CN101965399A

CN101965399A - 用于生产乙醇酸的固定的微生物腈水解酶的改进

Info

Publication number: CN101965399A
Application number: CN200880124121XA
Authority: CN
Inventors: R·迪科西莫; A·本-巴萨特
Original assignee: EI Du Pont de Nemours and Co
Current assignee: High Purity Technology Science Co.,Ltd.
Priority date: 2007-10-31
Filing date: 2008-10-31
Publication date: 2011-02-02
Anticipated expiration: 2028-10-31
Also published as: JP5702604B2; DK2215225T3; PL2215225T3; CN101965399B; JP2011501969A; US7695945B2; ES2563482T3; US20090111158A1; EP2215225B1; EP2215225A1; WO2009059101A1

Abstract

本发明提供了制备具有将乙醇腈水解为乙醇酸的腈水解酶活性的酶催化剂的方法，所述酶催化剂在具有催化剂循环的连续批式反应中其回收的催化剂活性的保留得到改进，所述方法包括以戊二醛预处理酶催化剂。与未经戊二醛预处理的酶催化剂相比，戊二醛预处理的酶催化剂具有改进的比活性，从而具有改进的总的催化剂活性和生产力。

Description

用于生产乙醇酸的固定的微生物腈水解酶的改进

技术领域

本发明涉及有机酸的合成和微生物学领域。更具体地，本发明提供了制备酶催化剂的方法，所述酶催化剂在乙醇腈水解为乙醇酸过程中的初始腈水解酶活性的保留得到改进，所述方法包括在固定和交联之前以戊二醛预处理酶催化剂。如上所述，与未经戊二醛预处理的固定的和交联的酶催化剂相比，戊二醛预处理的固定的和交联的酶催化剂具有改进的比活性，从而具有将乙醇腈转化为乙醇酸的改进的总的催化剂活性、催化剂生产力和单位体积生产力。

背景技术

乙醇酸(HOCH₂COOH；CAS注册号为79-14-1)是羧酸的α-羟基酸家族的最简单的成员。乙醇酸的性质使其具有理想的广范围的消费和工业应用，包括用于水井复原、皮革工业、油气工业、洗衣和纺织工业、作为单体制备聚乙醇酸(PGA)，以及作为个人护理产品的成分。乙醇酸还是多种工业上的清洁剂(奶制品和食品加工设备清洁剂，家居和机构清洁剂，工业清洁剂[用于运输设备、石工技术、印刷电路板、不锈钢锅炉和处理设备、冷却塔/热交换器]和金属加工[用于金属酸洗、铜的抛光、蚀刻、电镀、电解法抛光])的主要成分。也有报道指出乙醇酸可作为气体屏障材料(即显示出高度的氧气屏障特性)用于包装食物和碳酸饮料(WO 2005/106005A1)。但是，乙醇酸的传统化学合成产生大量的杂质，在使用前必须除去这些杂质。工业界迫切需要商业化生产乙醇酸的新技术，特别是以低成本生产高纯度乙醇酸的新技术。

微生物酶催化剂能够使用腈水解酶(EC 3.5.5.7)将腈(例如乙醇腈)直接水解为相应的羧酸(例如乙醇酸)，其中不产生相应的酰胺中间体(反应式1)；或者可通过腈水合酶(EC 4.2.1.84)与酰胺酶(EC 3.5.1.4)的组合将腈(例如乙醇腈)水解为相应的羧酸(例如乙醇酸)，其中腈水合酶(NHase)首先将腈转化为酰胺，然后酰胺被酰胺酶转化成相应的羧酸(反应式2))：

已经证明：在具有生物催化剂循环的连续批式反应中使用一次以后，酶催化剂的比活性(以催化剂的每克干细胞重每分钟产生的乙醇酸的微摩尔数测定)比初始活性降低35％至50％。这代表批式反应中酶催化剂的比活性的显著损失，进而代表总的酶活性和生产力的显著损失。对于生产乙醇酸的商业上可行的酶学方法来说，这种酶活性的损失需要加以解决。

酶活性损失的一个方面可能是由于酶催化剂与乙醇腈反应以产生乙醇酸时杂质和其它成分的存在。以前曾经报道过通过甲醛和氰化氢的水溶液的反应合成乙醇腈的方法(US 2,175,805；US 2,890,238；和US 5,187,301；反应反应式3)。

但是，这些方法通常产生水性的乙醇腈反应产物，其需要进行深度纯化(例如蒸馏纯化)，因为很多杂质和/或反应的副产物(包括多余的反应物甲醛)可能干扰乙醇腈至乙醇酸的酶法转化，包括抑制催化剂活性(即降低比活性)。特别地，众所周知甲醛能够通过与来自N-末端氨基酸残基和精氨酸、半胱氨酸、组氨酸和赖氨酸残基的侧链的氨基基团发生反应而在蛋白中产生不想要的修饰(Metz等人，J.Biol.Chem.，279(8)：6235-6243(2004))。催化剂活性的抑制降低催化剂的总体生产力(即每克催化剂形成的乙醇酸的总克数)，这显著增加了总体过程的成本，使得与化学合成相比酶法生产可能在经济上不可行。因此，需要这样的反应条件：其能够保护酶活性以抵抗那些降低催化剂活性的不想要的杂质。

已经报道了通过在乙醇腈合成反应之前将甲醛进行加热处理从而产生高纯度乙醇腈的方法(US 11/3143865和US 11/314905；反应式3)。但是，乙醇腈能够可逆地分解成甲醛和氰化氢。因此，仍然需要保护腈水解酶活性以抵抗由乙醇腈分解产生的甲醛和氰化氢的不良效应。

US 5,508,181还描述了将腈化合物转化为α-羟基酸时与酶催化剂的迅速灭活相关的类似困难。具体地，US 5,508,181记载：根据分解平衡，α-羟基腈化合物部分分解为相应的醛。据报道，这些醛通过与蛋白结合而在短时间内将酶灭活，因此难以从α-羟基腈以高生产力获得高浓度的α-羟基酸或α-羟基酰胺(第2栏，第16-29行)。作为防止由于醛的累积而使酶灭活的手段，向反应混合物中加入磷酸盐或次磷酸盐离子。类似地，US 5,326,702描述了使用亚硫酸盐、二亚硫酸盐或连二亚硫酸盐离子捕获醛并防止酶灭活，但是其得出结论：即使使用这样的添加物，所产生和积累的α-羟基酸的浓度也不足以满足多数商业目的。

此外，U.S.6,037,155教导：α-羟基酸产物的低积累与分解的醛的积累造成的短时间内的酶灭活有关。这些发明人提示在氰化氢(在水以及相应的醛或酮中α-羟基腈部分分解产生氰化氢(Mowry，David T.，Chemical Reviews，Vol.42，189-283页(1948)))存在时酶活性被抑制(Asano等人，Agricultural Biological Chemistry，Vol.46，1165-1174页(1982))。发明人通过使用微生物(其酶活性可以通过向反应混合物中加入氰物质而被改进)而解决了醛诱导的酶灭活的问题。氰物质的加入限制了α-羟基腈向醛和氰化氢的分解。虽然这个策略对系统提供了益处，但是其仅解决了与乙醇腈转化为乙醇酸中的酶灭活相关的一个方面，即，如上所述，已知乙醇腈可逆地分解为氰化氢和甲醛，二者均已知对酶催化剂活性具有消极效应。

已经开发了一种单独的方法以在存在甲醛的情况下将乙醇腈转化为乙醇酸时保护具有腈水解酶活性的酶催化剂的比活性(参见同一申请人的美国专利申请XX/XXXXXX(CL3584)，通过引用并入本文)，其中通过向反应混合物中加入胺保护剂或者通过将腈水解酶催化剂固定于基质(含有胺保护剂，例如PEI、聚烯丙胺、PVOH/聚乙烯胺等)中或基质上而获得了催化剂活性和稳定性的显著改进。在该系统中，改进了甲醛存在时的催化剂的比活性，但是没有完全解决与具有催化剂循环的连续批式反应中回收的催化剂活性的比活性损失相关的问题。

U.S.4,288,552记载(第1栏，第46-49行和第2栏，第50-55行)：戊二醛敏感性酶(例如巯基酶(例如腈水解酶)和其它在酶分子的活性位点或非常接近活性位点的地方具有SH基团的酶)被巯基反应性试剂例如戊二醛灭活。因此，直到现在，使用戊二醛改进乙醇腈水解为乙醇酸过程中初始催化剂活性的保留是不可预见的。本文证明了所述的不可预见的益处。

因此，需要一种方法，其提供乙醇腈水解为乙醇酸过程中初始腈水解酶活性的改进的保留，从而改进将乙醇腈转化为乙醇酸的总的催化剂活性、催化剂生产力和单位体积生产力。

发明内容

本发明通过提供制备具有腈水解酶活性的酶催化剂的方法而解决了上述需要，所述酶催化剂的将乙醇腈水解为乙醇酸的初始催化剂活性的保留得到改进，所述方法包括在固定和交联之前以戊二醛预处理酶催化剂。与未经戊二醛预处理而制备的固定的和交联的酶催化剂相比，戊二醛预处理的固定的和交联的酶催化剂在乙醇腈转化为乙醇酸过程中具有改进的比活性，从而具有将乙醇腈转化为乙醇酸的改进的总的催化剂活性、催化剂生产力和单位体积生产力。

在一个方面，本发明提供了生产具有腈水解酶活性的酶催化剂的方法，所述酶催化剂在将乙醇腈转化为乙醇酸的过程中其初始比活性的保留得到改进，所述方法包括：

(a)通过发酵产生具有腈水解酶活性的酶催化剂；

(b)以戊二醛预处理所述酶催化剂；

(c)任选地在戊二醛预处理之后以亚硫酸氢盐将未反应的戊二醛灭活；

(d)从(b)或(c)回收酶催化剂，并将所述酶催化剂固定于角叉菜胶中；和

(e)以戊二醛和聚乙烯亚胺将(d)中产生的角叉菜胶-固定的酶催化剂进行交联，

从而产生戊二醛预处理的固定的和交联的酶催化剂，并且其中所述戊二醛预处理的固定的和交联的酶催化剂在相同反应条件下与未经戊二醛预处理的固定的和交联的酶催化剂相比，在乙醇腈转化为乙醇酸过程中的初始比活性的保留得到改进。

本发明的另一个方面包含：在合适的反应条件下，在水溶液中将以上(e)的戊二醛预处理的固定的和交联的酶催化剂与乙醇腈接触，从而产生乙醇酸。另外，回收所述乙醇酸。

本发明的另一个方面涉及通过以上的步骤a)至e)的方法产生的戊二醛预处理的固定的和交联的酶催化剂。另外，所述戊二醛预处理的固定的和交联的具有腈水解酶活性的酶催化剂与未经过戊二醛预处理的相当的酶催化剂相比，当用于乙醇腈至乙醇酸(作为铵盐)的转化时，保留了显著较高百分率的其初始比活性(每克催化剂每分钟水解的乙醇腈的μmole数)。

本发明的另一个方面涉及通过以上的步骤a)至e)的方法产生的戊二醛预处理的固定的和交联的具有腈水解酶活性的酶催化剂，其中所述酶催化剂在至少4个连续批式循环之后，保留至少大约70％，至少大约75％，至少大约80％，至少大约85％，至少大约90％，至少大约95％，至少大约96％，或至少大约97％的其初始比活性。

本发明的另一个方面涉及通过以上的步骤a)至e)的方法产生的戊二醛预处理的固定的和交联的具有腈水解酶活性的酶催化剂，其中所述酶催化剂在戊二醛预处理的固定的和交联的酶催化剂的每克干细胞重产生至少40g乙醇酸之后(在从乙醇腈生产乙醇酸的连续反应中，例如在连续搅拌槽式反应器(CSTR)或固定床活塞流反应器或流化床或半流化床反应器中进行反应)，保留至少大约70％，至少大约75％，至少大约80％，至少大约85％，至少大约90％，至少大约95％，至少大约96％，或至少大约97％的其初始比活性。

本发明的另一个方面涉及将乙醇腈水解为乙醇酸的改进的方法，其包括改进具有腈水解酶活性的固定的酶催化剂在乙醇腈转化为乙醇酸过程中的初始比活性的保留，所述方法包括：

(a)通过发酵产生具有腈水解酶活性的酶催化剂；

(b)以戊二醛预处理所述酶催化剂；

(d)从(b)或(c)回收酶催化剂并将所述酶催化剂固定于角叉菜胶中；

(e)以戊二醛和聚乙烯亚胺将(d)中产生的角叉菜胶-固定的酶催化剂进行交联；和

(f)在合适的反应条件下，在水溶液中将(e)的交联的固定的酶催化剂与乙醇腈接触，从而产生乙醇酸，

其中步骤(f)在至少两个连续批式循环中进行。

另外，该方法可以包括回收通过所述改进的方法产生的乙醇酸。

附图、序列表和生物保藏说明

可以从附图、序列表、生物保藏和一起构成本申请的详细说明书更全面地理解本发明。

附图

图1中图板A-G是各种腈水解酶序列的CLUSTALW比对(版本1.83，使用缺省参数)。以灰色阴影标出了催化剂半胱氨酸残基周围的保守性催化剂标志(signature)序列。用下划线标出了代表催化三元组(triad)(Glu₄₈、Lys₁₃₀和Cys₁₆₄；编号基于SEQ ID NO：4的氨基酸序列)的氨基酸。

序列表

以下序列描述和所附序列表符合37C.F.R.§1.821-1.825列出的关于专利申请中的核苷酸和/或氨基酸序列公开的规则规定。序列描述中对于核苷酸序列符号使用单字母代码，对于氨基酸使用三字母代码，符合IUPAC-IYUB标准(如Nucleic Acids Research 13：3021-3030(1985)和Biochemical Journal 219(No.2)：345-373(1984)所描述，通过引用并入本文)。用于核苷酸和氨基酸序列数据的符号和格式符合37C.F.R.§1.822.列出的规定。

SEQ ID NO：1是包含腈水解酶的必需半胱氨酸残基的催化标志基序的氨基酸序列(式1)。

SEQ ID NO：2是包含腈水解酶的必需半胱氨酸残基的优选催化剂标志基序的氨基酸序列(式2)。

SEQ ID NO：3是敏捷食酸菌(Acidovorax facilis)72W腈水解酶编码序列的核苷酸序列，其中包含起始密码子从TTG至ATG的改变以促进在大肠杆菌中的重组表达。

SEQ ID NO：4是敏捷食酸菌72W腈水解酶(ATCC 55746)的推导氨基酸序列。

SEQ ID NO：5是固氮粪产碱菌(Alcaligenes faecalis)JM3腈水解酶(

BAA02684.1)的氨基酸序列。

SEQ ID NO：6是紫红红球菌J1腈水解酶(

Q03217)的氨基酸序列。

SEQ ID NO：7是紫红红球菌K22腈水解酶(

Q02068)的氨基酸序列。

SEQ ID NO：8是诺卡氏菌属(Nocardia sp.)C-14-1腈水解酶(

AAX18182.1)的氨基酸序列。

SEQ ID NO：9是支气管败血波氏杆菌(Bordetella bronchiseptica)RB50腈水解酶(NP_887662.1)的氨基酸序列。

SEQ ID NO：10是拟南芥(Arabidopsis thaliana)腈水解酶(

AAB60275.1和AAA19627.1)的氨基酸序列。

SEQ ID NO：11是细长聚球藻(Synechococcus elongatus)PCC 7942腈水解酶(YP_399857.1)的氨基酸序列。

SEQ ID NO：12是细长聚球藻PCC 6301腈水解酶(

YP_171411.1)的氨基酸序列。

SEQ ID NO：13是集胞藻属(Synechocystis sp.)PCC 6803腈水解酶(

NP_442646.1)的氨基酸序列。

SEQ ID NO：14是嗜虫假单胞菌(Pseudomonas entomophila)L48腈水解酶(YP_690481.1)的氨基酸序列。

SEQ ID NO：15是运动发酵单胞菌(Zymomonas moblis)腈水解酶的氨基酸序列(

YP_162942.1)。

SEQ ID NO：16是芽孢杆菌属(Bacillus sp.)OxB-1腈水解酶(

BAA90460.1)的氨基酸序列。

SEQ ID NO：17是睾丸酮丛毛单胞菌(Comamonas testosteroni)腈水解酶(

AAA82085.1)的氨基酸序列。

SEQ ID NO：18是蓝细菌属(Synechococcus sp.)CC9605腈水解酶(

YP_381420.1)的氨基酸序列。

SEQ ID NO：19是荧光假单胞菌(Pseudomonas fluorescens)Pf-5腈水解酶(

YP_260015.1)的氨基酸序列。

SEQ ID NO：20是鼻疽诺卡菌(Nocardia farcinica)IFM 10152腈水解酶(

YP_119480.1)的氨基酸序列。

SEQ ID NO：21是固氮粪产碱菌1650腈水解酶(AAY06506.1)的氨基酸序列。

SEQ ID NO：22是丁香假单胞杆菌丁香致病变种(Pseudomonas syringaepv.syringae)B728a腈水解酶(AAY35081.1)的氨基酸序列。

SEQ ID NO：23是慢生根瘤菌属(Bradyrhizobium sp.)BTAil腈水解酶(

ZP_00859948.1)的氨基酸序列。

SEQ ID NO：24是紫红红球菌NCIMB 11216腈水解酶(

CAC88237)的氨基酸序列。

SEQ ID NO：25是紫红红球菌ATCC^TM 39484的氨基酸序列。

SEQ ID NO：26是敏捷食酸菌72W腈水解酶突变体的核苷酸序列，其包含引起201位残基上单一氨基酸取代的密码子改变(L201Q；Leu→Gln)。

SEQ ID NO：27是突变的腈水解酶(SEQ ID NO：26)的推导氨基酸序列，其在敏捷食酸菌72W腈水解酶的201位残基含有单一氨基酸取代(Leu201→Gln)。

SEQ ID NO：28是敏捷食酸菌72W腈水解酶突变体的核苷酸序列，其包含引起201位残基上单一氨基酸取代的密码子改变(L201A；Leu→Ala)。

SEQ ID NO：29是突变的腈水解酶(SEQ ID NO：28)的推导氨基酸序列，其在敏捷食酸菌72W腈水解酶的201位残基含有单一氨基酸取代(Leu201→Ala)。

SEQ ID NO：30是敏捷食酸菌72W腈水解酶突变体的核苷酸序列，其包含引起201位残基上单一氨基酸取代的密码子改变(L201C；Leu→Cys)。

SEQ ID NO：31是突变的腈水解酶(SEQ ID NO：30)的推导氨基酸序列，其在敏捷食酸菌72W腈水解酶的201位残基含有单一氨基酸取代(Leu201→Cys)。

SEQ ID NO：32是敏捷食酸菌72W腈水解酶突变体的核苷酸序列，其包含引起201位残基上单一氨基酸取代的密码子改变(L201T；Leu→Thr)。

SEQ ID NO：33是突变的腈水解酶(SEQ ID NO：32)的推导氨基酸序列，其在敏捷食酸菌72W腈水解酶的201位残基含有单一氨基酸取代(Leu201→Thr)。

SEQ ID NO：34是敏捷食酸菌72W腈水解酶突变体的核苷酸序列，其包含引起201位残基上单一氨基酸取代的密码子改变(L201G；Leu→Gly)。

SEQ ID NO：35是突变的腈水解酶(SEQ ID NO：34)的推导氨基酸序列，其在敏捷食酸菌72W腈水解酶的201位残基含有单一氨基酸取代(Leu201→Gly)。

SEQ ID NO：36是敏捷食酸菌72W腈水解酶突变体的核苷酸序列，其包含引起201位残基上单一氨基酸取代的密码子改变(L201H；Leu→His)。

SEQ ID NO：37是突变的腈水解酶(SEQ ID NO：36)的推导氨基酸序列，其在敏捷食酸菌72W腈水解酶的201位残基含有单一氨基酸取代(Leu201→His)。

SEQ ID NO：38是敏捷食酸菌72W腈水解酶突变体的核苷酸序列，其包含引起201位残基上单一氨基酸取代的密码子改变(L201K；Leu→Lys)。

SEQ ID NO：39是突变的腈水解酶(SEQ ID NO：38)的推导氨基酸序列，其在敏捷食酸菌72W腈水解酶的201位残基含有单一氨基酸取代(Leu201→Lys)。

SEQ ID NO：40是敏捷食酸菌72W腈水解酶突变体的核苷酸序列，其包含引起201位残基上单一氨基酸取代的密码子改变(L201N；Leu→Asn)。

SEQ ID NO：41是突变的腈水解酶(SEQ ID NO：40)的推导氨基酸序列，其在敏捷食酸菌72W腈水解酶的201位残基含有单一氨基酸取代(Leu201→Asn)。

SEQ ID NO：42是敏捷食酸菌72W腈水解酶突变体的核苷酸序列，其包含引起201位残基上单一氨基酸取代的密码子改变(L201S；Leu→Ser)。

SEQ ID NO：43是突变的腈水解酶(SEQ ID NO：42)的推导氨基酸序列，其在敏捷食酸菌72W腈水解酶的201位残基含有单一氨基酸取代(Leu201→Ser)。

SEQ ID NO：44是敏捷食酸菌72W腈水解酶突变体的核苷酸序列，其包含引起168位残基上单一氨基酸取代的密码子改变(F168K；Phe→Lys)。

SEQ ID NO：45是突变的腈水解酶(SEQ ID NO：44)的推导氨基酸序列，其在敏捷食酸菌72W腈水解酶的168位残基含有单一氨基酸取代(Phe168→Lys)。

SEQ ID NO：46是敏捷食酸菌72W腈水解酶突变体的核苷酸序列，其包含引起168位残基上单一氨基酸取代的密码子改变(F168M；Phe→Met)。

SEQ ID NO：47是突变的腈水解酶(SEQ ID NO：46)的推导氨基酸序列，其在敏捷食酸菌72W腈水解酶的168位残基含有单一氨基酸取代(Phe168→Met)。

SEQ ID NO：48是敏捷食酸菌72W腈水解酶突变体的核苷酸序列，其包含引起168位残基上单一氨基酸取代的密码子改变(F168T；Phe→Thr)。

SEQ ID NO：49是突变的腈水解酶(SEQ ID NO：48)的推导氨基酸序列，其在敏捷食酸菌72W腈水解酶的168位残基含有单一氨基酸取代(Phe168→Thr)。

SEQ ID NO：50是敏捷食酸菌72W腈水解酶突变体的核苷酸序列，其包含引起168位残基上单一氨基酸取代的密码子改变(F168V；Phe→Val)。

SEQ ID NO：51是突变的腈水解酶(SEQ ID NO：50)的推导氨基酸序列，其在敏捷食酸菌72W腈水解酶的168位残基含有单一氨基酸取代(Phe168→Val)。

SEQ ID NO：52是敏捷食酸菌72W腈水解酶突变体的核苷酸序列，其包含引起168位残基上单一氨基酸取代的密码子改变(T210A；Thr→Ala)。

SEQ ID NO：53是突变的腈水解酶(SEQ ID NO：52)的推导氨基酸序列，其在敏捷食酸菌72W腈水解酶的210位残基含有单一氨基酸取代(Thr210→Ala)。

SEQ ID NO：54是敏捷食酸菌72W腈水解酶突变体的核苷酸序列，其包含引起168位残基上单一氨基酸取代的密码子改变(T210C；Thr→Cys)。

SEQ ID NO：55是突变的腈水解酶(SEQ ID NO：54)的推导氨基酸序列，其在敏捷食酸菌72W腈水解酶的210位残基含有单一氨基酸取代(Thr210→Cys)。

SEQ ID NO：56是在大肠杆菌菌株SS1001(ATCC PTA-1177)中表达的敏捷食酸菌72W腈水解酶的核苷酸序列。

SEQ ID NO：57是在大肠杆菌菌株SS1001(ATCC PTA-1177)中表达的敏捷食酸菌72W腈水解酶的推导氨基酸序列。

生物保藏

根据国际承认用于专利程序目的的微生物保存布达佩斯条约的条款作出以下生物保藏：

本文所用的“ATCC”是指位于ATCC，10801University Blvd.，Manassas，VA 201102209，USA的American Type Culture Collection InternationalDepository Authority。“国际保藏机构指定”是ATCC保藏的培养物的编号。

所列的保藏将在指明的国际保藏机构保持至少三十(30)年，在公开这些保藏的专利授权之后公众可以获得这些保藏。保藏的获得不构成实施本发明(损害由政府行为授予的专利权)的许可证。

具体实施方式

本发明提供了制备具有将乙醇腈水解为乙醇酸的腈水解酶活性的固定的和交联的酶催化剂的方法，所述酶催化剂在将乙醇腈转化为乙醇酸过程中的初始酶催化剂活性的保留得到改进，所述方法包括在固定之前以戊二醛预处理酶催化剂。如上所述，与未经戊二醛预处理的固定的和交联的酶催化剂的初始比活性的保留相比，戊二醛预处理的固定的酶催化剂在乙醇腈转化为乙醇酸过程中的初始比活性的保留得到改进，从而具有改进的总的催化剂活性、催化剂生产力和单位体积生产力。

定义

在本公开中使用了一些术语和缩写。除非另有特别指明，否则适用以下定义。

本文所用的术语“包含”意思是存在如权利要求中提到的指明的特征、整体、步骤或成分，但是不排除另外的一个或多个其它特征、整体、步骤、成分或其组合的存在。

本文所用的修饰本发明所用的成分或反应试剂的数量的术语“大约”是指数值量的差异，所述差异可以来自于，例如，在客观世界中配制浓度或使用溶液时的典型的测量和液体操作程序；这些程序中不经意的错误；制备组合物或实施方法时所用的成分的生产商、来源或纯度；等等。术语“大约”还包括由来自特定的初始混合物的组合物的不同平衡条件所产生的数量上的差异。不论是否以术语“大约”进行修饰，权利要求均包括数量的等同物。在一个实施方式中，术语“大约”意思是在报告的数值的10％以内，优选在报告的数值的5％以内.

本文所用的术语“乙醇腈”缩写为“GLN”，其与羟基乙腈、2-羟基乙腈、羟甲基腈和CAS注册号107-16-4的所有其它同义物是同义的。

本文所用的术语“乙醇酸”缩写为“GLA”，其与羟基乙酸(hydroxyacetic acid)、羟基乙酸(hydroxyethanoic acid)和CAS注册号79-14-1的所有其它同义物是同义的。根据本发明的方法产生的乙醇酸可以是质子化羧酸和/或相应的铵盐的形式。

本文所用的术语“乙醇酸铵”缩写为“NH₄GLA”。

本文所用的术语“羟乙酰胺”是氨与乙醇酸反应产生的酰胺，指具有CAS注册号598-42-5的化合物的所有其它同义物。

本文所用的术语“乙交酯”是指CAS注册号502-97-6的化合物。

本文所用的术语“甲醛”缩写为“FA”，其与蚁醛、甲基醛、氧甲烷和CAS注册号50-00-0的所有其它同义物是同义的。商售的甲醛通常包含单体甲醛(“自由甲醛”)和各种甲醛与一些甲醇的寡聚物(通常为大约1wt％至大约15wt％)。

本文所用的术语“氰化氢”与氰酸、氢氰酸和CAS注册号200-821-6的所有其它同义物是同义的。

本文所用的术语“戊二醛”缩写为“GA”，其与戊二醛(pentanedial)、1，5-戊二醛(1，5-pentanedial)、1，5-戊二酮(1，5-pentanedione)、戊二醛(diglutaric aldehyde)、戊二醛(glutaral)、戊二醛(glutardialdehyde)、戊二酸二醛(glutaric acid dialdehyde)、戊二醛(glutaric dialdehyde)和CAS注册号111-30-8的所有其它同义物是同义的。

本文所用的术语“亚硫酸氢盐”或“亚硫酸氢钠”与亚硫酸钠盐、亚硫酸单钠盐、亚硫酸氢钠(hydrogen sodium sulfite)、亚硫酸氢钠(hydrogen sulfite sodium)、亚硫酸单钠、酸式亚硫酸钠、亚硫酸氢钠(sodium bisulfite)、硫酸氢钠(sodium bisulphate)、亚硫酸氢钠(sodium hydrogen sulfite)、亚硫酸钠(NaHSO3)和CAS注册号7631-90-5的所有其它同义物是同义的。

本文所用的术语“回收”意思是分离、纯化或转移通过本发明的方法形成的产物。从反应混合物中分离和纯化产物的方法在本领域是熟知的，包括但不限于选择性沉淀、结晶、过滤、反应性溶剂提取、离子交换、电渗析、聚合化、蒸馏、热分解、醇解、柱层析及其组合。在一个实施方式中，术语“回收”还可以包括将产物混合物(通常在过滤掉酶催化剂之后)转移至另一个反应以产生一个或多个另外的产物。在优选的实施方式中，使用离子交换来回收乙醇酸。

本文所用的术语“酶催化剂”、“腈水解酶催化剂”或“微生物细胞催化剂”是指这样的催化剂，其特征在于具有将乙醇腈转化为乙醇酸和氨的腈水解酶活性(即包含至少一个具有腈水解酶活性的多肽)。腈水解酶将腈(优选为脂肪族的腈)直接转化为相应的羧酸，不形成相应的作为中间体的酰胺(见反应式1)。腈水解酶共同具有几个本领域已知的保守性标志结构域，包括在本文中称为“催化标志序列”或“标志序列”的标志结构域。该区域包含必需的半胱氨酸残基(例如SEQ ID NO：4的Cys₁₆₄)。因此，可以通过是否存在催化结构域标志序列(SEQ ID NO：1)而鉴定具有腈水解酶活性的多肽。在优选的实施方式中，标志序列是SEQ ID NO：2。酶催化剂可以是整个微生物细胞或透化的(permeabilized)微生物细胞的形式。本文所用的“循环使用的酶催化剂”是指在批式或连续反应中作为酶催化剂重新使用的酶催化剂。取决于本文描述的产生或使用酶催化剂的方法的步骤，酶催化剂可以是戊二醛预处理的固定的和交联的。

本文所用的术语“敏捷食酸菌(Acidovorax facilis)”和“敏捷食酸菌(A.facilis)”可替换使用，是指保藏于American Type Culture Collection(一个国际保藏机构)的保藏号为55746(“ATCC 55746”)的敏捷食酸菌72W。以前已经报道了源自敏捷食酸菌72W的突变的腈水解酶，其特征在于将乙醇腈转化为乙醇酸时具有改进的腈水解酶活性(见同一申请人拥有的美国专利7,198,927)。这些源自敏捷食酸菌72W的突变的腈水解酶的例子由SEQ IDNO：27、29、31、33、35、37、39、41、43、45、47、49、51、53和55提供。

本文所用的术语“大肠杆菌(Escherichia coli)”和“大肠杆菌(E.coli)”可替换使用。本文描述了适合用于重组表达的几个大肠杆菌菌株，包括但不限于，国际保藏号为ATCC 47076的大肠杆菌MG1655、国际保藏号为ATCC53911的大肠杆菌FM5、国际保藏号为ATCC 27325的大肠杆菌W3110、国际保藏号为ATCC 35695的大肠杆菌MC4100、国际保藏号为ATCC 12435的大肠杆菌W1485。在一个实施方式中，合适的大肠杆菌菌株包括大肠杆菌FM5(ATCC 53911)和大肠杆菌MG1655(ATCC 47076)。

本文所用的术语“大肠杆菌SS1001”或“SS1001”是指ATCC保藏号为PTA-1177的表达敏捷食酸菌72W腈水解酶的转化的大肠杆菌菌株(见美国专利6,870,038，通过引用全文并入本文)。重组表达的大肠杆菌SS1001腈水解酶(SEQ ID NO：57)与野生型72W腈水解酶序列(SEQ ID NO：4)相比具有两个微小序列改变。起始密码子从GTG变为ATG以促进重组表达，并且在克隆时引入了引起接近C-末端的单一氨基酸改变(Pro367[CCA]→Ser[TCA])的人造物。

本文所用的术语“合适的水性乙醇腈反应混合物”和“合适的水性反应混合物”是指材料(包括至少一种胺保护剂)和水，其中乙醇腈和酶催化剂相互接触。本文提供了合适的水性反应混合物的成分，本领域技术人员将认识到适合于本方法的成分变化的范围。

本文所用的术语“水性乙醇酸铵溶液”、“包含乙醇酸铵的水溶液”和“乙醇酸铵的水溶液”用于描述包含乙醇酸铵(在通常的酶反应条件下(即大约6至大约8的pH范围)通过乙醇腈酶法水解产生)的水溶液。乙醇酸铵的水溶液包含浓度为至少大约0.1重量％(wt％)至大约99wt％的乙醇酸铵。在另一个实施方式中，乙醇酸铵的水溶液包含至少大约10wt％至大约75wt％的乙醇酸铵。在另一个实施方式中，乙醇酸铵的水溶液包含至少大约20wt％至大约50wt％的乙醇酸铵。乙醇酸铵的水溶液的pH可以是大约2至大约12，优选为5至大约10，更优选为6至大约8。可以在启动与从乙醇酸铵的水溶液中回收乙醇酸(酸或盐的形式)相关的方法步骤之前根据需要调整pH。

本文所用的术语“催化剂生产力”和“酶催化剂生产力”是指每克酶催化剂干细胞重产生的产物的总量。在本发明中，酶催化剂包含腈水解酶(EC3.5.5.7)，形成的产物是乙醇酸和/或乙醇酸铵(取决于反应的pH)。一般地，与产生乙醇酸相关的生产过程在基本上中性pH的条件下进行，从而产生的乙醇酸主要是相应的乙醇酸盐的形式(即乙醇酸铵)。一般地，在具有催化剂循环的批式反应中，催化剂活性随着每个循环反应的进行而降低(酶灭活)。

本文所用的术语“单位体积生产力”是指反应中乙醇酸的单位体积产量，其表示为单位时间内每体积的反应混合物所产生的乙醇酸的克数。通常来讲，单位体积生产力表示为乙醇酸克数/L/h。

术语“腈水解酶活性”或“比活性”是指：将乙醇腈转化为乙醇酸(或相应的乙醇酸铵)时，每单位质量(例如毫克)的蛋白、干细胞重或珠子重量(固定的催化剂)的酶活性。腈水解酶活性的比较根据干细胞重或珠子重量成比例地测定。

本文所用的术语“一单位的酶活性”或“一单位的腈水解酶活性”或“U”定义为在指定温度(例如25℃)每分钟生产1μmol的乙醇酸产物所需要的酶活性的数量(GLA U/g干细胞重或珠子重量)。

本文所用的术语“相对腈水解酶活性”、“改进的腈水解酶活性”和“腈水解酶活性的相对改进”是指以参考(对照)腈水解酶活性的倍数(或分数)表示的腈水解酶活性。本文描述的腈水解酶相对于原态(native)敏捷食酸菌72W腈水解酶中观察到的腈水解酶活性显示出显著性改进的腈水解酶活性。相对腈水解酶活性的“显著性改进”是指在相同反应条件下相对于对照的腈水解酶活性至少提高1.5倍的腈水解酶活性的改进。在另一个实施方式中，改进是指在相同反应条件下相对于对照的腈水解酶活性至少提高2倍的腈水解酶活性。在另一个实施方式中，改进是指在相同反应条件下相对于对照的腈水解酶活性至少提高4倍的腈水解酶活性。

本文所用的术语“初始反应速率”是在指定的反应条件下乙醇腈转化为乙醇酸的速率的度量，其中在最初向反应混合物中加入乙醇腈后开始测定反应速率，并且在反应过程中乙醇腈的浓度保持在约50毫摩(mM)的时间段内测定反应速率。按照单位时间内产生的乙醇酸的浓度变化来测定反应速率(例如乙醇酸摩尔数/L/分钟或乙醇酸毫摩尔数/小时)。

本文所用的术语“初始比活性的改进的保留”是指：在指定的反应条件下将乙醇腈转化为乙醇酸的过程中，戊二醛预处理的固定的交联的酶催化剂与未经戊二醛预处理的固定的交联的酶催化剂(二者均具有腈水解酶活性)的比较，以每分钟每克酶催化剂的干细胞重所产生的乙醇酸的微摩尔数或每分钟每克固定的交联的酶催化剂所产生的乙醇酸的微摩尔数表示，其中，对于一个或多个后续反应而言，戊二醛预处理的固定的交联的酶催化剂在第一个或“初始”反应中测定的比活性比未经戊二醛预处理的固定的交联的酶催化剂的保留程度高。本文描述的最显著的改进是紧接初始的批式反应之后的那个反应所保留的活性数量(在具有催化剂循环的一个或多个后续批式反应中测定)。本文描述的第二显著的改进是：在连续搅拌槽式反应器(CSTR)或固定床活塞流反应器或流化床或半流化床反应器中的反应进行过程中所保留的活性数量。

本文所用的术语“重组生物”、“转化的宿主”、“转化子”、“转基因生物”和“转化的微生物宿主”是指经过异源或外源DNA转化的宿主生物。本发明的重组生物表达外源编码序列或编码活性腈水解酶的基因。“转化”是指将DNA片段转移进入宿主生物。转移的DNA片段可以整合入宿主生物的染色体或以非染色体方式进入宿主生物(即通过载体)。本文所用的术语“转化盒”是指含有一组遗传元件(其进行方便的排列以插入宿主细胞)的特异性DNA片段，通常是作为质粒的一部分。本文所用的术语“表达盒”是指含有一组遗传元件(其方便的排列以插入宿主细胞)的特异性DNA片段，通常是作为质粒的一部分，还允许宿主中增强的基因表达。

本文所用的术语“核酸片段”和“核酸分子”是指可以编码整个基因的DNA分子、编码序列和/或编码序列前面(5’，上游)或后面(3’，下游)的调控序列。在一个方面，本发明的核酸分子编码具有腈水解酶活性的多肽。

本文所用的术语“基因”是指表达特定蛋白的核酸分子。如本文所用，基因可以包括或不包括编码序列前面的调控序列(5’非编码序列)和编码序列后面的调控序列(3’非编码序列)。“嵌合基因”是指任何不是原态的基因，其包含不一起存在于原态中的调控和编码序列。因此，嵌合基因可以包含来自不同来源的调控序列和编码序列，或来自相同来源但是按照与原态中发现的排列方式不同的方式排列的调控序列和编码序列。“内源基因”是指位于生物基因组的天然位置的原态基因。“外源”基因是指通常不存在于宿主生物中，但是通过基因转移被导入宿主生物中的基因。外源基因可以包含插入非原态生物的原态基因，或嵌合基因。“转基因”是通过转化程序被导入基因组的基因。

本文所用的术语“编码序列”是指编码特异氨基酸序列的DNA序列。本文所用的“合适的调控序列”是指位于编码序列上游(5’非编码序列)、编码序列内，或编码序列下游(3’非编码序列)的核苷酸序列，其影响转录、RNA的加工或稳定性，或相连的编码序列的翻译。调控序列可以包括启动子、翻译引导序列、内含子、多腺苷化识别序列、RNA加工位点、效应子结合位点和茎环结构。

“启动子”是指能够控制编码序列或功能性RNA的表达的DNA序列。一般地，编码序列位于启动子序列的3’端。启动子可以以完整形式来源于原态基因，或者由源自天然发现的不同启动子的不同元件组成，甚至可以包含合成的DNA片段。在多数细胞类型中在多数时候或多数环境条件下引起基因表达的启动子通常被称为“组成型启动子”。仅在特定化合物或环境条件存在时引起基因表达的启动子通常被称为“可诱导启动子”。由于在多数情况下调控序列的精确边界已经被完全确定，所以不同长度的DNA片段可以具有相同的启动子活性。

本文所用的术语“可操作地连接”是指单个核酸分子上的核酸序列按照一个序列的功能被另一个所影响的方式连接。例如，当启动子能够影响编码序列的表达时(即编码序列在启动子的转录控制之下)，启动子与编码序列是可操作地连接。编码序列可以按照正义或反义方向与调控序列可操作地连接。

本文所用的术语“3’非编码序列”是指位于编码序列下游的DNA序列，包括多腺苷化识别序列(通常限于真核细胞)和其它编码能够影响mRNA加工或基因表达的调控信号的序列。多腺苷化信号(通常限于真核细胞)的特征通常是影响向mRNA前体的3’末端添加多腺苷酸束。

本领域技术人员完全明白在使用核苷酸密码子指定给定的氨基酸时特异的宿主细胞显示出的“密码子偏向性”。因此，当为了在宿主细胞中改进的表达而合成基因时，需要将该基因设计为其密码子的使用反映宿主细胞中优选的密码子偏向性。对于源自宿主细胞的基因的调查(其中序列信息是可以获得的)可以确定其密码子偏向性。密码子的最优化是本领域熟知的，已经描述了对于各种系统中的密码子的最优化，包括但不限于酵母(Outchkourov等人，Protein Expr Purif，24(1)：18-24(2002))和大肠杆菌(Feng等人，Biochemistry，39(50)：15399-15409(2000))。

具有腈水解酶活性的酶催化剂

所有的腈水解酶(EC 3.5.5.7)均具有保守性催化三元组(Glu，Lys和Cys)(Chauhan等人，Appl.Microbiol.Biotechnol.61：118-122(2003)；Pace，H.and Brenner，C.，Genome Biol.[online computer file]2(1)：reviews0001.1-0001.9(2001))。所有已知的腈水解酶均在酶活性位点具有亲核的半胱氨酸(Cowan等人，Extremophiles，2：207-216(1998)；Pace，H.and Brenner，C.，见上；和Chauhan等人，见上)并且都对巯基反应剂的灭活敏感(1.0mM浓度的氯化铜、硝酸银、醋酸汞或氯化铁每个均引起敏捷食酸菌72W腈水解酶酶活性的大幅下降)。半胱氨酸残基还能够被不可逆地氧化为亚磺酸，导致酶活性的损失。虽然腈水解酶对各种灭活机制有敏感性，但是固定的敏捷食酸菌72W细胞是强壮的(robust)，能够在多个循环反应后保留其大部分腈水解酶活性(US 6,870,038；U.S.7,148,051；U.S.7,198,927；和Chauhan等人，见上)。也已经显示源自敏捷食酸菌72W腈水解酶的腈水解酶催化剂催化α-羟基腈(即乙醇腈)向α-羟基羧酸(即乙醇酸)的转化(参见US 6,383,786；US6,416,980；和U.S.7,198,927)。

已经报道了敏捷食酸菌72W腈水解酶与其它细菌腈水解酶的序列比较(US 6,870,038；Chauhan等人，见上)。72W腈水解酶具有几个保守性标志结构域，包括靠近氨基末端处的16个氨基酸的区域(SEQ ID NO：4的氨基酸残基40-55)和含有必需的半胱氨酸残基的12个氨基酸的催化区域(SEQ IDNO：4的氨基酸残基160-171)。这个必需的半胱氨酸残基(SEQ ID NO：4的Cys₁₆₄)以及保守性的谷氨酸(SEQ ID NO：4的Glu₄₈)和赖氨酸残基(SEQID NO：4的Lys₁₃₀)构成在所有腈水解酶中发现的催化三元组基序(Pace，H.，and Brenner，C.，见上)。

每个催化三元组残基周围的区域是高度保守的，特别是催化性半胱氨酸残基周围的区域。必需的催化性半胱氨酸残基位于被称为“催化标志基序”或“标志基序”的高度保守性区域内。因此，本方法可用于保护包含式1(粗体代表严格保守性氨基酸残基，斜体残基是显示极小可变性的残基[即3个或更少的氨基酸残基的极小变化]，催化性半胱氨酸残基以下划线标出)定义的催化标志基序的任意腈水解酶的酶活性：

式1(SEQ ID NO：1)

Gly-Xaa₁-Xaa₂-Xaa₃-Cys-Trp-Glu-Xaa₄-Xaa₅-Xaa₆-Xaa₇-Xaa₈

其中

Xaa1＝Ala或Gly；

Xaa2＝Leu，Val或Ala；

Xaa3＝Ala，Asn，Ile，Cys，Val或Gln；

Xaa4＝His或Asn；

Xaa5＝Leu，Tyr，Phe，Ala，Met，Lys，Val，Thr或Arg；

Xaa6＝Asn，Gln，Met，Leu或Ser；

Xaa7＝Pro或Thr；和

Xaa8＝Leu或Val。

在优选的实施方式中，式1的腈水解酶标志基序是Xaa₁＝Ala或Gly；Xaa₂＝Leu；Xaa₃＝Ala，Asn，Ile，Cys，Val或Gln；Xaa₄＝His；Xaa₅＝Leu，Tyr，Phe，Ala，Met，Lys，Val，Thr或Arg；Xaa₆＝Ser，Gln，Asn或Met；Xaa₇＝Pro；和Xaa₈＝Leu；从而产生由下式代表的催化标志基序：

Gly-Xaa₁-Leu-Xaa₃-Cys-Trp-Glu-His-Xaa₅-Xaa₆-Pro-Leu(SEQ ID NO：2)。

在表1中提供了腈水解酶的例子，包括相应的催化标志基序序列的序列和位置。

表1.保守性催化性半胱氨酸区域-催化标志基序

在一个实施方式中，腈水解酶催化剂包含具有腈水解酶活性的多肽，所述多肽分离自下列属：食酸菌属、红球菌属、诺卡氏菌属、芽孢杆菌属和产碱杆菌属。在一个实施方式中，腈水解酶催化剂包含具有腈水解酶活性的多肽，所述多肽分离自下列属：食酸菌属和红球菌属。

在另一个实施方式中，具有腈水解酶活性的多肽源自敏捷食酸菌72W(ATCC 55746)或者与敏捷食酸菌72W腈水解酶(SEQ ID NO：4)或源自敏捷食酸菌72W的酶(由SEQ ID NO：51代表)实质上相似的多肽(具有腈水解酶活性)。

在一个实施方式中，腈水解酶催化剂是经过转化以表达至少一个具有腈水解酶活性的多肽的微生物宿主细胞。在一个实施方式中，转化的宿主细胞选自丛毛单胞菌属(Comamonas sp.)、棒状杆菌属(Corynebacterium sp.)、短杆菌属(Brevibacterium sp.)、红球菌属(Rhodococcus sp.)、固氮菌属(Azotobacter sp.)、柠檬酸杆菌属(Citrobacter sp.)、肠杆菌属(Enterobacter sp.)、梭菌属(Clostridium sp.)、克雷伯菌属(Klebsiella sp.)、沙门氏菌属(Salmonella sp.)、乳杆菌属(Lactobacillus sp.)、曲霉属(Aspergillus sp.)、酵母菌属(Saccharomyces sp.)、耶氏酵母属(Yarrowia sp.)、接合酵母属(Zygosaccharomyces sp.)、毕赤酵母属(Pichia sp.)、克鲁维酵母属(Kluyveromyces sp.)、假丝酵母属(Candida sp.)、汉逊酵母属(Hansenulasp.)、杜氏藻属(Dunaliella sp.)、德巴利酵母属(Debaryomyces sp.)、毛霉属(Mucor sp、)、球拟酵母属(Torulopsis sp.)、甲基杆菌属(Methylobacteria sp.)、芽孢杆菌属(Bacillus sp.)、埃希氏菌属(Escherichia sp.)、假单胞菌属(Pseudomonas sp.)、根瘤菌属(Rhizobium sp.)和链霉菌属(Streptomyces sp.)。在优选的实施方式中，微生物宿主细胞选自芽孢杆菌属、假单胞菌属和埃希氏菌属。在优选的实施方式中，催化剂是重组表达一个或多个具有腈水解酶活性的多肽的大肠杆菌宿主细胞。

在另一个实施方式中，腈水解酶催化剂包含具有腈水解酶活性的多肽，其中所述具有腈水解酶活性的多肽与SEQ ID NO：51具有至少60％的同一性，优选与SEQ ID NO：51具有至少70％的同一性，更优选与SEQ ID NO：51具有至少80％的同一性，再更优选与SEQ ID NO：51具有至少90％的同一性，最优选与SEQ ID NO：51具有至少95％的同一性。

本文描述了源自各种来源的具有腈水解酶活性的几个催化剂的工作实例，包括源自敏捷食酸菌72W腈水解酶的催化剂。本领域已经报道了源自敏捷食酸菌72W腈水解酶的各种突变体(美国专利7,148,051和美国专利7,198,927)。

在一个实施方式中，具有腈水解酶活性的多肽选自SEQ ID NO：4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、27、29、31、33、35、37、39、41、43、45、47、49、51、53、55和57。在另一个实施方式中，具有腈水解酶活性的多肽选自4、24、25、27、29、31、33、35、37、39、41、43、45、47、49、51、53、55和57。在另一个实施方式中，具有腈水解酶活性的多肽选自4、27、29、31、33、35、37、39、41、43、45、47、49、51、53、55和57。在另一个实施方式中，具有腈水解酶活性的多肽选自4、24、25和51。在另一个实施方式中，腈水解酶催化剂包含SEQ ID NO：51的多肽。

敏捷食酸菌72W(ATCC 55746)腈水解酶

敏捷食酸菌72W腈水解酶(EC 3.5.5.1)是从脂肪族或芳香族的腈产生羧酸的强烈催化剂(WO 01/75077；US 6,870,038；和Chauhan等人，见上)。还已经显示它催化α-羟基腈(即乙醇腈)向α-羟基羧酸(即乙醇酸)的转化(参见US 6,383,786；和US 6,416,980)。但是，在将乙醇腈转化成乙醇酸时具有改进的腈水解酶活性和/或稳定性(相对于敏捷食酸菌72W腈水解酶)的腈水解酶催化剂将降低生产乙醇酸的成本。因此，使用改进的腈水解酶催化剂生产乙醇酸的方法可用于降低生产乙醇酸的成本。但是，敏捷食酸菌72W腈水解酶是用于本方法目的的酶催化剂，以及上文详细描述的所述的改进的腈水解酶。

酶催化剂的工业生产

当需要进行本文描述的酶催化剂的商业生产时，可以使用多种培养方法。可以使用本领域熟知的方法以批式、补料-分批或连续模式进行发酵运行(Thomas D.Brock in Biotechnology：A Textbook of Industrial Microbiology，Second Edition(1989)Sinauer Associates，Inc.，Sunderland，MA，(1989)；Deshpande，Mukund V.，Appl.Biochem.Biotechnol.36(3)：227-234(1992))。

经典的批式培养方法是封闭的系统，其中在培养开始时设定培养基的成分，在培养过程中不进行人工改变。因此，在培养过程开始时将需要的生物接种到培养基并允许其生长或产生代谢活性(不向系统中加入任何物质)。但是，通常来讲，“批式”培养是添加碳源意义上的批，经常需要尝试控制一些因素例如pH和氧气浓度。在批式系统中，系统的代谢物和生物质成分持续变化，直至培养结束。在批式培养内，细胞从静态延迟期缓慢到达高速生长对数期，最终到达稳定期，在稳定期生长速率降低或停止。如果不处理的话，处于稳定期的细胞最终将死亡。在一些系统中，处于对数期的细胞通常负责大量生产终产物或中间体。在其它系统中可以在稳定期或指数期之后获得生产。

补料-分批系统是标准批式系统的变体。补料-分批培养过程也适合于本发明，其包含典型的批式系统，只不过随着培养过程的进行逐渐增加底物的投入。当分解代谢物阻遏倾向于抑制细胞的代谢并且需要限制培养基中底物的数量时，补料-分批系统是有用的。补料-分批系统中的实际底物浓度是难以测定的，因此基于可测因素(例如pH、溶解氧和废气(例如CO₂)分压)的变化来评估。批式和补料-分批培养方法是常见的也是本领域熟知的，可以在Brock(见上)和Deshpande(见上)中找到例子。

还可以通过连续培养实现具有腈水解酶的本发明的酶催化剂的商业生产。连续培养是开放的系统，其中向生物反应器中连续加入确定的培养基，同时为了处理除掉等量的条件化培养基。连续培养通常将细胞保持在恒定的高液相密度，其中细胞主要处于对数生长期。或者，可以以固定的细胞进行连续培养，其中连续加入碳和营养物并且从细胞物质中连续除掉有用的产物、副产物或废弃产物。可以使用多种固体支持物(由天然和/或合成材料组成)进行细胞的固定。

连续或半连续培养允许调节影响细胞生长或最终细胞浓度的一个因子或任意数目的因子。例如，一种方法将限制性营养物(例如碳源或氮水平)保持在固定速率并允许所有其它参数变缓。在其它系统中，可以持续改变一些影响生长的因子但保持细胞浓度(根据培养液浑浊度测定)恒定。连续系统努力保持稳定态生长条件，因此在培养物中必须保持由于培养液排除而造成的细胞损失与细胞生长速率之间的平衡。调节连续培养过程的营养物和生长因子的方法以及使细胞的生成速率最大化的技术在工业微生物领域是熟知的，Brock(见上)中详细描述了多种这样的方法。

本发明的发酵培养基必须含有合适的碳底物。合适的底物可以包括但不限于：单糖例如葡萄糖和果糖，二糖例如乳糖或蔗糖，多糖例如淀粉或纤维素或其混合物，以及来自可再生原料(例如干酪乳清渗透物、玉米浸泡液、甜菜糖浆和大麦麦芽)的未纯化的混合物。因此，考虑到本发明中使用的碳源可以包括很多种含碳底物，这将仅由生物的选择所限定。

在固定之前以戊二醛预处理酶催化剂

以戊二醛处理酶催化剂发酵培养物是杀灭培养物中的微生物从而避免污染和与活体重组培养物相关的操作、储存和运输方面的安全问题的简便方法。现在已经发现以戊二醛预处理或者在以戊二醛预处理之后以亚硫酸氢盐处理能够保持悬浮液和固定形式的细胞中的腈水解酶活性。

通过以戊二醛预处理酶催化剂而保持腈水解酶活性受到时间、温度、戊二醛浓度、pH和抑制性产物(例如氨和培养液中与戊二醛相互作用的其它胺(例如氨基酸和肽))浓度的影响。优选的戊二醛预处理方法以水中的5-10wt％的戊二醛处理来自高密度发酵的细胞(100-150OD₅₅₀)，所述水中的戊二醛优选在充分混合下以50mg至500mg戊二醛/L-min输送，更优选在充分混合下以50mg至200mg戊二醛/L-min输送，最优选在充分混合下以50mg至100mg戊二醛/L-min输送，从而产生大约3g至大约5g戊二醛/L的终浓度(大约0.025g至大约0.042g戊二醛/OD₅₅₀)，更优选大约3.6g至大约5g戊二醛/L的终浓度(大约0.030g至大约0.042g戊二醛/OD₅₅₀)。戊二醛预处理的培养物可以保持于发酵器中大约1至5小时。然后任选地以1g/L加入10wt％的亚硫酸氢钠水溶液以将残余戊二醛灭活。

发酵液或细胞悬浮液中酶催化剂的戊二醛预处理的优选pH为pH5.0至9.0，更优选为pH5.0至8.0，再更优选为pH5.0至7.0，再更优选为pH5.0至6.0，最候选为pH5.0至5.5。戊二醛预处理后的残余戊二醛浓度通常较低，处于10-200ppm范围内，可以按照如上所述通过加入亚硫酸氢盐至大约1g/L的终浓度来将残余戊二醛灭活。发现戊二醛和亚硫酸氢盐预处理对于腈水解酶活性没有显著性有害效应。任选地，将戊二醛或戊二醛/亚硫酸氢盐预处理的细胞悬浮液冷却至5-10℃，任选地以水或合适的储存缓冲液洗涤(通过浓缩和重新稀释细胞悬浮液或发酵液进行)以除去残余的亚硫酸氢盐和未反应的戊二醛。

戊二醛预处理的酶催化剂的固定和化学交联

已经大量报道了固定酶催化剂的方法，这些方法对本领域技术人员是熟知的(Methods in Biotechnology，Vol.1：Immobilization of Enzymes and Cells；Gordon F.Bickerstaff，Editor；Humana Press，Totowa，NJ，USA；1997)。以前也已经报道了敏捷食酸菌72W腈水解酶催化剂的固定(US 6,870,038)。

此外，还有在角叉菜胶中固定然后以戊二醛/聚乙烯亚胺交联固定的酶催化剂的方法(如在US 6,870,038中公开和在US 6,551,804B中详细描述的那样，通过引用并入本文)，但是，本领域普通技术人员将认识到并能容易地适用改变以完成固定和交联。所述改变考虑在本文之内并且落入本方法的范围。此外，用于固定和化学交联的成分的数量或浓度将根据酶催化剂和酶催化剂的发酵产物的数量和类型改变。本领域普通技术人员将认识到这些因素并相应地调整固定和化学交联程序。关于以戊二醛和聚乙烯亚胺进行化学交联，US 6,551,804(见上)描述了化学交联藻酸盐固定的细胞的方法和程序。所述描述也适用于本文的角叉菜胶固定的细胞。

使用腈水解酶催化剂将乙醇腈水解为乙醇酸

乙醇腈向乙醇酸(酸和/或相应的铵盐的形式)的酶法转化可以这样进行：在如下所述的合适的反应条件下(即在一定的pH范围、温度、浓度等等的水性反应混合物中)与具有腈水解酶活性的酶催化剂、固定的酶催化剂或交联的固定的酶催化剂接触。在一个实施方式中，将整个重组微生物细胞在角叉菜胶中固定、交联，产生的酶催化剂直接用于乙醇腈向乙醇酸的转化，或者，可以使用中空纤维膜卷或超滤膜从大宗反应混合物中单独保留非固定的细胞。在第二个实施方式中，将整个重组微生物细胞固定于聚丙烯酰胺凝胶中，所产生的酶催化剂直接用于乙醇腈向乙醇酸的转化。

水性反应混合物中的酶催化剂的浓度取决于酶催化剂的比活性并根据想要获得的反应速率来进行选择。水解反应中用作催化剂的微生物细胞的湿细胞重通常介于每毫升总反应体积中有0.001克至0.250克的湿细胞，优选为每毫升0.002克至0.050克的湿细胞。指明的每体积的总反应体积中的湿细胞重量百分比可以以固定的酶催化剂(按照如上所述制备(见上文))的形式存在于反应混合物中，其中从制备固定的酶催化剂的方法中可以得知作为固定的酶催化剂总重量百分率的湿细胞的重量。

将乙醇腈水解反应的温度选定为既控制反应速率又控制酶催化剂活性的稳定性。反应的温度可以是：稍微高于反应混合物的凝固点(大约0℃)至大约65℃，优选的反应温度范围是大约5℃至大约35℃。可以通过将固定的细胞悬浮于蒸馏水或缓冲剂的水溶液(其将会把反应的初始pH保持在大约5.0至大约10.0，优选大约5.5至大约8.0，更优选大约5.5至大约7.7，最优选大约6.0至大约7.7)中而制备酶催化剂悬浮液。随着反应的进行，反应混合物的pH将变化，这是由于从相应的腈官能团形成了羧酸的铵盐。可以将反应进行到完全实现乙醇腈的转化而不控制pH，或者可以随着反应的进程加入合适的酸或碱以保持想要的pH。

发现乙醇腈在25℃时以任意比例与水完全混溶。在反应条件被选定为底物(即α-羟基腈)的溶解性也依赖于溶液的温度和/或水相中盐的浓度(缓冲剂或产物乙醇酸铵盐，也称为乙醇酸铵)的情况下，初始的反应混合物可以由两相组成：含有酶催化剂和溶解的α-羟基腈的水相，和有机相(未溶解的α-羟基腈)。随着反应的进行，α-羟基腈溶解到水相中，最终获得单一相的产物混合物。反应还可以这样进行：以大约等于酶法水解反应速率的速率向反应混合物中加入α-羟基腈，从而保持单一相的水性反应混合物，并避免在高浓度的起始材料时酶的底物抑制的潜在问题。

乙醇酸可以作为质子化羧酸和/或其相应铵盐(取决于产物混合物的pH；乙醇酸的pKa是大约3.83)的混合物存在于产物混合物中，另外可以作为羧酸与产物混合物中可能另外存在的任何缓冲剂的盐而存在。通常，产生的乙醇酸主要是铵盐的形式(乙醇腈水解反应的pH通常为大约5.5至大约7.7)。根据需要，可以以质子化羧酸或羧酸盐的形式将乙醇酸产物从反应混合物中分离出来。

乙醇腈完全转化时产物混合物中乙醇酸的终浓度为0.001M至乙醇酸产物的溶解度上限。在一个实施方式中，乙醇酸的浓度为大约0.10M至大约5.0M。在另一个实施方式中，乙醇酸的浓度为大约0.2M至大约3.0M。

可以使用多种技术(包括但不限于，离子交换、电渗析、反应性溶剂提取、聚合化、热分解、醇解，及其组合)以酸或对应的盐的形式回收乙醇酸。

另外，当以范围、优选的范围或优选值的上限和优选值的下限给出数量、浓度或其它数值或参数的时候，应理解为具体公开了任意一对任意范围上限或优选值与任意范围下限或优选值之间形成的所有的范围，不论是否单独公开了这样的范围。除非另有指明，否则当本文中提到数值的范围时，该范围旨在包括其端点值和该范围内的所有整数和分数。当定义一个范围时，其目的不是将本发明的范围限定为具体的数值。

一般性方法

提供了以下实施例以证明本发明的优选实施方式。本领域技术人员应该认识到，以下实施例中公开的技术代表本发明人发现的在实施本发明时很好地发挥作用的技术，因此其被认为构成本发明实施的优选模式。但是，根据本公开，本领域技术人员应认识到可以不脱离本发明的精神和范围对所公开的特异性实施方式作出很多修改并仍然获得相同或相似的结果。

适合用于维持细菌培养物和细菌培养物生长的材料和方法在本领域是熟知的。可以在以下文献中找到适合用于以下实施例的技术：Manual of Methods for General Bacteriology (1994)(Phillipp Gerhardt，R.G.E.Murray，Ralph N.Costilow，Eugene W.Nester，Willis A.Wood，Noel R.Krieg and G.Briggs Phillips，eds.)，American Society for Microbiology，Washington，DC.)或Thomas D.Brock，in Biotechnology：A Textbook of Industrial Microbiology，(1989)Second Edition，(Sinauer Associates，Inc.，Sunderland，MA).Methods to immobilize enzymatic catalysts can be found in Bickerstaff，G.F.，见上)。

制备基因组DNA、PCR扩增、通过内切和外切核酸酶修饰DNA以产生想要的DNA克隆的末端、连接和细菌转化所需的程序在本领域中是熟知的。本文所用的标准重组DNA和分子克隆技术在本领域中是熟知的，见以下描述：Maniatis，上文；和T.J.Silhavy，M.L.Bennan，and L.W.Enquist，Experiments with Gene Fusions，(1984)Cold Spring Harbor Laboratory Press，Cold Spring，NY；and by Ausubel，F.M.等人，Current Protocols in Molecular Biology，(1994-1998)John Wiley&Sons，Inc.，New York。

除非另有指明，否则所有的反应试剂和材料均购自Aldrich Chemicals(Milwaukee，WI)，DIFCO Laboratories (Detroit，MI)，GIBCO/BRL(Gaithersburg，MD)或Sigma/Aldrich Chemical Company(St.Louis，MO)。

说明书中对应于测定单位、技术、性质或化合物的缩写如下：“sec”意思是秒，“min”意思是分钟，“h”或“hr”意思是小时，“d”意思是以g/mL表示的密度，“μL”意思是微升，“mL”意思是毫升，“L”意思是升，“mM”意思是毫摩尔，“M”意思是摩尔，“mmol”意思是毫摩，“wt”意思是重量，“wt％”意思是重量百分率，“g”意思是克，“μg”意思是微克，“HPLC”意思是高效液相色谱，“O.D.”意思是在指定波长时的光密度，“dcw”意思是干细胞重，“U”意思是腈水解酶活性单位，“EDTA”意思是乙二胺四乙酸，“DTT”意思是二硫苏糖醇。一单位的腈水解酶活性对应于每分钟水解1μmol的乙醇腈。

分析方法

HPLC分析

除非另有指明，否则使用以下的HPLC方法。通过以下HPLC方法分析反应的产物混合物。向1.50mL水中加入反应混合物的等份(0.01mL)并通过HPLC分析(HPX 87H柱，30cm x 7.8mm；0.01N H₂SO₄移动相；50℃，流速1.0mL/min；10μL注射体积；RI检测器，20分钟分析时间)。使用从Aldrich购买的商售乙醇腈的系列稀释将方法进行针对乙醇腈的校准。

定量¹³CNMR分析

使用Varian Unity Inova分光计(Varian，Inc.，Palo Alto，CA)在400MHz获得定量¹³CNMR光谱。样品制备：在10mm的NMR管中加入3.0mL反应产物和0.5mL D₂O。通常使用26KHz的谱宽(发射器位于100ppm，128Kpoints)和90度脉冲(发射能量为56db时pw90＝10.7微秒)获得¹³CNMR光谱。最长的13C T1(23sec)与GLN腈碳相关，总循环时间设定为比该数值大10倍(循环延迟d1＝240sec，获取时间at＝2.52sec)。信号平均为360个扫描时给出26.3小时的总实验时间。仅在获取时间(at)过程中对Waltz-调节的1H去耦设门，从而抑制Nuclear Overhauser Enhancement(NOE)。

实施例1大肠杆菌MG1655/pSW138-168V的发酵

在接种到发酵器之前，大肠杆菌MG1655/pSW138-168V的种子培养物在30℃摇动的(300rpm)500mL LB培养基(每毫升补充0.1mg氨比西林)中生长6-10h(OD₅₅₀＝1-2)。大肠杆菌MG1655/pSW138-168V腈水解酶菌株生长于14-L Braun Biostat C发酵器(B.Braun Biotech International Gmbh，Melsungen，Germany)中，使用最简培养基(其中含有葡萄糖、氨和盐以及用于诱导的乳糖)。在表2中描述了灭菌前发酵培养基(7.5L)。灭菌后的添加物包括过滤灭菌的痕量元素(表3)、每毫升0.1mg氨比西林、每升2g酪蛋白氨基酸(Difco)、每升4g葡萄糖和500mL种子培养物。

表4描述了发酵设定点。NH₄OH(40％w/v)和H₃PO₄(20％w/v)用于控制pH。将溶解氧浓度控制在空气饱和的25％，随着氧气需求的增加首先提高搅拌设定，然后通气。在表5中给出了乳糖诱导所用的发酵补料程序。如果葡萄糖累积超过5g/L，则降低葡萄糖补料速率。40-56小时后，将发酵液冷却至5-10℃，通过离心收集细胞。将细胞糊冷冻并储存于-70℃。将细胞糊命名为NIT 60(1910GLN U/g dcw)。

表2.灭菌前的发酵培养基

(NH4)₂SO₄	5.0g/L
		K₂HPO₄	4.0g/L
KH₂PO₄	3.5g/L

MgSO₄*7H₂O	0.6g/L
		柠檬酸三钠(Na₃Citrate)*2H₂O	1.0g/L
NZ Amine AS(Quest)	2.5g/L
		消泡剂(Antifoam)-Biospumex 153K	0.25ml/L

表3.发酵痕量元素

	浓度
		柠檬酸	10g/L
CaCl₂*2H₂O	1.5g/L
		FeSO₄*7H₂O	5g/L
ZnSO₄*7H₂O	0.39g/L
		CuSO₄*5H₂O	0.38g/L
CoCl₂*6H₂O	0.2g/L
		MnCl₂*4H₂O	0.3g/L

表4.发酵设定点

	初始设定点	最小值	最大值
				搅拌器(rpm)	400	400	1000
气流(slpm)	2	2	10
				pH	6.8	6.8	6.8
压力(kPa)	0.5	0.5	0.5
				DO	25％	25％	25％

温度℃

30

表5.乳糖诱导所用的发酵补料程序

EFT(h)	补料速率(g/min)	底物
			0	0	葡萄糖(批式的)
5	0.27	葡萄糖(50％w/w)
			14	1.3	乳糖(25％w/w)

实施例2大肠杆菌MG1655/pSW138-168V在GA/PEI交联的角叉菜胶珠子中的固定

在50℃，边迅速搅拌边向228g去离子蒸馏水中缓慢加入12g角叉菜胶(FMC GP911)，将得到的混合物加热至80℃直至角叉菜胶完全溶解，将得到的溶液搅拌冷却至52℃。在单独的具有搅拌子的烧杯中，在约25℃，向84.8g 0.35M Na₂HPO₄(pH 7.3)中加入83.2g冷冻的大肠杆菌MG1655/pNM18-168V细胞(25.2％dcw)并混合直至细胞悬浮，然后加入脱氧核糖核酸酶I溶液(10μL 12,500U/mL DNase(Sigma)/100mL细胞悬浮液)。将细胞悬浮液连续通过230微米和140微米Nupro TF strainer element filter过滤，并搅拌加热至50℃。在搅拌的同时，将50℃的160.0g大肠杆菌MG1655/pNM18-168V细胞悬浮液加入到52℃的角叉菜胶溶液中，在47℃将得到的细胞/角叉菜胶悬浮液抽入电加热的20G的针头，在约37-38℃在搅拌的同时滴入0.25M KHCO₃(pH＝7.3)；通过针头的流速设定为5-8mL/min。使得到的珠子在相同的缓冲液中于室温在搅拌的同时硬化1小时，将其储存于0.25M碳酸氢钾(pH 7.3)。

固定的细胞/角叉菜胶珠子的化学交联这样进行：将水中的0.5g 25％的戊二醛(GA)(Sigma M752-07)加入到悬浮于48mL 0.25M碳酸氢钾(pH 7.3)中的20g珠子中，并在室温搅拌1个小时。然后向珠子悬浮液中加入水中的2.0g 12.5wt％的聚乙烯亚胺(PEI，BASF LUPASOL PS)，在室温将珠子悬浮液再搅拌18个小时。从悬浮液中回收GA/PEI交联的珠子，在48mL 0.25M碳酸氢钾(pH 7.3)中搅拌两次(15分钟)；然后储存于5℃1.0M碳酸氢铵(pH 7.3)中。在用作乙醇腈转化为乙醇酸(作为铵盐)的催化剂之前，在室温以180mL 0.1M乙醇酸铵(pH 7.3)将珠子洗涤两次(15分钟)以除去1.0M碳酸氢铵(pH 7.3)储存缓冲液。得到的固定的细胞催化剂被称为固定的NIT 60。

实施例3固定之前以戊二醛预处理大肠杆菌MG1655/pSW138-168V

进行200-L的发酵以产生含有大肠杆菌MG1655/pSW138-168V细胞的发酵液，然后在固定之前在原位以戊二醛预处理细胞。首先制备预种子培养物：在2-L的摇瓶中加入0.5L种子培养基(含有酵母提取物(Ambrex 695，5.0g/L)，K₂HPO₄(10.0g/L)，KH₂PO₄(7.0g/L)，二水合柠檬酸钠(1.0g/L)，(NH₄)₂SO₄(4.0g/L)，MgSO₄·7H₂O(1.0g/L)和柠檬酸铁铵(0.10g/L))。将培养基的pH调整为6.8，将培养基在瓶中灭菌。灭菌后添加物包括葡萄糖(10mL，50wt％)和1mL氨比西林(25mg/mL)。将20％甘油中的1-mL冷冻的大肠杆菌MG1655/pSW138-168V贮存培养物接种到预种子培养基，并在35℃和300rpm培养。在约2OD₅₅₀将种子培养物转移到14L的种子发酵器(Braun)(其中含有8L的培养基，培养基中含有KH₂PO₄(3.50g/L)，FeSO₄·7H₂O(0.05g/L)，MgSO₄·7H₂O(2.0g/L)，二水合柠檬酸钠(1.90g/L)，酵母提取物(Ambrex 695，5.0g/L)，Biospumex153K除泡剂(0.25mL/L，CognisCorporation)，NaCl(1.0g/L)，CaCl₂·2H₂O(10g/L)和NIT痕量元素溶液(10mL/L))。痕量元素溶液含有一水合柠檬酸(10g/L)，水合MnSO₄(2g/L)，NaCl(2g/L)，FeSO₄·7H₂O(0.5g/L)，ZnSO₄·7H₂O(0.2g/L)，CuSO₄·5H₂O(0.02g/L)和NaMoO₄·2H₂O(0.02g/L)。灭菌后添加物包括120g葡萄糖溶液(50％w/w)和氨比西林16mL贮存液(25mg/mL)。

将溶解氧(dO)的浓度控制在空气饱和的25％。首先通过叶轮搅拌速率(400至1400rpm)然后通过通气速率(2至10slpm)控制dO。将pH控制在6.8。NH₄OH(29％w/w)和H₂SO₄(20％w/v)用于控制pH。将温度控制在35℃，头压力为0.5bars。在ca 6OD₅₅₀时将培养物转移到200L的Biostat-D Braun发酵器。所用的培养基与种子发酵器中的相同，初始工作体积为140L，加入50％w/w葡萄糖至8g/L。首先以批式操作启动发酵，一旦葡萄糖消耗至＜0.5g/L，则以50％w/w葡萄糖按照预先确定的速率(表6)启动补料分批操作。在ca 25OD₅₅₀时，将补料转换为按照预先确定的速率的25％D-乳糖溶液(表7)。

将温度控制在35.0℃，头压力控制在0.5bar，第一阶段(葡萄糖阶段)的pH控制在6.8，第二阶段(乳糖阶段)的pH控制在7.2。NH₄OH(29％w/w)和H₂SO₄(20％w/v)用于控制pH，第一阶段的dO控制在空气饱和的25％，第二阶段的dO控制在10％。首先通过搅拌(250至450rpm)然后通过通气(25至35slpm)控制dO。在补料操作过程中监视葡萄糖和乳糖水平，如果葡萄糖水平超过0.1g/L或者乳糖超过1g/L，则临时停止补料程序或降低补料。操作在启动乳糖补料40小时之后结束，通过离心或微过滤收集细胞或者保持在容器中进行戊二醛处理。产生的发酵大约为8kg干细胞重，具有2819BZN U/g dcw(1788GLN U/g dcw)的腈水解酶比活性。

表7：补料程序

补料时间间隔(h)	补料速率g/min	底物	阶段
				0	6.13	50％w/w葡萄糖	第一个
1	7.13	50％w/w葡萄糖	第一个
				2	8.28	50％w/w葡萄糖	第一个
3	9.62	50％w/w葡萄糖	第一个
				4	11.18	50％w/w葡萄糖	第一个
5	11.18	50％w/w葡萄糖	第一个
				6	11.18	50％w/w葡萄糖	第一个
7	11.18	50％w/w葡萄糖	第一个

8	11.18	50％w/w葡萄糖	第一个

0	11.22	25％w/w乳糖	第二个
				2	24.42	25％w/w乳糖	第二个
20	16.72	25％w/w乳糖	第二个
				30	18.7	25％w/w乳糖	第二个
40	18.7	25％w/w乳糖	第二个

在发酵末尾，搅拌减慢至150rpm，停止通气，将温度保持在35℃。取出部分发酵液，在发酵器中留下约180kg。将该遗留的液体滴定至pH 5.2并以20％H₂SO₄(20％w/w)和NaOH(50％w/w)保持在该pH，同时边搅拌边以～90mL/min的速度加入9.0L的水性戊二醛(GA，10％w/w)；这个添加速率等于50mg戊二醛/升发酵液/min，戊二醛的终浓度为约5g戊二醛/L(0.035g戊二醛/OD₅₅₀)。最初向液体中加入戊二醛5个小时后，将pH调整为7.0，边搅拌边加入1.8L的水性亚硫酸氢钠(10％w/w，pH 7)(终浓度为约1g亚硫酸氢钠/L)，将液体再搅拌15分钟。然后将液体的温度降低至10℃，搅拌降低至100rpm。使用Diskstack离心机(Alfa Laval)将液体浓缩至40kg的细胞悬浮液，然后向悬浮液中加入50kg DI水(20℃)，通过离心将混合物浓缩以产生40kg洗涤的细胞悬浮液。将悬浮液(称为NIT188A-C2)储存于5℃，将一部分细胞悬浮液直接用于制备固定的细胞催化剂(实施例6)。每个加工步骤中的腈水解酶比活性总结于表8。

表8：GA和亚硫酸氢盐处理过程中的不同阶段的腈水解酶活性

发酵阶段	BZN U/g dcw
		GA处理前GA处理后NaHSO3处理后	281933002493

实施例4戊二醛预处理的大肠杆菌MG1655/pNM18-168V在GA/PEI交联的角叉菜胶珠子中的固定

在5℃将从戊二醛和亚硫酸氢钠处理的实施例5的发酵液中回收的最终细胞悬浮液浓缩物离心。将产生的细胞沉淀重悬于5倍重量的0.35M磷酸钾缓冲液(pH 7.2)中，将得到的细胞悬浮液在5℃离心，从而产生如实施例2所述的固定的和GA与PEI化学交联的湿细胞糊。产生的固定的细胞催化剂被称为固定的NIT 188A-C2。

实施例5使用戊二醛/聚乙烯亚胺交联的角叉菜胶固定的大肠杆菌 MG1655/pSW138-F168V转化子的具有催化剂循环的连续批式反应中生物催化剂的比活性的改进

在典型的程序中，在50-mL的夹套反应器(具有顶部搅拌装置和温度控制)中进行乙醇腈向乙醇酸转化的批式反应(每组一式两份)。每个反应器加入8g GA/PEI交联的大肠杆菌MG1655/pSW138-168V/角叉菜胶珠子(根据实施例1所述方法(固定之前不进行GA预处理)或实施例4所述方法(固定之前进行GA预处理)制备，含有5％(dcw)表达敏捷食酸菌突变体F168V(SEQ ID NO：51)的转化子)。然后向容器中加入14.78mL蒸馏水和6.0mL的水性乙醇酸铵(4.0M，pH 7.0)，以氮气冲洗反应器。在25℃搅拌混合物，同时使用程序化注射器泵同时加入水中的1.07mL 60wt％乙醇腈(GLN)(12.0mmol GLN，0.084mmol甲醛；Fluka(重蒸馏的，以0.5wt％乙醇酸稳定))和0.150mL的水性氢氧化铵(1.875wt％)；同时每2个小时加入1份等体积的GLN和氢氧化铵溶液(总共加入8次等量的GLN溶液和水性氢氧化铵)以保持GLN的浓度≤400mM并且pH在6.5-7.5的范围内。在最初加入GLN之后预先确定的时间取出4个0.050-mL的反应样品，通过HPLC分析以确定初始反应速率和催化剂的比活性(μmol乙醇酸/min/g dcw生物催化剂)。在反应完成时，100％的GLN被转化以产生乙醇酸(作为铵盐的形式)，产率＞99％。

在第一个反应的末尾，从催化剂中轻轻倒出(在氮气下)水性产物混合物，留下约10.3g固定的细胞催化剂(8.0g)和残余产物混合物(ca 2.3g)的混合物。然后向反应器中加入20.78mL去离子蒸馏水，通过加入水性GLN和氢氧化铵的等份(如紧接的上一段所述)在25℃进行第二个反应。在表7中列出了具有催化剂循环的连续批式反应中回收的生物催化剂的比活性。

表7.具有生物催化剂循环的连续批式反应中回收的生物催化剂的比活性对于在固定之前以戊二醛预处理细胞的依赖性

实施例6使用戊二醛预处理的细胞制备的戊二醛/聚乙烯亚胺交联的角叉菜胶固定的大肠杆菌MG1655/pSW138-F168V的储存稳定性

将使用戊二醛预处理的细胞新鲜制备的戊二醛/聚乙烯亚胺交联的角叉菜胶固定的大肠杆菌MG1655/pSW138-F168V(如实施例4所述)在5℃在1.0M碳酸氢铵(pH 7.3)中储存32天。使用之前在室温以180mL 0.1M乙醇酸铵(pH 7.0)将珠子洗涤两次(15分钟)。然后使用按照实施例5中描述的程序在具有生物催化剂循环的连续批式反应(每组一式两份)中评估在5℃储存了4天或32天的生物催化剂(表8)。

表8.使用在固定前用戊二醛预处理的细胞制备的生物催化剂在具有催化剂循环的连续批式反应中的比活性；生物催化剂在5℃储存4天或32天。

序列表

<110>纳幕尔杜邦公司(E.I.duPont de Nemours and Company，Inc.)

<120>用于生产乙醇酸的固定的微生物腈水解酶的改进

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Gly Asp Asp Arg Met Arg Arg Leu Gln Leu Ala Ala Arg Arg Asn Lys

85 90 95

Ile Ala Leu Val Met Gly Tyr Ser Glu Arg Glu Ala Gly Ser Arg Tyr

100 105 110

Leu Ser Gln Val Phe Ile Asp Glu Arg Gly Glu Ile Val Ala Asn Arg

115 120 125

Arg Lys Leu Lys Pro Thr His Val Glu Arg Thr Ile Tyr Gly Glu Gly

130 135 140

Asn Gly Thr Asp Phe Leu Thr His Asp Phe Ala Phe Gly Arg Val Gly

145 150 155 160

Gly Leu Asn Cys Trp Glu His Phe Gln Pro Leu Ser Lys Phe Met Met

165 170 175

Tyr Ser Leu Gly Glu Gln Val His Val Ala Ser Trp Pro Ala Met Ser

180 185 190

Pro Leu Gln Pro Asp Val Phe Gln Leu Ser Ile Glu Ala Asn Ala Thr

195 200 205

Val Thr Arg Ser Tyr Ala Ile Glu Gly Gln Thr Phe Val Leu Cys Ser

210 215 220

Thr Gln Val Ile Gly Pro Ser Ala Ile Glu Thr Phe Cys Leu Asn Asp

225 230 235 240

Glu Gln Arg Ala Leu Leu Pro Gln Gly Cys Gly Trp Ala Arg Ile Tyr

245 250 255

Gly Pro Asp Gly Ser Glu Leu Ala Lys Pro Leu Ala Glu Asp Ala Glu

260 265 270

Gly Ile Leu Tyr Ala Glu Ile Asp Leu Glu Gln Ile Leu Leu Ala Lys

275 280 285

Ala Gly Ala Asp Pro Val Gly His Tyr Ser Arg Pro Asp Val Leu Ser

290 295 300

Val Gln Phe Asp Pro Arg Asn His Thr Pro Val His Arg Ile Gly Ile

305 310 315 320

Asp Gly Arg Leu Asp Val Asn Thr Arg Ser Arg Val Glu Asn Phe Arg

325 330 335

Leu Arg Gln Ala Ala Glu Gln Glu Arg Gln Ala Ser Lys Arg Leu Gly

340 345 350

Thr Lys Leu Phe Glu Gln Ser Leu Leu Ala Glu Glu Pro Val Pro Ala

355 360 365

Lys

<210>5

<211>356

<212>PRT

<213>固氮粪产碱菌LM3

<400>5

Met Gln Thr Arg Lys Ile Val Arg Ala Ala Ala Val Gln Ala Ala Ser

1 5 10 15

Pro Asn Tyr Asp Leu Ala Thr Gly Val Asp Lys Thr Ile Glu Leu Ala

20 25 30

Arg Gln Ala Arg Asp Glu Gly Cys Asp Leu Ile Val Phe Gly Glu Thr

35 40 45

Trp Leu Pro Gly Tyr Pro Phe His Val Trp Leu Gly Ala Pro Ala Trp

50 55 60

Ser Leu Lys Tyr Ser Ala Arg Tyr Tyr Ala Asn Ser Leu Ser Leu Asp

65 70 75 80

Ser Ala Glu Phe Gln Arg Ile Ala Gln Ala Ala Arg Thr Leu Gly Ile

85 90 95

Phe Ile Ala Leu Gly Tyr Ser Glu Arg Ser Gly Gly Ser Leu Tyr Leu

100 105 110

Gly Gln Cys Leu Ile Asp Asp Lys Gly Gln Met Leu Trp Ser Arg Arg

115 120 125

Lys Leu Lys Pro Thr His Val Glu Arg Thr Val Phe Gly Glu Gly Tyr

130 135 140

Ala Arg Asp Leu Ile Val Ser Asp Thr Glu Leu Gly Arg Val Gly Ala

145 150 155 160

Leu Cys Cys Trp Glu His Leu Ser Pro Leu Ser Lys Tyr Ala Leu Tyr

165 170 175

Ser Gln His Glu Ala Ile His Ile Ala Ala Trp Pro Ser Phe Ser Leu

180 185 190

Tyr Ser Glu Gln Ala His Ala Leu Ser Ala Lys Val Asn Met Ala Ala

195 200 205

Ser Gln Ile Tyr Ser Val Glu Gly Gln Cys Phe Thr Ile Ala Ala Ser

2l0 215 220

Ser Val Val Thr Gln Glu Thr Leu Asp Met Leu Glu Val Gly Glu His

225 230 235 240

Asn Ala Ser Leu Leu Lys Val Gly Gly Gly Ser Ser Met Ile Phe Ala

245 250 255

Pro Asp Gly Arg Thr Leu Ala Pro Tyr Leu Pro His Asp Ala Glu Gly

260 265 270

Leu Ile Ile Ala Asp Leu Asn Met Glu Glu Ile Ala Phe Ala Lys Ala

275 280 285

Ile Asn Asp Pro Val Gly His Tyr Ser Lys Pro Glu Ala Thr Arg Leu

290 295 300

Val Leu Asp Leu Gly His Arg Glu Pro Met Thr Arg Val His Ser Lys

305 310 315 320

Ser Val Ile Gln Glu Glu Ala Pro Glu Pro His Val Gln Ser Thr Ala

325 330 335

Ala Pro Val Ala Val Ser Gln Thr Gln Asp Ser Asp Thr Leu Leu Val

340 345 350

Gln Glu Pro Ser

355

<210>6

<211>366

<212>PRT

<213>紫红红球菌J1

<400>6

Met Val Glu Tyr Thr Asn Thr Phe Lys Val Ala Ala Val Gln Ala Gln

1 5 10 15

Pro Val Trp Phe Asp Ala Ala Lys Thr Val Asp Lys Thr Val Ser Ile

20 25 30

Ile Ala Glu Ala Ala Arg Asn Gly Cys Glu Leu Val Ala Phe Pro Glu

35 40 45

Val Phe Ile Pro Gly Tyr Pro Tyr His Ile Trp Val Asp Ser Pro Leu

50 55 60

Ala Gly Met Ala Lys Phe Ala Val Arg Tyr His Glu Asn Ser Leu Thr

65 70 75 80

Met Asp Ser Pro His Val Gln Arg Leu Leu Asp Ala Ala Arg Asp His

85 90 95

Asn Ile Ala Val Val Val Gly Ile Ser Glu Arg Asp Gly Gly Ser Leu

100 105 110

Tyr Met Thr Gln Leu Val Ile Asp Ala Asp Gly Gln Leu Val Ala Arg

115 120 125

Arg Arg Lys Leu Lys Pro Thr His Val Glu Arg Ser Val Tyr Gly Glu

130 135 140

Gly Asn Gly Ser Asp Ile Ser Val Tyr Asp Met Pro Phe Ala Arg Leu

145 150 155 160

Gly Ala Leu Asn Cys Trp Glu His Phe Gln Thr Leu Thr Lys Tyr Ala

165 170 175

Met Tyr Ser Met His Glu Gln Val His Val Ala Ser Trp Pro Gly Met

180 185 190

Ser Leu Tyr Gln Pro Glu Val Pro Ala Phe Gly Val Asp Ala Gln Leu

195 200 205

Thr Ala Thr Arg Met Tyr Ala Leu Glu Gly Gln Thr Phe Val Val Cys

210 215 220

Thr Thr Gln Val Val Thr Pro Glu Ala His Glu Phe Phe Cys Asp Asn

225 230 235 240

Asp Glu Gln Arg Lys Leu Ile Gly Arg Gly Gly Gly Phe Ala Arg Ile

245 250 255

Ile Gly Pro Asp Gly Arg Asp Leu Ala Thr Pro Leu Ala Glu Asp Glu

260 265 270

Glu Gly Ile Leu Tyr Ala Asp Ile Asp Leu Ser Ala Ile Thr Leu Ala

275 280 285

Lys Gln Ala Ala Asp Pro Val Gly His Tyr Ser Arg Pro Asp Val Leu

290 295 300

Ser Leu Asn Phe Asn Gln Arg His Thr Thr Pro Val Asn Thr Ala Ile

305 310 315 320

Ser Thr Ile His Ala Thr His Thr Leu Val Pro Gln Ser Gly Ala Leu

325 330 335

Asp Gly Val Arg Glu Leu Asn Gly Ala Asp Glu Gln Arg Ala Leu Pro

340 345 350

Ser Thr His Ser Asp Glu Thr Asp Arg Ala Thr Ala Ser Ile

355 360 365

<210>7

<211>383

<212>PRT

<213>紫红红球菌K22

<400>7

Met Ser Ser Asn Pro Glu Leu Lys Tyr Thr Gly Lys Val Lys Val Ala

1 5 10 15

Thr Val Gln Ala Glu Pro Val Ile Leu Asp Ala Asp Ala Thr Ile Asp

20 25 30

Lys Ala Ile Gly Phe Ile Glu Glu Ala Ala Lys Asn Gly Ala Glu Phe

35 40 45

Leu Ala Phe Pro Glu Val Trp Ile Pro Gly Tyr Pro Tyr Trp Ala Trp

50 55 60

Ile Gly Asp Val Lys Trp Ala Val Ser Asp Phe Ile Pro Lys Tyr His

65 70 75 80

Glu Asn Ser Leu Thr Leu Gly Asp Asp Arg Met Arg Arg Leu Gln Leu

85 90 95

Ala Ala Arg Gln Asn Asn Ile Ala Leu Val Met Gly Tyr Ser Glu Lys

100 105 110

Asp Gly Ala Ser Arg Tyr Leu Ser Gln Val Phe Ile Asp Gln Asn Gly

115 120 125

Asp Ile Val Ala Asn Arg Arg Lys Leu Lys Pro Thr His Val Glu Arg

130 135 140

Thr Ile Tyr Gly Glu Gly Asn Gly Thr Asp Phe Leu Thr His Asp Phe

145 150 155 160

Gly Phe Gly Arg Val Gly Gly Leu Asn Cys Trp Glu His Phe Gln Pro

165 170 175

Leu Ser Lys Tyr Met Met Tyr Ser Leu Asn Glu Gln Ile His Val Ala

180 185 190

Ser Trp Pro Ala Met Phe Ala Leu Thr Pro Asp Val His Gln Leu Ser

195 200 205

Val Glu Ala Asn Asp Thr Val Thr Arg Ser Tyr Ala Ile Glu Gly Gln

210 215 220

Thr Phe Val Leu Ala Ser Thr His Val Ile Gly Lys Ala Thr Gln Asp

225 230 235 240

Leu Phe Ala Gly Asp Asp Asp Ala Lys Arg Ala Leu Leu Pro Leu Gly

245 250 255

Gln Gly Trp Ala Arg Ile Tyr Gly Pro Asp Gly Lys Ser Leu Ala Glu

260 265 270

Pro Leu Pro Glu Asp Ala Glu Gly Leu Leu Tyr Ala Glu Leu Asp Leu

275 280 285

Glu Gln Ile Ile Leu Ala Lys Ala Ala Ala Asp Pro Ala Gly His Tyr

290 295 300

Ser Arg Pro Asp Val Leu Ser Leu Lys Ile Asp Thr Arg Asn His Thr

305 310 315 320

Pro Val Gln Tyr Ile Thr Ala Asp Gly Arg Thr Ser Leu Asn Ser Asn

325 330 335

Ser Arg Val Glu Asn Tyr Arg Leu His Gln Leu Ala Asp Ile Glu Lys

340 345 350

Tyr Glu Asn Ala Glu Ala Ala Thr Leu Pro Leu Asp Ala Pro Ala Pro

355 360 365

Ala Pro Ala Pro Glu Gln Lys Ser Gly Arg Ala Lys Ala Glu Ala

370 375 380

<210>8

<211>381

<212>PRT

<213>诺卡氏菌属C-14-1

<400>8

Met Lys Val Ala Thr Val Gln Ala Glu Pro Val Ile Leu Asp Ala Asp

1 5 10 15

Ala Thr Ile Asp Lys Ala Ile Gly Tyr Ile Glu Glu Ala Ser Lys Asn

20 25 30

Gly Ala Glu Phe Ile Ala Phe Pro Glu Val Trp Ile Pro Gly Tyr Pro

35 40 45

Tyr Trp Ala Trp Ile Gly Asp Val Lys Trp Ala Val Ser Glu Phe Ile

50 55 60

Pro Lys Tyr His Glu Asn Ser Leu Thr Leu Gly Asp Asp Arg Met Arg

65 70 75 80

Arg Leu Gln Leu Ala Ala Arg Gln His Asn Ile Ala Met Val Val Gly

85 90 95

Tyr Ser Glu Lys Asp Gly Ala Ser Arg Tyr Leu Ser Gln Val Phe Ile

100 105 110

Asp Gln Asn Gly Asp Ile Val Ala Asn Arg Arg Lys Leu Lys Pro Thr

115 120 125

His Val Glu Arg Thr Ile Tyr Gly Glu Gly Asn Gly Thr Asp Phe Leu

130 135 140

Thr His Asp Phe Gly Phe Gly Arg Val Gly Gly Leu Asn Cys Trp Glu

145 150 155 160

His Phe Gln Pro Leu Ser Lys Tyr Met Met Tyr Ser Leu Asn Glu Gln

165 170 175

Ile His Val Ala Ser Trp Pro Ala Met Phe Ala Leu Thr Pro Asp Val

180 185 190

His Gln Leu Ser Val Glu Ala Asn Asp Thr Val Thr Arg Ser Tyr Ala

195 200 205

Ile Glu Gly Gln Thr Phe Val Leu Ala Ala Thr His Val Ile Gly Lys

210 215 220

Ala Thr Gln Asp Leu Phe Ala Gly Asp Asp Glu Ala Lys Arg Ala Leu

225 230 235 240

Leu Pro Leu Gly Gln Gly Trp Ala Arg Ile Tyr Gly Pro Asp Gly Lys

245 250 255

Ser Leu Ala Glu Pro Leu Ala Glu Asn Ala Glu Gly Leu Leu Tyr Ala

260 265 270

Glu Leu Asp Leu Glu Gln Ile Ile Val Ala Lys Ala Ala Ala Asp Pro

275 280 285

Ala Gly His Tyr Ser Arg Pro Asp Val Leu Ser Leu Lys Val Asp Thr

290 295 300

Arg Asn His Thr Pro Val Gln Tyr Val Thr Glu Asp Gly Gly Ser Ser

305 310 315 320

Leu Asn Ser Asn Ser Arg Val Glu Asn Tyr Arg Leu Arg Gln Leu Ala

325 330 335

Asp Ile Glu Lys Tyr Glu Asn Ala Asp Ser Ala Thr Val Pro Leu Asp

340 345 350

Val Thr Thr Pro Glu Lys Gln Ser Gly Asp Val Asn Ala Asn Gly Asn

355 360 365

Ala Lys Val Asn Thr Asn Pro Ser Ala Lys Ala Lys Ala

370 375 380

<210>9

<211>310

<212>PRT

<213>支气管败血波氏杆菌RB50

<400>9

Met Thr Thr His Arg Ile Ala Val Ile Gln Asp Gly Pro Val Pro Gly

1 5 10 15

Asp Ala Met Ala Thr Ala Glu Lys Met Ser Arg Leu Ala Ala Ser Ala

20 25 30

Lys Ala Gln Gly Ala Arg Leu Ala Leu Phe Pro Glu Ala Phe Val Gly

35 40 45

Gly Tyr Pro Lys Gly Ala Asp Phe His Ile Phe Leu Gly Gly Arg Thr

50 55 60

Pro Gln Gly Arg Ala Gln Tyr Gln Arg Tyr Ala Glu Thr Ala Ile Ala

65 70 75 80

Val Pro Gly Pro Val Thr Glu Arg Ile Gly Gln Ile Ala Ala Glu Gln

85 90 95

Asp Met Phe Ile Val Val Gly Val Ile Glu Arg Asp Gly Gly Thr Leu

100 105 110

Tyr Cys Thr Ile Leu Phe Phe Ser Pro Glu Gly Glu Leu Leu Gly Lys

115 120 125

His Arg Lys Leu Met Pro Thr Ala Leu Glu Arg Leu Leu Trp Gly Tyr

130 135 140

Gly Asp Gly Ser Thr Phe Pro Val Tyr Asp Thr Pro Leu Gly Lys Leu

145 150 155 160

Gly Ala Val Val Cys Trp Glu Asn Tyr Met Pro Leu Leu Arg Met Ala

165 170 175

Met Tyr Gly Lys Gln Ile Gln Ile Tyr Cys Ala Pro Thr Ala Asp Asp

180 185 190

Lys Pro Thr Trp Val Ser Thr Met Gln His Val Ala Leu Glu Gly Arg

195 200 205

Cys Phe Val Leu Ser Ala Cys Gln His Leu Arg Gly Lys Asp Phe Pro

210 215 220

Pro Glu Phe His Asn Ala Leu Asp Val Gln Pro Asp Thr Val Leu Met

225 230 235 240

Arg Gly Gly Ser Cys Ile Val Asp Pro Met Gly Gln Leu Leu Ala Gly

245 250 255

Pro Val Tyr Asp Glu Asp Ala Ile Leu Val Ala Asp Ile Asp Leu Asp

260 265 270

Ala Val Thr Arg Gly Lys Met Asp Phe Asp Val Val Gly His Tyr Ala

275 280 285

Arg Pro Asp Ile Phe Ser Leu Thr Val Asp Glu Arg Pro Lys Pro Pro

290 295 300

Val Thr Thr Leu Lys Pro

305 310

<210>10

<211>339

<212>PRT

<213>拟南芥

<400>10

Met Ser Thr Ser Glu Asn Thr Pro Phe Asn Gly Val Ala Ser Ser Thr

1 5 10 15

Ile Val Arg Ala Thr Ile Val Gln Ala Ser Thr Val Tyr Asn Asp Thr

20 25 30

Pro Ala Thr Leu Glu Lys Ala Asn Lys Phe Ile Val Glu Ala Ala Ser

35 40 45

Lys Gly Ser Glu Leu Val Val Phe Pro Glu Ala Phe Ile Gly Gly Tyr

50 55 60

Pro Arg Gly Phe Arg Phe Gly Leu Gly Val Gly Val His Asn Glu Glu

65 70 75 80

Gly Arg Asp Glu Phe Arg Lys Tyr His Ala Ser Ala Ile Lys Val Pro

85 90 95

Gly Pro Glu Val Glu Lys Leu Ala Glu Leu Ala Gly Lys Asn Asn Val

100 105 110

Tyr Leu Val Met Gly Ala Ile Glu Lys Asp Gly Tyr Thr Leu Tyr Cys

115 120 125

Thr Ala Leu Phe Phe Ser Pro Gln Gly Gln Phe Leu Gly Lys His Arg

130 135 140

Lys Leu Met Pro Thr Ser Leu Glu Arg Cys Ile Trp Gly Gln Gly Asp

145 150 155 160

Gly Ser Thr Ile Pro Val Tyr Asp Thr Pro Ile Gly Lys Leu Gly Ala

165 170 175

Ala Ile Cys Trp Glu Asn Arg Met Pro Leu Tyr Arg Thr Ala Leu Tyr

180 185 190

Ala Lys Gly Ile Glu Leu Tyr Cys Ala Pro Thr Ala Asp Gly Ser Lys

195 200 205

Glu Trp Gln Ser Ser Met Leu His Ile Ala Ile Glu Gly Gly Cys Phe

210 215 220

Val Leu Ser Ala Cys Gln Phe Cys Leu Arg Lys Asp Phe Pro Asp His

225 230 235 240

Pro Asp Tyr Leu Phe Thr Asp Trp Tyr Asp Asp Lys Glu Pro Asp Ser

245 250 255

Ile Val Ser Gln Gly Gly Ser Val Ile Ile Ser Pro Leu Gly Gln Val

260 265 270

Leu Ala Gly Pro Asn Phe Glu Ser Glu Gly Leu Ile Thr Ala Asp Leu

275 280 285

Asp Leu Gly Asp Val Ala Arg Ala Lys Leu Tyr Phe Asp Ser Val Gly

290 295 300

His Tyr Ser Arg Pro Asp Val Leu His Leu Thr Val Asn Glu His Pro

305 310 315 320

Lys Lys Pro Val Thr Phe Ile Ser Lys Val Glu Lys Ala Glu Asp Asp

325 330 335

Ser Asn Lys

<210>11

<211>334

<212>PRT

<213>细长聚球藻PCC 7942

<400>11

Met Ala Asp Lys Ile Ile Val Ala Ala Ala Gln Ile Arg Pro Val Leu

1 5 10 15

Phe Ser Leu Glu Gly Ser Val Ala Arg Val Leu Ala Ala Met Ala Glu

20 25 30

Ala Ala Ala Ala Gly Val Gln Leu Ile Val Phe Pro Glu Thr Phe Leu

35 40 45

Pro Tyr Tyr Pro Tyr Phe Ser Phe Val Glu Pro Pro Val Leu Met Gly

50 55 60

Arg Ser His Leu Lys Leu Tyr Glu Gln Ala Phe Thr Met Thr Gly Pro

65 70 75 80

Glu Leu Gln Gln Ile Ala Arg Ala Ala Arg Gln His Arg Leu Phe Val

85 90 95

Leu Leu Gly Val Asn Glu Arg Asp Gly Gly Ser Leu Tyr Asn Thr Gln

100 105 110

Leu Leu Ile Ser Asp Gln Gly Asp Leu Leu Leu Lys Arg Arg Lys Ile

115 120 125

Thr Pro Thr Tyr His Glu Arg Met Val Trp Gly Gln Gly Gly Gly Ala

130 135 140

Gly Leu Thr Val Val Glu Thr Val Leu Gly Lys Val Gly Ala Leu Ala

145 150 155 160

Cys Trp Glu His Tyr Asn Pro Leu Ala Arg Phe Ser Leu Met Thr Gln

165 170 175

Gly Glu Glu Ile His Cys Ala Gln Phe Pro Gly Ser Leu Val Gly Pro

180 185 190

Ile Phe Ser Glu Gln Thr Ala Val Thr Leu Arg His His Ala Leu Glu

195 200 205

Ala Gly Cys Phe Val Leu Ser Ser Thr Ala Trp Leu Asp Pro Ala Asp

210 215 220

Tyr Asp Thr Ile Thr Pro Asp Arg Ser Leu His Lys Ala Phe Gln Gly

225 230 235 240

Gly Cys His Thr Ala Ile Ile Ser Pro Glu Gly Arg Tyr Leu Ala Gly

245 250 255

Pro Leu Pro Glu Gly Glu Gly Leu Ala Ile Ala Glu Leu Asp Lys Ser

260 265 270

Leu Ile Thr Lys Arg Lys Arg Met Met Asp Ser Val Gly His Tyr Ser

275 280 285

Arg Pro Asp Leu Leu Ser Leu Arg Ile Asn Arg Ser Pro Ala Thr Gln

290 295 300

Val Gln Ala Ile Gly Ser Ala Ala Ala Leu Pro Glu Leu Pro Asn Leu

305 310 315 320

Glu Ala Ala Pro Ala Glu Thr Ala Glu Asp Tyr Leu His Ala

325 330

<210>12

<211>334

<212>PRT

<213>细长聚球藻PCC 6301

<400>12

Met Ala Asp Lys Ile Ile Val Ala Ala Ala Gln Ile Arg Pro Val Leu

1 5 10 15

Phe Ser Leu Glu Gly Ser Val Ala Arg Val Leu Ala Ala Met Ala Glu

20 25 30

Ala Ala Ala Ala Gly Val Gln Leu Ile Val Phe Pro Glu Thr Phe Leu

35 40 45

Pro Tyr Tyr Pro Tyr Phe Ser Phe Val Glu Pro Pro Val Leu Met Gly

50 55 60

Arg Ser His Leu Lys Leu Tyr Glu Gln Ala Phe Thr Met Thr Gly Pro

65 70 75 80

Glu Leu Gln Gln Ile Ala Arg Ala Ala Arg Gln His Arg Leu Phe Val

85 90 95

Leu Leu Gly Val Asn Glu Arg Asp Gly Gly Ser Leu Tyr Asn Thr Gln

100 105 110

Leu Leu Ile Ser Asp Gln Gly Asp Leu Leu Leu Lys Arg Arg Lys Ile

115 120 125

Thr Pro Thr Tyr His Glu Arg Met Val Trp Gly Gln Gly Gly Gly Ala

130 135 140

Gly Leu Thr Val Val Glu Thr Val Leu Gly Lys Val Gly Ala Leu Ala

145 150 155 160

Cys Trp Glu His Tyr Asn Pro Leu Ala Arg Phe Ser Leu Met Thr Gln

165 170 175

Gly Glu Glu Ile His Cys Ala Gln Phe Pro Gly Ser Leu Val Gly Pro

180 185 190

Ile Phe Ser Glu Gln Thr Ala Val Thr Leu Arg His His Ala Leu Glu

195 200 205

Ala Gly Cys Phe Val Leu Ser Ser Thr Ala Trp Leu Asp Pro Ala Asp

210 215 220

Tyr Asp Thr Ile Thr Pro Asp Arg Ser Leu His Lys Ala Phe Gln Gly

225 230 235 240

Gly Cys His Thr Ala Ile Ile Ser Pro Glu Gly Arg Tyr Leu Ala Gly

245 250 255

Pro Leu Pro Glu Gly Glu Gly Leu Ala Ile Ala Glu Leu Asp Lys Ser

260 265 270

Leu Ile Thr Lys Arg Lys Arg Met Met Asp Ser Val Gly His Tyr Ser

275 280 285

Arg Pro Asp Leu Leu Ser Leu Arg Ile Asn Arg Ser Pro Ala Thr Gln

290 295 300

Val Gln Ala Ile Gly Ser Ala Ala Ala Leu Pro Glu Leu Pro Asn Leu

305 310 315 320

Glu Ala Ala Pro Ala Glu Thr Ala Glu Asp Tyr Leu His Ala

325 330

<210>13

<211>346

<212>PRT

<213>集胞藻属PCC 6803

<400>13

Met Leu Gly Lys Ile Met Leu Asn Tyr Thr Lys Asn Ile Arg Ala Ala

1 5 10 15

Ala Ala Gln Ile Ser Pro Val Leu Phe Ser Gln Gln Gly Thr Met Glu

20 25 30

Lys Val Leu Asp Ala Ile Ala Asn Ala Ala Lys Lys Gly Val Glu Leu

35 40 45

Ile Val Phe Pro Glu Thr Phe Val Pro Tyr Tyr Pro Tyr Phe Ser Phe

50 55 60

Val Glu Pro Pro Val Leu Met Gly Lys Ser His Leu Lys Leu Tyr Gln

65 70 75 80

Glu Ala Val Thr Val Pro Gly Lys Val Thr Gln Ala Ile Ala Gln Ala

85 90 95

Ala Lys Thr His Gly Met Val Val Val Leu Gly Val Asn Glu Arg Glu

100 105 110

Glu Gly Ser Leu Tyr Asn Thr Gln Leu Ile Phe Asp Ala Asp Gly Ala

115 120 125

Leu Val Leu Lys Arg Arg Lys Ile Thr Pro Thr Tyr His Glu Arg Met

130 135 140

Val Trp Gly Gln Gly Asp Gly Ala Gly Leu Arg Thr Val Asp Thr Thr

145 150 155 160

Val Gly Arg Leu Gly Ala Leu Ala Cys Trp Glu His Tyr Asn Pro Leu

165 170 175

Ala Arg Tyr Ala Leu Met Ala Gln His Glu Gln Ile His Cys Gly Gln

180 185 190

Phe Pro Gly Ser Met Val Gly Gln Ile Phe Ala Asp Gln Met Glu Val

195 200 205

Thr Met Arg His His Ala Leu Glu Ser Gly Cys Phe Val Ile Asn Ala

210 215 220

Thr Gly Trp Leu Thr Ala Glu Gln Lys Leu Gln Ile Thr Thr Asp Glu

225 230 235 240

Lys Met His Gln Ala Leu Ser Gly Gly Cys Tyr Thr Ala Ile Ile Ser

245 250 255

Pro Glu Gly Lys His Leu Cys Glu Pro Ile Ala Glu Gly Glu Gly Leu

260 265 270

Ala Ile Ala Asp Leu Asp Phe Ser Leu Ile Ala Lys Arg Lys Arg Met

275 280 285

Met Asp Ser Val Gly His Tyr Ala Arg Pro Asp Leu Leu Gln Leu Thr

290 295 300

Leu Asn Asn Gln Pro Trp Ser Ala Leu Glu Ala Asn Pro Val Thr Pro

305 310 315 320

Asn Ala Ile Pro Ala Val Ser Asp Pro Glu Leu Thr Glu Thr Ile Glu

325 330 335

Ala Leu Pro Asn Asn Pro Ile Phe Ser His

340 345

<210>14

<211>307

<212>PRT

<213>嗜虫假单胞菌L48

<400>14

Met Pro Lys Ser Ile Val Ala Ala Leu Gln Val Gly Ser Leu Pro Glu

1 5 10 15

Gly Lys Ala Ala Thr Leu Glu Gln Ile Leu Gly Tyr Glu Gln Ala Ile

20 25 30

Arg Glu Ala Gly Ala Arg Leu Val Val Met Pro Glu Ala Leu Leu Gly

35 40 45

Gly Tyr Pro Lys Gly Glu Gly Phe Gly Thr Gln Leu Gly Tyr Arg Leu

50 55 60

Pro Glu Gly Arg Glu Ala Phe Ala Arg Tyr Phe Ala Asn Ala Ile Asp

65 70 75 80

Val Pro Gly Ser Glu Thr Ala Ala Leu Ala Gly Leu Ser Ala Arg Thr

85 90 95

Gly Ala Ser Leu Val Leu Gly Val Ile Glu Arg Ser Gly Asn Thr Leu

100 105 110

Tyr Cys Thr Val Leu Phe Phe Glu Pro Glu Gly Gly Leu Val Ala Lys

115 120 125

His Arg Lys Leu Met Pro Thr Gly Thr Glu Arg Leu Ile Trp Gly Lys

130 135 140

Gly Asp Gly Ser Thr Leu Pro Val Val Asp Gly Arg Ala Gly Arg Ile

145 150 155 160

Gly Ala Ala Val Cys Trp Glu Asn Tyr Met Pro Leu Leu Arg Thr Ala

165 170 175

Met Tyr Ala Lys Gly Val Gln Leu Trp Cys Ala Pro Thr Val Asp Glu

180 185 190

Arg Glu Leu Trp Gln Val Ser Met Arg His Val Ala Ala Glu Gly Arg

195 200 205

Cys Phe Val Ile Ser Ala Cys Gln Val Gln Asp Ser Pro Ala Ala Leu

210 215 220

Gly Met Glu Val Ala Asn Trp Pro Ala Glu Arg Pro Leu Ile Asn Gly

225 230 235 240

Gly Ser Leu Ile Val Gly Pro Leu Gly Asp Val Leu Ala Gly Pro Leu

245 250 255

Leu Gly Ala Arg Gly Leu Val Cys Ala Glu Val Asp Thr Asp Glu Leu

260 265 270

Val Arg Ala Arg Tyr Asp Phe Asp Val Val Gly His Tyr Ala Arg Pro

275 280 285

Asp Val Phe Glu Leu Ser Val Asp Glu Arg Pro Arg Pro Gly Val Arg

290 295 300

Phe Ile Gly

305

<210>15

<211>329

<212>PRT

<213>运动发酵单胞菌运动亚种ZM4

<400>15

Met Ser Cys His Arg Val Ala Val Ile Gln Ala Gly Thr Ser Leu Phe

1 5 10 15

Asp Thr Glu Lys Thr Leu Asp Arg Met Glu Ala Leu Cys Arg Gln Ala

20 25 30

Ala Glu Gln Asn Val Glu Leu Ala Val Phe Pro Glu Ala Tyr Ile Gly

35 40 45

Gly Tyr Pro Lys Gly Leu Asp Phe Gly Ala Arg Met Gly Thr Arg Thr

50 55 60

Glu Ala Gly Arg Glu Asp Phe Leu Arg Tyr Trp Lys Ala Ala Ile Asp

65 70 75 80

Val Pro Gly Lys Glu Thr Ala Arg Ile Gly Ser Phe Ala Ala Lys Met

85 90 95

Lys Ala Tyr Leu Val Val Gly Val Ile Glu Arg Ser Glu Ala Thr Leu

100 105 110

Tyr Cys Thr Ala Leu Phe Phe Ala Pro Asp Gly Thr Leu Ile Gly Lys

115 120 125

His Arg Lys Leu Met Pro Thr Ala Thr Glu Arg Leu Val Trp Gly Gln

130 135 140

Gly Asp Gly Ser Thr Ile Glu Ile Leu Asp Thr Ala Val Gly Lys Leu

145 150 155 160

Gly Ala Ala Ile Cys Trp Glu Asn Tyr Met Pro Val Leu Arg Gln Val

165 170 175

Met Tyr Ala Gly Gly Val Asn Ile Trp Cys Ala Pro Thr Val Asp Gln

180 185 190

Arg Glu Ile Trp Gln Val Ser Met Arg His Ile Ala Tyr Glu Gly Arg

195 200 205

Leu Phe Val Leu Ser Ala Cys Gln Tyr Met Thr Arg Ala Asp Ala Pro

210 215 220

Ala Asp Tyr Asp Cys Ile Gln Gly Asn Asp Pro Glu Thr Glu Leu Ile

225 230 235 240

Ala Gly Gly Ser Val Ile Ile Asp Pro Met Gly Asn Ile Leu Ala Gly

245 250 255

Pro Leu Tyr Gly Gln Glu Gly Val Leu Val Ala Asp Ile Asp Leu Ser

260 265 270

Asp Thr Ile Lys Ala Arg Tyr Asp Leu Asp Val Ser Gly His Tyr Gly

275 280 285

Arg Pro Asp Ile Phe Glu Ile Lys Val Asp Arg Gln Ser His Gln Val

290 295 300

Ile Thr Asp Gln Phe Ser Arg Asp Gln Ala Thr Glu Lys Lys Pro Val

305 310 315 320

Ser Asp Ser Glu Ile Ser Gln Leu Asp

325

<210>16

<211>339

<212>PRT

<213>芽孢杆菌属OxB-1

<400>16

Met Ser Asn Tyr Pro Lys Tyr Arg Val Ala Ala Val Gln Ala Ser Pro

1 5 10 15

Val Leu Leu Asp Leu Asp Ala Thr Ile Asp Lys Thr Cys Arg Leu Val

20 25 30

Asp Glu Ala Ala Ala Asn Gly Ala Lys Val Ile Ala Phe Pro Glu Ala

35 40 45

Phe Ile Pro Gly Tyr Pro Trp Trp Ile Trp Leu Gly Asn Ala Asp Tyr

50 55 60

Gly Met Lys Tyr Tyr Ile Gln Leu Tyr Lys Asn Ser Val Glu Ile Pro

65 70 75 80

Ser Leu Ala Val Gln Lys Leu Ser Ser Ala Gly Thr Asn Lys Val Tyr

85 90 95

Phe Cys Val Ser Val Thr Glu Lys Asp Gly Gly Ser Leu Tyr Leu Thr

100 105 110

Gln Leu Trp Phe Asp Pro Asn Gly Asp Leu Ile Gly Lys His Arg Lys

115 120 125

Leu Lys Ala Thr Asn Ala Glu Lys Thr Ile Trp Gly Asp Gly Asp Gly

130 135 140

Ser Met Met Pro Val Phe Glu Thr Glu Phe Gly Asn Leu Gly Gly Leu

145 150 155 160

Gln Cys Trp Glu His Phe Leu Pro Leu Asn Val Ala Ala Met Ala Ser

165 170 175

Met Asn Glu Gln Val His Val Ala Ser Trp Pro Ile Gly Met Pro Gln

180 185 190

Glu Gly His Leu Phe Gly Pro Glu Gln Cys Val Thr Ala Thr Lys Tyr

195 200 205

Tyr Ala Ile Ser Asn Gln Val Phe Cys Leu Leu Ser Ser Gln Ile Trp

210 215 220

Thr Glu Glu Gln Arg Asp Lys Ile Cys Glu Thr Glu Glu Gln Arg Asn

225 230 235 240

Phe Met Lys Val Gly His Gly Phe Ser Lys Ile Ile Ala Pro Asn Gly

245 250 255

Met Glu Ile Gly Asn Lys Leu Ala His Asp Glu Glu Gly Ile Thr Tyr

260 265 270

Ala Asp Ile Asp Leu Glu Gln Ile Ile Pro Gly Lys Phe Leu Ile Asp

275 280 285

Ser Ala Gly His Tyr Ser Thr Pro Gly Phe Leu Ser Leu Ser Phe Asp

290 295 300

Arg Thr Glu Lys Lys Pro Ile Lys His Ile Gly Glu Ser Ala Gln Glu

305 310 315 320

Thr Val Thr Tyr Glu Glu Ile Gln Tyr Gly Asn Lys Ala Asn Val Lys

325 330 335

Val His Ser

<210>17

<211>354

<212>PRT

<213>睾丸酮丛毛单胞菌

<400>17

Met Lys Asn Tyr Pro Thr Val Lys Val Ala Ala Val Gln Ala Ala Pro

1 5 10 15

Val Phe Met Asn Leu Glu Ala Thr Val Asp Lys Thr Cys Lys Leu Ile

20 25 30

Ala Glu Ala Ala Ser Met Gly Ala Lys Val Ile Gly Phe Pro Glu Ala

35 40 45

Phe Ile Pro Gly Tyr Pro Tyr Trp Ile Trp Thr Ser Asn Met Asp Phe

50 55 60

Thr Gly Met Met Trp Ala Val Leu Phe Lys Asn Ala Ile Glu Ile Pro

65 70 75 80

Ser Lys Glu Val Gln Gln Ile Ser Asp Ala Ala Lys Lys Asn Gly Val

85 90 95

Tyr Val Cys Val Ser Val Ser Glu Lys Asp Asn Ala Ser Leu Tyr Leu

100 105 110

Thr Gln Leu Trp Phe Asp Pro Asn Gly Asn Leu Ile Gly Lys His Arg

115 120 125

Lys Phe Lys Pro Thr Ser Ser Glu Arg Ala Val Trp Gly Asp Gly Asp

130 135 140

Gly Ser Met Ala Pro Val Phe Lys Thr Glu Tyr Gly Asn Leu Gly Gly

145 150 155 160

Leu Gln Cys Trp Glu His Ala Leu Pro Leu Asn Ile Ala Ala Met Gly

165 170 175

Ser Leu Asn Glu Gln Val His Val Ala Ser Trp Pro Ala Phe Val Pro

180 185 190

Lys Gly Ala Val Ser Ser Arg Val Ser Ser Ser Val Cys Ala Ser Thr

195 200 205

Asn Ala Met His Gln Ile Ile Ser Gln Phe Tyr Ala Ile Ser Asn Gln

210 215 220

Val Tyr Val Ile Met Ser Thr Asn Leu Val Gly Gln Asp Met Ile Asp

225 230 235 240

Met Ile Gly Lys Asp Glu Phe Ser Lys Asn Phe Leu Pro Leu Gly Ser

245 250 255

Gly Asn Thr Ala Ile Ile Ser Asn Thr Gly Glu Ile Leu Ala Ser Ile

260 265 270

Pro Gln Asp Ala Glu Gly Ile Ala Val Ala Glu Ile Asp Leu Asn Gln

275 280 285

Ile Ile Tyr Gly Lys Trp Leu Leu Asp Pro Ala Gly His Tyr Ser Thr

290 295 300

Pro Gly Phe Leu Ser Leu Thr Phe Asp Gln Ser Glu His Val Pro Val

305 310 315 320

Lys Lys Ile Gly Glu Gln Thr Asn His Phe Ile Ser Tyr Glu Asp Leu

325 330 335

His Glu Asp Lys Met Asp Met Leu Thr Ile Pro Pro Arg Arg Val Ala

340 345 350

Thr Ala

<210>18

<211>332

<212>PRT

<213>蓝细菌属CC9605

<400>18

Met Thr Thr Val Lys Val Ala Ala Ala Gln Ile Arg Pro Val Leu Phe

1 5 10 15

Ser Leu Asp Gly Ser Leu Gln Lys Val Leu Asp Ala Met Ala Glu Ala

20 25 30

Ala Ala Gln Gly Val Glu Leu Ile Val Phe Pro Glu Thr Phe Leu Pro

35 40 45

Tyr Tyr Pro Tyr Phe Ser Phe Val Glu Pro Pro Val Leu Met Gly Arg

50 55 60

Ser His Leu Ala Leu Tyr Glu Gln Ala Val Val Val Pro Gly Pro Val

65 70 75 80

Thr Asp Ala Val Ala Ala Ala Ala Ser Gln Tyr Gly Met Gln Val Leu

85 90 95

Leu Gly Val Asn Glu Arg Asp Gly Gly Thr Leu Tyr Asn Thr Gln Leu

100 105 110

Leu Phe Asn Ser Cys Gly Glu Leu Val Leu Lys Arg Arg Lys Ile Thr

115 120 125

Pro Thr Tyr His Glu Arg Met Val Trp Gly Gln Gly Asp Gly Ser Gly

130 135 140

Leu Lys Val Val Gln Thr Pro Leu Ala Arg Val Gly Ala Leu Ala Cys

145 150 155 160

Trp Glu His Tyr Asn Pro Leu Ala Arg Tyr Ala Leu Met Ala Gln Gly

165 170 175

Glu Glu Ile His Cys Ala Gln Phe Pro Gly Ser Leu Val Gly Pro Ile

180 185 190

Phc Thr Glu Gln Thr Ala Val Thr Met Arg His His Ala Leu Glu Ala

195 200 205

Gly Cys Phe Val Ile Cys Ser Thr Gly Trp Leu His Pro Asp Asp Tyr

210 215 220

Ala Ser Ile Thr Ser Glu Ser Gly Leu His Lys Ala Phe Gln Gly Gly

225 230 235 240

Cys His Thr Ala Val Ile Ser Pro Glu Gly Arg Tyr Leu Ala Gly Pro

245 250 255

Leu Pro Asp Gly Glu Gly Leu Ala Ile Ala Asp Leu Asp Leu Ala Leu

260 265 270

Ile Thr Lys Arg Lys Arg Met Met Asp Ser Val Gly His Tyr Ser Arg

275 280 285

Pro Glu Leu Leu Ser Leu Gln Ile Asn Ser Ser Pro Ala Val Pro Val

290 295 300

Gln Asn Met Ser Thr Ala Ser Val Pro Leu Glu Pro Ala Thr Ala Thr

305 310 315 320

Asp Ala Leu Ser Ser Met Glu Ala Leu Asn His Val

325 330

<210>19

<211>306

<212>PRT

<213>荧光假单胞菌Pf-5

<400>19

Met Pro Lys Ser Val Val Ala Ala Leu Gln Ile Gly Ala Leu Pro Glu

1 5 10 15

Gly Lys Ala Ala Thr Leu Glu Gln Ile Leu Ser Tyr Glu Ala Ala Ile

20 25 30

Ile Glu Ala Gly Ala Gln Leu Val Val Met Pro Glu Ala Leu Leu Gly

35 40 45

Gly Tyr Pro Lys Gly Glu Gly Phe Gly Thr Gln Leu Gly Tyr Arg Leu

50 55 60

Pro Glu Gly Arg Glu Ala Phe Ala Arg Tyr Phe Ala Asn Ala Ile Glu

65 70 75 80

Val Pro Gly Val Glu Thr Asp Ala Leu Ala Ala Leu Ser Ala Arg Thr

85 90 95

Gly Ala Asn Leu Val Leu Gly Val Ile Glu Arg Ser Gly Ser Thr Leu

100 105 110

Tyr Cys Thr Ala Leu Tyr Phe Asp Pro Gln Gln Gly Leu Ser Gly Lys

115 120 125

His Arg Lys Leu Met Pro Thr Gly Thr Glu Arg Leu Ile Trp Gly Lys

130 135 140

Gly Asp Gly Ser Thr Leu Pro Val Leu Asp Thr Gln Val Gly Arg Val

145 150 155 160

Gly Ala Val Ile Cys Trp Glu Asn Met Met Pro Leu Leu Arg Thr Ala

165 170 175

Met Tyr Ala Gln Gly Ile Glu Val Trp Cys Ala Pro Thr Val Asp Glu

180 185 190

Arg Glu Met Trp Gln Val Ser Met Arg His Ile Ala His Glu Gly Arg

195 200 205

Cys Phe Val Val Ser Ala Cys Gln Val Gln Ala Ser Pro Glu Glu Leu

210 215 220

Gly Leu Glu Ile Ala Asn Trp Pro Ala Gln Arg Pro Leu Ile Ala Gly

225 230 235 240

Gly Ser Val Ile Val Gly Pro Met Gly Asp Val Leu Ala Gly Pro Leu

245 250 255

Val Gly Arg Ala Gly Leu Ile Ser Ala Gln Ile Asp Thr Ala Asp Leu

260 265 270

Val Arg Ala Arg Tyr Asp Tyr Asp Val Val Gly His Tyr Ala Arg Pro

275 280 285

Asp Val Phe Glu Leu Thr Val Asp Gln Arg Pro Arg Pro Gly Val Arg

290 295 300

Phe Thr

305

<210>20

<211>336

<212>PRT

<213>鼻疽诺卡菌IFM 10152]

<400>20

Met Ser Gln Arg Asp Ser Phe Arg Ala Ala Ala Val Gln Ala Ala Pro

1 5 10 15

Val Trp Leu Asp Gly Ala Ala Thr Val Asp Lys Cys Val Ala Leu Ile

20 25 30

Glu Glu Ala Ala Asp Asn Gly Ala Ala Leu Ile Ala Phe Pro Glu Thr

35 40 45

Phe Val Pro Gly Tyr Pro Trp Trp Leu Trp Leu Asp Ser Pro Ala Trp

50 55 60

Gly Met Gln Phe Val Ala Arg Tyr Phe Asp Asn Ser Leu Ala Leu Asp

65 70 75 80

Gly Pro Leu Phe Ala Arg Leu Arg Glu Ala Ala Arg Arg Ser Ala Ile

85 90 95

Thr Val Val Thr Gly His Ser Glu Arg Asp Gly Gly Ser Leu Tyr Met

100 105 110

Gly Gln Ala Ile Ile Gly Ala Asp Gly Glu Val Leu Ala Ala Arg Arg

115 120 125

Lys Leu Lys Pro Thr His Val Glu Arg Thr Val Phe Gly Glu Ser Asp

130 135 140

Gly Ser Asn Leu Thr Val Val Asp Thr Glu Leu Gly Arg Leu Gly Ala

145 150 155 160

Leu Cys Cys Trp Glu His Leu Gln Pro Leu Thr Lys Tyr Ala Met Tyr

165 170 175

Ser Gln His Glu Gln Ile His Val Ala Ala Trp Pro Ser Phe Ser Val

180 185 190

Tyr Arg Gly Ala Ala Tyr Ala Leu Gly Pro Glu Val Asn Thr Gly Ala

195 200 205

Ala Arg Gln Tyr Ala Val Glu Gly Gln Cys Phe Val Leu Ser Pro Cys

210 215 220

Ala Val Ile Asp Glu Ala Gly Val Glu Leu Phe Cys Asp Thr Pro Ala

225 230 235 240

Lys Arg Glu Leu Leu Leu Pro Gly Gly Gly Phe Ala Gln Ile Tyr Gly

245 250 255

Pro Asp Gly Arg Glu Leu Gly Thr Ala Leu Pro Glu Thr Glu Glu Gly

260 265 270

Leu Val Tyr Ala Asp Leu Glu Ala Ser Ala Val Ala Val Ala Lys Ser

275 280 285

Ala Ala Asp Pro Val Gly His Tyr Ser Arg Pro Asp Val Leu Gln Leu

290 295 300

Leu Trp Asp Pro Arg Pro Arg Ser Val Val Arg Gln Val Ala Leu Ser

305 310 315 320

Val Ala Ser Pro Ala Glu Ser Ala Asp Asp Ala Glu Pro Ala Val Arg

325 330 335

<210>21

<211>356

<212>PRT

<213>固氮粪产碱菌1650

<400>21

Met Gln Thr Arg Lys Ile Val Arg Ala Ala Ala Val Gln Ala Ala Ser

1 5 10 15

Pro Asn Tyr Asp Leu Ala Thr Gly Val Asp Lys Thr Ile Glu Leu Ala

20 25 30

Arg Gln Ala Arg Asp Glu Gly Cys Asp Leu Ile Val Phe Gly Glu Thr

35 40 45

Trp Leu Pro Gly Tyr Pro Phe His Val Trp Leu Gly Ala Pro Ala Trp

50 55 60

Ser Leu Lys Tyr Ser Ala Arg Tyr Tyr Ala Asn Ser Leu Ser Leu Asp

65 70 75 80

Ser Ala Glu Phe Gln Arg Ile Ala Gln Ala Ala Arg Thr Leu Gly Ile

85 90 95

Phe Ile Ala Leu Gly Tyr Ser Glu Arg Ser Gly Gly Ser Leu Tyr Leu

100 105 110

Gly Gln Cys Leu Ile Asp Asp Lys Gly Glu Met Leu Trp Ser Arg Arg

115 120 125

Lys Leu Lys Pro Thr His Val Glu Arg Thr Val Phe Gly Glu Gly Tyr

130 135 140

Ala Arg Asp Leu Ile Val Ser Asp Thr Glu Leu Gly Arg Val Gly Ala

145 150 155 160

Leu Cys Cys Trp Glu His Leu Ser Pro Leu Ser Lys Tyr Ala Leu Tyr

165 170 175

Ser Gln His Glu Ala Ile His Ile Ala Ala Trp Pro Ser Phe Ser Leu

180 185 190

Tyr Ser Glu Gln Ala His Ala Leu Ser Ala Lys Val Asn Met Ala Ala

195 200 205

Ser Gln Ile Tyr Ser Val Glu Gly Gln Cys Phe Thr Ile Ala Ala Ser

210 215 220

Ser Val Val Thr Gln Glu Thr Leu Asp Met Leu Glu Val Gly Glu His

225 230 235 240

Asn Ala Pro Leu Leu Lys Val Gly Gly Gly Ser Ser Met Ile Phe Ala

245 250 255

Pro Asp Gly Arg Thr Leu Ala Pro Tyr Leu Pro His Asp Ala Glu Gly

260 265 270

Leu Ile Ile Ala Asp Leu Asn Met Glu Glu Ile Ala Phe Ala Lys Ala

275 280 285

Ile Asn Asp Pro Val Gly His Tyr Ser Lys Pro Glu Ala Thr Arg Leu

290 295 300

Val Leu Asp Leu Gly His Arg Asp Pro Met Thr Arg Val His Ser Lys

305 310 315 320

Ser Val Thr Arg Glu Glu Ala Pro Glu Gln Gly Val Gln Ser Lys Ile

325 330 335

Ala Ser Val Ala Ile Ser His Pro Gln Asp Ser Asp Thr Leu Leu Val

340 345 350

Gln Glu Pro Ser

355

<210>22

<211>336

<212>PRT

<213>丁香假单胞杆菌丁香致病变种B728a

<400>22

Met Lys Glu Pro Leu Lys Val Ala Cys Val Gln Ala Ala Pro Val Phe

1 5 10 15

Leu Asp Leu Asp Ala Thr Val Asp Lys Thr Ile Thr Leu Met Glu Gln

20 25 30

Ala Ala Ala Ala Gly Ala Gly Leu Ile Ala Phe Pro Glu Thr Trp Ile

35 40 45

Pro Gly Tyr Pro Trp Phe Leu Trp Leu Asp Ala Pro Ala Trp Asn Met

50 55 60

Pro Leu Val Gln Arg Tyr His Gln Gln Ser Leu Val Leu Asp Ser Val

65 70 75 80

Gln Ala Arg Arg Ile Ser Asp Ala Ala Arg His Leu Gly Leu Tyr Val

85 90 95

Val Leu Gly Tyr Ser Glu Arg Asn Lys Ala Ser Leu Tyr Ile Gly Gln

100 105 110

Trp Ile Ile Asp Asp His Gly Glu Thr Val Gly Val Arg Arg Lys Leu

115 120 125

Lys Ala Thr His Val Glu Arg Thr Met Phe Gly Glu Gly Asp Gly Ala

130 135 140

Ser Leu Arg Thr Phe Glu Thr Pro Val Gly Val Leu Gly Ala Leu Cys

145 150 155 160

Cys Trp Glu His Leu Gln Pro Leu Ser Lys Tyr Ala Met Tyr Ala Gln

165 170 175

Asn Glu Gln Ile His Val Ala Ala Trp Pro Ser Phe Ser Leu Tyr Arg

180 185 190

Asn Ala Thr Ser Ala Leu Gly Pro Glu Val Asn Thr Ala Ala Ser Arg

195 200 205

Val Tyr Ala Ala Glu Gly Gln Cys Phe Val Leu Ala Pro Cys Ala Ile

210 215 220

Val Ser Pro Glu Met Ile Glu Met Leu Cys Asp Ser Asp Ala Lys Arg

225 230 235 240

Ser Leu Leu Gln Ala Gly Gly Gly His Ala Arg Ile Phe Gly Pro Asp

245 250 255

Gly Ser Asp Leu Ala Thr Pro Leu Gly Glu His Glu Glu Gly Leu Leu

260 265 270

Tyr Ala Thr Leu Asp Pro Ala Ala Leu Thr Leu Ala Lys Val Ala Ala

275 280 285

Asp Pro Ala Gly His Tyr Ser Arg Pro Asp Val Thr Arg Leu Met Phe

290 295 300

Asn Pro Asn Pro Thr Pro Cys Val Val Asp Leu Pro Asp Leu Pro Ile

305 310 315 320

Ser Ser Glu Ser Ile Glu Leu Leu Arg Pro Asp Ile Ala Leu Glu Val

325 330 335

<210>23

<211>345

<212>PRT

<213>慢生根瘤菌属BTAi1

<400>23

Met Gly Leu Ala His Pro Lys Tyr Lys Val Ala Val Val Gln Ala Ala

1 5 10 15

Pro Ala Trp Leu Asp Leu Asp Ala Ser Ile Lys Lys Thr Ile Ala Leu

20 25 30

Ile Glu Glu Ala Ala Asp Lys Gly Ala Lys Leu Ile Ala Phe Pro Glu

35 40 45

Val Phe Ile Pro Gly Tyr Pro Trp His Ile Trp Met Asp Ser Pro Ala

50 55 60

Trp Cys Ile Gly Arg Gly Phe Val Gln Arg Tyr Phe Asp Asn Ser Leu

65 70 75 80

Ala Tyr Asp Ser Pro Gln Ala Glu Ala Leu Arg Ala Ala Val Arg Lys

85 90 95

Ala Gln Leu Thr Ala Val Leu Gly Leu Ser Glu Arg Asp Gly Gly Ser

100 105 110

Leu Tyr Ile Ala Gln Trp Leu Ile Gly Ala Asp Gly Glu Thr Ile Ala

115 120 125

Lys Arg Arg Lys Leu Arg Pro Thr His Ala Glu Arg Thr Val Tyr Gly

130 135 140

Glu Gly Asp Gly Ser Asp Leu Ala Val His Glu Arg Pro Asp Ile Gly

145 150 155 160

Arg Ile Gly Ala Leu Cys Cys Trp Glu His Leu Gln Pro Leu Ser Lys

165 170 175

Tyr Ala Met Tyr Ala Gln Asn Glu Gln Val His Val Ala Ala Trp Pro

180 185 190

Ser Phe Ser Leu Tyr Asp Pro Phe Ala Pro Ala Leu Gly Ala Glu Val

195 200 205

Asn Asn Ala Ala Ser Arg Val Tyr Ala Val Glu Gly Ser Cys Phe Val

210 215 220

Leu Ala Pro Cys Ala Thr Val Ser Gln Ala Met Ile Asp Glu Leu Cys

225 230 235 240

Asp Arg Pro Asp Lys His Ala Leu Leu His Ala Gly Gly Gly His Ala

245 250 255

Ala Ile Phe Gly Pro Asp Gly Ser Ala Leu Ala Ala Gln Leu Pro Pro

260 265 270

Asp Gln Glu Gly Leu Leu Ile Ala Glu Ile Asp Leu Gly Met Ile Gly

275 280 285

Ile Ala Lys Asn Ala Ala Asp Pro Ala Gly His Tyr Ser Arg Pro Asp

290 295 300

Val Thr Arg Leu Leu Leu Asn Lys Lys Pro Leu Asn Arg Val Glu His

305 310 315 320

Phe Ser Leu Pro Val Asp Ser Ala Ala Ala Ala Leu Pro Gly Glu Ala

325 330 335

Ala Val Ala Arg Pro Asp Gln Ser Ile

340 345

<210>24

<211>366

<212>PRT

<213>紫红红球菌NCIMB 11216

<400>24

Met Val Glu Tyr Thr Asn Thr Phe Lys Val Ala Ala Val Gln Ala Gln

1 5 10 15

Pro Val Trp Phe Asp Ala Ala Lys Thr Val Asp Lys Thr Val Ser Ile

20 25 30

Ile Ala Glu Ala Ala Arg Asn Gly Cys Glu Leu Val Ala Phe Pro Glu

35 40 45

Val Phe Ile Pro Gly Tyr Pro Tyr His Ile Trp Val Asp Ser Pro Leu

50 55 60

Ala Gly Met Ala Lys Phe Ala Val Arg Tyr His Glu Asn Ser Leu Thr

65 70 75 80

Met Asp Ser Pro His Val Gln Arg Leu Leu Asp Ala Ala Arg Asp His

85 90 95

Asn Ile Ala Val Val Val Gly Ile Ser Glu Arg Asp Gly Gly Ser Leu

100 105 110

Tyr Met Thr Gln Leu Ile Ile Asp Ala Asp Gly Gln Leu Val Ala Arg

115 120 125

Arg Arg Lys Leu Lys Pro Thr His Val Glu Arg Ser Val Tyr Gly Glu

130 135 140

Gly Asn Gly Ser Asp Ile Ser Val Tyr Asp Met Pro Phe Ala Arg Leu

145 150 155 160

Gly Ala Leu Asn Cys Trp Glu His Phe Gln Thr Leu Thr Lys Tyr Ala

165 170 175

Met Tyr Ser Met His Glu Gln Val His Val Ala Ser Trp Pro Gly Met

180 185 190

Ser Leu Tyr Gln Pro Glu Val Pro Ala Phe Gly Val Asp Ala Gln Leu

195 200 205

Thr Ala Thr Arg Met Tyr Ala Leu Glu Gly Gln Thr Phe Val Val Cys

210 215 220

Thr Thr Gln Val Val Thr Pro Glu Ala His Glu Phe Phe Cys Glu Asn

225 230 235 240

Glu Glu Gln Arg Lys Leu Ile Gly Arg Gly Gly Gly Phe Ala Arg Ile

245 250 255

Ile Gly Pro Asp Gly Arg Asp Leu Ala Thr Pro Leu Ala Glu Asp Glu

260 265 270

Glu Gly Ile Leu Tyr Ala Asp Ile Asp Leu Ser Ala Ile Thr Leu Ala

275 280 285

Lys Gln Ala Ala Asp Pro Val Gly His Tyr Ser Arg Pro Asp Val Leu

290 295 300

Ser Leu Asn Phe Asn Gln Arg Arg Thr Thr Pro Val Asn Thr Pro Leu

305 310 315 320

Ser Thr Ile His Ala Thr His Thr Phe Val Pro Gln Phe Gly Ala Leu

325 330 335

Asp Gly Val Arg Glu Leu Asn Gly Ala Asp Glu Gln Arg Ala Leu Pro

340 345 350

Ser Thr His Ser Asp Glu Thr Asp Arg Ala Thr Ala Thr Leu

355 360 365

<210>25

<211>366

<212>PRT

<213>紫红红球菌ATCC 39484

<400>25

Met Val Glu Tyr Thr Asn Thr Phe Lys Val Ala Ala Val Gln Ala Gln

1 5 10 15

Pro Val Trp Phe Asp Ala Ala Lys Thr Val Asp Lys Thr Val Ser Ile

20 25 30

Ile Ala Glu Ala Ala Arg Asn Gly Cys Glu Leu Val Ala Phe Pro Glu

35 40 45

Val Phe Ile Pro Gly Tyr Pro Tyr His Ile Trp Val Asp Ser Pro Leu

50 55 60

Ala Gly Met Ala Lys Phe Ala Val Arg Tyr His Glu Asn Ser Leu Thr

65 70 75 80

Met Asp Ser Pro His Val Gln Arg Leu Leu Asp Ala Ala Arg Asp His

85 90 95

Asn Ile Ala Val Val Val Gly Ile Ser Glu Arg Asp Gly Gly Ser Leu

100 105 110

Tyr Met Thr Gln Leu Ile Ile Asp Ala Asp Gly Gln Leu Val Ala Arg

115 120 125

Arg Arg Lys Leu Lys Pro Thr His Val Glu Arg Ser Val Tyr Gly Glu

130 135 140

Gly Asn Gly Ser Asp Ile Ser Val Tyr Asp Met Pro Phe Ala Arg Leu

145 150 155 160

Gly Ala Leu Asn Cys Trp Glu His Phe Gln Thr Leu Thr Lys Tyr Ala

165 170 175

Met Tyr Ser Met His Glu Gln Val His Val Ala Ser Trp Pro Gly Met

180 185 190

Ser Leu Tyr Gln Pro Glu Val Pro Ala Phe Gly Val Asp Ala Gln Leu

195 200 205

Thr Ala Thr Arg Met Tyr Ala Leu Glu Gly Gln Thr Phe Val Val Cys

210 215 220

Thr Thr Gln Val Val Thr Pro Glu Ala His Glu Phe Phe Cys Glu Asn

225 230 235 240

Glu Glu Gln Arg Met Leu Ile Gly Arg Gly Gly Gly Phe Ala Arg Ile

245 250 255

Ile Gly Pro Asp Gly Arg Asp Leu Ala Thr Pro Leu Ala Glu Asp Glu

260 265 270

Glu Gly Ile Leu Tyr Ala Asp Ile Asp Leu Ser Ala Ile Thr Leu Ala

275 280 285

Lys Gln Ala Ala Asp Pro Val Gly His Tyr Ser Arg Pro Asp Val Leu

290 295 300

Ser Leu Asn Phe Asn Gln Arg Arg Thr Thr Pro Val Asn Thr Pro Leu

305 310 315 320

Ser Thr Ile His Ala Thr His Thr Phe Val Pro Gln Phe Gly Ala Leu

325 330 335

Asp Gly Val Arg Glu Leu Asn Gly Ala Asp Glu Gln Arg Ala Leu Pro

340 345 350

Ser Thr His Ser Asp Glu Thr Asp Arg Ala Thr Ala Thr Leu

355 360 365

<210>26

<211>1110

<212>DNA

<213>敏捷食酸菌72W

<220>

<221>CDS

<222>(1)..(1110)

<400>26

atg gtt tcg tat aac agc aag ttc ctc gcg gca acc gtt cag gca gag 48

Met Val Ser Tyr Asn Ser Lys Phe Leu Ala Ala Thr Val Gln Ala Glu

1 5 10 15

ccg gta tgg ctc gac gca gac gca acg atc gac aag tcg atc ggc atc 96

Pro Val Trp Leu Asp Ala Asp Ala Thr Ile Asp Lys Ser Ile Gly Ile

20 25 30

ate gaa gaa get gcc caa aag ggc gcg agt ctg atc gct ttc ccg gaa 144

Ile Glu Glu Ala Ala Gln Lys Gly Ala Ser Leu Ile Ala Phe Pro Glu

35 40 45

gta ttc att ccg ggc tac ccc tat tgg gcg tgg ctc ggc gac gtg aag 192

Val Phe Ile Pro Gly Tyr Pro Tyr Trp Ala Trp Leu Gly Asp Val Lys

50 55 60

tac agc cta agc ttt act tca cgc tat cac gag aat tcg ttg gag cta 240

Tyr Ser Leu Ser Phe Thr Ser Arg Tyr His Glu Asn Ser Leu Glu Leu

65 70 75 80

ggt gac gac cgt atg cgt cgc ctc cag ctg gcc gcg cgc cgc aac aaa 288

Gly Asp Asp Arg Met Arg Arg Leu Gln Leu Ala Ala Arg Arg Asn Lys

85 90 95

atc gca ctc gtc atg ggc tat tcg gag cgg gaa gcc gga tcg cgc tat 336

Ile Ala Leu Val Met Gly Tyr Ser Glu Arg Glu Ala Gly Ser Arg Tyr

100 105 110

ctg agc cag gtg ttc atc gac gag cgt ggc gag atc gtt gcc aat cgg 384

Leu Ser Gln Val Phe Ile Asp Glu Arg Gly Glu Ile Val Ala Asn Arg

115 120 125

cgc aag ctg aag ccc aca cac gtt gag cgt acg atc tac ggc gaa ggc 432

Arg Lys Leu Lys Pro Thr His Val Glu Arg Thr Ile Tyr Gly Glu Gly

130 135 140

aac gga acc gat ttc ctc acg cac gac ttc gcg ttc gga cgc gtc ggt 480

Asn Gly Thr Asp Phe Leu Thr His Asp Phe Ala Phe Gly Arg Val Gly

145 150 155 160

gga ttg aac tgc tgg gaa cat ttc caa ccg ctc agc aag ttc atg atg 528

Gly Leu Asn Cys Trp Glu His Phe Gln Pro Leu Ser Lys Phe Met Met

165 170 175

tac agc ctc ggt gag cag gtc cac gtt gca tcg tgg ccg gcg atg tcc 576

Tyr Ser Leu Gly Glu Gln Val His Val Ala Ser Trp Pro Ala Met Ser

180 185 190

cct ctt cag ccg gat gtt ttc caa cag agc atc gaa gcc aac gcg acg 624

Pro Leu Gln Pro Asp Val Phe Gln Gln Ser Ile Glu Ala Asn Ala Thr

195 200 205

gtc acc cgc tcg tac gca atc gaa ggc caa acc ttt gtg ctt tgc tcg 672

Val Thr Arg Ser Tyr Ala Ile Glu Gly Gln Thr Phe Val Leu Cys Ser

210 215 220

acg cag gtg atc gga cct agc gcg atc gaa acg ttc tgc ctc aac gac 720

Thr Gln Val Ile Gly Pro Ser Ala Ile Glu Thr Phe Cys Leu Asn Asp

225 230 235 240

gaa cag cgc gca ctg ttg ccg caa gga tgt ggc tgg gcg cgc att tac 768

Glu Gln Arg Ala Leu Leu Pro Gln Gly Cys Gly Trp Ala Arg Ile Tyr

245 250 255

ggc ccg gat gga agc gag ctt gcg aag cct ctg gcg gaa gat gct gag 816

Gly Pro Asp Gly Ser Glu Leu Ala Lys Pro Leu Ala Glu Asp Ala Glu

260 265 270

ggg atc ttg tac gca gag atc gat ctg gag cag att ctg ctg gcg aag 864

Gly Ile Leu Tyr Ala Glu Ile Asp Leu Glu Gln Ile Leu Leu Ala Lys

275 280 285

gct gga gcc gat ccg gtc ggg cac tat tcg cgg cct gac gtg ctg tcg 912

Ala Gly Ala Asp Pro Val Gly His Tyr Ser Arg Pro Asp Val Leu Ser

290 295 300

gtc cag ttc gac ccg cgc aat cat acg cca gtt cat cgc atc ggc att 960

Val Gln Phe Asp Pro Arg Asn His Thr Pro Val His Arg Ile Gly Ile

305 310 315 320

gac ggt cgc ttg gat gtg aat acc cgc agt cgc gtg gag aat ttc cga 1008

Asp Gly Arg Leu Asp Val Asn Thr Arg Ser Arg Val Glu Asn Phe Arg

325 330 335

ctg cga caa gcg gct gag cag gag cgt cag gca tcc aag cgg ctc gga 1056

Leu Arg Gln Ala Ala Glu Gln Glu Arg Gln Ala Ser Lys Arg Leu Gly

340 345 350

acg aaa ctc ttt gaa caa tcc ctt ctg gct gaa gaa ccg gtc cca gca 1104

Thr Lys Leu Phe Glu Gln Ser Leu Leu Ala Glu Glu Pro Val Pro Ala

355 360 365

aag tag 1110

Lys

<210>27

<211>369

<212>PRT

<213>敏捷食酸菌72W

<400>27

Met Val Ser Tyr Asn Ser Lys Phe Leu Ala Ala Thr Val Gln Ala Glu

1 5 10 15

Pro Val Trp Leu Asp Ala Asp Ala Thr Ile Asp Lys Ser Ile Gly Ile

20 25 30

Ile Glu Glu Ala Ala Gln Lys Gly Ala Ser Leu Ile Ala Phe Pro Glu

35 40 45

Val Phe Ile Pro Gly Tyr Pro Tyr Trp Ala Trp Leu Gly Asp Val Lys

50 55 60

Tyr Ser Leu Ser Phe Thr Ser Arg Tyr His Glu Asn Ser Leu Glu Leu

65 70 75 80

Gly Asp Asp Arg Met Arg Arg Leu Gln Leu Ala Ala Arg Arg Asn Lys

85 90 95

Ile Ala Leu Val Met Gly Tyr Ser Glu Arg Glu Ala Gly Ser Arg Tyr

100 105 110

Leu Ser Gln Val Phe Ile Asp Glu Arg Gly Glu Ile Val Ala Asn Arg

115 120 125

Arg Lys Leu Lys Pro Thr His Val Glu Arg Thr Ile Tyr Gly Glu Gly

130 135 140

Asn Gly Thr Asp Phe Leu Thr His Asp Phe Ala Phe Gly Arg Val Gly

145 150 155 160

Gly Leu Asn Cys Trp Glu His Phe Gln Pro Leu Ser Lys Phe Met Met

165 170 175

Tyr Ser Leu Gly Glu Gln Val His Val Ala Ser Trp Pro Ala Met Ser

180 185 190

Pro Leu Gln Pro Asp Val Phe Gln Gln Ser Ile Glu Ala Asn Ala Thr

195 200 205

Val Thr Arg Ser Tyr Ala Ile Glu Gly Gln Thr Phe Val Leu Cys Ser

210 215 220

Thr Gln Val Ile Gly Pro Ser Ala Ile Glu Thr Phe Cys Leu Asn Asp

225 230 235 240

Glu Gln Arg Ala Leu Leu Pro Gln Gly Cys Gly Trp Ala Arg Ile Tyr

245 250 255

Gly Pro Asp Gly Ser Glu Leu Ala Lys Pro Leu Ala Glu Asp Ala Glu

260 265 270

Gly Ile Leu Tyr Ala Glu Ile Asp Leu Glu Gln Ile Leu Leu Ala Lys

275 280 285

Ala Gly Ala Asp Pro Val Gly His Tyr Ser Arg Pro Asp Val Leu Ser

290 295 300

Val Gln Phe Asp Pro Arg Asn His Thr Pro Val His Arg Ile Gly Ile

305 310 315 320

Asp Gly Arg Leu Asp Val Asn Thr Arg Ser Arg Val Glu Asn Phe Arg

325 330 335

Leu Arg Gln Ala Ala Glu Gln Glu Arg Gln Ala Ser Lys Arg Leu Gly

340 345 350

Thr Lys Leu Phe Glu Gln Ser Leu Leu Ala Glu Glu Pro Val Pro Ala

355 360 365

Lys

<210>28

<211>1110

<212>DNA

<213>敏捷食酸菌72W

<220>

<221>CDS

<222>(1)..(1110)

<400>28

atg gtt tcg tat aac agc aag ttc ctc gcg gca acc gtt cag gca gag 48

Met Val Ser Tyr Asn Ser Lys Phe Leu Ala Ala Thr Val Gln Ala Glu

1 5 10 15

ccg gta tgg ctc gac gca gac gca acg atc gac aag tcg atc ggc atc 96

Pro Val Trp Leu Asp Ala Asp Ala Thr Ile Asp Lys Ser Ile Gly Ile

20 25 30

atc gaa gaa gct gcc caa aag ggc gcg agt ctg atc gct ttc ccg gaa 144

Ile Glu Glu Ala Ala Gln Lys Gly Ala Ser Leu Ile Ala Phe Pro Glu

35 40 45

gta ttc att ccg ggc tac ccc tat tgg gcg tgg ctc ggc gac gtg aag 192

Val Phe Ile Pro Gly Tyr Pro Tyr Trp Ala Trp Leu Gly Asp Val Lys

50 55 60

tac agc cta agc ttt act tca cgc tat cac gag aat tcg ttg gag cta 240

Tyr Ser Leu Ser Phe Thr Ser Arg Tyr His Glu Asn Ser Leu Glu Leu

65 70 75 80

ggt gac gac cgt atg cgt cgc ctc cag ctg gcc gcg cgc cgc aac aaa 288

Gly Asp Asp Arg Met Arg Arg Leu Gln Leu Ala Ala Arg Arg Asn Lys

85 90 95

atc gca ctc gtc atg ggc tat tcg gag cgg gaa gcc gga tcg cgc tat 336

Ile Ala Leu Val Met Gly Tyr Ser Glu Arg Glu Ala Gly Ser Arg Tyr

100 105 110

ctg agc cag gtg ttc atc gac gag cgt ggc gag atc gtt gcc aat cgg 384

Leu Ser Gln Val Phe Ile Asp Glu Arg Gly Glu Ile Val Ala Asn Arg

115 120 125

cgc aag ctg aag ccc aca cac gtt gag cgt acg atc tac ggc gaa ggc 432

Arg Lys Leu Lys Pro Thr His Val Glu Arg Thr Ile Tyr Gly Glu Gly

130 135 140

aac gga acc gat ttc ctc acg cac gac ttc gcg ttc gga cgc gtc ggt 480

Asn Gly Thr Asp Phe Leu Thr His Asp Phe Ala Phe Gly Arg Val Gly

145 150 155 160

gga ttg aac tgc tgg gaa cat ttc caa ccg ctc agc aag ttc atg atg 528

Gly Leu Asn Cys Trp Glu His Phe Gln Pro Leu Ser Lys Phe Met Met

165 170 175

tac agc ctc ggt gag cag gtc cac gtt gca tcg tgg ccg gcg atg tcc 576

Tyr Ser Leu Gly Glu Gln Val His Val Ala Ser Trp Pro Ala Met Ser

180 185 190

cct ctt cag ccg gat gtt ttc caa gct agc atc gaa gcc aac gcg acg 624

Pro Leu Gln Pro Asp Val Phe Gln Ala Ser Ile Glu Ala Asn Ala Thr

195 200 205

gtc acc cgc tcg tac gca atc gaa ggc caa acc ttt gtg ctt tgc tcg 672

Val Thr Arg Ser Tyr Ala Ile Glu Gly Gln Thr Phe Val Leu Cys Ser

210 215 220

acg cag gtg atc gga cct agc gcg atc gaa acg ttc tgc ctc aac gac 720

Thr Gln Val Ile Gly Pro Ser Ala Ile Glu Thr Phe Cys Leu Asn Asp

2252 30 235 240

gaa cag cgc gca ctg ttg ccg caa gga tgt ggc tgg gcg cgc att tac 768

Glu Gln Arg Ala Leu Leu Pro Gln Gly Cys Gly Trp Ala Arg Ile Tyr

245 250 255

ggc ccg gat gga agc gag ctt gcg aag cct ctg gcg gaa gat gct gag 816

Gly Pro Asp Gly Ser Glu Leu Ala Lys Pro Leu Ala Glu Asp Ala Glu

260 265 270

ggg atc ttg tac gca gag atc gat ctg gag cag att ctg ctg gcg aag 864

Gly Ile Leu Tyr Ala Glu Ile Asp Leu Glu Gln Ile Leu Leu Ala Lys

275 280 285

gct gga gcc gat ccg gtc ggg cac tat tcg cgg cct gac gtg ctg tcg 912

Ala Gly Ala Asp Pro Val Gly His Tyr Ser Arg Pro Asp Val Leu Ser

290 295 300

gtc cag ttc gac ccg cgc aat cat acg cca gtt cat cgc atc ggc att 960

Val Gln Phe Asp Pro Arg Asn His Thr Pro Val His Arg Ile Gly Ile

305 310 315 320

gac ggt cgc ttg gat gtg aat acc cgc agt cgc gtg gag aat ttc cga 1008

Asp Gly Arg Leu Asp Val Asn Thr Arg Ser Arg Val Glu Asn Phe Arg

325 330 335

ctg cga caa gcg gct gag cag gag cgt cag gca tcc aag cgg ctc gga 1056

Leu Arg Gln Ala Ala Glu Gln Glu Arg Gln Ala Ser Lys Arg Leu Gly

340 345 350

acg aaa ctc ttt gaa caa tcc ctt ctg gct gaa gaa ccg gtc cca gca 1104

Thr Lys Leu Phe Glu Gln Ser Leu Leu Ala Glu Glu Pro Val Pro Ala

355 360 365

aag tag 1110

Lys

<210>29

<211>369

<212>PRT

<213>敏捷食酸菌72W

<400>29

Met Val Ser Tyr Asn Ser Lys Phe Leu Ala Ala Thr Val Gln Ala Glu

1 5 10 15

Pro Val Trp Leu Asp Ala Asp Ala Thr Ile Asp Lys Ser Ile Gly Ile

20 25 30

Ile Glu Glu Ala Ala Gln Lys Gly Ala Ser Leu Ile Ala Phe Pro Glu

35 40 45

Val Phe Ile Pro Gly Tyr Pro Tyr Trp Ala Trp Leu Gly Asp Val Lys

50 55 60

Tyr Ser Leu Ser Phe Thr Ser Arg Tyr His Glu Asn Ser Leu Glu Leu

65 70 75 80

Gly Asp Asp Arg Met Arg Arg Leu Gln Leu Ala Ala Arg Arg Asn Lys

85 90 95

Ile Ala Leu Val Met Gly Tyr Ser Glu Arg Glu Ala Gly Ser Arg Tyr

100 105 110

Leu Ser Gln Val Phe Ile Asp Glu Arg Gly Glu Ile Val Ala Asn Arg

115 120 125

Arg Lys Leu Lys Pro Thr His Val Glu Arg Thr Ile Tyr Gly Glu Gly

130 135 140

Asn Gly Thr Asp Phe Leu Thr His Asp Phe Ala Phe Gly Arg Val Gly

145 150 155 160

Gly Leu Asn Cys Trp Glu His Phe Gln Pro Leu Ser Lys Phe Met Met

165 170 175

Tyr Ser Leu Gly Glu Gln Val His Val Ala Ser Trp Pro Ala Met Ser

180 185 190

Pro Leu Gln Pro Asp Val Phe Gln Ala Ser Ile Glu Ala Asn Ala Thr

195 200 205

Val Thr Arg Ser Tyr Ala Ile Glu Gly Gln Thr Phe Val Leu Cys Ser

210 215 220

Thr Gln Val Ile Gly Pro Ser Ala Ile Glu Thr Phe Cys Leu Asn Asp

225 230 235 240

Glu Gln Arg Ala Leu Leu Pro Gln Gly Cys Gly Trp Ala Arg Ile Tyr

245 250 255

Gly Pro Asp Gly Ser Glu Leu Ala Lys Pro Leu Ala Glu Asp Ala Glu

260 265 270

Gly Ile Leu Tyr Ala Glu Ile Asp Leu Glu Gln Ile Leu Leu Ala Lys

275 280 285

Ala Gly Ala Asp Pro Val Gly His Tyr Ser Arg Pro Asp Val Leu Ser

290 295 300

Val Gln Phe Asp Pro Arg Asn His Thr Pro Val His Arg Ile Gly Ile

305 310 315 320

Asp Gly Arg Leu Asp Val Asn Thr Arg Ser Arg Val Glu Asn Phe Arg

325 330 335

Leu Arg Gln Ala Ala Glu Gln Glu Arg Gln Ala Ser Lys Arg Leu Gly

340 345 350

Thr Lys Leu Phe Glu Gln Ser Leu Leu Ala Glu Glu Pro Val Pro Ala

355 360 365

Lys

<210>30

<211>1110

<212>DNA

<213>敏捷食酸菌72W

<220>

<221>CDS

<222>(1)..(1110)

<400>30

atg gtt tcg tat aac agc aag ttc ctc gcg gca acc gtt cag gca gag 48

Met Val Ser Tyr Asn Ser Lys Phe Leu Ala Ala Thr Val Gln Ala Glu

1 5 10 15

ccg gta tgg ctc gac gca gac gca acg atc gac aag tcg atc ggc atc 96

Pro Val Trp Leu Asp Ala Asp Ala Thr Ile Asp Lys Ser Ile Gly Ile

20 25 30

atc gaa gaa gct gcc caa aag ggc gcg agt ctg atc gct ttc ccg gaa 144

Ile Glu Glu Ala Ala Gln Lys Gly Ala Ser Leu Ile Ala Phe Pro Glu

35 40 45

gta ttc att ccg ggc tac ccc tat tgg gcg tgg ctc ggc gac gtg aag 192

Val Phe Ile Pro Gly Tyr Pro Tyr Trp Ala Trp Leu Gly Asp Val Lys

50 55 60

tac agc cta agc ttt act tca cgc tat cac gag aat tcg ttg gag cta 240

Tyr Ser Leu Ser Phe Thr Ser Arg Tyr His Glu Asn Ser Leu Glu Leu

65 70 75 80

gg t gac gac cgt atg cgt cgc ctc cag ctg gcc gcg cgc cgc aac aaa 288

Gly Asp Asp Arg Met Arg Arg Leu Gln Leu Ala Ala Arg Arg Asn Lys

85 90 95

atc gca ctc gtc atg ggc tat tcg gag cgg gaa gcc gga tcg cgc tat 336

Ile Ala Leu Val Met Gly Tyr Ser Glu Arg Glu Ala Gly Ser Arg Tyr

100 105 110

ctg agc cag gtg ttc atc gac gag cgt ggc gag atc gtt gcc aat cgg 384

Leu Ser Gln Val Phe Ile Asp Glu Arg Gly Glu Ile Val Ala Asn Arg

115 120 125

cgc aag ctg aag ccc aca cac gtt gag cgt acg atc tac ggc gaa ggc 432

Arg Lys Leu Lys Pro Thr His Val Glu Arg Thr Ile Tyr Gly Glu Gly

130 135 140

aac gga acc gat ttc ctc acg cac gac ttc gcg ttc gga cgc gtc ggt 480

Asn Gly Thr Asp Phe Leu Thr His Asp Phe Ala Phe Gly Arg Val Gly

145 150 155 160

gga ttg aac tgc tgg gaa cat ttc caa ccg ctc agc aag ttc atg atg 528

Gly Leu Asn Cys Trp Glu His Phe Gln Pro Leu Ser Lys Phe Met Met

165 170 175

tac agc ctc ggt gag cag gtc cac gtt gca tcg tgg ccg gcg atg tcc 576

Tyr Ser Leu Gly Glu Gln Val His Val Ala Ser Trp Pro Ala Met Ser

180 185 190

cct ctt cag ccg gat gtt ttc caa tgt agc atc gaa gcc aac gcg acg 624

Pro Leu Gln Pro Asp Val Phe Gln Cys Ser Ile Glu Ala Asn Ala Thr

195 200 205

gtc acc cgc tcg tac gca atc gaa ggc caa acc ttt gtg ctt tgc tcg 672

Val Thr Arg Ser Tyr Ala Ile Glu Gly Gln Thr Phe Val Leu Cys Ser

210 215 220

acg cag gtg atc gga cct agc gcg atc gaa acg ttc tgc ctc aac gac 720

Thr Gln Val Ile Gly Pro Ser Ala Ile Glu Thr Phe Cys Leu Asn Asp

225 230 235 240

gaa cag cgc gca ctg ttg ccg caa gga tgt ggc tgg gcg cgc att tac 768

Glu Gln Arg Ala Leu Leu Pro Gln Gly Cys Gly Trp Ala Arg Ile Tyr

245 250 255

ggc ccg gat gga agc gag ctt gcg aag cct ctg gcg gaa gat gct gag 816

Gly Pro Asp Gly Ser Glu Leu Ala Lys Pro Leu Ala Glu Asp Ala Glu

260 265 270

ggg atc ttg tac gca gag atc gat ctg gag cag att ctg ctg gcg aag 864

Gly Ile Leu Tyr Ala Glu Ile Asp Leu Glu Gln Ile Leu Leu Ala Lys

275 280 285

gct gga gcc gat ccg gtc ggg cac tat tcg cgg cct gac gtg ctg tcg 912

Ala Gly Ala Asp Pro Val Gly His Tyr Ser Arg Pro Asp Val Leu Ser

290 295 300

gtc cag ttc gac ccg cgc aat cat acg cca gtt cat cgc atc ggc att 960

Val Gln Phe Asp Pro Arg Asn His Thr Pro Val His Arg Ile Gly Ile

305 310 315 320

gac ggt cgc ttg gat gtg aat acc cgc agt cgc gtg gag aat ttc cga 1008

Asp Gly Arg Leu Asp Val Asn Thr Arg Ser Arg Val Glu Asn Phe Arg

325 330 335

ctg cga caa gcg gct gag cag gag cgt cag gca tcc aag cgg ctc gga 1056

Leu Arg Gln Ala Ala Glu Gln Glu Arg Gln Ala Ser Lys Arg Leu Gly

340 345 350

acg aaa ctc ttt gaa caa tcc ctt ctg gct gaa gaa ccg gtc cca gca 1104

Thr Lys Leu Phe Glu Gln Ser Leu Leu Ala Glu Glu Pro Val Pro Ala

355 360 365

aag tag 1110

Lys

<210>31

<211>369

<212>PRT

<213>敏捷食酸菌72W

<400>31

Met Val Ser Tyr Asn Ser Lys Phe Leu Ala Ala Thr Val Gln Ala Glu

1 5 10 15

Pro Val Trp Leu Asp Ala Asp Ala Thr Ile Asp Lys Ser Ile Gly Ile

20 25 30

Ile Glu Glu Ala Ala Gln Lys Gly Ala Ser Leu Ile Ala Phe Pro Glu

35 40 45

Val Phe Ile Pro Gly Tyr Pro Tyr Trp Ala Trp Leu Gly Asp Val Lys

50 55 60

Tyr Ser Leu Ser Phe Thr Ser Arg Tyr His Glu Asn Ser Leu Glu Leu

65 70 75 80

Gly Asp Asp Arg Met Arg Arg Leu Gln Leu Ala Ala Arg Arg Asn Lys

85 90 95

Ile Ala Leu Val Met Gly Tyr Ser Glu Arg Glu Ala Gly Ser Arg Tyr

100 105 110

Leu Ser Gln Val Phe Ile Asp Glu Arg Gly Glu Ile Val Ala Asn Arg

115 120 125

Arg Lys Leu Lys Pro Thr His Val Glu Arg Thr Ile Tyr Gly Glu Gly

130 135 140

Asn Gly Thr Asp Phe Leu Thr His Asp Phe Ala Phe Gly Arg Val Gly

145 150 155 160

Gly Leu Asn Cys Trp Glu His Phe Gln Pro Leu Ser Lys Phe Met Met

165 170 175

Tyr Ser Leu Gly Glu Gln Val His Val Ala Ser Trp Pro Ala Met Ser

180 185 190

Pro Leu Gln Pro Asp Val Phe Gln Cys Ser Ile Glu Ala Asn Ala Thr

195 200 205

Val Thr Arg Ser Tyr Ala Ile Glu Gly Gln Thr Phe Val Leu Cys Ser

210 215 220

Thr Gln Val Ile Gly Pro Ser Ala Ile Glu Thr Phe Cys Leu Asn Asp

225 230 235 240

Glu Gln Arg Ala Leu Leu Pro Gln Gly Cys Gly Trp Ala Arg Ile Tyr

245 250 255

Gly Pro Asp Gly Ser Glu Leu Ala Lys Pro Leu Ala Glu Asp Ala Glu

260 265 270

Gly Ile Leu Tyr Ala Glu Ile Asp Leu Glu Gln Ile Leu Leu Ala Lys

275 280 285

Ala Gly Ala Asp Pro Val Gly His Tyr Ser Arg Pro Asp Val Leu Ser

290 295 300

Val Gln Phe Asp Pro Arg Asn His Thr Pro Val His Arg Ile Gly Ile

305 310 315 320

Asp Gly Arg Leu Asp Val Asn Thr Arg Ser Arg Val Glu Asn Phe Arg

325 330 335

Leu Arg Gln Ala Ala Glu Gln Glu Arg Gln Ala Ser Lys Arg Leu Gly

340 345 350

Thr Lys Leu Phe Glu Gln Ser Leu Leu Ala Glu Glu Pro Val Pro Ala

355 360 365

Lys

<210>32

<211>1110

<212>DNA

<213>敏捷食酸菌72W

<220>

<221>CDS

<222>(1)..(1110)

<400>32

atg gtt tcg tat aac agc aag ttc ctc gcg gca acc gtt cag gca gag 48

Met Val Ser Tyr Asn Ser Lys Phe Leu Ala Ala Thr Val Gln Ala Glu

1 5 10 15

ccg gta tgg ctc gac gca gac gca acg atc gac aag tcg atc ggc atc 96

Pro Val Trp Leu Asp Ala Asp Ala Thr Ile Asp Lys Ser Ile Gly Ile

20 25 30

atc gaa gaa gct gcc caa aag ggc gcg agt ctg atc gct ttc ccg gaa 144

Ile Glu Glu Ala Ala Gln Lys Gly Ala Ser Leu Ile Ala Phe Pro Glu

35 40 45

gta ttc att ccg ggc tac ccc tat tgg gcg tgg ctc ggc gac gtg aag 192

Val Phe Ile Pro Gly Tyr Pro Tyr Trp Ala Trp Leu Gly Asp Val Lys

50 55 60

tac agc cta agc ttt act tca cgc tat cac gag aat tcg ttg gag cta 240

Tyr Ser Leu Ser Phe Thr Ser Arg Tyr His Glu Asn Ser Leu Glu Leu

65 70 75 80

ggt gac gac cgt atg cgt cgc ctc cag ctg gcc gcg cgc cgc aac aaa 288

Gly Asp Asp Arg Met Arg Arg Leu Gln Leu Ala Ala Arg Arg Asn Lys

85 90 95

atc gca ctc gtc atg ggc tat tcg gag cgg gaa gcc gga tcg cgc tat 336

Ile Ala Leu Val Met Gly Tyr Ser Glu Arg Glu Ala Gly Ser Arg Tyr

100 105 110

ctg agc cag gtg ttc atc gac gag cgt ggc gag atc gtt gcc aat cgg 384

Leu Ser Gln Val Phe Ile Asp Glu Arg Gly Glu Ile Val Ala Asn Arg

115 120 125

cgc aag ctg aag ccc aca cac gtt gag cgt acg atc tac ggc gaa ggc 432

Arg Lys Leu Lys Pro Thr His Val Glu Arg Thr Ile Tyr Gly Glu Gly

130 135 140

aac gga acc gat ttc ctc acg cac gac ttc gcg ttc gga cgc gtc ggt 480

Asn Gly Thr Asp Phe Leu Thr His Asp Phe Ala Phe Gly Arg Val Gly

145 150 155 160

gga ttg aac tgc tgg gaa cat ttc caa ccg ctc agc aag ttc atg atg 528

Gly Leu Asn Cys Trp Glu His Phe Gln Pro Leu Ser Lys Phe Met Met

165 170 175

tac agc ctc ggt gag cag gtc cac gtt gca tcg tgg ccg gcg atg tcc 576

Tyr Ser Leu Gly Glu Gln Val His Val Ala Ser Trp Pro Ala Met Ser

180 185 190

cct ctt cag ccg gat gtt ttc caa acc agc atc gaa gcc aac gcg acg 624

Pro Leu Gln Pro Asp Val Phe Gln Thr Ser Ile Glu Ala Asn Ala Thr

195 200 205

gtc acc cgc tcg tac gca atc gaa ggc caa acc ttt gtg ctt tgc tcg 672

Val Thr Arg Ser Tyr Ala Ile Glu Gly Gln Thr Phe Val Leu Cys Ser

210 215 220

acg cag gtg atc gga cct agc gcg atc gaa acg ttc tgc ctc aac gac 720

Thr Gln Val Ile Gly Pro Ser Ala Ile Glu Thr Phe Cys Leu Asn Asp

225 230 235 240

gaa cag cgc gca ctg ttg ccg caa gga tgt ggc tgg gcg cgc att tac 768

Glu Gln Arg Ala Leu Leu Pro Gln Gly Cys Gly Trp Ala Arg Ile Tyr

245 250 255

ggc ccg gat gga agc gag ctt gcg aag cct ctg gcg gaa gat gct gag 816

Gly Pro Asp Gly Ser Glu Leu Ala Lys Pro Leu Ala Glu Asp Ala Glu

260 265 270

ggg atc ttg tac gca gag atc gat ctg gag cag att ctg ctg gcg aag 864

Gly Ile Leu Tyr Ala Glu Ile Asp Leu Glu Gln Ile Leu Leu Ala Lys

275 280 285

gct gga gcc gat ccg gtc ggg cac tat tcg cgg cct gac gtg ctg tcg 912

Ala Gly Ala Asp Pro Val Gly His Tyr Ser Arg Pro Asp Val Leu Ser

290 295 300

gtc cag ttc gac ccg cgc aat cat acg cca gtt cat cgc atc ggc att 960

Val Gln Phe Asp Pro Arg Asn His Thr Pro Val His Arg Ile Gly Ile

305 310 315 320

gac ggt cgc ttg gat gtg aat acc cgc agt cgc gtg gag aat ttc cga 1008

Asp Gly Arg Leu Asp Val Asn Thr Arg Ser Arg Val Glu Asn Phe Arg

325 330 335

ctg cga caa gcg gct gag cag gag cgt cag gca tcc aag cgg ctc gga 1056

Leu Arg Gln Ala Ala Glu Gln Glu Arg Gln Ala Ser Lys Arg Leu Gly

340 345 350

acg aaa ctc ttt gaa caa tcc ctt ctg gct gaa gaa ccg gtc cca gca 1104

Thr Lys Leu Phe Glu Gln Ser Leu Leu Ala Glu Glu Pro Val Pro Ala

355 360 365

aag tag 1110

Lys

<210>33

<211>369

<212>PRT

<213>敏捷食酸菌72W

<400>33

Met Val Ser Tyr Asn Ser Lys Phe Leu Ala Ala Thr Val Gln Ala Glu

1 5 10 15

Pro Val Trp Leu Asp Ala Asp Ala Thr Ile Asp Lys Ser Ile Gly Ile

20 25 30

Ile Glu Glu Ala Ala Gln Lys Gly Ala Ser Leu Ile Ala Phe Pro Glu

35 40 45

Val Phe Ile Pro Gly Tyr Pro Tyr Trp Ala Trp Leu Gly Asp Val Lys

50 55 60

Tyr Ser Leu Ser Phe Thr Ser Arg Tyr His Glu Asn Ser Leu Glu Leu

65 70 75 80

Gly Asp Asp Arg Met Arg Arg Leu Gln Leu Ala Ala Arg Arg Asn Lys

85 90 95

Ile Ala Leu Val Met Gly Tyr Ser Glu Arg Glu Ala Gly Ser Arg Tyr

100 105 110

Leu Ser Gln Val Phe Ile Asp Glu Arg Gly Glu Ile Val Ala Asn Arg

115 120 125

Arg Lys Leu Lys Pro Thr His Val Glu Arg Thr Ile Tyr Gly Glu Gly

130 135 140

Asn Gly Thr Asp Phe Leu Thr His Asp Phe Ala Phe Gly Arg Val Gly

145 150 155 160

Gly Leu Asn Cys Trp Glu His Phe Gln Pro Leu Ser Lys Phe Met Met

165 170 175

Tyr Ser Leu Gly Glu Gln Val His Val Ala Ser Trp Pro Ala Met Ser

180 185 190

Pro Leu Gln Pro Asp Val Phe Gln Thr Ser Ile Glu Ala Asn Ala Thr

195 200 205

Val Thr Arg Ser Tyr Ala Ile Glu Gly Gln Thr Phe Val Leu Cys Ser

210 215 220

Thr Gln Val Ile Gly Pro Ser Ala Ile Glu Thr Phe Cys Leu Asn Asp

225 230 235 240

Glu Gln Arg Ala Leu Leu Pro Gln Gly Cys Gly Trp Ala Arg Ile Tyr

245 250 255

Gly Pro Asp Gly Ser Glu Leu Ala Lys Pro Leu Ala Glu Asp Ala Glu

260 265 270

Gly Ile Leu Tyr Ala Glu Ile Asp Leu Glu Gln Ile Leu Leu Ala Lys

275 280 285

Ala Gly Ala Asp Pro Val Gly His Tyr Ser Arg Pro Asp Val Leu Ser

290 295 300

Val Gln Phe Asp Pro Arg Asn His Thr Pro Val His Arg Ile Gly Ile

305 310 315 320

Asp Gly Arg Leu Asp Val Asn Thr Arg Ser Arg Val Glu Asn Phe Arg

325 330 335

Leu Arg Gln Ala Ala Glu Gln Glu Arg Gln Ala Ser Lys Arg Leu Gly

340 345 350

Thr Lys Leu Phe Glu Gln Ser Leu Leu Ala Glu Glu Pro Val Pro Ala

355 360 365

Lys

<210>34

<211>1110

<212>DNA

<213>敏捷食酸菌72W

<220>

<221>CDS

<222>(1)..(1110)

<400>34

atg gtt tcg tat aac agc aag ttc ctc gcg gca acc gtt cag gca gag 48

Met Val Ser Tyr Asn Ser Lys Phe Leu Ala Ala Thr Val Gln Ala Glu

1 5 10 15

ccg gta tgg ctc gac gca gac gca acg atc gac aag tcg atc ggc atc 96

Pro Val Trp Leu Asp Ala Asp Ala Thr Ile Asp Lys Ser Ile Gly Ile

20 25 30

atc gaa gaa gct gcc caa aag ggc gcg agt ctg atc gct ttc ccg gaa 144

Ile Glu Glu Ala Ala Gln Lys Gly Ala Ser Leu Ile Ala Phe Pro Glu

35 40 45

gta ttc att ccg ggc tac ccc tat tgg gcg tgg ctc ggc gac gtg aag 192

Val Phe Ile Pro Gly Tyr Pro Tyr Trp Ala Trp Leu Gly Asp Val Lys

50 55 60

tac agc cta agc ttt act tca cgc tat cae gag aat tcg ttg gag cta 240

Tyr Ser Leu Ser Phe Thr Ser Arg Tyr His Glu Asn Ser Leu Glu Leu

65 70 75 80

ggt gac gac cgt atg cgt cgc ctc cag ctg gcc gcg cgc cgc aac aaa 288

Gly Asp Asp Arg Met Arg Arg Leu Gln Leu Ala Ala Arg Arg Asn Lys

85 90 95

atc gca ctc gtc atg ggc tat tcg gag cgg gaa gcc gga tcg cgc tat 336

Ile Ala Leu Val Met Gly Tyr Ser Glu Arg Glu Ala Gly Ser Arg Tyr

100 105 110

ctg agc cag gtg ttc atc gac gag cgt ggc gag atc gtt gcc aat cgg 384

Leu Ser Gln Val Phe Ile Asp Glu Arg Gly Glu Ile Val Ala Asn Arg

115 120 125

cgc aag ctg aag ccc aca cac gtt gag cgt acg atc tac ggc gaa ggc 432

Arg Lys Leu Lys Pro Thr His Val Glu Arg Thr Ile Tyr Gly Glu Gly

130 135 140

aac gga acc gat ttc ctc acg cac gac ttc gcg ttc gga cgc gtc ggt 480

Asn Gly Thr Asp Phe Leu Thr His Asp Phe Ala Phe Gly Arg Val Gly

145 150 155 160

gga ttg aac tgc tgg gaa cat ttc caa ccg ctc agc aag ttc atg atg 528

Gly Leu Asn Cys Trp Glu His Phe Gln Pro Leu Ser Lys Phe Met Met

165 170 175

tac agc ctc ggt gag cag gtc cac gtt gca tcg tgg ccg gcg atg tcc 576

Tyr Ser Leu Gly Glu Gln Val His Val Ala Ser Trp Pro Ala Met Ser

180 185 190

cct ctt cag ccg gat gtt ttc caa gga agc atc gaa gcc aac gcg acg 624

Pro Leu Gln Pro Asp Val Phe Gln Gly Ser Ile Glu Ala Asn Ala Thr

195 200 205

gtc acc cgc tcg tac gca atc gaa ggc caa acc ttt gtg ctt tgc tcg 672

Val Thr Arg Ser Tyr Ala Ile Glu Gly Gln Thr Phe Val Leu Cys Ser

210 215 220

acg cag gtg atc gga cct agc gcg atc gaa acg ttc tgc ctc aac gac 720

Thr Gln Val Ile Gly Pro Ser Ala Ile Glu Thr Phe Cys Leu Asn Asp

225 230 235 240

gaa cag cgc gca ctg ttg ccg caa gga tgt ggc tgg gcg cgc att tac 768

Glu Gln Arg Ala Leu Leu Pro Gln Gly Cys Gly Trp Ala Arg Ile Tyr

245 250 255

ggc ccg gat gga agc gag ctt gcg aag cct ctg gcg gaa gat gct gag 816

Gly Pro Asp Gly Ser Glu Leu Ala Lys Pro Leu Ala Glu Asp Ala Glu

260 265 270

ggg atc ttg tac gca gag atc gat ctg gag cag att ctg ctg gcg aag 864

Gly Ile Leu Tyr Ala Glu Ile Asp Leu Glu Gln Ile Leu Leu Ala Lys

275 280 285

gct gga gcc gat ccg gtc ggg cac tat tcg cgg cct gac gtg ctg tcg 912

Ala Gly Ala Asp Pro Val Gly His Tyr Ser Arg Pro Asp Val Leu Ser

290 295 300

gtc cag ttc gac ccg cgc aat cat acg cca gtt cat cgc atc ggc att 960

Val Gln Phe Asp Pro Arg Asn His Thr Pro Val His Arg Ile Gly Ile

305 310 315 320

gac ggt cgc ttg gat gtg aat acc cgc agt cgc gtg gag aat ttc cga 1008

Asp Gly Arg Leu Asp Val Asn Thr Arg Ser Arg Val Glu Asn Phe Arg

325 330 335

ctg cga caa gcg gct gag cag gag cgt cag gca tcc aag cgg ctc gga 1056

Leu Arg Gln Ala Ala Glu Gln Glu Arg Gln Ala Ser Lys Arg Leu Gly

340 345 350

acg aaa ctc ttt gaa caa tcc ctt ctg gct gaa gaa ccg gtc cca gca 1104

Thr Lys Leu Phe Glu Gln Ser Leu Leu Ala Glu Glu Pro Val Pro Ala

355 360 365

aag tag 1110

Lys

<210>35

<211>369

<212>PRT

<213>敏捷食酸菌72W

<400>35

Met Val Ser Tyr Asn Ser Lys Phe Leu Ala Ala Thr Val Gln Ala Glu

1 5 10 15

Pro Val Trp Leu Asp Ala Asp Ala Thr Ile Asp Lys Ser Ile Gly Ile

20 25 30

Ile Glu Glu Ala Ala Gln Lys Gly Ala Ser Leu Ile Ala Phe Pro Glu

35 40 45

Val Phe Ile Pro Gly Tyr Pro Tyr Trp Ala Trp Leu Gly Asp Val Lys

50 55 60

Tyr Ser Leu Ser Phe Thr Ser Arg Tyr His Glu Asn Ser Leu Glu Leu

65 70 75 80

Gly Asp Asp Arg Met Arg Arg Leu Gln Leu Ala Ala Arg Arg Asn Lys

85 90 95

Ile Ala Leu Val Met Gly Tyr Ser Glu Arg Glu Ala Gly Ser Arg Tyr

100 105 110

Leu Ser Gln Val Phe Ile Asp Glu Arg Gly Glu Ile Val Ala Asn Arg

115 120 125

Arg Lys Leu Lys Pro Thr His Val Glu Arg Thr Ile Tyr Gly Glu Gly

130 135 140

Asn Gly Thr Asp Phe Leu Thr His Asp Phe Ala Phe Gly Arg Val Gly

145 150 155 160

Gly Leu Asn Cys Trp Glu His Phe Gln Pro Leu Ser Lys Phe Met Met

165 170 175

Tyr Ser Leu Gly Glu Gln Val His Val Ala Ser Trp Pro Ala Met Ser

180 185 190

Pro Leu Gln Pro Asp Val Phe Gln Gly Ser Ile Glu Ala Asn Ala Thr

195 200 205

Val Thr Arg Ser Tyr Ala Ile Glu Gly Gln Thr Phe Val Leu Cys Ser

210 215 220

Thr Gln Val Ile Gly Pro Ser Ala Ile Glu Thr Phe Cys Leu Asn Asp

225 230 235 240

Glu Gln Arg Ala Leu Leu Pro Gln Gly Cys Gly Trp Ala Arg Ile Tyr

245 250 255

Gly Pro Asp Gly Ser Glu Leu Ala Lys Pro Leu Ala Glu Asp Ala Glu

260 265 270

Gly Ile Leu Tyr Ala Glu Ile Asp Leu Glu Gln Ile Leu Leu Ala Lys

275 280 285

Ala Gly Ala Asp Pro Val Gly His Tyr Ser Arg Pro Asp Val Leu Ser

290 295 300

Val Gln Phe Asp Pro Arg Asn His Thr Pro Val His Arg Ile Gly Ile

305 310 315 320

Asp Gly Arg Leu Asp Val Asn Thr Arg Ser Arg Val Glu Asn Phe Arg

325 330 335

Leu Arg Gln Ala Ala Glu Gln Glu Arg Gln Ala Ser Lys Arg Leu Gly

340 345 350

Thr Lys Leu Phe Glu Gln Ser Leu Leu Ala Glu Glu Pro Val Pro Ala

355 360 365

Lys

<210>36

<211>1110

<212>DNA

<213>敏捷食酸菌72W

<220>

<221>CDS

<222>(1)..(1110)

<400>36

atg gtt tcg tat aac agc aag ttc ctc gcg gca acc gtt cag gca gag 48

Met Val Ser Tyr Asn Ser Lys Phe Leu Ala Ala Thr Val Gln Ala Glu

1 5 10 15

ccg gta tgg ctc gac gca gac gca acg atc gac aag tcg atc ggc atc 96

Pro Val Trp Leu Asp Ala Asp Ala Thr Ile Asp Lys Ser Ile Gly Ile

20 25 30

atc gaa gaa gct gcc caa aag ggc gcg agt ctg atc gct ttc ccg gaa 144

Ile Glu Glu Ala Ala Gln Lys Gly Ala Ser Leu Ile Ala Phe Pro Glu

35 40 45

gta ttc att ccg ggc tac ccc tat tgg gcg tgg ctc ggc gac gtg aag 192

Val Phe Ile Pro Gly Tyr Pro Tyr Trp Ala Trp Leu Gly Asp Val Lys

50 55 60

tac agc cta agc ttt act tca cgc tat cac gag aat tcg ttg gag cta 240

Tyr Ser Leu Ser Phe Thr Ser Arg Tyr His Glu Asn Ser Leu Glu Leu

65 70 75 80

ggt gac gac cgt atg cgt cgc ctc cag ctg gcc gcg cgc cgc aac aaa 288

Gly Asp Asp Arg Met Arg Arg Leu Gln Leu Ala Ala Arg Arg Asn Lys

85 90 95

atc gca ctc gtc atg ggc tat tcg gag cgg gaa gcc gga tcg cgc tat 336

Ile Ala Leu Val Met Gly Tyr Ser Glu Arg Glu Ala Gly Ser Arg Tyr

100 105 110

ctg agc cag gtg ttc atc gac gag cgt ggc gag atc gtt gcc aat cgg 384

Leu Ser Gln Val Phe Ile Asp Glu Arg Gly Glu Ile Val Ala Asn Arg

115 120 125

cgc aag ctg aag ccc aca cac gtt gag cgt acg atc tac ggc gaa ggc 432

Arg Lys Leu Lys Pro Thr His Val Glu Arg Thr Ile Tyr Gly Glu Gly

130 135 140

aac gga acc gat ttc ctc acg cac gac ttc gcg ttc gga cgc gtc ggt 480

Asn Gly Thr Asp Phe Leu Thr His Asp Phe Ala Phe Gly Arg Val Gly

145 150 155 160

gga ttg aac tgc tgg gaa cat ttc caa ccg ctc agc aag ttc atg atg 528

Gly Leu Asn Cys Trp Glu His Phe Gln Pro Leu Ser Lys Phe Met Met

165 170 175

tac agc ctc ggt gag cag gtc cac gtt gca tcg tgg ccg gcg atg tcc 576

Tyr Ser Leu Gly Glu Gln Val His Val Ala Ser Trp Pro Ala Met Ser

180 185 190

cct ctt cag ccg gat gtt ttc caa cac agc atc gaa gcc aac gcg acg 624

Pro Leu Gln Pro Asp Val Phe Gln His Ser Ile Glu Ala Asn Ala Thr

195 200 205

gtc acc cgc tcg tac gca atc gaa ggc caa acc ttt gtg ctt tgc tcg 672

Val Thr Arg Ser Tyr Ala Ile Glu Gly Gln Thr Phe Val Leu Cys Ser

210 215 220

acg cag gtg atc gga cct agc gcg atc gaa acg ttc tgc ctc aac gac 720

Thr Gln Val Ile Gly Pro Ser Ala Ile Glu Thr Phe Cys Leu Asn Asp

225 230 235 240

gaa cag cgc gca ctg ttg ccg caa gga tgt ggc tgg gcg cgc att tac 768

Glu Gln Arg Ala Leu Leu Pro Gln Gly Cys Gly Trp Ala Arg Ile Tyr

245 250 255

ggc ccg gat gga agc gag ctt gcg aag cct ctg gcg gaa gat gct gag 816

Gly Pro Asp Gly Ser Glu Leu Ala Lys Pro Leu Ala Glu Asp Ala Glu

260 265 270

ggg atc ttg tac gca gag atc gat ctg gag cag att ctg ctg gcg aag 864

Gly Ile Leu Tyr Ala Glu Ile Asp Leu Glu Gln Ile Leu Leu Ala Lys

275 280 285

gct gga gcc gat ccg gtc ggg cac tat tcg cgg cct gac gtg ctg tcg 912

Ala Gly Ala Asp Pro Val Gly His Tyr Ser Arg Pro Asp Val Leu Ser

290 295 300

gtc cag ttc gac ccg cgc aat cat acg cca gtt cat cgc atc ggc att 960

Val Gln Phe Asp Pro Arg Asn His Thr Pro Val His Arg Ile Gly Ile

305 310 315 320

gac ggt cgc ttg gat gtg aat acc cgc agt cgc gtg gag aat ttc cga 1008

Asp Gly Arg Leu Asp Val Asn Thr Arg Ser Arg Val Glu Asn Phe Arg

325 330 335

ctg cga caa gcg gct gag cag gag cgt cag gca tcc aag cgg ctc gga 1056

Leu Arg Gln Ala Ala Glu Gln Glu Arg Gln Ala Ser Lys Arg Leu Gly

340 345 350

acg aaa ctc ttt gaa caa tcc ctt ctg gct gaa gaa ccg gtc cca gca 1104

Thr Lys Leu Phe Glu Gln Ser Leu Leu Ala Glu Glu Pro Val Pro Ala

355 360 365

aag tag 1110

Lys

<210>37

<211>369

<212>PRT

<213>敏捷食酸菌72W

<400>37

Met Val Ser Tyr Asn Ser Lys Phe Leu Ala Ala Thr Val Gln Ala Glu

1 5 10 15

Pro Val Trp Leu Asp Ala Asp Ala Thr Ile Asp Lys Ser Ile Gly Ile

20 25 30

Ile Glu Glu Ala Ala Gln Lys Gly Ala Ser Leu Ile Ala Phe Pro Glu

35 40 45

Val Phe Ile Pro Gly Tyr Pro Tyr Trp Ala Trp Leu Gly Asp Val Lys

50 55 60

Tyr Ser Leu Ser Phe Thr Ser Arg Tyr His Glu Asn Ser Leu Glu Leu

65 70 75 80

Gly Asp Asp Arg Met Arg Arg Leu Gln Leu Ala Ala Arg Arg Asn Lys

85 90 95

Ile Ala Leu Val Met Gly Tyr Ser Glu Arg Glu Ala Gly Ser Arg Tyr

100 105 110

Leu Ser Gln Val Phe Ile Asp Glu Arg Gly Glu Ile Val Ala Asn Arg

115 120 125

Arg Lys Leu Lys Pro Thr His Val Glu Arg Thr Ile Tyr Gly Glu Gly

130 135 140

Asn Gly Thr Asp Phe Leu Thr His Asp Phe Ala Phe Gly Arg Val Gly

145 150 155 160

Gly Leu Asn Cys Trp Glu His Phe Gln Pro Leu Ser Lys Phe Met Met

165 170 175

Tyr Ser Leu Gly Glu Gln Val His Val Ala Ser Trp Pro Ala Met Ser

180 185 190

Pro Leu Gln Pro Asp Val Phe Gln His Ser Ile Glu Ala Asn Ala Thr

195 200 205

Val Thr Arg Ser Tyr Ala Ile Glu Gly Gln Thr Phe Val Leu Cys Ser

210 215 220

Thr Gln Val Ile Gly Pro Ser Ala Ile Glu Thr Phe Cys Leu Asn Asp

225 230 235 240

Glu Gln Arg Ala Leu Leu Pro Gln Gly Cys Gly Trp Ala Arg Ile Tyr

245 250 255

Gly Pro Asp Gly Ser Glu Leu Ala Lys Pro Leu Ala Glu Asp Ala Glu

260 265 270

Gly Ile Leu Tyr Ala Glu Ile Asp Leu Glu Gln Ile Leu Leu Ala Lys

275 280 285

Ala Gly Ala Asp Pro Val Gly His Tyr Ser Arg Pro Asp Val Leu Ser

290 295 300

Val Gln Phe Asp Pro Arg Asn His Thr Pro Val His Arg Ile Gly Ile

305 310 315 320

Asp Gly Arg Leu Asp Val Asn Thr Arg Ser Arg Val Glu Asn Phe Arg

325 330 335

Leu Arg Gln Ala Ala Glu Gln Glu Arg Gln Ala Ser Lys Arg Leu Gly

340 345 350

Thr Lys Leu Phe Glu Gln Ser Leu Leu Ala Glu Glu Pro Val Pro Ala

355 360 365

Lys

<210>38

<211>1110

<212>DNA

<213>敏捷食酸菌72W

<220>

<221>CDS

<222>(1)..(1110)

<400>38

atg gtt tcg tat aac agc aag ttc ctc gcg gca acc gtt cag gca gag 48

Met Val Ser Tyr Asn Ser Lys Phe Leu Ala Ala Thr Val Gln Ala Glu

1 5 10 15

ccg gta tgg ctc gac gca gac gca acg atc gac aag tcg atc ggc atc 96

Pro Val Trp Leu Asp Ala Asp Ala Thr Ile Asp Lys Ser Ile Gly Ile

20 25 30

atc gaa gaa gct gcc caa aag ggc gcg agt ctg atc gct ttc ccg gaa 144

Ile Glu Glu Ala Ala Gln Lys Gly Ala Ser Leu Ile Ala Phe Pro Glu

35 40 45

gta ttc att ccg ggc tac ccc tat tgg gcg tgg ctc ggc gac gtg aag 192

Val Phe Ile Pro Gly Tyr Pro Tyr Trp Ala Trp Leu Gly Asp Val Lys

50 55 60

tac agc cta agc ttt act tca cgc tat cac gag aat tcg ttg gag cta 240

Tyr Ser Leu Ser Phe Thr Ser Arg Tyr His Glu Asn Ser Leu Glu Leu

65 70 75 80

ggt gac gac cgt atg cgt cgc ctc cag ctg gcc gcg cgc cgc aac aaa 288

Gly Asp Asp Arg Met Arg Arg Leu Gln Leu Ala Ala Arg Arg Asn Lys

85 90 95

atc gca ctc gtc atg ggc tat tcg gag cgg gaa gcc gga tcg cgc tat 336

Ile Ala Leu Val Met Gly Tyr Ser Glu Arg Glu Ala Gly Ser Arg Tyr

100 105 110

ctg agc cag gtg ttc atc gac gag cgt ggc gag atc gtt gcc aat cgg 384

Leu Ser Gln Val Phe Ile Asp Glu Arg Gly Glu Ile Val Ala Asn Arg

115 120 125

cgc aag ctg aag ccc aca cac gtt gag cgt acg atc tac ggc gaa ggc 432

Arg Lys Leu Lys Pro Thr His Val Glu Arg Thr Ile Tyr Gly Glu Gly

130 135 140

aac gga acc gat ttc ctc acg cac gac ttc gcg ttc gga cgc gtc ggt 480

Asn Gly Thr Asp Phe Leu Thr His Asp Phe Ala Phe Gly Arg Val Gly

145 150 155 160

gga ttg aac tgc tgg gaa cat ttc caa ccg ctc agc aag ttc atg atg 528

Gly Leu Asn Cys Trp Glu His Phe Gln Pro Leu Ser Lys Phe Met Met

165 170 175

tac agc ctc ggt gag cag gtc cac gtt gca tcg tgg ccg gcg atg tcc 576

Tyr Ser Leu Gly Glu Gln Val His Val Ala Ser Trp Pro Ala Met Ser

180 185 190

cct ctt cag ccg gat gtt ttc caa aag agc atc gaa gcc aac gcg acg 624

Pro Leu Gln Pro Asp Val Phe Gln Lys Ser Ile Glu Ala Asn Ala Thr

195 200 205

gtc acc cgc tcg tac gca atc gaa ggc caa acc ttt gtg ctt tgc tcg 672

Val Thr Arg Ser Tyr Ala Ile Glu Gly Gln Thr Phe Val Leu Cys Ser

210 215 220

acg cag gtg atc gga cct agc gcg atc gaa acg ttc tgc ctc aac gac 720

Thr Gln Val Ile Gly Pro Ser Ala Ile Glu Thr Phe Cys Leu Asn Asp

225 230 235 240

gaa cag cgc gca ctg ttg ccg caa gga tgt ggc tgg gcg cgc att tac 768

Glu Gln Arg Ala Leu Leu Pro Gln Gly Cys Gly Trp Ala Arg Ile Tyr

245 250 255

ggc ccg gat gga agc gag ctt gcg aag cct ctg gcg gaa gat gct gag 816

Gly Pro Asp Gly Ser Glu Leu Ala Lys Pro Leu Ala Glu Asp Ala Glu

260 265 270

ggg atc ttg tac gca gag atc gat ctg gag cag att ctg ctg gcg aag 864

Gly Ile Leu Tyr Ala Glu Ile Asp Leu Glu Gln Ile Leu Leu Ala Lys

275 280 285

gct gga gcc gat ccg gtc ggg cac tat tcg cgg cct gac gtg ctg tcg 912

Ala Gly Ala Asp Pro Val Gly His Tyr Ser Arg Pro Asp Val Leu Ser

290 295 300

gtc cag ttc gac ccg cgc aat cat acg cca gtt cat cgc atc ggc att 960

Val Gln Phe Asp Pro Arg Asn His Thr Pro Val His Arg Ile Gly Ile

305 310 315 320

gac ggt cgc ttg gat gtg aat acc cgc agt cgc gtg gag aat ttc cga 1008

Asp Gly Arg Leu Asp Val Asn Thr Arg Ser Arg Val Glu Asn Phe Arg

325 330 335

ctg cga caa gcg gct gag cag gag cgt cag gca tcc aag cgg ctc gga 1056

Leu Arg Gln Ala Ala Glu Gln Glu Arg Gln Ala Ser Lys Arg Leu Gly

340 345 350

acg aaa ctc ttt gaa caa tcc ctt ctg gct gaa gaa ccg gtc cca gca 1104

Thr Lys Leu Phe Glu Gln Ser Leu Leu Ala Glu Glu Pro Val Pro Ala

355 360 365

aag tag 1110

Lys

<210>39

<211>369

<212>PRT

<213>敏捷食酸菌72W

<400>39

Met Val Ser Tyr Asn Ser Lys Phe Leu Ala Ala Thr Val Gln Ala Glu

1 5 10 15

Pro Val Trp Leu Asp Ala Asp Ala Thr Ile Asp Lys Ser Ile Gly Ile

20 25 30

Ile Glu Glu Ala Ala Gln Lys Gly Ala Ser Leu Ile Ala Phe Pro Glu

35 40 45

Val Phe Ile Pro Gly Tyr Pro Tyr Trp Ala Trp Leu Gly Asp Val Lys

50 55 60

Tyr Ser Leu Ser Phe Thr Ser Arg Tyr His Glu Asn Ser Leu Glu Leu

65 70 75 80

Gly Asp Asp Arg Met Arg Arg Leu Gln Leu Ala Ala Arg Arg Asn Lys

85 90 95

Ile Ala Leu Val Met Gly Tyr Ser Glu Arg Glu Ala Gly Ser Arg Tyr

100 105 110

Leu Ser Gln Val Phe Ile Asp Glu Arg Gly Glu Ile Val Ala Asn Arg

115 120 125

Arg Lys Leu Lys Pro Thr His Val Glu Arg Thr Ile Tyr Gly Glu Gly

130 135 140

Asn Gly Thr Asp Phe Leu Thr His Asp Phe Ala Phe Gly Arg Val Gly

145 150 155 160

Gly Leu Asn Cys Trp Glu His Phe Gln Pro Leu Ser Lys Phe Met Met

165 170 175

Tyr Ser Leu Gly Glu Gln Val His Val Ala Ser Trp Pro Ala Met Ser

180 185 190

Pro Leu Gln Pro Asp Val Phe Gln Lys Ser Ile Glu Ala Asn Ala Thr

195 200 205

Val Thr Arg Ser Tyr Ala Ile Glu Gly Gln Thr Phe Val Leu Cys Ser

210 215 220

Thr Gln Val Ile Gly Pro Ser Ala Ile Glu Thr Phe Cys Leu Asn Asp

225 230 235 240

Glu Gln Arg Ala Leu Leu Pro Gln Gly Cys Gly Trp Ala Arg Ile Tyr

245 250 255

Gly Pro Asp Gly Ser Glu Leu Ala Lys Pro Leu Ala Glu Asp Ala Glu

260 265 270

Gly Ile Leu Tyr Ala Glu Ile Asp Leu Glu Gln Ile Leu Leu Ala Lys

275 280 285

Ala Gly Ala Asp Pro Val Gly His Tyr Ser Arg Pro Asp Val Leu Ser

290 295 300

Val Gln Phe Asp Pro Arg Asn His Thr Pro Val His Arg Ile Gly Ile

305 310 315 320

Asp Gly Arg Leu Asp Val Asn Thr Arg Ser Arg Val Glu Asn Phe Arg

325 330 335

Leu Arg Gln Ala Ala Glu Gln Glu Arg Gln Ala Ser Lys Arg Leu Gly

340 345 350

Thr Lys Leu Phe Glu Gln Ser Leu Leu Ala Glu Glu Pro Val Pro Ala

355 360 365

Lys

<210>40

<211>1110

<212>DNA

<213>敏捷食酸菌72W

<220>

<221>CDS

<222>(1)..(1110)

<400>40

atg gtt tcg tat aac agc aag ttc ctc gcg gca acc gtt cag gca gag 48

Met Val Ser Tyr Asn Ser Lys Phe Leu Ala Ala Thr Val Gln Ala Glu

1 5 10 15

ccg gta tgg ctc gac gca gac gca acg atc gac aag tcg atc ggc atc 96

Pro Val Trp Leu Asp Ala Asp Ala Thr Ile Asp Lys Ser Ile Gly Ile

20 25 30

atc gaa gaa gct gcc caa aag ggc gcg agt ctg atc gct ttc ccg gaa 144

Ile Glu Glu Ala Ala Gln Lys Gly Ala Ser Leu Ile Ala Phe Pro Glu

35 40 45

gta ttc att ccg ggc tac ccc tat tgg gcg tgg ctc ggc gac gtg aag 192

Val Phe Ile Pro Gly Tyr Pro Tyr Trp Ala Trp Leu Gly Asp Val Lys

50 55 60

tac agc cta agc ttt act tca cgc tat cac gag aat tcg ttg gag cta 240

Tyr Ser Leu Ser Phe Thr Ser Arg Tyr His Glu Asn Ser Leu Glu Leu

65 70 75 80

ggt gac gac cgt atg cgt cgc ctc cag ctg gcc gcg cgc cgc aac aaa 288

Gly Asp Asp Arg Met Arg Arg Leu Gln Leu Ala Ala Arg Arg Asn Lys

85 90 95

atc gca ctc gtc atg ggc tat tcg gag cgg gaa gcc gga tcg cgc tat 336

Ile Ala Leu Val Met Gly Tyr Ser Glu Arg Glu Ala Gly Ser Arg Tyr

100 105 110

ctg agc cag gtg ttc atc gac gag cgt ggc gag atc gtt gcc aat cgg 384

Leu Ser Gln Val Phe Ile Asp Glu Arg Gly Glu Ile Val Ala Asn Arg

115 120 125

cgc aag ctg aag ccc aca cac gtt gag cgt acg atc tac ggc gaa ggc 432

Arg Lys Leu Lys Pro Thr His Val Glu Arg Thr Ile Tyr Gly Glu Gly

130 135 140

aac gga acc gat ttc ctc acg cac gac ttc gcg ttc gga cgc gtc ggt 480

Asn Gly Thr Asp Phe Leu Thr His Asp Phe Ala Phe Gly Arg Val Gly

145 150 155 160

gga ttg aac tgc tgg gaa cat ttc caa ccg ctc agc aag ttc atg atg 528

Gly Leu Asn Cys Trp Glu His Phe Gln Pro Leu Ser Lys Phe Met Met

165 170 175

tac agc ctc ggt gag cag gtc cac gtt gca tcg tgg ccg gcg atg tcc 576

Tyr Ser Leu Gly Glu Gln Val His Val Ala Ser Trp Pro Ala Met Ser

180 185 190

cct ctt cag ccg gat gtt ttc caa aat agc atc gaa gcc aac gcg acg 624

Pro Leu Gln Pro Asp Val Phe Gln Asn Ser Ile Glu Ala Asn Ala Thr

195 200 205

gtc acc cgc tcg tac gca atc gaa ggc caa acc ttt gtg ctt tgc tcg 672

Val Thr Arg Ser Tyr Ala Ile Glu Gly Gln Thr Phe Val Leu Cys Ser

210 215 220

acg cag gtg atc gga cct agc gcg atc gaa acg ttc tgc ctc aac gac 720

Thr Gln Val Ile Gly Pro Ser Ala Ile Glu Thr Phe Cys Leu Asn Asp

225 230 235 240

gaa cag cgc gca ctg ttg ccg caa gga tgt ggc tgg gcg cgc att tac 768

Glu Gln Arg Ala Leu Leu Pro Gln Gly Cys Gly Trp Ala Arg Ile Tyr

245 250 255

ggc ccg gat gga agc gag ctt gcg aag cct ctg gcg gaa gat gct gag 816

Gly Pro Asp Gly Ser Glu Leu Ala Lys Pro Leu Ala Glu Asp Ala Glu

260 265 270

ggg atc ttg tac gca gag atc gat ctg gag cag att ctg ctg gcg aag 864

Gly Ile Leu Tyr Ala Glu Ile Asp Leu Glu Gln Ile Leu Leu Ala Lys

275 280 285

gct gga gcc gat ccg gtc ggg cae tat tcg cgg cct gac gtg ctg tcg 912

Ala Gly Ala Asp Pro Val Gly His Tyr Ser Arg Pro Asp Val Leu Ser

290 295 300

gtc cag ttc gac ccg cgc aat cat acg cca gtt cat cgc atc ggc att 960

Val Gln Phe Asp Pro Arg Asn His Thr Pro Val His Arg Ile Gly Ile

305 310 315 320

gac ggt cgc ttg gat gtg aat acc cgc agt cgc gtg gag aat ttc cga 1008

Asp Gly Arg Leu Asp Val Asn Thr Arg Ser Arg Val Glu Asn Phe Arg

325 330 335

ctg cga caa gcg gct gag cag gag cgt cag gca tcc aag cgg ctc gga 1056

Leu Arg Gln Ala Ala Glu Gln Glu Arg Gln Ala Ser Lys Arg Leu Gly

340 345 350

acg aaa ctc ttt gaa caa tcc ctt ctg gct gaa gaa ccg gtc cca gca 1104

Thr Lys Leu Phe Glu Gln Ser Leu Leu Ala Glu Glu Pro Val Pro Ala

355 360 365

aag tag 1110

Lys

<210>41

<211>369

<212>PRT

<213>敏捷食酸菌72W

<400>41

Met Val Ser Tyr Asn Ser Lys Phe Leu Ala Ala Thr Val Gln Ala Glu

1 5 10 15

Pro Val Trp Leu Asp Ala Asp Ala Thr Ile Asp Lys Ser Ile Gly Ile

20 25 30

Ile Glu Glu Ala Ala Gln Lys Gly Ala Ser Leu Ile Ala Phe Pro Glu

35 40 45

Val Phe Ile Pro Gly Tyr Pro Tyr Trp Ala Trp Leu Gly Asp Val Lys

50 55 60

Tyr Ser Leu Ser Phe Thr Ser Arg Tyr His Glu Asn Ser Leu Glu Leu

65 70 75 80

Gly Asp Asp Arg Met Arg Arg Leu Gln Leu Ala Ala Arg Arg Asn Lys

85 90 95

Ile Ala Leu Val Met Gly Tyr Ser Glu Arg Glu Ala Gly Ser Arg Tyr

100 105 110

Leu Ser Gln Val Phe Ile Asp Glu Arg Gly Glu Ile Val Ala Asn Arg

115 120 125

Arg Lys Leu Lys Pro Thr His Val Glu Arg Thr Ile Tyr Gly Glu Gly

130 135 140

Asn Gly Thr Asp Phe Leu Thr His Asp Phe Ala Phe Gly Arg Val Gly

145 150 155 160

Gly Leu Asn Cys Trp Glu His Phe Gln Pro Leu Ser Lys Phe Met Met

165 170 175

Tyr Ser Leu Gly Glu Gln Val His Val Ala Ser Trp Pro Ala Met Ser

180 185 190

Pro Leu Gln Pro Asp Val Phe Gln Asn Ser Ile Glu Ala Asn Ala Thr

195 200 205

Val Thr Arg Ser Tyr Ala Ile Glu Gly Gln Thr Phe Val Leu Cys Ser

210 215 220

Thr Gln Val Ile Gly Pro Ser Ala Ile Glu Thr Phe Cys Leu Asn Asp

225 230 235 240

Glu Gln Arg Ala Leu Leu Pro Gln Gly Cys Gly Trp Ala Arg Ile Tyr

245 250 255

Gly Pro Asp Gly Ser Glu Leu Ala Lys Pro Leu Ala Glu Asp Ala Glu

260 265 270

Gly Ile Leu Tyr Ala Glu Ile Asp Leu Glu Gln Ile Leu Leu Ala Lys

275 280 285

Ala Gly Ala Asp Pro Val Gly His Tyr Ser Arg Pro Asp Val Leu Ser

290 295 300

Val Gln Phe Asp Pro Arg Asn His Thr Pro Val His Arg Ile Gly Ile

305 310 315 320

Asp Gly Arg Leu Asp Val Asn Thr Arg Ser Arg Val Glu Asn Phe Arg

325 330 335

Leu Arg Gln Ala Ala Glu Gln Glu Arg Gln Ala Ser Lys Arg Leu Gly

340 345 350

Thr Lys Leu Phe Glu Gln Ser Leu Leu Ala Glu Glu Pro Val Pro Ala

355 360 365

Lys

<210>42

<211>1110

<212>DNA

<213>敏捷食酸菌72W

<220>

<221>CDS

<222>(1)..(1110)

<400>42

atg gtt tcg tat aac agc aag ttc ctc gcg gca acc gtt cag gca gag 48

Met Val Ser Tyr Asn Ser Lys Phe Leu Ala Ala Thr Val Gln Ala Glu

1 5 10 15

ccg gta tgg ctc gac gca gac gca acg atc gac aag tcg atc ggc atc 96

Pro Val Trp Leu Asp Ala Asp Ala Thr Ile Asp Lys Ser Ile Gly Ile

20 25 30

atc gaa gaa gct gcc caa aag ggc gcg agt ctg atc gct ttc ccg gaa 144

Ile Glu Glu Ala Ala Gln Lys Gly Ala Ser Leu Ile Ala Phe Pro Glu

35 40 45

gta ttc att ccg ggc tac ccc tat tgg gcg tgg ctc ggc gac gtg aag 192

Val Phe Ile Pro Gly Tyr Pro Tyr Trp Ala Trp Leu Gly Asp Val Lys

50 55 60

tac agc cta agc ttt act tca cgc tat cac gag aat tcg ttg gag cta 240

Tyr Ser Leu Ser Phe Thr Ser Arg Tyr His Glu Asn Ser Leu Glu Leu

65 70 75 80

ggt gac gac cgt atg cgt cgc ctc cag ctg gcc gcg cgc cgc aac aaa 288

Gly Asp Asp Arg Met Arg Arg Leu Gln Leu Ala Ala Arg Arg Asn Lys

85 90 95

atc gca ctc gtc atg ggc tat tcg gag cgg gaa gcc gga tcg cgc tat 336

Ile Ala Leu Val Met Gly Tyr Ser Glu Arg Glu Ala Gly Ser Arg Tyr

100 105 110

ctg agc cag gtg ttc atc gac gag cgt ggc gag atc gtt gcc aat cgg 384

Leu Ser Gln Val Phe Ile Asp Glu Arg Gly Glu Ile Val Ala Asn Arg

115 120 125

cgc aag ctg aag ccc aca cac gtt gag cgt acg atc tac ggc gaa ggc 432

Arg Lys Leu Lys Pro Thr His Val Glu Arg Thr Ile Tyr Gly Glu Gly

130 135 140

aac gga acc gat ttc ctc acg cac gac ttc gcg ttc gga cgc gtc ggt 480

Asn Gly Thr Asp Phe Leu Thr His Asp Phe Ala Phe Gly Arg Val Gly

145 150 155 160

gga ttg aac tgc tgg gaa cat ttc caa ccg ctc agc aag ttc atg atg 528

Gly Leu Asn Cys Trp Glu His Phe Gln Pro Leu Ser Lys Phe Met Met

165 170 175

tac agc ctc ggt gag cag gtc cac gtt gca tcg tgg ccg gcg atg tcc 576

Tyr Ser Leu Gly Glu Gln Val His Val Ala Ser Trp Pro Ala Met Ser

180 185 190

cct ctt cag ccg gat gtt ttc caa tct agc atc gaa gcc aac gcg acg 624

Pro Leu Gln Pro Asp Val Phe Gln Ser Ser Ile Glu Ala Asn Ala Thr

195 200 205

gtc acc cgc tcg tac gca atc gaa ggc caa acc ttt gtg ctt tgc tcg 672

Val Thr Arg Ser Tyr Ala Ile Glu Gly Gln Thr Phe Val Leu Cys Ser

210 215 220

acg cag gtg atc gga cct agc gcg atc gaa acg ttc tgc ctc aac gac 720

Thr Gln Val Ile Gly Pro Ser Ala Ile Glu Thr Phe Cys Leu Asn Asp

225 230 235 240

gaa cag cgc gca ctg ttg ccg caa gga tgt ggc tgg gcg cgc att tac 768

Glu Gln Arg Ala Leu Leu Pro Gln Gly Cys Gly Trp Ala Arg Ile Tyr

245 250 255

ggc ccg gat gga agc gag ctt gcg aag cct ctg gcg gaa gat gct gag 816

Gly Pro Asp Gly Ser Glu Leu Ala Lys Pro Leu Ala Glu Asp Ala Glu

260 265 270

ggg atc ttg tac gca gag atc gat ctg gag cag att ctg ctg gcg aag 864

Gly Ile Leu Tyr Ala Glu Ile Asp Leu Glu Gln Ile Leu Leu Ala Lys

275 280 285

gct gga gcc gat ccg gtc ggg cac tat tcg cgg cct gac gtg ctg tcg 912

Ala Gly Ala Asp Pro Val Gly His Tyr Ser Arg Pro Asp Val Leu Ser

290 295 300

gtc cag ttc gac ccg cgc aat cat acg cca gtt cat cgc atc ggc att 960

Val Gln Phe Asp Pro Arg Asn His Thr Pro Val His Arg Ile Gly Ile

305 310 315 320

gac ggt cgc ttg gat gtg aat acc cgc agt cgc gtg gag aat ttc cga 1008

Asp Gly Arg Leu Asp Val Asn Thr Arg Ser Arg Val Glu Asn Phe Arg

325 330 335

ctg cga caa gcg gct gag cag gag cgt cag gca tcc aag cgg ctc gga 1056

Leu Arg Gln Ala Ala Glu Gln Glu Arg Gln Ala Ser Lys Arg Leu Gly

340 345 350

acg aaa ctc ttt gaa caa tcc ctt ctg gct gaa gaa ccg gtc cca gca 1104

Thr Lys Leu Phe Glu Gln Ser Leu Leu Ala Glu Glu Pro Val Pro Ala

355 360 365

aag tag 1110

Lys

<210>43

<211>369

<212>PRT

<213>敏捷食酸菌72W

<400>43

Met Val Ser Tyr Asn Ser Lys Phe Leu Ala Ala Thr Val Gln Ala Glu

1 5 10 15

Pro Val Trp Leu Asp Ala Asp Ala Thr Ile Asp Lys Ser Ile Gly Ile

20 25 30

Ile Glu Glu Ala Ala Gln Lys Gly Ala Ser Leu Ile Ala Phe Pro Glu

35 40 45

Val Phe Ile Pro Gly Tyr Pro Tyr Trp Ala Trp Leu Gly Asp Val Lys

50 55 60

Tyr Ser Leu Ser Phe Thr Ser Arg Tyr His Glu Asn Ser Leu Glu Leu

65 70 75 80

Gly Asp Asp Arg Met Arg Arg Leu Gln Leu Ala Ala Arg Arg Asn Lys

85 90 95

Ile Ala Leu Val Met Gly Tyr Ser Glu Arg Glu Ala Gly Ser Arg Tyr

100 105 110

Leu Ser Gln Val Phe Ile Asp Glu Arg Gly Glu Ile Val Ala Asn Arg

115 120 125

Arg Lys Leu Lys Pro Thr His Val Glu Arg Thr Ile Tyr Gly Glu Gly

130 135 140

Asn Gly Thr Asp Phe Leu Thr His Asp Phe Ala Phe Gly Arg Val Gly

145 150 155 160

Gly Leu Asn Cys Trp Glu His Phe Gln Pro Leu Ser Lys Phe Met Met

165 170 175

Tyr Ser Leu Gly Glu Gln Val His Val Ala Ser Trp Pro Ala Met Ser

180 185 190

Pro Leu Gln Pro Asp Val Phe Gln Ser Ser Ile Glu Ala Asn Ala Thr

195 200 205

Val Thr Arg Ser Tyr Ala Ile Glu Gly Gln Thr Phe Val Leu Cys Ser

210 215 220

Thr Gln Val Ile Gly Pro Ser Ala Ile Glu Thr Phe Cys Leu Asn Asp

225 230 235 240

Glu Gln Arg Ala Leu Leu Pro Gln Gly Cys Gly Trp Ala Arg Ile Tyr

245 250 255

Gly Pro Asp Gly Ser Glu Leu Ala Lys Pro Leu Ala Glu Asp Ala Glu

260 265 270

Gly Ile Leu Tyr Ala Glu Ile Asp Leu Glu Gln Ile Leu Leu Ala Lys

275 280 285

Ala Gly Ala Asp Pro Val Gly His Tyr Ser Arg Pro Asp Val Leu Ser

290 295 300

Val Gln Phe Asp Pro Arg Asn His Thr Pro Val His Arg Ile Gly Ile

305 310 315 320

Asp Gly Arg Leu Asp Val Asn Thr Arg Ser Arg Val Glu Asn Phe Arg

325 330 335

Leu Arg Gln Ala Ala Glu Gln Glu Arg Gln Ala Ser Lys Arg Leu Gly

340 345 350

Thr Lys Leu Phe Glu Gln Ser Leu Leu Ala Glu Glu Pro Val Pro Ala

355 360 365

Lys

<210>44

<211>1110

<212>DNA

<213>敏捷食酸菌72W

<220>

<221>CDS

<222>(1)..(1110)

<400>44

atg gtt tcg tat aac agc aag ttc ctc gcg gca acc gtt cag gca gag 48

Met Val Ser Tyr Asn Ser Lys Phe Leu Ala Ala Thr Val Gln Ala Glu

1 5 10 15

ccg gta tgg ctc gac gca gac gca acg atc gac aag tcg atc ggc atc 96

Pro Val Trp Leu Asp Ala Asp Ala Thr Ile Asp Lys Ser Ile Gly Ile

20 25 30

atc gaa gaa gct gcc caa aag ggc gcg agt ctg atc gct ttc ccg gaa 144

Ile Glu Glu Ala Ala Gln Lys Gly Ala Ser Leu Ile Ala Phe Pro Glu

35 40 45

gta ttc att ccg ggc tac ccc tat tgg gcg tgg ctc ggc gac gtg aag 192

Val Phe Ile Pro Gly Tyr Pro Tyr Trp Ala Trp Leu Gly Asp Val Lys

50 55 60

tac agc cta agc ttt act tca cgc tat cac gag aat tcg ttg gag cta 240

Tyr Ser Leu Ser Phe Thr Ser Arg Tyr His Glu Asn Ser Leu Glu Leu

65 70 75 80

ggt gac gac cgt atg cgt cgc ctc cag ctg gcc gcg cgc cgc aac aaa 288

Gly Asp Asp Arg Met Arg Arg Leu Gln Leu Ala Ala Arg Arg Asn Lys

85 90 95

atc gca ctc gtc atg ggc tat tcg gag cgg gaa gcc gga tcg cgc tat 336

Ile Ala Leu Val Met Gly Tyr Ser Glu Arg Glu Ala Gly Ser Arg Tyr

100 105 110

ctg agc cag gtg ttc atc gac gag cgt ggc gag atc gtt gcc aat cgg 384

Leu Ser Gln Val Phe Ile Asp Glu Arg Gly GluIle Val Ala Asn Arg

115 120 125

cgc aag ctg aag ccc aca cac gtt gag cgt acg atc tac ggc gaa ggc 432

Arg Lys Leu Lys Pro Thr His Val Glu Arg Thr Ile Tyr Gly Glu Gly

130 135 140

aac gga acc gat ttc ctc acg cac gac ttc gcg ttc gga cgc gtc ggt 480

Asn Gly Thr Asp Phe Leu Thr His Asp Phe Ala Phe Gly Arg Val Gly

145 150 155 160

gga ttg aac tgc tgg gaa cat aaa caa ccg ctc agc aag ttc atg atg 528

Gly Leu Asn Cys Trp Glu His Lys Gln Pro Leu Ser Lys Phe Met Met

165 170 175

tac agc ctc ggt gag cag gtc cac gtt gca tcg tgg ccg gcg atg tcc 576

Tyr Ser Leu Gly Glu Gln Val His Val Ala Ser Trp Pro Ala Met Ser

180 185 190

cct ctt cag ccg gat gtt ttc caa ctg agc atc gaa gcc aac gcg acg 624

Pro Leu Gln Pro Asp Val Phe Gln Leu Ser Ile Glu Ala Asn Ala Thr

195 200 205

gtc acc cgc tcg tac gca atc gaa ggc caa acc ttt gtg ctt tgc tcg 672

Val Thr Arg Ser Tyr Ala Ile Glu Gly Gln Thr Phe Val Leu Cys Ser

210 215 220

acg cag gtg atc gga cct agc gcg atc gaa acg ttc tgc ctc aac gac 720

Thr Gln Val Ile Gly Pro Ser Ala Ile Glu Thr Phe Cys Leu Asn Asp

225 230 235 240

gaa cag cgc gca ctg ttg ccg caa gga tgt ggc tgg gcg cgc att tac 768

Glu Gln Arg Ala Leu Leu Pro Gln Gly Cys Gly Trp Ala Arg Ile Tyr

245 250 255

ggc ccg gat gga agc gag ctt gcg aag cct ctg gcg gaa gat gct gag 816

Gly Pro Asp Gly Ser Glu Leu Ala Lys Pro Leu Ala Glu Asp Ala Glu

260 265 270

ggg atc ttg tac gca gag atc gat ctg gag cag att ctg ctg gcg aag 864

Gly Ile Leu Tyr Ala Glu Ile Asp Leu Glu Gln Ile Leu Leu Ala Lys

275 280 285

gct gga gcc gat ccg gtc ggg cac tat tcg cgg cct gac gtg ctg tcg 912

Ala Gly Ala Asp Pro Val Gly His Tyr Ser Arg Pro Asp Val Leu Ser

290 295 300

gtc cag ttc gac ccg cgc aat cat acg cca gtt cat cgc atc ggc att 960

Val Gln Phe Asp Pro Arg Asn His Thr Pro Val His Arg Ile Gly Ile

305 310 315 320

gac ggt cgc ttg gat gtg aat acc cgc agt cgc gtg gag aat ttc cga 1008

Asp Gly Arg Leu Asp Val Asn Thr Arg Ser Arg Val Glu Asn Phe Arg

325 330 335

ctg cga caa gcg gct gag cag gag cgt cag gca tcc aag cgg ctc gga 1056

Leu Arg Gln Ala Ala Glu Gln Glu Arg Gln Ala Ser Lys Arg Leu Gly

340 345 350

acg aaa ctc ttt gaa caa tcc ctt ctg gct gaa gaa ccg gtc cca gca 1104

Thr Lys Leu Phe Glu Gln Ser Leu Leu Ala Glu Glu Pro Val Pro Ala

355 360 365

aag tag 1110

Lys

<210>45

<211>369

<212>PRT

<213>敏捷食酸菌72W

<400>45

Met Val Ser Tyr Asn Ser Lys Phe Leu Ala Ala Thr Val Gln Ala Glu

1 5 10 15

Pro Val Trp Leu Asp Ala Asp Ala Thr Ile Asp Lys Ser Ile Gly Ile

20 25 30

Ile Glu Glu Ala Ala Gln Lys Gly Ala Ser Leu Ile Ala Phe Pro Glu

35 40 45

Val Phe Ile Pro Gly Tyr Pro Tyr Trp Ala Trp Leu Gly Asp Val Lys

50 55 60

Tyr Ser Leu Ser Phe Thr Ser Arg Tyr His Glu Asn Ser Leu Glu Leu

65 70 75 80

Gly Asp Asp Arg Met Arg Arg Leu Gln Leu Ala Ala Arg Arg Asn Lys

85 90 95

Ile Ala Leu Val Met Gly Tyr Ser Glu Arg Glu Ala Gly Ser Arg Tyr

100 105 110

Leu Ser Gln Val Phe Ile Asp Glu Arg Gly Glu Ile Val Ala Asn Arg

115 120 125

Arg Lys Leu Lys Pro Thr His Val Glu Arg Thr Ile Tyr Gly Glu Gly

130 135 140

Asn Gly Thr Asp Phe Leu Thr His Asp Phe Ala Phe Gly Arg Val Gly

145 150 155 160

Gly Leu Asn Cys Trp Glu His Lys Gln Pro Leu Ser Lys Phe Met Met

165 170 175

Tyr Ser Leu Gly Glu Gln Val His Val Ala Ser Trp Pro Ala Met Ser

180 185 190

Pro Leu Gln Pro Asp Val Phe Gln Leu Ser Ile Glu Ala Asn Ala Thr

195 200 205

Val Thr Arg Ser Tyr Ala Ile Glu Gly Gln Thr Phe Val Leu Cys Ser

210 215 220

Thr Gln Val Ile Gly Pro Ser Ala Ile Glu Thr Phe Cys Leu Asn Asp

225 230 235 240

Glu Gln Arg Ala Leu Leu Pro Gln Gly Cys Gly Trp Ala Arg Ile Tyr

245 250 255

Gly Pro Asp Gly Ser Glu Leu Ala Lys Pro Leu Ala Glu Asp Ala Glu

260 265 270

Gly Ile Leu Tyr Ala Glu Ile Asp Leu Glu Gln Ile Leu Leu Ala Lys

275 280 285

Ala Gly Ala Asp Pro Val Gly His Tyr Ser Arg Pro Asp Val Leu Ser

290 295 300

Val Gln Phe Asp Pro Arg Asn His Thr Pro Val His Arg Ile Gly Ile

305 310 315 320

Asp Gly Arg Leu Asp Val Asn Thr Arg Ser Arg Val Glu Asn Phe Arg

325 330 335

Leu Arg Gln Ala Ala Glu Gln Glu Arg Gln Ala Ser Lys Arg Leu Gly

340 345 350

Thr Lys Leu Phe Glu Gln Ser Leu Leu Ala Glu Glu Pro Val Pro Ala

355 360 365

Lys

<210>46

<211>1110

<212>DNA

<213>敏捷食酸菌72W

<220>

<221>CDS

<222>(1)..(1110)

<400>46

atg gtt tcg tat aac agc aag ttc ctc gcg gca acc gtt cag gca gag 48

Met Val Ser Tyr Asn Ser Lys Phe Leu Ala Ala Thr Val Gln Ala Glu

1 5 10 15

ccg gta tgg ctc gac gca gac gca acg atc gac aag tcg atc ggc atc 96

Pro Val Trp Leu Asp Ala Asp Ala Thr Ile Asp Lys Ser Ile Gly Ile

20 25 30

atc gaa gaa gct gcc caa aag ggc gcg agt ctg atc gct ttc ccg gaa 144

Ile Glu Glu Ala Ala Gln Lys Gly Ala Ser Leu Ile Ala Phe Pro Glu

35 40 45

gta ttc att ccg ggc tac ccc tat tgg gcg tgg ctc ggc gac gtg aag 192

Val Phe Ile Pro Gly Tyr Pro Tyr Trp Ala Trp Leu Gly Asp Val Lys

50 55 60

tac agc cta agc ttt act tca cgc tat cac gag aat tcg ttg gag cta 240

Tyr Ser Leu Ser Phe Thr Ser Arg Tyr His Glu Asn Ser Leu Glu Leu

65 70 75 80

ggt gac gac cgt atg cgt cgc ctc cag ctg gcc gcg cgc cgc aac aaa 288

GLy Asp Asp Arg Met Arg Arg Leu Gln Leu Ala Ala Arg Arg Asn Lys

85 90 95

atc gca ctc gtc atg ggc tat tcg gag cgg gaa gcc gga tcg cgc tat 336

Ile Ala Leu Val Met Gly Tyr Ser Glu Arg Glu Ala Gly Ser Arg Tyr

100 105 110

ctg agc cag gtg ttc atc gac gag cgt ggc gag atc gtt gcc aat cgg 384

Leu Ser Gln Val PheIle Asp Glu Arg Gly Glu Ile Val Ala Asn Arg

115 120 125

cgc aag ctg aag ccc aca cac gtt gag cgt acg atc tac ggc gaa ggc 432

Arg Lys Leu Lys Pro Thr His Val Glu Arg Thr Ile Tyr Gly Glu Gly

130 135 140

aac gga acc gat ttc ctc acg cac gac ttc gcg ttc gga cgc gtc ggt 480

Asn Gly Thr Asp Phe Leu Thr His Asp Phe Ala Phe Gly Arg Val Gly

145 150 155 160

gga ttg aac tgc tgg gaa cat atg caa ccg ctc agc aag ttc atg atg 528

Gly Leu Asn Cys Trp Glu His Met Gln Pro Leu Ser Lys Phe Met Met

165 170 175

tac agc ctc ggt gag cag gtc cac gtt gca tcg tgg ccg gcg atg tcc 576

Tyr Ser Leu Gly Glu Gln Val His Val Ala Ser Trp Pro Ala Met Ser

180 185 190

cct ctt cag ccg gat gtt ttc caa ctg agc atc gaa gcc aac gcg acg 624

Pro Leu Gln Pro Asp Val Phe Gln Leu Ser Ile Glu Ala Asn Ala Thr

195 200 205

gtc acc cgc tcg tac gca atc gaa ggc caa acc ttt gtg ctt tgc tcg 672

Val Thr Arg Ser Tyr Ala Ile Glu Gly Gln Thr Phe Val Leu Cys Ser

210 215 220

acg cag gtg atc gga cct agc gcg atc gaa acg ttc tgc ctc aac gac 720

Thr Gln Val Ile Gly Pro Ser Ala Ile Glu Thr Phe Cys Leu Asn Asp

225 230 235 240

gaa cag cgc gca ctg ttg ccg caa gga tgt ggc tgg gcg cgc att tac 768

Glu Gln Arg Ala Leu Leu Pro Gln Gly Cys Gly Trp Ala Arg Ile Tyr

245 250 255

ggc ccg gat gga agc gag ctt gcg aag cct ctg gcg gaa gat gct gag 816

Gly Pro Asp Gly Ser Glu Leu Ala Lys Pro Leu Ala Glu Asp Ala Glu

260 265 270

ggg atc ttg tac gca gag atc gat ctg gag cag att ctg ctg gcg aag 864

Gly Ile Leu Tyr Ala Glu Ile Asp Leu Glu Gln Ile Leu Leu Ala Lys

275 280 285

gct gga gcc gat ccg gtc ggg cac tat tcg cgg cct gac gtg ctg tcg 912

Ala Gly Ala Asp Pro Val Gly His Tyr Ser Arg Pro Asp Val Leu Ser

290 295 300

gtc cag ttc gac ccg cgc aat cat acg cca gtt cat cgc atc ggc att 960

Val Gln Phe Asp Pro Arg Asn His Thr Pro Val His Arg Ile Gly Ile

305 310 315 320

gac ggt cgc ttg gat gtg aat acc cgc agt cgc gtg gag aat ttc cga 1008

Asp Gly Arg Leu Asp Val Asn Thr Arg Ser Arg Val Glu Asn Phe Arg

325 330 335

ctg cga caa gcg gct gag cag gag cgt cag gca tcc aag cgg ctc gga 1056

Leu Arg Gln Ala Ala Glu Gln Glu Arg Gln Ala Ser Lys Arg Leu Gly

340 345 350

acg aaa ctc ttt gaa caa tcc ctt ctg gct gaa gaa ccg gtc cca gca 1104

Thr Lys Leu Phe Glu Gln Ser Leu Leu Ala Glu Glu Pro Val Pro Ala

355 360 365

aag tag 1110

Lys

<210>47

<211>369

<212>PRT

<213>敏捷食酸菌72W

<400>47

Met Val Ser Tyr Asn Ser Lys Phe Leu Ala Ala Thr Val Gln Ala Glu

1 5 10 15

Pro Val Trp Leu Asp Ala Asp Ala Thr Ile Asp Lys Ser Ile Gly Ile

20 25 30

Ile Glu Glu Ala Ala Gln Lys Gly Ala Ser Leu Ile Ala Phe Pro Glu

35 40 45

Val Phe Ile Pro Gly Tyr Pro Tyr Trp Ala Trp Leu Gly Asp Val Lys

50 55 60

Tyr Ser Leu Ser Phe Thr Ser Arg Tyr His Glu Asn Ser Leu Glu Leu

65 70 75 80

Gly Asp Asp Arg Met Arg Arg Leu Gln Leu Ala Ala Arg Arg Asn Lys

85 90 95

Ile Ala Leu Val Met Gly Tyr Ser Glu Arg Glu Ala Gly Ser Arg Tyr

100 105 110

Leu Ser Gln Val Phe Ile Asp Glu Arg Gly Glu Ile Val Ala Asn Arg

115 120 125

Arg Lys Leu Lys Pro Thr His Val Glu Arg Thr Ile Tyr Gly Glu Gly

130 135 140

Asn Gly Thr Asp Phe Leu Thr His Asp Phe Ala Phe Gly Arg Val Gly

145 150 155 160

Gly Leu Asn Cys Trp Glu His Met Gln Pro Leu Ser Lys Phe Met Met

165 170 175

Tyr Ser Leu Gly Glu Gln Val His Val Ala Ser Trp Pro Ala Met Ser

180 185 190

Pro Leu Gln Pro Asp Val Phe Gln Leu Ser Ile Glu Ala Asn Ala Thr

195 200 205

Val Thr Arg Ser Tyr Ala Ile Glu Gly Gln Thr Phe Val Leu Cys Ser

210 215 220

Thr Gln Val Ile Gly Pro Ser Ala Ile Glu Thr Phe Cys Leu Asn Asp

225 230 235 240

Glu Gln Arg Ala Leu Leu Pro Gln Gly Cys Gly Trp Ala Arg Ile Tyr

245 250 255

Gly Pro Asp Gly Ser Glu Leu Ala Lys Pro Leu Ala Glu Asp Ala Glu

260 265 270

Gly Ile Leu Tyr Ala Glu Ile Asp Leu Glu Gln Ile Leu Leu Ala Lys

275 280 285

Ala Gly Ala Asp Pro Val Gly His Tyr Ser Arg Pro Asp Val Leu Ser

290 295 300

Val Gln Phe Asp Pro Arg Asn His Thr Pro Val His Arg Ile Gly Ile

305 310 315 320

Asp Gly Arg Leu Asp Val Asn Thr Arg Ser Arg Val Glu Asn Phe Arg

325 330 335

Leu Arg Gln Ala Ala Glu Gln Glu Arg Gln Ala Ser Lys Arg Leu Gly

340 345 350

Thr Lys Leu Phe Glu Gln Ser Leu Leu Ala Glu Glu Pro Val Pro Ala

355 360 365

Lys

<210>48

<211>1110

<212>DNA

<213>敏捷食酸菌72W

<220>

<221>CDS

<222>(1)..(1110)

<400>48

atg gtt tcg tat aac agc aag ttc ctc gcg gca acc gtt cag gca gag 48

Met Val Ser Tyr Asn Ser Lys Phe Leu Ala Ala Thr Val Gln Ala Glu

1 5 10 15

ccg gta tgg ctc gac gca gac gca acg atc gac aag tcg atc ggc atc 96

Pro Val Trp Leu Asp Ala Asp Ala Thr Ile Asp Lys Ser Ile Gly Ile

20 25 30

atc gaa gaa gct gcc caa aag ggc gcg agt ctg atc gct ttc ccg gaa 144

Ile Glu Glu Ala Ala Gln Lys Gly Ala Ser Leu Ile Ala Phe Pro Glu

35 40 45

gta ttc att ccg ggc tac ccc tat tgg gcg tgg ctc ggc gac gtg aag 192

Val Phe Ile Pro Gly Tyr Pro Tyr Trp Ala Trp Leu Gly Asp Val Lys

50 55 60

tac agc cta agc ttt act tca cgc tat cac gag aat tcg ttg gag cta 240

Tyr Ser Leu Ser Phe Thr Ser Arg Tyr His Glu Asn Ser Leu Glu Leu

65 70 75 80

ggt gac gac cgt atg cgt cgc ctc cag ctg gcc gcg cgc cgc aac aaa 288

Gly Asp Asp Arg Met Arg Arg Leu Gln Leu Ala Ala Arg Arg Asn Lys

85 90 95

atc gca ctc gtc atg ggc tat tcg gag cgg gaa gcc gga tcg cgc tat 336

Ile Ala Leu Val Met Gly Tyr Ser Glu Arg Glu Ala Gly Ser Arg Tyr

100 105 110

ctg agc cag gtg ttc atc gac gag cgt ggc gag atc gtt gcc aat cgg 384

Leu Ser Gln Val Phe Ile Asp Glu Arg Gly Glu Ile Val Ala Asn Arg

115 120 125

cgc aag ctg aag ccc aca cac gtt gag cgt acg atc tac ggc gaa ggc 432

Arg Lys Leu Lys Pro Thr His Val G1u Arg Thr Ile Tyr G1y Glu G1y

130 135 140

aac gga acc gat ttc ctc acg cac gac ttc gcg ttc gga cgc gtc ggt 480

Asn Gly Thr Asp Phe Leu Thr His Asp Phe Ala Phe G1y Arg Val G1y

145 150 155 160

gga ttg aac tgc tgg gaa cat acc caa ccg ctc agc aag ttc atg atg 528

Gly Leu Asn Cys Trp Glu His Thr Gln Pro Leu Ser Lys Phe Met Met

165 170 175

tac agc ctc ggt gag cag gtc cac gtt gca tcg tgg ccg gcg atg tcc 576

Tyr Ser Leu Gly Glu Gln Val His Val Ala Ser Trp Pro Ala Met Ser

180 185 190

cct ctt cag ccg gat gtt ttc caa ctg agc atc gaa gcc aac gcg acg 624

Pro Leu Gln Pro Asp Val Phe Gln Leu Ser Ile Glu Ala Asn Ala Thr

195 200 205

gtc acc cgc tcg tac gca atc gaa ggc caa acc ttt gtg ctt tgc tcg 672

Val Thr Arg Ser Tyr Ala Ile Glu Gly Gln Thr Phe Val Leu Cys Ser

210 215 220

acg cag gtg atc gga cct agc gcg atc gaa acg ttc tgc ctc aac gac 720

Thr Gln Val Ile Gly Pro Ser Ala Ile Glu Thr Phe Cys Leu Asn Asp

225 230 235 240

gaa cag cgc gca ctg ttg ccg caa gga tgt ggc tgg gcg cgc att tac 768

Glu Gln Arg Ala Leu Leu Pro Gln Gly Cys Gly Trp Ala Arg Ile Tyr

245 250 255

ggc ccg gat gga agc gag ctt gcg aag cct ctg gcg gaa gat gct gag 816

Gly Pro Asp Gly Ser Glu Leu Ala Lys Pro Leu Ala Glu Asp Ala Glu

260 265 270

ggg atc ttg tac gca gag atc gat ctg gag cag att ctg ctg gcg aag 864

Gly Ile Leu Tyr Ala Glu Ile Asp Leu Glu Gln Ile Leu Leu Ala Lys

275 280 285

gct gga gcc gat ccg gtc ggg cac tat tcg cgg cct gac gtg ctg tcg 912

Ala Gly Ala Asp Pro Val Gly His Tyr Ser Arg Pro Asp Val Leu Ser

290 295 300

gtc cag ttc gac ccg cgc aat cat acg cca gtt cat cgc atc ggc att 960

Val Gln Phe Asp Pro Arg Asn His Thr Pro Val His Arg Ile Gly Ile

305 310 315 320

gac ggt cgc ttg gat gtg aat acc cgc agt cgc gtg gag aat ttc cga 1008

Asp Gly Arg Leu Asp Val Asn Thr Arg Ser Arg Val Glu Asn Phe Arg

325 330 335

ctg cga caa gcg gct gag cag gag cgt cag gca tcc aag cgg ctc gga 1056

Leu Arg Gln Ala Ala Glu Gln Glu Arg Gln Ala Ser Lys Arg Leu Gly

340 345 350

acg aaa ctc ttt gaa caa tcc ctt ctg gct gaa gaa ccg gtc cca gca 1104

Thr Lys Leu Phe Glu Gln Ser Leu Leu Ala Glu Glu Pro Val Pro Ala

355 360 365

aag tag 1110

Lys

<210>49

<211>369

<212>PRT

<213>敏捷食酸菌72W

<400>49

Met Val Ser Tyr Asn Ser Lys Phe Leu Ala Ala Thr Val Gln Ala Glu

1 5 10 15

Pro Val Trp Leu Asp Ala Asp Ala Thr Ile Asp Lys Ser Ile Gly Ile

20 25 30

Ile Glu Glu Ala Ala Gln Lys Gly Ala Ser Leu Ile Ala Phe Pro Glu

35 40 45

Val Phe Ile Pro Gly Tyr Pro Tyr Trp Ala Trp Leu Gly Asp Val Lys

50 55 60

Tyr Ser Leu Ser Phe Thr Ser Arg Tyr His Glu Asn Ser Leu Glu Leu

65 70 75 80

Gly Asp Asp Arg Met Arg Arg Leu Gln Leu Ala Ala Arg Arg Asn Lys

85 90 95

Ile Ala Leu Val Met Gly Tyr Ser Glu Arg Glu Ala Gly Ser Arg Tyr

100 105 110

Leu Ser Gln Val Phe Ile Asp Glu Arg Gly Glu Ile Val Ala Asn Arg

115 120 125

Arg Lys Leu Lys Pro Thr His Val Glu Arg Thr Ile Tyr Gly Glu Gly

130 135 140

Asn Gly Thr Asp Phe Leu Thr His Asp Phe Ala Phe Gly Arg Val Gly

145 150 155 160

Gly Leu Asn Cys Trp Glu His Thr Gln Pro Leu Ser Lys Phe Met Met

165 170 175

Tyr Ser Leu Gly Glu Gln Val His Val Ala Ser Trp Pro Ala Met Ser

180 185 190

Pro Leu Gln Pro Asp Val Phe Gln Leu Ser Ile Glu Ala Asn Ala Thr

195 200 205

Val Thr Arg Ser Tyr Ala Ile Glu Gly Gln Thr Phe Val Leu Cys Ser

210 215 220

Thr Gln Val Ile Gly Pro Ser Ala Ile Glu Thr Phe Cys Leu Asn Asp

225 230 235 240

Glu Gln Arg Ala Leu Leu Pro Gln Gly Cys Gly Trp Ala Arg Ile Tyr

245 250 255

Gly Pro Asp Gly Ser Glu Leu Ala Lys Pro Leu Ala Glu Asp Ala Glu

260 265 270

Gly Ile Leu Tyr Ala Glu Ile Asp Leu Glu Gln Ile Leu Leu Ala Lys

275 280 285

Ala Gly Ala Asp Pro Val Gly His Tyr Ser Arg Pro Asp Val Leu Ser

290 295 300

Val Gln Phe Asp Pro Arg Asn His Thr Pro Val His Arg Ile Gly Ile

305 310 315 320

Asp Gly Arg Leu Asp Val Asn Thr Arg Ser Arg Val Glu Asn Phe Arg

325 330 335

Leu Arg Gln Ala Ala Glu Gln Glu Arg Gln Ala Ser Lys Arg Leu Gly

340 345 350

Thr Lys Leu Phe Glu Gln Ser Leu Leu Ala Glu Glu Pro Val Pro Ala

355 360 365

Lys

<210>50

<211>1110

<212>DNA

<213>敏捷食酸菌72W

<220>

<221>CDS

<222>(1)..(1110)

<400>50

atg gtt tcg tat aac agc aag ttc ctc gcg gca acc gtt cag gca gag 48

Met Val Ser Tyr Asn Ser Lys Phe Leu Ala Ala Thr Val Gln Ala Glu

1 5 10 15

ccg gta tgg ctc gac gca gac gca acg atc gac aag tcg atc ggc atc 96

Pro Val Trp Leu Asp Ala Asp Ala Thr Ile Asp Lys Ser Ile Gly Ile

20 25 30

atc gaa gaa gct gcc caa aag ggc gcg agt ctg atc gct ttc ccg gaa 144

Ile Glu Glu Ala Ala Gln Lys Gly Ala Ser Leu Ile Ala Phe Pro Glu

35 40 45

gta ttc att ccg ggc tac ccc tat tgg gcg tgg ctc ggc gac gtg aag 192

Val Phe Ile Pro Gly Tyr Pro Tyr Trp Ala Trp Leu Gly Asp Val Lys

50 55 60

tac agc cta agc ttt act tca cgc tat cac gag aat tcg ttg gag cta 240

Tyr Ser Leu Ser Phe Thr Ser Arg Tyr His Glu Asn Ser Leu Glu Leu

65 70 75 80

ggt gac gac cgt atg cgt cgc ctc cag ctg gcc gcg cgc cgc aac aaa 288

Gly Asp Asp Arg Met Arg Arg Leu Gln Leu Ala Ala Arg Arg Asn Lys

85 90 95

atc gca ctc gtc atg ggc tat tcg gag cgg gaa gcc gga tcg cgc tat 336

Ile Ala Leu Val Met Gly Tyr Ser Glu Arg Glu Ala Gly Ser Arg Tyr

100 105 110

ctg agc cag gtg ttc atc gac gag cgt ggc gag atc gtt gcc aat cgg 384

Leu Ser Gln Val Phe Ile Asp Glu Arg Gly Glu Ile Val Ala Asn Arg

115 120 125

cgc aag ctg aag ccc aca cac gtt gag cgt acg atc tac ggc gaa ggc 432

Arg Lys Leu Lys Pro Thr His Val Glu Arg Thr Ile Tyr Gly Glu Gly

130 135 140

aac gga acc gat ttc ctc acg cac gac ttc gcg ttc gga cgc gtc ggt 480

Asn Gly Thr Asp Phe Leu Thr His Asp Phe Ala Phe Gly Arg Val Gly

145 150 155 160

gga ttg aac tgc tgg gaa cat gtg caa ccg ctc agc aag ttc atg atg 528

Gly Leu Asn Cys Trp Glu His Val Gln Pro Leu Ser Lys Phe Met Met

165 170 175

tac agc ctc ggt gag cag gtc cac gtt gca tcg tgg ccg gcg atg tcc 576

Tyr Ser Leu Gly Glu Gln Val His Val Ala Ser Trp Pro Ala Met Ser

180 185 190

cct ctt cag ccg gat gtt ttc caa ctg agc atc gaa gcc aac gcg acg 624

Pro Leu Gln Pro Asp Val Phe Gln Leu Ser Ile Glu Ala Asn Ala Thr

195 200 205

gtc acc cgc tcg tac gca atc gaa ggc caa acc ttt gtg ctt tgc tcg 672

Val Thr Arg Ser Tyr Ala Ile Glu Gly Gln Thr Phe Val Leu Cys Ser

210 215 220

acg cag gtg atc gga cct agc gcg atc gaa acg ttc tgc ctc aac gac 720

Thr Gln Val Ile Gly Pro Ser Ala Ile Glu Thr Phe Cys Leu Asn Asp

225 230 235 240

gaa cag cgc gca ctg ttg ccg caa gga tgt ggc tgg gcg cgc att tac 768

Glu Gln Arg Ala Leu Leu Pro Gln Gly Cys Gly Trp Ala Arg Ile Tyr

245 250 255

ggc ccg gat gga agc gag ctt gcg aag cct ctg gcg gaa gat gct gag 816

Gly Pro Asp Gly Ser Glu Leu Ala Lys Pro Leu Ala Glu Asp Ala Glu

260 265 270

ggg atc ttg tac gca gag atc gat ctg gag cag att ctg ctg gcg aag 864

Gly Ile Leu Tyr Ala Glu Ile Asp Leu Glu Gln Ile Leu Leu Ala Lys

275 280 285

gct gga gcc gat ccg gtc ggg cac tat tcg cgg cct gac gtg ctg tcg 912

Ala Gly Ala Asp Pro Val Gly His Tyr Ser Arg Pro Asp Val Leu Ser

290 295 300

gtc cag ttc gac ccg cgc aat cat acg cca gtt cat cgc atc ggc att 960

Val Gln Phe Asp Pro Arg Asn His Thr Pro Val His Arg Ile Gly Ile

305 310 315 320

gac ggt cgc ttg gat gtg aat acc cgc agt cgc gtg gag aat ttc cga 1008

Asp Gly Arg Leu Asp Val Asn Thr Arg Ser Arg Val Glu Asn Phe Arg

325 330 335

ctg cga caa gcg gct gag cag gag cgt cag gca tcc aag cgg ctc gga 1056

Leu Arg Gln Ala Ala Glu Gln Glu Arg Gln Ala Ser Lys Arg Leu Gly

340 345 350

acg aaa ctc ttt gaa caa tcc ctt ctg gct gaa gaa ccg gtc cca gca 1104

Thr Lys Leu Phe Glu Gln Ser Leu Leu Ala Glu Glu Pro Val Pro Ala

355 360 365

aag tag 1110

Lys

<210>51

<211>369

<212>PRT

<213>敏捷食酸菌72W

<400>51

Met Val Ser Tyr Asn Ser Lys Phe Leu Ala Ala Thr Val Gln Ala Glu

1 5 10 15

Pro Val Trp Leu Asp Ala Asp Ala Thr Ile Asp Lys Ser Ile Gly Ile

20 25 30

Ile Glu Glu Ala Ala Gln Lys Gly Ala Ser Leu Ile Ala Phe Pro Glu

35 40 45

Val Phe Ile Pro Gly Tyr Pro Tyr Trp Ala Trp Leu Gly Asp Val Lys

50 55 60

Tyr Ser Leu Ser Phe Thr Ser Arg Tyr His Glu Asn Ser Leu Glu Leu

65 70 75 80

Gly Asp Asp Arg Met Arg Arg Leu Gln Leu Ala Ala Arg Arg Asn Lys

85 90 95

Ile Ala Leu Val Met Gly Tyr Ser Glu Arg Glu Ala Gly Ser Arg Tyr

100 105 110

Leu Ser Gln Val Phe Ile Asp Glu Arg Gly Glu Ile Val Ala Asn Arg

115 120 125

Arg Lys Leu Lys Pro Thr His Val Glu Arg Thr Ile Tyr Gly Glu Gly

130 135 140

Asn Gly Thr Asp Phe Leu Thr His Asp Phe Ala Phe Gly Arg Val Gly

145 150 155 160

Gly Leu Asn Cys Trp Glu His Val Gln Pro Leu Ser Lys Phe Met Met

165 170 175

Tyr Ser Leu Gly Glu Gln Val His Val Ala Ser Trp Pro Ala Met Ser

180 185 190

Pro Leu Gln Pro Asp Val Phe Gln Leu Ser Ile Glu Ala Asn Ala Thr

195 200 205

Val Thr Arg Ser Tyr Ala Ile Glu Gly Gln Thr Phe Val Leu Cys Ser

210 215 220

Thr Gln Val Ile Gly Pro Ser Ala Ile Glu Thr Phe Cys Leu Asn Asp

225 230 235 240

Glu Gln Arg Ala Leu Leu Pro Gln Gly Cys Gly Trp Ala Arg Ile Tyr

245 250 255

Gly Pro Asp Gly Ser Glu Leu Ala Lys Pro Leu Ala Glu Asp Ala Glu

260 265 270

Gly Ile Leu Tyr Ala Glu Ile Asp Leu Glu Gln Ile Leu Leu Ala Lys

275 280 285

Ala Gly Ala Asp Pro Val Gly His Tyr Ser Arg Pro Asp Val Leu Ser

290 295 300

Val Gln Phe Asp Pro Arg Asn His Thr Pro Val His Arg Ile Gly Ile

305 310 315 320

Asp Gly Arg Leu Asp Val Asn Thr Arg Ser Arg Val Glu Asn Phe Arg

325 330 335

Leu Arg Gln Ala Ala Glu Gln Glu Arg Gln Ala Ser Lys Arg Leu Gly

340 345 350

Thr Lys Leu Phe Glu Gln Ser Leu Leu Ala Glu Glu Pro Val Pro Ala

355 360 365

Lys

<210>52

<211>1110

<212>DNA

<213>敏捷食酸菌72W

<220>

<221>CDS

<222>(1)..(1110)

<400>52

atg gtt tcg tat aac agc aag ttc ctc gcg gca acc gtt cag gca gag 48

Met Val Ser Tyr Asn Ser Lys Phe Leu Ala Ala Thr Val Gln Ala Glu

1 5 10 15

ccg gta tgg ctc gac gca gac gca acg atc gac aag tcg atc ggc atc 96

Pro Val Trp Leu Asp Ala Asp Ala Thr Ile Asp Lys Ser Ile Gly Ile

20 25 30

atc gaa gaa gct gcc caa aag ggc gcg agt ctg atc gct ttc ccg gaa 144

Ile Glu Glu Ala Ala Gln Lys Gly Ala Ser Leu Ile Ala Phe Pro Glu

35 40 45

gta ttc att ccg ggc tac ccc tat tgg gcg tgg ctc ggc gac gtg aag 192

Val Phe Ile Pro Gly Tyr Pro Tyr Trp Ala Trp Leu Gly Asp Val Lys

50 55 60

tac agc cta agc ttt act tca cgc tat cac gag aat tcg ttg gag cta 240

Tyr Ser Leu Ser Phe Thr Ser Arg Tyr His Glu Asn Ser Leu Glu Leu

65 70 75 80

ggt gac gac cgt atg cgt cgc ctc cag ctg gcc gcg cgc cgc aac aaa 288

Gly Asp Asp Arg Met Arg Arg Leu Gln Leu Ala Ala Arg Arg Asn Lys

85 90 95

atc gca ctc gtc atg ggc tat tcg gag cgg gaa gcc gga tcg cgc tat 336

Ile Ala Leu Val Met Gly Tyr Ser Glu Arg Glu Ala Gly Ser Arg Tyr

100 105 110

ctg agc cag gtg ttc atc gac gag cgt ggc gag atc gtt gcc aat cgg 384

Leu Ser Gln Val Phe Ile Asp Glu Arg Gly Glu Ile Val Ala Asn Arg

115 120 125

cgc aag ctg aag ccc aca cac gtt gag cgt acg atc tac ggc gaa ggc 432

Arg Lys Leu Lys Pro Thr His Val Glu Arg Thr Ile Tyr Gly Glu Gly

130 135 140

aac gga acc gat ttc ctc acg cac gac ttc gcg ttc gga cgc gtc ggt 480

Asn Gly Thr Asp Phe Leu Thr His Asp Phe Ala Phe Gly Arg Val Gly

145 150 155 160

gga ttg aac tgc tgg gaa cat ttc caa ccg ctc agc aag ttc atg atg 528

Gly Leu Asn Cys Trp Glu His Phe Gln Pro Leu Ser Lys Phe Met Met

165 170 175

tac agc ctc ggt gag cag gtc cac gtt gca tcg tgg ccg gcg atg tcc 576

Tyr Ser Leu Gly Glu Gln Val His Val Ala Ser Trp Pro Ala Met Ser

180 185 190

cct ctt cag ccg gat gtt ttc caa ctg agc atc gaa gcc aac gcg acg 624

Pro Leu Gln Pro Asp Val Phe Gln Leu Ser Ile Glu Ala Asn Ala Thr

195 200 205

gtc gcc cgc tcg tac gca atc gaa ggc caa acc ttt gtg ctt tgc tcg 672

Val Ala Arg Ser Tyr Ala Ile Glu Gly Gln Thr Phe Val Leu Cys Ser

210 215 220

acg cag gtg atc gga cct agc gcg atc gaa acg ttc tgc ctc aac gac 720

Thr Gln Val Ile Gly Pro Ser Ala Ile Glu Thr Phe Cys Leu Asn Asp

225 230 235 240

gaa cag cgc gca ctg ttg ccg caa gga tgt ggc tgg gcg cgc att tac 768

Glu Gln Arg Ala Leu Leu Pro Gln Gly Cys Gly Trp Ala Arg Ile Tyr

245 250 255

ggc ccg gat gga agc gag ctt gcg aag cct ctg gcg gaa gat gct gag 816

Gly Pro Asp Gly Ser Glu Leu Ala Lys Pro Leu Ala Glu Asp Ala Glu

260 265 270

ggg atc ttg tac gca gag atc gat ctg gag cag att ctg ctg gcg aag 864

Gly Ile Leu Tyr Ala Glu Ile Asp Leu Glu Gln Ile Leu Leu Ala Lys

275 280 285

gct gga gcc gat ccg gtc ggg cac tat tcg cgg cct gac gtg ctg tcg 912

Ala Gly Ala Asp Pro Val Gly His Tyr Ser Arg Pro Asp Val Leu Ser

290 295 300

gtc cag ttc gac ccg cgc aat cat acg cca gtt cat cgc atc ggc att 960

Val Gln Phe Asp Pro Arg Asn His Thr Pro Val His Arg Ile Gly Ile

305 310 315 320

gac ggt cgc ttg gat gtg aat acc cgc agt cgc gtg gag aat ttc cga 1008

Asp Gly Arg Leu Asp Val Asn Thr Arg Ser Arg Val Glu Asn Phe Arg

325 330 335

ctg cga caa gcg gct gag cag gag cgt cag gca tcc aag cgg ctc gga 1056

Leu Arg Gln Ala Ala Glu Gln Glu Arg Gln Ala Ser Lys Arg Leu Gly

340 345 350

acg aaa ctc ttt gaa caa tcc ctt ctg gct gaa gaa ccg gtc cca gca 1104

Thr Lys Leu Phe Glu Gln Ser Leu Leu Ala Glu Glu Pro Val Pro Ala

355 360 365

aag tag 1110

Lys

<210>53

<211>369

<212>PRT

<213>敏捷食酸菌72W

<400>53

Met Val Ser Tyr Asn Ser Lys Phe Leu Ala Ala Thr Val Gln Ala Glu

1 5 10 15

Pro Val Trp Leu Asp Ala Asp Ala Thr Ile Asp Lys Ser Ile Gly Ile

20 25 30

Ile Glu Glu Ala Ala Gln Lys Gly Ala Ser Leu Ile Ala Phe Pro Glu

35 40 45

Val Phe Ile Pro Gly Tyr Pro Tyr Trp Ala Trp Leu Gly Asp Val Lys

50 55 60

Tyr Ser Leu Ser Phe Thr Ser Arg Tyr His Glu Asn Ser Leu Glu Leu

65 70 75 80

Gly Asp Asp Arg Met Arg Arg Leu Gln Leu Ala Ala Arg Arg Asn Lys

85 90 95

Ile Ala Leu Val Met Gly Tyr Ser Glu Arg Glu Ala Gly Ser Arg Tyr

100 105 110

Leu Ser Gln Val Phe Ile Asp Glu Arg Gly Glu Ile Val Ala Asn Arg

115 120 125

Arg Lys Leu Lys Pro Thr His Val Glu Arg Thr Ile Tyr Gly Glu Gly

130 135 140

Asn Gly Thr Asp Phe Leu Thr His Asp Phe Ala Phe Gly Arg Val Gly

145 150 155 160

Gly Leu Asn Cys Trp Glu His Phe Gln Pro Leu Ser Lys Phe Met Met

165 170 175

Tyr Ser Leu Gly Glu Gln Val His Val Ala Ser Trp Pro Ala Met Ser

180 185 190

Pro Leu Gln Pro Asp Val Phe Gln Leu Ser Ile Glu Ala Asn Ala Thr

195 200 205

Val Ala Arg Ser Tyr Ala Ile Glu Gly Gln Thr Phe Val Leu Cys Ser

210 215 220

Thr Gln Val Ile Gly Pro Ser Ala Ile Glu Thr Phe Cys Leu Asn Asp

225 230 235 240

Glu Gln Arg Ala Leu Leu Pro Gln Gly Cys Gly Trp Ala Arg Ile Tyr

245 250 255

Gly Pro Asp Gly Ser Glu Leu Ala Lys Pro Leu Ala Glu Asp Ala Glu

260 265 270

Gly Ile Leu Tyr Ala Glu Ile Asp Leu Glu Gln Ile Leu Leu Ala Lys

275 280 285

Ala Gly Ala Asp Pro Val Gly His Tyr Ser Arg Pro Asp Val Leu Ser

290 295 300

Val Gln Phe Asp Pro Arg Asn His Thr Pro Val His Arg Ile Gly Ile

305 310 315 320

Asp Gly Arg Leu Asp Val Asn Thr Arg Ser Arg Val Glu Asn Phe Arg

325 330 335

Leu Arg Gln Ala Ala Glu Gln Glu Arg Gln Ala Ser Lys Arg Leu Gly

340 345 350

Thr Lys Leu Phe Glu Gln Ser Leu Leu Ala Glu Glu Pro Val Pro Ala

355 360 365

Lys

<210>54

<211>1110

<212>DNA

<213>敏捷食酸菌72W

<220>

<221>CDS

<222>(1)..(1110)

<400>54

atg gtt tcg tat aac agc aag ttc ctc gcg gca acc gtt cag gca gag 48

Met Val Ser Tyr Asn Ser Lys Phe Leu Ala Ala Thr Val Gln Ala Glu

1 5 10 15

ccg gta tgg ctc gac gca gac gca acg atc gac aag tcg atc ggc atc 96

Pro Val Trp Leu Asp Ala Asp Ala Thr Ile Asp Lys Ser Ile Gly Ile

20 25 30

atc gaa gaa gct gcc caa aag ggc gcg agt ctg atc gct ttc ccg gaa 144

Ile Glu Glu Ala Ala Gln Lys Gly Ala Ser Leu Ile Ala Phe Pro Glu

35 40 45

gta ttc att ccg ggc tac ccc tat tgg gcg tgg ctc ggc gac gtg aag 192

Val Phe Ile Pro Gly Tyr Pro Tyr Trp Ala Trp Leu Gly Asp Val Lys

50 55 60

tac agc cta agc ttt act tca cgc tat cac gag aat tcg ttg gag cta 240

Tyr Ser Leu Ser Phe Thr Ser Arg Tyr His Glu Asn Ser Leu Glu Leu

65 70 75 80

ggt gac gac cgt atg cgt cgc ctc cag ctg gcc gcg cgc cgc aac aaa 288

Gly Asp Asp Arg Met Arg Arg Leu Gln Leu Ala Ala Arg Arg Asn Lys

85 90 95

atc gca ctc gtc atg ggc tat tcg gag cgg gaa gcc gga tcg cgc tat 336

Ile Ala Leu Val Met Gly Tyr Ser Glu Arg Glu Ala Gly Ser Arg Tyr

100 105 110

ctg agc cag gtg ttc atc gac gag cgt ggc gag atc gtt gcc aat cgg 384

Leu Ser Gln Val Phe Ile Asp Glu Arg Gly Glu Ile Val Ala Asn Arg

115 120 125

cgc aag ctg aag ccc aca cac gtt gag cgt acg atc tac ggc gaa ggc 432

Arg Lys Leu Lys Pro Thr His Val Glu Arg Thr Ile Tyr Gly Glu Gly

130 135 140

aac gga acc gat ttc ctc acg cac gac ttc gcg ttc gga cgc gtc ggt 480

Asn Gly Thr Asp Phe Leu Thr His Asp Phe Ala Phe Gly Arg Val Gly

145 150 155 160

gga ttg aac tgc tgg gaa cat ttc caa ccg ctc agc aag ttc atg atg 528

Gly Leu Asn Cys Trp Glu His Phe Gln Pro Leu Ser Lys Phe Met Met

165 170 175

tac agc ctc ggt gag cag gtc cac gtt gca tcg tgg ccg gcg atg tcc 576

Tyr Ser Leu Gly Glu Gln Val His Val Ala Ser Trp Pro Ala Met Ser

180 185 190

cct ctt cag ccg gat gtt ttc caa ctg agc atc gaa gcc aac gcg acg 624

Pro Leu Gln Pro Asp Val Phe Gln Leu Ser Ile Glu Ala Asn Ala Thr

195 200 205

gtc tgc cgc tcg tac gca atc gaa ggc caa acc ttt gtg ctt tgc tcg 672

Val Cys Arg Ser Tyr Ala Ile Glu Gly Gln Thr Phe Val Leu Cys Ser

210 215 220

acg cag gtg atc gga cct agc gcg atc gaa acg ttc tgc ctc aac gac 720

Thr Gln Val Ile Gly Pro Ser Ala Ile Glu Thr Phe Cys Leu Asn Asp

225 230 235 240

gaa cag cgc gca ctg ttg ccg caa gga tgt ggc tgg gcg cgc att tac 768

Glu Gln Arg Ala Leu Leu Pro Gln Gly Cys Gly Trp Ala Arg Ile Tyr

245 250 255

ggc ccg gat gga agc gag ctt gcg aag cct ctg gcg gaa gat gct gag 816

Gly Pro Asp Gly Ser Glu Leu Ala Lys Pro Leu Ala Glu Asp Ala Glu

260 265 270

ggg atc ttg tac gca gag atc gat ctg gag cag att ctg ctg gcg aag 864

Gly Ile Leu Tyr Ala Glu Ile Asp Leu Glu Gln Ile Leu Leu Ala Lys

275 280 285

gct gga gcc gat ccg gtc ggg cac tat tcg cgg cct gac gtg ctg tcg 912

Ala Gly Ala Asp Pro Val Gly His Tyr Ser Arg Pro Asp Val Leu Ser

290 295 300

gtc cag ttc gac ccg cgc aat cat acg cca gtt cat cgc atc ggc att 960

Val Gln Phe Asp Pro Arg Asn His Thr Pro Val His Arg Ile Gly Ile

305 310 315 320

gac ggt cgc ttg gat gtg aat acc cgc agt cgc gtg gag aat ttc cga 1008

Asp Gly Arg Leu Asp Val Asn Thr Arg Ser Arg Val Glu Asn Phe Arg

325 330 335

ctg cga caa gcg gct gag cag gag cgt cag gca tcc aag cgg ctc gga 1056

Leu Arg Gln Ala Ala Glu Gln Glu Arg Gln Ala Ser Lys Arg Leu Gly

340 345 350

acg aaa ctc ttt gaa caa tcc ctt ctg gct gaa gaa ccg gtc cca gca 1104

Thr Lys Leu Phe Glu Gln Ser Leu Leu Ala Glu Glu Pro Val Pro Ala

355 360 365

aag tag 1110

Lys

<210>55

<211>369

<212>PRT

<213>敏捷食酸菌72W

<400>55

Met Val Ser Tyr Asn Ser Lys Phe Leu Ala Ala Thr Val Gln Ala Glu

1 5 10 15

Pro Val Trp Leu Asp Ala Asp Ala Thr Ile Asp Lys Ser Ile Gly Ile

20 25 30

Ile Glu Glu Ala Ala Gln Lys Gly Ala Ser Leu Ile Ala Phe Pro Glu

35 40 45

Val Phe Ile Pro Gly Tyr Pro Tyr Trp Ala Trp Leu Gly Asp Val Lys

50 55 60

Tyr Ser Leu Ser Phe Thr Ser Arg Tyr His Glu Asn Ser Leu Glu Leu

65 70 75 80

Gly Asp Asp Arg Met Arg Arg Leu Gln Leu Ala Ala Arg Arg Asn Lys

85 90 95

Ile Ala Leu Val Met Gly Tyr Ser Glu Arg Glu Ala Gly Ser Arg Tyr

100 105 110

Leu Ser Gln Val Phe Ile Asp Glu Arg Gly Glu Ile Val Ala Asn Arg

115 120 125

Arg Lys Leu Lys Pro Thr His Val Glu Arg Thr Ile Tyr Gly Glu Gly

130 135 140

Asn Gly Thr Asp Phe Leu Thr His Asp Phe Ala Phe Gly Arg Val Gly

145 150 155 160

Gly Leu Asn Cys Trp Glu His Phe Gln Pro Leu Ser Lys Phe Met Met

165 170 175

Tyr Ser Leu Gly Glu Gln Val His Val Ala Ser Trp Pro Ala Met Ser

180 185 190

Pro Leu Gln Pro Asp Val Phe Gln Leu Ser Ile Glu Ala Asn Ala Thr

195 200 205

Val Cys Arg Ser Tyr Ala Ile Glu Gly Gln Thr Phe Val Leu Cys Ser

210 215 220

Thr Gln Val Ile Gly Pro Ser Ala Ile Glu Thr Phe Cys Leu Asn Asp

225 230 235 240

Glu Gln Arg Ala Leu Leu Pro Gln Gly Cys Gly Trp Ala Arg Ile Tyr

245 250 255

Gly Pro Asp Gly Ser Glu Leu Ala Lys Pro Leu Ala Glu Asp Ala Glu

260 265 270

Gly Ile Leu Tyr Ala Glu Ile Asp Leu Glu Gln Ile Leu Leu Ala Lys

275 280 285

Ala Gly Ala Asp Pro Val Gly His Tyr Ser Arg Pro Asp Val Leu Ser

290 295 300

Val Gln Phe Asp Pro Arg Asn His Thr Pro Val His Arg Ile Gly Ile

305 310 315 320

Asp Gly Arg Leu Asp Val Asn Thr Arg Ser Arg Val Glu Asn Phe Arg

325 330 335

Leu Arg Gln Ala Ala Glu Gln Glu Arg Gln Ala Ser Lys Arg Leu Gly

340 345 350

Thr Lys Leu Phe Glu Gln Ser Leu Leu Ala Glu Glu Pro Val Pro Ala

355 360 365

Lys

<210>56

<211>1110

<212>DNA

<213>大肠杆菌SS1001

<220>

<221>CDS

<222>(1)..(1110)

<400>56

atg gtt tcg tat aac agc aag ttc ctc gcg gca acc gtt cag gca gag 48

Met Val Ser Tyr Asn Ser Lys Phe Leu Ala Ala Thr Val Gln Ala Glu

1 5 10 15

ccg gta tgg ctc gac gca gac gca acg atc gac aag tcg atc ggc atc 96

Pro Val Trp Leu Asp Ala Asp Ala Thr Ile Asp Lys Ser Ile Gly Ile

20 25 30

atc gaa gaa gct gcc caa aag ggc gcg agt ctg atc gct ttc ccg gaa 144

Ile Glu Glu Ala Ala Gln Lys Gly Ala Ser Leu Ile Ala Phe Pro Glu

35 40 45

gta ttc att ccg ggc tac ccc tat tgg gcg tgg ctc ggc gac gtg aag 192

Val Phe Ile Pro Gly Tyr Pro Tyr Trp Ala Trp Leu Gly Asp Val Lys

50 55 60

tac agc cta agc ttt act tca cgc tat cac gag aat tcg ttg gag cta 240

Tyr Ser Leu Ser Phe Thr Ser Arg Tyr His Glu Asn Ser Leu Glu Leu

65 70 75 80

ggt gac gac cgt atg cgt cgc ctc cag ctg gcc gcg cgc cgc aac aaa 288

Gly Asp Asp Arg Met Arg Arg Leu Gln Leu Ala Ala Arg Arg Asn Lys

85 90 95

atc gca ctc gtc atg ggc tat tcg gag cgg gaa gcc gga tcg cgc tat 336

Ile Ala Leu Val Met Gly Tyr Ser Glu Arg Glu Ala Gly Ser Arg Tyr

100 105 110

ctg agc cag gtg ttc atc gac gag cgt ggc gag atc gtt gcc aat cgg 384

Leu Ser Gln Val Phe Ile Asp Glu Arg Gly Glu Ile Val Ala Asn Arg

115 120 125

cgc aag ctg aag ccc aca cac gtt gag cgt acg atc tac ggc gaa ggc 432

Arg Lys Leu Lys Pro Thr His Val Glu Arg Thr Ile Tyr Gly Glu Gly

130 135 140

aac gga acc gat ttc ctc acg cac gac ttc gcg ttc gga cgc gtc ggt 480

Asn Gly Thr Asp Phe Leu Thr His Asp Phe Ala Phe Gly Arg Val Gly

145 150 155 160

gga ttg aac tgc tgg gaa cat ttc caa ccg ctc agc aag ttc atg atg 528

Gly Leu Asn Cys Trp Glu His Phe Gln Pro Leu Ser Lys Phe Met Met

165 170 175

tac agc ctc ggt gag cag gtc cac gtt gca tcg tgg ccg gcg atg tcc 576

Tyr Ser Leu Gly Glu Gln Val His Val Ala Ser Trp Pro Ala Met Ser

180 185 190

cct ctt cag ccg gat gtt ttc caa ctg agc atc gaa gcc aac gcg acg 624

Pro Leu Gln Pro Asp Val Phe Gln Leu Ser Ile Glu Ala Asn Ala Thr

195 200 205

gtc acc cgc tcg tac gca atc gaa ggc caa acc ttt gtg ctt tgc tcg 672

Val Thr Arg Ser Tyr Ala Ile Glu Gly Gln Thr Phe Val Leu Cys Ser

210 215 220

acg cag gtg atc gga cct agc gcg atc gaa acg ttc tgc ctc aac gac 720

Thr Gln Val Ile Gly Pro Ser Ala Ile Glu Thr Phe Cys Leu Asn Asp

225 230 235 240

gaa cag cgc gca ctg ttg ccg caa gga tgt ggc tgg gcg cgc att tac 768

Glu Gln Arg Ala Leu Leu Pro Gln Gly Cys Gly Trp Ala Arg Ile Tyr

245 250 255

ggc ccg gat gga agc gag ctt gcg aag cct ctg gcg gaa gat gct gag 816

Gly Pro Asp Gly Ser Glu Leu Ala Lys Pro Leu Ala Glu Asp Ala Glu

260 265 270

ggg atc ttg tac gca gag atc gat ctg gag cag att ctg ctg gcg aag 864

Gly Ile Leu Tyr Ala Glu Ile Asp Leu Glu GlnIle Leu Leu Ala Lys

275 280 285

gct gga gcc gat ccg gtc ggg cac tat tcg cgg cct gac gtg ctg tcg 912

Ala Gly Ala Asp Pro Val Gly His Tyr Ser Arg Pro Asp Val Leu Ser

290 295 300

gtc cag ttc gac ccg cgc aat cat acg cca gtt cat cgc atc ggc att 960

Val Gln Phe Asp Pro Arg Asn His Thr Pro Val His Arg Ile Gly Ile

305 310 315 320

gac ggt cgc ttg gat gtg aat acc cgc agt cgc gtg gag aat ttc cga 1008

Asp Gly Arg Leu Asp Val Asn Thr Arg Ser Arg Val Glu Asn Phe Arg

325 330 335

ctg cga caa gcg gct gag cag gag cgt cag gca tcc aag cgg ctc gga 1056

Leu Arg Gln Ala Ala Glu Gln Glu Arg Gln Ala Ser Lys Arg Leu Gly

340 345 350

acg aaa ctc ttt gaa caa tcc ctt ctg gct gaa gaa ccg gtc tca gca 1104

Thr Lys Leu Phe Glu Gln Ser Leu Leu Ala Glu Glu Pro Val Ser Ala

355 360 365

aag tag 1110

Lys

<210>57

<211>369

<212>PRT

<213>大肠杆菌SS1001

<400>57

Met Val Ser Tyr Asn Ser Lys Phe Leu Ala Ala Thr Val Gln Ala Glu

1 5 10 15

Pro Val Trp Leu Asp Ala Asp Ala Thr Ile Asp Lys Ser Ile Gly Ile

20 25 30

Ile Glu Glu Ala Ala Gln Lys Gly Ala Ser Leu Ile Ala Phe Pro Glu

35 40 45

Val Phe Ile Pro Gly Tyr Pro Tyr Trp Ala Trp Leu Gly Asp Val Lys

50 55 60

Tyr Ser Leu Ser Phe Thr Ser Arg Tyr His Glu Asn Ser Leu Glu Leu

65 70 75 80

Gly Asp Asp Arg Met Arg Arg Leu Gln Leu Ala Ala Arg Arg Asn Lys

85 90 95

Ile Ala Leu Val Met Gly Tyr Ser Glu Arg Glu Ala Gly Ser Arg Tyr

100 105 110

Leu Ser Gln Val Phe Ile Asp Glu Arg Gly Glu lle Val Ala Asn Arg

115 120 125

Arg Lys Leu Lys Pro Thr His Val Glu Arg Thr Ile Tyr Gly Glu Gly

130 135 140

Asn Gly Thr Asp Phe Leu Thr His Asp Phe Ala Phe Gly Arg Val Gly

145 150 155 160

Gly Leu Asn Cys Trp Glu His Phe Gln Pro Leu Ser Lys Phe Met Met

165 170 175

Tyr Ser Leu Gly Glu Gln Val His Val Ala Ser Trp Pro Ala Met Ser

180 185 190

Pro Leu Gln Pro Asp Val Phe Gln Leu Ser Ile Glu Ala Asn Ala Thr

195 200 205

Val Thr Arg Ser Tyr Ala Ile Glu Gly Gln Thr Phe Val Leu Cys Ser

210 215 220

Thr Gln Val Ile Gly Pro Ser Ala Ile Glu Thr Phe Cys Leu Asn Asp

225 230 235 240

Glu Gln Arg Ala Leu Leu Pro Gln Gly Cys Gly Trp Ala Arg Ile Tyr

245 250 255

Gly Pro Asp Gly Ser Glu Leu Ala Lys Pro Leu Ala Glu Asp Ala Glu

260 265 270

Gly Ile Leu Tyr Ala Glu Ile Asp Leu Glu Gln Ile Leu Leu Ala Lys

275 280 285

Ala Gly Ala Asp Pro Val Gly His Tyr Ser Arg Pro Asp Val Leu Ser

290 295 300

Val Gln Phe Asp Pro Arg Asn His Thr Pro Val His Arg Ile Gly Ile

305 310 315 320

Asp Gly Arg Leu Asp Val Asn Thr Arg Ser Arg Val Glu Asn Phe Arg

325 330 335

Leu Arg Gln Ala Ala Glu Gln Glu Arg Gln Ala Ser Lys Arg Leu Gly

340 345 350

Thr Lys Leu Phe Glu Gln Ser Leu Leu Ala Glu Glu Pro Val Ser Ala

355 360 365

Lys

Claims

1.一种方法，用于改进乙醇腈转化为乙醇酸过程中具有腈水解酶活性的酶催化剂的初始比活性的保留，所述方法包括：

(a)通过发酵产生具有腈水解酶活性的酶催化剂；

(b)以戊二醛预处理所述酶催化剂；

(d)从(b)或(c)回收酶催化剂并将所述酶催化剂固定于角叉菜胶中；和

(e)以戊二醛和聚乙烯亚胺将(d)中产生的角叉菜胶-固定的酶催化剂进行交联，从而产生戊二醛预处理的固定的和交联的酶催化剂，并且其中所述戊二醛预处理的固定的和交联的酶催化剂在相同反应条件下与未经戊二醛预处理的固定的和交联的酶催化剂相比，在乙醇腈转化为乙醇酸过程中的初始比活性的保留得到改进。

2.根据权利要求1所述的方法，其中(a)的酶催化剂包含氨基酸序列标志基序SEQ ID NO：1或SEQ ID NO：2。

3.根据权利要求2所述的方法，其中所述酶催化剂包含具有选自SEQID NO：4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、27、29、31、33、35、37、39、41、43、45、47、49、51、53、55和57的氨基酸序列的多肽。

4.根据权利要求3所述的方法，其中所述酶催化剂是敏捷食酸菌72W。

5.根据权利要求1所述的方法，其中在以戊二醛进行预处理的过程中将pH保持在5.0至9.0。

6.根据权利要求1所述的方法，其中步骤(b)中以戊二醛进行预处理包括向步骤(a)产生的发酵液中加入大约3g/L(0.025g GA/OD₅₅₀)至大约5g/L(0.042g GA/OD₅₅₀)的戊二醛。

7.根据权利要求1所述的方法，其中步骤(b)中以戊二醛进行预处理包括以50mg/L/h至500mg/L/h的速率向步骤(a)产生的发酵液中加入戊二醛。

8.根据权利要求1所述的方法产生的戊二醛预处理的固定的和交联的酶催化剂。

9.根据权利要求8所述的酶催化剂，其中所述催化剂在至少4个连续批式循环反应之后，保留其初始比活性的至少大约70％。

10.一种将乙醇腈水解为乙醇酸的改进的方法，包括改进乙醇腈转化为乙醇酸过程中具有腈水解酶活性的固定的酶催化剂的初始比活性的保留，所述方法包括：

(a)通过发酵产生具有腈水解酶活性的酶催化剂；

(b)以戊二醛预处理所述酶催化剂；

(e)以戊二醛和聚乙烯亚胺将(d)中产生的角叉菜胶-固定的酶催化剂进行交联，从而产生交联的固定的酶催化剂；和

(f)在合适的反应条件下，在水溶液中将(e)的交联的固定的酶催化剂与乙醇腈接触，从而产生乙醇酸。

11.根据权利要求10所述的方法，还包括步骤(g)：回收步骤(f)中产生的乙醇酸。

12.根据权利要求10所述的方法，其中在固定的酶催化剂的每克干细胞重产生至少40g乙醇酸之后，所述交联的固定的酶催化剂保留其初始比活性的至少大约70％，并且与另一种产生乙醇酸的方法相比，所述方法具有改进的生产乙醇酸的生产力，所述另一种产生乙醇酸的方法使用包含未经过戊二醛预处理的具有腈水解酶活性的酶催化剂的交联的固定的酶催化剂。