CN110678479A - 性能改善的乳糖酶 - Google Patents
性能改善的乳糖酶 Download PDFInfo
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- CN110678479A CN110678479A CN201880035256.2A CN201880035256A CN110678479A CN 110678479 A CN110678479 A CN 110678479A CN 201880035256 A CN201880035256 A CN 201880035256A CN 110678479 A CN110678479 A CN 110678479A
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Abstract
本发明涉及表现出β‑半乳糖苷酶活性的新的改善的肽或二聚肽，以及降低诸如乳制品等组合物中乳糖含量的改善的方法。
Description
技术领域
本发明涉及表现出β-半乳糖苷酶活性的新的改善的肽或二聚肽以及降低诸如乳制品等组合物中乳糖含量的改善的方法。
背景技术
为了在乳中生长，乳糖水解是乳酸菌获得作为碳源的葡萄糖和半乳糖的好方式。乳糖酶(β-半乳糖苷酶；EC 3.2.1.23)是将乳中的糖乳糖水解为单糖的酶。乳糖酶的商业用途是分解乳制品中的乳糖。乳糖不耐受人士难以消化高乳糖水平的乳制品。据估计，全世界约有70％的人消化乳糖的能力有限。因此，对不含或仅含低水平乳糖的乳制食品的需求正在增长。
乳糖酶已从多种生物中分离出来，包括克鲁维酵母菌属(Kluyveromyces)和芽孢杆菌属(Bacillus)等微生物。克鲁维酵母菌属，特别是脆壁克鲁维酵母(K.fragilis)和乳酸克鲁维酵母(K.lactis)，以及其他真菌，例如念珠菌属(Candida)、串酵母属(Torula)和球拟酵母属(Torulopsis)的真菌，是真菌乳糖酶的常见来源，而凝结芽孢杆菌(B.coagulans)和环状芽孢杆菌(B.circulans)是众所周知的细菌乳糖酶来源。来自这些生物的几种商业乳糖酶制剂是可用的，例如
(可从丹麦的Novozymes获得)、HA-乳糖酶(可从丹麦的Chr.Hansen(科.汉森有限公司)获得)和
(可从荷兰的DSM获得)，所有这些制剂均来自乳酸克鲁维酵母。所有这些乳糖酶都是所谓的中性乳糖酶，其最适pH在pH 6至pH 8之间，最适温度在37℃左右。当此类乳糖酶用于生产例如于低乳糖酸奶时，在发酵前必须在单独步骤中进行酶处理，或者必须使用高剂量的酶，因为它们的活性会随着发酵过程中pH值的降低而降低。而且，这些乳糖酶不适合在高温下进行的乳中乳糖的水解，这在某些情况下有利于保持微生物低计数并因此确保高乳质量。此外，已知的乳糖酶不适合用于生产超热处理(UHT)乳的所需方法中，其中在UHT处理之前添加了酶。
WO2010092057和WO0104276涉及冷活性β-半乳糖苷酶。WO07110619涉及具有高转半乳糖基化活性的β-半乳糖苷酶，而WO2009071539涉及具有较低的转半乳糖基化活性的β-半乳糖苷酶。
发明目的
本发明实施方案的目的是，提供具有使得能够生产改善的无乳糖或低乳糖产品的性能的β-半乳糖苷酶。
本发明实施方案的另一个目的是，提供具有以下性能的β-半乳糖苷酶，所述性能使得用于降低产品(例如无乳糖产品或低乳糖产品)中的乳糖的改善的生产方法成为可能，例如更容易、更快、更可靠或更便宜的生产方法。
发明内容
本发明人已经鉴定出具有以前未描述的性能的β-半乳糖苷酶，所述性能使得能够生产改善的无乳糖或低乳糖产品，并且使得用于此类无乳糖或低乳糖产品的改善的生产方法成为可能。特别是，已证明这些β-半乳糖苷酶在非常宽的温度范围和pH值范围内都非常稳定并且具有相对高的活性。它们还可以在特定温度下使用，例如在高温和在利用这些酶时通常见不到的pH值下。首先，这使得能够在不知为可能的特定pH值和温度下使用β-半乳糖苷酶。它还使得能在几种不同的应用中使用相同的特定酶，这在工业上是非常需要的。
因此，在第一方面，本发明涉及表现出β-半乳糖苷酶活性的肽，其具有SEQ ID NO:1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33所示的氨基酸序列，或上述氨基酸序列的具有不超过1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21或22个氨基酸取代、添加或缺失的氨基酸序列。
在第二方面，本发明涉及表现出β-半乳糖苷酶活性的二聚肽(dimeric peptide)，所述二聚肽的组成为具有SEQ ID NO:2和3、5和6、20和21、23和24、26和27或28和29所示氨基酸序列的两个肽，或其酶促活性片段，或任一上述序列的具有不超过1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21或22个氨基酸取代、添加或缺失的氨基酸序列。
在第三方面，本发明涉及编码根据本发明的表现出β-半乳糖苷酶活性的肽或二聚肽的核苷酸序列。
在另一方面，本发明涉及包含编码本发明的表现出β-半乳糖苷酶活性的肽或二聚肽的核苷酸序列的宿主细胞。
在另一方面，本发明涉及产生本发明的表现出β-半乳糖苷酶活性的肽或二聚肽的方法，所述方法包括在合适的宿主细胞中表达包含本发明的核苷酸序列的载体；和从所述宿主细胞的表达产物中纯化所述肽或二聚肽。
在另一方面，本发明涉及降低诸如乳制品等含乳糖的组合物中的乳糖含量的方法，所述方法包括使所述组合物在3-10的pH和0℃-140℃的温度下与表现出β-半乳糖苷酶活性的肽或二聚肽接触的步骤，所述肽具有SEQ ID NO:1-33所示的氨基酸序列；或所述二聚肽的组成为具有SEQ ID NO:2和3、5和6、20和21、23和24、26和27或28和29所示氨基酸序列的两个肽；或与任一所述序列具有至少80％序列同一性的序列；或与表达任一所述肽的宿主细胞接触的步骤。
在另一方面，本发明涉及表现出β-半乳糖苷酶活性的肽或二聚肽在生产乳糖含量降低的乳制品中的用途，所述肽具有SEQ ID NO:1-33所示的氨基酸序列，或者所述二聚肽的组成为具有SEQ ID NO:2和3、5和6、20和21、23和24、26和27或28和29所示氨基酸序列的两个肽；或与任一所述序列具有至少80％序列同一性的序列；或表达任一所述肽的宿主细胞在生产乳糖含量降低的乳制品中的用途。
在一些实施方案中，这种含有乳糖的组合物或这种乳制品选自由以下各项组成的组：无乳糖乳、低乳糖乳、酸奶，包括未巴氏灭菌的以及巴氏灭菌前后的酸奶、奶酪、发酵乳制品、饮食补充剂和益生菌饮食制品。在一些其他实施方案中，这种宿主细胞是选自以下的一种：双歧杆菌属(Bifidobacterium)的细菌，例如青春双歧杆菌(Bifidobacteriumadolescentis)、两歧双歧杆菌(Bifidobacterium bifidum)、短双歧杆菌(Bifidobacterium breve)、链状双歧杆菌(Bifidobacterium catenulatum)、长双歧杆菌(Bifidobacterium longum)，或乳杆菌属(Lactobacillus)，例如清酒乳杆菌(L.sakei)、噬淀粉乳杆菌(L.amylovorus)、德氏乳杆菌保加利亚亚种(L.delbrueckiisubsp.bulgaricus)、德氏乳杆菌乳酸亚种(L.delbrueckii subsp.lactis)、德氏乳杆菌印度亚种(L.delbrueckii subsp.Indicus)、卷曲乳杆菌(L.crispatus)、罗伊氏乳杆菌(L.reuteri)、瑞士乳杆菌(L.helveticus)，或嗜热链球菌(Streptococcusthermophilus)。在一些其他实施方案中，乳糖浓度降低至小于约1％，例如小于约0.1％或更低，例如小于约0.01％。
在另一方面，本发明涉及生产乳制品的方法，所述方法包括以下步骤：
a)提供包含乳糖的乳基基质；
b)向所述包含乳糖的乳基基质添加表现出β-半乳糖苷酶活性的肽或二聚肽，所述肽具有SEQ ID NO:1-33所示的氨基酸序列；所述二聚肽的组成为具有SEQ ID NO:2和3、5和6、20和21、23和24、26和27或28和29所示氨基酸序列的两个肽；或与任一所述序列具有至少80％序列同一性的序列；和
c)用所述表现出β-半乳糖苷酶活性的肽或二聚肽处理所述乳基基质。
在另一方面，本发明涉及通过本发明的方法制备的乳制品。
在另一方面，本发明涉及包含表现出β-半乳糖苷酶的酶活性的肽或二聚肽的食品，例如乳制品，所述肽具有SEQ ID NO:1-33所示的氨基酸序列，或者所述二聚肽的组成为具有SEQ ID NO:2和3、5和6、20和21、23和24、26和27或28和29所示氨基酸序列的两个肽；或与任一所述序列具有至少80％序列同一性的序列。
在另一方面，本发明涉及包含表达表现出β-半乳糖苷酶活性的肽或二聚肽的宿主细胞的食品，例如乳制品，所述肽具有SEQ ID NO:1-33所示的氨基酸序列，或所述二聚肽的组成为具有SEQ ID NO:2和3、5和6、20和21、23和24、26和27或28和29所示氨基酸序列的两个肽；或与任一所述序列具有至少80％序列同一性的序列。在一些具体实施方案中，这样的食品选自饮料、婴儿食品、谷物、面包、饼干、糖果、蛋糕、食品补充剂、膳食补充剂、益生菌可食用产品、益生元可食用产品、动物饲料、家禽饲料和药物，或选自由无乳糖乳、低乳糖乳、奶粉、婴儿乳、酸奶、冰淇淋、奶酪、发酵乳制品、膳食补充剂和益生菌饮食产品组成的乳制品。
附图说明
图1.在pH 6.7、37℃下以乳糖为底物测定的纯化酶的比活性,描述为SUAL-1，实施例6中所讨论。在给定条件下测得的标准偏差小于6％。
图2.在半乳糖存在的情况下，在pH 6.7、37℃测定的纯化酶的比活性，描述为SUAG，实施例7中所讨论。在给定条件下测得的标准偏差小于15％。
图3.在pH 6.7、4℃下以乳糖为底物测定的纯化酶的比活性，描述为SUAL-2，实施例8中所讨论。在给定条件下测得的标准偏差小于5％。
图4.在pH 6.7、43℃下以乳糖为底物测定的纯化酶的比活性,描述为SUAL-3，实施例9中所讨论。在给定条件下测得的标准偏差小于5％。
图5.在pH 5.5、4℃下以乳糖为底物测定的纯化酶的比活性,描述为SUAL-4，实施例10中所讨论。在给定条件下测得的标准偏差小于5％。
图6.在pH 5.5、37℃下以乳糖为底物测定的纯化酶的比活性,描述为SUAL-5，实施例11中所讨论。在给定条件下测得的标准偏差小于5％。
图7.在pH 5.5、43℃下以乳糖为底物测定的纯化酶的比活性,描述为SUAL-6，实施例12中所讨论。在给定条件下测得的标准偏差小于5％。
图8.在pH 4.5、4℃下以乳糖为底物测定的纯化酶的比活性,描述为SUAL-7，实施例13中所讨论。在给定条件下测得的标准偏差小于5％。
图9.在pH 4.5、37℃下以乳糖为底物测定的纯化酶的比活性,描述为SUAL-8，实施例14中所讨论。在给定条件下测得的标准偏差小于5％。
图10.在pH 4.5、43℃下以乳糖为底物测定的纯化酶的比活,描述为SUAL-9，实施例15中所讨论。在给定条件下测得的标准偏差小于5％。
图11.用固定量的酶于5℃下处理24h后，使用HPLC测定的巴氏灭菌乳中残留乳糖的百分比。
图12.用固定量的酶于25℃下处理24h后，使用HPLC测定的UHT乳中残留乳糖的百分比。
图13.以乳糖为底物测定的纯化酶在升高的温度下的残留活性百分比。在pH 6.7、37℃下的活性视为为100％。
图14.在pH 6.7下于4℃、37℃和43℃测定的纯化酶的比活性。测得的比活性描述为每毫克(mg)酶每分钟形成的葡萄糖的微摩尔数(μmol)。半乳糖抑制方法描述于实施例7中，并基于每mg酶每分钟形成的葡萄糖的微摩尔数来计算。
图15.在pH 5.5下于4℃、37℃和43℃测定的纯化酶的比活性。测得的比活性描述为每毫克酶每分钟形成的葡萄糖的微摩尔数。
图16.在pH 4.5下于4℃、37℃和43℃测定的纯化酶的比活性。测得的比活性描述为每毫克酶每分钟形成的葡萄糖的微摩尔数。
具体实施方式
本发明人发现，表现出β-半乳糖苷酶活性的某些肽和二聚肽在许多不同的物理条件下令人惊讶地稳定，提供相对高的活性，超出对于这类酶通常认为是最佳的范围之外。
因此，本发明人鉴定的这些酶在约4℃或5℃具有相对高的活性，并且因此可在生产例如鲜乳时用于乳糖水解。此外，这些酶在10℃至25℃的范围内也具有相对高的活性，因此完全相同的酶可用于UHT乳中的乳糖水解。酶的这种即使在宽的温度范围内的可行性是高度相关的，因为乳可能会在室温/环境温度下储存，而室温/环境温度可能会因世界的不同地区而异，也会因季节而异。对于UHT处理，温度通常为约135℃或约140℃。高度希望这些酶在高达140℃的温度范围具有活性，以便可以在UHT步骤之前将酶添加到生乳中。在当前的实践中，在UHT步骤之后添加酶，这是因为本领域已知的酶的功能活性在高温热处理步骤之后显著降低，例如低至可测量活性以下的值。同时乳保存在室温下，在世界不同地区室温可能会有很大差异。
还发现这些表现出β-半乳糖苷酶活性的新型改善的肽在通常用于巴氏灭菌的温度范围内具有活性。因此，可以在巴氏灭菌前将这些酶添加到生乳中。应当理解，在巴氏灭菌步骤之后，本领域已知的酶的功能活性显著降低，例如降低到可测量活性以下的值。
这些表现出β-半乳糖苷酶活性的新型改善的肽的另一个优点是，它们具有相对低的半乳糖抑制程度。这些新型酶的较低半乳糖抑制对于需要极低乳糖浓度的应用具有重要意义。
就发酵产品的适用性而言，非常有利的是本文所述的酶在4℃-43℃相对宽的温度范围内具有高β-半乳糖苷酶酶促活性，例如在约37℃，其中发酵通常是最佳的，但是β-半乳糖苷酶的这种活性在低pH值下也是存在的,例如低至4.5，或低至4.0，或低至3.5，甚至低至pH 3。
总而言之，本发明人发现，表现出β-半乳糖苷酶活性的某些肽在宽温度范围内具有活性，在宽pH范围内具有活性，具有通常的高水解活性而无副活性，这些肽没有或具有很小的半乳糖抑制(例如小于60％)，并且它们在长期储存中稳定。
所述β-半乳糖苷酶活性可以通过在设定条件下与乳糖一起孵育后测量释放的葡萄糖量来测定。释放的葡萄糖可以通过显色反应来检测。
定义
本文和本发明上下文中使用的术语“乳”应理解为通过对任何哺乳动物，例如奶牛、绵羊、山羊、水牛或骆驼挤奶而获得的乳状分泌物。
如本文所用，术语“含有乳糖的组合物”是指含有明显可测量程度的乳糖，例如乳糖含量高于0.002％(0.002g/100ml)的任何组合物，例如任何液体。该术语涵盖乳和乳基基质。
在本发明的上下文中，术语“乳基基质”可以是任何生乳材料和/或加工的乳材料。有用的乳基基质包括但不限于任何包含乳糖的乳或类似乳的产品的溶液/悬浮液，例如全脂乳或低脂乳、脱脂乳、酪乳、低乳糖乳、复原乳粉(reconstituted milk powder)、炼乳，乳粉溶液、UHT乳、乳清、乳清渗透物、酸乳清、奶油、发酵乳制品的溶液，发酵乳制品例如酸奶、干酪、膳食补充剂、益生菌膳食制品。通常，术语乳基基质是指为了生产乳制品而被进一步加工的生乳材料或加工乳材料。
本文所用的“巴氏灭菌”是指降低或消除乳基基质中活的有机体例如微生物存在的方法。优选，通过将指定温度保持指定的时间段来实现巴氏灭菌。通常通过加热来达到指定的温度。可以选择温度和持续时间，以杀死或灭活某些细菌，例如有害细菌，和/或灭活乳中的酶。随后可以进行快速冷却步骤。
本文所用的术语“乳制品”可以是其中主要成分之一是基于乳的任何食品。通常，所述主要成分是基于乳的，并且在一些实施方案中，所述主要成分是已经根据本发明的方法用具有β-半乳糖苷酶活性的酶处理的乳基基质。
本发明的乳制品可以是例如脱脂乳、低脂乳、全脂乳、奶油、UHT乳、具有延长的保存期限的乳、发酵乳制品、奶酪、酸奶、黄油、乳制涂抹酱(dairy spread)、酪乳(buttermilk)、酸化的乳饮料、酸奶油(sour cream)、基于乳清的饮料、冰淇淋、炼乳、炼奶焦糖酱(dulce de leche)或调味乳饮料。
乳制品可以另外包含非乳成分，例如，植物成分，例如植物油、植物蛋白质和/或植物碳水化合物。乳制品还可以包含其他添加剂，例如酶、调味剂、微生物培养物(例如益生菌培养物)、盐、甜味剂、糖、酸、水果、水果制剂、果汁或本领域已知的任何其他成分，作为乳制品的成分或添加剂。
本文所用的术语“发酵乳制品(fermented dairy product)”或“发酵乳产品(fermented milk product)”应理解为任何类型的发酵构成生产过程的一部分的任何乳制品。发酵乳制品的实例是酸奶、酪乳、鲜奶油(creme fraiche)、夸克(quark)和清爽干酪(fromage frais)等产品。发酵乳制品可以通过本领域已知的任何方法来生产或包括本领域已知的任何方法的步骤。
本文所用的术语“发酵”是指通过微生物的作用将碳水化合物转化为醇或酸。在一些实施方案中，本发明的发酵包括将乳糖转化为乳酸。在本发明的上下文中，“微生物”可以包括能够使乳基质发酵的任何细菌或真菌。
本文所用的术语“增加的β-半乳糖苷酶活性”是指与参考序列相比，β-半乳糖苷酶的相对较高的比活性。
本文所用的术语“表现出β-半乳糖苷酶活性的肽”是指具有催化二糖乳糖水解成其组分单糖即葡萄糖和半乳糖的酶促活性的任何肽。这种肽也可以称为乳糖酶或简称为β-半乳糖苷酶(EC:3.2.1.23)。
在本申请的上下文中使用的术语“肽”和“寡肽”应认为是同义的(如通常所承认的)，并且当上下文需要指示通过肽基键偶联的至少两个氨基酸的链时，每个术语可以互换使用。本文使用的“多肽”一词用于含有超过十个氨基酸残基的链。本文中所有肽和多肽的式或序列按从左至右并按从氨基末端至羧基末端的方向书写。本文所用“蛋白质”是指由某些宿主生物产生的肽序列，并且可以包括翻译后修饰，例如添加的聚糖。
本文所用的术语“氨基酸”或“氨基酸序列”是指寡肽、肽、多肽或蛋白质序列，或任何上述物质的片段，以及天然存在的分子或合成的分子。在本文中，“片段”是指表现出β-半乳糖苷酶活性的肽的片段，其保留了一些酶促活性。在本文中提及“氨基酸序列”以表示天然存在的蛋白质分子的氨基酸序列时，“氨基酸序列”和类似术语并不意味着将所述氨基酸序列限制为与所述肽分子相关的完整天然氨基酸序列。
本发明的示例性肽还包括酶的长度为至少约50、100、150、200、250、300、350、400、450、500、550、600、650、700、750、800或更多个残基的片段或酶的全长。因此，本发明的“肽片段”或“酶促活性片段”是保留至少一些功能性酶促活性的片段。通常，本发明的肽片段将会仍然含有表现出β-半乳糖苷酶活性的肽的功能性催化结构域或其他必要活性位点。其他域可以缺失。
通常会测量β-半乳糖苷酶的比活性，并将其表示为每毫克所用的酶每分钟形成的葡萄糖的微摩尔。但是，该比值会根据所应用的条件例如温度和pH而变化。因此，β-半乳糖苷酶活性的值也可以称为相对于参考已知酶，例如由SEQ ID NO:34或SEQ ID NO:35定义的β-半乳糖苷酶。
可选地，可以测量β-半乳糖苷酶的比活性，并表示为每μM所用的酶每秒形成的葡萄糖μM数。但是，该比值会根据所应用的条件例如温度和pH而变化。
除非另有说明，否则本文针对氨基酸所用的术语“序列同一性”是指计算为(nref-ndif)·100/nref的序列同一性，其中ndif是当比对时两条序列中不相同残基的总数，其中nref是序列之一中残基的数目。
在一些实施方案中，序列同一性通过常规方法测定，例如Smith and Waterman,1981,Adv.Appl.Math.2:482，Pearson&Lipman,1988,Proc.Natl.Acad.Sci.USA 85:2444的相似性搜索法，使用Thompson et al.,1994,Nucleic Acids Res 22:467380的CLUSTAL W算法，通过计算机执行这些算法(Genetics Computer Group的Wisconsin Genetics软件包中的GAP、BESTFIT、FASTA和TFASTA)。也可以使用BLAST算法(Altschul et al.,1990,Mol.Biol.215:403-10)，针对其软件可以通过国家生物技术信息中心www.ncbi.nlm.nih.gov/获得)。当使用任何上述算法时，将使用“窗口”长度、空位罚分等的默认参数。
本文所述的具有具体氨基酸序列的肽可以通过氨基酸取代、添加/插入或缺失中的任何一种而不同于参考肽序列。
本发明的一些实施方案涉及具有SEQ ID NO:1-33所示的氨基酸序列或与任一所述序列具有至少80％序列同一性的序列的肽的用途。在一些实施方案中，这种序列同一性可以为至少约81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％，例如与SEQ ID NO:1-33所示的任一参考氨基酸序列相比，具有不超过1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、15、16、17、18、19、20、21或22个氨基酸取代、添加或缺失的肽。本发明的特征还在于本发明的肽的生物活性片段。本发明的肽的生物活性片段包括这样的肽，所述肽包含与本发明的肽的氨基酸序列充分相同或由其衍生的氨基酸序列，其包含比全长蛋白质少的氨基酸，但是表现出相应全长肽的相当大一部分的生物学活性。通常，生物活性片段包含具有本发明变异蛋白质的至少一种活性的结构域或基序。本发明的肽的生物活性片段可以是例如长度为100、200、300、400、500、600、700、800、900、1000或更多个氨基酸的肽。
如本文所用，术语“宿主细胞”包括易于用包含编码本发明肽的多核苷酸的核酸构建体或表达载体转化、转染、转导等的任何细胞类型。宿主细胞可以是衍生特定酶的细胞类型，或者其可以是对特定酶的产生敏感的替代细胞类型。该术语包括野生型和减毒株。
合适的宿主细胞可以是任何细菌，包括“乳杆菌目(Lactobacillales)”中的乳酸菌，包括乳球菌属种(Lactococcus spp.)、链球菌属种(Streptococcus spp.)、乳杆菌属种(Lactobacillus spp)、明串珠菌属种(Leuconostoc spp.)、假明串珠菌属种(PseudoLeuconostoc spp.)、片球菌属种(Pediococcus spp.)、短杆菌属种(Brevibacterium spp.)、肠球菌属种(Enterococcus spp.)和丙酸杆菌属种(Propionibacterium spp.)。还包括属于厌氧细菌双歧杆菌即双歧杆菌属种(Bifidobacterium spp.)的产乳酸细菌，其经常单独或与乳酸菌组合用作食品培养物。该定义还包括乳酸乳球菌(Lactococcus lactis)、乳酸乳球菌乳脂亚种(Lactococcuslactis subsp.cremoris)、肠膜明串珠菌乳脂亚种(Leuconostoc mesenteroidessubsp.cremoris)、肠膜假明串珠菌乳脂亚种(Pseudoleuconostoc mesenteroidessubsp.cremoris)、戊糖片球菌(Pediococcus pentosaceus)、乳酸乳球菌乳酸亚种丁二酮生物变种(Lactococcus lactis subsp.lactis biovar.diacetylactis)、干酪乳杆菌干酪亚种(Lactobacillus casei subsp.casei)和副干酪乳杆菌副干酪亚种(Lactobacillusparacasei subsp.Paracasei)，嗜热乳酸菌物种包括例如嗜热链球菌(Streptococcusthermophilus)、屎肠球菌(Enterococcus faecium)、德氏乳杆菌乳酸亚种(Lactobacillusdelbrueckii subsp.lactis)、瑞士乳杆菌(Lactobacillus helveticus)、德氏乳杆菌保加利亚亚种(Lactobacillus delbrueckii subsp.bulgaricus)和嗜酸乳杆菌(Lactobacillus acidophilus)。在该定义内的其他具体细菌包括双歧杆菌科(Bifidobacteriaceae)的细菌，例如来自双歧杆菌属(Bifidobacterium)，例如来自动物双歧杆菌(bifidobacterium animalis)或长双歧杆菌(bifidobacterium longum)、青春双歧杆菌(bifidobacterium adolescentis)、两歧双歧杆菌(bifidobacterium bifodum)、短双歧杆菌(bifidobacterium breve)、链状双歧杆菌(bifidobacterium catenulatum)、婴儿双歧杆菌(bifidobacterium infantus)的菌株，或来自乳杆菌属(Lactobacillus)，例如清酒乳杆菌、噬淀粉乳杆菌、德氏乳杆菌乳酸亚种和瑞士乳杆菌。
宿主细胞的这种定义还包括以下的菌株：蘑菇属(Agaricus)，例如，双孢蘑菇(A.bisporus)；Ascovaginospora；曲霉属(Aspergillus)，例如黑曲霉(A.niger)、泡盛曲霉(A.awamori)、臭曲霉(A.foetidus)、日本曲霉(A.japonicus)、米曲霉(A.oryzae)；念珠菌属(Candida)；毛壳属(Chaetomium)；Chaetotomastia；网柄菌属(Dictyostelium)，例如盘基网柄菌(D.discoideum)；克鲁维酵母属(Kluveromyces)，例如脆壁克鲁维酵母(K.fragilis)、乳酸克鲁维酵母(K.lactis)；毛霉属(Mucor)，例如爪哇毛霉(M.javanicus)、高大毛霉(M.mucedo)、细孢毛霉(M.subtilissimus)；脉孢菌属(Neurospora)，例如粗糙脉孢菌(N.crassa)；根毛霉属(Rhizomucor)，例如微小根毛霉(R.pusillus)；根霉属(Rhizopus)，例如少根根霉(R.arrhizus)、日本根霉(R.japonicus)、匍枝根霉(R.stolonifer)；核盘菌属(Sclerotinia)，例如大豆核盘菌(S.libertiana)；串酵母属(Torula)；球拟酵母属(Torulopsis)；毛癣菌属(Trichophyton)，例如红色毛癣菌(Trichophyton rubrum)；维氏核盘菌属(Whetzelinia)，例如，图维氏核盘菌(W.sclerotiorum)；芽孢杆菌属(Bacillus)，例如凝结芽孢杆菌(B.coagulans)、环状芽胞杆菌(B.circulans)、巨大芽孢杆菌(B.megaterium)、休闲地芽胞杆菌(B.novalis)、枯草芽孢杆菌(B.subtilis)、短小芽孢杆菌(B.pumilus)、嗜热脂肪芽孢杆菌(B.stearothermophilus)、苏云金芽孢杆菌(B.thuringiensis)；双歧杆菌属(Bifidobactgerium)，例如长双歧杆菌(B.longum)、两歧双歧杆菌(B.bifidum)、动物双歧杆菌(B.animalis)；金黄杆菌属(Chryseobacterium)；柠檬酸杆菌属(Citrobacter)，例如弗氏柠檬酸杆菌(C.freundii)；梭菌属(Clostridium)，例如产气荚膜梭菌(C.perfringens)；色二孢属(Diplodia)，例如棉色二孢(D.gossypina)；肠杆菌属(Enterobacter)，例如产气肠杆菌(E.aerogenes)、阴沟肠杆菌(E.cloacae)；爱德华菌属(Edwardsiella)，迟缓爱德华菌(E.tarda)；欧文氏菌属(Erwinia)，例如草生欧文氏菌(E.herbicola)；埃希氏菌属(Escherichia)，例如大肠杆菌(E.coli)；克雷伯氏菌属(Klebsiella)，例如肺炎克雷伯氏菌(K.pneumoniae)；Miriococcum；Myrothesium；毛霉属(Mucor)；脉孢霉属(Neurospora)，例如粗糙脉孢霉(N.crassa)；变形杆菌属(Proteus)，例如普通变形杆菌(P.vulgaris)；普罗维登斯菌属(Providencia)，例如斯氏普罗维登斯菌(P.stuartii)；密孔菌属(Pycnoporus)，例如朱红密孔菌(Pycnoporus cinnabarinus)、血红密孔菌(Pycnoporus sanguineus)；瘤胃球菌属(Ruminococcus)，例如扭链瘤胃球菌(R.torques)；沙门氏菌(Salmonella)，例如鼠伤寒沙门氏菌(S.typhimurium)；沙雷氏菌属(Serratia)，例如液化沙雷氏菌(S.liquefasciens)、粘质沙雷氏菌(S.marcescens)；志贺氏菌属(Shigella)，例如福氏志贺菌(S.flexneri)；链霉菌属(Streptomyces)，例如抗生素链霉菌(S.antibioticus)、栗色浑圆链霉菌(S.castaneoglobisporus)、紫红链霉菌(S.violeceoruber)；栓菌属(Trametes)；木霉属(Trichoderma)，例如里氏木霉(T.reesei)、绿色木霉(T.viride)；耶尔森氏菌属(Yersinia)，例如小肠结肠炎耶尔森氏菌(Y.enterocolitica)。
本发明的具体实施方案
如上所述，本发明涉及表现出β-半乳糖苷酶活性的肽，其具有SEQ ID NO:1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33所示的氨基酸序列，或上述序列的具有不超过1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21或22个氨基酸取代、添加或缺失的氨基酸序列。
因此，在一个实施方案中，本发明涉及表现出β-半乳糖苷酶活性的肽，其具有SEQID NO:1所示的氨基酸序列，或上述序列的具有不超过1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21或22个氨基酸取代、添加或缺失的氨基酸序列。在一个实施方案中，本发明涉及表现出β-半乳糖苷酶活性的肽，其具有SEQ ID NO:2所示的氨基酸序列，或上述序列的具有不超过1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21或22个氨基酸取代、添加或缺失的氨基酸序列。在一个实施方案中，本发明涉及表现出β-半乳糖苷酶活性的肽，其具有SEQ ID NO:3所示的氨基酸序列，或上述序列的具有不超过1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21或22个氨基酸取代、添加或缺失的氨基酸序列。在一个实施方案中，本发明涉及表现出β-半乳糖苷酶活性的肽，其具有SEQ IDNO:4所示的氨基酸序列，或上述序列的具有不超过1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21或22个氨基酸取代、添加或缺失的氨基酸序列。在一个实施方案中，本发明涉及表现出β-半乳糖苷酶活性的肽，其具有SEQ ID NO:5所示的氨基酸序列，或上述序列的具有不超过1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21或22个氨基酸取代、添加或缺失的氨基酸序列。在一个实施方案中，本发明涉及表现出β-半乳糖苷酶活性的肽，其具有SEQ ID NO:6所示的氨基酸序列，或上述序列的具有不超过1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21或22个氨基酸取代、添加或缺失的氨基酸序列。在一个实施方案中，本发明涉及表现出β-半乳糖苷酶活性的肽，其具有SEQ ID NO:7所示的氨基酸序列，或上述序列的具有不超过1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21或22个氨基酸取代、添加或缺失的氨基酸序列。在一个实施方案中，本发明涉及表现出β-半乳糖苷酶活性的肽，其具有SEQ ID NO:8所示的氨基酸序列，或上述序列的具有不超过1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21或22个氨基酸取代、添加或缺失的氨基酸序列。在一个实施方案中，本发明涉及表现出β-半乳糖苷酶活性的肽，其具有SEQ ID NO:9所示的氨基酸序列，或上述序列的具有不超过1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21或22个氨基酸取代、添加或缺失的氨基酸序列。在一个实施方案中，本发明涉及表现出β-半乳糖苷酶活性的肽，其具有SEQ ID NO:10所示的氨基酸序列，或上述序列的具有不超过1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21或22个氨基酸取代、添加或缺失的氨基酸序列。在一个实施方案中，本发明涉及表现出β-半乳糖苷酶活性的肽，其具有SEQ ID NO:11所示的氨基酸序列，或上述序列的具有不超过1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21或22个氨基酸取代、添加或缺失的氨基酸序列。在一个实施方案中，本发明涉及表现出β-半乳糖苷酶活性的肽，其具有SEQ ID NO:12所示的氨基酸序列，或上述序列的具有不超过1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21或22个氨基酸取代、添加或缺失的氨基酸序列。在一个实施方案中，本发明涉及表现出β-半乳糖苷酶活性的肽，其具有SEQ ID NO:13所示的氨基酸序列，或上述序列的具有不超过1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21或22个氨基酸取代、添加或缺失的氨基酸序列。在一个实施方案中，本发明涉及表现出β-半乳糖苷酶活性的肽，其具有SEQ ID NO:14所示的氨基酸序列，或上述序列的具有不超过1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21或22个氨基酸取代、添加或缺失的氨基酸序列。在一个实施方案中，本发明涉及表现出β-半乳糖苷酶活性的肽，其具有SEQ ID NO:15所示的氨基酸序列，或上述序列的具有不超过1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21或22个氨基酸取代、添加或缺失的氨基酸序列。在一个实施方案中，本发明涉及表现出β-半乳糖苷酶活性的肽，其具有SEQ ID NO:16所示的氨基酸序列，或上述序列的具有不超过1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21或22个氨基酸取代、添加或缺失的氨基酸序列。在一个实施方案中，本发明涉及表现出β-半乳糖苷酶活性的肽，其具有SEQ ID NO:17所示的氨基酸序列，或上述序列的具有不超过1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21或22个氨基酸取代、添加或缺失的氨基酸序列。在一个实施方案中，本发明涉及表现出β-半乳糖苷酶活性的肽，其具有SEQ ID NO:18所示的氨基酸序列，或上述序列的具有不超过1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21或22个氨基酸取代、添加或缺失的氨基酸序列。在一个实施方案中，本发明涉及表现出β-半乳糖苷酶活性的肽，其具有SEQ ID NO:19所示的氨基酸序列，或上述序列的具有不超过1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21或22个氨基酸取代、添加或缺失的氨基酸序列。在一个实施方案中，本发明涉及表现出β-半乳糖苷酶活性的肽，其具有SEQ ID NO:20所示的氨基酸序列，或上述序列的具有不超过1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21或22个氨基酸取代、添加或缺失的氨基酸序列。在一个实施方案中，本发明涉及表现出β-半乳糖苷酶活性的肽，其具有SEQ ID NO:21所示的氨基酸序列，或上述序列的具有不超过1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21或22个氨基酸取代、添加或缺失的氨基酸序列。在一个实施方案中，本发明涉及表现出β-半乳糖苷酶活性的肽，其具有SEQ ID NO:22所示的氨基酸序列，或上述序列的具有不超过1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21或22个氨基酸取代、添加或缺失的氨基酸序列。在一个实施方案中，本发明涉及表现出β-半乳糖苷酶活性的肽，其具有SEQ ID NO:23所示的氨基酸序列，或上述序列的具有不超过1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21或22个氨基酸取代、添加或缺失的氨基酸序列。在一个实施方案中，本发明涉及表现出β-半乳糖苷酶活性的肽，其具有SEQ ID NO:24所示的氨基酸序列，或上述序列的具有不超过1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21或22个氨基酸取代、添加或缺失的氨基酸序列。在一个实施方案中，本发明涉及表现出β-半乳糖苷酶活性的肽，其具有SEQ ID NO:25所示的氨基酸序列，或上述序列的具有不超过1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21或22个氨基酸取代、添加或缺失的氨基酸序列。在一个实施方案中，本发明涉及表现出β-半乳糖苷酶活性的肽，其具有SEQ ID NO:26所示的氨基酸序列，或上述序列的具有不超过1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21或22个氨基酸取代、添加或缺失的氨基酸序列。在一个实施方案中，本发明涉及表现出β-半乳糖苷酶活性的肽，其具有SEQ ID NO:27所示的氨基酸序列，或上述序列的具有不超过1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21或22个氨基酸取代、添加或缺失的氨基酸序列。在一个实施方案中，本发明涉及表现出β-半乳糖苷酶活性的肽，其具有SEQ ID NO:28所示的氨基酸序列，或上述序列的具有不超过1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21或22个氨基酸取代、添加或缺失的氨基酸序列。在一个实施方案中，本发明涉及表现出β-半乳糖苷酶活性的肽，其具有SEQ ID NO:29所示的氨基酸序列，或上述序列的具有不超过1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21或22个氨基酸取代、添加或缺失的氨基酸序列。在一个实施方案中，本发明涉及表现出β-半乳糖苷酶活性的肽，其具有SEQ ID NO:30所示的氨基酸序列，或上述序列的具有不超过1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21或22个氨基酸取代、添加或缺失的氨基酸序列。在一个实施方案中，本发明涉及表现出β-半乳糖苷酶活性的肽，其具有SEQ ID NO:31所示的氨基酸序列，或上述序列的具有不超过1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21或22个氨基酸取代、添加或缺失的氨基酸序列。在一个实施方案中，本发明涉及表现出β-半乳糖苷酶活性的肽，其具有SEQ ID NO:32所示的氨基酸序列，或上述序列的具有不超过1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21或22个氨基酸取代、添加或缺失的氨基酸序列。在一个实施方案中，本发明涉及表现出β-半乳糖苷酶活性的肽，其具有SEQ ID NO:33所示的氨基酸序列，或上述序列的具有不超过1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21或22个氨基酸取代、添加或缺失的氨基酸序列。
在一些实施方案中，本发明的肽具有在本文所述实施例8给出的条件下，在约4℃的温度和pH 6.7下，以每μM酶每秒形成的葡萄糖的μM测量的β-半乳糖苷酶活性，所述活性超过SEQ ID NO:34或SEQ ID NO:35定义的β-半乳糖苷酶活性至少约20％，例如至少约30％，例如至少约40％，例如至少约50％，例如至少约60％，例如至少约70％，例如至少约80％，例如至少约90％，例如至少约100％，例如至少约200％，例如至少约300％，例如至少约400％，例如至少约500％。
在一些实施方案中，本发明的肽具有在约4℃的温度和pH 6.7下以每μM酶每秒形成的葡萄糖的微摩尔(μM)测量的β-半乳糖苷酶活性，所述活性超过SEQ ID NO:34或SEQ IDNO:35定义的β-半乳糖苷酶活性至少约20％，例如至少约30％，例如至少约40％，例如至少约50％，例如至少约60％，例如至少约70％，例如至少约80％，例如至少约90％，例如至少约100％，例如至少约200％，例如至少约300％，例如至少约400％，例如至少约500％。在一些实施方案中，本发明的肽具有在约4℃的温度和pH 6.7下以每μM酶每秒形成的葡萄糖的μM测量的β-半乳糖苷酶活性，所述活性高于每μM酶每秒形成的约2μM葡萄糖，例如高于约4μM，例如高于约6μM，例如高于约8μM，例如高于约10μM，例如高于约12μM，例如高于约14μM，例如高于约16μM。
在一些实施方案中，本发明的肽具有在本文所述实施例10给出的条件下，在约4℃的温度和pH 5.5下，以每μM酶每秒形成的葡萄糖的μM测量的β-半乳糖苷酶活性，所述活性超过SEQ ID NO:34或SEQ ID NO:35定义的β-半乳糖苷酶的活性至少约20％，例如至少约30％，例如至少约40％，例如至少约50％，例如至少约60％，例如至少约70％，例如至少约80％，例如至少约90％，例如至少约100％，例如至少约200％，例如至少约300％，例如至少约400％，例如至少约500％。
在一些实施方案中，本发明的肽具有在约4℃的温度和pH 5.5下以每μM酶每秒形成的葡萄糖的μM测量的β-半乳糖苷酶活性，所述活性超过SEQ ID NO:34或SEQ ID NO:35定义的β-半乳糖苷酶的活性至少约20％，例如至少约30％，例如至少约40％，例如至少约50％，例如至少约60％，例如至少约70％，例如至少约80％，例如至少约90％，例如至少约100％，例如至少约200％，例如至少约300％，例如至少约400％，例如至少约500％。在一些实施方案中，本发明的肽具有在约4℃的温度和pH 5.5下，以每μM酶每秒形成的葡萄糖的μM测量的β-半乳糖苷酶活性，所述活性高于每μM酶每秒形成的约1μM葡萄糖，例如高于约2μM，例如高于约3μM，例如高于约4μM，例如高于约5μM，例如高于约6μM，例如高于约7μM，例如高于约8μM。
在一些实施方案中，本发明的肽具有在本文所述实施例13给出的条件下，在约4℃的温度和pH 4.5下，以每μM酶每秒形成的葡萄糖的μM测量的β-半乳糖苷酶活性，所述活性超过SEQ ID NO:34或SEQ ID NO:35定义的β-半乳糖苷酶的活性至少约20％，例如至少约30％，例如至少约40％，例如至少约50％，例如至少约60％，例如至少约70％，例如至少约80％，例如至少约90％，例如至少约100％，例如至少约200％，例如至少约300％，例如至少约400％，例如至少约500％。
在一些实施方案中，本发明的肽具有在约4℃的温度和pH 4.5下以每μM酶每秒形成的葡萄糖的μM测量的β-半乳糖苷酶活性，所述活性超过SEQ ID NO:34或SEQ ID NO:35定义的β-半乳糖苷酶的活性至少约20％，例如至少约30％，例如至少约40％，例如至少约50％，例如至少约60％，例如至少约70％，例如至少约80％，例如至少约90％，例如至少约100％，例如至少约200％，例如至少约300％，例如至少约400％，例如至少约500％。在一些实施方案中，本发明的肽具有在约4℃的温度和pH 4.5下以每μM酶每秒形成的葡萄糖的μM测量的β-半乳糖苷酶活性，所述活性高于每μM酶每秒形成的约0.5μM葡萄糖，例如高于约1.0μM，例如高于约1.5μM，例如高于约2.0μM，例如高于约2.5μM，例如高于约3.0μM，例如高于约3.5μM，例如高于约4.0μM。
在一些实施方案中，本发明的肽具有在本文所述实施例9给出的条件下，在约43℃的温度和pH 6.7下以每μM每秒形成的葡萄糖的μM测量的β-半乳糖苷酶活性，所述活性超过SEQ ID NO:34或SEQ ID NO:35定义的β-半乳糖苷酶的活性至少约20％，例如至少约30％，例如至少约40％，例如至少约50％，例如至少约60％，例如至少约70％，例如至少约80％，例如至少约90％，例如至少约100％，例如至少约200％，例如至少约300％，例如至少约400％，例如至少约500％。
在一些实施方案中，本发明的肽具有在约43℃的温度和pH 6.7下以每μM酶每秒形成的葡萄糖的μM测量的β-半乳糖苷酶活性，所述活性超过SEQ ID NO:34或SEQ ID NO:35定义的β-半乳糖苷酶的活性至少约20％，例如至少约30％，例如至少约40％，例如至少约50％，例如至少约60％，例如至少约70％，例如至少约80％，例如至少约90％，例如至少约100％，例如至少约200％，例如至少约300％，例如至少约400％，例如至少约500％。在一些实施方案中，本发明的肽具有在约43℃的温度和pH 6.7下以每μM酶每秒形成的葡萄糖的μM测量的β-半乳糖苷酶活性，所述活性高于每μM酶每秒形成的约10μM葡萄糖，例如高于约20μM，例如高于约40μM，例如高于约60μM，例如高于约80μM，例如高于约100μM，例如高于约120μM，例如高于约140μM，例如高于约160μM。
在一些实施方案中，本发明的肽具有在本文所述实施例12给出的条件下，在约43℃的温度和pH 5.5下，以每μM酶每秒形成的葡萄糖的μM测量的β-半乳糖苷酶活性，所述活性超过SEQ ID NO:34或SEQ ID NO:35定义的β-半乳糖苷酶的活性至少约20％，例如至少约30％，例如至少约40％，例如至少约50％，例如至少约60％，例如至少约70％，例如至少约80％，例如至少约90％，例如至少约100％，例如至少约200％，例如至少约300％，例如至少约400％，例如至少约500％。
在一些实施方案中，本发明的肽具有在约43℃的温度和pH 5.5下，以每μM酶每秒形成的葡萄糖的μM测量的β-半乳糖苷酶活性，所述活性超过SEQ ID NO:34或SEQ ID NO:35定义的β-半乳糖苷酶的活性至少约20％，例如至少约30％，例如至少约40％，例如至少约50％，例如至少约60％，例如至少约70％，例如至少约80％，例如至少约90％，例如至少约100％，例如至少约200％，例如至少约300％，例如至少约400％，例如至少约500％。在一些实施方案中，本发明的肽具有在约43℃的温度和pH 5.5下以每μM酶每秒形成的葡萄糖的μM测量的β-半乳糖苷酶活性，所述活性高于每μM酶每秒形成的约5μM葡萄糖，例如高于约10μM，例如高于约15μM，例如高于约20μM，例如高于约25μM，例如高于约30μM，例如高于约35μM，例如高于约40μM，例如高于约45μM，例如高于约50μM，例如高于约55μM，例如高于约60μM。
在一些实施方案中，本发明的肽具有在本文所述实施例15给出的条件下，在约43℃的温度和pH 4.5下，以每μM酶每秒形成的葡萄糖的μM测量的β-半乳糖苷酶活性，所述活性超过SEQ ID NO:34或SEQ ID NO:35定义的β-半乳糖苷酶的活性至少约20％，例如至少约30％，例如至少约40％，例如至少约50％，例如至少约60％，例如至少约70％，例如至少约80％，例如至少约90％，例如至少约100％，例如至少约200％，例如至少约300％，例如至少约400％，例如至少约500％。
在一些实施方案中，本发明的肽具有在约43℃的温度和pH 4.5下以每μM酶每秒形成的葡萄糖的μM测量的β-半乳糖苷酶活性，所述活性超过SEQ ID NO:34或SEQ ID NO:35定义的β-半乳糖苷酶的活性至少约20％，例如至少约30％，例如至少约40％，例如至少约50％，例如至少约60％，例如至少约70％，例如至少约80％，例如至少约90％，例如至少约100％，例如至少约200％，例如至少约300％，例如至少约400％，例如至少约500％。在一些实施方案中，本发明的肽具有在约43℃的温度和pH 4.5下以每μM酶每秒形成的葡萄糖的μM测量的β-半乳糖苷酶活性，所述活性高于每μM酶每秒形成的约1μM葡萄糖，例如高于约2μM，例如高于约3μM，例如高于约4μM，例如高于约5μM，例如高于约6μM，例如高于约7μM，例如高于约8μM，例如高于约9μM，例如高于约10μM，例如高于约11μM，例如高于约12μM。
在一些实施方案中，本发明的肽具有在本文所述实施例6给出的条件下，在约37℃的温度和pH 6.7下，以每μM酶每秒形成的葡萄糖的μM测量的β-半乳糖苷酶活性，所述活性超过SEQ ID NO:34或SEQ ID NO:35定义的β-半乳糖苷酶的活性至少约20％，例如至少约30％，例如至少约40％，例如至少约50％，例如至少约60％，例如至少约70％，例如至少约80％，例如至少约90％，例如至少约100％，例如至少约200％，例如至少约300％，例如至少约400％，例如至少约500％。
在一些实施方案中，本发明的肽具有在约37℃的温度和pH 6.7下以每μM酶每秒形成的葡萄糖的μM测量的β-半乳糖苷酶活性，所述活性超过SEQ ID NO:34或SEQ ID NO:35定义的β-半乳糖苷酶的活性至少约20％，例如至少约30％，例如至少约40％，例如至少约50％，例如至少约60％，例如至少约70％，例如至少约80％，例如至少约90％，例如至少约100％，例如至少约200％，例如至少约300％，例如至少约400％，例如至少约500％。在一些实施方案中，本发明的肽具有在约37℃的温度和pH 6.7下以每μM酶每秒形成的葡萄糖的μM测量的β-半乳糖苷酶活性，所述活性高于每μM酶每秒形成的约10μM葡萄糖，例如高于约20μM，例如高于约30μM，例如高于约40μM，例如高于约50μM，例如高于约60μM，例如高于约70μM，例如高于约80μM，例如高于约90μM，例如高于约100μM，例如高于约110μM，例如高于约120μM，例如高于约130μM。
在一些实施方案中，本发明的肽具有在本文所述实施例11给出的条件下，在约37℃的温度和pH 5.5下，以每μM酶每秒形成的葡萄糖的μM测量的β-半乳糖苷酶活性，所述活性超过SEQ ID NO:34或SEQ ID NO:35定义的β-半乳糖苷酶的活性至少约20％，例如至少约30％，例如至少约40％，例如至少约50％，例如至少约60％，例如至少约70％，例如至少约80％，例如至少约90％，例如至少约100％，例如至少约200％，例如至少约300％，例如至少约400％，例如至少约500％。
在一些实施方案中，本发明的肽具有在约37℃的温度和pH 5.5下以每μM酶每秒形成的葡萄糖的μM测量的β-半乳糖苷酶活性，所述活性超过SEQ ID NO:34或SEQ ID NO:35定义的β-半乳糖苷酶的活性至少约20％，例如至少约30％，例如至少约40％，例如至少约50％，例如至少约60％，例如至少约70％，例如至少约80％，例如至少约90％，例如至少约100％，例如至少约200％，例如至少约300％，例如至少约400％，例如至少约500％。在一些实施方案中，本发明的肽具有在约37℃的温度和pH 5.5下以每μM酶每秒形成的葡萄糖的μM测量的β-半乳糖苷酶活性，所述活性高于每μM酶每秒形成的约5μM葡萄糖，例如高于约10μM，例如高于约15μM，例如高于约20μM，例如高于约25μM，例如高于约30μM，例如高于约35μM，例如高于约40μM，例如高于约45μM，例如高于约50μM，例如高于约55μM，例如高于约60μM。
在一些实施方案中，本发明的肽具有在本文所述实施例14给出的条件下，在约37℃的温度和pH 4.5下，以每μM酶每秒形成的葡萄糖的μM测量的β-半乳糖苷酶活性，所述活性超过SEQ ID NO:34或SEQ ID NO:35定义的β-半乳糖苷酶的活性至少约20％，例如至少约30％，例如至少约40％，例如至少约50％，例如至少约60％，例如至少约70％，例如至少约80％，例如至少约90％，例如至少约100％，例如至少约200％，例如至少约300％，例如至少约400％，例如至少约500％。
在一些实施方案中，本发明的肽具有在约37℃的温度和pH 4.5下以每μM酶每秒形成的葡萄糖的μM的测量的β-半乳糖苷酶活性，所述活性超过SEQ ID NO:34或SEQ ID NO:35定义的β-半乳糖苷酶的活性至少约20％，例如至少约30％，例如至少约40％，例如至少约50％，例如至少约60％，例如至少约70％，例如至少约80％，例如至少约90％，例如至少约100％，例如至少约200％，例如至少约300％，例如至少约400％，例如至少约500％。在一些实施方案中，本发明的肽具有在约37℃的温度和pH 4.5下，以每μM酶每秒形成的葡萄糖的μM测量的β-半乳糖苷酶活性，所述活性高于每μM酶每秒形成的约1μM葡萄糖，例如高于约2μM，例如高于约3μM，例如高于约4μM，例如高于约5μM，例如高于约6μM，例如高于约7μM，例如高于约8μM，例如高于约9μM，例如高于约10μM，例如高于约11μM，例如高于约12μM，例如高于约13μM，例如高于约14μM，例如高于约15μM，例如高于约16μM，例如高于约17μM，例如高于约18μM。
在一些实施方案中，本发明的肽衍生自双歧杆菌属的细菌，例如青春双歧杆菌、两歧双歧杆菌、短双歧杆菌、链状双歧杆菌、长双歧杆菌，或衍生自乳杆菌属的细菌，例如清酒乳杆菌、噬淀粉乳杆菌、德氏乳杆菌保加利亚亚种、德氏乳杆菌乳酸亚种、德氏乳杆菌印度亚种、卷曲乳杆菌、罗伊氏乳杆菌、瑞士乳杆菌，或衍生自嗜热链球菌。
在一些实施方案中，根据本发明的肽表现出的半乳糖抑制小于60％，例如小于55％，例如小于50％，例如小于约45％，例如小于约40％。
如上所述，本发明的一部分涉及用于生产乳制品的方法，所述方法包括以下步骤：
a)提供包含乳糖的乳基基质；
b)将表现出β-半乳糖苷酶活性并具有SEQ ID NO:1-33所示的氨基酸序列或与任一所述序列具有至少80％序列同一性的序列的肽添加到所述包含乳糖的乳基基质中；和
c)用所述表现出β-半乳糖苷酶活性的肽处理所述乳基基质。
在本发明的一些实施方案中，这种肽衍生自双歧杆菌属的任一种细菌，例如青春双歧杆菌、两歧双歧杆菌、短双歧杆菌、链状双歧杆菌、长双歧杆菌，或衍生自乳杆菌属，例如清酒乳杆菌、噬淀粉乳杆菌、德氏乳杆菌保加利亚亚种、德氏乳杆菌乳酸亚种、德氏乳杆菌印度亚种、卷曲乳杆菌、罗伊氏乳杆菌、瑞士乳杆菌，或衍生自嗜热链球菌。
在本发明的一些实施方案中，进行步骤c)的pH在3-10的范围内，例如在3-9的范围内，例如在3-8的范围内，例如在3-7的范围内,例如在3-6的范围内，例如在3-5的范围内，例如在3-4的范围内，例如在4-10的范围内，例如在4-9的范围内，例如在4-8的范围内，例如在4-7的范围内，例如在4-6的范围内，例如在4-5的范围内，例如在5-10的范围内，例如在5-9的范围内，例如在5-8的范围内，例如在5-7的范围内，例如在5-6的范围内，例如在6-10的范围内，例如在6-9的范围内，例如在6-8的范围内，例如在6-7的范围内。
在本发明的一些实施方案中，进行步骤c)或步骤c)的一部分的温度不超过约25℃，例如不超过约20℃，例如不超过约18℃，例如不超过约16℃，例如不超过约14℃，例如不超过约12℃，例如不超过约10℃，例如不超过约8℃，例如不超过约7℃，例如不超过约6℃，例如不超过约5℃，例如不超过约4℃，例如不超过约3℃，例如不超过约2℃。
在本发明的一些实施方案中，进行步骤c)或步骤c)的一部分的温度为至少约25℃，例如至少约30℃，例如至少约35℃，例如至少约40℃，例如至少约45℃，例如至少约50℃，例如至少约55℃，例如至少约60℃，例如至少约65℃，例如至少约70℃，例如至少约75℃，例如至少约80℃，例如至少约85℃，例如至少约90℃，例如至少约95℃，例如至少约100℃，例如至少约110℃，例如至少约120℃，例如至少约130℃，例如至少约120℃，例如至少约130℃，例如至少约135℃，例如至少约140℃。
在本发明的一些实施方案中，乳制品选自由以下组成的组：无乳糖乳、低乳糖乳、酸奶、奶酪、发酵乳制品、膳食补充剂和益生菌膳食制品。
在本发明的一些实施方案中，所述乳基基质选自鲜乳或生乳(直接由巴氏灭菌步骤获得)、直接在超热处理(UHT)步骤之后获得的乳或直接在发酵步骤之后获得的乳。
在本发明的一些实施方案中，所用肽的半乳糖抑制小于60％，例如小于55％，例如小于50％，例如小于约45％，例如小于约40％。
在本发明的一些实施方案中，乳制品是发酵乳制品，并且在发酵期间或之前进行所述步骤b)。
在本发明的一些实施方案中，所述方法不需要在发酵后添加其他酶。
在本发明的一些实施方案中，乳制品是发酵乳制品，并且在发酵后立即进行所述步骤b)。
在本发明的一些实施方案中，乳制品是鲜乳，并且在巴氏灭菌步骤之前、同时或之后立即进行所述步骤b)。
在本发明的一些实施方案中，乳制品是超热处理(UHT)乳，并且在超热处理步骤之前、同时或之后立即进行所述步骤b)。
在本发明的一些实施方案中，开始步骤c)的温度为40℃至100℃，例如50℃至100℃，例如60℃至100℃，例如70℃至100℃，例如80℃至100℃，例如40℃至90℃，例如40℃至80℃，例如40℃至70℃，例如40℃至60℃，例如40℃至50℃。
在本发明的一些实施方案中，当水解乳基基质中的乳糖时，肽的乳糖酶与转半乳糖基酶活性之比大于1:1。
在本发明的一些实施方案中，当步骤c)完成时，少于80％的乳糖已被水解，并且其中超过90％的乳糖在一周后已被水解。
编号的实施方案
1.表现出β-半乳糖苷酶活性的肽，所述肽具有SEQ ID NO:1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33所示的氨基酸序列或其酶促活性片段的氨基酸序列或任一上述序列的具有不超过1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21或22个氨基酸取代、添加或缺失的氨基酸序列。
2.表现出β-半乳糖苷酶活性的二聚肽，所述二聚肽的组成为具有SEQ ID NO:2和3、5和6、20和21、23和24、26和27或28和29所示氨基酸序列的两个肽；或其酶促活性片段，或任一上述序列的具有不超过1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21或22个氨基酸取代、添加或缺失的氨基酸序列。
3.根据实施方案1或2的所述肽或二聚肽，其具有在本文所述实施例8给出的条件下，在约4℃的温度和pH 6.7下以每μM酶每秒形成的葡萄糖的μM测量的β-半乳糖苷酶活性，所述活性超过SEQ ID NO:34或SEQ ID NO:35定义的β-半乳糖苷酶的活性至少约20％，例如至少约30％，例如至少约40％，例如至少约50％，例如至少约60％，例如至少约70％，例如至少约80％，例如至少约90％，例如至少约100％，例如至少约200％，例如至少约300％，例如至少约400％，例如至少约500％。
4.根据实施方案1-3中任一项所述的肽或二聚肽，其具有在本文所述实施例10给出的条件下，在约4℃的温度和pH 5.5下，以每μM酶每秒形成的葡萄糖的μM测量的β-半乳糖苷酶活性，所述活性超过SEQ ID NO:34或SEQ ID NO:35定义的β-半乳糖苷酶的活性至少约20％，例如至少约30％，例如至少约40％，例如至少约50％，例如至少约60％，例如至少约70％，例如至少约80％，例如至少约90％，例如至少约100％，例如至少约200％，例如至少约300％，例如至少约400％，例如至少约500％。
5.根据实施方案1-4中任一项所述的肽或二聚肽，其具有在本文所述实施例13给出的条件下，在约4℃的温度和pH 4.5下以每μM酶每秒形成的葡萄糖的μM测量的β-半乳糖苷酶活性，所述活性超过SEQ ID NO:34或SEQ ID NO:35定义的β-半乳糖苷酶的活性至少约20％，例如至少约30％，例如至少约40％，例如至少约50％，例如至少约60％，例如至少约70％，例如至少约80％，例如至少约90％，例如至少约100％，例如至少约200％，例如至少约300％，例如至少约400％，例如至少约500％。
6.根据实施方案1-5中任一项所述的肽或二聚肽，其具有在本文所述实施例9给出的条件下，在约43℃的温度和pH 6.7下以每μM酶每秒形成的葡萄糖的μM测量的β-半乳糖苷酶活性，所述活性超过SEQ ID NO:34或SEQ ID NO:35定义的β-半乳糖苷酶的活性至少约20％，例如至少约30％，例如至少约40％，例如至少约50％，例如至少约60％，例如至少约70％，例如至少约80％，例如至少约90％，例如至少约100％，例如至少约200％，例如至少约300％，例如至少约400％，例如至少约500％。
7.根据实施方案1-6中任一项所述的肽或二聚肽，其具有在本文所述实施例12给出的条件下，在约43℃的温度和pH 5.5下以每μM酶每秒形成的葡萄糖的μM测量的β-半乳糖苷酶活性，所述活性超过SEQ ID NO:34或SEQ ID NO:35定义的β-半乳糖苷酶的活性至少约20％，例如至少约30％，例如至少约40％，例如至少约50％，例如至少约60％，例如至少约70％，例如至少约80％，例如至少约90％，例如至少约100％，例如至少约200％，例如至少约300％，例如至少约400％，例如至少约500％。
8.根据实施方案1-7中任一项所述的肽或二聚肽，其具有在本文所述实施例15给出的条件下，在约43℃的温度和pH 4.5下以每μM酶每秒形成的葡萄糖的μM测量的β-半乳糖苷酶活性，所述活性超过SEQ ID NO:34或SEQ ID NO:35定义的β-半乳糖苷酶的活性至少约20％，例如至少约30％，例如至少约40％，例如至少约50％，例如至少约60％，例如至少约70％，例如至少约80％，例如至少约90％，例如至少约100％，例如至少约200％，例如至少约300％，例如至少约400％，例如至少约500％。
9.根据实施方案1-8中任一项所述的肽或二聚肽，其具有在本文所述实施例6中给出的条件下，在约37℃的温度和pH 6.7下以每μM酶每秒形成的葡萄糖的μM测量的β-半乳糖苷酶活性，所述活性超过SEQ ID NO:34或SEQ ID NO:35定义的β-半乳糖苷酶的活性至少约20％，例如至少约30％，例如至少约40％，例如至少约50％，例如至少约60％，例如至少约70％，例如至少约80％，例如至少约90％，例如至少约100％，例如至少约200％，例如至少约300％，例如至少约400％，例如至少约500％。
10.根据实施方案1-9中任一项所述的肽或二聚肽，其具有在本文所述实施例11给出的条件下，在约37℃的温度和pH 5.5下以每μM酶每秒形成的葡萄糖的μM测量的β-半乳糖苷酶活性，所述活性超过SEQ ID NO:34或SEQ ID NO:35定义的β-半乳糖苷酶的活性至少约20％，例如至少约30％，例如至少约40％，例如至少约50％，例如至少约60％，例如至少约70％，例如至少约80％，例如至少约90％，例如至少约100％，例如至少约200％，例如至少约300％，例如至少约400％，例如至少约500％。
11.根据实施方案1-10中任一项的所述肽或二聚肽，其具有在本文所述实施例14给出的条件下，在约37℃的温度和pH 4.5下以每μM酶每秒形成的葡萄糖的μM测量的β-半乳糖苷酶活性，所述活性超过SEQ ID NO:34或SEQ ID NO:35定义的β-半乳糖苷酶的活性至少约20％，例如至少约30％，例如至少约40％，例如至少约50％，例如至少约60％，例如至少约70％，例如至少约80％，例如至少约90％，例如至少约100％，例如至少约200％，例如至少约300％，例如至少约400％，例如至少约500％。
12.根据实施方案1-11中任一项所述的肽或二聚肽，其衍生自双歧杆菌属的细菌，例如青春双歧杆菌、两歧双歧杆菌、短双歧杆菌、链状双歧杆菌、长双歧杆菌，或衍生自乳杆菌属，例如清酒乳杆菌、噬淀粉乳杆菌、德氏乳杆菌保加利亚亚种、德氏乳杆菌乳酸亚种、德氏乳杆菌印度亚种、卷曲乳杆菌、罗伊氏乳杆菌、瑞士乳杆菌，或衍生自嗜热链球菌。
13.根据实施方案1-12中任一项所述的肽或二聚肽，其中所述肽或二聚肽表现出的半乳糖抑制小于60％，例如小于55％，例如小于50％，例如小于约45％，例如小于约40％。
14.核苷酸序列，其编码实施方案1-13中任一项所定义的肽或二聚肽。
15.宿主细胞，其包含实施方案14所定义的核苷酸序列。
16.用于产生实施方案1-13中任一项所定义的肽或二聚肽的方法，所述方法包括在合适的宿主细胞中表达含有实施方案14所定义的核苷酸序列的载体；和从所述宿主细胞的表达产物中纯化所述肽或二聚肽。
17.降低诸如乳制品等含有乳糖的组合物中乳糖含量的方法，包括使所述组合物在3-10的pH和0℃-140℃的温度下与表现出β-半乳糖苷酶活性的肽或二聚肽接触的步骤，所述肽具有SEQ ID NO:1-33所示的氨基酸序列，或所述二聚肽的组成为具有SEQ ID NO:2和3、5和6、20和21、23和24、26和27或28和29所示氨基酸序列的两个肽；或其酶促活性片段，或与任一所述序列或酶促活性片段具有至少80％序列同一性的任何序列；或与表达所述肽或二聚肽中任一种的宿主细胞接触的步骤。
18.根据实施方案17所述的方法，其中所述组合物是选自由无乳糖乳、低乳糖乳、酸奶、奶酪、发酵乳制品、膳食补充剂和益生菌膳食制品组成的组的乳制品。
19.根据实施方案17-18中任一项所述的方法，其中所述宿主细胞是选自双歧杆菌属的细菌的任一种，例如青春双歧杆菌、两歧双歧杆菌、短双歧杆菌、链状双歧杆菌、长双歧杆菌，或选自乳杆菌属的任一种，例如清酒乳杆菌、噬淀粉乳杆菌、德氏乳杆菌保加利亚亚种、德氏乳杆菌乳酸亚种、德氏乳杆菌印度亚种、卷曲乳杆菌、罗伊氏乳杆菌、瑞士乳杆菌，或选自嗜热链球菌。
20.根据实施方案17-19中任一项所述的方法，其中乳糖浓度降低至小于约1％，例如至小于约0.1％或更低，例如至小于约0.01％。
21.表现出β-半乳糖苷酶活性的肽或二聚肽，所述肽具有SEQ ID NO:1-33所示的氨基酸序列，或所述二聚肽的组成为具有SEQ ID NO:2和3、5和6、20和21、23和24、26和27或28和29所示氨基酸序列的两个肽；或与任一所述序列具有至少80％序列同一性的序列；或表达所述肽或二聚肽中任一种的宿主细胞，用于生产乳糖含量降低的乳制品的用途。
22.根据实施方案21所述的用途，其中所述乳制品选自由无乳糖乳、低乳糖乳、酸奶、奶酪、发酵乳制品、膳食补充剂和益生菌膳食制品组成的组。
23.根据实施方案21-22中任一项所述的用途，其中所述宿主细胞是选自下列的任何一种：双歧杆菌属的细菌，例如青春双歧杆菌、两歧双歧杆菌、短双歧杆菌、链状双歧杆菌、长双歧杆菌，或乳杆菌属，例如清酒乳杆菌、噬淀粉乳杆菌、德氏乳杆菌保加利亚亚种、德氏乳杆菌乳酸亚种、德氏乳杆菌印度亚种、卷曲乳杆菌、罗伊氏乳杆菌、瑞士乳杆菌，或嗜热链球菌。
24.生产乳制品的方法，所述方法包括以下步骤：
a)提供包含乳糖的乳基基质；
b)向所述包含乳糖的乳基基质添加表现出β-半乳糖苷酶活性的肽或二聚肽，所述肽具有SEQ ID NO:1-33所示的氨基酸序列；或所述二聚肽的组成为具有SEQ ID NO:2和3、5和6、20和21、23和24、26和27或28和29所示氨基酸序列的两个肽；或与任一所述序列具有至少80％序列同一性的序列；和
c)用所述表现出β-半乳糖苷酶活性的肽或二聚肽处理所述乳基基质。
25.根据实施方案24所述的方法，其中所述肽或二聚肽衍生自双歧杆菌属的任一种细菌，例如青春双歧杆菌、两歧双歧杆菌、短双歧杆菌、链状双歧杆菌、长双歧杆菌，或衍生自乳杆菌属，例如清酒乳杆菌、噬淀粉乳杆菌、德氏乳杆菌保加利亚亚种、德氏乳杆菌乳酸亚种、德氏乳杆菌印度亚种、卷曲乳杆菌、罗伊氏乳杆菌、瑞士乳杆菌，或衍生自嗜热链球菌。
26.根据实施方案24-25中任一项的方法，其中进行步骤c)的pH在3-10的范围内，例如在3-9的范围内，例如在3-8的范围内，例如在3-7的范围内，例如在3-6的范围内，例如在3-5的范围内，例如在3-4的范围内，例如在4-10的范围内，例如在4-9的范围内，例如在4-8的范围内，例如在4-7的范围内，例如在4-6的范围内，例如在4-5的范围内，例如在5-10的范围内，例如在5-9的范围内，例如在5-8的范围内，例如在5-7的范围内，例如在5-6的范围内，例如在6-10的范围内，例如在6-9的范围内，例如在6-8的范围内，例如在6-7的范围内。
27.根据实施方案24-26中任一项所述的方法，其中进行步骤c)或步骤c)的一部分的温度不超过约25℃，例如不超过约20℃，例如不超过约18℃，例如不超过约16℃，例如不超过约14℃，例如不超过约12℃，例如不超过约10℃，例如不超过约8℃，例如不超过约7℃，例如不超过约6℃，例如不超过约5℃，例如不超过约4℃，例如不超过约3℃，例如不超过约2℃。
28.根据实施方案24-27中任一项所述的方法，其中进行步骤c)或步骤c)的一部分的温度为至少约25℃，例如至少约30℃，例如至少约35℃，例如至少约40℃，例如至少约45℃，例如至少约50℃，例如至少约55℃，例如至少约60℃，例如至少约65℃例如，例如至少约70℃，例如至少约75℃，例如至少约80℃，例如至少约85℃，例如至少约90℃，例如至少约95℃，例如至少约100℃，例如至少约110℃，例如至少约120℃，例如至少约130℃，例如至少约120℃，例如至少约130℃，例如至少约135℃，例如至少约140℃。
29.根据实施方案24-28中任一项所述的方法，其中所述乳制品选自由以下组成的组：无乳糖乳、低乳糖乳、酸奶、奶酪、发酵乳制品、膳食补充剂和益生菌膳食制品。
30.根据实施方案24-29中任一项所述的方法，其中所述乳基基质选自直接从巴氏灭菌步骤获得的鲜乳或生乳，直接在超热处理(UHT)步骤后获得的乳或直接在发酵步骤后获得的乳。
31.根据实施方案24-30中任一项所述的方法，其中所述肽或二聚肽的半乳糖抑制小于60％，例如小于55％，例如小于50％，例如小于约45％，例如小于约40％。
32.根据实施方案24-31中任一项所述的方法，其中所述乳制品是发酵乳制品，并且在发酵期间或发酵前进行所述步骤b)。
33.根据实施方案32所述的方法，所述方法不需要在发酵后添加其他酶。
34.根据实施方案24-31中任一项所述的方法，其中所述乳制品是发酵乳制品，并且在发酵后立即进行所述步骤b)。
35.根据实施方案24-31中任一项所述的方法，其中，所述乳制品是鲜乳，并且在巴氏灭菌步骤之前、同时或之后立即进行所述步骤b)。
36.根据实施例24-31中任一项所述的方法，其中，所述乳制品是超热处理(UHT)乳，并且在超热步骤处理之前、同时或之后立即进行所述步骤b)。
37.根据实施方案24-36中任一项所述的方法，其中开始步骤c)的温度为40℃至100℃，例如温度为50℃至100℃，例如温度为60℃至100℃，例如温度为70℃至100℃，例如温度为80℃至100℃，例如温度为40℃至90℃，例如温度为40℃至80℃，例如温度为40℃至70℃，例如温度为40℃至60℃，例如温度为40℃至50℃。
38.根据实施方案24-37中任一项所述的方法，其中当水解乳基基质中的乳糖时，所述肽或二聚肽的乳糖酶与转半乳糖基酶活性之比大于1:1。
39.根据实施方案24-38中任一项所述的方法，其中当步骤c)完成时，少于80％的乳糖已被水解，并且其中一周后超过90％的乳糖已被水解。
40.乳制品，其通过实施方案24-39中任一项所定义的方法制备。
41.食品，例如乳制品，其包含表现出β-半乳糖苷酶活性的肽或二聚肽，所述肽具有SEQ ID NO:1-33所示的氨基酸序列，或所述二聚肽的组成为具有SEQ ID NO:2和3、5和6、20和21、23和24、26和27或28和29所示氨基酸序列的两个肽；或与任一所述序列具有至少80％序列同一性的序列。
42.食品，例如乳制品，其包含表达表现出β-半乳糖苷酶活性的肽或二聚肽的宿主细胞，所述肽具有SEQ ID NO:1-33所示的氨基酸序列，或所述二聚肽的组成为具有SEQ IDNO:2和3、5和6、20和21、23和24、26和27或28和29所示氨基酸序列的两个肽；或与任一所述序列具有至少80％序列同一性的序列。
43.根据实施方案42所述的食品，其选自饮料、婴儿食品、谷物、面包、饼干、糖果、蛋糕、食品补充剂、膳食补充剂、益生菌可食用产品、益生元可食用产品、动物饲料、家禽饲料和药物，或选自由无乳糖乳、低乳糖乳、奶粉、婴儿乳、酸奶、冰淇淋、奶酪、发酵乳制品、膳食补充剂和益生菌膳食制品组成的组的乳制品。
序列
表1.基因编号和相应的序列识别号。
SEQ ID No.1
MADTAELAIVHATTASASWLTDPTVFAANRKPAHSSHRYVIGETSEPKQSLDGEWKVRIEQARNVDVESAPFAAVDFEDGDFGAIEVPGHLQMAGYLKNKYVNIQYPWDGHEDPQAPNIPENNHVAIYRRRFALDAQLARTLENDGTVSLTFHGAATAIYVWLDGTFVGYGEDGFTPSEFDVTEALRNGNGNAADSPEAEHTLTVACYEYSSASWLEDQDFWRLHGLFRTVELAAQPHTHVETVQLEADYTAADTAGTADTAELNAALTLRNSADAMTIESTLRDGDGNVVWESTQACNGEIALNSGKMTNIAPWSAESPTLYTLTVRVVGHDGAIIETVTQKIGFRTFRIENGIMTLNGKRIVFKGADRHEFDAKRGRAITREDMLSDVVFCKRHNINAIRTSHYPNQEYWYDLCDEYGLYLIDETNMETHGTWVANNVERPEDGIPGSRPEWEGACVDRINSMMRRDYNHPSVLIWSLGNESSAGEVFRAMYRHAHTIDPNRPVHYEGSVHMREFEDVTDIESRMYAHADEIERYLNDGSPAHTDGPKKPYISCEYMHAMGNSCGNMDEYTALERYPMYQGGFIWDFIDQAIETKLPDGTTRMCYGGDFGDRPSDYEFSGDGLLFADRTPSPKAQEVKQLYANVKIVVSVDEARITNDNLFVSTGDYRFVLRILADGKPVWSTTRRFDVAAGESASFEVDWPVDDYRSNAEELVLEVSQQLGNACDWAPAGYELAFGQCVVAGAKTTADAVDAAGAPADGTVTLGRWNAGVRGQGREALFSRTQGGMVSYTFGEREFVLRRPSITTFRPLTDNDRGAGHAFERAAWAVAGKYARCVDCAIANRGENAVEATYTYELAIPQRTKVTVRYVADTAGLVSLDVEYPGEKNGDLPTIPAFGIEWALPVEYANLRFYGAGPEETYADRRHAKLGVWSTTAGDDCAPYLLPQETGNHEDVRWAEITDDSGHGVRVKRGAGAKPFAMSLLPYSSTMLEEALHQDELPKPRHMFLRLLAAQMGVGGDDSWMSPVHEQYQLPADQPLSLNVQLKLF
SEQ ID No.2(G16结构域a)
MQPNIQWLDTPAVFRVGQLPAHSDHRYYATLAEMAQQQSSFEQSLNGTWQFHYSVNAASRPKSFYELAFDAQDFEPITVPQHIELAGYEQLHYINTMYPWEGHYYRRPAFSTSDDKQHLGMFSEADYNPVGSYLHHFDLTPALRNQRVIIRFEGVEQAMYVWLNGQFIGYAEDSFTPSEFDLTPYLKETDNCLAVEVHKRSSAAFIEDQDFFRFFGIFRDVKLLAKPRTHLEDLWVIPEYDVVQQTGQVKLRLQFSGDENRVHLRIRDQHQIILTADLTSAAQVNGLYKMPELVQAWSNQTPNLYTLELEVVDQAGETIEISQQPFGFRKIEIKDKVMLLNGKRLVINGVNRHEWHPETGRTITAEDEAWDIACMQRNHINAVRTSHYPDRLSFYNGCDQAGIYMMAETNLESHGSWQKMGAVEPSWNVPGSYDEWEAATLDRARTNFETFKNHVSILFWSLGNESYAGSVLEKMNAYYKQQDPTRLVHYEGVFRAPEYKATISDVESRMYATPAEIKAYLDNAPQKPFILCEYMHDMGNSLGGMQSYIDLLSQYDMYQGGFIWDFIDQALLVTDPVTGQRELRYGGDFDDRPSDYEFSGDGLVFATRDEKPAMQEVRYYYGEHK
SEQ ID No.3(G16结构域b)
MKNQQCRRLDTIMANTNKRLAVIFGDVTLGLKGPDFHYLFSYQTGGPESLRIQGKEWLYRSPKPTFWRATTDNDRGNQFPLKSGMWLAADQFIACQSITVAIDGQTIPLPIAPENNRYSGQETAQEVTVTYTYQTITTPQTTVEVSYTIQASGKIRVAVTYHGQAGLPSLPVFGLRFVMPTPATRFIYQGLSGETYPDRMAGGMAGEYEVTGLPVTPYLVPQDCGVHMATDWVTIYRQAVLDNRLREPVETGLKFKMVDQPFAFSCLPYTAEELENATHHSELPAPHRTVLSLLGAVRGVGGIDSWGSDVEAAYQIDATQDHHLEFEISF
SEQ ID No.4
MADTAELAIVHATTASASWLTDPTVFAANRKPAHSSHRYVIGETSEPKQSLDGEWKVRIEQARNVDVESAPFAAVDFEDGDFGAIEVPGHLQMAGYLKNKYVNIQYPWDGHEDPQAPNIPENNHVAIYRRRFALDAQLARTLENDGTVSLTFHGAATAIYVWLDGTFVGYGEDGFTPSEFDVTEALRNGNGNAADSPEAEHTLTVACYEYSSASWLEDQDFWRLHGLFRTVELAAQPHTHVETVQLEADYTAADTAGTADTAELNAALTLRNPADAMTIESTLRDGDGNVVWESTQACNGEIALNSGKMTNIAPWSAESPTLYTLTVRVVGHDGAIIETVTQKIGFRTFRIENGIMTLNGKRIVFKGADRHEFDAKRGRAITREDMLSDVVFCKRHNINAIRTSHYPNQEYWYDLCDEYGLYLIDETNMETHGTWVANNVERPEDGIPGSRPEWEGACVDRINSMMRRDYNHPSVLIWSLGNESSAGEVFRAMYRHAHTIDPNRPVHYEGSVHMREFEDVTDIESRMYAHADEIERYLNDGSPAHTDGPKKPYISCEYMHAMGNSCGNMDEYTALERYPMYQGGFIWDFIDQAIETKLPDGTTRMCYGGDFGDRPSDYEFSGDGLLFADRTPSPKAQEVKQLYANVKIAVSVDEARITNDNLFVSTGDYRFVLRILADGKPVWSTTRRFDVAAGESASFEVDWPVDDYRSNAEELVLEVSQQLGNACDWAPAGYELAFGQCVVAGAKTTADAVDAAGAPADGTVTLGRWNAGVRGQGREALFSRTQGGMVSYTFGEREFVLRRPSITTFRPLTDNDRGAGHAFERAAWAVAGKYARCVDCAIANRGENAVEATYTYELAIPQRTKVTVRYVADTAGLVSLDVEYPGEKNGDLPTIPAFGIEWALPVEYANLRFYGAGPEETYADRRHAKLGVWSTTAGDDCAPYLLPQETGNHEDVRWAEITDDSGHGVRVKRGAGAKPFAMSLLPYSSTMLEEALHQDELPKPRHMFLRLLAAQMGVGGDDSWMSPVHEQYQLPADQPLSLNVQLKLF
SEQ ID No.5(G40结构域a)
MKANIKWLDDPEVFRINQLPAHSDHPFYKDYREWQNHSSSFKQSLNGAWQFHFSKDPQSRPIDFYKRSFDSSSFDTIPVPSEIELNGYAQNQYTNILYPWESKIYRKPAYTLGRGIKDGDFSQGKDNTVGSYLKHFDLNPALAGHDIHIQFEGVERAMYVYLNGHFIGYAEDSFTPSEFDLTPYIQAKDNILAVEVFKHSTASWLEDQDMFRFSGIFRSVELLALPRTHLMDLDIKPTVVNDYHDGVFNAKLHFMGKTSGNVHVLIEDIDGKTLLNKKLPLKSTVEIENETFANVHLWDNHDPYLYQLIIEVHDQDGKLVELIPYQFGFRKIEITKDHVVLLNGKRLIINGVNRHEWDAKRGRSITLADMKQDIATFKHNNINAVRTCHYPNQIPWYYLCDQNGIYMMAENNLESHGTWQKLGQVEATSNVPGSIPEWREVVVDRARSNYETFKNHTAILFWSLGNESYAGSNIAAMNKLYKDHDSSRLTHYEGVFHAPEFKKEISDLESCMYLPPKEAEEYLQNPKKPLVECEYMHDMGTPDGGMGSYIKLIDKYPQYMGGFIWDFIDQALLVHDPVSGQDVLRYGGDFDDRHSDYEFSGDGLMFADRTPKPAMQEVRYYYGLHK
SEQ ID No.6(G40结构域b)
MAYTNNLHVVYGEASLGVNGQDFAYLFSYERGGLESLKIKDKEWLYRTPTPTFWRATTDNDRGSGFNQKAAQWLGADMFTKCVGIHVQVDDHRFDELPVAPINNQFSNQEFAHEVKVAFDYETLTTPATKVKIIYNINDFGHMTITMHYFGKKGLPPLPVIGMRFIMPTKAKSFDYTGLSGETYPDRMAGAERGTFHIDGLPVTKYLVPQENGMHMQTNELVITRNSTQNNADKDGDFSLKITQTKQPFNFSLLPYTAEELENATHIEELPLARRSVLVIAGAVRGVGGIDSWGSDVEEQYHIDPEQDHEFSFTLN
SEQ ID No.7
MNTTDDQRKNGDPIVSPSIPTTAWLADPRVYAVHRLDAHSDHACWSRSPVDGESTDLRQSLDGEWRVRVETAPTGRFPDGTSDGPDWISDVSPLFAAPGFDDSSFSRVQVPSHLETAGLLAPQYVNVQYPWDGHEDPKAPAIPEHGHVAVYRREFDADGEVAQAVREGRPVTLTFQGAATAIYVWLNGSFIGYAEDSFTPSEFDVTDAIKVDGNVLAVACYEYSSASWLEDQDFWRLHGLFRSVELNARPAAHVADLHADADWDLATSRGSLSLDVLIDGAANAATADFALRDKNGTIVWRTATKADGTLHAEAEIDDAAPWSAERPDLYELSVTLLDADGKVLETARTRIGFRHVAIEDGILKLNGKRLVFRGVNRHEFDCRRGRAITEEDMLWDIRFMKRHNINAVRTSHYPNQSRWYELCDEYGIYLIDETNLETHGSWNSPGDIPVGTSVPGDDEAWLGACIDRLDSMILRDRNHPSVLVWSLGNESYAGEVLKAMSAHAHQLDPGRPVHYEGVNWNHAYDGISDFESRMYAKPAEIQDWLEHGDERGEASKPFVSCEYMHAMGNSCGGLSEFIDLERYERYSGGFIWDYIDQGLVQRLPDGSERLSVGGEWGDRPTDYEFVGNGIVFADRTPSPKAQEVKQLYSPVKLAPDGHGVTIENRNLFAGTDGYVFAARLLEDGHEIWHADYRFDVAAGDTQHHDIAFPDIDADGDTREVTYEVDLLLAEATAWAPAGYELAFGQLTGTLNPEQDITETSHDDDGRATRTLSRWNAGIRRDDEEILLSRTQGGIVSWKRDDREMVIRRPELVTFRPLTDNDRGNHSGFDRAAWFAAGRYAIVTETKIHESDDGLVAEYQYELADPNHTPVSVTYHVNSDMRMQLTVEYPGNATDMASLPAFGIEWELPGEYDRLRYYGPGPEETYRDRKQGGKLGIWDATAKASMAPYLMVQETGSHEDVRWLEATDIQGHGLRVTQRGDRHFTASLLPWNTYTIEAARRHEDLPKPRHNYLRLLAAQMGVGGDDSWGAPVHTAYQLPAGRPLTLDVNLELI
SEQ ID No.8
MNTTDDQRKNGDPIVSPSIPTTAWLADPRVYAVHRLDAHSDHACWSRSPVDGESTDLRQSLDGEWRVRVETAPTGRFPDGTSDGPDWISDVSPLFAAPGFDDSSFSRVQVPSHLETAGLLAPQYVNVQYPWDGHEDPKAPAIPEHGHVAVYRREFDADGEVAQAVREGRPVTLTFQGAATAIYVWLNGSFIGYAEDSFTPSEFDVTDAIKVDGNVLAVACYEYSSASWLEDQDFWRLHGLFRSVELNARPAAHVADLHADADWDLATSRGSLSLDVLIDGAANAATADFALWDKNGTIVWHIVTKADGTLHAEAEIDDAAPWSAERPDLYELSVTLLDADGKVLETARTRIGFRHVAIEDGILKLNGKRLVFRGVNRHEFDCRRGRAITEEDMLWDIRFMKRHNINAVRTSHYPNQSRWYELCDEYGIYLIDETNLETHGSWNSPGDIPVGTSVPGDDEAWLGACIDRLDSMILRDRNHPSVLVWSLGNESYAGEVLKAMSAHAHRLDPGRPVHYEGVNWNHAYDGISDFESRMYAKPAEIQDWLEHGDERGEASKPFVSCEYMHAMGNSCGGLSEFIDLERYERYSGGFIWDYIDQGLVQRLPDGSERLSVGGEWGDRPTDYEFVGNGIVFADRTPSPKAQEVKQLYSPVKLAPDGHGVTIENRNLFAGTDGYVFAARLLEDGHEIWHADYRFDVAAGDTQHHDIAFPDIDADGDTREVTYEVDLLLAEATAWAPAGYELAFGQLTGTLNPEQDITETSHDDDGRATRTLSRWNAGIRRDDKEILLSRTQGGIVSWKRDDREMVIRRPELVTFRPLTDNDRGNHSGFDRAAWFAAGRYAIVTETKIHESDDGLVAEYQYELADPNHTPVSVTYHVNSDMRMQLTVEYPGNATDMASLPAFGIEWELPGEYDRLRYYGPGPEETYRDRKQGGKLGIWDATAKASMAPYLMVQETGSHEDVRWLEATDIQGHGLRVTQRGDRHFTASLLPWNTYMIEAARRHEDLPEPRHNYLRLLAAQMGVGGDDSWGAPVHTAYQLPAGRPLTLDVNLELI
SEQ ID No.9
MTNSMQGKAKTIMTNLQSAQQFSQAWLTDPRVFAVNRLAAHSSHKFYDHSPQCGEAMDLKQSLDGQWRVQMLDLADLADNELAEAAFAQPGYDAAGFSPIEVPSALETKGFLNHQYVNQQYPWSGHESPVAPDVPKHNHVALYRHEFSLEPKAAAVLEANKTAADDAAKRRVTLTFQGAATAIVVWLNGAFIGYAEDSFTPSEFDVTDVLRDGVNTLAVACFEFSSASWLEDQDFWRLHGIFRSVELEAQPLVHVNDLRVLADYDHTTGEGSLDVVALLRNAGTAAAVAATVLDAAGNTVWHSKLTAGADAETLTVKANVGKVNPWSAEEPTLYTLQVVATDAAGQVIEAALQRIGFRHFAIEDGLMKLNGKRIVFKGVDRHEFDARTGRTIAEADMIEDIHSFKRLNINAVRTSHYPNETRWYELCDEYGIYVLDETNLETHGSWTDPGDVFQPARAIPGSKDEWRAACVDRTASMVRRDYNHPSVVIWSLGNEAFGGDVFYSMRDFVHENDPFRPVHYEGTFNDPEFSAATDIMSRMYAKPDEIVKLYLGEDGKKPYISCEYSHSMGNSTGGLHLYTELERYPLYQGGFIWDYVDQALWQDCGNGTERLAYGGDFEDRPNDYEFSGDGVMFADRTPSPKAQEVKQLYANVKLVPDESGVTITNDNLFISTASSLFTARVLVDGAERWHANYRFDVPAGETVREPIAFPKVTDLVALSGSAEVTYEVDQRLAEATDWAPAGYELTFGQYVAAVSFDDGAADAVVAGDAEVAADGFNAGIHTDFGEVLLSKTQGGMVSFKRDGREMVIRRPNLTTFRALTDNDRGNGSGFERAQWMAAGRYARVTGTSVEETADGKGLKATYSYELADAKHTPVTVHYEVDAALRVHLTVEYPGEADAATLPAFGLEWILPKQYDRLRFYGLGPEETYADRLHGAKLGVFSRTAAEDCAPYLLPQETGNHEQVRWAEITDEYGHGMRVTAAGGTRFATSLLPYSSLMFEDALHQNELPKPRHTFLRLLAAQMGVGGDDTWGAPVHDEFQVPADQPLKLDVTLELI
SEQ ID No.10
MTQRRSYRWPQPLAGQQARIWYGGDYNPDQWPEEVWDDDVRLMKKAGVNLVSVGIFSWAKIETSEGVYDFDWLDRIIDKLGEAGIAVDLASATASPPMWLTQAHPEVLWKDYRGDVCQPGARQHWRPTSPVFREYALKLCRAMAEHYKGNPYVVAWHVSNEYGCHNRFDYSEDAERAFRKWCEERYGTIDAVNDAWGTAFWAQRMNDFTEIVPPRFIGDGNFMNPGKLLDFKRFSSDALKAFYVAERDALAEITPDLPLTTNFMVSAAGSVLDYDDWGREVDFVSNDHYFIPGEAHLDELAFSASLVDGIARKDPWFLMEHSTSAVNWRPVNYRKEPGQLVRDSLAHVAMGADAVCYFQWRQSKAGAEKFHSAMVPHTGEDSAVFRDVCELGADLNTLADNGLLGTKLAKSKVAVVFDYESEWATEHTATPTQKVHHVDEPLQWFRALADHGVTADVVPVSSNWDEYEVVVLPSVYILSEETTRRVRDYVVNGGRLIVTYYTGLSDEKDHVWLGGYPGSIRDVVGVRVEEFMPMGDDFPGVPDCLGLSNGAVAHDIADVIGSVDGTATVLETFRDDPWTGMDGAPAIVANTFGEGRSVYVGARLGRDGIAKSLPEIFESLGMAETGENDSRVLRVEREGSDGSRFVFSFNRTHEAVQIPFEGKIVVSSFAEVSGENVSIKPNGVIVTKQ
SEQ ID No.11
MANSNRVEHASETWLTDATVFEVNRTPAHSNHKCFTHDPQSGEHSDLTQSLDGEWRVEIVQASDIDFNEEPFVAENFDDSSFCRAQVPGHLQMAGLLKNKYVNIQYPWDGHENPLEPNVPENNHVALYRRKFVVSKRLADTKESEGSVSIVFHGMATAIYVWVNGLFAGYGEDGFTPNEFDITDLLHDGENVVAVACYEYSSASWLEDQDFWRLHGLFRSVELTAQPHVHVENMQLEADWDAESGTASLDAALSVRNASDAATISATLKDSEGNVVWEASTNADANTTFASGSLQGLEPWSAESPSLYELEVNVIDQAGNIVEAAVQKVGFRRFRIENGIMTLNGKRIVFKGADRHEFDAKRGRSITEQDMIDDVIFCKRHNINAIRTSHYPNQERWYDLCDEYGIYLIDETNLETHGSWCLPGDVVTAETAVPGSKAHWEGACVDRVNSMVRRDYNHPSVVIWSLGNESYTGDVFRAMYKHVHDIDPNRPVHYEGVTKNRDYDDVTDIETRMYEHADVVEEYLKNDPQKPYISCEYMHAMGNSVGNLDEYTALERYPHYQGGFIWDFIDQAIYATQPDGSTRLCYGGDFGDRPSDYEFSGNGLVFADRTPTPKAQEVKQLYSNVHIDVTDRSVSIKNDNLFISTGGYQFVLRILADGEPVWQSERRFDVPADSACTFDVEWPVDLYRANADELVLEVSQRLAEATDWAPAGYELAFGQTIVAGTKAAEDAALPADGIVTVGRWNAGVQGSGREILLSRTQGGLVSYTFDGHEFVLRRPAITTFRALTDNDRGAGHGFERAQWMVAGRYARCVDNVIEQVDEDTLKAVYTYELATPQCTKVTVGYTADTTGRLNLHVEYPGESGELPTIPAFGIEWTLPVQYSNLRFFGAGPEETYQDRKHAKLGVWSTDAFKDHAPYLMPQETGNHEEVRWAEITDENGHGLRVSRANGAAPFAVSLQPYSSFMIEEAQHQDELPAPKHMFLRVLAAQMGVGGDDSWMSPVHSQYHITADQPISLDVNLELI
SEQ ID No.12
MSNKLVKEKRVDQADLAWLTDPEVYEVNTIPPHSDHESFQSQEELEEGKSSLVQSLDGDWLIDYAENGQGPVNFYAEDFDDSNFKSVKVPGNLELQGFGQPQYVNVQYPWDGSEEIFPPQIPSKNPLASYVRYFDLDEAFWDKEVSLKFDGAATAIYVWLNGHFVGYGEDSFTPSEFMVTKFLKKENNRLAVALYKYSSASWLEDQDFWRMSGLFRSVTLQAKPRLHLEDLKLTASLTDNYQKGKLEVEANIAYRLPNASFKLEVRDSEGDLVAEKLGPIRSEQLEFTLADLPVAAWSAEKPNLYQVRLYLYQAGSLLEVSRQEVGFRNFELKDGIMYLNGQRIVFKGANRHEFDSKLGRAITEEDMIWDIKTMKRSNINAVRCSHYPNQSLFYRLCDKYGLYVIDEANLESHGTWEKVGGHEDPSFNVPGDDQHWLGASLSRVKNMMARDKNHASILIWSLGNESYAGTVFAQMADYVRKADPTRVQHYEGVTHNRKFDDATQIESRMYAPAKVIEEYLTNKPAKPFISVEYAHAMGNSVGDLAAYTALEKYPHYQGGFIWDWIDQGLEKDGHLLYGGDFDDRPTDYEFCGNGLVFADRTESPKLANVKALYANLKLEVKDGQLFLKNDNLFTNSSSYYFLTSLLVDGKLTYQSRPLTFGLEPGESGTFALPWPEVADEKGEVVYRVTAHLKEDLPWADEGFTVAEAEEVAQKLPEFKPEGRPDLVDSDYNLGLKGNNFQILFSKVKGWPVSLKYAGREYLKRLPEFTFWRALTDNDRGAGYGYDLARWENAGKYARLKDISCEVKEDSVLVKTAFTLPVALKGDLTVTYEVDGRGKIAVTADFPGAEEAGLLPAFGLNLALPKELTDYRYYGLGPNESYPDRLEGNYLGIYQGAVKKNFSPYLRPQETGNRSKVRWYQLFDEKGGLEFTANGADLNLSALPYSAAQIEAADHAFDLTNNYTWVRALSAQMGVGGDDSWGQKVHPEFCLDAQKARQLRLVIQPLLLK
SEQ ID No.13
MSNKLVKEKRVDQADLAWLTDPEVYEVNTIPPHSDHESFQSQEELEEGKSSLVQSLDGNWLIDYAENGQGPINFYAEDFDDSNFKSVKVPGNLELQGFGQPQYVNIQYPWDGSEEIFPPQVPSKNPLASYVRYFDLDEALWDKEVSLKFAGAATAIYVWLNGHFVGYGEDSFTPSEFMVTKFLKKEGNRLAVALYKYSSASWLEDQDFWRLSGLFRSVTLEAKPLLHLEDLKLTASLTDNYQKGKLEVEANIAYRLPNASFKLEVRDSEGDLVAEKVGPIRSEKLGFSLADLPVAAWSAEKPNLYQVRLYLYQAGSLLEVSRQEVGFRNFELKDGIMYLNGQRIVFKGVNRHEFDSKLGRAITEADMIWDIKTMKQSNINAVRCSHYPNQSLFYRLCDKYGLYVIDEANLESHGTWEKVGHEDPSFNVPGDDQHWLGASLSRVKNMMARDKNHASILIWSLGNESYAGTVFAQMADYVRKADPTRVQHYEGVTHNRKFDDATQIESRMYAPAKEIEEYLTKKPAKPFISVEYAHAMGNSVGDLAAYTALEKYPHYQGGFIWDWIDQGLEKDGHLLYGGDFDDRPTDYEFCGDGLVFADRTTSPKLANVKALYSNLKLEVKDGQLFIKNDNLFTNSSAYYFLASLLVDGKLTYQSQPLTFGLEPGESGTFVLPWPEVEDEKGEIVYQVTAHLKEDLPWADEGFTVAEAEEAVTKLPEFYPAGRPELVDSDFNLGLKGNGFRILFSKAKGWPVSIKYAGREYLKRLPEFTFWRALTDNDRGAGYGYDLAKWENAGKYARLQDISYEIKENSALVKTTFTLPVALKGDLTITYEVDSLGKIAVTANFPGAVENGLLPAFGLNFALPKELSDYRYYGLGPNESYADRLEGSYLGIYQGAVEKNFTPYLRPQEAGNRSKVRYYQLFDEEGGLEFTANGADLNLSALPYSAAQIEAADHAFELTNNYTWVRALAAQMGVGGDDSWGQKVHPEFCLDAQEARQLKLVIQPLLLK
SEQ ID No.14
MSNKLVKEKRVDQADLAWLTDPEVYEVNTIPPHFDHESFQSQEELEEGKSSLVQSLDGDWLIDYAENGQGPVNFYAEDFDDSNFKSVKVPGNLELQGFGQPQYVNVQYPWDGSEEIFPPQIPSKNPLASYVRYFDLDEAFWDKEVSLKFDGAATAIYVWLNGHFVGYGEDSFTPSEFMVTKFLKKENNRLAVALYKYSSASWLEDQDFWRMSGLFRSVTLQAKPRLHLEDLKLTASLTDNYQKGKLEVEANIAYRLPNASFKLEVRDSEGDLVAEKLGPIRSEQLEFTLADLPVAAWSAEKPNLYQVRLYLYQAGSLLEVSRQEVGFRNFELKDGIMYLNGQRIVFKGANRHEFDSKLGRAITEEDMIWDIKTMKRSNINAVRCSHYPNQSLFYRLCDKYGLYVIDEANLESHGTWEKVGGHEDPSFNVPGDDQHWLGASLSRVKNMMARDKNHASILIWSLGNESYAGTVFAQMADYVRKADPTRVQHYEGVTHNRKFDDATQIESRMYAPAKVIEEYLTNKPAKPFISVEYAHAMGNSVGDLAAYTALEKYPHYQGGFIWDWIDQGLEKDGHLLYGGDFDDRPTDYEFCGNGLVFADRTESPKLANVKALYANLKLEVKDGQLFLKNDNLFTNSSSYYFLTSLLVDGKLTYQSRPLTFGLEPGESGTFALPWPEVADEKGEVVYRVTAHLKEDLPWADEGFTVAEAEEVAQKLPEFKPEGRPDLVDSDYNLGLKGNNFQILFSKVKGWPVSLKYAGREYLKRLPEFTFWRALTDNDRGAGYGYDLARWENAGKYARLKDISCEVKEDSVLVKTAFTLPVALKGDLTVTYEVDGRGKIAVTADFPGAEEAGLLPAFGLNLALPKELTDYRYYGLGPNESYPDRLEGNYLGIYQGAVKKNFSPYLRPQETGNRSKVRWYQLFDEKGGLEFTANGADLNLSALPYSAAQIEAADHAFELTNNYTWVRALSAQMGVGGDDSWGQKVHPEFCLDAQKARQLRLVIQPLLLK
SEQ ID No.15
MSNKLVKEKRVDQADLAWLTDPEVYEVNTIPPHSDHESFQSQEELEEGKSSLVQSLDGDWLIDYAENGQGPVNFYAEDFDDSNFKSVKVPGNLELQGFGQPQYVNVQYPWDGSEEIFPPQIPSKNPLASYVRYFDLDEAFWDKEVSLKFDGAATAIYVWLNGHFVGYGEDSFTPSEFMVTKFLKKENNRLAVALYKYSSASWLEDQDFWRMSGLFRSVTLQAKPRLHLEDLKLTASLTDNYQKGKLEVEANIAYRLPNASFKLEVRDSEGDLVAEKLGPIRSEQLEFTLADLPVAAWSAEKPNLYQVRLYLYQAGSLLEVSRQEVGFRNFELKDGIMYLNGQRIVFKGANRHEFDSKLGRAITEEDMIWDIKTMKRSNINAVRCSHYPNQSLFYRLCDKYGLYVIDEANLESHGTWEKVGGHEDPSFNVPGDDQHWLGASLSRVKNMMARDKNHASILIWSLGNESYAGTVFAQMADYVRKADPTRVQHYEGVTHNRKFDDATQIESRMYAPAKVIEEYLTNKPAKPFISVEYAHAMGNSVGDLAAYTALEKYPHYQGGFIWDWIDQGLEKDGHLLYGGDFDDRPTDYEFCGNGLVFADRTESPKLANVKALYANLKLEVKDGQLFLKNDNLFTNSSSYYFLTSLLVDGKLTYQSRPLTFGLEPGESGTFALPWPEVADEKGEVVYRVTAHLKEDLPWADEGFTVAEAEEVAQKLPEFKPEGRPDLVDSDYNLGLKGNNFQILFSKVKGWPVSLKYAGREYLKRLPEFTFWRALTDNDRGAGYGYDLARWENAGKYARLKDISCEVKEDSVLVKTAFTLPVALKGDLTVTYEVDGRGKIAVTADFPGAEEAGLLPAFGLNLALPKELTDYRYYGLGPNESYPDRLEGNYLGIYQGAVKKNFSPYLRPQETGNRSKVRWYQLFDEKGGLEFTANGADLNLSALPYSAAQIEAADHAFELTNNYTWVRALSAQMGVGGDDSWGQKVHPEFCLDAQKARQLRLVIQPLLLK
SEQ ID No.16
MSNKLVKEKRVDQADLAWLTDPEVYEVNTIPPHSDHESFQSQEELEEGKSSLVQSLDGDWLIDYAENGQGPVNFYAEDFDDSNFKSVKVPGNLELQGFGQPQYVNIQYPWDGSEEIFPPQVPSKNPLASYVRYFDLDEAFWDKEVSLKFAGAATAIYVWLNGHFVGYGEDSFTPSEFMVTKFLKKENNRLAVALYKYSSASWLEDQDFWRLSGLFRSVTLQAKPLLHLEDLKLTASLTDNYQKGKLEVEANIAYRLPNASFKLEVRDSEGDLVAEKLGPIRSEQLEFTLADLPVAAWSAEKPNLYQVRLYLYQAGSLLEVSRQEVGFRNFELKDGIMYLNGQRIVFKGVNRHEFDSKLGRAITEEDMIWDIKTMKRSNINAVRCSHYPNQSLFYRLCDKYGLYVIDEANLESHGTWEKVGHEDPSFNVPGDDQHWLGASLSRVKNMMARDKNHASILIWSLGNESYAGTVFAQMADYVRKADPTRVQHYEGVTHNRKFDDATQIESRMYAPAKEIEEYLTKKPAKPFISVEYAHAMGNSVGDLAAYTALEKYPHYQGGFIWDWIDQGLEKDGHLLYGGGDFDDRPTDYEFCGNGLVFADRTTSPKLANVKALYSNLKLEVKDGQLFLKNDNLFTNSSAYYFLTSLLVDGKLTYQSQPLTFGLEPGESGTFVLPWPEVEDEKGEIVYQVTAHLKEDLPWADEGFTVAEAEEAVTKLPEFYPAGRPELVDSDFNLGLKGNGFRILFSKAKGWPVSIKYAGREYLKRLPEFTFWRALTDNDRGAGYGYDLAKWENAGKYARLQDISYEIKENSVLVKTAFTLPVALKGDLTITYEVDSLGKIAVTANFPGAVENGLLPAFGLNFALPKELSDYRYYGLGPNESYADRLEGSYLGIYQGAVEKNFTPYLRPQEAGNRSKVRYYQLFDEESGLEFTANGADLNLSALPYSAAQIEAADHAFELSNNYTWVRALAAQMGVGGDDSWGQKVHPEFCLDAQEARQLKLVIQPLLLK
SEQ ID No.17
MSNKLVKEKRVDQADLAWLTDPEVYEVNTIPPHSDHESFQSQEELEEGKSSLVQSLDGDWLIDYAENGQGPVNFYAEDFDDSNFKSVKVPGNLELQGFGQPQYVNVQYPWDGSEEIFPPQIPSKNPLASYVRYFDLDEAFWDKEVSLKFDGAATAIYVWLNGHFVGYGEDSFTPSEFMVTKFLKKENNRLAVALYKYSSASWLEDQDFWRMSGLFRSVTLQAKPRLHLEDLKLTASLTDNYQKGKLEVEANIAYRLPNASFKLEVRDSEGDLVAEKLGPIRSEQLEFTLADLPVAAWSAEKPNLYQVRLYLYQAGSLLEVSRQEVGFRNFELKDGIMYLNGQRIVFKGVNRHEFDSKLGRAITEEDMIWDIKTIKRSNINAVRCSHYPNQSLFYRLCDKYGLYVIDEANLESHGTWEKVGGHEDPSFNVPGDDQHWLGASLSRVKNMMARDKNHASILIWSLGNESYAGTVFAQMADYVRKADPTRVQHYEGVTHNRKFDDATQIESRMYAPAKVIEEYLTNKPAKPFISVEYAHAMGNSVGDLAAYTALEKYPHYQGGFIWDWIDQGLEKDGHLLYGGDFDDRPTDYEFCGNGLVFADRTESPKLANVKALYANLKLEVKDGQLFLKNDNLFTNSSSYYFLTSLLVDGKLTYQSRPLTFGLEPGESGTFALPWPEVADEKGEVVYRVTAHLKEDLPWADEGFTVAEAEEVAQKLPEFKPEGRPDLVDSDYNLGLKGNNFQILFSKVKGWPVSLKYAGREYLKRLPEFTFWRALTDNDRGAGYGYDLARWENAGKYARLKDISCEVKEDSVLVKTAFTLPVALKGDLTVTYEVDGRGKIAVTADFPGAEEAGLLPAFGLNLALPKELTDYRYYGLGPNESYPDRLEGNYLGIYQGAVKKNFSPYLRPQETGNRSKVRWYQLFDEKGGLEFTANGADLNLSALPYSAAQIEAADHAFDLTNNYTWVRALSAQMGVGGDDSWGQKVHPEFCLDAQKARQLRLVIQPLLLK
SEQ ID No.18
MSNKLVKEKRVDQADLAWLTDPEVYEVNTIPPHSDHESFQSQEELEEGKSSLVQSLDGDWLIDYAENGQGPVNFYAEDFDDSNFKSVKVPGNLELQGFGQPQYVNVQYPWDGSEEIFPPQIPSKNPLASYVRYFDLDEAFWDKEVSLKFDGAATAIYVWLNGHFVGYGEDSFTPSEFMVTKFLKKENNRLAVALYKYSSASWLEDQDFWRMSGLFRSVTLQAKPRLHLEDLKLTASLTDNYQKGKLEVEANIAYRLPNASFKLEVRDSEGDLVAEKLGPIGSEQLEFTLADLPVAAWSAEKPNLYQVRLYLYQAGSLLEVSRQEVGFRNFELKDGIMYLNGQRIVFKGVNRHEFDSKLGRAITEEDMIWDIKTIKRSNINAVRCSHYPNQSLFYRLCDKYGLYVIDEANLESHGTWEKVGGHEDPSFNVPGDDQHWLGASLSRVKNMMARDKNHASILIWSLGNESYAGTVFAQMADYVRKADPTRVQHYEGVTHNRKFDDATQIESRMYAPAKVIEEYLTNKPAKPFISVEYAHAMGNSVGDLAAYTALEKYPHYQGGFIWDWIDQGLEKDGHLLYGGDFDDRPTDYEFCGNGLVFADRTESPKLANVKALYANLKLEVKDGQLFLKNDNLFTNSSSYYFLTSLLVDGKLTYQSRPLTFGLEPGESGTFALPWPEVADEKGEVVYRVTAHLKEDLPWADEGFTVAEAEEVAQKLPEFKPEGRPDLVDSDYNLGLKGNNFQILFSKVKGWPVSLKYAGREYLKRLPEFTFWRALTDNDRGAGYGYDLARWENAGKYARLKDISCEVKEDSVLVKTAFTLPVALKGDLTVTYEVDGRGKIAVTADFPGAEEAGLLPAFGLNLALPKELTDYRYYGLGPNESYPDRLEGNYLGIYQGAVKKNFSPYLRPQETGNRSKVRWYQLFDEKGGLEFTANGADLNLSALPYSAAQIEAADHAFELTNNYTWVRALSAQMGVGGDDSWGQKVHPEFCLDAQKARQLRLVIQPLLLK
SEQ ID No.19
MSNKLVKEKRVDQADLAWLTDPEVYEVNTIPPHSDHESFQSQEELEEGKSSLVQSLDGNWLIDYAENGQGPINFYAEDFDDSNFKSVKVPGNLELQGFGQPQYVNIQYPWDGSEEIFPPQVPSKNPLASYVRYFDLDEALWDKEVSLKFAGAATAIYVWLNGHFVGYGEDSFTPSEFMVTKFLKKEGNRLAVALYKYSSASWLEDQDFWRLSGLFRSVTLEAKPLLHLEDLKLTASLTDNYQKGKLEVEANIAYRLPNASFKLEVRDSEGDLVAEKVGPIRSEKLDFSLADLPVAAWSAEKPNLYQVRLYLYQAGSLLEVSRQEVGFRNFELKDGIMYLNGQRIVFKGVNRHEFDSKLGRAITEADMIWDIKTMKQSNINAVRCSHYPNQSLFYRLCDKYGLYVIDEANLESHGTWEKVGHEDPSFNVPGDDQHWLGASLSRVKNMMARDKNHASILIWSLGNESYAGTVFAQMADYVRKADPTRVQHYEGVTHNRKFDDATQIESRMYAPAKEIEEYLTKKPAKPFISVEYAHAMGNSVGDLAAYTALEKYPHYQGGFIWDWIDQGLEKDGHLLYGGDFDDRPTDYEFCGDGLVFADRTTSPKLANVKALYSNLKLEVKDGQLFIKNDNLFTNSSAYYFLTSLLVDGKLTYQSQPLTFGLEPGESGTFALPWPEVEDEKGEIVYQVTAHLKEDLPWADEGFTVAEAEEAVTKLPEFYPAGRPELVDSDFNLGLKGNGFRILFSKAKGWPVSIKYAGREYLKRLPEFTFWRALTDNDRGAGYGYDLAKWENAGKYARLQDISYEIKENSALVKTAFTLPVALKGDLTITYEVDSLGKIAVTANFPGAVENGLLPAFGLNFALPKELSDYRYYGLGPNESYADRLEGSYLGIYQGMVEKNFTPYLRPQEAGNRSKVRYYQLFDEEGGLEFTANGADLNLSALPYSAAQIEAADHAFELTNNYTWVRALAAQMGVGGDDSWGQKVHPEFCLDAQEARQLKLVIQPLLLK
SEQ ID No.20(G145结构域a)
MQANINWLDNPEVFRVNQLPAHSDHPFFRDYREWQKQHSSYQQSLNGKWKFHFSANPMDRPQDFYQRDFDSSNFDSIPVPSEIELSNYTQNQYINVLFPWEGKIFRRPAYALDPNDHEEGSFSKGADNTVGSYLKRFDLSSALIGKDVHIKFEGVEQAMYVWLNGHFVGYAEDSFTPSEFDLTPYIQDKDNLLAVEVFKHSTASWLEDQDMFRFSGIFRSVELLGIPATHLMDMDLKPRVADNYQDGIFNLKLHFIGKKAGSFHLLVKDIKGHTLLEKNEDIKENVQINNEKFENVHLWNNHDPYLYQLLIEVYDEQQNLLELIPFQFGFRRIEISPEKVVLLNGKRLIINGVNRHEWDAKRGRSITMSDMTTDINTFKENNINAVRTCHYPNQIPWYYLCDQNGIYVMAENNLESHGTWQKMGEIEPSDNVPGSIPQWKEAVIDRARNNYETFKNHTSILFWSLGNESYAGDNIIAMNEFYKSHDDTRLVHYEGVVHRPELKDKISDVESCMYLPPKKVEEYLQNDPPKPFMECEYMHDMGNSDGGMGSYIKLLDKYPQYFGGFIWDFIDQALLVHDEISGHDVLRYGGDFDDRHSDYEFSGDGLMFADRTPKPAMQEVRYYYGLHK
SEQ ID No.21(G145结构域b)
MDYTNNQLHIIYGDATFGVNGKDFQYIFSYERGGLESLKVHGKEWLYRVPTPTFWRATTDNDRGSGFNLKAAQWLGADMFTKCTDIHLKVDRHDFAELPIAPFNNKFSNHEYAKSAEISFTYQTLTTPATNAKIIYNIDDVGHIKVTMRYYGKKGLPPLPVIGIRLIMPTAATGFDYEGLSGETYPDRMAGAKEGKFHIDGLPVTEYLVPQENGMHMQTKKLTINRETTQNNVDRTNEKFSLSIQQAEKPFNFSCLPYTAEELENATHIEELPLVRRTVLVIAGAVRGVGGIDSWGTDVESAYHINPELDHEFSFILN
SEQ ID No.22
MTDVTHVDRASQAWLTDPTVFEVNRTPAHSSHKWYARDPQSGQWSDLKQSLDGEWRVEVVQAADINLEEEPATAESFDDSSFERIQVPGHLQTAGLMNHKYVNVQYPWDGHENPLEPNIPENNHVALYRRKFTVSAPVANAKQAGGSVSIVFHGMATAIYVWVNGAFVGYGEDGFTPNEFDITELLHDGENVVAVACYEYSSASWLEDQDFWRLHGLFRSVELAARPHVHIENTQIEADWDPEAGTASLDAALTVLNAADAATVRATLKDADGNTVWQTTGDAEAQTAISSGPLQGIAPWSAESPTLYELDVDVIDQAGDVIECTSQKVGFRRFRIEDGILTINGKRIVFKGADRHEFDAEQGRAITEQDMIDDVVFCKRHNINSIRTSHYPNQERWYELCDEYGIYLIDEANLEAHGSWSLPGDVLTEDTIVPGSKREWEGACVDRVNSMMRRDYNHPSVLIWSLGNESYVGDVFRAMYKHVHDIDPNRPVHYEGVTHNRDYDDVTDIETRMYSHADEIEKYLKDDPKKPYLSCEYMHAMGNSVGNMDEYTALERYPKYQGGFIWDFIDQAIYATQPDGTRSLRYGGDFGDRPSDYEFSGDGLLFANRKPSPKAQEVKQLYSNVHIDVTKDSVSVKNDNLFTATGDYVFVLSVLADGKPVWQSTRRFDVPAGETRTFDVAWPVAAYRADARELVLQVSQRLAKATDWAESGYELAFGQTVVPADATATPDTKPADGTITVGRWNAGVRGAGREVLLSRTQGGMVSYTFAGNEFVLRRPAITTFRPLTDNDRGAGHGFERVQWLGAGRYARCVDNVLEQIDDSTLKGTYTYELATAQRTKVTVSYTAHTDGRVNLHVEYPGEQGDLPTIPAFGIEWTLPVQYTNLRFFGTGPAETYLDRKHAKLGVWSTNAFADHAPYLMPQETGNHEDVRWAEITDDHGHGMRVSRADGAAPFAVSLLPYSSFMLEEAQHQDELPKPKHMFLRVLAAQMGVGGDDSWMSPVHPQYHIPADKPISLDVDLELI
SEQ ID No.23(G224结构域a)
MDADIKWLDEPETFRVNQLPAHSDHYYYGNYDEWRHNNSRFAQNLDGQWQFNFAENLRERENDFYKMDYDSSSFGTIEVPSEIELNNYAQNNYINTLIPWEGKIYRRPAYTLSPDDAQEGSFSDGDDNTIGEYLKHFDLDPSLRGKQVRIRFDGVERAMYVWLNGHFIGYAEDSFTPSEFDLTPYIQDEGNVLAVEVFKHSTASWIEDQDMFRFSGIFRSVNLLAQPLVHVEDLNIRPIVTDNYQDGIFNVELQLHGEKTGNVNVRVIDNDGNTLVNETHPVDSTVKVQDQFLENVHLWDNHDPYLYQLLIEIRDDEGNLVELVPYRFGFRRIEINKDHVVLLNGQRLIINGVNRHEWDARRGRAITMDDMTSDIHTFKENNINAVRTCHYPDQIPWYYLCDDNGIYMMAENNLESHATWQKMGAIEPSYNVPGSVPQWRDVVVDRARTNYETFKNHPSILFWSLGNESYAGDNIVKMNEFYKKHDDSRLVHYEGVCHTPEYRDRISDVESWMYLPPKEVEEYLKNNPDKPFMECEYMHDMGNSDGGMGSYISLLDKYPQYFGGFIWDFIDQALLVKDPVSGQEVMRYGGDFDDRHSDYEFSGDGLMFADRTPKPAMQEVRYYYGLHK
SEQ ID No.24(G224结构域b)
MAYTNKLRVIYGDATLGLSGDGFHYIFSYERGGLESLKLNGKEWLYREPMPTFWRATTDNDRGSGFNIRSAQWLAADTFHKCVGIDLTVDNQHFAELPIAPITNEFSDPVSAESVKIKYTFATLTVPATQVTVIYEVNGQGEIKVTMHYYGHEDLPGLPVVGMRFIMPTVATGFDYQGLSGETYPDRMAGATEGTFHVDGLPVTKYLVPQENGMHMATHALTITRDSTQNNADHSREPFSLTVKQDAQPFAFSCLPYTAEELENATHIEELPLARRTVLVVAGAVRGVGGIDSWGADVEEQYHIPADRDVEFSFVLNAK
SEQ ID No.25
MSNKLVKEKRVDQADLAWLTDPEVYEVNTIPPHSDHESFQSQEELEEGKSSLVQSLDGNWLIDYAENGQGPINFYAEDFDDSNFKSVKVPGNLELQGFGQPQYVNIQYPWDGSEEIFPPQVPSKIPLASYVRYFDLDEALWDKEVSLKFAGAATAIYVWLNGHFVGYGEDSFTPSEFMVTKFLKKEGNRLAVALYKYSSASWLEDQDFWRLSGLFRSVTLEAKPLLHLEDLKLTASLTDNYQKGKLEVEANIAYRLPNASFKLEVRDSEGDLVAEKVGPIRSEKLDFSLADLPVAAWSAEKPNLYQVRLYLYQAGSLLEVSRQEVGFRNFELKDGIMYLNGQRIVFKGVNRHEFDSKLGRAITEADMIWDIKTMKQSNINAVRCSHYPNQSLFYRLCDKYGLYVIDEANLESHGTWEKVGHEDPSFNVPGDDQHWLGASLSRVKNMMARDKNHASILIWSLGNESYAGTVFAQMADYVRKADPTRVQHYEGVTHNRKFDDATQIESRMYAPAKEIEEYLTKKPAKPFISVEYAHAMGNSVGDLAAYTALEKYPHYQGGFIWDWIDQGLEKDGHLLYGGDFDDRPTDYEFCGDGLVFADRTTSPKLANVKALYSNLKLEVKDGQLFIKNDNLFTNSSAYYFLTSLLVDGKLTYQSQPLTFGLEPGESGTFALPWPEVEDEKGEIVYQVTAHLKEDLPWADEGFTVAEAEEAVTKLPEFYPAGRPELVDSDFNLGLKGNGFRILFSKAKGWPVSIKYAGREYLKRLPEFTFWRALTDNDRGAGYGYDLAKWENAGKYARLQDISYEIKENSALVKTTFTLPVALKGDLTITYEVDSLGKIAVTANFPGAVENGLLPAFGLNFALPKELSDYRYYGLGPNESYADRLEGSYLGIYQGMVEKNFTPYLRPQEAGNRSKVRYYQLFDEEGGLEFTANGADLNLSALPYSAAQIEAADHAFELTNNYTWVRALAAQMGVGGDDSWGQKVHPEFCLDAQEARQLKLVIQPLLLK
SEQ ID No.26(G282结构域a)
MQANINWLDNPEVFRVNQLPAHSDHPFFRDYREWQKQHSSYQQSLNGKWKFHFSANPMDRPQDFYQRDFDSSNFDSIPVPSEIELSNYTQNQYINVLFPWEGKIFRRPAYALDPNDHEEGSFSKGADNTVGSYLKRFDLSSALIGKDVHIKFEGVEQAMYVWLNGHFVGYAEDSFTPSEFDLTPYIQEKDNLLAVEVFKHSTASWLEDQDMFRFSGIFRSVELLGIPATHLMDMDLKPRVADNYQDGIFNLKLHFIGKKAGSFHLLVKDIKGHTLLEKNEDIKENVQINNEKFENVHLWNNHDPYLYQLLIEVYDEQQNLLELIPFQFGFRRIEISPEKVVLLNGKRLIINGVNRHEWDAKRGRSITMSDMTTDINTFKENNINAVRTCHYPNQIPWYYLCDQNGIYVMAENNLESHGTWQKMGEIEPSDNVPGSIPQWKEAVIDRARNNYETFKNHTSILFWSLGNESYAGDNIIAMNEFYKSHDDTRLVHYEGVVHRPELKDKISDVESCMYLPPKKVEEYLQNDPPKPFMECEYMHDMGNSNGGMDSYIKLLDKYPQYFGGFIWDFIDQALLVHDEISGHDVLRYGGDFDDRHSDYEFSGDGLMFADRKPKPAMQEVRYYYGLHK
SEQ ID No.27(G282结构域b)
MDYTNNQLHIIYGDATFGVNGKDFQYIFSYERGGLESLKVHGKEWLYRVPTPTFWRATTDNDRGSGFNLKAAQWLGADMFTKCTDIHLKVDRHDFAELPIAPFNNKFSNHEYAKSAEISFTYQTLTTPATNAKIIYNIDDGGHIKVTMRYYGKKGLPPLPVIGIRLIMPTAATGFDYEGLSGETYPDRMAGAKEGKFHIDGLPVTEYLVPQENGMHMQTKKLTINRETTQNNVDRTNEKFSLSIQQAEKPFNFSCLPYTAEELENATHIEELPLVRRTVLVIAGAVRGVGGIDSWGTDVESAYHINPDLDHEFSFILN
SEQ ID No.28(G334结构域a)
MKANIKWLDDPEVFRINQLPAHSDHPFYKDYREWQKHSSSFKQSLNGAWQFHFSKDPQSRPIDFYKLSFDSSSFDTIPVPSEIELNGYAQNQYTNILYPWESKIYRKPAYTLGRGIKDGDFSQGKDNTVGSYLKHFDLNPALAGHDIHIQFEGVERAMYVYLNGHFIGYAEDSFTPSEFDLTPYIQAKDNILAVEVFKHSTASWLEDQDMFRFSGIFRSVELLALPRTHLMDLDIKPTVVNDYHDGVFNAKLHFMGKTSGNVHVLIEDIDGKTLLNKKLPLKSTVEIENETFANVHLWDNHDPYLYQLIIEVHDQDGKLVELIPYQFGFRKIEITKDHVVLLNGKRLIINGVNRHEWDAKRGRSITLADMKQDIATFKHNNINAVRTCHYPNQIPWYYLCDQNGIYMMAENNLESHGTWQKLGQVEATSNVPGSIPEWREVVVDRARSNYETFKNHTAILFWSLGNESYAGSNIAAMNKLYKDHDSSRLTHYEGVFHAPEFKKEISDLESCMYLPPKEAEEYLQNPKKPLVECEYMHDMGNSDGGIGSYIKLIDKYPQYMGGFIWDFIDQALLVHDPVSGQDVLRYGGDFDDRHSDYEFSGDGLMFADRTPKPAMQEVRYYYGLHK
SEQ ID No.29(G334结构域b)
MAYTNNLHVVYGEASLGVNGQDFAYLFSYERGVLESLKIKDKEWLYRTPTPTFWRATTDNDRGSGFNQKAAQWLGADMFTKCVGIHVQVDDHQFDELPIAPINNQFSNQEFAHEVKVAFDYETLTTPATKVKIIYNINDAGHMTITMHYFGKKGLPPLPVIGMRFIMPTKAKSFDYTGLSGETYPDRMAGAERGTFHIDGLPVTKYLVPQENGMHMQTNELVITRNSTQNNADKDGDFSLKITQTKQPFNFSLLPYTAEELENATHIEELPLARRSVLVIAGAVRGVGGIDSWGSDVEEQYHIDPEQDHEFSFTLN
SEQ ID No 30
MNMTKIQTYLNDPKIVSVNTVDAHSDHKYFESLEEFSEGEMKLRQSLNGKWKIHYAQNTNQVLKDFYKTEFDETDLNFINVPGHLELQGFGSPQYVNTQYPWDGKEFLRPPQVPQESNAVASYVKHFTLNDALKDKKVFISFQGVATSIFVWVNGNFVGYSEDSFTPSEFEISDYLVEGDNKLAVAVYRYSTASWLEDQDFWRLYGIFRDVYLYAIPKVHVQDLFVKGDYDYQTKAGQLDIDLKTVGDYEDKKIKYVLSDYEGIVTEGDASVNGDGELSVSLENLKIKPWSAESPKLYDLILHVLDDDQVVEVVPVKVGFRRFEIKDKLMLLNGKRIVFKGVNRHEFNARTGRCITEEDMLWDIKVMKQHNINAVRTSHYPNQTRWYELCDEYGLYVIDEANLETHGTWQKLGLCEPSWNIPASEPEWLPACLDRANNMFQRDKNHASVIIWSCGNESYAGKDIADMADYFRSVDNTRPVHYEGVAWCREFDYITDIESRMYAKPADIEEYLTTGKLVDLSSVSDKHFASGNLTNKPQKPYISCEYMHTMGNSGGGLQLYTDLEKYPEYQGGFIWDFIDQAIYKTLPNGSEFLSYGGDWHDRPSDYEFCGNGIVFADRTLTPKLQTVKHLYSNIKIAVDEKSVTIKNDNLFEDLSAYTFLARVYEDGRKVSESEYHFDVKPGEEATFPVNFVVEASNSEQIYEVACVLREATKWAPKGHEIVRGQYVVEKISTETPVKAPLNVVEGDFNIGIQGQNFSILLSRAQNTLVSAKYNGVEFIEKGPKLSFTRAYTDNDRGAGYPFEMAGWKVAGNYSKVTDTQIQIEDDSVKVTYVHELPGLSDVEVKVTYQVDYKGRIFVTANYDGKAGLPNFPEFGLEFAIGSQFTNLSYYGYGAEESYRDKLPGAYLGRYETSVEKTFAPYLMPQESGNHYGTREFTVSDDNHNGLKFTALNKAFEFSALRNSTEQIENARHQYELQESDATWIKVLAAQMGVGGDDSWGAPVHDEFLLSSADSYQLSFMIEPLN
SEQ ID No 31
MNNKLAQVKRVDQADLAWLTDPEIYEVNTIPPHSDHESFQSLEELEEGKSSLVQSLDGDWLIDYAENGEGPANFYEENFDDSSFKSVKVPGNLELQGFGQPQYVNVQYPWDGSDEIFPPMIPSKNPVASYVRYFDLEEAFWDKEVSLKFAGAATAIYVWLNGHFVGYGEDSFTPSEFMVTKFLKKEGNRLAVALYKYSSASWLEDQDFWRMSGLFRSVTLEAKPLLHLQDLKLTASLTNDYQKGSLQVEADIDYRLPNSSFKLELRDSAGELVAEKVGPIRSEKLDFSLADLPVAAWSAEEPNLYQVRLSLYQQGSLLEVSRQEVGFRNFELKDGIMYLNGKRIVFKGVNRHEFDSKLGRAITEADMIWDIKTMKQSNINAVRCSHYPNQSIFYHLCDKYGLYVIDEANLESHGTWEKVGGHEDPSFNVPGDDQRWLGASLSRVKNMMARDKNHASILIWSLGNESYAGKVFAQMADYVRQADPTRVQHYEGVTHNRKFDDATQIESRMYAPAKEIEEYLTKKPAKPFVSCEYAHAMGNSVGDLAAYTALEKYPHYQGGFIWDWIDQGLEKEGHLLYGGDFDDRPSDYEFCGDGLVFADRTTSPKLANVKALYSNLKLELKDGQLFLKNDNLFTNSSAYYFLTSLLVDGKLTYQSQPLTFALEPGESGTFALPWPEVEDEKGEIVYQVTAHLKEDLPWADEGFTVAEAEEAVTKLPEFYPAGRPELVDSDYNLGIKGNGFRILFSKAKGWPVSIKYAGREYLKRLPEFTFWRALTDNDRGAGYGYDLAKWENAGKYARLQDISYEIKENSVLVKTAFTLPVALKGDLTITYEVDSLGKIAVTANFPGAVENGLLPAFGLNFALPKELSDYRYYGLGPNESYADRLEGSYLGIYQGAVEKNFTPYLRPQEVGNRSKVRYYQLFDEEGGLEFTANGANLNLSALPYSAAQIEAADHAFELTNNYTWVRALAAQMGVGGDDSWGQKVHPEFCLDAQEARQLKLVIQPLFTE
SEQ ID NO:32
MADTAELAIVHATTASASWLTDPTVFAANRKPAHSSHRYVIGETSEPKQSLDGEWKVRIEQARNVDVESAPFAAVDFEDGDFGAIEVPGHLQMAGYLKNKYVNIQYPWDGHEDPQAPNIPENNHVAIYRRRFALDAQLARTLENDGTVSLTFHGAATAIYVWLDGTFVGYGEDGFTPSEFDVTEALRNGNGNAADSPEAEHTLTVACYEYSSASWLEDQDFWRLHGLFRTVELAAQPHTHVETVQLEADYTAADTAGTADTAELNAALTLRNPADAMTIESTLRDGDGNVVWESTQACNGEIALNSGKMTNIAPWSAESPTLYTLTVRVVGHDGAIIETVTQKIGFRTFRIENGIMTLNGKRIVFKGADRHEFDAKRGRAITREDMLSDVVFCKRHNINAIRTSHYPNQEYWYDLCDEYGLYLIDETNMETHGTWVANNVERPEDGIPGSRPEWEDACVDRINSMMRRDYNHPSVLIWSLGNESSAGEVFRAMYRHAHTIDPNRPVHYEGSVHMREFEDVTDIESRMYAHADEIERYLNDGSPAHTDGPKKPYISCEYMHAMGNSCGNMDEYTALERYPMYQGGFIWDFIDQAIETKLPDGTTRMCYGGDFGDRPSDYEFSGDGLLFADRTPSPKAQEVKQLYANVKIAVSVDEARITNDNLFVSTGDYRFVLRILADGKPVWSTTRRFDVAAGESASFEVDWPVDDYRSNAEELVLEVSQQLGNACDWAPAGYELAFGQCVVAGAKTTADAVDAAGAPADGTVTLGRWNAGVRGQGREALFSRTQGGMVSYTFGEREFVLRRPSITTFRPLTDNDRGAGHAFERAAWAVAGKYARCVDCAIANRGENAVEATYTYELAIPQRTKVTVRYVADTAGLVSLDVEYPGEKNGDLPTIPAFGIEWALPVEYANLRFYGAGPEETYADRRHAKLGVWSTTAGDDCAPYLLPQETGNHEDVRWAEITDDSGHGVRVKRGAGAKPFAMSLLPYSSTMLEEALHQDELPKPRHMFLRLLAAQMGVGGDDSWMSPVHEQYQLPADQPLSLNVQLKLF
SEQ ID NO:33
MANETRIEHASETWLADSTVFEVNRVPAHSDHKCYAHDSQTNEWSDLRQSLDGEWRVEVVQASDIEFNEEPFVRENFDDSAFERIQVPGHLQMAGLMNNKYVNIQYPWDGHENPAEPNIPENNHVALYRKTFTMANRLADTKNAGGTVSIVFHGMATAIYVWVNGMFVGYGEDGFTPNEFDITEMLHDGENVVAVACYEYSSASWLEDQDFWRLHGLFRSVELAAQPHVHIENMQIESDWDPESGSASLDAALTVRNAADAATISATLKDSDGNVVWETANCADPDTSISTGSLNGIRPWSAEDPVLYEFEVTVIDHAGNIAEVAVQKVGFRRFRIEDGIMTINGKRIVFKGADRHEFDPKRGRAITEQDMIDDVVFCKRHNLNAIRTSHYPNQERWYELCDEYGIYLIDETNLETHGSWCLPGDVLTEETAVPGSKAHWEGACVDRVNSMVRRDYNHPSVLIWSLGNESYTGDVFRAMYKRVHDIDPNRPVHYEGVTHNRDYNDVTDIETRMYAHADAIEEYLKNDPQKPYISCEYMHAMGNSCGNMDEYTALERYPKYQGGFIWDFIDQAIYATQPDGTTSLRYGGDFGDRPSDYEFSGNGLVFADRKPTPKAQEVKQLYSNVHIDVAEDSVTIKNDNLFTSTGEYTFVLRVLADGEPVWQSERRFDVPAGSTEKLDVDWPLDLYRDGASELVLEVSQRLAKATNWAVAGYELAFGQTVVAGSKKASAPVKPVDGIVTVGRWNVGVQGSGREVLLSRTQGGLVSYTFNNREFVLRRPAVTTFRALTDNDRGAGHGFERAQWLGAGRYARCIGNEIEQIDENTVKASYTYELATPQRTKVTVSYTADTTGRVNLHVEYPGEPGDLPTIPAFGIEWTLPVQYSNLRFFGAGPEETYQDRKHAKLGVWSTDAFKDHAPYLMPQETGNHEDVRWAEITDEKGHGLRISRAEGAEPFAMSLQPYSSFMLEEAQHQDELPAPKHMFLRVLAEQMGVGGDDSWMSPVHPQYHIPADQPISLDVDLDLI
SEQ ID NO:34(参考酶)
MVEDATRSDSTTQMSSTPEVVYSSAVDSKQNRTSDFDANWKFMLSDSVQAQDPAFDDSAWQQVDLPHDYSITQKYSQSNEAESAYLPGGTGWYRKSFTIDRDLAGKRIAINFDGVYMNATVWFNGVKLGTHPYGYSPFSFDLTGNAKFGGENTIVVKVENRLPSSRWYSGSGIYRDVTLTVTDGVHVGNNGVAIKTPSLATQNGGNVTMNLTTKVANDTEAAANITLKQTVFPKGGKTDAAIGTVTTASKSIAAGASADVTSTITAASPKLWSIKNPNLYTVRTEVLNGDTVLDTYDTEYGFRWTGFDATSGFSLNGEKVKLKGVSMHHDQGSLGAVANRRAIERQVEILQKMGVNSIRTTHNPAAKALIDVCNEKGVLVVEEVFDMWNRSKNGNTEDYGKWFGQTIAGDNAVLGGDKDETWAKFDLTSTINRDRNAPSVIMWSLGNEMMEGISGSVSDFPATSAKLVAWTKAADSTRPMTYGDNKIKANWNESNTMGDNLTANGGVVGTNYSDGANYDKIRTTHPSWAIYGSETASAINSRGIYNRTTGGAQSSDKQLTSYDNSAVGWGAVASSAWYDVVQRDFVAGTYVWTGFDYLGEPTPWNGTGSGAVGSWPSPKNSYFGIVDTAGFPKDTYYFYQSQWNDDVHTLHILPAWNENVVAKGSGNKVPVVVYTDAAKVKLYFTPKGSTEKRLIGEKSFTKKTTAAGYTYQVYEGTDKDSTAHKNMYLTWNVPWAEGTISAEAYDENNRLIPEGSTEGNASVTTTGKAAKLKADADRKTITADGKDLSYIEVDVTDANGHIVPDAANRVTFDVKGAGKLVGVDNGSSPDHDSYQADNRKAFSGKVLAIVQSTKEAGEITVTAKADGLQSSTVKIATTAVPGTSTEKTVRSFYYSRNYYVKTGNKPILPSDVEVRYSDGTSDRQNVTWDAVSDDQIAKAGSFSVAGTVAGQKISVRVTMIDEIGALLNYSASTPVGTPAVLPGSRPAVLPDGTVTSANFAVHWTKPADTVYNTAGTVKVPGTATVFGKEFKVTATIRVQRSQVTIGSSVSGNALRLTQNIPADKQSDTLDAIKDGSTTVDANTGGGANPSAWTNWAYSKAGHNTAEITFEYATEQQLGQIVMYFFRDSNAVRFPDAGKTKIQISADGKNWTDLAATETIAAQESSDRVKPYTYDFAPVGATFVKVTVTNADTTTPSGVVCAGLTEIELKTATSKFVTNTSAALSSLTVNGTKVSDSVLAAGSYNTPAIIADVKAEGEGNASVTVLPAHDNVIRVITESEDHVTRKTFTINLGTEQEFPADSDERD
SEQ ID No:35(参考酶)
MSCLIPENLRNPKKVHENRLPTRAYYYDQDIFESLNGPWAFALFDAPLDAPDAKNLDWETAKKWSTISVPSHWELQEDWKYGKPIYTNVQYPIPIDIPNPPTVNPTGVYARTFELDSKSIESFEHRLRFEGVDNCYELYVNGQYVGFNKGSRNGAEFDIQKYVSEGENLVVVKVFKWSDSTYIEDQDQWWLSGIYRDVSLLKLPKKAHIEDVRVTTTFVDSQYQDAELSVKVDVQGSSYDHINFTLYEPEDGSKVYDASSLLNEENGNTTFSTKEFISFSTKKNEETAFKINVKAPEHWTAENPTLYKYQLDLIGSDGSVIQSIKHHVGFRQVELKDGNITVNGKDILFRGVNRHDHHPRFGRAVPLDFVVRDLILMKKFNINAVRNSHYPNHPKVYDLFDKLGFWVIDEADLETHGVQEPFNRHTNLEAEYPDTKNKLYDVNAHYLSDNPEYEVAYLDRASQLVLRDVNHPSIIIWSLGNEACYGRNHKAMYKLIKQLDPTRLVHYEGDLNALSADIFSFMYPTFEIMERWRKNHTDENGKFEKPLILCEYGHAMGNGPGSLKEYQELFYKEKFYQGGFIWEWANHGIEFEDVSTADGKLHKAYAYGGDFKEEVHDGVFIMDGLCNSEHNPTPGLVEYKKVIEPVHIKIAHGSVTITNKHDFITTDHLLFIDKDTGKTIDVPSLKPEESVTIPSDTTYVVAVLKDDAGVLKAGHEIAWGQAELPLKVPDFVTETAEKAAKINDGKRYVSVESSGLHFILDKLLGKIESLKVKGKEISSKFEGSSITFWRPPTNNDEPRDFKNWKKYNIDLMKQNIHGVSVEKGSNGSLAVVTVNSRISPVVFYYGFETVQKYTIFANKINLNTSMKLTGEYQPPDFPRVGYEFWLGDSYESFEWLGRGPGESYPDKKESQRFGLYDSKDVEEFVYDYPQENGNHTDTHFLNIKFEGAGKLSIFQKEKPFNFKISDEYGVDEAAHACDVKRYGRHYLRLDHAIHGVGSEACGPAVLDQYRLKAQDFNFEFDLAFE
实施例
一般材料和方法
分子克隆与遗传技术
限制性酶消化、连接、转化和其他标准分子生物学操作的技术基于文献(Maniatiset al.“Molecular cloning:a laboratory manual,2nd edition(分子克隆：实验室手册，第二版)”Cold Spring Harbor Laboratory Press,Cold Spring Harbor,NY,1989；Sambrook and Russell“Molecular Cloning:A Laboratory Manual,3rd edition(分子克隆：实验室手册，第三版)”Cold Spring Harbor Laboratory Press,Cold Spring Harbor,NY 2001；Miller“Experiment in molecular genetics(分子遗传学实验)”Cold SpringHarbor Laboratory Press,1972)中所述的方法；或按照制造商的建议。PCR在获自(Bio-Rad,USA)的DNA热循环仪中进行。DNA测序由德国柏林的LGC进行。在变性条件下，使用十二烷基硫酸钠在含有10％SDS的凝胶(Mini-
TGX stain-freeTM gel,Biorad,USA)上通过聚丙烯酰胺凝胶电泳(PAGE)分析蛋白质。遵循试剂盒提供的操作方案，使用BCA方法确定蛋白质浓度。
细菌菌株、质粒和生长条件
大肠杆菌(Escherichia coli)菌株TOP10(Invitrogen)用于克隆和分离质粒。β-半乳糖苷酶缺陷型大肠杆菌菌株BW25113(Δ(araD-araB)567,ΔlacZ4787(::rrnB-3),λ-,rph-1,Δ(rhaD-rhaB)568,hsdR514)(Datsenko KA,Wanner BL；2000,Proc Natl Acad SciU.S.A.97:6640-6645)与pBAD/His载体(从InvitrogenTM Life Technologies CorporationEurope BV获得)联合用于重组蛋白生产。
蛋白质表达的生长培养基
包含(16g/L BD BBLTM Phyton TM蛋白胨、10g/L酵母提取物、5g/L NaCl)的2xPY培养基用于重组蛋白生产。生长培养基补充有氨苄青霉素(100μg/ml)以维持质粒。通过向培养基中添加0.05％的阿拉伯糖来开始蛋白质生产。
实施例1：用于产生乳糖酶的表达载体的构建
使用商业基因组提取试剂盒，按照提供的方案(DNeasy,Qaigen,德国)提取乳酸菌或双歧杆菌的基因组DNA。使用两个合成引物，以纯化的基因组DNA源作为生物质，通过PCR扩增乳糖酶基因，PCR试剂以Phusion U Hot start DNA聚合酶(Thermo Scientific,美国)试剂盒提供。使用USER克隆方法(Nour-Eldin HH,Geu-Flores F,Halkier BA,PlantSecondary Metabolism Engineering,Methods in Molecular Biology,643；2010)将乳糖酶基因克隆到表达载体pBAD/His的起始密码子中，产生表达构建体。使用USER克隆方法，在PCR产物和目的载体上都产生了长的互补的突出端。这些突出端可以彼此退火形成稳定的杂交产物，该杂交产物用于无需连接而转化到大肠杆菌中。为了在PCR产物中产生突出端，在每个引物的上游区域中包括单个脱氧尿苷残基以扩增靶DNA。使用在第9位上含有尿苷(如粗体显示)然后是乳糖酶基因序列的正向引物
和反向引物
扩增乳糖酶基因。平行地，使用在第9位上含有单个脱氧尿苷残基(粗体突出显示)，随后是载体DNA序列的正向
和反向引物
对，通过PCR扩增载体DNA。使用商业PCR纯化试剂盒(Qiagen，丹麦)纯化PCR产物。按照提供的方案，将纯化的PCR产物(乳糖酶基因和载体DNA)等摩尔量混合，并与商业USER酶混合物(New EnglandBiolabs，美国)一起孵育。这些酶去除尿嘧啶残基以及尿苷上游的短片段，从而在PCR产物中产生互补的突出端。这些互补的突出端彼此退火，产生pBAD-乳糖酶表达载体。将连接混合物的等分试样转化至有化学感受态的大肠杆菌TOP 10细胞。在LB-Amp平板(LB；Luria-Bertani，Amp；100μg/ml氨苄青霉素)上于37℃选择转化体。第二天，使用载体引物(引物1：5’-CGGCGTCACACTTTGCTATGCC-3’，和引物2：5’-CCGCGCTACTGCCGCCAGGC-3’)，使用过夜生长的转化体中的少量生物质进行菌落PCR。将来自菌落PCR的阳性克隆在5mL LB-Amp培养基中培养，并从细胞中分离质粒DNA。对克隆的乳糖酶基因进行测序，以验证在基因扩增过程中没有引入其他突变。将质粒DNA转化到表达宿主大肠杆菌菌株BW25113中。
实施例2：乳糖酶在大肠杆菌表达宿主中的表达
使用pBAD表达系统在大肠杆菌BW25113中产生乳糖酶。使用无菌环从Lb-Amp板中收集携带质粒DNA的新鲜转化的大肠杆菌BW25113细胞，并用于接种5mL Lb-Amp培养基。用过夜生长的培养物(200μL)在摇床(42)中的250mL烧瓶中接种50mL 2x PY培养基(含有100μg/mL氨苄青霉素)。培养物在37℃、220rpm下生长，直到OD600达到0.6-0.8。通过向培养基中添加0.05％阿拉伯糖来开始乳糖酶表达，并将细胞在18℃、180rpm下再培养16-20小时。通过离心(5000rpm，在4℃下10分钟)收集细胞，并在-20℃下保存直至进一步使用。
实施例3：使用固定金属亲和色谱法进行蛋白质纯化
将来自50mL培养物的细胞在冰上融化，并使用10mL裂解缓冲液的混合物(
(Novagen)，含有2mg/mL溶菌酶(Sigma Aldrich)、1单位全能核酸酶(Benzonase)(Sigma Aldrich)和1X完全蛋白酶抑制剂混合物(Complete Proteaseinhibitor cocktail)(不含EDTA,Roche))，通过将细胞在室温下孵育30分钟来裂解细胞。30分钟后，通过在4℃下16000rpm离心20分钟去除细胞碎片。将所得的上清液用0.45μm的孔径过滤器过滤。通过洗出乙醇和水，用1mL浆液制备重力流Ni-Sepharose(GE Healthcare)柱。然后用洗涤缓冲液(50mM NaH2PO4，pH 8.0，含有300mM NaCl和20mM咪唑)平衡所述柱。将无细胞的提取物上样至所述柱，并从所述柱上洗脱未结合的蛋白质。用20mL洗涤缓冲液洗涤所述柱，并用3.5mL洗脱缓冲液(50mM NaH2PO4，pH8.0，含有300mM NaCl和250mM咪唑)洗脱保留的蛋白质。通过SDS-PAGE在含有10％丙烯酰胺的凝胶上分析收集的级分，并将含有纯化的乳糖酶的凝胶合并在一起。使用预装的PD-10脱盐G-25凝胶过滤柱(GE Healthcare)，将缓冲液换成存储缓冲液(50mM KH2PO4缓冲液，pH 7.0，含10mM NaCl、1mM MgCl2)。将纯化的酶保存在4℃下直至进一步使用。
实施例4：使用凝胶过滤色谱法的蛋白质纯化
将来自50mL培养物的细胞在冰上融化，并使用10mL裂解缓冲液的混合物(
(Novagen)，含有2mg/mL溶菌酶、1单位全能核酸酶(Sigma Aldrich)和1X完全蛋白酶抑制剂混合物(不含EDTA,Roche))，通过将细胞在室温下(25℃)孵育30分钟来裂解细胞。30分钟后，通过在4℃下16000rpm离心20分钟除去细胞碎片。将所得的上清液用0.45μm的孔径过滤器过滤。遵循提供的方案，通过用30000道尔顿过滤器(Vivaspin 20,GEHealthcare)过滤，浓缩澄清的无细胞提取物。用1g柱材料制备重力流Sephadex G50超细柱(Pharmacia Chemicals，瑞典)(通过在100mL水中煮沸1小时，冷却至室温而制备)。通过将20mL冷却的浆液施加到30mL过滤柱上来制备柱。将该柱用MilliQ水洗涤，并用洗涤缓冲液B(50mM的NaH2PO4缓冲液，pH 7.0)平衡。将500μL浓缩的上清液施加到所述柱上，使上清液进入柱床。将洗涤缓冲液(50mM NaH2PO4缓冲液，pH 7.0)施加到柱顶部，并分别收集洗脱液级分。在SDS-PAGE凝胶(含有10％丙烯酰胺)上分析收集的级分。将蛋白质级分合并在一起，并如前一节所述，用脱盐柱将缓冲液换成储存缓冲液(50mM KH2PO4缓冲液，pH 7.0，含有10mMNaCl、1mM MgCl2)。将纯化的酶保存在4℃下直至进一步使用。
实施例5：使用BCA测定的蛋白质浓度测量
使用PierceTM BCA蛋白测定试剂盒(Thermo Fisher Scientific,德国)，按照试剂盒提供的操作方案测定纯化的乳糖酶的浓度。
实施例6：在pH 6.7、37℃下，以乳糖为底物的使用纯化酶的活性测定
为了测量β-半乳糖苷酶活性，将纯化的乳糖酶在缓冲液A(50mM NaH2PO4缓冲液pH6.7，含有100μM MgSO4)中稀释至40x。在另一个反应中，将稀释的酶与在缓冲液B中制备的乳糖溶液(在100mM pH 6.7的柠檬酸钠缓冲液中制备140mM乳糖，含有100μM MgSO4)一起孵育。通过在PCR管中混合13μL稀释的酶和37μL乳糖溶液来制备反应混合物。将反应混合物在DNA热循环仪中以下述孵育参数(反应时间：37℃下10分钟，酶失活：95℃下10分钟，4℃冷却)孵育。将反应混合物储存在-20℃下直至进一步使用。为了测定反应过程中形成的葡萄糖量，将10μL反应混合物转移至标准微量滴定板(Thermo Fischer Scientific，丹麦)的一个含有80μL缓冲液C(100mM NaH2PO4缓冲液，pH 7.0，含有葡萄糖氧化酶；0.6g/L(SigmaAdrich)、2,2'-连氮基-双(3-乙基苯并噻唑啉-6-磺酸二铵盐)；ABTS：1.0g/L(SigmaAdrich)，辣根过氧化物酶；0.02g/L(Sigma Adrich))的孔中，并在30℃下孵育40分钟。40分钟后，使用FLUOStar Omega UV酶标仪(德国BMG Labtech)测定610nm处的吸光度。使用0.1-1.5之间的吸光度值进行计算，如果A610 nm值>1.5，则将反应混合物用缓冲液A稀释至10倍。对于每种纯化的酶，均一式三份进行反应，三次测量的平均值用于计算。将用空pBAD/His转化的大肠杆菌细胞进行的蛋白纯化用于标准化。使用已知浓度的葡萄糖(0–2.5mM)，绘制标准曲线，曲线的斜率用于计算反应过程中形成的葡萄糖。用每种乳糖酶的最大吸光度值测定每秒形成的葡萄糖的微摩尔数(μM)，描述为在37℃、pH 6.7下，在乳糖情况下的1单位活性(UAL-1)。在37℃、pH 6.7时的比活性定义为每μM酶每秒形成的葡萄糖的微摩尔数(μM葡萄糖/秒/μM酶)，并通过将UAL-1除以以μM的蛋白质浓度来测定，描述为SUAL-1。在相似条件下测定SEQ ID NO:34和SEQ ID NO:35的比活性。为了酶在鲜乳和发酵奶应用中的稳健性，非常需要在pH 6.7下的高比活性。图1描述了酶在37℃、pH 6.7下的比活性的详细结果。
另外，活性描述为每毫克所添加的酶每分钟形成的葡萄糖的μM数。结果如图14所示。
实施例7：在pH 6.7、37℃下，在半乳糖存在的情况下，使用纯化的酶进行活性测定
将纯化的乳糖酶在缓冲液A(50mM NaH2PO4缓冲液，pH 6.7，含有100μM MgSO4)中稀释至40x。在单独的反应中，将稀释的酶与缓冲液D(140mM乳糖和140mM半乳糖，在100mM、pH6.7的柠檬酸钠缓冲液中制备，含有100μM MgSO4)一起孵育。在PCR管中反应混合物由13μL的稀释的酶和37μL缓冲液D组成。将反应混合物在热循环仪中按照以下孵育参数(反应时间：37℃下10分钟，酶灭活：95℃下10分钟，冷却：4℃)进行孵育。将反应混合物储存在-20℃下直至进一步使用。为了测定反应过程中形成的葡萄糖量，将10μL反应混合物转移至标准微量滴定板(Thermo Fischer Scientific，丹麦)的一个含有80μL缓冲液C(100mM NaH2PO4缓冲液，pH 7.0，含有葡萄糖氧化酶；0.6g/L(Sigma Aldrich)，2,2'-连氮基-双(3-乙基苯并噻唑啉-6-磺酸二铵盐)；ABTS：1.0g/L(Sigma Aldrich)，辣根过氧化物酶；0.02g/L(Sigma Aldrich))的孔中，并在30℃下孵育40分钟。40分钟后，使用FLUOStar Omega UV酶标仪(德国BMG Labtech)测定610nm处的吸光度。使用0.1-1.5之间的吸光度值进行计算，如果A610 nm值>1.5，则将反应混合物用缓冲液A稀释至10倍。对于每种纯化的酶，反应均进行一式三份，三次测量的平均值用于计算。将用空pBAD/His转化的大肠杆菌细胞进行的蛋白质纯化用于标准化。使用已知浓度的葡萄糖(0–2.5mM)，绘制标准曲线，曲线的斜率用于计算与反应过程中形成的1μM葡萄糖相对应的吸光度。使用每种乳糖酶的最大吸光度值测定每秒形成的葡萄糖的μM，描述为在pH 6.7、37℃下，在半乳糖情况下的1个活性单位(UAG)。在半乳糖存在的情况下，在37℃、pH 6.7时的比活性定义为每μM酶每秒形成的葡萄糖的μM数(μM葡萄糖/秒/μM酶)，并通过将UAG除以以μM的蛋白质浓度来测定，描述为SUAG。
半乳糖对酶的抑制百分比可通过以下公式计算：
％抑制＝100*(A-B)/A
其中，A是如实施例6所述的在37℃、pH 6.7下、在乳糖的情况下酶的比活性(SUAL)，并且如实例7所述，B代表在37℃、pH 6.7下，在半乳糖存在的情况下，酶的比活性(SUAG)。图2和图14描述了半乳糖抑制百分比的详细结果。较低的半乳糖抑制与需要非常低的乳糖浓度的应用高度相关。另外，活性描述为每毫克所添加的酶每分钟形成的葡萄糖的微摩尔。结果如图14所示。
注意：半乳糖抑制测量中相对较高的标准偏差是由于购买的半乳糖中的痕量葡萄糖杂质引起的。
实施例8：在4℃、pH 6.7下使用纯化的酶基于乳糖的活性测定
将纯化的乳糖酶在缓冲液A(50mM NaH2PO4缓冲液pH 6.7，含有100μM MgSO4的)中稀释至40x。在另一个反应中，将稀释的酶与在缓冲液B中制备的乳糖溶液(在100mM、pH 6.7的柠檬酸钠缓冲液中制备的140mM乳糖，含有100μM MgSO4)一起孵育。通过在PCR管中混合13μL稀释的纯化酶和37μL乳糖溶液来制备反应混合物。使用以下孵育参数(反应时间：4℃下60分钟，酶灭活：95℃下10分钟，储存：4℃)在DNA热循环仪中孵育反应混合物。将反应混合物储存在-20℃的冰箱中直至进一步使用。按照实施例6所述的方案测定反应过程中形成的葡萄糖的量。用每种乳糖酶的最大吸光度值测定每秒形成的葡萄糖的μM数，描述为在4℃、pH 6.7下，在乳糖情况下的1个活性单位(UAL-2)。在4℃、pH 6.7下的比活性定义为每μM酶每秒形成的葡萄糖的μM数(μM葡萄糖/秒/μM酶)，并通过将UAL-2除以以μM的蛋白质浓度来测定，描述为SUAL-2。对于新鲜/巴氏灭菌乳应用中的乳糖水解，在4℃、pH 6.7下的高比活性是非常需要的。图3描述了在4℃、pH 6.7下酶比活的详细结果。另外，以每毫克所添加的酶每分钟形成的葡萄糖的μmole数来描述活性。结果如图14中所示。
实施例9：在43℃、pH 6.7下，使用纯化的酶以乳糖为底物的活性测定
将纯化的乳糖酶在缓冲液A(50mM NaH2PO4缓冲液pH 6.7，含有100μM MgSO4)中稀释至40x。在另一个反应中，将稀释的酶与在缓冲液B中制备的乳糖溶液(在100mM、pH 6.7的柠檬酸钠缓冲液中制备的140mM乳糖，含有100μM MgSO4)一起孵育。通过在PCR管中混合13μL稀释的纯化酶和37μL乳糖溶液来制备反应混合物。使用以下孵育参数(反应时间：43℃下10分钟，酶灭活：95℃下10分钟，储存：4℃)在DNA热循环仪中孵育反应混合物。将反应混合物储存在-20℃的冰箱中直至进一步使用。按照实施例6所述的方案测定反应过程中形成的葡萄糖的量。用每种乳糖酶的最大吸光度值测定每秒形成的葡萄糖的μM数，描述为在43℃、pH 6.7下，在乳糖情况下的1个活性单位(UAL-3)。在43℃、pH 6.7下的比活性定义为每μM酶每秒形成的葡萄糖的μM数(μM葡萄糖/秒/μM酶)，并通过将UAL-3除以以μM的蛋白质浓度来测定，描述为SUAL-3。对于发酵乳应用中的乳糖水解，非常期望在43℃、pH 6.7下的高比活性。图4描述了在43℃、pH 6.7下酶比活性的详细结果。另外，以每毫克添加的酶每分钟形成的葡萄糖的微摩尔数来描述活性。结果如图14所示。
实施例10：在4℃、pH 5.5下，使用纯化的酶在乳糖为底物上的活性测定
将纯化的乳糖酶在缓冲液A(50mM NaH2PO4缓冲液pH 6.7，含有100μM MgSO4)中稀释至40x。在另一个反应中，将稀释的酶与在缓冲液E中制备的乳糖溶液(在100mM、pH 5.5的柠檬酸钠缓冲液中制备的140mM乳糖，含有100μM MgSO4)一起孵育。通过在PCR管中混合13μL稀释的纯化酶和37μL乳糖溶液来制备反应混合物。使用以下孵育参数(反应时间：4℃下60分钟，酶灭活：95℃下10分钟，储存：4℃)在DNA热循环仪中孵育反应混合物。在pH 5.5的缓冲液中制备底物溶液，并且酶溶液的pH为6.7。为了启动反应，将13μL酶添加到37μL底物溶液中。这两种缓冲液的这种混合最终使反应pH从5.5增加到5.7。将反应混合物储存在-20℃的冰箱中直至进一步使用。为了测定反应过程中形成的葡萄糖的量，将10μL反应混合物转移至标准微量滴定板的一个含有80μL缓冲液C的孔中，并在30℃下孵育40分钟。40分钟后，使用FLUOStar Omega UV酶标仪(德国BMG Labtech)测定610nm处的吸光度。使用0.1-1.5之间的吸光度值进行计算，如果A610 nm值>1.5，则将反应混合物用缓冲液A稀释至5x。对于每种纯化的酶，反应均按一式三份进行，三次重复测量的平均值用于计算。每种乳糖酶的最大吸光度值用于确定每秒形成的葡萄糖的μM数，描述为在4℃、pH 5.5下，在乳糖情况下的1个活性单位(UAL-4)。在4℃、pH 5.5下的比活性定义为每μM酶每秒形成的葡萄糖的μM数(μM葡萄糖/秒/μM酶)，并通过将UAL-4除以μM的蛋白质浓度来测定，描述为SUAL-4。在pH 5.5、4℃下的高比活性对发酵乳应用中的乳糖水解是相关的。图5描述了在4℃、pH 5.5下酶比活性的详细结果。另外，以每毫克添加的酶每分钟形成的葡萄糖的微摩尔数来描述活性。结果如图15所示。
实施例11：在37℃、pH 5.5下，使用纯化酶在乳糖为底物上的活性测定
将纯化的乳糖酶在缓冲液A(50mM NaH2PO4缓冲液pH 6.7，含有100μM MgSO4)中稀释至40x。在另一个反应中，将稀释的酶与在缓冲液E中制备的乳糖溶液(在100mM、pH 5.5的柠檬酸钠缓冲液中制备的140mM乳糖，含有100μM MgSO4)一起孵育。通过在PCR管中混合13μL稀释的纯化酶和37μL乳糖溶液来制备反应混合物。在pH 5.5的缓冲液中制备底物溶液，并且酶溶液的pH为6.7。为了启动反应，将13μL酶添加到37μL底物溶液中。这两种缓冲液的这种混合最终使反应pH从5.5增加到5.7。使用以下孵育参数(反应时间：37℃下10分钟，酶灭活：95℃下10分钟，储存：4℃)在DNA热循环仪中孵育反应混合物。将反应混合物储存在-20℃下直至进一步使用。按照实施例10所述的方案测定反应过程中形成的葡萄糖的量。用每种乳糖酶的最大吸光度值测定每秒形成的葡萄糖的μM数，描述为在37℃、pH 5.5下，在乳糖情况下的1个活性单位(UAL-5)。在37℃、pH 5.5下的比活性定义为每μM酶每秒形成的葡萄糖的μM数(μM葡萄糖/秒/μM酶)，并通过将UAL-5除以以μM的蛋白质浓度来确定，描述为SUAL-5。在pH 5.5、37℃下的高比活性对发酵乳应用中的乳糖水解和甜乳清乳糖水解是相关的。图6描述了在37℃、pH 5.5下酶比活性的详细结果。另外，以每毫克添加的酶每分钟形成的葡萄糖微摩尔数来描述活性。结果如图15所示。
实施例12：在43℃、pH 5.5下，使用纯化的酶在乳糖为底物上的活性测定
将纯化的乳糖酶在缓冲液A(50mM NaH2PO4缓冲液pH 6.7，含有100μM MgSO4)中稀释至40x。在另一个反应中，将稀释的酶与在缓冲液E中制备的乳糖溶液(在100mM、pH 5.5的柠檬酸钠缓冲液中制备的140mM乳糖，含有100μM MgSO4)一起孵育。通过在PCR管中混合13μL稀释的纯化酶和37μL乳糖溶液来制备反应混合物。在pH 5.5的缓冲液中制备底物溶液，并且酶溶液的pH为6.7。为了启动反应，将13μL酶添加到37μL底物溶液中。这两种缓冲液的这种混合最终使反应pH从5.5增加到5.7。
使用以下孵育参数(反应时间：43℃下10分钟，酶灭活：95℃下10分钟，储存：4℃)在DNA热循环仪中孵育反应混合物。将反应混合物储存在-20℃下直至进一步使用。按照实施例10所述的方案测定反应过程中形成的葡萄糖的量。用每种乳糖酶的最大吸光度值测定每秒形成的葡萄糖的μM数，描述为在43℃、pH 5.5下，在乳糖情况下的1个活性单位(UAL-6)。在43℃、pH 5.5下的比活性定义为每μM酶每秒形成的葡萄糖的μM数(μM葡萄糖/秒/μM酶)，并通过将UAL-6除以以μM的蛋白质浓度来测定，描述为SUAL-6。在pH 5.5、43℃下的高比活性对于发酵乳应用中的乳糖水解和甜乳清乳糖水解是相关的。图7描述了在pH 5.5、43℃下酶比活性的详细结果。此外，活性描述为每毫克添加的酶每分钟形成的葡萄糖微摩尔数。结果如图15所示。
实施例13：在4℃、pH 4.5下，使用纯化酶在乳糖为底物上的活性测定
将纯化的乳糖酶在缓冲液A(50mM NaH2PO4缓冲液pH 6.7，含有100μM MgSO4)中稀释至40x。在另一个反应中，将稀释的酶与在缓冲液F中制备的乳糖溶液(在100mM、pH 4.5的柠檬酸钠缓冲液中制备的140mM乳糖，含有100μM MgSO4)一起孵育。通过在PCR管中混合13μL稀释的纯化酶和37μL乳糖溶液来制备反应混合物。在pH 4.5的缓冲液中制备底物溶液，并且酶溶液的pH为6.7。为了启动反应，将13μL酶添加到37μL底物溶液中。这两种缓冲液的这种混合最终使反应pH从4.5增加到4.7。
使用以下孵育参数(反应时间：4℃下60分钟，酶灭活：95℃下10分钟，储存：4℃)在DNA热循环仪中孵育反应混合物。为了测定反应过程中形成的葡萄糖的量，将10μL反应混合物转移至标准微量滴定板的一个含有80μL缓冲液C的孔中(如实施例6所述)，并在30℃下孵育40分钟。40分钟后，使用FLUOStar Omega UV酶标仪测定在610nm处的吸光度。使用0.1-1.5之间的吸光度值进行计算，如果A610 nm值>1.5，则将反应混合物用缓冲液A稀释至5x。对于每种纯化的酶，反应均按一式三份进行，三次重复测量的平均值用于计算。每种乳糖酶的最大吸光度值用于确定每秒形成的葡萄糖的μM数，描述为在4℃、pH 4.5下，在乳糖情况下的1个活性单位(UAL-7)。在4℃、pH 4.5下的比活性定义为每μM酶每秒形成的葡萄糖的μM数(μM葡萄糖/秒/μM酶)，并通过将UAL-7除以以μM的蛋白质浓度来测定，描述为SUAL-7。pH4.5、4℃下的高比活性对于发酵乳应用中的乳糖水解是相关的。图8描述了在4℃、pH 4.5下酶比活的详细结果。另外，以每毫克添加的酶每分钟形成的葡萄糖微摩尔数来描述活性。结果如图16所示。
实施例14：在37℃、pH 4.5下，使用纯化的酶在乳糖为底物上的活性测定
将纯化的乳糖酶在缓冲液A(50mM NaH2PO4缓冲液pH 6.7，含有100μM MgSO4)中稀释至40x。在另一个反应中，将稀释的酶与在缓冲液F中制备的乳糖溶液(在100mM、pH 4.5的柠檬酸钠缓冲液中制备的140mM乳糖，含有100μM MgSO4)一起孵育。通过在PCR管中混合13μL稀释的纯化酶和37μL乳糖溶液来制备反应混合物。在pH 4.5的缓冲液中制备底物溶液，并且酶溶液的pH为6.7。为了启动反应，将13μL酶添加到37μL底物溶液中。这两种缓冲液的这种混合最终使反应pH从4.5增加到4.7。
使用以下孵育参数(反应时间：37℃下10分钟，酶灭活：95℃下10分钟，储存：4℃)在DNA热循环仪中孵育反应混合物。将反应混合物储存在-20℃下直至进一步使用。按照实施例13所述的方案测定反应过程中形成的葡萄糖的量。用每种乳糖酶的最大吸光度值测定每秒形成的葡萄糖的μM数，描述为在37℃、pH 4.5下，在乳糖情况下的1个活性单位(UAL-8)。在37℃、pH 4.5下的比活性定义为每μM酶每秒形成的葡萄糖的μM数(μM葡萄糖/秒/μM酶)，并通过将UAL-8除以以μM的蛋白质浓度来测定，描述为SUAL-8。在37℃、pH 4.5下的高比活对于发酵乳应用中的乳糖水解和酸性乳清乳糖水解是相关的。图9描述了在37℃、pH4.5下酶比活性的详细结果。另外，以每毫克添加的酶每分钟形成的葡萄糖的微摩尔数来描述活性。结果如图16所示。
实施例15：在43℃、pH 4.5下，使用纯化酶在乳糖为底物上的活性测定
将纯化的乳糖酶在缓冲液A(50mM NaH2PO4缓冲液pH 6.7，含有100μM MgSO4)中稀释至40x。在另一个反应中，将稀释的酶与在缓冲液F中制备的乳糖溶液(在100mM、pH 4.5的柠檬酸钠缓冲液中制备的140mM乳糖，含有100μM MgSO4)一起孵育。通过在PCR管中混合13μL稀释的纯化酶和37μL乳糖溶液来制备反应混合物。在pH 4.5的缓冲液中制备底物溶液，并且酶溶液的pH为6.7。为了启动反应，将13μL酶添加到37μL底物溶液中。这两种缓冲液的这种混合最终使反应pH从4.5增加到4.7。使用以下孵育参数(反应时间：43℃下10分钟，酶灭活：95℃下10分钟，储存：4℃)在DNA热循环仪中孵育反应混合物。将反应混合物储存在-20℃下直至进一步使用。按照实施例13所述的方案测定反应过程中形成的葡萄糖的量。用每种乳糖酶的最大吸光度值测定每秒形成的葡萄糖的μM数，描述为在43℃、pH 4.5下，在乳糖情况下的1个活性单位(UAL-9)。在43℃、pH 4.5下的比活性定义为每μM酶每秒形成的葡萄糖的μM数(μM葡萄糖/秒/μM酶)，并通过将UAL-9除以以μM的蛋白质浓度来测定，描述为SUAL-9。在43℃、pH 4.5下的高比活对于发酵乳应用中的乳糖水解和酸性乳清乳糖水解是相关的。图10描述了在43℃、pH为4.5下酶比活性的详细结果。此外，以每毫克添加的酶每分钟形成的葡萄糖的微摩尔数描述活性。结果如图16所示。
实施例16：以BLU为单位的活性测定
在缓冲液G(50mM NaH2PO4 pH 7.0缓冲液，含有100μM MgSO4、0.045％Brij，SigmaAldrich)中，将商购可获得的NOLATM Fit酶(Chr-Hansen，丹麦)稀释为0.5BLU/mL至2.5BLU/mL。将稀释的酶与在缓冲液H中制备的乳糖溶液(在100mM、pH 6.7的柠檬酸钠缓冲液中制备的105mM乳糖，含有100μM MgSO4)一起孵育。通过在PCR管中混合13μL稀释的纯化酶和37μL乳糖溶液来制备反应混合物。使用以下孵育参数(反应时间：37℃下10分钟，酶灭活：95℃下10分钟，储存：4℃)在DNA热循环仪中孵育反应混合物。通过将10μL反应混合物转移至标准微量滴定板的含有80μL缓冲液C的单个孔中，并在30℃下孵育40分钟，来测定葡萄糖转化量。40分钟后，使用FLUOStar Omega UV酶标仪(德国BMG Labtech)测定610nm处的吸光度。使用测得的吸光度值绘制针对BLU/mL的标准曲线。曲线的最大斜率用于测定新酶以BLU/mL为单位的活性。
实施例17：使用乳糖作为底物以BLU/mL进行新乳糖酶的活性测定
将纯化的乳糖酶在缓冲液A(50mM NaH2PO4缓冲液pH 6.7，含有100μM MgSO4)中稀释至50x。在另一个反应中，将稀释的酶与在缓冲液H中制备的乳糖溶液(在100mM、pH 6.7的柠檬酸钠缓冲液中制备的105mM乳糖，含有100μM MgSO4)一起孵育。通过在PCR管中混合13μL稀释的纯化酶和37μL乳糖溶液来制备反应混合物。使用以下孵育参数(反应时间：37℃下10分钟，酶灭活：95℃下10分钟，储存：4℃)在DNA热循环仪中孵育反应混合物。反应后，将10μL反应混合物转移至标准微量滴定板的一个含有80μL缓冲液C的孔中(如实施例6所述)，并在30℃下孵育40分钟。40分钟后，使用FLUOStar Omega UV酶标仪在610nm处测定吸光度。使用0.1-1.5之间的吸光度值进行计算，如果A610nm值>1.5，则将反应混合物用缓冲液A稀释至5x。最大吸光度值用于利用实施例16所述的标准曲线计算以BLU/mL的酶活性。
实施例18：低温下鲜乳中残留乳糖百分比的测量
在10mL玻璃管中，将2mL商业巴氏灭菌乳(1.5％脂肪的巴氏灭菌乳，Arla Food)与10-125μL如实施例17所测定的酶(相当于10BLU/mL)混合。将样品在恒定条件下于4℃孵育24小时。孵育后，通过在95℃下热灭活7分钟来终止反应，然后在-20℃储存直至进一步使用。使用HPLC测定法分析乳中残留乳糖的量。将分析样品用1.8mL蛋白质沉淀溶液(0.083MPCA和2mM Na-EDTA)和2mL MQW处理，然后在4℃下以2800rpm离心30分钟。使用Janus稀释机器人(PerkinElmer，沃尔瑟姆，马萨诸塞州，美国)将上清液的等分试样总共稀释200倍。稀释后的样品在Dionex ICS-5000系统(Thermo Fischer Scientific，沃尔瑟姆(马萨诸塞州)，美国)上使用4x 250mm CarboPac SA20分析柱(Thermo Fischer Scientific，沃尔瑟姆，马萨诸塞州，美国)和脉冲安培检测器进行分析。将检测器设置为选择性检测碳水化合物的简单的三步电势波形。洗脱液设定为1mM KOH，并通过捕集柱(CR-TC，Thermo FischerScientific，沃尔瑟姆(马萨诸塞州)，美国)连续再生。洗脱液的流速为1.2mL/min，分析时间为每次进样10分钟。使用三点外部校准曲线对每个样品中的乳糖进行定量，该校准曲线是通过将已知量的乳糖一水合物(Sigma-Aldrich，美国密苏里州圣路易斯)添加到MQW中制得的。根据色谱峰高度计算浓度。鲜乳中测量的残留乳糖百分比如图11所示。
实施例19：室温下UHT乳中的活性测定
在10mL玻璃管中将2mL UHT乳(1.5％脂肪UHT乳，Arla Food)与2-25μL如实施例17中所测定的酶(相当于2BLU/mL)混合。将样品在恒定条件下于25℃孵育24小时。孵育后，通过在95℃下热灭活7分钟来终止反应，然后在-20℃储存直至进一步使用。按照如实施例18所述的方案，使用HPLC分析UHT乳中残留乳糖的量。图12列出了水解后鲜乳中残留乳糖的百分比。
实施例20：缓冲液中高温下的酶性能
将纯化的酶在缓冲液A(50mM NaH2PO4缓冲液pH 6.7，含有100μM MgSO4)中稀释至5BLU/mL。在另一个反应中，将13μL稀释酶在DNA热循环仪中与乳糖溶液(在100mM、pH 6.7的柠檬酸钠缓冲液中制备的105mM乳糖，含有100μM MgSO4)一起孵育。通过在PCR管中混合13μL酶和37μL乳糖溶液来制备反应混合物。使用以下孵育参数(反应时间：37℃下10分钟，酶灭活：95℃下10分钟，储存：4℃)在DNA热循环仪中孵育反应混合物。反应后，将10μL反应混合物转移至标准微量滴定板的一个含有80μL缓冲液C的孔中(如实施例6所述)，并在30℃下孵育40分钟。40分钟后，使用FLUOStar Omega UV酶标仪在610nm处测定吸光度。使用0.1-1.5之间的吸光度值进行计算，如果A610 nm值>1.5，则将反应混合物用缓冲液A稀释至5x。测得的吸光度称为Abs37℃，并视为计算的参考值。
为了测量热处理对酶活性的影响，在单独的反应中，使用以下孵育参数(72℃下15秒，或74℃15秒，或76℃下6秒，或78℃下6秒，或80℃下4秒，或85℃下5秒，或90℃下5秒，或95℃下5秒，然后在4℃下储存)在DNA热循环仪中孵育13μL稀释的酶(5BLU/mL)。如上所述，通过与乳糖溶液(在100mM、pH 6.7的100mM柠檬酸钠缓冲液中制备的105mM乳糖，含有100μMMgSO4)一起孵育来测定热处理的酶的活性。在不同温度下(例如在72℃、74℃、76℃、78℃、80℃、85℃、90℃或95℃下)测得的吸光度称为Abs72℃、Abs74℃、Abs76℃、Abs78℃、Abs80℃、Abs85℃、Abs90℃、Abs95℃。
使用以下公式测定高温下的残留活性百分比：
％残留活性＝(Abs72℃/Abs37℃)*100
图13描述了不同酶在不同温度下的残留活性百分比。
实施例21：高热处理后的残留乳糖百分比
通过在乳中孵育固定量的酶，然后进行热处理，测定热处理对巴氏灭菌乳中酶性能的影响。在单独的反应中，在PCR管中将50μL的巴氏灭菌乳与10BLU/mL的纯化酶(如实施例17所测定)混合。将乳样品在72℃下孵育15秒，或在76℃下孵育10秒，或在85℃下孵育5秒，和在90℃下孵育5秒，然后在5℃孵育24小时。在5℃下24h(小时)后，通过在95℃下将反应加热7分钟来终止反应，然后在-20℃下储存。按照提供的方案，使用
测定试剂盒(Chr.Hansen，丹麦)，测量残留乳糖。在不同温度下以g/L测定测量的残留乳糖。
试剂盒的检出限为0.2g/L至10g/L。结果如表2所述。
表2：用固定量的纯化酶处理，然后在不同温度下孵育(72℃下15秒，76℃下10秒，85℃下5秒和90℃下5秒，然后4℃下孵育24h)的巴氏灭菌乳中残留乳糖的百分比，其使用测定试剂盒测定。
试剂盒的检出限为0.2g/L至10g/L乳糖。这里ND；未测定到。
序列表
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Asp Phe Gly Ala Ile Glu Val Pro Gly His Leu Gln Met Ala Gly Tyr
85 90 95
Leu Lys Asn Lys Tyr Val Asn Ile Gln Tyr Pro Trp Asp Gly His Glu
100 105 110
Asp Pro Gln Ala Pro Asn Ile Pro Glu Asn Asn His Val Ala Ile Tyr
115 120 125
Arg Arg Arg Phe Ala Leu Asp Ala Gln Leu Ala Arg Thr Leu Glu Asn
130 135 140
Asp Gly Thr Val Ser Leu Thr Phe His Gly Ala Ala Thr Ala Ile Tyr
145 150 155 160
Val Trp Leu Asp Gly Thr Phe Val Gly Tyr Gly Glu Asp Gly Phe Thr
165 170 175
Pro Ser Glu Phe Asp Val Thr Glu Ala Leu Arg Asn Gly Asn Gly Asn
180 185 190
Ala Ala Asp Ser Pro Glu Ala Glu His Thr Leu Thr Val Ala Cys Tyr
195 200 205
Glu Tyr Ser Ser Ala Ser Trp Leu Glu Asp Gln Asp Phe Trp Arg Leu
210 215 220
His Gly Leu Phe Arg Thr Val Glu Leu Ala Ala Gln Pro His Thr His
225 230 235 240
Val Glu Thr Val Gln Leu Glu Ala Asp Tyr Thr Ala Ala Asp Thr Ala
245 250 255
Gly Thr Ala Asp Thr Ala Glu Leu Asn Ala Ala Leu Thr Leu Arg Asn
260 265 270
Pro Ala Asp Ala Met Thr Ile Glu Ser Thr Leu Arg Asp Gly Asp Gly
275 280 285
Asn Val Val Trp Glu Ser Thr Gln Ala Cys Asn Gly Glu Ile Ala Leu
290 295 300
Asn Ser Gly Lys Met Thr Asn Ile Ala Pro Trp Ser Ala Glu Ser Pro
305 310 315 320
Thr Leu Tyr Thr Leu Thr Val Arg Val Val Gly His Asp Gly Ala Ile
325 330 335
Ile Glu Thr Val Thr Gln Lys Ile Gly Phe Arg Thr Phe Arg Ile Glu
340 345 350
Asn Gly Ile Met Thr Leu Asn Gly Lys Arg Ile Val Phe Lys Gly Ala
355 360 365
Asp Arg His Glu Phe Asp Ala Lys Arg Gly Arg Ala Ile Thr Arg Glu
370 375 380
Asp Met Leu Ser Asp Val Val Phe Cys Lys Arg His Asn Ile Asn Ala
385 390 395 400
Ile Arg Thr Ser His Tyr Pro Asn Gln Glu Tyr Trp Tyr Asp Leu Cys
405 410 415
Asp Glu Tyr Gly Leu Tyr Leu Ile Asp Glu Thr Asn Met Glu Thr His
420 425 430
Gly Thr Trp Val Ala Asn Asn Val Glu Arg Pro Glu Asp Gly Ile Pro
435 440 445
Gly Ser Arg Pro Glu Trp Glu Gly Ala Cys Val Asp Arg Ile Asn Ser
450 455 460
Met Met Arg Arg Asp Tyr Asn His Pro Ser Val Leu Ile Trp Ser Leu
465 470 475 480
Gly Asn Glu Ser Ser Ala Gly Glu Val Phe Arg Ala Met Tyr Arg His
485 490 495
Ala His Thr Ile Asp Pro Asn Arg Pro Val His Tyr Glu Gly Ser Val
500 505 510
His Met Arg Glu Phe Glu Asp Val Thr Asp Ile Glu Ser Arg Met Tyr
515 520 525
Ala His Ala Asp Glu Ile Glu Arg Tyr Leu Asn Asp Gly Ser Pro Ala
530 535 540
His Thr Asp Gly Pro Lys Lys Pro Tyr Ile Ser Cys Glu Tyr Met His
545 550 555 560
Ala Met Gly Asn Ser Cys Gly Asn Met Asp Glu Tyr Thr Ala Leu Glu
565 570 575
Arg Tyr Pro Met Tyr Gln Gly Gly Phe Ile Trp Asp Phe Ile Asp Gln
580 585 590
Ala Ile Glu Thr Lys Leu Pro Asp Gly Thr Thr Arg Met Cys Tyr Gly
595 600 605
Gly Asp Phe Gly Asp Arg Pro Ser Asp Tyr Glu Phe Ser Gly Asp Gly
610 615 620
Leu Leu Phe Ala Asp Arg Thr Pro Ser Pro Lys Ala Gln Glu Val Lys
625 630 635 640
Gln Leu Tyr Ala Asn Val Lys Ile Ala Val Ser Val Asp Glu Ala Arg
645 650 655
Ile Thr Asn Asp Asn Leu Phe Val Ser Thr Gly Asp Tyr Arg Phe Val
660 665 670
Leu Arg Ile Leu Ala Asp Gly Lys Pro Val Trp Ser Thr Thr Arg Arg
675 680 685
Phe Asp Val Ala Ala Gly Glu Ser Ala Ser Phe Glu Val Asp Trp Pro
690 695 700
Val Asp Asp Tyr Arg Ser Asn Ala Glu Glu Leu Val Leu Glu Val Ser
705 710 715 720
Gln Gln Leu Gly Asn Ala Cys Asp Trp Ala Pro Ala Gly Tyr Glu Leu
725 730 735
Ala Phe Gly Gln Cys Val Val Ala Gly Ala Lys Thr Thr Ala Asp Ala
740 745 750
Val Asp Ala Ala Gly Ala Pro Ala Asp Gly Thr Val Thr Leu Gly Arg
755 760 765
Trp Asn Ala Gly Val Arg Gly Gln Gly Arg Glu Ala Leu Phe Ser Arg
770 775 780
Thr Gln Gly Gly Met Val Ser Tyr Thr Phe Gly Glu Arg Glu Phe Val
785 790 795 800
Leu Arg Arg Pro Ser Ile Thr Thr Phe Arg Pro Leu Thr Asp Asn Asp
805 810 815
Arg Gly Ala Gly His Ala Phe Glu Arg Ala Ala Trp Ala Val Ala Gly
820 825 830
Lys Tyr Ala Arg Cys Val Asp Cys Ala Ile Ala Asn Arg Gly Glu Asn
835 840 845
Ala Val Glu Ala Thr Tyr Thr Tyr Glu Leu Ala Ile Pro Gln Arg Thr
850 855 860
Lys Val Thr Val Arg Tyr Val Ala Asp Thr Ala Gly Leu Val Ser Leu
865 870 875 880
Asp Val Glu Tyr Pro Gly Glu Lys Asn Gly Asp Leu Pro Thr Ile Pro
885 890 895
Ala Phe Gly Ile Glu Trp Ala Leu Pro Val Glu Tyr Ala Asn Leu Arg
900 905 910
Phe Tyr Gly Ala Gly Pro Glu Glu Thr Tyr Ala Asp Arg Arg His Ala
915 920 925
Lys Leu Gly Val Trp Ser Thr Thr Ala Gly Asp Asp Cys Ala Pro Tyr
930 935 940
Leu Leu Pro Gln Glu Thr Gly Asn His Glu Asp Val Arg Trp Ala Glu
945 950 955 960
Ile Thr Asp Asp Ser Gly His Gly Val Arg Val Lys Arg Gly Ala Gly
965 970 975
Ala Lys Pro Phe Ala Met Ser Leu Leu Pro Tyr Ser Ser Thr Met Leu
980 985 990
Glu Glu Ala Leu His Gln Asp Glu Leu Pro Lys Pro Arg His Met Phe
995 1000 1005
Leu Arg Leu Leu Ala Ala Gln Met Gly Val Gly Gly Asp Asp Ser
1010 1015 1020
Trp Met Ser Pro Val His Glu Gln Tyr Gln Leu Pro Ala Asp Gln
1025 1030 1035
Pro Leu Ser Leu Asn Val Gln Leu Lys Leu Phe
1040 1045
<210> 5
<211> 626
<212> PRT
<213> 噬淀粉乳杆菌(Lactobacillus amylovorus)
<400> 5
Met Lys Ala Asn Ile Lys Trp Leu Asp Asp Pro Glu Val Phe Arg Ile
1 5 10 15
Asn Gln Leu Pro Ala His Ser Asp His Pro Phe Tyr Lys Asp Tyr Arg
20 25 30
Glu Trp Gln Asn His Ser Ser Ser Phe Lys Gln Ser Leu Asn Gly Ala
35 40 45
Trp Gln Phe His Phe Ser Lys Asp Pro Gln Ser Arg Pro Ile Asp Phe
50 55 60
Tyr Lys Arg Ser Phe Asp Ser Ser Ser Phe Asp Thr Ile Pro Val Pro
65 70 75 80
Ser Glu Ile Glu Leu Asn Gly Tyr Ala Gln Asn Gln Tyr Thr Asn Ile
85 90 95
Leu Tyr Pro Trp Glu Ser Lys Ile Tyr Arg Lys Pro Ala Tyr Thr Leu
100 105 110
Gly Arg Gly Ile Lys Asp Gly Asp Phe Ser Gln Gly Lys Asp Asn Thr
115 120 125
Val Gly Ser Tyr Leu Lys His Phe Asp Leu Asn Pro Ala Leu Ala Gly
130 135 140
His Asp Ile His Ile Gln Phe Glu Gly Val Glu Arg Ala Met Tyr Val
145 150 155 160
Tyr Leu Asn Gly His Phe Ile Gly Tyr Ala Glu Asp Ser Phe Thr Pro
165 170 175
Ser Glu Phe Asp Leu Thr Pro Tyr Ile Gln Ala Lys Asp Asn Ile Leu
180 185 190
Ala Val Glu Val Phe Lys His Ser Thr Ala Ser Trp Leu Glu Asp Gln
195 200 205
Asp Met Phe Arg Phe Ser Gly Ile Phe Arg Ser Val Glu Leu Leu Ala
210 215 220
Leu Pro Arg Thr His Leu Met Asp Leu Asp Ile Lys Pro Thr Val Val
225 230 235 240
Asn Asp Tyr His Asp Gly Val Phe Asn Ala Lys Leu His Phe Met Gly
245 250 255
Lys Thr Ser Gly Asn Val His Val Leu Ile Glu Asp Ile Asp Gly Lys
260 265 270
Thr Leu Leu Asn Lys Lys Leu Pro Leu Lys Ser Thr Val Glu Ile Glu
275 280 285
Asn Glu Thr Phe Ala Asn Val His Leu Trp Asp Asn His Asp Pro Tyr
290 295 300
Leu Tyr Gln Leu Ile Ile Glu Val His Asp Gln Asp Gly Lys Leu Val
305 310 315 320
Glu Leu Ile Pro Tyr Gln Phe Gly Phe Arg Lys Ile Glu Ile Thr Lys
325 330 335
Asp His Val Val Leu Leu Asn Gly Lys Arg Leu Ile Ile Asn Gly Val
340 345 350
Asn Arg His Glu Trp Asp Ala Lys Arg Gly Arg Ser Ile Thr Leu Ala
355 360 365
Asp Met Lys Gln Asp Ile Ala Thr Phe Lys His Asn Asn Ile Asn Ala
370 375 380
Val Arg Thr Cys His Tyr Pro Asn Gln Ile Pro Trp Tyr Tyr Leu Cys
385 390 395 400
Asp Gln Asn Gly Ile Tyr Met Met Ala Glu Asn Asn Leu Glu Ser His
405 410 415
Gly Thr Trp Gln Lys Leu Gly Gln Val Glu Ala Thr Ser Asn Val Pro
420 425 430
Gly Ser Ile Pro Glu Trp Arg Glu Val Val Val Asp Arg Ala Arg Ser
435 440 445
Asn Tyr Glu Thr Phe Lys Asn His Thr Ala Ile Leu Phe Trp Ser Leu
450 455 460
Gly Asn Glu Ser Tyr Ala Gly Ser Asn Ile Ala Ala Met Asn Lys Leu
465 470 475 480
Tyr Lys Asp His Asp Ser Ser Arg Leu Thr His Tyr Glu Gly Val Phe
485 490 495
His Ala Pro Glu Phe Lys Lys Glu Ile Ser Asp Leu Glu Ser Cys Met
500 505 510
Tyr Leu Pro Pro Lys Glu Ala Glu Glu Tyr Leu Gln Asn Pro Lys Lys
515 520 525
Pro Leu Val Glu Cys Glu Tyr Met His Asp Met Gly Thr Pro Asp Gly
530 535 540
Gly Met Gly Ser Tyr Ile Lys Leu Ile Asp Lys Tyr Pro Gln Tyr Met
545 550 555 560
Gly Gly Phe Ile Trp Asp Phe Ile Asp Gln Ala Leu Leu Val His Asp
565 570 575
Pro Val Ser Gly Gln Asp Val Leu Arg Tyr Gly Gly Asp Phe Asp Asp
580 585 590
Arg His Ser Asp Tyr Glu Phe Ser Gly Asp Gly Leu Met Phe Ala Asp
595 600 605
Arg Thr Pro Lys Pro Ala Met Gln Glu Val Arg Tyr Tyr Tyr Gly Leu
610 615 620
His Lys
625
<210> 6
<211> 316
<212> PRT
<213> 噬淀粉乳杆菌
<400> 6
Met Ala Tyr Thr Asn Asn Leu His Val Val Tyr Gly Glu Ala Ser Leu
1 5 10 15
Gly Val Asn Gly Gln Asp Phe Ala Tyr Leu Phe Ser Tyr Glu Arg Gly
20 25 30
Gly Leu Glu Ser Leu Lys Ile Lys Asp Lys Glu Trp Leu Tyr Arg Thr
35 40 45
Pro Thr Pro Thr Phe Trp Arg Ala Thr Thr Asp Asn Asp Arg Gly Ser
50 55 60
Gly Phe Asn Gln Lys Ala Ala Gln Trp Leu Gly Ala Asp Met Phe Thr
65 70 75 80
Lys Cys Val Gly Ile His Val Gln Val Asp Asp His Arg Phe Asp Glu
85 90 95
Leu Pro Val Ala Pro Ile Asn Asn Gln Phe Ser Asn Gln Glu Phe Ala
100 105 110
His Glu Val Lys Val Ala Phe Asp Tyr Glu Thr Leu Thr Thr Pro Ala
115 120 125
Thr Lys Val Lys Ile Ile Tyr Asn Ile Asn Asp Phe Gly His Met Thr
130 135 140
Ile Thr Met His Tyr Phe Gly Lys Lys Gly Leu Pro Pro Leu Pro Val
145 150 155 160
Ile Gly Met Arg Phe Ile Met Pro Thr Lys Ala Lys Ser Phe Asp Tyr
165 170 175
Thr Gly Leu Ser Gly Glu Thr Tyr Pro Asp Arg Met Ala Gly Ala Glu
180 185 190
Arg Gly Thr Phe His Ile Asp Gly Leu Pro Val Thr Lys Tyr Leu Val
195 200 205
Pro Gln Glu Asn Gly Met His Met Gln Thr Asn Glu Leu Val Ile Thr
210 215 220
Arg Asn Ser Thr Gln Asn Asn Ala Asp Lys Asp Gly Asp Phe Ser Leu
225 230 235 240
Lys Ile Thr Gln Thr Lys Gln Pro Phe Asn Phe Ser Leu Leu Pro Tyr
245 250 255
Thr Ala Glu Glu Leu Glu Asn Ala Thr His Ile Glu Glu Leu Pro Leu
260 265 270
Ala Arg Arg Ser Val Leu Val Ile Ala Gly Ala Val Arg Gly Val Gly
275 280 285
Gly Ile Asp Ser Trp Gly Ser Asp Val Glu Glu Gln Tyr His Ile Asp
290 295 300
Pro Glu Gln Asp His Glu Phe Ser Phe Thr Leu Asn
305 310 315
<210> 7
<211> 1052
<212> PRT
<213> 两歧双歧杆菌(Bifidobacterium bifidum)
<400> 7
Met Asn Thr Thr Asp Asp Gln Arg Lys Asn Gly Asp Pro Ile Val Ser
1 5 10 15
Pro Ser Ile Pro Thr Thr Ala Trp Leu Ala Asp Pro Arg Val Tyr Ala
20 25 30
Val His Arg Leu Asp Ala His Ser Asp His Ala Cys Trp Ser Arg Ser
35 40 45
Pro Val Asp Gly Glu Ser Thr Asp Leu Arg Gln Ser Leu Asp Gly Glu
50 55 60
Trp Arg Val Arg Val Glu Thr Ala Pro Thr Gly Arg Phe Pro Asp Gly
65 70 75 80
Thr Ser Asp Gly Pro Asp Trp Ile Ser Asp Val Ser Pro Leu Phe Ala
85 90 95
Ala Pro Gly Phe Asp Asp Ser Ser Phe Ser Arg Val Gln Val Pro Ser
100 105 110
His Leu Glu Thr Ala Gly Leu Leu Ala Pro Gln Tyr Val Asn Val Gln
115 120 125
Tyr Pro Trp Asp Gly His Glu Asp Pro Lys Ala Pro Ala Ile Pro Glu
130 135 140
His Gly His Val Ala Val Tyr Arg Arg Glu Phe Asp Ala Asp Gly Glu
145 150 155 160
Val Ala Gln Ala Val Arg Glu Gly Arg Pro Val Thr Leu Thr Phe Gln
165 170 175
Gly Ala Ala Thr Ala Ile Tyr Val Trp Leu Asn Gly Ser Phe Ile Gly
180 185 190
Tyr Ala Glu Asp Ser Phe Thr Pro Ser Glu Phe Asp Val Thr Asp Ala
195 200 205
Ile Lys Val Asp Gly Asn Val Leu Ala Val Ala Cys Tyr Glu Tyr Ser
210 215 220
Ser Ala Ser Trp Leu Glu Asp Gln Asp Phe Trp Arg Leu His Gly Leu
225 230 235 240
Phe Arg Ser Val Glu Leu Asn Ala Arg Pro Ala Ala His Val Ala Asp
245 250 255
Leu His Ala Asp Ala Asp Trp Asp Leu Ala Thr Ser Arg Gly Ser Leu
260 265 270
Ser Leu Asp Val Leu Ile Asp Gly Ala Ala Asn Ala Ala Thr Ala Asp
275 280 285
Phe Ala Leu Arg Asp Lys Asn Gly Thr Ile Val Trp Arg Thr Ala Thr
290 295 300
Lys Ala Asp Gly Thr Leu His Ala Glu Ala Glu Ile Asp Asp Ala Ala
305 310 315 320
Pro Trp Ser Ala Glu Arg Pro Asp Leu Tyr Glu Leu Ser Val Thr Leu
325 330 335
Leu Asp Ala Asp Gly Lys Val Leu Glu Thr Ala Arg Thr Arg Ile Gly
340 345 350
Phe Arg His Val Ala Ile Glu Asp Gly Ile Leu Lys Leu Asn Gly Lys
355 360 365
Arg Leu Val Phe Arg Gly Val Asn Arg His Glu Phe Asp Cys Arg Arg
370 375 380
Gly Arg Ala Ile Thr Glu Glu Asp Met Leu Trp Asp Ile Arg Phe Met
385 390 395 400
Lys Arg His Asn Ile Asn Ala Val Arg Thr Ser His Tyr Pro Asn Gln
405 410 415
Ser Arg Trp Tyr Glu Leu Cys Asp Glu Tyr Gly Ile Tyr Leu Ile Asp
420 425 430
Glu Thr Asn Leu Glu Thr His Gly Ser Trp Asn Ser Pro Gly Asp Ile
435 440 445
Pro Val Gly Thr Ser Val Pro Gly Asp Asp Glu Ala Trp Leu Gly Ala
450 455 460
Cys Ile Asp Arg Leu Asp Ser Met Ile Leu Arg Asp Arg Asn His Pro
465 470 475 480
Ser Val Leu Val Trp Ser Leu Gly Asn Glu Ser Tyr Ala Gly Glu Val
485 490 495
Leu Lys Ala Met Ser Ala His Ala His Gln Leu Asp Pro Gly Arg Pro
500 505 510
Val His Tyr Glu Gly Val Asn Trp Asn His Ala Tyr Asp Gly Ile Ser
515 520 525
Asp Phe Glu Ser Arg Met Tyr Ala Lys Pro Ala Glu Ile Gln Asp Trp
530 535 540
Leu Glu His Gly Asp Glu Arg Gly Glu Ala Ser Lys Pro Phe Val Ser
545 550 555 560
Cys Glu Tyr Met His Ala Met Gly Asn Ser Cys Gly Gly Leu Ser Glu
565 570 575
Phe Ile Asp Leu Glu Arg Tyr Glu Arg Tyr Ser Gly Gly Phe Ile Trp
580 585 590
Asp Tyr Ile Asp Gln Gly Leu Val Gln Arg Leu Pro Asp Gly Ser Glu
595 600 605
Arg Leu Ser Val Gly Gly Glu Trp Gly Asp Arg Pro Thr Asp Tyr Glu
610 615 620
Phe Val Gly Asn Gly Ile Val Phe Ala Asp Arg Thr Pro Ser Pro Lys
625 630 635 640
Ala Gln Glu Val Lys Gln Leu Tyr Ser Pro Val Lys Leu Ala Pro Asp
645 650 655
Gly His Gly Val Thr Ile Glu Asn Arg Asn Leu Phe Ala Gly Thr Asp
660 665 670
Gly Tyr Val Phe Ala Ala Arg Leu Leu Glu Asp Gly His Glu Ile Trp
675 680 685
His Ala Asp Tyr Arg Phe Asp Val Ala Ala Gly Asp Thr Gln His His
690 695 700
Asp Ile Ala Phe Pro Asp Ile Asp Ala Asp Gly Asp Thr Arg Glu Val
705 710 715 720
Thr Tyr Glu Val Asp Leu Leu Leu Ala Glu Ala Thr Ala Trp Ala Pro
725 730 735
Ala Gly Tyr Glu Leu Ala Phe Gly Gln Leu Thr Gly Thr Leu Asn Pro
740 745 750
Glu Gln Asp Ile Thr Glu Thr Ser His Asp Asp Asp Gly Arg Ala Thr
755 760 765
Arg Thr Leu Ser Arg Trp Asn Ala Gly Ile Arg Arg Asp Asp Glu Glu
770 775 780
Ile Leu Leu Ser Arg Thr Gln Gly Gly Ile Val Ser Trp Lys Arg Asp
785 790 795 800
Asp Arg Glu Met Val Ile Arg Arg Pro Glu Leu Val Thr Phe Arg Pro
805 810 815
Leu Thr Asp Asn Asp Arg Gly Asn His Ser Gly Phe Asp Arg Ala Ala
820 825 830
Trp Phe Ala Ala Gly Arg Tyr Ala Ile Val Thr Glu Thr Lys Ile His
835 840 845
Glu Ser Asp Asp Gly Leu Val Ala Glu Tyr Gln Tyr Glu Leu Ala Asp
850 855 860
Pro Asn His Thr Pro Val Ser Val Thr Tyr His Val Asn Ser Asp Met
865 870 875 880
Arg Met Gln Leu Thr Val Glu Tyr Pro Gly Asn Ala Thr Asp Met Ala
885 890 895
Ser Leu Pro Ala Phe Gly Ile Glu Trp Glu Leu Pro Gly Glu Tyr Asp
900 905 910
Arg Leu Arg Tyr Tyr Gly Pro Gly Pro Glu Glu Thr Tyr Arg Asp Arg
915 920 925
Lys Gln Gly Gly Lys Leu Gly Ile Trp Asp Ala Thr Ala Lys Ala Ser
930 935 940
Met Ala Pro Tyr Leu Met Val Gln Glu Thr Gly Ser His Glu Asp Val
945 950 955 960
Arg Trp Leu Glu Ala Thr Asp Ile Gln Gly His Gly Leu Arg Val Thr
965 970 975
Gln Arg Gly Asp Arg His Phe Thr Ala Ser Leu Leu Pro Trp Asn Thr
980 985 990
Tyr Thr Ile Glu Ala Ala Arg Arg His Glu Asp Leu Pro Lys Pro Arg
995 1000 1005
His Asn Tyr Leu Arg Leu Leu Ala Ala Gln Met Gly Val Gly Gly
1010 1015 1020
Asp Asp Ser Trp Gly Ala Pro Val His Thr Ala Tyr Gln Leu Pro
1025 1030 1035
Ala Gly Arg Pro Leu Thr Leu Asp Val Asn Leu Glu Leu Ile
1040 1045 1050
<210> 8
<211> 1052
<212> PRT
<213> 两歧双歧杆菌
<400> 8
Met Asn Thr Thr Asp Asp Gln Arg Lys Asn Gly Asp Pro Ile Val Ser
1 5 10 15
Pro Ser Ile Pro Thr Thr Ala Trp Leu Ala Asp Pro Arg Val Tyr Ala
20 25 30
Val His Arg Leu Asp Ala His Ser Asp His Ala Cys Trp Ser Arg Ser
35 40 45
Pro Val Asp Gly Glu Ser Thr Asp Leu Arg Gln Ser Leu Asp Gly Glu
50 55 60
Trp Arg Val Arg Val Glu Thr Ala Pro Thr Gly Arg Phe Pro Asp Gly
65 70 75 80
Thr Ser Asp Gly Pro Asp Trp Ile Ser Asp Val Ser Pro Leu Phe Ala
85 90 95
Ala Pro Gly Phe Asp Asp Ser Ser Phe Ser Arg Val Gln Val Pro Ser
100 105 110
His Leu Glu Thr Ala Gly Leu Leu Ala Pro Gln Tyr Val Asn Val Gln
115 120 125
Tyr Pro Trp Asp Gly His Glu Asp Pro Lys Ala Pro Ala Ile Pro Glu
130 135 140
His Gly His Val Ala Val Tyr Arg Arg Glu Phe Asp Ala Asp Gly Glu
145 150 155 160
Val Ala Gln Ala Val Arg Glu Gly Arg Pro Val Thr Leu Thr Phe Gln
165 170 175
Gly Ala Ala Thr Ala Ile Tyr Val Trp Leu Asn Gly Ser Phe Ile Gly
180 185 190
Tyr Ala Glu Asp Ser Phe Thr Pro Ser Glu Phe Asp Val Thr Asp Ala
195 200 205
Ile Lys Val Asp Gly Asn Val Leu Ala Val Ala Cys Tyr Glu Tyr Ser
210 215 220
Ser Ala Ser Trp Leu Glu Asp Gln Asp Phe Trp Arg Leu His Gly Leu
225 230 235 240
Phe Arg Ser Val Glu Leu Asn Ala Arg Pro Ala Ala His Val Ala Asp
245 250 255
Leu His Ala Asp Ala Asp Trp Asp Leu Ala Thr Ser Arg Gly Ser Leu
260 265 270
Ser Leu Asp Val Leu Ile Asp Gly Ala Ala Asn Ala Ala Thr Ala Asp
275 280 285
Phe Ala Leu Trp Asp Lys Asn Gly Thr Ile Val Trp His Ile Val Thr
290 295 300
Lys Ala Asp Gly Thr Leu His Ala Glu Ala Glu Ile Asp Asp Ala Ala
305 310 315 320
Pro Trp Ser Ala Glu Arg Pro Asp Leu Tyr Glu Leu Ser Val Thr Leu
325 330 335
Leu Asp Ala Asp Gly Lys Val Leu Glu Thr Ala Arg Thr Arg Ile Gly
340 345 350
Phe Arg His Val Ala Ile Glu Asp Gly Ile Leu Lys Leu Asn Gly Lys
355 360 365
Arg Leu Val Phe Arg Gly Val Asn Arg His Glu Phe Asp Cys Arg Arg
370 375 380
Gly Arg Ala Ile Thr Glu Glu Asp Met Leu Trp Asp Ile Arg Phe Met
385 390 395 400
Lys Arg His Asn Ile Asn Ala Val Arg Thr Ser His Tyr Pro Asn Gln
405 410 415
Ser Arg Trp Tyr Glu Leu Cys Asp Glu Tyr Gly Ile Tyr Leu Ile Asp
420 425 430
Glu Thr Asn Leu Glu Thr His Gly Ser Trp Asn Ser Pro Gly Asp Ile
435 440 445
Pro Val Gly Thr Ser Val Pro Gly Asp Asp Glu Ala Trp Leu Gly Ala
450 455 460
Cys Ile Asp Arg Leu Asp Ser Met Ile Leu Arg Asp Arg Asn His Pro
465 470 475 480
Ser Val Leu Val Trp Ser Leu Gly Asn Glu Ser Tyr Ala Gly Glu Val
485 490 495
Leu Lys Ala Met Ser Ala His Ala His Arg Leu Asp Pro Gly Arg Pro
500 505 510
Val His Tyr Glu Gly Val Asn Trp Asn His Ala Tyr Asp Gly Ile Ser
515 520 525
Asp Phe Glu Ser Arg Met Tyr Ala Lys Pro Ala Glu Ile Gln Asp Trp
530 535 540
Leu Glu His Gly Asp Glu Arg Gly Glu Ala Ser Lys Pro Phe Val Ser
545 550 555 560
Cys Glu Tyr Met His Ala Met Gly Asn Ser Cys Gly Gly Leu Ser Glu
565 570 575
Phe Ile Asp Leu Glu Arg Tyr Glu Arg Tyr Ser Gly Gly Phe Ile Trp
580 585 590
Asp Tyr Ile Asp Gln Gly Leu Val Gln Arg Leu Pro Asp Gly Ser Glu
595 600 605
Arg Leu Ser Val Gly Gly Glu Trp Gly Asp Arg Pro Thr Asp Tyr Glu
610 615 620
Phe Val Gly Asn Gly Ile Val Phe Ala Asp Arg Thr Pro Ser Pro Lys
625 630 635 640
Ala Gln Glu Val Lys Gln Leu Tyr Ser Pro Val Lys Leu Ala Pro Asp
645 650 655
Gly His Gly Val Thr Ile Glu Asn Arg Asn Leu Phe Ala Gly Thr Asp
660 665 670
Gly Tyr Val Phe Ala Ala Arg Leu Leu Glu Asp Gly His Glu Ile Trp
675 680 685
His Ala Asp Tyr Arg Phe Asp Val Ala Ala Gly Asp Thr Gln His His
690 695 700
Asp Ile Ala Phe Pro Asp Ile Asp Ala Asp Gly Asp Thr Arg Glu Val
705 710 715 720
Thr Tyr Glu Val Asp Leu Leu Leu Ala Glu Ala Thr Ala Trp Ala Pro
725 730 735
Ala Gly Tyr Glu Leu Ala Phe Gly Gln Leu Thr Gly Thr Leu Asn Pro
740 745 750
Glu Gln Asp Ile Thr Glu Thr Ser His Asp Asp Asp Gly Arg Ala Thr
755 760 765
Arg Thr Leu Ser Arg Trp Asn Ala Gly Ile Arg Arg Asp Asp Lys Glu
770 775 780
Ile Leu Leu Ser Arg Thr Gln Gly Gly Ile Val Ser Trp Lys Arg Asp
785 790 795 800
Asp Arg Glu Met Val Ile Arg Arg Pro Glu Leu Val Thr Phe Arg Pro
805 810 815
Leu Thr Asp Asn Asp Arg Gly Asn His Ser Gly Phe Asp Arg Ala Ala
820 825 830
Trp Phe Ala Ala Gly Arg Tyr Ala Ile Val Thr Glu Thr Lys Ile His
835 840 845
Glu Ser Asp Asp Gly Leu Val Ala Glu Tyr Gln Tyr Glu Leu Ala Asp
850 855 860
Pro Asn His Thr Pro Val Ser Val Thr Tyr His Val Asn Ser Asp Met
865 870 875 880
Arg Met Gln Leu Thr Val Glu Tyr Pro Gly Asn Ala Thr Asp Met Ala
885 890 895
Ser Leu Pro Ala Phe Gly Ile Glu Trp Glu Leu Pro Gly Glu Tyr Asp
900 905 910
Arg Leu Arg Tyr Tyr Gly Pro Gly Pro Glu Glu Thr Tyr Arg Asp Arg
915 920 925
Lys Gln Gly Gly Lys Leu Gly Ile Trp Asp Ala Thr Ala Lys Ala Ser
930 935 940
Met Ala Pro Tyr Leu Met Val Gln Glu Thr Gly Ser His Glu Asp Val
945 950 955 960
Arg Trp Leu Glu Ala Thr Asp Ile Gln Gly His Gly Leu Arg Val Thr
965 970 975
Gln Arg Gly Asp Arg His Phe Thr Ala Ser Leu Leu Pro Trp Asn Thr
980 985 990
Tyr Met Ile Glu Ala Ala Arg Arg His Glu Asp Leu Pro Glu Pro Arg
995 1000 1005
His Asn Tyr Leu Arg Leu Leu Ala Ala Gln Met Gly Val Gly Gly
1010 1015 1020
Asp Asp Ser Trp Gly Ala Pro Val His Thr Ala Tyr Gln Leu Pro
1025 1030 1035
Ala Gly Arg Pro Leu Thr Leu Asp Val Asn Leu Glu Leu Ile
1040 1045 1050
<210> 9
<211> 1055
<212> PRT
<213> 短双歧杆菌(Bifidobacterium breve)
<400> 9
Met Thr Asn Ser Met Gln Gly Lys Ala Lys Thr Ile Met Thr Asn Leu
1 5 10 15
Gln Ser Ala Gln Gln Phe Ser Gln Ala Trp Leu Thr Asp Pro Arg Val
20 25 30
Phe Ala Val Asn Arg Leu Ala Ala His Ser Ser His Lys Phe Tyr Asp
35 40 45
His Ser Pro Gln Cys Gly Glu Ala Met Asp Leu Lys Gln Ser Leu Asp
50 55 60
Gly Gln Trp Arg Val Gln Met Leu Asp Leu Ala Asp Leu Ala Asp Asn
65 70 75 80
Glu Leu Ala Glu Ala Ala Phe Ala Gln Pro Gly Tyr Asp Ala Ala Gly
85 90 95
Phe Ser Pro Ile Glu Val Pro Ser Ala Leu Glu Thr Lys Gly Phe Leu
100 105 110
Asn His Gln Tyr Val Asn Gln Gln Tyr Pro Trp Ser Gly His Glu Ser
115 120 125
Pro Val Ala Pro Asp Val Pro Lys His Asn His Val Ala Leu Tyr Arg
130 135 140
His Glu Phe Ser Leu Glu Pro Lys Ala Ala Ala Val Leu Glu Ala Asn
145 150 155 160
Lys Thr Ala Ala Asp Asp Ala Ala Lys Arg Arg Val Thr Leu Thr Phe
165 170 175
Gln Gly Ala Ala Thr Ala Ile Val Val Trp Leu Asn Gly Ala Phe Ile
180 185 190
Gly Tyr Ala Glu Asp Ser Phe Thr Pro Ser Glu Phe Asp Val Thr Asp
195 200 205
Val Leu Arg Asp Gly Val Asn Thr Leu Ala Val Ala Cys Phe Glu Phe
210 215 220
Ser Ser Ala Ser Trp Leu Glu Asp Gln Asp Phe Trp Arg Leu His Gly
225 230 235 240
Ile Phe Arg Ser Val Glu Leu Glu Ala Gln Pro Leu Val His Val Asn
245 250 255
Asp Leu Arg Val Leu Ala Asp Tyr Asp His Thr Thr Gly Glu Gly Ser
260 265 270
Leu Asp Val Val Ala Leu Leu Arg Asn Ala Gly Thr Ala Ala Ala Val
275 280 285
Ala Ala Thr Val Leu Asp Ala Ala Gly Asn Thr Val Trp His Ser Lys
290 295 300
Leu Thr Ala Gly Ala Asp Ala Glu Thr Leu Thr Val Lys Ala Asn Val
305 310 315 320
Gly Lys Val Asn Pro Trp Ser Ala Glu Glu Pro Thr Leu Tyr Thr Leu
325 330 335
Gln Val Val Ala Thr Asp Ala Ala Gly Gln Val Ile Glu Ala Ala Leu
340 345 350
Gln Arg Ile Gly Phe Arg His Phe Ala Ile Glu Asp Gly Leu Met Lys
355 360 365
Leu Asn Gly Lys Arg Ile Val Phe Lys Gly Val Asp Arg His Glu Phe
370 375 380
Asp Ala Arg Thr Gly Arg Thr Ile Ala Glu Ala Asp Met Ile Glu Asp
385 390 395 400
Ile His Ser Phe Lys Arg Leu Asn Ile Asn Ala Val Arg Thr Ser His
405 410 415
Tyr Pro Asn Glu Thr Arg Trp Tyr Glu Leu Cys Asp Glu Tyr Gly Ile
420 425 430
Tyr Val Leu Asp Glu Thr Asn Leu Glu Thr His Gly Ser Trp Thr Asp
435 440 445
Pro Gly Asp Val Phe Gln Pro Ala Arg Ala Ile Pro Gly Ser Lys Asp
450 455 460
Glu Trp Arg Ala Ala Cys Val Asp Arg Thr Ala Ser Met Val Arg Arg
465 470 475 480
Asp Tyr Asn His Pro Ser Val Val Ile Trp Ser Leu Gly Asn Glu Ala
485 490 495
Phe Gly Gly Asp Val Phe Tyr Ser Met Arg Asp Phe Val His Glu Asn
500 505 510
Asp Pro Phe Arg Pro Val His Tyr Glu Gly Thr Phe Asn Asp Pro Glu
515 520 525
Phe Ser Ala Ala Thr Asp Ile Met Ser Arg Met Tyr Ala Lys Pro Asp
530 535 540
Glu Ile Val Lys Leu Tyr Leu Gly Glu Asp Gly Lys Lys Pro Tyr Ile
545 550 555 560
Ser Cys Glu Tyr Ser His Ser Met Gly Asn Ser Thr Gly Gly Leu His
565 570 575
Leu Tyr Thr Glu Leu Glu Arg Tyr Pro Leu Tyr Gln Gly Gly Phe Ile
580 585 590
Trp Asp Tyr Val Asp Gln Ala Leu Trp Gln Asp Cys Gly Asn Gly Thr
595 600 605
Glu Arg Leu Ala Tyr Gly Gly Asp Phe Glu Asp Arg Pro Asn Asp Tyr
610 615 620
Glu Phe Ser Gly Asp Gly Val Met Phe Ala Asp Arg Thr Pro Ser Pro
625 630 635 640
Lys Ala Gln Glu Val Lys Gln Leu Tyr Ala Asn Val Lys Leu Val Pro
645 650 655
Asp Glu Ser Gly Val Thr Ile Thr Asn Asp Asn Leu Phe Ile Ser Thr
660 665 670
Ala Ser Ser Leu Phe Thr Ala Arg Val Leu Val Asp Gly Ala Glu Arg
675 680 685
Trp His Ala Asn Tyr Arg Phe Asp Val Pro Ala Gly Glu Thr Val Arg
690 695 700
Glu Pro Ile Ala Phe Pro Lys Val Thr Asp Leu Val Ala Leu Ser Gly
705 710 715 720
Ser Ala Glu Val Thr Tyr Glu Val Asp Gln Arg Leu Ala Glu Ala Thr
725 730 735
Asp Trp Ala Pro Ala Gly Tyr Glu Leu Thr Phe Gly Gln Tyr Val Ala
740 745 750
Ala Val Ser Phe Asp Asp Gly Ala Ala Asp Ala Val Val Ala Gly Asp
755 760 765
Ala Glu Val Ala Ala Asp Gly Phe Asn Ala Gly Ile His Thr Asp Phe
770 775 780
Gly Glu Val Leu Leu Ser Lys Thr Gln Gly Gly Met Val Ser Phe Lys
785 790 795 800
Arg Asp Gly Arg Glu Met Val Ile Arg Arg Pro Asn Leu Thr Thr Phe
805 810 815
Arg Ala Leu Thr Asp Asn Asp Arg Gly Asn Gly Ser Gly Phe Glu Arg
820 825 830
Ala Gln Trp Met Ala Ala Gly Arg Tyr Ala Arg Val Thr Gly Thr Ser
835 840 845
Val Glu Glu Thr Ala Asp Gly Lys Gly Leu Lys Ala Thr Tyr Ser Tyr
850 855 860
Glu Leu Ala Asp Ala Lys His Thr Pro Val Thr Val His Tyr Glu Val
865 870 875 880
Asp Ala Ala Leu Arg Val His Leu Thr Val Glu Tyr Pro Gly Glu Ala
885 890 895
Asp Ala Ala Thr Leu Pro Ala Phe Gly Leu Glu Trp Ile Leu Pro Lys
900 905 910
Gln Tyr Asp Arg Leu Arg Phe Tyr Gly Leu Gly Pro Glu Glu Thr Tyr
915 920 925
Ala Asp Arg Leu His Gly Ala Lys Leu Gly Val Phe Ser Arg Thr Ala
930 935 940
Ala Glu Asp Cys Ala Pro Tyr Leu Leu Pro Gln Glu Thr Gly Asn His
945 950 955 960
Glu Gln Val Arg Trp Ala Glu Ile Thr Asp Glu Tyr Gly His Gly Met
965 970 975
Arg Val Thr Ala Ala Gly Gly Thr Arg Phe Ala Thr Ser Leu Leu Pro
980 985 990
Tyr Ser Ser Leu Met Phe Glu Asp Ala Leu His Gln Asn Glu Leu Pro
995 1000 1005
Lys Pro Arg His Thr Phe Leu Arg Leu Leu Ala Ala Gln Met Gly
1010 1015 1020
Val Gly Gly Asp Asp Thr Trp Gly Ala Pro Val His Asp Glu Phe
1025 1030 1035
Gln Val Pro Ala Asp Gln Pro Leu Lys Leu Asp Val Thr Leu Glu
1040 1045 1050
Leu Ile
1055
<210> 10
<211> 689
<212> PRT
<213> 链状双歧杆菌(Bifidobacterium catenulatum)
<400> 10
Met Thr Gln Arg Arg Ser Tyr Arg Trp Pro Gln Pro Leu Ala Gly Gln
1 5 10 15
Gln Ala Arg Ile Trp Tyr Gly Gly Asp Tyr Asn Pro Asp Gln Trp Pro
20 25 30
Glu Glu Val Trp Asp Asp Asp Val Arg Leu Met Lys Lys Ala Gly Val
35 40 45
Asn Leu Val Ser Val Gly Ile Phe Ser Trp Ala Lys Ile Glu Thr Ser
50 55 60
Glu Gly Val Tyr Asp Phe Asp Trp Leu Asp Arg Ile Ile Asp Lys Leu
65 70 75 80
Gly Glu Ala Gly Ile Ala Val Asp Leu Ala Ser Ala Thr Ala Ser Pro
85 90 95
Pro Met Trp Leu Thr Gln Ala His Pro Glu Val Leu Trp Lys Asp Tyr
100 105 110
Arg Gly Asp Val Cys Gln Pro Gly Ala Arg Gln His Trp Arg Pro Thr
115 120 125
Ser Pro Val Phe Arg Glu Tyr Ala Leu Lys Leu Cys Arg Ala Met Ala
130 135 140
Glu His Tyr Lys Gly Asn Pro Tyr Val Val Ala Trp His Val Ser Asn
145 150 155 160
Glu Tyr Gly Cys His Asn Arg Phe Asp Tyr Ser Glu Asp Ala Glu Arg
165 170 175
Ala Phe Arg Lys Trp Cys Glu Glu Arg Tyr Gly Thr Ile Asp Ala Val
180 185 190
Asn Asp Ala Trp Gly Thr Ala Phe Trp Ala Gln Arg Met Asn Asp Phe
195 200 205
Thr Glu Ile Val Pro Pro Arg Phe Ile Gly Asp Gly Asn Phe Met Asn
210 215 220
Pro Gly Lys Leu Leu Asp Phe Lys Arg Phe Ser Ser Asp Ala Leu Lys
225 230 235 240
Ala Phe Tyr Val Ala Glu Arg Asp Ala Leu Ala Glu Ile Thr Pro Asp
245 250 255
Leu Pro Leu Thr Thr Asn Phe Met Val Ser Ala Ala Gly Ser Val Leu
260 265 270
Asp Tyr Asp Asp Trp Gly Arg Glu Val Asp Phe Val Ser Asn Asp His
275 280 285
Tyr Phe Ile Pro Gly Glu Ala His Leu Asp Glu Leu Ala Phe Ser Ala
290 295 300
Ser Leu Val Asp Gly Ile Ala Arg Lys Asp Pro Trp Phe Leu Met Glu
305 310 315 320
His Ser Thr Ser Ala Val Asn Trp Arg Pro Val Asn Tyr Arg Lys Glu
325 330 335
Pro Gly Gln Leu Val Arg Asp Ser Leu Ala His Val Ala Met Gly Ala
340 345 350
Asp Ala Val Cys Tyr Phe Gln Trp Arg Gln Ser Lys Ala Gly Ala Glu
355 360 365
Lys Phe His Ser Ala Met Val Pro His Thr Gly Glu Asp Ser Ala Val
370 375 380
Phe Arg Asp Val Cys Glu Leu Gly Ala Asp Leu Asn Thr Leu Ala Asp
385 390 395 400
Asn Gly Leu Leu Gly Thr Lys Leu Ala Lys Ser Lys Val Ala Val Val
405 410 415
Phe Asp Tyr Glu Ser Glu Trp Ala Thr Glu His Thr Ala Thr Pro Thr
420 425 430
Gln Lys Val His His Val Asp Glu Pro Leu Gln Trp Phe Arg Ala Leu
435 440 445
Ala Asp His Gly Val Thr Ala Asp Val Val Pro Val Ser Ser Asn Trp
450 455 460
Asp Glu Tyr Glu Val Val Val Leu Pro Ser Val Tyr Ile Leu Ser Glu
465 470 475 480
Glu Thr Thr Arg Arg Val Arg Asp Tyr Val Val Asn Gly Gly Arg Leu
485 490 495
Ile Val Thr Tyr Tyr Thr Gly Leu Ser Asp Glu Lys Asp His Val Trp
500 505 510
Leu Gly Gly Tyr Pro Gly Ser Ile Arg Asp Val Val Gly Val Arg Val
515 520 525
Glu Glu Phe Met Pro Met Gly Asp Asp Phe Pro Gly Val Pro Asp Cys
530 535 540
Leu Gly Leu Ser Asn Gly Ala Val Ala His Asp Ile Ala Asp Val Ile
545 550 555 560
Gly Ser Val Asp Gly Thr Ala Thr Val Leu Glu Thr Phe Arg Asp Asp
565 570 575
Pro Trp Thr Gly Met Asp Gly Ala Pro Ala Ile Val Ala Asn Thr Phe
580 585 590
Gly Glu Gly Arg Ser Val Tyr Val Gly Ala Arg Leu Gly Arg Asp Gly
595 600 605
Ile Ala Lys Ser Leu Pro Glu Ile Phe Glu Ser Leu Gly Met Ala Glu
610 615 620
Thr Gly Glu Asn Asp Ser Arg Val Leu Arg Val Glu Arg Glu Gly Ser
625 630 635 640
Asp Gly Ser Arg Phe Val Phe Ser Phe Asn Arg Thr His Glu Ala Val
645 650 655
Gln Ile Pro Phe Glu Gly Lys Ile Val Val Ser Ser Phe Ala Glu Val
660 665 670
Ser Gly Glu Asn Val Ser Ile Lys Pro Asn Gly Val Ile Val Thr Lys
675 680 685
Gln
<210> 11
<211> 1023
<212> PRT
<213> 链状双歧杆菌
<400> 11
Met Ala Asn Ser Asn Arg Val Glu His Ala Ser Glu Thr Trp Leu Thr
1 5 10 15
Asp Ala Thr Val Phe Glu Val Asn Arg Thr Pro Ala His Ser Asn His
20 25 30
Lys Cys Phe Thr His Asp Pro Gln Ser Gly Glu His Ser Asp Leu Thr
35 40 45
Gln Ser Leu Asp Gly Glu Trp Arg Val Glu Ile Val Gln Ala Ser Asp
50 55 60
Ile Asp Phe Asn Glu Glu Pro Phe Val Ala Glu Asn Phe Asp Asp Ser
65 70 75 80
Ser Phe Cys Arg Ala Gln Val Pro Gly His Leu Gln Met Ala Gly Leu
85 90 95
Leu Lys Asn Lys Tyr Val Asn Ile Gln Tyr Pro Trp Asp Gly His Glu
100 105 110
Asn Pro Leu Glu Pro Asn Val Pro Glu Asn Asn His Val Ala Leu Tyr
115 120 125
Arg Arg Lys Phe Val Val Ser Lys Arg Leu Ala Asp Thr Lys Glu Ser
130 135 140
Glu Gly Ser Val Ser Ile Val Phe His Gly Met Ala Thr Ala Ile Tyr
145 150 155 160
Val Trp Val Asn Gly Leu Phe Ala Gly Tyr Gly Glu Asp Gly Phe Thr
165 170 175
Pro Asn Glu Phe Asp Ile Thr Asp Leu Leu His Asp Gly Glu Asn Val
180 185 190
Val Ala Val Ala Cys Tyr Glu Tyr Ser Ser Ala Ser Trp Leu Glu Asp
195 200 205
Gln Asp Phe Trp Arg Leu His Gly Leu Phe Arg Ser Val Glu Leu Thr
210 215 220
Ala Gln Pro His Val His Val Glu Asn Met Gln Leu Glu Ala Asp Trp
225 230 235 240
Asp Ala Glu Ser Gly Thr Ala Ser Leu Asp Ala Ala Leu Ser Val Arg
245 250 255
Asn Ala Ser Asp Ala Ala Thr Ile Ser Ala Thr Leu Lys Asp Ser Glu
260 265 270
Gly Asn Val Val Trp Glu Ala Ser Thr Asn Ala Asp Ala Asn Thr Thr
275 280 285
Phe Ala Ser Gly Ser Leu Gln Gly Leu Glu Pro Trp Ser Ala Glu Ser
290 295 300
Pro Ser Leu Tyr Glu Leu Glu Val Asn Val Ile Asp Gln Ala Gly Asn
305 310 315 320
Ile Val Glu Ala Ala Val Gln Lys Val Gly Phe Arg Arg Phe Arg Ile
325 330 335
Glu Asn Gly Ile Met Thr Leu Asn Gly Lys Arg Ile Val Phe Lys Gly
340 345 350
Ala Asp Arg His Glu Phe Asp Ala Lys Arg Gly Arg Ser Ile Thr Glu
355 360 365
Gln Asp Met Ile Asp Asp Val Ile Phe Cys Lys Arg His Asn Ile Asn
370 375 380
Ala Ile Arg Thr Ser His Tyr Pro Asn Gln Glu Arg Trp Tyr Asp Leu
385 390 395 400
Cys Asp Glu Tyr Gly Ile Tyr Leu Ile Asp Glu Thr Asn Leu Glu Thr
405 410 415
His Gly Ser Trp Cys Leu Pro Gly Asp Val Val Thr Ala Glu Thr Ala
420 425 430
Val Pro Gly Ser Lys Ala His Trp Glu Gly Ala Cys Val Asp Arg Val
435 440 445
Asn Ser Met Val Arg Arg Asp Tyr Asn His Pro Ser Val Val Ile Trp
450 455 460
Ser Leu Gly Asn Glu Ser Tyr Thr Gly Asp Val Phe Arg Ala Met Tyr
465 470 475 480
Lys His Val His Asp Ile Asp Pro Asn Arg Pro Val His Tyr Glu Gly
485 490 495
Val Thr Lys Asn Arg Asp Tyr Asp Asp Val Thr Asp Ile Glu Thr Arg
500 505 510
Met Tyr Glu His Ala Asp Val Val Glu Glu Tyr Leu Lys Asn Asp Pro
515 520 525
Gln Lys Pro Tyr Ile Ser Cys Glu Tyr Met His Ala Met Gly Asn Ser
530 535 540
Val Gly Asn Leu Asp Glu Tyr Thr Ala Leu Glu Arg Tyr Pro His Tyr
545 550 555 560
Gln Gly Gly Phe Ile Trp Asp Phe Ile Asp Gln Ala Ile Tyr Ala Thr
565 570 575
Gln Pro Asp Gly Ser Thr Arg Leu Cys Tyr Gly Gly Asp Phe Gly Asp
580 585 590
Arg Pro Ser Asp Tyr Glu Phe Ser Gly Asn Gly Leu Val Phe Ala Asp
595 600 605
Arg Thr Pro Thr Pro Lys Ala Gln Glu Val Lys Gln Leu Tyr Ser Asn
610 615 620
Val His Ile Asp Val Thr Asp Arg Ser Val Ser Ile Lys Asn Asp Asn
625 630 635 640
Leu Phe Ile Ser Thr Gly Gly Tyr Gln Phe Val Leu Arg Ile Leu Ala
645 650 655
Asp Gly Glu Pro Val Trp Gln Ser Glu Arg Arg Phe Asp Val Pro Ala
660 665 670
Asp Ser Ala Cys Thr Phe Asp Val Glu Trp Pro Val Asp Leu Tyr Arg
675 680 685
Ala Asn Ala Asp Glu Leu Val Leu Glu Val Ser Gln Arg Leu Ala Glu
690 695 700
Ala Thr Asp Trp Ala Pro Ala Gly Tyr Glu Leu Ala Phe Gly Gln Thr
705 710 715 720
Ile Val Ala Gly Thr Lys Ala Ala Glu Asp Ala Ala Leu Pro Ala Asp
725 730 735
Gly Ile Val Thr Val Gly Arg Trp Asn Ala Gly Val Gln Gly Ser Gly
740 745 750
Arg Glu Ile Leu Leu Ser Arg Thr Gln Gly Gly Leu Val Ser Tyr Thr
755 760 765
Phe Asp Gly His Glu Phe Val Leu Arg Arg Pro Ala Ile Thr Thr Phe
770 775 780
Arg Ala Leu Thr Asp Asn Asp Arg Gly Ala Gly His Gly Phe Glu Arg
785 790 795 800
Ala Gln Trp Met Val Ala Gly Arg Tyr Ala Arg Cys Val Asp Asn Val
805 810 815
Ile Glu Gln Val Asp Glu Asp Thr Leu Lys Ala Val Tyr Thr Tyr Glu
820 825 830
Leu Ala Thr Pro Gln Cys Thr Lys Val Thr Val Gly Tyr Thr Ala Asp
835 840 845
Thr Thr Gly Arg Leu Asn Leu His Val Glu Tyr Pro Gly Glu Ser Gly
850 855 860
Glu Leu Pro Thr Ile Pro Ala Phe Gly Ile Glu Trp Thr Leu Pro Val
865 870 875 880
Gln Tyr Ser Asn Leu Arg Phe Phe Gly Ala Gly Pro Glu Glu Thr Tyr
885 890 895
Gln Asp Arg Lys His Ala Lys Leu Gly Val Trp Ser Thr Asp Ala Phe
900 905 910
Lys Asp His Ala Pro Tyr Leu Met Pro Gln Glu Thr Gly Asn His Glu
915 920 925
Glu Val Arg Trp Ala Glu Ile Thr Asp Glu Asn Gly His Gly Leu Arg
930 935 940
Val Ser Arg Ala Asn Gly Ala Ala Pro Phe Ala Val Ser Leu Gln Pro
945 950 955 960
Tyr Ser Ser Phe Met Ile Glu Glu Ala Gln His Gln Asp Glu Leu Pro
965 970 975
Ala Pro Lys His Met Phe Leu Arg Val Leu Ala Ala Gln Met Gly Val
980 985 990
Gly Gly Asp Asp Ser Trp Met Ser Pro Val His Ser Gln Tyr His Ile
995 1000 1005
Thr Ala Asp Gln Pro Ile Ser Leu Asp Val Asn Leu Glu Leu Ile
1010 1015 1020
<210> 12
<211> 1008
<212> PRT
<213> 德氏乳杆菌保加利亚亚种(Lactobacillus delbrueckii subsp. bulgaricus)
<400> 12
Met Ser Asn Lys Leu Val Lys Glu Lys Arg Val Asp Gln Ala Asp Leu
1 5 10 15
Ala Trp Leu Thr Asp Pro Glu Val Tyr Glu Val Asn Thr Ile Pro Pro
20 25 30
His Ser Asp His Glu Ser Phe Gln Ser Gln Glu Glu Leu Glu Glu Gly
35 40 45
Lys Ser Ser Leu Val Gln Ser Leu Asp Gly Asp Trp Leu Ile Asp Tyr
50 55 60
Ala Glu Asn Gly Gln Gly Pro Val Asn Phe Tyr Ala Glu Asp Phe Asp
65 70 75 80
Asp Ser Asn Phe Lys Ser Val Lys Val Pro Gly Asn Leu Glu Leu Gln
85 90 95
Gly Phe Gly Gln Pro Gln Tyr Val Asn Val Gln Tyr Pro Trp Asp Gly
100 105 110
Ser Glu Glu Ile Phe Pro Pro Gln Ile Pro Ser Lys Asn Pro Leu Ala
115 120 125
Ser Tyr Val Arg Tyr Phe Asp Leu Asp Glu Ala Phe Trp Asp Lys Glu
130 135 140
Val Ser Leu Lys Phe Asp Gly Ala Ala Thr Ala Ile Tyr Val Trp Leu
145 150 155 160
Asn Gly His Phe Val Gly Tyr Gly Glu Asp Ser Phe Thr Pro Ser Glu
165 170 175
Phe Met Val Thr Lys Phe Leu Lys Lys Glu Asn Asn Arg Leu Ala Val
180 185 190
Ala Leu Tyr Lys Tyr Ser Ser Ala Ser Trp Leu Glu Asp Gln Asp Phe
195 200 205
Trp Arg Met Ser Gly Leu Phe Arg Ser Val Thr Leu Gln Ala Lys Pro
210 215 220
Arg Leu His Leu Glu Asp Leu Lys Leu Thr Ala Ser Leu Thr Asp Asn
225 230 235 240
Tyr Gln Lys Gly Lys Leu Glu Val Glu Ala Asn Ile Ala Tyr Arg Leu
245 250 255
Pro Asn Ala Ser Phe Lys Leu Glu Val Arg Asp Ser Glu Gly Asp Leu
260 265 270
Val Ala Glu Lys Leu Gly Pro Ile Arg Ser Glu Gln Leu Glu Phe Thr
275 280 285
Leu Ala Asp Leu Pro Val Ala Ala Trp Ser Ala Glu Lys Pro Asn Leu
290 295 300
Tyr Gln Val Arg Leu Tyr Leu Tyr Gln Ala Gly Ser Leu Leu Glu Val
305 310 315 320
Ser Arg Gln Glu Val Gly Phe Arg Asn Phe Glu Leu Lys Asp Gly Ile
325 330 335
Met Tyr Leu Asn Gly Gln Arg Ile Val Phe Lys Gly Ala Asn Arg His
340 345 350
Glu Phe Asp Ser Lys Leu Gly Arg Ala Ile Thr Glu Glu Asp Met Ile
355 360 365
Trp Asp Ile Lys Thr Met Lys Arg Ser Asn Ile Asn Ala Val Arg Cys
370 375 380
Ser His Tyr Pro Asn Gln Ser Leu Phe Tyr Arg Leu Cys Asp Lys Tyr
385 390 395 400
Gly Leu Tyr Val Ile Asp Glu Ala Asn Leu Glu Ser His Gly Thr Trp
405 410 415
Glu Lys Val Gly Gly His Glu Asp Pro Ser Phe Asn Val Pro Gly Asp
420 425 430
Asp Gln His Trp Leu Gly Ala Ser Leu Ser Arg Val Lys Asn Met Met
435 440 445
Ala Arg Asp Lys Asn His Ala Ser Ile Leu Ile Trp Ser Leu Gly Asn
450 455 460
Glu Ser Tyr Ala Gly Thr Val Phe Ala Gln Met Ala Asp Tyr Val Arg
465 470 475 480
Lys Ala Asp Pro Thr Arg Val Gln His Tyr Glu Gly Val Thr His Asn
485 490 495
Arg Lys Phe Asp Asp Ala Thr Gln Ile Glu Ser Arg Met Tyr Ala Pro
500 505 510
Ala Lys Val Ile Glu Glu Tyr Leu Thr Asn Lys Pro Ala Lys Pro Phe
515 520 525
Ile Ser Val Glu Tyr Ala His Ala Met Gly Asn Ser Val Gly Asp Leu
530 535 540
Ala Ala Tyr Thr Ala Leu Glu Lys Tyr Pro His Tyr Gln Gly Gly Phe
545 550 555 560
Ile Trp Asp Trp Ile Asp Gln Gly Leu Glu Lys Asp Gly His Leu Leu
565 570 575
Tyr Gly Gly Asp Phe Asp Asp Arg Pro Thr Asp Tyr Glu Phe Cys Gly
580 585 590
Asn Gly Leu Val Phe Ala Asp Arg Thr Glu Ser Pro Lys Leu Ala Asn
595 600 605
Val Lys Ala Leu Tyr Ala Asn Leu Lys Leu Glu Val Lys Asp Gly Gln
610 615 620
Leu Phe Leu Lys Asn Asp Asn Leu Phe Thr Asn Ser Ser Ser Tyr Tyr
625 630 635 640
Phe Leu Thr Ser Leu Leu Val Asp Gly Lys Leu Thr Tyr Gln Ser Arg
645 650 655
Pro Leu Thr Phe Gly Leu Glu Pro Gly Glu Ser Gly Thr Phe Ala Leu
660 665 670
Pro Trp Pro Glu Val Ala Asp Glu Lys Gly Glu Val Val Tyr Arg Val
675 680 685
Thr Ala His Leu Lys Glu Asp Leu Pro Trp Ala Asp Glu Gly Phe Thr
690 695 700
Val Ala Glu Ala Glu Glu Val Ala Gln Lys Leu Pro Glu Phe Lys Pro
705 710 715 720
Glu Gly Arg Pro Asp Leu Val Asp Ser Asp Tyr Asn Leu Gly Leu Lys
725 730 735
Gly Asn Asn Phe Gln Ile Leu Phe Ser Lys Val Lys Gly Trp Pro Val
740 745 750
Ser Leu Lys Tyr Ala Gly Arg Glu Tyr Leu Lys Arg Leu Pro Glu Phe
755 760 765
Thr Phe Trp Arg Ala Leu Thr Asp Asn Asp Arg Gly Ala Gly Tyr Gly
770 775 780
Tyr Asp Leu Ala Arg Trp Glu Asn Ala Gly Lys Tyr Ala Arg Leu Lys
785 790 795 800
Asp Ile Ser Cys Glu Val Lys Glu Asp Ser Val Leu Val Lys Thr Ala
805 810 815
Phe Thr Leu Pro Val Ala Leu Lys Gly Asp Leu Thr Val Thr Tyr Glu
820 825 830
Val Asp Gly Arg Gly Lys Ile Ala Val Thr Ala Asp Phe Pro Gly Ala
835 840 845
Glu Glu Ala Gly Leu Leu Pro Ala Phe Gly Leu Asn Leu Ala Leu Pro
850 855 860
Lys Glu Leu Thr Asp Tyr Arg Tyr Tyr Gly Leu Gly Pro Asn Glu Ser
865 870 875 880
Tyr Pro Asp Arg Leu Glu Gly Asn Tyr Leu Gly Ile Tyr Gln Gly Ala
885 890 895
Val Lys Lys Asn Phe Ser Pro Tyr Leu Arg Pro Gln Glu Thr Gly Asn
900 905 910
Arg Ser Lys Val Arg Trp Tyr Gln Leu Phe Asp Glu Lys Gly Gly Leu
915 920 925
Glu Phe Thr Ala Asn Gly Ala Asp Leu Asn Leu Ser Ala Leu Pro Tyr
930 935 940
Ser Ala Ala Gln Ile Glu Ala Ala Asp His Ala Phe Asp Leu Thr Asn
945 950 955 960
Asn Tyr Thr Trp Val Arg Ala Leu Ser Ala Gln Met Gly Val Gly Gly
965 970 975
Asp Asp Ser Trp Gly Gln Lys Val His Pro Glu Phe Cys Leu Asp Ala
980 985 990
Gln Lys Ala Arg Gln Leu Arg Leu Val Ile Gln Pro Leu Leu Leu Lys
995 1000 1005
<210> 13
<211> 1007
<212> PRT
<213> 德氏乳杆菌乳酸亚种(Lactobacillus delbrueckii subsp. lactis)
<400> 13
Met Ser Asn Lys Leu Val Lys Glu Lys Arg Val Asp Gln Ala Asp Leu
1 5 10 15
Ala Trp Leu Thr Asp Pro Glu Val Tyr Glu Val Asn Thr Ile Pro Pro
20 25 30
His Ser Asp His Glu Ser Phe Gln Ser Gln Glu Glu Leu Glu Glu Gly
35 40 45
Lys Ser Ser Leu Val Gln Ser Leu Asp Gly Asn Trp Leu Ile Asp Tyr
50 55 60
Ala Glu Asn Gly Gln Gly Pro Ile Asn Phe Tyr Ala Glu Asp Phe Asp
65 70 75 80
Asp Ser Asn Phe Lys Ser Val Lys Val Pro Gly Asn Leu Glu Leu Gln
85 90 95
Gly Phe Gly Gln Pro Gln Tyr Val Asn Ile Gln Tyr Pro Trp Asp Gly
100 105 110
Ser Glu Glu Ile Phe Pro Pro Gln Val Pro Ser Lys Asn Pro Leu Ala
115 120 125
Ser Tyr Val Arg Tyr Phe Asp Leu Asp Glu Ala Leu Trp Asp Lys Glu
130 135 140
Val Ser Leu Lys Phe Ala Gly Ala Ala Thr Ala Ile Tyr Val Trp Leu
145 150 155 160
Asn Gly His Phe Val Gly Tyr Gly Glu Asp Ser Phe Thr Pro Ser Glu
165 170 175
Phe Met Val Thr Lys Phe Leu Lys Lys Glu Gly Asn Arg Leu Ala Val
180 185 190
Ala Leu Tyr Lys Tyr Ser Ser Ala Ser Trp Leu Glu Asp Gln Asp Phe
195 200 205
Trp Arg Leu Ser Gly Leu Phe Arg Ser Val Thr Leu Glu Ala Lys Pro
210 215 220
Leu Leu His Leu Glu Asp Leu Lys Leu Thr Ala Ser Leu Thr Asp Asn
225 230 235 240
Tyr Gln Lys Gly Lys Leu Glu Val Glu Ala Asn Ile Ala Tyr Arg Leu
245 250 255
Pro Asn Ala Ser Phe Lys Leu Glu Val Arg Asp Ser Glu Gly Asp Leu
260 265 270
Val Ala Glu Lys Val Gly Pro Ile Arg Ser Glu Lys Leu Gly Phe Ser
275 280 285
Leu Ala Asp Leu Pro Val Ala Ala Trp Ser Ala Glu Lys Pro Asn Leu
290 295 300
Tyr Gln Val Arg Leu Tyr Leu Tyr Gln Ala Gly Ser Leu Leu Glu Val
305 310 315 320
Ser Arg Gln Glu Val Gly Phe Arg Asn Phe Glu Leu Lys Asp Gly Ile
325 330 335
Met Tyr Leu Asn Gly Gln Arg Ile Val Phe Lys Gly Val Asn Arg His
340 345 350
Glu Phe Asp Ser Lys Leu Gly Arg Ala Ile Thr Glu Ala Asp Met Ile
355 360 365
Trp Asp Ile Lys Thr Met Lys Gln Ser Asn Ile Asn Ala Val Arg Cys
370 375 380
Ser His Tyr Pro Asn Gln Ser Leu Phe Tyr Arg Leu Cys Asp Lys Tyr
385 390 395 400
Gly Leu Tyr Val Ile Asp Glu Ala Asn Leu Glu Ser His Gly Thr Trp
405 410 415
Glu Lys Val Gly His Glu Asp Pro Ser Phe Asn Val Pro Gly Asp Asp
420 425 430
Gln His Trp Leu Gly Ala Ser Leu Ser Arg Val Lys Asn Met Met Ala
435 440 445
Arg Asp Lys Asn His Ala Ser Ile Leu Ile Trp Ser Leu Gly Asn Glu
450 455 460
Ser Tyr Ala Gly Thr Val Phe Ala Gln Met Ala Asp Tyr Val Arg Lys
465 470 475 480
Ala Asp Pro Thr Arg Val Gln His Tyr Glu Gly Val Thr His Asn Arg
485 490 495
Lys Phe Asp Asp Ala Thr Gln Ile Glu Ser Arg Met Tyr Ala Pro Ala
500 505 510
Lys Glu Ile Glu Glu Tyr Leu Thr Lys Lys Pro Ala Lys Pro Phe Ile
515 520 525
Ser Val Glu Tyr Ala His Ala Met Gly Asn Ser Val Gly Asp Leu Ala
530 535 540
Ala Tyr Thr Ala Leu Glu Lys Tyr Pro His Tyr Gln Gly Gly Phe Ile
545 550 555 560
Trp Asp Trp Ile Asp Gln Gly Leu Glu Lys Asp Gly His Leu Leu Tyr
565 570 575
Gly Gly Asp Phe Asp Asp Arg Pro Thr Asp Tyr Glu Phe Cys Gly Asp
580 585 590
Gly Leu Val Phe Ala Asp Arg Thr Thr Ser Pro Lys Leu Ala Asn Val
595 600 605
Lys Ala Leu Tyr Ser Asn Leu Lys Leu Glu Val Lys Asp Gly Gln Leu
610 615 620
Phe Ile Lys Asn Asp Asn Leu Phe Thr Asn Ser Ser Ala Tyr Tyr Phe
625 630 635 640
Leu Ala Ser Leu Leu Val Asp Gly Lys Leu Thr Tyr Gln Ser Gln Pro
645 650 655
Leu Thr Phe Gly Leu Glu Pro Gly Glu Ser Gly Thr Phe Val Leu Pro
660 665 670
Trp Pro Glu Val Glu Asp Glu Lys Gly Glu Ile Val Tyr Gln Val Thr
675 680 685
Ala His Leu Lys Glu Asp Leu Pro Trp Ala Asp Glu Gly Phe Thr Val
690 695 700
Ala Glu Ala Glu Glu Ala Val Thr Lys Leu Pro Glu Phe Tyr Pro Ala
705 710 715 720
Gly Arg Pro Glu Leu Val Asp Ser Asp Phe Asn Leu Gly Leu Lys Gly
725 730 735
Asn Gly Phe Arg Ile Leu Phe Ser Lys Ala Lys Gly Trp Pro Val Ser
740 745 750
Ile Lys Tyr Ala Gly Arg Glu Tyr Leu Lys Arg Leu Pro Glu Phe Thr
755 760 765
Phe Trp Arg Ala Leu Thr Asp Asn Asp Arg Gly Ala Gly Tyr Gly Tyr
770 775 780
Asp Leu Ala Lys Trp Glu Asn Ala Gly Lys Tyr Ala Arg Leu Gln Asp
785 790 795 800
Ile Ser Tyr Glu Ile Lys Glu Asn Ser Ala Leu Val Lys Thr Thr Phe
805 810 815
Thr Leu Pro Val Ala Leu Lys Gly Asp Leu Thr Ile Thr Tyr Glu Val
820 825 830
Asp Ser Leu Gly Lys Ile Ala Val Thr Ala Asn Phe Pro Gly Ala Val
835 840 845
Glu Asn Gly Leu Leu Pro Ala Phe Gly Leu Asn Phe Ala Leu Pro Lys
850 855 860
Glu Leu Ser Asp Tyr Arg Tyr Tyr Gly Leu Gly Pro Asn Glu Ser Tyr
865 870 875 880
Ala Asp Arg Leu Glu Gly Ser Tyr Leu Gly Ile Tyr Gln Gly Ala Val
885 890 895
Glu Lys Asn Phe Thr Pro Tyr Leu Arg Pro Gln Glu Ala Gly Asn Arg
900 905 910
Ser Lys Val Arg Tyr Tyr Gln Leu Phe Asp Glu Glu Gly Gly Leu Glu
915 920 925
Phe Thr Ala Asn Gly Ala Asp Leu Asn Leu Ser Ala Leu Pro Tyr Ser
930 935 940
Ala Ala Gln Ile Glu Ala Ala Asp His Ala Phe Glu Leu Thr Asn Asn
945 950 955 960
Tyr Thr Trp Val Arg Ala Leu Ala Ala Gln Met Gly Val Gly Gly Asp
965 970 975
Asp Ser Trp Gly Gln Lys Val His Pro Glu Phe Cys Leu Asp Ala Gln
980 985 990
Glu Ala Arg Gln Leu Lys Leu Val Ile Gln Pro Leu Leu Leu Lys
995 1000 1005
<210> 14
<211> 1008
<212> PRT
<213> 德氏乳杆菌保加利亚亚种
<400> 14
Met Ser Asn Lys Leu Val Lys Glu Lys Arg Val Asp Gln Ala Asp Leu
1 5 10 15
Ala Trp Leu Thr Asp Pro Glu Val Tyr Glu Val Asn Thr Ile Pro Pro
20 25 30
His Phe Asp His Glu Ser Phe Gln Ser Gln Glu Glu Leu Glu Glu Gly
35 40 45
Lys Ser Ser Leu Val Gln Ser Leu Asp Gly Asp Trp Leu Ile Asp Tyr
50 55 60
Ala Glu Asn Gly Gln Gly Pro Val Asn Phe Tyr Ala Glu Asp Phe Asp
65 70 75 80
Asp Ser Asn Phe Lys Ser Val Lys Val Pro Gly Asn Leu Glu Leu Gln
85 90 95
Gly Phe Gly Gln Pro Gln Tyr Val Asn Val Gln Tyr Pro Trp Asp Gly
100 105 110
Ser Glu Glu Ile Phe Pro Pro Gln Ile Pro Ser Lys Asn Pro Leu Ala
115 120 125
Ser Tyr Val Arg Tyr Phe Asp Leu Asp Glu Ala Phe Trp Asp Lys Glu
130 135 140
Val Ser Leu Lys Phe Asp Gly Ala Ala Thr Ala Ile Tyr Val Trp Leu
145 150 155 160
Asn Gly His Phe Val Gly Tyr Gly Glu Asp Ser Phe Thr Pro Ser Glu
165 170 175
Phe Met Val Thr Lys Phe Leu Lys Lys Glu Asn Asn Arg Leu Ala Val
180 185 190
Ala Leu Tyr Lys Tyr Ser Ser Ala Ser Trp Leu Glu Asp Gln Asp Phe
195 200 205
Trp Arg Met Ser Gly Leu Phe Arg Ser Val Thr Leu Gln Ala Lys Pro
210 215 220
Arg Leu His Leu Glu Asp Leu Lys Leu Thr Ala Ser Leu Thr Asp Asn
225 230 235 240
Tyr Gln Lys Gly Lys Leu Glu Val Glu Ala Asn Ile Ala Tyr Arg Leu
245 250 255
Pro Asn Ala Ser Phe Lys Leu Glu Val Arg Asp Ser Glu Gly Asp Leu
260 265 270
Val Ala Glu Lys Leu Gly Pro Ile Arg Ser Glu Gln Leu Glu Phe Thr
275 280 285
Leu Ala Asp Leu Pro Val Ala Ala Trp Ser Ala Glu Lys Pro Asn Leu
290 295 300
Tyr Gln Val Arg Leu Tyr Leu Tyr Gln Ala Gly Ser Leu Leu Glu Val
305 310 315 320
Ser Arg Gln Glu Val Gly Phe Arg Asn Phe Glu Leu Lys Asp Gly Ile
325 330 335
Met Tyr Leu Asn Gly Gln Arg Ile Val Phe Lys Gly Ala Asn Arg His
340 345 350
Glu Phe Asp Ser Lys Leu Gly Arg Ala Ile Thr Glu Glu Asp Met Ile
355 360 365
Trp Asp Ile Lys Thr Met Lys Arg Ser Asn Ile Asn Ala Val Arg Cys
370 375 380
Ser His Tyr Pro Asn Gln Ser Leu Phe Tyr Arg Leu Cys Asp Lys Tyr
385 390 395 400
Gly Leu Tyr Val Ile Asp Glu Ala Asn Leu Glu Ser His Gly Thr Trp
405 410 415
Glu Lys Val Gly Gly His Glu Asp Pro Ser Phe Asn Val Pro Gly Asp
420 425 430
Asp Gln His Trp Leu Gly Ala Ser Leu Ser Arg Val Lys Asn Met Met
435 440 445
Ala Arg Asp Lys Asn His Ala Ser Ile Leu Ile Trp Ser Leu Gly Asn
450 455 460
Glu Ser Tyr Ala Gly Thr Val Phe Ala Gln Met Ala Asp Tyr Val Arg
465 470 475 480
Lys Ala Asp Pro Thr Arg Val Gln His Tyr Glu Gly Val Thr His Asn
485 490 495
Arg Lys Phe Asp Asp Ala Thr Gln Ile Glu Ser Arg Met Tyr Ala Pro
500 505 510
Ala Lys Val Ile Glu Glu Tyr Leu Thr Asn Lys Pro Ala Lys Pro Phe
515 520 525
Ile Ser Val Glu Tyr Ala His Ala Met Gly Asn Ser Val Gly Asp Leu
530 535 540
Ala Ala Tyr Thr Ala Leu Glu Lys Tyr Pro His Tyr Gln Gly Gly Phe
545 550 555 560
Ile Trp Asp Trp Ile Asp Gln Gly Leu Glu Lys Asp Gly His Leu Leu
565 570 575
Tyr Gly Gly Asp Phe Asp Asp Arg Pro Thr Asp Tyr Glu Phe Cys Gly
580 585 590
Asn Gly Leu Val Phe Ala Asp Arg Thr Glu Ser Pro Lys Leu Ala Asn
595 600 605
Val Lys Ala Leu Tyr Ala Asn Leu Lys Leu Glu Val Lys Asp Gly Gln
610 615 620
Leu Phe Leu Lys Asn Asp Asn Leu Phe Thr Asn Ser Ser Ser Tyr Tyr
625 630 635 640
Phe Leu Thr Ser Leu Leu Val Asp Gly Lys Leu Thr Tyr Gln Ser Arg
645 650 655
Pro Leu Thr Phe Gly Leu Glu Pro Gly Glu Ser Gly Thr Phe Ala Leu
660 665 670
Pro Trp Pro Glu Val Ala Asp Glu Lys Gly Glu Val Val Tyr Arg Val
675 680 685
Thr Ala His Leu Lys Glu Asp Leu Pro Trp Ala Asp Glu Gly Phe Thr
690 695 700
Val Ala Glu Ala Glu Glu Val Ala Gln Lys Leu Pro Glu Phe Lys Pro
705 710 715 720
Glu Gly Arg Pro Asp Leu Val Asp Ser Asp Tyr Asn Leu Gly Leu Lys
725 730 735
Gly Asn Asn Phe Gln Ile Leu Phe Ser Lys Val Lys Gly Trp Pro Val
740 745 750
Ser Leu Lys Tyr Ala Gly Arg Glu Tyr Leu Lys Arg Leu Pro Glu Phe
755 760 765
Thr Phe Trp Arg Ala Leu Thr Asp Asn Asp Arg Gly Ala Gly Tyr Gly
770 775 780
Tyr Asp Leu Ala Arg Trp Glu Asn Ala Gly Lys Tyr Ala Arg Leu Lys
785 790 795 800
Asp Ile Ser Cys Glu Val Lys Glu Asp Ser Val Leu Val Lys Thr Ala
805 810 815
Phe Thr Leu Pro Val Ala Leu Lys Gly Asp Leu Thr Val Thr Tyr Glu
820 825 830
Val Asp Gly Arg Gly Lys Ile Ala Val Thr Ala Asp Phe Pro Gly Ala
835 840 845
Glu Glu Ala Gly Leu Leu Pro Ala Phe Gly Leu Asn Leu Ala Leu Pro
850 855 860
Lys Glu Leu Thr Asp Tyr Arg Tyr Tyr Gly Leu Gly Pro Asn Glu Ser
865 870 875 880
Tyr Pro Asp Arg Leu Glu Gly Asn Tyr Leu Gly Ile Tyr Gln Gly Ala
885 890 895
Val Lys Lys Asn Phe Ser Pro Tyr Leu Arg Pro Gln Glu Thr Gly Asn
900 905 910
Arg Ser Lys Val Arg Trp Tyr Gln Leu Phe Asp Glu Lys Gly Gly Leu
915 920 925
Glu Phe Thr Ala Asn Gly Ala Asp Leu Asn Leu Ser Ala Leu Pro Tyr
930 935 940
Ser Ala Ala Gln Ile Glu Ala Ala Asp His Ala Phe Glu Leu Thr Asn
945 950 955 960
Asn Tyr Thr Trp Val Arg Ala Leu Ser Ala Gln Met Gly Val Gly Gly
965 970 975
Asp Asp Ser Trp Gly Gln Lys Val His Pro Glu Phe Cys Leu Asp Ala
980 985 990
Gln Lys Ala Arg Gln Leu Arg Leu Val Ile Gln Pro Leu Leu Leu Lys
995 1000 1005
<210> 15
<211> 1008
<212> PRT
<213> 德氏乳杆菌保加利亚亚种
<400> 15
Met Ser Asn Lys Leu Val Lys Glu Lys Arg Val Asp Gln Ala Asp Leu
1 5 10 15
Ala Trp Leu Thr Asp Pro Glu Val Tyr Glu Val Asn Thr Ile Pro Pro
20 25 30
His Ser Asp His Glu Ser Phe Gln Ser Gln Glu Glu Leu Glu Glu Gly
35 40 45
Lys Ser Ser Leu Val Gln Ser Leu Asp Gly Asp Trp Leu Ile Asp Tyr
50 55 60
Ala Glu Asn Gly Gln Gly Pro Val Asn Phe Tyr Ala Glu Asp Phe Asp
65 70 75 80
Asp Ser Asn Phe Lys Ser Val Lys Val Pro Gly Asn Leu Glu Leu Gln
85 90 95
Gly Phe Gly Gln Pro Gln Tyr Val Asn Val Gln Tyr Pro Trp Asp Gly
100 105 110
Ser Glu Glu Ile Phe Pro Pro Gln Ile Pro Ser Lys Asn Pro Leu Ala
115 120 125
Ser Tyr Val Arg Tyr Phe Asp Leu Asp Glu Ala Phe Trp Asp Lys Glu
130 135 140
Val Ser Leu Lys Phe Asp Gly Ala Ala Thr Ala Ile Tyr Val Trp Leu
145 150 155 160
Asn Gly His Phe Val Gly Tyr Gly Glu Asp Ser Phe Thr Pro Ser Glu
165 170 175
Phe Met Val Thr Lys Phe Leu Lys Lys Glu Asn Asn Arg Leu Ala Val
180 185 190
Ala Leu Tyr Lys Tyr Ser Ser Ala Ser Trp Leu Glu Asp Gln Asp Phe
195 200 205
Trp Arg Met Ser Gly Leu Phe Arg Ser Val Thr Leu Gln Ala Lys Pro
210 215 220
Arg Leu His Leu Glu Asp Leu Lys Leu Thr Ala Ser Leu Thr Asp Asn
225 230 235 240
Tyr Gln Lys Gly Lys Leu Glu Val Glu Ala Asn Ile Ala Tyr Arg Leu
245 250 255
Pro Asn Ala Ser Phe Lys Leu Glu Val Arg Asp Ser Glu Gly Asp Leu
260 265 270
Val Ala Glu Lys Leu Gly Pro Ile Arg Ser Glu Gln Leu Glu Phe Thr
275 280 285
Leu Ala Asp Leu Pro Val Ala Ala Trp Ser Ala Glu Lys Pro Asn Leu
290 295 300
Tyr Gln Val Arg Leu Tyr Leu Tyr Gln Ala Gly Ser Leu Leu Glu Val
305 310 315 320
Ser Arg Gln Glu Val Gly Phe Arg Asn Phe Glu Leu Lys Asp Gly Ile
325 330 335
Met Tyr Leu Asn Gly Gln Arg Ile Val Phe Lys Gly Ala Asn Arg His
340 345 350
Glu Phe Asp Ser Lys Leu Gly Arg Ala Ile Thr Glu Glu Asp Met Ile
355 360 365
Trp Asp Ile Lys Thr Met Lys Arg Ser Asn Ile Asn Ala Val Arg Cys
370 375 380
Ser His Tyr Pro Asn Gln Ser Leu Phe Tyr Arg Leu Cys Asp Lys Tyr
385 390 395 400
Gly Leu Tyr Val Ile Asp Glu Ala Asn Leu Glu Ser His Gly Thr Trp
405 410 415
Glu Lys Val Gly Gly His Glu Asp Pro Ser Phe Asn Val Pro Gly Asp
420 425 430
Asp Gln His Trp Leu Gly Ala Ser Leu Ser Arg Val Lys Asn Met Met
435 440 445
Ala Arg Asp Lys Asn His Ala Ser Ile Leu Ile Trp Ser Leu Gly Asn
450 455 460
Glu Ser Tyr Ala Gly Thr Val Phe Ala Gln Met Ala Asp Tyr Val Arg
465 470 475 480
Lys Ala Asp Pro Thr Arg Val Gln His Tyr Glu Gly Val Thr His Asn
485 490 495
Arg Lys Phe Asp Asp Ala Thr Gln Ile Glu Ser Arg Met Tyr Ala Pro
500 505 510
Ala Lys Val Ile Glu Glu Tyr Leu Thr Asn Lys Pro Ala Lys Pro Phe
515 520 525
Ile Ser Val Glu Tyr Ala His Ala Met Gly Asn Ser Val Gly Asp Leu
530 535 540
Ala Ala Tyr Thr Ala Leu Glu Lys Tyr Pro His Tyr Gln Gly Gly Phe
545 550 555 560
Ile Trp Asp Trp Ile Asp Gln Gly Leu Glu Lys Asp Gly His Leu Leu
565 570 575
Tyr Gly Gly Asp Phe Asp Asp Arg Pro Thr Asp Tyr Glu Phe Cys Gly
580 585 590
Asn Gly Leu Val Phe Ala Asp Arg Thr Glu Ser Pro Lys Leu Ala Asn
595 600 605
Val Lys Ala Leu Tyr Ala Asn Leu Lys Leu Glu Val Lys Asp Gly Gln
610 615 620
Leu Phe Leu Lys Asn Asp Asn Leu Phe Thr Asn Ser Ser Ser Tyr Tyr
625 630 635 640
Phe Leu Thr Ser Leu Leu Val Asp Gly Lys Leu Thr Tyr Gln Ser Arg
645 650 655
Pro Leu Thr Phe Gly Leu Glu Pro Gly Glu Ser Gly Thr Phe Ala Leu
660 665 670
Pro Trp Pro Glu Val Ala Asp Glu Lys Gly Glu Val Val Tyr Arg Val
675 680 685
Thr Ala His Leu Lys Glu Asp Leu Pro Trp Ala Asp Glu Gly Phe Thr
690 695 700
Val Ala Glu Ala Glu Glu Val Ala Gln Lys Leu Pro Glu Phe Lys Pro
705 710 715 720
Glu Gly Arg Pro Asp Leu Val Asp Ser Asp Tyr Asn Leu Gly Leu Lys
725 730 735
Gly Asn Asn Phe Gln Ile Leu Phe Ser Lys Val Lys Gly Trp Pro Val
740 745 750
Ser Leu Lys Tyr Ala Gly Arg Glu Tyr Leu Lys Arg Leu Pro Glu Phe
755 760 765
Thr Phe Trp Arg Ala Leu Thr Asp Asn Asp Arg Gly Ala Gly Tyr Gly
770 775 780
Tyr Asp Leu Ala Arg Trp Glu Asn Ala Gly Lys Tyr Ala Arg Leu Lys
785 790 795 800
Asp Ile Ser Cys Glu Val Lys Glu Asp Ser Val Leu Val Lys Thr Ala
805 810 815
Phe Thr Leu Pro Val Ala Leu Lys Gly Asp Leu Thr Val Thr Tyr Glu
820 825 830
Val Asp Gly Arg Gly Lys Ile Ala Val Thr Ala Asp Phe Pro Gly Ala
835 840 845
Glu Glu Ala Gly Leu Leu Pro Ala Phe Gly Leu Asn Leu Ala Leu Pro
850 855 860
Lys Glu Leu Thr Asp Tyr Arg Tyr Tyr Gly Leu Gly Pro Asn Glu Ser
865 870 875 880
Tyr Pro Asp Arg Leu Glu Gly Asn Tyr Leu Gly Ile Tyr Gln Gly Ala
885 890 895
Val Lys Lys Asn Phe Ser Pro Tyr Leu Arg Pro Gln Glu Thr Gly Asn
900 905 910
Arg Ser Lys Val Arg Trp Tyr Gln Leu Phe Asp Glu Lys Gly Gly Leu
915 920 925
Glu Phe Thr Ala Asn Gly Ala Asp Leu Asn Leu Ser Ala Leu Pro Tyr
930 935 940
Ser Ala Ala Gln Ile Glu Ala Ala Asp His Ala Phe Glu Leu Thr Asn
945 950 955 960
Asn Tyr Thr Trp Val Arg Ala Leu Ser Ala Gln Met Gly Val Gly Gly
965 970 975
Asp Asp Ser Trp Gly Gln Lys Val His Pro Glu Phe Cys Leu Asp Ala
980 985 990
Gln Lys Ala Arg Gln Leu Arg Leu Val Ile Gln Pro Leu Leu Leu Lys
995 1000 1005
<210> 16
<211> 1008
<212> PRT
<213> 德氏乳杆菌乳酸亚种
<400> 16
Met Ser Asn Lys Leu Val Lys Glu Lys Arg Val Asp Gln Ala Asp Leu
1 5 10 15
Ala Trp Leu Thr Asp Pro Glu Val Tyr Glu Val Asn Thr Ile Pro Pro
20 25 30
His Ser Asp His Glu Ser Phe Gln Ser Gln Glu Glu Leu Glu Glu Gly
35 40 45
Lys Ser Ser Leu Val Gln Ser Leu Asp Gly Asp Trp Leu Ile Asp Tyr
50 55 60
Ala Glu Asn Gly Gln Gly Pro Val Asn Phe Tyr Ala Glu Asp Phe Asp
65 70 75 80
Asp Ser Asn Phe Lys Ser Val Lys Val Pro Gly Asn Leu Glu Leu Gln
85 90 95
Gly Phe Gly Gln Pro Gln Tyr Val Asn Ile Gln Tyr Pro Trp Asp Gly
100 105 110
Ser Glu Glu Ile Phe Pro Pro Gln Val Pro Ser Lys Asn Pro Leu Ala
115 120 125
Ser Tyr Val Arg Tyr Phe Asp Leu Asp Glu Ala Phe Trp Asp Lys Glu
130 135 140
Val Ser Leu Lys Phe Ala Gly Ala Ala Thr Ala Ile Tyr Val Trp Leu
145 150 155 160
Asn Gly His Phe Val Gly Tyr Gly Glu Asp Ser Phe Thr Pro Ser Glu
165 170 175
Phe Met Val Thr Lys Phe Leu Lys Lys Glu Asn Asn Arg Leu Ala Val
180 185 190
Ala Leu Tyr Lys Tyr Ser Ser Ala Ser Trp Leu Glu Asp Gln Asp Phe
195 200 205
Trp Arg Leu Ser Gly Leu Phe Arg Ser Val Thr Leu Gln Ala Lys Pro
210 215 220
Leu Leu His Leu Glu Asp Leu Lys Leu Thr Ala Ser Leu Thr Asp Asn
225 230 235 240
Tyr Gln Lys Gly Lys Leu Glu Val Glu Ala Asn Ile Ala Tyr Arg Leu
245 250 255
Pro Asn Ala Ser Phe Lys Leu Glu Val Arg Asp Ser Glu Gly Asp Leu
260 265 270
Val Ala Glu Lys Leu Gly Pro Ile Arg Ser Glu Gln Leu Glu Phe Thr
275 280 285
Leu Ala Asp Leu Pro Val Ala Ala Trp Ser Ala Glu Lys Pro Asn Leu
290 295 300
Tyr Gln Val Arg Leu Tyr Leu Tyr Gln Ala Gly Ser Leu Leu Glu Val
305 310 315 320
Ser Arg Gln Glu Val Gly Phe Arg Asn Phe Glu Leu Lys Asp Gly Ile
325 330 335
Met Tyr Leu Asn Gly Gln Arg Ile Val Phe Lys Gly Val Asn Arg His
340 345 350
Glu Phe Asp Ser Lys Leu Gly Arg Ala Ile Thr Glu Glu Asp Met Ile
355 360 365
Trp Asp Ile Lys Thr Met Lys Arg Ser Asn Ile Asn Ala Val Arg Cys
370 375 380
Ser His Tyr Pro Asn Gln Ser Leu Phe Tyr Arg Leu Cys Asp Lys Tyr
385 390 395 400
Gly Leu Tyr Val Ile Asp Glu Ala Asn Leu Glu Ser His Gly Thr Trp
405 410 415
Glu Lys Val Gly His Glu Asp Pro Ser Phe Asn Val Pro Gly Asp Asp
420 425 430
Gln His Trp Leu Gly Ala Ser Leu Ser Arg Val Lys Asn Met Met Ala
435 440 445
Arg Asp Lys Asn His Ala Ser Ile Leu Ile Trp Ser Leu Gly Asn Glu
450 455 460
Ser Tyr Ala Gly Thr Val Phe Ala Gln Met Ala Asp Tyr Val Arg Lys
465 470 475 480
Ala Asp Pro Thr Arg Val Gln His Tyr Glu Gly Val Thr His Asn Arg
485 490 495
Lys Phe Asp Asp Ala Thr Gln Ile Glu Ser Arg Met Tyr Ala Pro Ala
500 505 510
Lys Glu Ile Glu Glu Tyr Leu Thr Lys Lys Pro Ala Lys Pro Phe Ile
515 520 525
Ser Val Glu Tyr Ala His Ala Met Gly Asn Ser Val Gly Asp Leu Ala
530 535 540
Ala Tyr Thr Ala Leu Glu Lys Tyr Pro His Tyr Gln Gly Gly Phe Ile
545 550 555 560
Trp Asp Trp Ile Asp Gln Gly Leu Glu Lys Asp Gly His Leu Leu Tyr
565 570 575
Gly Gly Gly Asp Phe Asp Asp Arg Pro Thr Asp Tyr Glu Phe Cys Gly
580 585 590
Asn Gly Leu Val Phe Ala Asp Arg Thr Thr Ser Pro Lys Leu Ala Asn
595 600 605
Val Lys Ala Leu Tyr Ser Asn Leu Lys Leu Glu Val Lys Asp Gly Gln
610 615 620
Leu Phe Leu Lys Asn Asp Asn Leu Phe Thr Asn Ser Ser Ala Tyr Tyr
625 630 635 640
Phe Leu Thr Ser Leu Leu Val Asp Gly Lys Leu Thr Tyr Gln Ser Gln
645 650 655
Pro Leu Thr Phe Gly Leu Glu Pro Gly Glu Ser Gly Thr Phe Val Leu
660 665 670
Pro Trp Pro Glu Val Glu Asp Glu Lys Gly Glu Ile Val Tyr Gln Val
675 680 685
Thr Ala His Leu Lys Glu Asp Leu Pro Trp Ala Asp Glu Gly Phe Thr
690 695 700
Val Ala Glu Ala Glu Glu Ala Val Thr Lys Leu Pro Glu Phe Tyr Pro
705 710 715 720
Ala Gly Arg Pro Glu Leu Val Asp Ser Asp Phe Asn Leu Gly Leu Lys
725 730 735
Gly Asn Gly Phe Arg Ile Leu Phe Ser Lys Ala Lys Gly Trp Pro Val
740 745 750
Ser Ile Lys Tyr Ala Gly Arg Glu Tyr Leu Lys Arg Leu Pro Glu Phe
755 760 765
Thr Phe Trp Arg Ala Leu Thr Asp Asn Asp Arg Gly Ala Gly Tyr Gly
770 775 780
Tyr Asp Leu Ala Lys Trp Glu Asn Ala Gly Lys Tyr Ala Arg Leu Gln
785 790 795 800
Asp Ile Ser Tyr Glu Ile Lys Glu Asn Ser Val Leu Val Lys Thr Ala
805 810 815
Phe Thr Leu Pro Val Ala Leu Lys Gly Asp Leu Thr Ile Thr Tyr Glu
820 825 830
Val Asp Ser Leu Gly Lys Ile Ala Val Thr Ala Asn Phe Pro Gly Ala
835 840 845
Val Glu Asn Gly Leu Leu Pro Ala Phe Gly Leu Asn Phe Ala Leu Pro
850 855 860
Lys Glu Leu Ser Asp Tyr Arg Tyr Tyr Gly Leu Gly Pro Asn Glu Ser
865 870 875 880
Tyr Ala Asp Arg Leu Glu Gly Ser Tyr Leu Gly Ile Tyr Gln Gly Ala
885 890 895
Val Glu Lys Asn Phe Thr Pro Tyr Leu Arg Pro Gln Glu Ala Gly Asn
900 905 910
Arg Ser Lys Val Arg Tyr Tyr Gln Leu Phe Asp Glu Glu Ser Gly Leu
915 920 925
Glu Phe Thr Ala Asn Gly Ala Asp Leu Asn Leu Ser Ala Leu Pro Tyr
930 935 940
Ser Ala Ala Gln Ile Glu Ala Ala Asp His Ala Phe Glu Leu Ser Asn
945 950 955 960
Asn Tyr Thr Trp Val Arg Ala Leu Ala Ala Gln Met Gly Val Gly Gly
965 970 975
Asp Asp Ser Trp Gly Gln Lys Val His Pro Glu Phe Cys Leu Asp Ala
980 985 990
Gln Glu Ala Arg Gln Leu Lys Leu Val Ile Gln Pro Leu Leu Leu Lys
995 1000 1005
<210> 17
<211> 1008
<212> PRT
<213> 德氏乳杆菌保加利亚亚种
<400> 17
Met Ser Asn Lys Leu Val Lys Glu Lys Arg Val Asp Gln Ala Asp Leu
1 5 10 15
Ala Trp Leu Thr Asp Pro Glu Val Tyr Glu Val Asn Thr Ile Pro Pro
20 25 30
His Ser Asp His Glu Ser Phe Gln Ser Gln Glu Glu Leu Glu Glu Gly
35 40 45
Lys Ser Ser Leu Val Gln Ser Leu Asp Gly Asp Trp Leu Ile Asp Tyr
50 55 60
Ala Glu Asn Gly Gln Gly Pro Val Asn Phe Tyr Ala Glu Asp Phe Asp
65 70 75 80
Asp Ser Asn Phe Lys Ser Val Lys Val Pro Gly Asn Leu Glu Leu Gln
85 90 95
Gly Phe Gly Gln Pro Gln Tyr Val Asn Val Gln Tyr Pro Trp Asp Gly
100 105 110
Ser Glu Glu Ile Phe Pro Pro Gln Ile Pro Ser Lys Asn Pro Leu Ala
115 120 125
Ser Tyr Val Arg Tyr Phe Asp Leu Asp Glu Ala Phe Trp Asp Lys Glu
130 135 140
Val Ser Leu Lys Phe Asp Gly Ala Ala Thr Ala Ile Tyr Val Trp Leu
145 150 155 160
Asn Gly His Phe Val Gly Tyr Gly Glu Asp Ser Phe Thr Pro Ser Glu
165 170 175
Phe Met Val Thr Lys Phe Leu Lys Lys Glu Asn Asn Arg Leu Ala Val
180 185 190
Ala Leu Tyr Lys Tyr Ser Ser Ala Ser Trp Leu Glu Asp Gln Asp Phe
195 200 205
Trp Arg Met Ser Gly Leu Phe Arg Ser Val Thr Leu Gln Ala Lys Pro
210 215 220
Arg Leu His Leu Glu Asp Leu Lys Leu Thr Ala Ser Leu Thr Asp Asn
225 230 235 240
Tyr Gln Lys Gly Lys Leu Glu Val Glu Ala Asn Ile Ala Tyr Arg Leu
245 250 255
Pro Asn Ala Ser Phe Lys Leu Glu Val Arg Asp Ser Glu Gly Asp Leu
260 265 270
Val Ala Glu Lys Leu Gly Pro Ile Arg Ser Glu Gln Leu Glu Phe Thr
275 280 285
Leu Ala Asp Leu Pro Val Ala Ala Trp Ser Ala Glu Lys Pro Asn Leu
290 295 300
Tyr Gln Val Arg Leu Tyr Leu Tyr Gln Ala Gly Ser Leu Leu Glu Val
305 310 315 320
Ser Arg Gln Glu Val Gly Phe Arg Asn Phe Glu Leu Lys Asp Gly Ile
325 330 335
Met Tyr Leu Asn Gly Gln Arg Ile Val Phe Lys Gly Val Asn Arg His
340 345 350
Glu Phe Asp Ser Lys Leu Gly Arg Ala Ile Thr Glu Glu Asp Met Ile
355 360 365
Trp Asp Ile Lys Thr Ile Lys Arg Ser Asn Ile Asn Ala Val Arg Cys
370 375 380
Ser His Tyr Pro Asn Gln Ser Leu Phe Tyr Arg Leu Cys Asp Lys Tyr
385 390 395 400
Gly Leu Tyr Val Ile Asp Glu Ala Asn Leu Glu Ser His Gly Thr Trp
405 410 415
Glu Lys Val Gly Gly His Glu Asp Pro Ser Phe Asn Val Pro Gly Asp
420 425 430
Asp Gln His Trp Leu Gly Ala Ser Leu Ser Arg Val Lys Asn Met Met
435 440 445
Ala Arg Asp Lys Asn His Ala Ser Ile Leu Ile Trp Ser Leu Gly Asn
450 455 460
Glu Ser Tyr Ala Gly Thr Val Phe Ala Gln Met Ala Asp Tyr Val Arg
465 470 475 480
Lys Ala Asp Pro Thr Arg Val Gln His Tyr Glu Gly Val Thr His Asn
485 490 495
Arg Lys Phe Asp Asp Ala Thr Gln Ile Glu Ser Arg Met Tyr Ala Pro
500 505 510
Ala Lys Val Ile Glu Glu Tyr Leu Thr Asn Lys Pro Ala Lys Pro Phe
515 520 525
Ile Ser Val Glu Tyr Ala His Ala Met Gly Asn Ser Val Gly Asp Leu
530 535 540
Ala Ala Tyr Thr Ala Leu Glu Lys Tyr Pro His Tyr Gln Gly Gly Phe
545 550 555 560
Ile Trp Asp Trp Ile Asp Gln Gly Leu Glu Lys Asp Gly His Leu Leu
565 570 575
Tyr Gly Gly Asp Phe Asp Asp Arg Pro Thr Asp Tyr Glu Phe Cys Gly
580 585 590
Asn Gly Leu Val Phe Ala Asp Arg Thr Glu Ser Pro Lys Leu Ala Asn
595 600 605
Val Lys Ala Leu Tyr Ala Asn Leu Lys Leu Glu Val Lys Asp Gly Gln
610 615 620
Leu Phe Leu Lys Asn Asp Asn Leu Phe Thr Asn Ser Ser Ser Tyr Tyr
625 630 635 640
Phe Leu Thr Ser Leu Leu Val Asp Gly Lys Leu Thr Tyr Gln Ser Arg
645 650 655
Pro Leu Thr Phe Gly Leu Glu Pro Gly Glu Ser Gly Thr Phe Ala Leu
660 665 670
Pro Trp Pro Glu Val Ala Asp Glu Lys Gly Glu Val Val Tyr Arg Val
675 680 685
Thr Ala His Leu Lys Glu Asp Leu Pro Trp Ala Asp Glu Gly Phe Thr
690 695 700
Val Ala Glu Ala Glu Glu Val Ala Gln Lys Leu Pro Glu Phe Lys Pro
705 710 715 720
Glu Gly Arg Pro Asp Leu Val Asp Ser Asp Tyr Asn Leu Gly Leu Lys
725 730 735
Gly Asn Asn Phe Gln Ile Leu Phe Ser Lys Val Lys Gly Trp Pro Val
740 745 750
Ser Leu Lys Tyr Ala Gly Arg Glu Tyr Leu Lys Arg Leu Pro Glu Phe
755 760 765
Thr Phe Trp Arg Ala Leu Thr Asp Asn Asp Arg Gly Ala Gly Tyr Gly
770 775 780
Tyr Asp Leu Ala Arg Trp Glu Asn Ala Gly Lys Tyr Ala Arg Leu Lys
785 790 795 800
Asp Ile Ser Cys Glu Val Lys Glu Asp Ser Val Leu Val Lys Thr Ala
805 810 815
Phe Thr Leu Pro Val Ala Leu Lys Gly Asp Leu Thr Val Thr Tyr Glu
820 825 830
Val Asp Gly Arg Gly Lys Ile Ala Val Thr Ala Asp Phe Pro Gly Ala
835 840 845
Glu Glu Ala Gly Leu Leu Pro Ala Phe Gly Leu Asn Leu Ala Leu Pro
850 855 860
Lys Glu Leu Thr Asp Tyr Arg Tyr Tyr Gly Leu Gly Pro Asn Glu Ser
865 870 875 880
Tyr Pro Asp Arg Leu Glu Gly Asn Tyr Leu Gly Ile Tyr Gln Gly Ala
885 890 895
Val Lys Lys Asn Phe Ser Pro Tyr Leu Arg Pro Gln Glu Thr Gly Asn
900 905 910
Arg Ser Lys Val Arg Trp Tyr Gln Leu Phe Asp Glu Lys Gly Gly Leu
915 920 925
Glu Phe Thr Ala Asn Gly Ala Asp Leu Asn Leu Ser Ala Leu Pro Tyr
930 935 940
Ser Ala Ala Gln Ile Glu Ala Ala Asp His Ala Phe Asp Leu Thr Asn
945 950 955 960
Asn Tyr Thr Trp Val Arg Ala Leu Ser Ala Gln Met Gly Val Gly Gly
965 970 975
Asp Asp Ser Trp Gly Gln Lys Val His Pro Glu Phe Cys Leu Asp Ala
980 985 990
Gln Lys Ala Arg Gln Leu Arg Leu Val Ile Gln Pro Leu Leu Leu Lys
995 1000 1005
<210> 18
<211> 1008
<212> PRT
<213> 德氏乳杆菌保加利亚亚种
<400> 18
Met Ser Asn Lys Leu Val Lys Glu Lys Arg Val Asp Gln Ala Asp Leu
1 5 10 15
Ala Trp Leu Thr Asp Pro Glu Val Tyr Glu Val Asn Thr Ile Pro Pro
20 25 30
His Ser Asp His Glu Ser Phe Gln Ser Gln Glu Glu Leu Glu Glu Gly
35 40 45
Lys Ser Ser Leu Val Gln Ser Leu Asp Gly Asp Trp Leu Ile Asp Tyr
50 55 60
Ala Glu Asn Gly Gln Gly Pro Val Asn Phe Tyr Ala Glu Asp Phe Asp
65 70 75 80
Asp Ser Asn Phe Lys Ser Val Lys Val Pro Gly Asn Leu Glu Leu Gln
85 90 95
Gly Phe Gly Gln Pro Gln Tyr Val Asn Val Gln Tyr Pro Trp Asp Gly
100 105 110
Ser Glu Glu Ile Phe Pro Pro Gln Ile Pro Ser Lys Asn Pro Leu Ala
115 120 125
Ser Tyr Val Arg Tyr Phe Asp Leu Asp Glu Ala Phe Trp Asp Lys Glu
130 135 140
Val Ser Leu Lys Phe Asp Gly Ala Ala Thr Ala Ile Tyr Val Trp Leu
145 150 155 160
Asn Gly His Phe Val Gly Tyr Gly Glu Asp Ser Phe Thr Pro Ser Glu
165 170 175
Phe Met Val Thr Lys Phe Leu Lys Lys Glu Asn Asn Arg Leu Ala Val
180 185 190
Ala Leu Tyr Lys Tyr Ser Ser Ala Ser Trp Leu Glu Asp Gln Asp Phe
195 200 205
Trp Arg Met Ser Gly Leu Phe Arg Ser Val Thr Leu Gln Ala Lys Pro
210 215 220
Arg Leu His Leu Glu Asp Leu Lys Leu Thr Ala Ser Leu Thr Asp Asn
225 230 235 240
Tyr Gln Lys Gly Lys Leu Glu Val Glu Ala Asn Ile Ala Tyr Arg Leu
245 250 255
Pro Asn Ala Ser Phe Lys Leu Glu Val Arg Asp Ser Glu Gly Asp Leu
260 265 270
Val Ala Glu Lys Leu Gly Pro Ile Gly Ser Glu Gln Leu Glu Phe Thr
275 280 285
Leu Ala Asp Leu Pro Val Ala Ala Trp Ser Ala Glu Lys Pro Asn Leu
290 295 300
Tyr Gln Val Arg Leu Tyr Leu Tyr Gln Ala Gly Ser Leu Leu Glu Val
305 310 315 320
Ser Arg Gln Glu Val Gly Phe Arg Asn Phe Glu Leu Lys Asp Gly Ile
325 330 335
Met Tyr Leu Asn Gly Gln Arg Ile Val Phe Lys Gly Val Asn Arg His
340 345 350
Glu Phe Asp Ser Lys Leu Gly Arg Ala Ile Thr Glu Glu Asp Met Ile
355 360 365
Trp Asp Ile Lys Thr Ile Lys Arg Ser Asn Ile Asn Ala Val Arg Cys
370 375 380
Ser His Tyr Pro Asn Gln Ser Leu Phe Tyr Arg Leu Cys Asp Lys Tyr
385 390 395 400
Gly Leu Tyr Val Ile Asp Glu Ala Asn Leu Glu Ser His Gly Thr Trp
405 410 415
Glu Lys Val Gly Gly His Glu Asp Pro Ser Phe Asn Val Pro Gly Asp
420 425 430
Asp Gln His Trp Leu Gly Ala Ser Leu Ser Arg Val Lys Asn Met Met
435 440 445
Ala Arg Asp Lys Asn His Ala Ser Ile Leu Ile Trp Ser Leu Gly Asn
450 455 460
Glu Ser Tyr Ala Gly Thr Val Phe Ala Gln Met Ala Asp Tyr Val Arg
465 470 475 480
Lys Ala Asp Pro Thr Arg Val Gln His Tyr Glu Gly Val Thr His Asn
485 490 495
Arg Lys Phe Asp Asp Ala Thr Gln Ile Glu Ser Arg Met Tyr Ala Pro
500 505 510
Ala Lys Val Ile Glu Glu Tyr Leu Thr Asn Lys Pro Ala Lys Pro Phe
515 520 525
Ile Ser Val Glu Tyr Ala His Ala Met Gly Asn Ser Val Gly Asp Leu
530 535 540
Ala Ala Tyr Thr Ala Leu Glu Lys Tyr Pro His Tyr Gln Gly Gly Phe
545 550 555 560
Ile Trp Asp Trp Ile Asp Gln Gly Leu Glu Lys Asp Gly His Leu Leu
565 570 575
Tyr Gly Gly Asp Phe Asp Asp Arg Pro Thr Asp Tyr Glu Phe Cys Gly
580 585 590
Asn Gly Leu Val Phe Ala Asp Arg Thr Glu Ser Pro Lys Leu Ala Asn
595 600 605
Val Lys Ala Leu Tyr Ala Asn Leu Lys Leu Glu Val Lys Asp Gly Gln
610 615 620
Leu Phe Leu Lys Asn Asp Asn Leu Phe Thr Asn Ser Ser Ser Tyr Tyr
625 630 635 640
Phe Leu Thr Ser Leu Leu Val Asp Gly Lys Leu Thr Tyr Gln Ser Arg
645 650 655
Pro Leu Thr Phe Gly Leu Glu Pro Gly Glu Ser Gly Thr Phe Ala Leu
660 665 670
Pro Trp Pro Glu Val Ala Asp Glu Lys Gly Glu Val Val Tyr Arg Val
675 680 685
Thr Ala His Leu Lys Glu Asp Leu Pro Trp Ala Asp Glu Gly Phe Thr
690 695 700
Val Ala Glu Ala Glu Glu Val Ala Gln Lys Leu Pro Glu Phe Lys Pro
705 710 715 720
Glu Gly Arg Pro Asp Leu Val Asp Ser Asp Tyr Asn Leu Gly Leu Lys
725 730 735
Gly Asn Asn Phe Gln Ile Leu Phe Ser Lys Val Lys Gly Trp Pro Val
740 745 750
Ser Leu Lys Tyr Ala Gly Arg Glu Tyr Leu Lys Arg Leu Pro Glu Phe
755 760 765
Thr Phe Trp Arg Ala Leu Thr Asp Asn Asp Arg Gly Ala Gly Tyr Gly
770 775 780
Tyr Asp Leu Ala Arg Trp Glu Asn Ala Gly Lys Tyr Ala Arg Leu Lys
785 790 795 800
Asp Ile Ser Cys Glu Val Lys Glu Asp Ser Val Leu Val Lys Thr Ala
805 810 815
Phe Thr Leu Pro Val Ala Leu Lys Gly Asp Leu Thr Val Thr Tyr Glu
820 825 830
Val Asp Gly Arg Gly Lys Ile Ala Val Thr Ala Asp Phe Pro Gly Ala
835 840 845
Glu Glu Ala Gly Leu Leu Pro Ala Phe Gly Leu Asn Leu Ala Leu Pro
850 855 860
Lys Glu Leu Thr Asp Tyr Arg Tyr Tyr Gly Leu Gly Pro Asn Glu Ser
865 870 875 880
Tyr Pro Asp Arg Leu Glu Gly Asn Tyr Leu Gly Ile Tyr Gln Gly Ala
885 890 895
Val Lys Lys Asn Phe Ser Pro Tyr Leu Arg Pro Gln Glu Thr Gly Asn
900 905 910
Arg Ser Lys Val Arg Trp Tyr Gln Leu Phe Asp Glu Lys Gly Gly Leu
915 920 925
Glu Phe Thr Ala Asn Gly Ala Asp Leu Asn Leu Ser Ala Leu Pro Tyr
930 935 940
Ser Ala Ala Gln Ile Glu Ala Ala Asp His Ala Phe Glu Leu Thr Asn
945 950 955 960
Asn Tyr Thr Trp Val Arg Ala Leu Ser Ala Gln Met Gly Val Gly Gly
965 970 975
Asp Asp Ser Trp Gly Gln Lys Val His Pro Glu Phe Cys Leu Asp Ala
980 985 990
Gln Lys Ala Arg Gln Leu Arg Leu Val Ile Gln Pro Leu Leu Leu Lys
995 1000 1005
<210> 19
<211> 1007
<212> PRT
<213> 德氏乳杆菌乳酸亚种
<400> 19
Met Ser Asn Lys Leu Val Lys Glu Lys Arg Val Asp Gln Ala Asp Leu
1 5 10 15
Ala Trp Leu Thr Asp Pro Glu Val Tyr Glu Val Asn Thr Ile Pro Pro
20 25 30
His Ser Asp His Glu Ser Phe Gln Ser Gln Glu Glu Leu Glu Glu Gly
35 40 45
Lys Ser Ser Leu Val Gln Ser Leu Asp Gly Asn Trp Leu Ile Asp Tyr
50 55 60
Ala Glu Asn Gly Gln Gly Pro Ile Asn Phe Tyr Ala Glu Asp Phe Asp
65 70 75 80
Asp Ser Asn Phe Lys Ser Val Lys Val Pro Gly Asn Leu Glu Leu Gln
85 90 95
Gly Phe Gly Gln Pro Gln Tyr Val Asn Ile Gln Tyr Pro Trp Asp Gly
100 105 110
Ser Glu Glu Ile Phe Pro Pro Gln Val Pro Ser Lys Asn Pro Leu Ala
115 120 125
Ser Tyr Val Arg Tyr Phe Asp Leu Asp Glu Ala Leu Trp Asp Lys Glu
130 135 140
Val Ser Leu Lys Phe Ala Gly Ala Ala Thr Ala Ile Tyr Val Trp Leu
145 150 155 160
Asn Gly His Phe Val Gly Tyr Gly Glu Asp Ser Phe Thr Pro Ser Glu
165 170 175
Phe Met Val Thr Lys Phe Leu Lys Lys Glu Gly Asn Arg Leu Ala Val
180 185 190
Ala Leu Tyr Lys Tyr Ser Ser Ala Ser Trp Leu Glu Asp Gln Asp Phe
195 200 205
Trp Arg Leu Ser Gly Leu Phe Arg Ser Val Thr Leu Glu Ala Lys Pro
210 215 220
Leu Leu His Leu Glu Asp Leu Lys Leu Thr Ala Ser Leu Thr Asp Asn
225 230 235 240
Tyr Gln Lys Gly Lys Leu Glu Val Glu Ala Asn Ile Ala Tyr Arg Leu
245 250 255
Pro Asn Ala Ser Phe Lys Leu Glu Val Arg Asp Ser Glu Gly Asp Leu
260 265 270
Val Ala Glu Lys Val Gly Pro Ile Arg Ser Glu Lys Leu Asp Phe Ser
275 280 285
Leu Ala Asp Leu Pro Val Ala Ala Trp Ser Ala Glu Lys Pro Asn Leu
290 295 300
Tyr Gln Val Arg Leu Tyr Leu Tyr Gln Ala Gly Ser Leu Leu Glu Val
305 310 315 320
Ser Arg Gln Glu Val Gly Phe Arg Asn Phe Glu Leu Lys Asp Gly Ile
325 330 335
Met Tyr Leu Asn Gly Gln Arg Ile Val Phe Lys Gly Val Asn Arg His
340 345 350
Glu Phe Asp Ser Lys Leu Gly Arg Ala Ile Thr Glu Ala Asp Met Ile
355 360 365
Trp Asp Ile Lys Thr Met Lys Gln Ser Asn Ile Asn Ala Val Arg Cys
370 375 380
Ser His Tyr Pro Asn Gln Ser Leu Phe Tyr Arg Leu Cys Asp Lys Tyr
385 390 395 400
Gly Leu Tyr Val Ile Asp Glu Ala Asn Leu Glu Ser His Gly Thr Trp
405 410 415
Glu Lys Val Gly His Glu Asp Pro Ser Phe Asn Val Pro Gly Asp Asp
420 425 430
Gln His Trp Leu Gly Ala Ser Leu Ser Arg Val Lys Asn Met Met Ala
435 440 445
Arg Asp Lys Asn His Ala Ser Ile Leu Ile Trp Ser Leu Gly Asn Glu
450 455 460
Ser Tyr Ala Gly Thr Val Phe Ala Gln Met Ala Asp Tyr Val Arg Lys
465 470 475 480
Ala Asp Pro Thr Arg Val Gln His Tyr Glu Gly Val Thr His Asn Arg
485 490 495
Lys Phe Asp Asp Ala Thr Gln Ile Glu Ser Arg Met Tyr Ala Pro Ala
500 505 510
Lys Glu Ile Glu Glu Tyr Leu Thr Lys Lys Pro Ala Lys Pro Phe Ile
515 520 525
Ser Val Glu Tyr Ala His Ala Met Gly Asn Ser Val Gly Asp Leu Ala
530 535 540
Ala Tyr Thr Ala Leu Glu Lys Tyr Pro His Tyr Gln Gly Gly Phe Ile
545 550 555 560
Trp Asp Trp Ile Asp Gln Gly Leu Glu Lys Asp Gly His Leu Leu Tyr
565 570 575
Gly Gly Asp Phe Asp Asp Arg Pro Thr Asp Tyr Glu Phe Cys Gly Asp
580 585 590
Gly Leu Val Phe Ala Asp Arg Thr Thr Ser Pro Lys Leu Ala Asn Val
595 600 605
Lys Ala Leu Tyr Ser Asn Leu Lys Leu Glu Val Lys Asp Gly Gln Leu
610 615 620
Phe Ile Lys Asn Asp Asn Leu Phe Thr Asn Ser Ser Ala Tyr Tyr Phe
625 630 635 640
Leu Thr Ser Leu Leu Val Asp Gly Lys Leu Thr Tyr Gln Ser Gln Pro
645 650 655
Leu Thr Phe Gly Leu Glu Pro Gly Glu Ser Gly Thr Phe Ala Leu Pro
660 665 670
Trp Pro Glu Val Glu Asp Glu Lys Gly Glu Ile Val Tyr Gln Val Thr
675 680 685
Ala His Leu Lys Glu Asp Leu Pro Trp Ala Asp Glu Gly Phe Thr Val
690 695 700
Ala Glu Ala Glu Glu Ala Val Thr Lys Leu Pro Glu Phe Tyr Pro Ala
705 710 715 720
Gly Arg Pro Glu Leu Val Asp Ser Asp Phe Asn Leu Gly Leu Lys Gly
725 730 735
Asn Gly Phe Arg Ile Leu Phe Ser Lys Ala Lys Gly Trp Pro Val Ser
740 745 750
Ile Lys Tyr Ala Gly Arg Glu Tyr Leu Lys Arg Leu Pro Glu Phe Thr
755 760 765
Phe Trp Arg Ala Leu Thr Asp Asn Asp Arg Gly Ala Gly Tyr Gly Tyr
770 775 780
Asp Leu Ala Lys Trp Glu Asn Ala Gly Lys Tyr Ala Arg Leu Gln Asp
785 790 795 800
Ile Ser Tyr Glu Ile Lys Glu Asn Ser Ala Leu Val Lys Thr Ala Phe
805 810 815
Thr Leu Pro Val Ala Leu Lys Gly Asp Leu Thr Ile Thr Tyr Glu Val
820 825 830
Asp Ser Leu Gly Lys Ile Ala Val Thr Ala Asn Phe Pro Gly Ala Val
835 840 845
Glu Asn Gly Leu Leu Pro Ala Phe Gly Leu Asn Phe Ala Leu Pro Lys
850 855 860
Glu Leu Ser Asp Tyr Arg Tyr Tyr Gly Leu Gly Pro Asn Glu Ser Tyr
865 870 875 880
Ala Asp Arg Leu Glu Gly Ser Tyr Leu Gly Ile Tyr Gln Gly Met Val
885 890 895
Glu Lys Asn Phe Thr Pro Tyr Leu Arg Pro Gln Glu Ala Gly Asn Arg
900 905 910
Ser Lys Val Arg Tyr Tyr Gln Leu Phe Asp Glu Glu Gly Gly Leu Glu
915 920 925
Phe Thr Ala Asn Gly Ala Asp Leu Asn Leu Ser Ala Leu Pro Tyr Ser
930 935 940
Ala Ala Gln Ile Glu Ala Ala Asp His Ala Phe Glu Leu Thr Asn Asn
945 950 955 960
Tyr Thr Trp Val Arg Ala Leu Ala Ala Gln Met Gly Val Gly Gly Asp
965 970 975
Asp Ser Trp Gly Gln Lys Val His Pro Glu Phe Cys Leu Asp Ala Gln
980 985 990
Glu Ala Arg Gln Leu Lys Leu Val Ile Gln Pro Leu Leu Leu Lys
995 1000 1005
<210> 20
<211> 628
<212> PRT
<213> 瑞士乳杆菌(Lactobacillus helvaticus)
<400> 20
Met Gln Ala Asn Ile Asn Trp Leu Asp Asn Pro Glu Val Phe Arg Val
1 5 10 15
Asn Gln Leu Pro Ala His Ser Asp His Pro Phe Phe Arg Asp Tyr Arg
20 25 30
Glu Trp Gln Lys Gln His Ser Ser Tyr Gln Gln Ser Leu Asn Gly Lys
35 40 45
Trp Lys Phe His Phe Ser Ala Asn Pro Met Asp Arg Pro Gln Asp Phe
50 55 60
Tyr Gln Arg Asp Phe Asp Ser Ser Asn Phe Asp Ser Ile Pro Val Pro
65 70 75 80
Ser Glu Ile Glu Leu Ser Asn Tyr Thr Gln Asn Gln Tyr Ile Asn Val
85 90 95
Leu Phe Pro Trp Glu Gly Lys Ile Phe Arg Arg Pro Ala Tyr Ala Leu
100 105 110
Asp Pro Asn Asp His Glu Glu Gly Ser Phe Ser Lys Gly Ala Asp Asn
115 120 125
Thr Val Gly Ser Tyr Leu Lys Arg Phe Asp Leu Ser Ser Ala Leu Ile
130 135 140
Gly Lys Asp Val His Ile Lys Phe Glu Gly Val Glu Gln Ala Met Tyr
145 150 155 160
Val Trp Leu Asn Gly His Phe Val Gly Tyr Ala Glu Asp Ser Phe Thr
165 170 175
Pro Ser Glu Phe Asp Leu Thr Pro Tyr Ile Gln Asp Lys Asp Asn Leu
180 185 190
Leu Ala Val Glu Val Phe Lys His Ser Thr Ala Ser Trp Leu Glu Asp
195 200 205
Gln Asp Met Phe Arg Phe Ser Gly Ile Phe Arg Ser Val Glu Leu Leu
210 215 220
Gly Ile Pro Ala Thr His Leu Met Asp Met Asp Leu Lys Pro Arg Val
225 230 235 240
Ala Asp Asn Tyr Gln Asp Gly Ile Phe Asn Leu Lys Leu His Phe Ile
245 250 255
Gly Lys Lys Ala Gly Ser Phe His Leu Leu Val Lys Asp Ile Lys Gly
260 265 270
His Thr Leu Leu Glu Lys Asn Glu Asp Ile Lys Glu Asn Val Gln Ile
275 280 285
Asn Asn Glu Lys Phe Glu Asn Val His Leu Trp Asn Asn His Asp Pro
290 295 300
Tyr Leu Tyr Gln Leu Leu Ile Glu Val Tyr Asp Glu Gln Gln Asn Leu
305 310 315 320
Leu Glu Leu Ile Pro Phe Gln Phe Gly Phe Arg Arg Ile Glu Ile Ser
325 330 335
Pro Glu Lys Val Val Leu Leu Asn Gly Lys Arg Leu Ile Ile Asn Gly
340 345 350
Val Asn Arg His Glu Trp Asp Ala Lys Arg Gly Arg Ser Ile Thr Met
355 360 365
Ser Asp Met Thr Thr Asp Ile Asn Thr Phe Lys Glu Asn Asn Ile Asn
370 375 380
Ala Val Arg Thr Cys His Tyr Pro Asn Gln Ile Pro Trp Tyr Tyr Leu
385 390 395 400
Cys Asp Gln Asn Gly Ile Tyr Val Met Ala Glu Asn Asn Leu Glu Ser
405 410 415
His Gly Thr Trp Gln Lys Met Gly Glu Ile Glu Pro Ser Asp Asn Val
420 425 430
Pro Gly Ser Ile Pro Gln Trp Lys Glu Ala Val Ile Asp Arg Ala Arg
435 440 445
Asn Asn Tyr Glu Thr Phe Lys Asn His Thr Ser Ile Leu Phe Trp Ser
450 455 460
Leu Gly Asn Glu Ser Tyr Ala Gly Asp Asn Ile Ile Ala Met Asn Glu
465 470 475 480
Phe Tyr Lys Ser His Asp Asp Thr Arg Leu Val His Tyr Glu Gly Val
485 490 495
Val His Arg Pro Glu Leu Lys Asp Lys Ile Ser Asp Val Glu Ser Cys
500 505 510
Met Tyr Leu Pro Pro Lys Lys Val Glu Glu Tyr Leu Gln Asn Asp Pro
515 520 525
Pro Lys Pro Phe Met Glu Cys Glu Tyr Met His Asp Met Gly Asn Ser
530 535 540
Asp Gly Gly Met Gly Ser Tyr Ile Lys Leu Leu Asp Lys Tyr Pro Gln
545 550 555 560
Tyr Phe Gly Gly Phe Ile Trp Asp Phe Ile Asp Gln Ala Leu Leu Val
565 570 575
His Asp Glu Ile Ser Gly His Asp Val Leu Arg Tyr Gly Gly Asp Phe
580 585 590
Asp Asp Arg His Ser Asp Tyr Glu Phe Ser Gly Asp Gly Leu Met Phe
595 600 605
Ala Asp Arg Thr Pro Lys Pro Ala Met Gln Glu Val Arg Tyr Tyr Tyr
610 615 620
Gly Leu His Lys
625
<210> 21
<211> 318
<212> PRT
<213> 瑞士乳杆菌
<400> 21
Met Asp Tyr Thr Asn Asn Gln Leu His Ile Ile Tyr Gly Asp Ala Thr
1 5 10 15
Phe Gly Val Asn Gly Lys Asp Phe Gln Tyr Ile Phe Ser Tyr Glu Arg
20 25 30
Gly Gly Leu Glu Ser Leu Lys Val His Gly Lys Glu Trp Leu Tyr Arg
35 40 45
Val Pro Thr Pro Thr Phe Trp Arg Ala Thr Thr Asp Asn Asp Arg Gly
50 55 60
Ser Gly Phe Asn Leu Lys Ala Ala Gln Trp Leu Gly Ala Asp Met Phe
65 70 75 80
Thr Lys Cys Thr Asp Ile His Leu Lys Val Asp Arg His Asp Phe Ala
85 90 95
Glu Leu Pro Ile Ala Pro Phe Asn Asn Lys Phe Ser Asn His Glu Tyr
100 105 110
Ala Lys Ser Ala Glu Ile Ser Phe Thr Tyr Gln Thr Leu Thr Thr Pro
115 120 125
Ala Thr Asn Ala Lys Ile Ile Tyr Asn Ile Asp Asp Val Gly His Ile
130 135 140
Lys Val Thr Met Arg Tyr Tyr Gly Lys Lys Gly Leu Pro Pro Leu Pro
145 150 155 160
Val Ile Gly Ile Arg Leu Ile Met Pro Thr Ala Ala Thr Gly Phe Asp
165 170 175
Tyr Glu Gly Leu Ser Gly Glu Thr Tyr Pro Asp Arg Met Ala Gly Ala
180 185 190
Lys Glu Gly Lys Phe His Ile Asp Gly Leu Pro Val Thr Glu Tyr Leu
195 200 205
Val Pro Gln Glu Asn Gly Met His Met Gln Thr Lys Lys Leu Thr Ile
210 215 220
Asn Arg Glu Thr Thr Gln Asn Asn Val Asp Arg Thr Asn Glu Lys Phe
225 230 235 240
Ser Leu Ser Ile Gln Gln Ala Glu Lys Pro Phe Asn Phe Ser Cys Leu
245 250 255
Pro Tyr Thr Ala Glu Glu Leu Glu Asn Ala Thr His Ile Glu Glu Leu
260 265 270
Pro Leu Val Arg Arg Thr Val Leu Val Ile Ala Gly Ala Val Arg Gly
275 280 285
Val Gly Gly Ile Asp Ser Trp Gly Thr Asp Val Glu Ser Ala Tyr His
290 295 300
Ile Asn Pro Glu Leu Asp His Glu Phe Ser Phe Ile Leu Asn
305 310 315
<210> 22
<211> 1023
<212> PRT
<213> 长双歧杆菌(Bifidobacterium longum)
<400> 22
Met Thr Asp Val Thr His Val Asp Arg Ala Ser Gln Ala Trp Leu Thr
1 5 10 15
Asp Pro Thr Val Phe Glu Val Asn Arg Thr Pro Ala His Ser Ser His
20 25 30
Lys Trp Tyr Ala Arg Asp Pro Gln Ser Gly Gln Trp Ser Asp Leu Lys
35 40 45
Gln Ser Leu Asp Gly Glu Trp Arg Val Glu Val Val Gln Ala Ala Asp
50 55 60
Ile Asn Leu Glu Glu Glu Pro Ala Thr Ala Glu Ser Phe Asp Asp Ser
65 70 75 80
Ser Phe Glu Arg Ile Gln Val Pro Gly His Leu Gln Thr Ala Gly Leu
85 90 95
Met Asn His Lys Tyr Val Asn Val Gln Tyr Pro Trp Asp Gly His Glu
100 105 110
Asn Pro Leu Glu Pro Asn Ile Pro Glu Asn Asn His Val Ala Leu Tyr
115 120 125
Arg Arg Lys Phe Thr Val Ser Ala Pro Val Ala Asn Ala Lys Gln Ala
130 135 140
Gly Gly Ser Val Ser Ile Val Phe His Gly Met Ala Thr Ala Ile Tyr
145 150 155 160
Val Trp Val Asn Gly Ala Phe Val Gly Tyr Gly Glu Asp Gly Phe Thr
165 170 175
Pro Asn Glu Phe Asp Ile Thr Glu Leu Leu His Asp Gly Glu Asn Val
180 185 190
Val Ala Val Ala Cys Tyr Glu Tyr Ser Ser Ala Ser Trp Leu Glu Asp
195 200 205
Gln Asp Phe Trp Arg Leu His Gly Leu Phe Arg Ser Val Glu Leu Ala
210 215 220
Ala Arg Pro His Val His Ile Glu Asn Thr Gln Ile Glu Ala Asp Trp
225 230 235 240
Asp Pro Glu Ala Gly Thr Ala Ser Leu Asp Ala Ala Leu Thr Val Leu
245 250 255
Asn Ala Ala Asp Ala Ala Thr Val Arg Ala Thr Leu Lys Asp Ala Asp
260 265 270
Gly Asn Thr Val Trp Gln Thr Thr Gly Asp Ala Glu Ala Gln Thr Ala
275 280 285
Ile Ser Ser Gly Pro Leu Gln Gly Ile Ala Pro Trp Ser Ala Glu Ser
290 295 300
Pro Thr Leu Tyr Glu Leu Asp Val Asp Val Ile Asp Gln Ala Gly Asp
305 310 315 320
Val Ile Glu Cys Thr Ser Gln Lys Val Gly Phe Arg Arg Phe Arg Ile
325 330 335
Glu Asp Gly Ile Leu Thr Ile Asn Gly Lys Arg Ile Val Phe Lys Gly
340 345 350
Ala Asp Arg His Glu Phe Asp Ala Glu Gln Gly Arg Ala Ile Thr Glu
355 360 365
Gln Asp Met Ile Asp Asp Val Val Phe Cys Lys Arg His Asn Ile Asn
370 375 380
Ser Ile Arg Thr Ser His Tyr Pro Asn Gln Glu Arg Trp Tyr Glu Leu
385 390 395 400
Cys Asp Glu Tyr Gly Ile Tyr Leu Ile Asp Glu Ala Asn Leu Glu Ala
405 410 415
His Gly Ser Trp Ser Leu Pro Gly Asp Val Leu Thr Glu Asp Thr Ile
420 425 430
Val Pro Gly Ser Lys Arg Glu Trp Glu Gly Ala Cys Val Asp Arg Val
435 440 445
Asn Ser Met Met Arg Arg Asp Tyr Asn His Pro Ser Val Leu Ile Trp
450 455 460
Ser Leu Gly Asn Glu Ser Tyr Val Gly Asp Val Phe Arg Ala Met Tyr
465 470 475 480
Lys His Val His Asp Ile Asp Pro Asn Arg Pro Val His Tyr Glu Gly
485 490 495
Val Thr His Asn Arg Asp Tyr Asp Asp Val Thr Asp Ile Glu Thr Arg
500 505 510
Met Tyr Ser His Ala Asp Glu Ile Glu Lys Tyr Leu Lys Asp Asp Pro
515 520 525
Lys Lys Pro Tyr Leu Ser Cys Glu Tyr Met His Ala Met Gly Asn Ser
530 535 540
Val Gly Asn Met Asp Glu Tyr Thr Ala Leu Glu Arg Tyr Pro Lys Tyr
545 550 555 560
Gln Gly Gly Phe Ile Trp Asp Phe Ile Asp Gln Ala Ile Tyr Ala Thr
565 570 575
Gln Pro Asp Gly Thr Arg Ser Leu Arg Tyr Gly Gly Asp Phe Gly Asp
580 585 590
Arg Pro Ser Asp Tyr Glu Phe Ser Gly Asp Gly Leu Leu Phe Ala Asn
595 600 605
Arg Lys Pro Ser Pro Lys Ala Gln Glu Val Lys Gln Leu Tyr Ser Asn
610 615 620
Val His Ile Asp Val Thr Lys Asp Ser Val Ser Val Lys Asn Asp Asn
625 630 635 640
Leu Phe Thr Ala Thr Gly Asp Tyr Val Phe Val Leu Ser Val Leu Ala
645 650 655
Asp Gly Lys Pro Val Trp Gln Ser Thr Arg Arg Phe Asp Val Pro Ala
660 665 670
Gly Glu Thr Arg Thr Phe Asp Val Ala Trp Pro Val Ala Ala Tyr Arg
675 680 685
Ala Asp Ala Arg Glu Leu Val Leu Gln Val Ser Gln Arg Leu Ala Lys
690 695 700
Ala Thr Asp Trp Ala Glu Ser Gly Tyr Glu Leu Ala Phe Gly Gln Thr
705 710 715 720
Val Val Pro Ala Asp Ala Thr Ala Thr Pro Asp Thr Lys Pro Ala Asp
725 730 735
Gly Thr Ile Thr Val Gly Arg Trp Asn Ala Gly Val Arg Gly Ala Gly
740 745 750
Arg Glu Val Leu Leu Ser Arg Thr Gln Gly Gly Met Val Ser Tyr Thr
755 760 765
Phe Ala Gly Asn Glu Phe Val Leu Arg Arg Pro Ala Ile Thr Thr Phe
770 775 780
Arg Pro Leu Thr Asp Asn Asp Arg Gly Ala Gly His Gly Phe Glu Arg
785 790 795 800
Val Gln Trp Leu Gly Ala Gly Arg Tyr Ala Arg Cys Val Asp Asn Val
805 810 815
Leu Glu Gln Ile Asp Asp Ser Thr Leu Lys Gly Thr Tyr Thr Tyr Glu
820 825 830
Leu Ala Thr Ala Gln Arg Thr Lys Val Thr Val Ser Tyr Thr Ala His
835 840 845
Thr Asp Gly Arg Val Asn Leu His Val Glu Tyr Pro Gly Glu Gln Gly
850 855 860
Asp Leu Pro Thr Ile Pro Ala Phe Gly Ile Glu Trp Thr Leu Pro Val
865 870 875 880
Gln Tyr Thr Asn Leu Arg Phe Phe Gly Thr Gly Pro Ala Glu Thr Tyr
885 890 895
Leu Asp Arg Lys His Ala Lys Leu Gly Val Trp Ser Thr Asn Ala Phe
900 905 910
Ala Asp His Ala Pro Tyr Leu Met Pro Gln Glu Thr Gly Asn His Glu
915 920 925
Asp Val Arg Trp Ala Glu Ile Thr Asp Asp His Gly His Gly Met Arg
930 935 940
Val Ser Arg Ala Asp Gly Ala Ala Pro Phe Ala Val Ser Leu Leu Pro
945 950 955 960
Tyr Ser Ser Phe Met Leu Glu Glu Ala Gln His Gln Asp Glu Leu Pro
965 970 975
Lys Pro Lys His Met Phe Leu Arg Val Leu Ala Ala Gln Met Gly Val
980 985 990
Gly Gly Asp Asp Ser Trp Met Ser Pro Val His Pro Gln Tyr His Ile
995 1000 1005
Pro Ala Asp Lys Pro Ile Ser Leu Asp Val Asp Leu Glu Leu Ile
1010 1015 1020
<210> 23
<211> 628
<212> PRT
<213> 罗伊氏乳杆菌(Lactobacillus reuteri)
<400> 23
Met Asp Ala Asp Ile Lys Trp Leu Asp Glu Pro Glu Thr Phe Arg Val
1 5 10 15
Asn Gln Leu Pro Ala His Ser Asp His Tyr Tyr Tyr Gly Asn Tyr Asp
20 25 30
Glu Trp Arg His Asn Asn Ser Arg Phe Ala Gln Asn Leu Asp Gly Gln
35 40 45
Trp Gln Phe Asn Phe Ala Glu Asn Leu Arg Glu Arg Glu Asn Asp Phe
50 55 60
Tyr Lys Met Asp Tyr Asp Ser Ser Ser Phe Gly Thr Ile Glu Val Pro
65 70 75 80
Ser Glu Ile Glu Leu Asn Asn Tyr Ala Gln Asn Asn Tyr Ile Asn Thr
85 90 95
Leu Ile Pro Trp Glu Gly Lys Ile Tyr Arg Arg Pro Ala Tyr Thr Leu
100 105 110
Ser Pro Asp Asp Ala Gln Glu Gly Ser Phe Ser Asp Gly Asp Asp Asn
115 120 125
Thr Ile Gly Glu Tyr Leu Lys His Phe Asp Leu Asp Pro Ser Leu Arg
130 135 140
Gly Lys Gln Val Arg Ile Arg Phe Asp Gly Val Glu Arg Ala Met Tyr
145 150 155 160
Val Trp Leu Asn Gly His Phe Ile Gly Tyr Ala Glu Asp Ser Phe Thr
165 170 175
Pro Ser Glu Phe Asp Leu Thr Pro Tyr Ile Gln Asp Glu Gly Asn Val
180 185 190
Leu Ala Val Glu Val Phe Lys His Ser Thr Ala Ser Trp Ile Glu Asp
195 200 205
Gln Asp Met Phe Arg Phe Ser Gly Ile Phe Arg Ser Val Asn Leu Leu
210 215 220
Ala Gln Pro Leu Val His Val Glu Asp Leu Asn Ile Arg Pro Ile Val
225 230 235 240
Thr Asp Asn Tyr Gln Asp Gly Ile Phe Asn Val Glu Leu Gln Leu His
245 250 255
Gly Glu Lys Thr Gly Asn Val Asn Val Arg Val Ile Asp Asn Asp Gly
260 265 270
Asn Thr Leu Val Asn Glu Thr His Pro Val Asp Ser Thr Val Lys Val
275 280 285
Gln Asp Gln Phe Leu Glu Asn Val His Leu Trp Asp Asn His Asp Pro
290 295 300
Tyr Leu Tyr Gln Leu Leu Ile Glu Ile Arg Asp Asp Glu Gly Asn Leu
305 310 315 320
Val Glu Leu Val Pro Tyr Arg Phe Gly Phe Arg Arg Ile Glu Ile Asn
325 330 335
Lys Asp His Val Val Leu Leu Asn Gly Gln Arg Leu Ile Ile Asn Gly
340 345 350
Val Asn Arg His Glu Trp Asp Ala Arg Arg Gly Arg Ala Ile Thr Met
355 360 365
Asp Asp Met Thr Ser Asp Ile His Thr Phe Lys Glu Asn Asn Ile Asn
370 375 380
Ala Val Arg Thr Cys His Tyr Pro Asp Gln Ile Pro Trp Tyr Tyr Leu
385 390 395 400
Cys Asp Asp Asn Gly Ile Tyr Met Met Ala Glu Asn Asn Leu Glu Ser
405 410 415
His Ala Thr Trp Gln Lys Met Gly Ala Ile Glu Pro Ser Tyr Asn Val
420 425 430
Pro Gly Ser Val Pro Gln Trp Arg Asp Val Val Val Asp Arg Ala Arg
435 440 445
Thr Asn Tyr Glu Thr Phe Lys Asn His Pro Ser Ile Leu Phe Trp Ser
450 455 460
Leu Gly Asn Glu Ser Tyr Ala Gly Asp Asn Ile Val Lys Met Asn Glu
465 470 475 480
Phe Tyr Lys Lys His Asp Asp Ser Arg Leu Val His Tyr Glu Gly Val
485 490 495
Cys His Thr Pro Glu Tyr Arg Asp Arg Ile Ser Asp Val Glu Ser Trp
500 505 510
Met Tyr Leu Pro Pro Lys Glu Val Glu Glu Tyr Leu Lys Asn Asn Pro
515 520 525
Asp Lys Pro Phe Met Glu Cys Glu Tyr Met His Asp Met Gly Asn Ser
530 535 540
Asp Gly Gly Met Gly Ser Tyr Ile Ser Leu Leu Asp Lys Tyr Pro Gln
545 550 555 560
Tyr Phe Gly Gly Phe Ile Trp Asp Phe Ile Asp Gln Ala Leu Leu Val
565 570 575
Lys Asp Pro Val Ser Gly Gln Glu Val Met Arg Tyr Gly Gly Asp Phe
580 585 590
Asp Asp Arg His Ser Asp Tyr Glu Phe Ser Gly Asp Gly Leu Met Phe
595 600 605
Ala Asp Arg Thr Pro Lys Pro Ala Met Gln Glu Val Arg Tyr Tyr Tyr
610 615 620
Gly Leu His Lys
625
<210> 24
<211> 319
<212> PRT
<213> 罗伊氏乳杆菌
<400> 24
Met Ala Tyr Thr Asn Lys Leu Arg Val Ile Tyr Gly Asp Ala Thr Leu
1 5 10 15
Gly Leu Ser Gly Asp Gly Phe His Tyr Ile Phe Ser Tyr Glu Arg Gly
20 25 30
Gly Leu Glu Ser Leu Lys Leu Asn Gly Lys Glu Trp Leu Tyr Arg Glu
35 40 45
Pro Met Pro Thr Phe Trp Arg Ala Thr Thr Asp Asn Asp Arg Gly Ser
50 55 60
Gly Phe Asn Ile Arg Ser Ala Gln Trp Leu Ala Ala Asp Thr Phe His
65 70 75 80
Lys Cys Val Gly Ile Asp Leu Thr Val Asp Asn Gln His Phe Ala Glu
85 90 95
Leu Pro Ile Ala Pro Ile Thr Asn Glu Phe Ser Asp Pro Val Ser Ala
100 105 110
Glu Ser Val Lys Ile Lys Tyr Thr Phe Ala Thr Leu Thr Val Pro Ala
115 120 125
Thr Gln Val Thr Val Ile Tyr Glu Val Asn Gly Gln Gly Glu Ile Lys
130 135 140
Val Thr Met His Tyr Tyr Gly His Glu Asp Leu Pro Gly Leu Pro Val
145 150 155 160
Val Gly Met Arg Phe Ile Met Pro Thr Val Ala Thr Gly Phe Asp Tyr
165 170 175
Gln Gly Leu Ser Gly Glu Thr Tyr Pro Asp Arg Met Ala Gly Ala Thr
180 185 190
Glu Gly Thr Phe His Val Asp Gly Leu Pro Val Thr Lys Tyr Leu Val
195 200 205
Pro Gln Glu Asn Gly Met His Met Ala Thr His Ala Leu Thr Ile Thr
210 215 220
Arg Asp Ser Thr Gln Asn Asn Ala Asp His Ser Arg Glu Pro Phe Ser
225 230 235 240
Leu Thr Val Lys Gln Asp Ala Gln Pro Phe Ala Phe Ser Cys Leu Pro
245 250 255
Tyr Thr Ala Glu Glu Leu Glu Asn Ala Thr His Ile Glu Glu Leu Pro
260 265 270
Leu Ala Arg Arg Thr Val Leu Val Val Ala Gly Ala Val Arg Gly Val
275 280 285
Gly Gly Ile Asp Ser Trp Gly Ala Asp Val Glu Glu Gln Tyr His Ile
290 295 300
Pro Ala Asp Arg Asp Val Glu Phe Ser Phe Val Leu Asn Ala Lys
305 310 315
<210> 25
<211> 1007
<212> PRT
<213> 德氏乳杆菌乳酸亚种
<400> 25
Met Ser Asn Lys Leu Val Lys Glu Lys Arg Val Asp Gln Ala Asp Leu
1 5 10 15
Ala Trp Leu Thr Asp Pro Glu Val Tyr Glu Val Asn Thr Ile Pro Pro
20 25 30
His Ser Asp His Glu Ser Phe Gln Ser Gln Glu Glu Leu Glu Glu Gly
35 40 45
Lys Ser Ser Leu Val Gln Ser Leu Asp Gly Asn Trp Leu Ile Asp Tyr
50 55 60
Ala Glu Asn Gly Gln Gly Pro Ile Asn Phe Tyr Ala Glu Asp Phe Asp
65 70 75 80
Asp Ser Asn Phe Lys Ser Val Lys Val Pro Gly Asn Leu Glu Leu Gln
85 90 95
Gly Phe Gly Gln Pro Gln Tyr Val Asn Ile Gln Tyr Pro Trp Asp Gly
100 105 110
Ser Glu Glu Ile Phe Pro Pro Gln Val Pro Ser Lys Ile Pro Leu Ala
115 120 125
Ser Tyr Val Arg Tyr Phe Asp Leu Asp Glu Ala Leu Trp Asp Lys Glu
130 135 140
Val Ser Leu Lys Phe Ala Gly Ala Ala Thr Ala Ile Tyr Val Trp Leu
145 150 155 160
Asn Gly His Phe Val Gly Tyr Gly Glu Asp Ser Phe Thr Pro Ser Glu
165 170 175
Phe Met Val Thr Lys Phe Leu Lys Lys Glu Gly Asn Arg Leu Ala Val
180 185 190
Ala Leu Tyr Lys Tyr Ser Ser Ala Ser Trp Leu Glu Asp Gln Asp Phe
195 200 205
Trp Arg Leu Ser Gly Leu Phe Arg Ser Val Thr Leu Glu Ala Lys Pro
210 215 220
Leu Leu His Leu Glu Asp Leu Lys Leu Thr Ala Ser Leu Thr Asp Asn
225 230 235 240
Tyr Gln Lys Gly Lys Leu Glu Val Glu Ala Asn Ile Ala Tyr Arg Leu
245 250 255
Pro Asn Ala Ser Phe Lys Leu Glu Val Arg Asp Ser Glu Gly Asp Leu
260 265 270
Val Ala Glu Lys Val Gly Pro Ile Arg Ser Glu Lys Leu Asp Phe Ser
275 280 285
Leu Ala Asp Leu Pro Val Ala Ala Trp Ser Ala Glu Lys Pro Asn Leu
290 295 300
Tyr Gln Val Arg Leu Tyr Leu Tyr Gln Ala Gly Ser Leu Leu Glu Val
305 310 315 320
Ser Arg Gln Glu Val Gly Phe Arg Asn Phe Glu Leu Lys Asp Gly Ile
325 330 335
Met Tyr Leu Asn Gly Gln Arg Ile Val Phe Lys Gly Val Asn Arg His
340 345 350
Glu Phe Asp Ser Lys Leu Gly Arg Ala Ile Thr Glu Ala Asp Met Ile
355 360 365
Trp Asp Ile Lys Thr Met Lys Gln Ser Asn Ile Asn Ala Val Arg Cys
370 375 380
Ser His Tyr Pro Asn Gln Ser Leu Phe Tyr Arg Leu Cys Asp Lys Tyr
385 390 395 400
Gly Leu Tyr Val Ile Asp Glu Ala Asn Leu Glu Ser His Gly Thr Trp
405 410 415
Glu Lys Val Gly His Glu Asp Pro Ser Phe Asn Val Pro Gly Asp Asp
420 425 430
Gln His Trp Leu Gly Ala Ser Leu Ser Arg Val Lys Asn Met Met Ala
435 440 445
Arg Asp Lys Asn His Ala Ser Ile Leu Ile Trp Ser Leu Gly Asn Glu
450 455 460
Ser Tyr Ala Gly Thr Val Phe Ala Gln Met Ala Asp Tyr Val Arg Lys
465 470 475 480
Ala Asp Pro Thr Arg Val Gln His Tyr Glu Gly Val Thr His Asn Arg
485 490 495
Lys Phe Asp Asp Ala Thr Gln Ile Glu Ser Arg Met Tyr Ala Pro Ala
500 505 510
Lys Glu Ile Glu Glu Tyr Leu Thr Lys Lys Pro Ala Lys Pro Phe Ile
515 520 525
Ser Val Glu Tyr Ala His Ala Met Gly Asn Ser Val Gly Asp Leu Ala
530 535 540
Ala Tyr Thr Ala Leu Glu Lys Tyr Pro His Tyr Gln Gly Gly Phe Ile
545 550 555 560
Trp Asp Trp Ile Asp Gln Gly Leu Glu Lys Asp Gly His Leu Leu Tyr
565 570 575
Gly Gly Asp Phe Asp Asp Arg Pro Thr Asp Tyr Glu Phe Cys Gly Asp
580 585 590
Gly Leu Val Phe Ala Asp Arg Thr Thr Ser Pro Lys Leu Ala Asn Val
595 600 605
Lys Ala Leu Tyr Ser Asn Leu Lys Leu Glu Val Lys Asp Gly Gln Leu
610 615 620
Phe Ile Lys Asn Asp Asn Leu Phe Thr Asn Ser Ser Ala Tyr Tyr Phe
625 630 635 640
Leu Thr Ser Leu Leu Val Asp Gly Lys Leu Thr Tyr Gln Ser Gln Pro
645 650 655
Leu Thr Phe Gly Leu Glu Pro Gly Glu Ser Gly Thr Phe Ala Leu Pro
660 665 670
Trp Pro Glu Val Glu Asp Glu Lys Gly Glu Ile Val Tyr Gln Val Thr
675 680 685
Ala His Leu Lys Glu Asp Leu Pro Trp Ala Asp Glu Gly Phe Thr Val
690 695 700
Ala Glu Ala Glu Glu Ala Val Thr Lys Leu Pro Glu Phe Tyr Pro Ala
705 710 715 720
Gly Arg Pro Glu Leu Val Asp Ser Asp Phe Asn Leu Gly Leu Lys Gly
725 730 735
Asn Gly Phe Arg Ile Leu Phe Ser Lys Ala Lys Gly Trp Pro Val Ser
740 745 750
Ile Lys Tyr Ala Gly Arg Glu Tyr Leu Lys Arg Leu Pro Glu Phe Thr
755 760 765
Phe Trp Arg Ala Leu Thr Asp Asn Asp Arg Gly Ala Gly Tyr Gly Tyr
770 775 780
Asp Leu Ala Lys Trp Glu Asn Ala Gly Lys Tyr Ala Arg Leu Gln Asp
785 790 795 800
Ile Ser Tyr Glu Ile Lys Glu Asn Ser Ala Leu Val Lys Thr Thr Phe
805 810 815
Thr Leu Pro Val Ala Leu Lys Gly Asp Leu Thr Ile Thr Tyr Glu Val
820 825 830
Asp Ser Leu Gly Lys Ile Ala Val Thr Ala Asn Phe Pro Gly Ala Val
835 840 845
Glu Asn Gly Leu Leu Pro Ala Phe Gly Leu Asn Phe Ala Leu Pro Lys
850 855 860
Glu Leu Ser Asp Tyr Arg Tyr Tyr Gly Leu Gly Pro Asn Glu Ser Tyr
865 870 875 880
Ala Asp Arg Leu Glu Gly Ser Tyr Leu Gly Ile Tyr Gln Gly Met Val
885 890 895
Glu Lys Asn Phe Thr Pro Tyr Leu Arg Pro Gln Glu Ala Gly Asn Arg
900 905 910
Ser Lys Val Arg Tyr Tyr Gln Leu Phe Asp Glu Glu Gly Gly Leu Glu
915 920 925
Phe Thr Ala Asn Gly Ala Asp Leu Asn Leu Ser Ala Leu Pro Tyr Ser
930 935 940
Ala Ala Gln Ile Glu Ala Ala Asp His Ala Phe Glu Leu Thr Asn Asn
945 950 955 960
Tyr Thr Trp Val Arg Ala Leu Ala Ala Gln Met Gly Val Gly Gly Asp
965 970 975
Asp Ser Trp Gly Gln Lys Val His Pro Glu Phe Cys Leu Asp Ala Gln
980 985 990
Glu Ala Arg Gln Leu Lys Leu Val Ile Gln Pro Leu Leu Leu Lys
995 1000 1005
<210> 26
<211> 628
<212> PRT
<213> 瑞士乳杆菌
<400> 26
Met Gln Ala Asn Ile Asn Trp Leu Asp Asn Pro Glu Val Phe Arg Val
1 5 10 15
Asn Gln Leu Pro Ala His Ser Asp His Pro Phe Phe Arg Asp Tyr Arg
20 25 30
Glu Trp Gln Lys Gln His Ser Ser Tyr Gln Gln Ser Leu Asn Gly Lys
35 40 45
Trp Lys Phe His Phe Ser Ala Asn Pro Met Asp Arg Pro Gln Asp Phe
50 55 60
Tyr Gln Arg Asp Phe Asp Ser Ser Asn Phe Asp Ser Ile Pro Val Pro
65 70 75 80
Ser Glu Ile Glu Leu Ser Asn Tyr Thr Gln Asn Gln Tyr Ile Asn Val
85 90 95
Leu Phe Pro Trp Glu Gly Lys Ile Phe Arg Arg Pro Ala Tyr Ala Leu
100 105 110
Asp Pro Asn Asp His Glu Glu Gly Ser Phe Ser Lys Gly Ala Asp Asn
115 120 125
Thr Val Gly Ser Tyr Leu Lys Arg Phe Asp Leu Ser Ser Ala Leu Ile
130 135 140
Gly Lys Asp Val His Ile Lys Phe Glu Gly Val Glu Gln Ala Met Tyr
145 150 155 160
Val Trp Leu Asn Gly His Phe Val Gly Tyr Ala Glu Asp Ser Phe Thr
165 170 175
Pro Ser Glu Phe Asp Leu Thr Pro Tyr Ile Gln Glu Lys Asp Asn Leu
180 185 190
Leu Ala Val Glu Val Phe Lys His Ser Thr Ala Ser Trp Leu Glu Asp
195 200 205
Gln Asp Met Phe Arg Phe Ser Gly Ile Phe Arg Ser Val Glu Leu Leu
210 215 220
Gly Ile Pro Ala Thr His Leu Met Asp Met Asp Leu Lys Pro Arg Val
225 230 235 240
Ala Asp Asn Tyr Gln Asp Gly Ile Phe Asn Leu Lys Leu His Phe Ile
245 250 255
Gly Lys Lys Ala Gly Ser Phe His Leu Leu Val Lys Asp Ile Lys Gly
260 265 270
His Thr Leu Leu Glu Lys Asn Glu Asp Ile Lys Glu Asn Val Gln Ile
275 280 285
Asn Asn Glu Lys Phe Glu Asn Val His Leu Trp Asn Asn His Asp Pro
290 295 300
Tyr Leu Tyr Gln Leu Leu Ile Glu Val Tyr Asp Glu Gln Gln Asn Leu
305 310 315 320
Leu Glu Leu Ile Pro Phe Gln Phe Gly Phe Arg Arg Ile Glu Ile Ser
325 330 335
Pro Glu Lys Val Val Leu Leu Asn Gly Lys Arg Leu Ile Ile Asn Gly
340 345 350
Val Asn Arg His Glu Trp Asp Ala Lys Arg Gly Arg Ser Ile Thr Met
355 360 365
Ser Asp Met Thr Thr Asp Ile Asn Thr Phe Lys Glu Asn Asn Ile Asn
370 375 380
Ala Val Arg Thr Cys His Tyr Pro Asn Gln Ile Pro Trp Tyr Tyr Leu
385 390 395 400
Cys Asp Gln Asn Gly Ile Tyr Val Met Ala Glu Asn Asn Leu Glu Ser
405 410 415
His Gly Thr Trp Gln Lys Met Gly Glu Ile Glu Pro Ser Asp Asn Val
420 425 430
Pro Gly Ser Ile Pro Gln Trp Lys Glu Ala Val Ile Asp Arg Ala Arg
435 440 445
Asn Asn Tyr Glu Thr Phe Lys Asn His Thr Ser Ile Leu Phe Trp Ser
450 455 460
Leu Gly Asn Glu Ser Tyr Ala Gly Asp Asn Ile Ile Ala Met Asn Glu
465 470 475 480
Phe Tyr Lys Ser His Asp Asp Thr Arg Leu Val His Tyr Glu Gly Val
485 490 495
Val His Arg Pro Glu Leu Lys Asp Lys Ile Ser Asp Val Glu Ser Cys
500 505 510
Met Tyr Leu Pro Pro Lys Lys Val Glu Glu Tyr Leu Gln Asn Asp Pro
515 520 525
Pro Lys Pro Phe Met Glu Cys Glu Tyr Met His Asp Met Gly Asn Ser
530 535 540
Asn Gly Gly Met Asp Ser Tyr Ile Lys Leu Leu Asp Lys Tyr Pro Gln
545 550 555 560
Tyr Phe Gly Gly Phe Ile Trp Asp Phe Ile Asp Gln Ala Leu Leu Val
565 570 575
His Asp Glu Ile Ser Gly His Asp Val Leu Arg Tyr Gly Gly Asp Phe
580 585 590
Asp Asp Arg His Ser Asp Tyr Glu Phe Ser Gly Asp Gly Leu Met Phe
595 600 605
Ala Asp Arg Lys Pro Lys Pro Ala Met Gln Glu Val Arg Tyr Tyr Tyr
610 615 620
Gly Leu His Lys
625
<210> 27
<211> 318
<212> PRT
<213> 瑞士乳杆菌
<400> 27
Met Asp Tyr Thr Asn Asn Gln Leu His Ile Ile Tyr Gly Asp Ala Thr
1 5 10 15
Phe Gly Val Asn Gly Lys Asp Phe Gln Tyr Ile Phe Ser Tyr Glu Arg
20 25 30
Gly Gly Leu Glu Ser Leu Lys Val His Gly Lys Glu Trp Leu Tyr Arg
35 40 45
Val Pro Thr Pro Thr Phe Trp Arg Ala Thr Thr Asp Asn Asp Arg Gly
50 55 60
Ser Gly Phe Asn Leu Lys Ala Ala Gln Trp Leu Gly Ala Asp Met Phe
65 70 75 80
Thr Lys Cys Thr Asp Ile His Leu Lys Val Asp Arg His Asp Phe Ala
85 90 95
Glu Leu Pro Ile Ala Pro Phe Asn Asn Lys Phe Ser Asn His Glu Tyr
100 105 110
Ala Lys Ser Ala Glu Ile Ser Phe Thr Tyr Gln Thr Leu Thr Thr Pro
115 120 125
Ala Thr Asn Ala Lys Ile Ile Tyr Asn Ile Asp Asp Gly Gly His Ile
130 135 140
Lys Val Thr Met Arg Tyr Tyr Gly Lys Lys Gly Leu Pro Pro Leu Pro
145 150 155 160
Val Ile Gly Ile Arg Leu Ile Met Pro Thr Ala Ala Thr Gly Phe Asp
165 170 175
Tyr Glu Gly Leu Ser Gly Glu Thr Tyr Pro Asp Arg Met Ala Gly Ala
180 185 190
Lys Glu Gly Lys Phe His Ile Asp Gly Leu Pro Val Thr Glu Tyr Leu
195 200 205
Val Pro Gln Glu Asn Gly Met His Met Gln Thr Lys Lys Leu Thr Ile
210 215 220
Asn Arg Glu Thr Thr Gln Asn Asn Val Asp Arg Thr Asn Glu Lys Phe
225 230 235 240
Ser Leu Ser Ile Gln Gln Ala Glu Lys Pro Phe Asn Phe Ser Cys Leu
245 250 255
Pro Tyr Thr Ala Glu Glu Leu Glu Asn Ala Thr His Ile Glu Glu Leu
260 265 270
Pro Leu Val Arg Arg Thr Val Leu Val Ile Ala Gly Ala Val Arg Gly
275 280 285
Val Gly Gly Ile Asp Ser Trp Gly Thr Asp Val Glu Ser Ala Tyr His
290 295 300
Ile Asn Pro Asp Leu Asp His Glu Phe Ser Phe Ile Leu Asn
305 310 315
<210> 28
<211> 626
<212> PRT
<213> 卷曲乳杆菌(Lactobacillus crispatus)
<400> 28
Met Lys Ala Asn Ile Lys Trp Leu Asp Asp Pro Glu Val Phe Arg Ile
1 5 10 15
Asn Gln Leu Pro Ala His Ser Asp His Pro Phe Tyr Lys Asp Tyr Arg
20 25 30
Glu Trp Gln Lys His Ser Ser Ser Phe Lys Gln Ser Leu Asn Gly Ala
35 40 45
Trp Gln Phe His Phe Ser Lys Asp Pro Gln Ser Arg Pro Ile Asp Phe
50 55 60
Tyr Lys Leu Ser Phe Asp Ser Ser Ser Phe Asp Thr Ile Pro Val Pro
65 70 75 80
Ser Glu Ile Glu Leu Asn Gly Tyr Ala Gln Asn Gln Tyr Thr Asn Ile
85 90 95
Leu Tyr Pro Trp Glu Ser Lys Ile Tyr Arg Lys Pro Ala Tyr Thr Leu
100 105 110
Gly Arg Gly Ile Lys Asp Gly Asp Phe Ser Gln Gly Lys Asp Asn Thr
115 120 125
Val Gly Ser Tyr Leu Lys His Phe Asp Leu Asn Pro Ala Leu Ala Gly
130 135 140
His Asp Ile His Ile Gln Phe Glu Gly Val Glu Arg Ala Met Tyr Val
145 150 155 160
Tyr Leu Asn Gly His Phe Ile Gly Tyr Ala Glu Asp Ser Phe Thr Pro
165 170 175
Ser Glu Phe Asp Leu Thr Pro Tyr Ile Gln Ala Lys Asp Asn Ile Leu
180 185 190
Ala Val Glu Val Phe Lys His Ser Thr Ala Ser Trp Leu Glu Asp Gln
195 200 205
Asp Met Phe Arg Phe Ser Gly Ile Phe Arg Ser Val Glu Leu Leu Ala
210 215 220
Leu Pro Arg Thr His Leu Met Asp Leu Asp Ile Lys Pro Thr Val Val
225 230 235 240
Asn Asp Tyr His Asp Gly Val Phe Asn Ala Lys Leu His Phe Met Gly
245 250 255
Lys Thr Ser Gly Asn Val His Val Leu Ile Glu Asp Ile Asp Gly Lys
260 265 270
Thr Leu Leu Asn Lys Lys Leu Pro Leu Lys Ser Thr Val Glu Ile Glu
275 280 285
Asn Glu Thr Phe Ala Asn Val His Leu Trp Asp Asn His Asp Pro Tyr
290 295 300
Leu Tyr Gln Leu Ile Ile Glu Val His Asp Gln Asp Gly Lys Leu Val
305 310 315 320
Glu Leu Ile Pro Tyr Gln Phe Gly Phe Arg Lys Ile Glu Ile Thr Lys
325 330 335
Asp His Val Val Leu Leu Asn Gly Lys Arg Leu Ile Ile Asn Gly Val
340 345 350
Asn Arg His Glu Trp Asp Ala Lys Arg Gly Arg Ser Ile Thr Leu Ala
355 360 365
Asp Met Lys Gln Asp Ile Ala Thr Phe Lys His Asn Asn Ile Asn Ala
370 375 380
Val Arg Thr Cys His Tyr Pro Asn Gln Ile Pro Trp Tyr Tyr Leu Cys
385 390 395 400
Asp Gln Asn Gly Ile Tyr Met Met Ala Glu Asn Asn Leu Glu Ser His
405 410 415
Gly Thr Trp Gln Lys Leu Gly Gln Val Glu Ala Thr Ser Asn Val Pro
420 425 430
Gly Ser Ile Pro Glu Trp Arg Glu Val Val Val Asp Arg Ala Arg Ser
435 440 445
Asn Tyr Glu Thr Phe Lys Asn His Thr Ala Ile Leu Phe Trp Ser Leu
450 455 460
Gly Asn Glu Ser Tyr Ala Gly Ser Asn Ile Ala Ala Met Asn Lys Leu
465 470 475 480
Tyr Lys Asp His Asp Ser Ser Arg Leu Thr His Tyr Glu Gly Val Phe
485 490 495
His Ala Pro Glu Phe Lys Lys Glu Ile Ser Asp Leu Glu Ser Cys Met
500 505 510
Tyr Leu Pro Pro Lys Glu Ala Glu Glu Tyr Leu Gln Asn Pro Lys Lys
515 520 525
Pro Leu Val Glu Cys Glu Tyr Met His Asp Met Gly Asn Ser Asp Gly
530 535 540
Gly Ile Gly Ser Tyr Ile Lys Leu Ile Asp Lys Tyr Pro Gln Tyr Met
545 550 555 560
Gly Gly Phe Ile Trp Asp Phe Ile Asp Gln Ala Leu Leu Val His Asp
565 570 575
Pro Val Ser Gly Gln Asp Val Leu Arg Tyr Gly Gly Asp Phe Asp Asp
580 585 590
Arg His Ser Asp Tyr Glu Phe Ser Gly Asp Gly Leu Met Phe Ala Asp
595 600 605
Arg Thr Pro Lys Pro Ala Met Gln Glu Val Arg Tyr Tyr Tyr Gly Leu
610 615 620
His Lys
625
<210> 29
<211> 316
<212> PRT
<213> 卷曲乳杆菌
<400> 29
Met Ala Tyr Thr Asn Asn Leu His Val Val Tyr Gly Glu Ala Ser Leu
1 5 10 15
Gly Val Asn Gly Gln Asp Phe Ala Tyr Leu Phe Ser Tyr Glu Arg Gly
20 25 30
Val Leu Glu Ser Leu Lys Ile Lys Asp Lys Glu Trp Leu Tyr Arg Thr
35 40 45
Pro Thr Pro Thr Phe Trp Arg Ala Thr Thr Asp Asn Asp Arg Gly Ser
50 55 60
Gly Phe Asn Gln Lys Ala Ala Gln Trp Leu Gly Ala Asp Met Phe Thr
65 70 75 80
Lys Cys Val Gly Ile His Val Gln Val Asp Asp His Gln Phe Asp Glu
85 90 95
Leu Pro Ile Ala Pro Ile Asn Asn Gln Phe Ser Asn Gln Glu Phe Ala
100 105 110
His Glu Val Lys Val Ala Phe Asp Tyr Glu Thr Leu Thr Thr Pro Ala
115 120 125
Thr Lys Val Lys Ile Ile Tyr Asn Ile Asn Asp Ala Gly His Met Thr
130 135 140
Ile Thr Met His Tyr Phe Gly Lys Lys Gly Leu Pro Pro Leu Pro Val
145 150 155 160
Ile Gly Met Arg Phe Ile Met Pro Thr Lys Ala Lys Ser Phe Asp Tyr
165 170 175
Thr Gly Leu Ser Gly Glu Thr Tyr Pro Asp Arg Met Ala Gly Ala Glu
180 185 190
Arg Gly Thr Phe His Ile Asp Gly Leu Pro Val Thr Lys Tyr Leu Val
195 200 205
Pro Gln Glu Asn Gly Met His Met Gln Thr Asn Glu Leu Val Ile Thr
210 215 220
Arg Asn Ser Thr Gln Asn Asn Ala Asp Lys Asp Gly Asp Phe Ser Leu
225 230 235 240
Lys Ile Thr Gln Thr Lys Gln Pro Phe Asn Phe Ser Leu Leu Pro Tyr
245 250 255
Thr Ala Glu Glu Leu Glu Asn Ala Thr His Ile Glu Glu Leu Pro Leu
260 265 270
Ala Arg Arg Ser Val Leu Val Ile Ala Gly Ala Val Arg Gly Val Gly
275 280 285
Gly Ile Asp Ser Trp Gly Ser Asp Val Glu Glu Gln Tyr His Ile Asp
290 295 300
Pro Glu Gln Asp His Glu Phe Ser Phe Thr Leu Asn
305 310 315
<210> 30
<211> 1025
<212> PRT
<213> 嗜热链球菌(Streptococcus thermophilus)
<400> 30
Met Asn Met Thr Lys Ile Gln Thr Tyr Leu Asn Asp Pro Lys Ile Val
1 5 10 15
Ser Val Asn Thr Val Asp Ala His Ser Asp His Lys Tyr Phe Glu Ser
20 25 30
Leu Glu Glu Phe Ser Glu Gly Glu Met Lys Leu Arg Gln Ser Leu Asn
35 40 45
Gly Lys Trp Lys Ile His Tyr Ala Gln Asn Thr Asn Gln Val Leu Lys
50 55 60
Asp Phe Tyr Lys Thr Glu Phe Asp Glu Thr Asp Leu Asn Phe Ile Asn
65 70 75 80
Val Pro Gly His Leu Glu Leu Gln Gly Phe Gly Ser Pro Gln Tyr Val
85 90 95
Asn Thr Gln Tyr Pro Trp Asp Gly Lys Glu Phe Leu Arg Pro Pro Gln
100 105 110
Val Pro Gln Glu Ser Asn Ala Val Ala Ser Tyr Val Lys His Phe Thr
115 120 125
Leu Asn Asp Ala Leu Lys Asp Lys Lys Val Phe Ile Ser Phe Gln Gly
130 135 140
Val Ala Thr Ser Ile Phe Val Trp Val Asn Gly Asn Phe Val Gly Tyr
145 150 155 160
Ser Glu Asp Ser Phe Thr Pro Ser Glu Phe Glu Ile Ser Asp Tyr Leu
165 170 175
Val Glu Gly Asp Asn Lys Leu Ala Val Ala Val Tyr Arg Tyr Ser Thr
180 185 190
Ala Ser Trp Leu Glu Asp Gln Asp Phe Trp Arg Leu Tyr Gly Ile Phe
195 200 205
Arg Asp Val Tyr Leu Tyr Ala Ile Pro Lys Val His Val Gln Asp Leu
210 215 220
Phe Val Lys Gly Asp Tyr Asp Tyr Gln Thr Lys Ala Gly Gln Leu Asp
225 230 235 240
Ile Asp Leu Lys Thr Val Gly Asp Tyr Glu Asp Lys Lys Ile Lys Tyr
245 250 255
Val Leu Ser Asp Tyr Glu Gly Ile Val Thr Glu Gly Asp Ala Ser Val
260 265 270
Asn Gly Asp Gly Glu Leu Ser Val Ser Leu Glu Asn Leu Lys Ile Lys
275 280 285
Pro Trp Ser Ala Glu Ser Pro Lys Leu Tyr Asp Leu Ile Leu His Val
290 295 300
Leu Asp Asp Asp Gln Val Val Glu Val Val Pro Val Lys Val Gly Phe
305 310 315 320
Arg Arg Phe Glu Ile Lys Asp Lys Leu Met Leu Leu Asn Gly Lys Arg
325 330 335
Ile Val Phe Lys Gly Val Asn Arg His Glu Phe Asn Ala Arg Thr Gly
340 345 350
Arg Cys Ile Thr Glu Glu Asp Met Leu Trp Asp Ile Lys Val Met Lys
355 360 365
Gln His Asn Ile Asn Ala Val Arg Thr Ser His Tyr Pro Asn Gln Thr
370 375 380
Arg Trp Tyr Glu Leu Cys Asp Glu Tyr Gly Leu Tyr Val Ile Asp Glu
385 390 395 400
Ala Asn Leu Glu Thr His Gly Thr Trp Gln Lys Leu Gly Leu Cys Glu
405 410 415
Pro Ser Trp Asn Ile Pro Ala Ser Glu Pro Glu Trp Leu Pro Ala Cys
420 425 430
Leu Asp Arg Ala Asn Asn Met Phe Gln Arg Asp Lys Asn His Ala Ser
435 440 445
Val Ile Ile Trp Ser Cys Gly Asn Glu Ser Tyr Ala Gly Lys Asp Ile
450 455 460
Ala Asp Met Ala Asp Tyr Phe Arg Ser Val Asp Asn Thr Arg Pro Val
465 470 475 480
His Tyr Glu Gly Val Ala Trp Cys Arg Glu Phe Asp Tyr Ile Thr Asp
485 490 495
Ile Glu Ser Arg Met Tyr Ala Lys Pro Ala Asp Ile Glu Glu Tyr Leu
500 505 510
Thr Thr Gly Lys Leu Val Asp Leu Ser Ser Val Ser Asp Lys His Phe
515 520 525
Ala Ser Gly Asn Leu Thr Asn Lys Pro Gln Lys Pro Tyr Ile Ser Cys
530 535 540
Glu Tyr Met His Thr Met Gly Asn Ser Gly Gly Gly Leu Gln Leu Tyr
545 550 555 560
Thr Asp Leu Glu Lys Tyr Pro Glu Tyr Gln Gly Gly Phe Ile Trp Asp
565 570 575
Phe Ile Asp Gln Ala Ile Tyr Lys Thr Leu Pro Asn Gly Ser Glu Phe
580 585 590
Leu Ser Tyr Gly Gly Asp Trp His Asp Arg Pro Ser Asp Tyr Glu Phe
595 600 605
Cys Gly Asn Gly Ile Val Phe Ala Asp Arg Thr Leu Thr Pro Lys Leu
610 615 620
Gln Thr Val Lys His Leu Tyr Ser Asn Ile Lys Ile Ala Val Asp Glu
625 630 635 640
Lys Ser Val Thr Ile Lys Asn Asp Asn Leu Phe Glu Asp Leu Ser Ala
645 650 655
Tyr Thr Phe Leu Ala Arg Val Tyr Glu Asp Gly Arg Lys Val Ser Glu
660 665 670
Ser Glu Tyr His Phe Asp Val Lys Pro Gly Glu Glu Ala Thr Phe Pro
675 680 685
Val Asn Phe Val Val Glu Ala Ser Asn Ser Glu Gln Ile Tyr Glu Val
690 695 700
Ala Cys Val Leu Arg Glu Ala Thr Lys Trp Ala Pro Lys Gly His Glu
705 710 715 720
Ile Val Arg Gly Gln Tyr Val Val Glu Lys Ile Ser Thr Glu Thr Pro
725 730 735
Val Lys Ala Pro Leu Asn Val Val Glu Gly Asp Phe Asn Ile Gly Ile
740 745 750
Gln Gly Gln Asn Phe Ser Ile Leu Leu Ser Arg Ala Gln Asn Thr Leu
755 760 765
Val Ser Ala Lys Tyr Asn Gly Val Glu Phe Ile Glu Lys Gly Pro Lys
770 775 780
Leu Ser Phe Thr Arg Ala Tyr Thr Asp Asn Asp Arg Gly Ala Gly Tyr
785 790 795 800
Pro Phe Glu Met Ala Gly Trp Lys Val Ala Gly Asn Tyr Ser Lys Val
805 810 815
Thr Asp Thr Gln Ile Gln Ile Glu Asp Asp Ser Val Lys Val Thr Tyr
820 825 830
Val His Glu Leu Pro Gly Leu Ser Asp Val Glu Val Lys Val Thr Tyr
835 840 845
Gln Val Asp Tyr Lys Gly Arg Ile Phe Val Thr Ala Asn Tyr Asp Gly
850 855 860
Lys Ala Gly Leu Pro Asn Phe Pro Glu Phe Gly Leu Glu Phe Ala Ile
865 870 875 880
Gly Ser Gln Phe Thr Asn Leu Ser Tyr Tyr Gly Tyr Gly Ala Glu Glu
885 890 895
Ser Tyr Arg Asp Lys Leu Pro Gly Ala Tyr Leu Gly Arg Tyr Glu Thr
900 905 910
Ser Val Glu Lys Thr Phe Ala Pro Tyr Leu Met Pro Gln Glu Ser Gly
915 920 925
Asn His Tyr Gly Thr Arg Glu Phe Thr Val Ser Asp Asp Asn His Asn
930 935 940
Gly Leu Lys Phe Thr Ala Leu Asn Lys Ala Phe Glu Phe Ser Ala Leu
945 950 955 960
Arg Asn Ser Thr Glu Gln Ile Glu Asn Ala Arg His Gln Tyr Glu Leu
965 970 975
Gln Glu Ser Asp Ala Thr Trp Ile Lys Val Leu Ala Ala Gln Met Gly
980 985 990
Val Gly Gly Asp Asp Ser Trp Gly Ala Pro Val His Asp Glu Phe Leu
995 1000 1005
Leu Ser Ser Ala Asp Ser Tyr Gln Leu Ser Phe Met Ile Glu Pro
1010 1015 1020
Leu Asn
1025
<210> 31
<211> 1008
<212> PRT
<213> 德氏乳杆菌印度亚种(Lactobacillus delbrueckii subsp. indicus)
<400> 31
Met Asn Asn Lys Leu Ala Gln Val Lys Arg Val Asp Gln Ala Asp Leu
1 5 10 15
Ala Trp Leu Thr Asp Pro Glu Ile Tyr Glu Val Asn Thr Ile Pro Pro
20 25 30
His Ser Asp His Glu Ser Phe Gln Ser Leu Glu Glu Leu Glu Glu Gly
35 40 45
Lys Ser Ser Leu Val Gln Ser Leu Asp Gly Asp Trp Leu Ile Asp Tyr
50 55 60
Ala Glu Asn Gly Glu Gly Pro Ala Asn Phe Tyr Glu Glu Asn Phe Asp
65 70 75 80
Asp Ser Ser Phe Lys Ser Val Lys Val Pro Gly Asn Leu Glu Leu Gln
85 90 95
Gly Phe Gly Gln Pro Gln Tyr Val Asn Val Gln Tyr Pro Trp Asp Gly
100 105 110
Ser Asp Glu Ile Phe Pro Pro Met Ile Pro Ser Lys Asn Pro Val Ala
115 120 125
Ser Tyr Val Arg Tyr Phe Asp Leu Glu Glu Ala Phe Trp Asp Lys Glu
130 135 140
Val Ser Leu Lys Phe Ala Gly Ala Ala Thr Ala Ile Tyr Val Trp Leu
145 150 155 160
Asn Gly His Phe Val Gly Tyr Gly Glu Asp Ser Phe Thr Pro Ser Glu
165 170 175
Phe Met Val Thr Lys Phe Leu Lys Lys Glu Gly Asn Arg Leu Ala Val
180 185 190
Ala Leu Tyr Lys Tyr Ser Ser Ala Ser Trp Leu Glu Asp Gln Asp Phe
195 200 205
Trp Arg Met Ser Gly Leu Phe Arg Ser Val Thr Leu Glu Ala Lys Pro
210 215 220
Leu Leu His Leu Gln Asp Leu Lys Leu Thr Ala Ser Leu Thr Asn Asp
225 230 235 240
Tyr Gln Lys Gly Ser Leu Gln Val Glu Ala Asp Ile Asp Tyr Arg Leu
245 250 255
Pro Asn Ser Ser Phe Lys Leu Glu Leu Arg Asp Ser Ala Gly Glu Leu
260 265 270
Val Ala Glu Lys Val Gly Pro Ile Arg Ser Glu Lys Leu Asp Phe Ser
275 280 285
Leu Ala Asp Leu Pro Val Ala Ala Trp Ser Ala Glu Glu Pro Asn Leu
290 295 300
Tyr Gln Val Arg Leu Ser Leu Tyr Gln Gln Gly Ser Leu Leu Glu Val
305 310 315 320
Ser Arg Gln Glu Val Gly Phe Arg Asn Phe Glu Leu Lys Asp Gly Ile
325 330 335
Met Tyr Leu Asn Gly Lys Arg Ile Val Phe Lys Gly Val Asn Arg His
340 345 350
Glu Phe Asp Ser Lys Leu Gly Arg Ala Ile Thr Glu Ala Asp Met Ile
355 360 365
Trp Asp Ile Lys Thr Met Lys Gln Ser Asn Ile Asn Ala Val Arg Cys
370 375 380
Ser His Tyr Pro Asn Gln Ser Ile Phe Tyr His Leu Cys Asp Lys Tyr
385 390 395 400
Gly Leu Tyr Val Ile Asp Glu Ala Asn Leu Glu Ser His Gly Thr Trp
405 410 415
Glu Lys Val Gly Gly His Glu Asp Pro Ser Phe Asn Val Pro Gly Asp
420 425 430
Asp Gln Arg Trp Leu Gly Ala Ser Leu Ser Arg Val Lys Asn Met Met
435 440 445
Ala Arg Asp Lys Asn His Ala Ser Ile Leu Ile Trp Ser Leu Gly Asn
450 455 460
Glu Ser Tyr Ala Gly Lys Val Phe Ala Gln Met Ala Asp Tyr Val Arg
465 470 475 480
Gln Ala Asp Pro Thr Arg Val Gln His Tyr Glu Gly Val Thr His Asn
485 490 495
Arg Lys Phe Asp Asp Ala Thr Gln Ile Glu Ser Arg Met Tyr Ala Pro
500 505 510
Ala Lys Glu Ile Glu Glu Tyr Leu Thr Lys Lys Pro Ala Lys Pro Phe
515 520 525
Val Ser Cys Glu Tyr Ala His Ala Met Gly Asn Ser Val Gly Asp Leu
530 535 540
Ala Ala Tyr Thr Ala Leu Glu Lys Tyr Pro His Tyr Gln Gly Gly Phe
545 550 555 560
Ile Trp Asp Trp Ile Asp Gln Gly Leu Glu Lys Glu Gly His Leu Leu
565 570 575
Tyr Gly Gly Asp Phe Asp Asp Arg Pro Ser Asp Tyr Glu Phe Cys Gly
580 585 590
Asp Gly Leu Val Phe Ala Asp Arg Thr Thr Ser Pro Lys Leu Ala Asn
595 600 605
Val Lys Ala Leu Tyr Ser Asn Leu Lys Leu Glu Leu Lys Asp Gly Gln
610 615 620
Leu Phe Leu Lys Asn Asp Asn Leu Phe Thr Asn Ser Ser Ala Tyr Tyr
625 630 635 640
Phe Leu Thr Ser Leu Leu Val Asp Gly Lys Leu Thr Tyr Gln Ser Gln
645 650 655
Pro Leu Thr Phe Ala Leu Glu Pro Gly Glu Ser Gly Thr Phe Ala Leu
660 665 670
Pro Trp Pro Glu Val Glu Asp Glu Lys Gly Glu Ile Val Tyr Gln Val
675 680 685
Thr Ala His Leu Lys Glu Asp Leu Pro Trp Ala Asp Glu Gly Phe Thr
690 695 700
Val Ala Glu Ala Glu Glu Ala Val Thr Lys Leu Pro Glu Phe Tyr Pro
705 710 715 720
Ala Gly Arg Pro Glu Leu Val Asp Ser Asp Tyr Asn Leu Gly Ile Lys
725 730 735
Gly Asn Gly Phe Arg Ile Leu Phe Ser Lys Ala Lys Gly Trp Pro Val
740 745 750
Ser Ile Lys Tyr Ala Gly Arg Glu Tyr Leu Lys Arg Leu Pro Glu Phe
755 760 765
Thr Phe Trp Arg Ala Leu Thr Asp Asn Asp Arg Gly Ala Gly Tyr Gly
770 775 780
Tyr Asp Leu Ala Lys Trp Glu Asn Ala Gly Lys Tyr Ala Arg Leu Gln
785 790 795 800
Asp Ile Ser Tyr Glu Ile Lys Glu Asn Ser Val Leu Val Lys Thr Ala
805 810 815
Phe Thr Leu Pro Val Ala Leu Lys Gly Asp Leu Thr Ile Thr Tyr Glu
820 825 830
Val Asp Ser Leu Gly Lys Ile Ala Val Thr Ala Asn Phe Pro Gly Ala
835 840 845
Val Glu Asn Gly Leu Leu Pro Ala Phe Gly Leu Asn Phe Ala Leu Pro
850 855 860
Lys Glu Leu Ser Asp Tyr Arg Tyr Tyr Gly Leu Gly Pro Asn Glu Ser
865 870 875 880
Tyr Ala Asp Arg Leu Glu Gly Ser Tyr Leu Gly Ile Tyr Gln Gly Ala
885 890 895
Val Glu Lys Asn Phe Thr Pro Tyr Leu Arg Pro Gln Glu Val Gly Asn
900 905 910
Arg Ser Lys Val Arg Tyr Tyr Gln Leu Phe Asp Glu Glu Gly Gly Leu
915 920 925
Glu Phe Thr Ala Asn Gly Ala Asn Leu Asn Leu Ser Ala Leu Pro Tyr
930 935 940
Ser Ala Ala Gln Ile Glu Ala Ala Asp His Ala Phe Glu Leu Thr Asn
945 950 955 960
Asn Tyr Thr Trp Val Arg Ala Leu Ala Ala Gln Met Gly Val Gly Gly
965 970 975
Asp Asp Ser Trp Gly Gln Lys Val His Pro Glu Phe Cys Leu Asp Ala
980 985 990
Gln Glu Ala Arg Gln Leu Lys Leu Val Ile Gln Pro Leu Phe Thr Glu
995 1000 1005
<210> 32
<211> 1049
<212> PRT
<213> 青春双歧杆菌
<400> 32
Met Ala Asp Thr Ala Glu Leu Ala Ile Val His Ala Thr Thr Ala Ser
1 5 10 15
Ala Ser Trp Leu Thr Asp Pro Thr Val Phe Ala Ala Asn Arg Lys Pro
20 25 30
Ala His Ser Ser His Arg Tyr Val Ile Gly Glu Thr Ser Glu Pro Lys
35 40 45
Gln Ser Leu Asp Gly Glu Trp Lys Val Arg Ile Glu Gln Ala Arg Asn
50 55 60
Val Asp Val Glu Ser Ala Pro Phe Ala Ala Val Asp Phe Glu Asp Gly
65 70 75 80
Asp Phe Gly Ala Ile Glu Val Pro Gly His Leu Gln Met Ala Gly Tyr
85 90 95
Leu Lys Asn Lys Tyr Val Asn Ile Gln Tyr Pro Trp Asp Gly His Glu
100 105 110
Asp Pro Gln Ala Pro Asn Ile Pro Glu Asn Asn His Val Ala Ile Tyr
115 120 125
Arg Arg Arg Phe Ala Leu Asp Ala Gln Leu Ala Arg Thr Leu Glu Asn
130 135 140
Asp Gly Thr Val Ser Leu Thr Phe His Gly Ala Ala Thr Ala Ile Tyr
145 150 155 160
Val Trp Leu Asp Gly Thr Phe Val Gly Tyr Gly Glu Asp Gly Phe Thr
165 170 175
Pro Ser Glu Phe Asp Val Thr Glu Ala Leu Arg Asn Gly Asn Gly Asn
180 185 190
Ala Ala Asp Ser Pro Glu Ala Glu His Thr Leu Thr Val Ala Cys Tyr
195 200 205
Glu Tyr Ser Ser Ala Ser Trp Leu Glu Asp Gln Asp Phe Trp Arg Leu
210 215 220
His Gly Leu Phe Arg Thr Val Glu Leu Ala Ala Gln Pro His Thr His
225 230 235 240
Val Glu Thr Val Gln Leu Glu Ala Asp Tyr Thr Ala Ala Asp Thr Ala
245 250 255
Gly Thr Ala Asp Thr Ala Glu Leu Asn Ala Ala Leu Thr Leu Arg Asn
260 265 270
Pro Ala Asp Ala Met Thr Ile Glu Ser Thr Leu Arg Asp Gly Asp Gly
275 280 285
Asn Val Val Trp Glu Ser Thr Gln Ala Cys Asn Gly Glu Ile Ala Leu
290 295 300
Asn Ser Gly Lys Met Thr Asn Ile Ala Pro Trp Ser Ala Glu Ser Pro
305 310 315 320
Thr Leu Tyr Thr Leu Thr Val Arg Val Val Gly His Asp Gly Ala Ile
325 330 335
Ile Glu Thr Val Thr Gln Lys Ile Gly Phe Arg Thr Phe Arg Ile Glu
340 345 350
Asn Gly Ile Met Thr Leu Asn Gly Lys Arg Ile Val Phe Lys Gly Ala
355 360 365
Asp Arg His Glu Phe Asp Ala Lys Arg Gly Arg Ala Ile Thr Arg Glu
370 375 380
Asp Met Leu Ser Asp Val Val Phe Cys Lys Arg His Asn Ile Asn Ala
385 390 395 400
Ile Arg Thr Ser His Tyr Pro Asn Gln Glu Tyr Trp Tyr Asp Leu Cys
405 410 415
Asp Glu Tyr Gly Leu Tyr Leu Ile Asp Glu Thr Asn Met Glu Thr His
420 425 430
Gly Thr Trp Val Ala Asn Asn Val Glu Arg Pro Glu Asp Gly Ile Pro
435 440 445
Gly Ser Arg Pro Glu Trp Glu Asp Ala Cys Val Asp Arg Ile Asn Ser
450 455 460
Met Met Arg Arg Asp Tyr Asn His Pro Ser Val Leu Ile Trp Ser Leu
465 470 475 480
Gly Asn Glu Ser Ser Ala Gly Glu Val Phe Arg Ala Met Tyr Arg His
485 490 495
Ala His Thr Ile Asp Pro Asn Arg Pro Val His Tyr Glu Gly Ser Val
500 505 510
His Met Arg Glu Phe Glu Asp Val Thr Asp Ile Glu Ser Arg Met Tyr
515 520 525
Ala His Ala Asp Glu Ile Glu Arg Tyr Leu Asn Asp Gly Ser Pro Ala
530 535 540
His Thr Asp Gly Pro Lys Lys Pro Tyr Ile Ser Cys Glu Tyr Met His
545 550 555 560
Ala Met Gly Asn Ser Cys Gly Asn Met Asp Glu Tyr Thr Ala Leu Glu
565 570 575
Arg Tyr Pro Met Tyr Gln Gly Gly Phe Ile Trp Asp Phe Ile Asp Gln
580 585 590
Ala Ile Glu Thr Lys Leu Pro Asp Gly Thr Thr Arg Met Cys Tyr Gly
595 600 605
Gly Asp Phe Gly Asp Arg Pro Ser Asp Tyr Glu Phe Ser Gly Asp Gly
610 615 620
Leu Leu Phe Ala Asp Arg Thr Pro Ser Pro Lys Ala Gln Glu Val Lys
625 630 635 640
Gln Leu Tyr Ala Asn Val Lys Ile Ala Val Ser Val Asp Glu Ala Arg
645 650 655
Ile Thr Asn Asp Asn Leu Phe Val Ser Thr Gly Asp Tyr Arg Phe Val
660 665 670
Leu Arg Ile Leu Ala Asp Gly Lys Pro Val Trp Ser Thr Thr Arg Arg
675 680 685
Phe Asp Val Ala Ala Gly Glu Ser Ala Ser Phe Glu Val Asp Trp Pro
690 695 700
Val Asp Asp Tyr Arg Ser Asn Ala Glu Glu Leu Val Leu Glu Val Ser
705 710 715 720
Gln Gln Leu Gly Asn Ala Cys Asp Trp Ala Pro Ala Gly Tyr Glu Leu
725 730 735
Ala Phe Gly Gln Cys Val Val Ala Gly Ala Lys Thr Thr Ala Asp Ala
740 745 750
Val Asp Ala Ala Gly Ala Pro Ala Asp Gly Thr Val Thr Leu Gly Arg
755 760 765
Trp Asn Ala Gly Val Arg Gly Gln Gly Arg Glu Ala Leu Phe Ser Arg
770 775 780
Thr Gln Gly Gly Met Val Ser Tyr Thr Phe Gly Glu Arg Glu Phe Val
785 790 795 800
Leu Arg Arg Pro Ser Ile Thr Thr Phe Arg Pro Leu Thr Asp Asn Asp
805 810 815
Arg Gly Ala Gly His Ala Phe Glu Arg Ala Ala Trp Ala Val Ala Gly
820 825 830
Lys Tyr Ala Arg Cys Val Asp Cys Ala Ile Ala Asn Arg Gly Glu Asn
835 840 845
Ala Val Glu Ala Thr Tyr Thr Tyr Glu Leu Ala Ile Pro Gln Arg Thr
850 855 860
Lys Val Thr Val Arg Tyr Val Ala Asp Thr Ala Gly Leu Val Ser Leu
865 870 875 880
Asp Val Glu Tyr Pro Gly Glu Lys Asn Gly Asp Leu Pro Thr Ile Pro
885 890 895
Ala Phe Gly Ile Glu Trp Ala Leu Pro Val Glu Tyr Ala Asn Leu Arg
900 905 910
Phe Tyr Gly Ala Gly Pro Glu Glu Thr Tyr Ala Asp Arg Arg His Ala
915 920 925
Lys Leu Gly Val Trp Ser Thr Thr Ala Gly Asp Asp Cys Ala Pro Tyr
930 935 940
Leu Leu Pro Gln Glu Thr Gly Asn His Glu Asp Val Arg Trp Ala Glu
945 950 955 960
Ile Thr Asp Asp Ser Gly His Gly Val Arg Val Lys Arg Gly Ala Gly
965 970 975
Ala Lys Pro Phe Ala Met Ser Leu Leu Pro Tyr Ser Ser Thr Met Leu
980 985 990
Glu Glu Ala Leu His Gln Asp Glu Leu Pro Lys Pro Arg His Met Phe
995 1000 1005
Leu Arg Leu Leu Ala Ala Gln Met Gly Val Gly Gly Asp Asp Ser
1010 1015 1020
Trp Met Ser Pro Val His Glu Gln Tyr Gln Leu Pro Ala Asp Gln
1025 1030 1035
Pro Leu Ser Leu Asn Val Gln Leu Lys Leu Phe
1040 1045
<210> 33
<211> 1023
<212> PRT
<213> 青春双歧杆菌
<400> 33
Met Ala Asn Glu Thr Arg Ile Glu His Ala Ser Glu Thr Trp Leu Ala
1 5 10 15
Asp Ser Thr Val Phe Glu Val Asn Arg Val Pro Ala His Ser Asp His
20 25 30
Lys Cys Tyr Ala His Asp Ser Gln Thr Asn Glu Trp Ser Asp Leu Arg
35 40 45
Gln Ser Leu Asp Gly Glu Trp Arg Val Glu Val Val Gln Ala Ser Asp
50 55 60
Ile Glu Phe Asn Glu Glu Pro Phe Val Arg Glu Asn Phe Asp Asp Ser
65 70 75 80
Ala Phe Glu Arg Ile Gln Val Pro Gly His Leu Gln Met Ala Gly Leu
85 90 95
Met Asn Asn Lys Tyr Val Asn Ile Gln Tyr Pro Trp Asp Gly His Glu
100 105 110
Asn Pro Ala Glu Pro Asn Ile Pro Glu Asn Asn His Val Ala Leu Tyr
115 120 125
Arg Lys Thr Phe Thr Met Ala Asn Arg Leu Ala Asp Thr Lys Asn Ala
130 135 140
Gly Gly Thr Val Ser Ile Val Phe His Gly Met Ala Thr Ala Ile Tyr
145 150 155 160
Val Trp Val Asn Gly Met Phe Val Gly Tyr Gly Glu Asp Gly Phe Thr
165 170 175
Pro Asn Glu Phe Asp Ile Thr Glu Met Leu His Asp Gly Glu Asn Val
180 185 190
Val Ala Val Ala Cys Tyr Glu Tyr Ser Ser Ala Ser Trp Leu Glu Asp
195 200 205
Gln Asp Phe Trp Arg Leu His Gly Leu Phe Arg Ser Val Glu Leu Ala
210 215 220
Ala Gln Pro His Val His Ile Glu Asn Met Gln Ile Glu Ser Asp Trp
225 230 235 240
Asp Pro Glu Ser Gly Ser Ala Ser Leu Asp Ala Ala Leu Thr Val Arg
245 250 255
Asn Ala Ala Asp Ala Ala Thr Ile Ser Ala Thr Leu Lys Asp Ser Asp
260 265 270
Gly Asn Val Val Trp Glu Thr Ala Asn Cys Ala Asp Pro Asp Thr Ser
275 280 285
Ile Ser Thr Gly Ser Leu Asn Gly Ile Arg Pro Trp Ser Ala Glu Asp
290 295 300
Pro Val Leu Tyr Glu Phe Glu Val Thr Val Ile Asp His Ala Gly Asn
305 310 315 320
Ile Ala Glu Val Ala Val Gln Lys Val Gly Phe Arg Arg Phe Arg Ile
325 330 335
Glu Asp Gly Ile Met Thr Ile Asn Gly Lys Arg Ile Val Phe Lys Gly
340 345 350
Ala Asp Arg His Glu Phe Asp Pro Lys Arg Gly Arg Ala Ile Thr Glu
355 360 365
Gln Asp Met Ile Asp Asp Val Val Phe Cys Lys Arg His Asn Leu Asn
370 375 380
Ala Ile Arg Thr Ser His Tyr Pro Asn Gln Glu Arg Trp Tyr Glu Leu
385 390 395 400
Cys Asp Glu Tyr Gly Ile Tyr Leu Ile Asp Glu Thr Asn Leu Glu Thr
405 410 415
His Gly Ser Trp Cys Leu Pro Gly Asp Val Leu Thr Glu Glu Thr Ala
420 425 430
Val Pro Gly Ser Lys Ala His Trp Glu Gly Ala Cys Val Asp Arg Val
435 440 445
Asn Ser Met Val Arg Arg Asp Tyr Asn His Pro Ser Val Leu Ile Trp
450 455 460
Ser Leu Gly Asn Glu Ser Tyr Thr Gly Asp Val Phe Arg Ala Met Tyr
465 470 475 480
Lys Arg Val His Asp Ile Asp Pro Asn Arg Pro Val His Tyr Glu Gly
485 490 495
Val Thr His Asn Arg Asp Tyr Asn Asp Val Thr Asp Ile Glu Thr Arg
500 505 510
Met Tyr Ala His Ala Asp Ala Ile Glu Glu Tyr Leu Lys Asn Asp Pro
515 520 525
Gln Lys Pro Tyr Ile Ser Cys Glu Tyr Met His Ala Met Gly Asn Ser
530 535 540
Cys Gly Asn Met Asp Glu Tyr Thr Ala Leu Glu Arg Tyr Pro Lys Tyr
545 550 555 560
Gln Gly Gly Phe Ile Trp Asp Phe Ile Asp Gln Ala Ile Tyr Ala Thr
565 570 575
Gln Pro Asp Gly Thr Thr Ser Leu Arg Tyr Gly Gly Asp Phe Gly Asp
580 585 590
Arg Pro Ser Asp Tyr Glu Phe Ser Gly Asn Gly Leu Val Phe Ala Asp
595 600 605
Arg Lys Pro Thr Pro Lys Ala Gln Glu Val Lys Gln Leu Tyr Ser Asn
610 615 620
Val His Ile Asp Val Ala Glu Asp Ser Val Thr Ile Lys Asn Asp Asn
625 630 635 640
Leu Phe Thr Ser Thr Gly Glu Tyr Thr Phe Val Leu Arg Val Leu Ala
645 650 655
Asp Gly Glu Pro Val Trp Gln Ser Glu Arg Arg Phe Asp Val Pro Ala
660 665 670
Gly Ser Thr Glu Lys Leu Asp Val Asp Trp Pro Leu Asp Leu Tyr Arg
675 680 685
Asp Gly Ala Ser Glu Leu Val Leu Glu Val Ser Gln Arg Leu Ala Lys
690 695 700
Ala Thr Asn Trp Ala Val Ala Gly Tyr Glu Leu Ala Phe Gly Gln Thr
705 710 715 720
Val Val Ala Gly Ser Lys Lys Ala Ser Ala Pro Val Lys Pro Val Asp
725 730 735
Gly Ile Val Thr Val Gly Arg Trp Asn Val Gly Val Gln Gly Ser Gly
740 745 750
Arg Glu Val Leu Leu Ser Arg Thr Gln Gly Gly Leu Val Ser Tyr Thr
755 760 765
Phe Asn Asn Arg Glu Phe Val Leu Arg Arg Pro Ala Val Thr Thr Phe
770 775 780
Arg Ala Leu Thr Asp Asn Asp Arg Gly Ala Gly His Gly Phe Glu Arg
785 790 795 800
Ala Gln Trp Leu Gly Ala Gly Arg Tyr Ala Arg Cys Ile Gly Asn Glu
805 810 815
Ile Glu Gln Ile Asp Glu Asn Thr Val Lys Ala Ser Tyr Thr Tyr Glu
820 825 830
Leu Ala Thr Pro Gln Arg Thr Lys Val Thr Val Ser Tyr Thr Ala Asp
835 840 845
Thr Thr Gly Arg Val Asn Leu His Val Glu Tyr Pro Gly Glu Pro Gly
850 855 860
Asp Leu Pro Thr Ile Pro Ala Phe Gly Ile Glu Trp Thr Leu Pro Val
865 870 875 880
Gln Tyr Ser Asn Leu Arg Phe Phe Gly Ala Gly Pro Glu Glu Thr Tyr
885 890 895
Gln Asp Arg Lys His Ala Lys Leu Gly Val Trp Ser Thr Asp Ala Phe
900 905 910
Lys Asp His Ala Pro Tyr Leu Met Pro Gln Glu Thr Gly Asn His Glu
915 920 925
Asp Val Arg Trp Ala Glu Ile Thr Asp Glu Lys Gly His Gly Leu Arg
930 935 940
Ile Ser Arg Ala Glu Gly Ala Glu Pro Phe Ala Met Ser Leu Gln Pro
945 950 955 960
Tyr Ser Ser Phe Met Leu Glu Glu Ala Gln His Gln Asp Glu Leu Pro
965 970 975
Ala Pro Lys His Met Phe Leu Arg Val Leu Ala Glu Gln Met Gly Val
980 985 990
Gly Gly Asp Asp Ser Trp Met Ser Pro Val His Pro Gln Tyr His Ile
995 1000 1005
Pro Ala Asp Gln Pro Ile Ser Leu Asp Val Asp Leu Asp Leu Ile
1010 1015 1020
<210> 34
<211> 1305
<212> PRT
<213> 两歧双歧杆菌
<400> 34
Met Val Glu Asp Ala Thr Arg Ser Asp Ser Thr Thr Gln Met Ser Ser
1 5 10 15
Thr Pro Glu Val Val Tyr Ser Ser Ala Val Asp Ser Lys Gln Asn Arg
20 25 30
Thr Ser Asp Phe Asp Ala Asn Trp Lys Phe Met Leu Ser Asp Ser Val
35 40 45
Gln Ala Gln Asp Pro Ala Phe Asp Asp Ser Ala Trp Gln Gln Val Asp
50 55 60
Leu Pro His Asp Tyr Ser Ile Thr Gln Lys Tyr Ser Gln Ser Asn Glu
65 70 75 80
Ala Glu Ser Ala Tyr Leu Pro Gly Gly Thr Gly Trp Tyr Arg Lys Ser
85 90 95
Phe Thr Ile Asp Arg Asp Leu Ala Gly Lys Arg Ile Ala Ile Asn Phe
100 105 110
Asp Gly Val Tyr Met Asn Ala Thr Val Trp Phe Asn Gly Val Lys Leu
115 120 125
Gly Thr His Pro Tyr Gly Tyr Ser Pro Phe Ser Phe Asp Leu Thr Gly
130 135 140
Asn Ala Lys Phe Gly Gly Glu Asn Thr Ile Val Val Lys Val Glu Asn
145 150 155 160
Arg Leu Pro Ser Ser Arg Trp Tyr Ser Gly Ser Gly Ile Tyr Arg Asp
165 170 175
Val Thr Leu Thr Val Thr Asp Gly Val His Val Gly Asn Asn Gly Val
180 185 190
Ala Ile Lys Thr Pro Ser Leu Ala Thr Gln Asn Gly Gly Asn Val Thr
195 200 205
Met Asn Leu Thr Thr Lys Val Ala Asn Asp Thr Glu Ala Ala Ala Asn
210 215 220
Ile Thr Leu Lys Gln Thr Val Phe Pro Lys Gly Gly Lys Thr Asp Ala
225 230 235 240
Ala Ile Gly Thr Val Thr Thr Ala Ser Lys Ser Ile Ala Ala Gly Ala
245 250 255
Ser Ala Asp Val Thr Ser Thr Ile Thr Ala Ala Ser Pro Lys Leu Trp
260 265 270
Ser Ile Lys Asn Pro Asn Leu Tyr Thr Val Arg Thr Glu Val Leu Asn
275 280 285
Gly Asp Thr Val Leu Asp Thr Tyr Asp Thr Glu Tyr Gly Phe Arg Trp
290 295 300
Thr Gly Phe Asp Ala Thr Ser Gly Phe Ser Leu Asn Gly Glu Lys Val
305 310 315 320
Lys Leu Lys Gly Val Ser Met His His Asp Gln Gly Ser Leu Gly Ala
325 330 335
Val Ala Asn Arg Arg Ala Ile Glu Arg Gln Val Glu Ile Leu Gln Lys
340 345 350
Met Gly Val Asn Ser Ile Arg Thr Thr His Asn Pro Ala Ala Lys Ala
355 360 365
Leu Ile Asp Val Cys Asn Glu Lys Gly Val Leu Val Val Glu Glu Val
370 375 380
Phe Asp Met Trp Asn Arg Ser Lys Asn Gly Asn Thr Glu Asp Tyr Gly
385 390 395 400
Lys Trp Phe Gly Gln Thr Ile Ala Gly Asp Asn Ala Val Leu Gly Gly
405 410 415
Asp Lys Asp Glu Thr Trp Ala Lys Phe Asp Leu Thr Ser Thr Ile Asn
420 425 430
Arg Asp Arg Asn Ala Pro Ser Val Ile Met Trp Ser Leu Gly Asn Glu
435 440 445
Met Met Glu Gly Ile Ser Gly Ser Val Ser Asp Phe Pro Ala Thr Ser
450 455 460
Ala Lys Leu Val Ala Trp Thr Lys Ala Ala Asp Ser Thr Arg Pro Met
465 470 475 480
Thr Tyr Gly Asp Asn Lys Ile Lys Ala Asn Trp Asn Glu Ser Asn Thr
485 490 495
Met Gly Asp Asn Leu Thr Ala Asn Gly Gly Val Val Gly Thr Asn Tyr
500 505 510
Ser Asp Gly Ala Asn Tyr Asp Lys Ile Arg Thr Thr His Pro Ser Trp
515 520 525
Ala Ile Tyr Gly Ser Glu Thr Ala Ser Ala Ile Asn Ser Arg Gly Ile
530 535 540
Tyr Asn Arg Thr Thr Gly Gly Ala Gln Ser Ser Asp Lys Gln Leu Thr
545 550 555 560
Ser Tyr Asp Asn Ser Ala Val Gly Trp Gly Ala Val Ala Ser Ser Ala
565 570 575
Trp Tyr Asp Val Val Gln Arg Asp Phe Val Ala Gly Thr Tyr Val Trp
580 585 590
Thr Gly Phe Asp Tyr Leu Gly Glu Pro Thr Pro Trp Asn Gly Thr Gly
595 600 605
Ser Gly Ala Val Gly Ser Trp Pro Ser Pro Lys Asn Ser Tyr Phe Gly
610 615 620
Ile Val Asp Thr Ala Gly Phe Pro Lys Asp Thr Tyr Tyr Phe Tyr Gln
625 630 635 640
Ser Gln Trp Asn Asp Asp Val His Thr Leu His Ile Leu Pro Ala Trp
645 650 655
Asn Glu Asn Val Val Ala Lys Gly Ser Gly Asn Lys Val Pro Val Val
660 665 670
Val Tyr Thr Asp Ala Ala Lys Val Lys Leu Tyr Phe Thr Pro Lys Gly
675 680 685
Ser Thr Glu Lys Arg Leu Ile Gly Glu Lys Ser Phe Thr Lys Lys Thr
690 695 700
Thr Ala Ala Gly Tyr Thr Tyr Gln Val Tyr Glu Gly Thr Asp Lys Asp
705 710 715 720
Ser Thr Ala His Lys Asn Met Tyr Leu Thr Trp Asn Val Pro Trp Ala
725 730 735
Glu Gly Thr Ile Ser Ala Glu Ala Tyr Asp Glu Asn Asn Arg Leu Ile
740 745 750
Pro Glu Gly Ser Thr Glu Gly Asn Ala Ser Val Thr Thr Thr Gly Lys
755 760 765
Ala Ala Lys Leu Lys Ala Asp Ala Asp Arg Lys Thr Ile Thr Ala Asp
770 775 780
Gly Lys Asp Leu Ser Tyr Ile Glu Val Asp Val Thr Asp Ala Asn Gly
785 790 795 800
His Ile Val Pro Asp Ala Ala Asn Arg Val Thr Phe Asp Val Lys Gly
805 810 815
Ala Gly Lys Leu Val Gly Val Asp Asn Gly Ser Ser Pro Asp His Asp
820 825 830
Ser Tyr Gln Ala Asp Asn Arg Lys Ala Phe Ser Gly Lys Val Leu Ala
835 840 845
Ile Val Gln Ser Thr Lys Glu Ala Gly Glu Ile Thr Val Thr Ala Lys
850 855 860
Ala Asp Gly Leu Gln Ser Ser Thr Val Lys Ile Ala Thr Thr Ala Val
865 870 875 880
Pro Gly Thr Ser Thr Glu Lys Thr Val Arg Ser Phe Tyr Tyr Ser Arg
885 890 895
Asn Tyr Tyr Val Lys Thr Gly Asn Lys Pro Ile Leu Pro Ser Asp Val
900 905 910
Glu Val Arg Tyr Ser Asp Gly Thr Ser Asp Arg Gln Asn Val Thr Trp
915 920 925
Asp Ala Val Ser Asp Asp Gln Ile Ala Lys Ala Gly Ser Phe Ser Val
930 935 940
Ala Gly Thr Val Ala Gly Gln Lys Ile Ser Val Arg Val Thr Met Ile
945 950 955 960
Asp Glu Ile Gly Ala Leu Leu Asn Tyr Ser Ala Ser Thr Pro Val Gly
965 970 975
Thr Pro Ala Val Leu Pro Gly Ser Arg Pro Ala Val Leu Pro Asp Gly
980 985 990
Thr Val Thr Ser Ala Asn Phe Ala Val His Trp Thr Lys Pro Ala Asp
995 1000 1005
Thr Val Tyr Asn Thr Ala Gly Thr Val Lys Val Pro Gly Thr Ala
1010 1015 1020
Thr Val Phe Gly Lys Glu Phe Lys Val Thr Ala Thr Ile Arg Val
1025 1030 1035
Gln Arg Ser Gln Val Thr Ile Gly Ser Ser Val Ser Gly Asn Ala
1040 1045 1050
Leu Arg Leu Thr Gln Asn Ile Pro Ala Asp Lys Gln Ser Asp Thr
1055 1060 1065
Leu Asp Ala Ile Lys Asp Gly Ser Thr Thr Val Asp Ala Asn Thr
1070 1075 1080
Gly Gly Gly Ala Asn Pro Ser Ala Trp Thr Asn Trp Ala Tyr Ser
1085 1090 1095
Lys Ala Gly His Asn Thr Ala Glu Ile Thr Phe Glu Tyr Ala Thr
1100 1105 1110
Glu Gln Gln Leu Gly Gln Ile Val Met Tyr Phe Phe Arg Asp Ser
1115 1120 1125
Asn Ala Val Arg Phe Pro Asp Ala Gly Lys Thr Lys Ile Gln Ile
1130 1135 1140
Ser Ala Asp Gly Lys Asn Trp Thr Asp Leu Ala Ala Thr Glu Thr
1145 1150 1155
Ile Ala Ala Gln Glu Ser Ser Asp Arg Val Lys Pro Tyr Thr Tyr
1160 1165 1170
Asp Phe Ala Pro Val Gly Ala Thr Phe Val Lys Val Thr Val Thr
1175 1180 1185
Asn Ala Asp Thr Thr Thr Pro Ser Gly Val Val Cys Ala Gly Leu
1190 1195 1200
Thr Glu Ile Glu Leu Lys Thr Ala Thr Ser Lys Phe Val Thr Asn
1205 1210 1215
Thr Ser Ala Ala Leu Ser Ser Leu Thr Val Asn Gly Thr Lys Val
1220 1225 1230
Ser Asp Ser Val Leu Ala Ala Gly Ser Tyr Asn Thr Pro Ala Ile
1235 1240 1245
Ile Ala Asp Val Lys Ala Glu Gly Glu Gly Asn Ala Ser Val Thr
1250 1255 1260
Val Leu Pro Ala His Asp Asn Val Ile Arg Val Ile Thr Glu Ser
1265 1270 1275
Glu Asp His Val Thr Arg Lys Thr Phe Thr Ile Asn Leu Gly Thr
1280 1285 1290
Glu Gln Glu Phe Pro Ala Asp Ser Asp Glu Arg Asp
1295 1300 1305
<210> 35
<211> 1025
<212> PRT
<213> 乳酸克鲁维酵母(Kluyveromyces lactis)
<400> 35
Met Ser Cys Leu Ile Pro Glu Asn Leu Arg Asn Pro Lys Lys Val His
1 5 10 15
Glu Asn Arg Leu Pro Thr Arg Ala Tyr Tyr Tyr Asp Gln Asp Ile Phe
20 25 30
Glu Ser Leu Asn Gly Pro Trp Ala Phe Ala Leu Phe Asp Ala Pro Leu
35 40 45
Asp Ala Pro Asp Ala Lys Asn Leu Asp Trp Glu Thr Ala Lys Lys Trp
50 55 60
Ser Thr Ile Ser Val Pro Ser His Trp Glu Leu Gln Glu Asp Trp Lys
65 70 75 80
Tyr Gly Lys Pro Ile Tyr Thr Asn Val Gln Tyr Pro Ile Pro Ile Asp
85 90 95
Ile Pro Asn Pro Pro Thr Val Asn Pro Thr Gly Val Tyr Ala Arg Thr
100 105 110
Phe Glu Leu Asp Ser Lys Ser Ile Glu Ser Phe Glu His Arg Leu Arg
115 120 125
Phe Glu Gly Val Asp Asn Cys Tyr Glu Leu Tyr Val Asn Gly Gln Tyr
130 135 140
Val Gly Phe Asn Lys Gly Ser Arg Asn Gly Ala Glu Phe Asp Ile Gln
145 150 155 160
Lys Tyr Val Ser Glu Gly Glu Asn Leu Val Val Val Lys Val Phe Lys
165 170 175
Trp Ser Asp Ser Thr Tyr Ile Glu Asp Gln Asp Gln Trp Trp Leu Ser
180 185 190
Gly Ile Tyr Arg Asp Val Ser Leu Leu Lys Leu Pro Lys Lys Ala His
195 200 205
Ile Glu Asp Val Arg Val Thr Thr Thr Phe Val Asp Ser Gln Tyr Gln
210 215 220
Asp Ala Glu Leu Ser Val Lys Val Asp Val Gln Gly Ser Ser Tyr Asp
225 230 235 240
His Ile Asn Phe Thr Leu Tyr Glu Pro Glu Asp Gly Ser Lys Val Tyr
245 250 255
Asp Ala Ser Ser Leu Leu Asn Glu Glu Asn Gly Asn Thr Thr Phe Ser
260 265 270
Thr Lys Glu Phe Ile Ser Phe Ser Thr Lys Lys Asn Glu Glu Thr Ala
275 280 285
Phe Lys Ile Asn Val Lys Ala Pro Glu His Trp Thr Ala Glu Asn Pro
290 295 300
Thr Leu Tyr Lys Tyr Gln Leu Asp Leu Ile Gly Ser Asp Gly Ser Val
305 310 315 320
Ile Gln Ser Ile Lys His His Val Gly Phe Arg Gln Val Glu Leu Lys
325 330 335
Asp Gly Asn Ile Thr Val Asn Gly Lys Asp Ile Leu Phe Arg Gly Val
340 345 350
Asn Arg His Asp His His Pro Arg Phe Gly Arg Ala Val Pro Leu Asp
355 360 365
Phe Val Val Arg Asp Leu Ile Leu Met Lys Lys Phe Asn Ile Asn Ala
370 375 380
Val Arg Asn Ser His Tyr Pro Asn His Pro Lys Val Tyr Asp Leu Phe
385 390 395 400
Asp Lys Leu Gly Phe Trp Val Ile Asp Glu Ala Asp Leu Glu Thr His
405 410 415
Gly Val Gln Glu Pro Phe Asn Arg His Thr Asn Leu Glu Ala Glu Tyr
420 425 430
Pro Asp Thr Lys Asn Lys Leu Tyr Asp Val Asn Ala His Tyr Leu Ser
435 440 445
Asp Asn Pro Glu Tyr Glu Val Ala Tyr Leu Asp Arg Ala Ser Gln Leu
450 455 460
Val Leu Arg Asp Val Asn His Pro Ser Ile Ile Ile Trp Ser Leu Gly
465 470 475 480
Asn Glu Ala Cys Tyr Gly Arg Asn His Lys Ala Met Tyr Lys Leu Ile
485 490 495
Lys Gln Leu Asp Pro Thr Arg Leu Val His Tyr Glu Gly Asp Leu Asn
500 505 510
Ala Leu Ser Ala Asp Ile Phe Ser Phe Met Tyr Pro Thr Phe Glu Ile
515 520 525
Met Glu Arg Trp Arg Lys Asn His Thr Asp Glu Asn Gly Lys Phe Glu
530 535 540
Lys Pro Leu Ile Leu Cys Glu Tyr Gly His Ala Met Gly Asn Gly Pro
545 550 555 560
Gly Ser Leu Lys Glu Tyr Gln Glu Leu Phe Tyr Lys Glu Lys Phe Tyr
565 570 575
Gln Gly Gly Phe Ile Trp Glu Trp Ala Asn His Gly Ile Glu Phe Glu
580 585 590
Asp Val Ser Thr Ala Asp Gly Lys Leu His Lys Ala Tyr Ala Tyr Gly
595 600 605
Gly Asp Phe Lys Glu Glu Val His Asp Gly Val Phe Ile Met Asp Gly
610 615 620
Leu Cys Asn Ser Glu His Asn Pro Thr Pro Gly Leu Val Glu Tyr Lys
625 630 635 640
Lys Val Ile Glu Pro Val His Ile Lys Ile Ala His Gly Ser Val Thr
645 650 655
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<212> DNA
<213> 引物
<220>
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Claims (15)
1.表现出β-半乳糖苷酶活性的肽，其具有SEQ ID NO:1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33所示的氨基酸序列或其酶促活性片段，或任一所述序列的具有不超过1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21或22个氨基酸取代、添加或缺失的氨基酸序列。
2.表现出β-半乳糖苷酶活性的二聚肽，所述二聚肽的组成为具有SEQ ID NO:2和3、5和6、20和21、23和24、26和27或28和29所示氨基酸序列的两个肽；或其酶促活性片段，或任一所述序列的具有不超过1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21或22个氨基酸取代、添加或缺失的氨基酸序列。
3.降低诸如乳制品等含有乳糖的组合物中乳糖含量的方法，包括使所述组合物在范围为3-10的pH和0℃-140℃的温度下与表现出β-半乳糖苷酶活性的肽或二聚肽接触的步骤，所述肽具有SEQ ID NO:1-33所示的氨基酸序列，或所述二聚肽的组成为具有SEQ ID NO:2和3、5和6、20和21、23和24、26和27或28和29所示氨基酸序列的两个肽；或其酶促活性片段，或与任一所述序列或酶促活性片段具有至少80％序列同一性的任何序列；或与表达所述肽或二聚肽中任一种的宿主细胞接触的步骤。
4.表现出β-半乳糖苷酶活性的肽或二聚肽或表达所述肽或二聚肽中任一种的宿主细胞在生产乳糖含量降低的乳制品中的用途，所述肽具有SEQ ID NO:1-33所示的氨基酸序列，或所述二聚肽的组成为具有SEQ ID NO:2和3、5和6、20和21、23和24、26和27或28和29所示氨基酸序列的两个肽；或与任一所述序列具有至少80％序列同一性的序列。
5.生产乳制品的方法，所述方法包括以下步骤：
a)提供包含乳糖的乳基基质；
b)向所述包含乳糖的乳基基质添加表现出β-半乳糖苷酶活性的肽或二聚肽，所述肽具有SEQ ID NO:1-33所示的氨基酸序列；或所述二聚肽的组成为具有SEQ ID NO:2和3、5和6、20和21、23和24、26和27或28和29所示氨基酸序列的两个肽；或与任一所述序列具有至少80％序列同一性的序列；和
c)用所述表现出β-半乳糖苷酶活性的肽或二聚肽处理所述乳基基质。
6.根据权利要求5所述的方法，其中，进行步骤c)的pH在3-10的范围内，例如在3-9的范围内，例如在3-8的范围内，例如在3-7的范围内，例如在3-6的范围内，例如在3-5的范围内，例如在3-4的范围内，例如在4-10的范围内，例如在4-9的范围内，例如在4-8的范围内，例如在4-7的范围内，例如在4-6的范围内，例如在4-5的范围内，例如在5-10的范围内，例如在5-9的范围内，例如在5-8的范围内，例如在5-7的范围内，例如在5-6的范围内，例如在6-10的范围内，例如在6-9的范围内，例如在6-8的范围内，例如在6-7的范围内。
7.根据权利要求5-6中任一项所述的方法，其中，进行步骤c)或步骤c)的一部分的温度不超过约25℃，例如不超过约20℃，例如不超过约18℃，例如不超过约16℃，例如不超过约14℃，例如不超过约12℃，例如不超过约10℃，例如不超过约8℃，例如不超过约7℃，例如不超过约6℃，例如不超过约5℃，例如不超过约4℃，例如不超过约3℃，例如不超过约2℃。
8.根据权利要求5-7中任一项所述的方法，其中进行步骤c)或步骤c)的一部分的温度为至少约25℃，例如至少约30℃，例如至少约35℃，例如至少约40℃，例如至少约45℃，例如至少约50℃，例如至少约55℃，例如至少约60℃，例如至少约65℃，例如至少约70℃，例如至少约75℃，例如至少约80℃，例如至少约85℃，例如至少约90℃，例如至少约95℃，例如至少约100℃，例如至少约110℃，例如至少约120℃，例如至少约130℃，例如至少约120℃，例如至少约130℃，例如至少约135℃，例如至少约140℃。
9.根据权利要求5-8中任一项所述的方法，其中：所述肽或二聚肽的半乳糖抑制小于60％，例如小于55％，，例如小于50％，例如小于约45％，例如小于约40％。
10.根据权利要求5-9中任一项所述的方法，其中，所述乳制品是发酵乳制品，并且在发酵期间或发酵之前进行所述步骤b)。
11.根据权利要求10所述的方法，所述方法不需要在发酵后添加其他酶。
12.根据权利要求5-10中任一项所述的方法，其中所述乳制品是发酵乳制品，并且在发酵后立即进行所述步骤b)。
13.根据权利要求5-10中任一项所述的方法，其中，所述乳制品是鲜乳，并且在巴氏灭菌步骤之前、同时或之后立即进行所述步骤b)。
14.根据权利要求5-10所述的方法，其中所述乳制品是超热处理(UHT)乳，并且在超热处理步骤之前、同时或之后立即进行所述步骤b)。
15.根据权利要求5-14中任一项所述的方法，其中，开始步骤c)的温度为40℃至100℃，例如50℃至100℃，例如60℃至100℃，例如70℃至100℃，例如80℃至100℃，例如40℃至90℃，例如40℃至80℃，例如40℃至70℃，例如40℃至60℃，例如40℃至50℃。
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| US20220304323A1 (en) * | 2019-08-30 | 2022-09-29 | Chr. Hansen A/S | Production of lactase enzymes using altered regulation strains |
| WO2021170559A1 (en) * | 2020-02-26 | 2021-09-02 | Novozymes A/S | Polypeptide variants |
| AU2022422537A1 (en) * | 2021-12-22 | 2024-06-06 | Novozymes A/S | Method of producing a milk-based product |
| WO2023237360A1 (en) | 2022-06-07 | 2023-12-14 | Chr. Hansen A/S | Improving filterability of lactase by adding anions selected from malate, tartrate, citrate, gluconate, edta or combinations thereof and sterile filtered lactase product obtained |
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| AR111462A1 (es) | 2019-07-17 |
| BR112019021142A2 (pt) | 2020-05-12 |
| AR111589A1 (es) | 2019-07-31 |
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