CN1452656A - 脂肪氧合酶 - Google Patents
脂肪氧合酶 Download PDFInfo
- Publication number
- CN1452656A CN1452656A CN01815183A CN01815183A CN1452656A CN 1452656 A CN1452656 A CN 1452656A CN 01815183 A CN01815183 A CN 01815183A CN 01815183 A CN01815183 A CN 01815183A CN 1452656 A CN1452656 A CN 1452656A
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- ala
- leu
- lipoxygenase
- gly
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Abstract
本发明提供了有脂肪氧合酶活性的微生物蛋白质的序列信息,及通过重组DNA技术产生该蛋白质的方法。更具体地,本发明人从禾顶囊壳分离了编码脂肪氧合酶的基因,将其克隆入大肠杆菌株系,并对其进行了测序。比较表明其与已知脂肪氧合酶序列有不到25%的同一性,最接近人15S脂肪氧合酶。本发明人已重组表达了该脂肪氧合酶,而且发现重组脂肪氧合酶是糖基化的。
Description
发明领域
本发明涉及编码脂肪氧合酶的多核苷酸及它用于脂肪氧合酶重组生产的用途。本发明也涉及通过用特异探针筛选DNA文库获得脂肪氧合酶的方法。
发明背景
脂肪氧合酶是催化亚油酸氧合和产生氢过氧化物的酶。它在酶命名法中分类为EC.1.13.11.12。这种酶广泛分布在植物和动物中。已从各种来源分离了编码基因,而且已公布了序列。因此,GENESEQP W93832和Genbank U78294给出了人15S脂肪氧合酶的序列。
已知微生物脂肪氧合酶可来源于酿酒酵母(saccharomyces cerevisiae),嗜热放线菌类普通高温放线菌(Thermoactinomyces vulgaris),来源于真菌尖镰孢(Fusarium oxysporum),百合苗枯病菌(Fusarium proliferatum)和禾顶囊壳(Gaeumannomyces graminis)(Su和Oliw,J.BiologicalChemistry,273(21),13072-13079(1998))。尚没有编码微生物脂肪氧合酶的分离的基因的描述。
现有技术描述了脂肪氧合酶的多种用途,例如,做为发酵面团或面条的食物添加剂。
发明概述
在此我们第一次提供了有脂肪氧合酶活性的微生物蛋白质的序列信息和以工业规模生产这种蛋白质的方法。更具体地,本发明人从禾顶囊壳分离了编码脂肪氧合酶的基因,将其克隆入大肠杆菌(E.coli)菌株并对其进行了测序。禾顶囊壳基因组包含约60%的G和C核苷酸,这使得这项工作的进行非常困难。该基因与已知脂肪氧合酶序列比较显示不到25%的同一性,最接近人的15S脂肪氧合酶。本发明人已重组表达了此脂肪氧合酶。
因此,本发明提供了有脂肪氧合酶活性的多肽,其
a)含有与SEQ ID NO:2或23的成熟多肽有至少50%同一性的氨基酸序列;
b)是由如下核酸序列编码的,在55℃,所述核酸序列与编码SEQ IDNO:1的成熟多肽的核酸序列或其至少有100个核苷酸的亚序列的互补链杂交;
c)具有能从SEQ ID NO:2或23的成熟多肽通过一个或多个氨基酸置换,缺失,和/或插入获得的氨基酸序列;或
d)是由克隆在大肠杆菌(E.coli)的质粒中的DNA序列的脂肪氧合酶编码部分编码的,其中所述大肠杆菌的保藏号为DSM 13586;本发明也提供了多核苷酸,其包含:
a)克隆在大肠杆菌DSM 13586的质粒中的编码成熟脂肪氧合酶的部分DNA序列;
b)编码SEQ ID NO:2或23所示成熟脂肪氧合酶的部分DNA序列;
c)a)或b)中定义的序列的类似物,其编码脂肪氧合酶并且
i)与所述DNA序列有至少50%同一性,或
ii)在低严紧条件下,与所述DNA序列或其具有至少100个核苷酸的亚序列的互补链杂交;
iii)是其等位基因变体,或
d)a),b)或c)的互补链。
本发明的其它方面提供了包含该多核苷酸的核酸构建体,包含该核酸构建体的重组表达载体,和用该核酸构建体转化的重组宿主细胞。本发明也提供了产生脂肪氧合酶的重组方法,基于SEQ ID NO:2或23的寡核苷酸探针和通过用基于SEQ ID NO:2的探针筛选真核DNA文库获得脂肪氧合酶的方法。
进一步,本发明提供了包含锰脂肪氧合酶的面团组合物和制备面团或面团的焙烤产品的方法,所述方法包括添加锰脂肪氧合酶到面团中。本发明也提供了氧化底物的方法,所述底物选自亚麻酸,花生四烯酸,亚油醇和亚油酸酯,该方法包括在氧存在的情况下,用锰脂肪氧合酶接触底物。最后,本发明提供了包含锰脂肪氧合酶和表面活性剂的去污剂组合物。发明详述
基因组DNA来源
可以从真菌中获得编码脂肪氧合酶(LOX)的DNA,所述真菌特别是子囊菌门(Ascomycota),更特别的是Ascomycota incertae sedis,例如Magnaporthaceae如顶囊壳属(Gaeumannomyces),或无性型Magnaporthaceae如梨孢属(Pyricularia),或可选地,无性型子囊菌门,如地霉属(Geotrichum)。实例是禾顶囊壳(G. graminis),例如禾顶囊壳禾谷变种(G.graminis var.graminis),禾顶囊壳燕麦变种(G.graminis var.avenae)或禾顶囊壳小麦变种(G.graminis var.tritici),特别是菌株禾顶囊壳禾谷变种CBS 903.73,禾顶囊壳燕麦变种CBS 870.73或禾顶囊壳小麦变种CBS 905.73。从Centraalbureau voor Schimmelcultures,Baarn,荷兰,可以商业上得到CBS株系。
本发明人从禾顶囊壳菌株获得两个编码LOX的DNA序列,发现它们有SEQ ID NO:1和SEQ ID NO:22所示的序列。他们将编码LOX的基因插入大肠杆菌株系,而且根据布达佩斯条约在2000年7月5日在DSMZ,即德意志微生物保藏中心(Mascheroder Weg 1b,D-38124 BraunschweigDE,德国)将其保藏为大肠杆菌DSM 13586。Novo Nordisk A/S进行了这个保藏,后来转让给了Novozymes A/S。
脂肪氧合酶
本发明的脂肪氧合酶是锰脂肪氧合酶,即它有脂肪氧合酶活性(EC1.13.11.12)并且在辅基中有锰存在。它是糖基化的,而且可以有范围为90-110kDa的分子量,特别是95-105kDa。在65-90℃,特别是75-85℃的最适温度下,它是热稳定的。脂肪氧合酶在pH10及高达400ppm的LAS(直链烷基苯磺酸盐)中是稳定的。锰脂肪氧合酶在pH5-12具有酶活性,并具有pH6-8的宽最适pH范围。
重组脂肪氧合酶可以有更高的糖基化和更高的热稳定性。重组脂肪氧合酶可以有范围为90-110kDa,特别是95-105kDa的分子量。它可以有65-90℃,特别是75-85℃的最适温度。
重组体表达载体
本发明的表达载体通常包括编码启动子,操纵基因,核糖体结合位点,翻译起始信号,和可选地,选择标记,转录终止子,阻抑蛋白基因或各种激活蛋白基因的控制序列。载体可以是自主复制载体,或可以整合进宿主细胞基因组。
通过培养转化体进行生产
可以用编码脂肪氧合酶的DNA序列转化适宜的宿主细胞,在允许该酶产生的条件下培养转化的生物体,从培养物中回收该酶,以产生本发明的脂肪氧合酶。
宿主生物体可以是真核细胞,特别是真菌细胞,如酵母细胞或丝状真菌细胞,如曲霉属(Aspergillus),镰孢属(Fusarium),木霉属(Trichoderma)或酵母属(Saccharomyces)的菌株,特别是黑曲霉(A.niger),米曲霉(A.oryzae),禾本科镰孢(F.graminearum),接骨木镰孢(F.sambucinum),F.cerealis或酿酒酵母。可以通过EP 238,023(Novo Nordisk),WO96/00787(Novo Nordisk)或EP 244,234(Alko)所述的一般方法在这些宿主生物体中产生脂肪氧合酶。
核苷酸探针
可以基于SEQ ID NO:1的DNA序列或SEQ ID NO:2的多肽序列,特别是成熟肽部分的多肽序列设计核苷酸探针。如下所述,可以用探针筛选编码LOX的DNA。
可以通过标准技术(例如在Sambrook J,Fritsch EF,Maniatis T(1989)分子克隆:实验室手册(Molecular cloning:a laboratory manual)(第二版),Cold Spring Harbor Laboratory,Cold Spring Harbor,纽约中所述),以SEQ ID NO:2中成熟部分的氨基酸序列或对应的DNA序列部分为基础制备合成的寡核苷酸引物。它可以是简并探针,且通常将包含至少20个核苷酸。
真核DNA文库的筛选
通过包括下列步骤的方法,可以获得有脂肪氧合酶活性的多肽:
a)制备真核DNA文库,
b)筛选文库以选择与上述探针杂交的DNA分子,
c)用选定的DNA分子转化宿主细胞,
d)培养转化宿主细胞以表达该DNA分子编码的多肽,和
e)分析表达的多肽以选择有脂肪氧合酶活性的多肽。
可以通过常规方法制备真核DNA文库。它可以包括从适当来源,如上述来源得到的基因组DNA或双链cDNA。
可以通过聚合酶链式反应(PCR),接着通过杂交进行DNA序列的分子筛选。
根据公知的方法,可以分离分子筛选中产生的PCR片段,并将其亚克隆入适当的载体。可以通过,例如集落或噬斑杂交用此PCR片段筛选DNA文库。
杂交
可以用杂交来指明给定DNA序列与对应于本发明DNA序列的核苷酸探针是类似的。可以在低度、中度或高度严紧条件下完成杂交。在下文将详述一个杂交条件的实例。
用于检测核苷酸探针和同源DNA或RNA序列间杂交的适宜条件包括在5×SSC(标准柠檬酸盐水)中预浸泡含有DNA片段或RNA的滤膜10分钟,滤膜在5×SSC(Sambrook等,1989),5×Denhardt溶液(Sambrook等,1989),0.5%SDS和100μg/ml变性超声鲑鱼精子DNA(Sambrook等,1989)的溶液中预杂交,随后在约45℃,在含有随机引发的(Feinberg,A.P.和Vogelstein,B.(1983)Anal. Biochem.132:6-13)32p-dCTP标记的(比活性>1×109cpm/μg)探针的相同溶液中进行杂交12小时。然后在至少55℃温度,特别是在60℃,更特别是在65℃,例如至少70℃,或至少75℃,在2×SSC,0.5%SDS中洗滤膜2次30分钟。
用X射线胶片检测在这些条件下与寡核苷酸探针杂交的分子。
比对和同一性
本发明的核苷酸序列与本文公开的序列可以有至少75%,或至少85%,特别地至少90%,或至少95%的同一性,例如至少98%的同一性。
对于本发明的目的,用对蛋白质和DNA比对均有用的Needleman-Wunsch比对(即整体比对(global alignment))进行序列比对和同一性分值的计算。分别用默认评分矩阵BLOSUM50和同一性矩阵进行蛋白质和DNA的比对。缺口(gap)中第一个残基的罚分对于蛋白质是-12,对于DNA是-16,而缺口中其它残基的罚分对于蛋白质是-2,对于DNA是-4。比对利用FASTA软件包版本v20u6(W.R.Pearson和D.J.Lipman(1988),“用于生物学序列分析的改良工具”,PNAS 85:2444-2448,和W.R.Pearson(1990)“用FASTP和FASTA进行的快速灵敏序列比较”,Methods inEnzymology,183:63-98)进行。
脂肪氧合酶的用途
锰脂肪氧合酶,如上述的锰脂肪氧合酶可以用于下列应用,例如以和所指出的出版物中所述类似的方式应用。
脂肪氧合酶可以用做制备焙烤产品,如面包,饼干和蛋糕的面团的添加剂。因此脂肪氧合酶可以用于面包的生产过程,包括将脂肪氧合酶添加到面团中,揉面及焙烤面团以生产焙烤产品。SU 426640 A,JP58190346A[SLK1],JP 1165332 A[SLK2],JP 8322456[SLK3],JP10028516[SLK4],JP 08322456,JP 2964215。如JP 11299440 A所述它也可以用于面条的生产中。
脂肪氧合酶可以用于漂白,例如β-胡萝卜素,小麦面粉和小麦面团的漂白。US1,957,333-1,957,337。
它也可以用于氧化脂肪酸混合物产生氢过氧化脂肪酸,用作脂质过氧化的加速剂,以及用作分析工具以估算某些油中亚油酸和亚麻酸的含量。
本发明提供了包含脂肪氧合酶和表面活性剂,特别是阴离子表面活性剂,如LAS(直链烷基苯磺酸盐)的去污剂组合物。有利的是,在这种表面活性剂存在的情况下,本发明脂肪氧合酶有好的稳定性。可以如US3635828[SLK5]或US 5789362[SLK6]所述配制此去污剂。有利地,如DK9800352[SLK7]所述,脂肪氧合酶也可以用于漂白织物或硬表面上的污迹。
如JP 09163953,EP 772980,JP 2000-106832所述,脂肪氧合酶也可以用于淀粉的修饰。如EP 947142,DE 19840069或JP 61078361所述,它也可以用于蛋白质的修饰,或如JP 5905128,US 3729379所述用于油的修饰(共轭脂肪酸的生产)。
通过氧化酶,如漆酶,胆红素氧化酶等,可以使用脂肪氧合酶交联蛋白质。EP947142。
通过将脂肪氧合酶加到鱼肉中,脂肪氧合酶可以用于获得具有改进的粘性和改进的味道的糊状海产品,如Kamaboko,Hanpen。JP 61078361.
脂肪氧合酶也可以用于生产番茄加工产品。它可以用于番茄糊,番茄调料(salsa),调味番茄酱等等。EP 983725。
通过脂肪氧合酶与不饱和的4-24C脂肪酸反应,脂肪氧合酶可以用于氢过氧化脂肪酸的产生。JP 11029410。
亚油酸和亚麻酸的氢过氧化物可以进一步转化成例如生长调节激素茉莉酮酸,而来自于花生四烯酸的产物可以转化成生理学效应物白三烯和脂毒素。
脂肪氧合酶的应用不应该限于上述的例子。因为氢过氧化物,脂肪氧合酶反应的产物是产生自由基的良好氧化剂,故脂肪氧合酶可以用于利用氧化反应的任何其它应用,如漂白食物材料或纺织品染料,或化学化合物的聚合以生产塑料材料或纤维。
脂肪氧合酶活性的测定法
在25℃,通过监测氢过氧化物的形成以分光光度法检测脂肪氧合酶的活性。对于标准分析,加10μL酶到含有980μL 25mM磷酸缓冲液(pH7.0)和10μL底物溶液(用0.2%(体积/体积,v/v)吐温20分散的10mM亚麻酸)的1mL石英比色杯中。通常充分稀释酶以确保在第一分钟内最多转化10%的所加底物。随后,测定234nm处的吸光度,从曲线的线性部分估计转化速率。在234nm,一个单位的酶引起0.001/min的吸光度增加。
底物特异性的检测
用标准测定条件并使用大量不同化合物作为底物研究脂肪氧合酶的底物特异性。用0.2%(体积/体积)吐温20将所有底物制成分散体。因为粘稠使得不可能精确测量体积,故通过质量确定配制这些母液时添加的化合物的量。通过变化加入测定中的底物的量测定极限速率常数和特异性常数。由此得到的速率对所用的底物浓度作图。最后,通过非线性最小二乘方回归对此图进行Michaelis-Menten方程式的理论双曲线拟合。其中假定顺式-反式-共轭氢(过氧)化脂肪酸有23,000M-1cm-1的分子消光系数。
实施例
材料和方法
分子克隆技术见Sambrook等(1989)所述。
用下面的商业质粒和大肠杆菌株系进行亚克隆和DNA文库构建:
pT7Blue(Novagen)
pUC19(TOYOBO,日本)
大肠杆菌JM109(TOYOBO,日本)
大肠杆菌DH12S(GIBCO BRL,Life Technologies,美国)
用下面的商业试剂盒进行cDNA克隆:
cDNA合成试剂盒(Takara,日本)
Marathon cDNA扩增试剂盒(Clontech,美国)
寡聚dT纤维素粉(Invitrogen荷兰)
用DIG-标记和检测试剂盒(Boehringer Manheim)进行杂交探针的标记和检测。尼龙膜Hybond-N+(Amersham,英国)用于Southern印迹和菌落杂交的DNA转移。
培养基和缓冲溶液
COVE-ar:每升342.3g蔗糖,20ml COVE盐溶液,10mM丙烯酰胺,15mM CsCl2,30g纯净琼脂(Difco)
COVE2-ar:每升30g蔗糖,20ml COVE盐溶液,10mM丙烯酰胺,30g纯净琼脂(Difco)
COVE盐溶液:每升26g KCl,26g MgSO4-7H2O,76g KH2PO4,50mlCove痕量金属。
Cove痕量金属:每升0.04g NaB4O7-10H2O,0.4g CuSO4-5H2O,1.2gFeSO4-7H2O,0.7g MnSO4-H2O,0.7g Na2MoO2-2H2O,0.7g ZnSO4-7H2O。
AMG痕量金属:每升14.3g ZnSO4-7H2O,2.5g CuSO4-5H2O,0.5gNiCl2,13.8g FeSO4,8.5g MnSO4,3.0g柠檬酸。
YPG:每升4g酵母提取物,1g KH2PO4,0.5g MgSO4-7H2O,15g葡萄糖,pH6.0。
STC:0.8M山梨醇,25mM Tris pH8,25mM CaCl2。
STPC:在STC缓冲液中40%PEG4000。
Cove顶层琼脂糖:每升342.3g蔗糖,20ml COVE盐溶液,10mM乙酰胺,10g低熔点琼脂糖。
MS-9:每升30g大豆粉,20g甘油,pH6.0。
MDU-2Bp:每升45g麦芽糖-1H2O,7g酵母提取物,12g KH2PO4,1g MgSO4-7H2O,2g K2SO4,5g尿素,1g NaCl,0.5ml AMG痕量金属溶液pH5.0。材料
α-32P-dCTP(3000μCi/mmol),dNTP,α-33p-ddNTP,Hybond-N膜,和DNA标记珠(-dCTP),T-引发的第一链试剂盒,和Thermo Sequenase试剂盒来自于Amersham Pharmacia Biotech(Uppsala,瑞典)。TA克隆试剂盒来自于Invitrogen(Groningen,荷兰)。Taq DNA聚合酶和增强的禽RT-PCR试剂盒来自于Sigma(St,Louis,MO)。限制性酶来自于New England Biolabs(Beverly,MA)。如Su和Oliw(见上)所述,获得和生长禾顶囊壳。Qiagen Plant RNeasy mini和QIAquick凝胶提取试剂盒来自于Merck Eurolab(Stockholm,瑞典)。从TIB Molbiol(Berlin,德国)获得PCR简并引物,而从CyberGene(Huddinge,瑞典)购买测序引物。用Life Technologies(Tby,瑞典)的试剂盒(用于快速扩增cDNA末端的5’-RACE系统)进行总RNA的5’-RACE和反转录。
实施例1:源于禾顶囊壳的LOX的部分肽序列的测定
基本上如Chao Su和Ernst H.Oliw(J.Biological Chemistry,273(21),13072-13079(1998))所述,培养禾顶囊壳小麦变种真菌株系并回收脂肪氧合酶。
为了从酶的N-末端部分获得数据,根据制造商的说明,在494蛋白质测序仪上(Applied Biosystems)用传统的edman降解法直接分析约10mg酶。
冻干处理另外40μg样品使之降低到约20μl,加入20μl含有DTT的SDS样品缓冲液后37℃温育30分钟,然后煮沸样品3分钟。然后加入5μl 0.5M在1 M Tris-HCl(pH7.5)中的碘乙酰胺,室温下温育样品20分钟,然后根据制造商的说明,在SDS-PAGE(4-20%,Novex)上电泳样品。根据Novex的标准方法染色凝胶。
随后切下凝胶条带(60kDa),用刀片切碎。在37℃,在0.5M tris pH9.2/ACN(1:1)中洗凝胶碎片2次45分钟。用100%ACN处理凝胶碎片10分钟以使凝胶碎片收缩。除去ACN,凝胶碎片在speed-vac.中干燥。加入200ml 0.1M NH4CO3(AMBIC),温育15分钟。除去AMBIC,加入100mlACN。再一次温育10分钟,随后除去ACN,在speed-vac.中干燥。将使用AMBIC的循环重复2次。最后的干燥步骤后,加入20ml 0.05mg/ml在0.1M tris pH9.2和10%ACN中的胰蛋白酶。温育10分钟。然后,加入300ml 0.1M tris pH9.2,10%CAN。在37℃持续温育过夜。之后除去上清液(留做对照),通过加30ml 10%TFA从凝胶中提取出肽。5分钟后,抽取TFA并收集。通过加150ml 0.1%TFA,60%ACN到凝胶碎片中,并在37℃温育30分钟进行进一步的提取,共2次。收集所有的提取物(30ml+150ml+150ml),在speed-vac.中浓缩到50ml。用TFA/异丙醇溶剂系统,使浓缩物样品(5ml)在Vydac C-18柱上进行RP-HPLC,以观察是否有任何肽存在。其余的样品过柱以收集肽。平行操作空白凝胶条对照。为了使肽的损失最小化,没有进行任何再纯化就直接进行了选定级分的测序。
由此得到的N-末端序列如SEQ ID NO:21所示,两个内部的肽(表示为fr29和34)如SEQ ID NOS:19和20所示。
进一步,加约100μg脂肪氧合酶到40μl 0.05M磷酸钾,10mM EDTA,1%Triton X-100,0.05%SDS,pH7.3中,加热到90℃,4分钟,然后使之冷却。然后向样品中加入25mU O-糖苷酶(无BSA)和800mU EndoF糖苷酶(Boehringer),37℃放置过夜。然后,根据制造商的说明,样品中加入75μl SDS样品缓冲液,在SDS-PAGE(Novex 4-20%)上7条泳道中电泳。
从凝胶上切下60kDa条带,在eppendorf微量离心管中切碎,并在37℃,用400μl 0.5M Tris-HCl,pH9.2:ACN 1∶1洗两次45分钟。然后用200μl ACN处理凝胶碎片10分钟,接着在speed-vac.中干燥。加入400μlNH4CO3,放置10分钟,除去上清液,然后用另外200μl ACN处理碎片10分钟,然后干燥。加入400μl H2O,放置样品10分钟,之后再一次用ACN重复这个过程。然后凝胶碎片中加入25μl 0.1mg/ml胰蛋白酶+300μl0.1M Tris-HCl,10%ACN,pH9.2,37℃放置过夜。温育后,加35μl 10 TFA,30分钟后取上清液进行HPLC(Vydac C 18,在0.1%TFA中达到80%的乙腈梯度)。然后,用150μl 0.1%TFA,60%乙腈进一步提取凝胶碎片2次。取上清液,在speed-vac.中蒸发到约50μl,再加入100μl 0.1%TFA,然后再一次蒸发达到50μl,然后用这50μl进行HPLC。
得到了3个氨基酸序列(表示为fr20,21和25),如SEQ ID NO:16,17和18所示。
实施例2:从禾顶囊壳克隆LOX的基因组克隆和cDNA克隆真菌染色体DNA的制备
在25℃,在YPG(每升组成为:4g酵母提取物,1g KH2PO4,0.5gMgSO4-7H2O,15g葡萄糖,pH6.0)中,温和搅拌培养禾顶囊壳小麦变种真菌株系6天。用Miracloth(Calbiochem,美国)过滤收集菌丝体,用去离子水洗两次,在滤纸上短时干燥后,用液氮冷冻菌丝体,在干冰上用研钵磨碎。将约0.2g磨碎的菌丝体放入1.5ml eppendorf管中,而且在0.5ml由100mM NaCl,25Mm EDTA,1%SDS和50mM Tris-HCl(pH8)组成的缓冲溶液中重悬浮。加入3μl 25mg/ml蛋白酶K后,在65℃温育管子30-60分钟。用相同体积的苯酚抽提溶液,用0.7体积的异丙醇在-20℃沉淀DNA。在0.5ml无菌水中重悬浮沉淀,用50μg RNase在37℃消化留下的RNA 30分钟。再一次用苯酚抽提DNA和进行乙醇沉淀。在适量的无菌水中重悬浮沉淀。
mRNA的制备和cDNA的合成
在25℃,在YPG中,温和搅拌培养禾顶囊壳小麦变种真菌株系6天。确定脂肪氧合酶活性后,如上所述,收集菌丝体,在干冰上磨碎以用于按苯酚-氯仿方法制备总RNA。用寡聚dT纤维素粉(Invitrogen,荷兰)从总RNA中进行mRNA纯化。
用cDNA合成试剂盒(Takara,日本)进行cDNA的合成。在含有每种dNTP各1.0mM,4μg寡聚(dT)18和2μg随机引物及100U反转录酶和第一链合成缓冲液的混合物中,用5-6μg热变性mRNA做为模板,合成第一链cDNA。在室温下,将总共50μl反应混合物放置10分钟,然后在42℃温育1小时。温育后,反应混合物在冰上急冷2分钟,之后用于第二链cDNA的合成。将1138U大肠杆菌DNA聚合酶及5μl大肠杆菌RNase H/大肠杆菌DNA连接酶混合物和第二链DNA合成缓冲液加入第一链合成混合物中,用DEPC-H2O稀释到240μl。在12℃温育反应混合物1小时,22℃温育1小时及70℃温育10分钟。随后加10U T4 DNA聚合酶到反应混合物中,37℃温育10分钟。合成的cDNA进行琼脂糖凝胶电泳以确定质量。
通过PCR进行LOX基因的部分克隆的分离
以实施例1中测定的氨基酸序列为基础设计和合成下面引物。
将禾顶囊壳的亚油酸二醇酯(linoleate diol)合成酶(Genbank登记号#AF124979)的核苷酸序列作为密码子使用的参照。
N-末端侧的引物1:SEQ ID NO:9(与SEQ ID NO:21 N-末端氨基酸第1-5位对应)。
C-末端侧1的引物2:SEQ ID NO:10(与fr34,即SEQ ID NO:20的氨基酸第18-25位对应)。
C-末端侧2的引物3:SEQ ID NO:11(与fr34,即SEQ ID NO:20的氨基酸第6-15位对应)。
在50μl含有每种dNTP各2.5mM,引物1和引物2各20pmol,2.5ULA taq聚合酶(Takara,日本)和Takara为LA Taq提供的GC缓冲液I的反应混合物中,用0.6μg禾顶囊壳的染色体DNA做模板进行聚合酶链式反应(PCR)。步骤1后,加LA taq聚合酶到反应混合物中。
步骤 | 温度 | 时间 |
1 | 98℃ | 10分钟 |
2 | 96℃ | 20秒 |
3 | 53℃ | 45秒 |
4 | 72℃ | (27+3×循环)秒 |
5 | 72℃ | 10分钟 |
*重复50次步骤2到步骤4。
在上述反应混合物中进行第二次PCR反应,但是用2μl第一次PCR产物做为模板,用引物3代替引物2。除了步骤2到步骤4重复30次外,反应条件与上述相同。
用QIAquickTM凝胶提取试剂盒(Qiagen)凝胶纯化扩增的1kb片段,将其亚克隆至pT7Blue。测定PCR克隆的序列,见SEQ ID NO:3所示。从推导的此PCR片段的氨基酸序列,证明引物1与预料之外的别的位置杂交,然而,在PCR片段(SEQ ID NO:8)的连续216个氨基酸的序列中发现实施例1中测定的氨基酸序列250599Bfr25(SEQ ID NO:18)。同一性检索表明此216个氨基酸序列与人15S脂肪氧合酶(Genbank U78294,GENESEQP W93832),人花生四烯酸12-脂肪氧合酶(Swiss-ProtP18054)和贵重珊瑚(Plexaura homomalla)8R-脂肪氧合酶(GenBankAF003692,SPTREMBL O16025)有最高同一性。结果表明得到的PCR片段含有脂肪氧合酶基因。与人15S获得了同一性的最高分值,此同一性的最高分值不到25%。
基因组LOX基因的克隆
为了获得全长基因组克隆,用PCR片段做探针,在禾顶囊壳基因组DNA上进行southern印迹分析。以上述结果为基础,用SalI消化基因组DNA,在1.0%琼脂糖凝胶上分离。从凝胶上回收约6kb的DNA消化产物,与用BAP处理的通过SalI线性化的pUC19连接。转化连接混合物进入大肠杆菌(E.coli)DH12S中以构建部分基因组文库。用PCR片段做为探针,通过菌落杂交进行筛选,分离阳性大肠杆菌菌落,回收到称为pSG16的质粒。质粒pSG16含有源于禾顶囊壳的6kb SalI片段。在这个6kb片段外,还确定到包括此PCR克隆的4.1kb长序列,见SEQ ID NO:4所示。最大的开放阅读框(ORF)含有上述216个氨基酸的序列及与fr20,21,29和34(SEQ ID NOS:16,17,19和20)类似的序列,但是没有实施例1中检测到的N-末端序列(SEQ ID NO:21)。在此最大的开放阅读框上游发现了两个其它的小ORF,但是它们中没有一个有该N-末端序列。为了发现正确的起始ATG密码子,克隆cDNA是必要的。
LOX基因的cDNA克隆的分离
从产生脂肪氧合酶的菌丝体中提取总RNA,并通过寡聚dT纤维素粉进行mRNA的制备。以获得全长cDNA为目的用cDNA合成试剂盒(Takara,日本)从mRNA合成cDNA,凝胶纯化1-4kb的cDNA用于进行部分cDNA文库的构建。通过与Marathon cDNA扩增试剂盒(Clontech,美国)的衔接子连接构建文库,这使得可以用衔接子引物(AP1)和设计针对目标克隆内部序列的定制引物进行目的cDNA的扩增。
对于LOX cDNA的扩增,根据基因组克隆的序列设计两个引物,引物4(SEQ ID NO:12)和引物5(SEQ ID NO:13)。用引物4和AP1扩增C-末端部分,用引物5和AP1扩增N-末端部分。
PCR反应混合物由2.5mM dNTP,引物4和AP1或引物5和AP1各30pmol,5单位LA taq聚合酶(Takara)和提供的GC缓冲液I。反应条件如下所示,步骤1后,加LA taq聚合酶到反应混合物中。
步骤 | 温度 | 时间 |
1 | 98℃ | 5分钟 |
2 | 95℃ | 30秒 |
3 | 74℃ | 15秒 |
4 | 68℃ | 3分钟 |
5 | 95℃ | 30秒 |
6 | 95℃ | 5分钟 |
7 | 54℃ | 30秒 |
8 | 68℃ | 15秒 |
*重复15次步骤2到步骤4,每3次重复后,步骤3的温度降低到4℃。重复20次步骤6到步骤8。
结果,对于5’-末端和3’-末端分别扩增到0.6kb和1.6kb的片段,测定序列,如SEQ ID NO:5和SEQ ID NO:6所示。基于预测的起始ATG和终止密码子TAA周围的序列,设计用于扩增末端-到-末端cDNA的引物6(SEQ ID NO:14)和引物7(SEQ ID NO:15)。还在两个末端引入期望的限制性酶位点以用于进一步的质粒构建。
反应混合物含有0.08μg cDNA文库,2.5mM dNTP,引物6和7各30pmol,1单位的LA taq聚合酶(Takara)和GC缓冲液。反应条件如下所示,步骤1后,加LA taq聚合酶到反应混合物中。
步骤 | 温度 | 时间 |
1 | 98℃ | 10分钟 |
2 | 96℃ | 20秒 |
3 | 53℃ | 45秒 |
4 | 72℃ | (27+3×循环)秒 |
5 | 72℃ | 10分钟 |
*重复50次步骤2到步骤4。
分离PCR扩增的1.9kb片段,将其克隆入pT7Blue中,产生pSG26。测定全长cDNA的序列。推导出的开放阅读框由1857bp组成,与618个氨基酸和67600Da分子量相符。与基因组序列的比较证明LOX基因在N-末端一侧含有一个内含子。通过信号序列测定程序预测的N-末端序列是“ALPLAAEDAAAT”。用全长氨基酸序列进行的同一性检索表明它与人15S脂肪氧合酶(Genbank登记号W93832)有最高同一性(不到25%)。
转化质粒pSG26到大肠杆菌JM109中,在DSMZ保藏为DSM 13586,登记日期是2000年7月5日。
实施例3:在米曲霉(A.oryzae)中表达禾顶囊壳的LOX
宿主生物体
在丹麦专利申请PA 1999 01726中描述了米曲霉(A.oryzae)BECh2。它是JaL228(在WO98/123000中有描述)的突变体,而JaL228是IFO4177的突变体。
米曲霉(A.oryzae)的转化
在100ml YPG培养基中接种米曲霉(A.oryzae)菌株BECh2,并在32℃温育16小时,其间以80rpm进行搅拌。过滤收集生长的菌丝体,随后用0.6M KCl冲洗,在30ml含有浓度为30μg/ml的Glucanex(NovoNordisk)的0.6M KCl中重悬浮。在32℃,60rpm搅拌下,温育混合物直到形成原生质体。过滤除去残留的菌丝体后,通过离心收集原生质体,用STC缓冲液洗两次。用血细胞计数器(hematitometer)计数原生质体,在STC∶STPC∶DMSO(8∶2∶0.1)溶液中重悬浮到1.2×107个原生质体/ml的最终浓度。加约4μg DNA到100μl原生质体溶液中,轻轻混合,在冰上温育30分钟。加1μSTPC缓冲液到混合物中,37℃温育另外30分钟。加入10ml50℃预温的Cove顶层琼脂糖后,将反应混合物倒在COVE-ar琼脂平板上。在32℃温育平板5天。
SDS-PAGE
根据提供的方法,用商业的凝胶PAGEL AE6000 NPU-7.5L(7.5T%)及仪器AE-6400(Atto,日本)进行SDS聚丙烯酰胺电泳。在2×浓度的15μl加样缓冲液(100mM Tris-HCl(pH6.8),200mM二硫苏糖醇,4%SDS,0.2%溴酚蓝和20%甘油)中重悬浮15μl样品,煮沸5分钟。加20μl样品溶液到聚丙烯酰胺凝胶上,在每块凝胶20mA下,在电泳缓冲液(25mM Tris,0.1%SDS,192mM甘氨酸)中电泳。用考马斯亮蓝染色由此得到的凝胶。
表达质粒的构建
用BglII和XhoI消化含有禾顶囊壳LOX cDNA的质粒pSG26,用BamHI和XhoI消化过的pMT2188与含有LOX基因的1.9kb片段连接。质粒pMT2188具有修饰的黑曲霉中性淀粉酶启动子,构巢曲霉(Aspergillus nidulans)TP1前导序列,黑曲霉葡萄糖淀粉酶终止子,做为真菌转化的标记的构巢曲霉amdS基因和做为大肠杆菌(E.coli)转化的标记的酿酒酵母ura3。用pyrF基因缺陷但可以用酿酒酵母Ura3弥补的大肠杆菌(E.coli)DB6507进行转化。由此产生的质粒称为pSG27。
在米曲霉(A.oryzae)中表达禾顶囊壳LOX
用质粒pSG27转化米曲霉(A.oryzae)BECh2,分离筛选出的阳性转化体。转化体在32℃,在COVE2-ar上生长5天,之后接种到100ml MS-9摇瓶中。在32℃强力搅拌下培养1天后,转移每个培养物3ml到摇瓶的100ml MDU-2Bp中,在32℃培养3天。在3500rpm,离心培养液10分钟,收集上清液。如前所述,分光光度法检测上清液的脂肪氧合酶活性。阳性转化体显示了约50,000U/ml培养液,而未转化的米曲霉(A.oryzae)BECh2没有显示活性。也用培养上清液进行SDS-PAGE分析。阳性转化体显示了90-110kDa成片不清晰条带,表明蛋白质高度糖基化。未转化的米曲霉(A.oryzae)BECh2没有显示任何主要条带。
实施例4:重组脂肪氧合酶的纯化
在40mL 25mM Tris-HCl(pH8.0)中溶解如前面实施例所述制备的1g冻干的粗脂肪氧合酶,然后过滤(0.45μm,Millex-HV型,Millipore)。在室温下进行所有的上述和随后步骤。以1mL/分钟用25mM Tris-HCl(pH8.0)将滤液加载到SP-Sepharose Fast Flow(2.6×14cm)上。然后用相同缓冲液以2.5mL/分钟洗柱,直至达到基线(约4个柱体积)。然后用2个柱体积的,25mM Tris-HCl(pH8.0)中从0到330mM的线性梯度NaCl洗脱结合的蛋白质。收集10mL的级分。用25mM Tris-HCl(pH8.0)中的1M NaCl清洗柱子。汇合含有多数纯化的脂肪氧合酶的级分(按SDS-PAGE和活性测定法所估计的),并用Amicon室(10,000 NMWL,YM10,Millipore)浓缩。通过透析,最后将酶转移到50mM磷酸钠(pH7.0)中,使用前在-20℃等份贮藏。
SDS-PAGE分析表明脂肪氧合酶已纯化达到同质。发现这个酶有估算的90-110kDa的分子量,稍高于基于氨基酸序列的理论值(65.6kDa)。这被认为指示了糖基化。正如利用阳离子交换层析实现的成功纯化所证明的,此蛋白质被发现有非常高的等电点。
实施例5:锰-脂肪氧合酶基因和推导的蛋白质序列的确定
1.锰脂肪氧合酶的内部肽氨基酸序列和C-端氨基酸
用禾顶囊壳株系(与前面的实施例不同),按Su和Oliw所述(见上),纯化锰脂肪氧合酶达到同质。产生内部肽,纯化,并基本上如用于另一种禾顶囊壳蛋白质的所述方法(Hornsten L. Su C,Osbourn AE,Garosi P,Hellman U,Wernstedt C和Oliw EH,亚油酸二醇酯合成酶的克隆揭示了与前列腺H合成酶同源,J Bio Chem 274(40):28219-24,1999)通过Sanger法测序。尽管封闭了锰脂肪氧合酶的N-末端氨基酸,但是通过C-末端测序获得了4个C-末端氨基酸。
(i)C末端氨基酸序列
这些C-末端氨基酸是FLSV。
(ii)内部氨基酸序列
获得了下面8个内部氨基酸序列(其中(K),(K/R)和(E)表示Lys-C,胰蛋白酶和V8分别在K残基,K或R残基,和E残基的C-末端一侧切割肽的事实):
(K)LYTPQPGRYAAACQGLFYLDARSNQFLPLAIK(相应于SEQ ID NO:23的氨基酸第205-237位,但具有K206L替代)
(K/R)HPVMGVLNR(相应于SEQ ID NO:23的氨基酸第295-304位,但在第295位为Lys或Arg)(相应于SEQ ID NO:23的氨基酸第196-205位,但在196位为Lys或Arg)(E)M?AGRGFDGKGLSQG(W/M)PFV(相应于SEQ ID NO:23的氨基酸569-587位,但氨基酸第507位是不确定的Met且氨基酸第584位是Trp或Met)
(K/R)GLVGEDSGPR(相应于SEQ ID NO:23的氨基酸第365-375位,但发现氨基酸第365位是Lys或Arg,而368位是Val)
(K)TNVGADLTYTPLD/AD/WK/LP/ND/NE(相应于SEQ ID NO:23的氨基酸第237-255位,但发现氨基酸第242位是Ala,250位是Asp或Ala,251位是Asp或Trp)
(K)G/F SGVLPLHPAw(相应于SEQ ID NO:23的氨基酸第472-483位,但发现氨基酸第473位是Gly或Phe,氨基酸第483位是不确定的Trp)
(K)QTVDDAFAAPDLLAGNGPGRA(相应于SEQ ID NO:23的氨基酸第532-553位,但发现氨基酸第536位是Asp,552位是Arg)
2.用简并引物经RT-PCT产生锰-脂肪氧合酶cDNA
由于禾顶囊壳基因组的高GC含量,本发明这部分的实施是困难的。
必须优化从禾顶囊壳分离总RNA以及将mRNA转录为cDNA的方法。尽管用DNAses和其它预防措施,cDNA仍常常有基因组DNA的污染。
用超过30个的简并引物以各种组合进行大量试验后,通过RT-PCR获得锰-脂肪氧合酶的第一个cDNA克隆。它是通过下面的简并引物获得的,这些引物是以内部肽1和2及上述为基础的。
Mn60(5′-AACCAGTTCCTSCCSCTCGCSATCAA)
Mn15R(5′-GTCGAGGTAGAAGAGGCCCTGRCAVGC),
EO3a(5′-CATCCSGTSATGGGYGTSCTBAA)
EOr3a(5′-CGGTTSAGGACRCCCATVACVGGRTG).
引物Mn60和EOr3A产生了约230bp的RT-PCR条带,而引物EO3A和Mn15R产生了约220bp的RT-PCR条带。来自于这个序列(MnS2:5’-CCGTTCAGCGTCGAGAGCAAGG)的有义引物和来自其它序列(MnS1,5’-TCTCGGGGATCGTGTGGAAGAGCA)的反义引物扩增了337bp的片段。测序扩增子,它在其中一个开放阅读框中含有肽1的氨基酸序列。用扩增子做为Northern印迹分析和筛选基因组文库的探针(Hornsten等,见上)。
3.禾顶囊壳基因组文库的筛选
如Bowyer P等,Science 267(5196):371-4,1955所述,在λZAPII中得到禾顶囊壳的基因组文库。用来自cDNA序列的0.33kb探针筛选它。对超过100 000个噬菌斑的筛选产生了11个阳性克隆,所述阳性克隆通过另外2-3轮噬菌体筛选循环进行了噬斑纯化。根据公知的方法,用辅助噬菌体切除Bluescript SK噬菌粒。限制性酶分析表明,拯救的所有噬菌粒都含有相同的8kb插入片段。
4.禾顶囊壳的Mn-LO基因和编码区的测序
基于循环测序,用两种不同方法进行两条链的测序。由于基因中高GC含量(超过60%GC),测序是困难的。
测序3.4kb禾顶囊壳基因组,锰-脂肪氧合酶基因的2725个核苷酸的序列中包括133bp的内含子。通过5’RACE,从2mRNA的转录起始位点(a1gcaggttc)和蛋白质翻译起始点A72TG(核苷酸第72位)鉴定锰-脂肪氧合酶的基因。发现了在位置2060-2062有终止密码子和C-末端氨基酸FLSV。测序大于0.6kb的3’-非翻译区,发现了如下假定的聚腺苷酸化信号:
用5’-RACE和cDNA测序以证实推导的开放阅读框和外显子-内含子边界。确定了转录起始点,翻译起始点和翻译终点,见SEQ ID NO:22和23所示。
发现内含子长度是133bp,而且有如SEQ ID NO:24所示的序列。发现其定位于核苷酸第372-373位间,即SEQ ID NO:22的Ser108和Arg109间。
实施例6:天然和遗传修饰的锰-脂肪氧合酶的表达
用限制性酶SpeI和NsiI,我们从Bluescript SK噬菌粒中将含有锰-脂肪氧合酶基因编码区的基因组片段(3kb)亚克隆到质粒pGEM-5Zf(Promega)的多克隆位点(有SpeI和NsiI位点)中。
按如下修饰5’-端和内含子。用SpeI和BseRI切割带有插入片段的pGEM-5Z,切下基因5’-端和带有内含子(1323bp)的部分基因组序列。在pGEM中将这一部分用约405bp的cDNA序列置换,而所述cDNA序列是通过用SpeI和BseRI切割448bp的PCR产物得到的。这个载体命名为pGEM_Met。所述PCR产物是用对翻译起始区特异的有义引物(在引物5’-末端有SpeI和NdeI位点,5’-TTACTAGTCATATGCGCTCCAGGATCCTTGCT)和位于BseRI位点3’-末端的基因特异性反义引物产生的。故如此插入的这个cDNA部分含有ORF的起始部分(没有位于核苷酸第372-373位,即Ser108和Arg109间的内含子,如上表所示),以致在载体pGEM_Met中得到了整个ORF。
利用距离终止信号约130bp的BbvCI位点,用PCR修饰3’-末端。有义引物是基因特异的,而且位于该限制性位点的5’侧,而反义引物是根据末端氨基酸的核苷酸设计的并含有NdeI和NsiI限制性位点。用NsiI和BbvCI切割pGEM Met载体,用以相同方式切割的PCR产物置换切下的片段。这样产生了载体pGEM-Met-ter。因此能用NdeI切下这个载体中的经修饰的锰—脂肪氧合酶编码区。通过表达构建体的测序,已证实了所有的修饰。
1.锰-脂肪氧合酶在原核细胞(大肠杆菌)中的表达
如pET表达载体制造商(Stratagene)所建议,用NdeI线性化表达载体pET-19b,而且用NdeI切下修饰的锰-脂肪氧合酶编码区,将其连接入这个载体以便在大肠杆菌(E.coli)中实现表达。现在正在进行大肠杆菌(E.coli)中表达的重组锰-脂肪氧合酶的研究。
2.锰-脂肪氧合酶在真核细胞(巴斯德毕赤酵母(pichia pastoris),酿
酒酵母,构巢曲霉,禾顶囊壳)中的表达
我们计划使用带有pCIC9或相关载体的毕赤酵母表达试剂盒(Invitrogen),这些载体必须进行轻微修饰以适于我们的修饰的锰-脂肪氧合酶编码区。不同宿主间重组体锰-脂肪氧合酶的糖基化可以不同,这是可能的。因此我们计划在酿酒酵母,构巢曲霉,禾顶囊壳中研究一系列真核表达系统。糖基化可以改进重组酶的稳定性。
3.
锰-脂肪氧合酶在真核细胞(昆虫细胞)中的表达
我们计划使用Invitrogen的果蝇表达系统(Schneider 2细胞)和在C-末端没有His尾的表达载体。
4.
用于表达的遗传修饰的锰-脂肪氧合酶
我们发现锰-脂肪氧合酶属于脂肪氧合酶基因家族,这个发现为结构的合理修饰提供了巨大的可能性。几种脂肪氧合酶的3D序列是已知的,而且锰-脂肪氧合酶与大豆脂肪氧合酶-1的许多α-螺旋(Prigge ST,BoyingtonJC,Gaffney BJ和Amzel LM,脂肪氧合酶中的结构保守性:大豆脂肪氧合酶-1的结构分析和人脂肪氧合酶的模建。Proteins 24(3):275-91,1996)显示了显著的氨基酸同一性,其中的大豆脂肪氧合酶-1已用于许多脂肪氧合酶的模建。可以突变脂肪氧合酶的金属配体和其它结构重要的氨基酸,以增加酶的漂白特性和氧化特性。
4.1重要α-螺旋的氨基酸的定向诱变
比对锰-脂肪氧合酶与α-螺旋9(Prigge等,上文)的氨基酸序列,α-螺旋9含有可能是锰配体的WLLAK序列和两个His残基。这个螺旋中氨基酸的系统改变可能对酶活性和漂白特性有深远影响。以相同方式,比对锰-脂肪氧合酶的氨基酸序列与α-螺旋18,α-螺旋18含有铁配体和可能的锰配体(His和Asn)。应该突变锰-脂肪氧合酶中与大豆脂肪氧合酶-1(Prigge等,上文)的α-螺旋7,8,10-17,19-22对应的其它预测的α-螺旋。我们预测这些遗传修饰的锰-脂肪氧合酶中的一些可能有完全不同的特性,从而可能增强漂白效果。因此预测的锰配体是3个His残基,一个Asp残基和一个Val残基。锰-脂肪氧合酶可能属于具有“2-His-1-羧基面三联体”的酶。
4.2 C-末端氨基酸的定向诱变
我们计划将末端Val突变为Ile或其它残基,然后检测突变形式的漂白特性。
4.3锰脂肪氧合酶的嵌合形式
为了改进锰-脂肪氧合酶的特性,我们计划利用上述α-螺旋信息,将多个部分置换为大豆脂肪氧合酶中的对应序列。
实施例7:真核DNA的筛选
为了筛选真核真菌株系中的同源脂肪氧合酶基因,用禾顶囊壳LOX基因的cDNA做探针,对来源于几个真菌株系的基因组DNA进行Southern杂交。检测了下面种属的株系:稻梨孢霉(Pyricularia oryzae),Psaliotacampestris,娄地青霉(Penicillium roqueforti)和白地霉(Geotrichumcandidum)ATCC34614。如实施例2所述分离基因组DNA。
按如下利用DIG DNA标记混合物(Boehringer Mannheim),用地高辛配基-dUTP标记探针:用引物6(SEQ ID NO:14)和引物7(SEQ IDNO:15),通过PCR制备禾顶囊壳LOX全长cDNA形式的DIG标记探针。PCR反应混合物包含做为模板的0.1μg pSG26,1.25Mm dNTP,8%DIGDNA标记混合物,引物6和7各30pmol,1单位的LA taq聚合酶(Takara)和GC缓冲液。反应条件如下所示。步骤1后,加LA taq聚合酶到反应混合物中。
步骤 | 温度 | 时间 |
1 | 98℃ | 10分钟 |
2 | 94℃ | 2分钟 |
3 | 60℃ | 30秒 |
4 | 72℃ | 2分钟 |
5 | 72℃ | 10分钟 |
*重复30次步骤2到4。
使用前,凝胶纯化PCR产物,而且通过在98℃加热变性PCR产物。
在1.0%琼脂糖凝胶上分离用限制性酶消化的约5μg DNA,而且通过在0.2N HCl中浸泡凝胶30分钟和在0.5N NaOH+1.5M NaCl中浸泡凝胶30分钟使DNA变性,然后在1M Tris(Ph7.5)+1.5M NaCl中中和30分钟。然后,用2×SSC,通过真空转移15分钟,将变性的DNA转移到尼龙膜上。通过UV照射固定后,用尼龙膜进行杂交。杂交溶液由5×SSC,0.5%封闭试剂(Boehringer Mannheim),0.1%N-月桂酰肌氨酸和0.02%SDS组成。在60℃,用杂交溶液预杂交尼龙膜1小时。之后,加热变性的DIG-标记的探针到杂交溶液中,在60℃温育过夜。室温下,用包含2×SSC+0.1%SDS的冲洗缓冲液洗所得膜5分钟2次,随后在杂交温度下,用由0.1×SSC+0.1%SDS组成的冲洗缓冲液2洗15分钟2次。空气干燥洗过的膜,根据DNA检测试剂盒(Boehringer Mannheim)提供的方法,将膜用于DIG-标记DNA的检测。
结果,稻梨孢霉显示了清晰的阳性信号,而白地霉显示了非常弱的信号。结果表明稻梨孢霉有与禾顶囊壳LOX具有高度同一性的脂肪氧合酶基因,而白地霉有与禾顶囊壳LOX具有低同一性的基因。
实施例8:pH对锰-脂肪氧合酶的影响
在各种pH值下,检测如实施例4所产生的脂肪氧合酶的活性。发现亚油酸或亚麻酸做底物时,酶有宽的pH最适值,在pH6-10或7-11范围内有高活性。
在40℃,在各种pH下温育1小时后,检测酶的稳定性。发现酶在pH4-10范围内有好的稳定性。
实施例9:脂肪氧合酶的底物特异性
按上述方法,检测如实施例4所产生的脂肪氧合酶对各种底物的活性。根据Michaelis-Menten方程式,将结果表示为:kcat(或Vmax),KM和kcat/KM。
底物 | kcatμmol/min/mg | KMMM | kcat/KM |
亚油酸 | 5.63 | 0.0068 | 828 |
花生四烯酸 | 0.296 | 0.0175 | 16.9 |
亚油醇 | 3.32 | 0.0034 | 982 |
亚油酸甲酯 | 1.37 | 0.164 | 8.39 |
单亚油精 | 85.4 | ||
1,3-二亚油精 | 12.4 | ||
三亚油精 | 9.15 |
在pH7时,脂肪氧合酶对亚麻酸比对亚油酸显示了高约2倍的活性。
实施例10:通过天然锰-脂肪氧合酶进行的β-胡萝卜素的漂白
在pH4.5,6.5和9.5,用纯化的锰-脂肪氧合酶漂白β-胡萝卜素。发现在pH6.5有最高漂白活性。
实施例11:LAS对锰-脂肪氧合酶的影响
在pH7.0和pH10,用高达400ppm的LAS检测如实施例4所产生的禾顶囊壳脂肪氧合酶的活性。发现在pH7.0和10,该脂肪氧合酶可以抵抗高达400ppm(检测的最高浓度)LAS仍是充分稳定的。这表明在正常洗涤条件下(通常pH10和200ppm LAS),该脂肪氧合酶是足够稳定的。
0-10-1-1 | 表格-PCT/RO/134(EASY)与保藏的微生物或其它生物材料有关的声明(PCT细则第13条之2)制备时利用 | PCT-EASY版本2.92(01.03.2001更新) |
0-2 | 国际申请号 | PCT/DK01/00574 |
0-3 | 申请人或代理人的参考卷号 | 10092-WO |
11-11-2 | 下面列出与说明书中提及的保藏的微生物或其它生物材料有关的声明:页行 | 321-24 |
1-31-3-11-3-21-3-31-3-4 | 保藏鉴定保藏机构名称保藏机构地址保藏日期保藏号 | DSMZ-德意志微生物保藏中心Mascheroder weg 1b,D-38124 Braunschweig,德国2000年7月5日(05.07.2000 )DSMZ 13586 |
1-4 | 其它声明 | 无 |
1-5 | 声明适用的指定国 | 所有指定国 |
1-6 | 声明的单独提供这些声明之后将提交给国际局 | 无 |
序列表<110>Novo Nordisk A/S<120>脂肪氧合酶<130>10072<160>24<170>PateRtIn version 3.1<210>1<211>1857<212>DNA<213>禾顶囊壳(Gaeumannomyces graminis)<220><221>CDS<222>(1)..(1854)<223><220><221>mat_peptide<222>(49)..()<223><400>1atg cgc tcc agg atc ctt gcc ata gtc ttc gcg gca cgc cat gtg gca 48Met Arg Ser Arg Ile Leu Ala Ile Val Phe Ala Ala Arg His Val Ala
-15 -10 -5 -1gcg ctg cca ctc gct gcc gaa gac gct gcg gcg acg ctg tct ttg acg 96Ala Leu Pro Leu Ala Ala Glu Asp Ala Ala Ala Thr Leu Ser Leu Thr1 5 10 15tcc agc gcc tcc agc acc acc gtg ctc ccg tct ccg acc cag tac acg 144Ser Ser Ala Ser Ser Thr Thr Val Leu Pro Ser Pro Thr Gln Tyr Thr
20 25 30ctg ccc aac aac gac ccc aac cag ggg gca cgc aac gcc agt ata gct 192Leu Pro Asn Asn Asp Pro Asn Gln Gly Ala Arg Asn Ala Ser Ile Ala
35 40 45cgg aag cgg gag ttg ttc ctc tac ggc cca tcc act ctc ggg cag acg 240Arg Lys Arg Glu Leu Phe Leu Tyr Gly Pro Ser Thr Leu Gly Gln Thr
50 55 60acc ttc tac cct acc gga gag ctg ggg aac aac atc tcg gcc cgc gac 288Thr Phe Tyr Pro Thr Gly Glu Leu Gly Asn Asn Ile Ser Ala Arg Asp65 70 75 80gtg cta ctt tgg cgc caa gat gcg gcg aac cag acg gca acg gcg tac 336Val Leu Leu Trp Arg Gln Asp Ala Ala Asn Gln Thr Ala Thr Ala Tyr
85 90 95cgc gaa gcc aat gag acg ttt gca gat att acc agc cgt ggc ggt ttc 384Arg Glu Ala Asn Glu Thr Phe Ala Asp Ile Thr Ser Arg Gly Gly Phe
100 105 110aaa acg ctc gac gac ttt gcg ctc ctc tac aat ggt cac tgg aag gag 432Lys Thr Leu Asp Asp Phe Ala Leu Leu Tyr Asn Gly His Trp Lys Glu
115 120 125tcg gtt ccg gag ggc ata tcg aag ggc atg ttg agc aac tac acc tcg 480Ser Val Pro Glu Gly Ile Ser Lys Gly Met Leu Ser Asn Tyr Thr Ser
130 135 140gac ctt ctc ttt tcc atg gag cgg ctg tcc tcc aac cct tac gtc ctc 528Asp Leu Leu Phe Ser Met Glu Arg Leu Ser Ser Asn Pro Tyr Val Leu145 150 155 160aag cgc ctc cac cca gcc aag gac aaa ctg ccg ttc agc gtc gag agc 576Lys Arg Leu His Pro Ala Lys Asp Lys Leu Pro Phe Ser Val Glu Ser
165 170 175aag gtg gtg aag aag ctg acg gcc acc acg ctt gag gcg ctc cac aag 624Lys Val Val Lys Lys Leu Thr Ala Thr Thr Leu Glu Ala Leu His Lys
180 185 190ggc ggc cgc ctg ttc ctc gtg gac cac agc tac cag aag aag tac acc 672Gly Gly Arg Leu Phe Leu Val Asp His Ser Tyr Gln Lys Lys Tyr Thr
195 200 205ccc cag cca gga cgg tac gcc gcg gcc tgc cag ggg ctt ttc tac ctg 720Pro Gln Pro Gly Arg Tyr Ala Ala Ala Cys Gln Gly Leu Phe Tyr Leu
210 215 220gac gcg cgg tcc aac caa ttc ctg cct ctg gca atc aag acc aac gtg 768Asp Ala Arg Ser Asn Gln Phe Leu Pro Leu Ala Ile Lys Thr Asn Val225 230 235 240ggg gcg gac ctg acg tac acg ccc ctc gac gac aag aac gac tgg ctg 816Gly Ala Asp Leu Thr Tyr Thr Pro Leu Asp Asp Lys Asn Asp Trp Leu
245 250 255ctg gcc aag atc atg ttc aac aac aac gac ctg ttc tac tcc cag atg 864Leu Ala Lys Ile Met Phe Asn Asn Asn Asp Leu Phe Tyr Ser Gln Met
260 265 270tac cac gtg ctc ttc cac acg atc ccc gag atc gtg cac gag gcc gcc 912Tyr His Val Leu Phe His Thr Ile Pro Glu Ile Val His Glu Ala Ala
275 280 285ttc cgg acg ctg agc gac agg cac ccg gtc atg ggc gtg ctc aac cgc 960Phe Arg Thr Leu Ser Asp Arg His Pro Val Met Gly Val Leu Asn Arg
290 295 300ctc atg tac cag gcc tac gcc atc cgg ccc gtg ggc ggg gct gtg ctc 1008Leu Met Tyr Gln Ala Tyr Ala Ile Arg Pro Val Gly Gly Ala Val Leu305 310 315 320ttc aac ccc ggc ggg ttc tgg gac caa aac ttt ggc ctg ccc gcc tcg 1056Phe Asn Pro Gly Gly Phe Trp Asp Gln Asn Phe Gly Leu Pro Ala Ser
325 330 335gcc gcc atc gac ttc ccc ggc tcc gtg tac gcg cag ggc ggg ggc ggg 1104Ala Ala Ile Asp Phe Pro Gly Ser Val Tyr Ala Gln Gly Gly Gly Gly
340 345 350ttc cag gcc ggc tac ctg gag aag gac ctg cgg agc cgg ggg ctg gtc 1152Phe Gln Ala Gly Tyr Leu Glu Lys Asp Leu Arg Ser Arg Gly Leu Val
355 360 365ggc gag gac agc ggc ccg cgg ctg ccg cac ttc ccc ttc tac gag gac 1200Gly Glu Asp Ser Gly Pro Arg Leu Pro His Phe Pro Phe Tyr Glu Asp
370 375 380gcg cac cgc ctg atc ggg gcg atc cgg cgc ttc atg cag gcg ttc gtg 1248Ala His Arg Leu Ile Gly Ala Ile Arg Arg Phe Met Gln Ala Phe Val385 390 395 400gac tcg acg tac ggt gcc gac gac ggc gac gac ggg gcg ctg ctg cgc 1296Asp Ser Thr Tyr Gly Ala Asp Asp Gly Asp Asp Gly Ala Leu Leu Arg
405 410 415gac tac gag ctg cag aac tgg atc gcc gag gcc aac ggg ccg gcg cag 1344Asp Tyr Glu Leu Gln Asn Trp Ile Ala Glu Ala Asn Gly Pro Ala Gln
420 425 430gtg cgc gac ttc ccc gcg gcg ccg ctg cgg cgg cgc gca cag ctg gtg 1392Val Arg Asp Phe Pro Ala Ala Pro Leu Arg Arg Arg Ala Gln Leu Val
435 440 445gac gtg ctg acg cac gtg gcc tgg gtc acg ggc ggg gcg cac cac gtc 1440Asp Val Leu Thr His Val Ala Trp Val Thr Gly Gly Ala His His Val
450 455 460atg aac cag ggc tcg ccc gtc aag ttc tcg ggg gtg ctg ccg ctg cac 1488Met Asn Gln Gly Ser Pro Val Lys Phe Ser Gly Val Leu Pro Leu His465 470 475 480ccg gcg gcg ctg tac gcg ccc atc ccg acg acc aag ggc gcc acc ggc 1536Pro Ala Ala Leu Tyr Ala Pro Ile Pro Thr Thr Lys Gly Ala Thr Gly
485 490 495aac ggg acg agg gcg ggc ctg ctg gcg tgg ctg ccc aac gag cgg cag 1584Asn Gly Thr Arg Ala Gly Leu Leu Ala Trp Leu Pro Asn Glu Arg Gln
500 505 510gcc gtg gag cag gtc tcg ctg ctc gcg cgc ttc aac cgt gcg cag gtc 1632Ala Val Glu Gln Val Ser Leu Leu Ala Arg Phe Asn Arg Ala Gln Val
515 520 525ggg gac agg aag cag acg gtg cgc gac gcc ttc gcc gcg ccc gac ctg 1680Gly Asp Arg Lys Gln Thr Val Arg Asp Ala Phe Ala Ala Pro Asp Leu
530 535 540ctg gcc ggc aac ggg ccg ggg tac gcg gcg gcc aac gcg agg ttc gtc 1728Leu Ala Gly Asn Gly Pro Gly Tyr Ala Ala Ala Asn Ala Arg Phe Val545 550 555 560gag gac acg ggc cgt ata agt cgc gag atg gcg ggc aga ggg ttc gac 1776Glu Asp Thr Gly Arg Ile Ser Arg Glu Met Ala Gly Arg Gly Phe Asp
565 570 575ggc aag ggc ctc agc cag ggc atg ccg ttc gtc tgg acc gcg ctc aat 1824Gly Lys Gly Leu Ser Gln Gly Met Pro Phe Val Trp Thr Ala Leu Asn
580 585 590ccc gcc gtc aac cct ttt ttc cta agc gtc taa 1857Pro Ala Val Asn Pro Phe Phe Leu Ser Val
595 600<210>2<211>618<212>PRT<213>禾顶囊壳<400>2Met Arg Ser Arg Ile Leu Ala Ile Val Phe Ala Ala Arg His Val Ala
-15 -10 -5 -1Ala Leu Pro Leu Ala Ala Glu Asp Ala Ala Ala Thr Leu Ser Leu Thr1 5 10 15Ser Ser Ala Ser Ser Thr Thr Val Leu Pro Ser Pro Thr Gln Tyr Thr
20 25 30Leu Pro Asn Asn Asp Pro Asn Gln Gly Ala Arg Asn Ala Ser Ile Ala
35 40 45Arg Lys Arg Glu Leu Phe Leu Tyr Gly Pro Ser Thr Leu Gly Gln Thr
50 55 60Thr Phe Tyr Pro Thr Gly Glu Leu Gly Asn Asn Ile Ser Ala Arg Asp65 70 75 80Val Leu Leu Trp Arg Gln Asp Ala Ala Asn Gln Thr Ala Thr Ala Tyr
85 90 95Arg Glu Ala Asn Glu Thr Phe Ala Asp Ile Thr Ser Arg Gly Gly Phe
100 105 110Lys Thr Leu Asp Asp Phe Ala Leu Leu Tyr Asn Gly His Trp Lys Glu
115 120 125Ser Val Pro Glu Gly Ile Ser Lys Gly Met Leu Ser Asn Tyr Thr Ser
130 135 140Asp Leu Leu Phe Ser Met Glu Arg Leu Ser Ser Asn Pro Tyr Val Leu145 150 155 160Lys Arg Leu His Pro Ala Lys Asp Lys Leu Pro Phe Ser Val Glu Ser
165 170 175Lys Val Val Lys Lys Leu Thr Ala Thr Thr Leu Glu Ala Leu His Lys
180 185 190Gly Gly Arg Leu Phe Leu Val Asp His Ser Tyr Gln Lys Lys Tyr Thr
195 200 205Pro Gln Pro Gly Arg Tyr Ala Ala Ala cys Gln Gly Leu Phe Tyr Leu
210 215 220Asp Ala Arg Ser Asn Gln Phe Leu Pro Leu Ala Ile Lys Thr Asn Val225 230 235 240Gly Ala Asp Leu Thr Tyr Thr Pro Leu Asp Asp Lys Asn Asp Trp Leu
245 250 255Leu Ala Lys Ile Met Phe Asn Asn Asn Asp Leu Phe Tyr Ser Gln Met
260 265 270Tyr His Val Leu Phe His Thr Ile Pro Glu Ile Val His Glu Ala Ala
275 280 285Phe Arg Thr Leu Ser Asp Arg His Pro Val Met Gly Val Leu Asn Arg
290 295 300Leu Met Tyr Gln Ala Tyr Ala Ile Arg Pro Val Gly Gly Ala Val Leu305 310 315 320Phe Asn Pro Gly Gly Phe Trp Asp Gln Asn Phe Gly Leu Pro Ala Ser
325 330 335Ala Ala Ile Asp Phe Pro Gly Ser Val Tyr Ala Gln Gly Gly Gly Gly
340 345 350Phe Gln Ala Gly Tyr Leu Glu Lys Asp Leu Arg Ser Arg Gly Leu Val
355 360 365Gly Glu Asp Ser Gly Pro Arg Leu Pro His Phe Pro Phe Tyr Glu Asp
370 375 380Ala His Arg Leu Ile Gly Ala Ile Arg Arg Phe Met Gln Ala Phe Val385 390 395 400Asp Ser Thr Tyr Gly Ala Asp Asp Gly Asp Asp Gly Ala Leu Leu Arg
405 410 415Asp Tyr Glu Leu Gln Asn Trp Ile Ala Glu Ala Asn Gly Pro Ala Gln
420 425 430Val Arg Asp Phe Pro Ala Ala Pro Leu Arg Arg Arg Ala Gln Leu Val
435 440 445Asp Val Leu Thr His Val Ala Trp Val Thr Gly Gly Ala His His Val
450 455 460Met Asn Gln Gly Ser Pro Val Lys Phe Ser Gly Val Leu Pro Leu His465 470 475 480Pro Ala Ala Leu Tyr Ala Pro Ile Pro Thr Thr Lys Gly Ala Thr Gly
485 490 495Asn Gly Thr Arg Ala Gly Leu Leu Ala Trp Leu Pro Asn Glu Arg Gln
500 505 510Ala Val Glu Gln Val Ser Leu Leu Ala Arg Phe Asn Arg Ala Gln Val
515 520 525Gly Asp Arg Lys Gln Thr Val Arg Asp Ala Phe Ala Ala Pro Asp Leu
530 535 540Leu Ala Gly Asn Gly Pro Gly Tyr Ala Ala Ala Asn Ala Arg Phe Val545 550 555 560Glu Asp Thr Gly Arg Ile Ser Arg Glu Met Ala Gly Arg Gly Phe Asp
565 570 575Gly Lys Gly Leu Ser Gln Gly Met Pro Phe Val Trp Thr Ala Leu Asn
580 585 590Pro Ala Val Asn Pro Phe Phe Leu Ser Val
595 600<210>3<211>1013<212>DNA<213>禾顶囊壳<400>3gccctgccga acaacgaccc caaccagggg gcacgcaacg ccagtatagc tcggaagcgg 60gagttgttcc tctacggccc atccactctc gggcagacga ccttctaccc taccggagag 120ctggggaaca acatctcggc ccgcgacgtg ctactttggc gccaagatgc ggcgaaccag 180acggcaacgg cgtaccgcga agccaatgag acgtttgcag atattaccag cgtatgtgct 240gatcacatct atgcgtgtag tggccagtct gtttaggagg ctgccagttc ttcctttcgc 300acttggtatt ggtacctacc tacccaccta acctaggtac taacacgtct cgttgggcta 360tagcgtggcg gtttcaaaac gctcgacgac tttgcgctcc tctacaatgg tcactggaag 420gagtcggttc cggagggcat atcgaagggc atgttgagca actacacctc ggaccttctc 480ttttccatgg agcggctgtc ctccaaccct tacgtcctca agcgcctcca cccagccaag 540gacaaactgc cgttcagcgt cgagagcaag gtggtgaaga agctgacggc caccacgctt 600gaggcgctcc acaagggcgg ccgcctgttc ctcgtggacc acagctacca gaagaagtgc 660accccccagc caggacggta cgccgcggcc tgccaggggc ttttctacct ggacgcgcgg 720tccaaccaat tcctgcctct ggcaatcaag accaacgtgg gggcggacct gacgtacacg 780cccctcgacg acaagaacga ctggctgctg gccaagatca tgttcaacaa caacgacctg 840ttctactccc agatgtacca cgtgctcttc cacacgatcc ccgagatcgt gcacgaggcc 900gccttccgga cgctgagcga caggcacccg gtcatgggcg tgctcaaccg cctcatgtac 960caggcctacg ccatccggcc cgtgggcggg gccgtgctct tcaaccccgg cgg 1013<210>4<211>4098<212>DNA<21 3>禾顶囊壳<400>4gtcgactcgg cgatgcacgg gccatgtcga attaattcaa ttccatcgag tcctgcacgc 60actttaggaa gctccaagcc aaggcactat gaaagttcac aatcgggcat ttgactacca 120cggcgatttg acgccccagc cgagccgaca ggagcctcaa tatcactcat gtgtctgcac 180atgggcaggc agaccacagc atcccactat ctcttgcgca ccttcttctc acatcagcca 240aaacactcca ctatcggacc acccgatcag ccctgtacaa atcaaaagaa ccataacaag 300gtcgctttac caggaatatc cccctcggtg gctgtaagag gttgggtgcc ttgcagagta 360taagacgttt gtgttcatgt tcctagtctc cctttcctcc attcacgctg ccagctgaca 420ccaagccata tgtctgacta ttcgactgct acactatgcc cattgtgata agcccgcgcc 480gcttaatacc acggaccata catcgaaaac ctcaacttcc aagtcggtaa atacgttgtc 540atgtgatggt agaaggatgc ctcgccgttt ggatcaataa actgtccctt ctgtggtgcg 600gcccgagacc ccaggattac tcaggctgga taataatatc tagctcctcc cccattattt 660gtgttacttc aaattcgata gatggatggt tcgggcaccc tcgtcgctgg aatggcgatc 720tgcagaaaat ccacacagga ggaacagagc tgacatggaa attgtgaagg agtcggcctg 780tctgatggcg atggcgaaat tatctcaact agatctctcg gtccaacgtc agcctcgtac 840cagtgatatc gccgtctaca ggtgcctagg aagtactgcg ccccgatcat ccgctgtcac 900agcttcaatg tttcggtctc gccgacatat attgcccatg aaaacgattc aacgtgaggc 960ggcaacccag tcaagcttcc tattgtcgcc atgaccggtg caagatgtca ccgcgccggg 1020cacacgatat ttcttaggca tgccacacac agattgtggc atactagcaa aatctgcctc 1080tgtttgtgat ccgatggctt gcatcaaaat gcagttcccg tccgtcccgg gctgacagct 1140ggggtgtcat tggacggatc ggtgcggcca ccacctacta ggtgcgatta ttgatactca 1200acgtgaccaa taagcccagc aatttttccg aacaccctct cgggcatatc caactggagc 1260taagggggcg gcctgtagga ttcctccgtg acctcatgag agctgagaga gctcagctct 1320cagctcggtt gagcataagc ccgaagcctt gaccgaggct ggaggtgggc gcagtgagac 1380acccttgagg gccgtgtcct ttagtggcta gaaggatagt gagtatttaa aagtcgagga 1440aaggctgcat cagcaccatc atgatttccc tttacctcta aggcatttgt gcagtagttc 1500gctcgttgtt tgcttcttag cccggtagac gctcacgacc aaggctccac cttcgctcga 1560cgaaatgcgc tccaggatcc ttgccatagt cttcgcggca cgccatgtgg cagcgctgcc 1620actcgctgcc gaagacgctg cggcgacgct gtctttgacg tccagcgcct ccagcaccac 1680cgtgctcccg tctccgaccc agtacacgct gcccaacaac gaccccaacc agggggcacg 1740caacgccagt atagctcgga agcgggagtt gttcctctac ggcccatcca ctctcggca 1800gacgaccttc taccctaccg gagagctggg gaacaacatc tcggcccgcg acgtgctact 1860ttggcgccaa gatgcggcga accagacggc aacggcgtac cgcgaagcca atgagacgtt 1920tgcagatatt accagcgtat gtgctgatca catctatgcg tgtagtggcc agtctgttta 1980ggaggctgcc agttctttct ttcgcacttg gtattggtac ctacctaccc acctaaccta 2040ggtactaaca cgtctcgttg ggctatagcg tggcggtttc aaaacgctcg acgactttgc 2100gctcctctac aatggtcact ggaaggagtc ggttccggag ggcatatcga agggcatgtt 2160gagcaactac acctcggacc ttctcttttc catggagcgg ctgtcctcca acccttacgt 2220cctcaagcgc ctccacccag ccaaggacaa actgccgttc agcgtcgaga gcaaggtggt 2280gaagaagctg acggccacca cgcttgaggc gctccacaag ggcggccgcc tgttcctcgt 2340ggaccacagc taccagaaga agtacacccc ccagccagga cggtacgccg cggcctgcca 2400ggggcttttc tacctggacg cgcggtccaa ccaattcctg cctctggcaa tcaagaccaa 2460cgtgggggcg gacctgacgt acacgcccct cgacgacaag aacgactggc tgctggccaa 2520gatcatgttc aacaacaacg acctgttcta ctcccagatg taccacgtgc tcttccacac 2580gatccccgag atcgtgcacg aggccgcctt ccggacgctg agcgacaggc acccggtcat 2640gggcgtgctc aaccgcctca tgtaccaggc ctacgccatc cggcccgtgg gcggggctgt 2700gctcttcaac cccggcgggt tctgggacca aaactttggc ctgcccgcct cggccgccat 2760cgacttcccc ggctccgtgt acgcgcaggg cgggggcggg ttccaggccg gctacctgga 2820gaaggacctg cggagccggg ggctggtcgg cgaggacagc ggcccgcggc tgccgcactt 2880ccccttctac gaggacgcgc accgcctgat cggggcgatc cggcgcttca tgcaggcgtt 2940cgtggactcg acgtacggtg ccgacgacgg cgacgacggg gcgctgctgc gcgactacga 3000gctgcagaac tggatcgccg aggccaacgg gccggcgcag gtgcgcgact tccccgcggc 3060gccgctgcgg cggcgcgcac agctggtgga cgtgctgacg cacgtggcct gggtcacggg 3120cggggcgcac cacgtcatga accagggctc gcccgtcaag ttctcggggg tgctgccgct 3180gcacccggcg gcgctgtacg cgcccatccc gacgaccaag ggcgccaccg gcaacgggac 3240gagggcgggc ctgctggcgt ggctgcccaa cgagcggcag gccgtggagc aggtctcgct 3300gctcgcgcgc ttcaaccgtg cgcaggtcgg ggacaggaag cagacggtgc gcgacgcctt 3360cgccgcgccc gacctgctgg ccggcaacgg gccggggtac gcggcggcca acgcgaggtt 3420cgtcgaggac acgggccgta taagtcgcga gatggcgggc agagggttcg acggcaaggg 3480cctcagccag ggcatgccgt tcgtctggac cgcgctcaat cccgccgtca accctttttt 3540cctaagcgtc taaaaggcct ggccaaagct cagctaattg tggattcggt gtcaaggcct 3600gtcgccctcg gcgacctgag acgggagatg gggtttatga agagcgagga tggacattgg 3660aggtattggg tggtaattaa cagcatgtgg agggagggct acacgagcca aactctgtaa 3720tggatggcca ccagctgcta gtcagcagtt cccacattcc ccagaatcac ggctaccgaa 3780tcgaatgttc acagcacccg actttccatg catatgttca tgtcgccggc ctggttgctt 3840gcatgcatcc acgtgcgtgc ctggccatgc gagccatgcg agcagtagcc ctggcgacgc 3900caagggggga caaagcaggc agtgatggag gatggtaaca accataatgt actttagtct 3960ggatgcaagt ccgtggctag ggaggaaaaa ggacgtgtct cgcccgcagg aggtagggcg 4020cggacttttt ggcgaggatg atccaccccc gagcttttcc aaatgaagtc atgaccttgg 4080cataaaatgt gtctcaca 4098<210>5<211>575<212>DNA<213>禾顶囊壳<400>5agacgctcac gaccaaggct ccaccttcgc tcgacgaaat gcgctccagg atccttgcca 60tagtcttcgc ggcacgccat gtggcagcgc tgccactcgc tgccgaagac gctgcggcga 120cgctgtcttt gacgtccagc gcctccagca ccaccgtgct cccgtctccg acccagtaca 180cgctgcccaa caacgacccc aaccaggggg cacgcaacgc cagtatagct cggaagcggg 240agttgttcct ctacggccca tccactctcg ggcagacgac cttctaccct accggagagc 300tggggaacaa catctcggcc cgcgacgtgc tactttggcg ccaagatgcg gcgaaccaga 360cggcaacggc gtaccgcgaa gccaatgaga cgtttgcaga tattaccagc cgtggcggtt 420tcaaaacgct cgacgacttt gcgctcctct acaatggtca ctggaaggag tcggttccgg 480agggcatatc gaagggcatg ttgagcaact acacctcgga ccttctcttt tccatggagc 540ggctgtcctc caacccttac gtcctcaagc gcctc 575<210>6<211>1611<212>DNA<213>禾顶囊壳<400>6cggctgtcct ccaaccctta cgtcctcaag cgcctccacc cagccaagga caaactgccg 60ttcagcgtcg agagcaaggt ggtgaagaag ctgacggcca ccacgcttga ggcgctccac 120aagggcggcc gcctgttcct cgtggaccac agctaccaga agaagtacac cccccagcca 180ggacggtacg ccgcggcctg ccaggggctt ttctacctgg acgcgcggtc caaccaattc 240ctgcctctgg caatcaagac caacgtgggg gcggacctga cgtacacgcc cctcgacgac 300aagaacgact ggctgctggc caagatcatg ttcaacaaca acgacctgtt ctactcccag 360atgtaccacg tgctcttcca cacgatcccc gagatcgtgc acgaggccgc cttccggacg 420ctgagcgaca ggcacccggt catgggcgtg ctcaaccgcc tcatgtacca ggcctacgcc 480atccggcccg tgggcggggc tgtgctcttc aaccccggcg ggttctggga ccaaaacttt 540ggcctgcccg cctcggccgc catcgacttc cccggctccg tgtacgcgca gggcgggggc 600gggttccagg ccggctacct ggagaaggac ctgcggagcc gggggctggt cggcgaggac 660agcggcccgc ggctgccgca cttccccttc tacgaggacg cgcaccgcct gatcggggcg 720atccggcgct tcatgcaggc gttcgtggac tcgacgtacg gtgccgacga cggcgacgac 780ggggcgctgc tgcgcgacta cgagctgcag aactggatcg ccgaggccaa cgggccggcg 840caggtgcgcg acttccccgc ggcgccgctg cggcggcgcg cacagctggt ggacgtgctg 900acgcacgtgg cctgggtcac gggcggggcg caccacgtca tgaaccaggg ctcgcccgtc 960aagttctcgg gggtgctgcc gctgcacccg gcggcgctgt acgcgcccat cccgacgacc 1020aagggcgcca ccggcaacgg gacgagggcg ggcctgctgg cgtggctgcc caacgagcgg 1080caggccgtgg agcaggtctc gctgctcgcg cgcttcaacc gtgcgcaggt cggggacagg 1140aagcagacgg tgcgcgacgc cttcgccgcg cccgacctgc tggccggcaa cgggccgggg 1200tacgcggcgg ccaacgcgag gttcgtcgag gacacgggcc gtataagtcg cgagatggcg 1260ggcagagggt tcgacggcaa gggcctcagc cagggcatgc cgttcgtctg gaccgcgctc 1320aatcccgccg tcaacccttt tttcctaagc gtctaaaagg cctggccaaa gctcagctaa 1380ttgtggattc ggtgtcaagg cctgtcgccc tcggcgacct gagacgggag atggggttta 1440tgaagagcga ggatggacat tggaggtatt gggtggtaat taacagcatg tggagggagg 1500gctacacgag ccaaactctg taatggatgg ccaccagctg ctagtcagca gttcccacat 1560tccccagaat cacggctacc gaatcgaatg ttcacagcaa aaaaaaaaaa a 1611<210>7<211>1857<212>DNA<213>禾顶囊壳<400>7atgcgctcca ggatccttgc catagtcttc gcggcacgcc atgtggcagc gctgccactc 60gctgccgaag acgctgcggc gacgctgtct ttgacgtcca gcgcctccag caccaccgtg 120ctcccgtctc cgacccagta cacgctgccc aacaacgacc ccaaccaggg ggcacgcaac 180gccagtatag ctcggaagcg ggagttgttc ctctacggcc catccactct cgggcagacg 240accttctacc ctaccggaga gctggggaac aacatctcgg cccgcgacgt gctactttgg 300cgccaagatg cggcgaacca gacggcaacg gcgtaccgcg aagccaatga gacgtttgca 360gatattacca gccgtggcgg tttcaaaacg ctcgacgact ttgcgctcct ctacaatggt 420cactggaagg agtcggttcc ggagggcata tcgaagggca tgttgagcaa ctacacctcg 480gaccttctct tttccatgga gcggctgtcc tccaaccctt acgtcctcaa gcgcctccac 540ccagccaagg acaaactgcc gttcagcgtc gagagcaagg tggtgaagaa gctgacggcc 600accacgcttg aggcgctcca caagggcggc cgcctgttcc tcgtggacca cagctaccag 660aagaagtaca ccccccagcc aggacggtac gccgcggcct gccaggggct tttctacctg 720gacgcgcggt ccaaccaatt cctgcctctg gcaatcaaga ccaacgtggg ggcggacctg 780acgtacacgc ccctcgacga caagaacgac tggctgctgg ccaagatcat gttcaacaac 840aacgacctgt tctactccca gatgtaccac gtgctcttcc acacgatccc cgagatcgtg 900cacgaggccg ccttccggac gctgagcgac aggcacccgg tcatgggcgt gctcaaccgc 960ctcatgtacc aggcctacgc catccggccc gtgggcgggg ctgtgctctt caaccccggc 1020gggttctggg accaaaactt tggcctgccc gcctcggccg ccatcgactt ccccggctcc 1080gtgtacgcgc agggcggggg cgggttccag gccggctacc tggagaagga cctgcggagc 1140cgggggctgg tcggcgagga cagcggcccg cggctgccgc acttcccctt ctacgaggac 1200gcgcaccgcc tgatcggggc gatccggcgc ttcatgcagg cgttcgtgga ctcgacgtac 1260ggtgccgacg acggcgacga cggggcgctg ctgcgcgact acgagctgca gaactggatc 1320gccgaggcca acgggccggc gcaggtgcgc gacttccccg cggcgccgct gcggcggcgc 1380gcacagctgg tggacgtgct gacgcacgtg gcctgggtca cgggcggggc gcaccacgtc 1440atgaaccagg gctcgcccgt caagttctcg ggggtgctgc cgctgcaccc ggcggcgctg 1500tacgcgccca tcccgacgac caagggcgcc accggcaacg ggacgagggc gggcctgctg 1560gcgtggctgc ccaacgagcg gcaggccgtg gagcaggtct cgctgctcgc gcgcttcaac 1620cgtgcgcagg tcggggacag gaagcagacg gtgcgcgacg ccttcgccgc gcccgacctg 1680ctggccggca acgggccggg gtacgcggcg gccaacgcga ggttcgtcga ggacacgggc 1740cgtataagtc gcgagatggc gggcagaggg ttcgacggca agggcctcag ccagggcatg 1800ccgttcgtct ggaccgcgct caatcccgcc gtcaaccctt ttttcctaag cgtctaa 1857<210>8<211>216<212>PRT<213>禾顶囊壳<400>8Arg Gly Gly Phe Lys Thr Leu Asp Asp Phe Ala Leu Leu Tyr Asn Gly1 5 10 15His Trp Lys Glu Ser Val Pro Glu Gly Ile Ser Lys Gly Met Leu Ser
20 25 30Asn Tyr Thr Ser Asp Leu Leu Phe Ser Met Glu Arg Leu Ser Ser Asn
35 40 45Pro Tyr Val Leu Lys Arg Leu His Pro Ala Lys Asp Lys Leu Pro Phe
50 55 60Ser Val Glu Ser Lys Val Val Lys Lys Leu Thr Ala Thr Thr Leu Glu65 70 75 80Ala Leu His Lys Gly Gly Arg Leu Phe Leu Val Asp His Ser Tyr Gln
85 90 95Lys Lys Cys Thr Pro Gln Pro Gly Arg Tyr Ala Ala Ala Cys Gln Gly
100 105 110Leu Phe Tyr Leu Asp Ala Arg Ser Asn Gln Phe Leu Pro Leu Ala Ile
115 120 125Lys Thr Asn Val Gly Ala Asp Leu Thr Tyr Thr Pro Leu Asp Asp Lys
130 135 140Asn Asp Trp Leu Leu Ala Lys Ile Met Phe Asn Asn Asn Asp Leu Phe145 150 155 160Tyr Ser Gln Met Tyr His Val Leu Phe His Thr Ile Pro Glu Ile Val
165 170 175His Glu Ala Ala Phe Arg Thr Leu Ser Asp Arg His Pro Val Met Gly
180 185 190Val Leu Asn Arg Leu Met Tyr Gln Ala Tyr Ala Ile Arg Pro Val Gly
195 200 205Gly Ala Val Leu Phe Asn Pro Gly
210 215<210>9<211>15<212>DNA<213>人工<220><221>misc feature<223>引物1<220><221>修饰的碱基<222>(9)..(9)<223>I<220><221>misc feature<222>(9)..(9)<223>I<400>9gccctsccna acaac 15<210>10<211>23<212>DNA<213>人工<220><221>misc feature<223>引物2<400>10gcsggsaggc cgaagttctg gtc 23<210>11<211>29<212>DNA<213>人工<220><221>修饰的碱基<222>(3)..(6)<223>I<220><221>misc feature<222>(3)..(6)<223>I<400>11ccnccngggt traasagsac sgcsccscc 29<210>12<211>36<212>DNA<213>人工<220><221>misc feature<223>引物4<400>12cggctgtcct ccaaccctta cgtcctcaag cgcctc 36<210>13<211>36<212>DNA<213>人工<220><221>misc feature<223>引物5<400>13gaggcgcttg aggacgtaag ggttggagga cagccg 36<210>14<211>36<212>DNA<213>人工<220><221>misc feature<222>(4)..(9)<223>Bgl II位点<220><221>misc feature<223>引物6<400>14ggaagatcta tgcgctccag gatccttgcc atagtc 36<210>15<211>38<212>DNA<213>人工<220><221>misc feature<222>(4)..(9)<223>Xho I位点<220><221>misc feature<223>引物7<400>15ccgctcgagt tagacgctta ggaaaaaagg gttgacgg 38<210>16<211>24<212>PRT<213>禾顶囊壳<400>16Gly Leu Ser Gln Gly Met Pro Phe Val Trp Thr Ala Leu Asn Pro Ala1 5 10 15Val Asn Pro Phe Phe Leu Ser Val
20<210>17<211>27<212>PRT<213>禾顶囊壳<400>17Gly Ala Thr Gly Asp Gly Thr Arg Ala Gly Leu Leu Ala Trp Leu Pro1 5 10 15Asp Glu Arg Gln Ala Val Glu Gln Val Ser Leu
20 25<210>18<211>22<212>PRT<213>禾顶囊壳<400>18Gly Met Leu Ser Asp Tyr Thr Ser Asp Leu Leu Phe Ser Met Glu Arg1 5 10 15Leu Ser Ser Asn Pro Tyr
20<210>19<211>20<212>PRT<213>禾顶囊壳<400>19Phe Ser Gly Val Leu Pro Leu His Pro Ala Ala Leu Tyr Ala Pro Ile1 5 10 15Ile Thr Thr Lys
20<210>20<211>25<212>PRT<213>禾顶囊壳<220><221>misc feature<222>(17)..(17)<223>未知<400>20Ala Ile Arg Pro Val Gly Gly Ala Val Leu Phe Asn Pro Gly Gly Phe1 5 10 15Xaa Asp Gln Asn Phe Gly Leu Pro Ala
20 25<210>21<211>19<212>PRT<213>禾顶囊壳<220><221>misc_feature<222>(6)..(18)<223>未知<400>21Ala Leu Pro Asn Asn Xaa Pro Ala Ala Arg Thr Ala Lys Leu His Xaa1 5 10 15Leu Xaa Leu<210>22<211>1857<212>DNA<213>禾顶囊壳<220><221>CDS<222>(1)..(1854)<223><220><221>mat_peptide<222>(49) .. ()<223><400>22atg cgc tcc agg atc ctt gct ata gtc ttc gca gca cgc cat gtg gca 48Met Arg Ser Arg Ile Leu Ala Ile Val Phe Ala Ala Arg His Val Ala
-15 -10 -5 -1gcg ctg cca ctc gct gcc gaa gac gct gcg gcg acg ctg tct ttg acg 96Ala Leu Pro Leu Ala Ala Glu Asp Ala Ala Ala Thr Leu Ser Leu Thr1 5 10 15tcc agc gcc tcc agc acc acc gtg ctc ccg tct ccg acc cag tac acg 144Ser Ser Ala Ser Ser Thr Thr Val Leu Pro Ser Pro Thr Gln Tyr Thr
20 25 30ctg ccc aac aaa gac ccc aac cag ggg gca cgc aac gcc agt ata gcg 192Leu Pro Asn Lys Asp Pro Asn Gln Gly Ala Arg Asn Ala Ser Ile Ala
35 40 45cgg aag cgg gag ttg ttc ctc tac ggc cca tcc acg ctc ggg cag acg 240Arg Lys Arg Glu Leu Phe Leu Tyr Gly Pro Ser Thr Leu Gly Gln Thr
50 55 60acc ttc tac cct acc gga gag cta ggg aac aat atc tcg gcc cgc gac 288Thr Phe Tyr Pro Thr Gly Glu Leu Gly Asn Asn Ile Ser Ala Arg Asp65 70 75 80gtg ctg ctt tgg cgc caa gat gcg gcg aac cag acg gca acg gcg tac 336Val Leu Leu Trp Arg Gln Asp Ala Ala Asn Gln Thr Ala Thr Ala Tyr
85 90 95cgc gaa gcc aat gag acg ttt gca gat att acc agc cgt ggc ggt ttc 384Arg Glu Ala Asn Glu Thr Phe Ala Asp Ile Thr Ser Arg Gly Gly Phe
100 105 110aaa acg ctc gac gac ttt gcg ctc ctc tac aat ggt cac tgg aag gag 432Lys Thr Leu Asp Asp Phe Ala Leu Leu Tyr Asn Gly His Trp Lys Glu
115 120 125tcg gtt ccg gag ggc ata tcg aag ggc atg ttg agc aac tac acc tcg 480Ser Val Pro Glu Gly Ile Ser Lys Gly Met Leu Ser Asn Tyr Thr Ser
130 135 140gac ctt ctc ttt tcc atg gag cgg ctg tcc tcc aac cct tac gtc ctc 528Asp Leu Leu Phe Ser Met Glu Arg Leu Ser Ser Asn Pro Tyr Val Leu145 150 155 160aag cgc ctc cac cca acc aag gac aaa ctg ccg ttc agc gtc gag agc 576Lys Arg Leu His Pro Thr Lys Asp Lys Leu Pro Phe Ser Val Glu Ser
165 170 175aag gtg gtg aag aag ctg acg gcc acc acg ctt gag gcg ctc cac aag 624Lys Val Val Lys Lys Leu Thr Ala Thr Thr Leu Glu Ala Leu His Lys
180 185 190ggc ggc cgc ctg ttc ctc gtg gac cac agc tac cag aag aag tac acc 672Gly Gly Arg Leu Phe Leu Val Asp His Ser Tyr Gln Lys Lys Tyr Thr
195 200 205ccc cag cca gga cgg tac gcc gcg gcc tgc cag ggg ctt ttc tac ctg 720Pro Gln Pro Gly Arg Tyr Ala Ala Ala Cys Gln Gly Leu Phe Tyr Leu
210 215 220gac gcg cgg tcc aac cag ttc ctg cct ctg gca atc aag acc aac gtg 768Asp Ala Arg Ser Asn Gln Phe Leu Pro Leu Ala Ile Lys Thr Asn Val225 230 235 240ggg gtg gat ctg acg tac acg ccc ctc gac gac aag gac gac tgg ctg 816Gly Val Asp Leu Thr Tyr Thr Pro Leu Asp Asp Lys Asp Asp Trp Leu
245 250 255ctg gcc aag atc atg ttc aac aac aac gac ctg ttc tac tcc cag atg 864Leu Ala Lys Ile Met Phe Asn Asn Asn Asp Leu Phe Tyr Ser Gln Met
260 265 270tac cac gtg ctc ttc cac acg atc ccc gag atc gtg cac gag gcc gcc 912Tyr His Val Leu Phe His Thr Ile Pro Glu Ile Val His Glu Ala Ala
275 280 285ttc cgg acg ctg agc gac agg cac ccg gtc atg ggc gtg ctc aac cgc 960Phe Arg Thr Leu Ser Asp Arg His Pro Val Met Gly Val Leu Asn Arg
290 295 300ctc atg tac cag gcc tac gcc atc cgg ccc gtg ggc ggg gct gtg ctc 1008Leu Met Tyr Gln Ala Tyr Ala Ile Arg Pro Val Gly Gly Ala Val Leu305 310 315 320ttc aac ccc ggc ggg ttc tgg gac caa aac ttt ggc ctg ccc gcc tcg 1056Phe Asn Pro Gly Gly Phe Trp Asp Gln Asn Phe Gly Leu Pro Ala Ser
325 330 335gcc gcc atc gac ttc ccc ggc tcc gtg tac gcg cag ggc ggg ggc ggg 1104Ala Ala Ile Asp Phe Pro Gly Ser Val Tyr Ala Gln Gly Gly Gly Gly
340 345 350ttc cag gcc ggc tac ctg gag aag gac ctg cgg agc cgg ggg ctg atc 1152Phe Gln Ala Gly Tyr Leu Glu Lys Asp Leu Arg Ser Arg Gly Leu Ile
355 360 365ggc gag gac agc ggc ccg cgg ctg ccg cac ttc ccc ttc tac gag gac 1200Gly Glu Asp Ser Gly Pro Arg Leu Pro His Phe Pro Phe Tyr Glu Asp
370 375 380gcg cac cgc ctg atc ggg gcg atc cgg cgc ttc atg cag gcg ttc gtg 1248Ala His Arg Leu Ile Gly Ala Ile Arg Arg Phe Met Gln Ala Phe Val385 390 395 400gac tcg acg tac ggt gcc gac gac ggc gac gac ggg gcg ctg ctg cgc 1296Asp Ser Thr Tyr Gly Ala Asp Asp Gly Asp Asp Gly Ala Leu Leu Arg
405 410 415gac tat gag cta cag aac tgg atc gcc gag gcc aac ggg ccg gcg cag 1344Asp Tyr Glu Leu Gln Asn Trp Ile Ala Glu Ala Asn Gly Pro Ala Gln
420 425 430gtg cgc gac ttc ccc gcg gcg ccg ctg cga cgg cgc gcg cag ctg gtg 1392Val Arg Asp Phe Pro Ala Ala Pro Leu Arg Arg Arg Ala Gln Leu Val
435 440 445gac gtg ctg acg cac gtg gcc tgg atc acg ggc ggg gcg cac cac gtc 1440Asp Val Leu Thr His Val Ala Trp Ile Thr Gly Gly Ala His His Val
450 455 460atg aac cag ggc tcg ccc gtc aag ttc tcg ggg gtg ctg ccg ctg cac 1488Met Asn Gln Gly Ser Pro Val Lys Phe Ser Gly Val Leu Pro Leu His465 470 475 480ccg gcg gcg ctg tac gcg ccc atc ccg acg gcc aag ggc gcc acc ggc 1536Pro Ala Ala Leu Tyr Ala Pro Ile Pro Thr Ala Lys Gly Ala Thr Gly
485 490 495aac ggg acg agg gcg ggc ctg ctg gcg tgg ctg ccc aac gag cgg cag 1584Asn Gly Thr Arg Ala Gly Leu Leu Ala Trp Leu Pro Asn Glu Arg Gln
500 505 510gcc gtg gag cag gtc tcg ctg ctc gcg cgc ttc aac cgt gcc cag gtc 1632Ala Val Glu Gln Val Ser Leu Leu Ala Arg Phe Asn Arg Ala Gln Val
515 520 525ggg gac agg aag cag acg gtg cgc gac gcc ttc gcc gcg ccc gac ctg 1680Gly Asp Arg Lys Gln Thr Val Arg Asp Ala Phe Ala Ala Pro Asp Leu
530 535 540ctg gcc ggc aac ggg ccg ggg tac gcg gcg gcc aac gcg agg ttc gtc 1728Leu Ala Gly Asn Gly Pro Gly Tyr Ala Ala Ala Asn Ala Arg Phe Val545 550 555 560gag gac acg ggc cgt ata agt cgc gag att gcg ggc aga ggg ttt gac 1776Glu Asp Thr Gly Arg Ile Ser Arg Glu Ile Ala Gly Arg Gly Phe Asp
565 570 575ggc aag ggc ctc agc cag ggc atg ccg ttc gtc tgg acc gcg ctc aat 1824Gly Lys Gly Leu Ser Gln Gly Met Pro Phe Val Trp Thr Ala Leu Asn
580 585 590ccc gcc gtc aac cct ttt ttc ctg agc gtc taa 1857Pro Ala Val Asn Pro Phe Phe Leu Ser Val
595 600<210>23<211>618<212>PRT<213>禾顶囊壳<400>23Met Arg Ser Arg Ile Leu Ala Ile Val Phe Ala Ala Arg His Val Ala
-15 -10 -5 -1Ala Leu Pro Leu Ala Ala Glu Asp Ala Ala Ala Thr Leu Ser Leu Thr1 5 10 15Ser Ser Ala Ser Ser Thr Thr Val Leu Pro Ser Pro Thr Gln Tyr Thr
20 25 30Leu Pro Asn Lys Asp Pro Asn Gln Gly Ala Arg Asn Ala Ser Ile Ala
35 40 45Arg Lys Arg Glu Leu Phe Leu Tyr Gly Pro Ser Thr Leu Gly Gln Thr
50 55 60Thr Phe Tyr Pro Thr Gly Glu Leu Gly Asn Asn Ile Ser Ala Arg Asp65 70 75 80Val Leu Leu Trp Arg Gln Asp Ala Ala Asn Gln Thr Ala Thr Ala Tyr
85 90 95Arg Glu Ala Asn Glu Thr Phe Ala Asp Ile Thr Ser Arg Gly Gly Phe
100 105 110Lys Thr Leu Asp Asp Phe Ala Leu Leu Tyr Asn Gly His Trp Lys Glu
115 120 125Ser Val Pro Glu Gly Ile Ser Lys Gly Met Leu Ser Asn Tyr Thr Ser
130 135 140Asp Leu Leu Phe Ser Met Glu Arg Leu Ser Ser Asn Pro Tyr Val Leu145 150 155 160Lys Arg Leu His Pro Thr Lys Asp Lys Leu Pro Phe Ser Val Glu Ser
165 170 175Lys Val Val Lys Lys Leu Thr Ala Thr Thr Leu Glu Ala Leu His Lys
180 185 190Gly Gly Arg Leu Phe Leu Val Asp His Ser Tyr Gln Lys Lys Tyr Thr
195 200 205Pro Gln Pro Gly Arg Tyr Ala Ala Ala Cys Gln Gly Leu Phe Tyr Leu
210 215 220Asp Ala Arg Ser Asn Gln Phe Leu Pro Leu Ala Ile Lys Thr Asn Val225 230 235 240Gly Val Asp Leu Thr Tyr Thr Pro Leu Asp Asp Lys Asp Asp Trp Leu
245 250 255Leu Ala Lys Ile Met Phe Asn Asn Asn Asp Leu Phe Tyr Ser Gln Met
260 265 270Tyr His Val Leu Phe His Thr Ile Pro Glu Ile Val His Glu Ala Ala
275 280 285Phe Arg Thr Leu Ser Asp Arg His Pro Val Met Gly Val Leu Asn Arg
290 295 300Leu Met Tyr Gln Ala Tyr Ala Ile Arg Pro Val Gly Gly Ala Val Leu305 310 315 320Phe Asn Pro Gly Gly Phe Trp Asp Gln Asn Phe Gly Leu Pro Ala Ser
325 330 335Ala Ala Ile Asp Phe Pro Gly Ser Val Tyr Ala Gln Gly Gly Gly Gly
340 345 350Phe Gln Ala Gly Tyr Leu Glu Lys Asp Leu Arg Ser Arg Gly Leu Ile
355 360 365Gly Glu Asp Ser Gly Pro Arg Leu Pro His Phe Pro Phe Tyr Glu Asp
370 375 380Ala His Arg Leu Ile Gly Ala Ile Arg Arg Phe Met Gln Ala Phe Val385 390 395 400Asp Ser Thr Tyr Gly Ala Asp Asp Gly Asp Asp Gly Ala Leu Leu Arg
405 410 415Asp Tyr Glu Leu Gln Asn Trp Ile Ala Glu Ala Asn Gly Pro Ala Gln
420 425 430Val Arg Asp Phe Pro Ala Ala Pro Leu Arg Arg Arg Ala Gln Leu Val
435 440 445Asp Val Leu Thr His Val Ala Trp Ile Thr Gly Gly Ala His His Val
450 455 460Met Asn Gln Gly Ser Pro Val Lys Phe Ser Gly Val Leu Pro Leu His465 470 475 480Pro Ala Ala Leu Tyr Ala Pro Ile Pro Thr Ala Lys Gly Ala Thr Gly
485 490 495Asn Gly Thr Arg Ala Gly Leu Leu Ala Trp Leu Pro Asn Glu Arg Gln
500 505 510Ala Val Glu Gln Val Ser Leu Leu Ala Arg Phe Asn Arg Ala Gln Val
515 520 525Gly Asp Arg Lys Gln Thr Val Arg Asp Ala Phe Ala Ala Pro Asp Leu
530 535 540Leu Ala Gly Asn Gly Pro Gly Tyr Ala Ala Ala Asn Ala Arg Phe Val545 550 555 560Glu Asp Thr Gly Arg Ile Ser Arg Glu Ile Ala Gly Arg Gly Phe Asp
565 570 575Gly Lys Gly Leu Ser Gln Gly Met Pro Phe Val Trp Thr Ala Leu Asn
580 585 590Pro Ala Val Asn Pro Phe Phe Leu Ser Val
595 600<210>24<211>133<212>DNA<213>禾顶囊壳<400>24gtatgtgctg atcacatcta tgcgtgtggt gaccggtctg ctttaggagg ctgccagttc 60tttctttcgc acttggtatt ggtacctacc tacccaccta acctaggtgc taacacgtct 120cgttgggcta tag 133
Claims (17)
1.有脂肪氧合酶活性的多肽,其:
a)具有与SEQ ID NO:2或23的成熟多肽有至少50%同一性的氨基酸序列;
b)是如下核酸序列编码的,所述核酸序列在55℃与编码SEQ IDNO:1的成熟多肽的核酸序列或其至少有100个核苷酸的亚序列的互补链杂交;
c)具有能通过一个或多个氨基酸替换,缺失,和/或插入从SEQ IDNO:2或23的成熟多肽获得的氨基酸序列;或
d)是克隆在如下质粒中的DNA序列的脂肪氧合酶编码部分编码的,其中所述质粒存在于保藏号为DSM 13586的大肠杆菌中。
2.多核苷酸,其包含:
a)克隆在大肠杆菌DSM 13586的质粒中的编码成熟脂肪氧合酶的部分DNA序列;
b)编码SEQ ID NO:2或23中所示成熟脂肪氧合酶的部分DNA序列;
c)a)或b)中定义的序列的类似物,它编码脂肪氧合酶且
i)与所述DNA序列有至少50%同一性,或
ii)在低严紧条件下,与所述DNA序列或其有至少100个核苷酸的亚序列的互补链杂交;
iii)是其等位基因变体,或
d)a),b)或c)的互补链。
3.前一项权利要求的多核苷酸,其中所述部分DNA序列是SEQ IDNO:1或22中显示的编码成熟肽的序列。
4.核酸构建体,其包含可操作地连接一个或多个控制序列的权利要求2或3的多核苷酸,所述控制序列能在适宜的表达宿主中指导脂肪氧合酶的表达。
5.重组表达载体,其包含权利要求4的核酸构建体,启动子和转录及翻译终止信号。
6.用权利要求4的核酸构建体或权利要求5的载体转化的重组宿主细胞。
7.生产脂肪氧合酶的方法,包括:
a)在利于脂肪氧合酶产生的条件下,培养权利要求6的宿主细胞,和
b)回收脂肪氧合酶。
8.寡核苷酸探针,其由至少20个核苷酸组成,并且编码SEQ ID NO:2或23的部分多肽序列。
9.获得具有脂肪氧合酶活性的多肽的方法,包括:
a)制备真核DNA文库,
b)筛选文库以选择与权利要求8的探针杂交的DNA分子,
c)用选定的DNA分子转化宿主细胞,
d)培养转化的宿主细胞以表达该DNA分子编码的多肽,和
e)分析表达的多肽以选择有脂肪氧合酶活性的多肽。
10.面团组合物,其包含锰脂肪氧合酶。
11.前一项权利要求的组合物,其中脂肪氧合酶是权利要求1的多肽。
12.制备面团或面团的焙烤产品的方法,包括向面团中添加锰脂肪氧合酶。
13.氧化选自亚麻酸,花生四烯酸,亚油醇和亚油酸酯的底物的方法,包括在氧存在的情况下,用锰脂肪氧合酶接触底物。
14.前一项权利要求的方法,其中亚油酸酯是亚油酸甲酯,单亚油精,双亚油精或三亚油精。
15.权利要求12-14之任一项的方法,其中脂肪氧合酶是权利要求1的多肽。
16.去污剂组合物,其包含锰脂肪氧合酶和表面活性剂。
17.前一项权利要求的组合物,其中表面活性剂包括阴离子表面活性剂,特别是直链烷基苯磺酸盐。
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SE0004790A SE0004790D0 (sv) | 2000-12-22 | 2000-12-22 | Manganese lipoxygenase sequences and use thereof |
DKPA200100322 | 2001-02-27 | ||
DKPA200100322 | 2001-02-27 | ||
PCT/DK2001/000574 WO2002020730A2 (en) | 2000-09-05 | 2001-09-05 | Manganese lipoxygenase |
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JPH11299440A (ja) | 1998-04-22 | 1999-11-02 | Nisshin Flour Milling Co Ltd | 麺類の製造方法 |
EP0983725B1 (en) | 1998-09-02 | 2003-05-02 | Unilever N.V. | Tomato products enriched in beta-cyclocitral |
DE19840069A1 (de) | 1998-09-03 | 2000-03-09 | Basf Ag | Wirkstoffzubereitungen sowie ein Verfahren zu deren Herstellung |
JP3702401B2 (ja) | 1998-10-06 | 2005-10-05 | 株式会社J−オイルミルズ | 油脂加工澱粉及びその製造方法 |
-
2001
- 2001-09-05 EP EP01964942A patent/EP1317528A2/en not_active Withdrawn
- 2001-09-05 BR BR0113681-0A patent/BR0113681A/pt not_active Application Discontinuation
- 2001-09-05 US US10/362,776 patent/US7456001B2/en not_active Expired - Fee Related
- 2001-09-05 AU AU8571901A patent/AU8571901A/xx active Pending
- 2001-09-05 CN CN018151833A patent/CN1452656B/zh not_active Expired - Fee Related
- 2001-09-05 AU AU2001285719A patent/AU2001285719B2/en not_active Ceased
- 2001-09-05 JP JP2002525737A patent/JP5230889B2/ja not_active Expired - Fee Related
- 2001-09-05 CA CA002419577A patent/CA2419577A1/en not_active Abandoned
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Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102268400A (zh) * | 2011-07-28 | 2011-12-07 | 江南大学 | 一种产脂肪氧合酶的基因工程菌及其构建方法和应用 |
CN111278969A (zh) * | 2017-09-12 | 2020-06-12 | 银杏生物制品公司 | 保护性酶 |
CN113980922A (zh) * | 2021-11-29 | 2022-01-28 | 广东省农业科学院蚕业与农产品加工研究所 | 一种重组脂肪氧合酶及其诱导表达方法和用途 |
Also Published As
Publication number | Publication date |
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CA2419577A1 (en) | 2002-03-14 |
AU8571901A (en) | 2002-03-22 |
JP2004508039A (ja) | 2004-03-18 |
BR0113681A (pt) | 2003-07-08 |
US7456001B2 (en) | 2008-11-25 |
JP5230889B2 (ja) | 2013-07-10 |
AU2001285719B2 (en) | 2007-08-23 |
EP1317528A2 (en) | 2003-06-11 |
WO2002020730A3 (en) | 2002-06-20 |
WO2002020730A2 (en) | 2002-03-14 |
US20040029225A1 (en) | 2004-02-12 |
AU2001285719B8 (en) | 2002-03-22 |
CN1452656B (zh) | 2011-06-15 |
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