CN115087741A - 由寡糖和/或多糖制成果糖 - Google Patents

由寡糖和/或多糖制成果糖 Download PDF

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CN115087741A
CN115087741A CN202080095806.7A CN202080095806A CN115087741A CN 115087741 A CN115087741 A CN 115087741A CN 202080095806 A CN202080095806 A CN 202080095806A CN 115087741 A CN115087741 A CN 115087741A
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安德列·皮克
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Archer Daniels Midland Co
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Abstract

本发明涉及将至少一种寡糖和/或多糖转化为果糖的方法,包括以下步骤:a)向包含水、磷酸盐和至少一种寡糖和/或多糖的组合物中加入至少四种酶,和b)随后在存在所述至少四种酶的情况下,将所述至少一种寡糖和/或多糖酶促转化为果糖,其中在步骤a)中加入至少一种另外的糖,其中所述至少一种另外的糖选自由以下糖组成的组,糖中包含20个或更少的单糖残基和/或其组合;其中在步骤a)中,所述至少四种酶,优选至少五种酶,选自转移酶、磷酸化酶、变位酶、异构酶、水解酶、磷酸酶及其组合;并且其中所述步骤a)中的至少一种酶是磷酸酶。

Description

由寡糖和/或多糖制成果糖
技术领域
本发明涉及将至少一种寡糖和/或多糖转化为果糖的方法、用于将至少一种寡糖和/或多糖转化为果糖的组合物以及包含果糖的水性组合物。此外,本发明涉及一种磷酸酶和该磷酸酶在转化至少一种寡糖和/或多糖中的用途。
发明内容
有关D-果糖的目前工业生产工艺尤其基于两阶段工艺。第一步,多糖或寡糖,例如淀粉,通过水解裂解成单糖。第二步,进行异构化,由此获得D-果糖,产率(对应于百分比)约为42%。用于制备纯D-果糖的后续工作是通过昂贵的色谱纯化技术展开的。
另一种工业生产是用酶转化水解蔗糖。由此获得D-葡萄糖和D-果糖的等摩尔混合物。为了丰富D-果糖,必须通过昂贵的色谱工艺再次对其进行清洁。
工业生产存在明显缺点,因为仅形成D-果糖溶液,其中D-果糖占溶液中存在的糖的最大50%。为了获得更高含量的D-果糖溶液,要么必须进行昂贵的色谱纯化,要么必须添加昂贵的纯D-果糖,例如果糖含量为55%的高果糖玉米糖浆(HFCS55)。
使用D-葡萄糖作为底物生产纯D-果糖的替代方法是已知的并且在EP0028136B1和AT513928B1中有详细描述。在这种情况下,D-葡萄糖被吡喃糖-2-氧化酶氧化成D-葡萄糖酮,然后被还原成D-果糖。可以通过化学方式,即均相或多相催化,或通过使用生物催化剂进行还原。
这种工艺在技术上还没有实现。它的缺点是氧化和还原必须彼此分开进行,因此需要额外的工艺步骤。此外,在化学还原的情况下,需要高纯度的底物并且需要高压和高温,这会产生不希望产生的副产物。在酶促还原的情况下,还原剂和所使用的不稳定辅酶如NADH的成本非常高。
生产D-果糖的已知方法具有各种缺点。为了实现底物的有效转化,部分地需要高压和高温。或者,取决于底物,只能实现50%的最大产率,或者必须使用清洁底物来减少副产物。进一步浓缩需要非常干净的工艺流,以确保色谱设备的重复使用。
需要一种将寡糖和/或多糖转化为D-果糖的方法,该方法可以克服上述缺点,例如使用昂贵的辅因子或需要进一步色谱纯化和浓缩步骤的不利平衡。
发明内容
本发明的目的是提供一种能够相对于底物生产高产率D-果糖且没有或存在很少的副产物的方法。此外,应简化后续工作,无需使用有机辅酶。
该目的通过根据权利要求1所述的用于将至少一种寡糖和/或多糖转化为果糖的方法、根据权利要求8所述的用于将至少一种寡糖和/或多糖转化为果糖的组合物和根据权利要求10所述的包含果糖的水性组合物解决。此外,该目的通过根据权利要求13所述的磷酸酶和根据权利要求15所述的磷酸酶在转化至少一种寡糖和/或多糖中的用途来解决。
第一方面,本发明涉及将至少一种寡糖和/或多糖转化为果糖的方法,包括以下步骤:
a)向包含水、磷酸盐和至少一种寡糖和/或多糖的组合物中加入至少四种酶,优选至少五种酶,和
b)随后在至少四种酶,优选至少五种酶存在的情况下,将至少一种寡糖和/或多糖酶促转化为果糖,
其中在步骤a)中加入至少一种另外的糖,其中至少一种另外的糖选自包含20个或更少的单糖残基的糖和/或其组合,优选包含17个或更少的单糖残基的糖和/或其组合;
其中在步骤a)中,所述至少四种酶,优选至少五种酶,选自转移酶、磷酸化酶、变位酶、异构酶、水解酶、磷酸酶及其组合;并且其中步骤a)中的至少一种酶是磷酸酶。
从属权利要求以及说明书中描述了进一步的优选实施例。
本发明的上下文中提及的果糖应理解为提及D-果糖。
根据本发明所述的寡糖是包含至少两个单糖残基的糖,其中这些单糖残基可以相同或不同。
在磷酸酶下,属于分类EC3.1.3的任何酶都应该理解。在转移酶下,属于分类EC2.4.的任何酶都应该理解。在磷酸化酶下,属于分类EC2.4.1.的任何酶都应该理解。在变位酶下,属于分类EC5.4.2.的任何酶都应该理解。在异构酶下,属于分类EC5.3.1.的任何酶都应该理解。在水解酶下,属于分类EC3.2.的任何酶都应该理解。
可以使用寡糖和多糖作为本发明的底物。提高的产率源于糖磷酸盐,它可以通过糖类裂解和可能已经存在于糖类中的果糖单元来制备。
令人惊讶的是,发现了一种D-果糖的生产方法,该方法无需高压和高温即可工作,并且可以更好地耐受过程中污染物的存在。催化剂的选择可以通过形成中间体糖磷酸盐在该过程中获得高产率的D-果糖。因此,与目前使用的工业生产方法相比,可以实现基于底物(糖)的更高比例,并且后续工作变得多余。这省去了昂贵的清洁步骤。糖可以是寡糖和/或多糖。
优选的,以干重计,步骤a)中的组合物包含等于或小于35%的至少一种寡糖和/或多糖。优选的,以干重计,该组合物包含0.5%至35%的至少一种寡糖和/或多糖。
进一步优选的,寡糖和/或多糖选自基于葡萄糖的寡糖和/或多糖,优选选自淀粉及其衍生物、半纤维素及其衍生物、纤维素及其衍生物和/或其组合。
相应的寡糖和/或多糖可以通过淀粉生产或通过生物质的水解获得,例如在纸浆生产中或在秸秆加工中获得。
优选的,在步骤a)中,至少一种另外的糖选自十四糖、十三糖、十二糖、十一糖、十糖、九糖、八糖、七糖、六糖、五糖、四糖、三糖、二糖和/或其组合,更多优选四糖、三糖、二糖及其组合,甚至更优选选自麦芽糖、麦芽三糖、麦芽四糖及其组合。
在步骤a)中进一步优选的,至少一种另外的糖是麦芽糖糊精。麦芽糖糊精是由D-葡萄糖单元组成的糖,主要通过α-1,4-糖苷键连接,以可变长度的链连接。通常,麦芽糖糊精由长度从3到17个葡萄糖单位不等的链混合物组成。麦芽糖糊精按DE(葡萄糖当量)分类,DE介于3和20之间。DE值越高,葡萄糖链越短。
进一步优选的,至少一种另外的糖具有与至少一种寡糖和/或多糖至少部分相同的糖苷键。
优选的,磷酸酶包含与根据SEQ ID NO:13所述的序列至少90%、优选至少95%、更优选至少97%并且甚至更优选至少98.5%相同的氨基酸序列。
在一个优选的实施例中,磷酸酶包含与根据SEQ ID NO:41所述的序列至少98%、优选至少98.5%、更优选至少99%并且甚至更优选至少99.5%相同的氨基酸序列。
根据一个优选的实施例,磷酸酶选自具有以下氨基酸序列的磷酸化酶组:SEQ IDNO:15、SEQ ID NO:17、SEQ ID NO:19、SEQ ID NO:21、SEQ ID NO:23、SEQ ID NO:25、SEQ IDNO:27、SEQ ID NO:29、SEQ ID NO:31,SEQ ID NO:33、SEQ ID NO:35、SEQ ID NO:37、SEQ IDNO:39、SEQ ID NO:41、SEQ ID NO:43、SEQ ID NO:45、SEQ ID NO:47或SEQ ID NO:49。
此外,在步骤a)中,优选向组合物中添加至少六种酶。
优选的,至少四种酶、更优选地五种酶和甚至更优选地六种酶选自磷酸酶、转移酶、磷酸化酶、变位酶、异构酶和/或水解酶。
优选的,在步骤a)中加入至少一种转移酶,优选糖基转移酶,更优选葡聚糖转移酶,甚至更优选α-葡聚糖转移酶;和/或
优选的,在步骤a)中加入至少一种磷酸化酶,优选葡聚糖磷酸化酶;和/或
优选的,在步骤a)中加入至少一种变位酶,优选加入磷酸葡萄糖变位酶;和/或
优选的,在步骤a)中加入至少一种异构酶,优选加入磷酸葡萄糖异构酶;和/或
优选的,在步骤a)中加入至少一种水解酶,优选葡聚糖水解酶,更优选支链淀粉酶。
根据在步骤a)中将至少一种寡糖和/或多糖转化为果糖的方法的一个优选实施例,所述至少一种另外的糖选自四糖、三糖、二糖及其组合,更优选选自麦芽糖、麦芽三糖、麦芽四糖及其组合;和/或步骤a)中的组合物以干重计包含等于或小于35%的所述至少一种寡糖和/或多糖,优选所述组合物以干重计包含0.5%至35%的至少一种寡糖和/或多糖;和/或至少一种寡糖和/或多糖选自基于葡萄糖的寡糖和/或多糖,优选选自淀粉及其衍生物、半纤维素及其衍生物、纤维素及其衍生物和/或其组合;和/或在步骤a)中向组合物中加入至少六种酶;和/或至少一种另外的糖具有与至少一种寡糖和/或多糖至少部分相同的糖苷键。
优选的,在步骤b)中中间产生糖磷酸。
在步骤b)中进一步优选的酶促转化是一锅法反应。
根据步骤b)中的优选实施例,中间产生了糖磷酸,并且酶促转化是一锅法反应。
本发明的方法允许通过几个催化步骤将寡糖和多糖转化为D-果糖。这产生了糖磷酸盐形式的中间体,其最终被裂解为D-果糖和磷酸盐。
优选的,步骤a)中添加的寡糖和/或多糖包含多个葡萄糖单元,更优选D-葡萄糖单位。甚至更优选的,步骤a)中添加的寡糖和/或多糖由多个葡萄糖单位组成,更优选D-葡萄糖单位组成。
在根据一个实施例的本发明方法中,应用了一种反应序列的酶促过程,使得能够以D-葡萄糖-1-磷酸的形式裂解单个D-葡萄糖分子。随后,这些释放的D-葡萄糖-1-磷酸分子通过进一步的糖-磷酸中间体转化为D-果糖-6-磷酸。D-果糖-6-磷酸随后被分裂,得到D-果糖和磷酸。
该转化过程中的反应步骤包括以下步骤:糖分子被酶促D-葡萄糖1-磷酸裂解,D-葡萄糖1-磷酸酶促转化为D-葡萄糖6-磷酸,以及D-葡萄糖6-磷酸被酶促转化为D-果糖-6-磷酸,并且D-果糖-6-磷酸被酶促裂解为D-果糖和磷酸。
磷酸盐仅以少量催化量用于中间形成糖磷酸盐,例如D-葡萄糖-1-磷酸、D-葡萄糖-6-磷酸、D-果糖-6-磷酸。在初始步骤中,将磷酸盐添加到含有D-葡萄糖单元的混合物中,在最后一步中,磷酸盐被裂解掉并再次用于初始步骤。
本发明的这种方法提供了本领域已知的生产D-果糖的酶替代方法,它极大地简化了分离和纯化残留中间体的需要。因此,与目前使用的技术相比,本发明代表了由糖生产D-果糖方面的显著改进。与现有方法相比,所使用的生物催化剂系统实现了产品D-果糖的增强富集。
该方法基本上适用于寡糖和多糖的使用。
优选的,可以由纤维二糖、麦芽糖或淀粉生产富含D-果糖的水解产物。在这种情况下,初始步骤使用纤维二糖磷酸化酶、麦芽糖磷酸化酶或淀粉磷酸化酶进行,然后是几个催化步骤。该方法在使用蔗糖作为底物时特别有用,该底物可以从甜菜或甘蔗中获得。在这种情况下,使用蔗糖磷酸化酶进行初始步骤。在这种特殊情况下,获得了产率>53%(基于质量)或>1摩尔D-果糖/摩尔蔗糖的D-果糖。
本发明的方法可以在各种溶剂系统中进行。优选在水性溶剂系统中进行。
也可以通过缓冲系统补充水性系统。合适的缓冲剂(系统)是已知的并且包括常规缓冲剂(系统),例如乙酸盐、磷酸钾、Tris-HCl、甘氨酰甘氨酸和甘氨酸缓冲剂,或这些的混合物。
优选的,在本发明的方法中使用的缓冲剂具有3至12、优选4至12、更优选4至11的pH。为了实现生物催化剂离子的最佳活性,待添加例如Mg2+。稳定剂、甘油等的使用可以允许更长时间地使用生物催化剂。
在本发明的方法中,需要磷酸盐。为了确保该过程中有足够量的磷酸盐,以磷酸、磷酸盐、多磷酸盐或其组合形式的外部添加是必要的。
优选的,本发明的方法在合适的温度下进行,例如可能取决于使用的酶。合适的温度包括10至100℃,优选10至90℃,更优选20至90℃,甚至更优选20至80℃。
优选的,步骤b)中的温度在10-100℃之间,更优选在20-90℃之间,甚至更优选在20-80℃之间。
进一步优选的,组合物的pH为3至12,更优选4至10。
本发明方法的一个优点是它在同一反应批次中进行所有催化步骤而不需要分离中间体。该方法可以分批或连续操作。在这种情况下,可以同时添加所有涉及的酶,或者仅添加一部分酶,例如可以在步骤b)期间添加一种或多种酶,并且可以在时间或局部延迟的情况下稍后添加另一部分酶。
在添加酶的第二部分之前,可以例如物理地(过滤或通过固定)除去已经存在于反应混合物中的酶或使该酶灭活。可以通过将温度短期10分钟升高到例如80℃来完成这种灭活。或者,反应溶液也可以流过反应混合物以提高转化率和产率数倍。
本发明方法中糖类裂解成D-葡萄糖-1-磷酸是酶促的,即通过酶催化,并且可以根据已知方法进行。裂解优选通过磷酸化酶催化进行。
优选的,步骤a)的组合物中存在的至少50%的糖在酶促转化24小时后转化为果糖。
还优选步骤a)的组合物中存在的至少70%的糖在酶促转化48小时后转化为果糖。
本发明的方法允许进一步改进对高浓度寡糖和/或多糖例如溶胀淀粉的处理。例如,可以以35%的干物质淀粉的起始浓度运行本发明的方法。
优选的,步骤b)在室温(25℃)下进行至少24小时。
另一方面,本发明涉及一种将至少一种寡糖和/或多糖转化为果糖的方法,包括以下步骤:a)向包含水、磷酸盐和至少一种寡糖和/或多糖的组合物中加入至少一种酶,和b)随后在所述至少一种酶存在的情况下,将所述至少一种寡糖和/或多糖酶促转化为果糖,其中在步骤a)中添加至少一种另外的糖,由此所述至少一种另外的糖选自由以下糖组成的组,糖中包含20个或更少的单糖残基和/或其组合,优选包含17个或更少的单糖残基和/或其组合。
关于用于将至少一种寡糖和/或多糖转化为果糖的方法,特别是关于如上文提到的酶和寡糖和/或多糖的所有说明在适用的情况下也适用于该方法。
优选的,步骤a)中的至少一种酶是磷酸酶。
此外,在步骤a)中,优选向组合物中加入至少四种酶,更优选至少五种酶,甚至更优选至少六种酶。
第二方面,本发明涉及用于将至少一种寡糖和/或多糖转化为果糖的组合物,其包含
-水;
-磷酸盐;
-至少四种酶,优选至少五种酶,其中至少一种酶是磷酸酶;
-至少一种寡糖和/或多糖;
其中所述至少四种酶,优选至少五种酶,选自转移酶、磷酸化酶、变位酶、异构酶、水解酶、磷酸酶及其组合;和
其中所述组合物还包含至少一种另外的糖,其中所述至少一种另外的糖选自由以下糖组成的组,糖中包含20个或更少的单糖残基和/或其组合,优选包含17个或更少的单糖残基和/或其组合。
所有关于将至少一种寡糖和/或多糖转化为果糖的方法,特别是关于酶和如上文提及的寡糖和/或多糖的所有说明在适用的情况下也适用于将至少一种寡糖和/或多糖转化为果糖的组合物。
根据一个优选的实施例,磷酸酶包含与根据SEQ ID NO:13所述的序列至少90%、优选至少95%、更优选至少97%并且甚至更优选至少98.5%相同的氨基酸序列。
根据另一个优选实施例,磷酸酶包含与根据SEQ ID NO:41所述的序列至少98%、优选至少98.5%、更优选至少99%并且甚至更优选至少99.5%相同的氨基酸序列。
根据一个优选的实施例,磷酸酶选自具有以下氨基酸序列的磷酸化酶组:SEQ IDNO:15、SEQ ID NO:17、SEQ ID NO:19、SEQ ID NO:21、SEQ ID NO:23、SEQ ID NO:25、SEQ IDNO:27、SEQ ID NO:29、SEQ ID NO:31,SEQ ID NO:33、SEQ ID NO:35、SEQ ID NO:37、SEQ IDNO:39、SEQ ID NO:41、SEQ ID NO:43、SEQ ID NO:45、SEQ ID NO:47或SEQ ID NO:49。
优选地,至少四种酶、更优选地五种酶和甚至更优选地六种酶选自磷酸酶、转移酶、磷酸化酶、变位酶、异构酶和/或水解酶。
根据一个优选的实施例,所述组合物包含转移酶,优选糖基转移酶,更优选葡聚糖转移酶,甚至更优选α-葡聚糖转移酶;和/或
所述组合物还包含磷酸化酶,优选添加葡聚糖磷酸化酶;和/或
所述组合物包含变位酶,优选磷酸葡萄糖变位酶;和/或
所述组合物包含异构酶,优选磷酸葡萄糖异构酶;和/或
该组合物包含水解酶,优选葡聚糖水解酶,更优选支链淀粉酶。
另一方面,本发明涉及用于将至少一种寡糖和/或多糖转化为果糖的组合物,其包含水;磷酸盐;至少五种酶,其中至少一种酶是磷酸酶;至少一种寡糖和/或多糖;其中磷酸酶包含与根据SEQ ID NO:13的序列至少90%、优选至少95%、更优选至少97%并且甚至更优选至少98.5%相同的氨基酸序列。
所有关于将至少一种寡糖和/或多糖转化为果糖的方法,特别是关于如上所所述的酶和寡糖和/或多糖和用于将至少一种寡糖和/或多糖转化为果糖的组合物的所有说明在适用的情况下也适用于该方面。
优选的,至少五种酶选自磷酸酶、转移酶、磷酸化酶、变位酶、异构酶和/或水解酶。
第三方面,本发明涉及一种水性组合物,其包含
-至少50%的果糖,以干重计;
-0.001至25%的葡萄糖,优选0.005至20%的葡萄糖,以干重计;
-0.01至22%的磷酸盐,优选0.05至20%的磷酸盐,以干重计;和
-0.001至2%的至少四种酶,优选至少五种酶,优选0.005至1%的至少四种酶,优选至少五种酶,以干重计,
其中所述至少四种酶,优选至少五种酶,选自转移酶、磷酸化酶、变位酶、异构酶、水解酶、磷酸酶及其组合;和
其中至少一种酶是磷酸酶。
所有关于将至少一种寡糖和/或多糖转化为果糖的方法,特别是关于如上所述的酶和寡糖和/或多糖的所有说明在适用的情况下也适用于水性组合物。
根据一个优选的实施例,磷酸酶包含与根据SEQ ID NO:13所述的序列至少90%、优选至少95%、更优选至少97%并且甚至更优选至少98.5%相同的氨基酸序列。
根据另一个优选实施例,磷酸酶包含与根据SEQ ID NO:41所述的序列至少98%、优选至少98.5%、更优选至少99%并且甚至更优选至少99.5%相同的氨基酸序列。
根据一个优选的实施例,磷酸酶选自具有以下氨基酸序列的磷酸化酶组:SEQ IDNO:15、SEQ ID NO:17、SEQ ID NO:19、SEQ ID NO:21、SEQ ID NO:23、SEQ ID NO:25、SEQ IDNO:27、SEQ ID NO:29、SEQ ID NO:31,SEQ ID NO:33、SEQ ID NO:35、SEQ ID NO:37、SEQ IDNO:39、SEQ ID NO:41、SEQ ID NO:43、SEQ ID NO:45、SEQ ID NO:47或SEQ ID NO:49。
优选地,至少四种酶、更优选地五种酶和甚至更优选地六种酶选自磷酸酶、转移酶、磷酸化酶、变位酶、异构酶和/或水解酶。
根据一个优选的实施例,所述组合物包含转移酶,优选糖基转移酶,更优选葡聚糖转移酶,甚至更优选α-葡聚糖转移酶;和/或
所述组合物还包含磷酸化酶,优选添加葡聚糖磷酸化酶;和/或
所述组合物包含变位酶,优选磷酸葡萄糖变位酶;和/或
所述组合物包含异构酶,优选磷酸葡萄糖异构酶;和/或
所述组合物包含水解酶,优选葡聚糖水解酶,更优选支链淀粉酶。
第四方面,本发明涉及一种水性组合物,其包含
-至少50%的果糖,以干重计;
-0.001至25%的葡萄糖,优选0.005至20%的葡萄糖,以干重计;
-0.01至22%的磷酸盐,优选0.05至20%的磷酸盐,以干重计;和
-0.001至2%的至少四种酶,优选至少五种酶,优选0.005至1%的至少四种酶,优选至少五种酶,以干重计,
其中所述至少四种酶,优选至少五种酶,选自转移酶、磷酸化酶、变位酶、异构酶、水解酶、磷酸酶及其组合;和
其中至少一种酶是磷酸酶,
可通过上述本发明的方法获得这种磷酸酶。
所有关于用于将至少一种寡糖和/或多糖转化为果糖的方法,特别是关于上文提及的酶和寡糖和/或多糖的说明也在适用的情况下适用于可通过本发明方法获得的水性组合物。
根据一个优选的实施例,磷酸酶包含与根据SEQ ID NO:13所述的序列至少90%、优选至少95%、更优选至少97%、甚至更优选至少98.5%相同的氨基酸序列。
根据另一个优选的实施例,其中磷酸酶包含与根据SEQ ID NO:41所述的序列至少98%、优选至少98.5%、更优选至少99%并且甚至更优选至少99.5%相同的氨基酸序列。
根据一个优选的实施例,磷酸酶选自具有以下氨基酸序列的磷酸化酶组:SEQ IDNO:15、SEQ ID NO:17、SEQ ID NO:19、SEQ ID NO:21、SEQ ID NO:23、SEQ ID NO:25、SEQ IDNO:27、SEQ ID NO:29、SEQ ID NO:31,SEQ ID NO:33、SEQ ID NO:35、SEQ ID NO:37、SEQ IDNO:39、SEQ ID NO:41、SEQ ID NO:43、SEQ ID NO:45、SEQ ID NO:47或SEQ ID NO:49。
优选的,至少四种酶、更优选地五种酶和甚至更优选地六种酶选自磷酸酶、转移酶、磷酸化酶、变位酶、异构酶和/或水解酶。
根据一个优选的实施例,所述组合物包含转移酶,优选糖基转移酶,更优选葡聚糖转移酶,甚至更优选α-葡聚糖转移酶;和/或
所述组合物还包含磷酸化酶,优选添加葡聚糖磷酸化酶;和/或
所述组合物包含变位酶,优选磷酸葡萄糖变位酶;和/或
所述组合物包含异构酶,优选磷酸葡萄糖异构酶;和/或
所述组合物包含水解酶,优选葡聚糖水解酶,更优选支链淀粉酶。
另一方面,本发明涉及一种水性组合物,以干重计,其包含至少50%的果糖;以干重计,少于25%的葡萄糖,优选少于20%的葡萄糖;以干重计,少于22%的磷酸盐,优选少于20%的磷酸盐;以干重计,少于2%的至少四种酶,优选少于1%的至少四种酶,其中一种酶是磷酸酶。
所有关于用于将至少一种寡糖和/或多糖转化为果糖的方法,特别是关于如上文提到的酶和寡糖和/或多糖的所有说明在适用的情况下也适用于水性组合物。
根据一个优选的实施例,磷酸酶包含与根据SEQ ID NO:13所述的序列至少90%、优选至少95%、更优选至少97%并且甚至更优选至少98.5%相同的氨基酸序列。
根据一个优选的实施例,磷酸酶选自具有以下氨基酸序列的磷酸化酶组:SEQ IDNO:15、SEQ ID NO:17、SEQ ID NO:19、SEQ ID NO:21、SEQ ID NO:23、SEQ ID NO:25、SEQ IDNO:27、SEQ ID NO:29、SEQ ID NO:31,SEQ ID NO:33、SEQ ID NO:35、SEQ ID NO:37、SEQ IDNO:39、SEQ ID NO:41、SEQ ID NO:43、SEQ ID NO:45、SEQ ID NO:47或SEQ ID NO:49。
根据一个优选的实施例,所述组合物包含转移酶,优选糖基转移酶,更优选葡聚糖转移酶,甚至更优选α-葡聚糖转移酶;和/或所述组合物还包含磷酸化酶,优选添加葡聚糖磷酸化酶;和/或所述组合物包含变位酶,优选磷酸葡萄糖变位酶;和/或所述组合物包含异构酶,优选磷酸葡萄糖异构酶;和/或所述组合物包含水解酶,优选葡聚糖水解酶,更优选支链淀粉酶。
另一方面,本发明涉及一种水性组合物,以干重计,其包含至少50%的果糖;以干重计,少于25%的葡萄糖,优选少于20%的葡萄糖;以干重计,少于22%的磷酸盐,优选少于20%的磷酸盐;以干重计,少于2%的至少四种酶,优选少于1%的至少四种酶,其中一种酶是可通过上述本发明方法获得的磷酸酶。
所有关于用于将至少一种寡糖和/或多糖转化为果糖的方法,特别是关于上文提到的酶和寡糖和/或多糖的说明在适用的情况下也适用于可通过本发明方法获得的水性组合物。
根据一个优选的实施例,磷酸酶包含与根据SEQ ID NO:13所述的序列至少90%、优选至少95%、更优选至少97%并且甚至更优选至少98.5%相同的氨基酸序列。
根据一个优选的实施例,磷酸酶选自具有以下氨基酸序列的磷酸化酶组:SEQ IDNO:15、SEQ ID NO:17、SEQ ID NO:19、SEQ ID NO:21、SEQ ID NO:23、SEQ ID NO:25、SEQ IDNO:27、SEQ ID NO:29、SEQ ID NO:31,SEQ ID NO:33、SEQ ID NO:35、SEQ ID NO:37、SEQ IDNO:39、SEQ ID NO:41、SEQ ID NO:43、SEQ ID NO:45、SEQ ID NO:47或SEQ ID NO:49。
根据一个优选的实施例,所述组合物包含转移酶,优选糖基转移酶,更优选葡聚糖转移酶,甚至更优选α-葡聚糖转移酶;和/或所述组合物还包含磷酸化酶,优选添加葡聚糖磷酸化酶;和/或所述组合物包含变位酶,优选磷酸葡萄糖变位酶;和/或所述组合物包含异构酶,优选磷酸葡萄糖异构酶;和/或所述组合物包含水解酶,优选葡聚糖水解酶,更优选支链淀粉酶。
另一方面,本发明涉及可通过上述本发明方法获得的果糖。
关于用于将至少一种寡糖和/或多糖转化为果糖的方法,特别是关于如上文提到的酶和寡糖和/或多糖的所有说明在适用的情况下也适用于可通过本发明的方法获得的果糖。
第五方面,本发明涉及一种磷酸酶,其包含与根据SEQ IDNO:41所述的序列至少98%、优选至少98.5%、更优选至少99%、甚至更优选至少99.5%相同的氨基酸序列。
所有关于用于将至少一种寡糖和/或多糖转化为果糖的方法,特别是关于如上提到的酶和寡糖和/或多糖的所有说明在适用的情况下也适用于磷酸酶。
优选的,磷酸酶具有以下氨基酸序列:SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:19、SEQ ID NO:21、SEQ ID NO:23、SEQ ID NO:25、SEQ ID NO:27、SEQ ID NO:29、SEQ IDNO:31,SEQ ID NO:33、SEQ ID NO:35、SEQ ID NO:37、SEQ ID NO:39、SEQ ID NO:41、SEQ IDNO:43、SEQ ID NO:45、SEQ ID NO:47或SEQ ID NO:49。
这些磷酸酶使D-果糖具有更高的产量。本发明的磷酸酶提供了关于果糖-6-磷酸的改进的选择性。由于它们具有较高的活性,因此可以降低要应用的酶的量。因此,这些磷酸酶特别适用于工业生产。
另一方面,本发明涉及一种磷酸酶,其包含与根据SEQ ID NO:13所述的序列至少90%、优选至少95%、更优选至少97%、甚至更优选至少98.5%相同的氨基酸序列。
优选的,磷酸酶具有如下所示的氨基酸序列:SEQ ID NO:15、SEQ ID NO:17、SEQID NO:19、SEQ ID NO:21、SEQ ID NO:23、SEQ ID NO:25、SEQ ID NO:27、SEQ ID NO:29、SEQID NO:31、SEQ ID NO:33、SEQ ID NO:35、SEQ ID NO:37、SEQ ID NO:39、SEQ ID NO:41、SEQID NO:43、SEQ ID NO:45、SEQ ID NO:47或SEQ ID NO:49。
所有关于将至少一种寡糖和/或多糖转化为果糖的方法,特别是关于如上提到的酶和寡糖和/或多糖的所有说明在适用的情况下也适用于磷酸酶。
第六方面,本发明涉及本发明的磷酸酶在将至少一种寡糖和/或多糖转化为果糖中的用途。
所有关于将至少一种寡糖和/或多糖转化为果糖的方法,特别是关于如上提到的酶和寡糖和/或多糖的说明在适用的情况下也适用于本发明的磷酸酶的用途。
附图说明
图1以淀粉开始并产生果糖的本发明方法的示意图;
图2图1所示方法的中间步骤;葡聚糖磷酸化酶(3)使用无机磷酸盐(G)切割末端葡萄糖残基与聚合物其余部分(B-A)之间的α-1,4键,从而释放出1-磷酸葡萄糖(C);
图3图1所示方法的中间步骤;1-磷酸葡萄糖(C)在葡萄糖磷酸变位酶(4)的作用下转化为6-磷酸葡萄糖(D);
图4图1所示方法的中间步骤;使用磷酸葡萄糖异构酶(5)将6-磷酸葡萄糖转化为6-磷酸果糖(E);
图5图1所示方法的中间步骤;6-磷酸果糖在磷酸酶(6)的作用下裂解为果糖(F)和磷酸(G);
图6图1所示方法的中间步骤;淀粉中包含的所有α-1,6键(A–直链淀粉(B)和支链淀粉(H)的混合物)都被支链淀粉酶切割;
图7图1所示方法的中间步骤;α-葡聚糖转移酶催化1,4-α-D-葡聚糖(B)的片段转移到受体碳水化合物(B)中的新位置,产生新的α-1,4键;
图8显示了使用改进的磷酸酶即P46T和麦芽糖(0.1mM)在48小时内更快地转化天然淀粉的优化工艺的进展;
图9显示了证明短寡糖对使用支链淀粉酶、α-葡聚糖转移酶和葡聚糖磷酸化酶分解天然淀粉的有益影响以及检测产生的G1 P的实验设置的结果;
图10显示了野生型磷酸酶与九种变体(P46T、P46TE47Y、P46TE47YG50L、Y23HP46TE47Y、P46TE47YG50I、Y23HP46TE47YG50L、Y23HP46TE47YG50I、Y23SP46TE47YG50L、Y23SP46TE47YG50I)在该过程中发生的三种不同糖磷酸盐(G6P和F6P)的情况下改善的催化性能的比较的示意图;
图11显示了野生型磷酸酶与六种变体(HTYL、Y47T、Y47F、V45M、V45Q和V45R)在该过程中出现的三种不同的糖-磷酸盐(G6P和F6P)的情况下改善的催化性能的比较的示意图;
图12显示了六种磷酸酶变体(V45R、HTFL、HTTLV45R、HTFLV45R、HTFLV45M和HTFLV45Q)在该过程中出现的三种不同糖-磷酸盐(G6P和F6P)的情况下改善的催化性能的比较的示意图;
图13显示了野生型磷酸酶和两种变体(TY和HTYL)在蔗糖转化为果糖的过程中改善的催化性能的比较的示意图。
下文解释了图和/或表中使用的缩写和酶:
1 支链淀粉没
2 α-葡聚糖转移酶
3 葡聚糖磷酸化酶
4 磷酸葡萄糖变位酶
5 磷酸葡萄糖异构酶
6 磷酸酶
A 淀粉
B 直链淀粉
B-A 直链淀粉-1葡萄糖单位
C 1-磷酸葡萄糖(G1P)
D 6-磷酸葡萄糖(G6P)
E 6-磷酸果糖(F6P)
F 果糖
G 磷酸盐
H 支链淀粉
WT 野生型;SEQ ID NO:13;
T 磷酸酶变体(P46T),在第46位氨基酸从脯氨酸交换到苏氨酸;SEQ ID NO.15;
TY 磷酸酶变体,在第46位氨基酸从脯氨酸交换到苏氨酸,在第47位氨基酸从谷氨酸交换到酪氨酸(P46TE47Y);SEQ ID NO.17;
TYL 磷酸酶变体,在第46位氨基酸从脯氨酸交换到苏氨酸,在第47位氨基酸从谷氨酸交换到酪氨酸,在第50位氨基酸从甘氨酸交换到亮氨酸(P46TE47YG50L);SEQ IDNO.19;
HTY 磷酸酶变体,在第46位氨基酸从脯氨酸交换到苏氨酸,在第47位氨基酸从谷氨酸交换到酪氨酸,在第23位氨基酸从酪氨酸交换到组氨酸(Y23HP46TE47Y);SEQ IDNO.23;
TYI 磷酸酶变体,在第46位氨基酸从脯氨酸交换到苏氨酸,在第47位氨基酸从谷氨酸交换到酪氨酸,在第50位氨基酸从甘氨酸交换到异亮氨酸(P46TE47YG50I);SEQ IDNO.21;
HTYL 磷酸酶变体,在第46位氨基酸从脯氨酸交换到苏氨酸,在第47位氨基酸从谷氨酸交换到酪氨酸,在第50位氨基酸从甘氨酸交换到亮氨酸,在第23位氨基酸从酪氨酸交换到组氨酸(Y23HP46TE47YG50L);SEQ ID NO.25;
HTYI 磷酸酶变体,在第46位氨基酸从脯氨酸交换到苏氨酸,在第47位氨基酸从谷氨酸交换到酪氨酸,在第50位氨基酸从甘氨酸交换到异亮氨酸,在第23位氨基酸从酪氨酸交换到组氨酸(Y23HP46TE47YG50I);SEQ ID NO.27;
STYL 磷酸酶变体,在第46位氨基酸从脯氨酸交换到苏氨酸,在第47位氨基酸从谷氨酸交换到酪氨酸,在第50位氨基酸从甘氨酸交换到亮氨酸,在第23位氨基酸从酪氨酸交换到丝氨酸(Y23SP46TE47YG50L);SEQ ID NO.29;
STYI 磷酸酶变体在第46位氨基酸从脯氨酸交换到苏氨酸,在第47位氨基酸从谷氨酸交换到酪氨酸,在第50位氨基酸从甘氨酸交换到异亮氨酸,在第23位氨基酸从酪氨酸交换到丝氨酸(Y23SP46TE47YG50I);SEQ ID NO.31;
Y47T/HTTL 磷酸酶变体,在第46位氨基酸从脯氨酸交换到苏氨酸,在第47位氨基酸从谷氨酸交换到苏氨酸,在第50位氨基酸从甘氨酸交换到亮氨酸,在第23位氨基酸从酪氨酸交换到组氨酸(Y23HP46TE47TG50L);SEQ ID NO.33;
Y47F/HTFL 磷酸酶变体,在第46位氨基酸从脯氨酸交换到苏氨酸,在第47位氨基酸从谷氨酸交换到苯丙氨酸,在第50位氨基酸从甘氨酸交换到亮氨酸,在第23位氨基酸从酪氨酸交换到组氨酸(Y23HP46TE47FG50L);SEQ ID NO.35;
V45M 磷酸酶变体,在第46位氨基酸从脯氨酸交换到苏氨酸,在第47位氨基酸从谷氨酸交换到酪氨酸,在第50位氨基酸从甘氨酸交换到亮氨酸,在第23位氨基酸从酪氨酸交换到组氨酸,在第45位氨基酸从缬氨酸交换到蛋氨酸(Y23HV45MP46TE47YG50L);SEQ IDNO.37;
V45Q 磷酸酶变体,在第46位氨基酸从脯氨酸交换到苏氨酸,在第47位氨基酸从谷氨酸交换到酪氨酸,在第50位氨基酸从甘氨交换到亮氨酸,在第23位氨基酸从酪氨酸交换到组氨酸,在第45位氨基酸从缬氨酸交换到谷氨酰胺(Y23HV45QP46TE47YG50L);SEQ IDNO.39;
V45R 磷酸酶变体,在第46位氨基酸从脯氨酸交换到苏氨酸,在第47位氨基酸从谷氨酸交换到酪氨酸,在第50位氨基酸从甘氨酸交换到亮氨酸,在第23位氨基酸从酪氨酸交换到组氨酸,在第45位氨基酸从缬氨酸交换到精氨酸(Y23HV45RP46TE47YG50L);SEQ IDNO.41;
HTTLV45R 磷酸酶变体,在第46位氨基酸从脯氨酸交换到苏氨酸,在第47位氨基酸从谷氨酸到苏氨酸,在第50位氨基酸从甘氨酸交换到亮氨酸,在第23位氨基酸从酪氨酸交换到组氨酸,在第45位氨基酸从缬氨酸交换到精氨酸(Y23HV45RP46TE47TG50L);SEQ IDNO.43;
HTFLV45R 磷酸酶变体,在第46位氨基酸从脯氨酸交换到苏氨酸,在第47位氨基酸从谷氨酸交换到苯丙氨酸,在第50位氨基酸从甘氨酸交换到亮氨酸,在第23位氨基酸从酪氨酸交换到组氨酸,在第45位氨基酸从缬氨酸交换到精氨酸(Y23HV45RP46TE47FG50L);SEQID NO.45;
HTFLV45M 磷酸酶变体,在第46位氨基酸从脯氨酸交换到苏氨酸,在第47位氨基酸从谷氨酸交换到苯丙氨酸,在第50位氨基酸从甘氨酸交换到亮氨酸,在第23位氨基酸从酪氨酸交换到组氨酸,在第45位氨基酸从缬氨酸交换到蛋氨酸(Y23HV45MP46TE47FG50L);SEQID NO.47;
HTFLV45Q磷酸酶变体,在第46位氨基酸从脯氨酸交换到苏氨酸,在第47位氨基酸从谷氨酸交换到苯丙氨酸,在第50位氨基酸从甘氨酸交换到亮氨酸,在第23位氨基酸从酪氨酸交换到组氨酸,在第45位氨基酸从缬氨酸交换到谷氨酰胺(Y23HV45QP46TE47FG50L);SEQ ID NO.49。
具体实施方式
图1显示了以淀粉(A–直链淀粉和支链淀粉的混合物)开始的本发明方法的示意图,该淀粉通过使用支链淀粉酶(1)和α-葡聚糖转移酶(2)转化为直链淀粉(B),以下步骤由使用无机磷酸盐(G)的磷酸化酶(葡聚糖磷酸化酶(3))催化,以切断末端葡萄糖残基和聚合物其余部分之间的α-1,4键,释放1-磷酸葡萄糖(C),C通过磷酸葡萄糖变位酶(4)的作用转化为6-磷酸葡萄糖(D),在最后一步中,使用磷酸葡萄糖异构酶(5)将6-磷酸葡萄糖转化为6-磷酸果糖(E),6-磷酸果糖通过磷酸酶(6)的作用裂解为果糖(F)和磷酸(G)。
图2示出了图1所示的一般过程的中间过程步骤。葡聚糖磷酸化酶(3)和无机磷酸盐(G)用于切割末端葡萄糖残基与聚合物(B-A)其余部分之间的α-1,4键,以释放1-磷酸葡萄糖(C)。
图3至图7示出了图1所示过程的进一步中间步骤。
图8至13给出了实验数据图,下面的实验部分将进一步讨论这些实验数据。
尽管已经参考某些优选实施例描述了本发明,但是本领域技术人员将理解,在不脱离本发明的范围的情况下,可以进行各种改变并且可以用等效物代替其元件。此外,可以进行许多修改以使特定情况或材料适应本发明的教导而不脱离本发明的基本范围。因此,本发明不限于特定实施例,而是本发明将包括落入所附权利要求范围内的所有实施例。
实验部分
1.材料
所有化学品均为分析级或具有更高质量,购自Sigma-Aldrich、Carbosynth或VWR,无需进一步纯化即可使用。
Glucidex 12和Glucidex 19是麦芽糖糊精化合物,购自Roquette Freres(法国)。可以使用公式(180*n-18*(n-1))计算葡萄糖聚合物的分子量,其中n是葡萄糖聚合物的DP(聚合度)。DE(葡萄糖当量)可以计算为100*(180/分子量(葡萄糖聚合物))。DP可以使用等式DP=111/DE来计算(Balto,Amy S.,et al.“On the use of differential solubilityin aqueous ethanol solutions to narrow the DP range of food-grade starchhydrolysis products."Food chemistry 197(2016):872-880.)。Glucidex 12的DE在11–14之间,假设平均DP为8–10。
Glucidex 19的DE在18–20之间,假设平均DP为5–6。但是由于这些制剂是通过酶处理得到的,因此还包括越来越小的麦芽糖糊精。
在这项工作中使用了以下菌株:E.coli XL1 Blue、E.coli BL21(DE3),每个构建体都使用自诱导培养基在37℃下成功表达。
2.方法
分析
使用配备自动进样器(WPS 3000TRS)、柱温箱(TCC3000RS)和光散射检测器的HPLCDionex Ultimate 3000系统对图1所示的整个过程进行分析。YMC Triart Diol Hilic色谱柱(100*2mm,1.9μm,12nm)在7℃下用于通过梯度洗脱(15-85%)进行分离,其中含有0.1%甲酸pH 4.5和0.1%甲酸的乙腈溶液作为0.45ml min-1的流动相。样品在水/乙腈中以3:7的比例稀释。使用Dionex Chromeleon软件分析数据。
使用基于氯化三苯基四氮唑(TTC)的测定法检测总酮糖(果糖和6-磷酸果糖)。该检测方法基于醛糖和酮糖之间的不同还原率。向2ml试管中加入0.05ml样品、0.01ml 1%TTC水溶液和0.04ml 6 N NaOH。恰好5分钟后,加入1.5ml乙酸:乙醇(1:9)并涡旋试管内容物。用水作为空白对照组,使用分光光度计(Multiskan GO,Thermo Fisher)在480nm处测量吸光度。葡萄糖和6-磷酸葡萄糖将TTC还原为红色色素-三苯基甲臜-比等量的果糖慢约100倍。
使用基于葡萄糖氧化酶的测定来检测葡萄糖。将50μl样品或稀释样品与50μl预混液(0.75mM ABTS、2U/ml葡萄糖氧化酶、0.1U/ml过氧化物酶、20mM KPi pH 6.0)混合,并在30℃下孵育30分钟,然后使用分光光度计(Multiskan GO,Thermo Fisher)在418nm处测量。
蛋白质表达
针对一种酶示例性地描述了优化的蛋白质表达,并通过相同的步骤对所有蛋白质进行。
含有感兴趣的质粒(pET28衍生物,其中一个基因编码该过程中的所述其中一个酶)的大肠杆菌BL21(DE3)在250ml自诱导培养基中生长(Studier,F.William."Proteinproduction by auto-induction in high-density shaking cultures."Proteinexpression and purification 41.1(2005):207-234.)。将预培养物在4ml含有100μg/ml卡那霉素的LB培养基中在37℃下在旋转振荡器(180rpm)上孵育过夜。用1:100稀释的过夜培养物接种表达培养物。在37℃下孵育24小时。通过离心收获细胞并重新悬浮在50mMTRIS-HCl(pH=8.0)中。通过使用Basic-Z细胞破碎仪(IUL Constant Systems)或超声波装置制备粗提取物,随后添加MgCl2至2.5mM的最终浓度,并与DNaseI(1μg/ml)结合,随后在室温(25℃)下孵育20分钟,从而降解DNA。之后,所有粗提取物在70℃下热处理30分钟。通过离心(在4℃下,在20,000rpm下进行40分钟)去除裂解物中的不溶部分。将上清液通过0.45μm注射式过滤器过滤并用作纯化的酶制剂。每次纯化的等分试样都经过Laemmli Ulrich K描述的12%SDS-Page。“Cleavage of structural proteins during the assembly of thehead of bacteriophage T4."nature 227.5259(1970):680.).”
酶对应于以下序列SEQ ID NO:1,SEQ ID NO:3,SEQ ID NO:5,SEQ ID NO:7,SEQID NO:9,SEQ ID NO:11,SEQ ID NO:13,SEQ ID NO:ID NO:15,SEQ ID NO:17,SEQ ID NO:19,SEQ ID NO:21,SEQ ID NO:23,SEQ ID NO:25,SEQ ID NO:27,SEQ ID NO:29,SEQ IDNO:31,SEQ ID NO:33,SEQ ID NO:35,SEQ ID NO:37,SEQ ID NO:39,SEQ ID NO:41,SEQ IDNO:43,SEQ ID NO:45,SEQ ID NO:47,SEQ ID NO:49。
酶活性测试
使用支链淀粉作为底物和用于检测的3,5-二硝基水杨酸(DNS)测试了黄热芽孢杆菌(SEQ ID NO:1)的支链淀粉酶的活性。
支链淀粉酶与1%(w/v)支链淀粉在50mM磷酸钾缓冲液pH 7.0和5mM MgCl2中于65℃下孵育。在不同的时间点,将24μl等分试样转移到96μl DNS试剂中(10g 3,5-二硝基水杨酸、300g酒石酸钾钠、16g氢氧化钠,1升)。加热至95℃5分钟,然后将100μl转移到平底微量滴定板中,并在分光光度计(Multiskan GO,Thermo Fisher)中在540nm处测量。
使用麦芽三糖作为底物测试了Meiothermus ruber(SEQ ID NO:3)的α-葡糖转移酶的活性和构建较大寡糖的能力。使用TLC(异丙醇:乙酸乙酯:水(3:1:1))和百里酚喷雾试剂(0.5g百里酚,95ml乙醇,5ml 97%硫酸)进行活性检测。Meiothermus ruber的α-葡糖转移酶与50mM麦芽三糖在50mM磷酸钾缓冲液pH 7.0和5mM MgCl2中在65℃下孵育。
使用酶的组合来检测嗜热嗜热袍菌(SEQ ID NO:5)的α-葡聚糖磷酸化酶的活性以检测产物α-D-葡萄糖-1-磷酸。α-D-葡萄糖-1-磷酸转化为α-D-葡萄糖-6-磷酸,随后α-D-葡萄糖-6-磷酸被α-D-葡萄糖-6-磷酸脱氢酶氧化为α-D-葡萄糖-6-磷酸。产生的NADH用于通过使用1-甲氧基-5-甲基吩嗪鎓甲基硫酸盐(PMS)作为电子介体来转化WST-1。α-葡聚糖磷酸化酶与1%(w/v)可溶性淀粉在50mM磷酸钾缓冲液pH 7.0和5mM MgCl2和0.1mM磷酸吡哆醛中在65℃下孵育。在不同的时间点,将10μl等分试样转移到190μl含有磷酸葡萄糖变位酶0.1U、α-D-葡萄糖-6-磷酸脱氢酶0.1U、0.1mM WST-1、0.005mM PMS的检测溶液中。将混合物孵育30分钟并在分光光度计(Multiskan GO,Thermo Fisher)中在440nm处测量。
以蔗糖为底物,和用于检测的3,5-二硝基水杨酸测得青春双歧杆菌(SEQ ID NO:51)的蔗糖磷酸化酶的活性。在使用3,5-二硝基水杨酸进行检测的分析中,只有产品果糖产生信号。将蔗糖磷酸化酶与100mM蔗糖在100mM磷酸钾缓冲液(pH 7.0)和5mM MgCl2中于50℃下孵育。在不同的时间点,将24μl的等份转移到96μl DNS试剂中(10g 3,5-二硝基水杨酸,300g酒石酸钾钠,16g氢氧化钠,1升)。加热至95℃5分钟,然后将100μl转移到平底微滴定板中,并在540nm处用分光光度计(Multiskan GO,Thermo Fisher)测量。
以α-D-葡萄糖-1-磷酸为底物,和用于检测的3,5-二硝基水杨酸,对Saccharolobus sulfataricus P2(SEQ ID NO:7)或Chlostridium thermocellum(SEQ IDNO:9)的磷酸葡萄糖变位酶进行活性检测。在使用3,5-二硝基水杨酸进行检测的分析中,只有产物α-D-葡萄糖-6-磷酸产生信号。在65℃下,将磷酸葡萄糖变位酶与50mMα-D-葡萄糖-1-磷酸盐在50mM磷酸钾缓冲液(pH 7.0)和5mM MgCl2中孵育。在不同的时间点,将24μl的等份转移到96μl DNS试剂中(10g 3,5-二硝基水杨酸,300g酒石酸钾钠,16g氢氧化钠,1升)。加热至95℃5分钟,然后100μl转移到平底微滴定板中,并在540nm处用分光光度计(Multiskan GO,Thermo Fisher)测量。
以果糖-6-磷酸为底物,和用于检测的α-D-葡萄糖-6-磷酸脱氢酶,对海栖热袍菌(SEQ ID NO:11)的磷酸葡萄糖异构酶进行活性检测。通过使用1-甲氧基-5-甲基吩嗪甲基硫酸盐(PMS)作为电子介质,将产生的NADH转化为WST-1。
在65℃下,将磷酸葡萄糖异构酶与50mM果糖-6-磷酸盐在50mM磷酸钾缓冲液(pH7.0)和5mM MgCl2中孵育。在不同的时间点,将10μl的等分试样转移到190μl含有α-D-葡萄糖-6-磷酸脱氢酶0.1U、0.1mM WST-1、0.005mM PMS的检测溶液中。将混合物培养30分钟,并用分光光度计(Multiskan GO,Thermo Fisher)在440nm处测量。
使用6-磷酸果糖为底物和磷酸盐测定法,对嗜热菌(SEQ ID NOs:13、15、17、19、21、23、25、27、29、31、33、35、37、39、41、43、45、47、49)的磷酸酶进行活性测试。磷酸盐测定由三种溶液组成,溶液A(12%(w/v)l-抗坏血酸在1N HCl溶液中)、溶液B(2%(w/v)Na2MoO4x 2H2O在ddH2O中)、溶液F(2%(w/v)柠檬酸和2%(w/v)乙酸在ddH2O中)。溶液D是溶液a和溶液B的2:1混合物,在测量前现做的。用移液管将75μl溶液D移到平底微量滴定板的所需孔中。下一步,添加25μl样品或规定浓度的标准品,并在室温下培养5分钟。最后一步,通过添加75μl溶液F和在室温下额外培养15分钟,使反应停止。用分光光度计(Multiskan GO,Thermo Fisher)在655nm处测量样品。
所有测试的酶都满足催化工艺中其中一个步骤的要求,并且在工艺条件下(65℃,50mM磷酸钾缓冲液pH 7.0,5mM MgCl2 0.005mM PLP)保持稳定。
3.淀粉转化为果糖(发明实施例1;IE1)
使用50ml falcon试管和7g天然淀粉以15ml的比例进行转化实验。用容积为6ml的水和缓冲液预处理天然淀粉,并将其加热至95℃30分钟,以使其几乎完全膨胀。然后,将膨胀的淀粉冷却至65℃并添加酶。向7g天然淀粉中加入容积为10.5ml的缓冲液、盐和酶。此外,添加麦芽糖至10mM的终浓度。最终混合物含有50mM KPi pH 7.0、5mM MgCl2、0.1mMPLP、10mM麦芽糖、0.4mg支链淀粉酶、1.5mgα-葡聚糖转移酶、30mgα-葡聚糖磷酸化酶、1.4mg磷酸葡萄糖变位酶、1.2mg磷酸葡萄糖异构酶和14mg磷酸酶。使用来自Bacillusflavocaldarius,Meiothermus ruber,Thermotoga naphthophila(磷酸化酶和磷酸酶)、Chlostridium thermocellum和Thermotoga maritima的酶。从定期培养0’小时开始,使用10kDa旋滤器(VWR 82031-350)采集样品并将其过滤。如有必要,将样品在水/乙腈中以3:7的比例稀释,以便进一步分析。
根据本发明实施例1(IE1)将淀粉转化为果糖的结果(35%干物质淀粉对应于2153.5mM浓度的单糖)如表1和图8所示。
表1
Figure BDA0003784171380000221
从表1可以得出,48小时后可获得约70%产率的果糖。
4.在麦芽糖(IE2-IE5)、麦芽三糖(IE6-IE9)、麦芽四糖(IE10-IE12)、麦芽糊精葡糖苷酶12(IE13-IE15)或麦芽糊精葡糖苷酶19(IE16-IE18)存在下,淀粉转化为果糖。
磷酸钾缓冲液50mM pH 7.0,MgCl2 5mM,PLP 0.005mM,膨胀的天然淀粉1%(w/v),麦芽糖(0.01mM,0.1mM,1.0mM或10mM;IE2–IE5)或麦芽三糖(0.01mM,0.1mM,1.0mM或10mM;IE6–IE9)或麦芽四糖(0.01mM,0.1mM或1.0mM;IE10–IE12)或麦芽糊精葡糖苷酶12(0.0342%(w/v),0.00342%(w/v)或0.000342%(w/v);IE13–IE15)或麦芽糊精葡糖苷酶19(0.0342%(w/v)、0.00342%(w/v)或0.000342%(w/v);IE16-IE18),葡聚糖磷酸化酶(0.0025mg/ml),α-葡聚糖转移酶(0.075mg/ml),支链淀粉酶(0.02mg/ml),总容积为5ml,在65℃下培养1小时,并取下等份试样以检查1-磷酸葡萄糖。1-磷酸葡萄糖测定在上述酶的活性测试中进行了描述。
通过仅分别添加1mM麦芽糖、麦芽三糖、麦芽四糖、葡糖苷酶12或葡糖苷酶19,而不添加淀粉来执行比较实施例1至5(CE1–CE5)。执行比较实施例6而不添加额外的糖,例如麦芽糖、麦芽三糖、麦芽四糖、葡糖苷酶12或葡糖苷酶19。
表2显示了额外的糖(如短寡糖,如麦芽糖、麦芽三糖、麦芽三糖、麦芽四糖、Glucidex12或Glucidex19)对1-磷酸葡萄糖转化率影响的试验结果。相应的示意图如图9所示。
表2
Figure BDA0003784171380000222
Figure BDA0003784171380000231
在该过程中,大多糖(如淀粉)或类似底物(如纤维素等)的转化包含一个主要的限速步骤,即生成1-磷酸葡萄糖分子,例如由葡聚糖磷酸化酶催化得到。
磷酸化酶需要多糖的易接近端来催化末端葡萄糖残基和聚合物其余部分之间的无机磷酸盐对α-1,4键的裂解,以释放1-磷酸葡萄糖。支链淀粉酶、α-葡聚糖转移酶和短寡糖(如麦芽糖(IE2-IE5)、麦芽三糖(IE6-IE9)、麦芽四糖(IE10-IE12)、MaltodextrinGlucidex 12(IE13-IE15)或Maltodextrin Glucidex 19(IE16-IE18))的联合作用,参见表2,表明添加短寡糖可提高转化率。这导致更高的1-磷酸葡萄糖产量,因此允许在本发明工艺中更快地将淀粉转化为果糖。
与发明实施例2至18相比,所有比较实施例1至6的转化率更低。
5.磷酸盐分析
为了监测不同磷酸酶的不同活性以及过程中出现的两种糖磷酸中间产物的变体,进行了磷酸盐分析。
反应混合物中含有50mM TRIS-HCl缓冲液pH 7.0、2.5mM MgCl2、10mM G1P、G6P或F6P和一种磷酸酶(0.003mg/ml),总容积为1ml。混合物在65℃下孵育,定期取出一份等分试样并测试释放的磷酸盐。上文对磷酸盐分析进行了更详细的描述。
磷酸盐测定结果如表3和图10所示。检测G6P和F6P的含量,并测定F6P/G6P的比率。F6P/G6P的比率是指被测磷酸酶的选择性。
表3
Figure BDA0003784171380000241
如可从表3和图10中得出,将突变P46T(IE19(T))引入hermotoga naphthophila磷酸酶可增加酶的整体活性,并降低酶对6-磷酸葡萄糖(G6P)的活性。这使得在减少酶量和提高果糖总产量的情况下,可以实现更好的工艺经济性。与野生型(CE7(WT))相比,P46TE47Y(IE20(TY))对F6P的活性略有提高,对G6P的活性大致相同,可减少了不需要的副产物,如葡萄糖。与野生型(CE7(WT))相比,P46TE47YG50L(IE21(TYL))对F6P的活性提高,对G6P的活性降低,也同样减少了不需要的副产物,如葡萄糖。与CE7(WT)相比,Y23HP46TE47Y(IE23(HTY))对F6P的活性也有所提高,对G6P的活性略有增加。但就这两种活性而言,该变体还显示了本发明工艺的改进性能,从而允许改进工艺减少不需要的副产物,如葡萄糖。P46TE47YG50I(IE22(TYI))对F6P有更好的选择性。在总体活性增加的情况下,与CE7(WT)相比,Y23HP46TE47YG50L(IE24(HTYL))对F6P的活性提高,而对G6P的活性降低。在总活性增加的情况下,Y23HP46TE47YG50I(IE25(HTYI))对F6P的活性有所提高,而对G6P的活性则有所降低。在总活性增加的情况下,与CE7(WT)相比。Y23SP46TE47YG50L(IE26(STYL))对F6P的活性提高,而对对G6P的活性则有所降低。Y23SP46TE47YG50I(IE27(STYI))对F6P有更好的选择性。
本发明的磷酸酶(参见表3和图10)显示活性增加和/或选择性增加。
与野生型磷酸酶相比,进一步磷酸盐分析测试额外磷酸酶的结果如表4和图11所示。由于所用磷酸酶的活性有所提高,因此缩短了测量的时间间隔。
表4
Figure BDA0003784171380000251
在总体活性增加的情况下,与野生型相比,Y23HP46TE47YG50L(HTYL)对F6P的活性提高,而对G6P的活性降低。因此,这种酶可以改进工艺,减少不需要的副产品,如葡萄糖。这同样适用于变体Y23HP46TE47TG50L(Y47T)、Y23HP46TE47TG50L(Y47F)、Y23HV45MP46TE47TG50L(V45M)、Y23HV45QP46TE47TG50L(V45Q)、Y23HV45RP46TE47TG50L(V45R)。
表5和图12示出了对其他磷酸酶进行磷酸盐测定的结果。由于所用磷酸酶的活性有所提高,因此测量的时间间隔也缩短了。
表5
Figure BDA0003784171380000261
在总活性增加的情况下,变体Y23HV45RP46TE47TG50L(V45R)、Y23HP46TE47FG50L(HTFL)、Y23HV45RP46TE47TG50L(HTTLV45R)、Y23HV45MP46TE47FG50L(V45M)和Y23HV45QP46TE47FG50L(V45Q)对F6P的活性提高,而对G6P的活性降低。因此,这些酶允许一种改进的工艺来减少不需要的副产品,如葡萄糖。
6.利用改良磷酸酶将蔗糖转化为果糖
磷酸钾缓冲液50mM pH 7.0、MgCl2 5mM、蔗糖1000mM、蔗糖磷酸化酶(0.25mg/ml)、磷酸葡萄糖变位酶(0.04mg/ml)、磷酸葡萄糖异构酶(0.075mg/ml)和磷酸酶(0.18mg/ml),总容积为10ml,在50℃下培养48小时,并移除等分试样以检查葡萄糖和果糖浓度。使用来自青春双歧杆菌(蔗糖磷酸化酶)、Thermotoga naphthophila(磷酸酶)、Chlostridiumthermocellum(磷酸葡萄糖变位酶)和Thermotoga maritima(磷酸葡萄糖异构体酶)的酶。按照方法中所述测定果糖。
表6和图13示出了使用三种不同磷酸酶将蔗糖转化为果糖的结果。
表6
Figure BDA0003784171380000271
从表6中可得出,本发明磷酸酶TY(IE40)和HTYL(IE41)的转化率高于野生型磷酸酶WT(CE9)。
序列表
<110> 卡斯凯特公司
<120> 由寡糖和/或多糖制成果糖
<130> PC03722
<160> 52
<170> SIPOSequenceListing 1.0
<210> 1
<211> 475
<212> PRT
<213> Bacillus flavocaldarius KP 1228
<220>
<223> Pullulanase
<400> 1
Met Ala Trp Tyr Glu Gly Ala Phe Phe Tyr Gln Ile Phe Pro Asp Arg
1 5 10 15
Phe Phe Arg Ala Gly Pro Pro Gly Arg Pro Ala Pro Ala Gly Pro Phe
20 25 30
Glu Pro Trp Glu Asp Pro Pro Thr Leu Arg Gly Phe Lys Gly Gly Thr
35 40 45
Leu Trp Gly Val Ala Glu Lys Leu Pro Tyr Leu Leu Asp Leu Gly Val
50 55 60
Glu Ala Ile Tyr Leu Asn Pro Val Phe Ala Ser Thr Ala Asn His Arg
65 70 75 80
Tyr His Thr Val Asp Tyr Phe Gln Val Asp Pro Ile Leu Gly Gly Asn
85 90 95
Glu Ala Leu Arg Tyr Leu Leu Glu Val Ala His Ala His Gly Val Arg
100 105 110
Val Ile Leu Asp Gly Val Phe Asn His Thr Gly Arg Gly Phe Phe Ala
115 120 125
Phe Gln His Leu Met Glu Asn Gly Glu Gln Ser Pro Tyr Arg Asp Trp
130 135 140
Tyr His Val Lys Gly Phe Pro Leu Lys Ala Tyr Thr Ala His Pro Asn
145 150 155 160
Tyr Glu Ala Trp Trp Gly Asn Pro Glu Leu Pro Lys Leu Lys Val Glu
165 170 175
Thr Pro Ala Val Arg Glu Tyr Leu Leu Ala Val Ala Glu His Trp Ile
180 185 190
Arg Phe Gly Val Asp Gly Trp Arg Leu Asp Val Pro Asn Glu Ile Pro
195 200 205
Asp Pro Thr Phe Trp Arg Glu Phe Arg Gln Arg Val Lys Gly Ala Asn
210 215 220
Pro Glu Ala Tyr Ile Val Gly Glu Ile Trp Glu Glu Ala Asp Phe Trp
225 230 235 240
Leu Gln Gly Asp Met Phe Asp Ala Val Met Asn Tyr Pro Leu Ala Arg
245 250 255
Ala Val Leu Gly Phe Val Gly Gly Glu Ala Leu Asp Arg Asp Leu Ala
260 265 270
Ala Gln Thr Gly Leu Gly Arg Ile Glu Pro Leu Gln Ala Leu Ala Phe
275 280 285
Ser His Arg Leu Glu Asp Leu Phe Gly Arg Tyr Arg Pro Glu Val Val
290 295 300
Arg Ala Gln Met Asn Leu Leu Thr Ser His Asp Thr Pro Arg Leu Leu
305 310 315 320
Ser Leu Met Arg Gly Ser Val Glu Arg Ala Arg Leu Ala Leu Ala Leu
325 330 335
Leu Phe Leu Leu Pro Gly Asn Pro Thr Val Tyr Tyr Gly Glu Glu Val
340 345 350
Gly Met Ala Gly Gly Lys Asp Pro Glu Asn Arg Gly Gly Met Val Trp
355 360 365
Glu Glu Ala Arg Trp Gln Lys Asp Leu Arg Glu Thr Val Lys Arg Leu
370 375 380
Ala Arg Leu Arg Lys Glu His Pro Ala Leu Arg Thr Ala Pro Tyr Leu
385 390 395 400
Arg Ile Tyr Ala Gln Asp Gly His Leu Ala Phe Ala Arg Gly Pro Tyr
405 410 415
Leu Val Val Val Asn Ala Ser Pro His Pro Phe Arg Gln Asp Phe Pro
420 425 430
Leu His Gly Val Phe Pro Arg Gly Gly Arg Ala Val Asp Leu Leu Ser
435 440 445
Gly Glu Val Cys Thr Pro Gln Gly Gly Arg Leu Cys Gly Pro Val Leu
450 455 460
Pro Pro Phe Ser Leu Ala Leu Trp Arg Glu Ala
465 470 475
<210> 2
<211> 1428
<212> DNA
<213> Bacillus flavocaldarius KP 1228
<220>
<223> coding for Pullulanase
<400> 2
atggcatggt atgaaggtgc ctttttctat cagatttttc cggatcgttt ttttcgtgca 60
ggtccgcctg gtcgtccggc accggcaggt ccgtttgaac cgtgggaaga tccgcctaca 120
ctgcgtggtt ttaaaggtgg caccctgtgg ggtgttgcag aaaaactgcc gtatctgctg 180
gatctgggtg ttgaagcaat ttatctgaat ccggtttttg caagcaccgc caatcatcgt 240
tatcataccg ttgattattt tcaggtggac ccgattcttg gtggtaatga agcactgcgt 300
tacctgctgg aagttgcaca tgcacatggt gttcgtgtta ttctggatgg tgtttttaat 360
cataccggtc gtggtttttt tgcctttcag catctgatgg aaaatggtga acagagcccg 420
tatcgtgatt ggtatcatgt taaaggtttt ccgctgaaag catataccgc acatccgaat 480
tatgaagcat ggtggggtaa tccggaactg ccgaaactga aagttgaaac accggcagtt 540
cgtgaatatt tactggcagt tgcagaacat tggattcgtt ttggtgttga tggttggcgt 600
ctggatgttc cgaatgaaat tcctgatccg accttttggc gtgaatttcg tcagcgtgtg 660
aaaggtgcaa atccggaagc atatattgtt ggcgaaattt gggaagaagc agatttttgg 720
ctgcagggtg atatgtttga tgccgttatg aattatccgc tggcacgtgc agttctgggt 780
tttgttggtg gtgaagccct ggatcgtgat ctggcagcac agacaggtct gggtcgtatt 840
gaaccgctgc aggcactggc atttagccat cgtctggaag atttatttgg tcgttatcgt 900
ccggaagttg ttcgtgcaca gatgaatctg ctgaccagcc atgatacacc gcgtctgctg 960
agcctgatgc gtggtagcgt tgaacgtgcc cgtctggcac tggccctgct gtttctgctg 1020
cctggtaatc cgaccgttta ttatggtgaa gaggttggta tggcaggcgg taaagatcct 1080
gaaaatcgtg gtggtatggt gtgggaagaa gcccgttggc agaaagatct gcgtgaaacc 1140
gttaaacgcc tggcacgtct gcgtaaagaa catccggcac tgcgtaccgc accttatctg 1200
cgtatttatg cacaggatgg tcatctggca tttgcacgtg gtccgtatct ggttgttgtt 1260
aatgcaagtc cgcatccgtt tcgtcaggat tttcctctgc atggtgtgtt tccgcgtggt 1320
ggtcgtgcag ttgatctgct gagtggtgaa gtttgtacac cgcaaggtgg tcgtctgtgt 1380
ggtccggttc tgcctccgtt tagcctggca ctgtggcgtg aagcataa 1428
<210> 3
<211> 501
<212> PRT
<213> Meiothermus ruber DSM 1279
<220>
<223> alpha-Glucanotransferase
<400> 3
Met Gln Leu Gln Arg Ala Phe Gly Ile Leu Leu His Pro Thr Ser Phe
1 5 10 15
Pro Gly Arg Trp Gly Ile Gly Ala Leu Gly Arg Glu Ala Glu Arg Phe
20 25 30
Leu Asp Trp Leu Ala Asp Ala Gly Ala Arg Trp Trp Gln Val Leu Pro
35 40 45
Leu Gly Pro Thr Ser Tyr Gly Asp Ser Pro Tyr Gln Ser Phe Ser Ala
50 55 60
Phe Ala Gly Asn Pro Tyr Leu Val Asp Pro Glu Met Leu Ile Glu Lys
65 70 75 80
Gly Trp Leu Glu Gln Ser Glu Ala Pro Pro Pro Tyr Pro Thr Gln Arg
85 90 95
Val Asp Tyr Gly Trp Leu Tyr Gln Thr Arg Trp Pro Leu Leu Arg Arg
100 105 110
Ala Phe Ala Gly Phe Arg Ala Arg Ala Ser Ala Gln Asp Lys Thr Arg
115 120 125
Leu Glu Ala Phe Ile Glu Ala Glu Arg Phe Trp Leu Glu Asp Tyr Ala
130 135 140
Leu Phe Met Ala Leu Lys Thr Arg Phe Asp Gly Lys Pro Trp Asn Glu
145 150 155 160
Trp Ser Pro Glu Leu Arg Asp Arg Glu Pro Ala Ala Leu Ala Arg Ala
165 170 175
Arg Glu Glu Leu Ala Glu Glu Val Ala Leu Tyr Glu Trp Ile Gln Trp
180 185 190
Leu Phe Tyr Leu Glu Trp Gly Gln Thr Lys Ala Tyr Ala Glu Ser Lys
195 200 205
Gly Ile Gln Ile Ile Gly Asp Met Pro Ile Phe Val Ala Phe Asp Ser
210 215 220
Ser Asp Val Trp Ala Asn Pro Gln Tyr Phe Tyr Leu Glu Ala Asp Gly
225 230 235 240
Asn Pro Thr Val Val Ala Gly Val Pro Pro Asp Tyr Phe Ser Glu Thr
245 250 255
Gly Gln Leu Trp Gly Asn Pro Leu Tyr Arg Trp Asp Val Met Glu Arg
260 265 270
Asp Asn Phe Ala Trp Trp Ile Ala Arg Ile Arg Gln Ser Leu Lys Gln
275 280 285
Cys His Leu Val Arg Ile Asp His Phe Arg Gly Phe Glu Ala Tyr Trp
290 295 300
Glu Val Pro Phe Gly Arg Pro Asn Ala Val Glu Gly Arg Trp Val Lys
305 310 315 320
Ala Pro Gly Glu Lys Leu Phe Ala Ala Val Arg Ala Gln Leu Ser Asp
325 330 335
Ala Pro Ile Ile Ala Glu Asp Leu Gly Val Ile Thr Pro Glu Val Glu
340 345 350
Ala Leu Arg Asp Gly Phe Gly Phe Pro Gly Met Lys Ile Leu Gln Phe
355 360 365
Ala Phe Ser Gly Glu Asp Asn Ala Phe Leu Pro His Asn Tyr Pro Ala
370 375 380
His Gly Asn Val Val Val Tyr Ser Gly Thr His Asp Asn Asp Thr Thr
385 390 395 400
Leu Gly Trp Phe Arg Thr Ala Pro Glu Ala Glu Arg Ala Phe Met Arg
405 410 415
Ala Tyr Leu Ala Arg Tyr Gly Ile Arg Cys Leu Ser Glu Tyr Glu Val
420 425 430
Ala Gly Ala Leu Ile Glu Leu Ala Phe Lys Ser Pro Ala Lys Leu Ala
435 440 445
Ile Val Pro Leu Gln Asp Val Leu Gly Leu Gly Pro Glu Ala Arg Met
450 455 460
Asn Phe Pro Gly Arg Leu Gly Asp Asn Trp Ala Trp Arg Tyr Ala Glu
465 470 475 480
Gly Asp Leu Glu Pro Gly Leu Ala Ala Gly Leu Arg Ala Leu Ala Glu
485 490 495
Ala Ser Gln Arg Ala
500
<210> 4
<211> 1506
<212> DNA
<213> Meiothermus ruber DSM 1279
<220>
<223> coding for alpha-Glucanotransferase
<400> 4
atgcaactcc aacgcgcttt tggaattttg ctccacccca ccagttttcc gggtcgctgg 60
gggattgggg ctctgggccg cgaggccgag cggtttttgg actggctggc cgatgcggga 120
gcccgctggt ggcaggtctt accgctgggc cctaccagtt acggcgactc gccgtaccag 180
tccttctcgg cttttgccgg taacccgtat ttggttgacc ccgagatgct gattgaaaaa 240
ggctggctgg aacaaagcga agcgcccccg ccgtatccga cccagcgcgt ggattatggc 300
tggctttacc agacccgctg gcccctgttg cggcgggctt tcgcggggtt tcgggcaagg 360
gcttcggccc aggataagac ccgactggaa gcctttatcg aggccgagcg cttctggctg 420
gaagactatg cgctctttat ggccctcaag acccggtttg acggcaagcc ctggaacgag 480
tggagccccg agctgcgcga ccgtgaaccg gctgccctgg ccagggcccg tgaggagctg 540
gccgaggagg tggcccttta cgagtggatt cagtggcttt tttatctgga atggggccag 600
accaaggcct atgccgaatc caaggggatt cagattatcg gcgatatgcc catctttgtg 660
gccttcgatt cctcagatgt ctgggccaac ccgcagtact tctacctcga ggccgatggc 720
aaccccacgg tggtggcggg cgttccgccg gactacttct ccgaaaccgg ccagctctgg 780
ggcaatccgc tctatcgctg ggatgtgatg gaaagggaca actttgcctg gtggattgcc 840
cgcataaggc agtcgctcaa gcagtgccac ctggtgcgca tcgaccactt ccgcgggttt 900
gaagcctact gggaggttcc gtttggccgg cccaatgctg tggaggggcg ctgggtcaaa 960
gccccagggg agaagctgtt tgctgcggtg cgggcccaac tgagcgatgc gcccatcatt 1020
gccgaagacc tgggggtgat cacccccgag gtggaggctt tgcgcgatgg cttcgggttc 1080
cccggcatga agattttgca gtttgctttt tccggtgagg acaacgcctt tttgccccac 1140
aactaccccg cgcacggcaa tgtggtggtg tacagcggaa cccacgacaa cgacaccacc 1200
ctgggatggt tccgcaccgc gccggaggcc gagcgggcct tcatgcgggc ctacctggcc 1260
cgctatggca tccgttgttt gtcggaatac gaggtcgcgg gcgctttgat cgagctggcc 1320
ttcaaaagcc cggccaagct ggctattgtg cctttgcagg acgtgctggg gctgggcccc 1380
gaggcccgca tgaacttccc cggacggctg ggggacaact gggcgtggcg ctacgccgaa 1440
ggcgacctcg agcccggtct ggccgcggga ctgcgggccc tggccgaggc cagccagcgc 1500
gcttga 1506
<210> 5
<211> 822
<212> PRT
<213> Thermotoga naphthophila DSM 13996
<220>
<223> alpha-Glucan Phosphorylase
<400> 5
Met Leu Glu Lys Leu Pro Glu Asn Leu Lys Glu Leu Glu Ser Leu Ala
1 5 10 15
Tyr Asn Leu Trp Trp Ser Trp Ser Arg Pro Ala Gln Arg Leu Trp Arg
20 25 30
Met Ile Asp Ser Glu Lys Trp Glu Glu His Arg Asn Pro Val Lys Ile
35 40 45
Leu Arg Glu Val Ser Lys Glu Arg Leu Glu Glu Leu Ser Lys Asp Glu
50 55 60
Asp Phe Ile Ala Leu Tyr Glu Leu Thr Leu Glu Arg Phe Thr Asp Tyr
65 70 75 80
Met Glu Arg Glu Asp Thr Trp Phe Asn Val Asn Tyr Pro Glu Trp Asp
85 90 95
Glu Lys Ile Val Tyr Met Cys Met Glu Tyr Gly Leu Thr Lys Ala Leu
100 105 110
Pro Ile Tyr Ser Gly Gly Leu Gly Ile Leu Ala Gly Asp His Leu Lys
115 120 125
Ser Ala Ser Asp Leu Gly Leu Pro Leu Ile Ala Val Gly Leu Leu Tyr
130 135 140
Lys His Gly Tyr Phe Thr Gln Gln Ile Asp Ser Asp Gly Arg Gln Ile
145 150 155 160
Glu Ile Phe Pro Glu Tyr Asp Ile Glu Glu Leu Pro Met Lys Pro Leu
165 170 175
Arg Asp Glu Asp Gly Asn Gln Val Ile Val Glu Val Pro Ile Asp Asn
180 185 190
Asp Thr Val Lys Ala Arg Val Phe Glu Val Gln Val Gly Arg Val Lys
195 200 205
Leu Tyr Leu Leu Asp Thr Asp Phe Glu Glu Asn Glu Asp Arg Phe Arg
210 215 220
Lys Ile Cys Asp Tyr Leu Tyr Asn Pro Glu Pro Asp Val Arg Val Ser
225 230 235 240
Gln Glu Ile Leu Leu Gly Ile Gly Gly Met Lys Leu Leu Lys Thr Leu
245 250 255
Lys Ile Lys Pro Gly Val Ile His Leu Asn Glu Gly His Pro Ala Phe
260 265 270
Ser Ser Leu Glu Arg Ile Lys Ser Tyr Met Glu Glu Gly Tyr Ser Phe
275 280 285
Thr Glu Ala Leu Glu Ile Val Arg Gln Thr Thr Val Phe Thr Thr His
290 295 300
Thr Pro Val Pro Ala Gly His Asp Arg Phe Pro Phe Asp Phe Val Glu
305 310 315 320
Lys Lys Leu Thr Lys Phe Phe Glu Gly Phe Glu Ser Lys Glu Leu Leu
325 330 335
Met Asn Leu Gly Lys Asp Glu Asp Gly Asn Phe Asn Met Thr Tyr Leu
340 345 350
Ala Leu Arg Thr Ser Ser Phe Ile Asn Gly Val Ser Lys Leu His Ala
355 360 365
Asp Val Ser Arg Arg Met Phe Lys Asn Val Trp Lys Gly Val Pro Val
370 375 380
Glu Glu Ile Pro Ile Glu Gly Ile Thr Asn Gly Val His Met Gly Thr
385 390 395 400
Trp Ile Asn Arg Glu Met Arg Lys Leu Phe Asp Arg Tyr Leu Gly Arg
405 410 415
Val Trp Arg Glu His Thr Asp Leu Glu Gly Ile Trp Tyr Gly Val Asp
420 425 430
Arg Ile Pro Asp Glu Glu Leu Trp Glu Ala His Leu Asn Ala Lys Lys
435 440 445
Arg Phe Ile Asp Tyr Ile Arg Glu Ser Ile Lys Arg Arg Asn Glu Arg
450 455 460
Leu Gly Ile Asn Glu Pro Leu Pro Glu Ile Ser Glu Asn Val Leu Ile
465 470 475 480
Ile Gly Phe Ala Arg Arg Phe Ala Thr Tyr Lys Arg Ala Val Leu Leu
485 490 495
Phe Ser Asp Leu Glu Arg Leu Lys Arg Ile Val Asn Asn Ser Glu Arg
500 505 510
Pro Val Tyr Ile Val Tyr Ala Gly Lys Ala His Pro Arg Asp Glu Gly
515 520 525
Gly Lys Glu Phe Leu Arg Arg Ile Tyr Glu Val Ser Gln Met Pro Asp
530 535 540
Phe Lys Asn Lys Ile Ile Val Leu Glu Asn Tyr Asp Ile Gly Met Ala
545 550 555 560
Arg Leu Met Val Ser Gly Val Asp Val Trp Leu Asn Asn Pro Arg Arg
565 570 575
Pro Met Glu Ala Ser Gly Thr Ser Gly Met Lys Ala Ala Ala Asn Gly
580 585 590
Val Leu Asn Ala Ser Val Tyr Asp Gly Trp Trp Val Glu Gly Tyr Asn
595 600 605
Gly Arg Asn Gly Trp Val Ile Gly Asp Glu Ser Val Leu Pro Glu Thr
610 615 620
Glu Ala Asp Asp Pro Lys Asp Ala Glu Ala Leu Tyr Glu Leu Leu Glu
625 630 635 640
Asn Glu Ile Ile Pro Thr Tyr Tyr Glu Asn Arg Glu Lys Trp Ile Phe
645 650 655
Met Met Lys Glu Ser Ile Lys Ser Val Ala Pro Lys Phe Ser Thr Thr
660 665 670
Arg Met Leu Lys Glu Tyr Thr Glu Lys Phe Tyr Ile Lys Gly Leu Val
675 680 685
Asn Arg Glu Trp Leu Glu Arg Arg Glu Asn Val Glu Lys Ile Gly Ala
690 695 700
Trp Lys Glu Arg Ile Leu Lys Asn Trp Glu Asn Val Ser Ile Glu Arg
705 710 715 720
Ile Val Leu Glu Asp Ser Lys Ser Val Glu Val Thr Val Lys Leu Gly
725 730 735
Asp Leu Thr Pro Asn Asp Val Ile Val Glu Leu Val Ala Gly Arg Gly
740 745 750
Glu Gly Met Glu Asp Leu Glu Val Trp Lys Val Ile His Ile Arg Arg
755 760 765
Tyr Arg Lys Glu Asn Asp Leu Phe Val Tyr Thr Tyr Thr Asn Gly Val
770 775 780
Leu Gly His Leu Gly Ser Pro Gly Trp Phe Tyr Ala Val Arg Val Ile
785 790 795 800
Pro Tyr His Pro Arg Leu Pro Ile Lys Phe Leu Pro Glu Val Pro Val
805 810 815
Val Trp Lys Lys Val Leu
820
<210> 6
<211> 2469
<212> DNA
<213> Thermotoga naphthophila DSM 13996
<220>
<223> coding for alpha-Glucan Phosphorylase
<400> 6
atgctggaga aacttcccga gaacctgaaa gagctcgaga gccttgccta caacctctgg 60
tggagctggt ccagacctgc tcagagactc tggagaatga tcgattcaga aaagtgggag 120
gaacacagaa atcccgtcaa aatactgaga gaagtctcaa aggaaagact ggaagaacta 180
tcgaaagacg aggacttcat cgctctctac gaactgacgc tcgagagatt cacagactac 240
atggaaaggg aagacacctg gttcaacgtg aactatcccg aatgggacga aaagatagtt 300
tacatgtgta tggaatacgg actgacgaaa gcacttccga tctactctgg aggactcggt 360
atccttgccg gagaccacct caaatcagcc agtgatcttg gccttcctct catagccgta 420
ggtcttcttt acaaacacgg gtatttcact caacagatag acagtgacgg aagacagatc 480
gagatctttc cagaatacga catcgaagaa ctcccgatga aacctctcag ggatgaagac 540
ggaaaccagg tgatcgtaga agtacccata gacaacgata ctgtaaaagc gcgtgtgttc 600
gaggtacagg tcggaagggt gaaactgtat cttctcgaca ctgacttcga ggaaaacgag 660
gatagattca gaaagatctg cgactatctc tacaatcccg agcctgatgt gagagtttcc 720
caggaaattc tgctcggcat tggtggaatg aaactcctga agactctcaa gataaaacct 780
ggagtcatcc acctgaacga aggtcatccc gctttttcat ccctcgaaag gataaagagc 840
tacatggaag aaggatattc cttcaccgag gcccttgaga tcgtcagaca gaccacagtt 900
ttcacgacac acacccccgt ccccgcaggt cacgacaggt tcccgttcga tttcgtggaa 960
aagaagctga caaagttctt cgaaggattc gaatccaaag aactgcttat gaaccttgga 1020
aaagacgaag acggaaattt caacatgacg tatcttgctt tgagaacctc ctcctttata 1080
aacggagtga gcaaacttca cgctgacgta tcgagaagga tgttcaaaaa tgtctggaag 1140
ggagttccgg tggaggagat ccccattgaa ggcatcacga atggtgtcca catgggaacc 1200
tggatcaacc gcgagatgag aaaactgttc gacaggtacc tcggtagagt ctggagggaa 1260
cacactgacc tcgaaggaat atggtacgga gttgacagaa tacccgatga agaactctgg 1320
gaagcgcatc tgaacgcaaa gaaacgattc atagattaca taagagaatc catcaaaagg 1380
agaaacgaaa ggcttggaat caacgaacca ctgccggaga tcagtgaaaa cgtgctcatc 1440
ataggttttg ccagaaggtt cgcaacttac aagagagccg tcctgctctt cagcgatctg 1500
gaaagactca agagaattgt caataattcc gagaggccgg tttacattgt gtacgctgga 1560
aaggcccacc cgagagacga aggtggaaag gagtttctca gaaggatcta cgaagtttca 1620
cagatgcccg atttcaagaa caaaatcatc gtactcgaaa actatgacat cggaatggct 1680
cgactcatgg tgtcgggtgt tgacgtgtgg ttgaacaatc caaggaggcc catggaggca 1740
agcgggacaa gtggtatgaa agctgcagcg aacggtgttc tgaacgcgag tgtatacgat 1800
ggctggtggg ttgagggata caacggcaga aacggatggg tgataggtga tgaaagcgtg 1860
cttccagaaa cagaagcgga tgatccaaag gatgccgagg ctctgtatga gcttctcgaa 1920
aacgaaataa tccccaccta ctacgaaaac agagaaaagt ggatcttcat gatgaaagaa 1980
agcataaaga gcgtggctcc aaaattcagc accacccgca tgctcaaaga gtacacggag 2040
aaattctaca taaagggact tgtgaacagg gaatggctgg agagaagaga aaacgtcgaa 2100
aaaatcggag cctggaaaga aagaatcctc aagaactggg agaatgtttc catagagcgc 2160
atcgttcttg aagattcgaa gagcgtagaa gtaactgtaa aactgggcga tctcacaccg 2220
aacgacgtga tagtcgaact tgtggctgga agaggagagg gaatggaaga tctcgaagtg 2280
tggaaagtga tacacatcag aaggtacagg aaagagaacg atctattcgt ttacacttac 2340
accaatggtg ttcttggtca tcttggatct cccggatggt tctacgcggt gagagtcata 2400
ccgtaccatc ccaggcttcc catcaagttc ctgcccgaag taccggttgt ctggaagaag 2460
gttctctga 2469
<210> 7
<211> 455
<212> PRT
<213> Saccharolobus solfataricus
<220>
<223> Phosphoglucomutase
<400> 7
Met Gly Lys Leu Phe Gly Thr Asp Gly Ile Arg Gly Val Thr Asn Thr
1 5 10 15
Glu Leu Gln Pro Glu Phe Ala Leu Lys Ile Gly Lys Ala Ile Gly Thr
20 25 30
Tyr Phe Gly Lys Gly Ala Arg Ile Leu Ile Gly Arg Asp Val Arg Ala
35 40 45
Gly Gly Asp Met Leu Leu Lys Ala Val Glu Ser Gly Leu Leu Ser Ser
50 55 60
Gly Val Leu Val Tyr Glu Ala Gly Met Ala Pro Thr Pro Ala Phe Gln
65 70 75 80
Tyr Gly Val Lys Ala Leu Gly Tyr Asp Gly Gly Val Ile Ile Thr Ala
85 90 95
Ser His Asn Pro Ala Glu Tyr Asn Gly Ile Lys Val Leu Ser Pro His
100 105 110
Gly Ile Glu Ile Ser Arg Glu Asp Glu Asp Lys Ile Glu Asp Ile Tyr
115 120 125
Phe Asn Ser Arg Phe His Val Val Glu Trp Asn Gly Leu Val Asn Asp
130 135 140
Val Lys Arg Glu Asp Lys Val Ile Glu Thr Tyr Leu Arg Gly Ile Leu
145 150 155 160
Ser His Val Asp Thr Glu Lys Ile Lys Ser Lys Lys Tyr Lys Val Leu
165 170 175
Ile Asp Pro Ala Asn Ser Val Gly Thr Leu Val Thr Pro Ile Val Ala
180 185 190
Arg Glu Leu Gly Cys Lys Val Phe Thr Ile Asn Gly Asn Leu Asp Pro
195 200 205
Leu Phe Ser Ala Arg Thr Pro Glu Pro Thr Phe Glu Ser Leu Thr Glu
210 215 220
Ser Ala Lys Val Ala Lys Gln Leu Gly Val Asp Leu Ala Val Ala His
225 230 235 240
Asp Gly Asp Ala Asp Arg Ala Ile Phe Ile Asp Ser Met Gly Arg Val
245 250 255
Gln Trp Gly Asp Arg Ser Gly Thr Leu Leu Ser Tyr Trp Ala Ser Val
260 265 270
Lys Ala Pro Asn Leu Pro Arg Arg Ile Phe Thr Ala Val Ser Ser Ser
275 280 285
Ser Leu Val Glu Glu Tyr Leu Lys Lys Tyr Asn Ile Glu Val Lys Trp
290 295 300
Thr Lys Val Gly Ser Ile Asp Ile Ala His Met Leu Phe Lys Glu Lys
305 310 315 320
Gly Val Ala Gly Phe Glu Glu Asn Gly Gly Phe Met Tyr Pro Pro His
325 330 335
Gln Thr Val Arg Asp Gly Ala Met Ser Phe Ala Leu Met Leu Asp Met
340 345 350
Met Ala Ser Glu Asn Glu Ser Ser Ala Glu Leu Phe Asn Arg Leu Pro
355 360 365
Val Tyr Tyr Leu Val Lys Thr Lys Val Arg Val Thr Glu Lys Ser Asp
370 375 380
Ile Lys Lys Ile Tyr Asp Glu Val Met Asn Lys Tyr Gly Asn Tyr Gly
385 390 395 400
Asn Thr Val Thr Ile Asp Gly Val Lys Ile Ile Gly Ser Asp Phe Trp
405 410 415
Leu Leu Val Arg Lys Ser Gly Thr Glu Pro Ile Ile Arg Ile Leu Val
420 425 430
Glu Ala Lys Asp Glu Asn Lys Ser Lys Glu Leu Ala Lys Glu Leu Glu
435 440 445
Lys Leu Val Ser Glu Leu Val
450 455
<210> 8
<211> 1368
<212> DNA
<213> Saccharolobus solfataricus
<220>
<223> coding for Phosphoglucomutase
<400> 8
atgggtaagt tgtttggaac tgatggaata agaggagtta ccaatactga attacaacca 60
gagtttgcgt taaagattgg aaaagctata ggaacatatt ttggtaaagg tgctagaatt 120
ctaataggta gggatgtaag agcaggagga gatatgctac ttaaagctgt tgagagtgga 180
ttgttaagta gtggtgtact ggtttatgaa gcagggatgg ctcctactcc agctttccaa 240
tatggtgtga aggctttagg atatgacggt ggtgttataa taactgccag tcataatcca 300
gctgaatata atggtatcaa agtcctttct ccacacggta ttgagatatc tagagaagat 360
gaggataaga tagaagatat atactttaat agcaggtttc atgtggtaga gtggaatggt 420
ttagtaaacg atgtgaaaag agaagacaag gttattgaga cttatttaag aggtattctc 480
tctcatgttg atactgaaaa aattaagagt aaaaagtata aagttctcat agatcctgct 540
aatagtgtag gtacattagt aacgcccatt gttgcaaggg agttgggatg taaagttttt 600
actattaatg ggaatttaga tcctttattt tcagctagaa ctcctgaacc tactttcgag 660
agccttaccg aatccgcaaa agttgctaaa caattaggag ttgatttagc agtggctcat 720
gatggtgatg cagatagggc aatttttata gattccatgg gtagggtcca atggggtgat 780
aggagcggta ctctattatc atattgggcc tcggttaaag ctccaaattt acctagacgc 840
atatttactg cagtttctag ctctagcttg gttgaggagt atctgaaaaa gtataatata 900
gaggttaagt ggactaaggt aggtagcata gatattgcgc atatgttatt taaagaaaaa 960
ggagtagctg gatttgagga aaatggtggg ttcatgtatc cccctcatca gacggtaaga 1020
gatggcgcaa tgtcgttcgc cttaatgctg gatatgatgg catctgagaa cgaaagttct 1080
gctgagctat ttaatagact ccccgtttat tatttggtaa aaacgaaagt tagagttacg 1140
gaaaagagtg atataaagaa gatttatgat gaagtaatga ataagtacgg aaattacggg 1200
aatactgtta caatagatgg tgttaagatt ataggtagtg atttctggct cttggttagg 1260
aaaagtggta cggagccgat aataagaata ctagttgagg caaaagatga gaataaatct 1320
aaagaattag ctaaagaatt ggaaaagtta gtgagtgaac tagtatga 1368
<210> 9
<211> 578
<212> PRT
<213> Clostridium thermocellum
<220>
<223> Phosphoglucomutase
<400> 9
Met Arg Ser Ser Ala Leu Tyr Lys Phe Trp Val Glu Asn Asp Tyr Phe
1 5 10 15
Asp Ala Glu Thr Lys Lys Glu Leu Leu Ser Ile Lys Asp Asn Pro Lys
20 25 30
Glu Ile Glu Glu Arg Phe Tyr Lys Asp Leu Glu Phe Gly Thr Gly Gly
35 40 45
Leu Arg Gly Ile Ile Gly Ala Gly Thr Asn Arg Ile Asn Ile Tyr Thr
50 55 60
Val Arg Lys Ala Ser Gln Gly Leu Ala Asp Tyr Ile Lys Ser Leu Gly
65 70 75 80
Leu Gln Asp Arg Gly Ile Ala Ile Ala Tyr Asp Ser Arg Tyr Lys Ser
85 90 95
Pro Glu Phe Ala Leu Glu Ala Ala Lys Val Phe Ala Gly Asn Gly Ile
100 105 110
Lys Ala Phe Leu Phe Asp Glu Leu Arg Pro Thr Pro Glu Leu Ser Phe
115 120 125
Thr Val Arg His Leu Asn Ala Ala Ala Gly Val Val Ile Thr Ala Ser
130 135 140
His Asn Pro Lys Glu Tyr Asn Gly Tyr Lys Val Tyr Gly Glu Asp Gly
145 150 155 160
Gly Gln Leu Pro Val Glu Ala Ser Asn Lys Val Ile Ser Tyr Ile Asn
165 170 175
Lys Ile Glu Asp Ile Thr Gln Val Lys Val Met Glu Lys Asp Glu Ala
180 185 190
Ile Glu Lys Gly Leu Leu Arg Ile Ile Gly Lys Glu Ile Asp Asp Glu
195 200 205
Tyr Ile Ser Lys Leu Lys Thr Leu Ser Ala Asn Pro Glu Leu Ala Ala
210 215 220
Glu Ile Gly Lys Thr Phe Lys Ile Val Tyr Thr Pro Leu His Gly Ala
225 230 235 240
Gly Asn Lys Pro Val Arg Arg Ile Leu Asp Glu Ile Gly Phe Lys Asn
245 250 255
Val Leu Val Val Lys Glu Gln Glu Leu Pro Asp Ser Glu Phe Ser Thr
260 265 270
Val Lys Ser Pro Asn Pro Glu Glu Arg Glu Ala Phe Glu Leu Ala Ile
275 280 285
Glu Leu Ala Lys Lys Glu Asn Val Asp Leu Ile Ile Gly Thr Asp Pro
290 295 300
Asp Cys Asp Arg Val Gly Ile Val Val Arg Asn Lys Glu Gly Glu Tyr
305 310 315 320
Val Pro Leu Thr Gly Asn Gln Thr Gly Cys Leu Leu Leu Glu Tyr Ile
325 330 335
Leu Ser Gln Lys Lys Gln Arg Gly Glu Leu Pro Glu Asn Gly Phe Val
340 345 350
Val Lys Thr Ile Val Thr Thr Glu Leu Ala Arg Ala Ile Thr Asp Ala
355 360 365
Tyr Asn Val Glu Leu Val Glu Val Leu Thr Gly Phe Lys Phe Ile Gly
370 375 380
Glu Lys Ile Lys Gln Leu Asp Glu Phe Gly Asp Lys Lys Tyr Leu Phe
385 390 395 400
Gly Phe Glu Glu Ser Tyr Gly Tyr Leu Ala Gly Thr Phe Ala Arg Asp
405 410 415
Lys Asp Ala Val Val Ala Ser Met Leu Ile Ala Glu Met Ala Ala Tyr
420 425 430
Tyr Lys Ser Arg Gly Leu Thr Leu Tyr Glu Gly Leu Met Glu Leu Leu
435 440 445
Glu Lys Tyr Gly Tyr Thr Leu Glu Gly Ile Thr Ser Phe Thr Leu Lys
450 455 460
Gly Lys Asp Gly Val Glu Lys Ile Lys Ser Ala Met Lys Asn Leu Arg
465 470 475 480
Glu Asn Arg Val Val Lys Phe Gly Glu Tyr Glu Ala Val Ala Val Arg
485 490 495
Asp Tyr Leu Thr Ser Glu Arg Tyr Glu Val Ala Thr Gly Ala Lys Glu
500 505 510
Lys Leu Thr Leu Val Glu Ser Asp Val Leu Tyr Tyr Glu Leu Lys Asp
515 520 525
Lys Ala Trp Phe Cys Ile Arg Pro Ser Gly Thr Glu Pro Lys Ile Lys
530 535 540
Ile Tyr Tyr Gly Val Thr Glu Lys Ser Met Asp Ala Ala Lys Glu Lys
545 550 555 560
Leu Lys His Leu Gln Asp Asn Val Leu Ser Val Ile Glu Pro Leu Leu
565 570 575
Lys Asp
<210> 10
<211> 1737
<212> DNA
<213> Chlostridium thermocellum
<220>
<223> coding for Phosphoglucomutase
<400> 10
atgcgaagta gcgcgcttta taaattttgg gtggagaatg attattttga tgcggaaaca 60
aagaaagaac ttttgagtat taaagacaat ccgaaggaaa tagaagagag gttttacaag 120
gatttggaat ttggtaccgg cggacttagg ggaattatcg gtgccgggac caacagaatc 180
aatatttaca ccgtcagaaa agcttcccag ggacttgcgg attatataaa gagtcttgga 240
ttgcaagaca ggggaatagc aattgcttat gattcccgtt acaaatcccc ggaatttgct 300
ttggaagctg ccaaggtttt tgcgggaaac ggcataaaag cgtttctttt tgatgaattg 360
agaccaacac cggaactttc ttttaccgta aggcatttaa atgcagctgc cggtgtggtt 420
attacggcga gccataatcc caaggagtat aatggataca aagtttatgg agaagacggg 480
ggacaacttc ctgtagaggc atcaaacaaa gttatatcct atataaataa aattgaggat 540
ataactcagg ttaaggttat ggaaaaggac gaagcgatag agaagggttt gctcaggatt 600
ataggcaagg aaattgacga tgagtatata tcgaaattaa aaactctttc tgcaaaccct 660
gaactggctg cagaaatagg caaaactttc aaaatagtgt acacgccgtt gcatggcgca 720
ggaaacaaac ctgtaagaag aatacttgac gaaattggat ttaaaaatgt acttgtggtc 780
aaagaacagg agcttccgga cagcgaattt tccacggtaa aatcacccaa tccggaggaa 840
agggaagcgt ttgagcttgc cattgagctg gcaaaaaaag aaaatgttga ccttatcata 900
ggtaccgacc ctgactgtga cagagtgggt attgtcgtaa gaaacaaaga aggcgagtat 960
gtgcctctta cgggtaatca gacgggttgt ttgctgcttg agtacatact gtctcaaaag 1020
aagcaaagag gagagcttcc tgaaaacgga tttgtcgtaa agacaatagt tacaacggag 1080
cttgccagag ctattactga tgcttacaat gtggaacttg tggaagtttt gacgggattt 1140
aagtttatag gcgagaaaat taagcagctg gatgagtttg gagacaaaaa atatcttttt 1200
ggttttgaag aaagctatgg ataccttgca ggaacttttg caagagacaa ggacgctgtt 1260
gttgcatcca tgcttattgc cgaaatggcg gcttattaca aatcaagagg tctgactctt 1320
tacgagggac tgatggagtt acttgagaaa tatggataca ctcttgaagg aattacttcg 1380
tttactctca agggaaaaga cggcgtggaa aagataaagt cggctatgaa aaaccttaga 1440
gaaaacagag ttgtaaaatt tggagaatat gaagccgttg ccgtaaggga ctatttgaca 1500
agtgaacggt atgaagtggc aacgggtgcg aaagagaaac ttacacttgt tgagtcggat 1560
gtgttgtatt atgagcttaa ggataaagcc tggttctgta taagaccttc cggcacagag 1620
ccgaaaataa agatttatta tggtgttaca gaaaagagta tggatgctgc aaaagagaaa 1680
ttgaaacatt tgcaggataa tgtactgtca gttatagaac cgcttcttaa agactga 1737
<210> 11
<211> 448
<212> PRT
<213> Thermotoga maritima
<220>
<223> Phosphoglucoisomerase
<400> 11
Met Ser Leu Lys Phe Asp Phe Ser Asn Leu Phe Glu Pro Asn Ile Ser
1 5 10 15
Gly Gly Leu Thr Asp Glu Asp Val Lys Ser Val Glu Glu Lys Val Thr
20 25 30
Ser Ala Val Arg Asn Phe Val Glu Asn Thr Pro Asp Phe Ala Lys Leu
35 40 45
Asp Arg Ser Trp Ile Asp Ser Val Lys Ser Leu Glu Asp Trp Ile Ile
50 55 60
Asn Phe Asp Thr Val Val Val Leu Gly Ile Gly Gly Ser Gly Leu Gly
65 70 75 80
Asn Leu Ala Leu His Tyr Ser Leu Arg Pro Leu Asn Trp Asn Glu Met
85 90 95
Thr Arg Glu Glu Arg Asn Gly Tyr Ala Arg Val Phe Val Val Asp Asn
100 105 110
Val Asp Pro Asp Leu Met Ser Ser Val Leu Asp Arg Ile Asp Pro Lys
115 120 125
Thr Thr Leu Phe Asn Val Ile Ser Lys Ser Gly Ser Thr Ala Glu Val
130 135 140
Met Ala Thr Tyr Ser Ile Ala Arg Gly Ile Leu Glu Ala Tyr Gly Leu
145 150 155 160
Asp Pro Arg Glu His Met Leu Ile Thr Thr Asp Pro Glu Lys Gly Phe
165 170 175
Leu Arg Lys Leu Val Lys Glu Glu Gly Phe Arg Ser Leu Glu Val Pro
180 185 190
Pro Gly Val Gly Gly Arg Phe Ser Val Leu Thr Pro Val Gly Leu Leu
195 200 205
Ser Ala Met Ala Glu Gly Ile Asp Ile Asp Glu Leu His Glu Gly Ala
210 215 220
Lys Asp Ala Phe Glu Lys Ser Met Lys Glu Asn Ile Leu Glu Asn Pro
225 230 235 240
Ala Ala Met Ile Ala Leu Thr His Tyr Leu Tyr Leu Asn Lys Gly Lys
245 250 255
Ser Ile Ser Val Met Met Ala Tyr Ser Asn Arg Met Ile Tyr Leu Val
260 265 270
Asp Trp Tyr Arg Gln Leu Trp Ala Glu Ser Leu Gly Lys Arg Tyr Asn
275 280 285
Leu Lys Gly Glu Glu Val Phe Thr Gly Gln Thr Pro Val Lys Ala Leu
290 295 300
Gly Ala Thr Asp Gln His Ser Gln Ile Gln Leu Tyr Asn Glu Gly Pro
305 310 315 320
Asn Asp Lys Val Ile Thr Phe Leu Arg Val Glu Asn Phe Asp Arg Glu
325 330 335
Ile Val Ile Pro Glu Thr Gly Arg Ala Glu Leu Ser Tyr Leu Ala Arg
340 345 350
Lys Lys Leu Ser Glu Leu Leu Leu Ala Glu Gln Thr Gly Thr Glu Glu
355 360 365
Ala Leu Arg Glu Asn Asn Arg Pro Asn Met Arg Val Thr Phe Asp Gly
370 375 380
Leu Thr Pro Tyr Asn Val Gly Gln Phe Phe Ala Tyr Tyr Glu Ala Ala
385 390 395 400
Thr Ala Phe Met Gly Tyr Leu Leu Glu Ile Asn Pro Phe Asp Gln Pro
405 410 415
Gly Val Glu Leu Gly Lys Lys Ile Thr Phe Ala Leu Met Gly Arg Glu
420 425 430
Gly Tyr Thr Tyr Glu Ile Lys Glu Arg Ser Lys Lys Val Ile Ile Glu
435 440 445
<210> 12
<211> 1347
<212> DNA
<213> Thermotoga maritima
<220>
<223> coding for Phosphoglucoisomerase
<400> 12
atgagcctga aatttgattt tagcaacctg tttgaaccga atattagcgg tggtctgacc 60
gatgaagatg ttaaaagcgt ggaagaaaaa gttaccagcg cagtgcgtaa ttttgttgaa 120
aacacaccgg attttgcaaa actggatcgt agctggattg atagcgtgaa aagcctggaa 180
gattggatca ttaactttga taccgttgtt gttctgggta ttggtggtag cggtctgggt 240
aatctggcac tgcattatag cctgcgtccg ctgaattgga atgaaatgac ccgtgaagaa 300
cgtaatggtt atgcccgtgt gtttgttgtt gataatgtgg atccggatct gatgagcagc 360
gttctggatc gtattgatcc gaaaaccacc ctgtttaacg ttattagcaa aagcggtagc 420
accgcagaag ttatggcaac ctatagcatt gcacgtggta ttctggaagc ctatggtctg 480
gatccgcgtg aacacatgct gattaccacc gatccggaaa aaggttttct gcgcaaactg 540
gtgaaagaag aaggttttcg tagcctggaa gttccgcctg gtgttggtgg tcgttttagc 600
gttctgacac cggttggtct gctgagcgca atggcagaag gtattgatat cgacgaactg 660
catgaaggtg caaaagatgc ctttgaaaaa agcatgaaag aaaacatcct ggaaaatccg 720
gcagcaatga ttgcactgac ccattatctg tatctgaaca aaggcaaaag cattagcgtt 780
atgatggcct atagcaaccg tatgatttat ctggttgatt ggtatcgtca gctgtgggca 840
gaaagcctgg gtaaacgcta taatctgaaa ggcgaagaag tttttaccgg tcagacaccg 900
gttaaagcac tgggtgcaac cgatcagcat agccagattc agctgtataa tgaaggtccg 960
aacgataaag ttattacctt tctgcgcgtg gaaaattttg atcgcgaaat tgttattccg 1020
gaaaccggtc gtgcagaact gagctatctg gcacgtaaaa aactgagcga actgctgctg 1080
gcagaacaga ccggcaccga agaagcactg cgcgaaaaca atcgtccgaa tatgcgtgtt 1140
acctttgatg gtctgacccc gtataacgtt ggtcagtttt ttgcctatta cgaagcagca 1200
accgcattta tgggttatct gctggaaatt aatccgtttg atcagcctgg tgttgaactg 1260
ggcaaaaaaa tcacctttgc actgatgggt cgtgaaggct atacctatga aatcaaagaa 1320
cgcagcaaaa aagtgattat tgaataa 1347
<210> 13
<211> 216
<212> PRT
<213> Thermotoga naphthophila DSM 13996
<220>
<223> Phosphatase
<400> 13
Met Glu Ala Val Ile Phe Asp Met Asp Gly Val Leu Met Asp Thr Glu
1 5 10 15
Pro Leu Tyr Phe Glu Ala Tyr Arg Arg Val Ala Glu Ser Tyr Gly Lys
20 25 30
Pro Tyr Thr Glu Asp Leu His Arg Arg Ile Met Gly Val Pro Glu Arg
35 40 45
Glu Gly Leu Pro Ile Leu Met Glu Ala Leu Glu Ile Lys Asp Ser Leu
50 55 60
Glu Asn Phe Lys Lys Arg Val His Glu Glu Lys Lys Arg Val Phe Ser
65 70 75 80
Glu Leu Leu Lys Glu Asn Pro Gly Val Arg Glu Ala Leu Glu Phe Val
85 90 95
Lys Ser Lys Arg Ile Lys Leu Ala Leu Ala Thr Ser Thr Pro Gln Arg
100 105 110
Glu Ala Leu Glu Arg Leu Arg Arg Leu Asp Leu Glu Lys Tyr Phe Asp
115 120 125
Val Met Val Phe Gly Asp Gln Val Lys Asn Gly Lys Pro Asp Pro Glu
130 135 140
Ile Tyr Leu Leu Val Leu Glu Arg Leu Asn Val Val Pro Glu Lys Val
145 150 155 160
Val Val Phe Glu Asp Ser Lys Ser Gly Val Glu Ala Ala Lys Ser Ala
165 170 175
Gly Ile Glu Arg Ile Tyr Gly Val Val His Ser Leu Asn Asp Gly Lys
180 185 190
Ala Leu Leu Glu Ala Gly Ala Val Ala Leu Val Lys Pro Glu Glu Ile
195 200 205
Leu Asn Val Leu Lys Glu Val Leu
210 215
<210> 14
<211> 651
<212> DNA
<213> Thermotoga naphthophila DSM 13996
<220>
<223> coding for Phosphatase
<400> 14
atggaagcgg tgattttcga catggatgga gtgctcatgg acacagagcc tctctacttc 60
gaagcttaca gaagagtcgc ggaaagctat ggaaaacctt acacggagga tctccacagg 120
agaataatgg gagttcctga aagagaaggt cttcccatcc tcatggaagc tctggagata 180
aaagattctc tggagaactt caaaaagagg gtccacgaag aaaaaaagcg cgttttctct 240
gagcttctca aggaaaatcc gggtgtaaga gaggcgctcg agttcgtaaa gagcaaaaga 300
ataaaactcg cgctcgcaac ctccacacca cagcgagaag cgctggagag attgagaaga 360
ctcgatctcg aaaagtactt cgacgtcatg gtgttcggtg atcaggtgaa gaacggaaag 420
cctgatccag agatatacct tctcgttctg gaaaggttga atgtggtccc agagaaggtt 480
gtggtcttcg aagactcaaa gagcggtgtt gaagccgcaa aaagcgccgg catagaaaga 540
atctatggag tcgttcactc tttgaacgac ggtaaagcgc ttcttgaagc gggtgcggtt 600
gctctggtga aacccgagga aatcctgaac gttctcaaag aggttcttta a 651
<210> 15
<211> 216
<212> PRT
<213> Thermotoga naphthophila DSM 13996
<220>
<223> Phosphatase-variant P46T
<400> 15
Met Glu Ala Val Ile Phe Asp Met Asp Gly Val Leu Met Asp Thr Glu
1 5 10 15
Pro Leu Tyr Phe Glu Ala Tyr Arg Arg Val Ala Glu Ser Tyr Gly Lys
20 25 30
Pro Tyr Thr Glu Asp Leu His Arg Arg Ile Met Gly Val Thr Glu Arg
35 40 45
Glu Gly Leu Pro Ile Leu Met Glu Ala Leu Glu Ile Lys Asp Ser Leu
50 55 60
Glu Asn Phe Lys Lys Arg Val His Glu Glu Lys Lys Arg Val Phe Ser
65 70 75 80
Glu Leu Leu Lys Glu Asn Pro Gly Val Arg Glu Ala Leu Glu Phe Val
85 90 95
Lys Ser Lys Arg Ile Lys Leu Ala Leu Ala Thr Ser Thr Pro Gln Arg
100 105 110
Glu Ala Leu Glu Arg Leu Arg Arg Leu Asp Leu Glu Lys Tyr Phe Asp
115 120 125
Val Met Val Phe Gly Asp Gln Val Lys Asn Gly Lys Pro Asp Pro Glu
130 135 140
Ile Tyr Leu Leu Val Leu Glu Arg Leu Asn Val Val Pro Glu Lys Val
145 150 155 160
Val Val Phe Glu Asp Ser Lys Ser Gly Val Glu Ala Ala Lys Ser Ala
165 170 175
Gly Ile Glu Arg Ile Tyr Gly Val Val His Ser Leu Asn Asp Gly Lys
180 185 190
Ala Leu Leu Glu Ala Gly Ala Val Ala Leu Val Lys Pro Glu Glu Ile
195 200 205
Leu Asn Val Leu Lys Glu Val Leu
210 215
<210> 16
<211> 650
<212> DNA
<213> Thermotoga naphthophila DSM 13996
<220>
<223> coding for Phosphatase-variant P46T
<400> 16
atggaagcgg tgattttcga catggatgga gtgctcatgg acacagagcc tctctacttc 60
gaagcttaca gaagagtcgc ggaaagctat ggaaaacctt acacggagga tctccacagg 120
agaataatgg gagttactga aagagaaggt cttcccatcc tcatggaagc tctggagata 180
aaagattctc tggagaactt caaaaagagg gtccacgaag aaaaaaagcg cgttttctct 240
gagcttctca aggaaaatcc gggtgtaaga gaggcgctcg agttcgtaaa gagcaaaaga 300
ataaaactcg cgctcgcaac ctccacacca cagcgagaag cgctggagag attgagaaga 360
ctcgatctcg aaaagtactt cgacgtcatg gtgttcggtg atcaggtgaa gaacggaaag 420
cctgatccag agatatacct tctcgttctg gaaaggttga atgtggtccc agagaaggtt 480
gtggtcttcg aagactcaaa gagcggtgtt gaagccgcaa aaagcgccgg catagaaaga 540
atctatggag tcgttcactc tttgaacgac ggtaaagcgc ttcttgaagc gggtgcggtt 600
gctctggtga aacccgagga aatcctgaac gttctcaaag aggttcttta 650
<210> 17
<211> 216
<212> PRT
<213> Thermotoga naphthophila DSM 13996
<220>
<223> Phosphatase-variant P46TE47Y
<400> 17
Met Glu Ala Val Ile Phe Asp Met Asp Gly Val Leu Met Asp Thr Glu
1 5 10 15
Pro Leu Tyr Phe Glu Ala Tyr Arg Arg Val Ala Glu Ser Tyr Gly Lys
20 25 30
Pro Tyr Thr Glu Asp Leu His Arg Arg Ile Met Gly Val Thr Tyr Arg
35 40 45
Glu Gly Leu Pro Ile Leu Met Glu Ala Leu Glu Ile Lys Asp Ser Leu
50 55 60
Glu Asn Phe Lys Lys Arg Val His Glu Glu Lys Lys Arg Val Phe Ser
65 70 75 80
Glu Leu Leu Lys Glu Asn Pro Gly Val Arg Glu Ala Leu Glu Phe Val
85 90 95
Lys Ser Lys Arg Ile Lys Leu Ala Leu Ala Thr Ser Thr Pro Gln Arg
100 105 110
Glu Ala Leu Glu Arg Leu Arg Arg Leu Asp Leu Glu Lys Tyr Phe Asp
115 120 125
Val Met Val Phe Gly Asp Gln Val Lys Asn Gly Lys Pro Asp Pro Glu
130 135 140
Ile Tyr Leu Leu Val Leu Glu Arg Leu Asn Val Val Pro Glu Lys Val
145 150 155 160
Val Val Phe Glu Asp Ser Lys Ser Gly Val Glu Ala Ala Lys Ser Ala
165 170 175
Gly Ile Glu Arg Ile Tyr Gly Val Val His Ser Leu Asn Asp Gly Lys
180 185 190
Ala Leu Leu Glu Ala Gly Ala Val Ala Leu Val Lys Pro Glu Glu Ile
195 200 205
Leu Asn Val Leu Lys Glu Val Leu
210 215
<210> 18
<211> 651
<212> DNA
<213> Thermotoga naphthophila DSM 13996
<220>
<223> coding for Phosphatase-variant P46TE47Y
<400> 18
atggaagcgg tgattttcga catggatgga gtgctcatgg acacagagcc tctctacttc 60
gaagcttaca gaagagtcgc ggaaagctat ggaaaacctt acacggagga tctccacagg 120
agaataatgg gagttactta tagagaaggt cttcccatcc tcatggaagc tctggagata 180
aaagattctc tggagaactt caaaaagagg gtccacgaag aaaaaaagcg cgttttctct 240
gagcttctca aggaaaatcc gggtgtaaga gaggcgctcg agttcgtaaa gagcaaaaga 300
ataaaactcg cgctcgcaac ctccacacca cagcgagaag cgctggagag attgagaaga 360
ctcgatctcg aaaagtactt cgacgtcatg gtgttcggtg atcaggtgaa gaacggaaag 420
cctgatccag agatatacct tctcgttctg gaaaggttga atgtggtccc agagaaggtt 480
gtggtcttcg aagactcaaa gagcggtgtt gaagccgcaa aaagcgccgg catagaaaga 540
atctatggag tcgttcactc tttgaacgac ggtaaagcgc ttcttgaagc gggtgcggtt 600
gctctggtga aacccgagga aatcctgaac gttctcaaag aggttcttta a 651
<210> 19
<211> 216
<212> PRT
<213> Thermotoga naphthophila DSM 13996
<220>
<223> Phosphatase-variant P46TE47YG50L
<400> 19
Met Glu Ala Val Ile Phe Asp Met Asp Gly Val Leu Met Asp Thr Glu
1 5 10 15
Pro Leu Tyr Phe Glu Ala Tyr Arg Arg Val Ala Glu Ser Tyr Gly Lys
20 25 30
Pro Tyr Thr Glu Asp Leu His Arg Arg Ile Met Gly Val Thr Tyr Arg
35 40 45
Glu Leu Leu Pro Ile Leu Met Glu Ala Leu Glu Ile Lys Asp Ser Leu
50 55 60
Glu Asn Phe Lys Lys Arg Val His Glu Glu Lys Lys Arg Val Phe Ser
65 70 75 80
Glu Leu Leu Lys Glu Asn Pro Gly Val Arg Glu Ala Leu Glu Phe Val
85 90 95
Lys Ser Lys Arg Ile Lys Leu Ala Leu Ala Thr Ser Thr Pro Gln Arg
100 105 110
Glu Ala Leu Glu Arg Leu Arg Arg Leu Asp Leu Glu Lys Tyr Phe Asp
115 120 125
Val Met Val Phe Gly Asp Gln Val Lys Asn Gly Lys Pro Asp Pro Glu
130 135 140
Ile Tyr Leu Leu Val Leu Glu Arg Leu Asn Val Val Pro Glu Lys Val
145 150 155 160
Val Val Phe Glu Asp Ser Lys Ser Gly Val Glu Ala Ala Lys Ser Ala
165 170 175
Gly Ile Glu Arg Ile Tyr Gly Val Val His Ser Leu Asn Asp Gly Lys
180 185 190
Ala Leu Leu Glu Ala Gly Ala Val Ala Leu Val Lys Pro Glu Glu Ile
195 200 205
Leu Asn Val Leu Lys Glu Val Leu
210 215
<210> 20
<211> 651
<212> DNA
<213> Thermotoga naphthophila DSM 13996
<220>
<223> coding for Phosphatase-variant P46TE47YG50L
<400> 20
atggaagcgg tgattttcga catggatgga gtgctcatgg acacagagcc tctctacttc 60
gaagcttaca gaagagtcgc ggaaagctat ggaaaacctt acacggagga tctccacagg 120
agaataatgg gagttactta tagagaattg cttcccatcc tcatggaagc tctggagata 180
aaagattctc tggagaactt caaaaagagg gtccacgaag aaaaaaagcg cgttttctct 240
gagcttctca aggaaaatcc gggtgtaaga gaggcgctcg agttcgtaaa gagcaaaaga 300
ataaaactcg cgctcgcaac ctccacacca cagcgagaag cgctggagag attgagaaga 360
ctcgatctcg aaaagtactt cgacgtcatg gtgttcggtg atcaggtgaa gaacggaaag 420
cctgatccag agatatacct tctcgttctg gaaaggttga atgtggtccc agagaaggtt 480
gtggtcttcg aagactcaaa gagcggtgtt gaagccgcaa aaagcgccgg catagaaaga 540
atctatggag tcgttcactc tttgaacgac ggtaaagcgc ttcttgaagc gggtgcggtt 600
gctctggtga aacccgagga aatcctgaac gttctcaaag aggttcttta a 651
<210> 21
<211> 216
<212> PRT
<213> Thermotoga naphthophila DSM 13996
<220>
<223> Phosphatase-variant P46TE47YG50I
<400> 21
Met Glu Ala Val Ile Phe Asp Met Asp Gly Val Leu Met Asp Thr Glu
1 5 10 15
Pro Leu Tyr Phe Glu Ala Tyr Arg Arg Val Ala Glu Ser Tyr Gly Lys
20 25 30
Pro Tyr Thr Glu Asp Leu His Arg Arg Ile Met Gly Val Thr Tyr Arg
35 40 45
Glu Leu Leu Pro Ile Leu Met Glu Ala Leu Glu Ile Lys Asp Ser Leu
50 55 60
Glu Asn Phe Lys Lys Arg Val His Glu Glu Lys Lys Arg Val Phe Ser
65 70 75 80
Glu Leu Leu Lys Glu Asn Pro Gly Val Arg Glu Ala Leu Glu Phe Val
85 90 95
Lys Ser Lys Arg Ile Lys Leu Ala Leu Ala Thr Ser Thr Pro Gln Arg
100 105 110
Glu Ala Leu Glu Arg Leu Arg Arg Leu Asp Leu Glu Lys Tyr Phe Asp
115 120 125
Val Met Val Phe Gly Asp Gln Val Lys Asn Gly Lys Pro Asp Pro Glu
130 135 140
Ile Tyr Leu Leu Val Leu Glu Arg Leu Asn Val Val Pro Glu Lys Val
145 150 155 160
Val Val Phe Glu Asp Ser Lys Ser Gly Val Glu Ala Ala Lys Ser Ala
165 170 175
Gly Ile Glu Arg Ile Tyr Gly Val Val His Ser Leu Asn Asp Gly Lys
180 185 190
Ala Leu Leu Glu Ala Gly Ala Val Ala Leu Val Lys Pro Glu Glu Ile
195 200 205
Leu Asn Val Leu Lys Glu Val Leu
210 215
<210> 22
<211> 651
<212> DNA
<213> Thermotoga naphthophila DSM 13996
<220>
<223> coding for Phosphatase-variant P46TE47YG50I
<400> 22
atggaagcgg tgattttcga catggatgga gtgctcatgg acacagagcc tctctacttc 60
gaagcttaca gaagagtcgc ggaaagctat ggaaaacctt acacggagga tctccacagg 120
agaataatgg gagttactta tagagaaatt cttcccatcc tcatggaagc tctggagata 180
aaagattctc tggagaactt caaaaagagg gtccacgaag aaaaaaagcg cgttttctct 240
gagcttctca aggaaaatcc gggtgtaaga gaggcgctcg agttcgtaaa gagcaaaaga 300
ataaaactcg cgctcgcaac ctccacacca cagcgagaag cgctggagag attgagaaga 360
ctcgatctcg aaaagtactt cgacgtcatg gtgttcggtg atcaggtgaa gaacggaaag 420
cctgatccag agatatacct tctcgttctg gaaaggttga atgtggtccc agagaaggtt 480
gtggtcttcg aagactcaaa gagcggtgtt gaagccgcaa aaagcgccgg catagaaaga 540
atctatggag tcgttcactc tttgaacgac ggtaaagcgc ttcttgaagc gggtgcggtt 600
gctctggtga aacccgagga aatcctgaac gttctcaaag aggttcttta a 651
<210> 23
<211> 216
<212> PRT
<213> Thermotoga naphthophila DSM 13996
<220>
<223> Phosphatase-variant Y23HP46TE47Y
<400> 23
Met Glu Ala Val Ile Phe Asp Met Asp Gly Val Leu Met Asp Thr Glu
1 5 10 15
Pro Leu Tyr Phe Glu Ala His Arg Arg Val Ala Glu Ser Tyr Gly Lys
20 25 30
Pro Tyr Thr Glu Asp Leu His Arg Arg Ile Met Gly Val Thr Tyr Arg
35 40 45
Glu Gly Leu Pro Ile Leu Met Glu Ala Leu Glu Ile Lys Asp Ser Leu
50 55 60
Glu Asn Phe Lys Lys Arg Val His Glu Glu Lys Lys Arg Val Phe Ser
65 70 75 80
Glu Leu Leu Lys Glu Asn Pro Gly Val Arg Glu Ala Leu Glu Phe Val
85 90 95
Lys Ser Lys Arg Ile Lys Leu Ala Leu Ala Thr Ser Thr Pro Gln Arg
100 105 110
Glu Ala Leu Glu Arg Leu Arg Arg Leu Asp Leu Glu Lys Tyr Phe Asp
115 120 125
Val Met Val Phe Gly Asp Gln Val Lys Asn Gly Lys Pro Asp Pro Glu
130 135 140
Ile Tyr Leu Leu Val Leu Glu Arg Leu Asn Val Val Pro Glu Lys Val
145 150 155 160
Val Val Phe Glu Asp Ser Lys Ser Gly Val Glu Ala Ala Lys Ser Ala
165 170 175
Gly Ile Glu Arg Ile Tyr Gly Val Val His Ser Leu Asn Asp Gly Lys
180 185 190
Ala Leu Leu Glu Ala Gly Ala Val Ala Leu Val Lys Pro Glu Glu Ile
195 200 205
Leu Asn Val Leu Lys Glu Val Leu
210 215
<210> 24
<211> 651
<212> DNA
<213> Thermotoga naphthophila DSM 13996
<220>
<223> coding for Phosphatase-variant Y23HP46TE47Y
<400> 24
atggaagcgg tgattttcga catggatgga gtgctcatgg acacagagcc tctctacttc 60
gaagctcata gaagagtcgc ggaaagctat ggaaaacctt acacggagga tctccacagg 120
agaataatgg gagttactta tagagaaggt cttcccatcc tcatggaagc tctggagata 180
aaagattctc tggagaactt caaaaagagg gtccacgaag aaaaaaagcg cgttttctct 240
gagcttctca aggaaaatcc gggtgtaaga gaggcgctcg agttcgtaaa gagcaaaaga 300
ataaaactcg cgctcgcaac ctccacacca cagcgagaag cgctggagag attgagaaga 360
ctcgatctcg aaaagtactt cgacgtcatg gtgttcggtg atcaggtgaa gaacggaaag 420
cctgatccag agatatacct tctcgttctg gaaaggttga atgtggtccc agagaaggtt 480
gtggtcttcg aagactcaaa gagcggtgtt gaagccgcaa aaagcgccgg catagaaaga 540
atctatggag tcgttcactc tttgaacgac ggtaaagcgc ttcttgaagc gggtgcggtt 600
gctctggtga aacccgagga aatcctgaac gttctcaaag aggttcttta a 651
<210> 25
<211> 216
<212> PRT
<213> Thermotoga naphthophila DSM 13996
<220>
<223> Phosphatase-variant Y23HP46TE47YG50L
<400> 25
Met Glu Ala Val Ile Phe Asp Met Asp Gly Val Leu Met Asp Thr Glu
1 5 10 15
Pro Leu Tyr Phe Glu Ala His Arg Arg Val Ala Glu Ser Tyr Gly Lys
20 25 30
Pro Tyr Thr Glu Asp Leu His Arg Arg Ile Met Gly Val Thr Tyr Arg
35 40 45
Glu Leu Leu Pro Ile Leu Met Glu Ala Leu Glu Ile Lys Asp Ser Leu
50 55 60
Glu Asn Phe Lys Lys Arg Val His Glu Glu Lys Lys Arg Val Phe Ser
65 70 75 80
Glu Leu Leu Lys Glu Asn Pro Gly Val Arg Glu Ala Leu Glu Phe Val
85 90 95
Lys Ser Lys Arg Ile Lys Leu Ala Leu Ala Thr Ser Thr Pro Gln Arg
100 105 110
Glu Ala Leu Glu Arg Leu Arg Arg Leu Asp Leu Glu Lys Tyr Phe Asp
115 120 125
Val Met Val Phe Gly Asp Gln Val Lys Asn Gly Lys Pro Asp Pro Glu
130 135 140
Ile Tyr Leu Leu Val Leu Glu Arg Leu Asn Val Val Pro Glu Lys Val
145 150 155 160
Val Val Phe Glu Asp Ser Lys Ser Gly Val Glu Ala Ala Lys Ser Ala
165 170 175
Gly Ile Glu Arg Ile Tyr Gly Val Val His Ser Leu Asn Asp Gly Lys
180 185 190
Ala Leu Leu Glu Ala Gly Ala Val Ala Leu Val Lys Pro Glu Glu Ile
195 200 205
Leu Asn Val Leu Lys Glu Val Leu
210 215
<210> 26
<211> 651
<212> DNA
<213> Thermotoga naphthophila DSM 13996
<220>
<223> coding for Phosphatase-variant Y23HP46TE47YG50L
<400> 26
atggaagcgg tgattttcga catggatgga gtgctcatgg acacagagcc tctctacttc 60
gaagctcaca gaagagtcgc ggaaagctat ggaaaacctt acacggagga tctccacagg 120
agaataatgg gagttactta tagagaattg cttcccatcc tcatggaagc tctggagata 180
aaagattctc tggagaactt caaaaagagg gtccacgaag aaaaaaagcg cgttttctct 240
gagcttctca aggaaaatcc gggtgtaaga gaggcgctcg agttcgtaaa gagcaaaaga 300
ataaaactcg cgctcgcaac ctccacacca cagcgagaag cgctggagag attgagaaga 360
ctcgatctcg aaaagtactt cgacgtcatg gtgttcggtg atcaggtgaa gaacggaaag 420
cctgatccag agatatacct tctcgttctg gaaaggttga atgtggtccc agagaaggtt 480
gtggtcttcg aagactcaaa gagcggtgtt gaagccgcaa aaagcgccgg catagaaaga 540
atctatggag tcgttcactc tttgaacgac ggtaaagcgc ttcttgaagc gggtgcggtt 600
gctctggtga aacccgagga aatcctgaac gttctcaaag aggttcttta a 651
<210> 27
<211> 216
<212> PRT
<213> Thermotoga naphthophila DSM 13996
<220>
<223> Phosphatase-variant Y23HP46TE47YG50I
<400> 27
Met Glu Ala Val Ile Phe Asp Met Asp Gly Val Leu Met Asp Thr Glu
1 5 10 15
Pro Leu Tyr Phe Glu Ala His Arg Arg Val Ala Glu Ser Tyr Gly Lys
20 25 30
Pro Tyr Thr Glu Asp Leu His Arg Arg Ile Met Gly Val Thr Tyr Arg
35 40 45
Glu Ile Leu Pro Ile Leu Met Glu Ala Leu Glu Ile Lys Asp Ser Leu
50 55 60
Glu Asn Phe Lys Lys Arg Val His Glu Glu Lys Lys Arg Val Phe Ser
65 70 75 80
Glu Leu Leu Lys Glu Asn Pro Gly Val Arg Glu Ala Leu Glu Phe Val
85 90 95
Lys Ser Lys Arg Ile Lys Leu Ala Leu Ala Thr Ser Thr Pro Gln Arg
100 105 110
Glu Ala Leu Glu Arg Leu Arg Arg Leu Asp Leu Glu Lys Tyr Phe Asp
115 120 125
Val Met Val Phe Gly Asp Gln Val Lys Asn Gly Lys Pro Asp Pro Glu
130 135 140
Ile Tyr Leu Leu Val Leu Glu Arg Leu Asn Val Val Pro Glu Lys Val
145 150 155 160
Val Val Phe Glu Asp Ser Lys Ser Gly Val Glu Ala Ala Lys Ser Ala
165 170 175
Gly Ile Glu Arg Ile Tyr Gly Val Val His Ser Leu Asn Asp Gly Lys
180 185 190
Ala Leu Leu Glu Ala Gly Ala Val Ala Leu Val Lys Pro Glu Glu Ile
195 200 205
Leu Asn Val Leu Lys Glu Val Leu
210 215
<210> 28
<211> 651
<212> DNA
<213> Thermotoga naphthophila DSM 13996
<220>
<223> coding for Phosphatase-variant Y23HP46TE47YG50I
<400> 28
atggaagcgg tgattttcga catggatgga gtgctcatgg acacagagcc tctctacttc 60
gaagctcaca gaagagtcgc ggaaagctat ggaaaacctt acacggagga tctccacagg 120
agaataatgg gagttactta tagagaaatt cttcccatcc tcatggaagc tctggagata 180
aaagattctc tggagaactt caaaaagagg gtccacgaag aaaaaaagcg cgttttctct 240
gagcttctca aggaaaatcc gggtgtaaga gaggcgctcg agttcgtaaa gagcaaaaga 300
ataaaactcg cgctcgcaac ctccacacca cagcgagaag cgctggagag attgagaaga 360
ctcgatctcg aaaagtactt cgacgtcatg gtgttcggtg atcaggtgaa gaacggaaag 420
cctgatccag agatatacct tctcgttctg gaaaggttga atgtggtccc agagaaggtt 480
gtggtcttcg aagactcaaa gagcggtgtt gaagccgcaa aaagcgccgg catagaaaga 540
atctatggag tcgttcactc tttgaacgac ggtaaagcgc ttcttgaagc gggtgcggtt 600
gctctggtga aacccgagga aatcctgaac gttctcaaag aggttcttta a 651
<210> 29
<211> 216
<212> PRT
<213> Thermotoga naphthophila DSM 13996
<220>
<223> Phosphatase-variant Y23SP46TE47YG50L
<400> 29
Met Glu Ala Val Ile Phe Asp Met Asp Gly Val Leu Met Asp Thr Glu
1 5 10 15
Pro Leu Tyr Phe Glu Ala Ser Arg Arg Val Ala Glu Ser Tyr Gly Lys
20 25 30
Pro Tyr Thr Glu Asp Leu His Arg Arg Ile Met Gly Val Thr Tyr Arg
35 40 45
Glu Leu Leu Pro Ile Leu Met Glu Ala Leu Glu Ile Lys Asp Ser Leu
50 55 60
Glu Asn Phe Lys Lys Arg Val His Glu Glu Lys Lys Arg Val Phe Ser
65 70 75 80
Glu Leu Leu Lys Glu Asn Pro Gly Val Arg Glu Ala Leu Glu Phe Val
85 90 95
Lys Ser Lys Arg Ile Lys Leu Ala Leu Ala Thr Ser Thr Pro Gln Arg
100 105 110
Glu Ala Leu Glu Arg Leu Arg Arg Leu Asp Leu Glu Lys Tyr Phe Asp
115 120 125
Val Met Val Phe Gly Asp Gln Val Lys Asn Gly Lys Pro Asp Pro Glu
130 135 140
Ile Tyr Leu Leu Val Leu Glu Arg Leu Asn Val Val Pro Glu Lys Val
145 150 155 160
Val Val Phe Glu Asp Ser Lys Ser Gly Val Glu Ala Ala Lys Ser Ala
165 170 175
Gly Ile Glu Arg Ile Tyr Gly Val Val His Ser Leu Asn Asp Gly Lys
180 185 190
Ala Leu Leu Glu Ala Gly Ala Val Ala Leu Val Lys Pro Glu Glu Ile
195 200 205
Leu Asn Val Leu Lys Glu Val Leu
210 215
<210> 30
<211> 651
<212> DNA
<213> Thermotoga naphthophila DSM 13996
<220>
<223> coding for Phosphatase-variant Y23SP46TE47YG50L
<400> 30
atggaagcgg tgattttcga catggatgga gtgctcatgg acacagagcc tctctacttc 60
gaagcttcca gaagagtcgc ggaaagctat ggaaaacctt acacggagga tctccacagg 120
agaataatgg gagttactta tagagaattg cttcccatcc tcatggaagc tctggagata 180
aaagattctc tggagaactt caaaaagagg gtccacgaag aaaaaaagcg cgttttctct 240
gagcttctca aggaaaatcc gggtgtaaga gaggcgctcg agttcgtaaa gagcaaaaga 300
ataaaactcg cgctcgcaac ctccacacca cagcgagaag cgctggagag attgagaaga 360
ctcgatctcg aaaagtactt cgacgtcatg gtgttcggtg atcaggtgaa gaacggaaag 420
cctgatccag agatatacct tctcgttctg gaaaggttga atgtggtccc agagaaggtt 480
gtggtcttcg aagactcaaa gagcggtgtt gaagccgcaa aaagcgccgg catagaaaga 540
atctatggag tcgttcactc tttgaacgac ggtaaagcgc ttcttgaagc gggtgcggtt 600
gctctggtga aacccgagga aatcctgaac gttctcaaag aggttcttta a 651
<210> 31
<211> 216
<212> PRT
<213> Thermotoga naphthophila DSM 13996
<220>
<223> Phosphatase-variant Y23SP46TE47YG50I
<400> 31
Met Glu Ala Val Ile Phe Asp Met Asp Gly Val Leu Met Asp Thr Glu
1 5 10 15
Pro Leu Tyr Phe Glu Ala Ser Arg Arg Val Ala Glu Ser Tyr Gly Lys
20 25 30
Pro Tyr Thr Glu Asp Leu His Arg Arg Ile Met Gly Val Thr Tyr Arg
35 40 45
Glu Ile Leu Pro Ile Leu Met Glu Ala Leu Glu Ile Lys Asp Ser Leu
50 55 60
Glu Asn Phe Lys Lys Arg Val His Glu Glu Lys Lys Arg Val Phe Ser
65 70 75 80
Glu Leu Leu Lys Glu Asn Pro Gly Val Arg Glu Ala Leu Glu Phe Val
85 90 95
Lys Ser Lys Arg Ile Lys Leu Ala Leu Ala Thr Ser Thr Pro Gln Arg
100 105 110
Glu Ala Leu Glu Arg Leu Arg Arg Leu Asp Leu Glu Lys Tyr Phe Asp
115 120 125
Val Met Val Phe Gly Asp Gln Val Lys Asn Gly Lys Pro Asp Pro Glu
130 135 140
Ile Tyr Leu Leu Val Leu Glu Arg Leu Asn Val Val Pro Glu Lys Val
145 150 155 160
Val Val Phe Glu Asp Ser Lys Ser Gly Val Glu Ala Ala Lys Ser Ala
165 170 175
Gly Ile Glu Arg Ile Tyr Gly Val Val His Ser Leu Asn Asp Gly Lys
180 185 190
Ala Leu Leu Glu Ala Gly Ala Val Ala Leu Val Lys Pro Glu Glu Ile
195 200 205
Leu Asn Val Leu Lys Glu Val Leu
210 215
<210> 32
<211> 651
<212> DNA
<213> Thermotoga naphthophila DSM 13996
<220>
<223> coding for Phosphatase-variant Y23SP46TE47YG50I
<400> 32
atggaagcgg tgattttcga catggatgga gtgctcatgg acacagagcc tctctacttc 60
gaagcttcca gaagagtcgc ggaaagctat ggaaaacctt acacggagga tctccacagg 120
agaataatgg gagttactta tagagaaatt cttcccatcc tcatggaagc tctggagata 180
aaagattctc tggagaactt caaaaagagg gtccacgaag aaaaaaagcg cgttttctct 240
gagcttctca aggaaaatcc gggtgtaaga gaggcgctcg agttcgtaaa gagcaaaaga 300
ataaaactcg cgctcgcaac ctccacacca cagcgagaag cgctggagag attgagaaga 360
ctcgatctcg aaaagtactt cgacgtcatg gtgttcggtg atcaggtgaa gaacggaaag 420
cctgatccag agatatacct tctcgttctg gaaaggttga atgtggtccc agagaaggtt 480
gtggtcttcg aagactcaaa gagcggtgtt gaagccgcaa aaagcgccgg catagaaaga 540
atctatggag tcgttcactc tttgaacgac ggtaaagcgc ttcttgaagc gggtgcggtt 600
gctctggtga aacccgagga aatcctgaac gttctcaaag aggttcttta a 651
<210> 33
<211> 216
<212> PRT
<213> Thermotoga naphthophila DSM 13996
<220>
<223> Phosphatase-variant Y23HP46TE47TG50L
<400> 33
Met Glu Ala Val Ile Phe Asp Met Asp Gly Val Leu Met Asp Thr Glu
1 5 10 15
Pro Leu Tyr Phe Glu Ala His Arg Arg Val Ala Glu Ser Tyr Gly Lys
20 25 30
Pro Tyr Thr Glu Asp Leu His Arg Arg Ile Met Gly Val Thr Thr Arg
35 40 45
Glu Leu Leu Pro Ile Leu Met Glu Ala Leu Glu Ile Lys Asp Ser Leu
50 55 60
Glu Asn Phe Lys Lys Arg Val His Glu Glu Lys Lys Arg Val Phe Ser
65 70 75 80
Glu Leu Leu Lys Glu Asn Pro Gly Val Arg Glu Ala Leu Glu Phe Val
85 90 95
Lys Ser Lys Arg Ile Lys Leu Ala Leu Ala Thr Ser Thr Pro Gln Arg
100 105 110
Glu Ala Leu Glu Arg Leu Arg Arg Leu Asp Leu Glu Lys Tyr Phe Asp
115 120 125
Val Met Val Phe Gly Asp Gln Val Lys Asn Gly Lys Pro Asp Pro Glu
130 135 140
Ile Tyr Leu Leu Val Leu Glu Arg Leu Asn Val Val Pro Glu Lys Val
145 150 155 160
Val Val Phe Glu Asp Ser Lys Ser Gly Val Glu Ala Ala Lys Ser Ala
165 170 175
Gly Ile Glu Arg Ile Tyr Gly Val Val His Ser Leu Asn Asp Gly Lys
180 185 190
Ala Leu Leu Glu Ala Gly Ala Val Ala Leu Val Lys Pro Glu Glu Ile
195 200 205
Leu Asn Val Leu Lys Glu Val Leu
210 215
<210> 34
<211> 651
<212> DNA
<213> Thermotoga naphthophila DSM 13996
<220>
<223> coding for Phosphatase-variant Y23HP46TE47TG50L
<400> 34
atggaagcgg tgattttcga catggatgga gtgctcatgg acacagagcc tctctacttc 60
gaagctcaca gaagagtcgc ggaaagctat ggaaaacctt acacggagga tctccacagg 120
agaataatgg gagttactac gagagaattg cttcccatcc tcatggaagc tctggagata 180
aaagattctc tggagaactt caaaaagagg gtccacgaag aaaaaaagcg cgttttctct 240
gagcttctca aggaaaatcc gggtgtaaga gaggcgctcg agttcgtaaa gagcaaaaga 300
ataaaactcg cgctcgcaac ctccacacca cagcgagaag cgctggagag attgagaaga 360
ctcgatctcg aaaagtactt cgacgtcatg gtgttcggtg atcaggtgaa gaacggaaag 420
cctgatccag agatatacct tctcgttctg gaaaggttga atgtggtccc agagaaggtt 480
gtggtcttcg aagactcaaa gagcggtgtt gaagccgcaa aaagcgccgg catagaaaga 540
atctatggag tcgttcactc tttgaacgac ggtaaagcgc ttcttgaagc gggtgcggtt 600
gctctggtga aacccgagga aatcctgaac gttctcaaag aggttcttta a 651
<210> 35
<211> 216
<212> PRT
<213> Thermotoga naphthophila DSM 13996
<220>
<223> Phosphatase-variant Y23HP46TE47FG50L
<400> 35
Met Glu Ala Val Ile Phe Asp Met Asp Gly Val Leu Met Asp Thr Glu
1 5 10 15
Pro Leu Tyr Phe Glu Ala His Arg Arg Val Ala Glu Ser Tyr Gly Lys
20 25 30
Pro Tyr Thr Glu Asp Leu His Arg Arg Ile Met Gly Val Thr Tyr Arg
35 40 45
Glu Leu Leu Pro Ile Leu Met Glu Ala Leu Glu Ile Lys Asp Ser Leu
50 55 60
Glu Asn Phe Lys Lys Arg Val His Glu Glu Lys Lys Arg Val Phe Ser
65 70 75 80
Glu Leu Leu Lys Glu Asn Pro Gly Val Arg Glu Ala Leu Glu Phe Val
85 90 95
Lys Ser Lys Arg Ile Lys Leu Ala Leu Ala Thr Ser Thr Pro Gln Arg
100 105 110
Glu Ala Leu Glu Arg Leu Arg Arg Leu Asp Leu Glu Lys Tyr Phe Asp
115 120 125
Val Met Val Phe Gly Asp Gln Val Lys Asn Gly Lys Pro Asp Pro Glu
130 135 140
Ile Tyr Leu Leu Val Leu Glu Arg Leu Asn Val Val Pro Glu Lys Val
145 150 155 160
Val Val Phe Glu Asp Ser Lys Ser Gly Val Glu Ala Ala Lys Ser Ala
165 170 175
Gly Ile Glu Arg Ile Tyr Gly Val Val His Ser Leu Asn Asp Gly Lys
180 185 190
Ala Leu Leu Glu Ala Gly Ala Val Ala Leu Val Lys Pro Glu Glu Ile
195 200 205
Leu Asn Val Leu Lys Glu Val Leu
210 215
<210> 36
<211> 651
<212> DNA
<213> Thermotoga naphthophila DSM 13996
<220>
<223> coding for Phosphatase-variant Y23HP46TE47FG50L
<400> 36
atggaagcgg tgattttcga catggatgga gtgctcatgg acacagagcc tctctacttc 60
gaagctcaca gaagagtcgc ggaaagctat ggaaaacctt acacggagga tctccacagg 120
agaataatgg gagttacttt tagagaattg cttcccatcc tcatggaagc tctggagata 180
aaagattctc tggagaactt caaaaagagg gtccacgaag aaaaaaagcg cgttttctct 240
gagcttctca aggaaaatcc gggtgtaaga gaggcgctcg agttcgtaaa gagcaaaaga 300
ataaaactcg cgctcgcaac ctccacacca cagcgagaag cgctggagag attgagaaga 360
ctcgatctcg aaaagtactt cgacgtcatg gtgttcggtg atcaggtgaa gaacggaaag 420
cctgatccag agatatacct tctcgttctg gaaaggttga atgtggtccc agagaaggtt 480
gtggtcttcg aagactcaaa gagcggtgtt gaagccgcaa aaagcgccgg catagaaaga 540
atctatggag tcgttcactc tttgaacgac ggtaaagcgc ttcttgaagc gggtgcggtt 600
gctctggtga aacccgagga aatcctgaac gttctcaaag aggttcttta a 651
<210> 37
<211> 216
<212> PRT
<213> Thermotoga naphthophila DSM 13996
<220>
<223> Phosphatase-variant Y23HV45MP46TE47YG50L
<400> 37
Met Glu Ala Val Ile Phe Asp Met Asp Gly Val Leu Met Asp Thr Glu
1 5 10 15
Pro Leu Tyr Phe Glu Ala His Arg Arg Val Ala Glu Ser Tyr Gly Lys
20 25 30
Pro Tyr Thr Glu Asp Leu His Arg Arg Ile Met Gly Met Thr Tyr Arg
35 40 45
Glu Leu Leu Pro Ile Leu Met Glu Ala Leu Glu Ile Lys Asp Ser Leu
50 55 60
Glu Asn Phe Lys Lys Arg Val His Glu Glu Lys Lys Arg Val Phe Ser
65 70 75 80
Glu Leu Leu Lys Glu Asn Pro Gly Val Arg Glu Ala Leu Glu Phe Val
85 90 95
Lys Ser Lys Arg Ile Lys Leu Ala Leu Ala Thr Ser Thr Pro Gln Arg
100 105 110
Glu Ala Leu Glu Arg Leu Arg Arg Leu Asp Leu Glu Lys Tyr Phe Asp
115 120 125
Val Met Val Phe Gly Asp Gln Val Lys Asn Gly Lys Pro Asp Pro Glu
130 135 140
Ile Tyr Leu Leu Val Leu Glu Arg Leu Asn Val Val Pro Glu Lys Val
145 150 155 160
Val Val Phe Glu Asp Ser Lys Ser Gly Val Glu Ala Ala Lys Ser Ala
165 170 175
Gly Ile Glu Arg Ile Tyr Gly Val Val His Ser Leu Asn Asp Gly Lys
180 185 190
Ala Leu Leu Glu Ala Gly Ala Val Ala Leu Val Lys Pro Glu Glu Ile
195 200 205
Leu Asn Val Leu Lys Glu Val Leu
210 215
<210> 38
<211> 651
<212> DNA
<213> Thermotoga naphthophila DSM 13996
<220>
<223> coding for Phosphatase-variant Y23HV45MP46TE47YG50L
<400> 38
atggaagcgg tgattttcga catggatgga gtgctcatgg acacagagcc tctctacttc 60
gaagctcaca gaagagtcgc ggaaagctat ggaaaacctt acacggagga tctccacagg 120
agaataatgg gaatgactta tagagaattg cttcccatcc tcatggaagc tctggagata 180
aaagattctc tggagaactt caaaaagagg gtccacgaag aaaaaaagcg cgttttctct 240
gagcttctca aggaaaatcc gggtgtaaga gaggcgctcg agttcgtaaa gagcaaaaga 300
ataaaactcg cgctcgcaac ctccacacca cagcgagaag cgctggagag attgagaaga 360
ctcgatctcg aaaagtactt cgacgtcatg gtgttcggtg atcaggtgaa gaacggaaag 420
cctgatccag agatatacct tctcgttctg gaaaggttga atgtggtccc agagaaggtt 480
gtggtcttcg aagactcaaa gagcggtgtt gaagccgcaa aaagcgccgg catagaaaga 540
atctatggag tcgttcactc tttgaacgac ggtaaagcgc ttcttgaagc gggtgcggtt 600
gctctggtga aacccgagga aatcctgaac gttctcaaag aggttcttta a 651
<210> 39
<211> 216
<212> PRT
<213> Thermotoga naphthophila DSM 13996
<220>
<223> Phosphatase-variant Y23HV45QP46TE47YG50L
<400> 39
Met Glu Ala Val Ile Phe Asp Met Asp Gly Val Leu Met Asp Thr Glu
1 5 10 15
Pro Leu Tyr Phe Glu Ala His Arg Arg Val Ala Glu Ser Tyr Gly Lys
20 25 30
Pro Tyr Thr Glu Asp Leu His Arg Arg Ile Met Gly Gln Thr Tyr Arg
35 40 45
Glu Leu Leu Pro Ile Leu Met Glu Ala Leu Glu Ile Lys Asp Ser Leu
50 55 60
Glu Asn Phe Lys Lys Arg Val His Glu Glu Lys Lys Arg Val Phe Ser
65 70 75 80
Glu Leu Leu Lys Glu Asn Pro Gly Val Arg Glu Ala Leu Glu Phe Val
85 90 95
Lys Ser Lys Arg Ile Lys Leu Ala Leu Ala Thr Ser Thr Pro Gln Arg
100 105 110
Glu Ala Leu Glu Arg Leu Arg Arg Leu Asp Leu Glu Lys Tyr Phe Asp
115 120 125
Val Met Val Phe Gly Asp Gln Val Lys Asn Gly Lys Pro Asp Pro Glu
130 135 140
Ile Tyr Leu Leu Val Leu Glu Arg Leu Asn Val Val Pro Glu Lys Val
145 150 155 160
Val Val Phe Glu Asp Ser Lys Ser Gly Val Glu Ala Ala Lys Ser Ala
165 170 175
Gly Ile Glu Arg Ile Tyr Gly Val Val His Ser Leu Asn Asp Gly Lys
180 185 190
Ala Leu Leu Glu Ala Gly Ala Val Ala Leu Val Lys Pro Glu Glu Ile
195 200 205
Leu Asn Val Leu Lys Glu Val Leu
210 215
<210> 40
<211> 651
<212> DNA
<213> Thermotoga naphthophila DSM 13996
<220>
<223> coding for Phosphatase-variant Y23HV45QP46TE47YG50L
<400> 40
atggaagcgg tgattttcga catggatgga gtgctcatgg acacagagcc tctctacttc 60
gaagctcaca gaagagtcgc ggaaagctat ggaaaacctt acacggagga tctccacagg 120
agaataatgg gacagactta tagagaattg cttcccatcc tcatggaagc tctggagata 180
aaagattctc tggagaactt caaaaagagg gtccacgaag aaaaaaagcg cgttttctct 240
gagcttctca aggaaaatcc gggtgtaaga gaggcgctcg agttcgtaaa gagcaaaaga 300
ataaaactcg cgctcgcaac ctccacacca cagcgagaag cgctggagag attgagaaga 360
ctcgatctcg aaaagtactt cgacgtcatg gtgttcggtg atcaggtgaa gaacggaaag 420
cctgatccag agatatacct tctcgttctg gaaaggttga atgtggtccc agagaaggtt 480
gtggtcttcg aagactcaaa gagcggtgtt gaagccgcaa aaagcgccgg catagaaaga 540
atctatggag tcgttcactc tttgaacgac ggtaaagcgc ttcttgaagc gggtgcggtt 600
gctctggtga aacccgagga aatcctgaac gttctcaaag aggttcttta a 651
<210> 41
<211> 216
<212> PRT
<213> Thermotoga naphthophila DSM 13996
<220>
<223> Phosphatase-variant Y23HV45RP46TE47YG50L
<400> 41
Met Glu Ala Val Ile Phe Asp Met Asp Gly Val Leu Met Asp Thr Glu
1 5 10 15
Pro Leu Tyr Phe Glu Ala His Arg Arg Val Ala Glu Ser Tyr Gly Lys
20 25 30
Pro Tyr Thr Glu Asp Leu His Arg Arg Ile Met Gly Arg Thr Tyr Arg
35 40 45
Glu Leu Leu Pro Ile Leu Met Glu Ala Leu Glu Ile Lys Asp Ser Leu
50 55 60
Glu Asn Phe Lys Lys Arg Val His Glu Glu Lys Lys Arg Val Phe Ser
65 70 75 80
Glu Leu Leu Lys Glu Asn Pro Gly Val Arg Glu Ala Leu Glu Phe Val
85 90 95
Lys Ser Lys Arg Ile Lys Leu Ala Leu Ala Thr Ser Thr Pro Gln Arg
100 105 110
Glu Ala Leu Glu Arg Leu Arg Arg Leu Asp Leu Glu Lys Tyr Phe Asp
115 120 125
Val Met Val Phe Gly Asp Gln Val Lys Asn Gly Lys Pro Asp Pro Glu
130 135 140
Ile Tyr Leu Leu Val Leu Glu Arg Leu Asn Val Val Pro Glu Lys Val
145 150 155 160
Val Val Phe Glu Asp Ser Lys Ser Gly Val Glu Ala Ala Lys Ser Ala
165 170 175
Gly Ile Glu Arg Ile Tyr Gly Val Val His Ser Leu Asn Asp Gly Lys
180 185 190
Ala Leu Leu Glu Ala Gly Ala Val Ala Leu Val Lys Pro Glu Glu Ile
195 200 205
Leu Asn Val Leu Lys Glu Val Leu
210 215
<210> 42
<211> 651
<212> DNA
<213> Thermotoga naphthophila DSM 13996
<220>
<223> coding for Phosphatase-variant Y23HV45RP46TE47YG50L
<400> 42
atggaagcgg tgattttcga catggatgga gtgctcatgg acacagagcc tctctacttc 60
gaagctcaca gaagagtcgc ggaaagctat ggaaaacctt acacggagga tctccacagg 120
agaataatgg gacgcactta tagagaattg cttcccatcc tcatggaagc tctggagata 180
aaagattctc tggagaactt caaaaagagg gtccacgaag aaaaaaagcg cgttttctct 240
gagcttctca aggaaaatcc gggtgtaaga gaggcgctcg agttcgtaaa gagcaaaaga 300
ataaaactcg cgctcgcaac ctccacacca cagcgagaag cgctggagag attgagaaga 360
ctcgatctcg aaaagtactt cgacgtcatg gtgttcggtg atcaggtgaa gaacggaaag 420
cctgatccag agatatacct tctcgttctg gaaaggttga atgtggtccc agagaaggtt 480
gtggtcttcg aagactcaaa gagcggtgtt gaagccgcaa aaagcgccgg catagaaaga 540
atctatggag tcgttcactc tttgaacgac ggtaaagcgc ttcttgaagc gggtgcggtt 600
gctctggtga aacccgagga aatcctgaac gttctcaaag aggttcttta a 651
<210> 43
<211> 216
<212> PRT
<213> Thermotoga naphthophila DSM 13996
<220>
<223> Phosphatase-variant Y23HV45RP46TE47TG50L
<400> 43
Met Glu Ala Val Ile Phe Asp Met Asp Gly Val Leu Met Asp Thr Glu
1 5 10 15
Pro Leu Tyr Phe Glu Ala His Arg Arg Val Ala Glu Ser Tyr Gly Lys
20 25 30
Pro Tyr Thr Glu Asp Leu His Arg Arg Ile Met Gly Arg Thr Thr Arg
35 40 45
Glu Leu Leu Pro Ile Leu Met Glu Ala Leu Glu Ile Lys Asp Ser Leu
50 55 60
Glu Asn Phe Lys Lys Arg Val His Glu Glu Lys Lys Arg Val Phe Ser
65 70 75 80
Glu Leu Leu Lys Glu Asn Pro Gly Val Arg Glu Ala Leu Glu Phe Val
85 90 95
Lys Ser Lys Arg Ile Lys Leu Ala Leu Ala Thr Ser Thr Pro Gln Arg
100 105 110
Glu Ala Leu Glu Arg Leu Arg Arg Leu Asp Leu Glu Lys Tyr Phe Asp
115 120 125
Val Met Val Phe Gly Asp Gln Val Lys Asn Gly Lys Pro Asp Pro Glu
130 135 140
Ile Tyr Leu Leu Val Leu Glu Arg Leu Asn Val Val Pro Glu Lys Val
145 150 155 160
Val Val Phe Glu Asp Ser Lys Ser Gly Val Glu Ala Ala Lys Ser Ala
165 170 175
Gly Ile Glu Arg Ile Tyr Gly Val Val His Ser Leu Asn Asp Gly Lys
180 185 190
Ala Leu Leu Glu Ala Gly Ala Val Ala Leu Val Lys Pro Glu Glu Ile
195 200 205
Leu Asn Val Leu Lys Glu Val Leu
210 215
<210> 44
<211> 651
<212> DNA
<213> Thermotoga naphthophila DSM 13996
<220>
<223> coding for Phosphatase-variant Y23HV45RP46TE47TG50L
<400> 44
atggaagcgg tgattttcga catggatgga gtgctcatgg acacagagcc tctctacttc 60
gaagctcaca gaagagtcgc ggaaagctat ggaaaacctt acacggagga tctccacagg 120
agaataatgg gacgcactac cagagaattg cttcccatcc tcatggaagc tctggagata 180
aaagattctc tggagaactt caaaaagagg gtccacgaag aaaaaaagcg cgttttctct 240
gagcttctca aggaaaatcc gggtgtaaga gaggcgctcg agttcgtaaa gagcaaaaga 300
ataaaactcg cgctcgcaac ctccacacca cagcgagaag cgctggagag attgagaaga 360
ctcgatctcg aaaagtactt cgacgtcatg gtgttcggtg atcaggtgaa gaacggaaag 420
cctgatccag agatatacct tctcgttctg gaaaggttga atgtggtccc agagaaggtt 480
gtggtcttcg aagactcaaa gagcggtgtt gaagccgcaa aaagcgccgg catagaaaga 540
atctatggag tcgttcactc tttgaacgac ggtaaagcgc ttcttgaagc gggtgcggtt 600
gctctggtga aacccgagga aatcctgaac gttctcaaag aggttcttta a 651
<210> 45
<211> 216
<212> PRT
<213> Thermotoga naphthophila DSM 13996
<220>
<223> Phosphatase-variant Y23HV45RP46TE47FG50L
<400> 45
Met Glu Ala Val Ile Phe Asp Met Asp Gly Val Leu Met Asp Thr Glu
1 5 10 15
Pro Leu Tyr Phe Glu Ala His Arg Arg Val Ala Glu Ser Tyr Gly Lys
20 25 30
Pro Tyr Thr Glu Asp Leu His Arg Arg Ile Met Gly Val Thr Tyr Arg
35 40 45
Glu Leu Leu Pro Ile Leu Met Glu Ala Leu Glu Ile Lys Asp Ser Leu
50 55 60
Glu Asn Phe Lys Lys Arg Val His Glu Glu Lys Lys Arg Val Phe Ser
65 70 75 80
Glu Leu Leu Lys Glu Asn Pro Gly Val Arg Glu Ala Leu Glu Phe Val
85 90 95
Lys Ser Lys Arg Ile Lys Leu Ala Leu Ala Thr Ser Thr Pro Gln Arg
100 105 110
Glu Ala Leu Glu Arg Leu Arg Arg Leu Asp Leu Glu Lys Tyr Phe Asp
115 120 125
Val Met Val Phe Gly Asp Gln Val Lys Asn Gly Lys Pro Asp Pro Glu
130 135 140
Ile Tyr Leu Leu Val Leu Glu Arg Leu Asn Val Val Pro Glu Lys Val
145 150 155 160
Val Val Phe Glu Asp Ser Lys Ser Gly Val Glu Ala Ala Lys Ser Ala
165 170 175
Gly Ile Glu Arg Ile Tyr Gly Val Val His Ser Leu Asn Asp Gly Lys
180 185 190
Ala Leu Leu Glu Ala Gly Ala Val Ala Leu Val Lys Pro Glu Glu Ile
195 200 205
Leu Asn Val Leu Lys Glu Val Leu
210 215
<210> 46
<211> 651
<212> DNA
<213> Thermotoga naphthophila DSM 13996
<220>
<223> coding for Phosphatase-variant Y23HV45RP46TE47FG50L
<400> 46
atggaagcgg tgattttcga catggatgga gtgctcatgg acacagagcc tctctacttc 60
gaagctcaca gaagagtcgc ggaaagctat ggaaaacctt acacggagga tctccacagg 120
agaataatgg gacgcacttt cagagaattg cttcccatcc tcatggaagc tctggagata 180
aaagattctc tggagaactt caaaaagagg gtccacgaag aaaaaaagcg cgttttctct 240
gagcttctca aggaaaatcc gggtgtaaga gaggcgctcg agttcgtaaa gagcaaaaga 300
ataaaactcg cgctcgcaac ctccacacca cagcgagaag cgctggagag attgagaaga 360
ctcgatctcg aaaagtactt cgacgtcatg gtgttcggtg atcaggtgaa gaacggaaag 420
cctgatccag agatatacct tctcgttctg gaaaggttga atgtggtccc agagaaggtt 480
gtggtcttcg aagactcaaa gagcggtgtt gaagccgcaa aaagcgccgg catagaaaga 540
atctatggag tcgttcactc tttgaacgac ggtaaagcgc ttcttgaagc gggtgcggtt 600
gctctggtga aacccgagga aatcctgaac gttctcaaag aggttcttta a 651
<210> 47
<211> 216
<212> PRT
<213> Thermotoga naphthophila DSM 13996
<220>
<223> Phosphatase-variant Y23HV45MP46TE47FG50L
<400> 47
Met Glu Ala Val Ile Phe Asp Met Asp Gly Val Leu Met Asp Thr Glu
1 5 10 15
Pro Leu Tyr Phe Glu Ala His Arg Arg Val Ala Glu Ser Tyr Gly Lys
20 25 30
Pro Tyr Thr Glu Asp Leu His Arg Arg Ile Met Gly Met Thr Phe Arg
35 40 45
Glu Leu Leu Pro Ile Leu Met Glu Ala Leu Glu Ile Lys Asp Ser Leu
50 55 60
Glu Asn Phe Lys Lys Arg Val His Glu Glu Lys Lys Arg Val Phe Ser
65 70 75 80
Glu Leu Leu Lys Glu Asn Pro Gly Val Arg Glu Ala Leu Glu Phe Val
85 90 95
Lys Ser Lys Arg Ile Lys Leu Ala Leu Ala Thr Ser Thr Pro Gln Arg
100 105 110
Glu Ala Leu Glu Arg Leu Arg Arg Leu Asp Leu Glu Lys Tyr Phe Asp
115 120 125
Val Met Val Phe Gly Asp Gln Val Lys Asn Gly Lys Pro Asp Pro Glu
130 135 140
Ile Tyr Leu Leu Val Leu Glu Arg Leu Asn Val Val Pro Glu Lys Val
145 150 155 160
Val Val Phe Glu Asp Ser Lys Ser Gly Val Glu Ala Ala Lys Ser Ala
165 170 175
Gly Ile Glu Arg Ile Tyr Gly Val Val His Ser Leu Asn Asp Gly Lys
180 185 190
Ala Leu Leu Glu Ala Gly Ala Val Ala Leu Val Lys Pro Glu Glu Ile
195 200 205
Leu Asn Val Leu Lys Glu Val Leu
210 215
<210> 48
<211> 651
<212> DNA
<213> Thermotoga naphthophila DSM 13996
<220>
<223> coding for Phosphatase-variant Y23HV45MP46TE47FG50L
<400> 48
atggaagcgg tgattttcga catggatgga gtgctcatgg acacagagcc tctctacttc 60
gaagctcaca gaagagtcgc ggaaagctat ggaaaacctt acacggagga tctccacagg 120
agaataatgg gaagtacttt cagagaattg cttcccatcc tcatggaagc tctggagata 180
aaagattctc tggagaactt caaaaagagg gtccacgaag aaaaaaagcg cgttttctct 240
gagcttctca aggaaaatcc gggtgtaaga gaggcgctcg agttcgtaaa gagcaaaaga 300
ataaaactcg cgctcgcaac ctccacacca cagcgagaag cgctggagag attgagaaga 360
ctcgatctcg aaaagtactt cgacgtcatg gtgttcggtg atcaggtgaa gaacggaaag 420
cctgatccag agatatacct tctcgttctg gaaaggttga atgtggtccc agagaaggtt 480
gtggtcttcg aagactcaaa gagcggtgtt gaagccgcaa aaagcgccgg catagaaaga 540
atctatggag tcgttcactc tttgaacgac ggtaaagcgc ttcttgaagc gggtgcggtt 600
gctctggtga aacccgagga aatcctgaac gttctcaaag aggttcttta a 651
<210> 49
<211> 216
<212> PRT
<213> Thermotoga naphthophila DSM 13996
<220>
<223> Phosphatase-variant Y23HV45QP46TE47FG50L
<400> 49
Met Glu Ala Val Ile Phe Asp Met Asp Gly Val Leu Met Asp Thr Glu
1 5 10 15
Pro Leu Tyr Phe Glu Ala His Arg Arg Val Ala Glu Ser Tyr Gly Lys
20 25 30
Pro Tyr Thr Glu Asp Leu His Arg Arg Ile Met Gly Val Thr Tyr Arg
35 40 45
Glu Leu Leu Pro Ile Leu Met Glu Ala Leu Glu Ile Lys Asp Ser Leu
50 55 60
Glu Asn Phe Lys Lys Arg Val His Glu Glu Lys Lys Arg Val Phe Ser
65 70 75 80
Glu Leu Leu Lys Glu Asn Pro Gly Val Arg Glu Ala Leu Glu Phe Val
85 90 95
Lys Ser Lys Arg Ile Lys Leu Ala Leu Ala Thr Ser Thr Pro Gln Arg
100 105 110
Glu Ala Leu Glu Arg Leu Arg Arg Leu Asp Leu Glu Lys Tyr Phe Asp
115 120 125
Val Met Val Phe Gly Asp Gln Val Lys Asn Gly Lys Pro Asp Pro Glu
130 135 140
Ile Tyr Leu Leu Val Leu Glu Arg Leu Asn Val Val Pro Glu Lys Val
145 150 155 160
Val Val Phe Glu Asp Ser Lys Ser Gly Val Glu Ala Ala Lys Ser Ala
165 170 175
Gly Ile Glu Arg Ile Tyr Gly Val Val His Ser Leu Asn Asp Gly Lys
180 185 190
Ala Leu Leu Glu Ala Gly Ala Val Ala Leu Val Lys Pro Glu Glu Ile
195 200 205
Leu Asn Val Leu Lys Glu Val Leu
210 215
<210> 50
<211> 651
<212> DNA
<213> Thermotoga naphthophila DSM 13996
<220>
<223> coding for Phosphatase-variant Y23HV45QP46TE47FG50L
<400> 50
atggaagcgg tgattttcga catggatgga gtgctcatgg acacagagcc tctctacttc 60
gaagctcaca gaagagtcgc ggaaagctat ggaaaacctt acacggagga tctccacagg 120
agaataatgg gacagacttt cagagaattg cttcccatcc tcatggaagc tctggagata 180
aaagattctc tggagaactt caaaaagagg gtccacgaag aaaaaaagcg cgttttctct 240
gagcttctca aggaaaatcc gggtgtaaga gaggcgctcg agttcgtaaa gagcaaaaga 300
ataaaactcg cgctcgcaac ctccacacca cagcgagaag cgctggagag attgagaaga 360
ctcgatctcg aaaagtactt cgacgtcatg gtgttcggtg atcaggtgaa gaacggaaag 420
cctgatccag agatatacct tctcgttctg gaaaggttga atgtggtccc agagaaggtt 480
gtggtcttcg aagactcaaa gagcggtgtt gaagccgcaa aaagcgccgg catagaaaga 540
atctatggag tcgttcactc tttgaacgac ggtaaagcgc ttcttgaagc gggtgcggtt 600
gctctggtga aacccgagga aatcctgaac gttctcaaag aggttcttta a 651
<210> 51
<211> 504
<212> PRT
<213> Bifidobacterium adolescentis
<220>
<223> Sucrose Phosphorylase variant Q331ER393ND445PD446TQ460EE485H
<400> 51
Met Lys Asn Lys Val Gln Leu Ile Thr Tyr Ala Asp Arg Leu Gly Asp
1 5 10 15
Gly Thr Ile Lys Ser Met Thr Asp Ile Leu Arg Thr Arg Phe Asp Gly
20 25 30
Val Tyr Asp Gly Val His Ile Leu Pro Phe Phe Thr Pro Phe Asp Gly
35 40 45
Ala Asp Ala Gly Phe Asp Pro Ile Asp His Thr Lys Val Asp Glu Arg
50 55 60
Leu Gly Ser Trp Asp Asp Val Ala Glu Leu Ser Lys Thr His Asn Ile
65 70 75 80
Met Val Asp Ala Ile Val Asn His Met Ser Trp Glu Ser Lys Gln Phe
85 90 95
Gln Asp Val Leu Ala Lys Gly Glu Glu Ser Glu Tyr Tyr Pro Met Phe
100 105 110
Leu Thr Met Ser Ser Val Phe Pro Asn Gly Ala Thr Glu Glu Asp Leu
115 120 125
Ala Gly Ile Tyr Arg Pro Arg Pro Gly Leu Pro Phe Thr His Tyr Lys
130 135 140
Phe Ala Gly Lys Thr Arg Leu Val Trp Val Ser Phe Thr Pro Gln Gln
145 150 155 160
Val Asp Ile Asp Thr Asp Ser Asp Lys Gly Trp Glu Tyr Leu Met Ser
165 170 175
Ile Phe Asp Gln Met Ala Ala Ser His Val Ser Tyr Ile Arg Leu Asp
180 185 190
Ala Val Gly Tyr Gly Ala Lys Glu Ala Gly Thr Ser Cys Phe Met Thr
195 200 205
Pro Lys Thr Phe Lys Leu Ile Ser Arg Leu Arg Glu Glu Gly Val Lys
210 215 220
Arg Gly Leu Glu Ile Leu Ile Glu Val His Ser Tyr Tyr Lys Lys Gln
225 230 235 240
Val Glu Ile Ala Ser Lys Val Asp Arg Val Tyr Asp Phe Ala Leu Pro
245 250 255
Pro Leu Leu Leu His Ala Leu Ser Thr Gly His Val Glu Pro Val Ala
260 265 270
His Trp Thr Asp Ile Arg Pro Asn Asn Ala Val Thr Val Leu Asp Thr
275 280 285
His Asp Gly Ile Gly Val Ile Asp Ile Gly Ser Asp Gln Leu Asp Arg
290 295 300
Ser Leu Lys Gly Leu Val Pro Asp Glu Asp Val Asp Asn Leu Val Asn
305 310 315 320
Thr Ile His Ala Asn Thr His Gly Glu Ser Glu Ala Ala Thr Gly Ala
325 330 335
Ala Ala Ser Asn Leu Asp Leu Tyr Gln Val Asn Ser Thr Tyr Tyr Ser
340 345 350
Ala Leu Gly Cys Asn Asp Gln His Tyr Ile Ala Ala Arg Ala Val Gln
355 360 365
Phe Phe Leu Pro Gly Val Pro Gln Val Tyr Tyr Val Gly Ala Leu Ala
370 375 380
Gly Lys Asn Asp Met Glu Leu Leu Asn Lys Thr Asn Asn Gly Arg Asp
385 390 395 400
Ile Asn Arg His Tyr Tyr Ser Thr Ala Glu Ile Asp Glu Asn Leu Lys
405 410 415
Arg Pro Val Val Lys Ala Leu Asn Ala Leu Ala Lys Phe Arg Asn Glu
420 425 430
Leu Asp Ala Phe Asp Gly Thr Phe Ser Tyr Thr Thr Pro Thr Asp Thr
435 440 445
Ser Ile Ser Phe Thr Trp Arg Gly Glu Thr Ser Glu Ala Thr Leu Thr
450 455 460
Phe Glu Pro Lys Arg Gly Leu Gly Val Asp Asn Thr Thr Pro Val Ala
465 470 475 480
Met Leu Glu Trp His Asp Ser Ala Gly Asp His Arg Ser Asp Asp Leu
485 490 495
Ile Ala Asn Pro Pro Val Val Ala
500
<210> 52
<211> 1515
<212> DNA
<213> Bifidobacterium adolescentis
<220>
<223> coding for Sucrose Phosphorylase variant
Q331ER393ND445PD446TQ460EE485H
<400> 52
atgaagaaca aagtgcagct gattacctat gcagatcgtt taggtgatgg caccattaaa 60
agcatgaccg atattctgcg tacccgtttt gatggtgtgt atgatggtgt tcatatcctg 120
ccgtttttta ccccgtttga tggcgcagat gcaggttttg atccgattga tcataccaaa 180
gtggatgaac gtttaggtag ctgggatgat gttgcagaac tgagcaaaac ccataacatt 240
atggttgatg ccattgtgaa tcacatgagc tgggaaagca aacagtttca ggatgttctg 300
gcaaaaggtg aagaaagcga atattatccg atgtttctga ccatgagcag cgtttttccg 360
aatggtgcaa ccgaagaaga tctggcaggt atttatcgtc cgcgtccggg tctgccgttt 420
acacattaca aatttgcagg taaaacccgt ctggtttggg ttagctttac accgcagcag 480
gttgatattg ataccgatag cgataaaggt tgggaatatc tgatgagcat ctttgatcag 540
atggcagcaa gccatgttag ctatattcgt ctggatgcag ttggttatgg tgcaaaagaa 600
gcaggcacca gctgttttat gaccccgaaa acctttaaac tgattagccg tctgcgtgaa 660
gaaggtgtta aacgcggtct ggaaattctg attgaagtgc acagctacta caagaaacag 720
gttgaaattg ccagcaaagt tgatcgcgtt tatgattttg cactgcctcc gctgctgctg 780
catgcactga gcaccggtca tgttgaaccg gttgcacatt ggacagatat tcgtccgaat 840
aatgcagtta ccgttctgga tacccatgat ggtattggtg ttattgatat tggtagcgat 900
cagctggatc gtagcctgaa aggtctggtt ccggatgaag atgttgataa tctggtgaat 960
accattcatg ccaatacaca tggtgaaagc gaggcagcaa ccggtgcagc agccagcaat 1020
ctggatctgt atcaggttaa tagcacctat tatagcgcac tgggttgtaa cgatcagcat 1080
tatattgcag cacgtgccgt tcagtttttt ctgcctggtg ttccgcaggt ttattatgtt 1140
ggtgcactgg caggcaaaaa tgatatggaa ctgctgaata aaaccaataa cggtcgtgat 1200
attaaccgcc attattacag caccgcagaa attgatgaaa atctgaaacg tccggttgtg 1260
aaagcactga atgcactggc caaatttcgc aatgaactgg atgcatttga tggcacattt 1320
agctatacca cccctactga taccagcatt tcatttacct ggcgtggtga aaccagcgag 1380
gcgaccctga cctttgaacc gaaacgtggt ctgggcgttg ataataccac accggtggca 1440
atgctggaat ggcacgatag tgccggtgat catcgtagtg atgatctgat tgcaaatccg 1500
cctgttgttg cataa 1515

Claims (15)

1.将至少一种寡糖和/或多糖转化为果糖的方法,包括以下步骤:
a)向包含水、磷酸盐和至少一种寡糖和/或多糖的组合物中加入至少四种酶,优选至少五种酶,和
b)随后在存在所述至少四种酶,优选至少五种酶的情况下,将所述至少一种寡糖和/或多糖酶促转化为果糖,
其中在步骤a)中加入至少一种另外的糖,其中所述至少一种另外的糖选自由以下糖组成的组,糖中包含20个或更少的单糖残基和/或其组合,优选包含17个或更少的单糖残基和/或其组合;
其中在步骤a)中,所述至少四种酶,优选至少五种酶,选自转移酶、磷酸化酶、变位酶、异构酶、水解酶、磷酸酶及其组合;并且
其中所述步骤a)中的至少一种酶是磷酸酶。
2.根据权利要求1所述的方法,其特征在于,
在步骤a)中,所述至少一种另外的糖选自十四糖、十三糖、十二糖、十一糖、十糖、九糖、八糖、七糖、六糖、五糖、四糖、三糖、二糖和/或其组合,优选选自四糖、三糖、二糖及其组合,更优选选自麦芽糖、麦芽三糖、麦芽四糖及其组合;和/或
其中,以干重计,步骤a)中的所述组合物包含等于或小于35%的至少一种寡糖和/或多糖;和/或
其中,所述至少一种寡糖和/或多糖选自基于葡萄糖的寡糖和/或多糖,优选选自淀粉及其衍生物、半纤维素及其衍生物、纤维素及其衍生物和/或其组合;和/或
其中,在步骤a)中,将至少六种酶添加到所述组合物中;和/或
其中,所述至少一种另外的糖具有与所述至少一种寡糖和/或多糖至少部分相同的糖苷键。
3.根据权利要求1或2所述的方法,其特征在于,
优选地,在步骤b)中,糖磷酸盐是中间产物;和/或
其中,在步骤b)中,酶转化为一锅反应;和/或
其中,所述磷酸酶包含与根据SEQ ID NO:13所述的序列至少90%、优选至少95%、更优选至少97%相同的氨基酸序列。
4.根据前述权利要求中任一项所述的方法,其特征在于,
在步骤a)中添加至少一种转移酶,优选糖基转移酶,更优选葡聚糖转移酶,甚至更优选α-葡聚糖转移酶;和/或
其中,在步骤a)中添加至少一种磷酸化酶,优选葡聚糖磷酸化酶;和/或
其中,在步骤a)中添加至少一种变位酶,优选磷酸葡萄糖变位酶;和/或
其中,在步骤a)中添加至少一种异构酶,优选磷酸葡萄糖异构酶;和/或
其中,在步骤a)中添加至少一种水解酶,优选葡聚糖水解酶,更优选支链淀粉酶。
5.根据前述权利要求中任一项所述的方法,其特征在于,
步骤b)在室温(25℃)下进行至少24小时。
6.根据前述权利要求中任一项所述的方法,其特征在于,
步骤a)的所述组合物中至少50%的糖在酶转化24小时后转化为果糖;和/或
其中步骤a)的所述组合物中至少70%的糖在酶转化48小时后转化为果糖。
7.根据前述权利要求中任一项所述的方法,其特征在于,
步骤b)中的温度在10–100℃之间,优选在20–90℃之间;和/或
其中,所述组合物的pH值在3到12之间,优选在4到10之间。
8.将至少一种寡糖和/或多糖转化为果糖的组合物,其包含
-水;
-磷酸盐;
-至少四种酶,优选至少五种酶,其中至少一种酶是磷酸酶;
-至少一种寡糖和/或多糖;
其中所述至少四种酶,优选至少五种酶,选自转移酶、磷酸化酶、变位酶、异构酶、水解酶、磷酸酶及其组合;和
其中所述组合物还包含至少一种另外的糖,其中所述至少一种另外的糖选自由以下糖组成的组,糖中包含20个或更少的单糖残基和/或其组合,优选包含17个或更少的单糖残基和/或其组合。
9.根据权利要求8所述的组合物,其特征在于,
所述磷酸酶包含与根据SEQ ID NO:41所述的序列至少98%、优选至少98.5%、更优选至少99%相同的氨基酸序列;或
其中,所述磷酸酶具有以下氨基酸序列:SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:19、SEQ ID NO:21、SEQ ID NO:23、SEQ ID NO:25、SEQ ID NO:27、SEQ ID NO:29、SEQ ID NO:31,SEQ ID NO:33、SEQ ID NO:35、SEQ ID NO:37、SEQ ID NO:39、SEQ ID NO:41、SEQ IDNO:43、SEQ ID NO:45、SEQ ID NO:47或SEQ ID NO:49。
10.一种水性组合物,其包含
-至少50%的果糖,以干重计;
-0.001至25%的葡萄糖,以干重计;
-0.01至22%的磷酸盐,以干重计;和
-0.001至2%的至少四种酶,优选至少五种酶,以干重计,
其中所述至少四种酶,优选至少五种酶,选自转移酶、磷酸化酶、变位酶、异构酶、水解酶、磷酸酶及其组合;和
其中至少一种酶是磷酸酶。
11.根据权利要求10所述的水性组合物,其特征在于,
所述磷酸酶包含与根据SEQ ID NO:41所述的序列至少98%、优选至少98.5%、更优选至少99%相同的氨基酸序列;或
其中,磷酸酶具有以下氨基酸序列:SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:19、SEQID NO:21、SEQ ID NO:23、SEQ ID NO:25、SEQ ID NO:27、SEQ ID NO:29、SEQ ID NO:31,SEQID NO:33、SEQ ID NO:35、SEQ ID NO:37、SEQ ID NO:39、SEQ ID NO:41、SEQ ID NO:43、SEQID NO:45、SEQ ID NO:47或SEQ ID NO:49。
12.根据权利要求10或11所述的水性组合物,可通过如权利要求1至7所述的方法获得。
13.一种磷酸酶,其包含与根据SEQ ID NO:41所述的序列至少98%、优选至少95%、更优选至少98.5%并且甚至更优选至少99%相同的氨基酸序列。
14.根据权利要求13所述的磷酸酶,其具有根据以下所述的氨基酸序列:SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:19、SEQ ID NO:21、SEQ ID NO:23、SEQ ID NO:25、SEQ IDNO:27、SEQ ID NO:29、SEQ ID NO:31,SEQ ID NO:33、SEQ ID NO:35、SEQ ID NO:37、SEQ IDNO:39、SEQ ID NO:41、SEQ ID NO:43、SEQ ID NO:45、SEQ ID NO:47或SEQ ID NO:49。
15.根据权利要求13或14所述的磷酸酶在将至少一种寡糖和/或多糖转化为果糖中的用途。
CN202080095806.7A 2019-12-20 2020-12-21 由寡糖和/或多糖制成果糖 Pending CN115087741A (zh)

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