CZ20001503A3 - Vícenásobně substituované varianty proteáz - Google Patents
Vícenásobně substituované varianty proteáz Download PDFInfo
- Publication number
- CZ20001503A3 CZ20001503A3 CZ20001503A CZ20001503A CZ20001503A3 CZ 20001503 A3 CZ20001503 A3 CZ 20001503A3 CZ 20001503 A CZ20001503 A CZ 20001503A CZ 20001503 A CZ20001503 A CZ 20001503A CZ 20001503 A3 CZ20001503 A3 CZ 20001503A3
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- CZ
- Czechia
- Prior art keywords
- protease variant
- positions
- amino acid
- residue
- substitution
- Prior art date
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Classifications
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- C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
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- C—CHEMISTRY; METALLURGY
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- C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
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- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
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- C11D3/38609—Protease or amylase in solid compositions only
-
- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
- C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
- C11D3/16—Organic compounds
- C11D3/38—Products with no well-defined composition, e.g. natural products
- C11D3/386—Preparations containing enzymes, e.g. protease or amylase
- C11D3/38618—Protease or amylase in liquid compositions only
-
- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
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- C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
- C11D3/39—Organic or inorganic per-compounds
- C11D3/3902—Organic or inorganic per-compounds combined with specific additives
- C11D3/3905—Bleach activators or bleach catalysts
- C11D3/3907—Organic compounds
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- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
- C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
- C11D3/39—Organic or inorganic per-compounds
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- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
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- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/21—Serine endopeptidases (3.4.21)
- C12Y304/21062—Subtilisin (3.4.21.62)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
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- A61K2800/86—Products or compounds obtained by genetic engineering
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Description
Oblast techniky
Serinové proteázy jsou podskupiny karbonylových hydroláz.
které vykazují' ‘široké funkcí (popisuje se 131: 74-88). Navzdory
Obsahují diverzní skupinu enzymu, rozmezí specifit a biologických v publikaci Stroud, R. Sci. Amer., jejich funkční diverzitě katalitického mechanizmu serinových proteáz se dosáhlo alespoň u dvou geneticky odlišných rodin 7 enzymů: 1)' subtiliziny a 2.) serinové. proteázy příbuzné savčímu chymotrypsinu a homologní bakteriální serinové proteázy (například trypsin a trypsin S. gresius) . Tyto dvě rodiny serinových proteáz vykazují značně stejné mechanizmy katalýzy (popisuje se v publikaci Kraut, J. (1977) , .' Annu. Rev. Biochem., 46: 331-358. .Dále, ačkoli proteázy nemají příbuznou primární strukturu, terciální . struktura .těchto dvou enzymatických rodin tvoří konzervativní katalytickou triádu aminokyselin, které obsahují serin, histidin. a aspartát.
Subtiliziny jsou serinové proteázy (s . přibližnou molekulovou hmotností 27 500), které se vylučují ve velkém množství z širokého rozmezí druhů Bacillus j iných mikroorganizmů. Proteinová sekvence subtilizínu se stanovila alespoň z devíti různých druhů Bacillus (Markand, F. S., et al., (1983), Hoppe-Seyler's Z. Physiol. Chem. 364: 1537-1540).
V literatuře se zmiňuje struktura subtilizínů amyloliquefaciens, Bacillus trojrozměrná krystalografická z mikroorganizmu Bacillus licheniformis a několik Tyto studie přirozených variant mikroorganizmu B. lentus. ukazují, že ačkoli subtilizín' není geneticky příbuzný se proteázami, vykazuje podobnou aktivní místní strukturu. Analýza krystalických struktur subtilizínu pomocí X-záření, přičemž subtilizín obsahuje- kovalentně vázané peptidové inhibitory (popisuje se v.publikaci Robertus, J. D., et al., (1972), Biochemistry, 11: 2439-2449) nebo komplexy savčími serinovými • · · · • ·
• '.·.·· 2 produktů (popisuje se v publikaci Robertus, J. D. et al/, (1976), J. . Biol. Chem. 251:' ' 1097-1103) , poskytuje také informace o aktivním místě a 'putativním vazebném štěpení f. substrátu subtilizínu.' Publikuje se velké množství studií /kinetických a chemických úprav molekul (popisuje se v publikaci Svedsen, B., '(1976) Cerlsberg Res. Commun., 41:
237-291, Marklánd, F. S. Id.;) a existuje’alespoň jedna zpráva, kde vedlejší řetězec methioninu subtilizínu s 222 zbytky .'se | V přeměnil peroxidem vodíku na methioninsulf oxid (popisuje se I v publikaci Stauffer, D. C., et al., (1965), J. Biol. Chem.,
Λ ' ,
244: 5333-5338) a provedla se rozsáhlá místně specifická /•mutageneze (Wells and Estell (1.988) TIBS 13: 291-297)'.
Podstata vynálezu ,-Vynález popisuje varianty proteáz. obsahující substituci aminokyseliny . v jedné nebo ;více poloh . zbytků odpovídající polohám zbytků vybraných ze 'skupiny zahrnující 62, 212, 230,
232, / 252 'a -257 •amyloliquefaciens.
Zatímco se může uvedených substitucí subtilizínu mikroorganizmu Bacillus ze shora proteázové použít libovolná kombinace aminokyselin, preferované varianty enzymů použitelné podle vynálezu obsahují substituci aminokýselinových zbytků v následujících kombinacích poloh. Všechny polohy zbytků odpovídají polohám subtilizínu mikroorganizmu Bacillus amyloliquefaciens:
1) varianta,'. proteázy zahrnující substituce 'aminokyselinových zbytků v poloze 62 a v jedné nebo více z následujících poloh .103, 104, 109, 159, 213,
-232,. 236, 245, 248 -a 252,
2) .. varianta proteázy zahrnující . aminokyselinových zbytků· v poloze nebo . více .z následuj ících . poloh 12, ' ·' 104, 159, 183, 185, 205, 209, 210,
215, 217, 232, 236, 245, 248.a 252,
substituce | |
212 | a. v jedné |
.98, | 102, 1.03, |
211, | 212, 213, |
• ·
3) varianta proteázy zahrnující substituce aminokyselinových zbytků v. poloze 230 a v-jedné nebo více z následujících poloh. 68, 103, 104, 159,
232, 236 a 245,
4) -. varianta proteázy, - .zahrnující substituce aminokyselinových zbytků 'v poloze. 232 a v jedné nebo více z následujících poloh 1,· 9, 12, 61’, 62,
68, 76, 97, /98, 101, .102, 103, 104., 109, -130, 131, 159, 183, 185, 205, 209, 210, ,212, .213, 217, 230,
236, 245, 248-,-252, 257, 260, 270 a 275,
5) varianta-.· proteázy. \ , zahrnující substituce aminokyselinových -zbytků. v poloze 232 a v jedné nebo více 'z následujících poloh 103, 104, 236 a
245, . -· .. . . '
6) varianta proteázy zahrnující substituce • aminokyselinových .zbytků v poloze 232 a.. 103 a v-jedné nebo více z následujících -poloh .1, .9, .12,.
-/ l... 61, 62, 68, 76, 97, 98, 101, 102, 103, . 104, 109,
130, 131, 159, 183, 185, 205, 209, 210, 212, 213,.
, ‘ 217’, 230, 236, ' 245, 243, 252, 257, 260, 270 a 275,
7) varianta. proteázy zahrnující substituce aminokyselinových zbytků v poloze 232 a 104 a v jedné nebo více z následujících poloh 1, 9, 12,
61', 62, 68, 76, 97, 98, 101, 102, 103, 104, 109,
130, 131, 159, 183, 185, 205, 209, 210, 212, 213,
217, 230, 236, 245, 248, 252, 257, 260, 270 a 275,
8) varianta proteázy zahrnující substituce aminokyselinových zbytků v poloze 232 a 236 a v jedné nebo více z následujících poloh 1, 9, 12,
61, 62,. 68, 76, 97, 98, 101, 102, 103, 104, 109,
130, 131, 159,-.183, 185, 205, 209, 210, 212, 213,
217, . varianta ‘proteázy zahrnující substituce aminokyselinových zbytků v poloze 232 a 245 a - v jedné nebo více zj následujících poloh 1, 9, 12, • ·
61., 62, 68, 76, 97,. 98, 101, 102, 103, 104, 109,
130, 131, 159, 183, 185, 205, 209, 2.10, 212,' 213,
217, 230, 236, 245, 248, 252, 257, 260, 270 a 275,'
9) ', varianta · proteázy zahrnující substituce aminokyselinových zbytků v poloze 232 a 245- a v jedné nebo, více z následujících poloh 1, 9, 12,
61,’ 62, 68, 76, 97, 98, 101, 102, 103, 104, 'l09, '130,' 131,' 159, 183, 18.5, 205, 209, 210-, 212, 213;
21-7,-230, 236, 245, 248, 2 52, 257, 2 60, 270 -a 275,
10) varianta proteázy zahrnující , substituce aminokyselinových zbytků v poloze . 232, 103, 104,
236 a 245-a v jedné nebo více z následujících poloh
1, 9, 12, 61, 62, 68, . 76 | , 97, 98, 101 | , 102, -103, | |
' 104, 109,' 130, 131, 159, | 183, 185, 205 | ,. 209, 210, | |
‘ 212, 213, 217, 230, 236, | 245, 248, 252 | , 257, 260, | |
.. 270 a 275, - .. . | 3 · .- | ||
11) | varianta proteázy | zahrnující.\ . | substituce |
- aminokyselinových zbytků | v poloze 252 | . a v jedné | |
nebo více z následujících | poloh 1, 9, | 12, 61, 62, | |
68, -98, 101, 102, 103, . : | 104, 109., 130, | 131, 159, | |
.183, 185, 210, 212, 213, | 217, 232, 236, | 245, 248 a | |
270,' | |||
12) | varianta proteázy | zahrnuj ící | substituce |
aminokyselinových zbytků | v poloze 252 | a v jedné | |
nebo více z následuj ících poloh 103, | 104, 236 a | ||
245, | |||
13) | varianta proteázy | zahrnuj ící | substituce |
aminokyselinových zbytků | v poloze 252 | : a 103 a |
v jedné nebo . více z následujících poloh 1, 9, 12,
61, 62, | 68, '97, 98, 101, | 102, 103, | 104, | 10 9, 130, | |
. 131, 159 | , 183, 185, 210, | 212, 213, | 217, | 232, 236, | |
245, 248, | , a 270, | ||||
14) | varianta | proteázy | zahrnující | substituce | |
aminokyselinových zbytků | v poloze | 252 | a 104 a |
16)
17)
18) ξ
• to 5- 1. | ···· ·· ··· • · · · • to · · · to. · · to · - · ·· ·· · | • · · •' · to • • to | • · ♦ · ' · ·· · ·· · • · | |
v jedné nebo·více z následujících poloh | 1, 9 | , 12, | ||
61, 62, 68, 97, 98, 101, -i | 102, 103, | 104, . | 109, | 130, |
.131,’ 159,. 183, 185, 210, | 212, 213, | ,217, | 232, | 236, |
245, 248, a 270, | ||||
varianta proteázy | zahrnující | substituce | ||
aminokyselinových zbytků | v poloze ' | 252 | a 236 a | |
v. jedné nebo více z následujících poloh | 1,- 9 | , 12, | ||
61, 62,' ‘68, 97, 98, 101, | 102, 103,- | 104, | 109, | 130, |
131, .159, 183, 185, 210,, | 212, 213, | .217, | 12 32, | 236, |
245, 248, a 270, ' ' | ||||
varianta proteázy | zahrnuj ící | substituce | ||
aminokyselinových zbytků | v poloze | 252 | a 245 a | |
v jedné nebo více z následujících poloh | .1, 9 | , 12, | ||
61, 62,- 68, 97, 98, 101, | 102, 103, | 104, | 10 9, | 130, |
131, 159, .183, 185, 210, | 212, 213, | 217, | 232, | 236, |
2 4 5’, 24 8, a 270, . | ||||
varianta ' ' proteázy | -zahrnuj ící | ..substituce | ||
aminokyselinových zbytků | .v poloze 252, | 103-, | 104, | |
236 a 245 a v jedné nebo více z následují | .cích | poloh | ||
1, 9, 12, 61, 62, 68,. 97, | , 98.,. 101, | 102, | ‘ 103, | 104, |
109, 130, 131, .159, 183, | 185, 210, . | 212, | 213, | 217, |
232, 236, 24 5, '24 8,' a 270 | a | - | ||
varianta proteázy | zahrnuj ící | substituce | ||
aminokyselinových zbytků | v poloze | 257 | a v | j edné |
nebo více z následujících | poloh 68, | 103, | 104, | 205, |
209, 210, 232, : 236, 245 a 275. Více preferované proteázové varianty jsou sada substitucí vybrané ze skupiny obsahující polohy zbytků odpovídající polohám v tabulce č. 1 subtilizínu mikroorganizmu Bacillus amyloliquefaciens:
<?
)339
7 9
7 j
7
7 9 9
3 3 3
7995» • 9 «3 9 3 y 3 9
3 J 5
3 3 9
9 3 9
3-7 9 9
o ! | I | i ΐ ! I í i | i ! ! i i ! ’ ! ! ; | |||||||||||||||
! i | i ! | I i I í , i L | 1 | |||||||||||||||
i I I | i | |||||||||||||||||
I ί I | LT7 TT 04 | 1 1 1 259 | I 260 | 245 | ||||||||||||||
U7 «Τ OJ | 257 | 275 | P- to OJ | I I 10 i s J | 07 Of | 236 | i í ď71 ι-C i U7 ”7 I ί *7 οι 1 ojí oj í i | 245 | 261 | 245 | 236 | |||||||
236 | co •«r OJ | 257 | tn TT OJ | m OJ | co 07 OJ | 236 232 } 236 | 236 236 | 236 | tO 7 OJ | 236 | 232 | |||||||
232 | 236 | 257 | m v OJ | 236 | 245 | OJ 07 OJ | OJ 07 Ol | ΤΓ Ol | i 1 OJI OJ I Ol 07 1 071 07 Ol i Ol | OJ í | 232 | 236 | | 245 | 232 | 159 | ||||
210 | 232 | in τ OJ | 236 | OJ 07 OJ | 236 | O) o OJ | Ol | 159 I | 215 | 159 159 | 159 | j 232 | 242 | | 210 | 104 | 245 | ||
159 | 159 | 236 | OJ 07 OJ | 275 | ’Τ OJ OJ | 232 | 03 U7 | 03 U7 | o | 159 | ! 7 ί TT cl o Ί | 7 o | 159 | 236 | I- 159 | 103 | 236 I | |
104 | 104 | 232 | 03 U7 V“ | 257 | 159 | 159 | O τ— | ,:·ΤΓ o | 103 | 7* .o | 103 | } 103 | 103 | 104 | 232 | 104 | 76 | I 232 |
103 | 103 | 104 | XT O | o 5— | *T O τ— | ^r o | 07 O | 07 ? O | .CO | 103 | C fs. | 76 | 76 | 103 | 104 | 103 | 68 | 104 |
76 | 76 | 103 | 103 I | 103 | 103 | 07 O v- | 76 | 76 | I 68 | 76 | •o o | 68 | 87 | 76 | | 103 | co r- | co 7 | 103 ' |
I 68 | 68 | 76- | 68 | 76 | 68 | 76 | I 68 | ---1 68 I | OJ | 89 | .Oj | O OJ | 89 | 68 | 76 | 68 | Ol | 76 |
99··
9···
1 _!__ | 1 : ! ! H I | |||||||||||||||||
• | 1 | . | ||||||||||||||||
tn | CM | |||||||||||||||||
•o· | m | |||||||||||||||||
CM . | CM | |||||||||||||||||
. CM | tn | CO | <N | CM | « | CM | CM | CM | CM | 4— | CM | |||||||
ib | Γ- | XT | co | tn | IO | LO | xr 1 | LO | tn | LO | io | CO | m | |||||
CM | CM | CM | CM | CM | CM | .CM | CM | CM | CM | CM | CM | CM | CM | |||||
00 | tO | CO | co | m | tO | CM | CO | CO | oo | tn | CO | 00 | 00 | CO | CM | 00 | ||
xr | xr | CO | LC | o- | O* | CO | o* | XT | XT | XT | xr | xr | XT | xr | tn | xr | ||
CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | ||
ΙΟ | CO | CO | CM | lO | co | CO | LO | tO | tn | LO | co | CM | LO | IO | to | tn | CO | tn |
XT | XT | CO | CO | θ’ | CO | CO | co | θ' | XT | XT | (O | in | xr | xr | xr | XT | xr | xr |
CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | |
CO | co | CM | CD | CO | CM | CM | O | CO | co | co | CM | co | co | co | co | co | tn | co. |
CO | CO | CO | O | CO | CO | CO | r*- | CO | co | CO | CO | xr | co | co | co | co | xr | CO |
CM | CM | CM | CM | CM | CM | CM | T-_ | CM | CM | CM | t\l | CM | CM | CM | CM | CM | CM | CM |
CM | CM | cn | CO | CM | CO | cn | <n | CM | CM | CM | xr | tn | CM | CM | CM | CM | CO | CM |
co | co | to | 00 | CO | O | tn | IO | CO | CO | CO | <50 | XT | CO | CO | CO | co | co | CO |
CM | CM | T— | CM | CM | . | CM | CM | CM | 4— | CM | CM | CM | CM | CM | CM | CM | ||
CM | cn | XT | cn | cn | cn | XT | CO | cn | cn | CM | cn | CO | cn | cn | cn | cn | CM | LO |
cn | IO | O | to | to | tO | O | 4— | lO | IO | 4— | tn | CO | o | to | to | o | CO | oo |
4“ | ** | 4“ | 4— | 4— | T— | CM | CM | CM | 4— | CM | CM | |||||||
cn | r^- | CO | xr | XT | XT | co | XT | XT | XT | cn | CM | cn | cn | 4T | cn | cn | cn | |
tn | XT | o | o | O | O | o | o | o | o | LO | CO | to | o | o | to | to | to | |
’Η’ | r^· | 4“ | CM | 4— | ||||||||||||||
•θ’ | xr | co | co | CO | co. | COl | co | XT | xr | cn | xr | xr | co | xr | xr | xr | ||
o | o | fs. | o | o | O | o | co | o | o | o | in | o | o | o | o | o | o | |
T“ | 4— | V“ | .4“ | 4“ | 4— | 4“ | ' 4“ | |||||||||||
03 | 03 | CO co | 1 76 | 1 76 | 76 | oo co | co h* | oo co | oo CD | 03 | 03 | 04 | 03 | 03 | oo co | 03 | 03 | 03 1 |
4“ | 5 | V“ | ||||||||||||||||
CO | CO | CM | 00 | 00 | OO | N. | OO | co | 00 | CO | CO | 00 | 00 | o | oo | CO | 00 | |
r- | r* | co | co | co | CM | CO | CD | xr | co | co | o | co | co | CM | co | CO | co |
···· ··
·♦ φφφφ ·· ···· • Φ . · φ φ . · • φ φφφ · φ ♦ φφφφφ < φφφ φ · · φφ φφ φφ >·
I -1 1 • 1 | j . i I l· ! | i Ί I | I | I ' í l I z | l | I | ||||||||||||
o | o | CM | ||||||||||||||||
co | co | to | ||||||||||||||||
CM | CM | CM | ||||||||||||||||
Ο | A- | lO | CM | to | Csl | CM | CO ! | |||||||||||
CO | O- | to | TT | to | to | T i | ||||||||||||
CM | CM | CM | CM | CM | CM | Csl | | ||||||||||||
CM | φ· | T“ | CM | CM | CO | CM | o | CO | CO | CO | to | CM | ||||||
ΙΟ | <Ο | (O | LO | LO | co | lO. | O· | CO | rr | O* | TT | to | ||||||
CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | Csl | |||||||
CO | CM | CM | 00 | CM | CO | Csl | CM | CO | - | Csl | to | to | CO | co | CM | |||
θ’ | to | tO | M*. | to | O | LO | co | **T | O* | CO | M· | CO | - M* | to | ||||
CN | CM | CM | CM | * | CM | CM | CM | CM | Csl | CM | CM | CM | CM | CM | CM | . CM | ||
ιο | co | CO | to | CM | CM | CO | to | ‘oo | CO | LO | • Ό | CO | CO | tO | co | CM | to | oo |
μ- | Μ | M | to | to | M* | o* | TT | M* | CO | T— | CO | θ’ | co | CO | M* | •^r | ||
Csl | CM | CM | CM | CM | Csl | CM | CM | Csl | CM | Csl | CM | CM | CM | CM | Csl | CM | CM | |
'. to | to | · tO | CO | oo | CO | to | CO | to | CO | cd | CM | O | CM | CO | CM | cn | co | to |
.·<ο | O* | CO | rr | TT | co | TT | *— | co | CO | CO | fO | CO | to | <o | TT | |||
CM | CM | Csl | CM | CM | CM | CM | CM | Csl | Csl | CM | CM | CM | CM | CM | CM | CM | ||
CM | CO | CO | CM | to | to | co | CM | Co | σ> | CM | o | O | CJ> | Csl | cn | >- | CM | co |
CO | co | co | CO | o- | M* | co | CO | co | m | CO I o | to | LO | CO | to | co | CO | co | |
CM | CM | Csl | CM | CM | CM | CM | CM | CM | CM | - | CM | CM | CM | |||||
σ> | CM | CM | O) | CO | CO | CM | CT> | CM | •'θ’ | CO | T* | ‘ θ’ | Μ | O | O | co | O | CM |
ΙΟ | CO | CO | to | CO | co | CO | io | CO | o | CM | O | o | O | co | co | to | CO | |
Csl | CM | CM | CM | CM | CM | Csl | T— | CM | Csl | |||||||||
co | cn | O | tt | CM | CM | O | M* | cn | co | O | ΓΟ | CO | CO | to | θ’ | co | σ> | |
tO | tO | O | CO | CO | to | O | to | O | uo | o | O | o | o | o | o | co | to | |
T“ | Τ’* | ' CM | CM | T“ | T“ | CM | ||||||||||||
. ο* | θ’ | TT | CO | rr | cn | TT | CO | θ’ | co | 89 | <o | cn | co | co | ΤΓ | tt | ||
ο | o | O | o | o | to | o | o | o | o | O | to | o | o | o | o | |||
T“ | ▼— | T· | V“ | |||||||||||||||
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O | co | 05 | to | to | to | to | o | t— | to | tO | tO | co | tO | tO | O | |||
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o | > | o | < | __ | _ | < | Ω | > | n | X | Ω | |||||||
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TT | co | co | co | CO | CO | CO | K co | CO | CO | CO | TT | CO | co | co | co | co | CO | CO |
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co | CO | CO | CO | CO | tt— | CO | CO | CO | CO | CO | co | CO | CO | CO | CO | CO | CO | CO |
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Dálé vynález popisuje ' sekvence DNA kódující takovéproteázové varianty, stejně jako expresívní vektory obsahující takové, sekvence DNA. .
' Dále vynález popisuje hostitelské buňky transformované takovými vektory, stejně jako hostitelské buňky, které jsou schopny exprimovat takovou -DNA za intracelulární nebo extracelulární produkce proteázových variant.
Dále · vynález popisuje.· čistící kompozice obsahující proteázovou variantu podle vynálezu.
Dále vynalez zahrnuje - zvířecí’ krmivo obsahující proteázovou variantu podle vynálezu. . .
. , . Dále vynález popisuje kompozici pro ošetření textilie obsahující proteázovou variantu podle vynálezu.
Proteázy jsou karbonylové hydrolázy, které v obecném případě štěpí peptidové vazby'.proteinů a' peptidů'. Termín „proteáza znamená přirozené se .vyskytující proteázu nebo,. r.ekombinantní proteázu. Přirozeně se vyskytující, proteázy zahrnují aaminoacylp'epti'dovqu hydroiázu, hydrolázu ' peptidylaminové kyseliny,·, acyiaminovou.^.hydrolázu, serinovou - karboxypeptidázu, metalokarboxypept idázu’, thiolovou proteinázu, karboxylpro.teinázu a metaloproteinázu. Dále zahrnují serinovou proteázu, metalopřoteázu, thiolovou proteázu a kyselou proteázu. stejně jako exogenní proteázy.
Vynález popisuje proteázové enzymy, kterými jsou varianty karbonylových hydroláz (varianty proteáz) , které se přirozeně nevyskytují a které mají různou proteolytickou aktivitu., stabilitu, specifitu substrátu, profil pH a/nebo charakteristiku provedení ve srovnání s prekurzorovou karbonylovou hydřolázou, ze které se odvodila aminokyselinová sekvence.. Takové varianty proteáz mají specificky aminokyselinovou sekvenci, která se nenachází v přírodě, která se získala , ‘substitucí velkého množství aminokyselinových zbytků prekurzorové proteázy různými aminokyselinami.
····
Prekurzorovou proteázou může být přirozeně se vyskytující proteáza nebo rekombinantní proteáza.
Varianty proteáz, které jsou použitelné' podle vynálezu, zdůrazňují substituci libovolné ,ζ Ί9 přirozeně se vyskytujících L-aminokyselin v označených polohách aminokyselinových . zbytků. Takové substituce se mohou provést v libovolném prekurzorovém subtilizínu (prokaryontní, eukaryontní, savčí, atd...). V této přihlášce se aminokyseliny, popisují jednopísmenným nebo. třípísmenným kódem. Takové kódy se popisují v publikaci Dále, M. W.. (1989), Molecular Genetics of Bacterie) -John Wiley and Sons, Ltd., Appendix B.
, Použité' varianty proteáz se mohou získat, ze subtilizínu mikroorganizmu Bacillus. Více. se upřednostňuje,, aby se pro.fceázové /varianty odvodily' ze. subtilizínu „ mikroorganizmu Bacillus 'lentus a/nebo subtilizínu 309.
Subtilizíny jsou bakteriální a houbové -proteázy, které v obecném případě štěpí pept-idové vazby- proteinů. a peptidů. Termín' - .„subtilizín znamená přirozeně se. vyskytující subtilizín nebo rekombinantní subtilizín. Série přiro'zeně se výskytujících subtilizínů je známa, že se produkuje a často se vylučuje různými mikrobiálními druhy. Aminokyselinové sekvence členů uvedených - sérií 'nejsou celé homologní. . Subtilizíny těchto sérií vykazují stejný nebo podobný typ proteolytické aktivity. Tato třída serinových proteáz sdílí běžnou aminokyselinovou sekvenci definující katalytickou triádu, která je odlišuje od třídy serinových proteáz příbuzných chymotripsinu. Subtilizíny a serinové proteázy příbuzné chymotripsinu mají katalytickou -triádu obsahující aspartát, histidin a serin. U proteáz příbuzných subtilizínu je relativní pořadí aminokyselin, které se čtou od N-konce k Ckonci, aspartát-histidin-serin.. U proteáz příbuzných chymotrypsinu je relativní pořadí histidin-aspartát-serin. Subtilizín zde vystupuje jako serinováproteáza vykazující katalytickou, triádu proteáz příbuzných 'subtilizínu. Příklady ·· «· ► · · 4 » 4 · 1 »· ···· je . kódující se mohou eukaryontních organizmů, zahrnují, ale nejsou omezeny na subtilizíny uvedené na obrázku č. 3. Číslování aminokyselin v proteázách odpovídá číslům· přiřazených sekvencím subtilizínu mikroorganizmu Bacillus amyloliquefaciens přítomných ňa obrázku č. 1.
Termín „rekombinantní subtilizín . nebo „rekombinantní proteáza znamená subtilizín,’ ve kterém sekvence DNA kódující subtilizín nebo proteázu . je upravena tak, áby se produkovala varianta (nebo mutant) sekvence DNA, která' kóduje substituci, deleci nebo inzerci jedné nebo více aminokyselin v přirozeně se , vyskytující ’ aminokyselinové sekvenci. Vhodné metody· produkce takové úpravy 'jsou popsány v dokumentu US patent RE 34,606, US patent 5,204,015 a US patent 5,185,258, US patent 5,700,676, US patent 5,801,038 a US patent 5,763,257.
• - Termín „subtilizíny, které nepocházejí z člověka, a DNA .získat, . z mnoha prokaryontních . a Vhodné ' příklady prokaryontních organizmů zahrnují gram-negativní .organizmy, jako je E. coli nebo Pseudomonas a grám pozitivní bakterie jako je. Micrococcus nebo Bacillus. Příklady eukaryontních organizmů, ze kterých je možné., .získat subtilizín a< jejich .geny, mohou zahrnovat kvasinky, jakp j sou Saccharomyces. cerevisiae, houby jako je Aspergillus sp..
.Termín „varianta proteázy má .aminokyselinovou sekvenci, která se získala z aminokyselinové sekvence „prekurzorové proteázy. Prekurzorové proteázy zahrnují přirozeně se vyskytující proteázy a rekombinantní proteázy. Aminokyselinová sekvence varianty proteázy se „získala z aminokyselinové sekvence prekurzorové proteázy substitucí, deleci nebo inzercí jedné nebo více aminokyselin prekurzorové aminokyselinové sekvence. Taková modifikace je spíše modifikace „sekvence prekurzorové DNA, která kóduje aminokyselinovou sekvenci prekurzorové proteázy, než úprava enzymy prekurzorové proteázy, per se. Vhodné metody takových úprav prekurzorové sekvence DNA zahrnují, zde popsané metody, stejně jako metody známé v oboru ·· ···· • · · · · · · · · ·· · ·· · · ·· (popisuje se například v dokumentu EP 0 328299, WO89/06279 a v už uvedených US patentech a přihláškách).
Identifikovaly se specifické substituce aminokyselin v jedné nebo více poloh zbytků odpovídající polohám zbytků vybraných ze skupiny zahrnující 62, 212, 230, 232, 252 a 257 subtilizínu mikroorganismu Bacillus amyloliquefaciens.
- Preferované varianty jsou ty, které vykazují kombinace substitucí v polohách zbytků odpovídající polohám subtilizínu mikroorganizmu Bacillus amyloliquefaciens v tabulce č. 1.
• - Více se preferují varianty, které vykazují kombinace substitucí v polohách zbytků, 'které odpovídají polohám .'subtilizínu mikroorganizmu Bacillus amyloliquefaciens uvedené v tabulce č. 2.
Dále se preferují varianty, ..které. mají . kombinace substitucí v polohách; zbytků, jenž odpovídají polohám subtilizínu uvedených v tabulce .č.' 3. '
Tabulka č, 3
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Tyto čísla poloh aminokyselin odpovídají číslům označujícím sekvenci - subtilizínu mikroorganizmu Bacillus amyloliquefaciens, která je uvedena na obrázku č. 1. Vynález se neomezuje pouze na mutaci tohoto určitého subtilizínu, ale také zahrnuje prekurzorové .proteázy, které obsahují aminokyselinové zbytky v polohách,, ^které jsou „ekvivalentní s určitými identifikovanými zbytky v subtilizínu organizmu Bacillus amyloliquefaciens. V preferovaném provedení vynálezu prekurzorové proteáza je subtilizín organizmu Bacillus lentus a substituce se provádí v ekvivalentních polohách aminokyselinových zbytku iv organizmu B. · - lentus, které odpovídají těm uvedeným shora v textu.
Poloha zbytku (aminokyseliny) prekurzorové proteázy je ekvivalentní -zbytku subtilizínu mikroorganizmu Bacillus amyloliquefaciens v případě, že je buď homologní (to znamená, že odpovídá poloze buď v primární nebo terciální struktury) nebo analogická se specifickým zbytkem nebo částí uvedeného zbytku v subtilizínu organizmu Bacillus amyloliquefaciens ( to znamená, že vykazuje stejnou nebo podobnou funkční kapacitu kombinovat, reagovat nebo chemicky interagovat) .,
Za účelem dosáhnout homologie s primární strukturou aminokyselinová sekvence . prekurzorové proteázy . se přímo porovnává s primární sekvencí subtilizínu a zvláště se sadou zbytků, které jsou známy, že jsou v subtilizínech se známou sekvencí neměnné. Například obrázek č.. 2 znázorňuje konzervativní zbytky subtilizínu organizmu B. amyloliquefaciens a B. lentus. Po uspořádání konzervativních zbytků, což umožňuje nezbytné začlenění a delece za účelem udržet uspořádání (to znamená zabránit eliminaci konzervativních zbytků pomocí doplňkové delece a inzerce), se definuji zbytky ekvivalentní pro určité aminokyseliny v primární sekvenci subtilizínu organizmu Bacillus amyloliquefaciens. Uspořádání konzervativních zbytků by mělo přednostně konzervovat 100 % takových zbytků. Avšak uspořádání
Φ φ φ φ φ φ vyšší než 75 % nebo 50 % konzervativních zbytků je také adekvátní pro definování ekvivalentních zbytků. Měla by se udržovat konzervativnost katalytické triády
Asp32/His 64/Ser221. V publikaci Siezen et ál. , (1991) Protein
Eng·. 4(7) : serinových ^rozdělení
719-737 se popisuje uspořádání velkého množství .proteáz. V této publikaci - se také •popisuje' šubtiláz- nebo serinových proteáz podobných subtilizínu do jednotlivých skupin.
Například na obrázku- č. 3 jsou uspořádány aminokyselinové sekvence subtilizínu , z mikroorganizmů Bacillus amyloliquefaciens, Bacilus ' subtilis, Bacilus iichenoformis (carlsbergensis) a. Becillus lentus, přičemž· se stanoví maximální '‘množství homologie' .mezí aminokyselinovými^ sekvencemi. Porovnání těchto sekvencí naznačuje, že- v každé sekvenci existuje řada konzervativních zbytků. ' Tyto konzervativní zbytky (mezi· BPN'a B. lentus} jsou .uvedeny na obrázku č. 2. ...... š ,.*.···' . - - ,
Tyto konzervativní- zbytky se tak mohou použít k definici odpovídajících, ’ ekvivalentních aminokyselinových’ zbytků subtilizínu mikroorganizmu Bacillus amyloliquefaciens' v jiných subtilízínech, jako je subtilizín 'mikroorganizmu Bacillus lentus (popisuje· se v přihlášce PCT č. W089/06279, uveřejněné 13. .července 1989), který · je preferovaným proteázovým prekurozořovým enzymem nebo subtilizín popsaný jako PB92 (popsaný v dokumentu EP 0 328 299), který vykazuje vysokou homologii .s preferovaným subtilizínem mikroorganizmu Bacillus lentus. Aminokyselinové sekvence jistého z těchto subtilizínu jsou uspořádány na obrázku č. 3A a 3B se sekvencí subtilizínu Bacillus amyloliquefaciens tak, aby se dosáhlo maximální homologie konzervativních zbytků. Jak je možné vidět, v sekvenci mikroorganizmu Bacillus lentus ve srovnání se subtilizínem Bacillus amyloliquefaciens existuje řada delecí. Tak -například ekvivalentní aminokyselinou v.případě Val 165
• · · · • · · · • · · · v subtilizínu Bacillus amyloliquefaciens u jiných subtilizínu mikroorganizmů je izoleucin B. lentus a B. licheniformis.
Termín „ekvivalentní zbytky se mohou také definovat stanovenou homologii prekurzorové proteázy na úrovni ..terciální struktury, přičemž. terciální struktura se stanovila krystalografií pomocí rentgenového záření. Ekvivalentní zbytky se definují jako ty, pro které, atomové koordináty dvou' nebo více hlavních řetězcových atomů určitého aminokyselinového zbytku prekurzorové proteázy a subtilizín .mikroorganizmu Bacillus amyloliquefaciens (N na N, CA na CA, C na C a 0 na 0) jsou. 0,13 nm, přičemž se upřednostňuje, aby tyto atomové koordináty po uspořádání byly 0, 1 nm. Uspořádání—se. dosáhne po té, co se zorientuje a umístí nejlepší model, .přičemž dojde k maximálnímu přesahu atomových koordinát atomů vodíku proteinu proteázy vůči subtilizínu Bacillus amyloliquefaciens. Nejlepším modelem je krystalografický model vykazující nejnižší faktor R v případě experimentálních «difrakčních ' dat při nejvyšším možném rozlišení.
R factor lb\Fo(h)\-\Fc(h)[
7ih\Fo(h)\
Ekvivalentní zbytky, které jsou funkčními analogy ke specifickému zbytku subtilizínu Bacillus amyloliquefaciens jsou definovány jako aminokyseliny prekurzorové roteázy, které mohou přizpůsobit konfořmaci tak, že buď změní nebo se podílejí definovaným způsobem na struktuře proteinu, navázání substrátu nebo katalýze a přiřazují se ke specifickému zbytku subtilizínu mikroorganizmu Bacillus amyloliquefaciens. Jsou to ty zbytky prekurzorové proteázy (jejichž terciální struktura se získala rentgenovou krystalografií), které obsazují analogickou polohu, ačkoli atomy hlavního řetězce daného zbytku nemohou uspokojit kriterium ekvivalence na základě obsazení homologické polohy, přičemž atomové koordináty
9 »»·· · 9 9 • « · · ·· 44
9 9 9
9 9 9
9 9 9 • 9 9 9
9 « 9
99
4 «·94 alespoň dvou atomů vedlejšího řetězce zbytku leží 0,13 nm od odpovídajících atomů vedlejšího řetězce subtilizínu mikroorganizmu- Bacillus amyloliquefaciens. Koordináty trojrozměrné . struktury subtilizínu mikroorganizmu Bacillus. amyloliquefaciens jsou uvedeny v dokumentu přihláška EPO č. 0 251 446 (ekvivalent US patentu č. .5/182,204) a' může se použít ke stanovení ekvivalentních zbytků na . úrovni terciální struktury.' . .
. Některé z těchto · zbytků určených· .pr.o substituci,· jsou konzervativní zbytky, zatímco ostatní nejsou konzervativní.
V případě7 zbytku/ které nej sou ' konzervativní ,—·—substituce jednoho nebo více aminokyselin je limitována na substituci, která 'produkuje variantu, která, vykazuje aminokyselinovou sekvenci, která neodpovídá ' sekvenci’ zjištěné v přírodě.
IV-případě konzervativních zbytků výsledkem takových substitucí nebude přirozeně se vyskytující sekvence. Varianty proteáz podle vynálezu zahrnují zralé .formy; variant proteáz, stejně jako pro-formý -a prepro-formy takových' variant proteáz. * *·.· .-'.Prepro-formy jsou preferované konstrukce,' p.rotože umožňují •expresi,' sekreci variant proteáz. ·' * 4 t, '
Termín' „pro-sekvence znamená sekvence aminokyselin vázaných na N-terminální část zralé formy proteázy, která když se odstraní vede k objevení „zralé formy proteázy. Řada proteolytických enzymů se nachází v přírodě jako translační pro-enzymové produkty a v nepřítomnosti post-translačního zpracování se exprimují tímto způsobem. Preferované' prosekvence produkující varianty proteáz jsou putativní prosekvence subtilizínu mikroorganizmu Bacillus amyloliquefaciens, ačkoli se mohou použít jiné protéázové prosekvence.
Termín „signální sekvence nebo.· „presekvence znamenají libovolnou sekvenci aminokyselin vázaných na N-termínální Části proteázy nebo N-terminální části pro-proteázy, která se může podílet na sekreci zralé formy nebo pro-formy proteázy.
·· ···« ·· ···♦ ·· ·· « · ··· ··»·· • € · · · · · ··· • · · · · · · · · · · » • · · · · · · ···· ·· ·· ·· » ·· ··
Tato·· definice signální sekvence je funkční, jestliže zahrnuje všechny uvedené aminokyselinové sekvence kódované N-terminální částí genu proteázy, která se podílí na uskutečnění sekrece proteázy za nativních podmínek. Vynález popisuje takové sekvence, které . se používají pro ovlivnění sekrece' zde definovaných variant proteáz. Jedna možná signální sekvence obsahuje prvních sedm aminokyselinových zbytků signální sekvence, které pochází ze subtilizínu Bacillus .subtillis fúzovaného se zbytkem signální „sekvence . .subtilizínu z mikroorganizmu Bacillus lentus (ATCC 21536).
Termín „prepro-’ forma varianty proteázy zadrrnOýe zrale·; formu proteázy obsahující prosekvenci operativně spojenou s Nkoncem proteáz a s pre-sekvencí nebo se signální sekvencí operativně spojenou s amino-koncem prosekvence.
Termín „expresívní vektor znamená konstrukci DNA obsahující sekvenci DNA, která je operativně spojená :s vhodnou kontrolní sekvencí schopnou ovlivnit expresi uvedené DNA ve vhodném, hostiteli. Takové kontrolní sekvence zahrnují promotor, který ovlivňuje transkripci, operátorovou sekvenci, která řídí takovou-transkripci, sekvenci kódující vhodná místa na ribozóm.u, kam se váže mRNA a sekvence , které řídí ukončení transkripce a translace. Vektorem může být plazmid, fágové částice nebo jednoduše potencionální inzert v genomu. Po té, co je vhodný hostitel transformován, vektor se může replikovat a funguje nezávisle na genomu hostitele nebo se může v některých případech sám začlenit do genomu hostitele. U některých specifikacích termín „plazmid a „vektor jsou vnitřně zaměnitelné, přičemž plazmid je nejběžněji užívanou formou vektoru. Vynález dále popisuje také jiné formy expresívních vektorů, které vykazují ekvivalentní funkce a stávají se dobře známy v oboru.
Termín „hostitelská buňka podle vynálezu je v obecném případě prokaryontní nebo eukaryontní hostitel, který se přednostně upravuje metodami popsanými, v US patentu RE 34,606 tak, aby nebyl schopen vylučovat enzymaticky aktivní endoproteázy. Preferovaná hostitelská buňka pro ’expresi proteázy je mikroorganizmus Bacillus kmen BG2036., 'který je nedostatečný v případě enzymaticky aktivní'neutrální proteázy a alkalické proteázy (subtilizínu) . Konstrukce kmene, BG2036 se popisuje v_ US patentu 5,264,366. Jiné hostitelské buňky vhodné pro expresi proteázy. zahrnují Bacillus subtilis‘16Q (popisuje se v publikaci US patent č. 34,606 a US patent ,č. 5,264,366) stejně jako vhodný kmen mikroorganizmu Bacillus jako je B. licheniformis, B. lentus.atd. - ' · Hostitelské. buňky -se . transformovaly ' nebo l transfekovaly vektory konstruovanými Takové ' transformované za použití metod rekombinace DNA. .hostitelské (buňky ’ jsou schopny replikovat vektorý. .kódující· varianty proteáz. .nebo' exprimovat požadovanou variantu proteáz,. V případě vektorů, .které kódují pre- nebo přepro-formu· varianty prot-eázy, se pak tyto varianty v typickém případě vylučují .z hostitelské/ buňky do média hostitelské buňky. ’ ' ' ,
Termín „operativně spojený znamená- vztah . mezí dvěmi oblastmi DNA. Jednoduše znamená, „že jsou vzájemně' funkčně příbuzné. Například -pre-sekvence je operativně ' spojená s peptidem, jestliže funguje jako signální sekvence, 'která se podílí na vylučování zralé formy proteinu; přičemž ne j pravděpodobně j i zahrnuje štěpení.· signální
Promotor je operativně 'spojen řídí· transkripci sekvence.
sekvence. s kódující sekvencí', jestliže Vazebné' místo ribozómu je operativně spojeno s kódující sekvencí, jestliže je umístěno tak, že umožňuje translaci.
Geny kódující přirozeně se vyskytující, prekurzorpvoú proteázu se mohou získat pomocí obecných metod, které jsou známy v oboru. Metody v, obecném případě obsahují syntézu značených sond, které vykazují putativní sekvence' kódující oblasti proteázy, přípravu genomových knihoveri genu
99 9 • · · ··· · » · 9
9 1 1 1 9 9 9 9 9 9 9 • · ·· ·· · ·· ·· hybridizací se sondami. Pozitivně hybridizující klony se pak mapují a sekvenují.
Klonované proteázy se pak používají pro transformaci T hostitelské buňky za účelem exprese proteázy. -Gen proteázy se pak liguje do plazmidu s vysokým počtem .kopií. Tento plazmid » se replikuje v hostitelech ve smyslu, že obsahuje dobře známé elementy nezbytné pro plazmidovou replikaci: promotor je ' operativně spojený s'genem (který se -může začlenit jako . vlastní homologní promotor genu, jestliže je '.rozeznán, to znamená transkribován hostitelem), ukončení transkripce a » polyadenylační . oblast (nezbytnou pro stabilitu mRNA transkribovanou-.hostitelem z genu. proteázy v jistých . buňkách eukaryontního hostitele), která je exog.enní nebo se dodává .endogenní' terminační oblastí- genu proteázy a je-li to nutné, zahrnuje selekční gen , jako je gen nesoucí rezistenci na antibiotika , který umožňuje kontinuální -kultivaci buněk . hostitele infikovaných plazmidem růstem v médiu, které obsahuje .antibiotika. Plazmidy s vysokým počtem kopií také obsahují počátek replikace, který je pro hostitele vhodný. Plazmidy s vysokým,, počtem kopií také obsahují počátek replikace, což umožňuje vytvořit velké množství plazmidů v cytoplazmě bez omezení chromozomu. Avšak vynález také popisuje začlenění více kopií genu proteázy do hostitelského genomu. To 'umožňují prokaryontní a eukaryontní organizmy, které jsou zvláště náchylné k homologní rekombinaci.
Gen může být přirozený gen organizmu B. lentus. V jiném případě je možné produkovat syntetický gen kódující přirozeně se vyskytující nebo mutantní prekurzorovou proteázu. V takovém případě je možné stanovit sekvenci DNA a/nebo aminokyselinovou sekvenci prekurzorové proteázy. Syntetizovalo se několik přesahujících syntetických jednořetězcových fragmentů DNA, které po hybridizací a ligaci produkují syntetickou DNA kódující prekurzorovou proteázu. Příklad syntetické genové • ·· · konstrukce se uvádí v příkladu 3 dokumentu US patent č. 5,204,015.
Klonoval se přirozeně se vyskytující nebo syntetický gen prekurzorové proteázy. - Provedla se řada modifikací, .aby se zvýšila možnost použití genu při syntéze . .přirozeně se vyskytující prekurzorové proteázy. Takové.'modifikace. zahrnují produkcirekombinantních proteáz, jak se -popisuje . .v publikaci .US patent RE 34,606 a přihláška EPO č. 0.2.51 .446, a produkci zde popsaných variant proteáz.
Následující způsob kazetové mutageneze se může použít pro konstrukci variant proteáz. pbdTě vynálezu, ačkoli se mohou použít i jiné . metody. Nejdříve se získá přirozeně se vyskytující .gen kódující proteázu a celý nebo jeho část se sekvenuj e.. Pak se v sekvenci hledá bod, ve . kterém je nutné provést mutaci (deleci, inzerci nebo substituci) jedné nebo více aminokyselin v.kódovaném enzymu. V.sekvenci, která sousedí s tímto bodem se hodnotila přítomnost restrikčních míst vhodných pro nahrazení krátkého segmentu genu s oligonukleotidy, které v případě, že se exprimují budou kódovat různé mutanty. .Upřednostňuje .se, aby taková restrikční místa byla jediná restrikční místa v proteázovém genu, což umožňuje nahrazení segmentu libovolné restrikční místo, genu. Může se .však použít které se v genu proteázy neopakuje, přičemž štěpením restrikčními enzymy mohou vznikat genové · fragmenty, které se sestaví do správné sekvence. Jestliže. restrikční místa nejsou v polohách s vhodnou vzdáleností od vybraného bodu (10 až 15 nukleotidů), pak se taková místa vytvoří substitucí nukleotidů v genu takovým způsobem, že. se v konečné konstrukci změní čtecí rámec nebo kódované · aminokyseliny. Extenzí primeru M13 podle obecně známých metod se · provedla mutace genu za účelem změny sekvence. Úkol lokalizovat vhodné lemující oblasti a zhodnotit potřebné změny pro získání dvou vhodných sekvencí restrikčních míst se provádí rutině redundancí genetického kódu pomocí map ii mi
genu restrikčními enzymy a použitím velkého počtu různých restrikčních enzymů. Je nutné poznamenat, že jestliže -je dostupné vhodné lemující restrikční místo, -pak shora popsaná metoda se může použít pouze ve spojení s lemující oblastí, které neobsahuje místo. .
Jestliže . jednou dojde ke klonování' přirozeně se vyskytující DNA nebo syntetické DNA, pak .restrikční ' místa lemující místa·, kde se provede mutace, se ...štěpí ; restrikční my enzymy a do genu seliguje pluralita oligonukleotidových kazet s . komplementárním'.· koncem. Tímto způsobem se zjednoduší mutageneze, protože všechny oligonukleotidy se syntetbzOvadry tak,· aby. měly stejná restrikční místa..a pro vznik, restrikčních míst- jsou nezbytné syntetické linkery.
Proteolytická. aktivita se definuje jako rychlost hydrolýzy peptidových vazeb na miligram aktivního 'enzymu. Za účelem měření proteolytické aktivity existuje, řada 'známých postupů (popisuje se v publikaci K. Kalisz, . „Micřobial Proteinases, Advances in - Biochemic.al
Engineering/Biotechnology, A.
Fiechtěr ed,,
1988) . Vedle upravené ' proteolytické aktivity řada . enzymů · podle vynálezu může mít jiné 'upravené vlastnosti, jako je poměr km, kcat, kcat/Fm a/nebo upravenou specifitu *substrátu a/nebo upravený profil aktivity' v závislosti na pH. Tyto enzymy se mohou upravit pro' určitý substrát, který předchází přípravě peptidů nebo je přítomen při .hydrolytickém zpracování, jako je použití při praní prádla.
Vynález dále popisuje zabezpečení varianty, proteázy, která vykazuje ve srovnání ,s prekurzorovou proteázou změněnou, přednostně zvýšenou, účinnost praní alespoň v jedné detergentové. formulaci a/nebo alespoň při jedné sadě podmínek praní. ' . - <·
Existuje řada podmínek praní, které zahrnují různé detergentové formulace, objem prací vody, teplotu prací vody a délku praní,- po kterou- působí, varianta proteázy, Detrge.ntové » · · · · · formulace například, které se používají, v 'různých oblastech mají různé koncentrace jejich relevantních komponentů, které jsou přítomny v prací . vodě. Evropské detergenty například v typickém' případě, obsahují v prací vodě přibližně 4 500 až 5 000 ppm komponentů detergentů, zatímco v Japonsku detergenty ..v typickém případě obsahují přibližně 667 ppm detergentů V Severní Americe, * zvláště . pak ve státech,, detergent v typickém případě obsahuje přibližně 975 ppm komponent detergentů přítomných v prací vodě...
Systém ,s nízkou koncentrací . detergentů zahrnuje komponentů Spoj ených detergenty,, kde v prací vodě; je přítomno méně než přibližně 800 ,ppm detergentových 'komponentů. Japonské detergenty se v obecném případě považují za .systém s nízkou koncentrací, detergentů,. který obsahuje v prací, vodě . přibližně 667 ppm detergentových komponentů. ,·.·'
Střední koncentrace detergentů zahrnuje detergenty, 'kde v prací vodě je 'obsaženo přibližně detergentových komponentů, v obecném případě považují
800 ppm a; 2000 ppm Detergénty v Severní Americe , se za systémy se střední koncentrací detergentů, které v prací vodě obsahují přibližně 975 ppm detergenty 500 ppm
V Brazílii obsahují vodě' přibližně 1 detergentových komponentů, v typickém případě v prací detergentových komponentů.
Sytém s vysokou koncentrací . detergentů zahrnuje detergenty, kde v promývací vodě je obsaženo více jak 2 00Ó ppm detergentových komponentů. Evropské detergenty . se v obecném případě považují za systémy s vysokou koncentrací detergentů, což znamená, že v prací vodě obsahují přibližně 4 500 až 5 000 ppm detergentových komponentů.
Detergenty Latinské Ameriky v obecném případě obsahují vysoké množství fosfátů a rozmezí detergentů používaných v Latinské Americe spadá do systému se střední nebo vysokou koncentrací,, detergentů, což je. rozmezí mezi 1 500 ppm až 6 0.00 ppm detergentových komponentů v prací vodě.·. Jak se uvádí -shora • · • « v textu brazilské detergenty v typickém případě obsahují v prací vodě 1 500 ppm detergentových komponentů. Avšak výskyt detergentů . s vysokým obsahem fosfátů není' omezen ná další státy Latinské Ameriky, kde se mohou používat systémy s vysokou koncentrací detergentu, které. .obsahují v promývací vodě až přibližně 6 000 ppm detergentových komponentů.
: Z uvedených_ skutečností je zřejmé, že koncentrace detergentových sloučenin v typických pracích roztocích v celém světě kolísá od hodnoty nižší než přibližně 800. ppm (což .je nízká koncentrace ,detergentu), například přibližně 667 ppm v Japonsku,. . přes hodnotu 80Ό ppm Idhibdhbte 2 0ΌΌ ppm fcož—j-e— střední, koncentrace detergentu), například přibližně 975 ppm v US a přibližně 1 500 v Brazílii až. k hodnotě 2. 000 ppm (což je vysoká koncentrace detergentu). Příkladem je .koncentrace 4 .500' ppm.až přibližně 5 000 ppm v Evropě a 6 000. ppm v místech, kde se používají detergenty s vysokým obsahem fosfátů.
Koncentrace v typických pracích .roztocích se stanoví empiricky. V USA v typickém přípačě v pračce udržuje objem 64,4 1 pracího roztoku. Aby se získala přibližná hodnota koncentrace detergentu v'pracím roztoku 975, ppm, pak se musí do pracího roztoku přidat přibližně 62,79 g detergentové kompozice. Toto množství je typické množství odměřované uživatelem do prací vody .odměrkcu, která je přibalena u detergentu. ,
V různých geografickým místech se používá různé teploty praní. Teplota prací vody v Japonsku je v typickém případě nižší než teplota užívaná v Evropě.
Vynález popisuje variantu proteázy, která vykazuje zvýšenou účinnost praní alespoň v jedné sadě podmínek praní.
V jiném provedení vynálezu se stanovilo, že substituceaminokyseliny v jedné nebo více poloh zbytků odpovídající polohám zbytků vybraných ze skupiny zahrnující 62, 212, 230,
232, 252 a 257 subtilizínu mikroorganizmu Bacillus » ···· .· · • · • · • · · • · amyloliquefaciens jsou důležité pro . zlepšení účinnosti praní enzymu. ..
Tyto substituce se přednostně vytvořily v subtilizínu mikroorganizmu Bacillus amyloliquefaciens (rekombinantním nebo přirozeném), ačkoli substituce je možné provést v libovolné proteáze mikroorganizmu Bacillus.
. Na základě výsledků testování získaných s variantami proteáz, jsou zmíněné mutace v subtilizínu v mikroorganizmu Bacillus amyloliquefaciens důležité pro ...„proteolytickou aktivitu, účinnost a/nebo stabilitu těchto enzymů a účinnosti čištění nebo praní' za použití takových variant enzymůu
Řada proteázových variant podle vynálezu je. použitelná při vytváření různých kompozic detergentu' nebo formulací osobní potřeby, jako jsou šampony nebo pleťová voda. Řada známých látek jsou vhodná povrchově aktivní. činidla, která se používají v kompozicích obsahujících mutantý proteáz podle vynálezu. Tyto látky zahrnují neiontové, aniontové, kationtové nebo obojetné. detergenty, jak - se popisuje v dokumentu US 4,404,128 (Barry J. Anderson) a US 4,261,868 ' (Jiri Flora, et al·., ) . Vhodná detergentová formulace se popisuje v příkladu 7 dokumentu US patent 5,204,015. V oboru jsou známy různé formulace, které se používají jako čistící kompozice. Vedle typických .čistících kompozic se pro libovolný účel, kde se používá přirozená proteáza nebo proteáza divokého typu, je možné použít varianty proteáz podle vynálezu. Tyto varianty se mohou použít například při aplikacích kusového nebo kapalného mýdla, při aplikací prostředků na nádobí, čistících roztoků pro kontaktní čočky, produktů hydrolýzy peptidů, při zpracování odpadů, aplikací na textilie, jako enzymy štěpící fúzi při produkci proteinů. Varianty podle vynálezu mohou vykazovat zvýšenou účinnost detergentové srovnání s prekurzorem). Zvýšená účinnost kompozice (ve detergentu · se definuje jako zesílení schopnosti čistit skvrny citlivé vůči »· 999· »· 9999 • · • · • · jistému enzymu, jako jsou skvrny od trávy -a krve, jak se definuje obvyklým hodnocením po standardním cyklu praní.
Proteázy podle vynálezu se mohou vnést db.práškových nebo kapalných - detergentů, jejichž pH je mezi .6,5 a 12,0 š procentovým . zastoupením' přibližně 0,01- až 5% hmot. (upřednostňuje se -0,1 % až 0,5 %) . Tyto detergentové .čistící kompozice mohou 'také zahrnovat- jiné enzymy, jako 'jsou proteázy, amylázycelulázy, lipázy nebo endoglykolázy . stej ně jako plnidla nebo stabilizátory. . ·., .
Vedle proteáz'- podle ' vynálezu neexistuje žádné specifické využití běžných čistících prostředkům Jinými siovy—i-i-bov-oin-áteplota a pH vhodné pro detergent je také vhodný .pro uvedenou kompozici, pokud pH je ve shora uvedeném rozmezí a.'teplota“ je nižší než teplota denaturující proteázy. Proteázy podle vynálezu se mohou použít jako -čistící kompozice bez detergentů buď samotné nebo ·ν. kombinaci s ^plnidly a stabilizátory.
Vynález také popisuječistící kompozice obsahující proteázové. varianty podle vynálezu. -Čistící kompozice mohou dále obsahovat aditiva, která se : v těchto' čistících kompozicích běžně používají. Tyto aditiva ,se mohou vybrat,.ale nejsou omezena,·, na ..bělidla, povrchově aktivní činidla, plnidla, enzymy a bělící katalytická činidla. V oboru je známo, že aditiva jsou vhodná pro inkluzi do kompozic. Zde uvedený seznam není vyčerpávající a je nutné ho brát jako příklady vhodných aditiv. V oboru je také známo, že:je možné používat pouze ty aditiva,· která. j sou kompatibilní s enzymy a jinými komponenty . v kompozici, jako je například povrchově aktivní činidlo.
Množství aditiva, které je přítomno v-’čistící kompozici je přibližně 0,01 % až 99,9 %, upřednostňuje se přibližně 1 % %, více se upřednostňuje 1 % až 80 I.
Varianty proteáz podle vynálezu se mohou, přidat zvířecího krmení, jak se popisuje.v dokumentu US 5,612,055, US 5,314, 692 a US 5,147,642. . ' az do • · · ·· · • ·· · ·· ·♦ • · · <
• · · ·
·. · · · · ··
Dále vynález popis-uje kompozici pro ošetření textilií, které zahrnují varianty proteáz podle vynálezu. Kompozice se může použít při ošetření například hedvábí nebo vlny, ;jak se popisuje v dokumentech ŘD 216,034, EP 134,267, US 4,533,359 a EP 344,259. .
Přehled obrázků na výkrese
Na. obrázku č. 1 A až C je znázorněna sekvence DNA a aminokyselinová sekvence' subtilizínu. mikroorganizmu Bacillus
-~a-m-v-l-ol-i~o--u-efa-ci^ns—a-ř;Ásf.ečná restrikční mapa tohoto genu.
Na obrázku · . č. 2 jsou znázorněny konzervativní aminokyselinové, zbytky subtilizínu mikroorganizmů Bacillus amyloliquefaciens (BPNj 'a Bacillus lentus (divoký typ) .
; Na obrázcích· 3A až 3B je znázorněna aminokyselinová sekvence.. čtyř subtilizínu. 'Horní .řádek reprezentuje aminokyselinovou sekvenci . subtilizínu z mikroorganizmu Bacillus amyloliquefaciens (také ·’ se někdy nazývá jako subtilizín ,BPN'). Druhý řádek znázorňuje aminokyselinovou sekvenci' subtilizínu ,z mikroorganizmu Bacillus subtilis. Třetí řádek;· znázorňuje aminokyselinovou . sekvenci subtilizínu z mikroorganizmu' B. licheniformis. Čtvrtý řádek znázorňuje aminokyselinovou sekvenci subtilizínu z mikroorganizmu Bacillus lentus- (který se v dokumentu PCT WO89/06276 nazývá subtilizín 309) . Symbol * znázorňuje nepřítomnost specifických aminokyselinových zbytků-ve srovnání se subtilizínem BPN'.
i
Příklady provedení vynálezu
Příklad 1:
Produkovalo se a čistilo se velké množství variant proteáz za použití metod dobře známých v oboru. Všechny mutace, se provedly v subtilizínu organizmu Bacillus lentus GG36. Varianty jsou .uvedeny v'tabulce č. 4.
·· «··· ♦ e · · · · • · • · * » · · · · · · • · · .·· · « · · t • · · · · · ·· · *·· ··
Tabulka č. 4
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co | |||||||||||||||||
CM | |||||||||||||||||
co | |||||||||||||||||
• | τ | σ | > | ||||||||||||||
co | co | CD | x— | ||||||||||||||
co | xr | r- | |||||||||||||||
_ | CM | CM | CM | ||||||||||||||
z | > | I | LU | ||||||||||||||
> | > | ř— | X. | X | co | *> | X | > | > | X | |||||||
X“ | T— | CM | CD | CM | CM- | <o | co | 00 | co | CM | |||||||
r~ | co | xr | X“ | 00 | r·* | o | co | o | 00 | ||||||||
CM | CM | CM | X—* ' | X— | CM | CM | CM | CM | X“ | ||||||||
LU | 2 | co | 2 | σ | < | σ | 2 | o | |||||||||
Q. | X. | l·— | n | M | IX | X | X | __ | X | X | — | ||||||
χί* | CM | CM | CM | xr | xr | 00 | xr | xr | CM | o | xr | Γ- | CM | xr | |||
ο | LT5 | xr | r* | o | O | LO | r— | o | o | CO | o | o | co | co | o | ||
χ— > | S2 | CM Z | S2 | CM LU | > | > | G2 | CM LU | > | > | σ | X~“ σ | X*-> > | S | a | > | |
< | _ | __ | < | < | < | < | < | b- | < | ||||||||
co | xr | xr | xr | xr | co | co | ’Γ | co | co | o· | xr | co | xr | 00 | xr | co | |
ο | o | o | O | o | o | o | o | o | o | o | O- | o | O - | o | CM | o | o |
CO | > | > | > | X“ > | ω | co | > | > | co | co | T” . > | > | ω | >- | A2 | > | V“ ω |
Q9ZN | S103A | S103A L , | S103A | ! S103A I | N76D | N76D I | S103A | i S103A I | N76D | N76D | S103A | S103A | S87G | I S103A | V104I | 8103 A | o co r~ 2 |
X | Q | O | Q | Q | X. | co | Q | Q | x | co | Q | O | □ | Q | < n | Q | 2 |
CM | co | co | co | co | CM | cn | co | co | X“* | 00 | co | co | co | CO | co | X— | |
X~“ | r-» | xr | c- „ | h- | co | co | r·» | Γ· | CM | ||||||||
σ | 2 | 2 | 2 | z | σ | 2 | 2 | 2 | O | h— | 2 | 2 | 2 | 2 | CO | 2 | _l |
5* • · · · · ·
- | * | 1 1 . 1 | |||||||||||||||
• | |||||||||||||||||
·* . | • | ||||||||||||||||
. - | σ | □ | R | ||||||||||||||
CN | a— | ||||||||||||||||
LO | LO | LO | |||||||||||||||
CN | CN | CN | |||||||||||||||
X | z | X | |||||||||||||||
LU | LU | Q | < | ||||||||||||||
b | b | O | TT | ||||||||||||||
a— | 00 | TT | |||||||||||||||
CN | CN | r— | CN | ||||||||||||||
-J | —; | z | > | ||||||||||||||
cr | co | Cd | Cd | 0 | Q | LU | LU | £2 | c | (— | LU | tu | tu | Q | |||
b- | co | CD | co . | CO | CO | o | o | CO | to | 00 | G0 | LO | |||||
co . | T | O | LO | b | CO | O | o | LO | JO | co | o | co | LO | 03 | |||
a— · | CN | CM | CN | CN | CN | CN | CN | a“ | a— | CN | a— | T— | V“· | ||||
σ | Z | a | o | LU | z | σ | a | O | O | < | σ | ω | < | z | |||
__ | Cd | Cd | X | _ | UJ | LU | LU | Q | |||||||||
o | b- | TT | co | CN | TT | tn | TT | T | CO | <0 | TT | TT | O- | 05 | TT | tt | tt |
co | O | o | o | o | O | o | LO | o | o | o | o | LO | o | O | O | ||
i | T“ σ | CN a | S2 | aa· > | Q2 | > | a“ > | < | CN O | > | > | > | 5 | > | > | T· > | |
_· | < | <f | — | < | < | < | < | < | < | < | < | ||||||
TT. | ΤΓ | co | TT | TT | co | TT | co | co | tj· | co | co | co | co | co | co | ||
o | o | o | O | O | o | o | o | o | o | o | o | o | o | o | a | o | o |
T“ | a-“ | a~ | a— | T“ | > | a— | a~ | ||||||||||
> | > | ω | > | > | co | > | co | co | > | > | ω | CO | co | co | ω | co | |
03A | 03A | 76D | I03A | I03A | 76D | I03AΊ | 76D | 77D | 03 A | 03A | 76D | 76D | 77D | I03A | 76D | 76D | Q b b |
ω | co | z | ω | ω | Z | ω | z | z | ω | w | Z | Z | Z | ω | Z | Z | z |
Q | o | w | Q | Q | co | Q | LU | Q | Q | Q | LU | LU | Q | Q | LU | LU | d |
CO | to | n | to | to | 00 | co | to | to | to | to | co | co | |||||
b- Z | b- Z | a— < | b- z | b- Z | LO 1- | b- z | > | N. Z | N7 | b z | > | > | b z | b z | > | > | z |
··· ·
1 i 1 | |||||||||||||||||
X | |||||||||||||||||
LO | |||||||||||||||||
TT | |||||||||||||||||
CM | |||||||||||||||||
σ | --1 | ||||||||||||||||
> | X | ll | > | ||||||||||||||
tt— | CO | CO | CO | O) | |||||||||||||
r^. | co | LO | co | TT | |||||||||||||
CM | CM | CM | CM | CM | |||||||||||||
LU | σ | 1- | σ | X | |||||||||||||
> | X | X | X | o | X | X | cn | - | t | X | O | ||||||
to | CO | co | 03 | CO | co | TT | co | LO | co | 03 | |||||||
r^. | T | CO | co | LO | CO | co | 00 | T | TT | co | tt | ||||||
CM | CM | CM | CM | tt— | CM | CM | tt“ | CM | CM | CM | CM | ||||||
lil | σ | σ | O | 0 | σ | σ | 2 | 2 | σ | a . | X | ||||||
I | X | 0 | o | CD | O | X | -r | o | 0 | X | |||||||
co | CD | Γ-*- | CD | cn | T” | 03 | CD | 03 | CO | co | 03 | 03 ' | CO | ||||
CO | CO | τ* | n | IC) | CM | io | o | to | co | co | LO | LO | co | ||||
CM | CM | CM | CM | tt— | t-” | CM | tt— | CM | CM | tt— | tt— | CN | |||||
σ | a | -J | σ | 0 | > | 0 | >. | 0 | σ | σ | 0 | 0 | a | ||||
X | 0 | 0 | o | a | > | a | L4 | o | o | > | CD. | o | X | ||||
CO | 03 | cn | cd | TT | 03 | ^T | TT | TT | 03 | r- | 03 | 03 | CM | CO | cn | LO | |
CO | to | LO | LO | o | LO | o | tt— | o | LO | T— | LO | LO | tt | CM | LO | Τ | |
CN | tt“ | tt— | τ— | τ— | tt“ | ’r“ | T— | τ— | τ— | tt* | tt— | CM | |||||
σ | o | 0 | 0 | > | 0 | > | < | > | 0 | 2 | 0 | 0 | < | 2 | 0 | σ | |
_ | _ | < | < | < | < | CD | |||||||||||
TT | TT | T | TT | co | rr | T | co | co | co | TT | TT | ’Τ | TT | TT | ’Τ | TT | CM |
o | o | co | O | o | o | O | o | o | o | o | o | o | o | o | o | a | CN |
tt— | tt“ | tt— | tt” | τ— | tt— | tt— | tt— | tt— | tt— | tt“ | tt— | CM | |||||
> | > | > | > | ω | > | > | cd | CD | cn | > | > | > | > | > | > | > | 2 |
< | < | < | < | Q | < | < | Q | n | Q | < | < | < | < | < | < | < | |
co | co | co | co | co | co | co | co | co | co | co | co | co | |||||
o | o | o | o | o | o „ | o | o | o | o | o | o | o | |||||
tt“ | tt— | tt— | tt— | tt— | tt— | tt“ . | tt— | tt— | tt— | tt— | tt— | ||||||
in | co | co | co | X | ω | CD | X | z | X | CD | CD | CD | CD | CD | CD | ω | > |
o | Q | 0 | □ | < | O | o | X | 0 | < | O | a | a | Q | 0 | 0 | o | < |
co | co | CD | cu | co | co | co | to | co | co | co | co | co | co | CO | co | co | o |
h- | r^- | f*“ - | r-* | co | >- | Γ*- | r> | co | >- | r- | r* | r·— | r* | r- | |||
2 | 2 | 2 | 2 | > | 2 | 2 | _J | 2 | > | 2 | 2 | 2 | 2 | 2 | 2 | 2 | CD |
< | < | < | < | a | < | < | < | < | X | < | < | < | < | < | < | <ť | 0 |
ao | co | CO | co | r- | ao | co | co | co | CN | co | CO | co | CO | CO | co | CO | co |
co | co | co | co | τ— | co | co | co | cn | tt” | co | co | co | <O | co | co | co | |
> | > | > | > | X | > | > | > | > | σ | > | > | > | > | > | > | > | 2 |
Μ' • ·
to o
• | — | ||||||||||||||||
1 . ί l i 1 | |||||||||||||||||
' | |||||||||||||||||
co | X | X | X | ||||||||||||||
m | LO | LO | LO | ||||||||||||||
• | LO | xr | XT | xr | |||||||||||||
CM | CM | CM | CM | ||||||||||||||
(- | σ | σ | a | ||||||||||||||
Q | X | I | X | X | X | X | X | ||||||||||
LO | b- | co | LO | co | lO | co | |||||||||||
CO | xr | xr · | co | xr | CO | xr | co | ||||||||||
CM | CM | CM | CM | 04 | CM | 04 | 04 | ||||||||||
z | σ | X | σ- | σ | σ | a | a | ||||||||||
14. | IX | X | X | Q | X | Q | -r | < | |||||||||
co | LO | co | LO | xr | co | CO | cp | CO | |||||||||
co | xr | co | xr | o | co | T— | co | o | |||||||||
CM | CM | CM | CM | CM | CM | CM | CM | CM | |||||||||
> | σ | σ | □ | z | σ | z | σ | > | |||||||||
X | IX . | o | co | 2 | Q | X | > | Q | Q | > | |||||||
05 | b- | 03 | co | O) | co | xr | xr | 05 | CM | xr o | |||||||
xr | co | o- | xr | co | LO | co | b- | o | LO | co | |||||||
CM | CM | CM | CM | CM | t— | CM | CM | -r— | CM | ||||||||
-X | • | UJ | z | σ | O | σ | < | z | O | < | < | ||||||
co | co | 14. | co | co | co | co | co | co | IX | o | z | Q | Q | Q | > | o | a |
CM | CM | CO | CM | CM | CM | CM | CM | CM | O) | 05 | O) | O | O | co | C5 | 05 | |
CM | CM | co | CM | CM | CM | CM | CM | CM | LO | xr | LO | co | LO | co | LO | LO | |
CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | t— | t— | ||||||
2 | > | 2 | ΞΕ | 2 | z | O | co | O | o | O | < | O | O | ||||
X | IX | co | _„ | IX | IX | x | tx | co | |||||||||
co | CM | xr | CJ) | o | xr | b- | o | CM | xr | xr | xr | XT | xr | xr | xr | XT | |
CM | 1^. | CM | o | o | o | o | co | o | CM | o | o | o | o | o | o | o | o |
CM | CM | T“ | T*- | T·— | T“ | >c“ | v | ||||||||||
σ | z | Σ | > | σ | σ | > | o | σ | 2 | > | > | > | > | > | > | > | > |
041 | M o | o | 03A j | xr O | o | 03A | xr o | o | o | < co o | < co o | < co o | < co o | < co o | < CO σ | <·' co o | 03A 1 1 |
> | > | > | co | > | > | co | > | > | > | co | CO | co | X*“ ω | ω | co | co | ω |
< CO | < co | < co | Q | < m | < co | O | < co | < co | < co | O | Q | Q | o | Q | O | □ | □ |
o ω | o ω | o ω | co b- 2 | OIS | o co | co b- 2 | o w | o ω | o *r— co | CO b* 2 | co Z | CO Γ- Z | N76 | N76 1 . | CO b· 2 | CO ri- Z | co b- 2 |
Q | o | Q | Q | Q | x | Q | Q | o | < | < | < | < | < | < | < | < | |
CO | CO | co | co | CD | V“ | co | CD | co | co | co | CD | CD | OO | CD | CD | co | |
b- | r* | b- | CM | o- | b- | CO | b- | b* | b- | CO | co | CO | CO | CD | CO | CO | co |
z | z | z | _J | Z | z | o | z | z | z | > | > | > | > | > | > | > | > |
• · ··· ·
i i í' | |||||||||||||||||
----- | — | -— | ·—— | — | —,__ | ___ | |||||||||||
< | cn | ||||||||||||||||
o | to | ||||||||||||||||
co | b- | ||||||||||||||||
CM | CM | ||||||||||||||||
b“ | X | ||||||||||||||||
x | X | p—— | ze | ze | 2 | ||||||||||||
to | LO | CM | CM | CN | CM | CN | CM | ||||||||||
7 | 7 | LO | to | LO | to | lO | CD | ||||||||||
CM | CM | CM | CM | CM | CM | CM | CM | ||||||||||
σ | a | z | z | z | z | z | -J | ||||||||||
X | ze | X | 0 | X | X | X | X . | X | cn | ||||||||
CD | CA | CD | co | • | to | to | to | to | to | oo | |||||||
CO | to | co | 7 | 7 | 7 | 7 | 7 | 7 | 7 | ||||||||
CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | ||||||||
σ | z | σ | z | a | a | a | a | a | z | ||||||||
> | X | > | X | X | X | X | X | X | X | X | X | X | |||||
CM | to | CM | tO | to | LO | to | CD | CD | CD | CD | co | to | |||||
CO | 7 | CO | 7 | 7 | 7 | 7 | CO | CO | CO | CO | co | 7 | |||||
CM , | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | |||||
< | σ | < | σ | σ | σ | σ | σ | σ | σ | σ | σ | a | |||||
X | 0 | X | X | X | ω | X | > | > | > | > | > | cn | |||||
to | cn | CD | co | 7 | LO | CM | co | co | CM | CM | CM | CM | CM | CM | |||
7 | to | CO | 7 | CM | co | co | CO | CO | CO | CO | CO | CM | |||||
CM | x— | CM | CM | CM | CM | CM | CM | CM | CN | CM | CM | CN | CM | ||||
σ | o | σ | H | > | σ | 2 | Ό | σ | < | < | < | < | < | 2 | |||
ω | cc | > | O | cn | t- | b— | > | > | 0 . | 0 | 0 | 0 | O | b— | |||
CM | to | 7 - | CM | cn | CM | o | o | 7 | CM | CM | σ> | cn | o | cn | cn | o | |
CM | 7 | O | CO | to | CM | ▼— | co | O | CO | CO | tO | to | to | to | to | co | |
CM | CN | CM | CM | CM | CM | CM | x— | •V“ | x— | X— | X— | x— | |||||
2 | a | > | < | o | 2 | X | cn | > | < | < | O | O | o | o | 0 | cn | |
p o > | A232V | S103A I | G159D | V104I I | I104T I | M222S | I104T | S103A | Q 7 CO X— z | 0 Í2 0 | G159D | 0 o TT Z | V104I | V104I | V104I | V104I | I104T |
< | *· | < | < | < | < | O | < | __ | <f | < | < | < | < | ||||
CO | 7 | co | co | co | CO | CO | CO | x. | 7 | 7 | co | CO | CO | CO | CO. | ||
o | O | o | o | o | o | o | co | o | c | O | O | o | o | o | o | o | |
cn | > | Z | > | cn | cn | cn | cn | z | cn | > | > | > | cn | cn | cn | cn | cn |
O | < cn | < | < cn | □ | 0 | 0 | Q | < | Q | < | < | < cn | < | < | < | 0 | 0 |
CD | CO | CD | CD | co | co | co | CD | 03 | CO | co | r- | cO | |||||
b- | co | b- | b- | b- | b- | CD | b- | cn | co | CD | 00 | b* | |||||
z | čn | > | čň | z | z | z | z | > | z | cn | čň | čn | > | > | > | cn | z |
cc | 0 | 1- | < | < | X | X | X | ze | < | < | < | < | cn | a | < | X | |
CN | co | 7 | CD | co | CM | CN | CN | CM | as | CO | ca | cn | CO | CO | CO | CO | CM |
b* | CN | cn | cn | X— | χ— | CM | CO | CD | co | CD | 7 | 7 | 7 | co | |||
σ | z | ω | > | > | a | σ | a | ř— | > | > | > | > | z | z | z | > | σ |
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I t . I | |||||||||||||||||
____ | I . I | ||||||||||||||||
---.— | -- | ||||||||||||||||
X | |||||||||||||||||
tn | |||||||||||||||||
7 | |||||||||||||||||
04 | |||||||||||||||||
• | a | ||||||||||||||||
X | cc | > | > | X | X | X | X | ||||||||||
tn | tn | o- | tn | tn | tn | tn | co | ||||||||||
7 | 7 | tn | o- | m | 7 | 7 | 7 | co | |||||||||
CN | 04 | 04 | 04 | OJ | 04 | 04 | 04 | OJ | |||||||||
σ | σ | -j | X | -j | σ | σ | σ | σ | |||||||||
X | X | cc | cc | CC | ω | X | 7 | X | > | X | > | 7 | X | > | |||
co | tn | tn | to | lO | 00 | tn | co | tn | tn | o. | CO | CO | co | 04 | |||
CO | 7 | 7 | 7 | 7 | 7 | 7 | CO | 7 | tn | 7 | tn | m | co | CO | CO | ||
04 | 04 | 04 | 04 | 04 | 04 | 04 | 04 | 04 | 04 | OJ | 04 | 04 | CN | CN | CN | ||
σ | σ | σ | σ | σ | X | σ | σ | σ | _i - | σ | —i | σ | σ | σ | < | ||
> | X | X | X | 7 | cc | 7 | > | X | > | X | X | X | > | > | > | _J | |
04 | CO | co | co | co | tn | co | 04 | co | tn | co | tn | 04 | 04 | OJ | 7 | ||
CO | co | CO | CO | co | 7 | CO | co . | co | tn | 7 | co | 7 . | CO | CO | co | ||
04 | C\l | 04 | 04 | 04 | OJ | 04 | OJ | OJ | 04 | 04 | 04 | 04. | 04 | 04 | 04 | 04 | |
< | σ | σ | σ | σ | σ | σ | < | σ | _J - | σ | σ | σ | < | < | < | > | |
_1 | > | > | • ' > | > | 7 | > | cc | > | X | > | -r | -T-j $ | X | > | Q | ||
CO | 04 | 04 | 04 | OJ | co | 04 | o | OJ | tn | co | 04 | cn | t— | cn | |||
O | CO | CO | CO | co | co | CO | co | 7 | co | CO | CO | V“ | tn | ||||
04 | 04 | 04 | 04 | 04 | 04 | 04 | OJ | OJ | 04 | 04 | 04 | 04 | 04 | OJ | v— | ||
σ | < | < | < | < | σ | < | X | < | σ | σ | < | σ | > | O | o | o | |
> | o | H* | Q | l·— | > | Q | Q | Q | X | > | -- | < | > | Q | Q | Q | - |
TT | oo | o | cn | tn | 04 | cn | 05 | 05 | co | OJ | tn | 7 | 04 | cn | cn | cn | 7 |
r- | 00 | co | tn | CO | tn | tn | tn | co | co | r- | 04 | CO | tn | tn | tn | ||
04 | 04 | 04 | T— | ▼— | <r· | 04 | OJ | 04 | 04 | 04 | T“ | φ-» | r— | ||||
< | co | < | o | < | < | o | o | O | σ | < | X | H | < | o | o | O | > |
Q | Q | o | Q | Q | - | > | □ | > | Q | Q | < | ||||||
cn | cn | cn | 7 | cn | CD | 7 | 7 | 7 | 04 | CD | CD | CD | 7 | 7 | 7 | co | |
tn | tn | tn | O | tO | tn | O | O | O | CO | tn | LřY | tn | tn | O | O | O | o |
δ | δ | G1 | > | δ | δ | > | > | 04 < | δ | L2í | δ | δ | > | > | T” > | T“ ω | |
< | __ | < | < | < | < | < | < | Q | |||||||||
7 | 7 | 7 | co | 7 | 7 | co | co | co | 7 | 7 | 7 | 7 | 7 | co | co | co | |
o | o | o | o | O | O | o | o | o | o | o | o | o | C5 | o | o | o | co |
> | T“ > | > | δ | > | > | δ | T“ ω | δ | > | T“ > | > | > | T* > | co | δ | 7 CO | 2 |
03A | 03A | 03A | 98T I | I03A | I03A | 76D I | 76D | Q co r— | 03A | 03A | 03A | 03A | 03A | Q co | 76D | 76D | 68A I |
ω | ω | ω | < | ω | ώ | 2 | 2 | 2 | δ | δ | Ύ— ω | δ- | ω | 2 | 2 | 2 | > |
<· | <f | < | < | < | < | < | < | < | Q | <· | o | < | Q | < | <f | < | X |
αο | eo | CD | co | 00 | co | CD | CO | CD | CD | co | co | 03 | co | CD | CD | co | 04 |
co | co | CO | co | CD | co | CO | co | CD | O- | cn | o- | cn | CO | cn | co | T~ | |
> | > | > | > | > | > | > | > | 2 | > | 2 | > | 2 | > | > | > | σ |
* · · * c · ·· *··· w · • « • · * ·
* | |||||||||||||||||
• | |||||||||||||||||
--.— | — | — | - | QZ— | |||||||||||||
~ - | |||||||||||||||||
LO | |||||||||||||||||
XT | |||||||||||||||||
cu | |||||||||||||||||
0 | |||||||||||||||||
o | > | CZ | X | co | QZ | X | |||||||||||
05 | σ | LO | r— | CM | LO | co | |||||||||||
io | co | xr | LO | S | XT | CO | |||||||||||
CM | CM | CM | CM | CM | CM | cu | |||||||||||
co | H | 0 | < | 0 | |||||||||||||
QZ | cz | oz | x . | 0 | X | X · | co | QZ | X | CZ | QZ | OZ | > | ||||
LO | 10 | LO | LO | v- . | LO | co | QO | LO | to | to | LO | cn | cu | ||||
χτ | χτ | XT | xr | to | (O | XT | co | XT- | XT | co | LO | XT | XT | co | |||
CM | cu | CM | CM | cu | CM | CU | CM | cu | cu | CM | CM | CM | CM | CM | |||
σ | a | 0 | 0 | z | z | 0 | 0 | z | 0 | 0 | co | 0 | a | < | |||
X | X | X_ | X | QZ | CZ | x | > | CZ | CZ | X | > | CZ | X | X | co | ||
co | co | co | co | LO | LO | to | CM | to | LO | co | CM | LO | co | to | to | ||
co | co | co | CO | xT ’ | •Ν’ | σ) | cn | XT | .xr | CO | cn | XT | CO | CO | ao | ||
CM | CM | cu | cu | CU . | CU | CU | CU | CM | cu | cu | CM | CM | CN | CN | · | ||
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Příklad 2:
U velkého množství . variant . proteáz produkovaných v příkladu 1 se testovala účinnost ve dvou typech detergentu a při podmínkách praní za použití' mikrotestu popsaném v publikaci „An improved .method of assaying . for a preffered enzyme and/or preffered detergent composition, U.S. Seriál No. 60/068,796.
,- V tabulce č. 5 jsou uvedeny varianty testovaných proteáz a, výsledky testování . ve dvou různých detergentech. V případě sloupce A ’ detergentem bylo’ 0, 67 g/1 filtrovaného Ariei Ultra (Procter and Gamble, Cincinnati, OH, USA) v roztoku, který obsahuje 0/Í95 g smíšené tvrdosti Ca2+/Mg2+ ha 4,54 1. V každé prohlubni se použilo. 0,3 ppm enzymu při teplotě 20 °C. V případě sloupce B detergentem bylo 3,38 g/1 filtrovaného
Ariel Futur.e (Procter' and Gamble, Cincinnati, . OH, USA) v roztoku, který 'obsahuje 0,975 g smíšené tvrdosti Ca2+/Mg2+ na .4,54 1. V každé . prohlubni se použilo 0,3. ppm enzymu při teplotě 40 °C.
·« ···· ·* ··*· • · · · · · • · · · · · • ' · · · · · ·· ·· ·· ·
Tabulka č. 5
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CM | T- | T“ | T“1· | T“ | ||||||||||||
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CO | r*. | co | CD | co | co | -r- | co | r*· | l>- | r- | tn | ICM | CM | tn | CM | o | r- |
cd | co | CD | CM | CM | CM | co | o | CO | n | o | CD | CN | CD | T— | |||
b | o | b | b | b | o | b | Τ’* | o | <” | Ί— | CT | r· | |||||
O | o . | CM | CO | 00 | CD | CM ' | co | T“ | v— | CD | 00 | CO | CD | 05 | V | ||
o | ít | ΊΡ· | r— . | 00 | CM | tn | CM | CM | CM | to | CO | co | cn | co | CD | co | |
Τ’* | T“ • | o | b. | CT;. | b | b | CT | b | b | o - . | o | o | o | ||||
í ' 1 i ! ! 1 ! | I. i I . } | ||||||||||||||||
— | • · Ί ! i i I l i i I . i I ! I | • | |||||||||||||||
Ϊ . I | i | ||||||||||||||||
co | |||||||||||||||||
co | |||||||||||||||||
Τ’” | |||||||||||||||||
z | |||||||||||||||||
Q | < | LU | 1— | > | >- | H | X | ||||||||||
CO | v | Γ- | O | CM | co | ||||||||||||
00 | r*. | CD | o | co | T“* | ||||||||||||
t— | CM | CM | CM | CM | CM | CM | T“ | ||||||||||
z | H | X | z | LU | z | co | z | ||||||||||
LU | > | Q | _J | cc. | Z | CL | X | (— | o | - | X | ||||||
TT | Γ'-* | co | CD | 'T | CT | CD | T | '’Τ | CM | CM | CM | v | co | ||||
O | o | CO | CM | tn | o | co | CT | o | OO | o | tn | o | o | tn | |||
> | > | CM | A2 | δ | > | w | δ | > | s | > | CM ω | CM z: | CM ω | CM UJ | > | > | G2 |
< | < | - | < | - | < | _ | < | < | < | ||||||||
co | TT | Ό” | co | O | co | rr- | co | ΤΓ | TT | TT | co | co | |||||
o | o | o | o | o | o | o | o | CT | CT | CT | o | CT | o | CT | CT | o | o |
ω | ω | T— > | > | > | ω | > | > | ω | > | W | > | r— > | > | > | cň | T- ω | > |
P86S | N76D | S103A | S103A | S103A | N76D | S103A | S103A | S99R | S103A | N76D | S103A | S103A | , S103A | S103A | N76D | Q co r- Z | S103A |
Q | Q | O | Q | _1 | Q | Q | Q ' | O | X | Q | Q | a | Q | X | co | O | |
co | CM | co | CD | co | r*- | CD | co | CD | co | CM | co | to | (O | co | CN | co | co |
h- | r** | r>* | r-- | T“· | r- | ř*. | N. | Ν» | T· | r*. | r*· | r*· | t— | r- | |||
z | z | z | Z | z | z | z | z | z | a | z | z | z | Z | σ | z | Z |
• ·· 9
• · · • ·
Ή • · ··
LO | ΙΛ ICO | Y- | )CN | Y— | CN | icp | co | XT | LO | XT | co | 00 | co | ”T | XT | XT | |
CN | 05 | 0 | 05 | 0 | 0 | o | 0 | CM | o | 0 | o | T | co | 0 | 0 | 0 | 0 |
τ· | 0' | 0 | d | T | *“ | d | d | d | d | d | d | d | d | d | |||
fx- | CN | XT | co | co | T·» | co | co | h- | γ- | 0 | LO | Y— | XT | αο | co | Y— | |
LO | CN | CN | τ | T | co | co | co | co | 05 | LO | XT | 05 | XT | co | fx- | X* | 03 |
d | d | 0. | d | d | d | d | d | d | 7* | r· | ·“ | CN | X | CN | x” | ||
1 | |||||||||||||||||
I I ' I | I I 'i | ! | |||||||||||||||
I I I ! | |||||||||||||||||
' | |||||||||||||||||
H- | |||||||||||||||||
Υ» | |||||||||||||||||
10 | |||||||||||||||||
CN | |||||||||||||||||
X. | |||||||||||||||||
a | < | ||||||||||||||||
LO | XT | ||||||||||||||||
00 | T | ||||||||||||||||
τ— | CN | ||||||||||||||||
z | > | ||||||||||||||||
> | (X | £X | co | QC | DL | O | Q | LU | LU | l·“ | Q | LU | LU | ||||
co σ» | CN | r^· | co | CO | 00 | T | γ— | CD | CD | co | Y— | CD | 00 | ||||
co | co | χτ | 0 | 10 | rx- | CD | O | O | co | co | 0 | co | |||||
Τ’ | Y·» | CM | CM | CM | CN | CN | CN | CN | Y- | CN | CN | Y— | |||||
— | σ | σ | Z | σ | O | LU | z | σ | σ | < | z | σ | ω | ||||
o | r. | __ | CC | tr | CL | LU | LU | LU | LU | Q | |||||||
CN | XT | XT | co | CN | XT | CO | χτ | XT | CO | CD | XT | CD | 05 | XT | |||
co | o | co | 0 | 0 | 0 | 0 | o | 0 | 10 | 0 | 0 | O | LO | σ | |||
CN LU | T— σ | > | σ | Q2 | CN ω | > | CN σ | r— > | >_ | Y— < | CN σ | Τ’ > | CN a | 0 | > | ||
_ | _ | < | L_ | < | - | < | < | < | < | < | |||||||
χτ | XT | CO | XT | XT | co | XT | XT | co | XT | <0 | XT | χτ | co | XT | XT | co | |
o | 0 | 0 | σ | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | o | 0 | 0 | 0 | o | 0 |
>~ | δ | > | T· > | δ | > | > | T ω | > | δ | δ | > | > | T— CO | > | Y“ > | δ | |
S103A | S103A | N76D | S103A | S103A | N76D | S103A | S103A | [ N76D | S103A | N76D L | N77D | S103A | S103A | N77D | S103A | S103A | Q co i· Z |
O | Q | 2 | Q | Q | t- | α | α | co | Q | UJ | Q | Q | Q | Q | O | Q | LLÍ |
co | co | co | co | CO | co | co | co | co | co | co | co | co | co | CD | |||
fx» | r** | CN | r- | h- | τ | r- | fx-. | tn | h* | > | f- | fx* | (x- | fx- | fx* | T— | > |
z | z | z | z | < | z | z | H | z | Z | z | z | z | z | z |
• · · ·
····
xr | CO | CD | 03 | r»· | xr | 00 | o> | U3 | >- | CN | co | 05 | oo. | Y“ | co | xr | tn |
o | o | T- | O | γ— | Y— | Y— | Y— | o | Ύ | o | r- | CN | c- | xr | xr | ||
o | o | o | O | o | 03 | o | o | Ó | o | Y· | CN | Y“ | ó | o | o | ||
co | co | xr | O | co | CN | co | r>- | O | Y— | xr | 05 | CN | Y— | CN | co | h* | c- |
o | o | CN | co | XT | co | CD | 03 | co | xr | cn | co | T“ | Y~ | co | co | 05 | |
c\i | CN | CO | -Y“ | Y— | Y~ | Y*“ | - | -1- | o | O | o | o | o’ | CN | CN | ||
i 1 , 1 . | |||||||||||||||||
> | |||||||||||||||||
γ— | |||||||||||||||||
r- | |||||||||||||||||
CN | |||||||||||||||||
LU | |||||||||||||||||
Q | X | a | > | *> | > | X | |||||||||||
05 | co | O) | Y— | Y·- | co | ||||||||||||
tn | co | tn | r*. | r- | co | ||||||||||||
Y” | CN | Y—· | CN | CN | CN | CN | |||||||||||
o | σ | o | UJ | UJ | LU | a | |||||||||||
UJ | 1- | Q | a | Q | Q | cn | < | U. | 0. | -J | Cd | _J | X | a | |||
00 | Y— | 03 | 03 | xr | 05 | cn | CO | xr | CO | CN | tn | tn | o | xr | co | 03 | |
m | m | tn | tn | o | tn | tn | co | CN | co | xr | xr | Y— | o | co | tn | ||
Ύ— | CN | Y“ | Y“ | Y“ | r— | CN | CN | CN | CN | CN | CN | CN | CN | Y“ | |||
< | SC | o | o | > | o | o | z | F— | > | cn | σ | σ | Q_ | > | σ | o | |
UJ | .. | < | cn | o | Q | Q. | Q_ | CC | Od | < | |||||||
xr | co | xr | XT | XT | co | XT | CO | o | 05 | CN | xr | CN | 05 | 05 | co | xr | xr |
o | a | o | o | o | o | o | xr | tn | cn | o | Y— | O | o | o | o | o | |
> | Q2 | > | > | > | cň | > | δ | δ | δ | S2 | Y— > | CM cn | δ | δ | Y— cn | > | Y“ > |
< | < | < | < ' | Q | < | _ | - | < | < | < | |||||||
co | xr | co | co | CO | co | xr | xr | xr | xr | co | xr | XT | xr | UJ | co | co | |
o | o | o | o | o | co | o | o | o | o | o | o | o | o | o | CO | o | o |
ω | > | ω | γ— cn | cň | z | cň | > | > | > | > | ω | Y“ > | > | > | z | ω | δ |
76D | 03A | 76D | 76D | 76D | 62H | 76D | I03A | I03A | I03A | I03A | B7R | 03A | νεο | Í03A | 62S | 76D | 76D |
Z | cn | Z | Z | Z | z | Z | cn | cn | cn | cn | co | cn | cn | cn | z | z | z |
UJ | Q | l·— | < | > | I | a | Q | Q | Q | o | Q | a | Q | > | < | < | |
co | co | 00 | CN | CN | CN | co | co | co | co | CD | co | co | CO | o | ca | co | |
r- | ’Τ | co | xr | Y— | xr | Γ*- | r·'- | r* | Γ— | r** | CN | co | CO | ||||
z | < | > | σ | —j | Z | z | Z | z | z | z | z | z | O | > | > |
• · • · · ·
• ·
Ύ“* | 04' | 00 | «Τ- | Ύ— | XT | o | XT | [cn | (cp | cn | cn | ”T | cn | 04 | cn | 04 | |
co | CO | CD | τ— | x* | XT | cn | o | o | kO | o | o | o | o | co | o | o | |
ο | ο | ό | Ο | Ο | o - | o | o | d | o | d | d | d | o | o | d | d | d |
ο | CD | CD | τ— | co | ▼- | 04 | co | co | O | Γ-» | 5— | m | T— | CD | CO | 00 . | |
ο | Ο- | Ν’ | χτ | CO | σ | X— | x— | T— | r** | xr | CO | T- | X“ | cn | 04 | o* | Ol |
cn | ΟΙ | CM | OÍ | cn | cn | cn | cn | cn ’ | oi | oi | cn | cn | cn | cn | cn | 04 | cn |
• | z , 1 | - | |||||||||||||||
1 i ) | |||||||||||||||||
X | to | X | |||||||||||||||
LO | in | tn | |||||||||||||||
xr | in | xr | |||||||||||||||
04 | Ol | 04 | |||||||||||||||
σ | i- | σ | |||||||||||||||
> | TT | X | X | > | Q | X | X | X | |||||||||
co | cn | co | cn | tn | O- | co | |||||||||||
ο* | cn | kO | cn | xr | co | xr | xr | cn | |||||||||
04 | 04 | 04 | 04 | 104 | 04 | 04 | CM | CM | |||||||||
LU | σ | t— | σ | X | X | σ | X | a | |||||||||
X | X | n- | Q | X | X | co | _J | X | σ | X | X | X | c | ||||
ΙΟ | co. | co | ,O | co | co | xr | tn | co | O) | tn | co | tn | xr | ||||
Ν’ | cn | cn | kO | cn | cn | co | xr | cn | xr | cn | χτ | o | |||||
04 | ιΟΙ | 04 | T— | 04 | Ol | ·»— | 04 | 04 | 04 | 04 | 04 | 04 | 04 | ||||
σ | σ | a | o | σ | σ | X | σ | σ | X | σ | a | σ | X | ||||
χ | _ | X | Cl | Q . | co | Q | X | Q | X | X | o | X | > | ||||
co | ο» | co | cn | cn | cn | O | co | cd | co | CD | cn | co | xr | ||||
CO | «τ- | CO | kO | 04 | kO | o | IO | co | m | cn | cn | tn | cn | o- | |||
04 | 04 | 04 | T— | 04 | r— | 04 | 04 | 04 | Τ’* | 04 | T“ | ||||||
σ | a | o | > | o | > | O | σ | O | o | σ | o | σ | < | ||||
α | α | Q | __ | > | - | Q | X | Q | co | Q | o | X | Q | Q | |||
cn | cn | xr | cn | XT | xr | XT | cn | co | σ> | cn | cd | CD | cn | 05 | |||
ΙΟ | ΙΟ | σ | to | o | o | kO | T— | cn | tn | 04 | tn | tn | xr | m | n | ||
ν— | τ— | v· | v- | x~ | T“ | •^· | Ol | tt* | T“ | T— | y— | ▼— | T· | ||||
Ο | ο | > | o | > | < | > | o | X | σ | O | X | o | o | to | o | o | |
< | _ | < | _ | < | |||||||||||||
xr | xr | Ν | xr | cn | XT | m | xr | xr | xr | xr | xr | xr | XT | N | xr | xr | |
ο | σ | ο | ο | o | o | o | o | o | o | o | o | o | o | o | o | o | o |
Τ·· | T“ | T“ | T“ | T | γ— | γ— | Y“” | T” | |||||||||
> | > | ω | > | > | to | > | co | > | > | > | > | > | > | > | > | > | > |
< | < | «Ο | < | < | Q | < | Q | < | < | < | < | < | < | < | < | < | < |
ση | CO | CO | cn | cn | cn | cn | cn | cn | cn | cn | cn | cn | cn | cn | |||
ο | ο | ο | o | í— | o | o | o | o | o | o | o | o | o | o | o | ||
T·” | T*~ | γ— | T“ | T— | T“ | γ» | |||||||||||
ω | ω | X . | ω | ω | 00 | X | co | co | co | co | co | to | to | to | to | to | |
Q | Q | < | α | Q | X | Q | < | Q | Q | Q | Q | Q | Q | Q | Q | c | Q |
CO | CO | co | CO | CD | lO | <o | co | co | co | co | co | CD | co | co | co | co | CD |
Ο* | Ο* | CO | ο- | >- | O- | r- | co | o* | r* | r- | r- | r*. | í^. | o- | o- | O- | |
X | X | > | X | X | _J | X . | > | X | X | X | z | X | X | X | z | z | X |
< | < | σ | < | < | < | < | X | < | < | < | < | < | < | < | <c | < | |
CO | 00 | Γ*» | CO | co | co | co | 04 | co | co | co | co | co | co | co | co | co | co |
<ο | <0 | τ· | CD | co | CO | co | Ύ— | co | cn | co | co | co | CO | co | <0 | co | CD |
> | > | X | > | > | > | > | σ | > | > | > | > | > | > | > | > | > | > |
• aa a /2 ’ a a a a a a
CM | tn | CD | CO | CO | CO | TT | co | to | tn | cn | 05 | co | CO I CM | LO | CN | 05 | |
O | o | tn | a— | b | a— | cn | o | v | o | 05 | b | O | 05 | R | b | LO | |
O | o | O | o | *“* | o | a— | O | a— | R— | t— | |||||||
TT | co | T— | co | o | CO | co | CN | CD | t— | CN | b | TT · | to | CO | b | CO | b |
co | CN | o> | oo | CO | 00 | CN | b | O | 05 | 05 | LO | b | 7“ | cn | co | T— | b |
co | CO | CN - | CN | a- | a— | CO | CO | T— | CO | CN | CN | CN | |||||
I I i. | |||||||||||||||||
< | X | 0 | |||||||||||||||
o | to | 05 | |||||||||||||||
co | TT | to | |||||||||||||||
CN | CN | CN | |||||||||||||||
R | 0 | ω | |||||||||||||||
cd | X | Cd | cd | > | X | X | X | Z | X | X | X | ||||||
LO | to | LO | to | b | LO | to | to | co | to | LO | to | ||||||
O“ | TT | tt | TT | to | tt | Ν’ | ΤΓ | co | TT | TT | TT | ||||||
CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | ||||||
a | 0 | 0 | 0 | _J | 0 | 0 | 0 | σ | 0 | 0 | 0 | ||||||
Cd | -r . | Cd | X | X | X | X | > | X | X | ——I X : | > | X | X | X | |||
to | CO | IO | co | co | CO | LO | b | CO | co | CD | CM | co | CD | CD | |||
TT | co | T | co | CO | co | ΤΓ ' | to | CO. | cn | cn | cn | cn | CO - | CO | |||
CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | |||
σ | σ | 0 | 0 | 0 | 0 | 0 | _1 | 0 | 0 | 0 | < | 0 | 0 | 0 | |||
X | 0 | X | < | > | > | X | > | X | > | > | > | —1 | > | > | > | ||
cd | ao | CD | co | CN | CN | co | b | to | CN | CN | CN | TT | CN | CN | CM | ||
<o | a*-· | CO | o | CO | CO | co | LO | TT | CO | CO | CO | a— | CO | CO | m | ||
CN | CN · | CN | CM | CM | CM | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | ||
σ | z | 0 | > | < | < | 0 | _1 | 0 | < | < | < | >· | < | < | < | ||
0 | O | > | __ | Cd | X | > | X | X ' | $ | Cd | > | 0 | X | 0 | 0 | ||
TJ· | 05 | CN | co | o | CN | 10 | co | 05 | a— | a* | 05 | to | 05 | 05 | |||
o | LO | CO | a— | ar* | CO | TT | co | r- | v— | tn | T“ | tn | LO | ||||
CN | a— | CN | CN | CN | CN | CN | CN | CN | CN | a— | CN | a— | T· | ||||
z | 0 | < | < | R | X | < | 0 | 0 | > | 0 | 0 | 0 | < | 0 | 0 | ||
0 | > | 0 | Q | 0 | 0 | O | X | -r | > | 0 | O | o | 0 | — - | |||
05 | co | σ> | 05 | 05 ' | TT | 05 | 05 | CD | to | CN | 05 | 05 | 05 | TT | 05 | TT | TT |
tn | co | lo | to | LO | O | to | LO | CO | b | co | LO | LO | LO | o | LO | CD | O |
a— | T“ | a“ | a- | a— | CM | CN | CN | a— | t— | T“ | |||||||
O | < | 0 | 0 | 0 | > | 0 | 0 | 0 | X | < | 0 | 0 | 0 | > | 0 | > | > |
< | __ | - | > | > | 0 | < | < | < | |||||||||
Tf | TT | tt | TT | TT | co | tt | TT | CN | 05 | O“ | tt | R | co | TT | co | co | |
O | O | O | O | o | o | O | o | CO | LO | LO | o | o | O | o | o | o | o |
> | > | T“ > | > | > | ω | > | > | CN < | L2! | 0 | > | > | T“ > | čň | > | čň | čň |
S103A | S103A | S103A ! I | S103A | S103A | N76D | S103A | | | S103A | V104I | I V104I i | TT O a- > | S103A | S103A | S103A I | N76D | S103A | N76D | | N76D |
O | 0 | □ | 0 | O | < | 0 | 0 | < co | )3A | I03A | 0 | Q | 0 | < | 0 | < | < |
co | co | co | co | co | CD | co | co | co | CD | co | CO | co | 00 | co | |||
b | b | b | b | b | CO | b | b | b | b | b | co | b | co | to | |||
z | z | z | z | z | > | z | z | ω | čo | w | z | z | z | > | z | > | > |
< | < | < | < | < | X | < | < | Q | O | 0 | < | < | < | od | < | X | Cd |
03 | ao | co | co | co | CM | CD | CD | co | co | CD | ao | co | 00 | CM | 00 | CN | o |
CO | co | co | co | co | CM | CO | CO | R | b | b | co | co | CO | co | r— | CN | |
> | > | > | > | > | R | > | > | Z | z | z | > | > . | > | σ | > | 0 | 0 |
** **
Q12R I N76D I S103A I I104T I S130T M222S Q245R V268A 0.77 1.60
CO | co |
co | 03 |
X- | Ó |
co | <D |
r·*· | o |
ci | c\i |
X | X |
in | in |
xr | xr |
CM | CM |
σ | σ |
co | X |
o | co |
r— | co |
CM | CM |
0_ | σ |
ω | > |
CM | CM |
CM | CO |
CM | CM |
2 | < |
(- | Q |
o | σ> |
co | |
T— | |
co | O |
ι— τ
o <
CO o
W o
co r-X
CM <
CO o
T“ co
Q.
co r<
CD >
999 • · • · • · · · · · · · 9
Příklad 3: ., ' .
V tabulce č. 6 jsou uvedeny varianty testovaných proteáz popsané v-příkladu 1 a výsledky testování ve dvou 'různých detergentech. Stejné testy 'jako v příkladu- 2 se uskutečnily s uvedenými variantami, proteáz s následujícími detergenty. V případě sloupce A detergentem bylo 0,67' g/1 filtrovaného Ariel Ultra (Procter and 'Gamble, Cincinnati, OH, USA) v roztoku, který obsahuje .0,195 g smíšené.. tvrdosti Ca2+/Mg2 + na· 4,54 1. V každé prohlubni se použilo 0,3 ppm enzymu při teplotě 20 °C. V případě sloupce B detergentem bylo 3,38 g/1 filtrovaného Ariel Future (Procter ' and Gamble, .Cincinnati, OH, USA) '''v roztoku, který obsahuje 0,97 g smíšené tvrdosti Ca2 + /Mg2+ na' '4,54 1. V každé, prohlubni se použilo .0,3 ppm enzymu při teplotě ,40 °C. 'V případě sloupce C detergentem bylo 3/5 g/1 detergentů HS.Pl (Procter and Gamble',, Cincinnati, OH,
USA) v. roztoku, který obsahuje 0,52 g smíšené , tvrdosti Ca2 + /Mg2+ na 4,54 1. V každé prohlubni se použilo 0,3 ppm enzymu pří teplotě 20 °C. V případě sloupce D detergentem bylo 1,5 ml/1 detergentů' Tide KT (Procter and Gamble, Cincinnati, OH, USA) v roztoku, který obsahuje 0,195 g smíšené tvrdosti Ca2 + /Mg2+ na 5 4,54 1. V každé prohlubni se použilo 0,3 ppm enzymu při teplotě 20 °C.
·· ·«·· ·· ·*·· • · · · · .· · · · · • · · · · · ···· • · ·♦· ·· · · · · · • · · · ··· · · · · • · · * tt · 9 9 9 9
CO kO >υ
Tabulka
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o | t— | 05 | LO | O | CO | O | CO | CO | LO | CO | CM | CO | b- | Y— | |
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7 | 7 . | 7 | 7 | 7 | 7 | 7 | 7 | 7 | 7 | 7 | co | CO | CO | CO | 7 | 7 | 7 |
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co | CO | CO | o | CO | CO | CO | CO | CO | CO | CO | xr | ||||||
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U3 | IO | LO | o | LO | IO | LO | tn | O | o | tn | O | o | C3 | tn | o | CO | co |
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co | co | co | co | co | co | co | CM | CM | co | CN | CM | CM | co | co | XT | ||
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Z | Z | z | z | Z | Z | Z | Z | Z | Z | Z | Z | Z | Z | Z | Z | Z | |
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Γ- | CO | CM | c- | co | co | o | 4— | co | xr | co | X— | CM | co | LO | CO | 03 | c- |
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xr | LO | co | 05 | o | CN | 03 | T“ | c* | x— | xr | 03 | w | 05 | xr | 04 | ||
có | CM | -- | CM | co | CM | co | CM | X— | CM | 4“· | -- | -- | X>“ | <N | χτ | ||
X | id | bť | 30 | ||||||||||||||
CM | CM | CM | CM | CM | CM | ||||||||||||
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z | z | z | z | • | z | z | |||||||||||
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CO | CM | CM | CM | CO | CO | CO | CM | CM | CM | cv | CM | CM | CM | CM | CO | CO | |
xr | to | LO | LO | xr | χτ | xr | LO | LO | LO | to . | LO | LO | LO | LO | xr | χτ | |
CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | |
z | z | z | z | z | z | z | z | z | z | z | z | z | z | z | z | z | |
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to | ao | ao | ao | LO | LO | LO | 03 | 03 | ao | 00 | CO | CO | co | 00 | LO | LO | |
xr | χτ | xr | xr | xr | xr · | xr | xr | xr | xr | XT | xr | xr | xr | xr | xr | xr | |
CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | |
a | z | z | z | σ | σ | σ | z | z | z | z | z | z | z | z | a | 0 | |
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CO | xr | χτ | χτ | co | co | CO | xr | xr | xr | xr | xr | xr | *r | co | co | ||
CN | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | |
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CO | co | CO | co | CO | CO | CO | co | co | co | CO | CO | co | co | v | CO | CO | CO |
CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CN |
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co | CM | CM | CM | co | co | co | CM | CM | CM | CM | CM | CM | CM | co | CM | xr | O |
X“. | CO | CO | CO | X— | v— | CO | CO | CO | CO | CO | CO | CO | co | CO | <35 | co | |
CN | CM | CM | CM | CN | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | X- | 4^ |
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lo | LO | LO | LO | LO | LO | LO | LO | LO | in | T— | o | co | 05 | LO | LO | ||
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0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | co | X | > | < | < | 0 | 0 | |
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co | co | CM | CM | CM | CM | CO | o | O | LO | LO | LO | LO | LO | LO | o | ||
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co | co | co | co | co | co | co | 4-“ | co | co | co | co | co | cn | ao co | |||
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co | co | CO | co | co | co | co | co | co | co | co | co | CO | co | CO | co | co | |
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CM | CM | CM | CM | CM | CM | CM | CM | tt— | CN | CM | |||||||
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cn | TT | CM | CM | CM | CM | CM | CM | CM | r*- | IO | tt | CM | ΤΓ | xn | LO | CM | v |
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CM | CM | CM | CN | CM | |||||||||||||
cd | 2 | 2 | 2 | 2 | |||||||||||||
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CM | CM | CM | co | LO | CM | CM | CM | CM | CM | CM | CO | 03 | |||||
LO | LO | LO | TT | TT | LO | LO | LO | LO | LO | LO | TT | TT | |||||
CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | |||||
2 | 2 | 2 | 2 | 0 | 2 | 2 | 2 | 2 | 2 | 2 | 2 | 2 | |||||
O | O | Li | Li | Li | Li | Li | 0 | X | X | 0 | 0 | 0 | 0 | 0 | 0 | X | X |
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TT | TT | LO | LO | LO | LO | LO | tt | TT | cn | TT | TT | TT | tt | TT | TT | ΤΓ | |
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LO | IO | CO | CO | CO | co | CO | LO | co | CM | LO | LO | LO | LO | LO | LO | CO | <o |
TT | TT | TT | -Τ | TT | TT | TT | TT | cn | cn | TT | TT | TT | TT | TT | TT | cn | cn |
CM | CN | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM |
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cn | cn | TT | TT | tt | TT | TT | cn | cn | cn | cn | cn | cn | cn | cn | cn | cn | |
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Claims (9)
- PATENTOVÉ NÁROKY1. Varianta proteázy obsahující nahrazení aminokyseliny v jedné nebo -více poloh zbytků odpovídajících polohám zbytků vybraných ze' skupiny zahrnující <212, 232, .252. a 257 subtilizínu mikroorganizmu Bacillus amyloliquefaciens.
- 2. Varianta proteázy podle nároku 1, která se získala ze subtilizínu mikroorganizmu Bacillus.
- 3. Varianta proteázy podle nároku 2, která se získala ze subtilizínu mikroorganizmu Bacillus lentus.
- 4. DNA. kódující variantu proteázy podle nároku 1. '5. Expresívní vektor kódující DNA podle nároku 4.
6. Hostitelská buňka, v y z n se transformuje expresívním a č u j i c i i. vektorem podle s e nárc t >ku i 5 m, ž e 7. Čistící kompozice, v y z n a č u j í c i s e t i m, ž e obsahuje variantu proteázy podle nároku 1. 8 . Zvířecí krmivo, v y z n a č u j i* c i s e t i m, ž e zahrnuje variantu proteázy podle nároku 1. - 9. Kompozice vhodná pro - ošetření textilie, vyznačují* cí se tím, že záhrnuje variantu proteázy podle nároku 1.
- 10. Varianta proteázy podle nároku 1, která obsahuje sadu substitucí vybraných ze skupiny zahrnující polohy zbytků odpovídající polohám v tabulce č. 1 subtilizínu mikroorganizmu Bacillus amyloliquefaciens.
- 11. Varianta proteázy podle nároku 10, která obsahuje sadu substitucí vybraných ze skupiny zahrnující polohy zbytků odpovídající polohám v tabulce č. 2 subtilizínu mikroorganizmu Bacillus amyloliquefaciens.
- 12. Varianta proteázy podle nároku 10, která obsahuje sadu substitucí vybraných ze skupiny zahrnující polohy zbytků • to ·· *·'·· ϊ·'· ···· • to ,· to · · · · · odpovídající polohám v-tabuice - č. 3 subtilizínu mikroorganizmu Bacillus amyloliquefaciens.
- 13. Variantá proteázy podle nároku 1, která dále obsahuje sadu substitucí aminokyselinových zbytků v jedné nebo obou polohách 62 a 230. · .. „ -
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US95632497A | 1997-10-23 | 1997-10-23 | |
US95632397A | 1997-10-23 | 1997-10-23 | |
US95656497A | 1997-10-23 | 1997-10-23 |
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CZ20001493A CZ300347B6 (cs) | 1997-10-23 | 1998-10-23 | Zpusob zvýšení úcinnosti subtilizinu a zpusob výroby pracího prostredku |
CZ20001504A CZ302287B6 (cs) | 1997-10-23 | 1998-10-23 | Varianta subtilisinu |
CZ20001479A CZ20001479A3 (cs) | 1997-10-23 | 1998-10-23 | Bělící prostředky obsahující mnohočetné substituované proteázové varianty |
CZ20001478A CZ20001478A3 (cs) | 1997-10-23 | 1998-10-23 | Ćistící prostředky obsahující mnohonásobně substituované proteázové a amylázové varianty |
CZ20001503A CZ301469B6 (cs) | 1997-10-23 | 1998-10-23 | Subtilizín a prostredek s jeho obsahem |
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CZ20001477A CZ20001477A3 (cs) | 1997-10-23 | 1998-10-23 | Čistící prostředky obsahující mnohočetně substituované proteázové varianty |
CZ20001493A CZ300347B6 (cs) | 1997-10-23 | 1998-10-23 | Zpusob zvýšení úcinnosti subtilizinu a zpusob výroby pracího prostredku |
CZ20001504A CZ302287B6 (cs) | 1997-10-23 | 1998-10-23 | Varianta subtilisinu |
CZ20001479A CZ20001479A3 (cs) | 1997-10-23 | 1998-10-23 | Bělící prostředky obsahující mnohočetné substituované proteázové varianty |
CZ20001478A CZ20001478A3 (cs) | 1997-10-23 | 1998-10-23 | Ćistící prostředky obsahující mnohonásobně substituované proteázové a amylázové varianty |
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1998
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Effective date: 20161023 |