U.S. patent application number 15/969954 was filed with the patent office on 2018-09-06 for thermolysin variants.
The applicant listed for this patent is DANISCO US INC. Invention is credited to DAVID A. ESTELL, RONALDUS W.J. HOMMES, AMY D. LIU, ANDREW SHAW.
Application Number | 20180251742 15/969954 |
Document ID | / |
Family ID | 40345178 |
Filed Date | 2018-09-06 |
United States Patent
Application |
20180251742 |
Kind Code |
A1 |
ESTELL; DAVID A. ; et
al. |
September 6, 2018 |
THERMOLYSIN VARIANTS
Abstract
The present invention provides methods and compositions
comprising at least one thermolysin-like neutral protease enzyme
with improved storage stability and/or catalytic activity. In some
embodiments, the thermolysin finds use in cleaning and other
applications comprising detergent. In some particularly preferred
embodiments, the present invention provides methods and
compositions comprising thermolysin formulated and/or engineered to
resist detergent-induced inactivation.
Inventors: |
ESTELL; DAVID A.; (PALO
ALTO, CA) ; LIU; AMY D.; (SUNNYVALE, CA) ;
HOMMES; RONALDUS W.J.; (HAARLEM, NL) ; SHAW;
ANDREW; (SAN FRANCISCO, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
DANISCO US INC |
Palo Alto |
CA |
US |
|
|
Family ID: |
40345178 |
Appl. No.: |
15/969954 |
Filed: |
May 3, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14794687 |
Jul 8, 2015 |
9976134 |
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15969954 |
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14035441 |
Sep 24, 2013 |
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14794687 |
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12740782 |
Dec 22, 2010 |
8569034 |
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PCT/US2008/012276 |
Oct 28, 2008 |
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14035441 |
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60984664 |
Nov 1, 2007 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C11D 3/38618 20130101;
C12N 9/54 20130101; C11D 3/386 20130101; C11D 3/38636 20130101;
C12Y 304/24027 20130101 |
International
Class: |
C12N 9/54 20060101
C12N009/54; C11D 3/386 20060101 C11D003/386 |
Claims
1. A composition comprising an isolated thermolysin and a neutral
metalloprotease inhibitor, wherein said thermolysin is a
Geobacillus thermolysin or a Bacillus thermolysin.
2. The composition of claim 1, wherein said neutral metalloprotease
inhibitor is phosphoramidon or galardin.
3. The composition of claim 1, wherein said Geobacillus is G.
caldoproteolyticus or G. stearothermophilus.
4. The composition of claim 1, wherein said Bacillus is B.
thermoproteolyticus.
5. The composition of claim 1, wherein said thermolysin has at
least 50% amino acid identity with the amino acid sequence set
forth in SEQ ID NO:3.
6. The composition of claim 1, wherein said thermolysin comprises
the amino acid sequence set forth in SEQ ID NO:3.
7. An isolated thermolysin variant having improved stability and/or
performance.
8. The isolated thermolysin variant of claim 7, wherein said
thermolysin variant is a Geobacillus thermolysin variant having an
amino acid sequence comprising one or more substitutions at
positions chosen from positions equivalent to positions 6, 7, 49,
56, 58, 61, 63, 65, 75, 128, 151, 156, 196, 273, 278, and 280 of
the amino acid sequence set forth as SEQ ID NO:3.
9. The isolated thermolysin variant of claim 8, wherein said one or
more substitutions comprise one, two, three, four or five
substitutions at positions chosen from positions equivalent to
positions 6, 7, 49, 56, 58, 61, 63, 65, 75, 128, 151, 156, 196,
273, 278, and 280 of the amino acid sequence set forth as SEQ ID
NO:3.
10. The isolated thermolysin variant of claim 7, wherein said
thermolysin variant is a Geobacillus thermolysin variant having an
amino acid sequence comprising one or more substitutions at
positions chosen from positions equivalent to positions 4, 6, 7,
36, 49, 53, 56, 58, 61, 63, 65, 75, 85, 108, 128, 129, 151, 156,
194, 195, 196, 261, 265, 273, 278, 280 and 297 of the amino acid
sequence set forth as SEQ ID NO:3.
11. The isolated thermolysin variant of claim 10, wherein said one
or more substitutions comprise one, two, three, four or five
substitutions at positions chosen from positions equivalent to
positions 4, 6, 7, 36, 49, 53, 56, 58, 61, 63, 65, 75, 85, 108,
128, 129, 151, 156, 194, 195, 196, 261, 265, 273, 278, 280 and 297
of the amino acid sequence set forth as SEQ ID NO:3.
12. The isolated thermolysin variant of claim 7, wherein said
thermolysin variant comprises one or more substitutions chosen from
substitutions T006G, T006H, T006I, T006K, T006M, T006N, T006P,
T006Q, T006R, T006V, T006W, T006Y, V007F, V007H, V007K, V007L,
V007M, V007P, V007Q, V007R, V007T, V007Y, T049G, T049H, T049I,
T049K, T049L, T049N, T049P, T049Q, T049W, A058I, A058P, A058R,
F063I, F063L, F063P, S065K, S065Y, Y075G, Y075M, Y075T, Q128H,
Q128I, Q128L, Q128M, Q128V, Q128Y, Y151D, Y151E, Y151H, Y151K,
Y151M, Y151N, Y151Q, Y151R, Y151T, Y151V, Y151W, I156M, I156R,
I156T, I156W, G196R, Q273I, Q273P, Q273Y, T278K, T278M, T278P,
N280K, N280R, T006A, T006C, T049D, T049I, T049L, T049M, T049N,
T049S, A056C, A056R, A056Y, A058S, S065C, S065E, S0651, S065T,
S065V, S065Y, Q128C, Q128I, Q128M, Q128T, Q128V, Q128Y, Y151A,
Y151C, Y151D, Y151E, Y151H, Y151M, Y151N, Y151Q, Y151R, Y151S,
Y151T, Y151V, Y151W, I156E, I156H, I156K, I156M, I156R, I156T,
I156W, G196D, G196H, Q273A, Q273N, Q273T, Q273W, Q273Y, T278C,
T278H, T278M, T278N, T278S, T278Y, N280E, N280I, N280L, N280M,
N280S, T006C, T049D, T049N, T049Q, T049S, A056C, A056E, A058C,
A058E, Q061E, Q061M, S065C, S065D, S065E, S065P, S065V, S065W,
S065Y, Q128C, Q128I, Q128M, Q128T, Q128V, Q128Y, Y151A, Y151C,
Y151N, Y151S, Y151T, and I156E.
13. The isolated thermolysin variant of claim 12, wherein said one
or more substitutions comprise one, two, three, four or five
substitutions chosen from substitutions T006G, T006H, T006I, T006K,
T006M, T006N, T006P, T006Q, T006R, T006V, T006W, T006Y, V007F,
V007H, V007K, V007L, V007M, V007P, V007Q, V007R, V007T, V007Y,
T049G, T049H, T049I, T049K, T049L, T049N, T049P, T049Q, T049W,
A058I, A058P, A058R, F063I, F063L, F063P, S065K, S065Y, Y075G,
Y075M, Y075T, Q128H, Q128I, Q128L, Q128M, Q128V, Q128Y, Y151D,
Y151E, Y151H, Y151K, Y151M, Y151N, Y151Q, Y151R, Y151T, Y151V,
Y151W, I156M, I156R, I156T, I156W, G196R, Q273I, Q273P, Q273Y,
T278K, T278M, T278P, N280K, N280R, T006A, T006C, T049D, T049I,
T049L, T049M, T049N, T049S, A056C, A056R, A056Y, A058S, S065C,
S065E, S0651, S065T, S065V, S065Y, Q128C, Q128I, Q128M, Q128T,
Q128V, Q128Y, Y151A, Y151C, Y151D, Y151E, Y151H, Y151M, Y151N,
Y151Q, Y151R, Y151S, Y151T, Y151V, Y151W, I156E, I156H, I156K,
I156M, I156R, I156T, I156W, G196D, G196H, Q273A, Q273N, Q273T,
Q273W, Q273Y, T278C, T278H, T278M, T278N, T278S, T278Y, N280E,
N280I, N280L, N280M, N280S, T006C, T049D, T049N, T049Q, T049S,
A056C, A056E, A058C, A058E, Q061E, Q061M, S065C, S065D, S065E,
S065P, S065V, S065W, S065Y, Q128C, Q128I, Q128M, Q128T, Q128V,
Q128Y, Y151A, Y151C, Y151N, Y151S, Y151T, and I156E.
14. An isolated thermolysin variant having an improvement in
stability and/or performance as compared to wild-type Geobacillus
sp. thermolysin (e.g., thermolysin comprising the amino acid
sequence set forth as SEQ ID NO:3).
15. The isolated thermolysin variant of claim 14, wherein said
improvement comprises one or more of improved thermostability,
improved performance under lower pH conditions, improved
performance under higher pH conditions, and improved autolytic
stability.
16. A method for producing an enzyme having thermolysin activity,
comprising: i) transforming a host cell with an expression vector
comprising a polynucleotide encoding a thermolysin variant having
50 to 99% amino acid identity with the amino acid sequence set
forth in SEQ ID NO:3; and ii) cultivating said transformed host
cell under conditions suitable for the production of said
thermolysin.
17. The method of claim 16, wherein said method further comprises
the step of harvesting the produced thermolysin.
18. The method of claim 16, wherein said host cell is a Bacillus
species (e.g., B subtilis).
19. A composition comprising at least one thermolysin variant
obtained from the recombinant Bacillus sp. host cell of claim
18.
20. The composition of claim 7, wherein said composition further
comprises at least one calcium ion and/or zinc ion.
21. The composition of claim 7, wherein said composition further
comprises at least one stabilizer.
22. The composition of claim 21, wherein said stabilizer is chosen
from borax, glycerol, zinc ions, calcium ions, and calcium
formate.
23. The composition of claim 21, wherein said stabilizer is at
least one competitive inhibitor that stabilizes the at least one
thermolysin in the presence of an anionic surfactant.
24. The composition of claim 7, wherein said composition is a
cleaning composition.
25. The composition of claim 24, wherein said cleaning composition
is a detergent.
26. The composition of claim 7, further comprising at least one
additional enzyme or enzyme derivative chosen from proteases,
amylases, lipases, mannanases, pectinases, cutinases,
oxidoreductases, hemicellulases, and cellulases.
27. The composition of claim 7, wherein said composition comprises
at least about 0.0001 weight percent of said thermolysin
variant.
28. The composition of claim 7, wherein said composition comprises
from about 0.001 to about 0.5 weight percent of said thermolysin
variant.
29. The composition of claim 7, further comprising at least one
adjunct ingredient.
30. The composition of claim 7, further comprising a sufficient
amount of a pH modifier to provide the composition with a neat pH
of from about 3 to about 5, the composition being essentially free
of materials that hydrolyze at a pH of from about pH 3 to about pH
5.
31. The composition of claim 30, wherein said materials that
hydrolyze at a pH of from about pH 3 to about pH 5 comprise at
least one surfactant.
32. The composition of claim 31, wherein said surfactant is a
sodium alkyl sulfate surfactant comprising an ethylene oxide
moiety.
33. The composition of claim 7, wherein said composition is a
liquid.
34. An animal feed composition comprising the isolated thermolysin
variant of claim 7.
35. A textile processing composition comprising the isolated
thermolysin variant of claim 7.
36. A leather processing composition comprising the isolated
thermolysin variant of claim 7.
37. A method of cleaning, comprising the step of contacting a
surface and/or an article comprising a fabric with a cleaning
composition comprising the isolated thermolysin variant of claim
7.
38. The method of claim 37, further comprising the step of rinsing
the surface and/or material after contacting the surface or
material with the cleaning composition.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a Divisional of U.S. application
Ser. No. 14/794,687, filed Jul. 8, 2015, which is a Divisional of
U.S. application Ser. No. 14/035,441, filed on Sep. 24, 2013, which
is a Divisional of U.S. application Ser. No. 12/740,782, filed Dec.
22, 2010, now U.S. Pat. No. 8,569,034, which is a 371 National
Phase application of PCT/US2008/012276, filed Oct. 28, 2008, which
claims the benefit of U.S. Provisional Application No. 60/984,664,
filed on Nov. 1, 2007, the disclosure of each application is
incorporated herein in its entirety.
SEQUENCE LISTING
[0002] The sequence listing submitted via EFS, in compliance with
37 C.F.R. .sctn. 1.52(e), is incorporated herein by reference. The
sequence listing text file submitted via EFS contains the file
20180426_NB31056USDIV3_SeqLst.txt, created on Apr. 26, 2018, which
is 20 kb in size.
FIELD OF THE INVENTION
[0003] The present invention provides methods and compositions
comprising at least one thermolysin-like neutral protease enzyme
with improved storage stability and/or catalytic activity. In some
embodiments, the thermolysin finds use in cleaning and other
applications comprising detergent. In some particularly preferred
embodiments, the present invention provides methods and
compositions comprising thermolysin formulated and/or engineered to
resist detergent-induced inactivation.
BACKGROUND OF THE INVENTION
[0004] Bacilli are gram-positive bacteria that secrete a number of
industrially useful enzymes, which can be produced cheaply in high
volume by fermentation. Examples of secreted Bacillus enzymes are
the subtilisin serine proteases, zinc containing neutral proteases,
alpha-amylases, and cellulases. Bacillus proteases are widely used
in the textile, laundry and household industries (Galante, Current
Organic Chemistry, 7:1399-1422, 2003; and Showell, Handbook of
Detergents, Part D: Formulation, Hubbard (ed.), NY: Taylor and
Francis Group, 2006). Highly efficient color and stain removal from
laundry require proteases. However, liquid preparations of cleaning
and washing reagents typically contain builders, surfactants, and
metal chelators, which have a destabilizing effect on most
proteases.
[0005] Detergent and other cleaning compositions typically include
a complex combination of active ingredients. For example, most
cleaning products include a surfactant system, enzymes for
cleaning, bleaching agents, builders, suds suppressors,
soil-suspending agents, soil-release agents, optical brighteners,
softening agents, dispersants, dye transfer inhibition compounds,
abrasives, bactericides, and perfumes. Despite the complexity of
current detergents, there are many stains that are difficult to
completely remove. Furthermore, there is often residue build-up,
which results in discoloration (e.g., yellowing) and diminished
aesthetics due to incomplete cleaning. These problems are
compounded by the increased use of low (e.g., cold water) wash
temperatures and shorter washing cycles. Moreover, many stains are
composed of complex mixtures of fibrous material, mainly
incorporating carbohydrates and carbohydrate derivatives, fiber,
and cell wall components (e.g., plant material, wood, mud/clay
based soil, and fruit). These stains present difficult challenges
to the formulation and use of cleaning compositions.
[0006] In addition, colored garments tend to wear and show
appearance losses. A portion of this color loss is due to abrasion
in the laundering process, particularly in automated washing and
drying machines. Moreover, tensile strength loss of fabric appears
to be an unavoidable result of mechanical and chemical action due
to use, wearing, and/or washing and drying. Thus, a means to
efficiently and effectively wash colored garments so that these
appearance losses are minimized is needed.
[0007] In sum, despite improvements in the capabilities of cleaning
compositions, there remains a need in the art for detergents that
remove stains, maintain fabric color and appearance, and prevent
dye transfer. In addition, there remains a need for detergent
and/or fabric care compositions that provide and/or restore tensile
strength, as well as provide anti-wrinkle, anti-bobbling, and/or
anti-shrinkage properties to fabrics, as well as provide static
control, fabric softness, maintain the desired color appearance,
and fabric anti-wear properties and benefits. In particular, there
remains a need for the inclusion of compositions that are capable
of removing the colored components of stains, which often remain
attached to the fabric being laundered. In addition, there remains
a need for improved methods and compositions suitable for textile
bleaching.
SUMMARY OF THE INVENTION
[0008] The present invention provides methods and compositions
comprising at least one thermolysin-like neutral protease enzyme
with improved storage stability and/or catalytic activity. In some
embodiments, the thermolysin finds use in cleaning and other
applications comprising detergent. In some particularly preferred
embodiments, the present invention provides methods and
compositions comprising thermolysin formulated and/or engineered to
resist detergent-induced inactivation.
[0009] The present invention provides compositions comprising an
isolated thermolysin and a neutral metalloprotease inhibitor,
wherein the thermolysin is a Geobacillus thermolysin or a Bacillus
thermolysin. In some embodiments, the compositions of the invention
comprise an isolated thermolysin and a neutral metalloprotease
inhibitor chosen from phosphoramidon and galardin. In some
embodiments, the thermolysin of the compositions of the invention
is a G. caldoproteolyticus or a G. stearothermophilus, thermolysin,
while in other embodiments, the thermolysin of the compositions of
the invention is a B. thermoproteolyticus thermolysin. In some
particular embodiments, the compositions of the invention comprise
a thermolysin has at least 50% (50 to 100%, preferably at least
55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%)
amino acid identity with the thermolysin comprising the amino acid
sequence set forth in SEQ ID NO:3. In some other embodiments, the
compositions of the invention comprise a thermolysin that comprises
the amino acid sequence set forth in SEQ ID NO:3. In yet other
embodiments, the compositions of the invention comprise a
thermolysin of SEQ ID NO:3.
[0010] In addition the present invention provides an isolated
thermolysin variant having improved stability and/or performance.
In some preferred embodiments, the thermolysin variant is a
Geobacillus thermolysin variant having an amino acid sequence
comprising one or more substitutions at positions chosen from
positions equivalent to positions 6, 7, 49, 56, 58, 61, 63, 65, 75,
128, 151, 156, 196, 273, 278, and 280 of the amino acid sequence
set forth as SEQ ID NO:3. In a subset of these embodiments, the one
or more substitutions comprise one, two, three, four or five
substitutions at positions chosen from positions equivalent to
positions 6, 7, 49, 56, 58, 61, 63, 65, 75, 128, 151, 156, 196,
273, 278, and 280 of the amino acid sequence set forth as SEQ ID
NO:3. In further embodiments, the invention provides an isolated
Geobacillus thermolysin variant having an amino acid sequence
comprising one or more substitutions at positions chosen from
positions equivalent to positions 4, 6, 7, 36, 49, 53, 56, 58, 61,
63, 65, 75, 85, 108, 128, 129, 151, 156, 194, 195, 196, 261, 265,
273, 278, 280 and 297 of the amino acid sequence set forth as SEQ
ID NO:3, and having improved stability and/or performance. In a
subset of these embodiments, the one or more substitutions comprise
one, two, three, four or five substitutions at positions chosen
from positions equivalent to positions 4, 6, 7, 36, 49, 53, 56, 58,
61, 63, 65, 75, 85, 108, 128, 129, 151, 156, 194, 195, 196, 261,
265, 273, 278, 280 and 297 of the amino acid sequence set forth as
SEQ ID NO:3.
[0011] In some other embodiments, the invention provides a
thermolysin variant that comprises one or more substitutions chosen
from the group of the substitutions T006G, T006H, T006I, T006K,
T006M, T006N, T006P, T006Q, T006R, T006V, T006W, T006Y, V007F,
V007H, V007K, V007L, V007M, V007P, V007Q, V007R, V007T, V007Y,
T049G, T049H, T049I, T049K, T049L, T049N, T049P, T049Q, T049W,
A058I, A058P, A058R, F063I, F063L, F063P, S065K, S065Y, Y075G,
Y075M, Y075T, Q128H, Q128I, Q128L, Q128M, Q128V, Q128Y, Y151D,
Y151E, Y151H, Y151K, Y151M, Y151N, Y151Q, Y151R, Y151T, Y151V,
Y151W, I156M, I156R, I156T, I156W, G196R, Q273I, Q273P, Q273Y,
T278K, T278M, T278P, N280K, N280R, T006A, T006C, T049D, T049I,
T049L, T049M, T049N, T049S, A056C, A056R, A056Y, A058S, S065C,
S065E, S0651, S065T, S065V, S065Y, Q128C, Q128I, Q128M, Q128T,
Q128V, Q128Y, Y151A, Y151C, Y151D, Y151E, Y151H, Y151M, Y151N,
Y151Q, Y151R, Y151S, Y151T, Y151V, Y151W, I156E, I156H, I156K,
I156M, I156R, I156T, I156W, G196D, G196H, Q273A, Q273N, Q273T,
Q273W, Q273Y, T278C, T278H, T278M, T278N, T278S, T278Y, N280E,
N280I, N280L, N280M, N280S, T006C, T049D, T049N, T049Q, T049S,
A056C, A056E, A058C, A058E, Q061E, Q061M, S065C, S065D, S065E,
S065P, S065V, S065W, S065Y, Q128C, Q128I, Q128M, Q128T, Q128V,
Q128Y, Y151A, Y151C, Y151N, Y151S, Y151T, and I156E, as listed in
Table 7-1, Table 8-1 and Table 8-2, and that has improved stability
and/or performance. In a subset of these embodiments, the one or
more substitutions comprise one, two, three, four or five
substitutions chosen from the group of substitutions T006G, T006H,
T006I, T006K, T006M, T006N, T006P, T006Q, T006R, T006V, T006W,
T006Y, V007F, V007H, V007K, V007L, V007M, V007P, V007Q, V007R,
V007T, V007Y, T049G, T049H, T049I, T049K, T049L, T049N, T049P,
T049Q, T049W, A058I, A058P, A058R, F063I, F063L, F063P, S065K,
S065Y, Y075G, Y075M, Y075T, Q128H, Q128I, Q128L, Q128M, Q128V,
Q128Y, Y151D, Y151E, Y151H, Y151K, Y151M, Y151N, Y151Q, Y151R,
Y151T, Y151V, Y151W, I156M, I156R, I156T, I156W, G196R, Q273I,
Q273P, Q273Y, T278K, T278M, T278P, N280K, N280R, T006A, T006C,
T049D, T049I, T049L, T049M, T049N, T049S, A056C, A056R, A056Y,
A058S, S065C, S065E, S0651, S065T, S065V, S065Y, Q128C, Q128I,
Q128M, Q128T, Q128V, Q128Y, Y151A, Y151C, Y151D, Y151E, Y151H,
Y151M, Y151N, Y151Q, Y151R, Y151S, Y151T, Y151V, Y151W, I156E,
I156H, I156K, I156M, I156R, I156T, I156W, G196D, G196H, Q273A,
Q273N, Q273T, Q273W, Q273Y, T278C, T278H, T278M, T278N, T278S,
T278Y, N280E, N280I, N280L, N280M, N280S, T006C, T049D, T049N,
T049Q, T049S, A056C, A056E, A058C, A058E, Q061E, Q061M, S065C,
S065D, S065E, S065P, S065V, S065W, S065Y, Q128C, Q128I, Q128M,
Q128T, Q128V, Q128Y, Y151A, Y151C, Y151N, Y151S, Y151T, and I156E,
as listed in Table 7-1, Table 8-1 and Table 8-2.
[0012] Moreover the present invention provides an isolated
thermolysin variant having improved stability and/or performance as
compared to wild-type Geobacillus sp. thermolysin (e.g.,
thermolysin comprising the amino acid sequence set forth as SEQ ID
NO:3). In some embodiments, the invention provides an isolated
thermolysin variant having improvements that comprise one or more
of improved thermostability, improved performance under lower or
higher pH conditions, and improved autolytic stability.
[0013] In some embodiments, the invention provides a Bacillus sp.
host cell transformed with a polynucleotide encoding a thermolysin
variant having 50 to 99% (at least 55%, 60%, 65%, 70%, 75%, 80%,
85%, 90%, 95%, 96%, 97%, 98%, or 99%) amino acid identity with the
amino acid sequence of SEQ ID NO:3.
[0014] Also provided by the present invention are methods for
producing an enzyme having thermolysin activity, comprising: i)
transforming a host cell with an expression vector comprising a
polynucleotide encoding a thermolysin variant having 50 to 99%
identity (at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,
96%, 97%, 98%, or 99%) amino acid identity with the thermolysin
comprising the amino acid sequence set forth in SEQ ID NO:3, and
ii) cultivating the transformed host cell under conditions suitable
for the production of the thermolysin. Optionally, the method of
the invention further comprises harvesting the produced
thermolysin. In some embodiments, the invention provides for
methods for producing an enzyme having thermolysin activity,
comprising: i) transforming a host cell with an expression vector
comprising a polynucleotide encoding a thermolysin variant having
polynucleotide encoding the thermolysin variant has at least 55%,
60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino
acid identity with the thermolysin comprising the amino acid
sequence set forth in SEQ ID NO:3, and ii) cultivating the
transformed host cell under conditions suitable for the production
of the thermolysin. Optionally, the methods further comprise the
step of harvesting the produced thermolysin. In some other
embodiments, the invention provides a method for producing an
enzyme having thermolysin activity, comprising: i) transforming a
Bacillus species (e.g., B subtilis) host cell with an expression
vector comprising a polynucleotide encoding a thermolysin variant
having 50 to 99% identity (at least 55%, 60%, 65%, 70%, 75%, 80%,
85%, 90%, 95%, 96%, 97%, 98%, or 99%) amino acid identity with the
thermolysin comprising the amino acid sequence set forth in SEQ ID
NO:3, and ii) cultivating the transformed host cell under
conditions suitable for the production of the thermolysin.
Optionally, the methods further comprise the step of harvesting the
produced thermolysin.
[0015] In some embodiments, the present invention provides
compositions comprising at least one thermolysin variant obtained
from the recombinant Bacillus sp. host cell of the present
invention. In some embodiments, the composition comprising at least
one thermolysin variant further comprises at least one calcium ion
and/or zinc ion. In some alternative embodiments, the composition
comprising at least one thermolysin variant further comprises at
least one stabilizer. In a subset of these embodiments, the
stabilizer is chosen from borax, glycerol, zinc ions, calcium ions,
and calcium formate. In some embodiments, the stabilizer is at
least one competitive inhibitor that stabilizes the thermolysin in
the presence of an anionic surfactant. Alternatively, the
compositions comprising at least one thermolysin variant, comprise
at least one calcium ion and/or zinc ion, in combination with at
least one stabilizer. Any one of the stabilizers recited above may
be combined with the at least one calcium ion and/or zinc ion to
provide the compositions comprising at least one thermolysin
variant. In a subset of these embodiments, the stabilizer is chosen
from borax, glycerol, zinc ions, calcium ions, and calcium formate.
In some other embodiments, the stabilizer is at least one
competitive inhibitor that stabilizes the thermolysin in the
presence of an anionic surfactant.
[0016] In other embodiments, the invention provides a composition
comprising at least one thermolysin variant obtained from the
recombinant Bacillus sp. host cell of the present invention, in
combination with at least one additional enzyme or enzyme
derivative chosen from proteases, amylases, lipases, mannanases,
pectinases, cutinases, oxidoreductases, hemicellulases, and
cellulases. In some embodiments, the compsiton comprising at least
one thermolysin variant and at least one additional enzyme or
enzyme derivative chosen from proteases, amylases, lipases,
mannanases, pectinases, cutinases, oxidoreductases, hemicellulases,
and cellulases further comprises at least one stabilizer. In a
subset of these embodiments, the stabilizer is chosen from borax,
glycerol, zinc ions, calcium ions, and calcium formate. In some
embodiments, the stabilizer is at least one competitive inhibitor
that stabilizes the thermolysin in the presence of an anionic
surfactant. Alternatively, the compositions comprising at least one
thermolysin variant, comprise at least one calcium ion and/or zinc
ion, in combination with at least one stabilizer. Any one of the
stabilizers recited above may be combined with the at least one
calcium ion and/or zinc ion to provide the compositions comprising
at least one thermolysin variant. In a subset of these embodiments,
the stabilizer is chosen from borax, glycerol, zinc ions, calcium
ions, and calcium formate. In some other embodiments, the
stabilizer is at least one competitive inhibitor that stabilizes
the thermolysin in the presence of an anionic surfactant.
[0017] In some embodiments, present invention provides a cleaning
composition comprising at least one thermolysin variant obtained
from the recombinant Bacillus sp. host cell of the present
invention. In some embodiments, the cleaning composition comprising
at least one thermolysin variant, further comprises at least one
calcium ion and/or zinc ion. In some alternative embodiments, the
cleaning composition comprising at least one thermolysin variant,
further comprises at least one stabilizer. In a subset of these
embodiments, the stabilizer is chosen from borax, glycerol, zinc
ions, calcium ions, and calcium formate. In some embodiments, the
stabilizer is at least one competitive inhibitor that stabilizes
the thermolysin in the presence of an anionic surfactant.
Alternatively, the cleaning compositions comprising at least one
thermolysin variant, comprise at least one calcium ion and/or zinc
ion, in combination with at least one stabilizer. Any one of the
stabilizers recited above may be combined with the at least one
calcium ion and/or zinc ion to provide the compositions comprising
at least one thermolysin variant. In some embodiments, the
stabilizer is at least one competitive inhibitor that stabilizes
the thermolysin in the presence of an anionic surfactant.
[0018] In other embodiments, the invention provides a cleaning
composition comprising at least one thermolysin variant obtained
from the recombinant Bacillus sp. host cell of the present
invention, in combination with at least one additional enzyme or
enzyme derivative chosen from proteases, amylases, lipases,
mannanases, pectinases, cutinases, oxidoreductases, hemicellulases,
and cellulases. In some embodiments, the cleaning compsiton
comprising at least one thermolysin variant and at least one
additional enzyme or enzyme derivative chosen from proteases,
amylases, lipases, mannanases, pectinases, cutinases,
oxidoreductases, hemicellulases, and cellulases further comprises
at least one stabilizer. In a subset of these embodiments, the
stabilizer is chosen from borax, glycerol, zinc ions, calcium ions,
and calcium formate. In some embodiments, the stabilizer is at
least one competitive inhibitor that stabilizes the thermolysin in
the presence of an anionic surfactant. Alternatively, the cleaning
compositions comprising at least one thermolysin variant, comprise
at least one calcium ion and/or zinc ion, in combination with at
least one stabilizer. Any one of the stabilizers recited above may
be combined with the at least one calcium ion and/or zinc ion to
provide the compositions comprising at least one thermolysin
variant. In a subset of these embodiments, the stabilizer is chosen
from borax, glycerol, zinc ions, calcium ions, and calcium formate.
In some other embodiments, the stabilizer is at least one
competitive inhibitor that stabilizes the thermolysin in the
presence of an anionic surfactant.
[0019] In some embodiments, the present invention provides
compositions comprising an isolated thermolysin variant having
improved stability and/or performance. In some embodiments, the
composition comprising the isolated thermolysin variant having
improved stability and/or performance, further comprises at least
one calcium ion and/or zinc ion. In some alternative embodiments,
the composition comprising the isolated thermolysin variants having
improved stability and/or performance, further comprises at least
one stabilizer. In a subset of these embodiments, the stabilizer is
chosen from borax, glycerol, zinc ions, calcium ions, and calcium
formate. In some embodiments, the stabilizer is at least one
competitive inhibitor that stabilizes the thermolysin in the
presence of an anionic surfactant. Alternatively, the compositions
comprising the isolated thermolysin variant having improved
stability and/or performance, comprise at least one calcium ion
and/or zinc ion, in combination with at least one stabilizer. Any
one of the stabilizers recited above may be combined with the at
least one calcium ion and/or zinc ion to provide the compositions
comprising at least one thermolysin variant. In a subset of these
embodiments, the stabilizer is chosen from borax, glycerol, zinc
ions, calcium ions, and calcium formate. In some other embodiments,
the stabilizer is at least one competitive inhibitor that
stabilizes the thermolysin in the presence of an anionic
surfactant. In some embodiments, the thermolysin variant having
improved stability and/or performance is a Geobacillus thermolysin
variant having an amino acid sequence comprising one or more
substitutions at positions chosen from positions equivalent to
positions 6, 7, 49, 56, 58, 61, 63, 65, 75, 128, 151, 156, 196,
273, 278, and 280 of the amino acid sequence set forth as SEQ ID
NO:3. In a subset of these embodiments, the one or more
substitutions comprise one, two, three, four or five substitutions
at positions chosen from positions equivalent to positions 6, 7,
49, 56, 58, 61, 63, 65, 75, 128, 151, 156, 196, 273, 278, and 280
of the amino acid sequence set forth as SEQ ID NO:3. In further
embodiments, the invention provides an isolated Geobacillus
thermolysin variant having an amino acid sequence comprising one or
more substitutions at positions chosen from positions equivalent to
positions 4, 6, 7, 36, 49, 53, 56, 58, 61, 63, 65, 75, 85, 108,
128, 129, 151, 156, 194, 195, 196, 261, 265, 273, 278, 280 and 297
of the amino acid sequence set forth as SEQ ID NO:3, and having
improved stability and/or performance. In a subset of these
embodiments, the one or more substitutions comprise one, two,
three, four or five substitutions at positions chosen from
positions equivalent to positions 4, 6, 7, 36, 49, 53, 56, 58, 61,
63, 65, 75, 85, 108, 128, 129, 151, 156, 194, 195, 196, 261, 265,
273, 278, 280 and 297 of the amino acid sequence set forth as SEQ
ID NO:3. In some other embodiments, the thermolysin variant having
improved stability and/or performance comprises one or more
substitutions chosen from the group of the substitutions T006G,
T006H, T006I, T006K, T006M, T006N, T006P, T006Q, T006R, T006V,
T006W, T006Y, V007F, V007H, V007K, V007L, V007M, V007P, V007Q,
V007R, V007T, V007Y, T049G, T049H, T049I, T049K, T049L, T049N,
T049P, T049Q, T049W, A058I, A058P, A058R, F063I, F063L, F063P,
S065K, S065Y, Y075G, Y075M, Y075T, Q128H, Q128I, Q128L, Q128M,
Q128V, Q128Y, Y151D, Y151E, Y151H, Y151K, Y151M, Y151N, Y151Q,
Y151R, Y151T, Y151V, Y151W, I156M, I156R, I156T, I156W, G196R,
Q273I, Q273P, Q273Y, T278K, T278M, T278P, N280K, N280R, T006A,
T006C, T049D, T049I, T049L, T049M, T049N, T049S, A056C, A056R,
A056Y, A058S, S065C, S065E, S0651, S065T, S065V, S065Y, Q128C,
Q128I, Q128M, Q128T, Q128V, Q128Y, Y151A, Y151C, Y151D, Y151E,
Y151H, Y151M, Y151N, Y151Q, Y151R, Y151S, Y151T, Y151V, Y151W,
I156E, I156H, I156K, I156M, I156R, I156T, I156W, G196D, G196H,
Q273A, Q273N, Q273T, Q273W, Q273Y, T278C, T278H, T278M, T278N,
T278S, T278Y, N280E, N280I, N280L, N280M, N280S, T006C, T049D,
T049N, T049Q, T049S, A056C, A056E, A058C, A058E, Q061E, Q061M,
S065C, S065D, S065E, S065P, S065V, S065W, S065Y, Q128C, Q128I,
Q128M, Q128T, Q128V, Q128Y, Y151A, Y151C, Y151N, Y151S, Y151T, and
I156E, as listed in Table 7-1, Table 8-1 and Table 8-2. In a subset
of these embodiments, the one or more substitutions comprise one,
two, three, four or five substitutions chosen from the group of
substitutions T006G, T006H, T006I, T006K, T006M, T006N, T006P,
T006Q, T006R, T006V, T006W, T006Y, V007F, V007H, V007K, V007L,
V007M, V007P, V007Q, V007R, V007T, V007Y, T049G, T049H, T049I,
T049K, T049L, T049N, T049P, T049Q, T049W, A058I, A058P, A058R,
F063I, F063L, F063P, S065K, S065Y, Y075G, Y075M, Y075T, Q128H,
Q128I, Q128L, Q128M, Q128V, Q128Y, Y151D, Y151E, Y151H, Y151K,
Y151M, Y151N, Y151Q, Y151R, Y151T, Y151V, Y151W, I156M, I156R,
I156T, I156W, G196R, Q273I, Q273P, Q273Y, T278K, T278M, T278P,
N280K, N280R, T006A, T006C, T049D, T049I, T049L, T049M, T049N,
T049S, A056C, A056R, A056Y, A058S, S065C, S065E, S0651, S065T,
S065V, S065Y, Q128C, Q128I, Q128M, Q128T, Q128V, Q128Y, Y151A,
Y151C, Y151D, Y151E, Y151H, Y151M, Y151N, Y151Q, Y151R, Y151S,
Y151T, Y151V, Y151W, I156E, I156H, I156K, I156M, I156R, I156T,
I156W, G196D, G196H, Q273A, Q273N, Q273T, Q273W, Q273Y, T278C,
T278H, T278M, T278N, T278S, T278Y, N280E, N280I, N280L, N280M,
N280S, T006C, T049D, T049N, T049Q, T049S, A056C, A056E, A058C,
A058E, Q061E, Q061M, S065C, S065D, S065E, S065P, S065V, S065W,
S065Y, Q128C, Q128I, Q128M, Q128T, Q128V, Q128Y, Y151A, Y151C,
Y151N, Y151S, Y151T, and I156E, as listed in Table 7-1, Table 8-1
and Table 8-2.
[0020] In other embodiments, the invention provides a composition
comprising an isolated thermolysin variant having improved
stability and/or performance, in combination with at least one
additional enzyme or enzyme derivative chosen from proteases,
amylases, lipases, mannanases, pectinases, cutinases,
oxidoreductases, hemicellulases, and cellulases. In some
embodiments, the composition comprising the isolated thermolysin
variant having improved stability and/or performance, and at least
one additional enzyme or enzyme derivative chosen from proteases,
amylases, lipases, mannanases, pectinases, cutinases,
oxidoreductases, hemicellulases, and cellulases, further comprises
at least one stabilizer. In a subset of these embodiments, the
stabilizer is chosen from borax, glycerol, zinc ions, calcium ions,
and calcium formate. In some embodiments, the stabilizer is at
least one competitive inhibitor that stabilizes the thermolysin in
the presence of an anionic surfactant. Alternatively, the
compositions comprising isolated thermolysin variants having
improved stability and/or performance, comprise at least one
calcium ion and/or zinc ion, in combination with at least one
stabilizer. Any one of the stabilizers recited above may be
combined with the at least one calcium ion and/or zinc ion to
provide the compositions comprising at least one thermolysin
variant. In a subset of these embodiments, the stabilizer is chosen
from borax, glycerol, zinc ions, calcium ions, and calcium formate.
In some other embodiments, the stabilizer is at least one
competitive inhibitor that stabilizes the thermolysin in the
presence of an anionic surfactant. In some embodiments, the
thermolysin variant having improved stability and/or performance is
a Geobacillus thermolysin variant having an amino acid sequence
comprising one or more substitutions at positions chosen from
positions equivalent to positions 6, 7, 49, 56, 58, 61, 63, 65, 75,
128, 151, 156, 196, 273, 278, and 280 of the amino acid sequence
set forth as SEQ ID NO:3. In a subset of these embodiments, the one
or more substitutions comprise one, two, three, four or five
substitutions at positions chosen from positions equivalent to
positions 6, 7, 49, 56, 58, 61, 63, 65, 75, 128, 151, 156, 196,
273, 278, and 280 of the amino acid sequence set forth as SEQ ID
NO:3. In further embodiments, the invention provides an isolated
Geobacillus thermolysin variant having an amino acid sequence
comprising one or more substitutions at positions chosen from
positions equivalent to positions 4, 6, 7, 36, 49, 53, 56, 58, 61,
63, 65, 75, 85, 108, 128, 129, 151, 156, 194, 195, 196, 261, 265,
273, 278, 280 and 297 of the amino acid sequence set forth as SEQ
ID NO:3, and having improved stability and/or performance. In a
subset of these embodiments, the one or more substitutions comprise
one, two, three, four or five substitutions at positions chosen
from positions equivalent to positions 4, 6, 7, 36, 49, 53, 56, 58,
61, 63, 65, 75, 85, 108, 128, 129, 151, 156, 194, 195, 196, 261,
265, 273, 278, 280 and 297 of the amino acid sequence set forth as
SEQ ID NO:3. In some other embodiments, the thermolysin variant
having improved stability and/or performance comprises one or more
substitutions chosen from the group of the substitutions T006G,
T006H, T006I, T006K, T006M, T006N, T006P, T006Q, T006R, T006V,
T006W, T006Y, V007F, V007H, V007K, V007L, V007M, V007P, V007Q,
V007R, V007T, V007Y, T049G, T049H, T049I, T049K, T049L, T049N,
T049P, T049Q, T049W, A058I, A058P, A058R, F063I, F063L, F063P,
S065K, S065Y, Y075G, Y075M, Y075T, Q128H, Q128I, Q128L, Q128M,
Q128V, Q128Y, Y151D, Y151E, Y151H, Y151K, Y151M, Y151N, Y151Q,
Y151R, Y151T, Y151V, Y151W, I156M, I156R, I156T, I156W, G196R,
Q273I, Q273P, Q273Y, T278K, T278M, T278P, N280K, N280R, T006A,
T006C, T049D, T049I, T049L, T049M, T049N, T049S, A056C, A056R,
A056Y, A058S, S065C, S065E, S0651, S065T, S065V, S065Y, Q128C,
Q128I, Q128M, Q128T, Q128V, Q128Y, Y151A, Y151C, Y151D, Y151E,
Y151H, Y151M, Y151N, Y151Q, Y151R, Y151S, Y151T, Y151V, Y151W,
I156E, I156H, I156K, I156M, I156R, I156T, I156W, G196D, G196H,
Q273A, Q273N, Q273T, Q273W, Q273Y, T278C, T278H, T278M, T278N,
T278S, T278Y, N280E, N280I, N280L, N280M, N280S, T006C, T049D,
T049N, T049Q, T049S, A056C, A056E, A058C, A058E, Q061E, Q061M,
S065C, S065D, S065E, S065P, S065V, S065W, S065Y, Q128C, Q128I,
Q128M, Q128T, Q128V, Q128Y, Y151A, Y151C, Y151N, Y151S, Y151T, and
I156E, as listed in Table 7-1, Table 8-1 and Table 8-2. In a subset
of these embodiments, the one or more substitutions comprise one,
two, three, four or five substitutions chosen from the group of
substitutions T006G, T006H, T006I, T006K, T006M, T006N, T006P,
T006Q, T006R, T006V, T006W, T006Y, V007F, V007H, V007K, V007L,
V007M, V007P, V007Q, V007R, V007T, V007Y, T049G, T049H, T049I,
T049K, T049L, T049N, T049P, T049Q, T049W, A058I, A058P, A058R,
F063I, F063L, F063P, S065K, S065Y, Y075G, Y075M, Y075T, Q128H,
Q128I, Q128L, Q128M, Q128V, Q128Y, Y151D, Y151E, Y151H, Y151K,
Y151M, Y151N, Y151Q, Y151R, Y151T, Y151V, Y151W, I156M, I156R,
I156T, I156W, G196R, Q273I, Q273P, Q273Y, T278K, T278M, T278P,
N280K, N280R, T006A, T006C, T049D, T049I, T049L, T049M, T049N,
T049S, A056C, A056R, A056Y, A058S, S065C, S065E, S0651, S065T,
S065V, S065Y, Q128C, Q128I, Q128M, Q128T, Q128V, Q128Y, Y151A,
Y151C, Y151D, Y151E, Y151H, Y151M, Y151N, Y151Q, Y151R, Y151S,
Y151T, Y151V, Y151W, I156E, I156H, I156K, I156M, I156R, I156T,
I156W, G196D, G196H, Q273A, Q273N, Q273T, Q273W, Q273Y, T278C,
T278H, T278M, T278N, T278S, T278Y, N280E, N280I, N280L, N280M,
N280S, T006C, T049D, T049N, T049Q, T049S, A056C, A056E, A058C,
A058E, Q061E, Q061M, S065C, S065D, S065E, S065P, S065V, S065W,
S065Y, Q128C, Q128I, Q128M, Q128T, Q128V, Q128Y, Y151A, Y151C,
Y151N, Y151S, Y151T, and I156E, as listed in Table 7-1, Table 8-1
and Table 8-2.
[0021] In some embodiments, the present invention provides cleaning
compositions comprising an isolated thermolysin variant having
improved stability and/or performance. In some embodiments, the
cleaning composition comprising the isolated thermolysin variant
having improved stability and/or performance, further comprises at
least one calcium ion and/or zinc ion. In some alternative
embodiments, the cleaning composition comprising the isolated
thermolysin variant having improved stability and/or performance,
further comprises at least one stabilizer. In a subset of these
embodiments, the stabilizer is chosen from borax, glycerol, zinc
ions, calcium ions, and calcium formate. In some embodiments, the
stabilizer is at least one competitive inhibitor that stabilizes
the thermolysin in the presence of an anionic surfactant.
Alternatively, the cleaning composition comprising the isolated
thermolysin variant having improved stability and/or performance,
comprises at least one calcium ion and/or zinc ion, in combination
with at least one stabilizer. Any one of the stabilizers recited
above may be combined with the at least one calcium ion and/or zinc
ion to provide the compositions comprising at least one thermolysin
variant. In a subset of these embodiments, the stabilizer is chosen
from borax, glycerol, zinc ions, calcium ions, and calcium formate.
In some other embodiments, the stabilizer is at least one
competitive inhibitor that stabilizes the thermolysin in the
presence of an anionic surfactant. In some embodiments, the
thermolysin variant comprised in the cleaning compositions and
having improved stability and/or performance is a Geobacillus
thermolysin variant having an amino acid sequence comprising one or
more substitutions at positions chosen from positions equivalent to
positions 6, 7, 49, 56, 58, 61, 63, 65, 75, 128, 151, 156, 196,
273, 278, and 280 of the amino acid sequence set forth as SEQ ID
NO:3. In a subset of these embodiments, the one or more
substitutions comprise one, two, three, four or five substitutions
at positions chosen from positions equivalent to positions 6, 7,
49, 56, 58, 61, 63, 65, 75, 128, 151, 156, 196, 273, 278, and 280
of the amino acid sequence set forth as SEQ ID NO:3. In further
embodiments, the thermolysin variant comprised in the cleaning
compositions and having improved stability and/or performance is a
Geobacillus thermolysin variant having an amino acid sequence
comprising one or more substitutions at positions chosen from
positions equivalent to positions 4, 6, 7, 36, 49, 53, 56, 58, 61,
63, 65, 75, 85, 108, 128, 129, 151, 156, 194, 195, 196, 261, 265,
273, 278, 280 and 297 of the amino acid sequence set forth as SEQ
ID NO:3, and having improved stability and/or performance. In a
subset of these embodiments, the one or more substitutions comprise
one, two, three, four or five substitutions at positions chosen
from positions equivalent to positions 4, 6, 7, 36, 49, 53, 56, 58,
61, 63, 65, 75, 85, 108, 128, 129, 151, 156, 194, 195, 196, 261,
265, 273, 278, 280 and 297 of the amino acid sequence set forth as
SEQ ID NO:3. In some other embodiments, the thermolysin variant
comprised in the cleaning compositions and having improved
stability and/or performance is a Geobacillus thermolysin variant
having an amino acid sequence comprising one or more substitutions
chosen from the group of the substitutions T006G, T006H, T006I,
T006K, T006M, T006N, T006P, T006Q, T006R, T006V, T006W, T006Y,
V007F, V007H, V007K, V007L, V007M, V007P, V007Q, V007R, V007T,
V007Y, T049G, T049H, T049I, T049K, T049L, T049N, T049P, T049Q,
T049W, A058I, A058P, A058R, F063I, F063L, F063P, S065K, S065Y,
Y075G, Y075M, Y075T, Q128H, Q128I, Q128L, Q128M, Q128V, Q128Y,
Y151D, Y151E, Y151H, Y151K, Y151M, Y151N, Y151Q, Y151R, Y151T,
Y151V, Y151W, I156M, I156R, I156T, I156W, G196R, Q273I, Q273P,
Q273Y, T278K, T278M, T278P, N280K, N280R, T006A, T006C, T049D,
T049I, T049L, T049M, T049N, T049S, A056C, A056R, A056Y, A058S,
S065C, S065E, S0651, S065T, S065V, S065Y, Q128C, Q128I, Q128M,
Q128T, Q128V, Q128Y, Y151A, Y151C, Y151D, Y151E, Y151H, Y151M,
Y151N, Y151Q, Y151R, Y151S, Y151T, Y151V, Y151W, I156E, I156H,
I156K, I156M, I156R, I156T, I156W, G196D, G196H, Q273A, Q273N,
Q273T, Q273W, Q273Y, T278C, T278H, T278M, T278N, T278S, T278Y,
N280E, N280I, N280L, N280M, N280S, T006C, T049D, T049N, T049Q,
T049S, A056C, A056E, A058C, A058E, Q061E, Q061M, S065C, S065D,
S065E, S065P, S065V, S065W, S065Y, Q128C, Q128I, Q128M, Q128T,
Q128V, Q128Y, Y151A, Y151C, Y151N, Y151S, Y151T, and I156E, as
listed in Table 7-1, Table 8-1 and Table 8-2. In a subset of these
embodiments, the one or more substitutions comprise one, two,
three, four or five substitutions chosen from the group of
substitutions T006G, T006H, T006I, T006K, T006M, T006N, T006P,
T006Q, T006R, T006V, T006W, T006Y, V007F, V007H, V007K, V007L,
V007M, V007P, V007Q, V007R, V007T, V007Y, T049G, T049H, T049I,
T049K, T049L, T049N, T049P, T049Q, T049W, A058I, A058P, A058R,
F063I, F063L, F063P, S065K, S065Y, Y075G, Y075M, Y075T, Q128H,
Q128I, Q128L, Q128M, Q128V, Q128Y, Y151D, Y151E, Y151H, Y151K,
Y151M, Y151N, Y151Q, Y151R, Y151T, Y151V, Y151W, I156M, I156R,
I156T, I156W, G196R, Q273I, Q273P, Q273Y, T278K, T278M, T278P,
N280K, N280R, T006A, T006C, T049D, T049I, T049L, T049M, T049N,
T049S, A056C, A056R, A056Y, A058S, S065C, S065E, S0651, S065T,
S065V, S065Y, Q128C, Q128I, Q128M, Q128T, Q128V, Q128Y, Y151A,
Y151C, Y151D, Y151E, Y151H, Y151M, Y151N, Y151Q, Y151R, Y151S,
Y151T, Y151V, Y151W, I156E, I156H, I156K, I156M, I156R, I156T,
I156W, G196D, G196H, Q273A, Q273N, Q273T, Q273W, Q273Y, T278C,
T278H, T278M, T278N, T278S, T278Y, N280E, N280I, N280L, N280M,
N280S, T006C, T049D, T049N, T049Q, T049S, A056C, A056E, A058C,
A058E, Q061E, Q061M, S065C, S065D, S065E, S065P, S065V, S065W,
S065Y, Q128C, Q128I, Q128M, Q128T, Q128V, Q128Y, Y151A, Y151C,
Y151N, Y151S, Y151T, and I156E, as listed in Table 7-1, Table 8-1
and Table 8-2.
[0022] In other embodiments, the invention provides a cleaning
composition comprising an isolated thermolysin variant having
improved stability and/or performance, in combination with at least
one additional enzyme or enzyme derivative chosen from proteases,
amylases, lipases, mannanases, pectinases, cutinases,
oxidoreductases, hemicellulases, and cellulases. In some
embodiments, the cleaning composition comprises the isolated
thermolysin variant having improved stability and/or performance,
and at least one additional enzyme or enzyme derivative chosen from
proteases, amylases, lipases, mannanases, pectinases, cutinases,
oxidoreductases, hemicellulases, and cellulases further comprises
at least one stabilizer. In a subset of these embodiments, the
stabilizer is chosen from borax, glycerol, zinc ions, calcium ions,
and calcium formate. In some embodiments, the stabilizer is at
least one competitive inhibitor that stabilizes the thermolysin in
the presence of an anionic surfactant. Alternatively, the cleaning
compositions comprising the isolated thermolysin variant having
improved stability and/or performance, comprise at least one
calcium ion and/or zinc ion, in combination with at least one
stabilizer. Any one of the stabilizers recited above may be
combined with the at least one calcium ion and/or zinc ion to
provide the compositions comprising at least one thermolysin
variant. In a subset of these embodiments, the stabilizer is chosen
from borax, glycerol, zinc ions, calcium ions, and calcium formate.
In some other embodiments, the stabilizer is at least one
competitive inhibitor that stabilizes the thermolysin in the
presence of an anionic surfactant. In some embodiments, the
thermolysin variant comprised in the cleaning compositions and
having improved stability and/or performance is a Geobacillus
thermolysin variant having an amino acid sequence comprising one or
more substitutions at positions chosen from positions equivalent to
positions 6, 7, 49, 56, 58, 61, 63, 65, 75, 128, 151, 156, 196,
273, 278, and 280 of the amino acid sequence set forth as SEQ ID
NO:3. 9 In a subset of these embodiments, the one or more
substitutions comprise one, two, three, four or five substitutions
at positions chosen from positions equivalent to positions 6, 7,
49, 56, 58, 61, 63, 65, 75, 128, 151, 156, 196, 273, 278, and 280
of the amino acid sequence set forth as SEQ ID NO:3. In further
embodiments, the thermolysin variant comprised in the cleaning
compositions and having improved stability and/or performance is a
Geobacillus thermolysin variant having an amino acid sequence
comprising one or more substitutions at positions chosen from
positions equivalent to positions 4, 6, 7, 36, 49, 53, 56, 58, 61,
63, 65, 75, 85, 108, 128, 129, 151, 156, 194, 195, 196, 261, 265,
273, 278, 280 and 297 of the amino acid sequence set forth as SEQ
ID NO:3, and having improved stability and/or performance. In a
subset of these embodiments, the one or more substitutions comprise
one, two, three, four or five substitutions at positions chosen
from positions equivalent to positions 4, 6, 7, 36, 49, 53, 56, 58,
61, 63, 65, 75, 85, 108, 128, 129, 151, 156, 194, 195, 196, 261,
265, 273, 278, 280 and 297 of the amino acid sequence set forth as
SEQ ID NO:3. In some other embodiments, the thermolysin variant
comprised in the cleaning compositions and having improved
stability and/or performance is a Geobacillus thermolysin variant
having an amino acid sequence comprising one or more substitutions
chosen from the group of the substitutions T006G, T006H, T006I,
T006K, T006M, T006N, T006P, T006Q, T006R, T006V, T006W, T006Y,
V007F, V007H, V007K, V007L, V007M, V007P, V007Q, V007R, V007T,
V007Y, T049G, T049H, T049I, T049K, T049L, T049N, T049P, T049Q,
T049W, A058I, A058P, A058R, F063I, F063L, F063P, S065K, S065Y,
Y075G, Y075M, Y075T, Q128H, Q128I, Q128L, Q128M, Q128V, Q128Y,
Y151D, Y151E, Y151H, Y151K, Y151M, Y151N, Y151Q, Y151R, Y151T,
Y151V, Y151W, I156M, I156R, I156T, I156W, G196R, Q273I, Q273P,
Q273Y, T278K, T278M, T278P, N280K, N280R, T006A, T006C, T049D,
T049I, T049L, T049M, T049N, T049S, A056C, A056R, A056Y, A058S,
S065C, S065E, 50651, S065T, S065V, S065Y, Q128C, Q128I, Q128M,
Q128T, Q128V, Q128Y, Y151A, Y151C, Y151D, Y151E, Y151H, Y151M,
Y151N, Y151Q, Y151R, Y151S, Y151T, Y151V, Y151W, I156E, I156H,
I156K, I156M, I156R, I156T, I156W, G196D, G196H, Q273A, Q273N,
Q273T, Q273W, Q273Y, T278C, T278H, T278M, T278N, T278S, T278Y,
N280E, N280I, N280L, N280M, N280S, T006C, T049D, T049N, T049Q,
T049S, A056C, A056E, A058C, A058E, Q061E, Q061M, S065C, S065D,
S065E, S065P, S065V, S065W, S065Y, Q128C, Q128I, Q128M, Q128T,
Q128V, Q128Y, Y151A, Y151C, Y151N, Y151S, Y151T, and I156E, as
listed in Table 7-1, Table 8-1 and Table 8-2. In a subset of these
embodiments, the one or more substitutions comprise one, two,
three, four or five substitutions chosen from the group of
substitutions T006G, T006H, T006I, T006K, T006M, T006N, T006P,
T006Q, T006R, T006V, T006W, T006Y, V007F, V007H, V007K, V007L,
V007M, V007P, V007Q, V007R, V007T, V007Y, T049G, T049H, T049I,
T049K, T049L, T049N, T049P, T049Q, T049W, A058I, A058P, A058R,
F063I, F063L, F063P, S065K, S065Y, Y075G, Y075M, Y075T, Q128H,
Q128I, Q128L, Q128M, Q128V, Q128Y, Y151D, Y151E, Y151H, Y151K,
Y151M, Y151N, Y151Q, Y151R, Y151T, Y151V, Y151W, I156M, I156R,
I156T, I156W, G196R, Q273I, Q273P, Q273Y, T278K, T278M, T278P,
N280K, N280R, T006A, T006C, T049D, T049I, T049L, T049M, T049N,
T049S, A056C, A056R, A056Y, A058S, S065C, S065E, 50651, S065T,
S065V, S065Y, Q128C, Q128I, Q128M, Q128T, Q128V, Q128Y, Y151A,
Y151C, Y151D, Y151E, Y151H, Y151M, Y151N, Y151Q, Y151R, Y151S,
Y151T, Y151V, Y151W, I156E, I156H, I156K, I156M, I156R, I156T,
I156W, G196D, G196H, Q273A, Q273N, Q273T, Q273W, Q273Y, T278C,
T278H, T278M, T278N, T278S, T278Y, N280E, N280I, N280L, N280M,
N280S, T006C, T049D, T049N, T049Q, T049S, A056C, A056E, A058C,
A058E, Q061E, Q061M, S065C, S065D, S065E, S065P, S065V, S065W,
S065Y, Q128C, Q128I, Q128M, Q128T, Q128V, Q128Y, Y151A, Y151C,
Y151N, Y151S, Y151T, and I156E, as listed in Table 7-1, Table 8-1
and Table 8-2.
[0023] In some embodiments, any one of the cleaning compositions
comprising a thermolysin variant having improved stability and/or
performance as recited herein, is a detergent. In some embodiments,
the compositions are detergent compositions. In other embodiments,
the compositions are liquid.
[0024] In some embodiments, the present invention provides a
composition comprising a thermolysin variant having improved
stability and/or performance e.g. a cleaning composition,
comprising at least about 0.0001 weight percent of the thermolysin
variant; or from about 0.001 to about 0.5 weight percent of the
same thermolysin variant. Optionally, the composition of the
present invention, which comprises a thermolysin variant having
improved stability and/or performance e.g. a cleaning composition,
further comprises at least one adjunct ingredient. Alternatively,
in some other embodiments, the composition e.g. a cleaning
composition, further comprises a sufficient amount of a pH modifier
to provide the composition with a neat pH of from about 3 to about
5, the composition being essentially free of materials that
hydrolyze at a pH of from about pH 3 to about pH 5. In some
embodiments, the materials that hydrolyze at a pH of from about pH
3 to about pH 5 comprise at least one surfactant. In some preferred
embodiments, the surfactant is a sodium alkyl sulfate surfactant
comprising an ethylene oxide moiety. In some embodiments, the
composition comprising a thermolysin variant having improved
stability and/or performance e.g. a cleaning composition, is a
detergent.
[0025] In addition, the present invention provides animal feed
compositions comprising an isolated thermolysin variant having
improved stability and/or performance. In further embodiments
textile processing compositions are provided comprising an isolated
thermolysin variant having improved stability and/or performance.
In still further embodiments leather processing compositions are
provided comprising an isolated thermolysin variant having improved
stability and/or performance.
[0026] Moreover, the present invention provides methods of
cleaning, comprising the step of contacting a surface and/or an
article comprising a fabric with a cleaning composition comprising
an isolated thermolysin variant having improved stability and/or
performance. In some embodiments, the methods of cleaning further
comprise the step of rinsing the surface and/or material after
contacting the surface or material with the cleaning
composition.
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] FIG. 1 provides the amino acid sequence (SEQ ID NO:3) of the
mature form of Geobacillus caldoproteolyticus thermolysin-like
neutral metalloprotease enzyme (also referred to herein as
thermolysin, Proteinase-T or PrT).
[0028] FIG. 2 provides a map of the pHPLT plasmid.
[0029] FIG. 3 provides a map of the pHPLT-thermolysin expression
vector.
[0030] FIG. 4A-B provides the nucleic acid sequence (SEQ ID NO:8)
of the pHPLT-thermolysin expression vector.
[0031] FIG. 5 provides a graph comparing protease activity of
thermolysin and NprE after incubation at room temperature in
Unilever ALL Small and Mighty 3.times. detergent.
[0032] FIG. 6 provides a graph comparing protease activity of
thermolysin and NprE after incubation at room temperature in
Proctor & Gamble TIDE.RTM. Fresh Breeze 1.times. detergent.
[0033] FIG. 7 provides a graph comparing protease activity of
thermolysin and NprE after incubation at room temperature in
Proctor & Gamble TIDE.RTM. Fresh Breeze 2.times. detergent.
[0034] FIG. 8 shows an SDS-PAGE analysis of thermolysin stability
after prolonged incubation in Unilever ALL small and mighty
detergent in the presence and absence of known metalloproteinase
inhibitors.
[0035] FIG. 9 provides an alignment of the thermolysin (T) and NprE
amino acid sequences. The thermolysin sequence is set forth as SEQ
ID NO:3, while the NprE sequence is set forth as SEQ ID NO:9.
GENERAL DESCRIPTION OF THE INVENTION
[0036] The present invention provides methods and compositions
comprising at least one thermolysin-like neutral protease enzyme
with improved storage stability and/or catalytic activity. In some
embodiments, the thermolysin finds use in cleaning and other
applications comprising detergent. In some particularly preferred
embodiments, the present invention provides methods and
compositions comprising thermolysin formulated and/or engineered to
resist detergent-induced inactivation.
[0037] Unless otherwise indicated, the practice of the present
invention involves conventional techniques commonly used in
molecular biology, microbiology, and recombinant DNA, which are
within the skill of the art. Such techniques are known to those of
skill in the art and are described in numerous texts and reference
works (See e.g., Sambrook et al., "Molecular Cloning: A Laboratory
Manual," Second Edition, Cold Spring Harbor, 1989; and Ausubel et
al., "Current Protocols in Molecular Biology," 1987). All patents,
patent applications, articles and publications mentioned herein,
both supra and infra, are hereby expressly incorporated herein by
reference.
[0038] Unless defined otherwise herein, 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
invention pertains. For example, Singleton and Sainsbury,
Dictionary of Microbiology and Molecular Biology, 2d Ed., John
Wiley and Sons, NY (1994); and Hale and Marham, The Harper Collins
Dictionary of Biology, Harper Perennial, N.Y. (1991) provide those
of skill in the art with a general dictionaries of many of the
terms used in the invention. Although any methods and materials
similar or equivalent to those described herein find use in the
practice of the present invention, the preferred methods and
materials are described herein. Accordingly, the terms defined
immediately below are more fully described by reference to the
Specification as a whole.
[0039] Also, as used herein, the singular "a", "an" and "the"
includes the plural reference unless the context clearly indicates
otherwise. Numeric ranges are inclusive of the numbers defining the
range. Unless otherwise indicated, nucleic acids are written left
to right in 5' to 3' orientation; amino acid sequences are written
left to right in amino to carboxy orientation, respectively. It is
to be understood that this invention is not limited to the
particular methodology, protocols, and reagents described, as these
may vary, depending upon the context they are used by those of
skill in the art.
[0040] Furthermore, the headings provided herein are not
limitations of the various aspects or embodiments of the invention,
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. Nonetheless,
in order to facilitate understanding of the invention, a number of
terms are defined below.
Definitions
[0041] Unless defined otherwise herein, 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
invention pertains. Although any methods and materials similar or
equivalent to those described herein find use in the practice of
the present invention, the preferred methods and materials are
described herein. Accordingly, the terms defined immediately below
are more fully described by reference to the Specification as a
whole. Also, as used herein, the singular terms "a," "an," and
"the" include the plural reference unless the context clearly
indicates otherwise. Unless otherwise indicated, nucleic acids are
written left to right in 5' to 3' orientation; amino acid sequences
are written left to right in amino to carboxy orientation,
respectively. It is to be understood that this invention is not
limited to the particular methodology, protocols, and reagents
described, as these may vary, depending upon the context they are
used by those of skill in the art.
[0042] It is intended that every maximum numerical limitation given
throughout this specification includes every lower numerical
limitation, as if such lower numerical limitations were expressly
written herein. Every minimum numerical limitation given throughout
this specification will include every higher numerical limitation,
as if such higher numerical limitations were expressly written
herein. Every numerical range given throughout this specification
will include every narrower numerical range that falls within such
broader numerical range, as if such narrower numerical ranges were
all expressly written herein.
[0043] All documents cited are, in relevant part, incorporated
herein by reference; the citation of any document is not to be
construed as an admission that it is prior art with respect to the
present invention.
[0044] As used herein, the terms "protease," and "proteolytic
activity" refer to a protein or peptide exhibiting the ability to
hydrolyze peptides or substrates having peptide linkages. Many well
known procedures exist for measuring proteolytic activity (Kalisz,
"Microbial Proteinases," In: Fiechter (ed.), Advances in
Biochemical Engineering/Biotechnology, 1988). For example,
proteolytic activity may be ascertained by comparative assays,
which analyze the respective protease's ability to hydrolyze a
commercial substrate. Exemplary substrates useful in such analysis
of protease or proteolytic activity, include, but are not limited
to di-methyl casein (Sigma C-9801), bovine collagen (Sigma C-9879),
bovine elastin (Sigma E-1625), and bovine keratin (ICN Biomedical
902111). Colorimetric assays utilizing these substrates are well
known in the art (See e.g., WO 99/34011; and U.S. Pat. No.
6,376,450, both of which are incorporated herein by reference. The
pNA assay (See e.g., Del Mar et al., Anal Biochem, 99:316-320,
1979) also finds use in determining the active enzyme concentration
for fractions collected during gradient elution. This assay
measures the rate at which p-nitroaniline is released as the enzyme
hydrolyzes the soluble synthetic substrate,
succinyl-alanine-alanine-proline-phenylalanine-p-nitroanilide
(sAAPF-pNA). The rate of production of yellow color from the
hydrolysis reaction is measured at 410 nm on a spectrophotometer
and is proportional to the active enzyme concentration. In
addition, absorbance measurements at 280 nm can be used to
determine the total protein concentration. The active
enzyme/total-protein ratio gives the enzyme purity.
[0045] As used herein, the terms "NprE protease," and "NprE," refer
to the neutral metalloproteases described herein. In some preferred
embodiments, the NprE protease is the protease designated herein as
purified MULTIFECT.RTM. Neutral or PMN obtained from Bacillus
amyloliquefaciens. Thus, in some embodiments, the term "PMN
protease" refers to a naturally occurring mature protease derived
from Bacillus amyloliquefaciens. In alternative embodiments, the
present invention provides portions of the NprE protease.
[0046] The term "Bacillus protease homologues" refers to naturally
occurring proteases having substantially identical amino acid
sequences to the mature protease derived from Bacillus
thermoproteolyticus thermolysin or polynucleotide sequences which
encode for such naturally occurring proteases, and which proteases
retain the functional characteristics of a neutral metalloprotease
encoded by such nucleic acids.
[0047] As used herein, the term "thermolysin variant," is used in
reference to proteases that are similar to the wild-type
thermolysin, particularly in their function, but have mutations in
their amino acid sequence that make them different in sequence from
the wild-type protease.
[0048] As used herein, "Bacillus ssp." refers to all of the species
within the genus "Bacillus," which are Gram-positive bacteria
classified as members of the Family Bacillaceae, Order Bacillales,
Class Bacilli. 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
stearothermophilus." The production of resistant endospores in the
presence of oxygen is considered the defining feature of the genus
Bacillus, although this characteristic also applies to the recently
named Alicyclobacillus, Amphibacillus, Aneurinibacillus,
Anoxybacillus, Brevibacillus, Filobacillus, Gracilibacillus,
Halobacillus, Paenibacillus, Salibacillus, Thermobacillus,
Ureibacillus, and Virgibacillus.
[0049] Related (and derivative) proteins comprise "variant
proteins." In some preferred embodiments, variant proteins differ
from a parent protein and one another by a small number of amino
acid residues. The number of differing amino acid residues may be
one or more, preferably 1, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, or
more amino acid residues. In some preferred embodiments, the number
of different amino acids between variants is between 1 and 10. In
some particularly preferred embodiments, related proteins and
particularly variant proteins comprise at least 35%, 40%, 45%, 50%,
55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% amino
acid sequence identity. Additionally, a related protein or a
variant protein as used herein, refers to a protein that differs
from another related protein or a parent protein in the number of
prominent regions. For example, in some embodiments, variant
proteins have 1, 2, 3, 4, 5, or 10 corresponding prominent regions
that differ from the parent protein.
[0050] Several methods are known in the art that are suitable for
generating variants of the enzymes of the present invention,
including but not limited to site-saturation mutagenesis, scanning
mutagenesis, insertional mutagenesis, random mutagenesis,
site-directed mutagenesis, and directed-evolution, as well as
various other recombinatorial approaches.
[0051] Characterization of wild-type and mutant proteins is
accomplished via any means or "test" suitable and is preferably
based on the assessment of properties of interest. For example, pH
and/or temperature, as well as detergent and/or oxidative stability
is/are determined in some embodiments of the present invention.
Indeed, it is contemplated that enzymes having various degrees of
stability in one or more of these characteristics (pH, temperature,
proteolytic stability, detergent stability, and/or oxidative
stability) will find use.
[0052] The terms "polynucleotide" and "nucleic acid", used
interchangeably herein, refer to a polymeric form of nucleotides of
any length, either ribonucleotides or deoxyribonucleotides. These
terms include, but are not limited to, a single-, double- or
triple-stranded DNA, genomic DNA, cDNA, RNA, DNA-RNA hybrid, or a
polymer comprising purine and pyrimidine bases, or other natural,
chemically, biochemically modified, non-natural or derivatized
nucleotide bases. The following are non-limiting examples of
polynucleotides: genes, gene fragments, chromosomal fragments,
ESTs, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA,
recombinant polynucleotides, branched polynucleotides, plasmids,
vectors, isolated DNA of any sequence, isolated RNA of any
sequence, nucleic acid probes, and primers. In some embodiments,
polynucleotides comprise modified nucleotides, such as methylated
nucleotides and nucleotide analogs, uracil, other sugars and
linking groups such as fluororibose and thioate, and nucleotide
branches. In alternative embodiments, the sequence of nucleotides
is interrupted by non-nucleotide components.
[0053] As used herein, the terms "DNA construct" and "transforming
DNA" are used interchangeably to refer to DNA used to introduce
sequences into a host cell or organism. The DNA may be generated in
vitro by PCR or any other suitable technique(s) known to those in
the art. In particularly preferred embodiments, the DNA construct
comprises a sequence of interest (e.g., as an incoming sequence).
In some embodiments, the sequence is operably linked to additional
elements such as control elements (e.g., promoters, etc.). The DNA
construct may further comprise a selectable marker. It may further
comprise an incoming sequence flanked by homology boxes. In a
further embodiment, the transforming DNA comprises other
non-homologous sequences, added to the ends (e.g., stuffer
sequences or flanks). In some embodiments, the ends of the incoming
sequence are closed such that the transforming DNA forms a closed
circle. The transforming sequences may be wild-type, mutant or
modified. In some embodiments, the DNA construct comprises
sequences homologous to the host cell chromosome. In other
embodiments, the DNA construct comprises non-homologous sequences.
Once the DNA construct is assembled in vitro it may be used to: 1)
insert heterologous sequences into a desired target sequence of a
host cell, and/or 2) mutagenize a region of the host cell
chromosome (i.e., replace an endogenous sequence with a
heterologous sequence), 3) delete target genes; and/or introduce a
replicating plasmid into the host.
[0054] As used herein, the terms "expression cassette" and
"expression vector" refer to nucleic acid constructs generated
recombinantly or synthetically, with a series of specified nucleic
acid elements that permit transcription of a particular nucleic
acid in a target cell. The recombinant expression cassette can be
incorporated into a plasmid, chromosome, mitochondrial DNA, plastid
DNA, virus, or nucleic acid fragment. Typically, the recombinant
expression cassette portion of an expression vector includes, among
other sequences, a nucleic acid sequence to be transcribed and a
promoter. In preferred embodiments, expression vectors have the
ability to incorporate and express heterologous DNA fragments in a
host cell. Many prokaryotic and eukaryotic expression vectors are
commercially available. Selection of appropriate expression vectors
is within the knowledge of those of skill in the art. The term
"expression cassette" is used interchangeably herein with "DNA
construct," and their grammatical equivalents. Selection of
appropriate expression vectors is within the knowledge of those of
skill in the art.
[0055] As used herein, the term "vector" refers to a polynucleotide
construct designed to introduce nucleic acids into one or more cell
types. Vectors include cloning vectors, expression vectors, shuttle
vectors, plasmids, cassettes and the like. In some embodiments, the
polynucleotide construct comprises a DNA sequence encoding the
protease (e.g., precursor or mature protease) that is operably
linked to a suitable prosequence (e.g., secretory, etc.) capable of
effecting the expression of the DNA in a suitable host.
[0056] As used herein, the term "plasmid" refers to a circular
double-stranded (ds) DNA construct used as a cloning vector, and
which forms an extrachromosomal self-replicating genetic element in
some eukaryotes or prokaryotes, or integrates into the host
chromosome.
[0057] As used herein in the context of introducing a nucleic acid
sequence into a cell, the term "introduced" refers to any method
suitable for transferring the nucleic acid sequence into the cell.
Such methods for introduction include but are not limited to
protoplast fusion, transfection, transformation, conjugation, and
transduction (See e.g., Ferrari et al., "Genetics," in Hardwood et
al, (eds.), Bacillus, Plenum Publishing Corp., pages 57-72,
1989).
[0058] As used herein, the terms "transformed" and "stably
transformed" refers to a cell that has a non-native (heterologous)
polynucleotide sequence integrated into its genome or as an
episomal plasmid that is maintained for at least two
generations.
[0059] As used herein, the term "selectable marker-encoding
nucleotide sequence" refers to a nucleotide sequence, which is
capable of expression in the host cells and where expression of the
selectable marker confers to cells containing the expressed gene
the ability to grow in the presence of a corresponding selective
agent or lack of an essential nutrient.
[0060] As used herein, the terms "selectable marker" and "selective
marker" refer to a nucleic acid (e.g., a gene) capable of
expression in host cell, which allows for ease of selection of
those hosts containing the vector. Examples of such selectable
markers include but are not limited to antimicrobials. Thus, the
term "selectable marker" refers to genes that provide an indication
that a host cell has taken up an incoming DNA of interest or some
other reaction has occurred.
[0061] Typically, selectable markers are genes that confer
antimicrobial resistance or a metabolic advantage on the host cell
to allow cells containing the exogenous DNA to be distinguished
from cells that have not received any exogenous sequence during the
transformation. A "residing selectable marker" is one that is
located on the chromosome of the microorganism to be transformed. A
residing selectable marker encodes a gene that is different from
the selectable marker on the transforming DNA construct. Selective
markers are well known to those of skill in the art. As indicated
above, preferably the marker is an antimicrobial resistant marker
(e.g., amp.sup.R; phleo.sup.R; spec.sup.R; kan.sup.R; ery.sup.R;
tee; cmp.sup.R; and nee (See e.g., Guerot-Fleury, Gene,
167:335-337, 1995; Palmeros et al., Gene 247:255-264, 2000; and
Trieu-Cuot et al., Gene, 23:331-341, 1983). Other markers useful in
accordance with the invention include, but are not limited to
auxotrophic markers, such as tryptophan; and detection markers,
such as .beta.-galactosidase.
[0062] As used herein, the term "promoter" refers to a nucleic acid
sequence that functions to direct transcription of a downstream
gene. In preferred embodiments, the promoter is appropriate to the
host cell in which the target gene is being expressed. The
promoter, together with other transcriptional and translational
regulatory nucleic acid sequences (also termed "control sequences")
is necessary to express a given gene. In general, the
transcriptional and translational regulatory sequences include, but
are not limited to, promoter sequences, ribosomal binding sites,
transcriptional start and stop sequences, translational start and
stop sequences, and enhancer or activator sequences.
[0063] A nucleic acid is "operably linked" when it is placed into a
functional relationship with another nucleic acid sequence. For
example, DNA encoding a secretory leader (i.e., a signal peptide),
is operably linked to DNA for a polypeptide if it is expressed as a
preprotein that participates in the secretion of the polypeptide; a
promoter or enhancer is operably linked to a coding sequence if it
affects the transcription of the sequence; or a ribosome binding
site is operably linked to a coding sequence if it is positioned so
as to facilitate translation. Generally, "operably linked" means
that the DNA sequences being linked are contiguous, and, in the
case of a secretory leader, contiguous and in reading phase.
However, enhancers do not have to be contiguous. Linking is
accomplished by ligation at convenient restriction sites. If such
sites do not exist, the synthetic oligonucleotide adaptors or
linkers are used in accordance with conventional practice.
[0064] As used herein the term "gene" refers to a polynucleotide
(e.g., a DNA segment) that encodes a polypeptide and includes
regions preceding and following the coding regions as well as
intervening sequences (introns) between individual coding segments
(exons).
[0065] As used herein, "homologous genes" refers to a pair of genes
from different, but usually related species, which correspond to
each other and which are identical or very similar to each other.
The term encompasses genes that are separated by speciation (i.e.,
the development of new species) (e.g., orthologous genes), as well
as genes that have been separated by genetic duplication (e.g.,
paralogous genes).
[0066] As used herein, "ortholog" and "orthologous genes" refer to
genes in different species that have evolved from a common
ancestral gene (i.e., a homologous gene) by speciation. Typically,
orthologs retain the same function during the course of evolution.
Identification of orthologs finds use in the reliable prediction of
gene function in newly sequenced genomes.
[0067] As used herein, "paralog" and "paralogous genes" refer to
genes that are related by duplication within a genome. While
orthologs retain the same function through the course of evolution,
paralogs evolve new functions, even though some functions are often
related to the original one. Examples of paralogous genes include,
but are not limited to genes encoding trypsin, chymotrypsin,
elastase, and thrombin, which are all serine proteinases and occur
together within the same species.
[0068] As used herein, "homology" refers to sequence similarity or
identity, with identity being preferred. This homology is
determined using standard techniques known in the art (See e.g.,
Smith and Waterman, Adv Appl Math, 2:482, 1981; Needleman and
Wunsch, J Mol Biol, 48:443, 1970; Pearson and Lipman, Proc Natl
Acad Sci USA, 85:2444, 1988; programs such as GAP, BESTFIT, FASTA,
and TFASTA in the Wisconsin Genetics Software Package, Genetics
Computer Group, Madison, Wis.; and Devereux et al., Nucl Acid Res,
12:387-395, 1984).
[0069] As used herein, an "analogous sequence" is one wherein the
function of the gene is essentially the same as the gene based on
the Geobacillus caldoproteolyticus thermolysin. Additionally,
analogous genes include at least 45%, 50%, 55%, 60%, 65%, 70%, 75%,
80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% sequence identity with
the sequence of the Geobacillus caldoproteolyticus thermolysin. In
additional embodiments more than one of the above properties
applies to the sequence. Analogous sequences are determined by
known methods of sequence alignment. A commonly used alignment
method is BLAST, although as indicated above and below, there are
other methods that also find use in aligning sequences. One example
of a useful algorithm is PILEUP. PILEUP creates a multiple sequence
alignment from a group of related sequences using progressive,
pair-wise alignments. It can also plot a tree showing the
clustering relationships used to create the alignment. PILEUP uses
a simplification of the progressive alignment method of Feng and
Doolittle (Feng and Doolittle, J Mol Evol, 35:351-360, 1987). The
method is similar to that described by Higgins and Sharp (Higgins
and Sharp, CABIOS 5:151-153, 1989). Useful PILEUP parameters
including a default gap weight of 3.00, a default gap length weight
of 0.10, and weighted end gaps.
[0070] Another example of a useful algorithm is the BLAST
algorithm, described by Altschul et al., (Altschul et al., J Mol
Biol, 215:403-410, 1990; and Karlin et al., Proc Natl Acad Sci USA,
90:5873-5787, 1993). A particularly useful BLAST program is the
WU-BLAST-2 program (See, Altschul et al., Meth Enzymol,
266:460-480, 1996). WU-BLAST-2 uses several search parameters, most
of which are set to the default values. The adjustable parameters
are set with the following values: overlap span=1, overlap
fraction=0.125, word threshold (T)=11. The HSP S and HSP S2
parameters are dynamic values and are established by the program
itself depending upon the composition of the particular sequence
and composition of the particular database against which the
sequence of interest is being searched. However, the values may be
adjusted to increase sensitivity. A % amino acid sequence identity
value is determined by the number of matching identical residues
divided by the total number of residues of the "longer" sequence in
the aligned region. The "longer" sequence is the one having the
most actual residues in the aligned region (gaps introduced by
WU-Blast-2 to maximize the alignment score are ignored).
[0071] Thus, "percent (%) nucleic acid sequence identity" is
defined as the percentage of nucleotide residues in a candidate
sequence that are identical to the nucleotide residues of the
starting sequence (i.e., the sequence of interest). A preferred
method utilizes the BLASTN module of WU-BLAST-2 set to the default
parameters, with overlap span and overlap fraction set to 1 and
0.125, respectively.
[0072] As used herein, the term "hybridization" refers to the
process by which a strand of nucleic acid joins with a
complementary strand through base pairing, as known in the art.
[0073] A nucleic acid sequence is considered to be "selectively
hybridizable" to a reference nucleic acid sequence if the two
sequences specifically hybridize to one another under moderate to
high stringency hybridization and wash conditions. Hybridization
conditions are based on the melting temperature (Tm) of the nucleic
acid binding complex or probe. For example, "maximum stringency"
typically occurs at about Tm-5.degree. C. (5.degree. below the Tm
of the probe); "high stringency" at about 5-10.degree. C. below the
Tm; "intermediate stringency" at about 10-20.degree. C. below the
Tm of the probe; and "low stringency" at about 20-25.degree. C.
below the Tm. Functionally, maximum stringency conditions may be
used to identify sequences having strict identity or near-strict
identity with the hybridization probe; while intermediate or low
stringency hybridization can be used to identify or detect
polynucleotide sequence homologs.
[0074] Moderate and high stringency hybridization conditions are
well known in the art. An example of high stringency conditions
includes hybridization at about 42.degree. C. in 50% formamide,
5.times.SSC, 5.times.Denhardt's solution, 0.5% SDS and 100 .mu.g/ml
denatured carrier DNA followed by washing two times in 2.times.SSC
and 0.5% SDS at room temperature and two additional times in
0.1.times.SSC and 0.5% SDS at 42.degree. C. An example of moderate
stringent conditions include an overnight incubation at 37.degree.
C. in a solution comprising 20% formamide, 5.times.SSC (150 mM
NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6),
5.times.Denhardt's solution, 10% dextran sulfate and 20 mg/ml
denatured sheared salmon sperm DNA, followed by washing the filters
in 1.times.SSC at about 37-50.degree. C. Those of skill in the art
know how to adjust the temperature, ionic strength, etc. as
necessary to accommodate factors such as probe length and the
like.
[0075] As used herein, "recombinant" includes reference to a cell
or vector, that has been modified by the introduction of a
heterologous nucleic acid sequence or that the cell is derived from
a cell so modified. Thus, for example, recombinant cells express
genes that are not found in identical form within the native
(non-recombinant) form of the cell or express native genes that are
otherwise abnormally expressed, under expressed or not expressed at
all as a result of deliberate human intervention. "Recombination,"
"recombining," and generating a "recombined" nucleic acid are
generally the assembly of two or more nucleic acid fragments
wherein the assembly gives rise to a chimeric gene.
[0076] In a preferred embodiment, mutant DNA sequences are
generated with site saturation mutagenesis in at least one codon.
In another preferred embodiment, site saturation mutagenesis is
performed for two or more codons. In a further embodiment, mutant
DNA sequences have more than 50%, more than 55%, more than 60%,
more than 65%, more than 70%, more than 75%, more than 80%, more
than 85%, more than 90%, more than 95%, or more than 98% homology
with the wild-type sequence. In alternative embodiments, mutant DNA
is generated in vivo using any known mutagenic procedure such as,
for example, radiation, nitrosoguanidine and the like. The desired
DNA sequence is then isolated and used in the methods provided
herein.
[0077] As used herein, the term "target sequence" refers to a DNA
sequence in the host cell that encodes the sequence where it is
desired for the incoming sequence to be inserted into the host cell
genome. In some embodiments, the target sequence encodes a
functional wild-type gene or operon, while in other embodiments the
target sequence encodes a functional mutant gene or operon, or a
non-functional gene or operon.
[0078] As used herein, a "flanking sequence" refers to any sequence
that is either upstream or downstream of the sequence being
discussed (e.g., for genes A-B-C, gene B is flanked by the A and C
gene sequences). In a preferred embodiment, the incoming sequence
is flanked by a homology box on each side. In another embodiment,
the incoming sequence and the homology boxes comprise a unit that
is flanked by stuffer sequence on each side. In some embodiments, a
flanking sequence is present on only a single side (either 3' or
5'), but in preferred embodiments, it is on each side of the
sequence being flanked. In some embodiments, a flanking sequence is
present on only a single side (either 3' or 5'), while in preferred
embodiments it is present on each side of the sequence being
flanked.
[0079] As used herein, the term "stuffer sequence" refers to any
extra DNA that flanks homology boxes (typically vector sequences).
However, the term encompasses any non-homologous DNA sequence. Not
to be limited by any theory, a stuffer sequence provides a
noncritical target for a cell to initiate DNA uptake.
[0080] As used herein, the terms "amplification" and "gene
amplification" refer to a process by which specific DNA sequences
are disproportionately replicated such that the amplified gene
becomes present in a higher copy number than was initially present
in the genome. In some embodiments, selection of cells by growth in
the presence of a drug (e.g., an inhibitor of an inhibitable
enzyme) results in the amplification of either the endogenous gene
encoding the gene product required for growth in the presence of
the drug or by amplification of exogenous (i.e., input) sequences
encoding this gene product, or both.
[0081] "Amplification" is a special case of nucleic acid
replication involving template specificity. It is to be contrasted
with non-specific template replication (i.e., replication that is
template-dependent but not dependent on a specific template).
Template specificity is here distinguished from fidelity of
replication (i.e., synthesis of the proper polynucleotide sequence)
and nucleotide (ribo- or deoxyribo-) specificity. Template
specificity is frequently described in terms of "target"
specificity. Target sequences are "targets" in the sense that they
are sought to be sorted out from other nucleic acid. Amplification
techniques have been designed primarily for this sorting out.
[0082] As used herein, the term "co-amplification" refers to the
introduction into a single cell of an amplifiable marker in
conjunction with other gene sequences (i.e., comprising one or more
non-selectable genes such as those contained within an expression
vector) and the application of appropriate selective pressure such
that the cell amplifies both the amplifiable marker and the other,
non-selectable gene sequences. The amplifiable marker may be
physically linked to the other gene sequences or alternatively two
separate pieces of DNA, one containing the amplifiable marker and
the other containing the non-selectable marker, may be introduced
into the same cell.
[0083] As used herein, the terms "amplifiable marker," "amplifiable
gene," and "amplification vector" refer to a gene or a vector
encoding a gene, which permits the amplification of that gene under
appropriate growth conditions.
[0084] "Template specificity" is achieved in most amplification
techniques by the choice of enzyme. Amplification enzymes are
enzymes that, under conditions they are used, will process only
specific sequences of nucleic acid in a heterogeneous mixture of
nucleic acid. For example, in the case of Q.beta. replicase, MDV-1
RNA is the specific template for the replicase (See e.g., Kacian et
al., Proc Natl Acad Sci USA 69:3038, 1972) and other nucleic acids
are not replicated by this amplification enzyme. Similarly, in the
case of T7 RNA polymerase, this amplification enzyme has a
stringent specificity for its own promoters (See, Chamberlin et
al., Nature 228:227, 1970). In the case of T4 DNA ligase, the
enzyme will not ligate the two oligonucleotides or polynucleotides,
where there is a mismatch between the oligonucleotide or
polynucleotide substrate and the template at the ligation junction
(See, Wu and Wallace, Genomics 4:560, 1989). Finally, Taq and Pfu
polymerases, by virtue of their ability to function at high
temperature, are found to display high specificity for the
sequences bounded and thus defined by the primers; the high
temperature results in thermodynamic conditions that favor primer
hybridization with the target sequences and not hybridization with
non-target sequences.
[0085] As used herein, the term "amplifiable nucleic acid" refers
to nucleic acids, which may be amplified by any amplification
method. It is contemplated that "amplifiable nucleic acid" will
usually comprise "sample template."
[0086] As used herein, the term "sample template" refers to nucleic
acid originating from a sample, which is analyzed for the presence
of "target" (defined below). In contrast, "background template" is
used in reference to nucleic acid other than sample template, which
may or may not be present in a sample. Background template is most
often inadvertent. It may be the result of carryover, or it may be
due to the presence of nucleic acid contaminants sought to be
purified away from the sample. For example, nucleic acids from
organisms other than those to be detected may be present as
background in a test sample.
[0087] As used herein, the term "primer" refers to an
oligonucleotide, whether occurring naturally as in a purified
restriction digest or produced synthetically, which is capable of
acting as a point of initiation of synthesis when placed under
conditions in which synthesis of a primer extension product which
is complementary to a nucleic acid strand is induced, (i.e., in the
presence of nucleotides and an inducing agent such as DNA
polymerase and at a suitable temperature and pH). The primer is
preferably single stranded for maximum efficiency in amplification,
but may alternatively be double stranded. If double stranded, the
primer is first treated to separate its strands before being used
to prepare extension products. Preferably, the primer is an
oligodeoxyribonucleotide. The primer must be sufficiently long to
prime the synthesis of extension products in the presence of the
inducing agent. The exact lengths of the primers will depend on
many factors, including temperature, source of primer and the use
of the method.
[0088] As used herein, the term "probe" refers to an
oligonucleotide (i.e., a sequence of nucleotides), whether
occurring naturally as in a purified restriction digest or produced
synthetically, recombinantly or by PCR amplification, which is
capable of hybridizing to another oligonucleotide of interest. A
probe may be single-stranded or double-stranded. Probes are useful
in the detection, identification and isolation of particular gene
sequences. It is contemplated that any probe used in the present
invention will be labeled with any "reporter molecule," so that is
detectable in any detection system, including, but not limited to
enzyme (e.g., ELISA, as well as enzyme-based histochemical assays),
fluorescent, radioactive, and luminescent systems. It is not
intended that the present invention be limited to any particular
detection system or label.
[0089] As used herein, the term "target," when used in reference to
the polymerase chain reaction, refers to the region of nucleic acid
bounded by the primers used for polymerase chain reaction. Thus,
the "target" is sought to be sorted out from other nucleic acid
sequences. A "segment" is defined as a region of nucleic acid
within the target sequence.
[0090] As used herein, the term "polymerase chain reaction" ("PCR")
refers to the methods of U.S. Pat. Nos. 4,683,195, 4,683,202, and
4,965,188, hereby incorporated by reference, which include methods
for increasing the concentration of a segment of a target sequence
in a mixture of genomic DNA without cloning or purification. This
process for amplifying the target sequence consists of introducing
a large excess of two oligonucleotide primers to the DNA mixture
containing the desired target sequence, followed by a precise
sequence of thermal cycling in the presence of a DNA polymerase.
The two primers are complementary to their respective strands of
the double stranded target sequence. To effect amplification, the
mixture is denatured and the primers then annealed to their
complementary sequences within the target molecule. Following
annealing, the primers are extended with a polymerase so as to form
a new pair of complementary strands. The steps of denaturation,
primer annealing and polymerase extension can be repeated many
times (i.e., denaturation, annealing and extension constitute one
"cycle"; there can be numerous "cycles") to obtain a high
concentration of an amplified segment of the desired target
sequence. The length of the amplified segment of the desired target
sequence is determined by the relative positions of the primers
with respect to each other, and therefore, this length is a
controllable parameter. By virtue of the repeating aspect of the
process, the method is referred to as the "polymerase chain
reaction" (hereinafter "PCR"). Because the desired amplified
segments of the target sequence become the predominant sequences
(in terms of concentration) in the mixture, they are said to be
"PCR amplified".
[0091] As used herein, the term "amplification reagents" refers to
those reagents (deoxyribonucleotide triphosphates, buffer, etc.),
needed for amplification except for primers, nucleic acid template
and the amplification enzyme. Typically, amplification reagents
along with other reaction components are placed and contained in a
reaction vessel (test tube, microwell, etc.).
[0092] With PCR, it is possible to amplify a single copy of a
specific target sequence in genomic DNA to a level detectable by
several different methodologies (e.g., hybridization with a labeled
probe; incorporation of biotinylated primers followed by
avidin-enzyme conjugate detection; incorporation of
.sup.32P-labeled deoxynucleotide triphosphates, such as dCTP or
dATP, into the amplified segment). In addition to genomic DNA, any
oligonucleotide or polynucleotide sequence can be amplified with
the appropriate set of primer molecules. In particular, the
amplified segments created by the PCR process itself are,
themselves, efficient templates for subsequent PCR
amplifications.
[0093] As used herein, the terms "PCR product," "PCR fragment," and
"amplification product" refer to the resultant mixture of compounds
after two or more cycles of the PCR steps of denaturation,
annealing and extension are complete. These terms encompass the
case where there has been amplification of one or more segments of
one or more target sequences.
[0094] As used herein, the term "RT-PCR" refers to the replication
and amplification of RNA sequences. In this method, reverse
transcription is coupled to PCR, most often using a one enzyme
procedure in which a thermostable polymerase is employed, as
described in U.S. Pat. No. 5,322,770, herein incorporated by
reference. In RT-PCR, the RNA template is converted to cDNA due to
the reverse transcriptase activity of the polymerase, and then
amplified using the polymerizing activity of the polymerase (i.e.,
as in other PCR methods).
[0095] As used herein, the terms "restriction endonucleases" and
"restriction enzymes" refer to bacterial enzymes, each of which cut
double-stranded DNA at or near a specific nucleotide sequence.
[0096] A "restriction site" refers to a nucleotide sequence
recognized and cleaved by a given restriction endonuclease and is
frequently the site for insertion of DNA fragments. In certain
embodiments of the invention restriction sites are engineered into
the selective marker and into 5' and 3' ends of the DNA
construct.
[0097] As used herein, the term "chromosomal integration" refers to
the process whereby an incoming sequence is introduced into the
chromosome of a host cell. The homologous regions of the
transforming DNA align with homologous regions of the chromosome.
Subsequently, the sequence between the homology boxes is replaced
by the incoming sequence in a double crossover (i.e., homologous
recombination). In some embodiments of the present invention,
homologous sections of an inactivating chromosomal segment of a DNA
construct align with the flanking homologous regions of the
indigenous chromosomal region of the Bacillus chromosome.
Subsequently, the indigenous chromosomal region is deleted by the
DNA construct in a double crossover (i.e., homologous
recombination).
[0098] "Homologous recombination" means the exchange of DNA
fragments between two DNA molecules or paired chromosomes at the
site of identical or nearly identical nucleotide sequences. In a
preferred embodiment, chromosomal integration is homologous
recombination.
[0099] "Homologous sequences" as used herein means a nucleic acid
or polypeptide sequence having 100%, 99%, 98%, 97%, 96%, 95%, 94%,
93%, 92%, 91%, 90%, 88%, 85%, 80%, 75%, or 70% sequence identity to
another nucleic acid or polypeptide sequence when optimally aligned
for comparison. In some embodiments, homologous sequences have
between 85% and 100% sequence identity, while in other embodiments
there is between 90% and 100% sequence identity, and in more
preferred embodiments, there is 95% and 100% sequence identity.
[0100] As used herein "amino acid" refers to peptide or protein
sequences or portions thereof. The terms "protein," "peptide," and
"polypeptide" are used interchangeably.
[0101] As used herein, the term "heterologous protein" refers to a
protein or polypeptide that does not naturally occur in the host
cell. Examples of heterologous proteins include enzymes such as
hydrolases including proteases. In some embodiments, the gene
encoding the proteins are naturally occurring genes, while in other
embodiments, mutated and/or synthetic genes are used.
[0102] As used herein, "homologous protein" refers to a protein or
polypeptide native or naturally occurring in a cell. In preferred
embodiments, the cell is a Gram-positive cell, while in
particularly preferred embodiments the cell is a Bacillus host
cell. In alternative embodiments, the homologous protein is a
native protein produced by other organisms, including but not
limited to E. coli, Streptomyces, Trichoderma, and Aspergillus. The
invention encompasses host cells producing the homologous protein
via recombinant DNA technology.
[0103] As used herein, an "operon region" comprises a group of
contiguous genes that are transcribed as a single transcription
unit from a common promoter, and are thereby subject to
co-regulation. In some embodiments, the operon includes a regulator
gene. In most preferred embodiments, operons that are highly
expressed as measured by RNA levels, but have an unknown or
unnecessary function are used.
[0104] As used herein, an "antimicrobial region" is a region
containing at least one gene that encodes an antimicrobial
protein.
[0105] A polynucleotide is said to "encode" an RNA or a polypeptide
if, in its native state or when manipulated by methods known to
those of skill in the art, it can be transcribed and/or translated
to produce the RNA, the polypeptide or a fragment thereof. The
anti-sense strand of such a nucleic acid is also said to encode the
sequences.
[0106] As is known in the art, a DNA can be transcribed by an RNA
polymerase to produce RNA, but an RNA can be reverse transcribed by
reverse transcriptase to produce a DNA. Thus a DNA can encode a RNA
and vice versa.
[0107] The term "regulatory segment" or "regulatory sequence" or
"expression control sequence" refers to a polynucleotide sequence
of DNA that is operatively linked with a polynucleotide sequence of
DNA that encodes the amino acid sequence of a polypeptide chain to
effect the expression of the encoded amino acid sequence. The
regulatory sequence can inhibit, repress, or promote the expression
of the operably linked polynucleotide sequence encoding the amino
acid.
[0108] "Host strain" or "host cell" refers to a suitable host for
an expression vector comprising DNA according to the present
invention.
[0109] An enzyme is "overexpressed" in a host cell if the enzyme is
expressed in the cell at a higher level that the level at which it
is expressed in a corresponding wild-type cell.
[0110] The terms "protein" and "polypeptide" are used
interchangeability herein. The 3-letter code for amino acids as
defined in conformity with the IUPAC-IUB Joint Commission on
Biochemical Nomenclature (JCBN) is used through out this
disclosure. It is also understood that a polypeptide may be coded
for by more than one nucleotide sequence due to the degeneracy of
the genetic code.
[0111] A "prosequence" is an amino acid sequence between the signal
sequence and mature protease that is necessary for the secretion of
the protease. Cleavage of the pro sequence will result in a mature
active protease.
[0112] The term "signal sequence" or "signal peptide" refers to any
sequence of nucleotides and/or amino acids that participate in the
secretion of the mature or precursor forms of the protein. This
definition of signal sequence is a functional one, meant to include
all those amino acid sequences encoded by the N-terminal portion of
the protein gene, which participate in the effectuation of the
secretion of protein. They are often, but not universally, bound to
the N-terminal portion of a protein or to the N-terminal portion of
a precursor protein. The signal sequence may be endogenous or
exogenous. The signal sequence may be that normally associated with
the protein (e.g., protease), or may be from a gene encoding
another secreted protein. One exemplary exogenous signal sequence
comprises the first seven amino acid residues of the signal
sequence from Bacillus subtilis subtilisin fused to the remainder
of the signal sequence of the subtilisin from Bacillus lentus (ATCC
21536).
[0113] The term "hybrid signal sequence" refers to signal sequences
in which part of sequence is obtained from the expression host
fused to the signal sequence of the gene to be expressed. In some
embodiments, synthetic sequences are utilized.
[0114] The term "mature" form of a protein or peptide refers to the
final functional form of the protein or peptide. To exemplify, a
mature form of thermolysin includes the amino acid sequence of SEQ
ID NO:3.
[0115] The term "precursor" form of a protein or peptide refers to
a mature form of the protein having a prosequence operably linked
to the amino or carbonyl terminus of the protein. The precursor may
also have a "signal" sequence operably linked, to the amino
terminus of the prosequence. The precursor may also have additional
polynucleotides that are involved in post-translational activity
(e.g., polynucleotides cleaved therefrom to leave the mature form
of a protein or peptide).
[0116] "Naturally occurring enzyme" refers to an enzyme having the
unmodified amino acid sequence identical to that found in nature.
Naturally occurring enzymes include native enzymes, those enzymes
naturally expressed or found in the particular microorganism.
[0117] The terms "derived from" and "obtained from" refer to not
only a protease produced or producible by a strain of the organism
in question, but also a protease encoded by a DNA sequence isolated
from such strain and produced in a host organism containing such
DNA sequence. Additionally, the term refers to a protease that is
encoded by a DNA sequence of synthetic and/or cDNA origin and which
has the identifying characteristics of the protease in question. To
exemplify, "proteases derived from Bacillus sp." refers to those
enzymes having proteolytic activity which are naturally-produced by
Bacillus sp., as well as to neutral metalloproteases like those
produced by Bacillus sp. sources but which through the use of
genetic engineering techniques are produced by non-Geobacillus
caldoproteolyticus organisms transformed with a nucleic acid
encoding said neutral metalloproteases.
[0118] A "derivative" within the scope of this definition generally
retains the characteristic proteolytic activity observed in the
wild-type, native or parent form to the extent that the derivative
is useful for similar purposes as the wild-type, native or parent
form. Functional derivatives of neutral metalloprotease encompass
naturally occurring, synthetically or recombinantly produced
peptides or peptide fragments having the general characteristics of
the neutral metalloprotease of the present invention.
[0119] The term "functional derivative" refers to a derivative of a
nucleic acid having the functional characteristics of a nucleic
acid encoding a neutral metalloprotease. Functional derivatives of
a nucleic acid, which encode neutral metalloprotease of the present
invention encompass naturally occurring, synthetically or
recombinantly produced nucleic acids or fragments and encode
neutral metalloprotease characteristic of the present invention.
Wild type nucleic acid encoding neutral metalloprotease according
to the invention include naturally occurring alleles and homologues
based on the degeneracy of the genetic code known in the art.
[0120] The term "identical" in the context of two nucleic acids or
polypeptide sequences refers to the residues in the two sequences
that are the same when aligned for maximum correspondence, as
measured using one of the following sequence comparison or analysis
algorithms.
[0121] The term "optimal alignment" refers to the alignment giving
the highest percent identity score.
[0122] "Percent sequence identity," "percent amino acid sequence
identity," "percent gene sequence identity," and/or "percent
nucleic acid/polynucloetide sequence identity," with respect to two
amino acid, polynucleotide and/or gene sequences (as appropriate),
refer to the percentage of residues that are identical in the two
sequences when the sequences are optimally aligned. Thus, 80% amino
acid sequence identity means that 80% of the amino acids in two
optimally aligned polypeptide sequences are identical.
[0123] The phrase "substantially identical" in the context of two
nucleic acids or polypeptides thus refers to a polynucleotide or
polypeptide that comprising at least 70% sequence identity,
preferably at least 75%, preferably at least 80%, preferably at
least 85%, preferably at least 90%, preferably at least 95%,
preferably at least 97%, preferably at least 98% and preferably at
least 99% sequence identity as compared to a reference sequence
using the programs or algorithms (e.g., BLAST, ALIGN, CLUSTAL)
using standard parameters. One indication that two polypeptides are
substantially identical is that the first polypeptide is
immunologically cross-reactive with the second polypeptide.
Typically, polypeptides that differ by conservative amino acid
substitutions are immunologically cross-reactive. Thus, a
polypeptide is substantially identical to a second polypeptide, for
example, where the two peptides differ only by a conservative
substitution. Another indication that two nucleic acid sequences
are substantially identical is that the two molecules hybridize to
each other under stringent conditions (e.g., within a range of
medium to high stringency).
[0124] The term "isolated" or "purified" refers to a material that
is removed from its original environment (e.g., the natural
environment if it is naturally occurring). For example, the
material is said to be "purified" when it is present in a
particular composition in a higher or lower concentration than
exists in a naturally occurring or wild type organism or in
combination with components not normally present upon expression
from a naturally occurring or wild type organism. For example, a
naturally-occurring polynucleotide or polypeptide present in a
living animal is not isolated, but the same polynucleotide or
polypeptide, separated from some or all of the coexisting materials
in the natural system, is isolated. Such polynucleotides could be
part of a vector, and/or such polynucleotides or polypeptides could
be part of a composition, and still be isolated in that such vector
or composition is not part of its natural environment. In preferred
embodiments, a nucleic acid or protein is said to be purified, for
example, if it gives rise to essentially one band in an
electrophoretic gel or blot.
[0125] The term "isolated", when used in reference to a DNA
sequence, refers to a DNA sequence that has been removed from its
natural genetic milieu and is thus free of other extraneous or
unwanted coding sequences, and is in a form suitable for use within
genetically engineered protein production systems. Such isolated
molecules are those that are separated from their natural
environment and include cDNA and genomic clones. Isolated DNA
molecules of the present invention are free of other genes with
which they are ordinarily associated, but may include naturally
occurring 5' and 3' untranslated regions such as promoters and
terminators. The identification of associated regions will be
evident to one of ordinary skill in the art (See e.g., Dynan and
Tijan, Nature 316:774-78, 1985). The term "an isolated DNA
sequence" is alternatively referred to as "a cloned DNA
sequence".
[0126] The term "isolated," when used in reference to a protein,
refers to a protein that is found in a condition other than its
native environment. In a preferred form, the isolated protein is
substantially free of other proteins, particularly other homologous
proteins. An isolated protein is more than 10% pure, preferably
more than 20% pure, and even more preferably more than 30% pure, as
determined by SDS-PAGE. Further aspects of the invention encompass
the protein in a highly purified form (i.e., more than 40% pure,
more than 60% pure, more than 80% pure, more than 90% pure, more
than 95% pure, more than 97% pure, and even more than 99% pure), as
determined by SDS-PAGE.
[0127] The following cassette mutagenesis method may be used to
facilitate the construction of the enzyme variants of the present
invention, although other methods may be used. First, as described
herein, a naturally-occurring gene encoding the enzyme is obtained
and sequenced in whole or in part. Then, the sequence is scanned
for a point at which it is desired to make a mutation (deletion,
insertion or substitution) of one or more amino acids in the
encoded enzyme. The sequences flanking this point are evaluated for
the presence of restriction sites for replacing a short segment of
the gene with an oligonucleotide pool which when expressed will
encode various mutants. Such restriction sites are preferably
unique sites within the protein gene so as to facilitate the
replacement of the gene segment. However, any convenient
restriction site that is not overly redundant in the enzyme gene
may be used, provided the gene fragments generated by restriction
digestion can be reassembled in proper sequence. If restriction
sites are not present at locations within a convenient distance
from the selected point (from 10 to 15 nucleotides), such sites are
generated by substituting nucleotides in the gene in such a fashion
that neither the reading frame nor the amino acids encoded are
changed in the final construction. Mutation of the gene in order to
change its sequence to conform to the desired sequence is
accomplished by M13 primer extension in accord with generally known
methods. The task of locating suitable flanking regions and
evaluating the needed changes to arrive at two convenient
restriction site sequences is made routine by the redundancy of the
genetic code, a restriction enzyme map of the gene and the large
number of different restriction enzymes. Note that if a convenient
flanking restriction site is available, the above method need be
used only in connection with the flanking region that does not
contain a site.
[0128] Once the naturally-occurring DNA and/or synthetic DNA is
cloned, the restriction sites flanking the positions to be mutated
are digested with the cognate restriction enzymes and a plurality
of end termini-complementary oligonucleotide cassettes are ligated
into the gene. The mutagenesis is simplified by this method because
all of the oligonucleotides can be synthesized so as to have the
same restriction sites, and no synthetic linkers are necessary to
create the restriction sites.
[0129] As used herein, "corresponding to," refers to a residue at
the enumerated position in a protein or peptide, or a residue that
is analogous, homologous, or equivalent to an enumerated residue in
a protein or peptide.
[0130] As used herein, "corresponding region," generally refers to
an analogous position along related proteins or a parent
protein.
[0131] As used herein, the term, "combinatorial mutagenesis" refers
to methods in which libraries of variants of a starting sequence
are generated. In these libraries, the variants contain one or
several mutations chosen from a predefined set of mutations. In
addition, the methods provide means to introduce random mutations,
which were not members of the predefined set of mutations. In some
embodiments, the methods include those set forth in U.S.
application Ser. No. 09/699,250, filed Oct. 26, 2000, hereby
incorporated by reference. In alternative embodiments,
combinatorial mutagenesis methods encompass commercially available
kits (e.g., QUIKCHANGE.RTM. Multisite, Stratagene, San Diego,
Calif.).
[0132] As used herein, the term "library of mutants" refers to a
population of cells which are identical in most of their genome but
include different homologues of one or more genes. Such libraries
can be used, for example, to identify genes or operons with
improved traits.
[0133] As used herein, the terms "starting gene" and "parent gene"
refer to a gene of interest that encodes a protein of interest that
is to be improved and/or changed using the present invention.
[0134] As used herein, the terms "multiple sequence alignment" and
"MSA" refer to the sequences of multiple homologs of a starting
gene that are aligned using an algorithm (e.g., Clustal W).
[0135] As used herein, the terms "consensus sequence" and
"canonical sequence" refer to an archetypical amino acid sequence
against which all variants of a particular protein or sequence of
interest are compared. The terms also refer to a sequence that sets
forth the nucleotides that are most often present in a DNA sequence
of interest. For each position of a gene, the consensus sequence
gives the amino acid that is most abundant in that position in the
MSA.
[0136] As used herein, the term "consensus mutation" refers to a
difference in the sequence of a starting gene and a consensus
sequence. Consensus mutations are identified by comparing the
sequences of the starting gene and the consensus sequence obtained
from a MSA. In some embodiments, consensus mutations are introduced
into the starting gene such that it becomes more similar to the
consensus sequence. Consensus mutations also include amino acid
changes that change an amino acid in a starting gene to an amino
acid that is more frequently found in an MSA at that position
relative to the frequency of that amino acid in the starting gene.
Thus, the term consensus mutation comprises all single amino acid
changes that replace an amino acid of the starting gene with an
amino acid that is more abundant than the amino acid in the
MSA.
[0137] The terms "modified sequence" and "modified genes" are used
interchangeably herein to refer to a sequence that includes a
deletion, insertion or interruption of naturally occurring nucleic
acid sequence. In some preferred embodiments, the expression
product of the modified sequence is a truncated protein (e.g., if
the modification is a deletion or interruption of the sequence). In
some particularly preferred embodiments, the truncated protein
retains biological activity. In alternative embodiments, the
expression product of the modified sequence is an elongated protein
(e.g., modifications comprising an insertion into the nucleic acid
sequence). In some embodiments, an insertion leads to a truncated
protein (e.g., when the insertion results in the formation of a
stop codon). Thus, an insertion may result in either a truncated
protein or an elongated protein as an expression product.
[0138] As used herein, the terms "mutant sequence" and "mutant
gene" are used interchangeably and refer to a sequence that has an
alteration in at least one codon occurring in a host cell's
wild-type sequence. The expression product of the mutant sequence
is a protein with an altered amino acid sequence relative to the
wild-type. The expression product may have an altered functional
capacity (e.g., enhanced enzymatic activity).
[0139] The terms "mutagenic primer" or "mutagenic oligonucleotide"
(used interchangeably herein) are intended to refer to
oligonucleotide compositions which correspond to a portion of the
template sequence and which are capable of hybridizing thereto.
With respect to mutagenic primers, the primer will not precisely
match the template nucleic acid, the mismatch or mismatches in the
primer being used to introduce the desired mutation into the
nucleic acid library. As used herein, "non-mutagenic primer" or
"non-mutagenic oligonucleotide" refers to oligonucleotide
compositions that match precisely to the template nucleic acid. In
one embodiment of the invention, only mutagenic primers are used.
In another preferred embodiment of the invention, the primers are
designed so that for at least one region at which a mutagenic
primer has been included, there is also non-mutagenic primer
included in the oligonucleotide mixture. By adding a mixture of
mutagenic primers and non-mutagenic primers corresponding to at
least one of the mutagenic primers, it is possible to produce a
resulting nucleic acid library in which a variety of combinatorial
mutational patterns are presented. For example, if it is desired
that some of the members of the mutant nucleic acid library retain
their parent sequence at certain positions while other members are
mutant at such sites, the non-mutagenic primers provide the ability
to obtain a specific level of non-mutant members within the nucleic
acid library for a given residue. The methods of the invention
employ mutagenic and non-mutagenic oligonucleotides which are
generally between 10-50 bases in length, more preferably about
15-45 bases in length. However, it may be necessary to use primers
that are either shorter than 10 bases or longer than 50 bases to
obtain the mutagenesis result desired. With respect to
corresponding mutagenic and non-mutagenic primers, it is not
necessary that the corresponding oligonucleotides be of identical
length, but only that there is overlap in the region corresponding
to the mutation to be added.
[0140] Primers may be added in a pre-defined ratio according to the
present invention. For example, if it is desired that the resulting
library have a significant level of a certain specific mutation and
a lesser amount of a different mutation at the same or different
site, by adjusting the amount of primer added, it is possible to
produce the desired biased library. Alternatively, by adding lesser
or greater amounts of non-mutagenic primers, it is possible to
adjust the frequency with which the corresponding mutation(s) are
produced in the mutant nucleic acid library.
[0141] The terms "wild-type sequence," or "wild-type gene" are used
interchangeably herein, to refer to a sequence that is native or
naturally occurring in a host cell. In some embodiments, the
wild-type sequence refers to a sequence of interest that is the
starting point of a protein-engineering project. The wild-type
sequence may encode either a homologous or heterologous protein. A
homologous protein is one the host cell would produce without
intervention. A heterologous protein is one that the host cell
would not produce but for the intervention.
[0142] As used herein, the term "equivalent" when used in reference
to the position of an amino acid residue in a thermolysin protein
refers to the position of an amino acid residue in a thermolysin
variant that corresponds in position in the primary sequence of the
unmodified precursor e.g. wild-type thermolysin. In order to
establish the position of equivalent amino acid positions in a
thermolysin, the amino acid sequence of the thermolysin that is
modified to generate the thermolysin variant is directly compared
to the thermolysin of SEQ ID NO:3. After aligning the residues,
allowing for insertions and deletions in order to maintain
alignment (i.e. avoiding the elimination of conserved residues
through arbitrary deletion or insertion), the residues at positions
equivalent to particular amino acid positions in the sequence of
the thermolysin of SEQ ID NO:3 are defined.
[0143] The term "oxidation stable" refers to proteases of the
present invention that retain a specified amount of enzymatic
activity over a given period of time under conditions prevailing
during the proteolytic, hydrolyzing, cleaning or other process of
the invention, for example while exposed to or contacted with
bleaching agents or oxidizing agents. In some embodiments, the
proteases retain at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 92%,
95%, 96%, 97%, 98% or 99% proteolytic activity after contact with a
bleaching or oxidizing agent over a given time period, for example,
at least 1 minute, 3 minutes, 5 minutes, 8 minutes, 12 minutes, 16
minutes, 20 minutes, etc.
[0144] The term "chelator stable" refers to proteases of the
present invention that retain a specified amount of enzymatic
activity over a given period of time under conditions prevailing
during the proteolytic, hydrolyzing, cleaning or other process of
the invention, for example while exposed to or contacted with
chelating agents. In some embodiments, the proteases retain at
least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or
99% proteolytic activity after contact with a chelating agent over
a given time period, for example, at least 10 minutes, 20 minutes,
40 minutes, 60 minutes, 100 minutes, etc.
[0145] The terms "thermally stable" and "thermostable" refer to
proteases of the present invention that retain a specified amount
of enzymatic activity after exposure to identified temperatures
over a given period of time under conditions prevailing during the
proteolytic, hydrolyzing, cleaning or other process of the
invention, for example while exposed altered temperatures. Altered
temperatures include increased or decreased temperatures. In some
embodiments, the proteases retain at least 50%, 60%, 70%, 75%, 80%,
85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% proteolytic activity after
exposure to altered temperatures over a given time period, for
example, at least 60 minutes, 120 minutes, 180 minutes, 240
minutes, 300 minutes, etc.
[0146] As used herein, the term "chemical stability" refers to the
stability of a protein (e.g., an enzyme) towards chemicals that
adversely affect its activity. In some embodiments, such chemicals
include, but are not limited to hydrogen peroxide, peracids,
anionic detergents, cationic detergents, non-ionic detergents,
chelants, etc. However, it is not intended that the present
invention be limited to any particular chemical stability level nor
range of chemical stability. In particular, the terms "detergent
stable" and "LAS stable" refer to proteases of the present
invention that retain a specified amount of enzymatic activity
after exposure to a detergent composition over a given period of
time under conditions prevailing during the proteolytic,
hydrolyzing, cleaning or other process of the invention. In some
embodiments, the proteases retain at least 50%, 60%, 70%, 75%, 80%,
85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% proteolytic activity after
exposure to detergent over a given time period, for example, at
least 60 minutes, 120 minutes, 180 minutes, 240 minutes, 300
minutes, etc.
[0147] The term "enhanced stability" in the context of an
oxidation, chelator, thermal and/or pH stable protease refers to a
higher retained proteolytic activity over time as compared to other
neutral metalloproteases and/or wild-type enzymes.
[0148] The term "diminished stability" in the context of an
oxidation, chelator, thermal and/or pH stable protease refers to a
lower retained proteolytic activity over time as compared to other
neutral metalloproteases and/or wild-type enzymes.
[0149] As used herein, the term "cleaning composition" includes,
unless otherwise indicated, granular or powder-form all-purpose or
"heavy-duty" washing agents, especially cleaning detergents;
liquid, gel or paste-form all-purpose washing agents, especially
the so-called heavy-duty liquid types; liquid fine-fabric
detergents; hand dishwashing agents or light duty dishwashing
agents, especially those of the high-foaming type; machine
dishwashing agents, including the various tablet, granular, liquid
and rinse-aid types for household and institutional use; liquid
cleaning and disinfecting agents, including antibacterial hand-wash
types, cleaning bars, mouthwashes, denture cleaners, car or carpet
shampoos, bathroom cleaners; hair shampoos and hair-rinses; shower
gels and foam baths and metal cleaners; as well as cleaning
auxiliaries such as bleach additives and "stain-stick" or pre-treat
types.
[0150] Unless otherwise noted, all component or composition levels
are in reference to the active level of that component or
composition, and are exclusive of impurities, for example, residual
solvents or by-products, which may be present in commercially
available sources.
[0151] Enzyme components weights are based on total active protein.
All percentages and ratios are calculated by weight unless
otherwise indicated. All percentages and ratios are calculated
based on the total composition unless otherwise indicated.
[0152] The term "cleaning activity" refers to the cleaning
performance achieved by the protease under conditions prevailing
during the proteolytic, hydrolyzing, cleaning or other process of
the invention. In some embodiments, cleaning performance is
determined by the application of various cleaning assays concerning
enzyme sensitive stains, for example grass, blood, milk, or egg
protein as determined by various chromatographic,
spectrophotometric or other quantitative methodologies after
subjection of the stains to standard wash conditions. Exemplary
assays include, but are not limited to those described in WO
99/34011, and U.S. Pat. No. 6,605,458 (both of which are herein
incorporated by reference), as well as those methods included in
the Examples.
[0153] The term "cleaning effective amount" of a protease refers to
the quantity of protease described hereinbefore that achieves a
desired level of enzymatic activity in a specific cleaning
composition. Such effective amounts are readily ascertained by one
of ordinary skill in the art and are based on many factors, such as
the particular protease used, the cleaning application, the
specific composition of the cleaning composition, and whether a
liquid or dry (e.g., granular, bar) composition is required,
etc.
[0154] The term "cleaning adjunct materials" as used herein, means
any liquid, solid or gaseous material selected for the particular
type of cleaning composition desired and the form of the product
(e.g., liquid, granule, powder, bar, paste, spray, tablet, gel; or
foam composition), which materials are also preferably compatible
with the protease enzyme used in the composition. In some
embodiments, granular compositions are in "compact" form, while in
other embodiments, the liquid compositions are in a "concentrated"
form.
[0155] As used herein, a "low detergent concentration" system
includes detergents where less than about 800 ppm of detergent
components are present in the wash water. Japanese detergents are
typically considered low detergent concentration systems, as they
have usually have approximately 667 ppm of detergent components
present in the wash water.
[0156] As used herein, a "medium detergent concentration" systems
includes detergents wherein between about 800 ppm and about 2000
ppm of detergent components are present in the wash water. North
American detergents are generally considered to be medium detergent
concentration systems as they have usually approximately 975 ppm of
detergent components present in the wash water. Brazilian
detergents typically have approximately 1500 ppm of detergent
components present in the wash water.
[0157] As used herein, "high detergent concentration" systems
includes detergents wherein greater than about 2000 ppm of
detergent components are present in the wash water. European
detergents are generally considered to be high detergent
concentration systems as they have approximately 3000-8000 ppm of
detergent components in the wash water.
[0158] As used herein, "fabric cleaning compositions" include hand
and machine laundry detergent compositions including laundry
additive compositions and compositions suitable for use in the
soaking and/or pretreatment of stained fabrics (e.g., clothes,
linens, and other textile materials).
[0159] As used herein, "non-fabric cleaning compositions" include
non-textile (i.e., fabric) surface cleaning compositions, including
but not limited to dishwashing detergent compositions, oral
cleaning compositions, denture cleaning compositions, and personal
cleansing compositions.
[0160] The "compact" form of the cleaning compositions herein is
best reflected by density and, in terms of composition, by the
amount of inorganic filler salt. Inorganic filler salts are
conventional ingredients of detergent compositions in powder form.
In conventional detergent compositions, the filler salts are
present in substantial amounts, typically 17-35% by weight of the
total composition. In contrast, in compact compositions, the filler
salt is present in amounts not exceeding 15% of the total
composition. In some embodiments, the filler salt is present in
amounts that do not exceed 10%, or more preferably, 5%, by weight
of the composition. In some embodiments, the inorganic filler salts
are selected from the alkali and alkaline-earth-metal salts of
sulfates and chlorides. A preferred filler salt is sodium
sulfate.
DETAILED DESCRIPTION OF THE INVENTION
[0161] Neutral metalloendopeptidases (i.e., neutral
metalloproteases) (EC 3.4.24.4) belong to a protease class that has
an absolute requirement for zinc ions for catalytic activity. These
enzymes are optimally active at neutral pH and are in the 30 to 40
kDa size range. Neutral metalloproteases bind between two and four
calcium ions that contribute to the structural stability of the
protein. The bound metal ion at the active site of metalloproteases
is an essential feature that allows the activation of a water
molecule. The water molecule then functions as the nucleophile and
cleaves the carbonyl group of the peptide bond.
[0162] The neutral zinc-binding metalloprotease family includes the
bacterial enzyme thermolysin, and thermolysin-like proteases
(TLPs), as well as carboxypeptidase A (a digestive enzyme), and the
matrix metalloproteases that catalyze the reactions in tissue
remodeling and degradation. The only well characterized of these
proteases, with respect to stability and function is thermolysin,
which hydrolyzes protein bonds on the amino-terminal side of
hydrophobic amino acid residues. Thermolysin is a thermostable
neutral zinc metalloproteinase first identified in the culture
broth of Bacillus thermoproteolyticus Rokko. Subsequently, a
similar neutral metalloprotease was identified in Geobacillus
caldoprotelyticus, and this enzyme is also referred to herein as
thermolysin. Natural and engineered proteases, such as thermolysin
are often expressed in Bacillus subtilis (O'Donohue et al., Biochem
J, 300:599-603, 1994), and several have been applied in detergent
formulations to remove proteinaceous stains. Today, thermolysin is
used in industry, especially for the enzymatic synthesis of
N-carbobenzoxy 1-Asp-1-Phe methyl ester, a precursor of the
artificial sweetener aspartame.
[0163] In general however, the serine proteases have been more
widely utilized in detergents, at least partially due to the
relative ease with which these proteases can be stabilized.
[0164] Indeed, metalloproteases are less frequently used in
industry, and particularly in the detergent industry for a number
of reasons. These enzymes involve more complex protein systems, as
the enzymes have the absolute requirement for calcium and zinc ions
for stability and function, respectively. Further, the detergent
solution as well as the water used in the laundry process often
contains components that interfere with the binding of the ions by
the enzyme, or chelate these ions, resulting in a decrease or loss
of proteolytic function and destabilization of the protease.
Detailed Description of Cleaning and Detergent Formulations of the
Present Invention
[0165] Unless otherwise noted, all component or composition levels
provided herein are made in reference to the active level of that
component or composition, and are exclusive of impurities, for
example, residual solvents or by-products, which may be present in
commercially available sources. Enzyme components weights are based
on total active protein. All percentages and ratios are calculated
by weight unless otherwise indicated. All percentages and ratios
are calculated based on the total composition unless otherwise
indicated.
[0166] In the exemplified detergent compositions, the enzymes
levels are expressed by pure enzyme by weight of the total
composition and unless otherwise specified, the detergent
ingredients are expressed by weight of the total compositions.
Cleaning Compositions Comprising Neutral Metalloprotease
[0167] The neutral metalloproteases of the present invention are
useful in formulating various detergent compositions. The cleaning
composition of the present invention may be advantageously employed
for example, in laundry applications, hard surface cleaning,
automatic dishwashing applications, as well as cosmetic
applications such as dentures, teeth, hair and skin. However, due
to the unique advantages of increased effectiveness in lower
temperature solutions and the superior color-safety profile, the
enzymes of the present invention are ideally suited for laundry
applications such as the bleaching of fabrics. Furthermore, the
enzymes of the present invention find use in both granular and
liquid compositions.
[0168] The enzymes of the present invention also find use in
cleaning additive products. A cleaning additive product including
at least one enzyme of the present invention is ideally suited for
inclusion in a wash process when additional bleaching effectiveness
is desired. Such instances include, but are not limited to low
temperature solution cleaning applications. The additive product
may be, in its simplest form, one or more neutral metalloprotease
enzyme as provided by the present invention. In some embodiments,
the additive is packaged in dosage form for addition to a cleaning
process where a source of peroxygen is employed and increased
bleaching effectiveness is desired. In some embodiments, the single
dosage form comprises a pill, tablet, gelcap or other single dosage
unit including pre-measured powders and/or liquids. In some
embodiments, filler and/or carrier material(s) are included, in
order to increase the volume of such composition. Suitable filler
or carrier materials include, but are not limited to, various salts
of sulfate, carbonate and silicate as well as talc, clay and the
like. In some embodiments filler and/or carrier materials for
liquid compositions include water and/or low molecular weight
primary and secondary alcohols including polyols and diols.
Examples of such alcohols include, but are not limited to,
methanol, ethanol, propanol and isopropanol. In some embodiments,
the compositions comprise from about 5% to about 90% of such
materials. In additional embodiments, acidic fillers are used to
reduce the pH of the composition. In some alternative embodiments
the cleaning additive includes at least one activated peroxygen
source as described below and/or adjunct ingredients as more fully
described below.
[0169] The cleaning compositions and cleaning additives of the
present invention require an effective amount of neutral
metalloprotease enzyme as provided in the present invention. In
some embodiments, the required level of enzyme is achieved by the
addition of one or more species of neutral metalloprotease provided
by the present invention. Typically, the cleaning compositions of
the present invention comprise at least 0.0001 weight percent, from
about 0.0001 to about 1, from about 0.001 to about 0.5, or even
from about 0.01 to about 0.1 weight percent of at least one neutral
metalloprotease provided by the present invention.
[0170] In some preferred embodiments, the cleaning compositions
provided herein are typically formulated such that, during use in
aqueous cleaning operations, the wash water has a pH of from about
5.0 to about 11.5, or in alternative embodiments, even from about
6.0 to about 10.5. In some preferred embodiments, liquid product
formulations are typically formulated to have a neat pH from about
3.0 to about 9.0, while in some alternative embodiments the
formulation has a neat pH from about 3 to about 5. In some
preferred embodiments, granular laundry products are typically
formulated to have a pH from about 8 to about 11. Techniques for
controlling pH at recommended usage levels include the use of
buffers, alkalis, acids, etc., and are well known to those skilled
in the art.
[0171] In some particularly preferred embodiments, when at least
one neutral metalloprotease is employed in a granular composition
or liquid, the neutral metalloprotease is in the form of an
encapsulated particle to protect the enzyme from other components
of the granular composition during storage. In addition,
encapsulation also provides a means of controlling the availability
of the neutral metalloprotease(s) during the cleaning process and
may enhance performance of the neutral metalloprotease(s). It is
contemplated that the encapsulated neutral metalloproteases of the
present invention will find use in various settings. It is also
intended that the neutral metalloprotease be encapsulated using any
suitable encapsulating material(s) and method(s) known in the
art.
[0172] In some preferred embodiments, the encapsulating material
typically encapsulates at least part of the neutral metalloprotease
catalyst. In some embodiments, the encapsulating material is
water-soluble and/or water-dispersible. In some additional
embodiments, the encapsulating material has a glass transition
temperature (Tg) of 0.degree. C. or higher (See e.g., WO 97/11151,
particularly from page 6, line 25 to page 7, line 2, for more
information regarding glass transition temperatures).
[0173] In some embodiments, the encapsulating material is chosen
from carbohydrates, natural or synthetic gums, chitin and chitosan,
cellulose and cellulose derivatives, silicates, phosphates,
borates, polyvinyl alcohol, polyethylene glycol, paraffin waxes and
combinations thereof. In some embodiments in which the
encapsulating material is a carbohydrate, it is chosen from
monosaccharides, oligosaccharides, polysaccharides, and
combinations thereof. In some preferred embodiments, the
encapsulating material is a starch (See e.g., EP 0 922 499; U.S.
Pat. No. 4,977,252. U.S. Pat. No. 5,354,559, and U.S. Pat. No.
5,935,826, for descriptions of some exemplary starches).
[0174] In additional embodiments, the encapsulating material
comprises a microsphere made from plastic (e.g., thermoplastics,
acrylonitrile, methacrylonitrile, polyacrylonitrile,
polymethacrylonitrile and mixtures thereof; commercially available
microspheres that find use include, but are not limited to
EXPANCEL.RTM. [Casco Products, Stockholm, Sweden], PM 6545, PM
6550, PM 7220, PM 7228, EXTENDOSPHERES.RTM., and Q-CEL.RTM. [PQ
Corp., Valley Forge, Pa.], LUXSIL.RTM. and SPHERICEL1.RTM. [Potters
Industries, Inc., Carlstadt, N.J. and Valley Forge, Pa.]).
Processes of Making and Using of Applicants' Cleaning
Composition
[0175] In some preferred embodiments compositions of the present
invention are formulated into any suitable form and prepared by any
process chosen by the formulator, (See e.g., U.S. 5,879,584, U.S.
Pat. No. 5,691,297, U.S. Pat. No. 5,574,005, U.S. Pat. No.
5,569,645, U.S. Pat. No. 5,565,422, U.S. Pat. No. 5,516,448, U.S.
Pat. No. 5,489,392, and U.S. Pat. No. 5,486,303, for some
non-limiting examples). In some embodiments in which a low pH
cleaning composition is desired, the pH of such composition is
adjusted via the addition of an acidic material such as HCl.
Adjunct Materials
[0176] While not essential for the purposes of the present
invention, in some embodiments, the non-limiting list of adjuncts
described herein are suitable for use in the cleaning compositions
of the present invention. Indeed, in some embodiments, adjuncts are
incorporated into the cleaning compositions of the present
invention. In some embodiments, adjunct materials assist and/or
enhance cleaning performance, treat the substrate to be cleaned,
and/or modify the aesthetics of the cleaning composition (e.g.,
perfumes, colorants, dyes, etc.). It is understood that such
adjuncts are in addition to the neutral metalloproteases of the
present invention. The precise nature of these additional
components, and levels of incorporation thereof, depends on the
physical form of the composition and the nature of the cleaning
operation for which it is to be used. Suitable adjunct materials
include, but are not limited to, surfactants, builders, chelating
agents, dye transfer inhibiting agents, deposition aids,
dispersants, additional enzymes, and enzyme stabilizers, catalytic
materials, bleach activators, bleach boosters, hydrogen peroxide,
sources of hydrogen peroxide, preformed peracids, polymeric
dispersing agents, clay soil removal/anti-redeposition agents,
brighteners, suds suppressors, dyes, perfumes, structure
elasticizing agents, fabric softeners, carriers, hydrotropes,
processing aids and/or pigments. In addition to those provided
explicitly herein, additional examples are known in the art (See
e.g., U.S. Pat. Nos. 5,576,282, 6,306,812 B1 and 6,326,348 B1). In
some embodiments, the aforementioned adjunct ingredients constitute
the balance of the cleaning compositions of the present
invention.
[0177] Surfactants--
[0178] In some embodiments, the cleaning compositions of the
present invention comprise at least one surfactant or surfactant
system, wherein the surfactant is chosen from nonionic surfactants,
anionic surfactants, cationic surfactants, ampholytic surfactants,
zwitterionic surfactants, semi-polar nonionic surfactants, and
mixtures thereof. In some low pH cleaning composition embodiments
(e.g., compositions having a neat pH of from about 3 to about 5),
the composition typically does not contain alkyl ethoxylated
sulfate, as it is believed that such surfactant may be hydrolyzed
in acidic compositions.
[0179] In some embodiments, the surfactant is present at a level of
from about 0.1% to about 60%, while in alternative embodiments, the
level is from about 1% to about 50%, while in still further
embodiments, the level is from about 5% to about 40%, by weight of
the cleaning composition.
[0180] Builders--
[0181] In some embodiments, the cleaning compositions of the
present invention comprise one or more detergent builders or
builder systems. In some embodiments incorporating at least one
builder, the cleaning compositions comprise at least about 1%, from
about 3% to about 60% or even from about 5% to about 40% builder by
weight of the cleaning composition.
[0182] Builders include, but are not limited to, the alkali metal,
ammonium and alkanolammonium salts of polyphosphates, alkali metal
silicates, alkaline earth and alkali metal carbonates,
aluminosilicate builders polycarboxylate compounds. ether
hydroxypolycarboxylates, copolymers of maleic anhydride with
ethylene or vinyl methyl ether, 1,3,5-trihydroxy
benzene-2,4,6-trisulphonic acid, and carboxymethyloxysuccinic acid,
the various alkali metal, ammonium and substituted ammonium salts
of polyacetic acids such as ethylenediamine tetraacetic acid and
nitrilotriacetic acid, as well as polycarboxylates such as mellitic
acid, succinic acid, citric acid, oxydisuccinic acid, polymaleic
acid, benzene 1,3,5-tricarboxylic acid, carboxymethyloxysuccinic
acid, and soluble salts thereof. Indeed, it is contemplated that
any suitable builder will find use in various embodiments of the
present invention.
[0183] Chelating Agents--
[0184] In some embodiments, the cleaning compositions of the
present invention contain at least one chelating agent. Suitable
chelating agents include, but are not limited to copper, iron
and/or manganese chelating agents and mixtures thereof. In
embodiments in which at least one chelating agent is used, the
cleaning compositions of the present invention comprise from about
0.1% to about 15% or even from about 3.0% to about 10% chelating
agent by weight of the subject cleaning composition.
[0185] Deposition Aid--
[0186] In some embodiments, the cleaning compositions of the
present invention include at least one deposition aid. Suitable
deposition aids include, but are not limited to polyethylene
glycol, polypropylene glycol, polycarboxylate, soil release
polymers such as polytelephthalic acid, clays such as kaolinite,
montmorillonite, atapulgite, illite, bentonite, halloysite, and
mixtures thereof.
[0187] Dye Transfer Inhibiting Agents--
[0188] In some embodiments, the cleaning compositions of the
present invention include one or more dye transfer inhibiting
agents. Suitable polymeric dye transfer inhibiting agents include,
but are not limited to, polyvinylpyrrolidone polymers, polyamine
N-oxide polymers, copolymers of N-vinylpyrrolidone and
N-vinylimidazole, polyvinyloxazolidones and polyvinylimidazoles or
mixtures thereof.
[0189] In embodiments in which at least one dye transfer inhibiting
agent is used, the cleaning compositions of the present invention
comprise from about 0.0001% to about 10%, from about 0.01% to about
5%, or even from about 0.1% to about 3% by weight of the cleaning
composition.
[0190] Dispersants--
[0191] In some embodiments, the cleaning compositions of the
present invention contains at least one dispersants. Suitable
water-soluble organic materials include, but are not limited to the
homo- or co-polymeric acids or their salts, in which the
polycarboxylic acid comprises at least two carboxyl radicals
separated from each other by not more than two carbon atoms.
[0192] Enzymes--
[0193] In some embodiments, the cleaning compositions of the
present invention comprise one or more detergent enzymes, which
provide cleaning performance and/or fabric care benefits. Examples
of suitable enzymes include, but are not limited to,
hemicellulases, peroxidases, proteases, cellulases, xylanases,
lipases, phospholipases, esterases, cutinases, pectinases,
keratinases, reductases, oxidases, phenoloxidases, lipoxygenases,
ligninases, pullulanases, tannases, pentosanases, malanases,
B-glucanases, arabinosidases, hyaluronidase, chondroitinase,
laccase, and amylases, or mixtures thereof. In some embodiments, a
combination of enzymes is used (i.e., a "cocktail") comprising
conventional applicable enzymes like protease, lipase, cutinase
and/or cellulase in conjunction with amylase is used.
[0194] Enzyme Stabilizers--
[0195] In some embodiments of the present invention, the enzymes
used in the detergent formulations of the present invention are
stabilized. It is contemplated that various techniques for enzyme
stabilization will find use in the present invention. For example,
in some embodiments, the enzymes employed herein are stabilized by
the presence of water-soluble sources of zinc (II), calcium (II)
and/or magnesium (II) ions in the finished compositions that
provide such ions to the enzymes, as well as. other metal ions
(e.g., barium (II), scandium (II), iron (II), manganese (II),
aluminum (III), Tin (II), cobalt (II), copper (II), Nickel (II),
and oxovanadium (IV)).
[0196] Catalytic Metal Complexes--
[0197] In some embodiments, the cleaning compositions of the
present invention contain one or more catalytic metal complexes. In
some embodiments, a metal-containing bleach catalyst finds use. In
some preferred embodiments, the metal bleach catalyst comprises a
catalyst system comprising a transition metal cation of defined
bleach catalytic activity, (e.g., copper, iron, titanium,
ruthenium, tungsten, molybdenum, or manganese cations), an
auxiliary metal cation having little or no bleach catalytic
activity (e.g., zinc or aluminum cations), and a sequestrate having
defined stability constants for the catalytic and auxiliary metal
cations, particularly ethylenediaminetetraacetic acid,
ethylenediaminetetra (methylenephosphonic acid) and water-soluble
salts thereof are used (See e.g., U.S. Pat. No. 4,430,243).
[0198] In some embodiments, the cleaning compositions of the
present invention are catalyzed by means of a manganese compound.
Such compounds and levels of use are well known in the art (See
e.g., U.S. Pat. No. 5,576,282).
[0199] In additional embodiments, cobalt bleach catalysts find use
in the cleaning compositions of the present invention. Various
cobalt bleach catalysts are known in the art (See e.g., U.S. Pat.
No. 5,597,936, and U.S. Pat. No. 5,595,967). Such cobalt catalysts
are readily prepared by known procedures (See e.g., U.S. Pat. No.
5,597,936, and U.S. Pat. No. 5,595,967).
[0200] In additional embodiments, the cleaning compositions of the
present invention include a transition metal complex of a
macropolycyclic rigid ligand ("MRL"). As a practical matter, and
not by way of limitation, in some embodiments, the compositions and
cleaning processes provided by the present invention are adjusted
to provide on the order of at least one part per hundred million of
the active MRL species in the aqueous washing medium, and in some
preferred embodiments, provide from about 0.005 ppm to about 25
ppm, more preferably from about 0.05 ppm to about 10 ppm, and most
preferably from about 0.1 ppm to about 5 ppm, of the MRL in the
wash liquor.
[0201] Preferred transition-metals in the instant transition-metal
bleach catalyst include, but are not limited to manganese, iron and
chromium. Preferred MRLs also include, but are not limited to
special ultra-rigid ligands that are cross-bridged (e.g.,
5,12-diethyl-1,5,8,12-tetraazabicyclo[6.6.2]hexadecane). Suitable
transition metal MRLs are readily prepared by known procedures (See
e.g., WO 00/32601, and U.S. Pat. No. 6,225,464).
Processes of Making and Using Cleaning Compositions
[0202] The cleaning compositions of the present invention are
formulated into any suitable form and prepared by any suitable
process chosen by the formulator, (See e.g., U.S. Pat. No.
5,879,584, U.S. Pat. No. 5,691,297, U.S. Pat. No. 5,574,005, U.S.
Pat. No. 5,569,645, U.S. Pat. No. 5,565,422, U.S. Pat. No.
5,516,448, U.S. Pat. No. 5,489,392, U.S. Pat. No. 5,486,303, U.S.
Pat. No. 4,515,705, U.S. Pat. No. 4,537,706, U.S. Pat. No.
4,515,707, U.S. Pat. No. 4,550,862, U.S. Pat. No. 4,561,998, U.S.
Pat. No. 4,597,898, U.S. Pat. No. 4,968,451, U.S. Pat. No.
5,565,145, U.S. Pat. No. 5,929,022, U.S. Pat. No. 6,294,514, and
U.S. Pat. No. 6,376,445, all of which are incorporated herein by
reference for some non-limiting examples).
Method of Use
[0203] In preferred embodiments, the cleaning compositions of the
present invention find use in cleaning surfaces and/or fabrics. In
some embodiments, at least a portion of the surface and/or fabric
is contacted with at least one embodiment of the cleaning
compositions of the present invention, in neat form or diluted in a
wash liquor, and then the surface and/or fabric is optionally
washed and/or rinsed. For purposes of the present invention,
"washing" includes, but is not limited to, scrubbing, and
mechanical agitation. In some embodiments, the fabric comprises any
fabric capable of being laundered in normal consumer use
conditions. In preferred embodiments, the cleaning compositions of
the present invention are used at concentrations of from about 500
ppm to about 15,000 ppm in solution. In some embodiments in which
the wash solvent is water, the water temperature typically ranges
from about 5.degree. C. to about 90.degree. C. In some preferred
embodiments for fabric cleaning, the water to fabric mass ratio is
typically from about 1:1 to about 30:1.
EXPERIMENTAL
[0204] The following examples are provided in order to demonstrate
and further illustrate certain preferred embodiments and aspects of
the present invention and are not to be construed as limiting the
scope thereof.
[0205] In the experimental disclosure which follows, the following
abbreviations apply: .degree. C. (degrees Centigrade); rpm
(revolutions per minute); H.sub.2O (water); HCl (hydrochloric
acid); aa and AA (amino acid); bp (base pair); kb (kilobase pair);
kD (kilodaltons); gm (grams); .mu.g and ug (micrograms); mg
(milligrams); ng (nanograms); .mu.l and ul (microliters); ml
(milliliters); mm (millimeters); nm (nanometers); .mu.m and um
(micrometer); M (molar); mM (millimolar); .mu.M and uM
(micromolar); U (units); V (volts); MW (molecular weight); sec
(seconds); min(s) (minute/minutes); hr(s) (hour/hours); MgCl.sub.2
(magnesium chloride); NaCl (sodium chloride); OD.sub.280 (optical
density at 280 nm); OD.sub.405 (optical density at 405 nm);
OD.sub.600 (optical density at 600 nm); PAGE (polyacrylamide gel
electrophoresis); EtOH (ethanol); PBS (phosphate buffered saline
[150 mM NaCl, 10 mM sodium phosphate buffer, pH 7.2]); LAS (lauryl
sodium sulfonate); SDS (sodium dodecyl sulfate); Tris
(tris(hydroxymethyl)aminomethane); TAED
(N,N,N'N'-tetraacetylethylenediamine); BES (polyesstersulfone); MES
(2-morpholinoethanesulfonic acid, monohydrate; f.w. 195.24; Sigma #
M-3671); CaCl.sub.2 (calcium chloride, anhydrous; f.w. 110.99;
Sigma # C-4901); DMF (N,N-dimethylformamide, f.w. 73.09, d=0.95);
Abz-AGLA-Nba
(2-aminobenzoyl-L-alanyl-glycyl-L-leucyl-L-alanino-4-nitrobenzylamide,
f.w. 583.65; Bachem # H-6675, VWR catalog #100040-598); SBG1%
(Super Broth with Glucose; 6 g Soytone [Difco], 3 g yeast extract,
6 g NaCl, 6 g glucose); the pH was adjusted to 7.1 with NaOH prior
to sterilization using methods known in the art; w/v (weight to
volume); v/v (volume to volume); SEQUEST.RTM. (SEQUEST database
search program, University of Washington); MS (mass spectroscopy);
BMI (blood, milk, ink); SRI (Stain Removal Index); Npr and npr
(neutral metalloprotease gene); Npr and npr (neutral
metalloprotease enzyme); NprE and nprE (B. amyloliquefaciens
neutral metalloprotease); PrT and prt (proteinase-T enzyme); and
TLP (thermolysin-like protease).
[0206] The following abbreviations apply to companies whose
products or services may have been referred to in the experimental
examples: TIGR (The Institute for Genomic Research, Rockville,
Md.); AATCC (American Association of Textile and Coloring
Chemists); Amersham (Amersham Life Science, Inc. Arlington Heights,
Ill.); Corning (Corning International, Corning, N.Y.); ICN (ICN
Pharmaceuticals, Inc., Costa Mesa, Calif.); Pierce (Pierce
Biotechnology, Rockford, Ill.); Equest (Equest, Warwick
International Group, Inc., Flintshire, UK); EMPA (Eidgenossische
Material Prufungs and Versuch Anstalt, St. Gallen, Switzerland);
CFT (Center for Test Materials, Vlaardingen, The Netherlands);
Amicon (Amicon, Inc., Beverly, Mass.); ATCC (American Type Culture
Collection, Manassas, Va.); Becton Dickinson (Becton Dickinson
Labware, Lincoln Park, N.J.); Perkin-Elmer (Perkin-Elmer,
Wellesley, Mass.); Rainin (Rainin Instrument, LLC, Woburn, Mass.);
Eppendorf (Eppendorf AG, Hamburg, Germany); Waters (Waters, Inc.,
Milford, Mass.); Perseptive Biosystems (Perseptive Biosystems,
Ramsey, Minn.); Molecular Probes (Molecular Probes, Eugene, Oreg.);
BioRad (BioRad, Richmond, Calif.); Clontech (CLONTECH Laboratories,
Palo Alto, Calif.); Cargill (Cargill, Inc., Minneapolis, Minn.);
Difco (Difco Laboratories, Detroit, Mich.); GIBCO BRL or Gibco BRL
(Life Technologies, Inc., Gaithersburg, Md.); New Brunswick (New
Brunswick Scientific Company, Inc., Edison, N.J.); Thermoelectron
(Thermoelectron Corp., Waltham, Mass.); BMG (BMG Labtech, GmbH,
Offenburg, Germany); Greiner (Greiner Bio-One, Kremsmuenster,
Austria); Novagen (Novagen, Inc., Madison, Wis.); Novex (Novex, San
Diego, Calif.); Finnzymes (Finnzymes OY, Finland) Qiagen (Qiagen,
Inc., Valencia, Calif.); Invitrogen (Invitrogen Corp., Carlsbad,
Calif.); Sigma (Sigma Chemical Co., St. Louis, Mo.); DuPont
Instruments (Asheville, N.Y.); Global Medical Instrumentation or
GMI (Global Medical Instrumentation; Ramsey, Minn.); MJ Research
(MJ Research, Waltham, Mass.); Infors (Infors AG, Bottmingen,
Switzerland); Stratagene (Stratagene Cloning Systems, La Jolla,
Calif.); Roche (Hoffmann La Roche, Inc., Nutley, N.J.); Agilent
(Agilent Technologies, Palo Alto, Calif.); S-Matrix (S-Matrix
Corp., Eureka, Calif.); US Testing (United States Testing Co.,
Hoboken, N.Y.); West Coast Analytical Services (West Coast
Analytical Services, Inc., Santa Fe Springs, Calif.); Ion Beam
Analysis Laboratory (Ion Bean Analysis Laboratory, The University
of Surrey Ion Beam Centre (Guildford, UK); BaChem (BaChem AG,
Bubendorf, Switzerland); Molecular Devices (Molecular Devices,
Inc., Sunnyvale, Calif.); MicroCal (Microcal, Inc., Northhampton,
Mass.); Chemical Computing (Chemical Computing Corp., Montreal,
Canada); NCBI (National Center for Biotechnology Information,
Bethesda, Md.); Argo Bioanalytica (Argo Bioanalytica. Inc, New
Jersey); Vydac (Grace Vydac, Hesperia, Calif.); Minolta (Konica
Minolta, Ramsey, N.J.); Zeiss (Carl Zeiss, Inc., Thornwood, N.Y.);
Sloning BioTechnology GmbH (Puchheim, Germany); and Procter and
Gamble (Cincinnati, Ohio).
Example 1
Assays
[0207] The following assays were used in the examples described
below. Any deviations from the protocols provided below are
indicated in the examples. In these experiments, a
spectrophotometer was used to measure the absorbance of the
products formed after the completion of the reactions.
A. Bradford Assay for Protein Content Determination in 96-Well
Plates
[0208] The Bradford Dye reagent (Quick Start) assay was used to
determine the protein concentration in thermolysin samples on a
microtiter plate (MTP) scale.
[0209] In this assay system, the chemical and reagent solutions
used were: [0210] Bradford Quick Start Dye Reagent.TM. (BIO-RAD
Catalogue No. 500-0205) [0211] Dilution Buffer (10 mM NaCl, 0.1 mM
CaCl.sub.2, 0.005% TWEEN.RTM.-80)
[0212] The equipment used was a Biomek FX Robot (Beckman Coulter)
and a SpectraMAX MTP Reader (type 340; Molecular Devices). MTPs
were obtained from Costar (type 9017).
[0213] In the test, 200 .mu.l Bradford Dye Reagent was pipetted
into each well, followed by the addition of 15 .mu.l dilution
buffer. Finally, 10 .mu.l of the thermolysin containing filtered
culture supernatants was added to the wells. After thorough mixing,
the MTPs were incubated for at least 10 minutes at room
temperature. Possible air bubbles were blown away and the
absorbance of the wells was read at 595 nm.
[0214] To determine the protein concentration, the background
reading (i.e., from uninoculated wells) was subtracted from the
sample readings. The resulting OD.sub.595 values provided a
relative measure of the protein content in the samples. The
Bradford results were linear with respect to thermolysin protein
concentrations between 10 to 100 .mu.g protein per ml.
B. Microswatch Assay for Testing Protease Performance
[0215] The stain removal performance of thermolysin and variants
thereof was determined using microswatches (EMPA 116) on a MTP
scale. Thermolysin containing protease samples were obtained from
filtered broth of cultures grown in microtiter plates for 3 days at
37.degree. C. with shaking at 280 rpm under humidified
aeration.
[0216] In this assay system, the chemical and reagent solutions
used were: [0217] Thermolysin containing culture supernatants
(.about.100-200 .mu.g protein per ml) [0218] TIDE.RTM. 2.times.
(nil enzymes) detergent (P&G) [0219] Dilution Buffer (10 mM
NaCl, 0.1 mM CaCl.sub.2, 0.005% TWEEN.RTM.-80)
[0220] The equipment used was a Biomek FX Robot (Beckman Coulter),
a SpectraMAX MTP Reader (type 340; Molecular Devices), and an iEMS
incubator/shaker (Thermo/Labsystems). MTPs were obtained from
Costar (type 9017).
TIDE.RTM. 2.times. Liquid Detergent Preparation (US
Conditions):
[0221] Milli-Q water was adjusted to 6 gpg water hardness using a
(Ca/Mg 3:1) hardness stock solution (282.3 g/L
CaCl.sub.2.2H.sub.20, 130.1 g/L MgCl.sub.2.6H.sub.2O), 0.78 g/l
detergent TIDE.RTM. 2.times. was added, and the detergent solution
was stirred vigorously for at least 15 minutes. Then, 5 mM HEPES
was added and the pH adjusted to 8.2.
Microswatches:
[0222] Microswatches of 1/4 inch circular diameter were obtained
from CFT (Vlaardingen, The Netherlands). Before cutting the
swatches, the fabric (EMPA 116) was pre-washed in de-ionised water
for 20 minutes at ambient temperature, and subsequently
air-dried.
[0223] Two microswatches were placed vertically into each well of a
96-well microtiter plate to expose the whole surface area (i.e.,
not flat on the bottom of the well).
Test Method:
[0224] The incubator was set to 20.degree. C. The filtered culture
broth samples were tested at an appropriate concentration by
dilution with a mixture of 10 mM NaCl, 0.1 mM CaCl.sub.2, 0.005%
TWEEN.RTM.-80 solution. The detergent solution was prepared as
described above. Then, 190 .mu.l of detergent solution were added
to each well of the MTP, containing microswatches. To this mixture,
10 .mu.l of diluted enzyme solution were added to each well (to
provide a total volume of 200 .mu.l/well). The MTP was covered with
a plate seal and placed in an incubator for 30 minutes at
20.degree. C., with agitation at 1400 rpm (iEMS incubator).
Following incubation under the appropriate conditions, 100 .mu.l of
solution from each well was removed and placed into a new MTP.
Subsequently this MTP, containing 100 .mu.l of solution/well, was
read at 405 nm in a MTP-Reader. Blank controls, containing 2
microswatches/well and detergent, without the addition of
thermolysin containing samples, were also included in the test.
Calculation of the BMI (Blood/Milk/Ink) Performance:
[0225] The observed absorbance value was corrected for the blank
value (obtained after incubation of microswatches in the absence of
added enzyme). The resulting absorbance was a measure for the
hydrolytic activity. For each sample (thermolysin or a variant) the
performance index (PI) was calculated. The performance index is a
comparison of the performance of the variant (actual value) and the
standard thermolysin enzyme (theoretical value) at the same protein
concentration. In addition, the theoretical values were calculated,
using the parameters of the Langmuir equation of the standard
enzyme.
[0226] A performance index greater than 1 (PI>1) identified a
better variant (as compared to the standard [e.g., wild-type]),
while a PI of 1 (PI=1) identified a variant that performs the same
as the standard, and a PI less than 1 (PI<1) identified a
variant that performs worse than the standard. Thus, the PI
identified winners, as well as variants that are less desirable for
use under certain circumstances.
C. Stability Assay in the Presence of Detergent
[0227] The stability of thermolysin and variants thereof was
measured after incubation under defined conditions in the presence
of 25% TIDE.RTM. 2.times. detergent. The initial and residual
activity was determined.
[0228] In this assay system, the chemical and reagent solutions
used were: [0229] thermolysin containing culture supernatants
(.about.100-200 .mu.g protein per ml) [0230] TIDE.RTM. 2.times.
liquid detergent with and without DTPA chelator (P&G) [0231]
27.5% TIDE.RTM. 2.times. detergent solution with DTPA in 5.5 mM
HEPES buffer, pH 8.2 (TIDE.RTM.+ solution) [0232] 27.5% TIDE.RTM.
2.times. detergent solution w/o DTPA in 5.5 mM HEPES buffer, pH 8.2
(TIDE.RTM.- solution) [0233] MES assay buffer (55.5 mM MES/NaOH,
2.6 mM CaCl.sub.2, 0.005% TWEEN.RTM.-80, pH 6.5)
[0234] The equipment used was a Biomek FX Robot (Beckman Coulter),
a fluorescence spectrophotometer (FLUOstar Optima; BMG), an iEMS
incubator/shaker (Thermo/Labsystems). MTPs were obtained from
Costar (type 9017) and from Greiner (black plate, type 655076).
Test Method
Unstressed Conditions:
[0235] First, 20 .mu.l thermolysin containing culture supernatant
was diluted with 180 .mu.l MES assay buffer. Then, 20 .mu.l diluted
supernatant was diluted further with 180 .mu.l MES assay buffer.
Subsequently 10 .mu.l of this dilution was transferred into 190
.mu.l AGLA-substrate solution in a pre-warmed plate (Greiner
655076) at 25.degree. C. Any air bubbles present were blown away
and the plate was measured according to the AGLA protease assay
protocol described below.
Stressed Conditions:
[0236] First, 20 .mu.l of culture supernatant was diluted with 180
.mu.l 27.5% TIDE.RTM.+ detergent solution and placed in the iEMS
shaker. The plate covered with a plate seal was incubated for a
total of 60 minutes at 32.degree. C. at 900 rpm. In addition, 20
.mu.l of culture supernatant was diluted with 180 .mu.l 27.5%
TIDE.RTM.- solution and placed in the iEMS shaker. This plate
covered with a plate seal was incubated for a total of 180 minutes
at 50.degree. C. at 900 rpm.
[0237] Subsequently after the respective incubations, 20 .mu.l of
either of these solutions were diluted with 180 .mu.l MES assay
buffer and 10 .mu.l of this dilution were diluted with 190 .mu.l
AGLA-substrate solution in a pre-warmed plate (Greiner 655076) at
25.degree. C.
[0238] Any air bubbles present were blown away and the plate was
measured according to the AGLA protease assay protocol described
below.
Calculations of TIDE.RTM. 2.times. Stability
[0239] Fluorescence measurements were taken at excitation of 350 nm
and emission of 420 nm. The spectrofluorometer software calculated
the reaction rates (=slope) of the increase in fluorescence for
each well to a linearly regressed line of (milli-) RFU/min. The
ratio of the residual and initial AGLA activity was used to express
the 25% TIDE.RTM. 2.times. stability as follows:
Percentage of residual activity=[slope of stressed]*100/[slope of
unstressed]
[0240] For each sample (thermolysin and variants thereof) the
performance index was calculated by dividing the residual activity
of the variant by the residual activity of thermolysin. The
performance index compared the stability of the variant and the
standard thermolysin enzyme (e.g., wild type or parental enzyme),
determined under the same conditions.
[0241] A performance index (PI) greater than 1 (PI>1) identified
a better variant (as compared to the standard [e.g., wild-type]),
while a PI of 1 (PI=1) identified a variant that displayed the same
stability as the standard, and a PI less than 1 (PI<1)
identified a variant that was less stable as compared to the
standard. Thus, the PI identified winners, as well as variants that
are less desirable for use under certain circumstances.
D.
2-Aminobenzoyl-L-alanyl-L-glycyl-L-leucyl-L-alanino-4-nitrobenzylamide
(Abz-AGLA-Nba) Protease Assay
[0242] The method described herein provides a degree of technical
detail that yields reproducible protease assay data independent of
time and place. While the assay can be adapted to a given
laboratory condition, any data obtained through a modified
procedure must be reconciled with results produced by the original
method. Neutral metallo-proteases cleave the peptide bond between
glycyl- and leucyl- of
2-Aminobenzoyl-L-alanyl-L-glycyl-L-leucyl-L-alanino-4-nitrobenzylamide
(Abz-AGLA-Nba). Free 2-Aminobenzoyl-L-alanylglycine (Abz-AG) in
solution has a fluorescence emission maximum at 415 nm with an
excitation maximum of 340 nm. Fluorescence of Abz-AG is quenched by
nitrobenzylamide in the intact Abz-AGLA-Nba molecule.
[0243] In these experiments, the liberation of Abz-AG by protease
cleavage of Abz-AGLA-Nba was monitored by fluorescence spectrometry
(Ex. 350/Em. 420). The rate of appearance of Abz-AG was a measure
of proteolytic activity.
[0244] In this assay system, the chemical and reagent solutions
used were: [0245] MES substrate buffer--52.5 mM MES, 2.5 mM
CaCl.sub.2, 0.005% TWEEN.RTM.-80, pH 6.5 [0246] MES assay
buffer--55.5 mM MES, 2.6 mM CaCl.sub.2, 0.005% TWEEN.RTM.-80, pH
6.5 [0247] Abz-AGLA-Nba stock solution--48 mM Abz-AGLA-Nba in
dimethylformamid (28.2 mg/ml DMF)
[0248] The equipment used was a Biomek FX Robot (Beckman Coulter),
a spectrofluorometer (FLUOstar Optima; BMG), an iEMS
incubator/shaker (Thermo/Labsystems) and Innova incubator
(Innova-4230; New Brunswick). MTPs were obtained from Costar (type
9017) and from Greiner (black plate, type 655076).
Test Method
[0249] The Abz-AGLA-Nba assay solution was prepared by adding 1 ml
of the Abz-AGLA-Nba stock to 19 ml MES substrate buffer and mixed
well for at least 2 minutes. Subsequently the thermolysin or
variants thereof containing culture supernatants were diluted with
MES assay buffer to a concentration of 1-6 .mu.g protein per
ml.
[0250] The assay was performed by adding 10 .mu.l of diluted
protease solution to each well, followed by the addition of 190
.mu.l Abz-AGLA-Nba assay solution that was pre-equilibrated for at
least 15 minutes at 25.degree. C. The solutions were vigorously
mixed, and the liberation of Abz-AG by protease cleavage of
Abz-AGLA-Nba was monitored by fluorescence spectrometry at
25.degree. C. in kinetic mode with excitation set at 350 nm and
emission set at 420 nm. The rate of appearance of Abz-AG was a
measure of proteolytic activity in the samples. The protease
activity was expressed as RFU (relative fluorescence
unitsmin.sup.-1).
Example 2
Thermolysin Production in B. subtilis
[0251] In this Example, experiments conducted to produce
thermolysin in B. subtilis are described. The full-length
thermolysin of Geobacillus caldoproteolyticus is greater than 99%
identical to the thermolysin precursor of Bacillus
thermoproteolyticus Rokko, and to the bacillolysin (NprS) precursor
of Bacillus stearothermophilus. As such the terms "thermolysin,"
"bacillolysin," "proteinase-T" and "PrT" are used interchangeably
herein to refer to the neutral metalloprotease enzyme of G.
caldoproteolyticus. The DNA sequence (thermolysin leader,
thermolysin pro and thermolysin mature from Geobacillus
caldoproteolyticus) provided below, encodes the thermolysin
precursor protein:
TABLE-US-00001 (SEQ ID NO: 1)
ATGAAAATGAAAATGAAATTAGCATCGTTTGGTCTTGCAGCAGGACT
AGCGGCCCAAGTATTTTTACCTTACAATGCGCTGGCTTCAACGGAAC
ACGTTACATGGAACCAACAATTTCAAACCCCTCAATTCATCTCCGGT
GATCTGCTGAAAGTGAATGGCACATCCCCAGAAGAACTCGTCTATCA
ATATGTTGAAAAAAACGAAAACAAGTTTAAATTTCATGAAAACGCTA
AGGATACTCTACAATTGAAAGAAAAGAAAAATGATAACCTTGGTTTT
ACGTTTATGCGCTTCCAACAAACGTATAAAGGGATTCCTGTGTTTGG
AGCAGTAGTAACTGCGCACGTGAAAGATGGCACGCTGACGGCGCTAT
CAGGGACACTGATTCCGAATTTGGACACGAAAGGATCCTTAAAAAGC
GGGAAGAAATTGAGTGAGAAACAAGCGCGTGACATTGCTGAAAAAGA
TTTAGTGGCAAATGTAACAAAGGAAGTACCGGAATATGAACAGGGAA
AAGACACCGAGTTTGTTGTTTATGTCAATGGGGACGAGGCTTCTTTA
GCGTACGTTGTCAATTTAAACTTTTTAACTCCTGAACCAGGAAACTG
GCTGTATATCATTGATGCCGTAGACGGAAAAATTTTAAATAAATTTA
ACCAACTTGACGCCGCAAAACCAGGTGATGTGAAGTCGATAACAGGA
ACATCAACTGTCGGAGTGGGAAGAGGAGTACTTGGTGATCAAAAAAA
TATTAATACAACCTACTCTACGTACTACTATTTACAAGATAATACGC
GTGGAAATGGGATTTTCACGTATGATGCGAAATACCGTACGACATTG
CCGGGAAGCTTATGGGCAGATGCAGATAACCAATTTTTTGCGAGCTA
TGATGCTCCAGCGGTTGATGCTCATTATTACGCTGGTGTGACATATG
ACTACTATAAAAATGTTCATAACCGTCTCAGTTACGACGGAAATAAT
GCAGCTATTAGATCATCCGTTCATTATAGCCAAGGCTATAATAACGC
ATTTTGGAACGGTTCGCAAATGGTGTATGGCGATGGTGATGGTCAAA
CATTTATTCCACTTTCTGGTGGTATTGATGTGGTCGCACATGAGTTA
ACGCATGCGGTAACCGATTATACAGCCGGACTCATTTATCAAAACGA
ATCTGGTGCAATTAATGAGGCAATATCTGATATTTTTGGAACGTTAG
TCGAATTTTACGCTAACAAAAATCCAGATTGGGAAATTGGAGAGGAT
GTGTATACACCTGGTATTTCAGGGGATTCGCTCCGTTCGATGTCCGA
TCCGGCAAAGTATGGTGATCCAGATCACTATTCAAAGCGCTATACAG
GCACGCAAGATAATGGCGGGGTTCATATCAATAGCGGAATTATCAAC
AAAGCCGCTTATTTGATTAGCCAAGGCGGTACGCATTACGGTGTGAG
TGTTGTCGGAATCGGACGCGATAAATTGGGGAAAATTTTCTATCGTG
CATTAACGCAATATTTAACACCAACGTCCAACTTTAGCCAACTTCGT
GCTGCCGCTGTTCAATCAGCCACTGACTTGTACGGTTCGACAAGCCA
GGAAGTCGCTTCTGTGAAGCAGGCCTTTGATGCGGTAGGGGTGAAAT AA
[0252] In the above sequence, bold indicates the DNA encoding the
mature thermolysin protease, standard font indicates the DNA
encoding the leader sequence (thermolysin leader), and underlined
text indicates DNA encoding the pro sequence (thermolysin pro). The
amino acid sequence (thermolysin leader, thermolysin pro and
thermolysin mature DNA sequence) provided below (SEQ ID NO:2),
corresponds to the full length thermolysin precursor protein. In
this sequence, underlined indicates the pro sequence and bold
indicates the mature thermolysin protease.
TABLE-US-00002 (SEQ ID NO: 2)
MKMKMKLASFGLAAGLAAQVFLPYNALASTEHVTWNQQFQTPQFISG
DLLKVNGTSPEELVYQYVEKNENKFKFHENAKDTLQLKEKKNDNLGF
TFMRFQQTYKGIPVFGAVVTAHVKDGTLTALSGTLIPNLDTKGSLKS
GKKLSEKQARDIAEKDLVANVTKEVPEYEQGKDTEFVVYVNGDEASL
AYVVNLNFLTPEPGNWLYIIDAVDGKILNKFNQLDAAKPGDVKSITG
TSTVGVGRGVLGDQKNINTTYSTYYYLQDNTRGNGIFTYDAKYRTTL
PGSLWADADNQFFASYDAPAVDAHYYAGVTYDYYKNVHNRLSYDGNN
AAIRSSVHYSQGYNNAFWNGSQMVYGDGDGQTFIPLSGGIDVVAHEL
THAVTDYTAGLIYQNESGAINEAISDIFGTLVEFYANKNPDWEIGED
VYTPGISGDSLRSMSDPAKYGDPDHYSKRYTGTQDNGGVHINSGIIN
KAAYLISQGGTHYGVSVVGIGRDKLGKIFYRALTQYLTPTSNFSQLR
AAAVQSATDLYGSTSQEVASVKQAFDAVGVK
The mature thermolysin sequence is set forth as SEQ ID NO:3 and
shown in FIG. 1. This sequence was used as the basis for making the
variant libraries describe herein.
TABLE-US-00003 (SEQ ID NO: 3)
ITGTSTVGVGRGVLGDQKNINTTYSTYYYLQDNTRGNGIFTYDAKYR
TTLPGSLWADADNQFFASYDAPAVDAHYYAGVTYDYYKNVHNRLSYD
GNNAAIRSSVHYSQGYNNAFWNGSQMVYGDGDGQTFIPLSGGIDVVA
HELTHAVTDYTAGLIYQNESGAINEAISDIFGTLVEFYANKNPDWEI
GEDVYTPGISGDSLRSMSDPAKYGDPDHYSKRYTGTQDNGGVHINSG
IINKAAYLISQGGTHYGVSVVGIGRDKLGKIFYRALTQYLTPTSNFS
QLRAAAVQSATDLYGSTSQEVASVKQAFDAVGVK
[0253] The pHPLT-thermolysin expression vector was constructed by
amplifying the thermolysin gene from genomic DNA of Geobacillus
caldoproteolyticus (Chen et al., Extremophiles, 8:489-498, 2004)
and from pHPLT plasmid DNA (van Solingen et al., Extremophiles,
5:333-341, 2001). A map for the pHPLT plasmid is provided in FIG.
2. This plasmid contains the thermostable amylase LAT promoter
(P.sub.LAT) of Bacillus licheniformis to drive expression of
thermolysin. The thermolysin gene was amplified from the genomic
DNA using Finnzymes (Finnzymes OY, Espoo, Finland) Phusion
High-Fidelity DNA Polymerase (Catalog No. F-530L) and the following
primers:
TABLE-US-00004 pHPLT-ProT-FW: (SEQ ID NO: 4)
GAGAGGGTAAAGAATGAAAATGAAAATGAAATTAGCATC proT-EcoRI-RV: (SEQ ID NO:
5) GTTAACCTGCAGGAATTCTTATTTCACCCCTACCGCATCAAAGGCC
[0254] The pHPLT fragment was amplified from the plasmid pHPLT
using Finnzymes Phusion High-Fidelity DNA Polymerase and the
following primers:
TABLE-US-00005 pHPLT-ProT-RV: (SEQ ID NO: 6)
CATTTTCATTTTCATTCTTTACCCTCTCCTTTTGCTAGAC proT-EcoRI-FW: (SEQ ID NO:
7) CCATAAGAATTCCTGCAGGTTAACAGAGGACGGATTTCCTGAAGG
[0255] The following PCR conditions were used to amplify both
pieces:
[0256] 98.degree. C. for 30 sec, 30.times. (98.degree. C. for 10
sec, 55.degree. C. for 20 sec, and 72.degree. C. for 45 sec
(thermolysin) or 72.degree. C. for 80 sec (pHPLT)), followed by
72.degree. C. for 5 min. The resulting PCR products were run on an
E-gel (Invitrogen), excised, and purified with a gel extraction kit
(Qiagen). In addition, a PCR overlap extension fusion (Ho, Gene,
15:51-59, 1989) was used to fuse the above gene fragments with High
fidelity platinum Taq DNA polymerase (Invitrogen) using the
following primers:
TABLE-US-00006 proT-EcoRI-FW: (SEQ ID NO: 7)
CCATAAGAATTCCTGCAGGTTAACAGAGGACGGATTTCCTGAAGG proT-EcoRI-RV: (SEQ
ID NO: 5) GTTAACCTGCAGGAATTCTTATTTCACCCCTACCGCATCAAAGGCC
[0257] The following conditions were used for these reactions:
[0258] 94.degree. C. for 2 min, 25.times. (94.degree. C. for 30
sec, 55.degree. C. for 30 sec, and 68.degree. C. for 5 min)
followed by 68.degree. C. for 5 min. The resulting PCR fusion
product was run on an E-gel (Invitrogen), excised, and purified
with a gel extraction kit (Qiagen). The purified fusion product was
cut (PstI) and self-ligated (T4 DNA Ligase, Invitrogen). A map of
the pHPLT-thermolysin expression vector is provided in FIG. 3,
while the DNA sequence of the pHPLT-thermolysin expression vector
(SEQ ID NO:8) is provided in FIG. 4.
[0259] The ligation mixture was used to transform B. subtilis SC6.1
(phenotype: .DELTA.aprE, .DELTA.nprE, oppA, .DELTA.spoIIE,
degUHy32, .DELTA.amyE:(xylR,pxylA-comK). Transformation of B.
subtilis SC6.1 strain was performed as described in WO 02/14490,
incorporated herein by reference. Selective growth of B. subtilis
transformants containing the pHPLT-thermolysin vector was done in
shake flasks containing 25 ml MBD medium (a MOPS based defined
medium), with 20 mg/L neomycin. Culturing resulted in the
production of secreted mature thermolysin enzyme having proteolytic
activity. Gel analysis was performed using NuPage Novex 10%
Bis-Tris gels (Invitrogen, Catalog No. NP0301BOX). To prepare
samples for analysis, 2 volumes of supernatant were mixed with 1
volume 1M HCl, 1 volume 4.times.LDS sample buffer (Invitrogen,
Catalog No. NP0007), and 1% PMSF (20 mg/ml), and subsequently
heated for 10 minutes at 70.degree. C. Then, 25 .mu.L of each
sample was loaded onto the gel, adjacent to 10 .mu.L of SeeBlue
plus 2 pre-stained protein standards (Invitrogen, Catalog No.
LC5925). The results clearly demonstrated that the thermolysin
cloning strategy described in this example is suitable for
production of active recombinant thermolysin in B. subtilis.
Example 3
Generation of Thermolysin Site Evaluation Libraries (SELs)
[0260] In this Example, methods used in the construction of
thermolysin SELs are described. As previously indicated, the terms
"thermolysin," "bacillolysin," "proteinase-T" and "PrT" are used
interchangeably throughout to refer to the neutral metalloprotease
enzyme of G. caldoproteolyticus. The pHPLT-thermolysin vector (FIG.
3) contains the thermolysin expression cassette, which served as a
template DNA for the site evaluation libraries. Every thermolysin
site evaluation library contains a collection of B. subtilis
clones, all expressing a specific thermolysin variant. Each library
contains B. subtilis clones, maximally including 20 different
variants. For example, thermolysin SEL 27 contains variants in
which the DNA triplet coding for tyrosine at position 27 of the
mature thermolysin enzyme is replaced by another DNA triplet
encoding: Alanine, Aspartic acid, Cysteine, Glutamic acid,
Phenylalanine, Glycine, Histidine, Isoleucine, Lysine, Leucine,
Methionine, Asparagine, Proline, Glutamine, Arginine, Serine,
Threonine, Valine, Tryptophan or Tyrosine.
[0261] Briefly, DNA triplets of specific positions in the DNA
coding strand of the mature thermolysin are replaced. The mutated
thermolysin fragments are subsequently ligated to pHPLT. The
pHPLT-thermolysin variant plasmids are used to transform B.
subtilis SC6.1 The production of prt variants was done using the
gene synthesis products and services of Sloning BioTechnology GmbH
(Puchheim, Germany). The specific mutation of each variant was
confirmed by DNA sequencing.
Example 4
Preparation of Crude Thermolysin Samples
[0262] The thermolysin (also referred to as Proteinase-T or PrT)
variant proteins were produced by culturing the B. subtilis
transformants in 96 well MTP at 37.degree. C. for 68 hours in MBD
medium (a MOPS based defined medium) including 10 mg/L neomycin.
MBD medium was made essentially as known in the art (See, Neidhardt
et al., J Bacteriol, 119: 736-747, 1974), except that NH.sub.4Cl,
FeSO.sub.4, and CaCl.sub.2 were omitted from the base medium, 3 mM
K.sub.2HPO.sub.4 was used, and the base medium was supplemented
with 60 mM urea, 75 g/L glucose, and 1% soytone. Also, the
micronutrients were made up as a 100.times. stock containing in one
liter, 400 mg FeSO.sub.4.7H.sub.2O, 100 mg MnSO.sub.4.H.sub.2O, 100
mg ZnSO4.7H.sub.2O, 50 mg CuCl.sub.2.2H.sub.2O, 100 mg
CoCl.sub.2.6H.sub.2O, 100 mg NaMoO.sub.4.2H.sub.2O, 100 mg
Na.sub.2B.sub.4O.sub.7.10H.sub.2O, 10 ml of 1M CaCl.sub.2, and 10
ml of 0.5 M sodium citrate.
Example 5
Stability of Thermolysin in Heavy Duty Liquid (HDL) Detergent
[0263] Unilever detergent ALL Small and Mighty, P&G TIDE.RTM.
Fresh Breeze, P&G TIDE.RTM. 2.times. Fresh Breeze were
purchased from Walmart. The commercially available detergents were
heated at 90.degree. C. for 1 hour and then cooled to room
temperature, to inactivate the proteases in these cleaning
compositions. Thermolysin (also referred to as Proteinase-T or PrT)
in lyophilized powder was purchased from Sigma, and dissolved in
100 mM Tris pH 7 and 50% propylene glycol at 20 mg/ml. NprE was
purified from Bacillus sp. supernatant through ion-exchange
chromatography. To lml of heat-treated detergent in an eppendorf
tube, 800 .quadrature.g of thermolysin or NprE was added. The tube
was mixed well on a rocker for 15 min at room temperature, and then
incubated at 25.degree. C. or 32.degree. C. At different time
points, remaining proteinase activity was measured using an AGLA
assay as described above in Example 1. Briefly, 10 .quadrature.l of
sample was diluted 441 fold in AGLA buffer (50 mM MES, pH 6.5,
0.005% Tween 80, 2.5 mM CaCl.sub.2), then 10 .quadrature.l of
diluted sample was added into 200 .quadrature.l of AGLA substrate
(2.4 mM Abz-AGLA-Nba in AGLA buffer). Excitation at 350 nm and
emission at 415 nm was monitored for the first 100 seconds, and the
initial slope was recorded as enzyme activity. The enzyme activity
was plotted against time, and curves were fitted with exponential
decay.
[0264] As shown in FIG. 5, thermolysin is 140 fold more stable than
NprE in Unilever All Small & Mighty at room temperature.
Similarly as shown in FIG. 6, thermolysin is 68 fold more stable
than NprE in P&G TIDE.RTM. at 32.degree. C., while FIG. 7 shows
that thermolysin is 98 fold more stable than NprE in P&G
TIDE.RTM. 2.times. at 32.degree. C. Thus, thermolysin is much more
stable than NprE in Unilever detergent ALL (3.times.), P&G
TIDE.RTM. 1.times. Fresh Breeze and P&G TIDE.RTM. 2.times.
Fresh Breeze.
Example 6
Metalloproteinase Inhibitors can Improve Thermolysin Stability in
Heavy Duty Liquid (HDL) Detergent
[0265] Zinc Chloride, Phosphoramidon, Galardin are known
metalloproteinase inhibitors. They were purchased from Sigma and
dissolved in water or DMSO. Different concentrations of the
inhibitors were premixed with thermolysin (also referred to as
Proteinase-T or PrT) for 10 min at room temperature. Then the
inhibitors were added into Unilever detergent ALL Small and Mighty
so that the final concentration of thermolysin was 800
.quadrature.g/ml in a total volume of 1 ml. At different time
points, samples were taken and precipitated with TCA. Briefly, 10
.quadrature.l sample of detergent with enzyme was added into 500
.quadrature.l of 0.2 N HCl on ice, and then 500 .quadrature.l of
20% TCA was added. The tubes were mixed and incubated on ice for 20
min. The pellet was collected and washed with 90% ice-cold acetone.
The pellet was dissolved in sample loading buffer (Invitrogen) for
SDS-PAGE analysis. As shown in FIG. 8, both 500 .quadrature.M
Phosphoramidon and 1 mM Galardin significantly stabilize
thermolysin in detergent.
Example 7
Stain Removal Performance of Thermolysin Variants in a TIDE.RTM.
2.times. Microswatch Assay
[0266] In this example, experiments were conducted to determine the
stain removal performance of various singly substituted thermolysin
(also referred to herein as Proteinase-T or PrT) variants. As
described in Example 1, the stain cleaning performance of
thermolysin variants was done utilizing a blood/milk/ink (BMI)
microswatch assay. Briefly the cleaning performance of chosen
single-substitution thermolysin variants was assessed in a
TIDE.RTM. 2.times. microswatch assay. Table 7-1 provides
performance indices for the tested variants (e.g., showing improved
performance as compared to wild-type thermolysin enzyme). Those
variants with a performance index greater than 1 (PI>1) have
improved performance. As indicated by these results, numerous
variants having single amino acid substitutions performed better
than wild-type enzyme in this assay system.
TABLE-US-00007 TABLE 7-1 Stain Removal For Variants With PI > 1
Variant PI T006G 1.13 T006H 1.01 T006I 1.27 T006K 1.76 T006M 1.05
T006N 1.23 T006P 1.05 T006Q 1.19 T006R 1.58 T006V 1.04 T006W 1.14
T006Y 1.06 V007F 1.08 V007H 1.32 V007K 1.60 V007L 1.16 V007M 1.01
V007P 1.27 V007Q 1.20 V007R 1.53 V007T 1.23 V007Y 1.11 T049G 1.01
T049H 1.25 T049I 1.24 T049K 1.01 T049L 1.25 T049N 1.10 T049P 1.24
T049Q 1.30 T049W 1.10 A058I 1.04 A058P 1.10 A058R 1.04 F063I 1.11
F063L 1.03 F063P 1.20 S065K 1.29 S065Y 1.05 Y075G 1.04 Y075M 1.14
Y075T 1.01 Q128H 1.39 Q128I 1.34 Q128L 1.04 Q128M 1.10 Q128V 1.07
Q128Y 1.13 Y151D 1.08 Y151E 1.11 Y151H 1.17 Y151K 1.03 Y151M 1.06
Y151N 1.19 Y151Q 1.29 Y151R 1.75 Y151T 1.13 Y151V 1.25 Y151W 1.22
I156M 1.11 I156R 1.22 I156T 1.03 I156W 1.16 G196R 1.13 Q273I 1.18
Q273P 1.13 Q273Y 1.09 T278K 1.09 T278M 1.02 T278P 1.07 N280K 1.02
N280R 1.04
Example 8
Stability of Thermolysin Variants in TIDE.RTM. 2.times. Liquid
Detergent
[0267] In this example, experiments were conducted to assess the
stability of various singly substituted thermolysin (also referred
to herein as Proteinase-T or PrT) variants in the presence of
liquid detergent. As described in Example 1, the stability of
thermolysin variants was measured by determining the AGLA activity
before and after incubation in the presence of TIDE.RTM. 2.times.
heavy duty liquid (HDL) detergent at an elevated temperature. The
tables contain the relative stability values compared to wild-type
thermolysin, which is the quotient of the variant residual activity
divided by the wild-type residual activity. A value greater than
one indicates higher stability in the presence of detergent. In
Table 8-1 and Table 8-2, data are provided showing the relative
stability of single-substitution variants of thermolysin relative
to the stability of wild-type thermolysin in HDL detergent in the
presence and absence of DTPA.
TABLE-US-00008 TABLE 8-1 Stability Of Variants In 25% TIDE .RTM. 2X
With DTPA Variant PI T006A 1.01 T006C 1.03 T049D 1.05 T049I 1.01
T049L 1.02 T049M 1.02 T049N 1.03 T049S 1.08 A056C 1.01 A056R 1.10
A056Y 1.05 A058S 1.02 S065C 1.05 S065E 1.08 S065I 1.05 S065T 1.04
S065V 1.08 S065Y 1.05 Q128C 1.01 Q128I 1.32 Q128M 1.06 Q128T 1.18
Q128V 1.45 Q128Y 1.09 Y151A 1.15 Y151C 1.25 Y151D 1.12 Y151E 1.10
Y151H 1.11 Y151M 1.09 Y151N 1.25 Y151Q 1.03 Y151R 1.26 Y151S 1.23
Y151T 1.18 Y151V 1.11 Y151W 1.02 I156E 1.58 I156H 1.21 I156K 1.07
I156M 1.19 I156R 1.15 I156T 1.08 I156W 1.12 G196D 1.02 G196H 1.19
Q273A 1.03 Q273N 1.25 Q273T 1.08 Q273W 1.05 Q273Y 1.05 T278C 1.05
T278H 1.07 T278M 1.09 T278N 1.07 T278S 1.08 T278Y 1.05 N280E 1.13
N280I 1.16 N280L 1.21 N280M 1.16 N280S 1.19
TABLE-US-00009 TABLE 8-2 Stability Of Variants In 25% TIDE .RTM. 2X
Without DTPA Variant PI T006C 1.07 T049D 1.28 T049N 1.07 T049Q 1.07
T049S 1.26 A056C 1.19 A056E 1.07 A058C 1.01 A058E 1.24 Q061E 1.05
Q061M 1.01 S065C 1.14 S065D 1.20 S065E 1.34 S065P 1.18 S065V 1.08
S065W 1.09 S065Y 1.05 Q128C 1.05 Q128I 1.19 Q128M 1.09 Q128T 1.15
Q128V 1.20 Q128Y 1.05 Y151A 1.24 Y151C 1.09 Y151N 1.05 Y151S 1.17
Y151T 1.10 I156E 1.09
[0268] All publications and patents mentioned in the above
specification are herein incorporated by reference. Various
modifications and variations of the described method and system of
the invention will be apparent to those skilled in the art without
departing from the scope and spirit of the invention. Although the
invention has been described in connection with specific preferred
embodiments, it should be understood that the invention as claimed
should not be unduly limited to such specific embodiments. Indeed,
various modifications of the described modes for carrying out the
invention, which are obvious to those skilled in the relevant
fields are intended to be within the scope of the following claims.
Sequence CWU 1
1
1011647DNAGeobacillus caldoproteolyticus 1atgaaaatga aaatgaaatt
agcatcgttt ggtcttgcag caggactagc ggcccaagta 60tttttacctt acaatgcgct
ggcttcaacg gaacacgtta catggaacca acaatttcaa 120acccctcaat
tcatctccgg tgatctgctg aaagtgaatg gcacatcccc agaagaactc
180gtctatcaat atgttgaaaa aaacgaaaac aagtttaaat ttcatgaaaa
cgctaaggat 240actctacaat tgaaagaaaa gaaaaatgat aaccttggtt
ttacgtttat gcgcttccaa 300caaacgtata aagggattcc tgtgtttgga
gcagtagtaa ctgcgcacgt gaaagatggc 360acgctgacgg cgctatcagg
gacactgatt ccgaatttgg acacgaaagg atccttaaaa 420agcgggaaga
aattgagtga gaaacaagcg cgtgacattg ctgaaaaaga tttagtggca
480aatgtaacaa aggaagtacc ggaatatgaa cagggaaaag acaccgagtt
tgttgtttat 540gtcaatgggg acgaggcttc tttagcgtac gttgtcaatt
taaacttttt aactcctgaa 600ccaggaaact ggctgtatat cattgatgcc
gtagacggaa aaattttaaa taaatttaac 660caacttgacg ccgcaaaacc
aggtgatgtg aagtcgataa caggaacatc aactgtcgga 720gtgggaagag
gagtacttgg tgatcaaaaa aatattaata caacctactc tacgtactac
780tatttacaag ataatacgcg tggaaatggg attttcacgt atgatgcgaa
ataccgtacg 840acattgccgg gaagcttatg ggcagatgca gataaccaat
tttttgcgag ctatgatgct 900ccagcggttg atgctcatta ttacgctggt
gtgacatatg actactataa aaatgttcat 960aaccgtctca gttacgacgg
aaataatgca gctattagat catccgttca ttatagccaa 1020ggctataata
acgcattttg gaacggttcg caaatggtgt atggcgatgg tgatggtcaa
1080acatttattc cactttctgg tggtattgat gtggtcgcac atgagttaac
gcatgcggta 1140accgattata cagccggact catttatcaa aacgaatctg
gtgcaattaa tgaggcaata 1200tctgatattt ttggaacgtt agtcgaattt
tacgctaaca aaaatccaga ttgggaaatt 1260ggagaggatg tgtatacacc
tggtatttca ggggattcgc tccgttcgat gtccgatccg 1320gcaaagtatg
gtgatccaga tcactattca aagcgctata caggcacgca agataatggc
1380ggggttcata tcaatagcgg aattatcaac aaagccgctt atttgattag
ccaaggcggt 1440acgcattacg gtgtgagtgt tgtcggaatc ggacgcgata
aattggggaa aattttctat 1500cgtgcattaa cgcaatattt aacaccaacg
tccaacttta gccaacttcg tgctgccgct 1560gttcaatcag ccactgactt
gtacggttcg acaagccagg aagtcgcttc tgtgaagcag 1620gcctttgatg
cggtaggggt gaaataa 16472548PRTGeobacillus caldoproteolyticus 2Met
Lys Met Lys Met Lys Leu Ala Ser Phe Gly Leu Ala Ala Gly Leu 1 5 10
15 Ala Ala Gln Val Phe Leu Pro Tyr Asn Ala Leu Ala Ser Thr Glu His
20 25 30 Val Thr Trp Asn Gln Gln Phe Gln Thr Pro Gln Phe Ile Ser
Gly Asp 35 40 45 Leu Leu Lys Val Asn Gly Thr Ser Pro Glu Glu Leu
Val Tyr Gln Tyr 50 55 60 Val Glu Lys Asn Glu Asn Lys Phe Lys Phe
His Glu Asn Ala Lys Asp 65 70 75 80 Thr Leu Gln Leu Lys Glu Lys Lys
Asn Asp Asn Leu Gly Phe Thr Phe 85 90 95 Met Arg Phe Gln Gln Thr
Tyr Lys Gly Ile Pro Val Phe Gly Ala Val 100 105 110 Val Thr Ala His
Val Lys Asp Gly Thr Leu Thr Ala Leu Ser Gly Thr 115 120 125 Leu Ile
Pro Asn Leu Asp Thr Lys Gly Ser Leu Lys Ser Gly Lys Lys 130 135 140
Leu Ser Glu Lys Gln Ala Arg Asp Ile Ala Glu Lys Asp Leu Val Ala 145
150 155 160 Asn Val Thr Lys Glu Val Pro Glu Tyr Glu Gln Gly Lys Asp
Thr Glu 165 170 175 Phe Val Val Tyr Val Asn Gly Asp Glu Ala Ser Leu
Ala Tyr Val Val 180 185 190 Asn Leu Asn Phe Leu Thr Pro Glu Pro Gly
Asn Trp Leu Tyr Ile Ile 195 200 205 Asp Ala Val Asp Gly Lys Ile Leu
Asn Lys Phe Asn Gln Leu Asp Ala 210 215 220 Ala Lys Pro Gly Asp Val
Lys Ser Ile Thr Gly Thr Ser Thr Val Gly 225 230 235 240 Val Gly Arg
Gly Val Leu Gly Asp Gln Lys Asn Ile Asn Thr Thr Tyr 245 250 255 Ser
Thr Tyr Tyr Tyr Leu Gln Asp Asn Thr Arg Gly Asn Gly Ile Phe 260 265
270 Thr Tyr Asp Ala Lys Tyr Arg Thr Thr Leu Pro Gly Ser Leu Trp Ala
275 280 285 Asp Ala Asp Asn Gln Phe Phe Ala Ser Tyr Asp Ala Pro Ala
Val Asp 290 295 300 Ala His Tyr Tyr Ala Gly Val Thr Tyr Asp Tyr Tyr
Lys Asn Val His 305 310 315 320 Asn Arg Leu Ser Tyr Asp Gly Asn Asn
Ala Ala Ile Arg Ser Ser Val 325 330 335 His Tyr Ser Gln Gly Tyr Asn
Asn Ala Phe Trp Asn Gly Ser Gln Met 340 345 350 Val Tyr Gly Asp Gly
Asp Gly Gln Thr Phe Ile Pro Leu Ser Gly Gly 355 360 365 Ile Asp Val
Val Ala His Glu Leu Thr His Ala Val Thr Asp Tyr Thr 370 375 380 Ala
Gly Leu Ile Tyr Gln Asn Glu Ser Gly Ala Ile Asn Glu Ala Ile 385 390
395 400 Ser Asp Ile Phe Gly Thr Leu Val Glu Phe Tyr Ala Asn Lys Asn
Pro 405 410 415 Asp Trp Glu Ile Gly Glu Asp Val Tyr Thr Pro Gly Ile
Ser Gly Asp 420 425 430 Ser Leu Arg Ser Met Ser Asp Pro Ala Lys Tyr
Gly Asp Pro Asp His 435 440 445 Tyr Ser Lys Arg Tyr Thr Gly Thr Gln
Asp Asn Gly Gly Val His Ile 450 455 460 Asn Ser Gly Ile Ile Asn Lys
Ala Ala Tyr Leu Ile Ser Gln Gly Gly 465 470 475 480 Thr His Tyr Gly
Val Ser Val Val Gly Ile Gly Arg Asp Lys Leu Gly 485 490 495 Lys Ile
Phe Tyr Arg Ala Leu Thr Gln Tyr Leu Thr Pro Thr Ser Asn 500 505 510
Phe Ser Gln Leu Arg Ala Ala Ala Val Gln Ser Ala Thr Asp Leu Tyr 515
520 525 Gly Ser Thr Ser Gln Glu Val Ala Ser Val Lys Gln Ala Phe Asp
Ala 530 535 540 Val Gly Val Lys 545 3316PRTGeobacillus
caldoproteolyticus 3Ile Thr Gly Thr Ser Thr Val Gly Val Gly Arg Gly
Val Leu Gly Asp 1 5 10 15 Gln Lys Asn Ile Asn Thr Thr Tyr Ser Thr
Tyr Tyr Tyr Leu Gln Asp 20 25 30 Asn Thr Arg Gly Asn Gly Ile Phe
Thr Tyr Asp Ala Lys Tyr Arg Thr 35 40 45 Thr Leu Pro Gly Ser Leu
Trp Ala Asp Ala Asp Asn Gln Phe Phe Ala 50 55 60 Ser Tyr Asp Ala
Pro Ala Val Asp Ala His Tyr Tyr Ala Gly Val Thr 65 70 75 80 Tyr Asp
Tyr Tyr Lys Asn Val His Asn Arg Leu Ser Tyr Asp Gly Asn 85 90 95
Asn Ala Ala Ile Arg Ser Ser Val His Tyr Ser Gln Gly Tyr Asn Asn 100
105 110 Ala Phe Trp Asn Gly Ser Gln Met Val Tyr Gly Asp Gly Asp Gly
Gln 115 120 125 Thr Phe Ile Pro Leu Ser Gly Gly Ile Asp Val Val Ala
His Glu Leu 130 135 140 Thr His Ala Val Thr Asp Tyr Thr Ala Gly Leu
Ile Tyr Gln Asn Glu 145 150 155 160 Ser Gly Ala Ile Asn Glu Ala Ile
Ser Asp Ile Phe Gly Thr Leu Val 165 170 175 Glu Phe Tyr Ala Asn Lys
Asn Pro Asp Trp Glu Ile Gly Glu Asp Val 180 185 190 Tyr Thr Pro Gly
Ile Ser Gly Asp Ser Leu Arg Ser Met Ser Asp Pro 195 200 205 Ala Lys
Tyr Gly Asp Pro Asp His Tyr Ser Lys Arg Tyr Thr Gly Thr 210 215 220
Gln Asp Asn Gly Gly Val His Ile Asn Ser Gly Ile Ile Asn Lys Ala 225
230 235 240 Ala Tyr Leu Ile Ser Gln Gly Gly Thr His Tyr Gly Val Ser
Val Val 245 250 255 Gly Ile Gly Arg Asp Lys Leu Gly Lys Ile Phe Tyr
Arg Ala Leu Thr 260 265 270 Gln Tyr Leu Thr Pro Thr Ser Asn Phe Ser
Gln Leu Arg Ala Ala Ala 275 280 285 Val Gln Ser Ala Thr Asp Leu Tyr
Gly Ser Thr Ser Gln Glu Val Ala 290 295 300 Ser Val Lys Gln Ala Phe
Asp Ala Val Gly Val Lys 305 310 315 439DNAArtificialsynthetic
primer 4gagagggtaa agaatgaaaa tgaaaatgaa attagcatc
39546DNAArtificialsynthetic primer 5gttaacctgc aggaattctt
atttcacccc taccgcatca aaggcc 46640DNAArtificialsynthetic primer
6cattttcatt ttcattcttt accctctcct tttgctagac
40745DNAArtificialsynthetic primer 7ccataagaat tcctgcaggt
taacagagga cggatttcct gaagg 4585335DNAArtificialsynthetic
expression vector 8agcttggaga caaggtaaag gataaaacag cacaattcca
agaaaaacac gatttagaac 60ctaaaaagaa cgaatttgaa ctaactcata accgagaggt
aaaaaaagaa cgaagtcgag 120atcagggaat gagtttataa aataaaaaaa
gcacctgaaa aggtgtcttt ttttgatggt 180tttgaacttg ttctttctta
tcttgataca tatagaaata acgtcatttt tattttagtt 240gctgaaaggt
gcgttgaagt gttggtatgt atgtgtttta aagtattgaa aacccttaaa
300attggttgca cagaaaaacc ccatctgtta aagttataag tgactaaaca
aataactaaa 360tagatggggg tttcttttaa tattatgtgt cctaatagta
gcatttattc agatgaaaaa 420tcaagggttt tagtggacaa gacaaaaagt
ggaaaagtga gaccatggag agaaaagaaa 480atcgctaatg ttgattactt
tgaacttctg catattcttg aatttaaaaa ggctgaaaga 540gtaaaagatt
gtgctgaaat attagagtat aaacaaaatc gtgaaacagg cgaaagaaag
600ttgtatcgag tgtggttttg taaatccagg ctttgtccaa tgtgcaactg
gaggagagca 660atgaaacatg gcattcagtc acaaaaggtt gttgctgaag
ttattaaaca aaagccaaca 720gttcgttggt tgtttctcac attaacagtt
aaaaatgttt atgatggcga agaattaaat 780aagagtttgt cagatatggc
tcaaggattt cgccgaatga tgcaatataa aaaaattaat 840aaaaatcttg
ttggttttat gcgtgcaacg gaagtgacaa taaataataa agataattct
900tataatcagc acatgcatgt attggtatgt gtggaaccaa cttattttaa
gaatacagaa 960aactacgtga atcaaaaaca atggattcaa ttttggaaaa
aggcaatgaa attagactat 1020gatccaaatg taaaagttca aatgattcga
ccgaaaaata aatataaatc ggatatacaa 1080tcggcaattg acgaaactgc
aaaatatcct gtaaaggata cggattttat gaccgatgat 1140gaagaaaaga
atttgaaacg tttgtctgat ttggaggaag gtttacaccg taaaaggtta
1200atctcctatg gtggtttgtt aaaagaaata cataaaaaat taaaccttga
tgacacagaa 1260gaaggcgatt tgattcatac agatgatgac gaaaaagccg
atgaagatgg attttctatt 1320attgcaatgt ggaattggga acggaaaaat
tattttatta aagagtagtt caacaaacgg 1380gccagtttgt tgaagattag
atgctataat tgttattaaa aggattgaag gatgcttagg 1440aagacgagtt
attaatagct gaataagaac ggtgctctcc aaatattctt atttagaaaa
1500gcaaatctaa aattatctga aaagggaatg agaatagtga atggaccaat
aataatgact 1560agagaagaaa gaatgaagat tgttcatgaa attaaggaac
gaatattgga taaatatggg 1620gatgatgtta aggctattgg tgtttatggc
tctcttggtc gtcagactga tgggccctat 1680tcggatattg agatgatgtg
tgtcatgtca acagaggaag cagagttcag ccatgaatgg 1740acaaccggtg
agtggaaggt ggaagtgaat tttgatagcg aagagattct actagattat
1800gcatctcagg tggaatcaga ttggccgctt acacatggtc aatttttctc
tattttgccg 1860atttatgatt caggtggata cttagagaaa gtgtatcaaa
ctgctaaatc ggtagaagcc 1920caaacgttcc acgatgcgat ttgtgccctt
atcgtagaag agctgtttga atatgcaggc 1980aaatggcgta atattcgtgt
gcaaggaccg acaacatttc taccatcctt gactgtacag 2040gtagcaatgg
caggtgccat gttgattggt ctgcatcatc gcatctgtta tacgacgagc
2100gcttcggtct taactgaagc agttaagcaa tcagatcttc cttcaggtta
tgaccatctg 2160tgccagttcg taatgtctgg tcaactttcc gactctgaga
aacttctgga atcgctagag 2220aatttctgga atgggattca ggagtggaca
gaacgacacg gatatatagt ggatgtgtca 2280aaacgcatac cattttgaac
gatgacctct aataattgtt aatcatgttg gttacgtatt 2340tattaacttc
tcctagtatt agtaattatc atggctgtca tggcgcatta acggaataaa
2400gggtgtgctt aaatcgggcc attttgcgta ataagaaaaa ggattaatta
tgagcgaatt 2460gaattaataa taaggtaata gatttacatt agaaaatgaa
aggggatttt atgcgtgaga 2520atgttacagt ctatcccggc attgccagtc
ggggatatta aaaagagtat aggtttttat 2580tgcgataaac taggtttcac
tttggttcac catgaagatg gattcgcagt tctaatgtgt 2640aatgaggttc
ggattcatct atgggaggca agtgatgaag gctggcgctc tcgtagtaat
2700gattcaccgg tttgtacagg tgcggagtcg tttattgctg gtactgctag
ttgccgcatt 2760gaagtagagg gaattgatga attatatcaa catattaagc
ctttgggcat tttgcacccc 2820aatacatcat taaaagatca gtggtgggat
gaacgagact ttgcagtaat tgatcccgac 2880aacaatttga ttagcttttt
tcaacaaata aaaagctaaa atctattatt aatctgttca 2940gcaatcgggc
gcgattgctg aataaaagat acgagagacc tctcttgtat cttttttatt
3000ttgagtggtt ttgtccgtta cactagaaaa ccgaaagaca ataaaaattt
tattcttgct 3060gagtctggct ttcggtaagc tagacaaaac ggacaaaata
aaaattggca agggtttaaa 3120ggtggagatt ttttgagtga tcttctcaaa
aaatactacc tgtcccttgc tgatttttaa 3180acgagcacga gagcaaaacc
cccctttgct gaggtggcag agggcaggtt tttttgtttc 3240ttttttctcg
taaaaaaaag aaaggtctta aaggttttat ggttttggtc ggcactgccg
3300acagcctcgc agagcacaca ctttatgaat ataaagtata gtgtgttata
ctttacttgg 3360aggtggttgc cggaaagagc gaaaatgcct cacatttgtg
ccacctaaaa aggagcgatt 3420tacatatgag ttatgcagtt tgtagaatgc
aaaaagtgaa atcaggggga tcctaatcgg 3480cgcttttctt ttggaagaaa
atatagggaa aatggtactt gttaaaaatt cggaatattt 3540atacaatatc
atatgtttca cattgaaagg ggaggaaaat cgtgaaacaa caaaaacggc
3600tttagtctag caaaaggaga gggtaaagaa tgaaaatgaa aatgaaatta
gcatcgtttg 3660gtcttgcagc aggactagcg gcccaagtat ttttacctta
caatgcgctg gcttcaacgg 3720aacacgttac atggaaccaa caatttcaaa
cccctcaatt catctccggt gatctgctga 3780aagtgaatgg cacatcccca
gaagaactcg tctatcaata tgttgaaaaa aacgaaaaca 3840agtttaaatt
tcatgaaaac gctaaggata ctctacaatt gaaagaaaag aaaaatgata
3900accttggttt tacgtttatg cgcttccaac aaacgtataa agggattcct
gtgtttggag 3960cagtagtaac tgcgcacgtg aaagatggca cgctgacggc
gctatcaggg acactgattc 4020cgaatttgga cacgaaagga tccttaaaaa
gcgggaagaa attgagtgag aaacaagcgc 4080gtgacattgc tgaaaaagat
ttagtggcaa atgtaacaaa ggaagtaccg gaatatgaac 4140agggaaaaga
caccgagttt gttgtttatg tcaatgggga cgaggcttct ttagcgtacg
4200ttgtcaattt aaacttttta actcctgaac caggaaactg gctgtatatc
attgatgccg 4260tagacggaaa aattttaaat aaatttaacc aacttgacgc
cgcaaaacca ggtgatgtga 4320agtcgataac aggaacatca actgtcggag
tgggaagagg agtacttggt gatcaaaaaa 4380atattaatac aacctactct
acgtactact atttacaaga taatacgcgt ggaaatggga 4440ttttcacgta
tgatgcgaaa taccgtacga cattgccggg aagcttatgg gcagatgcag
4500ataaccaatt ttttgcgagc tatgatgctc cagcggttga tgctcattat
tacgctggtg 4560tgacatatga ctactataaa aatgttcata accgtctcag
ttacgacgga aataatgcag 4620ctattagatc atccgttcat tatagccaag
gctataataa cgcattttgg aacggttcgc 4680aaatggtgta tggcgatggt
gatggtcaaa catttattcc actttctggt ggtattgatg 4740tggtcgcaca
tgagttaacg catgcggtaa ccgattatac agccggactc atttatcaaa
4800acgaatctgg tgcaattaat gaggcaatat ctgatatttt tggaacgtta
gtcgaatttt 4860acgctaacaa aaatccagat tgggaaattg gagaggatgt
gtatacacct ggtatttcag 4920gggattcgct ccgttcgatg tccgatccgg
caaagtatgg tgatccagat cactattcaa 4980agcgctatac aggcacgcaa
gataatggcg gggttcatat caatagcgga attatcaaca 5040aagccgctta
tttgattagc caaggcggta cgcattacgg tgtgagtgtt gtcggaatcg
5100gacgcgataa attggggaaa attttctatc gtgcattaac gcaatattta
acaccaacgt 5160ccaactttag ccaacttcgt gctgccgctg ttcaatcagc
cactgacttg tacggttcga 5220caagccagga agtcgcttct gtgaagcagg
cctttgatgc ggtaggggtg aaataagaat 5280tcctgcaggt taacagagga
cggatttcct gaaggaaatc cgttttttta tttta 53359300PRTBacillus
amyloliquefaciens 9Ala Ala Thr Thr Gly Thr Gly Thr Thr Leu Lys Gly
Lys Thr Val Ser 1 5 10 15 Leu Asn Ile Ser Ser Glu Ser Gly Lys Tyr
Val Leu Arg Asp Leu Ser 20 25 30 Lys Pro Thr Gly Thr Gln Ile Ile
Thr Tyr Asp Leu Gln Asn Arg Glu 35 40 45 Tyr Asn Leu Pro Gly Thr
Leu Val Ser Ser Thr Thr Asn Gln Phe Thr 50 55 60 Thr Ser Ser Gln
Arg Ala Ala Val Asp Ala His Tyr Asn Leu Gly Lys 65 70 75 80 Val Tyr
Asp Tyr Phe Tyr Gln Lys Phe Asn Arg Asn Ser Tyr Asp Asn 85 90 95
Lys Gly Gly Lys Ile Val Ser Ser Val His Tyr Gly Ser Arg Tyr Asn 100
105 110 Asn Ala Ala Trp Ile Gly Asp Gln Met Ile Tyr Gly Asp Gly Asp
Gly 115 120 125 Ser Phe Phe Ser Pro Leu Ser Gly Ser Met Asp Val Thr
Ala His Glu 130 135 140 Met Thr His Gly Val Thr Gln Glu Thr Ala Asn
Leu Asn Tyr Glu Asn 145 150 155 160 Gln Pro Gly Ala Leu Asn Glu Ser
Phe Ser Asp Val Phe Gly Tyr Phe 165 170 175 Asn Asp Thr Glu Asp Trp
Asp Ile Gly Glu Asp Ile Thr Val Ser Gln 180 185 190 Pro Ala Leu Arg
Ser Leu Ser Asn Pro Thr Lys Tyr Gly Gln Pro Asp 195 200 205 Asn Phe
Lys Asn Tyr Lys Asn Leu Pro Asn Thr Asp Ala Gly Asp Tyr 210 215 220
Gly Gly Val His Thr Asn Ser Gly Ile Pro Asn Lys Ala Ala Tyr Asn 225
230 235 240 Thr Ile Thr Lys Ile Gly Val Asn Lys Ala Glu Gln Ile Tyr
Tyr Arg 245 250 255 Ala Leu Thr Val Tyr Leu Thr Pro Ser Ser Thr Phe
Lys Asp Ala Lys 260 265 270 Ala Ala Leu Ile Gln Ser Ala Arg Asp Leu
Tyr Gly Ser Gln Asp Ala 275
280 285 Ala Ser Val Glu Ala Ala Trp Asn Ala Val Gly Leu 290 295 300
104PRTArtificialsynthetic reagentMISC_FEATURE(1)..(1)aminobenzoyl
modificationMISC_FEATURE(4)..(4)nitrobenzylamide modification 10Ala
Gly Leu Ala 1
* * * * *