CN100547077C - 淀粉水解的方法 - Google Patents
淀粉水解的方法 Download PDFInfo
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- CN100547077C CN100547077C CNB2004800180051A CN200480018005A CN100547077C CN 100547077 C CN100547077 C CN 100547077C CN B2004800180051 A CNB2004800180051 A CN B2004800180051A CN 200480018005 A CN200480018005 A CN 200480018005A CN 100547077 C CN100547077 C CN 100547077C
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Abstract
本发明涉及将颗粒状淀粉在低于所述颗粒状淀粉的初始凝胶化温度的温度，酶法水解为可溶的淀粉水解产物的方法。
Description
技术领域
本发明涉及在低于颗粒状淀粉的初始凝胶化温度的温度，将所述颗粒状淀粉水解为可溶的淀粉水解产物的方法。
背景技术
已经描述了大量的方法用于将淀粉转化为淀粉水解产物，诸如麦芽糖、葡萄糖或特制糖浆，用作甜味剂(sweetener)或用作其他糖类如果糖的前体。葡萄糖也可以被发酵变为乙醇或其他发酵产物，诸如柠檬酸、谷氨酸一钠、葡糖酸、葡糖酸钠、葡糖酸钙、葡糖酸钾、葡糖酸delta内酯、异抗坏血酸钠(sodiumerythorbate)、衣康酸、乳酸、葡糖酸；酮类；氨基酸，谷氨酸(单谷氨酸钠(sodiummonoglutaminate))、青霉素、四环素；酶；维生素，如核黄素、B12、beta-胡萝卜素或激素。
淀粉是高分子量聚合物，其由葡萄糖单元的链组成。它通常由大约80％支链淀粉和20％直链淀粉组成。支链淀粉是分枝的多糖，其中alpha-1，4D-葡萄糖残基的线状链通过alpha-1，6糖苷键连接。
直链淀粉是线状的多糖，其由通过alpha-1，4糖苷键连接在一起的D-吡喃型葡萄糖构建而成。在将淀粉转化为可溶的淀粉水解产物的情况下，所述的淀粉被解聚(depolymerization)。常规的解聚方法由凝胶化步骤和两个连续的处理步骤，即液化(liquefaction)处理和糖化(saccharification)处理组成。
颗粒状淀粉由微观的颗粒组成，其在室温不溶于水。当将水性的淀粉浆体加热时，所述的颗粒膨胀并最终胀破，将所述的淀粉分子分散到所述溶液中。在此“凝胶化”(gelatinization)处理过程中，粘度有显著的提高。由于在通常的工业过程中，所述的固体水平为30-40％，就必须将所述的淀粉变稀薄和“液化”，以使其能被处理。此粘度的降低现今主要通过酶法降解获得。在液化步骤中，所述的长链淀粉被alpha-淀粉酶降解为较小的分枝的和线状的单元(麦芽糖糊精)。所述的液化步骤通常在大约105-110℃进行大约5至10分钟，然后在大约95℃进行大约1-2小时。然后将所述的温度降低到60℃，加入葡糖淀粉酶或beta-淀粉酶和任选的脱支酶，诸如异淀粉酶或支链淀粉酶，而且所述糖化处理进行大约24至72小时。
由前面的讨论显然，由于依据不同的步骤中温度的不同要求，常规的淀粉转化方法是非常消耗能量的。因此就需要能够选出用于此方法的酶，可以进行全过程而无需将所述淀粉凝胶化。这样的方法是专利US4591560、US4727026和US4009074和EP0171218的主题。
本发明涉及一步方法，用于在低于所述颗粒状淀粉的初始凝胶化温度的温度，将颗粒状淀粉转化为可溶的淀粉水解产物。
发明内容
在第一个方面，本发明提供了制备可溶的淀粉水解产物的方法，所述方法包括将水性(aqueous)的颗粒状淀粉浆体(slurry)在低于所述颗粒状淀粉的初始凝胶化温度的温度，与第一种酶作用，其中酶；是所述的糖苷水解酶家族13的成员；具有alpha-1，4-糖苷水解(glucosidic hydrolysis)活性，和；包括属于CBM家族20的功能的糖-结合组件(CBM)，其中CBM具有氨基酸序列，所述氨基酸序列与选自SEQ ID NO：1、SEQ ID NO：2或SEQ ID NO：3的氨基酸序列具有至少60％同源性；而其中第二种酶选自包括真菌alpha-淀粉酶(E.C.3.2.1.1)、beta-淀粉酶(E.C.3.2.1.2)或葡糖淀粉酶(E.C.3.2.1.3)的列表。
本方面的第一个方面的方法可以作为一步方法和/或作为包含一步或多步的方法实施。
在第二个方面，本发明提供了制备基于高果糖淀粉糖浆(high fructosestarch-based syrup)(HFSS)的方法，所述的方法包括通过本发明的第一个方面的方法制备可溶的淀粉水解产物，并进一步包括将所述的可溶的淀粉水解产物转化为基于高果糖淀粉糖浆(HFSS)的步骤。
在第三个方面，本发明提供了制备燃料乙醇或饮料(potable)乙醇的方法；其包括通过本发明的第一个方面的方法制备可溶的淀粉水解产物，并进一步包括将所述的可溶的淀粉水解产物发酵成为乙醇的步骤，其中所述的发酵步骤同时或分别地/依次地进行所述颗粒状淀粉的水解。
在第四个方面，本发明提供了具有alpha-淀粉酶活性的酶在淀粉水解的方法中的用途，所述的酶包含功能的CBM，其具有氨基酸序列，所述的氨基酸序列与选自SEQ ID NO：1、SEQ ID NO：2或SEQ ID NO：3的氨基酸序列具有至少60％同源性。
在第五个方面，本发明提供了具有alpha-淀粉酶活性的酶在颗粒状淀粉水解的方法中的用途，所述的酶包含氨基酸序列，所述的氨基酸序列与选自SEQ ID NO：4、SEQ ID NO：5、SEQ ID NO：6、SEQ ID NO：7、SEQ ID NO：8、SEQ ID NO：9、SEQ ID NO：10、SEQ ID NO：11、SEQ ID NO：12、SEQ ID NO：13、SEQ ID NO：14、SEQ ID NO：15、SEQ ID NO：16、SEQ ID NO：17或SEQ IDNO：18的氨基酸序列具有至少75％、至少80％、至少85％、至少90％、至少95％、至少98％、如至少99％同源性。
在第六个方面，本发明提供了具有alpha-淀粉酶活性和功能的CBM的酶在颗粒状淀粉水解的方法中的用途，所述的酶包含氨基酸序列，所述的氨基酸序列与选自SEQ ID NO：19、SEQ ID NO：20、SEQ ID NO：21和SEQ IDNO：22的氨基酸序列具有至少75％、至少80％、至少85％、至少90％、至少95％、至少98％、如至少99％同源性。
发明详述
定义
术语“颗粒状淀粉”理解为生的未烹调过的淀粉，即未经过凝胶化的淀粉。淀粉作为不可溶于水的微小颗粒在植物内形成。这些颗粒在低于初始凝胶化温度的温度保留在淀粉中，当置于冷水中时，谷物颗粒可吸收少量的液体并膨胀。在温度高达50℃至70℃时，所述的膨胀是可逆的。可逆性的程度依赖于具体的淀粉。在更高的温度下，开始被称为凝胶化的不可逆膨胀。
术语“初始凝胶化温度”理解为淀粉的凝胶化开始发生的最低温度。在水中加热的淀粉在50℃-75℃开始凝胶化；凝胶化的准确温度依赖于具体的淀粉，并能够由本领域的技术人员容易地确定。这样，初始凝胶化温度可根据植物种类、植物种类的具体品种以及生长条件而变化。在本发明的上下文中，给出的淀粉的初始凝胶化温度是应用Gorinstein.S.和Lii.C.，Starch/
Vol.44(12)pp.461-466(1992)描述的方法，淀粉颗粒的双折射(birefringence)损失5％时的温度。
术语“可溶的淀粉水解产物”理解为本发明的方法的可溶的产物，并可以包括单糖、二糖和寡糖，如葡萄糖、麦芽糖、麦芽糖糊精、环糊精和这些的任意混合物。优选至少90％、91％、92％、93％、94％、95％、96％、97％或98％的颗粒状淀粉的干固体被转化为可溶的淀粉水解产物。
术语“特制糖浆”(speciality syrups)，是本技术领域中公认术语，并根据DE和糖谱(carbohydrate spectrum)(参见由G.G.Birch和L.F.Green编辑的教科书“Molecular Structure and Function of Food Carbohydrate”，Applied SciencePublishers LTD.，London中的文章“New Speciality Glucose Syrups”，p.50+)进行表征。通常特制糖浆具有范围在35至45的DE。
所述的“糖苷水解酶家族13”在本发明的上下文中定义为水解酶的组，其包括具有(beta/alpha)8或TIM筒状结构的催化组件(module)，并通过alpha-保留反应机理(alpha-retaining reacting mechanism)作用于淀粉和相关的物质(Koshland，1953，Biol.Rev.Camp.Philos.Soc 28，416-436)。
具有“alpha-1，4-糖苷水解活性”的酶在本发明的上下文中定义为包括酶的组，所述的酶催化alpha-1，4-糖苷键的水解和/或合成，如由Takata(Takata etal，1992，J.Biol.Chem.267，18447-18452)和由Koshland(Koshland，1953，Biol.Rev.Camp.Philos.Soc 28，416-436)所定义的。
所述的“家族20的糖-结合组件”或CBM-20组件，在本发明的上下文中定义为大约100个氨基酸的序列，其与由Joergensen et al.(1997)在Biotechnol.Lett.19：1027-1031，在图1中公开的多肽的糖-结合组件(CBM)具有至少45％同源性。所述的CBM包括所述多肽的最后102个氨基酸，即来自氨基酸582至氨基酸683的亚序列。此公开中应用的糖苷水解酶家族的编号依据Coutinho，P.M.& Henrissat，B.(1999)CAZy-Carbohydrate-ActiveEnzymes server在URL：http://afmb.cnrs-mrs.fr/-cazy/CAZY/index.html或可替换地Coutinho，P.M.& Henrissat，B.1999；纤维素酶和其他糖类-活性酶的组件结构：综合的数据库方法，在“Genetics，Biochemistry and Ecology of CelluloseDegradation”，K.Ohmiya，K.Hayashi，K.Sakka，Y.Kobayashi，S.Karita和T.Kimura eds.，Uni Publishers Co.，Tokyo，pp.15-23，和Bourne，Y.Henrissat，B.2001；糖苷水解酶和糖基转移酶：家族和功能组件，Current Opinion inStructural Biology 11：593-600的概念。
糖-结合组件(CBM)是多肽氨基酸序列，其优先连接于多糖或寡糖(碳水化合物)，常常-但不一定排他地-连接于其不溶于水(包括晶体)的形式。
虽然许多类型的CBM已经在专利和科学文献中描述，其大多数-源自纤维素分解酶(纤维素酶)的多种-通常被称作“纤维素-结合组件”；通常的纤维素-结合组件由此为出现在纤维素酶中的CBM。同样地，其他CBM的亚-类可包含，例如，壳多糖-结合组件(通常出现在壳多糖酶(chitinase)中的CBM)、木聚糖-结合组件(通常出现在木聚糖酶中的CBM)、甘露聚糖-结合组件(通常出现在甘露聚糖酶中的CBM)、淀粉-结合组件(通常出现在一些淀粉分解酶，如一些葡糖淀粉酶中，或在如环糊精葡聚糖转移酶的酶中，或在alpha-淀粉酶中的CBM)。
发现CBM作为大多肽或蛋白的组成部分，所述蛋白由两个或多个多肽氨基酸序列区组成，尤其在水解酶(水解酶)中，其通常包括含有用于底物水解的活性位点的催化组件和用于与所述糖底物结合的糖-结合组件(CBM)。这些酶可包括一个以上的催化组件和一个、两个或三个CBM，并任选的进一步包括一个或多个多肽氨基酸序列区，它将所述的CBM与所述的催化组件相连接，后一种类型的区域通常表示为“接头”。包括CBM的水解酶的实例-其中的一些已经在前面提及-为纤维素酶、木聚糖酶、甘露聚糖酶、阿拉伯呋喃糖酶(arabinofuranosidases)、乙酰酯酶(acetylesterase)和壳聚糖酶。CBM也在藻类，例如在红藻Porphyra purpura中以非-水解多糖-结合蛋白的形式被发现。
在出现CBM的蛋白质/多肽中(例如酶，通常为水解酶)，CBM可位于N或C末端或位于内部位点。
构成CBM的那部分多肽或蛋白(例如水解酶)本身，通常由多于大约30个并且少于大约250个氨基酸残基组成。
本发明优选的是包含CBM的酶，所述的CBM包括选自氨基酸序列SEQID NO：1、SEQ ID NO：2或SEQ ID NO：3的氨基酸序列，以及包含CBM的酶，所述的CBM含有氨基酸序列，该氨基酸序列与选自氨基酸序列SEQ IDNO：1、SEQ ID NO：2或SEQ ID NO：3的氨基酸序列具有至少50％同源性。
本公开中所指的多肽“同源性”理解为指示第一个序列与第二个序列差异的两个序列之间的同一性(identity)程度。同源性可通过本领域已知的计算机程序适合地确定，所述的计算机程序如GCG程序包中提供的GAP(ProgramManual for the Wisconsin Package，Version 8，August 1994，Genetics ComputerGroup，575 Science Drive，Madison，Wisconsin，USA 53711)(Needleman，S.B.and Wunsch，C.D.，(1970)，Journal of Molecular Biology，48，443-453)。在氨基酸序列比较中采用下列设置：GAP产生罚分3.0和GAP延伸罚分0.1。
用作本发明的第一种酶的酶是四组件alpha-淀粉酶，其由三个组件淀粉酶核心和单独的(separate)家族20的糖结合组件组成。所述的alpha-淀粉酶可为源自细菌或真菌来源的野生型alpha-淀粉酶，或它可以为突变体、蛋白质工程变体或该野生型的其他变体，或者它可以为变体或野生型的杂合体。
所述的alpha-淀粉酶优选为野生型酶。所述alpha-淀粉酶更优选为上述alpha-淀粉酶的变体和/或杂合体，其包括氨基酸修饰，所述的氨基酸修饰导致活性增加、在低pH和/或高pH蛋白稳定性增加、对于钙损耗的稳定性增加，和/或在高温下稳定性增加。
本公开中所指的术语“酶杂合体”理解为修饰的酶，其包括淀粉分解酶的氨基酸序列[所述的淀粉分解酶在本发明的上下文中可为，例如alpha-淀粉酶(EC 3.2.1.1)、异淀粉酶(EC 3.2.1.68)或支链淀粉酶(EC 3.2.1.41)]，所述的淀粉分解酶连接(即共价结合)于包括CBM的氨基酸序列。所述的CBM优选但不排他地融合于所述的N-末端。所述的杂合体可包含一种以上的CBM。
含CBM的酶杂合体，及其制备和纯化的详细描述是本技术领域公知的[参见，例如，WO 90/00609、WO94/24158和WO 95/16782，以及Greenwoodet al.，Biotechnology and Bioengineering 44(1994)pp.1295-1305]。它们可，例如通过将DNA构建体转化入宿主细胞来制备，所述DNA构建体包括至少DNA的片段，其编码所述的纤维素-结合组件，所述组件用或不用接头连接于编码目标酶的DNA序列，并使转化的宿主细胞生长以表达所述的融合的基因。
构建糖-结合组件(CBM)和alpha-淀粉酶之间的杂合体蛋白需要一个或多个下列步骤，以获得稳定的、可以表达和应用的酶。
1)应用常规方法，将所述的CBM-供体分子与所述催化组件的供体进行比对，常常需要鉴定可能的交叉点。如果所述的同源性相对高，则也许有几个可能的交叉点。然而如果所述的同源性低或者只能分别得到所述催化组件和所述CBM的序列，可将所述的CBM作为延长附加于所述催化组件，或在所述序列的开始，即在最终的信号序列之后插入的N-末端中，或在终止信号之前的C-末端中。无论所述的CBM是否位于所述的N-末端或C-末端，都有益于缺失几个氨基酸或插入几个氨基酸作为接头，以得到可表达和应用的稳定的酶。
2)根据1)的考虑，构建编码所述CBM和淀粉分解组件的基因的DNA杂合体，可以通过本领域的技术人员公知的方法进行。这些方法其中包括，PCR反应，其应用引物，所述引物设计为在得到的DNA杂交(crossing)点杂交，DNA消化，然后是例如通过酵母实例的连接或体内组合。
3)CBM与淀粉分解组件的简单连接常常产生杂合体蛋白，所述的杂合体蛋白由于折叠或稳定性问题，或由于在杂合体蛋白中在设定的应用中缺乏足够的稳定性和/或活性而表达不足。为了克服上述问题，将所述的杂合体蛋白或通过定点突变方法，或通过更随机的方法经受蛋白质工程改造。这包括所述CBM的组件中的氨基酸和所述淀粉分解组件中的氨基酸，以及优化从淀粉分解组件到CBM的转换，关于长度和氨基酸序列。
作为本发明的第一种酶优选的是包含CBM的杂合体酶，其包括选自氨基酸序列SEQ ID NO：1、SEQ ID NO：2或SEQ ID NO：3的氨基酸序列，以及包含氨基酸序列的酶，所述的氨基酸序列与选自氨基酸序列SEQ ID NO：1、SEQ ID NO：2或SEQ ID NO：3的氨基酸序列具有至少50％、至少55％、至少60％、至少65％、至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少98％、如至少99％同源性。
作为本发明的第一种酶还优选包含氨基酸序列的杂合体酶，所述的氨基酸序列具有alpha-淀粉酶活性并包含选自氨基酸序列SEQ ID NO：4、SEQ IDNO：5、SEQ ID NO：6、SEQ ID NO：7、SEQ ID NO：8、SEQ ID NO：9、SEQ IDNO：10、SEQ ID NO：11、SEQ ID NO：12、SEQ ID NO：13、SEQ ID NO：14、SEQID NO：15、SEQ ID NO：16、SEQ ID NO：17或SEQ ID NO：18的氨基酸序列，以及包含氨基酸序列的酶，所述的氨基酸序列与选自SEQ ID NO：4、SEQ IDNO：5、SEQ ID NO：6、SEQ ID NO：7、SEQ ID NO：8、SEQ ID NO：9、SEQ IDNO：10、SEQ ID NO：11、SEQ ID NO：12、SEQ ID NO：13、SEQ ID NO：14、SEQID NO：15、SEQ ID NO：16、SEQ ID NO：17或SEQ ID NO：18的氨基酸序列具有至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少98％、如至少99％同源性。
本发明的第一种酶优选包含CBM和/或alpha-淀粉分解序列，其源自真菌，诸如来自属于踝节菌属(Talaromyces sp.)的菌株，或来自属于曲霉属(Aspergillus)的菌株，如泡盛曲霉(A.awamori)、A.kawachii、黑曲霉(A.niger)、米曲霉(A.oryzae)等，或源自细菌，诸如来自属于芽孢杆菌属(Bacillus sp)的菌株，如来自属于解淀粉芽孢杆菌(B.amyloliquefacience)、黄热芽孢杆菌(B.flavothermus)、地衣芽孢杆菌(B.licheniformis)或嗜热脂肪芽孢杆菌(B.stearothermophilus)的菌株。
作为本发明的第一种酶更优选四组件alpha-淀粉酶，其由三个组件淀粉酶核心和单独的家族20的糖结合组件组成。最优选四组件alpha-淀粉酶，其包含氨基酸序列，所述氨基酸序列与选自氨基酸序列SEQ ID NO：19、SEQ IDNQ：20、SEQ ID NO：21或SEQ ID NO：22的氨基酸序列具有至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少98％、如至少99％同源性。
本发明的第一种酶优选四组件alpha-淀粉酶，其分离自真菌或细菌，如来自芽孢杆菌属的菌种，如SEQ ID NO：20和SEQ ID NO：21中显示的多肽；或来自黄热芽孢杆菌的菌株，如SEQ ID NO：19中显示的多肽；或来自泡盛曲霉，如SEQ ID NO：22中显示的多肽。
作为本发明的第一种酶最优选alpha-淀粉酶，其包含氨基酸序列，所述的氨基酸序列与选自氨基酸序列SEQ ID NO：19、SEQ ID NO：20、SEQ IDNO：21或SEQ ID NO：22的氨基酸序列具有至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少98％、如至少99％同源性。
上述的alpha-淀粉酶可以以0.001-1.0KNU/g DS的量加入，优选0.002-0.5KNU/g DS，优选0.02-0.1KNU/g DS。
真菌alpha-淀粉酶
用作本发明的方法的第二种酶的具体的酶是真菌alpha-淀粉酶(E.C.3.2.1.1)，如fungamyl-样alpha-淀粉酶。在本公开中，术语“fungamyl-样alpha-淀粉酶”表示alpha-淀粉酶，其与WO96/23874的SEQ ID NO：10所示的氨基酸序列显示高同源性，即高于50％、55％、60％、65％、70％、75％、80％、85％或甚至90％同源性。真菌alpha-淀粉酶可以0.001-1.0AFAU/g DS的量加入，优选0.002-0.5AFAU/g DS，优选0.02-0.1AFAU/g DS。
beta-淀粉酶
用作本发明的方法的第二种酶的另一种具体的酶可为beta-淀粉酶(E.C.3.2.1.2)。beta-淀粉酶是传统上给予外-作用(exo-acting)产麦芽糖淀粉酶的名字，其在直链淀粉、支链淀粉和相关的葡萄糖聚合物中催化1，4-alpha-糖苷键的水解，由此释放麦芽糖。
beta-淀粉酶已经从多种植物和微生物中(W.M.Fogarty and C.T.Kelly，Progress in Industrial Microbiology，vol.15，pp.112-115，1979)分离。这些beta-淀粉酶以具有在40℃-65℃范围内的最适温度和在4.5-7.0范围内的最适pH为特征。预期的beta-淀粉酶包括来自大麦
BBA 1500、
DBA和来自Genencor Int.的OptimaltTM ME、OptimaltTM BBA以及来自NovozymesA/S的NOVOZYMTM WBA的beta-淀粉酶。
葡糖淀粉酶
用作本发明的方法的第二种酶的又一种具体的酶也可为源自微生物或植物的葡糖淀粉酶(glucoamylase)(E.C.3.2.1.3)。优选真菌或细菌来源的葡糖淀粉酶选自曲霉属葡糖淀粉酶，特别为黑曲霉菌G1或G2葡糖淀粉酶(Boel et al.，(1984)，EMBO J.3(5)，p.1097-1102)，或其变体，如在WO92/00381和WO00/04136中所公开的；泡盛曲霉葡糖淀粉酶(WO84/02921)，米曲霉菌(Agric.Biol.Chem.(1991)，55(4)，p.941-949)，或其片段或变体。
其他预期的曲霉属葡糖淀粉酶变体包括热稳定性增加的变体：G137A和G139A(Chen et al.(1996)，Prof.Engng.9，499-505)；D257E和D293E/Q(Chenet al.(1995)，Prot.Engng.8，575-582)；N182(Chen et al.(1994)，Biochem.J.301，275-281)；二硫化物键(disulphide bond)，A246C(Fierobe et al.(1996)，Biochemistry，35，8698-8704；和在位点A435和S436导入Pro残基(Li et al.(1997)，Protein Engng.10，1199-1204)。其他预期的葡糖淀粉酶包括踝节菌属葡糖淀粉酶，尤其源自Talaromyces emersonii(WO99/28448)、Talaromycesleycettanus(美国专利no.Re.32，153)、Talaromyces duponti、嗜热踝节菌(Talaromyces thermophilus)(美国专利no.4,587,215)。预期的细菌葡糖淀粉酶包括来自梭菌属(Clostridium)的葡糖淀粉酶，尤其是C.thermoamylolyticum(EP135,138)和热硫化氢梭菌(C.thermohydrosulfuricum)(WO86/01831)。优选的葡糖淀粉酶包括源自米曲霉菌的葡糖淀粉酶，如葡糖淀粉酶，其与在WO00/04136的SEQ ID NO：2中所示的氨基酸序列具有50％、55％、60％、65％、70％、75％、80％、85％或甚至90％同源性。预期的也为商业产品AMG 200L、AMG 300L、SANTM SUPER和AMGTM E(来自Novozymes)；OPTIDEXTM 300(来自Genencor Int.)；AMIGASETM和AMIGASETM PLUS(来自DSM)；G-ZYMETM G900(来自Enzyme Bio-Systems)；G-ZYMETM G990ZR(黑曲霉葡糖淀粉酶和低蛋白酶含量)。
葡糖淀粉酶可以0.02-2.0AGU/gDS的量加入，优选0.1-1.0AGU/g DS，如0.2AGU/g DS。
附加的酶
本发明的方法可以在第三种酶存在的条件下进行。具体的第三种酶可为芽孢杆菌alpha-淀粉酶(常常被成作“Termamyl-样alpha-淀粉酶”)。众所周知的Termamyl-样alpha-淀粉酶包括alpha-淀粉酶，其源自地衣芽孢杆菌(可作为Termamyl商业上得到)、解淀粉芽孢杆菌和嗜热脂肪芽孢杆菌的菌株的alpha-淀粉酶。其他Termamyl-样alpha-淀粉酶包括源自芽孢杆菌NCIB 12289，NCIB12512，NCIB 12513或DSM 9375的菌株的alpha-淀粉酶，所述的所有菌株在WO95/26397中详细描述，而且所述的alpha-淀粉酶由Tsukamoto et al.，Biochemical and Biophysical Research Communications，151(1988)，pp.25-31描述。在本发明的上下文中，Termamyl-样alpha-淀粉酶是如在WO99/19467的第3页第18行至第6页第27行定义的alpha-淀粉酶。预期的变体和杂合体在WO96/23874、WO97/41213和WO99/19467中描述。尤其预期的是重组嗜热脂肪芽孢杆菌alpha-淀粉酶变体，其具有突变：I181*+G182*+N193F。芽孢杆菌alpha-淀粉酶可以以本领域的技术人员熟知的有效量加入。
本发明的又一种具体的第三种酶可为脱支酶，诸如异淀粉酶(E.C.3.2.1.68)或支链淀粉酶(E.C.3.2.1.41)。异淀粉酶水解支链淀粉和beta-极限糊精中的alpha-1，6-D-支链糖苷键，并可通过异淀粉酶不能攻击支链淀粉，及通过对于alpha-极限糊精的有限作用与支链淀粉酶相区别。脱支酶可以以本领域的技术人员熟知的有效量加入。
本发明的实施方案
经过本发明的处理的淀粉浆体可具有20-55％干固体颗粒状淀粉，优选25-40％干固体颗粒状淀粉，更优选30-35％干固体颗粒状淀粉。
经过本发明的第一个方面的处理之后，至少85％、至少86％、至少87％、至少88％、至少89％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或优选至少99％所述颗粒状淀粉的干固体被转化为可溶的淀粉水解产物。
根据本发明，第一和第二方面的处理在低于初始凝胶化温度的温度进行。优选地，进行所述处理的温度为至少30℃、至少31℃、至少32℃、至少33℃、至少34℃、至少35℃、至少36℃、至少37℃、至少38℃、至少39℃、至少40℃、至少41℃、至少42℃、至少43℃、至少44℃、至少45℃、至少46℃、至少47℃、至少48℃、至少49℃、至少50℃、至少51℃、至少52℃、至少53℃、至少54℃、至少55℃、至少56℃、至少57℃、至少58℃、至少59℃或优选至少60℃。
本发明的第一个方面的处理进行的pH可为3.0-7.0的范围，优选3.5至6.0，或更优选4.0-5.0。
本发明第一个方面的方法的产物的精确组成，所述可溶的淀粉水解产物，依赖于应用的酶的组合以及处理的颗粒状淀粉的类型。优选地，所述可溶的淀粉水解产物为麦芽糖，其具有至少85％、至少90％、至少95.0％、至少95.5％、至少96.0％、至少96.5％、至少97.0％、至少97.5％、至少98.0％、至少98.5％、至少99.0％或至少99.5％的纯度。还更优选所述可溶的淀粉水解产物为葡萄糖，并最优选所述可溶的淀粉水解产物具有至少94.5％、至少95.0％、至少95.5％、至少96.0％、至少96.5％、至少97.0％、至少97.5％、至少98.0％、至少98.5％、至少99.0％或至少99.5％的DX(总溶解的干固体中葡萄糖百分比)。然而，同样地预期的是所述的方法，其中本发明的方法的产物，所述可溶的淀粉水解产物，是特制糖浆，诸如含有葡萄糖、麦芽糖、DP3和DPn的混合物的特制糖浆，用于制造冰淇淋、蛋糕、糖果、水果罐头。
在本发明的处理中加工的颗粒状淀粉尤其可以从块茎、根、茎、豆类、谷类或整粒谷物(whole grain)中得到。更具体地，所述的颗粒状淀粉可以从玉米、粗玉米粉(com grits)、穗轴(cob)、小麦、大麦、黑麦、蜀黍类(milo)、西米(sago)、木薯(cassava)、木薯淀粉(tapioca)、高粱、稻米、豌豆、大豆、香蕉或马铃薯中得到。具体预期蜡质(waxy)和非蜡质(non-waxy)类型的玉米和大麦。待处理的颗粒状淀粉可具有高度精制的淀粉质量，优选至少90％、至少95％、至少97％或至少99.5％纯度或其可为含有原料较粗的淀粉，所述的原料包括磨碎的整粒谷物，其包括非-淀粉部分，如胚芽残余物(germ residue)和纤维。将所述的原料，如整粒谷物磨碎以打开其结构并允许进一步的处理。根据本发明优选两种磨碎方法：湿磨(wet milling)和干磨(dry milling)。进行干磨时，研磨并应用所述的整个谷粒。湿磨使胚芽和粗磨粉(meal)(淀粉颗粒和蛋白)良好的分离，并在所述的淀粉水解产物用于制备糖浆的应用中具有少数例外。干磨和湿磨在淀粉加工的技术领域中都是熟知的，并同样预期用于本发明的方法。本发明的第一个方面的方法在超滤系统中实施，而且其中所述的渗余物在酶、生淀粉和水存在的回流(recirculation)条件下保留，而且其中所述的渗透物是可溶的淀粉水解产物。同样预期的是本方法在具有超滤膜的连续膜反应器中实施，而且其中所述的渗余物在酶、生淀粉和水存在的回流条件下保留，而且其中所述的渗透物是可溶的淀粉水解产物。也预期的是本方法在具有微滤膜的连续膜反应器中实施，而且其中所述的渗余物在酶、生淀粉和水存在的回流条件下保留，而且其中所述的渗透物是可溶的淀粉水解产物。
在本发明的第二个方面的方法中，将本发明的第一个方面的方法的可溶的淀粉水解产物经过转化成为基于高果糖淀粉糖浆(HFSS)，诸如高果糖浆(HFCS)。此转化优选应用葡萄糖异构酶完成，并更优选通过在固体支持物支持的固定化的葡萄糖异构酶进行。预期的葡萄糖异构酶包括下列商业产品，来自Novozymes A/S的SweetzymeTMIT、来自Rhodia的G-zymeTM IMGI和G-zymeTM G993、KetomaxTM和G-zymeTM G993、来自Genencor Int的G-zymeTMG993液体和GenSweetTM IGI。
在本发明的第三个方面的方法中，本发明的第一个方面的方法的可溶的淀粉水解产物用于制备燃料或饮料乙醇。在第三个方面的方法中，所述的发酵可同时或分别地/依次地进行所述颗粒状淀粉的水解。当所述的发酵与所述的水解同时进行时，所述的温度优选微30℃-35℃，并更优选31℃-34℃。本发明的第三方面的方法可以在超滤系统中实施，其中所述的渗余物(retentate)在酶、生淀粉、酵母、酵母营养素和水存在的回流条件下保留，而且其中所述的渗透物(permeate)是含有乙醇的液体(ethanol containing liquid)。同样预期的是所述的方法在具有超滤膜的连续膜反应器中实施，其中所述的渗余物在酶、生淀粉、酵母、酵母营养素和水存在的回流条件下保留，而且其中所述的渗透物是含有乙醇的液体。
本发明的第一个方面的方法的可溶的淀粉水解产物也可用于产生发酵产品，所述的发酵产品包括将处理过的淀粉发酵成为发酵产品，诸如柠檬酸、谷氨酸一钠、葡糖酸、葡糖酸钠、葡糖酸钙、葡糖酸钾、葡糖酸内酯、异抗坏血酸钠。
在另一个实施方案中，将所述的淀粉浆体与包含CBM，但无淀粉分解组件的多肽接触，即疏松的CBM。所述疏松的CBM可为淀粉结合组件、纤维素结合组件、壳多糖结合组件、木聚糖结合组件、甘露聚糖结合组件和其他结合组件。本上下文中优选的CBM是微生物CBM，尤其是细菌或真菌CBM。尤其优选的是在本公开中所示的淀粉结合组件，如SEQ ID NO：1、SEQ IDNO：2和SEQ ID NO：3的多肽序列，或在美国临时申请No.60/511044中公开的淀粉结合组件，如SEQ ID NO：12；来自Hormoconis sp.，如来自Hormoconisresinae(同物异名为木馏油真菌(Creosote fungus)或Amorphotheca resinae)的葡糖淀粉酶的CBM(SWISSPROT：Q03045)，SEQ ID NO：13；来自Lentinula sp.，如来自Lentinula edodes(shiitake mushroom)的CBM(SPTREMBL：Q9P4C5)，SEQ ID NO：14；来自脉孢菌属(Neurospora sp.)，如来自粗糙脉孢菌(Neurosporacrassa)的CBM(SWISSPROT：P14804)，SEQ ID NO：15；来自踝节菌属，如来自Talaromyces byssochlamydioides的CBM，SEQ ID NO：16；来自Geosmithiasp.，如来自Geosmithia cylindrospora的CBM，SEQ ID NO：17；来自Scorias sp.，如来自Scorias spongiosa的CBM，SEQ ID NO：18；来自Eupenicillium sp.，如来自Eupenicillium ludwigii的CBM，SEQ ID NO：19；来自曲霉属，如来自日本曲霉(Asperigillus japonicaus)的CBM，SEQ ID NO：20；来自青霉属(Penicillium sp.)，如来自Penicillium cf.miczynskii的CBM，SEQ ID NO：21；来自Mz1青霉属的CBM，SEQ ID NO：22；来自Thysanophora sp.的CBM，SEQ ID NO：23；来自腐质霉属(Humicola sp.)，如来自灰腐质霉thermoidea变种(Humicola grisea var.thermoidea)的CBM。最优选的CBM包括在美国临时申请No.60/511044中公开的CBM，如SEQ ID NO：24；来自曲霉属，如来自黑曲霉的葡糖淀粉酶的CBM，而且如SEQ ID NO：25；来自Athelia sp.，如来自Athelia rolfsii的葡糖淀粉酶的CBM。本发明也优选应用任意CBM，其与上述提及的CBM氨基酸序列具有至少50％、60％、70％、80％或甚至至少90％同源性。
所述疏松的CBMs可以以有效量应用于所述的颗粒状淀粉。
材料和方法
alpha-淀粉酶活性(KNU)
所述的淀粉分解活性可以应用马铃薯淀粉作为底物进行测定。此方法基于通过酶破坏变性的马铃薯淀粉，而且所述的反应之后将所述的淀粉/酶溶液的样品与碘溶液混合。起初，形成蓝黑色，但是在破坏淀粉的过程中，蓝色变弱而且逐渐变为棕红色，其与有色玻璃标准物相比。
将1Kilo Novo alpha淀粉酶单位(KNU)作为酶量的定义，其在标准条件下(即，在37℃+/-0.05；0.0003M Ca2+；和pH5.6)将5.26g淀粉干底物MerckAmylum solubile转化成糊精。
更详细地描述此分析方法的文件夹(folder)AF 9/6，可根据要求从Novozymes A/S，Denmark得到，其文件夹在此包括作为参考。
葡糖淀粉酶活性(AGU)
Novo葡糖淀粉酶单位(AGU)定义为其每分钟在37℃和pH 4.3水解1微摩尔麦芽糖的酶量。
所述的活性通过根据(AEL-SM-0131，可根据要求从Novozymes得到)修改的方法测定为AGU/ml，应用来自Boehringer Mannheim，124036的葡萄糖GOD-Perid试剂盒。标准：AMG-标准物，批号7-1195，195AGU/ml。将375μL底物(50mM乙酸钠中的1％麦芽糖，pH 4.3)在37℃温育5分钟。加入25μL稀释于乙酸钠中的酶。所述的反应10分钟后通过加入100μL 0.25M NaOH而终止。将20μL转移到96孔微量滴定板，并加入200μL GOD-Perid溶液(124036，Boehringer Mannheim)。在室温30分钟后，在650nm测定吸光度，并且从AMG-标准计算以AGU/ml表示的活性。更详细地描述此分析方法的文件夹(AEL-SM-0131)，可根据要求从Novozymes A/S，Denmark得到，其文件夹在此包括作为参考。
真菌alpha-淀粉酶活性(FAU)
真菌alpha-淀粉酶活性可以以FAU(真菌Alpha-淀粉酶单位)测定。一个(1)FAU是在标准条件下(即在37℃和pH4.7)，每小时破坏5260mg固体淀粉(Amylum solubile，Merck)的酶量。更详细地描述此FAU测定的文件夹AF9.1/3，可根据要求从Novozymes A/S，Denmark得到，其文件夹在此包括作为参考。
酸性alpha-淀粉酶活性(AFAU)
酸性alpha-淀粉酶活性可以以AFAU(酸性真菌alpha-淀粉酶单位)测定，其是相对于酶标准物测定的。
所述的标准物为AMG 300L(来自Novozymes A/S，葡糖淀粉酶野生型黑曲霉菌G1，也公开于Boel et al.(1984)，EMBO J.3(5)，p.1097-1102和WO92/00381中)。此AMG中的中性alpha-淀粉酶，在室温贮藏3个星期之后，从大约1FAU/ml降低为0.05FAU/ml。
此AMG标准物中的酸性alpha-淀粉酶活性根据如下描述进行测定。此方法中，1AFAU定义为每小时在下述标准条件下降解5.26mg淀粉干物质的酶量。
碘与淀粉形成蓝色复合物，但与淀粉的降解产物则不能形成。因此颜色的强度直接与淀粉的浓度成正比。淀粉酶活性在具体的分析条件下，应用淀粉浓度的降低的反向比色法进行测定。
蓝/紫 t＝23秒 脱色
标准条件/反应条件：(每分钟)
底物：可溶性淀粉，大约0.17g/L
缓冲液：柠檬酸盐，大约0.03M
碘(I2)：0.03g/L
CaCl2：1.85mM
pH：2.50-0.05
温育温度：40℃
反应时间：23秒
波长：lambda＝590nm
酶浓度：0.025AFAU/mL
酶工作范围：0.01-0.04AFAU/mL
更详细地描述此分析方法的文件夹EB-SM-0259.02/01，可根据要求从Novozymes A/S，Denmark得到，而且并入作为参考。
beta-淀粉酶活性(DP°)
支链淀粉酶活性(新支链淀粉酶单位Novo)(NPUN)
支链淀粉酶活性可以相对于支链淀粉底物进行测定。支链淀粉是线状的D-葡萄糖聚合物，其主要由麦芽三糖基单位通过1，6-alpha-键连接组成。内-支链淀粉酶随机地水解1，6-alpha-键，释放麦芽三糖、63-alpha-麦芽三糖基-麦芽三糖、63-alpha-(63-alpha-麦芽三糖基-麦芽三糖基)-麦芽三糖。
一个新支链淀粉酶单位Novo(NPUN)是内-支链淀粉酶活性的单位，并相对于Novozymes A/S Promozyme D标准物测定。标准条件为在40℃和pH 4.5反应30分钟，并以0.7％支链淀粉作为底物。红色底物降解产物的量通过分光光度法在510nm测定，并与样品中的内-支链淀粉酶活性成比例。更详细地描述此分析方法的文件夹(EB-SM.0420.02/01)，可根据要求从NovozymesA/S，Denmark得到，其文件夹在此包括作为参考。
在标准条件下，一个NPUN大约与释放具有相当于2.86μmole葡萄糖每分钟的还原力的还原糖的酶量相等。
测定糖分布(profile)和溶解的干固体
所述淀粉水解产物的糖的组成通过HPLC进行测定，并随后计算葡萄糖产量为DX.°BRIX，所述淀粉水解产物的溶解的(可溶的)干固体通过折射率(refractive index)测定方法进行测定。
原料
应用如下的酶活性。具有CBD的细菌alpha-淀粉酶，其具有SEQ IDNO：19中描述的序列和同样的但不具有所述CBD组件的细菌alpha-淀粉酶(SEQ ID NO：4)。源自黑曲霉菌的葡糖淀粉酶，其具有WO00/04136的SEQ IDNO：2中所示的氨基酸序列或所公开的变体中的一个。源自黑曲霉菌的酸性真菌alpha-淀粉酶。
小麦淀粉(S-5127)从Sigma-Aldrich得到。
实施例1
本实施例说明，应用细菌四组件alpha-淀粉酶和葡糖淀粉酶和酸性真菌淀粉酶，将颗粒状小麦淀粉转化为葡萄糖。具有33％干固体(DS)颗粒状淀粉的浆体，通过在将247.5g小麦淀粉在搅拌的条件下加入502.5ml水中来进行制备。所述的pH用HCl调节到4.5。将所述的颗粒状淀粉浆体分配到100ml蓝色加盖摇瓶中，每个摇瓶中加75g。将所述的摇瓶用磁力搅拌在60℃水浴中保温。在0小时，将表1中设定的酶活性配入所述的摇瓶中。在24、48、72和96小时后取样。
表1.采用的酶活性水平
全部干固体淀粉应用如下方法进行测定。所述的淀粉通过加入过量的alpha-淀粉酶(300KNU/Kg干固体)并将所述的样品在95℃油浴中放置45分钟进行完全地水解。随后将所述的样品冷却到60℃，并加入过量的葡糖淀粉酶(600AGU/kg DS)，然后在60℃温育2小时。
所述的淀粉水解产物中可溶的干固体，通过检测通过0.22microM过滤器过滤后的样品的折射率进行测定。所述的糖分布模式通过HPLC进行测定。计算葡萄糖的量为DX。所述的结果显示在表2和表3中。
表2.alpha-淀粉酶用量为100KNU/kg DS时，可溶的干固体占全部干物质的百分比
表3.alpha-淀粉酶用量为100KNU/kg DS时，所述可溶的水解产物的DX
实施例2
本实施例说明，应用葡糖淀粉酶和酸性真菌淀粉酶，将颗粒状淀粉部分转化为葡萄糖。
制备含33％DS颗粒状淀粉的摇瓶，并如实施例1中所述进行温育。在0小时，将表4中设定的酶活性配入所述的摇瓶中。在24、48、72和96小时后取样。所述的样品如实施例1中所述进行分析。所述的结果显示在表5和表6中。
表4.采用的酶活性水平
表5.可溶的干固体占全部干物质的百分比
表6.所述水解产物的DX
实施例3
在实施例3中，应用葡糖淀粉酶(200AGU/kgDS)，酸性真菌淀粉酶(50AFAU/kg DS)和在实施例1中采用的完整的细菌四组件alpha-淀粉酶(SEQ IDNO：19)或同样的但不具有所述CBD组件的细菌四组件alpha-淀粉酶(SEQ IDNO：4)(100KNU/kg DS)，进行颗粒状小麦淀粉向葡萄糖的转化。制备含33％干固体(DS)颗粒状淀粉的浆体，并如实施例1中所述进行温育。在24、46、70和90小时后取样。
全部干固体淀粉如实施例1中所述进行测定。所述的淀粉水解产物中可溶的干固体和所述的糖分布如实施例1中所述进行分析。所述的结果显示在表7和表8中。
表7.可溶的干固体占全部干物质的百分比。酶：葡糖淀粉酶、真菌酸性淀粉酶和具有所述CBD组件的细菌alpha-淀粉酶(SEQ ID NO：19)或不具有所述CBD组件的细菌alpha-淀粉酶(SEQ ID NO：4)。
表8.所述可溶的水解产物的DX：酶：葡糖淀粉酶、真菌酸性淀粉酶和具有所述CBD组件的细菌alpha-淀粉酶(SEQ ID NO：19)或不具有所述CBD组件的细菌alpha-淀粉酶(SEQ ID NO：4)。
序列表
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<120>四域的alpha淀粉酶的冷液化(Cold Liquefaction with four-domain alpha-amylase)
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Gln Pro Ala Leu Ala Val Thr Leu Val Asp Asn His Asp Thr Gln Pro
325 330 335
Gly Gln Ser Leu Gln Ser Trp Val Glu Pro Trp Phe Lys Pro Leu Ala
340 345 350
Tyr Ala Phe Ile Leu Thr Arg Gln Glu Gly Tyr Pro Cys Val Phe Tyr
355 360 365
Gly Asp Tyr Tyr Gly Ile Pro Lys Tyr Asn Ile Pro Gly Leu Lys Ser
370 375 380
Lys Ile Asp Pro Leu Leu Ile Ala Arg Arg Asp Tyr Ala Tyr Gly Thr
385 390 395 400
Gln Arg Asp Tyr Ile Asp His Gln Asp Ile Ile Gly Trp Thr Arg Glu
405 410 415
Gly Ile Asp Ala Lys Pro Asn Ser Gly Leu Ala Ala Leu Ile Thr Asp
420 425 430
Gly Pro Gly Gly Ser Lys Trp Met Tyr Val Gly Lys Arg His Ala Gly
435 440 445
Lys Val Phe Tyr Asp Leu Thr Gly Asn Arg Ser Asp Thr Val Thr Ile
450 455 460
Asn Ala Asp Gly Trp Gly Glu Phe Lys Val Asn Gly Gly Ser Val Ser
465 470 475 480
Ile Trp Val Ala Lys
485
<210>6
<211>484
<212>PRT
<213>Alkaliphilic bacillus
<400>6
Gly Ser Val Pro Val Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr
1 5 10 15
Leu Pro Asp Asp Gly Thr Leu Trp Thr Lys Val Ala Asn Asn Ala Gln
20 25 30
Ser Leu Ala Asn Leu Gly Ile Thr Ala Leu Trp Leu Pro Pro Ala Tyr
35 40 45
Lys Gly Thr Ser Ser Ser Asp Val Gly Tyr Gly Val Tyr Asp Leu Tyr
50 55 60
Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly
65 70 75 80
Thr Lys Thr Gln Tyr Ile Gln Ala Ile Gln Ala Ala His Thr Ala Gly
85 90 95
Met Gln Val Tyr Ala Asp Val Val Phe Asn His Lys Ala Gly Ala Asp
100 105 110
Gly Thr Glu Leu Val Asp Ala Val Glu Val Asn Pro Ser Asp Arg Asn
115 120 125
Gln Glu Ile Ser Gly Thr Tyr Gln Ile Gln Ala Trp Thr Lys Phe Asp
130 135 140
Phe Pro Gly Arg Gly Asn Thr Tyr Ser Ser Phe Lys Trp Arg Trp Tyr
145 150 155 160
His Phe Asp Gly Thr Asp Trp Asp Glu Ser Arg Lys Leu Asn Arg Ile
165 170 175
Tyr Lys Phe Arg Gly Thr Gly Lys Ala Trp Asp Trp Glu Val Asp Thr
180 185 190
Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Leu Asp Met Asp
195 200 205
His Pro Glu Val Val Ser Glu Leu Lys Asn Trp Gly Lys Trp Tyr Val
210 215 220
Ile Thr Thr Asn Ile Asp Gly Phe Arg Leu Asp Ala Val Lys His Ile
225 230 35 240
Lys Tyr Ser Phe Phe Pro Asp Trp Leu Ser Tyr Leu Arg Thr Gln Thr
245 250 255
Gln Lys Pro Leu Phe Ala Val Gly Glu Phe Trp Ser Tyr Asp Ile Asn
260 265 270
Lys Leu His Asn Tyr Ile Thr Lys Thr Asn Gly Ser Met Ser Leu Phe
275 280 285
Asp Ala Pro Leu His Asn Asn Phe Tyr Ile Ala Ser Lys Ser Gly Gly
290 295 300
Tyr Phe Asp Met Arg Thr Leu Leu Asn Asn Thr Leu Met Lys Glu Gln
305 310 315 320
Pro Thr Leu Ser Val Thr Leu Val Asp Asn His Asp Thr Glu Pro Gly
325 330 335
Gln Ser Leu Gln Ser Trp Val Glu Pro Trp Phe Lys Pro Leu Ala Tyr
340 345 350
Ala Phe Ile Leu Thr Arg Gln Glu Gly Tyr Pro Cys Val Phe Tyr Gly
355 360 365
Asp Tyr Tyr Gly Ile Pro Lys Tyr Asn Ile Pro Ala Leu Lys Ser Lys
370 375 380
Leu Asp Pro Leu Leu Ile Ala Arg Arg Asp Tyr Ala Tyr Gly Thr Gln
385 390 395 400
His Asp Tyr Ile Asp Asn Ala Asp Ile Ile Gly Trp Thr Arg Glu Gly
405 410 415
Val Ala Glu Lys Ala Asn Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly
420 425 430
Pro Gly Gly Ser Lys Trp Met Tyr Val Gly Lys Gln His Ala Gly Lys
435 440 445
Thr Phe Tyr Asp Leu Thr Gly Asn Arg Ser Asp Thr Val Thr Ile Asn
450 455 460
Ala Asp Gly Trp Gly Glu Phe Lys Val Asn Gly Gly Ser Val Ser Ile
465 470 475 480
Trp Val Pro Lys
<210>7
<211>517
<212>PRT
<213>芽孢杆菌属的菌种(Bacillus sp.)：
<400>7
Met Ser Leu Phe Lys Lys Ile Phe Pro Trp Ile Leu Ser Leu Leu Leu
1 5 10 15
Leu Phe Leu Phe Ile Ala Pro Phe Ser Ile Gln Thr Glu Lys Val Arg
20 25 30
Ala Gly Ser Val Pro Val Asn Gly Thr Met Met Gln Tyr Phe Glu Trp
35 40 45
Tyr Leu Pro Asp Asp Gly Thr Leu Trp Thr Lys Val Ala Asn Asn Ala
50 55 60
Gln Ser Leu Ala Asn Leu Gly Ile Thr Ala Leu Trp Leu Pro Pro Ala
65 70 75 80
Tyr Lys Gly Thr Ser Ser Ser Asp Val Gly Tyr Gly Val Tyr Asp Leu
85 90 95
Tyr Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr
100 105 110
Gly Thr Lys Thr Gln Tyr Ile Gln Ala Ile Gln Ala Ala His Thr Ala
115 120 125
Gly Met Gln Val Tyr Ala Asp Val Val Phe Asn His Lys Ala Gly Ala
130 135 140
Asp Gly Thr Glu Leu Val Asp Ala Val Glu Val Asn Pro Ser Asp Arg
145 150 155 160
Asn Gln Glu Ile Ser Gly Thr Tyr Gln Ile Gln Ala Trp Thr Lys Phe
165 170 175
Asp Phe Pro Gly Arg Gly Asn Thr Tyr Ser Ser Phe Lys Trp Arg Trp
180 185 190
Tyr His Phe Asp Gly Thr Asp Trp Asp Glu Ser Arg Lys Leu Asn Arg
195 200 205
Ile Tyr Lys Phe Arg Gly Thr Gly Lys Ala Trp Asp Trp Glu Val Asp
210 215 220
Thr Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Leu Asp Met
225 230 235 240
Asp His Pro Glu Val Val Ser Glu Leu Lys Asn Trp Gly Lys Trp Tyr
245 250 255
Val Thr Thr Thr Asn Ile Asp Gly Phe Arg Leu Asp Ala Val Lys His
260 265 270
Ile Lys Tyr Ser Phe Phe Pro Asp Trp Leu Ser Tyr Val Arg Thr Gln
275 280 285
Thr Gln Lys Pro Leu Phe Ala Val Gly Glu Phe Trp Ser Tyr Asp Ile
290 295 300
Ser Lys Leu His Asn Tyr Ile Thr Lys Thr Asn Gly Ser Met Ser Leu
305 310 315 320
Phe Asp Ala Pro Leu His Asn Asn Phe Tyr Ile Ala Ser Lys Ser Gly
325 330 335
Gly Tyr Phe Asp Met Arg Thr Leu Leu Asn Asn Thr Leu Met Lys Asp
340 345 350
Gln Pro Thr Leu Ala Val Thr Leu Val Asp Asn His Asp Thr Glu Pro
355 360 365
Gly Gln Ser Leu Gln Ser Trp Val Glu Pro Trp Phe Lys Pro Leu Ala
370 375 380
Tyr Ala Phe Ile Leu Thr Arg Gln Glu Gly Tyr Pro Cys Val Phe Tyr
385 390 395 400
Gly Asp Tyr Tyr Gly Ile Pro Lys Tyr Asn Ile Pro Ala Leu Lys Ser
405 410 415
Lys Leu Asp Pro Leu Leu Ile Ala Arg Arg Asp Tyr Ala Tyr Gly Thr
420 425 430
Gln His Asp Tyr Ile Asp Ser Ala Asp Ile Ile Gly Trp Thr Arg Glu
435 440 445
Gly Val Ala Glu Lys Ala Asn Ser Gly Leu Ala Ala Leu Ile Thr Asp
450 455 460
Gly Pro Gly Gly Ser Lys Trp Met Tyr Val Gly Lys Gln His Ala Gly
465 470 475 480
Lys Thr Phe Tyr Asp Leu Thr Gly Asn Arg Ser Asp Thr Val Thr Ile
485 490 495
Asn Ala Asp Gly Trp Gly Glu Phe Lys Val Asn Gly Gly Ser Val Ser
500 505 510
Ile Trp Val Pro Lys
515
<210>8
<211>550
<212>PRT
<213>未知
<220>
<223>来源未知
<400>8
Met Ser Leu Phe Lys Lys Ile Phe Pro Trp IIe Val Ser Leu Leu Leu
1 5 10 15
Leu Phe Ser Phe Ile Ala Pro Phe Ser Ile Gln Thr Glu Lys Val Arg
20 25 30
Ala Gly Ser Val Pro Val Asn Gly Thr Met Met Gln Tyr Phe Glu Trp
35 40 45
Tyr Leu Pro Asp Asp Gly Thr Leu Trp Thr Lys Val Ala Asn Asn Ala
50 55 60
Gln Ser Leu Ala Asn Leu Gly Ile Thr Ala Leu Trp Leu Pro Pro Ala
65 70 75 80
Tyr Lys Gly Thr Ser Ser Ser Asp Val Gly Tyr Gly Val Tyr Asp Leu
85 90 95
Tyr Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr
100 105 110
Gly Thr Lys Thr Gln Tyr Ile Gln Ala Ile Gln Ala Ala His Thr Ala
115 120 125
Gly Met Gln Val Tyr Ala Asp Val Val Phe Asn His Lys Ala Gly Ala
130 135 140
Asp Gly Thr Glu Leu Val Asp Ala Val Glu Val Asn Pro Ser Asp Arg
145 150 155 160
Asn Gln Glu Ile Ser Gly Thr Tyr Gln Ile Gln Ala Trp Thr Lys Phe
165 170 175
Asp Phe Pro Gly Arg Gly Asn Thr Tyr Ser Ser Phe Lys Trp Arg Trp
180 185 190
Tyr His Phe Asp Gly Thr Asp Trp Asp Glu Ser Arg Lys Leu Asn Arg
195 200 205
Ile Tyr Lys Phe Arg Gly Thr Gly Lys Ala Trp Asp Trp Glu Val Asp
210 215 220
Thr Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Leu Asp Met
225 230 235 240
Asp His Pro Glu Val Val Ser Glu Leu Lys Asn Trp Gly Lys Trp Tyr
245 250 255
Val Thr Thr Thr Asn Ile Asp Gly Phe Arg Leu Asp Ala Val Lys His
260 265 270
Ile Lys Tyr Ser Phe Phe Pro Asp Trp Leu Ser Tyr Val Arg Thr Gln
275 280 285
Thr Gln Lys Pro Leu Phe Ala Val Gly Glu Phe Trp Ser Tyr Asp Ile
290 295 300
Asn Lys Leu His Asn Tyr Ile Thr Lys Thr Asn Gly Ser Met Ser Leu
305 310 315 320
Phe Asp Ala Pro Leu His Asn Asn Phe Tyr Ile Ala Ser Lys Ser Gly
325 330 335
Gly Tyr Phe Asp Met Arg Thr Leu Leu Asn Asn Thr Leu Met Lys Asp
340 345 350
Gln Pro Thr Leu Ser Val Thr Leu Val Asp Asn His Asp Thr Glu Pro
355 360 365
Gly Gln Ser Leu Gln Ser Trp Val Glu Pro Trp Phe Lys Pro Leu Ala
370 375 380
Tyr Ala Phe Ile Leu Thr Arg Gln Glu Gly Tyr Pro Cys Ile Phe Tyr
385 390 395 400
Gly Asp Tyr Tyr Gly Ile Pro Lys Tyr Asn Ile Pro Ala Leu Lys Ser
405 410 415
Lys Leu Asp Pro Leu Leu Ile Ala Arg Arg Asp Tyr Ala Tyr Gly Thr
420 425 430
Gln His Asp Tyr Ile Asp Asn Ala Asp Ile Ile Gly Trp Thr Arg Glu
435 440 445
Gly Val Ala Glu Lys Ala Asn Ser Gly Leu Ala Ala Leu Ile Thr Asp
450 455 460
Gly Pro Gly Gly Ser Lys Trp Met Tyr Val Gly Lys Gln His Ala Gly
465 470 475 480
Lys Thr Phe Tyr Asp Leu Thr Gly Asn Arg Ser Asp Thr Val Thr Ile
485 490 495
Asn Ala Asp Gly Trp Gly Glu Phe Lys Val Asn Gly Gly Ser Val Ser
500 505 510
Ile Trp Val Pro Lys Thr Ser Thr Thr Ser Gln Ile Thr Phe Thr Val
515 520 525
Asn Asn Ala Thr Thr Val Trp Gly Gln Asn Val Tyr Val Val Gly Asn
530 535 540
Ile Ser Gln Leu Gly Asn
545 550
<210>9
<211>482
<212>PRT
<213>未知
<220>
<223>未知来源
<400>9
Ala Pro Val Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Asp Leu Pro
1 5 10 15
Asn Asp Gly Thr Leu Trp Thr Lys Val Lys Asn Glu Ala Thr Asn Leu
20 25 30
Ser Ser Leu Gly Ile Thr Ala Leu Trp Leu Pro Pro Ala Tyr Lys Gly
35 40 45
Thr Ser Gln Ser Asp Val Gly Tyr Gly Val Tyr Asp Leu Tyr Asp Leu
50 55 60
Gly Glu Phe Asn Gln Lys Gly Thr Ile Arg Thr Lys Tyr Gly Thr Lys
65 70 75 80
Ala Gln Tyr Ile Gln Ala Ile Gln Ala Ala Lys Ala Ala Gly Met Gln
85 90 95
Val Tyr Ala Asp Val Val Phe Asn His Lys Ala Gly Ala Asp Gly Thr
100 105 110
Glu Phe Val Asp Ala Val Glu Val Asn Pro Ser Asn Arg Asn Gln Glu
115 120 125
Thr Ser Gly Thr Tyr Gln Ile Gln Ala Trp Thr Lys Phe Asp Phe Pro
130 135 140
Gly Arg Gly Asn Thr Tyr Ser Ser Phe Lys Trp Arg Trp Tyr His Phe
145 150 155 160
Asp Gly Thr Asp Trp Asp Glu Ser Arg Lys Leu Asn Arg Ile Tyr Lys
165 170 175
Phe Arg Gly Thr Gly Lys Ala Trp Asp Trp Glu Val Asp Thr Glu Asn
180 185 190
Gly Asn Tyr Asp Tyr Leu Met Phe Ala Asp Leu Asp Met Asp His Pro
195 200 205
Glu Val Val Thr Glu Leu Lys Asn Trp Gly Lys Trp Tyr Val Asn Thr
210 215 220
Thr Asn Val Asp Gly Phe Arg Leu Asp Ala Val Lys His Ile Lys Tyr
225 230 235 240
Ser Phe Phe Pro Asp Trp Leu Thr Tyr Val Arg Asn Gln Thr Gly Lys
245 250 255
Asn Leu Phe Ala Val Gly Glu Phe Trp Ser Tyr Asp Val Asn Lys Leu
260 265 270
His Asn Tyr lle Thr Lys Thr Asn Gly Ser Met Ser Leu Phe Asp Ala
275 280 285
Pro Leu His Asn Asn Phe Tyr Ile Ala Ser Lys Ser Ser Gly Tyr Phe
290 295 300
Asp Met Arg Tyr Leu Leu Asn Asn Thr Leu Met Lys Asp Gln Pro Ser
305 310 315 320
Leu Ala Val Thr Leu Val Asp Asn His Asp Thr Gln Pro Gly Gln Ser
325 330 335
Leu Gln Ser Trp Val Glu Ala Trp Phe Lys Pro Leu Ala Tyr Ala Phe
340 345 350
Ile Leu Thr Arg Gln Glu Gly Tyr Pro Cys Val Phe Tyr Gly Asp Tyr
355 360 365
Tyr Gly Ile Pro Lys Tyr Asn Ile Pro Gly Leu Lys Ser Lys Ile Asp
370 375 380
Pro Leu Leu Ile Ala Arg Arg Asp Tyr Ala Tyr Gly Thr Gln Arg Asp
385 390 395 400
Tyr Ile Asp His Gln Asp Ile Ile Gly Trp Thr Arg Glu Gly Ile Asp
405 410 415
Ala Lys Pro Asn Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro Gly
420 425 430
Gly Ser Lys Trp Met Tyr Val Gly Lys Lys His Ala Gly Lys Val Phe
435 440 445
Tyr Asp Leu Thr Gly Asn Arg Ser Asp Thr Val Thr Ile Asn Ala Asp
450 455 460
Gly Trp Gly Glu Phe Lys Val Asn Gly Gly Ser Val Ser Ile Trp Val
465 470 475 480
Ala Lys
<210>10
<211>482
<212>PRT
<213>未知
<220>
<223>来源未知
<400>10
Ala Pro Val Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Asp Leu Pro
1 5 10 15
Asn Asp Gly Thr Leu Trp Thr Lys Val Lys Asn Glu Ala Thr Asn Leu
20 25 30
Ser Ser Leu Gly Ile Thr Ala Leu Trp Leu Pro Pro Ala Tyr Lys Gly
35 40 45
Thr Ser Gln Ser Asp Val Gly Tyr Gly Val Tyr Asp Leu Tyr Asp Leu
50 55 60
Gly Glu Phe Asn Gln Lys Gly Thr Ile Arg Thr Lys Tyr Gly Thr Lys
65 70 75 80
Thr Gln Tyr Ile Gln Ala Ile Gln Thr Ala Gln Ala Ala Gly Met Gln
85 90 95
Val Tyr Ala Asp Val Val Phe Asn His Lys Ala Gly Ala Asp Ser Thr
100 105 110
Glu Phe Val Asp Ala Val Glu Val Asn Pro Ser Asn Arg Asn Gln Glu
115 120 125
Thr Ser Gly Thr Tyr Gln Ile Gln Ala Trp Thr Lys Phe Asp Phe Pro
130 135 140
Gly Arg Gly Asn Thr Tyr Ser Ser Phe Lys Trp Arg Trp Tyr His Phe
145 150 155 160
Asp Gly Thr Asp Trp Asp Glu Ser Arg Lys Leu Asn Arg Ile Tyr Lys
165 170 175
Phe Arg Gly Thr Gly Lys Ala Trp Asp Trp Glu Val Asp Thr Glu Asn
180 185 190
Gly Asn Tyr Asp Tyr Leu Met Phe Ala Asp Leu Asp Met Asp His Pro
195 200 205
Glu Val Val Thr Glu Leu Lys Asn Trp Gly Thr Trp Tyr Val Asn Thr
210 215 220
Thr Asn Ile Asp Gly Phe Arg Leu Asp Ala Val Lys His Ile Lys Tyr
225 230 235 240
Ser Phe Phe Pro Asp Trp Leu Thr Tyr Val Arg Asn Gln Thr Gly Lys
245 250 255
Asn Leu Phe Ala Val Gly Glu Phe Trp Ser Tyr Asp Val Asn Lys Leu
260 265 270
His Asn Tyr Ile Thr Lys Thr Asn Gly Ser Met Ser Leu Phe Asp Ala
275 280 285
Pro Leu His Asn Asn Phe Tyr Thr Ala Ser Lys Ser Ser Gly Tyr Phe
290 295 300
Asp Met Arg Tyr Leu Leu Asn Asn Thr Leu Met Lys Asp Gln Pro Ser
305 310 315 320
Leu Ala Val Thr Leu Val Asp Asn His Asp Thr Gln Pro Gly Gln Ser
325 330 335
Leu Gln Ser Trp Val Glu Pro Trp Phe Lys Gln Leu Ala Tyr Ala Phe
340 345 350
Ile Leu Thr Arg Gln Glu Gly Tyr Pro Cys Val Phe Tyr Gly Asp Tyr
355 360 365
Tyr Gly Ile Pro Lys Tyr Asn Ile Pro Gly Leu Lys Ser Lys Ile Asp
370 375 380
Pro Leu Leu Ile Ala Arg Arg Asp Tyr Ala Tyr Gly Thr Gln Arg Asp
385 390 395 400
Tyr Ile Asp His Gln Asp Ile Ile Gly Trp Thr Arg Glu Gly Ile Asp
405 410 415
Ala Lys Pro Asn Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro Gly
420 425 430
Gly Ser Lys Trp Met Tyr Val Gly Lys Lys His Ala Gly Lys Val Phe
435 440 445
Tyr Asp Leu Thr Gly Asn Arg Ser Asp Thr Val Thr Ile Asn Ala Asp
450 455 460
Gly Trp Gly Glu Phe Lys Val Asn Gly Gly Ser Val Ser Ile Trp Val
465 470 475 480
Ala Lys
<210>11
<211>482
<212>PRT
<213>未知
<220>
<223>来源未知
<400>11
Ala Pro Val Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Asp Leu Pro
1 5 10 15
Asn Asp Gly Thr Leu Trp Thr Lys Val Lys Asn Glu Ala Ser Ser Leu
20 25 30
Ser Ser Leu Gly Ile Thr Ala Leu Trp Leu Pro Pro Ala Tyr Lys Gly
35 40 45
Thr Ser Gln Gly Asp Val Gly Tyr Gly Val Tyr Asp Leu Tyr Asp Leu
50 55 60
Gly Glu Phe Asn Gln Lys Gly Thr Ile Arg Thr Lys Tyr Gly Thr Lys
65 70 75 80
Thr Gln Tyr Leu Gln Ala Ile Gln Ala Ala Lys Ser Ala Gly Met Gln
85 90 95
Val Tyr Ala Asp Val Val Phe Asn His Lys Ala Gly Ala Asp Ser Thr
100 105 110
Glu Trp Val Asp Ala Val Glu Val Asn Pro Ser Asn Arg Asn Gln Glu
115 120 125
Thr Ser Gly Thr Tyr Gln Ile Gln Ala Trp Thr Lys Phe Asp Phe Pro
130 135 140
Gly Arg Gly Asn Thr Tyr Ser Ser Phe Lys Trp Arg Trp Tyr His Phe
145 150 155 160
Asp Gly Thr Asp Trp Asp Glu Ser Arg Lys Leu Asn Arg Ile Tyr Lys
165 170 175
Phe Arg Gly Thr Gly Lys Ala Trp Asp Trp Glu Val Asp Thr Glu Asn
180 185 190
Gly Asn Tyr Asp Tyr Leu Met Phe Ala Asp Leu Asp Met Asp His Pro
195 200 205
Glu Val Val Thr Glu Leu Lys Asn Trp Gly Thr Trp Tyr Val Asn Thr
210 215 220
Thr Asn Val Asp Gly Phe Arg Leu Asp Ala Val Lys His Ile Lys Tyr
225 230 235 240
Ser Phe Phe Pro Asp Trp Leu Thr His Val Arg Ser Gln Thr Arg Lys
245 250 255
Asn Leu Phe Ala Val Gly Glu Phe Trp Ser Tyr Asp Val Asn Lys Leu
260 265 270
His Asn Tyr Ile Thr Lys Thr Ser Gly Thr Met Ser Leu Phe Asp Ala
275 280 285
Pro Leu His Asn Asn Phe Tyr Thr Ala Ser Lys Ser Ser Gly Tyr Phe
290 295 300
Asp Met Arg Tyr Leu Leu Asn Asn Thr Leu Met Lys Asp Gln Pro ser
305 310 315 320
Leu Ala Val Thr Leu Val Asp Asn His Asp Thr Gln Pro Gly Gln Ser
325 330 335
Leu Gln Ser Trp Val Glu Pro Trp Phe Lys Pro Leu Ala Tyr Ala Phe
340 345 350
Ile Leu Thr Arg Gln Glu Gly Tyr Pro Cys Val Phe Tyr Gly Asp Tyr
355 360 365
Tyr Gly Ile Pro Lys Tyr Asn Ile Pro Gly Leu Lys Ser Lys Ile Asp
370 375 380
Pro Leu Leu Ile Ala Arg Arg Asp Tyr Ala Tyr Gly Thr Gln Arg Asp
385 390 395 400
Tyr Ile Asp His Gln Asp Ile Ile Gly Trp Thr Arg Glu Gly Ile Asp
405 410 415
Ser Lys Pro Asn Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro Gly
420 425 430
Gly Ser Lys Trp Met Tyr Val Gly Lys Lys His Ala Gly Lys Val Phe
435 440 445
Tyr Asp Leu Thr Gly Asn Arg Ser Asp Thr Val Thr Ile Asn Ala Asp
450 455 460
Gly Trp Gly Glu Phe Lys Val Asn Gly Gly Ser Val Ser Ile Trp Val
465 470 475 480
Ala Lys
<210>12
<211>482
<212>PRT
<213>未知
<220>
<223>来源未知
<400>12
Ala Pro Val Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Asp Leu Pro
1 5 10 15
Asn Asp Gly Thr Leu Trp Thr Lys Val Lys Asn Glu Ala Ser Ser Leu
20 25 30
Ser Ser Leu Gly Ile Thr Ala Leu Trp Leu Pro Pro Ala Tyr Lys Gly
35 40 45
Thr Ser Gln Gly Asp Val Gly Tyr Gly Val Tyr Asp Leu Tyr Asp Leu
50 55 60
Gly Glu Phe Asn Gln Lys Gly Thr Ile Arg Thr Lys Tyr Gly Thr Lys
65 70 75 80
Thr Gln Tyr Leu Gln Ala Ile Gln Ala Ala Lys Ser Ala Gly Met Gln
85 90 95
Val Tyr Ala Asp Val Val Phe Asn His Lys Ala Gly Ala Asp Ser Thr
100 105 110
Glu Trp Val Asp Ala Val Glu Val Asn Pro Ser Asn Arg Asn Gln Glu
115 120 125
Thr Ser Gly Thr Tyr Gln Ile Gln Ala Trp Thr Lys Phe Asp Phe Pro
130 135 140
Asp Arg Gly Asn Thr Tyr Ser Ser Phe Lys Trp Arg Trp Tyr His Phe
145 150 155 160
Asp Gly Thr Asp Trp Asp Glu Ser Arg Lys Leu Asn Arg Ile Tyr Lys
165 170 175
Phe Arg Gly Thr Gly Lys Ala Trp Asp Trp Glu Val Asp Thr Glu Asn
180 185 190
Gly Asn Tyr Asp Tyr Leu Met Phe Ala Asp Leu Asp Met Asp His Pro
195 200 205
Glu Val Val Thr Glu Leu Lys Asn Trp Gly Thr Trp Tyr Val Asn Thr
210 215 220
Thr Asn Val Asp Gly Phe Arg Leu Asp Ala Val Lys His Ile Lys Tyr
225 230 235 240
Ser Phe Phe Pro Asp Trp Leu Thr Tyr Val Arg Ser Gln Thr Gln Lys
245 250 255
Asn Leu Phe Ala Val Gly Glu Phe Trp Ser Tyr Asp Val Asn Lys Leu
260 265 270
His Asn Tyr Ile Thr Lys Thr Ser Gly Thr Met Ser Leu Phe Asp Ala
275 280 285
Pro Leu His Asn Asn Phe Tyr Thr Ala Ser Lys Ser Ser Gly Tyr Phe
290 295 300
Asp Met Arg Tyr Leu Leu Asn Asn Thr Leu Met Lys Asp Gln Pro Ser
305 310 315 320
Leu Ala Val Thr Leu Val Asp Asn His Asp Thr Gln Pro Gly Gln Ser
325 330 335
Leu Gln Ser Trp Val Glu Pro Trp Phe Lys Pro Leu Ala Tyr Ala Phe
340 345 350
Ile Leu Thr Arg Gln Glu Gly Tyr Pro Cys Val Phe Tyr Gly Asp Tyr
355 360 365
Tyr Gly Ile Pro Lys Tyr Asn Ile Pro Gly Leu Lys Ser Lys Ile Asp
370 375 380
Pro Leu Leu Ile Ala Arg Arg Asp Tyr Ala Tyr Gly Thr Gln Arg Asp
385 390 395 400
Tyr Ile Asp His Gln Asp Ile Ile Gly Trp Thr Arg Glu Gly Ile Asp
405 410 415
Ser Lys Pro Asn Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro Gly
420 425 430
Gly Ser Lys Trp Met Tyr Val Gly Lys Lys His Ala Gly Lys Val Phe
435 440 445
Tyr Asp Leu Thr Gly Asn Arg Ser Asp Thr Val Thr Ile Asn Ala Asp
450 455 460
Gly Trp Gly Glu Phe Lys Val Asn Gly Gly Ser Val Ser Ile Trp Val
465 470 475 480
Ala Lys
<210>13
<211>483
<212>PRT
<213>地衣芽孢杆菌(Bacillus licheniformis)
<400>13
Ala Asn Leu Asn Gly Thr Leu Met Gln Tyr Phe Glu Trp Tyr Met Pro
1 5 10 15
Asn Asp Gly Gln His Trp Arg Arg Leu Gln Asn Asp Ser Ala Tyr Leu
20 25 30
Ala Glu His Gly Ile Thr Ala Val Trp Ile Pro Pro Ala Tyr Lys Gly
35 40 45
Thr Ser Gln Ala Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr Asp Leu
50 55 60
Gly Glu Phe His Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr Lys
65 70 75 80
Gly Glu Leu Gln Ser Ala Ile Lys Ser Leu His Ser Arg Asp Ile Asn
85 90 95
Val Tyr Gly Asp Val Val Ile Asn His Lys Gly Gly Ala Asp Ala Thr
100 105 110
Glu Asp Val Thr Ala Val Glu Val Asp Pro Ala Asp Arg Asn Arg Val
115 120 125
Ile Ser Gly Glu His Leu Ile Lys Ala Trp Thr His Phe His Phe Pro
130 135 140
Gly Arg Gly Ser Thr Tyr Ser Asp Phe Lys Trp His Trp Tyr His Phe
145 150 155 160
Asp Gly Thr Asp Trp Asp Glu Ser Arg Lys Leu Asn Arg Ile Tyr Lys
165 170 175
Phe Gln Gly Lys Ala Trp Asp Trp Glu Val Ser Asn Glu Asn Gly Asn
180 185 190
Tyr Asp Tyr Leu Met Tyr Ala Asp Ile Asp Tyr Asp His Pro Asp Val
195 200 205
Ala Ala Glu Ile Lys Arg Trp Gly Thr Trp Tyr Ala Asn Glu Leu Gln
210 215 220
Leu Asp Gly Phe Arg Leu Asp Ala Val Lys His Ile Lys Phe Ser Phe
225 230 235 240
Leu Arg Asp Trp Val Asn His Val Arg Glu Lys Thr Gly Lys Glu Met
245 250 255
Phe Thr Val Ala Glu Tyr Trp Gln Asn Asp Leu Gly Ala Leu Glu Asn
260 265 270
Tyr Leu Asn Lys Thr Asn Phe Asn His Ser Val Phe Asp Val Pro Leu
275 280 285
His Tyr Gln Phe His Ala Ala Ser Thr Gln Gly Gly Gly Tyr Asp Met
290 295 300
Arg Lys Leu Leu Asn Gly Thr Val Val Ser Lys His Pro Leu Lys Ser
305 310 315 320
Val Thr Phe Val Asp Asn His Asp Thr Gln Pro Gly Gln Ser Leu Glu
325 330 335
Ser Thr Val Gln Thr Trp Phe Lys Pro Leu Ala Tyr Ala Phe Ile Leu
340 345 350
Thr Arg Glu Ser Gly Tyr Pro Gln Val Phe Tyr Gly Asp Met Tyr Gly
355 360 365
Thr Lys Gly Asp Ser Gln Arg Glu Ile Pro Ala Leu Lys His Lys Ile
370 375 380
Glu Pro Ile Leu Lys Ala Arg Lys Gln Tyr Ala Tyr Gly Ala Gln His
385 390 395 400
Asp Tyr Phe Asp His His Asp Ile Val Gly Trp Thr Arg Glu Gly Asp
405 410 415
Ser Ser Val Ala Asn Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro
420 425 430
Gly Gly Ala Lys Arg Met Tyr Val Gly Arg Gln Asn Ala Gly Glu Thr
435 440 445
Trp His Asp Ile Thr Gly Asn Arg Ser Glu Pro Val Val Ile Asn Ser
450 455 460
Glu Gly Trp Gly Glu Phe His Val Asn Gly Gly Ser Val Ser Ile Tyr
465 470 475 480
Val Gln Arg
<210>14
<211>483
<212>PRT
<213>解淀粉芽孢杆菌(Bacillus amyloliquefacience)
<400>14
Val Asn Gly Thr Leu Met Gln Tyr Phe Glu Trp Tyr Thr Pro Asn Asp
1 5 10 15
Gly Gln His Trp Lys Arg Leu Gln Asn Asp Ala Glu His Leu Ser Asp
20 25 30
Ile Gly Ile Thr Ala Val Trp Ile Pro Pro Ala Tyr Lys Gly Leu Ser
35 40 45
Gln Ser Asp Asn Gly Tyr Gly Pro Tyr Asp Leu Tyr Asp Leu Gly Glu
50 55 60
Phe Gln Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr Lys Ser Glu
65 70 75 80
Leu Gln Asp Ala Ile Gly Ser Leu His Ser Arg Asn Val Gln Val Tyr
85 90 95
Gly Asp Val Val Leu Asn His Lys Ala Gly Ala Asp Ala Thr Glu Asp
100 105 110
Val Thr Ala Val Glu Val Asn Pro Ala Asn Arg Asn Gln Glu Thr Ser
115 120 125
Glu Glu Tyr Gln Ile Lys Ala Trp Thr Asp Phe Arg Phe Pro Gly Arg
130 135 140
Gly Asn Thr Tyr Ser Asp Phe Lys Trp His Trp Tyr His Phe Asp Gly
145 150 155 160
Ala Asp Trp Asp Glu Ser Arg Lys Ile Ser Arg Ile Phe Lys Phe Arg
165 170 175
Gly Glu Gly Lys Ala Trp Asp Trp Glu Val Ser Ser Glu Asn Gly Asn
180 185 190
Tyr Asp Tyr Leu Met Tyr Ala Asp Val Asp Tyr Asp His Pro Asp Val
195 200 205
Val Ala Glu Thr Lys Lys Trp Gly Ile Trp Tyr Ala Asn Glu Leu Ser
210 215 220
Leu Asp Gly Phe Arg Ile Asp Ala Ala Lys His Ile Lys Phe Ser Phe
225 230 235 240
Leu Arg Asp Trp Val Gln Ala Val Arg Gln Ala Thr Gly Lys Glu Met
245 250 255
Phe Thr Val Ala Glu Tyr Trp Gln Asn Asn Ala Gly Lys Leu Glu Asn
260 265 270
Tyr Leu Asn Lys Thr Ser Phe Asn Gln Ser Val Phe Asp Val Pro Leu
275 280 285
His Phe Asn Leu Gln Ala Ala Ser Ser Gln Gly Gly Gly Tyr Asp Met
290 295 300
Arg Arg Leu Leu Asp Gly Thr Val Val Ser Arg His Pro Glu Lys Ala
305 310 315 320
Val Thr Phe Val Glu Asn His Asp Thr Gln Pro Gly Gln Ser Leu Glu
325 330 335
Ser Thr Val Gln Thr Trp Phe Lys Pro Leu Ala Tyr Ala Phe Ile Leu
340 345 350
Thr Arg Glu Ser Gly Tyr Pro Gln Val Phe Tyr Gly Asp Met Tyr Gly
355 360 365
Thr Lys Gly Thr Ser Pro Lys Glu Ile Pro Ser Leu Lys Asp Asn Ile
370 375 380
Glu Pro Ile Leu Lys Ala Arg Lys Glu Tyr Ala Tyr Gly Pro Gln His
385 390 395 400
Asp Tyr Ile Asp His Pro Asp Val Ile Gly Trp Thr Arg Glu Gly Asp
405 410 415
Ser Ser Ala Ala Lys Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro
420 425 430
Gly Gly Ser Lys Arg Met Tyr Ala Gly Leu Lys Asn Ala Gly Glu Thr
435 440 445
Trp Tyr Asp Ile Thr Gly Asn Arg Ser Asp Thr Val Lys Ile Gly Ser
450 455 460
Asp Gly Trp Gly Glu Phe His Val Asn Asp Gly Ser Val Ser Ile Tyr
465 470 475 480
Val Gln Lys
<210>15
<211>483
<212>PRT
<213>嗜热脂肪芽孢杆菌(Bacillus stearothermophilus)
<400>15
Ala Ala Pro Phe Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr Leu
1 5 10 15
Pro Asp Asp Gly Thr Leu Trp Thr Lys Val Ala Asn Glu Ala Asn Asn
20 25 30
Leu Ser Ser Leu Gly Ile Thr Ala Leu Trp Leu Pro Pro Ala Tyr Lys
35 40 45
Gly Thr Ser Arg Ser Asp Val Gly Tyr Gly Val Tyr Asp Leu Tyr Asp
50 55 60
Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr
65 70 75 80
Lys Ala Gln Tyr Leu Gln Ala Ile Gln Ala Ala His Ala Ala Gly Met
85 90 95
Gln Val Tyr Ala Asp Val Val Phe Asp His Lys Gly Gly Ala Asp Gly
100 105 110
Thr Glu Trp Val Asp Ala Val Glu Val Asn Pro Ser Asp Arg Asn Gln
115 120 125
Glu Ile Ser Gly Thr Tyr Gln Ile Gln Ala Trp Thr Lys Phe Asp Phe
130 135 140
Pro Gly Arg Gly Asn Thr Tyr Ser Ser Phe Lys Trp Arg Trp Tyr His
145 150 155 160
Phe Asp Gly Val Asp Trp Asp Glu Ser Arg Lys Leu Ser Arg Ile Tyr
165 170 175
Lys Phe Arg Gly Ile Gly Lys Ala Trp Asp Trp Glu Val Asp Thr Glu
180 185 190
Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Leu Asp Met Asp His
195 200 205
Pro Glu Val Val Thr Glu Leu Lys Asn Trp Gly Lys Trp Tyr Val Asn
210 215 220
Thr Thr Asn Ile Asp Gly Phe Arg Leu Asp Ala Val Lys His Ile Lys
225 230 235 240
Phe Ser Phe Phe Pro Asp Trp Leu Ser Tyr Val Arg Ser Gln Thr Gly
245 250 255
Lys Pro Leu Phe Thr Val Gly Glu Tyr Trp Ser Tyr Asp Ile Asn Lys
260 265 270
Leu His Asn Tyr Ile Thr Lys Thr Asn Gly Thr Met Ser Leu Phe Asp
275 280 285
Ala Pro Leu His Asn Lys Phe Tyr Thr Ala Ser Lys Ser Gly Gly Ala
290 295 300
Phe Asp Met Arg Thr Leu Met Thr Asn Thr Leu Met Lys Asp Gln Pro
305 310 315 320
Thr Leu Ala Val Thr Phe Val Asp Asn His Asp Thr Glu Pro Gly Gln
325 330 335
Ala Leu Gln Ser Trp Val Asp Pro Trp Phe Lys Pro Leu Ala Tyr Ala
340 345 350
Phe Ile Leu Thr Arg Gln Glu Gly Tyr Pro Cys Val Phe Tyr Gly Asp
355 360 365
Tyr Tyr Gly Ile Pro Gln Tyr Asn Ile Pro Ser Leu Lys Ser Lys Ile
370 375 380
Asp Pro Leu Leu Ile Ala Arg Arg Asp Tyr Ala Tyr Gly Thr Gln His
385 390 395 400
Asp Tyr Leu Asp His Ser Asp Ile Ile Gly Trp Thr Arg Glu Gly Val
405 410 415
Thr Glu Lys Pro Gly Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro
420 425 430
Gly Gly Ser Lys Trp Met Tyr Val Gly Lys Gln His Ala Gly Lys Val
435 440 445
Phe Tyr Asp Leu Thr Gly Asn Arg Ser Asp Thr Val Thr Ile Asn Ser
450 455 460
Asp Gly Trp Gly Glu Phe Lys Val Asn Gly Gly Ser Val Ser Val Trp
465 470 475 480
Val Pro Arg
<210>16
<211>485
<212>PRT
<213>未知
<220>
<223>来源未知
<400>16
His His Asn Gly Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr
1 5 10 15
Leu Pro Asn Asp Gly Asn His Trp Asn Arg Leu Arg Ser Asp Ala Ser
20 25 30
Asn Leu Lys Asp Lys Gly Ile Ser Ala Val Trp Ile Pro Pro Ala Trp
35 40 45
Lys Gly Ala Ser Gln Asn Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr
50 55 60
Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Ile Arg Thr Lys Tyr Gly
65 70 75 80
Thr Arg Asn Gln Leu Gln Ala Ala Val Asn Ala Leu Lys Ser Asn Gly
85 90 95
Ile Gln Val Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp
100 105 110
Ala Thr Glu Met Val Arg Ala Val Glu Val Asn Pro Asn Asn Arg Asn
115 120 125
Gln Glu Val Ser Gly Glu Tyr Thr Ile Glu Ala Trp Thr Lys Phe Asp
130 135 140
Phe Pro Gly Arg Gly Asn Thr His Ser Asn Phe Lys Trp Arg Trp Tyr
145 150 155 160
His Phe Asp Gly Val Asp Trp Asp Gln Ser Arg Lys Leu Asn Asn Arg
165 170 175
Ile Tyr Lys Phe Arg Gly Asp Gly Lys Gly Trp Asp Trp Glu Val Asp
l80 185 190
Thr Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Ile Asp Met
195 200 205
Asp His Pro Glu Val Val Asn Glu Leu Arg Asn Trp Gly Val Trp Tyr
210 215 220
Thr Asn Thr Leu Gly Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His
225 230 235 240
Ile Lys Tyr Ser Phe Thr Arg Asp Trp Ile Asn His Val Arg Ser Ala
245 250 255
Thr Gly Lys Asn Met Phe Ala Val Ala Glu Phe Trp Lys Asn Asp Leu
260 265 270
Gly Ala Ile Glu Asn Tyr Leu Asn Lys Thr Asn Trp Asn His Ser Val
275 280 285
Phe Asp Val Pro Leu His Tyr Asn Leu Tyr Asn Ala Ser Lys Ser Gly
290 295 300
Gly Asn Tyr Asp Met Arg Gln Ile Phe Asn Gly Thr Val Val Gln Arg
305 310 315 320
His Pro Met His Ala Val Thr Phe Val Asp Asn His Asp Ser Gln Pro
325 330 335
Glu Glu Ala Leu Glu Ser Phe Val Glu Glu Trp Phe Lys Pro Leu Ala
340 345 350
Tyr Ala Leu Thr Leu Thr Arg Glu Gln Gly Tyr Pro Ser Val Phe Tyr
355 360 365
Gly Asp Tyr Tyr Gly Ile Pro Thr His Gly Val Pro Ala Met Lys Ser
370 375 380
Lys Ile Asp Pro Ile Leu Glu Ala Arg Gln Lys Tyr Ala Tyr Gly Arg
385 390 395 400
Gln Asn Asp Tyr Leu Asp His His Asn Ile Ile Gly Trp Thr Arg Glu
405 410 415
Gly Asn Thr Ala His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp
420 425 430
Gly Ala Gly Gly Asn Lys Trp Met Phe Val Gly Arg Asn Lys Ala Gly
435 440 445
Gln Val Trp Thr Asp Ile Thr Gly Asn Arg Ala Gly Thr Val Thr Ile
450 455 460
Asn Ala Asp Gly Trp Gly Asn Phe Ser Val Asn Gly Gly Ser Val Ser
465 470 475 480
Ile Trp Val Asn Lys
485
<210>17
<211>484
<212>PRT
<213>未知
<220>
<223>来源未知
<400>17
Gly Ser Val Pro Val Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr
1 5 10 15
Leu Pro Asp Asp Gly Thr Leu Trp Thr Lys Val Ala Asn Asn Ala Gln
20 25 30
Ser Leu Ala Asn Leu Gly Ile Thr Ala Leu Trp Leu Pro Pro Ala Tyr
35 40 45
Lys Gly Thr Ser Ser Ser Asp Val Gly Tyr Gly Val Tyr Asp Leu Tyr
50 55 60
Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly
65 70 75 80
Thr Lys Thr Gln Tyr Ile Gln Ala Ile Gln Ala Ala His Thr Ala Gly
85 90 95
Met Gln Val Tyr Ala Asp Val Val Phe Asn His Lys Ala Gly Ala Asp
100 105 110
Gly Thr Glu Leu Val Asp Ala Val Glu Val Asn Pro Ser Asp Arg Asn
115 120 125
Gln Glu Ile Ser Gly Thr Tyr Gln Ile Gln Ala Trp Thr Lys Phe Asp
130 135 140
Phe Pro Gly Arg Gly Asn Thr Tyr Ser Ser Phe Lys Trp Arg Trp Tyr
145 150 155 160
His Phe Asp Gly Thr Asp Trp Asp Glu Ser Arg Lys Leu Asn Arg Ile
165 170 175
Tyr Lys Phe Arg Gly Thr Gly Lys Ala Trp Asp Trp Glu Val Asp Thr
180 185 190
Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Leu Asp Met Asp
195 200 205
His Pro Glu Val Val Ser Glu Leu Lys Asn Trp Gly Lys Trp Tyr Val
210 215 220
Thr Thr Thr Asn Ile Asp Gly Phe Arg Leu Asp Ala Val Lys His Ile
225 230 235 240
Lys Tyr Ser Phe Phe Pro Asp Trp Leu Ser Tyr Val Arg Thr Gln Thr
245 250 255
Gln Lys Pro Leu Phe Ala Val Gly Glu Phe Trp Ser Tyr Asp Ile Asn
260 265 270
Lys Leu His Asn Tyr Ile Thr Lys Thr Asn Gly Ser Met Ser Leu Phe
275 280 285
Asp Ala Pro Leu His Asn Asn Phe Tyr Ile Ala Ser Lys Ser Gly Gly
290 295 300
Tyr Phe Asp Met Arg Thr Leu Leu Asn Asn Thr Leu Met Lys Asp Gln
305 310 315 320
Pro Thr Leu Ser Val Thr Leu Val Asp Asn His Asp Thr Glu Pro Gly
325 330 335
Gln Ser Leu Gln Ser Trp Val Glu Pro Trp Phe Lys Pro Leu Ala Tyr
340 345 350
Ala Phe Ile Leu Thr Arg Gln Glu Gly Tyr Pro Cys Ile Phe Tyr Gly
355 360 365
Asp Tyr Tyr Gly Ile Pro Lys Tyr Asn Ile Pro Ala Leu Lys Ser Lys
370 375 380
Leu Asp Pro Leu Leu Ile Ala Arg Arg Asp Tyr Ala Tyr Gly Thr Gln
385 390 395 400
His Asp Tyr Ile Asp Asn Ala Asp Ile Ile Gly Trp Thr Arg Glu Gly
405 410 415
Val Ala Glu Lys Ala Asn Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly
420 425 430
Pro Gly Gly Ser Lys Trp Met Tyr Val Gly Lys Gln His Ala Gly Lys
435 440 445
Thr Phe Tyr Asp Leu Thr Gly Asn Arg Ser Asp Thr Val Thr Ile Asn
450 455 460
Ala Asp Gly Trp Gly Glu Phe Lys Val Asn Gly Gly Ser Val Ser Ile
465 470 475 480
Trp Val Pro Lys
<210>18
<211>485
<212>PRT
<213>未知
<220>
<223>来源未知
<400>18
Ala Asn Thr Ala Pro Ile Asn Glu Thr Met Met Gln Tyr Phe Glu Trp
1 5 10 15
Asp Leu Pro Asn Asp Gly Thr Leu Trp Thr Lys Val Lys Asn Glu Ala
20 25 30
Ala Asn Leu Ser Ser Leu Gly Ile Thr Ala Leu Trp Leu Pro Pro Ala
35 40 45
Tyr Lys Gly Thr Ser Gln Ser Asp Val Gly Tyr Gly Val Tyr Asp Leu
50 55 60
Tyr Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Ile Arg Thr Lys Tyr
65 70 75 80
Gly Thr Lys Thr Gln Tyr Ile Gln Ala Ile Gln Ala Ala Lys Ala Ala
85 90 95
Gly Met Gln Val Tyr Ala Asp Val Val Phe Asn His Lys Ala Gly Ala
100 105 110
Asp Gly Thr Glu Phe Val Asp Ala Val Glu Val Asp Pro Ser Asn Arg
115 120 125
Asn Gln Glu Thr Ser Gly Thr Tyr Gln Ile Gln Ala Trp Thr Lys Phe
130 135 140
Asp Phe Pro Gly Arg Gly Asn Thr Tyr Ser Ser Phe Lys Trp Arg Trp
145 150 155 160
Tyr His Phe Asp Gly Thr Asp Trp Asp Glu Ser Arg Lys Leu Asn Arg
165 170 175
Ile Tyr Lys Phe Arg Ser Thr Gly Lys Ala Trp Asp Trp Glu Val Asp
180 185 190
Thr Glu Asn Gly Asn Tyr Asp Tyr Leu Met Phe Ala Asp Leu Asp Met
195 200 205
Asp His Pro Glu Val Val Thr Glu Leu Lys Asn Trp Gly Thr Trp Tyr
210 215 220
Val Asn Thr Thr Asn Ile Asp Gly Phe Arg Leu Asp Ala Val Lys His
225 230 235 240
Ile Lys Tyr Ser Phe Phe Pro Asp Trp Leu Thr Tyr Val Arg Asn Gln
245 250 255
Thr Gly Lys Asn Leu Phe Ala Val Gly Glu Phe Trp Ser Tyr Asp Val
260 265 270
Asn Lys Leu His Asn Tyr Ile Thr Lys Thr Asn Gly Ser Met Ser Leu
275 280 285
Phe Asp Ala Pro Leu His Asn Asn Phe Tyr Thr Ala Ser Lys Ser Ser
290 295 300
Gly Tyr Phe Asp Met Arg Tyr Leu Leu Asn Asn Thr Leu Met Lys Asp
305 310 315 320
Gln Pro Ser Leu Ala Val Thr Leu Val Asp Asn His Asp Thr Gln Pro
325 330 335
Gly Gln Ser Leu Gln Ser Trp Val Glu Pro Trp Phe Lys Pro Leu Ala
340 345 350
Tyr Ala Phe Ile Leu Thr Arg Gln Glu Gly Tyr Pro Cys Val Phe Tyr
355 360 365
Gly Asp Tyr Tyr Gly Ile Pro Lys Tyr Asn Ile Pro Gly Leu Lys Ser
370 375 380
Lys Ile Asp Pro Leu Leu Ile Ala Arg Arg Asp Tyr Ala Tyr Gly Thr
385 390 395 400
Gln Arg Asp Tyr Ile Asp His Gln Asp Ile Ile Gly Trp Thr Arg Glu
405 410 415
Gly Ile Asp Thr Lys Pro Asn Ser Gly Leu Ala Ala Leu Ile Thr Asp
420 425 430
Gly Pro Gly Gly Ser Lys Trp Met Tyr Val Gly Lys Lys His Ala Gly
435 440 445
Lys Val Phe Tyr Asp Leu Thr Gly Asn Arg Ser Asp Thr Val Thr Ile
450 455 460
Asn Ala Asp Gly Trp Gly Glu Phe Lys Val Asn Gly Gly Ser Val Ser
465 470 475 480
Ile Trp Val Ala Lys
485
<210>19
<211>619
<212>PRT
<213>黄热芽孢杆菌(Bacillus flavothermus)
<400>19
Met Ser Leu Phe Lys Lys Ser Phe Pro Trp Ile Leu Ser Leu Leu Leu
1 5 10 15
Leu Phe Ser Phe Ile Ala Pro Phe Ser Ile Gln Thr Glu Lys Val Arg
20 25 30
Ala Gly Ser Val Pro Val Asn Gly Thr Met Met Gln Tyr Phe Glu Trp
35 40 45
Tyr Leu Pro Asp Asp Gly Thr Leu Trp Thr Lys Val Ala Asn Asn Ala
50 55 60
Gln Ser Leu Ala Asn Leu Gly Ile Thr Ala Leu Trp Leu Pro Pro Ala
65 70 75 80
Tyr Lys Gly Thr Ser Ser Ser Asp Val Gly Tyr Gly Val Tyr Asp Leu
85 90 95
Tyr Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr
100 105 110
Gly Thr Lys Thr Gln Tyr Ile Gln Ala Ile Gln Ala Ala His Thr Ala
115 120 125
Gly Met Gln Val Tyr Ala Asp Val Val Phe Asn His Lys Ala Gly Ala
130 135 140
Asp Gly Thr Glu Leu Val Asp Ala Val Glu Val Asn Pro Ser Asp Arg
145 150 155 160
Asn Gln Glu Ile Ser Gly Thr Tyr Gln Ile Gln Ala Trp Thr Lys Phe
165 170 175
Asp Phe Pro Gly Arg Gly Asn Thr Tyr Ser Ser Phe Lys Trp Arg Trp
180 185 190
Tyr His Phe Asp Gly Thr Asp Trp Asp Glu Ser Arg Lys Leu Asn Arg
195 200 205
Ile Tyr Lys Phe Arg Gly Thr Gly Lys Ala Trp Asp Trp Glu Val Asp
210 215 220
Thr Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Leu Asp Met
225 230 235 240
Asp His Pro Glu Val Val Ser Glu Leu Lys Asn Trp Gly Lys Trp Tyr
245 250 255
Val Thr Thr Thr Asn Ile Asp Gly Phe Arg Leu Asp Ala Val Lys His
260 265 270
Ile Lys Tyr Ser Phe Phe Pro Asp Trp Leu Ser Tyr Val Arg Thr Gln
275 280 285
Thr Gln Lys Pro Leu Phe Ala Val Gly Glu Phe Trp Ser Tyr Asp Ile
290 295 300
Ser Lys Leu His Asn Tyr Ile Thr Lys Thr Asn Gly Ser Met Ser Leu
305 310 315 320
Phe Asp Ala Pro Leu His Asn Asn Phe Tyr Ile Ala Ser Lys Ser Gly
325 330 335
Gly Tyr Phe Asp Met Arg Thr Leu Leu Asn Asn Thr Leu Met Lys Asp
340 345 350
Gln Pro Thr Leu Ala Val Thr Leu Val Asp Asn His Asp Thr Glu Pro
355 360 365
Gly Gln Ser Leu Gln Ser Trp Val Glu Pro Trp Phe Lys Pro Leu Ala
370 375 380
Tyr Ala Phe Ile Leu Thr Arg Gln Glu Gly Tyr Pro Cys Val Phe Tyr
385 390 395 400
Gly Asp Tyr Tyr Gly Ile Pro Lys Tyr Asn Ile Pro Ala Leu Lys Ser
405 410 415
Lys Leu Asp Pro Leu Leu Ile Ala Arg Arg Asp Tyr Ala Tyr Gly Thr
420 425 430
Gln His Asp Tyr Ile Asp Ser Ala Asp Ile Ile Gly Trp Thr Arg Glu
435 440 445
Gly Val Ala Glu Lys Ala Asn Ser Gly Leu Ala Ala Leu Ile Thr Asp
450 455 460
Gly Pro Gly Gly Ser Lys Trp Met Tyr Val Gly Lys Gln His Ala Gly
465 470 475 480
Lys Thr Phe Tyr Asp Leu Thr Gly Asn Arg Ser Asp Thr Val Thr Ile
485 490 495
Asn Ala Asp Gly Trp Gly Glu Phe Lys Val Asn Gly Gly Ser Val Ser
500 505 510
Ile Trp Val Pro Lys Ile Ser Thr Thr Ser Gln Ile Thr Phe Thr Val
515 520 525
Asn Asn Ala Thr Thr Val Trp Gly Gln Asn Val Tyr Val Val Gly Asn
530 535 540
Ile Ser Gln Leu Gly Asn Trp Asp Pro Val His Ala Val Gln Met Thr
545 550 555 560
Pro Ser Ser Tyr Pro Thr Trp Thr Val Thr Ile Pro Leu Leu Gln Gly
565 570 575
Gln Asn Ile Gln Phe Lys Phe Ile Lys Lys Asp Ser Ala Gly Asn Val
580 585 590
Ile Trp Glu Asp Ile Ser Asn Arg Thr Tyr Thr Val Pro Thr Ala Ala
595 600 605
Ser Gly Ala Tyr Thr Ala Ser Trp Asn Val Pro
610 615
<210>20
<211>613
<212>PRT
<213>芽孢杆菌属(Bacillus)
<400>20
Met Ser Tyr Leu Lys Lys Val Trp Leu Tyr Tyr Thr Ile Ile Ala Thr
1 5 10 15
Leu Ile Ile Ser Phe Phe Thr Pro Phe Ser Thr Ala Gln Ala Asn Thr
20 25 30
Ala Pro Val Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Asp Leu Pro
35 40 45
Asn Asp Gly Thr Leu Trp Thr Lys Val Lys Asn Glu Ala Ser Ser Leu
50 55 60
Ser Ala Leu Gly Ile Thr Ala Leu Trp Leu Pro Pro Ala Tyr Lys Gly
65 70 75 80
Thr Ser Gln Ala Asp Val Gly Tyr Gly Val Tyr Asp Leu Tyr Asp Leu
85 90 95
Gly Glu Phe Asn Gln Lys Gly Thr Ile Arg Thr Lys Tyr Gly Thr Lys
100 105 110
Thr Gln Tyr Leu Gln Ala Ile Gln Ala Ala Lys Ser Ala Gly Met Gln
115 120 125
Val Tyr Ala Asp Val Val Phe Asn His Lys Ala Gly Ala Asp Ser Thr
130 135 140
Glu Trp Val Asp Ala Val Glu Val Asn Pro Ser Asn Arg Asn Gln Glu
145 150 155 160
Thr Ser Gly Thr Tyr Gln Ile Gln Ala Trp Thr Lys Phe Asp Phe Pro
165 170 175
Gly Arg Gly Asn Thr Tyr Ser Ser Phe Lys Trp Arg Trp Tyr His Phe
180 185 190
Asp Gly Thr Asp Trp Asp Glu Ser Arg Lys Leu Asn Arg Ile Tyr Lys
195 200 205
Phe Arg Gly Thr Gly Lys Ala Trp Asp Trp Glu Val Asp Thr Glu Asn
210 215 220
Gly Asn Tyr Asp Tyr Leu Met Phe Ala Asp Leu Asp Met Asp His Pro
225 230 235 240
Glu Val Val Ala Glu Leu Lys Asn Trp Gly Lys Trp Tyr Val Asn Thr
245 250 255
Thr Asn Val Asp Gly Phe Arg Leu Asp Ala Val Lys His Ile Lys Tyr
260 265 270
Ser Phe Phe Pro Asp Trp Leu Ser Tyr Val Arg Asn Gln Thr Gly Lys
275 280 285
Asn Leu Phe Ala Val Gly Glu Phe Trp Gly Tyr Asp Val Asn Lys Leu
290 295 300
His Asn Tyr Ile Thr Lys Thr Asn Gly Ala Met Ser Leu Phe Asp Ala
305 310 315 320
Pro Leu His Asn Asn Phe Tyr Ile Ala Ser Lys Ser Ser Gly Tyr Phe
325 330 335
Asp Met Arg Tyr Leu Leu Asn Asn Thr Leu Met Lys Asp Gln Pro Ala
340 345 350
Leu Ala Val Thr Leu Val Asp Asn His Asp Thr Gln Pro Gly Gln Ser
355 360 365
Leu Gln Ser Trp Val Glu Pro Trp Phe Lys Pro Leu Ala Tyr Ala Phe
370 375 380
Ile Leu Thr Arg Gln Glu Gly Tyr Pro Cys Val Phe Tyr Gly Asp Tyr
385 390 395 400
Tyr Gly Ile Pro Lys Tyr Asn Ile Pro Gly Leu Lys Ser Lys Ile Asp
405 410 415
Pro Leu Leu Ile Ala Arg Arg Asp Tyr Ala Tyr Gly Thr Gln Arg Asp
420 425 430
Tyr Ile Asp His Gln Asp Ile Ile Gly Trp Thr Arg Glu Gly Ile Asp
435 440 445
Ala Lys Pro Asn Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro Gly
450 455 460
Gly Ser Lys Trp Met Tyr Val Gly Lys Arg His Ala Gly Lys Val Phe
465 470 475 480
Tyr Asp Leu Thr Gly Asn Arg Ser Asp Thr Val Thr Ile Asn Ala Asp
485 490 495
Gly Trp Gly Glu Phe Lys Val Asn Gly Gly Ser Val Ser Ile Trp Val
500 505 510
Ala Lys Thr Ser Asn Val Thr Phe Thr Val Asn Asn Ala Thr Thr Val
515 520 525
Tyr Gly Gln Asn Val Tyr Val Val Gly Asn Ile Pro Glu Leu Gly Asn
530 535 540
Trp Asn Ile Ala Asn Ala Ile Gln Met Thr Pro Ser Ser Tyr Pro Thr
545 550 555 560
Trp Lys Thr Thr Val Ser Leu Pro Gln Gly Lys Ala Ile Glu Phe Lys
565 570 575
Phe Ile Lys Lys Asp Ser Ala Gly Asn Val Ile Trp Glu Asn Ile Ala
580 585 590
Asn Arg Thr Tyr Thr Val Pro Phe Ser Ser Thr Gly Ser Tyr Thr Ala
595 600 605
Asn Trp Asn Val Pro
610
<210>21
<211>619
<212>PRT
<213>Alkaliphilic bacillus
<400>21
Met Ser Leu Phe Lys Lys Ile Phe Pro Trp Ile Leu Ser Leu Leu Leu
1 5 10 15
Leu Phe Ser Phe Ile Ala Pro Phe Ser Ile Gln Thr Glu Lys Val Arg
20 25 30
Ala Gly Ser Val Pro Val Asn Gly Thr Met Met Gln Tyr Phe Glu Trp
35 40 45
Tyr Leu Pro Asp Asp Gly Thr Leu Trp Thr Lys Val Ala Asn Asn Ala
50 55 60
Gln Ser Leu Ala Asn Leu Gly Ile Thr Ala Leu Trp Leu Pro Pro Ala
65 70 75 80
Tyr Lys Gly Thr Ser Ser Ser Asp Val Gly Tyr Gly Val Tyr Asp Leu
85 90 95
Tyr Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr
100 105 110
Gly Thr Lys Thr Gln Tyr Ile Gln Ala Ile Gln Ala Ala His Thr Ala
115 120 125
Gly Met Gln Val Tyr Ala Asp Val Val Phe Asn His Lys Ala Gly Ala
130 135 140
Asp Gly Thr Glu Leu Val Asp Ala Val Glu Val Asn Pro Ser Asp Arg
145 150 155 160
Asn Gln Glu Ile Ser Gly Thr Tyr Gln Ile Gln Ala Trp Thr Lys Phe
165 170 175
Asp Phe Pro Gly Arg Gly Asn Thr Tyr Ser Ser Phe Lys Trp Arg Trp
180 185 190
Tyr His Phe Asp Gly Thr Asp Trp Asp Glu Ser Arg Lys Leu Asn Arg
195 200 205
Ile Tyr Lys Phe Arg Gly Thr Gly Lys Ala Trp Asp Trp Glu Val Asp
210 215 220
Thr Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Leu Asp Met
225 230 235 240
Asp His Pro Glu Val Val Ser Glu Leu Lys Asn Trp Gly Lys Trp Tyr
245 250 255
Val Ile Thr Thr Asn Ile Asp Gly Phe Arg Leu Asp Ala Val Lys His
260 265 270
Ile Lys Tyr Ser Phe Phe Pro Asp Trp Leu Ser Tyr Leu Arg Thr Gln
275 280 285
Thr Gln Lys Pro Leu Phe Ala Val Gly Glu Phe Trp Ser Tyr Asp Ile
290 295 300
Asn Lys Leu His Asn Tyr Ile Thr Lys Thr Asn Gly Ser Met Ser Leu
305 310 315 320
Phe Asp Ala Pro Leu His Asn Asn Phe Tyr Ile Ala Ser Lys Ser Gly
325 330 335
Gly Tyr Phe Asp Met Arg Thr Leu Leu Asn Asn Thr Leu Met Lys Glu
340 345 350
Gln Pro Thr Leu Ser Val Thr Leu Val Asp Asn His Asp Thr Glu Pro
355 360 365
Gly Gln Ser Leu Gln Ser Trp Val Glu Pro Trp Phe Lys Pro Leu Ala
370 375 380
Tyr Ala Phe Ile Leu Thr Arg Gln Glu Gly Tyr Pro Cys Val Phe Tyr
385 390 395 400
Gly Asp Tyr Tyr Gly Ile Pro Lys Tyr Asn Ile Pro Ala Leu Lys Ser
405 410 415
Lys Leu Asp Pro Leu Leu Ile Ala Arg Arg Asp Tyr Ala Tyr Gly Thr
420 425 430
Gln His Asp Tyr Ile Asp Asn Ala Asp Ile Ile Gly Trp Thr Arg Glu
435 440 445
Gly Val Ala Glu Lys Ala Asn Ser Gly Leu Ala Ala Leu Ile Thr Asp
450 455 460
Gly Pro Gly Gly Ser Lys Trp Met Tyr Val Gly Lys Gln His Ala Gly
465 470 475 480
Lys Thr Phe Tyr Asp Leu Thr Gly Asn Arg Ser Asp Thr Val Thr Ile
485 490 495
Asn Ala Asp Gly Trp Gly Glu Phe Lys Val Asn Gly Gly Ser Val Ser
500 505 510
Ile Trp Val Pro Lys Thr Ser Thr Thr Ser Gln Ile Thr Phe Thr Val
515 520 525
Asn Asn Ala Thr Thr Val Trp Gly Gln Asn Val Tyr Val Val Gly Asn
530 535 540
Ile Ser Gln Leu Gly Asn Trp Asp Pro Val Asn Ala Val Gln Met Thr
545 550 555 560
Pro Ser Ser Tyr Pro Thr Trp Val Val Thr Val Pro Leu Pro Gln Ser
565 570 575
Gln Asn Ile Gln Phe Lys Phe Ile Lys Lys Asp Gly Ser Gly Asn Val
580 585 590
Ile Trp Glu Asn Ile Ser Asn Arg Thr Tyr Thr Val Pro Thr Ala Ala
595 600 605
Ser Gly Ala Tyr Thr Ala Asn Trp Asn Val Pro
610 615
<210>22
<211>640
<212>PRT
<213>Aspergillus kawachii
<400>22
Met Arg Val Ser Thr Ser Ser Ile Ala Leu Ala Val Ser Leu Phe Gly
1 5 10 15
Lys Leu Ala Leu Gly Leu Ser Ala Ala Glu Trp Arg Thr Gln Ser Ile
20 25 30
Tyr Phe Leu Leu Thr Asp Arg Phe Gly Arg Thr Asp Asn Ser Thr Thr
35 40 45
Ala Thr Cys Asn Thr Gly Asp Gln Ile Tyr Cys Gly Gly Ser Trp Gln
50 55 60
Gly Ile Ile Asn His Leu Asp Tyr Ile Gln Gly Met Gly Phe Thr Ala
65 70 75 80
Ile Trp Ile Ser Pro Ile Thr Glu Gln Leu Pro Gln Asp Thr Ser Asp
85 90 95
Gly Glu Ala Tyr His Gly Tyr Trp Gln Gln Lys Ile Tyr Tyr Val Asn
100 105 110
Ser Asn Phe Gly Thr Ala Asp Asp Leu Lys Ser Leu Ser Asp Ala Leu
115 120 125
His Ala Arg Gly Met Tyr Leu Met Val Asp Val Val Pro Asn His Met
130 135 140
Gly Tyr Ala Gly Asn Gly Asn Asp Val Asp Tyr Ser Val Phe Asp Pro
145 150 155 160
Phe Asp Ser Ser Ser Tyr Phe His Pro Tyr Cys Leu Ile Thr Asp Trp
165 170 175
Asp Asn Leu Thr Met Val Gln Asp Cys Trp Glu Gly Asp Thr Ile Val
180 185 190
Ser Leu Pro Asp Leu Asn Thr Thr Glu Thr Ala Val Arg Thr Ile Trp
195 200 205
Tyr Asp Trp Val Ala Asp Leu Val Ser Asn Tyr Ser Val Asp Gly Leu
210 215 220
Arg Ile Asp Ser Val Glu Glu Val Glu Pro Asp Phe Phe Pro Gly Tyr
225 230 235 240
Gln Glu Ala Ala Gly Val Tyr Cys Val Gly Glu Val Asp Asn Gly Asn
245 250 255
Pro Ala Leu Asp Cys Pro Tyr Gln Lys Tyr Leu Asp Gly Val Leu Asn
260 265 270
Tyr Pro Ile Tyr Trp Gln Leu Leu Tyr Ala Phe Glu Ser Ser Ser Gly
275 280 285
Ser Ile Ser Asn Leu Tyr Asn Met Ile Lys Ser Val Ala Ser Asp Cys
290 295 300
Ser Asp Pro Thr Leu Leu Gly Asn Phe Ile Glu Asn His Asp Asn Pro
305 310 315 320
Arg Phe Ala Ser Tyr Thr Ser Asp Tyr Ser Gln Ala Lys Asn Val Leu
325 330 335
Ser Tyr Ile Phe Leu Ser Asp Gly Ile Pro Ile Val Tyr Ala Gly Glu
340 345 350
Glu Gln His Tyr Ser Gly Gly Asp Val Pro Tyr Asn Arg Glu Ala Thr
355 360 365
Trp Leu Ser Gly Tyr Asp Thr Ser Ala Glu Leu Tyr Thr Trp Ile Ala
370 375 380
Thr Thr Asn Ala Ile Arg Lys Leu Ala Ile Ser Ala Asp Ser Asp Tyr
385 390 395 400
Ile Thr Tyr Lys Ash Asp Pro Ile Tyr Thr Asp Ser Asn Thr Ile Ala
405 410 415
Met Arg Lys Gly Thr Ser Gly Ser Gln Ile Ile Thr Val Leu Ser Asn
420 425 430
Lys Gly Ser Ser Gly Ser Ser Tyr Thr Leu Thr Leu Ser Gly Ser Gly
435 440 445
Tyr Thr Ser Gly Thr Lys Leu Ile Glu Ala Tyr Thr Cys Thr Ser Val
450 455 460
Thr Val Asp Ser Asn Gly Asp Ile Pro Val Pro Met Ala Ser Gly Leu
465 470 475 480
Pro Arg Val Leu Leu Pro Als Ser Val Val Asp Ser Ser Ser Leu Cys
485 490 495
Gly Gly Ser Gly Asn Thr Thr Thr Thr Thr Thr Ala Ala Thr Ser Thr
500 505 510
Ser Lys Ala Thr Thr Ser Ser Ser Ser Ser Ser Ala Ala Ala Thr Thr
515 520 525
Ser Ser Ser Cys Thr Ala Thr Ser Thr Thr Leu Pro Ile Thr Phe Glu
530 535 540
Glu Leu Val Thr Thr Thr Tyr Gly Glu Glu Val Tyr Leu Ser Gly Ser
545 550 555 560
Ile Ser Gln Leu Gly Glu Trp His Thr Ser Asp Ala Val Lys Leu Ser
565 570 575
Ala Asp Asp Tyr Thr Ser Ser Asn Pro Glu Trp Ser Val Thr Val Ser
580 585 590
Leu Pro Val Gly Thr Thr Phe Glu Tyr Lys Phe Ile Lys Val Asp Glu
595 600 605
Gly Gly Ser Val Thr Trp Glu Ser Asp Pro Asn Arg Glu Tyr Thr Val
610 615 620
Pro Glu Cys Gly Ser Gly Ser Gly Glu Thr Val Val Asp Thr Trp Arg
625 630 635 640
Claims (25)
1.制备可溶的淀粉水解产物的方法，所述方法包括将水性的颗粒状淀粉浆体在低于所述颗粒状淀粉的初始凝胶化温度的温度，与第一种酶和第二种酶作用，其中第一种酶；
(a)是所述的糖苷水解酶家族13的成员；
(b)具有alpha-1，4-糖苷水解活性，和；
(c)包括属于CBM家族20的功能的糖-结合组件(CBM)，其中CBM具有氨基酸序列，所述氨基酸序列选自SEQ ID NO：1、SEQ ID NO：2和SEQ IDNO：3的氨基酸序列；
而其中第二种酶选自包括真菌alpha-淀粉酶(E.C.3.2.1.1)、beta-淀粉酶(E.C.3.2.1.2)和葡糖淀粉酶(E.C.3.2.1.3)的列表。
2.权利要求1的方法，其中所述的alpha-淀粉酶包括氨基酸序列，其选自SEQ ID NO：4、SEQ ID NO：5、SEQ ID NO：6、SEQ ID NO：7、SEQ ID NO：8、SEQ ID NO：9、SEQ ID NO：10、SEQ ID NO：11、SEQ ID NO：12、SEQ ID NO：13、SEQ ID NO：14、SEQ ID NO：15、SEQ ID NO：16、SEQ ID NO：17和SEQ IDNO：18的氨基酸序列。
3.权利要求1或2的方法，其中所述的alpha-淀粉酶包括氨基酸序列，其选自SEQ ID NO：19、SEQ ID NO：20、SEQ ID NO：21和SEQ ID NO：22的氨基酸序列。
4.权利要求1或2任一项的方法，其中所述的淀粉浆体具有20-55％干固体颗粒状淀粉。
5.权利要求1或2的方法，其中至少85％、86％、87％、88％、89％、至少90％、91％、92％、93％94％、95％、96％、97％、98％或至少99％的所述颗粒状淀粉的干固体被转化为可溶的淀粉水解产物。
6.权利要求1或2的方法，其包括将所述的颗粒状淀粉浆体与异淀粉酶和/或支链淀粉酶反应。
7.权利要求1或2的方法，其中所述的温度为至少58℃或59℃。
8.权利要求1或2的方法，其中所述的pH在3.0-7.0的范围。
9.权利要求1或2的方法，其中所述的可溶的淀粉水解产物具有至少94.5％、95.0％、95.5％、96.0％、96.5％、97.0％、97.5％、98.0％、98.5、99.0％或至少99.5％的DX。
10.权利要求1或2的方法，其中所述的颗粒状淀粉是从根茎、根、茎或整粒谷物中得到的。
11.权利要求1或2的方法，其中所述的颗粒状淀粉是从谷类得到的。
12.权利要求1或2的方法，其中所述的颗粒状淀粉是从玉米、穗轴、小麦、大麦、黑麦、蜀黍类、西米、木薯、木薯淀粉、高梁、稻米或马铃薯中得到的。
13.权利要求1或2的方法，其中所述的颗粒状淀粉是从整粒谷物的干磨物或从整粒谷物的湿磨物或从磨碎的粗玉米粉中得到的。
14.权利要求1或2的方法，其中所述的方法在超滤系统中实施，而且其中所述的渗余物在酶、生淀粉和水存在的回流条件下保留，而且其中所述的渗透物是可溶的淀粉水解产物。
15.权利要求1或2的方法，其中所述的方法在具有超滤膜的连续膜反应器中实施，而且其中所述的渗余物在酶、生淀粉和水存在的回流条件下保留，并且其中所述的渗透物是可溶的淀粉水解产物。
16.权利要求1或2的方法，其中所述的方法在具有微滤膜的连续膜反应器中实施，而且其中所述的渗余物在酶、生淀粉和水存在的回流条件下保留，而且其中所述的渗透物是可溶的淀粉水解产物。
17.制备基于高果糖淀粉糖浆(HFSS)的方法，其中权利要求1-16的方法的可溶的淀粉水解产物经过转化成为基于高果糖淀粉糖浆(HFSS)。
18.权利要求17的制备基于高果糖淀粉糖浆(HFSS)的方法，其中所述高果糖淀粉糖浆(HFSS)为高果糖浆(HFCS)。
19.制备发酵产物的方法，其中权利要求1-16中任一项的方法的可溶的淀粉水解产物经过发酵成为发酵产物。
20.权利要求19的方法，其中所述发酵产物是乙醇。
21.权利要求20的方法，其中所述的发酵步骤同时或分别地/依次地进行所述颗粒状淀粉的水解。
22.权利要求1或2的方法，其中所述的过程在超滤系统中实施，其中所述的渗余物在酶、生淀粉、酵母、酵母营养素和水存在的回流条件下保留，而且其中所述的渗透物是含有乙醇的液体。
23.权利要求1或2的方法，其中所述的过程在具有超滤膜的连续膜反应器中实施，其中所述的渗余物在酶、生淀粉、酵母、酵母营养素和水存在的回流条件下保留，而且其中所述的渗透物是含有乙醇的液体。
24.权利要求1或2的方法，其中将所述的淀粉浆体与包括CBM但无催化组件的多肽接触，即疏松的CBM。
25.具有alpha-淀粉酶活性的酶在淀粉水解的方法中的用途，所述的酶包含功能的CBM，其具有氨基酸序列，所述的氨基酸序列选自SEQ ID NO：1、SEQ ID NO：2和SEQ ID NO：3的氨基酸序列。
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| WO2013055676A1 (en) * | 2011-10-11 | 2013-04-18 | Novozymes North America, Inc. | Processes for producing fermentation products |
| CN103907809A (zh) * | 2013-01-09 | 2014-07-09 | 卢建军 | 粉条生产过程中淀粉水解工艺的酸碱调节方法 |
| EP2984174A4 (en) * | 2013-04-10 | 2016-11-30 | Novozymes As | PROCESS FOR STARCH HYDROLYSIS |
| MX2015014257A (es) * | 2013-04-10 | 2016-03-01 | Novozymes As | Proceso para la preparacion de azucares y jarabes. |
| CN105264058B (zh) * | 2013-06-06 | 2022-03-29 | 诺维信公司 | α-淀粉酶变体以及对其进行编码的多核苷酸 |
| WO2015150457A1 (en) * | 2014-04-01 | 2015-10-08 | Novozymes A/S | Polypeptides having alpha amylase activity |
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| EP0171218B1 (en) * | 1984-08-06 | 1993-10-13 | Genencor, Inc. | Enzymatic hydrolysis of granular starch directly to glucose |
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| EP0171218B1 (en) * | 1984-08-06 | 1993-10-13 | Genencor, Inc. | Enzymatic hydrolysis of granular starch directly to glucose |
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| Title |
|---|
| 非蜡质淀粉水解物的生产方法. 袁光和，丁积善.四川食品与发酵，第2期. 2001 |
| 非蜡质淀粉水解物的生产方法. 袁光和，丁积善.四川食品与发酵，第2期. 2001 * |
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