TW562859B - Multiply-substituted subtilisin variants - Google Patents
Multiply-substituted subtilisin variants Download PDFInfo
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- TW562859B TW562859B TW087117606A TW87117606A TW562859B TW 562859 B TW562859 B TW 562859B TW 087117606 A TW087117606 A TW 087117606A TW 87117606 A TW87117606 A TW 87117606A TW 562859 B TW562859 B TW 562859B
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Description
562859 五、發明說明(i) 發明之背景 絲胺酸蛋白酶為羰基水靜酶之亞群。彼等包含不同類具 廣泛圍特異性及生物功能之之酶。S ΐr 〇u d,R. Sc i A m e r ·, 1 3 1 : 7 4 - 8 8。不論其在功能上之歧異性,絲铵酸 蛋白I每之催化機制已藉下列至少二種遺傳上不同族群之_ 達成·· 1 )枯草桿菌素,以及2)哺乳動物胰凝乳蛋白酶-相 關絲胺酸蛋白酶及同等的細菌絲胺酸蛋白酶(例如胰蛋白 酶及S. g r e s i u s姨蛋白酶)。此二族群之絲胺酸蛋白i每顯 示顯著相似之催化機制。K r a u t, J · ( 1 9 7 7 ),Annu. Rev. B i ochem.,4 6 : 3 3 1 -3 5 8。再者,餘然此二酶族群之一級 結構不相關,但其三級結構均帶有甴絲胺酸、組胺酸及天 冬胺酸組成之胺基酸之守恆催化三元組。 5 枯草桿菌素為由廣泛種類之枯草芽孢桿菌及其他微生物j 所大量分泌之絲铵酸皰内蛋白酶(分子量約2 7, 5 0 0 )。至少; 來自四不同種芽孢桿菌之枯草芽孢桿菌素之蛋白質序列曾 被測定。M a r k 1 a n d,F . S . e t a 1 . ( 1 9 8 3 ),
Hoppe — Seyler’s Z. Physiol. Chem. , 364: 1537-1540。解 | 殿粉枯草芽抱桿g型枯草芽孢桿菌素之解析度為2 . 5 A之三|
I 度空間結晶圖結構已被報告。Wnght, C. S., et ! al.(1969), Nature, 221:235-242 ;Drenth, J., et al. j j ( 1 9 7 2 ), Eur . J. Biochem., 2 6 : 1 7 7 - 1 8 1。此等研究顯示 雖然枯草芽孢桿g素在遺傳上與哺乳動物絲胺酸蛋白i每不 相關,其具有相似的活性部位結構。含有共價鍵結之肽抑 制劑之枯草芽抱桿菌素(R〇b e r t u s, J · D., e t a i. ( 1 9 7 2 ), 562859 五、發明說明(2)
Biochemistry,11:2439-2449)或產物複合物(Robert us, J, D·,et al · ( 1 9 7 6 ),J. Biol· Chem·,2 5 1:1 0 9 7- 1 1 0 3 ) 之X -光結晶結構亦提供有關枯草芽孢桿菌素之活性部位及! 推定之受質結合裂口之資料。此外,已報告大量有關枯草 芽孢桿菌之動力及化學修改研究(Ph i 1 ipp,iM·, ei: a 1. ( 1 9 83 ),Mol. Cell. Bi ochem·,51:5-32 ; Svendsen, B.(1976), Carlsberg Res. Commun., 41:237-291 ;
Markland, F.S. Id.),以及在至少一報告中,枯草芽孢 ^ 桿菌素之殘基2 2 2上之甲硫胺酸之支鏈藉過氧化氫轉變成 j 曱硫胺酸-亞碾(Stauffer, D. C·,el: al. ( 1 9 6 5 ), J·
Biol.Chen^, 2 44 : 5 3 3 3 - 5 3 3 8 ),以及在殘棊 2 2 1 上之絲胺 酸之支鏈藉化學修改轉變成胱膝酸(Polgar,et a 1. (19 81),B i o ch i m. e t B i oph v s. Acta, 667:351-354)。 發明之概j i 本發明之一目的為提供一種蛋白酶變異體,其在對應於I 解激粉芽孢桿菌型枯草芽孢桿菌素之位置1 0 3之殘基位置 上含有铵基酸之取代、刪除或***,以及在選自對應於解 ;殿粉芽跑桿菌型枯草芽孢桿菌素之下列位置之殘基位置所 組成族群中之殘基位置上,取代、刪除或***一個或以上j 之胺基醆:1,3,4,8,10,12,13,15-18 ,20-22, | j 24 ,25 ,27 ,33 ,37 ,38 ,42 ,43 ,48 ,55 ,57 ,61 , | 62 ’ 68 ’ 72 , 75-79 , 86 , 87 , 89 , 97-99 , 101 , 102 , 104 ’106 ,1〇7 ,1〇9 ,111 , 114-117 , 119 , 121 , 123 ,
I—
第10頁 562859 五、發明說明(3) 128,130,131,133,134,137,140-142,146,147, 158,159,160,161,166,167,170,173,174,177, 181-185 ,188 ,192 ,194 ,198 ,203-206 ,209-218 , 222,2 24,2 27,2 28,2 3 0,232,2 36-23 8,24 0, 242-249 , 251-263 , 265 , 268-272 , 274及275 ; 其中當對應於殘基位置1 0 3之位置上之取代與對應於殘基 位置7 6之位置上之取代組合時,在對應於解澱粉芽孢桿菌 型枯草芽孢桿菌素之位置27,99,1〇1,1〇4,107,109, 123,128,166,204,206,210,216,217,218,222, · 260,265或274之殘基位置以外之一個或以上殘基位置上 有一取代。 雖然可以使用上列胺基酸取代之任何組合,但對本發明 而言’較佳之蛋白酶變異體包含在下列組合中之胺基酸殘 基之取代。所有殘基位置對應於解澱粉芽孢桿菌型枯草芽 孢桿菌素之位置: (1 )在位置1 〇 3以及位置2 3 6及2 4 5中之一個或以上之胺基 酸殘基被取代之蛋白酶變異體; (2 )在位置1 〇 3及2 3 6以及一個或以上之下列位置之胺基 | 酸殘基被取代之蛋白酶變異體·· 1,9,1 2,61,6 2,6 8, | 76 ’ 97 , 98 , 1〇1 , 1〇2 , 104 , 109 , 130 , 131 , 159 , | 183 ’185,205,209,210,2Π,212,213,215,217,! 2 3 0 ’ 2 32 ’ 2 48,2 52,2 57,2 6 0,2 70 及 2 7 5 ; ί (3 )在位置丨q 3及2 4 5以及一個或以上之下列位置之胺基 | 毁殘基被取代之蛋白酶變異體:1,9,1 2,6 1,6 2,6 8,
第11頁 562859 五、發明說明(4) 7 6,9 7,9 8,1 0 1,1 0 2,1 0 4,1 0 9,1 3 0,1 3 1,I 5 9, 170,183,185,205,209,210,211,212,213,215, 217,2 22,2 3 0,2 32,2 48,2 52,2 57,2 6 0,261,2 7 0 及 2 7 5 ;以及 (4)在位置103,236及245以及一個或以上之下列位置之 胺基酸殘基被取代之蛋白_變異體:1,9,12,61,62, 6 8,7 6,9 7,9 8,1 0 1,1 0 2,1 0 4,1 0 9,1 3 0 ’ 1 3 1 ’ 1 5 9,1 8 3,1 8 5,2 0 5,2 0 9,2 1 0,2 1 1 ’ 2 1 2 ’ 2 1 3 ’ 2 1 5 ’ 217,230,232,243,248,252,257,260,270 及275 ° 更佳之蛋白酶變異體被示於下列表I中:
第12頁 562859 五、發明說明(5)
〇〇 CD CO G) O) G) —k G) S) S) Ο O) o 〇 CO —λ o —X 〇 U) G) G) —x o CJ —λ o u> o OJ CO CO »λ 〇 CO -k Ο OJ o OJ 〇 oo s ^X o o ~k o s __^ 〇 CO _X o _X o CO o X. 〇 CO _k. o —k o OJ o 〇L> —w o o λ. o —^ o ·>L o fO CJ) —X g ro cr> ^JL 't —X o k o CD CO _k o ro CD —X. o ^k o _λ o —X o ro ro ro ~X fO CD ro CO
苐13頁 562859
562859 五、發明說明(7) ro Ο) ο σ) Ο) Ο) ro 05 O) M O) cn CO s O) 0 S CD CO ο 0J ο C0 Ο) —k ο C0 S 2 o CO 〇 CO —x 〇 CJ O) o OJ o CO —Jl 〇 CO ο —X Ο ω —k ο —X ο —X ο C0 —k ο V. o OJ _^ O OJ λ. 〇 _^ 〇 CO _X o _λ. o _X O OJ s —X o —k 〇 CO o _λ. ο CD ~k —X Ο 8 ο CD CO —1 o —λ. 〇 A ro CD N) —X o __i> 00 cn ro 'vl 〇 _X o — 〇 cn CO —k o ro o Μ Ο Μ CO ro GJ CD -vj nj Ol _k
第15頁 562859 五、發明說明(8) cn ΓΟ -νΐ ο v. 〇 CO G) CD 'vl 〇 〇 〇J CD CD CD CD CO ro CD -vl o •vj σ) 〇 S -vl Ο) S —X o s CO —^ O CJ —k 〇 CO -vl cn CD _k 〇 CO —k 〇 CO 〜 G) CD X 〇 CO —X o 〇J —k o 0J 〇 0J 〇 λ. Ο GO ο ro CD —1 o X. o CO k o o A —>λ o 03 __x. 〇 GO __λ 〇 —k o 〇 CO s _k o o —X o _k o —k —X CD Ο λ. CD ro —1 CO X o I —k CD ro X σ o ro __^ ro CJl CD —X o s ro fO ro ro cn ro ro _V ro _k CO V. CD ΓΟ —^ CD OD ro CO OD ro 〇 G) CD fO ro k CD ro _X CTi ro ro ro G) —λ. ro _k ro ~vj X. ro CO ro CD co λ. CD CJ ro CD un _1 1
第16頁 562859 五、發明說明(9)
第丨7頁 562859 五、發明說明(ίο)
第18頁 562859 五、發明說明(11) Μ O) cn 03 CD CD O CD σ> CD 05 CD G) CD CD CO ro CD CD CD G) CD CD CD CD CD CD CO CD Ο) »λ o OJ o O) O) o CD CD s G) O σ> CO O G) 〇 0J o _X. o GJ s X 〇 CJ __k o CO s s 〇 U) 〇) 〇) <_x o CJ »1 〇 CO O) 〇 CJ _x 〇 CO _^ O 0J ο ro ro ro o ~k o —X o -JL o X o o —X 〇 —X 〇 CO —X o CO —k 〇 c〇 o o o CO o οX o —w o ro ro ro N) CJl U1 CD ro CJ _1 A ro —X cn CD X cn CO —λ —i. cn CD _X o _X _X A _^ o _k cn CD —X o CJl CD _k cn CD —X. on CO ro CD ro 00 CD —V cn CD —x cn CD M M O) —X cn CO M CD λ. cn CD ro X. —^ cn CD ro 0J CD NJ __λ. ro CJ G) NJ GJ G) CD ro CD M cn ro CJ _λ. s ro o ro CD —λ cn CO ro co CD M 03 〇 M cn ro _^ ro CD ro CJ CD ro cn c〇 ro CJ CD fvj -vl _k M cn
第19頁 562859 五、發明說明(12) ro CD CD CD CO cn CD cn 00 O) 00 CD CD CD CO 〇 CD -vl 〇) -Nl CD CD CD —λ CD 'vl σ> ro -vj CD -vj cn Ο) G) 'Vl cn G) CD CD -vl o O) -vj G) k o O·) —λ. o CJ V. o OJ CD —k o OJ 〇 0J O) _λ. o OJ o CO 〇 OJ o CJ o CO o 00 o 00 __x o LO X 〇 CO o OJ —i. o CJ _X : o —k o o 〇 _X. o —k o 〇 CJ k o k o _JL Ο L o —k o —X o —X o —X o o —k o o ro N) ro k 〇 CD k CO -JL o —k. 〇 CD i. 〇 CO k o ro ro ro ·_k OJ N3 ro ro __X cn CD —X cn CD λ CO 0J _X C71 CD __λ. cn CD —X cn CD 公 cn CD N) CD ro ro ro M ro ro M ΓΟ ro ro ro M ro ro ro ro ro N3 M 〇) c〇 N3 ro N3 M Ol _k s ro CO ro cn CD ro o _X ro CJ CD Ol CD ro co CD ro CD ro ro GJ -vl ro s ro CO CD ro —X CO ro ω σ> ro σ ro cn ro CO CD ro 公 on ro cn OJ I 1 ro CO CD M cn ro cn ro cn ro CJ CD M ro Ol N) CD —X ro on M Oi ro cn ro cn cn 562859
第21頁 562859 五、發钥說明(14)
〇 CD CD cr> CD CD CO cn CD O) OD o σ> CD CD CD cn CD f〇 ro ro ro ro S —λ o 0J _X. o OJ s o U) —JL o CO σ> CD —λ o ω -A o CJ __V. o 0J G) O) O) Oj _x 〇 0J O) O k ο o o _k o o s —k o —5*. o _k o s —3l 〇 CO —X o u> —V· 〇 CO —k o —X o CO _λ. 〇 03 CJ1 CO —Jk. cn CD Ol CD 〇 CO _k Ol CD _k (J\ CD o V. cn CO X ^k CD -_X cn CD λ. o o —i. o _k o 0J o —i. 0J o _L 〇 -Cx _i. o —k OD 00 〇k k OD CO _X. o M ro ro s Ol CD ro 03 ro _ Un CD ro 〇J ro _^ _k CO 〇 —^ CJ 〇 _^ o _X CO 〇 ro ro ro _λ CJ o _λ. CO o ro OJ ro ro § ro U) ro k cn CD ro 0J CD ro ro ro OJ ro ro c〇 CD ro co ro ro CO CD N) fO ro ro ro ro 〇 __V. 〇 ro _^ 00 ro cn ro ro ro ro ro ro ro CO CD M OJ ro ro OJ CD ro GJ ro M CO ro CO CD ro c〇 CD ΓΟ cn ro 0J CD f〇 cn ro _k 〇 ro 么 cn _k CD CJ1 ro ro ro ro N3 ro ro 厶 cn ro ro 'Nl ro cn ro 0J CD Ol M OJ CD CJl ro CJl N) cn ro CD M cn ro cn -^1 fO cn ro CD CD ro ro ro ro ΟΊ cn M G) _ M ΟΊ ro cn M CJl ro CO ro cn __v ro CD ro ro o ro M 么 cn NJ CD CD
562859 _ ___-— ----------------------!
五、發明說明(15) I
Μ NJ O) CO ro CD CD CD CD O) G) CD 5) CD CO C) O) CD CD CD O) CD cn CO CD CD CD CO CD CD Ο) CO CD 00 O) CD CD O) —X o CO o CO »X o CO __^ 〇 00 λ 〇 CO O) xV o CO >_λ 〇 CO CD 〇 CJ Ο) s 〇 0J —k o CO —k o —λ o o —X 〇 —X o —X 〇 v. 〇 CO —X o Cx) —^ o —k o —X s —k o s 〇 CO o ~X 〇 CO _k o _k o —X cn CD V. cn CD ro aj V. cn CD ro GJ ro —λ o _X o _k o cn CD on CD _x o cn CD ο —k o —k Ol CD —k o »λ cn CD —Jk cn CD ro CO ro ro ro ro cn ro co ro ro LO CJ) cn CD cn CD _k cn CO ro CO ro ro __ cn —X cn CjO ro CJ o —i. cn C£> cn CO ΓΟ _V cn _X cn CD ro _V g CO ro 0J CD ro CO M ro co O) ro cn ro c〇 ro ro —^ 〇 JNJ CO N) ro U) G) ro OJ ro ro OJ ro ro CJ ro ro CO ro ro N) ro ro ro ro to ro ro c〇 ro ro cn ΓΟ 00 CD ro cn ro cn ro oo cn NJ GO ro NJ OJ CD N) cn NJ GJ σ> INJ CJ CD N) OJ σ> ro CO σ) ro 0J CD ro CJ CD ro CO ro ro GO cn ro c〇 cn ro cn cn ro cn ro cn N3 OJ CD ro 0*1 ro 么 CD ro 二 cn M cn M cn M σι ro cn ro cn ro 0J CD N) cn ro cn ro Oj ro CJl ro CJI M cn ro cn CD ro cn 第23頁 562859 五、發明說明(16) G) C0 CJ) 00 6 2 CD CO ro CJ) CD CD CD C7) CD ro O) 5) O) NJ CJ) CD C) CD CD G) CD —X 〇 GJ __V. 〇 OJ cn CD G) CD G) cn 03 o s o O) CD 〇 CO 〇 CJ 〇 CO 会 G) _k o OJ G) a k Ο ^k o _^ o GJ s —λ 〇 CO O) X 〇 Cx) _X. o CO _X 〇 CO O) o _X σ X. o CD CD s OJ k o k 〇 CJ 〇 CD CD _JL cn CO —X 〇 o —Λ. o 〇 CO o —X o —X o _x 〇 CO —Jl 么 λ. (Si CD ro GJ ro o o ro GJ ro _V. o 〇 CJ __k cn CD M — ro cn CD —k CJ1 CD k CJ) X o ·__λ cn CD cn CD λ. cn CD o —k cn CD —A. CD ro ro CO σ> 〇 OJ V. cn CO ro CJ cn —k cn CD o s ro OJ ro N3 OJ ΓΟ ro co ro __k CJ1 CO cn CD ro o CD ro OJ ro __λ. CO —k cn CD ro co ro ro CJ ro M cn o ro 〇 M a ro M CJ ro __λ cn CD ro M ro c〇 CD ro OJ CD ro CO CD -vl o ro c〇 ro ro c〇 M N3 CO CD ro 〇 CJ) ro co ro ro co CD ro CO CD —λ. CTl CD ro OJ ro M cn OJ CD ΓΟ CJ ro ro OJ O) fO cn M cn M cn __X CD cn ro OJ cn N· OJ σ> ro cn ro OJ ro ro OJ CD ro 么 cn M cn ro OJ N) ro CJ σ> ro 二 on ro CJ G) N) CJl ro CD ro 么 CD M CD ro GJ M M cn ro Ol ro O) ro OJ CD ro cn ro CD ro OJ CD ro cn ro 〇 __ ro cr. M CD ro Ol ro ro cn ro M (Si N) ro co C7) ro cn ro ro cn __^ M 二 cn ro o 〇 ro cn N3 M cn
562859 五、發明說明(π) CD CD CD CO O) 03 CO CD 〇 CD CD CD σ> CD O) ca CD co CD co σ> co o co CD CD CD CO CD CD _λ. 〇 GJ __λ Ο CO »k 〇 00 V. 〇 CO G) CD —X 〇 OJ _x 〇 CO -A o CO —A. o CO v. 〇 co _k o —A. o 03 --λ o CO _k 〇 CO __x o CO co CD —λ. 〇 GJ _^ o OJ —>> 〇 二 —k ο o o —V. 〇 OJ _k o k o λ. o —X o —λ. o —X o _k o —^ o o λ 〇 _V. o CJ —X. o X , o _λ. cn CO k CJ1 CO —λ. cn CD λ cn CD ·_·λ. o A —λ. σι CD cn CD ro x CJ — ur CO —Jk. cn CD cn CD —3>· cn co __X cn CD —V. cn CD Ik cn CD _k o —k o CD _k cn CD 0J NJ r〇 o CD ro 00 ro __^ -vj CO k cn CO ro CD ro V. un ro CO ro fO CJ ro X ro _λ 03 cn ro _k 〇 >_λ 03 cn ro OJ ro fO 〇 CO __X cn co —X. cn CD ro § ro CO o ro a> ro ro O) ro co ro M CJ ro ro CJ ro ro OJ ro ro GJ cn ro CO M ro CJ M ro _x 〇 ro OJ ro ro 0J ro ro 03 CD ro co ro ro 0J M ro 0J ro CO ro ro cn ro OJ CD ro 公 cn ro CO O) ?NJ CO CD ro CO O) ro CJ O) N) cn ro co CD ro CO G) ro OJ ro NJ CJ CJ) ro 0J CD CJl ro 0J G) r\j OJ CD inj CJ CJ) μ 0J o ro CD M cn NJ CD M cn ro 厶 cn ro cn ro cn ro OD ro cn ro ΟΛ ro 0J cn ro 么 cn M cn ro CO ro 厶 CJl l\J cn NJ 么 cn cn ro cn NJ M CO ro cn _k. N) CD ro 二 CD M CO fO CD ro CJl N) ro 03 N) CD ro cn ro 二 0D ro 么 00 ro cn M ro Tx 00 ro CO ro CO NJ CD ΓΌ cn ro ro cn ro N3 cn ro N3 cn ro ro cn ro ro cn ro ro cn ro ro cn ro M CD ro cn ro ro cn NJ ro 〇 —X cn ro ro C71 ro M cn ro ΓΌ cn ro INJ cn ro
苐25頁 562859 五、發明說明(18)
〇 C0 CD CD CD G) CD 〇 CJ cn CD CD CO CD CD σ> CO CD CO CD O) CD CD CD CD CD CD CD σ) CD 0*1 cn G) CO σ) __v. o CO CD 03 —1 o CO o —X o CO o _x o CO —X o co —k 〇 CO CD CD _3k 〇 CO o 〇-> ·_·X 〇 CO —X o CO _k o CO G) CD __λ. o CO 〇 o —^ 〇 CO —X o cn CD —X o _X o CO —V o o — o __k. 〇 OJ _X ' o o o o —X 〇 _V 〇 CJ _^ o 〇 CJ cn CO —X o —X cn CQ ro CO ro M CO NJ —X o — cn CO —X cn CD k —k CD —X o _^ cn CD k CJl CD λ CJl CD —λ cn CD _k 〇i to _λ o k cn CD k ο ro CO ro _k cn CD ro CO fO ro OJ O) ro OJ CD —V, cn CO ro OJ N) ro ro k cn CD _λ cn CD ro OJ ro OJ ro ΓΟ OJ NJ ro OJ M ro OJ ro cr· CD OJ M _X cn CD ro OJ cn ro c〇 ro ro CD ro cn ro 么 CJ1 ro CO ro ro OJ CD ro CO CD ro CO ro N) GJ ro ro GJ cn ro CJ G) ro GJ ① ro CO O) ro OJ σ> ro CO ro ro OJ CD ro —v. ro cn ro ω σ> ro cn ro CD M CD ro OJ σ> ro cn ro cn ro OJ CD M CO o ro cn ro -C^ CJl Oi N) cn M ΟΊ ro CJ O N) ro —^ CO ro CD ro cn ro 么 CD ro CJl ro ro CJl ro ro cn ro CD ro CD ro Ol NJ 么 cn ro CD ro CO ro CD M CD ro 厶 CD ro 二 cn l\3 CD ro CO ro ro C71 ro M CD ro cn M ro 么 CD N3 cn M ro cn ro ro CO M CD ΓΟ cn ro ro cn ro ro cn ro ro ΟΊ ro (SJ on ro NJ 二 CO ΓΟ cn INJ N3 OJ CD ro cn ro ro cn ro ro o ro CD __λ. ro cn ro ro CJl ro ro cn CD ro cn M CT) 〇 ro cn G) M cn cn ,NJ (-Π i 1 M ΟΊ ro CD 〇 M ① CD 1 丨 ro CD o ! 1 第26頁
562859 五、發明說明(19) _λ. 〇 ΓΟ o ~k CD CD CD CD CO CD CO cn cn CD O) CD G) cn CD _λ. O 0J CD O) CO CJ 03 CJ) CO —λ ο CJ o OJ _x 〇 OJ 〇 CO 〇 CD CD CD CO 00 _X 〇 o CO o co cn co o CO _k o 〇 CO G) CJ) CD _k 〇 OJ ο _k o o —k o —Λ o —X. o OJ cn CD s o o o k o 0J o OJ —k on CO CD CD O) —k o cn CD _k cn CD cn CD —X cn CD —λ cn CO ~k o 5) —k o —k cn CD _λ. cn CD _k U) 0J o k o ro 〇 (j\ —X o CO —X 〇 CJ —X 〇 00 _k cn CD M CO ro ro CO ro ro 03 ro ro ro ro CJ ro —X cn CO _X. o OJ _X cn CO ro _ CD ro CJ ro _X cn CD _V CO U) —k GJ 〇 ro _Ik. 〇 _w o —k o _k o ro ro CO ΓΟ OJ G) ro to CD ro CO CD Μ GJ CD ro CO G) —k o —k CD 〇 ΓΟ 0J ro ro 0J cn ro CJ N3 _k 0J -vl _X cn CO M CJ M _x cn CO —V cn CD cn CD ro 0J ro M cn ro cn ro on ro A cn cn ro CO ro ro OJ ro ro CO ro ro CJ cn ro cn ro 0J CD X cn CD ro 0J ro ro CO G) ro CJ ro ro 〇 ro CJ ro NJ 0J 二 M CD M CD CD CD ro CD M GJ CD fO GJ cn ro :CO CD ro 么 CJl ro CD ro cn ro co ro ro OJ CD ro 二 αι ro CJ O) M _X 0J ro CJ G) ro 二 ΟΊ ro CJi N) ro cn ro ΓΟ ΟΊ ro ro cn ro ro on ro ro cn ro cn ro 公 cn ro CD N) cn ro M CD ro 0J cn ro cn ro 二 CD ro CO NJ ro a cn CO M CO ro CD M 00 ro cn ro Μ cn ro ro cn ro 00 ΓΟ CD ro co CD ro CO N3 ΟΊ ΓΟ M cn N3 M cn N3 ro cn M ro CO ro cn ro NJ cn NJ cn ro on ro ro Ol ro ro G) o
第27頁 562859 五、發明說明(20) 〇 CD 〇 03 OJ CD CJ CD ro __λ. o OJ 〇 c〇 G) ro —k 〇 OJ cn ro _k 〇 CO o 0J —Λ. 〇 0J CD ΓΟ ·_k 〇 0J —^ 〇 CO __X 〇 CJ Ο) C) O) —^ o 03 __v. 〇 0J o o _λ o CJ _k o —1. 〇 CO cn ro —k o _ o —X o 〇 OJ —X 〇 •ix —^ o o 3 λ. o CO _X o OJ X o X o _k OJ -«>k 0J 〇 λ o —X cn CD o —k 〇 Cv) cn CD CJ1 CD cn CD 〇 —X or CD 〇 CD s _X 〇 _X 〇 —X o λ cn CO X cn CD λ cn CD cn CD __X CJ1 CO ro o CD __X. cn CD _λ o ro _k. cn CD _1 g V. cn CO N3 OJ ISJ ΟΊ CO _λ. cn CO —k cn CD —X cn CD —V. cn CD ro OJ ro ro OJ ro fO OJ ro ro CJ ro ro o CD ro __^ ro _X CO k cn CD ro 0J ro NJ OJ M ro CO ro ro CO ro ro CJ CJ) NJ 0J ro ro CO ro ro § ro _X CO ro _k OJ ro G) ro GJ G) ro σ> ro CJ cn M LO ro ro 〇J ro ro GJ ro ro —k CO ro CO CD ro CO CD ro OJ ro CO CD ro 二 cn ro CO CD ro OJ CD M —k CJ ro co ro ro CO M ro cn ro cn ro cn N3 办 cn ro GJ CD ro 0J ① ro CO CD ro ro ro cn M cn ro 么 On ro cn ro 二 CD ro cn ro 二 cn ro CO ro ro OJ CD ro OJ cn ro CO N) CD ro CD ro CO M cn ro ΟΊ ro cn ro GJ CJ) ro CO ro CO ΓΟ CD ro CO M cn ro ro cn ro 二 CD ro GJ CD M cn M cn ro cn ro ro cn ro M un ro ro (SI ro M CD ro CO ro CD M cn ro cn ro ro ΟΊ ro ro cn ro N3 ΟΊ ΓΟ M CD _λ. ro cn M ro on ro ro cn ro CD 〇 ro g ro cn ro ro cn ro M cn ro ro 么 CO ro g ro -nI _k ro cn ro
苐28頁 562859 五、發明說明(21) k 〇 OJ G) 00 O) CD cn CD __x o CJ CD CD —^ 〇 0J CD CD CD CO CD 00 CD CD cn CD CD CO CD CD CD G) CD CD CD 〇 X. 〇 OJ —A. o w X 〇 CO _X o k 〇 u> _λ. o 〇 CO _λ. o CJ _JL 〇 CO —V 〇 CJ o 3k, o 〇 CO 〇 GJ G) S G) X cn 〇 _X o o —X 〇 __λ cn CO _X o —k cn CD X o o ««λ. o _k o —X o o ^k. o _k 〇 s -k 〇 CJ _x 〇 CJ ro co ro —X cn CO —X cn CD cn CD ro co —x CJ] CD ro —X cn CO k. cn CD —i. CJ1 (SD Ol CD — cn CD cn co cn CD __k CJl CD _X 〇 A k o o ro c〇 σ> ro 〇 CD X CO 公 —X ro CO CJ) ro OJ ro ro 0J CD ro 〇 ro o cn —乂 M CD ro 0J 〇 ro o ro o co ro _«x. OJ ro OJ _X Ol CO cn co cn CO ro 0*1 ro CO M ro 03 ro ro CO ro ro cn ro co CD cn M CO ro ro CO ro ro OJ ro ro CO fO ro CO ro ro OJ ro ro U) ro ro co ro ro _>Ik. o ro o Cn ro s ro Oj ro CO σ> ro GJ cn ro CO CD ro Ol ro CO CD N) CJ CO M CO CD M 〇L> CD ro c〇 CD ro GJ 〇 ro CJ CD ro CO CD CJ NJ ro _k CJ 1 ro _x co ! ro cn M cn ro cn N3 CD 〇 ro cn ro cn ro cn ro 么 cn ro cn NJ cn ro 么 cn ro 二 cn fO CJ CO ro CJ M ro OJ ro ro un ro cn ro cn ro CD 〇 ro CD 〇 cn ro CO G) ro OJ CD ro CD o M A cn 二 cn hJ σ> 〇 ro G) 〇
第29頁 562859 五、發明說明(22) 〇 C0 〇 CJ O CJ o OJ O) CD —k o CJ —k o CJ CD CO —k 〇 OJ —k 〇 CO o 〇J CD CO 〇 CJ o OJ 〇 cu CD CD G) CD X 〇 C0 o 二 X o —X. o o CO —^ o _k o s o _3k o _λ. o —k 〇 CJ ci o o _k o s C) —V 〇 —λ. on CD —X cn CD — ΟΊ CD —X o X cn CO _X on CD 1 o λ CJ; CO cn CD ro o CD 3L 〇 _x o CO cn CO X cn CD V. CJl CO k o _X 〇 CO ro CD ro 〇 cn N3 〇 CD CJl cn CD CD ro 〇 〇l —V. cn CD ro o cn M cn ro CJ ro —x ΟΊ CD —X o N3 〇 CO ro _k 〇 ro CJ —i. cn CD —k 〇 CJ1 CD ro O M 〇 CD ro 〇 G1 ro o CD ro 〇 CD ro s CD ro _k 〇 ro CO cn ro 〇 CD —X cn CO ro 〇J ro ro CJ ro ro CJ M l\J OJ M cn CO Μ ω Ν3 ro CJ ro M CO ro ro —Ik o ro 〇 CO ro LO ro ro —_V. o ro s ro CO ro ro _X OJ M cn ro CO _k 〇 M σ> ro CJ CD ΓΟ O·) G) ro GJ G) ro GJ ro 〇J CD ro OJ G) ro CO cn ro OJ M ro —v o ro 〇〇 〇) ro CJ M ro k 〇 ro OJ CJ) ro CO ro ro cn ro OJ ro ro _k OJ N: 二 cn M CJl cr\ 0*1 ro OJ ro ro 二 cn M 二 cn N) cn ro c〇 O) ro CO N3 ro a CJl CO CD ro CO ro ro on M CO CD ro OJ CD ro OJ N3 ro LTi ro a CD ro CJ) 〇 ro cn ro CJ C7) N3 公 αι NJ OJ CD ro Ol ro C71 ro CJ CD M CD 〇 on ro 么 cn ro CO CJ) ro cn ro ro cn «_^ NJ cn K> A cn ro Ol cn ro CD o ro CD 〇 ro cn M CD 〇 ro O) 〇 ro CD 〇 ΓΟ G) _k 562859 五、發明說明(23) CD CD —X 〇 _k 〇 _k s «.X ro O) ro O OJ 〇 CJ __>>. o CJ _X 〇 ro CD CD 〇 k _X ro NJ A CD CD -Dx CO CD s _X o GO S —X 〇 03 O) ro k o U) o 3L o X 〇 —^ 〇 CO _3L o NJ s s M ^.Λ O CO CD CD CD CD CD OD —k O CJ Ο o o —^ o s o X cn CD cn CO CO —X o —x 〇 03 —w o CO —k 〇 OJ k. o —k O CO _v. 〇 OJ 〇 CO :_V o _X ο —Λ cn CD cn CD ·_k cn CD o —k cn CD ro CJ ro «.X s i. cn CD _*· cn CD —X o _X o o X cn CD —k o _k o o —X cn CO λ U1 CD ro _k CO ro § —^ CD cn —X CJ1 CO ro —x. CO ro OJ cn ro OJ ro ro CJ ro ro — 03 On CD _X cn CO cn CO ro —V. NJ —^ cn CD —X cn CD —X cn CD ro OJ o ro OJ ro N3 CJ hJ INJ OJ ro ro GJ ro M —λ. CO ro CO ro ro 公 ro OJ CD ro CJ cn ro OJ ro ro _x. ro N3 V. ro ro —X ro ΓΟ CJ ro ro OJ ro ro CO NJ ro o CD NJ OJ G) ro OJ CD ro CO cr> ro CO CD ro CO cn ro OJ ro M OJ CD N) Ol 〇1 ai ro 03 CD ro CJ ro ro CO ro OJ ro ro CO G) ro CJ G) M CJ G) M CO ro ro 二 cn ro 公 cn M 么 cn M cn N) cn ro CO CD M cn ro 么 OD N) CD ro CD ro 办 cn M CO G) ro Cx3 cn ro CO cn ro cn. M cn M cn NJ CO O) ro CO N3 么 CO ro CD M CD ro cn ro CD ΓΟ cn M ro cn ro INJ cn M N) CD ro cn M cn ro 〇l M CD M cn M CD ro 二 σι ro cn M ro cn ro ro cn ro ro cn ro CD N3 cn ro ro cn ro ro CD ro CO ro CD ΓΟ cn ro ro CJ) _^ ro cn ro ro cn ro ro cn CD ΓΟ cn ro ro cn ro ro cn ro
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苐31頁 562859
第32頁 562859 五、發明說明(25) CD ΓΟ o — 〇 —^ o —Jl —X o —X _λ. 〇 ^k —x o —i G) —^ o k 〇 〇J 〇 c〇 o λ o 〇J —x o 〇〇 _X o OJ —_X o OJ —k o CO __k 〇 OJ —Λ. o CO λ. ο _k o —λ. ο 〇J o o —k 〇 A —X o 公 __V. 〇 —Jl o —λ. o _λ cn CD —V. cn CD o λ C71 CD —X CJi CD _>k 0*1 CD __1 CJl CD __λ cn CD V. cr> _λ. cn CD »k CD cn ro ω —k U1 CD λ CD 办 ro ro 〇 Ol ro —^ o ro § o ro —λ ro ro 〇 G) ro c〇 ro CD ro OJ ro ro OJ 〇) ro OJ ro ro f〇 M 0J ro X CD ro OJ ro ro —X CO ro 00 CD ro c〇 ro ro 00 cn ro on ro CO ① ro c〇 CD ro 0J CD NJ co o ro OJ ro f〇 un ro OJ G) ro cn ro 二 cn ro 么 cn ro 么 cn ro 二 cn ro CO cn M CO M un ro CD ro CO ro CD 二 CO ro 二 CO ro cn ro Ol ro ro 二 CD ro cn ro ro cn ro ro cn ro ro cn ro ro cn ro ro 二 CD ro cn ro ro cn ro ro ·_k 圓圓圈 第33頁 562859 五、發明說明(26) 1 最佳蛋白_變異體為表2所示者。 | 本發明之另一 g的為提供一種蛋白酶變異體,其在對應 j 1 於選自解澱粉芽孢桿菌型枯草芽孢桿菌素之62,212, ! 2 3 0,2 3 2,2 5 2及2 5 7所組成族群中之一個或以上殘基位置 上,铵基酸被取代。 雖然可以使周上列胺基酸取代之任何組合,但本發明之 較佳蛋白_變異體包含在下列組合中之胺基酸殘基之取 代。所有殘基位置對應於解澱粉芽孢桿菌型枯草芽孢桿菌I 素之位置: i (1 )在位置6 2以及一個或以上之下列位置上胺基酸殘基 | _ i被取代之蛋白酶變異體:103,104,109,159,213, 232 , 236 , 245 , 248 及252 ; (2 )在位置2 1 2以及一個或以上之下列位置上胺基酸殘基 被取代之蛋白酶變異體·· 1 2,9 8,1 0 2,1 0 3,1 04,1 5 9, | 2 3 2,2 3 6,2 4 5,2 4 8 及 2 5 2 ; (3 )在位置2 3 0以及一個或以上之下列位置上胺基酸殘基 被取代之蛋白酶變異體:6 8,1 0 3,1 0 4,15 9,2 3 2,2 3 6
I 及 2 4 5 ; (4 )在位置2 3 2以及一個或以上之下列位置上胺基酸殘基 | ί · 被取代之蛋白酶變異體:1,9,12,61,62, 68, 76, | 9 7,9 8,1 0 1,1 0 2,1 0 3,1 0 4,1 0 9,1 3 0,1 3 1,1 5 9, | 1 8 3,1 8 5,2 Q 5,2 0 9,2 1 ◦,2 1 2,2 1 3,2 1 7,2 3 0,2 3 6, | 2 4 5,2 4 8,2 5 2,2 5 7,2 6 0,2 7 0 及 2 7 5 ;
('5 )在位置2 3 2以及一個或以上之下列位置上胺基酸殘基I
第34頁 562859 五、發明說明(27) 被取代之蛋白酶變異體:103,1 04,2 3 6及245 ; (6 )在位置2 3 2及1 0 3以及一個或以上之下列位置上胺基 | 酸殘基被取代之蛋白酶變異體:1,9,1 2,61,6 2,6 8, 76 ’97 , 98 , 101 , 102 , 103 , 104 , 109 , 130 , 131 , 150 , 183 , 185 , 205 , 209 , 210 , 212 , 213 , 217 , 230 , 236 ’245 , 248 ,252 ,257 ,260 ,270及275 ; (7 )在位置2 3 2及1 0 4以及一個或以上之下列位置上胺基 酸殘基被取代之蛋白酶變異體:1,9,1 2,6 1,6 2,6 8, 76 ’ 97 , 98 , 101 , 102 , 103 , 104 , 109 , 130 , 131 , 159 , 183 , 185 , 205 , 209 , 210 , 212 , 213 , 217 , 230 , 236 ’245 , 248 ,252 ,257 ,260 ,270 及275 ; (8 )在位置2 3 2及2 3 6以及一個或以上之下列位置上胺基 | 酸殘基被取代之蛋白酶變異體:1,9,1 2,61,6 2,6 8, | 76 ’97,98,101,102,103,104,109,130,131, 159,183,185,2 05,209,210,212,213,217,2 30, 2 3 6,245 > 2 48,252,257,260,270 及275 ; (9)在位置232及245以及一個或以上之下列位置上胺基 | 酸殘基被取代之蛋白_變異體:1,9,1 2,6 1,6 2,6 8, | 7 6 ’ 9 7,9 8,1 0 1,1 0 2,1 0 3,1 0 4,1 0 9,1 3 0,1 3 1, ! i 1 5 9,1 8 3,1 8 5,2 0 5,2 0 9,2 1 0,2 1 2,2 1 3,2 1 7,2 3 0 ’ | ( 236 ’245, 248, 252, 257, 260 , 270 及275; ! (1 〇 )在位置2 3 2,1 0 3,1 0 4,2 3 6及2 4 5以及一個或以上 | 之下列位置之胺基酸殘基被取代之蛋白酶變異體:1,9 ’ ! i 1 2,61,62,68,76,97,98,101,102,103,104 ’ !
第35頁 562859 五、發明說明(28) 109,130,131,159,183,185,205,209,210,212, 213,217,230,236,2 45,248,252,2 57,260,270 及 275 ; ()在位置2 5 2以及一個或以上之下列位置之胺基酸殘 基被取代之蛋白i每變異體:1,9,12,61,62,68,97, 98,101,102,103,104,109,130,131,159,183, 185 , 210 , 212 , 213 , 217 , 232 , 236 , 245 , 248 及270 ; (1 2 )在位置2 5 2以及一個或以上之下列位置上按基酸殘 I 基被取代之蛋白酶變異體:1 0 3,1 0 4,2 3 6及2 4 5 ; | (1 3 )在位置2 5 2及1 0 3以及一個或以上之下列位置上胺基 酸殘基被取代之蛋白酶變異體:1,9,1 2,6 1,6 2,6 8, 9 7,9 8,1 0 1,1 0 2,1 0 3,1 0 4,1 0 9,1 3 0,1 3 1,1 5 9, 183,185,210,212,213,217,232,236,245,24 8 及 2 70 ; (1 4 )在位置2 5 2及1 0 4以及一個或以上之下列位置上胺基 酸殘基被取代之蛋白酶變異體:1,9,1 2,61,6 2,6 8, 9 7,9 8,丨01,10 2,10 3,10 4,10 9,13 0,131,15 9, 1 8 3,1 8 5 ’ 2 1 0,2 1 2,2 1 3,2 1 7,2 3 2,2 3 6,2 4 5,2 4 8 及 2 70 ; ! (1 5 )在位置2 5 2及2 3 6以及一個或以上之下列位置上胺基i 酸殘基被取代之蛋白酶變異體:1,9,1 2,61,6 2,6 8, 9 7,9 8 ’ 丨 0 1,I 0 2,1 0 3,1 0 4,丨 0 9,1 3 0,1 3 1,1 5 9,
183,185,2 10,2 12,2 13,217,2 3 2,2 3 6,2 45,24 8 及 I
2 7 0 ; I
第36頁 562859 五、發明說明(29) (16) 在位置252及245以及一個或以上之下列位置上胺基 酸殘基被取代之蛋白酶變異體:1,9,1 2,6 1,6 2,6 8, 97,98,101,102,103,104,109,130,131,159, 183,185,210,212,2 13,217,232,236,245,248& 2 70 ; (17) 在位置2 5 2,103,104,2 3 6及245以及一個或以上 .之下列位置上胺基酸殘基被取代之蛋白酶變異體:1,9, 12,61,62,68,97,98,101,102,103,104,109, 130,131,159,183,185,210,212,213,217,2 32, 236 , 245 , 248及270 ;以及 (1 8 )在位置2 5 7以及一個或以上之下列位置上胺基狻殘 丨基被取代之蛋白酶變異體:6 8,1 0 3,1 0 4,2 0 5,2 0 9, 210,232,236,245及275。本發明之再一目的為提供編 碼該蛋白I每變異體之DNA序列,以及含有該變異DNA序列之 表現載體。 1
再者,本發明之另一目的為提供用該載體轉型之宿主細I
I 胞,以及能表現該D N A而在細胞内或細跑外製造蛋白酶變 | 異體之宿主細胞。 丨 1 本發明尚提供一種清潔組合物,其包含本發明之蛋白酶! 變異體。 另外,提供一種動物飼斜,其包含本發明之蛋白i每變異|
I 體。 丨 本發明亦提供一種3於處理織物之組合物,其包含本發| 明之蛋白歸變異體。 !
第37頁 562859 五、發明說明(30) 式之簡單說明 圖! A-B描緣解殿粉芽跑椁 胺基酸序列以及該基因之邱八Λ,闽^ 亍囷$之山以及 4分限制圖譜。 圖2描繪來自解褒又粉芽跑# ν虹化样囷(ΒΡΝ)及遲緩芽胞桿菌
BaClllus lentus)(野生型)之枯草芽孢桿菌 (conserved)胺基酸殘基。 ' 圖3A及3B描繪四種枯草; —▲ ‘ 行代表來自解殿粉芽孢桿菌‘ :::t : $暖序列♦。第- 稱為枯草芽孢桿菌素BPN,)之膝1於牙也旱菌素(有時亦被 自枯草芽孢桿菌(BaciU 乂广-文序列。第二行描繪來
u s s u b t 1 1 1 s )刮妊苜玆%』曰y 士 之胺基酸序列。第三行描繪一括早牙孢柃囷I licheniformis)型括草芽~ 、牙化桿菌(B. 描緣來自遲緩芽孢桿菌°己素之妆基酸序列。第四行 • v D* 1 ent us ) ^ l PCT WO 8 9/ 0 62 7 6中亦被稱為枯早牙孢桿菌素(在 酸序列。代號*表示與枯草 1菌素3〇9)之装基 特異性胺基酸殘基。 U 〃、βρΝ相較,不存在 發明之詳細說明 蛋白酶為羰基水解酶,其之作两、 之肽鐽。如在本文中所用者,「蛋,二為打斷蛋白質或肽| 白酶或重組蛋白_。天铁芦 玄=瞎」意指天然產之晷! 解酶、肽基胺基竣水解酶、酲胺某:-胺酲基肽水 酶、金屬致基肽ϋ、硫醇蛋=了二=晦、絲胺酸叛基狀 酶。絲胺酸、I屬、硫醇及酸類…:蛋白酶及金屬蛋白 外-蛋白酶均被包括。 一 ^ ’以及胞内-及胞
562859 五、發明說明(31) 本發明包括屬於非天然產之羰基水解酶變異體(蛋白酶 變異體)之蛋白酶,其與同於衍生該變異體之胺基酸序列 之先質羰基水解酶相較,具有不同的蛋白分解活性、安定 性、受質特異性、pH特性及/或表現特徵。更特定而言, 該蛋白質變異體具有在天然中未曾被發現之胺基酸序列, 其藉著使周不同胺基酸取代先質蛋白酶之多個胺基酸殘基 而衍生得。先質蛋白籐可為天然產蛋白酶或重組蛋白I每。 在本發明中有用之蛋白酶變異體包含在指定之胺基酸殘 基位置,兩1 9個天然產L -胺基酸之任一者取代。此等取代 可在任何先質枯草芽孢桿菌素(來自原核、真核、哺乳動 物等)中進行。在本案之全文中,以通用之一字母及三字 母代碼來表示胺基酸。該代碼被述於Dale, M. W. ( 1 9 8 9 ), Molecular Genetics of Bacteria, John W iley & Sons, Ltd., Appendix B 。 在本發明中有周的蛋白i每變異體較佳衍生自芽孢桿g之I 枯草芽孢桿菌素。蛋白酶變異體更佳衍生自遲緩芽胞桿菌 型枯草芽抱桿菌素及/或枯草芽孢桿菌素3 0 9。 i |
枯草芽孢桿菌素為細菌性或真菌性蛋白酶,其作罔通常I 為打斷蛋白質或肽之肽鍵。如在本文中所闬者,「枯草芽! * 孢桿菌素」意指天然產之枯草芽孢桿菌素或重組枯草穿孢|
桿菌素。已知一系列之枯草芽孢桿菌素被製造及被各種微 I ϊ 生物分泌。該系列成員之胺基酸序列並非完全具同等性。| 不過,在該系列中之枯草穿孢桿菌素顯示相同或近似類型 I 之蛋白分解活性。該類絲胺酸蛋白酶共有界定催化三元組
第39頁 562859 五、發明說明(32) !
之共同胺基酸序列,其中該催化三元組使彼等與胰凝乳蛋I 白酶相關類之絲胺酸蛋白誨區別。枯草芽孢桿菌素-相關 | 之絲胺酸蛋白酶及胰凝乳蛋白酶-相關之絲胺酸蛋白_皆 I
I 具有包含天冬胺酸、組胺酸及絲胺酸之催化三元組。在枯| 草芽孢桿菌素-相關蛋白酶中,此等胺基酸之相對次序,| 若從胺基端讀至羧基端,為天冬胺酸-組胺酸-絲胺酸。不 | 過在胰凝乳蛋白酶-相關蛋白酶中,相對次序為組胺酸-天 冬胺酸鹽-絲胺酸。因此,在本文中枯草芽孢桿菌素係指 具有枯草芽孢桿菌素-相關蛋白酶之催化三元組之絲铵酸 : 蛋白酶。其例非限定性地包括圖3所示之枯草芽孢桿菌 素。一般而言,就本發明之目的言之,在蛋白酶中胺基酸 之編號對應於圖1所示之成熟解澱粉芽孢桿菌型枯草芽孢 I 桿菌素序列之編號。 j 「重組枯草芽孢桿g素」或「重組蛋白酶」係指一種枯| 草牙抱桿菌素或隻白而母,其中分別編5馬天然產蛋白酸或枯1丨 草穿孢桿菌素之DNA序列被修改,以產生編碼天然產胺基 ! 酸序列中一種或以上胺基酸之取代、***或删除之變異 (或突變)DNA序列。產生此等修改之適當方法(可與本文所 | 揭示之方法合闬)包括美國專利RE 34,606,5,204, 0 15 , | 5,1 8 5,2 5 8,美國專利 5,7 0 0,6 7 6 ,美國專利 5,8 0 1,0 3 8 及 | 美國專利5 , 7 6 3,2 5 7 所揭示者。 | 「非人類枯草芽孢桿菌素」以及編碼彼等之D NA可得自 許多原核及真核生物。原核生物之適當例子包括袼蘭氏陰 i 性細菌諸如大腸桿菌或假單跑_ ,以及格蘭氏陽性細i諸丨
第40頁 562859 五、發明說明(33) 如微球菌或芽孢桿菌。可以得到羰基水解酶及其基因之真 核生物之例子包括酵母菌諸如釀酒酵母菌,真菌諸如麴 菌。 「蛋白酶變異體」所具有之胺基酸序列衍生自「先質蛋 白酶」之胺基酸序列。先質蛋白酶包括天然產蛋白酶及重 組蛋白酶。蛋白酶變異體之胺基酸序列藉先質胺基酸序列 之一種或以上胺基酸之取代、刪除或***而衍生自先質蛋: ! 白酶胺基酸序列。該修改係針對編碼先質蛋白酶之胺基酸 序列之「先質DNA序列」進行,而非針對先質蛋白酶本身 進行。此等處理先質DNA序列之適當方法包括本文所揭示 之方法,以及熟悉本技藝者已知之方法(例如見 EP 0 3 2 8 2 9 9,\ί〇8 9/ 0 6 2 7 9及已被列為本文參考文獻之 美國專利及申請案)。 | 在本發明中,對應於解澱粉芽孢桿菌型枯草芽孢桿g素i
I 之位置1 0 3之特異性取代與一個或以上對應於解澱粉芽孢 i 桿菌型枯草芽孢桿菌素之下列位置之取代之組合被鑑別 | 出:1,3,4,8,10,1 2,13,16,17,18,20,21, | 22 ,24 ,27 ,33 ,37 ,38 ,42 ,43 ,48 ,55 ,57 ,58 , | 61,62,68,72,75,76,77,78,79,86,8'7,89, ; 9 7,9 8,9 9,1 0 1 ,1 0 2,1 0 4,1 0 6,1 0 7,1 0 9,1 1 1 , ! {
114, 116, 117, 119, 121, 123, 126, 128, 130, 131, I 1 3 3,1 3 4,1 3 7,1 4 0,1 4 1,1 4 2,1 4 6,1 4 7,1 5 8,1 5 9, | » 160,166,167,170,173,174,177,18 卜 185,188, j 192,194,198,2 0 3,2 04,2 0 5,2 0 6,2 0 9,2 10,21 1, |
苐41頁 562859 五、發明說明(34) j 212,2 13,214,215,216,217,218,222,224,227, j j 228,2 30,2 32,2 3 6,2 37,238,2 40,242,2 43,24 4,j 245,246,247,2 48,2 49,251,252,253,2 54,255, 256,257,258,259,260,261,262,263,265,2 68, 269,270 ’271,272,2 74及275。上胺基酸殘基位置上, 腔基酸殘基亦被取代。 更佳之蛋白酶變異體被示於下列表I中。最佳蛋白酶變 異體為表2所示者。 本發明之另一目的為提供一種蛋白酶變異體,其在對應 於選自解澱粉芽孢桿菌型枯草芽孢桿菌素之62,21 2, 230,232,252及257所組成族群中之一個或以上殘基位置 上,胺基酸被取代。 雖然可以使用上列胺基酸取代之任何組合,但本發明之 較佳蛋白酶變異體包含在下列組合中之胺基酸殘基之取 代、刪除或***: (1 )在位置6 2以及一個或以上之下列位置上胺基酸殘基 被取代之蛋白酶變異體:1 〇 3,1 0 4,1 0 9,1 5 9,2 1 3, 2 32,2 3 6,2 4 5,2 48 及 2 5 2 ; (2)在位置2 1 2以及一個或以上之下列位置上胺基酸殘基 被取代之蛋白酶變異體·· 1·2 ’ 9 8,1 0 2,1 0 3,1 0 4,1 5 9 ’ 2 3 2,2 3 6,2 45,2 48 及 2 5 2 ; (3 )在位置2 3 0以及一個或以上之下列位置上胺基酸殘基 被取代之蛋白酶變異體:68,1〇3,104,159,232,236 及 24 5 ;
第42頁 562859 五、發明說明(35) (4)在位置2 3 2以及一個或以上之下列位置上胺基酸殘基 被取代之蛋白酶變異體:1,9,12,61,62,68,76, 97,98,101,102,103,104,109,130,131,159, 183,185,205,209,210,212,213,217,230,236, i 245,248,2 52,2 57,2 6 0,270 及2 75 ; (5 )在位置2 3 2以及一個或以上之下列位置上胺基酸殘基 被取代之蛋白酶變異體:103,104,236及245 ; (6 )在位置2 3 2及1 0 3以及一個或以上之下列位置上胺基 酸殘基被取代之蛋白酶變異體:1,9,1 2,6 1,6 2,· 6 8, 7 6,9 7,9 8,1 0 1,1 0 2,1 0 3,1 0 4,1 0 9,1 3 0,1 3 1, 1 5 9,1 8 3,1 8 5,2 0 5,2 0 9,2 1 0,2 1 2,2 1 3,2 1 7,2 3 0, 2 3 6,2 45,2 48,2 5 2,2 57,2 6 0,2 7 0 及275 ; (7 )在位置2 3 2及1 0 4以及一個或以上之下列位置上胺基 酸殘基被取代之蛋白酶變異體:1,9,1 2,61,6 2,6 8, | 7 6 ’ 9 7,9 8,1 0 1,1 0 2,1 0 3,1 0 4,1 0 9,1 3 0,1 3 1, j 159,183,185,205,209,210,212,213,217,230, | 236,245,248,252,257,260,2 70 及275 ; (8 )在位置2 3 2及2 3 6以及一個或以上之下列位置上胺基 酸殘基被取代之蛋白酶變異體:1,9,1 2,6 1,6 2,6 8, 7 6,9 7,9 8,1〇1,1〇 2,10 3,1 0 4,1 0 9,13 0,131, j 159 ’183 , 185 , 205 , 209 , 210 , 212 , 213 , 217 , 230 , ! 2 3 6,2 4 5,2 4 8,2 5 2,2 5 7,2 6 0,2 7 0 及 2 7 5 ; ;
I (9 )在位置2 3 2及2 4 5以及一個或以上之下列位置上胺基 |
I 酸殘基被取代之蛋白酶變異體:1,9,1 2,6 1,6 2,6 8,
苐43頁 562859 五、發明說明(36) 7 6,9 7,9 8,1 0 1,1 0 2,1 0 3,1 0 4,1 0 9,1 3 0,1 3 1, 1 5 9,1 8 3,1 8 5,2 0 5,2 0 9,2 1 0,2 1 2,2 1 3,2 Π,2 3 0, 23 6,245,248,2 52,2 57,2 6 0,2 70 及275 ; (10)在位置232,103,104,236及24 5以及一個或以上 之下列位置之胺基酸殘基被取代之蛋白酶變異體:1,9, 12,61,62,68,76,97,98,1 01,1 02,103,I04, 1 0 9,1 3 0,1 3 1,1 5 9,1 8 3,1 8 5,2 0 5,2 0 9,2 1 0,2 1 2, 213,217,2 3 0,2 3 6,2 45,248,2 52,257,2 60,27 0 & 275 ; (1 1 )在位置2 5 2以及一個或以上之下列位置之胺基酸殘 I基被取代之蛋白酶變異體·· 1,9,1 2,61,6 2,6 8,9 7, 98,1〇1,1〇2,103,104,109,130,131,159,183, 185 ,2i〇 ,212 ,213 ,217 ,232 ,236 ,245 ,248 及270 ; (1 2 )在位置2 5 2以及一個或以上之下列位置上胺基酸殘 基被取代之蛋白酶變異體:1 〇 3,1 0 4,2 3 6及2 4 5 ; (1 3 )在位置2 5 2及1 0 3以及一個或以上之下列位置上胺基 酸殘基被取代之蛋白_變異體·· 1,9,12,61,6 2,6 8,:
I 9 7,9 8,1 〇 1,1 〇 2,1 0 3,1 0 4,1 0 9,1 3 0,1 3 1,1 5 9 ’ | 1 8 3,1 8 5,2 1 0,2 1 2,2 1 3,2 1 7,2 3 2,2 3 6,2 4 5,2 4 8 及 j 2 70 ; (1 4 )在位置2 5 2及1 0 4以及一個或以上之下列位置上胺基 酸殘基被取代之蛋白酶變異體:1,9,1 2,61,6 2,6 8, | ? 97,98,101,102,103,104,109,130,131 ’159, |
183 ’185,2 10,212,213,217,232,236,245,248 及 I
I
第44頁 pB2859 五、發明說明(37) ] 270 ; (1 5 )在位置2 5 2及2 3 6以及一個或以上之下列位置上胺基 酸殘基被取代之蛋白酶變異體:1,9,12,61,62,68, 97,98,101,102,103,104,109,130,131,159, ! ⑻,185,210,212,213,217,232,236,245,248 & ! 270 ; 〇6)在位置25 2及245以及一個或以上之下列位置上胺基 酸殘基被取代之蛋白酶變異體:1,9,1 2,6 1,6 2,6 8, 97 ’98,1〇1,1〇2,103,104,109,130,131,159,
183,185,210,2 12,213,217,232,236,245,248 及 2 70 ; (17)在位置252,103,104,236及245以及一個或以上 之下列位置上胺基酸殘基被取代之蛋白酶變異體:1,9, 12 ’61 ’62,68,97,98,101,102,103,104,109, 130 ’131,159,183,185,210,212,213,217,23 2, 236 ’245 , 248及270 ;以及 08)在位置257以及一個或以上之下列位置上胺基竣殘 I 基被取代之蛋白_變異體:6 8,1 0 3,1 0 4,2 0 5,2 0 9, | 21 0,2 3 2,2 3 6,245 及 2 75。 | 此等胺基酸位置編號參照圖1所示之成熟澱粉芽孢桿菌 | 型枯草芽孢桿菌素序列之編號。不過,本發明非限於使闬 該成熟之特定枯草芽孢桿g素,而係延伸至在上述位置上 含有與澱粉芽孢桿菌型枯草芽孢桿菌素之特定殘基為「對
第45頁 562859 五、發明說明(38) 例中,先質蛋白酶為遲缓芽孢桿菌型枯草芽孢桿g素,以 及對於遲缓芽孢桿菌中對應於上列者之對等胺基酸殘基可 進行取代、刪除或***。 先質蛋白酶之殘基(胺基酸)若與解澱粉芽孢桿菌型枯草 芽孢桿菌素中之特異性殘基或該殘基之一部份為同等(即 在位置以及一級或三級結構為對應)或類似(即具有相同或 相似之結合、反應或化學交互作闬之功能),則與解澱粉 芽孢桿菌型枯草芽孢桿菌素之殘基為對等。 為了確證與一級結構為同等,將先質蛋白酶之胺基酸序 列與解澱粉芽孢桿菌型枯草芽孢桿菌素一級序列(尤其是 序列為已知之枯草芽孢桿菌素中之一組不會變異之殘基) 直接比較。舉例言之,本文之圖2顯示解;殿粉芽孢桿菌型 枯草芽孢桿菌素與遲缓芽孢桿菌型枯草芽孢桿菌素間之守 十亙殘基(c ο n s e r v e d r e s i d u e s )。將此等守怪殘基對準後, 進行必需之***及刪除以保持對準(即避免經由任意之刪 除及***而除去守恆殘基),以及界定與解澱粉芽孢桿菌 型枯草芽孢桿菌素之一級序列中之特定胺基酸為對等之殘 基。守恆殘基之對準較佳應保留1 0 0 %之此等殘基。不過, 大於75%或低達50%之守恆殘基之對準亦足以界定對等殘 基。催化三元組Asp32/His64/Ser221應被保持。Siezen et a 1. (19 9 1) Protein Engineering 4(7):719-737 顯示
大量絲胺酸蛋白酶之雜列。S i e z e n等人稱該群組為枯草桿 菌酶或枯草桿菌素類之絲胺酸蛋白酶。 I 舉例言之,在圖3中來自解澱粉芽孢桿菌、枯草芽孢桿 |
562859 五、發明說明(39) 菌、地衣芽胞桿菌及遲緩芽胞桿菌之括草芽抱桿菌素之# 基酸序列被對準,以在胺基酸序列間提供最大量之奴 性。此等2=較顯示在各序列中含有許多守值殘:。 此等守恆“(BPN肖遲緩芽胞桿菌之間)被示於 。
因此,此等守恆殘基可被两於界定遲缓芽對 對等胺基酸殘基( 1 98 9年7月13日公開之pCT公土 tM 8 9/0 62 79號),在本發明中較佳之蛋白酶先質二枯宜笑游 桿菌素被稱,PB92(EP 0 32 8 29 9 ),其與較佳之遲緩芽抱 桿菌型枯草牙抱桿菌言具高度之同等性。在圖3A及⑽中, 此等枯草芽抱桿菌素中某些之胺基酸序列與解澱粉芽孢桿 ,型枯草芽孢桿菌素之序列對準以產生守悝殘基之最大同 寻性。如所見者,遲缓牙孢桿菌型枯草芽孢桿菌素,盘解 殿粉芽抱桿菌型括草芽抱桿菌素相較,有許多刪除。因 =,舉例言之,在其他枯草芽孢桿菌素中,與解殿粉芽孢 才于菌型括草芽抱桿菌素中之731165為對等之胺基酸,就 緩芽孢桿菌及地衣芽孢桿i言之為異白胺酸。 0 「對等殘基」亦可藉在先質蛋白酶之三級結構層 (稭X光結晶照相測定)同等性而界定1等殘基被界定為 ^貝蛋白酶及解;&粉芽孢柃菌型祜草芽孢桿菌素二者之 义胺基酸殘基之二個或以上主要鐽原子之原子座標在〇 = nm之内以及較佳為〇· 1 nm。對準於最佳模型被定向及定位 ^破測定之蛋白酶之非氫蛋白質原子之原子座標與解毁从 =炮桿g型枯草芽孢桿菌素者得到最大重疊而達成。最: 偉型為在可獲得之最高解析度下,得到實驗繞射數據之最
562859 五、發明說明(40) 低R因子之結晶照相模型。 _ lh\Fo(h)\r\Fc(h)\
Rf—二Σ:涵厂
功能上與解澱粉芽孢桿菌型枯草穿孢桿菌素之特異性殘 基類似之對等殘基,被界定為「先質蛋白酶之胺基酸殘基 中所採納之結構係按照對解澱粉芽孢桿菌型枯草芽孢桿菌: 素之特定殘基所界定之方式,變化、修改或獲致蛋白質結| 構、受質結合性或催化特性者」。再者,該功能對等殘基 係指先質蛋白酶(三級結構已藉X -光結晶照相得到)之此等 殘基佔據類似位置之程度為:雖然既定殘基之主鏈原子若 就其所佔據位置之同等性而言無法滿足對等之標準,但該 殘基之至少二支鏈原子之原子座標在解澱粉芽孢桿菌型枯 草芽孢桿菌素之對應支鏈原子之0. 1 3 nm之内。解澱粉芽 孢桿菌型枯草芽孢桿菌素之三度空間結構之座標被記載於 EPO公告0 2 5 1 4 4 6號(相當於美國專利5,1 8 2,2 0 4號,該案 之内容被列為本文之參考文獻),以及如上文所摘要者,
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被用於測定在三級結構層次之對等殘基。 I 被鑑定供取代之殘基中,一些為守恆殘基而另一些則 否。在非為守恆殘基之場合,一種或以上胺基酸之取代被 限於所產生之變異體之胺基酸序列與在天然中所發現者不 對應。在守恆殘基之場合,該取代不應會產生天然產序 列。本發明之蛋白酶變異體包括蛋白酶變異體之成熟形
第48頁 562859 五、發明說明(41) 式,以及該蛋白酶變異體之原型及前-原-型。前原型既然 利於蛋白·變異體之表現、分泌及成熟,因此為較佳之構 築體。 「原序列」(p r 〇 s e q u e n c e )係指與蛋白酶成熟形式之N -端部分結合之胺基酸序列,當其被移除時,產生蛋白酶之| 「成熟」形式。許多蛋白分解酶在自然界係以未經轉譯後 加工之轉譯原酶產物形式被發現。供製造蛋白酶變異體之 較佳原序列為解澱粉芽孢桿菌型枯草芽孢桿菌素之推定原 序列,惟亦可使闬其他蛋白酶原序列。 「訊號序列」或「前導序列」係指與蛋白酶之N-端部分 或原蛋白酶之N-端部分結合之任何胺基酸序列,其可能參 與蛋白酶之成熟或原形式之分泌。訊號序列之該定義為一 種功能性定義,意欲包括被蛋白酶基因之N-端部分編碼且 參與在自然條件下蛋白i每之分泌之所有胺基酸序列。本發 明利用該序列進行本文定義之蛋白酶變異體之分泌。一可 i 能的訊號序列包含來自枯草芽孢桿菌型枯草芽孢桿菌素之 訊號序列之最初7個胺基酸殘基與來自遲缓芽抱桿囷(A T C C 2 1 5 3 6 )之枯草芽孢桿菌素之訊號序列之其餘部分融合所成 者。 蛋白酶變異體之「前原」形式由蛋白酵之成熟形式,與 該蛋白酶之胺基端可運作地連接之原序列以及與該原序列| 之胺基端可運作地連接之「前導」或「訊號」序列組成。I 「表現載體」意指一種DNA構築體,其含有一DNA序列且 其與在適當宿主中能表現該DNA之適當控制序列可運作地
第49頁 562859 五、發明說明(42) 連接在一起。此等控制序列包括進行轉錄之啟動子,可選 擇存在之周於控制該轉錄之操縱基因序列,編碼適當m R N A 核糖體結合部位之序列以及控制轉錄及轉譯之終止之序 ; 列。載體可為質體,噬菌體粒子,或僅是潛在的染色體組 插子。該載體一旦被轉型入適當宿主中,可獨立地複製及 運作,或者在一些例子中,可被整合入宿主之染色體組 中。在本說明書中,「質體」及「載體」有時被交換使 兩,因為目前質體為載體中最常被使周之形式。不過,本 發明意欲包括具有同等功能且為本技藝已知之其他形式之 表現載體。 本發明所使周之「宿主細胞」通常為原核或真核宿主, 其較佳經美國專利RE 3 4,6 0 6所揭示之方法處理,以使其 不會分泌具_活性之胞内酶。表現蛋白酶之較佳宿主為去夬 乏具酶活性之中性蛋白酶及鹼性蛋白酶(枯草芽孢桿g素) 之芽孢桿菌菌株BG 2 0 3 6。BG 2 0 3 6之構築被詳述於美國專利 5, 264, 366中。表現蛋白酶之其他宿主細胞包括枯草芽孢 桿菌1 6 8 (亦被記載於美國專利RE 3 4,6 0 6及美國專利 5,264, 366中,其之内容被列為本文參考文獻),以及任何 適當的芽孢桿菌菌株諸如地衣芽孢桿菌、遲緩芽孢桿i j
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宿主細胞以使周重組D ΝΑ技術構築之載體而予以轉型或 轉染。該被轉型之宿主細胞能複製編碼蛋白酶變異體之載 體或表現所期望之蛋白酶變異體。在編碼蛋白酶變異體之 前-或前原-形式之載體之場合,此等變異體當被表現時,I
第50頁 562859 五、發明說明(43) 通常從宿主細胞分泌入宿主細胞培養基中。 「可運作地連接」,當被周於描述二DNA區域間之關係 時,僅是表示彼此具功能相關性。舉例言之,原序列連接 至肽後若能做為訊號序列,參與蛋白質之成熟形式之分泌 (最可能涉及訊號序列之斷裂),即為可運作地連接至肽。 啟動子若能控制編碼序列之轉錄,即為可運作地連接至編 碼序列;核糖體結合部位若被定位成能允許轉譯,即為可 運作地連接至編碼序列。 編碼天然產先質蛋白酶之基因可按照熟悉本技藝者已知 之一般方法獲得。該方法通常包含合成被標記之探子(具 有編碼所欲蛋白酶之區域之推定序列),製備來自表現該 蛋白酶之生物之基因庫,以及藉與探子雜交而從基因庫中 篩選所欲之基因。然後製作陽性雜交純系之圖譜及予以定 序。 然後將選殖之蛋白酶周於轉型宿主細胞以表現蛋白酶。 繼而將蛋白酶基因粘接入高套數之質體中。該質體在宿主 中複製,若其含有質體複製所需之已知要件:一可運作地 連接至上述基因之啟動子(其可為基因本身之同源啟動 j 子,只要其能被宿主識別(即轉錄));一轉錄終止及聚腺 苷醯化區域(在某些原核宿主細胞中,為被宿主從蛋白酶 基因轉錄之in RN A之安定性所必需),其為外源性或由蛋白 | 酶基因之内源终止區域供應;以及一期望存在之選擇基 I 因,諸如抗生素抗性基因,其能藉著在含抗生素之培養基 I 中生長而連續培養及保持被質體感染之宿主細胞。高套數 |
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第51頁 562859 五、發明說明(44) 質體亦含有供宿主複製之源頭,藉此能使大數目之質體在 細胞質中被產生而不受到染色體之限制。不過,將蛋白酶 基因之多套整合入宿主染色體組内亦在本發明之範圍之 内。此易.藉對同源重組具特別感受性之原核及真核生物達; 成。 該基因可為天然的遲缓芽孢桿菌基因。另一選擇為可以^ 製造編碼天然產或突變先質蛋白酶之合成基因。在該法 中,決定先質蛋白酶之DNA及/或胺基酸序列。繼而合成多 個交疊之合成單股DNA片段,其經雜交及粘接後製造編碼 先質蛋白酶之合成DNA。合成基因構築體之一例被記載於 美國專利5, 2 04, 1 0 5之實例3,其内容被列為本文之參考文 獻。 一旦天然產或合成先質蛋白酶基因已被選殖,可以進行i 許多修改以增加基因在合成天然產先質蛋白酶以外之罔 途。該修改包括美國專利RE 34, 606及EPO公告0 251 446 號所揭示之重組蛋白_之製造以及本文所述之蛋白酶變異 體之製造。 下列套裝突變誘發方法利於本發明蛋白酶變異體之構 i 築,惟亦可使用其他方法。首先得到編碼蛋白酶之天然產i 基因以及將其全部或部分定序。然後掃描該序列,以找出| 在被編碼之酶中一個或以上期望發生胺基酸突變(刪除、 ***或取代)之點。評估在該點側翼之序列是否存在限制 _切割部位以供寡核苷酸匯集物取代基因之一短段,而於I 被表現時編碼各種突變體。該限制酶切割部位較佳為蛋白|
第52頁 562859 五、發明說明(45) 酉每基因中之唯一之部位,以利於基因片段之取代。不過, 任何在蛋白酶基因中非屬過多之便利限制酶切割部位皆可 被使用,只要限制酶消化所產生之基因片段可以適當順序 重新組合。若限制酶切割部位在距選定點方便距離(1 0至 1 5個核苷酸)内之位置上不存在,該部位可以在不改變最 終構築體之譯讀框或被編碼之胺基酸之方式下藉取代基因 中之核苷酸而產生。基因為改變其序列以符合期望序列所 進行之突變,係按照一般已知之方法進行Μ1 3引子延伸而 I達成。適當側翼區域之定位以及形成二便利限制酶切割部 !位序列所需改變之評估,係藉基因密碼之冗餘、基因之限 制酶切割圖譜以及大量不同的限制酶而達成。應注意是否 有可利用之便利側翼限制酶切割部位;上述方法只有在側 翼區不含該部位時才需要被使用。 一旦天然產或合成先質蛋白酶基因被選殖,在欲突變位 置側翼之限制酶切割部位用同種限制酶消化,以及將多個 終端-互補性寡核苷酸匣粘接入基因中。突變誘發藉該法 單純化,因為所有寡核苷酸可被合成為具有相同限制i每切 割部位,以及該限制_切割部位之產生不需要合成粘結 子。 如本文所周者,蛋白酶分解活性被界定為每毫克活性酶 肽鍵水解之速率。已知存在許多測量蛋白分解活性之方法 (K. M.· Kal isz,,’微生物蛋白酶",生化工程/生物技術之 進步,A. Fiechter ed·, 1988)。除了具有修改的蛋白分 解活性之外,本發明之變異酶尚具有其他經修改的性質或
第53頁 562859 五、發明說明(46) 以該修改性質替代蛋白分解活性,諸如ΚΏ、Keat、UK, 比,及/或經修改之受質特異性,及/或經修改的pH活性模 式。此等_可依照例如在肽之製備或水解過程(諸如洗衣 使兩時)中預期存在之特定受質而量身訂作。 本發明在一方面之目的為確保變異蛋白酶在至少一清潔 調配物中及/或於至少一組洗滌條件下,與先質蛋白酶相 較,具有改變(較佳改善)之洗滌效能。 蛋白酶變異體可能暴露於各種洗滌條件下,此等洗滌條 件包括各種清潔劑調配物、洗蘇水體積、洗蘇水溫度及洗 滌時間長度。舉例言之,在不同區域使罔之清潔劑調配 物,其相關成分在洗蘇水中之濃度不同。例如,歐洲清潔 劑在洗蘇水中通常具有4 5 0 0 - 5 0 0 0 p p m之清潔劑成分,而 j 曰本清潔劑在洗蘇水中通常具有約6 6 7 p p m之清潔劑成 分。在北美,尤其是美國,清潔劑在洗滌水中通常具有約 9 7 5 p p m之清潔劑成分。 低清潔劑系統包括清潔劑成分在洗滌水中之濃度低於約 8 0 0 p p m之清潔劑。曰本清潔劑通常被認為屬低清潔劑濃 1
度系統,因為彼等之清潔劑成分在洗滌水中之濃度為約 I 6 6 7 pp m 〇 ! 中清潔劑系統包括清潔劑成分在洗條水中之濃度在約 | 8 0 0 p p m與2 0 0 0 p p m間之清潔劑。北美清潔劑通常被認為 | 屬中清潔劑濃度系統,因為彼等之清潔劑成分在洗滌水中 之濃度為約9 7 5 p p m。巴西清潔劑之清潔劑成分在洗務水
I 中之濃度為約1500ppm。 :
苐54頁 562859 五、發明說明(47)
高清潔劑系統包括清潔劑成分在洗滌水中之.濃度大於約 2 0 0 0 ppm之清潔劑。歐洲清潔劑通常被認為屬高清潔劑濃 度系統,因為彼等之清潔劑成分在洗務水中之濃度為約 4500-5000ppm° I 拉丁美洲清潔劑通常為高發泡磷酸鹽助潔清潔劑,以及 拉丁美洲所周之清潔劑落在中與高清潔劑濃度二者之範圍 内,因為清潔劑成分在洗滌水中之濃度在1 5 0 0 ppm至6 0 0 0 ppm之範圍内。如上文所述,巴西清潔劑之清潔劑成分在 1 洗滌水中之濃度為約1 5 0 0 ppm。不過,其他高發泡磷酸鹽| 助潔清潔劑(非限於其他拉丁美洲國家)可具有高清潔劑濃I春 度,清潔劑成分在洗滌水中之濃度高達約6 0 0 0 ppm。 從前述可知,在全世界,清潔劑組合物在典型洗液中之 濃度可從低於約8 0 0 ppm之清潔劑組合物(低清潔劑濃度系 統,例如在日本為約6 6 7 p p m ),變化至約8 0 0 p p m至約 2 0 0 0 p p m (中清潔劑濃度系統,例如在美國約9 7 5 p p m及在 巴西約1 5 0 0 p p m ),再變化至大於約2 0 0 0 ρ p m (高清潔劑濃; ) 度系統,例如在歐洲約4 5 0 0 p p m至約5 0 0 0 p p m,以及在高| ; 發泡鱗酸鹽助潔劑之場合為約6 0 0 0 p p m)。
典型洗液之濃度憑實驗測定。舉例言之,在美國,典型 φ 之洗滌機器保持約6 4. 4公升體積之洗液。所以,為了在洗 液中得到濃度為約9 7 5 p pm之清潔劑,必須將約6 2 . 7 9公克I 之清潔劑組合物加到6 4. 4公升洗液中。該量為消費者罔清i 潔劑附設量杯測量而加到洗滌水中之典型量。 I ί 再一例子為不同系統使周不同洗滌溫度。在日本洗滌水|
I
第55頁 562859 五、發明說明(48) 之溫度通常低於歐洲使周者 所以本發明之一方面包括在至少一組洗務條件下顧示?文| 善之洗滌效能之蛋白酶變異體。 — 在本發明之另一方面,已測知在對應於解殿粉芽孢桿菌 型枯草芽孢桿菌素(subtilisin)之位置103上之取代,與 在一個或以上選自對應於下列者之位置所組成族群中之位 置上之取代之組合,在增進酶之洗滌效能上重要:丨,3 , 4 ,8 ,1〇 ,12 ,13 ,16 , 17 ,18 ,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 , 1 0 1,1 0 2,10 4,10 6,10 7,10 9,111,114,116,117 119 , 121 , 123 , 126 , 128 , 130 , 131 , 133 , 134 , 137 140 , 141 , 142 , 146 , 147 , 158 , 159 , 160 , 166 , 167 170 , 173 , 174 , 177 , 181-185 , 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 2 72 ,2 74,及 2 7 5。 更佳之蛋白酶變異體為表1所示者。最佳之蛋白藤變異 體為衣2所不者。 本發明.之另一目的為提供一種蛋白酶變異體,其在對應 於選自解殿粉芽?包桿菌型枯草芽抱桿_素之6 2 ’ 2 1 2 ’
苐56頁 562859 五、發明說明(49) 230,232,252及257所組成族群中之一個或以上殘基位置 上,胺基酸被取代。 雖然可以使同上列胺基酸取代之任何組合,但本發明之 較佳蛋白酶變異體包含在下列組合中之胺基酸殘基之取 代、删除或***: (1 )在位置6 2以及一個或以上之下列位置上胺基酸殘基 被取代之蛋白酶變異體:103,104,109,159,213, 232,2 3 6,245,248 及2 52 ; (2)在位置2 1 2以及一個或以上之下列位置上胺基酸殘基 被取代之蛋白酶變異體:12,98,1 02,103,1 04,159, 232,2 3 6,2 45,248 及2 52 ; (3 )在位置2 3 0以及一個或以上之下列位置上胺基酸殘基 被取代之蛋白酶變異體:68,103,104,159,232,236 及245 ; ! (4 )在位置2 3 2以及一個或以上之下列位置上胺基酸殘基| 被取代之蛋白酶變異體:1,9,12,61,62,68,76, 9 7,9 8,i 0 1,1 0 2,1 0 3,1 0 4,1 0 9,1 3 0,1 3 1,1 5 9, 183,185,205,2 09,210,212,213,217,23 0 , 236, 245,248,2 5 2,2 5 7,2 6 0,2 7 0 及2 75 ; (5) 在位置232以及一個或以上之下列位置上胺基酸殘基 | 被取代之蛋白酶變異體:丨〇3,1 04,2 3 6及24 5 ; | (6) 在位置232及103以及一個或以上之下列位置上胺基 i 酸殘基被取代之蛋白酶變異體:1,9,1 2,6 1,6 2,6 8 ’ 76,97,98,101,102,103,104,109,.130,131 ’
第57頁 562859 五、發明說明(50) 159,183,185,2 05,209,210,212,213,217,230, 2 3 6,245 > 248,252 > 2 57,260,270 及275 ; (7 )在位置2 3 2及1 0 4以及一個或以上之下列位置上胺基 酸殘基被取代之蛋白酶變異體:1,9,12,61,62,68, 76 ,97 ,98 ,101 ,102 ,103 ,104 ,109 ,130 ,131 , 159 , 183 , 185 , 205 , 209 , 210 , 212 , 213 , 217 , 230 , 236 ,245 ,248 >252 ,257 > 260 , 270 及275 ; (8) 在位置232及236以及一個或以上之下列位置上胺基 酸殘基被取代之蛋白酶變異體:1,9,1 2,61,6 2,6 8 ’ ; 76,97,98,101,102,103,104,109,130,131, ; 159,183,185,205,209,210,212,213,217,230, 2 38,245,2 48,252,257,260,270 及275 ; (9) 在位置23 2及245以及一個或以上之下列位置上胺基 酸殘基被取代之蛋白酶變異體:1 ’9 ’12 ’61 ’62 ’68 ’ 76 ,97 ,98 ,101 ,102 ,103 ,104 ,109 ,130 ,131 , 159 , 183 , 185 , 205 , 209 , 210 ’ 212 , 213 , 217 ’ 230 , ! 236 , 245 , 248 , 252 , 257 , 260 , 270 及275 ; | (10) 在位置2 3 2,103,104,2 3 6及245以及一個或以上 | 之下列位置之胺基酸殘基被取代之蛋白隨變異體:1 ’ 9 ’ 12 ,61 ,62 ,68 ,76 ,97 ,98 ,101 ,102 ,103 ,104 ,
1 〇 9,1 3 0,1 3 1,1 5 9,1 8 3,1 8 5,2 0 5,2 0 9,2 1 0,2 1 2,j 213,217,230,236,245,248,252,257,260,270 及 I i 2 75 ; !
I
(1 1 )在位置2 5 2以及一個致以上之卜列位置之胺基毁殘 I
第58頁 562859 五、發明說明(51) 基被取代之蛋白酶變異體:1,9,1 2,6 1,6 2,6 8,9 7, 9 8,1 〇 1,1 〇 2,1 0 3,1 0 4,1 0 9,1 3 0,1 3 1,1 5 9,1 8 3, • 185,210,212,213,217,232,236,245,248 及270 ; (1 2 )在位置2 5 2以及一個或以上之下列位置上胺基酸殘 基被取代之蛋白酶變異體:103,104,2 3 6及24 5 ; , (1 3 )在位置2 5 2及1 0 3以及一個或以上之下列位置上胺基 酸殘基被取代之蛋白酶變異體:1,9,1 2,61,6 2 ’ 6 8, 9 7,9 8,i 〇 1,1 〇 2,1 0 3,1 0 4,1 0 9,1 3 0,1 3 1,1 5 9, 183,185,210 ’212 ’213,217 ’232 ’236 ’245 ’248 及 270 ; (1 4 )在位置2 5 2及1 0 4以及一個或以上之下列位置上踪基丨: 酸殘基被取代之蛋白酶變異體:1,9,12,61,62,68, | 97 , 98 , 101 , 102 , 103 , 104 , 109 , 130 , 131 , 159 , | 183 , 185 , 210 ’ 212 ’ 213 , 217 , 232 , 236 ’245 , 248 及 i 2 7 0 ; j
(15)在位置252及236以及一個或以上之下列位置上踪基I 酸殘基被取代之蛋白_變異體·· 1 ’ 9,1 2,61 ’ 6 2,6 8 ’ | 97 , 98 , 101 , 102 , 103 , 104 , 109 , 130 , 131 , 159 , j 183 , 185 , 210 , 212 , 213 , 217 , 232 , 236 , 245 , 248 及 j 2 7 0 ; | (1 6 )在位置2 5 2及2 4 5以及一個或以上之下列位置上胺基| 酸殘基被取代之蛋白酶變異體·· 1,9,1 2,61,6 2,6 8 ’ | 97,98,⑴,丨 02,103,1〇4,109,130,131,159 ’ | 1 8 3,1 8 5,2 1 0,2 1 2,2 1 3,2 1 7,2 3 2,2 3 6,2 4 5,2 4 8 及·|
562859 五、發明說明(52) 2 70 ;
(17)在位置2 5 2,103,104,2 3 6及24 5以及一個或以上 之下列位置上胺基酸殘基被取代之蛋白酶變異體:丨,9, 12,61,·6 2,68,97,98,1〇1,1〇2,1〇3,1〇4,109, 130,131,159,183,185,210,212,213,2 17,23 2, 236 ’245 , 248及270 ;以及 (1 8 )在位置2 5 7以及一個或以上之下列位置上鞍某竣殘 基被取代之蛋白酶變異體:6 8,1 0 3,1 〇 4,2 〇 5,Μ 210,232,2 36,245 及275。 此等取代較佳在遲缓芽孢桿菌型枯草芽孢桿菌素(舍兔 始型)中進行,惟該取代可在任何芽抱桿菌 根據周變異蛋白酶得到之篩選結果可知在 囷型知早牙孢桿菌素中之突變對於此等酶之I白八解、舌* 性、效能及/或安定性以及對於該變異酶之 刀〜’ 能重要。 % 或洗滌效 本♦月之許多蛋白i每變異體在調配各種清潔技人物十 : = 己物諸如洗髮精或洗劑上有同。許丄已:之: :在包S本發明之蛋白_變異體之組合物中為適罔界 。此等界面活性劑包括㈣子、陰籍子或兩性離 二…如頒予Barry J· Anders〇n之美國專利4 4〇4丄=及F1〇ra等人之美國專利4,261,8 68號所揭。㈣周配物被記載在美國專利5,2°4,。丨5, “丨?中(先前己被納為本文參考文獻)。本技藝者熟3
562859 五、發明說明(53) 可被兩作清潔组合物之不同調配物。除了典型的清潔組合 物外,易了解本發明之蛋白酶變異體可被用於原始或野生 型蛋白酶所闬之任何目的。因此,此等變異體例如可被兩 於條塊或液體肥皂、碗盤洗滌調配物、隱形眼鏡清潔溶液 或產品、肽水解、廢物處理、織物應用以及在蛋白製造中 做為融合-斷裂_等。本發明之變異體在清潔劑組合物中 可增加效能(與先質相較)。在本發明中,清潔劑之效能增丨 加被界定為,標準洗滌循環後藉常兩評估法測量時,某些 酶敏感污斑諸如草汁或血液之清潔增加。 本發明之蛋白酶可被調配成已知之粉末態及液態清潔 劑,其中pH值在6 . 5與1 2 . 0之間,以及濃度以重量計為約 0 . 0 1 %至約5 % (較佳0 . 1 %至0 . 5 % )。此等清潔劑清潔組合物 亦可包括其他酶諸如已知的蛋白酶、澱粉酶、纖維素酶、|
I 脂解酶或胞内葡萄糖苷酶、以及助潔劑及安定劑。 將本發明之蛋白酶加到習用清潔組合物不會造成任何特 殊周适之限制。換言之,任何適兩於清潔劑之溫度及pH | 值,只要pH值在上述範圍内以及溫度低於所述蛋白酶之變I 性溫度,則亦適周於本發明組合物。此外,本發明之蛋白| 酶可被用於不含清潔劑之清潔組合物中,該蛋白i每可被罝 ! 獨使闬或與助潔劑及安定劑合用。 | 本發明亦關於含有本發明之蛋白酶變異體之清潔組合 !
I 物。清潔組合物尚可含有清潔組合物常用之添加劑。此等| 添加劑可非限定性地選自漂白劑、界面活性劑、助潔劑、 酶及漂白觸媒。本技藝一般人士顯而易知添加劑是於被納ί
562859 五、發明說明(54) 入組合物中。本文提供之表絕非全部添加劑,而應被視做 適當添加劑之例子。本技藝一般人士顯而易知只可使闬與 組合物中之酶及其他成分(例如界面活性劑)相容之添加 劑。 當添加劑存在時,清潔組合物中存在之添加劑之量為約 0 . 0 1 %至約9 9 . 9%,較佳約1 %至約9 5%,更佳約U至約8 0 %。 本發明之變異蛋白酶可被包括在動物飼料,諸如部分之 動物飼料添加劑,例如美國專利5,6 1 2,0 5 5號,美國專利 5,3 1 4,6 9 2號及美國專利5,1 4 7,6 4 2號所述者。 本發明之一方靣為處理織物周之組合物,其包括本發明 之變異蛋白酶。該組合物可被用於處理,例如,絲或羊 毛,如RD 2 1 6,0 34、EP 1 34,2 6 7、US 4,5 3 3,3 5 9 &EP 344, 2 5 9等公告所述者。 下列實例僅係例示而非為本發明之申請專利範圍設限。 所有本文引述之刊物及專利均以全文列為本案參考文 獻。 實例1
大量之蛋白酶變異體用本技藝已知之方法製造及純化 變異體被示於表2中。
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苐64頁 562859 五、發明說明(57) 2 CD Ο z CD σ -2L O) Ό 2 G) Ό 〇 ro 刀 z 'vl CD o 2: -vl cn σ 2 -n! CD o Z: 〇 O X 「 z CD 〇 Cf) M no 2: O) a z O) a 7; ro Z 2: a 2 CD a CD CO 刀 —^ o X z -vl O) O cn __X ο cn O CO > cn I > ω —X 〇 CO > 2: 〇 σ CO _X o > cn CD CO 刀 cn o OJ > cn _^ 〇 OJ > Z • 05 〇 ω s > 2 -n4 CD ◦ cn CD T3 cn ^-x 〇 OJ > Z CD a GO —V 〇 GJ > 01 s > 2: CD 〇 Z -vj o a cn 〇 CO > < ο < o < o < o cn —X 〇 CO > < 〇 cn ^X o CJ > < o < o cn s > < o CO s > or o < —X 〇 ω σ CJ > < _k o < —X 〇 cn _k o a> > cn s > < —k o m r〇 'vl —V. D CO ro hJ 2: ro cn cn ΓΟ —X ro TJ < o 〇 CD < o D —x o CD 73 ω _^ CO o < ~3k o ω —x CD ⑦ O < _V o < —1 o 广 M O) ro 2 < ο z: —X CD cn a > ro 〇 < < σ < λ o CD cn CD a 2: ro cn —k m ro < - z ro o H δ GJ m o ro CO 〇 刀 r * < k -vj > •U δ cn _X 刀 1 •
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562859 五、發明說明(58) 2 σ> Ο CD GO z O) σ 2: 'vl cn 〇 > 〇J 2: -vl O a 2: -vj 〇) a CD 〇 cn o CJ > 2: •^i σ> 〇 2: CD σ 2: O) a 2: CD a d 03 GO 〇 CD —_k 刀 G) a 2: CD σ 2: OJ CO 〇 _k ro 73 01 〇 2: G) 〇 in k O cn —k o 2: -vl CD σ cn 〇 Cf) S > 2: -^1 ο a σ CJ > < o cn __^ o GJ > 0D CO -vl o ω ^λ 〇 CO > cn _x O 0J > 2: G) a Z CD a GO O 〇J > cn _λ 〇 ω CD 〇 Z 〇 a < 〇 (J) 〇 < o < o CO O < o < O 〇 G3 > < o > ro ro CD < 〇 cn λ. o < o < o ω O CO > cn __X o < —k o < —X Ο CO s > CO O CJ > Ο μ CD m < o cn ro »k ro TJ 〇 ro § < X o D CO 73 CD < 〇 〇 __^ CD 73 〇 CO < X o 〇 X o CD 刀 D —k CD N: 刀 < —X o < o m M -sj C) ο ro cn 00 a3 < o < _k o 2: NJ CD _k ◦ m ro O O ro cn 03 73 〇 ro § 73 2: ro CD CO 〇 _3k CJ 70 D —k CD ro 刀 _w CO CD < 2: 0J CD -< D ro § M CD < > M -nI ro cn —i. CD M 73 2: —X CD 7; cn M ro H m M < X ro CD D < NJ G) CJ 工 2: __v. CD CO ω ro CD cn O * \
第66頁 562859 五、發明說明(59) G) Ο 2: CD 〇 2: -si CD a N£ 〇 M X N) < < G) CD > > CD Z CD o 2: -vj CJ) 〇 m m z CD a 2: 〇 〇 m m 2: 〜 CD o 2: -vl cn 〇 2: G) 〇 m C0 ^k 〇 CD 〇 OJ > cn —^ o OJ > CD 〇 2: CD M X 2: CD 〇 σ z cn 〇 cn —^ O 00 > 2: 'vj o z -^4 cn 〇 2: -vl CD 〇 cn —x o CO > Z -si 〇 z a 2: 03 o cn s > cn o CJ > -^1 〇 z -vl cn a < ο < X o < o cn s > 2: σ cn -JW o GO O ω σ OJ > < k o ω ο OJ > in __ o 0J > cn s > < o CD 9k 〇 cn —^ 〇 CJ > ω σ OJ > < X o < —X o cn ^-X o OJ > cn 〇 CO > Q _X. CJ1 CD o Ώ »X CJ1 CD a • O V. CD cn < —k o cn O < o < .-λ Ο < X o 〇 CD m < ο < —X o < —X o 〇 __x cn CD 〇 < _X o: < —λ o < _k o 〇 M O m > cn CD m < —X o < o > z ro CO 03 in Q —x cn CD a O __x. cn CD a < o O cn CD σ ο X. cn CD Ο r k 7s ro CJl Z __^ CD σι σ > _λ. cn CD m cn —a CD CD m D ro § m > —1 GJ CJ <7) _X cn CD 〇 〇 — cn CD a 〇 M O) m D ro 〇 σ> m O cn CD 〇 Ο Μ CO ① 工 〇 _λ CJ1 CD o < M > Z: __^ C3 cn σ 广 ro _k 〜 m r~ ro _X ^nI m Μ cn _X Η z ro ΟΊ M 〇 ro CJl _k 〇 一
第67頁 562859 五、發明說明(60)
〇 < CD < o CO > 工 < CD < ω < GD < C7) 03 〇 ro 2: *-s| 〇T) 2: -^1 Q) 2: ⑦ 2 -vj 〇 CD 〇 2: 〇) Z: 〇 j i ΓΟ CD > > 03 > D > > > > o < a o Ο 〇 a □ σ σ 2: 备 03 > 2: 「 ai 73 Z Z < Z Z z σ> ro cn CO __λ cn CO cn —x cn -X ω m cn __x 〇 -vl O) a ◦ -nJ CD a CD 〜 cn a -vj cn a -vl ⑦ σ CD a O 〇 CO > o s > S > CD ->J 刀 CD CD > O OJ > ω 2 σί Ο z Z in CD X z cn —x cn ω ω 2: < < < —k < k < GO —k cn s > < —k S > -vl CD 〇 S a s > s > -^4 σ 〇 O CJ > ο CO > s > o a o o o o O 〇 0J > o < cn cn _k 00 —»X < < cn —k < < < < —k in _x D 〇 cn N) H k 03 ω GO M < —1. < _k cn ro J 〇 o 〇 o o o 〇 o o ο o 〇 s 73 o CD 刀 ro Tl ro Tl o o ro TJ X CD —X < > < Q < ~k 〇 C) Ο D ro < TJ ro D N3 A ro 00 X Ny 〇 ro cn 广 cn ro < NJ CJ1 CD ο o < 〇 ai CD a o cn CD 〇 cn CD a cn CD Ο c〇 ο 工 O —x. ο 二 ai 刀 CO CD 厂 X. 2: NJ "U CO Tl -< Μ CD —X cn < —X ro CD _X CJ1 D fO CO ro —a. o ro OJ D ro OJ M f k> cn l\J —x f\j ΠΊ fO m l\J 'vj m ro -vl k CD ω CD a CD o o A) o .工 CD X ω Tl < < < ο D Q D D K Λ o m ΓΟ ΓΤΊ |NJ m ro ΓΟ CO CD 工 N) CO G) cn CD ro GJ CD X CO cn X l、J CJ1 _x. -^J _k < -^1 _k < X 〇 刀 < i cn o ΓΟ CO m ro I 1 CO o 工 < I D 1 N3 cn 刀 ) i « j i 苐68頁 562859 五、發明說明(61) < cn 03 > < Ο) 03 > < 〇 CD > z o 〇 2: 〇 z G) o Q cn _k 刀 z CD o z CD 〇 Z: cn a 2 σ> 〇 Z CD 〇 2: ''vl Ο) σ < O CO > < ο CO > < G) CD > < σ> CD > < 〇 CD > < CJ) CO > 2: σ) Ο ζ -Nj CD Ο Z o σ cn —X o ω ω >_X o ω in —x o GJ > Z a CO λ o CO > ω —X 〇 CJ > 2 CD o cn v. 〇 (f) s > ω —x O ω cn —χ ο 0J > Z G) o Ο) Ο ζ 'vj Ο) ο -vj CD Ο Z O) 〇 z: o σ ω ο CO > ω s > cn 〇 c〇 > < k o < o < 〇 CO _x. O CO > < o < —X o: CO —X 〇 〇J > < o < o < ο < ο ω 〇 CO > ω —X ο OJ > ω C0 > CO —k Ο 0J > ω —x. σ OJ > ⑺ —χ σ' c〇 > < ο < _X ο < —k o nj ro ro cn o —x. O CD 73 D CO 7J < o D o CD 刀 D § ;〇 < o §: ro ro ro cn 2: __x ω Ο _^ ro 刀 ΓΟ ro ro cn < —^ o < k ο < —X Ο < k o < _λ. ο < o Q —、 cn CD α ω — 2: O __^ cn CD 〇 X CD 刀 ro ro N3 cn ro ro ro (f) NJ NJ M CD S: ro ro ro CO ro ro ro ω s N3 ro ro ω < ro cn 0J T1 ro ro M cn 乙 Μ ro Μ in 〇 ro cn 73 r * Q cn CO a 2: _^ Μ CJ cn > —V. Μ < 〇 k cn CO a Ο _^ cn CD a —A D Μ GJ Ο) X Ο λ. cn CD α 〇 ro CO CD 工 z ro CD cn m M _乂 σ ro CJ 73 工 Μ CD 73 O M CJ CD 工 Ο _k 〇Ί CD Ο Ο —V cn CO Ο O M CO 2 〇 ro CJ CD 工 Q —x cn CD 〇 ο ro cn 刀 ο ΓΟ 0J ο 〇 ro cn JJ -< ro CD OJ ΊΊ 二 CO Ο Ο ΓΟ CJ CD Τ 〇 N) cn 厂 Z M 么 GJ 2: —x g CO 刀 Μ 工 Ο ΓΟ 刀 Z M CD _X o 工 Μ CO -< o ro OJ o X d cn cn (f)
第69頁 562859 五、發明說明(62) Z: 公 2: 么 < Ο < CD CD > < 〇 D _λ, O _k D —λ. < cn < O) ω ro 'vj 〇 X < CD < G) < CD < CD I CJ cn CD > 03 > M ro 7J ro 刀 NJ 刀 CD > 赛 H CD 〇 N3 刀 CD > CO > CD > CO > 00 > < < ω cn ω _k < Z Z z 〜f 2: 〜1 Cf) ~λ < rr\ ω __^ z -vj z Z z Z Z cn CO > O) CD > ο ο s > G) 〇 G) CD > G) σ G) Ό CD 〇 CD o s > \jj O ω G) o CD 〇 O o G) σ G) a o σ cn —x cn —k < < < cn _k Z ω _k cn ω _X cn < < —^ 00 X cn _k cn —k cn S > Cf) _k ω —k ο s > ο ο o s > CD o 〇 CO > o 00 > o GJ > 〇 > o CD o σ o CJ > o OJ > o CO > 〇 c〇 > V_J CO > < < 2: Ο —k 〇 —k 2: cn —X ro ro ro _k σ 1 < _k 〇 _X cn k > ro < < < —k < _^ < ~X < _X ο o 么 Ο cn CO cn CO s s O o cn CO 〇 〇 0J > CO M O o o o o o α α σ ◦ > H an 1 r * Q Ο 〇 ο > > < CO Tl e: 〇 > < D Q Q > o cn co σ CJ1 CD o CJ1 ① Ο ro GJ ro 03 M o —X CO 〇 M —X o ro ro ro cn cn CD o M CO ro < —X o f|Q cn 刀 ro ro Μ ω CJl CD 〇 Ol CO a GJ CO < cn CO 〇 CJl CO 〇 > > > 〇 D D < d 〇 〇 〇 > > Q Z N. > ro GO ro OJ ro 03 ro CO PO GJ ro N) M N) ro CO ^x cn NJ _X. s M CO cn -vj N. Μ ro < Μ 工 cn 工 i ro cn cn 刀 -Cx Cx) 刀 CD 工 CO o (S\ Zl N) < CO a 4*^ a 4-^ < D 〇 Ο D 〇 Ω 〇 > D > < O z O 2: ro CO CJ) X ro O) I Μ OJ CT) M cn N) cn M CJ1 ro cn ro ro ro cn N) C〇 ro < 0J M CJ G) N3 _k 03 CJ 〇 NJ 〇 a X 73 73 73 刀 < 刀 > 工 a 工 i i D o Ο 2 〇 2: 〇 •.〇 o 〇 〇 o cn 刀 λ3 Μ cn 73 M CO a ro 03 CD X ro CJl ro τς M C〇 CJ) X M OJ CD 工 ro CJl 刀 INJ CO σ) cn 刀 ro GJ CJ) 工 2: 2: z o 〇 o 〇 〇 i Μ cn ro 7; Μ cn ro 7; ro cn ro 7; N) cn 73 M CJl 刀 M cn 刀 ΟΊ 刀 ro on 刀 i 1 1 ro G) 〇 > ) i i j I i 1 | X \ i i i j —i
第70頁 562859 五、發明說明(63) < 0D < G) 73 < CD < CD 〇 —λ 〇 —k Ω _k o —k D _k 2: 〇 —k o —k o «»·λ. 〇 —^ 〇 —k < CD 2: 00 > CD > O 〇 赛 CD > CD > ro 73 ro 刀 ro 刀 ro aJ ro 72 G) a ro 73 NJ 72 ro 刀 ro 刀 ro 刀 CO > co 〇 (J) cn 〇 ω cn __λ 〇 < CD CD CO X 〇 cn _x o cn s > Z -si CD z cn 2 CD cn aj 5 CD cn 〇 2: CD Z O) z G) z 'vl cn Z -vl cn cn CD 〇 > 〇J > a a a Ί3 〇 0J > a a σ 〇 a 〇 > < < cn 乂 < < < cn ^k cn ω 2: in __x. O H cn _λ. cn cn —X cn cn 3 > ω 〇 00 k Q CD 2: o ο o CO > o o o O CO > O 03 > 〇 CJ > (J) 〇 o CO > 〇 OJ > OJ > Vw> CJ > CO > CJ > (f) 〇 Ο < Q 〇 k cn ^X ω _k _k _λ X —X < < Ο cn CD σ cn co α —‘ o —_V cn co a —X CD 〇 X cn co a o H o H O H 〇 03 > o H ω ο o H O H o 么 H 〇 H o H o O < > ro Q Q _k > M cn —JL cn _x ω k o H cn w 1: ro cn r * CO —i. ω _^ <S) —i. cn _k 〇 _V. 〇 —k Ο GJ ro < S m cn CD 〇 CJ cn CD a CO fO < H 爸 H 00 〇 H H ro ro ω H co o H GJ 〇 c〇 〇 H GJ 〇 H cn co 〇 an CD 〇 Ο 〇 > > D > Ω §: Zl 00 〇 > > > > _^ CJ1 CD Ο ro co CD 工 m co ro < ro CJ M < ro CO CD ;工 ro co ro < fO CO σι ro ro ro ω ro ro ro in _x 〇 cn _x co o ro _k CD o ro cn 刀 ro ro ro cn fO —k cq ΓΟ ro NJ GO NJ — cn < ro ro l\J Cn >NJ CO N3 < ΓΌ CO M < > CO D o D 〇 D T) Ω z S: 〇 < 〇 o o Μ OJ ro 0J CO ro ro co N) ro _1L r—N M k CO f r> ro ro M M M cn ro M N Λ M ΓΟ -vj N) ro ro 二 cn NJ OJ (j) ro 00 CD Ν3 < ΓΠ G) 工 o cn i刀 CD X cn 73 cn cn 刀 □ N3 ω NJ cn 刀 1 vJ ω > 1 vJ Cf) 7J 工 工 D o D D D on 〇 < O 〇 z O D 〇 Z CD ;o o ΓΟ CO Ο) ro •cn ro cn ro 二 ΟΊ ro CD ro cn ro cn M G) CO ro N) ro M cn ro 公 cn NJ cr> _k cn M cn ro cn ro 二 cn ro ΟΊ 工 刀 73 刀 σ 刀 < 刀 > cn 73 刀 〇 刀 刀 刀 CO 刀 刀 Ο Μ cn 刀 2: N3 03 σ M cn —k 〇 ro cn ro CO i3 〇 ro cn 厂 NJ G) ΓΟ 2 2: NJ cn NJ 7; Z ro cn 7; O ro fs. 2: NJ ⑦ 刀 ro -'vl 4-^ CJl ;〇 CD a O'! an
562859 五、發明說明(64) < G) 03 > D < CD 2: < cn Z -^1 < cn rr\ z ΓΠ < CD CD < CD CD 00 < ο CD < CD 〇3 < CD CD do < CJ) CD < o 03 < CD CD ΓΟ a3 CO > CO > CD > G) a 03 > a > a v>J > > > > > > > > > > 2: < 2: z z CD cn (Λ ω cn .,> Z 2: 2: o D ω cn —x > cn —X ω —x ω λ (f) 〇 CD CD > cn 〇 〇 -si ① σ 〇 〇 CO > 〇 c〇 > 〇 CJ > O CJ > O) a -vj CJ) o σ ω o ω CD CD H s > 〇 GJ > σ CJ > σ 〇J > cn _k z cn cn CD < < < —X < < O) ω ω < —X < cn X < < < < 3 > G) o o ω s > S > O O 〇 〇 o O c〇 > ο CO > o ο o o CJ > o 〇 σ o < cn —k < < < 〇 —X 〇 ro o > ro < < < Q _χ Q __λ < —λ 〇 〇 Ο __λ o —X ο s > o o O cn CD a cn CD a cn U1 CO 〇 c〇 ro < o ο; o cn CD Ο cn CO 〇 o cn CO a cn CD 〇 cn CD Ο CJ1 CO O Ο λ ΟΊ CD < o Q cn CD G) —x cn CD O —X cn CD Ό > ro CO ro 1 N) 刀 ro CJl > M OJ M D ro CO o Q _k cn CD Ο λ. cn CO CD __k CJl CO > ro CO Μ ~k ΟΊ 〇 __k cn CO > ro CJ o cn —V CD CD > z λ co 〇J ο Ό 〇 < > < X 〇 σ 〇 < H a —1 o < a cn o Q ro 〇 M D > jNJ D fsj D ro > ro ΤΙ Μ > ro Ο ro > |NJ > ro CO fvj > ro > M Ο ro cn CD —A _k —X —λ 〇 CD CO cn 工 CO fO CJ CD cn OJ ro Ο CO ro CO CD CJ ro OJ ro CJ fvj § 「 CJ ro 刀 〇 < 刀 < X 刀 < 刀 < 工 < < < < < > > > > 〇 〇 〇 f3 D > ΓΟ D N、 D D ro 〇 ΓΌ D ro o N) > D ro Μ CO ro N) N) CJ ro NJ CO M ro OJ ro cn N) CJ 〇 X ro a cn aj fvJ CD 00 ΓΟ < I >J 0J ⑦ (SI CJ CD 0J CD GJ G) OJ CD GJ N3 CJ CD < r < < < 73 刀 < 工 工 7J X 工 X X < X Ο Μ > M D M o M 〇 ro f3 D fvj ίο D ro ο ro ο Μ cn 2: o 〇 NJ 〇 cn o ro o NJ 〇 ro OJ CD 工 OJ _ro CO CD 工 GO CD X OJ CD X CJj σι CJj 厶 cn co cn 么 CO cn cn 公 cn CO cn cn < < 刀 < 刀 X 刀 GO 刀 刀 刀 73 X 刀 Ο D Ω D D cn -^j 73 厂 D D Ν3 N3 CO M ro ro f rt cn N) 公 (π i\J cn cn ;〇 G) 工 cn cn 刀 cn AJ < 工 < 7J 刀 D 1 M cn 刀 562859 五、發明說明(65) CD > 2: 公 CO 〇 Q CD m < G) CD > -vj 73 < CD O CO > < 〇) CD > 〇 —k ro 刀 2: -^1 σ> Ο 2: CD o 2: cn σ 〇 —k ro 刀 < ① CO > 2: G) a CD > < o CD > < CD CO > 〇 ro o 7J o —k ro 刀 CO —k ο < C7) CD > < CO CD > -vl G) Ό < CJ) CD > -vj CD 〇 -vj O) a z -vl 〇) a < G) CD > ω _jk ο CO > cn —x. O CJ > cn S > > CO < 2: -vl 〇 a cn o GJ > O) a Z -sj CD a cn 73 < C7) CD > < cn < ο (f) —k. 〇 CO > w —λ o u > cn —X 〇 CO > 2: ⑦ σ cn s > cn 〇 ω 〇 Z CD a < Ο < _ o < O < cr> CD > cn 〇 ω < o cn —X s > ω 〇 c〇 > -nI 05 a -^1 CD 〇 2: 〜 CD a Ο X CTJ CO σ < o < o < o ω S > < o < o < 〇 (Ω o CO > < 〇 X cn CD 〇 > ro CO K) < Z O) a < o > ro CJ NJ < < O < —、 o CO __k O CJ > ω _X 〇 00 > ω 〇 (I) Μ __、 ΓΌ TJ G) V, cn CD 〇 〇 cn CO Ό z —k _k cn H < —X ο Q _λ cn CD a G) —x cn CO ◦ 〇 _ cn CO 〇 < ^k 〇 ο —X cn CD Ο -< CD fO *n D ro CJ σ> X ω 〇 0J > o cn CD a CO G) X 〇 cn CO o 〇 cn CO 〇 < _k o < k o < o > ro CO μ OJ M > ro 0J ro < Q ·_、 cn CD a Q _3k cn CD 〇 D ro § 刀 > ro 0J N) < 2: _X CD C〇 7; 〇 ci CO a > ro c〇 ro < > ro 0J ro < o M cn 刀 < _X O 刀 ro _λ o r CO ro TI > ro OJ M < > ro G3 NJ < CD —x cn C£) o 〇 cn CO a 〇 cn CD a D NJ CO CD 工 〇 ro CO CD X 〇 ro CO CD 工 73 —乂 -si o cn > N3 CO > ro c〇 M D N3 GJ cn X o ro o cn r > ro CO M < 〇 M CO cn X 〇 M C〇 CD 工 〇 —v cn CO 〇 > M CO fO < 〇 ΟΊ 刀 o M c〇 o 工 o »V) GJ G) 工 > M GJ M < > M CO M > ISJ OJ ro < D fO cn 73 D cn -72 D ro cn 刀 2: »X. CD cn (D D ro OJ cn X D ro 0J CD 工 D cn 73 > ro CO ro < D M 0J 〇 工 D M cn 刀 〇 cn 刀 > ro GO ro < o fO CO CD 工 〇 fO cn 73 o iNJ 二 cn 刀 D ro CO cn 工 D ro CO CD X D ro OJ G) 工 2: M 03 〇 Z 00 σ 2: NJ 厶 CD 〇 > ro CO ro < o ro cn 72 D ro cn 刀 CO ro cn CD 刀 〇 ro CJ a> IE 〇 ro on 73 Z M CO (f) D ro O·) CD X D ro 么 cn 刀 2: ro 〇 z ro 〇 Q 〇 ro cn 刀 o N3 cn 73 D ro 公 cn 刀 2: ro cn ro 2: ro cn N) 7; 2: ro ΟΊ ro 7; D ro CO Q) 工 〇 ro cn 刀 > ro ro ω ro cn 73 o cn 73 —I ro CD 〇 < CO M CJ\ CD CD 〇 ro en y〇 第73頁 562859 五、發明說明(66) S 1 ? < Ο) < CD CO > < CD CD > < cn CD > CD > < CD C〇 > < Ο CD > < G) CO > < G) QO > < CD CO > < cn CD > < CD CD > < CD 03 > < cn CO > < CD CO > o ro 〇 73 < cn CD > < CD CD > 01 o CO > < ① CO > ⑺ —X 〇 > 〇 cn s > s > cn 〇 ω cn s > cn _k 〇 CO > ω s > cn _X o CJ > cn 〇 0J > cn s > ω _^ O CJ > cn ~X o CJ > ω —k 〇 0J > ω _k 〇 CO > < CD CD > ω 〇 ω cn 〇 CO > < _k o ω —X o CJ > < 〇 < _k o < _ o < o < o < —k o < —x o < < o < k o < o < ^X o < o < X o < o CD 〇 < _x o < o Q «_λ cn CD a < —^ o Ο —X cn CD Ό 〇 __x on CD 〇 »k 0J cn Q —k CJl CD o Ω __^ cn CD a Q ~λ. cn CD Ό 〇 —X ai CD 〇 Q cji CO a O cn CD 〇 〇 —‘ cn CD a 〇 cn CO U 〇 cn CD a Ω cn CD a 〇 X cn CD a 〇 __^ cn CD σ < Ο 〇 § G) _—λ ai CD a δ C〇 ro < cn CD CD Z CO 70 d —V. CO m > f〇 GJ —X 0J m cn |N〇 X ro m cn ro ^k ro O CD NJ ro > TI ro o 厂 Z __λ CO an 〇 "0 ro —X 〇 cn Ό ro o u ro o 刀 2: —^ CO 0*1 〇 > ro CO ro < O CO ~n Ο _χ cn CD Ο ¢7) —X cn CO a M 〇 CD D NJ GJ O) X CD —X cn CD a > ΓΟ CO ΓΟ < > ro GJ 〇 ro c〇 O) X > ro GJ N3 < > ro CO ΓΌ < > ro c〇 ro < > ro CJ ro < > ro co ro < TI M _i. 〇 r* > N) CO N) < > M CJ M < > ro 0J ro < > ro 00 ro < 〇 ro CO 2 > M OJ ro < > CO »NJ < > »NJ CO ro < > ro OJ M < 〇 N3 cn 刀 2: X s o Ο ro CO cn 工 〇 ro Ca) CD 工 0 M 01 73 Ω ro CO CD 工 〇 ro CO 〇 X 〇 ro 03 CJ) 工 〇 ro CO CD 工 〇 ro GJ CD 工 > ro ⑴ ro < 〇 ro GJ CD X o N) GJ CD 工 D M C〇 CD 工 〇 ro co CD X 〇 M cn a3 〇 M CJ CD X D ro CJ G) 工 〇 fv〇 CJ CD 工 〇 M CO 〇 工 Z ro CD 〇 > ro CO ro < 〇 ΟΊ 刀 D M •cn 刀 2: ro CD o D ro cn 刀 〇 cn 刀 D M CJl 刀 D CJl 刀 〇 M Ol 刀 D ro GJ CD X 〇 M cn 刀 o M cn 刀 D ro cn 刀 〇 cn 2: N) CD 〇 o NJ 么 cn 73 〇 M A cn 73 〇 Cn 刀 o M cn 73 2: ro 〇-| ro 〇 Μ CJ o 工 2: M CO o 2: N) 03 〇 z ro cn ro 六 M CD 〇 ro CD σ ro 办 00 σ 2: ro CO σ z ro 00 σ o M cn 刀 2: ro 公 CD o 2: CD 〇 Z ro CD 〇 Z ro OD σ z M cn NJ Z M CO a Z M C3 o 2: CO 〇 Z M CD 〇 〇 M 二 cn 7J Z ro cn ro Z ro ΟΊ ro Z ro ΟΊ ro 7; 2: ro cn NJ 7; 2: ΓΟ ΟΊ ro 2: ro cn ro z ro cn ro 7; Z ro CO 〇 2: ro cn ro Z ro cn ro 2: M cn ro Ps z ro cn ro Z M G) _V. 〇 z M ai ro 7; z ro cn ro 7s 2: M cn ro 7; z ro cn ro Z CO 〇 Z ro cn ro 7; Z M cn ro 1画 111 1 第74頁 562859 五、發明說明(67) < Ο) 03 > < cn 案 < O CD > < σ> CD > < CD CD > < G) 03 > < CD CD > TJ cn cn cn < CD CD > < O) 03 > < CD CD > < O) < CD CD > o ro 〇 > < G) CO > < G) CO > < 〇) CO > CD > < CD CO > < G) CD > (/) 〇 (J) s > (J) s > cn 〇 CO > cn _k 〇 GJ > cn X 〇 in s > ω o 0J > ⑺ λ Ο CO > in k o CJ > cn __v o CO > cn λ o GJ > < o 00 > ω 〇 〇J > cn o CJ > cn «·k o CJ > in o CO > CO s > ω 〇 CJ > < —X 〇 < o < _k o < o < k 〇 < 〇 < _x o cn —k o OJ < o < o < —k ! o < o < X o ⑺ • ο CO < o < o < —k o < o < o < 〇 〇 cn CO α G) —X Ol CD a Q —i. cn CD a Q cn CD a o _X cn CD a Q _i. cn CD a 0 _)k 01 CD a < o Q —X CJl CD a 〇 _x CJl CD o Q —λ cn CD a 〇 _V cn CD a 〇 X cn CO 〇 < —X ο 〇 cn CD o 〇 cn CO 〇 ο —X cn CO 〇 Q —>>. on CO a G) cn co σ o —i. σι CD a CO ro < > INJ CJ ro < > ro ro < > N) CO > ro CO N3 < > ro oo ro < > ro c〇 ro < Ώ _v cn CD Ό > ro c〇 ro < > ro co M O ro o CD 73 > ro CO M 2: co a Ο —χ cn CD Ο V. o 00 N3 O) < (f) ro G) > ro cn 刀 > ro ^k cn < H ro —X OJ o Ο Μ CO 空 D M CO CD 工 〇 ro GJ cn 工 〇 M GO cn 工 o ro cn X 〇 ro OJ σ> X 〇 M C〇 CD X > ro CO ro < 〇 ro CO cn 工 D ro CO CD X > ro 〇J M < D ro CJ CD 工 > M 0J ro < > ro GJ ro < > ro OJ ro < > Μ OJ ro < > CJ NJ < > N) CJ ro < > (NJ GJ fSJ < > ro OJ M Ο ro cn 73 D CJl 刀 D ro 么 CJl D ro an 刀 D M cn Zl D cn 刀 D N) cn 刀 〇 ro CJ cn 工 〇 ro cn Zl D cn 刀 〇 ro CO CD X 〇 cn 刀 D ro 0J G) X 〇 ro 0J CD 工 D ro CJ U) 工 o ro OJ O) 工 o ro CO G) 工 〇 ro OJ CJ) ZE o N3 CO o 工 o ro G) X 2: ro 00 〇 2: ro CD Z ro CD 〇 2: ♦CD o 2: N) CD a ro CD a Z ro CD a 〇 ro cn 7J 2 ro 么 CD a 2: N3 C3 a 〇 N3 A cn 刀 z M CD a D ro cn 刀 〇 ro 二 cn 刀 〇 INJ 么 cn 刀 〇 NJ 二 on 刀 〇 ro cn 刀 〇 M cn 刀 D ro Cx cn 刀 〇 ro (SI 73 Z: ro cn ro 7; z ro cn 7; 2: M ΟΊ M 7; 2: N) cn ro 7; z N) cn ro 7; 2: (Jl M z: ro cn ro 刃 Z ro 03 〇 z M cn ro 「 z ro cn ro TI 2 CD a λ INJ cn _v. < Z ro -£^ CO a Z C3 o 2: IV) CO 〇 Z ro 二 CO σ Z N) CD a Z ro CO a Z M CO σ 2: f〇 CO a Q N3 CJ1 CD 〇 口 ai -vl 刀 d CD 〇 a3 cn M cn CD 刀 ω ro cn CD m cn ro cn σ> 2: Ol cn < Z: ro CJl ro T| z ro cn M 7; 2: M cn ro 7 z ro cn ro 7; 2: ro cn ro 7; 2: M cn ro 7; z ro cn •NJ 7n 2: ro <j) ro 7; z NJ cn M 7s z ro cn rvj z ro cn ro
562859 五、發明說明(68) Ο ① Ο Ο) o o cn **k 〇 o G) < CD < G) < CD 2: < CD cn X O m < 〇 03 CO < cn CD < cn δ 00 > m —>k m X m ω m CD > c〇 cn CD > c〇 > 00 > GO an CO > CJ > VjhJ > > > > > < ω k ⑺ < < < < z < cn X z 〜1 CD < ω < (/) __^ -vj ω ω —λ cn s > < CD 〇 > 〇 ω CD G) CD > O CD > o" o G) 03 > o CO > σ> a o OJ > CD > 〇 ω o O CO > O) a O ο CO > CO > < < cn k ω 〇 _x z m 00 CD a 2: < cn < k cn —X < «JL Q —^ < —k cn ^x < < _λ. < ~k ω 〇 ο s > ο ω > cn CD σ CD a O) 〇 o 〇 —x H o 〇 ω 〇 cn CD .〇 o U GJ > o ο o o OJ > 〇 > —χ < < O cn —k cn s cn »k o k ω CD —3k < 〇 —x > ro > ro < —λ. 〇 Q —k 2: _x < —^ cn CD ω CO o ο 〇 o CO CJ1 CO o CJ cn CD o cn CD CO NJ 〇〇 ro O CJl CD o CJl CO σ σί o ο < > > > Ό > 〇 〇 CO < CD > ο > ω T} < < < > ro < > ro 〇 ·_X > M 〇 ro rO ro 〇 —x > M > ro 〇 〇 __x OJ Μ ίί CD Ο CO OJ cn 0J σ > ro _k o O o o ro CD < o OJ ro < cn CD a CO ro < CJ CD 工 c〇 CJ) 工 CJl CO a OJ ΓΟ < CO ro < ΟΊ CD σ cn CD a Ο > 〇 Ο > 2 -\〇 ω > ro 〇 _k 〇 ro > ro D N) 〇 ro 〇 (SJ > ro 〇 ro 〇 ro > ro > ro ΓΌ C0 Ο) ΙΕ CO ΓΟ ω cn CD ro CO fsj cj\ CD cn Ό cn CD CJ ro cn CO CO OJ ro OJ CD A cn 上 cn 0J ro GJ CD CJ G) CJ ro < OJ ro < < 刀 Ο < ◦ a 〇 < a < 刀 刀 JJ < X 工 Ο ο Ο > D > T3 > 〇 —"1 〇 〇 〇 2: Z 〇 o o D 〇 μ ro 0J cn ro ro OJ CD X ro 0J ro k ro co M M 0J ro —Λ f . N ro N) CO f ro -Cx 03 M 么 CD ro CO CT) N3 cn ro 二 cn N3 0J CD N) OJ cn CJ1 刀 σ) X CO a ΓΟ < N3 < 〇 厂 CD X UJ 刀 cn < CJ) ZE Vjl 刀 a a X zi 73 工 X 2: D > Ο D D 〇 o z ~ZL ro o 2: z z D z Z 〇 o Μ Μ fO 0J ro CJ M ro CO M OJ ro ro _λ ro ro 么 M cn ro cn NJ 二 ro 么 ro ro f\j f 00 Ο οη •刀 cn X 3 CD X OJ 7J CD X CJl 刀 CD ω CD a cn 刀 CO a ro M 7; cn 刀 CD o CD σ cn 73 u】 73 ζ: D D o > o 2: > 2: Z: ro CO z z 2 z 2: N) CD Z ΓΟ cn Μ 7^ Μ CD Ο ro CO O) 工 M cn 刀 ro σι 刀 M CO f〇 < M cn 刀 M CD 〇 ro OJ ro < ro CJ1 ro 7; ro on M 7s NJ 公 CO o N) cn ro 7; NJ cn ro 7s iNJ 二 CO 〇 ◦ Ό 2: ΓΟ αι D ro Z 2: NJ 二 〇 ro CO 2: INJ 么 Z M cn 〇 ro CO Z M cn 2: ro cn M Z ro ω _λ. Z ro G) _l 2 ro cn (NJ Z ro cn M Ν3 7^ CJ1 刀 UJ a C〇 o C7) 工 vjj U ro 7s 工 7; 7^: σ 刀 7n 2: 2: z 〇 2: 〇 d CD 〇 Z ro CD ro ΟΊ ro ro cn ro M on ro cn M ai cn CO σ 刀 7; 刀 (TI 〇 z ro cn id CD —1 ro cr> M 〇 > o >
562859 五、發明說明(69) Z: Q cn CO cn 2: cn cn cn GO V Q cn k cn (D > CD o cn CJ 厂 〇 < CD ΓΟ α ro o 73 o CJ > o o OJ > 〇 M Ό o GJ > 〇 OJ > 〇 0J > o > o ro > o 〇 o m CO CO m CD C3 a CO m CD m G) m C7) CD > (f) Z < < < cn —x < < < ' < k cn GO cn cn __k GO X GO 〇 Cj) GO _^ ο G) ro a o o o o o o o o o ω > s > O O CJ > o CJ > 〇 CD > m CD > o co > < cn —k 〇 k O —k 〇 ^λ < D —3k 〇 D ~X ω —X < < —X < < —X < _k < cn _x. < O an _^ < ο s CJl CD CJl CD CJl CO o § cn co o CD § o 〇 o o o O s CD > o CJ > o > Ό o Ό 刀 〇 m m > ο < _k cn x 2 〇 o > ro Q 〇 CD G) _X 〇 _ 〇 _k Q _x 〇 < —k ' < _^ Q — cn co o CJ) CD s CJl CO cn CD c〇 ro cn CD cn CD ai co cn co σι CD CJl CO cn CD cn CO o o σ o CJl CD α m a σ a 〇 < a a a a a 〇; 〇 〇丨 o id ~X OJ ζι o —λ cn CD a > N3 CJ N) < > ro OJ ro < > ro co > ro OJ ro < > ro co ro < 〇 ro CO a> 工 > M 0J N) < > N) CO > ro OJ ro < > ro CJ > ro CJ > M CO ΓΟ < > iTO CJ < > ro Ca) ro < 〇 cn (Ώ 〇 ω __x ο Q CJl CD o z ro _x CD cn > d OJ D D D D 〇 D D D D D N、 〇 D 〇 .V i 〇 GO < cn _k > ro N3 0J Μ ro co Q) N3 OJ CJ) ro CO CD N3 C〇 cn o G) N3 CJl NJ 0J 〇) ro CO CD N) CO σ> INJ CJ CD 〇J CD P'J co CD 1 vJ OJ ο i VJ CO CD CD -vl ο 二 G) 〇 < co ro 73 X X X 工 73 X X 工 工 X 工 工 工 TI < D > ro GJ D D D D D Z o D D D D 〇 Ο 〇 > > > 〇 ro CO cn 工 ro NJ 公 M M ro M M M 广Tl M t rt M (rt N3 cn NJ cn N3 cn NJ OJ ro ΓΟ CO |S〇 ro CO ro N) OJ cn ro < CJl zi CJl 刀 cn 刀 CJl 刀 CJl 刀 CO Ό cn 刀 CTl 刀 Vwi 1 7J w ϊ 刀 w I 刀 刀 刀 刀 < < < X Ο 〇 ro CO 2: Z 2: z z z 2: z z z: z z z 2: o o O O ro ro ro ro M M ro CJl M M M ro 00 M CD M 〇3 C〇 CD (\J CO CD ro CO C7) M CO CJ) ro 二 cn C71 73 C7) CD a co 〇 00 a CD a 00 a NJ 7; LO 〇 CO o a o σ a a σ X 工 a) 2: 〇 2: 2: z z 2: Z 2: z z z 2: z z z o o D 2: Μ CD σ ro cn ZI INJ CJl ro 7; ro cn ro 7; ro cn ro 7; ro cn M ro cn M M G) _X 刀 M cn M 7; M cn ro 7; ro cn ro ro cn N3 7; ro cn f〇 7^: Μ cn M .nj CJl INJ NJ cn NJ 7; ro 二 cn aJ ro 二 cn 刀 M cn 刀 1*0 CD σ 2 fO cn z N3 Z 2: Z z NJ 么 N) N3 么 r>r\ ro cn ro 7s CD a 03 a C3 o σ inJ 7; z i\j z ,\J (N〇 M cn cn ro cn ro cn NJ Ps 7; 7n 7s 1111 第77頁 562859 五、發明說明(70)
< G) CD > < ο 03 > < 〇 CD > < O) CD > < CD CD > CJ) 〇 < ① CD > < G) CD > < CD CD > < CD CD > < ο CD > CD > d CD > d CD 〇 7; ro -vl Z CD 〇 OJ > ω —X o CO > 2: cn M 〇 an o OJ > ω ⑺ cn ω cn cn ω 2: 2: Ζ 2: z (I) _k cn k < < cn < ο S > s > 3 > 〇 CO > 〇 CO > 〇 CJ > s > -vj CJ) 〇 -v| 〇) a ^vj CJ> σ cn ο σ a s > 〇 OJ > o o o CO > o < ο < < < < < < < cn cn ω λ ω _χ 01 ~x cn < < TI ω —k < 〇 ο o 〇 —X 〇 λ 〇 X 〇 o o CO > o CJ > ο CO > S > O CO > 〇 Cx) > o o CO X < 岂 Q o cn CD σ Ο Q 〇 o Q o _k o k o k < < < —X < < < _X o 〇 〇 —^ G) —^ Q D cn 73 cn CD σ CJ1 CD σ cn CD σ cn CO σ cn CD a cn CD a cn CD a ai CD a o o ο ο o o cn CD a cn CD σ cn CO σ cn CO a cn CD 〇 ΤΙ Ν3 —X Ο r < Μ cn 「 —X ro OJ ~n > ro CO o < *a ro o -< CD _w zo id —x GJ 72 〇 cn CD 〇 〇 —^ ai CD a Ο __^ αι CO Ο Ο —χ. cn CO Ο CD cn CD a Q ΟΊ CD σ r> ro CO NJ < > ro OJ > ro CO ro < > ro CO ro < 〇 ro 〇 cn 73 f3 __w m > ΓΟ 0J Μ > ΓΟ CJ f〇 < > ro OJ ro < > f〇 GJ ΓΟ < > NJ CO NJ < > NJ GJ > ro OJ ro < > ro CO ro < T3 N) —X o < M cn ΤΙ ΓΟ _k ο < ΓΟ § —k CO 刀 —X OJ 73 〇 ro OJ O) 工 o r\J OJ cn 工 〇 NJ CJ CD 工 〇 CJ cn 工 > ro GJ ro < > ro OJ M < D Μ CO Oi X D fO CO σ) 工 D ro 0〇 CD X 〇 NJ GJ 空 o ro CO cn X D NJ GJ cn X 〇 ro CO CD X D ro OJ σ) 工 > ro CO fO < —1 ro _^ OJ 72 Η ΓΟ C0 73 —X CO > M OJ ro < > ro OJ ro < 〇 ro cn 刀 〇 ro cn 刀 o N) on XI 〇 ΟΊ 73 〇 ro GJ G) 工 〇 ro CJ G) Ο ΓΟ cn 73 D N) ^ cn 73 Ω N) CJl 7J o M cn D M cn 73 〇 M cn 73 D ro cn 刀 Ο fvj cn 刀 D CO CD X > fO OJ ro < > ΓΟ C0 ΓΟ < > ro c〇 ro < 〇 ro OJ cn X 〇 ro GJ CD X Z ru CO a Z 二 CO a Z NJ -Dw CD σ 2: CD 〇 D NJ 么 an 73 D ro 二 ΟΊ λ] CD 〇 > CD 〇 > D M cn 刀 〇 M GJ CD 工 ο ΓΟ OJ CD X D ro OJ CD X 〇 cn 刀 〇 ro ΟΊ 刀 z ro cn ro 7; 2: ro cn ro 7; ro cn ro 2: ro cn M 7; 2: ro CO σ 2: .NJ 二 C3 a CD 〇 > D ro cn 刀 ο ΓΟ ΟΊ 73 D (NJ cjl 刀 CD 〇 > 1 ro G) 〇 > 2: ro ai ro 7; z ro G1 NJ —1 ro CD 〇 > —"1 Μ CD 〇 > —"1 〇 > m r〇 Q
第78頁 562859 五、發明說明(71) C0 _k (3 cn 〇 CO s > cn k cx ω _X Q O —λ cn __x 〇 cn o cn __k o < CD < o GO —V. O < o rr\ < cn rr\ < CD Q3 cn _X o 03 cn —w o ω CO > GO > ro 72 CD > CJ > c〇 > 03 > CO > > VJU > > GJ > > OJ > < < cn < < < cn 2: < < < cn 2: < cn ^-X cn cn —k < —k 00 ««λ < —λ ο o 〇 o o _k o o CO > O) a o o o o C) a o o CO > s > s > o s > o Ο CD < o o o -< CD < ω O O O < cn Q < < < o —k < v, Q »λ CJI co cn c〇 CD CJ\ CD o 〇 OJ cn CO CJI CD cn CD o 2 cn CD o o o cn CD σ o cn co ο a a σ > a σ o: — > a D ο C£J < 〇 < < O _k < -< ro TI Κ. Λ -H ro O _^ < > ro O o »k Q __x > NJ 〇 _V > ro cn ai CD a NJ g ΓΟ C£] CO M ΟΊ CD a o o co $ |VJ —X ο CO 73 cn CD ◦ 〇 OJ ro < cn CD O CJI CD o cn CD a GJ ro < cn CD 〇 < -< g CD < 〇 O > CO > ro O O ro > > O ΓΌ > ro 〇 N) g § 03 CD S cn CJD CO ΓΌ co 03 ro Ol co co CD § s 'vj CJ G) CO ro CJ CT) CJI 〇 o < < a 工 CO < X < X > ro CJ ro < TI ro —k g > ro CO f. \ o M d x f .Λ Tl ro k D ro GJ D ro CO iT-pk D ro OJ CD O ro OJ CD ->>k CO D M cn > ro CO ro > ro co fsj > NJ CO |\J O cn 〇 ro OJ CJ) o ro cn o NJ < 73 CJI 刀 刀 o 「 CD 工 X X X 刀 刀 < < < 73 工 刀 〇 > D ro —k 〇 > αι > id — CO D o o o > r O o o 〇 ro ro CO INJ CO OD M CO p〇 fO CO fsj M cn cji fO cn N) cn ro CO ro ro CJj ro CJ CD ro OJ G) NJ CO cn N3 cn 工 o 工 < < < 73 刀 73 73 刀 < < X 工 工 刀 D 〇 CO D D D > 「 Z —1 ro ω o r o o o o id G) 〇 ro GJ NJ ro OJ CD X fO OJ N) OJ ro 0Ί ro cn -^J ro CJ cn ro cn ro A cn cn cn 刀, CD ,工 NJ < cn 刀 CD X ro < < UJ σ > < 工 刀 刀 刀 > :?3 丨3 o ro cn o ro GJ CJi o o M 另 D cn D ro co CD 2: M cn ro Z ro CD —^ o cn cn 「 ro CJI < ΟΊ < 刀 X > 刀 X 7; 刀 < < ; f3 cn o M ~~1 ro ⑦ d cn O M G) Q < cn 刀 o > o > cn ZI > H 2: ro ω ro CD o >
第79頁 562859 五、發明說明(72) 2: CD cn —k cn cn — cn _x cn o _X > cn 〇 _k CD > > A > > < CD cn —X CO cn < CD ro Ο o GJ > o a> > o CO > s > o CO > ◦ cn r o Q M ZI o N) > < CD < CO < CO > o OJ > o CJ > o CJ > CD > cn _X < < < < < X. ω 〇 k G) —k o —k Cf) 系 < 〇 ω an _k < < _^ < _k GO —X ο 〇 o o o o s > o M o N) > S > o CO > 〇 c〇 > 〇 CO > o o o o CJ > < 〇 〇 〇 〇 TI _X < cn —k cn CD —λ < cn cn _X cn —x < < o —_k. o —X Ώ __^ < ο CO a CD 〇 cn g cn CD σ c〇 —k < o 〇 CO > 〇 CJ > 〇 o o ω o CO > o ω o o cn CD a cn CO a cn CD a o Ο > > 2 —k z o 〇 < < < o __x < < < 〇 cn < ro < K ^ o cn CD α CJ OJ g Q g cn cn CD a (J1 CD o o o o cn CD a o o o 公 on CD 〇 ro CD 厂 o O] O CO rxj g cn CD 〇 id _k CO 〇 ro D N> > fS〇 > fSJ > ro 〇 _^ 〇 __X 〇 X cn ro 〇 —k Ώ —x Q —X r> N3 —X TI 刀 ro < CJ] 03 CD CO CD CO N> CO ro co —k CO cn CD ai CD cn CD ro O cn CD cn CO ai CO OJ 〇 < CJl CD — 〇 —k o o CD 刀 X X < < 刀 〇 a σ σ 〇 a σ < < 〇 D 〇 > cn 00 cn > > > —C M CD o > > > T) < ro ro N) M CO ro CO ro CO O) X ro CO ro —k M —X ro ro OJ ro CJ M ro CO ro OJ CO ro CO ro OJ ro § ro < > 么 H CD zr σ> 工 ro < ro G) N3 o ro CD ro < ro < CD 工 iXJ < fvj < o $ 〇 D D O D 〇 D > > > D D D > O D o o > TI ro 〇j cn X M M M M M fvj 0J ro GJ N Λ ro co K Λ ro OJ fsj ro 0J cn M C>J CD ro CJ Q) |\J G3 N) M cn NJ OJ CD NJ OJ 〇) M CJ G) ro OJ ro M —k cn 刀 CJ1 73 CJ1 刀 CJ1 刀 CJ1 73 \J) 工 1 NJ < InJ < < X X X < 刀 工 X < V) D z 2: 2: 2: 2: 〇 o 〇 〇 o o o o o D o 〇 > fO M M ro ro ro GJ ro CO ro M M M CJ M ro 么 二 ro OJ ro CJ cn 刀 CD XJ CD 〇 CD a CD 〇 CD o cn 刀 CD 工 CD 工 cn 工 cn 刀 cn 72 cn 刀 cn 工 cn 刀 cn 刀 cn 刀 〇 工 ro < 2: z 2 z z 2: o 〇 D 2: M CJl 2: o o o M 03 M cn ro NJ ΟΊ ro ro cn NJ M ΟΊ ro ro cn ro M C3 ro a cn cn M cn M 03 M CD ro 二 cn iNJ A cn 刀 ro CJ G) a 7; 7^: 7; a 刀 刀 刀 σ < a 73 z 2: Z Z 2: z Z: z o ro cn ro ai N) N3 么 ro 么 ro cn ro CJ) _X ro cn ro 二 CJi ro 7s ro 7; 00 o CO 〇 CD Ό N) 7; ro 7; CO K 1 2: 2: V Λ 2: K Λ —1 ro cn cn iXJ CJl M fvj cn ro fNJ CJl f〇 〇 〇 > 73 1 1
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第82頁 562859 五、發明說明(75) | 實例2 ! 按照美國專利申請案6 0/ 0 6 8, 7 9 6號「測定較佳酶及/或 較佳清潔劑組合物之改良方法」所述之微型開關測定法,
測試實例1製得之大量蛋白_變異體在二類型清潔劑及洗 滌條件中之效能。 I 表3列出被測定之變異蛋白酶以及在二不同清潔劑中之 | 測試結果。A攔:清潔劑為0.67公克/公升經過濾之Ariel VI(Procter & Gamble,美國俄亥俄州辛辛那提市),在含 3格令(gr a i n s ) /加合混合Ca2; / Mg2T硬度之溶液中,以及在 各孔中使兩0 · 3 ppm酶,溫度為25 t。B欄:清潔劑為 j 3 . 3 8 公克 / 公升經過遽之 A r i e 1 F u ΐ u r ( P r 〇 c t e r & |
Gamble,美國俄亥俄州辛辛那提市),在含15格令(grain) j /加舍混合Ca2+/ Mg2'硬度之溶液中,以及在各孔中使0 . 3 | p p m酶,温度為4 0 °C。 , i
第83頁 562859 五、發明說明(76)
2: 卜 O < < < < < G) < cn Z :Ζ < C7) < 〇 < G) CO > cn Z 0J σ G) —x m 03 > CD > CD > CJ σ 0J ω Ο CO > CO > o GJ > G) a < O) CD > CO 〇 GJ > cn X·X 〇 cn —^ O ω s > ω —^ ο CO > CD _k 〇 CO > < CD CD > 00 > < CD CD > ω λ ο C0 > CO S CO > GO —k O cn o GJ > < 〇 cn ~k O CJ > cn —k CO < < < < < λ. < —λ (f) x. ω —_X ω — < < λ < —k < < λ ο o 03 > o o o o ο o 〇 CO > Ο ο GJ > σ o o 〇 ro CD H o 二 < < O _x CD O —X 〇 —X Ο o —Jk < < < 2: _、 O _^ 〇 cn CD Q λ ο o cn CO cn CD cn CO cn CD cn CD CJl CD o ο Ο ο a CJl CD cn CD Ό 〇 〇 ◦ Ο o σ σ ◦ Q _k O id > Μ > ro 〇 Ο —X Ο —X ο __^ > ro > ro > r?o CJl CD cn CD ro 00 ro fNJ CO ro CO N3 GJ ro cn CD CJ1 CD cn CO cn CD ro < CO M < OJ M < O 〇 > < < < < < a Ο Ο σ > CJ ro > 〇 〇 〇 D D > > > > 〇 〇 〇 N3 GJ ro M GJ ro OJ CD N3 GJ 〇 N3 CO CO OJ CD ro ω cn N) CO ro ΓΟ OJ ΓΟ ΓΟ CO ro < ro 0J ro < ro CO CD ro OJ CD ro CJ CD < < < 工 X 工 工 工 < < X 工 工 o D O 〇 o 〇 〇 〇 ί ο Ο 〇 D D 〇 ro GO 〇 fO CO CD ro CO CD ro cn M cn ro c〇 ro cn cn ro CO G) ΓΟ CO ο Μ 0J ① ro CO CD M cn cn M cn 工 X X 刀 aJ m 刀 刀 工 ;X 工 工 刀 刀 刀 〇 D D CJj 2: D Z ΟΊ -vj o D Ο o Z Z M 广ΤΊ ,ΤΊ M ro ΟΊ V Λ M M m M / n Μ ' (η Μ cn cn ro 么 co ro cn ΓΌ vj 1 7J \J 1 •刀 cn 刀 < |NJ cn cn 刀 vJJ o < LJ 1 73 W ι 刀 73 刀 〇 7^: Z M cn 刀 Μ -vj Z ro cn 2: Μ cn 2: M cn 2 f〇 cn CD CO Ul ro Μ ro M 〇 O < X 7^ 7n Z ro f ή 2: ro r π \J I ro \j 1 ro 7; CJ an 〇 CO OJ cn CJ lo ro cn CD ro CD NJ CO OJ NJ ώ CO ro b ,o ro ο cn —3L CO _k ro CO CD ,NJ k) o M 'vj -^1 _k _X 〇 cn o —X > o o —k 〇 —k o o __l _k _X. _X _k _k _k —X _k b CD ro CD -vj 丄 _X ro 〇 cn CJ '〇 —J CO A 03 C3 Ο ro M 〇 CO ΟΠ _k _^ CD
第84頁 562859 五、發明說明(77) Z z Z 2: 2: 2: 刀 2 H CJ 2: Z: z -v! > > m Z 'Nsl < G) CD σ N3 < cn 〇 CD o G) Ό CD σ CJ) 〇 CD Ο CD r CD 〇 CD GO G) σ G) Ό CD o m ω —1 CJ) 〇 O) Ό CD > veois 'vl CJ) a ~U CD o ω cn o ω CO _k 〇 cn s > ω ο 03 > 2 Ο) Ο Z 〇 • 〇 cn o c〇 > Z CD o 2: -vj -Vi 〇 cn s > cn s > 2: G) a Z -vj G) a z a z 〇 a > CO CD m ω 〇 > < ο cn CJ) < < < < (I) ω < cn _k· cn —V. < < _)b. cn GO __λ CO —x CO cn —k < s > 〇 ω o o o Ο σ CJ > s > o 公 〇 c〇 > o 〇J > o o o ω 〇 CO > O ω s > s > 〇 f〇 CJ m < k o < o 7; N3 cn H CO M Ol CD 〇 2 M cn ro 〇 2: k s Ο < ~k ο < o 2: —λ. CD 〇J 〇 < —X o < o cn M —x CD o 2: M O) o < k o < ~X o < o < o < —k o Q6S10 d > 2: >Jk CD cn a δ CO D > -^4 < r * z M CD 〇 cn to M TJ > ro CJ M < O ro CJ 2 〇 NJ 二 cn 刀 2: CD a 2 ro ΟΊ .\J —k —X —k ~k 〇 _k —^ —λ. __v. _^ _^ _k _^ _X _k —k NJ _X ro o b 〇 CO _k CO ro <JD ΓΟ S g . _λ. O CD 〇 C^l N3 IV) N3 —^ CJ N) N3 •cn o CO _x. O _、 CD o CO -^4 〇 CD 00 〇 〇J ·◦ —k ro o Ln CO 〇 Μ CO 〇 _k CO o 二 CD o k) ΟΊ 〇 cn cn ◦ GJ cn O _>k GJ O GJ 0J 〇 OJ ro 〇 GJ ◦ C7) 〇 L) 0J 〇 μ CO __x 二 cn
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第86頁 562859 五、發明說明(79) Μ O ro 工 Γ~ -Dw ΓΟ < 03 > > 二 2: C7D Ό m < m 2: CD 〇 ζ CD ο ^ν| G) Ο 2: 'vj Ο) ο Ζ 〜 σ) Ο Ζ -ν| CD Ο m Z -^1 G) σ CD CO Z S a --vl O) σ > OJ 2: 'Vi Ο) α M 工 2: -vl G) o 2: CD a 〇 a ω s > 2: G) 〇 z CD a Cf) ~k 〇 CJ > ω —λ. ο CJ > Ζ -ν! α ω __X σ C0 > ω _X ο C0 > ζ -νΐ Ο ζ σ> Ο cn X o 0J > -si 〇 cn _k 〇 GO > cn __k. 〇 GJ > 2: 〇 〇 (η S > 2: cn Ό cn k. O CO > cn ·_^ O cn ~k 〇 ω < o cn —k O 0J > ω __^ O CO > < o < _X ο ω __X Ο CO > < σ < —λ. ο cn σ ω —λ σ C0 > < —k o cn __k 〇 CO > < o < _X o cn o > < ο cn _k o OJ > < _k o < o < o D ro § m < o < k o O ΟΊ CD 〇 D Μ 〇 Ο) m < 〇 Ο ΓΟ ο CD m > —λ CJ1 〇〇 m < ο < _^ 〇 O ro 〇 CJ> m < o cn ro ro 〇 〇 ro § < _k o Ο __k. cn CD α < ^3k o Ώ cn CD a 〇 ·__X cn CO a 二 cn —k H > σι CD m CO _λ CD CD ΠΊ D INJ 〇 G) m 2: Μ CD ^JW ο > __χ GJ CO D NJ g m 〇 cn m z ro CD _X σ m ro Q O ΓΌ cn 03 刀 〇 ro 〇 CD 刀 Q —_X Ol CO σ 〇 ro OJ σ> 工 O cn CO a < NJ > 2: C0 cn ο ΓΟ ΟΊ —X Η __v. CD o —V, ro 03 0J CO NJ 〇 M 〇 _JL CJ ro b CD _k '<JD _k ΓΟ _X ο _X '•^ι CD _k *0J Μ _1 _ν CD cn — •二 〇 _k cn _^ CO o 2 〇 0J CD 〇 0J 〇J o 〇J _)k o —V. CD 〇 X 二 〇 _X 〇 o CD p o 〇 b CD o o 〇 b a ο ο 二 Ο ·〇 二 ο ο 0J Ο CD 03 ο —X CO 〇 '〇 二 O b cn 〇 b 二 _ ro 〇 _X b G) _x b NJ _k b _k 562859
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90頁 562859 r 五、發明說明(83)
苐9丨頁 562859 五、發明說明(84) < 〇 CD > 〇 — ro 73 o ro 73 〇 ro 7J o M 刀 2: O) 〇 2: Ο Z G) 〇 2: -vl CD o Z CD o cn ΟΊ Tl cn s > veots CO o CJ > cn —X o CO > cn s > -vJ cn a —k o ~i < 〇 _k o H _λ o H —^ o H ω __X 〇 OJ > cn __v CO o r〇 ΟΊ CO Ο cn _^ OJ ◦ H cn _x CJ o H cn —^ OJ o o H <: ro ro N3 ω > ro OJ ro < 1: ro •NJ •NJ CO 1: ro ro ro CO 刀 -vj 〇 CO CO H D cn 刀 〇 ro OJ CD 工 TJ NJ _k 〇 ω D ro 厶 cn ZI z _V CO cn 〇 乙 ro ro f〇 CO o M cn :0 〇 ro cn 刀 < NJ 〇 CD > |NJ INJ tNJ CO 〇 ro CJl XI t z M CO o ro CJl _^ D 1 i 1 i f o ro A cn 刀 ro o CD o OJ o ro o _X _\ __V. nj CD 〇 OD CD _k CD 〇 —k O) o o Ό o _k CD _Jk 03 o
第92頁 562859 五、發明說明(85) ] 實例3 | 表4列出來自實例1之被測定變異蛋白酶以及在四種不同| 清潔劑中之測試結果。對於被記載之變異蛋白I每以及下列 清潔劑進行與實例2相同的效能試驗。A攔:清潔劑為0 . 6 7 公克/ 公升經過濾之Ariel Ultra(Procter & Gamble,美 | 國俄亥俄州辛辛那提市),在含3格令(g r a i η ) /加侖混合 I Ca2,/iMg2T硬度之溶液中,以及在各孔中使用0. 3 ppm _。Β ί 榻:清潔劑為3 . 3 8公克/公升經過濾之A r i e 1 F u t u r (Procter & Gamble,美國俄亥俄州辛辛那提市),在含15 I格令(grain) /加侖混合Ca2-/Mg2f硬度之溶液中,以及在各 | 孔中使闬0 . 3 p p m醒。C榻:清潔劑為3 . 5公克/公升H S P 1清| ! 潔劑(P r 〇 c t e r &· G a m b 1 e,美國俄亥俄州辛辛那提市),在 含8格令(gr a i η ) /加舍混合C a2_/Mg2T硬度之溶液中,以及在j 各孔中使周0 . 3 p p m S每。D欄:1. 5毫升/公升T i d e K 丁清潔 劑(P r〇c t e r G a m b 1 e,美國俄亥俄州辛辛那提市),3格
I
令(grain)/加侖,以及在各孔中使周0.3 ppm |每。 I
第93頁 562859 五、發明說明(86) ο a < C7) CD > < CD CO > < G) m < o 03 > < CD © © < O) © Q M 73 ro in s > Z C) a cn S > > s < cn o cn s > ω 8 > CD 1 > ω s > cn s > ω ^a. O ω s > cn s > ω 1 > < CD CD > tn s > cn _^ o ω < o cn S > < 〇 < o < o < o < O < —k o < o < o < 〇_ < o < o < o cn s < o < o G) ai CD a < O Ο cn CD Ο 〇 cn ,CO a d _λ. •GJ cn 〇 k. on ΧΩ a O _^ cn CD 〇 〇 —k cn CD 〇 O —V ai CD a 〇 CJl CD a Q — ai CO a Q —^ CJl CD o O —w cn CD 〇 〇 <_λ cn CD σ < o D g ;〇 〇 »k ai CD 〇 > ro CO ro < id _k id CJ m CJ ro < d —w GJ m ro — ro m cn ro N) 〇 ω ro ro 〇 〇 z ^w CD S _X o 70 2 _x. CD CO σ -< M CD 〇 — cn CD a o a -< ro g D ro OJ > ro CO M < > ro CO 〇 ro OJ σ> X > ro 03 > ro OJ M < CJ ro > ro OJ • < > ro CO ro < Ί0 ro _k p > ro CO ro < > M CO ro < > ro CO ro < > M CO ro < OJ ro < > M CO ro < 〇 M cn 73 \ D M CO CD X D ro co σ> 工 D ro cn 73 〇 ro OJ CD X 〇 ro OJ O) X D ro 03 cn X 〇 ro CJ CD 工 D ro CO cn X > M GJ M 〇 ro CJ cn 工 〇 N) OJ CD X D ro CO CD 工 D N) OJ o X D ro CJ cn 工 〇 NJ CJ CD 工 2: N3 CD 〇 D ro cn 73 D N) 另 2: M CD a o M CJl 73 o ro cn 刀 o M cn 73 〇 ro cn 刀 o N) cn 73 〇 fO CO CD 工 〇 M cn 7J O N) cn 〇 M s D M cn 刀 D M CJl 7J D ro cn 2: ro cn ro 7; M CD a 2: ro CD 〇 2: ro cn ro 2: M CD a 2: ro CD 〇 2 fO CD a 2: ro 03 a Z: N) CD o 〇 M s 2 M CD a z CD a z M 03 σ Z ro CD 〇 Z M CD 〇 Z fO CO 〇 2: ro on ro 7; 2 ro cn ro 7s z ro cn M 7; 2: ro on M z ro cn M 7; 2: M cn M 7; z M un ro 7; 2: ro CD σ 2: M cn ro 2: N3 cn M 7s 2: M cn ro 2: ro cn N3 7; 2 ro cn ro 7; Z cn M 7; z ro cn ro 7; CO -vj ro ro ~vj _k _k —k ro cn M b) CO NJ OJ —A. N3 03 〇 M ro M M 〇 CO CJ M CO __k ro —x CD _X CD _λ. b cn ro OJ —^ _k > —_乂 ro 〇 _κ CJ CD 〇 g 〇 cn CD —k 丄 k CJ ^-x CO 〇 CD CD —k 03 CD 〇 ώ _k —X L> CD _乂 Μ —k _X CD «k 丄 _k k CD OJ 'Vl _k _k ro _k •CO L —k ro _k. CJ o ^k '〇 to *k cn CJ —k cn cn _k. _k ro OJ —w 乂 CD —3l CT) 〇 —k cn o _L ro 〇 _1L cn cn _k CO CD _k o b O) ro b —w 〇 cn ro CD ro O) CD ro ·〇 ro b k CD ro CD CD 〇 CD o ro … o ro b M _L *0J k _k «_k CO ro L) cn _V. *M cn _k o
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第95頁 562859 五、發明說明(88) < on CO > < O) CD > o s < O) CD > < CD CO > O CD rri Q CO m < CD C3 > Z ο σ 2: 5) o cn __k 〇 cn s > ω s > CD • —k s > cn S > m CD CD a ω —k 〇 < o < o < o 0) s > < 〇 < Ο cn 〇 < 〇 O —k ai CD σ O _ cn CD σ > OJ ω < o o —JL cn CD a ο ^Λ. σ\ CD D < 1 〇 —1L CJ1 CD σ 2: ro CD ω CO ro < O _a cn CD a ω > > M ro > f〇 Μ 〇 k cn CD ◦ > ro CO ro < > ro OJ M D ro CJ CD j: CJ ro < O —a. CJ) CD D > M C〇 M Ο Μ 0J cn X T) ro —v 〇 厂 〇 ro OJ cn 工 O ro CO σί X D M cn 刀 D ro OJ cn 工 > ro CO ro < 〇 ro CO cn X Ο Μ cn 7] id OJ 73 D f〇 cn 刀 O fO U1 Zl 2: CD G) O N) CJl 7J 〇 ro CO cn X 〇 M ctj ZI 2: Μ 〇〇 Ο > M CJ M < 2: M 03 σ 2: M CD σ z ro cn ro 2: ro CO O D M cn 70 2 M CD 〇 Ζ Μ cn ro 7; o M GJ cn 工 2: NJ cn ro z M cn ro 7s Z M cn Z M CD 〇 2: ro cn ro D ro cn 刀 2: M cn ro 7; —! M cn 〇 > ro cn ο _k OJ M _k ro ro _k L) ro _^ 〇 ro 03 OJ ro CO _k 〇 CD OJ •OJ 〇 _^ M —k •cn 〇 —k •s o •CO 〇 ro cn _k L) CD k OJ »k k _k '〇 cn M 〇 CD CD —V. OJ CD _k. L) c〇 二 CJ M k) cn _Ik Kj CD —k k cn _k cn O) nj ro m CD M lew •二
Claims (1)
- 其衍生自 枯草芽抱桿菌。 3 ·根據申請專利範圍第2項之枯草芽孢桿菌素變異體, 其衍生自遲緩芽孢桿菌型枯草桿菌素。 ’ 〃O:\55\55452-920924.ptc 第98頁 562859 _案號87117606_年月曰 修正_ 六、申請專利範圍 4. 一種編碼申請專利範圍第1項之枯草芽孢桿菌變異體 之DNA。 . 5. —種包含申請專利範圍第4項之DNA之表現載體。 6. —種宿主細胞,其用申請專利範圍第5項之表現載體 轉型。 7. —種清潔組合物,其包含申請專利範圍第1項之枯草 芽孢桿菌變異體。 8. —種動物飼料,其包含申請專利範圍第1項之枯草芽 孢桿菌變異體。 9. 一種處理織物之組合物,其包含申請專利範圍第1項 之枯草芽孢桿菌變異體。 1 0. —種枯草芽孢桿菌素變異體,其包含在與選自解澱 粉芽孢桿菌型枯草芽孢桿菌素之2 3 2,2 5 2及2 5 7所組成族 群中之殘基位置對應之一或多個殘基位置上,取代胺基 酸。 1 1.根據申請專利範圍第1項之枯草芽孢桿菌素變異體, 其包括取代在對應於解澱粉芽孢桿菌型枯草芽孢桿菌素之 位置1 0 3及2 3 6及一或多個選自下列殘基位置之胺基酸所組 成群組之殘基位置上:1, 8, 9, 1 0, 1 2, 6 1, 6 2, 6 8, 76, 97, 98, 101, 102, 104, 109, 130, 131, 159, 183, 185, 205, 209, 210, 211, 212, 213, 215, 217, 2 3 0,2 3 2,2 48,2 5 2,2 5 7,2 6 0,2 7 0 & 2 7 5 ° 1 2.根據申請專利範圍第1 1項之變異體,進一步包括在 胺基酸殘基位置1 5 9取代。O:\55\55452-920924.ptc 第99頁 562859六、申請專利範圍 1 3.根據申請專利範圍第1項之枯草芽孢桿菌素變異體, 其包括取代.在對應於解殿粉芽孢桿菌型枯草芽孢菌' 位置i 03及245及一或多個選自下列殘基位置之所 成群組之殘基位置上:1,8, 9, 1〇, ι2, 6 1 62 6 8 76,97,98,101,102,104,1〇9,13〇: m ⑴’ 183,185,2 0 5,2 0 9,210,211,212,213 ⑴ 230, 232, 248, 252, 257, 260, 270 及275、 ’ ’ 1 4.根據申請專利範圍第1 3項之變異體,進一步包括在 胺基酸殘基位置1 5 9取代。 7 1 5 ·根據申請專利範圍第1項之枯草芽孢桿菌素變異體, 其包括取代在對應於解澱粉芽孢桿菌型括草芽?包桿菌' 素之 位置103、2 3 6及2 4 5及一或多個選自下列殘基位胺其 酸所組成群組之殘基位置上:1,8, 9, 1 l i 2 61 & 68,76,97,98,101,102,104,109,1’30 m ’ 159’ 183, 185, 205, 209, 210, 211, 212, 213,’215 ’217 ’ 2 3 0,2 3 2,2 48,2 5 2,2 57,2 6 0,2 7 0 及 2 7 5’。 ’ ’ 1 6 ·根據申請專利範圍第1 0項之枯草芽孢桿菌素變$ 體,其中該取代係在在對應於解澱粉芽孢桿菌型/括%草/' 桿菌素之位置2 3 2及一或多個選自下列殘基位置之胺基酸匕 所組成群組之殘基位置上:1, 9, 1 2, 6 1, 6 2 6 8 ^ 97,98,101,102,104,1 0 9, 1 3 0,131’,1 5 9 1 83 ’ 185,2 0 5,2 0 9,210,211,212,213,2’17,2^ 0 2 3 6 245, 252, 257, 260, 270及275。 ’ ’ ’ 1 7 ·根據申請專利範圍第1 0項之枯草芽孢桿菌素變異O:\55\55452-920924.ptc 第100頁 562859 _案號87117606_年月曰 修正_ 六、申請專利範圍 體,進一步包括在一或多個對應於胺基酸殘基位置62及 2 1 2置換。. 1 8.根據申請專利範圍第1項之枯草芽孢桿菌素變異體, 其中該取代進一步包括在對應於位置104及232及一或多個 選自下列殘基位置之胺基酸所組成群組之殘基位置上:1, 9,12 ,6 1, 62, 68, 76, 97, 98, 101, 102, 109, 130, 131, 159, 183, 185, 2 0 5, 209, 210, 212, 213, 217, 2 3 0, 2 4 8, 2 5 2, 2 5 7, 2 6 0, 2 7 0 及 2 7 5 〇 19. 根據 申請專 利範 圍第1 8項之變異體 ,其中該取 代進 一步包括在胺基酸殘基位置1 5 9取代。O:\55\55452-920924.ptc 第101頁
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MA23346A1 (fr) * | 1993-10-14 | 1995-04-01 | Genencor Int | Variantes de la subtilisine |
AU8798198A (en) * | 1997-08-29 | 1999-03-22 | Novo Nordisk A/S | Protease variants and compositions |
ES2368718T3 (es) * | 1997-10-23 | 2011-11-21 | Danisco Us Inc. | Variantes de subtilisina con múltiples sustituciones. |
MA25044A1 (fr) * | 1997-10-23 | 2000-10-01 | Procter & Gamble | Compositions de lavage contenant des variants de proteases multisubstituees. |
CA2331783C (en) | 1998-07-02 | 2009-12-29 | Genencor International, Inc. | Chemically modified proteins with a carbohydrate moiety |
WO2000024924A2 (en) * | 1998-10-23 | 2000-05-04 | The Procter & Gamble Company | Methods for screening protease variants for use in detergent compositions |
JP4932990B2 (ja) | 1998-11-10 | 2012-05-16 | ダニスコ・ユーエス・インコーポレイテッド | 化学修飾突然変異体セリンヒドロラーゼは改良された触媒活性およびキラル選択性を示す |
EP1185302B1 (en) * | 1999-04-28 | 2013-07-24 | Genencor International, Inc. | Specifically targeted catalytic antagonists and uses thereof |
US6627744B2 (en) | 1999-07-02 | 2003-09-30 | Genencor International, Inc. | Synthesis of glycodendrimer reagents |
KR20020021395A (ko) * | 1999-07-22 | 2002-03-20 | 데이비드 엠 모이어 | 입체적으로 보호된 clip 부위를 갖는 프로테아제콘쥬게이트 |
MXPA02000840A (es) * | 1999-07-22 | 2002-07-30 | Procter & Gamble | Variantes de proteasa de subtilisina que tienen substituciones de aminoacidos en regiones de epitopes definidas. |
CN1375000A (zh) * | 1999-09-09 | 2002-10-16 | 宝洁公司 | 含有蛋白酶的洗涤剂组合物 |
KR100387166B1 (ko) * | 1999-12-15 | 2003-06-12 | 닛뽄덴끼 가부시끼가이샤 | 유기 일렉트로 루미네선스 소자 |
US7067049B1 (en) * | 2000-02-04 | 2006-06-27 | Exxonmobil Oil Corporation | Formulated lubricant oils containing high-performance base oils derived from highly paraffinic hydrocarbons |
DE10007608A1 (de) * | 2000-02-18 | 2001-08-30 | Henkel Kgaa | Protease und Percarbonat enthaltende Wasch- und Reinigungsmittel |
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