CN105296445B - 脂肪酶变体 - Google Patents
脂肪酶变体 Download PDFInfo
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- CN105296445B CN105296445B CN201510608345.XA CN201510608345A CN105296445B CN 105296445 B CN105296445 B CN 105296445B CN 201510608345 A CN201510608345 A CN 201510608345A CN 105296445 B CN105296445 B CN 105296445B
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Abstract
本发明涉及脂肪酶变体，具体地提供变体脂肪酶，优选地，通过在亲本脂肪酶中鉴定的一个或多个区域中引入突变而获得的产生气味的趋势降低的变体。
Description
本发明申请是基于申请日为2007年1月22日，申请号为200780002901.2(国际申请号为PCT/US2007/060841)，名称为“脂肪酶变体”的发明专利申请的分案申请。
技术领域
本发明涉及脂肪酶变体。
背景技术
脂肪酶是有用的，例如，作为洗涤剂酶用于从衣物和其它纺织品去除脂质或脂肪沾污，作为用于面包和其它烘焙产品的生面团的添加剂。因此，源自细毛嗜热霉(Thermomyces lanuginosus)(同物异名疏棉状腐质霉(Humicola lanuginosa)，EP 258068和EP 305 216)的脂肪酶以商品名
(Novo Nordisk A/S的产品)以洗涤剂用途出售。WO 0060063描述了细毛嗜热霉脂肪酶的变体，其在洗涤剂溶液中具有特别优良的初次洗涤性能(first-wash performance)。WO 9704079、WO 9707202和WO 0032758也公开了细毛嗜热霉脂肪酶的变体。
在一些应用中，感兴趣的是将产生气味的短链脂肪酸的形成降至最低。因此，已知的是含有脂肪酶的洗衣洗涤剂有时可能在以牛奶污染的织物上留下残留的气味(EP430315)。WO 02062973公开了脂肪酶变体，其中通过附接(attaching)C末端延伸减少了气味的产生。
发明内容
发明人发现，通过在脂肪酶的某些区/位置引入突变，可改进脂肪酶的性质或特征。
在优选实施方案中，本发明涉及用于洗涤剂具有改进性能的脂肪酶。例如，本发明通过在亲本脂肪酶中鉴定的一个或多个区中引入突变而获得的产生气味的倾向降低的变体。在另一个优选实施方案中，本发明提供脂肪酶变体，与亲本脂肪酶相比，其产生气味的可能降低而无C末端延伸的附接。
在另外的方面中，本发明涉及编码本发明脂肪酶变体的DNA序列，携带所述DNA序列的表达载体和包含DNA序列或表达载体的转化宿主细胞。
在另一个方面，本发明提供产生本发明脂肪酶变体的方法。
具体地，本发明涉及如下各项：
1.亲本脂肪酶的变体，其中变体包含至少三个选自下组的取代(使用SEQ ID NO:2编号)：
a)I区中的至少两个取代，和
b)II区中的至少一个取代，和
c)III区中的至少一个取代，和
d)IV区中的至少一个取代；
和其中所述变体具有脂肪酶活性。
2.项1的脂肪酶变体，其中亲本脂肪酶的I区中至少两个取代包含在对应于位置231和233的位置的氨基酸的取代(使用SEQ ID NO:2编号)。
3.项2的脂肪酶变体，其中亲本脂肪酶的I区中至少两个取代包含在对应于位置231和233的位置的用氨基酸R的取代(使用SEQ ID NO:2编号)。
4.根据项1的脂肪酶变体，其变体脂肪酶与SEQ ID NO:2至少80％、85％、90％、95％、96％、97％、98％、99％相同。
5.根据项1的脂肪酶变体，其中亲本脂肪酶是具有SEQ ID NO:2的氨基酸序列的脂肪酶。
6.根据项2的脂肪酶变体，其中所述脂肪酶在对应于SEQ ID NO:2的位置4和/或位置227的位置包含其它取代。
7.根据项6的脂肪酶变体，其中脂肪酶具有对应于X4V和X227G的取代(使用SEQ IDNO:2编号)。
8.根据项1的脂肪酶变体，其中亲本脂肪酶的II区中至少一个取代包含如下取代，其选自由在对应于位置202、210、211、255和256的位置的取代组成的组(使用SEQ ID NO:2编号)。
9.根据项8的脂肪酶变体，其中亲本脂肪酶中至少一个取代选自下组：X202G、X210K、X211L、X255Y/V和X256K(使用SEQ ID NO:2编号)。
10.根据项1的脂肪酶变体，其中亲本脂肪酶的III区中至少一个取代包含如下取代，其选自由在对应于位置83、86和90的位置的取代组成的组(使用SEQ ID NO:2编号)。
11.根据项10的脂肪酶变体，其中亲本脂肪酶中至少一个取代选自下组：X83T、X86V和X90A/R(使用SEQ ID NO:2编号)。
12.根据项1的脂肪酶变体，其中亲本脂肪酶的IV区中至少一个取代包含如下取代，其选自由在对应于位置27、58和60的位置的取代组成的组(使用SEQ ID NO:2编号)。
13.根据项1的脂肪酶变体，其中亲本脂肪酶的IV区中至少一个取代包含选自下组的取代：X27R、X58N/A/G/P/T和X60S/V/G/N/R/K/A/L(使用SEQ ID NO:2编号)。
14.根据项1的脂肪酶变体，其中脂肪酶变体还包含亲本脂肪酶中的至少一个取代，其选自由在对应于如下位置的位置的取代组成的组：位置81、147、150、227和249(使用SEQ ID NO:2编号)。
15.根据项1的脂肪酶变体，其中所述脂肪酶变体还包含选自下组的至少一个取代：X81Q/E、X147M/Y、X150G和X249R/I/L(使用SEQ ID NO:2编号)。
16.项1的脂肪酶变体，其中所述脂肪酶包含选自下组取代的取代：
a)T231R+N233R+I255Y
b)I202G+T231R+N233R
c)I86V+L227G+T231R+N233R+P256K
d)Q4V+S58N+V60S+T231R+N233R
e)S58N+V60S+I90R+T231R+N233R
f)I90A+T231R+N233R+I255V
g)S58N+V60S+I86V+A150G+L227G+T231R+N233R+P256K
h)S58N+V60S+L147M+F211L+T231R+N233R
i)Q4V+S58A+V60S+S83T+I86V+A150G+E210K+L227G+T231R+N233R+P256K
j)S58N+V60S+I86V+A150G+L227G+T231R+N233R+P256K
17.编码项1-16的脂肪酶变体的DNA序列。
18.携带项17的DNA序列的表达载体。
19.包含项17的DNA序列的转化宿主细胞。
20.产生脂肪酶变体的方法，所述方法包含在利于产生脂肪酶变体的条件下培养项19的转化宿主细胞，和从得到的培养液中回收脂肪酶变体。
21.SEQ ID NO:2的变体，其包含Q4V、S58N/A/G/P/T、I90R或Q249I/L中的至少一个突变。
附图简述
图1显示脂肪酶的比对。
序列表
SEQ ID NO:1显示编码来自细毛嗜热霉的脂肪酶的DNA序列。
SEQ ID NO:2显示来自细毛嗜热霉的脂肪酶的氨基酸序列。
SEQ ID NO:3显示来自曲柄犁头霉(Absidia reflexa)的脂肪酶的氨基酸序列。
SEQ ID NO:4显示来自伞状犁头霉(Absidia corymbifera)的脂肪酶的氨基酸序列。
SEQ ID NO:5显示来自曼赫根毛霉(Rhizmucor miehei)的脂肪酶的氨基酸序列。
SEQ ID NO:6显示来自米根霉(Rhizopus oryzae)的脂肪酶的氨基酸序列。
SEQ ID NO:7显示来自黑曲霉(Aspergillus niger)的脂肪酶的氨基酸序列。
SEQ ID NO:8显示来自塔宾曲霉(Aspergillus tubigensis)的脂肪酶的氨基酸序列。
SEQ ID NO:9显示来自尖镰孢(Fusarium oxysporum)的脂肪酶的氨基酸序列。
SEQ ID NO:10显示来自异孢镰孢(Fusarium heterosporum)的脂肪酶的氨基酸序列。
SEQ ID NO:11显示来自米曲霉(Aspergillus oryzea)的脂肪酶的氨基酸序列。
SEQ ID NO:12显示来自沙门柏干酪青霉(Penicilium camembertii)的脂肪酶的氨基酸序列。
SEQ ID NO:13显示来自臭曲霉(Aspergillus foetidus)的脂肪酶的氨基酸序列。
SEQ ID NO:14显示来自黑曲霉的脂肪酶的氨基酸序列。
SEQ ID NO:15显示来自米曲霉的脂肪酶的氨基酸序列。
SEQ ID NO:16显示来自Landerina penisapora的脂肪酶的氨基酸序列
发明详述
亲本脂肪酶
可以使用任何合适的亲本脂肪酶。在优选实施方案中，亲本脂肪酶可以是真菌脂肪酶。在另一个优选实施方案中，亲本脂肪酶可以与SEQ ID NO:2中所示的细毛嗜热霉的序列具有至少50％、60％、70％、80％、85％、90％、95％、96％、97％、98％、99％或甚至100％的同源性，所述同源性如“同源性和比对”部分所定义。
亲本脂肪酶可以是酵母多肽，例如念珠菌属(Candida)、克鲁维酵母属(Kluyveromyces)、毕赤酵母属(Pichia)、酵母属(Saccharomyces)、裂殖酵母属(Schizosaccharomyces)或西洋蓍霉属(Yarrowia)多肽；或更优选是丝状真菌多肽，例如枝顶孢霉属(Acremonium)、曲霉属(Aspergillus)、短梗霉属(Aureobasidium)、隐球菌属(Cryptococcus)、Filobasidium、镰孢属(Fusarium)、腐质霉属(Humicola)、梨孢菌属(Magnaporthe)、毛霉属(Mucor)、毁丝霉属(Myceliophthora)、新考玛脂霉属(Neocallimastix)、脉孢菌属(Neurospora)、拟青霉属(Paecilomyces)、青霉属(Penicillium)、瘤胃壶菌属(Piromyces)、裂褶菌属(Schizophyllum)、踝节菌属(Talaromyces)、嗜热子囊菌属(Thermoascus)、梭孢壳属(Thielavia)、弯颈霉属(Tolypocladium)或木霉属(Trichoderma)多肽。
在优选的方面，亲本脂肪酶是卡尔酵母(Saccharomyces carlsbergensis)、酿酒酵母(Saccharomyces cerevisiae)、糖化酵母(Saccharomyces diastaticus)、道格拉氏酵母(Saccharomyces douglasii)、克鲁弗酵母(Saccharomyces kluyveri)、诺地酵母(Saccharomyces norbensis)或卵形酵母(Saccharomyces oviformis)多肽。
在另一个优选的方面，亲本脂肪酶是棘孢曲霉(Aspergillus aculeatus)、泡盛曲霉(Aspergillus awamori)、烟曲霉(Aspergillus fumigatus)、臭曲霉、日本曲霉(Aspergillus japonicus)、构巢曲霉(Aspergillus nidulans)、黑曲霉、米曲霉、塔宾曲霉、杆孢状镰孢(Fusarium bactridioides)、禾谷镰孢(Fusarium cerealis)、库威镰孢(Fusarium crookwellense)、大刀镰孢(Fusarium culmorum)、禾本科镰孢(Fusariumgraminearum)、禾赤镰孢(Fusarium graminum)、异孢镰孢、合欢木镰孢(Fusariumnegundi)、尖镰孢、多枝镰孢(Fusarium reticulatum)、粉红镰孢(Fusarium roseum)、接骨木镰孢(Fusarium sambucinum)、肤色镰孢(Fusarium sarcochroum)、拟分枝孢镰孢(Fusarium sporotrichioides)、硫色镰孢(Fusarium sulphureum)、圆镰孢(Fusariumtorulosum)、拟丝孢镰孢(Fusarium trichothecioides)、镶片镰孢(Fusariumvenenatum)、特异腐质霉(Humicola insolens)、细毛嗜热霉(同物异名：疏棉状腐质霉)、米赫毛霉(Mucor miehei)、嗜热毁丝霉(Myceliophthora thermophila)、粗糙脉孢菌(Neurospora crassa)、产紫青霉(Penicillium purpurogenum)、哈茨木霉(Trichodermaharzianum)、康宁木霉(Trichoderma koningii)、长枝木霉(Trichodermalongibrachiatum)、里氏木霉(Trichoderma reesei)或绿色木霉(Trichoderma viride)多肽。
在另一个优选方面，亲本脂肪酶是嗜热霉属脂肪酶。
在更优选的方面，亲本脂肪酶是细毛嗜热霉脂肪酶。在甚至更优选的实施方案中，亲本脂肪酶是SEQ ID NO:2的脂肪酶。
变体脂肪酶
与亲本脂肪酶相比，本发明的脂肪酶变体包含选自下组的至少三个取代：
a)I区中的至少两个取代，和
b)II区中的至少一个取代，和
c)III区中的至少一个取代，和
d)IV区中的至少一个取代；
且其中，变体具有脂肪酶活性。
在优选实施方案中，变体脂肪酶是嗜热霉属脂肪酶的变体，更优选地，细毛嗜热霉脂肪酶的变体，甚至更优选地，SEQ ID NO:2中所示的细毛嗜热霉脂肪酶的变体。在优选实施方案中，变体脂肪酶与SEQ ID NO:2具有至少50％、60％、70％、80％、85％、90％、95％、96％、97％、98％或99％的同一性。
变体脂肪酶可以是由从下述亲本生物之一衍生/获得的基因编码的亲本脂肪酶的变体：念珠菌属、克鲁维酵母属、毕赤酵母属、酵母属、裂殖酵母属或西洋蓍霉属，枝顶孢霉属、曲霉属、短梗霉属、隐球菌属、Filobasidium、镰孢属、腐质霉属、梨孢菌属、毛霉属、毁丝霉属、新考玛脂霉属、脉孢菌属、拟青霉属、青霉属、瘤胃壶菌属、裂褶菌属、踝节菌属、嗜热子囊菌属、梭孢壳属、弯颈霉属或木霉属。在优选实施方案中，变体脂肪酶与亲本脂肪酶具有至少50％、60％、70％、80％、85％、90％、95％、96％、97％、98％或99％的同一性，所述亲本脂肪酶由从下述亲本生物之一衍生/获得的基因编码：念珠菌属、克鲁维酵母属、毕赤酵母属、酵母属、裂殖酵母属或西洋蓍霉属，枝顶孢霉属、曲霉属、短梗霉属、隐球菌属、Filobasidium、镰孢属、腐质霉属、梨孢菌属、毛霉属、毁丝霉属、新考玛脂霉属、脉孢菌属、拟青霉属、青霉属、瘤胃壶菌属、裂褶菌属、踝节菌属、嗜热子囊菌属、梭孢壳属、弯颈霉属或木霉属。
在优选的方面，变体脂肪酶是卡尔酵母、酿酒酵母、糖化酵母、道格拉氏酵母、克鲁弗酵母、诺地酵母或卵形酵母的变体。在优选实施方案中，变体脂肪酶与亲本脂肪酶具有至少50％、60％、70％、80％、85％、90％、95％、96％、97％、98％或99％的同一性，所述亲本脂肪酶由从下述亲本生物之一衍生/获得的基因编码：卡尔酵母、酿酒酵母、糖化酵母、道格拉氏酵母、克鲁弗酵母、诺地酵母或卵形酵母。
变体脂肪酶可以是由从下述亲本生物之一衍生/获得的基因编码的亲本脂肪酶的变体：棘孢曲霉、泡盛曲霉、烟曲霉、臭曲霉、日本曲霉、构巢曲霉、黑曲霉、米曲霉、塔宾曲霉、杆孢状镰孢、禾谷镰孢、库威镰孢、大刀镰孢、禾本科镰孢、禾赤镰孢、异孢镰孢、合欢木镰孢、尖镰孢、多枝镰孢、粉红镰孢、接骨木镰孢、肤色镰孢、拟分枝孢镰孢、硫色镰孢、圆镰孢、拟丝孢镰孢、镶片镰孢、特异腐质霉、细毛嗜热霉(同物异名：疏棉状腐质霉)、米赫毛霉、嗜热毁丝霉、粗糙脉孢菌、产紫青霉、哈茨木霉、康宁木霉、长枝木霉、里氏木霉或绿色木霉。在优选实施方案中，变体脂肪酶与亲本脂肪酶具有至少50％、60％、70％、80％、85％、90％、95％、96％、97％、98％或99％的同一性，所述亲本脂肪酶由从下述亲本生物之一衍生/获得的基因编码：棘孢曲霉、泡盛曲霉、烟曲霉、臭曲霉、日本曲霉、构巢曲霉、黑曲霉、米曲霉、塔宾曲霉、杆孢状镰孢、禾谷镰孢、库威镰孢、大刀镰孢、禾本科镰孢、禾赤镰孢、异孢镰孢、合欢木镰孢、尖镰孢、多枝镰孢、粉红镰孢、接骨木镰孢、肤色镰孢、拟分枝孢镰孢、硫色镰孢、圆镰孢、拟丝孢镰孢、镶片镰孢、特异腐质霉、细毛嗜热霉(同物异名：疏棉状腐质霉)、米赫毛霉、嗜热毁丝霉、粗糙脉孢菌、产紫青霉、哈茨木霉、康宁木霉、长枝木霉、里氏木霉或绿色木霉。
在另一个优选方面，变体是嗜热霉属脂肪酶的变体。
在更优选的方面，亲本脂肪酶是细毛嗜热霉脂肪酶。在甚至更优选的实施方案中，亲本脂肪酶是SEQ ID NO:2的脂肪酶。
区和取代的鉴定
下面I区至IV区中所提及的位置是SEQ ID NO:2中氨基酸残基的位置。为了在不同的脂肪酶中发现相应的(或同源的)位置，使用“同源性与比对”中所述的过程。
I区中的取代
I区由N末端残基E1附近的氨基酸残基组成。在这个区中，优选用带更多正电的(more positive)氨基酸取代亲本脂肪酶的氨基酸。脂肪酶变体可以在I区中包含至少两个取代，比如在I区中的三个、四个、五个或六个取代。
I区中包含对应于下述位置的氨基酸残基：1,2至11和223-239。下述位置是特别感兴趣的：1、4、8、11、223、227、229、231、233、234、236。
具体而言，已经鉴定了以下取代：X1N/*X4V、X227G、X231R和X233R。
在优选的实施方案中，变体脂肪酶与SEQ ID NO:2具有至少80％、85％、90％、95％、96％、97％、98％、99％同一性。
在最优选的实施方案中，变体脂肪酶是具有SEQ ID NO:2的氨基酸序列的脂肪酶的变体。
II区中的取代
II区由在酰基链的一侧和醇部分的一侧与底物接触的氨基酸残基组成。在这个区中，优选用带更多正电的氨基酸或用疏水性较小的氨基酸来取代亲本脂肪酶的氨基酸。
脂肪酶变体可以在II区中包含至少一个取代，比如在II区中的两个、三个、四个、五个或六个取代。
II区中包含对应于下述位置的氨基酸残基：202至211和249至269。下述位置是特别感兴趣的：202、210、211、253、254、255、256。
具体而言，已经鉴定了以下取代：X202G、X210K/W/A、X255Y/V/A和X256K/R和X259G/M/Q/V。
在优选实施方案中，变体脂肪酶与SEQ ID NO:2具有至少80％、85％、90％、95％、96％、97％、98％或99％的同一性。
在最优选的实施方案中，变体脂肪酶是具有SEQ ID NO:2的氨基酸序列的脂肪酶的变体。
III区中的取代
III区由形成柔性结构(flexible structure)的氨基酸组成，因此允许底物进入活性部位。在这个区中，优选用带更多正电的氨基酸或疏水性较小的氨基酸取代亲本脂肪酶的氨基酸。
脂肪酶变体可以在III区中包含至少一个取代，比如在III区中的两个、三个、四个、五个或六个取代。
III区中包含对应于下述位置的氨基酸残基：82至102。下述位置是特别感兴趣的：83、86、87、90、91、95、96、99。
具体而言，已经鉴定了以下取代：X83T、X86V和X90A/R。
在优选实施方案中，变体脂肪酶与SEQ ID NO:2具有至少80％、85％、90％、95％、96％、97％、98％或99％的同一性。
在最优选的实施方案中，变体脂肪酶是具有SEQ ID NO:2的氨基酸序列的脂肪酶的变体。
IV区中的取代
IV区由与表面以静电结合的氨基酸残基组成。在这个区中，优选用带更多正电的氨基酸取代亲本脂肪酶的氨基酸。
脂肪酶变体可以在IV区中包含至少一个取代，比如在IV区中的两个、三个、四个、五个或六个取代。
IV区中包含对应于下述位置的氨基酸残基：27和54至62。下述位置是特别感兴趣的：27、56、57、58、60。
具体而言，已经鉴定了以下取代：X27R、X58N/AG/T/P和X60V/S/G/N/R/K/A/L。
在优选实施方案中，变体脂肪酶与SEQ ID NO:2具有至少80％、85％、90％、95％、96％、97％、98％或99％的同一性。
在最优选的实施方案中，变体脂肪酶是具有SEQ ID NO:2的氨基酸序列的脂肪酶的变体。
其它位置的氨基酸
亲本脂肪酶可以任选地包含其它变化，例如，其它氨基酸的取代，特别是与亲本脂肪酶相比，小于10个，小于9个，小于8个，小于7个，小于6个，小于5个变化。实例是对应于亲本脂肪酶下述位置中一个或多个位置的取代：24、37、38、46、74、81、83、115、127、131、137、143、147、150、199、200、203、206、211、263、264、265、267和269。在特定实施方案中，在对应于位置81、147、150、227和249的至少一个位置有取代。在优选实施方案中，至少一个取代选自下组：X38R、X81Q/E、X143S/C/N/D/A、X147M/Y、X150G/K、X227G和X249R/I/L。
变体可包含在定义的I至IV区之外取代；定义的I至IV区之外的取代数目优选小于六个，比如五个、四个、三个、两个或一个取代。
其它取代可以，例如，根据本领域已知的原则产生，例如WO 92/05249、WO 94/25577、WO 95/22615、WO 97/04079和WO 97/07202中所述取代。
亲本脂肪酶变体
亲本脂肪酶包括具有下面的表1列出的取代的亲本脂肪酶(使用SEQ ID NO:2编号)。
| I区 | II区 | III区 | IV区 | 区之外 |
| X4V+X227G+X231R+X233R | X210K+X256K | X83T+X86V | X58A+X60S | X150G |
| X227G+X231R+X233R | X256K | X86V | X58N+X60S | X150G |
| X231R+X233R | X255Y | |||
| X231R+X233R | X202G | |||
| X227G+X231R+X233R | X256K | X86V | ||
| X4V+X231R+X233R | X58N+X60S | |||
| X231R+X233R | X90R | X58N+X60S | ||
| X231R+X233R | X255V | X90A | ||
| X227G+X231R+X233R | X256K | X86V | X58N+X60S | X150G |
| X231R+X233R | X211L | X58N+X60S | X147M | |
| X231R+X233R | X150K |
表1：
在更特定的实施方案中，亲本脂肪酶与SEQ ID NO:2相同，而表1的变体因此是：
| I区 | II区 | III区 | IV区 | 区之外 |
| Q4V+L227G+T231R+N233R | E210K+P256K | S83T+I86V | S58A+V60S | A150G |
| L227G+T231R+N233R | P256K | I86V | S58N+V60S | A150G |
| T231R+N233R | I255Y | |||
| T231R+N233R | I202G | |||
| L227G+T231R+N233R | P256K | I86V | ||
| Q4V+T231R+N233R | S58N+V60S | |||
| T231R+N233R | I90R | S58N+V60S | ||
| T231R+N233R | I255V | I90A | ||
| L227G+T231R+N233R | P256K | I86V | S58N+V60S | A150G |
| T231R+N233R | F211L | S58N+V60S | L147M | |
| T231R+N233R | A150K |
表2：SEQ ID NO:2的一些特定变体
氨基酸修饰的命名法
为了易于参考，在描述本发明的脂肪酶变体时，采用以下命名法：
原始氨基酸：位置：取代氨基酸
根据这种命名法，例如，在位置195中用谷氨酸取代甘氨酸表示为G195E。在相同的位置缺失甘氨酸表示为G195*，而***额外的氨基酸残基例如赖氨酸表示为G195GK。
与其它脂肪酶相比，当特定脂肪酶含有“缺失”并且在该位置进行***的情况，就在位置36***天冬氨酸而言，将这种情况表示为*36D。
用加号分隔多个突变，即：R170Y+G195E，分别表示在位置170用酪氨酸取代精氨酸，和在位置195用谷氨酸取代甘氨酸。
当应用所述排列方法时，X231表示在亲本多肽中对应于位置231的氨基酸。X231R表示用R置换所述氨基酸。就SEQ ID NO:2而言，X是T，因此T231R表示用R取代位置231的T。当某个位置(例如231)中的氨基酸可以由选自一组氨基酸的另一个氨基酸取代的情况，例如由R和P和Y组成的组，将这种情况表示为X231R/P/Y。
在所有的情况下，采用公认的IUPAC单字母或三字母的氨基酸缩写。
氨基酸分组
在本说明中，根据氨基酸在pH 10的电荷，将它们分成带负电的、带正电的或电中性的。因此，带负电的(negative)氨基酸是E、D、C(半胱氨酸)和Y，特别是E和D。带正电的(positive)氨基酸是R、K和H，特别是R和K。电中性(neutral)氨基酸是G、A、V、L、I、P、F、W、S、T、M、N、Q和形成二硫桥的部分时的C。用同一组(带负电、带正电或电中性)中的另一个氨基酸取代称为保守取代。
可以将电中性氨基酸分成疏水或非极性(G、A、V、L、I、P、F、W和作为二硫桥的部分的C)和亲水或极性(S、T、M、N、Q)。
氨基酸同一性
参数“同一性”描述两个氨基酸序列之间或两个核苷酸序列之间的相关性。
就本发明而言，通过使用来自EMBOSS软件包(http://emboss.org)版本2.8.0的Needle程序来测定两个氨基酸序列之间的比对。Needle程序执行总体比对算法，其在Needleman,S.B.and Wunsch,C.D.(1970)J.Mol.Biol.48,443-453中描述。使用的取代矩阵是BLOSUM62，缺口开启罚分(gap opening penalty)是10，并且缺口延伸罚分(gapextension penalty)是0.5。
本发明的氨基酸序列(“发明序列”；例如SEQ ID NO:2的氨基酸1至269)和不同氨基酸序列(“外源序列”)之间的同一性程度如下计算：用两个序列比对中完全匹配的数目除以“发明序列”的长度或“外源序列”的长度中最短的一个。将结果表示为百分比同一性。
当“发明序列”和“外源序列”在重叠的相同位置中具有同一氨基酸残基时，发生完全匹配。序列的长度是序列中氨基酸残基的数目(例如SEQ ID NO:2的长度是269)。
可以使用上述方法计算同一性和同源性，并用于比对。在本发明的上下文中，同源性和比对如下所述计算。
同源性和比对
就本发明而言，可以通过本领域已知的计算机程序的方法适当地测定同源性的程度，所述程序例如GCG程序包中提供的GAP(Program Manual for the Wisconsin Package,Version 8,August 1994,Genetics Computer Group,575Science Drive,Madison,Wisconsin,USA 53711)(Needleman,S.B.and Wunsch,C.D.,(1970),Journal ofMolecular Biology,48,443-45)，按照用于多肽序列比较的以下设定来使用GAP：GAP产生罚分(GAP creation penalty)为3.0，和GAP延伸罚分(GAP extension penalty)为0.1。
在本发明中，通过图1所示的比对确定曲柄犁头霉、伞状犁头霉(Absidiacorymbefera)、曼赫根毛霉、德氏根霉、黑曲霉、塔宾曲霉、尖镰孢、异孢镰孢、米曲霉、沙门柏干酪青霉、臭曲霉、黑曲霉、细毛嗜热霉(同物异名：疏棉状腐质霉)和Landerinapenisapora的脂肪酶序列中相应(或同源)的位置。
为了发现所述比对中未显示的脂肪酶序列中的同源位置，将感兴趣的序列与图1中显示的序列进行比对。通过使用GAP将新序列与GAP程序发现的最同源的序列进行比对，来将新序列排入图1中的现有比对。GAP在GCG程序包(Program Manual for the WisconsinPackage,Version 8,August 1994,Genetics Computer Group,575Science Drive,Madison,Wisconsin,USA 53711)(Needleman,S.B.和Wunsch,C.D.,(1970),Journal ofMolecular Biology,48,443-45)中提供。使用以下设置用于多肽序列比较：GAP产生罚分为3.0，GAP延伸罚分为0.1。
杂交
本发明还涉及具有脂肪酶活性的分离的多肽，所述分离的多肽由多核苷酸编码，所述多核苷酸在非常低严紧性条件下，优选低严紧性条件下，更优选中严紧性条件下，更优选中-高严紧性条件下，甚至更优选高严紧性条件下，并且最优选非常高严紧性条件下，与以下杂交：(i)SEQ ID NO:1的核苷酸178至660，(ii)SEQ ID NO:1的核苷酸178至660中包含的cDNA序列，(iii)(i)或(ii)的亚序列，或(iv)(i)、(ii)或(iii)的互补链(J.Sambrook,E.F.Fritsch,and T.Maniatus,1989,Molecular Cloning,A Laboratory Manual,2dedition,Cold Spring Harbor,New York)。SEQ ID NO:1的亚序列含有至少100个连续的核苷酸或优选至少200个连续的核苷酸。此外，所述亚序列可编码具有脂肪酶活性的多肽片段。
对于长度至少100个核苷酸的长探针，将非常低至非常高的严紧条件定义为：在5×SSPE、0.3％SDS、200μg/ml剪切和变性的鲑精DNA中于42℃预杂交和杂交，对于非常低和低严紧性，使用25％甲酰胺，对于中和中-高严紧性，使用35％甲酰胺，或者对于高和非常高严紧性，使用50％甲酰胺，根据标准Southern印迹方法最佳进行12至24小时。
对于长度至少为100个核苷酸的长探针，使用2×SSC、0.2％SDS，优选至少在15℃(非常低严紧性)，更优选至少50℃(低严紧性)，更优选至少55℃(中等严紧性)，更优选至少60℃(中-高严紧性)，甚至更优选至少65℃(高严紧性)，和最优选至少70℃(非常高严紧性)将载体材料最终清洗三次，每次15分钟。
DNA序列、表达载体、宿主细胞、脂肪酶的产生
本发明提供编码本发明的脂肪酶的DNA序列，携带该DNA序列的表达载体，和包含DNA序列或表达载体的转化的宿主细胞。这些可以通过本领域已知的方法获得。
本发明还提供通过如下产生脂肪酶的方法：在利于产生脂肪酶的条件下培养转化的宿主细胞，并从所得的培养液中回收脂肪酶。可以根据本领域已知的原理实施所述方法。
脂肪酶活性
在中性pH对三丁精的脂肪酶活性(LU)
通过使用***树胶作为乳化剂将三丁酸甘油酯(tributyrin)(甘油三丁酸酯)乳化来制备用于脂肪酶的底物。在pH 7或9在30℃水解三丁酸甘油酯，之后进行pH恒定的滴定实验。1单位的脂肪酶活性(1LU)等于在pH 7能够释放1微摩尔丁酸/分钟的酶量。
效益风险(Benefit Risk)
效益风险因子描述的是与减少的气味风险相比的性能，将其定义为：BR＝RPavg/R，如下所述。
用途
本发明的酶可以具有工业用途，例如，包括在用于去除脂肪物质的洗涤剂组合物中。
实施例
用作缓冲剂和底物的化学品是至少试剂等级的商业产品。
培养基与溶液
产品
商品名
LAS： Surfac PS
沸石A Wessalith P
使用的其它组分是标准实验室试剂。
材料
产品
供应商
EMPA221 EMPA St.Gallen,Lerchfeldstrasse 5,CH-9014St.Gallen,Switzerland
实施例1
酶的产生
使用本领域的标准方法构建含有编码脂肪酶的基因的质粒，并且转化至合适的宿主细胞中。
使用温度为34℃的恒定培养基和1.2升的起始体积，按照补料分批发酵来进行发酵。将培养基的初始pH设为6.5。一旦pH增加至7.0，通过添加10％H3PO4来保持该值。通过改变搅拌速率和使用1.0升空气每升培养基每分钟的固定通气速率来控制培养基中的溶解氧水平。在整个补料分批阶段过程中，将补料添加速率保持在恒定水平。
分批培养基含有麦芽糖糖浆作为碳源，尿素和酵母提取物作为氮源，以及痕量金属和盐的混合物。在补料分批阶段期间连续添加的补料含有麦芽糖糖浆作为碳源，而添加酵母提取物和尿素以确保氮的充足供应。
可以通过使用本领域已知的标准方法来进行脂肪酶的纯化，例如通过过滤发酵上清，继之以疏水层析和离子交换层析，例如如EP 0851913EP，实施例3中所述。
实施例2
AMSA-自动化机械应力试验-用于计算相对性能(RP)
使用自动机械应力试验(Automatic Mechanical Stress Assay；AMSA)测试本申请的酶变体。使用AMSA测试，能够检查大量小体积酶洗涤剂溶液的洗涤性能。AMSA平板具有用于测试溶液的多个槽(slot)，和将待洗涤的纺织品样品相对于所有槽的开口(slotopening)压紧的盖。在洗涤时间中，剧烈摇动平板、测试溶液、纺织品和盖以使测试溶液与纺织品接触并且施以机械应力。进一步的描述参见WO 02/42740，特别是第23-24页的段落“特殊方法实施方式”。含有洗涤剂测试溶液的容器由金属平板中的圆柱形孔洞(直径6mm，深10mm)组成。沾污的织物(测试材料)放置在金属平板顶部，并将沾污的织物用作盖子密封在所述容器上。另一个金属平板放置在沾污织物的顶部以避免从各个容器中的任何溢出。以2mm振幅和30Hz的频率将两块金属平板连同沾污织物上下振动。
在下文指定的实验条件下进行所述试验：
表3
通过将5g姜黄(Santa Maria,Denmark)与100g奶油(38％脂肪,Arla,Denmark)在50℃混合来制备奶油-姜黄样品，将所述混合物在该温度保持大约20分钟，并且过滤(50℃)以去除任何未溶解的颗粒。将混合物冷却至20℃，并且将编织的棉布样品EMPA221浸入所述奶油-姜黄混合物，之后允许其在室温干燥过夜，并且冷冻直到使用。奶油-姜黄样品的制备在专利申请WO2006/125437中公开。
将酶变体的性能作为用特定酶变体洗涤的纺织品样品的颜色亮度来测量。也可以将亮度表示为当用白光照射时，从纺织品样品反射的光的强度。当纺织品被沾污时，反射光的强度与清洁纺织品的反射光强度相比较低。因此，能够使用反射光的强度来测量酶变体的洗涤性能。
使用专业平板扫描仪(PFU DL2400pro)进行颜色测量，使用该扫描仪来捕捉经洗涤纺织品样品的图像。使用200dpi的分辨率和24比特(bit)的输出色深度来进行扫描。为了获得精确的结果，经常性地使用Kodak反射IT8指标(target)校准扫描仪。
为了从扫描的图像提取光强度的值，使用特殊设计的软件应用程序(NovozymesColor Vector Analyzer)。该程序从所述图像恢复24比特像素值，并且将它们转化为红、绿和蓝的值(RGB)。通过将RGB值加在一起作为矢量，再取所得矢量的长度来计算强度值(Int)：
根据下式计算变体的洗涤性能(P)：
P＝Int(v)–Int(r)
其中
Int(v)是用酶洗涤的纺织品表面的光强度值，和
Int(r)是不用酶洗涤的纺织品表面的光强度值。
根据以下定义，给出相对性能评分作为AMSA洗涤的结果：
相对性能评分(RP)概括了测试酶变体相对于参照酶的性能(P)：
RP＝P(测试酶)/P(参照酶)
RPavg表示在全部四种酶浓度(0.125、0.25、0.5、1.0mg ep/l)时，与参照酶相比的平均相对性能。
RPavg＝avg(RP(0.125),RP(0.25)RP(0.5),RP(1.0))
如果变体表现优于参照，则认为变体呈现出改进的洗涤性能。
在本发明的上下文中，参照酶是具有取代T231R+N233R的SEQ ID NO:2的脂肪酶。
实施例3
GC-气相色谱—用于计算风险因子
使用以下方法通过Solid Phase Micro Extraction Gas Chromatography(SPME-GC)测量来自经脂肪酶洗涤的样品的丁酸释放。将在含有1mg/L脂肪酶的表3中指定溶液中洗涤的四个纺织品片(直径5mm)转移至气相层析(GC)瓶。在装备有Stabilwax-DA w/Integra-Guard柱(30m,0.32mm ID和0.25微米df)和Carboxen PDMS SPME纤维(75微米)的Varian 3800GC上分析样品。将各个样品在40℃预温育10分钟，接着用SPME纤维在纺织品片之上的顶部空间(head space)进行20分钟采样。继而将样品注射到柱上(注射器温度＝250℃)。柱流速＝2ml氦/分钟。柱加热炉温度梯度：0分钟＝40℃，2分钟＝40℃，22分钟＝240℃，32分钟＝240℃。由FID检测法检测出丁酸，并且基于丁酸标准曲线来计算丁酸的量。
脂肪酶变体的风险性能气味(Risk Performance Odour)R是：经脂肪酶变体洗涤的样品所释放的丁酸量和使用具有取代T231R+N233R的SEQ ID NO:2的脂肪酶洗涤的样品所释放的丁酸量之间的比例，在计算比例之前将这两个值根据无脂肪酶洗涤的样品所释放的丁酸量进行修正。根据下式计算变体的风险(R)：
气味＝以相对于空白校正的在1mg酶蛋白/升产生的微克丁酸测量
Alpha测试酶＝气味测试酶-空白
Alpha参照酶＝气味参照酶-空白
R＝Alpha测试酶/Alpha参照酶
如果R因子小于1，则认为变体与参照相比表现出减少的气味。
实施例4
相对于280nm吸光度的活性(LU)
通过以下试验来测定相对于280nm吸光度的脂肪酶活性：
LU/A280：
如上面脂肪酶活性部分所述，确定脂肪酶的活性。测量脂肪酶在280nm的吸光度(A280)，并且计算比例LU/A280。用变体的LU/A280除以参照酶的LU/A280来计算相对LU/A280。在本发明的上下文中，参照酶是具有取代T231R+N233R的SEQ ID NO:2的脂肪酶。
实施例5
BR-效益风险
效益风险因子描述的是与减少的气味风险相比的性能，因此将其定义为：
BR＝RPavg/R
如果BR因子高于1，则认为变体显示出改进的洗涤性能和减少的气味。
应用以上方法获得了以下结果：
表4
表4中的参照脂肪酶和变体7与8如WO 2000/060063中所述。
实施例6
BR-效益风险
测定列于表5中所列变体的效益风险。用与实施例5中所述相同的方法测定效益风险因子，并发现所有列出的变体，效益风险因子都大于1。
表5
参照酶如WO 2000/060063中所述。
Claims (8)
1.亲本脂肪酶的变体，其中变体是通过对SEQ ID NO: 2引入选自以下取代组的取代的氨基酸序列的变体：
- S58N+ V60S+ I86V+ A150G+ G156R+ E210K+ L227G+ T231R+ N233R+ I255A+P256K
- I202G + T231R + N233R
- I86V + L227G + T231R + N233R + P256K
- Q4V + S58N + V60S + T231R + N233R
- S58N + V60S + I90R + T231R + N233R
- I255Y + T231R + N233R
- I90A + T231R + N233R + I255V
- L97V+ T231R+N233R
- I90R+T231R+N233R
- I202V+T231R+N233R
- L227G+ T231R+ N233R+ P256K
- I90A+ T231R+ N233R
- T231R+N233R+ I255P
- I90V+I255V+T231R+N233R
- F211L+ L227G+ T231R+ N233R+ I255L+ P256K
- S58N+ V60S+ T231R+ N233R+ Q249L
- S58N+ V60S+ T231R+ N233R+ Q249I
- K46L+ S58N+ V60S+ T231R+ N233R+ Q249L+ D254I
- Q4L+ E43T+ K46I+ S58N+ V60S+ T231R+ N233R+ Q249L+ D254I
- Q4L+ S58N+ V60S+ T231R+ N233R+ Q249L+ D254I
- K46I+ S58N+ V60S+ T231R+ N233R+ Q249L+ D254L
- K46L+ S58N+ V60S+ K223I+ T231R+ N233R+ D254I
- E43T+ K46I+ S58N+ V60S+ T231R+ N233R+ Q249L+ D254I
- K24R+ K46R+ K74R+ I86V+ K98R+ K127R+ D137K+ A150G+ K223R+ T231R+ N233R
- S58A+V60A+ I86V+T231R+N233R
- K24R+ K46R+ S58N+ V60S+ K74R+ I86V+ K98R+ K127R+ D137K+ K223R+ T231R+N233R
- S58N+ V60V+ D62G+ T231R+ N233R
- Q4V+ S58N+ V60S+ I86V+ T231R+ N233R+ Q249L
- Y53A+ S58N+ V60S+ T231R+ N233R+ P256L
- I202L+ T231R+ N233R+ I255A
- E210V+ T231R+ N233R+ Q249R
- S58N+ V60S+ E210V+ T231R+ N233R+ Q249R
- Q4V+ V60K+ I90R+ T231R+ N233R+ I255V
- V60K+ S83T+ T231R+ N233R
- T231R+ N233G+ D234G
- G91S+ D96V+ D254R
- V60L+ G91M+ T231W+ Q249L
- T37A+ D96A+ T231R+ N233R+ Q249G
- E56G+E87D+T231R+N233R+D254A
- E210K+T231R+N233R
- D27H+E87Q+D96N+T231R+N233R+D254V
- F181L+E210V+T231R+N233R
- D27N+ D96G+ T231R+ N233R
- D96N+ T231R+ N233R
- T231R+ N233I+ D234G
- S58K+ V60L+ E210V+ Q249R
- S58H+ V60L+ E210V+ Q249R
- Q4V+ F55V+ I86V+ T231R+ N233R+ I255V
- Q4V+ S58T+ V60K+ T199L+ N200A+ E210K+ T231R+ N233R+ I255A+ P256K
- Q4V+ D27N+ V60K+ T231R+ N233R
- I90F+ I202P+ T231R+ N233R+ I255L
- S58N+ V60S+ D158N+ T231R+ N233R
- S58N+ V60S+ S115K+ T231R+ N233R
- I90V+L227G+T231R+N233R+ P256K
- T231R+N233R+ I255S
- I86G+ T231R+ N233R
- V60K+ I202V+ E210K+ T231R+ N233R+ I255A+ P256K
- I90G+ I202L+ T231R+ N233R+ I255S
- S58G+ V60G+ T231R+ N233R。
2.编码权利要求1的脂肪酶的变体的DNA序列。
3.携带权利要求2的DNA序列的表达载体。
4.包含权利要求2的DNA序列的转化宿主细胞。
5.产生脂肪酶变体的方法，所述方法包含在利于产生脂肪酶变体的条件下培养权利要求4的转化宿主细胞，和从得到的培养液中回收脂肪酶变体。
6.权利要求1的脂肪酶的变体在洗涤剂中的用途。
7.权利要求1的脂肪酶的变体在去除脂肪物质的洗涤剂组合物中的用途。
8.包含权利要求1的脂肪酶的变体的洗涤剂组合物。
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| US20090029440A1 (en) | 2009-01-29 |
| JP5133899B2 (ja) | 2013-01-30 |
| EP2371949A1 (en) | 2011-10-05 |
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| ES2628940T3 (es) | 2017-08-04 |
| DK2371948T3 (da) | 2017-06-19 |
| DK1979477T3 (en) | 2017-06-26 |
| BRPI0707202A2 (pt) | 2011-04-26 |
| ES2629334T3 (es) | 2017-08-08 |
| EP2371948A1 (en) | 2011-10-05 |
| EP2371949B1 (en) | 2017-04-19 |
| JP2009523455A (ja) | 2009-06-25 |
| ES2629332T3 (es) | 2017-08-08 |
| WO2007087508A3 (en) | 2008-01-24 |
| EP2371948B1 (en) | 2017-04-19 |
| DK2371949T3 (en) | 2017-06-26 |
| CN105296445A (zh) | 2016-02-03 |
| WO2007087508A8 (en) | 2009-07-30 |
| WO2007087508A2 (en) | 2007-08-02 |
| CN101370933B (zh) | 2015-11-25 |
| DK2371948T5 (da) | 2017-07-24 |
| US8187854B2 (en) | 2012-05-29 |
| CN101370933A (zh) | 2009-02-18 |
| EP1979477B1 (en) | 2017-04-19 |
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