CN112899261B

CN112899261B - 赖氨酸脱羧酶突变体、其编码基因及应用

Info

Publication number: CN112899261B
Application number: CN202110319193.7A
Authority: CN
Inventors: 张才达; 任丽梅; 高文杲; 闫利军; 李丽; 张建坤; 刘�东
Original assignee: Meibangmeihe Biotechnology Co ltd
Current assignee: Meibangmeihe Biotechnology Co ltd
Priority date: 2021-03-25
Filing date: 2021-03-25
Publication date: 2022-03-29
Anticipated expiration: 2041-03-25
Also published as: CN112899261A

Abstract

本发明公开了一种赖氨酸脱羧酶突变体、其编码基因及应用。所述突变体的氨基酸序列由SEQ：ID：NO:1所示序列经过一次或多次突变得来。包含其第69位(E69D)、170位(F170G)、180位(I180V)、648(M648K)位点中一个或多个突变位点。本发明还提供了包含赖氨酸脱羧酶突变体基因的重组质粒、重组菌株、酶的表达方法以及在1,5‑戊二胺酶法转化和尼龙56盐的制备中的应用。对比野生型赖氨酸脱羧酶，赖氨酸脱羧酶突变体的催化活性以及反应稳定性大大提高。利用本发明的赖氨酸脱羧酶，不仅底物残留极低，且能够直接液体回收重复使用，极大降低了生产成本。

Description

赖氨酸脱羧酶突变体、其编码基因及应用

技术领域

本发明涉及基因工程技术领域，具体地说是一种赖氨酸脱羧酶突变体、其编码基因及应用。

背景技术

L-赖氨酸脱羧酶(英文名称为Lysine Decarboxylase，简称LDC，酶分类号EC4.1.1.18)存在于各种微生物中，主要生物学功能是催化L-赖氨酸脱羧生成1,5-戊二胺。目前，已经分别从大肠杆菌(E.coli)、蜂房哈夫尼菌(Hafnia alvei)、反刍动物月形单胞菌(Selenomonas ruminatium)、鼠伤寒沙门氏菌(Salmonella typhimurium)等细菌中克隆出该酶基因。

1,5-戊二胺(1,5-pentanediamine)，尸胺，分子式为C₅H₁₄N₂，分子量为102.18。该物质易燃，有毒，具强刺激性。1,5-戊二胺具有各种各样的功能及用途。工业重要应用如下：作为一种重要的工业化工原料，是由可再生原料衍生得到的生物聚酰胺，广泛应用于各种聚酰胺产品。可以替代传统的化工方法生产的生物胺---已二胺，与二元酸进行聚合反应可合成优质的高分子材料--新型尼龙56。在农业上，可用于调控植物衰老过程、促进雌雄蕊的发育、改善植物果实发育、提高果实产量。在医学上，作为一种有效治疗痢疾的药物，也是一种重要的药物中间体。

迄今为止，工业上二胺类物质主要由石油基原材料经二羧酸中间体或者通过氨基酸的化学脱羧作用进行化学生产。1,5-戊二胺的化学合成法采用以不可再生的战略石油作为原料，环境污染严重，无法实现可持续发展。利用生物法转化可再生资源来生产1,5-戊二胺，具有污染小，环境友好，可持续发展等优点，预计会在缓解能源危机和遏制全球气候变暖等方面做出重要贡献。生物法早期工业生产工艺主要为生物发酵法，约10年前，生物法主要采取谷氨酸棒杆菌代谢通路改造或者大肠杆菌基因组改造的方法生产1,5-戊二胺，产量一般都在10g/L以下，谷棒系统主流，全细胞催化主流。例如美国专利报道：采用棒状杆菌，染色体重组基因，产量约为5g/L。后期改造：谷棒杆菌分泌型，基因组改造，产量可达7.5g/L。韩国也有报道，大肠杆菌代谢改造：9.61g/L。国内报道普遍低于3.5g/L。随着生物技术的发展，人类可以利用外源过量表达的赖氨酸脱羧酶催化底物赖氨酸脱羧，得到1,5-戊二胺。以下专利中对此进行了描述：CN 108795916B、CN 111748549A、CN 109652356 A、CN110004131A等现阶段主要为酶法催化为主，特点为底物浓度高可见200-450g/L等。

生物催化反应主要缺点为生物酶在体外工业环境下的稳定性差以及酶制剂的成本过高等缺陷，极大的限制了生物催化在工业领域的实际应用。近几年来，生物催化主要采用L-赖氨酸脱羧酶酶已知为大肠杆菌L-赖氨酸降解酶。其基因的核苷酸序列(被称为CadA)和这个基因编码的氨基酸序列已经公开(Meng S.1992)，结构也已经被解析。这条序列也是各个研究学者主要使用的出发序列。大肠杆菌赖氨酸脱羧酶CadA是二聚体的结构，在行使功能的时候则是一个十聚体的结构，而当pH逐渐上升的时候，CadA会自动发生一个解聚的过程，变为二聚体的结构，此时，CadA的催化活性极剧下降。因此选用合适的方法体外改造酶基因的序列，并通过合适的酶活性检测方法，来获得具有预期表型的突变是研究的重点。例如CN109652356A公开，进化后的LDC原始菌酶活力3.93倍，在30℃-50℃条件下，无机酸控制pH 6.0-8.0，转化3h，戊二胺含量最高达到326.3g/L，赖氨酸盐酸盐摩尔转化率达到99.98％，戊二胺生产效率得到极大的提高。但是仍有稳定性不足，如何重复利用等问题存在。目前研究学者们也在LDC的重复利用(固定化)、提高稳定性上面有持续的研究改进，力图最大程度提升该酶的各种性能，但仍有明显不足。

综上所述，本发明通过一种Klebsiella pneumoniae物种来源的、新型的L-赖氨酸脱羧酶作为初始序列，获得提升赖氨酸脱羧酶的活性、稳定性的突变体序列。可以减少催化剂的用量或缩短反应时间，进而降低生产成本，简化了1,5-戊二胺溶液和酶的分离步骤，完成了1,5-戊二胺、尼龙56盐的新工艺制备技术，推进了生物法生产1,5-戊二胺、尼龙56盐的产业化进程。因此本领域急需提升赖氨酸脱羧酶的性能，实现1,5-戊二胺的低成本工业化生产。

发明内容

本发明的目的就是提供一种赖氨酸脱羧酶突变体、其编码基因及应用，以解决现有赖氨酸脱羧酶在活性、稳定性等方面性能不理想的问题。

本发明的目的是通过以下技术方案实现的：一种赖氨酸脱羧酶突变体，其为突变体3，所述突变体3是在氨基酸序列如SEQ：ID：NO:1所示的赖氨酸脱羧酶的基础上，将其第170位的Phe突变为Gly、第648位的Met突变为Lys。

所述的赖氨酸脱羧酶突变体为突变体4、突变体5或突变体6，其中，所述突变体4在突变体3的基础上，将其第69位的Glu突变为Asp；所述突变体5是在突变体3的基础上，将其第180位的Ile突变为Val；所述突变体6是在突变体3的基础上，将其第69位的Glu突变为Asp、第180位的Ile突变为Val。

编码赖氨酸脱羧酶突变体3的基因，所述基因的序列如SEQ：ID：NO:4所示。

编码突变体4的基因序列如SEQ：ID：NO:6所示；编码突变体5的基因序列如SEQ：ID：NO:8所示；编码突变体6的基因序列如SEQ：ID：NO:10所示。

一种重组质粒，其包含有上述的任一编码突变体的基因，所述重组质粒选用pET系列载体。

优选地，所述重组质粒选用pET22b载体。

一种重组菌株，其包含上述的重组质粒，所述重组菌株为大肠杆菌。

一种表达赖氨酸脱羧酶突变体的方法，包括如下步骤：将上述的重组质粒转化至大肠杆菌菌株中，得到重组菌株；将重组菌株活化后接种于LB培养基或发酵培养基中，待菌体OD600值达到0.6-30时，加入IPTG诱导表达赖氨酸脱羧酶突变体；所述IPTG在培养基中终浓度为0.01-1mM，所述诱导条件为16-37℃，诱导时间为4-48h。

上述的赖氨酸脱羧酶突变体在合成1,5-戊二胺、尼龙56盐中的应用。

进一步地，通过固定化酶技术，将所述突变体制成固定化突变酶并应用于合成1,5-戊二胺、尼龙56盐中。

固定化酶的制备为：准备发酵纯化后的赖氨酸脱羧酶酶液，酶液浓度为100-1000mg/L。按照每克树脂投入100-500mg蛋白情况下固定化，固定化条件参考不同树脂(氨基树脂，环氧树脂，大孔树脂，离子交换树脂)适宜的条件，完成固定化。环氧树脂在200-600mM磷酸盐存在条件下常温缓慢搅拌24-48h，20mM磷酸盐缓冲液轻柔洗去表面吸附的酶。布氏漏斗抽滤制备固定化酶。

本发明提供了野生型赖氨酸脱羧酶的氨基酸序列SEQ：ID：NO:1，以及其密码子优化后核苷酸序列SEQ：ID：NO:2。三种赖氨酸脱羧酶突变体，分别为突变体1(E69D)、突变体2(I180V)、突变体3(F170G+M648K)，共计4个突变位置。三个突变体氨基酸序列均由SEQ：ID：NO:1突变而来，其中，突变体3的氨基酸序列如SEQ：ID：NO:3所示，其核苷酸序列如SEQ：ID：NO:4所示。

本发明进一步提供了赖氨酸脱羧酶突变体4、突变体5和突变体6，分别对应E69D+F170G+M648K、F170G+I180V+M648K、E69D+F170G+I180V+M648K突变组合。其氨基酸序列由突变体1-3中突变位置组合而来，其中，突变体4的氨基酸序列如SEQ：ID：NO:5所示，其核苷酸序列如SEQ：ID：NO:6所示；突变体5的氨基酸序列如SEQ：ID：NO:7所示，其核苷酸序列如SEQ：ID：NO:8所示；突变体6的氨基酸序列如SEQ：ID：NO:9所示，其核苷酸序列如SEQ：ID：NO:10所示。赖氨酸脱羧酶突变体突变位置说明见表1和图1所示。

表1 野生型与突变体

种类	获得方法	位置	名称
				野生型	---------	--------------------	LDC-W
突变体1	非理性设计	I180V	LDC-1
				突变体2	非理性设计	E69D	LDC-2
突变体3	非理性设计	F170G+M648K	LDC-3
				突变体4	理性设计	E69D+F170G+M648K	LDC-4
突变体5	理性设计	F170G+I180V+M648K	LDC-5
				突变体6	理性设计	E69D+F170G+I180V+M648K	LDC-6

本发明提供了一种高活性、高稳定性、耐碱性、不经过固定化可以回收再利用等全面提升的L-赖氨酸脱羧酶，从而大大提高了工业1,5戊二胺、尼龙56盐(1,5戊二胺己二酸盐)的生产效率。

本发明可以减少催化剂的用量或缩短反应时间，进而降低生产成本，简化了1,5-戊二胺溶液和酶的分离步骤，完成了1,5-戊二胺、尼龙56盐的新工艺制备技术，推进了生物法生产1,5-戊二胺、尼龙56盐的产业化进程。

附图说明

图1是赖氨酸脱羧酶突变位置示意图。

图2是尼龙56盐反应前(显示L-赖氨酸)色谱图。

图3是尼龙56盐反应后(显示1,5-戊二胺)色谱图。

具体实施方式

下面实施例用于进一步详细说明本发明，但不以任何形式限制本发明。

实施例1野生型赖氨酸脱羧酶片段获得

全基因合成Klebsiella pneumoniae物种LDC序列，密码子优化，两端设计引物，引入酶切位点进行PCR，将得到的片段克隆至载体pET22b，得到重组质粒LDC-W。两端引物如表2所示，PCR反应体系如表3所示。

表2：

表3：

PCR反应条件：(1)95℃预变性5min，(2)95℃变性15s，57℃退火15s，72℃延伸2min，30个循环，(3)72℃延伸10min。随后经限制性内切酶EcoR I消化后，CIP(NEB)处理后将PCR扩增产物连接入大肠杆菌表达载体pET-22b中相应的位点，酶切和连接的条件分别参见表4和表5选择方向正确连接产物转化入E.coli BL21(DE3)获得菌株，送测序公司进行测序，测序成功的质粒为LDC-W。

表4：酶切体系

酶切条件为37℃，60min。

表5：连接体系

连接条件为：25℃，30min。

实施例2 重组质粒LDC-1、LDC-2、LDC-3以及对应菌株的获得

利用LDC-W质粒进行易错PCR体系扩增。PCR引物同实施例1，为LDC-WF以及LDC-WR。易错PCR采用易错PCR试剂盒(货号BTN1010055)进行，后续操作步骤同实施例1。

通过相同条件下活力筛选方法获得高活性，高稳定性菌株，提取质粒送测序公司测序。根据近百株菌株测活结果，活性上升稳定性升高突变体共获得6个，选择显著变化序列命名为突变体1-3，测序获得4个突变位点(表1)，分别为第69位(E69D)、170位(F170G)、180位(I180V)、648(M648K)，对应活性结果以及稳定性结果参见表7，可见筛选出的突变体活性明显提升，尤其突变体3，可达野生型的4.02倍。

实施例3 重组质粒LDC-4、LDC-5、LDC-6以及对应菌株的获得

以实施例2制备的重组质粒LDC-3为模板，利用PCR反应获得定点突变质粒LDC-4、LDC-5、LDC-6，具体引物应用参见表6，突变位置设计参见表1。

PCR反应条件：(1)95℃预变性5min，(2)95℃变性15s，57℃退火15s，72℃延伸7.5min，18个循环，(3)72℃延伸10min。

结束后取PCR原液10μL，并加入1μL Dpn 1(NEB)，37℃反应2h，特异性切除甲基化DNA链。测序成功的质粒分别命名为LDC-4、LDC-5、LDC-6，对应活性结果以及稳定性结果参见表7。可见突变体4-6活力以及稳定性进一步提升，尤其突变体5，可以达到野生型的7.43倍。

表6：

实施例4 赖氨酸脱羧酶以及突变体的小量表达

将含L-赖氨酸脱羧酶及其突变体重组质粒分别各自转化至大肠杆菌BL21(DE3)中，得到重组菌株中，接种于LB培养基中，待菌体OD600值达到0.6时，加入适宜浓度(0.1mM)IPTG诱导表达赖氨酸脱羧酶及其突变体，诱导条件为16-37℃。诱导时间为20h以内。摇瓶水平收集菌体量为5g/L。菌体经破碎制备成粗酶液。

实施例5 赖氨酸脱羧酶以及突变体的大量培养

将含L-赖氨酸脱羧酶及其突变体重组质粒分别各自转化至大肠杆菌BL21(DE3)中，得到重组菌株中，接种于LB培养基中活化后接终于大肠杆菌发酵培养基(胰蛋白胨8g/L、酵母提取物3.2g/L、NaCl 1.6g/L、无水Na₂HPO₄ 0.95g/L、无水KH₂PO₄ 1.6g/L、甘油62.5g/L、Amp 100mg/L根据监控补加碳源与氨水，pH 7.2)，待菌体OD600值达到25时，加入适宜浓度(1mM)IPTG诱导表达赖氨酸脱羧酶及其突变体，诱导条件为16-37℃，诱导培养时间为48h以内。进行若干批次实验，在发酵补料条件下，菌体浓度在100-200g/L，平均菌体浓度为150g/L。菌体经破碎制备成粗酶液。

实施例6 赖氨酸脱羧酶活性评价和稳定性评价方法

活性测定：利用摇瓶制备野生型以及突变体1-6粗酶液，将等量赖氨酸脱羧酶及各种突变型赖氨酸脱羧酶(5mg/mL)加入到3mL PBS缓冲液中(100mM赖氨酸，1mM磷酸吡哆醛)，37℃反应30min。离子色谱法测定1,5-戊二胺生成量以及底物L-赖氨酸的减少(如图2和图3所示)，计算评估野生型和四种突变型酶活性。以野生型序列为100％，评估其各个突变体的活性变化。

温度稳定性测试：将等量赖氨酸脱羧酶及各种突变型赖氨酸脱羧酶(5mg/mL)在不同温度下(37℃，50℃，60℃，65℃)放置0.5h，恢复室温后，用上述方法测定酶活性。结果显示，37℃-65℃升温过程中，活力逐渐下降，到65℃保温处理0.5h时，野生型活力基本消失。而突变体4和5明显提升耐高温酶活性，到65℃保温处理0.5h时仍剩余20％部分酶活。

表7：不同突变体温度耐受性变化

实施例7 赖氨酸脱羧酶突变前后耐碱性对比

按照实施例6活性测定方法，分别在pH 6.0、pH 8.0、pH 9.0、pH 10.0条件下，40℃，220rpm震荡保存24h后反应测定5种处理酶活力，结果显示，在pH 6.0时，5种酶活力差异不大，在pH 8.0时野生型酶活力显著降低，而突变型5和6，酶活力基本保持不变，特别地，突变型5在pH 10.0时仍保持有较高酶活力(参见表8)，剩余50％左右。

表8：不同突变体的不同pH耐受性

实施例8 1,5-戊二胺的制备与底物残留测定

测定不同L-赖氨酸脱羧酶的对L-赖氨酸的反应能力，采用100mL反应体系，测试底物L-赖氨酸的浓度在200-450g/L变化，pH为6.0，温度为42℃，加入相同浓度的L-赖氨酸脱羧酶以及突变体(100mg/L)。搅拌反应，反应5h检测1,5-戊二胺的生成量，计算转化率(表9)，根据测试结果得到反应完全时各组对应的底物浓度范围，由表9可知，突变体4-6可以更好的完成各项反应，尤其是突变体5，即使在底物浓度较大时也能反应完全。

表9：酶促反应指标

实施例9 固定化酶制备

准备发酵纯化后的赖氨酸脱羧酶酶液，酶液浓度为800mg/L。按照每克环氧树脂投入400mg蛋白进行固定化，环氧树脂在300mM磷酸盐存在条件下常温缓慢搅拌36h，20mM磷酸盐缓冲液轻柔洗去表面吸附的酶，布氏漏斗抽滤制得固定化酶。

实施例10 尼龙56盐制备-1

准备赖氨酸质量分数为30％的溶液，缓慢加入己二酸调节溶液pH至7.0，加入制备好的赖氨酸脱羧酶(600mg/L突变体6固定化酶)，1.5％PLP，40℃，搅拌反应，取样监测反应速度，反应3h后，沉降收集固定化酶重复使用。该固定化酶可重复使用50-200次。滤液进一步收集，树脂纯化，脱水浓缩，结晶罐结晶。二次结晶后，尼龙56盐摩尔收率达80％。

实施例11 尼龙56盐制备-2

准备赖氨酸质量分数为25％的溶液，缓慢加入己二酸调节溶液pH至6.0，加入制备好的赖氨酸脱羧酶液酶(200mg/L突变体5液酶)，2％PLP，37℃，搅拌反应，取样监测反应速度，反应4h后，超滤收集酶液重复使用。该酶液可重复使用10-50次。滤液进一步收集，树脂纯化，脱水浓缩，结晶罐结晶。二次结晶后，尼龙56盐摩尔收率达85％。

序列表

<120> 赖氨酸脱羧酶突变体、其编码基因及应用

<160> 10

<170> SIPOSequenceListing 1.0

<210> 1

<211> 715

<212> PRT

<213> 肺炎克雷白杆菌(Klebsiella pneumoniae)

<400> 1

Met Asn Val Ile Ala Ile Met Asn His Met Gly Val Tyr Phe Lys Glu

1 5 10 15

Glu Pro Ile Arg Glu Leu His Arg Ala Leu Glu Arg Leu Asp Phe Arg

20 25 30

Ile Val Tyr Pro Asn Asp Arg Asp Asp Leu Leu Lys Leu Ile Glu Asn

35 40 45

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

50 55 60

Glu Leu Cys Glu Glu Ile Ser Lys Met Asn Glu Tyr Met Pro Leu Tyr

65 70 75 80

Ala Phe Ala Asn Thr Tyr Ser Thr Leu Asp Val Ser Leu Asn Asp Leu

85 90 95

Arg Met Gln Val Arg Phe Phe Glu Tyr Ala Leu Gly Ala Ala Glu Asp

100 105 110

Ile Ala Asn Lys Ile Lys Gln Asn Thr Asp Glu Tyr Ile Asp Thr Ile

115 120 125

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

130 135 140

Tyr Thr Phe Cys Thr Pro Gly His Met Gly Gly Thr Ala Phe Gln Lys

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Asn Ala Glu Arg Ser Tyr Met Val Thr Asn Gly Thr Ser Thr Ala Asn

210 215 220

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

225 230 235 240

Asp Arg Asn Cys His Lys Ser Leu Thr His Leu Met Met Met Ser Asp

245 250 255

Ile Thr Pro Ile Tyr Phe Arg Pro Thr Arg Asn Ala Tyr Gly Ile Leu

260 265 270

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

275 280 285

Val Lys Glu Thr Pro Asn Ala Thr Trp Pro Val His Ala Val Ile Thr

290 295 300

Asn Ser Thr Tyr Asp Gly Leu Leu Tyr Asn Thr Asp Phe Ile Lys Lys

305 310 315 320

Thr Leu Asp Val Lys Ser Ile His Phe Asp Ser Ala Trp Val Pro Tyr

325 330 335

Thr Asn Phe Ser Pro Ile Tyr Glu Gly Lys Cys Gly Met Ser Gly Gly

340 345 350

Arg Val Glu Gly Lys Val Ile Tyr Glu Thr Gln Ser Thr His Lys Leu

355 360 365

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

370 375 380

Asn Glu Glu Thr Phe Asn Glu Ala Tyr Met Met His Thr Thr Thr Ser

385 390 395 400

Pro His Tyr Gly Ile Val Ala Ser Thr Glu Thr Ala Ala Ala Met Met

405 410 415

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

420 425 430

Ile Lys Phe Arg Lys Glu Ile Lys Arg Leu Lys Gly Glu Ser Asp Gly

435 440 445

Trp Phe Phe Asp Val Trp Gln Pro Glu His Ile Asp Gly Pro Glu Cys

450 455 460

Trp Pro Leu Arg Ser Asp Ser Ala Trp His Gly Phe Lys Asn Ile Asp

465 470 475 480

Asn Glu His Met Tyr Leu Asp Pro Ile Lys Val Thr Leu Leu Thr Pro

485 490 495

Gly Met Lys Lys Asp Gly Thr Met Asp Asp Phe Gly Ile Pro Ala Ser

500 505 510

Ile Val Ala Lys Tyr Leu Asp Glu His Gly Ile Val Val Glu Lys Thr

515 520 525

Gly Pro Tyr Asn Leu Leu Phe Leu Phe Ser Ile Gly Ile Asp Lys Thr

530 535 540

Lys Ala Leu Ser Leu Leu Arg Ala Leu Thr Asp Phe Lys Arg Ala Phe

545 550 555 560

Asp Leu Asn Leu Arg Val Lys Asn Met Leu Pro Ser Leu Tyr Arg Glu

565 570 575

Asp Pro Glu Phe Tyr Glu Asn Met Arg Ile Gln Asp Leu Ala Gln Asn

580 585 590

Ile His Lys Leu Ile Glu His His Asn Leu Pro Asp Leu Met Phe Arg

595 600 605

Ala Phe Glu Val Leu Pro Ser Met Val Met Thr Pro Tyr Ala Ala Phe

610 615 620

Gln Lys Glu Leu His Gly Gln Thr Glu Glu Val Tyr Leu Glu Glu Met

625 630 635 640

Val Gly Arg Val Asn Ala Asn Met Ile Leu Pro Tyr Pro Pro Gly Val

645 650 655

Pro Leu Val Met Pro Gly Glu Met Ile Thr Glu Glu Ser Arg Pro Val

660 665 670

Leu Glu Phe Leu Gln Met Leu Cys Glu Ile Gly Ala His Tyr Pro Gly

675 680 685

Phe Glu Thr Asp Ile His Gly Ala Tyr Arg Gln Ala Asp Gly Arg Tyr

690 695 700

Thr Val Lys Val Leu Lys Glu Glu Asn Asn Lys

705 710 715

<210> 2

<211> 2148

<212> DNA

<213> 肺炎克雷白杆菌(Klebsiella pneumoniae)

<400> 2

atgaatgtga tcgcaatcat gaaccacatg ggtgtttact tcaaagagga accgatccgt 60

gaactgcacc gcgctctgga acgtctggat ttccgcatcg tgtaccctaa cgaccgtgat 120

gatctgctga aactgatcga aaacaactct cgtctgtgtg gcgttatctt cgattgggat 180

aaatacaacc tggaactgtg tgaagagatt tccaaaatga acgaatacat gccgctgtac 240

gcgttcgcca acacctacag caccctggat gtgagcctga acgacctgcg catgcaggta 300

cgtttttttg aatacgcact gggtgccgcg gaagacattg ctaacaaaat caaacagaac 360

accgacgaat acatcgatac catcctgcca ccgctgacca aggcgctgtt caagtacgtg 420

cgcgagggca aatacacctt ctgtacgccg ggtcacatgg gcggtactgc gtttcaaaaa 480

agcccggtgg gtagcatctt ctatgatttc ttcggcagca acacgatgaa atctgatatc 540

agcatctccg tatccgagct gggctctctg ctggatcatt ctggtccaca caaagaagcg 600

gaagaataca ttgcccgtgt atttaacgca gaacgttcct atatggtcac gaacggtacc 660

agcactgcga acaaaatcgt tggtatgtat tctgccccgg ccggttctac tgtcctgatc 720

gatcgtaact gccataaaag cctgactcac ctgatgatga tgagcgacat caccccgatc 780

tattttcgtc cgactcgcaa cgcgtacggc atcctgggtg gcattccgca gtctgagttc 840

cagcacgcaa ccatcgcgaa acgtgttaaa gaaaccccga acgctacctg gccggttcat 900

gcagtgatca ccaactctac ttacgatggc ctgctgtaca acaccgactt catcaaaaaa 960

accctggatg taaagtctat ccacttcgat tccgcgtggg taccgtatac taacttctct 1020

ccgatttatg agggcaaatg tggtatgtct ggtggtcgtg tagaaggtaa agtgatctac 1080

gaaactcagt ccacccacaa actgctggcg gctttcagcc aggcgtctat gatccacgtg 1140

aaaggcgacg taaatgaaga gaccttcaac gaagcgtaca tgatgcacac caccacctcc 1200

cctcattatg gtatcgtggc ttctaccgaa accgcggcgg cgatgatgaa aggtaatgct 1260

ggcaaacgtc tgattgacgg ttccatcgag cgttctatca aattccgtaa agaaatcaag 1320

cgtctgaagg gcgaatccga cggctggttc ttcgacgtat ggcagccgga gcacattgat 1380

ggcccagaat gttggcctct gcgttctgac agcgcttggc acggtttcaa aaatattgac 1440

aacgaacata tgtatctgga tccgatcaaa gtaacgctgc tgactccggg tatgaaaaag 1500

gatggcacca tggatgactt cggtattccg gcttccatcg ttgcaaaata cctggacgag 1560

catggcatcg tggtggaaaa aaccggcccg tacaatctgc tgttcctgtt cagcatcggc 1620

atcgataaaa ccaaagcact gtccctgctg cgtgcactga ccgactttaa acgtgctttt 1680

gatctgaacc tgcgtgtgaa aaacatgctg ccgtccctgt accgcgaaga tccggagttc 1740

tatgagaaca tgcgcattca ggacctggcg caaaacatcc acaaactgat tgagcaccac 1800

aacctgccgg atctgatgtt ccgcgcgttc gaagtactgc ctagcatggt catgactccg 1860

tatgctgcgt tccagaaaga gctgcacggc cagaccgagg aagtgtacct ggaagaaatg 1920

gtgggccgtg tcaatgcaaa catgatcctg ccgtatccgc caggtgttcc gctggttatg 1980

ccgggtgaaa tgattactga agaatctcgt ccggttctgg aatttctgca gatgctgtgc 2040

gaaatcggcg ctcactatcc gggctttgaa accgacatcc atggcgcata ccgtcaggct 2100

gatggtcgtt acaccgtaaa agtcctgaaa gaagaaaata acaaatga 2148

<210> 3

<211> 715

<212> PRT

<213> 人工序列(Artificial Sequence)

<400> 3

Met Asn Val Ile Ala Ile Met Asn His Met Gly Val Tyr Phe Lys Glu

1 5 10 15

Glu Pro Ile Arg Glu Leu His Arg Ala Leu Glu Arg Leu Asp Phe Arg

20 25 30

Ile Val Tyr Pro Asn Asp Arg Asp Asp Leu Leu Lys Leu Ile Glu Asn

35 40 45

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

50 55 60

Glu Leu Cys Glu Glu Ile Ser Lys Met Asn Glu Tyr Met Pro Leu Tyr

65 70 75 80

Ala Phe Ala Asn Thr Tyr Ser Thr Leu Asp Val Ser Leu Asn Asp Leu

85 90 95

Arg Met Gln Val Arg Phe Phe Glu Tyr Ala Leu Gly Ala Ala Glu Asp

100 105 110

Ile Ala Asn Lys Ile Lys Gln Asn Thr Asp Glu Tyr Ile Asp Thr Ile

115 120 125

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

130 135 140

Tyr Thr Phe Cys Thr Pro Gly His Met Gly Gly Thr Ala Phe Gln Lys

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Asn Ala Glu Arg Ser Tyr Met Val Thr Asn Gly Thr Ser Thr Ala Asn

210 215 220

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

225 230 235 240

Asp Arg Asn Cys His Lys Ser Leu Thr His Leu Met Met Met Ser Asp

245 250 255

Ile Thr Pro Ile Tyr Phe Arg Pro Thr Arg Asn Ala Tyr Gly Ile Leu

260 265 270

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

275 280 285

Val Lys Glu Thr Pro Asn Ala Thr Trp Pro Val His Ala Val Ile Thr

290 295 300

Asn Ser Thr Tyr Asp Gly Leu Leu Tyr Asn Thr Asp Phe Ile Lys Lys

305 310 315 320

Thr Leu Asp Val Lys Ser Ile His Phe Asp Ser Ala Trp Val Pro Tyr

325 330 335

Thr Asn Phe Ser Pro Ile Tyr Glu Gly Lys Cys Gly Met Ser Gly Gly

340 345 350

Arg Val Glu Gly Lys Val Ile Tyr Glu Thr Gln Ser Thr His Lys Leu

355 360 365

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

370 375 380

Asn Glu Glu Thr Phe Asn Glu Ala Tyr Met Met His Thr Thr Thr Ser

385 390 395 400

Pro His Tyr Gly Ile Val Ala Ser Thr Glu Thr Ala Ala Ala Met Met

405 410 415

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

420 425 430

Ile Lys Phe Arg Lys Glu Ile Lys Arg Leu Lys Gly Glu Ser Asp Gly

435 440 445

Trp Phe Phe Asp Val Trp Gln Pro Glu His Ile Asp Gly Pro Glu Cys

450 455 460

Trp Pro Leu Arg Ser Asp Ser Ala Trp His Gly Phe Lys Asn Ile Asp

465 470 475 480

Asn Glu His Met Tyr Leu Asp Pro Ile Lys Val Thr Leu Leu Thr Pro

485 490 495

Gly Met Lys Lys Asp Gly Thr Met Asp Asp Phe Gly Ile Pro Ala Ser

500 505 510

Ile Val Ala Lys Tyr Leu Asp Glu His Gly Ile Val Val Glu Lys Thr

515 520 525

Gly Pro Tyr Asn Leu Leu Phe Leu Phe Ser Ile Gly Ile Asp Lys Thr

530 535 540

Lys Ala Leu Ser Leu Leu Arg Ala Leu Thr Asp Phe Lys Arg Ala Phe

545 550 555 560

Asp Leu Asn Leu Arg Val Lys Asn Met Leu Pro Ser Leu Tyr Arg Glu

565 570 575

Asp Pro Glu Phe Tyr Glu Asn Met Arg Ile Gln Asp Leu Ala Gln Asn

580 585 590

Ile His Lys Leu Ile Glu His His Asn Leu Pro Asp Leu Met Phe Arg

595 600 605

Ala Phe Glu Val Leu Pro Ser Met Val Met Thr Pro Tyr Ala Ala Phe

610 615 620

Gln Lys Glu Leu His Gly Gln Thr Glu Glu Val Tyr Leu Glu Glu Met

625 630 635 640

Val Gly Arg Val Asn Ala Asn Lys Ile Leu Pro Tyr Pro Pro Gly Val

645 650 655

Pro Leu Val Met Pro Gly Glu Met Ile Thr Glu Glu Ser Arg Pro Val

660 665 670

Leu Glu Phe Leu Gln Met Leu Cys Glu Ile Gly Ala His Tyr Pro Gly

675 680 685

Phe Glu Thr Asp Ile His Gly Ala Tyr Arg Gln Ala Asp Gly Arg Tyr

690 695 700

Thr Val Lys Val Leu Lys Glu Glu Asn Asn Lys

705 710 715

<210> 4

<211> 2148

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 4

atgaatgtga tcgcaatcat gaaccacatg ggtgtttact tcaaagagga accgatccgt 60

gaactgcacc gcgctctgga acgtctggat ttccgcatcg tgtaccctaa cgaccgtgat 120

gatctgctga aactgatcga aaacaactct cgtctgtgtg gcgttatctt cgattgggat 180

aaatacaacc tggaactgtg tgaagagatt tccaaaatga acgaatacat gccgctgtac 240

gcgttcgcca acacctacag caccctggat gtgagcctga acgacctgcg catgcaggta 300

cgtttttttg aatacgcact gggtgccgcg gaagacattg ctaacaaaat caaacagaac 360

accgacgaat acatcgatac catcctgcca ccgctgacca aggcgctgtt caagtacgtg 420

cgcgagggca aatacacctt ctgtacgccg ggtcacatgg gcggtactgc gtttcaaaaa 480

agcccggtgg gtagcatctt ctatgatggc ttcggcagca acacgatgaa atctgatatc 540

agcatctccg tatccgagct gggctctctg ctggatcatt ctggtccaca caaagaagcg 600

gaagaataca ttgcccgtgt atttaacgca gaacgttcct atatggtcac gaacggtacc 660

agcactgcga acaaaatcgt tggtatgtat tctgccccgg ccggttctac tgtcctgatc 720

gatcgtaact gccataaaag cctgactcac ctgatgatga tgagcgacat caccccgatc 780

tattttcgtc cgactcgcaa cgcgtacggc atcctgggtg gcattccgca gtctgagttc 840

cagcacgcaa ccatcgcgaa acgtgttaaa gaaaccccga acgctacctg gccggttcat 900

gcagtgatca ccaactctac ttacgatggc ctgctgtaca acaccgactt catcaaaaaa 960

accctggatg taaagtctat ccacttcgat tccgcgtggg taccgtatac taacttctct 1020

ccgatttatg agggcaaatg tggtatgtct ggtggtcgtg tagaaggtaa agtgatctac 1080

gaaactcagt ccacccacaa actgctggcg gctttcagcc aggcgtctat gatccacgtg 1140

aaaggcgacg taaatgaaga gaccttcaac gaagcgtaca tgatgcacac caccacctcc 1200

cctcattatg gtatcgtggc ttctaccgaa accgcggcgg cgatgatgaa aggtaatgct 1260

ggcaaacgtc tgattgacgg ttccatcgag cgttctatca aattccgtaa agaaatcaag 1320

cgtctgaagg gcgaatccga cggctggttc ttcgacgtat ggcagccgga gcacattgat 1380

ggcccagaat gttggcctct gcgttctgac agcgcttggc acggtttcaa aaatattgac 1440

aacgaacata tgtatctgga tccgatcaaa gtaacgctgc tgactccggg tatgaaaaag 1500

gatggcacca tggatgactt cggtattccg gcttccatcg ttgcaaaata cctggacgag 1560

catggcatcg tggtggaaaa aaccggcccg tacaatctgc tgttcctgtt cagcatcggc 1620

atcgataaaa ccaaagcact gtccctgctg cgtgcactga ccgactttaa acgtgctttt 1680

gatctgaacc tgcgtgtgaa aaacatgctg ccgtccctgt accgcgaaga tccggagttc 1740

tatgagaaca tgcgcattca ggacctggcg caaaacatcc acaaactgat tgagcaccac 1800

aacctgccgg atctgatgtt ccgcgcgttc gaagtactgc ctagcatggt catgactccg 1860

tatgctgcgt tccagaaaga gctgcacggc cagaccgagg aagtgtacct ggaagaaatg 1920

gtgggccgtg tcaatgcaaa caagatcctg ccgtatccgc caggtgttcc gctggttatg 1980

ccgggtgaaa tgattactga agaatctcgt ccggttctgg aatttctgca gatgctgtgc 2040

gaaatcggcg ctcactatcc gggctttgaa accgacatcc atggcgcata ccgtcaggct 2100

gatggtcgtt acaccgtaaa agtcctgaaa gaagaaaata acaaatga 2148

<210> 5

<211> 715

<212> PRT

<213> 人工序列(Artificial Sequence)

<400> 5

Met Asn Val Ile Ala Ile Met Asn His Met Gly Val Tyr Phe Lys Glu

1 5 10 15

Glu Pro Ile Arg Glu Leu His Arg Ala Leu Glu Arg Leu Asp Phe Arg

20 25 30

Ile Val Tyr Pro Asn Asp Arg Asp Asp Leu Leu Lys Leu Ile Glu Asn

35 40 45

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

50 55 60

Glu Leu Cys Glu Asp Ile Ser Lys Met Asn Glu Tyr Met Pro Leu Tyr

65 70 75 80

Ala Phe Ala Asn Thr Tyr Ser Thr Leu Asp Val Ser Leu Asn Asp Leu

85 90 95

Arg Met Gln Val Arg Phe Phe Glu Tyr Ala Leu Gly Ala Ala Glu Asp

100 105 110

Ile Ala Asn Lys Ile Lys Gln Asn Thr Asp Glu Tyr Ile Asp Thr Ile

115 120 125

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

130 135 140

Tyr Thr Phe Cys Thr Pro Gly His Met Gly Gly Thr Ala Phe Gln Lys

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Asn Ala Glu Arg Ser Tyr Met Val Thr Asn Gly Thr Ser Thr Ala Asn

210 215 220

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

225 230 235 240

Asp Arg Asn Cys His Lys Ser Leu Thr His Leu Met Met Met Ser Asp

245 250 255

Ile Thr Pro Ile Tyr Phe Arg Pro Thr Arg Asn Ala Tyr Gly Ile Leu

260 265 270

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

275 280 285

Val Lys Glu Thr Pro Asn Ala Thr Trp Pro Val His Ala Val Ile Thr

290 295 300

Asn Ser Thr Tyr Asp Gly Leu Leu Tyr Asn Thr Asp Phe Ile Lys Lys

305 310 315 320

Thr Leu Asp Val Lys Ser Ile His Phe Asp Ser Ala Trp Val Pro Tyr

325 330 335

Thr Asn Phe Ser Pro Ile Tyr Glu Gly Lys Cys Gly Met Ser Gly Gly

340 345 350

Arg Val Glu Gly Lys Val Ile Tyr Glu Thr Gln Ser Thr His Lys Leu

355 360 365

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

370 375 380

Asn Glu Glu Thr Phe Asn Glu Ala Tyr Met Met His Thr Thr Thr Ser

385 390 395 400

Pro His Tyr Gly Ile Val Ala Ser Thr Glu Thr Ala Ala Ala Met Met

405 410 415

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

420 425 430

Ile Lys Phe Arg Lys Glu Ile Lys Arg Leu Lys Gly Glu Ser Asp Gly

435 440 445

Trp Phe Phe Asp Val Trp Gln Pro Glu His Ile Asp Gly Pro Glu Cys

450 455 460

Trp Pro Leu Arg Ser Asp Ser Ala Trp His Gly Phe Lys Asn Ile Asp

465 470 475 480

Asn Glu His Met Tyr Leu Asp Pro Ile Lys Val Thr Leu Leu Thr Pro

485 490 495

Gly Met Lys Lys Asp Gly Thr Met Asp Asp Phe Gly Ile Pro Ala Ser

500 505 510

Ile Val Ala Lys Tyr Leu Asp Glu His Gly Ile Val Val Glu Lys Thr

515 520 525

Gly Pro Tyr Asn Leu Leu Phe Leu Phe Ser Ile Gly Ile Asp Lys Thr

530 535 540

Lys Ala Leu Ser Leu Leu Arg Ala Leu Thr Asp Phe Lys Arg Ala Phe

545 550 555 560

Asp Leu Asn Leu Arg Val Lys Asn Met Leu Pro Ser Leu Tyr Arg Glu

565 570 575

Asp Pro Glu Phe Tyr Glu Asn Met Arg Ile Gln Asp Leu Ala Gln Asn

580 585 590

Ile His Lys Leu Ile Glu His His Asn Leu Pro Asp Leu Met Phe Arg

595 600 605

Ala Phe Glu Val Leu Pro Ser Met Val Met Thr Pro Tyr Ala Ala Phe

610 615 620

Gln Lys Glu Leu His Gly Gln Thr Glu Glu Val Tyr Leu Glu Glu Met

625 630 635 640

Val Gly Arg Val Asn Ala Asn Lys Ile Leu Pro Tyr Pro Pro Gly Val

645 650 655

Pro Leu Val Met Pro Gly Glu Met Ile Thr Glu Glu Ser Arg Pro Val

660 665 670

Leu Glu Phe Leu Gln Met Leu Cys Glu Ile Gly Ala His Tyr Pro Gly

675 680 685

Phe Glu Thr Asp Ile His Gly Ala Tyr Arg Gln Ala Asp Gly Arg Tyr

690 695 700

Thr Val Lys Val Leu Lys Glu Glu Asn Asn Lys

705 710 715

<210> 6

<211> 2148

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 6

atgaatgtga tcgcaatcat gaaccacatg ggtgtttact tcaaagagga accgatccgt 60

gaactgcacc gcgctctgga acgtctggat ttccgcatcg tgtaccctaa cgaccgtgat 120

gatctgctga aactgatcga aaacaactct cgtctgtgtg gcgttatctt cgattgggat 180

aaatacaacc tggaactgtg tgaagatatt tccaaaatga acgaatacat gccgctgtac 240

gcgttcgcca acacctacag caccctggat gtgagcctga acgacctgcg catgcaggta 300

cgtttttttg aatacgcact gggtgccgcg gaagacattg ctaacaaaat caaacagaac 360

accgacgaat acatcgatac catcctgcca ccgctgacca aggcgctgtt caagtacgtg 420

cgcgagggca aatacacctt ctgtacgccg ggtcacatgg gcggtactgc gtttcaaaaa 480

agcccggtgg gtagcatctt ctatgatggc ttcggcagca acacgatgaa atctgatatc 540

agcatctccg tatccgagct gggctctctg ctggatcatt ctggtccaca caaagaagcg 600

gaagaataca ttgcccgtgt atttaacgca gaacgttcct atatggtcac gaacggtacc 660

agcactgcga acaaaatcgt tggtatgtat tctgccccgg ccggttctac tgtcctgatc 720

gatcgtaact gccataaaag cctgactcac ctgatgatga tgagcgacat caccccgatc 780

tattttcgtc cgactcgcaa cgcgtacggc atcctgggtg gcattccgca gtctgagttc 840

cagcacgcaa ccatcgcgaa acgtgttaaa gaaaccccga acgctacctg gccggttcat 900

gcagtgatca ccaactctac ttacgatggc ctgctgtaca acaccgactt catcaaaaaa 960

accctggatg taaagtctat ccacttcgat tccgcgtggg taccgtatac taacttctct 1020

ccgatttatg agggcaaatg tggtatgtct ggtggtcgtg tagaaggtaa agtgatctac 1080

gaaactcagt ccacccacaa actgctggcg gctttcagcc aggcgtctat gatccacgtg 1140

aaaggcgacg taaatgaaga gaccttcaac gaagcgtaca tgatgcacac caccacctcc 1200

cctcattatg gtatcgtggc ttctaccgaa accgcggcgg cgatgatgaa aggtaatgct 1260

ggcaaacgtc tgattgacgg ttccatcgag cgttctatca aattccgtaa agaaatcaag 1320

cgtctgaagg gcgaatccga cggctggttc ttcgacgtat ggcagccgga gcacattgat 1380

ggcccagaat gttggcctct gcgttctgac agcgcttggc acggtttcaa aaatattgac 1440

aacgaacata tgtatctgga tccgatcaaa gtaacgctgc tgactccggg tatgaaaaag 1500

gatggcacca tggatgactt cggtattccg gcttccatcg ttgcaaaata cctggacgag 1560

catggcatcg tggtggaaaa aaccggcccg tacaatctgc tgttcctgtt cagcatcggc 1620

atcgataaaa ccaaagcact gtccctgctg cgtgcactga ccgactttaa acgtgctttt 1680

gatctgaacc tgcgtgtgaa aaacatgctg ccgtccctgt accgcgaaga tccggagttc 1740

tatgagaaca tgcgcattca ggacctggcg caaaacatcc acaaactgat tgagcaccac 1800

aacctgccgg atctgatgtt ccgcgcgttc gaagtactgc ctagcatggt catgactccg 1860

tatgctgcgt tccagaaaga gctgcacggc cagaccgagg aagtgtacct ggaagaaatg 1920

gtgggccgtg tcaatgcaaa caagatcctg ccgtatccgc caggtgttcc gctggttatg 1980

ccgggtgaaa tgattactga agaatctcgt ccggttctgg aatttctgca gatgctgtgc 2040

gaaatcggcg ctcactatcc gggctttgaa accgacatcc atggcgcata ccgtcaggct 2100

gatggtcgtt acaccgtaaa agtcctgaaa gaagaaaata acaaatga 2148

<210> 7

<211> 715

<212> PRT

<213> 人工序列(Artificial Sequence)

<400> 7

Met Asn Val Ile Ala Ile Met Asn His Met Gly Val Tyr Phe Lys Glu

1 5 10 15

Glu Pro Ile Arg Glu Leu His Arg Ala Leu Glu Arg Leu Asp Phe Arg

20 25 30

Ile Val Tyr Pro Asn Asp Arg Asp Asp Leu Leu Lys Leu Ile Glu Asn

35 40 45

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

50 55 60

Glu Leu Cys Glu Glu Ile Ser Lys Met Asn Glu Tyr Met Pro Leu Tyr

65 70 75 80

Ala Phe Ala Asn Thr Tyr Ser Thr Leu Asp Val Ser Leu Asn Asp Leu

85 90 95

Arg Met Gln Val Arg Phe Phe Glu Tyr Ala Leu Gly Ala Ala Glu Asp

100 105 110

Ile Ala Asn Lys Ile Lys Gln Asn Thr Asp Glu Tyr Ile Asp Thr Ile

115 120 125

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

130 135 140

Tyr Thr Phe Cys Thr Pro Gly His Met Gly Gly Thr Ala Phe Gln Lys

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Asn Ala Glu Arg Ser Tyr Met Val Thr Asn Gly Thr Ser Thr Ala Asn

210 215 220

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

225 230 235 240

Asp Arg Asn Cys His Lys Ser Leu Thr His Leu Met Met Met Ser Asp

245 250 255

Ile Thr Pro Ile Tyr Phe Arg Pro Thr Arg Asn Ala Tyr Gly Ile Leu

260 265 270

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

275 280 285

Val Lys Glu Thr Pro Asn Ala Thr Trp Pro Val His Ala Val Ile Thr

290 295 300

Asn Ser Thr Tyr Asp Gly Leu Leu Tyr Asn Thr Asp Phe Ile Lys Lys

305 310 315 320

Thr Leu Asp Val Lys Ser Ile His Phe Asp Ser Ala Trp Val Pro Tyr

325 330 335

Thr Asn Phe Ser Pro Ile Tyr Glu Gly Lys Cys Gly Met Ser Gly Gly

340 345 350

Arg Val Glu Gly Lys Val Ile Tyr Glu Thr Gln Ser Thr His Lys Leu

355 360 365

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

370 375 380

Asn Glu Glu Thr Phe Asn Glu Ala Tyr Met Met His Thr Thr Thr Ser

385 390 395 400

Pro His Tyr Gly Ile Val Ala Ser Thr Glu Thr Ala Ala Ala Met Met

405 410 415

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

420 425 430

Ile Lys Phe Arg Lys Glu Ile Lys Arg Leu Lys Gly Glu Ser Asp Gly

435 440 445

Trp Phe Phe Asp Val Trp Gln Pro Glu His Ile Asp Gly Pro Glu Cys

450 455 460

Trp Pro Leu Arg Ser Asp Ser Ala Trp His Gly Phe Lys Asn Ile Asp

465 470 475 480

Asn Glu His Met Tyr Leu Asp Pro Ile Lys Val Thr Leu Leu Thr Pro

485 490 495

Gly Met Lys Lys Asp Gly Thr Met Asp Asp Phe Gly Ile Pro Ala Ser

500 505 510

Ile Val Ala Lys Tyr Leu Asp Glu His Gly Ile Val Val Glu Lys Thr

515 520 525

Gly Pro Tyr Asn Leu Leu Phe Leu Phe Ser Ile Gly Ile Asp Lys Thr

530 535 540

Lys Ala Leu Ser Leu Leu Arg Ala Leu Thr Asp Phe Lys Arg Ala Phe

545 550 555 560

Asp Leu Asn Leu Arg Val Lys Asn Met Leu Pro Ser Leu Tyr Arg Glu

565 570 575

Asp Pro Glu Phe Tyr Glu Asn Met Arg Ile Gln Asp Leu Ala Gln Asn

580 585 590

Ile His Lys Leu Ile Glu His His Asn Leu Pro Asp Leu Met Phe Arg

595 600 605

Ala Phe Glu Val Leu Pro Ser Met Val Met Thr Pro Tyr Ala Ala Phe

610 615 620

Gln Lys Glu Leu His Gly Gln Thr Glu Glu Val Tyr Leu Glu Glu Met

625 630 635 640

Val Gly Arg Val Asn Ala Asn Lys Ile Leu Pro Tyr Pro Pro Gly Val

645 650 655

Pro Leu Val Met Pro Gly Glu Met Ile Thr Glu Glu Ser Arg Pro Val

660 665 670

Leu Glu Phe Leu Gln Met Leu Cys Glu Ile Gly Ala His Tyr Pro Gly

675 680 685

Phe Glu Thr Asp Ile His Gly Ala Tyr Arg Gln Ala Asp Gly Arg Tyr

690 695 700

Thr Val Lys Val Leu Lys Glu Glu Asn Asn Lys

705 710 715

<210> 8

<211> 2148

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 8

atgaatgtga tcgcaatcat gaaccacatg ggtgtttact tcaaagagga accgatccgt 60

gaactgcacc gcgctctgga acgtctggat ttccgcatcg tgtaccctaa cgaccgtgat 120

gatctgctga aactgatcga aaacaactct cgtctgtgtg gcgttatctt cgattgggat 180

aaatacaacc tggaactgtg tgaagagatt tccaaaatga acgaatacat gccgctgtac 240

gcgttcgcca acacctacag caccctggat gtgagcctga acgacctgcg catgcaggta 300

cgtttttttg aatacgcact gggtgccgcg gaagacattg ctaacaaaat caaacagaac 360

accgacgaat acatcgatac catcctgcca ccgctgacca aggcgctgtt caagtacgtg 420

cgcgagggca aatacacctt ctgtacgccg ggtcacatgg gcggtactgc gtttcaaaaa 480

agcccggtgg gtagcatctt ctatgatggc ttcggcagca acacgatgaa atctgatgtc 540

agcatctccg tatccgagct gggctctctg ctggatcatt ctggtccaca caaagaagcg 600

gaagaataca ttgcccgtgt atttaacgca gaacgttcct atatggtcac gaacggtacc 660

agcactgcga acaaaatcgt tggtatgtat tctgccccgg ccggttctac tgtcctgatc 720

gatcgtaact gccataaaag cctgactcac ctgatgatga tgagcgacat caccccgatc 780

tattttcgtc cgactcgcaa cgcgtacggc atcctgggtg gcattccgca gtctgagttc 840

cagcacgcaa ccatcgcgaa acgtgttaaa gaaaccccga acgctacctg gccggttcat 900

gcagtgatca ccaactctac ttacgatggc ctgctgtaca acaccgactt catcaaaaaa 960

accctggatg taaagtctat ccacttcgat tccgcgtggg taccgtatac taacttctct 1020

ccgatttatg agggcaaatg tggtatgtct ggtggtcgtg tagaaggtaa agtgatctac 1080

gaaactcagt ccacccacaa actgctggcg gctttcagcc aggcgtctat gatccacgtg 1140

aaaggcgacg taaatgaaga gaccttcaac gaagcgtaca tgatgcacac caccacctcc 1200

cctcattatg gtatcgtggc ttctaccgaa accgcggcgg cgatgatgaa aggtaatgct 1260

ggcaaacgtc tgattgacgg ttccatcgag cgttctatca aattccgtaa agaaatcaag 1320

cgtctgaagg gcgaatccga cggctggttc ttcgacgtat ggcagccgga gcacattgat 1380

ggcccagaat gttggcctct gcgttctgac agcgcttggc acggtttcaa aaatattgac 1440

aacgaacata tgtatctgga tccgatcaaa gtaacgctgc tgactccggg tatgaaaaag 1500

gatggcacca tggatgactt cggtattccg gcttccatcg ttgcaaaata cctggacgag 1560

catggcatcg tggtggaaaa aaccggcccg tacaatctgc tgttcctgtt cagcatcggc 1620

atcgataaaa ccaaagcact gtccctgctg cgtgcactga ccgactttaa acgtgctttt 1680

gatctgaacc tgcgtgtgaa aaacatgctg ccgtccctgt accgcgaaga tccggagttc 1740

tatgagaaca tgcgcattca ggacctggcg caaaacatcc acaaactgat tgagcaccac 1800

aacctgccgg atctgatgtt ccgcgcgttc gaagtactgc ctagcatggt catgactccg 1860

tatgctgcgt tccagaaaga gctgcacggc cagaccgagg aagtgtacct ggaagaaatg 1920

gtgggccgtg tcaatgcaaa caagatcctg ccgtatccgc caggtgttcc gctggttatg 1980

ccgggtgaaa tgattactga agaatctcgt ccggttctgg aatttctgca gatgctgtgc 2040

gaaatcggcg ctcactatcc gggctttgaa accgacatcc atggcgcata ccgtcaggct 2100

gatggtcgtt acaccgtaaa agtcctgaaa gaagaaaata acaaatga 2148

<210> 9

<211> 715

<212> PRT

<213> 人工序列(Artificial Sequence)

<400> 9

Met Asn Val Ile Ala Ile Met Asn His Met Gly Val Tyr Phe Lys Glu

1 5 10 15

Glu Pro Ile Arg Glu Leu His Arg Ala Leu Glu Arg Leu Asp Phe Arg

20 25 30

Ile Val Tyr Pro Asn Asp Arg Asp Asp Leu Leu Lys Leu Ile Glu Asn

35 40 45

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

50 55 60

Glu Leu Cys Glu Asp Ile Ser Lys Met Asn Glu Tyr Met Pro Leu Tyr

65 70 75 80

Ala Phe Ala Asn Thr Tyr Ser Thr Leu Asp Val Ser Leu Asn Asp Leu

85 90 95

Arg Met Gln Val Arg Phe Phe Glu Tyr Ala Leu Gly Ala Ala Glu Asp

100 105 110

Ile Ala Asn Lys Ile Lys Gln Asn Thr Asp Glu Tyr Ile Asp Thr Ile

115 120 125

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

130 135 140

Tyr Thr Phe Cys Thr Pro Gly His Met Gly Gly Thr Ala Phe Gln Lys

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Asn Ala Glu Arg Ser Tyr Met Val Thr Asn Gly Thr Ser Thr Ala Asn

210 215 220

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

225 230 235 240

Asp Arg Asn Cys His Lys Ser Leu Thr His Leu Met Met Met Ser Asp

245 250 255

Ile Thr Pro Ile Tyr Phe Arg Pro Thr Arg Asn Ala Tyr Gly Ile Leu

260 265 270

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

275 280 285

Val Lys Glu Thr Pro Asn Ala Thr Trp Pro Val His Ala Val Ile Thr

290 295 300

Asn Ser Thr Tyr Asp Gly Leu Leu Tyr Asn Thr Asp Phe Ile Lys Lys

305 310 315 320

Thr Leu Asp Val Lys Ser Ile His Phe Asp Ser Ala Trp Val Pro Tyr

325 330 335

Thr Asn Phe Ser Pro Ile Tyr Glu Gly Lys Cys Gly Met Ser Gly Gly

340 345 350

Arg Val Glu Gly Lys Val Ile Tyr Glu Thr Gln Ser Thr His Lys Leu

355 360 365

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

370 375 380

Asn Glu Glu Thr Phe Asn Glu Ala Tyr Met Met His Thr Thr Thr Ser

385 390 395 400

Pro His Tyr Gly Ile Val Ala Ser Thr Glu Thr Ala Ala Ala Met Met

405 410 415

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

420 425 430

Ile Lys Phe Arg Lys Glu Ile Lys Arg Leu Lys Gly Glu Ser Asp Gly

435 440 445

Trp Phe Phe Asp Val Trp Gln Pro Glu His Ile Asp Gly Pro Glu Cys

450 455 460

Trp Pro Leu Arg Ser Asp Ser Ala Trp His Gly Phe Lys Asn Ile Asp

465 470 475 480

Asn Glu His Met Tyr Leu Asp Pro Ile Lys Val Thr Leu Leu Thr Pro

485 490 495

Gly Met Lys Lys Asp Gly Thr Met Asp Asp Phe Gly Ile Pro Ala Ser

500 505 510

Ile Val Ala Lys Tyr Leu Asp Glu His Gly Ile Val Val Glu Lys Thr

515 520 525

Gly Pro Tyr Asn Leu Leu Phe Leu Phe Ser Ile Gly Ile Asp Lys Thr

530 535 540

Lys Ala Leu Ser Leu Leu Arg Ala Leu Thr Asp Phe Lys Arg Ala Phe

545 550 555 560

Asp Leu Asn Leu Arg Val Lys Asn Met Leu Pro Ser Leu Tyr Arg Glu

565 570 575

Asp Pro Glu Phe Tyr Glu Asn Met Arg Ile Gln Asp Leu Ala Gln Asn

580 585 590

Ile His Lys Leu Ile Glu His His Asn Leu Pro Asp Leu Met Phe Arg

595 600 605

Ala Phe Glu Val Leu Pro Ser Met Val Met Thr Pro Tyr Ala Ala Phe

610 615 620

Gln Lys Glu Leu His Gly Gln Thr Glu Glu Val Tyr Leu Glu Glu Met

625 630 635 640

Val Gly Arg Val Asn Ala Asn Lys Ile Leu Pro Tyr Pro Pro Gly Val

645 650 655

Pro Leu Val Met Pro Gly Glu Met Ile Thr Glu Glu Ser Arg Pro Val

660 665 670

Leu Glu Phe Leu Gln Met Leu Cys Glu Ile Gly Ala His Tyr Pro Gly

675 680 685

Phe Glu Thr Asp Ile His Gly Ala Tyr Arg Gln Ala Asp Gly Arg Tyr

690 695 700

Thr Val Lys Val Leu Lys Glu Glu Asn Asn Lys

705 710 715

<210> 10

<211> 2148

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 10

atgaatgtga tcgcaatcat gaaccacatg ggtgtttact tcaaagagga accgatccgt 60

gaactgcacc gcgctctgga acgtctggat ttccgcatcg tgtaccctaa cgaccgtgat 120

gatctgctga aactgatcga aaacaactct cgtctgtgtg gcgttatctt cgattgggat 180

aaatacaacc tggaactgtg tgaagatatt tccaaaatga acgaatacat gccgctgtac 240

gcgttcgcca acacctacag caccctggat gtgagcctga acgacctgcg catgcaggta 300

cgtttttttg aatacgcact gggtgccgcg gaagacattg ctaacaaaat caaacagaac 360

accgacgaat acatcgatac catcctgcca ccgctgacca aggcgctgtt caagtacgtg 420

cgcgagggca aatacacctt ctgtacgccg ggtcacatgg gcggtactgc gtttcaaaaa 480

agcccggtgg gtagcatctt ctatgatggc ttcggcagca acacgatgaa atctgatgtc 540

agcatctccg tatccgagct gggctctctg ctggatcatt ctggtccaca caaagaagcg 600

gaagaataca ttgcccgtgt atttaacgca gaacgttcct atatggtcac gaacggtacc 660

agcactgcga acaaaatcgt tggtatgtat tctgccccgg ccggttctac tgtcctgatc 720

gatcgtaact gccataaaag cctgactcac ctgatgatga tgagcgacat caccccgatc 780

tattttcgtc cgactcgcaa cgcgtacggc atcctgggtg gcattccgca gtctgagttc 840

cagcacgcaa ccatcgcgaa acgtgttaaa gaaaccccga acgctacctg gccggttcat 900

gcagtgatca ccaactctac ttacgatggc ctgctgtaca acaccgactt catcaaaaaa 960

accctggatg taaagtctat ccacttcgat tccgcgtggg taccgtatac taacttctct 1020

ccgatttatg agggcaaatg tggtatgtct ggtggtcgtg tagaaggtaa agtgatctac 1080

gaaactcagt ccacccacaa actgctggcg gctttcagcc aggcgtctat gatccacgtg 1140

aaaggcgacg taaatgaaga gaccttcaac gaagcgtaca tgatgcacac caccacctcc 1200

cctcattatg gtatcgtggc ttctaccgaa accgcggcgg cgatgatgaa aggtaatgct 1260

ggcaaacgtc tgattgacgg ttccatcgag cgttctatca aattccgtaa agaaatcaag 1320

cgtctgaagg gcgaatccga cggctggttc ttcgacgtat ggcagccgga gcacattgat 1380

ggcccagaat gttggcctct gcgttctgac agcgcttggc acggtttcaa aaatattgac 1440

aacgaacata tgtatctgga tccgatcaaa gtaacgctgc tgactccggg tatgaaaaag 1500

gatggcacca tggatgactt cggtattccg gcttccatcg ttgcaaaata cctggacgag 1560

catggcatcg tggtggaaaa aaccggcccg tacaatctgc tgttcctgtt cagcatcggc 1620

atcgataaaa ccaaagcact gtccctgctg cgtgcactga ccgactttaa acgtgctttt 1680

gatctgaacc tgcgtgtgaa aaacatgctg ccgtccctgt accgcgaaga tccggagttc 1740

tatgagaaca tgcgcattca ggacctggcg caaaacatcc acaaactgat tgagcaccac 1800

aacctgccgg atctgatgtt ccgcgcgttc gaagtactgc ctagcatggt catgactccg 1860

tatgctgcgt tccagaaaga gctgcacggc cagaccgagg aagtgtacct ggaagaaatg 1920

gtgggccgtg tcaatgcaaa caagatcctg ccgtatccgc caggtgttcc gctggttatg 1980

ccgggtgaaa tgattactga agaatctcgt ccggttctgg aatttctgca gatgctgtgc 2040

gaaatcggcg ctcactatcc gggctttgaa accgacatcc atggcgcata ccgtcaggct 2100

gatggtcgtt acaccgtaaa agtcctgaaa gaagaaaata acaaatga 2148

Claims

1.一种赖氨酸脱羧酶突变体，其特征在于，其为突变体3，所述突变体3是在氨基酸序列如SEQ：ID：NO:1所示的赖氨酸脱羧酶的基础上，将其第170位的Phe突变为Gly、第648位的Met突变为Lys。

2.根据权利要求1所述的赖氨酸脱羧酶突变体，其特征在于，其为突变体4、突变体5或突变体6，其中，所述突变体4在突变体3的基础上，将其第69位的Glu突变为Asp；所述突变体5是在突变体3的基础上，将其第180位的Ile突变为Val；所述突变体6是在突变体3的基础上，将其第69位的Glu突变为Asp、第180位的Ile突变为Val。

3.编码权利要求1所述的赖氨酸脱羧酶突变体的基因，其特征在于，编码突变体3的基因序列如SEQ：ID：NO:4所示。

4.编码权利要求2所述的赖氨酸脱羧酶突变体的基因，其特征在于，编码突变体4的基因序列如SEQ：ID：NO:6所示；编码突变体5的基因序列如SEQ：ID：NO:8所示；编码突变体6的基因序列如SEQ：ID：NO:10所示。

5.一种重组质粒，其特征在于，其包含有权利要求3或权利要求4中任一编码突变体的基因，所述重组质粒的起始载体选用pET系列载体。

6.根据权利要求5所述的重组质粒，其特征在于，所述重组质粒的起始载体选用pET22b载体。

7.一种重组菌株，其特征在于，其包含权利要求5或权利要求6所述的重组质粒，所述重组菌株为大肠杆菌。

8.一种表达赖氨酸脱羧酶突变体的方法，其特征在于，包括如下步骤：将权利要求5或权利要求6所述的重组质粒转化至大肠杆菌菌株中，得到重组菌株；将重组菌株活化后接种于LB培养基或发酵培养基中，待菌体OD600值达到0.6-30时，加入IPTG诱导表达赖氨酸脱羧酶突变体；所述IPTG在培养基中终浓度为0.1-1mM，所述诱导条件为16-37℃，诱导时间为4-48h。

9.权利要求1或2所述的赖氨酸脱羧酶突变体在合成1,5-戊二胺、尼龙56盐中的应用。

10.根据权利要求9所述的应用，其特征在于，通过固定化酶技术，将所述突变体制成固定化突变酶并应用于合成1,5-戊二胺、尼龙56盐中。