626 0.35 SSU 31 95.61 2.06 0.68 1.65 37 96.06 2.09
0.63 1.22 48.5 96.29 2.31 0.54 0.86 55 96.31 2.43 0.50 0.76 70
96.21 2.74 0.45 0.59
[0327] Table 4 showed that AfGA1TR resulted in >95.5% DP1 in 24
hours, compared to AnGA which took 48.5 hours under an equal dose
of protein. The data in Table 4 showed that AfGA1TR demonstrated an
improved performance over AnGA at 50% dose equivalent based on
protein (under the identical conditions of complimentary enzymes
dosage).
Example 7
Comparison of DP2 Levels
[0328] DP2 level from AfGA1TR treated liquified starch was compared
to the one from AnGA treated liquified starch based on the same DP1
level (96%). The comparison showed a statistically significant
reduction in DP2 level at equal DP1 level approx. by 0.2%, possibly
due to lower glucoamylase dose. Reversion reaction by AnGA and
AfGA1TR (as the triple blend) was measured by calculating
isomaltose/maltose ratio through ion chromatography.
TABLE-US-00010 TABLE 5 Product profile of fungal glucoamylases on
liquefied starch. % of the total composition after % of the
reversion glucoamylase total DP2 after reaction reaction
glucoamylase reaction Ratio Hour % DP1 % DP2 % IsoMaltose % Maltose
(IsoM/M) .DELTA.Ratio AnGA 48 96.08 2.26 55.6 44.4 1.25 70 95.91
2.77 64.9 35.1 1.85 AfGA1TR 48 96.16 2.13 54.8 45.2 1.21 0.04 70
96.10 2.60 63.4 36.6 1.73 0.13 .DELTA.Ratio = [AnGA ratio - AfGA1TR
ratio] at 48 and 70 hours
[0329] Table 5 shows that for both glucoamylases isomaltose is
accumulating over time according to increasing ratio of
Isomaltose:Maltose. However, isomaltose formation appears to be
slightly lower with AfGA1TR because the difference of ratio between
AnGA and AfGA1TR is increased from 48 hours to 70 hours, which may
support the lower reversion reaction by AfGA1TR.
Example 8
Titration of AkAA
[0330] To assay the products of fungal glucoamylase catalysis of
polysaccharides using varied doses of accessory alpha-amylase,
AfGA1TR (0.059 mg/gds) and Pullulanase (PU, OPTIMAX.RTM.
L-1000)(0.256 ASPU/gds) were incubated with different
concentrations of acid-stable alpha-amylase, GC626.RTM. (AkAA) from
Aspergillus kawachi. AkAA was added at 0 to 0.3 SSU/ds in
increments of 0.1 SSU and 34% DS LIQUOZYME.RTM. Supra liquefied
starch (CPI, Stockton, Calif.), at 60.degree. C., pH 4.5 for 2
days. Pullulanase (PU) and GC626.RTM. (AkAA) were dosed along with
purified AfGA1TR at 0.256 ASPU/gds and 0.35 SSU/gds, respectively.
Samples were taken at different intervals of time and analyzed for
sugar composition by HPLC.
[0331] Table 6 and FIG. 7 disclose profiles of oligosaccharides
saccharified by AfGA1TR, PU and varying doses of AkAA. Only
oligosaccharides with DP1, DP2, DP3 and HS are shown. The numbers
in Table 6 reflect the weight percentage of each DPn as a fraction
of the total DP1, DP2, DP3, and HS.
TABLE-US-00011 TABLE 6 Effect of AkAA during saccharification
enzyme liquefied starch by AfGA1TR. AkAA (GC626 .RTM.) Flask dose:
/gds Hr % DP1 % DP2 % DP3 % HS 1 0 6 64.43 8.51 0.68 26.37 21 88.93
1.60 0.46 9.01 29 92.19 1.80 0.45 5.56 45 94.60 2.22 0.40 2.78 53
94.99 2.40 0.38 2.23 68 95.55 2.77 0.37 1.30 2 0.1 SSU 6 63.10
10.00 0.77 26.12 21 91.07 2.01 0.59 6.33 29 94.33 2.01 0.58 3.09 45
96.13 2.23 0.49 1.15 53 96.24 2.39 0.46 0.91 68 96.20 2.72 0.42
0.66 3 0.2 SSU 6 63.66 10.47 0.92 24.96 21 91.75 2.18 0.65 5.43 29
95.03 2.05 0.64 2.28 45 96.19 2.25 0.52 1.03 53 96.28 2.41 0.48
0.83 68 96.22 2.73 0.43 0.62 4 0.3 SSU 6 59.97 11.76 1.74 26.53 21
91.52 2.44 0.71 5.33 29 95.03 2.08 0.70 2.19 45 96.21 2.20 0.57
1.01 53 96.31 2.34 0.52 0.83 68 96.27 2.64 0.46 0.63
Table 6 shows that AfGA1TR was able to reach >96% DP1 in 45
hours in presence of at least 0.1 SSU/gds, while lack of AkAA
resulted in a statistically significantly reduced rate of
saccharification. The result indicates that a significant increase
in the final glucose yield was achieved by the addition of AkAA
during saccharification of enzyme liquefied starch by AfGA1TR.
Example 9
Solubilization and Hydrolysis of Granular Starch by an Enzyme Blend
Containing Alpha-Amylase, AfGA1TR and Pullulanase
[0332] Granular corn starch slurry having 35% dry solid starch in
distilled water was prepared and the pH was adjusted to pH 5.0
using NaOH. 10 AAU/gds of alpha-amylase (SPEZYME.RTM. XTRA) and
purified protein of AfGA1TR were added at 0.047 mg/gds along with
0.15 ASPU/gds of pullulanase (OPTIMAX.RTM. L-1000) to the starch
slurry. Then, the starch slurry was kept in a water bath maintained
at 60.degree. C. with constant stirring. An aliquot was withdrawn
at different time intervals and centrifuged. The clear supernatant
was used for refractive index (RI) to calculate percent
solubilization and analyzed for sugar composition by HPLC.
TABLE-US-00012 TABLE 7 Product profile of fungal glucoamylases on
starch during liquefaction. % Dose: Solu- % % % % Enzymes /gds Hr
bility DP1 DP2 DP3 HS SPEZYME 10 5 52.5% 73.05 13.20 1.55 12.20
XTRA AAU 20.5 72.5% 93.00 2.38 1.86 2.76 29 76.2% 94.10 2.21 1.55
2.13 OPTIMAX 0.15 44.5 82.0% 95.14 2.16 1.16 1.54 L-1000 ASPU 52
83.5% 95.43 2.20 1.02 1.36 AfGA1TR 0.047 68 86.1% 95.75 2.36 0.81
1.08 mg
[0333] Table 7 shows that AfGA1TR was able to reach >95.5% DP1
in 68 hours using granular starch in presence of alpha-amylase and
PU, where granular starch was 86% solubilized.
Example 10
Effect of Residual Alpha-Amylase Activity on DP3 Level
[0334] The effect of single pH (5.5) and the effect of residual
alpha-amylase activity at pH 4.5 on both DP1 and DP3 with AfGA1TR
by adding 0.066 KG/MTds of LIQUOZYME.RTM. Supra (NZ) back to
alpha-killed starch liquefact was analyzed. 0.066 mg/gds of AfGA1TR
was blended with 0.25 ASPU/gds of OPTIMAX.RTM. L-1000 (pullulanase)
and 0.1 SS U/gds of AkAA. Table 8 and FIG. 5 disclose profiles of
oligosaccharides saccharified by AfGA1TR and OPTIMAX.RTM. 4060 VHP
(an AnGA/pullulanase blend).
TABLE-US-00013 TABLE 8 Product profile of fungal glucoamylase on
liquefied starch. Sugar composition at 48 hours % DP1 % DP2 % DP3 %
HS AfGA1TR triple 0.066 mg 95.12 1.99 0.71 2.19 (0.141 GAU),
Active, pH 5.5 AfGA1TR triple 0.066 mg 95.92 2.19 0.59 1.30 (0.141
GAU), Active, pH 4.5 AfGA1TR triple 0.066 mg 96.00 2.26 0.43 1.31
(0.141 GAU), Killed, pH 4.5 OPTIMAX .RTM. 4060 VHP 0.16 GAU, 95.93
2.27 0.55 1.25 Killed, pH 4.5 OPTIMAX .RTM. 4060 VHP 0.16 GAU,
95.86 2.27 0.68 1.18 Active, pH 4.5
[0335] AfGA1TR triple blend showed significant loss in the rate of
saccharification at pH 5.5 compared to pH 4.5 possibly due to
unfavorable pH to OPTIMAX.RTM. L-1000 but was able to achieve
>95.5% DP1 in 48 hours. Both AfGA1TR and OPTIMAX.RTM. 4060 VHP
were a bit negatively affected by residual alpha-amylase activity
to maximize glucose yield because of higher DP3 as expected. In the
case of alpha-amylase killed liquefact, AfGA1TR resulted in
significantly lower DP3 than OPTIMAX.RTM. 4060 VHP by 0.1%. Levels
of AfGA1TR with alpha-amylase active liquefact were as low as the
one of OPTIMAX.RTM. 4060 VHP with alpha-"killed" liquefact.
Example 11
Comparison of AfGA1TR with Wild-Type Aspergillus fumigatus
Glucoamylase
[0336] Starch liquefact was prepared to have 34% dry solids by
diluting with water and the saccharification was carried out using
the 2 different glucoamylases; 1) AfGA1TR at 0.067 mg/gds starch
and 2) purified protein of wild-type AfGA (expressed in Aspergillus
fumigatus) from GLUCOTEAM DB (Nagase Co. & Ltd., Japan) at
0.065 mg/gds at pH 4.4 and 60.degree. C. In addition, pullulanase
(PU) and acid-stable alpha-amylase, GC626.RTM. (AkAA) at 0.14
ASPU/gds and 0.9 SSU/gds, respectively, were dosed along with each
glucoamylase. Samples were taken at different intervals of time and
analyzed for sugar composition by HPLC.
[0337] Table 9 showed that AfGA1TR resulted in >95.5% DP1 in 48
hours, whereas commercial Aspergillus fumigatus took longer
saccharification time. Both glucoamylases were able to reach
>96% DP1 with DP2 less than 3%.
TABLE-US-00014 TABLE 9 Product profile of fungal glucoamylase
blends on liquefied starch. % % % % Enzymes Dose: /gds Hr DP1 DP2
DP3 HS AFGA1TR 0.067 mg 15 86.62 4.60 0.76 8.02 OPTIMAX L-1000 0.14
ASPU 26 94.78 2.11 0.77 2.34 GC626 .RTM. 0.9 SSU 39 96.12 2.07 0.55
1.26 48 96.27 2.26 0.45 1.02 63 96.22 2.61 0.34 0.83 71 96.16 2.77
0.31 0.76 Wild-type AfGA 0.065 mg 16 75.43 10.45 0.84 13.28 OPTIMAX
L-1000 0.14 ASPU 24 87.33 4.87 0.93 6.87 GC626 .RTM. 0.9 SSU 40
95.11 2.05 0.89 1.95 48 95.81 1.93 0.79 1.47 64 96.16 2.09 0.61
1.14 72 96.21 2.19 0.55 1.05
Example 12
Solubilization and Hydrolysis of Granular Starch by Enzyme Blend
Containing Alpha-Amylase, Pullulanase and Aspergillus fumigatus
GAs
[0338] In a typical example, granular corn starch slurry having 35%
dry solid starch in distilled water was prepared and the pH was
adjusted to pH 5.0 using sodium hydroxide. Ten AAU/gds of
SPEZYME.RTM. XTRA and purified protein of AfGA1TR or wild-type
Aspergillus fumigatus GA (AfGA) from GLUCOTEAM DB (Nagase Co. &
Ltd., Japan) were added along with 0.15 ASPU/gds of OPTIMAX.RTM.
L-1000 to the starch slurry. Then, the starch slurry was kept in a
water bath maintained at 60.degree. C. with constant stirring. An
aliquot was withdrawn at different time intervals and centrifuged.
The clear supernatant was used for refractive index (RI) to
calculate percent solubilization and analyzed for sugar composition
by HPLC. Table 10 shows that both Aspergillus fumigatus
glucoamylases were able to reach >95.5% DP1 by 68 hours using
granular starch in presence of alpha-amylase and PU, where granular
starch was >86% solubilized.
TABLE-US-00015 TABLE 10 Effect of alpha-amylase and pullulanase on
granular starch solubilization % Enzymes Dose: /gds Hr Solubility %
DP1 % DP2 % DP3 % HS SPEZYME XTRA 10 AAU 5 52.5% 73.05 13.20 1.55
12.20 OPTIMAX L-1000 0.15 ASPU 20.5 72.5% 93.00 2.38 1.86 2.76
AfGA1TR 0.047 mg 29 76.2% 94.10 2.21 1.55 2.13 44.5 82.0% 95.14
2.16 1.16 1.54 52 83.5% 95.43 2.20 1.02 1.36 68 86.1% 95.75 2.36
0.81 1.08 SPEZYME XTRA 10 AAU 15 72.3% 87.15 4.92 2.25 5.68 OPTIMAX
L-1000 0.14 ASPU 25 78.7% 91.93 2.68 2.15 3.24 Wild-type AfGA 0.075
mg 39 86.0% 94.24 2.00 1.70 2.06 50 87.3% 94.84 1.96 1.45 1.75 64
91.5% 95.42 2.04 1.15 1.39 73 92.1% 95.62 2.12 1.01 1.25
Example 13
Expression and Purification of AfGA2TR
[0339] The nucleic acid sequence for the AfGA2 gene (NCBI Reference
Sequence DS499595 from 145382 to 147441) was mined from Aspergillus
fumigatus A1163, and the amino acid sequence of the hypothetical
protein encoded by the AfGA2 gene was found in the NCBI Databases
(NCBI Accession No. EDP53734). The nucleotide sequence of the AfGA2
gene from Aspergillus fumigatus A1163 was optimized and synthesized
by Generay (Generay Biotech Co., Ltd, Shanghai, China).
[0340] The DNA sequence of AfGA2 was optimized for its expression
in Trichoderma reesei, then synthesized and inserted into the
pTrex3gM expression vector (described in U.S. Published Application
2011/0136197 A1) by Generay (Generay Biotech Co., Ltd, Shanghai,
China), resulting in pJG313 (FIG. 9).
[0341] The plasmid pJG313 was transformed into a quad-deleted
Trichoderma reesei strain (described in WO 05/001036) using
biolistic method (Te'o et al., J. Microbiol. Methods 51:393-99,
2002). Transformants were selected on a medium containing acetamide
as a sole source of nitrogen (acetamide 0.6 g/L; cesium chloride
1.68 g/L; glucose 20 g/L; potassium dihydrogen phosphate 15 g/L;
magnesium sulfate heptahydrate 0.6 g/L; calcium chloride dihydrate
0.6 g/L; iron (II) sulfate 5 mg/L; zinc sulfate 1.4 mg/L; cobalt
(II) chloride 1 mg/L; manganese (II) sulfate 1.6 mg/L; agar 20 g/L;
pH 4.25). Transformed colonies (about 50-100) appeared in about 1
week. After growth on acetamide plates, transformants were picked
and transferred individually to acetamide agar plates. After 5 days
of growth on acetamide plates, transformants displaying stable
morphology were inoculated into 200 .mu.l Glucose/Sophorose defined
media in 96-well microtiter plates. The microtiter plate was
incubated in an oxygen growth chamber at 28.degree. C. for 5 days.
Supernatants from these cultures were used to confirm the protein
expression by SDS-PAGE analysis and assay for enzyme activity. The
stable strains were subsequently grown in a 7 L fermentor in a
defined medium containing 60% glucose-sophorose feed.
Glucose/Sophorose defined medium (per liter) consists of
(NH.sub.4).sub.2SO.sub.4 5 g, PIPPS buffer 33 g, Casamino Acids 9
g, KH.sub.2PO.sub.4 4.5 g, CaCl.sub.2 (anhydrous) 1 g,
MgSO.sub.4.7H.sub.2O 1 g, pH to 5.5 adjusted with 50% NaOH with
Milli-Q H.sub.2O to bring to 966.5 mL. After sterilization, the
following were added: 26 mL 60% Glucose/Sophrose, and 400.times.T.
reesei Trace Metals 2.5 mL.
[0342] The protein, AfGA2TR, was purified via two steps of
chromatography. Ammonium sulfate was added to 600 mL fermentation
broth until the final concentration of ammonium sulfate reaches 1
M. The sample was loaded onto a 50 mL hydrophobic interaction
chromatography Phenyl HP column pre-equilibrated with 20 mM sodium
phosphate pH 7.0 containing 1 M Ammonium sulfate (buffer A). The
column was washed with a linear salt gradient from 1 to 0 M
ammonium sulfate. The active fractions were pooled and applied to
affinity chromatography. The sample after hydrophobic interaction
chromatography was exchanged into 25 mM pH4.3 sodium acetate buffer
(buffer B) and loaded onto .beta.-cyclodextrin coupled Sepharose 6B
column pre-equilibrated with buffer B. The target protein was
eluted by 25 mM pH 4.3 sodium acetate with 10 mM
.alpha.-cyclodextrin (Buffer C). The eluant was concentrated by
using a 10K Amicon Ultra-15 device. The final product was above 98%
pure and stored in 40% glycerol at -80.degree. C. for further
studies.
Example 14
Glucoamylase Activity of AfGA2TR
[0343] AfGA2TR belongs to the glycosyl hydrolase 15 family (GH15,
CAZy number). The glucoamylase activity of AfGA2TR was measured
using 1% w/w soluble starch (Sigma S9765) as a substrate. The assay
was performed in 50 mM sodium acetate buffer pH 5.0 at 50.degree.
C. for 10 minutes. The rate of glucose release was measured using
the glucose oxidase/peroxidase (GOX/HRP) technique disclosed in
Example 3. Glucose was quantified as the rate of oxidation of
2,2'-azino-bis 3-ethylbenzothiazoline-6-sulfonic acid (ABTS) via
excess coupled GOX/HRP enzymes. The enzyme activity was calculated
based on a glucose standard curve. In this assay, one glucoamylase
unit is defined as the amount of enzyme required to generate 1
.mu.mol of glucose per minute under the conditions of the assay.
The specific activity towards soluble starch of purified AfGA2TR
was determined to be 214 units/mg using the above method.
Example 15
pH Profile of AfGA2TR
[0344] The effect of pH (from 3.0 to 10.0) on AfGA2TR activity was
monitored by following the ABTS assay protocol as described in
Example 3. Buffer working solutions consisted of the combination of
glycine/sodium acetate/HEPES (250 mM), with pH variation from 3.0
to 10.0. Substrate solutions were prepared by mixing soluble starch
(1% in water, w/w) with 250 mM buffer solution at a ratio of 9:1.
Enzyme working solutions were prepared in water at a certain dose
(showing signal within linear range as per dose response curve).
All the incubations were done at 50.degree. C. for 10 min using the
same protocol as described for glucoamylase activity assay in
Example 3. Enzyme activity at each pH was reported as relative
activity towards enzyme activity at optimum pH. The pH profile of
AfGA2TR is shown in FIG. 10. AfGA2TR was found to have an optimum
pH at about 5.3, and retain greater than 70% of maximum activity
between pH 3.3 and 7.3.
Example 16
Temperature Profile of AfGA2TR
[0345] The effect of temperature (from 40.degree. C. to 84.degree.
C.) on AfGA2TR activity was monitored by following the ABTS assay
protocol as described in Example 3. Substrate solutions were
prepared by mixing 9 mL of soluble starch (1% in water, w/w) and 1
mL of 0.5 M buffer (pH 5.0 sodium acetate) into a 15-mL conical
tube. Enzyme working solutions were prepared in water at a certain
dose (showing signal within linear range as per dose response
curve). Incubations were carried out at temperatures from
40.degree. C. to 84.degree. C., respectively, for 10 min. After
incubation, the activities were determined following the same
protocol as described for glucoamylase activity assay in Example 3.
The activity was reported as relative activity towards the enzyme
activity at optimum temperature. The temperature profile of AfGA2TR
is shown in FIG. 11. AfGA2TR was found to have an optimum
temperature of 69.degree. C., and retain greater than 70% of
maximum activity between 61.degree. C. and 74.degree. C.
Example 17
Thermostability of AfGA2TR
[0346] The thermostability of AfGA2TR was determined in 50 mM
sodium acetate buffer pH 5.0. The enzyme was incubated at desired
temperature for 2 hours in a PCR machine prior to addition into
substrate. The remaining activity of the samples was measured as
described in Example 3. The activity of the sample kept on ice was
defined as 100% activity. As shown in FIG. 12, at temperature lower
than 63.degree. C., AfGA2TR retained over 50% activity during a
2-hour incubation period.
Example 18
Comparison of AfGA1TR with AfGA2TR
[0347] Starch liquefact was prepared to have 34% dry solids by
diluting with water and the saccharification was carried out using
the 2 different glucoamylases; 1) AfGA1TR at 0.06 mg/gds starch and
2) purified protein of AfGA2TR at 0.06 mg/gds at pH 4.4 and
60.degree. C. In addition, pullulanase (OPTIMAX.RTM. L-1000) and
acid-stable alpha-amylase, GC626.RTM. (AsAA) at 0.14 ASPU/gds and
0.9 SSU/gds, respectively, were dosed along with each glucoamylase
to enhance glucose production. Samples were taken at different
intervals of time and analyzed for sugar composition by HPLC.
TABLE-US-00016 TABLE 11 Product profile of AfGA1TR and AfGA2TR
blends on liquefied starch. % % % % Enzymes Dose: /gds Hr DP1 DP2
DP3 HS AFGA1TR 0.06 mg 16 82.99 6.76 1.14 9.11 OPTIMAX .RTM. 0.14
ASPU 24 91.65 3.30 1.21 3.84 L-1000 0.9 SSU 40 95.32 2.33 0.93 1.42
GC626 .RTM. 48 95.66 2.39 0.79 1.16 64 95.73 2.71 0.64 0.92 72
95.64 2.87 0.62 0.87 AfGA2TR 0.06 mg 16 86.40 4.32 1.05 8.23
OPTIMAX .RTM. 0.14 ASPU 24 93.34 2.34 1.05 3.27 L-1000 0.9 SSU 40
95.68 2.17 0.78 1.37 GC626 .RTM. 48 95.91 2.28 0.67 1.14 64 95.93
2.62 0.55 0.90 72 95.88 2.77 0.51 0.84
Table 11 showed that both glucoamylases resulted in >95.5% DP1
in 48 hours with a slightly faster saccharification using
AfGA2TR.
[0348] Although the compositions and methods of making and using
has been described in detail with reference to examples above, it
is understood that various modifications can be made without
departing from the spirit these compositions and methods, and would
be readily known to the skilled artisan.
[0349] All cited patents and publications referred to in this
application are herein incorporated by reference in their entirety
for all purposes.
SEQUENCE LISTING
TABLE-US-00017 [0350] AfGA1 precursor SEQ ID NO: 1
MPRLSYALCALSLGHAAIAAPQLSARATGSLDSWLGTETTVALNGILANI
GADGAYAKSAKPGIIIASPSTSEPDYYYTWTRDAALVTKVLVDLFRNGNL
GLQKVITEYVNSQAYLQTVSNPSGGLASGGLAEPKYNVDMTAFTGAWGRP
QRDGPALRATALIDFGNWLIDNGYSSYAVNNIWPIVRNDLSYVSQYWSQS
GFDLWEEVNSMSFFTVAVQHRALVEGSTFAKRVGASCSWCDSQAPQILCY
MQSFWTGSYINANTGGGRSGKDANTVLASIHTFDPEAGCDDTTFQPCSPR
ALANHKVYTDSFRSVYAINSGIPQGAAVSAGRYPEDVYYNGNPWFLTTLA
AAEQLYDAIYQWKKIGSISITSTSLAFFKDIYSSAAVGTYASSTSTFTDI
INAVKTYADGYVSIVQAHAMNNGSLSEQFDKSSGLSLSARDLTWSYAAFL
TANMRRNGVVPAPWGAASANSVPSSCSMGSATGTYSTATATSWPSTLTSG
SPGSTTTVGTTTSTTSGTAAETACATPTAVAVTFNEIATTTYGENVYIVG
SISELGNWDTSKAVALSASKYTSSNNLWYVSVTLPAGTTFEYKYIRKESD
GSIVWESDPNRSYTVPAACGVSTATENDTWQ AfGA2 precursor SEQ ID NO: 2
MPRLSYALCALSLGHAAIAAPQLSARATGSLDSWLGTETTVALNGILANI
GADGAYAKSAKPGIIIASPSTSEPDYYYTWTRDAALVTKVLVDLFRNGNL
GLQKVITEYVNSQAYLQTVSNPSGGLASGGLAEPKYNVDMTAFTGAWGRP
QRDGPALRATALIDFGNWLIDNGYSSYAVNNIWPIVRNDLSYVSQYWSQS
GFDLWEEVNSMSFFTVAVQHRALVEGSTFAKRVGASCSWCDSQAPQILCY
MQSFWTGSYINANTGGGRSGKDANTVLASIHTFDPEAGCDDTTFQPCSPR
ALANHKVYTDSFRSVYAINSGIPQGAAVSAGRYPEDVYYNGNPWFLTTLA
AAEQLYDAIYQWKKIGSISITSTSLAFFKDIYSSAAVGTYASSTSTFTDI
INAVKTYADGYVSIVQAHAMNNGSLSEQFDKSSGLSLSARDLTWSYAAFL
TANMRRNGVVPAPWGAASANSVPSSCSMGSATGTYSTATATSWPSTLTSG
SPGSTTTVGTTTSTTSGTATETACATPTAVAVTFNEIATTTYGENVYIVG
SISELGNWDTSKAVALSASKYTSSNNLWYVSVTLPAGTTFEYKYIRKESD
GSIVWESDPNRSYTVPAACGVSTATENDTWR Nf_NRRL_181_GA SEQ ID NO: 3
MPRLSYALCALSLGHAAIAAPQLSPRATGSLDSWLATESTVSLNGILANI
GADGAYAKSAKPGIIIASPSTSDPDYYYTWTRDAALVTKVLVDLFRNGNL
GLQKVITEYVNSQAYLQTVSTPSGGLSSGGLAEPKYNVDMTAFTGAWGRP
QRDGPALRATALIDFGNWLIDNGYSSYAVNNIWPIVRNDLSYVSQYWSQS
GFDLWEEVNSMSFFTVAVQHRALVEGSTFAKRVGASCSWCDSQAPQILCY
MQSFWTGSYINANTGGGRSGKDANTVLASIHTFDPEAGCDDTTFQPCSPR
ALANHKVYTDSFRSVYAINSGIPQGVAVSAGRYPEDVYYNGNPWFLTTLA
AAEQLYDAIYQWKKIGSISITSTSLAFFKDIYSSVAVGTYASSSSTFTAI
IDAVKTYADGYVSIVEAHAMTNGSLSEQFDKSSGMSLSARDLTWSYAALL
TANMRRNGVVPAPWGAASANSVPSSCSMGSATGTYSTATATSWPSTLTSG
SPSDTTSGTTPGTTTTTSACTTPTSVAVTFDEIATTTYGENVYTIGSISQ
LGSWDTSKAVPLSSSKYTSSNNLWYVTINLPAGTTFEYKYIRKESDGSIE
WESDPNRSYTVPSACGVSTATEKDTWR Ts_ATCC0_10500_GA SEQ ID NO: 4
MTRLSSVLCALAALGQTALAAPGLSPRASTSLDAWLATETTVSLSGILAN
IGADGAYSKSAKPGVVIASPSTDNPNYYYTWTRDSALTLKVLIDLFRNGN
LGLQTVIEEYVNAQAYLQTVSNPSGDLSSGAGLAEPKFNVDMSAFTGSWG
RPQRDGPALRAIALIDFGNWLIENGYTSLAANNIWPIVRNDLSYVAQYWS
QSGFDLWEEVNSMSFFTVANQHRSLVEGSTFAAKVGASCSWCDSQAPQIL
CYMQTFWTGSYMNANTGGGRSGKDANTVLTSIATFDPEATCDDVTFQPCS
PRALANHKVYTDSFRSVYGLNSGIAEGVAVAVGRYPEDSYYNGNPWFLSN
LAAAEQLYDAIYQWNKIGSITITSTSLAFFKDVYSSAAVGTYASGSSAFT
SIINAVKTYADGYISVVQSHAMNNGSLSEQFDKNTGAELSARDLTWSYAA
LLTANMRRNGVVPPSWGAASATSIPSSCTTGSAIGTYSTPTATSWPSTLT
SGTGSPGSTTSATGSVSTSVSATTTSAGSCTTPTSVAVTFDEIATTSYGE
NVYIVGSISQLGSWNTANAIALSASKYTTSNNLWYVTINLPAGTTFQYKY
IRKESDGTVKWESDPNRSYTVPSACGVSTATENDTWR Pm_ATCC_18224_GA SEQ ID NO: 5
MTFSRLSSSVLCALAALGHNALAAPQFSPRATVGLDAWLASETTFSLNGI
LANIGSSGAYSASAKPGVVIASPSTNNPNYYYTWTRDSALTLKVLIDLFG
NGNLSLQTVIEEYINAQAYLQTVSNPSGDLSSGAGLAEPKYNVDMSPFTG
GWGRPQRDGPALRAIALIEFGNWLIDNGYSSYAVNNIWPIVRNDLSYVSQ
YWSQSGFDLWEEVNSMSFFTVANQHRALVQGSTFAARVGASCSWCDSQAP
QILCYMQTFWTGSYINANTGGGRSGKDSNTVLTTIHTFDPEATCDDVTFQ
PCSPRALANHKVYTDSFRSIYGVNSGIAQGVAVSVGRYPEDSYYGGNPWF
LSNLAAAEQLYDAIYQWNKIGSITITSTSLAFFKDVYSSAAVGTYASGST
AFTSIISAVKTYADGYVSIVQGHAAANGSLSEQFDRNSGVEISARDLTWS
YAALLTANLRRNGVMPPSWGAASANSVPSSCSMGSATGTYSTPTATAWPS
TLTSATGIPVTTSATASVTKATSATSTTTSATTCTTPTSVAVTFDEIATT
TYGENVFIVGSISQLGSWDTSKAIALSASQYTSSNHLWFATLSLPAGTTF
QYKYIRKESNGSIVWESDPNRSYTVPSGCGVSTATENDTWR An_FGSC_A4_GA SEQ ID NO:
6 MPTTILKITLFPLIDSIFSVQLSPVRIAMLTLSKVLPVLALSHAVAAAPQ
LSARATASLNTWLSTEASFALDGILTNIGANGAYAKTAKAGADYYTWTRD
AALTVKVLVDLFHNGDLSLQTILEEYTNSQAYLQTVSNPSGGLASGGLAE
PKFYVDMTAFTGSWGRPQRDGPALRATTLIGFGNWLIDNGYSSYASNNIW
PIVRNDLTYVAQYWSKSGYDLWEEVNSMSFFTVAVQHRALVEGSTFAHRV
GASCPWCDSQAPQILCYMQNFWTGSYINANTGGGRSGKDANTVLASIHTF
DPDAACDDITFQPCSSRALANHKVYTDSFRSVYSLNTGIAQGVAVAAGRY
PEDSYYNGNPWFLTTLAAAEQLYDAIYQWQKARSISITSTSLAFFKDIYS
SAAVGTYASGSSAFTAIIDAVKTYADGYVSIVKAHAMANGSLSEQFDKTY
GTCVSARDLTWSYAALLTASMRRNGVVPPSWDAASANTLPSSCSTGSATG
TYSTATVTTWPSTLTSGSASATTTIMATSTATSSSTTTSTTTACTTPSTV
AVTFNVIATTTYGENVYIVGSISQLGNWDTGSAVALSASKNTSSNNLWYV
DINLPGGTAFEYKYIRKETDGSIVWESDPNRSYTVPSSCGVSTATESDTW RCTLETQSVRN
AfGA1 and AfGA2 CBM SEQ ID NO: 7
FNEIATTTYGENVYIVGSISELGNWDTSKAVALSASKYTSSNNLWYVSVT
LPAGTTFEYKYIRKESDGSIVWESDPNRSYTVPAACGVSTATENDTW AfGA1 gene of
pTrex3gM-AfGA1 SEQ ID NO: 8
ATGCCTCGCCTTTCCTACGCGCTCTGTGCGCTGTCTCTCGGGCATGCTGC
TATTGCAGCTCCTCAGTTATCCGCTCGTGCTACCGGCAGCTTGGACTCCT
GGTTGGGTACTGAGACCACCGTTGCGCTCAATGGTATTCTGGCCAACATC
GGTGCCGACGGTGCTTATGCGAAGAGCGCTAAGCCTGGCATAATCATTGC
CAGTCCGAGCACCAGCGAACCAGACTGTGAGAACCTTCCTGAACTGGCCC
TGTCCGGCAGTCATTGACCTCGGTAGACTACTATACCTGGACGAGAGATG
CTGCTCTCGTCACGAAAGTCCTGGTCGACCTCTTCCGCAACGGCAACCTG
GGTCTGCAGAAAGTCATTACCGAATACGTCAACTCTCAGGCGTACTTGCA
GACCGTGTCTAATCCGTCGGGTGGTCTTGCGAGCGGAGGTCTCGCGGAGC
CTAAGTACAACGTCGACATGACGGCCTTTACCGGAGCCTGGGGTCGTCCT
CAGCGTGATGGTCCGGCTCTGCGGGCCACCGCCCTCATCGACTTTGGCAA
CTGGCTGATTGTATGTTCTCCATACGAGCCCCAGGAAGCGTTGCTGACGT
CTACAGGACAACGGCTACTCCAGCTATGCTGTCAACAACATCTGGCCCAT
TGTGCGCAACGACTTGTCCTACGTTTCTCAGTACTGGAGCCAGAGTGGCT
TTGGTGAGTCCCGACTCTCTGGAAGTTTACAACGTGCATCGATTACTGAC
AATTGAGATTCTACGTGACAGATCTCTGGGAAGAAGTCAACTCCATGTCC
TTCTTCACCGTCGCTGTCCAGCACCGTGCCCTCGTGGAGGGAAGCACGTT
CGCTAAACGGGTGGGAGCGTCGTGCTCGTGGTGTGACTCGCAGGCCCCCC
AGATCCTCTGCTACATGCAGAGTTTCTGGACTGGCTCGTATATCAACGCC
AACACCGGTGGTGGCCGGTCCGGCAAGGATGCCAACACCGTCCTCGCCAG
CATCCATACCTTCGACCCCGAAGCCGGCTGCGACGATACTACTTTCCAGC
CCTGCTCTCCTCGGGCCCTTGCCAACCACAAGGTGTACACCGATTCGTTC
CGCTCGGTCTACGCGATCAACTCCGGCATCCCACAGGGCGCTGCCGTTTC
CGCTGGCCGCTACCCCGAGGACGTCTACTACAACGGCAACCCTTGGTTCC
TCACCACCCTCGCCGCTGCCGAGCAGCTCTACGACGCTATCTACCAGTGG
AAGAAGATCGGTTCCATCAGCATCACCAGCACCTCCCTCGCCTTCTTCAA
GGACATCTACAGCTCCGCCGCGGTCGGCACCTACGCCTCTAGCACCTCCA
CCTTCACGGACATCATCAACGCGGTCAAGACCTACGCAGACGGCTACGTG
AGCATCGTCCAGGCACACGCCATGAACAACGGCTCCCTTTCGGAGCAATT
CGACAAGTCCTCTGGGCTGTCCCTCTCCGCCCGCGATCTGACCTGGTCCT
ACGCCGCTTTCCTCACCGCCAACATGCGTCGTAACGGCGTGGTGCCTGCC
CCCTGGGGCGCCGCCTCCGCCAACTCCGTCCCCTCGTCTTGCTCCATGGG
CTCGGCCACGGGCACCTACAGCACCGCGACAGCCACCTCCTGGCCCAGCA
CGCTGACCAGCGGCTCGCCAGGCAGCACCACCACCGTGGGCACCACGACC
AGTACCACCTCTGGCACCGCCGCCGAGACCGCCTGTGCGACCCCTACCGC
CGTGGCCGTCACCTTTAACGAGATCGCCACCACCACCTACGGCGAGAATG
TTTACATTGTTGGGTCCATCTCCGAGCTCGGGAACTGGGATACCAGCAAA
GCAGTGGCCCTGAGTGCGTCCAAGTATACCTCCAGCAATAACCTCTGGTA
CGTGTCCGTCACCCTGCCGGCTGGCACGACATTCGAGTACAAGTATATCC
GCAAGGAAAGCGATGGCTCGATCGTGTGGGAGAGTGACCCCAACCGCTCG
TATACGGTGCCGGCAGCTTGTGGAGTGTCTACTGCGACCGAGAATGATAC TTGGCAGTGA AfGA1
SEQ ID NO: 9 GCGGCGGCCGCACCATGCCTCGCCTTTCCTACGC AfGA1 SEQ ID NO: 10
CCGGCGCGCCCTTATCACTGCCAAGTATCATTCTCG AfGA1 and AfGA2 signal peptide
SEQ ID NO: 11 MPRLSYALCALSLGHAAIA AfGA1 Mature form SEQ ID NO: 12
APQLSARATGSLDSWLGTETTVALNGILANIGADGAYAKSAKPGIIIASP
STSEPDYYYTWTRDAALVTKVLVDLFRNGNLGLQKVITEYVNSQAYLQTV
SNPSGGLASGGLAEPKYNVDMTAFTGAWGRPQRDGPALRATALIDFGNWL
IDNGYSSYAVNNIWPIVRNDLSYVSQYWSQSGFDLWEEVNSMSFFTVAVQ
HRALVEGSTFAKRVGASCSWCDSQAPQILCYMQSFWTGSYINANTGGGRS
GKDANTVLASIHTFDPEAGCDDTTFQPCSPRALANHKVYTDSFRSVYAIN
SGIPQGAAVSAGRYPEDVYYNGNPWFLTTLAAAEQLYDAIYQWKKIGSIS
ITSTSLAFFKDIYSSAAVGTYASSTSTFTDIINAVKTYADGYVSIVQAHA
MNNGSLSEQFDKSSGLSLSARDLTWSYAAFLTANMRRNGVVPAPWGAASA
NSVPSSCSMGSATGTYSTATATSWPSTLTSGSPGSTTTVGTTTSTTSGTA
AETACATPTAVAVTFNEIATTTYGENVYIVGSISELGNWDTSKAVALSAS
KYTSSNNLWYVSVTLPAGTTFEYKYIRKESDGSIVWESDPNRSYTVPAAC GVSTATENDTWQ
AfGA2 Mature form SEQ ID NO: 13
APQLSARATGSLDSWLGTETTVALNGILANIGADGAYAKSAKPGIIIASP
STSEPDYYYTWTRDAALVTKVLVDLFRNGNLGLQKVITEYVNSQAYLQTV
SNPSGGLASGGLAEPKYNVDMTAFTGAWGRPQRDGPALRATALIDFGNWL
IDNGYSSYAVNNIWPIVRNDLSYVSQYWSQSGFDLWEEVNSMSFFTVAVQ
HRALVEGSTFAKRVGASCSWCDSQAPQILCYMQSFWTGSYINANTGGGRS
GKDANTVLASIHTFDPEAGCDDTTFQPCSPRALANHKVYTDSFRSVYAIN
SGIPQGAAVSAGRYPEDVYYNGNPWFLTTLAAAEQLYDAIYQWKKIGSIS
ITSTSLAFFKDIYSSAAVGTYASSTSTFTDIINAVKTYADGYVSIVQAHA
MNNGSLSEQFDKSSGLSLSARDLTWSYAAFLTANMRRNGVVPAPWGAASA
NSVPSSCSMGSATGTYSTATATSWPSTLTSGSPGSTTTVGTTTSTTSGTA
TETACATPTAVAVTFNEIATTTYGENVYIVGSISELGNWDTSKAVALSAS
KYTSSNNLWYVSVTLPAGTTFEYKYIRKESDGSIVWESDPNRSYTVPAAC GVSTATENDTWR
AfGA2 gene of pTrex3gM-AfGA2 SEQ ID NO: 14
ATGCCTCGACTGAGCTACGCTCTCTGCGCTCTGTCCCTGGGTCACGCTGC
CATCGCCGCTCCCCAACTGAGCGCCCGAGCTACTGGCAGCCTCGATTCCT
GGCTGGGCACTGAGACCACCGTTGCTCTGAACGGCATCCTCGCTAACATC
GGCGCTGATGGTGCCTATGCCAAGAGCGCTAAACCTGGCATCATCATCGC
CAGCCCTAGCACCAGCGAGCCTGATTACTACTATACTTGGACCCGCGACG
CTGCTCTGGTCACCAAGGTCCTCGTTGACCTGTTCCGCAATGGTAACCTG
GGCCTCCAGAAAGTCATTACCGAGTACGTCAACAGCCAAGCTTATCTGCA
AACCGTTAGCAATCCCTCCGGTGGCCTCGCTTCCGGCGGCCTGGCCGAGC
CCAAATACAACGTCGACATGACCGCCTTTACCGGTGCCTGGGGTCGCCCC
CAGCGAGATGGCCCTGCCCTGCGCGCCACCGCTCTCATCGACTTCGGCAA
CTGGCTGATCGACAACGGCTATTCCAGCTATGCTGTCAACAACATTTGGC
CCATCGTCCGCAACGACCTGTCCTATGTTTCCCAATACTGGTCCCAGTCC
GGTTTCGACCTCTGGGAGGAGGTTAATTCCATGAGCTTTTTCACCGTCGC
TGTCCAACATCGAGCTCTCGTCGAGGGCTCCACTTTCGCTAAGCGCGTCG
GCGCCAGCTGTTCCTGGTGCGATTCCCAGGCCCCTCAGATTCTGTGCTAC
ATGCAGTCCTTTTGGACCGGTAGCTATATCAATGCCAATACCGGCGGTGG
TCGAAGCGGCAAGGACGCTAATACTGTTCTGGCTTCCATCCACACCTTCG
ATCCCGAGGCCGGCTGTGATGATACTACCTTTCAGCCCTGCTCCCCTCGC
GCTCTCGCCAACCATAAAGTTTACACCGACAGCTTTCGCAGCGTTTACGC
CATCAACTCCGGCATTCCTCAAGGCGCTGCTGTTTCCGCTGGTCGCTACC
CCGAGGACGTTTACTATAATGGCAACCCCTGGTTCCTCACTACTCTGGCT
GCTGCTGAGCAGCTCTATGACGCTATCTACCAATGGAAGAAAATCGGCAG
CATCAGCATTACTTCCACCTCCCTCGCCTTCTTCAAAGACATCTATAGCT
CCGCTGCCGTTGGCACTTATGCTTCCTCCACTAGCACTTTCACTGATATT
ATCAACGCTGTTAAAACCTACGCTGACGGCTACGTCAGCATCGTTCAAGC
CCACGCTATGAACAACGGTTCCCTCTCCGAGCAGTTCGACAAGTCCAGCG
GTCTGAGCCTCAGCGCTCGCGACCTCACCTGGTCCTACGCCGCCTTCCTG
ACTGCCAACATGCGCCGAAACGGCGTCGTTCCTGCCCCTTGGGGTGCCGC
CAGCGCCAATTCCGTCCCCAGCAGCTGTAGCATGGGCTCCGCCACTGGTA
CCTACAGCACCGCTACCGCTACTAGCTGGCCCAGCACCCTGACTAGCGGC
TCCCCCGGTTCCACTACTACCGTCGGCACCACTACCTCCACCACTTCCGG
TACTGCCACCGAGACTGCCTGTGCCACCCCTACCGCCGTCGCCGTCACCT
TTAACGAGATTGCTACCACCACCTACGGCGAGAACGTCTACATCGTCGGT
AGCATCTCCGAGCTCGGCAATTGGGACACTTCCAAGGCTGTCGCCCTGTC
CGCCTCCAAATATACTAGCAGCAACAACCTGTGGTATGTCTCCGTTACCC
TGCCTGCTGGTACTACTTTTGAGTACAAGTACATTCGCAAAGAGTCCGAT
GGCTCCATCGTTTGGGAGTCCGATCCCAACCGAAGCTACACCGTTCCCGC
TGCTTGTGGCGTCTCCACTGCTACTGAGAATGACACCTGGCGCTAA
Sequence CWU 1
1
141631PRTAspergillus fumigatusmisc_feature(1)..(631)AfGA1 precursor
1Met Pro Arg Leu Ser Tyr Ala Leu Cys Ala Leu Ser Leu Gly His Ala 1
5 10 15 Ala Ile Ala Ala Pro Gln Leu Ser Ala Arg Ala Thr Gly Ser Leu
Asp 20 25 30 Ser Trp Leu Gly Thr Glu Thr Thr Val Ala Leu Asn Gly
Ile Leu Ala 35 40 45 Asn Ile Gly Ala Asp Gly Ala Tyr Ala Lys Ser
Ala Lys Pro Gly Ile 50 55 60 Ile Ile Ala Ser Pro Ser Thr Ser Glu
Pro Asp Tyr Tyr Tyr Thr Trp 65 70 75 80 Thr Arg Asp Ala Ala Leu Val
Thr Lys Val Leu Val Asp Leu Phe Arg 85 90 95 Asn Gly Asn Leu Gly
Leu Gln Lys Val Ile Thr Glu Tyr Val Asn Ser 100 105 110 Gln Ala Tyr
Leu Gln Thr Val Ser Asn Pro Ser Gly Gly Leu Ala Ser 115 120 125 Gly
Gly Leu Ala Glu Pro Lys Tyr Asn Val Asp Met Thr Ala Phe Thr 130 135
140 Gly Ala Trp Gly Arg Pro Gln Arg Asp Gly Pro Ala Leu Arg Ala Thr
145 150 155 160 Ala Leu Ile Asp Phe Gly Asn Trp Leu Ile Asp Asn Gly
Tyr Ser Ser 165 170 175 Tyr Ala Val Asn Asn Ile Trp Pro Ile Val Arg
Asn Asp Leu Ser Tyr 180 185 190 Val Ser Gln Tyr Trp Ser Gln Ser Gly
Phe Asp Leu Trp Glu Glu Val 195 200 205 Asn Ser Met Ser Phe Phe Thr
Val Ala Val Gln His Arg Ala Leu Val 210 215 220 Glu Gly Ser Thr Phe
Ala Lys Arg Val Gly Ala Ser Cys Ser Trp Cys 225 230 235 240 Asp Ser
Gln Ala Pro Gln Ile Leu Cys Tyr Met Gln Ser Phe Trp Thr 245 250 255
Gly Ser Tyr Ile Asn Ala Asn Thr Gly Gly Gly Arg Ser Gly Lys Asp 260
265 270 Ala Asn Thr Val Leu Ala Ser Ile His Thr Phe Asp Pro Glu Ala
Gly 275 280 285 Cys Asp Asp Thr Thr Phe Gln Pro Cys Ser Pro Arg Ala
Leu Ala Asn 290 295 300 His Lys Val Tyr Thr Asp Ser Phe Arg Ser Val
Tyr Ala Ile Asn Ser 305 310 315 320 Gly Ile Pro Gln Gly Ala Ala Val
Ser Ala Gly Arg Tyr Pro Glu Asp 325 330 335 Val Tyr Tyr Asn Gly Asn
Pro Trp Phe Leu Thr Thr Leu Ala Ala Ala 340 345 350 Glu Gln Leu Tyr
Asp Ala Ile Tyr Gln Trp Lys Lys Ile Gly Ser Ile 355 360 365 Ser Ile
Thr Ser Thr Ser Leu Ala Phe Phe Lys Asp Ile Tyr Ser Ser 370 375 380
Ala Ala Val Gly Thr Tyr Ala Ser Ser Thr Ser Thr Phe Thr Asp Ile 385
390 395 400 Ile Asn Ala Val Lys Thr Tyr Ala Asp Gly Tyr Val Ser Ile
Val Gln 405 410 415 Ala His Ala Met Asn Asn Gly Ser Leu Ser Glu Gln
Phe Asp Lys Ser 420 425 430 Ser Gly Leu Ser Leu Ser Ala Arg Asp Leu
Thr Trp Ser Tyr Ala Ala 435 440 445 Phe Leu Thr Ala Asn Met Arg Arg
Asn Gly Val Val Pro Ala Pro Trp 450 455 460 Gly Ala Ala Ser Ala Asn
Ser Val Pro Ser Ser Cys Ser Met Gly Ser 465 470 475 480 Ala Thr Gly
Thr Tyr Ser Thr Ala Thr Ala Thr Ser Trp Pro Ser Thr 485 490 495 Leu
Thr Ser Gly Ser Pro Gly Ser Thr Thr Thr Val Gly Thr Thr Thr 500 505
510 Ser Thr Thr Ser Gly Thr Ala Ala Glu Thr Ala Cys Ala Thr Pro Thr
515 520 525 Ala Val Ala Val Thr Phe Asn Glu Ile Ala Thr Thr Thr Tyr
Gly Glu 530 535 540 Asn Val Tyr Ile Val Gly Ser Ile Ser Glu Leu Gly
Asn Trp Asp Thr 545 550 555 560 Ser Lys Ala Val Ala Leu Ser Ala Ser
Lys Tyr Thr Ser Ser Asn Asn 565 570 575 Leu Trp Tyr Val Ser Val Thr
Leu Pro Ala Gly Thr Thr Phe Glu Tyr 580 585 590 Lys Tyr Ile Arg Lys
Glu Ser Asp Gly Ser Ile Val Trp Glu Ser Asp 595 600 605 Pro Asn Arg
Ser Tyr Thr Val Pro Ala Ala Cys Gly Val Ser Thr Ala 610 615 620 Thr
Glu Asn Asp Thr Trp Gln 625 630 2631PRTAspergillus fumigatus
A1163misc_feature(1)..(631)AfGA2 precursor 2Met Pro Arg Leu Ser Tyr
Ala Leu Cys Ala Leu Ser Leu Gly His Ala 1 5 10 15 Ala Ile Ala Ala
Pro Gln Leu Ser Ala Arg Ala Thr Gly Ser Leu Asp 20 25 30 Ser Trp
Leu Gly Thr Glu Thr Thr Val Ala Leu Asn Gly Ile Leu Ala 35 40 45
Asn Ile Gly Ala Asp Gly Ala Tyr Ala Lys Ser Ala Lys Pro Gly Ile 50
55 60 Ile Ile Ala Ser Pro Ser Thr Ser Glu Pro Asp Tyr Tyr Tyr Thr
Trp 65 70 75 80 Thr Arg Asp Ala Ala Leu Val Thr Lys Val Leu Val Asp
Leu Phe Arg 85 90 95 Asn Gly Asn Leu Gly Leu Gln Lys Val Ile Thr
Glu Tyr Val Asn Ser 100 105 110 Gln Ala Tyr Leu Gln Thr Val Ser Asn
Pro Ser Gly Gly Leu Ala Ser 115 120 125 Gly Gly Leu Ala Glu Pro Lys
Tyr Asn Val Asp Met Thr Ala Phe Thr 130 135 140 Gly Ala Trp Gly Arg
Pro Gln Arg Asp Gly Pro Ala Leu Arg Ala Thr 145 150 155 160 Ala Leu
Ile Asp Phe Gly Asn Trp Leu Ile Asp Asn Gly Tyr Ser Ser 165 170 175
Tyr Ala Val Asn Asn Ile Trp Pro Ile Val Arg Asn Asp Leu Ser Tyr 180
185 190 Val Ser Gln Tyr Trp Ser Gln Ser Gly Phe Asp Leu Trp Glu Glu
Val 195 200 205 Asn Ser Met Ser Phe Phe Thr Val Ala Val Gln His Arg
Ala Leu Val 210 215 220 Glu Gly Ser Thr Phe Ala Lys Arg Val Gly Ala
Ser Cys Ser Trp Cys 225 230 235 240 Asp Ser Gln Ala Pro Gln Ile Leu
Cys Tyr Met Gln Ser Phe Trp Thr 245 250 255 Gly Ser Tyr Ile Asn Ala
Asn Thr Gly Gly Gly Arg Ser Gly Lys Asp 260 265 270 Ala Asn Thr Val
Leu Ala Ser Ile His Thr Phe Asp Pro Glu Ala Gly 275 280 285 Cys Asp
Asp Thr Thr Phe Gln Pro Cys Ser Pro Arg Ala Leu Ala Asn 290 295 300
His Lys Val Tyr Thr Asp Ser Phe Arg Ser Val Tyr Ala Ile Asn Ser 305
310 315 320 Gly Ile Pro Gln Gly Ala Ala Val Ser Ala Gly Arg Tyr Pro
Glu Asp 325 330 335 Val Tyr Tyr Asn Gly Asn Pro Trp Phe Leu Thr Thr
Leu Ala Ala Ala 340 345 350 Glu Gln Leu Tyr Asp Ala Ile Tyr Gln Trp
Lys Lys Ile Gly Ser Ile 355 360 365 Ser Ile Thr Ser Thr Ser Leu Ala
Phe Phe Lys Asp Ile Tyr Ser Ser 370 375 380 Ala Ala Val Gly Thr Tyr
Ala Ser Ser Thr Ser Thr Phe Thr Asp Ile 385 390 395 400 Ile Asn Ala
Val Lys Thr Tyr Ala Asp Gly Tyr Val Ser Ile Val Gln 405 410 415 Ala
His Ala Met Asn Asn Gly Ser Leu Ser Glu Gln Phe Asp Lys Ser 420 425
430 Ser Gly Leu Ser Leu Ser Ala Arg Asp Leu Thr Trp Ser Tyr Ala Ala
435 440 445 Phe Leu Thr Ala Asn Met Arg Arg Asn Gly Val Val Pro Ala
Pro Trp 450 455 460 Gly Ala Ala Ser Ala Asn Ser Val Pro Ser Ser Cys
Ser Met Gly Ser 465 470 475 480 Ala Thr Gly Thr Tyr Ser Thr Ala Thr
Ala Thr Ser Trp Pro Ser Thr 485 490 495 Leu Thr Ser Gly Ser Pro Gly
Ser Thr Thr Thr Val Gly Thr Thr Thr 500 505 510 Ser Thr Thr Ser Gly
Thr Ala Thr Glu Thr Ala Cys Ala Thr Pro Thr 515 520 525 Ala Val Ala
Val Thr Phe Asn Glu Ile Ala Thr Thr Thr Tyr Gly Glu 530 535 540 Asn
Val Tyr Ile Val Gly Ser Ile Ser Glu Leu Gly Asn Trp Asp Thr 545 550
555 560 Ser Lys Ala Val Ala Leu Ser Ala Ser Lys Tyr Thr Ser Ser Asn
Asn 565 570 575 Leu Trp Tyr Val Ser Val Thr Leu Pro Ala Gly Thr Thr
Phe Glu Tyr 580 585 590 Lys Tyr Ile Arg Lys Glu Ser Asp Gly Ser Ile
Val Trp Glu Ser Asp 595 600 605 Pro Asn Arg Ser Tyr Thr Val Pro Ala
Ala Cys Gly Val Ser Thr Ala 610 615 620 Thr Glu Asn Asp Thr Trp Arg
625 630 3627PRTNeosartorya fisheri NRRL
181misc_feature(1)..(627)Nf_NRRL_181_GA 3Met Pro Arg Leu Ser Tyr
Ala Leu Cys Ala Leu Ser Leu Gly His Ala 1 5 10 15 Ala Ile Ala Ala
Pro Gln Leu Ser Pro Arg Ala Thr Gly Ser Leu Asp 20 25 30 Ser Trp
Leu Ala Thr Glu Ser Thr Val Ser Leu Asn Gly Ile Leu Ala 35 40 45
Asn Ile Gly Ala Asp Gly Ala Tyr Ala Lys Ser Ala Lys Pro Gly Ile 50
55 60 Ile Ile Ala Ser Pro Ser Thr Ser Asp Pro Asp Tyr Tyr Tyr Thr
Trp 65 70 75 80 Thr Arg Asp Ala Ala Leu Val Thr Lys Val Leu Val Asp
Leu Phe Arg 85 90 95 Asn Gly Asn Leu Gly Leu Gln Lys Val Ile Thr
Glu Tyr Val Asn Ser 100 105 110 Gln Ala Tyr Leu Gln Thr Val Ser Thr
Pro Ser Gly Gly Leu Ser Ser 115 120 125 Gly Gly Leu Ala Glu Pro Lys
Tyr Asn Val Asp Met Thr Ala Phe Thr 130 135 140 Gly Ala Trp Gly Arg
Pro Gln Arg Asp Gly Pro Ala Leu Arg Ala Thr 145 150 155 160 Ala Leu
Ile Asp Phe Gly Asn Trp Leu Ile Asp Asn Gly Tyr Ser Ser 165 170 175
Tyr Ala Val Asn Asn Ile Trp Pro Ile Val Arg Asn Asp Leu Ser Tyr 180
185 190 Val Ser Gln Tyr Trp Ser Gln Ser Gly Phe Asp Leu Trp Glu Glu
Val 195 200 205 Asn Ser Met Ser Phe Phe Thr Val Ala Val Gln His Arg
Ala Leu Val 210 215 220 Glu Gly Ser Thr Phe Ala Lys Arg Val Gly Ala
Ser Cys Ser Trp Cys 225 230 235 240 Asp Ser Gln Ala Pro Gln Ile Leu
Cys Tyr Met Gln Ser Phe Trp Thr 245 250 255 Gly Ser Tyr Ile Asn Ala
Asn Thr Gly Gly Gly Arg Ser Gly Lys Asp 260 265 270 Ala Asn Thr Val
Leu Ala Ser Ile His Thr Phe Asp Pro Glu Ala Gly 275 280 285 Cys Asp
Asp Thr Thr Phe Gln Pro Cys Ser Pro Arg Ala Leu Ala Asn 290 295 300
His Lys Val Tyr Thr Asp Ser Phe Arg Ser Val Tyr Ala Ile Asn Ser 305
310 315 320 Gly Ile Pro Gln Gly Val Ala Val Ser Ala Gly Arg Tyr Pro
Glu Asp 325 330 335 Val Tyr Tyr Asn Gly Asn Pro Trp Phe Leu Thr Thr
Leu Ala Ala Ala 340 345 350 Glu Gln Leu Tyr Asp Ala Ile Tyr Gln Trp
Lys Lys Ile Gly Ser Ile 355 360 365 Ser Ile Thr Ser Thr Ser Leu Ala
Phe Phe Lys Asp Ile Tyr Ser Ser 370 375 380 Val Ala Val Gly Thr Tyr
Ala Ser Ser Ser Ser Thr Phe Thr Ala Ile 385 390 395 400 Ile Asp Ala
Val Lys Thr Tyr Ala Asp Gly Tyr Val Ser Ile Val Glu 405 410 415 Ala
His Ala Met Thr Asn Gly Ser Leu Ser Glu Gln Phe Asp Lys Ser 420 425
430 Ser Gly Met Ser Leu Ser Ala Arg Asp Leu Thr Trp Ser Tyr Ala Ala
435 440 445 Leu Leu Thr Ala Asn Met Arg Arg Asn Gly Val Val Pro Ala
Pro Trp 450 455 460 Gly Ala Ala Ser Ala Asn Ser Val Pro Ser Ser Cys
Ser Met Gly Ser 465 470 475 480 Ala Thr Gly Thr Tyr Ser Thr Ala Thr
Ala Thr Ser Trp Pro Ser Thr 485 490 495 Leu Thr Ser Gly Ser Pro Ser
Asp Thr Thr Ser Gly Thr Thr Pro Gly 500 505 510 Thr Thr Thr Thr Thr
Ser Ala Cys Thr Thr Pro Thr Ser Val Ala Val 515 520 525 Thr Phe Asp
Glu Ile Ala Thr Thr Thr Tyr Gly Glu Asn Val Tyr Ile 530 535 540 Ile
Gly Ser Ile Ser Gln Leu Gly Ser Trp Asp Thr Ser Lys Ala Val 545 550
555 560 Pro Leu Ser Ser Ser Lys Tyr Thr Ser Ser Asn Asn Leu Trp Tyr
Val 565 570 575 Thr Ile Asn Leu Pro Ala Gly Thr Thr Phe Glu Tyr Lys
Tyr Ile Arg 580 585 590 Lys Glu Ser Asp Gly Ser Ile Glu Trp Glu Ser
Asp Pro Asn Arg Ser 595 600 605 Tyr Thr Val Pro Ser Ala Cys Gly Val
Ser Thr Ala Thr Glu Lys Asp 610 615 620 Thr Trp Arg 625
4637PRTTalaromyces stipitatus ATCC
10500misc_feature(1)..(637)Ts_ATCC0_10500_GA 4Met Thr Arg Leu Ser
Ser Val Leu Cys Ala Leu Ala Ala Leu Gly Gln 1 5 10 15 Thr Ala Leu
Ala Ala Pro Gly Leu Ser Pro Arg Ala Ser Thr Ser Leu 20 25 30 Asp
Ala Trp Leu Ala Thr Glu Thr Thr Val Ser Leu Ser Gly Ile Leu 35 40
45 Ala Asn Ile Gly Ala Asp Gly Ala Tyr Ser Lys Ser Ala Lys Pro Gly
50 55 60 Val Val Ile Ala Ser Pro Ser Thr Asp Asn Pro Asn Tyr Tyr
Tyr Thr 65 70 75 80 Trp Thr Arg Asp Ser Ala Leu Thr Leu Lys Val Leu
Ile Asp Leu Phe 85 90 95 Arg Asn Gly Asn Leu Gly Leu Gln Thr Val
Ile Glu Glu Tyr Val Asn 100 105 110 Ala Gln Ala Tyr Leu Gln Thr Val
Ser Asn Pro Ser Gly Asp Leu Ser 115 120 125 Ser Gly Ala Gly Leu Ala
Glu Pro Lys Phe Asn Val Asp Met Ser Ala 130 135 140 Phe Thr Gly Ser
Trp Gly Arg Pro Gln Arg Asp Gly Pro Ala Leu Arg 145 150 155 160 Ala
Ile Ala Leu Ile Asp Phe Gly Asn Trp Leu Ile Glu Asn Gly Tyr 165 170
175 Thr Ser Leu Ala Ala Asn Asn Ile Trp Pro Ile Val Arg Asn Asp Leu
180 185 190 Ser Tyr Val Ala Gln Tyr Trp Ser Gln Ser Gly Phe Asp Leu
Trp Glu 195 200 205 Glu Val Asn Ser Met Ser Phe Phe Thr Val Ala Asn
Gln His Arg Ser 210 215 220 Leu Val Glu Gly Ser Thr Phe Ala Ala Lys
Val Gly Ala Ser Cys Ser 225 230 235 240 Trp Cys Asp Ser Gln Ala Pro
Gln Ile Leu Cys Tyr Met Gln Thr Phe 245 250 255 Trp Thr Gly Ser Tyr
Met Asn Ala Asn Thr Gly Gly Gly Arg Ser Gly 260 265 270 Lys Asp Ala
Asn Thr Val Leu Thr Ser Ile Ala Thr Phe Asp Pro Glu 275 280 285 Ala
Thr Cys Asp Asp Val Thr Phe Gln Pro Cys Ser Pro Arg Ala Leu 290 295
300 Ala Asn His Lys Val Tyr Thr Asp Ser Phe Arg Ser Val Tyr Gly Leu
305 310 315 320 Asn Ser Gly Ile Ala Glu Gly Val Ala Val Ala Val Gly
Arg Tyr Pro 325 330 335 Glu Asp Ser Tyr Tyr Asn Gly Asn Pro Trp Phe
Leu Ser Asn Leu Ala 340 345 350 Ala Ala Glu Gln Leu Tyr Asp Ala Ile
Tyr Gln Trp Asn Lys Ile Gly 355 360 365 Ser Ile Thr Ile Thr Ser Thr
Ser Leu Ala Phe Phe Lys Asp Val Tyr 370
375 380 Ser Ser Ala Ala Val Gly Thr Tyr Ala Ser Gly Ser Ser Ala Phe
Thr 385 390 395 400 Ser Ile Ile Asn Ala Val Lys Thr Tyr Ala Asp Gly
Tyr Ile Ser Val 405 410 415 Val Gln Ser His Ala Met Asn Asn Gly Ser
Leu Ser Glu Gln Phe Asp 420 425 430 Lys Asn Thr Gly Ala Glu Leu Ser
Ala Arg Asp Leu Thr Trp Ser Tyr 435 440 445 Ala Ala Leu Leu Thr Ala
Asn Met Arg Arg Asn Gly Val Val Pro Pro 450 455 460 Ser Trp Gly Ala
Ala Ser Ala Thr Ser Ile Pro Ser Ser Cys Thr Thr 465 470 475 480 Gly
Ser Ala Ile Gly Thr Tyr Ser Thr Pro Thr Ala Thr Ser Trp Pro 485 490
495 Ser Thr Leu Thr Ser Gly Thr Gly Ser Pro Gly Ser Thr Thr Ser Ala
500 505 510 Thr Gly Ser Val Ser Thr Ser Val Ser Ala Thr Thr Thr Ser
Ala Gly 515 520 525 Ser Cys Thr Thr Pro Thr Ser Val Ala Val Thr Phe
Asp Glu Ile Ala 530 535 540 Thr Thr Ser Tyr Gly Glu Asn Val Tyr Ile
Val Gly Ser Ile Ser Gln 545 550 555 560 Leu Gly Ser Trp Asn Thr Ala
Asn Ala Ile Ala Leu Ser Ala Ser Lys 565 570 575 Tyr Thr Thr Ser Asn
Asn Leu Trp Tyr Val Thr Ile Asn Leu Pro Ala 580 585 590 Gly Thr Thr
Phe Gln Tyr Lys Tyr Ile Arg Lys Glu Ser Asp Gly Thr 595 600 605 Val
Lys Trp Glu Ser Asp Pro Asn Arg Ser Tyr Thr Val Pro Ser Ala 610 615
620 Cys Gly Val Ser Thr Ala Thr Glu Asn Asp Thr Trp Arg 625 630 635
5641PRTPenicillium marneffei ATCC
18224misc_feature(1)..(641)Pm_ATCC_18224_GA 5Met Thr Phe Ser Arg
Leu Ser Ser Ser Val Leu Cys Ala Leu Ala Ala 1 5 10 15 Leu Gly His
Asn Ala Leu Ala Ala Pro Gln Phe Ser Pro Arg Ala Thr 20 25 30 Val
Gly Leu Asp Ala Trp Leu Ala Ser Glu Thr Thr Phe Ser Leu Asn 35 40
45 Gly Ile Leu Ala Asn Ile Gly Ser Ser Gly Ala Tyr Ser Ala Ser Ala
50 55 60 Lys Pro Gly Val Val Ile Ala Ser Pro Ser Thr Asn Asn Pro
Asn Tyr 65 70 75 80 Tyr Tyr Thr Trp Thr Arg Asp Ser Ala Leu Thr Leu
Lys Val Leu Ile 85 90 95 Asp Leu Phe Gly Asn Gly Asn Leu Ser Leu
Gln Thr Val Ile Glu Glu 100 105 110 Tyr Ile Asn Ala Gln Ala Tyr Leu
Gln Thr Val Ser Asn Pro Ser Gly 115 120 125 Asp Leu Ser Ser Gly Ala
Gly Leu Ala Glu Pro Lys Tyr Asn Val Asp 130 135 140 Met Ser Pro Phe
Thr Gly Gly Trp Gly Arg Pro Gln Arg Asp Gly Pro 145 150 155 160 Ala
Leu Arg Ala Ile Ala Leu Ile Glu Phe Gly Asn Trp Leu Ile Asp 165 170
175 Asn Gly Tyr Ser Ser Tyr Ala Val Asn Asn Ile Trp Pro Ile Val Arg
180 185 190 Asn Asp Leu Ser Tyr Val Ser Gln Tyr Trp Ser Gln Ser Gly
Phe Asp 195 200 205 Leu Trp Glu Glu Val Asn Ser Met Ser Phe Phe Thr
Val Ala Asn Gln 210 215 220 His Arg Ala Leu Val Gln Gly Ser Thr Phe
Ala Ala Arg Val Gly Ala 225 230 235 240 Ser Cys Ser Trp Cys Asp Ser
Gln Ala Pro Gln Ile Leu Cys Tyr Met 245 250 255 Gln Thr Phe Trp Thr
Gly Ser Tyr Ile Asn Ala Asn Thr Gly Gly Gly 260 265 270 Arg Ser Gly
Lys Asp Ser Asn Thr Val Leu Thr Thr Ile His Thr Phe 275 280 285 Asp
Pro Glu Ala Thr Cys Asp Asp Val Thr Phe Gln Pro Cys Ser Pro 290 295
300 Arg Ala Leu Ala Asn His Lys Val Tyr Thr Asp Ser Phe Arg Ser Ile
305 310 315 320 Tyr Gly Val Asn Ser Gly Ile Ala Gln Gly Val Ala Val
Ser Val Gly 325 330 335 Arg Tyr Pro Glu Asp Ser Tyr Tyr Gly Gly Asn
Pro Trp Phe Leu Ser 340 345 350 Asn Leu Ala Ala Ala Glu Gln Leu Tyr
Asp Ala Ile Tyr Gln Trp Asn 355 360 365 Lys Ile Gly Ser Ile Thr Ile
Thr Ser Thr Ser Leu Ala Phe Phe Lys 370 375 380 Asp Val Tyr Ser Ser
Ala Ala Val Gly Thr Tyr Ala Ser Gly Ser Thr 385 390 395 400 Ala Phe
Thr Ser Ile Ile Ser Ala Val Lys Thr Tyr Ala Asp Gly Tyr 405 410 415
Val Ser Ile Val Gln Gly His Ala Ala Ala Asn Gly Ser Leu Ser Glu 420
425 430 Gln Phe Asp Arg Asn Ser Gly Val Glu Ile Ser Ala Arg Asp Leu
Thr 435 440 445 Trp Ser Tyr Ala Ala Leu Leu Thr Ala Asn Leu Arg Arg
Asn Gly Val 450 455 460 Met Pro Pro Ser Trp Gly Ala Ala Ser Ala Asn
Ser Val Pro Ser Ser 465 470 475 480 Cys Ser Met Gly Ser Ala Thr Gly
Thr Tyr Ser Thr Pro Thr Ala Thr 485 490 495 Ala Trp Pro Ser Thr Leu
Thr Ser Ala Thr Gly Ile Pro Val Thr Thr 500 505 510 Ser Ala Thr Ala
Ser Val Thr Lys Ala Thr Ser Ala Thr Ser Thr Thr 515 520 525 Thr Ser
Ala Thr Thr Cys Thr Thr Pro Thr Ser Val Ala Val Thr Phe 530 535 540
Asp Glu Ile Ala Thr Thr Thr Tyr Gly Glu Asn Val Phe Ile Val Gly 545
550 555 560 Ser Ile Ser Gln Leu Gly Ser Trp Asp Thr Ser Lys Ala Ile
Ala Leu 565 570 575 Ser Ala Ser Gln Tyr Thr Ser Ser Asn His Leu Trp
Phe Ala Thr Leu 580 585 590 Ser Leu Pro Ala Gly Thr Thr Phe Gln Tyr
Lys Tyr Ile Arg Lys Glu 595 600 605 Ser Asn Gly Ser Ile Val Trp Glu
Ser Asp Pro Asn Arg Ser Tyr Thr 610 615 620 Val Pro Ser Gly Cys Gly
Val Ser Thr Ala Thr Glu Asn Asp Thr Trp 625 630 635 640 Arg
6661PRTAspergillus nidulans FGSC
A4misc_feature(1)..(661)An_FGSC_A4_GA 6Met Pro Thr Thr Ile Leu Lys
Ile Thr Leu Phe Pro Leu Ile Asp Ser 1 5 10 15 Ile Phe Ser Val Gln
Leu Ser Pro Val Arg Ile Ala Met Leu Thr Leu 20 25 30 Ser Lys Val
Leu Pro Val Leu Ala Leu Ser His Ala Val Ala Ala Ala 35 40 45 Pro
Gln Leu Ser Ala Arg Ala Thr Ala Ser Leu Asn Thr Trp Leu Ser 50 55
60 Thr Glu Ala Ser Phe Ala Leu Asp Gly Ile Leu Thr Asn Ile Gly Ala
65 70 75 80 Asn Gly Ala Tyr Ala Lys Thr Ala Lys Ala Gly Ala Asp Tyr
Tyr Thr 85 90 95 Trp Thr Arg Asp Ala Ala Leu Thr Val Lys Val Leu
Val Asp Leu Phe 100 105 110 His Asn Gly Asp Leu Ser Leu Gln Thr Ile
Leu Glu Glu Tyr Thr Asn 115 120 125 Ser Gln Ala Tyr Leu Gln Thr Val
Ser Asn Pro Ser Gly Gly Leu Ala 130 135 140 Ser Gly Gly Leu Ala Glu
Pro Lys Phe Tyr Val Asp Met Thr Ala Phe 145 150 155 160 Thr Gly Ser
Trp Gly Arg Pro Gln Arg Asp Gly Pro Ala Leu Arg Ala 165 170 175 Thr
Thr Leu Ile Gly Phe Gly Asn Trp Leu Ile Asp Asn Gly Tyr Ser 180 185
190 Ser Tyr Ala Ser Asn Asn Ile Trp Pro Ile Val Arg Asn Asp Leu Thr
195 200 205 Tyr Val Ala Gln Tyr Trp Ser Lys Ser Gly Tyr Asp Leu Trp
Glu Glu 210 215 220 Val Asn Ser Met Ser Phe Phe Thr Val Ala Val Gln
His Arg Ala Leu 225 230 235 240 Val Glu Gly Ser Thr Phe Ala His Arg
Val Gly Ala Ser Cys Pro Trp 245 250 255 Cys Asp Ser Gln Ala Pro Gln
Ile Leu Cys Tyr Met Gln Asn Phe Trp 260 265 270 Thr Gly Ser Tyr Ile
Asn Ala Asn Thr Gly Gly Gly Arg Ser Gly Lys 275 280 285 Asp Ala Asn
Thr Val Leu Ala Ser Ile His Thr Phe Asp Pro Asp Ala 290 295 300 Ala
Cys Asp Asp Ile Thr Phe Gln Pro Cys Ser Ser Arg Ala Leu Ala 305 310
315 320 Asn His Lys Val Tyr Thr Asp Ser Phe Arg Ser Val Tyr Ser Leu
Asn 325 330 335 Thr Gly Ile Ala Gln Gly Val Ala Val Ala Ala Gly Arg
Tyr Pro Glu 340 345 350 Asp Ser Tyr Tyr Asn Gly Asn Pro Trp Phe Leu
Thr Thr Leu Ala Ala 355 360 365 Ala Glu Gln Leu Tyr Asp Ala Ile Tyr
Gln Trp Gln Lys Ala Arg Ser 370 375 380 Ile Ser Ile Thr Ser Thr Ser
Leu Ala Phe Phe Lys Asp Ile Tyr Ser 385 390 395 400 Ser Ala Ala Val
Gly Thr Tyr Ala Ser Gly Ser Ser Ala Phe Thr Ala 405 410 415 Ile Ile
Asp Ala Val Lys Thr Tyr Ala Asp Gly Tyr Val Ser Ile Val 420 425 430
Lys Ala His Ala Met Ala Asn Gly Ser Leu Ser Glu Gln Phe Asp Lys 435
440 445 Thr Tyr Gly Thr Cys Val Ser Ala Arg Asp Leu Thr Trp Ser Tyr
Ala 450 455 460 Ala Leu Leu Thr Ala Ser Met Arg Arg Asn Gly Val Val
Pro Pro Ser 465 470 475 480 Trp Asp Ala Ala Ser Ala Asn Thr Leu Pro
Ser Ser Cys Ser Thr Gly 485 490 495 Ser Ala Thr Gly Thr Tyr Ser Thr
Ala Thr Val Thr Thr Trp Pro Ser 500 505 510 Thr Leu Thr Ser Gly Ser
Ala Ser Ala Thr Thr Thr Ile Met Ala Thr 515 520 525 Ser Thr Ala Thr
Ser Ser Ser Thr Thr Thr Ser Thr Thr Thr Ala Cys 530 535 540 Thr Thr
Pro Ser Thr Val Ala Val Thr Phe Asn Val Ile Ala Thr Thr 545 550 555
560 Thr Tyr Gly Glu Asn Val Tyr Ile Val Gly Ser Ile Ser Gln Leu Gly
565 570 575 Asn Trp Asp Thr Gly Ser Ala Val Ala Leu Ser Ala Ser Lys
Asn Thr 580 585 590 Ser Ser Asn Asn Leu Trp Tyr Val Asp Ile Asn Leu
Pro Gly Gly Thr 595 600 605 Ala Phe Glu Tyr Lys Tyr Ile Arg Lys Glu
Thr Asp Gly Ser Ile Val 610 615 620 Trp Glu Ser Asp Pro Asn Arg Ser
Tyr Thr Val Pro Ser Ser Cys Gly 625 630 635 640 Val Ser Thr Ala Thr
Glu Ser Asp Thr Trp Arg Cys Thr Leu Glu Thr 645 650 655 Gln Ser Val
Arg Asn 660 797PRTAspergillus fumigatusmisc_feature(1)..(97)AfGA1
and AfGA2 CBM 7Phe Asn Glu Ile Ala Thr Thr Thr Tyr Gly Glu Asn Val
Tyr Ile Val 1 5 10 15 Gly Ser Ile Ser Glu Leu Gly Asn Trp Asp Thr
Ser Lys Ala Val Ala 20 25 30 Leu Ser Ala Ser Lys Tyr Thr Ser Ser
Asn Asn Leu Trp Tyr Val Ser 35 40 45 Val Thr Leu Pro Ala Gly Thr
Thr Phe Glu Tyr Lys Tyr Ile Arg Lys 50 55 60 Glu Ser Asp Gly Ser
Ile Val Trp Glu Ser Asp Pro Asn Arg Ser Tyr 65 70 75 80 Thr Val Pro
Ala Ala Cys Gly Val Ser Thr Ala Thr Glu Asn Asp Thr 85 90 95 Trp
82060DNAAspergillus fumigatusmisc_feature(1)..(2060)AfGA1 gene of
pTrex3gM-AfGA1 8atgcctcgcc tttcctacgc gctctgtgcg ctgtctctcg
ggcatgctgc tattgcagct 60cctcagttat ccgctcgtgc taccggcagc ttggactcct
ggttgggtac tgagaccacc 120gttgcgctca atggtattct ggccaacatc
ggtgccgacg gtgcttatgc gaagagcgct 180aagcctggca taatcattgc
cagtccgagc accagcgaac cagactgtga gaaccttcct 240gaactggccc
tgtccggcag tcattgacct cggtagacta ctatacctgg acgagagatg
300ctgctctcgt cacgaaagtc ctggtcgacc tcttccgcaa cggcaacctg
ggtctgcaga 360aagtcattac cgaatacgtc aactctcagg cgtacttgca
gaccgtgtct aatccgtcgg 420gtggtcttgc gagcggaggt ctcgcggagc
ctaagtacaa cgtcgacatg acggccttta 480ccggagcctg gggtcgtcct
cagcgtgatg gtccggctct gcgggccacc gccctcatcg 540actttggcaa
ctggctgatt gtatgttctc catacgagcc ccaggaagcg ttgctgacgt
600ctacaggaca acggctactc cagctatgct gtcaacaaca tctggcccat
tgtgcgcaac 660gacttgtcct acgtttctca gtactggagc cagagtggct
ttggtgagtc ccgactctct 720ggaagtttac aacgtgcatc gattactgac
aattgagatt ctacgtgaca gatctctggg 780aagaagtcaa ctccatgtcc
ttcttcaccg tcgctgtcca gcaccgtgcc ctcgtggagg 840gaagcacgtt
cgctaaacgg gtgggagcgt cgtgctcgtg gtgtgactcg caggcccccc
900agatcctctg ctacatgcag agtttctgga ctggctcgta tatcaacgcc
aacaccggtg 960gtggccggtc cggcaaggat gccaacaccg tcctcgccag
catccatacc ttcgaccccg 1020aagccggctg cgacgatact actttccagc
cctgctctcc tcgggccctt gccaaccaca 1080aggtgtacac cgattcgttc
cgctcggtct acgcgatcaa ctccggcatc ccacagggcg 1140ctgccgtttc
cgctggccgc taccccgagg acgtctacta caacggcaac ccttggttcc
1200tcaccaccct cgccgctgcc gagcagctct acgacgctat ctaccagtgg
aagaagatcg 1260gttccatcag catcaccagc acctccctcg ccttcttcaa
ggacatctac agctccgccg 1320cggtcggcac ctacgcctct agcacctcca
ccttcacgga catcatcaac gcggtcaaga 1380cctacgcaga cggctacgtg
agcatcgtcc aggcacacgc catgaacaac ggctcccttt 1440cggagcaatt
cgacaagtcc tctgggctgt ccctctccgc ccgcgatctg acctggtcct
1500acgccgcttt cctcaccgcc aacatgcgtc gtaacggcgt ggtgcctgcc
ccctggggcg 1560ccgcctccgc caactccgtc ccctcgtctt gctccatggg
ctcggccacg ggcacctaca 1620gcaccgcgac agccacctcc tggcccagca
cgctgaccag cggctcgcca ggcagcacca 1680ccaccgtggg caccacgacc
agtaccacct ctggcaccgc cgccgagacc gcctgtgcga 1740cccctaccgc
cgtggccgtc acctttaacg agatcgccac caccacctac ggcgagaatg
1800tttacattgt tgggtccatc tccgagctcg ggaactggga taccagcaaa
gcagtggccc 1860tgagtgcgtc caagtatacc tccagcaata acctctggta
cgtgtccgtc accctgccgg 1920ctggcacgac attcgagtac aagtatatcc
gcaaggaaag cgatggctcg atcgtgtggg 1980agagtgaccc caaccgctcg
tatacggtgc cggcagcttg tggagtgtct actgcgaccg 2040agaatgatac
ttggcagtga 2060934DNAArtificial SequenceSynthetic primer AfGA1-Fw
9gcggcggccg caccatgcct cgcctttcct acgc 341036DNAArtificial
SequenceSynthetic primer AfGA1-Rv 10ccggcgcgcc cttatcactg
ccaagtatca ttctcg 361119PRTAspergillus
fumigatusmisc_feature(1)..(19)AfGA1 and AfGA2 signal peptide 11Met
Pro Arg Leu Ser Tyr Ala Leu Cys Ala Leu Ser Leu Gly His Ala 1 5 10
15 Ala Ile Ala 12612PRTAspergillus
fumigatusmisc_feature(1)..(612)AfGA1 Mature form 12Ala Pro Gln Leu
Ser Ala Arg Ala Thr Gly Ser Leu Asp Ser Trp Leu 1 5 10 15 Gly Thr
Glu Thr Thr Val Ala Leu Asn Gly Ile Leu Ala Asn Ile Gly 20 25 30
Ala Asp Gly Ala Tyr Ala Lys Ser Ala Lys Pro Gly Ile Ile Ile Ala 35
40 45 Ser Pro Ser Thr Ser Glu Pro Asp Tyr Tyr Tyr Thr Trp Thr Arg
Asp 50 55 60 Ala Ala Leu Val Thr Lys Val Leu Val Asp Leu Phe Arg
Asn Gly Asn 65 70 75 80 Leu Gly Leu Gln Lys Val Ile Thr Glu Tyr Val
Asn Ser Gln Ala Tyr 85 90 95 Leu Gln Thr Val Ser Asn Pro Ser Gly
Gly Leu Ala Ser Gly Gly Leu 100 105 110 Ala Glu Pro Lys Tyr Asn Val
Asp Met Thr Ala Phe Thr Gly Ala Trp 115 120 125 Gly Arg Pro Gln Arg
Asp Gly Pro Ala Leu Arg Ala Thr Ala Leu Ile 130 135 140 Asp Phe Gly
Asn Trp Leu Ile Asp Asn Gly Tyr Ser Ser Tyr Ala Val 145 150 155 160
Asn Asn Ile Trp Pro Ile Val Arg Asn Asp Leu Ser Tyr Val Ser Gln 165
170 175 Tyr Trp Ser Gln Ser Gly Phe Asp Leu Trp Glu Glu Val Asn Ser
Met 180 185 190 Ser Phe Phe Thr Val Ala Val Gln His Arg Ala Leu Val
Glu Gly Ser 195 200 205 Thr Phe Ala Lys Arg Val Gly
Ala Ser Cys Ser Trp Cys Asp Ser Gln 210 215 220 Ala Pro Gln Ile Leu
Cys Tyr Met Gln Ser Phe Trp Thr Gly Ser Tyr 225 230 235 240 Ile Asn
Ala Asn Thr Gly Gly Gly Arg Ser Gly Lys Asp Ala Asn Thr 245 250 255
Val Leu Ala Ser Ile His Thr Phe Asp Pro Glu Ala Gly Cys Asp Asp 260
265 270 Thr Thr Phe Gln Pro Cys Ser Pro Arg Ala Leu Ala Asn His Lys
Val 275 280 285 Tyr Thr Asp Ser Phe Arg Ser Val Tyr Ala Ile Asn Ser
Gly Ile Pro 290 295 300 Gln Gly Ala Ala Val Ser Ala Gly Arg Tyr Pro
Glu Asp Val Tyr Tyr 305 310 315 320 Asn Gly Asn Pro Trp Phe Leu Thr
Thr Leu Ala Ala Ala Glu Gln Leu 325 330 335 Tyr Asp Ala Ile Tyr Gln
Trp Lys Lys Ile Gly Ser Ile Ser Ile Thr 340 345 350 Ser Thr Ser Leu
Ala Phe Phe Lys Asp Ile Tyr Ser Ser Ala Ala Val 355 360 365 Gly Thr
Tyr Ala Ser Ser Thr Ser Thr Phe Thr Asp Ile Ile Asn Ala 370 375 380
Val Lys Thr Tyr Ala Asp Gly Tyr Val Ser Ile Val Gln Ala His Ala 385
390 395 400 Met Asn Asn Gly Ser Leu Ser Glu Gln Phe Asp Lys Ser Ser
Gly Leu 405 410 415 Ser Leu Ser Ala Arg Asp Leu Thr Trp Ser Tyr Ala
Ala Phe Leu Thr 420 425 430 Ala Asn Met Arg Arg Asn Gly Val Val Pro
Ala Pro Trp Gly Ala Ala 435 440 445 Ser Ala Asn Ser Val Pro Ser Ser
Cys Ser Met Gly Ser Ala Thr Gly 450 455 460 Thr Tyr Ser Thr Ala Thr
Ala Thr Ser Trp Pro Ser Thr Leu Thr Ser 465 470 475 480 Gly Ser Pro
Gly Ser Thr Thr Thr Val Gly Thr Thr Thr Ser Thr Thr 485 490 495 Ser
Gly Thr Ala Ala Glu Thr Ala Cys Ala Thr Pro Thr Ala Val Ala 500 505
510 Val Thr Phe Asn Glu Ile Ala Thr Thr Thr Tyr Gly Glu Asn Val Tyr
515 520 525 Ile Val Gly Ser Ile Ser Glu Leu Gly Asn Trp Asp Thr Ser
Lys Ala 530 535 540 Val Ala Leu Ser Ala Ser Lys Tyr Thr Ser Ser Asn
Asn Leu Trp Tyr 545 550 555 560 Val Ser Val Thr Leu Pro Ala Gly Thr
Thr Phe Glu Tyr Lys Tyr Ile 565 570 575 Arg Lys Glu Ser Asp Gly Ser
Ile Val Trp Glu Ser Asp Pro Asn Arg 580 585 590 Ser Tyr Thr Val Pro
Ala Ala Cys Gly Val Ser Thr Ala Thr Glu Asn 595 600 605 Asp Thr Trp
Gln 610 13612PRTAspergillus fumigatusmisc_feature(1)..(612)AfGA2
Mature form 13Ala Pro Gln Leu Ser Ala Arg Ala Thr Gly Ser Leu Asp
Ser Trp Leu 1 5 10 15 Gly Thr Glu Thr Thr Val Ala Leu Asn Gly Ile
Leu Ala Asn Ile Gly 20 25 30 Ala Asp Gly Ala Tyr Ala Lys Ser Ala
Lys Pro Gly Ile Ile Ile Ala 35 40 45 Ser Pro Ser Thr Ser Glu Pro
Asp Tyr Tyr Tyr Thr Trp Thr Arg Asp 50 55 60 Ala Ala Leu Val Thr
Lys Val Leu Val Asp Leu Phe Arg Asn Gly Asn 65 70 75 80 Leu Gly Leu
Gln Lys Val Ile Thr Glu Tyr Val Asn Ser Gln Ala Tyr 85 90 95 Leu
Gln Thr Val Ser Asn Pro Ser Gly Gly Leu Ala Ser Gly Gly Leu 100 105
110 Ala Glu Pro Lys Tyr Asn Val Asp Met Thr Ala Phe Thr Gly Ala Trp
115 120 125 Gly Arg Pro Gln Arg Asp Gly Pro Ala Leu Arg Ala Thr Ala
Leu Ile 130 135 140 Asp Phe Gly Asn Trp Leu Ile Asp Asn Gly Tyr Ser
Ser Tyr Ala Val 145 150 155 160 Asn Asn Ile Trp Pro Ile Val Arg Asn
Asp Leu Ser Tyr Val Ser Gln 165 170 175 Tyr Trp Ser Gln Ser Gly Phe
Asp Leu Trp Glu Glu Val Asn Ser Met 180 185 190 Ser Phe Phe Thr Val
Ala Val Gln His Arg Ala Leu Val Glu Gly Ser 195 200 205 Thr Phe Ala
Lys Arg Val Gly Ala Ser Cys Ser Trp Cys Asp Ser Gln 210 215 220 Ala
Pro Gln Ile Leu Cys Tyr Met Gln Ser Phe Trp Thr Gly Ser Tyr 225 230
235 240 Ile Asn Ala Asn Thr Gly Gly Gly Arg Ser Gly Lys Asp Ala Asn
Thr 245 250 255 Val Leu Ala Ser Ile His Thr Phe Asp Pro Glu Ala Gly
Cys Asp Asp 260 265 270 Thr Thr Phe Gln Pro Cys Ser Pro Arg Ala Leu
Ala Asn His Lys Val 275 280 285 Tyr Thr Asp Ser Phe Arg Ser Val Tyr
Ala Ile Asn Ser Gly Ile Pro 290 295 300 Gln Gly Ala Ala Val Ser Ala
Gly Arg Tyr Pro Glu Asp Val Tyr Tyr 305 310 315 320 Asn Gly Asn Pro
Trp Phe Leu Thr Thr Leu Ala Ala Ala Glu Gln Leu 325 330 335 Tyr Asp
Ala Ile Tyr Gln Trp Lys Lys Ile Gly Ser Ile Ser Ile Thr 340 345 350
Ser Thr Ser Leu Ala Phe Phe Lys Asp Ile Tyr Ser Ser Ala Ala Val 355
360 365 Gly Thr Tyr Ala Ser Ser Thr Ser Thr Phe Thr Asp Ile Ile Asn
Ala 370 375 380 Val Lys Thr Tyr Ala Asp Gly Tyr Val Ser Ile Val Gln
Ala His Ala 385 390 395 400 Met Asn Asn Gly Ser Leu Ser Glu Gln Phe
Asp Lys Ser Ser Gly Leu 405 410 415 Ser Leu Ser Ala Arg Asp Leu Thr
Trp Ser Tyr Ala Ala Phe Leu Thr 420 425 430 Ala Asn Met Arg Arg Asn
Gly Val Val Pro Ala Pro Trp Gly Ala Ala 435 440 445 Ser Ala Asn Ser
Val Pro Ser Ser Cys Ser Met Gly Ser Ala Thr Gly 450 455 460 Thr Tyr
Ser Thr Ala Thr Ala Thr Ser Trp Pro Ser Thr Leu Thr Ser 465 470 475
480 Gly Ser Pro Gly Ser Thr Thr Thr Val Gly Thr Thr Thr Ser Thr Thr
485 490 495 Ser Gly Thr Ala Thr Glu Thr Ala Cys Ala Thr Pro Thr Ala
Val Ala 500 505 510 Val Thr Phe Asn Glu Ile Ala Thr Thr Thr Tyr Gly
Glu Asn Val Tyr 515 520 525 Ile Val Gly Ser Ile Ser Glu Leu Gly Asn
Trp Asp Thr Ser Lys Ala 530 535 540 Val Ala Leu Ser Ala Ser Lys Tyr
Thr Ser Ser Asn Asn Leu Trp Tyr 545 550 555 560 Val Ser Val Thr Leu
Pro Ala Gly Thr Thr Phe Glu Tyr Lys Tyr Ile 565 570 575 Arg Lys Glu
Ser Asp Gly Ser Ile Val Trp Glu Ser Asp Pro Asn Arg 580 585 590 Ser
Tyr Thr Val Pro Ala Ala Cys Gly Val Ser Thr Ala Thr Glu Asn 595 600
605 Asp Thr Trp Arg 610 141896DNAAspergillus
fumigatusmisc_feature(1)..(1896)AfGA2 gene of pTrex3gM-AfGA2
14atgcctcgac tgagctacgc tctctgcgct ctgtccctgg gtcacgctgc catcgccgct
60ccccaactga gcgcccgagc tactggcagc ctcgattcct ggctgggcac tgagaccacc
120gttgctctga acggcatcct cgctaacatc ggcgctgatg gtgcctatgc
caagagcgct 180aaacctggca tcatcatcgc cagccctagc accagcgagc
ctgattacta ctatacttgg 240acccgcgacg ctgctctggt caccaaggtc
ctcgttgacc tgttccgcaa tggtaacctg 300ggcctccaga aagtcattac
cgagtacgtc aacagccaag cttatctgca aaccgttagc 360aatccctccg
gtggcctcgc ttccggcggc ctggccgagc ccaaatacaa cgtcgacatg
420accgccttta ccggtgcctg gggtcgcccc cagcgagatg gccctgccct
gcgcgccacc 480gctctcatcg acttcggcaa ctggctgatc gacaacggct
attccagcta tgctgtcaac 540aacatttggc ccatcgtccg caacgacctg
tcctatgttt cccaatactg gtcccagtcc 600ggtttcgacc tctgggagga
ggttaattcc atgagctttt tcaccgtcgc tgtccaacat 660cgagctctcg
tcgagggctc cactttcgct aagcgcgtcg gcgccagctg ttcctggtgc
720gattcccagg cccctcagat tctgtgctac atgcagtcct tttggaccgg
tagctatatc 780aatgccaata ccggcggtgg tcgaagcggc aaggacgcta
atactgttct ggcttccatc 840cacaccttcg atcccgaggc cggctgtgat
gatactacct ttcagccctg ctcccctcgc 900gctctcgcca accataaagt
ttacaccgac agctttcgca gcgtttacgc catcaactcc 960ggcattcctc
aaggcgctgc tgtttccgct ggtcgctacc ccgaggacgt ttactataat
1020ggcaacccct ggttcctcac tactctggct gctgctgagc agctctatga
cgctatctac 1080caatggaaga aaatcggcag catcagcatt acttccacct
ccctcgcctt cttcaaagac 1140atctatagct ccgctgccgt tggcacttat
gcttcctcca ctagcacttt cactgatatt 1200atcaacgctg ttaaaaccta
cgctgacggc tacgtcagca tcgttcaagc ccacgctatg 1260aacaacggtt
ccctctccga gcagttcgac aagtccagcg gtctgagcct cagcgctcgc
1320gacctcacct ggtcctacgc cgccttcctg actgccaaca tgcgccgaaa
cggcgtcgtt 1380cctgcccctt ggggtgccgc cagcgccaat tccgtcccca
gcagctgtag catgggctcc 1440gccactggta cctacagcac cgctaccgct
actagctggc ccagcaccct gactagcggc 1500tcccccggtt ccactactac
cgtcggcacc actacctcca ccacttccgg tactgccacc 1560gagactgcct
gtgccacccc taccgccgtc gccgtcacct ttaacgagat tgctaccacc
1620acctacggcg agaacgtcta catcgtcggt agcatctccg agctcggcaa
ttgggacact 1680tccaaggctg tcgccctgtc cgcctccaaa tatactagca
gcaacaacct gtggtatgtc 1740tccgttaccc tgcctgctgg tactactttt
gagtacaagt acattcgcaa agagtccgat 1800ggctccatcg tttgggagtc
cgatcccaac cgaagctaca ccgttcccgc tgcttgtggc 1860gtctccactg
ctactgagaa tgacacctgg cgctaa 1896
* * * * *
References