CZ302287B6 - Varianta subtilisinu - Google Patents
Varianta subtilisinu Download PDFInfo
- Publication number
- CZ302287B6 CZ302287B6 CZ20001504A CZ20001504A CZ302287B6 CZ 302287 B6 CZ302287 B6 CZ 302287B6 CZ 20001504 A CZ20001504 A CZ 20001504A CZ 20001504 A CZ20001504 A CZ 20001504A CZ 302287 B6 CZ302287 B6 CZ 302287B6
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- subtilisin
- bacillus amyloliquefaciens
- residue
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- variant
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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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Abstract
Varianta subtilisinu, obsahující zbytek alaninu v poloze, odpovídající zbytku 103 v subtilisinu Bacillus amyloliquefaciens a zbytek argininu, leucinu nebo valinu v poloze, odpovídající zbytku 245 v subtilisinu Bacillus amyloliquefaciens a mimoto jednu nebo vetší pocet substitucí zbytku aminokyselin, vybraných ze skupiny, tvorené zbytky v polohách, odpovídajících polohám 3, 12, 20, 27, 38, 43, 48, 57, 61, 62, 68, 76, 87, 89, 97, 98, 99, 101, 102, 104, 109, 114, 121, 128, 130, 131, 133, 140, 141, 147, 159, 160, 166, 167, 170, 173, 174, 184, 185, 192, 194, 203, 204, 205, 206, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 222, 224, 227, 228, 232, 236, 237, 242, 243, 244, 247, 248, 251, 252, 255, 256, 257, 259, 260, 261, 262, 268, 269, 271, 272 a 275 subtilisinu Bacillus amyloliquefaciens, pricemž v prípade, že substituce v poloze, odpovídající poloze 103 subtilisinu Bacillus amyloliquefaciens je kombinována se substitucí, odpovídající poloze 76, je obsažena rovnež substituce v jedné nebo vetším poctu poloh, odlišných od poloh 27, 99, 101, 104, 109, 128, 166, 204, 206, 210, 216, 217, 218, 222 nebo 260 subtilisinu Bacillus amyloliquefaciens za predpokladu, že aminokyselinou v poloze, odpovídající poloze 232 v subtilisinu Bacillus amyloliquefaciens je aminokyselina, odlišná od valinu, pricemž varianta subtilisinu má zvýšenou prací úcinnost ve srovnání s prekurzorovou proteázou bez uvedených substitucí.
Description
Oblast techniky
Šeřino vé proteázy jsou podskupinou karbonylových hydroláz. Obsahují diverzní skupinu enzy15 mů, které vykazují Široké rozmezí specifit a biologických funkcí (popisuje se v publikaci Stroud, R. Sci. Amer., 131: 74-88). Navzdory jejich funkční diverzitě katalytického mechanizmu serinových proteáz se dosáhlo alespoň u dvou geneticky odlišných rodin enzymů: 1) subtilisiny 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, terciární struktura těchto dvou enzymatických rodin tvoří konzervativní katalytickou triádu aminokyselin, které obsahují serin, histidin a aspartát.
Subtilisiny jsou serinové proteázy (s přibližnou molekulovou hmotností 27 500), které se vylučují ve velkém množství z širokého rozmezí druhů Bacillus a jiných mikroorganizmů. Proteinová sekvence subtilisinu se stanovila alespoň z devíti různých druhů Bacillus (Markand, F. S., et al., (1983), Hoppe-Seylers Z. Physiol. Chem. 364: 1537-1540). V literatuře se zmiňuje trojrozměrná krystalografická struktura subtilisinů z mikroorganizmu Bacillus amyloliquefaciens, Bacillus licheniformis a několik přirozených variant mikroorganizmu B. lentus. Tyto studie ukazují, že ačkoli subtilisin není geneticky příbuzný se savčími serinovými proteázami, vykazuje podobnou strukturu aktivního místa. Analýza krystalických struktur subtilisinu pomocí rentgenového záření, přičemž subtilisin obsahuje kovalentně vázané peptidové inhibitory (popisuje se v publikaci Robertus, J. D., et al., (1972), Biochemistry, 11: 2439-2449) nebo komplexy 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 substrátu subtilisinu. Publikuje se velké množství studií kinetických a chemických úprav molekul (popisuje se v publikaci Svedsen, B., (1976) Carlsberg Res. Commun., 41: 237-291, Markland, F. S. Id.) a existuje alespoň jedna zpráva, kde vedlejší řetězec methioninu subtilisinu s 222 zbytky se přeměnil peroxidem vodíku na methioninsulfoxid (popisuje se v publikaci Stauffer, D. C., et al., (1965), J. Biol. Chem., 244: 5333-5338) a provedla se rozsáhlá místně specifická mutageneze (Wells and Estell (1988) TIBS 13:291-297).
Podstata vynálezu
Podstatu vynálezu tvoří varianta subtilisinu, obsahující zbytek alaninu v poloze, odpovídající zbytku 103 v subtilisinu Bacillus amyloliquefaciens a zbytek argininu, leucinu nebo valinu v poloze, odpovídající zbytku 245 v subtilisinu Bacillus amyloliquefaciens a mimoto jednu nebo větší počet substitucí zbytků aminokyselin, vybraných ze skupiny, tvořené zbytky v polohách, odpovídajících polohám 3, 12, 20, 27, 38, 43, 48, 57, 61, 62, 68, 76, 87, 89, 97, 98, 99, 101,102,
104, 109, 114, 121, 128, 130, 131, 133, 140, 141, 147, 159, 160, 166, 167, 170, 173, 174, 184,
185, 192, 194, 203, 204, 205, 206, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 222, 224,
227, 228, 232, 236, 237, 242, 243, 244, 247, 248, 251, 252, 255, 256, 257, 259, 260, 261, 262,
268, 269, 271, 272 a 275 subtilisinu Bacillus amyloliquefaciens, přičemž v případě, že substituce
- 1 CZ 302287 B6 v poloze, odpovídající poloze 103 subtilisinu Bací Hus amyloliquefaciens je kombinována se substitucí, odpovídající poloze 76, je obsažena rovněž substituce v jedné nebo větším počtu poloh, odlišných od poloh 27, 99, 101, 104, 109, 128, 166, 204, 206,210,216,217,218, 222 nebo 260 subtilisinu Bacillus amyloliquefaciens za předpokladu, že aminokyselinou v poloze, odpovídající poloze 232 v subtilisinu Bacillus amyloliquefaciens je aminokyselina, odlišná od valinu, přičemž varianta subtilisinu má zvýšenou prací účinnost ve srovnání s prekurzorovou proteázou bez uvedených substitucí.
Více preferované proteázové varianty jsou sady substitucí vybrané ze skupiny obsahující polohy o zbytků odpovídající polohám v tabulce č. 1 subtilisinu mikroorganizmu Bacillus amyloliquefaciens:
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Tabulka č. 1
I | |||||||||||||||||||
[ | |||||||||||||||||||
218 | co o- CM | * *> > | M· r- | p*. co CM | |||||||||||||||
222 | 104 I | ·*τ o V“ | 107 | O | co ’τ CM | 3 T- | co co -t— | 104 | 104 | 261 | 160 | 216 | 104 | 104 L | 104 | 104 | M“ O i— | 104 | co co *— |
T o | 103 | 103 | 2 T- | (O O 7— | 104 | 103 | s | 103 | 103 | 104 | s | Μ- α | 103 | 103 | 103 | 103 | 103 | co O | 104 |
co a | 00 os | CD | 103 | 76 | co o Ύ— | 77 | 103 | g | co P- | 103 | 103 | 103 | co r-. | 76 l | 77 | 76 | s | 76 | 103 |
76 | 76 | 76 | to | v | 76 | 76 | 76 | CO | ** | 76 | 76 | 76 | r·* | 37 | co r- | CD co | 38 | ao | 76 |
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i | ||||||||||||||||||||
1 | ||||||||||||||||||||
185 | 274 | O θ’ CM | r. r*. r- | |||||||||||||||||
104 | 104 | 104 | ·*τ co | 252 | 259 | 251 | 104 | 104 | 237 | 160 | 228 | ΤΓ O | 254 | 204 | 204 | ΤΓ o | σ> m | 104 | o | 270 |
103 | 103 | 103 | 104 I ί ______ | ťOl | 104 | WH | co o | co o | 104 | 104 | s | co o v— | 104 | 104 | 104 | o | Ό- o | 103 | 103 | o |
co r- | 76 | 76 | 103 | 103 | 103 | 103 | 99 | 76 I | co o | 103 | 103 | co r*. | 103 | co o | 103 | fS | 103 | 76 | 76 | 103 |
o | CD | 19 | 76 | 76 | 76 | 76 | 76 | 72 i | 76 | 76 | 76 | in m | 76 | 76 ' | 76 | 43 | 76 | O | 58 | co r- |
251 | eo 00 | ( | ||||||||||||||||||
236 | 237 | 204 | l·» » | 271 | τ- Φ CM | 1 242 | <0 ν | |||||||||||||
ΙΟ 00 τ~ | 3 | 262 | 104 | ο | <Ο C0 ▼— | 3 | 130 | 109 ( | 104 | CO | 3 | 212 | 252 | <Ν ^Γ CM | ν Ν- CM | ί 104 | 3 V* | 258 | 271 | 104 |
104 | 103 | 104 | 103 | 103 | 3 | 103 | 3 τ— | 104 | σι ο V | fl· Ο | 103 | ο | 3 | 3 Τ’ | 'Τ Ο | 103 | 103 | 104 | 104 | 103 |
ο τ” | (Ο Ν' | (Ό ο ν- | 00 ν* | 76 | 103 | 76 | σϊ ο | <Ό Ο τ- | σ> σ> | Ο τ- | £ | | 103 | Π ο | 103 | 103 | CO Ν* | CO Ν'. | σ> ο | rt ο T“ | to Ν- |
I I | 27 | 76 | co r*. | CM | 76 | Ν' | (Ο Ν- | 76 I | 76 | 9Ζ | <Ν | C0 Ν' | C0 Ν. | CD Ν* | £ | <Ν | (Ό | C0 Ν' | C0 Γ'» | 5 |
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tn cO CN | 251 | 252 | ||||||||||||||||||
263 | 249 | 271 | 217 | 217 | ||||||||||||||||
182 | 272 | co ΤΓ CN | 206 | 238 | 198 | CN CO | 137 | CD ΤΓ CN | co in CN | 271 | 261 | 206 | co o CN | σ> m T- | 159 | | |||||
104 | 182 | ; 109 | 104 | 137 | 1B2 | 104 | CD | 1» m V“ | ^r o | 206 | CN CN | o- o | 206 | 2 | O | 158 | 206 | 104 | 104 I | |
1 103 | 104 | ΤΓ o T— | 103 l | TT O | 228 | 104 | 103 | 104 | o- O | co o t— | 104 | 2 i— | 103 | 104 | 103 | 103 | 2 T— | Ν’ O v- | 103 | 103 |
co r*- | 103 | co o T- | r- co | 103 | 104 | 103 | co r- | 103 | 103 | 76 | 103 | CO O | s | 103 | ÍS | 77 | 103 | <0 σ | 76 | 76 I |
38 | co | CO r- | co r*- | co b- | 103 | 76 | CN | 76 | co r-. | co | 9Z | co r* | 58 | co | ’Τ | ÍS | 76 | 76 | Ν’ | π |
τ- m CM | 271 | 271 | ||||||||||||||||||
185 | 244 | σ> tn | 236 | 159 | CM | 271 | 271 | I 212 l | 243 | |||||||||||
133 I | 206 | 00 00 | 158 | 185 | uň CM | q> tn ** | m tn | 104 | O) tn **► s | O) tO ** | 8 CM | 224 | ! 268 | 212 | tn ^r CM | 141 | . I | 236 | m M- CM | ||
wn | ; 159 | TT o | 3 | 3 | 208 | WH | o t— | T o | co o | 104 | ΘΗ | 159 | 159 | CM fŇ | 104 | 104 | CM CM | θ’ CO | CM t— CM | g |
103 | 104 | CO O | 103 | 103 | 104 | 103 | 103 | o | co r- | 103 _I | 3 | 104 | 104 | 104 | 103 | 103 | 104 | 104 | 3 | 3 |
77 | 103 | CO Γ-- | 76 | 103 | co r- | co Γ-. | S | 62 | 76 í I | 103 | 103 | 103 | co o | 69 | i*- CD | 103 | 103 | 103 | 103 | |
76 | 76 | o- | 76 | co r- | co | 89 | CM | CM | CM M* | s | co r*. | co r- | £ | co t*» | s | co | co r- | 76 | s |
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245 | ||||||||||||||||||||
r- 04 | 8 OJ | cn tn OJ | 236 | cn xr OJ | ||||||||||||||||
271 | 245 | 236 | 236 | 159 | 236 | to co OJ | xr co | 243 | tn xr OJ | 236 | 249 | |||||||||
236 | 236 | 217 | 236 | σ> tn | 04 | 159 | cn o OJ | 159 | 236 | 236 | Ol m | 159 I | 236 | 249 | ||||||
210 | XT o | 236 | cn to X** | cn tn •t“ | σ> tn | s T- | 159 | 104 | ^ř v* TS > | o t— | Oi tn r- | r- | O) tn T- | 159 | Ol XT T“ | co Ol x— | cn tn X— | 245 | 222 | |
109 | co o T— | WH | XT O T~ | 104 | XT O | 103 | § x— | Γ104 | [ 103 | 103 | 103 | xr o | : 104 | s r· | M04 | 104 | j 104 | xr o x— | 222 | 3 |
’Τ o | to r— | 103 | 103 | 103 | <o o T· | s | 103 | co o x— | 76 | 8 | to n- | CO o | ' 103 | CO O •r- | co o X— | co o | 103 | co o | 104 | 103 |
ί 103 | i 62 I | 76 | 76 | 76 | l 76 | 99 | s | to O- | 75 | 76 | 68 | CO o- | to Γ*- | 76 | 76 | 76 | 76 | 76 | 103 | 76 |
76 | 20 | co to | 99 | 66 | co co | o- | co to | 66 | | 99 | 8 | 04 | co to | 99 | 99 | 99 | 68 | 99 | 99 | 76 | OJ |
tn | tn | tn | tn | tn | ||||||||||||||||
tn | v | tt | M | |||||||||||||||||
CM | CM | CM | CM | CM | ||||||||||||||||
s | tn 'N* | h* M· | s | tn ’Τ | 8 | tn ΤΓ | CD CO | 8 | CM tn | |||||||||||
CM | CM | CM | CM | CM | CM | CM | CM | CM | CN | |||||||||||
eo | tn | CO | tn | TT | CD | 00 | CO | CO | CM | m | ||||||||||
CD | ^r | CO | ΤΓ | O | CO | T- | CO | O | CO | M- | ||||||||||
CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | ||||||||||
1- | CD | 8 | 05 | CD | ΤΓ | O) | CM | XT | m | 05 | co | |||||||||
CO | N. | 'V | tn | CO | Γ-- | O | tn | CO | 05 | M- | m | co | ||||||||
CM | CM | CM | CM | CM | CM | CM | CM | CM | ||||||||||||
CM | co | CM | CM | CM | CM | CM | CM | 05 | 05 | T* | 05 | 05 | 05 | eo | CD | 05 | CM | m | Μ | CM |
<M | CD | CM | CM | CM | CM | CM | CM | v | tn | tn | tn | tn | to | tn | u) | CM | TT | O | CO | |
CM | CM | CM | CM | CM | CM | CM | CM | CM | f“ | *> | f- | v- | T· | CM | CM | V» | CN | |||
CO | CM | 'M' | § | Φ | v | c- | 05 | CM | s | ΤΓ | v | M- | ^r | ΤΓ | 3 | CM | CO | 05 | ||
h- | CM | O | O | o | CO | O | CM | O | o | o | o | O | o | o | CO | o | m | |||
CM | <r- | ÍM | *” | r- | T— | T“ | CM | t- | ||||||||||||
ΤΓ | M- | CO | ΤΓ | ^r | eo | ’Τ | Ό* | M· | eo | eo | co | CO | eo | eo | eo | eo | eo | 3 | co | 3 |
o | O | o | O | O | O | o | O | O | o | o | σ | o | o | o | o | O | o | |||
v- | *“ | t— | τ- | *” | *“ | |||||||||||||||
co o T- | 103 | i 76 | co o t— | 103 | CD r*» | 103 | 103 | s | 76 | 76 | 76 | s | 76 | 76 | 76 | 76 | 76 | 103 | co CD | eo o Ύ— |
CD | co | CD | CD | V· | ř5 | CD | <p | 99 | CD | 05 | 05 | 05 | co | 05 | CD | CM | co | M· | CO | |
f- | Γ-- | CM | CO | CD | CD | CD | CO | to | to | CD | τ— | r*. | CM | co |
-9CZ 302287 B6
I | i | |||||||||||||||||||
o | <o | Ol | LO | |||||||||||||||||
tO | r- | <0 | Ν' | |||||||||||||||||
CM | CN | CN | CN | |||||||||||||||||
m | CN | CN | ÍN | CN | CN | CN | CN | CN | CO | |||||||||||
N | CO | CO | CO | lO | m | CO | CO | CO | ΙΟ | Ν' | ||||||||||
CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | ||||||||||
CO | CO | LO | CO | lO | CO | in | CD | to | UO | UO | LO | CN | ||||||||
CO | Ν' | N | Ν' | Ν' | Ν' | Ν' | Ν' | Ν' | N | co | N | XT | CN | |||||||
CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | |||||||
CM | in | co | CO | cn | CO | CO | CO | CO | CO | CO | CN | r- | CO | ÍN | CN | iO | ||||
CO | N | xp | Ν' | N | CO | CO | CO | co | CO | Ν' | CN | CN | Ν' | CN | CN | CO | ||||
CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | * | ||||
CO | n* | CO | CN | (0 | <0 | ťN | CN | CN | CN | CN | CN | m | CN | CN | LO | 00 | o | O | ||
N | Ν' | CN | CO | CO | CO | CO | co | CO | CO | CN | r* | CN | CN | Ν' | co | r- | ||||
CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN 1 | CN | CN | ÍN | x— | x— | |||
Ol | (N | O | O | N | Ν' | CN | CN | cn | cn | σ> | cn | O | o | o | O | CN | O | N | O | |
LO | CN | x— | CO | O | 00 | CO | CO | co | uO | CA | LO | to | CO | co | co | CO | CN | co | O | CO |
CN | CN | x— | *“ | CN | CN | X“ | * | X— | T- | CN | T- | |||||||||
Ν' | Ν' | CN | Ν' | CO | Ν' | Ol | 01 | O | N | N | Ν' | Ν’ | Ν' | Ν' | Ν' | N* | O | Ν' | CO | Ν’ |
O | O | CN | O | o | O | CO | CO | N | O | O | O | O | O | O | O | o | CO | O | O | O |
CN | * | x— | X“ | T— | x— | X“ | x— | x- | X“ | x— | ||||||||||
co | co | CO | CO | řrt | CO | ν- | 2 | N | CO | CO | CO | co | CO | CO | CO | CO | Ν' | CO | CO | |
o | o | O | O | CD Is_ | O | ο | o | O | o | O | o | o | O | o | o | o | O | o | ViJ | O |
r- | P* | *“ | T“ | x— | X” | x— | X— | X» | X— | x— | 1 | x— | ||||||||
co | CO | CO | co | co | CO | CO | rt Q | rt | CD | °S | 00 | £- | co | co | CO | co | CO | CO | h- | CO |
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-11CZ 302287 B6
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- 12CZ 302287 B6
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-13CZ 302287 B6
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- 15 CZ 302287 B6
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Ol | o | cn | cn | O | O | cn | cm | o> | oi | cn | CM | V | σ> | cn | o | cn | TT | Ol | CM | O |
IO | io | IO | IO | IO | cn | to | cn | to | to | to | CO | O | to | o | o | IO | cn | O | ||
r- | v- | <» | CM | •r- | CM | T— | CM | CM | CM | CM | T— | CM | CM | |||||||
<r | <r | n* | s | TT | cn | X | <5 | 'Μ' | n- | s | 01 | cn | Tf | cn | cn | cn | cn | ΤΓ | cn | 0) |
o | o | O | O | to | o | io | O | O | IO | o | O | IO | to | «2 | o | O | O | 10 | ||
r— | *“ | -r— | r— | T- | v* | T“ | CM | t— | ||||||||||||
tn O | cn o | cn o | cn o | cn o | TT O | cn o | s | cn o | s | 03 | 04 | CO | cn o | -n- o | n- o | 3 | <0 | cn o | M- O | 3 |
v- | T-. | Ί— | t- | Γ*» | T- | T“ | T- | |||||||||||||
69 ( | 69 | co CO | 89 | co co | 103 | 89 | 103 | 89 | 1 68 | | 68 | cn o | 99 | CO CO | 103 | 103 | O T~ | 68 | CM | 103 | 103 |
17CZ 302287 B6
1 | ||||||||||||||||||||
σ | CM | CM | CM | |||||||||||||||||
co | tn | ui | tn | |||||||||||||||||
CM | CM | CM | CM | |||||||||||||||||
m | tn | CM | CM | CO | 00 | cd | CM | |||||||||||||
TT | io | Μ- | m | co | tn | •n· | -T | XT | U1 | |||||||||||
CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | |||||||||||
o | CO | tn | h- | CO | tn | tn | CD | E- | CD | tn | tn | tn | 00 | CM | CM | CM | ||||
co | co | ’Τ | m | co | Μ- | Ό | XT | tn | Μ | XT | xf | •o· | •O· | m | tn | tn | ||||
CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | ||||
m | CM | CO | tn | CM | CD | tn | tn | tn | CO | tn | tn | tn | CO | CO | co | tn | 03 | 00 | CD | |
ΜΓ | <0 | co | Μ- | CO | CO | Ό | xr | TT | co | o- | XT | XT | CO | CO | CO | ΧΓ | v | •θ’ | XT | |
CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | |
CD | O | CM | CO | O | CM | CO | CO | CO | tn | CM | CO | co | CO | CM | CM | CM | co | tn | tn | tn |
cO | CO | co | CO | CO | co | eo | θ’ | CO | co | co | (0 | CO | CO | CO | CO | XT | XT | |||
CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM |
CM | CD | O | CM | CD | O | CM | CM | CM | CO | Ol | CM | cm ; | CM | CM | CM | CM | CM | co | CO | χχ |
CO | O | CO | O | V“ | CO | CO | CO | CO | O | CO | co | CO | T- | CO | co | CO | XT | |||
CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM |
CD | tn | o> | CD | tn | σι | O | O | Ol | O | Ol | 0> | Ol | CM | Ol | 01 | 01 | CO | CM | CM | CO |
O | o | o | O | o | o | T“ | tn | co | LO | LO | tn | tn | tn | tn | CO | CO | CO | |||
CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | Ύ“ | CM | CM | CM | CM | ||||||
tn | CD | tn | tn | Ol | tn | O) | tn | 00 | 01 | XT | XT | θ’ | 01 | xr | XT | xr | Ol | 01 | XT | CM |
o | m | o | o | tn | o | o | o | CM | m | o | O | o | tn | o | O | o | tn | tn | CD | CO |
CM | CM | CM | CM | CM | CM | T“ | T“ | T“ | T— | r™ | CM | |||||||||
CD | O | o | CD | Μ- | Ol | Ol | Ol | θ’ | T | CO | CO | to | XT | CO | co | co | xT | r· | 01 | Ol |
tn | O | tn | tn | O | to | tn | tn | o | O | o | o | o | o | o | o | o | O | CO | tn | tn |
v— | v | Ύ— | -r— | V | ||||||||||||||||
rr | co | 'T | Ό | CO | Ό· | TT | T | co | co | a? co | 89 | 89 | co | CM | CM | CM | CO | XT | XT | xT |
o | o | o | O | O | O | O | o | o | o | o | O | O | O | O | o | a | o | |||
r~ | T“ | T | V“ | v— | r- | |||||||||||||||
103 | 89 | 103 | 103 | 68 | 103 | 103 | co o | 89 | co Ό | CD -o- | co v | 48 | 102 | CM | τ- Ο | 86 | 102 | 103 | co o f— | 103 |
- 18CZ 302287 B6
04 | ||||||||||||||||||||
tn | ||||||||||||||||||||
OJ | ||||||||||||||||||||
to | Ol | OJ | Ol | OJ | to | OJ | 04 | 04 | 04 | |||||||||||
tn | tn | tn | m | tn | XT | tn | tn | tn | m | |||||||||||
Ol | OJ | Ol | Ol | Ol | Ol | Ol | Ol | Ol | OJ | |||||||||||
Ol | 00 | 04 | Ol | OJ | OJ | Ol | 00 | Ol | co | 00 | tn | co | OJ | OJ | 00 | 00 | co | o | ||
tn | XT | tn | tn | tn | tn | tn | XT | tn | xr | xr | xr | xr | tn | tn | XT | XT | xr | 8 | ||
Ol | OJ | OJ | Ol | Ol | Ol | Ol | OJ | 04 | OJ | Ol | Ol | Ol | Ol | 04 | OJ | 04 | Ol | |||
CO | tn | 00 | oo | oo | oo | tn | tn | oo | tn | tn | 36 | tn | oo | 00 | in | tn | m | 04 | ||
XT | XT | xr | xr | xr | xr | XT | XT | XT | XT | •v | XT | xr | xr | xr | XT | xr | tn | |||
Ol | Ol | OJ | OJ | Ol | Ol | Ol | OJ | Ol | OJ | Ol | Ol | Ol | OJ | Ol | 04 | Ol | Ol | Ol | ||
tn | co | to | m | n | m | tn | 8 | 8 | to | to | Ol | to | tn | to | co | to | to | co | ||
xr | cn | Xf | xr | XT | xr | xr | cn | cn | cn | tn | xr | XT | cn | tn | cn | xr | ||||
Ol | Ol | OJ | OJ | Ol | Ol | OJ | OJ | OJ | 04 | 04 | Ol | Ol | 04 | Ol | OJ | 04 | Ol | 04 | ||
co | 04 | to | 8 | CO | to | co | Ol | Ol | Ol | OJ | cn | Ol | Ol | to | to | 04 | 04 | 04 | tn | |
cn | cn | cn | cn | cn | cn | tn | cn | cn | cn | T* | m | cn | cn | cn | cn | cn | cn | XT | ||
OJ | OJ | 04 | Oí | OJ | Ol | Ol | Ol | 04 | Ol | OJ | Ol | Ol | OJ | OJ | Ol | Ol | 04 | Ol | 04 | |
OJ | cn | OJ | Ol | Ol | Ol | 04 | Ol | Ol | cn | cn | Ol | oo | cn | OJ | Ol | cn | <n | cn | CO | |
cn | V— | cn | cn | cn | cn | tn | X- | X» | xr | cn | tn | X“ | X- | tn | ||||||
OJ | Ol | O| | Ol | Ol | OJ | 04 | Ol | Ol | 04 | Ol | 04 | oi | 04 | 04 | O| | Ol | Ol | 04 | Ol | |
co | oi | in | co | cn | 01 | Ol | Ol | Ol | Ol | Ol | Ol | tn | tn | Ol | Ol | O1 | Ol | Ol | Ol | 04 |
tn | oo | o | tn | tn | tn | tn | tn | & | tn | XT | xr | tn | tn | tn | tn | tn | in | cn | ||
04 | x- | Oí | oi | *“ | X- | Ol | Ol | x- | X“ | OJ | ||||||||||
o | xr | Ol | Ol | 01 | xr | XT | 3 | 3 | Ol | 01 | 3 | OJ | o | o | O | 00 | 3 | co | 00 | Ol |
tn | O | tn | tn | in | O | o | o | tn | cn | cn | cn | cn | Ol | Ol | 04 | tn | ||||
x— | X- | v* | x— | X- | x~ | 04 | Ol | X“ | X“ | x- | ||||||||||
XT | cn | xf | xr | xr | cn | cn | cn | cn | xr | xr : | cn | Ol | Ol | xr | xr | xr | cn | XT | xr | XT |
O | o | O | o | o | o | o | o | o | O | O | O | tn | m | O | O | O | o | o | O | O |
X” | T- | x— | X· | *” | X“ | X— | ||||||||||||||
cn | OJ co | cn | cn | cn | Ol | Ol | oi | 04 | cn | cn | 3 | xr | cn | cn | cn | cn | cn | cn | ||
o | o | o | o | o | o | o | O | o | o | O | o | o | o | o | o | o | o | o | ||
X“ | x- | x— | X— | x- | X- | |||||||||||||||
62 | CM r— | T· o T* | 101 | 101 | 00 0> | o | 96 | 86 | Ol co | 62 | 1 62 1 | cn o | 103 | 62 | O V* | x- O X— | 62 | 62 | 62 | 101 |
- 19 CZ 302287 B6
271 | |||||||||||
CN | |||||||||||
tn | |||||||||||
CN | |||||||||||
CN | CO | ||||||||||
tn | T | ||||||||||
CN | ÍN | ||||||||||
CN | CN | CN | CN | CN | CN | CN | CN | co | CN | tn | |
tn | tn | tn | tn | tn | tO | tn | m | tn | ΤΓ | ||
CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | |
co | co | CO | CO | CO | CO | CO | CO | tn | 03 | CO | |
ΤΓ | ΤΓ | 'T | N | 'T | ΤΓ | Ό- | Ν’ | ΤΓ | N- | CO | |
CN | CN | CN | CN | CN | CN | <N | CN | CN | CN | CN | |
tn | tn | tn | tn | m | tn | tn | tn | CO | tn | CN | |
o· | •v | ΤΓ | ΤΓ | ΤΓ | Ν' | ΤΓ | CO | 'tr | CO | ||
CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | |
co | CO | CO | CO | CO | CO | CO | tn | CO | CN | CO | CO |
co | CO | CO | CO | CO | CO | CO | Ό | co | CO | co | |
<N | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN |
CN | CN | CN | CN | CN | CN | CN | CO | CN | θ' | CN | co |
CO | CO | CO | CO | CO | CO | CO | CO | CO | 03 | CO | o |
CN | CN | CN | CN | CN | CN | CN | CN | CN | T | CN | CN |
O) | σ» | 03 | CN | 03 | O | m | O | 03 | O | tn | |
U3 | tn | LO | O | o | CO | o> | tn | co | CO | ||
CN | CN | CN | CN | CN | Ύ— | r> | T- | ||||
*T | ΤΓ | 03 | CD | σ» | 03 | o> | 03 | Ν’ | 03 | 03 | |
CO | o | O | IO | IO | tn | tn | tn | tn | O | tn | tn |
T~ | T~ | T— | |||||||||
Ό | co | co | Ν’ | NT | *r | 'tf | T | co | 'tr | ||
O | o | o | O | O | o | O | o | O | o | o | o |
T“ | * | i- | |||||||||
co | τ- | CO | CO | co | CO | co | CO | 'T*· | CO | co | |
o | o | Ο | o | o | o | o | o | o | o | o | o |
T | Ύ· | T“ | T- | T- | T* | T- | |||||
v* | τ- | Ύ·* | 76 | ||||||||
O | OD ω | G) Φ | O | Ο | o | o | O | O | o | CN CO |
Nejvíce preferovanými proteázovými variantami jsou ty uvedené v tabulce č. 3.
Dále vynález popisuje sekvence DNA kódující takové proteázové varianty, stejně jako expresní vektory obsahující takové sekvence DNA.
Dále vynález popisuje hostitelské buňky transformované takovými vektory, stejně jako hostitelské buňky, které jsou schopny exprimovat takovou DNA za intracelulámí nebo extracelulámí produkce proteázových variant.
to
Dále vynález popisuje čisticí kompozice obsahující proteázovou variantu podle vynálezu.
Dáte vynález zahrnuje zvířecí krmivo obsahující proteázovou variantu podle vynálezu.
ts Dále vynález popisuje kompozici pro ošetření textilie obsahující proteázovou variantu podle vynálezu.
Proteázy jsou karbony lové hydrolázy, které v obecném případě štěpí peptidové vazby proteinů a peptidů. Termín „proteáza“ znamená přirozeně se vyskytující proteázu nebo rekombinantní pro20 teázu. Přirozeně se vyskytující proteázy zahrnují α-aminoacylpeptidovou hydrolázu, hydrolázu peptidylaminové kyseliny, acylaminovou hydrolázu, serinovou karboxypeptidázu, metalokarboxypeptidázu, thiolovou proteinázu, karboxylproteinázu a metaloproteinázu. Dále zahrnují serinovou proteázu, metaloproteázu, thiolovou proteázu a kyselou proteázu stejně jako exogenní proteázy. Vynález popisuje proteázové enzymy, kterými jsou varianty karbony lových hydroláz (varianty proteáz), které se přirozeně nevyskytují a které mají různou proteolytickou aktivitu, stabilitu, specifítu substrátu, profil pH a/nebo charakteristiku provedení ve srovnání s prekurzorovou karbonylovou hydrolázou, 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 amino30 kyselinami. Prekurzorovou proteázou může být přirozeně se vyskytující proteáza nebo rekombinantní proteáza.
Varianty proteáz, které jsou použitelné podle vynálezu, zdůrazňují substituci libovolné z 19 přirozeně se vyskytujících L-aminokyselin v označených polohách aminokyselinových zbytků. Tako35 vé substituce se mohou provést v libovolném prekurzorovém subtilisinu (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 subtilisinu mikroorganizmu Bacillus. Více se upřednostňuje, aby se proteázové varianty odvodily ze subtilizéru mikroorganizmu Bacillus lentus a/nebo subtilisinu 309.
Subtilisiny jsou bakteriální a houbové proteázy, které v obecném případě štěpí peptidové vazby proteinů a peptidů. Termín „subtilisin“ znamená přirozeně se vyskytující subtilisin nebo rekombínantní subtilisin. Série přirozeně se vyskytujících subtilisinů je známa, že se produkuje a často se vylučuje různými mikrobiálními druhy. Aminokyselinové sekvence členů uvedených sérií nejsou celé homologní. Subtilisiny 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 chymotrypsinu. Subtilisiny a serinové proteázy příbuzné chymotrypsinu mají katalytickou triádu obsahující aspartát, histidin a serin. U proteáz příbuzných subtilisinu je relativní pořadí aminokyselin, které se čtou od Nkonce k C-konci, aspartát-histidin-serin. U proteáz příbuzných chymotrypsinu je relativní pořadí histidin-aspartát-serin. Subtilisin zde vystupuje jako serinová proteáza vykazující katalytickou triádu proteáz příbuzných subtilisinu. Příklady zahrnují, ale nejsou omezeny na subtilisiny uvede-21 CZ 302287 B6 né na obrázku č. 3. Číslování aminokyselin v proteázách odpovídá číslům přiřazených sekvencím subtilisinu mikroorganizmu Bacillus amyloliquefaciens přítomných na obrázku č. 1.
Termín „rekombinantní subtilisin“ nebo „rekombinantní proteáza“ znamená subtilisin, ve kterém sekvence DNA kódující subtilisin nebo proteázu je upravena tak, aby 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.
io „Subtilisiny, které nepocházejí z člověka“ a DNA je kódující se mohou získat z mnoha prokaryontních a eukaryontních organizmů. Vhodné příklady prokaryontních organizmů zahrnují gram-negativní organizmy, jako je E. coli nebo Pseudomonas a gram pozitivní bakterie jako je Micrococcus nebo Bacillus. Příklady eukaryontních organizmů, ze kterých je možné získat subtilisin a jejich geny, mohou zahrnovat kvasinky, jako jsou Saccharomyces cerevisiae, houby jako je Aspergillus sp.
„Varianta proteázy“ má aminokyselinovou sekvenci, která se získala z aminokyselinové sekvence „prekurzorové proteázy“. Prekurzorové proteázy zahrnují přirozeně se vyskytující proteázy zo 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).
Specifické substituce odpovídající poloze 103 v kombinaci sjednou nebo více z následujících substitucí odpovídající polohám 1,3,4, 8, 10, 12, 13, 16, 17, 18, 19, 20, 21, 22, 24, 27, 33, 37, 38, 42,43,48, 55, 57, 58,61,62, 68, 72, 75, 76, 77, 78, 79, 86, 87, 89, 97,98,99, 101, 102, 103,
104, 106, 107, 109, 111, 114, 116, 117, 119, 121, 123, 126, 128, 130, 131, 133, 134, 137, 140,
141, 142, 146, 147, 158, 159, 160, 166, 167, 170, 173, 174, 177, 181, 182, 183, 184, 185, 188,
192, 194, 198, 203, 204, 205, 206, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 222, 224,
227, 228, 230, 232, 236, 237, 238, 240, 242, 243, 244, 245, 246, 247, 248, 249, 251, 252, 253,
254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 265, 268, 269, 270, 271, 272, 274 a 275 subtilisinu mikroorganizmu Bacillus amyloliquefaciens.
Preferované varianty jsou ty, které vykazují kombinace substitucí v polohách zbytků odpovídající polohám subtilisinu mikroorganizmu Bacillus amyloliquefaciens v tabulce č. 1. Více se preferují varianty, které vykazují kombinace substitucí v polohách zbytků, které odpovídají polohám subtilisinu mikroorganizmu Bacillus amyloliquefaciens uvedené v tabulce ě. 3.
Dále se preferují varianty, které mají kombinace substitucí v polohách zbytků, jenž odpovídají polohám subtilisinu uvedených v tabulce č. 2.
-22CZ 302287 B6
Tabulka č. 2
i > | 1 | |||||||||||||||||
I Γ | ||||||||||||||||||
| | I | I I I I | ||||||||||||||||
09 | tn -’Τ | |||||||||||||||||
CM | CM | |||||||||||||||||
m | CM* | CM | CM | tn | r- | tn | CO | m | ||||||||||
XT | m | m | m | xr | tn | xr | co | T | ||||||||||
CM | CM | CM | CM | CM | CM | CM | CM | CM | ||||||||||
CM tn | 36 | 48 | tn xr | tn T | tn O- | 5 | r^. t. | 8 | m Ό· | h- tn | 8 | CM CO | 8 | |||||
CM | CM | CM | CM | CM | CM | CM | 0, | CM | CM | CM | CM | CM | CM | |||||
tn | CM | ) | l tn | tn | 36 | CD | 36 | tn | tU. | CM | l CO | tn | CM | xr | CM | |||
-*r | co | M- | XT | CO | v | I | i *? | CO | CO | ΤΓ | CO | CO | ||||||
CM | CM | CM | CM | CM | CM | CM | CM | I | ICM | CM | CM | CM | CM | CM | CM | |||
<D | co | 36 | CD | CM | CM | (M | CM | I | I !C | O | CM | co | T- | 03 | tn | |||
co | Τ’» | CO | CO | CO | CO | CM | — | n | T“ | CO | co | V“ | tn | r- | ||||
CM | CM | CM | CM | CM | CM | CM | CM | CM l | líM | CM | CM | CM | CM | r- | CM | |||
UT3 | a> | CM | CT> | XT | CM | CM | d} | 03 | O | CM | 1 ÍÍM | cn | TT | CM | 03 | í | 03 | |
XT | T | CO | m | o | CO | CO | tn | tn | n | σι | CM | ICO | tn | CM | CO | tn | O | tn |
CM | CM | CM | T- | CM | CM | T- | C4 | ICM | CM | CM | ||||||||
CM | CM | 03 | Μ | CO | cn | o> | o | XT | s | í—* | to | 3 | CD | cn | xr | CO | 3 | |
CM | CM | tn | O | O | m | tn | XT | O | O | ?5 | i-n | m | tn | O | o | |||
CM | CM | P* | r· | ’ I | 1 | -í— | r» | T- | ||||||||||
ΧΓ | s | O* | CO | CO | Ό- | XT | xr | CO | co | CO | [tr ' | <0 | xr | •V | CO | CD | G | |
o | O | O | O | O | o | o | o | o | Ic | ; 3 | o | o | O | o | o | |||
t” | T“ | T— | *“ | |||||||||||||||
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-25 CZ 302287 B6
Tyto čísla poloh aminokyselin odpovídají číslům označujícím sekvenci subtilisinu mikroorganizmu Bacillus amyloliquefaciens, která je uvedena na obrázku č. 1. Vynález se neomezuje pouze na mutaci tohoto určitého subtilisinu, ale také zahrnuje prekurzorové proteázy, které obsahují aminokyselinové zbytky v polohách, kteréjsou „ekvivalentní“ s určitými identifikovanými zbytky v subtilisinu organizmu Bacillus amyloliquefaciens. V preferovaném provedení vynálezu prekurzorová proteáza je subtilisin organizmu Bacillus lentus a substituce se provádí v ekvivalentních polohách aminokyselinových zbytků v organizmu B. lentus, které odpovídají těm uvedeným shora v textu.
Poloha zbytku (aminokyseliny) prekurzorové proteázy je ekvivalentní zbytku subtilisinu mikroorganizmu Bacillus amyloliquefaciens v případě, zeje buď homologní (to znamená, že odpovídá poloze buď v primární nebo terciární struktury) nebo analogická se specifickým zbytkem nebo částí uvedeného zbytku v subtilisinu 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 subtilisinu a zvláště se sadou zbytků, kteréjsou známy, že jsou v subti li sinech se známou sekvencí neměnné. Například obrázek č. 2 znázorňuje konzervativní zbytky subtilisinu organizmu B. amyloliquefaciens a B. lentus. Po uspořádání konzervativních zbytků, dovolují nezbytné inzerce a delece za účelem udržet uspořádání (to znamená zabránit eliminaci konzervativních zbytků pomocí doplňkové delece a inzerce), se definují zbytky ekvivalentní pro určité aminokyseliny v primární sekvenci subtilisinu organizmu
Bacillus amyloliquefaciens. Uspořádání konzervativních zbytků by mělo přednostně konzervovat 100 % takových zbytků. Avšak uspořádání vyšší než 75 % nebo 50 % konzervativních zbytků je také adekvátní pro definování ekvivalentních zbytků. Měla by se udržovat konzervativnost katalytické triády Asp32/His64/Ser221. V publikaci Siezen et al., (1991) Protein Eng. 4(7): 719-737 se popisuje uspořádání velkého množství serinových proteáz. V této publikaci se také popisuje w rozdělení subti lás nebo serinových proteáz podobných subtilisinu do jednotlivých skupin.
Například na obrázku ě. 3 jsou uspořádány aminokyselinové sekvence subtilisinu z mikroorganizmů Bacillus amyloliquefaciens, Bacillus subtilis, Bacillus lichenoformis (carlsbergensis) a Becillus lentus, přičemž se stanoví maximální množství homologie mezi 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.
Tyto konzervativní zbytky se tak mohou použít k definici odpovídajících ekvivalentních aminokyselinových zbytků subtilisinu mikroorganizmu Bacillus amyloliquefaciens v jiných subtilisi40 nech, jako je subtilisin mikroorganizmu Bacillus lentus (popisuje se v přihlášce PCT WO89/06279, uveřejněné 13. července 1989), který je preferovaným proteázovým prekurzorovým enzymem nebo subtilisin popsaný jako PB92 (popsaný v dokumentu EP 0 328 299), kteiý vykazuje vysokou homologii s preferovaným subti!ismem mikroorganizmu Bacillus lentus. Aminokyselinové sekvence některých z těchto subtilisinů jsou porovnány na obrázku č. 3A a 3B se sekvencí subtilisinu Bacillus amyloliquefaciens tak, aby se dosáhlo maximální homologie konzervativních zbytků. Jak je možné vidět, v sekvenci mikroorganizmu Bacillus lentus ve srovnání se subti li sinem Bacillus amyloliquefaciens existuje řada deleci. Tak například ekvivalentní aminokyselinou v případě Val 165 v subtilisinu Bacillus amyloliquefaciens je u jiných subtilisinů izoleucin pro B. lentus a B. licheniformis.
„Ekvivalentní zbytky“ se mohou také definovat stanovenou homologii prekurzorové proteázy na úrovni terciární struktury, přičemž terciární 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 proteá55 zy a subtilisin mikroorganizmu Bacillus amyloliquefaciens (N na N, CA na CA, C na C a O na O)
- 26CZ 302287 B6 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 nej lepší model, přičemž dojde k maximálnímu přesahu atomových koordinát atomů vodíku proteinu proteázy vůči subtilisinu Bacillus amyloliquefaciens. Nejlepším modelem je krystalografický model vykazující nejnižší faktor R v případě experimentálních difrakčních dat pri nej vyšším možném rozlišení.
R faaor
Ekvivalentní zbytky, které jsou funkčními analogy ke specifickému zbytku subtilisinu Bacillus ιο amyloliquefaciens jsou definovány jako aminokyseliny prekurzorové proteázy, které mohou přizpůsobit konformaci tak, že bud’ změní nebo se podílejí definovaným způsobem na struktuře proteinu, navázání substrátu nebo katalýze a přiřazují se ke specifickému zbytku subtilisinu mikroorganizmu Bacillus amyloliquefaciens. Jsou to ty zbytky prekurzorové proteázy (jejichž terciární struktura se získala rentgenovou krystalografií), které obsazují analogickou polohu, is ačkoli atomy hlavního řetězce daného zbytku nemohou uspokojit kriterium ekvivalence na základě obsazení homologické polohy, přičemž atomové koordináty alespoň dvou atomů vedlejšího řetězce zbytku leží 0,13 nm od odpovídajících atomů vedlejšího řetězce subtilisinu mikroorganizmu Bacillus amyloliquefaciens. Koordináty trojrozměrné struktury subtilisinu mikroorganizmu
Bacillus amyloliquefaciens jsou uvedeny v dokumentu přihláška EPOO 251 446 (ekvivalent
US patentu 5 182 204) a může se použít ke stanovení ekvivalentních zbytků na úrovni terciární struktury.
Některé z těchto zbytků určených pro substituci jsou konzervativní zbytky, zatímco ostatní nejsou konzervativní. V případě zbytků, které nejsou konzervativní, substituce jednoho nebo více aminokyselin je limitována na substituci, která produkuje variantu, která vykazuje aminokyselinovou sekvenci, která neodpovídá sekvenci zjištěné v přírodě. V případě konzervativních zbytků výsledkem takových substitucí nebude přirozeně se vyskytující sekvence. Varianty proteáz podle vynálezu zahrnují zralé formy variant proteáz, stejně jako pro-formy a prepro-formy takových variant proteáz. Prepro-formy jsou preferované konstrukce, protože umožňují expresi, sekreci variant proteáz.
Termín „pro-sekvence“ znamená sekvence aminokyselin vázaných na N-terminální ěást zralé formy proteázy, která když se odstraní vede k objevení „zralé“ formy proteázy. Řada proteolyrických enzymů se nachází v přírodě jako translační pro-enzymové produkty a v nepřítomnosti post-translačního zpracování se exprimují tímto způsobem. Preferované pro-sekvence produkující varianty proteáz jsou putativní pro-sekvence subtilisinu mikroorganizmu Bacillus amyloliquefaciens, ačkoli se mohou použít jiné proteázové pro-sekvence.
Termín „signální sekvence“ nebo „presekvence“ znamenají libovolnou sekvenci aminokyselin vázaných na N-terminální 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 subtilisinu Bacillus subtillis fúzovaného se zbytkem signální sekvence subtilisinu z mikroorganizmu Bacillus lentus (ATCC 21536).
Termín „prepro-,, forma varianty proteázy zahrnuje zralou formu proteázy obsahující prosek50 věnci operativně spojenou s aminokoncem proteáz a s pre-sekvencí nebo se signální sekvencí operativně spojenou s amino-koncem prosekvence.
-27 CZ 302287 B6
Termín „expresní 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 ribozómu, kam se váže mRNA a sekvence, které řídí ukončení transkripce a translace. Vektorem může být plazmid, fágové částice nebo jednoduše potencionální inzert v genomu. Poté, 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 specifikací termín „plazmid“ a „vektor“ jsou vnitřně zaměnitelné, přičemž plazmid je nejběžněji užívanou formou vektoru. Vynález dále popisuje také jiné formy expresních 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 (subtilisinu). Konstrukce kmene BG2036 se popisuje v US patentu 5 264 366. Jiné hostitelské buňky vhodné pro expresi proteázy zahrnují Bacillus subtilis 168 (popisuje se v publikaci US patent 34 606 a US patent 5 264 366) stejně jako vhodný kmen mikroorganizmu Bacillus, jako je B. licheniformis, B. lentus atd.
Hostitelské buňky se transformovaly nebo transfekovaly vektory konstruovanými za použití metod rekombinace DNA. Takové transformované hostitelské buňky jsou schopny replikovat vektory kódující varianty proteáz nebo exprimovat požadovanou variantu proteáz. V případě vektorů, které kódují pre- nebo prepro-formu varianty proteázy, se pak tyto varianty v typickém případě vylučují z hostitelské buňky do média hostitelské buňky.
Termín „operativně spojený“ znamená vztah mezi 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 nej pravděpodobněji zahrnuje štěpení signální sekvence. Promotor je operativně spojen s kódující sekvencí, jestliže řídí transkripci sekvence. Vazebné místo ribozómu je operativně spojeno s kódující sekvencí, jestliže je umístěno tak, že umožňuje translaci.
Geny kódující přirozeně se vyskytující prekurzorovou 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 knihoven genu hybrid i žací se sondami. Pozitivně hybrid izující klony se pak mapují a sekvenují.
Klonované proteázy se pak používají pro transformaci hostitelské buňky za účelem exprese proteázy. Gen proteázy se pak liguje do plazmidu s vysokým počtem kopií. Tento plazmid se replikuje v hostitelích ve smyslu, že obsahuje dobře známé elementy nezbytné pro plazmidovou replikaci: promotor 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 transkribované hostitelem z genu proteázy v jistých buňkách eukaryontního hostitele), která je exogenní nebo se dodává s 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, kteiý 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 re kombinaci.
-28CZ 302287 B6
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 jednoretězcových fragmentů DNA, které po hybridizaci a ligaci produkují syntetickou DNA kódující prekurzorovou proteázu. Příklad syntetické genové konstrukce se uvádí v příkladu 3 dokumentu US patent 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 prekurio zorové proteázy. Takové modifikace zahrnují produkci rekombinantních proteáz, jak se popisuje v publikaci US patent RE 34 606 a přihláška EPO 0 251 446, a produkci zde popsaných variant proteáz.
Následující způsob kazetové mutageneze se může použít pro konstrukci variant proteáz podle vynálezu, ačkoli se mohou použít i jiné metody. Nejdříve se získá přirozeně se vyskytující gen kódující proteázu a celý nebo jeho část se sekvenuje. 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 oligonukleotidy, které v případě, že se exprimu20 jí budou kódovat různé mutanty. Upřednostňuje se, aby taková restrikční místa byla jediná restrikční místa v proteázovém genu, což umožňuje nahrazení segmentu genu. Může se však použít libovolné restrikční místo, 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 primerů Ml3 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 rutinně redundancí genetického kódu pomocí map 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, která 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 restrikčními enzymy a do genu se liguje pluralita oligonukleotidových kazet s komplementárním koncem. Tímto způsobem se zjednoduší mutageneze, protože všechny oligonukleotidy se syntetizovaly 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. M. Kalisz, „Microbial Proteinases“, Advances in Biochemical Engineering/Biotechnology, A. Fiechter ed., 1988), Vedle upravené proteolytické aktivity řada enzymů podle vynálezu může mít jiné upravené vlastnosti, jako je poměr km, k<.at, kkac/Km a/nebo upravenou spe45 cifitu 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, jakoje použití při praní prádla.
Vynález dále popisuje zabezpečení varianty proteázy, která vykazuje ve srovnání s prekurzoro50 vou proteázou změněnou, přednostně zvýšenou, účinnost praní alespoň v jedné detergentové formulaci a/nebo alespoň při jedné sadě podmínek praní.
Existuje rada podmínek praní, které zahrnují různé detergentové formulace, objem prací vody, teplotu prací vody a délku praní, po kterou působí varianta proteázy. Detergentové formulace například, které se používají v různých oblastech mají různé koncentrace jejich relevantních
-29 CZ 302287 B6 komponentů, které jsou přítomny v prací vodě. Evropské detergenty například v typickém případě obsahují v prací vodě přibližně 4 500 až 5 000 ppm komponent detergentu, zatímco v Japonsku detergenty v typickém případě obsahují přibližně 667 ppm komponent detergentu v Severní Americe, zvláště pak ve Spojených státech, detergent v typickém případě obsahuje přibližně
975 ppm komponent detergentu přítomných v prací vodě.
Systém s nízkou koncentrací detergentu zahrnuje 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 detergentu, který obsahuje v prací vodě přibližně io 667 ppm detergentových komponentů.
Střední koncentrace detergentu zahrnuje detergenty, kde v prací vodě je obsaženo přibližně 800 ppm a 2000 ppm detergentových komponentů. Detergenty v Severní Americe se v obecném případě považují za systémy se střední koncentrací detergentu, které v prací vodě obsahují pří15 bližně 975 ppm detergentových komponentů. V Brazílií obsahují detergenty v typickém případě v prací vodě přibližně 1500 ppm detergentových komponentů.
Systém s vysokou koncentrací detergentu zahrnuje detergenty, kde v promývací vodě je obsaženo více jak 2000 ppm detergentových komponentů. Evropské detergenty se v obecném případě považují za systémy s vysokou koncentrací detergentu, což znamená, že v prací vodě obsahují přibližně 4500 až 5000 ppm detergentových komponentů.
Detergenty Latinské Ameriky v obecném případě obsahují používaných v Latinské Americe spadá do systému se střední nebo vysokou koncentrací detergentu, což je rozmezí mezi 1500 ppm až 6000 ppm detergentových komponentů v prací vodě. Jak se uvádí shora v textu brazilské detergenty v typickém případě obsahují v prací vodě 1500 ppm detergentových komponentů. Avšak výskyt detergentů s vysokým obsahem fosfátů není omezen na další státy Latinské Ameriky, kde se mohou používat systémy s vysokou koncentrací detergentu, které obsahují v promývací vodě až přibližně 6000 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 800 ppm k hodnotě 2000 ppm (což je střední koncentrace detergentu), například přibližně 975 ppm v US a přibližně 1500 v Brazílii až k hodnotě 2000 ppm (což je vysoká koncentrace detergentu). Příkladem je koncentrace 4500 ppm až přibližně 5000 ppm v Evropě a 6000 ppm v místech, kde se používají detergenty s vysokým obsahem fosfátů.
Koncentrace v typických pracích roztocích se stanoví empiricky. V USA v typickém případě
4o v pračce udržuje objem 64,4 I 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 odměrkou, 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 substituce v poloze 103 v kombinaci s jednou nebo více substitucí vybraných ze skupiny zahrnující polohy odpovídající 1, 3, 4, 8, 10, 12, 13, 16, 17, 18, 19, 20, 21, 22, 24, 27, 33, 37, 38, 42, 43, 48, 55, 57, 58, 61, 62, 68, 72, 75, 76, 77, 78, 79, 86, 87, 89, 97,98, 99, 101, 102, 103, 104, 106, 107, 109, 111, 114, 116, 117, 119, 121, 123, 126, 128, 130, 131, 133, 134, 137, 140, 141, 142, 146, 147, 158, 159, 160, 166, 167, 170, 173, 174,
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177, 181, 182, 183, 184, 185, 188, 192, 194, 198, 203, 204, 205, 206, 209,210,211,212,213,
214, 215, 216, 217, 218, 222, 224, 227, 228, 230, 232, 236, 237, 238, 240, 242, 243, 244, 245,
246, 247, 248, 249, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 265, 268,
269, 270, 271, 272, 274 a 275 subtilisinu mikroorganizmu Bacillus amyloliquefaciens jsou důle5 žité pro zlepšení účinnosti praní enzymu.
Tyto substituce se přednostně vytvořily v subtilisinu mikroorganizmu Bacillus amyloliquefaciens (rekombinantním nebo přirozeném), ačkoli substituce je možné provést v libovolné proteáze mikroorganizmu Bacillus.
io
Na základě výsledků testování získaných s variantami proteáz, jsou zmíněné mutace v subtilisinu v mikroorganizmu Bacillus amyloliquefaciens důležité pro proteolytickou aktivitu, účinnost a/nebo stabilitu těchto enzymů a účinnosti čištění nebo praní za použití takových variant enzymů.
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 vhodné povrchově aktivní Činidla, která se používají v kompozicích obsahujících mutanty 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
2u 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 čisticí kompozice. Vedle typických čisticích 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 pří aplikacích kusového nebo kapalného mýdla, při aplikací prostředků na nádo25 bí, čisticích 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 kompozice (ve srovnání s prekurzorem). Zvýšená účinnost detergentu se definuje jako zesílení schopnosti čistit skvrny citlivé vůči jistému enzymu, jako jsou skvrny od trávy a krve, jak se definuje obvyklým hodnocením po standardním cyklu praní.
Proteázy podle vynálezu se mohou vnést do práškových nebo kapalných detergentů, jejichž pH je mezi 6,5 a 12,0 s procentovým zastoupením přibližně 0,01 až 5% hmotn, (upřednostňuje se 0,1 % až 0,5 %). Tyto detergentové čisticí kompozice mohou také zahrnovat jiné enzymy, jako jsou proteázy, amylázy, celulázy, lipázy nebo endoglykolázy stejně jako plnidla nebo stabilizátory.
Vedle proteáz podle vynálezu neexistuje žádné specifické využití běžných čisticích prostředků. Jinými slovy libovolná teplota a pH vhodné pro detergent je také vhodné pro uvedenou kompo40 zici, pokud pH je ve shora uvedeném rozmezí a teplota je nižší než teplota denaturující proteázy. Proteázy podle vynálezu se mohou použít jako čisticí kompozice bez detergentů buď samotné nebo v kombinaci s plnidly a stabilizátory.
Vynález také popisuje čisticí kompozice obsahující proteázové varianty podle vynálezu. Čisticí kompozice mohou dále obsahovat aditiva, která se v těchto čisticích 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 inkluži 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 ta aditiva, která jsou kom50 patibilní s enzymy a jinými komponenty v kompozici, jako je například povrchově aktivní činidlo.
Množství aditiva, které je přítomno v čisticí kompozici je přibližně 0,01 % až 99,9 %, upřednostňuje se přibližně 1 % až 95 %, více se upřednostňuje 1 % až 80 %.
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Varianty proteáz podle vynálezu se mohou přidat do zvířecího krmení, jak se popisuje v dokumentu US 5 612 055, US 5 3 14 692 a US 5 147 642.
Dále vynález popisuje kompozici pro ošetření textilií, která zahrnuje varianty proteáz podle vyná5 lezu. Kompozice se může použít při ošetření například hedvábí nebo vlny, jak se popisuje v dokumentech RD 216 034, EP 134 267, US 4 533 359 a EP 344 259.
Přehled obrázků na výkresech io
Na obrázku č. 1 A až C je znázorněna sekvence DNA a aminokyselinová sekvence subtilisinu mikroorganizmu Bacittus amyloliquefaciens a částečná restrikční mapa tohoto genu.
Na obrázku č. 2 jsou znázorněny konzervativní aminokyselinové zbytky subtilisinu mikroorga15 nizmů Bacittus amyloliquefaciens (BPN)' a Bacittus lentus (divoký typ).
Na obrázcích 3A až 3B je znázorněna aminokyselinová sekvence čtyř subtilisinu. Horní řádek reprezentuje aminokyselinovou sekvenci subtilisinu z mikroorganizmu Bacittus amyloliquefaciens (také se někdy nazývá jako subtilisin BPN'). Druhý řádek znázorňuje aminokyselinovou io sekvenci subtilisinu z mikroorganizmu Bacittus subtilis. Třetí řádek znázorňuje aminokyselinovou sekvenci subtilisinu z mikroorganizmu B. licheniformis. Čtvrtý řádek znázorňuje aminokyselinovou sekvenci subtilisinu z mikroorganizmu Bacittus lentus (který se v dokumentu PCT
WO98/06276 nazývá subtilisin 309). Symbol * znázorňuje nepřítomnost specifických aminokyselinových zbytků ve srovnání se substilisinem BPN'.
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 subtilisinu organizmu Bacittus lentus GG36. Varianty jsou uvedeny v tabulce č. 3
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Tabulka č. 3
00 Ol 2 | o co xr Ol 2 | > xr r» < | ||||||||||||||
M222S | V104I | ! V104I i | I107V | V104I | I246V ; __/ | V1041 | Q cn co 2 | v o > | 3 > | Q 5 Ol 2 | S160T | S216C | V104I | xr o > | O > | xr O > |
V104I | S103A | S103A | XT O > | S103A | xr O X“ > | S103A | O > | S103A | S O ω | xr o > | xr O X“ > | V1Q4I | S103A | < cn o x- ω | s o T“ trt | S103A |
< cn O ω | A98E | S78T | S103A | N76D | S103A | N77D | S103A | Q CD 2 | N76D | s o ω | S103A | S103A | Q ε 2 | § o. Z | O r- r-' 2 | O to o· 2 |
IŇ76D | O co r*- Z | Q ε 2 | O tO o- 2 | LLl 5 | O ε 2 | Q co n- 2 | Q9ZN | A16T | tu x— < | Q to o. 2 | Q to Ο» 2 | Q ε 2 | l H17Q | S37T I | O to o- 2 | ω co cn I- |
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K237Q | O © 03 T- z | T274A | S240T | |||||||||||||||
Ν' α > | 3 r- > | N183D | N O > | Ν- Ο T“ > | Ν- Ο > | □ Ν’ CO t— Z | N252D - -/_ | S259C | t— -r- © 2 | 3 > | 5 > | K237E | S160L | A228V | Ν' O > | A254T | H s CN Z | N204D |
S103A | S103A | ( V104I | S103A | S103A | S103A | Ň O > | V104I | V104I | V104I | S103A | s o *· cn | V104I | V104I | V104I | S103A | s > | z Ν- Ο | Ν' o > |
N76D | N76D | S103A I | N76D | N76D | N76D í _i | S103A| | S103A | S103A | S103A | w g 0. | Q 8 z | S103A | [S103A | S103A | Q © r— Z | S103AJ | S103A | S103A |
cn 8 1- | Š | o ÍO r- Z | R19L | > co < | O Φ T* ir | O co F'» z | | N76D | | N76D | 1 N76D | N76D | > CN | O to h* Z | N76D | N76D | P55S | N76D | O © r- Z | N76D |
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I | ||||||||||||||||||
K251R | ||||||||||||||||||
< 1— r- > | Q236R | ni 55 2 | N204T | E271V | N261Y | |||||||||||||
•ς- σ > | Ω Ol m δ | V1041 | T O > | A270V | Ω LO 00 z | 3 > | S CM SP CM -J | TT e > | V104J | S166G | o > | S130L | Q109R | 3 > | D161N | 3 > | S212P | X CM m CM Z |
S103A | mF o > | S103A | S103A | V1041 | o r— > | S103A | V104I | S103A | S103A | V104I | ω | V104I | 3 > | < cn o δ | V1041 | S103A | o »— > | o > |
Ω 8 z | S103A | N76D | O to t— z | S103A | S103A | N76D | S103A | S78P | Ω S Z | S103A | Ω CO r— Z | S103A | S103A | S99R | S103A | Q9ZN | S103A | S103A |
co cn •*r Z | Q CO r- Z | R10H | T58S | N76D | Ω CO F— z | K27N | Ω CO Γ- z | Q9ZN | cl τ CM co | N76D | -J | Ω CO r— Z | a CO Γ- Z | N76D | Ω CO r- Z | Z CM δ | N76D ! | Ω 8 z |
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S265G | ||||||||||||||||||
Ν1Θ3Ι ! | Y263H | H249Q | > P- CM UJ | |||||||||||||||
S242T | N116K 1 | Q182R | A272S | I246V | ce S CM σ | > 00 co CM 2 | > oo 05 T— | | Q182R | Q137R | CO 03 CM 2 | Q206R | |||||||
S242T | E271Q | xr o V > | V104I | G258R | E271G | xr σ ·»“ > | s. > | x CM <2 δ | Q109R | v o X— > | ce P- co σ | X CM 05 δ | V104I | O> 2 | Q137R | V104I | X s CM σ | |
V104I | s > | < CO O T“ ω | S103A | xr O > | χτ o > | S103A | S103A | X? o > | V104I | S.103A | Μ1 O T— > | l·- CD CM CM < | O > | S103A | o T- > | XT o > | S103A | xr O X- > |
S103A | S103A | N76D | O CO r- 2 | S103A | Š103A | Q co P- 2 | Q CO p— 2 | S103A | < co o 5> | O P- 00 co | < CO o ω | x? O > | S103A | N76D | S103A | S103A | N76D | S103A |
O CO p- Z | o CO P- 2 | X CM δ | CO co XT 2 | O tO P— Z | Q to p- 2 | G61R | T38S | N76D | Q co P— 2 | N76D | o co P- 2 | S103A | Q co p- 2 | 2 CM | N76D | O co Ρ» 2 | | A13T | □ CO P- 2 |
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σ ιΟ CM ií | N252D | K251T | ||||||||||||||||
LU Γ- CM _l | LU 5 | o m CO Z | < XT xr CM > | G159O | Q236H | 0 CT) tO δ | ||||||||||||
(Τ <Ό ΙΟ CM Ο | E271G | N261D | U1 S CM α | Q206E | 0 σ> to 5 | G159D i | A133T | Q206E | S188E | i A158E i | N185D | K251T | 5 X“ Zj | G159D | G159D | O T- > | ||
S212P | χτ ο > | Q206E | Š > | χτ ο > | Α158Ε | Q206E | x? o > | xr > | V104I | □ 05 tn δ | v o > | V104I | xr o > | Q206E | 3 > | xr o T- > | v o X— > | s o τ- ο) |
ο > | S103A | χτ ο τ— > | S103A | S103A | ΧΓ ο χ— > | V104I | s o ω | S103A | S103A | V104I | S103A | S103A | S103A | V104I | S103A | S103A | S103A | 0 8 z |
S103A | Ο CD ζ | S103A | Ο 8 ζ | Ο r>- Ν. Ζ | S103A | S103A | o CD r*- Z | 0 CD f*- Z | o r-- r*. Z | S103A | 0 CD Γ*· Z | 0 8 z | 0 b- řx. Z | S103A | 0 CD r- Z | N76D | N76D | CM CO Z |
Ο 8 ζ | T58S | N76D | ULI χτ > | N76D | N76D | ο CD Γ— Ζ | ω XT > | LU XT > | O CD r*- Z | □ (O r- Z | tu xr > | LU xr > | 0 8 z | 0 CD r- Z | l·— CO xr < | š § | > CM xr -J | X CM r- σ |
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> | ||||||||||||||||||
p- | ||||||||||||||||||
CM | ||||||||||||||||||
UJ | ||||||||||||||||||
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r- | r- | xr | co | |||||||||||||||
CM | CM | CM | CM | CM | ||||||||||||||
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r- | r- | p* | XT | |||||||||||||||
CM | CM | CM | CM | CM | CM | CM | CM | |||||||||||
LU | UJ | UJ | CO | 2 | σ | σ | _J | |||||||||||
O | Q | co i | < | U_ | X | _1 | 2 | _1 | X | —1 | _ | X | a | a | O | |||
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tn | m | co | CM | CO | M* | xr | co | X7 | O | CO | tn | tn | tn | O | ||||
Ί- | CM | CM | CM | CM | CM | x— | CM | CM | CM | T“ | CM | Τ- | ν- | Τ- | T“ | |||
o | Ο | 2 | > | co | σ | co | σ | σ | X | > | σ | Ο | α | Ο | > | |||
ω | Q | a | Q_ | X | co | x | X | X | < | < | ||||||||
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CM | v* | CM | CM | X— | X- | T“ | X— | |||||||||||
> | 5 | o | o | CO | > | > | CO | H | CO | 3 | σ | w | > | > | > | > | ω | > |
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O CO | < CO σ | < CO O | < cn o | < co o | < o CD | X r~ CD | < CO o T* | 103A | 103A | 103Á1 | < co o Μ» | CO CM CO | a CO Γ-. | a co P- | O co P- | □ co p- | < 00 co | □ £ |
2 | ω | ω | co | to | LU | CO | co | co | to | to | co | 2 | 2 | 2 | 2 | 2 | > | 2 |
o | o | o | a | Q | O | o | O | o | Q | Q | > | < | < | < | < | a | < | |
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M | Γ-- | r- | r- | r*. | D* | h- | P- | p- | r- | r-. | CM | CO | co | CO | co | |||
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-38CZ 302287 B6
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CN | CN | CN | CN | |||||||||||||||
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8 | 03 | co | CO | Ν* | CO | 0) | co | |||||||||||
tO | co | co | CO | Ν’ | co | XT | co | |||||||||||
CN | CN | CN | CN | CN | CN | CN | CN | |||||||||||
σ | o | σ | a | z | Z | σ | σ | X | I | I | > | |||||||
X | o | __ | Q | CO | a | X | X | O | o | X | X | X | Q | |||||
co | 03 | 03 | 03 | 03 | co | CO | 03 | 03 | (3 | Ol | Γ- | |||||||
co | tn | CM | m | O | tn | co | co | tn | tn | CO | Ν’ | η | f'- | |||||
CN | CN | ζϋ | CN | Τ- | CN | CN | CN | CN | ||||||||||
σ | δ | > | o | > | O | σ | σ | Ο | o | σ | X | Lil | ||||||
Q | > | Q | X | o | o | > | co | o | X | co | co | LL | co | co | CO | CO | ||
σ> | 3 | ΤΓ | 03 | h* | σ> | 03 | CN | co | 03 | m | CN | <N | CO | CN | CN | CN | CN | |
tn | T- | w | m | T“ | co | tn | 'T | CN | tn | NT | CN | CN | CO | CN | CN | CN | CN | |
r-’ | T— | T-** | T* | τ- | T- | T* | CN | CN | CN | CN | CN | CN | CN | CN | ||||
δ | > | < | > | o | z | Ο | o | < | Z | o | σ | 2 | 2 | >- | 2 | 2 | 2 | 2 |
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s | tn | co r- | o CO | cO N- | CO N- | £ | CO Γ | £ | CO r* | (0 r- | o | co h- | co r- | |||||
Z | Lj | Z | > | Z | Z | z | z | z | Z | Z | δ | δ | δ | δ | z | δ | δ | Z |
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CD | CO | co | co | cp | cp | CO | CD | CO | co | Γ | r*. | fx. | t*- | CN | r- | r- | CD | |
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co | T | ο- | T | co | m- | co | co | © | co | |||||||||
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ΤΓ | ’ΤΓ | α | co | co | © | CO | CO | T | τ | |||||||||
CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | ÍM | ||||||
σ | O | σ | Z | σ | Z | σ | > | < | σ | < | σ | O | ||||||
ω | X | Q | X | > | Q | Q | > | X | Q | -r- | X | X | CD | |||||
QD | CO | o | CD | Ο- | •*r | Oi- | CM | ΤΓ | © | cn | š | <o | M | © | CM | |||
*T | co | © | CO | Ι— | © | © | CO | a | -T | © | M | T | CM | |||||
CM | CM | CM | CM | t*» | CM | T— | CM | CM | CM | CM | CM | CM | ||||||
Z | σ | o | σ | < | Z | o | < | < | σ | 0 | σ | I— | > | σ | 2 | |||
cd | cd | ce | O | z | a | Q | o | > | O | Q | CD | X | _ | > | Q | CD | Ι- | Ι- |
CM | CM | cn | cn | © | cn | CT> | co | cn | cn | CM | © | ΤΓ | CM | cn | CM | Ο | ο | |
CM | CM | v | © | © | © | © | CO | © | © | CM | o· | O | CO | © | CM | co | ||
CM | CM | CM | T-“ | Τ» | ι- | ^r· | V“ | CM | CM | T“ | CM | r- | CM | CM | ||||
5 | 2 | X | o | ω | ϋ | 0 | o | < | o | o | 5 | a | > | < | 0 | s • | X | CD |
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V | < CO | < co | s | < co | < co | < co | < (O | s | T | Q | -T | < CO | < co | < co | 5 | |||
o | o | o | O | o | o | o | a | © | o | o | o | o | © | o | O | o | o | O |
r“ | v“ | T“ | t— | f— | ||||||||||||||
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< co | < co /“i | < co | o | Q | □ | O | □ | a | O | □ | a | < | < | < | Q | O | □ | o |
(0 | co | (0 | CD | CO | © | © | © | © | n λ | © | o | © | © | © | © | |||
r- | f— | Γ- | f— | 1— | 1— | i— | r— | r— | o | © | t-7 | 1— | r— | <— | 1— | |||
ω | CD | ω | Z | z | Z | Z | z | Z | Z | Z | z | CD | > | CO | Z | Z | z | Z |
□ | a | a | < | < | < | < | < | < | < | < | X | Q | H | < | < | X | X | X |
co | © | CO | ao | © | co | CD | oo | ao | © | CD | CM | © | ’Τ | § | oo | CM | CM | CM |
1— | i— | Γ— | CO | © | £ | £ | co | © | © | © | r- | CM | © | Τ- | ||||
z | Z | z | > | > | > | > | > | > | > | > | σ | Z | CD | > | > | δ | Ο | δ |
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04 | Ol | 04 | Ol | OJ | 04 | OJ | 04 | Τ” | O) | |||||||||
U3 | m | tn | tn | tn | co | co | CD | m | -T | |||||||||
04 | OJ | 04 | 04 | 04 | OJ | Ol | Ol | OJ | 04 | |||||||||
2 | 2 | Z | Z | Z | _l | 2 | ||||||||||||
Q | X | X | X | X | x | tn | X | X | X | Q | X | X | CO | < | ||||
co | tn | m | tn | tn | m | 03 | tn | m | m | tn | m | 04 | Φ | |||||
-T | ΤΓ | ΤΓ | -r | ΤΓ | ΤΓ | T | ΤΓ | ΤΓ | CO | -T | ΤΓ | 04 | CO | |||||
04 | OJ | OJ | 04 | 04 | 04 | 04 | 04 | OJ | OJ | OJ | OJ | 04 | 04 | OJ | ||||
2 | O | O | O | O | σ | 2 | σ | σ | σ | 2 | σ | σ | 2 | > | ||||
X | X | X | X | X | X | X | X | tn | < | co | X | co | co | Q | X | |||
in | tn | co | co | to | co | 8 | tn | Ol | 0- | Oi | tn | OJ | OJ | tn | in | |||
ΤΓ | ΤΓ | 03 | 03 | 03 | 03 | -T | OJ | 04 | OJ | T | Ol | 04 | co | v | ||||
04 | 04 | OJ | OJ | OJ | 04 | 04 | Ol | OJ | OJ | OJ | Oí | OJ | 04 | T— | 04 | |||
a | a | σ | σ | σ | σ | σ | a | 2 | > | 2 | σ | 2 | 2 | 2 | σ | |||
X | X | > | > | > | > | > | tn | > | co | )- | co | X | Q | Ι- | CO | CO | ||
to | to | CM | 04 | Ol | 04 | OJ | OJ | tn | 04 | tn | OJ | U3 | co | Ο | O | 04 | ||
03 | 03 | 03 | 03 | 03 | 03 | 03 | OJ | OJ | OJ | ’Γ | 03 | r-. | OJ | |||||
OJ | 04 | 04 | OJ | OJ | OJ | OJ | OJ | CM | OJ | 04 | 04 | 04 | OJ | 04 | ||||
a | a | < | < | < | < | < | 2 | < | 2 | < | 2 | σ | 2 | CO | X | 2 | ||
> | > | Q | Q | a | Q | Q | >- | ί- | H | ί- | Ι- | co | Ι- | Ι- | h- | |||
•'T | OJ | OJ | o | Ol | 03 | 03 | 03 | o | ο | o | ο | ο | OJ | Ο | Ο | O | ||
o | 03 | 03 | tn | tn | tn | tn | m | 03 | 03 | 03 | 03 | 03 | OJ | 03 | Ví Q | 03 | 03 | |
τ- | 04 | 04 | 1-· | r— | 04 | 'R· | ||||||||||||
> | < | < | o | o | g | G | G | tn | co | co | tň | « | 2 | tň | — | co | CO | |
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ω | 2 | δ | δ | 2 | > | > | >Ί | > | V“ | X | i— | tň | tň | X | *— | |||
α (0 Ο- ζ | | S103A | ír O > | O > | ΤΓ O > | S103A | S103A | < 03 O V* co | S103A | S103A | S103A | >S1O3A | S103A | S103A | I104T | S103A| | I N76D | S103A j | S103A |
< | Ο | < 03 | < 03 | <'' 03 O | < | < | < | G | Q | Q | Q | :Q | Q | < | Q | X | O | Q |
οο | to | co | co | co | o- | co | CO | CO | tn | co | <9 1 | CD | r-- | co | CD | |||
CO | ο- | <p | co | to | co | h-. | o- | o· | t*-. | o. | . O | m | 0* | r-- | ||||
> | Z | ω | ω | co | > | > | > | co | 2 | Z | 2 | Z. | 2 | tň | 2 | CO | 2 | 2 |
< | < | < | < | co | o | < | az | X | X | X | X | Q | X | X | X | X | ||
04 | 03 | co | co | oo | n | 03 | 03 | <33 | 04 | OJ | OJ | Ol | Ol | co | OJ | OJ | OJ | Ol |
CM | CO | to | to | to | ΤΓ | ’Γ | -T | tp | v· | T* | f** | T·- | ||||||
1— | > | > | > | > | 2 | 2 | 2 | > | σ | σ | σ | 5 | σ | 2 | 5 | σ | σ | a |
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X | X | X | > | |||||||||||||||
tn | tn | n | f— | |||||||||||||||
xr | xr | xr | tn | |||||||||||||||
CM | CM | CM | CM | |||||||||||||||
σ | σ | σ | _J | |||||||||||||||
x | > | x | Q | X | X | X | X | X | X | X | X | X | X | cn | X | X | X | |
tn | Γ- | tn | oo | tn | m | tn | s | tn | co | cn | tn | cn | tn | 00 | tn | co | to | |
xr | ΙΟ | v | T | M- | TT | ΤΓ | xr | co | xr | xr | xr | xr | xr | •x* | co | xr | ||
CM | CM | CM | ÍM | CM | CM | ÍM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | |
a | _1 | a | Z | a | σ | σ | σ | σ | σ | σ | O | σ | σ | Z | σ | σ | σ | |
co | x | X | X | — | X | tu | > | X | > | X | X | X | X | X | X | > | X | > |
o | tn | co | 1Λ | se | co | f- | CM | se | CM | co | co | CO | co | tn | CD | CM | co | r— |
’τ | co | -T | co | co | co | CO | co CM | CO | co | CO | cO | co | xr | CO | CO | co | tn | |
CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | |
X | a | σ | O | σ | O | < | σ | < | σ | σ | σ | σ | σ | σ | < | σ | -j | |
cn | ^- | > | X | > | > | Q | > | _! | > | > | > | > | X | > | X | > | X | |
CM | CD | CM | co | ÍM | CM | CO | Ol | CM | CO | CM | CM | CM | CM | co | ÍM | O | fM | tn |
CM | CO | CO | co | CO | CO | co | tn | CO | O | CO | CO | CO | CO | CO | CO | CO | <r | |
CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | |
s | σ | < | σ | < | < | σ | o | < | σ | < | < | < | < | σ | < | X | < | σ |
Ι- | > | O | > | C | LU | > | š | Q | > | O | h- | Q | > | Q | Q | Q | X | |
Ο | CM | oi | CM | o | CO | CM | co | Μ- | 00 | O | Ol | tn | CM | σι | Ol | o> | CO | |
ÍO | CO | tn | CO | tn | O | CO | 00 | r— | 00 | <0 | tn | CO | m | n | tn | co | ||
T— | CM | τ- | CM | Τ- | CM | CM | v* | V“ | CM | Τ- | CM | CM | Τ- | CM | ||||
CO | < | Ο | < | Ο | > | < | z | < | ω | < | Ο | < | < | Ο | o | o | σ | |
Q | Q | Q | □ | Q | < | o | O | Q | Q | Q | Q | _ | > | |||||
Ol | co | Ol | X | O) | 01 | to | 01 | O | 01 | xr | σι | 01 | 3 | xr | XT | CM | ||
X» O | to | tn | o | tn | tn | o | *O | tn | tn | m | o | tn | tn | O | o | CO | ||
T— | T** | “Ι- | τ- | T“ | T“ | ÍM | ||||||||||||
o | Z | o | > | o | Ο | cn | O | o | o | Ο | > | δ | δ | > | > | > | < | |
S103A | > | V o > | v o Τ'* > | S103A | v o > | v o > | z Ol | 3 Τ' > | v o > | v o > | o > | S103A | o > | xr o > | S103A | < o T* ω | S103A l | x? o T“ > |
Q £ Z | š o f- cn | S103A | S103A | < 00 co > | S103A | < co o cn | Q co r- Z | S103A | S103A | S103A | < co o r· tn | 1- 00 Ol < | S103A | S103A | O £ z | Q cO (— Z | Q CD r— Z | < CO o ω |
X | < | < | < | O | < | < | < | < | < | < | < | á | < | < | š | < | < | □ |
CM | 00 | 00 | co | o | co co | oo | 00 <p | 00 co | co | co | co | 00 | 00 | co | co | CO | ||
CO | co | tp | CD | co | <0 | CD | co | co | co | co | co | CD | f— | |||||
a | > | > | > | X | > | > | > | > | > | > 1 | > | > | > | > | > | > | > | Z |
-42 CZ 302287 B6
X | o | > | X | |||||||||||||||
to | 05 | o | to | |||||||||||||||
xr | to | to | xr | |||||||||||||||
CN | CN | CN | CN | |||||||||||||||
σ | CO | h- | σ | |||||||||||||||
χ | > | X | X | X | X | X | X | X | X | O | § | X | X | |||||
to | UD | to | to- | 8 | to | tO | to | tO | T“ | to | 8 | |||||||
to | Ν' | xr | xr | T | xr | xr | xr | co | s CM | XT | ||||||||
CM | 3 | ÍN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | ||||||
X | σ | σ | σ | σ | σ | σ | a | σ | Z | z | σ | σ | ||||||
> | X | > | z | X | X | > | X | X | 32 | x- | X | X | X | > | X | |||
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CM | CN | CN | CN | cn | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | |||
σ | _l | σ | o | σ | < | σ | σ | σ | σ | σ | σ | σ | < | σ | ||||
x | X | X | > | > | > | —1 | > | > | > | > | X | X | X | > | a | X | ||
to | 8 | to | CN | CN | CN | XT | CN | CN | CN | CN | co | 8 | to | CN | 05 | to | ||
XT | XT | CO | CO | CO | CO | CO | CO | CO | CO | xr | CO | to | co | |||||
CN | CN | CN | CN | cn | 5 | Sf | CN | CN | CN | CN | CN | CN | CN | CN | T“ | CN | ||
a | O | o | < | < | < | < | < | < | O | σ | σ | < | o | σ | ||||
X | > | X | X | > | o | X | a | O | O | > | > | X | -J | X | > | |||
to | CN | co | 05 | 05 | to | 05 | 05 | 05 | CN | CN | CN | O | to | CN | ||||
CO | co | co | o | to | r— | to | to | to | CO | CO | XT | o | XT | CO | ||||
ÍN | CN | CN | CN | CN | CN | CN | Τ- | Τ- | CN | CN | CN | CN | CN | CN | ||||
σ | < | a | > | O | O | o | < | O | Ο | Ο | < | < | ω | X | > | σ | < | |
> | X | < | > | O | Q | a | O | O | O | X | a | < | X | IX | ||||
CN CO | to N. | XT CN | CN CO | 05 to | g» to | 05 CO | s | <2 to | s | 3 | ΤΓ o | 05 to | 05 tO | to co | 05 to | co o | 8 | CN 05 |
CN | CN | CN | CN | V* | T“ | CN | Τ- | CN | T | |||||||||
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ω | w | ω | ω | Z | z | z | > | z | > | > | (Λ | z | z | ω | Z | < | ω | ω |
< | Q | < | Q | < | < | s | X | < | X | X | < | < | < | Q | < | X | o | o |
co | co | 00 | CO | <D | 8 | CN | CN | o | co | 03 | 03 | co | 8 | CN | <0 | to | ||
co | b* | co | b* | CO | SP | CO | Τ- | CN | CO | to | to | r- | X“ | b* | b- | |||
> | z | > | Z | > | > | > | σ | > | Ο | Q | > | > | > | Z | > | σ | Z | z |
X | X. | |||||||||||||||||
LO | CM | |||||||||||||||||
XT | LO | |||||||||||||||||
CM | CM | |||||||||||||||||
σ | X | |||||||||||||||||
x | 0 | X | X | ¥ | Q | ifí | a | |||||||||||
CM | to | 8 | CM | CM | CM | CO | CM | CM | CM | CM | CM | CM | ||||||
0 | Γ'- | XT | 40 | 40 | 40 | 'T | 0 | 0 | LO | 0 | 0 | 0 | 0 | |||||
2 | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | ||||
< | σ | a | X | X | X 1 | X | X | X | X | X | X | X | ||||||
0 | x | X | X | X | X | > | Q | a | a | X | Q | o | □ | o | v | a | o | |
OD | 0 | co | co | 40 | 0 | CM | co | co | co | 40 | CO | co | 03 | co | CM | 0 | 0 | |
xr | XT | co | m | χτ | xr | CO | xr | XT | xr | T | XT | xr | χτ | ΤΓ | to | M | XT | |
CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | |
X | a | σ | 0 | σ | σ | < | X | X | X | σ | X | X | X | X | X | X | X | |
X | X | > | X | X | X | ω | X | X | X | -ι- | vx | X | X | X | X | Q | X | X |
0 | co | CM | in | co | co | 40 | 40 | LO | 40 | ΙΟ | CM | LO | 40 | 0 | 0 | co | 0 | 0 |
XT | CO | CO | XT | co | co | CO | XT | XT | ’Τ | CO | LO | xr | XT | XJ“ | χτ | χτ | TT | XT |
CM | CM | CM | CM | CM | CM | ir* | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | ÍM | CM |
σ | O | < | σ | σ | σ | X | σ | a | σ | a | X | O | a | a | a | X | σ | σ |
X | > | X | > | > | ω | X | X | X | > | Q | X | X | X | -r | X | X | X | |
8 | CM CO | 8 | co co | CM CO | CM CO | o r- | co co | 8 | co <o | 32 | 0 -T | CO CO | co | 0 CO | 0 CO | 0 xr | 0 co | 8 |
CM | CM | CM | CM | CM | CM | CM | CM | ÍM | ÍM | CM | CM | CM | CM | CM | CM | CM | CM | |
a | < | σ | σ | < | < | X | σ | σ | σ | < | X | σ | O | a | O | a | σ | σ |
> | Q | > | X | Q | o | > | > | > | a | X | > | > | > | > | X | > | > | |
CM | os | co | CM | š | as | as | CM | CM | CM | XT | LO | CM | CM | CM | CM | 0 | CM | CM |
co | LO | co | CO | m | LO | CO | CO | CO | 03 | v | CO | cO | CO | CO | CO | CO | CO | |
CM | <T“ | CM | CM | CM | CM | CM | T— | CM | CM | CM | CM | CM | CM | CM | CM | |||
< | o | X | < | σ i | |o | 0 | < | < | < | X | a | < | < | < | < | σ | < | < |
Q | Q | Q | o | ί- | Q | a | X | a | — | $ | O | Q | LL | > | Q | X | ||
CD | os | O) | os | ''T | α | as | as | CM | os | (0 | O o | cs | 05 | as | CM | 0 | o | |
LO | o | LO | 0 | 0 | u | 40 | LO | 40 | co | 0 | 0 | o | CO | 0 | ||||
Ύ— | T“ | Γ* | T— | v- | CM | T“ | CM- | CN > | CM | CM | CM | |||||||
5 | > | o | 0 | 0 | > | X | 0 | 0 | 0 | 0 | σ | 0 | 0 | > | < | Z | X | |
V147I | S103A | V104I | o > | o r- > | < co O -r· 0 | o > | > | V104I | G159D | N66S | A232V | G159D | Q109R | v o i— > | G159D | G159D | Q as 0 t— 0 | G159D |
XT o | Q CO | < co o | < co o | < CO O | Q CO | < co o | < CO o | 03A | M- O | o | a os LO | XT o | XT O | < CO o | 5 | Ό- O | xr o | v o |
v— | r*·» | P*· | T— | |||||||||||||||
> | X | w | δ | 0 | X | 0 | 0 | 0 | > | > | 0 | > | > | 0 | > | > | > | > |
3 O | < co co | a co r- | O co r- | O co f'- | < co co | a co N. | < CO co | < 00 co | I03A | 03 A | xf O | 3 O | < co O | < CD (Q | 3 O | < co o | < co o | < CO O |
tň | > | X | X | X | > | X | > | > | 0 | 0 | > | 0 | 0 | > | 0 | T“ 0 | 0 | 0 |
Q | X | < | < | < | X | < | UJ | a | < | < | < co | < | < | X | < | < | < | < |
co | CM | co | 03 | co | r-- | co | co | CO | CO | co | CO | o | 0 | CO | 0 | 0 | ||
r- | co | CO | 0 | CM | 0 | co | XT | co | CD | o | CD | CO | CM | 0 | 0 | 0 | 0 | |
X | δ | > | > | > | > | 0 | X | > | > | 0 | > | 5 | 0 | > | > | > | > |
44CZ 302287 B6
CM | ||||||||||||||||||
m CM | ||||||||||||||||||
2 | ||||||||||||||||||
iíí | Q | íjS | X | X | hS | X | hí | X | 3sS | iS | X, | |||||||
CN | CM | 00 | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | |
un | tn | y | tn | tn | tn | tn | tn | tn | tn | tn | m | tn | tn | tn | tn | tn | tn | |
CM | 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 | 2 | Z | Z | Z | Z | Z | Z | Z | |
Q | O | OS | Q | Q | O | Q | O | X. | Q | a | a | □ | O | O | a | o | Q | O |
00 | 05 | m | co | co | 00 | 00 | 00 | CM | 00 | OD | oo | 00 | ao | oo | ao | 00 | ao | 00 |
y | O | y | y | y | y | y | tn | Y | Y | T | y | Y | Y | Y | y | y | y | |
CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM |
Z | Z | a | Z | Z | Z | Z | Z | Z | Z | Z | Z | Z | Z | Z | z | Z | Z | Z |
OS | QC | X | OS | os | os | OS | OS | Q | as | os | OS | os | as | □s | OS | as | as | as |
m | tn | co | m | tn | tn | tn | tn | co | tn | n | tn | m | tn | m | tn | tn | tn | tn |
y | y | CO | y | y | T | y | y | v | y | Y | Y | Y | Y | Y | γ | y | y | y |
CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM |
σ | σ | a | σ | σ | σ | σ | σ | Z | σ | σ | σ | O | σ | a | σ | σ | O | σ |
X | X | > | X | X | X | X | X | OS | X | X | y | X | y | X | X | X | X | X |
CD CO | 8 | CM CO | co co | 8 | CD eo | to co | co co | tn Y | 36 | to co | n co | 8 | 8 | co co | 8 | (0 co | co co | co co |
CM | CM | CM | CM | CM | CM | CM | ÍM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM |
a | a | < | σ | σ | σ | σ | σ | σ | σ | σ | O | σ | σ | σ | O | σ | σ | σ |
> | > | ~J | > | > | > | > | > | X | > | > | > | > | > | > | > | > | > | > |
CM | CM | O | CM | CM | CM | CM | CM | co | CM | CM | CM | CM | (M | CM | CM | CM | CM | CM |
(O | CO | CO | CO | CO | CO | CO | co | CO | CO | CO | CO | CO | CO | <O | CO | CO | CO | |
CM | CM | CM | CM | CM | CM | CM | CM | CM | ÍM | CM | CM | CM | CM | CM | CM | CM | CM | CM |
< | < | CL | < | < | < | < | < | σ | < | < | < | < | < | < | < | < | < | < |
Ι- | 0 | o | < | 0 | UJ | LU | > | LU | 2S | 1 0 | > | OS | Η | > | O | Q | Q | |
Ο | o | tn | O | CM | CM | CM | CO | CM | eo | co | co | tn | tn | CO | co | co | 05 | co |
V* | OO | 5— | T- | T— | Τ'* | 7“ | CO | r- | T- | T- | 7— | 7“ | y— | T- | tn | r- | ||
CM | ÍM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CN | CM | T· | ||
CL | CL | Z | CL | 0 | 0 | ω | 1— | < | l- | t- | l·- | < | < | CO | co | 0 | 0 | Z |
Q : | Q | o | Q | Q | □ | Q | Q | ω | O | a | Q | O | Q | Q | Q | Q | O | |
05 | 05 | 05 | 05 | 05 | 05 | 05 | 05 | CO | 05 | a | 05 | <35 | 05 | <35 | 05 | 05 | 05 | |
tn | tn | tn | tn | tn | m | tn | tns | tn | m | tn | tn | tn | tn | tn | tn | u | m | |
τ- | τ— | γ·» | y™ | Ύ* | <N | Y*· | γ» | γ»- | *ť“ | |||||||||
0 | O | o | o | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | > | 0 | |
y o | Μ O | y o | v o | y O | y o | y O | Y O | s | 3 | y C | Y O | 3 | Y O | y o | y O | O | < co o | y O |
•5— | τ- | Y-* | v | T* | T* | τ» | t™ | y— | Y | |||||||||
> | > | > | > | > | > | > | > | > | > | > | > | > | > | > | > | > | 0 | > |
< | < | < | < | < | < | < | < | < | > | < | < | < | < | < | < | < | < | |
cO | CO | co | co | co | co | CO | CO | co | co | CO | CO | co | co | co | co | co | 03 (O | co |
o | o | o | o | o | o | o | O | o | o | c | O | o | o | O | o | O | o | |
T— | T- | T“ | τ- | y» | T* | «r- | T | |||||||||||
0 | 0 | 0 | 0 | 0 | 0 | 0 | w | ω | < | cn | tn | w | CO | 0 | ω | 0 | 0 | |
< | < | < | < | < | < | á | < | < | < | < | < | < | < | < | < | < | < | < |
OD | 00 | 00 | 00 | 00 | 05 | co | 03 | 00 co | co | QO | s | 00 | 00 | oo | Q0 | a | CO | |
CO | CO | co | CO | co | CO | CO | CO | to | co | CD | CO | co | co | CO | CM | co | ||
> | > | > | > | > | > | > | > | > | > | > | > | > | > | > | > | > | 0 | > |
-45CZ 302287 B6
C | UJ | |||||||||||||||||
05 | o | |||||||||||||||||
CO | co | |||||||||||||||||
CM | CM | |||||||||||||||||
2 | ||||||||||||||||||
ϋ | u. | > | 2 | UJ | X | x | O | v | ϋ | X | Q | |||||||
CM | CM | CM | m | co | 8 | 8 | o | r-. | CD | CM | CM | CM | ||||||
<o | (O | tn | to | to | co | to | IO | tn | to | <0 | CD | tn | ||||||
CM | CM | CM | CM | CM | cm | CM | CM | CM | CM | CM | CM | CM | CM | CM | ||||
2 | 2 | 2 | f— | cn | cn | cn | 1— | O | z | Z | Z | Z | 2 | |||||
> | Q | Li_ | _1 | O | X. | ϋ | ϋ | í: | id | ϋ | X | O | O | ϋ | ϋ | a | ||
O? | CM | CM | co | CM | CM | CM | CM | CM | CM | CM | co | CD | CM | CM | co | |||
to | XT | tn | to | v | tO | tn | tn | to | tn | tO | tn | χτ | χτ | IO | IO | xr | ||
CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM , | CM | CM | CM | CM | CM | CM | ||
id | Z | 2 | 2 | Z | 2 | 2 | Z | 2 | 2 | 2 | 2 | 2 | Z | 2 | 2 | |||
Q | tr | Q | Q | x | Q | Q | Q | Q | Q | O | Q | X | X | Q | Q | X | ϋ | |
oo | to | (O | co | tn | CD | CD | CD | co | CD | OD | co | tn | to | co | co | tn | CM | CM |
XT | χτ | XT | xr | xr | XT | xr | xr | xr | χτ | XT | xr | xr | xr | xr | xr | tn | to | |
CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM |
Z | O | 2 | 2 | a | Z | 2 | 2 | Z | 2 | 2 | 2 | σ | σ | 2 | Z | σ | 2 | 2 |
x | -r | 01 | a | X | X | X | X | X | X | X | — | X | X | X | X | Q | a | |
to | <0 | tO | tO | co | tO | tO | to | tn | to | tO | tn | co | co | tO | tO | co | CD | co |
xr | cn | xr | xr | cn | xr | xr | xr | xr | xr | xr | XT | cn | cn | xr | xr | cn | xr | XT |
CM | CM | CM | CM | CM | CM | CM | CM | CM | <M | CM | CM | CM | CM | CM | CM | CM | CM | CM |
a | a | O | a | a | O | σ | σ | σ | σ | O | σ | O | σ | σ | σ | σ | 2 | z |
-I- | > | X I | lx | > | X | X | X | X | X | T | X | > | > | X | X | > | X | X |
co | CM | CO 1 | co | CM | co | CD | co | co | CO | cO | co | CM | CM | 8 | C0 | CM | to | IO |
CO | cn | cn i | cn | cn | cn | cn | cn | cn | cn | cn | cn | cn | cn | cn | cn | XT | xr | |
CM | CM | CM 1 | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM |
a | < | a i | Ισ | < | o | σ | σ | σ | O | O | σ | < | < | a | σ | < | σ | σ |
> | x | > | > | O | > | > | > | > | > | > | > | Q | Q | > | > | O | X | -r |
CM | co | CM | CM | 05 | CM | CM | CM | CM | CM | CM | CM | 05 | 05 | CM | CM | 05 | co | CO |
CQ | o | cn | cn | tn | cn | cn | CO | cn | cn | cn | cn | LO | IO | cn | cn | tn | cn | cn |
CM | CM | CM | CM | τ- | CM | CM | CM | CM | CM | CM | CM | T“ | CM | CM | V“ | CM | CM | |
< | σ | < | < | < | < | < | < | < | < | < | o | 5 | < | < | CD | σ | σ | |
Q | Q | Q | Q | a | a | O | Q | Q | Q | Q | cn | Q | O | - | > | cn | ||
05 | m | o | 05 | A | 05 | 05 | 05 | 05 | O) | 05 | 05 | xr | co | 05 | 05 | χτ | CM | CM |
to | to | IO | IO | o | to | to | tO x | JO | to | to | tn | o | T— | to | tO | o | cn | cn |
i“ | T* | Τ- | v*· | V | —— | CM | CM | |||||||||||
o | o | o | o | > | o | o | o | Ο | o | o | o | > | 2 | δ | δ | > | < | < |
x? | x? | v | < co | v | v | 2 | 1 XT | v | < cn o | ΜΓ | X? | x? | < cn | O 05 | ||||
o | o | o | o | o | o | o | o | o | o | o | σ | O | o | O | o | o | IO | |
x— | T- | X“ | v— | Ύ— | v- | r— | ||||||||||||
> | > | > | > | ώ | > | > | > | > | > | > | > | δ | > | > | > | δ | > | δ |
< | < | < | < | < | < | < | < | < | < | < | < | < | < | a co | < | |||
cn | cn | cn | cn | oo co | CO | cn | cn | cn | cn | en | cn | cn | tn | cn | cn | |||
o | o | o | o | o | o | o | o | c | O | o | 00 JO | o | o | o | o | 1 o | ||
γ-* | >* | X— | T— | T-* | 1^» | |||||||||||||
tn | cn | tn | CO | cn | CO- | cn | cn | cn | cn | cn | > | cň | cn | ω | 2 | cn | > | |
< | < | < | < | « | 5 | < | < | < | < | < | < | < | < | < | < | < | < | |
co <0 | § | co | 00 co | tn tn | co tp | to co | <x> cd | 68 | oo co | 00 co | co co | > | «0 co | ao CO | 00 co | CD CD | co co | cn o |
> | > | > | > | CL | > | > | > | > | > | > | > | > | > | > | > | δ |
-46 CZ 302287 B6
< | < | |||||||||||||||||
O CD | s | cn tn | ||||||||||||||||
CN | cn | CN | ||||||||||||||||
ř- | H | Z | ||||||||||||||||
££ | CZ | X | 3ťí | Q | ||||||||||||||
tn | CN | tn | CN | CN | oo | CN | CN | CN | ||||||||||
xr | tn | XT | tn | tn | XJ- | tn | tn | tn | ||||||||||
CN | CN | CN | CN | CN | CN | CN | CN | CN | ||||||||||
a | Z | a | Z | Z | Z | Z | Z | Z | ||||||||||
X | O | X | O | Q | o* | a | Q | Q | ||||||||||
CN | CD | CN | 00 | 8 | 00 | CD | tn | CN | CN | 00 | co | 00 | ||||||
ΙΩ | CO | to | XT | xr | XT | xr | tn | m | XT | XT | XT | |||||||
CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | ||||||
Z | a | Z | z | a | Z | Z | σ | Z | Z | Z | Z | Z | ||||||
χ | Q | > | o | CZ | > | CZ | X | x- | O | 'Z | Q | X | CZ | CZ | X. | |||
CN | CD | CN | CN | oo | tn | CN | tn | tn | CO | 03 | CN | CO | tn | tn | tn | CN | CN | |
tn | XT | tn | CO | XT | XT | CO | XT | xr | CO | xr | tn | χχ | xr | XT | T | tn | tn | |
CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | £2 | CN | CN | |
Z | Z | Z | < | Z | O | < | σ | σ | σ | Z | Z | Z | σ | σ | σ | Z | Z | |
Q | x | O | co | CZ | X | CZ | X | X | X | > | CZ | O | CZ | X | X | Q | Q | |
00 XT | tn xr | 00 XT | 00 | tn xr | 8 | CO | 8 | tn XT | 8 | CN CO | tn XT | 00 xr | tn xr | CD CO | co co | 8 | 00 xr | 00 xr |
CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | (N | CN | CN | CN | CN |
z | σ | Z | Z | σ | a | a | σ | σ | < | σ | Z | σ | σ | σ | σ | Z | Z | |
or | X | > | K | X | > | —í | > | X | > | O | X | (Z | X | > | > | > | K | Q£ |
LT5 | CD | m | (Ti | CD | CN | O | CN | 8 | CN | 05 | CO | tn | CO | CN | CN | CN | m | tn |
xr | CO | xr | CO | CO | Τ- | CO | CO | m | CO | xr | CO | CO | CO | CO | -θ' | xr | ||
CN | CN | CN | CN | CN | CN | ΟΝ | CN | CN | CN | V* | CN | CN | CN | CN | CN | CN | CN | CN |
a | σ | σ | h- | a | < | CL | < | a | < | o | σ | a | a | < | < | < | σ | σ |
cz | > | X | O | > | O | Q | Q | > | O | o: | > | X | > | > | __. | LL | X | X |
(O | CN | 8 | 05 | CN | 05 | 05 | 05 | CN | 05 | IX. | CN | CO | CN | s | XT | r- | CD | co |
CO | CO | in | CO | tn | tn | tn | CO | tn | co | CO | CO | CO | o | CO | CO | co | ||
CN | CN | CN | Τ- | CN | Τ- | τ- | CN | T· | CN | CN | CN | T- | x“ | T- | CN | CN | ||
a | < | σ | Ο | < | Ο | Ο | o | < | o | σ | < | σ | < | ω | > | ω | σ | σ |
> | Q | > | > | __ | < | <z> | O | > | W | Q | < | □ | > | > | ||||
CN | σι | CN | v | 00 | o | o | co | 05 | CN | 03 | 05 | co | □5 | CN | CN | |||
CO | un | CO | o | CN | o | o | o | co | co | tn | CO | tn | o | tn | CO | CO | ||
CN | CN | r· | CN | T* | CN | Τ' | T- | Τ- | CN | CN | χ- | CN | CN | |||||
< | δ | < | > | < | > | > | > | EL | tn | < | Ο | < | Z | Ο | δ | o | < | < |
Q | Q | < | O | < | < | < | > | Q | Q | __ | Q | O | ||||||
CT> Λ | xr o | o> m τ- | CO O | 05 tn | co o | co o | (O o | LO € | o | 5 v- | co co | 05 tn | 05 tn | XT σ | u CD b- | XT o | 05 tn | 05 tn |
δ | > | Ο | δ | δ | δ | δ | δ | > | > | > | < | δ | δ | > | z | > | δ | δ |
V1041 | S103A I | V104I 1 | S101T | V104I | | N76D | Q 05 00 UJ | Q co Z | G159D | S103A | S103A | o > | o > | o > | S103A | < co co > : | S103A | V104I | X? O > |
s o | < 03 CD | < co o | Q CD r*. | < co o | š co | o CD r- | š co | XT O | š co | 68A | < co o | s o | < co o | š co | LU CD | š CD | s o | < co o |
δ | > | ω | Z | δ | > | z | > | > | > | > | δ | δ | δ | > | O | > | δ | δ |
< | (Z) | < | š | < | ώ | < | UJ | < cn | Ul | LU | LU | < | < | UJ | _1 | LU | UJ | O |
00 | 00 | 00 | 00 | co | 00 | T- | rt | T- | T— | T“ | 00 | 03 | v- | n to | r* | 00 | ||
CD | co | CD | tp | co | co | CD | co | co | CD | co | CO | CD | co | 05 | 05 | |||
> | z | > | > | > | 1- | > | O | δ | O | o | O | > | > | O | O | O | < |
-47CZ 302287 B6
tM | ||||||||||||||||||
tn | ||||||||||||||||||
CM | ||||||||||||||||||
2 | ||||||||||||||||||
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co | CM | CM | CM | |||||||||||||||
xr | tn | tn | tn | |||||||||||||||
CM | CM | CM | CM | |||||||||||||||
2 | 2 | 2 | 2 | |||||||||||||||
X | X | X | x. | X | X | O | Q | Q | X | |||||||||
CM | CM | CM | CM | CM | CM | V | CM | CM | CM | CM | CM | tn | 00 | 00 | 03 | CM | CM | CM |
ID | m | tn | m | m | tn | co | tn | tn | tn | tn | tn | xr | xr | xr | XT | tn | tn | tn |
CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM |
2 | 2 | 2 | Z | z | 2 | 2 | 2 | 2 | 2 | 2 | 2 | σ | 2 | 2 | 2 | Z | 2 | 2 |
Q | Q | O | o | O | Q | X | Q | O | Q | Q | O | — | X | X | X | O | Q | O |
co | OO | <o | co | co | co | CM | co | 00 | co | oo | co | to | tn | tn | tn | 00 | oo | 03 |
XT | xr | XT | XT | xr | xr | tn | XT | XT | xr | xr | XT | tn | xr | xr | xr | XT | xr | xr |
CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | tM | CM |
2 | 2 | 2 | Z | 2 | 2 | 2 | Z | 2 | 2 | 2 | 2 | O | σ | σ | σ | 2 | 2 | 2 |
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m | m | tn | tn | m | tn | oo | tn | tn | tn | tn | tn | CM | co | to | co | tn | m | m |
T | XT | xr | xr | xr | xT | XT | xT | XT | xT | xr | xr | tn | tn | cn | cn | xr | XT | xr |
CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | |
a | a | σ | σ | O | σ | 2 | O | σ | □ | σ | O | < | σ | O | σ | σ | σ | σ |
X | X | X | X | x | X | X | X | X | X- | — | — | cr | > | > | > | X | X | X |
(O cn | to cn | S | tO cn | to tn | (O tn | tn xr | to tn | to cn | to cn | to tn | cn | CM tn | CM tn | CM cn | 8 | to tn | to tn | |
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a | O | σ | σ | O | O | σ | σ | O | a | σ | σ | I- | < | < | < | a | σ | σ |
> | > | > | > | > | > | X | > | > | > | > | > | O | X | LU | X | > | > | > |
CM | CM | CM | CM | CM | CM | CO | CM | cn | CM | CM | CM | 05 | cn | f-* | s | CM | CM | CM |
m | cn | cn | cn | cn | cn | tn | cn | cn | tn | cn | tn | tn | cn | cn | cn | |||
CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | Τ- | CM | CM | CM | CM | CM | CM |
< | < | < | < | < | < | σ | < | < | < | < | < | Ο | i- | —1 | σ | < | < | < |
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tn | m | tn | tn | tn | tn | tn | tn | 12 | to | tn | o | tn | tn | tn | tn | |||
T“ | CM | CM | CM | |||||||||||||||
o | o | O | o | o | < | 5 | O | 2 | δ | > | δ | o | δ | δ | o | o | ||
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S99E | UJ τ- α δ | O o δ | G102A | < cn o ω | S103A. | S103A | s o δ | o CM CO 2 | S103A | cn σ δ | < cn o δ | G20R | O CM <O 2 | < cn o δ | O cn to | S103A | S103A | 2 r- 2 |
-48CZ 302287 B6
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x | > | > | X | X | X | > | X | X | X | X | X | X | X | X | X | X | ||
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CN | CN | CN | CN | <N | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | ||
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co | rt | u CN | CO | CO | CO | CO | CO | CO | CO | CO | co | Ό | CO | |||||
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-49CZ 302287 B6
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tn | tn | Ν' | co | tn | IO | xr | xr | co | co | xr | tn | to | xr | |||||
C\l | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | 3 | CN | CN | |||||
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o- | XT | tn | co | XT | ΙΟ | CO | co | co | xr | CO | CO | xr | CO | CO | xr | |||
CN | CN | CN | CN | CN | CN | 3 | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | ||
a | a | a | Z | < | σ | σ | σ | < | σ | σ | < | σ | σ | |||||
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CO | co | Γ- | CN | tn | tn | tn | tn | co | CN | Γ- | CN | CO | O | CN | CO | o | CN | 8 |
n | co | ΙΟ | CO | xT | T | x? | XT | CO | ΙΟ | CO | co | T- | CO | co | CO | |||
IN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CM | CN | CN |
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> | > | 01 | cl | X | X | x | —1 | CL | X | X | > | § | — | > | § | > | ||
CN | CN | tn | co | CO | co | co | co | o | co | tn | co | CN | 05 | O | CN | 05 | o | CN |
CO | CO | Ν’ | T- | co | co | co | co | T* | xr | CO | o | x- | CO | o | <T' | CO | ||
CN | CN | CN | CN | CN | IN | CN | CN | CN | CN | CN | CN | CN | CN > | CN | CN | fN | CM Λ | CN |
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a | < | __ | Ω | Ω | Ω | < | «.- | 5 | Ω | Ω | _ 1 | Ω | Ω | Ω | Ω | |||
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O | o | tn | o | o | tn | tn | tn | o | O | m | tn | O i | .o | o | O | o | tn | |
*” | X“ | r- | Τ- | Τ» | CN > | y i | ||||||||||||
> | > | o | ω | > | Ο | o | O | δ | > | δ | o | > 1 | δ | δ | > | o | o | |
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δ | δ | > | z | ω | > | > | > | z | δ | > | > | δ | > | > | δ | > | > | |
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50CZ 302287 B6
X | X | |||||||||||||||||
CM | CM | CM | co | CM | ||||||||||||||
tn | m | m | tn | m | ||||||||||||||
CM | CM | CM | CM | CM | ||||||||||||||
Z | 2 | 2 | 05 | 2 | ||||||||||||||
X | $ | X | Q | Q | O | Q | ||||||||||||
CM | CM | CD | CD | co | CM | ÍM | 03 | CM | ||||||||||
tn | CO | tn | XT | xr | XT | tn | tn | χτ | tn | |||||||||
CM | CJ | CM | CM | CM | CM | CM | CM | CM | CM | |||||||||
X | 2 | 2 | 2 | 2 | 2 | 2 | 2 | 2 | 2 | |||||||||
X | Q | > | Q | X | X | X | O | X | a | X | O | |||||||
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XT | XT | tn | XT | xr | χτ | xr | xr | tn | tn | tn | tn | m | xr | xr | xr | |||
CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | £ | CM | |||
σ | Z | -J | 2 | σ | σ | σ | 2 | Z | Z | Z | 2 | 2 | Z | σ | 2 | |||
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xr | XT | xr | xr | χτ | co | co | xr | XT | xr | xr | XT | XT | xr | co | T | |||
Csl | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | |
σ | σ | O | σ | σ | O | σ | σ | σ | σ | Z | 2 | 2 | 2 | 2 | a | σ | σ | |
2 | X | X | > | X | X | X | > | > | > | X | X | X | X | X | X | X | > | X |
co | 8 | m | CM | co | co | co | CM | CM | CM | 8 | tn | tn | tn | tn | tn | co | CM | CO |
co | XT | CO | co | co | co | CO | CO | CO | xr | xr | xr | xr | xr | co | CO | co | ||
CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM |
σ | σ | σ | < | O | a | σ | < | < | < | σ | σ | σ | σ | σ | σ | a | < | σ |
> | > | X | $ | > | > | > | O | O | O | > | -r | X | X | I- | < | > | X | > |
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CO | CO | co | o | CO | CO | CO | T** | i— | CO | co | co | xr | xr | CO | CO | |||
CM | CM | CM | cu | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | ÍM | CM | CM |
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-51 CZ 302287 B6
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tn | LC | tn | LO | xr | tn | xr | χτ | xr | XT | tn | tn | tn | χτ | XT | xr | co | ||
CM | CM | CM | CM | CM | CM | CM | CM | CM | CM i | CM | CM | CM | CM | CM | CM | CM | ||
Z | Z | Z | Z | Z | z | Z | Z | o | Z | Z | Z | Z | Z | Z | Z | t- | ||
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co | co | ® | 00 | LO | 00 | to | tn | co | m | co | 00 | co | tn | tn | m | CM | ||
XT | XT | xr | xr | xr | xr | xr | xr | co | χτ | xr | XT | xr | χτ | χτ | xr | tn | ||
CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | ||
Z | Z | Z | Z | σ | Z | σ | σ | σ | σ | Z | Z | Z | σ | σ | σ | Z | ||
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xr | xr | χτ | xr | CO | co | co | co | CO | CO | xr | xr | XT | CO | xr | ||||
CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | ||
σ | σ | σ | σ | a | σ | σ | σ | < | σ | σ | σ | a | σ | σ | σ | Z | ||
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CO | CO | co | CO | CO | CO | CO | tn | CO | co | co | CO | CO | CO | xr | ||||
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σ | σ | σ | σ | < | < | < | < | 1— | Z | < | a | σ | σ | < | < | < | O | |
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to | co | σ> | Oi | o | cn | cn | CM | 05 | m | tn | 05 | 05 | 05 | 05 | 05 | 05 | σ> | CM |
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0 | O | _ | 0Č | o | > | > 1 | !0 | 0 | 0 | ~J | 0 | 0 | ||||||
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-52CZ 302287 B6
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Ul | ui | LO | tn | tn | <n | in | Ul | xr | Ul | xr | |
CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | |
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Q | Q | O | Q | O | Q | Q | O | X | Q | X | |
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xr | -v | xr | XT | xr | xr . | v | xr | χτ | χτ | CO | |
CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | |
2 | 2 | 2 | 2 | 2 | 2 | z | 2 | σ | 2 | σ | |
X | X | X | X | X | X | X | X | X | X | > | |
ui | tn | m | «1 | Ul | Ul | Ul | Ul | co | tn | CM | |
rr | xr | xr | xr | χτ | xr | xr | co | χτ | CO | ||
CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | |
a | σ | σ | σ | σ | σ | σ | σ | σ | σ | < | |
— | X | X | X | X | X | X | X | X | > | X | X |
CO | co | co | co | co | co | co | tn | CO | CM | co | co |
CO | co | eo | co | co | co | co | xr | co | CO | CO | |
CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM |
σ | σ | σ | σ | σ | σ | σ | σ | σ | < | σ | F— |
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CM | CM | CM | CM | CM | CM | CM | CM | CM | T— | CM | ÍM |
< | < | < | < | < | < | < | σ | < | < | < | σ |
O | Q | Q | 0 | $ | /A | tň O CM | > | X | Q | > | □ |
Ol m | Ol tn | Ol n | CM | O> o | o CM A | 30 | xr Ol | Ol Ul | O CO | tn co | |
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> | Q | Q | o | o | Q | Q | Q | □ | |||
’Τ | Ol | Ol | Ol | Ol | Ol | Ol | xr | Ol | Ol | ||
σΐ | o | O | in | tn | tn | tn | tn | «1 | o | Ul | tn |
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-53 CZ 302287 B6
Tabulka č. 4
m | T- | ** | tn 2“ | o > | r- 2* | CN Ν’ | o 2- | co f'- 'w | O co | 2- | co tn* o“ | O CN^ o“ | r-- o“ | 03 CN^ r- | 03 3· | r* 3- | CD | |
<r | 8 O* | T— | P- | co | CO | m | o | CD | co | r* | 8 cn | tn | r- | o | τ— | |||
’Π“ ** | r- CN* | cP | 03 CN* | 2* | A. CN | o CN* | CO CN* | co cn | co CN* | 03 co* | co co* | P* co | s- | A. CN | ||||
CN | CN | CN | ||||||||||||||||
tn | tn | tn | ||||||||||||||||
CN | CN | CN | ||||||||||||||||
Z | Z | Z | ||||||||||||||||
X | iť: | X | > | 0 | Q | 0 | ||||||||||||
CN | CN | CN | CN | tn | r-~ | 03 | CO | co | ||||||||||
tn | tn | tn | tn | Γ-- | tn | n | Ν’ | Ν’ | ||||||||||
CN | CN | CN | CN | CN | CN | CN | CN | CN | ||||||||||
Z | Z | Z | Z | X | _J | Z | Z | Z | ||||||||||
X | 0 | X | X | X | X | > | 0 | X | ω | > | X | X | X | X | ||||
CN | 03 | tn | tn | tn | tn | Γ. | ca | tn | CN | tn | tn | tn | tn | |||||
lO | Ν’ | ΤΓ | n· | Ν’ | N | tn | n- | tn | tn CN | o- | ΤΓ | Ν’ | Ν’ | |||||
CN | CN | CN | CN | CN | CN | CN | CM | CN | CN | CN | CN | ÍN | CN | |||||
Z | Z | σ | σ | σ | σ | 1 | z | 0 | Z | lj | 0 | 0 | 0 | 0 | ||||
X | X | X | X | i- | X | X | X 1 | IX | LJ | X | X | X | «·» | X | -τ- | |||
tn ΤΓ | U3 N* | tn Ν- | co co | 8 | co co | co co | tn i Ν’ | | [tn IT | Γ-- co | tn Ν’ | m N | CO co | CD CO | 8 | 8 | |||
CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | |||
o | σ | σ | O | σ | 0 | σ | σ | ισ | v | 0 | 0 | 0 | 0 | 0 | 0 | |||
J | 1 | X | X | > | > | > | > | T | — | X | -r | X | > | > | > | > | ||
8 | co | co | CN | CN | CN | CN | 8 | <o | co | CO | CO | CN | CN | CN | CN | |||
cn | co | CO | CO | CO | CO | cn | co | CO | CO | CO | CO | CO | CO | |||||
CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | |||
σ | σ | σ | < | < | < | < | 0 | 0 | 0 | 0 | 0 | < | < | < | < | |||
> | > | > | O | a | 0 | 0 | > | > | > | > | > | < | O | 0 | X | |||
CN | CN | CN | 03 | cn | 03 | 03 | CN | CN | CN | CN | CN | n- | 03 | 03 | CN | |||
CO | CO | CO | tn | tn | tn | tn | CO | CO | CO | CO | CO | CN | tn | tn | f- | |||
CN | CN | CN | t“ | CN | CN | CN | CN | Cn | CN | CN | ||||||||
< | < | < | 0 | 0 | 0 | 0 | < | < | < | < | < | J— | 0 | 0 | CO | |||
0 | 0 | 0 | 0 | _ | Ň> o | _ | 0 | 0 | □ | Q | 0 | 0 | MM | 0 | ||||
03 tn | 03 tn | 03 cn | O N | N* o | N O | 03 tn | 03 tn | 03 tn | 03 tn | 03 tn | 03 tn | s | 3 | 03 tn | ||||
T* | v* | V“* | ||||||||||||||||
o | 0 | 0 | z | > | > | > | 0 | 0 | 0 | 0 | 0 | 0 | > | > | 0 | |||
J“ | _ | _ | _ | MM | < | < | < | ί -n_ | _ | — | < | < | ||||||
C | ® CN | **r o | 'V O | N O | s | to o | CO o | co o | n- c | o | ΤΓ o | ΤΓ o | 'T O | ν- ο | co o | σϊ O | 3 | |
T- > | CN < | i— > | > | > | -T™ > | δ | δ | T* ω | T— > | > | T— > | T- > | > | τ— > | τ— ω | T- ω | > | |
S σ | n c | § | § | < co o | < co o | < co co | < ta CD | < <0 co | < CO o | < co o | < CO o | < O | s o | š § | š | < co σ | ||
δ | > | ω | v* ω | ω | δ | > | > | > | δ | 'V“ ω | δ | T“ ω | v- ω | δ | § | δ | ||
Q | < co | < | < | < | < | ω | O | < | < | < | < | < | š | 1±1 | 0 | < | ||
<0 | « | «0 | CO | co | co | co | CO | co | 03 | co | 03 | 03 | T- | CO | CD | |||
r·- | co | co | <o | N | N- | Ό- | CO | CO | co | CO | CO | CO | <D | N | CO | |||
Z | w | > | > | > | > | z | Z | z | > | > | > 1 | > | > | > | 0 | Z | > |
Příklad 2
U velkého množství variant proteáz produkovaných v příkladu 1 se testovala účinnost ve dvou 5 typech detergentů a podmínkách praní za použití mikrotestu popsaném v publikaci „An improved method of assaying for a preffered enzyme and/or prefered detergent composition,“ U.S. Seriál
No. 60/068 796.
V tabulce č. 4 jsou uvedeny varianty testovaných proteáz a výsledky testování ve dvou různých io detergentech. 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 0,975 g smíšené tvrdosti Ca2+/Mg2+ na 4,54 1. V každé i s prohlubni se použilo 0,3 ppm enzymu při teplotě 40 °C.
Příklad 3
V tabulce č. 5 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 pri teplotě 40 °C. V případě sloupce C detergentem bylo 3,5 g/1 detergentů HSP1 (Procter and Gamble, Cincinnati, OH, USA) v roztoku, který obsahuje 0,52 g smíšené tvrdosti Ca2+/Mg2+ na 4,54 I. V každé prohlubni se použilo 0,3 ppm enzymu při 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 4,54 1. V každé prohlubni se použilo 0,3 ppm enzymu při teplotě 20 °C.
-55 CZ 302287 B6
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- 57 CZ 302287 B6
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ω | δ | δ | CO | δ | w | δ | z | Z | Z | Z | Z | > | Z | Z | —i | Ζ | > | Ζ | Ζ |
Ω | Ω | Ω | Ω | Ω | Ω | Ω | 5 | < | š | < | < | σ | s | š | < | < | X | < | < |
© | © | © | © | © | © | © | o | © | © | © | r- | © | © | ΓΝ | © | © | |||
h- | r* | I*- | r- | r- | r- | h*· | CN | SP | SP | © | © | NF· | © | © | © | © | © | © | |
Ζ | Ζ | Ζ | Z | Z | Z | Z | O | > | > | > | > | > | > | > | > | σ | > | > |
-59CZ 302287 B6
Γ- ιο o“ | co o | CO o o” | y <□_ O | CO o o | CM 0 o* | CO O o | CM O cT | CM o“* | CO 0 | 0 tn o* | 0,13 | CO | co | y tn | 0 o | 0 cf* | 03 0e | 66*0 | |
Cl | fC Ί | 0 | X“ | 0 | oo | co | y | 0 | Y“ | 0 | CO | eo | 0 | CM | 0 | 7— | CM | Γ'- | |
γ | 0 | Y | Y^ | «1. | CM | CM | 0~ | oi_ | 0_ | co^ | CM | Γ- | O* | o>^ | a> —v ' | 0^ | |||
CM* | σΓ* | co | co | co | CO | CM | CO* | co* | co | CM | CM* | Y | CO* | <*f* | |||||
3 0 CM 1— | 1 | ||||||||||||||||||
0 | X | X | X | X | X | > | X | ||||||||||||
tn | to | 0 | tn | n | tn | 1— | 0 | ||||||||||||
m | y | y | y | y | y | 0 | y | ||||||||||||
CM | CM | CM | CM | CM | CM | CM | CM | ||||||||||||
H | σ | σ | σ | σ | σ | -J | σ | ||||||||||||
> | Q | CZ | X | X | X | Y | X | X | X | X | X | > | X | ||||||
m | T— | m | r·» | 0 | tn | 0 | tn | 0 | 0 | 0 | 0 | r- | 8 | ||||||
y | co | y | y | CO | y | CO | y | CO | CO | CO | y | 0 | |||||||
CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | ||||||
X | Z | σ | X | σ | σ | σ | a | σ | σ | σ | σ | _1 | σ | ||||||
_J | X | σ | X | X | X | o | X | o | X | < | > | > | X | > | X | > | |||
0 | 8 | 01 | to | 8 | tn | y | 0 | 0 | 0 | CO | CM | CM | 0 | r- | 0 | CM | |||
y | y | y | y | o | CO | co | O | CO | CO | CO | 0 | y | 0 | ||||||
CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | 3 | CM | <M | |||
σ | σ | X | σ | σ | a | Z | σ | Z | σ | > | < | < | σ | σ | < | ||||
X | o | X | X | Q | X | > | o | Q | > | X | X | > | X | X | § | ||||
co | 01 | co | 0 | Ol | 0 | y | y | oi | CM | co | 0 | CM | 0 | 0 | O) | ||||
co | to | co | co | tn | CO | h- | O | tn | CO | O) | Y | CO | y | co | O | ||||
CM | CM | CM | v- | CM | γ* | CM | CM | CM | CM | CM | CM | CM | CN | ||||||
σ | 0 | a | σ | 0 | σ | < | Z | 0 | < | < | 1- | X | < | σ | σ | > | |||
*—1 | Q | 0 | Q | o | z | O | Q | o | > | Q | Q | Q | Q | a | X | X | > | o | |
CO | σι | co | 01 | Ol | Y | Ol | Oi | Ol | CO | Ol | cn | CD | y | 05 | 05 | 0 | 0 | CM | Oi |
co | to | CM | IO | to | y | tn | to | tn | co | tn | tn | tn | 0 | tn | 0 | co | r- | 0 | 0 |
CM | Y* | Y | Y* | Y | V“ | Y** | Y | y | γ— | CM | CM | CM | |||||||
σ | 0 | Z | 0 | 0 | 0 | 0 | 0 | 0 | < | 0 | 0 | 0 | > | 0 | 0 | σ | X | < | 0 |
y o | y o | 3 | O | y o | 3 | 3 | y o | y o | o | y o | y o | y o | a o | y 0 | y o | > | > r- 0 | O Ol 0 | y O |
Y | Y*~ | γ· | γ· | Y | Y^ | Y | Y | CM < | Y·* | ||||||||||
> | > | > | > | > | > | > | > | > | > | > | > | > | 0 | > | > | CM | 0 | > | |
< | < | < | < | < | < | < | < | < | < | < | < | < | Q | < | < | __ | s | ||
co | CO | eo | co | CO | CO | co | co | co | co | CO | co | co | co | co | y | s | |||
o | o | o | o | O | O | o | o | O | o | o | o | 0 | O | o | o | o | o | ||
v* | T“ | γ·» | γ— | γ* | Y | P** | Y | ||||||||||||
0 | ω | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | z | 0 | 0 | > | > | > | 0 |
O | o | Q | Q | Q | Q | O | O | Q | o | Q | Q | Q | < | Q | O | < | < | < 0 | Q |
co | co | CO | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | Λ | co f—* | 0 | |
Γ- | r-. | r- | r- | r- | r- | Γ- | ř- | r- | Γ- | r- | Ν» | r- | 0 | r- | Γ*- | o | Γ- | ||
Z | Z | Z | Z | Z | Z | z | Z | z | z | Z | Z | Z | > | Z | Z | 0 | 0 | 0 | Z |
< | < | < | < | < | < | < | < | < | < | < | < | < | < | < | O | Q | Q | < | |
co | CD | CD | CD | co | CD | co | CD | CD | 0 | 0 | 0 | 0 | CM | 0 | 0 | 0 | 0 | 0 | CD |
co | CO | CO | (0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | CM | 0 | 0 | Γ- | Γ- | r- | 0 |
> | > | > | > | > | > | > | > | > | > | > | > | > | K | > | > | Z | Z | Z | > |
60CZ 302287 B6
CD | 8_ | CM | m | CM | <2 | 05 | CD | r- | CM | co | CD | XT | xr | 05 | co | CN | CN | ||
b. | σ> | b-_ | m -w | cq_ | co | b *· | CO. | xT. | CN •Wl, | <D^ | «L | «U | |||||||
T- | Y*“ | v | σ’ | o | T“ | χ- | t— . | T· | |||||||||||
xr | in | co | r- | CO | b- | CN | CN | b- | CD | CN | 05 | <5 | m | CN | b· | 05 | o | ||
b^ | CO_ | co__ | CO^ | 05. | 5- | 05 | S- | σ. | n. | »· | O | o •w | |||||||
co* | CN | *“ | CN | CM | CM | CN | CN | o | CN | CN | o* | T* | v- | ** | CM | cm | co | ||
X | 0 | > | X | X | CO | X | |||||||||||||
cn | 05 | O | in | T— | CN | U5 | |||||||||||||
xr | m | CO | T | tn | b- | XT | |||||||||||||
CM | CN | CN | CM | CN | CN | CM | |||||||||||||
σ | co | ť- | σ | X | < | σ | |||||||||||||
X | X | X | X | X | X | X | O | I | X | X | co | X | X | (X | X | X | |||
m | m | to | ΙΛ | in | m | m | m | CD | CD | m | CO | s | m | tn | |||||
xr | o· | co | XT | xr | xr | XT | co | co | xr | CO | XT | XT | CO | XT | XT | ||||
CN | CM | CN | ca | CM | CM | CN | CN | CN | CN | CM | CM | CN | CN | CN | CN | CN | |||
σ | σ | σ | σ | σ | σ | σ | Z | Z | σ | σ | Z | σ | σ | to | σ | a | |||
X | I | > | X | X | X | X | X | X | X | > | X | X | X | > | X | X | X | ||
8 | 8 | CN CO | 8 | co co | 8 | 8 | m xr | m XT | co co | CN CO | tn xr | m XT | co co | CN CO | in xr | 8 | co eo | ||
CM | CM | CN | CM | CN | CM | CN | CN | CN | £3 | CM | CN | CN | CN | CN | CM | CN | CM | ||
σ | a | < | σ | σ | σ | σ | σ | σ | σ | < | σ | σ | O | < | σ | σ | σ | ||
> | > | -J | > | > | > | > | X | X | X | > | 0 | X | X | > | _1 | X | > | > | |
CM | CM | XT | CN | CN | CN | ca | co | 8 | in | CN | 05 | co | 8 | CN | CO | co | CM | CM | |
CO | co | CO | CO | CO | co | co | xr | CO | cr> | eo | CO | o | co | CO | CO | ||||
CN | CM | CN | CM | CN | CN | CM | CN | CM | CN | CN | CN | CM | CN | CN | CM | CN | CN | ||
< | < | > | < | < | < | < | σ | σ | σ | < | o | σ | σ | < | σ | σ | < | < | |
X | > | O | X | a | 0 | 0 | > | > | X | _í | X | > | > | Q | > | X | a | ||
T— | o> | m | 05 | 0) | 05 | CN | cn | CN | O | in | CN | CN | 05 | eo | CM | 8 | 05 | ||
v | m | m | tn | LO | CO | co | XT | o | xT | CO | CO | U> | co | CO | m | ||||
CM | CM | V“ | CN | T* | Τ- | CN | CN | CM | CN | T- | CM | CN | CN | T- | CN | CN | |||
O | o | o | < | o | Ο | o | < | < | to | X | > | σ | < | < | o | Z | < | σ | o |
0 | 0 | 0 | 0 | O | X | o | 3 O | X | U_ | 0 | __ | Q | Q | Q | |||||
CD tn | 05 LO | s | Oi cn | xr o | S | S | 05 m | 05 m | co co | 59 | co co | CN 05 | 05 m | xr a | 05 m | 05 tn | 05 U5 | s | |
Τ- | T“ | T“ | Τ- | cm | CM | Τ- | Τ- | Τ- | Τ- | ||||||||||
δ | Ο | > | O | > | > | > | o | Ο | a | o | ω | σ | > | Ο | > | Ο | Ο | Ο | > |
o > | XT o > | S103A | V104I | S103A | | < co σ ω | |S103A | V104I | V104I | šr co CN < | XT o > | 0 co b. Z | A232V | 0 05 U5 δ | b XT > | < co o ω | a > | V1041 | V104I | < CO o ω |
| S103A | S103A | N76D | S103A | N76D | 0 tO b- Z | X b. co (0 | S103A | S103A | x? O > | S103A | 3 .§ | V104I | O > | X? O > | a co b. z | S103A | S103A | S103A | o co b- Z |
Q | 0 | < | Q | < | < | Q | O | O | O | O | > | < co Λ | < | < | < | Q | Q | Q | < |
CD | co | CD | CO | co | CD | co | co | co | co | CD | rt | rt | CD | CO | co | CO | CD | ||
b- | b- | <O | b- | CO | co | b- | b- | t*** | b. | XT | c? | o | o | CO | b- | b· | b· | co | |
Z | Z | > | Z | > | > | z | z | z | co | Z | < | δ | CO | w | > | Z | 2 | z | > |
< | 3 | X | < | X | X | < J | < | < | 0 | 3 | X | 0 | 0 | o | X | < | < | < | X |
00 | CN | 8 | CN | o | Φ | oo | 00 | co | CN | CO | co | Φ | ÍN | as | ao | eo | |b | ||
co | CO | Γ* | CM | CD | co | co | h*» | <p | b | b- | b. | co | CO | co | 2 | ||||
> | > | σ | > | σ | O | > | > | > | z | > | σ | Z | Z | z | σ | > | > | > |
-61 CZ 302287 B6
Q | CO | in | CO | r·— | (D | to | CN | 8 a· | tn | co | to | xr | co | tn | si | o | o | ||
Z | cn_ | <0. | r- | cn_ | xr_ | tn* | R. | n. | |||||||||||
-r* | -r* | o | O | o | T“ | t— | x“ | χ~ | T- | x- | T- | X“ | |||||||
O | r- | CD | xr | 00 | CN | to | co | CN | m | co | 05 | CD | CN | o | 05 | CD | m | 05 | |
Z | o | «k | X“L. | tn_ | tn_ | xr | xr | tn | «U | <R. | tn. | r- | CN_ | ||||||
X“ | o | o | o | θ' | o” | o | X* | o | O | o | o“ | o” | o | o | x“ | x- | |||
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σ | |||||||||||||||||||
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to | |||||||||||||||||||
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> | CO | ||||||||||||||||||
CN | CN | ||||||||||||||||||
CO | CO | ||||||||||||||||||
CN < | □ | ||||||||||||||||||
CO | X | CZ | a | ||||||||||||||||
tn | tn | tn | τ- | ||||||||||||||||
00 | XT | xr | Ο | ||||||||||||||||
CN | CN | CN | |||||||||||||||||
Z | σ | σ | Z | ||||||||||||||||
CO | LU | CZ | CZ | (X | co | < | CZ | ||||||||||||
o | CO | tn | tn | to | CN | tn | |||||||||||||
r*· | co | xr | XT | XT | CN | CN | xr | ||||||||||||
CN | CN | CN | CN | CN | CN | CN | |||||||||||||
cz | > | σ | σ | σ | 2 | > | σ | ||||||||||||
O | £K | X | Q | X | Ι- | co | > | CO | co | CZ | |||||||||
05 | 05 | Γ. | tn | xr | Ο | CN | tn | CN | CN | tn | |||||||||
tn | xr | co | xr | xr | CN | CN | CN | xr | |||||||||||
CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | |||||||||
o | X | LU | σ | > | CL | 2 | < | 2 | 2 | σ | |||||||||
cr | CO | co | LU | CO | CO | CO | CO | co | cz | CO | K | co | co | Ι- | h- | Ι- | Ι- | co | |
to | m | CN | CN | CO | CN | CN | CN | CN | CN | 05 | CN | tn | CN | CN | ο | O | Ο | Ο | CN |
xr | CN | CN | CD | CN | CN | (N | CN | CN | XT | CN | xr | CN | CN | co | CO | co | co | CN | |
T“ | CM | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | ||||
z | σ | 2 | 2 | > | 2 | 2 | 2 | 2 | 2 | X | 2 | σ | 2 | 2 | δ | co | δ | δ | 2 |
V104I | co CN CN CN 2 | V104I | N173R | CO CN CN CN 2 | V104I | Q109R | Q109R | š X— > | í CO r· σ | M222S | xr o > | A232V | I104T | o t— > | I— xr o | I104T | I104T | I104T | S130T |
S103A | V104I | S103A | V1041 | o > | S103A | V1041 | V104I | 3 O δ | o > | s > | S103A | V104I | S103A | S103A | S103A | S103A | S103A | S103A | I104T |
Q to F'- | 3 O v™ | Q to r- | 3 O | 3 O | O CD r*- | < co o | 3 O | Q to | 103A | 103A | Q CO N- | < CO o | o to fx- | Q s | Q to r- | o CD r* | o CD | o CD | < co o |
z | co | Z | δ | δ | Z | δ | δ | Z | W | CO | Z | δ | z | z | Z | Z | Z | Z | δ |
< | Q | CZ | Q | O | 2 | Q | Q | CZ | O | Q | cz | Q | cz | cz | X | cr | CZ | CZ | Q |
00 | to | CM | co | co | to | to | tp | to | CN | to | CN | CN | CN | CN | CN | CN | co | ||
to | r* | r*. | r^ | CN | fx- | r* | co | r·* | h» | χ- | T-* | V“ | r- | ||||||
> | Z | a | Z | Z | _J | z | Z | o | Z | Z | o | Z | σ | Ο | σ | σ | a | σ | Z |
-62CZ 302287 B6
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tn | CM | 00 | tn | tn |
xr | CM | co | XT | xr |
CM | CM | CM | CM | CM |
σ | 2 | > | σ | σ |
co | Q | X | co | X |
CM | tn | m | o | 8 |
<M | «0 | XT | ||
CM | CM | 04 | CM | |
s | Z | σ | X | σ |
h— | co | co | CO | > |
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co | P>- | CM | CM | cn |
CM | CM | CM | ||
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m | h- | Γ*· | fx- | |
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U | O | 0 | o | 0 | a | 0 | <o | 0 | fO | CM | 0 | 1— | CM | Γ- | eo | |||
eo | to | CM | to | 0 | -x- | 0 | 0 | 2* | to | %. | eo | η | 0 | |||||
CD | w* | σι | CM | 0 | CT> | 0 | (O | tO | P- | 0 | a | 0 | 0 | CM | O | |||
xT | xr | ř— | 2* | Ol | eo | 3- | Ό- | xr | 0 | 0 | e*S | xr | XT | |||||
a | o | o“ | o™ | O | ||||||||||||||
< | xr | xr | 0 | ** | <31 | to | r— | CM | c | eo | 0 | rx- | r— | xr | ||||
T | eo cT | ta | CM* | Ol CM™ | Ol o* | 0 CM* | CM | CM | to CM* | A· CM | r- CM* | z*· | 3- | eo^ | N* | |||
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CM | CM | CM | CM | CM | CM | CM | ÍM | CM | CM | CM | CM | ÍM | ÍM | CM | ||||
Z | Z | Z | Z | Z | Z | Z | Z | Z | Z | Z | Z | Z | Z | Z | ||||
G | Q | G | G | G | G | X | G | a | G | Q | G | 3£ | G | G | Q | |||
eo | ťO | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | CM | 0 | 0 | 0 | |||
xr | xr | XT | xr | xr | xr | xr | xr | xr | xr | XT | 0 | XT | T | θ’ | ||||
CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | ÍM | tM | CM | |||
Z | Z | Z | Z | Z | Z | σ | Z | Z | Z | Z | Z | Z | Z | Z | Z | |||
i | X | X | X | X | X | X | X | X | X | X | X | X | Q | X | X | X | ||
CM | tn | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | ||
1 | 0 | xr | XT | xr | xr | xr | xr | eo | xr | xr | xr | XT | xr | xr | xr | χτ | ||
j | CM | CM | CM | CM | CM | CM | CM | CM | ÍM | CM | CM | CM | CM | CM | CM | CM | CM | |
i | Z | σ | a | σ | σ | σ | σ | σ | □ | σ | σ | σ | σ | Z | a | σ | σ | |
i | G | z | X | X | X | X | > | — | X | -r | X | X | Z | -Τ- | X | |||
O | to | to | <o | 0 | 0 | 0 | CM | 0 | to | 0 | Š | 0 | 0 | 0 | Ι | 0 | ||
x»· | to | to | eo | eo | CO | eo | to | co | eo | to | CO | T | tO | eo | ||||
CM | CM | CM | CM | CM | CM | CM | CM | ÍM | CM | CM | CM | CM | CM | CM | ÍM | CM | ||
Z | σ | σ | σ | σ | σ | σ | < | σ | a | σ | O | σ | σ | σ | O | σ | ||
X | > | > | > | > | > | > | _J | > | > | > | > | > | X | > | > | > | ||
<n | CM | CM | CM | CM | CM | CM | O | CM | CM | CM | CM | CM | 0 | CM | CM | CM | ||
τ | eo | eo | CO | CO | (O | to | T- | tO | eo | eo | CO | CO | to | to | to | CO | ||
tM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | ||
σ | < | < | < | < | < | < 1 1 | X | < | < | < | < | < | σ | < | < | < | ||
χ- | 5 | G | G | U_ | G | OL | Q | _I | U | o | LU | LU | > | LU | X | > | ||
α? | Ol | Ol | O | O) | 0 | o | 0 | a | CM | CM | CM | to | CM | to | co | 0 | ||
eo | o | 0 | uo | o | 0 | 0 | to | |||||||||||
CM | CM | CM | *“ | CM | CM | CM | CM | CM | ÍM | CM | CM | CM | ||||||
σ | > | o | o | > | Z | X | z | X | co | co | CO | 1- | < | H | < | |||
f 1 | > | Q | X | — | G | G | O | G | G | G | G | G | Q | CO | O | a | Q | |
CM | Ol | Ol | Ol | Cl | ai | oi | Ol | Ol | Ol | Ol | Ol | <O | Ol | Ol | 0 | |||
eo | 0 | o | o | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | |||
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64CZ 302287 B6
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Claims (12)
1. Varianta subtilisinu, obsahující zbytek alaninu v poloze, odpovídající zbytku 103 v subtilisinu Bacillus amyloliquefaciens a zbytek arginínu, leucinu nebo valinu v poloze, odpovídající zbytku 245 v subtilisinu Bacillus amyloliquefaciens a mimoto jednu nebo větší počet substitucí zbytků aminokyselin, vybraných ze skupiny, tvořené zbytky v polohách, odpovídajících polohám 3, 12, 20, 27, 38, 43, 48, 57, 61, 62, 68, 76, 87, 89, 97, 98, 99, 101, 102, 104, 109, 114, 121, 128,
130, 131, 133, 140, 141, 147, 159, 160, 166, 167, 170, 173, 174, 184, 185, 192, 194, 203, 204,
205, 206, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 222, 224, 227, 228, 232, 236, 237,
242, 243, 244, 247, 248, 251, 252, 255, 256, 257, 259, 260, 261, 262, 268, 269, 271, 272 a 275 subtilisinu Bacillus amyloliquefaciens, přičemž v případě, že substituce v poloze, odpovídající poloze 103 subtilisinu Bacillus amyloliquefaciens je kombinována se substitucí, odpovídající poloze 76, je obsažena rovněž substituce v jedné nebo větším počtu poloh, odlišných od poloh 27, 99, 101, 104, 109, 128, 166, 204, 206, 210, 216, 217, 218, 222 nebo 260 subtilisinu Bacillus amyloliquefaciens za předpokladu, že aminokyselinou v poloze, odpovídající poloze 232 v subtilisinu Bacillus amyloliquefaciens je aminokyselina, odlišná od valinu, přičemž varianta subtilisinu má zvýšenou prací účinnost ve srovnání s prekurzorovou proteázou bez uvedených substitucí.
2. Varianta subtilisinu podle nároku 1, obsahující jednu nebo větší počet substitucí zbytků aminokyselin, vybraných ze skupiny, tvořené zbytky v polohách, odpovídajících polohám 12, 61, 62, 68, 76, 97, 98, 101, 102, 104, 109, 130, 131, 159, 170, 185, 205, 209, 210, 211, 212, 213, 215, 217, 222, 232,248, 252, 257, 260, 261 a 275 subtilisinu Bacillus amyloliquefaciens.
3. Varianta subtilisinu podle nároku 1, obsahující substituci v poloze, odpovídající poloze 236 a mimoto jednu nebo větší počet substitucí zbytků aminokyselin, vybraných ze skupiny, tvořené zbytky v polohách, odpovídajících polohám 12, 61, 62, 68, 76, 97, 98, 101, 102, 104, 109, 130,
131, 159, 185, 205, 209, 210, 211, 212, 213, 215, 217, 232, 243, 248, 252, 257, 260 a 275 subtilisinu Bacillus amyloliquefaciens.
4. Varianta subtilisinu podle kteréhokoliv z nároků 1 až 3, obsahující substituci zbytku aminokyseliny v poloze, odpovídající poloze 159 subtilisinu Bacillus amyloliquefaciens.
5. Varianta subtilisinu podle nároku 1, odvozená od subtilisinu Bacillus.
6. Varianta subtilisinu podle nároku 1, odvozená od subtilisinu Bacillus lentus.
7. Kódová DNA pro subtilisin podle nároku 1.
8. Vektor pro expresi, obsahující kódovou DNA podle nároku 7.
9. Hostitelská buňka, transformovaná vektorem pro expresi podle nároku 8.
10. Čisticí prostředek, vyznačující se tím, že obsahuje variantu subtilisinu podle nároku 1.
11. Živočišné krmivo, vyznačující se tím, že obsahuje variantu subtilisinu podle nároku 1.
12. Prostředek pro ošetření textilních materiálů, vyznačující se tím, že obsahuje variantu subtilisinu podle nároku 1.
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US95656497A | 1997-10-23 | 1997-10-23 |
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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 |
CZ20001503A CZ301469B6 (cs) | 1997-10-23 | 1998-10-23 | Subtilizín a prostredek s jeho obsahem |
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CZ20001493A CZ300347B6 (cs) | 1997-10-23 | 1998-10-23 | Zpusob zvýšení úcinnosti subtilizinu a zpusob výroby pracího prostredku |
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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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1998
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2001
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2002
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2006
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2012
- 2012-06-20 JP JP2012138496A patent/JP5612026B2/ja not_active Expired - Fee Related
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WO1989006279A1 (en) * | 1988-01-07 | 1989-07-13 | Novo-Nordisk A/S | Mutated subtilisin genes |
WO1991000345A1 (en) * | 1989-06-26 | 1991-01-10 | Novo Nordisk A/S | A mutated subtilisin protease |
WO1995010615A1 (en) * | 1993-10-14 | 1995-04-20 | Genencor International, Inc. | Subtilisin variants |
WO1995030010A1 (en) * | 1994-05-02 | 1995-11-09 | The Procter & Gamble Company | Subtilisin bpn' variants having decreased adsorption and increased hydrolysis |
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Legal Events
Date | Code | Title | Description |
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PD00 | Pending as of 2000-06-30 in czech republic | ||
MM4A | Patent lapsed due to non-payment of fee |
Effective date: 20161023 |