U.S. patent application number 15/574589 was filed with the patent office on 2018-06-21 for acetolactate decarboxylase.
The applicant listed for this patent is DUPONT NUTRITION BIOSCIENCES APS. Invention is credited to Tove Bladt, Jacob Flyvholm Cramer, Lene Bojsen Jensen, Anja Hemmingsen Kellett-Smith, Sang-Kyu Lee.
Application Number | 20180171323 15/574589 |
Document ID | / |
Family ID | 56404282 |
Filed Date | 2018-06-21 |
United States Patent
Application |
20180171323 |
Kind Code |
A1 |
Cramer; Jacob Flyvholm ; et
al. |
June 21, 2018 |
ACETOLACTATE DECARBOXYLASE
Abstract
The present disclosure provides methods, compositions,
apparatuses and kits comprising ALDC enzymes having a better
stability and/or activity, and, optionally, the yield of ALDC
enzymes which can be recovered from microorganisms is improved. In
some embodiments, the present disclosure provides methods,
apparatuses, compositions and kits for the use of metal ions to
increase stability and/or activity, and which further can be used
to recover the enzymes from microorganisms in improved yields.
Inventors: |
Cramer; Jacob Flyvholm;
(Brabrand, DK) ; Jensen; Lene Bojsen; (Hojbjerg,
DK) ; Kellett-Smith; Anja Hemmingsen; (Copenhagen V,
DK) ; Bladt; Tove; (Skanderborg, DK) ; Lee;
Sang-Kyu; (Mountain View, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
DUPONT NUTRITION BIOSCIENCES APS |
Copenhagen K |
|
DK |
|
|
Family ID: |
56404282 |
Appl. No.: |
15/574589 |
Filed: |
May 18, 2016 |
PCT Filed: |
May 18, 2016 |
PCT NO: |
PCT/US16/33028 |
371 Date: |
November 16, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62168406 |
May 29, 2015 |
|
|
|
62166610 |
May 26, 2015 |
|
|
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62165671 |
May 22, 2015 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12G 2200/15 20130101;
C12G 1/0203 20130101; C12C 5/00 20130101; C12C 5/004 20130101; C12Y
401/01005 20130101; C12N 9/96 20130101; C12N 9/88 20130101; C12N
1/38 20130101 |
International
Class: |
C12N 9/96 20060101
C12N009/96; C12N 1/38 20060101 C12N001/38; C12N 9/88 20060101
C12N009/88; C12C 5/00 20060101 C12C005/00; C12G 1/022 20060101
C12G001/022 |
Claims
1. A composition comprising an acetolactate decarboxylase (ALDC)
enzyme and zinc, wherein said zinc is present at a concentration of
about 1 .mu.M to about 200 mM.
2. The composition of claim 1, wherein the zinc is present at a
concentration of about 10 .mu.M to about 150 mM, or about 20 .mu.M
to about 120 mM, or about 25 .mu.M to about 100 mM, or about 25
.mu.M to about 50 mM, or about 25 .mu.M to about 20 mM, or about 25
.mu.M to about 50 .mu.M, or about 100 .mu.M to about 20 mM, or
about 250 .mu.M to about 20 mM, or about 500 .mu.M to about 20 mM,
or about 1 mM to about 20 mM, or about 1 mM to about 10 mM, or
about 1 mM to about 5 mM.
3. The composition of claim 2, wherein the zinc (i) is present at a
concentration of about 100 .mu.M to about 10 mM; or (ii) is present
at a concentration of about 1 mM to about 5 mM.
4. The composition of claim 3, wherein the molar ratio of zinc to
ALDC enzyme is (i) higher than 1; or (ii) 2:1 or higher; or (iii)
10:1 or higher; or (iv) 20:1 or higher; or (v) 30:1 or higher; or
(vi) 60:1 or higher.
5. The composition of claim 4, wherein said ALDC enzyme is an ALDC
derivative.
6. The composition of claim 5, wherein said ALDC derivative is an
ALDC enzyme treated with glutaraldehyde.
7. The composition of claim 6, wherein said ALDC enzyme is treated
with glutaraldehyde at a concentration corresponding to about 0.1
to about 5 g of glutaraldehyde per g of pure ALDC enzyme.
8. The composition of claim 7, wherein the activity of said ALDC
enzyme is in the range of 950 to 2500 Units per mg of protein.
9. The composition of claim 8, wherein the activity of said ALDC
enzyme is in the range of 1000 to 2500 Units per mg of protein.
10. The composition of claim 9 further comprising at least one
additional enzyme or enzyme derivative selected from the group
consisting of acetolactate reductoisomerases, acetolactate
isomerases, amylase, glucoamylase, hemicellulase, cellulase,
glucanase, pullulanase, isoamylase, endo-glucanase and related
beta-glucan hydrolytic accessory enzymes, xylanase, xylanase
accessory enzymes (for example, arabinofuranosidase, ferulic acid
esterase, and xylan acetyl esterase) and protease.
11. The composition of claim 1, wherein the ALDC enzyme is from
Lactobacillus casei, Brevibacterium acetylicum, Lactococcus lactis,
Leuconostoc lactis, Enterobacter aerogenes, Bacillus subtilis,
Bacillus brevis, Lactococcus lactis DX, or Bacillus
licheniformis.
12. The composition of claim 11, wherein the ALDC enzyme is from
Bacillus brevis or Bacillus licheniformis.
13. The composition of claim 12, wherein said ALDC enzyme has an
amino acid sequence having at least 80% identity with any one
selected from SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO:
7 and SEQ ID NO: 8 or any functional fragment thereof.
14. Use of the composition according to claim 13 in beer and/or
wine and/or cider and/or perry and/or sake fermentation.
15. A method for increasing the activity and/or stability of an
ALDC enzyme in a composition comprising ALDC wherein said method
comprises the step of adding zinc to the composition so that said
zinc is present in said composition at a concentration of about 1
.mu.M to about 200 mM.
16. The method of claim 15, wherein the zinc is present in said
composition at a concentration of about 10 .mu.M to about 150 mM,
or about 20 .mu.M to about 120 mM, or about 25 .mu.M to about 100
mM, or about 25 .mu.M to about 50 mM, or about 25 .mu.M to about 20
mM, or about 25 .mu.M to about 50 .mu.M, or about 100 .mu.M to
about 20 mM, or about 250 .mu.M to about 20 mM, or about 500 .mu.M
to about 20 mM, or about 1 mM to about 20 mM, or about 1 mM to
about 10 mM, or about 1 mM to about 5 mM.
17. The method of claim 16, wherein the zinc is present in said
composition at a concentration of (i) about 100 .mu.M to about 10
mM; or (ii) about 1 mM to about 5 mM.
18. The method of claim 16, wherein the molar ratio of zinc to ALDC
enzyme is (i) higher than 1; or (ii) 2:1 or higher; or (iii) 10:1
or higher; or (iv) 20:1 or higher; or (v) 30:1 or higher; or (vi)
60:1 or higher in said composition.
19. A method for increasing the activity and/or stability of an
ALDC enzyme in a cultivation media comprising an ALDC producing
host cell wherein said method comprises the step of adding zinc to
the cultivation media as a supplement during the production of said
ALDC enzyme by the ALDC producing host cell.
20. The method of claim 19, wherein said zinc is added at a
concentration of 1 .mu.M to about 1 mM.
21. The method of claim 20, wherein said zinc is added at a
concentration of 25 .mu.M to about 150 .mu.M, or 60 .mu.M to about
150 .mu.M.
22. The method of claim 21, wherein said host cell is a Bacillus
host cell.
23. The method of claim 22, wherein said Bacillus host cell is
Bacillus subtilis.
24. A method for increasing the activity and/or stability of an
ALDC enzyme in a fermentation media and/or maturation media
comprising an ALDC producing host cell wherein said method
comprises the step of adding zinc to the fermentation media and/or
maturation media during a beer and/or wine and/or cider and/or
perry and/or sake fermentation.
25. The method of claim 24, wherein said zinc: (i) is added at a
concentration of about 1 .mu.M to about 300 .mu.M, such as about 6
.mu.M to about 300 .mu.M; or (ii) is added at a concentration of
about 1 .mu.M to about 50 .mu.M; or (iii) is added at a
concentration of about 1 .mu.M to about 25 .mu.M, preferably about
6 .mu.M to about 25 .mu.M.
26. The method of claim 24, wherein said zinc is added as a
composition comprising ALDC and zinc, wherein said zinc is present
in said composition at a concentration of 1 mM to about 5 mM.
27. A cultivation media for an ALDC producing host cell comprising
zinc at a concentration of about 1 .mu.M to about 1 mM; preferably
said cultivation media comprises an ALDC producing host cell.
28. The cultivation media of claim 27, comprising zinc at
concentration of about 25 .mu.M to about 150 .mu.M.
29. The cultivation media of claim 27, wherein said zinc is added
at a concentration of 60 .mu.M to about 150 .mu.M.
30. A beer and/or wine and/or cider and/or perry and/or sake
fermentation media and/or maturation media comprising a composition
comprising an ALDC enzyme and zinc wherein said composition
comprises zinc at a concentration of about 1 .mu.M to about 200
mM.
31. The beer and/or wine and/or cider and/or perry and/or sake
fermentation media and/or maturation media of claim 30, wherein:
(i) zinc is present in the composition at a concentration of about
1 .mu.M to about 300 .mu.M, such as about 6 .mu.M to about 300
.mu.M; or (ii) zinc is present in the composition at a
concentration of about 1 .mu.M to about 50 .mu.M, such as about 6
.mu.M to about 50 .mu.M, or about 6 .mu.M to about 25 .mu.M; or
(iii) the zinc and the ALDC enzyme are added in a composition,
wherein zinc is present in the composition at a concentration of
about 1 mM to about 20 mM, such as 1 mM to about 5 mM; or (iv) the
zinc and the ALDC enzyme are added in a composition, where the
molar ratio of zinc to ALDC enzyme in the composition is higher
than 1; or 2:1 or higher; or 10:1 or higher; or 20:1 or higher; or
30:1 or higher; or 60:1 or higher.
32. The beer and/or wine and/or cider and/or perry and/or sake
fermentation media and/or maturation media of claim 31, wherein the
activity of said ALDC enzyme is in the range of 1000 to 2500 Units
per mg of protein.
33. The beer and/or wine and/or cider and/or perry and/or sake
fermentation media and/or maturation media of claim 32, further
comprising at least one additional enzyme or enzyme derivative
selected from the group consisting of acetolactate
reductoisomerases, acetolactate isomerases, amylase, glucoamylase,
hemicellulase, cellulase, glucanase, pullulanase, isoamylase,
endo-glucanase and related beta-glucan hydrolytic accessory
enzymes, xylanase, xylanase accessory enzymes (for example,
arabinofuranosidase, ferulic acid esterase, and xylan acetyl
esterase) and protease.
34. A composition comprising an ALDC enzyme, wherein said ALDC
enzyme is in the range of 1000 to 2500 Units per mg of protein.
35. The composition of claim 34, wherein the ALDC enzyme is from
Lactobacillus casei, Brevibacterium acetylicum, Lactococcus lactis,
Leuconostoc lactis, Enterobacter aerogenes, Bacillus subtilis,
Bacillus brevis, Lactococcus lactis DX, or Bacillus
licheniformis.
36. The composition of claim 34, wherein the ALDC enzyme is from
Bacillus brevis or Bacillus licheniformis.
37. The composition of claim 36, wherein said ALDC enzyme has an
amino acid sequence having at least 80% identity with any one
selected from SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO:
7 and SEQ ID NO: 8 or any functional fragment thereof.
38. The composition of claim 37, wherein: (i) zinc is present at a
concentration of about 1 .mu.M to about 200 mM; or (ii) the molar
ratio of zinc to ALDC enzyme in the composition is higher than 1;
or 2:1 or higher; or 10:1 or higher; or 20:1 or higher; or 30:1 or
higher; or 60:1 or higher.
39. A method for beer and/or wine and/or cider and/or perry and/or
sake production comprising adding an ALDC enzyme and adding zinc to
a media (such as a fermentation and/or a maturation media) for the
beer and/or wine and/or cider and/or perry and/or sake during said
beer and/or wine and/or cider and/or perry and/or sake production,
so that said zinc is present in said media at a concentration of
about 1 .mu.M to about 300 .mu.M, such as about 6 .mu.M to about
300 .mu.M.
40. The method of claim 39, wherein said zinc is present in said
media at a concentration of about 0.1 .mu.M to about 50 .mu.M, such
as about 1 .mu.M to about 50 .mu.M, or about 6 .mu.M to about 50
.mu.M, or about 6 .mu.M to about 25 .mu.M.
41. A method for beer and/or wine and/or cider and/or perry and/or
sake production comprising adding a composition comprising an ALDC
enzyme and zinc to a media (such as a fermentation and/or a
maturation media) for the beer and/or wine and/or cider and/or
perry and/or sake during said beer and/or wine and/or cider and/or
perry and/or sake production, wherein (i) zinc is present in the
composition at a concentration of about 1 mM to about 5 mM; or (ii)
the molar ratio of zinc to ALDC enzyme in the composition is higher
than 1; or 2:1 or higher; or 10:1 or higher; or 20:1 or higher; or
30:1 or higher; or 60:1 or higher.
42. The method of claim 41, wherein said ALDC enzyme and said zinc
are added during a fermentation process or a maturation
process.
43. The method of claim 42, wherein said ALDC enzyme is added at a
concentration of about 0.5 g to about 10 g per hectoliter of beer
and/or wine and/or cider and/or perry and/or sake ferment.
44. The method of claim 43, wherein said ALDC enzyme is added at a
concentration of about 1 g to about 5 g per hectoliter of beer
and/or wine and/or cider and/or perry and/or sake ferment.
45. The method of claim 44, wherein the activity of said ALDC
enzyme is in the range of 1000 to 2500 Units per mg of protein.
46. The method of claim 45, wherein the ALDC enzyme is from
Lactobacillus casei, Brevibacterium acetylicum, Lactococcus lactis,
Leuconostoc lactis, Enterobacter aerogenes, Bacillus subtilis,
Bacillus brevis, Lactococcus lactis DX, or Bacillus
licheniformis.
47. The method of claim 45, wherein the ALDC enzyme is from
Bacillus brevis or Bacillus licheniformis.
48. The method of claim 45, wherein said ALDC enzyme has an amino
acid sequence having at least 80% identity with any one selected
from SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 and SEQ
ID NO: 8 or any functional fragment thereof.
49. A method for increasing the activity and/or stability of an
ALDC enzyme in a composition comprising ALDC wherein said method
comprises the step of adding a metal ion to the composition at a
concentration of about 1 .mu.M to about 200 mM, preferably about
100 .mu.M to about 200 mM.
50. The method of claim 49, wherein the atomic radius for the metal
ion is about 140 pm to about 165 pm.
51. The method of claim 50, wherein the atomic radius for the metal
ion is about 140 pm to about 150 pm.
52. The method of claim 51, wherein the atomic radius for the metal
ion is about 142 pm to about 146 pm.
53. The method of claim 49, wherein the metal ion is selected from
the group consisting of Mg.sup.2+, Mn.sup.2+, Co.sup.2+, Cu.sup.2+,
Ca.sup.2+, Ba.sup.2+, and Fe.sup.2+ and combinations thereof.
54. The method of claim 53, wherein the metal ion is selected from
the group consisting of Zn.sup.2+, Mn.sup.2+, and Co.sup.2+.
55. The method of claim 54, wherein the metal ion is Zn.sup.2+.
56. The method of claim 55, wherein the ALDC enzyme is from
Lactobacillus casei, Brevibacterium acetylicum, Lactococcus lactis,
Leuconostoc lactis, Enterobacter aerogenes, Bacillus subtilis,
Bacillus brevis, Lactococcus lactis DX, or Bacillus
licheniformis.
57. The method of claim 55, wherein the ALDC enzyme is from
Bacillus brevis or Bacillus licheniformis.
58. The method of claim 55, wherein said ALDC enzyme has an amino
acid sequence having at least 80% identity with any one selected
from SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 and SEQ
ID NO: 8 or any functional fragment thereof.
59. A composition comprising an acetolactate decarboxylase (ALDC)
enzyme and a metal ion, wherein said metal ion is present at a
concentration of about 1 .mu.M to about 200 mM, preferably about
100 .mu.M to about 200 mM.
60. The composition of claim 59, wherein the atomic radius for the
metal ion is about 140 pm to about 165 pm.
61. The composition of claim 59, wherein the atomic radius for the
metal ion is about 140 pm to about 150 pm.
62. The composition of claim 61, wherein the atomic radius for the
metal ion is about 142 pm to about 146 pm.
63. The composition of claim 59, wherein the metal ion is selected
from the group consisting of Mg.sup.2+, Mn.sup.2+, Co.sup.2+,
Cu.sup.2+, Ca.sup.2+, Ba.sup.2+, and Fe.sup.2+ and and combinations
thereof.
64. The composition of claim 63, wherein the metal ion is selected
from the group consisting of Zn.sup.2+, Mn.sup.2+, and
Co.sup.2+.
65. The composition of claim 64, wherein the metal ion is
Zn.sup.2+.
66. A method for decomposing acetolactate comprising the step of
treating a substrate with a composition comprising an ALDC enzyme
and a metal ion, wherein the metal ion is present at a
concentration of about 1 .mu.M to about 200 mM in said composition;
preferably said substrate is a carbohydrate containing substrate
such as a wort or a fruit juice; preferably said substrate is a
fermentation and/or maturation media.
67. The method of claim 66, where the metal ion is present in said
composition at a concentration of about 10 .mu.M to about 150 mM,
or about 20 .mu.M to about 120 mM, or about 25 .mu.M to about 100
mM, or about 25 .mu.M to about 50 mM, or about 25 .mu.M to about 20
mM, or about 100 .mu.M to about 20 mM, or about 250 .mu.M to about
20 mM, or about 1 mM to about 20 mM, or about 1 mM to about 5
mM.
68. The method of claim 66, wherein the metal ion is present in
said substrate at a concentration of: (i) about 1 .mu.M to about
500 .mu.M, or about 1 .mu.M to about 300 .mu.M, such as about 6
.mu.M to about 300 .mu.M, or (ii) about 1 .mu.M to about 100 .mu.M
or about 1 .mu.M to about 50 .mu.M, or (iii) about 1 .mu.M to about
25 .mu.M, or about 6 .mu.M to about 50 .mu.M, or about 6 .mu.M to
about 25 .mu.M, or about 25 .mu.M to about 50 .mu.M.
69. The method of claim 66, wherein the metal ion and ALDC is added
in a composition, and wherein (i) said metal ion is present in the
composition at a concentration of about 1 mM to 5 mM; or (ii) the
molar ratio of said metal ion to ALDC enzyme in the composition is
higher than 1; or 2:1 or higher; or 10:1 or higher; or 20:1 or
higher; or 30:1 or higher; or 60:1 or higher.
70. The method of claim 69, wherein the metal ion is selected from
the group consisting of Mg.sup.2+, Mn.sup.2+, Co.sup.2+, Cu.sup.2+,
Ca.sup.2+, Ba.sup.2+, and Fe.sup.2+ and combinations thereof.
71. The method of claim 70, wherein the metal ion is selected from
the group consisting of Zn.sup.2+, Mn.sup.2+, and Co.sup.2+.
72. The method of claim 71, wherein the metal ion is Zn.sup.2+.
73. The method of claim 72, wherein the substrate is treated during
a beer and/or wine and/or cider and/or perry and/or sake
fermentation or maturation process.
74. The method of claim 72, wherein the ALDC enzyme is from
Lactobacillus casei, Brevibacterium acetylicum, Lactococcus lactis,
Leuconostoc lactis, Enterobacter aerogenes, Bacillus subtilis,
Bacillus brevis, Lactococcus lactis DX, or Bacillus
licheniformis.
75. The method of claim 72, wherein the ALDC enzyme is from
Bacillus brevis or Bacillus licheniformis.
76. The method of claim 72, wherein said ALDC enzyme has an amino
acid sequence having at least 80% identity with any one selected
from SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 and SEQ
ID NO: 8 or any functional fragment thereof.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This applications claims priority to and the benefit of
United States provisional patent application No. 62/165,671, filed
May 22, 2015; 62/166,610, filed May 26, 2015; and 62/168,406, filed
May 29, 2015; each provisional application titled "ALDC".
INCORPORATION BY REFERENCE OF THE SEQUENCE LISTING
[0002] The sequence listing provided in the file named
"20160505_NB40736_PCT_SEQS_ST25.txt" with a size of 16,666 bytes
which was created on May 5, 2016 and which is filed herewith, is
incorporated by reference herein in its entirety.
BACKGROUND
[0003] Diacetyl is sometimes an unwanted by-product of fermentation
processes of carbohydrate containing substances, e.g. wort or grape
juice. Formation of diacetyl is most disadvantageous because of its
strong and unpleasant smell and in case of beer even small amounts
of diacetyl of about 0.10 to 0.15 mg/liter has a negative effect on
the flavor and taste of the beer. During the maturation of beer,
diacetyl is converted into acetoin by reductases in the yeast
cells. Acetoin is with respect to taste and flavor acceptable in
beer in much higher concentrations than diacetyl.
[0004] Acetolactate decarboxylase (ALDC) can also be used as an
enzyme to prevent the formation of diacetyl. .alpha.-acetolactate
can be converted into acetoin by adding an ALDC enzyme during
fermentation. However, ALDC can be unstable at fermenting
conditions, especially those of fermenting worts with low malt
content.
[0005] The purpose of the present invention is to provide ALDC
enzymes having a better stability and/or activity, and, optionally,
the yield of ALDC enzymes which can be recovered from
microorganisms is improved.
SUMMARY OF THE INVENTION
[0006] The present disclosure provides compositions and processes
for ALDC enzymes.
[0007] Aspects and embodiments of the compositions and methods are
set forth in the following separately numbered paragraphs.
[0008] 1. A composition comprising an acetolactate decarboxylase
(ALDC) enzyme and zinc, where the zinc is present at a
concentration of about 1 .mu.M to about 200 mM.
[0009] 2. The composition of paragraph 1, where the zinc is present
at a concentration of about 10 .mu.M to about 150 mM, or about 20
.mu.M to about 120 mM, or about 25 .mu.M to about 100 mM, or about
25 .mu.M to about 50 mM, or about 25 .mu.M to about 20 mM, or about
25 .mu.M to about 50 .mu.M, or about 100 .mu.M to about 20 mM, or
about 250 .mu.M to about 20 mM, or about 500 .mu.M to about 20 mM,
or about 1 mM to about 20 mM, or about 1 mM to about 10 mM, or
about 1 mM to about 5 mM.
[0010] 3. The composition of any preceding paragraph, where the
zinc is present at a concentration of about 100 .mu.M to about 10
mM.
[0011] 4. The composition of any preceding paragraph, where the
zinc is present at a concentration of about 1 mM to about 5 mM. The
composition of any preceding paragraph, wherein the molar ratio of
zinc to ALDC enzyme is higher than 1; or 2:1 or higher; or 10:1 or
higher; or 20:1 or higher; or 30:1 or higher; or 60:1 or
higher.
[0012] 5. The composition of any preceding paragraph, where the
ALDC enzyme is an ALDC derivative.
[0013] 6. The composition of paragraph 5, where the ALDC derivative
is an ALDC enzyme treated with glutaraldehyde.
[0014] 7. The composition of paragraph 6, where the ALDC enzyme is
treated with glutaraldehyde at a concentration corresponding to
about 0.1 to about 5 g of glutaraldehyde per g of pure ALDC
enzyme.
[0015] 8. The composition of any preceding paragraph, where the
activity of the ALDC enzyme is in the range of 950 to 2500 Units
per mg of protein.
[0016] 9. The composition of any preceding paragraph, where the
activity of the ALDC enzyme is in the range of 1000 to 2500 Units
per mg of protein.
[0017] 10. The composition of any preceding paragraph further
comprising at least one additional enzyme or enzyme derivative
selected from the group consisting of acetolactate
reductoisomerases, acetolactate isomerases, amylase, glucoamylase,
hemicellulase, cellulase, glucanase, pullulanase, isoamylase,
endo-glucanase and related beta-glucan hydrolytic accessory
enzymes, xylanase, xylanase accessory enzymes (for example,
arabinofuranosidase, ferulic acid esterase, and xylan acetyl
esterase) and protease.
[0018] 11. The composition of any preceding paragraph, where the
ALDC enzyme is from Lactobacillus casei, Brevibacterium acetylicum,
Lactococcus lactis, Leuconostoc lactis, Enterobacter aerogenes,
Bacillus subtilis, Bacillus brevis, Lactococcus lactis DX, or
Bacillus licheniformis.
[0019] 12. The composition of any preceding paragraph, where the
ALDC enzyme is from Bacillus brevis or Bacillus licheniformis.
[0020] 13. The composition of any preceding paragraph, where the
ALDC enzyme has an amino acid sequence having at least 80% identity
with any one selected from SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO:
5, SEQ ID NO: 7 and SEQ ID NO: 8 or any functional fragment
thereof.
[0021] 14. Use of the composition according to any preceding
paragraph in fermentation (such as beer and/or wine and/or cider
and/or perry and/or sake fermentation).
[0022] 15. A method for increasing the activity and/or stability of
an ALDC enzyme comprising adding zinc at a concentration of about 1
.mu.M to about 200 mM. The method of paragraph 15 for increasing
the activity and/or stability of an ALDC enzyme in a composition
comprising ALDC wherein said method comprises the step of adding
zinc to the composition so that said zinc is present in said
composition at a concentration of about 1 .mu.M to about 200
mM.
[0023] 16. The method of paragraph 15, where the zinc is present in
said composition at a concentration of about 1 .mu.M to about 300
.mu.M, or about 6 .mu.M to about 300 .mu.M, or about 1 .mu.M to
about 50 .mu.M; or about 1 .mu.M to about 25 .mu.M, or about 10
.mu.M to about 150 mM, or about 20 .mu.M to about 120 mM, or about
25 .mu.M to about 100 mM, or about 25 .mu.M to about 50 mM, or
about 25 .mu.M to about 20 mM, or about 25 .mu.M to about 50 .mu.M,
or about 100 .mu.M to about 20 mM, or about 250 .mu.M to about 20
mM, or about 500 .mu.M to about 20 mM, or about 1 mM to about 20
mM, or about 1 mM to about 10 mM, or about 1 mM to about 5 mM.
[0024] 17. The method of paragraphs 15 or 16, where the zinc is
present in said composition at a concentration of about 100 .mu.M
to about 10 mM.
[0025] 18. The method of paragraphs 15 or 16, where the zinc is
present in said composition at a concentration of about 1 mM to
about 5 mM. The method of paragraphs 15 or 16, where the molar
ratio of zinc to ALDC enzyme is higher than 1; or 2:1 or higher; or
10:1 or higher; or 20:1 or higher; or 30:1 or higher; or 60:1 or
higher in said composition.
[0026] 19. The method of paragraph 15 where the zinc is added to a
cultivation media as a supplement during the production of the ALDC
enzyme by an ALDC producing host cell. A method for increasing the
activity and/or stability of an ALDC enzyme in a cultivation media
comprising an ALDC producing host cell wherein said method
comprises the step of adding zinc to the cultivation media as a
supplement during the production of the ALDC enzyme by the ALDC
producing host cell.
[0027] 20. The method of paragraph 19, where the zinc is added at a
concentration of 1 .mu.M to about 1 mM.
[0028] 21. The method of paragraph 20, where the zinc is added at a
concentration of 25 .mu.M to about 150 .mu.M, or about 60 .mu.M to
about 150 .mu.M.
[0029] 22. The method of any one of paragraphs 19-21, where the
host cell is a Bacillus host cell.
[0030] 23. The method of paragraph 22, where the Bacillus host cell
is Bacillus subtilis. 24. The method of paragraph 15, where the
zinc is added in a fermentation media and/or maturation media
during fermentation of a beverage (such as beer and/or wine and/or
cider and/or perry and/or sake fermentation). A method for
increasing the activity and/or stability of an ALDC enzyme in a
fermentation media and/or maturation media comprising an ALDC
producing host cell wherein said method comprises the step adding
zinc to the fermentation media and/or maturation media during
fermentation of a beverage (such as beer and/or wine and/or cider
and/or perry and/or sake fermentation).
[0031] 25. The method of paragraph 24, where the zinc is added at a
concentration of about 1 .mu.M to about 300 .mu.M, or about 6 .mu.M
to about 300 .mu.M, or about 1 .mu.M to about 50 .mu.M; or about 1
.mu.M to about 25 .mu.M.
[0032] 26. The method of any one of paragraphs 24, 25 and the
current paragraph, where the zinc is added as a composition
comprising ALDC and zinc, wherein the zinc is present in the
composition at a concentration of 1 mM to about 5 mM. The method of
any one of paragraphs 24, 25 and the current paragraph, where the
zinc is added as a composition comprising ALDC and zinc, where the
molar ratio of zinc to ALDC enzyme in the composition is higher
than 1; or 2:1 or higher; or 10:1 or higher; or 20:1 or higher; or
30:1 or higher; or 60:1 or higher. 27. A cultivation media for an
ALDC producing host cell comprising zinc at a concentration of
about 1 .mu.M to about 1 mM; preferably said cultivation media
comprises an ALDC producing host cell.
[0033] 28. The cultivation media of paragraph 27, comprising zinc
at concentration of about 25 .mu.M to about 150 .mu.M.
[0034] 29. The cultivation media of paragraph 27, where the zinc is
added at a concentration of 60 .mu.M to about 150 .mu.M.
[0035] 30. A beer and/or wine and/or cider and/or perry and/or sake
fermentation media and/or maturation media comprising an ALDC
enzyme and zinc at a concentration of about 0.1 .mu.M to about 200
mM, A beer and/or wine and/or cider and/or perry and/or sake
fermentation media and/or maturation media comprising a composition
comprising an ALDC enzyme and zinc wherein said composition
comprises zinc at a concentration of about 0.1 .mu.M to about 200
mM, preferably 1 .mu.M to about 200 mM.
[0036] 31. The beer and/or wine and/or cider and/or perry and/or
sake fermentation media and/or maturation of paragraph 30, wherein
said composition comprises zinc at a concentration of about 0.1
.mu.M to about 300 .mu.M, preferably 1 .mu.M to about 300 .mu.M.
The beer and/or wine and/or cider and/or perry and/or sake
fermentation media and/or maturation of paragraph 30, comprising
wherein said composition comprises zinc at a concentration of about
6 .mu.M to about 300 .mu.M, or about 1 .mu.M to about 50 .mu.M, or
about 6 .mu.M to about 50 .mu.M, or about 6 .mu.M to about 25
.mu.M. The beer and/or wine and/or cider and/or perry and/or sake
fermentation media and/or maturation of paragraph 30 and the
current paragraph, where the zinc and the ALDC enzyme are added in
a composition, wherein the zinc is present in the composition at a
concentration of about 1 mM to about 20 mM, such as 1 mM to about 5
mM. The beer and/or wine and/or cider and/or perry and/or sake
fermentation media and/or maturation of paragraph 30 or the current
paragraph, where the zinc and the ALDC enzyme are added in a
composition, where the molar ratio of zinc to ALDC enzyme in the
composition is higher than 1; or 2:1 or higher; or 10:1 or higher;
or 20:1 or higher; or 30:1 or higher; or 60:1 or higher.
[0037] 32. The beer and/or wine and/or cider and/or perry and/or
sake fermentation media and/or maturation of paragraph 30 or 31,
where the activity of the ALDC enzyme is in the range of 1000 to
2500 Units per mg of protein.
[0038] 33. The beer and/or wine and/or cider and/or perry and/or
sake fermentation media and/or maturation of any one of paragraphs
30-32, further comprising at least one additional enzyme or enzyme
derivative selected from the group consisting of acetolactate
reductoisomerases, acetolactate isomerases, amylase, glucoamylase,
hemicellulase, cellulase, glucanase, pullulanase, isoamylase,
endo-glucanase and related beta-glucan hydrolytic accessory
enzymes, xylanase, xylanase accessory enzymes (for example,
arabinofuranosidase, ferulic acid esterase, and xylan acetyl
esterase) and protease.
[0039] 34. A composition comprising an ALDC enzyme, where the ALDC
enzyme is in the range of 1000 to 2500 Units per mg of protein.
[0040] 35. The composition of paragraph 34, where the ALDC enzyme
is from Lactobacillus casei, Brevibacterium acetylicum, Lactococcus
lactis, Leuconostoc lactis, Enterobacter aerogenes, Bacillus
subtilis, Bacillus brevis, Lactococcus lactis DX, or Bacillus
licheniformis.
[0041] 36. The composition of paragraph 34, where the ALDC enzyme
is from Bacillus brevis or Bacillus licheniformis.
[0042] 37. The composition of any one of paragraphs 34-36, where
the ALDC enzyme has an amino acid sequence having at least 80%
identity with any one selected from SEQ ID NO: 2, SEQ ID NO: 3, SEQ
ID NO: 5, SEQ ID NO: 7 and SEQ ID NO: 8 or any functional fragment
thereof.
[0043] 38. The composition of any one of paragraphs 34-37, where
zinc is present at a concentration of about 1 .mu.M to about 200
mM, preferably about 100 .mu.M to about 200 mM. The composition of
any one of paragraphs 34-37, where the molar ratio of zinc to ALDC
enzyme in the composition is higher than 1; or 2:1 or higher; or
10:1 or higher; or 20:1 or higher; or 30:1 or higher; or 60:1 or
higher.
[0044] 39. A method for beer and/or wine and/or cider and/or perry
and/or sake production comprising adding an ALDC enzyme and zinc
during the beer and/or wine and/or cider and/or perry and/or sake
production, where the zinc is present at a concentration of about
0.1 .mu.M to about 300 .mu.M, preferably 1 .mu.M to about 300
.mu.M. The method of paragraph 39 for beer and/or wine and/or cider
and/or perry and/or sake production comprising adding an ALDC
enzyme and adding zinc to a media (such as a fermentation and/or a
maturation media) for the beer and/or wine and/or cider and/or
perry and/or sake during said beer and/or wine and/or cider and/or
perry and/or sake production so that said zinc is present in said
media at a concentration of about 0.1 .mu.M to about 300 .mu.M,
such as about 6 .mu.M to about 300 .mu.M.
[0045] 40. The method of paragraph 39, where the zinc is present in
said media at a concentration of about 0.1 .mu.M to about 50 .mu.M.
The method of paragraph 39, where the zinc is present in said media
at a concentration of about 6 .mu.M to about 300 .mu.M, or 1 .mu.M
to about 50 .mu.M, or about 6 .mu.M to about 50 .mu.M, or about 6
.mu.M to about 25 .mu.M.
[0046] 41. The method of paragraph 39, where the zinc and the ALDC
enzyme are added in a composition, wherein zinc is present in said
composition at a concentration of about 1 mM to about 5 mM. The
method of paragraph 39, where the zinc and the ALDC enzyme are
added in a composition, where the molar ratio of zinc to ALDC
enzyme in the composition is higher than 1; or 2:1 or higher; or
10:1 or higher; or 20:1 or higher; or 30:1 or higher; or 60:1 or
higher. A method for beer and/or wine and/or cider and/or perry
and/or sake production comprising adding a composition comprising
an ALDC enzyme and zinc to a media (such as a fermentation and/or a
maturation media) for the beer and/or wine and/or cider and/or
perry and/or sake during said beer and/or wine and/or cider and/or
perry and/or sake production wherein (i) zinc is present in the
composition at a concentration of about 1 .mu.M to about 200 mM,
preferably about 1 mM to about 5 mM; or (ii) the molar ratio of
zinc to ALDC enzyme in the composition is higher than 1; or 2:1 or
higher; or 10:1 or higher; or 20:1 or higher; or 30:1 or higher; or
60:1 or higher.
[0047] 42. The method of any one of paragraphs 39-41, where the
ALDC enzyme and the zinc are added during a fermentation process or
a maturation process.
[0048] 43. The method of any one of paragraphs 39-42, where the
ALDC enzyme is added at a concentration of about 0.5 g to about 10
g per hectoliter of beer and/or wine and/or cider and/or perry
and/or sake ferment.
[0049] 44. The method of any one of paragraphs 39-43, where the
ALDC enzyme is added at a concentration of about 1 g to about 5 g
per hectoliter of beer and/or wine and/or cider and/or perry and/or
sake ferment.
[0050] 45. The method of any one of paragraphs 39-44, where the
activity of the ALDC enzyme is in the range of 1000 to 2500 Units
per mg of protein.
[0051] 46. The method of any one of paragraphs 39-45, where the
ALDC enzyme is from Lactobacillus casei, Brevibacterium acetylicum,
Lactococcus lactis, Leuconostoc lactis, Enterobacter aerogenes,
Bacillus subtilis, Bacillus brevis, Lactococcus lactis DX, or
Bacillus licheniformis.
[0052] 47. The method of any one of paragraphs 39-45, where the
ALDC enzyme is from Bacillus brevis or Bacillus licheniformis.
[0053] 48. The method of any one of paragraphs 39-45, where the
ALDC enzyme has an amino acid sequence having at least 80% identity
with any one selected from SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO:
5, SEQ ID NO: 7 and SEQ ID NO: 8 or any functional fragment
thereof.
[0054] 49. A method for increasing the activity and/or stability of
an ALDC enzyme comprising adding a metal ion at a concentration of
about 1 .mu.M to about 200 mM, preferably about 100 .mu.M to about
200 mM. The method of paragraph 49 for increasing the activity
and/or stability of an ALDC enzyme in a composition comprising ALDC
wherein said method comprises the step of adding a metal ion to the
composition at a concentration of about 1 .mu.M to about 200 mM,
preferably about 100 .mu.M to about 200 mM.
[0055] 50. The method of paragraph 49, where the atomic radius for
the metal ion is about 140 pm to about 165 pm.
[0056] 51. The method of paragraph 50, where the atomic radius for
the metal ion is about 140 pm to about 150 pm.
[0057] 52. The method of paragraph 51, where the atomic radius for
the metal ion is about 142 pm to about 146 pm.
[0058] 53. The method of paragraph 49, where the metal ion is
selected from the group consisting of Zn.sup.2+, Mg.sup.2+,
Mn.sup.2+, Co.sup.2+, Cu.sup.2+, Ca.sup.2+, Ba.sup.2+ and Fe.sup.2+
and combinations thereof.
[0059] 54. The method of paragraph 53, where the metal ion is
selected from the group consisting of Zn.sup.2+, Mn.sup.2+, and
Co.sup.2+.
[0060] 55. The method of paragraph 54, where the metal ion is
Zn.sup.2+.
[0061] 56. The method of any one of paragraphs 49-55, where the
ALDC enzyme is from Lactobacillus casei, Brevibacterium acetylicum,
Lactococcus lactis, Leuconostoc lactis, Enterobacter aerogenes,
Bacillus subtilis, Bacillus brevis, Lactococcus lactis DX, or
Bacillus licheniformis.
[0062] 57. The method of any one of paragraphs 49-55, where the
ALDC enzyme is from Bacillus brevis or Bacillus licheniformis.
[0063] 58. The method of any one of paragraphs 49-55, where the
ALDC enzyme has an amino acid sequence having at least 80% identity
with any one selected from SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO:
5, SEQ ID NO: 7 and SEQ ID NO: 8 or any functional fragment
thereof.
[0064] 59. A composition comprising an acetolactate decarboxylase
(ALDC) enzyme and a metal ion, where the metal ion is present at a
concentration of about 1 .mu.M to about 200 mM, preferably about
100 .mu.M to about 200 mM.
[0065] 60. The composition of paragraph 59, where the atomic radius
for the metal ion is about 140 pm to about 165 pm.
[0066] 61. The composition of paragraphs 59, where the atomic
radius for the metal ion is about 140 pm to about 150 pm.
[0067] 62. The composition of paragraph 61, where the atomic radius
for the metal ion is about 142 pm to about 146 pm.
[0068] 63. The composition of paragraph 59, where the metal ion is
selected from the group consisting of Zn.sup.2+, Mg.sup.2+,
Mn.sup.2+, Co.sup.2+, Cu.sup.2+, Ca.sup.2+, Ba.sup.2+ and Fe.sup.2+
and combinations thereof.
[0069] 64. The composition of paragraph 63, where the metal ion is
selected from the group consisting of Zn.sup.2+, Mn.sup.2+, and
Co.sup.2+.
[0070] 65. The composition of paragraph 64, where the metal ion is
Zn.sup.2+.
[0071] 66. A method for decomposing acetolactate comprising the
step of treating a substrate with an ALDC enzyme and a metal ion,
where the metal ion is present at a concentration of about 1 .mu.M
to about 200 mM. The method of paragraph 66 for decomposing
acetolactate comprising the step of treating a substrate with a
composition comprising an ALDC enzyme and a metal ion so that the
metal ion is present in said composition at a concentration of
about 1 .mu.M to about 200 mM. Preferably said substrate is a
carbohydrate containing substrate such as a wort or a fruit juice.
Preferably said substrate is a fermentation and/or maturation
media.
[0072] 67. The method of paragraph 66, where the metal ion is
present in said composition at a concentration of about 10 .mu.M to
about 150 mM, or about 20 .mu.M to about 120 mM, or about 25 .mu.M
to about 100 mM, or about 25 .mu.M to about 50 mM, or about 25
.mu.M to about 20 mM, or about 100 .mu.M to about 20 mM, or about
250 .mu.M to about 20 mM, or about 1 mM to about 20 mM, or about 1
mM to about 5 mM.
[0073] 68. The method of paragraph 66, where the metal ion is
present in said substrate (such as a fermentation and/or maturation
media) at a concentration of about 1 .mu.M to about 500 .mu.M, or
about 1 .mu.M to about 300 .mu.M, or about 6 .mu.M to about 300
.mu.M, or about 1 .mu.M to about 100 .mu.M, or about 1 .mu.M to
about 50 .mu.M, or about 1 .mu.M to about 25 .mu.M, or about 6
.mu.M to about 50 .mu.M, or about 6 .mu.M to about 25 .mu.M, or
about 25 .mu.M to about 50 .mu.M.
[0074] 69. The method of paragraph 66, where the metal ion and the
ALDC are added in a composition, wherein said metal ion is present
in said composition at a concentration of about 1 mM to about 5 mM.
The method of paragraph 66, where the metal ion and the ALDC are
added in a composition, where the molar ratio of the metal ion to
ALDC enzyme in the composition is higher than 1; or 2:1 or higher;
or 10:1 or higher; or 20:1 or higher; or 30:1 or higher; or 60:1 or
higher.
[0075] 70. The method of any one of paragraphs 66-69, where the
metal ion is selected from the group consisting of Zn.sup.2+,
Mg.sup.2+, Mn.sup.2+, Co.sup.2+, Cu.sup.2+, Ca.sup.2+, Ba.sup.2+
and Fe.sup.2+ and combinations thereof.
[0076] 71. The method of any one of paragraphs 66-70, where the
metal ion is selected from the group consisting of Zn.sup.2+,
Mn.sup.2+, and Co.sup.2+.
[0077] 72. The method of any one of paragraphs 66-71, where the
metal ion is Zn.sup.2+.
[0078] 73. The method of any one of paragraphs 66-72, where the
substrate is treated during a beer and/or wine and/or cider and/or
perry and/or sake fermentation or maturation process.
[0079] 74. The method of any one of paragraphs 66-72, where the
ALDC enzyme is from Lactobacillus casei, Brevibacterium acetylicum,
Lactococcus lactis, Leuconostoc lactis, Enterobacter aerogenes,
Bacillus subtilis, Bacillus brevis, Lactococcus lactis DX, or
Bacillus licheniformis.
[0080] 75. The method of any one of paragraphs 66-72, where the
ALDC enzyme is from Bacillus brevis or Bacillus licheniformis.
[0081] 76. The method of any one of paragraphs 66-72, where the
ALDC enzyme has an amino acid sequence having at least 80% identity
with any one selected from SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO:
5, SEQ ID NO: 7 and SEQ ID NO: 8 or any functional fragment
thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
[0082] A better understanding of the features and advantages of the
present invention will be obtained by reference to the following
detailed description that sets forth illustrative embodiments, in
which the principles of the invention are utilized, and the
accompanying drawings of which:
[0083] FIG. 1 shows a plasmid map of alrA(CB)RIHI-aldB for
expression of Acetolactate Decarboxylase, aldB
[0084] FIG. 2 shows a graph depicting the activity of ALDC relative
to 0 mM zinc vs concentration of zinc in mM.
[0085] FIG. 3 shows SDS-PAGE with aldB samples containing varying
concentration of ZnSO.sub.4. Gel A--Lane 1) Molecular weight
marker; Lane 2-7) BSA standard; Lane 8-9) AldB with 0 mM
ZnSO.sub.4; Lane 10-11) AldB with 0.25 mM ZnSO.sub.4; Lane 12-13)
AldB with 0.5 mM ZnSO.sub.4; Lane 14-15) AldB with 1.0 mM
ZnSO.sub.4; Lane 16-17) AldB with 2 mM ZnSO.sub.4; Lane 18-19) AldB
with 5 mM ZnSO.sub.4; Lane 20-21) AldB with 7.5 mM ZnSO.sub.4; Lane
22-23) AldB with 10 mM ZnSO.sub.4 and Lane 24-25) AldB with 20 mM
ZnSO.sub.4. Gel B--Lane 1) Molecular weight marker; Lane 2-7) BSA
standard; Lane 8-11) AldB with 0 mM ZnSO.sub.4; Lane 12-15) AldB
with 20 mM ZnSO.sub.4; Lane 16-17) AldB with 40 mM ZnSO.sub.4; Lane
18-19) AldB with 60 mM ZnSO.sub.4; Lane 20-21) AldB with 80 mM
ZnSO.sub.4; Lane 22-23) AldB with 100 mM ZnSO.sub.4 and Lane 24-25)
AldB with 120 mM ZnSO.sub.4.
[0086] FIG. 4 shows SDS-PAGE with purification of aldB produced in
B. subtilis. Lane 1) crude aldB ferment; 2-3) purified aldB from
Source15Q. Molecular weight marker is shown to the left and
in-between lane 1 and 2.
[0087] FIG. 5 shows development of vicinal diketones (VDK) during
malt-based fermentations in the presence or absence of 0.03 U/mL
wort aldB enzyme variants with different specific activity: A) 919
U/mg, B) 1103 U/mg and C) 1552 U/mg aldB. The VDK development (sum
of diacetyl and 2,3 pentanedione) was followed during the 7 days of
fermentation at 14.degree. C. The average VDK values are calculated
from duplicate samples and labels are shown as insert in
figure.
[0088] FIG. 6 shows VDK development in presence aldB enzyme (0.04
U/mL wort) during malt-based fermentations with different levels of
Zn.sup.2+ in the wort (see labels). The VDK development (sum of
diacetyl and 2,3-pentanedione) was followed during the 7 days of
fermentation at 14.degree. C. The average VDK values are calculated
from duplicate samples and labels are shown as insert in
figure.
DETAILED DESCRIPTION OF THE INVENTION
[0089] The present disclosure provides methods, compositions,
apparatuses and kits comprising ALDC enzymes having a better
stability and/or activity, and, optionally, the yield of ALDC
enzymes which can be recovered from microorganisms is improved. In
some embodiments, the present disclosure provides methods,
apparatuses, compositions and kits for the use of metal ions to
increase stability and/or activity, and, optionally, which further
can be used to recover the enzymes (e.g. ALDC enzymes) from
microorganisms in improved yields.
[0090] 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 to which this disclosure belongs.
Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR
BIOLOGY, 20 ED., John Wiley and Sons, New York (1994), and Hale
& Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper
Perennial, N.Y. (1991) provide one of skill with a general
dictionary of many of the terms used in this disclosure.
[0091] This disclosure is not limited by the exemplary methods and
materials disclosed herein, and any methods and materials similar
or equivalent to those described herein can be used in the practice
or testing of embodiments of this disclosure. Numeric ranges are
inclusive of the numbers defining the range. Unless otherwise
indicated, any nucleic acid sequences are written left to right in
5' to 3' orientation; amino acid sequences are written left to
right in amino to carboxy orientation, respectively.
[0092] The headings provided herein are not limitations of the
various aspects or embodiments of this disclosure which can be had
by reference to the specification as a whole. Accordingly, the
terms defined immediately below are more fully defined by reference
to the specification as a whole.
[0093] It must be noted that as used herein and in the appended
claims, the singular forms "a", "an", and "the" include plural
referents unless the context clearly dictates otherwise. Thus, for
example, reference to "a protease" includes a plurality of such
enzymes and reference to "the feed" includes reference to one or
more feeds and equivalents thereof known to those skilled in the
art, and so forth.
[0094] The publications discussed herein are provided solely for
their disclosure prior to the filing date of the present
application. Nothing herein is to be construed as an admission that
such publications constitute prior art to the claims appended
hereto.
[0095] All patents and publications referred to herein are
incorporated by reference.
ALDC
[0096] In some aspects the invention provides ALDC enzymes having a
better stability and/or activity, and, optionally, the yield of
ALDC enzymes which can be recovered from microorganisms is
improved. The terms "better stability" and "increased stability" as
used herein refer to an ALDC enzyme whose ALDC activity is
maintained for a longer period of time when in the presence of a
metal ion (such as Zn.sup.2+) when compared to the ALDC activity of
the enzyme in the absence of the metal ion. The terms "better
activity" and "increased activity" as used herein refer to an ALDC
enzyme having an increased ALDC activity when in the presence of a
metal ion (such as Zn.sup.2+) when compared to the ALDC activity of
the enzyme in the absence of the metal ion. The term "improved" in
connection to the yield of ALDC enzyme refers to an increase in the
ALDC activity which is produced when a host microorganism is in the
presence of a metal ion (such as Zn.sup.2+) compared to the ALDC
activity produced when the host microorganism is in the absence of
the metal ion. Without wishing to be bound by theory, the metal ion
(such as Zn.sup.2+) can be added during and/or after the culture
process (e.g. ALDC production) in order to increase stability
and/or increase activity and/or to increase yield of ALDC enzymes.
The terms "host cell", "host microorganism", "strain" and
"microorganism" may be used interchangeably herein.
[0097] Acetolactate decarboxylase (ALDC) is an enzyme that belongs
to the family of carboxy lyases, which are responsible for cleaving
carbon-carbon bonds. Acetolactate decarboxylase catalyzes the
conversion of 2-acetolactate (also known as
2-hydroxy-2-methyl-3-oxobutanoate) to 2-acetoin and releases
CO.sub.2. The terms "ALDC" and "ALDC enzyme" may be used
interchangeably herein.
[0098] Acetolactate decarboxylase enzymes catalyze the enzymatic
reaction belonging to the classification EC 4.1.1.5 (acetolactate
decarboxylase activity) and gene ontology (GO) term ID of GO:
0047605. The GO term ID specifies that any protein characterized as
having this associated GO term encodes an enzyme with catalytic
acetolactate decarboxylase activity.
[0099] Various acetolactate decarboxylase genes (such as alsD or
aldB), which encode acetolactate decarboxylase enzymes, are known
in the art. The alsD gene, which encodes ALDC enzyme, may be
derived or derivable from Bacillus subtilis. The aldB gene, which
encodes ALDC enzyme, may be derived or derivable from Bacillus
brevis. The alsD gene, which encodes ALDC enzyme, may be derived or
derivable from Bacillus licheniformis. UNIPROT accession number
Q65E52.1 is an example of an ALDC enzyme. UNIPROT accession number
Q65E52.1 is an example of an ALDC enzyme derived or derivable from
Bacillus licheniformis. Examples of acetolactate decarboxylase
genes include but are not limited to
gi|375143627|ref|YP_005006068.11 acetolactate decarboxylase
[Niastella koreensis OR20-10];
gi|361057673|gb|AEV96664.1|acetolactate decarboxylase [Niastella
koreensis OR20-10]; gi|218763415|gb|ACL05881.1|acetolactate
decarboxylase [Desulfatibacillum alkenivorans AK-01];
gi|220909520|ref|YP 002484831.1| acetolactate decarboxylase
[Cyanothece sp. PCC 7425]; gi|218782031|ref|YP 002433349.1|
acetolactate decarboxylase [Desulfatibacillum alkenivorans AK-01];
gi|213693090|ref|YP_002323676.11 acetolactate decarboxylase
[Bifidobacterium longum subsp. infantis ATCC 15697=JCM 1222];
gi|189500297|ref|YP 001959767.1| acetolactate decarboxylase
[Chlorobium phaeobacteroides BS1]; gi|189423787|ref|YP 001950964.11
acetolactate decarboxylase [Geobacter lovleyi SZ];
gi|172058271|ref|YP 001814731.11 acetolactate decarboxylase
[Exiguobacterium sibiricum 255-15]; gi|163938775|ref|YP
001643659.11 acetolactate decarboxylase [Bacillus
weihenstephanensis KBAB4]; gi|158522304|ref|YP 001530174.11
acetolactate decarboxylase [Desulfococcus oleovorans Hxd3];
gi|57371670|ref|YP 001479659.11 acetolactate decarboxylase
[Serratia proteamaculans 568]; gi|150395111|ref|YP 001317786.11
acetolactate decarboxylase [Staphylococcus aureus subsp. aureus
JH1]; gi|150394715|ref|YP 001317390.11 acetolactate decarboxylase
[Staphylococcus aureus subsp. aureus JH1]; gi|146311679|ref|YP
001176753.1| acetolactate decarboxylase [Enterobacter sp. 638];
gi|109900061|ref|YP 663316.11 acetolactate decarboxylase
[Pseudoalteromonas atlantica T6c]; gi|219866131|gb|ACL46470.1|
acetolactate decarboxylase [Cyanothece sp. PCC 7425];
gi|213524551|gb|ACJ53298.1|acetolactate decarboxylase
[Bifidobacterium longum subsp. infantis ATCC 15697=JCM 1222];
gi|189420046|gb|ACD94444.1| acetolactate decarboxylase [Geobacter
lovleyi SZ]; gi|158511130|gb|ABW68097.1|acetolactate decarboxylase
[Desulfococcus oleovorans Hxd3];
gi|157323434|gb|ABV42531.1|acetolactate decarboxylase [Serratia
proteamaculans 568]; gi|145318555|gb|ABP60702.1|acetolactate
decarboxylase [Enterobacter sp. 638];
gi|149947563|gb|ABR53499.1|acetolactate decarboxylase
[Staphylococcus aureus subsp. aureus JH1];
gi|149947167|gb|ABR53103.1|acetolactate decarboxylase
[Staphylococcusaureus subsp. aureus JH1];
gi|163860972|gb|ABY42031.1|Acetolactate decarboxylase [Bacillus
weihenstephanensis KBAB4]; gi|109702342|gb|ABG42262.1|Acetolactate
decarboxylase [Pseudoalteromonas atlantica T6c];
gi|189495738|gb|ACE04286.1|acetolactate decarboxylase [Chlorobium
phaeobacteroides BS1]; gi|171990792|gb|ACB61714.1|acetolactate
decarboxylase [Exiguobacterium sibiricum 255-15];
gi|223932563|ref|ZP 03624564.1 acetolactate decarboxylase
[Streptococcus suis 89/1591]; gi|194467531|ref|ZP 03073518.11
acetolactate decarboxylase [Lactobacillus reuteri 100-23];
gi|223898834|gb|EEF65194.1|acetolactate decarboxylase
[Streptococcus suis 89/1591];
gi|194454567|gb|EDX43464.1|acetolactate decarboxylase
[Lactobacillus reuteri 100-23]; gi|384267135|ref|YP 005422842.11
acetolactate decarboxylase [Bacillus amyloliquefaciens subsp.
plantarum YAU B9601-Y2]; gi|375364037|ref YP_005132076.11
acetolactate decarboxylase [Bacillus amyloliquefaciens subsp.
plantarum CAU B946]; gi|34079323|ref|YP_004758694.11 acetolactate
decarboxylase [Corynebacterium variabile DSM 44702];
gi|336325119|ref|YP_004605085.11 acetolactate decarboxylase
[Corynebacterium resistens DSM 45100];
gi|148269032|ref|YP_001247975.1| acetolactate decarboxylase
[Staphylococcus aureus subsp. aureus JH9];
gi|148268650|ref|YP_001247593.11 acetolactate decarboxylase
[Staphylococcus aureus subsp. aureus JH9];
gi|1485433721|ref|YP_001270742.11 acetolactate decarboxylase
[Lactobacillus reuteri DSM 20016];
gi|380500488|emb|CCG51526.1|acetolactate decarboxylase [Bacillus
amyloliquefaciens subsp. plantarum YAU B9601-Y2];
gi|371570031|emb|CCF06881.1| acetolactate decarboxylase [Bacillus
amyloliquefaciens subsp. plantarum CAU B946];
gi|340533141|gb|AEK35621.1|acetolactate decarboxylase
[Corynebacterium variabile DSM 44702];
gi|336101101|gb|AE108921.1|acetolactate decarboxylase
[Corynebacterium resistens DSM 45100];
gi|148530406|gb|ABQ82405.1|acetolactate decarboxylase
[Lactobacillus reuteri DSM 20016];
gi|147742101|gb|ABQ50399.1|acetolactate decarboxylase
[Staphylococcus aureus subsp. aureus JH9];
gi|147741719|gb|ABQ50017.1|acetolactate decarboxylase
[Staphylococcus aureus subsp. aureus JH9]; gi|392529510|ref|ZP
10276647.11 acetolactate decarboxylase [Carnobacterium
maltaromaticum ATCC 35586]; gi|366054074|ref|ZP 09451796.11
acetolactate decarboxylase [Lactobacillus suebicus KCTC 3549];
gi|339624147|ref|ZP 08659936.11 acetolactate decarboxylase
[Fructobacillus jructosus KCTC 3544]; and gi|336393727|ref|ZP
08575126.11 acetolactate decarboxylase [Lactobacillus coryniformis
subsp. torquens KCTC 3535]. UNIPROT Accession No. P23616.1
(Diderichsen et al., J Bacteriol. (1990) 172(8): 4315) is an
example of an ALDC enzyme. UNIPROT accession number P23616.1 is an
example of an ALDC enzyme derived or derivable from Bacillus
brevis. Each sequence associated with the foregoing accession
numbers is incorporated herein by reference.
[0100] In some embodiments, the invention relates to ALDC enzymes
from Lactobacillus casei (Godtfredsen 1984), Brevibacterium
acetylicum (Oshiro, 1989), Lactococcus lactis (Vincent Phalip
1994), Leuconostoc lactis (0 sulivan, 2001), Enterobacter aerogenes
(Blomquist, 1993), Bacillus subtilis (Renna, 1993), Bacillus brevis
(Svendsen, 1989) and Lactococcus lactis DX (Yuxing, 2014). In some
embodiments, the ALDC enzyme is from Lactobacillus casei,
Brevibacterium acetylicum, Lactococcus lactis, Leuconostoc lactis,
Enterobacter aerogenes, Bacillus subtilis, Bacillus brevis,
Lactococcus lactis DX, or Bacillus licheniformis. As used herein,
the term "ALDC enzyme is from" refers to the ALDC enzyme being
derived or derivable from.
[0101] It is to be understood that any suitable ALDC enzymes, i.e.
ALDC produced from any microorganism which activity is dependent on
metal ions, can be used according to the invention. In some
embodiments, the ALDC used in the methods and compositions
described herein is an ALDC from Bacillus brevis or Bacillus
licheniformis.
[0102] The ALDC activity of the enzyme composition according to the
invention is measured by the ALDC assays as described herein or any
suitable assay known in the art. The standard assay is carried out
at pH 6.0, and it can be performed at different pH values and
temperatures for the additional characterization and specification
of enzymes.
[0103] One unit of ALDC activity is defined as the amount of enzyme
which produces 1 .mu.mole acetoin per minute under the conditions
of the assay (e.g., pH 6.0 (or as specified) and 30.degree.
C.).
[0104] In some embodiments, the ALDC is an ALDC derivative. In some
embodiments, the ALDC derivative is characterized by the fact that
ALDC in an aqueous medium is treated with or has been treated with
glutaraldehyde. In some embodiments, the ALDC is treated with or
has been treated with glutaraldehyde in a concentration
corresponding to between 0.1 and 5 g of glutaraldehyde per g of
pure ALDC protein, preferably corresponding to between 0.25 and 2 g
of glutaraldehyde per g of pure ALDC protein.
[0105] In some embodiments, the ALDC enzyme comprises an amino acid
sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%,
99% or 100% identity with SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5,
SEQ ID NO: 7, or SEQ ID NO: 8 or any functional fragment thereof.
One aspect of the invention relates to an enzyme exhibiting ALDC
activity, which enzyme comprises an amino acid sequence having at
least 80% identity with any one selected from SEQ ID NO: 2, SEQ ID
NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8 or any functional
fragment thereof. In some embodiments, the ALDC enzyme is encoded
by a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%,
95%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 1, SEQ ID NO:
4, or SEQ ID NO: 6 or any functional fragment thereof.
[0106] In some embodiments, the enzyme has a temperature optimum in
the range of 5-80.degree. C., such as in the range of 5-40.degree.
C. or 15-80.degree. C., such as in the range 20-80.degree. C., such
as in the range 5-15.degree. C., 10-40.degree. C., 10-50.degree.
C., 15-20.degree. C., 45-65.degree. C., 50-65.degree. C.,
55-65.degree. C. or 60-80.degree. C. In some embodiments, the
enzyme has a temperature optimum of about 60.degree. C.
[0107] In some embodiments, the enzyme has a total number of amino
acids of less than 350, such as less than 340, such as less than
330, such as less than 320, such as less than 310, such as less
than 300 amino acids, such as in the range of 200 to 350, such as
in the range of 220 to 345 amino acids.
[0108] In some embodiments, the amino acid sequence of the enzyme
has at least one, two, three, four, five, six, seven, eight, nine
or ten amino acid substitutions as compared to any one amino acid
sequence selected from SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5,
SEQ ID NO: 7, SEQ ID NO: 8, or any functional fragment thereof.
[0109] In some embodiments, the amino acid sequence of the enzyme
has a maximum of one, two, three, four, five, six, seven, eight,
nine or ten amino acid substitutions compared to any one amino acid
sequence selected from SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5,
SEQ ID NO: 7, SEQ ID NO: 8, or any functional fragment thereof.
[0110] In some embodiments, the enzyme comprises the amino acid
sequence identified by any one of SEQ ID NO: 2, SEQ ID NO: 3, SEQ
ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 8, or any functional fragment
thereof.
[0111] In some embodiments the compositions, media and methods
according to the invention comprise any one or more further enzyme.
In some embodiments the one or more further enzyme is selected from
list consisting of acetolactate reductoisomerases, acetolactate
isomerases, amylase, glucoamylase, hemicellulase, cellulase,
glucanase, pullulanase, isoamylase, endo-glucanase and related
beta-glucan hydrolytic accessory enzymes, xylanase, xylanase
accessory enzymes (for example, arabinofuranosidase, ferulic acid
esterase, xylan acetyl esterase) and protease.
[0112] In some embodiments the compositions, media and methods
according to the invention comprise an enzyme exhibiting ALDC
activity, wherein the activity of said ALDC enzyme is in the range
of 950 to 2500 Units per mg of protein. In some embodiments the
compositions, media and methods according to the invention comprise
an enzyme exhibiting ALDC activity, wherein the activity of said
ALDC enzyme is in the range of 1000 to 2500 Units per mg of
protein. In some embodiments the compositions, media and methods
according to the invention comprise an enzyme exhibiting ALDC
activity, wherein the activity of said ALDC enzyme is in the range
of 1500 to 2500 Units per mg of protein. In some embodiments, the
enzyme exhibiting ALDC activity is an enzyme comprising an amino
acid sequence having at least 80% identity with any one selected
from SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID
NO: 8, or any functional fragment thereof. In some embodiments, the
enzyme exhibiting ALDC activity is encoded by a nucleic acid
sequence having at least 80% identity with SEQ ID NO: 1, SEQ ID NO:
4, SEQ ID NO: 6 or any functional fragment thereof.
[0113] Metal Ions
[0114] In one aspect, the invention provides methods and
compositions comprising ALDC enzymes having a better stability
and/or activity. In another aspect the invention provides methods
and compositions comprising ALDC enzymes which can be recovered
from microorganisms in improved yields.
[0115] Surprisingly, it has been found by the present inventors
that treatment of ALDC compositions with certain metal ions at
certain concentrations provides ALDC enzymes having a better
stability and/or activity, and, optionally, the yield of ALDC
activity which can be recovered from microorganisms is
improved.
[0116] In some embodiments, the atomic radius for the metal ion is
about 140 pm to about 255 pm. In some embodiments, the atomic
radius for the metal ion is about 140 pm to about 165 pm. In some
embodiments, the atomic radius for the metal ion is about 140 pm to
about 150 pm. In some embodiments, the atomic radius for the metal
ion is about 142 pm to about 146 pm.
[0117] In some embodiments, the metal ion is selected from the
group consisting of Zn.sup.2+, Mg.sup.2+, Mn.sup.2+, Co.sup.2+,
Cu.sup.2+, Ba.sup.2+, Ca.sup.2+ and Fe.sup.2+ and combinations
thereof. In some embodiments, the metal ion is selected from the
group consisting of Zn.sup.2+, Cu.sup.2+, and Fe.sup.2+. In some
embodiments, the metal ion is selected from the group consisting of
Zn.sup.2+, Mn.sup.2+, and Co.sup.2+. In some embodiments, the metal
ion is Zn.sup.2+ or Mn.sup.2+. In some embodiments, the metal ion
is Zn.sup.2+. The term "zinc" as used herein may be interchangeable
with the term "Zn.sup.2+". The term "metal" as used herein may be
interchangeable with the term "metal ion". The term "metal" as used
herein may refer to compounds which comprise the metal selected
from the group consisting of zinc, magnesium, manganese, cobalt,
copper, barium, calcium and iron; compounds which comprise these
metals are a source of the respective ions. The term "zinc" as used
herein refers to compounds which comprise zinc, such compounds are
a source of Zn.sup.2+ ions. Zinc sulfate (ZnSO.sub.4) is example of
zinc as referred to herein and is an example of a source of
Zn.sup.2+ ions. Magnesium sulfate (MgSO.sub.4) is an example of
magnesium as referred to herein and is an example of a source of
Mg.sup.2+ ions. Manganese(II) sulfate (MnSO.sub.4) is an example of
manganese as referred to herein and is an example of a source of
Mn.sup.2+ ions. Cobalt(II)chloride (CoCl.sub.2) is an example of
cobalt as referred to herein and is an example of a source of
Co.sup.2+ ions. Copper(II) sulphate (CuSO.sub.4) is an example of
copper as referred to herein and is an example of a source of
Cu.sup.2+ ions. Barium sulfate (BaSO.sub.4) is an example of barium
as referred to herein and is an example of a source of Ba.sup.2+
ions. Calcium sulfate (CaSO.sub.4) is an example of calcium as
referred to herein and is example of a source of Ca.sup.2+ ions.
Iron(II) sulfate (FeSO.sub.4) is an example of iron as referred to
herein and is example of a source of Fe.sup.2+ ions.
[0118] The present inventors have found that metal ions such as
Zn.sup.2+, Mn.sup.2+, Co.sup.2+, Cu.sup.2+, and Fe.sup.2+ increase
the stability of ALDC enzyme in different formulations (see
Examples), and also improve the recovery yields from microorganisms
when the metal ions are used during the production of the enzyme in
the cultivation media. Thus, in some embodiments, the present
invention provides methods and compositions that increase the
recovery yields, stability and/or activity of ALDC enzymes that can
be then used, e.g., to produce fermented products such as in
brewing.
[0119] In one aspect, the present disclosure provides compositions
comprising an ALDC enzyme with increased stability and/or
activity.
[0120] In some embodiments, the ALDC enzyme has an specific
activity of at least about 900 units per mg of protein (U/mg), at
least about 1000 U/mg, at least about 1500 U/mg, at least about
2000 U/mg, at least about 3000 U/mg at least about 5000 U/mg, at
least about 6000 U/mg, at least about 7000 U/mg, at least about
8000 U/mg, at least about 8500 U/mg, at least about 9000 U/mg, at
least about 9500 U/mg, or at least about 10000 U/mg as measured by
the assays described herein or any suitable assay known in the art.
In some embodiments, the ALDC enzyme has an ALDC activity in the
range of about 950 to 2500 units per mg of protein (U/mg), about
1000 to 2500 U/mg, or about 1500 to 2500 U/mg as measured by the
assays described herein or any suitable assay known in the art. In
some embodiments, the ALDC compositions according to the invention
comprise an ALDC enzyme with ALDC activity of at least about 900
units per gram of product, at least about 1000 U/g, at least about
1500 U/g, at least about 2000 U/g, at least about 3000 U/g at least
about 5000 U/g, such as at least about 6000 U/g, such as at least
about 7000 U/g, such as at least about 8000 U/g, such as at least
about 8500 U/g, such as at least about 9000 U/g, such as at least
about 9500 U/g, such as at least about 10000 U/g as measured by in
the assays described herein or any suitable assay known in the art.
In some embodiments, a different ALDC activity is used, e.g.,
depending on the acetolactate content and conditions requirements,
e.g. for brewing. In some embodiments, the ALDC compositions
according to the invention comprise an ALDC enzyme with ALDC
activity of at least about 8000 U/g.
[0121] In some embodiments, the compositions comprise an ALDC
enzyme and a metal ion, where the metal ion is present at a
concentration of about 0.1 .mu.M to about 200 mM, such as about 1
.mu.M to about 200 mM, or about 1 .mu.M to about 500 .mu.M, or
about 1 .mu.M to about 300 .mu.M, or about 6 .mu.M to about 300
.mu.M, or about 10 .mu.M to about 100 .mu.M, or about 15 .mu.M to
about 50 .mu.M, or about 1 .mu.M to about 150 mM, or about 10 .mu.M
to about 150 mM, or about 20 .mu.M to about 120 mM, or about 25
.mu.M to about 100 mM, or about 25 .mu.M to about 50 mM, or about
25 .mu.M to about 20 mM, or about 25 .mu.M to about 50 .mu.M, or
about 100 .mu.M to about 20 mM, or about 250 .mu.M to about 20 mM,
or about 1 mM to about 20 mM, or about 1 mM to about 5 mM. In some
embodiments, the compositions comprise an ALDC enzyme and a metal
ion, where the metal ion is present at a concentration of about 1
.mu.M to about 300 .mu.M, such as about 6 .mu.M to about 300 .mu.M,
or about 6 .mu.M to about 50 .mu.M, or about 6 .mu.M to about 25
.mu.M. In some embodiments, the compositions comprise an ALDC
enzyme and a metal ion, where the metal ion is present at a
concentration of about 60 .mu.M to about 150 .mu.M, or about 25
.mu.M to about 150 .mu.M. In some embodiments, the compositions
comprise an ALDC enzyme and a metal ion, where the metal ion is
present at a concentration of about 100 .mu.M to about 200 mM. In
some embodiments, the compositions comprise an ALDC enzyme and a
metal ion, where the metal ion is present at a concentration of
about 100 .mu.M to about 20 mM. In some embodiments, the
compositions comprise an ALDC enzyme and a metal ion, where the
metal ion is present at a concentration of about 1 mM to about 5
mM. In some embodiments, the metal ion is selected from the group
consisting of Zn.sup.2+, Mg.sup.2+, Mn.sup.2+, Co.sup.2+,
Cu.sup.2+, Ba.sup.2+, Ca.sup.2+ and Fe.sup.2+ and combinations
thereof. In some embodiments, the metal ion is selected from the
group consisting of Zn.sup.2+, Cu.sup.2+, and Fe.sup.2+. In some
embodiments, the metal ion is selected from the group consisting of
Zn.sup.2+, Mn.sup.2+, and Co.sup.2+. In some embodiments, the metal
ion is Zn.sup.2+ or Mn.sup.2+. In some embodiments, the metal ion
is Zn.sup.2+.
[0122] In some embodiments, the compositions comprise an ALDC
enzyme and zinc where the zinc is present at a concentration of
about 1 .mu.M to about 200 mM, such as about 1 .mu.M to about 500
.mu.M, or about 1 .mu.M to about 300 .mu.M, or about 6 .mu.M to
about 300 .mu.M, or about 10 .mu.M to about 100 .mu.M, or about 15
.mu.M to about 50 .mu.M, or about 10 .mu.M to about 150 mM, or
about 20 .mu.M to about 120 mM, or about 25 .mu.M to about 100 mM,
or about 25 .mu.M to about 50 mM, or about 25 .mu.M to about 20 mM,
or about 25 .mu.M to about 50 .mu.M, or about 100 .mu.M to about 20
mM, or about 250 .mu.M to about 20 mM, or about 500 .mu.M to about
20 mM, or about 1 mM to about 20 mM, or about 1 mM to about 10 mM,
or about 1 mM to about 5 mM, or about 5 mM to about 20 mM, or about
5 mM to about 10 mM. In some embodiments, the compositions comprise
an ALDC enzyme and zinc, where the zinc is present at a
concentration of about 1 .mu.M to about 300 .mu.M, such about 6
.mu.M to about 300 .mu.M, or about 6 .mu.M to about 25 .mu.M. In
some embodiments, the compositions comprise an ALDC enzyme and
zinc, where the zinc is present at a concentration of about 25
.mu.M to about 150 .mu.M or about 60 .mu.M to about 150 .mu.M. In
some embodiments, the compositions comprise an ALDC enzyme and
zinc, where the zinc is present at a concentration of about 100
.mu.M to about 20 mM. In some embodiments, the compositions
comprise an ALDC enzyme and zinc, where the zinc is present at a
concentration of about 100 .mu.M to about 10 mM. In some
embodiments, the compositions comprise an ALDC enzyme and zinc,
where the zinc is present at a concentration of about 1 mM to about
5 mM.
[0123] In some embodiments, the compositions comprise an ALDC
enzyme and zinc where the zinc is present at a concentration of
about 1 mM to about 3 mM, or about 0.75 mM to about 4 mM, or about
0.5 mM to about 5 mM, or about 0.25 mM to about 7.5 mM, or about
0.1 mM to about 10 mM. In some embodiments, the activity of said
ALDC enzyme is in the range of 950 to 2500 Units per mg of protein,
or 1000 to 2500 Units per mg of protein, or 1500 to 2500 Units per
mg of protein.
[0124] In some embodiments, the compositions comprise an ALDC
enzyme and zinc, where the molar ratio of zinc to enzyme is higher
than 1 such as 2:1, or 3:1, or 5:1, or 10:1, or 20:1 or 30:1, or
50:1, or 60:1, or 100:1, or 150:1, or 200:1, or 250:1, or 500:1. In
some embodiments, the compositions comprise an ALDC enzyme and
zinc, where the molar ratio of zinc to enzyme is 2:1 or higher. In
some embodiments, the compositions comprise an ALDC enzyme and
zinc, where the molar ratio of zinc to enzyme is 5:1 or higher. In
some embodiments, the compositions comprise an ALDC enzyme and
zinc, where the molar ratio of zinc to enzyme is 10:1 or higher. In
some embodiments, the compositions comprise an ALDC enzyme and
zinc, where the molar ratio of zinc to enzyme is 20:1 or higher. In
some embodiments, the compositions comprise an ALDC enzyme and
zinc, where the molar ratio of zinc to enzyme is 30:1 or higher. In
some embodiments, the compositions comprise an ALDC enzyme and
zinc, where the molar ratio of zinc to enzyme is 60:1 or higher.
The molar concentration of, for example, Zn.sup.2+, Mn.sup.2+,
Co.sup.2+ or other metal ions in solution may be determined by
inductively coupled plasma optical emission spectrometry (ICP-OES)
or similar techniques. The molar concentration of the ALDC enzyme
may be determined using. Criterion SLS-PAGE system (such as
described in the examples) and the amino acid sequence.
[0125] In some embodiments, the ALDC enzyme is an ALDC derivative.
In some embodiments, the ALDC derivative is an ALDC enzyme treated
with glutaraldehyde. In some embodiments, the ALDC enzyme is
treated with glutaraldehyde at a concentration corresponding to
about 0.1 to about 5 g of glutaraldehyde per g of pure ALDC
enzyme.
[0126] In some embodiments, the activity of the ALDC enzyme is in
the range of 950 to 2500 Units per mg of protein. In some
embodiments, the activity of said ALDC enzyme is in the range of
1000 to 2500 Units per mg of protein. In some embodiments, the
activity of said ALDC enzyme is in the range of 1500 to 2500 Units
per mg of protein. Thus, in some embodiments, the compositions
comprise an ALDC enzyme, where the ALDC enzyme is in the range of
950 to 2500 Units per mg of protein or 1000 to 2500 Units per mg of
protein or 1500 to 2500 Units per mg of protein.
[0127] In some embodiments, the compositions comprise an ALDC
enzyme and zinc, where the zinc is present at a concentration of
about 100 .mu.M to about 20 mM, and the activity of said ALDC
enzyme is in the range of 1000 to 2500 Units per mg of protein. In
some embodiments, the compositions comprise an ALDC enzyme or an
ALDC derivative and zinc, where the zinc is present at a
concentration of about 250 .mu.M to about 20 mM, and the activity
of said ALDC enzyme is in the range of 1000 to 2500 Units per mg of
protein.
[0128] In some embodiments, the ALDC enzyme compositions further
comprise at least one additional enzyme or enzyme derivative
selected from the group consisting of acetolactate
reductoisomerases, acetolactate isomerases, amylase, glucoamylase,
hemicellulase, cellulase, glucanase, pullulanase, isoamylase,
endo-glucanase and related beta-glucan hydrolytic accessory
enzymes, xylanase, xylanase accessory enzymes (for example,
arabinofuranosidase, ferulic acid esterase, and xylan acetyl
esterase) and protease.
[0129] In some embodiments, the ALDC enzyme compositions described
herein are used during fermentation and/or maturation of a beverage
preparation process, e.g., beer and wine, to reduce diacetyl
levels. The terms "ALDC enzyme composition", "composition
comprising an ALDC enzyme" and "composition comprising ALDC" as
used herein refer to compositions comprising the ALDC enzyme. The
composition may be in the form of a solution. As used herein, the
terms "ALDC enzyme composition" and "compositions comprising ALDC"
are mutually exclusive with media (such as cultivation media,
fermentation media or maturation media) which comprise
microorganisms expressing ALDC and/or capable of expressing ALDC
when cultured under conditions permitting expression of the enzyme.
Examples of ALDC enzyme compositions and compositions comprising
ALDC include compositions comprising ALDC in a purified form. ALDC
may be purified from a media comprising microorganisms capable of
expressing ALDC wherein said media has been cultured under
conditions permitting expression of ALDC. The term "purified" means
that ALDC is present at a high level. Preferably, ALDC is the
predominant component present in the composition. Preferably, ALDC
is present at a level of at least about 90%, or at least about 95%
or at least about 98%, said level being determined on a dry
weight/dry weight basis with respect to the total composition under
consideration. In some embodiments, an ALDC enzyme composition
further comprises a metal ion such as zinc.
[0130] As used herein, the terms "beverage" and "beverage(s)
product" include such foam forming fermented beverages as beer
brewed with 100% malt, beer brewed under different types of
regulations, ale, dry beer, near beer, light beer, low alcohol
beer, low calorie beer, porter, bock beer, stout, malt liquor,
non-alcoholic beer, non-alcoholic malt liquor and the like. The
term "beverages" or "beverages product" also includes non-foaming
beer and alternative malt beverages such as fruit 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. The term "beverages" or "beverages product" also includes
beer made with alternative materials other than malted barley, such
as rye, corn, oats, rice, millet, triticale, cassava, sorghum,
barley, wheat and a combination thereof. The term "beverages" or
"beverages product" also includes other fermented products such as
wine or ciders or perry or sake.
[0131] Beer is traditionally referred to as an alcoholic beverage
derived from malt, such as malt derived from barley grain, and
optionally adjunct, such as starch containing plant material (e.g.
cereal grains) and optionally flavored, e.g. with hops. In the
context of the present invention, the term "beer" includes any
fermented wort, produced by fermentation/brewing of a
starch-containing plant material, thus in particular also beer
produced exclusively from adjunct, or any combination of malt and
adjunct. Beer can be made from a variety of starch-containing plant
material by essentially the same process, where the starch consists
mainly of glucose homopolymers in which the glucose residues are
linked by alpha-1, 4- or alpha-1,6-bonds, with the former
predominating. Beer can be made from alternative materials such as
rye, corn, oats, rice, millet, triticale, cassava, sorghum, wheat,
barley and a combination thereof.
[0132] In some embodiments, the invention provides a fermentation
media (e.g. beer and/or wine and/or cider and/or perry and/or sake
fermentation) comprising an ALDC enzyme and metal ion at a
concentration of about 0.1 .mu.M to about 200 mM, or about 1 .mu.M
to about 200 mM, such as about 1 .mu.M to about 500 .mu.M, or about
0.1 .mu.M to about 300 .mu.M, or about 1 .mu.M to about 300 .mu.M,
or about 6 .mu.M to about 300 .mu.M, or about 1 .mu.M to about 100
.mu.M, or about 1 .mu.M to about 50 .mu.M, or about 6 .mu.M to
about 50 .mu.M, or about 6 .mu.M to about 25 .mu.M. In some
embodiments, the invention provides a fermentation media (e.g. beer
and/or wine and/or cider and/or perry and/or sake fermentation)
comprising an ALDC enzyme and metal ion at a concentration of about
0.1 .mu.M to about 100 mM, such as about 0.1 .mu.M to about 10
.mu.M, or 1 .mu.M to about 100 mM, or 1 .mu.M to about 10 .mu.M, or
6 .mu.M to about 10 .mu.M, or about 10 .mu.M to about 200 .mu.M, or
about 50 .mu.M to about 1 mM, or about 100 .mu.M to about 10 mM, or
about 100 .mu.M to about 50 mM, or about 100 .mu.M to about 100 mM,
or about 100 .mu.M to about 200 mM, or about 250 .mu.M to about 120
mM, or about 500 .mu.M to about 100 mM, or about 1 mM to about 50
mM, or about 1 mM to about 20 mM, or about 1 mM to about 5 mM. In
some embodiments, the invention provides a fermentation media (e.g.
beer and/or wine and/or cider and/or perry and/or sake
fermentation) comprising an ALDC enzyme and metal ion at a
concentration of about 0.1 .mu.M to about 200 mM or about 1 .mu.M
to about 200 mM, such as about 1 .mu.M to about 500 .mu.M, or about
1 .mu.M to about 300 .mu.M, or about 6 .mu.M to about 300 .mu.M, or
about 1 .mu.M to about 100 .mu.M, or about 1 .mu.M to about 50
.mu.M, or about 6 .mu.M to about 50 .mu.M, or about 6 .mu.M to
about 25 .mu.M. In some embodiments, the invention provides a
fermentation media (e.g. beer and/or wine and/or cider and/or perry
and/or sake fermentation) comprising an ALDC enzyme and metal ion
at a concentration of about 1 .mu.M to about 300 .mu.M, or about 6
.mu.M to about 300 .mu.M, or about 1 .mu.M to about 100 .mu.M, or
about 1 .mu.M to about 50 .mu.M, or about 6 .mu.M to about 50 .mu.M
or about 6 .mu.M to about 25 .mu.M. In some embodiments, the metal
ion is selected from the group consisting of Zn.sup.2+, Mg.sup.2+,
Mn.sup.2+, Co.sup.2+, Cu.sup.2+, Ba.sup.2+, Ca.sup.2+ and Fe.sup.2+
and combinations thereof. In some embodiments, the metal ion is
selected from the group consisting of Zn.sup.2+, Cu.sup.2+, and
Fe.sup.2+. In some embodiments, the metal ion is selected from the
group consisting of Zn.sup.2+, Mn.sup.2+, and Co.sup.2+. In some
embodiments, the metal ion is Zn.sup.2+ or Mn.sup.2+. In some
embodiments, the metal ion is Zn.sup.2+. In some embodiments, the
activity of said ALDC enzyme is in the range of 950 to 2500 Units
per mg of protein, or 1000 to 2500 Units per mg of protein, or 1500
to 2500 Units per mg of protein. In some embodiments, the
fermentation media (e.g. beer and/or wine and/or cider and/or perry
and/or sake fermentation) further comprises at least one additional
enzyme or enzyme derivative selected from the group consisting of
acetolactate reductoisomerases, acetolactate isomerases, amylase,
glucoamylase, hemicellulase, cellulase, glucanase, pullulanase,
isoamylase, endo-glucanase and related beta-glucan hydrolytic
accessory enzymes, xylanase, xylanase accessory enzymes (for
example, arabinofuranosidase, ferulic acid esterase, and xylan
acetyl esterase) and protease.
[0133] In some embodiments, the invention provides a maturation
media (e.g. beer and/or wine and/or cider and/or perry and/or sake
fermentation) comprising an ALDC enzyme and metal ion at a
concentration of about 0.1 .mu.M to about 200 mM, or 1 .mu.M to
about 200 mM, such as about 1 .mu.M to about 500 .mu.M, or about
0.1 .mu.M to about 300 .mu.M, or about 1 .mu.M to about 300 .mu.M,
or about 6 .mu.M to about 300 .mu.M, or about 1 .mu.M to about 100
.mu.M, or about 1 .mu.M to about 50 .mu.M, or about 6 .mu.M to
about 50 .mu.M, or about 6 .mu.M to about 25 .mu.M. In some
embodiments, the invention provides a maturation media (e.g. beer
and/or wine and/or cider and/or perry and/or sake fermentation)
comprising an ALDC enzyme and metal ion at a concentration of about
0.1 .mu.M to about 100 mM, or 1 .mu.M to about 100 mM, such as
about 0.1 .mu.M to about 10 .mu.M, or 1 .mu.M to about 10 .mu.M, or
6 .mu.M to about 10 .mu.M, or about 10 .mu.M to about 200 .mu.M, or
about 50 .mu.M to about 1 mM, or about 100 .mu.M to about 10 mM, or
about 100 .mu.M to about 50 mM, or about 100 .mu.M to about 100 mM,
or about 100 .mu.M to about 200 mM, or about 250 .mu.M to about 120
mM, or about 500 .mu.M to about 100 mM, or about 1 mM to about 50
mM, or about 1 mM to about 20 mM, or about 1 mM to about 5 mM. In
some embodiments, the invention provides a maturation media (e.g.
beer and/or wine and/or cider and/or perry and/or sake
fermentation) comprising an ALDC enzyme and metal ion at a
concentration of about 1 .mu.M to about 500 .mu.M, or about 1 .mu.M
to about 300 .mu.M, or about 6 .mu.M to about 300 .mu.M, or about 1
.mu.M to about 100 .mu.M, or about 1 .mu.M to about 50 .mu.M, or
about 6 .mu.M to about 50 .mu.M, or about 6 .mu.M to about 25
.mu.M. In some embodiments, the invention provides a maturation
media (e.g. beer and/or wine and/or cider and/or perry and/or sake
fermentation) comprising an ALDC enzyme and metal ion at a
concentration of about 1 .mu.M to about 300 .mu.M, or about 6 .mu.M
to about 300 .mu.M, or about 1 .mu.M to about 100 .mu.M, or about 1
.mu.M to about 50 .mu.M, or about 6 .mu.M to about 50 .mu.M, or
about 6 .mu.M to about 25 .mu.M. In some embodiments, the metal ion
is selected from the group consisting of Zn.sup.2+, Mg.sup.2+,
Mn.sup.2+, Co.sup.2+, Cu.sup.2+, Ba.sup.2+, Ca.sup.2+ and Fe.sup.2+
and combinations thereof. In some embodiments, the metal ion is
selected from the group consisting of Zn.sup.2+, Cu.sup.2+, and
Fe.sup.2+. In some embodiments, the metal ion is selected from the
group consisting of Zn.sup.2+, Mn.sup.2+, and Co.sup.2+. In some
embodiments, the metal ion is Zn.sup.2+ or Mn.sup.2+. In some
embodiments, the metal ion is Zn.sup.2+. In some embodiments, the
activity of said ALDC enzyme is in the range of 950 to 2500 Units
per mg of protein, or 1000 to 2500 Units per mg of protein, or 1500
to 2500 Units per mg of protein. In some embodiments, the
maturation media (e.g. beer and/or wine maturation) further
comprises at least one additional enzyme or enzyme derivative
selected from the group consisting of acetolactate
reductoisomerases, acetolactate isomerases, amylase, glucoamylase,
hemicellulase, cellulase, glucanase, pullulanase, isoamylase,
endo-glucanase and related beta-glucan hydrolytic accessory
enzymes, xylanase, xylanase accessory enzymes (for example,
arabinofuranosidase, ferulic acid esterase, and xylan acetyl
esterase) and protease.
[0134] In some embodiments, metal ions such as Zn.sup.2+,
Mg.sup.2+, Mn.sup.2+, Co.sup.2+, Cu.sup.2+, Ba.sup.2+, Ca.sup.2+
and Fe.sup.2+ and combinations thereof are added to the cultivation
and/or fermentation media during and/or after ALDC production to
increase the recovered yields from microorganisms.
[0135] The term "cultivation media" as used herein refers to a
media which supports the growth of microorganisms such as an ALDC
producing host cell. Examples of a cultivation media include: media
based on MOPs buffer with, for instance, urea as the major nitrogen
source and maltrin as the main carbon source; and TSB broth. In
some embodiments, the invention provides a cultivation media for an
ALDC producing host cell comprising a metal ion at a concentration
of about 1 .mu.M to about 1 mM. In some embodiments, the invention
provides a cultivation media for an ALDC producing host cell
comprising a metal ion at a concentration of about 25 .mu.M to
about 150 .mu.M. In some embodiments, the invention provides a
cultivation media for an ALDC producing host cell comprising a
metal ion at a concentration of about 25 .mu.M to about 50 .mu.M.
In some embodiments, the invention provides a cultivation media for
an ALDC producing host cell comprising a metal ion at a
concentration of about 30 .mu.M to about 40 .mu.M. In some
embodiments, the invention provides a cultivation media for an ALDC
producing host cell comprising a metal ion at a concentration of
about 40 .mu.M to about 150 .mu.M. In some embodiments, the
invention provides a cultivation media for an ALDC producing host
cell comprising a metal ion at a concentration of about 60 .mu.M to
about 150 .mu.M. In some embodiments, the metal ion is selected
from the group consisting of Zn2+, Mg.sup.2+, Mn.sup.2+, Co.sup.2+,
Cu.sup.2+, Ba.sup.2+, Ca.sup.2+ and Fe.sup.2+ and combinations
thereof. In some embodiments, the metal ion is selected from the
group consisting of Zn.sup.2+, Cu.sup.2+, and Fe.sup.2+. In some
embodiments, the metal ion is selected from the group consisting of
Zn.sup.2+, Mn.sup.2+, and Co.sup.2+. In some embodiments, the metal
ion is Zn.sup.2+ or Mn.sup.2+. In some embodiments, the metal ion
is Zn.sup.2+. In some embodiments, the activity of said ALDC enzyme
is in the range of 950 to 2500 Units per mg of protein, or 1000 to
2500 Units per mg of protein, or 1500 to 2500 Units per mg of
protein.
[0136] Materials may be added to an enzyme-containing composition
to improve the properties of the composition. Non-limiting examples
of such additives include: salts (e.g., alkali salts, earth metal
salts, additional chloride salts, sulfate salts, nitrate salts,
carbonate salts, where exemplary counter ions are calcium,
potassium, and sodium), inorganic minerals or clays (e.g.,
zeolites, kaolin, bentonite, talcs and/or silicates), carbohydrates
(e.g., sucrose and/or starch), coloring pigments (e.g., titanium
dioxide), biocides (e.g., Rodalon.RTM., Proxel.RTM.), dispersants,
anti-foaming agents, reducing agents, acid agents, alkaline agents,
enzyme stabilizers (e.g. polyol such as glycerol, propylene glycol,
sorbitol, inorganic salts, sugars, sugar or a sugar alcohol, lactic
acid, boric acid, or a boric acid derivative and combinations
thereof), enzyme inhibitors, preservative (e.g. methyl paraben,
propyl paraben, benzoate, sorbate or other food approved
preservatives) and combinations thereof. Excipients which may be
used in the composition, or the preparation thereof, include
maltose, maltose syrup, sucrose, glucose (including glucose syrup
or dried glucose syrup), pre-cooked starch, gelatinised starch,
L-lactic, ascorbyl palmitate, tocopherols, lecithins, citric acid,
citrates, phosphoric, phosphates, sodium alginate, carrageenan,
locust bean gum, guar gum, xanthan gum, pectins, sodium
carboxymethylcellulose, mono- and diglycerides, citric acid esters
of mono- and diglycerides, sucrose esters, carbon dioxide, argon,
helium, nitrogen, nitrous oxide, oxygen, hydrogen, and starch
sodium octenylsuccinate.
Methods
[0137] In some aspects the invention provides methods to improve
stability and/or activity of ALDC enzymes. In some aspects the
invention provides methods to improve ALDC recovery from
microorganisms.
[0138] In some embodiments, the invention provides methods for
increasing the activity and/or stability of an ALDC enzyme in a
composition comprising ALDC wherein said method comprises the step
of adding a metal ion to the composition so that said metal ion is
present in said composition at a concentration of about 1 .mu.M to
about 200 mM, such as about 1 .mu.M to about 500 .mu.M, or about 1
.mu.M to about 300 .mu.M, or about 6 .mu.M to about 300 .mu.M, or
about 1 .mu.M to about 100 .mu.M, or about 1 .mu.M to about 50
.mu.M, or about 10 .mu.M to about 150 mM, or about 20 .mu.M to
about 120 mM, or about 25 .mu.M to about 100 mM, or about 25 .mu.M
to about 50 mM, or about 25 .mu.M to about 20 mM, or about 25 .mu.M
to about 50 .mu.M, or about 100 .mu.M to about 20 mM, or about 250
.mu.M to about 20 mM, or about 500 .mu.M to about 20 mM, or about 1
mM to about 20 mM, or about 1 mM to about 10 mM, or about 1 mM to
about 5 mM, or about 5 mM to about 20 mM, or about 5 mM to about 10
mM. In some embodiments, the invention provides methods for
increasing the activity and/or stability of an ALDC enzyme in a
cultivation media comprising an ALDC producing host cell wherein
said method comprises the step of adding a metal ion to the media
so that said metal ion is present in said media at a concentration
of about 1 .mu.M to about 1 mM, such as about 1 .mu.M to about 300
.mu.M, about 6 .mu.M to about 300 .mu.M, about 25 .mu.M to about
150 .mu.M, or about 60 .mu.M to about 150 .mu.M. In some
embodiments, the invention provides methods for increasing the
activity and/or stability of an ALDC enzyme in a fermentation
and/or maturation media comprising an ALDC enzyme wherein said
method comprises the step of adding a metal ion to the media so
that said metal ion is present in said media at a concentration of
about 1 .mu.M to about 300 .mu.M, such as about 6 .mu.M to about
300 .mu.M, about 1 .mu.M to about 100 .mu.M, about 1 .mu.M to about
50 .mu.M, about 1 .mu.M to about 25 .mu.M, or about 6 .mu.M to
about 25 .mu.M. In some embodiments, the invention provides methods
for increasing the activity and/or stability of an ALDC enzyme
comprising adding a metal ion at a concentration of about 25 .mu.M
to about 150 .mu.M in a media. In some embodiments, the invention
provides methods for increasing the activity and/or stability of an
ALDC enzyme comprising adding a metal ion at a concentration of
about 100 .mu.M to about 20 mM. In some embodiments, the invention
provides methods for increasing the activity and/or stability of an
ALDC enzyme comprising adding a metal ion at a concentration of
about 1 mM to about 5 mM. In some embodiments, the metal ion is
selected from the group consisting of Zn.sup.2+, Mg.sup.2+,
Mn.sup.2+, Co.sup.2+, Cu.sup.2+, Ba.sup.2+, Ca.sup.2+ and Fe.sup.2+
and combinations thereof. In some embodiments, the metal ion is
selected from the group consisting of Zn.sup.2+, Cu.sup.2+, and
Fe.sup.2+. In some embodiments, the metal ion is selected from the
group consisting of Zn.sup.2+, Mn.sup.2+, and Co.sup.2+. In some
embodiments, the metal ion is Zn.sup.2+ or Mn.sup.2+. In some
embodiments, the metal ion is Zn.sup.2+.
[0139] In some embodiments, the invention provides methods for
increasing the activity and/or stability of an ALDC enzyme in a
composition comprising ALDC wherein said method comprises the step
of adding a zinc to the composition so that said zinc is present in
said composition at a concentration of about 1 .mu.M to about 200
mM, such as about 1 .mu.M to about 500 .mu.M, or about 1 .mu.M to
about 300 .mu.M, or about 6 .mu.M to about 300 .mu.M, or about 1
.mu.M to about 100 .mu.M, or about 1 .mu.M to about 50 .mu.M, or
about 10 .mu.M to about 150 mM, or about 20 .mu.M to about 120 mM,
or about 25 .mu.M to about 100 mM, or about 25 .mu.M to about 50
mM, or about 25 .mu.M to about 20 mM, or about 25 .mu.M to about 50
.mu.M, or about 100 .mu.M to about 20 mM, or about 250 .mu.M to
about 20 mM, or about 500 .mu.M to about 20 mM, or about 1 mM to
about 20 mM, or about 1 mM to about 10 mM, or about 1 mM to about 5
mM, or about 5 mM to about 20 mM, or about 5 mM to about 10 mM. In
some embodiments, the invention provides methods for increasing the
activity and/or stability of an ALDC enzyme in a cultivation media
comprising an ALDC producing host cell wherein said method
comprises the step of adding a zinc at a concentration of about 1
.mu.M to about 1 mM, such as about 1 .mu.M to about 300 .mu.M,
about 6 .mu.M to about 300 .mu.M, about 25 .mu.M to about 150
.mu.M, or about 60 .mu.M to about 150 .mu.M. In some embodiments,
the invention provides methods for increasing the activity and/or
stability of an ALDC enzyme in a fermentation and/or maturation
media comprising an ALDC enzyme wherein said method comprises the
step of adding a zinc to the media so that said zinc is present in
said media at a concentration of about 1 .mu.M to about 300 .mu.M,
such as about 6 .mu.M to about 300 .mu.M, about 1 .mu.M to about
100 .mu.M, about 1 .mu.M to about 50 .mu.M, about 1 .mu.M to about
25 .mu.M, or about 6 .mu.M to about 25 .mu.M. In some embodiments,
methods for increasing the activity and/or stability of an ALDC
enzyme comprise adding a zinc to a media so that the zinc is at a
concentration of about 25 .mu.M to about 150 .mu.M in the media. In
some embodiments, methods for increasing the activity and/or
stability of an ALDC enzyme comprise adding a zinc at a
concentration of about 100 .mu.M to about 20 mM. In some
embodiments, methods for increasing the activity and/or stability
of an ALDC enzyme comprise adding a zinc at a concentration of
about 1 mM to about 5 mM. In some embodiments, methods for
increasing the activity and/or stability of an ALDC enzyme comprise
adding zinc at a molar ratio of zinc to ALDC enzyme that is higher
than 1 such as 2:1, or 3:1, or 5:1, or 10:1, or 20:1 or 30:1, or
50:1, or 60:1, or 100:1, or 150:1, or 200:1 or 250:1 in said
composition. In some embodiments, methods for increasing the
activity and/or stability of an ALDC enzyme comprise adding zinc at
a molar ratio of zinc to ALDC enzyme of 5:1 or higher in said
composition. In some embodiments, methods for increasing the
activity and/or stability of an ALDC enzyme comprise adding zinc at
a molar ratio of zinc to ALDC enzyme of 10:1 or higher in said
composition. In some embodiments, methods for increasing the
activity and/or stability of an ALDC enzyme comprise adding zinc at
a molar ratio of zinc to ALDC enzyme of 20:1 or higher in said
composition. In some embodiments, methods for increasing the
activity and/or stability of an ALDC enzyme comprise adding zinc at
a molar ratio of zinc to ALDC enzyme of 30:1 or higher in said
composition.
[0140] In some embodiments, the metal ion is added (e.g. as a
supplement) to a cultivation media during the production of said
ALDC enzyme by an ALDC producing host cell. In some embodiments,
the metal ion is added at a concentration of about 0.1 .mu.M to
about 1 mM, such as about 25 .mu.M to about 150 .mu.M, or about 40
.mu.M to about 150 .mu.M, or about 60 .mu.M to about 150 .mu.M, or
about 25 .mu.M to about 50 .mu.M, or 30 .mu.M to about 40 .mu.M. In
some embodiments, the metal ion is selected from the group
consisting of Zn.sup.2+, Mg.sup.2+, Mn.sup.2+, Co.sup.2+,
Cu.sup.2+, Ba.sup.2+, Ca.sup.2+ and Fe.sup.2+ and combinations
thereof. In some embodiments, the metal ion is selected from the
group consisting of Zn.sup.2+, Cu.sup.2+, and Fe.sup.2+. In some
embodiments, the metal ion is selected from the group consisting of
Zn.sup.2+, Mn.sup.2+, and Co.sup.2+. In some embodiments, the metal
ion is Zn.sup.2+ or Mn.sup.2+. In some embodiments, the metal ion
is Zn.sup.2+. Thus, in some embodiments zinc is added (e.g. as a
supplement) to a cultivation media during the production of said
ALDC enzyme by an ALDC producing host cell at a concentration of 1
.mu.M to about 1 mM, such as 25 .mu.M to about 150 .mu.M, or about
40 .mu.M to about 150 .mu.M, or 60 .mu.M to about 150 .mu.M.
[0141] In some embodiments, the host cell is a Bacillus host cell.
In some embodiments, Bacillus host cell is Bacillus subtilis.
[0142] In some embodiments, the metal ion is added in the
fermentation media during production of a fermented beverage. In
some embodiments, the metal ion is added in the fermentation media
during beer and/or wine and/or cider and/or perry and/or sake
fermentation. In some embodiments, the metal ion is selected from
the group consisting of Zn.sup.2+, Mg.sup.2+, Mn.sup.2+, CO.sup.2+,
Cu.sup.2+, Ba.sup.2+, Ca.sup.2+ and Fe.sup.2+ and combinations
thereof. In some embodiments, the metal ion is selected from the
group consisting of Zn.sup.2+, Cu.sup.2+, and Fe.sup.2+. In some
embodiments, the metal ion is selected from the group consisting of
Zn.sup.2+, Mn.sup.2+, and Co.sup.2+. In some embodiments, the metal
ion is Zn.sup.2+ or Mn.sup.2+. In some embodiments, the metal ion
is Zn.sup.2+. Thus, in some embodiments, zinc is added in a
fermentation media during beer and/or wine and/or cider and/or
perry and/or sake fermentation. In some embodiments, zinc is added
at a concentration of about 1 .mu.M to about 1 mM, such as about 1
.mu.M to about 300 .mu.M, or about 6 .mu.M to about 300 .mu.M, or
about 1 .mu.M to about 100 .mu.M, or 25 .mu.M to about 50 .mu.M, or
30 .mu.M to about 40 .mu.M, or 1 .mu.M to about 50 .mu.M, or 6
.mu.M to about 50 .mu.M, or 1 .mu.M to about 25 .mu.M, or 6 .mu.M
to about 25 .mu.M. In some embodiments zinc and the ALDC enzyme are
added in a composition, wherein zinc is present in said composition
at a concentration of 0.1 .mu.M to about 200 mM or 1 .mu.M to about
200 mM, or 0.1 mM to about 120 mM, such as 1 mM to about 20 mM, or
1 mM to about 10 mM, or 1 mM to 5 mM. In some embodiments zinc and
the ALDC enzyme are added in a composition, wherein the molar ratio
of zinc to ALDC enzyme in the composition is higher than 1 such as
2:1, or 3:1, or 5:1, or 10:1, or 20:1 or 30:1, or 50:1, or
60:1.
[0143] In some embodiments, the metal ion is added in the
maturation media during production of a fermented beverage. In some
embodiments, the metal ion is added the maturation media during
beer and/or wine and/or cider and/or perry and/or sake
fermentation. In some embodiments, the metal ion is selected from
the group consisting of Zn.sup.2+, Mg.sup.2+, Mn.sup.2+, Co.sup.2+,
Cu.sup.2+, Ba.sup.2+, Ca.sup.2+ and Fe.sup.2+ and combinations
thereof. In some embodiments, the metal ion is selected from the
group consisting of Zn.sup.2+, Cu.sup.2+, and Fe.sup.2+. In some
embodiments, the metal ion is selected from the group consisting of
Zn.sup.2+, Mn.sup.2+, and Co.sup.2+. In some embodiments, the metal
ion is Zn.sup.2+ or Mn.sup.2+. In some embodiments, the metal ion
is Zn.sup.2+. Thus, in some embodiments, zinc is added in a
maturation media during beer and/or wine and/or cider and/or perry
and/or sake fermentation. In some embodiments, zinc is added at a
concentration of 1 .mu.M to about 1 mM, such as 1 .mu.M to about
300 .mu.M, or about 6 .mu.M to about 300 .mu.M, or about 1 .mu.M to
about 100 .mu.M, or 25 .mu.M to about 50 .mu.M, or 30 .mu.M to
about 40 .mu.M, or 1 .mu.M to about 50 .mu.M, or 6 .mu.M to about
50 .mu.M, or 1 .mu.M to about 25 .mu.M, or 6 .mu.M to about 25
.mu.M. In some embodiments zinc and ALDC are added in a
composition, wherein zinc is present in said composition at a
concentration of 0.1 .mu.M to about 200 mM, or 1 .mu.M to about 200
mM, or 0.25 mM to about 120 mM, such as 1 mM to about 20 mM, or 1
mM to about 10 mM, or 1 mM to about 5 mM. In some embodiments zinc
and the ALDC enzyme are added in a composition, wherein the molar
ratio of zinc to ALDC enzyme in the composition is higher than 1
such as 2:1, or 3:1, or 5:1, or 10:1, or 20:1 or 30:1, or 50:1, or
60:1.
[0144] In some embodiments, a method of producing acetoin is
provided in the disclosure. In some embodiments, a method of
decomposing acetolactate is provided in the disclosure. In some
embodiments, acetolactate is decomposed to acetoin. The methods
involve the step of treating a substrate with an ALDC enzyme and a
metal ion, wherein the metal ion is present at a concentration of
about 1 .mu.M to about 200 mM, such as about 1 .mu.M to about 500
.mu.M, or about 1 .mu.M to about 300 .mu.M, or about 6 .mu.M to
about 300 .mu.M, or about 1 .mu.M to about 100 .mu.M, or about 1
.mu.M to about 50 .mu.M, or 6 .mu.M to about 50 .mu.M, or 6 .mu.M
to about 25 .mu.M, or about 10 .mu.M to about 150 mM, or about 20
.mu.M to about 120 mM, or about 25 .mu.M to about 100 mM, or about
25 .mu.M to about 50 mM, or about 25 .mu.M to about 20 mM, or about
25 .mu.M to about 50 .mu.M, or about 100 .mu.M to about 20 mM, or
about 250 .mu.M to about 20 mM, or about 1 mM to about 20 mM, or
about 1 mM to about 5 mM. In some embodiments the metal ion and the
ALDC enzyme are added in a composition, where the metal ion is
present in said composition at a concentration of 0.1 .mu.M to
about 200 mM, or 1 .mu.M to about 200 mM, or 0.25 mM to about 120
mM, such as 1 mM to about 20 mM, or 1 mM to about 5 mM. In some
embodiments the metal ion and the ALDC enzyme are added in a
composition, wherein the molar ratio of metal ion to ALDC enzyme in
the composition is higher than 1 such as 2:1, or 3:1, or 5:1, or
10:1, or 20:1 or 30:1, or 50:1, or 60:1. In some embodiments, the
metal ion is selected from the group consisting of Zn.sup.2+,
Mg.sup.2+, Mn.sup.2+, Co.sup.2+, Cu.sup.2+, Ba.sup.2+, Ca.sup.2+
and Fe.sup.2+ and combinations thereof. In some embodiments, the
metal ion is selected from the group consisting of Zn.sup.2+,
Cu.sup.2+, and Fe.sup.2+. In some embodiments, the metal ion is
selected from the group consisting of Zn.sup.2+, Mn.sup.2+, and
Co.sup.2+. In some embodiments, the metal ion is Zn.sup.2+ or
Mn.sup.2+. In some embodiments, the metal ion is Zn.sup.2+. Thus,
in some embodiments, the methods involve the step of treating a
substrate with an ALDC enzyme and zinc, wherein said zinc is
present at a concentration of about 1 .mu.M to about 1 mM, such as
1 .mu.M to about 300 .mu.M, or about 6 .mu.M to about 300 .mu.M, or
1 .mu.M to about 100 .mu.M, or 6 .mu.M to about 100 .mu.M, or 6
.mu.M to about 50 .mu.M, or 6 .mu.M to about 25 .mu.M. In some
embodiments zinc and the ALDC enzyme are added in a composition,
where zinc is present in said composition at a concentration of 0.1
.mu.M to about 200 mM, or 1 .mu.M to about 200 mM, or 0.25 mM to
about 120 mM, such as 1 mM to about 20 mM, or 1 mM to about 5 mM.
In some embodiments zinc and the ALDC enzyme are added in a
composition, wherein the molar ratio of zinc to ALDC enzyme in the
composition is higher than 1 such as 2:1, or 3:1, or 5:1, or 10:1,
or 20:1 or 30:1, or 50:1, or 60:1.
[0145] In some embodiments a method of producing acetoin during the
production of a fermented beverage is provided in the disclosure.
In some embodiments, a method of decomposing acetolactate during
the production of a fermented beverage is provided in the
disclosure. In some embodiments, acetolactate is decomposed to
acetoin.
[0146] Fermented Products
[0147] In one aspect the present invention relates to a process for
producing fermented alcoholic products with a low diacetyl content
by fermentation of a carbohydrate containing substrate with a
microorganism. As used herein, a fermented alcoholic product with
"low diacetyl content" refers to a fermented alcoholic product
(e.g. a beer and/or a wine and/or a cider and/or a perry and/or
sake) produced by fermentation of a carbohydrate containing
substrate with a composition comprising ALDC in the presence of a
metal ion (such as zinc) wherein the diacetyl levels are lower when
compared to the fermented alcoholic produced by fermentation of a
carbohydrate containing substrate with a composition comprising
ALDC in the absence of a metal ion (such as zinc) under the same
fermentation conditions (e.g. same temperature and for the same
length of time). Examples of fermented alcoholic products with low
diacetyl content are fermented alcoholic products in which the
levels of diacetyl are less than about 1 ppm and/or the diacetyl
levels are below about 0.5 mg/L. In one embodiment, the diacetyl
levels are less than about 0.5 ppm, or less than about 0.1 ppm, or
less than about 0.05 ppm, or less than about 0.01 ppm, or less than
about 0.001 ppm. In one embodiment, the diacetyl levels are about
less than 0.1 mg/L, or about less than 0.05 mg/L, or about less
than 0.01 mg/L or about less than 0.001 mg/L.
[0148] When carbohydrate containing substrates, such as wort (e.g.
worts with low malt content) or fruit juices (such as grape juice,
apple juice or pear juice), are fermented with yeast or other
microorganisms, various processes take place in addition to the
alcohol fermentation which may cause generation of undesired
by-products, e.g., the formation of diacetyl which has a strong and
unpleasant smell even in very low concentrations. Alcoholic
beverages, such as beer or wine or cider or perry or sake, may thus
have an unacceptable aroma and flavor if the content of diacetyl
considerably exceeds certain limits, e.g., in the case of some
beers about 0.1 ppm.
[0149] Formation of diacetyl is also disadvantageous in the
industrial production of ethanol because it is difficult to
separate diacetyl from ethanol by distillation. A particular
problem arises in the preparation of absolute ethanol where ethanol
is dehydrated by azeotropic distillation with benzene. Diacetyl
will accumulate in the benzene phase during the azeotropic
distillation which may give rise to mixtures of diacetyl and
benzene which makes it difficult to recover the benzene used for
the azeotropic distillation.
[0150] The conventional brewing of beer comprises fermenting the
wort with a suitable species of yeast, such as Saccharomyces
cerevisae or Saccharomyces carlsbergensis.
[0151] Typically in conventional brewing, the fermentation is
usually effected in two steps, a main fermentation of a duration of
normally 5 to 12 days and a secondary fermentation--a so-called
maturation process-which may take from up to 12 weeks. During the
main fermentation most of the carbohydrates in the wort are
converted to ethanol and carbon dioxide. Maturation is usually
effected at a low temperature in the presence of a small residual
amount of yeast. The purposes of the maturation are, inter alia, to
precipitate undesirable, high molecular weight compounds and to
convert undesirable compounds to compounds, such as diols, which do
not affect flavor and aroma. For example butanediol, the final
product of the conversion of .alpha.-acetolactate and diacetyl in
beer, is typically reported as a compound with neutral sensory
characteristics. The term "fermentation media" as used herein
refers to a medium comprising carbohydrate containing substrates
which can be fermented by yeast or other microorganisms to produce,
for example, beer or wine or cider or perry or sake. Examples of
fermentation media include: wort, and fruit juices (such as grape
juice, apple juice and pear juice). Example 9 details an example of
suitable wort. The term "maturation media" as used herein refers to
a medium comprising carbohydrate containing substrates which have
been fermented by yeast or other microorganisms to produce, for
example, beer or wine or cider or perry or sake. Examples of
maturation media include partially fermented wort and fruit juices
(such as grape juice, apple juice and pear juice). In some
embodiments, the invention provides a fermentation or maturation
media for an ALDC producing host cell comprising a metal ion at a
concentration of about 1 .mu.M to about 300 .mu.M. In some
embodiments, the invention provides a fermentation or maturation
media for an ALDC producing host cell comprising a metal ion at a
concentration of about 6 .mu.M to about 25 .mu.M.
[0152] In some aspects, the present invention relates to the use of
a composition as described herein in beer and/or wine and/or cider
and/or perry and/or sake fermentation. In some embodiments, the
present invention comprises the use of the ALDC compositions
described herein to decompose acetolactate during beer and/or wine
and/or cider and/or perry and/or sake fermentation or maturation.
Also, the invention comprises the use of ALDC derivative according
to the invention to decompose acetolactate during beer and/or wine
and/or cider and/or perry and/or sake fermentation or
maturation.
[0153] In some embodiments, the methods of the invention are thus
characterized by the treatment of a substrate with a composition
comprising ALDC or an ALDC derivative as described herein during or
in continuation of a fermentation process, e.g., maturation.
[0154] Thus, in some embodiments, acetolactate is enzymatically
decarboxylated to acetoin, the result being that when undesirable,
the formation of diacetyl from acetolactate is avoided. In some
embodiments, other enzymes are used in combination with ALDC for
the conversion of .alpha.-acetolactate. Examples of such enzymes
include, but are not limited to, acetolactate reductoisomerases or
isomerases.
[0155] In some embodiments, the ALDC and/or ALDC derivative
compositions described herein are used together with ordinary yeast
in batch fermentation.
[0156] Instead of using the enzyme in a free state, it may be used
in an immobilized state, the immobilized enzyme being added to the
wort during or in continuation of the fermentation (e.g., during
maturation). The immobilized enzyme may also be maintained in a
column through which the fermenting wort or the beer is passed. The
enzyme may be immobilized separately, or coimmobilized yeast cells
and acetolactate decarboxylase may be used.
[0157] In some embodiments, the ALDC and/or ALDC derivative
compositions described herein are used during beer and/or wine
and/or cider and/or perry and/or sake fermentation or maturation to
reduce the diacetyl levels to below about 1 ppm, or about less than
0.5 ppm, or about less than 0.1 ppm, or about less than 0.05 ppm or
about less than 0.01 ppm, or about less than 0.001 ppm. In some
embodiments, the ALDC and/or ALDC derivative compositions described
herein are used during beer and/or wine and/or cider and/or perry
and/or sake fermentation or maturation to reduce the diacetyl
levels to below 0.1 ppm.
[0158] In some embodiments, the ALDC and/or ALDC derivative
compositions described herein are used during beer and/or wine
and/or cider and/or perry and/or sake fermentation or maturation to
reduce VDK content below 0.1 mg/L, or about less than 0.05 mg/L, or
less than 0.01 mg/L or less than 0.001 mg/L. Total VDK refers to
the amount of Diacetyl plus 2,3-pentanedione. In some embodiments,
the ALDC and/or ALDC derivative compositions described herein are
used during beer and/or wine and/or cider and/or perry and/or sake
fermentation or maturation to reduce Total VDK content below 0.1
mg/L.
[0159] In some embodiments, the ALDC and/or ALDC derivative
compositions described herein are used during beer and/or wine
and/or cider and/or perry and/or sake fermentation or maturation to
reduce the diacetyl levels to below about 0.5 mg/L, or about less
than 0.1 mg/L, or about less than 0.05 mg/L, or about less than
0.01 mg/L or about less than 0.001 mg/L. In some embodiments, the
ALDC and/or ALDC derivative compositions described herein are used
during beer and/or wine and/or cider and/or perry and/or sake
fermentation or maturation to reduce the diacetyl levels to below
0.1 mg/L.
[0160] In some embodiments, the ALDC and/or ALDC derivative
compositions described herein are used during beer and/or wine
and/or cider and/or perry and/or sake fermentation or maturation to
reduce other vicinal diketones (such as 2,3 pentanedione) to below
0.1 mg/L, or about less than 0.05 mg/L, or less than 0.01 mg/L or
less than 0.001 mg/L.
[0161] The processes of the invention can not only be used in
connection with the brewing of beer, but is also suitable for the
production of any suitable alcoholic beverage where a reduction in
diacetyl levels or other vicinal diketones is desirable (e.g. wine,
sake, cider, perry, etc.). In some embodiments, the processes of
the invention can be used in the production of wine where similar
advantages are obtained, in particular a reduction in the
maturation period and a simplification of the process. Of special
interest in this context is the use of acetolactate converting
enzymes in connection with the so-called malo-lactic fermentation.
This process which is affected by microorganisms as species of
Leuconostoc, Lactobacillus or Pediococcus is carried out after the
main fermentation of wine in order to increase the pH of the
product as well as its biological stability and to develop the
flavor of the wine. Moreover, it is highly desirable to carry out
the fermentation since it makes possible rapid bottling and thereby
improves the cash-flow of wineries substantially. Unfortunately,
however, the process may give rise to off-flavors due to diacetyl,
the formation of which can be reduced with the aid of acetolactate
converting enzymes.
[0162] Thus, in some embodiments, the processes of the invention
provide for the production of alcoholic beverages with lower
content of diacetyl, wherein the time required for producing the
alcoholic beverages with lower content of diacetyl is reduced by at
least 10%, or at least 20% or at least 30%, or at least 40%, or at
least 50%, or at least 60%, or at least 70%, or at least 80%, or at
least 90% when compared to a process without the use of the ALDC
and/or ALDC derivative compositions described herein. In some
embodiments, the processes of the invention provide for the
production of alcoholic beverages with lower content of diacetyl
when compared to a process without the use of the ALDC and/or ALDC
derivative compositions described herein, wherein a maturation step
is completely eliminated.
[0163] In some embodiments, the ALDC and/or ALDC derivative
compositions described herein are used during a fermentation
process (e.g. beer and/or wine and/or cider and/or perry and/or
sake fermentation), such that the time required for the
fermentation process is reduced by at least 10%, or at least 20% or
at least 30%, or at least 40%, or at least 50%, or at least 60%, or
at least 70%, or at least 80%, or at least 90%, when compared to a
process without the use of the ALDC and/or ALDC derivative
compositions described herein. In some embodiments, the processes
of the invention provide for the production of alcoholic beverages
with lower content of diacetyl when compared to a process without
the use of the ALDC and/or ALDC derivative compositions described
herein, wherein a maturation step is completely eliminated.
[0164] In some embodiments, the ALDC and/or ALDC derivative
compositions described herein are used during a maturation or
conditioning process (e.g. beer maturation/conditioning), such that
the time required for the maturation or conditioning process is
reduced by at least 10%, or at least 20% or at least 30%, or at
least 40%, or at least 50%, or at least 60%, or at least 70%, or at
least 80%, or at least 90%, when compared to a process without the
use of the ALDC and/or ALDC derivative compositions described
herein. In some embodiments, the processes of the invention provide
for the production of alcoholic beverages with lower content of
diacetyl when compared to a process without the use of the ALDC
and/or ALDC derivative compositions described herein, wherein a
maturation step is completely eliminated.
[0165] Further, in some embodiments, the processes described herein
can be used to advantage for industrial preparation of ethanol as
fermentation products are obtained without or practically without
any content of diacetyl, which simplifies the distillation process,
especially in case of azeotropic for the preparation of absolute
ethanol, i.e. pure anhydrous ethanol.
[0166] In some embodiments, the invention provides methods for beer
and/or wine and/or cider and/or perry and/or sake production
comprising adding a composition comprising an ALDC enzyme and metal
ion to a media (such as a fermentation and/or a maturation media)
for the beer and/or wine and/or cider and/or perry and/or sake so
that the metal ion is present in said media at a concentration of
about 0.1 .mu.M to about 500 .mu.M, or about 0.1 .mu.M to about 300
.mu.M, or about 0.1 .mu.M to about 50 .mu.M, or about 1 .mu.M to
about 500 .mu.M, or about 1 .mu.M to about 300 .mu.M, or about 6
.mu.M to about 300 .mu.M, or about 1 .mu.M to about 100 .mu.M, or
about 1 .mu.M to about 50 .mu.M, or about 6 .mu.M to about 50
.mu.M, or about 6 .mu.M to about 25 .mu.M, or about 10 .mu.M to
about 150 mM, or about 20 .mu.M to about 120 mM, or about 25 .mu.M
to about 100 mM, or about 25 .mu.M to about 50 mM, or about 25
.mu.M to about 20 mM, or about 25 .mu.M to about 50 .mu.M, or about
100 .mu.M to about 20 mM, or about 250 .mu.M to about 20 mM, or
about 1 mM to about 20 mM, or about 1 mM to about 5 mM. In some
embodiments, the metal ion is selected from the group consisting of
Zn.sup.2+, Mg.sup.2+, Mn.sup.2+, Co.sup.2+, Cu.sup.2+, Ba.sup.2+,
Ca.sup.2+ and Fe.sup.2+ and combinations thereof. In some
embodiments, the metal ion is selected from the group consisting of
Zn.sup.2+, Cu.sup.2+, and Fe.sup.2+. In some embodiments, the metal
ion is selected from the group consisting of Zn.sup.2+, Mn.sup.2+,
and Co.sup.2+. In some embodiments, the metal ion is Zn.sup.2+ or
Mn.sup.2+. In some embodiments, the metal ion is Zn.sup.2+. In some
embodiments, the invention provides methods for beer and/or wine
and/or cider and/or perry and/or sake production comprising adding
a composition comprising an ALDC enzyme and zinc to a media (such
as a fermentation and/or a maturation media) for the beer and/or
wine and/or cider and/or perry and/or sake wherein said zinc is
present in the composition at a concentration of about 0.25 mM to
about 200 mM, such as about 1 mM to about 20 mM, or about 1 mM to
about 5 mM. In some embodiments, the invention provides methods for
beer and/or wine and/or cider and/or perry and/or sake production
comprising adding a composition comprising an ALDC enzyme and zinc
to a media (such as a fermentation and/or a maturation media) for
the beer and/or wine and/or cider and/or perry and/or sake, wherein
the molar ratio of zinc to ALDC enzyme that is higher than 1 such
as 2:1, or 3:1, or 5:1, or 10:1, or 20:1 or 30:1, or 50:1, or 60:1.
In some embodiments, the ALDC enzyme and said zinc are added during
a fermentation process and/or a maturation process. In some
embodiments, the ALDC enzyme is added at a concentration of about
0.01 g to about 10 g, or about 0.5 g to about 10 g, or about 1 g to
about 5 g per hectoliter of beer and/or wine and/or cider and/or
perry and/or sake ferment. As used herein the term "beer and/or
wine and/or cider and/or perry and/or sake ferment" may be
interchangeable with the term media. In some embodiments, the
activity of said ALDC enzyme is in the range of 1000 to 2500 Units
per mg of protein.
[0167] In some embodiments, the invention provides methods for beer
and/or wine and/or cider and/or perry and/or sake production
comprising adding a composition comprising an ALDC enzyme and metal
ion to a media (such as a fermentation and/or a maturation media)
for the beer and/or wine and/or cider and/or perry and/or sake,
wherein the metal ion is present in said composition at a
concentration of about 1 .mu.M to about 200 mM, or about 100 .mu.M
to about 200 mM, and the composition comprising the ALDC enzyme and
the metal ion are added at a concentration of about 0.01 g to about
10 g per hectoliter of beer and/or wine and/or cider and/or perry
and/or sake ferment. In some embodiments, the invention provides
methods for beer and/or wine and/or cider and/or perry and/or sake
production comprising adding a composition comprising ALDC enzyme
and metal ion to a media (such as a fermentation and/or a
maturation media) for the beer and/or wine and/or cider and/or
perry and/or sake, wherein the metal ion is present in said
composition at a concentration of about 1 .mu.M to about 200 mM, or
about 100 .mu.M to about 200 mM, and the composition comprising the
ALDC enzyme and the metal ion are added at a concentration of about
0.5 g to about 10 g per hectoliter of beer and/or wine and/or cider
and/or perry and/or sake ferment. In some embodiments, the
invention provides methods for beer and/or wine and/or cider and/or
perry and/or sake production comprising adding a composition
comprising an ALDC enzyme and metal ion to a media (such as a
fermentation and/or a maturation media) for the beer and/or wine
and/or cider and/or perry and/or sake, wherein the metal ion is
present in said composition at a concentration of about 1 .mu.M to
about 200 mM or about 100 .mu.M to about 200 mM, and the
composition comprising the ALDC enzyme and the metal ion are added
at a concentration of about 1 g to about 5 g per hectoliter of beer
and/or wine and/or cider and/or perry and/or sake ferment. In some
embodiments, the invention provides methods for beer and/or wine
and/or cider and/or perry and/or sake production comprising adding
a composition comprising an ALDC enzyme and metal ion to a media
(such as a fermentation and/or a maturation media) for the beer
and/or wine and/or cider and/or perry and/or sake, wherein the
metal ion is present in said composition at a concentration of
about 1 .mu.M to about 200 mM, or about 100 .mu.M to about 200 mM,
and the composition comprising the ALDC enzyme and the metal ion
are added at a concentration of about 1 g to about 2 g per
hectoliter of beer and/or wine and/or cider and/or perry and/or
sake ferment. In some embodiments the metal ion is present in the
composition at a concentration of about 1 mM to about 20 mM, or
about 1 mM to about 10 mM, or about 1 mM to about 5 mM. In some
embodiments, the metal ion is selected from the group consisting of
Zn.sup.2+, Mg.sup.2+, Mn.sup.2+, Co.sup.2+, Cu.sup.2+, Ba.sup.2+,
Ca.sup.2+ and Fe.sup.2+ and combinations thereof. In some
embodiments, the metal ion is selected from the group consisting of
Zn.sup.2+, Cu.sup.2+, and Fe.sup.2+. In some embodiments, the metal
ion is selected from the group consisting of Zn.sup.2+, Mn.sup.2+,
and Co.sup.2+. In some embodiments, the metal ion is Zn.sup.2+ or
Mn.sup.2+. In some embodiments, the metal ion is Zn.sup.2+. In some
embodiments, the activity of said ALDC enzyme is in the range of
950 to 2500 Units per mg of protein or 1000 to 2500 Units per mg of
protein or 1500 to 2500 Units per mg of protein.
[0168] In some embodiments, the invention provides methods for beer
and/or wine and/or cider and/or perry and/or sake production
comprising adding an ALDC enzyme and metal ion in a composition to
a media (such as a fermentation and/or a maturation media) for the
beer and/or wine and/or cider and/or perry and/or sake, wherein the
molar ratio of the metal ion to the ALDC enzyme is higher than 1,
and the composition comprising the ALDC enzyme and the metal ion
are added at a concentration of about 0.01 g to about 10 g per
hectoliter of beer and/or wine and/or cider and/or perry and/or
sake ferment. In some embodiments, the invention provides methods
for beer and/or wine and/or cider and/or perry and/or sake
production comprising adding an ALDC enzyme and metal ion in a
composition to a media (such as a fermentation and/or a maturation
media) for the beer and/or wine and/or cider and/or perry and/or
sake, wherein the molar ratio of the metal ion to the ALDC enzyme
is higher than 1, and the composition comprising the ALDC enzyme
and the metal ion are added at a concentration of about 0.5 g to
about 10 g per hectoliter of beer and/or wine and/or cider and/or
perry and/or sake ferment. In some embodiments, the invention
provides methods for beer and/or wine and/or cider and/or perry
and/or sake production comprising adding an ALDC enzyme and metal
ion in a composition to a media (such as a fermentation and/or a
maturation media) for the beer and/or wine and/or cider and/or
perry and/or sake, wherein the molar ratio of the metal ion to the
ALDC enzyme is higher than 1, and the composition comprising the
ALDC enzyme and the metal ion are added at a concentration of about
1 g to about 5 g per hectoliter of beer and/or wine and/or cider
and/or perry and/or sake ferment. In some embodiments, the
invention provides methods for beer and/or wine and/or cider and/or
perry and/or sake production comprising adding an ALDC enzyme and
metal ion in a composition to a media (such as a fermentation
and/or a maturation media) for the beer and/or wine and/or cider
and/or perry and/or sake, wherein the molar ratio of the metal ion
to the ALDC enzyme is higher than 1, and the composition comprising
the ALDC enzyme and the metal ion are added at a concentration of
about 1 g to about 2 g per hectoliter of beer and/or wine and/or
cider and/or perry and/or sake ferment. In some embodiments, the
molar ratio of the metal ion to the ALDC enzyme is 2:1, or 3:1, or
5:1, or 10:1, or 20:1 or 30:1, or 50:1, or 60:1, or higher. In some
embodiments, the metal ion is selected from the group consisting of
Zn.sup.2+, Mg.sup.2+, Mn.sup.2+, Co.sup.2+, Cu.sup.2+, Ba.sup.2+,
Ca.sup.2+ and Fe.sup.2+ and combinations thereof. In some
embodiments, the metal ion is selected from the group consisting of
Zn.sup.2+, Cu.sup.2+, and Fe.sup.2+. In some embodiments, the metal
ion is selected from the group consisting of Zn.sup.2+, Mn.sup.2+,
and Co.sup.2+. In some embodiments, the metal ion is Zn.sup.2+ or
Mn.sup.2+. In some embodiments, the metal ion is Zn.sup.2+. In some
embodiments, the activity of said ALDC enzyme is in the range of
950 to 2500 Units per mg of protein or 1000 to 2500 Units per mg of
protein or 1500 to 2500 Units per mg of protein.
[0169] Production of ALDC Enzymes
[0170] In one aspect, the description relates to a nucleic acid
capable of encoding an ALDC enzyme as described herein. In a
further aspect, the description relates to an expression vector or
plasmid comprising such a nucleic acid, or capable of expressing an
ALDC enzyme as described herein. In one aspect, the expression
vector or plasmid comprises a promoter derived from Trichoderma
such as a T. reesei cbh1-derived promoter. In a further aspect, the
expression vector or plasmid comprises a terminator derived from
Trichoderma such as a T. reesei cbh1-derived terminator. In yet a
further aspect, the expression vector or plasmid comprises one or
more selective markers such as Aspergillus nidulans amdS and pyrG.
In another aspect, the expression vector or plasmid comprises one
or more telomere regions allowing for a non-chromosomal plasmid
maintenance in a host cell.
[0171] In one aspect, the description relates to a host cell having
heterologous expression of an ALDC enzyme as herein described. In a
further aspect, the host cell is a fungal cell. In yet a further
aspect, the fungal cell is of the genus Trichoderma. In yet a
further aspect, the fungal cell is of the species Trichoderma
reesei or of the species Hypocrea jecorina. In another aspect, the
host cell comprises, preferably is transformed with, a plasmid or
an expression vector as described herein.
[0172] In some embodiments, the host cell is a bacterial host cell
such as Bacillus. In some embodiments the ALDC enzyme is produced
by cultivation of a Bacillus subtilis strain containing a gene
encoding and expressing an ALDC enzyme (e.g. ALDC of Bacillus
brevis). Examples of such host cells and cultivation thereof are
described in DK149335B.
[0173] Examples of suitable expression and/or integration vectors
are provided in Sambrook et al. (1989) supra, and Ausubel (1987)
supra, and van den Hondel et al. (1991) in Bennett and Lasure
(Eds.) More Gene Manipulations In Fungi, Academic Press pp. 396-428
and U.S. Pat. No. 5,874,276. Reference is also made to the Fungal
Genetics Stock Center Catalogue of Strains (FGSC,
http://www.fgsc.net) for a list of vectors. Particularly useful
vectors include vectors obtained from for example Invitrogen and
Promega. Suitable plasmids for use in bacterial cells include
pBR322 and pUC19 permitting replication in E. coli and pE194 for
example permitting replication in Bacillus. Other specific vectors
suitable for use in E. coli host cells include vectors such as
pFB6, pBR322, pUC18, pUC100, pDONR.TM.201, 10 pDONR.TM.221,
pENTR.TM., pGEM.RTM.3 Z and pGEM.RTM.4 Z.
[0174] Specific vectors suitable for use in fungal cells include
pRAX, a general purpose expression vector useful in Aspergillus,
pRAX with a glaA promoter, and in Hypocrea/Trichoderma includes
pTrex3 g with a cbh1 promoter.
[0175] In some embodiments, the host cells are fungal cells and
optionally filamentous fungal host cells. The term "filamentous
fungi" refers to all filamentous forms of the subdivision
Eumycotina (see, Alexopoulos, C. J. (1962), Introductory Mycology,
Wiley, New York). These fungi are characterized by a vegetative
mycelium with a cell wall composed of chitin, cellulose, and other
complex polysaccharides. The filamentous fungi of the present
disclosure are morphologically, physiologically, and genetically
distinct from yeasts. Vegetative growth by filamentous fungi is by
hyphal elongation and carbon catabolism is obligatory aerobic. In
the present disclosure, the filamentous fungal parent cell may be a
cell of a species of, but not limited to, Trichoderma (e.g.,
Trichoderma reesei, the asexual morph of Hypocrea jecorina,
previously classified as T. longibrachiatum, Trichoderma viride,
Trichoderma koningii, Trichoderma harzianum) (Sheir-Neirs et al.,
Appl. Microbiol. Biotechnol. 20:46-53 (1984); ATCC No. 56765 and
ATCC No. 26921), Penicilliurn sp., Humicola sp. (e.g., H. insolens,
H. lanuginosa and H. grisea), Chrysosporium sp. (e.g., C.
lucknowense), Gliocladium sp., Aspergillus sp. (e.g., A. oryzae, A.
niger, A sojae, A. japonicus, A. nidulans, and A. awamori) (Ward et
al., Appl. Microbiol. Biotechnol. 39:738-743 (1993) and Goedegebuur
et al., Curr. Genet. 41:89-98 (2002)), Fusarium sp., (e.g., F.
roseum, F. graminum, F. cerealis, F. oxysporum, and F. venenatum),
Neurospora sp., (N. crassa), Hypocrea sp., Mucor sp. (M miehei),
Rhizopus sp., and Emericella sp. (see also, Innis et al., Science
228:21-26 (1985)). The term "Trichoderma" or "Trichoderma sp." or
"Trichoderma spp." refer to any fungal genus previously or
currently classified as Trichoderma.
[0176] In some embodiments, the host cells will be gram-positive
bacterial cells. Non-limiting examples include strains of
Streptomyces (e.g., S. lividans, S. coelicolor, and S. griseus) and
Bacillus. As used herein, "the genus Bacillus" includes all species
within the genus "Bacillus," as known to those of skill in the art,
including but not limited to B. subtilis, B. licheniformis, B.
lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B.
amyloliquefaciens, B. clausii, B. halodurans, B. megaterium, B.
coagulans, B. circulans, B. lautus, and B. thuringiensis. It is
recognized that the genus Bacillus continues to undergo taxonomical
reorganization. Thus, it is intended that the genus include species
that have been reclassified, including but not limited to such
organisms as B. stearothermophilus, which is now named "Geobacillus
tearothermophilus."
[0177] In some embodiments, the host cell is a gram-negative
bacterial strain, such as E. coli or Pseudomonas sp. In other
embodiments, the host cells may be yeast cells such as
Saccharomyces sp., Schizosaccharomyces sp., Pichia sp., or Candida
sp. In other embodiments, the host cell will be a genetically
engineered host cell wherein native genes have been inactivated,
for example by deletion in bacterial or fungal cells. Where it is
desired to obtain a fungal host cell having one or more inactivated
genes known methods may be used (e.g., methods disclosed in U.S.
Pat. No. 5,246,853, U.S. Pat. No. 5,475,101, and WO 92/06209). 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). In some
embodiments, when the host cell is a Trichoderma cell and
particularly a T. reesei host cell, the cbh1, cbh2, egl1 and egl2
genes will be inactivated and/or deleted. Exemplary Trichoderma
reesei host cells having quad-deleted proteins are set forth and
described in U.S. Pat. No. 5,847,276 and WO 05/001036. In other
embodiments, the host cell is a protease deficient or protease
minus strain. The term "protease deficient" or a "protease minus
strain" as used herein refers to a host cell derived or derivable
from a parental cell wherein the host cell comprises one or more
genetic alterations that causes the host cells to produce a
decreased amount of one or more proteases (e.g. functional
proteases) when compared to the parental cell; preferably said host
cell is deficient in one or more proteases selected from the group
consisting of WprA, Vpr, Epr, IspA, Bpr, NprE, AprE, ampS, aprX,
bpf, clpCP, clpEP, clpXP, codWX, lonA, lonB, nprB, map, mlpA, mpr,
pepT, pepF, dppA, yqyE, tepA, yfiT, yflG, ymfF, ypwA, yrrN, yrrO,
and ywaD. a variant host cell derived from a parental cell is
provided, the variant host cell comprises one or more genetic
alterations that causes cells of the variant strain to produce a
decreased amount of one or more proteases when compared to the
parental cell.
[0178] 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., Ausubel et al. (1987) supra, chapter 9; and Sambrook et
al. (1989) supra, and Campbell et al., Curr. Genet. 16:53-56
(1989)).
[0179] Transformation methods for Bacillus are disclosed in
numerous references including Anagnostopoulos C. and J. Spizizen,
J. Bacteriol. 81:741-746 (1961) and WO 02/14490. Transformation
methods for Aspergillus are described in Yelton et al., Proc. Natl.
Acad. Sci. USA 81:1470-1474 (1984); Berka et al., (1991) in
Applications of Enzyme Biotechnology, Eds. Kelly and Baldwin,
Plenum Press (NY); Cao et al., Protein Sci. 9:991-1001 (2000);
Campbell et al., Curr. Genet. 16:53-56 (1989), and EP 238 023. The
expression of heterologous protein in Trichoderma is described in
U.S. Pat. No. 6,022,725; U.S. Pat. No. 6,268,328; Harkki et al.
Enzyme Microb. Technol. 13:227-233 (1991); Harkki et al.,
BioTechnol. 7:596-603 (1989); EP 244,234; EP 215,594; and
Nevalainen et al., "The Molecular Biology of Trichoderma and its
Application to the Expression of Both Homologous and Heterologous
Genes", in Molecular Industrial Mycology, Eds. Leong and Berka,
Marcel Dekker Inc., NY (1992) pp. 129-148). Reference is also made
to WO96/00787 and Bajar et al., Proc. Natl. Acad. Sci. USA
88:8202-8212 (1991) for transformation of Fusarium strains.
[0180] In one aspect, the description relates to a method of
isolating an ALDC enzyme as defined herein, the method comprising
the steps of inducing synthesis of the ALDC enzyme in a host cell
as defined herein having heterologous expression of said ALDC
enzyme and recovering extracellular protein secreted by said host
cell, and optionally purifying the ALDC enzyme. In a further
aspect, the description relates to a method for producing an ALDC
enzyme as defined herein, the method comprising the steps of
inducing synthesis of the ALDC enzyme in a host cell as defined
herein having heterologous expression of said ALDC enzyme, and
optionally purifying the ALDC enzyme. In a further aspect, the
description relates to a method of expressing an ALDC enzyme as
defined herein, the method comprising obtaining a host cell as
defined herein, or any suitable host cells as known by a person of
ordinary skill in the art, and expressing the ALDC enzyme from said
host cell, and optionally purifying the ALDC enzyme. In another
aspect, the ALDC enzyme as defined herein is the dominant secreted
protein. In some embodiments, metal ions such as Zn.sup.2+,
Mg.sup.2+, Mn.sup.2+, Co.sup.2+, Cu.sup.2+, Ba.sup.2+, Ca.sup.2+
and Fe.sup.2+ and combinations thereof are added to the media (such
as a cultivation and/or a fermentation and/or a maturation media)
during and/or after ALDC production to increase the recovered
yields from microorganisms.
[0181] In some embodiments, the invention provides a cultivation
media for an ALDC producing host cell comprising a metal ion at a
concentration of about 1 .mu.M to about 1 mM. In some embodiments,
the invention provides a cultivation media for an ALDC producing
host cell comprising a metal ion at a concentration of about 25
.mu.M to about 150 .mu.M. In some embodiments, the invention
provides a cultivation media for an ALDC producing host cell
comprising a metal ion at a concentration of about 25 .mu.M to
about 50 .mu.M. In some embodiments, the invention provides a
cultivation media for an ALDC producing host cell comprising a
metal ion at a concentration of about 30 .mu.M to about 40 .mu.M.
In some embodiments, the invention provides a cultivation media for
an ALDC producing host cell comprising a metal ion at a
concentration of about 40 .mu.M to about 150 .mu.M. In some
embodiments, the invention provides a cultivation media for an ALDC
producing host cell comprising a metal ion at a concentration of
about 60 .mu.M to about 150 .mu.M. In some embodiments, the metal
ion is selected from the group consisting of Zn.sup.2+, Mg.sup.2+,
Mn.sup.2+, Co.sup.2+, Cu.sup.2+, and Fe.sup.2+ and combinations
thereof. In some embodiments, the metal ion is selected from the
group consisting of Zn.sup.2+, Cu.sup.2+, and Fe.sup.2+. In some
embodiments, the metal ion is selected from the group consisting of
Zn.sup.2+, Mn.sup.2+, and Co.sup.2+. In some embodiments, the metal
ion is Zn.sup.2+or Mn.sup.2+. In some embodiments, the metal ion is
Zn.sup.2+. In some embodiments, the activity of said ALDC enzyme is
in the range of 950 to 2500 Units per mg of protein or 1000 to 2500
Units per mg of protein or 1500 to 2500 Units per mg of protein.
The term "ALDC producing host cell" as used herein refers to a host
cell capable of expressing ALDC enzyme when said host cell is
cultured under conditions permitting the expression of the nucleic
acid sequence encoding ALDC. The nucleic acid sequence encoding
ALDC enzyme may be heterologous or homologous to the host cell. In
some embodiments, the ALDC producing host cell is Bacillus
subtilis. In some embodiments, the ALDC producing host cell is
Bacillus subtilis comprising a gene encoding and expressing ALDC
enzyme wherein the ALDC enzyme comprises an amino acid sequence
having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%
identity with SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO:
7, SEQ ID NO: 8, or any functional fragment thereof. In some
embodiments, the ALDC producing host cell is Bacillus subtilis
comprising a nucleic acid sequence encoding ALDC wherein said
nucleic acid sequence encoding ALDC has at least 70%, 75%, 80%,
85%, 90%, 95%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 1,
SEQ ID NO: 4, SEQ ID NO: 6 or any functional fragment thereof. In
some embodiments, the ALDC producing host cell is Bacillus subtilis
comprising a gene encoding ALDC derived from Bacillus brevis.
EXAMPLES
[0182] The present disclosure is described in further detail in the
following examples, which are not in any way intended to limit the
scope of the disclosure as claimed. The attached figures are meant
to be considered as integral parts of the specification and
description of the disclosure. The following examples are offered
to illustrate, but not to limit the claimed disclosure.
Example 1--Heterologous Expression of Acetolactate Decarboxylase,
aldB
[0183] The Brevibacillus brevis (which may be referred to as
Bacillus brevis) acetolactate decarboxylases (ALDC) aldB gene was
previously identified (Diderichsen et al., J Bacteriol. (1990)
172(8): 4315), with the sequence set forth as UNIPROT Accession No.
P23616.1. The sequence of this gene, aldB, is depicted in SEQ ID
NO:1. The nucleotides highlighted in bold and underlined are the
nucleotides which encode the signal peptide.
TABLE-US-00001 sets forth the nucleotide sequence of the aldB gene:
SEQ ID NO: 1 atgaaaaaaaatatcatcacttctatcacatctctggctctggttgccgg
gctgtctttgactgcttttgcagctacaacggctactgtaccagcaccac
ctgccaagcaggaatccaaacctgcggttgccgctaatccggcaccaaaa
aatgtactgtttcaatactcaacgatcaatgcactcatgcttggacagtt
tgaaggggacttgactttgaaagacctgaagctgcgaggcgatatggggc
ttggtaccatcaatgatctcgatggagagatgattcagatgggtacaaaa
ttctaccagatcgacagcaccggaaaattatcggagctgccagaaagtgt
gaaaactccatttgcggttactacacatttcgagccgaaagaaaaaacta
cattaaccaatgtgcaagattacaatcaattaacaaaaatgcttgaggag
aaatttgaaaacaagaacgtcttttatgccgtaaagctgaccggtacctt
taagatggtaaaggctagaacagttccaaaacaaaccagaccttatccgc
agctgactgaagtaaccaaaaaacaatccgagtttgaatttaaaaatgtt
aagggaaccctgattggcttctatacgccaaattatgcagcagccctgaa
tgttcccggattccatctccacttcatcacagaggataaaacaagtggcg
gacacgtattaaatctgcaatttgacaacgcgaatctggaaatttctccg
atccatgagtttgatgtacaattgccgcacacagatgattttgcccactc
tgatctgacacaagttactactagccaagtacaccaagctgagtcagaaa gaaaataa sets
forth an example of a nucleotide sequence encoding an ace tolactate
decarboxylase-the nucleotides highlighted in bold and underlined
are the nucleotides whichencode the signal peptide: SEQ ID NO: 6
atgaaaaaaaatatcatcacttctatcacatctctggctctcgttgccgg
gctgctttgactgcttttgcagctacaacggctactgtaccagcaccacc
tgccaagcaggaatccaaacctgtggttgccgctaatccggcaccaaaaa
atgtactgtttcaatactcaacgatcaatgcactcatgcttggacagttt
gaaggggacttgactttgaaagacctgaagctacgaggcgatatggggct
tggtaccatcaatgatctcgatggagagatgattcagatgggtacaaaat
tctaccagatcgacagcaccggaaaattatccgagctgccagaaagtgtg
aaaactccatttgcggttactacacatttcgagccgaaagaaaaaactac
attaaccaatgtgcaagattacaatcaattaacaaaaatgcttgaggaga
aatttgaaaacaagaacgtcttttatgccgtaaagctgaccggtaccttt
aagatggtaaaggctagaacagttccaaaacaaaccagaccttatccgca
gctgactgaagtaaccaaaaaacaatccgagtttgaatttaaaaatgtta
agggaaccctgattggcttctatacgccaaattatgcagcagccctgaat
gttcccggattccatctccacttcatcacagaggataaaacaagtggcgg
acacgtattaaatctgcaatttgacaacgcgaatctggaaatttctccga
tccatgagtttgatgtacaattgccgcacacagatgattttgcccactct
gatctgacacaagttactactagccaagtacaccaagctgagtcagaaag aaaataa
[0184] The proenzyme encoded by the aldB gene is depicted in SEQ ID
NO: 2. At the N-terminus, the protein has a signal peptide with a
length of 24 amino acids as predicted by SignalP-NN (Emanuelsson et
al., Nature Protocols (2007) 2: 953-971). This signal peptide
sequence is underlined and is in bold in SEQ ID NO:2. The presence
of a signal peptide indicates that this acetolactate decarboxylase,
aldB is a secreted enzyme. The sequence of the predicted, fully
processed mature chain (aldB, 261 amino acids) is depicted in SEQ
ID NO: 3.
TABLE-US-00002 sets forth the amino acid sequence of the aceto-
lactate decarboxylase (ALDC) precursor aldB: SEQ ID NO: 2
MKKNIITSITSLALVAGLSLTAFAATTATVPAPPAKQESKPAVAANPAP
KNVLFQYSTINALMLGQFEGDLTLKDLKLRGDMGLGTINDLDGEMIQMG
TKFYQIDSTGKLSELPESVKTPFAVTTHFEPKEKTTLTNVQDYNQLTKM
LEEKFENKNVFYAVKLTGTFKMVKARTVPKQTRPYPQLTEVTKKQSEFE
FKNVKGTLIGFYTPNYAAALNVPGFHLHFITEDKTSGGHVLNLQFDNAN
LEISPIHEFDVQLPHTDDFAHSDLTQVTTSQVHQAESERK sets forth an example of
an amino acid sequence of the acetolactate decarboxylase (ALDC)
precursor aldB-the signal peptide sequence is underlined and is in
bold: SEQ ID NO: 7
MKKNIITSITSLALVAGLSLTAFAATTATVPAPPAKQESKPVVAANPAPK
NVLFQYSTINALMLGQFEGDLTLKDLKLRGDMGLGTINDLDGEMIQMGTK
FYQIDSTGKLSELPESVKTPFAVTTHFEPKEKTTLTNVQDYNQLTKMLEE
KFENKNVFYAVKLTGTFKMVKARTVPKQTRPYPQLTEVTKKQSEFEFKNV
KGTLIGFYTPNYAAALNVPGFHLHFITEDKTSGGHVLNLQFDNANLEISP
IHEFDVQLPHTDDFAHSDLTQVTTSQVHQAESERK sets forth the predicted amino
acid sequence of the mature acetolactate decarboxylase (ALDC) aldB
(261 amino acids): SEQ ID NO: 3
ATTATVPAPPAKQESKPAVAANPAPKNVLFQYSTINALMLGQFEGDLTLK
DLKLRGDMGLGTINDLDGEMIQMGTKFYQIDSTGKLSELPESVKTPFAVT
THFEPKEKTTLTNVQDYNQLTKMLEEKFENKNVFYAVKLTGTFKMVKART
VPKQTRPYPQLTEVTKKQSEFEFKNVKGTLIGFYTPNYAAALNVPGFHLH
FITEDKTSGGHVLNLQFDNANLEISPIHEFDVQLPHTDDFAHSDLTQVTT SQVHQAESERK sets
forth an example of the predicted amino acid sequence of the mature
acetolactate decarboxylase (ALDO aldB (261 amino acids): SEQ ID NO:
8 ATTATVPAPPAKQESKPVVAANPAPKNVLFQYSTINALMLGQFEGDLTLK
DLKLRGDMGLGTINDLDGEMIQMGTKFYQIDSTGKLSELPESVKTPFAVT
THFEPKEKTTLTNVQDYNQLTKMLEEKFENKNVFYAVKLTGTFKMVKART
VPKQTRPYPQLTEVTKKQSEFEFKNVKGTLIGFYTPNYAAALNVPGFHLH
FITEDKTSGGHVLNLQFDNANLEISPIHEFDVQLPHTDDFAHSDLTQVTT SQVHQAESERK
[0185] The aldB gene from the strain Brevibacillus brevis encoding
an acetolactate decarboxylases enzyme (ALDC) was produced in B.
subtilis using an integrated expression cassette consisting of the
B. subtilis aprE promoter, the B. subtilis aprE signal peptide
sequence, the mature aldB and a BPN' terminator. This cassette was
cloned in the head to head orientation with respect to the
expression cassette and the aldB expression cassette introduced
into B. subtilis by homologous recombination.
[0186] A map of the vector containing the aldB gene (RIHI-aldB) is
shown in FIG. 1. The nucleotide mature sequence of the aldB gene in
plasmid RIHI-aldB is depicted in SEQ ID NO:4.
TABLE-US-00003 gctacaacggctactgtaccagcaccacctgccaagcaggaatccaaacc
tgcggttgccgctaatccggcaccaaaaaatgtactgtttcaatactcaa
cgatcaatgcactcatgcttggacagtttgaaggggacttgactttgaaa
gacctgaagctgcgaggcgatatggggcttggtaccatcaatgatctcga
tggagagatgattcagatgggtacaaaattctaccagatcgacagcaccg
gaaaattatcggagctgccagaaagtgtgaaaactccatttgeggttact
acacatttcgagccgaaagaaaaaactacattaaccaatgtgcaagatta
caatcaattaacaaaaatgcttgaggagaaatttgaaaacaagaacgtct
tttatgccgtaaagctgaccggtacttttaagatggtaaaggctagaaca
gttccaaaacaaaccagaccttatccgcagctgactgaagtaaccaaaaa
acaatccgagtttgaatttaaaaatgttaagggaaccctgattggcttct
atacgccaaattatgcagcagccctgaatgttcccggattccatctccac
ttcatcacagaggataaaacaagtggcggacacgtattaaatctgcaatt
tgacaacgcgaatctggaaatttctccgatccatgagtttgatgttcaat
tgccgcacacagatgattttgcccactctgatctgacacaagttactact
agccaagtacaccaagctgagtcagaaagaaaa
[0187] The amino acid sequence of the aldB precursor protein
expressed from plasmid RIHI-aldB is depicted in SEQ ID NO:5.
TABLE-US-00004 ATTATVPAPPAKQESKPAV
AANPAPKNVLFQYSTINALMLGQFEGDLTLKDLKLRGDMGLGTINDLDG
EMIQMGTKFYQIDSTGKLSELPESVKTPFAVTTHFEPKEKTTLTNVQDY
NQLTKMLEEKFENKNVFYAVKLTGTFKMVKARTVPKQTRPYPQLTEVTK
KQSEFEFKNVKGTLIGFYTPNYAAALNVPGFHLHFITEDKTSGGHVLNL
QFDNANLEISPIHEFDVQLPHTDDFAHSDLTQVTTSQVHQAESERK
The signal peptide sequence is in bold lowercase italics in SEQ ID
NO:5.
Small Scale Culture Conditions
[0188] To produce aldB, a B. subtilis strain transformant
containing aldB expression cassette was cultured in 15 mL Falcon
tubes for 5 hours in TSB (broth) with 300 ppm beta-chloro-D-alanine
(CDA), and 300 .mu.L of this pre-culture was added to a 500 mL
flask filled with 30 mL of cultivation media (described below)
supplemented with 300 ppm CDA and 50 .mu.M Zn.sup.2+. The flasks
were incubated for 24, 48 and 72 hours at 33.degree. C. with
constant rotational mixing at 180 rpm. Cultures were harvested by
centrifugation at 14500 rpm for 20 minutes in conical tubes. The
culture supernatants were used for protein determination and
assays. The cultivation media was an enriched semi-defined media
based on MOPs buffer, with urea as major nitrogen source, maltrin
as the main carbon source.
Fed-Batch Fermentation Conditions
[0189] To produce aldB, a B. subtilis strain transformant
containing aldB expression cassette was cultured in a 250 mL flasks
containing 30 mL of complex medium with 300 ppm
beta-chloro-D-alanine (CDA). The flask was incubated for 6 hours at
37.degree. C. with constant rotational mixing at 180 rpm.
[0190] The culture was transferred to a stirred fermentor
containing 7 liters of sterilized media components as described in
table 1 below. Temperature was controlled to 37.degree. C.; pH was
controlled to 7,5 using ammonium hydroxide as alkaline titrant;
dissolved oxygen was maintained at 40% or higher by maintaining an
airflow of 7 liter/min, a constant overpressure of 1 bar and
adjusting stirring rate. When initial glucose was exhausted a
feeding profile feeding a 60% glucose solution into the fermentor
was initiated (initial feeding rate was 20 g/h linearly increasing
to 32.8 g/h over 7 hours and kept constant at that rate until
fermentation termination).
[0191] Total fermentation time was 44 hours.
TABLE-US-00005 TABLE 1 Media recipe for ALDC fermentation Recipe
Conc (g/kg Component broth) Soy Meal 50.0 Citric acid 0.10
Magnesium sulfate heptahydrate 2.29 Potassium Phosphate, Mono Basic
5.44 Ferrous sulfate, heptahydrate 0.029 Manganese Sulfate Mono
hydrate 0.051 Zinc sulphate heptahydrate 0.001 Glucose mono hydrate
1.10 Anti foam agent 3.00
Example 2--Protein Determination Methods
Protein Determination by Stain Free Imager Criterion
[0192] Protein was quantified by SDS-PAGE gel and densitometry
using Gel Doc.TM. EZ imaging system. Reagents used in the assay:
Concentrated (2.times.) Laemmli Sample Buffer (Bio-Rad, Catalogue
#161-0737); 26-well XT 4-12% Bis-Tris Gel (Bio-Rad, Catalogue
#345-0125); protein markers "Precision Plus Protein Standards"
(Bio-Rad, Catalogue #161-0363); protein standard BSA (Thermo
Scientific, Catalogue #23208) and SimplyBlue Safestain (Invitrogen,
Catalogue #LC 6060. The assay was carried out as follow: In a
96well-PCR plate 504, diluted enzyme sample were mixed with 50
.mu.L sample buffer containing 2.7 mg DTT. The plate was sealed by
Microseal `B` Film from Bio-Rad and was placed into PCR machine to
be heated to 70.degree. C. for 10 minutes. After that the chamber
was filled by running buffer, gel cassette was set. Then 10 .mu.L
of each sample and standard (0.125-1.00 mg/mL BSA) was loaded on
the gel and 5 .mu.L of the markers were loaded. After that the
electrophoresis was run at 200 V for 45 min. Following
electrophoresis the gel was rinsed 3 times 5 min in water, then
stained in Safestain overnight and finally destained in water. Then
the gel was transferred to Imager. Image Lab software was used for
calculation of intensity of each band. By knowing the protein
amount of the standard sample, the calibration curve can be made.
The amount of sample can be determined by the band intensity and
calibration curve. The protein quantification method was employed
to prepare samples of aldB acetolactate decarboxylases enzyme used
for assays shown in subsequent Examples.
Example 3--Activity Assay Method
Spectrophotometric Assay of .alpha.-Acetolactate Decarboxylase
[0193] .alpha.-Acetolactate decarboxylase (ALDC) catalyses the
decarboxylation of .alpha.-acetolactate to acetoin. The reaction
product acetoin can be quantified colourimetrically. Acetoin mixed
with .alpha.-naphtol and creatine forms a characteristic red color
absorbing at OD522 nm. ALDC activity was calculated based on
OD.sub.522 nm and an acetoin calibration curve. The assay was
carried out as follows: 20 mM acetolactate substrate was prepared
by mixing 100 .mu.L ethyl-2-acetoxy-2-methylacetoacetate (Sigma,
Catalogue#220396) with 3.6 mL 0.5 M NaOH at 10.degree. C. for 10
min. 20 mL 50 mM MES pH 6.0 was added, pH was adjusted to pH 6.0
and volume adjusted to 25 mL with 50 mM MES pH 6.0. 80 .mu.L 20 mM
acetolactate substrate was mixed with 20 .mu.L enzyme sample
diluted in 50 mM MES, pH 6.0, 0.6 M NaCl, 0.05% BRIJ 35 and 0.01%
BSA. The substrate/enzyme mixture was incubated at 30.degree. C.
for 10 min. Then 16 .mu.L substrate/enzyme mixture was transferred
to 200 .mu.L 1 M NaOH, 1.0% .alpha.-naphtol (Sigma,
Catalogue#33420) and 0.1% creatine (Sigma, Catalogue# C3630). The
substrate/enzyme/color reagent mixture was incubated at 30.degree.
C. for 20 min and then OD.sub.522 nm was read. One unit of ALDC
activity is defined as the amount of enzyme which produces 1
.mu.mole acetoin per minute under the conditions of the assay.
Example 4--Zinc Influence on Activity of ALDC
The Presence of Zn.sup.2+ During Shake Flask Fermentation
[0194] ZnSO.sub.4 was added to the cultivation media for small
scale fermentations as described in Example 1 at concentrations
ranging from 0 mM to 800 .mu.M. aldB. B. subtilis transformant
containing aldB expression cassette was cultured in 15 mL Falcon
tubes for 5 hours in TSB (broth) with 300 ppm beta-chloro-D-alanine
(CDA), and 300 .mu.L of this pre-culture was added to a each 500 mL
flask filled with 30 mL of cultivation media supplemented with 300
ppm CDA and containing 0, 5, 25, 50, 100, 200, 400 and 800 .mu.M
Zn.sup.2+. The flasks were incubated for 24 and 48 hours at
33.degree. C. with constant rotational mixing at 180 rpm. Cultures
were harvested by centrifugation at 14500 rpm for 20 minutes in
conical tubes. The culture supernatants were used for protein
determination and ALDC activity assays (Table 2 and 3). It is
clearly seen that increasing the concentration of Zn.sup.2+ in the
fermentation media increased the total ALDC activity and specific
activity of expressed aldB enzyme.
TABLE-US-00006 TABLE 2 ALDC activity of aldB fermentation samples
with varying levels of Zn.sup.2+ after 24 and 48 hours of
fermentation ALDC activity ZnSO.sub.4 in 24 hours of 48 hours of
sample fermentation fermentation .mu.M U/mL U/mL aldB 0 298.6 235.3
5 371.9 354.8 25 533.9 808.0 50 606.4 965.3 100 633.4 668.8 200
617.6 629.5 400 691.1 488.5 800 428.1 474.0
TABLE-US-00007 TABLE 3 ALDC activity, aldB protein and specific
activity of aldB fermentation samples with varying levels of
Zn.sup.2+ after 48 hours of fermentation ZnSO.sub.4 in sample ALDC
activity AldB protein Specific activity .mu.M U/mL mg/mL U/mg aldB
0 235.3 0.626 375.9 5 354.8 0.736 482.1 25 808.0 1.437 562.3 50
965.3 1.573 609.9 100 668.8 0.957 698.9
Example 5--Modulation of the Specific Activity of aldB Samples
Zinc Dose Response of ALDC at 50.degree. C.
[0195] ZnSO.sub.4 was added to ALDC fermentation sample to produce
concentrations ranging from 0 mM to 20 mM. The samples were kept at
50.degree. C. for 60 min and then activity was measured as
described in Example 3. Relative to a sample with no zinc added
samples with >0.5 mM zinc had activity >200% higher. FIG. 2
shows results.
Example 6--Modulation of the Specific Activity of aldB Ferment
[0196] AldB activity was produced as described in (Example 1) and
activity determined as described in (Example 3). An AldB ferment
sample was diluted in 50 mM MES pH 6.0, 0.6 M NaCl, 0.05% Brij,
0.01% BSA (bovine serum albumin) to proximately 1000 U/mL and then
incubated with varying concentration of ZnSO.sub.4 in the following
steps: 0, 0.25, 0.5, 1, 2, 5, 7.5, 10, 20, 40, 60, 80, 100, 120 mM.
All samples were incubated at 4.degree. C. for 68 hours, then
centrifuged and supernatant analyzed for activity and protein by
Criterion SDS-PAGE analysis. The results of the SDS-PAGE analysis
is shown in FIG. 3 and the determined activity, protein
concentration and specific activity are given in table 4. It can be
seen that the ALDC activity increases with increasing concentration
of ZnSO.sub.4 in the sample until approximately 5-10 mM from where
it is more or less constant with the higher concentrations of
ZnSO.sub.4. The concentration of aldB protein in the sample was
determined to be more or less constant in all samples indicating an
apparent increase in the specific activity from 968 U/mg in the
sample without ZnSO.sub.4 up to 2200-2400 U/mg in samples with 5 mM
ZnSO.sub.4 or more after incubation.
TABLE-US-00008 TABLE 4 ALDC activity, aldB protein concentration
and specific activity of aldB sample incubated with ZnSO.sub.4 at
4.degree. C. for 68 hours. ZnSO.sub.4 in ALDC AldB Specific sample
activity protein activity mM U/mL mg/mL U/mg aldB 0 556 0.57 968
0.25 875 0.63 1387 0.5 949 0.63 1512 1 1058 0.59 1792 2 1271 0.60
2124 5 1411 0.60 2354 7.5 1485 0.64 2311 10 1520 0.64 2368 20 1492
0.64 2344 40 1499 0.67 2239 60 1495 0.64 2345 80 1500 0.65 2324 100
1607 0.64 2520 120 1520 0.69 2209
Example 7--Purification of aldB
[0197] Production of aldB in B. subtilis was carried out as
described in example (1). Fermentation media was clarified by
centrifugation (4000 rpm at 15 min.) and filtration (VacuCap 90,
0.2 .mu.m). The sample was desalted on PD10 column, prepared as
described by the manufacturer and equilibrated with 20 mM Tris/HCl,
pH 8.0. The eluate was kept on ice afterwards before further
purified by anion exchange chromatography on a Source 15Q XK26/15.
The column was equipped on an AKTA explorer system used for protein
purification and according to the method described by the
manufacturer (GE healthcare, USA--Cat. No. 18-1112-41).
[0198] The column was prepared as described by the manufacturer and
equilibrated with 20 mM Tris/HCl, pH 8.0 (buffer A). The desalted
sample was applied to the column at a flow rate of 5 mL/min and the
column was washed with buffer A. The bound proteins were eluted
with a linear gradient of 0-0.30 M NaCl in 20 mM Tris/HCl, pH 8.0
(7 mL/min, 40 min). During the entire run, fractions of approx.
10-20 mL were collected.
[0199] Fractions from purification were analysed by SDS-PAGE using
Xcell Mini-cell and according to the method described by the
manufacturer (Invitrogen, USA--Cat. No. EI0001) using: Nu-PAGE gel
(Invitrogen, USA--Cat. No. NP0321), MES running buffer (Invitrogen,
USA--Cat. No. NP0002), See Blue standard marker (Invitrogen,
USA--Cat. No. LC5925) and Stained with Coomasie Brilliant-Blue.
[0200] AldB was observed to bind at the Source 15Q column and was
eluted with approximately 30 mM NaCl. AldB fractions were
collected, concentrated (on a 10 kDa UF Vivaspin) and desalted in
25 mM HEPES buffer containing 150 mM NaCl, pH 8.0. The estimated
purity of aldB was above 95% and the result of SDS-page analysis of
the purified aldB is shown in FIG. 4.
Example 8--Modulation of the Specific Activity of Purified aldB
[0201] The purified aldB protein was stripped of divalent ions by
incubation of aldB (approximately 2 mg/mL) with 20 mM EDTA in
0.2.times. assay buffer (50 mM IVIES pH 6.0, 0.6 M NaCl, 0.05%
Brij, 0.01% BSA) at 37.degree. C. overnight. The EDTA treated
material was desalted on a PD10 column prepared as described by the
manufacturer and equilibrated with 50 mM IVIES pH 6.0, 0.6 M NaCl,
0.05% Brij, 0.01% BSA. ALDC activity and the concentration of AldB
protein in the sample before and after EDTA treatment and the
desalting column were determined as described in example (2 and 3)
and the results are shown in table 5.
TABLE-US-00009 TABLE 5 ALDC activity, aldB protein concentration
and specific activity of purified aldB before and after the
EDTA-desalting procedure. ALDC AldB protein Specific Activity
concentration Activity U/mL mg/mL U/mg AldB purified 5187 5.6 928
AldB purified - EDTA treated and 9 1.0 9 desalted
[0202] 50 .mu.L of the EDTA treated and desalted aldB sample was
mixed with 4504, of 0.2.times. assay buffer (50 mM MES pH 6.0, 0.6
M NaCl, 0.05% Brij, 0.01% BSA) with varying ZnSO.sub.4 in the step:
0, 0.05, 0.1, 0.5, 1, 2, 5, 10 and 20 mM respectively. Samples were
incubated at 37.degree. C. overnight. The samples were centrifuged
at 13000 rpm for 10 min and ALDC activity and the concentration of
AldB protein were determined in the supernatant. The results are
shown in table 6. It can be seen that the ALDC activity increases
with increasing concentration of ZnSO.sub.4 during incubation. The
concentration of aldB protein in the sample was determined to be
constant in all samples indicating an increase in the specific
activity from 2 U/mg of aldB in the sample without ZnSO.sub.4 up to
1800 U/mg aldB in samples with 5 mM ZnSO.sub.4 or more after
incubation.
TABLE-US-00010 TABLE 6 ALDC activity, aldB protein concentration
and specific activity of purified aldB before and after incubation
with ZnSO.sub.4. Concentration of Final ZnSO.sub.4 in 0.2X
concentration ALDC aldB protein Specific assay buffer of ZnSO.sub.4
Activity concentration activity mM mM U/mL mg/mL U/mg 0 0 0.1 0.092
2 0.05 0.045 4.4 0.092 48 0.1 0.09 26.1 0.092 283 0.5 0.45 147.1
0.092 1599 1 0.9 157.7 0.092 1714 2 1.8 157.9 0.092 1716 5 4.5
166.4 0.092 1809 10 9 174.6 0.092 1898 20 18 173.5 0.092 1886
[0203] In addition, 204, of the EDTA treated and desalted aldB
sample was mixed with 1804, of 50 mM MES pH 6.0 supplemented with
either 10 mM ZnSO.sub.4, MnSO.sub.4 or CoCl.sub.2 respectively.
Samples were incubated at 37.degree. C. and assayed after 45
minutes, 1 day, 2 days and 3 days. Results are shown in table 7.
The activity increased for incubation with all three divalent ions
in the order Zn.sup.2+, Mn.sup.2+ and Co.sup.2+ compared to the
control (addition of H.sub.2O). After 45 min the activity was shown
to decrease over time. The addition of Zn.sup.2+, Mn.sup.2+ and
Co.sup.2+ all resulted in an increased stability (activity) after 3
days compared to the control with addition of H.sub.2O. The
residual activity measured after 3 days relative to the start were
76.9%, 73.5% and 70.7% upon addition of Zn.sup.2+, Mn.sup.2+ and
Co.sup.2+ compared to 56.0% obtained with H.sub.2O, as shown in
table 7.
TABLE-US-00011 TABLE 7 ALDC activity after incubation at pH 6.0
with MnSO.sub.4, CoCl.sub.2 or ZnSO.sub.4 at various times.
Activity U/mL H.sub.2O Mn.sup.2+ Co.sup.2+ Zn.sup.2+ 45 min 21.8
217.3 101.8 319.7 1 day 18.9 219.6 90.5 288.5 2 days 22.5 216.2
95.2 295.4 3 days 12.2 153.6 74.8 245.7
Example 9--Reduction in Diacetyl and 2,3-Pentanedione During Beer
Fermentation by Use of aldB with Different Specific ALDC
Activity
[0204] The objective of this analysis was to test different
formulation variants of aldB (acetolactate decarboxylase) ability
to reduce development of diacetyl and 2,3-pentanedione (Vicinal
di-ketones, VDK) during a 7 days fermentation at 14.degree. C.
Pure Malt Brew Analysis
[0205] 1100 g Munton's Light Malt Extract (Batch XB 35189, expiry
date 24 May 2014) extract was dissolved in 3000 mL warm tapwater
(45.degree. C.). This slurry was stirred for about 10 min until the
liquid was homogeneous and the pH was adjusted to 5.2 with 2.5 M
sulphuric acid. To the slurry was added 10 pellets of Bitter hops
from Hopfenveredlung, St. Johann: Alpha content of 16.0% (EBC 7.7 0
specific HPLC analysis, 1 Oct. 2013), then split in 500 mL blue-cap
bottles and boiled for 1 hour to ensure protein precipitation and
avoid potential microbial contamination. The filtered malt extract
(wort) was sampled for specific gravity and Free Amino Nitrogen
(FAN) determination. The final wort had an initial Specific Gravity
of 1048 (i.e. 12.degree. Plato). 200 g of the filtered wort were
added to a 500 mL conical flask (Fermenting Vessel; FV), and then
cooled to 13.degree. C. Each conical flask was dosed with 0.5%
W34/70 (Weihenstephan) freshly produced yeast (1.0 g yeast per 200
g wort). The enzymes were dosed on similar ALDC activity (0.03 U/mL
wort, 8 ALDC Units per 200 g wort). The control fermentation vessel
with no enzyme received an amount of deionized water corresponding
to the amount of enzyme sample.
[0206] The wort samples were fermented in 500 mL conical flasks
under standardised laboratory test conditions at 14.degree. C. with
gentle agitation at 150 rpm in an orbital incubator. When weight
loss was less than 0.25 g over 24 hours, fermentation temperature
was decreased to 7.degree. C. Fermentation was stopped after 7 days
in total. 10 mL samples were taken out for diacetyl and
2,3-pentanedione analysis two times a day, preferably with 11 to 14
hours in between; at the end of fermentation only 1 sample per day
was taken. Yeast was allowed to settle before take-out and each
sample was cooled at 10.degree. C. for 10 minutes and then
centrifuged at 4000 rpm for 10 minutes at 8.degree. C. to sediment
any residual yeast. The supernatant was separated from the yeast
sediment and samples for GC analysis were added 0.5 g NaCl per mL
of sample. This slurry was transferred to a headspace vial and
heat-treated at 65.degree. C. for 30 minutes before analysis for
diacetyl and 2,3-pentanedione were carried out by gas
chromatography with mass spectrometric detection (GCMS).
[0207] Analyses were carried out at an Agilent 6890N/5973N GC with
CombiPAL headspace autosampler and MSChemStation acquisition and
analysis software. The samples were equilibrated at 70.degree. C.
for 10 minutes before 500 .mu.l of the gas phase above the sample
was injected onto a J&W 122-0763 DB-1701column (60 m.times.0.25
mmID.times.1 .mu.m). The injection temperature was 260.degree. C.
and the system was operated with a constant helium flow of 2
mL/min. The oven temperature was: 50.degree. C. (2 min),
160.degree. C. (20.degree. C./min), 220.degree. C. (40.degree.
C./min), hold 2 min. MS detection were made with 500 .mu.L at a
split ratio of 5:1 at selected ions. All sample were run in
duplicates and standards were made using tap water with the
addition of diacetyl or 2,3-pentanedione.
[0208] The concentration of a compound is calculated as
Compound ( mg / L ) = R F .times. Area 1000 .times. W s
##EQU00001##
where,
[0209] RF is the response factor of acetic acid
[0210] Area is the GC-area of acetic acid
[0211] W.sub.s is the amount of sample used (in mL)
[0212] The limit of diacetyl quantification was determined to 0.016
mg/L and the limit of 2,3-pentanedione quantification was
determined to 0.012 mg/L.
[0213] To check that addition of ALDC enzymes did not influence the
Real Degree of Fermentation (RDF) and the produced alcohol by
volume: RDF was measured using an Anton Paar (DMA 5000) following
Standard Instruction Brewing, 23.8580-B28 and alcohol by Standard
Instruction Brewing, 23.8580-B28.
[0214] Real degree of fermentation (RDF) value may be calculated
according to the equation below:
R D F ( % ) = ( 1 - R E .degree. P initial ) .times. 100
##EQU00002##
Where: RE=real
extract=(0.1808.degree.P.sub.initial)+(0.8192.times..degree.
P.sub.final), V.sub.initial is the specific gravity of the
standardised worts before fermentation and .degree. P.sub.final is
the specific gravity of the fermented worts expressed in degree
Plato.
[0215] In the present context, Real degree of fermentation (RDF)
was determined from the specific gravity and alcohol
concentration.
[0216] Specific gravity and alcohol concentration was determined on
the fermented samples using a Beer Alcolyzer Plus and a DMA 5000
Density meter (both from Anton Paar, Gratz, Austria). Based on
these measurements, the real degree of fermentation (RDF) value was
calculated according to the equation below:
R D F ( % ) = O E - E ( r ) O E .times. 100 ##EQU00003##
Where: E(r) is the real extract in degree Plato (.degree. P) and OE
is the original extract in .degree. P.
[0217] The ability to reduce development of diacetyl and
2,3-pentanedione (Vicinal di-ketones, VDK) during a 7 days
fermentation at 14.degree. C. was studied by addition of aldB with
different specific ALDC activity (obtained by varying the
ZnSO.sub.4 concentration in the sample) is given in table 8. The
aldB variant constitute: A) a crude preparation of aldB, B) a crude
preparation of aldB including 20 mM ZnSO.sub.4 added to the
clarified broth and C) a crude preparation of aldB including 50
.mu.M ZnSO.sub.4 added to the fermentation media and 20 mM
ZnSO.sub.4 into the clarified broth.
TABLE-US-00012 TABLE 8 ALDC activity, aldB protein concentration
and specific activity of aldB sample A, B and C. ALDC aldB Specific
ALDC activity protein activity U/mL mg/mL U/mg aldB variant A 426
0.463 919 aldB variant B 511 0.463 1103 aldB variant C 719 0.463
1552
[0218] The enzymes variants were dosed on similar ALDC activity
0.03 U/mL wort resulting in a different concentration of aldB
protein in the wort, as seen in table 9. The calculated aldB
protein concentration in the wort was 33.4, 28.0 and 19.9 .mu.g/L
wort with addition of variant A, B and C respectively.
TABLE-US-00013 TABLE 9 ALDC activity and aldB protein in wort with
addition of aldB variant A, B and C. Volume AldB ALDC Amount sample
pre- Activity protein in activity for pre-dilution dilution in wort
wort U/mL g mL U/mL .mu.g/l AldB variant A 426 0.721 50 0.03 33.4
AldB variant B 511 0.604 50 0.03 28.0 AldB variant C 719 0.430 50
0.03 19.9
[0219] The fermentations were conducted and VDK development
analysed as described above. Fermentations with enzyme were
compared to a control without any enzyme added. The development of
VDK is shown in FIG. 5.
[0220] All three aldB variants reduced the VDK development during
fermentation compared to control. The data suggest that the three
variants performed very similar, however with less increase in VDK
during the fermentation period from 20 to 40 hours for variant C
compared to A and B, but this is close to the relative standard
deviation of the analysis (RSD of 7.5%). The relative reduction in
VDK after 40 hours of fermentation was 63.1%, 58.8% and 63.2% for
variant A, B and C respectively. In addition, the fermentation time
required to reach threshold level of 0.1 mg/ml VDK or lower, was
observed to be approximately 112 hours for all three variants.
Thus, the higher specific activity of variant C compared to A or B
may enable similar VDK reduction with less aldB protein.
Example 10--Reduction in Diacetyl and 2,3-Pentanedione by aldB
During Beer Fermentation with Various Concentration of Zinc in the
Wort
[0221] The objective of this analysis was to test reduction of
diacetyl and 2,3-pentanedione (Vicinal di-ketones, VDK) by aldB
during a 4 days fermentation at 14.degree. C. in presence of
various level of ZnSO.sub.4 in the wort.
[0222] The brew analysis setup was as described in example 9. A
stock solution of ZnSO.sub.4 was made in cooled wort (0.686 g in
1000 mL wort) and applied to the wort samples (prepared as
described in example 9) before fermentation resulting in the
following Zn.sup.2+ concentrations 0, 0.1, 0.5, 0.75, 1, 2.5, 5.0
and 20.0 ppm respectively. ALDC enzyme and yeast were subsequently
added to the samples. An ALDC enzymes preparation were dosed
similarly to all samples (0.04 U/mL wort, corresponding to 8 ALDC
units per 200 g of wort). Each conical fermentation flask was dosed
with 0.5% W34/70 (Weihenstephan) freshly produced yeast (1.0 g
yeast per 200 g of wort) and analyses were carried out as described
in example 9. Fermentations with enzyme were compared to a control
without any enzyme added. The development of VDK is shown in FIG.
6.
[0223] All aldB treated samples provided less increase in the VDK
development during fermentation compared to control. Increasing the
concentration of Zn.sup.2+ in the samples resulted in less and less
increase in VDK generation. Especially increasing the concentration
of Zn.sup.2+ to 0.5 ppm or more greatly affected the enzymatic
activity to avoid generation of VDK. Supplementing the wort with 1
ppm Zn.sup.2+ or more ensured a VDK level below the flavor
threshold of 0.1 mg/L throughout the whole fermentation period.
[0224] While preferred embodiments of the present invention have
been shown and described herein, it will be obvious to those
skilled in the art that such embodiments are provided by way of
example only. Numerous variations, changes, and substitutions will
now occur to those skilled in the art without departing from the
invention. It should be understood that various alternatives to the
embodiments of the invention described herein may be employed in
practicing the invention. It is intended that the following claims
define the scope of the invention and that methods and structures
within the scope of these claims and their equivalents be covered
thereby.
Sequence CWU 1
1
81858DNABrevibacillus brevis 1atgaaaaaaa atatcatcac ttctatcaca
tctctggctc tggttgccgg gctgtctttg 60actgcttttg cagctacaac ggctactgta
ccagcaccac ctgccaagca ggaatccaaa 120cctgcggttg ccgctaatcc
ggcaccaaaa aatgtactgt ttcaatactc aacgatcaat 180gcactcatgc
ttggacagtt tgaaggggac ttgactttga aagacctgaa gctgcgaggc
240gatatggggc ttggtaccat caatgatctc gatggagaga tgattcagat
gggtacaaaa 300ttctaccaga tcgacagcac cggaaaatta tcggagctgc
cagaaagtgt gaaaactcca 360tttgcggtta ctacacattt cgagccgaaa
gaaaaaacta cattaaccaa tgtgcaagat 420tacaatcaat taacaaaaat
gcttgaggag aaatttgaaa acaagaacgt cttttatgcc 480gtaaagctga
ccggtacctt taagatggta aaggctagaa cagttccaaa acaaaccaga
540ccttatccgc agctgactga agtaaccaaa aaacaatccg agtttgaatt
taaaaatgtt 600aagggaaccc tgattggctt ctatacgcca aattatgcag
cagccctgaa tgttcccgga 660ttccatctcc acttcatcac agaggataaa
acaagtggcg gacacgtatt aaatctgcaa 720tttgacaacg cgaatctgga
aatttctccg atccatgagt ttgatgtaca attgccgcac 780acagatgatt
ttgcccactc tgatctgaca caagttacta ctagccaagt acaccaagct
840gagtcagaaa gaaaataa 8582285PRTBrevibacillus brevis 2Met Lys Lys
Asn Ile Ile Thr Ser Ile Thr Ser Leu Ala Leu Val Ala 1 5 10 15 Gly
Leu Ser Leu Thr Ala Phe Ala Ala Thr Thr Ala Thr Val Pro Ala 20 25
30 Pro Pro Ala Lys Gln Glu Ser Lys Pro Ala Val Ala Ala Asn Pro Ala
35 40 45 Pro Lys Asn Val Leu Phe Gln Tyr Ser Thr Ile Asn Ala Leu
Met Leu 50 55 60 Gly Gln Phe Glu Gly Asp Leu Thr Leu Lys Asp Leu
Lys Leu Arg Gly 65 70 75 80 Asp Met Gly Leu Gly Thr Ile Asn Asp Leu
Asp Gly Glu Met Ile Gln 85 90 95 Met Gly Thr Lys Phe Tyr Gln Ile
Asp Ser Thr Gly Lys Leu Ser Glu 100 105 110 Leu Pro Glu Ser Val Lys
Thr Pro Phe Ala Val Thr Thr His Phe Glu 115 120 125 Pro Lys Glu Lys
Thr Thr Leu Thr Asn Val Gln Asp Tyr Asn Gln Leu 130 135 140 Thr Lys
Met Leu Glu Glu Lys Phe Glu Asn Lys Asn Val Phe Tyr Ala 145 150 155
160 Val Lys Leu Thr Gly Thr Phe Lys Met Val Lys Ala Arg Thr Val Pro
165 170 175 Lys Gln Thr Arg Pro Tyr Pro Gln Leu Thr Glu Val Thr Lys
Lys Gln 180 185 190 Ser Glu Phe Glu Phe Lys Asn Val Lys Gly Thr Leu
Ile Gly Phe Tyr 195 200 205 Thr Pro Asn Tyr Ala Ala Ala Leu Asn Val
Pro Gly Phe His Leu His 210 215 220 Phe Ile Thr Glu Asp Lys Thr Ser
Gly Gly His Val Leu Asn Leu Gln 225 230 235 240 Phe Asp Asn Ala Asn
Leu Glu Ile Ser Pro Ile His Glu Phe Asp Val 245 250 255 Gln Leu Pro
His Thr Asp Asp Phe Ala His Ser Asp Leu Thr Gln Val 260 265 270 Thr
Thr Ser Gln Val His Gln Ala Glu Ser Glu Arg Lys 275 280 285
3261PRTBrevibacillus brevis 3Ala Thr Thr Ala Thr Val Pro Ala Pro
Pro Ala Lys Gln Glu Ser Lys 1 5 10 15 Pro Ala Val Ala Ala Asn Pro
Ala Pro Lys Asn Val Leu Phe Gln Tyr 20 25 30 Ser Thr Ile Asn Ala
Leu Met Leu Gly Gln Phe Glu Gly Asp Leu Thr 35 40 45 Leu Lys Asp
Leu Lys Leu Arg Gly Asp Met Gly Leu Gly Thr Ile Asn 50 55 60 Asp
Leu Asp Gly Glu Met Ile Gln Met Gly Thr Lys Phe Tyr Gln Ile 65 70
75 80 Asp Ser Thr Gly Lys Leu Ser Glu Leu Pro Glu Ser Val Lys Thr
Pro 85 90 95 Phe Ala Val Thr Thr His Phe Glu Pro Lys Glu Lys Thr
Thr Leu Thr 100 105 110 Asn Val Gln Asp Tyr Asn Gln Leu Thr Lys Met
Leu Glu Glu Lys Phe 115 120 125 Glu Asn Lys Asn Val Phe Tyr Ala Val
Lys Leu Thr Gly Thr Phe Lys 130 135 140 Met Val Lys Ala Arg Thr Val
Pro Lys Gln Thr Arg Pro Tyr Pro Gln 145 150 155 160 Leu Thr Glu Val
Thr Lys Lys Gln Ser Glu Phe Glu Phe Lys Asn Val 165 170 175 Lys Gly
Thr Leu Ile Gly Phe Tyr Thr Pro Asn Tyr Ala Ala Ala Leu 180 185 190
Asn Val Pro Gly Phe His Leu His Phe Ile Thr Glu Asp Lys Thr Ser 195
200 205 Gly Gly His Val Leu Asn Leu Gln Phe Asp Asn Ala Asn Leu Glu
Ile 210 215 220 Ser Pro Ile His Glu Phe Asp Val Gln Leu Pro His Thr
Asp Asp Phe 225 230 235 240 Ala His Ser Asp Leu Thr Gln Val Thr Thr
Ser Gln Val His Gln Ala 245 250 255 Glu Ser Glu Arg Lys 260
4783DNAArtificial Sequencemature sequence of the aldB gene in
plasmid RIHI-aldB 4gctacaacgg ctactgtacc agcaccacct gccaagcagg
aatccaaacc tgcggttgcc 60gctaatccgg caccaaaaaa tgtactgttt caatactcaa
cgatcaatgc actcatgctt 120ggacagtttg aaggggactt gactttgaaa
gacctgaagc tgcgaggcga tatggggctt 180ggtaccatca atgatctcga
tggagagatg attcagatgg gtacaaaatt ctaccagatc 240gacagcaccg
gaaaattatc ggagctgcca gaaagtgtga aaactccatt tgcggttact
300acacatttcg agccgaaaga aaaaactaca ttaaccaatg tgcaagatta
caatcaatta 360acaaaaatgc ttgaggagaa atttgaaaac aagaacgtct
tttatgccgt aaagctgacc 420ggtactttta agatggtaaa ggctagaaca
gttccaaaac aaaccagacc ttatccgcag 480ctgactgaag taaccaaaaa
acaatccgag tttgaattta aaaatgttaa gggaaccctg 540attggcttct
atacgccaaa ttatgcagca gccctgaatg ttcccggatt ccatctccac
600ttcatcacag aggataaaac aagtggcgga cacgtattaa atctgcaatt
tgacaacgcg 660aatctggaaa tttctccgat ccatgagttt gatgttcaat
tgccgcacac agatgatttt 720gcccactctg atctgacaca agttactact
agccaagtac accaagctga gtcagaaaga 780aaa 7835290PRTArtificial
SequencealdB precursor protein expressed from plasmid RIHI-aldB
5Val Arg Ser Lys Lys Leu Trp Ile Ser Leu Leu Phe Ala Leu Thr Leu 1
5 10 15 Ile Phe Thr Met Ala Phe Ser Asn Met Ser Ala Gln Ala Ala Thr
Thr 20 25 30 Ala Thr Val Pro Ala Pro Pro Ala Lys Gln Glu Ser Lys
Pro Ala Val 35 40 45 Ala Ala Asn Pro Ala Pro Lys Asn Val Leu Phe
Gln Tyr Ser Thr Ile 50 55 60 Asn Ala Leu Met Leu Gly Gln Phe Glu
Gly Asp Leu Thr Leu Lys Asp 65 70 75 80 Leu Lys Leu Arg Gly Asp Met
Gly Leu Gly Thr Ile Asn Asp Leu Asp 85 90 95 Gly Glu Met Ile Gln
Met Gly Thr Lys Phe Tyr Gln Ile Asp Ser Thr 100 105 110 Gly Lys Leu
Ser Glu Leu Pro Glu Ser Val Lys Thr Pro Phe Ala Val 115 120 125 Thr
Thr His Phe Glu Pro Lys Glu Lys Thr Thr Leu Thr Asn Val Gln 130 135
140 Asp Tyr Asn Gln Leu Thr Lys Met Leu Glu Glu Lys Phe Glu Asn Lys
145 150 155 160 Asn Val Phe Tyr Ala Val Lys Leu Thr Gly Thr Phe Lys
Met Val Lys 165 170 175 Ala Arg Thr Val Pro Lys Gln Thr Arg Pro Tyr
Pro Gln Leu Thr Glu 180 185 190 Val Thr Lys Lys Gln Ser Glu Phe Glu
Phe Lys Asn Val Lys Gly Thr 195 200 205 Leu Ile Gly Phe Tyr Thr Pro
Asn Tyr Ala Ala Ala Leu Asn Val Pro 210 215 220 Gly Phe His Leu His
Phe Ile Thr Glu Asp Lys Thr Ser Gly Gly His 225 230 235 240 Val Leu
Asn Leu Gln Phe Asp Asn Ala Asn Leu Glu Ile Ser Pro Ile 245 250 255
His Glu Phe Asp Val Gln Leu Pro His Thr Asp Asp Phe Ala His Ser 260
265 270 Asp Leu Thr Gln Val Thr Thr Ser Gln Val His Gln Ala Glu Ser
Glu 275 280 285 Arg Lys 290 6858DNAArtificial Sequencesequence
encoding an acetolactate decarboxylase 6atgaaaaaaa atatcatcac
ttctatcaca tctctggctc tcgttgccgg gctgtctttg 60actgcttttg cagctacaac
ggctactgta ccagcaccac ctgccaagca ggaatccaaa 120cctgtggttg
ccgctaatcc ggcaccaaaa aatgtactgt ttcaatactc aacgatcaat
180gcactcatgc ttggacagtt tgaaggggac ttgactttga aagacctgaa
gctacgaggc 240gatatggggc ttggtaccat caatgatctc gatggagaga
tgattcagat gggtacaaaa 300ttctaccaga tcgacagcac cggaaaatta
tccgagctgc cagaaagtgt gaaaactcca 360tttgcggtta ctacacattt
cgagccgaaa gaaaaaacta cattaaccaa tgtgcaagat 420tacaatcaat
taacaaaaat gcttgaggag aaatttgaaa acaagaacgt cttttatgcc
480gtaaagctga ccggtacctt taagatggta aaggctagaa cagttccaaa
acaaaccaga 540ccttatccgc agctgactga agtaaccaaa aaacaatccg
agtttgaatt taaaaatgtt 600aagggaaccc tgattggctt ctatacgcca
aattatgcag cagccctgaa tgttcccgga 660ttccatctcc acttcatcac
agaggataaa acaagtggcg gacacgtatt aaatctgcaa 720tttgacaacg
cgaatctgga aatttctccg atccatgagt ttgatgtaca attgccgcac
780acagatgatt ttgcccactc tgatctgaca caagttacta ctagccaagt
acaccaagct 840gagtcagaaa gaaaataa 8587285PRTArtificial
Sequencesequence of acetolactate decarboxylase (ALDC) precursor
aldB 7Met Lys Lys Asn Ile Ile Thr Ser Ile Thr Ser Leu Ala Leu Val
Ala 1 5 10 15 Gly Leu Ser Leu Thr Ala Phe Ala Ala Thr Thr Ala Thr
Val Pro Ala 20 25 30 Pro Pro Ala Lys Gln Glu Ser Lys Pro Val Val
Ala Ala Asn Pro Ala 35 40 45 Pro Lys Asn Val Leu Phe Gln Tyr Ser
Thr Ile Asn Ala Leu Met Leu 50 55 60 Gly Gln Phe Glu Gly Asp Leu
Thr Leu Lys Asp Leu Lys Leu Arg Gly 65 70 75 80 Asp Met Gly Leu Gly
Thr Ile Asn Asp Leu Asp Gly Glu Met Ile Gln 85 90 95 Met Gly Thr
Lys Phe Tyr Gln Ile Asp Ser Thr Gly Lys Leu Ser Glu 100 105 110 Leu
Pro Glu Ser Val Lys Thr Pro Phe Ala Val Thr Thr His Phe Glu 115 120
125 Pro Lys Glu Lys Thr Thr Leu Thr Asn Val Gln Asp Tyr Asn Gln Leu
130 135 140 Thr Lys Met Leu Glu Glu Lys Phe Glu Asn Lys Asn Val Phe
Tyr Ala 145 150 155 160 Val Lys Leu Thr Gly Thr Phe Lys Met Val Lys
Ala Arg Thr Val Pro 165 170 175 Lys Gln Thr Arg Pro Tyr Pro Gln Leu
Thr Glu Val Thr Lys Lys Gln 180 185 190 Ser Glu Phe Glu Phe Lys Asn
Val Lys Gly Thr Leu Ile Gly Phe Tyr 195 200 205 Thr Pro Asn Tyr Ala
Ala Ala Leu Asn Val Pro Gly Phe His Leu His 210 215 220 Phe Ile Thr
Glu Asp Lys Thr Ser Gly Gly His Val Leu Asn Leu Gln 225 230 235 240
Phe Asp Asn Ala Asn Leu Glu Ile Ser Pro Ile His Glu Phe Asp Val 245
250 255 Gln Leu Pro His Thr Asp Asp Phe Ala His Ser Asp Leu Thr Gln
Val 260 265 270 Thr Thr Ser Gln Val His Gln Ala Glu Ser Glu Arg Lys
275 280 285 8261PRTArtificial Sequencepredicted amino acid sequence
of the mature acetolactate decarboxylase (ALDC) aldB 8Ala Thr Thr
Ala Thr Val Pro Ala Pro Pro Ala Lys Gln Glu Ser Lys 1 5 10 15 Pro
Val Val Ala Ala Asn Pro Ala Pro Lys Asn Val Leu Phe Gln Tyr 20 25
30 Ser Thr Ile Asn Ala Leu Met Leu Gly Gln Phe Glu Gly Asp Leu Thr
35 40 45 Leu Lys Asp Leu Lys Leu Arg Gly Asp Met Gly Leu Gly Thr
Ile Asn 50 55 60 Asp Leu Asp Gly Glu Met Ile Gln Met Gly Thr Lys
Phe Tyr Gln Ile 65 70 75 80 Asp Ser Thr Gly Lys Leu Ser Glu Leu Pro
Glu Ser Val Lys Thr Pro 85 90 95 Phe Ala Val Thr Thr His Phe Glu
Pro Lys Glu Lys Thr Thr Leu Thr 100 105 110 Asn Val Gln Asp Tyr Asn
Gln Leu Thr Lys Met Leu Glu Glu Lys Phe 115 120 125 Glu Asn Lys Asn
Val Phe Tyr Ala Val Lys Leu Thr Gly Thr Phe Lys 130 135 140 Met Val
Lys Ala Arg Thr Val Pro Lys Gln Thr Arg Pro Tyr Pro Gln 145 150 155
160 Leu Thr Glu Val Thr Lys Lys Gln Ser Glu Phe Glu Phe Lys Asn Val
165 170 175 Lys Gly Thr Leu Ile Gly Phe Tyr Thr Pro Asn Tyr Ala Ala
Ala Leu 180 185 190 Asn Val Pro Gly Phe His Leu His Phe Ile Thr Glu
Asp Lys Thr Ser 195 200 205 Gly Gly His Val Leu Asn Leu Gln Phe Asp
Asn Ala Asn Leu Glu Ile 210 215 220 Ser Pro Ile His Glu Phe Asp Val
Gln Leu Pro His Thr Asp Asp Phe 225 230 235 240 Ala His Ser Asp Leu
Thr Gln Val Thr Thr Ser Gln Val His Gln Ala 245 250 255 Glu Ser Glu
Arg Lys 260
* * * * *
References