CN1930285A - 脂环酸芽孢杆菌的多肽 - Google Patents
脂环酸芽孢杆菌的多肽 Download PDFInfo
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- CN1930285A CN1930285A CNA2005800070785A CN200580007078A CN1930285A CN 1930285 A CN1930285 A CN 1930285A CN A2005800070785 A CNA2005800070785 A CN A2005800070785A CN 200580007078 A CN200580007078 A CN 200580007078A CN 1930285 A CN1930285 A CN 1930285A
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Abstract
公开了分离的成熟功能性多肽,其与可得自以保藏号DSM 15716保藏的细菌脂环酸芽孢杆菌的相应分泌多肽有至少90%的同一性并表现相同的功能。
Description
技术领域
本发明涉及以保藏号DSM 15716保藏的细菌脂环酸芽孢杆菌的基因组包含的多核苷酸所编码的功能性和有效的多肽。本发明还涉及编码这些多肽或促进其表达的多核苷酸和这些多核苷酸的构建体以及制备多肽的方法。本发明还涉及包含该多肽和多核苷酸的组合物以及该多肽的用途。本发明还涉及以保藏号DSM 15716保藏的脂环酸芽孢杆菌属细菌。
背景技术
一些来源于脂环酸芽孢杆菌属的酶是已知的,例如Matzke等;Genecloning,nucleotide sequence and biochemical properties of a cytoplasmiccyclomaltodextrinase(neopullulanase)from Alicyclobacillusacidocaldarius ATCC 2700;reclassification of a group of enzymes,提交(MAR-1999)给EMBL/GenBank/DDBJ数据库或Koivula等,Cloning andsequencing of a gene encoding acidophilic amylase from Bacillusacidocaldarius.J.Gen.Microbiol.139:2399(1993)或Bartolucci等,Thioredoxin from Bacillus acidocaldarius:characterization,high-levelexpression in Escherichia coli and molecular modeling,Biochem.J.328:277(1997)或Tsuruoka等,Collagenolytic Serine-Carboxyl Proteinase fromAlicyclobacillus sendainensis Strain NTAP-1:Purification,Characterization,Gene Cloning,and Heterologous Expression,递交(MAY-2002)给EMBL/GenBank/DDBJ数据库;Eckert K.& Schneider E.,A thermoacidophilic endoglucanase(ceIB)from Alicyclobacillusacidocaldarius displays high sequence similarity to arabinofuranosidasesbelonging to family 51 of glycosyl hydrolases;Eur.J.Biochem.,270:3593-3602,2003所述。
寻找新酶时,还已知通过对可能的候选者进行特定的酶测定来筛选这类新酶。该方法受到酶可获得性的限制,而且不能鉴定活性尚不了解的功能性酶或多肽。
此外,全基因组测序是从给定的微生物获得所有基因信息的已知方法,例如Fleischmann等,Whole genome sequences and assembly ofHaemophilus influenzae Rd;Nature 269:496-512;(1995)所述。
大部分工业用途的酶是微生物分泌至培养基中的酶。然而,只有小部分微生物的基因组编码分泌蛋白。例如仅有约4%的枯草芽孢杆菌(Bacillussubtilis)基因组或其最近亲属编码分泌蛋白(Van Dijl等:Protein transportpathways in Bacillus subtilis:a genome-based road map;《“Bacillussubtilis and its closest relatives”-From genes to cells》;337-355页;A.L.Sonenshein编;ASM Press 2002)。
基因组测序的一个缺点是所获得序列的绝大多数编码非分泌蛋白。
还已知的是信号捕获——使用与缺乏自身信号的额外的细胞报道基因所形成的融合物来鉴定含有编码信号肽的核苷酸的基因的方法(WO01/77315)。
发明概述
本发明者发现了在低pH(约4-5)和高温(50-60℃)下生长的脂环酸芽孢杆菌菌株,即脂环酸芽孢杆菌DSM 15716。因为已知菌株和DSM15716菌株之间的系统发生距离是显著的,并且其生长条件与工业酶若干应用的条件类似,因此该菌株很有意义。
微生物基因组包含数千个不同基因,一些编码多肽,一些编码RNA。微生物基因组中仅有限数量的基因编码服务于微生物的外部目的并由微生物分泌至周围培养基中的功能性多肽。从这类多肽能够以可观数量在连续过程中产生而不破坏产生该多肽的细胞来看,这类多肽对工业是有意义的。
鉴定和提供由以保藏号DSM 15716保藏的脂环酸杆菌分泌的对脂环酸杆菌具有功能性目的的多肽是本发明的目的,因为这类多肽不仅可用于工业目的,而且可以以工业相关的方法和数量产生它们。
一方面本发明提供分离的成熟功能性多肽,其与可得自以保藏号DSM15716保藏的脂环酸芽孢杆菌属细菌的相应分泌多肽有至少90%的同一性并表现相同的功能。
另一方面本发明提供细菌谷氨酸肽酶(EC 3.4.23.19)。
另一方面本发明提供编码本发明多肽的多核苷酸、包含编码多肽的多核苷酸的核苷酸构建体,其中与一个或多个在宿主细胞中指导多肽产生的控制序列有效连接、包含本发明核苷酸构建体的重组体表达载体和包含本发明核苷酸构建体的重组宿主细胞。
另一方面本发明提供制备本发明多肽的方法,包括:
(a)培养包含编码本发明多肽的核苷酸序列的菌株,所述菌株能够表达并分泌多肽,和
(b)回收多肽。
另一方面本发明提供包含本发明多肽的组合物和制备这样的组合物的方法,包括将本发明的多肽与赋形剂混合。
另一方面本发明提供包含本发明多核苷酸的组合物和制备这样的组合物的方法,包括将本发明的多核苷酸与赋形剂混合。
另一方面本发明提供本发明多肽或包含所述多肽的组合物在多种应用中的用途。
另一方面本发明涉及以保藏号DSM 15716保藏的细菌脂环酸芽孢杆菌。
最后一方面本发明提供包括本发明多肽氨基酸序列和本发明多核苷酸核苷酸序列信息的电子储藏媒体。
序列列表
本申请包含序列列表形式的信息,其附属于申请并在伴随该申请的数据载体中提交。数据载体的内容在本文全部引入作为参考。SEQ ID NO:1到SEQ ID NO:25中编码成熟多肽的区域编码SEQ ID NO:26到SEQ IDNO:50的成熟多肽。因此SEQ ID NO:1的编码成熟多肽的区域编码SEQID NO:26中包含的成熟多肽序列,SEQ ID NO:2的编码成熟多肽的区域编码SEQ ID NO:27中包含的成熟多肽,等等。
发明详述
定义
本文使用的术语“同一性”旨在理解为两个氨基酸序列或两个核苷酸序列间的同源性。就本发明而言,通过使用Vector NTI程序7.1版中的AlignX测定两个氨基酸序列间的同一性程度(Informax inc.,7600Wisconsin Avenue,Suite#1100,Bethesda,MD 20814,USA)。使用ClustalW算法(Nucleic Acid Research,22(22):4673-4680,1994)进行氨基酸比对。使用以下的附加参数:缺口打开罚分为10,缺口延伸罚分为0.05,缺口分离罚分范围为8。配对比对参数为Ktuple=1,缺口罚分=3,缺口长度打开罚分=10,缺口延伸罚分=0.1,窗口大小=5和对角线=5。使用与上述相同的算法和软件包测定两个核苷酸序列间的同一性程度,例如使用下面的设定:缺口罚分为10且缺口长度罚分为10。配对比对参数为Ktuple=3,缺口罚分=3和窗口=20。
本发明上下文中使用的术语“功能性多肽”是指可由细胞表达并分泌的多肽,其组成能够按照其设计的由细胞来执行的功能运作的运作单元。任选的,多肽可能需要辅因子以实现预期的功能。功能性多肽的一个实例为催化活性多肽或在细胞周围环境中帮助细胞催化反应的酶。另一实例可以是作为信号物质的多肽。其他的实例为作为环境参数(细胞周围环境中的化学品)传感器(受体)的多肽,或是针对其他生物(抗微生物(多)肽)的多肽,或促进细胞结构完整性的多肽。
本文使用的术语氨基酸序列或多肽部分的“成熟区域”是指氨基酸序列或多肽的部分或区域或结构域或片段,其为成熟的功能性多肽。
本文使用的术语“编码成熟多肽的核苷酸序列区域”是指从编码成熟多肽第一个氨基酸的三联体算起到编码成熟多肽最后一个氨基酸的最后三联体的核苷酸序列区域。
本文使用的术语“分泌多肽”应理解为在细胞中表达后被转运并释放到周围细胞外培养基中的多肽或结合/嵌入细胞膜使得至少多肽的一部分暴露于周围细胞外培养基中的多肽。
本发明的多肽
本发明涉及与可从以保藏号DSM 15716保藏的脂环酸芽孢杆菌获得的分泌多肽相类似的多肽。具体而言,本发明提供成熟的功能性多肽,所述多肽与从以保藏号DSM 15716保藏的脂环酸芽孢杆菌获得的相应分泌多肽具有至少90%的同一性,并表现相同的功能。
此外,令人惊奇的是脂环酸芽孢杆菌DSM 15716表达的SEQ ID NO:27的谷氨酸肽酶是第一个从细菌中分离的谷氨酸肽酶。因此,本发明还提供细菌谷氨酸肽酶(EC 3.4.23.19)。
本发明的多肽特别是是由脂环酸芽孢杆菌DSM 15716为了对该特定细胞发挥功能的目的而分泌的多肽。
在脂环酸芽孢杆菌DSM 15716基因组的数千个可能的基因中,该基因组的多肽编码了包含在SEQ ID NO:26到SEQ ID NO:50中的25个分泌功能性成熟多肽,其被确定为功能性的,即由选定的宿主细胞翻译为功能性多肽。
因此,脂环酸芽孢杆菌DSM 15716表达并分泌SEQ ID NO:26到SEQNO:50中包含的功能性成熟多肽,并且在特定菌株的基因组中,SEQ IDNO:1到SEQ ID NO:25的编码成熟多肽的区域为编码SEQ ID NO:26到SEQ NO:50中包含的成熟多肽的基因。在另一个具体的实施方案中,可表达所有编码SEQ ID NO:26到SEQ NO:50中包含的成熟多肽的基因,并且可在培养大肠杆菌宿主时分泌其相应的成熟多肽,所述大肠杆菌用包含SEQ ID NO:1到SEQ ID NO:25的编码成熟多肽的区域的多核苷酸转化。通过比较这25个多肽序列与已知序列的序列同源性或同一性注释多肽的具体功能。25个分泌功能性多肽中至少15个确定为酶。
具体而言,分离的多肽选自:
(a)具有与选自SEQ ID NO:26到SEQ ID NO:50中所包含成熟多肽的氨基酸序列有至少90%同一性的氨基酸序列的多肽,和
(b)在高严格度条件下与选自下述多核苷酸探针杂交的核苷酸序列编码的多肽,所述多核苷酸探针选自:
(i)SEQ ID NO:1到SEQ ID NO:25编码成熟多肽的区域的核苷酸序列互补链,
(ii)SEQ ID NO:1到SEQ ID NO:25编码成熟多肽的区域的核苷酸序列中所包含的cDNA序列的互补链;
其中多肽显示SEQ ID NO:26到SEQ ID NO:50中相应成熟多肽的功能。
在一个具体的实施方案中,本发明多肽选自以DSM保藏号15716保藏的脂环酸芽孢杆菌分泌并由本发明者分离的酶,即由酸性内切葡聚糖酶、酸性纤维素酶、谷氨酸肽酶、多铜氧化酶、丝氨酸羧基蛋白酶、丝氨酸蛋白酶、HtrA样丝氨酸蛋白酶、二硫化物异构酶、γ-D-谷氨酰-L-二氨基酸内肽酶、内-β-N-乙酰氨基葡糖苷酶、肽酰脯氨酰异构酶、酸性磷酸酯酶、植酸酶、磷脂酶C、多糖脱乙酰酶、木聚糖脱乙酰酶和亚硫酸盐氧化酶组成的酶组。
本发明还提供选自以下的分离的酶:
(a)含有下述氨基酸序列的酶,所述氨基酸序列与以DSM保藏号15716保藏的脂环酸芽孢杆菌菌株分泌的选自酸性内切葡聚糖酶或酸性纤维素酶、谷氨酸肽酶、多铜氧化酶、丝氨酸羧基蛋白酶、丝氨酸蛋白酶或HtrA样丝氨酸蛋白酶、二硫化物异构酶、γ-D-谷氨酰-L-二氨基酸内肽酶、内-β-N-乙酰氨基葡糖苷酶、肽酰脯氨酰异构酶、酸性磷酸酯酶或植酸酶或磷脂酶C、多糖脱乙酰酶或木聚糖脱乙酰酶和亚硫酸盐氧化酶的成熟酶氨基酸序列具有至少90%同一性,和
(b)由在高严格度条件下与选自下述多核苷酸探针杂交的核苷酸序列所编码的酶,所述多核苷酸探针选自:
(i)以DSM保藏号15716保藏的脂环酸芽孢杆菌包含的核苷酸序列的互补链,所述核苷酸序列编码由该菌株分泌的选自酸性内切葡聚糖酶或酸性纤维素酶、谷氨酸肽酶、多铜氧化酶、丝氨酸羧基蛋白酶、丝氨酸蛋白酶或HtrA样丝氨酸蛋白酶、二硫化物异构酶、γ-D-谷氨酰-L-二氨基酸内肽酶、内-β-N-乙酰氨基葡糖苷酶、肽酰脯氨酰异构酶、酸性磷酸酯酶或植酸酶或磷脂酶C、多糖脱乙酰酶或木聚糖脱乙酰酶和亚硫酸盐氧化酶的成熟酶;
(ii)以DSM保藏号15716保藏的脂环酸芽孢杆菌包含的核苷酸序列包含的cDNA序列的互补链,所述核苷酸序列编码由该菌株分泌的选自酸性内切葡聚糖酶或酸性纤维素酶、谷氨酸肽酶、多铜氧化酶、丝氨酸羧基蛋白酶、丝氨酸蛋白酶或HtrA样丝氨酸蛋白酶、二硫化物异构酶、γ-D-谷氨酰-L-二氨基酸内肽酶、内-β-N-乙酰氨基葡糖苷酶、肽酰脯氨酰异构酶、酸性磷酸酯酶或植酸酶或磷脂酶C、多糖脱乙酰酶或木聚糖脱乙酰酶和亚硫酸盐氧化酶的成熟酶,
其中该酶具有选自酸性内切葡聚糖酶或酸性纤维素酶、谷氨酸肽酶、多铜氧化酶、丝氨酸羧基蛋白酶、丝氨酸蛋白酶或HtrA样丝氨酸蛋白酶、二硫化物异构酶、γ-D-谷氨酰-L-二氨基酸内肽酶、内-β-N-乙酰氨基葡糖苷酶、肽酰脯氨酰异构酶、酸性磷酸酯酶或植酸酶或磷脂酶C、多糖脱乙酰酶或木聚糖脱乙酰酶和亚硫酸盐氧化酶的功能。
在具体的实施方案中,该酶是选自以下的分离的酶:
(a)具有与选自SEQ ID NO:26到SEQ ID NO:40中所包含成熟酶的氨基酸序列具有至少90%同一性的氨基酸序列的酶,和
(b)在高严格度条件下与选自以下的多核苷酸探针杂交的核苷酸序列所编码的酶,所述多核苷酸探针选自:
(i)SEQ ID NO:1到SEQ ID NO:15编码成熟酶的区域的核苷酸序列互补链,
(ii)SEQ ID NO:1到SEQ ID NO:15编码成熟酶的区域的核苷酸序列中所包含的cDNA序列的互补链;
其中该酶具有SEQ ID NO:26到SEQ ID NO:40中包含的相应成熟多肽的功能。
本发明的多肽为分离的多肽,本发明的多肽制品优选包含以重量计最多90%的其天然结合的其他多肽材料(优选更低百分比的其他多肽材料,例如以重量计最多80%,以重量计最多60%,以重量计最多50%,以重量计最多40%,以重量计最多30%,以重量计最多20%,以重量计最多10%,以重量计最多9%,以重量计最多8%,以重量计最多6%,以重量计最多5%,以重量计最多4%,以重量计最多3%,以重量计最多2%,以重量计最多1%和以重量计最多1/2%)。因此,优选本发明分离的多肽至少92%纯度,即以重量计本发明多肽构成制品中存在的总多肽材料的至少92%,并优选更高的百分数,例如至少94%纯度,至少95%纯度,至少96%纯度,至少96%纯度,至少97%纯度,至少98%纯度,至少99%和最多99.5%纯度。具体而言,优选本发明多肽为“基本纯净的形式”,即多肽制品基本不含与之天然结合的其他多肽材料。这可通过例如用熟知的重组方法制备本发明多肽来实现。
本发明多肽可合成制造、天然产生或二者组合。在具体的实施方案中,本发明多肽可得自微生物例如原核细胞、古细菌细胞或真核细胞。还可通过遗传工程修饰细胞。
在具体的实施方案中,本发明多肽为在约10℃到约80℃范围内,特别是约20℃到60℃范围内的温度下显示最佳酶活性的酶。
在具体的实施方案中,本发明多肽为在高至100℃,特别是高至80℃,更特别高至60℃的温度下功能稳定的酶。
在具体的实施方案中,本发明多肽为表现选自SEQ ID NO:26到SEQID NO:50中所包含成熟酶的至少20%,特别是至少40%,例如至少50%,特别是至少60%,例如至少70%,更特别至少80%,例如至少90%,最特别至少95%,例如大约或至少100%酶活性的酶。
具体而言,分离的成熟功能性多肽与得自以保藏号DSM 15716保藏的脂环酸芽孢杆菌细菌的相应的分泌多肽具有至少90%同一性并显示相同的功能,且特别地,本发明的多肽包含、含有与选自SEQ ID NO:26到SEQID NO:50中所包含成熟多肽的多肽序列具有至少90%同一性的氨基酸序列或由该氨基酸序列组成。同一性百分比具体为至少95%,例如至少96%,例如至少97%,更特别至少98%,例如至少99%或甚至100%同一性。
在另一具体的实施方案中,同一性百分比为至少50%,特别是至少60%,特别是至少65%,特别是至少70%,特别是至少75%,特别是至少80%,甚至更特别至少85%同一性。
在具体的实施方案中,本发明多肽的氨基酸序列与SEQ ID NO:26到SEQ ID NO:50中所包含成熟多肽最多存在10个氨基酸(例如10个氨基酸)的差异,特别是最多5个氨基酸(例如5个氨基酸),例如最多4个氨基酸(例如4个氨基酸),例如最多3个氨基酸(例如3个氨基酸),特别是最多2个氨基酸(例如2个氨基酸),例如1个氨基酸的差异。
本发明多肽可以是分离自天然来源(如脂环酸芽孢杆菌DSM 15716菌株或另一野生型菌株)的野生型多肽,然而本发明还包括人工变体,其中例如通过在所述多肽上添加、替代和/或缺失一个或多个氨基酸突变本发明多肽,同时保留多肽功能和/或其他性质。因此,本发明多肽可以是人工变体,其中对含有SEQ ID NO:26和SEQ ID NO:50中所包含成熟多肽或由其组成的氨基酸序列进行至少一个氨基酸替换、缺失和/或插入。
本发明的多肽还包括本文所述氨基酸序列的功能性片段和编码本文所述氨基酸序列功能性片段的核酸,包括以DSM保藏号15716保藏的脂环酸芽孢杆菌菌株分泌的成熟酶片段,如本文所述,包括在DSM保藏号15716下保藏的脂环酸芽孢杆菌菌株分泌的选自酸性内切葡聚糖酶、酸性纤维素酶、谷氨酸肽酶、多铜氧化酶、丝氨酸羧基蛋白酶、丝氨酸蛋白酶、HtrA样丝氨酸蛋白酶、二硫化物异构酶、γ-D-谷氨酰-L-二氨基酸内肽酶、内-β-N-乙酰氨基葡糖苷酶、肽酰脯氨酰异构酶、酸性磷酸酯酶、植酸酶、磷脂酶C、多糖脱乙酰酶、木聚糖脱乙酰酶和亚硫酸盐氧化酶的酶片段。
可通过本领域公知的标准技术构建人工变体,通常接着进行筛选和/或鉴定。标准技术包括经典诱变,例如Gerhardt等(1994)所述通过UV辐射细胞或用化学诱变剂处理细胞;WO 97/07205所述的体内基因改组;Stemmer,(1994)或WO 95/17413所述的体外改组,如Eisenstadt E.等(1994)所述的随机诱变;Poulsen等(1991)所述的PCR技术;J.E.Ness等,NatureBiotechnology,卷17,893-896页(1999)所述的家族改组;Sambrook等(1989),Sambrook等,《Molecular Cloning,A Laboratory Manual》,ColdSpring Harbor,NY.所述的定向诱变。核苷酸替代概述可见于例如Ford等,1991,《Protein Expression and Purification 2》,95-107页。
这些标准遗传工程方法还可用于从编码本发明一个或多个亲本酶的基因制备变体核苷酸序列的变种文库、在适当的宿主细胞中表达酶变体和选择优选的变体。可使用本领域公知的一些技术(Reetz MT;Jaeger KE,《Biocatalysis-from Discovery to Application》,Fessner WD编,卷200,31-57页(1999);Stemmer,Nature,卷370,389-391页,1994;Zhao和Arnold,Proc.Natl.Acad.Sci.,USA,卷94,7997-8000页,1997或Yano等,Proc.Natl.Acad.Sci.,USA,卷95,5511-5515页,1998)建立变种文库。
在本发明具体的实施方案中,氨基酸变化(人工变体及野生型酶中)是次要的性质,即不显著影响蛋白质折叠和/或活性的保守氨基酸替代;一般1到约30个的小量缺失;氨基或羧基端的小量延伸,例如一个氨基端甲硫氨酸残基;上至约20-25个残基的小接头肽;或通过改变净电荷或另一功能促进纯化的小量延伸,例如多组氨酸束、抗原表位或结合结构域。
保守替代的实例在碱性氨基酸(精氨酸、赖氨酸和组氨酸)、酸性氨基酸(谷氨酸和天冬氨酸)、极性氨基酸(谷氨酰胺和天冬酰胺)、疏水性氨基酸(亮氨酸、异亮氨酸、缬氨酸和甲硫氨酸)、芳香族氨基酸(苯丙氨酸、色氨酸和酪氨酸)和小氨基酸(甘氨酸、丙氨酸、丝氨酸和苏氨酸)之内。通常不改变和/或损害蛋白质功能的氨基酸替代为本领域公知,并描述于例如H.Neurath和R.L.Hill,1979,《The Proteins》,AcademicPress,New York。最常出现的交换为丙氨酸/丝氨酸、缬氨酸/异亮氨酸、天冬氨酸/谷氨酸、苏氨酸/丝氨酸、丙氨酸/甘氨酸、丙氨酸/苏氨酸、丝氨酸/天冬酰胺、丙氨酸/缬氨酸、丝氨酸/甘氨酸、酪氨酸/苯丙氨酸、丙氨酸/脯氨酸、赖氨酸/精氨酸、天冬氨酸/天冬酰胺、亮氨酸/异亮氨酸、亮氨酸/缬氨酸、丙氨酸/谷氨酸和天冬氨酸/甘氨酸以及相反的交换。
在具体的实施方案中,氨基酸交换具有改变多肽理化性质的性质。例如优选进行改善酶热稳定性、改变底物特异性、改变最佳pH等的氨基酸变化。
具体而言,本发明多肽(特别是选自SEQ ID NO:26到SEQ ID NO:50中所包含的成熟多肽的多肽)中这类产生人工变体的替换、缺失和/或插入的数目为最多10,例如最多9,例如最多8,更优选最多7,例如最多6,例如最多5,最优选最多4,例如最多3,例如最多2,特别是最多1。
在具体的实施方案中,人工变体为与亲本酶相比在动物(包括人)中具有改变的(优选降低的)免疫原性,特别是变应原性的变体。本文中的术语“免疫原性”应理解为将人工变体施用(包括静脉、皮肤、皮下、口腔和气管内给药)于动物时能够引起改变的(特别是降低的)免疫应答。本文中的术语“免疫应答”是指施用人工变体引起动物体内免疫球蛋白例如IgE、IgG和IgM水平的改变或动物体内细胞因子水平的改变。定位蛋白质免疫原/抗原表位、制备具有改变免疫原性变体的方法和测定免疫应答的方法为本领域公知,并描述于例如WO 92/10755、WO 00/26230、WO00/26354和WO 01/31989。本文中的术语“变应原性”应理解为人工变体引起动物改变,特别是降低产生IgE的能力以及结合来自所述动物IgE的能力。特别是由对动物气管内施用多肽变体引起的变应原性是特别有意义的(亦称为呼吸变应原性)。
在另一实施方案中,本发明多肽为由至少在高严格度条件下,特别是非常高严格度条件下与选自以下的多核苷酸探针杂交的核苷酸序列编码的多肽:
(i)选自SEQ ID NO:1到SEQ ID NO:25编码成熟多肽的区域的核苷酸序列的互补链,
(ii)选自SEQ ID NO:1到SEQ ID NO:25编码成熟多肽的区域的核苷酸序列中所包含cDNA序列的互补链;
(iii)编码具有SEQ ID NO:26到SEQ ID NO:50中所包含相应成熟多肽的功能的分泌多肽的(i)或(ii)的片段
(J.Sambrook,E.F.Fritsch,和T.Maniatus,1989,《MolecularCloning,A Laboratory Manual》,第二版,Cold Spring Harbor,NewYork)。
具体而言,本发明的多肽由包含选自SEQ ID NO:1到SEQ ID NO:25编码成熟多肽区域的核苷酸序列或由于遗传密码简并性而与之不同的序列的多核苷酸编码。更具体地,本发明多肽由选自SEQ ID NO:1到SEQ IDNO:25编码成熟多肽区域的核苷酸序列或由于遗传密码简并性而与之不同的序列组成的多核苷酸编码。
SEQ ID NO:1到SEQ ID NO:25编码成熟多肽区域的核苷酸序列或其亚序列,以及SEQ ID NO:26到SEQ ID NO:50所包含的成熟多肽或其片段的氨基酸序列可用于设计根据本领域熟知的方法从不同种属菌株中鉴定并克隆编码本发明酶的DNA的多核苷酸探针。具体而言,这类探针可用于与目的属或种的基因组或cDNA杂交,随后进行标准Southern印迹以鉴定并分离其中相应的基因。这类探针可远短于整个序列,但是长度应至少为15个,优选至少25个,更优选至少35个核苷酸,例如长度至少为70个核苷酸。然而,多核苷酸探针长度优选为至少100个核苷酸。例如,多核苷酸探针长度可至少为200个核苷酸,长度至少为300个核苷酸,长度至少为400个核苷酸或长度至少为500个核苷酸。可使用更长的探针例如长度至少600个核苷酸,长度至少700个核苷酸,长度至少800个核苷酸或长度至少900个核苷酸长度的多核苷酸探针。DNA和RNA探针都可以使用。通常标记(例如用32P、3H、35S、生物素或抗生物素蛋白)探针以检测相应的基因。
因此,可从这些其他生物制备的基因组DNA或cDNA文库筛选与上述探针杂交并编码本发明酶的DNA。可通过琼脂糖或聚丙烯酰胺凝胶电泳或其他分离技术分离来自这些其他生物的基因组或其他DNA。可以将来自文库的DNA或分离的DNA转移并固定在硝酸纤维素或其他合适的载体材料上。为了鉴定克隆或DNA(所述DNA与选自SEQ ID NO:1到SEQ IDNO:25编码成熟多肽区域的核苷酸具有必须的同源性和/或同一性或与之同源和/或同一),在Southern印迹中使用带有固定的DNA的载体材料。
就本发明而言,杂交表明核苷酸序列在高到非常高严格度杂交条件下与标记的多核苷酸探针杂交,所述探针又与选自SEQ ID NO:1到SEQ IDNO:25编码成熟多肽的核苷酸序列杂交。可使用X射线胶片或本领域公知的其他方法检测在这些条件下与多核苷酸探针杂交的分子。本文使用术语“多核苷酸探针”时都应理解为这类探针包含至少15个核苷酸。
在一个值得注意的实施方案中,多核苷酸探针为选自SEQ ID NO:1到SEQ ID NO:25编码成熟多肽区域的核苷酸序列的互补链。
在另一个值得注意的实施方案中,多核苷酸探针为编码选自SEQ IDNO:26到SEQ ID NO:50酶的核苷酸序列的互补链。在另一值得注意的实施方案中,多核苷酸探针为选自SEQ ID NO:1到SEQ ID NO:25编码成熟多肽区域的核苷酸序列成熟多肽编码区的互补链。
对于长度至少为100个核苷酸的长探针,高到非常高严格度条件定义为42℃下根据标准Southern印迹操作在5×SSPE,1.0% SDS,5×Denhardt杂交溶液,100μg/ml剪切并变性的鲑精DNA中预杂交和杂交。优选地,至少100个核苷酸的长探针不含有多于1000个核苷酸。对至少100个核苷酸长度的长探针,载体材料最终用0.1×SSC,0.1%SDS在60℃(高严格度)洗涤三次,每次15分钟,特别是用0.1×SSC,0.1%SDS在68℃(非常高严格度)下洗涤三次,每次15分钟。
尽管并非特别优选,还可考虑使用较短的探针例如从约15个到99个核苷酸长度(如从约15到约70个核苷酸长度)的探针。对于这类短探针,严格条件定义为在比使用根据Bolton和McCarthy(1962,Proceedings ofthe National Academy of Sciences USA 48:1390)算法计算的Tm低5℃到10℃下,在0.9M NaCl、0.09M Tris-HCl pH 7.6、6mM EDTA、0.5%NP-40、1×Denhardt杂交溶液、1mM焦磷酸钠、1mM磷酸二氢钠、0.1mM ATP和0.2mg/ml酵母RNA中按照标准Southern印迹操作预杂交、杂交和杂交后洗涤。
对于约15个核苷酸到99个核苷酸长度的短探针,载体材料在6×SCC加0.1%SDS中洗涤一次(15分钟)并使用6×SSC在比计算的Tm低5℃到10℃下洗涤两次,每次15分钟。
SEQ ID NO:26酸性内切葡聚糖酶或酸性纤维素酶
在一具体的实施方案中,本发明多肽为酸性内切葡聚糖酶或酸性纤维素酶,所述内切葡聚糖酶或酸性纤维素酶包含与得自脂环酸芽孢杆菌特别是以DSM保藏号15716保藏的脂环酸芽孢杆菌菌株的酸性内切葡聚糖酶或酸性纤维素酶,更特别是SEQ ID NO:26包含的成熟酸性内切葡聚糖酶或酸性纤维素酶有至少90%,特别是至少95%,更特别至少96%,更特别至少97%,更特别至少98%,更特别至少99%或最特别100%同一性的氨基酸序列或由其组成。更具体地,成熟酸性内切葡聚糖酶或酸性纤维素酶包含SEQ ID NO:26位置25到959的序列或由其组成。本文中酸性内切葡聚糖酶定义为内水解(特别是在酸性条件下)纤维素、地衣淀粉、或谷物β-D-葡聚糖中1,4-β-D-糖苷键的酶。本文中酸性纤维素酶定义为内水解(特别是在酸性条件下)纤维素中1,4-β-D-糖苷键的酶。
SEQ ID NO:27谷氨酸肽酶
在一具体的实施方案中,本发明多肽为谷氨酸肽酶,所述谷氨酸肽酶包含与得自脂环酸芽孢杆菌特别是以DSM保藏号15716保藏的脂环酸芽孢杆菌菌株的谷氨酸肽酶,更特别是SEQ ID NO:27包含的成熟谷氨酸肽酶有至少90%,特别是至少95%,更特别至少96%,更特别至少97%,更特别至少98%,更特别至少99%或最特别100%同一性的氨基酸序列或由其组成。更具体地,成熟谷氨酸肽酶包含SEQ ID NO:27位置33到272的序列或由其组成。本文中谷氨酸肽酶定义为水解蛋白质或肽并包含保守活性位点残基Q和E的酶。
谷氨酸肽酶(PepG)(EC 3.4.23.19)先前归类为天冬氨酰蛋白酶(A4)但被MEROPS(http://merops.sanger.ac.uk/)重新归类,其公开了“作为Fujinaga,Cherney,Oyama,Oda & James(2004)出色文章The molecularstructure and catalytic mechanism of a novel carboxyl peptidase fromScytalidium lignicolum.PubMed的结果,我们目前认识到了第六种肽酶催化类型:谷氨酸肽酶。已知的谷氨酸肽酶均属于以前的A4家族,现在称为G1家族。”(Fujinaga M,Cherney MM,Oyama H,Oda K,James MN.;The molecular structure and catalytic mechanism of a novel carboxylpeptidase from Scy-talidium lignicolum;Proc.Natl.Acad.Sci.U.S.A.;101(10);3364-9页;Epub 01-Mar-2004;09-Mar-2004.)
SEQ ID NO:27多肽为谷氨酸肽酶还来自以下证实活性位点残基Q和E在SEQ ID NO:27中为保守的多序列比对。
CLUSTAL W(1.81)multiple sequence alignment
SWISSPROT_P24665 MKFSTILTGSLFATAALAAPLTEKRRA--RKEARAAGKRHS---NPPYIPGSDKEILK-L
TREMBL_Q9P8R1 MKFSIVAATALLAGSAVAAPGTALRQA--RAVKRAARTHGN---PVKYVEGPTN------
TREMBL_Q00551 MKYATVVAALLGANAALGARFTEKRRE--RNEARLARRSGSVRLPATNSEGVAIDAAESR
SWISSPROT_P15369 ------------------------------------------------------------
TREMBL_Q00550 MKYTAALAALVTLAAAAPTDGIIDIGDGVKLVPREPRAHTRLERLRTFRRGLMEGLESGE
TREMBL_Q8X1C5 ------------------------------------------------------------
SEQ ID NO.27 MNGTSVWKASGIAAASCLTAAALLAWP--HATSTLDASPAIFHAPRHALSPNTSPKPNSV
¤¤¤¤¤¤¤¤¤¤¤¤¤
SWISSPROT_P24665 NGTTNEEYSSNWAGAVLI----GDGYTKVTGEFTVPSVSAGSSGSSGYGGGYGYWKNKRQ
TREMBL_Q9P8R1 --KTDVSYSSNWAGAVLV----GTGYTSVTGTFTAPSPSTAGSGS---------------
TREMBL_Q00551 NDTTNVEYSSNWAGAVLI----GSGYKSVTGIFVVPTPKSPGSGN---------------
SWISSPROT_P15369 ------TVESNWGGAILI----SGDFDTVSATANVPSATGASGGSS--------------
TREMBL_Q00550 RNSSDVSYDSNWAGAVKI----GTGLNDVTGTIVVPTPSVPSGGSST-------------
TREMBL_Q8X1C5 ----------NWAGAVLTSPPSGSTFTSVSAQFTVPSPSLPQGSQQ--------------
SEQ ID NO.27 QAQNFGWSASNWSGYAVT----GSTYNDITGSWIVPAVSPSKRSTYS-------------
**.* * ::. .*: . .
TREMBL_Q9P8R1
SWISSPROT_P15369
TREMBL_Q00550
TREMBL_QBX1C5
SEQ ID NO.27
:*:**** . ::* * * : :*** :* * ::. ::. *
TREMBL_Q00551
SWISSPROT_P15369
TREMBL_Q8X1C5
* : . : .: : * . .. : : : .**:* *
SWISSPROT_P24665 ---LVAFADFG-SVTFTNAEATSGGSTVGPSDATVMDIEQDGSVLTETSVSG-DSVTVTY
TREMBL_Q9PBR1 ---LVQFANFG-TVTFTGASATQNGESVGVTGAQIIDLQQN-SVLTSVSTSS-NSVTVKY
TREMBL_Q00551 ---LVPFANFG-TVTFTGAEATTSSGTVTAADATLIDIEQNGEVLTSVTVSG-STVTVKY
SWISSPROT_P15369 SDEFVPFASFSPAVEFTDCSVTSDGESVSLDDAQITQVIINNQDVTDCSVSG-TTVSCSY
TREMBL_Q00550 --- ---LVNFADFD-TVTFKDCSPSVSG-------STIVDIRQSLEVLTECSTTGTTTVTCEY
TREMBL_Q8X1C5 ------------------------------------------------------------
SEQ ID NO.27 ---IATLANYG-ETTFDPGTVNGGNPGFTLSDAGYMVQNNAVVSVPSAPDSDTDGFNVAY
SWISSPROT_P24665 V---------
TREMBL_Q9P8R1 V---------
TREMBL_Q00551 V---------
SWISSPROT_P15369 V---------
TREMBL_Q00550 VG--------
TREMBL_Q8X1C5 ----------
SEQ ID NO.27 GSNQPSPPAS
SWISSPROTP24665
黑曲霉(Aspergillus niger)ASPERGILLOPEPSIN II;SEQ ID NO:55
TREMBL_Q9P8R1
核盘菌(Sclerotinia sclerotiorum)内肽酶EapC;SEQ ID NO:56
TREMBLQ00551
(Cryphonectria parasitica)内肽酶EapC;SEQ ID NO:57
SWISSPROTP15369
(Scytalidium lignicolum)scytalidoglutamic肽酶;SEQ ID NO:58
TREMBL-Q00550
(Cryphonectria parasitica)内肽酶EapB;SEQ ID NO:59
TREMBL_Q8X1C5
(Talaromyces emersonii)胃蛋白酶抑制品不敏感酸性蛋白酶(片段);SEQ ID NO:60
SEQ ID NO:27
本发明序列
o=形成Swissprot P24665活性位点的氨基酸
/=形成Swissprot P24665二硫键的半胱氨酸残基
¤=从Swissprot P24665酶原去除的前肽
因此,本发明者鉴定并分离了已知的第一个来自细菌的(G1)谷氨酸肽酶,特别是在低pH和高温下有活性的谷氨酸肽酶。最近亲缘为真菌G1蛋白酶(例如Aspergillopepsin II)。
另外,令人惊奇的是该谷氨酸肽酶由于分子中缺乏二硫键而与大多数已知的真菌谷氨酸肽酶不同。与例如公开了由两个通过二硫键交联的肽组成的已知真菌谷氨酸肽酶的SEQ ID NO:55相比,SEQ ID NO:27包含的谷氨酸肽酶仅含有一个半胱氨酸,因此蛋白酶结构中不含二硫键。因此,脂环酸芽孢杆菌特别是以DSM保藏号15716保藏的脂环酸芽孢杆菌谷氨酸肽酶缺少第二前肽,因此其产生少需要一个成熟步骤。这对细胞制备是有益的。
SEQ ID NO:28或SEQ ID NO:35多铜氧化酶
在一具体的实施方案中,本发明多肽为多铜氧化酶,所述多铜氧化酶包含与得自脂环酸芽孢杆菌特别是以DSM保藏号15716保藏的脂环酸芽孢杆菌菌株的多铜氧化酶,更特别是SEQ ID NO:28或35包含的成熟多铜氧化酶有至少90%,特别是至少95%,更特别至少96%,更特别至少97%,更特别至少98%,更特别至少99%或最特别100%同一性的氨基酸序列或由其组成。更具体地,成熟多铜氧化酶包含SEQ ID NO:28位置26到315或SEQ ID NO:35位置50到597的序列或由之组成。本文中多铜氧化酶定义为至少具有三个光谱差异铜中心的蛋白质。多铜氧化酶可以是氧化多种不同类型酚和二胺的漆酶、抗坏血酸氧化酶、氧化多种无机和有机物质的血浆铜蓝蛋白或失去结合铜能力从而通过细菌周质中重金属螯合介导重金属抗性的蛋白质部分。
SEQ ID NO:29或SEQ ID NO:30丝氨酸羧基蛋白酶
在一具体的实施方案中,本发明酶为丝氨酸羧基蛋白酶,所述丝氨酸羧基蛋白酶包含与得自脂环酸芽孢杆菌,特别是以DSM保藏号15716保藏的脂环酸芽孢杆菌菌株的丝氨酸羧基蛋白酶,更特别是SEQ ID NO:29或30包含的成熟丝氨酸羧基蛋白酶有至少90%,特别是至少95%,更特别至少96%,更特别至少97%,更特别至少98%,更特别至少99%或最特别100%同一性的氨基酸序列或由其组成。更具体地,成熟丝氨酸羧基蛋白酶包含SEQ ID NO:29位置190到626或SEQ ID NO:30位置25到533的序列或由其组成。本文中丝氨酸羧基蛋白酶定义为属于EC 3.4.21.100(pseudomonapepsin)类酶的蛋白酶,所述水解酶折叠类似枯草菌素的折叠,具有独特的丝氨酸-谷氨酸-天冬氨酸催化三联体以及氧阴离子洞中存在天冬氨酸残基。如果催化位点氨基酸存在于序列中且其显示与MEROPS丝氨酸蛋白酶家族53肽序列相似的肽序列,则该多肽序列可归类于丝氨酸羧基肽酶。
SEQ ID NO:31丝氨酸蛋白酶或HtrA样丝氨酸蛋白酶
在一具体的实施方案中,本发明多肽为丝氨酸蛋白酶或HtrA样丝氨酸蛋白酶,所述丝氨酸蛋白酶或HtrA样丝氨酸蛋白酶包含与得自脂环酸芽孢杆菌,特别是以DSM保藏号15716保藏的脂环酸芽孢杆菌菌株的丝氨酸蛋白酶或HtrA样丝氨酸蛋白酶,更特别是SEQ ID NO:31包含的成熟丝氨酸蛋白酶羧基蛋白酶有至少90%,特别是至少95%,更特别至少96%,更特别至少97%,更特别至少98%,更特别至少99%或最特别100%同一性的氨基酸序列或由其组成。更具体地,成熟丝氨酸蛋白酶包含SEQID NO:31位置42到411的序列或由其组成。本文中丝氨基蛋白酶定义为水解蛋白质或肽且催化位点包含丝氨酸残基的酶。HtrA样蛋白酶定义为在提高的温度下降解细菌细胞细胞外区室中受损蛋白质的酶。
SEQ ID NO:32二硫化物异构酶
在一具体的实施方案中,本发明多肽为二硫化物异构酶,所述二硫化物异构酶包含与得自脂环酸芽孢杆菌,特别是在DSM保藏号15716下保藏的脂环酸芽孢杆菌菌株的二硫化物异构酶,更特别是SEQ ID NO:32包含的成熟二硫化物异构酶有至少90%,特别是至少95%,更特别至少96%,更特别至少97%,更特别至少98%,更特别至少99%或最特别100%同一性的氨基酸序列或由其组成。更具体地,成熟二硫化物异构酶包含SEQ IDNO:32位置31到212的序列或由其组成。本文中二硫化物异构酶定义为催化蛋白质链内和链间二硫键重排以形成天然结构的酶。
SEQ ID NO:33γ-D-谷氨酰-L-二氨基酸内肽酶
在一具体的实施方案中,本发明多肽为γ-D-谷氨酰-L-二氨基酸内肽酶,所述γ-D-谷氨酰-L-二氨基酸内肽酶包含与得自脂环酸芽孢杆菌,特别是在DSM保藏号15716下保藏的脂环酸芽孢杆菌菌株的γ-D-谷氨酰-L-二氨基酸内肽酶,更特别是SEQ ID NO:33包含的成熟γ-D-谷氨酰-L-二氨基酸内肽酶有至少90%,特别是至少95%,更特别至少96%,更特别至少97%,更特别至少98%,更特别至少99%或最特别100%同一性的氨基酸序列或由其组成。更具体地,成熟γ-D-谷氨酰-L-二氨基酸内肽酶包含SEQ ID NO:33位置30到266序列或由其组成。本文中γ-D-谷氨酰-L-二氨基酸内肽酶定义为水解L-丙氨酸-γ-D-谷氨酰-l-(L)内消旋二胺基庚二酸-(L)-D-丙氨酸中与(L)内消旋二胺基庚二酸结合的γ-D-谷氨酰的酶。需要(L)内消旋二胺基庚二酸基团的ω氨基和ω羧基是未替代的。
SEQ ID NO:34内-β-N-乙酰氨基葡糖苷酶
在具体的实施方案中,本发明多肽为内-β-N-乙酰氨基葡糖苷酶,所述内-β-N-乙酰氨基葡糖苷酶包含与得自脂环酸芽孢杆菌,特别是在DSM保藏号15716下保藏的脂环酸芽孢杆菌菌株的内-β-N-乙酰氨基葡糖苷酶,更特别是SEQ ID NO:34包含的成熟内-β-N-乙酰氨基葡糖苷酶有至少90%,特别是至少95%,更特别至少96%,更特别至少97%,更特别至少98%,更特别至少99%或最特别100%同一性的氨基酸序列或由其组成。更具体地,成熟内-β-N-乙酰氨基葡糖苷酶包含SEQ ID NO:34位置27到768的序列或由其组成。本文中内-β-N-乙酰氨基葡糖苷酶定义为水解原核细胞壁肽聚糖杂聚物中N-乙酰-D-葡糖胺和N-乙酰胞壁酸间1,4-β-键的酶。
SEQ ID NO:36肽基脯氨酰异构酶
在一具体的实施方案中,本发明多肽为肽基脯氨酰异构酶,所述肽基脯氨酰异构酶包含与得自脂环酸芽孢杆菌,特别是在DSM保藏号15716下保藏的脂环酸芽孢杆菌菌株的肽基脯氨酰异构酶,更特别是SEQ ID NO:36包含的成熟肽基脯氨酰异构酶有至少90%,特别是至少95%,更特别至少96%,更特别至少97%,更特别至少98%,更特别至少99%或最特别100%同一性的氨基酸序列或由其组成。更具体地,成熟肽基脯氨酰异构酶包含SEQ ID NO:36位置30到246的序列或由其组成。本文中肽基脯氨酰异构酶定义为通过催化寡肽中脯氨酸亚胺肽键顺反异构化加速蛋白质折叠的酶。
SEQ ID NO:37酸性磷酸酯酶或植酸酶或磷脂酶C
在一具体的实施方案中,本发明多肽为酸性磷酸酯酶或植酸酶或磷脂酶C,所述酸性磷酸酯酶或植酸酶或磷脂酶C包含与得自脂环酸芽孢杆菌,特别是在DSM保藏号15716下保藏的脂环酸芽孢杆菌菌株的酸性磷酸酯酶或植酸酶或磷脂酶C,更特别是SEQ ID NO:37包含的成熟酸性磷酸酯酶或植酸酶或磷脂酶C有至少90%,特别是至少95%,更特别至少96%,更特别至少97%,更特别至少98%,更特别至少99%或最特别100%同一性的氨基酸序列或由其组成。更具体地,成熟酸性磷酸酯酶或植酸酶或磷脂酶C包含SEQ ID NO:37位置28到608序列或由其组成。酸性磷酸酯酶定义为将正磷酸单酯水解为醇和磷酸的酶。本文中植酸酶定义为从肌醇六磷酸上除去磷酸基团的酶。磷脂酶C定义为将磷酸卵磷脂水解为1,2-二酰基甘油和胆碱的酶。
SEQ ID NO:38或SEQ ID NO:39多糖脱乙酰酶
在一具体的实施方案中,本发明多肽为多糖脱乙酰酶或木聚糖脱乙酰酶,所述多糖脱乙酰酶或木聚糖脱乙酰酶包含与得自脂环酸芽孢杆菌,特别是在DSM保藏号15716下保藏的脂环酸芽孢杆菌菌株的多糖脱乙酰酶或木聚糖脱乙酰酶,更特别是SEQ ID NO:33或39包含的成熟多糖脱乙酰酶或木聚糖脱乙酰酶有至少90%,特别是至少95%,更特别至少96%,更特别至少97%,更特别至少98%,更特别至少99%或最特别100%同一性的氨基酸序列或由其组成。更具体地,成熟多糖脱乙酰酶或木聚糖脱乙酰酶包含SEQ ID NO:38位置26到251或SEQ ID NO:39位置22到324的序列或由其组成。本文中多糖脱乙酰酶定义为通过水解从特异的乙酰化多糖上去除乙酰残基的酶。木聚糖脱乙酰酶定义为从乙酰化木聚糖上去除乙酰基团的酶。
SEQ ID NO:40亚硫酸盐氧化酶
在一具体的实施方案中,本发明多肽为亚硫酸盐氧化酶,所述亚硫酸盐氧化酶包含与得自脂环酸芽孢杆菌,特别是在DSM保藏号15716下保藏的脂环酸芽孢杆菌菌株的亚硫酸盐氧化酶,更特别是SEQ ID NO:40包含的成熟亚硫酸盐氧化酶有至少90%,特别是至少95%,更特别至少96%,更特别至少97%,更特别至少98%,更特别至少99%或最特别100%同一性的氨基酸序列或由其组成。更具体地,成熟亚硫酸盐氧化酶包含SEQ IDNO:40位置30到214的序列或由其组成。亚硫酸盐氧化酶定义为将亚硫酸氧化为硫酸的酶。
SEQ ID NO:41功能性多肽
在一具体的实施方案中,本发明多肽为功能性多肽,所述功能性多肽包含与SEQ ID NO:41,特别是SEQ ID NO:41包含的成熟功能性多肽有至少90%,特别是至少95%,更特别至少96%,更特别至少97%,更特别至少98%,更特别至少99%或最特别100%同一性的氨基酸序列或由其组成。更具体地,成熟功能性多肽包含SEQ ID NO:41位置22到257的序列或由其组成。
SEQ ID NO:42功能性多肽
在一具体的实施方案中,本发明多肽为功能性多肽,所述功能性多肽包含与SEQ ID NO:42,特别是SEQ ID NO:42包含的成熟功能性多肽有至少90%,特别是至少95%,更特别至少96%,更特别至少97%,更特别至少98%,更特别至少99%或最特别100%同一性的氨基酸序列或由其组成。更具体地,成熟功能性多肽包含SEQ ID NO:42位置25到1130的序列或由其组成。
SEQ ID NO:43功能性多肽
在一具体的实施方案中,本发明多肽功能性多肽,所述功能性多肽为包含与SEQ ID NO:43,特别是SEQ ID NO:43包含的成熟功能性多肽有至少90%,特别是至少95%,更特别至少96%,更特别至少97%,更特别至少98%,更特别至少99%或最特别100%同一性的氨基酸序列或由其组成。更具体地,成熟功能性多肽包含SEQ ID NO:43位置42到248的序列或由其组成。
SEQ ID NO:44功能性多肽
在一具体的实施方案中,本发明多肽为功能性多肽,所述功能性多肽包含与SEQ ID NO:44,特别是SEQ ID NO:44包含的成熟功能性多肽有至少90%,特别是至少95%,更特别至少96%,更特别至少97%,更特别至少98%,更特别至少99%或最特别100%同一性的氨基酸序列或由其组成。更具体地,成熟功能性多肽包含SEQ ID NO:44位置26到172的序列或由其组成。
SEQ ID NO:45功能性多肽
在一具体的实施方案中,本发明多肽为功能性多肽,所述功能性多肽包含与SEQ ID NO:45,特别是SEQ ID NO:45包含的成熟功能性多肽有至少90%,特别是至少95%,更特别至少96%,更特别至少97%,更特别至少98%,更特别至少99%或最特别100%同一性的氨基酸序列或由其组成。更具体地,成熟功能性多肽包含SEQ ID NO:45位置31到242的序列或由其组成。
SEQ ID NO:46功能性多肽
在一具体的实施方案中,本发明多肽为功能性多肽,所述功能性多肽包含与SEQ ID NO:46,特别是SEQ ID NO:64包含的成熟功能性多肽有至少90%,特别是至少95%,更特别至少96%,更特别至少97%,更特别至少98%,更特别至少99%或最特别100%同一性的氨基酸序列或由其组成。更具体地,成熟功能性多肽包含SEQ ID NO:46位置25到280的序列或由其组成。
SEQ ID NO:47功能性多肽
在一具体的实施方案中,本发明多肽为功能性多肽,所述功能性多肽包含与SEQ ID NO:47,特别是SEQ ID NO:47包含的成熟功能性多肽有至少90%,特别是至少95%,更特别至少96%,更特别至少97%,更特别至少98%,更特别至少99%或最特别100%同一性的氨基酸序列或由其组成。更具体地,成熟功能性多肽包含SEQ ID NO:47位置26到478的序列或由其组成。
SEQ ID NO:48功能性多肽
在一具体的实施方案中,本发明多肽为功能性多肽,所述功能性多肽包含与SEQ ID NO:48,特别是SEQ ID NO:48包含的成熟功能性多肽有至少90%,特别是至少95%,更特别至少96%,更特别至少97%,更特别至少98%,更特别至少99%或最特别100%同一性的氨基酸序列或由其组成。更具体地,成熟功能性多肽包含SEQ ID NO:48位置20到340的序列或由其组成。
SEQ ID NO:49功能性多肽
在一具体的实施方案中,本发明多肽为功能性多肽,所述功能性多肽包含与SEQ ID NO:49,特别是SEQ ID NO:49包含的成熟功能性多肽有至少90%,特别是至少95%,更特别至少96%,更特别至少97%,更特别至少98%,更特别至少99%或最特别100%同一性的氨基酸序列或由其组成。更具体地,成熟功能性多肽包含SEQ ID NO:49位置30到341的序列或由其组成。
SEQ ID NO:50功能性多肽
在一具体的实施方案中,本发明多肽为功能性多肽,所述功能性多肽包含与SEQ ID NO:50,特别是SEQ ID NO:50包含的成熟功能性多肽有至少90%,特别是至少95%,更特别至少96%,更特别至少97%,更特别至少98%,更特别至少99%或最特别100%同一性的氨基酸序列或由其组成。更具体地,成熟功能性多肽包含SEQ ID NO:50位置29到400的序列或由其组成。
多核苷酸
本发明还涉及多核苷酸,特别是包含编码本发明多肽的核苷酸序列或由其组成的分离多核苷酸。在一具体的实施方案中,核苷酸序列在SEQ IDNO:1到SEQ ID NO:25中公开,包括由于遗传密码简并性而与之存在差异的核苷酸序列。在另一实施方案中,本发明多核苷酸为包含选自SEQ IDNO:1到SEQ ID NO:25编码成熟多肽区域的核苷酸序列或由其组成的修饰核苷酸序列,所述修饰核苷酸序列与SEQ ID NO:1到SEQ ID NO:25包含的亲本核苷酸序列相比包含至少一个修饰/突变。
用于分离和/或克隆编码酶的核苷酸序列的技术为本领域公知,包括从基因组DNA中分离、由cDNA制备或它们的组合。可通过例如使用熟知的聚合酶链式反应(PCR)或抗体筛选表达文库检测有共享结构特征的克隆DNA片段来从这些基因组DNA中克隆本发明的核苷酸序列。参阅如Innis等,1990,《PCR:A Guide to Methods and Application》,AcademicPress,New York。可使用其他扩增操作例如连接酶链反应(LCR)、连接激活转录(LAT)和基于核苷酸序列的扩增(NASBA)。
可通过基因工程中使用的将核苷酸序列从其天然位置重新定位于其再产生的不同位点的标准克隆方法获得核苷酸序列。克隆方法可包括切除并分离包含编码多肽的核苷酸序列的所需片段、将片段插入载体分子和将重组体载体整合进核苷酸序列的多拷贝或克隆在其中复制的宿主细胞。核苷酸序列可以是基因组、cDNA、RNA、半合成的、合成来源的或其任意组合。
具体的多核苷酸包含与选自SEQ ID NO:1到SEQ ID NO:25编码成熟多肽区域的核苷酸序列有至少50%同一性的核苷酸序列,优选由其组成。具体而言,核苷酸序列与选自SEQ ID NO:1到SEQ ID NO:25编码成熟多肽区域的核苷酸序列有至少65%同一性,更特别至少70%同一性,更特别至少80%同一性,更特别至少90%同一性,更特别至少95%同一性,更特别至少96%同一性,更特别至少97%同一性,更特别至少98%同一性,更特别至少99%同一性或最特别100%同一性。特别的,核苷酸序列包含选自SEQ ID NO:1到SEQ ID NO:25编码成熟多肽区域的核苷酸序列。在更特别的实施方案中,核苷酸序列由选自SEQ ID NO:1到SEQID NO:25编码成熟多肽区域的核苷酸序列组成。
具体而言,多核苷酸包括编码选自以下的成熟酶的核苷酸序列:酸性内切葡聚糖酶或酸性纤维素酶、谷氨酸肽酶、多铜氧化酶、丝氨酸羧基蛋白酶、丝氨酸蛋白酶或HtrA样丝氨酸蛋白酶、二硫化物异构酶、γ-D-谷氨酰-L-二氨基酸内肽酶、内-β-N-乙酰氨基葡糖苷酶、肽酰脯氨酰异构酶、酸性磷酸酯酶或植酸酶或磷脂酶C、多糖脱乙酰酶或木聚糖脱乙酰酶和亚硫酸盐氧化酶,或与编码选自以DSM保藏号15716保藏的脂环酸芽孢杆菌菌株所分泌的酸性内切葡聚糖酶或酸性纤维素酶、谷氨酸肽酶、多铜氧化酶、丝氨酸羧基蛋白酶、丝氨酸蛋白酶或HtrA样丝氨酸蛋白酶、二硫化物异构酶、γ-D-谷氨酰-L-二氨基酸内肽酶、内-β-N-乙酰氨基葡糖苷酶、肽酰脯氨酰异构酶、酸性磷酸酯酶或植酸酶或磷脂酶C、多糖脱乙酰酶或木聚糖脱乙酰酶和亚硫酸盐氧化酶的成熟酶的核苷酸序列有至少50%同一性,特别的至少65%同一性,更特别至少70%同一性,更特别至少80%同一性,更特别至少90%同一性,更特别至少95%同一性,更特别至少96%同一性,更特别至少97%同一性,更特别至少98%同一性,更特别至少99%同一性或最特别100%同一性的核苷酸序列,优选由其组成。
SEQ ID NO:1
在一具体的实施方案中,本发明的多核苷酸编码酸性内切葡聚糖酶或酸性纤维素酶,并包含与SEQ ID NO:1位置73到2877的核苷酸序列有至少70%同一性,更特别至少80%同一性,更特别至少90%同一性,更特别至少95%同一性,更特别至少96%同一性,更特别至少97%同一性,更特别至少98%同一性,更特别至少99%同一性或最特别100%同一性的核苷酸序列或由其组成。
SEQ ID NO:2
在一具体的实施方案中,本发明的多核苷酸编码谷氨酸肽酶,并包含与SEQ ID NO:2位置97到816的核苷酸序列有至少70%同一性,更特别至少80%同一性,更特别至少90%同一性,更特别至少95%同一性,更特别至少96%同一性,更特别至少97%同一性,更特别至少98%同一性,更特别至少99%同一性或最特别100%同一性的核苷酸序列或由其组成。
SEQ ID NO:3和10
在一具体的实施方案中,本发明的多核苷酸编码多铜氧化酶,并包含与SEQ ID NO:1位置76到945或SEQ ID NO:10位置148到1791的核苷酸序列有至少70%同一性,更特别至少80%同一性,更特别至少90%同一性,更特别至少95%同一性,更特别至少96%同一性,更特别至少97%同一性,更特别至少98%同一性,更特别至少99%同一性或最特别100%同一性的核苷酸序列或由其组成。
SEQ ID NO:4和5
在一具体的实施方案中,本发明的多核苷酸编码丝氨酸羧基蛋白酶,并包含与SEQ ID NO:4位置568到1878或SEQ ID NO:5位置73到1599的核苷酸序列有至少70%同一性,更特别至少80%同一性,更特别至少90%同一性,更特别至少95%同一性,更特别至少96%同一性,更特别至少97%同一性,更特别至少98%同一性,更特别至少99%同一性或最特别100%同一性的核苷酸序列或由其组成。
SEQ ID NO:6
在一具体的实施方案中,本发明的多核苷酸编码丝氨酸蛋白酶或HtrA样丝氨酸蛋白酶,并包含与SEQ ID NO:6位置124到1233的核苷酸序列有至少70%同一性,更特别至少80%同一性,更特别至少90%同一性,更特别至少95%同一性,更特别至少96%同一性,更特别至少97%同一性,更特别至少98%同一性,更特别至少99%同一性或最特别100%同一性的核苷酸序列或由其组成。
SEQ ID NO:7
在一具体的实施方案中,本发明的多核苷酸编码二硫化物异构酶,并包含与SEQ ID NO:7位置91到633的核苷酸序列有至少70%同一性,更特别至少80%同一性,更特别至少90%同一性,更特别至少95%同一性,更特别至少96%同一性,更特别至少97%同一性,更特别至少98%同一性,更特别至少99%同一性或最特别100%同一性的核苷酸序列或由其组成。
SEQ ID NO:8
在一具体的实施方案中,本发明的多核苷酸编码γ-D-谷氨酰-L-二氨基酸内肽酶,并包含与SEQ ID NO:8位置88到798的核苷酸序列有至少70%同一性,更特别至少80%同一性,更特别至少90%同一性,更特别至少95%同一性,更特别至少96%同一性,更特别至少97%同一性,更特别至少98%同一性,更特别至少99%同一性或最特别100%同一性的核苷酸序列或由其组成。
SEQ ID NO:9
在一具体的实施方案中,本发明的多核苷酸编码内-β-N-乙酰氨基葡糖苷酶,并包含与SEQ ID NO:9位置79到2304的核苷酸序列有至少70%同一性,更特别至少80%同一性,更特别至少90%同一性,更特别至少95%同一性,更特别至少96%同一性,更特别至少97%同一性,更特别至少98%同一性,更特别至少99%同一性或最特别100%同一性的核苷酸序列或由其组成。
SEQ ID NO:11
在一具体的实施方案中,本发明的多核苷酸编码肽酰脯氨酰异构酶,并包含与SEQ ID NO:9位置88到735的核苷酸序列有至少70%同一性,更特别至少80%同一性,更特别至少90%同一性,更特别至少95%同一性,更特别至少96%同一性,更特别至少97%同一性,更特别至少98%同一性,更特别至少99%同一性或最特别100%同一性的核苷酸序列或由其组成。
SEQ ID NO:12
在一具体的实施方案中,本发明的多核苷酸编码酸性磷酸酯酶或植酸酶或磷脂酶C,并包含与SEQ ID NO:12位置82到1824的核苷酸序列有至少70%同一性,更特别至少80%同一性,更特别至少90%同一性,更特别至少95%同一性,更特别至少96%同一性,更特别至少97%同一性,更特别至少98%同一性,更特别至少99%同一性或最特别100%同一性的核苷酸序列或由其组成。
SEQ ID NO:13和14
在一具体的实施方案中,本发明的多核苷酸编码多糖脱乙酰酶或木聚糖脱乙酰酶,并包含与SEQ ID NO:13位置76到750或SEQ ID NO:14位置64到972的核苷酸序列有至少70%同一性,更特别至少80%同一性,更特别至少90%同一性,更特别至少95%同一性,更特别至少96%同一性,更特别至少97%同一性,更特别至少98%同一性,更特别至少99%同一性或最特别100%同一性的核苷酸序列或由其组成。
SEQ ID NO:15
在一具体的实施方案中,本发明的多核苷酸编码亚硫酸盐氧化酶,并包含与SEQ ID NO:15位置88到642的核苷酸序列有至少70%同一性,更特别至少80%同一性,更特别至少90%同一性,更特别至少95%同一性,更特别至少96%同一性,更特别至少97%同一性,更特别至少98%同一性,更特别至少99%同一性或最特别100%同一性的核苷酸序列或由其组成。
SEQ ID NO:16
在一具体实施方案中,本发明的多核苷酸编码成熟功能性多肽,并包含与SEQ ID NO:16位置64到771的核苷酸序列具有至少70%同一性,更特别至少80%同一性,更特别至少90%同一性,更特别至少95%同一性,更特别至少96%同一性,更特别至少97%同一性,更特别至少98%同一性,更特别至少99%同一性或最特别100%同一性的核苷酸序列或由其组成。
SEQ ID NO:17
在一具体实施方案中,本发明的多核苷酸编码成熟功能性多肽,并包含与SEQ ID NO:17位置73到3390的核苷酸序列具有至少70%同一性,更特别至少80%同一性,更特别至少90%同一性,更特别至少95%同一性,更特别至少96%同一性,更特别至少97%同一性,更特别至少98%同一性,更特别至少99%同一性或最特别100%同一性的核苷酸序列或由其组成。
SEQ ID NO:18
在一具体实施方案中,本发明的多核苷酸编码成熟功能性多肽,并包含与SEQ ID NO:18位置124到744的核苷酸序列具有至少70%同一性,更特别至少80%同一性,更特别至少90%同一性,更特别至少95%同一性,更特别至少96%同一性,更特别至少97%同一性,更特别至少98%同一性,更特别至少99%同一性或最特别100%同一性的核苷酸序列或由其组成。
SEQ ID NO:19
在一具体实施方案中,本发明的多核苷酸编码成熟功能性多肽,并包含与SEQ ID NO:19位置76到516的核苷酸序列具有至少70%同一性,更特别至少80%同一性,更特别至少90%同一性,更特别至少95%同一性,更特别至少96%同一性,更特别至少97%同一性,更特别至少98%同一性,更特别至少99%同一性或最特别100%同一性的核苷酸序列或由其组成。
SEQ ID NO:20
在一具体实施方案中,本发明的多核苷酸编码成熟功能性多肽,并包含与SEQ ID NO:20位置91到726的核苷酸序列具有至少70%同一性,更特别至少80%同一性,更特别至少90%同一性,更特别至少95%同一性,更特别至少96%同一性,更特别至少97%同一性,更特别至少98%同一性,更特别至少99%同一性或最特别100%同一性的核苷酸序列或由其组成。
SEQ ID NO:21
在一具体实施方案中,本发明的多核苷酸编码成熟功能性多肽,并包含与SEQ ID NO:21位置73到540的核苷酸序列具有至少70%同一性,更特别至少80%同一性,更特别至少90%同一性,更特别至少95%同一性,更特别至少96%同一性,更特别至少97%同一性,更特别至少98%同一性,更特别至少99%同一性或最特别100%同一性的核苷酸序列或由其组成。
SEQ ID NO:22
在一具体实施方案中,本发明的多核苷酸编码成熟功能性多肽,并包含与SEQ ID NO:22位置76到1431的核苷酸序列具有至少70%同一性,更特别至少80%同一性,更特别至少90%同一性,更特别至少95%同一性,更特别至少96%同一性,更特别至少97%同一性,更特别至少98%同一性,更特别至少99%同一性或最特别100%同一性的核苷酸序列或由其组成。
SEQ ID NO:23
在一具体实施方案中,本发明的多核苷酸编码成熟功能性多肽,并包含与SEQ ID NO:23位置58到1020的核苷酸序列具有至少70%同一性,更特别至少80%同一性,更特别至少90%同一性,更特别至少95%同一性,更特别至少96%同一性,更特别至少97%同一性,更特别至少98%同一性,更特别至少99%同一性或最特别100%同一性的核苷酸序列或由其组成。
SEQ ID NO:24
在一具体实施方案中,本发明的多核苷酸编码成熟功能性多肽,并包含与SEQ ID NO:24位置88到1023的核苷酸序列具有至少70%同一性,更特别至少80%同一性,更特别至少90%同一性,更特别至少95%同一性,更特别至少96%同一性,更特别至少97%同一性,更特别至少98%同一性,更特别至少99%同一性或最特别100%同一性的核苷酸序列或由其组成。
SEQ ID NO:25
在一具体实施方案中,本发明的多核苷酸编码成熟功能性多肽,并包含与SEQ ID NO:25位置85到1197的核苷酸序列具有至少70%同一性,更特别至少80%同一性,更特别至少90%同一性,更特别至少95%同一性,更特别至少96%同一性,更特别至少97%同一性,更特别至少98%同一性,更特别至少99%同一性或最特别100%同一性的核苷酸序列或由其组成。
修饰编码本发明多肽的核苷酸序列对于合成多肽可能是必需的,所述多肽包含与选自SEQ ID NO:26到SEQ ID NO:50包含的成熟多肽的氨基酸序列相比至少有一个替代、缺失和/或插入的氨基酸序列。
对本领域技术人员显而易见的是,为保持酶的功能可制造这样的修饰,即修饰在对酶功能至关重要的区域外进行。因此对功能必需的氨基酸残基优选不受修饰(如替换)。可根据本领域已知的方法例如位点定向诱变或丙氨酸分区诱变(见例如Cunningham和Wells,1989,Science 244:1081-1085)鉴定功能必需的氨基酸残基。可通过对以核磁共振分析、晶体学或光亲和标记之类的技术(见例如de Vos等,1992,Science 255:306-312;Smith等,1992,Journal of Molecular Biology 224:899-904;Wlodaver等,1992,FEBS Letters 309:59-64)确定的三维结构进行分析来确定底物-酶相互作用位点。
此外,可通过引入核苷酸替代来修饰编码本发明酶的核苷酸序列,所述替代不产生由该核苷酸序列编码的酶的另一氨基酸序列,但是符合将用于产生该酶的宿主生物相应的密码子选择。
可使用任何本领域已知的方法通过位点定向诱变完成向核苷酸序列中引入将一个核苷酸与另一核苷酸交换的突变。特别有用的是利用带有目的插入片段的超螺旋、双链DNA载体和含有目的突变的两个合成引物的操作。分别与载体相反链互补的寡核苷酸引物通过Pfu DNA聚合酶在温度循环中延伸。引物整合产生含有交错切口的突变质粒。温度循环后用对甲基化和半甲基化DNA特异的Dpn I处理产物以消化亲代DNA模板并选择含有突变的合成DNA。也可使用其他本领域已知的方法。关于核苷酸替代的一般描述可查看例如Ford等,1991,《Protein Expression and Purification》2:95-107。
本发明还涉及含有编码本发明多肽的核苷酸序列并与选自:
(i)SEQ ID NO:1到SEQ ID NO:25编码成熟多肽的区域的核苷酸序列互补链,
(ii)包含在选自SEQ ID NO:1到SEQ ID NO:25编码成熟多肽的区域的核苷酸序列中的cDNA序列的互补链;
(iii)编码具有SEQ ID NO:26到SEQ ID NO:50中包含的成熟多肽相应功能的分泌成熟多肽的(i)或(ii)的片段的多核苷酸探针在高严格度条件下,特别是非常高严格度条件(J.Sambrook,E.F.Fritsch,和T.Maniatus,1989,《Molecular Cloning,ALaboratory Manual》,第二版,Cold Spring Harbor,New York)下杂交的核苷酸序列(优选由其组成)的多核苷酸。
应该理解关于核苷酸序列杂交的内容和细节将与本文题为“本发明多肽”的小节中讨论的杂交方面相同或类似。
本发明还包括适用于电子设备,优选数码设备的储存媒体,所述装置含有本发明多肽氨基酸序列或本发明多核苷酸核苷酸序列的信息,具体的为本发明任何多肽或多核苷酸序列为电子或数字形式,例如二进制代码或其他数字代码。合适的储存介质可以是用于电子设备和计算设备的磁盘或光盘,且信息可具体地以数字形式储存在储存媒体中。
核苷酸构建体
本发明还涉及含有与一个或多个控制序列有效连接的本发明核苷酸序列的核酸构建体,所述控制序列在与控制序列兼容的条件下指导编码序列在适当的宿主细胞内表达。
可以以多种方式操作编码本发明多肽的核苷酸序列,以便提供多肽的表达。插入载体前的核苷酸序列的操作可能是需要的或者是必要的,这取决于表达载体。利用重组DNA方法来修饰核苷酸序列的技术是本领域众所周知的。
控制序列可以是适当的启动子序列——被宿主细胞识别用于表达核苷酸序列的核苷酸序列。启动子序列含有介导多肽表达的转录控制序列。启动子可以是在所选择的宿主细胞内表现出转录活性的任何核苷酸序列,包括突变、截短和杂合启动子,并且可以从编码细胞外或者细胞内的与宿主细胞同源或者异源的的多肽的基因得到。
用于指导本发明核酸构建体转录(特别是在细菌宿主细胞中)的适当的启动子实例为从大肠杆菌乳糖操纵子、天蓝色链霉菌(Streptomycescoelicolor)琼脂糖酶基因(dagA)、枯草芽孢杆菌果聚糖蔗糖酶(sacB)、地衣芽孢杆菌(Bacillus licheniformis)α-淀粉酶基因(amyL)、嗜热脂肪芽孢杆菌(Bacillus stearothermophilus)麦芽糖(maltogenic)淀粉酶基因(amyM)、解淀粉芽孢杆菌(Bacillus amyloliquefaciens)α-淀粉酶基因(amyQ)、地衣芽孢杆菌青霉素酶基因(penP)、枯草芽孢杆菌xylA和xylB基因和原核β-内酰胺酶基因(Villa-Kamaroff等,1978,Proceedings of theNational Academy of Sciences USA 75:3727-3731)得到的启动子以及tac启动子(DeBoer等,1983,Proceedings of the National Academy of SciencesUSA 80:21-25)。其他的启动子描述于Scientific American,1980,242:74-94中″Useful proteins from recombinant bacteria″和Sambrook等,1989,上文。
用于指导本发明核酸构建体在丝状真菌宿主细胞内转录的适当启动子有从米曲霉(Aspergillus oryzae)TAKA淀粉酶、米赫根毛霉(Rhizomucormiehei)天冬氨酸蛋白酶、黑曲霉中性α-淀粉酶、黑曲霉酸稳定α-淀粉酶、黑曲霉或者泡盛曲霉(Aspergillus awamori)葡糖淀粉酶(glaA)、米赫根毛霉脂酶、米曲霉碱性蛋白酶、米曲霉丙糖磷酸异构酶、构巢曲霉(Aspergillusnidulans)乙酰胺酶和尖镰孢(Fusarium oxysporum)胰蛋白酶样蛋白酶(WO96/00787)基因得到的启动子,以及NA2-tpi启动子(从黑曲霉中性α-淀粉酶和米曲霉丙糖磷酸异构酶得到的杂合启动子)和它们的突变、截短和杂合启动子。
在酵母宿主中有用的启动子得自酿酒酵母(Saccharomyces cerevisiae)烯醇化酶(ENO-1)基因、酿酒酵母半乳糖激酶(GAL1)基因、酿酒酵母醇脱氢酶/甘油醛-3-磷酸脱氢酶(ADH2/GAP)基因和酿酒酵母3-磷酸甘油酸酯激酶基因。其他可用于酵母宿主细胞的启动子由Romanos等,1992,Yeast 8:423-488描述。
控制序列还可以是适当的转录终止序列——被宿主细胞识别以终止转录的序列。终止子序列与编码酶的核苷酸序列3’末端有效连接。在所选择的宿主细胞内具有功能的任何终止子均可用于本发明。
用于丝状真菌宿主细胞的优选的终止子得自米曲霉TAKA淀粉酶、黑曲霉葡糖淀粉酶、构巢曲霉临氨基苯甲酸核合酶、黑曲霉α-葡糖苷酶和尖镰孢胰蛋白酶样蛋白酶基因。
用于酵母宿主细胞的优选的终止子得自酿酒酵母烯醇化酶、酿酒酵母细胞色素C(CYC1)和酿酒酵母甘油醛-3-磷酸脱氢酶基因。其他可用于酵母宿主细胞的终止子由Romanos等,1992,上文,描述。
控制序列还可以是适宜的前导序列——对宿主细胞进行翻译而言重要的mRNA非翻译区。前导序列与编码多肽的核苷酸序列的5′末端有效连接。在所选择的宿主细胞内具有功能的任何前导序列均可用于本发明。
用于丝状真菌宿主细胞的优选的前导序列是从米曲霉TAKA淀粉酶和构巢曲霉丙糖磷酸异构酶基因得到的前导序列。
适用于酵母宿主细胞的前导序列可得自酿酒酵母烯醇化酶(ENO-1)、酿酒酵母3-磷酸甘油酸酯激酶、酿酒酵母α-因子和酿酒酵母醇脱氢酶/甘油醛-3-磷酸脱氢酶(ADH2/GAP)基因。
控制序列还可以是聚腺苷酸化序列,即与核苷酸序列的3′末端有效连接并且转录后作为向转录的mRNA添加聚腺苷酸残基的信号而被宿主细胞识别的序列。在所选择的宿主细胞内具有功能的任何聚腺苷酸化序列均可用于本发明。
用于丝状真菌宿主细胞的优选的聚核苷酸化序列得自米曲霉TAKA淀粉酶、黑曲霉葡糖淀粉酶、构巢曲霉邻氨基苯甲酸合酶、尖镰孢胰蛋白酶样蛋白酶和黑曲霉α-葡糖苷酶基因。
可用于酵母宿主细胞的聚腺苷酸化序列由Guo和Sherman,1995,Molecular Cellular Biology 15:5983-5990描述。
控制序列还可以是信号肽编码区,其编码的氨基酸序列与多肽的氨基末端连接并且指导编码的酶进入细胞的分泌途径。在核苷酸序列编码序列的5′末端可以天然的含有信号肽编码区,该信号肽编码区于翻译阅读框内与编码所分泌多肽的编码区片段天然连接。另外,编码序列的5′末端可以含有对于编码序列而言为外来的信号肽编码区。在编码区天然不含有信号肽编码区时,可能需要外来的信号肽编码区。另外,外来的信号肽编码区可简单地替换天然的信号肽编码区以增强酶的分泌。然而,能够指导表达的酶进入所选择宿主细胞的分泌途径的任何信号肽编码区都可用于本发明。
对于细菌宿主细胞,有效的信号肽编码区来自于芽孢杆菌NCIB 11837maltogenic淀粉酶、嗜热脂肪芽孢杆菌α-淀粉酶、地衣芽孢杆菌枯草杆菌蛋白酶、地衣芽孢杆菌β-内酰胺酶、嗜热脂肪芽孢杆菌中性蛋白酶(nprT、nprS、nprM)和枯草芽孢杆菌prsA基因的信号肽编码区。其他信号肽由Simonen和Palva,1993,Microbiological Reviews 57:109-137描述。
对于丝状真菌宿主细胞,有效的信号肽编码区来自于米曲霉TAKA淀粉酶、黑曲霉中性淀粉酶、黑曲霉葡糖淀粉酶、米赫根毛霉天冬氨酸蛋白酶、孤独腐质霉(Humicola insolens)纤维素酶和柔毛腐质霉(Humicolalanuginosa)脂酶基因的信号肽编码区。
可用于酵母宿主细胞的信号肽得自酿酒酵母α-因子和酿酒酵母转化酶。其他有用的信号肽编码区由Romanos等,1992,上文,描述。
控制序列还可以是编码位于酶氨基端的氨基酸序列的前肽编码区。产生的多肽可命名为酶原或多肽原。多肽原通常是非活性的并可通过从多肽原上催化或自身催化切除前肽转化为成熟活性多肽。前肽编码区可从枯草芽孢杆菌碱性蛋白酶(aprE)、枯草芽孢杆菌中性蛋白酶(nprT)、酿酒酵母α-因子、米赫根毛霉天冬氨酸蛋白酶和Myceliophthora thermophila漆酶(WO 95/33836)基因获得。
在多肽氨基端同时存在信号肽和前肽区时,前肽区位于紧邻多肽氨基端而信号肽区位于紧邻前肽区氨基端。在酵母中可使用ADH2系统或GAL1系统。在丝状真菌中,可使用TAKAα-淀粉酶启动子、黑曲霉葡糖淀粉酶启动子和米曲霉葡糖淀粉酶启动子作为调节序列。
调节序列的其他实例为允许基因扩增的调节序列。在真核系统中包括存在氨甲喋呤时扩增的二氢叶酸还原酶基因和有重金属时扩增的金属硫蛋白基因。在这些情况下,编码多肽的核苷酸序列应与调节序列有效连接。
重组表达载体
本发明还涉及包含发明的核酸构建体的重组表达载体。上述多种核苷酸和控制序列可连接在一起产生重组表达载体,其可以包括一个或多个便利的限制性酶切位点,以便允许编码多肽的核苷酸序列在此种位点进行插入或替代。另外,本发明的核苷酸序列可以通过将该核酸序列或者含有该序列的核酸构建体插入到用于表达的恰当载体中来表达。在构建表达载体时,编码序列定位于载体,以致于编码序列与用于表达的恰当控制序列有效连接。
重组表达载体可以是能够方便地进行重组DNA操作并且能够引起核苷酸序列表达的任何载体(例如质粒或者病毒)。载体的选择通常依赖于载体和欲导入载体的宿主细胞的兼容性。载体可以是线性的或者闭合环状质粒。
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,其复制不依赖于染色体的复制,例如质粒、染色体外遗传因子、微型染色体或者人工染色体。
载体可含有保证自我复制的任何手段。另外,载体可以是导入宿主细胞原整合进入基因组并且与整合进入的染色体一起复制的载体。此外,可以使用单一载体或质粒、或共同含有欲被导入宿主细胞基因组的全部DNA的两个或多个载体或质粒,或使用转座子。
本发明的载体优选含有便于选择转化细胞的一个或多个选择标记。选择标记是其产物能够提供杀生物剂或病毒抗性、重金属抗性、从原养型到自养型等等的基因。
细菌选择标记物的实例为来自枯草芽孢杆菌或地衣芽孢杆菌的dal基因,或赋予抗生素抗性如氨苄青霉素、卡那霉素、氯霉素或四环素抗性的标记物。适用于酵母宿主细胞的标记物为ADE2、HIS3、LEU2、LYS2、MET3、TRP1和URA3。在丝状真菌宿主细胞中使用的选择标记物包括(但不限于),amdS(乙酰胺酶)、argB(鸟氨酸氨甲酰基转移酶)、bar(膦丝菌素乙酰转移酶)、hygB(潮霉素磷酸转移酶)、niaD(硝酸盐还原酶)、pyrG(乳清酸核苷-5′-磷酸脱羧酶)、sC(硫酸腺苷酰转移酶)、trpC(邻氨基苯甲酸合酶),以及它们的等效物。
在曲霉属细胞中优先使用的是构巢曲霉或者米曲霉的amdS和pyrG基因和吸水链霉菌(Streptomyces hygroscopicus)的bar基因。
本发明的载体优选含有允许载体稳定整合到宿主细胞基因组中的元件或者允许载体在细胞内不依赖基因组而自主复制的元件。
为了整合进入宿主细胞基因组,载体可依赖于编码多肽的核苷酸序列或者载体中用于通过同源重组或者非同源重组将载体稳定整合进入基因组的任何其它元件。另外,载体可以包含用于通过同原重组指导整合进入宿主细胞基因组的附加核苷酸序列。附加核苷酸序列使载体能够在染色体上的精确位置整合进入宿主细胞基因组。为了提高在精确位置整合的可能性,整合元件应优选含有足够数量的核苷酸,例如100至1,500个碱基对,优选400至1,500个碱基对,并且最优选800至1,500个碱基对,它们与相应的靶序列高度同源以便增强同源重组的概率。整合元件可以是与宿主细胞基因组中的靶序列同源的任何序列。此外,整合元件可以是非编码的或者编码的核苷酸序列。另一方面,载体可以通过非同源重组的方式整合进入宿主细胞的基因组。
为了自主复制,载体还可包含使得载体能够在所述宿主细胞中自主复制的复制起点。细菌复制起点的实例为允许在大肠杆菌中复制的pBR322、pUC19、pACYC177和pACYC184和允许在芽孢杆菌中复制的pUB110、pE194、pTA1060和pAMβ1质粒复制起点。用于酵母宿主细胞的复制起点实例为两微米复制起点ARS1、ARS4,ARS1和CEN3的组合以及ARS4和CEN6的组合。
复制起点可以是具有突变的复制起点,所述突变使其在宿主细胞中温度敏感地发挥功能(见例如Ehrlich,1978,Proceedings of the NationalAcademy of Sciences USA 75:1433)。
可向宿主细胞中插入多于一个拷贝的本发明核苷酸序列以增加基因产物产生。可通过将至少一个附加拷贝的序列整合进宿主细胞基因组来提高核苷酸序列拷贝数,或通过在核苷酸序列中包含可扩增的选择标记基因使细胞中含有扩增拷贝的该选择标记,从而可通过在有适当选择剂时培养细胞选择核苷酸序列的附加拷贝。
用于连接上述元件以构建本发明重组表达载体的操作为本领域技术人员熟知(见例如Sambrook等,1989,上文)。
重组宿主细胞
本发明还涉及含有本发明核酸构建体的重组宿主细胞,该细胞有利于多肽的重组产生。将含有本发明核苷酸序列的载体导入宿主细胞,以便载体以作为先前描述的染色体整合体或者自主复制的染色体外载体而得以维持。
宿主细胞可以是单细胞微生物(如原核生物)或非单细胞微生物(如真核生物)。
有用的单细胞细胞是细菌细胞,例如革兰氏阳性菌,包括(但不仅限于)芽孢杆菌细胞,例如嗜碱芽孢杆菌(Bacillus alkalophilus)、解淀粉芽孢杆菌、短芽孢杆菌(Bacillus brevis)、环状芽孢杆菌(Bacilluscirculans)、Bacillus clausii、凝结芽孢杆菌(Bacillus coagulans)、Bacilluslautus、迟缓芽孢杆菌(Bacillus lentus)、地衣芽孢杆菌、巨大芽孢杆菌(Bacillus megaterium)、嗜热脂肪芽孢杆菌、枯草芽孢杆菌和苏云金芽孢杆菌(Bacillus thuringiensis)或链霉菌细胞,例如变铅青链霉菌(Streptomyces lividans)或鼠灰链霉菌(Streptomyces murinus)或革兰氏阴性菌例如大肠杆菌和假单胞菌属(Pseudomonas sp.)。在优选的实施方案中,细菌宿主细胞为迟缓芽孢杆菌、地衣芽孢杆菌、嗜热脂肪芽孢杆菌或枯草芽孢杆菌细胞。在另一优选的实施方案中,芽孢杆菌细胞为嗜碱芽孢杆菌。
可通过例如原生质体转化(见例如Chang和Cohen,1979,MolecularGeneral Genetics 168:111-115)、使用感受态细胞(见例如Young和Spizizin,1961,Journal of Bacteriology 81:823-829,或Dubnau和Davidoff-Abelson,1971,Journal of Molecular Biology 56:209-221)、电穿孔(见例如Shigekawa和Dower,1988,Biotechniques 6:742-751)或接合(见例如Koehler和Thorne,1987,Journal of Bacteriology 169:5771-5278)完成将载体引入细菌宿主细胞。
宿主细胞可以是真核生物,例如哺乳动物、昆虫、植物或真菌细胞。
在优选的实施方案中,宿主细胞为真菌细胞。本文使用“真菌”包括子囊菌门(Ascomycota)、壶菌门(Chytridiomycota)和接合菌门(Zygomycota)(如Hawksworth等在《Ainsworth and Bisby′s Dictionaryof The Fungi》,第8版,1995,CAB International,University Press,Cambridge,UK,中所定义),以及卵菌门(Oomycota)(如Hawksworth等1995,同前,171页,所引用)和所有的有丝分裂孢子真菌(Hawksworth等1995,同前)。在更优选的实施方案中,真菌宿主细胞为酵母细胞。本文使用“酵母”包括产子囊酵母(Endomycetales)、产担孢子酵母和属于半知菌类(Fungi imperfect)(酵母菌)的酵母。由于酵母的分类将来可能发生变化,就本发明而言,酵母如《Biology and Activities of Yeast》(Skinner,F.A.,Passmore,S.M.,和Davenport,R.R.编辑,Soc.App.Bacteriol.Symposium Series No.9,1980)所述定义。
在更优选的实施方案中,酵母宿主细胞为假丝酵母(Candida)、汉逊属(Hansenula)、克鲁维属(Kluyveromyces)、毕赤属(Pichia)、酵母属(Saccharomyces)、类酵母属(Schizosaccharomyces)或Yarrowia细胞。
在最优选的实施方案中,酵母宿主细胞为卡尔酵母(Saccharomycescarlsbergensis)、酿酒酵母、糖化酵母(Saccharomyces diastaticus)、Saccharomyces douglasii、克鲁弗酵母(Saccharomyces kluyveri)、诺地酵母(Saccharomyces norbensis)或Saccharomyces oviformis细胞。在另一最优选的实施方案中,酵母宿主细胞为乳酸克鲁维酵母(Kluyveromyceslactis)细胞。在另一最优选的实施方案中,酵母宿主细胞为Yarrowialipolytica细胞。
在另一个更加优选的实施方案中,真菌宿主细胞是丝状真菌细胞。“丝状真菌”包括真菌门(Eumycota)和卵菌门(如Hawksworth等1995,同前定义)细分的所有丝状形式。丝状真菌以由壳多糖、纤维素、葡聚糖、脱乙酰壳多糖、甘露聚糖和其它复合多糖组成的菌丝体壁为特征。通过菌丝延长进行营养生长并且碳的分解代谢是专性需氧的。相反,诸如酿酒酵母之类的酵母的营养生长是通过单细胞菌体的出芽完成并且碳的分解代谢可通过发酵进行。
在甚至更加优选的实施方案中,丝状真菌宿主细胞是(但不限于)枝顶孢属(Acremonium)、曲霉属(Aspergillus)、镰刀菌属(Fusarium)、腐质霉属(Humicola)、毛霉属(Mucor)、毁丝霉属(Myceliophthora)、脉孢菌属(Neurospora)、青霉属(Penicillium)、梭孢壳属(Thielavia)、Tolypocladium或者木霉属(Trichoderma)种类的细胞。
在最优选的实施方案中,丝状真菌宿主细胞是泡盛曲霉、臭曲霉(Aspergillus foetidus)、日本曲霉(Aspergillus japonicus)、构巢曲霉、黑曲霉或者米曲霉细胞。在另一个最优选的实施方案中,丝状真菌宿主细胞是杆孢状镰孢(Fusarium bactridioides)、Fusarium cerealis、Fusariumcrookwellense、大刀镰孢(Fusarium culmorum)、禾本科镰孢(Fusariumgraminearum)、禾赤镰孢(Fusarium graminum)、异孢镰孢(Fusariumheterosporum)、合欢木镰孢(Fusarium negundi)、尖镰孢、多枝镰孢(Fusarium reticulatum)、粉红镰孢(Fusarium roseum)、接骨木镰孢(Fusarium sambucinum)、肤色镰孢(Fusarium sarcochroum)、拟分枝孢镰孢(Fusarium sporotrichioides)、硫色镰胞(Fusarium sulphureum)、Fusarium torulosum、Fusarium trichothecioides或者Fusarium venenatum的细胞。在一个甚至最优选的实施方案中,丝状真菌母细胞是Fusariumvenenatum(Nirenberg sp.nov.)细胞。在另一个最优选的实施方案中,丝状真菌宿主细胞是孤独腐质霉、柔毛腐质霉、米黑毛霉(Mucor miehei)、Myceliophthora thermophila、粗糙脉孢菌(Neurospora crassa)、产紫青霉(Penicillium purpurogenum)、Thielavia terrestris、Trichodrma harzianum、康宁木霉(Trichoderma koningii)、Trichoderma longibrachiatum、Trichoderma reesei或者绿色木霉(Trichoderma viride)的细胞。
真菌细胞可以通过包括原生质体形成、原生质体转化和细胞壁回收的过程以本质上已知的方式进行转化。转化曲霉属细胞的适宜的操作在EP238023和Yelton等,1984,Proceedings of the National Academy ofSciences USA 81:1470-1474中有所描述。转化镰刀菌属物种的适宜的方法在Malardier等,1989,Gene 78:147-156和WO 96/00787中有所描述。可以使用Becker和Guarente,在Abelson,J.N.和Simon,M.I.,编,“Guideto Yeast Genetics and Molecular Biology”,《Methods in Enzymology》,194卷,182-187页,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中描述的操作转化酵母。
供体菌株
本发明还提供以保藏号DSM 15716保藏的脂环酸芽孢杆菌属细菌和含有该微生物的组合物。
制备酶多肽的方法本发明还涉及用于产生本发明酶的方法,该方法包括:(a)培养含有编码本发明酶的核苷酸序列的菌株,所述菌株能够表达并分泌该酶,和(b)回收该酶。在一具体的实施方案中,该菌株为野生型菌株,例如脂环酸芽孢杆菌DSM 15716,而在另一实施方案中该菌株为如上所述的重组宿主细胞。
在本发明这些方法中,使用本领域已知的方法将细胞在适于生产酶的营养培养基中培养。例如,细胞可以通过在实验室或者工业发酵罐中,在适宜的培养基上以及允许多肽表达和/或分离的条件下进行摇瓶培养、小规模或者大规模发酵(包括连续、批式、补料分批式或者固态发酵)。使用本领域已知的操作,在含有碳源和氮源和无机盐的适宜的营养培养基中进行培养。适宜的培养基可以从商品供应商得到或者根据公布的组合物(例如American Type Culture Collection的目录中所公布的)进行配制。如果酶分泌到营养培养基中,则可从培养基中直接回收酶。
可使用本领域已知的方法回收产生的多肽。例如可通过包括(但不限于)离心、过滤、抽提、喷雾干燥、蒸发或者沉淀的常规操作从营养培养基中回收多肽。
可通过本领域已知的多种方法,包括(但不限于)层析(例如离子交换层析、亲和层析、疏水层析、层析聚焦和大小排阻层析)、电泳方法(例如制备型等电聚焦电泳)、差异溶解性(如硫酸铵沉淀)、SDS-PAGE,或者抽提(例如见《蛋白质纯化》,J.-C.Janson和Lars Ryden编,VCHPublishers,New York,1989)对本发明多肽进行纯化。
本发明方法还包括在脂环酸芽孢杆菌DSM 15716样品上进行的WO01/77315A1的TAST方法,即通过将脂环酸芽孢杆菌DSM 15716基因组的基因(例如来自基因文库的基因)与编码无信号报告子的基因(例如β-内酰胺酶)经由转座子标签融合,在显示报告子存在的培养基(如含氨苄青霉素的培养基)上培养包含与编码无信号报告子的基因(例如β-内酰胺酶)经由转座子标签融合的脂环酸芽孢杆菌DSM 15716基因的宿主细胞克隆,检测分泌报告子的克隆并分离该克隆中含有的脂环酸芽孢杆菌DSM15716基因和多肽。
当在显示报告子存在的培养基(如含氨苄青霉素的培养基)上培养包含与编码无信号报告子的基因(例如β-内酰胺酶)经由转座子标签融合的脂环酸芽孢杆菌DSM 15716基因的宿主细胞克隆时,只有表达并分泌报告子(例如β-内酰胺酶)的克隆会被检测(例如存活)。然而,只有与报告子基因融合的基因具有在宿主菌株中能被识别的完整启动子和核糖体结合位点(即是真实生活中由细胞表达以产生多肽的基因),并且报告子被翻译从而合成的多肽被转运穿过细胞质膜并正确折叠时,才分泌报告子。因此,向选择的宿主细胞中插入融合基因时,检测到报告子存在的克隆(例如氨苄青霉素抗性)会含有来自脂环酸芽孢杆菌DSM 15716的编码功能性分泌多肽的基因。
转基因植物
本发明还涉及用编码本发明酶的核苷酸序列转化以表达并生产该酶的转基因植物、植物局部或植物细胞。在一个实施方案中,可以使用植物作为产生可回收数量酶的宿主。可以从植物或植物局部回收酶。另外,可以使用含有重组酶的植物或植物局部用于改善食物或饲料的品质(例如改善营养价值、味道和流变性质)或破坏抗营养因子。具体可以使用表达酶的植物或植物局部作为用于产生燃料醇或生物乙醇的改善的起始材料。
转基因植物可以是双子叶的(双子叶植物)或单子叶的(单子叶植物)。单子叶植物实例为草,例如草场草(青草,Poa),饲料草如羊茅属(festuca)、黑麦草属(Lolium)、temperate grass如Agrostis和谷类如小麦、燕麦、黑麦、大麦、稻、高粱和玉蜀黍(玉米)。
双子叶植物实例为烟草、豆类(如羽扇豆、马铃薯、甜菜、豌豆、豆和大豆)和十字花科植物(芸苔(Brassicaceae)科)(如花椰菜、rape seed和密切相关的模式生物拟南芥(Arabidopsis thaliana))。
植物局部的实例为茎、愈伤组织、叶、根、果实、种子和块茎。特定的植物组织例如叶绿体、质外体、线粒体、液泡、过氧化物酶体和细胞质也认为是植物局部。此外,认为无论来源于什么组织的植物细胞是植物局部。
本发明范围还包括这类植物、植物局部和植物细胞的后代。
可按照本领域已知方法构建表达本发明酶的转基因植物或植物细胞。简言之,通过将一个或多个编码本发明酶的表达构建体整合进植物宿主基因组并将产生的修饰的植物或植物细胞繁殖为转基因植物或植物细胞。
便利的表达构建体为包含与适当调节序列有效连接的编码本发明酶的核酸序列的核酸构建体,所述调节序列是在选择的植物或植物局部中表达核酸序列所必需的。此外,表达构建体还可包含用于鉴定整合入表达构建体的宿主细胞的选择标记和向所述植物中引入该构建体时必需的DNA序列(后者依赖于待使用的DNA引入方法)。
调节序列(如启动子和终止子序列和任选的信号或转运序列)的选择依赖于例如期望何时、何处和如何表达酶。例如,编码本发明酶的基因表达可以是组成型或诱导型的,或可以是发育特异、阶段或组织特异的,且基因产物可以靶向至特定组织或植物局部,例如种子或叶。调节序列由例如Tague等,1988,Plant Physiology 86:506描述。
关于组成型表达可使用35S-CaMV启动子(Franck等,1980,Cell 21:285-294)。器官特异启动子可以是例如来自储藏库组织,如种子、马铃薯块茎和果实(Edwards & Coruzzi,1990,Ann.Rev.Genet 24:275-303)或来自代谢库组织,如分生组织(Ito等,1994,Plant Mol.Biol.24:863-878)的启动子,种子特异启动子如来自稻的谷蛋白、谷醇溶蛋白、球蛋白或清蛋白启动子(Wu等,1998,Plant and Cell Physiology 39:885-889),来自豆球蛋白B4的蚕豆(Vicia faba)启动子和来自蚕豆的未知种子蛋白质基因(Con rad等,1998,Journal of Plant Physiology 152:708-711),来源于种子油体蛋白质的启动子(Chen等,1998,Plant and Cell Physiology 39:935-941),来源于欧洲油菜(Brassica napus)的储藏蛋白napA启动子和本领域已知的任何其他种子特异启动子(如描述于WO 91/14772的)。此外,启动子可以是叶特异的启动子,例如来源于稻或番茄的rbcs启动子(Kyozuka等,1993,Plant Physiology 102:991-1000)、小球藻病毒腺嘌呤甲基转移酶基因启动子(Mitra和Higgins,1994,Plant Molecular Biology26:85-93)或来源于稻的aldP基因启动子(Kagaya等,1995,Molecular andGeneral Genetics 248:668-674)或伤诱导启动子如马铃薯pin2启动子(Xu等,1993,Plant Molecular Biology 22:573-588)。
还可使用启动子增强子元件以便在植物中更高表达本发明的酶。例如,启动子增强子元件可以是置于启动子和编码本发明酶的核苷酸序列之间的内含子。例如Xu等,1993(上文)公开了稻肌动蛋白1基因的第一个内含子增强表达的用途。
标记基因和表达构建体的任何其他部分可选自可从本领域获得的那些。
根据本领域已知的常规技术将核酸构建体整合进植物基因组,所述技术包括农杆菌介导的转化、病毒介导的转化、显微注射、粒子轰击、生物射弹转化和电穿孔(Gasser等,1990,Science 244:1293;Potrykus,1990,BiolTechnology 8:535;Shimamoto等,1989,Nature 338:274)。
目前,根瘤农杆菌介导的基因转移是选择用于产生转基因双子叶植物的方法(综述见Hooykas和Schilperoort,1992,Plant Molecular Biology 19:15-38)。然而其也可用于转化单子叶植物,尽管通常优选其他转化方法用于单子叶植物。目前,选择用于产生转基因单子叶植物的方法为粒子轰击(转化DNA包被的显微金或钨颗粒)胚愈伤组织或发育中的胚(Christou,1992,Plant Journal 2:275-281;Shimamoto,1994,Current OpinionBiotechnology 5:158-162;Vasil等,1992,BiolTechnology 10:667-674)。用于转化单子叶植物的另一方法如Omirulleh等,1993,Plant MolecularBiology 21:415-428所述的基于原生质体转化。
转化之后,根据本领域熟知的方法选择其中整合了表达构建体的转化体并再生为完整的植株。
本发明还涉及生产本发明酶的方法,包括(a)在有助于酶产生的条件下培养含有编码本发明酶的核苷酸序列的转基因植物或植物细胞和(b)回收酶。
包含多肽的组合物及其制备方法
本发明提供包含本发明多肽并优选含有赋形剂的组合物以及制备这类组合物的方法,包括将本发明的多肽与赋形剂混和。组合物具体包含至少两种不同的本发明多肽,优选至少3种,更优选至少5种,更优选至少10种,更优选至少15种,更优选至少20种。最优选该组合物包含发酵脂环酸芽孢杆菌DSM 15716样品或其突变体(其中缺失或添加了一个或多个基因)时分泌的所有多肽。
在具体的实施方案中,本发明多肽为组合物的主要(多肽)组分,例如单一组分组合物。上下文中赋形剂应理解为用于配制组合物的任何辅助剂或化合物,包括溶剂、载体、稳定剂等。
组合物还可包含一个或多个额外的酶,例如氨肽酶、淀粉酶、糖酶、羧肽酶、过氧化氢酶、纤维素酶、壳多糖酶、角质酶(cutinase)、环糊精糖基转移酶、脱氧核糖核酸酶、酯酶、α-半乳糖苷酶、β-半乳糖苷酶、葡糖淀粉酶、α-葡糖苷酶、β-葡糖苷酶、haloperoxidase、转化酶、漆酶、脂肪酶、甘露糖苷酶、氧化酶、果胶分解酶、肽谷氨酰胺酶、过氧化物酶、植酸酶、多酚氧化酶、蛋白水解酶、核糖核酸酶、转谷氨酰胺酶或木聚糖酶。
可根据本领域已知方法制备组合物并且可以是液体或固体组合物的形式。例如,可以使用本领域已知的配制多肽和/或药品的方法配制酶组合物,例如配制成包衣或未包衣的颗粒或微粒。因此本发明多肽可以以颗粒(优选无尘颗粒)、液体(特别是稳定化的液体)、浆液或受保护的多肽形式提供。对于某些应用,优选将多肽固定在固体基质上。
可使用本领域已知方法稳定欲包含在组合物中的多肽,例如通过添加抗氧化剂或还原剂以限制多肽氧化来稳定组合物中的多肽,或可通过添加多聚体如PVP、PVA、PEG或其他已知有益于多肽在固体或液体组合物中稳定的合适的多聚体来稳定多肽。
在另一实施方案中,本发明组合物为洗涤剂组合物,其除本发明多肽外还包含表面活性剂和选自增量组分(如沸石)、漂白剂(如过碳酸盐)、漂白增强剂(如TAED或NOBS)、抑泡剂、芳香剂的任选化合物。
在另一实施方案中本发明组合物为饲料组合物,其除本发明多肽外还包含谷类或谷物产品。
在另一实施方案中本发明组合物为包含本发明多肽的食品组合物,例如面包师面粉组合物、酿造产品、果汁、油或猪油产品。
在另一实施方案中本发明组合物包含多糖或多糖混合物并包含本发明多肽。
在另一实施方案中本发明组合物为果浆组合物,其除本发明多肽外还还有果肉。
在另一实施方案中本发明组合物为杀生物组合物,其除本发明多肽外还含有氧化还原酶增强剂。
多肽或含有多肽的组合物用途
另一方面,本发明提供本发明多肽或多核苷酸或含有所述多肽或多核苷酸的组合物在多种应用中的用途,特别是(技术)方法例如用在工业或家庭的方法、下文为了商业研究目的进行的方法。因此本发明包含包括在(技术)工业、研究或家庭过程中使用本发明多肽或本发明多核苷酸的方法。
在一个实施方案中,本发明多肽或组合物用于清洁纤维织物。
在另一实施方案中,本发明多肽或组合物用于制备食品或饲料添加剂。
在另一实施方案中,本发明多肽或组合物用于处理lignolosic材料和果肉。
洗涤剂公开内容
本发明多肽可添加到洗涤剂组合物中从而成为其组分。
本发明洗涤剂组合物可配制为例如手工或机器洗衣洗涤剂组合物(包含适用于预处理脏污织物的洗衣添加剂组合物和漂洗添加的织物柔顺剂组合物),或可配制为用于一般家庭硬表面清洁操作的洗涤剂组合物,或为手工或机器洗碗机操作配制。
特别的,本发明提供包含本发明多肽的洗涤剂添加剂。该洗涤剂添加剂和洗涤剂组合物可包含一种或多种其他酶,例如蛋白酶、脂肪酶、cutinase、淀粉酶、糖酶、纤维素酶、果胶酶、甘露聚糖酶、阿拉伯糖酶、半乳聚糖酶、木聚糖酶、氧化酶例如漆酶和/或过氧化物酶。
通常所选择的酶的性质应与选择的洗涤剂兼容(即最适pH,与其他的酶或非酶成份兼容),且该酶应以有效数量存在。
蛋白酶:合适的蛋白酶包括动物、植物或微生物来源的蛋白酶。优选微生物来源。包括化学修饰或蛋白质工程的突变体。蛋白酶可以是丝氨酸蛋白酶或金属蛋白酶,优选碱性微生物蛋白酶或胰蛋白酶样蛋白酶。碱性蛋白酶的实例为枯草杆菌蛋白酶,特别是来自芽孢杆菌的蛋白酶,例如枯草杆菌蛋白酶Novo、枯草杆菌蛋白酶Carlsberg、枯草杆菌蛋白酶309、枯草杆菌蛋白酶147和枯草杆菌蛋白酶168(描述于WO 89/06279)。胰蛋白酶样蛋白酶为胰蛋白酶(如猪或牛来源的)和WO 89/06270和WO94/25583描述的镰刀菌蛋白酶。
有用的蛋白酶实例为WO 92/19729、WO 98/20115、WO 98/20116和WO 98/34946描述的变体,特别是在一个或多个下面位置具有替换的变体:27、36、57、76、87、97、101、104、120、123、167、170、194、206、218、222、224、235和274。
优选的市售蛋白酶包括Alcalase、Savinase、Primase、Duralase、Esperase和Kannase(Novozymes A/S)、Maxatase、Maxacal、Maxapem、Properase、Purafect、Purafect OxP、FN2和Fun3(Genencor International Inc.)。
脂肪酶:合适的脂肪酶包括细菌或真菌来源的脂肪酶。包括化学修饰或蛋白质工程突变体。有用的脂肪酶实例包括来自腐质霉属(同物异名Thermomyces)的脂肪酶(例如来自EP 258 068和EP 305 216所述柔毛腐质霉(T.lanuginosus)或来自WO 96/13580描述的孤独腐质霉),假单胞菌脂肪酶(例如来自产碱假单胞菌(P.alcaligenes)或类产碱假单胞菌(P.pseudoalcaligenes)(EP 218 272)、洋葱假单胞菌(P.cepacia)(EP 331 376)、施氏假单胞菌(P.stutzeri)(GB 1,372,034)、荧光假单胞菌(P.fluorescens)、假单胞菌属菌株SD 705(WO 95/06720和WO 96/27002)、P.wisconsinensis(WO 96/12012)),芽孢杆菌脂肪酶(例如来自枯草芽孢杆菌(Dartois等(1993),Biochemica et Biophysica Acta,1131,253-360)、嗜热脂肪芽孢杆菌(JP 64/744992)或短小芽孢杆菌(WO 91/16422))。
其他实例为脂肪酶变体例如WO 92/05249、WO 94/01541、EP 407225、EP 260105、WO 95/35381、WO 96/00292、WO 95/30744、WO 94/25578、WO 95/14783、WO 95/22615、WO 97/04079和WO 97/07202中所描述的脂肪酶变体。
优选的市售脂肪酶包括LipolaseTM、Lipolase UltraTM和Lipex(Novozymes A/S)。
淀粉酶:适合的淀粉酶(α和/或β)包括细菌或真菌来源的淀粉酶。包括化学修饰和蛋白质工程突变体。淀粉酶包括例如得自芽孢杆菌(例如地衣芽孢杆菌特定菌株,更详细描述于GB 1,296,839)的α-淀粉酶。
有用的淀粉酶实例为WO 94/02597、WO 94/18314、WO 96/23873和WO 97/43424描述的变体,特别是在一个或多个下面位置有替换的变体:15、23、105、106、124、128、133、154、156、181、188、190、197、202、208、209、243、264、304、305、391、408和444。
市售的淀粉酶为DuramylTM、TermamylTM、FungamylTM和BANTM(Novozymes A/S)、RapidaseTM和PurastarTM(来自Genencor InternationalInc.)。
纤维素酶:合适的纤维素酶包括细菌和真菌来源的纤维素酶。包括化学修饰或蛋白质工程突变体。合适的纤维素酶包括来自芽孢杆菌属、假单胞菌属、腐质霉属、镰刀菌属、梭孢壳属(Thielavia)、枝顶孢属(Acremonium)的纤维素酶,例如由US 4,435,307、US 5,648,263、US 5,691,178、US 5,776,757和WO 89/09259中公开的孤独腐质霉、Myceliophthorathermophila和尖镰孢产生的真菌纤维素酶。
特别合适的纤维素酶为具有颜色保护优点的碱性或中性纤维素酶。这类纤维素酶的实例为EP 0 495 257、EP 0 531 372、WO 96/11262、WO96/29397、WO 98/08940中描述的纤维素酶。其他实例为如WO 94/07998、EP 0 531 315、US 5,457,046、US 5,686,593、US 5,763,254、WO 95/24471、WO 98/12307和PCT/DK98/00299描述的纤维素酶变体。
市售纤维素酶包括Celluzyme和Carezyme(Novozymes)、Clazinase和Puradax HA(Genencor International Inc.)和KAC-500(B)(KaoCorporation)。
过氧化物酶/氧化酶:合适的过氧化物酶/氧化酶包括植物、细菌或真菌来源的过氧化物酶/氧化酶。包括化学修饰或蛋白质工程的突变体。有用的过氧化物酶实例包括来自鬼伞属(Coprinus)(例如来自C.cinereus)的过氧化物酶和WO 93/24618,WO 95/10602,and WO 98/15257中描述的它的变体。
市售过氧化物酶包括Guardzyme(Novozymes A/S)。
可通过添加含有一个或多个酶的独立添加剂或通过添加含有所有酶的组合添加剂使洗涤剂组合物中含有洗涤剂酶。本发明的洗涤添加剂(即独立添加剂或组合添加剂)可配制为颗粒、液体、浆体等。优选的洗涤添加剂制品为颗粒(特别是无尘颗粒)、液体(特别是稳定液体)或浆体。
可如US 4,106,991和4,661,452所公开产生无尘颗粒并可任选地通过本领域已知方法包被。蜡包衣材料的实例为平均分子量1000到2000的聚环氧乙烷产品(聚乙二醇,PEG);具有16到50环氧乙烷单元的乙氧基壬基苯酚;乙氧基脂肪醇(其中醇含有12到20碳原子且其中有15到80个环氧乙烷单位);脂肪醇;脂肪酸和单和双和三脂肪酸甘油酯。适于流化床技术应用的产膜包裹材料实例在GB 1483591中给出。可根据现有技术例如通过添加多元醇(如丙二醇)、糖或糖醇、乳酸或硼酸稳定液体酶制品。可根据EP 238,216描述的方法制备受保护的酶。
本发明的洗涤剂组合物可以是任何便利的形式,例如棒、片剂、粉末、颗粒剂、泥膏剂或液体。液体洗涤剂可以是水性的(通常含有上至70%的水和0-30%的有机溶剂)或非水性的。
洗涤剂组合物包含一种或多种表面活性剂,其可以是非离子(包括半极性)的和/或阴离子的和/或阳离子的和/或两性离子的。表面活性剂通常以按重量计从0.1%到60%的水平存在。
洗涤剂通常包含从约1%到约40%的阴离子表面活性剂,例如线性烷基笨磺酸盐、α-烯烃磺酸盐、磺基硫酸盐(脂肪醇硫酸盐)、醇乙氧基黄酸盐、仲链烷磺酸盐、α-磺基脂肪酸甲酯、烷基或烯基琥珀酸或肥皂。
洗涤剂通常包含从约0.2%到约40%非离子表面活性剂例如醇乙氧基化物、乙氧基壬基苯酚、烷基聚糖苷、烷基二甲基氧化胺、乙氧基脂肪酸单乙醇胺、脂肪酸单乙醇胺、多羟基烷基脂肪酰胺或葡糖胺N-酰基N-烷基衍生物(″glucamides″)。
洗涤剂可含有0-65%洗涤剂增量组分或络合剂,例如沸石、二磷酸盐、三磷酸盐、磷酸盐、碳酸盐、柠檬酸盐、次氮基三乙酸、乙二胺四乙酸、二乙烯三胺五乙酸、烷基或烯基琥珀酸、可溶硅酸盐或分层硅酸盐(例如来自Hoechst的SKS-6)。
洗涤剂可含有一种或多种多聚体。实例为羧甲基纤维素、聚乙烯替砒咯烷酮、聚乙二醇、聚乙烯醇、聚乙烯吡啶-N-氧化物、聚乙烯基咪唑、聚羧酸酯例如聚丙烯酸酯、马来酸/丙烯酸共聚物和十二烷基异丁烯酸/丙烯酸共聚物。
洗涤剂可包含漂白系统,其可包含与过酸形式的漂白激活剂(例如四乙酰乙二胺或壬酰氧苯磺酸盐)组合的H2O2来源(例如过硼酸盐或过碳酸盐)。另外,漂白系统可以包含例如酰胺、二酰亚胺或砜类型的过氧酸。
本发明洗涤剂组合物的酶可以使用常规稳定剂稳定,所述稳定剂例如多元醇(如丙二醇或甘油)、糖或糖醇、乳酸、硼酸或硼酸衍生物(如芳香族硼酸酯)或苯基硼酸衍生物(如4-甲酰苯基硼酸),且组合物可以如例如WO 92/19709和WO 92/19708所示配制。
洗涤剂还可以包含其他便利的洗涤成分,例如织物调节剂(包括粘土、发泡剂、抑泡剂、防蚀剂、土壤悬浮剂、抗土壤沉淀剂、染料、杀菌剂、增量剂、助水溶物、抑锈剂或香料)。
目前考虑可以向洗涤剂组合物中以对应于每升洗涤液0.01-100mg酶蛋白质,优选每升洗涤液0.05-5mg酶蛋白质,特别是每升洗涤液0.1-1mg酶蛋白质的数量添加任何酶,特别是本发明的酶。
本发明的酶可以附加地整合进WO 97/07202公开的洗涤制剂中,WO97/07202在本文中整体引入作为参考。保藏的微生物
申请人根据国际承认用于专利程序目的的微生物保藏的布达佩斯条约将下面的微生物保藏于德意志微生物保藏中心(Deutsche Sammlung vonMikroorganismen und Zellkulturen GmbH),MascheroderWeg 1b,D-38124 Braunschweig,德国:
2003年6月30:脂环酸芽孢杆菌CS81嗜热嗜酸菌;DSM保藏号15716。
实施例
实施例1:鉴定脂环酸芽孢杆菌DSM 15716分泌的功能性多肽
构建基因组文库
通过使用标准分子生物学技术(Ausuble等,1995《Current protocolsin molecular biology》,John Wiley and Sons出版)制备脂环酸芽孢杆菌DSM 15716的染色体DNA。用Sau3A部分酶切制备的DNA并在琼脂糖凝胶上分离。洗脱、沉淀并在适当的缓冲液中重悬3到8千碱基的片段。
通过使用Stratagene ZAP ExpressTM预消化载体试剂盒和StratageneZAP ExpressTM预消化Gigapack克隆试剂盒(Bam HI预消化的)(Stratagene Inc.,USA)按照制造商说明/推荐制备基因组文库。产生的λZAP包含38000pfu,选择其中10000用于大量酶切。混和产生的70000个大肠杆菌菌落并使用Qiagen Spin Mini prep试剂盒(Qiagen,德国)制备质粒。在离心管中用1体积份的醋酸钠pH 5和2体积份的96%乙醇在4℃以20000rpm沉淀约1ml含有质粒DNA的洗脱液,用70%v/v乙醇洗涤,在室温干燥并重悬于200μl TE缓冲液中。脂环酸芽孢杆菌基因组文库的质粒库DNA的DNA浓度为5.2μg/μl。
构建和制备转座子
WO 01/77315A1中描述的转座子辅助的信号捕获(TAST)方法的原理为通过转座子标签将选定的基因组中所有基因与编码无信号β-内酰胺酶的基因融合。因此当在含氨苄青霉素的培养基上培养包含与编码无信号β-内酰胺酶基因经由转座子标签融合的基因组基因的宿主细胞克隆时,只有表达并分泌β-内酰胺酶的克隆能够存活。然而,只有与β-内酰胺酶基因融合的基因具有在宿主细胞中能被识别的完整启动子和核糖体结合位点(即真实生活中由细胞表达以产生多肽的基因),并且β-内酰胺酶被翻译从而合成的多肽被转运穿过细胞质膜并正确折叠时,才分泌β-内酰胺酶。因此,当向选定的宿主细胞中插入融合基因时,具氨苄青霉素抗性的克隆含有编码功能性分泌多肽的基因。
通常使用TAST方法时,甚至不必须表达整个基因。当用转座子给基因加标签时,基因N末端部分作为为蛋白质融合物表达,显示该基因包含完整的转录、翻译和分泌序列。因此认为基因N末端部分作为蛋白质融合物的表达通常足够确保整个基因的表达和分泌。
因此可推断通过TAST方法获得的基因事实上的确编码分泌功的能性多肽。
构建含有β-内酰胺酶报告子基因的SigA4转座子
按照WO 01/77315A1的说明,使用标准分子生物学技术完成含有无信号β-内酰胺酶基因转座子的构建。最初使用proofreading聚合酶(PfuTurbo,Stratagene,USA)从载体pUC19中PCR扩增无信号β-内酰胺酶。产生的PCR片段含有NotI和EcoRI限制性位点以便于克隆。从Finnzymes,OY(Espo芬兰)获得含有Entranceposon和抗生素抗性标记物CAT(编码转座子氯霉素抗性)的质粒pEntranceposon(Camr)。质粒用限制酶NotI和EcoRI消化,凝胶纯化并与含有无信号β-内酰胺酶的片段连接。将连接物转化进电感受态DH10B细胞中并通过限制性分析鉴定含有有无信号β-内酰胺酶重组质粒的大肠杆菌克隆,命名为SigA2。
为了制备转座子,构建SigA2的更小的衍生物,其缺乏编码β-内酰胺酶的bla基因:使用两个寡核苷酸引物SigA2NotU-P 5′-TCG CGA TCCGTT TTC GCA TTT ATC GTG AAA CGC T-3′(SEQ ID NO:51)和SigA2NotD-P 5′-CCG CAA ACG CTG GTG AAA GTA AAA GAT GCTGAA-3′(SEQ ID NO:52)(其结合在SigA2的bla基因起点和末端指向外侧)PCR扩增不含有bla基因的SigA2。重连该PCR反应中产生的约3.6kb的扩增产物并转化进合适的大肠杆菌菌株。从能够在氯霉素LB上生长而不能在氨苄青霉素LB上生长的转化体中分离3.6kb的质粒。该质粒保留了所有两个BgIII位点并缺少活性bla基因并称为pSig4。
用BgIII消化60微升浓度为0.3微克/微升的pSig4质粒DNA制品并在琼脂糖凝胶上分离。洗脱2kb的SigA2转座子DNA条带并根据销售商说明使用GFXTMPCR、DNA和凝胶条带纯化试剂盒(Amersham PharmaciaBiotech Inc,美国)纯化并用200微升EB缓冲液洗脱。
C.转座子标记
从pSigA4制得的转座子带有5’截短的编码其分泌信号被去除的内酰胺酶的bla基因。只有当蛋白质分泌进入周质时,β-内酰胺酶才能给予大肠杆菌氨苄青霉素抗性,而β-内酰胺酶的细胞质表达不能赋予氨苄青霉素抗性。不含信号序列时,β-内酰胺酶不能转运到周质从而克隆不能在含有氨苄青霉素的培养基上生长。无信号β-内酰胺酶基因以转座子边缘和β-内酰胺酶编码区之间存在连续的开放读码框的方式包含在转座子内。由此,修饰的转座子转座进入编码分泌蛋白的基因时能够引起与靶基因的框内融合。这导致了分泌进入大肠杆菌周质时给予对氨苄青霉素抗性的融合基因产物。如果转座子甚至框内整合进入编码非分泌蛋白的基因,其各自的宿主不会成为氨苄青霉素抗性的。
为了体外转座子标记脂环酸芽孢杆菌文库,含有约2,6μg DNA的4或8微升SigA2转座子与1微升脂环酸芽孢杆菌基因组文库质粒库DNA的DNA浓缩物和2微升Finnzymes MuA转座酶(0,22微克/微升)和5微升Finnzymes OY(Espoo,芬兰)5×缓冲液以50微升的总体积混和并在30℃孵育3,5小时,然后在75℃热激10分钟。通过加入5微升3M醋酸钠pH5和110微升96%乙醇并在20000rpm离心30分钟沉淀DNA。洗涤并干燥沉淀并用10微升TE缓冲液重悬。
D.转化和选择
在Biorad Gene Pulse设备(50uF,25mAmp,1.8kV)中用5微升转座子标记的质粒库电穿孔转化电感受态大肠杆菌DH108细胞,与1ml SOC培养基混和,在37℃预孵育1小时并涂布在含有25微升/毫升氨苄青霉素、50微升/毫升卡那霉素、10微升/毫升氯霉素的LB上并孵育2-3天。从转化体中选择了1056个菌落并使用Qiaprep 96 Turbo Biorobot试剂盒根据供应商说明制备质粒。
E、质粒制备和测序
在一个反应中使用从上游读入转座子标记基因的A2up引物AGCGTTTGCGGCCGCGATCC(SEQ ID NO:53)并在另一反应中使用从下游读入转座子标记基因的B引物TTATTCGGTCGAAAAGGATCC(SEQ ID NO:54)测序1056个转座子标记的质粒。
F、序列组合和注解
使用PhredPhrap程序(Brent Ewing,LaDeana Hillier,Michael C.Wendl和Phil Green,Base-calling of automated sequencer traces usingphred 1.Accuracy assessment(1998)Genome Research 8:175-185;BrentEwing和Phil Green,Base-calling of automated sequencer traces usingphred 11.Error probabilities(1998)Genome Research 8:186-194)将得到的序列装配为重叠群。随后用BLASTX 2.0a19MP-WashU[1998年7月14日][Build linux-x86 18:51:441998年7月30日]程序比较获得的重叠群和得自标准公开DNA和蛋白质序列数据库的序列(Gish,Warren(1994-1997),未发表;Gish,Warren和David J.States(1993)。通过数据库相似度搜索鉴定蛋白质编码区。Nat.Genet.3:266-72)。
获得的序列是编码完整和功能性多肽的功能基因,如上文解释由于它们是作为氨苄青霉素抗性克隆获得的。
实施例2:通过同源性确定功能
通过与功能已知的基因或多肽序列比较注解SEQ ID NO:26到SEQID NO:50多肽的功能。将本发明多肽与来自公开和内部重叠群数据库的一列最相关序列比较。随后使用BLASTX 2.Oa19MP-WashU[1998年7月14]程序比较重叠群(SEQ ID NO:26到SEQ ID NO:50来自其中)和可以得自标准公开DNA和蛋白质序列数据库的序列。仔细分析SEQ IDNO:26到SEQ ID NO:40与它们最相关的来自其他数据库的功能已知序列的序列比对使得可能以氨基酸同一性程度为基础预测这些多肽的功能。甚至当总氨基酸同一性为40%时(通常难以进行好的预测),我们可以通过仔细分析并解读多肽序列催化位点或重要区域的氨基酸残基来预测SEQ ID NO:26到SEQ ID NO:40的功能。当已知序列催化位点的氨基酸残基也存在于本发明多肽中时,结合充分的总氨基酸同一性可以推出来自脂环酸芽孢杆菌DSM 15716的多肽具有与已知序列相同的功能。
实施例3:制备SEQ ID NO:26到SEQ ID NO:50的多肽
为了制备SEQ ID NO:26到SEQ ID NO:50的多肽,通过将编码开放读码框的DNA与适于在合适的宿主菌株(例如大肠杆菌、枯草芽孢杆菌、地衣芽孢杆菌或Bacillus clausii或脂环酸芽孢杆菌衍生物)中表达基因的启动子、核糖体结合位点和终止子融合以表达SEQ ID NO:1到SEQ IDNO:25包含的编码这些多肽的基因。启动子可以是诱导型启动子或组成型启动子。SEQ ID NO:26到SEQ ID NO:50的任何信号序列可以用另一细菌的适当信号肽交换。表达构建体可以是质粒或线性DNA的部分。可以通过重组将其整合进入宿主菌株的染色体或其可以以质粒存在于宿主细胞中。然后在适合的培养基中以所需体积培养带有目的基因的转化细胞。如果使用诱导型启动子,通过加入诱导剂起始基因表达。否则不需要诱导剂且培养细胞直到产生合适数量的来自目的基因的蛋白质。收集培养物并用标准方法回收蛋白质。
实施例4:测定丝氨酸羧基蛋白酶活性
可以对在适当缓冲液中合成并分泌丝氨酸羧基蛋白酶的宿主菌株的培养液或细胞裂解液测定该活性。将适当体积的这类样品点在琼脂糖平板上,所述平板含有不溶性显色底物AZCL胶原(MegazymeTM)或Azocoll(Sigma-Aldrich)和适合的酸性pH缓冲液,如pH3-5。平板在适当的温度(如55℃)孵育适当的时间(如一天)。活性显示为斑点周围蓝色的色圈。作为AZCL胶原或Azocoll的备选方案,向琼脂平板添加未标记的胶原,其中酶活性以亮区测定。添加胃蛋白酶抑制剂不能抑制丝氨酸羧基蛋白酶的蛋白酶活性。作为备选方案,可以如Tsuruoka N,Nakayama T,Ashida M,Hemmi H,Nakao M,Minakata H,Oyama H,Oda K,NishinoT;″Collagenolytic serine-carboxyl proteinase from Alicyclobacillussendaiensis strain NTAP-1:purification,characterization,gene cloning,and heterologous expression.″Appl Environ Microbiol.卷69(1);162-169页;2003年1月所述测量含有丝氨酸羧基蛋白酶样品的活性。
实施例5:测定多铜氧化酶活性
可以如Schneider等,《Enzyme and Microbial Technology 25》,(1999)502-508页所述用在适当缓冲液中合成并分泌多铜氧化酶的宿主菌株的培养液或细胞裂解液测定该活性。
例如将适当体积(可以是15微升)的这类样品点在琼脂糖平板上,所述平板含有在适当缓冲液(例如pH 5.5,0.1M醋酸钠缓冲液)中合适浓度(例如1mM)的ABTS(2,2′-联氮双-3-乙基苯并噻唑啉-6-硫酸)。将该平板在适当温度(如55℃)孵育适当时间(如16小时)。活性显示为样品周围绿色区域。该测定法用于上清液和提取物。
实施例6:测定丝氨酸蛋白酶活性
可以对在适当缓冲液中合成并分泌丝氨酸蛋白酶的宿主菌株的培养液或细胞裂解液测定该活性。将适当体积的这类样品点在琼脂糖平板上,所述平板含有不溶性显色底物AZCL酪蛋白(MegazymeTM)或AZCL-胶原(MegazymeTM)和适当pH的适当缓冲液。平板在适当的温度(如55℃)孵育适当的时间(如一天)。活性显示为斑点周围蓝色的色圈。作为AZCL酪蛋白或AZCL胶原(MegazymeTM)的备选方案,可以使用未标记的酪蛋白或未标记的胶原。在点了未标记胶原或未标记酪蛋白的平板上,存在丝氨酸蛋白酶时形成亮区。
实施例7:测定谷氨酸肽酶活性
可以对在适当缓冲液中合成并分泌谷氨酸肽酶的宿主菌株的培养液或细胞裂解液测定该活性。将适当体积的这类样品点在琼脂糖平板上,所述平板含有不溶性显色底物AZCL胶原(MegazymeTM)和适合的酸性pH缓冲液,如pH3-5。平板可以在适当的温度(如55℃)孵育适当的时间(如一天)。活性显示为斑点周围蓝色的色圈。作为AZCL胶原的备选方案,可以使用未标记胶原。在点了未标记胶原的平板上,存在谷氨酸肽酶时形成亮区。特定测试SEQ ID NO:27谷氨酸肽酶后,用20微升培养液在pH3.4的0.1%AZCL胶原(MegazymeTM)LB-PG琼脂平板上以斑点测试测定活性。将平板在55℃孵育(过夜)且活性显示为斑点周围蓝色的色圈。
SEQ ID NO:27包含的谷氨酸肽酶显示与属于现在被MEROPS重新分类为G1肽酶家族(PepG)(EC 3.4.23.19)的A4家族肽酶显著的序列相似性,参阅上文描述SEQ ID NO:27的小节和Fujinaga M,Cherney MM,Oyama H,Oda K,James MN.;The molecular structure and catalyticmechanism of a novel carboxyl peptidase from Scytalidium lignicolum;Proc.Natl.Acad.Sci.U.S.A.;101(10);3364-9页;Epub 2004年3月1日;2004年3月9日。
该家族包括其活性位点有保守的Q和E的肽酶序列。这两个残基在SEQ ID NO:27包含的谷氨酸肽酶中均保守。由此SEQ ID NO:27包含的谷氨酸肽酶是G1家族的第一个细菌多肽,该家族之前仅包括真菌肽酶。
SEQ ID NO:27特别与G1家族肽酶的参考序列比较:黑曲霉aspergillopepsin II(SEQ ID NO:55;Swissprot P24665;Takahashi,K.;Inoue,H.;Sakai,K.;Kohama,T.;Kitahara,S.;Takishima,K.;Tanji,M.;Athauda,S.B.P.;Takahashi,T.;Akanuma,H.;Mamiya,G.;Yamasaki,M;The primary structure of Aspergillus niger acid proteinase A.;J.Biol.Chem.;卷266;19480页;1991)。该多肽含有信号肽(aa1-aa18)和两个前肽(aa 19-58和aa 99-109),其在分泌后成熟时被去除。成熟时形成轻链和重链,其通过半胱氨酸残基间的二硫键交联。(Inoue,H.;Kimura,T.;Makabe,O.;Takahashi,K.;The gene and deduced protein sequences ofthe zymogene of Aspergillus niger acid proteinase A;J.Biol.Chem.;卷266;19484页;1991)。SEQ ID NO:27缺失了与第二个前肽(aa99-109)类似的氨基酸和对应于SEQ ID NO:55交联的半胱氨酸残基的氨基酸(见比对)。之前只描述有真菌G1肽酶缺失半胱氨酸残基(Maita,T.;Nagata,S.;Matsuda,G.;Maruta,S.;Oda,K.;Murao,S.;Tsuru,D.;Complete aminoacid sequence of Scytalidium lignicolum acid protease B;J.Biochem.;卷95;465页;1984)。
SEQ ID NO:55和SEQ ID NO:27的比对
SWISSPROT_P24665 MKFSTILTGS-LFATAALAAPLTEKRRARKEARAAGKRHSNPPYIPGSDKEILKLNGTTN
Seq ID No.27 MNGTSVWKASGIAAASCLTAAALLAWPHATSTLDASPAIFHAPRHALSPNTSPKPNSVQA
¤¤¤¤¤¤¤¤¤¤¤ :
SWISSPROT_P24665 EEY---SSNWAGAVLIGDGYTKVTGEFTVPSVSAGSSGSSGYGGGYGYWKNKRQSEEYCA
Seq ID No.27 QNFGWSASNWSGYAVTGSTYNDITGSWIVPAVSP----------------SKR--STYS-
: * :
SWISSPROT_P24665 SAWVGIDGDTCETAILQTGVDFCYEDGQTSYDAWYEWYPDYAYDFSDITISEGDSIKVTV
Seq ID No.27 SSWIGIDG-FNNSDLIQTGTEQDYVNGHAQYDAWWEILPAPETVISNMTIAPGDRMSAHI
: *
SWISSPROT_P24665 EATSKSSGSATVENLTTGQSVTHTFSGNVEGDLCETNAEWIVEDFESGDSLVAFADFGSV
Seq ID No.27 HNNGNGTWTITLTDVTRNETFSTTQSYSGPG----SSAEWIQEAPEIGGRIATLANYGET
SWISSPROT_P24665 TFTNAEATSG--GSTVGPSDAT--------------------------------------
Seq ID No.27 TFDPGTVNGGNPGFTLVPTRATWCRTTRSCLCRPHPTRIPTASTWPTAPTSRAHRPPDPR
SWISSPROT_P24665 -----VMDIEQDGSVLTETSVSGDSVTVTYV------------
Seq ID No.27 RSRRPCMEAQGPASFFARTLAPSRDVAAHAPQGHRPSALVRRA
*=形成Swissprot P24665活性位点的氨基酸
:=形成Swissprot P24665二硫键的半胱氨酸残基
¤=从Swissprot P24665酶原去除的前肽
实施例8:测定酸性β-葡聚糖酶活性
可以对在适当缓冲液中合成并分泌β-葡聚糖酶的宿主菌株的培养液或细胞裂解液测定该活性。将适当体积的这类样品点在琼脂糖平板上,所述平板含有不溶性显色底物AZCL β-葡聚糖(MegazymeTM)和适合的酸性pH缓冲液,如pH3-5。平板在适当的温度(如55℃)孵育适当的时间(如一天)。活性显示为斑点周围蓝色的色圈。
实施例9:测定酸性磷酸酶活性
可以将在适当缓冲液中在适当pH和适当温度(如55℃)合成并分泌酸性磷酸酶的宿主菌株的适当体积培养液或细胞裂解液与对硝基苯酚磷酸(pNPP)一起孵育以测定酶活性。酶促反应的产物为对硝基苯酚和无机磷酸盐或在合适的反应时间后加入Pi.NaOH以终止磷酸酶实验并形成对硝基苯酚盐。405nm处光学测量对硝基苯酚盐的吸光度。作为备选方案,使用在适当缓冲液中在适当pH和适当温度(如55℃)合成并分泌酸性磷酸酶的宿主菌株的适当体积培养液或细胞裂解液用EnzChek酸性磷酸酶测定试剂盒(E-12020)(Molecular Probes Europe BV;PoortGebouw,Rijnsburgerweg 10;2333 AA Leiden,The Netherlands)测量酶活性。
实施例10:测定多糖脱乙酰酶活性
使用在适当缓冲液中适当温度(如55℃)下合成并分泌多糖脱乙酰酶的宿主菌株的适当体积培养液或细胞裂解液测定活性。可使用细菌的胞壁质、N,N′-二乙酰壳二糖(Sigma)或半乳糖pentaacetate(Sigma)或/和乙酸纤维素(Sigma)作为该类型酶的底物。可以用适合酶物理需要的醋酸测定试剂盒(Biopharm)测量通过酶从底物中释放的醋酸(Kosugi A,Murashima K,和Doi RH;Xylanase and Acetyl Xylan Esterase Activities of XynA,a KeySubunit of the Clostridium cellulovorans Cellulosome for XylanDegradation;Appl.Environm.I Microbiol.;卷68;6399-6402页;2002)。
实施例11:测定内-β-N-乙酰氨基葡糖苷酶活性
使用在合适缓冲液(如pH3-5)适当温度(如55℃)下合成并分泌内-β-N-乙酰氨基葡糖苷酶的宿主菌株的适当体积培养液或细胞裂解液,根据MH Rashid,M Mori和J Sekiguchi;Glucosaminidase of Bacillus subtilis:cloning,regulation,primary structure and biochemical characterization;Microbiology;卷141;2391-2404页;1995测定该活性。
实施例12:测定肽酰脯氨酰异构酶活性
使用在合适缓冲液适当温度(如55℃)下合成并分泌多糖脱乙酰酶的宿主菌株的适当体积培养液或细胞裂解液测定该活性。可根据Fischer,G.,Bang,H.和Mech,C.;Determination of enzymatic catalysis for thecis-trans-isomerization of peptide binding in proline-containing peptides.;Biomed.Biochim.Acta;卷43;1101-1111页;1984测定活性。可对该实验进行适当的改进以适合特异的肽酰脯氨酰异构酶,如SEQ ID NO:36中包含的酶。
实施例13:测定酸性纤维素酶活性
可对在合适的缓冲液中合成并分泌酸性纤维素酶的宿主菌株培养液或细胞裂解液测试该活性。将适当体积的这类样品点在琼脂糖平板上,所述平板含有不溶性显色底物AZCL-HE纤维素(MegazymeTM)和酸性pH(如pH为3-5)的适当缓冲液。平板在适当的温度(如55℃)孵育适当的时间(如一天)。酸性纤维素酶的存在显示为斑点周围蓝色的色圈。
实施例14:测定木聚糖脱乙酰酶活性
使用在合适缓冲液中适当温度(如55℃)下合成并分泌多糖脱乙酰酶的宿主菌株的适当体积培养液或细胞裂解液测定木聚糖脱乙酰酶活性。木聚糖脱乙酰酶活性由从乙酰化木聚糖(通过Johnson等,1988(Johnson,K.G.,J.D.Fontana和C.R.Mackenzie.1988 Measurement of acetylxylanesterase in Streptomyces.Methods Enzymol.160:551-560)的方法由birchwood木聚糖制备)中释放的醋酸测量。可以用适合酶物理需要的醋酸测定试剂盒(Biopharm)测量通过酶从底物中释放的醋酸(Kosugi A,Murashima K,和Doi RH;Xylanase and Acetyl Xylan Esterase Activities ofXynA,a Key Subunit of the Clostridium cellulovorans Cellulosome forXylan Degradation;Appl.Environm.I Microbiol.;卷68;6399-6402页;2002)。
实施例15:测定植酸酶活性
可对在合适的缓冲液中合成并分泌植酸酶的宿主菌株培养液或细胞裂解液测试植酸酶活性。将适当体积的这类样品用0.1M醋酸钠和适当缓冲液中的0.01%Tween-20,pH 5.5稀释,该缓冲液可以是pH 3.0到3.5的HCl、pH 4.0到5.5的醋酸钠、pH 6.0到6.5的吗啉代乙磺酸(MES)和pH 7.0到9.0的Tris-HCl,并进一步在底物溶液(含5mM植酸钠[Sigma]的0.1M醋酸钠和0.01%Tween-20[pH 5.5],并在37℃预孵育)中稀释26倍,以起始反应。37℃30分钟后,加入等体积10%的三氯醋酸终止反应。通过加入等体积钼酸试剂测量游离的无机磷酸,100ml试剂含有7.3g FeSO4,1.0g(NH4)6Mo7O24·4H2O和3.2ml H2SO4。750nm处测量吸光度(Vmax微孔板读数器;Molecular Devices)(Lassen SF;Breinholt J;OstergaardPR;Brugger R;Bischoff A;Wyss M;Fuglsang CC;Expression,genecloning.and characterization of five novel phytases from fourbasidiomycete fungi:Peniophora Iycii,Agrocybe pediades,a Ceriporia sp.,and Trametes pubescens;Appl.Environ.Micr.;67;4701-4707页;2001)。
实施例16:测定磷脂酶活性
可对在合适的缓冲液中合成并分泌磷脂酶的宿主菌株培养液或细胞裂解液测试磷脂酶活性。向适当体积的这类样品中添加卵磷脂。在恒定的pH和温度下水解卵磷脂并根据中和释放的脂肪酸时的滴定剂(0.1N NaOH)消耗率测定磷脂酶活性。底物为大豆卵磷脂(L-α-Phosphotidyl-胆碱)且条件为pH 8.00,40.0℃,反应时间2分钟。单位(LEU)相对于标准定义。
实施例17:在枯草芽孢杆菌中表达谷氨酸肽酶基因(SEQ ID NO:2)
通过PCR将来自蛋白酶SAVINASETMTM(也称为地衣芽孢杆菌枯草杆菌蛋白酶309,来自Novozymes A/S)的信号肽与编码谷氨酸肽酶的基因(SEQ ID NO:2)框内融合。通过同源重组将编码产生的编码序列的DNA整合进入枯草芽孢杆菌宿主细胞基因组。在三元启动子体系(如WO99/43835所述)控制下表达基因构建体,所述体系由来自地衣芽孢杆菌α-淀粉酶基因(amyL)、解淀粉芽孢杆菌α-淀粉酶基因(amyQ)的启动子和包含稳定化序列的苏云金芽孢杆菌cryIIIa启动子组成。使用编码氯霉素乙酰转移酶的基因作为标记物(在例如Diderichsen等,A useful cloningvector for Bacillus subtilis.Plasmid,30,312页,1993中描述)。
通过DNA测序分析氯霉素抗性转化体以确认构建体的正确DNA序列。选择了一个这样的克隆。
在旋转摇床上于带有挡板的500ml Erlenmeyer摇瓶中发酵谷氨酸肽酶(SEQ ID NO:2)表达克隆,每个所述摇瓶含有100ml添加了6mg/l氯霉素的PS-1培养基。克隆在37℃发酵6天并在3、4、5和6天时取样并分析蛋白水解活性。用20微升培养液在pH3.4的0.1%AZCL胶原(MegazymeTM)LB-PG琼脂平板上以斑点测试测定活性(参阅实施例7)。将平板在55℃孵育(过夜)且活性显示为斑点周围的蓝色色圈。
实施例18:来自脂环酸芽孢杆菌的A4家族蛋白酶的纯化和表征
纯化
将培养液离心(20000xg,20分钟)并从沉淀上小心倒出上清液。组合的上清液通过Seitz EKS板过滤以去除剩余的芽孢杆菌宿主细胞。用柠檬酸将EKS滤出液调节至pH 4.0并在水浴中搅拌加热至70℃。溶液达到70℃时(从25℃到70℃大概用去15分钟),立刻将溶液置于冰上。该热激处理产生一些沉淀,其用另一Seitz EKS滤板过滤去除。向第二次EKS滤出液中添加硫酸铵至1.6M的终浓度并将混合物加至用20mMCH3COOH/NaOH,1.6M(NH4)2SO4,pH 4.5平衡的Butyl Toyopearl S柱上。用平衡缓冲液大致洗涤Butyl柱后,用相同缓冲液中的线性(NH4)2SO4梯度(1.6到0M)洗脱酶。分析来自柱的级分的蛋白酶活性(使用pH4.0测定缓冲液和37℃测定温度)并合并具有活性的级分。将合并的级分转移至20mM CH3COOH/NaOH,pH 5.5的G25葡聚糖凝胶柱并应用于相同缓冲液平衡的SOURCE 30Q柱。用平衡缓冲液大致洗涤SOURCE 30Q柱后,用相同缓冲液中的线性NaCl梯度(0到0.5M)洗脱蛋白酶。分析来自柱的级分的蛋白酶活性(pH4.0,37℃)并合并具有活性的级分。用1%(w/v)活性炭对带有轻微颜色的合并物处理5分钟,并用0.45pm的滤膜去除炭。滤出液的纯度用SDS-PAGE分析,其中在考马斯染色凝胶上只看到一个条带。
测定:
使用Protazyme OL(交联并染色的胶原)测定法。通过温和搅拌将Protazyme OL片剂(来自Megazyme)悬于2.0ml 0.01% Triton X-100。在Eppendorf离心管中混和500微升该悬液和500微升测定缓冲液并置于冰上。加入20微升蛋白酶样品(稀释于0.01%Triton X-100)。通过将Eppendorf离心管转移至Eppendorf热混和仪起始测定,混和仪设定为测定温度。管在Eppendorf热混和仪中最高摇动率(1400rpm)下孵育15分钟。通过将管移回冰浴上终止孵育。然后将管在冰冷的离心机中离心数分钟,将200微升上清液转移至微孔滴定板并在650nm处读出OD650。测定包括空白缓冲液(代替酶)。OD650(酶)-OD650(空白缓冲液)为蛋白酶活性测量值。
蛋白酶测定法:
底物:Protazyme OL片剂(Megazyme T-PROL)。
温度:受控的。
测定缓冲液:100mM琥珀酸、100mM HEPES、100mM CHES、100mMCABS、1mM CaCl2、150mM KCl、0.01%Triton X-100,用HCl或NaOH调节至pH值2.0、3.0、4.0、5.0、6.0、7.0、8.0、9.0、10.0、11.0和12.0。
表征:pH活性、pH稳定性和温度稳定性:
使用上述蛋白酶测定法获得pH活性谱、pH稳定性谱以及pH3.0的温度活性谱。为了测定pH稳定谱,用测定缓冲液5×稀释蛋白酶并在37℃孵育2小时。孵育后测定剩余活性前通过用pH3测定缓冲液稀释将样品转移到pH3.0。
37℃的pH活性谱
pH | 来自EXP00663的环状脂肪酸芽孢杆菌蛋白酶 |
2 | 0.90 |
3 | 0.98 |
4 | 1.00 |
5 | 0.93 |
6 | 0.77 |
7 | 0.28 |
8 | 0.04 |
9 | 0.02 |
PH稳定性谱(37℃2小时后的剩余活性)
pH | 来自EXP00663的环状脂肪酸芽孢杆菌蛋白酶 |
2.0 | 0.93 |
3.0 | 0.97 |
4.0 | 0.94 |
5.0 | 0.97 |
6.0 | 0.93 |
7.0 | 0.94 |
8.0 | 0.99 |
9.0 | 0.94 |
10.0 | 0.81 |
11.0 | 0.76 |
12.0 | 0.46 |
3.0 andafter 2hours at5℃ | 1.00 |
温度活性谱(pH 3.0时)
温度(℃) | 来自EXP00663的环状脂肪酸芽孢杆菌蛋白酶 |
15 | 0.08 |
25 | 0.19 |
37 | 0.60 |
50 | 0.94 |
60 | 1.00 |
70 | 0.89 |
80 | 0.45 |
其他特征:
由SDS-PAGE测定的A4蛋白酶的相对分子量为:Mr=26kDa.
实施例19:在枯草芽孢杆菌中表达酸性纤维素基因(SEQ ID NO:1)
通过PCR将来自TermamylTM(Novozymes)的信号肽与编码酸性纤维素酶的基因(SEQ ID NO:1)框内融合。通过同源重组将编码产生的编码序列的DNA整合进入枯草芽孢杆菌宿主细胞基因组。在三元启动子体系(如WO 99/43835所述)控制下表达基因构建体,所述体系由来自地衣芽孢杆菌α-淀粉酶基因(amyL)、解淀粉芽孢杆菌α-淀粉酶基因(amyQ)的启动子和包含稳定化序列的苏云金芽孢杆菌crailla启动子组成。使用编码氯霉素乙酰转移酶的基因作为标记物(在例如Diderichsen等,A usefulcloning vector for Bacillus subtilis.Plasmid,30,312页,1993中描述)。
通过DNA测序分析氯霉素抗性转化体以确认构建体的正确DNA序列。选择了一个这样的克隆。
在旋转摇床上于带有挡板的500ml Erlenmeyer摇瓶中发酵酸性纤维素酶(SEQ ID NO:1)表达克隆,每个所述摇瓶含有100ml添加了6mg/l氯霉素的PS-1培养基。克隆在37℃发酵3天并在1、2和3天时取样并分析纤维素酶活性。用20微升培养液在pH3.4的0.1%AZCL-HE纤维素酶(MegazymeTM)LB-PG琼脂平板上以斑点测试测定活性。将平板在55℃孵育(过夜)且活性显示为斑点周围的蓝色色圈。
附加说明(PCT/RO/134表)
关于“专家意见”的声名
保藏的微生物:
保藏号 保藏日期
DSM 15176 2003年6月30日
关于PCT/RO/134表所列的保藏微生物,我们需要所谓的专家意见:
在欧洲专利受权公告之前,或如果该申请已被驳回、撤消或视为撤消的情况下,自申请日期二十年内,保藏的微生物样品只提供给样品请求人指定的独立专家(参见EPC细则28(4))。并且就澳大利亚而言,同样请求专家意见,参见澳大利亚法规1991No 71细则3.25。同时,在加拿大我们请求只批准由委员会指定的独立专家有权获得保藏的微生物样品。
Bagsvaerd,2005年1月5日
诺和酶股份有限公司
Morten Birkeland,专利律师
序列表
序列表
<110>诺和酶股份有限公司
<120>脂环酸芽孢杆菌的多肽
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ttgaagactc gatggtcagg tgcgctggct gtgctcatcg ccctcggaac gggtgcctcg 60
cccgcttggg ccagtgtcca cagcgcggcc acgcacgcaa aggcgcacgt cggcgtgcgc 120
gctgcggata tggccgcagc gtccatgtcg gccgagattc agattctgca cgacgcgctc 180
acggcttccg agctgtcgtc cgtccaggcc gcggcacagg ccgccgccaa cctgcctgcc 240
tccacgtggg tgagctggct gtatccgagc gcctcctcgc cgagcgccgc acagacgcag 300
acggcgcagg ccctgggcgc gctcctcacc ttggtcacgt atggcgccgt cgcagacgat 360
ggccaaaaca tcgcacagaa tttgcaaacc cttcagtcga cttcgccgct cttatcgccc 420
gcggccgtct cgatgttcta tcaaaacttc ttcgtgctcg tcggccaatc gtccaaatcc 480
gtgctttcgg gccaggcaac cacctccacc gccggccacg ccctcgccca agcggccgcg 540
ctgacgccac agctcgccgc gtacctgcgc caatccggtc tttcgccgga cgatctcgcc 600
cgcgcctacg tgagctttgc ctccgccgtg gattcgcagg gcgcggcgca aacggctctc 660
ctgacgcgca tctgcaccaa catcctgggc tttggcgcgc cgacctccac ggcgaccatc 720
accgtcaacg ccgcggcgaa ccttggacag gtgccgacca ccgcgtttgg cctgaacgcg 780
gccgtgtggg acagcggtct caactcccag accgtcatct ccgaggtgca agcgctccac 840
cccgccctca tccgctggcc cggaggctcc atctcggacg tgtacaattg ggagaccaac 900
acgcggaacg acggcggcta cgtgaatccc gacgacacgt ttgatcactt catgcagttt 960
gtgaatgccg tcggctccac gcctatcatc acggtcaact acggcaccgg cacgccacag 1020
ctcgccgccg actgggtgaa gtacgccgac gtgacccacc acgacaacgt catgtattgg 1080
gaaattggca acgagattta cggcaacggt tactacaacg gcaacgggtg ggaggcggac 1140
gatcacgccg tggccggcca gccgcaaaaa ggcaaccctg gtttaagccc gcaggcgtac 1200
gcgcaaaacg ccctgcagtt catcaaggcg atgcgcgccg aggacccgtc catcaagatt 1260
ggggccgtgc tcacgatgcc gtacaactgg ccgtggggcg cgaccgtgaa cggcaacgac 1320
gactggaata ccgtcgtcct gaaggcgctc gggccctaca tcgattttgt ggacgtgcac 1380
tggtaccccg agacgcccgg gcaggagacc gacgccggcc tgctcgccga cacagatcaa 1440
atccccgcca tggtggcgga gctcaagcgc gaagtgaaca cctacgccgg atcgaacgcg 1500
aagaacatcc aaatctttgt gaccgagacc aacagcgtat cgtacaaccc cggcgagcag 1560
tcgaccaacc tgcctgaagc gctcttcttg gcggacgatc tcaccgggtt catccaggcc 1620
ggcgcggcca acgtcgactg gtgggatctg ttcaacggcg ccgaggacaa ctacacaagc 1680
ccgagcctct acggccagaa cctgtttggc gattatggac tcttgtcctc cggccagacc 1740
acgcaaaacg gttggcagga gccgcccgcc aacacgccgc ttccgcccta caatggcttc 1800
cagctggtct cggatttcgc gcagcccggc gacacgatgc tcggctccac cacgtcgcag 1860
agcgccatcg acgtgcacgc cgtgcgcaag ccgaatggcg acatttcgct catgctcgtc 1920
aatcgcagcc catccgccat ctacagcgcc aacctgaacg tgctcgggtt cgggccgttt 1980
gtcgtgacac atgcgctcgc gtacggtgaa ggctcgagcc gcgtggcgcc catgccggtt 2040
cttcccgtcc ccggcgcgcc catcaagctc atgccctaca gcgggatcga tctcaccctg 2100
cacccgctca ttccggcgcc acacgccgcc gcgcaggtga ccgatacgct cacgctgtct 2160
tcgcccacgg tgacggccgg cggtgcggag acgctctccg cctcgttcca ggcggatcga 2220
ccggttcatc acgccacggt ggagctcgag ctgtatgact cgacgaacga tctcgtcgcc 2280
acccacaccg tctcggatgt cgatcttcag ccgggatcgg ccacaagcga gacgtggagt 2340
ttcaccgcac cggccgcgaa cggcaattac cgcgttgagg cgtttgtgtt tgacccggtg 2400
acgggcgcga cgtacgacgc ggacacgcag ggcgcggttc tgaccgtcaa ccagccgcct 2460
caggcgacct acggcgacat cgtgacgaaa gacacggtca tcacggtgaa cgggacgacg 2520
tacgacgttc cggcacctga tgcgggcggg cactatccgt cggggacgaa tatttcggtg 2580
gcacccgggg acacggtgac cgtgcagacg acgtttgtca acgtctcatc gacggacgcg 2640
ctgcagaacg ggctcatcga catggaagtg gacggatcga acggggccat cctgcagaaa 2700
tactggccga gcacgactct tttgcctggc caatcggaga cggtgacggc gacgtggcaa 2760
gtgcctgcga atgtggcggc cggaacgtac ccgctcaact tccaggcctt caacacgagc 2820
agctggacgg gaaactgtta cttcacaaac ggtggcgtgg tcaacttcgt gatcagc 2877
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atgaacggca cctcagtctg gaaagcgtca ggcatcgcag ccgcctcgtg cctgacagcc 60
gcggcacttc tcgcctggcc ccacgccaca tccacgttgg acgcgtcgcc cgccatcttc 120
cacgcgccgc ggcacgcgct ctcgcccaac accagcccga aaccgaacag cgtccaggca 180
cagaactttg gttggtcggc gtcgaactgg tcgggatatg ccgtgaccgg cagcacgtac 240
aacgacatca caggcagttg gattgtgcct gcggtgagcc catccaagag aagcacgtac 300
tcttcgagct ggatcggcat cgacgggttc aacaacagcg atctcattca aaccggcacg 360
gagcaggact atgtcaacgg tcacgcgcag tacgacgcct ggtgggaaat cctccccgcc 420
cccgagacgg tcatctcgaa catgaccatc gccccgggcg accggatgag cgcgcacatc 480
cacaacaacg gcaacggaac ctggacgatt acgttgacgg acgtgacccg caacgagacg 540
ttctccacca cgcagtcgta ctcgggccct ggctcgtcgg ccgagtggat ccaggaggcg 600
ccggagatcg gcggccggat cgccacgctc gccaactacg gcgagaccac gttcgatccc 660
ggcaccgtaa acggcggcaa cccaggtttt accctgtccg acgcgggcta catggtgcag 720
aacaacgcgg tcgtgtctgt gccgtccgca cccgactcgg ataccgacgg cttcaacgtg 780
gcctacggct ccaaccagcc gagcccaccg gcctcc 816
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atgagaagac gcatgtcagg ctttgcgacg ggccttggca tcgcggcggg gctcgccctc 60
agttccgccc tcgccgcgcc gttcttccac gccgggaacg cgtccgcggc gtcgacgatg 120
tcgatggcgc cgacgagcac catgggcgcc ctgcccgcgc ccgaaggcgt gccggacgca 180
ggcccgctgt cgatcacgcc cgaggtcatt cgccaacaac aggctgacgc tgtccgggtc 240
atggacgaag aaggcctgaa gccacagatc ctctccggcg acatcaagcg attcaccctc 300
accgcgagcc aggtgaactg gtatttgtac cccggcaaag cggtcgtcgc gtgcggctac 360
aacggccaag tgcctggccc ggtcctccgc gtgcgcgtgg gcgatcgcgt ccaaatcctc 420
ctgagaaacg agctgaacga gcccaccacg ctgcacatcc agggcctcga tctgccggcg 480
tcgcagttgg gaatcggaga cgtcaccgaa tcccccatcc ctccgggcgg cgaacgcctg 540
tacagcttca ccgtgacgcc acagatggtg ggcacccacc tgtacgagag cggcacggat 600
atggccagcg agatcgaccc aaggactgca cggggtgctg ctcgtcgatc cggcccgggg 660
atccctttat ccccaggcga aggtggacgc gctcttcgag atcgacgcgt ggatggtgga 720
cggatcgacc accgaaaacg cgtttggcct ggacggcaag ccgtatcccg acgcgcccga 780
actgacggtg ccgtacggca gccgcgtggt gctgcgcatc gtcaacgcga gcgggatgtg 840
ctaccacgcc atgcacctgc acgagacgac gttttggctg ctggcggaag acgggcaccc 900
cctcgccaag ccgcggccga tgaacgtgct cgccatcgcg ccagg 945
<210>4
<211>1878
<212>DNA
<213>脂环酸芽孢杆菌属
<220>
<221>misc_feature
<222>(1)..(1878)
<223>CDS
<220>
<221>misc_feature
<222>(1)..(66)
<223>sig_peptide
<220>
<221>misc_feature
<222>(67)..(567)
<223>前肽
<220>
<221>misc_feature
<222>(568)..(1878)
<223>mat_peptide
<400>4
atgggcttgt ggaaacggct ggcgctcggc gtgcctgcgg cacttagcat gctcgcggtt 60
ggggtgcctg tgatgagcgc ggacaccgtg gaggctgcgc cgcttgcgaa tccttcaacg 120
gaaaatgcgc aggatatggg gccggcgagt gggagccaga cggtgacggc atccatcatt 180
ttgcgtgtgc agaatccgac ggcgctgcag aactacattc aagagacgga gacaccgggc 240
agtccgctgt accataagtt cttgacgacg gcgcagttcg ctcagcagta cgcgccttcg 300
gcggcgaccc ttcagcagat tgagcaggag cttcagggct atgggctcca ggtcgtgaat 360
gtcgacgcgg atcacctgga catgcaggtt cagggcacag ttcagcagtt tgacaacgcg 420
ttcaacaccg tgatcgacct gtttaaggca aacgggcaca tcttccgcgc gccgaagaag 480
ccgccgcaga tcccggtggc gcttctcacc aacgtgctcg ccgtggtggg actcgatacg 540
gcacaggcgg cgcagtcgct cacggtgaag acgccgaacg tcgcgggtgt gccttcgccc 600
aaggtggtgc ttccgcaggg aggcagcacg gcgacgggca cgccagggag ctacacggtt 660
ggggatacgg cgaatcgcta cgacatcaac ccactctatc agaagggtat cacgggcaag 720
ggcgagacca tcggcattgt gacgctgtcg agctttaatc cgcaggatgc ctacacctac 780
tggcagggca ttgggctgaa ggtggctcca aaccgcatcc agatggtgaa tgtggacggc 840
ggtggccaga tggatgatgg atcggtcgag acgacgctgg acgtggaaca gtcgggcggt 900
ttggcgccgg acgccaacgt cgtggtgtac gacgcgccga atacggatca gggcttcatc 960
gatgcgttct accaggcggt ctcggacaac caggcggatt ccctctcggt gagctgggga 1020
cagcctgaaa tcgattacct gccgcagatg aaccaaggcc agtcgtatgt ggatgagctc 1080
ctcgccttca cccaggcgtt catggaggcg gcggctcagg gcatttccat gtacgcggcc 1140
gcgggggatt caggcgccta cgacacggcg cgcgacttcc cgccctccga tggcttcacc 1200
acgccgctca gcgtggactt tcccgcctcc gacccgtaca tcacggctgc gggaggcacg 1260
acggtaccgt tcaccgcaaa gttctcgctc ggcacggtca acatcacgca ggagcagccc 1320
tggtcgtggc aataccttca aaacctcggc taccaagggc tcttctccgt gggcacaggc 1380
ggtggcgtga gcgtcatctt cccgcgcccg tggtatcagc tcggcgtggg cggcatgcaa 1440
aatagcgcgg ccaatcaggc cttcaccgac tcgcagggcg ttttgtacgg atcgcccttc 1500
acgtacaacc tgccgagcaa ttacgcgggc cggaatctgc cggacatctc catggatgct 1560
gatccggaga cgggctatct ggtctactgg agcgcgggcg gtggctggat tgcgggctac 1620
ggcgggacga gcttcgtggc gccgcagttg aacggtatca cggcgctcat tgatcaggag 1680
gtccatgggc gagtgggctt cctcaatccg ctgctgtaca ccctgttgac gcaaggggtc 1740
caaggtgggg cgcagccgtt ccacgacatc acgacgggga acaactggta ttggaatgcg 1800
gtgcctggtt acgatccggc ctcgggcgtg ggcacgccgg acgtcgcgaa cttggcgcag 1860
gacatcgcat cgctgaga 1878
<210>5
<211>1599
<212>DNA
<213>脂环酸芽孢杆菌属
<220>
<221>misc_feature
<222>(1)..(1599)
<223>CDS
<220>
<221>misc_feature
<222>(1)..(72)
<223>sig_peptide
<220>
<221>misc_feature
<222>(73)..(1599)
<223>mat_peptide
<400>5
atgcgagcgc tcgcacattt ggccattggc gccatcgcgt ccggcgtttt cgctgcacct 60
gtcgcttttg cctcgccggt tcaggaacgc gtggtggtgg cctcgcccga tccgcggacg 120
cgtagcgttc acgcggatgg cgaaatttca ccgtcgcagc cgatgcactt ggtcattacg 180
cttcgcctgc gccacgaggc gcagctcgag cagctgattc gagacctgta cacgccggga 240
tcgcccgatg caggtcactt cttgacgccc gcggcgttta acgcggcgta tgcaccgacg 300
gctgaggacg tgcaggccgt ggtccagggg ctgcgcgcat acggcctccg cgttgagccg 360
acggtaaatc ccatggtgct gaccgtgagt ggacgggccc gcgacgtcga gcgagcgttt 420
ggcgtgcatg agctccaatt tgggcgcgga gctggcgcat ggtacgcccc ggatggtgcg 480
gccacgctgc ctgcaccgct cgccgcgcgc gtgtcggccg tggtaggcct gacgagcgac 540
gcgatggagc gccacctcgt cctggcgcac gtcgcgccgg caggaggtgg ctacacgccc 600
gcgcaaattc agcgcgccta cgactatacg ccgctctaca gccaatacat ggggcgcgga 660
caggtcattg cggtggtgac ttccggctcg gtgctccgct ccgacctgct cgcgttcgat 720
cgcgccttcg ggcttccgaa tccggtggtg cgccagcggg tgatcgacgg atcgtccacg 780
tctcccgacg acgagaccac cctcgactgc gagtgggcgc atgccatcgc gccgacggca 840
tcgctcgccg tgtacgaggc cgctcaaccg gacgcgcagt cgttcatcga tgcgtttgcc 900
caggtggcgg cggacgatgg cgcgcatgtg gtcacgacga gttggggagc gcccgagtcg 960
gagaccgacg cggcgaccat gcaggcggag caccagatct tcatgcagat ggccgcccag 1020
gggcagagcg tgttcgccgc ggcgggcgac agcggatcgt cggacggaac aagcgggacg 1080
gacgtcgact atccgtcgtc ggatccgtat gtcaccgcgt gtggcgggac gaggctcgtt 1140
cttggtgcgg gtgcaaagcg gctgcaggag acggcgtggg ccgacacggg cggcggcgcg 1200
agctcggtgt acggagagcc gtggtggcaa tatggcccgg gcgtgccgca gacgggctat 1260
cggcagacgt gcgacgtcgc cctgaacgcc gatccggcca cgggctacga tttcatctat 1320
gagggtcagt gggaggtggc cggggggacg agctttgtcg cgccgatgat ggccgcgacg 1380
tttgcgctca tcgaccaagc gcgtgccctc gaaggtaagc cacccgttgg gctcgcagac 1440
gtcggcatct atgcgatggc gcgcaacgcg tcctacgcgc cgtacgcatt ccacgacatc 1500
acggccggat cgaacggcgc gtacagcgcg ggcccgggat gggatcatcc aaccggcttt 1560
ggttccatcg acgcgtacta ctttttgcac gggctcgac 1599
<210>6
<211>1233
<212>DNA
<213>脂环酸芽孢杆菌属
<220>
<221>misc_feature
<222>(1)..(1233)
<223>CDS
<220>
<221>misc_feature
<222>(1)..(123)
<223>sig_peptide
<220>
<221>misc_feature
<222>(124)..(1233)
<223>mat_peptide
<400>6
atgcggcgtc gacgttggga ttacgaggac tggccgagtg agaacaggcg tgtcggcgtg 60
tggctcgcga gcgggaccgc gctgcttgcc atctgctaca tcctcggcat ctggacgggt 120
gcggcgctca cgcgcggtca ttcccagacg accgtggaat acgttcctcc ccagacgggc 180
aacaccgcga gcacgtccgg atcgctcacg ccgatcccgg gcgtcgagga cacgaccata 240
gtgacgcaga tttataaccg agtgaaaaat agcatcttta ccattacggc cgtctccgga 300
ggcaagccga cgtcgagcga cgcagaagaa gatatcggca cggggttcct gatcgatcac 360
aacggcgatc tcttgaccaa cgcgcatgtc gtcggatcgg ccacaacggt ccaggtgtcc 420
ggggacaacc gccaattcgt cggccgcgtg attgacgccg accagctgga cgatctcgcc 480
atcgttcgca tcccggcgcc caaatcgctg gaaccgctgc cgttgggatc ggtgaagtcg 540
cttcagccgg gcagcctggt catcgccatc ggcaacccgt ttgagctgac ctcgagcgtc 600
agctcgggca tcgtgagcgg actcaaccgg tcgatgtccg agtcgaacgg gcacgtgatg 660
aacggcatga tccagacgga cgcgccgctc aaccctggaa attcgggagg cccgctgctc 720
aacgcggcag gacaggtcgt cggcatcaac acgctgatcg aaagccctat cgaggggtcc 780
atcggcattg gctttgccat tcctatcgac cggtttatcc agctcgagcc agaattgctc 840
gccggcaaac ccgtcgcgca cgcctggctc ggcatcgagg gaatggacat cgacaacctg 900
atgcgtcaag cgctgcactt gcctgtggcc tcgggcgtct atgtgaccga agtgaccccg 960
ggcggccccg ccgcgaaagc ggggctgcgc ggagattcga acgcggccaa gttgaacagt 1020
ctaagccagt cggccaatcc gtacgcgctg ctcaagggga acggggacat catcgtcggg 1080
attgacggca agcaggtctc cagcatcgaa cagttgacgc aggatatcaa ccaagatcaa 1140
ccgggtcaga cggtggtgct caccgtgttg cgcgcaggca aaaccctgca cgtgcgcgtc 1200
acgctcggga cctggccatc cagccaaaat ccg 1233
<210>7
<211>633
<212>DNA
<213>脂环酸芽孢杆菌属
<220>
<221>misc_feature
<222>(1)..(633)
<223>CDS
<220>
<221>misc_feature
<222>(1)..(90)
<223>sig_peptide
<220>
<221>misc_feature
<222>(91)..(633)
<223>mat_peptide
<400>7
atgcgcaggt cttggagcgt gctcatggcc gtttgcatgt cttggttggc ggtggggtgt 60
ggcacgcctg caaactcgtt gtcacaagcg accgctgcgt ctggaaggca cgcgccgcac 120
cccctcgtgt ttcagaacct cacaggtgcc atgaacgagg ggcaggatcc ccggtgggac 180
ccgaaagcgg ctcccacggg tgtctacgac gacgtgaccg tggtcacagc gagtggccga 240
caggaggtgc tctccgttcg ggatgcgccg ctcctgttcg cagcgtactg gtgccctcac 300
tgccagcgca cactgcagct tctcacgtcg attgaatcac gcctgaagca aaagcccatt 360
cttgtgaacg tcggctatcc tccgggcacg acactgcaga ccgcggcgcg catcgcgcgc 420
gaggagtctc aagttcttca cttggcgccg ttccaagagg tctttatctt gaatcctgat 480
gcaggggatc gatacgcccc gctagggtac ccaacactcg ctttttatcg cgccgggcga 540
gattggacgc tgtacggtga acatcgagcg tctatttggg aaaaggccct gtccgaatcg 600
acatcaaaag cgtacaatgg cagcgaggaa tca 633
<210>8
<211>798
<212>DNA
<213>脂环酸芽孢杆菌属
<220>
<221>misc_feature
<222>(1)..(798)
<223>CDS
<220>
<221>misc_feature
<222>(1)..(87)
<223>sig_peptide
<220>
<221>misc_feature
<222>(88)..(798)
<223>mat_peptide
<400>8
atggatgaga tgaacattcg atcttggtgt gtcgctgctt gtaccgtagc cttgacaagc 60
gccgtgggcg cgacgaccgc gttcgcgcag acggtgaccg tacaacccgg acaatcgctc 120
tggaccatcg cacgcgcaca cgggatgccc gttcagttgg tggcgtccgc caatccgcag 180
tacaatccgc tgaatctccc tgttggtgcg accgtcacac ttcccagtct caaggacgtg 240
gctgtgcagc cgggcgactc cctgtttctg atcggcaggc aatatggcgt gtcgctcgcc 300
gagatgttgg ccgcaaaccc gaacgtggat ccattgaatc tgcaagtggg ttcaagtgtg 360
cgtgttcccc ttgcatcatc ttcgaccaag agctccacag tttctgccca tgttgccgca 420
tccacgcccg aaaactccaa caacctgtac tggttggagc gcgtcattca cgcggaggcc 480
ggcggagaat cgctgcaggc acaaatcgcc gtggccgacg tcattctcca tcgcatggcc 540
gcgggtggat acgggagcac ggtgcaacaa gtggtcttcc aagtgagcga cgggcactac 600
caattcgaga gtgtcgcaaa cggttcgatt tacggtcagc cagacgcaca aaacgtgcag 660
gctgctctcg acgcgttgaa cggagacgat gtcgtcccag gcgcgttggt cttctacaac 720
cccgcgcaga cgccttccgg aagttgggtt tggcaacaac ctgtggtcgc tcatatcggt 780
catctcgtgt ttgcgaag 798
<210>9
<211>2304
<212>DNA
<213>脂环酸芽孢杆菌属
<220>
<221>misc_feature
<222>(1)..(2304)
<223>CDS
<220>
<221>misc_feature
<222>(1)..(78)
<223>sig_peptide
<220>
<221>misc_feature
<222>(79)..(2304)
<223>mat_peptide
<400>9
gtgaagacgc atcgcctgct cgcggtcgcg gcactgcctg caacagtgct gttgacaacg 60
ccggcgcccg cgctggctga gacctcgagc tcgcagagcg cttcggcgcc gtcgctgaac 120
gtgccggtcg ctgccctgac cctcgcgggt gttcaatcgt atcccatgct gagctacgga 180
tccacgggcg tgtacgtgga aattttgcag aacgccctga atgccctggg ctatgacgtg 240
ggacaagcca gcgggctgtt cgacgccacc acgcaggccg aagtgaaggc ttttcagcag 300
gcgatgggcc tgcagacgga cggcattgtg ggtcccctga cctggggggc tttggcgaag 360
gcggtggccg attatcgcca ggtgatgacc gtactctcca gtcgcagctc gctggttcag 420
caagtcgaat ggaagcgcat cgtatggaac ggcaggttga tttcgaagcc catcggcttc 480
acgtaccagg ggacagcgta catgcccatt tggtacgtca tgcaggcgct tagcaaggcg 540
ggcattgcga gcacgtggca gggaggggtt tggacgctca cgccgcccgg aggtcagacc 600
gtgaattacg gaaagatctc gtacgggccg ggcagtgcgg ccatcgccat cggccagacc 660
gtggtcgcca atgtgcccgc ggtggtgtac cctgatccgg catccggaaa gctcacgacc 720
ttcatgcctg tttggtacgt catgaacgcg ttgcagcggc tgggcatcgg ttcgacgtgg 780
cagggaaccg agtgggacat gaagccagct cccgtcgtga tcgagacggg cgatccgtcg 840
aacaacacca ccgggtcaga tcccgcgaac agcacgggca acggcaccgg gaactcgacg 900
ggcaacgcca cgggcgccgt gccaggcggc aataccgtga cgaacgtcac cacgggctcg 960
tccaacgtca ccggcaactc gacgggcaac agtttgggga actcgacggg caacagcttg 1020
ggcaacagca cgtcgaacgc gacgggcaat gccaccggca acaccaccgg gaatgcgacc 1080
ggcaattcca cgggcacgag cagcgggtcg ttcacgaatg tcgacctgcg ctatccggcg 1140
ccgtccaaca tcaatgcgca gagcatcaac cagtttctgc tgcagaacag ctcgccgctc 1200
aatgggctgg gcaattcgtt catggacgcc cagaacctgt acagcgtcga cgccaactac 1260
cttgtctcgc acgccatcct cgagagtgcg tgggggcaaa gccaaattgc ccttcagaag 1320
aacaatctgt ttggctacgg cgcttacgat tcgaaccccg gacaggatgc gggcgtattc 1380
ccgagcgacg actacgccat ccgattcgag gcgtggaccg tgcgcatgaa ctacctcacg 1440
ccgggcgcga gcttgtacgt gacgccgacg ctcagcggaa tgaacgtgaa ctacgccaca 1500
gccaagacct gggcaagcgg cattgcggcc atcatgacgc agtttgcgag ctccgtcgga 1560
tcgaacgtga atgcgtacgt gcagtacacg ccgtccaaca atccgcccgc tccgagatcg 1620
acagcggaac cggtgtacta catgaacggc gcgcaagggg taacgcagca ggatccgtat 1680
tacccgaatg gcggcgttcc gtactacccg accatcgcgc agggtgagaa tcagcagttc 1740
tttggccagc taagtgtcgg cagcttcggt caacccgtgg tggaggttca gcagttcctg 1800
aaccggacca tcaacgcggg gctgaccgtg gacgggcagt ttggcccgct gacgcaggcc 1860
gcggtcgaga agttccagtc gcaggtcatg cacatgtcga acccgaacgg catttggacg 1920
ttcagcatgt gggtccagta catccagcct tctcagtcga acgccaatct catcccggct 1980
gggaccaccg tgaaaattga ccaggtcgcc gagggcatgg cgggcccgta cgtcgtgcct 2040
tggtaccacg tggtgggcta tggctgggtc gactcgcagt atatcaagtt gaccaacgtg 2100
tatcgcgtca ttgtgcagaa cccggccgga acggccacca ccattcccgt ctaccaggtg 2160
ggcaacctgt cttcggtatt gctcaatctg cacagcggag actgggtggt tgccaactca 2220
gcgcagccct cgggcggcgt gtacaccatt cagattgcgg ctcaggatcc accgtgtcga 2280
acggctacgc cgccgggacg ctct 2304
<210>10
<211>1791
<212>DNA
<213>脂环酸芽孢杆菌属
<220>
<221>misc_feature
<222>(1)..(1791)
<223>CDS
<220>
<221>misc_feature
<222>(1)..(147)
<223>sig_peptide
<220>
<221>misc_feature
<222>(148)..(1791)
<223>mat_peptide
<400>10
atgatggccc acgatagatt ggacaggcga gtgaatgaga ggaggcaagc catgcgacgc 60
gcggcaaaat gggcaatcgc ccttggcacg acggcagtgg tggctggtgt cagcagcgtg 120
ttcgcacttc gcagtgtgcg agaagcaaac ctgaatccca acgcccctct cgcgaacgtg 180
cccgggcctc agggcgccta tacgcccatc agcgcgcttc agcccgtcgt tccgaaaaac 240
gcgcggatcg accactacac gctgacggcg gaatcccgca cactgaccgt cggcggccat 300
gccctgcaag ccatgacgtt caacggcacc gcgccagggc cgttgcttgt ggcccatcaa 360
ggcgacgtcg tgaaggtcac ggtgcacaac cgcctctccg tccctctgac cattcactgg 420
cacggcatcg cggtgcccgg cgcggaagac ggcgtccctg gtgtcacgca aaacccaatt 480
ccgcctggcg ggagctacac gtacgagttt caggttaacc agcccggaac gtactggtac 540
cactcgcacg aggcgagctt tgaagaggtg ggcctcgggt tgtacggcgc cttcgtcgtt 600
ctgcccaaac gggcggtcca tccggccgat cgcgactaca cgctcgtcct gcacgagtgg 660
ccgaccgcat ccaccgcgca gacgatgatg gcgaacctca aggctgggaa cttgggattc 720
tcagcgaaag gcgaatccgc aggcatgggc ggcatgggca tgcaacaaaa cggggacatg 780
aacggcatgg gcatgatggg cgcggcggac ggcacgggtc agggaggaaa tagcgcgagc 840
gacatcgcgc acgtgttgcc tggccccccg cttcaactga acggtttttc gccgaccgca 900
aacgattggg ctgcgcttga cgaaatggcg ggcatgtatg acgccttcac ggtgaatcag 960
aacgcgagcg gtacaacgct cttgccagcc aagccgggac agctcgttcg gcttcgcatc 1020
gtgaacagcg gcaacatgac acacctgttc acgctggtcg gcgcaccgtt tcgcgtcgtg 1080
gcgctcgacg gccacgacat tgccaacccc ggttggatcc gcggcgtctt gcttcccgtc 1140
ggcgctgcag agcgatacga catcgaattt cgcgtgccaa agtccggggc cgcattcctt 1200
gtgtgcgccg atcccgacac gactgcacag cgcgagcttc gcgccgccat cggtctgccc 1260
gacgcctggt cacaattcaa ggagacggat gcagcgagcc ttgaacgagc gccgtggttc 1320
gactttacac actatggcag cggcaggctg cccggcgaag ccgtgttccg cctgcatcag 1380
gcgtatcagg tacgctacaa catgaagctc accgtcggca tgtcgatgaa cggcatggtg 1440
tacgccatca acggcaaggt ctttccgaac atcccgccca tcgtcgtgcg aaagggcgac 1500
gccgtcctgg tccacatcgt gaacgacagc ccctacattc acccgatgca tctgcacgga 1560
cacgactttc aagtgctgac gcgcgatggg aaacctgtct ccggaagccc catcttcctg 1620
gacaccttgg acgtgttccc cggcgagagc tacgacatcg cgtttcgcgc cgacaacccg 1680
ggtttatgga tgtttcactg tcacgatctc gaacacgccg cggccggtat ggacgtcatg 1740
gtccagtacg cgggcatccg cgatccctac ccgatgagcg agatgtcgga g 1791
<210>11
<211>735
<212>DNA
<213>脂环酸芽孢杆菌属
<220>
<221>misc_feature
<222>(1)..(735)
<223>CDS
<220>
<221>misc_feature
<222>(1)..(87)
<223>sig_peptide
<220>
<221>misc_feature
<222>(88)..(735)
<223>mat_peptide
<400>11
atgaaacgtc ggaccttgct tgcgggcatc acgctggcgg cgctcgtcgc ggtggcgggc 60
tgtggcacgc cggccggtaa caccgcctcg ccggacaaca cagcgaactt gtcgaacacg 120
aacgcgccgg acacgctgtc caatgaaacc ggccagacgc tcgatacggc caacccgccg 180
tacctgcaca cgtcgaccga gcagtggaag agcatgccga agatgttcat caacccgaac 240
aagacctatg acgccattgt ccacaccaat tacgggacgt tcaccatcca gctgttcgcc 300
aaagacgcgc ccatcacggt gaacaacttc gtgttcctgg cagagcacaa cttctaccac 360
gattgcacgt tcttccgcat cgtgaagaac ttcgtgattc aaacgggcga tcctcgcaac 420
gacggtaccg gcggcccggg ctacaccatc ccagatgaac tcagccatca ggtgccattc 480
acgaagggca ttgtcgcgat ggccaacacg ggccagccgc acacgggcgg aagccagttt 540
ttcatctgca cggccaatga cacgcaggtc ttccagccgc ccaacaatcg ctatacggaa 600
ttcggccgcg tgatctccgg aatggacgtg atcgacaaga ttgccgccat cccggtgacc 660
gaaaacccca tgacgcagga agacagctat cctctgaaga ctgcgtacat cgagtcgatt 720
caaattcaag aatcg 735
<210>12
<211>1824
<212>DNA
<213>脂环酸芽孢杆菌属
<220>
<221>misc_feature
<222>(1)..(1824)
<223>CDS
<220>
<221>misc_feature
<222>(1)..(81)
<223>sig_peptide
<220>
<221>misc_feature
<222>(82)..(1824)
<223>mat_peptide
<400>12
gtgaagaagg gaaagagatg gtccgccgcg ctcgcgacgt ccgtggccct gtttgccacc 60
ctgtcgcccc aagcgctcgc cagcgacacc gtggttccgc aagtgaacac gctcacgccc 120
attcatcacc tcgtcgtcat cttcgacgag aacgtctcct ttgatcacta tttcgccacc 180
tatccgaacg ccgccaatcc agccggcgag ccgccctttt acgccgcgcc gggcaccccg 240
agcgtcaatg gcctgtccgg aagccttctc acgcacaatc ccaacggcgt gaatccgcag 300
cgcctcgacc gttcccaagc cgtgacgccg gacatgaacc acaactacac gccggagcag 360
caggccgtgg acgggggccg catggataac tttatcaata cggtcggccg cggaaatccc 420
atcgatctcg actactacga cggaaacacg gtcaccgcgc tctggtatta cgcgcaacac 480
ttcgccttga acgacaacgc gtactgcacg cagtacggcc cgtctacgcc tggcgccatc 540
aacctgattt cgggcgacac cgcgggagcg acggtttatt cttcaagtga gaccagcggc 600
gccgcacaag tcgtgccacc cggcagcaaa aactttccga atgccgtgac gccaaacggc 660
gtcgacatcg gcgacatcga tccctactac gacagcgcct ccaaaggcat gaccatggcg 720
atggccggca aaaacatcgg cgacctgtta aacgcgaagg gggtcacctg gggctggttc 780
cagggcggct ttgcaaatcc gaacgccaag gacaacaata tcgccggcac agatgaaacc 840
accgattaca gcgcacacca tgagccgttc cagtattatg cgtctacggc aaatccgaat 900
catctgccgc ctacgagcgt ggcgatgatc gggcgcacgg atcaggcaaa ccaccagtac 960
gacatcacga atttcttcca agcattgcaa aacggaaaca tgcccgccgt gagtttcctg 1020
aaagctcccg aatacgaaga cggtcacgcc ggctattccg atcccctcga cgaacagcgc 1080
tggctggtcc agaccatcaa tcaaatcgag gcgtcgcccg attggtcctc caccgccatc 1140
atcatcacct atgacgactc ggatggttgg tacgatcacg tcatgcctcc gctcgtgaac 1200
ggatcgagcg acaaggccgt ggacgtgctc ggtggcacgc cggttctgca aaacgggacc 1260
gacagggcgg gctatggacc gcgggtgccg ttcctcgtca tctcgcccta cgccaaacac 1320
aattttgtcg ataacacgct catcgaccag acttccgttc tgcggttcat cgaggagaac 1380
tggggcctcg gctcgttggg cccagcgtcg tacgactcgc tcgccggatc gatcatgaac 1440
atgtttgact ggaacacgca gaacccgcct gtgtttctcg atccgacgac cggtgaaccc 1500
gtgtccccag atatgcagcc ggaggtcatt cgcggcacca cgtatctcag cctgaatcac 1560
tacgctcaaa acctcgatgt cgtgctgcaa acctctcggg ggatggcgcg gttctcctac 1620
gaggggcacg aggtcgagat cgacgagcgt tccgggcttg tccgggtcga tggcgaagcg 1680
gtccatctca aggcgcctct tgtgcgggtg gacggcgtat ggatggtgcc cgtagaggaa 1740
atggattcgc tcattggggc cacgctgcac acctacaccg acggtcatct cacctactat 1800
ctcttttctc cgcaagacgc ccat 1824
<210>13
<211>750
<212>DNA
<213>脂环酸芽孢杆菌属
<220>
<221>misc_feature
<222>(1)..(750)
<223>CDS
<220>
<221>misc_feature
<222>(1)..(75)
<223>sig_peptide
<220>
<221>misc_feature
<222>(76)..(750)
<223>mat_peptide
<400>13
atgctgagct tgtggaagcg aatccgaacg ggaacactct cacttctggc tgcatgcgcg 60
tgcgcgctgt cggcgatggg cgctggggca ggatgggtgc atgcggctga gtcccaagcg 120
caagccccaa gggccattta caaggtggac acgaaggaaa aggtggtcgc tctcacgttc 180
gacatctcat gggggcaccg cacgcccgaa ccggttctcg agacactcaa gaagtgcggc 240
gtgaccaagg cgacgttttt cctgagcggt ccttggacca tgcaccacgc ggacatcgca 300
aagaaaatca aggcgatggg ctacgaaatt ggcagccatg ggtacctgca caaggactat 360
tccaattacc cggactcttg gattcgagaa caggcgatgc tcgcagacaa ggccattcaa 420
caggtcactg gggtcaagcc gaagctgttc aggacgccaa atggcgactt gaatccgcgc 480
gtcatccgct gcctgacgag catgggctac acggtggtcc aatggaacac cgattcgctt 540
gactggaaaa acccaggcgt cgacgcgatc gtcaaccgcg tcacgaagcg cgtggtgcct 600
ggcgatatca tcctgatgca cgcgagcgac tcgtccaaac agattgtgga ggccctgccg 660
cgcatcattg aatcgcttcg gcagcagggc taccggttcg tcaccgtctc cgagctgttg 720
gcgggcgcca gcgttcaatc caaggtccag 750
<210>14
<211>972
<212>DNA
<213>脂环酸芽孢杆菌属
<220>
<221>misc_feature
<222>(1)..(972)
<223>CDS
<220>
<221>misc_feature
<222>(1)..(63)
<223>sig_peptide
<220>
<221>misc_feature
<222>(64)..(972)
<223>mat_peptide
<400>14
atgcggaaga cggctgcagg cgcgtgcgcc ctggcgctga tgggggtctt gggcggttgg 60
gcgggcgcgg ccggcacggc ggtgaacgcg cacgcgccgg cggcgtcggc gccaagtgtt 120
tcggcacatg tgtgggaaga agtcagccgc acgtggggaa cgcttcccgt cgatgcccgc 180
cacgacggcg tgtggcacaa catccccggt ttgtcaggct ttgcgctcga cacggcggcg 240
agcgagcgcg agaccgcgcg gcgccatgac ggcgcgctcc acctggtatg gcgaaccctt 300
ccgccgaagc gaagactcgg agacctttcg cccgacgtga tttaccgcgg ccccgcgcag 360
gagaagtcgg tggcgctgat ggtgaatgtg tcctggggcg atgcgtacgt gcccaggatg 420
cttgaggtgc tgcgcagcgc gcacgtgaag gccacgtttt tcgtggacgg cgcgtttgcg 480
aagaagttcc ccgatctcgt ccgcgcgatg gcgcgagacg ggcacgcggt cgagtcccac 540
ggctttggac acccagactt tcgccggctg agcgacgcga agctcgccgc ccagcttgac 600
gagacgaatc gagtgctcgc cggcatcacg ggcaaggttc cacggctcat cgcgcctccg 660
gccggatcgt atgatgcgcg cctggctccg ctggcgcatt cgcggcgcat gtacgccatc 720
ctgtggaccg cggataccgt ggactggaaa aacccgcctg cggatgtcat cgtccaacgc 780
gttcagcgcg gtgcggaacc cggcgcgttg atcctgatgc atcccacggc gcccacggcg 840
gaggccctgc ctgatgtgat ccgctggctc gaggggcacg gttatcggct gaaaacggtg 900
gaggacgtga tcgacgaacg cccagcggtc acccctccga cgacgctggc gaacgagacg 960
ttccacagcg cg 972
<210>15
<211>642
<212>DNA
<213>脂环酸芽孢杆菌属
<220>
<221>misc_feature
<222>(1)..(642)
<223>CDS
<220>
<221>misc_feature
<222>(1)..(87)
<223>sig_peptide
<220>
<221>misc_feature
<222>(88)..(642)
<223>mat_peptide
<400>15
atgatgcgtt ggaattggaa ggttgctgtg ggatcgttgg cgttggccgc actgggcgca 60
ggggcggcgg tgtcgccggt gtttgcggcg gcgaagtcgt cgaaggccgc gcagtcccac 120
gcagaggcga gcgcggcagt cgtgatggct gggaagctgt acggcaacat tccgaacgtc 180
accattcgcg gcgtggaagc tgggaaggcg ccgtgggtcg tggacggatc gtaccagctg 240
aagagcaacc tgttcacggc gagtgggaag tggctcatca ttccgaagca gggctatatg 300
gagaacggtc agccggttcc ggccaaaatt ggcggcacga cgaacaacat tccggccgtc 360
ggggccgaaa tcacgtttgc aaacgcggcg cccattgtgt tgccgccggt caagctgtcg 420
agccaaggtg acttctcgtt ccacgacgcc atccagtggc cgaagggtgc cgcgcagccg 480
gtcatcctga ttgggcccga gaagaacggt cagctcgtcg cgtggtttgc ggcgtcggac 540
ttcctcgccg actacggcca ggcgacgggc atgggcggcg gatgggtgaa cgcggcgcat 600
ccagagactc ccgtgcggca cacccacctc gcttcgaaga ag 642
<210>16
<211>771
<212>DNA
<213>脂环酸芽孢杆菌属
<220>
<221>misc_feature
<222>(1)..(771)
<223>CDS
<220>
<221>misc_feature
<222>(1)..(63)
<223>sig_peptide
<220>
<221>misc_feature
<222>(64)..(771)
<223>mat_peptide
<400>16
atgaactggg cgcgtgtcgg cgcgtgggta tccacctggc tggtggctac ggcgcttgga 60
gctggctgtg ggacggcttc gcaagagcat ccgtccaaca cctccacgtc agatcaccgc 120
gttgcgcccg cggcgccagg cggctccgcc tcgatgcaaa accggcatat tctgcaggag 180
ccgctgccgc gtggcgtgaa aacggaaacg gatttgtaca actggctttt atggcagaga 240
ctcgccgaga tcaacaatcc ggcgcagggt gaaatctgcc tggacgccgc atgcaagatt 300
gcggccaccg tcttttctgg cccggccaag gccgcggccg gcacgcctgt cactctggtg 360
gcgttttcgc cgcgggcggg ttggcaggtg ctcgtgggtc cgctgcccca gtcggacaac 420
cctccgcgtc aagcacaatc catcacaggc cagtctgcgc gactacccgc gcaaagaggg 480
cgtatgcgtc gttcaaaccc acgaaatcga ctggtactgg attcaggacg gacacctgca 540
gctgatgcgt cagccgcgcg catgacgcgt cagctaaggc gatccgccag ctcgacgaac 600
gcgtcgagat cgcgcagggc aaagtcgatg gcgcgctgcc aaaagtcagg ttgcgtgaga 660
tccgcaccga tgtgtttttg ggccagatcc tcgacccgca tgcgaccggt gtcgcgaagc 720
aacgccacat acttgtccgc aaatcccgtg ccttccgctg aggccatggc a 771
<210>17
<211>3390
<212>DNA
<213>脂环酸芽孢杆菌属
<220>
<221>misc_feature
<222>(1)..(3390)
<223>CDS
<220>
<221>misc_feature
<222>(1)..(72)
<223>sig_peptide
<220>
<221>misc_feature
<222>(73)..(3390)
<223>mat_peptide
<400>17
ttgaaacgca cactgagtgg cattgcttca gctgcaattg ttctgggtgc gattagcccg 60
atggcgtttg cgcagacctc gtccagcggt ctcacgccgg ccggtcagtt gcctatcgtc 120
gtcaatggac aggttctgtc gaacccgtat gagatggtgg gcatggactc cggcaacaag 180
acgggcttct tcccgattta ctactttgac caggcgcttg aaaagattgg catcacggcg 240
acctggaatg gtgcaaccca cacctgggcg ctgacggact ccaacgtcaa tgcttcgaac 300
gtccaagtcg cgggtggtat gggcacgggg aacaccacgg tgaccctgaa cggcacgccg 360
attaagatgt tctacaccca ggttgcgaag gacccggcgg gtggcccggt cacgacgtat 420
atgccgattt actatatcaa caacatcctg agtgcgcttg ggatccatgg aacctttagc 480
ggacagacgg gtctcaacat taccaccggg cagacgcttg ccggtagcct gagtgccatc 540
acggtgacgg gggcgacgag cggtacgggg acctcttcga gcccggctgt ggcgttgaat 600
aacggcaagg ttacgctctc gacgactctg acggattcga atggcaatcc gattggcaac 660
gcggcggtca ccttcaactt ctctgaatat ggtgcgctgc cttcgaatgc gccgacggtc 720
accaatgcgt cgggtgcgac aattccggcg accaccggct cgacggctta tcagtacacg 780
gtctacacca actccagcgg tgtggcttcg atcacggtgt ctgggcccgt tggcttgacc 840
tacgcatacc aggtgactgc gacggcgccg atcagcaatg gcagcaatca aatgattagc 900
agccagccgg cgtatgtcga gtttgtcgcc aacaaccagg cgggtattgc gccgtacggc 960
acggcttctc aaccgtactc ggcttcgctg ggtaccgcag ttcccatcac ggtgattttg 1020
ccgccgggtg cgaacggtca gccgcaggcg aatgtgctcg tgaccctgtc gctgagcaac 1080
ccgaatggtg gcaccaacta tgcatacttc accaactcgt cgggtgcgaa tctgggcacg 1140
caaatccagg tgacgaccaa ctcgtcgggt gtggcgcaag cgtgggtcag cgacgcgaac 1200
gcgcagcctg ttgtcgtgac ggccaatgtg tcgaatgcga ccaatgtcag caacacttcg 1260
gtgagcacct acctgaactt tggtcaggca ggcgtgccag catcgatcgc caattacaac 1320
gatccgtatt cggctttggt ggccaacggt cagcagccgc tcgccggtac gacggtgacg 1380
attacgggta cgctcgtaga cgctgcaggc aacccggtgg ccaacggtca ggtgcttgta 1440
accggctcgt cgtccagcgg cgacttcggc tatgtcacga cgtccaacgg caagagcacg 1500
acgaccgact tcccgagcgt gggtacgttg cagcctggtc agcctgtgag ctccgcgctg 1560
ggtgacgtca tcacggcgga tgcgaacggc aacttctcgt tgcaagtcac agacacgcag 1620
aacgagcaag ccagcctgac gttctactcg gtgagcaacg gggtcattag cccggtgggg 1680
gtcattaaga ccgacacgct gaaattcgca gtgaacaatc agctgtcgac cattgcgctg 1740
ggtgcgacgg acgctcaagc ggacggcaac cagtacacga atctgacggg tctcacgggt 1800
tcggacaatg cgccggtgcc ggtgtatgtg gatccgcaga atccgtcggg cacaatggtg 1860
accaatcaga gcatcaccta tacgctcagc gtcagcagcg gcgacatcgt gggcattggc 1920
tctggtgcgt atctggcgcc gaccaatgcg aacaacagca cgattccgat caacagcggc 1980
aacggcctca gctccgtcca ggtcacggtc acggcattgg gcaacaacca ataccagatc 2040
tcggtgcccg gtcagcaagg cgtgttgacg acctcgtcgc ctgactttac ggtgctggtg 2100
aaaggctcga cgggttcgac gaagctgacg gtcagctccg gctcactctc gtcgacggca 2160
accatcacct tcacgtcgag caacccgacg gtggtggcta gcctgacgcc agtttcctcg 2220
gtgttggcgg ctggtcagaa cgagacggtc accttcaccg tggaagatgc agatggcaat 2280
ccggtgagcg gtaatacgca ggttgccatc acggcgcatg acagcaatga tccgttgtgg 2340
atcaccgcag tgaatggcac aaacttgagc gagtatgaga cgattaatgg tgctgcaacg 2400
tctgtcagca cgccgattcc gctcggtacg agttcgtatg caacctctgg tggttctacg 2460
ctctacccgg cttacacgaa cagcgggtac tttaagaatg gtgtgagcat cagcggtgtc 2520
gtatcgtggg atggtacggt gggcgatcca atctacgtca ccaccaactc gcaaggccaa 2580
gtcacgctga ccttgcaaaa cggcaacgtg acctattttg acggaaacaa caccacgttg 2640
tcgaatggca tcagcgttgc cggtacgagc ggaagtgaag ggttctacac atattcgagc 2700
gataccgcag cgacagcgtc ggatcttaca aatatgggcg tgttggtcat tggtcaagcc 2760
aatggtgacg cttcaacgtc gctcggaacg atttacatcg gcagtggtgg tgctacgcag 2820
acaccggccg ccttcaccta cgtggatgcc aataaccact cttacacgta ctcgaacacg 2880
agcgatacat ttacggtatc tagcacccag agtgttagcg gtggcaacta tgcgatcaca 2940
agcttcacgc cagttggagg tactgcaact tctacaatcc cgagtggcgt gagcgtaaat 3000
agctcgacgg gtacggtttc ggtgtcccaa aacgctgcag tcggtacgta caccgtgagc 3060
tattacctga acggcgtcac tgaatccact ggcacgttca aggtgtactc cggcagcggt 3120
gtggctccta cagagatcac tggctcgtca gtgacggttc ctgctgcaac gtactcgggt 3180
acgttgaaag tcacggtaag caacggtggt tcgccgctgt acgtgaacgt taccgctgga 3240
gaatcggcca atgcggtggc tgcagctatt tacaacgcgc ttgtcaatgc caatatcagc 3300
ggagatacct tctctgtttc gggttcgaca gtcagcgtga ccgctgcgag cggttcgccc 3360
acgctcacag ttgtcgatgc gaccaatttc 3390
<210>18
<211>744
<212>DNA
<213>脂环酸芽孢杆菌属
<220>
<221>misc_feature
<222>(1)..(744)
<223>CDS
<220>
<221>misc_feature
<222>(1)..(123)
<223>sig_peptide
<220>
<221>misc_feature
<222>(124)..(744)
<223>mat_peptide
<400>18
gtgcgaatta tgaaagtttt gggatggatt ttggtaccgt atatcatgct gtttattcag 60
tgggggcgaa tgaacagaat tctgcgtttt gccggttcat tgtgggcatt aattgtcttc 120
gcgaacacgg tgtatatgat tcgaggaaac acaccgcgga acgcatcaac ggtaagcgct 180
acaacttctt tggttaattc gacgaatagt tcacaggtag caaagcaaga gcaaaactcg 240
agtacgtctc ccgctcataa gtctacgaac tcattgcaac atgcgcaaca tcaagctgct 300
acgacttcat cttctcagtc gaagttacga tatatcccgt ttcacacata cgggaaggta 360
ggagacttgg aaattagagt taactccctg cagcaagtta agagtgtggg gtacgacggg 420
ataggtgaaa ccgcaaatgg tgcgttttgg gttatcaaca tcaccataag aaatgacgga 480
tccactccta tggaggtcgt tgatggcata ttccatttgc agaacttaaa cgggaacgtt 540
tatcagccgg attctactgc tgagatatat gcaaatacaa attcagggac tattccgacc 600
gacctcaacc ctggtgtgtc catgacgaca aatctcgtat ttgatatgcc ggattttatg 660
acatatggtc acgtcgggca gcattactca cttgtcgctt ccatgggttt cttcgggtca 720
gatgaaacga cgtatgctct tccg 744
<210>19
<211>516
<212>DNA
<213>脂环酸芽孢杆菌属
<220>
<221>misc_feature
<222>(1)..(516)
<223>CDS
<220>
<221>misc_feature
<222>(1)..(75)
<223>sig_peptide
<220>
<221>misc_feature
<222>(76)..(516)
<223>mat_peptide
<400>19
atgaaccgca aatccatgtt gtctgtgttg ggtgtggcag ccgcagtagc cctgatggtg 60
acgggctgtg gcacggccaa cagcacgaac aacacggcgt cgagcggtgc ggccagcaca 120
gccgtcacgg tgaagcacga gcacaagggg gccaatgctt cgaagacaga gacgaagcag 180
accgaagcga agtcgtcgaa caaggctgga gaaacggcga agtcgtcggt gaagctcacg 240
gccccggtgg caggcgcgac ggtgacggcc ggcggcacgc tgaaggtgag cggccaagtg 300
tcgtcgaacc tcgcgaagaa ggacgtgcaa attacgttga caaatagcgc gaagaaggtg 360
ctcgtgcagc agatcgtcgg tacgaatagc accggcgcat tcgtggacac gctcaagctt 420
ccaaagtacc ttgggaaagc cggaagcgac ctgacgctgt cggtgtccgt cgttggcgaa 480
aatggagtcg taagcacctt gtcgctgcac gtgaag 516
<210>20
<211>726
<212>DNA
<213>脂环酸芽孢杆菌属
<220>
<221>misc_feature
<222>(1)..(726)
<223>CDS
<220>
<221>misc_feature
<222>(1)..(90)
<223>sig_peptide
<220>
<221>misc_feature
<222>(91)..(726)
<223>mat_peptide
<400>20
atgaggcgcg cggttcgtat actagctgcg ctactgtttg ggctggcgac ggtaacagcc 60
acattgatgt tcgtgcctca ggcaagagcg gccacggtga caggagcgtt ggcgcaatcg 120
caagtggtgt ccattacggg cggctacaac acgacgacac agatgtatga gcagacgggt 180
cagcaaaccg tcgttacgaa ttggaccttt tctcttcaac aaactgtcaa ccaaaacaac 240
gagaatccgt cctacgctca atgcacagtc ttggcgggaa accagcaggt aacgtgcacg 300
tcggacgcta cgaataacgg tgcaatttgc acatccccct atcctggagc tattgacaag 360
caatgcacga acctgattgg gttcactgga aacatatcag tgagttcgca aaacggcaat 420
ccaacgttca ctttttctct tccgagcatc gacccgagta ccatgaagcc agttgggatc 480
tttgtgacgc ctgagacgat ctatggtcag atgggaacag ggtccgaaag ttatttaagc 540
tcaggtcaat ctggaggatg gtcatttaac ttttccaacg tctcagatcc tcaagattgg 600
tattttctcc ttgagttttt ggcgaatcca attgtcgcgg ccattgctgt gcccaccact 660
caaacggttc cgatttatag ctgggtcacc accacggttt ggcaccccgt tcaaatttcc 720
tacagc 726
<210>21
<211>540
<212>DNA
<213>脂环酸芽孢杆菌属
<220>
<221>misc_feature
<222>(1)..(540)
<223>CDS
<220>
<221>misc_feature
<222>(1)..(72)
<223>sig_peptide
<220>
<221>misc_feature
<222>(73)..(540)
<223>mat_peptide
<400>21
gtggttcgga tgcgcaagcg gttgggactt gttctgagta tggtgacatc tgtgttggtt 60
ggatgtggcg cttcacatcc gtctccattg aaccaagaca aatctttgtt gacgtggaac 120
gctgctaaac acgaggtgcg gtggaaagtg gtcgccggcg acggacgcgc aaacggcggt 180
atgaacttcg atggctatgc caatggcagt atgacactgg tcgtgccgat tgggtggcgc 240
gtcgtgatcg actttgacaa tgccagtttg atgccgcaca gcgcgatggt ggtgccttac 300
ggagatcgcg aacgctccaa cttcgacgca acgatggttg cgtttccagg cgcagaaacg 360
cccaatccgt cacagggaga ccctcaaggg acgcatcggg atgtcatctt cactgctgcg 420
aaggtgggaa cgtatgccct cgtctgcggg gtcccgggtc acgcgctggc gggaatgtgg 480
gatcagcttg tggtgtccga tgaagcgaaa cacccgtccc ttcgcgtgca acgcgactca 540
<210>22
<211>1431
<212>DNA
<213>脂环酸芽孢杆菌属
<220>
<221>misc_feature
<222>(1)..(1431)
<223>CDS
<220>
<221>misc_feature
<222>(1)..(75)
<223>sig_peptide
<220>
<221>misc_feature
<222>(76)..(1431)
<223>mat_peptide
<400>22
atggcggttc gtagagcgtg gcttctggcg cccttgtgcg cgagcagtct ggtcgtcccg 60
gcctcggtgc aggccggatt ggcccaggga catggcagct tttcgacggt tcgcgtgtcc 120
gtggggacgt cgagttccct gtccgtcccc gcgctgattc agggaaacga aacgtacatt 180
ccgctgtggg acctcatgca ggtgctccat cagctcggct tcaccgcgac gtgggcgaag 240
ggccaattca gcgtttcggc cccgccatcg gtgccgatgg acgaggcgcc tgggccagcg 300
ggcaaaggcg gggcgctcgt ggtgctcgac gggcaagtcg tggaacaggt gccgacggtc 360
atcgccacgc caccgggggc ggccacccct gaggtgtttc tgccgctcac gaacgcggag 420
gagatcctcg gtcggttggg cattcaggcc agcgcgaccg gcaatcaggt gaacctcgac 480
gcgtcggctg tgccccaggc gcttcccaac cagcaggtgg ctgtgtggaa cgtgcttgcc 540
gctgttgcgt ccgatctcgg cgtgtcgacc gcgccagccg ggccgagtcc ctacgccgac 600
ttgccgacag cctcgccggc gtggggcgcg gtggaggcgg ccattcgtct gggctggtat 660
tcgcccttat ccgcgtcgtc atccggcgcg tttcaaccca tcacgtgggc gcaaacggca 720
tccattctgt ggaatgcgct cggcatttca cagcaggacg cggcgtacca gccaggcgga 780
tcgccgacgg cgtgggcgag cgcccttggc cttgttccag aaaactggga tccagcgtcg 840
tacatgaccg cgcaggaatt ggacaccttg gcgtcgaatt tgcacgaatg tctgcaagga 900
gatgtcgaaa cgggcgccaa cacgtggcgg ctctggtatc cgccggctga cgaagtggag 960
gctaccctcc agtcgggagg cgggcagtcg ctgttcacct cgaccgctga cgcgcaggcc 1020
gccatctcgt cagcctacca attcttcaat cagcttgtgg tcacaagagt cggccaaggg 1080
tatgtcgtca ccgttccctc tgtgcctgag ggatatgggt ttgccacctt ttctgcgctc 1140
ggcggtgtgg cttaccagac gacacccggc ggtccgtgga cggtcgtgcc cgtgctggac 1200
acgcgcgacg tctccatccc ggccaagggc cgtctcagtg tcaaggttcc cgcgcagggc 1260
atcaccatca cgtggaatca gatgatgcca tcgctgggcg gaacggtggc catgggcgcg 1320
ctccaggtgt cgcctggacc cagcgggcct tcggtcgagc gcttgaatat cgtcacaccg 1380
aacttacctc cggtccttcc gtcgtccgtc acttctacgc aaccgcagtc a 1431
<210>23
<211>1020
<212>DNA
<213>脂环酸芽孢杆菌属
<220>
<221>misc_feature
<222>(1)..(1020)
<223>CDS
<220>
<221>misc_feature
<222>(1)..(57)
<223>sig_peptide
<220>
<221>misc_feature
<222>(58)..(1020)
<223>mat_peptide
<400>23
gtgaatcgac agtggaggct agcggtggcg acttctgccg tcgcggccag cctcgcgggg 60
tgtggagcac cggacctcgc ggcgatgcgg ccgacggtcc aaaagtctgc ggtactcgtg 120
gaggtcgtgg gcgcgccgcc gtttgcgccc tcagcttcac aactgggaac ggcaggggcc 180
acctccgtcg aggtggttca cgttgccctt ggcgaatggc agtctgtcgc ggcccacgca 240
ttggcgaagg ggcaattgac aggggtcatg gtcgtgtgcg acgacgcgaa cgccgtcgcg 300
tctggcctca accaacttgc tgccgaccat cccgacgttc gctttctcgt ggtcagcaac 360
tggccggctt cgcaaatcac ctccggaaac gtggaagacg tcgcacagga tcctgtggcc 420
gtcgcttaca gcattggcgc gctgtgcgga gactggatcg cgagctcaac gtcgacgagc 480
ggagcggtat acagcggcgt gcccagcatc gtctacgcgc cgcgcggtgc gaccgtggct 540
gaacaaaaag ccttcttcac gggtctgtat caggcgaacc ccaatgtccg ggtcgtcgcg 600
cttccgcagc ccgctgcgca gagcctgtcg agctatgggt acgcggtgga tttgggtgtg 660
gtaggcgggt ctcctgcggc aggggaactg tcggcgcttc gcagtgccgc ccccgcctgg 720
gctgcttttg gaacgtcgcc gatcgctggc tttgcgattt ctcctggcca tctgtcgtcg 780
tcggaggccg tgcaagcatt ccaggcgctc gtgtcgccgg acgcgtggca ctcgggtgag 840
catctcgtgc tcgacttgtc ttcggtggcc ttcgacgaca agcaggtgcc cgcgaccgtc 900
atcgcggcgt gggccaagct ggaggtcaac gcgatcgcgg ctgcagcgca atcgaacgcg 960
gccttcgcgt cactgccgcc gagcgtgcgc tcggacctcg ccaatgcgtt tcatttgtca 1020
<210>24
<211>1023
<212>DNA
<213>脂环酸芽孢杆菌属
<220>
<221>misc_feature
<222>(1)..(1023)
<223>CDS
<220>
<221>misc_feature
<222>(1)..(87)
<223>sig_peptide
<220>
<221>misc_feature
<222>(88)..(1023)
<223>mat_peptide
<400>24
atggtcatgc gcactcggtg gattcgatgg atggctttgg ctctcgcagt ctgtgtctgg 60
ctcagcccgt ttcccttctc gtggggcgcg acgagcctcg acgctgatct tccacaaccc 120
acgattccgc catccgcgtg gagcaacctc aatcaggact ggaaggacct tcagcgcttg 180
gcgcaaaaca cagtgccgcc ctcgaaagag agcagccaga cccacgcgcc cacacacaag 240
tcatcgcaac cgcctgccca agtcccgcaa gggccgctcg tcggggtcgg cgatacgggc 300
gaagcggccc ggtggttaaa cgaagccttg gccgtgctcg gctatttgcc cgccgtcttc 360
tctcccgcgg cgcagacgtc cacccgtcag gtgcggctcg cactcgcggc gagcgccgag 420
catcagacgc tcgtgcccat cccaggctcg tttcaacttc tgtatcacgc gccaagctcg 480
tgggtggcgc tctggtccgc cgacgaagac acgccgatca cggagggcgc cgtcatggcg 540
tttgaagcac aacatcacct gggcgtggat ggcattgccg ggccggacgt cattcatgcg 600
ctggcgcagg ccctcgccgg caatgagacg gcagaaaagg cgccctacag ctacatcctg 660
gtgaccacgt cgttgcccga gacgctcgaa ctctgggtga atggccagct tgtcctcaaa 720
tcgctgtgca acacaggcat cgcgcagtca cccacgccgt atggcacgta cggcgtctac 780
gtgcagtaca cgtcgcagga aatgaagggc aaggatccgg acggcacgcc ctacgacgat 840
cccggcgttc catgggtgag ctacttctac aaaggttgcg cggtccacgg tttcctgcgg 900
gcaaagtacg gctttcccca gagcctcggt tgcgtggaac tgccgtatgc cgcggccaaa 960
acggtgttct cctatacgca catcggcacg cttgtcaccg tcaccgcctc cccgctttcc 1020
gcg 1023
<210>25
<211>1197
<212>DNA
<213>脂环酸芽孢杆菌属
<220>
<221>misc_feature
<222>(1)..(1197)
<223>CDS
<220>
<221>misc_feature
<222>(1)..(84)
<223>sig_peptide
<220>
<221>misc_feature
<222>(85)..(1197)
<223>mat_peptide
<400>25
atggataggc tgctgaacaa caaggtggcg cttcgcctga ccgcgctcgt cctcgcgtgc 60
attctctggc tcgccgtgca cgcggagcag gggtcggggt cctccgcgtc cacgggagtg 120
accgagtcgt tcgagctgcc ggtgcgggtg gaaacctcgg ccgacgaggt gttggtgtct 180
caagttccga ccatcaccgc ccgggtgacg acgaacctgt tgagcctgcc gacgctggcc 240
tcggatatga tgaaagccga gatcgtcgcg gacgccgaaa atctgggccc gggcacgtac 300
acgttgcacg tggcggccgt caacatgcct gcaggggtgc gatcgtacac gctaacgcct 360
tccaccatca cggtgacgtt ggagcccaaa gtgacggtgg agcgaacggt gcgggtgaac 420
gtggtcggca cgccagggca gggatatgtc ctcggcaagc ccgagctcgg cgcgggggtc 480
gtcgaggtct cgggcgccga atccagtgtg caggccgtgg ccgaggtggc gggcgtcgtg 540
gacgcgagcg gcctgtcgca gacggcgacc aagctcgtcg agttgttgcc gcttgaccaa 600
gcgggcaagg cggtgccggg tgtgacggtc acgccatccg cgatttcggt cacgctgccg 660
atcacgtccg ccaatcaggc ggtgaagctg acgcctgcgg tcaccggcag ccctgcgcct 720
ggatacgccg tcgcctcggt gcacctggag cccgcgagcg ctgtggaaca ggggctagcg 780
gccagccagc ttccgcagcg cgggctcctc gtgcccatcg acgtcactgg attgaaccgg 840
cccacgacgg tgtcggtccc ggtgccgctt ttgccgggga tgacgagcgt ttcgcccacg 900
gcagtgacgg ccgtgatcga cgtggagccg tccgccgtct acaccgtttc gaacgtcccg 960
gtggccatca cgggcgcgac gggtgtcaag ctggtgacgc ctcggaccgt gaatgtcacg 1020
gtgacgggga tcgaggccga cgtgcgcgcg gtggagaggg atccggccgc ggtgcaggcg 1080
tttgtggacg cgaccgggtt gacacatggc tcggcgacgc tgcccgattc aaattcgtct 1140
gctgtcctgt ctcttgtgat ccggccacgg gaaaggcgta agcgaacaca tgtagtg 1197
<210>26
<211>959
<212>PRT
<213>脂环酸芽孢杆菌属
<220>
<221>SIGNAL
<222>(1)..(24)
<220>
<221>mat_peptide
<222>(25)..(959)
<223>酸性内切葡聚糖酶或酸性纤维素酶
<400>26
Met Lys Thr Arg Trp Ser Gly Ala Leu Ala Val Leu Ile Ala Leu Gly
-20 -15 -10
Thr Gly Ala Ser Pro Ala Trp Ala Ser Val His Ser Ala Ala Thr His
-5 -1 1 5
Ala Lys Ala His Val Gly Val Arg Ala Ala Asp Met Ala Ala Ala Ser
10 15 20
Met Ser Ala Glu Ile Gln Ile Leu His Asp Ala Leu Thr Ala Ser Glu
25 30 35 40
Leu Ser Ser Val Gln Ala Ala Ala Gln Ala Ala Ala Asn Leu Pro Ala
45 50 55
Ser Thr Trp Val Ser Trp Leu Tyr Pro Ser Ala Ser Ser Pro Ser Ala
60 65 70
Ala Gln Thr Gln Thr Ala Gln Ala Leu Gly Ala Leu Leu Thr Leu Val
75 80 85
Thr Tyr Gly Ala Val Ala Asp Asp Gly Gln Asn Ile Ala Gln Asn Leu
90 95 100
Gln Thr Leu Gln Ser Thr Ser Pro Leu Leu Ser Pro Ala Ala Val Ser
105 110 115 120
Met Phe Tyr Gln Asn Phe Phe Val Leu Val Gly Gln Ser Ser Lys Ser
125 130 135
Val Leu Ser Gly Gln Ala Thr Thr Ser Thr Ala Gly His Ala Leu Ala
140 145 150
Gln Ala Ala Ala Leu Thr Pro Gln Leu Ala Ala Tyr Leu Arg Gln Ser
155 160 165
Gly Leu Ser Pro Asp Asp Leu Ala Arg Ala Tyr Val Ser Phe Ala Ser
170 175 180
Ala Val Asp Ser Gln Gly Ala Ala Gln Thr Ala Leu Leu Thr Arg Ile
185 190 195 200
Cys Thr Asn Ile Leu Gly Phe Gly Ala Pro Thr Ser Thr Ala Thr Ile
205 210 215
Thr Val Asn Ala Ala Ala Asn Leu Gly Gln Val Pro Thr Thr Ala Phe
220 225 230
Gly Leu Asn Ala Ala Val Trp Asp Ser Gly Leu Asn Ser Gln Thr Val
235 240 245
Ile Ser Glu Val Gln Ala Leu His Pro Ala Leu Ile Arg Trp Pro Gly
250 255 260
Gly Ser Ile Ser Asp Val Tyr Asn Trp Glu Thr Asn Thr Arg Asn Asp
265 270 275 280
Gly Gly Tyr Val Asn Pro Asp Asp Thr Phe Asp His Phe Met Gln Phe
285 290 295
Val Asn Ala Val Gly Ser Thr Pro Ile Ile Thr Val Asn Tyr Gly Thr
300 305 310
Gly Thr Pro Gln Leu Ala Ala Asp Trp Val Lys Tyr Ala Asp Val Thr
315 320 325
His His Asp Asn Val Met Tyr Trp Glu Ile Gly Asn Glu Ile Tyr Gly
330 335 340
Asn Gly Tyr Tyr Asn Gly Asn Gly Trp Glu Ala Asp Asp His Ala Val
345 350 355 360
Ala Gly Gln Pro Gln Lys Gly Asn Pro Gly Leu Ser Pro Gln Ala Tyr
365 370 375
Ala Gln Asn Ala Leu Gln Phe Ile Lys Ala Met Arg Ala Glu Asp Pro
380 385 390
Ser Ile Lys Ile Gly Ala Val Leu Thr Met Pro Tyr Asn Trp Pro Trp
395 400 405
Gly Ala Thr Val Asn Gly Asn Asp Asp Trp Asn Thr Val Val Leu Lys
410 415 420
Ala Leu Gly Pro Tyr Ile Asp Phe Val Asp Val His Trp Tyr Pro Glu
425 430 435 440
Thr Pro Gly Gln Glu Thr Asp Ala Gly Leu Leu Ala Asp Thr Asp Gln
445 450 455
Ile Pro Ala Met Val Ala Glu Leu Lys Arg Glu Val Asn Thr Tyr Ala
460 465 470
Gly Ser Asn Ala Lys Asn Ile Gln Ile Phe Val Thr Glu Thr Asn Ser
475 480 485
Val Ser Tyr Asn Pro Gly Glu Gln Ser Thr Asn Leu Pro Glu Ala Leu
490 495 500
Phe Leu Ala Asp Asp Leu Thr Gly Phe Ile Gln Ala Gly Ala Ala Asn
505 510 515 520
Val Asp Trp Trp Asp Leu Phe Asn Gly Ala Glu Asp Asn Tyr Thr Ser
525 530 535
Pro Ser Leu Tyr Gly Gln Asn Leu Phe Gly Asp Tyr Gly Leu Leu Ser
540 545 550
Ser Gly Gln Thr Thr Gln Asn Gly Trp Gln Glu Pro Pro Ala Asn Thr
555 560 565
Pro Leu Pro Pro Tyr Asn Gly Phe Gln Leu Val Ser Asp Phe Ala Gln
570 575 580
Pro Gly Asp Thr Met Leu Gly Ser Thr Thr Ser Gln Ser Ala Ile Asp
585 590 595 600
Val His Ala Val Arg Lys Pro Asn Gly Asp Ile Ser Leu Met Leu Val
605 610 615
Asn Arg Ser Pro Ser Ala Ile Tyr Ser Ala Asn Leu Asn Val Leu Gly
620 625 630
Phe Gly Pro Phe Val Val Thr His Ala Leu Ala Tyr Gly Glu Gly Ser
635 640 645
Ser Arg Val Ala Pro Met Pro Val Leu Pro Val Pro Gly Ala Pro Ile
650 655 660
Lys Leu Met Pro Tyr Ser Gly Ile Asp Leu Thr Leu His Pro Leu Ile
665 670 675 680
Pro Ala Pro His Ala Ala Ala Gln Val Thr Asp Thr Leu Thr Leu Ser
685 690 695
Ser Pro Thr Val Thr Ala Gly Gly Ala Glu Thr Leu Ser Ala Ser Phe
700 705 710
Gln Ala Asp Arg Pro Val His His Ala Thr Val Glu Leu Glu Leu Tyr
715 720 725
Asp Ser Thr Asn Asp Leu Val Ala Thr His Thr Val Ser Asp Val Asp
730 735 740
Leu Gln Pro Gly Ser Ala Thr Ser Glu Thr Trp Ser Phe Thr Ala Pro
745 750 755 760
Ala Ala Asn Gly Asn Tyr Arg Val Glu Ala Phe Val Phe Asp Pro Val
765 770 775
Thr Gly Ala Thr Tyr Asp Ala Asp Thr Gln Gly Ala Val Leu Thr Val
780 785 790
Asn Gln Pro Pro Gln Ala Thr Tyr Gly Asp Ile Val Thr Lys Asp Thr
795 800 805
Val Ile Thr Val Asn Gly Thr Thr Tyr Asp Val Pro Ala Pro Asp Ala
810 815 820
Gly Gly His Tyr Pro Ser Gly Thr Asn Ile Ser Val Ala Pro Gly Asp
825 830 835 840
Thr Val Thr Val Gln Thr Thr Phe Val Asn Val Ser Ser Thr Asp Ala
845 850 855
Leu Gln Asn Gly Leu Ile Asp Met Glu Val Asp Gly Ser Asn Gly Ala
860 865 870
Ile Leu Gln Lys Tyr Trp Pro Ser Thr Thr Leu Leu Pro Gly Gln Ser
875 880 885
Glu Thr Val Thr Ala Thr Trp Gln Val Pro Ala Asn Val Ala Ala Gly
890 895 900
Thr Tyr Pro Leu Asn Phe Gln Ala Phe Asn Thr Ser Ser Trp Thr Gly
905 910 915 920
Asn Cys Tyr Phe Thr Asn Gly Gly Val Val Asn Phe Val Ile Ser
925 930 935
<210>27
<211>272
<212>PRT
<213>脂环酸芽孢杆菌属
<220>
<221>SIGNAL
<222>(1)..(32)
<220>
<221>mat_peptide
<222>(33)..(272)
<223>天冬氨酰蛋白酶
<400>27
Met Asn Gly Thr Ser Val Trp Lys Ala Ser Gly Ile Ala Ala Ala Ser
-30 -25 -20
Cys Leu Thr Ala Ala Ala Leu Leu Ala Trp Pro His Ala Thr Ser Thr
-15 -10 -5 -1
Leu Asp Ala Ser Pro Ala Ile Phe His Ala Pro Arg His Ala Leu Ser
1 5 10 15
Pro Asn Thr Ser Pro Lys Pro Asn Ser Val Gln Ala Gln Asn Phe Gly
20 25 30
Trp Ser Ala Ser Asn Trp Ser Gly Tyr Ala Val Thr Gly Ser Thr Tyr
35 40 45
Asn Asp Ile Thr Gly Ser Trp Ile Val Pro Ala Val Ser Pro Ser Lys
50 55 60
Arg Ser Thr Tyr Ser Ser Ser Trp Ile Gly Ile Asp Gly Phe Asn Asn
65 70 75 80
Ser Asp Leu Ile Gln Thr Gly Thr Glu Gln Asp Tyr Val Asn Gly His
85 90 95
Ala Gln Tyr Asp Ala Trp Trp Glu Ile Leu Pro Ala Pro Glu Thr Val
100 105 110
Ile Ser Asn Met Thr Ile Ala Pro Gly Asp Arg Met Ser Ala His Ile
115 120 125
His Asn Asn Gly Asn Gly Thr Trp Thr Ile Thr Leu Thr Asp Val Thr
130 135 140
Arg Asn Glu Thr Phe Ser Thr Thr Gln Ser Tyr Ser Gly Pro Gly Ser
145 150 155 160
Ser Ala Glu Trp Ile Gln Glu Ala Pro Glu Ile Gly Gly Arg Ile Ala
165 170 175
Thr Leu Ala Asn Tyr Gly Glu Thr Thr Phe Asp Pro Gly Thr Val Asn
180 185 190
Gly Gly Asn Pro Gly Phe Thr Leu Ser Asp Ala Gly Tyr Met Val Gln
195 200 205
Asn Asn Ala Val Val Ser Val Pro Ser Ala Pro Asp Ser Asp Thr Asp
210 215 220
Gly Phe Asn Val Ala Tyr Gly Ser Asn Gln Pro Ser Pro Pro Ala Ser
225 230 235 240
<210>28
<211>315
<212>PRT
<213>脂环酸芽孢杆菌属
<220>
<221>SIGNAL
<222>(1)..(25)
<220>
<221>mat_peptide
<222>(26)..(315)
<223>多铜氧化酶
<400>28
Met Arg Arg Arg Met Ser Gly Phe Ala Thr Gly Leu Gly Ile Ala Ala
-25 -20 -15 -10
Gly Leu Ala Leu Ser Ser Ala Leu Ala Ala Pro Phe Phe His Ala Gly
-5 -1 1 5
Asn Ala Ser Ala Ala Ser Thr Met Ser Met Ala Pro Thr Ser Thr Met
10 15 20
Gly Ala Leu Pro Ala Pro Glu Gly Val Pro Asp Ala Gly Pro Leu Ser
25 30 35
Ile Thr Pro Glu Val Ile Arg Gln Gln Gln Ala Asp Ala Val Arg Val
40 45 50 55
Met Asp Glu Glu Gly Leu Lys Pro Gln Ile Leu Ser Gly Asp Ile Lys
60 65 70
Arg Phe Thr Leu Thr Ala Ser Gln Val Asn Trp Tyr Leu Tyr Pro Gly
75 80 85
Lys Ala Val Val Ala Cys Gly Tyr Asn Gly Gln Val Pro Gly Pro Val
90 95 100
Leu Arg Val Arg Val Gly Asp Arg Val Gln Ile Leu Leu Arg Asn Glu
105 110 115
Leu Asn Glu Pro Thr Thr Leu His Ile Gln Gly Leu Asp Leu Pro Ala
120 125 130 135
Ser Gln Leu Gly Ile Gly Asp Val Thr Glu Ser Pro Ile Pro Pro Gly
140 145 150
Gly Glu Arg Leu Tyr Ser Phe Thr Val Thr Pro Gln Met Val Gly Thr
155 160 165
His Leu Tyr Glu Ser Gly Thr Asp Met Ala Ser Glu Ile Asp Pro Arg
170 175 180
Thr Ala Arg Gly Ala Ala Arg Arg Ser Gly Pro Gly Ile Pro Leu Ser
185 190 195
Pro Gly Glu Gly Gly Arg Ala Leu Arg Asp Arg Arg Val Asp Gly Gly
200 205 210 215
Arg Ile Asp His Arg Lys Arg Val Trp Pro Gly Arg Gln Ala Val Ser
220 225 230
Arg Arg Ala Arg Thr Asp Gly Ala Val Arg Gln Pro Arg Gly Ala Ala
235 240 245
His Arg Gln Arg Glu Arg Asp Val Leu Pro Arg His Ala Pro Ala Arg
250 255 260
Asp Asp Val Leu Ala Ala Gly Gly Arg Arg Ala Pro Pro Arg Gln Ala
265 270 275
Ala Ala Asp Glu Arg Ala Arg His Arg Ala Arg
280 285 290
<210>29
<211>626
<212>PRT
<213>脂环酸芽孢杆菌属
<220>
<221>SIGNAL
<222>(1)..(32)
<220>
<221>PROPEP
<222>(33)..(189)
<220>
<221>mat_peptide
<222>(190)..(626)
<223>丝氨酸羧基蛋白酶
<400>29
Met Gly Leu Trp Lys Arg Leu Ala Leu Gly Val Pro Ala Ala Leu
-185 -180 -175
Ser Met Leu Ala Val Gly Val Pro Val Met Ser Ala Asp Thr Val
-170 -165 -160
Glu Ala Ala Pro Leu Ala Asn Pro Ser Thr Glu Asn Ala Gln Asp
-155 -150 -145
Met Gly Pro Ala Ser Gly Ser Gln Thr Val Thr Ala Ser Ile Ile
-140 -135 -130
Leu Arg Val Gln Asn Pro Thr Ala Leu Gln Asn Tyr Ile Gln Glu
-125 -120 -115
Thr Glu Thr Pro Gly Ser Pro Leu Tyr His Lys Phe Leu Thr Thr
-110 -105 -100
Ala Gln Phe Ala Gln Gln Tyr Ala Pro Ser Ala Ala Thr Leu Gln Gln
-95 -90 -85
Ile Glu Gln Glu Leu Gln Gly Tyr Gly Leu Gln Val Val Asn Val Asp
-80 -75 -70
Ala Asp His Leu Asp Met Gln Val Gln Gly Thr Val Gln Gln Phe Asp
-65 -60 -55
Asn Ala Phe Asn Thr Val Ile Asp Leu Phe Lys Ala Asn Gly His Ile
-50 -45 -40
Phe Arg Ala Pro Lys Lys Pro Pro Gln Ile Pro Val Ala Leu Leu Thr
-35 -30 -25 -20
Asn Val Leu Ala Val Val Gly Leu Asp Thr Ala Gln Ala Ala Gln Ser
-15 -10 -5
Leu Thr Val Lys Thr Pro Asn Val Ala Gly Val Pro Ser Pro Lys Val
-1 1 5 10
Val Leu Pro Gln Gly Gly Ser Thr Ala Thr Gly Thr Pro Gly Ser Tyr
15 20 25
Thr Val Gly Asp Thr Ala Asn Arg Tyr Asp Ile Asn Pro Leu Tyr Gln
30 35 40 45
Lys Gly Ile Thr Gly Lys Gly Glu Thr Ile Gly Ile Val Thr Leu Ser
50 55 60
Ser Phe Asn Pro Gln Asp Ala Tyr Thr Tyr Trp Gln Gly Ile Gly Leu
65 70 75
Lys Val Ala Pro Asn Arg Ile Gln Met Val Asn Val Asp Gly Gly Gly
80 85 90
Gln Met Asp Asp Gly Ser Val Glu Thr Thr Leu Asp Val Glu Gln Ser
95 100 105
Gly Gly Leu Ala Pro Asp Ala Asn Val Val Val Tyr Asp Ala Pro Asn
110 115 120 125
Thr Asp Gln Gly Phe Ile Asp Ala Phe Tyr Gln Ala Val Ser Asp Asn
130 135 140
Gln Ala Asp Ser Leu Ser Val Ser Trp Gly Gln Pro Glu Ile Asp Tyr
145 150 155
Leu Pro Gln Met Asn Gln Gly Gln Ser Tyr Val Asp Glu Leu Leu Ala
160 165 170
Phe Thr Gln Ala Phe Met Glu Ala Ala Ala Gln Gly Ile Ser Met Tyr
175 180 185
Ala Ala Ala Gly Asp Ser Gly Ala Tyr Asp Thr Ala Arg Asp Phe Pro
190 195 200 205
Pro Ser Asp Gly Phe Thr Thr Pro Leu Ser Val Asp Phe Pro Ala Ser
210 215 220
Asp Pro Tyr Ile Thr Ala Ala Gly Gly Thr Thr Val Pro Phe Thr Ala
225 230 235
Lys Phe Ser Leu Gly Thr Val Asn Ile Thr Gln Glu Gln Pro Trp Ser
240 245 250
Trp Gln Tyr Leu Gln Asn Leu Gly Tyr Gln Gly Leu Phe Ser Val Gly
255 260 265
Thr Gly Gly Gly Val Ser Val Ile Phe Pro Arg Pro Trp Tyr Gln Leu
270 275 280 285
Gly Val Gly Gly Met Gln Asn Ser Ala Ala Asn Gln Ala Phe Thr Asp
290 295 300
Ser Gln Gly Val Leu Tyr Gly Ser Pro Phe Thr Tyr Asn Leu Pro Ser
305 310 315
Asn Tyr Ala Gly Arg Asn Leu Pro Asp Ile Ser Met Asp Ala Asp Pro
320 325 330
Glu Thr Gly Tyr Leu Val Tyr Trp Ser Ala Gly Gly Gly Trp Ile Ala
335 340 345
Gly Tyr Gly Gly Thr Ser Phe Val Ala Pro Gln Leu Asn Gly Ile Thr
350 355 360 365
Ala Leu Ile Asp Gln Glu Val His Gly Arg Val Gly Phe Leu Asn Pro
370 375 380
Leu Leu Tyr Thr Leu Leu Thr Gln Gly Val Gln Gly Gly Ala Gln Pro
385 390 395
Phe His Asp Ile Thr Thr Gly Asn Asn Trp Tyr Trp Asn Ala Val Pro
400 405 410
Gly Tyr Asp Pro Ala Ser Gly Val Gly Thr Pro Asp Val Ala Asn Leu
415 420 425
Ala Gln Asp Ile Ala Ser Leu Arg
430 435
<210>30
<211>533
<212>PRT
<213>脂环酸芽孢杆菌属
<220>
<221>SIGNAL
<222>(1)..(24)
<220>
<221>mat_peptide
<222>(25)..(534)
<223>丝氨酸羧基蛋白酶
<400>30
Met Arg Ala Leu Ala His Leu Ala Ile Gly Ala Ile Ala Ser Gly Val
-20 -15 -10
Phe Ala Ala Pro Val Ala Phe Ala Ser Pro Val Gln Glu Arg Val Val
-5 -1 1 5
Val Ala Ser Pro Asp Pro Arg Thr Arg Ser Val His Ala Asp Gly Glu
10 15 20
Ile Ser Pro Ser Gln Pro Met His Leu Val Ile Thr Leu Arg Leu Arg
25 30 35 40
His Glu Ala Gln Leu Glu Gln Leu Ile Arg Asp Leu Tyr Thr Pro Gly
45 50 55
Ser Pro Asp Ala Gly His Phe Leu Thr Pro Ala Ala Phe Asn Ala Ala
60 65 70
Tyr Ala Pro Thr Ala Glu Asp Val Gln Ala Val Val Gln Gly Leu Arg
75 80 85
Ala Tyr Gly Leu Arg Val Glu Pro Thr Val Asn Pro Met Val Leu Thr
90 95 100
Val Ser Gly Arg Ala Arg Asp Val Glu Arg Ala Phe Gly Val His Glu
105 110 115 120
Leu Gln Phe Gly Arg Gly Ala Gly Ala Trp Tyr Ala Pro Asp Gly Ala
125 130 135
Ala Thr Leu Pro Ala Pro Leu Ala Ala Arg Val Ser Ala Val Val Gly
140 145 150
Leu Thr Ser Asp Ala Met Glu Arg His Leu Val Leu Ala His Val Ala
155 160 165
Pro Ala Gly Gly Gly Tyr Thr Pro Ala Gln Ile Gln Arg Ala Tyr Asp
170 175 180
Tyr Thr Pro Leu Tyr Ser Gln Tyr Met Gly Arg Gly Gln Val Ile Ala
185 190 195 200
Val Val Thr Ser Gly Ser Val Leu Arg Ser Asp Leu Leu Ala Phe Asp
205 210 215
Arg Ala Phe Gly Leu Pro Asn Pro Val Val Arg Gln Arg Val Ile Asp
220 225 230
Gly Ser Ser Thr Ser Pro Asp Asp Glu Thr Thr Leu Asp Cys Glu Trp
235 240 245
Ala His Ala Ile Ala Pro Thr Ala Ser Leu Ala Val Tyr Glu Ala Ala
250 255 260
Gln Pro Asp Ala Gln Ser Phe Ile Asp Ala Phe Ala Gln Val Ala Ala
265 270 275 280
Asp Asp Gly Ala His Val Val Thr Thr Ser Trp Gly Ala Pro Glu Ser
285 290 295
Glu Thr Asp Ala Ala Thr Met Gln Ala Glu His Gln Ile Phe Met Gln
300 305 310
Met Ala Ala Gln Gly Gln Ser Val Phe Ala Ala Ala Gly Asp Ser Gly
315 320 325
Ser Ser Asp Gly Thr Ser Gly Thr Asp Val Asp Tyr Pro Ser Ser Asp
330 335 340
Pro Tyr Val Thr Ala Cys Gly Gly Thr Arg Leu Val Leu Gly Ala Gly
345 350 355 360
Ala Lys Arg Leu Gln Glu Thr Ala Trp Ala Asp Thr Gly Gly Gly Ala
365 370 375
Ser Ser Val Tyr Gly Glu Pro Trp Trp Gln Tyr Gly Pro Gly Val Pro
380 385 390
Gln Thr Gly Tyr Arg Gln Thr Cys Asp Val Ala Leu Asn Ala Asp Pro
395 400 405
Ala Thr Gly Tyr Asp Phe Ile Tyr Glu Gly Gln Trp Glu Val Ala Gly
410 415 420
Gly Thr Ser Phe Val Ala Pro Met Met Ala Ala Thr Phe Ala Leu Ile
425 430 435 440
Asp Gln Ala Arg Ala Leu Glu Gly Lys Pro Pro Val Gly Leu Ala Asp
445 450 455
Val Gly Ile Tyr Ala Met Ala Arg Asn Ala Ser Tyr Ala Pro Tyr Ala
460 465 470
Phe His Asp Ile Thr Ala Gly Ser Asn Gly Ala Tyr Ser Ala Gly Pro
475 480 485
Gly Trp Asp His Pro Thr Gly Phe Gly Ser Ile Asp Ala Tyr Tyr Phe
490 495 500
Leu His Gly Leu Asp
505
<210>31
<211>360
<212>PRT
<213>脂环酸芽孢杆菌属
<220>
<221>SIGNAL
<222>(1)..(41)
<220>
<221>mat_peptide
<222>(42)..(411)
<223>蛋白酶或HtrA样丝氨酸蛋白酶
<400>31
Met Arg Arg Arg Arg Trp Asp Tyr Glu Asp Trp Pro Ser Glu Asn Arg
-40 -35 -30
Arg Val Gly Val Trp Leu Ala Ser Gly Thr Ala Leu Leu Ala Ile Cys
-25 -20 -15 -10
Tyr Ile Leu Gly Ile Trp Thr Gly Ala Ala Leu Thr Arg Gly His Ser
-5 -1 1 5
Gln Thr Thr Val Glu Tyr Val Pro Pro Gln Thr Gly Asn Thr Ala Ser
10 15 20
Thr Ser Gly Ser Leu Thr Pro Ile Pro Gly Val Glu Asp Thr Thr Ile
25 30 35
Val Thr Gln Ile Tyr Asn Arg Val Lys Asn Ser Ile Phe Thr Ile Thr
40 45 50 55
Ala Val Ser Gly Gly Lys Pro Thr Ser Ser Asp Ala Glu Glu Asp Ile
60 65 70
Gly Thr Gly Phe Leu Ile Asp His Asn Gly Asp Leu Leu Thr Asn Ala
75 80 85
His Val Val Gly Ser Ala Thr Thr Val Gln Val Ser Gly Asp Asn Arg
90 95 100
Gln Phe Val Gly Arg Val Ile Asp Ala Asp Gln Leu Asp Asp Leu Ala
105 110 115
Ile Val Arg Ile Pro Ala Pro Lys Ser Leu Glu Pro Leu Pro Leu Gly
120 125 130 135
Ser Val Lys Ser Leu Gln Pro Gly Ser Leu Val Ile Ala Ile Gly Asn
140 145 150
Pro Phe Glu Leu Thr Ser Ser Val Ser Ser Gly Ile Val Ser Gly Leu
155 160 165
Asn Arg Ser Met Ser Glu Ser Asn Gly His Val Met Asn Gly Met Ile
170 175 180
Gln Thr Asp Ala Pro Leu Asn Pro Gly Asn Ser Gly Gly Pro Leu Leu
185 190 195
Asn Ala Ala Gly Gln Val Val Gly Ile Asn Thr Leu Ile Glu Ser Pro
200 205 210 215
Ile Glu Gly Ser Ile Gly Ile Gly Phe Ala Ile Pro Ile Asp Arg Phe
220 225 230
Ile Gln Leu Glu Pro Glu Leu Leu Ala Gly Lys Pro Val Ala His Ala
235 240 245
Trp Leu Gly Ile Glu Gly Met Asp Ile Asp Asn Leu Met Arg Gln Ala
250 255 260
Leu His Leu Pro Val Ala Ser Gly Val Tyr Val Thr Glu Val Thr Pro
265 270 275
Gly Gly Pro Ala Ala Lys Ala Gly Leu Arg Gly Asp Ser Asn Ala Ala
280 285 290 295
Lys Leu Asn Ser Leu Ser Gln Ser Ala Asn Pro Tyr Ala Leu Leu Lys
300 305 310
Gly Asn Gly Asp Ile Ile Val Gly
315
<210>32
<211>211
<212>PRT
<213>脂环酸芽孢杆菌属
<220>
<221>SIGNAL
<222>(1)..(30)
<220>
<221>mat_peptide
<222>(31)..(212)
<223>二硫化物异构酶
<400>32
Met Arg Arg Ser Trp Ser Val Leu Met Ala Val Cys Met Ser Trp Leu
-30 -25 -20 -15
Ala Val Gly Cys Gly Thr Pro Ala Asn Ser Leu Ser Gln Ala Thr Ala
-10 -5 -1 1
Ala Ser Gly Arg His Ala Pro His Pro Leu Val Phe Gln Asn Leu Thr
5 10 15
Gly Ala Met Asn Glu Gly Gln Asp Pro Arg Trp Asp Pro Lys Ala Ala
20 25 30
Pro Thr Gly Val Tyr Asp Asp Val Thr Val Val Thr Ala Ser Gly Arg
35 40 45 50
Gln Glu Val Leu Ser Val Arg Asp Ala Pro Leu Leu Phe Ala Ala Tyr
55 60 65
Trp Cys Pro His Cys Gln Arg Thr Leu Gln Leu Leu Thr Ser Ile Glu
70 75 80
Ser Arg Leu Lys Gln Lys Pro Ile Leu Val Asn Val Gly Tyr Pro Pro
85 90 95
Gly Thr Thr Leu Gln Thr Ala Ala Arg Ile Ala Arg Glu Glu Ser Gln
100 105 110
Val Leu His Leu Ala Pro Phe Gln Glu Val Phe Ile Leu Asn Pro Asp
115 120 125 130
Ala Gly Asp Arg Tyr Ala Pro Leu Gly Tyr Pro Thr Leu Ala Phe Tyr
135 140 145
Arg Ala Gly Arg Asp Trp Thr Leu Tyr Gly Glu His Arg Ala Ser Ile
150 155 160
Trp Glu Lys Ala Leu Ser Glu Ser Thr Ser Lys Ala Tyr Asn Gly Ser
165 170 175
Glu Glu Ser
180
<210>33
<211>266
<212>PRT
<213>脂环酸芽孢杆菌属
<220>
<221>SIGNAL
<222>(1)..(29)
<220>
<221>mat_peptide
<222>(30)..(266)
<223>γ-D-谷氨酰-L-二氨基酸内肽酶
<400>33
Met Asp Glu Met Asn Ile Arg Ser Trp Cys Val Ala Ala Cys Thr Val
-25 -20 -15
Ala Leu Thr Ser Ala Val Gly Ala Thr Thr Ala Phe Ala Gln Thr Val
-10 -5 -1 1
Thr Val Gln Pro Gly Gln Ser Leu Trp Thr Ile Ala Arg Ala His Gly
5 10 15
Met Pro Val Gln Leu Val Ala Ser Ala Asn Pro Gln Tyr Asn Pro Leu
20 25 30 35
Asn Leu Pro Val Gly Ala Thr Val Thr Leu Pro Ser Leu Lys Asp Val
40 45 50
Ala Val Gln Pro Gly Asp Ser Leu Phe Leu Ile Gly Arg Gln Tyr Gly
55 60 65
Val Ser Leu Ala Glu Met Leu Ala Ala Asn Pro Asn Val Asp Pro Leu
70 75 80
Asn Leu Gln Val Gly Ser Ser Val Arg Val Pro Leu Ala Ser Ser Ser
85 90 95
Thr Lys Ser Ser Thr Val Ser Ala His Val Ala Ala Ser Thr Pro Glu
100 105 110 115
Asn Ser Asn Asn Leu Tyr Trp Leu Glu Arg Val Ile His Ala Glu Ala
120 125 130
Gly Gly Glu Ser Leu Gln Ala Gln Ile Ala Val Ala Asp Val Ile Leu
135 140 145
His Arg Met Ala Ala Gly Gly Tyr Gly Ser Thr Val Gln Gln Val Val
150 155 160
Phe Gln Val Ser Asp Gly His Tyr Gln Phe Glu Ser Val Ala Asn Gly
165 170 175
Ser Ile Tyr Gly Gln Pro Asp Ala Gln Asn Val Gln Ala Ala Leu Asp
180 185 190 195
Ala Leu Asn Gly Asp Asp Val Val Pro Gly Ala Leu Val Phe Tyr Asn
200 205 210
Pro Ala Gln Thr Pro Ser Gly Ser Trp Val Trp Gln Gln Pro Val Val
215 220 225
Ala His Ile Gly His Leu Val Phe Ala Lys
230 235
<210>34
<211>768
<212>PRT
<213>脂环酸芽孢杆菌属
<220>
<221>SIGNAL
<222>(1)..(26)
<220>
<221>mat_peptide
<222>(27)..(768)
<223>内-β-N-乙酰氨基葡糖苷酶
<400>34
Met Lys Thr His Arg Leu Leu Ala Val Ala Ala Leu Pro Ala Thr Val
-25 -20 -15
Leu Leu Thr Thr Pro Ala Pro Ala Leu Ala Glu Thr Ser Ser Ser Gln
-10 -5 -1 1 5
Ser Ala Ser Ala Pro Ser Leu Asn Val Pro Val Ala Ala Leu Thr Leu
10 15 20
Ala Gly Val Gln Ser Tyr Pro Met Leu Ser Tyr Gly Ser Thr Gly Val
25 30 35
Tyr Val Glu Ile Leu Gln Asn Ala Leu Asn Ala Leu Gly Tyr Asp Val
40 45 50
Gly Gln Ala Ser Gly Leu Phe Asp Ala Thr Thr Gln Ala Glu Val Lys
55 60 65 70
Ala Phe Gln Gln Ala Met Gly Leu Gln Thr Asp Gly Ile Val Gly Pro
75 80 85
Leu Thr Trp Gly Ala Leu Ala Lys Ala Val Ala Asp Tyr Arg Gln Val
90 95 100
Met Thr Val Leu Ser Ser Arg Ser Ser Leu Val Gln Gln Val Glu Trp
105 110 115
Lys Arg Ile Val Trp Asn Gly Arg Leu Ile Ser Lys Pro Ile Gly Phe
120 125 130
Thr Tyr Gln Gly Thr Ala Tyr Met Pro Ile Trp Tyr Val Met Gln Ala
135 140 145 150
Leu Ser Lys Ala Gly Ile Ala Ser Thr Trp Gln Gly Gly Val Trp Thr
155 160 165
Leu Thr Pro Pro Gly Gly Gln Thr Val Asn Tyr Gly Lys Ile Ser Tyr
170 175 180
Gly Pro Gly Ser Ala Ala Ile Ala Ile Gly Gln Thr Val Val Ala Asn
185 190 195
Val Pro Ala Val Val Tyr Pro Asp Pro Ala Ser Gly Lys Leu Thr Thr
200 205 210
Phe Met Pro Val Trp Tyr Val Met Asn Ala Leu Gln Arg Leu Gly Ile
215 220 225 230
Gly Ser Thr Trp Gln Gly Thr Glu Trp Asp Met Lys Pro Ala Pro Val
235 240 245
Val Ile Glu Thr Gly Asp Pro Ser Asn Asn Thr Thr Gly Ser Asp Pro
250 255 260
Ala Asn Ser Thr Gly Asn Gly Thr Gly Asn Ser Thr Gly Asn Ala Thr
265 270 275
Gly Ala Val Pro Gly Gly Asn Thr Val Thr Asn Val Thr Thr Gly Ser
280 285 290
Ser Asn Val Thr Gly Asn Ser Thr Gly Asn Ser Leu Gly Asn Ser Thr
295 300 305 310
Gly Asn Ser Leu Gly Asn Ser Thr Ser Asn Ala Thr Gly Asn Ala Thr
315 320 325
Gly Asn Thr Thr Gly Asn Ala Thr Gly Asn Ser Thr Gly Thr Ser Ser
330 335 340
Gly Ser Phe Thr Asn Val Asp Leu Arg Tyr Pro Ala Pro Ser Asn Ile
345 350 355
Asn Ala Gln Ser Ile Asn Gln Phe Leu Leu Gln Asn Ser Ser Pro Leu
360 365 370
Asn Gly Leu Gly Asn Ser Phe Met Asp Ala Gln Asn Leu Tyr Ser Val
375 380 385 390
Asp Ala Asn Tyr Leu Val Ser His Ala Ile Leu Glu Ser Ala Trp Gly
395 400 405
Gln Ser Gln Ile Ala Leu Gln Lys Asn Asn Leu Phe Gly Tyr Gly Ala
410 415 420
Tyr Asp Ser Asn Pro Gly Gln Asp Ala Gly Val Phe Pro Ser Asp Asp
425 430 435
Tyr Ala Ile Arg Phe Glu Ala Trp Thr Val Arg Met Asn Tyr Leu Thr
440 445 450
Pro Gly Ala Ser Leu Tyr Val Thr Pro Thr Leu Ser Gly Met Asn Val
455 460 465 470
Asn Tyr Ala Thr Ala Lys Thr Trp Ala Ser Gly Ile Ala Ala Ile Met
475 480 485
Thr Gln Phe Ala Ser Ser Val Gly Ser Asn Val Asn Ala Tyr Val Gln
490 495 500
Tyr Thr Pro Ser Asn Asn Pro Pro Ala Pro Arg Ser Thr Ala Glu Pro
505 510 515
Val Tyr Tyr Met Asn Gly Ala Gln Gly Val Thr Gln Gln Asp Pro Tyr
520 525 530
Tyr Pro Asn Gly Gly Val Pro Tyr Tyr Pro Thr Ile Ala Gln Gly Glu
535 540 545 550
Asn Gln Gln Phe Phe Gly Gln Leu Ser Val Gly Ser Phe Gly Gln Pro
555 560 565
Val Val Glu Val Gln Gln Phe Leu Asn Arg Thr Ile Asn Ala Gly Leu
570 575 580
Thr Val Asp Gly Gln Phe Gly Pro Leu Thr Gln Ala Ala Val Glu Lys
585 590 595
Phe Gln Ser Gln Val Met His Met Ser Asn Pro Asn Gly Ile Trp Thr
600 605 610
Phe Ser Met Trp Val Gln Tyr Ile Gln Pro Ser Gln Ser Asn Ala Asn
615 620 625 630
Leu Ile Pro Ala Gly Thr Thr Val Lys Ile Asp Gln Val Ala Glu Gly
635 640 645
Met Ala Gly Pro Tyr Val Val Pro Trp Tyr His Val Val Gly Tyr Gly
650 655 660
Trp Val Asp Ser Gln Tyr Ile Lys Leu Thr Asn Val Tyr Arg Val Ile
665 670 675
Val Gln Asn Pro Ala Gly Thr Ala Thr Thr Ile Pro Val Tyr Gln Val
680 685 690
Gly Asn Leu Ser Ser Val Leu Leu Asn Leu His Ser Gly Asp Trp Val
695 700 705 710
Val Ala Asn Ser Ala Gln Pro Ser Gly Gly Val Tyr Thr Ile Gln Ile
715 720 725
Ala Ala Gln Asp Pro Pro Cys Arg Thr Ala Thr Pro Pro Gly Arg Ser
730 735 740
<210>35
<211>597
<212>PRT
<213>脂环酸芽孢杆菌属
<220>
<221>SIGNAL
<222>(1)..(49)
<220>
<221>mat_peptide
<222>(50)..(597)
<223>多铜氧化酶
<220>
<221>MISC_FEATURE
<222>(139)..(139)
<223>推定的铜结合位点
<220>
<221>MISC_FEATURE
<222>(141)..(141)
<223>推定的铜结合位点
<220>
<221>MISC_FEATURE
<222>(181)..(181)
<223>推定的铜结合位点
<220>
<221>MISC_FEATURE
<222>(183)..(183)
<223>推定的铜结合位点
<220>
<221>MISC_FEATURE
<222>(514)..(514)
<223>推定的铜结合位点
<220>
<221>MISC_FEATURE
<222>(566)..(566)
<223>推定的铜结合位点
<400>35
Met Met Ala His Asp Arg Leu Asp Arg Arg Val Asn Glu Arg Arg Gln
-45 -40 -35
Ala Met Arg Arg Ala Ala Lys Trp Ala Ile Ala Leu Gly Thr Thr Ala
-30 -25 -20
Val Val Ala Gly Val Ser Ser Val Phe Ala Leu Arg Ser Val Arg Glu
-15 -10 -5
Ala Asn Leu Asn Pro Asn Ala Pro Leu Ala Asn Val Pro Gly Pro Gln
-1 1 5 10 15
Gly Ala Tyr Thr Pro Ile Ser Ala Leu Gln Pro Val Val Pro Lys Asn
20 25 30
Ala Arg Ile Asp His Tyr Thr Leu Thr Ala Glu Ser Arg Thr Leu Thr
35 40 45
Val Gly Gly His Ala Leu Gln Ala Met Thr Phe Asn Gly Thr Ala Pro
50 55 60
Gly Pro Leu Leu Val Ala His Gln Gly Asp Val Val Lys Val Thr Val
65 70 75
His Asn Arg Leu Ser Val Pro Leu Thr Ile His Trp His Gly Ile Ala
80 85 90 95
Val Pro Gly Ala Glu Asp Gly Val Pro Gly Val Thr Gln Asn Pro Ile
100 105 110
Pro Pro Gly Gly Ser Tyr Thr Tyr Glu Phe Gln Val Asn Gln Pro Gly
115 120 125
Thr Tyr Trp Tyr His Ser His Glu Ala Ser Phe Glu Glu Val Gly Leu
130 135 140
Gly Leu Tyr Gly Ala Phe Val Val Leu Pro Lys Arg Ala Val His Pro
145 150 155
Ala Asp Arg Asp Tyr Thr Leu Val Leu His Glu Trp Pro Thr Ala Ser
160 165 170 175
Thr Ala Gln Thr Met Met Ala Asn Leu Lys Ala Gly Asn Leu Gly Phe
180 185 190
Ser Ala Lys Gly Glu Ser Ala Gly Met Gly Gly Met Gly Met Gln Gln
195 200 205
Asn Gly Asp Met Asn Gly Met Gly Met Met Gly Ala Ala Asp Gly Thr
210 215 220
Gly Gln Gly Gly Asn Ser Ala Ser Asp Ile Ala His Val Leu Pro Gly
225 230 235
Pro Pro Leu Gln Leu Asn Gly Phe Ser Pro Thr Ala Asn Asp Trp Ala
240 245 250 255
Ala Leu Asp Glu Met Ala Gly Met Tyr Asp Ala Phe Thr Val Asn Gln
260 265 270
Asn Ala Ser Gly Thr Thr Leu Leu Pro Ala Lys Pro Gly Gln Leu Val
275 280 285
Arg Leu Arg Ile Val Asn Ser Gly Asn Met Thr His Leu Phe Thr Leu
290 295 300
Val Gly Ala Pro Phe Arg Val Val Ala Leu Asp Gly His Asp Ile Ala
305 310 315
Asn Pro Gly Trp Ile Arg Gly Val Leu Leu Pro Val Gly Ala Ala Glu
320 325 330 335
Arg Tyr Asp Ile Glu Phe Arg Val Pro Lys Ser Gly Ala Ala Phe Leu
340 345 350
Val Cys Ala Asp Pro Asp Thr Thr Ala Gln Arg Glu Leu Arg Ala Ala
355 360 365
Ile Gly Leu Pro Asp Ala Trp Ser Gln Phe Lys Glu Thr Asp Ala Ala
370 375 380
Ser Leu Glu Arg Ala Pro Trp Phe Asp Phe Thr His Tyr Gly Ser Gly
385 390 395
Arg Leu Pro Gly Glu Ala Val Phe Arg Leu His Gln Ala Tyr Gln Val
400 405 410 415
Arg Tyr Asn Met Lys Leu Thr Val Gly Met Ser Met Asn Gly Met Val
420 425 430
Tyr Ala Ile Asn Gly Lys Val Phe Pro Asn Ile Pro Pro Ile Val Val
435 440 445
Arg Lys Gly Asp Ala Val Leu Val His Ile Val Asn Asp Ser Pro Tyr
450 455 460
Ile His Pro Met His Leu His Gly His Asp Phe Gln Val Leu Thr Arg
465 470 475
Asp Gly Lys Pro Val Ser Gly Ser Pro Ile Phe Leu Asp Thr Leu Asp
480 485 490 495
Val Phe Pro Gly Glu Ser Tyr Asp Ile Ala Phe Arg Ala Asp Asn Pro
500 505 510
Gly Leu Trp Met Phe His Cys His Asp Leu Glu His Ala Ala Ala Gly
515 520 525
Met Asp Val Met Val Gln Tyr Ala Gly Ile Arg Asp Pro Tyr Pro Met
530 535 540
Ser Glu Met Ser Glu
545
<210>36
<211>245
<212>PRT
<213>脂环酸芽孢杆菌属
<220>
<221>SIGNAL
<222>(1)..(29)
<220>
<221>mat_peptide
<222>(30)..(246)
<223>肽酰脯氨酰异构酶
<400>36
Met Lys Arg Arg Thr Leu Leu Ala Gly Ile Thr Leu Ala Ala Leu Val
-25 -20 -15
Ala Val Ala Gly Cys Gly Thr Pro Ala Gly Asn Thr Ala Ser Pro Asp
-10 -5 -1 1
Asn Thr Ala Asn Leu Ser Asn Thr Asn Ala Pro Asp Thr Leu Ser Asn
5 10 15
Glu Thr Gly Gln Thr Leu Asp Thr Ala Asn Pro Pro Tyr Leu His Thr
20 25 30 35
Ser Thr Glu Gln Trp Lys Ser Met Pro Lys Met Phe Ile Asn Pro Asn
40 45 50
Lys Thr Tyr Asp Ala Ile Val His Thr Asn Tyr Gly Thr Phe Thr Ile
55 60 65
Gln Leu Phe Ala Lys Asp Ala Pro Ile Thr Val Asn Asn Phe Val Phe
70 75 80
Leu Ala Glu His Asn Phe Tyr His Asp Cys Thr Phe Phe Arg Ile Val
85 90 95
Lys Asn Phe Val Ile Gln Thr Gly Asp Pro Arg Asn Asp Gly Thr Gly
100 105 110 115
Gly Pro Gly Tyr Thr Ile Pro Asp Glu Leu Ser His Gln Val Pro Phe
120 125 130
Thr Lys Gly Ile Val Ala Met Ala Asn Thr Gly Gln Pro His Thr Gly
135 140 145
Gly Ser Gln Phe Phe Ile Cys Thr Ala Asn Asp Thr Gln Val Phe Gln
150 155 160
Pro Pro Asn Asn Arg Tyr Thr Glu Phe Gly Arg Val Ile Ser Gly Met
165 170 175
Asp Val Ile Asp Lys Ile Ala Ala Ile Pro Val Thr Glu Asn Pro Met
180 185 190 195
Thr Gln Glu Asp Ser Tyr Pro Leu Lys Thr Ala Tyr Ile Glu Ser Ile
200 205 210
Gln Ile Gln Glu Ser
215
<210>37
<211>608
<212>PRT
<213>脂环酸芽孢杆菌属
<220>
<221>SIGNAL
<222>(1)..(27)
<220>
<221>mat_peptide
<222>(28)..(608)
<223>酸性磷酸酯酶或植酸酶或磷脂酶C
<400>37
Met Lys Lys Gly Lys Arg Trp Ser Ala Ala Leu Ala Thr Ser Val Ala
-25 -20 -15
Leu Phe Ala Thr Leu Ser Pro Gln Ala Leu Ala Ser Asp Thr Val Val
-10 -5 -1 1 5
Pro Gln Val Asn Thr Leu Thr Pro Ile His His Leu Val Val Ile Phe
10 15 20
Asp Glu Asn Val Ser Phe Asp His Tyr Phe Ala Thr Tyr Pro Asn Ala
25 30 35
Ala Asn Pro Ala Gly Glu Pro Pro Phe Tyr Ala Ala Pro Gly Thr Pro
40 45 50
Ser Val Asn Gly Leu Ser Gly Ser Leu Leu Thr His Asn Pro Asn Gly
55 60 65
Val Asn Pro Gln Arg Leu Asp Arg Ser Gln Ala Val Thr Pro Asp Met
70 75 80 85
Asn His Asn Tyr Thr Pro Glu Gln Gln Ala Val Asp Gly Gly Arg Met
90 95 100
Asp Asn Phe Ile Asn Thr Val Gly Arg Gly Asn Pro Ile Asp Leu Asp
105 110 115
Tyr Tyr Asp Gly Asn Thr Val Thr Ala Leu Trp Tyr Tyr Ala Gln His
120 125 130
Phe Ala Leu Asn Asp Asn Ala Tyr Cys Thr Gln Tyr Gly Pro Ser Thr
135 140 145
Pro Gly Ala Ile Asn Leu Ile Ser Gly Asp Thr Ala Gly Ala Thr Val
150 155 160 165
Tyr Ser Ser Ser Glu Thr Ser Gly Ala Ala Gln Val Val Pro Pro Gly
170 175 180
Ser Lys Asn Phe Pro Asn Ala Val Thr Pro Asn Gly Val Asp Ile Gly
185 190 195
Asp Ile Asp Pro Tyr Tyr Asp Ser Ala Ser Lys Gly Met Thr Met Ala
200 205 210
Met Ala Gly Lys Asn Ile Gly Asp Leu Leu Asn Ala Lys Gly Val Thr
215 220 225
Trp Gly Trp Phe Gln Gly Gly Phe Ala Asn Pro Asn Ala Lys Asp Asn
230 235 240 245
Asn Ile Ala Gly Thr Asp Glu Thr Thr Asp Tyr Ser Ala His His Glu
250 255 260
Pro Phe Gln Tyr Tyr Ala Ser Thr Ala Asn Pro Asn His Leu Pro Pro
265 270 275
Thr Ser Val Ala Met Ile Gly Arg Thr Asp Gln Ala Asn His Gln Tyr
280 285 290
Asp Ile Thr Asn Phe Phe Gln Ala Leu Gln Asn Gly Asn Met Pro Ala
295 300 305
Val Ser Phe Leu Lys Ala Pro Glu Tyr Glu Asp Gly His Ala Gly Tyr
310 315 320 325
Ser Asp Pro Leu Asp Glu Gln Arg Trp Leu Val Gln Thr Ile Asn Gln
330 335 340
Ile Glu Ala Ser Pro Asp Trp Ser Ser Thr Ala Ile Ile Ile Thr Tyr
345 350 355
Asp Asp Ser Asp Gly Trp Tyr Asp His Val Met Pro Pro Leu Val Asn
360 365 370
Gly Ser Ser Asp Lys Ala Val Asp Val Leu Gly Gly Thr Pro Val Leu
375 380 385
Gln Asn Gly Thr Asp Arg Ala Gly Tyr Gly Pro Arg Val Pro Phe Leu
390 395 400 405
Val Ile Ser Pro Tyr Ala Lys His Asn Phe Val Asp Asn Thr Leu Ile
410 415 420
Asp Gln Thr Ser Val Leu Arg Phe Ile Glu Glu Asn Trp Gly Leu Gly
425 430 435
Ser Leu Gly Pro Ala Ser Tyr Asp Ser Leu Ala Gly Ser Ile Met Asn
440 445 450
Met Phe Asp Trp Asn Thr Gln Asn Pro Pro Val Phe Leu Asp Pro Thr
455 460 465
Thr Gly Glu Pro Val Ser Pro Asp Met Gln Pro Glu Val Ile Arg Gly
470 475 480 485
Thr Thr Tyr Leu Ser Leu Asn His Tyr Ala Gln Asn Leu Asp Val Val
490 495 500
Leu Gln Thr Ser Arg Gly Met Ala Arg Phe Ser Tyr Glu Gly His Glu
505 510 515
Val Glu Ile Asp Glu Arg Ser Gly Leu Val Arg Val Asp Gly Glu Ala
520 525 530
Val His Leu Lys Ala Pro Leu Val Arg Val Asp Gly Val Trp Met Val
535 540 545
Pro Val Glu Glu Met Asp Ser Leu Ile Gly Ala Thr Leu His Thr Tyr
550 555 560 565
Thr Asp Gly His Leu Thr Tyr Tyr Leu Phe Ser Pro Gln Asp Ala His
570 575 580
<210>38
<211>250
<212>PRT
<213>脂环酸芽孢杆菌属
<220>
<221>SIGNAL
<222>(1)..(25)
<220>
<221>mat_peptide
<222>(26)..(251)
<223>多糖脱乙酰酶或术聚糖脱乙酰酶
<400>38
Met Leu Ser Leu Trp Lys Arg Ile Arg Thr Gly Thr Leu Ser Leu Leu
-25 -20 -15 -10
Ala Ala Cys Ala Cys Ala Leu Ser Ala Met Gly Ala Gly Ala Gly Trp
-5 -1 1 5
Val His Ala Ala Glu Ser Gln Ala Gln Ala Pro Arg Ala Ile Tyr Lys
10 15 20
Val Asp Thr Lys Glu Lys Val Val Ala Leu Thr Phe Asp Ile Ser Trp
25 30 35
Gly His Arg Thr Pro Glu Pro Val Leu Glu Thr Leu Lys Lys Cys Gly
40 45 50 55
Val Thr Lys Ala Thr Phe Phe Leu Ser Gly Pro Trp Thr Met His His
60 65 70
Ala Asp Ile Ala Lys Lys Ile Lys Ala Met Gly Tyr Glu Ile Gly Ser
75 80 85
His Gly Tyr Leu His Lys Asp Tyr Ser Asn Tyr Pro Asp Ser Trp Ile
90 95 100
Arg Glu Gln Ala Met Leu Ala Asp Lys Ala Ile Gln Gln Val Thr Gly
105 110 115
Val Lys Pro Lys Leu Phe Arg Thr Pro Asn Gly Asp Leu Asn Pro Arg
120 125 130 135
Val Ile Arg Cys Leu Thr Ser Met Gly Tyr Thr Val Val Gln Trp Asn
140 145 150
Thr Asp Ser Leu Asp Trp Lys Asn Pro Gly Val Asp Ala Ile Val Asn
155 160 165
Arg Val Thr Lys Arg Val Val Pro Gly Asp Ile Ile Leu Met His Ala
170 175 180
Ser Asp Ser Ser Lys Gln Ile Val Glu Ala Leu Pro Arg Ile Ile Glu
185 190 195
Ser Leu Arg Gln Gln Gly Tyr Arg Phe Val Thr Val Ser Glu Leu Leu
200 205 210 215
Ala Gly Ala Ser Val Gln Ser Lys Val Gln
220 225
<210>39
<211>324
<212>PRT
<213>脂环酸芽孢杆菌属
<220>
<221>SIGNAL
<222>(1)..(21)
<220>
<221>mat_peptide
<222>(22)..(324)
<223>多糖脱乙酰酶或木聚糖脱乙酰酶
<400>39
Met Arg Lys Thr Ala Ala Gly Ala Cys Ala Leu Ala Leu Met Gly Val
-20 -15 -10
Leu Gly Gly Trp Ala Gly Ala Ala Gly Thr Ala Val Asn Ala His Ala
-5 -1 1 5 10
Pro Ala Ala Ser Ala Pro Ser Val Ser Ala His Val Trp Glu Glu Val
15 20 25
Ser Arg Thr Trp Gly Thr Leu Pro Val Asp Ala Arg His Asp Gly Val
30 35 40
Trp His Asn Ile Pro Gly Leu Ser Gly Phe Ala Leu Asp Thr Ala Ala
45 50 55
Ser Glu Arg Glu Thr Ala Arg Arg His Asp Gly Ala Leu His Leu Val
60 65 70 75
Trp Arg Thr Leu Pro Pro Lys Arg Arg Leu Gly Asp Leu Ser Pro Asp
80 85 90
Val Ile Tyr Arg Gly Pro Ala Gln Glu Lys Ser Val Ala Leu Met Val
95 100 105
Asn Val Ser Trp Gly Asp Ala Tyr Val Pro Arg Met Leu Glu Val Leu
110 115 120
Arg Ser Ala His Val Lys Ala Thr Phe Phe Val Asp Gly Ala Phe Ala
125 130 135
Lys Lys Phe Pro Asp Leu Val Arg Ala Met Ala Arg Asp Gly His Ala
140 145 150 155
Val Glu Ser His Gly Phe Gly His Pro Asp Phe Arg Arg Leu Ser Asp
160 165 170
Ala Lys Leu Ala Ala Gln Leu Asp Glu Thr Asn Arg Val Leu Ala Gly
175 180 185
Ile Thr Gly Lys Val Pro Arg Leu Ile Ala Pro Pro Ala Gly Ser Tyr
190 195 200
Asp Ala Arg Leu Ala Pro Leu Ala His Ser Arg Arg Met Tyr Ala Ile
205 210 215
Leu Trp Thr Ala Asp Thr Val Asp Trp Lys Asn Pro Pro Ala Asp Val
220 225 230 235
Ile Val Gln Arg Val Gln Arg Gly Ala Glu Pro Gly Ala Leu Ile Leu
240 245 250
Met His Pro Thr Ala Pro Thr Ala Glu Ala Leu Pro Asp Val Ile Arg
255 260 265
Trp Leu Glu Gly His Gly Tyr Arg Leu Lys Thr Val Glu Asp Val Ile
270 275 280
Asp Glu Arg Pro Ala Val Thr Pro Pro Thr Thr Leu Ala Asn Glu Thr
285 290 295
Phe His Ser Ala
300
<210>40
<211>214
<212>PRT
<213>脂环酸芽孢杆菌属
<220>
<221>SIGNAL
<222>(1)..(29)
<220>
<221>mat_peptide
<222>(30)..(214)
<223>亚硫酸盐氧化酶
<400>40
Met Met Arg Trp Asn Trp Lys Val Ala Val Gly Ser Leu Ala Leu Ala
-25 -20 -15
Ala Leu Gly Ala Gly Ala Ala Val Ser Pro Val Phe Ala Ala Ala Lys
-10 -5 -1 1
Ser Ser Lys Ala Ala Gln Ser His Ala Glu Ala Ser Ala Ala Val Val
5 10 15
Met Ala Gly Lys Leu Tyr Gly Asn Ile Pro Asn Val Thr Ile Arg Gly
20 25 30 35
Val Glu Ala Gly Lys Ala Pro Trp Val Val Asp Gly Ser Tyr Gln Leu
40 45 50
Lys Ser Asn Leu Phe Thr Ala Ser Gly Lys Trp Leu Ile Ile Pro Lys
55 60 65
Gln Gly Tyr Met Glu Asn Gly Gln Pro Val Pro Ala Lys Ile Gly Gly
70 75 80
Thr Thr Asn Asn Ile Pro Ala Val Gly Ala Glu Ile Thr Phe Ala Asn
85 90 95
Ala Ala Pro Ile Val Leu Pro Pro Val Lys Leu Ser Ser Gln Gly Asp
100 105 110 115
Phe Ser Phe His Asp Ala Ile Gln Trp Pro Lys Gly Ala Ala Gln Pro
120 125 130
Val Ile Leu Ile Gly Pro Glu Lys Asn Gly Gln Leu Val Ala Trp Phe
135 140 145
Ala Ala Ser Asp Phe Leu Ala Asp Tyr Gly Gln Ala Thr Gly Met Gly
150 155 160
Gly Gly Trp Val Asn Ala Ala His Pro Glu Thr Pro Val Arg His Thr
165 170 175
His Leu Ala Ser Lys Lys
180 185
<210>41
<211>257
<212>PRT
<213>脂环酸芽孢杆菌属
<220>
<221>SIGNAL
<222>(1)..(21)
<220>
<221>mat_peptide
<222>(22)..(257)
<223>功能性多肽
<400>41
Met Asn Trp Ala Arg Val Gly Ala Trp Val Ser Thr Trp Leu Val Ala
-20 -15 -10
Thr Ala Leu Gly Ala Gly Cys Gly Thr Ala Ser Gln Glu His Pro Ser
-5 -1 1 5 10
Asn Thr Ser Thr Ser Asp His Arg Val Ala Pro Ala Ala Pro Gly Gly
15 20 25
Ser Ala Ser Met Gln Asn Arg His Ile Leu Gln Glu Pro Leu Pro Arg
30 35 40
Gly Val Lys Thr Glu Thr Asp Leu Tyr Asn Trp Leu Leu Trp Gln Arg
45 50 55
Leu Ala Glu Ile Asn Asn Pro Ala Gln Gly Glu Ile Cys Leu Asp Ala
60 65 70 75
Ala Cys Lys Ile Ala Ala Thr Val Phe Ser Gly Pro Ala Lys Ala Ala
80 85 90
Ala Gly Thr Pro Val Thr Leu Val Ala Phe Ser Pro Arg Ala Gly Trp
95 100 105
Gln Val Leu Val Gly Pro Leu Pro Gln Ser Asp Asn Pro Pro Arg Gln
110 115 120
Ala Gln Ser Ile Thr Gly Gln Ser Ala Arg Leu Pro Ala Gln Arg Gly
125 130 135
Arg Met Arg Arg Ser Asn Pro Arg Asn Arg Leu Val Leu Asp Ser Gly
140 145 150 155
Arg Thr Pro Ala Ala Asp Ala Ser Ala Ala Arg Met Thr Arg Gln Leu
160 165 170
Arg Arg Ser Ala Ser Ser Thr Asn Ala Ser Arg Ser Arg Arg Ala Lys
175 180 185
Ser Met Ala Arg Cys Gln Lys Ser Gly Cys Val Arg Ser Ala Pro Met
190 195 200
Cys Phe Trp Ala Arg Ser Ser Thr Arg Met Arg Pro Val Ser Arg Ser
205 210 215
Asn Ala Thr Tyr Leu Ser Ala Asn Pro Val Pro Ser Ala Glu Ala Met
220 225 230 235
Ala
<210>42
<211>1130
<212>PRT
<213>脂环酸芽孢杆菌属
<220>
<221>SIGNAL
<222>(1)..(24)
<220>
<221>mat_peptide
<222>(25)..(1130)
<223>功能性多肽
<400>42
Met Lys Arg Thr Leu Ser Gly Ile Ala Ser Ala Ala Ile Val Leu Gly
-20 -15 -10
Ala Ile Ser Pro Met Ala Phe Ala Gln Thr Ser Ser Ser Gly Leu Thr
-5 -1 1 5
Pro Ala Gly Gln Leu Pro Ile Val Val Asn Gly Gln Val Leu Ser Asn
10 15 20
Pro Tyr Glu Met Val Gly Met Asp Ser Gly Asn Lys Thr Gly Phe Phe
25 30 35 40
Pro Ile Tyr Tyr Phe Asp Gln Ala Leu Glu Lys Ile Gly Ile Thr Ala
45 50 55
Thr Trp Asn Gly Ala Thr His Thr Trp Ala Leu Thr Asp Ser Asn Val
60 65 70
Asn Ala Ser Asn Val Gln Val Ala Gly Gly Met Gly Thr Gly Asn Thr
75 80 85
Thr Val Thr Leu Asn Gly Thr Pro Ile Lys Met Phe Tyr Thr Gln Val
90 95 100
Ala Lys Asp Pro Ala Gly Gly Pro Val Thr Thr Tyr Met Pro Ile Tyr
105 110 115 120
Tyr Ile Asn Asn Ile Leu Ser Ala Leu Gly Ile His Gly Thr Phe Ser
125 130 135
Gly Gln Thr Gly Leu Asn Ile Thr Thr Gly Gln Thr Leu Ala Gly Ser
140 145 150
Leu Ser Ala Ile Thr Val Thr Gly Ala Thr Ser Gly Thr Gly Thr Ser
155 160 165
Ser Ser Pro Ala Val Ala Leu Asn Asn Gly Lys Val Thr Leu Ser Thr
170 175 180
Thr Leu Thr Asp Ser Asn Gly Asn Pro Ile Gly Asn Ala Ala Val Thr
185 190 195 200
Phe Asn Phe Ser Glu Tyr Gly Ala Leu Pro Ser Asn Ala Pro Thr Val
205 210 215
Thr Asn Ala Ser Gly Ala Thr Ile Pro Ala Thr Thr Gly Ser Thr Ala
220 225 230
Tyr Gln Tyr Thr Val Tyr Thr Asn Ser Ser Gly Val Ala Ser Ile Thr
235 240 245
Val Ser Gly Pro Val Gly Leu Thr Tyr Ala Tyr Gln Val Thr Ala Thr
250 255 260
Ala Pro Ile Ser Asn Gly Ser Asn Gln Met Ile Ser Ser Gln Pro Ala
265 270 275 280
Tyr Val Glu Phe Val Ala Asn Asn Gln Ala Gly Ile Ala Pro Tyr Gly
285 290 295
Thr Ala Ser Gln Pro Tyr Ser Ala Ser Leu Gly Thr Ala Val Pro Ile
300 305 310
Thr Val Ile Leu Pro Pro Gly Ala Asn Gly Gln Pro Gln Ala Asn Val
315 320 325
Leu Val Thr Leu Ser Leu Ser Asn Pro Asn Gly Gly Thr Asn Tyr Ala
330 335 340
Tyr Phe Thr Asn Ser Ser Gly Ala Asn Leu Gly Thr Gln Ile Gln Val
345 350 355 360
Thr Thr Asn Ser Ser Gly Val Ala Gln Ala Trp Val Ser Asp Ala Asn
365 370 375
Ala Gln Pro Val Val Val Thr Ala Asn Val Ser Asn Ala Thr Asn Val
380 385 390
Ser Asn Thr Ser Val Ser Thr Tyr Leu Asn Phe Gly Gln Ala Gly Val
395 400 405
Pro Ala Ser Ile Ala Asn Tyr Asn Asp Pro Tyr Ser Ala Leu Val Ala
410 415 420
Asn Gly Gln Gln Pro Leu Ala Gly Thr Thr Val Thr Ile Thr Gly Thr
425 430 435 440
Leu Val Asp Ala Ala Gly Asn Pro Val Ala Asn Gly Gln Val Leu Val
445 450 455
Thr Gly Ser Ser Ser Ser Gly Asp Phe Gly Tyr Val Thr Thr Ser Asn
460 465 470
Gly Lys Ser Thr Thr Thr Asp Phe Pro Ser Val Gly Thr Leu Gln Pro
475 480 485
Gly Gln Pro Val Ser Ser Ala Leu Gly Asp Val Ile Thr Ala Asp Ala
490 495 500
Asn Gly Asn Phe Ser Leu Gln Val Thr Asp Thr Gln Asn Glu Gln Ala
505 510 515 520
Ser Leu Thr Phe Tyr Ser Val Ser Asn Gly Val Ile Ser Pro Val Gly
525 530 535
Val Ile Lys Thr Asp Thr Leu Lys Phe Ala Val Asn Asn Gln Leu Ser
540 545 550
Thr Ile Ala Leu Gly Ala Thr Asp Ala Gln Ala Asp Gly Asn Gln Tyr
555 560 565
Thr Asn Leu Thr Gly Leu Thr Gly Ser Asp Asn Ala Pro Val Pro Val
570 575 580
Tyr Val Asp Pro Gln Asn Pro Ser Gly Thr Met Val Thr Asn Gln Ser
585 590 595 600
Ile Thr Tyr Thr Leu Ser Val Ser Ser Gly Asp Ile Val Gly Ile Gly
605 610 615
Ser Gly Ala Tyr Leu Ala Pro Thr Asn Ala Asn Asn Ser Thr Ile Pro
620 625 630
Ile Asn Ser Gly Asn Gly Leu Ser Ser Val Gln Val Thr Val Thr Ala
635 640 645
Leu Gly Asn Asn Gln Tyr Gln Ile Ser Val Pro Gly Gln Gln Gly Val
650 655 660
Leu Thr Thr Ser Ser Pro Asp Phe Thr Val Leu Val Lys Gly Ser Thr
665 670 675 680
Gly Ser Thr Lys Leu Thr Val Ser Ser Gly Ser Leu Ser Ser Thr Ala
685 690 695
Thr Ile Thr Phe Thr Ser Ser Asn Pro Thr Val Val Ala Ser Leu Thr
700 705 710
Pro Val Ser Ser Val Leu Ala Ala Gly Gln Asn Glu Thr Val Thr Phe
715 720 725
Thr Val Glu Asp Ala Asp Gly Asn Pro Val Ser Gly Asn Thr Gln Val
730 735 740
Ala Ile Thr Ala His Asp Ser Asn Asp Pro Leu Trp Ile Thr Ala Val
745 750 755 760
Asn Gly Thr Asn Leu Ser Glu Tyr Glu Thr Ile Asn Gly Ala Ala Thr
765 770 775
Ser Val Ser Thr Pro Ile Pro Leu Gly Thr Ser Ser Tyr Ala Thr Ser
780 785 790
Gly Gly Ser Thr Leu Tyr Pro Ala Tyr Thr Asn Ser Gly Tyr Phe Lys
795 800 805
Asn Gly Val Ser Ile Ser Gly Val Val Ser Trp Asp Gly Thr Val Gly
810 815 820
Asp Pro Ile Tyr Val Thr Thr Asn Ser Gln Gly Gln Val Thr Leu Thr
825 830 835 840
Leu Gln Asn Gly Asn Val Thr Tyr Phe Asp Gly Asn Asn Thr Thr Leu
845 850 855
Ser Asn Gly Ile Ser Val Ala Gly Thr Ser Gly Ser Glu Gly Phe Tyr
860 865 870
Thr Tyr Ser Ser Asp Thr Ala Ala Thr Ala Ser Asp Leu Thr Asn Met
875 880 885
Gly Val Leu Val Ile Gly Gln Ala Asn Gly Asp Ala Ser Thr Ser Leu
890 895 900
Gly Thr Ile Tyr Ile Gly Ser Gly Gly Ala Thr Gln Thr Pro Ala Ala
905 910 915 920
Phe Thr Tyr Val Asp Ala Asn Asn His Ser Tyr Thr Tyr Ser Asn Thr
925 930 935
Ser Asp Thr Phe Thr Val Ser Ser Thr Gln Ser Val Ser Gly Gly Asn
940 945 950
Tyr Ala Ile Thr Ser Phe Thr Pro Val Gly Gly Thr Ala Thr Ser Thr
955 960 965
Ile Pro Ser Gly Val Ser Val Asn Ser Ser Thr Gly Thr Val Ser Val
970 975 980
Ser Gln Asn Ala Ala Val Gly Thr Tyr Thr Val Ser Tyr Tyr Leu Asn
985 990 995 1000
Gly Val Thr Glu Ser Thr Gly Thr Phe Lys Val Tyr Ser Gly Ser
1005 1010 1015
Gly Val Ala Pro Thr Glu Ile Thr Gly Ser Ser Val Thr Val Pro
1020 1025 1030
Ala Ala Thr Tyr Ser Gly Thr Leu Lys Val Thr Val Ser Asn Gly
1035 1040 1045
Gly Ser Pro Leu Tyr Val Asn Val Thr Ala Gly Glu Ser Ala Asn
1050 1055 1060
Ala Val Ala Ala Ala Ile Tyr Asn Ala Leu Val Asn Ala Asn Ile
1065 1070 1075
Ser Gly Asp Thr Phe Ser Val Ser Gly Ser Thr Val Ser Val Thr
1080 1085 1090
Ala Ala Ser Gly Ser Pro Thr Leu Thr Val Val Asp Ala Thr Asn
1095 1100 1105
Phe
<210>43
<211>248
<212>PRT
<213>脂环酸芽孢杆菌属
<220>
<221>SIGNAL
<222>(1)..(41)
<220>
<221>mat_peptide
<222>(42)..(248)
<223>功能性多肽
<400>43
Met Arg Ile Met Lys Val Leu Gly Trp Ile Leu Val Pro Tyr Ile Met
-40 -35 -30
Leu Phe Ile Gln Trp Gly Arg Met Asn Arg Ile Leu Arg Phe Ala Gly
-25 -20 -15 -10
Ser Leu Trp Ala Leu Ile Val Phe Ala Asn Thr Val Tyr Met Ile Arg
-5 -1 1 5
Gly Asn Thr Pro Arg Asn Ala Ser Thr Val Ser Ala Thr Thr Ser Leu
10 15 20
Val Asn Ser Thr Asn Ser Ser Gln Val Ala Lys Gln Glu Gln Asn Ser
25 30 35
Ser Thr Ser Pro Ala His Lys Ser Thr Asn Ser Leu Gln His Ala Gln
40 45 50 55
His Gln Ala Ala Thr Thr Ser Ser Ser Gln Ser Lys Leu Arg Tyr Ile
60 65 70
Pro Phe His Thr Tyr Gly Lys Val Gly Asp Leu Glu Ile Arg Val Asn
75 80 85
Ser Leu Gln Gln Val Lys Ser Val Gly Tyr Asp Gly Ile Gly Glu Thr
90 95 100
Ala Asn Gly Ala Phe Trp Val Ile Asn Ile Thr Ile Arg Asn Asp Gly
105 110 115
Ser Thr Pro Met Glu Val Val Asp Gly Ile Phe His Leu Gln Asn Leu
120 125 130 135
Asn Gly Asn Val Tyr Gln Pro Asp Ser Thr Ala Glu Ile Tyr Ala Asn
140 145 150
Thr Asn Ser Gly Thr Ile Pro Thr Asp Leu Asn Pro Gly Val Ser Met
155 160 165
Thr Thr Asn Leu Val Phe Asp Met Pro Asp Phe Met Thr Tyr Gly His
170 175 180
Val Gly Gln His Tyr Ser Leu Val Ala Ser Met Gly Phe Phe Gly Ser
185 190 195
Asp Glu Thr Thr Tyr Ala Leu Pro
200 205
<210>44
<211>172
<212>PRT
<213>脂环酸芽孢杆菌属
<220>
<221>SIGNAL
<222>(1)..(25)
<220>
<221>mat_peptide
<222>(26)..(172)
<223>功能性多肽
<400>44
Met Asn Arg Lys Ser Met Leu Ser Val Leu Gly Val Ala Ala Ala Val
-25 -20 -15 -10
Ala Leu Met Val Thr Gly Cys Gly Thr Ala Asn Ser Thr Asn Asn Thr
-5 -1 1 5
Ala Ser Ser Gly Ala Ala Ser Thr Ala Val Thr Val Lys His Glu His
10 15 20
Lys Gly Ala Asn Ala Ser Lys Thr Glu Thr Lys Gln Thr Glu Ala Lys
25 30 35
Ser Ser Asn Lys Ala Gly Glu Thr Ala Lys Ser Ser Val Lys Leu Thr
40 45 50 55
Ala Pro Val Ala Gly Ala Thr Val Thr Ala Gly Gly Thr Leu Lys Val
60 65 70
Ser Gly Gln Val Ser Ser Asn Leu Ala Lys Lys Asp Val Gln Ile Thr
75 80 85
Leu Thr Asn Ser Ala Lys Lys Val Leu Val Gln Gln Ile Val Gly Thr
90 95 100
Asn Ser Thr Gly Ala Phe Val Asp Thr Leu Lys Leu Pro Lys Tyr Leu
105 110 115
Gly Lys Ala Gly Ser Asp Leu Thr Leu Ser Val Ser Val Val Gly Glu
120 125 130 135
Asn Gly Val Val Ser Thr Leu Ser Leu His Val Lys
140 145
<210>45
<211>242
<212>PRT
<213>脂环酸芽孢杆菌属
<220>
<221>SIGNAL
<222>(1)..(30)
<220>
<221>mat_peptide
<222>(31)..(242)
<223>功能性多肽
<400>45
Met Arg Arg Ala Val Arg Ile Leu Ala Ala Leu Leu Phe Gly Leu Ala
-30 -25 -20 -15
Thr Val Thr Ala Thr Leu Met Phe Val Pro Gln Ala Arg Ala Ala Thr
-10 -5 -1 1
Val Thr Gly Ala Leu Ala Gln Ser Gln Val Val Ser Ile Thr Gly Gly
5 10 15
Tyr Asn Thr Thr Thr Gln Met Tyr Glu Gln Thr Gly Gln Gln Thr Val
20 25 30
Val Thr Asn Trp Thr Phe Ser Leu Gln Gln Thr Val Asn Gln Asn Asn
35 40 45 50
Glu Asn Pro Ser Tyr Ala Gln Cys Thr Val Leu Ala Gly Asn Gln Gln
55 60 65
Val Thr Cys Thr Ser Asp Ala Thr Asn Asn Gly Ala Ile Cys Thr Ser
70 75 80
Pro Tyr Pro Gly Ala Ile Asp Lys Gln Cys Thr Asn Leu Ile Gly Phe
85 90 95
Thr Gly Asn Ile Ser Val Ser Ser Gln Asn Gly Asn Pro Thr Phe Thr
100 105 110
Phe Ser Leu Pro Ser Ile Asp Pro Ser Thr Met Lys Pro Val Gly Ile
115 120 125 130
Phe Val Thr Pro Glu Thr Ile Tyr Gly Gln Met Gly Thr Gly Ser Glu
135 140 145
Ser Tyr Leu Ser Ser Gly Gln Ser Gly Gly Trp Ser Phe Asn Phe Ser
150 155 160
Asn Val Ser Asp Pro Gln Asp Trp Tyr Phe Leu Leu Glu Phe Leu Ala
165 170 175
Asn Pro Ile Val Ala Ala Ile Ala Val Pro Thr Thr Gln Thr Val Pro
180 185 190
Ile Tyr Ser Trp Val Thr Thr Thr Val Trp His Pro Val Gln Ile Ser
195 200 205 210
Tyr Ser
<210>46
<211>180
<212>PRT
<213>脂环酸芽孢杆菌属
<220>
<221>SIGNAL
<222>(1)..(24)
<220>
<221>mat_peptide
<222>(25)..(180)
<223>功能性多肽
<400>46
Val Val Arg Met Arg Lys Arg Leu Gly Leu Val Leu Ser Met Val Thr
-20 -15 -10
Ser Val Leu Val Gly Cys Gly Ala Ser His Pro Ser Pro Leu Asn Gln
-5 -1 1 5
Asp Lys Ser Leu Leu Thr Trp Asn Ala Ala Lys His Glu Val Arg Trp
10 15 20
Lys Val Val Ala Gly Asp Gly Arg Ala Asn Gly Gly Met Asn Phe Asp
25 30 35 40
Gly Tyr Ala Asn Gly Ser Met Thr Leu Val Val Pro Ile Gly Trp Arg
45 50 55
Val Val Ile Asp Phe Asp Asn Ala Ser Leu Met Pro His Ser Ala Met
60 65 70
Val Val Pro Tyr Gly Asp Arg Glu Arg Ser Asn Phe Asp Ala Thr Met
75 80 85
Val Ala Phe Pro Gly Ala Glu Thr Pro Asn Pro Ser Gln Gly Asp Pro
90 95 100
Gln Gly Thr His Arg Asp Val Ile Phe Thr Ala Ala Lys Val Gly Thr
105 110 115 120
Tyr Ala Leu Val Cys Gly Val Pro Gly His Ala Leu Ala Gly Met Trp
125 130 135
Asp Gln Leu Val Val Ser Asp Glu Ala Lys His Pro Ser Leu Arg Val
140 145 150
Gln Arg Asp Ser
155
<210>47
<211>477
<212>PRT
<213>脂环酸芽孢杆菌属
<220>
<221>SIGNAL
<222>(1)..(25)
<220>
<221>mat_peptide
<222>(26)..(477)
<223>功能性多肽
<400>47
Met Ala Val Arg Arg Ala Trp Leu Leu Ala Pro Leu Cys Ala Ser Ser
-25 -20 -15 -10
Leu Val Val Pro Ala Ser Val Gln Ala Gly Leu Ala Gln Gly His Gly
-5 -1 1 5
Ser Phe Ser Thr Val Arg Val Ser Val Gly Thr Ser Ser Ser Leu Ser
10 15 20
Val Pro Ala Leu Ile Gln Gly Asn Glu Thr Tyr Ile Pro Leu Trp Asp
25 30 35
Leu Met Gln Val Leu His Gln Leu Gly Phe Thr Ala Thr Trp Ala Lys
40 45 50 55
Gly Gln Phe Ser Val Ser Ala Pro Pro Ser Val Pro Met Asp Glu Ala
60 65 70
Pro Gly Pro Ala Gly Lys Gly Gly Ala Leu Val Val Leu Asp Gly Gln
75 80 85
Val Val Glu Gln Val Pro Thr Val Ile Ala Thr Pro Pro Gly Ala Ala
90 95 100
Thr Pro Glu Val Phe Leu Pro Leu Thr Asn Ala Glu Glu Ile Leu Gly
105 110 115
Arg Leu Gly Ile Gln Ala Ser Ala Thr Gly Asn Gln Val Asn Leu Asp
120 125 130 135
Ala Ser Ala Val Pro Gln Ala Leu Pro Asn Gln Gln Val Ala Val Trp
140 145 150
Asn Val Leu Ala Ala Val Ala Ser Asp Leu Gly Val Ser Thr Ala Pro
155 160 165
Ala Gly Pro Ser Pro Tyr Ala Asp Leu Pro Thr Ala Ser Pro Ala Trp
170 175 180
Gly Ala Val Glu Ala Ala Ile Arg Leu Gly Trp Tyr Ser Pro Leu Ser
185 190 195
Ala Ser Ser Ser Gly Ala Phe Gln Pro Ile Thr Trp Ala Gln Thr Ala
200 205 210 215
Ser Ile Leu Trp Asn Ala Leu Gly Ile Ser Gln Gln Asp Ala Ala Tyr
220 225 230
Gln Pro Gly Gly Ser Pro Thr Ala Trp Ala Ser Ala Leu Gly Leu Val
235 240 245
Pro Glu Asn Trp Asp Pro Ala Ser Tyr Met Thr Ala Gln Glu Leu Asp
250 255 260
Thr Leu Ala Ser Asn Leu His Glu Cys Leu Gln Gly Asp Val Glu Thr
265 270 275
Gly Ala Asn Thr Trp Arg Leu Trp Tyr Pro Pro Ala Asp Glu Val Glu
280 285 290 295
Ala Thr Leu Gln Ser Gly Gly Gly Gln Ser Leu Phe Thr Ser Thr Ala
300 305 310
Asp Ala Gln Ala Ala Ile Ser Ser Ala Tyr Gln Phe Phe Asn Gln Leu
315 320 325
Val Val Thr Arg Val Gly Gln Gly Tyr Val Val Thr Val Pro Ser Val
330 335 340
Pro Glu Gly Tyr Gly Phe Ala Thr Phe Ser Ala Leu Gly Gly Val Ala
345 350 355
Tyr Gln Thr Thr Pro Gly Gly Pro Trp Thr Val Val Pro Val Leu Asp
360 365 370 375
Thr Arg Asp Val Ser Ile Pro Ala Lys Gly Arg Leu Ser Val Lys Val
380 385 390
Pro Ala Gln Gly Ile Thr Ile Thr Trp Asn Gln Met Met Pro Ser Leu
395 400 405
Gly Gly Thr Val Ala Met Gly Ala Leu Gln Val Ser Pro Gly Pro Ser
410 415 420
Gly Pro Ser Val Glu Arg Leu Asn Ile Val Thr Pro Asn Leu Pro Pro
425 430 435
Val Leu Pro Ser Ser Val Thr Ser Thr Gln Pro Gln Ser
440 445 450
<210>48
<211>340
<212>PRT
<213>脂环酸芽孢杆菌属
<220>
<221>SIGNAL
<222>(1)..(19)
<220>
<221>mat_peptide
<222>(20)..(340)
<223>功能性多肽
<400>48
Met Asn Arg Gln Trp Arg Leu Ala Val Ala Thr Ser Ala Val Ala Ala
-15 -10 -5
Ser Leu Ala Gly Cys Gly Ala Pro Asp Leu Ala Ala Met Arg Pro Thr
-1 1 5 10
Val Gln Lys Ser Ala Val Leu Val Glu Val Val Gly Ala Pro Pro Phe
15 20 25
Ala Pro Ser Ala Ser Gln Leu Gly Thr Ala Gly Ala Thr Ser Val Glu
30 35 40 45
Val Val His Val Ala Leu Gly Glu Trp Gln Ser Val Ala Ala His Ala
50 55 60
Leu Ala Lys Gly Gln Leu Thr Gly Val Met Val Val Cys Asp Asp Ala
65 70 75
Asn Ala Val Ala Ser Gly Leu Asn Gln Leu Ala Ala Asp His Pro Asp
80 85 90
Val Arg Phe Leu Val Val Ser Asn Trp Pro Ala Ser Gln Ile Thr Ser
95 100 105
Gly Asn Val Glu Asp Val Ala Gln Asp Pro Val Ala Val Ala Tyr Ser
110 115 120 125
Ile Gly Ala Leu Cys Gly Asp Trp Ile Ala Ser Ser Thr Ser Thr Ser
130 135 140
Gly Ala Val Tyr Ser Gly Val Pro Ser Ile Val Tyr Ala Pro Arg Gly
145 150 155
Ala Thr Val Ala Glu Gln Lys Ala Phe Phe Thr Gly Leu Tyr Gln Ala
160 165 170
Asn Pro Asn Val Arg Val Val Ala Leu Pro Gln Pro Ala Ala Gln Ser
175 180 185
Leu Ser Ser Tyr Gly Tyr Ala Val Asp Leu Gly Val Val Gly Gly Ser
190 195 200 205
Pro Ala Ala Gly Glu Leu Ser Ala Leu Arg Ser Ala Ala Pro Ala Trp
210 215 220
Ala Ala Phe Gly Thr Ser Pro Ile Ala Gly Phe Ala Ile Ser Pro Gly
225 230 235
His Leu Ser Ser Ser Glu Ala Val Gln Ala Phe Gln Ala Leu Val Ser
240 245 250
Pro Asp Ala Trp His Ser Gly Glu His Leu Val Leu Asp Leu Ser Ser
255 260 265
Val Ala Phe Asp Asp Lys Gln Val Pro Ala Thr Val Ile Ala Ala Trp
270 275 280 285
Ala Lys Leu Glu Val Asn Ala Ile Ala Ala Ala Ala Gln Ser Asn Ala
290 295 300
Ala Phe Ala Ser Leu Pro Pro Ser Val Arg Ser Asp Leu Ala Asn Ala
305 310 315
Phe His Leu Ser
320
<210>49
<211>341
<212>PRT
<213>脂环酸芽孢杆菌属
<220>
<221>SIGNAL
<222>(1)..(29)
<220>
<221>mat_peptide
<222>(30)..(341)
<223>功能性多肽
<400>49
Met Val Met Arg Thr Arg Trp Ile Arg Trp Met Ala Leu Ala Leu Ala
-25 -20 -15
Val Cys Val Trp Leu Ser Pro Phe Pro Phe Ser Trp Gly Ala Thr Ser
-10 -5 -1 1
Leu Asp Ala Asp Leu Pro Gln Pro Thr Ile Pro Pro Ser Ala Trp Ser
5 10 15
Asn Leu Asn Gln Asp Trp Lys Asp Leu Gln Arg Leu Ala Gln Asn Thr
20 25 30 35
Val Pro Pro Ser Lys Glu Ser Ser Gln Thr His Ala Pro Thr His Lys
40 45 50
Ser Ser Gln Pro Pro Ala Gln Val Pro Gln Gly Pro Leu Val Gly Val
55 60 65
Gly Asp Thr Gly Glu Ala Ala Arg Trp Leu Asn Glu Ala Leu Ala Val
70 75 80
Leu Gly Tyr Leu Pro Ala Val Phe Ser Pro Ala Ala Gln Thr Ser Thr
85 90 95
Arg Gln Val Arg Leu Ala Leu Ala Ala Ser Ala Glu His Gln Thr Leu
100 105 110 115
Val Pro Ile Pro Gly Ser Phe Gln Leu Leu Tyr His Ala Pro Ser Ser
120 125 130
Trp Val Ala Leu Trp Ser Ala Asp Glu Asp Thr Pro Ile Thr Glu Gly
135 140 145
Ala Val Met Ala Phe Glu Ala Gln His His Leu Gly Val Asp Gly Ile
150 155 160
Ala Gly Pro Asp Val Ile His Ala Leu Ala Gln Ala Leu Ala Gly Asn
165 170 175
Glu Thr Ala Glu Lys Ala Pro Tyr Ser Tyr Ile Leu Val Thr Thr Ser
180 185 190 195
Leu Pro Glu Thr Leu Glu Leu Trp Val Asn Gly Gln Leu Val Leu Lys
200 205 210
Ser Leu Cys Asn Thr Gly Ile Ala Gln Ser Pro Thr Pro Tyr Gly Thr
215 220 225
Tyr Gly Val Tyr Val Gln Tyr Thr Ser Gln Glu Met Lys Gly Lys Asp
230 235 240
Pro Asp Gly Thr Pro Tyr Asp Asp Pro Gly Val Pro Trp Val Ser Tyr
245 250 255
Phe Tyr Lys Gly Cys Ala Val His Gly Phe Leu Arg Ala Lys Tyr Gly
260 265 270 275
Phe Pro Gln Ser Leu Gly Cys Val Glu Leu Pro Tyr Ala Ala Ala Lys
280 285 290
Thr Val Phe Ser Tyr Thr His Ile Gly Thr Leu Val Thr Val Thr Ala
295 300 305
Ser Pro Leu Ser Ala
310
<210>50
<211>399
<212>PRT
<213>脂环酸芽孢杆菌属
<220>
<221>SIGNAL
<222>(1)..(28)
<220>
<221>mat_peptide
<222>(30)..(399)
<223>功能性多肽
<400>50
Met Asp Arg Leu Leu Asn Asn Lys Val Ala Leu Arg Leu Thr Ala Leu
-25 -20 -15
Val Leu Ala Cys Ile Leu Trp Leu Ala Val His Ala Glu Gln Gly Ser
-10 -5 -1 1
Gly Ser Ser Ala Ser Thr Gly Val Thr Glu Ser Phe Glu Leu Pro Val
5 10 15
Arg Val Glu Thr Ser Ala Asp Glu Val Leu Val Ser Gln Val Pro Thr
20 25 30 35
Ile Thr Ala Arg Val Thr Thr Asn Leu Leu Ser Leu Pro Thr Leu Ala
40 45 50
Ser Asp Met Met Lys Ala Glu Ile Val Ala Asp Ala Glu Asn Leu Gly
55 60 65
Pro Gly Thr Tyr Thr Leu His Val Ala Ala Val Asn Met Pro Ala Gly
70 75 80
Val Arg Ser Tyr Thr Leu Thr Pro Ser Thr Ile Thr Val Thr Leu Glu
85 90 95
Pro Lys Val Thr Val Glu Arg Thr Val Arg Val Asn Val Val Gly Thr
100 105 110 115
Pro Gly Gln Gly Tyr Val Leu Gly Lys Pro Glu Leu Gly Ala Gly Val
120 125 130
Val Glu Val Ser Gly Ala Glu Ser Ser Val Gln Ala Val Ala Glu Val
135 140 145
Ala Gly Val Val Asp Ala Ser Gly Leu Ser Gln Thr Ala Thr Lys Leu
150 155 160
Val Glu Leu Leu Pro Leu Asp Gln Ala Gly Lys Ala Val Pro Gly Val
165 170 175
Thr Val Thr Pro Ser Ala Ile Ser Val Thr Leu Pro Ile Thr Ser Ala
180 185 190 195
Asn Gln Ala Val Lys Leu Thr Pro Ala Val Thr Gly Ser Pro Ala Pro
200 205 210
Gly Tyr Ala Val Ala Ser Val His Leu Glu Pro Ala Ser Ala Val Glu
215 220 225
Gln Gly Leu Ala Ala Ser Gln Leu Pro Gln Arg Gly Leu Leu Val Pro
230 235 240
Ile Asp Val Thr Gly Leu Asn Arg Pro Thr Thr Val Ser Val Pro Val
245 250 255
Pro Leu Leu Pro Gly Met Thr Ser Val Ser Pro Thr Ala Val Thr Ala
260 265 270 275
Val Ile Asp Val Glu Pro Ser Ala Val Tyr Thr Val Ser Asn Val Pro
280 285 290
Val Ala Ile Thr Gly Ala Thr Gly Val Lys Leu Val Thr Pro Arg Thr
295 300 305
Val Asn Val Thr Val Thr Gly Ile Glu Ala Asp Val Arg Ala Val Glu
310 315 320
Arg Asp Pro Ala Ala Val Gln Ala Phe Val Asp Ala Thr Gly Leu Thr
325 330 335
His Gly Ser Ala Thr Leu Pro Asp Ser Asn Ser Ser Ala Val Leu Ser
340 345 350 355
Leu Val Ile Arg Pro Arg Glu Arg Arg Lys Arg Thr His Val Val
360 365 370
<210>51
<211>34
<212>DNA
<213>引物SigA2NotU-P
<400>51
tcgcgatccg ttttcgcatt tatcgtgaaa cgct 34
<210>52
<211>33
<212>DNA
<213>引物SigA2NotD-P
<400>52
ccgcaaacgc tggtgaaagt aaaagatgct gaa 33
<210>53
<211>20
<212>DNA
<213>引物A2up
<400>53
agcgtttgcg gccgcgatcc 20
<210>54
<211>21
<212>DNA
<213>引物B
<400>54
ttattcggtc gaaaaggatc c 21
<210>55
<211>282
<212>PRT
<213>黑曲霉
<220>
<221>SIGNAL
<222>(1)..(18)
<220>
<221>PROPEP
<222>(19)..(59)
<220>
<221>CHAIN
<222>(60)..(98)
<220>
<221>PROPEP
<222>(99)..(109)
<220>
<221>CHAIN
<222>(110)..(282)
<220>
<221>MOD_RES
<222>(110)..(110)
<220>
<221>DISULFID
<222>(115)..(139)
<220>
<221>DISULFID
<222>(127)..(210)
<400>55
Met Lys Phe Ser Thr Ile Leu Thr Gly Ser Leu Phe Ala Thr Ala Ala
1 5 10 15
Leu Ala Ala Pro Leu Thr Glu Lys Arg Arg Ala Arg Lys Glu Ala Arg
20 25 30
Ala Ala Gly Lys Arg His Ser Asn Pro Pro Tyr Ile Pro Gly Ser Asp
35 40 45
Lys Glu Ile Leu Lys Leu Asn Gly Thr Thr Asn Glu Glu Tyr Ser Ser
50 55 60
Asn Trp Ala Gly Ala Val Leu Ile Gly Asp Gly Tyr Thr Lys Val Thr
65 70 75 80
Gly Glu Phe Thr Val Pro Ser Val Ser Ala Gly Ser Ser Gly Ser Ser
85 90 95
Gly Tyr Gly Gly Gly Tyr Gly Tyr Trp Lys Asn Lys Arg Gln Ser Glu
100 105 110
Glu Tyr Cys Ala Ser Ala Trp Val Gly Ile Asp Gly Asp Thr Cys Glu
115 120 125
Thr Ala Ile Leu Gln Thr Gly Val Asp Phe Cys Tyr Glu Asp Gly Gln
130 135 140
Thr Ser Tyr Asp Ala Trp Tyr Glu Trp Tyr Pro Asp Tyr Ala Tyr Asp
145 150 155 160
Phe Ser Asp Ile Thr Ile Ser Glu Gly Asp Ser Ile Lys Val Thr Val
165 170 175
Glu Ala Thr Ser Lys Ser Ser Gly Ser Ala Thr Val Glu Asn Leu Thr
180 185 190
Thr Gly Gln Ser Val Thr His Thr Phe Ser Gly Asn Val Glu Gly Asp
195 200 205
Leu Cys Glu Thr Asn Ala Glu Trp Ile Val Glu Asp Phe Glu Ser Gly
210 215 220
Asp Ser Leu Val Ala Phe Ala Asp Phe Gly Ser Val Thr Phe Thr Asn
225 230 235 240
Ala Glu Ala Thr Ser Gly Gly Ser Thr Val Gly Pro Ser Asp Ala Thr
245 250 255
Val Met Asp Ile Glu Gln Asp Gly Ser Val Leu Thr Glu Thr Ser Val
260 265 270
Ser Gly Asp Ser Val Thr Val Thr Tyr Val
275 280
<210>56
<211>252
<212>PRT
<213>核盘菌
<220>
<221>MISC_FEATURE
<222>(1)..(252)
<223>内肽酶EapC
<400>56
Met Lys Phe Ser Ile Val Ala Ala Thr Ala Leu Leu Ala Gly Ser Ala
1 5 10 15
Val Ala Ala Pro Gly Thr Ala Leu Arg Gln Ala Arg Ala Val Lys Arg
20 25 30
Ala Ala Arg Thr His Gly Asn Pro Val Lys Tyr Val Glu Gly Pro Thr
35 40 45
Asn Lys Thr Asp Val Ser Tyr Ser Ser Asn Trp Ala Gly Ala Val Leu
50 55 60
Val Gly Thr Gly Tyr Thr Ser Val Thr Gly Thr Phe Thr Ala Pro Ser
65 70 75 80
Pro Ser Thr Ala Gly Ser Gly Ser Ala Trp Val Gly Ile Asp Gly Asp
85 90 95
Thr Cys Gly Thr Ala Ile Leu Gln Thr Gly Ile Asp Trp Asp Lys Ser
100 105 110
Gly Asn Ser Ile Thr Tyr Asp Ala Trp Tyr Glu Trp Tyr Pro Asp Tyr
115 120 125
Ala Tyr Asp Phe Ser Gly Ile Ser Ile Ser Ala Gly Asp Ser Ile Lys
130 135 140
Val Thr Val Thr Ala Ser Ser Lys Thr Thr Gly Thr Ala Thr Val Asp
145 150 155 160
Asn Leu Thr Lys Gly Lys Ser Val Thr His Thr Phe Ser Gly Gly Val
165 170 175
Asp Gly Asp Leu Cys Glu Tyr Asn Ala Glu Trp Ile Val Glu Asp Phe
180 185 190
Glu Glu Gly Ser Ser Leu Val Gln Phe Ala Asn Phe Gly Thr Val Thr
195 200 205
Phe Thr Gly Ala Ser Ala Thr Gln Asn Gly Glu Ser Val Gly Val Thr
210 215 220
Gly Ala Gln Ile Ile Asp Leu Gln Gln Asn Ser Val Leu Thr Ser Val
225 230 235 240
Ser Thr Ser Ser Asn Ser Val Thr Val Lys Tyr Val
245 250
<210>57
<211>269
<212>PRT
<213>Cryphonectria parasitica
<220>
<221>MISC_FEATURE
<222>(1)..(269)
<223>内肽酶EapC
<400>57
Met Lys Tyr Ala Thr Val Val Ala Ala Leu Leu Gly Ala Asn Ala Ala
1 5 10 15
Leu Gly Ala Arg Phe Thr Glu Lys Arg Arg Glu Arg Asn Glu Ala Arg
20 25 30
Leu Ala Arg Arg Ser Gly Ser Val Arg Leu Pro Ala Thr Asn Ser Glu
35 40 45
Gly Val Ala Ile Asp Ala Ala Glu Ser Arg Asn Asp Thr Thr Asn Val
50 55 60
Glu Tyr Ser Ser Asn Trp Ala Gly Ala Val Leu Ile Gly Ser Gly Tyr
65 70 75 80
Lys Ser Val Thr Gly Ile Phe Val Val Pro Thr Pro Lys Ser Pro Gly
85 90 95
Ser Gly Asn Thr Glu Tyr Ala Ala Ser Ala Trp Val Gly Ile Asp Gly
100 105 110
Asp Thr Ala Gln Asn Ser Ile Leu Gln Thr Gly Val Asp Phe Tyr Val
115 120 125
Glu Gly Ser Ser Val Ala Tyr Asp Ala Trp Tyr Glu Trp Tyr Pro Asp
130 135 140
Tyr Ala Tyr Asp Phe Ser Gly Ile Ser Ile Ser Ala Gly Asp Thr Ile
145 150 155 160
Lys Val Thr Val Thr Ala Thr Thr Thr Thr Ser Gly Thr Ala Val Val
165 170 175
Glu Asn Val Thr Lys Gly Thr Thr Val Thr His Thr Phe Thr Gly Gln
180 185 190
Ser Ala Ala Leu Gln Glu Leu Asn Ala Glu Trp Ile Val Glu Asp Phe
195 200 205
Glu Glu Gly Asp Glu Leu Val Pro Phe Ala Asn Phe Gly Thr Val Thr
210 215 220
Phe Thr Gly Ala Glu Ala Thr Thr Ser Ser Gly Thr Val Thr Ala Ala
225 230 235 240
Asp Ala Thr Leu Ile Asp Ile Glu Gln Asn Gly Glu Val Leu Thr Ser
245 250 255
Val Thr Val Ser Gly Ser Thr Val Thr Val Lys Tyr Val
260 265
<210>58
<211>204
<212>PRT
<213>Scytalidium lignicolum
<220>
<221>MISC_FEATURE
<222>(1)..(204)
<223>scytalidoglutamic肽酶
<400>58
Thr Val Glu Ser Asn Trp Gly Gly Ala Ile Leu Ile Gly Ser Asp Phe
1 5 10 15
Asp Thr Val Ser Ala Thr Ala Asn Val Pro Ser Ala Thr Gly Ala Ser
20 25 30
Gly Gly Ser Ser Ala Ala Trp Val Gly Ile Asp Gly Asp Thr Cys Gln
35 40 45
Thr Ala Ile Leu Gln Thr Gly Phe Asp Trp Tyr Gly Asp Gly Thr Tyr
50 55 60
Asp Ala Trp Tyr Glu Trp Tyr Pro Glu Val Ser Asp Asp Phe Ser Gly
65 70 75 80
Ile Thr Ile Ser Glu Gly Asp Ser Ile Gln Met Ser Val Thr Ala Thr
85 90 95
Ser Asp Thr Ser Gly Ser Ala Thr Leu Glu Asn Leu Thr Thr Gly Gln
100 105 110
Lys Val Ser Lys Ser Phe Ser Asn Glu Ser Ser Gly Leu Cys Arg Thr
115 120 125
Asn Ala Glu Phe Ile Ile Glu Asp Phe Glu Glu Cys Asn Ser Asp Gly
130 135 140
Ser Asp Glu Phe Val Pro Phe Ala Ser Phe Ser Pro Ala Val Glu Phe
145 150 155 160
Thr Asp Cys Ser Val Thr Ser Asp Gly Glu Ser Val Ser Leu Asp Asp
165 170 175
Ala Gln Ile Thr Gln Val Ile Ile Asn Asn Gln Asp Val Thr Asp Cys
180 185 190
Ser Val Ser Gly Thr Thr Val Ser Cys Ser Tyr Val
195 200
<210>59
<211>268
<212>PRT
<213>Cryphonectria parasitica
<220>
<221>MISC_FEATURE
<222>(1)..(268)
<223>内肽酶EapB
<400>59
Met Lys Tyr Thr Ala Ala Leu Ala Ala Leu Val Thr Leu Ala Ala Ala
1 5 10 15
Ala Pro Thr Asp Gly Ile Ile Asp Ile Gly Asp Gly Val Lys Leu Val
20 25 30
Pro Arg Glu Pro Arg Ala His Thr Arg Leu Glu Arg Leu Arg Thr Phe
35 40 45
Arg Arg Gly Leu Met Glu Gly Leu Glu Ser Gly Glu Arg Asn Ser Ser
50 55 60
Asp Val Ser Tyr Asp Ser Asn Trp Ala Gly Ala Val Lys Ile Gly Thr
65 70 75 80
Gly Leu Asn Asp Val Thr Gly Thr Ile Val Val Pro Thr Pro Ser Val
85 90 95
Pro Ser Gly Gly Ser Ser Thr Ala Lys Tyr Ala Ala Ser Ala Trp Val
100 105 110
Gly Ile Asp Gly Asp Thr Cys Thr Ser Ala Ile Leu Gln Thr Gly Val
115 120 125
Asp Phe Tyr Ala Gly Arg Gly Gly Val Ser Phe Asp Ala Trp Tyr Glu
130 135 140
Trp Tyr Pro Asn Tyr Ala Tyr Asp Phe Ser Gly Phe Ser Val Ser Ala
145 150 155 160
Gly Asp Thr Ile Val Met Thr Ala Ser Ala Ser Ser Leu Lys Ala Gly
165 170 175
Thr Val Thr Leu Glu Asn Ser Thr Thr Gly Lys Lys Val Thr Gln Ser
180 185 190
Phe Ser Ala Glu Ser Ser Glu Leu Cys Glu Tyr Asn Ala Glu Trp Ile
195 200 205
Val Glu Asp Phe Glu Ser Gly Ser Ser Leu Val Asn Phe Ala Asp Phe
210 215 220
Asp Thr Val Thr Phe Lys Asp Cys Ser Pro Ser Val Ser Gly Ser Thr
225 230 235 240
Ile Val Asp Ile Arg Gln Ser Leu Glu Val Leu Thr Glu Cys Ser Thr
245 250 255
Thr Gly Thr Thr Thr Val Thr Cys Glu Tyr Val Gly
260 265
<210>60
<211>147
<212>PRT
<213>埃默森篮状菌
<220>
<221>MISC_FEATURE
<222>(1)..(147)
<400>60
Asn Trp Ala Gly Ala Val Leu Thr Ser Pro Pro Ser Gly Ser Thr Phe
1 5 10 15
Thr Ser Val Ser Ala Gln Phe Thr Val Pro Ser Pro Ser Leu Pro Gln
20 25 30
Gly Ser Gln Gln Ala Ser Ser Ala Ser Ala Trp Val Gly Ile Asp Gly
35 40 45
Asp Thr Tyr Thr Asn Ala Ile Leu Gln Thr Gly Val Asp Phe Asn Val
50 55 60
Asp Thr Asn Gly Gln Val Ser Tyr Asp Ala Trp Tyr Glu Trp Tyr Pro
65 70 75 80
Asp Tyr Ala His Asp Phe Thr Gly Ile Ser Phe Gln Ser Gly Asp Val
85 90 95
Val Ser Val Ser Val Thr Ser Ser Ser Asn Ser Glu Gly Thr Ala Val
100 105 110
Ile Glu Asn Leu Thr Asn Gly Gln Lys Val Thr Lys Thr Leu Ser Ala
115 120 125
Pro Ser Ser Ser Ala Thr Leu Gly Gly Gln Asn Ala Glu Trp Ile Val
130 135 140
Glu Asp Phe
145
Claims (62)
1.分离的成熟功能性多肽,其与可从以保藏号DSM 15716保藏的脂环酸芽孢杆菌属细菌获得的相应分泌多肽具有至少90%的同一性并表现相同的功能。
2.细菌谷氨酸肽酶(EC 3.4.23.19)。
3.权利要求1的多肽,所述多肽选自:
(a)含有与SEQ ID NO:26到SEQ ID NO:50所包含成熟多肽的序列具有至少90%同一性的氨基酸序列的多肽;
(b)由核苷酸序列编码的多肽,所述核苷酸序列在高严格条件下与选自以下的多核苷酸探针杂交:
(i)编码成熟多肽的SEQ ID NO:1到SEQ ID NO:25区域的核苷酸序列的互补链;
(ii)编码成熟多肽的SEQ ID NO:1到SEQ ID NO:25区域的核苷酸序列中所包含cDNA序列的互补链;
(c)SEQ ID NO:26到SEQ ID NO:50中所包含成熟多肽的片段,
且其中该多肽具有SEQ ID NO:26到SEQ ID NO:50中所包含的相应成熟多肽的功能。
4.权利要求1的多肽,其中该多肽为具有选自以下功能的酶:酸性内切葡聚糖酶、酸性纤维素酶、天冬氨酰蛋白酶、多铜氧化酶、丝氨酸羧基蛋白酶、丝氨酸蛋白酶、HtrA样丝氨酸蛋白酶、二硫化物异构酶、γ-D-谷氨酰-L-二氨基酸内肽酶、内-β-N-乙酰氨基葡糖苷酶、肽酰脯氨酰异构酶、酸性磷酸酯酶、植酸酶、磷脂酶C、多糖脱乙酰酶、木聚糖脱乙酰酶和亚硫酸盐氧化酶。
5.权利要求4的酶,其选自:
(a)含有与SEQ ID NO:26到SEQ ID NO:40所包含成熟多肽的氨基酸序列具有至少90%同一性的氨基酸序列的酶;
(b)由核苷酸序列编码的酶,所述核苷酸序列在高严格条件下与选自以下的多核苷酸探针杂交,所述多核苷酸探针选自:
(i)编码成熟酶的SEQ ID NO:1到SEQ ID NO:15区域的核苷酸序列的互补链;
(ii)编码成熟多肽的SEQ ID NO:1到SEQ ID NO:15区域的核苷酸序列中所包含cDNA序列的互补链;
(c)SEQ ID NO:26到SEQ ID NO:40中所包含成熟酶的片段,
且其中该酶具有SEQ ID NO:26到SEQ ID NO:40中所包含的相应成熟多肽的功能。
6.权利要求1的多肽,其中严格度条件非常高。
7.权利要求1的多肽,其中编码多肽的多核苷酸由选自编码成熟多肽的SEQ ID NO:1到SEQ ID NO:25区域的核苷酸序列或由于遗传密码简并性而与之不同的序列组成。
8.权利要求4的酶,其表征为从以保藏号DSM 15716保藏的脂环酸芽孢杆菌菌株获得的酸性内切葡聚糖酶或酸性纤维素酶。
9.权利要求8的酸性内切葡聚糖酶或酸性纤维素酶,其包含SEQ IDNO:26包含的成熟酸性内切葡聚糖酶或酸性纤维素酶或由其组成。
10.权利要求9的酸性内切葡聚糖酶或酸性纤维素酶,其包含SEQ IDNO:26位置25到959的序列或由其组成。
11.权利要求2的谷氨酸肽酶,其表征为蛋白酶结构中不含二硫键。
12.权利要求2中的谷氨酸肽酶,可从以保藏号DSM 15716保藏的脂环酸芽孢杆菌菌株获得。
13.权利要求12的谷氨酸肽酶,其包含SEQ ID NO:27中包含的成熟天冬氨酰蛋白酶或由其组成。
14.权利要求13的谷氨酸肽酶,其包含SEQ ID NO:27位置33到272的序列或由其组成。
15.权利要求4的酶,其表征为可从以保藏号DSM 15716保藏的脂环酸芽孢杆菌菌株获得的多铜氧化酶。
16.权利要求15的多铜氧化酶,其包含SEQ ID NO:28或SEQ ID NO:35中包含的成熟多铜氧化酶或由其组成。
17.权利要求16的谷氨酸肽酶,其包含SEQ ID NO:28位置26到315或SEQ ID NO:35位置50到597的序列或由其组成。
18.权利要求4的酶,表征为可从以保藏号DSM 15716保藏的脂环酸芽孢杆菌菌株获得的丝氨酸羧基蛋白酶。
19.权利要求18的丝氨酸羧基蛋白酶,其包含SEQ ID NO:29或SEQID NO:30中包含的成熟丝氨酸羧基蛋白酶或由其组成。
20.权利要求19的丝氨酸羧基蛋白酶,其包含SEQ ID NO:29位置190到626或SEQ ID NO:30位置25到533的序列或由其组成。
21.权利要求4的酶,表征为可从以保藏号DSM 15716保藏的脂环酸芽孢杆菌菌株获得的氨酸蛋白酶或HtrA样丝氨酸蛋白酶。
22.权利要求21的丝氨酸蛋白酶或HtrA样丝氨酸蛋白酶,其包含SEQ ID NO:31中包含的成熟丝氨酸蛋白酶或HtrA样丝氨酸蛋白酶或由其组成。
23.权利要求22的丝氨酸蛋白酶或HtrA样丝氨酸蛋白酶,其包含SEQID NO:31位置42到411的序列或由其组成。
24.权利要求4的酶,表征为可从以保藏号DSM 15716保藏的脂环酸芽孢杆菌菌株获得的二硫化物异构酶。
25.权利要求24的二硫化物异构酶,其包含SEQ ID NO:32中包含的成熟二硫化物异构酶或由其组成。
26.权利要求25的二硫化物异构酶,其包含SEQ ID NO:32位置42到411的序列或由其组成。
27.权利要求4的酶,表征为可从以保藏号DSM 15716保藏的脂环酸芽孢杆菌菌株获得的γ-D-谷氨酰-L-二氨基酸。
28.权利要求27的γ-D-谷氨酰-L-二氨基酸,其包含SEQ ID NO:33中包含的成熟γ-D-谷氨酰-L-二氨基酸或由其组成。
29.权利要求28的γ-D-谷氨酰-L-二氨基酸,其包含SEQ ID NO:33从位置30到266的序列或由其组成。
30.权利要求4的酶,表征为可从以保藏号DSM 15716保藏的脂环酸芽孢杆菌菌株获得的内-β-N-乙酰氨基葡糖苷酶。
31.权利要求30的内-β-N-乙酰氨基葡糖苷酶,其包含SEQ ID NO:34中包含的成熟内-β-N-乙酰氨基葡糖苷酶或由其组成。
32.权利要求31的内-β-N-乙酰氨基葡糖苷酶,其包含SEQ ID NO:34位置27到768的序列或由其组成。
33.权利要求4的酶,表征为可从以保藏号DSM 15716保藏的脂环酸芽孢杆菌菌株获得的肽酰脯氨酰异构酶。
34.权利要求33的肽酰脯氨酰异构酶,其包含SEQ ID NO:36中包含的成熟肽酰脯氨酰异构酶或由其组成。
35.权利要求34的肽酰脯氨酰异构酶,其包含SEQ ID NO:36从位置30到246的序列或由其组成。
36.权利要求4的酶,表征为可从以保藏号DSM 15716保藏的脂环酸芽孢杆菌菌株获得的酸性磷酸酯酶或植酸酶或磷脂酶C。
37.权利要求36的酸性磷酸酯酶或植酸酶或磷脂酶C,其包含SEQ IDNO:37中包含的成熟酸性磷酸酯酶或植酸酶或磷脂酶C或由其组成。
38.权利要求37的酸性磷酸酯酶或植酸酶或磷脂酶C,其包含SEQ IDNO:37位置28到608的序列或由其组成。
39.权利要求4的酶,表征为可从以保藏号DSM 15716保藏的脂环酸芽孢杆菌菌株获得的多糖脱乙酰酶或木聚糖脱乙酰酶。
40.权利要求39的多糖脱乙酰酶或木聚糖脱乙酰酶,其包含SEQ IDNO:38或SEQ ID NO:39中包含的成熟多糖脱乙酰酶或木聚糖脱乙酰酶或由其组成。
41.权利要求40的多糖脱乙酰酶或木聚糖脱乙酰酶,其包含SEQ IDNO:38位置26到251或SEQ ID NO:39位置22到324的序列或由其组成。
42.分离的酶,其选自:
(a)含有下述氨基酸序列的酶,所述氨基酸序列与以DSM保藏号15716保藏的脂环酸芽孢杆菌菌株分泌的选自酸性内切葡聚糖酶或酸性纤维素酶、天冬氨酰蛋白酶、多铜氧化酶、丝氨酸羧基蛋白酶、丝氨酸蛋白酶或HtrA样丝氨酸蛋白酶、二硫化物异构酶、γ-D-谷氨酰-L-二氨基酸内肽酶、内-β-N-乙酰氨基葡糖苷酶、肽酰脯氨酰异构酶、酸性磷酸酯酶或植酸酶或磷脂酶C、多糖脱乙酰酶或木聚糖脱乙酰酶和亚硫酸盐氧化酶的成熟酶氨基酸序列具有至少90%同一性;
(b)由在高严格度条件下与下述多核苷酸探针杂交的核苷酸序列所编码的多肽,所述多核苷酸探针选自:
(i)以DSM保藏号15716保藏的脂环酸芽孢杆菌包含的核苷酸序列的互补链,所述核苷酸序列编码由该菌株分泌的选自酸性内切葡聚糖酶或酸性纤维素酶、天冬氨酰蛋白酶、多铜氧化酶、丝氨酸羧基蛋白酶、丝氨酸蛋白酶或HtrA样丝氨酸蛋白酶、二硫化物异构酶、γ-D-谷氨酰-L-二氨基酸内肽酶、内-β-N-乙酰氨基葡糖苷酶、肽酰脯氨酰异构酶、酸性磷酸酯酶或植酸酶或磷脂酶C、多糖脱乙酰酶或木聚糖脱乙酰酶和亚硫酸盐氧化酶的成熟酶;
(ii)以DSM保藏号15716保藏的脂环酸芽孢杆菌包含的核苷酸序列包含的cDNA序列的互补链,所述核苷酸序列编码由该菌株分泌的选自酸性内切葡聚糖酶或酸性纤维素酶、天冬氨酰蛋白酶、多铜氧化酶、丝氨酸羧基蛋白酶、丝氨酸蛋白酶或HtrA样丝氨酸蛋白酶、二硫化物异构酶、γ-D-谷氨酰-L-二氨基酸内肽酶、内-β-N-乙酰氨基葡糖苷酶、肽酰脯氨酰异构酶、酸性磷酸酯酶或植酸酶或磷脂酶C、多糖脱乙酰酶或木聚糖脱乙酰酶和亚硫酸盐氧化酶的成熟酶,
(c)成熟酶片段,所述酶为以DSM保藏号15716保藏的脂环酸芽孢杆菌菌株分泌的选自酸性内切葡聚糖酶或酸性纤维素酶、天冬氨酰蛋白酶、多铜氧化酶、丝氨酸羧基蛋白酶、丝氨酸蛋白酶或HtrA样丝氨酸蛋白酶、二硫化物异构酶、γ-D-谷氨酰-L-二氨基酸内肽酶、内-β-N-乙酰氨基葡糖苷酶、肽酰脯氨酰异构酶、酸性磷酸酯酶或植酸酶或磷脂酶C、多糖脱乙酰酶或木聚糖脱乙酰酶和亚硫酸盐氧化酶;
其中该酶具有选自酸性内切葡聚糖酶或酸性纤维素酶、天冬氨酰蛋白酶、多铜氧化酶、丝氨酸羧基蛋白酶、丝氨酸蛋白酶或HtrA样丝氨酸蛋白酶、二硫化物异构酶、γ-D-谷氨酰-L-二氨基酸内肽酶、内-β-N-乙酰氨基葡糖苷酶、肽酰脯氨酰异构酶、酸性磷酸酯酶或植酸酶或磷脂酶C、多糖脱乙酰酶或木聚糖脱乙酰酶和亚硫酸盐氧化酶的功能。
43.以保藏号DSM 15716保藏的细菌菌株。
44.含有权利要求1-42的多肽的组合物。
45.权利要求44的组合物,包含至少两种权利要求1-42的不同多肽,优选至少3种,更优选至少5种,更优选至少10种,更优选至少15种,更优选至少20种权利要求1-42的不同多肽。
46.权利要求44的组合物,包含发酵脂环酸芽孢杆菌DSM 15716或其突变体样品时分泌的所有多肽,所述突变体中缺失或添加了一个或多个基因。
47.权利要求44的组合物,其还包含一个或多个额外的酶。
48.权利要求44的组合物,表征为除多肽外还含有表面活性剂的洗涤剂组合物。
49.权利要求44的组合物,表征为除多肽外还含有谷类或谷物产品的饲料组合物。
50.权利要求44的组合物,表征为食品组合物。
51.权利要求44的组合物,还包含多糖或多糖混合物。
52.制备权利要求44的组合物的方法,包括将权利要求1-42的多肽与赋形剂混和。
53.多核苷酸,其具有编码权利要求1-42中任一项定义的多肽的核苷酸序列。
54.包含权利要求53的多核苷酸的组合物。
55.包含权利要求53定义的核苷酸序列的核酸构建体,所述核苷酸序列与一个或多个指导多肽在宿主细胞中产生的控制序列有效连接。
56.包含权利要求55的核酸构建体的重组表达载体。
57.包含权利要求55的核酸构建体的重组宿主细胞。
58.用于产生权利要求1-42的多肽的方法,包括:
(a)培养菌株以产生多肽,所述菌株的野生型形式能够产生该多肽;和
(b)回收该多肽。
59.用于产生权利要求1-42的多肽的方法,包括:
(a)在有助于产生多肽的条件下培养权利要求45定义的重组宿主细胞;和
(b)回收多肽。
60.权利要求59的方法,包括(i)将脂环酸芽孢杆菌DSM 15716基因组的基因与编码无信号报告子的基因通过转座子标签融合,(ii)在显示报告子存在的培养基中培养含有脂环酸芽孢杆菌DSM 15716融合基因的宿主细胞克隆,(iii)检测分泌报告子的克隆和(iv)分离该克隆中含有的脂环酸芽孢杆菌DSM 15716的基因和多肽。
61.适用于电子设备的存储媒体,其包含权利要求1-42的多肽的氨基酸序列或权利要求53的多核苷酸的核苷酸序列的信息。
62.包括在工业或家庭技术方法中使用权利要求1-42的多肽或权利要求53的多核苷酸的方法。
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CN102361973A (zh) * | 2009-01-21 | 2012-02-22 | 诺维信公司 | 具有酯酶活性的多肽和编码该多肽的核酸 |
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CN101701214B (zh) * | 2009-10-30 | 2011-09-21 | 中国农业科学院饲料研究所 | 一种宽pH适用性的木聚糖酶XYNA4及其基因和应用 |
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