[发明专利]包含枯草杆菌蛋白酶变体的组合物和方法

摘要

本发明提供由丝氨酸蛋白酶制备的丝氨酸蛋白酶变体。具体地讲，本发明提供与参照丝氨酸蛋白酶相比具有一个或多个置换的丝氨酸蛋白酶变体。此外，本发明提供包含这些丝氨酸蛋白酶变体的组合物。在一些实施例中，本发明提供包含这些丝氨酸蛋白酶变体中的至少一种的清洁组合物。

主权项

一种分离的枯草杆菌蛋白酶变体，其中所述枯草杆菌蛋白酶变体为具有蛋白水解活性的成熟形式并包含具有选自以下的氨基酸置换的组合的氨基酸序列：G020R‑N043R,N062E‑A158E,S103G‑A158E,S128N‑A158E,A016S‑A158E,V104L‑A158E,E089P‑A158E,L111V‑A158E,T022A‑A158E,S101A‑A158E,L148I‑A158E,P129E‑A158E,T022A‑E089P,A016S‑E089P,N062E‑E089P,N062E‑E271F,A158E‑E271F,R186H‑E271F,P129E‑E271F,L111V‑E271F,Y209E‑E271F,A016S‑E271F,S188D‑E271F,T022A‑E271F,G159E‑E271F,V104L‑E271F,S101A‑E271F,E089P‑E271F,S128N‑E271F,S103G‑E271F,L148I‑E271F,H249R‑E271F,N062E‑G159E,A016S‑G159E,S128N‑G159E,L148I‑G159E,L111V‑G159E,E089P‑G159E,T022A‑G159E,P129E‑G159E,S103G‑G159E,V104L‑G159E,A158E‑G159E,S101A‑G159E,A158E‑H249R,L111V‑H249R,P129E‑H249R,N062E‑H249R,A016S‑H249R,R186H‑H249R,L148I‑H249R,G159E‑H249R,S101A‑H249R,S188D‑H249R,V104L‑H249R,Y209E‑H249R,T022A‑H249R,S128N‑H249R,S103G‑H249R,E089P‑H249R,T022A‑L111V,S101A‑L111V,A016S‑L111V,V104L‑L111V,N062E‑L111V,S103G‑L111V,E089P‑L111V,A016S‑L148I,N062E‑L148I,T022A‑L148I,P129E‑L148I,V104L‑L148I,S103G‑L148I,S128N‑L148I,S101A‑L148I,E089P‑L148I,L111V‑L148I,A016S‑N062E,T022A‑N062E,N062E‑P129E,T022A‑P129E,S128N‑P129E,A016S‑P129E,S101A‑P129E,V104L‑P129E,E089P‑P129E,S103G‑P129E,L111V‑P129E,N062E‑R186H,S128N‑R186H,S101A‑R186H,T022A‑R186H,A016S‑R186H,A158E‑R186H,E089P‑R186H,P129E‑R186H,G159E‑R186H,S103G‑R186H,V104L‑R186H,L111V‑R186H,L148I‑R186H,N062E‑S101A,T022A‑S101A,A016S‑S101A,E089P‑S101A,N062E‑S103G,T022A‑S103G,A016S‑S103G,S101A‑S103G,E089P‑S103G,N062E‑S128N,A016S‑S128N,T022A‑S128N,S101A‑S128N,V104L‑S128N,E089P‑S128N,S103G‑S128N,L111V‑S128N,L111V‑S188D,N062E‑S188D,A016S‑S188D,L148I‑S188D,T022A‑S188D,S128N‑S188D,S101A‑S188D,V104L‑S188D,E089P‑S188D,P129E‑S188D,G159E‑S188D,R186H‑S188D,S103G‑S188D,A158E‑S188D,A016S‑T022A,A016S‑V104L,T022A‑V104L,S101A‑V104L,N062E‑V104L,S103G‑V104L,E089P‑V104L,G159E‑Y209E,L111V‑Y209E,S101A‑Y209E,A016S‑Y209E,S128N‑Y209E,L148I‑Y209E,P129E‑Y209E,N062E‑Y209E,T022A‑Y209E,S103G‑Y209E,A158E‑Y209E,S188D‑Y209E,V104L‑Y209E,E089P‑Y209E,R186H‑Y209E,N018R‑W241R,G020R‑W241R,S024R‑W241R,S009A‑W241R,G020R‑W241R,V004R‑W241R,N043R‑W241R,S078R‑W241R,T022R‑W241R,G115R‑W241R,A001R‑W241R,S212F‑W241R,L082R‑W241R,N018R‑V244R,S024R‑V244R,S078R‑V244R,G020R‑V244R,S212F‑V244R,S009A‑V244R,L082R‑V244R,A001R‑V244R,N043R‑V244R,T022R‑V244R,V004R‑V244R,G115R‑V244R,W241R‑V244R,S242R‑V244R,A001R‑V004R,S009A‑T022R,N018R‑T022R,G020R‑T022R,V004R‑T022R,A001R‑T022R,S024R‑S242R,N018R‑S242R,V004R‑S242R,G020R‑S242R,S212F‑S242R,L082R‑S242R,S078R‑S242R,A001R‑S242R,S009A‑S242R,T022R‑S242R,G115R‑S242R,N043R‑S242R,W241R‑S242R,N018R‑S212F,T022R‑S212F,V004R‑S212F,S024R‑S212F,A001R‑S212F,G115R‑S212F,G020R‑S212F,S009A‑S212F,N043R‑S212F,S078R‑S212F,L082R‑S212F,S009A‑S078R,G020R‑S078R,S024R‑S078R,T022R‑S078R,N018R‑S078R,V004R‑S078R,A001R‑S078R,N043R‑S078R,T022R‑S024R,G020R‑S024R,N018R‑S024R,A001R‑S024R,V004R‑S024R,S009A‑S024R,V004R‑S009A,A001R‑S009A,S242R‑N269R,S024R‑N269R,G020R‑N269R,T022R‑N269R,H249R‑N269R,S212F‑N269R,N043R‑N269R,V244R‑N269R,A001R‑N269R,N018R‑N269R,S078R‑N269R,S009A‑N269R,G115R‑N269R,W241R‑N269R,V004R‑N269R,L082R‑N269R,N018R‑N043R,G020R‑N043R,V004R‑N043R,T022R‑N043R,S009A‑N043R,A001R‑N043R,S024R‑N043R,S009A‑N018R,V004R‑N018R,A001R‑N018R,S024R‑L082R,S009A‑L082R,N018R‑L082R,A001R‑L082R,S078R‑L082R,G020R‑L082R,T022R‑L082R,V004R‑L082R,N043R‑L082R,N043R‑H249R,G020R‑H249R,V004R‑H249R,N018R‑H249R,S009A‑H249R,S212F‑H249R,T022R‑H249R,S024R‑H249R,G115R‑H249R,A001R‑H249R,L082R‑H249R,S242R‑H249R,W241R‑H249R,V244R‑H249R,S078R‑H249R,N018R‑G115R,G020R‑G115R,T022R‑G115R,S078R‑G115R,S009A‑G115R,V004R‑G115R,A001R‑G115R,L082R‑G115R,N043R‑G115R,S024R‑G115R,S009A‑G020R,N018R‑G020R,V004R‑G020R,A001R‑G020R,S009A‑E271L,G020R‑E271L,S024R‑E271L,V244R‑E271L,W241R‑E271L,N043R‑E271L,T022R‑E271L,H249R‑E271L,S212F‑E271L,G115R‑E271L,S242R‑E271L,S078R‑E271L,V004R‑E271L,N269R‑E271L,A001R‑E271L,N018R‑E271L，和L082R‑E271L，并且其中所述变体的总净电荷相对于迟缓芽孢杆菌(Bacillus lentus)枯草杆菌蛋白酶GG36蛋白酶的总净电荷为0、+1、+2、+3、+4、+5、‑1、‑2、‑3、‑4或‑5，并且其中所述总净电荷是通过一种或多种选自以下的置换来获得的：N43D,R45T,N62E,N76D,S101D,P129E,A158E,G159D,G159E,S166D,S188D,A230E,N18R,G20K,G20R,T22R,S24R,N43R,G118R,Q245R,H249R,N269R,E271F，和E271L，并且其中所述蛋白酶变体的氨基酸位置根据SEQ ID NO:1所示解淀粉芽孢杆菌(Bacillus amyloliquefaciens)枯草杆菌蛋白酶BPN’的氨基酸序列中对应的氨基酸位置的编号方式来进行编号。