CN1179041C - 改进的植酸酶 - Google Patents
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Abstract
本发明涉及改进的植酸酶，优选是增加了热稳定性的植酸酶，以及产生植酸酶的方法。具体的，将稳定的氨基酸突变导入到同源蛋白中，或使植酸酶的活性位点部分或全部被取代。本发明还公开了相应的DNA序列及其制备方法，以及生产改进的植酸酶的方法，及其应用。本发明还公开了烟曲霉植酸酶和共有植酸酶的特异性突变体。
Description
植酸酶是一种将植酸盐(肌醇六磷酸)水解为肌醇和无机磷酸的酶。已知其是一种有价值的饲料添加剂。
本发明涉及改进的植酸酶，即具有改良特性(如改良的活性特性)的植酸酶，其参照为例如其衍生源或已知的植酸酶。改良的活性特性是指在植酸酶活性的至少一个相关方面进行改良，例如(非-排它列举)：pH稳定性，温度稳定性，pH范围，温度范围，比活性(特别是相关于pH和温度的)，底物特异性，底物裂解模式，底物结合，位点特异性，磷酸从谷物中释放的速度和水平，反应速度，植酸盐降解速率)，释放的磷酸所达到的终水平。
改良的活性特性的例子为改良的比活性(如增加，如在pH3，4，5，或6时增加)；改良的pH或温度范围；和/或改良(如增加)的热稳定性，例如当使用差示扫描量热(DSC)测量时增加的解链温度。
本发明还涉及一种制备修饰型蛋白的方法，其中第一步是通过下述步骤a)，b)和c)从多个高度同源序列中测定出一个共有序列：
a)通过本领域已知的任何标准比对程序对至少3个，优选至少4个氨基酸序列进行比对；
b)在氨基酸序列比对的每一位点，评价氨基酸的进化相似性，而共有残基通过本领域公知的标准程序选择，其中为了预测(calculation)共有残基，至少应保证在某指定残基仅出现在比对序列中的两个序列上时，所述程序能确定一个共有残基。然而，如果在所比对的氨基酸序列中有一亚组序列，其彼此间的相似性远高于与其它序列之间的相似性，则仅当其共有序列可以相同于EP 897985所述的方式而确定时，或给该亚组中每一序列分配一个由1除以该亚组序列数所得的权重时，该亚组可用所述预测表示；
c)如果该程序未能在指定位置鉴定出共有序列，则可选择任何氨基酸，优选在此位置出现频率最高的氨基酸。
在本发明第二方面，使一个同源序列与该共有序列比对，并使该同源序列中一或多个非共有残基被相应共有残基取代。
优选的，该同源氨基酸序列仅在所述程序于中度严谨条件下可明确定义共有残基的位置上进行氨基酸残基的取代，而在未能于中度严谨条件下确定优选的共有氨基酸的所有位置上，该同源蛋白的氨基酸保持不变。
在发明第三方面，测定了目的蛋白的活性中心，包括共有序列中以及同源蛋白的序列中参与形成活性中心的所有氨基酸残基；随后，将同源蛋白的部分或全部趋异氨基酸残基插入共有序列的骨架中。
在此方法的一个实施方案中，用于比对特定位置之氨基酸的进化相似性的程序为程序＂PRETTY＂。
蛋白质活性中心可利用蛋白质三维结构分析测定。
同源蛋白如酶家族，限定的蛋白家族的实例为植酸酶家族，例如真菌来源的。
例如，植酸酶的氨基酸序列可通过引入至少一个选自下列的突变或取代被改变：
E58A F54Y
D69K I73V
D197N K94A
T214L R101A
E222T N153K
E267D V158I
R291I A203G
R329H S205G
S364T V217A
A379K A227V
G404A V234L
P238A
Q277E
A287H
A292Q
V366I
A396S
E415Q
G437A
R451E
为了解释这些缩写，举一个例子，突变E58A被解释如下：当从该数字减去26，可以得到在如图1所示共有植酸酶序列或另一被比对的植酸酶序列中的位置或残基号(对应于在图1所示序列中加入一段26个氨基酸的信号序列)。例如，在E58A中，数字58是指位置号32(58-26＝32)。该数字前的字母，即E，代表该植酸酶中被修饰的氨基酸，其被所述数字之后的氨基酸，即A，取代。
上述氨基酸取代，单独和/或联合，对氨基酸的稳定性有积极影响。
下组突变(即含有选自下组的至少一个突变的植酸酶)也是兴趣所在：
E58A，D69K，D197N，T214L，E222T，E267D，R291I，R329H，S364T，A379K，G404A；
F54Y，I73V，K94A，R101A，N153K，V158I，A203G，S205G，V217A，A227V，V234L，P238A，Q277E，A287H，A292Q，V366I，A396S，E415Q，G437A，R451E；
E58A，D69K，D197N，F54Y，I73V，K94A；
T214L，E222T，E267DR101A，N153K，V158I；
R291I，R329H，S364TA203G，S205G，V217A；
A379K，G404AA227V，V234L，P238A，Q277E；
A287H，A292Q，V366I，A396S，E415Q，G437A，R451E；
T214L，E222T，S364T，V158I，A203G，G404A，A227V，P238A，A396S，G437A，R451E。
宿主细胞的实例为植物细胞，动物细胞，和微生物细胞，例如原核或真核细胞，如细菌，真菌或酵母细胞。真菌宿主的实例为曲霉属菌株，酵母宿主的实例为糖酵母属菌株，汉逊氏酵母属(Hansenula)菌株。
本发明也涉及通过上述任一方法可获得的或已获得的修饰蛋白。
本发明也涉及植酸酶例如(但不限于)共有植酸酶-1的变体或突变蛋白，其中，在图2的氨基酸序列中，已进行至少一个下述取代：Q50L，Q50T，Q50G，Q50T-Y51N，Q50L-Y51N或Q50T-K91A。
在上述第三方面，如上所述从同源序列中确定一个共有序列；第二步，在所述共有序列及单个同源蛋白的序列中，测定蛋白活性中心，其包含参与形成活性中心的所有氨基酸残基。所述单个同源蛋白可具有优选的特性如高比活性或酶活性的不同pH依赖性。第三步，将参与形成该同源蛋白活性中心的部分或全部氨基酸残基插入所述共有序列的骨架中。结果产生嵌合蛋白，其具有来自单个蛋白的活性中心以及共有序列的骨架。
蛋白的活性中心可通过例如任何的蛋白三维结构分析来检测，例如通过基于已知蛋白的已知3D-结构的同源模拟。
本发明也提供了通过此方法可获得的或已获得的共有蛋白，特别是含有至少一个示于图2-6，10或21的氨基酸序列的蛋白，或其变体或突变蛋白。此类变体的实例见图7-9。
此类变体或突变蛋白可如欧洲专利申请EP0897010所述定义并制备，例如Q50L，Q50T，Q50G，Q50L-Y51N，或Q50T-Y51N。这些突变如上述定义，或参见图2。当参见图2时，不要求减去所述26个氨基酸的信号肽(例如，在图2中氨基酸序列第50位的“Q50L，”中，氨基酸Q被氨基酸L取代)。
含有本发明植酸酶的食品，饲料，或药物组合物是本发明的另一方面。
在此上下文及本发明的方法中，“此类指定蛋白质家族的至少3个，优选至少4个氨基酸序列”是指3，4，5，6至12，20，50个，或甚至更多的序列可用于比对和比较以产生共有蛋白的氨基酸序列。＂指定的蛋白家族的序列＂是指被折叠成三维结构的序列，其中α-螺旋，β-折叠和β-转角在同样的位置，使此结构被本领域技术人员称作可高度重叠。此外，这些序列定性了显示同类生物学活性的蛋白质，例如，特定酶类如植酸酶。这类蛋白的三维结构足以允许模拟此家族其它同源蛋白的结构。实施例1举例说明如何操作。本发明中“进化相似性”指根据结构相似性对氨基酸分类的方案，其允许一个氨基酸被另一个氨基酸取代且对整个结构影响最小，这可通过本领域公知的程序如“PRETTY”程序来进行。短语“此程序提供的相似程度...被设置到较小的严谨数”在本发明中指，可将确定本发明所用程序中相似性程度的参数值设定为，使该程序能针对完整氨基酸序列的最多数位置确定一个共有氨基酸，例如在程序PRETTY中，可将THRESHOLD的值选为2或3，PLURALITY的值为2。此外，“1除以这些序列的数目所得的权重”在本发明中指，能限定与用于确定共有序列之其它序列具有较高相似程度的一组序列的序列仅为所述确定提供一个系数，该系数等于1除以该组所有序列的数目。
如上所述，如果所述程序无法选出共有氨基酸，则将最常出现的氨基酸选为共有氨基酸；如果后一种情况不可能，本领域技术人员将从用于比较的所有序列中选出本领域已知其特性可改进目标蛋白热稳定性的氨基酸，方式如Janecek，S.(1993)，生物化学方法(Process Biochem.)28，435-445；Fersht，A.R.和Serrano，L.(1993)，结构生物学最新观点(Curr.Opin.Strruct.Biol.)3，75-83；Alber，T.(1989)，生物化学年鉴58，765-798；Matthews，B.W.(1987)，生物化学26，6885-6888；或Matthews，B.W.(1991)，结构生物学最新观点1，17-21。
酶的稳定性与多种工业应用相关。因此，已有大量成功或不太成功的尝试，其通过合理或随机的方法用来改进酶的稳定性，优选热稳定性。
在此我们提供了另一种改进蛋白热稳定性的方式。
本发明提供了一种制备共有蛋白的方法，包括对所谓共有蛋白的几乎所有位置的氨基酸残基进行预测的方法，以及从该序列合成能在原核或真核表达系统中表达的完整基因的方法。
本发明的DNA序列可从编码目的蛋白如植酸酶的基因组或cDNA序列开始构建。例如，可通过体外诱变构建[见，例如Sambrook等，分子克隆，冷泉港实验室出版社，纽约]。广泛使用的“定点诱变”最初由Hurchinson和Edgell[病毒学杂志8，181(1971)]所述，其包括使携有所需核苷酸取代的合成寡核苷酸与单链DNA序列中待导入突变的靶区域退火[见Smith，遗传学年鉴19，423(1985)，改良方法见Stanssen等，核酸研究17，4441-4454(1989)中文献2-6。对指定DNA序列的诱变还可通过聚合酶链式反应(PCR)进行。DNA作为起始材料可通过本领域公知的如Sambrook等(分子克隆)所述的方法从各个菌株中分离。
菌株信息参见如EP 684313或下述任何保藏单位。黑曲霉(Aspergillusniger)[ATCC 9142]，嗜热毁丝霉(Myceliophthora thermophila)[ATCC 48102]，嗜热踝节菌(Talaromyces thermophilus)[ATCC 20186]与烟曲霉(Aspergillusfumigatus)[ATCC 34625]已根据布达佩斯条约保藏在美国典型培养物保藏中心，保藏号分别为：ATCC 74337，ATCC 74340，ATCC 74338和ATCC74339。但应理解，编码本发明共有蛋白的DNA可以如EP 747483或EP897985，或实施例所述，通过本领域公知的方法合成。
序列信息参见如EP684313，或序列数据库如Genbank(Intelligenetics，加利福尼亚，美国)，欧洲生物信息协会(European Bioinformatics Institute)(HinstonHall，剑桥，英国)，NBRF(Georgetown大学，医疗中心，华盛顿特区，美国)和Vecbase(威斯康星大学，生物技术中心，Madison，威斯康星，美国)。
本发明的方法可例如用于改进植酸酶的热稳定性。
一旦获得本发明的全长DNA序列，可通过本领域公知以及Sambrook等(同上(s.a.))所述的方法将它们整合至载体，从而在适当宿主系统中过表达所编码的多肽。但本领域技术人员公知，DNA序列本身可用于转化本发明的适当宿主系统以获得所编码多肽的过表达。适当宿主系统有真菌，如黑曲霉[ATCC 9142]或无花果曲霉(Aspergillus ficuum)[NRRL 3135]等曲霉属或如Trichoderma reesei等木霉属；或酵母，如酿酒酵母等糖酵母属，或巴斯德毕赤酵母(Pichia pastoris)等毕赤酵母属，或多形汉逊氏酵母(DSM5215)等汉逊氏酵母多形体；或植物，如Pen等，生物/技术11，811-814(1994)所述。本领域技术人员公知此类微生物可来自保藏机构，如美国典型培养物保藏中心(ATCC)，真菌菌株保藏中心(CBS)或德意志微生物和细胞培养物保藏中心(DSM)或“Industrial Property”[(1991)1，29-40页]所列的任何其它保藏机构。可使用的细菌有大肠杆菌；枯草芽孢杆菌(Bacillus subtilis)等芽孢杆菌属；或变青链霉菌(Streptomyces lividans)等链霉菌属(参见如，Anne和Mallaert，FEMS微生物通讯114，121(1993)。优选大肠杆菌菌株，如大肠杆菌K12菌株，如M15[Villarejo，细菌学杂志120，466-474(1974)所述DZ 291]，HB 101[ATCC 33694]或大肠杆菌SG13009[Gottesman等，细菌学148，265-273(1981)]。
可用于真菌中表达的载体是本领域公知的且述于EP 420358，或Cullen等[生物/技术5，369-376(1987)]，Ward[分子工业真菌学，丝状真菌的系统和应用，Marcel Dekker，纽约(1991)]，Upshall等[生物/技术5，1301-1304(1987)]，Gwynne等[生物/技术5，71-79(1987)]，或Punt等[生物技术杂志17，19-34(1991)]；用于酵母中表达的载体见Sreekrishna等[基础微生物学杂志28，265-278(1988)，生物化学28，4117-4125(1989)]，Hitzemann等[自然293，717-722(1981)]或EP 183070，EP 183071，EP 248227，或EP263311。用于大肠杆菌中表达的合适载体见Sambrook等[同上]，Fiers等[第8次国际生物技术专题讨论会论文集，法国微生物协会(Soc.Franc.deMicrobiol.)，巴黎(Durand等编)，680-697页(1988)]，Bujard等[酶学方法155，416-433(1987)]，或Stuber等[免疫学方法，Lefkovits和Pernis编，AcademicPress，Inc.，卷IV，121-152(1990)]。用于芽孢杆菌中表达的载体是本领域公知的且述于如EP 207459，EP 405370，美国国家科学院院报，81，439(1984)或Yansura和Henner，酶学方法185，199-228(1990)。用于多形汉逊氏酵母中表达的载体是本领域公知的且述于如Gellissen等，生物技术9，291-295(1991)。
已携有调控元件如启动子的载体，或本发明的DNA序列可通过改造而含有这种元件。可使用的合适启动子元件是本领域公知的，例如Trichoderma reesei的cbhl-启动子[Haarki等，生物技术7，596-600(1989)]或pkil-启动子[Schindler等，基因130，271-275(1993)]；用于米曲霉的amy-启动子[Christensen等，Abstr.19th Lunteren Lectures on Molecular GeneticsF23(1987)，Christensen等，生物/技术6，1419-1422(1988)，Tada等，分子普通遗传学229，301(1991)]；用于黑曲霉的glaA-启动子[Cullen等，生物/技术5，369-376(1987)，Gwynne等，生物/技术5，713-719(1987)，Ward，分子工业真菌学，丝状真菌的系统和应用，Marcel Dekker，纽约，83-106(1991)]，alcA-启动子[Gwynne等，生物技术5，718-719(1987)]，sucl-启动子[Boddy等，现代遗传学24，60-66(1993)]，aphA-启动子[MacRae等，基因71，339-348(1988)，MacRae等，基因132，193-198(1993)]，tpiA-启动子[McKnight等，细胞46，143-147(1986)，Upshall等，生物/技术5，1301-1304(1987)]，gpdA-启动子[Punt等，基因69，49-57(1988)，Punt等，生物技术杂志17，19-37(1991)]以及pkiA-启动子[de Graaff等，现代遗传学22，21-27(1992)]。可用于酵母中表达的适当启动子是本领域已知的，例如pho5-启动子[Vogel等，分子细胞生物学，2050-2057(1989)；Rudolf和Hinnen，美国国家科学院院报(Proc.Nati.Acad.Sci.)84，1340-1344(1987)]或用于酿酒酵母中表达的gap-启动子；用于在巴斯德毕赤氏酵母中表达的aoxl-启动子[Koutz等，酵母5，167-177(1989)；Sreekrishna等，基础微生物学杂志28，265-278(1988)]；或用于在多形汉逊氏酵母中表达的FMD启动子[Hollenberg等，EPA 0299108]或MOX-启动子[Ledeboer等，核酸研究13，3063-3082(1985)]。
因此含有本发明DNA序列的载体，优选使所述DNA序列在细菌或真菌或酵母中表达的载体，以及所述转化的细菌或真菌或酵母，均是本发明的一部分。
本发明还提供一种允许蛋白，尤其本发明植酸酶，高表达的系统，例如重组汉逊氏酵母菌株。为此，可根据用于表达的生物如本案中的酵母(见实施例1)的密码子频率表来选择此类蛋白的密码子。随意地，信号序列的密码子可如实施例1中具体案例所述选择；这意味着密码子频率表是依据编码指定蛋白家族的氨基酸序列的DNA序列中所用的密码子来制备。然后从用于表达的宿主细胞的密码子频率表中选择用于信号序列之DNA序列的密码子，其中利用了在两个表中出现频率相当的密码子。
一旦这些DNA序列被适当培养基中的适当宿主细胞表达后，所编码的蛋白可通过本领域公知的蛋白纯化方法或就植酸酶而言EP 420358中的方法，在被分泌到培养基中的情况下从培养基中分离，或在蛋白存在于胞内的情况下从宿主细胞中分离。因此，本发明多肽的制备方法，所述方法产生的多肽，或由本发明DNA序列编码的多肽，都是本发明的一部分，其中所述方法包括将上述转化的细菌或宿主细胞在适当条件下培养，和从中回收所述多肽。
本发明的多肽一经获得，可通过本领域公知的任何试验，按其具有农业用途的特性进行鉴定。
通常，本发明的多肽可用于任何具体领域，例如植酸酶可用于将肌醇聚磷酸如植酸盐转化为肌醇和无机磷酸。
此外，本发明的多肽可在药用组合物或复合食品或饲料的制备方法中使用，其中将所述组合物的成分与至少一种本发明的多肽混合。因此，含有至少一种本发明多肽的复合食品或饲料或药用组合物也是本发明的一部分。本领域技术人员熟知其制备方法。这类药用组合物或复合食品或饲料可进一步含有通常用于此目的且本领域公知的添加剂或成分。
本发明进一步提供了减少动物肥料中植酸盐水平的方法，其中给动物喂食所述饲料组合物，其量可将饲料中所含植酸盐有效转化为低级肌醇磷酸和/或肌醇，和无机磷酸。
在上下文中，植酸酶是一种具有植酸酶活性的酶或多肽。植酸酶可为例如肌醇六磷酸的磷酸水解酶，例如(肌醇六磷酸3-磷酸水解酶，EC 3.1.3.8)与(肌醇六磷酸6-磷酸水解酶，EC 3.1.3.26)。
在一个实施方案中，通过SDS-PAGE评估，植酸酶被纯化，即纯度至少85％，优选至少86，87，88，89，90，91，92，93，94，95，96，97，98，99％。将植酸酶分离。植酸酶活性可通过本领域公知的任何植酸酶试验来测定，例如下述试验(见实施例9)。试验温度可以是真实(actual)植酸酶的最适温度，试验pH可以是真实植酸酶的最适pH。
试验温度可在例如20-90℃，或30-80℃，或35-75℃的范围内选择，例如37℃，50℃，60℃，或70℃。
试验pH可在例如pH 2-9，或3-8，或3-6的范围内选择，例如试验的pH值可选择3，4，5，6，或7。
测定氨基酸序列的同源性(或多肽或氨基酸同源性)作为两个序列相同的程度。这可通过本领域已知的预测机程序适当测定，例如GCG软件包所提供的GAP程序[威斯康星软件包程序手册，遗传学预测机组，575 ScienceDrive，Madison，威斯康星53711，美国]，也参见Needleman，S.B.和Wunsch，C.D.，(1970)，分子生物学杂志，48，443-453]。在9.1版本中，为了进行多肽序列对比，将长度权重设为0，缺口权重设为3.0。
两个DNA(核酸)序列之间的同一性或同源性的程度可通过本领域已知的预测机程序测定，例如GCG软件包所提供的GAP程序[威斯康星软件包程序手册，遗传学预测机组，575 Science Drive，Madison，威斯康星53711，美国]来测定，也参见Needleman，S.B.和Wunsch，C.D.，(1970)，分子生物学杂志，48，443-453]。在9.1版本中，为了进行DNA序列对比，使用GAP并使用下列设置：缺口产生罚分为50，缺口延伸罚分为3。
用于测定指定DNA或RNA序列是否与特定核苷酸和寡核苷酸探针杂交的适当实验条件包括，将含有所述DNA或RNA片段、用于检测杂交的膜在5xSSC(氯化钠/柠檬酸钠；(J.Sambrook，E.F.Fritsch，和T.Maniatis，1989，分子克隆，实验室手册，第二版，冷泉港，纽约)中预浸渍10min，并使该膜在5x SSC，5x Denhardt′s溶液，0.5％SDS和100μg/ml超声处理的变性鲑精DNA(Sambrook等1989)中预杂交，然后在含10ng/ml随机-引发的32P-dCTP-标记探针(比活性＞1×109cpm/μg)的相同溶液中于约45℃杂交12小时(Feinberg，A.P.和Vogelstein，B.(1983)生物化学年鉴132：6-13)。
然后将此膜在2xS SC，0.5％SDS中洗涤两次，每次30分钟，温度为至少55℃(低严谨度)，至少60℃(中度严谨)，至少65℃(中/高严谨度)，至少70℃(高严谨度)，或至少75℃(非常高严谨度)。
在这些条件下与所述寡核苷酸探针杂交的分子可通过x-线胶片检测。
具有改变的热稳定性的植酸酶，或热稳定植酸酶，均属于本发明。“热稳定”植酸酶是Tm(解链温度)——经差示扫描量热(DSC)对植酸酶蛋白质的测定——至少为65℃的一种植酸酶。DSC可使用恒定加热率，例如10℃/min。在另外的实施方案中，Tm至少为66，67，68，69，70，71，72，73，74或75℃。或，Tm等于或低于150℃，或等于或低于145，140，135，130，125，120，115或110℃。因此，Tm间距的实例为：65-150℃，66-150℃，-(等)-75-150℃；65-145℃，66-145℃，-(等)-75-145℃；65-140℃，-(等)-75-140℃；-(等)-65-110℃，66-110℃，-(等)-75-110℃。
Tm的具体范围如下：65-110℃；70-110℃；70-100℃；75-95℃，或80-90℃。
在以下实施例9和10中，描述了经DSC对Tm的测定，且显示了多种植酸酶的Tm。
还显示了最适pH，因为——作为另一种方式——热稳定的植酸酶可被定义为一种具有至少60℃的最适温度的植酸酶。优选该最适温度在pH 5.0或5.5时的底物植酸盐或植酸上测定。实施例9描述了一例植酸酶试验，其包括对单位的定义。
在另外的实施方案中，最适温度为至少61，62，63，64，65，66，67，68，69或70℃。在一个具体实施方案中，最适温度等于或低于140℃，或等于或低于135，130，125，120，115，110，105或100℃。因此，最适温度的间距可以为：60-140℃，61-140℃，-(等)-70-140℃；60-135℃，61-135℃，-(等)-70-135℃；60-130℃，-(等)-70-130℃；-(等)-60-100℃，61-100℃，-(等)-70-100℃。
在更具体地描述本发明之前，对所附图表的简单解释如下。
图1：共有植酸酶-1序列的图样。字母表示氨基酸残基的单字母代号。以下序列用于比对：土曲霉(Aspergillus terreus)9A-1的phyA[Mitchell，D.B.，Vogel，K.，Weimann，B.J.，Pasamontes，L.&van Loon，A.P.G.M.(1997)。组氨酸磷酸酶的植酸酶亚家族：来自土曲霉和嗜热毁丝霉的两个新植酸酶的基因的分离，微生物学143，245-252]；始于氨基酸(aa)27；SEQ ID NO：1]；土曲霉cbs116.46的phyA[EP 897985]。一种在动物实验中表现优良的烟曲霉耐热植酸酶。植酸酶的优化和天然变异性。在：植酸盐和植酸酶的生物化学(Rasmussen，S.K；Raboy，V.；Dalb～ge，H.和Loewus，F.编；KluwerAcademic Publishers)；始于aa 27；SEQ ID NO：2；黑曲霉泡盛变种的phyA(Piddington等(1993)基因133，55-62；始于aa27；SEQ ID NO：3)；黑曲霉T213的phyA(EP 897985)；始于aa27；SEQ ID NO：4)；黑曲霉NRRL3135株的phyA[van Hartingsveldt，W.，van Zeijl，C.M.F.，Harteveld，G.M.，Gouka，R.J.，Suykerbuyk，M.E.G.，Luiten，R.G.M.，van Paridon，P.A.，Selten，G.C.M.，Veenstra，A.E.，van Gorcom，R.F.M.和van den Hondel，C.A.M.J.J.(1993)，黑曲霉植酸酶编码基因(phyA)的克隆、鉴定和过表达。基因127，87-94；始于aa27；SEQ ID NO：5]；烟曲霉ATCC 13073的phyA(Pasamontes，L.，Haiker，M.，Wyss，M.，Tessier，M.和van Loon，A.P.G.M.(1997)烟曲霉耐热植酸酶的克隆、纯化和鉴定，应用环境微生物学63，1696-1700；始于aa25；SEQ ID NO：6)；烟曲霉ATCC 32722的phyA(EP897985)；始于aa27；SEQ ID NO：7)；烟曲霉ATCC 58128的phyA(EP897985)；始于aa27；SEQ ID NO：8)；烟曲霉ATCC 26906的phyA(EP897985)；始于aa27；SEQ ID NO：9)；烟曲霉ATCC 32239的phyA(EP897985)；始于aa 30；SEQ ID NO：10；Emericella nidulans的phyA[Pasamontes，L.，Haiker，M.，HenriquezHuecas，M.，Mitchell，D.B.和vanLoon，A.P.G.M.(1997a).Emericella nidulans和嗜热真菌嗜热踝节菌的植酸酶的克隆，生物化学和生物物理学学报1353，217-223；始于aa 25；SEQID NO：11]；嗜热踝节菌的phyA(Pasamontes等，1997a；始于aa 24；SEQ IDNO：12)；以及嗜热毁丝霉的phyA(Mitchell等，1997；始于aa 19；SEQ IDNO：13)。比对利用程序PILEUP预测。缺口的位置经手工修正(refine)。比对中指定位置上的大写氨基酸残基属于确立共有残基的氨基酸的集合。最终构建的共有植酸酶(Fcp)的氨基酸序列(SEQ ID NO：14)以粗体显示在预测的共有序列的下方。所预测的共有序列中的缺口根据实施例1所述原则手工补齐。
图2：共有植酸酶-1基因(fcp)的DNA序列(SEQ ID NO：15)和用于基因构建的引物序列。预测的氨基酸序列(图1，SEQ ID NO：14)用程序BACKTRANSLATE[Devereux，J.，Haeberli，P.&Smithies，O.(1984)，VAX序列分析程序组，核酸研究，12，387-395]转化为DNA序列，还有高表达酵母基因的密码子频率表(GCG程序包，9.0)。土曲霉cbs 116.46的植酸酶的信号肽被融合到N-末端。图2所示氨基酸序列为SEQ ID NO：16。粗体碱基表示用于产生基因的寡核苷酸序列。各个寡核苷酸的名字被交替注释在序列的上方或下方。加下划线的碱基代表基因的起始和终止密码子。斜体字表示的碱基代表被导入的两个EcoRI位点。
图3：五种担子菌植酸酶的比对和共有序列。字母表示氨基酸残基的单字母代号。来自Paxillus involutus的植酸酶的氨基酸序列，phyAl(始于aa21；SEQ ID NO：17)；以及phyA2(始于aa 21，WO 98/28409；SEQ ID NO：18)；绒毛栓菌(始于aa 24，WO 98/28409；SEQ ID NO：19)；平田头菇(始于aa 19，WO98/28409；SEQ ID NO：20)；和Peniophora lycii(始于aa 21，WO 98/28409；SEQ ID NO：21)，从圆括号中的氨基酸残基开始，用于称作“Basidio”的相应共有序列(实施例2；SEQ ID NO：22)的比对和预测。比对用程序PILEPUP来进行，缺口的位置经手工修正。共有序列用程序PRETTY预测。当两种P.involutus植酸酶的权重为0.5时，所有其它基因采用1.0的权重以便共有序列的预测。在所述程序不能测定共有残基之处，Basidio序列含有一个破折号(dash)。比对中指定位置的大写氨基酸残基代表能确定共有残基的氨基酸集和。
图4：共有植酸酶-10氨基酸序列的图样。将Thermomyces lanuginosus植酸酶的序列[Berka，R.M.，Rey，M.W.，Brown，K.M.，Byun，T.和Klotz，A.V.(1998)，嗜热真菌Thermomyces lanuginosus的一个植酸酶基因的分子鉴定和表达，应用环境微生物学64，4423-4427；SEQ ID NO：23]和五种担子菌植酸酶的共有序列(SEQ ID NO：22)添加至图1的比对中，由程序PRETTY预测出一个改进的共有序列。此外，略去黑曲霉T213的氨基酸序列，给剩余两种黑曲霉植酸酶序列分配权重0.5。详见实施例2。
图5：共有植酸酶-10的DNA和氨基酸序列(分别为SEQ ID NO：25，和SEQ ID NO：26)。氨基酸序列用单字母表示在相应DNA序列上方。用于装配基因的寡核苷酸的序列用粗体字母表示。不同于共有植酸酶-1的寡核苷酸和氨基酸带下划线。fcp10基因用以下寡核苷酸装配：CP-1，CP-2，CP-3.10，CP-4.10，CP-5.10，CP-6，CP-7.10，CP-8.10，CP-9.10，CP-10.10，CP-11.10，CP-12.10，ap13.10，CP-14.10，CP-15.10，CP-16.10，CP-17.10，CP18.10，CP19.10，CP-20.10，CP-21.10，以及CP-22.10。新合成的寡核苷酸进一步用数字10标记。植酸酶相对于共有植酸酶-1含有以下32个取代：Y54F，E58A，D69K，D70G，A94K，N134Q，I158V，S187A，Q188N，
D197N，S204A，T214L，D220E，L234V，A238P，D246H，T251N，Y259N，
E267D，E277Q，A283D，
R291I，A320V，
R329H，
S364T，I366V，
A379K，S396A，G404A，Q415E，A437G，A463E。带下划线的突变显示了当作为共有植酸酶-1中的单突变被测试时对共有植酸酶-1的稳定效应。
图6：共有植酸酶-11(SEQ ID NO：27)的比对。与共有植酸酶-10对照，设计共有植酸酶-11的氨基酸序列时，所有担子菌植酸酶均作为独立序列使用，每一序列分配一个0.2的权重。此外，黑曲霉T213的氨基酸序列再次用于该比对中。
图7：共有植酸酶-1-thermo[8]-Q50T-K91A的DNA和氨基酸序列(分别为SEQ ID NO：28和SEQ ID NO：29)。氨基酸序列用单字母代码表示在DNA序列之上。被取代的氨基酸残基(相对于共有植酸酶-1)带下划线。基因的终止密码子用星号(*)标记。
图8：共有植酸酶-10-thermo[3]-Q50T-K91A的DNA和氨基酸序列(分别为SEQ ID NO：30和SEQ ID NO：31)。氨基酸序列用单字母代码表示在DNA序列之上。被取代的氨基酸残基(相对于共有植酸酶-10)带下划线。基因的终止密码子用星号(*)标记。
图9：烟曲霉ATCC 13073植酸酶α-突变体Q51T的DNA和氨基酸序列(分别为SEQ ID NO：32和SEQ ID NO：33)。氨基酸序列用单字母代码表示在DNA序列之上。被取代的氨基酸残基(相对于烟曲霉ATCC13073的植酸酶)带下划线。基因的终止密码子用星号(*)标记。
图10：共有植酸酶-7的DNA和氨基酸序列(分别为SEQ ID NO：34和SEQ ID NO：35)。氨基酸序列用单字母代码表示在DNA序列之上。用于装配基因的寡核苷酸的序列用粗体字母表示。被取代的寡核苷酸和氨基酸(相对于共有植酸酶-1)带下划线，相应的三联密码用小写字母表示。fcp7基因由下列寡核苷酸装配：CP-1，CP-2，CP-3，CP-4.7，CP5.7，CP-6，CP-7，CP-8.7，CP-9，CP-10.7，CP-11.7，CP-12.7，CP13.7，CP-14.7，CP-15.7，CP-16，CP-17.7，CP-18.7，CP-19.7，CP-20，CP-21，和CP-22。新合成的寡核苷酸进一步用数字7标记。与原始的共有植酸酶-1相比，共有植酸酶-7含有以下24个取代：S89D，S92G，A94K，D164S，P201S，G203A，G205S，H212P，G224A，D226T，E255T，D256E，V258T，P265S，Q292H，G300K，Y305H，A314T，S364G，M365I，A397S，S398A，G404A，和A405S。
图11：共有植酸酶-1和共有植酸酶-10的差示扫描量热(DSC)。将蛋白样品浓缩至约50-60mg/ml，对10mM乙酸钠，pH5.0强力透析。以10℃/min的恒定加热速率加热到95℃。共有植酸酶-10(上图)的DSC产生85.4℃的解链温度，其比共有植酸酶-1(78.1℃，下图)的解链温度高7.3℃。
图12：共有植酸酶-10-thermo[3]-Q50T和共有植酸酶-10-thermo[3]-Q50T-K91A的差示扫描量热(DSC)。将蛋白样品浓缩至约50-60mg/ml，对10mM乙酸钠，pH5.0强力透析。以10℃/min的恒定加热速率加热到95℃。共有植酸酶-10-thermo[3]-Q50T(上图)的DSC产生88.6℃的解链温度，而共有植酸酶-10-thermo[3]-Q50T-K91A的解链温度被测定为89.3℃。
图13：共有植酸酶-1、共有植酸酶-10和共有植酸酶-10-thermo[3]-Q50T的最适温度比较。为了测定最适温度，在37-86℃之间的一系列温度下进行实施例9的植酸酶标准试验。用酿酒酵母的稀释上清进行检测。上清的其它组份对最适温度的测定没有影响：△，共有植酸酶-1；◇，共有植酸酶-10；■，共有植酸酶-10-thermo[3]-Q50T。
图14：共有植酸酶-10及其突变体thermo[3]-Q50T和thermo[3]-Q50T-K91A的依赖pH的活性图谱和底物特异性。植酸酶活性通过在适当缓冲液中不同pH的标准试验测定(见实施例9)。图a)显示了共有植酸酶-10(□)，共有植酸酶-10-thermo[3]-Q50T(●)和共有植酸酶-10-thermo[3]-Q50T-K91A(△)的依赖pH的活性图谱，图b)显示了通过在标准试验中用所述化合物置换植酸测得的相应底物特异性；空白栏，共有植酸酶-10；灰色栏，共有植酸酶-10-thermo[3]-Q50T；黑色栏，共有植酸酶-10-thermo[3]-Q50T-K91A。数字对应下列底物：1.植酸；2.对硝基苯磷酸；3.苯基磷酸；4.果糖-1，6-二磷酸；5.果糖-6-磷酸；6.葡萄糖-6-磷酸；7.核糖-5-磷酸；8.DL-甘油-3-磷酸；9.甘油-2-磷酸；10.3-磷酸甘油酸；11.磷酸烯醇丙酮酸；12.AMP；13.ADP；14.ATP。
图15：共有植酸酶-1-thermo[8]-Q50T和共有植酸酶-1-thermo[8]-Q50T-K91A的依赖pH的活性图谱和底物特异性。植酸酶活性通过在适当缓冲液中不同pH的标准试验测定(见实施例9)。图a)显示了Q50T变体(■)和Q50T-K91A变体(●)的依赖pH的活性图谱，图b)显示了通过在标准试验中用所述化合物置换植酸测得的相应底物特异性(空白栏，共有植酸酶-1-thermo[8]-Q50T；实心栏，共有植酸酶-1-thermo[8]-Q50T-K91A)。底物列在图14的图说明中。
图16：共有植酸酶-1-thermo[8]-Q50T和共有植酸酶-1-thermo[8]-Q50T-K91A的差示扫描量热(DSC)。将蛋白样品浓缩至约50-60mg/ml，对10mM乙酸钠，pH5.0强力透析。以10℃/min的恒定加热速率加热到95℃。共有植酸酶-1-thermo[8]-Q50T(上图)的DSC产生84.7℃的解链温度，而共有植酸酶-1-thermo[8]-Q50T-K91A的解链温度被测定为85.7℃。
图17：共有植酸酶-1，共有植酸酶-1和共有植酸酶-1-thermo-[3]和共有植酸酶-1-thermo[8]的最适温度的比较。为了测定最适温度，在37-86℃之间的一系列温度下进行植酸酶标准试验。用来自转化的酿酒酵母菌株的纯化蛋白进行检测。○，共有植酸酶-1；□，共有植酸酶-1-thermo[3]；▲，共有植酸酶1-thermo[8]。
图18：共有植酸酶-1，共有植酸酶-7，和黑曲霉NRRL 3135植酸酶的依赖pH的活性和底物特异性的比较。植酸酶活性通过在适当缓冲液中不同pH的标准试验测定(见实施例9)。图a)显示了植酸酶-1(■)，黑曲霉NRRL3135植酸酶(○)，和共有植酸酶-7(▲)的依赖pH的活性图谱，图表b)显示了通过在标准试验中用所述化合物置换植酸测得的相应底物特异性(黑色栏，黑曲霉NRRL 3135植酸酶；空白栏，共有植酸酶-1；灰色栏，共有植酸酶-7)。底物列在图14的图说明中。
图19：烟曲霉ATCC 13073的植酸酶及其含有氨基酸置换(F55Y，V100I，F114Y，A243L，S265P，和N294D)的稳定型α-突变体的差示扫描量热(DSC)。
将蛋白样品浓缩至约50-60mg/ml，对10mM乙酸钠，pH5.0强力透析。以10℃/min的恒定加热速率加热到95℃。烟曲霉ATCC 13073植酸酶(上图)的DSC显示为62.5℃的解链温度，而所述α-突变体的解链温度被测定为67.0℃。
图20：烟曲霉13073的野生型植酸酶，其α-突变体，以及其进一步稳定的α-突变体(E59A-S154N-R329H-S364T-G404A)的最适温度比较。为了测定最适温度，在37-75℃之间的一系列温度下进行植酸酶标准试验。用转化的酿酒酵母菌株的稀释上清进行检测。上清的其它组份对最适温度的测得没有影响。○，烟曲霉ATCC 13073植酸酶；▲，烟曲霉ATCC 13073α-突变体；□，烟曲霉ATCC 13073植酸酶α-突变体-(E59A-S154N-R329H-S364T-G404A)；■，烟曲霉ATCC 13073α-突变体(E59A-S154N-R329H-S364T-G404A)-Q51T-K92A。Q51T和K92A分别对应于共有植酸酶-1的取代Q50T和K91A。
图21：共有植酸酶-12的氨基酸序列(consphyl2；SEQ ID NO：36)，其含有从＂basidio＂共有序列转移到共有植酸酶-10-thermo[3]-Q50T-K91A的多个活性位点残基(加下划线)。
图22：共有植酸酶-3-thermo[11]-Q50T的DNA和氨基酸序列。氨基酸用单字母代码表示在相应DNA序列之下。
图23：共有植酸酶-3-thermo[11]-Q50T-K91A的DNA和氨基酸序列。氨基酸用单字母代码表示在相应DNA序列之下。
图24：共有植酸酶-10-thermo[5]-Q50T的DNA和氨基酸序列。氨基酸用单字母代码表示在相应DNA序列之下。
图25：共有植酸酶-10-thermo[5]-Q50T-K91A的DNA和氨基酸序列。氨基酸用单字母代码表示在相应DNA序列之下。
本文所述的生产植酸酶的微生物菌株，即Paxillus involutus CBS100231；Peniophora lycii CBS 686.96；平田头菇CBS 900.96；以及绒毛栓菌CBS100232分别分离于来自丹麦；丹麦；丹麦；和瑞典的天然样品(Uppsala保藏中心。分别于1992年11月；1993年10月；1995年6月；和1995年11月保藏。
实施例1 共有植酸酶-1
共有植酸酶-1(真菌共有植酸酶，fcp)的氨基酸序列如EP 897985中实施例1和2所述设计并预测。下表1显示了用于设计共有植酸酶-1的植酸酶氨基酸序列的起点和权重。进一步将共有植酸酶-1序列如EP 897985的实施例3所述转化为DNA序列，并如EP897985的实施例4所述构建和克隆共有植酸酶-1基因。
表1 植酸酶氨基酸序列的起点和权重
土曲霉9A-1的phyA，aa 27，权重0.5(Mitchell等，1997)
土曲霉cbs116.46 phyA，aa 27，权重0.5(EP 897985)
黑曲霉泡盛变种phyA，aa 27，权重0.33[Piddington，C.S.，Houston，C.S.，Paloheimo M.，Cantrell，M.，Miettinen-Oinonen，A.，Nevalainen，H.，&Rambosek，J.(1993)，编码黑曲霉泡盛变种的植酸酶(phy)和pH2.5最适型酸性磷酸酶(aph)的基因的克隆和测序，基因133，55-62)
黑曲霉T213 phyA(EP 897985)，aa 27，权重0.33
黑曲霉NRRL3135株phyA，aa 27，权重0.33(van Hartingsveldt等，1993)
烟曲霉ATCC 13073 phyA，aa 26，权重0.2(Pasamontes等，1997)
烟曲霉ATCC 32722 phyA，aa 26，权重0.2(EP 897985)
烟曲霉ATCC 58128 phyA，aa 26，权重0.2(EP 897985)
烟曲霉ATCC 26906 phyA，aa 26，权重0.2(EP 897985)
烟曲霉ATCC 32239 phyA，aa 30，权重0.2(EP 897985)
Emericella nidulans phyA，aa 25，权重1.0(Pasamontes等，1997a)
嗜热踝节菌ATCC 20186 phyA，aa 24，权重1.0(Pasamontes等，1997a)
嗜热毁丝霉phyA，aa 19，权重1.0(Mitchell等，1997)
实施例2 一种改进型共有植酸酶(共有植酸酶-10)氨基酸序列的设计
用于设计共有植酸酶-10的比对通过使用GCG序列分析软件包9.0版本(Devereux等，1984)的程序PILEUP以标准参数(缺口产生罚分12，缺口延伸罚分4)进行预测。缺口的定位使用文本编辑器(text editor)修正。
用以下序列对始于表2所述氨基酸(aa)的担子菌植酸酶进行比对：
表2
5种担子菌植酸酶用于预测相应共有氨基酸序列(basidio)的起点和权重
Paxillus involutus CBS 100231 phyA1，aa 21，权重0.5(WO 98/28409)
Paxillus involutus CBS 100231 phyA2，aa 21，权重0.5(WO 98/28409)
绒毛栓菌CBS 100232 phyA，aa 24，权重1.0(WO 98/28409)
平田头菇CBS 900.96 phyA，aa 19，权重1.0(WO 98/28409)
Peniophora lycii CBS 686.96 phyA，aa 21，权重1.0(WO 98/28409)
该比对显示在图3中。
表3中列出了用于最终比对的基因。该比对中所用序列的第一氨基酸(aa)述于生物命名之后。
表3 用于设计共有植酸酶-10的植酸酶序列的起点和权重
土曲霉9A-1 phyA，aa 27，权重0.5(Mitchell等，1997)
土曲霉cbs116.46 phyA，aa 27，权重0.5(EP 897985)
黑曲霉泡盛变种phyA，aa27，权重0.5(Piddington等，1993)
黑曲霉NRRL3135株phyA，aa 27，权重0.5(van Hartingsveldt等，1993)
烟曲霉ATCC 13073 phyA，aa 26，权重0.2(Pasamontes等，1997)
烟曲霉ATCC 32722 phyA，aa 26，权重0.2(EP 897985)
烟曲霉ATCC 58128 phyA，aa 26，权重0.2(EP 897985)
烟曲霉ATCC 26906 phyA，aa 26，权重0.2(EP 897985)
烟曲霉ATCC 32239 phyA，aa 30，权重0.2(EP 897985)
Emericella nidulans phyA，aa 25，权重1.0(Pasamontes等，1997a)
嗜热踝节菌ATCC 20186 phyA，aa 24，权重1.0(Pasamontes等，1997a)
嗜热毁丝霉phyA，aa 19，权重1.0(Mitchell等，1997)
Thermomyces lanuginosus phyA，aa 36，权重1.0(Berka等，1998)
五种担子菌植酸酶的共有序列，权重1.0(Basidio，图3)
相应比对显示于图4。
共有植酸酶-10的氨基酸序列的预测
为了改进比对，我们增加了来自4种不同担子菌之5种植酸酶的最初共有序列(称为Basidio；仍含有未确定的序列位置；见图3)，用于该最初共有植酸酶序列之预测的几乎所有植酸酶序列和一种新的来自子囊菌纲Thermomyces lanuginosus的植酸酶序列，以便进行更大的比对。
我们设置复数性(plurality)为2.0和阈值为3。所用权重列于表3。比对及相应共有序列见表4。新的共有植酸酶序列和最初的共有植酸酶-1相比具有32个不同的氨基酸。程序PRETTY不能预测共有氨基酸残基之处按实施例1所述规则补齐。该程序所提示的残基无一被取代。
此外，在另一预测中，我们将所有担子菌植酸酶分别作为个氨基酸序列配以权重0.2来进行预测。相应的比对示于图6。预测得到的共有氨基酸序列(共有植酸酶-11)与共有植酸酶-10的序列有以下差异。字母X指该程序不能预测共有氨基酸；圆括号中的氨基酸对应于最终包括在共有植酸酶-10内的氨基酸。
D35X(第一个字母是共有植酸酶-10的，最后一个字母是共有植酸酶-11的)，X(K)69K，X(E)100E，A101R，Q134N，X(K)153N，X(H)190H，X(A)204S，X(E)220D，E222T，V227A，X(R)271R，H287A，X(D)288D，X(K)379K，X(I)389I，E390X，X(E)415E，X(A)416A，X(R)446L，E463A.编号如图5。
我们也检查了改进型共有序列10和11所示单个氨基酸取代对最初共有植酸酶之稳定性的影响。该方法见实施例3。
共有植酸酶-10的氨基酸序列到DNA序列的转换
用土曲霉cbs116.46植酸酶的最初26个氨基酸残基作为信号肽，将其融合至共有植酸酶-10的N-末端。所用方法进一步描述在实施例1中。
所得fcp10基因的序列见图5。
共有植酸酶-10(fcp10)基因的构建和克隆
任选用正义链和反义链将预测的fcp10DNA序列分为85bp的寡核苷酸。每一寡核苷酸与相对链的前、后寡核苷酸重叠20bp。所有购自Microsynth，Balgach(瑞士)的PAGE-纯化型引物参见图5。
PCR-反应
在三个PCR反应中，将合成寡核苷酸被编入完整基因中。用来自Boehringer Mannheim(Boehringer Mannheim，Mannheim，德国)的HighFidelity试剂盒和来自AMS生物技术(欧洲)有限公司(Lugano，瑞士)的热循环仪“The ProtokolTM”实施PCR。所用以下寡核苷酸浓度为0.2pMol/ml。
Mix 1.10：CP-1，CP-2，CP-3.10，CP-4.10，CP-5.10，CP-6，
CP-7.10，CP-8.10，CP-9.10，CP-10.10
Mix 2.10：CP-9.10，Cp-1 1.10，CP-12.10，CP-13.10，Cp-14.10，
CP-15.10，CP-16.10，CP-17.10，CP18.10，CP-19.10，
CP-20.10，CP-21.10，CP-22.10
新合成的寡核苷酸用数字10标记。共有植酸酶-10与最初的共有植酸酶-1相比含有下面的32个取代，其在图5中被加下划线：Y54F，E58A，D69K，D70G，A94K，N134Q，I158V，S187A，Q188N，D197N，S204A，T214L，D220E，L234V，A238P，D246H，T251N，Y259N，E267D，E277Q，A283D，R291I，A320V，R329H，S364T，I366V，A379K，S396A，G404A，Q415E，A437G，A463E。
4个短PCR引物用于寡核苷酸的合成：
CP-a：Eco RI
5′-TATATGAATTCATGGGCGTGTTCGTC-3′SEQ ID NO：37)
CP-b：
5′-TGAAAAGTTCATTGAAGGTITC-3′(SEQ ID NO：38)
CP-c.10：
5′-TCTTCGAAAGCAGTACACAAAC-3′(SEQ ID NO：39)
CP-e：Eco RI
5′-TATATGAATTCTTAAGCGAAAC-3′(SEQ ID NO：40)
PCR反应a：10μl Mix 1.10(每一寡核苷酸2.0pmol)
2μl核苷酸(每一寡核苷酸10mM)
2μl引物CP-a(10pmol/ml)
2μl引物CP-c.10(10pmol/ml)
10.0μl PCR缓冲液
0.75μl聚合酶混合物(2.6U)
73.25μl H2O
PCR反应b：10μl Mix 2.10(每一寡核苷2.0pmol)
2μl核苷酸(每一核苷酸10mM)
2μl引物CP-b(10pmol/ml)
2μl引物CP-e(10pmol/ml)
10.0μl PCR缓冲液
0.75μl聚合酶混合物(2.6U)
73.25μl H2O
PCR反应a和b的反应条件：
步骤1 2min-45℃
步骤2 30sec-72℃
步骤3 30sec-94℃
步骤4 30sec-52℃
步骤5 1min-72℃
将步骤3到5重复40次。
PCR产物(670和905 bp)经琼脂糖凝胶电泳(0.9％琼脂糖)，然后经凝胶提取(QIAEX II凝胶提取试剂盒，Qiagen，Hilden，德国)纯化。用纯化的DNA片段进行PCR反应c。
PCR反应c：6μl PCR反应a的产物≈50ng)
6μl PCR反应b的产物≈50ng)
2μl 引物CP-a(10pmol/ml)
2μl 引物CP-e(10pmol/ml)
10.0μl PCR缓冲液
0.75μl聚合酶混合物(2.6U)
73.25μl H2O
PCR反应c的反应条件：
步骤1 2min-94℃
步骤2 30sec-94℃
步骤3 30sec-55℃
步骤4 1min-55℃
将步骤2到4重复31次。
所得PCR产物(1.4kb)如上述纯化，用EeoRI降解，并连接至EcoRI-消化并去磷酸化的pBsk(-)-载体(Stratagene，La Jolla，CA，美国)。用1μl连接混合物转化大肠杆菌XL-1感受态细胞(Stratagene，La Jolla，CA，美国)。所有标准方法如Sambrook等(1987)所述。所构建的基因(fcp10)的DNA序列通过本领域公知的测序方法检查。
实施例3 通过导入共有植酸酶-10和共有植酸酶-11的氨基酸序列所
显示的单个突变增加共有植酸酶-1的热稳定性
为了增加同源基因的热稳定性，也可检验目的蛋白与预测的共有序列之间每一差异氨基酸残基的稳定性效应，并将所有稳定突变组合至目的蛋白中。我们利用共有植酸酶-1作为目的蛋白，测试以单位点定点突变区别于共有植酸酶-10和/或-11的34个氨基酸残基的蛋白稳定性效应。
为构建在黑曲霉，酿酒酵母，或多形汉逊氏酵母中表达的突变蛋白，用含有共有植酸酶-1基因的相应表达质粒作为模板进行定点诱变(见实施例6-8)。突变用Stratagene(La Jolla，CA，美国)的＂快速交换(quick exchange)TM定点诱变试剂盒＂按说明书指示并使用相应引物来导入。所有获得的突变及相应引物概述于表4。携有所需突变的质粒用本领域公知的DNA序列分析来鉴定。
表4 用于共有植酸酶-1定点诱变的引物
所取代的碱基用粗体来突出。导入的限制性位点在序列上方标出。当限制性位点被写在圆括号中时，所述位点通过导入突变而破坏。
突变 引物系列
Kpn I
Q50T 5′-CACTTGTGGGGTACCTACTCTCCATACTTCTC-3′(SEQ ID NO：41)
5′-GAGAAGTATGGAGAGTAGGTACCCCACAAGTG-3′
Y54F 5′-GGTCAATACTCTCCATTCTTCTCTTTGGAAG-3′(SEQ ID NO：42)
5′-CTTCCAAAGAGAAGAATGGAGAGTATTGACC-3′
E58A 5′-CATACTTCTCTTTGGCAGACGAATCTGC-3′(SEQ ID NO：43)
5′-GCAGATTCGTCTGCCAAAGAGAAGTATG-3′
Aat II
D69K 5′-CTCCAGACGTCCCAAAGGACTGTAGAGTTAC-3′(SEQ ID NO：44)
5′-GTAACTCTACAGTCCTTTGGGACGTCTGGAG-3′
Aat II
D70G 5′-CTCCAGACGTCCCAGACGGCTGTAGAGTTAC-3′(SEQ ID NO：45)
5′-GTAACTCTACAGCCGTCTGGGACGTCTGGAG-3′
K91A 5′-GATACCCAACTTCTTCTGCGTCTAAGGCTTACTCTG-3′
(SEQ ID NO：46)
5′-CAGAGTAAGCCTTAGACGCAGAAGAAGTTGGGTATC-3′
Sca I
A94K 5′-CTTCTAAGTCTAAGAAGTACTCTGCTTTG-3′(SEQ ID NO：47)
5′-CAAAGCAGAGTACTTCTTAGACTTAGAAG-3′
A101R 5′-GCTTACTCTGCTTTGATTGAACGGATTCAAAAGAACGCTAC-3′
(SEQ ID NO：48)
5′-GTAGCGTTCTTTTGAATCCGTTCAATCAAAGCAGAGTAAGC-3′
N134Q 5′-CCATTCGGTGAACAGCAAATGGTTAACTC-3′(SEQID NO：49)
5′-GAGTTAACCATTTGCTGTTCACCGAATGG-3′
Nru I
K153N 5′-GATACAAGGCTCTCGCGAGAAACATTGTTC -3′(SEQ ID NO：50)
5′-GGAACAATGTTTCTCGCGAGAGCCTTGTATC-3′
Bss HI
I158V 5′-GATTGTTCCATTCGTGCGCGCTTCTGGTTC-3′(SEQ ID NO：51)
5′-GAACCAGAAGCGCGCACGAATGGAACAATC-3′
Apa I
S187A 5′-GGCTGACCCAGGGGCCCAACCACACCAAGC-3′(SEQ ID NO：53)
5′-GCTTGGTGTGGTTGGGCCCCTGGGTCAGCC-3′
Bcl I
D197N 5′-CTCCAGTTATTAACGTGATCATTCCAGAAGG-3′(SEQ ID NO：52)
5′-CCTTCTGGAATGATCACGTTAATAACTGGAG-3′
Nco I
T214L 5′-CACTTTGGACCATGGTCTTTGTACTGCTTTCG-3′(SEQ ID NO：54)
5′-CGAAAGCAGTACAAAGACCATGGTCCAAAGTG-3′
Avr II
E222T 5′-GCTTTCGAAGACTCTACCCTAGGTGACGACGTTG-3′
(SEQ ID NO：55)
5′-CAACGTCGTCACCTAGGGTAGAGTCTTCGAAAGC-3′
V227A 5′-GGTGACGACGCTGAAGCTAACTTCAC-3′(SEQ ID NO：56)
5′-GTGAAGTTAGCTTCAGCGTCGTCACC-3′
Sac II
L234V 5′-CTAACTTCACCGCGGTGTTCGCTCCAG-3′(SEQ ID NO：57)
5′-CTGGAGCGAACACCGCGGTGAAGTTAG-3′
A238P 5′-GCTTTGTTCGCTCCACCTATTAGAGCTAGATTGG-3′
(SEQ ID NO：58)
5′-CCAATCTAGCTCTAATAGGTGGAGCGAACAAAGC-3′
Hpa I
T251N 5′-GCCAGGTGTTAACTTGACTGACGAAG-3′(SEQ ID NO：59)
5′-TTCGTCAGTCAAGTTAACACCTGGC-3′
Aat II
Y259N 5′-GACGAAGACGTCGTTAACTTGATGGAC-3′(SEQ ID NO：60)
5′-GTCCATCAAGTTAACGACGTCTTCGTC-3′
Asp I
E267D 5′-GTCCATTCGACACTGTCGCTAGAACTTC-3′(SEQ ID NO：61)
5′-GAAGTTCTAGCGACAGTGTCGAATGGAC-3′
E277Q 5′-CTGACGCTACTCAGCTGTCTCCATTC-3′(SEQ ID NO：62)
5′-GAATGGAGACAGCTGAGTAGCGTCAG-3′
A283D 5′-GTCTCCATTCTGTGATTTGTTCACTCAC-3′(SEQ ID NO：63)
5′-GTGAGTGAACAAATCACAGAATGGAGAC-3′
Ksp I
H287A 5′-GCTTTGTTCACCGCGGACGAATGGAG-3′(SEQ ID NO：64)
5′-CTCCATTCGTCCGCGGTGAACAAAGC-3′
Bam HI
R291I 5′-CACGACGAATGGATCCAATACGACTAC-3′(SEQ ID NO：65)
5′-GTAGTCGTATTGGATCCATTCGTCGTG-3′
Bsi WI
Q292A 5′-GACGAATGGAGAGCGTACGACTACTTG-3′(SEQ ID NO：66)
5′-CAAGTAGTCGTACGCTCTCCATTCGTC-3′
Hpa I
A320V 5′-GGTGTTGGTTTCGTTAACGAATTGATTGC-3′(SEQ ID NO：67)
5′-GCAATCAATTCGTTAACGAAACCAACACC-3′
(Bgl II)
R329H 5′-GCTAGATTGACTCACTCTCCAGTTCAAG-3′(SEQ ID NO：68)
5′-CTTGAACTGGAGAGTGAGTCAATCTAGC-3′
Eco RV
S364T 5′-CTCACGACAACACTATGATATCTATTTTCTTC-3′(SEQ ID NO：69)
5′-GAAGAAAATAGATATCATAGTGTTGTCGTGAG-3′
Nco I
I366V 5′-CGACAACTCCATGGTTTCTATTTTCTTCGC-3′(SEQ ID NO：70)
5′-GCGAAGAAAATAGAAACCATGGAGTTGTCG-3′
Kpn I
A379K 5′-GTACAACGGTACCAAGCCATTGTCTAC-3′(SEQ ID NO：71)
5′-GTAGACAATGGCTTGGTACCGTTGTAC-3′
S396A 5′-CTGACGGTTACGCTGCTTCTTGGAC-3′(SEQ ID NO：72)
5′-GTCCAAGAAGCAGCGTAACCGTCAG-3′
G404A 5′-CTGTTCCATTCGCTGCTAGAGCTTAC-3′(SEQ ID NO：73)
5′-GTAAGCTCTAGCAGCGAATGGAACAG-3′
Q415E 5′-GATGCAATGTGAAGCTGAAAAGGAACC-3′(SEQ ID NO：74)
5′-GGTTCCTTTTCAGCTTCACATTGCATC-3′
Sal I
A437G 5′-CACGGTTGTGGTGTCGACAAGTTGGG-3′(SEQ ID NO：75)
5′-CCCAACTTGTCGACACCACAACCGTG-3′
Mun I
A463E 5′-GATCTGGTGGCAATTGGGAGGAATGTTTCG-3′(SEQID NO：76)
5′-CGAAACATTCCTCCCAATTGCCACCAGATC-3′
以及，相应地，可用于其它突变。
如实施例9所述测定于酿酒酵母中表达的纯化植酸酶(实施例7)的最适温度。表5显示所导入的每一突变对共有植酸酶-1稳定性的影响。
表5 单独氨基酸在共有植酸酶-1中的取代对稳定性的影响
+或-分别指蛋白稳定性变动最多1℃的正效应或负效应，++和--是指蛋白稳定性变动1℃至3℃的正效应或负效应；圆括号中的10或11指提示氨基酸取代的共有植酸酶序列。
稳定 中性 不稳定
| 突变 效应 | 突变 效应 | 突变 效应 |
| E58A (10) +D69K (11) +D197N (10) +T214L (10) ++E222T (11) ++E267D (10) +R291I +R329H (10) +S364T (10) ++A379K (11) +G404A (10) ++ | D69A ±D70G (10) ±N134Q (10) ±G186H ±S187A (10) ±T214V ±T251N (10) ±Y259N (10) ±A283D (10) ±A320V (10) ±K445T ±A463E (10) ± | Y54F (10) -V73I -A94K (10) -A101R (11) -K153N (11) -I158V (10) --G203A --G205S -A217V -V227A (11) --L234V (10) -A238P (10) --E277Q (10) -H287A (11) -Q292A -I366V (10) -S396A (10) --Q415E (11) -A437G (10) --E451R -- |
本实施例用上述引物和技术将8种正突变(E58A，D197N，E267D，R291I，R329H，S364T，A379K，G404A)组合在共有植酸酶-1-thermo[8]中。此外，导入主要影响植酸酶催化特性的突变Q50T和/或K91A(见专利申请EP 897010和EP 897985，以及实施例9)。所得植酸酶(共有植酸酶-1-thermo[8]-Q50T-K91A)的DNA和氨基酸序列示于图7。该共有植酸酶的最适温度和解链温度通过这种方式增加了7℃(图15，16，17)。
在另一个共有蛋白中，我们在共有植酸酶-1-thermo[11]中组合了11个正突变(E58A，D69K，D197N，T214L，E222T，E267D，R291I，R329H，S364T，A379K，G404A)。此外，导入突变Q50T和/或K91A。通过这种方式使解链温度比共有植酸酶-1-thermo[8]又增加3-4℃。
利用表5的结果，我们通过回复突变K94A，V158I，和A396S进一步改善了共有植酸酶-10的热稳定性，(A94K，I158V，和S396A)的回复显示对共有植酸酶-1的稳定性的强烈负效应，所得蛋白称为共有植酸酶-10-thermo[3]。SEQ ID NO：26加上3个突变K94A，V158I，和A396S。此外，我们导入了主要影响共有植酸酶催化特性的突变Q50T和K91A(参见专利申请EP 897010和EP 897985，以及实施例9和图14和15)。所得DNA和氨基酸序列见图8。所优化的植酸酶显示出比共有植酸酶-10高4℃的最适温度和解链温度(图12和13)。此外该植酸酶液在pH5.5对250U/mg植酸底物的比活性大大增加(图14)。
在另一种共有蛋白中，两个另外的突变被导入到产生共有植酸酶-10thermo[5]的共有植酸酶-10 thermo[3](E222T，G437A)中。此外，导入突变Q50T和/或K91A。通过这种方式使解链温度比共有植酸酶-10 thermo[3]又增加了1-2℃。
实施例4
烟曲霉ATCC 13073的植酸酶通过将其氨基酸残基用共有植酸酶-1和/
或共有植酸酶-10的相关残基取代而稳定
在图1的比对中的6个氨基酸序列位置上，仅有或几乎仅有烟曲霉13073植酸酶不含有相应的共有植酸酶氨基酸残基，将这些非共有氨基酸残基用共有残基取代。以下氨基酸在烟曲霉13073植酸酶中被取代，另含有Q51(24)T取代(其影响催化特性且对应于共有植酸酶中的Q50T取代)和土曲霉cbs116.46植酸酶的信号序列(参见欧洲专利申请0897010，和图9)：F55(28)Y，V100(73)I，F114(87)Y，A243(220)L，S265(242)P，N294(282)D。圆括号中的数字指图1中的编号。
在第二轮中，进一步将在共有植酸酶-10中鉴定并检验为共有植酸酶-1(表5)中单一突变的11个稳定氨基酸取代中的4个(E58A，R329H，S364T，G404A)导入烟曲霉α-突变体中。此外，还导入氨基酸取代S154N，其显示植酸酶的蛋白酶敏感性降低。
如实施例3所述导入突变(见表6)，如实施例6-8所述使之表达。产生的烟曲霉13073植酸酶变体被称为α突变体(例如带有Q24T，F28Y，V73I，F87Y，A220L，S242P，N282D取代的烟曲霉ATCC 13073植酸酶)以及＂优化的＂α-突变体(例如进一步具有E59A-S1S4N-R329H-S364T-G404A取代的烟曲霉α-突变体)。K92A是一个额外的优选突变。
烟曲霉13073α-突变体的植酸酶的最适温度(60℃，图20)和解链温度(67.0℃，图9)比野生型植酸酶的(最适温度：55℃，Tm：60℃)提高5-7℃。另5个氨基酸取代进一步使最适温度提高3℃(图20)。
表6 用于稳定烟曲霉ATCC 13073植酸酶的诱变引物
突变 引物
F55Y 5′-CACGTACTCGCCATACTTTTCGCTCGAG-3′(SEQ ID NO：77)
5′-CTCGAGCGAAAAGTATGGCGAGTACGTG-3′
(Xho I)
E58A 5′-CCATACTTTTCGCTCGCGGACGAGCTGTCCGTG-3′
(SEQ ID NO：78)
5′-CACGGACAGCTCGTCCGCGAGCGAAAAGTAGG-3′
V100I 5′-GTATAAGAAGCTTATTACGGCGATCCAGGCC-3′
(SEQ ID NO：79)
5′-GGCCTGGATCGCCGTAATAAGCTTCTTATAC-3′
F114Y 5′-CTTCAAGGGCAAGTACGCCTTTTTGAAGACG-3′
(SEQ ID NO：80)
5′-CGTCTTCAAAAAGGCGTACTTGCCCTTGAAG-3′
A243L 5′-CATCCGAGCTCGCCTCGAGAAGCATCTTC-3′(SEQID NO：81)
5′-GAAGATGCTTCTCGAGGCGAGCTCGGATG-3′
S265P 5′-CTAATGGA TGTGTCCGTTTGATACGGTAG-3′(SEQ ID NO：82)
5′-CTACCGTATCAAACGGACACATGTCCATTAG-3′
N294D 5′-GTGGAAGAAGTACGACTACCTTCAGTC-3′(SEQ ID NO：83)
5′-GACTGAAGGTAGTCGTACTTCTTCCAC-3′
(Mlu I)
R329H 5′-GCCCGGTTGACGCATTCGCCAGTGCAGG-3′(SEQ ID NO：84)
5′-CCTGCACTGGCGAATGCGTCAACCGGGC-3′
Nco I
S364T 5′-CACACGACAACACCATGGTTTCCATCTTC-3′(SEQ ID NO：85)
5′-GAAGATGGAAACCATGGTGTTGTCGTGTG-3′
(Bss HI)
G404A 5′-GTGGTGCCTTTCGCCGCGCGAGCCTACTTC-3′(SEQ ID NO：86)
5′-GAAGTAGGCTCGCGCGGCGAAAGGCACCAC-3′
实施例5
将黑曲霉NRRL3135植酸酶的活性位点氨基酸残基导入共有植酸酶-1
我们使用黑曲霉NRRL 3135植酸酶的晶体结构来限定所有活性位点氨基酸残基(见实施例1和EP 897010)。根据图1的比对，我们将共有植酸酶-1的下述活性位点残基以及不相同的邻近残基用黑曲霉植酸酶的那些取代：S89D，S92G，A94K，D164S，P201S，G203A，G205S，H212P，G224A，D226T，E255T，D256E，V258T，P265S，Q292H，G300K，Y305H，A314T，S364G，M365I，A397S，S398A，G404A，和A405S。
如实施例1所述将新的共有植酸酶-7蛋白序列反译为DNA序列(图10)。相应的基因(fcp7)如实施例1所述用下述寡核苷酸混合物来产生：
Mix 1.7：CP-1，CP-2，CP-3，CP-4.7，CP-5.7，CP-6，CP-7，CP8.7，
CP-9，CP-10.7
Mix 2.7：CP-9，CP-10.7，CP-11.7，CP-12.7，CP-13.7，CP-14.7，CP-15.7，
CP-16，CP-17.7，CP-18.7，CP-19.7，CP-20，CP-21，CP-22。
上述寡核苷酸的DNA序列见图10。新合成的寡核苷酸另用数字7标记。用如实施例1所述的相同PCR引物合成寡核苷酸后，实施例6-8所述将该基因克隆至表达载体中。
在多形汉逊氏酵母中表达和纯化后，所检测的该酶的pH-谱非常相似于黑曲霉植酸酶的(见图18)。
实施例6 共有植酸酶基因在多形汉逊氏酵母中的表达
用于转化多形汉逊氏酵母RB11的植酸酶表达载体[Gellissen，G.，Hollenberg，C.P.，Janowicz，Z.A.(1994)，甲基营养型酵母中的基因表达，在Smith，A.(编)，重组微生物中的基因表达。Dekker，纽约，PP395-439]通过将pBsk-fcp或其变体的Eco RI片段插入多形汉逊氏酵母表达载体pFPMT121的多克隆位点中而构建，其中pFPMT121基于酿酒酵母的ura3选择标记，多形汉逊氏酵母的甲酸脱氢酶(FMD)启动子元件和甲醇氧化酶(MO)终止子元件。将Fcp基因的5`末端融合至FMD启动子，3`末端融合至MOX终止子(Gellissen等，应用微生物学生物技术(Appl.Microbiol.Biotechnol.)46，46-54，1996；EP 299108)。所产生的表达载体命名为pFPMTfcp，pFPMTfcp10，和pFPMTfcp7。
使所构建的质粒在大肠杆菌中增殖。质粒DNA用现有技术中的常规方法纯化。利用如Gellissen等(1996)所述的感受态细胞制备方法和酵母转化方法将所述表达质粒转化至乳清苷-5`-磷酸脱羧酶(ura3)缺陷型多形汉逊氏酵母菌株RB11中。将每一种转化混合物涂布于含有2％葡萄糖和1.8％琼脂的YNB培养基(0.14％w/v Difco YNB和0.5％硫酸铵)上，37℃培养。4-5天后挑出单个转化菌落，如上述在液体培养基中37℃培养2天。随后，将此培养物的等分试样接种到装有含2％葡萄糖之YNB培养基的新鲜小瓶中。在选择培养基上进一步传7代后，所述表达载体以多聚体形式整合到酵母基因组中。然后，通过两个另外的在3ml非选择性液体培养基(YPD，2％葡萄糖，10g/l酵母提取物，和20g/l蛋白胨)中另两步培养，获得有丝分裂稳定型转化子。为了获得遗传同源性重组菌株，将最终的稳定培养物的等分试样涂布于选择平皿中。分离单菌落，分析在含有2％甘油(其阻遏FMD启动子)而非葡萄糖的YNB中表达的植酸酶。共有植酸酶的纯化如实施7所述。
实施例7
在酿酒酵母中表达共有植酸酶基因并从培养物上清中纯化植酸酶
共有植酸酶基因从相应蓝斑(Bluescript)-质粒(pBsk-fcp，pBSK-fcp10，pBskfcp7)中分离，将其连接至酿酒酵母表达载体pYES2(Invitrogen，SanDiego，CA，美国)的表达盒的Eco RI位点，或亚克隆至缩短的GAPFL(甘油醛-3-磷酸脱氢酶)启动子和pho5终止子之间，如Janes等，现代遗传学18，97-103所述。基因的正确方向通过PCR核对。酿酒酵母菌株，例如INVSc1(Invitrogen，San Diego，CA，USA)如Hinnen等，美国国家科学院院报75，1929-1933(1978)所述进行转化。挑出携有受控于GAPFL启动子之植酸酶基因的单菌落，在5ml选择培养基[SD-尿嘧啶；Sherman，J.P.，Finck，G.R.和Hicks，J.B.(1986)，酵母遗传学方法实验室手册.冷泉港大学]中30℃强烈振荡(250rpm)培养一天。再将预培养物加到500ml YPD培养基(Sherman等，1986)中并在相同的条件下培养。按说明书对gall启动子进行诱导。培养4天后，离心细胞培养物(7000rpm，GS3转头，15mm，5℃)以除去细胞，上清通过Amicon 8400 cell(PM3O膜；Grace AG，Wallizeller，瑞士)和ultrafree-15离心过滤装置(Biomax-30K，Millipore，Bedford，MA，美国)超滤浓缩。浓缩液(10ml)上样至40ml Sephadex G25 Superfine柱(PharmaciaBiotech，Freiburg，德国)，用10mM乙酸钠，pH 5.0洗脱以脱盐。将脱盐样品加至2M (NH4)2SO4中，直接上样至1ml丁基Sepharose凝胶4 Fast Flow疏水作用层析柱(Pharmacia Biotech，Feiburg，德国)，用在10mM乙酸钠，pH5.0中从2M到0M(NH4)2SO4的线性梯度洗脱。植酸酶在临界点洗脱，浓缩并上样至120ml Sephacryl S-300凝胶渗透层析柱(Pharmacia Biotech，Freiburg，德国)。共有植酸酶-1，-7和-10以均一对称峰形式洗脱，经SDS-PAGE证实纯度约95％。
实施例8 共有植酸酶基因在黑曲霉中的表达
以蓝斑质粒pBsk-fcp，pBsk-fcp10，和pBskfcp7为模板，在基因的起始密码子上游导入Bsp HI-位点，终止密码子下游导入Eco RV-位点。使用ExpandTM高保真PCR试剂盒(Boehringer Mannheim，Mannheim，德国)及以下引物：
引物Asp-1：
Bsp HI
5`-TATATCATGAGCGTGTTCGTCGTGCTACTGTTC-3`(SEQ ID NO：87)
用于克隆fcp和fcp7的引物Asp-2：
Eco RV
3`-ACCCGACTTACAAAGCGAATTCTATAGATATAT-5`(SEQ ID NO：88)
用于克隆fcp10的引物Asp-3：
Eco RV
3`-ACCCTTCTTACAAAGCGAATTCTATAGATATAT-5`(SEQ ID NO：89)
反应按厂家所述进行。经PCR-扩增的fcp-基因在起始密码子处有一个新的Bsp HI位点，其通过引物Asp-1导入，导致第二个氨基酸残基甘氨酸被丝氨酸取代。随后，将该DNA-片段用Bsp HI和Eco RV消化并连接至黑曲霉葡糖淀粉酶启动子(glaA)下游的Nco I位点和构巢曲霉(Aspergillusnidulans)色氨酸C终止子(trpC)上游的Eco RV位点(Mullaney等，1985)。在此克隆步骤后，对该基因测序以检测可能由PCR导入的错误。所得表达质粒(基本对应于EP 684313之实施例9所述PGLAC载体)含粗糙脉孢霉(Neurospora crassa)的乳清苷-5`-磷酸脱羧酶基因(pyr4)作为筛选标记。黑曲霉的转化和共有植酸酶基因的表达如EP 684313所述。共有植酸酶如实施例7所述被纯化。
实施例9 植酸酶活性和最适温度及pH的检测
此实施例涉及植酸酶活性和最适温度的检测。各种植酸酶都被检测。
如EP 420358以及van Hartingsveldt等(基因127，87-94，1993)所述制备黑曲霉NRRL 3135的植酸酶。
烟曲霉ATCC13073，土曲霉9A-1，土曲霉cbs116.46，Emericellanidulans，嗜热毁丝霉，及嗜热踝节菌的植酸酶如EP-0897985及其引用文献所述制备。
所测的其它植酸酶如本文所述制备。
共有植酸酶-1-thermo(8)是共有植酸酶-1的一个变体，其进一步包括8个突变，如图5中下划线所示。共有植酸酶-1的无信号肽形式示于图1(SEQID NO：14)，有信号肽形式示于图2(SEQ ID NO：16)。
植酸酶活性的测定基本上如Mitchell等(1997)所述。在含有0.5％植酸(≈5mM)的200mM乙酸钠，pH 5.0的试验混合物中进行活性测定。37℃培养15min后，通过加入等体积的15％三氯乙酸来终止反应。所释放的无机磷酸根通过使100μl试验混合物与900μl H2O和1ml 0.6M H2SO4，2％抗坏血酸和0.5％钼酸铵混合而定量测定。用磷酸钾的标准溶液作参照。1个酶活单位定义为37℃时每分钟释放1μmol磷酸根的酶量。蛋白浓度通过如Pace等人所述计算的280nm处酶消光系数来测定[Pace N.C.，Vajdos，F.，Fee，L.，Grimsley，G.和Gray，T.(1995)，如何测量和预测蛋白质的摩尔吸收系数，蛋白质科学，4，2411-2423)：280nm处的1吸收单位(1OD)相当于1.101mg/ml共有植酸酶-1，1.068mg/ml共有植酸酶-7，以及1.039mg/ml共有植酸酶-10。
在pH-最适曲线中，纯化的酶用10mM乙酸钠，pH 5.0稀释。将稀释蛋白质的试样与等体积的1％植酸(约10mM)在一系列的不同缓冲液中混合以进行培养：0.4M甘氨酸/HCl，pH 2.5；0.4M乙酸盐/NaOH，pH 3.0，3.5，4.0，4.5，5.0，5.5；0.4M咪唑/HCl，pH 6.0，6.5；0.4M Tris/HCl pH 7.0，7.5，8.0，8.5，9.0。对照试验表明pH仅受混合步骤的影响。如上所述37℃培养15分钟。
为了测定植酸酶的底物特异性，将试验混合物中的植酸用5mM浓度的各种磷酸盐化合物代替。此外，活性的测试如上所述。
为了测定最适温度，将酶(100μl)和底物溶液(100μl)在指定温度下预温5min。通过将酶加入底物溶液中开始反应。保温15分钟后，用三氯乙酸终止反应，检测磷酸根的释放量。
共有植酸酶-1的最适pH在pH 6.0-6.5(70U/mg)左右。导入Q50T突变使得最适pH渐变为pH 6.0(130U/mg)。导入K91A突变进一步使最适pH渐变至更酸性范围。Q50T和K91A突变对共有植酸酶-10和进一步稳定的共有植酸酶变体也有相当的效应(图14和15)。
共有植酸酶-7(其构建为使黑曲霉NRRL 3135植酸酶的催化特性转移至共有植酸酶-1)具有与黑曲霉NRRL 3135植酸酶(见图18)非常相似的pH-谱。其底物特异性与黑曲霉NRRL 3135植酸酶的相似程度更甚于共有植酸酶-1的。
共有植酸酶-1的最适温度(71℃)比用来预测共有序列的野生型植酸酶的(45-55℃，表7)高16-26℃。改进型共有植酸酶-10显示出其最适温度进一步增加到80℃(图13)。
当使用酿酒酵母菌株过度生产的上清时，发现共有植酸酶-1-thermo[8]的最适温度在相同的范围(78℃)。经测定共有植酸酶-10-thermo[3]-Q50T-K91A具有最高的最适温度为82℃。
表7 共有植酸酶以及来自烟曲霉，黑曲霉，E.nidulans，和嗜热毁丝
霉的植酸酶的最适温度和Tm-值。
最适温度的测定如实施例9所述。Tm-值通过实施例10所述差示扫描量热来测定。
| 植酸酶 | 最适温度(℃) | Tm(℃) |
| 黑曲霉 NRRL 3135 | 55 | 63.3 |
| 烟曲霉 ATCC 13073 | 55 | 62.5 |
| 土曲霉 9A-1 | 49 | 57.5 |
| 土曲霉 cbs116.46 | 45 | 58.5 |
| Emericella niduldns | 45 | 55.7 |
| 嗜热毁丝霉 | 55 | - |
| 嗜热踝节菌 | 45 | - |
| 共有植酸酶-10-thermo[5]-Q50T-K91A | - | 90.4 |
| 共有植酸酶-10-thermo[3]-Q50T-K91A | 82 | 89.3 |
| 共有植酸酶-10-thermo[3]-Q50T | 82 | 88.6 |
| 共有植酸酶-10 | 80 | 85.4 |
| 共有植酸酶-1-thermo[11]-Q50T-K91A | - | 88.0 |
| 共有植酸酶-1-thermo[11]-Q50T | - | 88.5 |
| 共有植酸酶-1-thermo[8]-Q50T-K91A | - | 85.7 |
| 共有植酸酶-1-thermo[8]-Q50T | 78 | 84.7 |
| 共有植酸酶-1-thermo[8] | 81 | - |
| 共有植酸酶-1-thermo[3] | 75 | - |
| 共有植酸酶-1-Q50T | - | 78.9 |
| 共有植酸酶-1 | 71 | 78.1 |
| 烟曲霉α-突变体Q51T | 60 | 67.0 |
| 烟曲霉α-突变体，加突变E59A，S154N，R329H，S364T，G404A | 63 | - |
| 烟曲霉＂优化的＂α突变体，加突变K92A | 63 | - |
实施例10
通过差示扫描量热(DSC)测定解链温度
为了测定植酸酶的伸展温度，如Brugger等，1997[Brugger，R.，Mascarello，F.，Augem，S.，van Loon，A.P.G.M.和Wyss，M.(1997)，通过差示扫描量热研究植酸酶和pH2.5酸性植酸酶的热变性。植酸和植酸酶的生物化学(Rasmussen，S.K.；Raboy，V.；
，H.和Loewus，E.编；Kluwer Academic Publishers，Dordrecht，荷兰]所述使用差示扫描量热法。用50-60mg/ml植酸酶均一溶液进行检验。以10℃/min的恒定加热速率加热到90-95℃。
所测的解链点证实了最适温度检测结果(表7)。迄今最稳定的共有植酸酶是共有植酸酶-10-mermo[3]-Q50T-K91A，其在所选条件下显示出解链温度89.3℃。这比所用的野生型植酸酶的解链温度高26.0到33.6℃。
实施例11
将担子菌植酸酶活性位点转移到植酸酶-10-thermo[3]-Q50T-K91A
如上所述(实施例5)，将衍生自担子菌植酸酶活性位点的突变导入共有植酸酶-10中。制备下述a)-e)的5个构建体：
a)称作共有植酸酶-12的构建物，其含有担子菌共有序列中选定数目的活性位点残基。其氨基酸序列被显示于图21(前26个氨基酸组成信号肽；不同于共有植酸酶-10-thermo[3]-Q50T-K91A的位置带下划线)；
b)将一簇突变(第II簇)转移到共有植酸酶-1和-10的序列中，即：S80Q，Y86F，S9OG，K91A，S92A，K93T，A94R，Y95I；
c)以类似的方式转移另一簇突变(第III簇)，即：T129V，E133A，Q134N，M136S，V137S，N138Q，S139A；
d)以类似的方式转移另一簇突变(第IV簇)，即：A168D，E171T，K172N，F173W：
e)最后，转移另一簇突变(第V簇)被，即：Q297G，S298D，G300D，Y305T。
这些构建物如实施例6到8所述被表达。
实施例12 利用GAP进行植酸酶比对
使本文所述植酸酶—即氨基酸序列及相应DNA序列—相互比对。此外，对其它植酸酶也进行相应比对，它们是：
-EP 897985所述的共有植酸酶-1；
-EP 420358所述的黑曲霉(ficuum)NRRL 3135(黑曲霉NRRL3135)的植酸酶；
-EP 897010所述的烟曲霉ATCC 13073(烟曲霉13073)；烟曲霉ATCC32239(烟曲霉32239)；土曲霉cbs116.46(土曲霉cbs)；Emericella nidulans(E.nidulans)；和嗜热踝节菌(T.thermophilus)的植酸酶；
-EP 684313所述的嗜热毁丝霉和土曲霉9-A1的植酸酶；
-WO 9735017(PCT/US97/04559)所述的Thermomyces lanuginosus(T.lanuginosus)的植酸酶；
-WO98/28409所述的平田头菇，Paxillus involutus 1和2(P.involutusphyA1和phyA2)；以及绒毛栓菌的植酸酶；
-WO 98/28408所述的Peniophora lycii(P.lycii)的植酸酶。
用GAP程序及如上所述设置进行比对。
在多肽比对中，除了与共有植酸酶-11比对外，还包括信号肽。
氨基酸序列比对的结果被显示于下面表8中。每一小格的第一个数字是氨基酸的相似性，第二个数字是氨基酸的同源性。
在DNA序列比对中，也总是包括信号肽。结果示于下表9。
本发明包括以外实施方案(A)-(J)。
在这些实施方案中，当测定另一种植酸酶在氨基酸水平的％同一性或％相似性时，使用上述GAP等比对程序将其氨基酸序列与参照序列(例如在实施方案(A)中的共有植酸酶-10氨基酸序列)相比对。同一性百分比和相似性百分比均通过此程序计算。其它植酸酶的氨基酸序列可包括或不包括信号肽。
当测定另一种编码植酸酶的DNA在DNA水平的％同一性时，使用上述GAP等比对程序将该DNA序列与参照序列[例如在实施方案(A)中的SEQ ID NO：25至核苷酸12-1412(如图5所示的共有植酸酶-10(Fcp10)的DNA序列]相比对。同一性百分比通过此程序计算。编码另一种植酸酶的DNA序列可以是包含内含子的基因组DNA序列，或cDNA序列。其可以包括或不包括信号肽编码部分。
当测定杂交时，所用探针是特异性DNA序列[例如在实施方案(A)中SEQID NO：25的核苷酸12-1412(如图5所示的共有植酸酶-10(Fcp10)的DNA序列]。编码另一种植酸酶的DNA序列可以是含有植酸酶DNA序列的基因组DNA；纯化的基因组DNA序列(就植酸酶DNA序列而言为纯化的)；或其可以是编码植酸酶的cDNA序列，优选纯化型或扩增型，例如PCR-扩增型。编码植酸酶的DNA，无论什么类型，可包括或不包括信号肽编码部分。合适的杂交条件如上所述。
术语＂DNA序列＂包括在此例示的DNA序列的片段或部分，只要其能够编码活性酶(例如植酸酶)。
术语＂氨基酸序列＂包括在此例示的氨基酸序列，只要其具有酶活性(例如表现植酸酶活性)。
(A)与共有植酸酶-10(CP10，Fcp10)相关的植酸酶及相应DNA序列
一种植酸酶，其含有与图5所示共有植酸酶-10(Fcp10)中1-467位的氨基酸序列有至少93.80％；或至少94，94.5，95，95.5，96，96.5，97，97.5，98，98.5，99，99.5％同一性的氨基酸序列。
一种植酸酶，含有与共有植酸酶-10中1-467位的氨基酸序列有至少95.09％；或至少95.5，96，96.5，97，97.5，98，98.5，99，99.5％相似性的氨基酸序列。
一种植酸酶，其由与图5所示共有植酸酶-10(Fcp10)之DNA序列中核苷酸12-1412有至少95.88％；或至少96，96.5，97，97.5，98，98.5，99，99.5％同一性的DNA序列编码。
一种编码植酸酶的DNA序列，其与图5所示共有植酸酶-10(Fcp10)之DNA序列的核苷酸12-1412：(i)有至少95.88％；或至少96，96.5，97，97.5，98，98.5，99，99.5％相同；或(ii)在低度，或中度，中/高度，高度，或极高度严谨条件下可杂交。合适的阴性对照是编码共有植酸酶-1的DNA。合适的阳性对照是编码CP10，CP10-thermo[3)-Q50T，K91A，CP1-thermo[8]，CP1-thermo[83]Q50T，K91A中任一个的DNA。
一种编码植酸酶的DNA序列，所述酶含有与图5所示共有植酸酶-10(Fcp10)中1-467位的氨基酸序列有至少93.80％；或至少94，94.5，95，95.5，96，96.5，97，97.5，98，98.5，99，99.5％同一性的氨基酸序列。
(B)与共有植酸酶-10-thermo[3]-Q50T-K91A相关的植酸酶及相应DNA
序列
一种植酸酶，其含有与图8所示共有植酸酶-10-thermo[3]-Q50T-K91A中1-467位的氨基酸序列有至少93.37％；或至少93.5，94，94.5，95，95.5，96，96.5，97，97.5，98，98.5，99，99.5％同一性的氨基酸序列。
一种植酸酶，其含有与图8所示共有植酸酶-10-thermo[3]-Q50T-K91A中1-467位的氨基酸序列有至少94.66％；或至少95.0，95.5，96，96.5，97，97.5，98，98.5，99，99.5％相似性的氨基酸序列。
一种植酸酶，其由与图8所示共有植酸酶-10-thermo[3]-Q50T-K91A之DNA序列的核苷酸12-1412有至少95.88％；或至少96，96.5，97，97.5，98，98.5，99，99.5％同一性的DNA序列编码。
一种编码植酸酶的DNA序列，其与图8所示共有植酸酶-10-thermo[3]-Q50T-K91A之DNA序列的核苷酸12-1412：(i)有至少95.88％；或至少96，96.5，97，97.5，98，98.5，99，99.5％相同；或(ii)在低度，或中度，中/高度，高度，或极高度严谨条件下可杂交。合适的阴性对照是编码共有植酸酶-1的DNA。合适的阳性对照是编码CP10，CP10-thermo[3)-Q50T-K91A，CP1-thermo[8]，或CP1-thermo[8]-Q50T-K91A中任一种的DNA。
一种编码植酸酶的DNA序列，所述酶含有与图8所示共有植酸酶-10-thermo[3]-Q50T-K91A中1-467位的氨基酸序列有至少93.37％；或至少93.5，94，94.5，95，95.5，96，96.5，97，97.5，98，98.5，99，99.5％同一性的氨基酸序列。
(C)与共有植酸酶-1-thermo[8]相关的植酸酶及相应DNA序列
一种植酸酶，其含有与共有植酸酶-1-thermo[8](示于图7；已添加回复突变T50Q和A91K)中1-467位的氨基酸序列有至少98.30％；或至少98.5，99，99.5％同一性的氨基酸序列。
一种植酸酶，其含有与共有植酸酶-1-thermo[8](示于图7；已添加回复突变T50Q和A91K)中1-467位的氨基酸序列有至少98.51％；或至少99，99.5％相似性的氨基酸序列。
一种植酸酶，其由与共有植酸酶-1-thermo[8](示于图7；已添加回复突变T50Q和A91K)之DNA序列的核苷酸1-1407有至少98.73％；或至少99，99.5％同一性的DNA序列编码。
一种编码植酸酶的DNA序列，其与共有植酸酶-1-thermo[8](示于图7；已添加回复突变T50Q和A91K)之DNA序列的核苷酸1-1407：(i)有至少98.73％；或至少99，99.5％相同；或(ii)在低度，或中度，中/高度，高度，或极高度严谨条件下可杂交。合适的阴性对照是编码共有植酸酶-1的DNA。合适的阳性对照是编码CP1-thermo[8]，或CP1-thermo[8]-Q50T-K91A中任一个的DNA。
一种编码植酸酶的DNA序列，所述酶含有与共有植酸酶-1-thermo[8](示于图7；已添加回复突变T50Q和A91K)中1-467位的氨基酸序列有至少98.30％；或至少98.5，99，99.5％同一性的氨基酸序列。
(D)与共有植酸酶-1-thermo[8]-Q50T-K91A相关的植酸酶及相应DNA
序列
一种植酸酶，其含有与图7所示共有植酸酶-1-thermo[8]-Q50T-K91A中1-467位的氨基酸序列有至少97.87％；或至少98，98.5，99，99.5％同一性的氨基酸序列。
一种植酸酶，其含有与图7所示共有植酸酶-1-thermo[8]-Q50T-K91A中1-467位的氨基酸序列有至少98.08％；或至少98.5，99，99.5％相似性的氨基酸序列。
一种植酸酶，其由与图7所示共有植酸酶-1-thermo[8]-Q50T-K91A之DNA序列的核苷酸1-1407有至少98.37％；或至少98.5，99，99.5％同一性的DNA序列编码。
一种编码植酸酶的DNA序列，其与图7所示共有植酸酶-1-thermo[8]-Q50T-K91A之DNA序列的核苷酸1-1407：(i)至少98.37％；或至少98.5，99，99.5％相同；或(ii)在低度，或中度，中/高度，高度，或极高度严谨条件下可杂交。合适的阴性对照是编码共有植酸酶-1的DNA。合适的阳性对照是编码CP1-thermo[8]，或CP1-thermo[8]-Q50T-K91A中任一种的DNA。
一种编码植酸酶的DNA序列，所述酶含有与图7所示的共有植酸酶-1-thermo[8]-Q50T-K91A中1-467位的氨基酸序列有至少97.87％；或至少98，98.5，99，99.5％同一性的氨基酸序列。
(E)与共有植酸酶-11相关的植酸酶及相应DNA序列
一种植酸酶，其含有与图6所示共有植酸酶-11中1-482位的氨基酸序列有至少90.71％；或至少91，91.5，92，92.5，93，93.5，94，94.5，95，95.5，96，96.5，97，97.5，98，98.5，99，99.5％同一性的氨基酸序列。
一种植酸酶，其含有与图6所示共有植酸酶-11中1-482位的氨基酸序列有至少92.07％；或至少92.5，93，93.5，94，94.5，95，95.5，96，96.5，97，97.5，98，98.5，99，99.5％相似性的氨基酸序列。
一种编码植酸酶的DNA序列，所述酶含有与图6所示共有植酸酶-11中1-482位的氨基酸序列有至少90.71％；或至少91，91.5，92，92.5，93，93.5，94，94.5，95，95.5，96，96.5，97，97.5，98，98.5，99，99.5％同一性的氨基酸序列。
(F)与烟曲霉α-突变体相关的植酸酶及相应DNA序列
一种植酸酶，其含有与图9所示烟曲霉α-突变体(植酸酶)中1-467位的氨基酸序列有至少97.17％；或至少97.5，98，98.5，99，99.5％同一性的氨基酸序列。
一种植酸酶，其含有与图9所示烟曲霉α-突变体(植酸酶)中1-467位的氨基酸序列有至少97.82％；或至少98，98.5，99，99.5％相似性的氨基酸序列。
一种植酸酶，其由与图9所示烟曲霉ATCC 13073α-突变体(植酸酶)之DNA序列的核苷酸1-1401有至少96.13％；或至少96.5，97，97.5，98，98.5，99，99.5％同一性的DNA序列编码。
一种编码植酸酶的DNA序列，所述酶含有与图9所示烟曲霉ATCC13073α-突变体(植酸酶)中1-467位的氨基酸序列有至少97.17％；或至少97.5，98，98.5，99，99.5％同一性的氨基酸序列。
一种编码植酸酶的DNA序列，其与图9所示烟曲霉ATCC 13073α-突变体(植酸酶)之DNA序列的核苷酸1-1401：(i)有至少96.13％；或至少96.5，97，97.5，98，98.5，99，99.5％相同；或(ii)在低度，或中度，中/高度，高度，或极高度严谨条件下可杂交。合适的阴性对照是编码烟曲霉13073的植酸酶的DNA。合适的阳性对照是编码烟曲霉ATCC 13073α-突变体的植酸酶，或优化型α-突变体的DNA。
(G)与优化型烟曲霉α-突变体相关的植酸酶及相应DNA序列
一种植酸酶，其含有与优化型烟曲霉α-突变体的植酸酶序列至少96.08％；或至少96.5，97，97.5，98，98.5，99，99.5％相同的氨基酸序列。
一种植酸酶，其含有与优化型烟曲霉α-突变体的植酸酶序列有至少96.74％；或至少97，97.5，98，98.5，99，99.5％相似性的氨基酸序列。
一种植酸酶，其由与编码优化型烟曲霉α-突变体植酸酶的DNA序列中核苷酸1-1401有至少95.63％；或至少96，96.5，97，97.5，98，98.5，99，99.5％同一性的DNA序列编码。
一种DNA序列，其编码的植酸酶含有与优化型烟曲霉α-突变体植酸酶有至少96.08％；或至少96.5，97，97.5，98，98.5，99，99.5％同一性的氨基酸序列。
一种编码植酸酶的DNA序列，其与编码优化型烟曲霉α-突变体植酸酶的DNA序列中核苷酸1-1401：(i)有至少95.63％；或至少96，96.5，97，97.5，98，98.5，99，99.5％相同；或(ii)在低度，或中度，中/高度，高度，或极高度严谨条件下可杂交。
合适的阴性对照是编码烟曲霉13073植酸酶的DNA。合适的阳性对照是编码烟曲霉ATCC 13073α-突变体植酸酶或优化型α-突变体的DNA。
(H)与共有植酸酶-7相关的植酸酶及相应DNA序列
一种植酸酶，其含有与图10所示共有植酸酶-7中1-467位的氨基酸序列有至少94.87％；或至少95，95.5，96，96.5，97，97.5，98，98.5，99，99.5％同一性的氨基酸序列。
一种植酸酶，其含有与图10所示共有植酸酶-7中1-467位的氨基酸序列有至少95.30％；或至少95.5，96，96.5，97，97.5，98，98.5，99，99.5％相似性的氨基酸序列。
一种植酸酶，其由与图10所示共有植酸酶-7之DNA序列的核苷酸12-1412有至少96.38％；或至少96.5，97，97.5，98，98.5，99，99.5％同一性的DNA序列编码。
一种编码植酸酶的DNA序列，其与图10所示共有植酸酶-7之DNA序列的核苷酸12-1412：(i)有至少96.38％；或至少96.5，97，97.5，98，98.5，99，99.5％相同；或(ii)在低度，或中度，中/高度，高度，或极高度严谨条件下可杂交。
一种编码植酸酶的DNA序列，所述酶含有与图10所示共有植酸酶-7中1-467位的氨基酸序列有至少94.87％；或至少95，95.5，96，96.5，97，97.5，98，98.5，99，99.5％同一性的氨基酸序列。
(I)与担子菌共有植酸酶相关的植酸酶
一种植酸酶，其含有与(i)图3所示担子菌共有植酸酶的氨基酸1-441，和(ii)图5所示氨基酸1-26的组合序列(将序列(ii)加在序列(i)的N-末端)有76.23％；或至少77，78，79，80，81，82，83，84，85，86，87，88，89，90，91，92，93，94，94.5，95，95.5，96，96.5，97，97.5，98，98.5，99，99.5％同一性的氨基酸序列。
一种植酸酶，其含有与图3所示担子菌共有植酸酶中1-441位的氨基酸序列有至少79.50％；或至少80，81，82，83，84，85，86，87，88，89，90，91，92，93，94，95，95.5，96，96.5，97，97.5，98，98.5，99，99.5％相似性的氨基酸序列。
(J)与共有植酸酶-12相关的植酸酶
一种植酸酶，其含有与图21所示共有植酸酶-12的1-467位的氨基酸序列有至少70，75，76，77，78，79，80，81，82，83，84，85，86，87，88，89，90，91，92，93，94，95，95.5，96，96.5，97，97.5，98，98.5，99，99.5％同一性的氨基酸序列。
一种植酸酶，其含有与图21所示共有植酸酶-12的1-467位的氨基酸序列有至少75，76，77，78，79，80，81，82，83，84，85，86，87，88，89，90，91，92，93，94，95，95.5，96，96.5，97，97.5，98，98.5，99，99.5％相似性的氨基酸序列。
表8
植酸酶氨基酸序列的比较
| 植酸酶 | CP10 | CP10-thermo[3]-Q50T-K91A | CP1-thermo[8] | CP1-thermo[8]-Q50T-K91A | CP11 | CP7 | Basidio | 烟曲霉α-突变体 | 烟曲霉α-突变体(优化) |
| 共有植酸酶-1 | 95.08/93.79 | 94.65/93.36 | 98.50/98.29 | 98.07/97.86 | 92.06/90.70 | 95.29/94.86 | 69.42/62.16 | 85.59/82.58 | 84.73/81.72 |
| 黑曲霉NRRL3135 | 79.48/76.46 | 79.05/76.03 | 80.35/77.75 | 79.91/77.32 | 79.27/76.31 | 84.02/81.64 | 67.19/59.32 | 74.07/70.11 | 74.95/70/99 |
| 土曲霉9-A1 | 76.04/72.11 | 75.82/71.90 | 76.47/72.33 | 76.25/72.11 | 76.51/73.02 | 75.76/71.18 | 65.39/58.02 | 69.67/64.84 | 69.45/64.84 |
| 土曲霉cbs | 79.04/75.11 | 78.82/74.89 | 79.48/75.76 | 79.26/75.55 | 77.19/73.27 | 79.17/75.00 | 66.92/59.65 | 72.59/67.76 | 72.37/67.76 |
| E.nidulans | 78.70/74.35 | 78.26/73.91 | 79.78/75.87 | 79.35/75.44 | 80.56/76.62 | 76.96/73.04 | 67.20/58.13 | 72.39/67.83 | 72.11/67.54 |
| 烟曲霉13073 | 82.93/80.31 | 82.50/79.87 | 82.31/79.04 | 81.88/78.60 | 81.36/78.64 | 80.13/76.20 | 63.54/57.91 | 97.82/97.16 | 96.73/96.07 |
| 烟曲霉32239 | 81.30/77.39 | 80.87/76.96 | 81.09/77.61 | 80.65/77.17 | 79.95/76.08 | 79.13/75.22 | 63.61/54.97 | 90.22/86.52 | 89.57/85.87 |
| 嗜热踝节菌 | 77.83/73.84 | 77.38/73.39 | 78.67/74.89 | 78.22/74.44 | 78.47/74.76 | 76.51/73.15 | 61.54/54.36 | 72.01/66.82 | 72.69/67.49 |
| 嗜热毁丝霉 | 69.16/62.81 | 69.48/63.33 | 69.27/62.84 | 69.59/63.36 | 69.65/63.06 | 68.82/62.13 | 65.56/57.91 | 66.21/58.45 | 66.44/58.68 |
| T.lanuginosus | 73.52/66.70 | 73.06/66.44 | 71.92/64.61 | 71.46/64.16 | 74.21/68.86 | 69.50/62.62 | 67.20/57.41 | 68.91/61.02 | 69.61/61.72 |
| P.lycii | 64.92/59.10 | 64.91/59.37 | 64.46/58.09 | 64.46/58.36 | 65.03/59.84 | 63.13/56.50 | 77.75/73.07 | 64.08/57.11 | 62.47/55.91 |
| 平田头菇 | 64.51/51.81 | 64.86/51.94 | 62.98/51.41 | 63.33/51.54 | 64.50/52.30 | 63.05/51.15 | 78.92/74.71 | 61.64/52.38 | 62.13/53.07 |
| P.involutus 1 | 66.67/58.07 | 66.67/58.33 | 64.84/56.51 | 64.84/56.77 | 63.30/54.52 | 65.33/56.53 | 79.49/76.22 | 59.59/51.81 | 59.95/52.20 |
| P.involutus 2 | 65.54/55.70 | 65.30/55.53 | 66.85/56.87 | 66.58/56.68 | 66.30/56.35 | 64.27/54.13 | 78.09/74.59 | 61.26/52.62 | 61.04/52.47 |
| 绒毛栓菌 | 65.46/57.22 | 65.72/57.47 | 62.89/55.67 | 63.14/55.93 | 65.03/57.65 | 63.28/56.51 | 78.34/75.12 | 64.08/57.11 | 62.30/55.24 |
| CP10 | - | 99.57/99.57 | 96.57/95.50 | 96.15/95.08 | 95.02/94.56 | 91.01/89.29 | 70.22/62.28 | 85.13/82.76 | 85.99/83.62 |
| CP10t[3]Q50TK91A | 99.57/99.57 | - | 96.15/95.08 | 96.57/95.50 | 94.56/94.10 | 90.58/88.87 | 70.47/62.28 | 85.13/82.76 | 85.99/83.62 |
| CP1thermo[8] | 96.57/95.50 | 96.15/95.08 | - | 99.57/99.57 | 93.42/92.29 | 94.43/93.79 | 68.40/60.74 | 84.52/81.94 | 85.38/82.80 |
| CP1t[8]Q50TK91A | 96.15/95.08 | 96.57/95.50 | 99.57/99.57 | - | 92.97/91.84 | 94.00/93.36 | 68.64/60.74 | 84.52/81.94 | 85.38/82.80 |
| CP11 | 95.02/94.56 | 94.56/94.10 | 93.42/92.29 | 92.97/91.84 | - | 88.44/86.62 | 68.27/59.73 | 82.23/79.73 | 83.37/80.87 |
| CP7 | 91.01/89.29 | 90.58/88.87 | 94.43/93.79 | 94.00/93.36 | 88.44/86.62 | - | 69.80/62.69 | 81.94/78.71 | 81.72/78.50 |
| Basidio | 70.22/62.28 | 70.47/62.28 | 68.40/60.74 | 68.64/60.74 | 68.27/59.73 | 69.80/62.69 | - | 65.97/60.52 | 66.41/60.68 |
| 烟曲霉α-突变 | 85.13/82.76 | 85.13/82.76 | 84.52/81.94 | 84.52/81.94 | 82.23/79.73 | 81.94/78.71 | 65.97/60.52 | - | 98.93/98.93 |
| 烟曲霉α-突变-优化 | 85.99/83.62 | 85.99/83.62 | 85.38/82.80 | 85.38/82.80 | 83.37/80.87 | 81.72/78.50 | 66.41/60.68 | 98.93/98.93 | - |
表9
编码植酸酶的DNA序列的比较
| 植酸酶 | CP10 | CP10-thermo[3]-Q50T-K91A | CP1-thermo[8] | CP1-thermo[8]-Q50T-K91A | CP7 | Basidio | 烟曲霉α-突变体 | 烟曲霉α-突变体(优化) |
| 共有植酸酶-1 | 95.87 | 95.87 | 98.72 | 98.36 | 96.37 | 65.46 | 66.88 | 66.88 |
| 黑曲霉NRRL3135 | 65.10 | 64.82 | 66.10 | 65.74 | 67.52 | 50.68 | 65.88 | 66.17 |
| 土曲霉9-A1 | 61.74 | 61.53 | 62.17 | 62.03 | 60.53 | 49.40 | 66.24 | 66.31 |
| 土曲霉cbs | 62.52 | 62.30 | 63.02 | 62.88 | 61.45 | 49.74 | 68.17 | 68.24 |
| E.nidulans | 65.08 | 64.94 | 65.30 | 65.01 | 64.22 | 49.92 | 64.90 | 65.44 |
| 烟曲霉13073 | 65.66 | 65.38 | 64.19 | 64.08 | 63.65 | 48.27 | 96.12 | 95.62 |
| 嗜热踝节菌 | 62.52 | 62.50 | 62.53 | 62.66 | 62.00 | 52.19 | 61.77 | 61.92 |
| 嗜热毁丝霉 | 55.51 | 55.15 | 55.36 | 55.22 | 53.91 | 48.44 | 58.17 | 58.24 |
| T.lanuginosus | 57.56 | 57.20 | 56.76 | 56.47 | 62.00 | 44.66 | 59.71 | 60.07 |
| P.lycii | 45.76 | 46.51 | 45.14 | 55.21 | 55.46 | 58.50 | 48.91 | 49.44 |
| 平田头菇 | 49.89 | 49.89 | 49.89 | 50.11 | 45.54 | 61.66 | 47.49 | 47.56 |
| P.involutus 1 | 48.32 | 49.03 | 47.81 | 47.96 | 49.59 | 59.80 | 49.96 | 50.19 |
| P.involutus 2 | 48.24 | 49.00 | 48.08 | 48.63 | 47.94 | 60.16 | 47.56 | 47.63 |
| 绒毛栓菌 | 47.00 | 47.17 | 46.46 | 47.62 | 46.83 | 60.37 | 49.89 | 49.96 |
| CP10 | - | 99.43 | 96.40 | 96.05 | 93.73 | 66.40 | 67.81 | 68.24 |
| CP10t[3]Q50TK91A | 99.43 | - | 96.37 | 96.58 | 93.45 | 66.29 | 67.81 | 68.24 |
| Cp1thermo[8] | 96.40 | 96.37 | - | 99.65 | 95.30 | 65.40 | 66.74 | 67.17 |
| CP1t[8]Q50TK91A | 96.05 | 96.58 | 99.65 | - | 94.94 | 65.47 | 66.74 | 67.17 |
| CP7 | 93.73 | 93.45 | 95.30 | 94.94 | - | 64.56 | 65.88 | 65.88 |
| Basidio | 66.40 | 66.29 | 65.40 | 65.47 | 64.56 | - | 50.41 | 50.49 |
| 烟曲霉α-突变 | 67.81 | 67.81 | 66.74 | 66.74 | 65.88 | 50.41 | - | 99.50 |
| 烟曲霉α-突变-优化 | 68.24 | 68.24 | 67.17 | 67.17 | 65.88 | 50.49 | 99.50 | - |
序列表
<110>Novo Nordisk A/S
<120>改进的植酸酶
<130>seqlist171299
<140>
<141>
<150>DK 99/00092
<151>1999-01-22
<150>DK 99/01340
<151>1999-09-21
<160>89
<170>PatentIn Ver.2.1
<210>1
<211>440
<212>PRT
<213>土曲霉9A-1
<400>1
Lys His Ser Asp Cys Asn Ser Val Asp His Gly Tyr Gln Cys Phe Pro
1 5 10 15
Glu Leu Ser His Lys Trp Gly Leu Tyr Ala Pro Tyr Phe Ser Leu Gln
20 25 30
Asp Glu Ser Pro Phe Pro Leu Asp Val Pro Glu Asp Cys His Ile Thr
35 40 45
Phe Val Gln Val Leu Ala Arg His Gly Ala Arg Ser Pro Thr His Ser
50 55 60
Lys Thr Lys Ala Tyr Ala Ala Thr Ile Ala Ala Ile Gln Lys Ser Ala
65 70 75 80
Thr Ala Phe Pro Gly Lys Tyr Ala Phe Leu Gln Ser Tyr Asn Tyr Ser
85 90 95
Leu Asp Ser Glu Glu Leu Thr Pro Phe Gly Arg Asn Gln Leu Arg Asp
100 105 110
Leu Gly Ala Gln Phe Tyr Glu Arg Tyr Asn Ala Leu Thr Arg His Ile
115 120 125
Asn Pro Phe Val Arg Ala Thr Asp Ala Ser Arg Val His Glu Ser Ala
130 135 140
Glu Lys Phe Val Glu Gly Phe Gln Thr Ala Arg Gln Asp Asp His His
145 150 155 160
Ala Asn Pro His Gln Pro Ser Pro Arg Val Asp Val Ala Ile Pro Glu
165 170 175
Gly Ser Ala Tyr Asn Asn Thr Leu Glu His Ser Leu Cys Thr Ala Phe
180 185 190
Glu Ser Ser Thr Val Gly Asp Asp Ala Val Ala Asn Phe Thr Ala Val
195 200 205
Phe Ala Pro Ala Ile Ala Gln Arg Leu Glu Ala Asp Leu Pro Gly Val
210 215 220
Gln Leu Ser Thr Asp Asp Val Val Asn Leu Met Ala Met Cys Pro Phe
225 230 235 240
Glu Thr Val Ser Leu Thr Asp Asp Ala His Thr Leu Ser Pro Phe Cys
245 250 255
Asp Leu Phe Thr Ala Thr Glu Trp Thr Gln Tyr Asn Tyr Leu Leu Ser
260 265 270
Leu Asp Lys Tyr Tyr Gly Tyr Gly Gly Gly Asn Pro Leu Gly Pro Val
275 280 285
Gln Gly Val Gly Trp Ala Asn Glu Leu Met Ala Arg Leu Thr Arg Ala
290 295 300
Pro Val His Asp His Thr Cys Val Asn Asn Thr Leu Asp Ala Ser Pro
305 310 315 320
Ala Thr Phe Pro Leu Asn Ala Thr Leu Tyr Ala Asp Phe Ser His Asp
325 330 335
Ser Asn Leu Val Ser Ile Phe Trp Ala Leu Gly Leu Tyr Asn Gly Thr
340 345 350
Ala Pro Leu Ser Gln Thr Ser Val Glu Ser Val Ser Gln Thr Asp Gly
355 360 365
Tyr Ala Ala Ala Trp Thr Val Pro Phe Ala Ala Arg Ala Tyr Val Glu
370 375 380
Met Met Gln Cys Arg Ala Glu Lys Glu Pro Leu Val Arg Val Leu Val
385 390 395 400
Asn Asp Arg Val Met Pro Leu His Gly Cys Pro Thr Asp Lys Leu Gly
405 410 415
Arg Cys Lys Arg Asp Ala Phe Val Ala Gly Leu Ser Phe Ala Gln Ala
420 425 430
Gly Gly Asn Trp Ala Asp Cys Phe
435 440
<210>2
<211>440
<212>PRT
<213>土曲霉cbs
<400>2
Asn His Ser Asp Cys Thr Ser Val Asp Arg Gly Tyr Gln Cys Phe Pro
1 5 10 15
Glu Leu Ser His Lys Trp Gly Leu Tyr Ala Pro Tyr Phe Ser Leu Gln
20 25 30
Asp Glu Ser Pro Phe Pro Leu Asp Val Pro Asp Asp Cys His Ile Thr
35 40 45
Phe Val Gln Val Leu Ala Arg His Gly Ala Arg Ser Pro Thr Asp Ser
50 55 60
Lys Thr Lys Ala Tyr Ala Ala Thr Ile Ala Ala Ile Gln Lys Asn Ala
65 70 75 80
Thr Ala Leu Pro Gly Lys Tyr Ala Phe Leu Lys Ser Tyr Asn Tyr Ser
85 90 95
Met Gly Ser Glu Asn Leu Thr Pro Phe Gly Arg Asn Gln Leu Gln Asp
100 105 110
Leu Gly Ala Gln Phe Tyr Arg Arg Tyr Asp Thr Leu Thr Arg His Ile
115 120 125
Asn Pro Phe Val Arg Ala Ala Asp Ser Ser Arg Val His Glu Ser Ala
130 135 140
Glu Lys Phe Val Glu Gly Phe Gln Asn Ala Arg Gln Gly Asp Pro His
145 150 155 160
Ala Asn Pro His Gln Pro Ser Pro Arg Val Asp Val Val Ile Pro Glu
165 170 175
Gly Thr Ala Tyr Asn Asn Thr Leu Glu His Ser Ile Cys Thr Ala Phe
180 185 190
Glu Ala Ser Thr Val Gly Asp Ala Ala Ala Asp Asn Phe Thr Ala Val
195 200 205
Phe Ala Pro Ala Ile Ala Lys Arg Leu Glu Ala Asp Leu Pro Gly Val
210 215 220
Gln Leu Ser Ala Asp Asp Val Val Asn Leu Met Ala Met Cys Pro Phe
225 230 235 240
Glu Thr Val Ser Leu Thr Asp Asp Ala His Thr Leu Ser Pro Phe Cys
245 250 255
Asp Leu Phe Thr Ala Ala Glu Trp Thr Gln Tyr Asn Tyr Leu Leu Ser
260 265 270
Leu Asp Lys Tyr Tyr Gly Tyr Gly Gly Gly Asn Pro Leu Gly Pro Val
275 280 285
Gln Gly Val Gly Trp Ala Asn Glu Leu Ile Ala Arg Leu Thr Arg Ser
290 295 300
Pro Val His Asp His Thr Cys Val Asn Asn Thr Leu Asp Ala Asn Pro
305 310 315 320
Ala Thr Phe Pro Leu Asn Ala Thr Leu Tyr Ala Asp Phe Ser His Asp
325 330 335
Ser Asn Leu Val Ser Ile Phe Trp Ala Leu Gly Leu Tyr Asn Gly Thr
340 345 350
Lys Pro Leu Ser Gln Thr Thr Val Glu Asp Ile Thr Arg Thr Asp Gly
355 360 365
Tyr Ala Ala Ala Trp Thr Val Pro Phe Ala Ala Arg Ala Tyr Ile Glu
370 375 380
Met Met Gln Cys Arg Ala Glu Lys Gln Pro Leu Val Arg Val Leu Val
385 390 395 400
Asn Asp Arg Val Met Pro Leu His Gly Cys Ala Val Asp Asn Leu Gly
405 410 415
Arg Cys Lys Arg Asp Asp Phe Val Glu Gly Leu Ser Phe Ala Arg Ala
420 425 430
Gly Gly Asn Trp Ala Glu Cys Phe
435 440
<210>3
<211>441
<212>PRT
<213>黑曲霉泡盛变种
<400>3
Asn Gln Ser Thr Cys Asp Thr Val Asp Gln Gly Tyr Gln Cys Phe Ser
1 5 10 15
Glu Thr Ser His Leu Trp Gly Gln Tyr Ala Pro Phe Phe Ser Leu Ala
20 25 30
Asn Glu Ser Ala Ile Ser Pro Asp Val Pro Ala Gly Cys Arg Val Thr
35 40 45
Phe Ala Gln Val Leu Ser Arg His Gly Ala Arg Tyr Pro Thr Glu Ser
50 55 60
Lys Gly Lys Lys Tyr Ser Ala Leu Ile Glu Glu Ile Gln Gln Asn Val
65 70 75 80
Thr Thr Phe Asp Gly Lys Tyr Ala Phe Leu Lys Thr Tyr Asn Tyr Ser
85 90 95
Leu Gly Ala Asp Asp Leu Thr Pro Phe Gly Glu Gln Glu Leu Val Asn
100 105 110
Ser Gly Ile Lys Phe Tyr Gln Arg Tyr Glu Ser Leu Thr Arg Asn Ile
115 120 125
Ile Pro Phe Ile Arg Ser Ser Gly Ser Ser Arg Val Ile Ala Ser Gly
130 135 140
Glu Lys Phe Ile Glu Gly Phe Gln Ser Thr Lys Leu Lys Asp Pro Arg
145 150 155 160
Ala Gln Pro Gly Gln Ser Ser Pro Lys Ile Asp Val Val Ile Ser Glu
165 170 175
Ala Ser Ser Ser Asn Asn Thr Leu Asp Pro Gly Thr Cys Thr Val Phe
180 185 190
Glu Asp Ser Glu Leu Ala Asp Thr Val Glu Ala Asn Phe Thr Ala Thr
195 200 205
Phe Ala Pro Ser Ile Arg Gln Arg Leu Glu Asn Asp Leu Ser Gly Val
210 215 220
Thr Leu Thr Asp Thr Glu Val Thr Tyr Leu Met Asp Met Cys Ser Phe
225 230 235 240
Asp Thr Ile Ser Thr Ser Thr Val Asp Thr Lys Leu Ser Pro Phe Cys
245 250 255
Asp Leu Phe Thr His Asp Glu Trp Ile His Tyr Asp Tyr Leu Gln Ser
260 265 270
Leu Lys Lys Tyr Tyr Gly His Gly Ala Gly Asn Pro Leu Gly Pro Thr
275 280 285
Gln Gly Val Gly Tyr Ala Asn Glu Leu Ile Ala Arg Leu Thr His Ser
290 295 300
Pro Val His Asp Asp Thr Ser Ser Asn His Thr Leu Asp Ser Asn Pro
305 310 315 320
Ala Thr Phe Pro Leu Asn Ser Thr Leu Tyr Ala Asp Phe Ser His Asp
325 330 335
Asn Gly Ile Ile Ser Ile Leu Phe Ala Leu Gly Leu Tyr Asn Gly Thr
340 345 350
Lys Pro Leu Ser Thr Thr Thr Val Glu Asn Ile Thr Gln Thr Asp Gly
355 360 365
Phe Ser Ser Ala Trp Thr Val Pro Phe Ala Ser Arg Leu Tyr Val Glu
370 375 380
Met Met Gln Cys Gln Ala Glu Gln Glu Pro Leu Val Arg Val Leu Val
385 390 395 400
Asn Asp Arg Val Val Pro Leu His Gly Cys Pro Ile Asp Ala Leu Gly
405 410 415
Arg Cys Thr Arg Asp Ser Phe Val Arg Gly Leu Ser Phe Ala Arg Ser
420 425 430
Gly Gly Asp Trp Ala Glu Cys Ser Ala
435 440
<210>4
<211>441
<212>PRT
<213>黑曲霉T213
<400>4
Asn Gln Ser Ser Cys Asp Thr Val Asp Gln Gly Tyr Gln Cys Phe Ser
1 5 10 15
Glu Thr Ser His Leu Trp Gly Gln Tyr Ala Pro Phe Phe Ser Leu Ala
20 25 30
Asn Glu Ser Val Ile Ser Pro Asp Val Pro Ala Gly Cys Arg Val Thr
35 40 45
Phe Ala Gln Val Leu Ser Arg His Gly Ala Arg Tyr Pro Thr Glu Ser
50 55 60
Lys Gly Lys Lys Tyr Ser Ala Leu Ile Glu Glu Ile Gln Gln Asn Val
65 70 75 80
Thr Thr Phe Asp Gly Lys Tyr Ala Phe Leu Lys Thr Tyr Asn Tyr Ser
85 90 95
Leu Gly Ala Asp Asp Leu Thr Pro Phe Gly Glu Gln Glu Leu Val Asn
100 105 110
Ser Gly Ile Lys Phe Tyr Gln Arg Tyr Glu Ser Leu Thr Arg Asn Ile
115 120 125
Ile Pro Phe Ile Arg Ser Ser Gly Ser Ser Arg Val Ile Ala Ser Gly
130 135 140
Glu Lys Phe Ile Glu Gly Phe Gln Ser Thr Lys Leu Lys Asp Pro Arg
145 150 155 160
Ala Gln Pro Gly Gln Ser Ser Pro Lys Ile Asp Val Val Ile Ser Glu
165 170 175
Ala Ser Ser Ser Asn Asn Thr Leu Asp Pro Gly Thr Cys Thr Val Phe
180 185 190
Glu Asp Ser Glu Leu Ala Asp Thr Val Glu Ala Asn Phe Thr Ala Thr
195 200 205
Phe Ala Pro Ser Ile Arg Gln Arg Leu Glu Asn Asp Leu Ser Gly Val
210 215 220
Thr Leu Thr Asp Thr Glu Val Thr Tyr Leu Met Asp Met Cys Ser Phe
225 230 235 240
Asp Thr Ile Ser Thr Ser Thr Val Asp Thr Lys Leu Ser Pro Phe Cys
245 250 255
Asp Leu Phe Thr His Asp Glu Trp Ile His Tyr Asp Tyr Leu Arg Ser
260 265 270
Leu Lys Lys Tyr Tyr Gly His Gly Ala Gly Asn Pro Leu Gly Pro Thr
275 280 285
Gln Gly Val Gly Tyr Ala Asn Glu Leu Ile Ala Arg Leu Thr His Ser
290 295 300
Pro Val His Asp Asp Thr Ser Ser Asn His Thr Leu Asp Ser Asn Pro
305 310 315 320
Ala Thr Phe Pro Leu Asn Ser Thr Leu Tyr Ala Asp Phe Ser His Asp
325 330 335
Asn Gly Ile Ile Ser Ile Leu Phe Ala Leu Gly Leu Tyr Asn Gly Thr
340 345 350
Lys Pro Leu Ser Thr Thr Thr Val Glu Asn Ile Thr Gln Thr Asp Gly
355 360 365
Phe Ser Ser Ala Trp Thr Val Pro Phe Ala Ser Arg Leu Tyr Val Glu
370 375 380
Met Met Gln Cys Gln Ala Glu Gln Glu Pro Leu Val Arg Val Leu Val
385 390 395 400
Asn Asp Arg Val Val Pro Leu His Gly Cys Pro Ile Asp Ala Leu Gly
405 410 415
Arg Cys Thr Arg Asp Ser Phe Val Arg Gly Leu Ser Phe Ala Arg Ser
420 425 430
Gly Gly Asp Trp Ala Glu Cys Phe Ala
435 440
<210>5
<211>441
<212>PRT
<213>黑曲霉NRRL3135
<400>5
Asn Gln Ser Ser Cys Asp Thr Val Asp Gln Gly Tyr Gln Cys Phe Ser
1 5 10 15
Glu Thr Ser His Leu Trp Gly Gln Tyr Ala Pro Phe Phe Ser Leu Ala
20 25 30
Asn Glu Ser Val Ile Ser Pro Glu Val Pro Ala Gly Cys Arg Val Thr
35 40 45
Phe Ala Gln Val Leu Ser Arg His Gly Ala Arg Tyr Pro Thr Asp Ser
50 55 60
Lys Gly Lys Lys Tyr Ser Ala Leu Ile Glu Glu Ile Gln Gln Asn Ala
65 70 75 80
Thr Thr Phe Asp Gly Lys Tyr Ala Phe Leu Lys Thr Tyr Asn Tyr Ser
85 90 95
Leu Gly Ala Asp Asp Leu Thr Pro Phe Gly Glu Gln Glu Leu Val Asn
100 105 110
Ser Gly Ile Lys Phe Tyr Gln Arg Tyr Glu Ser Leu Thr Arg Asn Ile
115 120 125
Val Pro Phe Ile Arg Ser Ser Gly Ser Ser Arg Val Ile Ala Ser Gly
130 135 140
Lys Lys Phe Ile Glu Gly Phe Gln Ser Thr Lys Leu Lys Asp Pro Arg
145 150 155 160
Ala Gln Pro Gly Gln Ser Ser Pro Lys Ile Asp Val Val Ile Ser Glu
165 170 175
Ala Ser Ser Ser Asn Asn Thr Leu Asp Pro Gly Thr Cys Thr Val Phe
180 185 190
Glu Asp Ser Glu Leu Ala Asp Thr Val Glu Ala Asn Phe Thr Ala Thr
195 200 205
Phe Val Pro Ser Ile Arg Gln Arg Leu Glu Asn Asp Leu Ser Gly Val
210 215 220
Thr Leu Thr Asp Thr Glu Val Thr Tyr Leu Met Asp Met Cys Ser Phe
225 230 235 240
Asp Thr Ile Ser Thr Ser Thr Val Asp Thr Lys Leu Ser Pro Phe Cys
245 250 255
Asp Leu Phe Thr His Asp Glu Trp Ile Asn Tyr Asp Tyr Leu Gln Ser
260 265 270
Leu Lys Lys Tyr Tyr Gly His Gly Ala Gly Asn Pro Leu Gly Pro Thr
275 280 285
Gln Gly Val Gly Tyr Ala Asn Glu Leu Ile Ala Arg Leu Thr His Ser
290 295 300
Pro Val His Asp Asp Thr Ser Ser Asn His Thr Leu Asp Ser Ser Pro
305 310 315 320
Ala Thr Phe Pro Leu Asn Ser Thr Leu Tyr Ala Asp Phe Ser His Asp
325 330 335
Asn Gly Ile Ile Ser Ile Leu Phe Ala Leu Gly Leu Tyr Asn Gly Thr
340 345 350
Lys Pro Leu Ser Thr Thr Thr Val Glu Asn Ile Thr Gln Thr Asp Gly
355 360 365
Phe Ser Ser Ala Trp Thr Val Pro Phe Ala Ser Arg Leu Tyr Val Glu
370 375 380
Met Met Gln Cys Gln Ala Glu Gln Glu Pro Leu Val Arg Val Leu Val
385 390 395 400
Asn Asp Arg Val Val Pro Leu His Gly Cys Pro Val Asp Ala Leu Gly
405 410 415
Arg Cys Thr Arg Asp Ser Phe Val Arg Gly Leu Ser Phe Ala Arg Ser
420 425 430
Gly Gly Asp Trp Ala Glu Cys Phe Ala
435 440
<210>6
<211>440
<212>PRT
<213>烟曲霉13073
<400>6
Gly Ser Lys Ser Cys Asp Thr Val Asp Leu Gly Tyr Gln Cys Ser Pro
1 5 10 15
Ala Thr Ser His Leu Trp Gly Gln Tyr Ser Pro Phe Phe Ser Leu Glu
20 25 30
Asp Glu Leu Ser Val Ser Ser Lys Leu Pro Lys Asp Cys Arg Ile Thr
35 40 45
Leu Val Gln Val Leu Ser Arg His Gly Ala Arg Tyr Pro Thr Ser Ser
50 55 60
Lys Ser Lys Lys Tyr Lys Lys Leu Val Thr Ala Ile Gln Ala Asn Ala
65 70 75 80
Thr Asp Phe Lys Gly Lys Phe Ala Phe Leu Lys Thr Tyr Asn Tyr Thr
85 90 95
Leu Gly Ala Asp Asp Leu Thr Pro Phe Gly Glu Gln Gln Leu Val Asn
100 105 110
Ser Gly Ile Lys Phe Tyr Gln Arg Tyr Lys Ala Leu Ala Arg Ser Val
115 120 125
Val Pro Phe Ile Arg Ala Ser Gly Ser Asp Arg Val Ile Ala Ser Gly
130 135 140
Glu Lys Phe Ile Glu Gly Phe Gln Gln Ala Lys Leu Ala Asp Pro Gly
145 150 155 160
Ala Thr Asn Arg Ala Ala Pro Ala Ile Ser Val Ile Ile Pro Glu Ser
165 170 175
Glu Thr Phe Asn Asn Thr Leu Asp His Gly Val Cys Thr Lys Phe Glu
180 185 190
Ala Ser Gln Leu Gly Asp Glu Val Ala Ala Asn Phe Thr Ala Leu Phe
195 200 205
Ala Pro Asp Ile Arg Ala Arg Ala Glu Lys His Leu Pro Gly Val Thr
210 215 220
Leu Thr Asp Glu Asp Val Val Ser Leu Met Asp Met Cys Ser Phe Asp
225 230 235 240
Thr Val Ala Arg Thr Ser Asp Ala Ser Gln Leu Ser Pro Phe Cys Gln
245 250 255
Leu Phe Thr His Asn Glu Trp Lys Lys Tyr Asn Tyr Leu Gln Ser Leu
260 265 270
Gly Lys Tyr Tyr Gly Tyr Gly Ala Gly Asn Pro Leu Gly Pro Ala Gln
275 280 285
Gly Ile Gly Phe Thr Asn Glu Leu Ile Ala Arg Leu Thr Arg Ser Pro
290 295 300
Val Gln Asp His Thr Ser Thr Asn Ser Thr Leu Val Ser Asn Pro Ala
305 310 315 320
Thr Phe Pro Leu Asn Ala Thr Met Tyr Val Asp Phe Ser His Asp Asn
325 330 335
Ser Met Val Ser Ile Phe Phe Ala Leu Gly Leu Tyr Asn Gly Thr Glu
340 345 350
Pro Leu Ser Arg Thr Ser Val Glu Ser Ala Lys Glu Leu Asp Gly Tyr
355 360 365
Ser Ala Ser Trp Val Val Pro Phe Gly Ala Arg Ala Tyr Phe Glu Thr
370 375 380
Met Gln Cys Lys Ser Glu Lys Glu Pro Leu Val Arg Ala Leu Ile Asn
385 390 395 400
Asp Arg Val Val Pro Leu His Gly Cys Asp Val Asp Lys Leu Gly Arg
405 410 415
Cys Lys Leu Asn Asp Phe Val Lys Gly Leu Ser Trp Ala Arg Ser Gly
420 425 430
Gly Asn Trp Gly Glu Cys Phe Ser
435 440
<210>7
<211>440
<212>PRT
<213>烟曲霉32722
<400>7
Gly Ser Lys Ser Cys Asp Thr Val Asp Leu Gly Tyr Gln Cys Ser Pro
1 5 10 15
Ala Thr Ser His Leu Trp Gly Gln Tyr Ser Pro Phe Phe Ser Leu Glu
20 25 30
Asp Glu Leu Ser Val Ser Ser Lys Leu Pro Lys Asp Cys Arg Ile Thr
35 40 45
Leu Val Gln Val Leu Ser Arg His Gly Ala Arg Tyr Pro Thr Ser Ser
50 55 60
Lys Ser Lys Lys Tyr Lys Lys Leu Val Thr Ala Ile Gln Ala Asn Ala
65 70 75 80
Thr Asp Phe Lys Gly Lys Phe Ala Phe Leu Lys Thr Tyr Asn Tyr Thr
85 90 95
Leu Gly Ala Asp Asp Leu Thr Pro Phe Gly Glu Gln Gln Leu Val Asn
100 105 110
Ser Gly Ile Lys Phe Tyr Gln Arg Tyr Lys Ala Leu Ala Arg Ser Val
115 120 125
Val Pro Phe Ile Arg Ala Ser Gly Ser Asp Arg Val Ile Ala Ser Gly
130 135 140
Glu Lys Phe Ile Glu Gly Phe Gln Gln Ala Lys Leu Ala Asp Pro Gly
145 150 155 160
Ala Thr Asn Arg Ala Ala Pro Ala Ile Ser Val Ile Ile Pro Glu Ser
165 170 175
Glu Thr Phe Asn Asn Thr Leu Asp His Gly Val Cys Thr Lys Phe Glu
180 185 190
Ala Ser Gln Leu Gly Asp Glu Val Ala Ala Asn Phe Thr Ala Leu Phe
195 200 205
Ala Pro Asp Ile Arg Ala Arg Ala Glu Lys His Leu Pro Gly Val Thr
210 215 220
Leu Thr Asp Glu Asp Val Val Ser Leu Met Asp Met Cys Ser Phe Asp
225 230 235 240
Thr Val Ala Arg Thr Ser Asp Ala Ser Gln Leu Ser Pro Phe Cys Gln
245 250 255
Leu Phe Thr His Asn Glu Trp Lys Lys Tyr Asn Tyr Leu Gln Ser Leu
260 265 270
Gly Lys Tyr Tyr Gly Tyr Gly Ala Gly Asn Pro Leu Gly Pro Ala Gln
275 280 285
Gly Ile Gly Phe Thr Asn Glu Leu Ile Ala Arg Leu Thr Arg Ser Pro
290 295 300
Val Gln Asp His Thr Ser Thr Asn Ser Thr Leu Val Ser Asn Pro Ala
305 310 315 320
Thr Phe Pro Leu Asn Ala Thr Met Tyr Val Asp Phe Ser His Asp Asn
325 330 335
Ser Met Val Ser Ile Phe Phe Ala Leu Gly Leu Tyr Asn Gly Thr Gly
340 345 350
Pro Leu Ser Arg Thr Ser Val Glu Ser Ala Lys Glu Leu Asp Gly Tyr
355 360 365
Ser Ala Ser Trp Val Val Pro Phe Gly Ala Arg Ala Tyr Phe Glu Thr
370 375 380
Met Gln Cys Lys Ser Glu Lys Glu Pro Leu Val Arg Ala Leu Ile Asn
385 390 395 400
Asp Arg Val Val Pro Leu His Gly Cys Asp Val Asp Lys Leu Gly Arg
405 410 415
Cys Lys Leu Asn Asp Phe Val Lys Gly Leu Ser Trp Ala Arg Ser Gly
420 425 430
Gly Asn Trp Gly Glu Cys Phe Ser
435 440
<210>8
<211>440
<212>PRT
<213>烟曲霉58128
<400>8
Gly Ser Lys Ser Cys Asp Thr Val Asp Leu Gly Tyr Gln Cys Ser Pro
1 5 10 15
Ala Thr Ser His Leu Trp Gly Gln Tyr Ser Pro Phe Phe Ser Leu Glu
20 25 30
Asp Glu Leu Ser Val Ser Ser Lys Leu Pro Lys Asp Cys Arg Ile Thr
35 40 45
Leu Val Gln Val Leu Ser Arg His Gly Ala Arg Tyr Pro Thr Ser Ser
50 55 60
Lys Ser Lys Lys Tyr Lys Lys Leu Val Thr Ala Ile Gln Ala Asn Ala
65 70 75 80
Thr Asp Phe Lys Gly Lys Phe Ala Phe Leu Lys Thr Tyr Asn Tyr Thr
85 90 95
Leu Gly Ala Asp Asp Leu Thr Pro Phe Gly Glu Gln Gln Leu Val Asn
100 105 110
Ser Gly Ile Lys Phe Tyr Gln Arg Tyr Lys Ala Leu Ala Arg Ser Val
115 120 125
Val Pro Phe Ile Arg Ala Ser Gly Ser Asp Arg Val Ile Ala Ser Gly
130 135 140
Glu Lys Phe Ile Glu Gly Phe Gln Gln Ala Lys Leu Ala Asp Pro Gly
145 150 155 160
Ala Thr Asn Arg Ala Ala Pro Ala Ile Ser Val Ile Ile Pro Glu Ser
165 170 175
Glu Thr Phe Asn Asn Thr Leu Asp His Gly Val Cys Thr Lys Phe Glu
180 185 190
Ala Ser Gln Leu Gly Asp Glu Val Ala Ala Asn Phe Thr Ala Leu Phe
195 200 205
Ala Pro Asp Ile Arg Ala Arg Ala Glu Lys His Leu Pro Gly Val Thr
210 215 220
Leu Thr Asp Glu Asp Val Val Ser Leu Met Asp Met Cys Ser Phe Asp
225 230 235 240
Thr Val Ala Arg Thr Ser Asp Ala Ser Gln Leu Ser Pro Phe Cys Gln
245 250 255
Leu Phe Thr His Asn Glu Trp Lys Lys Tyr Asn Tyr Leu Gln Ser Leu
260 265 270
Gly Lys Tyr Tyr Gly Tyr Gly Ala Gly Asn Pro Leu Gly Pro Ala Gln
275 280 285
Gly Ile Gly Phe Thr Asn Glu Leu Ile Ala Arg Leu Thr Arg Ser Pro
290 295 300
Val Gln Asp His Thr Ser Thr Asn Ser Thr Leu Val Ser Asn Pro Ala
305 310 315 320
Thr Phe Pro Leu Asn Ala Thr Met Tyr Val Asp Phe Ser His Asp Asn
325 330 335
Ser Met Val Ser Ile Phe Phe Ala Leu Gly Leu Tyr Asn Gly Thr Glu
340 345 350
Pro Leu Ser Arg Thr Ser Val Glu Ser Ala Lys Glu Leu Asp Gly Tyr
355 360 365
Ser Ala Ser Trp Val Val Pro Phe Gly Ala Arg Ala Tyr Phe Glu Thr
370 375 380
Met Gln Cys Lys Sar Glu Lys Glu Ser Leu Val Arg Ala Leu Ile Asn
385 390 395 400
Asp Arg Val Val Pro Leu His Gly Cys Asp Val Asp Lys Leu Gly Arg
405 410 415
Cys Lys Leu Asn Asp Phe Val Lys Gly Leu Ser Trp Ala Arg Ser Gly
420 425 430
Gly Asn Trp Gly Glu Cys Phe Ser
435 440
<210>9
<211>440
<212>PRT
<213>烟曲霉26906
<400>9
Gly Ser Lys Ser Cys Asp Thr Val Asp Leu Gly Tyr Gln Cys Ser Pro
1 5 10 15
Ala Thr Ser His Leu Trp Gly Gln Tyr Ser Pro Phe Phe Ser Leu Glu
20 25 30
Asp Glu Leu Ser Val Ser Ser Lys Leu Pro Lys Asp Cys Arg Ile Thr
35 40 45
Leu Val Gln Val Leu Ser Arg His Gly Ala Arg Tyr Pro Thr Ser Ser
50 55 60
Lys Ser Lys Lys Tyr Lys Lys Leu Val Thr Ala Ile Gln Ala Asn Ala
65 70 75 80
Thr Asp Phe Lys Gly Lys Phe Ala Phe Leu Lys Thr Tyr Asn Tyr Thr
85 90 95
Leu Gly Ala Asp Asp Leu Thr Ala Phe Gly Glu Gln Gln Leu Val Asn
100 105 110
Ser Gly Ile Lys Phe Tyr Gln Arg Tyr Lys Ala Leu Ala Arg Ser Val
115 120 125
Val Pro Phe Ile Arg Ala Ser Gly Ser Asp Arg Val Ile Ala Ser Gly
130 135 140
Glu Lys Phe Ile Glu Gly Phe Gln Gln Ala Lys Leu Ala Asp Pro Gly
145 150 155 160
Ala Thr Asn Arg Ala Ala Pro Ala Ile Ser Val Ile Ile Pro Glu Ser
165 170 175
Glu Thr Phe Asn Asn Thr Leu Asp His Gly Val Cys Thr Lys Phe Glu
180 185 190
Ala Ser Gln Leu Gly Asp Glu Val Ala Ala Asn Phe Thr Ala Leu Phe
195 200 205
Ala Pro Asp Ile Arg Ala Arg Ala Lys Lys His Leu Pro Gly Val Thr
210 215 220
Leu Thr Asp Glu Asp Val Val Ser Leu Met Asp Met Cys Ser Phe Asp
225 230 235 240
Thr Val Ala Arg Thr Ser Asp Ala Ser Gln Leu Ser Pro Phe Cys Gln
245 250 255
Leu Phe Thr His Asn Glu Trp Lys Lys Tyr Asn Tyr Leu Gln Ser Leu
260 265 270
Gly Lys Tyr Tyr Gly Tyr Gly Ala Gly Asn Pro Leu Gly Pro Ala Gln
275 280 285
Gly Ile Gly Phe Thr Asn Glu Leu Ile Ala Arg Leu Thr Arg Ser Pro
290 295 300
Val Gln Asp His Thr Ser Thr Asn Ser Thr Leu Val Ser Asn Pro Ala
305 310 315 320
Thr Phe Pro Leu Asn Ala Thr Met Tyr Val Asp Phe Ser His Asp Asn
325 330 335
Ser Met Val Ser Ile Phe Phe Ala Leu Gly Leu Tyr Asn Gly Thr Glu
340 345 350
Pro Leu Ser Arg Thr Ser Val Glu Ser Ala Lys Glu Leu Asp Gly Tyr
355 360 365
Ser Ala Ser Trp Val Val Pro Phe Gly Ala Arg Ala Tyr Phe Glu Thr
370 375 380
Met Gln Cys Lys Ser Glu Lys Glu Pro Leu Val Arg Ala Leu Ile Asn
385 390 395 400
Asp Arg Val Val Pro Leu His Gly Cys Asp Val Asp Lys Leu Gly Arg
405 410 415
Cys Lys Leu Asn Asp Phe Val Lys Gly Leu Ser Trp Ala Arg Ser Gly
420 425 430
Gly Asn Trp Gly Glu Cys Phe Ser
435 440
<210>10
<211>440
<212>PRT
<213>烟曲霉32239
<400>10
Gly Ser Lys Ala Cys Asp Thr Val Glu Leu Gly Tyr Gln Cys Ser Pro
1 5 10 15
Gly Thr Ser His Leu Trp Gly Gln Tyr Ser Pro Phe Phe Ser Leu Glu
20 25 30
Asp Glu Leu Ser Val Ser Ser Asp Leu Pro Lys Asp Cys Arg Val Thr
35 40 45
Phe Val Gln Val Leu Ser Arg His Gly Ala Arg Tyr Pro Thr Ala Ser
50 55 60
Lys Ser Lys Lys Tyr Lys Lys Leu Val Thr Ala Ile Gln Lys Asn Ala
65 70 75 80
Thr Glu Phe Lys Gly Lys Phe Ala Phe Leu Glu Thr Tyr Asn Tyr Thr
85 90 95
Leu Gly Ala Asp Asp Leu Thr Pro Phe Gly Glu Gln Gln Met Val Asn
100 105 110
Ser Gly Ile Lys Phe Tyr Gln Lys Tyr Lys Ala Leu Ala Gly Ser Val
115 120 125
Val Pro Phe Ile Arg Ser Ser Gly Ser Asp Arg Val Ile Ala Ser Gly
130 135 140
Glu Lys Phe Ile Glu Gly Phe Gln Gln Ala Asn Val Ala Asp Pro Gly
145 150 155 160
Ala Thr Asn Arg Ala Ala Pro Val Ile Ser Val Ile Ile Pro Glu Ser
165 170 175
Glu Thr Tyr Asn Asn Thr Leu Asp His Ser Val Cys Thr Asn Phe Glu
180 185 190
Ala Ser Glu Leu Gly Asp Glu Val Glu Ala Asn Phe Thr Ala Leu Phe
195 200 205
Ala Pro Ala Ile Arg Ala Arg Ile Glu Lys His Leu Pro Gly Val Gln
210 215 220
Leu Thr Asp Asp Asp Val Val Ser Leu Met Asp Met Cys Ser Phe Asp
225 230 235 240
Thr Val Ala Arg Thr Ala Asp Ala Ser Glu Leu Ser Pro Phe Cys Ala
245 250 255
Ile Phe Thr His Asn Glu Trp Lys Lys Tyr Asp Tyr Leu Gln Ser Leu
260 265 270
Gly Lys Tyr Tyr Gly Tyr Gly Ala Gly Asn Pro Leu Gly Pro Ala Gln
275 280 285
Gly Ile Gly Phe Thr Asn Glu Leu Ile Ala Arg Leu Thr Asn Ser Pro
290 295 300
Val Gln Asp His Thr Ser Thr Asn Ser Thr Leu Asp Ser Asp Pro Ala
305 310 315 320
Thr Phe Pro Leu Asn Ala Thr Ile Tyr Val Asp Phe Ser His Asp Asn
325 330 335
Gly Met Ile Pro Ile Phe Phe Ala Met Gly Leu Tyr Asn Gly Thr Glu
340 345 350
Pro Leu Ser Gln Thr Ser Glu Glu Ser Thr Lys Glu Ser Asn Gly Tyr
355 360 365
Ser Ala Ser Trp Ala Val Pro Phe Gly Ala Arg Ala Tyr Phe Glu Thr
370 375 380
Met Gln Cys Lys Ser Glu Lys Glu Pro Leu Val Arg Ala Leu Ile Asn
385 390 395 400
Asp Arg Val Val Pro Leu His Gly Cys Ala Val Asp Lys Leu Gly Arg
405 410 415
Cys Lys Leu Lys Asp Phe Val Lys Gly Leu Ser Trp Ala Arg Ser Gly
420 425 430
Gly Asn Ser Glu Gln Ser Phe Ser
435 440
<210>11
<211>439
<212>PRT
<213>Emericella nidulans
<400>11
Gln Asn His Ser Cys Asn Thr Ala Asp Gly Gly Tyr Gln Cys Phe Pro
1 5 10 15
Asn Val Ser His Val Trp Gly Gln Tyr Ser Pro Tyr Phe Ser Ile Glu
20 25 30
Gln Glu Ser Ala Ile Ser Glu Asp Val Pro His Gly Cys Glu Val Thr
35 40 45
Phe Val Gln Val Leu Ser Arg His Gly Ala Arg Tyr Pro Thr Glu Ser
50 55 60
Lys Ser Lys Ala Tyr Sar Gly Leu Ile Glu Ala Ile Gln Lys Asn Ala
65 70 75 80
Thr Ser Phe Trp Gly Gln Tyr Ala Phe Leu Glu Ser Tyr Asn Tyr Thr
85 90 95
Leu Gly Ala Asp Asp Leu Thr Ile Phe Gly Glu Asn Gln Met Val Asp
100 105 110
Ser Gly Ala Lys Phe Tyr Arg Arg Tyr Lys Asn Leu Ala Arg Lys Asn
115 120 125
Thr Pro Phe Ile Arg Ala Ser Gly Ser Asp Arg Val Val Ala Ser Ala
130 135 140
Glu Lys Phe Ile Asn Gly Phe Arg Lys Ala Gln Leu His Asp His Gly
145 150 155 160
Ser Gly Gln Ala Thr Pro Val Val Asn Val Ile Ile Pro Glu Ile Asp
165 170 175
Gly Phe Asn Asn Thr Leu Asp His Ser Thr Cys Val Ser Phe Glu Asn
180 185 190
Asp Glu Arg Ala Asp Glu Ile Glu Ala Asn Phe Thr Ala Ile Met Gly
195 200 205
Pro Pro Ile Arg Lys Arg Leu Glu Asn Asp Leu Pro Gly Ile Lys Leu
210 215 220
Thr Asn Glu Asn Val Ile Tyr Leu Met Asp Met Cys Ser Phe Asp Thr
225 230 235 240
Met Ala Arg Thr Ala His Gly Thr Glu Leu Ser Pro Phe Cys Ala Ile
245 250 255
Phe Thr Glu Lys Glu Trp Leu Gln Tyr Asp Tyr Leu Gln Ser Leu Ser
260 265 270
Lys Tyr Tyr Gly Tyr Gly Ala Gly Ser Pro Leu Gly Pro Ala Gln Gly
275 280 285
Ile Gly Phe Thr Asn Glu Leu Ile Ala Arg Leu Thr Gln Ser Pro Val
290 295 300
Gln Asp Asn Thr Ser Thr Asn His Thr Leu Asp Ser Asn Pro Ala Thr
305 310 315 320
Phe Pro Leu Asp Arg Lys Leu Tyr Ala Asp Phe Ser His Asp Asn Ser
325 330 335
Met Ile Ser Ile Phe Phe Ala Met Gly Leu Tyr Asn Gly Thr Gln Pro
340 345 350
Leu Ser Met Asp Ser Val Glu Ser Ile Gln Glu Met Asp Gly Tyr Ala
355 360 365
Ala Ser Trp Thr Val Pro Phe Gly Ala Arg Ala Tyr Phe Glu Leu Met
370 375 380
Gln Cys Glu Lys Lys Glu Pro Leu Val Arg Val Leu Val Asn Asp Arg
385 390 395 400
Val Val Pro Leu His Gly Cys Ala Val Asp Lys Phe Gly Arg Cys Thr
405 410 415
Leu Asp Asp Trp Val Glu Gly Leu Asn Phe Ala Arg Ser Gly Gly Asn
420 425 430
Trp Lys Thr Cys Phe Thr Leu
435
<210>12
<211>443
<212>PRT
<213>嗜热踝节菌
<400>12
Asp Ser His Ser Cys Asn Thr Val Glu Gly Gly Tyr Gln Cys Arg Pro
1 5 10 15
Glu Ile Ser His Ser Trp Gly Gln Tyr Ser Pro Phe Phe Ser Leu Ala
20 25 30
Asp Gln Ser Glu Ile Ser Pro Asp Val Pro Gln Asn Cys Lys Ile Thr
35 40 45
Phe Val Gln Leu Leu Ser Arg His Gly Ala Arg Tyr Pro Thr Ser Ser
50 55 60
Lys Thr Glu Leu Tyr Ser Gln Leu Ile Ser Arg Ile Gln Lys Thr Ala
65 70 75 80
Thr Ala Tyr Lys Gly Tyr Tyr Ala Phe Leu Lys Asp Tyr Arg Tyr Gln
85 90 95
Leu Gly Ala Asn Asp Leu Thr Pro Phe Gly Glu Asn Gln Met Ile Gln
100 105 110
Leu Gly Ile Lys Phe Tyr Asn His Tyr Lys Ser Leu Ala Arg Asn Ala
115 120 125
Val Pro Phe Val Arg Cys Ser Gly Ser Asp Arg Val Ile Ala Ser Gly
130 135 140
Arg Leu Phe Ile Glu Gly Phe Gln Ser Ala Lys Val Leu Asp Pro His
145 150 155 160
Ser Asp Lys His Asp Ala Pro Pro Thr Ile Asn Val Ile Ile Glu Glu
165 170 175
Gly Pro Ser Tyr Asn Asn Thr Leu Asp Thr Gly Ser Cys Pro Val Phe
180 185 190
Glu Asp Ser Ser Gly Gly His Asp Ala Gln Glu Lys Phe Ala Lys Gln
195 200 205
Phe Ala Pro Ala Ile Leu Glu Lys Ile Lys Asp His Leu Pro Gly Val
210 215 220
Asp Leu Ala Val Ser Asp Val Pro Tyr Leu Met Asp Leu Cys Pro Phe
225 230 235 240
Glu Thr Leu Ala Arg Asn His Thr Asp Thr Leu Ser Pro Phe Cys Ala
245 250 255
Leu Ser Thr Gln Glu Glu Trp Gln Ala Tyr Asp Tyr Tyr Gln Ser Leu
260 265 270
Gly Lys Tyr Tyr Gly Asn Gly Gly Gly Asn Pro Leu Gly Pro Ala Gln
275 280 285
Gly Val Gly Phe Val Asn Glu Leu Ile Ala Arg Met Thr His Ser Pro
290 295 300
Val Gln Asp Tyr Thr Thr Val Asn His Thr Leu Asp Ser Asn Pro Ala
305 310 315 320
Thr Phe Pro Leu Asn Ala Thr Leu Tyr Ala Asp Phe Ser His Asp Asn
325 330 335
Thr Met Thr Ser Ile Phe Ala Ala Leu Gly Leu Tyr Asn Gly Thr Ala
340 345 350
Lys Leu Ser Thr Thr Glu Ile Lys Ser Ile Glu Glu Thr Asp Gly Tyr
355 360 365
Ser Ala Ala Trp Thr Val Pro Phe Gly Gly Arg Ala Tyr Ile Glu Met
370 375 380
Met Gln Cys Asp Asp Ser Asp Glu Pro Val Val Arg Val Leu Val Asn
385 390 395 400
Asp Arg Val Val Pro Leu His Gly Cys Glu Val Asp Ser Leu Gly Arg
405 410 415
Cys Lys Arg Asp Asp Phe Val Arg Gly Leu Ser Phe Ala Arg Gln Gly
420 425 430
Gly Asn Trp Glu Gly Cys Tyr Ala Ala Ser Glu
435 440
<210>13
<211>466
<212>PRT
<213>嗜热毁丝霉
<400>13
Glu Ser Arg Pro Cys Asp Thr Pro Asp Leu Gly Phe Gln Cys Gly Thr
1 5 10 15
Ala Ile Ser His Phe Trp Gly Gln Tyr Ser Pro Tyr Phe Ser Val Pro
20 25 30
Ser Glu Leu Asp Ala Ser Ile Pro Asp Asp Cys Glu Val Thr Phe Ala
35 40 45
Gln Val Leu Ser Arg His Gly Ala Arg Ala Pro Thr Leu Lys Arg Ala
50 55 60
Ala Ser Tyr Val Asp Leu Ile Asp Arg Ile His His Gly Ala Ile Ser
65 70 75 80
Tyr Gly Pro Gly Tyr Glu Phe Leu Arg Thr Tyr Asp Tyr Thr Leu Gly
85 90 95
Ala Asp Glu Leu Thr Arg Thr Gly Gln Gln Gln Met Val Asn Ser Gly
100 105 110
Ile Lys Phe Tyr Arg Arg Tyr Arg Ala Leu Ala Arg Lys Ser Ile Pro
115 120 125
Phe Val Arg Thr Ala Gly Gln Asp Arg Val Val His Ser Ala Glu Asn
130 135 140
Phe Thr Gln Gly Phe His Ser Ala Leu Leu Ala Asp Arg Gly Ser Thr
145 150 155 160
Val Arg Pro Thr Leu Pro Tyr Asp Met Val Val Ile Pro Glu Thr Ala
165 170 175
Gly Ala Asn Asn Thr Leu His Asn Asp Leu Cys Thr Ala Phe Glu Glu
180 185 190
Gly Pro Tyr Ser Thr Ile Gly Asp Asp Ala Gln Asp Thr Tyr Leu Ser
195 200 205
Thr Phe Ala Gly Pro Ile Thr Ala Arg Val Asn Ala Asn Leu Pro Gly
210 215 220
Ala Asn Leu Thr Asp Ala Asp Thr Val Ala Leu Met Asp Leu Cys Pro
225 230 235 240
Phe Glu Thr Val Ala Ser Ser Ser Ser Asp Pro Ala Thr Ala Asp Ala
245 250 255
Gly Gly Gly Asn Gly Arg Pro Leu Ser Pro Phe Cys Arg Leu Phe Ser
260 265 270
Glu Ser Glu Trp Arg Ala Tyr Asp Tyr Leu Gln Ser Val Gly Lys Trp
275 280 285
Tyr Gly Tyr Gly Pro Gly Asn Pro Leu Gly Pro Thr Gln Gly Val Gly
290 295 300
Phe Val Asn Glu Leu Leu Ala Arg Leu Ala Gly Val Pro Val Arg Asp
305 310 315 320
Gly Thr Ser Thr Asn Arg Thr Leu Asp Gly Asp Pro Arg Thr Phe Pro
325 330 335
Leu Gly Arg Pro Leu Tyr Ala Asp Phe Ser His Asp Asn Asp Met Met
340 345 350
Gly Val Leu Gly Ala Leu Gly Ala Tyr Asp Gly Val Pro Pro Leu Asp
355 360 365
Lys Thr Ala Arg Arg Asp Pro Glu Glu Leu Gly Gly Tyr Ala Ala Ser
370 375 380
Trp Ala Val Pro Phe Ala Ala Arg Ile Tyr Val Glu Lys Met Arg Cys
385 390 395 400
Ser Gly Gly Gly Gly Gly Gly Gly Gly Gly Glu Gly Arg Gln Glu Lys
405 410 415
Asp Glu Glu Met Val Arg Val Leu Val Asn Asp Arg Val Met Thr Leu
420 425 430
Lys Gly Cys Gly Ala Asp Glu Arg Gly Met Cys Thr Leu Glu Arg Phe
435 440 445
Ile Glu Ser Met Ala Phe Ala Arg Gly Asn Gly Lys Trp Asp Leu Cys
450 455 460
Phe Ala
465
<210>14
<211>441
<212>PRT
<213>人工序列
<220>
<223>人工序列的描述：共有植酸酶
<400>14
Asn Ser His Ser Cys Asp Thr Val Asp Gly Gly Tyr Gln Cys Phe Pro
1 5 10 15
Glu Ile Ser His Leu Trp Gly Gln Tyr Ser Pro Tyr Phe Ser Leu Glu
20 25 30
Asp Glu Ser Ala Ile Ser Pro Asp Val Pro Asp Asp Cys Arg Val Thr
35 40 45
Phe Val Gln Val Leu Ser Arg His Gly Ala Arg Tyr Pro Thr Ser Ser
50 55 60
Lys Ser Lys Ala Tyr Ser Ala Leu Ile Glu Ala Ile Gln Lys Asn Ala
65 70 75 80
Thr Ala Phe Lys Gly Lys Tyr Ala Phe Leu Lys Thr Tyr Asn Tyr Thr
85 90 95
Leu Gly Ala Asp Asp Leu Thr Pro Phe Gly Glu Asn Gln Met Val Asn
100 105 110
Ser Gly Ile Lys Phe Tyr Arg Arg Tyr Lys Ala Leu Ala Arg Lys Ile
115 120 125
Val Pro Phe Ile Arg Ala Ser Gly Ser Asp Arg Val Ile Ala Ser Ala
130 135 140
Glu Lys Phe Ile Glu Gly Phe Gln Ser Ala Lys Leu Ala Asp Pro Gly
145 150 155 160
Ser Gln Pro His Gln Ala Ser Pro Val Ile Asp Val Ile Ile Pro Glu
165 170 175
Gly Ser Gly Tyr Asn Asn Thr Leu Asp His Gly Thr Cys Thr Ala Phe
180 185 190
Glu Asp Ser Glu Leu Gly Asp Asp Val Glu Ala Asn Phe Thr Ala Leu
195 200 205
Phe Ala Pro Ala Ile Arg Ala Arg Leu Glu Ala Asp Leu Pro Gly Val
210 215 220
Thr Leu Thr Asp Glu Asp Val Val Tyr Leu Met Asp Met Cys Pro Phe
225 230 235 240
Glu Thr Val Ala Arg Thr Ser Asp Ala Thr Glu Leu Ser Pro Phe Cys
245 250 255
Ala Leu Phe Thr His Asp Glu Trp Arg Gln Tyr Asp Tyr Leu Gln Ser
260 265 270
Leu Gly Lys Tyr Tyr Gly Tyr Gly Ala Gly Asn Pro Leu Gly Pro Ala
275 280 285
Gln Gly Val Gly Phe Ala Asn Glu Leu Ile Ala Arg Leu Thr Arg Ser
290 295 300
Pro Val Gln Asp His Thr Ser Thr Asn His Thr Leu Asp Ser Asn Pro
305 310 315 320
Ala Thr Phe Pro Leu Asn Ala Thr Leu Tyr Ala Asp Phe Ser His Asp
325 330 335
Asn Ser Met Ile Ser Ile Phe Phe Ala Leu Gly Leu Tyr Asn Gly Thr
340 345 350
Ala Pro Leu Ser Thr Thr Ser Val Glu Ser Ile Glu Glu Thr Asp Gly
355 360 365
Tyr Ser Ala Ser Trp Thr Val Pro Phe Gly Ala Arg Ala Tyr Val Glu
370 375 380
Met Met Gln Cys Gln Ala Glu Lys Glu Pro Leu Val Arg Val Leu Val
385 390 395 400
Asn Asp Arg Val Val Pro Leu His Gly Cys Ala Val Asp Lys Leu Gly
405 410 415
Arg Cys Lys Arg Asp Asp Phe Val Glu Gly Leu Ser Phe Ala Arg Ser
420 425 430
Gly Gly Asn Trp Ala Glu Cys Phe Ala
435 440
<210>15
<211>1426
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述：共有植酸酶
<220>
<221>CDS
<222>(12)..(1412)
<220>
<221>单肽
<222>(12)..(89)
<220>
<221>成熟肽
<222>(90)..(1412)
<400>15
tatatgaatt c atg ggc gtg ttc gtc gtg cta ctg tcc att gcc acc ttg 50
Met Gly Val Phe Val Val Leu Leu Ser Ile Ala Thr Leu
-25 -20 -15
ttc ggt tcc aca tcc ggt acc gcc ttg ggt cct cgt ggt aat tct cac 98
Phe Gly Ser Thr Ser Gly Thr Ala Leu Gly Pro Arg Gly Asn Ser His
-10 -5 -1 1
tct tgt gac act gtt gac ggt ggt tac caa tgt ttc cca gaa att tct 146
Ser Cys Asp Thr Val Asp Gly Gly Tyr Gln Cys Phe Pro Glu Ile Ser
5 10 15
cac ttg tgg ggt caa tac tct cca tac ttc tct ttg gaa gac gaa tct 194
His Leu Trp Gly Gln Tyr Ser Pro Tyr Phe Ser Leu Glu Asp Glu Ser
20 25 30 35
gct att tct cca gac gtt cca gac gac tgt aga gtt act ttc gtt caa 242
Ala Ile Ser Pro Asp Val Pro Asp Asp Cys Arg Val Thr Phe Val Gln
40 45 50
gtt ttg tct aga cac ggt gct aga tac cca act tct tct aag tct aag 290
Val Leu Ser Arg His Gly Ala Arg Tyr Pro Thr Ser Ser Lys Ser Lys
55 60 65
gct tac tct gct ttg att gaa gct att caa aag aac gct act gct ttc 338
Ala Tyr Ser Ala Leu Ile Glu Ala Ile Gln Lys Asn Ala Thr Ala Phe
70 75 80
aag ggt aag tac gct ttc ttg aag act tac aac tac act ttg ggt gct 386
Lys Gly Lys Tyr Ala Phe Leu Lys Thr Tyr Asn Tyr Thr Leu Gly Ala
85 90 95
gac gac ttg act cca ttc ggt gaa aac caa atg gtt aac tct ggt att 434
Asp Asp Leu Thr Pro Phe Gly Glu Asn Gln Met Val Asn Ser Gly Ile
100 105 110 115
aag ttc tac aga aga tac aag gct ttg gct aga aag att gtt cca ttc 482
Lys Phe Tyr Arg Arg Tyr Lys Ala Leu Ala Arg Lys Ile Val Pro Phe
120 125 130
att aga gct tct ggt tct gac aga gtt att gct tct gct gaa aag ttc 530
Ile Arg Ala Ser Gly Ser Asp Arg Val Ile Ala Ser Ala Glu Lys Phe
135 140 145
att gaa ggt ttc caa tct gct aag ttg gct gac cca ggt tct caa cca 578
Ile Glu Gly Phe Gln Ser Ala Lys Leu Ala Asp Pro Gly Ser Gln Pro
150 155 160
cac caa gct tct cca gtt att gac gtt att att cca gaa gga tcc ggt 626
His Gln Ala Ser Pro Val Ile Asp Val Ile Ile Pro Glu Gly Ser Gly
165 170 175
tac aac aac act ttg gac cac ggt act tgt act gct ttc gaa gac tct 674
Tyr Asn Asn Thr Leu Asp His Gly Thr Cys Thr Ala Phe Glu Asp Ser
180 185 190 195
gaa ttg ggt gac gac gtt gaa gct aac ttc act gct ttg ttc gct cca 722
Glu Leu Gly Asp Asp Val Glu Ala Asn Phe Thr Ala Leu Phe Ala Pro
200 205 210
gct att aga gct aga ttg gaa gct gac ttg cca ggt gtt act ttg act 770
Ala Ile Arg Ala Arg Leu Glu Ala Asp Leu Pro Gly Val Thr Leu Thr
215 220 225
gac gaa gac gtt gtt tac ttg atg gac atg tgt cca ttc gaa act gtt 818
Asp Glu Asp Val Val Tyr Leu Met Asp Met Cys Pro Phe Glu Thr Val
230 235 240
gct aga act tct gac gct act gaa ttg tct cca ttc tgt gct ttg ttc 866
Ala Arg Thr Ser Asp Ala Thr Glu Leu Ser Pro Phe Cys Ala Leu Phe
245 250 255
act cac gac gaa tgg aga caa tac gac tac ttg caa tct ttg ggt aag 914
Thr His Asp Glu Trp Arg Gln Tyr Asp Tyr Leu Gln Ser Leu Gly Lys
260 265 270 275
tac tac ggt tac ggt gct ggt aac cca ttg ggt cca gct caa ggt gtt 962
Tyr Tyr Gly Tyr Gly Ala Gly Asn Pro Leu Gly Pro Ala Gln Gly Val
280 285 290
ggt ttc gct aac gaa ttg att gct aga ttg act aga tct cca gtt caa 1010
Gly Phe Ala Asn Glu Leu Ile Ala Arg Leu Thr Arg Ser Pro Val Gln
295 300 305
gac cac act tct act aac cac act ttg gac tct aac cca gct act ttc 1058
Asp His Thr Ser Thr Asn His Thr Leu Asp Ser Asn Pro Ala Thr Phe
310 315 320
cca ttg aac gct act ttg tac gct gac ttc tct cac gac aac tct atg 1106
Pro Leu Asn Ala Thr Leu Tyr Ala Asp Phe Ser His Asp Asn Ser Met
325 330 335
att tct att ttc ttc gct ttg ggt ttg tac aac ggt act gct cca ttg 1154
Ile Ser Ile Phe Phe Ala Leu Gly Leu Tyr Asn Gly Thr Ala Pro Leu
340 345 350 355
tct act act tct gtt gaa tct att gaa gaa act gac ggt tac tct gct 1202
Ser Thr Thr Ser Val Glu Ser Ile Glu Glu Thr Asp Gly Tyr Ser Ala
360 365 370
tct tgg act gtt cca ttc ggt gct aga gct tac gtt gaa atg atg caa 1250
Ser Trp Thr Val Pro Phe Gly Ala Arg Ala Tyr Val Glu Met Met Gln
375 380 385
tgt caa gct gaa aag gaa cca ttg gtt aga gtt ttg gtt aac gac aga 1298
Cys Gln Ala Glu Lys Glu Pro Leu Val Arg Val Leu Val Asn Asp Arg
390 395 400
gtt gtt cca ttg cac ggt tgt gct gtt gac aag ttg ggt aga tgt aag 1346
Val Val Pro Leu His Gly Cys Ala Val Asp Lys Leu Gly Arg Cys Lys
405 410 415
aga gac gac ttc gtt gaa ggt ttg tct ttc gct aga tct ggt ggt aac 1394
Arg Asp Asp Phe Val Glu Gly Leu Ser Phe Ala Arg Ser Gly Gly Asn
420 425 430 435
tgg gct gaa tgt ttc gct taagaattca tata 1426
Trp Ala Glu Cys Phe Ala
440
<210>16
<211>467
<212>PRT
<213>人工序列
<223>人工序列的描述：共有植酸酶
<400>16
Met Gly Val Phe Val Val Leu Leu Ser Ile Ala Thr Leu Phe Gly Ser
-25 -20 -15
Thr Ser Gly Thr Ala Leu Gly Pro Arg Gly Asn Ser His Ser Cys Asp
-10 -5 -1 1 5
Thr Val Asp Gly Gly Tyr Gln Cys Phe Pro Glu Ile Ser His Leu Trp
10 15 20
Gly Gln Tyr Ser Pro Tyr Phe Ser Leu Glu Asp Glu Ser Ala Ile Ser
25 30 35
Pro Asp Val Pro Asp Asp Cys Arg Val Thr Phe Val Gln Val Leu Ser
40 45 50
Arg His Gly Ala Arg Tyr Pro Thr Ser Ser Lys Ser Lys Ala Tyr Ser
55 60 65 70
Ala Leu Ile Glu Ala Ile Gln Lys Asn Ala Thr Ala Phe Lys Gly Lys
75 80 85
Tyr Ala Phe Leu Lys Thr Tyr Asn Tyr Thr Leu Gly Ala Asp Asp Leu
90 95 100
Thr Pro Phe Gly Glu Asn Gln Met Val Asn Ser Gly Ile Lys Phe Tyr
105 110 115
Arg Arg Tyr Lys Ala Leu Ala Arg Lys Ile Val Pro Phe Ile Arg Ala
120 125 130
Ser Gly Ser Asp Arg Val Ile Ala Ser Ala Glu Lys Phe Ile Glu Gly
135 140 145 150
Phe Gln Ser Ala Lys Leu Ala Asp Pro Gly Ser Gln Pro His Gln Ala
155 160 165
Ser Pro Val Ile Asp Val Ile Ile Pro Glu Gly Ser Gly Tyr Asn Asn
170 175 180
Thr Leu Asp His Gly Thr Cys Thr Ala Phe Glu Asp Ser Glu Leu Gly
185 190 195
Asp Asp Val Glu Ala Asn Phe Thr Ala Leu Phe Ala Pro Ala Ile Arg
200 205 210
Ala Arg Leu Glu Ala Asp Leu Pro Gly Val Thr Leu Thr Asp Glu Asp
215 220 225 230
Val Val Tyr Leu Met Asp Met Cys Pro Phe Glu Thr Val Ala Arg Thr
235 240 245
Ser Asp Ala Thr Glu Leu Ser Pro Phe Cys Ala Leu Phe Thr His Asp
250 255 260
Glu Trp Arg Gln Tyr Asp Tyr Leu Gln Ser Leu Gly Lys Tyr Tyr Gly
265 270 275
Tyr Gly Ala Gly Asn Pro Leu Gly Pro Ala Gln Gly Val Gly Phe Ala
280 285 290
Asn Glu Leu Ile Ala Arg Leu Thr Arg Ser Pro Val Gln Asp His Thr
295 300 305 310
Ser Thr Asn His Thr Leu Asp Ser Asn Pro Ala Thr Phe Pro Leu Asn
315 320 325
Ala Thr Leu Tyr Ala Asp Phe Ser His Asp Asn Ser Met Ile Ser Ile
330 335 340
Phe Phe Ala Leu Gly Leu Tyr Asn Gly Thr Ala Pro Leu Ser Thr Thr
345 350 355
Ser Val Glu Ser Ile Glu Glu Thr Asp Gly Tyr Ser Ala Ser Trp Thr
360 365 370
Val Pro Phe Gly Ala Arg Ala Tyr Val Glu Met Met Gln Cys Gln Ala
375 380 385 390
Glu Lys Glu Pro Leu Val Arg Val Leu Val Asn Asp Arg Val Val Pro
395 400 405
Leu His Gly Cys Ala Val Asp Lys Leu Gly Arg Cys Lys Arg Asp Asp
410 415 420
Phe Val Glu Gly Leu Ser Phe Ala Arg Ser Gly Gly Asn Trp Ala Glu
425 430 435
Cys Phe Ala
440
<210>17
<211>422
<212>PRT
<213>Paxillus involutus phyA1
<400>17
Ser Val Pro Lys Asn Thr Ala Pro Thr Phe Pro Ile Pro Glu Ser Glu
1 5 10 15
Gln Arg Asn Trp Ser Pro Tyr Ser Pro Tyr Phe Pro Leu Ala Glu Tyr
20 25 30
Lys Ala Pro Pro Ala Gly Cys Gln Ile Asn Gln Val Asn Ile Ile Gln
35 40 45
Arg His Gly Ala Arg Phe Pro Thr Ser Gly Ala Thr Thr Arg Ile Lys
50 55 60
Ala Gly Leu Thr Lys Leu Gln Gly Val Gln Asn Phe Thr Asp Ala Lys
65 70 75 80
Phe Asn Phe Ile Lys Ser Phe Lys Tyr Asp Leu Gly Asn Ser Asp Leu
85 90 95
Val Pro Phe Gly Ala Ala Gln Ser Phe Asp Ala Gly Gln Glu Ala Phe
100 105 110
Ala Arg Tyr Ser Lys Leu Val Ser Lys Asn Asn Leu Pro Phe Ile Arg
115 120 125
Ala Asp Gly Ser Asp Arg Val Val Asp Ser Ala Thr Asn Trp Thr Ala
130 135 140
Gly Phe Ala Ser Ala Ser His Asn Thr Val Gln Pro Lys Leu Asn Leu
145 150 155 160
Ile Leu Pro Gln Thr Gly Asn Asp Thr Leu Glu Asp Asn Met Cys Pro
165 170 175
Ala Ala Gly Asp Ser Asp Pro Gln Val Asn Ala Trp Leu Ala Val Ala
180 185 190
Phe Pro Ser Ile Thr Ala Arg Leu Asn Ala Ala Ala Pro Ser Val Asn
195 200 205
Leu Thr Asp Thr Asp Ala Phe Asn Leu Val Ser Leu Cys Ala Phe Leu
210 215 220
Thr Val Ser Lys Glu Lys Lys Ser Asp Phe Cys Thr Leu Phe Glu Gly
225 230 235 240
Ile Pro Gly Ser Phe Glu Ala Phe Ala Tyr Gly Gly Asp Leu Asp Lys
245 250 255
Phe Tyr Gly Thr Gly Tyr Gly Gln Glu Leu Gly Pro Val Gln Gly Val
260 265 270
Gly Tyr Val Asn Glu Leu Ile Ala Arg Leu Thr Asn Ser Ala Val Arg
275 280 285
Asp Asn Thr Gln Thr Asn Arg Thr Leu Asp Ala Ser Pro Val Thr Phe
290 295 300
Pro Leu Asn Lys Thr Phe Tyr Ala Asp Phe Ser His Asp Asn Leu Met
305 310 315 320
Val Ala Val Phe Ser Ala Met Gly Leu Phe Arg Gln Pro Ala Pro Leu
325 330 335
Ser Thr Ser Val Pro Asn Pro Trp Arg Thr Trp Arg Thr Ser Ser Leu
340 345 350
Val Pro Phe Ser Gly Arg Met Val Val Glu Arg Leu Ser Cys Phe Gly
355 360 365
Thr Thr Lys Val Arg Val Leu Val Gln Asp Gln Val Gln Pro Leu Glu
370 375 380
Phe Cys Gly Gly Asp Arg Asn Gly Leu Cys Thr Leu Ala Lys Phe Val
385 390 395 400
Glu Ser Gln Thr Phe Ala Arg Ser Asp Gly Ala Gly Asp Phe Glu Lys
405 410 415
Cys Phe Ala Thr Ser Ala
420
<210>18
<211>422
<212> PRT
<213>Paxillus involutus phyA2
<400>18
Ser Val Pro Arg Asn Ile Ala Pro Lys Phe Ser Ile Pro Glu Ser Glu
1 5 10 15
Gln Arg Asn Trp Ser Pro Tyr Ser Pro Tyr Phe Pro Leu Ala Glu Tyr
20 25 30
Lys Ala Pro Pro Ala Gly Cys Glu Ile Asn Gln Val Asn Ile Ile Gln
35 40 45
Arg His Gly Ala Arg Phe Pro Thr Ser Gly Ala Ala Thr Arg Ile Lys
50 55 60
Ala Gly Leu Ser Lys Leu Gln Ser Val Gln Asn Phe Thr Asp Pro Lys
65 70 75 80
Phe Asp Phe Ile Lys Ser Phe Thr Tyr Asp Leu Gly Thr Ser Asp Leu
85 90 95
Val Pro Phe Gly Ala Ala Gln Ser Phe Asp Ala Gly Leu Glu Val Phe
100 105 110
Ala Arg Tyr Ser Lys Leu Val Ser Ser Asp Asn Leu Pro Phe Ile Arg
115 120 125
Ser Asp Gly Ser Asp Arg Val Val Asp Thr Ala Thr Asn Trp Thr Ala
130 135 140
Gly Phe Ala Ser Ala Ser Arg Asn Ala Ile Gln Pro Lys Leu Asp Leu
145 150 155 160
Ile Leu Pro Gln Thr Gly Asn Asp Thr Leu Glu Asp Asn Met Cys Pro
165 170 175
Ala Ala Gly Glu Ser Asp Pro Gln Val Asp Ala Trp Leu Ala Ser Ala
180 185 190
Phe Pro Ser Val Thr Ala Gln Leu Asn Ala Ala Ala Pro Gly Ala Asn
195 200 205
Leu Thr Asp Ala Asp Ala Phe Asn Leu Val Ser Leu Cys Pro Phe Met
210 215 220
Thr Val Ser Lys Glu Gln Lys Ser Asp Phe Cys Thr Leu Phe Glu Gly
225 230 235 240
Ile Pro Gly Ser Phe Glu Ala Phe Ala Tyr Ala Gly Asp Leu Asp Lys
245 250 255
Phe Tyr Gly Thr Gly Tyr Gly Gln Ala Leu Gly Pro Val Gln Gly Val
260 265 270
Gly Tyr Ile Asn Glu Leu Leu Ala Arg Leu Thr Asn Ser Ala Val Asn
275 280 285
Asp Asn Thr Gln Thr Asn Arg Thr Leu Asp Ala Ala Pro Asp Thr Phe
290 295 300
Pro Leu Asn Lys Thr Met Tyr Ala Asp Phe Ser His Asp Asn Leu Met
305 310 315 320
Val Ala Val Phe Ser Ala Met Gly Leu Phe Arg Gln Ser Ala Pro Leu
325 330 335
Ser Thr Ser Thr Pro Asp Pro Asn Arg Thr Trp Leu Thr Ser Ser Val
340 345 350
Val Pro Phe Ser Ala Arg Met Ala Val Glu Arg Leu Ser Cys Ala Gly
355 360 365
Thr Thr Lys Val Arg Val Leu Val Gln Asp Gln Val Gln Pro Leu Glu
370 375 380
Phe Cys Gly Gly Asp Gln Asp Gly Leu Cys Ala Leu Asp Lys Phe Val
385 390 395 400
Glu Ser Gln Ala Tyr Ala Arg Ser Gly Gly Ala Gly Asp Phe Glu Lys
405 410 415
Cys Leu Ala Thr Thr Val
420
<210>19
<211>420
<212>PRT
<213>绒毛栓菌
<400>19
His Ile Pro Leu Arg Asp Thr Ser Ala Cys Leu Asp Val Thr Arg Asp
1 5 10 15
Val Gln Gln Ser Trp Ser Met Tyr Ser Pro Tyr Phe Pro Ala Ala Thr
20 25 30
Tyr Val Ala Pro Pro Ala Ser Cys Gln Ile Asn Gln Val His Ile Ile
35 40 45
Gln Arg His Gly Ala Arg Phe Pro Thr Ser Gly Ala Ala Lys Arg Ile
50 55 60
Gln Thr Ala Val Ala Lys Leu Lys Ala Ala Ser Asn Tyr Thr Asp Pro
65 70 75 80
Leu Leu Ala Phe Val Thr Asn Tyr Thr Tyr Ser Leu Gly Gln Asp Ser
85 90 95
Leu Val Glu Leu Gly Ala Thr Gln Ser Ser Glu Ala Gly Gln Glu Ala
100 105 110
Phe Thr Arg Tyr Ser Ser Leu Val Ser Ala Asp Glu Leu Pro Phe Val
115 120 125
Arg Ala Ser Gly Ser Asp Arg Val Val Ala Thr Ala Asn Asn Trp Thr
130 135 140
Ala Gly Phe Ala Leu Ala Ser Ser Asn Ser Ile Thr Pro Val Leu Ser
145 150 155 160
Val Ile Ile Ser Glu Ala Gly Asn Asp Thr Leu Asp Asp Asn Met Cys
165 170 175
Pro Ala Ala Gly Asp Ser Asp Pro Gln Val Asn Gln Trp Leu Ala Gln
180 185 190
Phe Ala Pro Pro Met Thr Ala Arg Leu Asn Ala Gly Ala Pro Gly Ala
195 200 205
Asn Leu Thr Asp Thr Asp Thr Tyr Asn Leu Leu Thr Leu Cys Pro Phe
210 215 220
Glu Thr Val Ala Thr Glu Arg Arg Ser Glu Phe Cys Asp Ile Tyr Glu
225 230 235 240
Glu Leu Gln Ala Glu Asp Ala Phe Ala Tyr Asn Ala Asp Leu Asp Lys
245 250 255
Phe Tyr Gly Thr Gly Tyr Gly Gln Pro Leu Gly Pro Val Gln Gly Val
260 265 270
Gly Tyr Ile Asn Glu Leu Ile Ala Arg Leu Thr Ala Gln Asn Val Ser
275 280 285
Asp His Thr Gln Thr Asn Ser Thr Leu Asp Ser Ser Pro Glu Thr Phe
290 295 300
Pro Leu Asn Arg Thr Leu Tyr Ala Asp Phe Ser His Asp Asn Gln Met
305 310 315 320
Val Ala Ile Phe Ser Ala Met Gly Leu Phe Asn Gln Ser Ala Pro Leu
325 330 335
Asp Pro Thr Thr Pro Asp Pro Ala Arg Thr Phe Leu Val Lys Lys Ile
340 345 350
Val Pro Phe Ser Ala Arg Met Val Val Glu Arg Leu Asp Cys Gly Gly
355 360 365
Ala Gln Ser Val Arg Leu Leu Val Asn Asp Ala Val Gln Pro Leu Ala
370 375 380
Phe Cys Gly Ala Asp Thr Ser Gly Val Cys Thr Leu Asp Ala Phe Val
385 390 395 400
Glu Ser Gln Ala Tyr Ala Arg Asn Asp Gly Glu Gly Asp Phe Glu Lys
405 410 415
Cys Phe Ala Thr
420
<210>20
<211>435
<212>PRT
<213>平田头菇
<400>20
Gly Gly Val Val Gln Ala Thr Phe Val Gln Pro Phe Phe Pro Pro Gln
1 5 10 15
Ile Gln Asp Ser Trp Ala Ala Tyr Thr Pro Tyr Tyr Pro Val Gln Ala
20 25 30
Tyr Thr Pro Pro Pro Lys Asp Cys Lys Ile Thr Gln Val Asn Ile Ile
35 40 45
Gln Arg His Gly Ala Arg Phe Pro Thr Ser Gly Ala Gly Thr Arg Ile
50 55 60
Gln Ala Ala Val Lys Lys Leu Gln Ser Ala Lys Thr Tyr Thr Asp Pro
65 70 75 80
Arg Leu Asp Phe Leu Thr Asn Tyr Thr Tyr Thr Leu Gly His Asp Asp
85 90 95
Leu Val Pro Phe Gly Ala Leu Gln Ser Ser Gln Ala Gly Glu Glu Thr
100 105 110
Phe Gln Arg Tyr Ser Phe Leu Val Ser Lys Glu Asn Leu Pro Phe Val
115 120 125
Arg Ala Ser Ser Ser Asn Arg Val Val Asp Ser Ala Thr Asn Trp Thr
130 135 140
Glu Gly Phe Ser Ala Ala Ser His His Val Leu Asn Pro Ile Leu Phe
145 150 155 160
Val Ile Leu Ser Glu Ser Leu Asn Asp Thr Leu Asp Asp Ala Met Cys
165 170 175
Pro Asn Ala Gly Ser Ser Asp Pro Gln Thr Gly Ile Trp Thr Ser Ile
180 185 190
Tyr Gly Thr Pro Ile Ala Asn Arg Leu Asn Gln Gln Ala Pro Gly Ala
195 200 205
Asn Ile Thr Ala Ala Asp Val Ser Asn Leu Ile Pro Leu Cys Ala Phe
210 215 220
Glu Thr Ile Val Lys Glu Thr Pro Ser Pro Phe Cys Asn Leu Phe Thr
225 230 235 240
Pro Glu Glu Phe Ala Gln Phe Glu Tyr Phe Gly Asp Leu Asp Lys Phe
245 250 255
Tyr Gly Thr Gly Tyr Gly Gln Pro Leu Gly Pro Val Gln Gly Val Gly
260 265 270
Tyr Ile Asn Glu Leu Leu Ala Arg Leu Thr Glu Met Pro Val Arg Asp
275 280 285
Asn Thr Gln Thr Asn Arg Thr Leu Asp Ser Ser Pro Leu Thr Phe Pro
290 295 300
Leu Asp Arg Ser Ile Tyr Ala Asp Leu Ser His Asp Asn Gln Met Ile
305 310 315 320
Ala Ile Phe Ser Ala Met Gly Leu Phe Asn Gln Ser Ser Pro Leu Asp
325 330 335
Pro Ser Phe Pro Asn Pro Lys Arg Thr Trp Val Thr Ser Arg Leu Thr
340 345 350
Pro Phe Ser Ala Arg Met Val Thr Glu Arg Leu Leu Cys Gln Arg Asp
355 360 365
Gly Thr Gly Ser Gly Gly Pro Ser Arg Ile Met Arg Asn Gly Asn Val
370 375 380
Gln Thr Phe Val Arg Ile Leu Val Asn Asp Ala Leu Gln Pro Leu Lys
385 390 395 400
Phe Cys Gly Gly Asp Met Asp Ser Leu Cys Thr Leu Glu Ala Phe Val
405 410 415
Glu Ser Gln Lys Tyr Ala Arg Glu Asp Gly Gln Gly Asp Phe Glu Lys
420 425 430
Cys Phe Asp
435
<210>21
<211>419
<212>PRT
<213>Peniophora lycii
<400>21
Ser Thr Gln Phe Ser Phe Val Ala Ala Gln Leu Pro Ile Pro Ala Gln
1 5 10 15
Asn Thr Ser Asn Trp Gly Pro Tyr Asp Pro Phe Phe Pro Val Glu Pro
20 25 30
Tyr Ala Ala Pro Pro Glu Gly Cys Thr Val Thr Gln Val Asn Leu Ile
35 40 45
Gln Arg His Gly Ala Arg Trp Pro Thr Ser Gly Ala Arg Ser Arg Gln
50 55 60
Val Ala Ala Val Ala Lys Ile Gln Met Ala Arg Pro Phe Thr Asp Pro
65 70 75 80
Lys Tyr Glu Phe Leu Asn Asp Phe Val Tyr Lys Phe Gly Val Ala Asp
85 90 95
Leu Leu Pro Phe Gly Ala Asn Gln Ser His Gln Thr Gly Thr Asp Met
100 105 110
Tyr Thr Arg Tyr Ser Thr Leu Phe Glu Gly Gly Asp Val Pro Phe Val
115 120 125
Arg Ala Ala Gly Asp Gln Arg Val Val Asp Ser Ser Thr Asn Trp Thr
130 135 140
Ala Gly Phe Gly Asp Ala Ser Gly Glu Thr Val Leu Pro Thr Leu Gln
145 150 155 160
Val Val Leu Gln Glu Glu Gly Asn Cys Thr Leu Cys Asn Asn Met Cys
165 170 175
Pro Asn Glu Val Asp Gly Asp Glu Ser Thr Thr Trp Leu Gly Val Phe
180 185 190
Ala Pro Asn Ile Thr Ala Arg Leu Asn Ala Ala Ala Pro Ser Ala Asn
195 200 205
Leu Ser Asp Ser Asp Ala Leu Thr Leu Met Asp Met Cys Pro Phe Asp
210 215 220
Thr Leu Ser Ser Gly Asn Ala Ser Pro Phe Cys Asp Leu Phe Thr Ala
225 230 235 240
Glu Glu Tyr Val Ser Tyr Glu Tyr Tyr Tyr Asp Leu Asp Lys Tyr Tyr
245 250 255
Gly Thr Gly Pro Gly Asn Ala Leu Gly Pro Val Gln Gly Val Gly Tyr
260 265 270
Val Asn Glu Leu Leu Ala Arg Leu Thr Gly Gln Ala Val Arg Asp Glu
275 280 285
Thr Gln Thr Asn Arg Thr Leu Asp Ser Asp Pro Ala Thr Phe Pro Leu
290 295 300
Asn Arg Thr Phe Tyr Ala Asp Phe Ser His Asp Asn Thr Met Val Pro
305 310 315 320
Ile Phe Ala Ala Leu Gly Leu Phe Asn Ala Thr Ala Leu Asp Pro Leu
325 330 335
Lys Pro Asp Glu Asn Arg Leu Trp Val Asp Ser Lys Leu Val Pro Phe
340 345 350
Ser Gly His Met Thr Val Glu Lys Leu Ala Cys Ser Gly Lys Glu Ala
355 360 365
Val Arg Val Leu Val Asn Asp Ala Val Gln Pro Leu Glu Phe Cys Gly
370 375 380
Gly Val Asp Gly Val Cys Glu Leu Ser Ala Phe Val Glu Ser Gln Thr
385 390 395 400
Tyr Ala Arg Glu Asn Gly Gln Gly Asp Phe Ala Lys Cys Gly Phe Val
405 410 415
Pro Ser Glu
<210>22
<211>369
<212>PRT
<213>人工序列
<220>
<223>人工序列的描述：担子菌共有序列
<400>22
Ser Pro Arg Thr Ala Ala Gln Leu Pro Ile Pro Gln Gln Trp Ser Pro
1 5 10 15
Tyr Ser Pro Tyr Phe Pro Val Ala Tyr Ala Pro Pro Ala Gly Cys Gln
20 25 30
Ile Gln Val Asn Ile Ile Gln Arg His Gly Ala Arg Phe Pro Thr Ser
35 40 45
Gly Ala Ala Thr Arg Ile Gln Ala Ala Val Ala Lys Leu Gln Ser Ala
50 55 60
Thr Asp Pro Lys Leu Asp Phe Leu Asn Thr Tyr Leu Gly Asp Asp Leu
65 70 75 80
Val Pro Phe Gly Ala Gln Ser Ser Gln Ala Gly Gln Glu Ala Phe Thr
85 90 95
Arg Tyr Ser Leu Val Ser Asp Asn Leu Pro Phe Val Arg Ala Ser Gly
100 105 110
Ser Asp Arg Val Val Asp Ser Ala Thr Asn Trp Thr Ala Gly Phe Ala
115 120 125
Ala Ser Asn Thr Pro Leu Val Ile Leu Ser Glu Gly Asn Asp Thr Leu
130 135 140
Asp Asp Asn Met Cys Pro Ala Gly Asp Ser Asp Pro Gln Asn Trp Leu
145 150 155 160
Ala Val Phe Ala Pro Pro Ile Thr Ala Arg Leu Asn Ala Ala Ala Pro
165 170 175
Gly Ala Asn Leu Thr Asp Asp Ala Asn Leu Leu Cys Pro Phe Glu Thr
180 185 190
Val Ser Glu Ser Phe Cys Asp Leu Phe Glu Pro Glu Glu Phe Ala Phe
195 200 205
Tyr Gly Asp Leu Asp Lys Phe Tyr Gly Thr Gly Tyr Gly Gln Pro Leu
210 215 220
Gly Pro Val Gln Gly Val Gly Tyr Ile Asn Glu Leu Leu Ala Arg Leu
225 230 235 240
Thr Gln Ala Val Arg Asp Asn Thr Gln Thr Asn Arg Thr Leu Asp Ser
245 250 255
Ser Pro Thr Phe Pro Leu Asn Arg Thr Phe Tyr Ala Asp Phe Ser His
260 265 270
Asp Asn Gln Met Val Ala Ile Phe Ser Ala Met Gly Leu Phe Asn Gln
275 280 285
Ser Ala Pro Leu Asp Pro Ser Pro Asp Pro Asn Arg Thr Trp Val Thr
290 295 300
Ser Lys Leu Val Pro Phe Ser Ala Arg Met Val Val Glu Arg Leu Cys
305 310 315 320
Gly Thr Val Arg Val Leu Val Asn Asp Ala Val Gln Pro Leu Glu Phe
325 330 335
Cys Gly Gly Asp Asp Gly Cys Thr Leu Asp Ala Phe Val Glu Ser Gln
340 345 350
Tyr Ala Arg Glu Asp Gly Gln Gly Asp Phe Glu Lys Cys Phe Ala Thr
355 360 365
Pro
<210>23
<211>440
<212>PRT
<213>Thermomyces lanuginosus
<400>23
Asn Val Asp Ile Ala Arg His Trp Gly Gln Tyr Ser Pro Phe Phe Ser
1 5 10 15
Leu Ala Glu Val Ser Glu Ile Ser Pro Ala Val Pro Lys Gly Cys Arg
20 25 30
Val Glu Phe Val Gln Val Leu Ser Arg His Gly Ala Arg Tyr Pro Thr
35 40 45
Ala His Lys Ser Glu Val Tyr Ala Glu Leu Leu Gln Arg Ile Gln Asp
50 55 60
Thr Ala Thr Glu Phe Lys Gly Asp Phe Ala Phe Leu Arg Asp Tyr Ala
65 70 75 80
Tyr His Leu Gly Ala Asp Asn Leu Thr Arg Phe Gly Glu Glu Gln Met
85 90 95
Met Glu Ser Gly Arg Gln Phe Tyr His Arg Tyr Arg Glu Gln Ala Arg
100 105 110
Glu Ile Val Pro Phe Val Arg Ala Ala Gly Ser Ala Arg Val Ile Ala
115 120 125
Ser Ala Glu Phe Phe Asn Arg Gly Phe Gln Asp Ala Lys Asp Arg Asp
130 135 140
Pro Arg Ser Asn Lys Asp Gln Ala Glu Pro Val Ile Asn Val Ile Ile
145 150 155 160
Ser Glu Glu Thr Gly Ser Asn Asn Thr Leu Asp Gly Leu Thr Cys Pro
165 170 175
Ala Ala Glu Glu Ala Pro Asp Pro Thr Gln Pro Ala Glu Phe Leu Gln
180 185 190
Val Phe Gly Pro Arg Val Leu Lys Lys Ile Thr Lys His Met Pro Gly
195 200 205
Val Asn Leu Thr Leu Glu Asp Val Pro Leu Phe Met Asp Leu Cys Pro
210 215 220
Phe Asp Thr Val Gly Ser Asp Pro Val Leu Phe Pro Arg Gln Leu Ser
225 230 235 240
Pro Phe Cys His Leu Phe Thr Ala Asp Asp Trp Met Ala Tyr Asp Tyr
245 250 255
Tyr Tyr Thr Leu Asp Lys Tyr Tyr Ser His Gly Gly Gly Ser Ala Phe
260 265 270
Gly Pro Ser Arg Gly Val Gly Phe Val Asn Glu Leu Ile Ala Arg Met
275 280 285
Thr Gly Asn Leu Pro Val Lys Asp His Thr Thr Val Asn His Thr Leu
290 295 300
Asp Asp Asn Pro Glu Thr Phe Pro Leu Asp Ala Val Leu Tyr Ala Asp
305 310 315 320
Phe Ser His Asp Asn Thr Met Thr Gly Ile Phe Ser Ala Met Gly Leu
325 330 335
Tyr Asn Gly Thr Lys Pro Leu Ser Thr Ser Lys Ile Gln Pro Pro Thr
340 345 350
Gly Ala Ala Ala Asp Gly Tyr Ala Ala Ser Trp Thr Val Pro Phe Ala
355 360 365
Ala Arg Ala Tyr Val Glu Leu Leu Arg Cys Glu Thr Glu Thr Ser Ser
370 375 380
Glu Glu Glu Glu Glu Gly Glu Asp Glu Pro Phe Val Arg Val Leu Val
385 390 395 400
Asn Asp Arg Val Val Pro Leu His Gly Cys Arg Val Asp Arg Trp Gly
405 410 415
Arg Cys Arg Arg Asp Glu Trp Ile Lys Gly Leu Thr Phe Ala Arg Gln
420 425 430
Gly Gly His Trp Asp Arg Cys Phe
435 440
<210>24
<211>441
<212>PRT
<213>人工序列
<220>
<223>人工序列的描述：共有序列10
<400>24
Asn Ser His Ser Cys Asp Thr Val Asp Gly Gly Tyr Gln Cys Phe Pro
1 5 10 15
Glu Ile Ser His Leu Trp Gly Gln Tyr Ser Pro Phe Phe Ser Leu Ala
20 25 30
Asp Glu Ser Ala Ile Ser Pro Asp Val Pro Lys Gly Cys Arg Val Thr
35 40 45
Phe Val Gln Val Leu Ser Arg His Gly Ala Arg Tyr Pro Thr Ser Ser
50 55 60
Lys Ser Lys Lys Tyr Ser Ala Leu Ile Glu Ala Ile Gln Lys Asn Ala
65 70 75 80
Thr Ala Phe Lys Gly Lys Tyr Ala Phe Leu Lys Thr Tyr Asn Tyr Thr
85 90 95
Leu Gly Ala Asp Asp Leu Thr Pro Phe Gly Glu Gln Gln Met Val Asn
100 105 110
Ser Gly Ile Lys Phe Tyr Arg Arg Tyr Lys Ala Leu Ala Arg Lys Ile
115 120 125
Val Pro Phe Val Arg Ala Ser Gly Ser Asp Arg Val Ile Ala Ser Ala
130 135 140
Glu Lys Phe Ile Glu Gly Phe Gln Ser Ala Lys Leu Ala Asp Pro Gly
145 150 155 160
Ala Asn Pro His Gln Ala Ser Pro Val Ile Asn Val Ile Ile Pro Glu
165 170 175
Gly Ala Gly Tyr Asn Asn Thr Leu Asp His Gly Leu Cys Thr Ala Phe
180 185 190
Glu Glu Ser Glu Leu Gly Asp Asp Val Glu Ala Asn Phe Thr Ala Val
195 200 205
Phe Ala Pro Pro Ile Arg Ala Arg Leu Glu Ala His Leu Pro Gly Val
210 215 220
Asn Leu Thr Asp Glu Asp Val Val Asn Leu Met Asp Met Cys Pro Phe
225 230 235 240
Asp Thr Val Ala Arg Thr Ser Asp Ala Thr Gln Leu Ser Pro Phe Cys
245 250 255
Asp Leu Phe Thr His Asp Glu Trp Ile Gln Tyr Asp Tyr Leu Gln Ser
260 265 270
Leu Gly Lys Tyr Tyr Gly Tyr Gly Ala Gly Asn Pro Leu Gly Pro Ala
275 280 285
Gln Gly Val Gly Phe Val Asn Glu Leu Ile Ala Arg Leu Thr His Ser
290 295 300
Pro Val Gln Asp His Thr Ser Thr Asn His Thr Leu Asp Ser Asn Pro
305 310 315 320
Ala Thr Phe Pro Leu Asn Ala Thr Leu Tyr Ala Asp Phe Ser His Asp
325 330 335
Asn Thr Met Val Ser Ile Phe Phe Ala Leu Gly Leu Tyr Asn Gly Thr
340 345 350
Lys Pro Leu Ser Thr Thr Ser Val Glu Ser Ile Glu Glu Thr Asp Gly
355 360 365
Tyr Ala Ala Ser Trp Thr Val Pro Phe Ala Ala Arg Ala Tyr Val Glu
370 375 380
Met Met Gln Cys Glu Ala Glu Lys Glu Pro Leu Val Arg Val Leu Val
385 390 395 400
Asn Asp Arg Val Val Pro Leu His Gly Cys Gly Val Asp Lys Leu Gly
405 410 415
Arg Cys Lys Arg Asp Asp Phe Val Glu Gly Leu Ser Phe Ala Arg Ser
420 425 430
Gly Gly Asn Trp Glu Glu Cys Phe Ala
435 440
<210>25
<211>1426
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述：共有植酸酶10
<220>
<221>CDS
<222>(12)..(1412)
<220>
<221>成熟肽
<222>(90)..(1412)
<220>
<221>单肽
<222>(12)..(89)
<400>25
tatatgaatt c atg ggc gtg ttc gtc gtg cta ctg tcc att gcc acc ttg 50
Met Gly Val Phe Val Val Leu Leu Ser Ile Ala Thr Leu
-25 -20 -15
ttc ggt tcc aca tcc ggt acc gcc ttg ggt cct cgt ggt aat tct cac 98
Phe Gly Ser Thr Ser Gly Thr Ala Leu Gly Pro Arg Gly Asn Ser His
-10 -5 -1 1
tct tgt gac act gtt gac ggt ggt tac caa tgt ttc cca gaa att tct 146
Ser Cys Asp Thr Val Asp Gly Gly Tyr Gln Cys Phe Pro Glu Ile Ser
5 10 15
cac ttg tgg ggt caa tac tct cca ttc ttc tct ttg gct gac gaa tct 194
His Leu Trp Gly Gln Tyr Ser Pro Phe Phe Ser Leu Ala Asp Glu Ser
20 25 30 35
gct att tct cca gac gtt cca aag ggt tgt aga gtt act ttc gtt caa 242
Ala Ile Ser Pro Asp Val Pro Lys Gly Cys Arg Val Thr Phe Val Gln
40 45 50
gtt ttg tct aga cac ggt gct aga tac cca act tct tct aag tct aag 290
Val Leu Ser Arg His Gly Ala Arg Tyr Pro Thr Ser Ser Lys Ser Lys
55 60 65
aag tac tct gct ttg att gaa gct att caa aag aac gct act gct ttc 338
Lys Tyr Ser Ala Leu Ile Glu Ala Ile Gln Lys Asn Ala Thr Ala Phe
70 75 80
aag ggt aag tac gct ttc ttg aag act tac aac tac act ttg ggt gct 386
Lys Gly Lys Tyr Ala Phe Leu Lys Thr Tyr Asn Tyr Thr Leu Gly Ala
85 90 95
gac gac ttg act cca ttc ggt gaa caa caa atg gtt aac tct ggt att 434
Asp Asp Leu Thr Pro Phe Gly Glu Gln Gln Met Val Asn Ser Gly Ile
100 105 110 115
aag ttc tac aga aga tac aag gct ttg gct aga aag att gtt cca ttc 482
Lys Phe Tyr Arg Arg Tyr Lys Ala Leu Ala Arg Lys Ile Val Pro Phe
120 125 130
gtt aga gct tct ggt tct gac aga gtt att gct tct gct gaa aag ttc 530
Val Arg Ala Ser Gly Ser Asp Arg Val Ile Ala Ser Ala Glu Lys Phe
135 140 145
att gaa ggt ttc caa tct gct aag ttg gct gac cca ggt gct aac cca 578
Ile Glu Gly Phe Gln Ser Ala Lys Leu Ala Asp Pro Gly Ala Asn Pro
150 155 160
cac caa gct tct cca gtt att aac gtt att att cca gaa ggt gct ggt 626
His Gln Ala Ser Pro Val Ile Asn Val Ile Ile Pro Glu Gly Ala Gly
165 170 175
tac aac aac act ttg gac cac ggt ttg tgt act gct ttc gaa gaa tct 674
Tyr Asn Asn Thr Leu Asp His Gly Leu Cys Thr Ala Phe Glu Glu Ser
180 185 190 195
gaa ttg ggt gac gac gtt gaa gct aac ttc act gct gtt ttc gct cca 722
Glu Leu Gly Asp Asp Val Glu Ala Asn Phe Thr Ala Val Phe Ala Pro
200 205 210
cct att aga gct aga ttg gaa gct cac ttg cca ggt gtt aac ttg act 770
Pro Ile Arg Ala Arg Leu Glu Ala His Leu Pro Gly Val Asn Leu Thr
215 220 225
gac gaa gac gtt gtt aac ttg atg gac atg tgt cca ttc gac act gtt 818
Asp Glu Asp Val Val Asn Leu Met Asp Met Cys Pro Phe Asp Thr Val
230 235 240
gct aga act tct gac gct act caa ttg tct cca ttc tgt gac ttg ttc 866
Ala Arg Thr Ser Asp Ala Thr Gln Leu Ser Pro Phe Cys Asp Leu Phe
245 250 255
act cac gac gaa tgg att caa tac gac tac ttg caa tct ttg ggt aag 914
Thr His Asp Glu Trp Ile Gln Tyr Asp Tyr Leu Gln Ser Leu Gly Lys
260 265 270 275
tac tac ggt tac ggt gct ggt aac cca ttg ggt cca gct caa ggt gtt 962
Tyr Tyr Gly Tyr Gly Ala Gly Asn Pro Leu Gly Pro Ala Gln Gly Val
280 285 290
ggt ttc gtt aac gaa ttg att gct aga ttg act cac tct cca gtt caa 1010
Gly Phe Val Asn Glu Leu Ile Ala Arg Leu Thr His Ser Pro Val Gln
295 300 305
gac cac act tct act aac cac act ttg gac tct aac cca gct act ttc 1058
Asp His Thr Ser Thr Asn His Thr Leu Asp Ser Asn Pro Ala Thr Phe
310 315 320
cca ttg aac gct act ttg tac gct gac ttc tct cac gac aac act atg 1106
Pro Leu Asn Ala Thr Leu Tyr Ala Asp Phe Ser His Asp Asn Thr Met
325 330 335
gtt tct att ttc ttc gct ttg ggt ttg tac aac ggt act aag cca ttg 1154
Val Ser Ile Phe Phe Ala Leu Gly Leu Tyr Asn Gly Thr Lys Pro Leu
340 345 350 355
tct act act tct gtt gaa tct att gaa gaa act gac ggt tac gct gct 1202
Ser Thr Thr Ser Val Glu Ser Ile Glu Glu Thr Asp Gly Tyr Ala Ala
360 365 370
tct tgg act gtt cca ttc gct gct aga gct tac gtt gaa atg atg caa 1250
Ser Trp Thr Val Pro Phe Ala Ala Arg Ala Tyr Val Glu Met Met Gln
375 380 385
tgt gaa gct gaa aag gaa cca ttg gtt aga gtt ttg gtt aac gac aga 1298
Cys Glu Ala Glu Lys Glu Pro Leu Val Arg Val Leu Val Asn Asp Arg
390 395 400
gtt gtt cca ttg cac ggt tgt ggt gtt gac aag ttg ggt aga tgt aag 1346
Val Val Pro Leu His Gly Cys Gly Val Asp Lys Leu Gly Arg Cys Lys
405 410 415
aga gac gac ttc gtt gaa ggt ttg tct ttc gct aga tct ggt ggt aac 1394
Arg Asp Asp Phe Val Glu Gly Leu Ser Phe Ala Arg Ser Gly Gly Asn
420 425 430 435
tgg gaa gaa tgt ttc gct taagaattca tata 1426
Trp Glu Glu Cys Phe Ala
440
<210>26
<211>467
<212>PRT
<213>人工序列
<223>人工序列的描述：共有植酸酶10
<400> 26
Met Gly Val Phe Val Val Leu Leu Ser Ile Ala Thr Leu Phe Gly Ser
-25 -20 -15
Thr Ser Gly Thr Ala Leu Gly Pro Arg Gly Asn Ser His Ser Cys Asp
-10 -5 -1 1 5
Thr Val Asp Gly Gly Tyr Gln Cys Phe Pro Glu Ile Ser His Leu Trp
10 15 20
Gly Gln Tyr Ser Pro Phe Phe Ser Leu Ala Asp Glu Ser Ala Ile Ser
25 30 35
Pro Asp Val Pro Lys Gly Cys Arg Val Thr Phe Val Gln Val Leu Ser
40 45 50
Arg His Gly Ala Arg Tyr Pro Thr Ser Ser Lys Ser Lys Lys Tyr Ser
55 60 65 70
Ala Leu Ile Glu Ala Ile Gln Lys Asn Ala Thr Ala Phe Lys Gly Lys
75 80 85
Tyr Ala Phe Leu Lys Thr Tyr Asn Tyr Thr Leu Gly Ala Asp Asp Leu
90 95 100
Thr Pro Phe Gly Glu Gln Gln Met Val Asn Ser Gly Ile Lys Phe Tyr
105 110 115
Arg Arg Tyr Lys Ala Leu Ala Arg Lys Ile Val Pro Phe Val Arg Ala
120 125 130
Ser Gly Ser Asp Arg Val Ile Ala Ser Ala Glu Lys Phe Ile Glu Gly
135 140 145 150
Phe Gln Ser Ala Lys Leu Ala Asp Pro Gly Ala Asn Pro His Gln Ala
155 160 165
Ser Pro Val Ile Asn Val Ile Ile Pro Glu Gly Ala Gly Tyr Asn Asn
170 175 180
Thr Leu Asp His Gly Leu Cys Thr Ala Phe Glu Glu Ser Glu Leu Gly
185 190 195
Asp Asp Val Glu Ala Asn Phe Thr Ala Val Phe Ala Pro Pro Ile Arg
200 205 210
Ala Arg Leu Glu Ala His Leu Pro Gly Val Asn Leu Thr Asp Glu Asp
215 220 225 230
Val Val Asn Leu Met Asp Met Cys Pro Phe Asp Thr Val Ala Arg Thr
235 240 245
Ser Asp Ala Thr Gln Leu Ser Pro Phe Cys Asp Leu Phe Thr His Asp
250 255 260
Glu Trp Ile Gln Tyr Asp Tyr Leu Gln Ser Leu Gly Lys Tyr Tyr Gly
265 270 275
Tyr Gly Ala Gly Asn Pro Leu Gly Pro Ala Gln Gly Val Gly Phe Val
280 285 290
Asn Glu Leu Ile Ala Arg Leu Thr His Ser Pro Val Gln Asp His Thr
295 300 305 310
Ser Thr Asn His Thr Leu Asp Ser Asn Pro Ala Thr Phe Pro Leu Asn
315 320 325
Ala Thr Leu Tyr Ala Asp Phe Ser His Asp Asn Thr Met Val Ser Ile
330 335 340
Phe Phe Ala Leu Gly Leu Tyr Asn Gly Thr Lys Pro Leu Ser Thr Thr
345 350 355
Ser Val Glu Ser Ile Glu Glu Thr Asp Gly Tyr Ala Ala Ser Trp Thr
360 365 370
Val Pro Phe Ala Ala Arg Ala Tyr Val Glu Met Met Gln Cys Glu Ala
375 380 385 390
Glu Lys Glu Pro Leu Val Arg Val Leu Val Asn Asp Arg Val Val Pro
395 400 405
Leu His Gly Cys Gly Val Asp Lys Leu Gly Arg Cys Lys Arg Asp Asp
410 415 420
Phe Val Glu Gly Leu Ser Phe Ala Arg Ser Gly Gly Asn Trp Glu Glu
425 430 435
Cys Phe Ala
440
<210>27
<211>437
<212>PRT
<213>人工序列
<220>
<223>人工序列的描述：共有植酸酶ll
<400>27
Asn Ser His Ser Cys Asp Thr Val Asp Gly Tyr Gln Cys Pro Glu Ile
1 5 10 15
Ser His Leu Trp Gly Gln Tyr Ser Pro Phe Phe Ser Leu Ala Asp Glu
20 25 30
Ser Ala Ile Ser Pro Asp Val Pro Lys Gly Cys Arg Val Thr Phe Val
35 40 45
Gln Val Leu Ser Arg His Gly Ala Arg Tyr Pro Thr Ser Ser Lys Ser
50 55 60
Lys Lys Tyr Ser Ala Leu Ile Glu Arg Ile Gln Lys Asn Ala Thr Phe
65 70 75 80
Lys Gly Lys Tyr Ala Phe Leu Lys Thr Tyr Asn Tyr Thr Leu Gly Ala
85 90 95
Asp Asp Leu Thr Pro Phe Gly Glu Asn Gln Met Val Asn Ser Gly Ile
100 105 110
Lys Phe Tyr Arg Arg Tyr Lys Ala Leu Ala Arg Asn Ile Val Pro Phe
115 120 125
Val Arg Ala Ser Gly Ser Asp Arg Val Ile Ala Ser Ala Glu Lys Phe
130 135 140
Ile Glu Gly Phe Gln Ser Ala Lys Leu Ala Asp Pro Ala His Gln Ala
145 150 155 160
Ser Pro Val Ile Asn Val Ile Ile Pro Glu Gly Ser Gly Tyr Asn Asn
165 170 175
Thr Leu Asp His Gly Leu Cys Thr Ala Phe Glu Asp Ser Thr Leu Gly
180 185 190
Asp Asp Ala Glu Ala Asn Phe Thr Ala Val Phe Ala Pro Pro Ile Arg
195 200 205
Ala Arg Leu Glu Ala Leu Pro Gly Val Asn Leu Thr Asp Glu Asp Val
210 215 220
Val Asn Leu Met Asp Met Cys Pro Phe Asp Thr Val Ala Arg Thr Ser
225 230 235 240
Asp Ala Thr Gln Leu Ser Pro Phe Cys Asp Leu Phe Thr Ala Asp Glu
245 250 255
Trp Gln Tyr Asp Tyr Leu Gln Ser Leu Lys Tyr Tyr Gly Tyr Gly Ala
260 265 270
Gly Asn Pro Leu Gly Pro Ala Gln Gly Val Gly Phe Asn Glu Leu Ile
275 280 285
Ala Arg Leu Thr His Ser Pro Val Gln Asp His Thr Ser Thr Asn His
290 295 300
Thr Leu Asp Ser Asn Pro Ala Thr Phe Pro Leu Asn Ala Thr Leu Tyr
305 310 315 320
Ala Asp Phe Ser His Asp Asn Thr Met Val Ser Ile Phe Phe Ala Leu
325 330 335
Gly Leu Tyr Asn Gly Thr Lys Pro Leu Ser Thr Thr Ser Val Glu Ser
340 345 350
Ile Glu Thr Asp Gly Tyr Ala Ala Ser Trp Thr Val Pro Phe Ala Ala
355 360 365
Arg Ala Tyr Val Glu Met Met Gln Cys Glu Ala Gly Gly Gly Gly Gly
370 375 380
Glu Gly Glu Lys Glu Pro Leu Val Arg Val Leu Val Asn Asp Arg Val
385 390 395 400
Val Pro Leu His Gly Cys Gly Val Asp Lys Leu Gly Arg Cys Lys Leu
405 410 415
Asp Asp Phe Val Glu Gly Leu Ser Phe Ala Arg Ser Gly Gly Asn Trp
420 425 430
Ala Glu Cys Phe Ala
435
<210>28
<211>1404
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述：共有植酸酶1 thermo 8 q50t，k91a
<220>
<221>CDS
<222>(1)..(1401)
<220>
<221>成熟肽
<222>(79)..(1401)
<220>
<221>单肽
<222>(1)..(78)
<400>28
atg ggc gtg ttc gtc gtg cta ctg tcc att gcc acc ttg ttc ggt tcc 48
Met Gly Val Phe Val Val Leu Leu Ser Ile Ala Thr Leu Phe Gly Ser
-25 -20 -15
aca tcc ggt acc gcc ttg ggt cct cgt ggt aat tct cac tct tgt gac 96
Thr Ser Gly Thr Ala Leu Gly Pro Arg Gly Asn Ser His Ser Cys Asp
-10 -5 -1 1 5
act gtt gac ggt ggt tac caa tgt ttc cca gaa att tct cac ttg tgg 144
Thr Val Asp Gly Gly Tyr Gln Cys Phe Pro Glu Ile Ser His Leu Trp
10 15 20
ggt acc tac tct cca tac ttc tct ttg gca gac gaa tct gct att tct 192
Gly Thr Tyr Ser Pro Tyr Phe Ser Leu Ala Asp Glu Ser Ala Ile Ser
25 30 35
cca gac gtt cca gac gac tgt aga gtt act ttc gtt caa gtt ttg tct 240
Pro Asp Val Pro Asp Asp Cys Arg Val Thr Phe Val Gln Val Leu Ser
40 45 50
aga cac ggt gct aga tac cca act tct tct gcg tct aag gct tac tct 288
Arg His Gly Ala Arg Tyr Pro Thr Ser Ser Ala Ser Lys Ala Tyr Ser
55 60 65 70
gct ttg att gaa gct att caa aag aac gct act gct ttc aag ggt aag 336
Ala Leu Ile Glu Ala Ile Gln Lys Asn Ala Thr Ala Phe Lys Gly Lys
75 80 85
tac gct ttc ttg aag act tac aac tac act ttg ggt gct gac gac ttg 384
Tyr Ala Phe Leu Lys Thr Tyr Asn Tyr Thr Leu Gly Ala Asp Asp Leu
90 95 100
act cca ttc ggt gaa aac caa atg gtt aac tct ggt att aag ttc tac 432
Thr Pro Phe Gly Glu Asn Gln Met Val Asn Ser Gly Ile Lys Phe Tyr
105 110 115
aga aga tac aag gct ttg gct aga aag att gtt cca ttc att aga gct 480
Arg Arg Tyr Lys Ala Leu Ala Arg Lys Ile Val Pro Phe Ile Arg Ala
120 125 130
tct ggt tct gac aga gtt att gct tct gct gaa aag ttc att gaa ggt 528
Ser Gly Ser Asp Arg Val Ile Ala Ser Ala Glu Lys Phe Ile Glu Gly
135 140 145 150
ttc caa tct gct aag ttg gct gac cca ggt tct caa cca cac caa gct 576
Phe Gln Ser Ala Lys Leu Ala Asp Pro Gly Ser Gln Pro His Gln Ala
155 160 165
tct cca gtt att aac gtg atc att cca gaa gga tcc ggt tac aac aac 624
Ser Pro Val Ile Asn Val Ile Ile Pro Glu Gly Ser Gly Tyr Asn Asn
170 175 180
act ttg gac cac ggt act tgt act gct ttc gaa gac tct gaa tta ggt 672
Thr Leu Asp His Gly Thr Cys Thr Ala Phe Glu Asp Ser Glu Leu Gly
185 190 195
gac gac gtt gaa gct aac ttc act gct ttg ttc gct cca gct att aga 720
Asp Asp Val Glu Ala Asn Phe Thr Ala Leu Phe Ala Pro Ala Ile Arg
200 205 210
gct aga ttg gaa gct gac ttg cca ggt gtt act ttg act gac gaa gac 768
Ala Arg Leu Glu Ala Asp Leu Pro Gly Val Thr Leu Thr Asp Glu Asp
215 220 225 230
gtt gtt tac ttg atg gac atg tgt cca ttc gac act gtc gct aga act 816
Val Val Tyr Leu Met Asp Met Cys Pro Phe Asp Thr Val Ala Arg Thr
235 240 245
tct gac gct act gaa ttg tct cca ttc tgt gct ttg ttc act cac gac 864
Ser Asp Ala Thr Glu Leu Ser Pro Phe Cys Ala Leu Phe Thr His Asp
250 255 260
gaa tgg atc caa tac gac tac ttg caa agc ttg ggt aag tac tac ggt 912
Glu Trp Ile Gln Tyr Asp Tyr Leu Gln Ser Leu Gly Lys Tyr Tyr Gly
265 270 275
tac ggt gct ggt aac cca ttg ggt cca gct caa ggt gtt ggt ttc gct 960
Tyr Gly Ala Gly Asn Pro Leu Gly Pro Ala Gln Gly Val Gly Phe Ala
280 285 290
aac gaa ttg att gct aga ttg act cac tct cca gtt caa gac cac act 1008
Asn Glu Leu Ile Ala Arg Leu Thr His Ser Pro Val Gln Asp His Thr
295 300 305 310
tct act aac cac act ttg gac tct aac cca gct act ttc cca ttg aac 1056
Ser Thr Asn His Thr Leu Asp Ser Asn Pro Ala Thr Phe Pro Leu Asn
315 320 325
gct act ttg tac gct gac ttc tct cac gac aac act atg ata tct att 1104
Ala Thr Leu Tyr Ala Asp Phe Ser His Asp Asn Thr Met Ile Ser Ile
330 335 340
ttc ttc gct ttg ggt ttg tac aac ggt acc aag cca ttg tct act act 1152
Phe Phe Ala Leu Gly Leu Tyr Asn Gly Thr Lys Pro Leu Ser Thr Thr
345 350 355
tct gtt gaa tct att gaa gaa act gac ggt tac tct gct tct tgg act 1200
Ser Val Glu Ser Ile Glu Glu Thr Asp Gly Tyr Ser Ala Ser Trp Thr
360 365 370
gtt cca ttc gct gct aga gct tac gtt gaa atg atg caa tgt caa gct 1248
Val Pro Phe Ala Ala Arg Ala Tyr Val Glu Met Met Gln Cys Gln Ala
375 380 385 390
gaa aag gaa cca ttg gtt aga gtt ttg gtt aac gac aga gtt gtt cca 1296
Glu Lys Glu Pro Leu Val Arg Val Leu Val Asn Asp Arg Val Val Pro
395 400 405
ttg cac ggt tgt gct gtt gac aag ttg ggt aga tgt aag aga gac gac 1344
Leu His Gly Cys Ala Val Asp Lys Leu Gly Arg Cys Lys Arg Asp Asp
410 415 420
ttc gtt gaa ggt ttg tct ttc gct aga tct ggt ggt aac tgg gct gaa 1392
Phe Val Glu Gly Leu Ser Phe Ala Arg Ser Gly Gly Asn Trp Ala Glu
425 430 435
tgt ttc gct taa 1404
Cys Phe Ala
440
<210>29
<211>467
<212>PRT
<213>人工序列
<223>人工序列的描述：共有植酸酶1 thermo 8 q50t，k91a
<400>29
Met Gly Val Phe Val Val Leu Leu Ser Ile Ala Thr Leu Phe Gly Ser
-25 -20 -15
Thr Ser Gly Thr Ala Leu Gly Pro Arg Gly Asn Ser His Ser Cys Asp
-10 -5 -1 1 5
Thr Val Asp Gly Gly Tyr Gln Cys Phe Pro Glu Ile Ser His Leu Trp
10 15 20
Gly Thr Tyr Ser Pro Tyr Phe Ser Leu Ala Asp Glu Ser Ala Ile Ser
25 30 35
Pro Asp Val Pro Asp Asp Cys Arg Val Thr Phe Val Gln Val Leu Ser
40 45 50
Arg His Gly Ala Arg Tyr Pro Thr Ser Ser Ala Ser Lys Ala Tyr Ser
55 60 65 70
Ala Leu Ile Glu Ala Ile Gln Lys Asn Ala Thr Ala Phe Lys Gly Lys
75 80 85
Tyr Ala Phe Leu Lys Thr Tyr Asn Tyr Thr Leu Gly Ala Asp Asp Leu
90 95 100
Thr Pro Phe Gly Glu Asn Gln Met Val Asn Ser Gly Ile Lys Phe Tyr
105 110 115
Arg Arg Tyr Lys Ala Leu Ala Arg Lys Ile Val Pro Phe Ile Arg Ala
120 125 130
Ser Gly Ser Asp Arg Val Ile Ala Ser Ala Glu Lys Phe Ile Glu Gly
135 140 145 150
Phe Gln Ser Ala Lys Leu Ala Asp Pro Gly Ser Gln Pro His Gln Ala
155 160 165
Ser Pro Val Ile Asn Val Ile Ile Pro Glu Gly Ser Gly Tyr Asn Asn
170 175 180
Thr Leu Asp His Gly Thr Cys Thr Ala Phe Glu Asp Ser Glu Leu Gly
185 190 195
Asp Asp Val Glu Ala Asn Phe Thr Ala Leu Phe Ala Pro Ala Ile Arg
200 205 210
Ala Arg Leu Glu Ala Asp Leu Pro Gly Val Thr Leu Thr Asp Glu Asp
215 220 225 230
Val Val Tyr Leu Met Asp Met Cys Pro Phe Asp Thr Val Ala Arg Thr
235 240 245
Ser Asp Ala Thr Glu Leu Ser Pro Phe Cys Ala Leu Phe Thr His Asp
250 255 260
Glu Trp Ile Gln Tyr Asp Tyr Leu Gln Ser Leu Gly Lys Tyr Tyr Gly
265 270 275
Tyr Gly Ala Gly Asn Pro Leu Gly Pro Ala Gln Gly Val Gly Phe Ala
280 285 290
Asn Glu Leu Ile Ala Arg Leu Thr His Ser Pro Val Gln Asp His Thr
295 300 305 310
Ser Thr Asn His Thr Leu Asp Ser Asn Pro Ala Thr Phe Pro Leu Asn
315 320 325
Ala Thr Leu Tyr Ala Asp Phe Ser His Asp Asn Thr Met Ile Ser Ile
330 335 340
Phe Phe Ala Leu Gly Leu Tyr Asn Gly Thr Lys Pro Leu Sar Thr Thr
345 350 355
Ser Val Glu Ser Ile Glu Glu Thr Asp Gly Tyr Ser Ala Ser Trp Thr
360 365 370
Val Pro Phe Ala Ala Arg Ala Tyr Val Glu Met Met Gln Cys Gln Ala
375 380 385 390
Glu Lys Glu Pro Leu Val Arg Val Leu Val Asn Asp Arg Val Val Pro
395 400 405
Leu His Gly Cys Ala Val Asp Lys Leu Gly Arg Cys Lys Arg Asp Asp
410 415 420
Phe Val Glu Gly Leu Ser Phe Ala Arg Ser Gly Gly Asn Trp Ala Glu
425 430 435
Cys Phe Ala
440
<210>30
<211>1404
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述：共有植酸酶10 thermo 3 q50t，k91a
<220>
<221>CDS
<222>(1)..(1401)
<220>
<221>成熟肽
<222>(79)..(1401)
<220>
<221>单肽
<222>(1)..(78)
<400>30
atg ggc gtg ttc gtc gtg cta ctg tcc att gcc acc ttg ttc ggt tcc 48
Met Gly Val Phe Val Val Leu Leu Ser Ile Ala Thr Leu Phe Gly Ser
-25 -20 -15
aca tcc ggt acc gcc ttg ggt cct cgt ggt aac tct cac tct tgt gac 96
Thr Ser Gly Thr Ala Leu Gly Pro Arg Gly Asn Ser His Ser Cys Asp
-10 -5 -1 1 5
act gtt gac ggt ggt tac caa tgt ttc cca gaa att tct cac ttg tgg 144
Thr Val Asp Gly Gly Tyr Gln Cys Phe Pro Glu Ile Ser His Leu Trp
10 15 20
ggt aca tac tct cca ttc ttc tct ttg gct gac gaa tct gct att tct 192
Gly Thr Tyr Ser Pro Phe Phe Ser Leu Ala Asp Glu Ser Ala Ile Ser
25 30 35
cca gac gtt cca aag ggt tgt aga gtt act ttc gtt caa gtt ttg tct 240
Pro Asp Val Pro Lys Gly Cys Arg Val Thr Phe Val Gln Val Leu Ser
40 45 50
aga cac ggt gct aga tac cca act tct tct gcg tct aag gcg tac tct 288
Arg His Gly Ala Arg Tyr Pro Thr Ser Ser Ala Ser Lys Ala Tyr Ser
55 60 65 70
gct ttg att gaa gct att caa aag aac gct act gct ttc aag ggt aag 336
Ala Leu Ile Glu Ala Ile Gln Lys Asn Ala Thr Ala Phe Lys Gly Lys
75 80 85
tac gct ttc ttg aag act tac aac tac act ttg ggt gct gac gac ttg 384
Tyr Ala Phe Leu Lys Thr Tyr Asn Tyr Thr Leu Gly Ala Asp Asp Leu
90 95 100
act cca ttc ggt gaa caa caa atg gtt aac tct ggt att aag ttc tac 432
Thr Pro Phe Gly Glu Gln Gln Met Val Asn Ser Gly Ile Lys Phe Tyr
105 110 115
aga aga tac aag gct ttg gct aga aag att gtt cca ttc att aga gct 480
Arg Arg Tyr Lys Ala Leu Ala Arg Lys Ile Val Pro Phe Ile Arg Ala
120 125 130
tct ggt tct gac aga gtt att gct tct gct gaa aag ttc att gaa ggt 528
Ser Gly Ser Asp Arg Val Ile Ala Ser Ala Glu Lys Phe Ile Glu Gly
135 140 145 150
ttc caa tct gct aag ttg gct gac cca ggt gct aac cca cac caa gct 576
Phe Gln Ser Ala Lys Leu Ala Asp Pro Gly Ala Asn Pro His Gln Ala
155 160 165
tct cca gtt att aac gtt att att cca gaa ggt gct ggt tac aac aac 624
Ser Pro Val Ile Asn Val Ile Ile Pro Glu Gly Ala Gly Tyr Asn Asn
170 175 180
act ttg gac cac ggt ttg tgt act gct ttc gaa gaa tct gaa ttg ggt 672
Thr Leu Asp His Gly Leu Cys Thr Ala Phe Glu Glu Ser Glu Leu Gly
185 190 195
gac gac gtt gaa gct aac ttc act gct gtt ttc gct cca cca att aga 720
Asp Asp Val Glu Ala Asn Phe Thr Ala Val Phe Ala Pro Pro Ile Arg
200 205 210
gct aga ttg gaa gct cac ttg cca ggt gtt aac ttg act gac gaa gac 768
Ala Arg Leu Glu Ala His Leu Pro Gly Val Asn Leu Thr Asp Glu Asp
215 220 225 230
gtt gtt aac ttg atg gac atg tgt cca ttc gac act gtt gct aga act 816
Val Val Asn Leu Met Asp Met Cys Pro Phe Asp Thr Val Ala Arg Thr
235 240 245
tct gac gct act caa ttg tct cca ttc tgt gac ttg ttc act cac gac 864
Ser Asp Ala Thr Gln Leu Ser Pro Phe Cys Asp Leu Phe Thr His Asp
250 255 260
gaa tgg att caa tac gac tac ttg caa tct ttg ggt aag tac tac ggt 912
Glu Trp Ile Gln Tyr Asp Tyr Leu Gln Ser Leu Gly Lys Tyr Tyr Gly
265 270 275
tac ggt gct ggt aac cca ttg ggt cca gct caa ggt gtt ggt ttc gtt 960
Tyr Gly Ala Gly Asn Pro Leu Gly Pro Ala Gln Gly Val Gly Phe Val
280 285 290
aac gaa ttg att gct aga ttg act cac tct cca gtt caa gac cac act 1008
Asn Glu Leu Ile Ala Arg Leu Thr His Ser Pro Val Gln Asp His Thr
295 300 305 310
tct act aac cac act ttg gac tct aac cca gct act ttc cca ttg aac 1056
Ser Thr Asn His Thr Leu Asp Ser Asn Pro Ala Thr Phe Pro Leu Asn
315 320 325
gct act ttg tac gct gac ttc tct cac gac aac act atg gtt tct att 1104
Ala Thr Leu Tyr Ala Asp Phe Ser His Asp Asn Thr Met Val Ser Ile
330 335 340
ttc ttc gct ttg ggt ttg tac aac ggt act aag cca ttg tct act act 1152
Phe Phe Ala Leu Gly Leu Tyr Asn Gly Thr Lys Pro Leu Ser Thr Thr
345 350 355
tct gtt gaa tct att gaa gaa act gac ggt tac tct gct tct tgg act 1200
Ser Val Glu Ser Ile Glu Glu Thr Asp Gly Tyr Ser Ala Ser Trp Thr
360 365 370
gtt cca ttc gct gct aga gct tac gtt gaa atg atg caa tgt gaa gct 1248
Val Pro Phe Ala Ala Arg Ala Tyr Val Glu Met Met Gln Cys Glu Ala
375 380 385 390
gaa aag gaa cca ttg gtt aga gtt ttg gtt aac gac aga gtt gtt cca 1296
Glu Lys Glu Pro Leu Val Arg Val Leu Val Asn Asp Arg Val Val Pro
395 400 405
ttg cac ggt tgt ggt gtt gac aag ttg ggt aga tgt aag aga gac gac 1344
Leu His Gly Cys Gly Val Asp Lys Leu Gly Arg Cys Lys Arg Asp Asp
410 415 420
ttc gtt gaa ggt ttg tct ttc gct aga tct ggt ggt aac tgg gaa gaa 1392
Phe Val Glu Gly Leu Ser Phe Ala Arg Ser Gly Gly Asn Trp Glu Glu
425 430 435
tgt ttc gct taa 1404
Cys Phe Ala
440
<210>31
<211>467
<212>PRT
<213>人工序列
<223>人工序列的描述：共有植酸酶10 thermo 3 q50t，k91a
<400>31
Met Gly Val Phe Val Val Leu Leu Ser Ile Ala Thr Leu Phe Gly Ser
-25 -20 -15
Thr Ser Gly Thr Ala Leu Gly Pro Arg Gly Asn Ser His Ser Cys Asp
-10 -5 -1 1 5
Thr Val Asp Gly Gly Tyr Gln Cys Phe Pro Glu Ile Ser His Leu Trp
10 15 20
Gly Thr Tyr Ser Pro Phe Phe Ser Leu Ala Asp Glu Ser Ala Ile Ser
25 30 35
Pro Asp Val Pro Lys Gly Cys Arg Val Thr Phe Val Gln Val Leu Ser
40 45 50
Arg His Gly Ala Arg Tyr Pro Thr Ser Ser Ala Ser Lys Ala Tyr Ser
55 60 65 70
Ala Leu Ile Glu Ala Ile Gln Lys Asn Ala Thr Ala Phe Lys Gly Lys
75 80 85
Tyr Ala Phe Leu Lys Thr Tyr Asn Tyr Thr Leu Gly Ala Asp Asp Leu
90 95 100
Thr Pro Phe Gly Glu Gln Gln Met Val Asn Ser Gly Ile Lys Phe Tyr
105 110 115
Arg Arg Tyr Lys Ala Leu Ala Arg Lys Ile Val Pro Phe Ile Arg Ala
120 125 130
Ser Gly Ser Asp Arg Val Ile Ala Ser Ala Glu Lys Phe Ile Glu Gly
135 140 145 150
Phe Gln Ser Ala Lys Leu Ala Asp Pro Gly Ala Asn Pro His Gln Ala
155 160 165
Ser Pro Val Ile Asn Val Ile Ile Pro Glu Gly Ala Gly Tyr Asn Asn
170 175 180
Thr Leu Asp His Gly Leu Cys Thr Ala Phe Glu Glu Ser Glu Leu Gly
185 190 195
Asp Asp Val Glu Ala Asn Phe Thr Ala Val Phe Ala Pro Pro Ile Arg
200 205 210
Ala Arg Leu Glu Ala His Leu Pro Gly Val Asn Leu Thr Asp Glu Asp
215 220 225 230
Val Val Asn Leu Met Asp Met Cys Pro Phe Asp Thr Val Ala Arg Thr
235 240 245
Ser Asp Ala Thr Gln Leu Ser Pro Phe Cys Asp Leu Phe Thr His Asp
250 255 260
Glu Trp Ile Gln Tyr Asp Tyr Leu Gln Ser Leu Gly Lys Tyr Tyr Gly
265 270 275
Tyr Gly Ala Gly Asn Pro Leu Gly Pro Ala Gln Gly Val Gly Phe Val
280 285 290
Asn Glu Leu Ile Ala Arg Leu Thr His Ser Pro Val Gln Asp His Thr
295 300 305 310
Ser Thr Asn His Thr Leu Asp Ser Asn Pro Ala Thr Phe Pro Leu Asn
315 320 325
Ala Thr Leu Tyr Ala Asp Phe Ser His Asp Asn Thr Met Val Ser Ile
330 335 340
Phe Phe Ala Leu Gly Leu Tyr Asn Gly Thr Lys Pro Leu Ser Thr Thr
345 350 355
Ser Val Glu Ser Ile Glu Glu Thr Asp Gly Tyr Ser Ala Ser Trp Thr
360 365 370
Val Pro Phe Ala Ala Arg Ala Tyr Val Glu Met Met Gln Cys Glu Ala
375 380 385 390
Glu Lys Glu Pro Leu Val Arg Val Leu Val Asn Asp Arg Val Val Pro
395 400 405
Leu His Gly Cys Gly Val Asp Lys Leu Gly Arg Cys Lys Arg Asp Asp
410 415 420
Phe Val Glu Gly Leu Ser Phe Ala Arg Ser Gly Gly Asn Trp Glu Glu
425 430 435
Cys Phe Ala
440
<210>32
<211>1404
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述：烟曲霉α-突变体
<220>
<221>CDS
<222>(1)..(1401)
<220>
<221>成熟肽
<222>(79)..(1401)
<220>
<221>单肽
<222>(1)..(78)
<400>32
atg ggg gtt ttc gtc gtt cta tta tct atc gcg act ctg ttc ggc agc 48
Met Gly Val Phe Val Val Leu Leu Ser Ile Ala Thr Leu Phe Gly Ser
-25 -20 -15
aca tcg ggc act gcg ctg ggc ccc cgt gga aat cac tcc aag tcc tgc 96
Thr Ser Gly Thr Ala Leu Gly Pro Arg Gly Asn His Ser Lys Ser Cys
-10 -5 -1 1 5
gat acg gta gac cta ggg tac cag tgc tcc cct gcg act tct cat cta 144
Asp Thr Val Asp Leu Gly Tyr Gln Cys Ser Pro Ala Thr Ser His Leu
10 15 20
tgg ggc acg tac tcg cca tac ttt tcg ctc gag gac gag ctg tcc gtg 192
Trp Gly Thr Tyr Ser Pro Tyr Phe Ser Leu Glu Asp Glu Leu Ser Val
25 30 35
tcg agt aag ctt ccc aag gat tgc cgg atc acc ttg gta cag gtg cta 240
Ser Ser Lys Leu Pro Lys Asp Cys Arg Ile Thr Leu Val Gln Val Leu
40 45 50
tcg cgc cat gga gcg cgg tac cca acc agc tcc aag agc aaa aag tat 288
Ser Arg His Gly Ala Arg Tyr Pro Thr Ser Ser Lys Ser Lys Lys Tyr
55 60 65 70
aag aag ctt att acg gcg atc cag gcc aat gcc acc gac ttc aag ggc 336
Lys Lys Leu Ile Thr Ala Ile Gln Ala Asn Ala Thr Asp Phe Lys Gly
75 80 85
aag tac gcc ttt ttg aag acg tac aac tat act ctg ggt gcg gat gac 384
Lys Tyr Ala Phe Leu Lys Thr Tyr Asn Tyr Thr Leu Gly Ala Asp Asp
90 95 100
ctc act ccc ttt ggg gag cag cag ctg gtg aac tcg ggc atc aag ttc 432
Leu Thr Pro Phe Gly Glu Gln Gln Leu Val Asn Ser Gly Ile Lys Phe
105 110 115
tac cag agg tac aag gct ctg gcg cgc agt gtg gtg ccg ttt att cgc 480
Tyr Gln Arg Tyr Lys Ala Leu Ala Arg Ser Val Val Pro Phe Ile Arg
120 125 130
gcc tca ggc tcg gac cgg gtt att gct tcg gga gag aag ttc atc gag 528
Ala Ser Gly Ser Asp Arg Val Ile Ala Ser Gly Glu Lys Phe Ile Glu
135 140 145 150
ggg ttc cag cag gcg aag ctg gct gat cct ggc gcg acg aac cgc gcc 576
Gly Phe Gln Gln Ala Lys Leu Ala Asp Pro Gly Ala Thr Asn Arg Ala
155 160 165
gct ccg gcg att agt gtg att att ccg gag agc gag acg ttc aac aat 624
Ala Pro Ala Ile Ser Val Ile Ile Pro Glu Ser Glu Thr Phe Asn Asn
170 175 180
acg ctg gac cac ggt gtg tgc acg aag ttt gag gcg agt cag ctg gga 672
Thr Leu Asp His Gly Val Cys Thr Lys Phe Glu Ala Ser Gln Leu Gly
185 190 195
gat gag gtt gcg gcc aat ttc act gcg ctc ttt gca ccc gac atc cga 720
Asp Glu Val Ala Ala Asn Phe Thr Ala Leu Phe Ala Pro Asp Ile Arg
200 205 210
gct cgc ctc gag aag cat ctt cct ggc gtg acg ctg aca gac gag gac 768
Ala Arg Leu Glu Lys His Leu Pro Gly Val Thr Leu Thr Asp Glu Asp
215 220 225 230
gtt gtc agt cta atg gac atg tgt ccg ttt gat acg gta gcg cgc acc 816
Val Val Ser Leu Met Asp Met Cys Pro Phe Asp Thr Val Ala Arg Thr
235 240 245
agc gac gca agt cag ctg tca ccg ttc tgt caa ctc ttc act cac aat 864
Ser Asp Ala Ser Gln Leu Ser Pro Phe Cys Gln Leu Phe Thr His Asn
250 255 260
gag tgg aag aag tac gac tac ctt cag tcc ttg ggc aag tac tac ggc 912
Glu Trp Lys Lys Tyr Asp Tyr Leu Gln Ser Leu Gly Lys Tyr Tyr Gly
265 270 275
tac ggc gca ggc aac cct ctg gga ccg gct cag ggg ata ggg ttc acc 960
Tyr Gly Ala Gly Asn Pro Leu Gly Pro Ala Gln Gly Ile Gly Phe Thr
280 285 290
aac gag ctg att gcc cgg ttg acg cgt tcg cca gtg cag gac cac acc 1008
Asn Glu Leu Ile Ala Arg Leu Thr Arg Ser Pro Val Gln Asp His Thr
295 300 305 310
agc act aac tcg act cta gtc tcc aac ccg gcc acc ttc ccg ttg aac 1056
Ser Thr Asn Ser Thr Leu Val Ser Asn Pro Ala Thr Phe Pro Leu Asn
315 320 325
gct acc atg tac gtc gac ttt rca cac gac aac agc atg gtt tcc atc 1104
Ala Thr Met Tyr Val Asp Phe Ser His Asp Asn Ser Met Val Ser Ile
330 335 340
ttc ttt gca ttg ggc ctg tac aac ggc act gaa ccc ttg tcc cgg acc 1152
Phe Phe Ala Leu Gly Leu Tyr Asn Gly Thr Glu Pro Leu Ser Arg Thr
345 350 355
tcg gtg gaa agc gcc aag gaa ttg gat ggg tat tct gca tcc tgg gtg 1200
Ser Val Glu Ser Ala Lys Glu Leu Asp Gly Tyr Ser Ala Ser Trp Val
360 365 370
gtg cct ttc ggc gcg cga gcc tac ttc gag acg atg caa tgc aag tcg 1248
Val Pro Phe Gly Ala Arg Ala Tyr Phe Glu Thr Met Gln Cys Lys Ser
375 380 385 390
gaa aag gag cct ctt gtt cgc gct ttg att aat gac cgg gtt gtg cca 1296
Glu Lys Glu Pro Leu Val Arg Ala Leu Ile Asn Asp Arg Val Val Pro
395 400 405
ctg cat ggc tgc gat gtg gac aag ctg ggg cga tgc aag ctg aat gac 1344
Leu His Gly Cys Asp Val Asp Lys Leu Gly Arg Cys Lys Leu Asn Asp
410 415 420
ttt gtc aag gga ttg agt tgg gcc aga tct ggg ggc aac tgg gga gag 1392
Phe Val Lys Gly Leu Ser Trp Ala Arg Ser Gly Gly Asn Trp Gly Glu
425 430 435
tgc ttt agt tga 1404
Cys Phe Ser
440
<210>33
<211>467
<212>PRT
<213>人工序列
<223>人工序列的描述：烟曲霉α-突变体
<400>33
Met Gly Val Phe Val Val Leu Leu Ser Ile Ala Thr Leu Phe Gly Ser
-25 -20 -15
Thr Ser Gly Thr Ala Leu Gly Pro Arg Gly Asn His Ser Lys Ser Cys
-10 -5 -1 1 5
Asp Thr Val Asp Leu Gly Tyr Gln Cys Ser Pro Ala Thr Ser His Leu
10 15 20
Trp Gly Thr Tyr Ser Pro Tyr Phe Ser Leu Glu Asp Glu Leu Ser Val
25 30 35
Ser Ser Lys Leu Pro Lys Asp Cys Arg Ile Thr Leu Val Gln Val Leu
40 45 50
Ser Arg His Gly Ala Arg Tyr Pro Thr Ser Ser Lys Ser Lys Lys Tyr
55 60 65 70
Lys Lys Leu Ile Thr Ala Ile Gln Ala Asn Ala Thr Asp Phe Lys Gly
75 80 85
Lys Tyr Ala Phe Leu Lys Thr Tyr Asn Tyr Thr Leu Gly Ala Asp Asp
90 95 100
Leu Thr Pro Phe Gly Glu Gln Gln Leu Val Asn Ser Gly Ile Lys Phe
105 110 115
Tyr Gln Arg Tyr Lys Ala Leu Ala Arg Ser Val Val Pro Phe Ile Arg
120 125 130
Ala Ser Gly Ser Asp Arg Val Ile Ala Ser Gly Glu Lys Phe Ile Glu
135 140 145 150
Gly Phe Gln Gln Ala Lys Leu Ala Asp Pro Gly Ala Thr Asn Arg Ala
155 160 165
Ala Pro Ala Ile Ser Val Ile Ile Pro Glu Ser Glu Thr Phe Asn Asn
170 175 180
Thr Leu Asp His Gly Val Cys Thr Lys Phe Glu Ala Ser Gln Leu Gly
185 190 195
Asp Glu Val Ala Ala Asn Phe Thr Ala Leu Phe Ala Pro Asp Ile Arg
200 205 210
Ala Arg Leu Glu Lys His Leu Pro Gly Val Thr Leu Thr Asp Glu Asp
215 220 225 230
Val Val Ser Leu Met Asp Met Cys Pro Phe Asp Thr Val Ala Arg Thr
235 240 245
Ser Asp Ala Ser Gln Leu Ser Pro Phe Cys Gln Leu Phe Thr His Asn
250 255 260
Glu Trp Lys Lys Tyr Asp Tyr Leu Gln Ser Leu Gly Lys Tyr Tyr Gly
265 270 275
Tyr Gly Ala Gly Asn Pro Leu Gly Pro Ala Gln Gly Ile Gly Phe Thr
280 285 290
Asn Glu Leu Ile Ala Arg Leu Thr Arg Ser Pro Val Gln Asp His Thr
295 300 305 310
Ser Thr Asn Ser Thr Leu Val Ser Asn Pro Ala Thr Phe Pro Leu Asn
315 320 325
Ala Thr Met Tyr Val Asp Phe Ser His Asp Asn Ser Met Val Ser Ile
330 335 340
Phe Phe Ala Leu Gly Leu Tyr Asn Gly Thr Glu Pro Leu Ser Arg Thr
345 350 355
Ser Val Glu Ser Ala Lys Glu Leu Asp Gly Tyr Ser Ala Ser Trp Val
360 365 370
Val Pro Phe Gly Ala Arg Ala Tyr Phe Glu Thr Met Gln Cys Lys Ser
375 380 385 390
Glu Lys Glu Pro Leu Val Arg Ala Leu Ile Asn Asp Arg Val Val Pro
395 400 405
Leu His Gly Cys Asp Val Asp Lys Leu Gly Arg Cys Lys Leu Asn Asp
410 415 420
Phe Val Lys Gly Leu Ser Trp Ala Arg Ser Gly Gly Asn Trp Gly Glu
425 430 435
Cys Phe Ser
440
<210>34
<211>1426
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述：共有植酸酶7
<220>
<221>CDS
<222>(12)..(1412)
<220>
<221>成熟肽
<222>(90)..(1412)
<220>
<221>单肽
<222>(12)..(89)
<400>34
tatatgaatt c atg ggc gtg ttc gtc gtg cta ctg tcc att gcc acc ttg 50
Met Gly Val Phe Val Val Leu Leu Ser Ile Ala Thr Leu
-25 -20 -15
ttc ggt tcc aca tcc ggt acc gcc ttg ggt cct cgt ggt aat tct cac 98
Phe Gly Ser Thr Ser Gly Thr Ala Leu Gly Pro Arg Gly Asn Ser His
-10 -5 -1 1
tct tgt gac act gtt gac ggt ggt tac caa tgt ttc cca gaa att tct 146
Ser Cys Asp Thr Val Asp Gly Gly Tyr Gln Cys Phe Pro Glu Ile Ser
5 10 15
cac ttg tgg ggt caa tac tct cca tac ttc tct ttg gaa gac gaa tct 194
His Leu Trp Gly Gln Tyr Ser Pro Tyr Phe Ser Leu Glu Asp Glu Ser
20 25 30 35
gct att tct cca gac gtt cca gac gac tgt aga gtt act ttc gtt caa 242
Ala Ile Ser Pro Asp Val Pro Asp Asp Cys Arg Val Thr Phe Val Gln
40 45 50
gtt ttg tct aga cac ggt gct aga tac cca act gac tct aag ggt aag 290
Val Leu Ser Arg His Gly Ala Arg Tyr Pro Thr Asp Ser Lys Gly Lys
55 60 65
aag tac tct gct ttg att gaa gct att caa aag aac gct act gct ttc 338
Lys Tyr Ser Ala Leu Ile Glu Ala Ile Gln Lys Asn Ala Thr Ala Phe
70 75 80
aag ggt aag tac gct ttc ttg aag act tac aac tac act ttg ggt gct 386
Lys Gly Lys Tyr Ala Phe Leu Lys Thr Tyr Asn Tyr Thr Leu Gly Ala
85 90 95
gac gac ttg act cca ttc ggt gaa aac caa atg gtt aac tct ggt att 434
Asp Asp Leu Thr Pro Phe Gly Glu Asn Gln Met Val Asn Ser Gly Ile
100 105 110 115
aag ttc tac aga aga tac aag gct ttg gct aga aag att gtt cca ttc 482
Lys Phe Tyr Arg Arg Tyr Lys Ala Leu Ala Arg Lys Ile Val Pro Phe
120 125 130
att aga gct tct ggt tct tct aga gtt att gct tct gct gaa aag ttc 530
Ile Arg Ala Ser Gly Ser Ser Arg Val Ile Ala Ser Ala Glu Lys Phe
135 140 145
att gaa ggt ttc caa tct gct aag ttg gct gac cca ggt tct caa cca 578
Ile Glu Gly Phe Gln Ser Ala Lys Leu Ala Asp Pro Gly Ser Gln Pro
150 155 160
cac caa gct tct cca gtt att gac gtt att att tct gac gct tct tct 626
His Gln Ala Ser Pro Val Ile Asp Val Ile Ile Ser Asp Ala Ser Ser
165 170 175
tac aac aac act ttg gac cca ggt act tgt act gct ttc gaa gac tct 674
Tyr Asn Asn Thr Leu Asp Pro Gly Thr Cys Thr Ala Phe Glu Asp Ser
180 185 190 195
gaa ttg gct gac act gtt gaa gct aac ttc act gct ttg ttc gct cca 722
Glu Leu Ala Asp Thr Val Glu Ala Asn Phe Thr Ala Leu Phe Ala Pro
200 205 210
gct att aga gct aga ttg gaa gct gac ttg cca ggt gtt act ttg act 770
Ala Ile Arg Ala Arg Leu Glu Ala Asp Leu Pro Gly Val Thr Leu Thr
215 220 225
gac act gaa gtt act tac ttg atg gac atg tgt tct ttc gaa act gtt 818
Asp Thr Glu Val Thr Tyr Leu Met Asp Met Cys Ser Phe Glu Thr Val
230 235 240
gct aga act tct gac gct act gaa ttg tct cca ttc tgt gct ttg ttc 866
Ala Arg Thr Ser Asp Ala Thr Glu Leu Ser Pro Phe Cys Ala Leu Phe
245 250 255
act cac gac gaa tgg aga cac tac gac tac ttg caa tct ttg aag aag 914
Thr His Asp Glu Trp Arg His Tyr Asp Tyr Leu Gln Ser Leu Lys Lys
260 265 270 275
tac tac ggt cac ggt gct ggt aac cca ttg ggt cca act caa ggt gtt 962
Tyr Tyr Gly His Gly Ala Gly Asn Pro Leu Gly Pro Thr Gln Gly Val
280 285 290
ggt ttc gct aac gaa ttg att gct aga ttg act aga tct cca gtt caa 1010
Gly Phe Ala Asn Glu Leu Ile Ala Arg Leu Thr Arg Ser Pro Val Gln
295 300 305
gac cac act tct act aac cac act ttg gac tct aac cca gct act ttc 1058
Asp His Thr Ser Thr Asn His Thr Leu Asp Ser Asn Pro Ala Thr Phe
310 315 320
cca ttg aac gct act ttg tac gct gac ttc tct cac gac aac ggt att 1106
Pro Leu Asn Ala Thr Leu Tyr Ala Asp Phe Ser His Asp Asn Gly Ile
325 330 335
att tct att ttc ttc gct ttg ggt ttg tac aac ggt act gct cca ttg 1154
Ile Ser Ile Phe Phe Ala Leu Gly Leu Tyr Asn Gly Thr Ala Pro Leu
340 345 350 355
tct act act tct gtt gaa tct att gaa gaa act gac ggt tac tct tct 1202
Ser Thr Thr Ser Val Glu Ser Ile Glu Glu Thr Asp Gly Tyr Ser Ser
360 365 370
gct tgg act gtt cca ttc gct tct aga gct tac gtt gaa atg atg caa 1250
Ala Trp Thr Val Pro Phe Ala Ser Arg Ala Tyr Val Glu Met Met Gln
375 380 385
tgt caa gct gaa aag gaa cca ttg gtt aga gtt ttg gtt aac gac aga 1298
Cys Gln Ala Glu Lys Glu Pro Leu Val Arg Val Leu Val Asn Asp Arg
390 395 400
gtt gtt cca ttg cac ggt tgt gct gtt gac aag ttg ggt aga tgt aag 1346
Val Val Pro Leu His Gly Cys Ala Val Asp Lys Leu Gly Arg Cys Lys
405 410 415
aga gac gac ttc gtt gaa ggt ttg tct ttc gct aga tct ggt ggt aac 1394
Arg Asp Asp Phe Val Glu Gly Leu Ser Phe Ala Arg Ser Gly Gly Asn
420 425 430 435
tgg gct gaa tgt ttc gct taagaattca tata 1426
Trp Ala Glu Cys Phe Ala
440
<210>35
<211>467
<212>PRT
<213>人工序列
<223>人工序列的描述：共有植酸酶7
<400>35
Met Gly Val Phe Val Val Leu Leu Ser Ile Ala Thr Leu Phe Gly Ser
-25 -20 -15
Thr Ser Gly Thr Ala Leu Gly Pro Arg Gly Asn Ser His Ser Cys Asp
-10 -5 -1 1 5
Thr Val Asp Gly Gly Tyr Gln Cys Phe Pro Glu Ile Ser His Leu Trp
10 15 20
Gly Gln Tyr Ser Pro Tyr Phe Ser Leu Glu Asp Glu Ser Ala Ile Ser
25 30 35
Pro Asp Val Pro Asp Asp Cys Arg Val Thr Phe Val Gln Val Leu Ser
40 45 50
Arg His Gly Ala Arg Tyr Pro Thr Asp Ser Lys Gly Lys Lys Tyr Ser
55 60 65 70
Ala Leu Ile Glu Ala Ile Gln Lys Asn Ala Thr Ala Phe Lys Gly Lys
75 80 85
Tyr Ala Phe Leu Lys Thr Tyr Asn Tyr Thr Leu Gly Ala Asp Asp Leu
90 95 100
Thr Pro Phe Gly Glu Asn Gln Met Val Asn Ser Gly Ile Lys Phe Tyr
105 110 115
Arg Arg Tyr Lys Ala Leu Ala Arg Lys Ile Val Pro Phe Ile Arg Ala
120 125 130
Ser Gly Ser Ser Arg Val Ile Ala Ser Ala Glu Lys Phe Ile Glu Gly
135 140 145 150
Phe Gln Ser Ala Lys Leu Ala Asp Pro Gly Ser Gln Pro His Gln Ala
155 160 165
Ser Pro Val Ile Asp Val Ile Ile Ser Asp Ala Ser Ser Tyr Asn Asn
170 175 180
Thr Leu Asp Pro Gly Thr Cys Thr Ala Phe Glu Asp Ser Glu Leu Ala
185 190 195
Asp Thr Val Glu Ala Asn Phe Thr Ala Leu Phe Ala Pro Ala Ile Arg
200 205 210
Ala Arg Leu Glu Ala Asp Leu Pro Gly Val Thr Leu Thr Asp Thr Glu
215 220 225 230
Val Thr Tyr Leu Met Asp Met Cys Ser Phe Glu Thr Val Ala Arg Thr
235 240 245
Ser Asp Ala Thr Glu Leu Ser Pro Phe Cys Ala Leu Phe Thr His Asp
250 255 260
Glu Trp Arg His Tyr Asp Tyr Leu Gln Ser Leu Lys Lys Tyr Tyr Gly
265 270 275
His Gly Ala Gly Asn Pro Leu Gly Pro Thr Gln Gly Val Gly Phe Ala
280 285 290
Asn Glu Leu Ile Ala Arg Leu Thr Arg Ser Pro Val Gln Asp His Thr
295 300 305 310
Ser Thr Asn His Thr Leu Asp Ser Asn Pro Ala Thr Phe Pro Leu Asn
315 320 325
Ala Thr Leu Tyr Ala Asp Phe Ser His Asp Asn Gly Ile Ile Ser Ile
330 335 340
Phe Phe Ala Leu Gly Leu Tyr Asn Gly Thr Ala Pro Leu Ser Thr Thr
345 350 355
Ser Val Glu Ser Ile Glu Glu Thr Asp Gly Tyr Ser Ser Ala Trp Thr
360 365 370
Val Pro Phe Ala Ser Arg Ala Tyr Val Glu Met Met Gln Cys Gln Ala
375 380 385 390
Glu Lys Glu Pro Leu Val Arg Val Leu Val Asn Asp Arg Val Val Pro
395 400 405
Leu His Gly Cys Ala Val Asp Lys Leu Gly Arg Cys Lys Arg Asp Asp
410 415 420
Phe Val Glu Gly Leu Ser Phe Ala Arg Ser Gly Gly Asn Trp Ala Glu
425 430 435
Cys Phe Ala
440
<210>36
<211>467
<212>PRT
<213>人工序列
<220>
<223>人工序列的描述：共有植酸酶12
<400>36
Met Gly Val Phe Val Val Leu Leu Ser Ile Ala Thr Leu Phe Gly Ser
1 5 10 15
Thr Ser Gly Thr Ala Leu Gly Pro Arg Gly Asn Ser His Ser Cys Asp
20 25 30
Thr Val Asp Gly Gly Tyr Gln Cys Phe Pro Glu Ile Ser Ser Asn Trp
35 40 45
Ser Pro Tyr Ser Pro Tyr Phe Ser Leu Ala Asp Glu Ser Ala Ile Ser
50 55 60
Pro Asp Val Pro Lys Gly Cys Arg Val Thr Phe Val Gln Val Leu Gln
65 70 75 80
Arg His Gly Ala Arg Phe Pro Thr Ser Gly Ala Ala Thr Arg Ile Ser
85 90 95
Ala Leu Ile Glu Ala Ile Gln Lys Asn Ala Thr Ala Phe Lys Gly Lys
100 105 110
Tyr Ala Phe Leu Lys Thr Tyr Asn Tyr Thr Leu Gly Ala Asp Asp Leu
115 120 125
Val Pro Phe Gly Ala Asn Gln Ser Ser Gln Ala Gly Ile Lys Phe Tyr
130 135 140
Arg Arg Tyr Lys Ala Leu Ala Arg Lys Ile Val Pro Phe Ile Arg Ala
145 150 155 160
Ser Gly Ser Asp Arg Val Ile Asp Ser Ala Thr Asn Trp Ile Glu Gly
165 170 175
Phe Gln Ser Ala Lys Leu Ala Asp Pro Gly Ala Asn Pro His Gln Ala
180 185 190
Ser Pro Val Ile Asn Val Ile Ile Pro Glu Gly Ala Gly Tyr Asn Asn
195 200 205
Thr Leu Asp His Gly Leu Cys Thr Ala Phe Glu Glu Ser Glu Leu Gly
210 215 220
Asp Asp Val Glu Ala Asn Phe Thr Ala Val Phe Ala Pro Pro Ile Arg
225 230 235 240
Ala Arg Leu Glu Ala His Leu Pro Gly Val Asn Leu Thr Asp Glu Asp
245 250 255
Val Val Asn Leu Met Asp Met Cys Pro Phe Asp Thr Val Ala Arg Thr
260 265 270
Ser Asp Ala Thr Glu Leu Ser Pro Phe Cys Asp Leu Phe Thr His Asp
275 280 285
Glu Trp Ile Gln Tyr Asp Tyr Leu Gly Asp Leu Asp Lys Tyr Tyr Gly
290 295 300
Thr Gly Ala Gly Asn Pro Leu Gly Pro Ala Gln Gly Val Gly Phe Val
305 310 315 320
Asn Glu Leu Ile Ala Arg Leu Thr His Ser Pro Val Gln Asp His Thr
325 330 335
Ser Thr Asn His Thr Leu Asp Ser Asn Pro Ala Thr Phe Pro Leu Asn
340 345 350
Ala Thr Leu Tyr Ala Asp Phe Ser His Asp Asn Thr Met Val Ala Ile
355 360 365
Phe Phe Ala Leu Gly Leu Tyr Asn Gly Thr Lys Pro Leu Ser Thr Thr
370 375 380
Ser Val Glu Ser Ile Glu Glu Thr Asp Gly Tyr Ser Ala Ser Trp Leu
385 390 395 400
Val Pro Phe Ser Ala Arg Met Tyr Val Glu Met Met Gln Cys Glu Ala
405 410 415
Glu Lys Glu Pro Leu Val Arg Val Leu Val Asn Asp Arg Val Val Pro
420 425 430
Leu His Gly Cys Gly Val Asp Lys Leu Gly Arg Cys Lys Arg Asp Asp
435 440 445
Phe Val Glu Gly Leu Ser Phe Ala Arg Ser Gly Gly Asn Trp Glu Glu
450 455 460
Cys Phe Ala
465
<210>37
<211>26
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述：引物
<400>37
tatatgaatt catgggcgtg ttcgtc 26
<210>38
<211>22
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述：引物
<400>38
tgaaaagttc attgaaggtt tc 22
<210>39
<211>22
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述：引物
<400>39
tcttcgaaag cagtacacaa ac 22
<210>40
<211>22
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述：引物
<400>40
tatatgaatt cttaagcgaa ac 22
<210>41
<211>32
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述：引物
<400>41
cacttgtggg gtacctactc tccatacttc tc 32
<210>42
<211>31
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述：引物
<400>42
ggtcaatact ctccattctt ctctttggaa g 31
<210>43
<211>28
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述：引物
<400>43
catacttctc tttggcagac gaatctgc 28
<210>44
<211>31
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述：引物
<400>44
ctccagacgt cccaaaggac tgtagagtta c 31
<210>45
<211>31
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述：引物
<400>45
ctccagacgt cccagacggc tgtagagtta c 31
<210>46
<211>36
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述：引物
<400>46
gatacccaac ttcttctgcg tctaaggctt actctg 36
<210>47
<211>29
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述：引物
<400>47
cttctaagtc taagaagtac tctgctttg 29
<210>48
<211>41
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述：引物
<400>48
gcttactctg ctttgattga acggattcaa aagaacgcta c 41
<210>49
<211>29
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述：引物
<400>49
ccattcggtg aacagcaaat ggttaactc 29
<210>50
<211>30
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述：引物
<400>50
gatacaaggc tctcgcgaga aacattgttc 30
<210>51
<211>30
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述：引物
<400>51
gattgttccattcgtgcgcg cttctggttc 30
<210>52
<211>31
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述：引物
<400>52
ctccagttat taacgtgatc attccagaag g 31
<210>53
<211>30
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述：引物
<400>53
ggctgaccca ggggcccaac cacaccaagc 30
<210>54
<211>32
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述：引物
<400>54
cactttggac catggtcttt gtactgcttt cg 32
<210>55
<211>34
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述：引物
<400>55
gctttcgaag actctaccct aggtgacgac gttg 34
<210>56
<211>26
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述：引物
<400>56
ggtgacgacg ctgaagctaa cttcac 26
<210>57
<211>27
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述：引物
<400>57
ctaacttcac cgcggtgttc gctccag 27
<210>58
<211>34
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述：引物
<400>58
gctttgttcg ctccacctat tagagctagattgg 34
<210>59
<211>26
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述：引物
<400>59
gccaggtgtt aact tgactg acgaag 26
<210>60
<211>27
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述：引物
<400>60
gacgaagacg tcgttaactt gatggac 27
<210>61
<211>28
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述：引物
<400>61
gtccattcga cactgtcgct agaacttc 28
<210>62
<211>26
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述：引物
<400>62
ctgacgctac tcagctgtct ccattc 26
<210>63
<211>28
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述：引物
<400>63
gtctccattc tgtgatttgt tcactcac 28
<210>64
<211>26
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述：引物
<400>64
gctttgttca ccgcggacga atggag 26
<210>65
<211>27
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述：引物
<400>65
cacgacgaat ggatccaata cgactac 27
<210>66
<211>27
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述：引物
<400>66
gacgaatgga gagcgtacga ctacttg 27
<210>67
<211>29
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述：引物
<400>67
ggtgttggtt tcgttaacga attgattgc 29
<210>68
<211>28
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述：引物
<400>68
gctagattga ctcactctcc agttcaag 28
<210>69
<211>32
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述：引物
<400>69
ctcacgacaa cactatgata tctattttcttc 32
<210>70
<211>30
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述：引物
<400>70
cgacaactcc atggtttcta ttttcttcgc 30
<210>71
<211>27
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述：引物
<400>71
gtacaacggt accaagccat tgtctac 27
<210>72
<211>25
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述：引物
<400>72
ctgacggtta cgctgcttct tggac 25
<210>73
<211>26
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述：引物
<400>73
ctgttccatt cgctgctaga gcttac 26
<210>74
<211>27
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述：引物
<400>74
gatgcaatgt gaagctgaaa aggaacc 27
<210>75
<211>26
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述：引物
<400>75
cacggttgtg gtgtcgacaa gttggg 26
<210>76
<211>30
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述：引物
<400>76
gatctggtgg caattgggag gaatgtttcg 30
<210>77
<211>28
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述：引物
<400>77
cacgtactcg ccatactttt cgctcgag 28
<210>78
<211>33
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述：引物
<400>78
ccatactttt cgctcgcgga cgagctgtcc gtg 33
<210>79
<211>31
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述：引物
<400>79
gtataagaag cttattacgg cgatccaggc c 31
<210>80
<211>31
<212>DNA
<211>人工序列
<220>
<223>人工序列的描述：引物
<400>80
cttcaagggc aagtacgcct ttttgaagac g 31
<210>81
<211>29
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述：引物
<400>81
catccgagct cgcctcgaga agcatcttc 29
<210>82
<211>29
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述：引物
<400>82
ctaatggatg tgtccgtttg atacggtag 29
<210>83
<211>27
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述：引物
<400>83
gtggaagaag tacgactacc ttcagtc 27
<210>84
<211>28
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述：引物
<400>84
gcccggttga cgcattcgcc agtgcagg 28
<210>85
<211>29
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述：引物
<400>85
cacacgacaa caccatggtt tccatcttc 29
<210>86
<211>30
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述：引物
<400>86
gtggtgcctt tcgccgcgcg agcctacttc 30
<210>87
<211>33
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述：引物
<400>87
tatatcatga gcgtgttcgt cgtgctactg ttc 33
<210>88
<211>33
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述：引物
<400>88
acccgactta caaagcgaat tctatagata tat 33
<210>89
<211>33
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述：引物
<400>89
acccttctta caaagcgaat tctatagata tat 33
<210>90
<211>1404
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述：共有植酸酶-3-thermo-11-Q50T
<220>
<221>CDS
<222>(1)..(1401)
<220>
<221>单肽
<222>(1)..(69)
<220>
<221>成熟肽
<222>(70)..(1401)
<400>90
atg ggc gtg ttc gtc gtg cta ctg tcc att gcc acc ttg ttc ggt tcc 48
Met Gly Val Phe Val Val Leu Leu Ser Ile Ala Thr Leu Phe Gly Ser
-20 -15 -10
aca tcc ggt acc gcc ttg ggt cct cgt ggt aat tct cac tct tgt gac 96
Thr Ser Gly Thr Ala Leu Gly Pro Arg Gly Asn Ser His Ser Cys Asp
-5 -1 1 5
act gtt gac ggt ggt tac caa tgt ttc cca gaa att tct cac ttg tgg 144
Thr Val Asp Gly Gly Tyr Gln Cys Phe Pro Glu Ile Ser His Leu Trp
10 15 20 25
ggt acc tac tct cca tac ttc tct ttg gca gac gaa tct gct att tct 192
Gly Thr Tyr Ser Pro Tyr Phe Ser Leu Ala Asp Glu Ser Ala Ile Ser
30 35 40
cca gac gtc cca aag gac tgt aga gtt act ttc gtt caa gtt ttg tct 240
Pro Asp Val Pro Lys Asp Cys Arg Val Thr Phe Val Gln Val Leu Ser
45 50 55
aga cac ggt gct aga tac cca act tct tct aag tct aag gct tac tct 288
Arg His Gly Ala Arg Tyr Pro Thr Ser Ser Lys Ser Lys Ala Tyr Ser
60 65 70
gct ttg att gaa gct att caa aag aac gct act gct ttc aag ggt aag 336
Ala Leu Ile Glu Ala Ile Gln Lys Asn Ala Thr Ala Phe Lys Gly Lys
75 80 85
tac gct ttc ttg aag act tac aac tac act ttg ggt gct gac gac ttg 384
Tyr Ala Phe Leu Lys Thr Tyr Asn Tyr Thr Leu Gly Ala Asp Asp Leu
90 95 100 105
act cca ttc ggt gaa aac caa atg gtt aac tct ggt att aag ttc tac 432
Thr Pro Phe Gly Glu Asn Gln Met Val Asn Ser Gly Ile Lys Phe Tyr
110 115 120
aga aga tac aag gct ttg gct aga aag att gtt cca ttc att aga gct 480
Arg Arg Tyr Lys Ala Leu Ala Arg Lys Ile Val Pro Phe Ile Arg Ala
125 130 135
tct ggt tct gac aga gtt att gct tct gct gaa aag ttc att gaa ggt 528
Ser Gly Ser Asp Arg Val Ile Ala Ser Ala Glu Lys Phe Ile Glu Gly
140 145 150
ttc caa tct gct aag ttg gct gac cca ggt tct caa cca cac caa gct 576
Phe Gln Ser Ala Lys Leu Ala Asp Pro Gly Ser Gln Pro His Gln Ala
155 160 165
tct cca gtt att aac gtg atc att cca gaa gga tcc ggt tac aac aac 624
Ser Pro Val Ile Asn Val Ile Ile Pro Glu Gly Ser Gly Tyr Asn Asn
170 175 180 185
act ttg gac cat ggt ctt tgt act gct ttc gaa gac tct acc cta ggt 672
Thr Leu Asp His Gly Leu Cys Thr Ala Phe Glu Asp Ser Thr Leu Gly
190 195 200
gac gac gtt gaa gct aac ttc act gct ttg ttc gct cca gct att aga 720
Asp Asp Val Glu Ala Asn Phe Thr Ala Leu Phe Ala Pro Ala Ile Arg
205 210 215
gct aga ttg gaa gct gac ttg cca ggt gtt act ttg act gac gaa gac 768
Ala Arg Leu Glu Ala Asp Leu Pro Gly Val Thr Leu Thr Asp Glu Asp
220 225 230
gtt gtt tac ttg atg gac atg tgt cca ttc gac act gtc gct aga act 816
Val Val Tyr Leu Met Asp Met Cys Pro Phe Asp Thr Val Ala Arg Thr
235 240 245
tct gac gct act gaa ttg tct cca ttc tgt gct ttg ttc act cac gac 864
Ser Asp Ala Thr Glu Leu Ser Pro Phe Cys Ala Leu Phe Thr His Asp
250 255 260 265
gaa tgg atc caa tac gac tac ttg caa agc ttg ggt aag tac tac ggt 912
Glu Trp Ile Gln Tyr Asp Tyr Leu Gln Ser Leu Gly Lys Tyr Tyr Gly
270 275 280
tac ggt gct ggt aac cca ttg ggt cca gct caa ggt gtt ggt ttc gct 960
Tyr Gly Ala Gly Asn Pro Leu Gly Pro Ala Gln Gly Val Gly Phe Ala
285 290 295
aac gaa ttg att gct aga ttg act cac tct cca gtt caa gac cac act 1008
Asn Glu Leu Ile Ala Arg Leu Thr His Ser Pro Val Gln Asp His Thr
300 305 310
tct act aac cac act ttg gac tct aac cca gct act ttc cca ttg aac 1056
Ser Thr Asn His Thr Leu Asp Ser Asn Pro Ala Thr Phe Pro Leu Asn
315 320 325
gct act ttg tac gct gac ttc tct cac gac aac act atg ata tct att 1104
Ala Thr Leu Tyr Ala Asp Phe Ser His Asp Asn Thr Met Ile Ser Ile
330 335 340 345
ttc ttc gct ttg ggt ttg tac aac ggt acc aag cca ttg tct act act 1152
Phe Phe Ala Leu Gly Leu Tyr Asn Gly Thr Lys Pro Leu Ser Thr Thr
350 355 360
tct gtt gaa tct att gaa gaa act gac ggt tac tct gct tct tgg act 1200
Ser Val Glu Ser Ile Glu Glu Thr Asp Gly Tyr Ser Ala Ser Trp Thr
365 370 375
gtt cca ttc gct gct aga gct tac gtt gaa atg atg caa tgt caa gct 1248
Val Pro Phe Ala Ala Arg Ala Tyr Val Glu Met Met Gln Cys Gln Ala
380 385 390
gaa aag gaa cca ttg gtt aga gtt ttg gtt aac gac aga gtt gtt cca 1296
Glu Lys Glu Pro Leu Val Arg Val Leu Val Asn Asp Arg Val Val Pro
395 400 405
ttg cac ggt tgt gct gtt gac aag ttg ggt aga tgt aag aga gac gac 1344
Leu His Gly Cys Ala Val Asp Lys Leu Gly Arg Cys Lys Arg Asp Asp
410 415 420 425
ttc gtt gaa ggt ttg tct ttc gct aga tct ggt ggt aac tgg gct gaa 1392
Phe Val Glu Gly Leu Ser Phe Ala Arg Ser Gly Gly Asn Trp Ala Glu
430 435 440
tgt ttc gct taa 1404
Cys Phe Ala
<210>91
<211>467
<212>PRT
<213>人工序列
<223>人工序列的描述：共有植酸酶-3-thermo-11-Q50T
<400>91
Met Gly Val Phe Val Val Leu Leu Ser Ile Ala Thr Leu Phe Gly Ser
-20 -15 -10
Thr Ser Gly Thr Ala Leu Gly Pro Arg Gly Asn Ser His Ser Cys Asp
-5 -1 1 5
Thr Val Asp Gly Gly Tyr Gln Cys Phe Pro Glu Ile Ser His Leu Trp
10 15 20 25
Gly Thr Tyr Ser Pro Tyr Phe Ser Leu Ala Asp Glu Ser Ala Ile Ser
30 35 40
Pro Asp Val Pro Lys Asp Cys Arg Val Thr Phe Val Gln Val Leu Ser
45 50 55
Arg His Gly Ala Arg Tyr Pro Thr Ser Ser Lys Ser Lys Ala Tyr Ser
60 65 70
Ala Leu Ile Glu Ala Ile Gln Lys Asn Ala Thr Ala Phe Lys Gly Lys
75 80 85
Tyr Ala Phe Leu Lys Thr Tyr Asn Tyr Thr Leu Gly Ala Asp Asp Leu
90 95 100 105
Thr Pro Phe Gly Glu Asn Gln Met Val Asn Ser Gly Ile Lys Phe Tyr
110 115 120
Arg Arg Tyr Lys Ala Leu Ala Arg Lys Ile Val Pro Phe Ile Arg Ala
125 130 135
Ser Gly Ser Asp Arg Val Ile Ala Ser Ala Glu Lys Phe Ile Glu Gly
140 145 150
Phe Gln Ser Ala Lys Leu Ala Asp Pro Gly Ser Gln Pro His Gln Ala
155 160 165
Ser Pro Val Ile Asn Val Ile Ile Pro Glu Gly Ser Gly Tyr Asn Asn
170 175 180 185
Thr Leu Asp His Gly Leu Cys Thr Ala Phe Glu Asp Ser Thr Leu Gly
190 195 200
Asp Asp Val Glu Ala Asn Phe Thr Ala Leu Phe Ala Pro Ala Ile Arg
205 210 215
Ala Arg Leu Glu Ala Asp Leu Pro Gly Val Thr Leu Thr Asp Glu Asp
220 225 230
Val Val Tyr Leu Met Asp Met Cys Pro Phe Asp Thr Val Ala Arg Thr
235 240 245
Ser Asp Ala Thr Glu Leu Ser Pro Phe Cys Ala Leu Phe Thr His Asp
250 255 260 265
Glu Trp Ile Gln Tyr Asp Tyr Leu Gln Ser Leu Gly Lys Tyr Tyr Gly
270 275 280
Tyr Gly Ala Gly Asn Pro Leu Gly Pro Ala Gln Gly Val Gly Phe Ala
285 290 295
Asn Glu Leu Ile Ala Arg Leu Thr His Ser Pro Val Gln Asp His Thr
300 305 310
Ser Thr Asn His Thr Leu Asp Ser Asn Pro Ala Thr Phe Pro Leu Asn
315 320 325
Ala Thr Leu Tyr Ala Asp Phe Ser His Asp Asn Thr Met Ile Ser Ile
330 335 340 345
Phe Phe Ala Leu Gly Leu Tyr Asn Gly Thr Lys Pro Leu Ser Thr Thr
350 355 360
Ser Val Glu Ser Ile Glu Glu Thr Asp Gly Tyr Ser Ala Ser Trp Thr
365 370 375
Val Pro Phe Ala Ala Arg Ala Tyr Val Glu Met Met Gln Cys Gln Ala
380 385 390
Glu Lys Glu Pro Leu Val Arg Val Leu Val Asn Asp Arg Val Val Pro
395 400 405
Leu His Gly Cys Ala Val Asp Lys Leu Gly Arg Cys Lys Arg Asp Asp
410 415 420 425
Phe Val Glu Gly Leu Ser Phe Ala Arg Ser Gly Gly Asn Trp Ala Glu
430 435 440
Cys Phe Ala
<210>92
<211>1404
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述：共有植酸酶-3-thermo-11-Q50T-K91A
<220>
<221>单肽
<222>(1)..(69)
<220>
<221>CDS
<222>(1)..(1401)
<220>
<221>成熟肽
<222>(70)..(1401)
<400>92
atg ggc gtg ttc gtc gtg cta ctg tcc att gcc acc ttg ttc ggt tcc 48
Met Gly Val Phe Val Val Leu Leu Ser Ile Ala Thr Leu Phe Gly Ser
-20 -15 -10
aca tcc ggt acc gcc ttg ggt cct cgt ggt aat tct cac tct tgt gac 96
Thr Ser Gly Thr Ala Leu Gly Pro Arg Gly Asn Ser His Ser Cys Asp
-5 -1 1 5
act gtt gac ggt ggt tac caa tgt ttc cca gaa att tct cac ttg tgg 144
Thr Val Asp Gly Gly Tyr Gln Cys Phe Pro Glu Ile Ser His Leu Trp
10 15 20 25
ggt acc tac tct cca tac ttc tct ttg gca gac gaa tct gct att tct 192
Gly Thr Tyr Ser Pro Tyr Phe Ser Leu Ala Asp Glu Ser Ala Ile Ser
30 35 40
cca gac gtc cca aag gac tgt aga gtt act ttc gtt caa gtt ttg tct 240
Pro Asp Val Pro Lys Asp Cys Arg Val Thr Phe Val Gln Val Leu Ser
45 50 55
aga cac ggt gct aga tac cca act tct tct gcg tct aag gct tac tct 288
Arg His Gly Ala Arg Tyr Pro Thr Ser Ser Ala Ser Lys Ala Tyr Ser
60 65 70
gct ttg att gaa gct att caa aag aac gct act gct ttc aag ggt aag 336
Ala Leu Ile Glu Ala Ile Gln Lys Asn Ala Thr Ala Phe Lys Gly Lys
75 80 85
tac gct ttc ttg aag act tac aac tac act ttg ggt gct gac gac ttg 384
Tyr Ala Phe Leu Lys Thr Tyr Asn Tyr Thr Leu Gly Ala Asp Asp Leu
90 95 100 105
act cca ttc ggt gaa aac caa atg gtt aac tct ggt att aag ttc tac 432
Thr Pro Phe Gly Glu Asn Gln Met Val Asn Ser Gly Ile Lys Phe Tyr
110 115 120
aga aga tac aag gct ttg gct aga aag att gtt cca ttc att aga gct 480
Arg Arg Tyr Lys Ala Leu Ala Arg Lys Ile Val Pro Phe Ile Arg Ala
125 130 135
tct ggt tct gac aga gtt att gct tct gct gaa aag ttc att gaa ggt 528
Ser Gly Ser Asp Arg Val Ile Ala Ser Ala Glu Lys Phe Ile Glu Gly
140 145 150
ttc caa tct gct aag ttg gct gac cca ggt tct caa cca cac caa gct 576
Phe Gln Ser Ala Lys Leu Ala Asp Pro Gly Ser Gln Pro His Gln Ala
155 160 165
tct cca gtt att aac gtg atc att cca gaa gga tcc ggt tac aac aac 624
Ser Pro Val Ile Asn Val Ile Ile Pro Glu Gly Ser Gly Tyr Asn Asn
170 175 180 185
act ttg gac cat ggt ctt tgt act gct ttc gaa gac tct acc cta ggt 672
Thr Leu Asp His Gly Leu Cys Thr Ala Phe Glu Asp Ser Thr Leu Gly
190 195 200
gac gac gtt gaa gct aac ttc act gct ttg ttc gct cca gct att aga 720
Asp Asp Val Glu Ala Asn Phe Thr Ala Leu Phe Ala Pro Ala Ile Arg
205 210 215
gct aga ttg gaa gct gac ttg cca ggt gtt act ttg act gac gaa gac 768
Ala Arg Leu Glu Ala Asp Leu Pro Gly Val Thr Leu Thr Asp Glu Asp
220 225 230
gtt gtt tac ttg atg gac atg tgt cca ttc gac act gtc gct aga act 816
Val Val Tyr Leu Met Asp Met Cys Pro Phe Asp Thr Val Ala Arg Thr
235 240 245
tct gac gct act gaa ttg tct cca ttc tgt gct ttg ttc act cac gac 864
Ser Asp Ala Thr Glu Leu Ser Pro Phe Cys Ala Leu Phe Thr His Asp
250 255 260 265
gaa tgg atc caa tac gac tac ttg caa agc ttg ggt aag tac tac ggt 912
Glu Trp Ile Gln Tyr Asp Tyr Leu Gln Ser Leu Gly Lys Tyr Tyr Gly
270 275 280
tac ggt gct ggt aac cca ttg ggt cca gct caa ggt gtt ggt ttc gct 960
Tyr Gly Ala Gly Asn Pro Leu Gly Pro Ala Gln Gly Val Gly Phe Ala
285 290 295
aac gaa ttg att gct aga ttg act cac tct cca gtt caa gac cac act 1008
Asn Glu Leu Ile Ala Arg Leu Thr His Ser Pro Val Gln Asp His Thr
300 305 310
tct act aac cac act ttg gac tct aac cca gct act ttc cca ttg aac 1056
Ser Thr Asn His Thr Leu Asp Ser Asn Pro Ala Thr Phe Pro Leu Asn
315 320 325
gct act ttg tac gct gac ttc tct cac gac aac act atg ata tct att 1104
Ala Thr Leu Tyr Ala Asp Phe Ser His Asp Asn Thr Met Ile Ser Ile
330 335 340 345
ttc ttc gct ttg ggt ttg tac aac ggt acc aag cca ttg tct act act 1152
Phe Phe Ala Leu Gly Leu Tyr Asn Gly Thr Lys Pro Leu Ser Thr Thr
350 355 360
tct gtt gaa tct att gaa gaa act gac ggt tac tct gct tct tgg act 1200
Ser Val Glu Ser Ile Glu Glu Thr Asp Gly Tyr Ser Ala Ser Trp Thr
365 370 375
gtt cca ttc gct gct aga gct tac gtt gaa atg atg caa tgt caa gct 1248
Val Pro Phe Ala Ala Arg Ala Tyr Val Glu Met Met Gln Cys Gln Ala
380 385 390
gaa aag gaa cca ttg gtt aga gtt ttg gtt aac gac aga gtt gtt cca 1296
Glu Lys Glu Pro Leu Val Arg Val Leu Val Asn Asp Arg Val Val Pro
395 400 405
ttg cac ggt tgt gct gtt gac aag ttg ggt aga tgt aag aga gac gac 1344
Leu His Gly Cys Ala Val Asp Lys Leu Gly Arg Cys Lys Arg Asp Asp
410 415 420 425
ttc gtt gaa ggt ttg tct ttc gct aga tct ggt ggt aac tgg gct gaa 1392
Phe Val Glu Gly Leu Ser Phe Ala Arg Ser Gly Gly Asn Trp Ala Glu
430 435 440
tgt ttc gct taa 1404
Cys Phe Ala
<210>93
<211>467
<212>PRT
<213>人工序列
<223>人工序列的描述：共有植酸酶-3-thermo-11-Q50T-K91A
<400>93
Met Gly Val Phe Val Val Leu Leu Ser Ile Ala Thr Leu Phe Gly Ser
-20 -15 -10
Thr Ser Gly Thr Ala Leu Gly Pro Arg Gly Asn Ser His Ser Cys Asp
-5 -1 1 5
Thr Val Asp Gly Gly Tyr Gln Cys Phe Pro Glu Ile Ser His Leu Trp
10 15 20 25
Gly Thr Tyr Ser Pro Tyr Phe Ser Leu Ala Asp Glu Ser Ala Ile Ser
30 35 40
Pro Asp Val Pro Lys Asp Cys Arg Val Thr Phe Val Gln Val Leu Ser
45 50 55
Arg His Gly Ala Arg Tyr Pro Thr Ser Ser Ala Ser Lys Ala Tyr Ser
60 65 70
Ala Leu Ile Glu Ala Ile Gln Lys Asn Ala Thr Ala Phe Lys Gly Lys
75 80 85
Tyr Ala Phe Leu Lys Thr Tyr Asn Tyr Thr Leu Gly Ala Asp Asp Leu
90 95 100 105
Thr Pro Phe Gly Glu Asn Gln Met Val Asn Ser Gly Ile Lys Phe Tyr
110 115 120
Arg Arg Tyr Lys Ala Leu Ala Arg Lys Ile Val Pro Phe Ile Arg Ala
125 130 135
Ser Gly Ser Asp Arg Val Ile Ala Ser Ala Glu Lys Phe Ile Glu Gly
140 145 150
Phe Gln Ser Ala Lys Leu Ala Asp Pro Gly Ser Gln Pro His Gln Ala
155 160 165
Ser Pro Val Ile Asn Val Ile Ile Pro Glu Gly Ser Gly Tyr Asn Asn
170 175 180 185
Thr Leu Asp His Gly Leu Cys Thr Ala Phe Glu Asp Ser Thr Leu Gly
190 195 200
Asp Asp Val Glu Ala Asn Phe Thr Ala Leu Phe Ala Pro Ala Ile Arg
205 210 215
Ala Arg Leu Glu Ala Asp Leu Pro Gly Val Thr Leu Thr Asp Glu Asp
220 225 230
Val Val Tyr Leu Met Asp Met Cys Pro Phe Asp Thr Val Ala Arg Thr
235 240 245
Ser Asp Ala Thr Glu Leu Ser Pro Phe Cys Ala Leu Phe Thr His Asp
250 255 260 265
Glu Trp Ile Gln Tyr Asp Tyr Leu Gln Ser Leu Gly Lys Tyr Tyr Gly
270 275 280
Tyr Gly Ala Gly Asn Pro Leu Gly Pro Ala Gln Gly Val Gly Phe Ala
285 290 295
Asn Glu Leu Ile Ala Arg Leu Thr His Ser Pro Val Gln Asp His Thr
300 305 310
Ser Thr Asn His Thr Leu Asp Ser Asn Pro Ala Thr Phe Pro Leu Asn
315 320 325
Ala Thr Leu Tyr Ala Asp Phe Ser His Asp Asn Thr Met Ile Ser Ile
330 335 340 345
Phe Phe Ala Leu Gly Leu Tyr Asn Gly Thr Lys Pro Leu Ser Thr Thr
350 355 360
Ser Val Glu Ser Ile Glu Glu Thr Asp Gly Tyr Ser Ala Ser Trp Thr
365 370 375
Val Pro Phe Ala Ala Arg Ala Tyr Val Glu Met Met Gln Cys Gln Ala
380 385 390
Glu Lys Glu Pro Leu Val Arg Val Leu Val Asn Asp Arg Val Val Pro
395 400 405
Leu His Gly Cys Ala Val Asp Lys Leu Gly Arg Cys Lys Arg Asp Asp
410 415 420 425
Phe Val Glu Gly Leu Ser Phe Ala Arg Ser Gly Gly Asn Trp Ala Glu
430 435 440
Cys Phe Ala
<210>94
<211>1404
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述：共有植酸酶-10-thermo-5-Q50T
<220>
<221>单肽
<222>(1)..(69)
<220>
<221>CDS
<222>(1)..(1401)
<220>
<221>成熟肽
<222>(70)..(1401)
<400>94
atg ggc gtg ttc gtc gtg cta ctg tcc att gcc acc ttg ttc ggt tcc 48
Met Gly Val Phe Val Val Leu Leu Ser Ile Ala Thr Leu Phe Gly Ser
-20 -15 -10
aca tcc ggt acc gcc ttg ggt cct cgt ggt aat tct cac tct tgt gac 96
Thr Ser Gly Thr Ala Leu Gly Pro Arg Gly Asn Ser His Ser Cys Asp
-5 -1 1 5
act gtt gac ggt ggt tac caa tgt ttc cca gaa att tct cac ttg tgg 144
Thr Val Asp Gly Gly Tyr Gln Cys Phe Pro Glu Ile Ser His Leu Trp
10 15 20 25
ggt aca tac tct cca ttc ttc tct ttg gct gac gaa tct gct att tct 192
Gly Thr Tyr Ser Pro Phe Phe Ser Leu Ala Asp Glu Ser Ala Ile Ser
30 35 40
cca gac gtt cca aag ggt tgt aga gtt act ttc gtt caa gtt ttg tct 240
Pro Asp Val Pro Lys Gly Cys Arg Val Thr Phe Val Gln Val Leu Ser
45 50 55
aga cac ggt gct aga tac cca act tct tct aag tct aag gct tac tct 288
Arg His Gly Ala Arg Tyr Pro Thr Ser Ser Lys Ser Lys Ala Tyr Ser
60 65 70
gct ttg att gaa gct att caa aag aac gct act gct ttc aag ggt aag 336
Ala Leu Ile Glu Ala Ile Gln Lys Asn Ala Thr Ala Phe Lys Gly Lys
75 80 85
tac gct ttc ttg aag act tac aat tac act ttg ggt gct gac gac ttg 384
Tyr Ala Phe Leu Lys Thr Tyr Asn Tyr Thr Leu Gly Ala Asp Asp Leu
90 95 100 105
act cca ttc ggt gaa caa caa atg gtt aac tct ggt att aag ttc tac 432
Thr Pro Phe Gly Glu Gln Gln Met Val Asn Ser Gly Ile Lys Phe Tyr
110 115 120
aga aga tac aag gct ttg gct aga aag att gtt cca ttc att aga gct 480
Arg Arg Tyr Lys Ala Leu Ala Arg Lys Ile Val Pro Phe Ile Arg Ala
125 130 135
tct ggt tct gac aga gtt att gct tct gcc gaa aag ttc att gaa ggt 528
Ser Gly Ser Asp Arg Val Ile Ala Ser Ala Glu Lys Phe Ile Glu Gly
140 145 150
ttc caa tct gct aag ttg gct gac cca ggt gct aac cca cac caa gct 576
Phe Gln Ser Ala Lys Leu Ala Asp Pro Gly Ala Asn Pro His Gln Ala
155 160 165
tct cca gtt att aac gtt att att cca gaa ggt gct ggt tac aac aac 624
Ser Pro Val Ile Asn Val Ile Ile Pro Glu Gly Ala Gly Tyr Asn Asn
170 175 180 185
act ttg gac cac ggt ttg tgt act gct ttc gaa gaa tct acc cta ggt 672
Thr Leu Asp His Gly Leu Cys Thr Ala Phe Glu Glu Ser Thr Leu Gly
190 195 200
gac gac gtt gaa gct aac ttc act gct gtt ttc gct cca cca att aga 720
Asp Asp Val Glu Ala Asn Phe Thr Ala Val Phe Ala Pro Pro Ile Arg
205 210 215
gct aga ttg gaa gct cac ttg cca ggt gtt aac ttg act gac gaa gac 768
Ala Arg Leu Glu Ala His Leu Pro Gly Val Asn Leu Thr Asp Glu Asp
220 225 230
gtt gtt aac ttg atg gac atg tgt cca ttc gac act gtt gct aga act 816
Val Val Asn Leu Met Asp Met Cys Pro Phe Asp Thr Val Ala Arg Thr
235 240 245
tct gac gct act caa ttg tct cca ttc tgt gac ttg ttc act cac gac 864
Ser Asp Ala Thr Gln Leu Ser Pro Phe Cys Asp Leu Phe Thr His Asp
250 255 260 265
gaa tgg att caa tac gac tac ttg caa tct ttg ggt aag tac tac ggt 912
Glu Trp Ile Gln Tyr Asp Tyr Leu Gln Ser Leu Gly Lys Tyr Tyr Gly
270 275 280
tac ggt gct ggt aac cca ttg ggt cca gct caa ggt gtt ggt ttc gtt 960
Tyr Gly Ala Gly Asn Pro Leu Gly Pro Ala Gln Gly Val Gly Phe Val
285 290 295
aac gaa ttg att gct aga ttg act cac tct cca gtt caa gac cac act 1008
Asn Glu Leu Ile Ala Arg Leu Thr His Ser Pro Val Gln Asp His Thr
300 305 310
tct act aac cac act ttg gac tct aac cca gct act ttc cca ttg aac 1056
Ser Thr Asn His Thr Leu Asp Ser Asn Pro Ala Thr Phe Pro Leu Asn
315 320 325
gct act ttg tac gct gac ttc tct cac gac aac act atg gtt tct att 1104
Ala Thr Leu Tyr Ala Asp Phe Ser His Asp Asn Thr Met Val Ser Ile
330 335 340 345
ttc ttc gct ttg ggt ttg tac aac ggt act aag cca ttg tct act act 1152
Phe Phe Ala Leu Gly Leu Tyr Asn Gly Thr Lys Pro Leu Ser Thr Thr
350 355 360
tct gtt gaa tct att gaa gaa act gac ggt tac tct gct tct tgg act 1200
Ser Val Glu Ser Ile Glu Glu Thr Asp Gly Tyr Ser Ala Ser Trp Thr
365 370 375
gtt cca ttc gct gct aga gct tac gtt gaa atg atg caa tgt gaa gct 1248
Val Pro Phe Ala Ala Arg Ala Tyr Val Glu Met Met Gln Cys Glu Ala
380 385 390
gaa aag gaa cca ttg gtt aga gtt ttg gtt aac gac aga gtt gtt cca 1296
Glu Lys Glu Pro Leu Val Arg Val Leu Val Asn Asp Arg Val Val Pro
395 400 405
ttg cac ggt tgt gct gtt gac aag ttg ggt aga tgt aag aga gac gac 1344
Leu His Gly Cys Ala Val Asp Lys Leu Gly Arg Cys Lys Arg Asp Asp
410 415 420 425
ttc gtt gaa ggt ttg tct ttc gct aga tct ggt ggt aac tgg gaa gaa 1392
Phe Val Glu Gly Leu Ser Phe Ala Arg Ser Gly Gly Asn Trp Glu Glu
430 435 440
tgt ttc gct taa 1404
Cys Phe Ala
<210>95
<211>467
<212>PRT
<213>人工序列
<223>人工序列的描述：共有植酸酶-10-thermo-5-Q50T
<400>95
Met Gly Val Phe Val Val Leu Leu Ser Ile Ala Thr Leu Phe Gly Ser
-20 -15 -10
Thr Ser Gly Thr Ala Leu Gly Pro Arg Gly Asn Ser His Ser Cys Asp
-5 -1 1 5
Thr Val Asp Gly Gly Tyr Gln Cys Phe Pro Glu Ile Ser His Leu Trp
10 15 20 25
Gly Thr Tyr Ser Pro Phe Phe Ser Leu Ala Asp Glu Ser Ala Ile Ser
30 35 40
Pro Asp Val Pro Lys Gly Cys Arg Val Thr Phe Val Gln Val Leu Ser
45 50 55
Arg His Gly Ala Arg Tyr Pro Thr Ser Ser Lys Ser Lys Ala Tyr Ser
60 65 70
Ala Leu Ile Glu Ala Ile Gln Lys Asn Ala Thr Ala Phe Lys Gly Lys
75 80 85
Tyr Ala Phe Leu Lys Thr Tyr Asn Tyr Thr Leu Gly Ala Asp Asp Leu
90 95 100 105
Thr Pro Phe Gly Glu Gln Gln Met Val Asn Ser Gly Ile Lys Phe Tyr
110 115 120
Arg Arg Tyr Lys Ala Leu Ala Arg Lys Ile Val Pro Phe Ile Arg Ala
125 130 135
Ser Gly Ser Asp Arg Val Ile Ala Ser Ala Glu Lys Phe Ile Glu Gly
140 145 150
Phe Gln Ser Ala Lys Leu Ala Asp Pro Gly Ala Asn Pro His Gln Ala
155 160 165
Ser Pro Val Ile Asn Val Ile Ile Pro Glu Gly Ala Gly Tyr Asn Asn
170 175 180 185
Thr Leu Asp His Gly Leu Cys Thr Ala Phe Glu Glu Ser Thr Leu Gly
190 195 200
Asp Asp Val Glu Ala Asn Phe Thr Ala Val Phe Ala Pro Pro Ile Arg
205 210 215
Ala Arg Leu Glu Ala His Leu Pro Gly Val Asn Leu Thr Asp Glu Asp
220 225 230
Val Val Asn Leu Met Asp Met Cys Pro Phe Asp Thr Val Ala Arg Thr
235 240 245
Ser Asp Ala Thr Gln Leu Ser Pro Phe Cys Asp Leu Phe Thr His Asp
250 255 260 265
Glu Trp Ile Gln Tyr Asp Tyr Leu Gln Ser Leu Gly Lys Tyr Tyr Gly
270 275 280
Tyr Gly Ala Gly Asn Pro Leu Gly Pro Ala Gln Gly Val Gly Phe Val
285 290 295
Asn Glu Leu Ile Ala Arg Leu Thr His Ser Pro Val Gln Asp His Thr
300 305 310
Ser Thr Asn His Thr Leu Asp Ser Asn Pro Ala Thr Phe Pro Leu Asn
315 320 325
Ala Thr Leu Tyr Ala Asp Phe Ser His Asp Asn Thr Met Val Ser Ile
330 335 340 345
Phe Phe Ala Leu Gly Leu Tyr Asn Gly Thr Lys Pro Leu Ser Thr Thr
350 355 360
Ser Val Glu Ser Ile Glu Glu Thr Asp Gly Tyr Ser Ala Ser Trp Thr
365 370 375
Val Pro Phe Ala Ala Arg Ala Tyr Val Glu Met Met Gln Cys Glu Ala
380 385 390
Glu Lys Glu Pro Leu Val Arg Val Leu Val Asn Asp Arg Val Val Pro
395 400 405
Leu His Gly Cys Ala Val Asp Lys Leu Gly Arg Cys Lys Arg Asp Asp
410 415 420 425
Phe Val Glu Gly Leu Ser Phe Ala Arg Ser Gly Gly Asn Trp Glu Glu
430 435 440
Cys Phe Ala
<210>96
<211>1404
<212>DNA
<2l3>人工序列
<220>
<223>人工序列的描述：共有植酸酶-10-thermo-5-Q50T-K91A
<220>
<221>单肽
<222>(1)..(69)
<220>
<221>CDS
<222>(1)..(1401)
<220>
<221>成熟肽
<222>(70)..(1401)
<400>96
atg ggc gtg ttc gtc gtg cta ctg tcc att gcc acc ttg ttc ggt tcc 48
Met Gly Val Phe Val Val Leu Leu Ser Ile Ala Thr Leu Phe Gly Ser
-20 -15 -10
aca tcc ggt acc gcc ttg ggt cct cgt ggt aat tct cac tct tgt gac 96
Thr Ser Gly Thr Ala Leu Gly Pro Arg Gly Asn Ser His Ser Cys Asp
-5 -1 1 5
act gtt gac ggt ggt tac caa tgt ttc cca gaa att tct cac ttg tgg 144
Thr Val Asp Gly Gly Tyr Gln Cys Phe Pro Glu Ile Ser His Leu Trp
10 15 20 25
ggt aca tac tct cca ttc ttc tct ttg gct gac gaa tct gct att tct 192
Gly Thr Tyr Ser Pro Phe Phe Ser Leu Ala Asp Glu Ser Ala Ile Ser
30 35 40
cca gac gtt cca aag ggt tgt aga gtt act ttc gtt caa gtt ttg tct 240
Pro Asp Val Pro Lys Gly Cys Arg Val Thr Phe Val Gln Val Leu Ser
45 50 55
aga cac ggt gct aga tac cca act tct tct gcg tct aag gct tac tct 288
Arg His Gly Ala Arg Tyr Pro Thr Ser Ser Ala Ser Lys Ala Tyr Ser
60 65 70
gct ttg att gaa gct att caa aag aac gct act gct ttc aag ggt aag 336
Ala Leu Ile Glu Ala Ile Gln Lys Asn Ala Thr Ala Phe Lys Gly Lys
75 80 85
tac gct ttc ttg aag act tac aat tac act ttg ggt gct gac gac ttg 384
Tyr Ala Phe Leu Lys Thr Tyr Asn Tyr Thr Leu Gly Ala Asp Asp Leu
90 95 100 105
act cca ttc ggt gaa caa caa atg gtt aac tct ggt att aag ttc tac 432
Thr Pro Phe Gly Glu Gln Gln Met Val Asn Ser Gly Ile Lys Phe Tyr
110 115 120
aga aga tac aag gct ttg gct aga aag att gtt cca ttc att aga gct 480
Arg Arg Tyr Lys Ala Leu Ala Arg Lys Ile Val Pro Phe Ile Arg Ala
125 130 135
tct ggt tct gac aga gtt att gct tct gcc gaa aag ttc att gaa ggt 528
Ser Gly Ser Asp Arg Val Ile Ala Ser Ala Glu Lys Phe Ile Glu Gly
140 145 150
ttc caa tct gct aag ttg gct gac cca ggt gct aac cca cac caa gct 576
Phe Gln Ser Ala Lys Leu Ala Asp Pro Gly Ala Asn Pro His Gln Ala
155 160 165
tct cca gtt att aac gtt att att cca gaa ggt gct ggt tac aac aac 624
Ser Pro Val Ile Asn Val Ile Ile Pro Glu Gly Ala Gly Tyr Asn Asn
170 175 180 185
act ttg gac cac ggt ttg tgt act gct ttc gaa gaa tct acc cta ggt 672
Thr Leu Asp His Gly Leu Cys Thr Ala Phe Glu Glu Ser Thr Leu Gly
190 195 200
gac gac gtt gaa gct aac ttc act gct gtt ttc gct cca cca att aga 720
Asp Asp Val Glu Ala Asn Phe Thr Ala Val Phe Ala Pro Pro Ile Arg
205 210 215
gct aga ttg gaa gct cac ttg cca ggt gtt aac ttg act gac gaa gac 768
Ala Arg Leu Glu Ala His Leu Pro Gly Val Asn Leu Thr Asp Glu Asp
220 225 230
gtt gtt aac ttg atg gac atg tgt cca ttc gac act gtt gct aga act 816
Val Val Asn Leu Met Asp Met Cys Pro Phe Asp Thr Val Ala Arg Thr
235 240 245
tct gac gct act caa ttg tct cca ttc tgt gac ttg ttc act cac gac 864
Ser Asp Ala Thr Gln Leu Ser Pro Phe Cys Asp Leu Phe Thr His Asp
250 255 260 265
gaa tgg att caa tac gac tac ttg caa tct ttg ggt aag tac tac ggt 912
Glu Trp Ile Gln Tyr Asp Tyr Leu Gln Ser Leu Gly Lys Tyr Tyr Gly
270 275 280
tac ggt gct ggt aac cca ttg ggt cca gct caa ggt gtt ggt ttc gtt 960
Tyr Gly Ala Gly Asn Pro Leu Gly Pro Ala Gln Gly Val Gly Phe Val
285 290 295
aac gaa ttg att gct aga ttg act cac tct cca gtt caa gac cac act 1008
Asn Glu Leu Ile Ala Arg Leu Thr His Ser Pro Val Gln Asp His Thr
300 305 310
tct act aac cac act ttg gac tct aac cca gct act ttc cca ttg aac 1056
Ser Thr Asn His Thr Leu Asp Ser Asn Pro Ala Thr Phe Pro Leu Asn
315 320 325
gct act ttg tac gct gac ttc tct cac gac aac act atg gtt tct att 1104
Ala Thr Leu Tyr Ala Asp Phe Ser His Asp Asn Thr Met Val Ser Ile
330 335 340 345
ttc ttc gct ttg ggt ttg tac aac ggt act aag cca ttg tct act act 1152
Phe Phe Ala Leu Gly Leu Tyr Asn Gly Thr Lys Pro Leu Ser Thr Thr
350 355 360
tct gtt gaa tct att gaa gaa act gac ggt tac tct gct tct tgg act 1200
Ser Val Glu Ser Ile Glu Glu Thr Asp Gly Tyr Ser Ala Ser Trp Thr
365 370 375
gtt cca ttc gct gct aga gct tac gtt gaa atg atg caa tgt gaa gct 1248
Val Pro Phe Ala Ala Arg Ala Tyr Val Glu Met Met Gln Cys Glu Ala
380 385 390
gaa aag gaa cca ttg gtt aga gtt ttg gtt aac gac aga gtt gtt cca 1296
Glu Lys Glu Pro Leu Val Arg Val Leu Val Asn Asp Arg Val Val Pro
395 400 405
ttg cac ggt tgt gct gtt gac aag ttg ggt aga tgt aag aga gac gac 1344
Leu His Gly Cys Ala Val Asp Lys Leu Gly Arg Cys Lys Arg Asp Asp
410 415 420 425
ttc gtt gaa ggt ttg tct ttc gct aga tct ggt ggt aac tgg gaa gaa 1392
Phe Val Glu Gly Leu Ser Phe Ala Arg Ser Gly Gly Asn Trp Glu Glu
430 435 440
tgt ttc gct taa 1404
Cys Phe Ala
<210>97
<211>467
<212>PRT
<213>人工序列
<223>人工序列的描述：共有植酸酶-10-thermo-5-Q50T-K91A
<400>97
Met Gly Val Phe Val Val Leu Leu Ser Ile Ala Thr Leu Phe Gly Ser
-20 -15 -10
Thr Ser Gly Thr Ala Leu Gly Pro Arg Gly Asn Ser His Ser Cys Asp
-5 -1 1 5
Thr Val Asp Gly Gly Tyr Gln Cys Phe Pro Glu Ile Ser His Leu Trp
10 15 20 25
Gly Thr Tyr Ser Pro Phe Phe Ser Leu Ala Asp Glu Ser Ala Ile Ser
30 35 40
Pro Asp Val Pro Lys Gly Cys Arg Val Thr Phe Val Gln Val Leu Ser
45 50 55
Arg His Gly Ala Arg Tyr Pro Thr Ser Ser Ala Ser Lys Ala Tyr Ser
60 65 70
Ala Leu Ile Glu Ala Ile Gln Lys Asn Ala Thr Ala Phe Lys Gly Lys
75 80 85
Tyr Ala Phe Leu Lys Thr Tyr Asn Tyr Thr Leu Gly Ala Asp Asp Leu
90 95 100 105
Thr Pro Phe Gly Glu Gln Gln Met Val Asn Ser Gly Ile Lys Phe Tyr
110 115 120
Arg Arg Tyr Lys Ala Leu Ala Arg Lys Ile Val Pro Phe Ile Arg Ala
125 130 135
Ser Gly Ser Asp Arg Val Ile Ala Ser Ala Glu Lys Phe Ile Glu Gly
140 145 150
Phe Gln Ser Ala Lys Leu Ala Asp Pro Gly Ala Asn Pro His Gln Ala
155 160 165
Ser Pro Val Ile Asn Val Ile Ile Pro Glu Gly Ala Gly Tyr Asn Asn
170 175 180 185
Thr Leu Asp His Gly Leu Cys Thr Ala Phe Glu Glu Ser Thr Leu Gly
190 195 200
Asp Asp Val Glu Ala Asn Phe Thr Ala Val Phe Ala Pro Pro Ile Arg
205 210 215
Ala Arg Leu Glu Ala His Leu Pro Gly Val Asn Leu Thr Asp Glu Asp
220 225 230
Val Val Asn Leu Met Asp Met Cys Pro Phe Asp Thr Val Ala Arg Thr
235 240 245
Ser Asp Ala Thr Gln Leu Ser Pro Phe Cys Asp Leu Phe Thr His Asp
250 255 260 265
Glu Trp Ile Gln Tyr Asp Tyr Leu Gln Ser Leu Gly Lys Tyr Tyr Gly
270 275 280
Tyr Gly Ala Gly Asn Pro Leu Gly Pro Ala Gln Gly Val Gly Phe Val
285 290 295
Asn Glu Leu Ile Ala Arg Leu Thr His Ser Pro Val Gln Asp His Thr
300 305 310
Ser Thr Asn His Thr Leu Asp Ser Asn Pro Ala Thr Phe Pro Leu Asn
315 320 325
Ala Thr Leu Tyr Ala Asp Phe Ser His Asp Asn Thr Met Val Ser Ile
330 335 340 345
Phe Phe Ala Leu Gly Leu Tyr Asn Gly Thr Lys Pro Leu Ser Thr Thr
350 355 360
Ser Val Glu Ser Ile Glu Glu Thr Asp Gly Tyr Ser Ala Ser Trp Thr
365 370 375
Val Pro Phe Ala Ala Arg Ala Tyr Val Glu Met Met Gln Cys Glu Ala
380 385 390
Glu Lys Glu Pro Leu Val Arg Val Leu Val Asn Asp Arg Val Val Pro
395 400 405
Leu His Gly Cys Ala Val Asp Lys Leu Gly Arg Cys Lys Arg Asp Asp
410 415 420 425
Phe Val Glu Gly Leu Ser Phe Ala Arg Ser Gly Gly Asn Trp Glu Glu
430 435 440
Cys Phe Ala
Claims (16)
1.一种植酸酶，其含有与SEQ ID NO：26中氨基酸-26到+441的序列具有至少93.80％一致性的氨基酸序列，所述一致性用GCG软件包9.1版所提供的GAP程序以下述设置确定：长度权重为0，缺口权重为3.0。
2.一种植酸酶，其由与SEQ ID NO：25之核苷酸12-1412具有至少95.88％一致性的DNA序列编码，所述一致性用GCG软件包9.1版所提供的GAP程序以下述设置确定：GAP产生罚分为50，GAP延伸罚分为3。
3.一种植酸酶，其含有选自下组的氨基酸序列
(i)SEQ ID NO：26的序列-26至+441，+1至+441，或+1至+438，或
(ii)由SEQ ID NO：25的核苷酸12-1412或90-1412编码的氨基酸序列。
4.一种植酸酶，其包含选自下组的氨基酸序列
(i)SEQ ID NO：26的变体K94A+V158I+A396S，或
(ii)(i)的变体Q50T，K91A，或Q50T+K91A，或
(iii)序列(i)和(ii)之任一的序列+1至441，或
(iv)SEQ ID NO：31的序列-26至+441，或+1至+441；或
(v)由SEQ ID NO：30的核苷酸1-1401，或79-1401编码的氨基酸序列。
5.一种植酸酶，其包含选自下组的氨基酸序列
(i)SEQ ID NO：29的变体T50Q+A91K，或
(ii)(i)的变体Q50T，K91A，或Q50T+K91A，或
(iii)序列(i)和(ii)之任一的序列+1至441，或
(iv)SEQ ID NO：29的序列-26至+441，或+1至+441；或
(v)由SEQ ID NO：28的核苷酸1-1401，或79-1401编码的氨基酸序列。
6.一种植酸酶，其含有SEQ ID NO：27的氨基酸序列。
7.一种DNA序列，其含有编码权利要求1-6之任一植酸酶的DNA序列。
8.一种含有编码植酸酶之DNA序列的DNA序列，所述编码植酸酶的DNA序列与SEQ ID NO：25中核苷酸12-1412具有至少95.88％一致性，所述一致性用GCG软件包9.1版所提供的GAP程序以下述设置确定：GAP产生罚分为50，GAP延伸罚分为3。
9.一种含有编码植酸酶之DNA序列的DNA序列，所述植酸酶含有与SEQ ID NO：26之氨基酸-26至+441具有至少93.80％一致性的氨基酸序列，所述一致性用GCG软件包9.1版所提供的GAP程序以下述设置确定：GAP产生罚分为50，GAP延伸罚分为3。
10.一种含有编码植酸酶之DNA序列的DNA序列，其中所述编码植酸酶的DNA序列含有：
(i)SEQ ID NO：25的核苷酸12-1412，或90-1412；或
(ii)SEQ ID NO：30的核苷酸1-1401，或79-1401；或
(iii)SEQ ID NO：28的核苷酸1-1401，或79-1401。
11.一种载体，其含有权利要求7-10之任一的DNA序列。
12.一种微生物宿主细胞，其含有权利要求7-10之任一的DNA序列，或权利要求11的载体。
13.一种生产植酸酶的方法，该方法包括在允许植酸酶产生的条件下培养权利要求12的宿主细胞，并从培养基中回收植酸酶。
14.一种食品，其含有权利要求1-6之任一的植酸酶。
15.一种饲料，其含有权利要求1-6之任一的植酸酶。
16.一种药物组合物，其含有权利要求1-6之任一的植酸酶。
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DKPA199900092 | 1999-01-22 | ||
| DKPA199900092 | 1999-01-22 | ||
| DKPA199901340 | 1999-09-21 | ||
| DKPA199901340 | 1999-09-21 |
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| CN1344315A CN1344315A (zh) | 2002-04-10 |
| CN1179041C true CN1179041C (zh) | 2004-12-08 |
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| CNB00805245XA Expired - Fee Related CN1179041C (zh) | 1999-01-22 | 2000-01-21 | 改进的植酸酶 |
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| EP (1) | EP1151088B1 (zh) |
| JP (1) | JP2002534976A (zh) |
| CN (1) | CN1179041C (zh) |
| AT (1) | ATE338110T1 (zh) |
| AU (1) | AU781520B2 (zh) |
| BR (1) | BR0007655A (zh) |
| CA (1) | CA2356642A1 (zh) |
| DE (1) | DE60030412T2 (zh) |
| WO (1) | WO2000043503A1 (zh) |
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| WO2021078839A1 (en) | 2019-10-22 | 2021-04-29 | Novozymes A/S | Animal feed composition |
| US20230059825A1 (en) | 2020-01-14 | 2023-02-23 | Evonik Operations Gmbh | Bacillus strains with the ability to degrade inorganic nitrogen compounds |
| WO2021233936A1 (en) | 2020-05-18 | 2021-11-25 | Dsm Ip Assets B.V. | Animal feed compositions |
| WO2022058322A1 (en) | 2020-09-15 | 2022-03-24 | Novozymes A/S | Animal feed comprising insects or insect meal |
| EP4130258A1 (en) | 2021-08-02 | 2023-02-08 | Evonik Operations GmbH | Microorganisms displaying viral decoy receptors |
| EP4166002A1 (en) | 2021-10-12 | 2023-04-19 | Evonik Operations GmbH | Microbial preparations containing specific cryoprotectants |
| WO2023110957A1 (en) | 2021-12-15 | 2023-06-22 | Dsm Ip Assets B.V. | Methods and uses for improving egg production and egg quality involving administering feed comprising muramidase (ec 3.2.1.17) |
| WO2023131629A1 (en) | 2022-01-06 | 2023-07-13 | Novozymes A/S | Animal feed composition and use thereof |
| EP4230721A1 (en) | 2022-02-17 | 2023-08-23 | Evonik Operations GmbH | Microorganisms with reduced competence |
| EP4230722A1 (en) | 2022-02-17 | 2023-08-23 | Evonik Operations GmbH | Bacillota strains with improved outgrowth |
| WO2023180282A1 (en) | 2022-03-24 | 2023-09-28 | Evonik Operations Gmbh | Co-precipitates of methionylmethionine with organic compounds |
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| US6936694B1 (en) * | 1982-05-06 | 2005-08-30 | Intermune, Inc. | Manufacture and expression of large structural genes |
| CZ289014B6 (cs) * | 1989-09-27 | 2001-10-17 | Dsm N. V. | Vyčiątěná a izolovaná sekvence DNA kódující fungální fytázu, konstrukt pro expresi, vektory a transformované hostitelské buňky a způsob produkce fytázy |
| WO1997035016A1 (en) * | 1996-03-18 | 1997-09-25 | Novo Nordisk Biotech Inc | Polypeptides having phytase activity and nucleic acids encoding same |
| NZ330940A (en) * | 1997-07-24 | 2000-02-28 | F | Production of consensus phytases from fungal origin using computer programmes |
| ES2321047T3 (es) * | 1998-03-23 | 2009-06-01 | Novozymes A/S | Variantes de fitasa. |
| WO1999048380A1 (en) * | 1998-03-23 | 1999-09-30 | Novo Nordisk A/S | Thermostable phytases in feed preparation and plant expression |
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2000
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- 2000-01-21 BR BR0007655-4A patent/BR0007655A/pt not_active Application Discontinuation
- 2000-01-21 CA CA002356642A patent/CA2356642A1/en not_active Abandoned
- 2000-01-21 EP EP00901049A patent/EP1151088B1/en not_active Expired - Lifetime
- 2000-01-21 AU AU20928/00A patent/AU781520B2/en not_active Ceased
- 2000-01-21 DE DE60030412T patent/DE60030412T2/de not_active Expired - Lifetime
- 2000-01-21 CN CNB00805245XA patent/CN1179041C/zh not_active Expired - Fee Related
- 2000-01-21 AT AT00901049T patent/ATE338110T1/de not_active IP Right Cessation
- 2000-01-21 JP JP2000594911A patent/JP2002534976A/ja active Pending
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| CN1344315A (zh) | 2002-04-10 |
| BR0007655A (pt) | 2001-11-06 |
| EP1151088B1 (en) | 2006-08-30 |
| DE60030412D1 (de) | 2006-10-12 |
| WO2000043503A1 (en) | 2000-07-27 |
| AU781520B2 (en) | 2005-05-26 |
| AU2092800A (en) | 2000-08-07 |
| ATE338110T1 (de) | 2006-09-15 |
| EP1151088A1 (en) | 2001-11-07 |
| CA2356642A1 (en) | 2000-07-27 |
| DE60030412T2 (de) | 2007-08-30 |
| JP2002534976A (ja) | 2002-10-22 |
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