CN1875097B - 杂交酶 - Google Patents
杂交酶 Download PDFInfo
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- CN1875097B CN1875097B CN2004800320254A CN200480032025A CN1875097B CN 1875097 B CN1875097 B CN 1875097B CN 2004800320254 A CN2004800320254 A CN 2004800320254A CN 200480032025 A CN200480032025 A CN 200480032025A CN 1875097 B CN1875097 B CN 1875097B
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Abstract
本发明涉及杂交酶,其包含至少一种碳水化合物结合模块氨基酸序列和至少葡糖淀粉酶氨基酸序列的催化模块。本发明还涉及这种杂交酶在淀粉处理以及尤其是乙醇产生中的用途。
Description
参考序列表
该申请包含计算机可读形式的序列表。其在此处一并作为参考。
发明背景
发明领域
本发明涉及至少一种碳水化合物结合模块(carbohydrate-bindingmodule,″CBM″)和至少葡糖淀粉酶的催化模块(catalytic module,CM)的杂交体及其它。本发明也涉及杂交酶在淀粉处理中的用途,在此处理中,颗粒淀粉降解为糖,例如,浆液(slurry)、或者可以用作产生发酵产物,尤其是乙醇所用的酵母的营养。
相关现有技术描述
已经描述了大量的将淀粉转化为淀粉水解产物,如麦芽糖、葡萄糖或特制浆液以用作甜味剂或作为其它糖类如果糖的前体的方法。葡萄糖也可发酵为乙醇或其它的发酵产物。
淀粉是由葡萄糖单位的链所组成的高分子量多聚体。其通常由约80%的支链淀粉和20%直链淀粉组成。支链淀粉为支链多糖,其中α-1,4D-葡萄糖残基的线性链通过α-1,6糖苷键连接起来。
直链淀粉是由D-吡喃葡萄糖单位通过α-1,4糖苷键连接在一起而构成的线性多糖。在将淀粉转化为可溶性淀粉水解产物的例子中,所述淀粉被解聚化。传统的解聚方法由糊化步骤(gelatinization step)和两个连续的处理步骤即液化处理和糖化处理所组成。
颗粒淀粉由显微镜下可见的颗粒组成,其在室温下不溶于水。水性淀粉浆液加热时,颗粒膨胀并最终破裂,将淀粉分子分散到溶液中。在此“糊化”处理期间粘性急剧增加。由于典型工业处理中固体水平为30-40%,淀粉必须稀释或“液化”以使之能够处理。目前粘性的降低主要通过酶学降解获得。在液化步骤期间,长链淀粉被α-淀粉酶降解为更小的支链和线性单位(麦芽糊精)。通常,液化处理在约105-110℃实施约5到10分钟,接下来约95℃实施约1-2小时。然后将温度降至60℃,加入葡糖淀粉酶或β-淀粉酶和可选的脱支酶如异淀粉酶或支链淀粉酶(pullulanase),糖化处理进行约24至72小时。
传统的淀粉转化处理是非常耗能的,因为许多步骤中在温度方面有不同的需求。因此需要能够选择和/或设计此处理中所使用的酶以使整个过程能够在无需糊化淀粉的情况下进行。
发明概述
第一个方面本发明提供了包含具有葡糖淀粉酶活性的催化模块的氨基酸序列和碳水化合物结合模块的氨基酸序列的杂交酶。优选催化模块可以是真菌、细菌、或植物起源的。
更多方面,本发明提供编码第一方面所述杂交酶的分离的DNA序列,包含编码第一方面所述杂交酶的DNA序列的DNA构建体,包含编码第一方面所述杂交酶的DNA序列的表达载体,用载体转化的宿主细胞,其中宿主细胞能够表达编码第一方面所述杂交酶的DNA序列。
最后一方面,本发明提供由颗粒淀粉产生浆液或发酵产物的方法,包括用水性介质中的α-淀粉酶和本发明具有葡糖淀粉酶活性的杂交酶处理天然淀粉(raw starch)。当需要发酵产物时,所述方法包括发酵生物的存在。
附图简述
图1比较了包含埃默森篮状菌(Talaromyces emersonii)催化结构域和黑曲霉(A.niger)SBM的两个葡糖淀粉酶-SBM(淀粉结合模块)杂交体(TEAN-1和TEAN-3)与野生型埃默森篮状菌葡糖淀粉酶的每g DS的乙醇产量。
发明详述
术语“颗粒淀粉(granular starch)”理解为天然(raw)的未煮过的淀粉,即未被糊化的淀粉。淀粉作为不溶于水的微小颗粒在植物中形成。这些颗粒在低于起始糊化温度的温度中储存在淀粉中。当置于冷水中时,谷物可以吸收少量的液体。达到50℃至70℃时膨胀是可逆的,可逆程度依赖于具体淀粉。当温度更高时,称为糊化作用的不可逆膨胀开始。
术语“起始糊化温度”理解为淀粉糊化作用开始时的最低温度。在水中加热的淀粉在50℃和75℃之间开始糊化;糊化的确切温度依赖于具体淀粉,熟练技术人员可以很容易地测定。因此,根据植物种类、植物种类的具体品种以及生长条件,起始糊化温度可以不同。在本发明的上下文中,给定淀粉的起始糊化温度为使用Gorinstein.S.和Lii.C.,Starch/,Vol.44(12)pp.461-466(1992)所描述的方法测定时,5%的淀粉颗粒中双折射(birefringence)消失时的温度。
术语“可溶性淀粉水解产物”理解为本发明方法的可溶性产物,可以包含单糖、二糖、和寡糖,例如葡萄糖、麦芽糖、麦芽糊精、环糊精及其任何混合物。优选至少90%,91%,92%,93%,94%,95%,96%,97%,98%或至少99%的颗粒淀粉的干燥固体转化为可溶性淀粉水解产物。
术语多肽“同源性”理解为两条序列之间的一致性程度,其表明第一条序列与第二条序列的衍生关系。可以使用本领域已知的电脑方法如GCG方法包中提供的GAP(Program Manual for the Wisconsin Package,Version 8,August 1994,Genetics Computer Group,575Science Drive,Madison,Wisconsin,USA 53711)(Needleman,S.B.和Wunsch,C.D.,(1970),Journal ofMolecular Biology,48,443-453)测定同源性。使用以下的氨基酸序列比对设置:GAP产生罚分为3.0,GAP延伸罚分为0.1。
杂交酶
本说明书及权利要求中的酶分类号(EC编号)与生物化学与分子生物学国际联合会的命名委员会,Academic Press Inc.,1992所推荐(1992)的一致。
此处所称杂交酶包括包含葡糖淀粉酶(EC 3.2.1.3)的氨基酸序列且其连接(即共价结合)至包含碳水化合物结合模块(CBM)的氨基酸序列的那些。术语“碳水化合物结合模块(CBM)”也可称“碳水化合物结合结构域(CBD)”。
含CBM的杂交酶,及其制备和纯化的详述描述是本领域已知的[参见,例如WO 90/00609,WO 94/24158和WO 95/16782,以及Greenwood等,Biotechnology and Bioengineering 44(1994)pp.1295-1305]。其可以通过例如将DNA构建体转化到宿主细胞中并培养该转化的宿主细胞以表达融合基因来制备,所述DNA构建体包含至少一个编码碳水化合物结合模块的DNA片段,该DNA片段通过或不通过连接子连接至编码目标葡糖淀粉酶的DNA序列,此DNA序列具有或不具有其自身的天然碳水化合物结合模块。所得的重组产物(杂交酶)-本领域通常称为“融合蛋白”-可以用下述的通式描述:
A-CBM-MR-X
在后者的通式中,A-CBM为至少包含碳水化合物结合模块(CBM)本身的氨基酸序列的N端或C端区域。MR为中间区域(“连接子”),X为由编码CBM所连接的酶(或其它蛋白)的DNA序列所编码的多肽的氨基酸残基序列。
A部分可以不存在(这样A-CBM为CBM本身,即不包含除构成CBM的氨基酸残基之外的其它氨基酸残基),或者可以为具有一个或多个氨基酸残基的序列(作为CBM本身的末端延伸体而发挥功能)。连接子(MR)可以不存在,或者是键,或者为包含约2至约100个碳原子,特别地2至40个碳原子的短的连接基团。然而,MR优选为约2至约100个氨基酸残基的序列,更优选2至40个氨基酸残基,例如2至15个氨基酸残基。
X部分可以构成整个杂交酶的N端或C端区域。
这样,由以上所述很明显所讨论的杂交酶类型中的CBM可以位于杂交酶的C端、N端或内部。
在CBM位于内部的实施方案中,CBM可以通过两个连接子连接。
应了解,本发明的杂交酶可以具有一个以上CBM,例如两个或三个CBM。本发明涉及加入一个以上CBM的实施方案:例如,N或C端具有两个或多个CBD的串联构建体,具有N端+C端CBM的构建体。
因此,本发明杂交体的例子也包括下述通式的杂交体:
A-CBM1-MR1-X-MR2-CBM2-B
A-CBM1-MR1-B-CBM2-MR2-X
A-CBM1-MR1-CBM2-X-MR3-CBM3-C
CBM1和CBM2可以不同或相同。B和C可以不存在(这样,例如,B-CBM2为CBM2本身,即不包含除构成CBM2的氨基酸残基之外的其它氨基酸残基),或者可以(如A)为具有一个或多个氨基酸残基的序列(作为CBM2本身的末端延伸体而发挥功能)。连接子可以不存在也可以存在。
连接子序列
连接子序列可以为任何适合的连接子序列。在优选实施方案中,连接子序列衍生于罗耳阿太菌(Athelia rolfsii)葡糖淀粉酶、黑曲霉葡糖淀粉酶、埃默森篮状菌葡糖淀粉酶、或白曲霉(A.kawachii)α-淀粉酶。这样的连接子序列的具体例子包括:
-黑曲霉AMG连接子:
TGGTTTTATPTGSGSVTSTSKTTATASKTSTSTSSTSA(SEQ ID NO:20),
-白曲霉α-淀粉酶连接子:
TTTTTTAAATSTSKATTSSSSSSAAATTSSS(SEQ ID NO:21),
-罗耳阿太菌AMG连接子:
STGATSPGGSSGS(SEQ ID NO:27),
PEPT连接子:
PEPTPEPT(SEQ ID NO:22)。
所述连接子也可以是上述连接子的片段。
在另一优选实施方案中,所述杂交酶具有在不超过10个位置、不超过9个位置、不超过8个位置、不超过7个位置、不超过6个位置、不超过5个位置、不超过4个位置、不超过3个位置、不超过2个位置、或者甚至不超过1个位置不同于SEQ ID NO:20、SEQ ID NO:21、SEQ ID NO:27、或SEQID NO:22所示氨基酸序列的连接子序列。
碳水化合物结合模块(CBM)
碳水化合物结合模块(CBM)为优先结合于多糖或寡糖(碳水化合物)的多肽氨基酸序列,经常-但不一定仅仅-结合于其水不溶性(包括晶体)形式。
衍生于淀粉降解酶的CBM通常称为淀粉结合模块或SBM(可见于一些水解淀粉的酶的CBM,例如一些葡糖淀粉酶、或见于例如环糊精糖基转移酶(glucanotransferase)、或见于α-淀粉酶)。同样,其它的CBM亚类将包含例如,纤维素结合模块(源于水解纤维素的酶的CBM)、几丁质结合模块(通常见于几丁质酶的CBM)、木聚糖结合模块(通常见于木聚糖酶的CBM)、甘露聚糖结合模块(通常见于甘露聚糖酶的CBM)。SBM通常也称为SBD(淀粉结合结构域)。
可以发现CBM为由两个或多个多肽氨基酸序列区域组成的大型多肽或蛋白质的内在部分(integral parts),尤其是在水解性酶(水解酶)中,这样的酶通常包含催化模块,其中含有底物水解活性位点和结合目标碳水化合物底物的碳水化合物结合模块(CBM)。这样的酶可以包含不止一个催化模块,例如一个、两个或三个CBM,可选地进一步包含与具有催化模块的CBM连接的一个或多个多肽氨基酸序列区域,后一类型的区域通常称为“连接子”。包含CBM的水解性酶的例子-其中一些在上面已经提到-为纤维素酶、α-淀粉酶、木聚糖酶、甘露聚糖酶、阿拉伯呋喃糖苷酶、乙酰酯酶和几丁质酶。CBM也已发现以非水解多糖结合蛋白的形式存在于藻类例如红藻Porphyrapurpurea中。
在有CBM的蛋白质/多肽中(例如,酶、通常水解性酶),CBM可以位于N或C端或位于内部位置。
构成CBM本身的多肽或蛋白质(例如,水解性酶)部分通常由约30个以上但少于约250个氨基酸残基组成。
“碳水化合物结合模块家族20”或CBM-20模块在本发明上下文中定义为与Joergensen等(1997)在Biotechnol.Lett.19:1027-1031图1的多肽的碳水化合物结合模块(CBM)具有至少45%同源性的大约100个氨基酸的序列。所述CBM包含所述多肽的至少102个氨基酸,即从氨基酸582至氨基酸683的子序列。本说明书中应用的糖苷水解酶家族的编号遵循URL:afmb.cnrs-mrs.fr/~cazy/CAZY/index.html上Coutinho,P.M.&Henrissat,B.(1999)CAZy-Carbohydrate-Active Enzymes server或Coutinho,P.M.&Henrissat,B.1999;The modular structure of cellulases and othercarbohydrate-active enzymes:an integrated database approach.In″Genetics,Biochemistry and Ecology of Cellulose Degradation″.,K.Ohmiya,K.Hayashi,K.Sakka,Y.Kobayashi,S.Karita and T.Kimura eds.,Uni Publishers Co.,Tokyo,pp.15-23,和Bourne,Y.&Henrissat,B.2001;Glycoside hydrolases andglycosyltransferases:families and functional modules,Current Opinion inStructural Biology 11:593-600的概念。
包含适合用于本发明上下文中的CBM的酶的例子为α-淀粉酶、产麦芽糖α-淀粉酶、纤维素酶、木聚糖酶、甘露聚糖酶、阿拉伯呋喃糖苷酶、乙酰酯酶和几丁质酶。与本发明有关的更多的目标CBM包括衍生于葡糖淀粉酶(EC 3.2.1.3)或CGT酶(EC 2.4.1.19)的CBM。
衍生于真菌、细菌或植物来源的CBM一般会适合用于本发明上下文中。优选的为源于曲霉(Aspergillus sp.)、阿太菌(Athelia sp.)、篮状菌(Talaromycessp.)、芽孢杆菌(Bacillus sp.)、克雷伯杆菌(Klebsiella sp.)、或根霉(Rhizopus sp.)物种的CBM。优选的为真菌来源的CBM。就此而论,适合于分离相关基因的技术是本领域所熟知的。
本发明优选的为碳水化合物结合模块家族20的CBM。“碳水化合物结合模块家族20”或CBM-20模块在本发明上下文中定义为与Joergensen等(1997)在Biotechnol.Lett.19:1027-1031图1的多肽的碳水化合物结合模块(CBM)具有至少45%同源性的大约100个氨基酸的序列。所述CBM包含所述多肽的最后102个氨基酸,即从氨基酸582至氨基酸683的子序列。本说明书中应用的CMB编号遵循Coutinho&Henrissat 1999的概念(Coutinho,P.M.&Henrissat,B.The modular structure of cellulases and othercarbohydrate-active enzymes:an integrated database approach.In ″Genetics,Biochemistry and Ecology of Cellulose Degradation″.,K.Ohmiya,K.Hayashi,K.Sakka,Y.Kobayashi,S.Karita and T.Kimura eds.,Uni Publishers Co.,Tokyo,pp.15-23 or altematively:Coutinho,P.M.&Henrissat,B.(1999)Carbohydrate-Active Enzymes server at URL:afmb.cnrs-mrs.fr/~cazy/CAZY/index.html)。
适合于本发明的碳水化合物结合模块家族20的CBM可以衍生自泡盛曲霉(Aspergillus awamori)(SWISSPROT Q12537)、白曲霉(SWISSPROTP23176)、黑曲霉(SWISSPROT P04064)、米曲霉(Aspergillus oryzae)(SWISSPROT P36914)的葡糖淀粉酶,衍生自白曲霉(EMBL:#AB008370)、构巢曲霉(Aspergillus nidulans)(NCBI AAF17100.1)的α-淀粉酶,衍生自腊状芽孢杆菌(Bacillus cereus)(SWISSPROT P36924)的β-淀粉酶、或者衍生自环状芽孢杆菌(Bacillus circulans)(SWISSPROT P43379)的CGT酶。优选为衍生自白曲霉(EMBL:#AB008370)α-淀粉酶的CBM以及与白曲霉(EMBL:#AB008370)α-淀粉酶的CBM有至少50%,60%,70%,80%,90%,95%,97%或者甚至至少99%同源性的CBM,即与SEQ ID NO:2的氨基酸序列有至少50%,60%,70%,80%,90%,95%,97%或者甚至至少99%同源性的CBM。本发明还优选具有SEQ ID NO:3(黄热芽孢杆菌(Bacillusflavorthermus)CBM)、SEQ ID NO:4(芽孢杆菌CBM)、和SEQ ID NO:5(芽孢嗜碱菌(Alcaliphilic Bacillus)CBM)所示氨基酸序列的碳水化合物结合模块家族20的CBM。更多优选的CBM包括来自Hormoconis sp.例如来自Hormoconis resinae(与杂酚真菌或脂暗膜菌(Amorphotheca resinae)同义)的葡糖淀粉酶的CBM,例如CBMSWISSPROT:Q03045(SEQ ID NO:6),来自香菇(Lentinula sp.)如埃杜香菇(Lentinula edodes)(花菇(Shiitakemushroom))的葡糖淀粉酶的CBM如CBM SPTREMBL:Q9P4C5(SEQ IDNO:7)、来自脉孢霉(Neurospora sp.)如粗糙脉孢霉(Neurospora crassa)的葡糖淀粉酶的CBM如CBM SWISSPROT:P14804(SEQ ID NO:8)、来自篮状菌如Talaromyces byssochlamydioides的葡糖淀粉酶的CBM如CBMNN005220(SEQ ID NO:9),来自Geosmithia sp.如Geosmithia cylindrospora的葡糖淀粉酶的CBM如CBM NN48286(SEQ ID NO:10),来自Scorias sp.如Scorias spongiosa的葡糖淀粉酶的CBM如CBM NN007096(SEQ ID NO:11),来自正青霉(Eupenicillium sp.)如Eupenicillium ludwigii的葡糖淀粉酶的CBM如CBM NN005968(SEQ ID NO:12),来自曲霉如日本曲霉(Aspergillus japonicus)的葡糖淀粉酶的CBM如CBM NN001136(SEQ ID NO:13),来自青霉(Penicillium sp.)如米舒青霉,enicillium cf.miczynskii)的葡糖淀粉酶的CBM如CBM NN48691(SEQ ID NO:14),来自Mz1青霉的葡糖淀粉酶的CBM如CBM NN48690(SEQ ID NO:15),来自Thysanophora sp.的葡糖淀粉酶的CBM如CBM NN48711(SEQ ID NO:16),和来自腐质霉(Humicola sp.)如灰腐质霉高温变种(Humicola grisea var.thermoidea)的葡糖淀粉酶的CBM如CBM SPTREMBL:Q12623(SEQ ID NO:17)。最优选的CBM包括来自曲霉例如黑曲霉葡糖淀粉酶的CBM,如SEQ ID NO:18,和来自阿太菌如罗耳阿太菌葡糖淀粉酶的CBM,如SEQ ID NO:19。本发明还优选与前述任何CBD氨基酸序列有至少50%,60%,70%,80%,90%,95%,97%或者甚至至少99%同源性的任何CBD。
更多合适的碳水化合物结合模块家族20的CBM可见于URL:afmb.cnrs-mrs.fr/~cazy/CAZY/index.html)的碳水化合物活性酶服务器。
其它的CBM可以在来自卷枝毛霉(Mucor circinelloides)、米根霉(Rhizopus oryzae)、Arxula adeninivorans的葡糖淀粉酶中找到。
一旦鉴定了编码底物结合(碳水化合物结合)区域的核苷酸序列(它们为cDNA或染色体DNA),接下来就可以以许多方式操作而将其融合到编码目标酶的DNA序列中。然后编码结合碳水化合物的氨基酸序列的DNA片段和编码目标酶的DNA用或不用连接子而连接起来。然后所得的连接DNA可以以许多方式操作而获得表达。
葡糖淀粉酶序列
适合作为本发明的CBM/葡糖淀粉酶杂交体的基础的葡糖淀粉酶包括例如衍生自真菌生物、细菌或植物的葡糖淀粉酶。优选的葡糖淀粉酶为真菌或细菌起源,选自曲霉葡糖淀粉酶,特别是黑曲霉G1或G2葡糖淀粉酶(Boel等(1984),EMBO J.3(5),p.1097-1102),SEQ ID NO:24所示),或其变体,例如WO 92/00381,WO 00/04136和WO 01/04273(来自Novozymes,Denmark)中所公开的;泡盛曲霉葡糖淀粉酶(WO 84/02921),米曲霉(Agric.Biol.Chem.(1991),55(4),p.941-949),或其变体或片段。其它的葡糖淀粉酶包括罗耳阿太菌葡糖淀粉酶(美国专利4,727,046),见SEQ ID NO:26,篮状菌葡糖淀粉酶,特别是衍生自埃默森篮状菌(WO 99/28448),见SEQ ID NO:25)、Talaromyces leycettanus(美国再版专利32,153)、Talaromyces duponti、Talaromyces thermophilus(美国专利4,587,215)的葡糖淀粉酶。本发明涉及的细菌葡糖淀粉酶包括源于梭菌属(Clostridium),特别是热解淀粉梭菌(C.thermoamylolyticum)(EP 135,138),和热硫化氢梭菌(C.thermohydrosulfuricum)(WO 86/01831)的葡糖淀粉酶。葡糖淀粉酶可以具有或不具有天然CBM,但至少包含所述催化模块(CM)。
优选的葡糖淀粉酶为SEQ ID NO:24所公开的黑曲霉葡糖淀粉酶,或者与SEQ ID NO:24所示氨基酸序列具有大于50%,例如60%,70%,80%,90%,95%,96%,97%,98%或99%同源性(一致性)的葡糖淀粉酶。
应了解葡糖淀粉酶(催化模块)在一个实施方案中可以为具有葡糖淀粉酶活性的活性片段。
另一优选的葡糖淀粉酶为SEQ ID NO:26所示的罗耳阿太菌葡糖淀粉酶,或者与SEQ ID NO:26所示氨基酸序列具有大于50%,例如60%,70%,80%,90%,95%,96%,97%,98%或99%同源性(一致性)的葡糖淀粉酶。
第三个优选的葡糖淀粉酶为SEQ ID NO:25所示的埃默森篮状菌葡糖淀粉酶,或者与SEQ ID NO:25所示氨基酸序列具有大于50%,例如60%,70%,80%,90%,95%,96%,97%,98%或99%同源性(一致性)的葡糖淀粉酶。
杂交体
一方面本发明涉及杂交酶,其包含具有葡糖淀粉酶活性的催化模块的氨基酸序列和碳水化合物结合模块的氨基酸序列。在优选的实施方案中,所述催化模块为真菌起源的。在更优选的实施方案中,所述催化模块衍生于篮状菌菌株(优选埃默森篮状菌),曲霉菌株(优选黑曲霉),或阿太菌菌株(优选罗耳阿太菌)。
在优选实施方案中,本发明的杂交酶包含衍生自埃默森篮状菌的具有葡糖淀粉酶活性的催化模块和衍生自黑曲霉或罗耳阿太菌的碳水化合物结合模块。在一个实施方案中,所述杂交体在催化模块和碳水化合物结合模块之间可以包括连接子序列,优选来自黑曲霉、罗耳阿太菌、白曲霉或埃默森篮状菌。
在优选实施方案中,本发明的杂交酶包含衍生自黑曲霉的具有葡糖淀粉酶活性的催化模块和衍生自罗耳阿太菌或埃默森篮状菌的碳水化合物结合模块。在一个实施方案中,所述杂交体在催化模块和碳水化合物结合模块之间可以包括连接子序列,优选来自黑曲霉、罗耳阿太菌、白曲霉或埃默森篮状菌。
在另一优选实施方案中,本发明的杂交酶包含衍生自罗耳阿太菌的具有葡糖淀粉酶活性的催化模块和衍生自黑曲霉或埃默森篮状菌的碳水化合物结合模块。在一个实施方案中,所述杂交体在催化模块和碳水化合物结合模块之间可以包括连接子序列,优选来自黑曲霉、罗耳阿太菌、白曲霉或埃默森篮状菌。
优选的黑曲霉,罗耳阿太菌或埃默森篮状菌分别为SEQ ID NOS:24,25,和26中所示。
优选所述杂交酶包含与SEQ ID NO:3,SEQ ID NO:4,SEQ ID NO:5,SEQ ID NO:6,SEQ ID NO:7,SEQ ID N0:8,SEQ ID NO:9,SEQ ID NO:10,SEQ ID NO:11,SEQ ID NO:12,SEQ ID NO:13,SEQ ID NO:14,SEQ IDNO:15,SEQ ID NO:16,SEQ ID NO:17,SEQ ID NO:18,或SEQ ID NO:19任一所示的氨基酸序列具有至少50%,60%,70%,80%,90%,95%,97%或者甚至至少99%同源性的CBM序列。
更优选所述杂交酶包含具有SEQ ID NO:28所示氨基酸序列的CBM序列。在又一优选实施方案中,所述CBM序列具有与SEQ ID N0:28所示的氨基酸序列,或其它CBM序列中的任何一个,在不超过10个氨基酸位置、不超过9个位置、不超过8个位置、不超过7个位置、不超过6个位置、不超过5个位置、不超过4个位置、不超过3个位置、不超过2个位置、或者甚至不超过1个位置不同的氨基酸序列。在最优选的实施方案中,所述杂交酶包含衍生于来自罗耳阿太菌的葡糖淀粉酶的CBM,例如美国专利4,727,026中所述的来自罗耳阿太菌AHU 9627的AMG或者来自黑曲霉的CBM。
根据本发明考虑的具体杂交体包括下述:
融合体 | 杂交体名称 | CM | SBM | 融合连接点(SBM起点-下划线) |
SEQ ID NO:29 | ANTE1 | AN | TE | SSVPGTCSATSATGPYSTATNTVWPSSGSGSST |
SEQ ID NO:30 | ANTE2 | AN | TE | SSVPGTCAATSAIGTYSTATNTVWPSSGSGSST |
SEQ ID NO:3l | ANTE3 | AN | TE | SSVPGTCAATSAIGTYSSVTVTSWPSSGSGSST |
SEQ ID NO:32 | ANAR1 | AN | AR | SSVPGTCSTGATSPGGSSGSVEVTFDVYATTVY |
SEQ ID NO:33 | ANAR2 | AN | AR | SSVPGTCAATSAIGTGSSGSVEVTFDVYATTVY |
SEQ ID NO:34 | ANAR3 | AN | AR | SSVPGTCAATSAIGTYSSVTVTSWFDVYATTVY |
SEQ ID NO:35 | ARTE1 | AR | TE | GVSTSCSATSATGPYSTATNTVWPSSGSGSSTT |
SEQ ID NO:36 | ARTE2 | AR | TE | GVSTSCSTGATSPGYSTATNTVWPSSGSGSSTT |
SEQ ID NO:37 | ARTE3 | AR | TE | GVSTSCSTGATSPGGSSGSVEVTPSSGSGSGTT |
SEQ ID NO:38 | ARAN1 | AR | AN | GVSTSCAATSAIGTYSSVTVTSWPSIVATGGTT |
SEQ ID NO:39 | ARAN2 | AR | AN | GVSTSCSTGATSPGYSSVTVTSWPSIVATGGTT |
SEQ ID NO:40 | ARAN3 | AR | AN | GVSTSVSTGATSPGGSSGSVEVTPSIVATGGTT |
SEQ ID NO:41 | TEAN1 | TE | AN | SVPAVCAATSAIGTYSSVTVTSWPSIVATGGTT |
SEQ ID NO:42 | TEAN2 | TE | AN | SVPAVCSATSATGPYSSVTVTSWPSIVATGGTT |
SEQ ID NO:43 | TEAN3 | TE | AN | SVPAVCSATSATGPYSTATNTVWPSIVATGGTT |
SEQ ID NO:44 | TEAR1 | TE | AR | SSVPAVCSTGATSPGGSSGSVEVTFDVYATTVY |
SEQ ID NO:45 | TEAR2 | TE | AR | SSVPAVCSATSATGPYSSGSVEVTFDVYATTVY |
SEQ ID NO:46 | TEAR3 | TE | AR | SSVPAVCSATSATGPYSTATNTVWFDVYATTVY |
CM:催化模块;SBM:淀粉结合模块;AN:黑曲霉;TE:埃默森篮状菌;AR:罗耳阿太菌。
表达载体
本发明还涉及可包含编码杂交酶、启动子、信号肽序列、和转录及翻译终止信号的DNA序列的重租表达载俸。上述的多种DNA各控制序列可以结合在一起来制备重组表达载体,其可以包括一个或多个便利的限制性位点以允许在这些位点插入或取代编码所述多肽的DNA序列。可选地,本发明的DNA序列可以通过将所述DNA序列或包含该序列的DNA构建体插入适当的用于表达的载体来表达。在创建表达载体过程中,将编码序列定位于载体中,使编码序列与用于表达以及可能用于分泌的适当控制序列可操作相连。
所述重组表达载体可以是任何载体(例如,质粒或病毒),其可以方便地用于重组DNA方法,能够引发DNA序列的表达。载体的选择一般依赖于载体与所述载体所要插入的宿主细胞的兼容性。所述载体可以是线性的或者是封闭的环状质粒。该载体可以是自主复制载体,即作为染色体外实体存在的载体,其复制独立于染色体的复制,例如,质粒,染色体外成分,微小染色体、粘粒或人工染色体。该载体可以包含确保自我复制的任何手段。可选地,该载体可以是当引入到所述宿主细胞中时整合到基因组中,并与其所整合的染色体一起复制的一种载体。该载体系统可以是单一载体或质粒或总体上包含要引入到宿主细胞基因组中的全部DNA的两个或多个载体或质粒,或转座子。
标记
本发明的载体优选包含一个或多个可选择的标记,其允许容易地选择转化的细胞。可选择标记为基因,其产物提供杀生物剂或病毒抗性,重金属抗性,针对营养缺陷型的原养型等。
用于丝状真菌宿主细胞的可选择标记的例子可以选自包括但不限于以下的组:amdS(乙酰胺酶)、argB(鸟氨酸氨甲酰基转移酶)、bar(草铵膦(phosphinothricin)乙酰转移酶)、hygB(潮霉素磷酸转移酶)、niaD(硝酸还原酶)、pyrG(乳清酸核苷-5’-磷酸脱羧酶)、sC(硫酸腺苷酰基转移酶)、trpC(邻氨基苯甲酸合成酶)、和草丁膦(glufosinate)抗性标记、以及来自其它物种的等同物。优选用于曲霉细胞的为构巢曲霉或米曲霉的amdS和pyrG标记以及吸水链霉菌(Streptomyces hygroscopicus)的bar标记。另外,选择可通过共转化完成,例如见WO 91/17243,其中可选择标记在分离的载体上。
本发明的载体优选包含允许载体稳定地整合于宿主细胞基因组或者允许载体在细胞中不依赖于基因组而自主复制的元件。
当引入到宿主细胞时,本发明的载体可以整合到宿主细胞基因组中。为了整合,载体可依赖于编码目标多肽的DNA序列,或者用于通过同源和非同源重组将载体稳定地整合于基因组的载体的其它任何元件。可选地,载体可以包含通过同源重组引导向宿主细胞基因组整合的额外的DNA序列。额外的DNA序列使载体能够整合于宿主细胞基因组中染色体的精确位置。为了增加在精确位点整合的可能性,整合元件应优选包含足够数量的DNA,例如100至1,500个碱基对,优选400至1,500个碱基对,最优选800至1,500个碱基对,这些碱基对与相应的目标序列高度同源,以增加同源重组的概率。整合元件可以是与宿主细胞基因组中的目标序列同源的任何序列。另外,整合元件可以是非编码或编码DNA序列。另一方面,载体可以通过非同源重组整合至宿主细胞基因组。这些DNA序列可以是与宿主细胞基因组中的目标序列同源的任何序列,另外,可以是非编码或编码序列。
为了自主复制,载体可以进一步包含使载体能够在所讨论的宿主细胞中自主复制的复制起点。可以使用WO 00/24883中所公开的复制AMA1质粒载体的附加体。
可以将超过一个拷贝的编码目标多肽的DNA序列插入宿主细胞以增强DNA序列的表达。可以使用本领域已知的方法通过将至少一个额外的序列拷贝整合于宿主细胞基因组中以获得所述DNA序列的稳定扩增,并选择转化体。
用于连接上述元件以构建本发明的重组表达载体的方法是本领域技术人员所熟知的(参见,例如,Sambrook等,1989,分子克隆,实验指南,第二版,冷泉港,纽约)。
宿主细胞
宿主细胞可以是真菌例如丝状真菌或酵母来源,或者为细菌来源,例如芽孢杆菌来源。
包含含有编码杂交酶的DNA序列的DNA构建体或表达载体的本发明的宿主细胞,在杂交酶的重组产生中方便地用作宿主细胞。所述细胞可以用表达载体转化。可选地,可以方便地通过将编码所述杂交酶的本发明DNA构建体(以一个或多个拷贝)整合到宿主染色体中而用该DNA构建体转化所述细胞。DNA构建体向宿主染色体的整合可以根据传统的方法进行,例如通过同源或异源重组。
在优选的实施方案中,宿主细胞为下述子囊菌门(Ascomycota)组的丝状真菌,包括例如,脉胞菌、正青霉(Eupenicillium)(=青霉(Penicillium))、裸孢壳(Emericella)(=曲霉)、散囊菌(Eurotium)(=曲霉)。在更优选的实施方案中,丝状真菌包括真菌门(Eumycota)、卵菌门(Oomycota)的亚门(如Hawksworth等,在Ainsworth and Bisby’s Dictionary of The Fungi,第八版,1995,CABInternational,University Press,Cambridge,UK中定义)的所有丝状形式。丝状真菌通过由几丁质、纤维素、葡聚糖、壳聚糖、甘露聚糖和其它复合多糖构成的营养菌丝体鉴别。营养生长通过菌丝延长进行,碳代谢为需氧型。
在更加优选的实施方案中,丝状真菌宿主细胞是但不限于选自由属于曲霉物种的菌株优选米曲霉、黑曲霉、泡盛曲霉、白曲霉、或镰刀霉(Fusarium)菌株如尖孢镰刀霉(Fusarium oxysporium)、禾谷镰刀霉(Fusariumgraminearum)(更准确的说法叫作玉蜀黍赤霉(Gibberella zeae),以前为Sphaeria zeae,与玉米赤霉(Gibberella zeae)和Gibberella roseum f.sp.cerealis同义)、或硫色镰刀霉(Fusarium sulphureum)(更准确的说法叫作Gibberellapuricaris,与Fusarium trichothecioides、杆孢状镰刀霉(Fusariumbactridioides)、接骨木镰刀霉(Fusarium sambucium)、玫瑰色镰刀霉(Fusariumroseum)、和玫瑰色镰刀霉禾谷变种(Fusarium roseum var.graminearum)同义)、Fusarium cerealis(与Fusarium crookwellense同义)、或Fusariumvenenatum菌株所组成的组的细胞。
在最优选的实施方案中,丝状真菌宿主细胞为属于曲霉的菌株优选米曲霉或黑曲霉的细胞。
宿主细胞可以是野生型丝状真菌宿主细胞或变体、突变体或遗传改造的丝状真菌宿主细胞。在本发明的优选实施方案中,宿主细胞为蛋白酶缺陷或蛋白酶阴性菌株。特别预期的是曲霉菌株,例如经遗传改造以破坏或减少葡糖淀粉酶、酸稳定性α-淀粉酶、α-1,6转葡糖苷酶和蛋白酶活性表达的黑曲霉菌株。
在另一优选实施方案中,真菌宿主细胞是酵母细胞。此处所用“酵母”包括产子囊酵母(ascosporogenous)(内孢霉目(Endomycetales))、产担子孢子囊酵母(basidiosporogenous)、和属于半知菌纲(Fungi Imperfecti)(芽孢纲(Blastomycetes))的酵母。由于酵母的分类将来可能会有所变化,为了本发明的目的,应该按照Biololgy and Activities of Yeast(Skinner,F.A.,Passmore,S.M.,and Davenport,R.R.,eds,Soc.App.Bacteriol.Symposium Series No.9,1980)中所述定义酵母。
在更为优选的实施方案中,酵母宿主细胞为假丝酵母(Candida)、汉逊酵母(Hansenula)、克鲁维酵母(Kluyveromyces)、毕赤酵母(Pichia)、糖酵母(Saccharomyces)、裂殖酵母(Schizosaccharomyces)、或者亚罗威阿酵母(Yarrowia)细胞。
在最优选的实施方案中,酵母宿主细胞为卡尔糖酵母(Saccharomycescarlsbergensis)、啤酒糖酵母(Saccharomyces cerevisiae)、糖化糖酵母(Saccharomyces diastaticus)、道格拉斯酵母(Saccharomyces douglasii)、克鲁弗糖酵母(Saccharomyces kluyveri)、诺地糖酵母(Saccharomyces norbensis)或卵形糖酵母(Saccharomyces oviformis)细胞。在另一最优选实施方案中,酵母宿主细胞为乳克鲁维酵母(Kluyveromyceslactis)细胞。在另一最优选实施方案中,酵母宿主细胞是解脂亚罗威阿酵母(Yarrowia lipolytica)细胞。
转化真菌宿主细胞
真菌细胞可以通过涉及原生质体形成、原生质体转化、以及细胞壁再生等的方法以其原本已知的方式来转化。EP 238023和Yelton等,1984,Proceedings of the National Academy of Sciences USA 81:1470-1474中描述了转化曲霉宿主细胞的适当的方法。Malardier等,1989,Gene 78:147-156和WO96/00787中描述了转化镰刀霉物种的适当方法。
酵母可以用Becker and Guarente,Abelson,J.N.和Simon,M.I.,编著的Guide to Yeast Genetics and Molecular Biology,Methods in Enzymology,Volume 194,pp 182-187,Academic Press,Inc.,New York;Ito等,1983,Journalof Bacteriology 153:163;和Hinnen等,1978,Proceedings of the NationalAcademy of Sciences USA 75:1920中描述的方法转化。
分离并克隆DNA序列
用于分离或克隆编码目标多肽的DNA序列的技术是本领域已知的,包括从基因组DNA中分离,从cDNA制备,或其组合。从这样的基因组DNA中克隆本发明的DNA序列可以使用熟知的聚合酶链式反应(PCR)或者用抗体筛选表达文库以检测具有共有结构特征的克隆化DNA片段来实现。参见,例如,Innis等,1990,PCR:A Guide to Methods and Application,AcademicPress,New York。可以使用其它的DNA扩增方法例如连接酶链式反应法(ligase chain reaction,LCR)、连接激活转录(ligated activated transcription)(LAT)和基于DNA序列的扩增(DNA sequence-based amplification,NASBA)。
分离的DNA序列
本发明涉及包含编码杂交酶的DNA序列的分离的DNA序列及其它,所述杂交酶包含具有葡糖淀粉酶活性的催化模块的氨基酸序列和碳水化合物结合模块的氨基酸序列,其中催化模块。
此处所用术语“分离的DNA序列”指DNA序列,其基本不含有其它DNA序列,例如,当用琼脂糖电泳测定时至少约20%纯,优选至少约40%纯,更优选至少约60%纯,再优选至少约80%纯,最优选至少约90%纯。
例如,分离的DNA序列可用遗传工程的标准克隆方法将DNA序列从其天然位置重新定位到不同位置、并在该位置使其再生来获得。该克隆方法可涉及切除并分离包含编码目标多肽的DNA序列的所需DNA片段,将该片段插入载体分子,将重组载体掺入宿主细胞并在其中复制DNA序列的多个拷贝或克隆。可以以多种方法操作分离的DNA序列,以供目标多肽表达之用。DNA序列在插入载体之前可能需要或必需进行操作,这取决于表达载体。利用重组DNA方法修饰DNA序列的技术是本领域熟知的。
DNA构建体
本发明涉及包含编码杂交酶的DNA序列的DNA构建体,所述杂交酶包含具有葡糖淀粉酶活性的催化模块的氨基酸序列和碳水化合物结合模块的氨基酸序列。在一实施方案中,所述催化模块是真菌起源的。此处“DNA构建体”定义为单链或双链的DNA分子,其分离于天然发生的基因,或者是经过修饰而包含了以自然界不存在的方式组合和邻接(juxtaposed)的DNA片段。当DNA构建体包含本发明编码序列的表达所需的所有控制序列时,术语DNA构建体与术语表达盒同义。
制备方法
本发明的杂交体可以用任何方法制备,例如包括(a)在有利于制备杂交体的条件下培养宿主细胞;和(b)回收杂交体。
在本发明的制备方法中,用本领域已知的方法将所述细胞培养于适于所述杂交体产生的营养培养基中。例如,可以在实验室规模或者工业规模发酵罐中,在适当的培养基中以及允许杂交体表达和/或分离的条件下,通过摇瓶培养、小规模或大规模发酵(包括连续的、分批的、补料-分批的、或者固态发酵)来培养细胞。培养在包含碳源和氮源以及无机盐的适当营养培养基中,以本领域已知的方法进行。合适的培养基可以从供应商处获得,或者可以用公开的组成(例如,美国典型培养物保藏中心的目录中所述)制备。当杂交体分泌到营养培养基中,可以直接从培养基回收杂交体。当杂交体未被分泌,可从细胞裂解产物来回收。
杂交体可以用本领域已知的专门用于所述杂交体的方法检测。这些检测方法可包括使用特异性抗体、形成酶产物、或使酶底物消失。例如,可以用酶试验来测定此处所述杂交体的活性。
可以用本领域已知的方法回收所得杂交体。例如,可以通过传统方法包括但不限于离心、过滤、提取、喷雾干燥、蒸发、或沉淀从营养培养基中回收杂交体。
可以用本领域已知的多种方法来纯化本发明的杂交体,包括但不限于层析(例如,离子交换层析、亲合层析、疏水层析、聚焦层析、和大小排阻层析)、电泳方法(例如,制备性等电聚焦)、差异溶解(differential solubility)(例如,硫酸铵沉淀)、SDS-PAGE、或提取(参见,例如,Protein Purification,J.-C.Janson and Lars Ryden,editors,VCH Publishers,New York,1989)。
淀粉处理
本发明第一方面的杂交酶可用于产生浆液或发酵产物(例如尤其是乙醇)的产生过程中,其中颗粒淀粉在水性介质中用具有葡糖淀粉酶活性的本发明的杂交酶处理。在具体实施方案中,可以以0.02-20AGU/g干燥固体(DS),优选0.1-10AGU/g DS,如约0.1,0.3,0.5,1或2AGU/g DS,如0.1-0.5AGU/gDS的量加入具有葡糖淀粉酶活性的杂交酶。颗粒淀粉可进一步用α-淀粉酶(优选下述的一种)处理。
α-淀粉酶
根据本发明,α-淀粉酶可以是任何来源。优选为真菌或细胞来源的α-淀粉酶。α-淀粉酶可以是芽孢杆菌α-淀粉酶,例如,来源于地衣芽孢杆菌(B.licheniformis)、解淀粉芽孢杆菌(B.amyloliquefaciens)、嗜热脂肪芽孢杆菌(B.stearothermophilus)、和枯草芽孢杆菌(B.subtilis)菌株。其它的α-淀粉酶包括源于WO 95/26397中详细记载的芽孢杆菌NCIB 12289,NCIB 12512,NCIB12513或DSM 9375菌株的α-淀粉酶,和Tsukamoto等,Biochemical andBiophysical Research Communications,151(1988),pp.25-31所描述的α-淀粉酶。其它的α-淀粉酶变体和杂交体描述于WO 96/23874,WO 97/41213,和WO 99/19467。
其它的α-淀粉酶包括源于曲霉菌株的α-淀粉酶,例如米曲霉和黑曲霉α-淀粉酶。在优选实施方案中,所述α-淀粉酶为酸性α-淀粉酶。在更优选的实施方案中,所述酸性α-淀粉酶为酸性真菌α-淀粉酶或酸性细菌α-淀粉酶。更优选地,所述酸性α-淀粉酶为源于曲霉属的酸性真菌α-淀粉酶。商业上可获得的酸性真菌淀粉酶为SP288(可由Novozymes A/S,Denmark获得)。在优选实施方案中,所述α-淀粉酶为酸性α-淀粉酶。术语“酸性α-淀粉酶”意思是以有效量添加时,在3.0至7.0、优选3.5-6.0、或更优选4.0-5.0的pH范围具有活性的α-淀粉酶(E.C.3.2.1.1)。优选的酸性真菌α-淀粉酶为Fungamyl样α-淀粉酶。在本说明书中,术语“Fungamyl样α-淀粉酶”指显示与WO 96/23874中SEQ ID NO:10所示氨基酸序列有高度同源性,例如超过50%,55%,60%,65%,70%,75%,80%,85%90%,95%,96%,97%,98%,99%或者甚至为100%同源性(一致性)的α-淀粉酶。
优选α-淀粉酶为酸性α-淀粉酶,优选来自曲霉属,优选来自黑曲霉物种。在优选实施方案中,酸性真菌α-淀粉酶为Swiss-prot/TeEMBL数据库中以原始登录号P56271公开为“AMYA ASPNG”的来自黑曲霉的α-淀粉酶。也包括与所述酸性真菌淀粉酶具有至少70%一致性,如至少80%或甚至至少90%一致性,如至少95%,96%,97%,98%,或至少99%一致性的该酶的变体。
所述淀粉酶也可以是产麦芽糖α-淀粉酶。“产麦芽糖α-淀粉酶”(葡聚糖1,4-α-麦芽糖水解酶,E.C.3.2.1.133)能够将直链淀粉和支链淀粉水解为α-构型的麦芽糖。来自嗜热脂肪芽孢杆菌菌株NCIB 11837的产麦芽糖α-淀粉酶商业上可从Novozymes A/S获得,商用名为NOVAMYLTM。产麦芽糖α-淀粉酶记载于美国专利4,598,048,4,604,355和6,162,628,此处一并作为参考。优选地,产麦芽糖α-淀粉酶用于天然淀粉水解方法中,例如见WO 95/10627,此处一并作为参考。
当α-淀粉酶用作例如产麦芽糖的酶时,真菌α-淀粉酶可以以0.001-1.0AFAU/g DS,优选0.002-0.5AFAU/g DS,优选0.02-0.1AFAU/g DS的量添加。
优选的包含α-淀粉酶的商用组合物包括来自DSM(Gist Brocades)的MYCOLASE,BANTM,TERMAMYLTM SC,FUNGAMYETM,LIQUOZYMETMX和SANTM SUPER,SANTM EXTRA L(Novozymes A/S)和CLARASETML-40,000,DEX-LOTM,SPEYME FRED,SPEZYMETM AA,和SPEZYMETMDELTA AA (Genencor Int.),以及出售的商用名为SP288(可由NovozymesA/S,Denmark获得)的酸性真菌α-淀粉酶。
α-淀粉酶可以以本领域熟知的量添加。当以AAU单位测定时,酸性α-淀粉酶活性优选以5-50,0000AAU/kg DS的量,以500-50,000AAU/kg DS的量,或更优选以100-10,000AAU/kg DS的量,例如500-1,000AAU/kg DS的量提供。真菌酸性α-淀粉酶优选以10-10,000AFAU/kg DS的量,以500-2,500AFAU/kg DS的量,或更优选以100-1,000AFAU/kgDS的量,例如约500AFAU/kg DS的量添加。
处理
在一个实施方案中,本发明的处理包括颗粒淀粉浆的水解,特别是在低于所述颗粒淀粉的起始糊化温度的温度中将颗粒淀粉水解为可溶性淀粉水解产物。
欲用本发明的方法处理的颗粒淀粉浆可以具有20-55%的干燥固体颗粒淀粉,优选25-40%干燥固体颗粒淀粉,更优选30-35%干燥固体颗粒淀粉。
在本发明的第七个方面的处理之后,至少85%,至少86%,至少87%,至少88%,至少89%,至少90%,至少91%,至少92%,至少93%,至少94%,至少95%,至少96%,至少97%,至少98%,或优选至少99%的颗粒淀粉的干燥固体转化为可溶性淀粉水解产物。
根据本发明,所述处理在低于起始糊化温度的温度中进行。优选进行处理的温度在30-60℃,例如至少30℃,至少31℃,至少32℃,至少33℃,至少34℃,至少35℃,至少36℃,至少37℃,至少38℃,至少39℃,至少40℃,至少41℃,至少42℃,至少43℃,至少44℃,至少45℃,至少46℃,至少47℃,至少48℃,至少49℃,至少50℃,至少51℃,至少52℃,至少53℃,至少54℃,至少55℃,至少56℃,至少57℃,至少58℃,至少59℃,或优选至少60℃。
本发明第七个方面进行处理时的pH可以在3.0至7.0范围内,优选3.5至6.0,或更优选4.0-5.0。
特别地,将要在本发明的方法中处理的颗粒淀粉可以从块茎、根、茎、豆类、谷类或谷物整体获得。更特别地,颗粒淀粉可以从玉米、玉米芯(cob)、小麦、大麦、黑麦、买罗高粱(milo)、西米(sago)、木薯(cassava)、木薯粉(taouica)、高粱、水稻、豌豆、大豆(bean)、香蕉或马铃薯获得。特别还考虑糯性和非糯性类型的玉米和大麦。所要处理的淀粉可以具有高度精制的淀粉质量,优选至少90%,至少95%,至少97%或至少99.5%纯,或者可以是更为粗制的含有包含碾碎的谷物整体的材料的玉米,所述谷物整体包括非淀粉成分如胚芽残渣和纤维。将天然材料如谷物整体碾碎以打开结构并允许进一步处理。根据本发明,两种碾磨处理是优选的:湿磨和干磨。在干磨中,整个谷粒被碾碎后使用。湿磨使胚芽和粗粉(淀粉颗粒和蛋白质)很好地分离开,在淀粉水解产物用于产生浆液的情况下有几个例外。干磨和湿磨在淀粉处理领域都是熟知的,且都同样考虑用于本发明处理中。
发酵产物的产生
最后一个方面,本发明涉及具有葡糖淀粉酶活性的杂交酶在产生发酵产物尤其是乙醇的方法中的用途。该方法包括在发酵生物存在下用α-淀粉酶和本发明的杂交酶处理水性介质中的颗粒淀粉。所述α-淀粉酶可以是任何的α-淀粉酶,优选以上提到的一种。优选为酸性真菌α-淀粉酶,尤其是曲霉来源的。
优选的发酵生物是酵母。优选所述方法包括在用α-淀粉酶和本发明的杂交酶水解颗粒淀粉浆的同时,用酵母实施发酵。与水解同时进行的发酵的温度在30℃和35℃之间,更优选在31℃和34℃之间。
“发酵生物”指适合用于所需发酵过程的任何微生物。根据本发明,合适的发酵生物能够将糖如葡萄糖和/麦芽糖直接或间接地发酵,即转化为所需的发酵产物。发酵生物的例子包括真菌生物,如酵母。优选的酵母包括糖酵母(Sacchromyces sp.)菌株,特别是啤酒糖酵母。商业上可获得的酵母包括,例如/Lesaffre Ethanol Red(可由Red Star/Lesaffre,USA获得),FALI(可由Fleischmann’s Yeast,a division of Burns Philp Food Inc.,USA获得),SUPERSTART(可由Alltech获得),GERT STRAND(可由Gert StrandAB,Sweden获得)和FERMIOL(可由DSM Specialties获得)。
本发明杂交体的用途
最后一个方面本发明涉及本发明的杂交酶产生发酵产物,例如特别是乙醇,或浆液(优选葡萄糖或麦芽糖)的用途。本发明的杂交体可以用于本发明的方法中。
本发明此处所描述和要求的不受此处公开的具体实施方案范围的限制,因为这些实施方案的目的是作为本发明几个方面的例示。任何等同的实施方案都将落在本发明的范围内。事实上,由前面所描述的,除此处所列举和描述的那些之外,对本发明的许多修改对本领域熟练技术人员来说都将是显而易见的。这样的修改也将落入所附的权利要求的保护范围内。有不清楚的情况,本说明书包括定义将用于解释。
此处引用了许多参考文献和序列表,其全文整体一并作为参考。
材料和方法
酵母:
/Lesaffre Ethanol Red(可由Red Star/Lesaffre,USA获得)
酸稳定性α-淀粉酶活性
根据本发明使用时,可以测定任何酸稳定性α-淀粉酶的AFAU(酸性真菌α-淀粉酶单位)活性,其相对于酶标准来测定。1FAU定义为在下面所提到的标准条件下每小时降解5.260mg淀粉干物质的酶量。
酸稳定性α-淀粉酶、内切-α-淀粉酶(1,4-α-D-葡聚糖-葡聚糖基-水解酶,E.C.3.2.1.1)水解淀粉分子内部区域的α-1,4-糖苷键,形成不同链长的糊精和寡糖。与碘形成的颜色的强度直接与淀粉的浓度成比例。用反向比色法测定,在具体分析条件下,随淀粉浓度的降低的淀粉酶活性。
蓝/紫 t=23秒 脱色
标准条件/反应条件:
底物: 可溶性淀粉,约0.17g/L
缓冲液: 柠檬酸盐,约0.03M
碘(I2): 0.03g/L
CaCl2: 1.85mM
pH: 2.50±0.05
保温温度: 40℃
反应时间: 23秒
波长: 590nm
酶浓度: 0.025AFAU/mL
酶工作浓度: 0.01-0.04AFAU/mL
文件包EB-SM-0259.02/01更详细描述了此分析方法,可以向Novozymes A/S,Denmark请求提供而获得,该文件包包括在参考文献中。
葡糖淀粉酶活性
可以测定葡糖淀粉酶的活性,以淀粉葡萄糖苷酶单位(AmyloGlucosidaseUnits,AGU)表示。AGU定义为在37℃,pH4.3,底物:麦芽糖23.2mM,缓冲液:乙酸盐0.1M,反应时间5分钟的标准条件下,每分钟水解1微摩尔麦芽糖的酶量。
可以使用自动分析仪系统。向葡萄糖脱氢酶试剂中加入变旋酶,使存在的任何α-D-葡萄糖转化为β-D-葡萄糖。在以上提及的反应中,葡萄糖脱氢酶特异性地与β-D-葡萄糖反应,形成NADH,用光度计在340nm处测定NADH,作为初始葡萄糖浓度的测量值。
AMG保温: | |
底物: | 麦芽糖23.2mM |
缓冲液: | 乙酸盐0.1M |
pH: | 4.30±0.05 |
保温温度: | 37℃±1 |
反应时间: | 5分钟 |
酶工作浓度: | 0.5-4.0AGU/mL |
颜色反应: | |
葡萄糖脱氢酶: | 430U/L |
变旋酶: | 9U/L |
NAD: | 0.21mM |
缓冲液: | 磷酸盐0.12M;0.15M NaCl |
pH: | 7.60±0.05 |
保温温度: | 37℃±1 |
反应时间: | 5分钟 |
波长: | 340nm |
文件包EB-SM-0131.02/01更详细描述了此分析方法,可以向Novozymes A/S,Denmark请求提供而获得,该文件包包括在参考文献中。
DNA操作
除非另有说明,DNA操作和转化使用Sambrook等(1989)分子克隆:实验指南,冷泉港实验室,冷泉港,纽约;Ausubel,F.M.等(编)″Currentprotocols in Molecular Biology″,John Wiley and Sons,1995;Harwood,C.R.,和Cutting,S.M.(编)中所记载的标准方法进行。
实施例
实施例1
葡糖淀粉酶催化结构域-淀粉结合结构域杂交体的构建与表达
构建表达18种不同AMG杂交体(表1)的质粒,所述杂交体是黑曲霉(AN),埃默森篮状菌(TE),和罗耳阿太菌(AR)的葡糖淀粉酶的催化结构域(CD)和淀粉结合结构域(SBD)之间的杂交体。将质粒转化到啤酒糖酵母中以表达重组蛋白,或者随后将构建的葡糖淀粉酶杂交体重新克隆到黑曲霉表达载体中以在黑曲霉中表达杂交蛋白。
表1:葡糖淀粉酶杂交体中的融合邻接。
融合体 | 质粒名称 | CD | SBD | 融合连接点(SBD起点-下划线) |
SEQ ID NO:29 | pANTE1 | AN | TE | SSVPGTCSATSATGPYSTATNTVWPSSGSGSST |
SEQ ID NO:30 | pANTE2 | AN | TE | SSVPGTCAATSAIGTYSTATNTVWPSSGSGSST |
SEQ ID NO:31 | pANTE3 | AN | TE | SSVPGTCAATSAIGTYSSVTVTSWPSSGSGSST |
SEQ ID NO:32 | pANAR1 | AN | AR | SSVPGTCSTGATSPGGSSGSVEVTFDVYATTVY |
SEQ ID NO:33 | pANAR2 | AN | AR | SSVPGTCAATSAIGTGSSGSVEVTFDVYATTVY |
SEQ ID NO:34 | pANAR3 | AN | AR | SSVPGTCAATSAIGTYSSVTVTSWFDVYATTVY |
SEQ ID NO:35 | pARTE1 | AR | TE | GVSTSCSATSATGPYSTATNTVWPSSGSGSSTT |
SEQ ID NO:36 | pARTE2 | AR | TE | GVSTSCSTGATSPGYSTATNTVWPSSGSGSSTT |
SEQIDNO:37 | pARTE3 | AR | TE | GVSTSCSTGATSPGGSSGSVEVTPSSGSGSSTT |
SEQ ID NO:38. | pARAN1 | AR | AN | GVSTSCAATSAIGTYSSVTVTSWPSIVATGGTT |
SEQ ID NO:39. | pARAN2 | AR | AN | GVSTSCSTGATSPGYSSVTVTSWPSIVATGGTT |
SEQ ID NO:40. | pARAN3 | AR | AN | GVSTSCSTGATSPGGSSGSVEVTPSIVATGGTT |
SEQ ID NO:41 | pTEAN1 | TE | AN | SVPAVCAATSAIGTYSSVTVTSWPSIVATGGTT |
SEQ ID NO:42 | pTEAN2 | TE | AN | SVPAVCSATSATGPYSSVTVTSWPSIVATGGTT |
SEQ ID NO:43 | pTEAN3 | TE | AN | SVPAVCSATSATGPYSTATNTVWPSIVATGGTT |
SEQ ID NO:44 | pTEAR1 | TE | AR | SSVPAVCSTGATSPGGSSGSVEVTFDVYATTVY |
SEQ ID NO:45 | pTEAR2 | TE | AR | SSVPAVCSATSATGPYSSGSVEVTFDVYATTVY |
SEQ ID NO:46 | pTEAR3 | TE | AR | SSVPAVCSATSATGPYSTATNTVWFKVYATTVY |
试验方法
细菌各和真菌菌株各质粒
大肠杆菌(E.coli)DH10B(mcrA(mrr-hsdRMS-mcrBC)80dlacZM15lacX74deoR recA1endA1 araD139(ara,leu)7697galU galK,rpsL nupG),啤酒糖酵母INVSc1(MATa,his3D1,leu2,trp1-289,ura3-52)和大肠杆菌/酵母质粒穿梭载体pYES2购自Invitrogen Inc.(San Diego,CA)。
DNA构建体
DNA操作基本上按照Sambrook等,(1989)Maniatis,T.,1989.分子克隆:实验指南(第二版),冷泉港实验室出版社,冷泉港,纽约,和Dan Burke,Dean Dawson,Tim Steams(2000)Methods in Yeast Genetics:A Cold SpringHarbor Laboratory Course Manual,冷泉港实验室,中的记载的方法进行。限制性核酸内切酶和T4DNA连接酶来自New England Biolabs。Pwo DNA聚合酶(Boehringer Mannheim)基本如提供商所述使用。为进行SOE pcr反应,使每种所需pcr片段100ng发生凝胶纯化,混合后进行25个PCR循环,不添加任何引物。反应产物用琼脂糖凝胶电泳分离,将迁移到预计大小的DNA条带从胶上切下,用旋转柱纯化,在新的pcr反应中用作模板,使用侧翼引物。在两个步骤中构建质粒pSteD226:首先将埃默森篮状菌葡糖淀粉酶(SEQID NO:80)的编码序列作为HindIII-XbaI片段克隆到pYES2中制得pSteD212。此后用包含组成型TPI启动子(Alber and Kawasaki(1982)Nucleotide sequenceof the triose phosphate isomerase gene of Saccharomyces cerevisiae.J.Mol.Appl.Gen.,1:419-434)的AgeI-HindIII片段置换包含半乳糖诱导型启动子的pSteD212的AgeI-HindIII片段,制得pSteD226。将G1形式的黑曲霉葡糖淀粉酶(SEQ ID NO:81)的编码序列克隆到酵母/大肠杆菌穿梭载体pMT742中,作为EcoRI-HinDIII片段,以此构建质粒pLac102。为扩增CD和SBD,使用下述的DNA模板:携带编码G1-形式黑曲霉G1葡糖淀粉酶的cDNA的质粒pLAc102;携带编码G1-形式埃默森篮状菌葡糖淀粉酶的cDNA的质粒pSteD226;从包含G1形式罗耳阿太菌葡糖淀粉酶的罗耳阿太菌合成的cDNA。
用于扩增CD和SBD pcr产物的寡聚物分别列于表2和表3。SOE PCR产物用凝胶电泳和Qiagen旋转柱纯化,用HindIII/XbaI消化并连接到已被HindIII/XbaI切割并经凝胶纯化的pSteD226中。连接过夜后将反应产物电泳到DH10B中,在添加有100微克/ml氨苄青霉素(Sigma)的LB琼脂板上铺板。将转化体平板纯化,提取的质粒用于测序。通过限制性分析和DNA测序验证整个克隆的HindIII-XbaI片段的完整性。然后将选择的质粒转化到感受态酵母InvScI中,在选择性培养基上铺板。将酵母转化体纯化为单一菌落,将等份样品-80℃储存在15%甘油中。
表2:用于扩增葡糖淀粉酶催化结构域的寡聚物;位于正向引物中的HinDIII位点带下划线;起始密码子ATG以粗体表示。
模板 | 正向引物(按5’-3’列出) | 反向引物 | PCR产物 |
pLac102 | TCGTAAGCTTCACCATCTCGTTCCGATCTCTACTCGCC(SEQ ID NO:47) | ACAGGTGCCGGGCACGCTGCTGGC(SEQ ID NO:48) | AN1 |
pLac102 | TCGTAAGCTTCACCATGTCGTTCCGATCTCTACTCGCC(SEQ ID NO:48) | GGTACCAATGGCAGATGTGGCCGC(SEQ ID NO:49) | AN2 |
pLac102 | TCGTAAGCTTCACCATGTCGTTCCGATCTCTACTCGCC(SEQ ID NO:48) | CCACGAGGTGACAGTCACACTGCTG(SEQ ID NO:50) | AN3 |
pSteD226 | TGCAAAGCTTCACCATGGCGTCCCTCGTTGCTGG(SEQ ID NO:51) | GCAGACGGCAGGGACGCTGCTTGC(SEQ IDNO:52) | TE1 |
pSteD226 | TGCAAAGCTTCACCATGGCGTCCCTCGTTGCTGG(SEQ ID NO:51) | TGGGCCCGTGGCATAGGTGGCAGAG(SEQ ID NO:53) | TE2 |
pSteD226 | TGCAAAGCTTCACCATGGCGTCCCTCGTTGCTGG(SEQ ID NO:51) | CCAGACGGTGTTGGTAGCCGTGCT(SEQ ID NO:54) | TE3 |
AR cDNA | AAGAAAGCTTCACCATGTTTCGTTCACTCCTGGCCTTGGC(SEQ ID NO:55) | GCAGGAGGTAGAGACTCCCTTAGCA(SEQ ID NO:56) | AR1 |
AR cDNA | AAGAAAGCTTCACCATGTTTCGTTCACTCCTGGCCTTGGC(SEQ ID NO:55) | ACCCGGGCTTGTAGCACCAGTCGAG(SEQ ID NO:57) | AR2 |
AR cDNA | AAGAAAGCTTCACCATGTTTCGTTCACTCCTGGCCTTGGC(SEQ ID NO:55) | AGTGACCTCGACACTACCCGAGGAG(SEQ ID NO:58) | AR3 |
表3.用于扩增葡糖淀粉酶淀粉结合结构域的寡聚物;位于反向引物5`端的XbaI位点带下划线。
通过SOE(重叠延伸拼接)PCR融合催化结构域和淀粉结合结构域。
用试验方法章节所述SOE PCR制备所需的CD-SBD融合体。SOE反应中使用的PCR产物组合和所得的SOE杂交体列于表4。
表4:SOE pcr反应。
CD片段 | SBD产物 | SOE杂交体名称 | SOE杂交体 |
TE1 | TEANsbd1 | ANTE1 | SEQ ID NO:82 |
TE2 | TEANsbd2 | ANTE2 | SEQ ID NO:83 |
TE3 | TEANsbd3 | ANTE3 | SEQ ID NO:84 |
AR1 | ARANsbd1 | ANAR1 | SEQ ID NO:85 |
AR2 | ARANsbd2 | ANAR2 | SEQ ID NO:86 |
AR3 | ARANsbd3 | ANAR3 | SEQ ID NO:87 |
AR1 | ARTEsbd1 | ARTE1 | SEQ ID NO:88 |
Ar2 | ARTEsbd2 | ARTE2 | SEQ ID NO:89 |
AR3 | ARTEsbd3 | ARTE3 | SEQ ID NO:90 |
AN1 | ANTEsbd1 | ARAN1 | SEQ ID NO:91 |
AN2 | ANTEsbd2 | ARAN2 | SEQ ID NO:92 |
AN3 | ANTEsbd3 | ARAN3 | SEQ ID NO:93 |
TE1 | TEARsbd1 | TEAN1 | SEQ ID NO:94 |
TE2 | TEARsbd2 | TEAN2 | SEQ ID NO:95 |
TE3 | TEARsbd3 | TEAN3 | SEQ ID NO:96 |
AN1 | ANARsbd1 | TEAR1 | SEQ ID NO:97 |
AN2 | ANARsbd2 | TEAR2 | SEQ ID NO:98 |
AN3 | ANARsbd3 | TEAR3 | SEQ ID NO:99 |
实施例2
在“一步”燃料乙醇发酵中评估葡糖淀粉酶-SBM杂交体
通过微小规模发酵评估葡糖淀粉酶-SBM杂交体(TEAN-1,TEAN-3)相对于纯化的埃默森篮状菌葡糖淀粉酶的相对性能。约380g磨碎的玉米(在高速锤磨(pilot scale hammer mill)中磨碎,通过1.65mm筛)加到约620g的自来水中。此混合物中添加3mL 1g/L的青霉素。用40%H2SO4将此浆液的pH调整到5.0。一式三份地测定干燥固体(DS)水平为32%。将约5g的这种浆液加至15mL试管中。该研究中使用的酶详情如下:
酶 |
纯化的埃默森篮状菌葡糖淀粉酶 |
TEAN-1(埃默森篮状菌催化模块和黑曲霉SBM的杂交体) |
TEAN-3(埃默森篮状菌催化模块和黑曲霉SBM的杂交体) |
用每种酶实施四个剂量的剂量反应。所用剂量为0.1,0.3,0.6和1.0AGU/g DS。每个处理有六份重复。
加料后,所述试管用0.04mL/g酵母繁殖物(Red StarTM酵母)糖化醪(mash)接种,所述酵母繁殖物糖化醪已在玉米糖化醪上培养22.5小时。用旋盖盖在试管上部,用小针在旋盖上穿孔以允许空气释放,在称重和32℃保温前简短涡旋。发酵处理后在不同时间对试管称重。称重前将试管简短涡旋。使发酵连续进行约200小时。试验结果如图1所示。
由图1可以看到,两种杂交体TEAN-1,TEAN-3给出了明显高于野生型埃默森篮状菌葡糖淀粉酶的乙醇产量/g DS。
实施例3
在“一步”燃料乙醇发酵中评估葡糖淀粉酶-SBM杂交体
通过小规模发酵评估葡糖淀粉酶-SBM杂交体(TEAR-1,TEAR-1)相对于纯化的埃默森篮状菌葡糖淀粉酶的相对性能。约380g磨碎的玉米(在高速锤磨中磨碎,通过1.65mm筛)加到约620g的自来水中。此混合物中添加3mL 1g/L的青霉素。用40%H2SO4将此浆液的pH调整到5.0。一式三份地测定干燥固体(DS)水平为32%。将约5g的这种浆液加至15mL试管中。每种酶用0.3AGU/g DS的量实施剂量反应。加料后,所述试管用0.04mL/g酵母繁殖物(Red StarTM酵母)糖化醪接种,所述酵母繁殖物糖化醪已在玉米糖化醪上培养22.5小时。用旋盖盖在试管上部,用小针在旋盖上穿孔以允许空气释放,在称重和32℃保温前简短涡旋。发酵处理后在不同时间对试管称重。称重前将试管简短涡旋。使发酵连续进行约70小时。试验结果如以下表1所示:
表1
酶 | 相对活性 |
纯化的埃默森篮状菌葡糖淀粉酶 | 100% |
TEAR-1(埃默森篮状菌催化模块和罗耳阿太菌SBM的杂交体) | 250% |
TEAR-2(埃默森篮状菌催化模块和罗耳阿太菌SBM的杂交体) | 215% |
由表1可以看到,两种杂交体TEAR-1,TEAR-3具有明显高于野生型埃默森篮状菌葡糖淀粉酶的相对活性。
序列表
<110>诺维信北美公司(Novoaymes North America,Inc.)
<120>杂交酶
<130>10554.200-US
<160>99
<170>PatentIn version 3.3
<210>1
<211>396
<212>DNA
<213>白曲霉(Aspergillus kawachii)
<220>
<221>CDS
<222>(1)..(396)
<400>1
act agt aca tcc aaa gcc acc acc tcc tct tct tct tct tct gct gct 48
Thr Ser Thr Ser Lys Ala Thr Thr Ser Ser Ser Ser Ser Ser Ala Ala
1 5 10 15
gct act act tct tca tca tgc acc gca aca agc acc acc ctc ccc atc 96
Ala Thr Thr Ser Ser Ser Cys Thr Ala Thr Ser Thr Thr Leu Pro Ile
20 25 30
acc ttc gaa gaa ctc gtc acc act acc tac ggg gaa gaa gtc tac ctc 144
Thr Phe Glu Glu Leu Val Thr Thr Thr Tyr Gly Glu Glu Val Tyr Leu
35 40 45
agc gga tct atc tcc cag ctc gga gag tgg gat acg agt gac gcg gtg 192
Ser Gly Ser Ile Ser Gln Leu Gly Glu Trp Asp Thr Ser Asp Ala Val
50 55 60
aag ttg tcc gcg gat gat tat acc tcg agt aac ccc gag tgg tct gtt 240
Lys Leu Ser Ala Asp Asp Tyr Thr Ser Ser Asn Pro Glu Trp Ser Val
65 70 75 80
act gtg tcg ttg ccg gtg ggg acg acc ttc gag tat aag ttt att aag 288
Thr Val Ser Leu Pro Val Gly Thr Thr Phe Glu Tyr Lys Phe Ile Lys
85 90 95
gtc gat gag ggt gga agt gtg act tgg gaa agt gat ccg aat agg gag 336
Val Asp Glu Gly Gly Ser Val Thr Trp Glu Ser Asp Pro Asn Arg Glu
100 105 110
tat act gtg cct gaa tgt ggg aat ggg agt ggg gag acg gtg gtt gat 384
Tyr Thr Val Pro Glu Cys Gly Asn Gly Ser Gly Glu Thr Val Val Asp
115 120 125
acg tgg agg tag 396
Thr Trp Arg
130
<210>2
<211>131
<212>PRT
<213>白曲霉(Aspergillus kawachii)
<400>2
Thr Ser Thr Ser Lys Ala Thr Thr Ser Ser Ser Ser Ser Ser Ala Ala
1 5 10 15
Ala Thr Thr Ser Ser Ser Cys Thr Ala Thr Ser Thr Thr Leu Pro Ile
20 25 30
Thr Phe Glu Glu Leu Val Thr Thr Thr Tyr Gly Glu Glu Val Tyr Leu
35 40 45
Ser Gly Ser Ile Ser Gln Leu Gly Glu Trp Asp Thr Ser Asp Ala Val
50 55 60
Lys Leu Ser Ala Asp Asp Tyr Thr Ser Ser Asn Pro Glu Trp Ser Val
65 70 75 80
Thr Val Ser Leu Pro Val Gly Thr Thr Phe Glu Tyr Lys Phe Ile Lys
85 90 95
Val Asp Glu Gly Gly Ser Val Thr Trp Glu Ser Asp Pro Asn Arg Glu
100 105 110
Tyr Thr Val Pro Glu Cys Gly Asn Gly Ser Gly Glu Thr Val Val Asp
115 120 125
Thr Trp Arg
130
<210>3
<211>102
<212>PRT
<213>黄热芽孢杆菌(Bacillus flavothermus)
<400>3
Ile Ser Thr Thr Ser Gln Ile Thr Phe Thr Val Asn Asn Ala Thr Thr
1 5 10 15
Val Trp Gly Gln Asn Val Tyr Val Val Gly Asn Ile Ser Gln Leu Gly
20 25 30
Asn Trp Asp Pro Val His Ala Val Gln Met Thr Pro Ser Ser Tyr Pro
35 40 45
Thr Trp Thr Val Thr Ile Pro Leu Leu Gln Gly Gln Asn Ile Gln Phe
50 55 60
Lys Phe Ile Lys Lys Asp Ser Ala Gly Asn Val Ile Trp Glu Asp Ile
65 70 75 80
Ser Asn Arg Thr Tyr Thr Val Pro Thr Ala Ala Ser Gly Ala Tyr Thr
85 90 95
Ala Ser Trp Asn Val Pro
100
<210>4
<211>99
<212>PRT
<213>芽孢杆菌(Bacillus sp.)
<400>4
Thr Ser Asn Val Thr Phe Thr Val Asn Asn Ala Thr Thr Val Tyr Gly
1 5 10 15
Gln Asn Val Tyr Val Val Gly Asn Ile Pro Glu Leu Gly Asn Trp Asn
20 25 30
Ile Ala Asn Ala Ile Gln Met Thr Pro Ser Ser Tyr Pro Thr Trp Lys
35 40 45
Thr Thr Val Ser Leu Pro Gln Gly Lys Ala Ile Glu Phe Lys Phe Ile
50 55 60
Lys Lys Asp Ser Ala Gly Asn Val Ile Trp Glu Asn Ile Ala Asn Arg
65 70 75 80
Thr Tyr Thr Val Pro Phe Ser Ser Thr Gly Ser Tyr Thr Ala Asn Trp
85 90 95
Asn Val Pro
<210>5
<211>102
<212>PRT
<213>芽孢嗜碱菌(Alcaliphilic Bacillus)
<400>5
Thr Ser Thr Thr Ser Gln Ile Thr Phe Thr Val Asn Asn Ala Thr Thr
1 5 10 15
Val Trp Gly Gln Asn Val Tyr Val Val Gly Asn Ile Ser Gln Leu Gly
20 25 30
Asn Trp Asp Pro Val Asn Ala Val Gln Met Thr Pro Ser Ser Tyr Pro
35 40 45
Thr Trp Val Val Thr Val Pro Leu Pro Gln Ser Gln Asn Ile Gln Phe
50 55 60
Lys Phe Ile Lys Lys Asp Gly Ser Gly Asn Val Ile Trp Glu Asn Ile
65 70 75 80
Ser Asn Arg Thr Tyr Thr Val Pro Thr Ala Ala Ser Gly Ala Tyr Thr
85 90 95
Ala Asn Trp Asn Val Pro
100
<210>6
<211>112
<212>PRT
<213>Hormoconis resinae
<400>6
Cys Gln Val Ser Ile Thr Phe Asn Ile Asn Ala Thr Thr Tyr Tyr Gly
1 5 10 15
Glu Asn Leu Tyr Val Ile Gly Asn Ser Ser Asp Leu Gly Ala Trp Asn
20 25 30
Ile Ala Asp Ala Tyr Pro Leu Ser Ala Ser Ala Tyr Thr Gln Asp Arg
35 40 45
Pro Leu Trp Ser Ala Ala Ile Pro Leu Asn Ala Gly Glu Val Ile Ser
50 55 60
Tyr Gln Tyr Val Arg Gln Glu Asp Cys Asp Gln Pro Tyr Ile Tyr Glu
65 70 75 80
Thr Val Asn Arg Thr Leu Thr Val Pro Ala Cys Gly Gly Ala Ala Val
85 90 95
Thr Thr Asp Asp Ala Trp Met Gly Pro Val Gly Ser Ser Gly Asn Cys
100 105 110
<210>7
<211>95
<212>PRT
<213>埃杜香菇(Lentinula edodes)
<400>7
Val Ser Val Thr Phe Asn Val Asp Ala Ser Thr Leu Glu Gly Gln Asn
1 5 10 15
Val Tyr Leu Thr Gly Ala Val Asp Ala Leu Glu Asp Trp Ser Thr Asp
20 25 30
Asn Ala Ile Leu Leu Ser Ser Ala Asn Tyr Pro Thr Trp Ser Val Thr
35 40 45
Val Asp Leu Pro Gly Ser Thr Asp Val Gln Tyr Lys Tyr Ile Lys Lys
50 55 60
Asp Gly Ser Gly Thr Val Thr Trp Glu Ser Asp Pro Asn Met Glu Ile
65 70 75 80
Thr Thr Pro Ala Asn Gly Thr Tyr Ala Thr Asn Asp Thr Trp Arg
85 90 95
<210>8
<211>107
<212>PRT
<213>粗糙脉孢霉(Neurospora crassa)
<400>8
Cys Ala Ala Asp His Glu Val Leu Val Thr Phe Asn Glu Lys Val Thr
1 5 10 15
Thr Ser Tyr Gly Gln Thr Val Lys Val Val Gly Ser Ile Ala Ala Leu
20 25 30
Gly Asn Trp Ala Pro Ala Ser Gly Val Thr Leu Ser Ala Lys Gln Tyr
35 40 45
Ser Ser Ser Asn Pro Leu Trp Ser Thr Thr Ile Ala Leu Pro Gln Gly
50 55 60
Thr Ser Phe Lys Tyr Lys Tyr Val Val Val Asn Ser Asp Gly Ser Val
65 70 75 80
Lys Trp Glu Asn Asp Pro Asp Arg Ser Tyr Ala Val Gly Thr Asp Cys
85 90 95
Ala Ser Thr Ala Thr Leu Asp Asp Thr Trp Arg
100 105
<210>9
<211>115
<212>PRT
<213>Talaromyces byssochlamydioides
<400>9
Thr Thr Thr Gly Ala Ala Pro Cys Thr Thr Pro Thr Thr Val Ala Val
1 5 10 15
Thr Phe Asp Glu Ile Val Thr Thr Thr Tyr Gly Glu Thr Val Tyr Leu
20 25 30
Ser Gly Ser Ile Pro Ala Leu Gly Asn Trp Asp Thr Ser Ser Ala Ile
35 40 45
Ala Leu Ser Ala Val Asp Tyr Thr Ser Ser Asn Pro Leu Trp Tyr Val
50 55 60
Thr Val Asn Leu Pro Ala Gly Thr Ser Phe Glu Tyr Lys Phe Phe Val
65 70 75 80
Gln Gln Thr Asp Gly Thr Ile Val Trp Glu Asp Asp Pro Asn Arg Ser
85 90 95
Tyr Thr Val Pro Ala Asn Cys Gly Gln Thr Thr Ala Ile Ile Asp Asp
100 105 110
Ser Trp Gln
115
<210>10
<211>115
<212>PRT
<213>Geosmithia cylindrospora:
<400>10
Thr Ser Thr Gly Ser Ala Pro Cys Thr Thr Pro Thr Thr Val Ala Val
1 5 10 15
Thr Phe Asp Glu Ile Val Thr Thr Ser Tyr Gly Glu Thr Val Tyr Leu
20 25 30
Ala Gly Ser Ile Ala Ala Leu Gly Asn Trp Asp Thr Asn Ser Ala Ile
35 40 45
Ala Leu Ser Ala Ala Asp Tyr Thr Ser Asn Asn Asn Leu Trp Tyr Val
50 55 60
Thr Val Asn Leu Ala Ala Gly Thr Ser Phe Gln Tyr Lys Phe Phe Val
65 70 75 80
Lys Glu Thr Asp Ser Thr Ile Val Trp Glu Asp Asp Pro Asn Arg Ser
85 90 95
Tyr Thr Val Pro Ala Asn Cys Gly Gln Thr Thr Ala Ile Ile Asp Asp
100 105 110
Thr Trp Gln
115
<210>11
<211>139
<212>PRT
<213>Scorias spongiosa
<400>11
Ala Lys Val Pro Ser Thr Cys Ser Ala Ser Ser Ala Thr Gly Thr Cys
1 5 10 15
Thr Thr Ala Thr Ser Thr Phe Gly Gly Ser Thr Pro Thr Thr Ser Cys
20 25 30
Ala Thr Thr Pro Thr Leu Thr Thr Val Leu Phe Asn Glu Arg Ala Thr
35 40 45
Thr Asn Phe Gly Gln Asn Val His Leu Thr Gly Ser Ile Ser Gln Leu
50 55 60
Gly Ser Trp Asp Thr Asp Ser Ala Val Ala Leu Ser Ala Val Asn Tyr
65 70 75 80
Thr Ser Ser Asp Pro Leu Trp Phe Val Arg Val Gln Leu Pro Ala Gly
85 90 95
Thr Ser Phe Gln Tyr Lys Tyr Phe Lys Lys Asp Ser Ser Asn Ala Val
100 105 110
Ala Trp Glu Ser Asp Pro Asn Arg Ser Tyr Thr Val Pro Leu Asn Cys
115 120 125
Ala Gly Thr Ala Thr Glu Asn Asp Thr Trp Arg
130 135
<210>12
<211>126
<212>PRT
<213>Eupenicillium ludwigii
<400>12
Ser Thr Thr Thr Thr Ser Thr Thr Lys Thr Thr Thr Thr Ser Thr Thr
1 5 10 15
Thr Ser Cys Thr Thr Pro Thr Ala Val Ala Val Thr Phe Asp Leu Ile
20 25 30
Ala Thr Thr Tyr Tyr Gly Glu Asn Ile Lys Ile Ala Gly Ser Ile Ser
35 40 45
Gln Leu Gly Asp Trp Asp Thr Ser Asn Ala Val Ala Leu Ser Ala Ala
50 55 60
Asp Tyr Thr Ser Ser Asp His Leu Trp Phe Val Asp Ile Asp Leu Pro
65 70 75 80
Ala Gly Thr Val Phe Glu Tyr Lys Tyr Ile Arg Ile Glu Ser Asp Gly
85 90 95
Ser Ile Glu Trp Glu Ser Asp Pro Asn Arg Ser Tyr Thr Val Pro Ala
100 105 110
Ala Cys Ala Thr Thr Ala Val Thr Glu Asn Asp Thr Trp Arg
115 120 125
<210>13
<211>116
<212>PRT
<213>日本曲霉(Aspergillus japonicus)
<400>13
Lys Thr Ser Thr Thr Thr Ser Ser Cys Ser Thr Pro Thr Ser Val Ala
1 5 10 15
Val Thr Phe Asp Val Ile Ala Thr Thr Thr Tyr Gly Glu Asn Val Tyr
20 25 30
Ile Ser Gly Ser Ile Ser Gln Leu Gly Ser Trp Asp Thr Ser Ser Ala
35 40 45
Ile Ala Leu Ser Ala Ser Gln Tyr Thr Ser Ser Asn Asn Leu Trp Tyr
50 55 60
Ala Thr Val His Leu Pro Ala Gly Thr Thr Phe Gln Tyr Lys Tyr Ile
65 70 75 80
Arg Lys Glu Thr Asp Gly Ser Val Thr Trp Glu Ser Asp Pro Asn Arg
85 90 95
Ser Tyr Thr Val Pro Ser Ser Cys Gly Val Ser Ser Ala Thr Glu Ser
100 105 110
Asp Thr Trp Arg
115
<210>14
<211>133
<212>PRT
<213>米舒青霉(Penicillium cf.miczynskii)
<400>14
Thr Thr Thr Gly Gly Thr Thr Thr Ser Gln Gly Ser Thr Thr Thr Thr
1 5 10 15
Ser Lys Thr Ser Thr Thr Thr Ser Ser Cys Thr Ala Pro Thr Ser Val
20 25 30
Ala Val Thr Phe Asp Leu Ile Ala Thr Thr Val Tyr Asp Glu Asn Val
35 40 45
Gln Leu Ala Gly Ser Ile Ser Ala Leu Gly Ser Trp Asp Thr Ser Ser
50 55 60
Ala Ile Arg Leu Ser Ala Ser Gln Tyr Thr Ser Ser Asn His Leu Trp
65 70 75 80
Tyr Val Ala Val Ser Leu Pro Ala Gly Gln Val Phe Gln Tyr Lys Tyr
85 90 95
Ile Arg Val Ala Ser Ser Gly Thr Ile Thr Trp Glu Ser Asp Pro Asn
100 105 110
Leu Ser Tyr Thr Val Pro Val Ala Cys Ala Ala Thr Ala Val Thr Ile
115 120 125
Ser Asp Thr Trp Arg
130
<210>15
<211>116
<212>PRT
<213>Mz1青霉(Penicillium sp.)
<400>15
Thr Lys Thr Ser Thr Ser Thr Ser Cys Thr Thr Pro Thr Ala Val Ala
1 5 10 15
Val Thr Phe Asp Leu Ile Ala Thr Thr Thr Tyr Gly Glu Asn Ile Lys
20 25 30
Ile Ala Gly Ser Ile Ala Ala Leu Gly Ala Trp Asp Thr Asp Asp Ala
35 40 45
Val Ala Leu Ser Ala Ala Asp Tyr Thr Asp Ser Asp His Leu Trp Phe
50 55 60
Val Thr Gln Ser Ile Pro Ala Gly Thr Val Phe Glu Tyr Lys Tyr Ile
65 70 75 80
Arg Val Glu Ser Asp Gly Thr Ile Glu Trp Glu Ser Asp Pro Asn Arg
85 90 95
Ser Tyr Thr Val Pro Ala Ala Cys Ala Thr Thr Ala Val Thr Glu Ser
100 105 110
Asp Thr Trp Arg
115
<210>16
<211>114
<212>PRT
<213>Thysanophora sp
<400>16
Phe Thr Ser Thr Thr Lys Thr Ser Cys Thr Thr Pro Thr Ser Val Ala
1 5 10 15
Val Thr Phe Asp Leu Ile Ala Thr Thr Thr Tyr Gly Glu Ser Ile Arg
20 25 30
Leu Val Gly Ser Ile Ser Glu Leu Gly Asp Trp Asp Thr Gly Ser Ala
35 40 45
Ile Ala Leu His Ala Thr Asp Tyr Thr Asp Ser Asp His Leu Trp Phe
50 55 60
Val Thr Val Gly Leu Pro Ala Gly Ala Ser Phe Glu Tyr Lys Tyr Ile
65 70 75 80
Arg Val Glu Ser Ser Gly Thr Ile Glu Trp Glu Ser Asp Pro Asn Arg
85 90 95
Ser Tyr Thr Val Pro Ala Ala Cys Ala Thr Thr Ala Val Thr Glu Ser
100 105 110
Asp Thr
<210>17
<211>111
<212>PRT
<213>灰腐质霉高温变种(Humicola grisea var.thermoidea)
<400>17
Ala Asp Ala Ser Glu Val Tyr Val Thr Phe Asn Glu Arg Val Ser Thr
1 5 10 15
Ala Trp Gly Glu Thr Ile Lys Val Val Gly Asn Val Pro Ala Leu Gly
20 25 30
Asn Trp Asp Thr Ser Lys Ala Val Thr Leu Ser Ala Ser Gly Tyr Lys
35 40 45
Ser Asn Asp Pro Leu Trp Ser Ile Thr Val Pro Ile Lys Ala Thr Gly
50 55 60
Ser Ala Val Gln Tyr Lys Tyr Ile Lys Val Gly Thr Asn Gly Lys Ile
65 70 75 80
Thr Trp Glu Ser Asp Pro Asn Arg Ser Ile Thr Leu Gln Thr Ala Ser
85 90 95
Ser Ala Gly Lys Cys Ala Ala Gln Thr Val Asn Asp Ser Trp Arg
100 105 110
<210>18
<211>108
<212>PRT
<213>黑曲霉(Aspergillus niger)
<400>18
Cys Thr Thr Pro Thr Ala Val Ala Val Thr Phe Asp Leu Thr Ala Thr
1 5 10 15
Thr Thr Tyr Gly Glu Asn Ile Tyr Leu Val Gly Ser Ile Ser Gln Leu
20 25 30
Gly Asp Trp Glu Thr Ser Asp Gly Ile Ala Leu Ser Ala Asp Lys Tyr
35 40 45
Thr Ser Ser Asp Pro Leu Trp Tyr Val Thr Val Thr Leu Pro Ala Gly
50 55 60
Glu Ser Phe Glu Tyr Lys Phe Ile Arg Ile Glu Ser Asp Asp Ser Val
65 70 75 80
Glu Trp Glu Ser Asp Pro Asn Arg Glu Tyr Thr Val Pro Gln Ala Cys
85 90 95
Gly Thr Ser Thr Ala Thr Val Thr Asp Thr Trp Arg
100 105
<210>19
<211>97
<212>PRT
<213>Aspergillus rolfsii
<400>19
Val Glu Val Thr Phe Asp Val Tyr Ala Thr Thr Val Tyr Gly Gln Asn
1 5 10 15
Ile Tyr Ile Thr Gly Asp Val Ser Glu Leu Gly Asn Trp Thr Pro Ala
20 25 30
Asn Gly Val Ala Leu Ser Ser Ala Asn Tyr Pro Thr Trp Ser Ala Thr
35 40 45
Ile Ala Leu Pro Ala Asp Thr Thr Ile Gln Tyr Lys Tyr Val Asn Ile
50 55 60
Asp Gly Ser Thr Val Ile Trp Glu Asp Ala Ile Ser Asn Arg Glu Ile
65 70 75 80
Thr Thr Pro Ala Ser Gly Thr Tyr Thr Glu Lys Asp Thr Trp Asp Glu
85 90 95
Ser
<210>20
<211>38
<212>PRT
<213>黑曲霉(Aspergillus niger)
<400>20
Thr Gly Gly Thr Thr Thr Thr Ala Thr Pro Thr Gly Ser Gly Ser Val
1 5 10 15
Thr Ser Thr Ser Lys Thr Thr Ala Thr Ala Ser Lys Thr Ser Thr Ser
20 25 30
Thr Ser Ser Thr Ser Ala
35
<210>21
<211>31
<212>PRT
<213>白曲霉(Aspergillus kawachii)
<400>21
Thr Thr Thr Thr Thr Thr Ala Ala Ala Thr Ser Thr Ser Lys Ala Thr
1 5 10 15
Thr Ser Ser Ser Ser Ser Ser Ala Ala Ala Thr Thr Ser Ser Ser
20 25 30
<210>22
<211>8
<212>PRT
<213>人工的
<220>
<223>人工的
<400>22
Pro Glu Pro Thr Pro Glu Pro Thr
1 5
<210>23
<211>1605
<212>DNA
<213>黑曲霉(Aspergillus niger)
<220>
<221>CDS
<222>(1)..(1602)
<220>
<221>sig_peptide
<222>(1)..(72)
<220>
<221>mat_peptide
<222>(73)..(1602)
<400>23
atg tcg ttc cga tct cta ctc gcc ctg agc ggc ctc gtc tgc aca ggg 48
Met Ser Phe Arg Ser Leu Leu Ala Leu Ser Gly Leu Val Cys Thr Gly
-20 -15 -10
ttg gca aat gtg att tcc aag cgc gcg acc ttg gat tca tgg ttg agc 96
Leu Ala Asn Val Ile Ser Lys Arg Ala Thr Leu Asp Ser Trp Leu Ser
-5 -1 1 5
aac gaa gcg acc gtg gct cgt act gcc atc ctg aat aac atc ggg gcg 144
Asn Glu Ala Thr Val Ala Arg Thr Ala Ile Leu Asn Asn Ile Gly Ala
10 15 20
gac ggt gct tgg gtg tcg ggc gcg gac tct ggc att gtc gtt gct agt 192
Asp Gly Ala Trp Val Ser Gly Ala Asp Ser Gly Ile Val Val Ala Ser
25 30 35 40
ccc agc acg gat aac ccg gac tac ttc tac acc tgg act cgc gac tct 240
Pro Ser Thr Asp Asn Pro Asp Tyr Phe Tyr Thr Trp Thr Arg Asp Ser
45 50 55
ggt ctc gtc ctc aag acc ctc gtc gat ctc ttc cga aat gga gat acc 288
Gly Leu Val Leu Lys Thr Leu Val Asp Leu Phe Arg Asn Gly Asp Thr
60 65 70
agt ctc ctc tcc acc att gag aac tac atc tcc gcc cag gca att gtc 336
Ser Leu Leu Ser Thr Ile Glu Asn Tyr Ile Ser Ala Gln Ala Ile Val
75 80 85
cag ggt atc agt aac ccc tct ggt gat ctg tcc agc ggc gct ggt ctc 384
Gln Gly Ile Ser Asn Pro Ser Gly Asp Leu Ser Ser Gly Ala Gly Leu
90 95 100
ggt gaa ccc aag ttc aat gtc gat gag act gcc tac act ggt tct tgg 432
Gly Glu Pro Lys Phe Asn Val Asp Glu Thr Ala Tyr Thr Gly Ser Trp
105 110 115 120
gga cgg ccg cag cga gat ggt ccg gct ctg aga gca act gct atg atc 480
Gly Arg Pro Gln Arg Asp Gly Pro Ala Leu Arg Ala Thr Ala Met Ile
125 130 135
ggc ttc ggg cag tgg ctg ctt gac aat ggc tac acc agc acc gca acg 528
Gly Phe Gly Gln Trp Leu Leu Asp Asn Gly Tyr Thr Ser Thr Ala Thr
140 145 150
gac att gtt tgg ccc ctc gtt agg aac gac ctg tcg tat gtg gct caa 576
Asp Ile Val Trp Pro Leu Val Arg Asn Asp Leu Ser Tyr Val Ala Gln
155 160 165
tac tgg aac cag aca gga tat gat ctc tgg gaa gaa gtc aat ggc tcg 624
Tyr Trp Asn Gln Thr Gly Tyr Asp Leu Trp Glu Glu Val Asn Gly Ser
170 175 180
tct ttc ttt acg att gct gtg caa cac cgc gcc ctt gtc gaa ggt agt 672
Ser Phe Phe Thr Ile Ala Val Gln His Arg Ala Leu Val Glu Gly Ser
185 190 195 200
gcc ttc gcg acg gcc gtc ggc tcg tcc tgc tcc tgg tgt gat tct cag 720
Ala Phe Ala Thr Ala Val Gly Ser Ser Cys Ser Trp Cys Asp Ser Gln
205 210 215
gca ccc gaa att ctc tgc tac ctg cag tcc ttc tgg acc ggc agc ttc 768
Ala Pro Glu Ile Leu Cys Tyr Leu Gln Ser Phe Trp Thr Gly Ser Phe
220 225 230
att ctg gcc aac ttc gat agc agc cgt tcc ggc aag gac gca aac acc 816
Ile Leu Ala Asn Phe Asp Ser Ser Arg Ser Gly Lys Asp Ala Asn Thr
235 240 245
ctc ctg gga agc atc cac acc ttt gat cct gag gcc gca tgc gac gac 864
Leu Leu Gly Ser Ile His Thr Phe Asp Pro Glu Ala Ala Cys Asp Asp
250 255 260
tcc acc ttc cag ccc tgc tcc ccg cgc gcg ctc gcc aac cac aag gag 912
Ser Thr Phe Gln Pro Cys Ser Pro Arg Ala Leu Ala Asn His Lys Glu
265 270 275 280
gtt gta gac tct ttc cgc tca atc tat acc ctc aac gat ggt ctc agt 960
Val Val Asp Ser Phe Arg Ser Ile Tyr Thr Leu Asn Asp Gly Leu Ser
285 290 295
gac agc gag gct gtt gcg gtg ggt cgg tac cct gag gac acg tac tac 1008
Asp Ser Glu Ala Val Ala Val Gly Arg Tyr Pro Glu Asp Thr Tyr Tyr
300 305 310
aac ggc aac ccg tgg ttc ctg tgc acc ttg gct gcc gca gag cag ttg 1056
Asn Gly Asn Pro Trp Phe Leu Cys Thr Leu Ala Ala Ala Glu Gln Leu
315 320 325
tac gat gct cta tac cag tgg gac aag cag ggg tcg ttg gag gtc aca 1104
Tyr Asp Ala Leu Tyr Gln Trp Asp Lys Gln Gly Ser Leu Glu Val Thr
330 335 340
gat gtg tcg ctg gac ttc ttc aag gca ctg tac agc gat gct gct act 1152
Asp Val Ser Leu Asp Phe Phe Lys Ala Leu Tyr Ser Asp Ala Ala Thr
345 350 355 360
ggc acc tac tct tcg tcc agt tcg act tat agt agc att gta gat gcc 1200
Gly Thr Tyr Ser Ser Ser Ser Ser Thr Tyr Ser Ser Ile Val Asp Ala
365 370 375
gtg aag act ttc gcc gat ggc ttc gtc tct att gtg gaa act cac gcc 1248
Val Lys Thr Phe Ala Asp Gly Phe Val Ser Ile Val Glu Thr His Ala
380 385 390
gca agc aac ggc tcc atg tcc gag caa tac gac aag tct gat ggc gag 1296
Ala Ser Asn Gly Ser Met Ser Glu Gln Tyr Asp Lys Ser Asp Gly Glu
395 400 405
cag ctt tcc gct cgc gac ctg acc tgg tct tat gct gct ctg ctg acc 1344
Gln Leu Ser Ala Arg Asp Leu Thr Trp Ser Tyr Ala Ala Leu Leu Thr
410 415 420
gcc aac aac cgt cgt aac tcc gtc gtg cct gct tct tgg ggc gag acc 1392
Ala Asn Asn Arg Arg Asn Ser Val Val Pro Ala Ser Trp Gly Glu Thr
425 430 435 440
tct gcc agc agc gtg ccc ggc acc tgt gcg gcc aca tct gcc att ggt 1440
Ser Ala Ser Ser Val Pro Gly Thr Cys Ala Ala Thr Ser Ala Ile Gly
445 450 455
acc tac agc agt gtg act gtc acc tcg tgg ccg agt atc gtg gct act 1488
Thr Tyr Ser Ser Val Thr Val Thr Ser Trp Pro Ser Ile Val Ala Thr
460 465 470
ggc ggc acc act acg acg gct acc ccc act gga tcc ggc agc gtg acc 1536
Gly Gly Thr Thr Thr Thr Ala Thr Pro Thr Gly Ser Gly Ser Val Thr
475 480 485
tcg acc agc aag acc acc gcg act gct agc aag acc agc acc acg acc 1584
Ser Thr Ser Lys Thr Thr Ala Thr Ala Ser Lys Thr Ser Thr Thr Thr
490 495 500
cgc tct ggt atg tca ctg tga 1605
Arg Ser Gly Met Ser Leu
505 510
<210>24
<211>534
<212>PRT
<213>黑曲霉(Aspergillus niger)
<400>24
Met Ser Phe Arg Ser Leu Leu Ala Leu Ser Gly Leu Val Cys Thr Gly
-20 -15 -10
Leu Ala Asn Val Ile Ser Lys Arg Ala Thr Leu Asp Ser Trp Leu Ser
-5 -1 1 5
Asn Glu Ala Thr Val Ala Arg Thr Ala Ile Leu Asn Asn Ile Gly Ala
10 15 20
Asp Gly Ala Trp Val Ser Gly Ala Asp Ser Gly Ile Val Val Ala Ser
25 30 35 40
Pro Ser Thr Asp Asn Pro Asp Tyr Phe Tyr Thr Trp Thr Arg Asp Ser
45 50 55
Gly Leu Val Leu Lys Thr Leu Val Asp Leu Phe Arg Asn Gly Asp Thr
60 65 70
Ser Leu Leu Ser Thr Ile Glu Asn Tyr Ile Ser Ala Gln Ala Ile Val
75 80 85
Gln Gly Ile Ser Asn Pro Ser Gly Asp Leu Ser Ser Gly Ala Gly Leu
90 95 100
Gly Glu Pro Lys Phe Asn Val Asp Glu Thr Ala Tyr Thr Gly Ser Trp
105 110 115 120
Gly Arg Pro Gln Arg Asp Gly Pro Ala Leu Arg Ala Thr Ala Met Ile
125 130 135
Gly Phe Gly Gln Trp Leu Leu Asp Asn Gly Tyr Thr Ser Thr Ala Thr
140 145 150
Asp Ile Val Trp Pro Leu Val Arg Asn Asp Leu Ser Tyr Val Ala Gln
155 160 165
Tyr Trp Asn Gln Thr Gly Tyr Asp Leu Trp Glu Glu Val Asn Gly Ser
170 175 180
Ser Phe Phe Thr Ile Ala Val Gln His Arg Ala Leu Val Glu Gly Ser
185 190 195 200
Ala Phe Ala Thr Ala Val Gly Ser Ser Cys Ser Trp Cys Asp Ser Gln
205 210 215
Ala Pro Glu Ile Leu Cys Tyr Leu Gln Ser Phe Trp Thr Gly Ser Phe
220 225 230
Ile Leu Ala Asn Phe Asp Ser Ser Arg Ser Gly Lys Asp Ala Asn Thr
235 240 245
Leu Leu Gly Ser Ile His Thr Phe Asp Pro Glu Ala Ala Cys Asp Asp
250 255 260
Ser Thr Phe Gln Pro Cys Ser Pro Arg Ala Leu Ala Asn His Lys Glu
26 5270 275 280
Val Val Asp Ser Phe Arg Ser Ile Tyr Thr Leu Asn Asp Gly Leu Ser
285 290 295
Asp Ser Glu Ala Val Ala Val Gly Arg Tyr Pro Glu Asp Thr Tyr Tyr
300 305 310
Asn Gly Asn Pro Trp Phe Leu Cys Thr Leu Ala Ala Ala Glu Gln Leu
315 320 325
Tyr Asp Ala Leu Tyr Gln Trp Asp Lys Gln Gly Ser Leu Glu Val Thr
330 335 340
Asp Val Ser Leu Asp Phe Phe Lys Ala Leu Tyr Ser Asp Ala Ala Thr
345 350 355 360
Gly Thr Tyr Ser Ser Ser Ser Ser Thr Tyr Ser Ser Ile Val Asp Ala
365 370 375
Val Lys Thr Phe Ala Asp Gly Phe Val Ser Ile Val Glu Thr His Ala
380 385 390
Ala Ser Asn Gly Ser Met Ser Glu Gln Tyr Asp Lys Ser Asp Gly Glu
395 400 405
Gln Leu Ser Ala Arg Asp Leu Thr Trp Ser Tyr Ala Ala Leu Leu Thr
410 415 420
Ala Asn Asn Arg Arg Asn Ser Val Val Pro Ala Ser Trp Gly Glu Thr
425 430 435 440
Ser Ala Ser Ser Val Pro Gly Thr Cys Ala Ala Thr Ser Ala Ile Gly
445 450 455
Thr Tyr Ser Ser Val Thr Val Thr Ser Trp Pro Ser Ile Val Ala Thr
460 465 470
Gly Gly Thr Thr Thr Thr Ala Thr Pro Thr Gly Ser Gly Ser Val Thr
475 480 485
Ser Thr Ser Lys Thr Thr Ala Thr Ala Ser Lys Thr Ser Thr Thr Thr
490 495 500
Arg Ser Gly Met Ser Leu
505 510
<210>25
<210>591
<212>PRT
<213>埃默森篮状菌(Talaromyces emersonii)
<400>25
Ala Thr Gly Ser Leu Asp Ser Phe Leu Ala Thr Glu Thr Pro Ile Ala
1 5 10 15
Leu Gln Gly Val Leu Asn Asn Ile Gly Pro Asn Gly Ala Asp Val Ala
20 25 30
Gly Ala Ser Ala Gly Ile Val Val Ala Ser Pro Ser Arg Ser Asp Pro
35 40 45
Asn Tyr Phe Tyr Ser Trp Thr Arg Asp Ala Ala Leu Thr Ala Lys Tyr
50 55 60
Leu Val Asp Ala Phe Asn Arg Gly Asn Lys Asp Leu Glu Gln Thr Ile
65 70 75 80
Gln Gln Tyr Ile Ser Ala Gln Ala Lys Val Gln Thr Ile Ser Asn Pro
85 90 95
Ser Gly Asp Leu Ser Thr Gly Gly Leu Gly Glu Pro Lys Phe Asn Val
100 105 110
Asn Glu Thr Ala Phe Thr Gly Pro Trp Gly Arg Pro Gln Arg Asp Gly
115 120 125
Pro Ala Leu Arg Ala Thr Ala Leu Ile Ala Tyr Ala Asn Tyr Leu Ile
130 135 140
Asp Asn Gly Glu Ala Ser Thr Ala Asp Glu Ile Ile Trp Pro Ile Val
145 150 155 160
Gln Asn Asp Leu Ser Tyr Ile Thr Gln Tyr Trp Asn Ser Ser Thr Phe
165 170 175
Asp Leu Trp Glu Glu Val Glu Gly Ser Ser Phe Phe Thr Thr Ala Val
180 185 190
Gln His Arg Ala Leu Val Glu Gly Asn Ala Leu Ala Thr Arg Leu Asn
195 200 205
His Thr Cys Ser Asn Cys Val Ser Gln Ala Pro Gln Val Leu Cys Phe
210 215 220
Leu Gln Ser Tyr Trp Thr Gly Ser Tyr Val Leu Ala Asn Phe Gly Gly
225 230 235 240
Ser Gly Arg Ser Gly Lys Asp Val Asn Ser Ile Leu Gly Ser Ile His
245 250 255
Thr Phe Asp Pro Ala Gly Gly Cys Asp Asp Ser Thr Phe Gln Pro Cys
260 265 270
Ser Ala Arg Ala Leu Ala Asn His Lys Val Val Thr Asp Ser Phe Arg
275 280 285
Ser Ile Tyr Ala Ile Asn Ser Gly Ile Ala Glu Gly Ser Ala Val Ala
290 295 300
Val Gly Arg Tyr Pro Glu Asp Val Tyr Gln Gly Gly Asn Pro Trp Tyr
305 310 315 320
Leu Ala Thr Ala Ala Ala Ala Glu Gln Leu Tyr Asp Ala Ile Tyr Gln
325 330 335
Trp Lys Lys Ile Gly Ser Ile Ser Ile Thr Asp Val Ser Leu Pro Phe
340 345 350
Phe Gln Asp Ile Tyr Pro Ser Ala Ala Val Gly Thr Tyr Asn Ser Gly
355 360 365
Ser Thr Thr Phe Asn Asp Ile Ile Ser Ala Val Gln Thr Tyr Gly Asp
370 375 380
Gly Tyr Leu Ser Ile Val Glu Lys Tyr Thr Pro Ser Asp Gly Ser Leu
385 390 395 400
Thr Glu Gln Phe Ser Arg Thr Asp Gly Thr Pro Leu Ser Ala Ser Ala
405 410 415
Leu Thr Trp Ser Tyr Ala Ser Leu Leu Thr Ala Ser Ala Arg Arg Gln
420 425 430
Ser Val Val Pro Ala Ser Trp Gly Glu Ser Ser Ala Ser Ser Val Leu
435 440 445
Ala Val Cys Ser Ala Thr Ser Ala Thr Gly Pro Tyr Ser Thr Ala Thr
450 455 460
Asn Thr Val Trp Pro Ser Ser Gly Ser Gly Ser Ser Thr Thr Thr Ser
465 470 475 480
Ser Ala Pro Cys Thr Thr Pro Thr Ser Val Ala Val Thr Phe Asp Glu
485 490 495
Ile Val Ser Thr Ser Tyr Gly Glu Thr Ile Tyr Leu Ala Gly Ser Ile
500 505 510
Pro Glu Leu Gly Asn Trp Ser Thr Ala Ser Ala Ile Pro Leu Arg Ala
515 520 525
Asp Ala Tyr Thr Asn Ser Asn Pro Leu Trp Tyr Val Thr Val Asn Leu
530 535 540
Pro Pro Gly Thr Ser Phe Glu Tyr Lys Phe Phe Lys Asn Gln Thr Asp
545 550 555 560
Gly Thr Ile Val Trp Glu Asp Asp Pro Asn Arg Ser Tyr Thr Val Pro
565 570 575
Ala Tyr Cys Gly Gln Thr Thr Ala Ile Leu Asp Asp Ser Trp Gln
580 585 590
<210>26
<211>451
<212>PRT
<213>罗耳阿太菌(Athelium rolfsii)
<400>26
Gln Ser Ala Ser Ala Thr Ala Tyr Leu Thr Lys Glu Ser Ala Val Ala
1 5 10 15
Lys Asn Gly Val Leu Cys Asn Ile Gly Ser Gln Gly Cys Met Ser Glu
20 25 30
Gly Ala Tyr Ser Gly Ile Val Ile Ala Ser Pro Ser Lys Thr Ser Pro
35 40 45
Asp Tyr Leu Tyr Thr Trp Thr Arg Asp Ser Ser Leu Val Phe Lys Met
50 55 60
Leu Ile Asp Gln Tyr Thr Asn Gly Leu Asp Thr Thr Leu Arg Thr Leu
65 70 75 80
Ile Asp Glu Phe Val Ser Ala Glu Ala Thr Ile Gln Gln Thr Ser Asn
85 90 95
Ser Ser Gly Thr Val Ser Thr Gly Gly Leu Gly Glu Pro Lys Phe Asn
100 105 110
Ile Asp Glu Thr Ala Phe Thr Gly Ala Trp Gly Arg Pro Gln Arg Asp
115 120 125
Gly Pro Ala Leu Arg Ala Thr Ala Ile Met Thr Tyr Ala Thr Tyr Leu
130 135 140
Tyr Asn Asn Gly Asn Thr Ser Tyr Val Thr Asn Thr Leu Trp Pro Ile
145 150 155 160
Ile Lys Leu Asp Leu Asp Tyr Val Asn Ser Asp Trp Asn Gln Thr Thr
165 170 175
Phe Asp Leu Trp Glu Glu Val Asp Ser Ser Ser Phe Phe Thr Thr Ala
180 185 190
Val Gln His Arg Ala Leu Val Gln Gly Ala Ala Phe Ala Thr Leu Ile
195 200 205
Gly Gln Thr Ser Ser Ala Ser Thr Tyr Ser Ala Thr Ala Pro Ser Ile
210 215 220
Leu Cys Phe Leu Gln Ser Tyr Trp Asn Thr Asn Gly Tyr Trp Thr Ala
225 230 235 240
Asn Thr Gly Gly Gly Arg Ser Gly Lys Asp Ala Asn Thr Ile Leu Ala
245 250 255
Ser Ile His Thr Phe Asp Ala Ser Ala Gly Cys Ser Ala Ala Thr Ser
260 265 270
Gln Pro Cys Ser Asp Val Ala Leu Ala Asn Leu Lys Val Tyr Val Asp
275 280 285
Ser Phe Arg Ser Ile Tyr Thr Ile Asn Ser Gly Ile Ser Ser Thr Ser
290 295 300
Gly Val Ala Thr Gly Arg Tyr Pro Glu Asp Ser Tyr Tyr Asn Gly Asn
305 310 315 320
Pro Trp Tyr Leu Cys Thr Leu Ala Val Ala Glu Gln Leu Tyr Asp Ala
325 330 335
Leu Ile Val Trp Lys Ala Ala Gly Glu Leu Asn Val Thr Ser Val Ser
340 345 350
Leu Ala Phe Phe Gln Gln Phe Asp Ser Ser Ile Thr Ala Gly Thr Tyr
355 360 365
Ala Ser Ser Ser Ser Val Tyr Thr Ser Leu Ile Ser Asp Ile Gln Ala
370 375 380
Phe Ala Asp Glu Phe Val Asp Ile Val Ala Lys Tyr Thr Pro Ser Ser
385 390 395 400
Gly Phe Leu Ser Glu Gln Tyr Asp Lys Ser Thr Gly Ala Gln Asp Ser
405 410 415
Ala Ala Asn Leu Thr Trp Ser Tyr Ala Ala Ala Ile Thr Ala Tyr Gln
420 425 430
Ala Arg Asn Gly Phe Thr Gly Ala Ser Trp Gly Ala Lys Gly Val Ser
435 440 445
Thr Ser Cys
450
<210>27
<211>17
<212>PRT
<213>罗耳阿太菌(Athelia rolfsii)
<220>
<221>MISC_FEATURE
<222>(1)..()
<223>Linker
<400>27
Ser Thr Gly Ala Thr Ser Pro Gly Gly Ser Ser Gly Ser Val Glu Val
1 5 10 15
Thr
<210>28
<211>93
<212>PRT
<213>罗耳阿太菌(Athelia rolfsii)
<220>
<221>MISC_FEATURE
<222>(1)..()
<223>罗耳阿太菌(Athelium rolfsii)CBM
<400>28
Phe Asp Val Tyr Ala Thr Thr Val Tyr Gly Gln Asn Ile Tyr Ile Thr
1 5 10 15
Gly Asp Val Ser Glu Leu Gly Asn Trp Thr Pro Ala Asn Gly Val Ala
20 25 30
Leu Ser Ser Ala Asn Tyr Pro Thr Trp Ser Ala Thr Ile Ala Leu Pro
35 40 45
Ala Asp Thr Thr Ile Gln Tyr Lys Tyr Val Asn Ile Asp Gly Ser Thr
50 55 60
Val Ile Trp Glu Asp Ala Ile Ser Asn Arg Glu Ile Thr Thr Pro Ala
65 70 75 80
Ser Gly Thr Tyr Thr Glu Lys Asp Thr Trp Asp Glu Ser
85 90
<210>29
<211>33
<212>PRT
<213>黑曲霉(Aspergillus niger)-埃默森篮状菌(Talaromyces emersonii)
<220>
<221>MISC_FEATURE
<222>(1)..(33)
<223>融合连接点在位置8
<400>29
Ser Ser Val Pro Gly Thr Cys Ser Ala Thr Ser Ala Thr Gly Pro Tyr
1 5 10 15
Ser Thr Ala Thr Asn Thr Val Trp Pro Ser Ser Gly Ser Gly Ser Ser
20 25 30
Thr
<210>30
<211>33
<212>PRT
<213>黑曲霉(Aspergillus niger)-埃默森篮状菌(Talaromyces emersonii)
<220>
<221>MISC_FEATURE
<222>(1)..(33)
<223>融合连接点在位置16
<400>30
Ser Ser Val Pro Gly Thr Cys Ala Ala Thr Ser Ala Ile Gly Thr Tyr
1 5 10 15
Ser Thr Ala Thr Asn Thr Val Trp Pro Ser Ser Gly Ser Gly Ser Ser
20 25 30
Thr
<210>31
<211>33
<212>PRT
<213>黑曲霉(Aspergillus niger)-埃默森篮状菌(Talaromyces emersonii)
<220>
<221>MISC_FEATURE
<222>(1)..(33)
<223>融合连接点在位置25
<400>31
Ser Ser Val Pro Gly Thr Cys Ala Ala Thr Ser Ala Ile Gly Thr Tyr
1 5 10 15
Ser Ser Val Thr Val Thr Ser Trp Pro Ser Ser Gly Ser Gly Ser Ser
20 25 30
Thr
<210>32
<211>33
<212>PRT
<213>黑曲霉(Aspergillus niger)-罗耳阿太菌(Athelia rolfsii)
<220>
<221>MISC_FEATURE
<222>(1)..(33)
<223>融合连接点在位置8
<400>32
Ser Ser Val Pro Gly Thr Cys Ser Thr Gly Ala Thr Ser Pro Gly Gly
1 5 10 15
Ser Ser Gly Ser Val Glu Val Thr Phe Asp Val Tyr Ala Thr Thr Val
20 25 30
Tyr
<210>33
<211>33
<212>PRT
<213>黑曲霉(Aspergillus niger)-罗耳阿太菌(Athelia rolfsii)
<220>
<221>MISC_FEATURE
<222>(1)..(33)
<223>融合连接点在位置16
<400>33
Ser Ser Val Pro Gly Thr Cys Ala Ala Thr Ser Ala Ile Gly Thr Gly
1 5 10 15
Ser Ser Gly Ser Val Glu Val Thr Phe Asp Val Tyr Ala Thr Thr Val
20 25 30
Tyr
<210>34
<211>33
<212>PRT
<213>黑曲霉(Aspergillus niger)-罗耳阿太菌(Athelia rolfsii)
<220>
<221>MISC_FEATURE
<222>(1)..(33)
<223>融合连接点在位置25
<400>34
Ser Ser Val Pro Gly Thr Cys Ala Ala Thr Ser Ala Ile Gly Thr Tyr
1 5 10 15
Ser Ser Val Thr Val Thr Ser Trp Phe Asp Val Tyr Ala Thr Thr Val
20 25 30
Tyr
<210>35
<211>33
<212>PRT
<213>罗耳阿太菌(Athelia rolfsii)-埃默森篮状菌(Talaromyces emersonii)
<220>
<221>MISC_FEATURE
<222>(1)..(33)
<223>融合连接点在位置8
<400>35
Gly Val Ser Thr Ser Cys Ser Ala Thr Ser Ala Thr Gly Pro Tyr Ser
1 5 10 15
Thr Ala Thr Asn Thr Val Trp Pro Ser Ser Gly Ser Gly Ser Ser Thr
20 25 30
Thr
<210>36
<211>33
<212>PRT
<213>罗耳阿太菌(Athelia rolfsii)-埃默森篮状菌(Talaromyces emersonii)
<220>
<221>MISC_FEATURE
<222>(1)..(33)
<223>融合连接点在位置15
<400>36
Gly Val Ser Thr Ser Cys Ser Thr Gly Ala Thr Ser Pro Gly Tyr Ser
1 5 10 15
Thr Ala Thr Asn Thr Val Trp Pro Ser Ser Gly Ser Gly Ser Ser Thr
20 25 30
Thr
<210>37
<211>33
<212>PRT
<213>罗耳阿太菌(Athelia rolfsii)-埃默森篮状菌(Talaromyces emersonii)
<220>
<221>MISC_FEATURE
<222>(1)..(33)
<223>融合连接点在位置24
<400>37
Gly Val Ser Thr Ser Cys Ser Thr Gly Ala Thr Ser Pro Gly Gly Ser
1 5 10 15
Ser Gly Ser Val Glu Val Thr Pro Ser Ser Gly Ser Gly Ser Ser Thr
20 25 30
Thr
<210>38
<211>33
<212>PRT
<213>罗耳阿太菌(Athelia rolfsii)-黑曲霉(Aspergillus niger)
<220>
<221>MISC_FEATURE
<222>(1)..(33)
<223>融合连接点在位置7
<400>38
Gly Val Ser Thr Ser Cys Ala Ala Thr Ser Ala Ile Gly Thr Tyr Ser
1 5 10 15
Ser Val Thr Val Thr Ser Trp Pro Ser Ile Val Ala Thr Gly Gly Thr
20 25 30
Thr
<210>39
<211>33
<212>PRT
<213>罗耳阿太菌(Athelia rolfsii)-黑曲霉(Aspergillus niger)
<220>
<221>MISC_FEATURE
<222>(1)..(33)
<223>融合连接点在位置15
<400>39
Gly Val Ser Thr Ser Cys Ser Thr Gly Ala Thr Ser Pro Gly Tyr Ser
1 5 10 15
Ser Val Thr Val Thr Ser Trp Pro Ser Ile Val Ala Thr Gly Gly Thr
20 25 30
Thr
<210>40
<211>33
<212>PRT
<213>罗耳阿太菌(Athelia rolfsii)-黑曲霉(Aspergillus niger)
<220>
<221>MISC_FEATURE
<222>(1)..(33)
<223>融合连接点在位置24
<400>40
Gly Val Ser Thr Ser Cys Ser Thr Gly Ala Thr Ser Pro Gly Gly Ser
1 5 10 15
Ser Gly Ser Val Glu Val Thr Pro Ser Ile Val Ala Thr Gly Gly Thr
20 25 30
Thr
<210>41
<211>33
<212>PRT
<213>埃默森篮状菌(Talaromyces emersonii)-黑曲霉(Aspergillus niger)
<220>
<221>MISC_FEATURE
<222>(1)..(33)
<223>融合连接点在位置7
<400>41
Ser Val Pro Ala Val Cys Ala Ala Thr Ser Ala Ile Gly Thr Tyr Ser
1 5 10 15
Ser Val Thr Val Thr Ser Trp Pro Ser Ile Val Ala Thr Gly Gly Thr
20 25 30
Thr
<210>42
<211>33
<212>PRT
<213>埃默森篮状菌(Talaromyces emersonii)-黑曲霉(Aspergillus niger)
<220>
<221>MISC_FEATURE
<222>(1)..(33)
<223>融合连接点在位置16
<400>42
Ser Val Pro Ala Val Cys Ser Ala Thr Ser Ala Thr Gly Pro Tyr Ser
1 5 10 15
Ser Val Thr Val Thr Ser Trp Pro Ser Ile Val Ala Thr Gly Gly Thr
20 25 30
Thr
<210>43
<211>33
<212>PRT
<213>埃默森篮状菌(Talaromyces emersonii)-黑曲霉(Aspergillus niger)
<220>
<221>MISC_FEATURE
<222>(1)..(33)
<223>fusion junction at 24
<400>43
Ser Val Pro Ala Val Cys Ser Ala Thr Ser Ala Thr Gly Pro Tyr Ser
1 5 10 15
Thr Ala Thr Asn Thr Val Trp Pro Ser Ile Val Ala Thr Gly Gly Thr
20 25 30
Thr
<210>44
<211>33
<212>PRT
<213>埃默森篮状菌(Talaromyces emersonii)-罗耳阿太菌(Athelia rolfsii)
<220>
<221>MISC_FEATURE
<222>(1)..(33)
<223>融合连接点在位置9
<400>44
Ser Ser Val Pro Ala Val Cys Ser Thr Gly Ala Thr Ser Pro Gly Gly
1 5 10 15
Ser Ser Gly Ser Val Glu Val Thr Phe Asp Val Tyr Ala Thr Thr Val
20 25 30
Tyr
<210>45
<211>33
<212>PRT
<213>埃默森篮状菌(Talaromyces emersonii)-罗耳阿太菌(Athelia rolfsii)
<220>
<221>MISC_FEATURE
<222>(1)..(33)
<223>融合连接点在位置18
<400>45
Ser Ser Val Pro Ala Val Cys Ser Ala Thr Ser Ala Thr Gly Pro Tyr
1 5 10 15
Ser Ser Gly Ser Val Glu Val Thr Phe Asp Val Tyr Ala Thr Thr Val
20 25 30
Tyr
<210>46
<211>33
<212>PRT
<213>埃默森篮状菌(Talaromyces emersonii)-罗耳阿太菌(Athelia rolfsii)
<220>
<221>MISC_FEATURE
<222>(1)..(33)
<223>融合连接点在位置25
<400>46
Ser Ser Val Pro Ala Val Cys Ser Ala Thr Ser Ala Thr Gly Pro Tyr
1 5 10 15
Ser Thr Ala Thr Asn Thr Val Trp Phe Asp Val Tyr Ala Thr Thr Val
20 25 30
Tyr
<210>47
<211>38
<212>DNA
<213>人工的
<220>
<223>引物
<220>
<221>misc_feature
<222>(1)..(38)
<223>引物plac102fwd
<400>47
tcgtaagctt caccatgtcg ttccgatctc tactcgcc 38
<210>48
<211>24
<212>DNA
<213>人工的
<220>
<223>引物
<220>
<221>misc_feature
<222>(1)..(24)
<223>plac102 rev引物
<400>48
acaggtgccg ggcacgctgc tggc 24
<210>49
<211>24
<212>DNA
<213>人工的
<220>
<223>引物
<220>
<221>misc_feature
<222>(1)..(24)
<223>pLac102 rev2引物
<400>49
ggtaccaatg gcagatgtgg ccgc 24
<210>50
<211>25
<212>DNA
<213>人工的
<220>
<223>引物
<220>
<221>misc_feature
<222>(1)..(25)
<223>pLac102 rev3引物
<400>50
ccacgaggtg acagtcacac tgctg 25
<210>51
<211>34
<212>DNA
<213>人工的
<220>
<223>引物
<220>
<221>misc_feature
<222>(1)..(34)
<223>pSteD226 fwd1引物
<400>51
tgcaaagctt caccatggcg tccctcgttg ctgg 34
<210>52
<211>24
<212>DNA
<213>人工的
<220>
<223>引物
<220>
<221>misc_feature
<222>(1)..(24)
<223>pSteD226 rev1引物
<400>52
gcagacggca gggacgctgc ttgc 24
<210>53
<211>25
<212>DNA
<213>人工的
<220>
<223>引物
<220>
<221>misc_feature
<222>(1)..(25)
<223>pSteD rev2引物
<400>53
tgggcccgtg gcagaggtgg cagag 25
<210>54
<211>24
<212>DNA
<213>人工的
<220>
<223>引物
<220>
<221>misc_feature
<222>(1)..(24)
<223>pSteD226 rev3引物
<400>54
ccagacggtg ttggtagccg tgct 24
<210>55
<211>40
<212>DNA
<213>人工的
<220>
<223>引物
<220>
<221>misc_feature
<222>(1)..(40)
<223>AR cDNA fwd1引物
<400>55
aagaaagctt caccatgttt cgttcactcc tggccttggc 40
<210>56
<211>25
<212>DNA
<213>人工的
<220>
<223>引物
<220>
<221>misc_feature
<222>(1)..(25)
<223>AR cDNA rev1引物
<400>56
gcaggaggta gagactccct tagca 25
<210>57
<211>25
<212>DNA
<213>人工的
<220>
<223>引物
<220>
<221>misc_feature
<222>(1)..(25)
<223>AR cDNA rev2引物
<400>57
acccgggctt gtagcaccag tcgag 25
<210>58
<211>25
<212>DNA
<213>人工的
<220>
<223>引物
<220>
<221>misc_feature
<222>(1)..(25)
<223>AR cDNA rev3引物
<400>58
agtgacctcg acactacccg aggag 25
<210>59
<211>48
<212>DNA
<213>人工的
<220>
<223>引物
<220>
<221>misc_feature
<222>(1)..(48)
<223>pLac fwd2引物
<400>59
gcaagcagcg tccctgccgt ctgcgcggcc acatctgcca ttggtacc 48
<210>60
<211>34
<212>DNA
<213>人工的
<220>
<223>引物
<220>
<221>misc_feature
<222>(1)..(34)
<223>pLac102 rev4引物
<400>60
tagtatctag atcaccgcca ggtgtcagtc accg 34
<210>61
<211>49
<212>DNA
<213>人工的
<220>
<223>引物
<220>
<221>misc_feature
<222>(1)..(49)
<223>pLac102 fwd3引物
<400>61
ctctgccacc tctgccacgg gcccatacag cagtgtgact gtcacctcg 49
<210>62
<211>48
<212>DNA
<213>人工的
<220>
<223>引物
<220>
<221>misc_feature
<222>(1)..(48)
<223>PLac102 fwd4引物
<400>62
agcacggcta ccaacaccgt ctggccgagt atcgtggcta ctggcggc 48
<210>63
<211>49
<212>DNA
<213>人工的
<220>
<223>引物
<220>
<221>misc_fea ture
<222>(1)..(49)
<223>pLac102 fwd5引物
<400>63
tgctaaggga gtctctacct cctgcgcggc cacatctgcc attggtacc 49
<210>64
<211>49
<212>DNA
<213>人工的
<220>
<223>引物
<220>
<221>misc_feature
<222>(1)..(49)
<223>pLac102 fwd6引物
<400>64
ctcgactggt gctacaagcc cgggttacag cagtgtgact gtcacctcg 49
<210>65
<211>49
<212>DNA
<213>人工的
<220>
<223>引物
<220>
<221>misc_feature
<222>(1)..(49)
<223>pLac102 fwd7引物
<400>65
ctcgactggt gctacaagcc cgggttacag cagtgtgact gtcacctcg 49
<210>66
<211>50
<212>DNA
<213>人工的
<220>
<223>引物
<220>
<221>misc_feature
<222>(1)..(50)
<223>pSteD fwd2引物
<400>66
tgctaaggga gtctctacct cctgctctgc cacctctgcc acgggcccat 50
<210>67
<211>40
<212>DNA
<213>人工的
<220>
<223>引物
<220>
<221>misc_feature
<222>(1)..(40)
<223>pSteD rev4引物
<400>67
tacctctaga atcgtcactg ccaactatcg tcaagaatgg 40
<210>68
<211>49
<212>DNA
<213>人工的
<220>
<223>引物
<220>
<221>misc_feature
<222>(1)..(49)
<223>pSteD226 fwd3引物
<400>68
ctcgactggt gctacaagcc cgggttacag cacggctacc aacaccgtc 49
<210>69
<211>49
<212>DNA
<213>人工的
<220>
<223>引物
<220>
<221>misc_feature
<222>(1)..(49)
<223>pSteD226 fwd4引物
<400>69
ctcctcgggt agtgtcgagg tcactccaag ctctggctct ggcagctca 49
<210>70
<211>45
<212>DNA
<213>人工的
<220>
<223>引物
<220>
<221>misc_feature
<222>(1)..(45)
<223>pSteD226 fwd5引物
<400>70
gccagcagcg tgcccggcac ctgttctgcc acctctgcca cgggc 45
<210>71
<211>48
<212>DNA
<213>人工的
<220>
<223>引物
<220>
<221>misc_feature
<222>(1)..(48)
<223>pSted226 fwd6引物
<400>71
gcggccacat ctgccattgg tacctacagc acggctacca acaccgtc 48
<210>72
<211>48
<212>DNA
<213>人工的
<220>
<223>引物
<220>
<221>misc_feature
<222>(1)..(48)
<223>pSteD226 fwd7引物
<400>72
cagcagtgtg actgtcacct cgtggccaag ctctggctct ggcagctc 48
<210>73
<211>49
<212>DNA
<213>人工的
<220>
<223>引物
<220>
<221>misc_feature
<222>(1)..(49)
<223>AR cDNA fwd2引物
<400>73
gcaagcagcg tccctgccgt ctgctcgact ggtgctacaa gcccgggtg 49
<210>74
<211>48
<212>DNA
<213>人工的
<220>
<223>引物
<220>
<221>misc_feature
<222>(1)..(48)
<223>AR cDNA rev4引物
<400>74
cggccctcta gaatcgtcat taagattcat cccaagtgtc tttttcgg 48
<210>75
<211>49
<212>DNA
<213>人工的
<220>
<223>引物
<220>
<221>misc_feature
<222>(1)..(49)
<223>AR cDNA fwd3引物
<400>75
ctctgccacc tctgccacgg gcccaggctc ctcgggtagt gtcgaggtc 49
<210>76
<211>49
<212>DNA
<213>人工的
<220>
<223>引物
<220>
<221>misc_feature
<222>(1)..(49)
<223>AR cDNA fwd4引物
<400>76
agcacggcta ccaacaccgt ctggttcgac gtttacgcta ccacagtat 49
<210>77
<211>49
<212>DNA
<213>人工的
<220>
<223>引物
<220>
<221>misc_feature
<222>(1)..(49)
<223>AR cDNA fwd5引物
<400>77
gccagcagcg tgcccggcac ctgttcgact ggtgctacaa gcccgggtg 49
<210>78
<211>48
<212>DNA
<213>人工的
<220>
<223>引物
<220>
<221>misc_feature
<222>(1)..(48)
<223>AR cDNA fwd6引物
<400>78
gcggccacat ctgccat tgg taccggctcc tcgggtagtg tcgaggtc 48
<210>79
<211>51
<212>DNA
<213>人工的
<220>
<223>引物
<220>
<221>misc_feature
<222>(1)..(51)
<223>AR cDNA fwd7引物
<400>79
cagcagtgtg actgtcacct cgtggttcga cgtttacgct accacagtat a 51
<210>80
<211>1877
<212>DNA
<213>埃默森篮状菌(Talaromyces emersonii)
<400>80
aagcttcacc atggcgtccc tcgttgctgg cgctctctgc atcctgggcc tgacgcctgc 60
tgcatttgca cgagcgcccg ttgcagcgcg agccaccggt tccctggact cctttctcgc 120
aaccgaaact ccaattgccc tccaaggcgt gctgaacaac atcgggccca atggtgctga 180
tgtggcagga gcaagcgccg gcattgtggt tgccagtccg agcaggagcg acccaaatta 240
tttctactcc tggacacgtg acgcagcgct cacggccaaa tacctcgtcg acgccttcat 300
cgcgggcaac aaggacctag agcagaccat ccagcagtac atcagcgcgc aggcgaaggt 360
gcaaactatc tccaatccgt ccggagattt atccaccggt ggcttaggtg agcccaagtt 420
caatgtgaat gagacggctt ttaccgggcc ctggggtcgt ccacagaggg acggaccagc 480
gttgagagcg acggccctca ttgcgtatgc gaactatctc atcgacaacg gcgaggcttc 540
gactgccgat gagatcatct ggccgattgt ccagaatgat ctgtcctaca tcacccaata 600
ctggaactca tccaccttcg acctctggga agaagtagaa ggatcctcat tcttcacaac 660
cgccgtgcaa caccgcgccc tggtcgaagg caatgcactg gcaacaaggc tgaaccacac 720
gtgctccaac tgcgtctctc aggcccctca ggtcctgtgt ttcctgcagt catactggac 780
cggatcgtat gttctggcca actttggtgg cagcggtcgt tccggcaagg acgtgaattc 840
gattttgggc agcatccaca cctttgatcc cgccggaggc tgtgacgact cgaccttcca 900
gccgtgttcg gcccgtgcct tggcaaatca caaggtggtc accgactcgt tccggagtat 960
ctatgcgatc aactcaggca tcgcagaggg atctgccgtg gcagtcggcc gctaccctga 1020
ggatgtctac cagggcggga acccctggta cctggccaca gcagcggctg cagagcagct 1080
ttacgacgcc atctaccagt ggaagaagat cggctcgata agtatcacgg acgttagtct 1140
gccatttttc caggatatct acccttctgc cgcggtgggc acctataact ctggctccac 1200
gactttcaac gacatcatct cggccgtcca gacgtatggt gatggatatc tgagtattgt 1260
cgagaaatat actccctcag acggctctct taccgaacaa ttctcccgta cagacggcac 1320
tccgctttct gcctctgccc tgacttggtc gtacgcttct ctcctaaccg cttcggcccg 1380
cagacagtcc gtcgtccctg cttcctgggg cgaaagctcc gcaagcagcg tccctgccgt 1440
ctgctctgcc acctctgcca cgggcccata cagcacggct accaacaccg tctggccaag 1500
ctctggctct ggcagctcaa caaccaccag tagcgcccca tgcaccactc ctacctctgt 1560
ggctgtgacc ttcgacgaaa tcgtcagcac cagttacggg gagacaatct acctggccgg 1620
ctcgatcccc gagctgggca actggtccac ggccagcgcg atccccctcc gcgcggatgc 1680
ttacaccaac agcaacccgc tctggtacgt gaccgtcaat ctgccccctg gcaccagctt 1740
cgagtacaag ttcttcaaga accagacgga cgggaccatc gtctgggagg acgacccgaa 1800
ccggtcgtac acggtcccag cgtactgtgg gcagactacc gccattcttg acgatagttg 1860
gcagtgacga ttctaga 1877
<210>81
<211>1920
<212>DNA
<213>黑曲霉(Aspergillus niger)
<400>81
atgtcgttcc gatctctact cgccctgagc ggcctcgtct gcacagggtt ggcaaatgtg 60
atttccaagc gcgcgacctt ggattcatgg ttgagcaacg aagcgaccgt ggctcgtact 120
gccatcctga ataacatcgg ggcggacggt gcttgggtgt cgggcgcgga ctctggcatt 180
gtcgttgcta gtcccagcac ggataacccg gactacttct acacctggac tcgcgactct 240
ggtctcgtcc tcaagaccct cgtcgatctc ttccgaaatg gagataccag tctcctctcc 300
accattgaga actacatctc cgcccaggca attgtccagg gtatcagtaa cccctctggt 360
gatctgtcca gcggcgctgg tctcggtgaa cccaagttca atgtcgatga gactgcctac 420
actggttctt ggggacggcc gcagcgagat ggtccggctc tgagagcaac tgctatgatc 480
ggcttcgggc agtggctgct tgacaatggc tacaccagca ccgcaacgga cattgtttgg 540
cccctcgtta ggaacgacct gtcgtatgtg gctcaatact ggaaccagac aggatatgat 600
ctctgggaag aagtcaatgg ctcgtctttc tttacgattg ctgtgcaaca ccgcgccctt 660
gtcgaaggta gtgccttcgc gacggccgtc ggctcgtcct gctcctggtg tgattctcag 720
gcacccgaaa ttctctgcta cctgcagtcc ttctggaccg gcagcttcat tctggccaac 780
ttcgatagca gccgttccgg caaggacgca aacaccctcc tgggaagcat ccacaccttt 840
gatcctgagg ccgcatgcga cgactccacc ttccagccct gctccccgcg cgcgctcgcc 900
aaccacaagg aggttgtaga ctctttccgc tcaatctata ccctcaacga tggtctcagt 960
gacagcgagg ctgttgcggt gggtcggtac cctgaggaca cgtactacaa cggcaacccg 1020
tggttcctgt gcaccttggc tgccgcagag cagttgtacg atgctctata ccagtgggac 1080
aagcaggggt cgttggaggt cacagatgtg tcgctggact tcttcaaggc actgtacagc 1140
gatgctgcta ctggcaccta ctcttcgtcc agttcgactt atagtagcat tgtagatgcc 1200
gtgaagactt tcgccgatgg cttcgtctct attgtggaaa ctcacgccgc aagcaacggc 1260
tccatgtccg agcaatacga caagtctgat ggcgagcagc tttccgctcg cgacctgacc 1320
tggtcttatg ctgctctgct gaccgccaac aaccgtcgta actccgtcgt gcctgcttct 1380
tggggcgaga cctctgccag cagcgtgccc ggcacctgtg cggccacatc tgccattggt 1440
acctacagca gtgtgactgt cacctcgtgg ccgagtatcg tggctactgg cggcaccact 1500
acgacggcta cccccactgg atccggcagc gtgacctcga ccagcaagac caccgcgact 1560
gctagcaaga ccagcaccag tacgtcatca acctcctgta ccactcccac cgccgtggct 1620
gtgactttcg atctgacagc taccaccacc tacggcgaga acatctacct ggtcggatcg 1680
atctctcagc tgggtgactg ggaaaccagc gacggcatag ctctgagtgc tgacaagtac 1740
acttccagcg acccgctctg gtatgtcact gtgactctgc cggctggtga gtcgtttgag 1800
tacaagttta tccgcattga gagcgatgac tccgtggagt gggagagtga tcccaaccga 1860
gaatacaccg ttcctcaggc gtgcggaacg tcgaccgcga cggtgactga cacctggcgg 1920
<210>82
<211>1954
<212>DNA
<213>黑曲霉(Aspergillus niger)-Talaromyces hybrid DNA
<400>82
aagcttcacc atggcgtccc tcgttgctgg cgctctctgc atcctgggcc tgacgcctgc 60
tgcatttgca cgagcgcccg ttgcagcgcg agccaccggt tccctggact cctttctcgc 120
aaccgaaact ccaattgccc tccaaggcgt gctgaacaac atcgggccca atggtgctga 180
tgtggcagga gcaagcgccg gcattgtggt tgccagtccg agcaggagcg acccaaatta 240
tttctactcc tggacacgtg acgcagcgct cacggccaaa tacctcgtcg acgccttcat 300
cgcgggcaac aaggacctag agcagaccat ccagcagtac atcagcgcgc aggcgaaggt 360
gcaaactatc tccaatccgt ccggagattt atccaccggt ggcttaggtg agcccaagtt 420
caatgtgaat gagacggctt ttaccgggcc ctggggtcgt ccacagaggg acggaccagc 480
gttgagagcg acggccctca ttgcgtatgc gaactatctc atcgacaacg gcgaggcttc 540
gactgccgat gagatcatct ggccgattgt ccagaatgat ctgtcctaca tcacccaata 600
ctggaactca tccaccttcg acctctggga agaagtagaa ggatcctcat tcttcacaac 660
cgccgtgcaa caccgcgccc tggtcgaagg caatgcactg gcaacaaggc tgaaccacac 720
gtgctccaac tgcgtctctc aggcccctca ggtcctgtgt ttcctgcagt catactggac 780
cggatcgtat gttctggcca actttggtgg cagcggtcgt tccggcaagg acgtgaattc 840
gattttgggc agcatccaca cctttgatcc cgccggaggc tgtgacgact cgaccttcca 900
gccgtgttcg gcccgtgcct tggcaaatca caaggtggtc accgactcgt tccggagtat 960
ctatgcgatc aactcaggca tcgcagaggg atctgccgtg gcagtcggcc gctaccctga 1020
ggatgtctac cagggcggga acccctggta cctggccaca gcagcggctg cagagcagct 1080
ttacgacgcc atctaccagt ggaagaagat cggctcgata agtatcacgg acgttagtct 1140
gccatttttc caggatatct acccttctgc cgcggtgggc acctataact ctggctccac 1200
gactttcaac gacatcatct cggccgtcca gacgtatggt gatggatatc tgagtattgt 1260
cgagaaatat actccctcag acggctctct taccgaacaa ttctcccgta cagacggcac 1320
tccgctttct gcctctgccc tgacttggtc gtacgcttct ctcctaaccg cttcggcccg 1380
cagacagtcc gtcgtccctg cttcctgggg cgaaagctcc gcaagcagcg tccctgccgt 1440
ctgcgcggcc acatctgcca ttggtaccta cagcagtgtg actgtcacct cgtggccgag 1500
tatcgtggct actggcggca ccactacgac ggctaccccc actggatccg gcagcgtgac 1560
ctcgaccagc aagaccaccg cgactgctag caagaccagc accagtacgt catcaacctc 1620
ctgtaccact cccaccgccg tggctgtgac tttcgatctg acagctacca ccacctacgg 1680
cgagaacatc tacctggtcg gatcgatctc tcagctgggt gactgggaaa ccagcgacgg 1740
catagctctg agtgctgaca agtacacttc cagcgacccg ctctggtatg tcactgtgac 1800
tctgccggct ggtgagtcgt ttgagtacaa gtttatccgc attgagagcg atgactccgt 1860
ggagtgggag agtgatccca accgagaata caccgttcct caggcgtgcg gaacgtcgac 1920
cgcgacggtg actgacacct ggcggtgatc taga 1954
<210>83
<211>1954
<212>DNA
<213>黑曲霉(Aspergillus niger)-埃默森篮状菌(Talaromyces emersonii)
<400>83
aagcttcacc atggcgtccc tcgttgctgg cgctctctgc atcctgggcc tgacgcctgc 60
tgcatttgca cgagcgcccg ttgcagcgcg agccaccggt tccctggact cctttctcgc 120
aaccgaaact ccaattgccc tccaaggcgt gctgaacaac atcgggccca atggtgctga 180
tgtggcagga gcaagcgccg gcattgtggt tgccagtccg agcaggagcg acccaaatta 240
tttctactcc tggacacgtg acgcagcgct cacggccaaa tacctcgtcg acgccttcat 300
cgcgggcaac aaggacctag agcagaccat ccagcagtac atcagcgcgc aggcgaaggt 360
gcaaactatc tccaatccgt ccggagattt atccaccggt ggcttaggtg agcccaagtt 420
caatgtgaat gagacggctt ttaccgggcc ctggggtcgt ccacagaggg acggaccagc 480
gttgagagcg acggccctca ttgcgtatgc gaactatctc atcgacaacg gcgaggcttc 540
gactgccgat gagatcatct ggccgattgt ccagaatgat ctgtcctaca tcacccaata 600
ctggaactca tccaccttcg acctctggga agaagtagaa ggatcctcat tcttcacaac 660
cgccgtgcaa caccgcgccc tggtcgaagg caatgcactg gcaacaaggc tgaaccacac 720
gtgctccaac tgcgtctctc aggcccctca ggtcctgtgt ttcctgcagt catactggac 780
cggatcgtat gttctggcca actttggtgg cagcggtcgt tccggcaagg acgtgaattc 840
gattttgggc agcatccaca cctttgatcc cgccggaggc tgtgacgact cgaccttcca 900
gccgtgttcg gcccgtgcct tggcaaatca caaggtggtc accgactcgt tccggagtat 960
ctatgcgatc aactcaggca tcgcagaggg atctgccgtg gcagtcggcc gctaccctga 1020
ggatgtctac cagggcggga acccctggta cctggccaca gcagcggctg cagagcagct 1080
ttacgacgcc atctaccagt ggaagaagat cggctcgata agtatcacgg acgttagtct 1140
gccatttttc caggatatct acccttctgc cgcggtgggc acctataact ctggctccac 1200
gactttcaac gacatcatct cggccgtcca gacgtatggt gatggatatc tgagtattgt 1260
cgagaaatat actccctcag acggctctct taccgaacaa ttctcccgta cagacggcac 1320
tccgctttct gcctctgccc tgacttggtc gtacgcttct ctcctaaccg cttcggcccg 1380
cagacagtcc gtcgtccctg cttcctgggg cgaaagctcc gcaagcagcg tccctgccgt 1440
ctgctctgcc acctctgcca cgggcccata cagcagtgtg actgtcacct cgtggccgag 1500
tatcgtggct actggcggca ccactacgac ggctaccccc actggatccg gcagcgtgac 1560
ctcgaccagc aagaccaccg cgactgctag caagaccagc accagtacgt catcaacctc 1620
ctgtaccact cccaccgccg tggctgtgac tttcgatctg acagctacca ccacctacgg 1680
cgagaacatc tacctggtcg gatcgatctc tcagctgggt gactgggaaa ccagcgacgg 1740
catagctctg agtgctgaca agtacacttc cagcgacccg ctctggtatg tcactgtgac 1800
tctgccggct ggtgagtcgt ttgagtacaa gtttatccgc attgagagcg atgactccgt 1860
ggagtgggag agtgatccca accgagaata caccgttcct caggcgtgcg gaacgtcgac 1920
cgcgacggtg actgacacct ggcggtgatc taga 1954
<210>84
<211>1954
<212>DNA
<213>黑曲霉(Aspergillus niger)-埃默森篮状菌(Talaromyces emersonii)
<400>84
aagcttcacc atggcgtccc tcgttgctgg cgctctctgc atcctgggcc tgacgcctgc 60
tgcatttgca cgagcgcccg ttgcagcgcg agccaccggt tccctggact cctttctcgc 120
aaccgaaact ccaattgccc tccaaggcgt gctgaacaac atcgggccca atggtgctga 180
tgtggcagga gcaagcgccg gcattgtggt tgccagtccg agcaggagcg acccaaatta 240
tttctactcc tggacacgtg acgcagcgct cacggccaaa tacctcgtcg acgccttcat 300
cgcgggcaac aaggacctag agcagaccat ccagcagtac atcagcgcgc aggcgaaggt 360
gcaaactatc tccaatccgt ccggagattt atccaccggt ggcttaggtg agcccaagtt 420
caatgtgaat gagacggctt ttaccgggcc ctggggtcgt ccacagaggg acggaccagc 480
gttgagagcg acggccctca ttgcgtatgc gaactatctc atcgacaacg gcgaggcttc 540
gactgccgat gagatcatct ggccgattgt ccagaatgat ctgtcctaca tcacccaata 600
ctggaactca tccaccttcg acctctggga agaagtagaa ggatcctcat tcttcacaac 660
cgccgtgcaa caccgcgccc tggtcgaagg caatgcactg gcaacaaggc tgaaccacac 720
gtgctccaac tgcgtctctc aggcccctca ggtcctgtgt ttcctgcagt catactggac 780
cggatcgtat gttctggcca actttggtgg cagcggtcgt tccggcaagg acgtgaattc 840
gattttgggc agcatccaca cctttgatcc cgccggaggc tgtgacgact cgaccttcca 900
gccgtgttcg gcccgtgcct tggcaaatca caaggtggtc accgactcgt tccggagtat 960
ctatgcgatc aactcaggca tcgcagaggg atctgccgtg gcagtcggcc gctaccctga 1020
ggatgtctac cagggcggga acccctggta cctggccaca gcagcggctg cagagcagct 1080
ttacgacgcc atctaccagt ggaagaagat cggctcgata agtatcacgg acgttagtct 1140
gccatttttc caggatatct acccttctgc cgcggtgggc acctataact ctggctccac 1200
gactttcaac gacatcatct cggccgtcca gacgtatggt gatggatatc tgagtattgt 1260
cgagaaatat actccctcag acggctctct taccgaacaa ttctcccgta cagacggcac 1320
tccgctttct gcctctgccc tgacttggtc gtacgcttct ctcctaaccg cttcggcccg 1380
cagacagtcc gtcgtccctg cttcctgggg cgaaagctcc gcaagcagcg tccctgccgt 1440
ctgctctgcc acctctgcca cgggcccata cagcacggct accaacaccg tctggccgag 1500
tatcgtggct actggcggca ccactacgac ggctaccccc actggatccg gcagcgtgac 1560
ctcgaccagc aagaccaccg cgactgctag caagaccagc accagtacgt catcaacctc 1620
ctgtaccact cccaccgccg tggctgtgac tttcgatctg acagctacca ccacctacgg 1680
cgagaacatc tacctggtcg gatcgatctc tcagctgggt gactgggaaa ccagcgacgg 1740
catagctctg agtgctgaca agtacacttc cagcgacccg ctctggtatg tcactgtgac 1800
tctgccggct ggtgagtcgt ttgagtacaa gtttatccgc attgagagcg atgactccgt 1860
ggagtgggag agtgatccca accgagaata caccgttcct caggcgtgcg gaacgtcgac 1920
cgcgacggtg actgacacct ggcggtgatc taga 1954
<210>85
<211>1927
<212>DNA
<213>黑曲霉(Aspergillus niger)-罗耳阿太菌(Athelia rolfsii)
<400>85
aagcttcacc atgtttcgtt cactcctggc cttggctgcg tgtgcagtcg cctctgtatc 60
tgcacagtct gcgtctgcga cagcatatct taccaaggaa tctgcagttg ccaagaatgg 120
cgtactttgc aacattggta gccagggatg catgtctgag ggtgcctata gcggtattgt 180
gatcgcatct ccctctaaaa ctagccctga ctatctctat acctggactc gcgactcgtc 240
gctcgtcttc aagatgttaa ttgaccaata cacaaatggc ctggatacga cactgcgcac 300
tctcattgac gagtttgtct ctgcggaagc caccattcaa caaaccagta actcatctgg 360
taccgtctct accggtggtc tcggcgaacc caaattcaat atcgacgaga cggcatttac 420
gggcgcatgg ggtcgtcccc aacgtgatgg tcccgccctc cgtgcaaccg caatcatgac 480
ctatgcgacg tatctgtaca acaatggcaa cacttcctac gtgaccaaca ccctttggcc 540
tatcatcaag ctcgaccttg actatgtcaa ctcggactgg aaccagacca cgtttgacct 600
ctgggaagaa gttgactcgt cttctttctt tacgactgcc gttcagcacc gtgctcttgt 660
tcagggcgca gcctttgcta ccctcatcgg ccaaacttcg tctgcttcga cttactccgc 720
cacggcccct agcattctct gcttcttgca gtcttactgg aacaccaacg gatactggac 780
ggccaacact ggtggcggac gttccggcaa ggacgccaac accatactcg cttctatcca 840
cacatttgac gccagcgccg gctgctctgc tgccacgtct caaccatgct ctgacgtagc 900
attggccaac ctgaaggtat acgttgactc tttccgtagt atttatacga tcaacagcgg 960
tatttcctct acctcgggtg ttgctactgg tcgctacccc gaagattcgt attacaatgg 1020
caacccctgg tacctctgca cactcgccgt cgccgagcag ctctatgatg ctctcatcgt 1080
atggaaggct gccggggagc tcaacgtcac ctccgtctcg ctcgcgttct tccagcaatt 1140
cgactcgagc atcaccgccg gcacttacgc ctcctcgtcg agcgtataca cttcgctcat 1200
ctctgacatc caggcgttcg cagacgagtt tgttgacatt gttgccaagt acacgccttc 1260
gtctggcttc ttgtctgagc agtatgataa gtccacgggt gctcaggatt cggctgctaa 1320
cttgacttgg tcctatgctg ctgctatcac cgcttaccaa gcccgcaatg gcttcacagg 1380
tgcttcgtgg ggtgctaagg gagtctctac ctcctgcgcg gccacatctg ccattggtac 1440
ctacagcagt gtgactgtca cctcgtggcc gagtatcgtg gctactggcg gcaccactac 1500
gacggctacc cccactggat ccggcagcgt gacctcgacc agcaagacca ccgcgactgc 1560
tagcaagacc agcaccagta cgtcatcaac ctcctgtacc actcccaccg ccgtggctgt 1620
gactttcgat ctgacagcta ccaccaccta cggcgagaac atctacctgg tcggatcgat 1680
ctctcagctg ggtgactggg aaaccagcga cggcatagct ctgagtgctg acaagtacac 1740
ttccagcgac ccgctctggt atgtcactgt gactctgccg gctggtgagt cgtttgagta 1800
caagtttatc cgcattgaga gcgatgactc cgtggagtgg gagagtgatc ccaaccgaga 1860
atacaccgtt cctcaggcgt gcggaacgtc gaccgcgacg gtgactgaca cctggcggtg 1920
atctaga 1927
<210>86
<211>1927
<212>DNA
<213>黑曲霉(Aspergillus niger)-罗耳阿太菌(Athelia rolfsii)
<400>86
aagcttcacc atgtttcgtt cactcctggc cttggctgcg tgtgcagtcg cctctgtatc 60
tgcacagtct gcgtctgcga cagcatatct taccaaggaa tctgcagttg ccaagaatgg 120
cgtactttgc aacattggta gccagggatg catgtctgag ggtgcctata gcggtattgt 180
gatcgcatct ccctctaaaa ctagccctga ctatctctat acctggactc gcgactcgtc 240
gctcgtcttc aagatgttaa ttgaccaata cacaaatggc ctggatacga cactgcgcac 300
tctcattgac gagtttgtct ctgcggaagc caccattcaa caaaccagta actcatctgg 360
taccgtctct accggtggtc tcggcgaacc caaattcaat atcgacgaga cggcatttac 420
gggcgcatgg ggtcgtcccc aacgtgatgg tcccgccctc cgtgcaaccg caatcatgac 480
ctatgcgacg tatctgtaca acaatggcaa cacttcctac gtgaccaaca ccctttggcc 540
tatcatcaag ctcgaccttg actatgtcaa ctcggactgg aaccagacca cgtttgacct 600
ctgggaagaa gttgactcgt cttctttctt tacgactgcc gttcagcacc gtgctcttgt 660
tcagggcgca gcctttgcta ccctcatcgg ccaaacttcg tctgcttcga cttactccgc 720
cacggcccct agcattctct gcttcttgca gtcttactgg aacaccaacg gatactggac 780
ggccaacact ggtggcggac gttccggcaa ggacgccaac accatactcg cttctatcca 840
cacatttgac gccagcgccg gctgctctgc tgccacgtct caaccatgct ctgacgtagc 900
attggccaac ctgaaggtat acgttgactc tttccgtagt atttatacga tcaacagcgg 960
tatttcctct acctcgggtg ttgctactgg tcgctacccc gaagattcgt attacaatgg 1020
caacccctgg tacctctgca cactcgccgt cgccgagcag ctctatgatg ctctcatcgt 1080
atggaaggct gccggggagc tcaacgtcac ctccgtctcg ctcgcgttct tccagcaatt 1140
cgactcgagc atcaccgccg gcacttacgc ctcctcgtcg agcgtataca cttcgctcat 1200
ctctgacatc caggcgttcg cagacgagtt tgttgacatt gttgccaagt acacgccttc 1260
gtctggcttc ttgtctgagc agtatgataa gtccacgggt gctcaggatt cggctgctaa 1320
cttgacttgg tcctatgctg ctgctatcac cgcttaccaa gcccgcaatg gcttcacagg 1380
tgcttcgtgg ggtgctaagg gagtttctac ctcctgctcg actggtgcta caagcccggg 1440
ttacagcagt gtgactgtca cctcgtggcc gagtatcgtg gctactggcg gcaccactac 1500
gacggctacc cccactggat ccggcagcgt gacctcgacc agcaagacca ccgcgactgc 1560
tagcaagacc agcaccagta cgtcatcaac ctcctgtacc actcccaccg ccgtggctgt 1620
gactttcgat ctgacagcta ccaccaccta cggcgagaac atctacctgg tcggatcgat 1680
ctctcagctg ggtgactggg aaaccagcga cggcatagct ctgagtgctg acaagtacac 1740
ttccagcgac ccgctctggt atgtcactgt gactctgccg gctggtgagt cgtttgagta 1800
caagtttatc cgcattgaga gcgatgactc cgtggagtgg gagagtgatc ccaaccgaga 1860
atacaccgtt cctcaggcgt gcggaacgtc gaccgcgacg gtgactgaca cctggcggtg 1920
atctaga 1927
<210>87
<211>1927
<212>DNA
<213>黑曲霉(Aspergillus niger)-罗耳阿太菌(Athelia rolfsii)
<400>87
aagcttcacc atgtttcgtt cactcctggc cttggctgcg tgtgcagtcg cctctgtatc 60
tgcacagtct gcgtctgcga cagcatatct taccaaggaa tctgcagttg ccaagaatgg 120
cgtactttgc aacattggta gccagggatg catgtctgag ggtgcctata gcggtattgt 180
gatcgcatct ccctctaaaa ctagccctga ctatctctat acctggactc gcgactcgtc 240
gctcgtcttc aagatgttaa ttgaccaata cacaaatggc ctggatacga cactgcgcac 300
tctcattgac gagtttgtct ctgcggaagc caccattcaa caaaccagta actcatctgg 360
taccgtctct accggtggtc tcggcgaacc caaattcaat atcgacgaga cggcatttac 420
gggcgcatgg ggtcgtcccc aacgtgatgg tcccgccctc cgtgcaaccg caatcatgac 480
ctatgcgacg tatctgtaca acaatggcaa cacttcctac gtgaccaaca ccctttggcc 540
tatcatcaag ctcgaccttg actatgtcaa ctcggactgg aaccagacca cgtttgacct 600
ctgggaagaa gttgactcgt cttctttctt tacgactgcc gttcagcacc gtgctcttgt 660
tcagggcgca gcctttgcta ccctcatcgg ccaaacttcg tctgcttcga cttactccgc 720
cacggcccct agcattctct gcttcttgca gtcttactgg aacaccaacg gatactggac 780
ggccaacact ggtggcggac gttccggcaa ggacgccaac accatactcg cttctatcca 840
cacatttgac gccagcgccg gctgctctgc tgccacgtct caaccatgct ctgacgtagc 900
attggccaac ctgaaggtat acgttgactc tttccgtagt atttatacga tcaacagcgg 960
tatttcctct acctcgggtg ttgctactgg tcgctacccc gaagattcgt attacaatgg 1020
caacccctgg tacctctgca cactcgccgt cgccgagcag ctctatgatg ctctcatcgt 1080
atggaaggct gccggggagc tcaacgtcac ctccgtctcg ctcgcgttct tccagcaatt 1140
cgactcgagc atcaccgccg gcacttacgc ctcctcgtcg agcgtataca cttcgctcat 1200
ctctgacatc caggcgttcg cagacgagtt tgttgacatt gttgccaagt acacgccttc 1260
gtctggcttc ttgtctgagc agtatgataa gtccacgggt gctcaggatt cggctgctaa 1320
cttgacttgg tcctatgctg ctgctatcac cgcttaccaa gcccgcaatg gcttcacagg 1380
tgcttcgtgg ggtgctaagg gagtttctac ctcctgctcg actggtgcta caagcccggg 1440
tggctcctcg ggtagtgtcg aggtcactcc gagtatcgtg gctactggcg gcaccactac 1500
gacggctacc cccactggat ccggcagcgt gacctcgacc agcaagacca ccgcgactgc 1560
tagcaagacc agcaccagta cgtcatcaac ctcctgtacc actcccaccg ccgtggctgt 1620
gactttcgat ctgacagcta ccaccaccta cggcgagaac atctacctgg tcggatcgat 1680
ctctcagctg ggtgactggg aaaccagcga cggcatagct ctgagtgctg acaagtacac 1740
ttccagcgac ccgctctggt atgtcactgt gactctgccg gctggtgagt cgtttgagta 1800
caagtttatc cgcattgaga gcgatgactc cgtggagtgg gagagtgatc ccaaccgaga 1860
atacaccgtt cctcaggcgt gcggaacgtc gaccgcgacg gtgactgaca cctggcggtg 1920
atctaga 1927
<210>88
<211>1850
<212>DNA
<213>罗耳阿太菌(Athelia rolfsii)-埃默森篮状菌(Talaromyces emersonii)
<400>88
aagcttcacc atgtttcgtt cactcctggc cttggctgcg tgtgcagtcg cctctgtatc 60
tgcacagtct gcgtctgcga cagcatatct taccaaggaa tctgcagttg ccaagaatgg 120
cgtactttgc aacattggta gccagggatg catgtctgag ggtgcctata gcggtattgt 180
gatcgcatct ccctctaaaa ctagccctga ctatctctat acctggactc gcgactcgtc 240
gctcgtcttc aagatgttaa ttgaccaata cacaaatggc ctggatacga cactgcgcac 300
tctcattgac gagtttgtct ctgcggaagc caccattcaa caaaccagta actcatctgg 360
taccgtctct accggtggtc tcggcgaacc caaattcaat atcgacgaga cggcatttac 420
gggcgcatgg ggtcgtcccc aacgtgatgg tcccgccctc cgtgcaaccg caatcatgac 480
ctatgcgacg tatctgtaca acaatggcaa cacttcctac gtgaccaaca ccctttggcc 540
tatcatcaag ctcgaccttg actatgtcaa ctcggactgg aaccagacca cgtttgacct 600
ctgggaagaa gttgactcgt cttctttctt tacgactgcc gttcagcacc gtgctcttgt 660
tcagggcgca gcctttgcta ccctcatcgg ccaaacttcg tctgcttcga cttactccgc 720
cacggcccct agcattctct gcttcttgca gtcttactgg aacaccaacg gatactggac 780
ggccaacact ggtggcggac gttccggcaa ggacgccaac accatactcg cttctatcca 840
cacatttgac gccagcgccg gctgctctgc tgccacgtct caaccatgct ctgacgtagc 900
attggccaac ctgaaggtat acgttgactc tttccgtagt atttatacga tcaacagcgg 960
tatttcctct acctcgggtg ttgctactgg tcgctacccc gaagattcgt attacaatgg 1020
caacccctgg tacctctgca cactcgccgt cgccgagcag ctctatgatg ctctcatcgt 1080
atggaaggct gccggggagc tcaacgtcac ctccgtctcg ctcgcgttct tccagcaatt 1140
cgactcgagc atcaccgccg gcacttacgc ctcctcgtcg agcgtataca cttcgctcat 1200
ctctgacatc caggcgttcg cagacgagtt tgttgacatt gttgccaagt acacgccttc 1260
gtctggcttc ttgtctgagc agtatgataa gtccacgggt gctcaggatt cggctgctaa 1320
cttgacttgg tcctatgctg ctgctatcac cgcttaccaa gcccgcaatg gcttcacagg 1380
tgcttcgtgg ggtgctaagg gagtctctac ctcctgctct gccacctctg ccacgggccc 1440
atacagcacg gctaccaaca ccgtctggcc aagctctggc tctggcagct caacaaccac 1500
cagtagcgcc ccatgcacca ctcctacctc tgtggctgtg accttcgacg aaatcgtcag 1560
caccagttac ggggagacaa tctacctggc cggctcgatc cccgagctgg gcaactggtc 1620
cacggccagc gcgatccccc tccgcgcgga tgcttacacc aacagcaacc cgctctggta 1680
cgtgaccgtc aatctgcccc ctggcaccag cttcgagtac aagttcttca agaaccagac 1740
ggacgggacc atcgtctggg aggacgaccc gaaccggtcg tacacggtcc cagcgtactg 1800
tgggcagact accgccattc ttgacgatag ttggcagtga cgattctaga 1850
<210>89
<211>3110
<212>DNA
<213>罗耳阿太菌(Athelia rolfsii)-埃默森篮状菌(Talaromyces emersonii)
<400>89
aagcttcacc atgtttcgtt cactcctggc cttggctgcg tgtgcagtcg cctctgtatc 60
tgcacagtct gcgtctgcga cagcatatct taccaaggaa tctgcagttg ccaagaatgg 120
cgtactttgc aacattggta gccagggatg catgtctgag ggtgcctata gcggtattgt 180
gatcgcatct ccctctaaaa ctagccctga ctatctctat acctggactc gcgactcgtc 240
gctcgtcttc aagatgttaa ttgaccaata cacaaatggc ctggatacga cactgcgcac 300
tctcattgac gagtttgtct ctgcggaagc caccattcaa caaaccagta actcatctgg 360
taccgtctct accggtggtc tcggcgaacc caaattcaat atcgacgaga cggcatttac 420
gggcgcatgg ggtcgtcccc aacgtgatgg tcccgccctc cgtgcaaccg caatcatgac 480
ctatgcgacg tatctgtaca acaatggcaa cacttcctac gtgaccaaca ccctttggcc 540
tatcatcaag ctcgaccttg actatgtcaa ctcggactgg aaccagacca cgtttgacct 600
ctgggaagaa gttgactcgt cttctttctt tacgactgcc gttcagcacc gtgctcttgt 660
tcagggcgca gcctttgcta ccctcatcgg ccaaacttcg tctgcttcga cttactccgc 720
cacggcccct agcattctct gcttcttgca gtcttactgg aacaccaacg gatactggac 780
ggccaacact ggtggcggac gttccggcaa ggacgccaac accatactcg cttctatcca 840
cacatttgac gccagcgccg gctgctctgc tgccacgtct caaccatgct ctgacgtagc 900
attggccaac ctgaaggtat acgttgactc tttccgtagt atttatacga tcaacagcgg 960
tatttcctct acctcgggtg ttgctactgg tcgctacccc gaagattcgt attacaatgg 1020
caacccctgg tacctctgca cactcgccgt cgccgagcag ctctatgatg ctctcatcgt 1080
atggaaggct gccggggagc tcaacgtcac ctccgtctcg ctcgcgttct tccagcaatt 1140
cgactcgagc atcaccgccg gcacttacgc ctcctcgtcg agcgtataca cttcgctcat 1200
ctctgacatc caggcgttcg cagacgagtt tgttgacatt gttgccaagt acacgccttc 1260
aagcttcacc atgtttcgtt cactcctggc cttggctgcg tgtgcagtcg cctctgtatc 1320
tgcacagtct gcgtctgcga cagcatatct taccaaggaa tctgcagttg ccaagaatgg 1380
cgtactttgc aacattggta gccagggatg catgtctgag ggtgcctata gcggtattgt 1440
gatcgcatct ccctctaaaa ctagccctga ctatctctat acctggactc gcgactcgtc 1500
gctcgtcttc aagatgttaa ttgaccaata cacaaatggc ctggatacga cactgcgcac 1560
tctcattgac gagtttgtct ctgcggaagc caccattcaa caaaccagta actcatctgg 1620
taccgtctct accggtggtc tcggcgaacc caaattcaat atcgacgaga cggcatttac 1680
gggcgcatgg ggtcgtcccc aacgtgatgg tcccgccctc cgtgcaaccg caatcatgac 1740
ctatgcgacg tatctgtaca acaatggcaa cacttcctac gtgaccaaca ccctttggcc 1800
tatcatcaag ctcgaccttg actatgtcaa ctcggactgg aaccagacca cgtttgacct 1860
ctgggaagaa gttgactcgt cttctttctt tacgactgcc gttcagcacc gtgctcttgt 1920
tcagggcgca gcctttgcta ccctcatcgg ccaaacttcg tctgcttcga cttactccgc 1980
cacggcccct agcattctct gcttcttgca gtcttactgg aacaccaacg gatactggac 2040
ggccaacact ggtggcggac gttccggcaa ggacgccaac accatactcg cttctatcca 2100
cacatttgac gccagcgccg gctgctctgc tgccacgtct caaccatgct ctgacgtagc 2160
attggccaac ctgaaggtat acgttgactc tttccgtagt atttatacga tcaacagcgg 2220
tatttcctct acctcgggtg ttgctactgg tcgctacccc gaagattcgt attacaatgg 2280
caacccctgg tacctctgca cactcgccgt cgccgagcag ctctatgatg ctctcatcgt 2340
atggaaggct gccggggagc tcaacgtcac ctccgtctcg ctcgcgttct tccagcaatt 2400
cgactcgagc atcaccgccg gcacttacgc ctcctcgtcg agcgtataca cttcgctcat 2460
ctctgacatc caggcgttcg cagacgagtt tgttgacatt gttgccaagt acacgccttc 2520
gtctggcttc ttgtctgagc agtatgataa gtccacgggt gctcaggatt cggctgctaa 2580
cttgacttgg tcctatgctg ctgctatcac cgcttaccaa gcccgcaatg gcttcacagg 2640
tgcttcgtgg ggtgctaagg gagtttctac ctcctgctcg actggtgcta caagcccggg 2700
ttacagcacg gctaccaaca ccgtctggcc aagctctggc tctggcagct caacaaccac 2760
cagtagcgcc ccatgcacca ctcctacctc tgtggctgtg accttcgacg aaatcgtcag 2820
caccagttac ggggagacaa tctacctggc cggctcgatc cccgagctgg gcaactggtc 2880
cacggccagc gcgatccccc tccgcgcgga tgcttacacc aacagcaacc cgctctggta 2940
cgtgaccgtc aatctgcccc ctggcaccag cttcgagtac aagttcttca agaaccagac 3000
ggacgggacc atcgtctggg aggacgaccc gaaccggtcg tacacggtcc cagcgtactg 3060
tgggcagact accgccattc ttgacgatag ttggcagtga cgattctaga 3110
<210>90
<211>1850
<212>DNA
<213>罗耳阿太菌(Athelia rolfsii)-埃默森篮状菌(Talaromyces emersonii)
<400>90
aagcttcacc atgtttcgtt cactcctggc cttggctgcg tgtgcagtcg cctctgtatc 60
tgcacagtct gcgtctgcga cagcatatct taccaaggaa tctgcagttg ccaagaatgg 120
cgtactttgc aacattggta gccagggatg catgtctgag ggtgcctata gcggtattgt 180
gatcgcatct ccctctaaaa ctagccctga ctatctctat acctggactc gcgactcgtc 240
gctcgtcttc aagatgttaa ttgaccaata cacaaatggc ctggatacga cactgcgcac 300
tctcattgac gagtttgtct ctgcggaagc caccattcaa caaaccagta actcatctgg 360
taccgtctct accggtggtc tcggcgaacc caaattcaat atcgacgaga cggcatttac 420
gggcgcatgg ggtcgtcccc aacgtgatgg tcccgccctc cgtgcaaccg caatcatgac 480
ctatgcgacg tatctgtaca acaatggcaa cacttcctac gtgaccaaca ccctttggcc 540
tatcatcaag ctcgaccttg actatgtcaa ctcggactgg aaccagacca cgtttgacct 600
ctgggaagaa gttgactcgt cttctttctt tacgactgcc gttcagcacc gtgctcttgt 660
tcagggcgca gcctttgcta ccctcatcgg ccaaacttcg tctgcttcga cttactccgc 720
cacggcccct agcattctct gcttcttgca gtcttactgg aacaccaacg gatactggac 780
ggccaacact ggtggcggac gttccggcaa ggacgccaac accatactcg cttctatcca 840
cacatttgac gccagcgccg gctgctctgc tgccacgtct caaccatgct ctgacgtagc 900
attggccaac ctgaaggtat acgttgactc tttccgtagt atttatacga tcaacagcgg 960
tatttcctct acctcgggtg ttgctactgg tcgctacccc gaagattcgt attacaatgg 1020
caacccctgg tacctctgca cactcgccgt cgccgagcag ctctatgatg ctctcatcgt 1080
atggaaggct gccggggagc tcaacgtcac ctccgtctcg ctcgcgttct tccagcaatt 1140
cgactcgagc atcaccgccg gcacttacgc ctcctcgtcg agcgtataca cttcgctcat 1200
ctctgacatc caggcgttcg cagacgagtt tgttgacatt gttgccaagt acacgccttc 1260
gtctggcttc ttgtctgagc agtatgataa gtccacgggt gctcaggatt cggctgctaa 1320
cttgacttgg tcctatgctg ctgctatcac cgcttaccaa gcccgcaatg gcttcacagg 1380
tgcttcgtgg ggtgctaagg gagtttctac ctcctgctcg actggtgcta caagcccggg 1440
tggctcctcg ggtagtgtcg aggtcactcc aagctctggc tctggcagct caacaaccac 1500
cagtagcgcc ccatgcacca ctcctacctc tgtggctgtg accttcgacg aaatcgtcag 1560
caccagttac ggggagacaa tctacctggc cggctcgatc cccgagctgg gcaactggtc 1620
cacggccagc gcgatccccc tccgcgcgga tgcttacacc aacagcaacc cgctctggta 1680
cgtgaccgtc aatctgcccc ctggcaccag cttcgagtac aagttcttca agaaccagac 1740
ggacgggacc atcgtctggg aggacgaccc gaaccggtcg tacacggtcc cagcgtactg 1800
tgggcagact accgccattc ttgacgatag ttggcagtga cgattctaga 1850
<210>91
<211>1862
<212>DNA
<213>罗耳阿太菌(Athelia rolfsii)-黑曲霉(Aspergillus niger)
<400>91
aagcttcacc atgtcgttcc gatctctact cgccctgagc ggcctcgtct gcacagggtt 60
ggcaaatgtg atttccaagc gcgcgacctt ggattcatgg ttgagcaacg aagcgaccgt 120
ggctcgtact gccatcctga ataacatcgg ggcggacggt gcttgggtgt cgggcgcgga 180
ctctggcatt gtcgttgcta gtcccagcac ggataacccg gactacttct acacctggac 240
tcgcgactct ggtctcgtcc tcaagaccct cgtcgatctc ttccgaaatg gagataccag 300
tctcctctcc accattgaga actacatctc cgcccaggca attgtccagg gtatcagtaa 360
cccctctggt gatctgtcca gcggcgctgg tctcggtgaa cccaagttca atgtcgatga 420
gactgcctac actggttctt ggggacggcc gcagcgagat ggtccggctc tgagagcaac 480
tgctatgatc ggcttcgggc agtggctgct tgacaatggc tacaccagca ccgcaacgga 540
cattgtttgg cccctcgtta ggaacgacct gtcgtatgtg gctcaatact ggaaccagac 600
aggatatgat ctctgggaag aagtcaatgg ctcgtctttc tttacgattg ctgtgcaaca 660
ccgcgccctt gtcgaaggta gtgccttcgc gacggccgtc ggctcgtcct gctcctggtg 720
tgattctcag gcacccgaaa ttctctgcta cctgcagtcc ttctggaccg gcagcttcat 780
tctggccaac ttcgatagca gccgttccgg caaggacgca aacaccctcc tgggaagcat 840
ccacaccttt gatcctgagg ccgcatgcga cgactccacc ttccagccct gctccccgcg 900
cgcgctcgcc aaccacaagg aggttgtaga ctctttccgc tcaatctata ccctcaacga 960
tggtctcagt gacagcgagg ctgttgcggt gggtcggtac cctgaggaca cgtactacaa 1020
cggcaacccg tggttcctgt gcaccttggc tgccgcagag cagttgtacg atgctctata 1080
ccagtgggac aagcaggggt cgttggaggt cacagatgtg tcgctggact tcttcaaggc 1140
actgtacagc gatgctgcta ctggcaccta ctcttcgtcc agttcgactt atagtagcat 1200
tgtagatgcc gtgaagactt tcgccgatgg cttcgtctct attgtggaaa ctcacgccgc 1260
aagcaacggc tccatgtccg agcaatacga caagtctgat ggcgagcagc tttccgctcg 1320
cgacctgacc tggtcttatg ctgctctgct gaccgccaac aaccgtcgta actccgtcgt 1380
gcctgcttct tggggcgaga cctctgccag cagcgtgccc ggcacctgtt ctgccacctc 1440
tgccacgggc ccatacagca cggctaccaa caccgtctgg ccaagctctg gctctggcag 1500
ctcaacaacc accagtagcg ccccatgcac cactcctacc tctgtggctg tgaccttcga 1560
cgaaatcgtc agcaccagtt acggggagac aatctacctg gccggctcga tccccgagct 1620
gggcaactgg tccacggcca gcgcgatccc cctccgcgcg gatgcttaca ccaacagcaa 1680
cccgctctgg tacgtgaccg tcaatctgcc ccctggcacc agcttcgagt acaagttctt 1740
caagaaccag acggacggga ccatcgtctg ggaggacgac ccgaaccggt cgtacacggt 1800
cccagcgtac tgtgggcaga ctaccgccat tcttgacgat agttggcagt gacgattcta 1860
ga 1862
<210>92
<211>1862
<212>DNA
<213>罗耳阿太菌(Athelia rolfsii)-黑曲霉(Aspergillus niger)
<400>92
aagcttcacc atgtcgttcc gatctctact cgccctgagc ggcctcgtct gcacagggtt 60
ggcaaatgtg atttccaagc gcgcgacctt ggattcatgg ttgagcaacg aagcgaccgt 120
ggctcgtact gccatcctga ataacatcgg ggcggacggt gcttgggtgt cgggcgcgga 180
ctctggcatt gtcgttgcta gtcccagcac ggataacccg gactacttct acacctggac 240
tcgcgactct ggtctcgtcc tcaagaccct cgtcgatctc ttccgaaatg gagataccag 300
tctcctctcc accattgaga actacatctc cgcccaggca attgtccagg gtatcagtaa 360
cccctctggt gatctgtcca gcggcgctgg tctcggtgaa cccaagttca atgtcgatga 420
gactgcctac actggttctt ggggacggcc gcagcgagat ggtccggctc tgagagcaac 480
tgctatgatc ggcttcgggc agtggctgct tgacaatggc tacaccagca ccgcaacgga 540
cattgtttgg cccctcgtta ggaacgacct gtcgtatgtg gctcaatact ggaaccagac 600
aggatatgat ctctgggaag aagtcaatgg ctcgtctttc tttacgattg ctgtgcaaca 660
ccgcgccctt gtcgaaggta gtgccttcgc gacggccgtc ggctcgtcct gctcctggtg 720
tgattctcag gcacccgaaa ttctctgcta cctgcagtcc ttctggaccg gcagcttcat 780
tctggccaac ttcgatagca gccgttccgg caaggacgca aacaccctcc tgggaagcat 840
ccacaccttt gatcctgagg ccgcatgcga cgactccacc ttccagccct gctccccgcg 900
cgcgctcgcc aaccacaagg aggttgtaga ctctttccgc tcaatctata ccctcaacga 960
tggtctcagt gacagcgagg ctgttgcggt gggtcggtac cctgaggaca cgtactacaa 1020
cggcaacccg tggttcctgt gcaccttggc tgccgcagag cagttgtacg atgctctata 1080
ccagtgggac aagcaggggt cgttggaggt cacagatgtg tcgctggact tcttcaaggc 1140
actgtacagc gatgctgcta ctggcaccta ctcttcgtcc agttcgactt atagtagcat 1200
tgtagatgcc gtgaagactt tcgccgatgg cttcgtctct attgtggaaa ctcacgccgc 1260
aagcaacggc tccatgtccg agcaatacga caagtctgat ggcgagcagc tttccgctcg 1320
cgacctgacc tggtcttatg ctgctctgct gaccgccaac aaccgtcgta actccgtcgt 1380
gcctgcttct tggggcgaga cctctgccag cagcgtgccc ggcacctgtg cggccacatc 1440
tgccattggt acctacagca cggctaccaa caccgtctgg ccaagctctg gctctggcag 1500
ctcaacaacc accagtagcg ccccatgcac cactcctacc tctgtggctg tgaccttcga 1560
cgaaatcgtc agcaccagtt acggggagac aatctacctg gccggctcga tccccgagct 1620
gggcaactgg tccacggcca gcgcgatccc cctccgcgcg gatgcttaca ccaacagcaa 1680
cccgctctgg tacgtgaccg tcaatctgcc ccctggcacc agcttcgagt acaagttctt 1740
caagaaccag acggacggga ccatcgtctg ggaggacgac ccgaaccggt cgtacacggt 1800
cccagcgtac tgtgggcaga ctaccgccat tcttgacgat agttggcagt gacgattcta 1860
ga 1862
<210>93
<211>1862
<212>DNA
<213>罗耳阿太菌(Athelia rolfsi)-黑曲霉(Aspergillus niger)
<400>93
aagcttcacc atgtcgttcc gatctctact cgccctgagc ggcctcgtct gcacagggtt 60
ggcaaatgtg atttccaagc gcgcgacctt ggattcatgg ttgagcaacg aagcgaccgt 120
ggctcgtact gccatcctga ataacatcgg ggcggacggt gcttgggtgt cgggcgcgga 180
ctctggcatt gtcgttgcta gtcccagcac ggataacccg gactacttct acacctggac 240
tcgcgactct ggtctcgtcc tcaagaccct cgtcgatctc ttccgaaatg gagataccag 300
tctcctctcc accattgaga actacatctc cgcccaggca attgtccagg gtatcagtaa 360
cccctctggt gatctgtcca gcggcgctgg tctcggtgaa cccaagttca atgtcgatga 420
gactgcctac actggttctt ggggacggcc gcagcgagat ggtccggctc tgagagcaac 480
tgctatgatc ggcttcgggc agtggctgct tgacaatggc tacaccagca ccgcaacgga 540
cattgtttgg cccctcgtta ggaacgacct gtcgtatgtg gctcaatact ggaaccagac 600
aggatatgat ctctgggaag aagtcaatgg ctcgtctttc tttacgattg ctgtgcaaca 660
ccgcgccctt gtcgaaggta gtgccttcgc gacggccgtc ggctcgtcct gctcctggtg 720
tgattctcag gcacccgaaa ttctctgcta cctgcagtcc ttctggaccg gcagcttcat 780
tctggccaac ttcgatagca gccgttccgg caaggacgca aacaccctcc tgggaagcat 840
ccacaccttt gatcctgagg ccgcatgcga cgactccacc ttccagccct gctccccgcg 900
cgcgctcgcc aaccacaagg aggttgtaga ctctttccgc tcaatctata ccctcaacga 960
tggtctcagt gacagcgagg ctgttgcggt gggtcggtac cctgaggaca cgtactacaa 1020
cggcaacccg tggttcctgt gcaccttggc tgccgcagag cagttgtacg atgctctata 1080
ccagtgggac aagcaggggt cgttggaggt cacagatgtg tcgctggact tcttcaaggc 1140
actgtacagc gatgctgcta ctggcaccta ctcttcgtcc agttcgactt atagtagcat 1200
tgtagatgcc gtgaagactt tcgccgatgg cttcgtctct attgtggaaa ctcacgccgc 1260
aagcaacggc tccatgtccg agcaatacga caagtctgat ggcgagcagc tttccgctcg 1320
cgacctgacc tggtcttatg ctgctctgct gaccgccaac aaccgtcgta actccgtcgt 1380
gcctgcttct tggggcgaga cctctgccag cagcgtgccc ggcacctgtg cggccacatc 1440
tgccattggt acctacagca gtgtgactgt cacctcgtgg ccaagctctg gctctggcag 1500
ctcaacaacc accagtagcg ccccatgcac cactcctacc tctgtggctg tgaccttcga 1560
cgaaatcgtc agcaccagtt acggggagac aatctacctg gccggctcga tccccgagct 1620
gggcaactgg tccacggcca gcgcgatccc cctccgcgcg gatgcttaca ccaacagcaa 1680
cccgctctgg tacgtgaccg tcaatctgcc ccctggcacc agcttcgagt acaagttctt 1740
caagaaccag acggacggga ccatcgtctg ggaggacgac ccgaaccggt cgtacacggt 1800
cccagcgtac tgtgggcaga ctaccgccat tcttgacgat agttggcagt gacgattcta 1860
ga 1862
<210>94
<211>1790
<212>DNA
<213>Talaromyce emersonii-黑曲霉(Aspergillus niger)
<400>94
aagcttcacc atggcgtccc tcgttgctgg cgctctctgc atcctgggcc tgacgcctgc 60
tgcatttgca cgagcgcccg ttgcagcgcg agccaccggt tccctggact cctttctcgc 120
aaccgaaact ccaattgccc tccaaggcgt gctgaacaac atcgggccca atggtgctga 180
tgtggcagga gcaagcgccg gcattgtggt tgccagtccg agcaggagcg acccaaatta 240
tttctactcc tggacacgtg acgcagcgct cacggccaaa tacctcgtcg acgccttcat 300
cgcgggcaac aaggacctag agcagaccat ccagcagtac atcagcgcgc aggcgaaggt 360
gcaaactatc tccaatccgt ccggagattt atccaccggt ggcttaggtg agcccaagtt 420
caatgtgaat gagacggctt ttaccgggcc ctggggtcgt ccacagaggg acggaccagc 480
gttgagagcg acggccctca ttgcgtatgc gaactatctc atcgacaacg gcgaggcttc 540
gactgccgat gagatcatct ggccgattgt ccagaatgat ctgtcctaca tcacccaata 600
ctggaactca tccaccttcg acctctggga agaagtagaa ggatcctcat tcttcacaac 660
cgccgtgcaa caccgcgccc tggtcgaagg caatgcactg gcaacaaggc tgaaccacac 720
gtgctccaac tgcgtctctc aggcccctca ggtcctgtgt ttcctgcagt catactggac 780
cggatcgtat gttctggcca actttggtgg cagcggtcgt tccggcaagg acgtgaattc 840
gattttgggc agcatccaca cctttgatcc cgccggaggc tgtgacgact cgaccttcca 900
gccgtgttcg gcccgtgcct tggcaaatca caaggtggtc accgactcgt tccggagtat 960
ctatgcgatc aactcaggca tcgcagaggg atctgccgtg gcagtcggcc gctaccctga 1020
ggatgtctac cagggcggga acccctggta cctggccaca gcagcggctg cagagcagct 1080
ttacgacgcc atctaccagt ggaagaagat cggctcgata agtatcacgg acgttagtct 1140
gccatttttc caggatatct acccttctgc cgcggtgggc acctataact ctggctccac 1200
gactttcaac gacatcatct cggccgtcca gacgtatggt gatggatatc tgagtattgt 1260
cgagaaatat actccctcag acggctctct taccgaacaa ttctcccgta cagacggcac 1320
tccgctttct gcctctgccc tgacttggtc gtacgcttct ctcctaaccg cttcggcccg 1380
cagacagtcc gtcgtccctg cttcctgggg cgaaagctcc gcaagcagcg tccctgccgt 1440
ctgctcgact ggtgctacaa gcccgggtgg ctcctcgggt agtgtcgagg tcactttcga 1500
cgtttacgct accacagtat atggccagaa catctatatc accggtgatg tgagtgagct 1560
cggcaactgg acacccgcca atggtgttgc actctcttct gctaactacc ccacctggag 1620
tgccacgatc gctctccccg ctgacacgac aatccagtac aagtatgtca acattgacgg 1680
cagcaccgtc atctgggagg atgctatcag caatcgcgag atcacgacgc ccgccagcgg 1740
cacatacacc gaaaaagaca cttgggatga atcttaatga cgattctaga 1790
<210>95
<211>1790
<212>DNA
<213>Talaromyce emersonii-黑曲霉(Aspergillus niger)
<400>95
aagcttcacc atggcgtccc tcgttgctgg cgctctctgc atcctgggcc tgacgcctgc 60
tgcatttgca cgagcgcccg ttgcagcgcg agccaccggt tccctggact cctttctcgc 120
aaccgaaact ccaattgccc tccaaggcgt gctgaacaac atcgggccca atggtgctga 180
tgtggcagga gcaagcgccg gcattgtggt tgccagtccg agcaggagcg acccaaatta 240
tttctactcc tggacacgtg acgcagcgct cacggccaaa tacctcgtcg acgccttcat 300
cgcgggcaac aaggacctag agcagaccat ccagcagtac atcagcgcgc aggcgaaggt 360
gcaaactatc tccaatccgt ccggagattt atccaccggt ggcttaggtg agcccaagtt 420
caatgtgaat gagacggctt ttaccgggcc ctggggtcgt ccacagaggg acggaccagc 480
gttgagagcg acggccctca ttgcgtatgc gaactatctc atcgacaacg gcgaggcttc 540
gactgccgat gagatcatct ggccgattgt ccagaatgat ctgtcctaca tcacccaata 600
ctggaactca tccaccttcg acctctggga agaagtagaa ggatcctcat tcttcacaac 660
cgccgtgcaa caccgcgccc tggtcgaagg caatgcactg gcaacaaggc tgaaccacac 720
gtgctccaac tgcgtctctc aggcccctca ggtcctgtgt ttcctgcagt catactggac 780
cggatcgtat gttctggcca actttggtgg cagcggtcgt tccggcaagg acgtgaattc 840
gattttgggc agcatccaca cctttgatcc cgccggaggc tgtgacgact cgaccttcca 900
gccgtgttcg gcccgtgcct tggcaaatca caaggtggtc accgactcgt tccggagtat 960
ctatgcgatc aactcaggca tcgcagaggg atctgccgtg gcagtcggcc gctaccctga 1020
ggatgtctac cagggcggga acccctggta cctggccaca gcagcggctg cagagcagct 1080
ttacgacgcc atctaccagt ggaagaagat cggctcgata agtatcacgg acgttagtct 1140
gccatttttc caggatatct acccttctgc cgcggtgggc acctataact ctggctccac 1200
gactttcaac gacatcatct cggccgtcca gacgtatggt gatggatatc tgagtattgt 1260
cgagaaatat actccctcag acggctctct taccgaacaa ttctcccgta cagacggcac 1320
tccgctttct gcctctgccc tgacttggtc gtacgcttct ctcctaaccg cttcggcccg 1380
cagacagtcc gtcgtccctg cttcctgggg cgaaagctcc gcaagcagcg tccctgccgt 1440
ctgctctgcc acctctgcca cgggcccagg ctcctcgggt agtgtcgagg tcactttcga 1500
cgtttacgct accacagtat atggccagaa catctatatc accggtgatg tgagtgagct 1560
cggcaactgg acacccgcca atggtgttgc actctcttct gctaactacc ccacctggag 1620
tgccacgatc gctctccccg ctgacacgac aatccagtac aagtatgtca acattgacgg 1680
cagcaccgtc atctgggagg atgctatcag caatcgcgag atcacgacgc ccgccagcgg 1740
cacatacacc gaaaaagaca cttgggatga atcttaatga cgattctaga 1790
<210>96
<211>1790
<212>DNA
<213>Talaromyce emersonii-黑曲霉(Aspergillus niger)
<400>96
aagcttcacc atggcgtccc tcgttgctgg cgctctctgc atcctgggcc tgacgcctgc 60
tgcatttgca cgagcgcccg ttgcagcgcg agccaccggt tccctggact cctttctcgc 120
aaccgaaact ccaattgccc tccaaggcgt gctgaacaac atcgggccca atggtgctga 180
tgtggcagga gcaagcgccg gcattgtggt tgccagtccg agcaggagcg acccaaatta 240
tttctactcc tggacacgtg acgcagcgct cacggccaaa tacctcgtcg acgccttcat 300
cgcgggcaac aaggacctag agcagaccat ccagcagtac atcagcgcgc aggcgaaggt 360
gcaaactatc tccaatccgt ccggagattt atccaccggt ggcttaggtg agcccaagtt 420
caatgtgaat gagacggctt ttaccgggcc ctggggtcgt ccacagaggg acggaccagc 480
gttgagagcg acggccctca ttgcgtatgc gaactatctc atcgacaacg gcgaggcttc 540
gactgccgat gagatcatct ggccgattgt ccagaatgat ctgtcctaca tcacccaata 600
ctggaactca tccaccttcg acctctggga agaagtagaa ggatcctcat tcttcacaac 660
cgccgtgcaa caccgcgccc tggtcgaagg caatgcactg gcaacaaggc tgaaccacac 720
gtgctccaac tgcgtctctc aggcccctca ggtcctgtgt ttcctgcagt catactggac 780
cggatcgtat gttctggcca actttggtgg cagcggtcgt tccggcaagg acgtgaattc 840
gattttgggc agcatccaca cctttgatcc cgccggaggc tgtgacgact cgaccttcca 900
gccgtgttcg gcccgtgcct tggcaaatca caaggtggtc accgactcgt tccggagtat 960
ctatgcgatc aactcaggca tcgcagaggg atctgccgtg gcagtcggcc gctaccctga 1020
ggatgtctac cagggcggga acccctggta cctggccaca gcagcggctg cagagcagct 1080
ttacgacgcc atctaccagt ggaagaagat cggctcgata agtatcacgg acgttagtct 1140
gccatttttc caggatatct acccttctgc cgcggtgggc acctataact ctggctccac 1200
gactttcaac gacatcatct cggccgtcca gacgtatggt gatggatatc tgagtattgt 1260
cgagaaatat actccctcag acggctctct taccgaacaa ttctcccgta cagacggcac 1320
tccgctttct gcctctgccc tgacttggtc gtacgcttct ctcctaaccg cttcggcccg 1380
cagacagtcc gtcgtccctg cttcctgggg cgaaagctcc gcaagcagcg tccctgccgt 1440
ctgctctgcc acctctgcca cgggcccata cagcacggct accaacaccg tctggttcga 1500
cgtttacgct accacagtat atggccagaa catctatatc accggtgatg tgagtgagct 1560
cggcaactgg acacccgcca atggtgttgc actctcttct gctaactacc ccacctggag 1620
tgccacgatc gctctccccg ctgacacgac aatccagtac aagtatgtca acattgacgg 1680
cagcaccgtc atctgggagg atgctatcag caatcgcgag atcacgacgc ccgccagcgg 1740
cacatacacc gaaaaagaca cttgggatga atcttaatga cgattctaga 1790
<210>97
<211>1775
<212>DNA
<213>Talaromyce emersonii-罗耳阿太菌(Athelia rolfsii)
<400>97
aagcttcacc atgtcgttcc gatctctact cgccctgagc ggcctcgtct gcacagggtt 60
ggcaaatgtg atttccaagc gcgcgacctt ggattcatgg ttgagcaacg aagcgaccgt 120
ggctcgtact gccatcctga ataacatcgg ggcggacggt gcttgggtgt cgggcgcgga 180
ctctggcatt gtcgttgcta gtcccagcac ggataacccg gactacttct acacctggac 240
tcgcgactct ggtctcgtcc tcaagaccct cgtcgatctc ttccgaaatg gagataccag 300
tctcctctcc accattgaga actacatctc cgcccaggca attgtccagg gtatcagtaa 360
cccctctggt gatctgtcca gcggcgctgg tctcggtgaa cccaagttca atgtcgatga 420
gactgcctac actggttctt ggggacggcc gcagcgagat ggtccggctc tgagagcaac 480
tgctatgatc ggcttcgggc agtggctgct tgacaatggc tacaccagca ccgcaacgga 540
cattgtttgg cccctcgtta ggaacgacct gtcgtatgtg gctcaatact ggaaccagac 600
aggatatgat ctctgggaag aagtcaatgg ctcgtctttc tttacgattg ctgtgcaaca 660
ccgcgccctt gtcgaaggta gtgccttcgc gacggccgtc ggctcgtcct gctcctggtg 720
tgattctcag gcacccgaaa ttctctgcta cctgcagtcc ttctggaccg gcagcttcat 780
tctggccaac ttcgatagca gccgttccgg caaggacgca aacaccctcc tgggaagcat 840
ccacaccttt gatcctgagg ccgcatgcga cgactccacc ttccagccct gctccccgcg 900
cgcgctcgcc aaccacaagg aggttgtaga ctctttccgc tcaatctata ccctcaacga 960
tggtctcagt gacagcgagg ctgttgcggt gggtcggtac cctgaggaca cgtactacaa 1020
cggcaacccg tggttcctgt gcaccttggc tgccgcagag cagttgtacg atgctctata 1080
ccagtgggac aagcaggggt cgttggaggt cacagatgtg tcgctggact tcttcaaggc 1140
actgtacagc gatgctgcta ctggcaccta ctcttcgtcc agttcgactt atagtagcat 1200
tgtagatgcc gtgaagactt tcgccgatgg cttcgtctct attgtggaaa ctcacgccgc 1260
aagcaacggc tccatgtccg agcaatacga caagtctgat ggcgagcagc tttccgctcg 1320
cgacctgacc tggtcttatg ctgctctgct gaccgccaac aaccgtcgta actccgtcgt 1380
gcctgcttct tggggcgaga cctctgccag cagcgtgccc ggcacctgtt cgactggtgc 1440
tacaagcccg ggtggctcct cgggtagtgt cgaggtcact ttcgacgttt acgctaccac 1500
agtatatggc cagaacatct atatcaccgg tgatgtgagt gagctcggca actggacacc 1560
cgccaatggt gttgcactct cttctgctaa ctaccccacc tggagtgcca cgatcgctct 1620
ccccgctgac acgacaatcc agtacaagta tgtcaacatt gacggcagca ccgtcatctg 1680
ggaggatgct atcagcaatc gcgagatcac gacgcccgcc agcggcacat acaccgaaaa 1740
agacacttgg gatgaatctt aatgacgatt ctaga 1775
<210>98
<211>1775
<212>DNA
<213>Talaromyce emersonii-罗耳阿太菌(Athelia rolfsii)
<400>98
aagcttcacc atgtcgttcc gatctctact cgccctgagc ggcctcgtct gcacagggtt 60
ggcaaatgtg atttccaagc gcgcgacctt ggattcatgg ttgagcaacg aagcgaccgt 120
ggctcgtact gccatcctga ataacatcgg ggcggacggt gcttgggtgt cgggcgcgga 180
ctctggcatt gtcgttgcta gtcccagcac ggataacccg gactacttct acacctggac 240
tcgcgactct ggtctcgtcc tcaagaccct cgtcgatctc ttccgaaatg gagataccag 300
tctcctctcc accattgaga actacatctc cgcccaggca attgtccagg gtatcagtaa 360
cccctctggt gatctgtcca gcggcgctgg tctcggtgaa cccaagttca atgtcgatga 420
gactgcctac actggttctt ggggacggcc gcagcgagat ggtccggctc tgagagcaac 480
tgctatgatc ggcttcgggc agtggctgct tgacaatggc tacaccagca ccgcaacgga 540
cattgtttgg cccctcgtta ggaacgacct gtcgtatgtg gctcaatact ggaaccagac 600
aggatatgat ctctgggaag aagtcaatgg ctcgtctttc tttacgattg ctgtgcaaca 660
ccgcgccctt gtcgaaggta gtgccttcgc gacggccgtc ggctcgtcct gctcctggtg 720
tgattctcag gcacccgaaa ttctctgcta cctgcagtcc ttctggaccg gcagcttcat 780
tctggccaac ttcgatagca gccgttccgg caaggacgca aacaccctcc tgggaagcat 840
ccacaccttt gatcctgagg ccgcatgcga cgactccacc ttccagccct gctccccgcg 900
cgcgctcgcc aaccacaagg aggttgtaga ctctttccgc tcaatctata ccctcaacga 960
tggtctcagt gacagcgagg ctgttgcggt gggtcggtac cctgaggaca cgtactacaa 1020
cggcaacccg tggttcctgt gcaccttggc tgccgcagag cagttgtacg atgctctata 1080
ccagtgggac aagcaggggt cgttggaggt cacagatgtg tcgctggact tcttcaaggc 1140
actgtacagc gatgctgcta ctggcaccta ctcttcgtcc agttcgactt atagtagcat 1200
tgtagatgcc gtgaagactt tcgccgatgg cttcgtctct attgtggaaa ctcacgccgc 1260
aagcaacggc tccatgtccg agcaatacga caagtctgat ggcgagcagc tttccgctcg 1320
cgacctgacc tggtcttatg ctgctctgct gaccgccaac aaccgtcgta actccgtcgt 1380
gcctgcttct tggggcgaga cctctgccag cagcgtgccc ggcacctgtg cggccacatc 1440
tgccattggt accggctcct cgggtagtgt cgaggtcact ttcgacgttt acgctaccac 1500
agtatatggc cagaacatct atatcaccgg tgatgtgagt gagctcggca actggacacc 1560
cgccaatggt gttgcactct cttctgctaa ctaccccacc tggagtgcca cgatcgctct 1620
ccccgctgac acgacaatcc agtacaagta tgtcaacatt gacggcagca ccgtcatctg 1680
ggaggatgct atcagcaatc gcgagatcac gacgcccgcc agcggcacat acaccgaaaa 1740
agacacttgg gatgaatctt aatgacgatt ctaga 1775
<210>99
<211>1775
<212>DNA
<213>Talaromyce emersonii-罗耳阿太菌(Athelia rolfsii)
<400>99
aagcttcacc atgtcgttcc gatctctact cgccctgagc ggcctcgtct gcacagggtt 60
ggcaaatgtg atttccaagc gcgcgacctt ggattcatgg ttgagcaacg aagcgaccgt 120
ggctcgtact gccatcctga ataacatcgg ggcggacggt gcttgggtgt cgggcgcgga 180
ctctggcatt gtcgttgcta gtcccagcac ggataacccg gactacttct acacctggac 240
tcgcgactct ggtctcgtcc tcaagaccct cgtcgatctc ttccgaaatg gagataccag 300
tctcctctcc accattgaga actacatctc cgcccaggca attgtccagg gtatcagtaa 360
cccctctggt gatctgtcca gcggcgctgg tctcggtgaa cccaagttca atgtcgatga 420
gactgcctac actggttctt ggggacggcc gcagcgagat ggtccggctc tgagagcaac 480
tgctatgatc ggcttcgggc agtggctgct tgacaatggc tacaccagca ccgcaacgga 540
cattgtttgg cccctcgtta ggaacgacct gtcgtatgtg gctcaatact ggaaccagac 600
aggatatgat ctctgggaag aagtcaatgg ctcgtctttc tttacgattg ctgtgcaaca 660
ccgcgccctt gtcgaaggta gtgccttcgc gacggccgtc ggctcgtcct gctcctggtg 720
tgattctcag gcacccgaaa ttctctgcta cctgcagtcc ttctggaccg gcagcttcat 780
tctggccaac ttcgatagca gccgttccgg caaggacgca aacaccctcc tgggaagcat 840
ccacaccttt gatcctgagg ccgcatgcga cgactccacc ttccagccct gctccccgcg 900
cgcgctcgcc aaccacaagg aggttgtaga ctctttccgc tcaatctata ccctcaacga 960
tggtctcagt gacagcgagg ctgttgcggt gggtcggtac cctgaggaca cgtactacaa 1020
cggcaacccg tggttcctgt gcaccttggc tgccgcagag cagttgtacg atgctctata 1080
ccagtgggac aagcaggggt cgttggaggt cacagatgtg tcgctggact tcttcaaggc 1140
actgtacagc gatgctgcta ctggcaccta ctcttcgtcc agttcgactt atagtagcat 1200
tgtagatgcc gtgaagactt tcgccgatgg cttcgtctct attgtggaaa ctcacgccgc 1260
aagcaacggc tccatgtccg agcaatacga caagtctgat ggcgagcagc tttccgctcg 1320
cgacctgacc tggtcttatg ctgctctgct gaccgccaac aaccgtcgta actccgtcgt 1380
gcctgcttct tggggcgaga cctctgccag cagcgtgccc ggcacctgtg cggccacatc 1440
tgccattggt acctacagca gtgtgactgt cacctcgtgg ttcgacgttt acgctaccac 1500
agtatatggc cagaacatct atatcaccgg tgatgtgagt gagctcggca actggacacc 1560
cgccaatggt gttgcactct cttctgctaa ctaccccacc tggagtgcca cgatcgctct 1620
ccccgctgac acgacaatcc agtacaagta tgtcaacatt gacggcagca ccgtcatctg 1680
ggaggatgct atcagcaatc gcgagatcac gacgcccgcc agcggcacat acaccgaaaa 1740
agacacttgg gatgaatctt aatgacgatt ctaga 1775
Claims (10)
1.一种包含具有葡糖淀粉酶活性的催化模块和碳水化合物结合模块的杂交酶,其中
(a)催化模块与SEQ ID NO:25的氨基酸序列的序列具有至少99%序列同一性:并且来源于埃默森篮状菌;(b)碳水化合物结合模块与SEQ ID NO:18具有至少99%序列同一性,并且来源于黑曲霉。
2.权利要求1的杂交酶,其中所述的催化模块由SEQ ID NO:25的氨基酸序列组成,且碳水化合物结构域由SEQ ID NO:18的氨基酸序列组成。
3.权利要求1的杂交酶,其由SEQ ID NOs:94-96中任一序列所编码。
4.一种产生乙醇的方法,包括在发酵生物存在时,用权利要求1-3任一项的杂交酶在水性介质中处理颗粒淀粉。
5.权利要求4的方法,另外其中对所述颗粒淀粉进行α-淀粉酶处理。
6.权利要求5的方法,其中所述α-淀粉酶是酸性α-淀粉酶。
7.权利要求6的方法,其中所述酸性α-淀粉酶源于黑曲霉菌株。
8.权利要求4的方法,其中杂交酶以0.02-20AGU/g DS的量加入。
9.权利要求4的方法,其中所述颗粒淀粉在低于该颗粒淀粉的初始糊化温度下被水解为可溶性淀粉水解产物。
10.权利要求4的方法,其中所述发酵生物是啤酒糖酵母的菌株。
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AR005601A1 (es) | 1996-01-29 | 1999-06-23 | Novozymes As | Proceso para la remocion o blanqueo de suciedad o manchas presentes en telas celulosicas y proceso para el lavado de telas celulosicas manchadas o sucias y composicion detergente |
CN1209838A (zh) | 1996-01-29 | 1999-03-03 | 诺沃挪第克公司 | 纤维素织物的脱浆方法 |
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AU2382197A (en) | 1996-04-19 | 1997-11-12 | Novo Nordisk A/S | Fabric treated with a cellulase and a hybrid enzyme comprising a phenol oxidizing enzyme |
AU2692897A (en) | 1996-04-30 | 1997-11-19 | Novo Nordisk A/S | Alpha-amylase mutants |
AU4550997A (en) | 1996-10-11 | 1998-05-11 | Novo Nordisk A/S | Cellulose binding domains (cbds) for oral care products |
AU4551097A (en) | 1996-10-11 | 1998-05-11 | Novo Nordisk A/S | Alpha-amylase fused to cellulose binding domain, for starch degradation |
SE9603788L (sv) | 1996-10-14 | 1998-04-15 | Combitech Traffic Syst Ab | Radiokommunikationsenhet för fordon |
ATE386799T1 (de) | 1996-10-28 | 2008-03-15 | Novozymes As | Extrazelluläre expression von zellulose bindenden domänen (cbd) unter verwendung von bacillus |
EP0941359A1 (en) | 1996-11-21 | 1999-09-15 | Novo Nordisk A/S | Use of carbohydrate-binding domain in starch processing |
EP1023439B1 (en) | 1997-10-13 | 2009-02-18 | Novozymes A/S | alpha-AMYLASE MUTANTS |
WO1999028448A1 (en) | 1997-11-26 | 1999-06-10 | Novo Nordisk A/S | Thermostable glucoamylase |
CN1292028B (zh) | 1998-02-27 | 2013-08-14 | 诺维信公司 | 麦芽α淀粉酶变体 |
WO2000004136A1 (en) | 1998-07-15 | 2000-01-27 | Novozymes A/S | Glucoamylase variants |
EP1124949B1 (en) | 1998-10-26 | 2006-07-12 | Novozymes A/S | Constructing and screening a dna library of interest in filamentous fungal cells |
JP2003504046A (ja) | 1999-07-09 | 2003-02-04 | ノボザイムス アクティーゼルスカブ | グルコアミラーゼ変異体 |
ES2425351T3 (es) * | 2003-06-25 | 2013-10-14 | Novozymes A/S | Polipéptidos que tienen actividad de alfa-amilasa y polinucleótidos que codifican los mismos |
US7618795B2 (en) * | 2003-06-25 | 2009-11-17 | Novozymes A/S | Starch process |
MXPA06000212A (es) * | 2003-06-25 | 2006-03-21 | Novozymes As | Enzimas para procesar almidon. |
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2004
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Also Published As
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MXPA06004463A (es) | 2006-06-27 |
CN103215239B (zh) | 2015-08-05 |
ATE457034T1 (de) | 2010-02-15 |
US20050158839A1 (en) | 2005-07-21 |
ES2490616T3 (es) | 2014-09-04 |
EP2213732A1 (en) | 2010-08-04 |
EP1687419A4 (en) | 2007-01-24 |
PL1687419T3 (pl) | 2010-07-30 |
ES2340390T3 (es) | 2010-06-02 |
DE602004025419D1 (de) | 2010-03-25 |
US20080241901A1 (en) | 2008-10-02 |
DK2213732T3 (da) | 2014-08-18 |
US7129069B2 (en) | 2006-10-31 |
WO2005045018A1 (en) | 2005-05-19 |
US7749744B2 (en) | 2010-07-06 |
EP1687419B1 (en) | 2010-02-03 |
CN1875097A (zh) | 2006-12-06 |
US20060257984A1 (en) | 2006-11-16 |
EP2213732B1 (en) | 2014-05-21 |
US7312055B2 (en) | 2007-12-25 |
EP1687419A1 (en) | 2006-08-09 |
CN103215239A (zh) | 2013-07-24 |
JP2007509630A (ja) | 2007-04-19 |
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