CN1133062A - 对过氧化氢稳定的过氧化物酶变体 - Google Patents

对过氧化氢稳定的过氧化物酶变体 Download PDF

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CN1133062A
CN1133062A CN94193742A CN94193742A CN1133062A CN 1133062 A CN1133062 A CN 1133062A CN 94193742 A CN94193742 A CN 94193742A CN 94193742 A CN94193742 A CN 94193742A CN 1133062 A CN1133062 A CN 1133062A
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hydrogen peroxide
peroxidase
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A·H·佩德森
J·文德
A·斯文德森
J·R·彻里
M·拉姆萨
P·施奈德
B·R·詹森
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Abstract

本发明涉及显示极佳过氧化氢稳定性的灰盖鬼伞过氧化物酶新变体。

Description

对过氧化氢稳定的 过氧化物酶变体
本发明涉及一种新的过氧化物酶变体,和含有这种过氧化物酶变体的漂白或洗涤剂组合物。
在洗涤过程中使用漂白剂并作为洗涤剂组合物的成分是本领域中熟知的。因此,在市售的大部分洗涤剂组合物中掺有漂白剂或作为其成分出售。掺入洗涤剂组合物中的重要常规漂白剂是那些在洗涤过程中形成过氧化氢的作为其前体的化合物。用作漂白剂并以此方式起漂白作用的化合物,最重要的例子是过硼酸盐和过碳酸盐。现在还不知道用这些漂白剂漂白的详细机制,但一般认为在洗涤过程中形成的过氧化氢通过氧化将有色物质(由其在织物上形成污渍)转化为无色物质,有色物质与过硼酸盐或过碳酸盐直接作用也发生一些氧化。
现已发现以过氧化氢为底物的过氧化物酶能够增强洗涤过程中过氧化氢的漂白效果。WO89/09813中描述了过氧化物酶漂白织物上污渍的应用。还发现从染色织物上沥出的有色物质可借助过氧化物酶和过氧化氢一起漂白。WO91/05839中描述了过氧化物酶以此方式抑制染料转移的应用。
现令人惊异地发现,可通过重组DNA技术制备在碱性条件下提高了对过氧化氢稳定性的过氧化物酶变体。
因此,本发明涉及在碱性条件下提高了对过氧化氢稳定性的过氧化物酶变体,其特征在于在SEQ ID.1中所示序列编码的灰盖鬼伞(Coprinus cinereus)过氧化物酶这一亲本过氧化物酶的48-56,76,109,214,239,258-262,264,266-272氨基酸残基区中***、缺失或取代一个或多个氨基酸残基。
经X线衍射获得亲本过氧化物酶结晶结构的信息,还通过亲本过氧化物酶的氨基酸序列与其他已知过氧化物酶的氨基酸序列排列比较来获得这些信息(K.G.Welinder等,″Structure and evolutionof peroxidases″in Plant Peroxidase Biochemistry and Physiology,K.G.Welinder等(编),University of Copenhagen and Geneva1993)。
在本文中术语“提高了对过氧化氢的稳定性”意指在高达20mMH2O2浓度的过氧化氢存在下过氧化物酶变体稳定性至少比亲本过氧化物酶的高10%。更具体讲,这是指在10-60℃温度范围内一个或多个温度下、在最高达20mM H2O2的一个或多个H2O2浓度下,过氧化物酶变体的半衰期比野生型过氧化物酶(亲本过氧化物酶)的至少长10%。(通过将残余活性与一级衰变相匹配来测定半衰期)。术语“碱性条件”指7-11的PH范围。
另一方面,本发明涉及含有本发明过氧化物酶变体和过氧化氢源,任选还含有可氧化底物的漂白组合物;涉及含有表面活性剂、本发明的过氧化物酶变体,过氧化氢源、任选还含有可氧化底物的洗涤剂组合物。
参照附图进一步说明本发明,其中
图1表示质粒pHD414,它是质粒p775(在EP238023中有述)的衍生体。按实施例1获得质粒pHD414。
在本说明书和权利要求中使用了下列缩写:氨基酸:A=Ala=丙氨酸V=Val=缬氨酸L=Leu=亮氨酸I=Ile=异亮氨酸P=Pro=脯氨酸F=Phe=苯丙氨酸W=Trp=色氨酸M=Met=蛋氨酸G=Gly=甘氨酸S=Ser=丝氨酸T=Thr=苏氨酸C=Cys=半胱氨酸Y=Tyr=酪氨酸N=Asn=天冬酰胺Q=Gln=谷氨酰胺D=Asp=天冬氨酸E=Glu=谷氨酸K=Lys=赖氨酸R=Arg=精氨酸H=His=组氨酸
在描述本发明过氧化物酶变体过程中,为了指称方便使用了下列命名原则:原始氨基酸:位置:取代后的氨基酸
根据这一命名原则,例如在48位由丝氨酸取代赖氨酸表示如下:K48S;在此位缺失赖氨酸表示为:K48*;***另外的氨基酸残基如酪氨酸则表示为:K48KY。多个取代用加号隔开,即:E214L+E239L代表在214和239位由亮氨酸取代谷氨酸的突变。
亲本过氧化物酶由SEQ ID No.1中所示氨基酸序列编码。所述序列可从灰盖鬼伞衍化而来。
在本发明过氧化物酶变体的一种实施方案中,在SEQ ID No.1中所示氨基酸序列编码的亲本过氧化物酶48-56,76,109,214,239,258-262,264,266-272氨基酸残基区中缺失、***或取代一个或多个氨基酸残基。
在本发明过氧化物酶变体另一实施方案中,一个或多个氨基酸残基可适当地取代如下:
K48S,
V53K,A,
G72Q,
A91C,
N92K,
H109K,
Q118E,
M125A,P,W,G,S,T,C,Y,N,D,E,K,R,H,
特别是             M125S,T,
S147Q,
I152C,
P155C,
M166A,P,W,G,S,T,C,Y,N,D,E,K,R,H,
特别是             M166S,T,N192K,I195K,V206R,E214L,K218R,F229G,A230C,E239A,V,L,I,P,F,W,M,G,S,T,C,Y,N,Q,D,K,R,H,
特别是         E239K,G,S,L,Q,M,M242A,P,W,G,S,T,C,Y,N,D,E,K,R,H,
特别是         M242S,T,S244C,S252P,W258F,H,M261A,V,L,I,P,F,W,G,S,T,C,Y,N,Q,D,E,K,R,H,
特别是         M261F,V,I,L,Q,M268A,V,L,I,P,F,W,G,S,T,C,Y,N,Q,D,E,K,R,H,
特别是:       M268F,V,I,L,Q,Y272A,V,L,I,P,F,W,G,S,T,C,M,N,Q,D,E,K,R,H,
特别是         Y272F,M276A,P,W,G,S,T,C,Y,N,D,E,K,R,H,
特别是         M276S,T,K278R,M279A,P,W,G,S,T,C,Y,N,D,E,K,R,H,
特别是         M279S,T,A304E,V314P。
在一替代方案中,本发明过氧化物酶变体可为上述过氧化物酶变体的片段。
根据本发明,过氧化物酶序列的2个或多个氨基酸残基可被取代如下:
K41R+K48R,
V53K+Q118E,
V53A+E239G,
I152C+A91C,
P155C+A230C,
M166F+E239K,
G167N+V176L,
E214L+E239L,
R241E+E239K,
S244C+P155C,
E239K+M242I+Y272F,
M166F+E239K+M242I+Y272F,
M125L+M166F+E239K+M242I+Y272F。
根据本发明,本发明公开的取代、***或缺失可与WO93/24618中公开的取代、***或缺失相结合,WO93/24618中在本发明中示为SEQ ID No.1的亲本过氧化物酶79-94,125,153-157,161-204,242,276-279氨基酸残基区中缺失、***或取代一个或多个氮基酸残基。这种组合的一个实例是M242I+Y272F+E239K变体,在实施例4中证明其对过氧化氢具有良好的稳定性。
可按本领域熟知的各种方法从任何产生目标过氧化物酶的微生物分离编码亲本过氧化物酶的DNA序列。首先,应利用来自产生待研究过氧化物酶的生物体的染色体DNA或信使RNA构建基因组DNA和/或cDNA文库。然后,如果过氧化物酶的氨基酸序列已知,可合成同源性标记寡核苷酸探针,用于从细菌DNA基因组文库或从真菌cDNA文库鉴定编码过氧化物酶的克隆。或者,可在低严紧性杂交和冲洗条件下使用含有来自另一真菌菌株过氧化物酶的同源序列的标记寡核苷酸探针作为鉴定过氧化物酶编码性克隆的探针。
另一种鉴定产生的过氧化物酶克隆的方法包括:将基因组DNA片段***诸如质粒的表达载体中,用形成的基因组DNA文库转化过氧化物酶阴性细菌,然后将转化的细菌铺在含过氧化物酶底物的琼酯上。加入过氧化氢时那些含有携带过氧化物酶之质粒的细菌将产生周围有清澈琼脂圈的菌落,这是因为底物被所分泌的过氧化物酶氧化的缘故。
或者,按已有标准方法合成制备编码酶的DNA序列,如S.L.Beaucage和M.H.Caruthers(Tetrahedron Letters22,1981,pp.1859-1869)描述的氨基亚磷酸酯法,或Matthes等(The EMBO J.3,1984,pp,801-805)所述方法。按照氨基亚磷酸酯法,在如自动DNA合仪上合成寡核苷酸,并径纯化、退火,连接并克隆在适当载体中。
最后,该DNA序列可以是基因组和合成的、合成的和cDNA或基因组和cDNA的混合来源,按标准技术将相当于整个DNA序列各个部分的合成、基因组或cDNA来源(适当时)的片段连接而成。还可用特异引物通过聚合酶链反应(PCR)制备该DNA序列,如US4,683,202或R.K.Saiki等Science239,1988,pp487-491所述。
一旦分离了编码过氧化物酶的DNA序列并确定了突变的适宜位点,就可用合成寡核苷酸引入突变。这些寡核苷酸含有目标突变位点的侧翼核苷酸序列;并在寡核苷酸合成中***突变核苷酸。在一特定方法中,在携带过氧化物酶基因载体中在过氧化物酶编码序列当中造成一单链DNA缺口。然后将带有目标突变的合成核苷酸退火到单链DNA的同源性部分。剩余的缺口随后用DNA聚合酶I(Klenow片段)填平,用T4连接酶连接该构建物。Morinage等(1984,Biotechnology2:646-639)描述了该方法的一个具体例子。美国专利4,760,025(Estell等,于1988年7月26日授权)描述了通过对DNA序列盒进行小的改变引入编码多个突变的寡核苷酸的方法,但用Morinage方法一次可引入更多种突变,因为能引入各种长度的多种寡核苷酸。
Nelson和Long(Analytical Biochemistry180,1989,pp147-151)描述了向过氧化物酶编码序列中引入突变的另一种方法。它包括含有目标突变的PCR片段的3步生成法,用化学合成的DNA链作为PCR反应的引物之一引入突变。通过限制性内切酶酶切可从PCR产生的片段分离出带突变的DNA片段,并再***表达质粒中。
根据本发明,按上述方法之一或本领域已知的任何替代方法产生的突变过氧化物酶编码序列可用表达载体表达成酶形式,表达载体典型地包括编码启动子、操纵子、核糖体结合位点、翻译起始信号和任选的抑制基因或各种激活基因的调控序列。为了使得所表达蛋白可分泌,可在过氧化物酶编码序列前***编码信号序列的核苷酸。为了在调控序列控制下表达,将靶基因在适当的读码框架中与调控序列有效地连接。可引入质粒载体并能支持突变过氧化物酶基因转录的启动子序列,包括但不限于原核β-内酰胺酶启动子(Villa-kamaroff等,1978,Proc.Natl.Acad.Sci.U.S.A.75:3727-3731)和tac启动子(DeBoer等,1983,Proc.Natl.Acad.Sci.U.S.A.80:21-25)。在Scientific American,1980,242:74-94中“Useful proteins from recombinant bacteria”一文中可找到其他参考文献。
根据一种实施方案,用携带突变DNA的表达载体转化枯草芽孢杆菌(B.subtilis)。如果在诸如枯草芽孢杆菌的分泌性微生物中没有表达,则在翻译起始区之后和目标DNA序列之前需要一种信号序列。信号序列的作用是将表达产物运送到细胞壁,并在此处分泌时将其从产物上切下来。如上所定义的“调控序列”包括存在的信号序列。
用编码本发明过氧化物酶变体的DNA序列转化的宿主微生物还可以是酵母,优选酵母属菌株,如酿酒酵母或pombe裂殖酵母,或毕赤酵母属,如巴斯德毕赤酵母。
在现优选的一种产生本发明过氧化物酶变体的方法中,使用丝状真菌作为宿主生物。这种丝状真菌宿主生物可方便地为以前已用于产生重组蛋白的宿主之一,例如曲霉属菌株,如黑曲霉、构巢曲霉或米曲霉。在如EP238023中广泛描述了米曲霉在生产重组蛋白中的应用。
为了在曲霉中表达过氧化物酶变体,编码过氧化物酶的DNA序列之前需有一启动子。该启动子可为在曲霉中显示强转录活性的任何DNA序列,并可衍生自编码细胞外或细胞内蛋白如淀粉酶、葡糖淀粉酶、蛋白酶、脂酶、过氧化物酶、纤维素酶或糖酵解酶的基因。
适宜启动子的例子是那些衍生自编码米曲酶TAKA淀粉酶、米黑毛霉(Rhizomucor miehei)天冬氨酸蛋白酶、黑曲霉中性α-淀粉酶、黑曲酶酸稳定性α-淀粉酶、黑曲霉葡糖淀粉酶、米黑毛霉脂酶、米曲霉碱性蛋白酶或米曲霉丙糖磷酸异构酶的基因。
特别当宿主生物为米曲霉时,用于本发明方法的优选启动子是米曲霉TAKA淀粉酶启动子,因为它在米曲霉中显示强转录活性。EP 238023中描述了此TAKA淀粉酶启动子的序列。
终止和多聚腺苷化序列可适当地衍生自启动子的同一来源。
在EP238023中描述了用于转化真菌宿主细胞的适当技术。
为了保证过氧化物酶变体从宿主细胞中分泌,编码过氧化物酶变体的DNA序列之前可有一信号序列,它可以是天然存在的序列或其功能性部分或一种使蛋白从细胞分泌的合成序列。尤其,该信号序列可衍生自编码曲霉淀粉酶或葡糖淀粉酶的基因,编码米黑毛霉脂酶或蛋白酶的基因,或编码腐质霉纤维素酶、木聚糖酶或脂酶的基因。该信号序列优选地衍生自编码米曲霉TAKA淀粉酶、黑曲霉中性α-淀粉酶、黑曲霉酸稳定性α-淀粉酶、灰盖鬼伞或macrorhizus过氧化物酶、或黑曲霉葡糖淀粉酶的基因。
用于培养转化的宿主细胞的培养基可以是适于曲霉细胞生长的任何常规培养基。转化子通常是稳定的并可在无任何筛选压力下培养。但如果发现转化子不稳定,可向细胞中引入筛选标记用于筛选。
从宿主细胞分泌的成熟过氧化物酶蛋白可按常规方法从培养基中回收,为人熟知的方法包括离心或过滤从培养基中分离细胞,用盐如硫酸铵沉淀培养基中蛋白成分,然后进行层析步骤,如离子交换层析、亲合层析等。
为了获得过氧化物酶变体的漂白效果,本发明漂白组合物中应存在过氧化氢或过氧化氢前体,优选过硼酸盐或过碳酸盐,或产生过氧化氢的酶***,如氧化酶和其底物,或过氧羧酸或其盐作为过氧化物酶变体的底物。
虽然还没有阐明过氧化物酶漂白织物上或洗涤液中有色物质的机制,但现今相信这种酶通过还原过氧化氢并氧化有色物质(电子供体底物)起作用,从而产生无色物质或不与织物吸附的物质。下述反应式1中描述了这一反应。反应式1:
通过使用对过氧化氢敏感性低的本发明过氧化物酶变体,可以向漂白/洗涤液中加入更少量的酶而仍然获得满意的漂白效果。
在漂白组合物中,过氧化物酶变体的量相当于洗涤液中浓度为0.01-20PODU/ml,过氧化氢或过氧化氢前体或产生过氧化氢的酶***或过氧羧酸或其盐的量相当于过氧化氢浓度最高达20mMH2O2
过氧化物酶活性的测定:
一个过氧化物酶单位(PODU)是在下列催化条件下每分钟催化1μmol过氧化氢转化的酶量:0.88mM过氧化氢,1.67mM 2.2′-连氮基双(3-乙基苯并噻唑啉-6-磺酸盐),0.1M磷酸缓冲液,PH7.0,30℃保温,然后在418nm处光度计测定。
对本发明过氧化物酶变体用作漂白组合物,现发现在洗涤和/或漂洗过程开始时或此过程中加入另一种(本发明过氧化物酶变体的)可氧化底物,可增强所用过氧化物酶变体的漂白效果。认为这是由于形成了该底物的自由基或其他氧化态,其参与有色物质的漂白或其他改性。这种可氧化物质的例子为有机化合物如酚类化合物,如对羟基苯磺酸盐。其他可用于此目的的酚类化合物见M.Kato和S.shimizu的Plant Cell Physiol.,26(7),1985,pp,1291-1301(特别参见表1)或B.C.Saunders等Peroxidase,London,1964,p.141 ff.WO94/12621中公开了其他类型增强剂,它们可用于此目的,例如吩噻嗪和吩、嗪和其衍生物,如10-甲基吩噻嗪,10-吩噻嗪-丙酸、N-羟基琥珀酰亚胺-10-吩噻嗪-丙酸酯、10-乙基-4-吩噻嗪-羧酸,10-乙基吩噻嗪,10-丙基吩噻嗪,10-异丙基吩噻嗪,10-吩噻嗪丙酸甲酯,10-苯基吩噻嗪,10-烯丙基吩噻嗪,10-(3-(4-甲基-1-哌嗪基)丙基吩噻嗪,10-(2-吡咯烷-1-基乙基)吩噻嗪,10-(3-二甲氨基丙基)吩噻嗪,2-氯-10-甲基吩噻嗪,2-乙酰-10-甲基吩噻嗪和10-甲基吩噁嗪。
可氧化底物的量相当于每升洗液中浓度为0.1μM到100μM。
洗涤剂组合物
本发明的过氧化物酶变体可作为一种成分加入洗涤剂组合物中。因此,它可作为一种洗涤剂添加剂包括在洗涤剂组合物中。洗涤剂组合物和洗涤剂添加剂通常还含有一种或多种其他酶如脂肪酶、淀粉酶、角质酶和纤维素酶。
在一个特定方面,本发明提供一种洗涤剂添加剂。可通过加入多个含有一种或多种酶的分离式添加剂或加入一种含有所有这些酶的联合式添加剂而将酶加入洗涤剂组合物中。本发明的洗涤剂添加剂,即分离式添加剂或联合式添加剂,可制成如粒状、液态、浆液等。优选的洗涤剂添加剂制品是颗粒状的(尤其是非粉尘颗粒)、液态的(尤其是稳定的液体)、浆状或被保护酶的形式。
可按US4,106,991及4,661,452的方法(均为Novo IndustriA/S专利)生产非粉尘颗粒,并选择性采用本领域中已知的方法对非粉尘颗粒进行包被。蜡样包被材料的例子有:平均摩尔分子量为1000至20000的聚(环氧乙烷)产物(聚乙二醇;PEG);含有16到50个环氧乙烷单位的乙氧基化壬基酚;含有15到80个环氧乙烷单位的乙氧基化脂肪醇(其中的醇含有12-20个碳原子);脂肪醇;脂肪酸;甘油单-,双-及三脂肪酸酯。专利GB1483591中给出了适用于流化床技术的涂膜包被材料的例子。根据已建立的方法在液态酶制剂中加入多羟基化合物如丙二醇、糖或糖醇、乳酸或硼酸可达到稳定作用。本领域的技术人员熟知其他酶稳定剂。可按EP238,216的方法制备被保护酶。
本发明的洗涤剂组合物可制成任何方便的形式,如粉状、颗粒状、糊状或液态。液态洗涤剂可以是典型的含高达70%的水及0-30%有机溶剂的水性洗涤剂,也可是非水性的。
该洗涤剂组合物中含一种或多种表面活性剂,它们可以是阴离子、非离子、阳离子或两性离子型。本洗涤剂中通常含有0-50%的阴离子表面活性剂,如线性烷基苯磺酸盐(LAS),α-烯属磺酸盐(AOS),烷基硫酸盐(脂肪醇硫酸酯)(AS),乙氧基化醇硫酸酯(AEOS或AES),二级烷磺酸盐(SAS),α-磺基脂肪酸甲酯,烷基或链烯基琥珀酸或皂。还可以含0-40%的非离子表面活性剂,如醇乙氧基化物(AEO或AE),羧基化醇的乙氧基化物,壬基酚乙氧基化物,烷基聚甘醇甙,烷基二甲胺氧化物,乙氧基化脂肪酸单乙醇酰胺,脂肪酸单乙醇酰胺,或多羟基烷基脂肪酸酰胺(如W092/06154中所述)。
本洗涤剂组合物中还可含有一种或多种其他酶,如淀粉酶、脂肪酶、角质酶、蛋白酶和纤维素酶。
本洗涤剂中可含有1-65%的洗涤剂助洗剂或配合剂,如沸石、二磷酸盐、三磷酸盐、膦酸盐、柠檬酸盐、次氮基三乙酸(NTA)、乙二胺四乙酸(EDTA)、二亚乙基三胺五乙酸(DTMPA)、烷基或烯基琥珀酸、可溶性硅酸盐或层叠式硅酸盐(如从Hoechst得到的SKS-6)。本洗涤剂还可以是不带助洗剂的,即基本上不含洗涤剂助洗剂。
本洗涤剂可含一种或多种聚合物。例如,羧甲基纤维素(CMC),聚(乙烯基吡咯烷酮)(PVP)、聚乙二醇(PEG),聚(乙烯基醇)(PVA),多羧基化合物如聚丙烯酸、马来酸/丙烯酸共聚物和甲基丙烯酸月桂基酯/丙烯酸共聚物。
本洗涤剂可含有漂白***,其中可包括H2O2源如过硼酸盐或过碳酸盐,它们可以和过酸形成型漂白活化剂如四乙酰乙二胺(TAED)或壬酰氧基苯磺酸盐结合使用。或者,漂白***可含有如酰胺、酰亚胺或砜型的过氧酸。
本发明的洗涤剂组合物中的酶可用常规的稳定剂来稳定,如丙二醇或甘油之类的多羟基化合物、糖或糖醇、乳酸、硼酸或硼酸衍生物如硼酸芳酯,此组合物可按WO92/19709和WO92/19708中所述方法配制。
该洗涤剂中还可含有其他常规洗涤剂组分,如包括粘土的织物调理剂、泡沫促进剂、抑泡剂、抗腐蚀剂、污物悬浮剂、防污物再沉剂、染料、杀菌剂、荧光增白剂或香味剂。
PH值(在使用浓度的水溶液中测定)通常为中性或碱性,如7-11。
本发明范围内的特定洗涤剂组合物包括:1).配制成堆积密度至少为600g/l的颗粒状洗涤剂组合物含有:
-线性烷基苯磺酸盐(按酸计)            7-12%
-乙氧基醇硫酸酯(如C12-18的醇,        1-4%
1-2EO)或烷基硫酸盐(如C16-18)
-醇乙氧基化物(如C14-15的醇,7EO)      5-9%
-碳酸钠(按Na2CO3计)               14-20%
-可溶性硅酸盐(按Na2O·2SiO2计)      2-6%
-沸石(按NaAlSiO4计)                15-22%
-硫酸钠(按Na2SO4计)                 0-6%
-柠檬酸钠/柠檬酸                     0-15%
(按C6H5Na3O7/C6H8O7计)
-过硼酸钠(按NaBO3H2O计)           11-18%
-TAED                                 2-6%
-羧甲基纤维素                         0-2%
-聚合物(如马来酸/                     0-3%
丙烯酸共聚物,PVP,PEG)
-酶                                   0-5%
-小成分(如抑泡剂,香味剂,荧光增白剂,光漂白剂)
                                                 0-5%2).配制成堆积密度至少为600g/L的颗粒状洗涤剂组合物含有:
-线性烷基苯磺酸盐(按酸计)                      6-11%
-乙氧基醇硫酸酯(如C12-18的醇,1-2EO)           1-3%
或烷基硫酸盐(如C16-18)
-醇乙氧基化物(如C14-15的醇,7EO)                5-9%
-碳酸钠(按Na2CO3计)                         15-21%
-可溶性硅酸盐(按Na2O·2SiO2计)                1-4%
-沸石(按NaAlSiO4计)                          24-34%
-硫酸钠(按Na2SO4计)                          4-10%
-柠檬酸钠/柠檬酸                               0-15%
(按C6H5Na3O7/C6H8O7计)
-羧甲基纤维素                                   0-2%
-聚合物(如马来酸/丙烯酸共聚物PVP,PEG)          1-6%
-酶                                             0-5%
-小成分(如抑泡剂、香味剂)                       0-5%3).配制成堆积密度至少为600g/L的颗粒状洗涤剂组合物含有:
-线性烷基苯磺酸盐(按酸计)                       5-9%
-醇乙氧基化物(如C12-15的醇,7EO)               7-14%
-作为脂肪酸的皂类(如C16-22)                     1-3%
-碳酸钠(按Na2CO3计)                         10-17%
-可溶性硅酸盐(按Na2O·2SiO2计)                3-9%
-沸石(按NaAlSiO4计)                          23-33%
-硫酸钠(按Na2SO4计)                       0-4%
-过硼酸钠(按NaBO3·H2O计)                8-16%
-TAED                                       2-8%
-膦酸盐(如EDTMPA)                           0-1%
-羧甲基纤维素                               0-2%
-聚合物(如马来酸/丙烯酸                     0-3%
共聚物,PVP,PEG)
-酶                                         0-5%
-小成分(如抑泡剂,香味剂,荧光增白剂)       0-5%4).配制成堆积密度至少为600g/L的颗粒状洗涤剂组合物含有:
-线性烷基苯磺酸盐(按酸计)                  8-12%
-醇乙氧基化物(如C12-15的醇,7EO)          10-25%
-碳酸钠(按Na2CO3计)                     14-22%
-可溶性硅酸盐(按Na2O·2SiO2计)            1-5%
-沸石(按NaAlSiO4计)                      25-35%
-硫酸钠(按Na2SO4计)                      0-10%
-羧甲基纤维素                               0-2%
-聚合物(如马来酸/丙烯酸共聚物,             1-3%
PVP,PEG)
-酶                                         0-5%
-小成分(如抑泡剂,香味剂)                   0-5%5).水性液态洗涤剂组合物含有:
-线性烷基苯磺酸盐(按酸计)                 15-21%
-醇乙氧基化物                             12-18%
(如C12-15的醇,7EO或C12-15的醇,5EO)
-作为脂肪酸的皂(如油酸)                        3-13%
-链烯基琥珀酸(C12-14)                          0-13%
-氨基乙醇                                      8-18%
-柠檬酸                                         2-8%
-膦酸盐                                         0-3%
-聚合物(如PVP,PEG)                             0-3%
-硼酸盐(按B4O7计)                             0-2%
-乙醇                                           0-3%
-丙二醇                                        8-14%
-酶                                             0-5%
-小成分(如分散剂,抑泡剂,香味剂,荧光增白剂)   0-5%6).水性结构化液态洗涤剂组合物含有:
-线性烷基苯磺酸盐(按酸计)                     15-21%
-醇乙氧基化物                                   3-9%
(如C12-15的醇,7EO或C12-15的醇,5EO)
-作为脂肪酸的皂(如油酸)                        3-10%
-沸石(按NaAlSiO4计)                          14-22%
-柠檬酸钾                                      9-18%
-硼酸盐(按B4O7计)                             0-2%
-羧甲基纤维素                                   0-2%
-聚合物(如PVP,PEG)                             0-3%
-结合聚合物(如甲基丙烯酸月桂基酯/               0-3%
丙烯酸共聚物,摩尔比25∶1,分子量3800)
-甘油                                      0-5%
-酶                                        0-5%
-小成分(如分散剂,抑泡剂,香味剂,         0-5%
荧光增白剂)7).配制成堆积密度至少为600g/L的颗粒状洗涤剂组合物含有:
-脂肪醇硫酸酯                             5-10%
-乙氧基化脂肪酸单乙醇酰胺                  3-9%
-作为脂肪酸的皂                            0-3%
-碳酸钠(按Na2CO3计)                     5-10%
-可溶性硅酸盐(按Na2O·2SiO2计)           1-4%
-沸石(按NaAlSiO4计)                     20-40%
-硫酸钠(按Na2SO4计)                      2-8%
-过硼酸钠(按NaBO3·H2O计)              12-18%
-TAEP                                      2-7%
-聚合物(如马来酸/丙烯酸共聚物,PEG)        1-5%
-酶                                        0-5%
-小成分(如荧光增白剂,抑泡剂,香味剂)      0-5%8).配制成颗粒状的洗涤剂组合物含有:
-线性烷基苯磺酸盐(按酸计)                 8-14%
-乙氧基化脂肪酸单乙醇酰胺                 5-11%
-作为脂肪酸的皂                            0-3%
-碳酸钠(按Na2CO3计)                     4-10%
-可溶性硅酸盐(按Na2O·2SiO2计)           1-4%
-沸石(按NaAlSiO4计)                     30-50%
-硫酸钠(按Na2SO4计)                         3-11%
-柠檬酸钠(按C6H5Na3O7计)                  5-12%
-聚合物(如PVP,马来酸/                         1-5%
丙烯酸共聚物,PEG)
-酶                                            0-5%
-小成分(如抑泡剂,香味剂)                      0-5%9).配制成颗粒状的洗涤剂组合物含有:
-线性烷基苯磺酸盐(按酸计)                     6-12%
-非离子表面活性剂                              1-4%
-作为脂肪酸的皂                                2-6%
-碳酸钠(按Na2CO3计)                        14-22%
-沸石(按NaAlSiO4计)                         18-32%
-硫酸钠(按Na2SO4计)                         5-20%
-柠檬酸钠(按C6H5Na3O7计)                  3-8%
-过硼酸钠(按NaBO3·H2O计)                    4-9%
-漂白活化剂(如NOBS或TAED)                      1-5%
-羧甲基纤维素                                  0-2%
-聚合物(如多羧基化合物或PEG)                   1-5%
-酶                                            0-5%
-小成分(如荧光增白利,香味利)                  0-5%10).水性液态洗涤剂组合物含有:
-线性烷基苯磺酸盐(按酸计)                    15-23%
-乙氧基醇硫酸酯(如C12-15的醇,2-3EO)          8-15%
-醇乙氧基化物                                 3-9%
(如C12-15的醇,7EO或C12-15的醇,5EO)
-作为脂肪酸的皂(如油酸)                      0-3%
-氨基乙醇                                    1-5%
-柠檬酸钠                                   5-10%
-水溶助长剂(如甲苯磺酸钠)                    2-6%
-硼酸盐(按B4O7计)                          0-2%
-羧甲基纤维素                                0-1%
-乙醇                                        1-3%
-丙二醇                                      2-5%
-酶                                          0-5%
-小成分(如聚合物,分散剂,香味剂,           0-5%
荧光增白剂)11).水性液态洗涤剂组合物含有:
-线性烷基苯磺酸盐(按酸计)                  20-32%
-醇乙氧基化物                               6-12%
(如C12-15的醇,7EO或C12-15的醇,5EO)
-氨基乙醇                                    2-6%
-柠檬酸                                     8-14%
-硼酸盐(按B4O7计)                          1-3%
-聚合物                                      0-3%
(如马来酸/丙烯酸共聚物,结合聚合物
如甲基丙烯酸月桂基酯/丙烯酸共聚物和CMC)
-甘油                                        3-8%
-酶                                          0-5%
-小成分(如水溶助长剂,                       0-5%
分散利,香味剂,荧光增白剂)12).配制成堆积密度至少为600g/L的颗粒状洗涤剂组合物含有:
-阴离子表面活性剂                          25-40%
(如线性烷基苯磺酸盐,烷基硫酸盐,α-烯属磺酸盐,
α-磺基脂肪酸甲酯,烷磺酸盐,皂)
-非离子表面活性剂(如醇乙氧基化物)           1-10%
-碳酸钠(按Na2CO2计)                       8-25%
-可溶性硅酸盐(按Na2O·2SiO2计)            5-15%
-硫酸钠(Na2SO4计)                          0-5%
-沸石(按NaAlSiO4计)                       15-28%
-过硼酸钠(按NaBO3·4H2O计)                0-20%
-漂白活化剂(TAED或NOBs)                      0-5%
-酶                                          0-5%
-小成分(如香味剂,荧光增白剂)                0-3%13).如1)-12)中所述洗涤剂配方,其中的线性烷基苯磺酸盐-或其一部分-被烷基硫酸盐(C12-18)所取代。14).如1)-13)中所述洗涤剂配方,其中含有作为附加成分或已指定的漂白***取代物的稳定化的或包囊的过酸。15).如1)、3)、7)、9)及12)中描述的洗涤剂组合物,其中的过硼酸盐被过碳酸盐所取代。16).如1)、3)、7)、9)、及12)中所述洗涤剂组合物,其另外含有锰催化剂,如可为Nature,369,1994,pp.637-639中的文章《低温漂白的高效锰催化剂》中所述化合物的一种。17).配制成含有液态非离子表面活性剂(如线性烷氧基化伯醇)、助洗***(如磷酸盐)、酶及碱的非水性洗涤液的洗涤剂组合物。该洗涤剂中还可含有阴离子表面活性剂和/或漂白***。
现在考虑,可向本发明洗涤剂组合物中加入一定量的过氧化物酶变体,该量相当于洗液中浓度为0.01-20PODU/ml。
在以下实施例中进一步说明本发明,但无意对所要求的本发明范围进行任何限制。
实施例1.
构建表达灰盖鬼伞过氧化物酶K48S变体的质粒
1.克隆编码灰盖鬼伞过氧化物酶的cDNA
用PCR方法构建探针
用特异寡核苷酸引物进行聚合酶链反应(PCR)制备过氧化物酶cDNA片段(R.K.Saiki等,Science 239,1988,pp.487-491),在灰盖鬼伞过氧化物酶的氨基酸序列基础上构建引物。用基因扩增(Gene Amp)试剂盒和仪器(从Perkin Elmer Cetus,Norwalk,CT,USA购得)按制造商的说明进行PCR,只是反应先在28℃进行3个循环以与第一条cDNA更好地杂交(从灰盖鬼伞IFO 8371获得的mRNA制得),随后在65℃进行30个PCR循环。
PCR中使用下列特异引物:
                       T  T1.5′-GCGCGAATTCGTNGGNATNAACCACGG-3′
          A  A2.3′-TACAGNTTGACGGGNGGCCTAGGCG-5′
                  A     T  T3.5′-GCGAATTCACNCCNCAGGTNTTCGACAC-3′
       A        T  A4.3’-GGNAAGGGNCCNCTCAAGCCTAGGCG-5′
                 A5.5′-GCGCGAATTCTGGCAGTCNAC-3′
                 A6.5′-GCGCGAATTCTGGCAGAGNATG-3′
             T7.3′-CGNTACCGNTTCTACAGCCTAAGG-5′“N”指所有四种核苷酸的混合物。
这些引物按如下配对:1与2,3与4,5与7,6与7,1与4,1与7及3与7。这样在5′末端以EcoRI位点和在3′一末端以BamHI位点延伸PCR片段。在1%琼脂糖凝胶上分析PCR反应。在所有反应中发现了预期大小的带。为了证实这些带相当于过氧化物酶特异序列,将凝肢进行Southern印迹,并与具有以下序列的寡核苷酸探针杂交:
  T  A  A  A  T5′-GTCTCGATGTAGAACTG-3′
    T该序列位于PCR引物3和4之间。发现该探针与约130bp、420bp、540bp、和240bp的带杂交,因此说明所观察到的DNA带相应于过氧化物酶序列。
来自各种PCR反应的DNA用EcoRI和BamHI消化并克隆到质粒pUC19中(C.Yanisch-Perron等,Gene33,1985,pp.103-119)。通过用上述寡核苷酸探针杂交鉴定有正确PCR片段的菌落。按Sanger等(Proc.Natl.Acad.Sci.USA74,1977,pp.5463-5467)所述的限制酶作图和部分DNA序列分析分析阳性菌落的DNA。来自用引物1和4获得的克隆之一的430bp片段用于按如下所述筛选灰盖鬼伞cDNA文库。
在大肠杆菌中构建灰盖鬼伞cDNA文库
按如Boel等(EMBO J.3:1097-1102,1984)和Chirgwin等(Biochemistry(wash),18:5294-5299,1979)所述方法在最高过氧化物酶活性时收集菌丝体,从匀浆化的灰盖鬼伞(IFO8371)菌丝体提取总RNA。如Aviv和Leder(PNAS,USA69:1408-1412,1972)所述在Oligo(dT)-纤维素上进行两轮亲和层析获得含Poly(A)的RNA。用购自Invitrogen的cDNA合成试剂盒按制造商的说明合成cDNA。将来自灰盖鬼伞cDNA文库的约50,000大肠杆菌重组子转移到Whatman540滤纸上。按Gergen等所述(NucleicAcids Res.7,2115-2135,1979)裂解及固定菌落。此滤纸在0.2×SSC,0.1%SDS中与32P—标记的430bp过氧化物酶特异探针杂交。在65℃进行滤纸杂交和洗涤,然后用增敏屏放射自显影24小时。放射自显影后,升高温度洗涤滤纸,然后再用增敏屏放射自显影24小时。按此方式鉴定了50个阳性克隆。按标准方法(Birnboim和DolyNucleic Acids Res.:7,1513-1523,1979)从杂交的菌落分离Miniprep质粒DNA,按Sanger二脱氧方法(Sanger等Proc.Natl.Acad.Sci.USA74,1977,PP.5463-5467)测定***的cDNA的DNA序列。通过用HindIII/XhoI酶切将过氧化物酶cDNA片段从载体上切下,并用琼脂糖凝胶电泳纯化,电洗脱并准备用于连接反应。将此cDNA连接到HindIII/XhoI酶切的pHD414中生成pCiP,其中cDNA处在米曲霉TAKA启动子和黑曲霉AMG终止子的转录调控之下。
曲霉表达载体pHD414的构建
载休pHD414是质粒p775(在EP238023中有述)的衍生体。与p775不同,pHD414中在启动子和终止子之间有一串单一的限制性位点。
通过在终止子3′末端除去约200bp长的片段(含有不希望的限制位点),然后在启动子5′末端除去约250bp长的片段(也含有不希望的限制位点)来制备该质粒。通过用NarI(于pUC载体中)和XbaI(正位于终止子的3′末端)酶切从p775除去200bp区域,然后用Klenow DNA聚合酶+dNTP补平所造成的末端,在凝胶上纯化该载体片段并再连接。按如上所述方法用此DNA转化大肠杆菌MC1061。挑选10个菌落(pHD413-1到pHD413-10)并用限制酶分析法进行分析。一个克隆在限制酶分析中显示所期望的带型,用于构建pHD414。
pHD413用StuI(于启动子的5′末端)和PvvII(于pUC载体中)酶切,并在凝胶上分离。纯化该载体片段,再连接并转化至大肠杆菌MC1061中。挑选12个菌落,按限制性酶分析法进行分析。所有12个克隆均显示所希望的带型。质粒pHD414示于图1中。
2.三步PCR诱变:
三步PCR诱变中使用四种引物:
诱变引物            (=A):5′CCT GTT CGA TCG ATT CTT AGA-3′pCR辅助引物1  (=B):5′-TGA TCA TAG TAC CAT CTA ATT ACA TCA AGC
                  GGC-3′pCR辅助引物2  (=C):5′-CTG TAA TAC GAC TCA CTA-3′pCR Handle  (=D):5′TGA TCA GAC TAG TAC CAT-3′
将引物A和B稀释至20pmol/μl。引物C和D稀释至100pmol/μl。
所有三步在10×PCR缓冲液中进行:100mM Tris-HCl,pH8.3,500mM KCl,15mM MgCl2,0.1%明胶,2mM dATP、2mMdCTP、2mM dGTP、2mM TTP各200μl,和200μl水。
第1步,反应混合物组成为:10μl 10×PCR缓冲液,50μl 2×核苷酸溶液,5μl引物A,5μl引物B,1μl pCiP(0.05μg/μl),30μl水,0.5μl Taq聚合酶,和80μl石蜡,反应在94℃2分钟进行1个循环,94℃1分钟、50℃1分钟和72℃2分钟15个循环,94℃1分钟、50℃1分钟、72℃3分钟15个循环,及72℃5分钟1个循环。
在琼脂糖凝胶上纯化10μl PCR产物,重溶于10μl水中。然后进行第2步。反应混合物组成为:10μl 10×PCR缓冲液,50μl 2X核苷酸溶液,5μl第1步纯化产物,1μl pCiP(0.05μg/μl),30μl水,0.5μlTaq聚合酶,和80μl石蜡,反应在94℃5分钟、50℃2分钟和72℃10分钟进行一个循环。
向第2步反应混合物中加入1μl引物C和1μl引物D。按上述第1步进行PCR反应。
3.分离突变的限制性片段
用琼脂糖凝胶分离第3步的产物,重新溶于20μl水中。在补有牛血清白蛋白(BSA)的NEBuffer2(New England Biolabs)中用限制性酶XbaI和KpnI以总体积20μl 37℃酶切过夜。用琼脂糖凝胶分离570bp XbaI/KpnI片段。4.与表达载体pCiP连接
表达质粒pCiP在上文所示条件下用XbaI和KpnI酶切,用琼脂糖凝胶分离该大片段。将以上分离的突变片段连接到该载体上,连接混合物用于转化大肠杆菌。通过用限制酶酶切来自一转化子的质粒制备物证实了所述片段的存在和方向。用Sanger的二脱氧链终止方法在此双链质粒上进行序列分析。除改变的密码子外,生成的质粒与pCiP相同。
实施例2构建表达其他鬼伞过氧化物酶变体的质粒
用实施例1中所述相同方法构建了下列突变,只是使用别的消化PCR产物的限制酶和用于再克隆突变片段的载体。用于这种改造的突变和引物如下:
突变           引物A序列
K48S           5′-CCT GTT CGA TCG ATT CTT AGA-3′
V53K           5′-CTT AGA ATT AAA TTC CAT GAC-3′
G72Q           5′-GAT GGA GCC ATC GGC GCC TCC TTG ACC GAA TTG ACC-3′
A91C           5′-GCC TTC CCG TGC AAT GGC GGC-3′
N92K           5′-TTC CCG GCT AAA GGA GGC CTC-3′
H109K          5′-GGT ATT AAT AAA GGT GTC TCT-3′
Q118E          5′-GAT CTC ATC GAA TTC GCC ACT-3′
M125G          5′-GCC GTC GGC GGG TCC AAC TGC-3′
M125A          5′-GCC GTC GGC GCC TCC AAC TGC-3′
S147Q          5′-ACC GGG GAT CAA GCT TGG AGG TTG GGG TTG GGA ACT-3′
I152C          5′-CCT TCG TTG TGT CCC GGG CCC-3′
P155C          5′-G ATC CCC GGG TGC GGA AAC ACT-3′
M166G          5′-TTG GAT CGT GGG GGC GAT GCA-3′
N192K          5′-GAG GGT TTA AAA TCG GCC ATC-3′
I195K          5′-G AAC TCG GCC AAA TTC AGG TCT-3′
V206R          5′-CTG GGT ATC GAA GCG CTG AGG GGT CGA-3′
K218R          5′-CTG AGT GGT GCC TCG GAG CAA GGT CTC-3′
E214L          5′-TCT ACA TTT TAA CCT TGC TC-3′
F229G          5′-GAG CTC CTC GGC GCC GCC GAG AGA AGG-3′
A230C          5′-CTC GGC TTT TGC GAG GAA CTC-3′
E239G          5′-TTC CCT GGC GGC TTC CGC ATG-3′
E239H          5′-TTC CCT GGC CAC TTC CGC ATG-3′
E239K          5′-TTC CCT GGC AAA TTC CGC ATG-3′
E239L          5′-TTC CCT GGC CTA TTC CGC ATG-3′
E239M          5′-TTC CCT GGC ATG TTC CGC ATG-3′
E239Q          5′-TTC CCT GGC CAA TTC CGC ATG-3′
E239S          5′-TTC CCT GGC TCA TTC CGC ATG-3′
E239T          5′-TTC CCT GGC ACA TTC CGC ATG-3′
E239W          5′-TTC CCT GGC TGG TTC CGC ATG-3′
E239R            5′-TTC CCT GGC CGA TTC CGC ATG-3′
M242G            5′-GAA TCC CGC GGG AGG TCC GAT-3′
S244C            5′-T CGC ATG AGG TGC GAT GCT CTC-3′
S252P            5′-CG GCA GGC GGT CCG CGG GTC GCG AGC-3′
W258F            5′-GCA TGC CGA TTT CAA TCC AT-3′
W258H            5′-GCA TGC CGA CAT CAA TCC AT-3′
M261F            5′-TGG CAA TCC TTT ACT AGT AGC-3′
M261G            5′-TGG CAA TCC GGG ACC AGC AGC-3′
M261Y            5′-TGG CAA TCC TAT ACC AGC AGC-3′
M261V            5′-TGG CAA TCC GTC ACC AGC AGC-3′
M268L            5′-AAT GAA GTC CTA GGC CAG CGA-3′
M268G            5′-AAT GAA GTT GGG GGC CAG CGA-3′
M268A            5′-AAT GAA GTT GCA GGC CAG CGA-3′
M268F            5′-AAT GAA GTT TTT GGC CAG CGA-3′
Y272F            5′-GGG CCA GCG CTT TCG CGC CGC C-3′
Y272G            5′-GGC CAG CGA GGG CGC GCC GCC-3′
Y272A            5′-GGC CAG CGA GCC CGC GCC GCC-3′
M276S            5′-CGC GCC GCC TTT GCC AAG ATG-3′
M276I            5′-CGC GCC GCC ATA GCC AAG ATG-3′
M276H            5′-CGC GCC GCC CAC GCC AAG ATG-3′
K278R            5′-AG AAC AGA CAT GCG CGC CAT GGC GGC-3′
M279G            5′-ATG GCC AAG GGG TCT GTT CTC-3′
M279A            5′-ATG GCC AAG GCC TCT GTT CTC-3′
A304E            5′-AT AAC AGG CGC CTC GTT GGA CAC-3′
V314P            5′-GGC CTT ACT CCC GAT GAT ATC-3′
K41R+K48R       5′-AAT TCT AAG AAT TCG GCG AAC AGG GCT CTC
ACA TCG GGA CCC TTG-3′
G167N+V176L     5′-AAG CAA GTC AAC GAG CTC ATC AGG GCT GAA
GCC TGC ATC GTT CAT ACG ATC CAA-3′
R241E+E239K     5′-TTC CCT GGC AAG TTC GAA ATG AGG TCC-3′。
应注意1-29位的变体是用BamHI和XbaI在补有BSA的NEBuffer3(New England Biolabs)中酶切,生成160bp的片段。30-219位变体是用Xba/KpnI在补有BSA的NEBuffer2中酶切,生成570bp的片段。220-277位变体是用KpnI/MscI在补有BSA的NEBuffer2中酶切,生成170bp的片段。278-363位变体是用MscI/XhoI在补有BSA的NEBuffer2中酶切,生成420bp的片段。
实施例3
在米曲霉中表达鬼伞过氢化物酶变体
转化米曲霉或黑曲霉(一般方法)
在100ml YPD培养基(Sherman等Methods in Yeast Genet-ics,Cold Spring Harbor Laboratory,1981)中接种米曲霉或黑曲霉的孢子,37℃摇瓶培养过夜。通过无纺布(mira-cloth)过滤收集菌丝体,用200ml 0.6M MgSO4冲洗。将菌丝体悬浮在15ml 1.2MMgSO4,10m M NaH2PO4,pH5.8中。在冰上冷却悬液,加入1ml含120mg Novozym_234(批号1687)的缓冲液。5分钟后加入1ml 12mg/ml BSA(Sigrna type H25),轻轻搅动下37℃继续孵育1.5-2.5小时,直到在显微镜下检查在样品中可见大量的原生质体。
用无纺布过滤悬液,将滤液转移到无菌管中,有5ml 0.5M山梨醇,100mM Tris-HCl,pH=7.0覆盖。以100×g离心15分钟,在MgSO4层顶部收集原生质体。向原生质体悬液中加入2体积STC(1.2M山梨醇、10mM Tris-HCl,pH=7.5,10mM CaCl2),在1000×g离心该混合物5分钟。将原生质体层重悬于3ml STC中再次离心。重复这一操作。最后将原生质体重悬于0.2-1ml STC中。
将100μl原生质体悬液与10μl STC中的5-25μg适当DNA混合。将来自米曲霉菌株A1560(IFO4177)的原生质体与pToC186(一种带有构巢曲霉amds基因的质粒)混合。将混合物在室温下放置25分钟。加入0.2ml 60%PEG 4000(BDH29576),10mM CaCl2和10mM Tris-HCl,pH=7.5,小心混合(两次),最后加入0.85ml相同溶液并小心混合。混合物在室温下放置25分钟,于2500xg离心15分钟,沉淀重悬于2ml 1.2M山梨醇中。再次沉降后,将原生质体铺在适当的培养板上。将来自pToC186转化的A1560菌株的原生质体辅在含1.0M蔗糖(pH=7.0)。10mM乙酰胺作为氮源和用于抑制背景生长的20mM CsCl的基本培养板上(Cove Biochem.Bio-phys.Acta113(1966)51-56)。37℃孵育4-7天后挑出孢子,悬浮在无菌水中并铺板以形成单个菌落。重复这一步骤,第二次分离后将单一菌落的孢子作为所述转化子储存。
在米曲霉菌株中产生重组灰盖鬼伞过氧化物酶变体。
将在过氧化物酶基因中合适当突变的质粒通过与含构巢曲霉amds基因的pToC186共转化而转化至米曲霉A1560中,如上所述用等量pCiP和pToC186(各约5μg)的混合物进行转化。再分离两次能够利用乙酰胺作为其唯一氮源的转化子。在YPD培养基上(Sherman等,1981)生长3天后,分析培养物上清液的过氧化物酶活性(PODU)。
实施例4
过氧化氢稳定性
在H2O2存在下测试野生型重组灰盖鬼伞过氧化物酶(rCiP)和如上所述方法制备的E239K、Y272F、M276S、M242I+Y272F+E239K变体的稳定性。
按如下方法纯化待测变体样品:
通过Propex布吸滤使5升培养液澄清。轻轻搅拌下向3.5升培养液中加入1378g硫酸铵(终浓度为2.5M)。于5℃静置16小时。于2500xg 5℃离心5分钟收集沉淀。将沉淀再溶于CaCl2溶液(5mM)中,终体积为200ml。将30ml该溶液对5升12.5mM Bis-tris pH6.0缓冲液透析4小时,再对5升透析16小时。终体积为68ml。
将23ml透析液上样于Q-Sepharose HP柱(Pharmacia,26mmφ,100mm床高)上。.用12.5mM Bis-tris pH6.0缓冲液平衡柱。400ml起始缓冲液通过该柱,然后进行0.25M NaCl在12.5mMBis-tris pH6.0缓冲液中的线性梯度洗脱。总梯度体积为540ml。每10ml一份收集。在0.16M NaCl时过氧化物酶在20ml中洗下。将4ml上样于一Sephadex G25 SF(16mmφ400mm)柱上,在0.1M磷酸钠pH7中以4ml/分流速装柱。每2ml一份进行收集。过氧化物酶被洗脱在6ml中。
过氧化物酶稳定性的测试条件:酶                 25nM过氧化氢          0.2mM温度               30℃pH                 10.5碳酸盐缓冲液       20mM
试剂:
22mM pH10.5碳酸盐缓冲液:将1.807g Na2CO3和0.416gNaHCO3溶于去离子水中达终体积1升。检查pH值。
H2O2:2.2mM
过氧化物酶溶液:从Sephedex G25 SF得到的合并流份用22mM pH10.5碳酸盐缓冲液稀释至OD404=0.0030。(这相当于27.5nM)。
操作:
将碳酸盐缓冲液预热至30℃。制备过氧化物酶溶液。立即取过氧化物酶溶液样品,稀释并进行ABTS分析。将5ml过氧化物酶溶液与0.5nml 2.2mM H2O2混合,将混合物置于30℃水浴中。4、8、12和16分钟后,取样、稀释并进行ABTS分析。将残余活性与一级衰变匹配,并计算半衰期。
过氧化氢稳定性试验的结果列于下表1中。
表1
       变体   T%(分)    指数
rCiPE239KY272FM276SM242I+Y272F+E239K    6.58.38.77.314.8   1.001.271.341.132.27
序列表(1)一般信息:
(i)申请人:
  (A)名称:Novo Nordisk A/S,NN
  (B)街道:Navo Alle
  (C)城市:Bagsvaerd
  (E)国家:丹麦
  (F)邮政编码:(ZIP):DK-2880
  (G)电话:+4544448888
  (H)传真:+4544490555
  (I)电传:37173
(ii)发明名称:过氧化物酶变体2
(iii)序列数:1
(iv)计算机可读形式:
  (A)媒体类型:软盘
  (B)计算机:IBM PC兼容机
  (C)操作***:PC-DOS/MS-DOS
  (D)软件:PatentIn Release#1.0,Version#1.25(EPO)
(v)本申请资料:
    申请号:(2)SEQ ID NO:1的信息:
(i)序列特征:
  (A)长度:343个氨基酸
  (B)类型:氨基酸
  (C)链型:单链
  (D)拓扑结构:线性
(ii)分子类型:cDNA
(vi)原始原源:
  (A)生物体:灰盖鬼伞
  (B)菌株:IFO8371
(xi)序列描述:SEQ ID NO:l:Gln Gly Pro Gly Gly Gly Gly Ser Val Thr Cys Pro Gly Gly Gln Ser1               5                   10                  15Thr Ser Asn Ser Gln Cys Cys Val Trp Phe Asp Val Leu Asp Asp Leu
        20                  25                  30Gln Thr Asn Phe Tyr Gln Gly Ser Lys Cys Glu Ser Pro Val Arg Lys
    35                  40                  45Ile Leu Arg Ile Val Phe His Asp Ala Ile Gly Phe Ser Pro Ala Leu
50                  55                  60Thr Ala Ala Gly Gln Phe Gly Gly Gly Gly Ala Asp Gly Ser Ile Ile65                  70                  75                  80Ala His Ser Asn Ile Glu Leu Ala Phe Pro Ala Asn Gly Gly Leu Thr
            85                  90                  95Asp Thr Val Glu Ala Leu Arg Ala Val Gly Ile Asn His Gly Val Ser
        100                 105                 110Phe Gly Asp Leu Ile Gln Phe Ala Thr Ala Val Gly Met Ser Asn Cys
    115                 120                 125Pro Gly Ser Pro Arg Leu Glu Phe Leu Thr Gly Arg Ser Asn Ser Ser
130                 135                 140Gln Pro Ser Pro Pro Ser Leu Ile Pro Gly Pro Gly Asn Thr Val Thr145                 150                 155                 160Ala Ile Leu Asp Arg Met Gly Asp Ala Gly Phe Ser Pro Asp Glu ValVal Asp Leu Leu Ala Ala His Ser Leu Ala Ser Gln Glu Gly Leu Asn
        180                 185                 190Ser Ala Ile Phe Arg Ser Pro Leu Asp Ser Thr Pro Gln Val Phe Asp
    195                 200                 205Thr Gln Phe Tyr Ile Glu Thr Leu Leu Lys Gly Thr Thr Gln Pro Gly
210                 215                 220Pro Ser Leu Gly phe Ala Glu Glu Leu Ser Pro Phe Pro Gly Glu Phe225                 230                 235                 240Arg Met Arg Ser Asp Ala Ieu Leu Ala Arg Asp Ser Arg Thr Ala Cys
            245                 250                 255Arg Trp Gln Ser Met Thr Ser Ser Asn Glu Val Met Gly Gln Arg Tyr
        260                 265                 270Arg Ala Ala Met Ala Lys Met Ser Val Leu Gly Phe Asp Arg Asn Ala
    275                 280                 285Leu Thr Asp Cys Ser Asp Val Ile Pro Ser Ala Val Ser Asn Asn Ala
290                 295                 300Ala Pro Val Ile Pro Gly Gly Leu Thr Val Asp Asp Ile Glu Val Ser305                 310                 315                 320Cys Pro Ser Glu Pro Phe Pro Glu Ile Ala Thr Ala Ser Gly Pro Leu
            325                 330                 335Pro Ser Leu Ala Pro Ala Pro
        340

Claims (12)

1.一种在碱性条件下提高了对过氧化氢稳定性的过氧化物酶变体,其特征在于在SEQ ID No:1中所示氨基酸序列编码的灰盖鬼伞过氧化物酶这一亲本过氧化物酶的48-56,76,109,214,239,258-262,264,266-272氨基酸残基区中***、缺失或取代一个或多个氨基酸残基。
2.根据权利要求1的过氧化物酶变体,其中按如下取代一个或多个氨基酸残基:
K48S,
V53K,A
H109K,
E214L,
E239A,V,L,I,P,F,W,M,G,S,T,C,Y,N,Q,D,K,R,H,
特别是             E239K,G,S,L,Q,M,
W258F,H,
M261A,V,L,I,P,F,W,G,S,T,C,Y,N,Q,D,E,K,R,H,
特别是             M261F,V,I,L,Q,
M268A,V,L,I,P,F,W,G,S,T,C,Y,N,Q,D,E,K,R,H,
特别是             M268F,V,I,L,Q,
Y272A,V,L,I,P,F,W,G,S,T,C,M,N,Q,D,E,K,R,H,
特别是             Y272F。
3.一种在碱性条件下提高了对过氧化氢稳定性的过氧化物酶变体,其中SEQ ID No:1中所示氨基酸序列编码的灰盖鬼伞过氧化物酶这一亲本过氧化作酶的一个或多个氨基酸残基被取代如下:
G72Q,
A91C,
N92K,
Q118E,
M125A,P,W,G,S,T,C,Y,N,D,E,K,R,H,
特别是             M125S,T,
S147Q,
I152C,
P155C,
M166A,P,W,G,S,T,C,Y,N,D,E,K,R,H,
特别是             M166S,T,
N192K,
I195K,
V206R,
K218R,
F229G,
A230C,
M242A,P,W,G,S,T,C,Y,N,D,E,K,R,H,
特别是             M242S,T,
S244C,
S252P,
M276A,P,W,G,S,T,C,Y,N,D,E,K,R,H,
特别是             M276S,T,
K278R,
M279A,P,W,G,S,T,C,Y,N,D,E,K,R,H,
特别是             M279S,T,
A304E,
V314P,
K41R+K48R,
V53K+Q118E,
V53A+E239G,
I152C+A91C,
P155C+A230C,
M166F+E239K,
G167N+V176L,
E214L+E239L,
R241E+E239K,
S244C+P155C,
E239K+M242I+Y272F,
M166F+E239K+M242I+Y272F,
M125L+M166F+E239K+M242I+Y272F。
4.一种过氧化物酶变体,它是权利要求1-3的任一项过氧化物酶变体的片段。
5.一种漂白组合物,其含有权利要求1-4任一项的过氧化酶变体和过氧化氢或过氧化氢前体,如过硼酸盐或过碳酸盐,或产生过氧化氢的酶***,如氧化酶或和其底物,或过氧羧酸或其盐。
6.根据权利要求5的漂白组合物,其中过氧化物酶变体的量相当于洗涤液中浓度为0.01到20PODU/ml,过氧化氢或过氧化氢前体或产生过氧化氢的酶***或过氧羧酸或其盐的量相当于洗涤液中过氧化氢的浓度最高为20mM H2O2
7.根据权利要求5或6的漂白组合物,其另外含有可氧化底物如有机化合物,如酚类化合物或吩噻嗪的衍生物或吩噁嗪的衍生物。
8.根据权利要求7的漂白组合物,其中可氧化底物的量相当于洗涤液中浓度为0.1μM到100μM。
9.一种洗涤剂组合物,其包含表面活性剂、权利要求1-4中任一项的过氧化物酶变体和过氧化氢或过氧化氢前体,如过硼酸盐或过碳酸盐,或产生过氧化氢的酶***,如氧化酶和其底物,或过氧羧酸或其盐。
10.根据权利要求9的洗涤剂组合物,其中过氧化物酶变体的量相当于洗涤液中浓度为0.01-20PODU/ml过氧化氢或过氧化氢前体或产生过氧化氢的酶***或过氧羧酸或其盐的量相当于洗涤液中过氧化氢的浓度为最高20mM H2O2
11.根据权利要求9或10的洗涤剂组合物,其另外含有可氧化底物如有机化合物,如酚类化合物,或吩噻嗪的衍生物或吩噁嗪的衍生物。
12.根据权利要求11的洗涤剂组合物,其中可氧化底物的量相当于洗涤液中浓度为0.1μM到100μM。
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