CN1157008A - 纯化的毁丝霉属漆酶及编码该酶的核酸 - Google Patents

纯化的毁丝霉属漆酶及编码该酶的核酸 Download PDF

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CN1157008A
CN1157008A CN95194119A CN95194119A CN1157008A CN 1157008 A CN1157008 A CN 1157008A CN 95194119 A CN95194119 A CN 95194119A CN 95194119 A CN95194119 A CN 95194119A CN 1157008 A CN1157008 A CN 1157008A
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R·M·伯卡
S·H·布朗
徐丰
P·施内德
D·A·奥斯林
K·M·奥克森博尔
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Abstract

本发明涉及已分离的包含编码毁丝霉菌漆酶序列的核酸构建体，及其编码的漆酶蛋白。

Description

纯化的毁丝霉属漆酶及编码该酶的核酸
发明领域
本发明涉及分离出的编码真菌氧化还原酶的核酸片段及其产生的纯化酶。更具体地说，本发明涉及编码嗜热子囊菌纲毁丝霉属(Myceliophthora)酚氧化酶，特别是漆酶的核酸片段。
发明背景
漆酶(苯二酚：氧的氧化还原酶)是含多个铜的酶，其催化酚醛塑料的氧化反应。漆酶介导的氧化反应导致从适宜的酚类底物中产生芳氧基基团中间体；所产生的中间体的最后偶联提供了二聚、寡聚、和多聚反应产物的结合。事实上这样的反应在生物合成途径中很重要。生物合成途径可形成黑素、生物碱、毒素、木素、和腐殖酸。漆酶由多种真菌产生，包括子囊菌细如曲霉属(Aspergillus)、脉孢菌属(Neurospora)、和柄孢壳属(Podospora)、半知菌纲葡萄孢属(Botrytis)、和担子菌纲如金钱菌属(Collybia)、层孔菌属(Fomes)、香菇属(Lentinus)、侧耳属(Pleurotus)、栓菌属(Trametes)、以及丝核菌属(Rhizoctonia)的完全形式。漆酶表现出很广的底物特异性，并且通常每种不同的真菌漆酶与其它真菌漆酶相比氧化酚类底物的能力仅有量上的差别。由于底物不同，已经发现通常漆酶具有许多潜在的工业用途。其中有木素改性、纸张强度的增加、洗涤剂中染料转移的抑制、酚聚合作用、饮料的生产、酚树脂的生产、以及废水处理。
尽管催化能力类似，由不同种类的真菌生产的漆酶确实具有不同的最适温度和pH，并且这些根据特异性底物也可不同。已经分离了许多真菌漆酶，并且克隆了一些酶的基团。例如：Choi等人(Mol.Plant-Microbe Interactions 5：119-128，1992)描述了编码栗枯萎病真菌(Cryphouectria parasitica)漆酶的基因的分子表征及其克隆。Kojima等人(J.Biol.Chem.265：15224-15230，1990；JP2-238885)描述了白色腐殖担子菌纲毛革盖菌(Coriolus hirsutus)漆酶的两种等位形式。Germann和Lerch(Experientia 41：801，1985；PNAS USA 83：8854-8858，1986)报道了粗糙脉孢菌(Neurospora crassa)漆酶基因的克隆和部分测序。Saloheimo等人(J.Gen.Microbiol 137：1537-1544，1985；WO 92/01046)公开了得自真菌射脉菌(Phlebia radiata)的漆酶基因的结构分析。
在异源真菌系统中表达漆酶基因通常产率很低(Kojima等人，见上文；Saloheimo等人，Bio/Technol.2：987-990，1991)。例如，射脉菌漆酶在Trichoderma recsei中的异源表达每升活性酶仅给出20mg(Saloheimo，1991，见上文)。尽管漆酶具有很大的商业潜力，但能够大量表达该酶对其商业应用是很重要的。目前还没有在商业用宿主如曲霉属中得以高水平表达的漆酶。因此需要能以商业用的量(即每升克或更多)生产的漆酶。本发明满足了这一需要。
发明概述
本发明涉及含有编码毁丝霉属漆酶的核酸序列的DNA构建体。本发明还涉及该核酸序列编码的分离的漆酶。优选漆酶是基本上纯的。“基本纯的”是指基本上(即≥90％)不含其它非漆酶蛋白的漆酶。
为了便于生产这种新的漆酶，本发明还提供了含所要求的核酸序列的载体和宿主细胞，该载体和宿主细胞适用于漆酶的重组生产。该序列可操作地连接于能在所选择的宿主细胞中指导漆酶蛋白表达的转录和转译信号上。优选的宿主细胞是真菌细胞，最优选曲霉属真菌细胞。本发明的的漆酶的重组生产是这样完成的：在有助于表达漆酶蛋白的条件下培养用本发明的构建体转化或转染的宿主细胞或其子代，并从培养物中回收漆酶蛋白。
本发明的漆酶适用于许多需要氧化酚醛塑料的工业流程中。这些流程包括木素改性、饮料的生产、酚聚合作用以及酚树脂的生产。
附图的简单说明
图1显示了pRaMB1中7.5 EcoRI片断的限制性酶切图谱。与粗糙脉孢菌的漆酶基因探针杂交的区域用阴影表示。
图2表明了嗜热毁丝霉菌(Myce liophthora thermophila)漆酶的核苷酸序列(SEQ ID NO：1)和氨基酸序列(SEQ ID NO：2)。核苷酸序列中的小写字母表示内含子的位置。启动子区域中推测的TATA和CAAT序列用黑体表示并在下面画线。内含子中保守套索结构(PuCTPUAT)下面画了线。
图3表明了质粒pRaMB5的构建。
发明的详细描述
嗜热毁丝霉菌是一种嗜热子囊菌，Apinis最先对其进行了描述(Nova Hedwigia 5 57-78，1963)并命名为嗜热侧孢霉菌(Sporotrichum thermophile)。随后的分类学修订本将这种生物列在Chrysosporium属(Von Klopotek，A.Arch.Microbiol 98：365-369，1974)，随后列在毁丝霉属(Van Oorschot，Persoonia 9：401-408，1977)。许多以其它名称命名的微生物似乎也属于这一种类。这些包括侧孢霉属cellulophilum(美国专利号4,106,989)；嗜热梭孢壳菌(Thielavia thermophila)(Fergus and Sinden，Can.J.Botany47：1635-1637，1968)；Chrysosporium fergussi和Corynascusthermophilus(Von Klopotek，见上文)。这一种类被认为是许多不同的工业用酶，如纤维素酶，β-糖苷酶和木聚糖酶的来源(参见，例如Oberson等人，Enzyme Microb.Technol.14：303-312，1992；Merchant等人，Biotechnol.Lett.10：513-516，1988；Breuil等人，Biotechnol.Lett.8：673-676，1986；Gilbert等人，Bioresource Technol.39：147-154，1992)。现在已经确定毁丝霉属产生pH中性的漆酶，并且编码这种漆酶的基因可用于在便利的宿主系统如曲霉属中大量生成该酶。
为了鉴定毁丝霉属中漆酶基因的存在，在不同真菌种类的全部基因组DNA Southern杂交中，在适度严格的条件下，用粗糙脉孢菌漆酶基因(lccl)的5’部分作为探针。在毁丝霉属DNA中检测约12Kb的漆酶特异性序列。然后用粗糙脉孢菌片段在λEMBL4噬菌体克隆载体中筛选嗜热毁丝霉菌(M.thermophila)基因组DNA文库的约20,000个噬斑。八个噬斑与探针稳固杂交；从这八个的三个中分离出DNA。这些克隆中的每一个均含有7.5 EcoRI片段，其也可以与探针杂交(图1)。将片段之一亚克隆到pBR 322中从而产生质粒p RaMB1。用lccl探针确定克隆编码区域的位置。整个嗜热毁丝霉菌编码区域似乎是包含在3.2KbNheI-BglII节段中，然后将该节段克隆到pUC119中并用引物步移(primer walking)法测序。
一旦确定了序列，便将基因中内含子和外显子的位置根据推断的氨基酸序列的排布分配给相应的粗糙脉孢菌漆酶基因产物。从这一比较可以看出嗜热毁丝霉菌的基因(lccM)含有七个外显子(246、79、12、70、973、69和411核苷酸)，其间夹杂六个内含子(85、84、102、72、147和93核苷酸)。编码区，不包括间插序列，富含GC碱基(65.5％G+C)，并且编码含620个氨基酸的前酶原含22个氨基酸的信号肽、含25个氨基酸的前肽，以及含573个氨基酸的成熟漆酶。嗜热毁丝霉菌基因序列及预测的氨基酸序列示于图2(SEQ ID NOS：1和2)。
然后用漆酶基因产生表达载体以转化曲霉属宿主细胞。载体pRaMB5含有米曲霉(A.oryzae)TAKA淀粉酶启动子和终止子区域。pRaMB5的构建示于图3。用表达载体和含pyrG或amdS选择性标记物的质柱共转化曲霉属细胞。在含有ABTS的适当选择性培养基上选择转化体。产生漆酶的菌落呈现出绿环并可被分离出来。选出的转化体在摇瓶中生长并用丁香醛连氮法测定培养液的漆酶活力。摇瓶培养物可产生0.2g/升或更多的漆酶，并且发现发酵罐中的产率超过1-2g/升。
根据本发明，可以用这里提供的信息按照上述方法或本领域中已知的任何其它方法获得编码漆酶的毁丝霉属基因。可用表达载体以活性形式表达基因。适用的表达载体含有使载体稳定地整合到宿主细胞基因组中或独立于宿主细胞基因组使载体在宿主细胞中自主复制的因子，并且优选含有一种或多种能容易选择转化的宿主细胞的表型标记物。表达载体还可包括编码启动子的控制序列，核糖体结合位点、翻译起始信号，以及，可选择地，阻遏物基因或各种激活物基因。为了使表达的蛋白分泌出来，可以在基因编码序列前插入编码信号序列的核苷酸。为了在控制序列指导下进行表达，可将所用的根据本发明的漆酶基因与适当的阅读框架中的控制序列可操作地连接。可以掺入质粒载体并且可以指导漆酶基因转录的启动子序列包括，但不限制于原核生物β-内酰胺酶启动子(Villa-Kamaroff等人，1978，Proc.Nati.Acad.Sci.U.S.A75：3727-3731)以及tac启动子(DeBoer等人，1983，Proc.Natl.Acad.Sci.U.S.A 80：21-25)。进一步的参考文献还可见于Scienfific American，1980，242：74-94中的“Useful proteins from recornbinant bacteria”，以及Sambrook等人的Molecuar Cloning，1989。
携带本发明DNA构建体的表达载体可以是任何能便利地用于重组DNA程序的载体，并且载体的选择通常是基于其所掺入的宿主细胞。因此，载体可以是自主复制的载体，即以染色体外实体存在的载体，其复制不依赖于染色体的复制，如质粒，或者染色体外因子、微型染色体或人工染色体。另外，可以是这样的载体，当其掺入宿主细胞时可整合到宿主细胞基因组中并与其所整合的染色体一起复制。
在载体中，漆酶DNA序列应与适宜的启动子序列可操作地相连。启动子可以是任何在所选择的宿主细胞中表现出转录活性的DNA序列，并且可从编码与宿主细胞同源或异源的蛋白的基因中衍生而来。指导本发明DNA构建体转录(特别是在细菌宿主中)的适宜启动子的例子是大肠杆菌lac操纵子的启动子、天蓝色莲霉菌(Streptomycescoelicolor)琼脂糖酶基因dagA启动子、地衣芽孢杆菌(Bacilluslicheniformis)α-淀粉酶基因(amyL)启动子、嗜热脂肪芽孢杆菌(Bacillus Stearothermophilus)生麦芽糖淀粉酶基因(amyM)启动子、解淀粉芽孢杆菌(Bacillus amyloliquefaciens)α-淀粉酶(amyQ)启动子、或者枯草芽孢杆菌(Bacillus subtilis)xylA和xylB基因启动子。在酵母宿主中，适用的启动子是eno-1启动子。为了在真菌宿主中转录，适用的启动子的例子是那些从编码下列物质的基因中衍生出来的启动子：米曲霉TAKA淀粉酶、Rhizomucor miehei天冬氨酸蛋白酶、黑曲霉(A.niger)中性α-淀粉酶、黑曲霉酸稳定性α-淀粉酶、黑曲霉或泡盛曲霉(A.awamori)葡糖淀粉酶(glaA)、Rhizomucormiehei脂肪酶、米曲霉碱性蛋白酶、米曲霉丙糖磷酸异构酶或构巢曲霉(A.nidulans)乙酰胺酶。优选TAKA-淀粉酶和glaA启动子。
本发明的表达载体还可含有适宜的转录终止子，并且在真核细胞中聚腺苷酸化序列可操作地连接于编码本发明的漆酶的DNA序列上。终止序列和聚腺苷酸化序列可以适宜地从与启动子相同的来源中衍生。载体可以进一步含有使载体在所研究的宿主细胞中进行复制的DNA序列。这样的序列的例子是质粒pUC19、pACYC177、pUB110、pE194、pAMB1和pIJ702的复制起点。
载体还可含有选择性标记物，例如其产物补偿宿主细胞缺陷的基因如来自枯草芽孢杆菌或地衣芽孢杆菌的dal基因，或者提供抗生素抗性如氨苄青霉素、卡那霉素、氯霉素或四环素抗性的基因。曲霉属选择标记物的例子包括amdS、pyrG、argB、niaD、sC以及hygB，一种产生潮霉素抗性的标记物。优选用于曲霉属宿主细胞的是构巢曲霉或米曲霉的amdS和pyrG标记物。常用的哺乳动物标记物是二氢叶酸还原酶(DHFR)基因。另外，可以通过共转化作用完成选择，如WO91/17243所述。
通常优选表达能产生细胞外产物。因而本发明的漆酶可以含有使所表达的蛋白质分泌到培养基中的前区域。如果需要，该前区域可以是本发明的漆酶本来具有的或者是被不同的前区域或信号序列取代的，一般通过编码各个前区域的DNA序列的取代来完成。例如，前区域可以从下列基因中衍生：来自曲霉菌种的葡糖淀粉酶或淀粉酶基因、来自芽孢杆菌种的淀粉酶基因、来自Rhizomucor miehei的脂肪酶或蛋白酶基因、来自酿酒酵母的α-因子基因或者牛前凝乳酶原基因。当宿主是真菌细胞时，特别优选下列物质的前区域：米曲霉TAKA淀粉酶、黑曲霉中性啶粉酶、来自芽孢杆菌NCIB11837的生麦芽糖淀粉酶、嗜热脂肪芽孢杆菌α-淀粉酶、或地衣芽孢杆菌枯草杆菌蛋白酶。有效的信号序列是米曲霉TAKA淀粉酶信号、Rhizomucor miehei天门氨酸蛋白酶信号和Rhizomucor miehei脂肪酶信号。
分别用于连接本发明DNA构建体、启动子、终止子和其它因子、并且把它们插入适宜的含复制所需信息的载体中的步骤是本领域技术人员已知的(参见，例如，Sambrook等人，Molecular Cloning，1989)。
在本发明酶的重组生产中，含上述本发明DNA构建体或表达载体的本发明细胞利于用作宿主细胞。一般可以通过将DNA构建体整合到宿主染色体中采用本发明的DNA构建体转化细胞。通常认为该整合是有利的，因为DNA序列似乎更稳定地保留在细胞中。可以按照常用的方法进行DNA构建体到宿主染色体中的整合，例如，通过同源或异源重组。另外，可以用上述与各种类型宿主细胞相关的表达载体转化细胞。
宿主细胞可以选自原核细胞，如细菌细胞。适宜的细菌的例子是革兰氏阳性菌如枯草芽孢杆菌、地衣芽孢杆菌、迟缓芽孢杆菌(Bacilluslentus)、短芽孢杆菌(Bacillus brevis)、嗜热脂肪芽孢杆菌、嗜碱芽孢杆菌(Bacillus alkalophilus)、解淀粉芽孢杆菌、凝结芽孢杆菌(B.coagulans)、环状芽孢杆菌(B.circulans)、Bacillus lautus、巨大芽孢杆菌(B.megaterium)、苏云金芽孢杆菌(B.thuringiensis)、或变铅青链霉菌(Streptomyces lividans)或鼠灰链霉菌(Streptomycesmurinus)、或革兰氏阴性菌如大肠杆菌。可以用本来已知的方法例如通过原生质体转化或用感受态细胞来完成细菌的转化。
宿主细胞还可以是真核细胞，如哺乳动物细胞、昆虫细胞、植物细胞或者优选地是真菌细胞，包括酵母菌和丝状真菌。例如，有用的哺乳动物细胞包括CHO或COS细胞。酵母菌宿主细胞可以选自酵母属或裂殖酵母属(Schizosaccharomyccs)的菌种，例如酿酒酵母。有用的丝状真菌可以选自曲霉属的菌种，例如米曲霉或黑曲霉。另外，可以用镰孢菌种的菌株如尖镰孢(Fusarium、Oxysporum)作为宿主细胞。可用下列方法转化真菌细胞，该方法包括原生质体的形成和原生质体的转化，然后按原本已知的方法使细胞壁再生。EP238023描述了转化曲霉属宿主细胞的适宜步骤。Malardier等人于1989年描述了转化镰孢菌种的适宜方法。
因而本发明提供了一种生产本发明重组漆酶的方法，该方法包括在益于酶的产生的条件下培养如上所述的宿主细胞，并且从细胞和/或培养基中回收。用于培养细胞的培养基可以是任何常用的适于生长所研究的宿主细胞以及获得本发明漆酶的表达的培养基。可以从供货厂商获得适宜的培养基，或者可以按发表的配方(例如，在美国典型培养物保藏中心的目录中)制备。
在优选的实施方案中，在过量铜的存在下完成漆酶在培养物中的重组生产。尽管加入培养基中的痕量金属一般含有少量的铜。但进行的与本发明相关的实验表明向培养基中加入铜添加物可以使活性酶的产率提高数倍。优选以溶解形式向培养基中加入铜，优选以溶解性铜盐的形式，如氯化铜、硫酸铜、或醋酸铜。培养基中铜的最终浓度应在0.2-2mM范围内，并且优选在0.05-0.5mM的范围内。该方法可以用于增加任何重组生产的真菌漆酶、以及其它含酮酶、特别是氧化还原酶的产率。
可以用一般方法从培养基中回收所得酶，这些方法包括通过离心或过滤从培养基中分离细胞、用盐，如硫酸铵法沉淀上清液或滤液的蛋白质成分，然后用各种色谱方法，如离子交换色谱、凝胶过滤色谱、亲合色谱等等进行纯化。优选SDS-PAGE测定的分离蛋白的纯度约为90％，在食品、饮料或清洗剂应用中纯度非常重要。
在特别优选的实施方案中，在真菌宿主细胞如曲霉中进行漆酶的表达。如下列实施例中详细描述的，漆酶基因与含米曲霉TAKAα-淀粉酶启动子的质粒，以及构巢曲霉amds选择性标记物相连。另外，amdS可以在独立的质粒上并用于共转化作用。该质粒(或这些质粒)用于转化曲霉菌种宿主细胞，如米曲霉或黑曲霉，按照Yelton等人所述的方法(PNAS USA 81：1470-1474，1984)。
本领域技术人员会认识到本发明不限于使用这里具体公开的核酸片段，例如图1中的核酸片段。显而易见地，本发明包括了那些编码与图1中描述相同的氨基酸序列的核苷酸序列，但其不同于因为遗传密码简并而特别描述的核苷酸序列。并且，说明书和权利要求中所指的图1中应理解为包括那里所描述的基因组序列以及相应的cDNA和RNA序列，并且这里所用的术语“DNA构建体”和“核酸序列”应被理解为包括所有这些变型。“DNA构建体”通常应被理解为是指单链或双链DNA分子，其可以部分形式从天然存在的基因中分离出来或者其已被修饰成含有以并非天然存在的方式结合且并置的DNA节段的DNA构建体。
与其它已知的子囊菌纲或半知菌纲细胞外漆酶相比(对其中酶的比活性已有描述)，这里所述的毁丝霉属漆酶对丁香醛连氮底物具有特别高的比活性。本发明序列提供了也能对其它这样的子囊菌纲或半知菌纲漆酶进行分离的工具用公众可以得到的子囊菌纲和半知菌纲菌株，可以使用本发明实施例中所述的方法学来从这里具体例举之外的来源中鉴别与分离漆酶基因。具体地说，通过标准PCR或Southern杂交技术可将这里公开的具体序列用于设计对分离类似漆酶基因有用的引物和/或探针。因此本发明包括了子囊菌纲和半知菌纲漆酶，其比活性至少约为30SOU/mg，优选至少约40SOU/mg，“SOU”被定义为在最适pH下以丁香醛连氮作为底物所测定的每分钟氧化的μmole底物。
另外，本发明还包括其它毁丝霉属漆酶，包括可于嗜热毁丝霉菌中发现的漆酶以及可于其它符合Van Oorschot(1977年，见上文)所定义的毁丝霉属定义的真菌中发现的漆酶的其它形式。用公众可以得到的毁丝霉属菌株，可以使用本发明实施例中所述的方法学来从这里具体例举之外的来源中鉴别与分离漆酶基因。另外，通过标准PCR或Southern杂交技术可将这里公开的序列用于设计对分离漆酶基因有用的引物和/或探针。其它被命名为毁丝霉的菌种包括hinnulea毁丝霉(Awao等人，Mycotaxon.16：436-440，1983)，vellerea毁丝霉(Guarro等人，Mycotaxon.23：419-427，1985)，以及黄毁丝霉(M.lutea Costatin)。还包括漆酶类似物，如毁丝霉属菌种或菌株的变形或完全态。公众可在许多培养物保藏中心获得毁丝霉属的菌株，如ATCC48102、48103、48104等；CBS117.65、131.65、379.65等；DSM1799(嗜热毁丝霉菌)、ATCC52474、CBS539.82、540.82等(hinnulea毁丝霉菌)、DSM62114，CBS146.50、147.50、157.51等(黄毁丝霉菌)，以及CBS 478.76、479.76和715.84(vellerea毁丝霉菌)。本发明还包括任何变种核苷酸序列及其编码的蛋白，该蛋白保留了与图1中所述氨基酸序列至少约80％，优选至少85％，最优选至少90-95％的同源性，并且其定性地保留了这里所述序列的漆酶活性。上述种类中的有用的变种包括，例如，进行了保守性氨基酸置换的变种，该置换不明显影响蛋白质的活性。保守性置换是指同类氨基酸可被该类中的其它氨基酸置换。例如，非极性脂肪族残基Ala、Val、Leu和Ile可以互换，碱性残基Lys和Arg可以互换，或者酸性残基Asp和Glu可以互换。类似地，Ser和Thr彼此保守性置换，Asn和Gln彼此保守性置换。对本领域技术人员来说显而易见地，这种置换可以在对分子功能很重要的区域之外的位置进行，并且仍然可产生活性酶。通过进行标准ABTS氧化法可以测定目的活性的保留量，如本发明实施例所述。
该蛋白可用于许多种工业生产过程。这些过程包括木素、牛皮纸和木素硫酸盐在溶液中的聚合反应，以生产较高分子量的木素。中性/碱性漆酶特别有利之外在于牛皮纸木素在较高pH下溶解性更好。这样的方法描述于，例如，Jin等人，Holzforschung 45(6)：467-468，1991；美国专利号4,432,921；EP0275544；PCT/DK93/00217，1992。
本发明的漆酶还可用于牛皮纸纸浆中木素的就地解聚，从而生产低木素含量的纸浆。漆酶的这一用途比目前使用氯进行木素解聚有所改进，用氯解聚木素会引起氯化芳香族化合物的产生，其为对环境不利的造纸厂的副产物。这样的用途描述于，例如，Curreut opinion inBiotechnology 3：261-266，1992；J.Biotechnol.25：333-339，1992；Hiroi等人，Svensk papperstidning 5：162-166，1976。由于造纸厂的环境通常是碱性的，本发明的漆酶比其它已知的在酸性条件下作用最好的漆酶更适用于这一目的。
染料或染料前体和其它发色化合物的氧化导致了化合物的褪色。漆酶可用于这一用途，其可以特别适用于不希望染料在纤维织物间转移的情况，例如在纺织工业和洗涤剂工业中。染料转移抑制和染料氧化的方法可见于WO92/01406；WO92/18683；EP0495836；Calvo，Mededelingenvan de Faculteit Landbouw-wetens chappen/Rijiksuniversitet Gent.56：1565-1567，1991；Tsujino等人，J.Soc.Chem.42：273-282，1991。
漆酶尤其适用于头发的染色。在这一应用中，漆酶与染料前体接触，优选在头发上接触，从而控制染料前体的氧化使前体转化为染料、或者生成色素的化合物，如醌型化合物。优选染料前体属于下列三大化学家族之一的芳香族化合物：二胺、氨基苯酚(或氨基萘酚)以及苯酚。染料前体可被单独使用或结合使用。共聚反应中的中间体至少一个必须是邻位或对位二胺或氨基苯酚(初始中间体)。这样的例子见于下面的第四部分，并且包括对苯二胺(pPD)、对甲代苯二胺、氯对苯二胺、对氨基苯酚、邻氨基苯酚、3，4-二氨基甲基；美国专利号3,251,742还描述了其它化合物，其内容作为参考文献并入本发明。在一实施方案中，原料不仅包括酶和初始中间体，还包括改性剂(偶联剂)(或几种改性剂的混合)，改性剂通常为间二胺，间一氨基苯酚，或多酚。改性剂化合物的例子包括间苯二胺、2，4-二氨基苯甲醚、α-萘酚、氢醌、焦儿萘酚、间苯二酚，以及4-氯间苯二酚。然后改性剂在漆酶的存在下与初始中间体反应，将其转化成着色化合物。在另一实施方案中，漆酶可直接用于初始中间体，将其氧化成着色化合物。在所有情况下，可以用一种或多种初始中间体进行染色过程，其可单独使用或与一种或多种改性剂结合使用。各组分的量符合类似组分的一般工业用量，并且各组分的比例可以相应改变。
这种漆酶的用途比传统上使用的H2O2有所改进之处在于后者会损伤头发，并且其使用通常需要高pH，这也会损伤头发。相反，与漆酶的反应可以在碱性、中性或者甚至是酸性的条件下进行，氧化反应所需的氧来自空气而不是苛刻的化学氧化反应。使用毁丝霉菌漆酶所得结果可与使用H2O2所得结果相比，不仅在显色上，而且在洗涤稳定性和耐晒性方面。其它工业上的优点是可以在无氧气氛中制成既含漆酶也含前体的单容器包装，这一对使用H2O2来说是不可能的。
本发明的漆酶还可用于聚合存在于液体中的苯酚类化合物。该用途的一个例子是处理饮料如苹果汁，漆酶会加速饮料中存在的苯酚类化合物的沉淀，从而生产出更稳定的饮料。这一应用已描述于Stutz，Fruitprocessing 7/93.248-252，1993；Maier等人，Dt.Lebensmittelrindschau86(5)：137-142，1990；Dietrich等人，Fluss.Obst 57(2)：67-73，1990。
漆酶如毁丝霉属漆酶还可用于土壤的去毒处理(Nannipieri等人，J.Environ.Qual.20：510-517，1991；Dec和Bollag，Arch.Environ.Contam.Toxicol.19：543-550，1990)。
用下列非限制性实施例进一步说明本发明。
                      实施例I.嗜热毁丝霉菌漆酶基因的分离A.材料和方法
1.DNA的提取与杂交分析
将嗜热毁丝霉菌菌株E421于25ml YEG培养基(0.5％酵母萃，2％葡萄糖)中生长24小时，用下列方法从这些真菌细胞中提取总的细胞DNA；通过Miracloth(Calbiochem)过滤收集菌丝体并用25ml TE缓冲液洗涤一次。从菌丝体中排出多余的缓冲液，随后将菌丝体冷冻在液氮中。使冷冻的菌丝体在电子咖啡粉碎器中研成细粉，将粉末加入一次性塑料离心管内的20ml TE缓冲液和5ml 20％SDS(w/v)中。使混合液轻轻翻转数次确保混匀，用等体积苯酚∶氯仿∶异戊醇(25∶24∶1)提取两次。加入醋酸钠(3M溶液)至终浓度为0.3M，用2.5体积冰冷的乙醇沉淀核酸。将试管在15,000×g离心30分钟，将沉淀物空气中干燥30分钟后再悬浮于0.5ml TE缓冲液中。加入不含DNA酶的核糖核酸酶A至浓度为100μg/ml，并将混合物于37℃保温30分钟。加入蛋白酶K(200μg/ml)并将每个试管再于37℃保温1小时。最后，将每份样品用苯酚∶氯仿∶异戊醇提取两次，再用醋酸钠和乙醇沉淀DNA。将DNA沉淀真空干燥，再悬浮于TE缓冲液中，并于4℃保存。
用Southern杂交技术分析得自转化体和非转化对照菌株的总的细胞DNA样品。用EcoRI消化约5μgDNA，并在1％琼脂糖凝胶上按大小分级分离。在短波长UV下拍摄凝胶，并在0.5M NaOH、1.5M NaCl中浸渍15分钟，然后在1M Tris-HCl，pH8，1.5M NaCl中浸渍15分钟。在20X SSPE中通过毛细管印迹法将凝胶中的DNA转移到Zeta-ProbeTM杂交膜(BioRad Laboratories)上(R.W.Davis等人，Advanced Bacterial Genetics，A Manual for Genetic Engineering.ColdSpring Harbor Press.1980)。将膜在80℃真空烘烤2小时，于45℃在下列杂交缓冲液中浸泡2小时并轻轻搅拌：5X SSPE，35％甲酰胺(V/V)，0.3％SCS，200μg/ml变性并剪切的鲑睾丸DNA。以下列一对引物用标准PCR条件(Perkin-Elmer Cetus，Emeryviile，CA)从粗糙脉孢菌基因组DNA扩增编码粗糙脉孢菌lccl基因5’-部分的漆酶特异性探针片段(约1.5Kb)：正向引物，5’CGAGACTGATAACTGGCTTGG3’；反向引物，5’ACGGCGCATTGTCAGGGAAGT 3’。先将扩增的DNA节段克隆到TA-克隆载体(Invitrogen，Inc.，San Diego.CA)中，用EcoRI水解后再用琼脂糖凝胶电泳纯化。用α〔32P〕dCTP(Amersham)通过切口平移放射性标记纯化的探针片段，并以每ml缓冲液约1×106cpm的活性加入杂交缓冲液中。在振荡水浴中于45℃使混合液保温过夜。保温后，将膜在45℃用含0.1％SDS的0.2×SSPE洗涤一次，然后在同样温度于0.2×SSPE(不含SDS)中洗涤两次。使膜在纸上干燥15分钟，然后卷在Saran WrapTM中并用增感屏(Kodak)暴露于X-光片在-70℃过夜。
2.DAN文库及漆酶克隆的鉴别
在噬菌体克隆载体λ-EMBL4中构建基因组DNA文库(J.A.Sorge，in Vectors，A Survey of Molecular Cloning Vectors andTheir Uses，Rodrigulz等人，eds，43-63页，Butterworths，Boston，1988)。简单地说，总的细胞DNA用Sau3A部分消化，并在低熔点琼脂糖凝胶上按大小分级分离。将在9Kb和23Kb之间迁移的DNA片段切除并用B-琼脂糖酶(New England Biolabs，Beverly MA)从凝胶中洗脱出来，将洗脱的DNA片段与用BamHI切割的且脱磷酸化的λ-EMBL4载体臂连接，用市售包装提取液(Stratagene，LaJolla，CA)包装连接混合物。将包装的DNA文库铺在大肠杆菌K8.02细胞上并扩增。通过在上述条件下与放射性标记的lccl DNA片段的噬斑杂交从每个文库中筛选出约10,000-20,000个噬斑。将给出探针杂交信号的噬斑在大肠杆菌K802细胞上纯化2次，并用Qiagen Lambda试剂盒(Qiagen，Inc，Chatsworth，CA)从高滴度溶胞产物中纯化相应的噬菌体DNA。
3.漆酶基因的分析
用标准方法(Lewin，Genes，2d ed Wiley & Sons，1985，纽约)作出漆酶克隆的限制性酶切图谱。使用关于染料一终止子化学的引物步移技术(H.Giesecke等人，J.Virol.Methods 38：47-60，1992)用AppliedBiosystems Model 373A自动DNA测序仪(Applied Biosystems’Inc.，Foster City，加拿大)进行DNA测序。在Applied Biosystems model 394DNA/RNA合成仪上合成寡聚核苷酸测序引物。B.结果与讨论
1.漆酶基因序列的鉴别
从粗糙脉孢菌、灰葡萄孢(Botrytis cinerea)和毁丝霉菌种制备所有细胞DNA样品。用BamHI消化这些DNA制品的等分试样，并用琼脂糖凝胶电泳分级分离。如上所述，将凝胶中的DNA吸印到Zeta-ProbeTM膜滤器(BioRad Laboratories，Hercules，CA)上并在适度严格的条件下用编码粗糙脉孢菌lccl基因部分的放射性标记片段探测。在嗜热毁丝霉菌和粗糙脉孢菌对照品的基因组中检测出了漆酶特异性序列，但用该探针未在灰葡萄孢基因组DNA中检测出漆酶特异性序列。
2.嗜热毁丝霉菌漆酶(MtL)基因的克隆和表征：
从在λ-EMBL4克隆载体中构建的嗜热毁丝霉菌基因组DNA文库中筛选出约20,000个噬斑。该文库包含约10,000个带大小范围从9Kb至23Kb的插入片段的独立的克隆。假设平均插入片段大小为10Kb而嗜热毁丝霉菌总的基因组大小为4×107bp，这一数字是代表整个基因组所需克隆数目的约2.5倍。鉴别出8个与粗糙脉孢菌漆酶基因探针紧密杂交的噬斑。从其中的三个噬斑分离DNA，用EcoRI切割，并用琼脂糖凝胶电泳和Southern杂交技术分析。这三个克隆均含有与漆酶特异性探针杂交的7.5Kb EcoRI片段。将这些EcoRI片段之一亚克隆到pBR322(Bolivar等人，Gene2：95-113，1977)中以产生质粒pRaMB1。该DNA节段的限制性酶切图谱示于图1。通过与上述lccl基因片段杂交测定该克隆上漆酶编码区域的位置。根据获得的图谱数据以及估算的漆酶蛋白的大小约80Kdal，可以推断，整个嗜热毁丝霉菌漆酶编码区域包含有3.2Kb NheI-BglII节段，然后将该节段亚克隆到pUC119中(Viera andMessing，Methods Encymol.153：3-11，1987)。用引物步移法(Giesecke等人，见上文)测定该节段的核苷酸序列。核酸序列示于图2和SEQ IDNo：1。
根据与粗糙脉孢菌漆酶同源的氨基酸序列获得MtL的推断的氨基酸序列。在氨基酸水平上，这两种漆酶具有大约60％的序列一致性。在参与三核铜簇形成的四个组氨酸和一个半胱氨酸的相应区域中相似性最高(Perry等人，J.Gen.Microbiol.139：1209-1218，1993；Messerschmidt等人J.Mol.Biol.206：513-530，1989)。在MtL的推断的氨基酸序列中有11个潜在的N-连接糖基化位点。MtL的前22个氨基酸含有规范信号肽，其在Ala残基后面有一个预计的切割位点(vonHeijine，J.Mol.Biol.173：243-251，1984)。尽管不知道天然MtL的氨基末端序列，但用焦谷氨酸残基封闭了米曲霉中产生的重组MtL的氨基末端。酶解除去该残基，之后进行氨基酸测序，表明成熟MtL以Gln残基开始(图2中的位置1；SEQ ID No：2)。因此，合成的MtL显然是含有22个氨基酸的信号肽和25个残基的前肽的620个氨基酸前酶原。类似地加工粗糙脉孢菌漆酶(NcL)的氨基末端。另外，在NcL的C-末端进行蛋白水解，除去了13个氨基酸(Germann等人，J.Biol.Chem.263：885-896，1988)。加工位点包含在序列Asp-Ser-Gly-Len*Arg558中(其中*代表切割位点)。在MtL C-末端附近存在类似序列(Asp-Ser-Gly-Leu-Lys 560)，这表明可以对毁丝霉菌酶进行C-末端加工(Asp-Ser-Gly-Len*Lys560)而除去12个氨基酸。
通过比较推断的MtL氨基酸序列与推断的NcL氨基酸序列并且应用适合于丝状真菌内含子特征的保守原则，测定了lccl编码区域中的六个内含子的位置(85、84、102、72、147和93位的核苷酸)(Gurr等人，Gene Structure in Eukaryotic Microbes，J.R.Kinghorn，ed.pp93-139，IRL Press，Oxford，1987)。编码序列的1860个核苷酸，不包括内含子，富含鸟苷和胞苷(65.5％G+C)。该基因的密码子使用模式反映出其DNA碱基组成具有对于以G或C结尾的密码子的强烈偏倚(89.7％)。II.毁丝霉菌漆酶在曲霉中的表达A.材料和方法
1.细菌和真菌宿主菌株
在这项研究中用作构建和常规增殖漆酶表达载体的宿主是大肠杆菌JM101(Messing等人，Nucl.Acids Res.9：309-321，1981)。用于漆酶表达的真菌宿主包括黑曲霉菌株Bo-1、AB4.1和AB1.13(Mattern等人，Mol.Gen.Genet.234：332-336)，以及α-淀粉酶缺陷型米曲霉菌株How B104的需要尿苷(pyrG)的突变株。
2.质粒
质粒pRaMB2是pUC119的衍生物，其含有编码MtL的嗜热毁丝霉菌基因组DNA的3.2Kb BglII-NheI片段。通过将米曲霉TAKA-淀粉酶启动子和来自pTAKA 17的终止子元件(Christensen等人，Bio/Technol.6：1419-1422，1988；EP238 023)插入pUC18中(Yanisch-Perron等人，Gene 33：103-119，1985)而构建载体pMWR。该载体中，在启动子元件末端有一个单一SwaI位点，在定向克隆编码序列的终止子开始处有一个单一NsiI位点。通过在pUC118(Vieira和MeSSing，见上文)邻近的BamHI和XbaI位点之间插入含NsiI、ClaI、XhoI、和BglII限制位点的小接头而产生克隆载体pUC518。质粒pTOC 68(WO91/17243)含米曲霉TAKA-淀粉酶启动子和黑曲霉glaA终止子并且pTOC90(WO91/17243)携带构巢曲霉amdS基因。
3.漆酶表达载体的构建
漆酶表达载体pRaMB5的构建方法概括于图3中。指导漆酶基因转录的启动子得自米曲霉α-淀粉酶(TAKA-淀粉酶)基因(Christensen等人，见上文)，以及TAKA-淀粉酶终止子区域。首先通过在SwaI和NsiI位点之间插入含ApaI位点的小接头来修饰pMWR3而构建质粒，形成称之为pMWR3-SAN的质柱。用pfuI聚合酶定向PCR技术(Stratagene，La Jolla，加拿大)扩增编码MtL 5’-部分的短DNA节段，从起始密码子至内部PstI位点(约0.5Kb)。设计的该PCR反应正向引物使产生的EcoRI位点正好位于起始密码子的上游。然后，用EcoRI和PstI消化扩增的片段〔在这一步，通过用dNTPs和DAN聚合酶I(Klenow片段)处理使EcoRI位点钝化〕，并用琼脂糖凝胶电泳纯化。从pRaMB2切除嗜热毁丝霉菌编码区的3’部分，成为-2Kb PstI-ApaI片段(该节段还含有来自3’-非翻译区的约110bp)。使这两个片段在三部分的连接反应中与SwaI-和ApaI-切割的pMWR3-SAN结合而产生漆酶表达载体pRaMB5。
4.曲霉宿主细胞的转化
共转化曲霉菌株的方法如Christensen等人，见上文，所述。为了将漆酶表达载体引入米曲霉How B104 pyrG，使用了等量的(分别约为5μg)漆酶表达载体和下列质柱中的一个：pPYRG(Fungal GeneticsStock Center，Kansas City，KS)，其含有构巢曲霉pyrG基因(Oakley等人，Gene 61 338-339，1987)；pSO2，其含有克隆末曲霉pyrG基因。pPRYG24，其含有无花果曲霉(A.ficuum)(＝黑曲霉)PyrG基因。在曲霉基本培养基(Rowland和Turner，Mol.Gen.Genet.126：201-216，1973)上选择原养型(Pyr+)转化体，并且这些转化体是所筛选的能在含1mM 2，2’-连氮基双(3-乙基苯并噻唑啉磺酸)〔ABTS〕的基本培养基上产生漆酶的转化体。分泌活性漆酶的细胞氧化ABTS，在菌落周围形成绿色环。最后，用等量(分别约5μg)漆酶表达载体和含构巢曲霉amds(乙酰胺酶)基因(Hynes等人，Mol.Cell Biol.3：1430-1439，1983)的pToC90共转化黑曲霉Bo-1原生质体。在以1％葡萄糖作为碳源，以乙酰胺作为唯一氮源的Cove基本培养基(Cove，Biochim.Biophys.Acta 113：51-56，1966)上选择AmdS+转化体，并在含1mM ABTS的Cove培养基上筛查漆酶的表达。
5.产生漆酶的转化体的分析
将在琼脂板上产生漆酶活性的转化体通过分生孢子纯化两次，并分别制备于无菌的0.01％吐温-80中的孢子悬浮液。用分光光度测定法(A595nm)估算每种悬浮液中的孢子密度。使用大约0.5吸收单位的孢子接种25ml ASPO4或MY50培养基于125ml塑料瓶中。将培养物在37℃剧烈通气下(约200rpm)温育4至5天。离心收集培养肉汤，以丁香醛连氮作底物测定上清液漆酶活性的量。简单地说，将800μl分析缓冲液(25mM醋酸钠，pH5.5，40μm CuSO4)与20μl培养物上清液和60μl 0.28mM丁香醛连氮(Sigma Chemical Co.，St.Louis，MO)于50％乙醇中的溶液混合。在Genesys 5紫外-可见分光光度计(Milton-Roy)中在一段时间内测定于530μm的吸收值。一个漆酶单位(LACU)被定义为在室温每分钟氧化1μmol底物的酶量。用来自Novex (SanDiego，加拿大)的预制10-27％梯度凝胶进行SDS-聚丙烯酰胺凝胶电泳(PAGE)。用考马斯亮蓝(Sigma)显示蛋白质带。B.结果与讨论
1.毁丝霉菌漆酶的表达
通过将ABTS掺入选择性培养基中检测产生漆酶的转化体。用pyrG或amdS作为选择性标记物，共转化频率变化为约30％至70％。MtL的异源表达似乎在米曲霉转化体中最高。另外，与MY50相比，在ASPO4培养基中的生产似乎较好，尽管其原因仍未知晓。培养肉汤样品约SDS-PAGE分析表明在约80Kdal有明显的漆酶带，其与从嗜热毁丝霉菌纯化的在天然酶的大小相似。对黑曲霉共转化体培养物滤液的类似分析表明漆酶带被很强的葡糖淀粉酶和对酸稳定的淀粉酶蛋白带遮蔽了。结果示于表1中。
表1在选择的米曲霉和黑曲霉转化体中MtL的表达
宿主菌株 转化体 转化的DNAS  MTLACU/MLASPO4    MY50
米曲霉HowB104pyrG 未转化的RaMB5.15RaMB5.30RaMB5.33RaMB5.108RaMB5.111RaMB5.121RaMB5.142 无pRaMB5+pPYRGpRaMB5+pPYRGpRaMB5+pPYRGpRaMB5+PSO2pRaMB5+PSO2pRaMB5+PSO2pRaMB5+PSO2   0.00    0.000.85    0.290.71    0.870.60    0.260.68    0.190.70    0.170.49    0.200.54    0.04
黑曲霉Bo-1 未转化的RaMB5.1RaMB5.25RaMB5.49RaMB5.51RaMB5.53RaMB5.62 无pRaMB5+pToC90pRaMB5+pToC90pRaMB5+pToC90pRaMB5+pToC90pRaMB5+pToC90pRaMB5+pToC90   0.00    0.00n.d.    0.20n.d.    0.09n.d.    0.06n.d.    0.12n.d.    0.21n.d.    0.16
n.d.＝未测出
2.在过量铜存在与否时的表达
将1ml米曲霉转化体HowB104-pRaMB5.30的孢子悬浮液等分试样(约109孢子/ml)经无菌操作加入500ml摇瓶中，摇瓶中含100ml无菌摇瓶培养基(麦芽糖，50g/l；MgSO4·7H2O，2g/l；KH2PO4，10g/l；K2SO4，2g/l；CaCl2·2H2O，0.5g/l；柠檬酸，2g/l；酵母苹，10g/l；痕量金属〔ZnSO4·7H2O，14.3g/l；CuSO4·5H2O，2.5g/l；NiCl2·6H2O，0.5g/l；FeSO4· 7H2O，13.8g/l，MnSO4·H2O，8.5g/l；柠檬酸，3.0g/l〕，0.5ml/l；脲，2g/l，加自来水制成，并在高压灭菌前调节至pH6.0)，在旋转摇床上以200转/分子37℃温育18小时。将50ml该培养液无菌操作下转移到3升发酵罐中，发酵罐中含有1.8升发酵罐培养基(MgSO4·7H2O，2g/l；KH2PO4，2g/l；柠檬酸4g/l；K2SO4，3g/l；CaCl2·2H2O，2g/l；痕量金属，0.5ml/l；普罗兰尼克(pluronic)消沫剂，1ml/l)。通过使冷却水在发酵罐夹套中循环保持发酵罐温度在34℃。将无菌空气以1.8升/分钟(1体积/体积/米)的速率喷入发酵罐。使搅拌速率大约在维持培养物中溶解氧的水平超过20％时所需的最低水平下维持在600至1300转/分钟之间。用蠕动泵向发酵罐中加入无菌进料液(Nutriose 725〔麦芽糖糖浆〕，225g/l；脲，30g/l；酵母萃，15g/l；普罗兰尼克消沫剂，1.5ml/l，加蒸馏水制成并高压灭菌)。发酵期间进料速率分布如下：接种前最先加入进料液30g；0-24小时，2g/1小时；24-48小时，4g/1小时，48小时-终了，6g/1小时。
将铜制备成在水或适宜缓冲液中的400倍贮备液，过滤灭菌并无菌操作下加入罐中至终浓度为0.5mM。也可以不向罐培养基中加入铜添加物来进行上述发酵。取出用于酶活力测定的样品并通过Miracloth过滤除去菌丝体。用上述LACU分析法测定这些样品的漆酶活力。发现在发酵过程中漆酶活力不断增加，在含有过量铜的发酵中180小时后其值达到约为45LACU/ml。在22LACU/mg比活性下，这相当于2g/l表达的重组漆酶。另一方面，在没有铜添加物的发酵中170小时后达到的最大漆酶活力约为10LACU/ml，或者是在附加的铜存在下发现的约25％的活力。III.毁丝霉菌漆酶的纯化与表征
A.材料与方法
1.材料
用作缓冲液和底物的化学品是至少为试剂级别的市售产品。内切/N-糖苷酶F和焦谷氨酸氨基肽酶从Boethringer Mannheim购买。在Pharmacia FPLC或一般的低压系统中进行色谱分析。在分光光度计(Shimadzu PC160)或微板读数器(Molecular Devices)上进行分光测定。按照Queele，Biochemisches Tasshenbuch，H.M.Raven，II.Teil，S.9.3u.102，1964中所述方法制备Britton & Robinson(B&R)缓冲液。
2.酶测定
通过在30℃于1厘米石英比色池中的丁香醛连氮氧化反应测定漆酶活力。将60μl丁香醛连氮贮备液(0.28m于50％乙醇中)和20μl样品与0.8ml预热的缓冲液混合。在530um监测氧化反应5分钟。以每分钟氧化的μmol底物表示活力。使用各种pH的B&R缓冲液。活力单位在此表示为“SOU”。还用25mM醋酸钠，40μM CuSO4，pH5.5的缓冲液测定了活力，表示为LACU，如前面的定义。在室温下用0.4mMABTS，B&R缓冲液，pH4.1，通过监测ΔA405测定了2，2’-连氮基双(3-乙基苯并噻唑啉-6-磺酸)(ABTS)氧化反应。通过将冷却的ABTS-琼脂糖(0.05g ABTS，1g琼脂糖，50ml水，加热至琼脂糖溶解)倾置在天然IEF凝胶上并于室温下温育来进行ABTS氧化酶活力重叠测定。在各种温度下，用在B&R缓冲液(pH6)中预温育的具有3SOU活力的样品进行3漆酶(r-MtL)热稳定性分析。在室温下将样品以400倍稀释到相同缓冲液中之后进行分析。
3.从发酵液纯化
通过Whatman#2滤纸过滤3.7升干酪一布过滤的发酵液(pH7.6，16ms)。用S1Y100膜(MWCO：100)在螺旋浓缩器(Amicon)上将发酵液从3700ml浓缩至200ml。将浓缩液稀释到水中来调节至0.75mS并在S1Y100上再浓缩至170ml。洗涤并浓缩的发酵液为深绿色。
将发酵液于-20℃冷冻过夜，冷日融化并加在Q-琼脂糖XK26柱上(120ml)；柱子预先用10mM Tris，pH7.5，0.7mS(缓冲液A)平衡。在上样过程中蓝色漆酶带沿着柱子缓慢向下迁移。一组蓝色组分在上样并用缓冲液A洗涤后流过柱子。第二组在用缓冲液B(缓冲液A加2M NaC)线性梯度洗脱时洗脱下来。之后用1M NaOH洗脱出没有漆酶活性的一些棕色物质。SDS-PAGE分析表明这一制备生成了纯漆酶。
4.分析氨基酸含量、糖基化程度、以及N-末端序列
在ABI 476A测序仪上进行N-末端测序。在HP Amino Quant仪器上进行总的氨基酸分析，从而确定r-MtL的消光系数。用内切/N-糖苷酶F按照厂方说明进行脱糖基化，并通过在SDS-PAGE上测定的迁移率的不同来估算碳氢化合物含量。按照厂方说明用焦谷氨酸氨基肽酶进行N-末端解封。在转印到PVDF膜上测序之前在有或无1M脲或0.1M盐酸胍存在下用4μg肽酶处理的80μgr-MtL。获得了约20pmol解封蛋白并测定了其序列。
在Nvex细胞或Mini Protean II和Model III Mini IEF细胞(Bio-Rad)上进行SDS-PAGE和天然IEF分析。在Sephacryl S-300(Pharmacia)上进行凝胶过滤分析，用蓝色葡聚糖(2000千道尔顿)，牛IgG(158千道尔顿)、牛血清白蛋白(66千道尔顿)、卵清蛋白(45千道尔顿)和马心肌红蛋白(17千道尔顿)校正柱子以估算其天然分子量。
B.结果与讨论
1.来自发酵液的r-MtL的纯化与表征
从3.7开发酵液中分离出约2-3g r-MtL。使用MWCO为100千道尔顿的膜进行的最初浓缩除去了大量的棕色物质和少量的污染蛋白。r-MtL对于用pH7.5的10mM Tris平衡的Q琼脂糖基质亲合力较低，这便于使其与其它较酸的或结合更紧密的杂质分离。如SDS-PAGE所示，这一制备使得峰附近最有活性的部分成为基本上纯的漆酶。其它活性较低的部分可以在梯度较平缓的Mono0Q上或者在凝胶过滤柱如S-300上进一步纯化，由于杂质分子量较小而使其分离出来。其纯化了18倍，回收率为67％。如下面所讨论的，Q-琼脂糖色谱上存在的两个r-MtL洗脱带可能是由于示差糖基化。
纯化的r-MtL通过S-300凝胶过滤表现出的分子量为100-140千道尔顿，通过SDS-PAGE表现出的分子量为85千道尔顿。r-MtL脱糖基化后经SDS-PAGE的迁移率的增加表明碳水化合物占其总量的14％。天然IEF在pI～4.2呈现出一条主带，其在ABTS重叠测定中有活性。
在脱盐的溶液中或者在PVDF膜上直接从样品测定纯化的r-MtL N-末端序列是不成功的。但是，用焦谷氨酸氨基肽酶处理r-MtL产生了N-末端解封的蛋白质。这表明在r-MtL成熟期间的前肽加工过程同粗糙脉孢菌漆酶一样是翻译后的加工过程，但在其它漆酶如立枯丝核菌(Rhizoctonia solani)中未发现这一现象。推荐的方案概述如下：
MKSFISAATLWIVGILTPSVAAAPPSTEPQRDLLVPITEREEAAVKARQQSCNTPS
|<-推定的信号肽->|<-推定的前肽->|<-N-末端
  蓝色r-MtL光谱在276和589nm有最大吸收。
  用丁香醛连氮和ABTS作底物测试了漆酶活力。以每Abs 276或每mgg表示，漆酶在pH6.5时的SOU测定值分别为20或45单位。LACU测定所得值为每AbS276或每mg10或22单位。
r-MtL活力的pH分布型与野生型的很接近，最适pH为6.5。观察到的r-MtL预温育20分钟后保持全部活力的上限温度约为60℃。纯化的r-MtL于Q-琼脂糖洗脱缓冲液中于-20℃冷冻保存5星期后无活力丧失。
当比较从在Q-琼脂糖上分离的发酵液中获得的r-MtL的两种形式时，就SDS-PAGE、天然PAGE、天然IEF、S-300凝胶过滤、紫外一可见光谱、对丁香醛连氮和ABTS的比活性、以及解封N-末端的序列测定来源，两者没有明显差别。同样，由某种示差糖基化可造成在Q-琼脂糖上不同的洗脱模式。IV.毁丝霉菌漆酶在染发中的应用
在各种染料前体上测试毁丝霉菌漆酶的染色效果，进而在0.1％对苯二胺上进行了测定，并与多种改性剂相比较。材料：染料前体：0.1％对苯二胺于0.1MK-磷酸盐缓冲液，pH＝7.0)0.1％邻氨基苯酚于0.1MK-磷酸盐缓冲液，pH＝7.0)酶：重组嗜热毁丝霉菌漆酶，16LACU/ml(于最终的染料溶液中)。仪器：Datacolor Textflash 2000(CIE-Lab)评估头发颜色：
在Datacolor Textflash 2000上用CIE-Lab参数L*(“ 0”＝黑色而“100”＝白色)与a*(“-”＝绿色而“+”＝红色)定量结合测定成绺长发的颜色。结果：染色效果
就氧化的头发染色来说，用金色欧洲发绺(1克)测试嗜热毁丝霉菌漆酶。用对苯二胺和邻氨基苯酚作为染料前体。染发
使4ml染料前体溶液与1ml漆酶在Whirley混合器上混合，施于发绺上并于30℃保持60分钟。然后用流动的水冲洗发绺约3分钟，在两指间压紧、梳理并空气干燥。
染色效果试验的结果示于表1和2中。表1
邻氨基苯酚     酶     L*     a*
未处理的金色头发     -     70.3     2.3
漆酶     +     57.7     15.3
L*：0＝黑色，100＝白色  a*：-＝绿色，+＝红色表2
对苯二胺     酶     L*     a*
未处理的金色头发     -     70.3     2.3
1.0ml漆酶     +     29.1     4.1
L*：0＝黑色，100＝白色，a*：-＝绿色，+＝红色试验结果：
从表1和表2可见嗜热毁丝霉菌漆酶可用于氧化染色头发。生物材料的保藏
下列生物材料已按照布达佩斯条约规定在1994年5月25日保藏于农业研究机构专利培养物保藏中心(Northern Regional ResearchCenter，1815 University Street，Peoria，Illinois，61604)，并得到了下列保藏号。保藏                                        保藏号含pRaMB5的大肠杆菌                       NRRL B-21261
                    序列表(1)一般信息(i)申请人：
(A)名称：Novo Nordisk Biotech，Inc.
(B)街道：1445 Drew Avenue
(C)城市：Davis，California
(D)国别：美国
(E)邮编(ZIP)：95616-4880
(F)电话：(916)757-8100
(G)传真：(916)758-0317(i)申请人：
(A)名称：Novo Nordisk A/S
(B)街道：Novo Alle
(C)城市：Bagavard
(D)国别：丹麦
(E)邮编(ZIP)：DK-2880
(F)电话：+45 4444 8888
(G)传真：+45 4449 3256(ii)发明名称：纯化的毁丝霉属漆酶及编码该酶的核酸(iii)序列数：2(iv)联系地址：
(A)联系人：Novo Nordisk of North America，Inc.
(B)街道：405 Lexington Avenue，Suite 6400
(C)城市和州：纽约市、纽约州
(D)国别：美国
(E)邮编：10174-6401(v)计算机可读形式：
(A)媒介类型：软盘
(B)计算机：IBM PC兼容机
(C)操作系统：PC-DOS/MS-DOS
(D)软件：PatentIn Release#1.0，Version#1.25(EPO)(vi)目前的申请资料
(A)申请号：待定
(B)申请日：1995年5月31日
(C)分类：(vii)在先申请资料
(A)申请号：US 08/253,781
(B)申请日：1994年6月3日(viii)代理人信息
(A)姓名：Lowney，Karen A.
(B)注册号：31，274
(C)文档号：4184.204-WO(ix)电讯信息
(A)电话：212 867 0123
(B)传真：212 867 0298(2)SEQ ID NO：1的信息(i)序列特征
(A)长度：3187个碱基对
(B)类型：核酸
(C)链型：双链
(D)拓扑学：线型(ii)分子类型：DNA(基因组)(vi)最初来源
(A)微生物：嗜热毁丝霉菌(ix)特征：
(A)名称/关键词：内含子
(B)位置：833...917(ix)特征：
(A)名称/关键词：内含子
(B)位置：996...1077(ix)特征：
(A)名称/关键词：内含子
(B)位置：1090...1188(ix)特征：
       (A)名称/关键词：内含子
       (B)位置：1261...1332
 (ix)特征：
       (A)名称/关键词：内含子
       (B)位置：2305...2451
 (ix)特征：
       (A)名称/关键词：内含子
       (B)位置：2521...2613
 (ix)特征：
       (A)名称/关键词：CDS
       (B)位置：join(587..832，918..995，1078..1089，1189..1260，1333..2304，2452..2520，2614..3024)
 (xi)序列描述：SEQ ID NO：1GCTAGCTTCT TTGGTCACCG TCGTTTTCGC CCGCCCCCTC CCTCCTTCAA CCCCCTGAGT    60AGTCGGCTAA GCGATCCTCA ATCTGGTCTT GTGAGGTCAC GTCCTCCAGC AGATGACAGT   120TCATCGAGCG AGTGATCTCC ACCACCCAGA AGGGAGGGGG GATGCGCGCA TGCTCCAACA   180TACCCTGGTG TCGCTAGAGA CGTCGCGGCA TCAGCCTTTT CATCACACCG AGCACGTCCA   240CGGACCGGCT CCTTTCACCC CCGCGTCCTC CGGAGGATTG AGTCACGATA TTTCGGGATG   300TGGGAAGGGG GAGAGAAAGG AGGGGGGAGG GGCGGAAACA TGTTGGATAC GAGCTGCGCC   360CCTTTTCCAA CATCGAGAAC AGGAAGTCGT TGGTGTCGGC CGTAATGTCT ATAAAACGAG   420GCTCCTTCTC GTCGTCGACT TGTCTCAGGT TCTCTCTCTC GTCCACACCA AGCCAGTCTT   480GCCTGAGCCA CCTGAGCCAC CTTCAACTCA TCATCTTCAG TCAAGTCGTT CATTGACATT   540GTGTCTCTC TTTCTATCGAG TCGGCTTCCC GGCCCTTCAC CACAAC ATG AAG TCC      595
                                               Met Lys Ser
                                               1TTC ATC AGC GCC GCG ACG CTT TTG GTG GGC ATT CTC ACC CCT AGC GTT     643Phe Ile Ser Ala Ala Thr Leu Leu Val Gly Ile Leu Thr Pro Ser Val
5                   10                  15GCT GCT GCC CCT CCA TCC ACC CCT GAG CAG CGC GAC CTG CTC GTC CCG     691Ala Ala Ala Pro Pro Ser Thr Pro Glu Gln Arg Asp Leu Leu Val Pro20                  25                  30                  35ATC ACG GAG AGG GAG GAG GCA GCC GTG AAG GCT CGC CAG CAG AGC TGC    739Ile Thr Glu Arg Glu Glu Ala Ala Val Lys Ala Arg Gln Gln Ser Cys
            40                  45                  50AAC ACC CCC AGC AAC CGG GCG TGC TGG ACT GAC GGA TAC GAC ATC AAC    787Asn Thr Pro Ser Asn Arg Ala Cys Trp Thr Asp Gly Tyr Asp Ile Asn
        55                  60                  65ACC GAC TAC GAA GTG GAC AGC CCG GAC ACG GGT GTT GTT CGG CCG        832Thr Asp Tyr Glu Val Asp Ser Pro Asp Thr Gly Val Val Arg Pro
    70                  75                  80GTGAGTGCTC TCGTTAATTA CGCTTCGGCG AGTTGCGCAG ATATATTAAA TACTGCAAAC  892CTAAGCAGGA GCTGACATGC GACAG TAC ACT CTG ACT CTC ACC GAA GTC GAC    944
                        Tyr Thr Leu Thr Leu Thr Glu Val Asp
                                85                  90AAC TGG ACC GGA CCT GAT GGC GTC GTC AAG GAG AAG GTC ATG CTG GTT    992Asn Trp Thr Gly Pro Asp Gly Val Val Lys Glu Lys Val Met Leu Val
        95                  100                 105AAC GTACGGCACC CCTTTTCTTG TCCTAGGATC TGGGTGATGT GCGTCGTTGC        1045AsnCCCTGAGAGA CTGACCGAGC CTTTGGCTGC AG AAT AGT ATA ATC GTAATTAATT    1099
                                Asn Ser Ile Ile
                                    110ATACCGCCCT GCCTCCAGCA GCCCCAGCAG CTCGAGAAGG GTATCTGAAG TTAGTCAGGC 1159CTGCTGACCT GACCGGGGCC AACCCATAG GGA CCA ACA ATC TTT GCG GAC TGG   1212
                            Gly Pro Thr Ile Phe Ala Asp Trp
                                    115                 120GGC GAC ACG ATC CAG GTA ACG GTC ATC AAC AAC CTC GAG ACC AAC GGC   1260Gly Asp Thr Ile Gln Val Thr Val Ile Asn Asn Leu Glu Thr Asn Gly
            125                 130                 135GTATGTCTGC TGCTTGCTCT CTTGCTCTCC TCGTCCGCGA CTAATAATAA TATCAACTCG 1320TGTGGAAAAC AG ACG TCG ATC CAC TGG CAC GGA CTG CAC CAG AAG GGC     1368
          Thr Ser Ile His Trp His Gly Leu His Gln Lys Gly
                      140                 145ACC AAC CTG CAC GAC GGC GCC AAC GGT ATC ACC GAG TGC CCG ATC CCC   1416Thr Asn Leu His Asp Gly Ala Asn Gly Ile Thr Glu Cys Pro Ile Pro
150                 155                 160CCC AAG GGA GGG AGG AAG GTG TAC CGG TTC AAG GCT CAG CAG TAC GGG   1464Pro Lys Gly Gly Arg Lys Val Tyr Arg Phe Lys Ala Gln Gln Tyr Gly165                 170                 175                 180ACG AGC TGG TAC CAC TCG CAC TTC TCG GCC CAG TAC GGC AAC GGC GTG   1512Thr Ser Trp Tyr His Ser His Phe Ser Ala Gln Tyr Gly Asn Gly Val
            185                 190                 195GTC GGG GCC ATT CAG ATC AAC GGA CCG GCC TCG CTG CCG TAC GAC ACC   1560Val Gly Ala Ile Gln Ile Asn Gly Pro Ala Ser Leu Pro Tyr Asp Thr
        200                 205                 210GAC CTG GGT GTG TTC CCC ATC AGC GAC TAC TAC TAC AGC TCG GCC GAC   1608Asp Leu Gly Val Phe Pro Ile Ser Asp Tyr Tyr Tyr Ser Ser Ala Asp
    215             220                 225GAG CTG GTG GAA CTC ACC AAG AAC TCG GGC GCG CCC TTC AGC GAC AAC    1656Glu Leu Val Glu Leu Thr Lys Asn Ser Gly Ala Pro Phe Ser Asp Asn230                 235                 240                 245GTC CTG TTC AAC GGC ACG GCC AAG CAC CCG GAG ACG GGC GAG GGC GAG    1704Val Leu Phe Asn Gly Thr Ala Lys His Pro Glu Thr Gly Glu Gly Glu
            250                 255                 260TAC GCC AAC GTG ACG CTC ACC CCG GGC CGG CGG CAC CGC CTG CGC CTG    1752Tyr Ala Asn Val Thr Leu Thr Pro Gly Arg Arg His Arg Leu Arg Leu
        265                 270                 275ATC AAC ACG TCG GTC GAG AAC CAC TTC CAG GTC TCG CTC GTC AAC CAC    1800Ile Asn Thr Ser Val Glu Asn His Phe Gln Val Ser Leu Val Asn His
    280                 285                 290ACC ATG TGC ATC ATC GCC GCC GAC ATG GTG CCC GTC AAC GCC ATG ACG    1848Thr Met Cys Ile Ile Ala Ala Asp Met Val Pro Val Asn Ala Met Thr
295                 300                 305GTC GAC AGC CTC TTC CTC GGC GTC GGC CAG CGT TAC GAT GTC GTC ATC    1896Val Asp Ser Leu Phe Leu Gly Val Gly Gln Arg Tyr Asp Val Val Ile310                 315                 320                 325GAA GCC AAC CGA ACG CCC GGG AAC TAC TGG TTT AAC GTC ACA TTT GGC    1944Glu Ala Asn Arg Thr Pro Gly Asn Tyr Trp Phe Asn Val Thr Phe Gly
            330                 335                 340GGC GGC CTG CTC TGC GGC GGC TCC AGG AAT CCC TAC CCG GCC GCC ATC    1992Gly Gly Leu Leu Cys Gly Gly Ser Arg Asn Pro Tyr Pro Ala Ala Ile
        345                 350                 355TTC CAC TAC GCC GGC GCC CCC GGC GGC CCG CCC ACG GAC GAG GGC AAG    2040Phe His Tyr Ala Gly Ala Pro Gly Gly Pro Pro Thr Asp Glu Gly Lys
    360                 365                 370GCC CCG GTC GAC CAC AAC TGC CTG GAC CTC CCC AAC CTC AAG CCC GTC    2088Ala Pro Val Asp His Asn Cys Leu Asp Leu Pro Asn Lau Lys Pro Val
375                 380                 385GTG GCC CGC GAC GTG CCC CTG AGC GGC TTC GCC AAG CGG GCC GAC AAC    2136Val Ala Arg Asp Val Pro Leu Ser Gly Phe Ala Lys Arg Ala Asp Asn390                 395                 400                 405ACG CTC GAC GTC ACC CTC GAC ACC ACG GGC ACG CCC CTG TTC GTC TGG    2184Thr Leu Asp Val Thr Leu Asp Thr Thr Gly Thr Pro Leu Phe Val Trp
            410                 415                 420AAG GTC AAC GGC AGC GCC ATC AAC ATC GAC TGG GGG AGG GCC GTC GTC    2232Lys Val Asn Gly Ser Ala Ile Asn Ile Asp Trp Gly Arg Ala Val Val
        425                 430                 435GAC TAC GTC CTC ACG CAG AAC ACC AGC TTC CCA CCC GGG TAC AAC ATT    2280Asp Tyr Val Lau Thr Gln Asn Thr Ser Phe Pro Pro Gly Tyr Asn Ile
    440                 445                 450GTC GAG GTG AAC GGA GCT GAT CAG GTAAGAAAAA GGGGACCGCA GGGGTGCTGC   2334Val Glu Val Asn Gly Ala Asp Gln
455                 460TGCAAGTACA CCTTGCTCGC CCTCCTGTTC TTCCTTAATA ACTACCTCCC AACCCTCCCC  2394CCTAATTAAT TCACTTTAAA GGCCGATCAA GACTGACCGA GCCCCCTCTC TTTGCAG     2451TGG TCG TAC TGG TTG ATC GAG AAC GAT CCC GGC GCA CCT TTC ACC CTA    2499Trp Ser Tyr Trp Leu Ile Glu Asn Asp Pro Gly Ala Pro Phe Thr Leu
        465                 470                 475CCG CAT CCG ATG CAC CTG CAC GTAAGTTGGA TACATATATA TATATATATA      2550Pro His Pro Met His Leu His
    480TACATTGCTT TCCTGGCTCG CTCCCTTAAA TAAAATTAAA TAACCAAAAA TAACAAAAAA 2610AAG GGC CAC GAC TTT TAC GTG CTG GGC CGC TCG CCC GAC GAG TCG CCG   2658
Gly His Asp Phe Tyr Val Leu Gly Arg Ser Pro Asp Glu Ser Pro
485                 490                 495GCA TCC AAC GAG CGG CAC GTG TTC GAT CCG GCG CGG GAC GCG GGC CTG   2706Ala Ser Asn Glu Arg His Val Phe Asp Pro Ala Arg Asp Ala Gly Leu500                 505                 510                 515CTG AGC GGG GCC AAC CCT GTG CGG CGG GAC GTG TCG ATG CTG CCG GCG   2754Leu Ser Gly Ala Asn Pro Val Arg Arg Asp Val Ser Met Leu Pro Ala
            520                 525                 530TTC GGG TGG GTG GTG CTG TCC TTC CGG GCC GAC AAC CCG GGC GCC TGG   2802Phe Gly Trp Val Val Leu Ser Phe Arg Ala Asp Asn Pro Gly Ala Trp
        535                 540                 545CTG TTC CAC TGC CAC ATC GCC TGG CAC GTC TCG GGC GGC CTG GGC GTC   2850Leu Phe His Cys His Ile Ala Trp His Val Ser Gly Gly Leu Gly Val
    550                 555                 560GTC TAC CTC GAG CGC GCC GAC GAC CTG CGC GGG GCC GTC TCG GAC GCC   2898Val Tyr Leu Glu Arg Ala Asp Asp Leu Arg Gly Ala Val Ser Asp Ala
565                 570                 575GAC GCC GAC GAC CTC GAC CGC CTC TGC GCC GAC TGG CGC CGC TAC TGG   2946Asp Ala Asp Asp Leu Asp Arg Leu Cys Ala Asp TrP Arg Arg Tyr Trp
    580                 585                 590CCT ACC AAC CCC TAC CCC AAG TCC GAC TCG GGC CTC AAA CAC CGC TGG   2994Pro Thr Asn Pro Tyr Pro Lys Ser Asp Ser Gly Leu Lys His Arg Trp
595                 600                 605GTC GAG GAG GGC GAG TGG CTG GTC AAG GCG TGAGCGAAGG AGGAAAAAGG     3044Val Glu Glu Gly Glu Trp Leu Val Lys Ala610                 615AAACAAAGAG GGGGGGGGGG GCTAGTTCCT ATTTTTGCTT TTTTTTTTTG TTCTTGTCCT 3104TGTGCTGGCG GTTCCCTGGT AAAGGAGAAG GGGGCCCCAA GTTCGAGTGG GTGTGTGATC 3164GGGTAAATAT TATCAAGAGA TCT                                         3187(2)SEQ ID NO：2的信息
(i)序列特征
    (A)长度：620个氨基酸
    (B)类型：氨基酸
    (C)链型：单链
    (D)拓扑学：线型
(ii)分子类型：蛋白质
(vi)最初来源
    (A)微生物：嗜热毁丝霉菌
(xi)序列描述：SEQ ID NO：2：Pro Ser Val Ala Ala Ala Pro Pro Ser Thr Pro Glu Gln Arg Asp Leu
        20                  25                  30Leu Val Pro Ile Thr Glu Arg Glu Glu Ala Ala Val Lys Ala Arg Gln
    35                  40                  45Gln Ser Cys Asn Thr Pro Ser Asn Arg Ala Cys Trp Thr Asp Gly Tyr
50                  55                  60Asp Ile Asn Thr Asp Tyr Glu Val Asp Ser Pro Asp Thr Gly Val Val65                  70                  75                  80Arg  Pro Tyr Thr Leu Thr Leu Thr Glu Val Asp Asn Trp Thr Gly Pro
             85                  90                  95Asp Gly Val Val Lys Glu Lys Val Met Leu Val Asn Asn Ser Ile Ile
        100                 105                 110Gly Pro Thr Ile Phe Ala Asp Trp Gly Asp Thr Ile Gln Val Thr Val
    115                 120                 125Ile Asn Asn Leu Glu Thr Asn Gly Thr Ser Ile His Trp His Gly Leu
130                 135                 140His Gln Lys Gly Thr Asn Leu His Asp Gly Ala Asn Gly Ile Thr Glu145                 150                 155                 160Cys Pro Ile Pro Pro Lys Gly Gly Arg Lys Val Tyr Arg Phe Lys Ala
            165                 170                 175Gln Gln Tyr Gly Thr Ser Trp Tyr His Ser His Phe Ser Ala Gln Tyr
        180                 185                 190Gly Asn Gly Val Val Gly Ala Ile Gln Ile Asn Gly Pro Ala Ser Leu
    195                 200                 205Pro Tyr Asp Thr Asp Leu Gly Val Phe Pro Ile Ser Asp Tyr Tyr Tyr
210                 215                 220Ser Ser Ala Asp Glu Leu Val Glu Leu Thr Lys Asn Ser Gly Ala Pro225                 230                 235                 240Phe Ser Asp Asn Val Leu Phe Asn Gly Thr Ala Lys His Pro Glu Thr
            245                 250                 255Gly Glu Gly Glu Tyr Ala Asn Val Thr Leu Thr Pro Gly Arg Arg His
        260                 265                 270Arg Leu Arg Leu Ile Asn Thr Ser Val Glu Asn His Phe Gln Val Ser
    275                 280                 285Leu Val Asn His Thr Met Cys Ile Ile Ala Ala Asp Met Val Pro Val
290                 295                 300Asn Ala Met Thr Val Asp Ser Leu Phe Leu Gly Val Gly Gln Arg Tyr305                 310                 315                 320Asp Val Val Ile Glu Ala Asn Arg Thr Pro Gly Asn Tyr Trp Phe Asn
            325                 330                 335Val Thr Phe Gly Gly Gly Leu Leu Cys Gly Gly Ser Arg Asn Pro Tyr
        340                 345                 350Pro Ala Ala Ile Phe His Tyr Ala Gly Ala Pro Gly Gly Pro Pro Thr
    355                 360                 365Asp Glu Gly Lys Ala Pro Val Asp His Asn Cys Leu Asp Leu Pro Asn
370                 375                 380Leu Lys Pro Val Val Ala Arg Asp Val  Pro Leu Ser Gly Phe Ala Lys385                 390                  395                 400Arg Ala Asp Asn Thr Leu Asp Val Thr Leu Asp Thr Thr Gly Thr Pro
            405                 410                 415Leu Phe Val Trp Lys Val Asn Gly Ser Ala Ile Asn Ile Asp Trp Gly
        420                 425                 430Arg Ala Val Val Asp Tyr Val Leu Thr Gln Asn Thr Ser Phe Pro Pro
    435                 440                 445Gly Tyr Asn Ile Val Glu Val Asn Gly Ala Asp Gln Trp Ser Tyr Trp
450                 455                 460Leu Ile Glu Asn Asp Pro Gly Ala Pro Phe Thr Leu Pro His Pro Met465                 470                 475                 480His Leu His Gly His Asp Phe Tyr Val Leu Gly Arg Ser Pro Asp Glu
            485                 490                 495Ser Pro Ala Ser Asn Glu Arg His Val Phe Asp Pro Ala Arg Asp Ala
        500                 505                 510Gly Leu Leu Ser Gly Ala Asn Pro Val Arg Arg Asp Val Ser Met Leu
    515                 520                 525Pro Ala Phe Gly Trp Val Val Leu Ser Phe Arg Ala Asp Asn Pro Gly
530                 535                 540Ala Trp Leu Phe His Cys His Ile Ala Trp His Val Ser Gly Gly Leu545                 550                 555                 560Gly Val Val Tyr Leu Glu Arg Ala Asp Asp Leu Arg Gly Ala Val Ser
            565                 570                 575Asp Ala Asp Ala Asp Asp Leu Asp Arg Leu Cys Ala Asp Trp Arg Arg
        580                 585                 590Tyr Trp Pro Thr Asn Pro Tyr Pro Lys Ser Asp Ser Gly Leu Lys His
    595                 600                 605Arg Trp Val Glu Glu Gly Glu Trp Leu Val Lys Ala
610                 615             620

Claims (42)

1.一种含有编码毁丝霉菌漆酶序列的DNA构建体。
2.权利要求1所述构建体，其包含编码嗜热毁丝霉菌漆酶的序列。
3.权利要求1所述构建体，其包含编码SEQ ID No.2中所描述的氨基酸序列的核酸序列。
4.权利要求1所述构建体，其包含SEQ ID No.1中所描述的核酸序列。
5.权利要求1所述构建体，其包含NRRL B-21261中含有的核酸序列。
6.基本上纯的毁丝霉菌漆酶。
7.权利要求6所述的酶，其为嗜热毁丝霉菌漆酶。
8.权利要求6所述的酶，其包含SEQ ID No.2中所描述的氨基酸序列，或者与其至少约80％同源的序列。
9.包含含编码毁丝霉菌漆酶序列的DNA构建体的重组载体。
10.权利要求9所述载体，其中的构建体可操作地连接于启动子序列上。
11.权利要求10所述载体，其中的启动子是真菌或酵母菌启动子。
12.权利要求11所述载体，其中的启动子是米曲霉TAKA淀粉酶启动子。
13.权利要求11所述载体，其中的启动子是黑曲霉或泡盛曲霉的葡糖淀粉酶(gluA)启动子。
14.权利要求9所述载体，其还含有选择性标记物。
15.权利要求14所述载体，其中的选择性标记物选自由amdS、pyrG、argB、niaD、sC和hygB组成的一组物质。
16.权利要求14所述载体，其中的选择性标记物是构巢曲霉或米曲霉的amdS标记物，或者是构巢曲霉、黑曲霉、泡盛曲霉或米曲霉的pyrG标记物。
17.权利要求14所述载体，其含有米曲霉的TAKA淀粉酶启动子以及构巢曲霉或米曲霉的amdS或pyrG标记物。
18.包含含有编码毁丝霉菌漆酶核酸序列的异源核酸构建体的重组宿主细胞。
19.权利要求18所述细胞，其为真菌细胞。
20.权利要求19所述细胞，其为曲霉细胞。
21.权利要求18所述细胞，其中的构建体被整合到宿主细胞基因组中。
22.权利要求18所述细胞，其中的构建体包含在载体上。
23.权利要求18所述细胞，其包含含有编码SEQ ID No.2中所述氨基酸序列的序列构建体。
24.一种获得漆酶的方法，其包括在有助于酶表达的条件下培养包含含有编码毁丝霉菌漆酶的核酸序列的DNA构建体的重组宿主细胞，并从培养物中回收酶。
25.按照权利要求24中的方法获得为毁丝霉菌漆酶。
26.子囊菌或半知菌漆酶，其在最适pH对丁香醛连氮的比活性至少为30SOU/mg。
27.一种提高活性重组含铜酶产率的方法，其包括在有助于酶表达的条件下，在至少约0.02mM铜存在下培养包含含编码含铜酶序列的DNA构建体的重组宿主细胞。
28.一种在溶液中聚合木素或木素硫酸盐底物的方法，其包括使底物与毁丝霉菌漆酶接触。
29.一种就地解聚牛皮纸纸浆的方法，其包括使纸浆与毁丝霉菌漆酶接触。
30.一种氧化染料或染料前体的方法，其包括使染料与毁丝霉菌漆酶接触。
31.一种染发的方法，其包括在有或无至少一种改性剂的存在下使毁丝霉菌漆酶与至少一种染料前体接触一段时间并处于足以使染料前体氧化成染料的条件下。
32.权利要求31所述方法，其中的染料前体选自由二胺、氨基苯酚、和苯酚组成的一种物质。
33.权利要求31所述方法，其中的改性剂，当使用时，是间二胺、间氨基苯酚或多酚。
34.权利要求32所述方法，其中的染料前体是选自由邻-或对-二胺或氨基苯酚组成的一组的最初中间体。
35.权利要求31所述方法，其中使用了一种以上的染料前体。
36.权利要求31所述方法，其中使用了一种以上的改性剂。
37.权利要求31所述方法，其中使用了最初中间体和改性剂。
38.一种染料组合物，其含有与至少一种染料前体结合的毁丝霉菌漆酶。
39.一种染料组合物，其含有与至少一种最初中间体和至少一种改性剂结合的毁丝霉菌漆酶。
40.一种含有染料组合物的容器，其在无氧气氛中包含毁丝霉菌漆酶和至少一种染料前体。
41.权利要求40所述容器，其含有与至少一种改性剂结合的至少一种最初中间体染料前体。
42.一种聚合或氧化酚类或苯胺化合物的方法，其包括使酚类或苯胺化合物与毁丝霉菌漆酶接触。
CNB951941194A 1994-06-03 1995-05-31 纯化的毁丝霉属漆酶及编码该酶的核酸 Expired - Fee Related CN1192108C (zh)

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CN103820401A (zh) * 2013-11-14 2014-05-28 李洋 一种碱性细菌漆酶在酵母中的高通量表达方法
CN105778550A (zh) * 2016-03-07 2016-07-20 周霞萍 基质膜、含腐殖酸组合物和制备方法及应用、制得的产品
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WO1995033836A1 (en) 1995-12-14
CN1192108C (zh) 2005-03-09
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FI964808A0 (fi) 1996-12-02
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FI964808A (fi) 1996-12-02
JP3649338B2 (ja) 2005-05-18
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MX9606013A (es) 1997-12-31
US5981243A (en) 1999-11-09
ES2165420T3 (es) 2002-03-16
US5795760A (en) 1998-08-18
ATE206460T1 (de) 2001-10-15
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AU2656595A (en) 1996-01-04
DK0765394T3 (da) 2001-12-10
CA2191718A1 (en) 1995-12-14

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