CN110938606A - HpaBC基因、突变体及其应用 - Google Patents

HpaBC基因、突变体及其应用 Download PDF

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CN110938606A
CN110938606A CN201911058209.2A CN201911058209A CN110938606A CN 110938606 A CN110938606 A CN 110938606A CN 201911058209 A CN201911058209 A CN 201911058209A CN 110938606 A CN110938606 A CN 110938606A
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陈伟
姚骏
唐双焱
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Abstract

本发明涉及生物技术领域，尤其涉及大肠杆菌生产羟基酪醇的技术领域。本发明提供大肠杆菌4‑羟基苯乙酸‑3‑羟化酶亚基HpaBC基因、突变体及其应用，所述HpaBC基因德核苷酸序列如SEQ ID NO.1所述或氨基酸序列如SEQ ID NO.2所述。本发明中的菌株BL21(DE3)(pETDuet‑1‑T7‑TyrB‑Aro10‑T7‑HpaBCMUT)通过突变体HpaBC基因实现了羟基酪醇生物合成量的显著提高。

Description

HpaBC基因、突变体及其应用
技术领域
本发明涉及生物技术领域，尤其涉及大肠杆菌生产羟基酪醇的技术领域。
背景技术
羟基酪醇(Hydroxytyrosol)，化学名为3,4-二羟基苯乙醇，主要存在于橄榄属植物的果实和枝叶中。羟基酪醇是一种天然的强抗氧化剂，具有很强的清除自由基能力，可预防自由基引起的直接损伤以及紫外线诱导的氧化应激等。研究报道羟基酪醇能显著降低氧化物对DNA的损伤，阻滞肿瘤细胞循环及诱发其凋亡，具有很好的抗肿瘤活性。另外，羟基酪醇也被报道具有明显降血脂、抗动脉硬化、治疗糖尿病和肥胖症、抗菌消炎等功效。
在植物中，天然羟基酪醇很少以游离的形式存在，主要以橄榄苦苷的形式存在。通过酸碱或生物转化法降解橄榄果渣、橄榄叶和橄榄油加工废水中的橄榄苦苷，再提取分离得到羟基酪醇。但由于这些原料中除了橄榄苦苷，还存在女贞苷等各种苷类及其他成分，降解液中化学成分十分复杂，羟基酪醇含量低，难以分离纯化，制备成本高。采用化学法合成羟基酪醇已有研究报道。以酪醇(Tyrosol)和3,4-二羟基苯乙酸为原料经化学催化剂可合成羟基酪醇；以邻苯二酚和3,4-二羟基苯甲醛等为原料，经多步化学催化反应亦可合成羟基酪醇。但存在催化剂成本昂贵，反应条件苛刻，反应得率低，合成成本高等问题。
采用生物转化法合成羟基酪醇，通常以酪醇或橄榄苦苷为底物通过微生物菌株发酵或专一酶催化生成羟基酪醇。研究表明一些细菌和真菌能使酪醇转化为羟基酪醇，但产量低、后期分离纯化较复杂。大鼠的酪氨酸羟化酶(Tyrosine hydroxylase，TH)和细胞色素P450可共同催化酪醇生成羟基酪醇，但蘑菇酪氨酸酶能继续催化羟基酪醇生成4-羟乙基邻苯醌。
研究亦发现一些微生物的4-羟基苯乙酸-3-羟化酶(4-hydroxyphenylaceticacid 3-hydroxylase，HPAH，EC 1.14.13.3)能催化酪醇生成羟基酪醇，但活性低，且大部分来源的HPAH能继续氧化羟基酪醇。该酶的天然活性是催化4-羟基苯乙酸合成3,4-二羟基苯乙酸，虽然其对酪醇有一定活性，但并非最佳底物。HPAH是由二条肽链组成的单氧化酶，在许多微生物中都存在。该酶由HpaB和HpaC两个亚基组成，其中HpaB亚基负责与底物的结合。我们比较了不同来源的HPAH，结果表明它们催化酪醇生成羟基酪醇的活性都不是很高。且有些来源的HPAH除了合成羟基酪醇外，另有副产物产生。大肠杆菌中编码该酶的基因簇为hpaBC，表达的HPAH酶(HpaBC)能够催化酪醇生产羟基酪醇且不会进一步氧化羟基酪醇，因此是用来生物合成羟基酪醇很好的候选酶之一。大肠杆菌HpaBC催化酪醇生成羟基酪醇的活性较低，因此对该酶进行改造获得高催化活性的工程酶具有非常重要的应用价值。
发明内容
本发明的第一个目的是提供大肠杆菌4-羟基苯乙酸-3-羟化酶亚基HpaBC基因，它的核苷酸序列如SEQ ID NO.1所述或氨基酸序列如SEQ ID NO.2所述。
本发明的第二个目的是提供包括HpaBC基因的载体。
本发明的第三个目的是提供HpaBC基因的突变体HpaBC-MUT基因及其获得方法，它的核苷酸序列如下表任一所述或氨基酸序列如下表任一所述：
<u>突变体名称</u> <u>核苷酸序列</u> <u>氨基酸序列</u>
<u>HpaBC-MUT1</u> <u>SEQ ID NO.3</u> <u>SEQ ID NO.12</u>
<u>HpaBC-MUT2</u> <u>SEQ ID NO.4</u> <u>SEQ ID NO.13</u>
<u>HpaBC-MUT3</u> <u>SEQ ID NO.5</u> <u>SEQ ID NO.14</u>
<u>HpaBC-MUT4</u> <u>SEQ ID NO.6</u> <u>SEQ ID NO.15</u>
<u>HpaBC-MUT5</u> <u>SEQ ID NO.7</u> <u>SEQ ID NO.16</u>
<u>HpaBC-MUT6</u> <u>SEQ ID NO.8</u> <u>SEQ ID NO.17</u>
<u>HpaBC-MUT7</u> <u>SEQ ID NO.9</u> <u>SEQ ID NO.18</u>
<u>HpaBC-MUT8</u> <u>SEQ ID NO.10</u> <u>SEQ ID NO.19</u>
<u>HpaBC-MUT9</u> <u>SEQ ID NO.11</u> <u>SEQ ID NO.20</u>
。
本发明的第四个目的是提供含HpaBC-MUT基因的载体。
本发明的第五个目的是提供一种羟基酪醇的制备方法，包括：
将序列表SEQ ID NO.3至SEQ ID NO.11的任一序列所示的DNA分子插入pETDuet-1的NdeI和XhoI双酶切位点间得到的pETDuet-1-T7-TyrB-Aro10-T7-HpaBCMUT；
将pETDuet-1-T7-TyrB-Aro10-T7-HpaBCMUT导入BL21(DE3)的感受态细胞中，得到BL21(DE3)(pETDuet-1-T7-TyrB-Aro10-T7-HpaBCMUT)；
将大肠杆菌BL21(DE3)(pETDuet-1-T7-TyrB-Aro10-T7-HpaBCMUT)的单菌落接种至含有卡那霉素(终浓度为50μg/ml)的LB液体培养基中，37℃培养12h，收集发酵液；
将发酵液按照1％(体积百分含量)的接种量转接至100ml新鲜的LB液体培养基中，37℃培养至OD600达到0.6；
在培养基中加入无菌的IPTG，使IPTG在培养基中的终浓度为0.4mM，在30℃进行诱导发酵；同时加入无菌的酪氨酸溶液至终浓度为10mM，16h后结束发酵，将发酵液12000rpm离心10min收集上清液。
发明中的菌株BL21(DE3)(pETDuet-1-T7-TyrB-Aro10-T7-HpaBCMUT)通过突变体HpaBC基因实现了羟基酪醇生物合成量的显著提高。
附图说明
图1是菌株BL21(DE3)(pETDuet-1-T7-TyrB-Aro10-T7-HpaBC)发酵产物HPLC检测结果图。其中，峰Ⅰ为羟基酪醇。
图2是菌株BL21(DE3)(pETDuet-1-T7-TyrB-Aro10-T7-HpaBCMUT)发酵产物HPLC检测结果图。其中，峰Ⅰ为羟基酪醇。
图3是羟基酪醇的标准品HPLC检测结果图。
图4是菌株BL21(DE3)(pETDuet-1-T7-TyrB-Aro10-T7-HpaBCMUT)发酵产物(峰Ⅰ)MS图谱。
图5是羟基酪醇标品MS图谱。
具体实施方式
以下结合附图和实施例对本发明作进一步的说明，应当理解的是，此处所描述的具体实施方式仅用于说明和解释本发明，并不用于限制本发明。
本发明中，对表达载体的种类没有特殊要求，可以为能够在大肠杆菌中表达目的基因的本领域常用的各种表达载体，例如质粒等。本领域技术人员应该理解的是，表达载体的构建方法可以采用本领域常用的各种方法，如将目的基因经过酶切处理后连接至载体中，在此不再赘述。
以下实施例中：
1)大肠杆菌菌株BL21(DE3)(购自Novagen，货号69450)和DH5α(购自ThermoFisher Scientific，货号18265017)为常用大肠杆菌菌株，均可市售获得，大肠杆菌菌株BL21(DE3)用于本发明中所有基因的表达和HpaBC基因的克隆，DH5α用于载体构建。
2)大肠杆菌表达载体pETDuet-1，购自Novagen，货号71146-3。
3)Phusion高保真DNA聚合酶、限制性内切酶购自Thermo公司。
4)细菌基因组DNA提取试剂盒购自上海捷瑞生物工程有限公司。
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1
1)、以序列表SEQ ID NO.21、SEQ ID NO.22为PCR引物，以大肠杆菌BL21(DE3)基因组为模板，PCR扩增TyrB基因的开放阅读框，利用NcoI和NotI双酶切将TyrB基因连入载体pETDuet-1，得到质粒pETDuet-1-T7-TyrB；以序列表SEQ ID NO.23、SEQ ID NO.24位PCR引物，以酵母基因组为模板，PCR扩增Aro10基因的开放阅读框，利用NheI和NotI双酶切将Aro10基因连入载体pETDuet-1-T7-TyrB，得到质粒pETDuet-1-T7-TyrB-Aro10；以序列表SEQ ID NO.25、SEQ ID NO.26为PCR引物，以大肠杆菌HpaBC突变体基因为模板，PCR扩增HpaBC突变体基因的开放阅读框架，利用NdeI和XhoI双酶切将HpaBC突变体基因连入载体pETDuet-1-T7-TyrB-Aro10，得到表达载体pETDuet-1-T7-TyrB-Aro10-T7-HpaBCMUT。将表达载体转入大肠杆菌菌株BL21(DE3)，得到重组大肠杆菌菌株BL21(DE3)(pETDuet-1-T7-TyrB-Aro10-T7-HpaBCMUT)。
2)、提取大肠杆菌BL21(DE3)的基因组DNA并以其为模板，以序列表SEQ ID NO.25、SEQ ID NO.26为引物进行PCR扩增。PCR扩增条件如下：先95℃预变性4min，然后95℃45s，55℃30s，72℃1min30s，共30个循环；最后72℃延伸10min。回收上述PCR反应产物，进行琼脂糖凝胶电泳检测，结果获得约1563bp大小的片段。将上述获得的PCR产物用NdeI和XhoI双酶切，酶切产物与经相同酶切的pETDuet-1-T7-TyrB-Aro10载体用T4连接酶(购自宝生物公司)16℃连接过夜，连接产物转化大肠杆菌DH5α感受态细胞，获得重组菌。提取重组菌的质粒测序验证，测序分析，其核苷酸序列为序列表SEQ ID NO.1所示，通过比对，其为大肠杆菌的4-羟基苯乙酸-3-羟化酶HpaBC的编码基因，该基因编码的蛋白命名为HpaBC，该蛋白的氨基酸序列为序列表SEQ ID NO.2所示。
将含有该PCR产物的质粒命名为pETDuet-1-T7-TyrB-Aro10-T7-HpaBC，该质粒为将序列表SEQ ID NO.1插入pETDuet-1-T7-TyrB-Aro10载体的NdeI和XhoI双酶切位点间得到的载体。
3)、在hpaBC基因的基础上，利用定点饱和突变PCR的方法，构建定点饱和突变文库。主要是通过重叠PCR从而在基因序列中的特定位点引入突变。在本实施例中，进行一轮突变文库构建，得到突变的PCR产物1。将上述突变的PCR产物1分别NdeI和XhoI双酶切，酶切产物分别与经相同酶切的pETDuet-1-T7-TyrB-Aro10载体连接，连接产物分别转化BL21(DE3)的感受态细胞中，得到突变文库。在无菌操作间用无菌牙签将包含着突变体的单菌落克隆挑入已分装有800ul LB培养基(胰蛋白胨10g/L，NaCl 10g/L,酵母提取物5g/L,添加抗生素卡那霉素至50μg/mL)37℃摇床培养2h后，加入诱导剂IPTG至终浓度0.4mM，同时加入酪氨酸至终浓度10mM，在30℃摇床诱导约16h。离心、取上清液180μL至96孔酶标板，向每孔加入20μL 100mM的高碘酸钠溶液，混合均匀，静置10min，检测400nm处的吸光值，间接体现转化生成羟基酪醇的能力。对于生成羟基酪醇比野生型的OD400数值高出50％及以上的重组菌，提取质粒送去测序，结果该质粒中的PCR产物的基因具有序列表SEQ ID NO.3至SEQ IDNO.11所示的核苷酸，突变基因被依次命名为hpaBC-MUT1至hpaBC-MUT9，该基因编码的蛋白命名为HpaBC-MUT1至HpaBC-MUT9，该蛋白的氨基酸序列为序列表SEQ ID NO.12至20所示的氨基酸：
Figure BDA0002257130860000051
Figure BDA0002257130860000061
将含有hpaBC-MUT的质粒命名为pETDuet-1-T7-TyrB-Aro10-T7-HpaBCMUT，该质粒为将序列表SEQ ID NO.3至SEQ ID NO.11的任一序列插入pETDuet-1-T7-TyrB-Aro10的NdeI和XhoI双酶切位点间得到的载体，将含有该质粒的重组菌命名为BL21(DE3)(pETDuet-1-T7-TyrB-Aro10-T7-HpaBCMUT)。
上述BL21(DE3)(pETDuet-1-T7-TyrB-Aro10-T7-HpaBCMUT)也可以按照如下方法制备：(1)将序列表SEQ ID NO.3至SEQ ID NO.11的任一序列所示的DNA分子插入pET28a的NdeI和XhoI双酶切位点间得到的pETDuet-1-T7-TyrB-Aro10-T7-HpaBCMUT；(2)再将pETDuet-1-T7-TyrB-Aro10-T7-HpaBCMUT导入BL21(DE3)的感受态细胞中，得到大肠杆菌BL21(DE3)(pETDuet-1-T7-TyrB-Aro10-T7-HpaBCMUT)。
采用同样的方法将质粒pETDuet-1-T7-TyrB-Aro10-T7-HpaBC转入BL21(DE3)的感受态细胞中，得到重组大肠杆菌BL21(DE3)(pETDuet-1-T7-TyrB-Aro10-T7-HpaBC)。
实施例2
大肠杆菌BL21(DE3)(pETDuet-1-T7-TyrB-Aro10-T7-HpaBC)和BL21(DE3)(pETDuet-1-T7-TyrB-Aro10-T7-HpaBCMUT)的发酵培养：(1)将上述获得的BL21(DE3)(pETDuet-1-T7-TyrB-Aro10-T7-HpaBC)和BL21(DE3)(pETDuet-1-T7-TyrB-Aro10-T7-HpaBCMUT)的单菌落分别接种至含有卡那霉素(终浓度为50μg/ml)的LB液体培养基中，37℃培养12h，收集发酵液；(2)将发酵液按照1％(体积百分含量)的接种量转接至100ml新鲜的LB液体培养基中，37℃培养至OD600达到0.6，得，得发酵后的LB液体培养基；(3)再在发酵后的LB液体培养基中加入无菌的IPTG，使IPTG在培养基中的终浓度为0.4mM在30℃进行诱导发酵；同时加入无菌的酪氨酸溶液至终浓度为10mM，16h后结束发酵，将发酵液12000rpm离心10min收集上清液。
实施例3
羟基酪醇的检测
(1)产物的HPLC检测
取实施例2中两种不同菌株的发酵液各1mL，12000rpm离心10min后，取上清，进行HPLC分析检测。
分析条件如下：仪器为：安捷伦液相色谱仪，测定条件包括：C18柱(4.6×250mm)；检测波长280nm；流动相A＝水(含1％体积乙酸)，B＝乙腈；流速＝1mL/min；梯度洗脱条件：0–15min，80％A和20％B；15–20min，80％A和20％B到100％B(线性梯度)；20–25min，100％B。进样量20μL。
发酵液及标准品的HPLC检测结果见图1。其中，1是菌株BL21(DE3)(pETDuet-1-T7-TyrB-Aro10-T7-HpaBC)发酵液，2是菌株BL21(DE3)(pETDuet-1-T7-TyrB-Aro10-T7-HpaBCMUT)发酵液，3是羟基酪醇的标准品。
如图1所示，菌株BL21(DE3)(pETDuet-1-T7-TyrB-Aro10-T7-HpaBC)的发酵液在4.542min时存在一个峰(1，峰Ⅰ)，与羟基酪醇标准品的出峰位置一致；而BL21(DE3)(pETDuet-1-T7-TyrB-Aro10-T7-HpaBCMUT)的发酵液中在4.512min时也存在一个峰(1，峰Ⅰ)，与羟基酪醇标准品的出峰时间一致。
(2)产物的LC-MS分析
对步骤(1)发酵液中看到的4.512min的峰Ⅰ进行LC-MS分析，其中，进行LC-MS分析的条件包括：C18柱(4.6×250mm)；检测波长280nm；流动相A＝水(含1体积％乙酸)，B＝乙腈；流速＝1mL/min；洗脱条件：0–15min，80％A和20％B；15–20min，80％A和20％B到100％B(线性梯度)；20–25min，100％B。进样量20μL。
ESI正离子源，分子量扫描范围50–800。两种发酵液的LC-MS检测结果见图2。峰Ⅰ的MS图谱上有羟基酪醇的MS特征峰153。
且经测定，菌株BL21(DE3)(pETDuet-1-T7-TyrB-Aro10-T7-HpaBCMUT)中羟基酪醇的产量为3550mg/L，而对比例BL21(DE3)(pETDuet-1-T7-TyrB-Aro10-T7-HpaBC)发酵液中羟基酪醇的产量仅为305mg/L。发明中的菌株BL21(DE3)(pETDuet-1-T7-TyrB-Aro10-T7-HpaBCMUT)通过突变体HpaBC实现了羟基酪醇生物合成量的显著提高。
序列表
<110> 中国科学院微生物研究所
<120> HpaBC基因、突变体及其应用
<150> 201811584195.3
<151> 2018-12-24
<160> 26
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1563
<212> DNA
<213> 大肠杆菌(Escherichia coli)
<400> 1
atgaaaccag aagatttccg cgccagtacc caacgtcctt tcaccgggga agagtatctg 60
aaaagcctgc aggatggtcg cgagatctat atctatggcg agcgagtgaa agacgtcacc 120
actcatccgg catttcgtaa tgcggcagcg tctgttgccc agctgtacga cgcactgcac 180
aaaccggaga tgcaggactc tctgtgttgg aacaccgaca ccggcagcgg cggctatacc 240
cataaattct tccgcgtggc gaaaagtgcc gacgacctgc gccagcaacg cgacgccatc 300
gctgagtggt cacgcctgag ctatggctgg atgggccgta ccccagacta caaagccgct 360
ttcggttgcg cactgggcgc gaatccgggc ttttacggtc agttcgagca gaacgcccgt 420
aactggtaca cccgtattca ggaaactggc ctctacttta accacgcgat tgttaaccca 480
ccgatcgatc gtcatttgcc gaccgataaa gtgaaagacg tttacatcaa gctggaaaaa 540
gagactgacg ccgggattat cgtcagcggt gcgaaagtgg ttgccaccaa ctcggcgctg 600
actcactaca acatgattgg cttcggctcg gcacaagtga tgggcgaaaa cccggacttc 660
gcactgatgt tcgttgcgcc aatggatgcc gatggcgtga aattaatctc ccgcgcctct 720
tatgagatgg tcgcgggtgc taccggctcg ccatacgact acccgctctc cagccgcttc 780
gatgagaacg atgcgattct ggtgatggat aacgtgctga ttccatggga aaacgtgctg 840
atctaccgcg attttgatcg ctgccgtcgc tggacgatgg aaggcggttt tgcccgtatg 900
tatccgctgc aagcctgtgt gcgcctggca gtgaaattag acttcattac ggcactgctg 960
aaaaaatcac tcgaatgtac cggcaccctg gagttccgtg gtgtgcaggc cgatctcggt 1020
gaagtggtag cgtggcgcaa caccttctgg gcattgagtg actcgatgtg ttcagaagca 1080
acgccgtggg tcaacggggc ttatttaccg gatcatgccg cactgcaaac ctatcgcgta 1140
ctggcaccaa tggcctacgc gaagatcaaa aacattatcg aacgcaacgt taccagtggc 1200
ctgatctatc tcccttccag tgcccgtgac ctgaataatc cgcagatcga ccagtatctg 1260
gcgaagtatg tgcgcggttc gaacggtatg gatcacgtcc agcgcatcaa gatcctcaaa 1320
ctgatgtggg atgctattgg cagcgaattt ggtggtcgtc acgaactgta tgaaatcaac 1380
tactccggta gccaggatga gattcgcctg cagtgtctgc gccaggcaca aaactccggc 1440
aatatggaca agatgatggc gatggttgat cgctgcctgt cggaatacga ccaggacggc 1500
tggactgtgc cgcacctgca caacaacgac gatatcaaca tgctggataa gctgctgaaa 1560
taa 1563
<210> 2
<211> 520
<212> PRT
<213> 大肠杆菌(Escherichia coli)
<400> 2
Met Lys Pro Glu Asp Phe Arg Ala Ser Thr Gln Arg Pro Phe Thr Gly
1 5 10 15
Glu Glu Tyr Leu Lys Ser Leu Gln Asp Gly Arg Glu Ile Tyr Ile Tyr
20 25 30
Gly Glu Arg Val Lys Asp Val Thr Thr His Pro Ala Phe Arg Asn Ala
35 40 45
Ala Ala Ser Val Ala Gln Leu Tyr Asp Ala Leu His Lys Pro Glu Met
50 55 60
Gln Asp Ser Leu Cys Trp Asn Thr Asp Thr Gly Ser Gly Gly Tyr Thr
65 70 75 80
His Lys Phe Phe Arg Val Ala Lys Ser Ala Asp Asp Leu Arg Gln Gln
85 90 95
Arg Asp Ala Ile Ala Glu Trp Ser Arg Leu Ser Tyr Gly Trp Met Gly
100 105 110
Arg Thr Pro Asp Tyr Lys Ala Ala Phe Gly Cys Ala Leu Gly Ala Asn
115 120 125
Pro Gly Phe Tyr Gly Gln Phe Glu Gln Asn Ala Arg Asn Trp Tyr Thr
130 135 140
Arg Ile Gln Glu Thr Gly Leu Tyr Phe Asn His Ala Ile Val Asn Pro
145 150 155 160
Pro Ile Asp Arg His Leu Pro Thr Asp Lys Val Lys Asp Val Tyr Ile
165 170 175
Lys Leu Glu Lys Glu Thr Asp Ala Gly Ile Ile Val Ser Gly Ala Lys
180 185 190
Val Val Ala Thr Asn Ser Ala Leu Thr His Tyr Asn Met Ile Gly Phe
195 200 205
Gly Ser Ala Gln Val Met Gly Glu Asn Pro Asp Phe Ala Leu Met Phe
210 215 220
Val Ala Pro Met Asp Ala Asp Gly Val Lys Leu Ile Ser Arg Ala Ser
225 230 235 240
Tyr Glu Met Val Ala Gly Ala Thr Gly Ser Pro Tyr Asp Tyr Pro Leu
245 250 255
Ser Ser Arg Phe Asp Glu Asn Asp Ala Ile Leu Val Met Asp Asn Val
260 265 270
Leu Ile Pro Trp Glu Asn Val Leu Ile Tyr Arg Asp Phe Asp Arg Cys
275 280 285
Arg Arg Trp Thr Met Glu Gly Gly Phe Ala Arg Met Tyr Pro Leu Gln
290 295 300
Ala Cys Val Arg Leu Ala Val Lys Leu Asp Phe Ile Thr Ala Leu Leu
305 310 315 320
Lys Lys Ser Leu Glu Cys Thr Gly Thr Leu Glu Phe Arg Gly Val Gln
325 330 335
Ala Asp Leu Gly Glu Val Val Ala Trp Arg Asn Thr Phe Trp Ala Leu
340 345 350
Ser Asp Ser Met Cys Ser Glu Ala Thr Pro Trp Val Asn Gly Ala Tyr
355 360 365
Leu Pro Asp His Ala Ala Leu Gln Thr Tyr Arg Val Leu Ala Pro Met
370 375 380
Ala Tyr Ala Lys Ile Lys Asn Ile Ile Glu Arg Asn Val Thr Ser Gly
385 390 395 400
Leu Ile Tyr Leu Pro Ser Ser Ala Arg Asp Leu Asn Asn Pro Gln Ile
405 410 415
Asp Gln Tyr Leu Ala Lys Tyr Val Arg Gly Ser Asn Gly Met Asp His
420 425 430
Val Gln Arg Ile Lys Ile Leu Lys Leu Met Trp Asp Ala Ile Gly Ser
435 440 445
Glu Phe Gly Gly Arg His Glu Leu Tyr Glu Ile Asn Tyr Ser Gly Ser
450 455 460
Gln Asp Glu Ile Arg Leu Gln Cys Leu Arg Gln Ala Gln Asn Ser Gly
465 470 475 480
Asn Met Asp Lys Met Met Ala Met Val Asp Arg Cys Leu Ser Glu Tyr
485 490 495
Asp Gln Asp Gly Trp Thr Val Pro His Leu His Asn Asn Asp Asp Ile
500 505 510
Asn Met Leu Asp Lys Leu Leu Lys
515 520
<210> 3
<211> 1563
<212> DNA
<213> 大肠杆菌(Escherichia coli)
<400> 3
atgaaaccag aagatttccg cgccagtacc caacgtcctt tcaccgggga agagtatctg 60
aaaagcctgc aggatggtcg cgagatctat atctatggcg agcgagtgaa agacgtcacc 120
actcatccgg catttcgtaa tgcggcagcg tctgttgccc agctgtacga cgcactgcac 180
aaaccggaga tgcaggactc tctgtgttgg aacaccgaca ccggcagcgg cggctatacc 240
cataaattct tccgcgtggc gaaaagtgcc gacgacctgc gccagcaacg cgacgccatc 300
gctgagtggt cacgcctgag ctatggctgg atgggccgta ccccagacta caaagccgct 360
ttcggttgcg cactgggcgc gaatccgggc ttttacggtc agttcgagca gaacgcccgt 420
aactggtaca cccgtattca ggaaactggc ctctacttta accacgcgat tgttaaccca 480
ccgatcgatc gtcatttgcc gaccgataaa gtgaaagacg tttacatcaa gctggaaaaa 540
gagactgacg ccgggattat cgtcagcggt gcgaaagtgg ttgccaccaa ctcggcgctg 600
actcactaca acatgattgg cttcggcact cttgaagtga tgggcgaaaa cccggacttc 660
gcactgatgt tcgttgcgcc aatggatgcc gatggcgtga aattaatctc ccgcgcctct 720
tatgagatgg tcgcgggtgc taccggctcg ccatacgact acccgctctc cagccgcttc 780
gatgagaacg atgcgattct ggtgatggat aacgtgctga ttccatggga aaacgtgctg 840
atctaccgcg attttgatcg ctgccgtcgc tggacgatgg aaggcggttt tgcccgtatg 900
tatccgctgc aagcctgtgt gcgcctggca gtgaaattag acttcattac ggcactgctg 960
aaaaaatcac tcgaatgtac cggcaccctg gagttccgtg gtgtgcaggc cgatctcggt 1020
gaagtggtag cgtggcgcaa caccttctgg gcattgagtg actcgatgtg ttcagaagca 1080
acgccgtggg tcaacggggc ttatttaccg gatcatgccg cactgcaaac ctatcgcgta 1140
ctggcaccaa tggcctacgc gaagatcaaa aacattatcg aacgcaacgt taccagtggc 1200
ctgatctatc tcccttccag tgcccgtgac ctgaataatc cgcagatcga ccagtatctg 1260
gcgaagtatg tgcgcggttc gaacggtatg gatcacgtcc agcgcatcaa gatcctcaaa 1320
ctgatgtggg atgctattgg cagcgaattt ggtggtcgtc acgaactgta tgaaatcaac 1380
tactccggta gccaggatga gattcgcctg cagtgtctgc gccaggcaca aaactccggc 1440
aatatggaca agatgatggc gatggttgat cgctgcctgt cggaatacga ccaggacggc 1500
tggactgtgc cgcacctgca caacaacgac gatatcaaca tgctggataa gctgctgaaa 1560
taa 1563
<210> 4
<211> 1563
<212> DNA
<213> 大肠杆菌(Escherichia coli)
<400> 4
atgaaaccag aagatttccg cgccagtacc caacgtcctt tcaccgggga agagtatctg 60
aaaagcctgc aggatggtcg cgagatctat atctatggcg agcgagtgaa agacgtcacc 120
actcatccgg catttcgtaa tgcggcagcg tctgttgccc agctgtacga cgcactgcac 180
aaaccggaga tgcaggactc tctgtgttgg aacaccgaca ccggcagcgg cggctatacc 240
cataaattct tccgcgtggc gaaaagtgcc gacgacctgc gccagcaacg cgacgccatc 300
gctgagtggt cacgcctgag ctatggctgg atgggccgta ccccagacta caaagccgct 360
ttcggttgcg cactgggcgc gaatccgggc ttttacggtc agttcgagca gaacgcccgt 420
aactggtaca cccgtattca ggaaactggc ctctacttta accacgcgat tgttaaccca 480
ccgatcgatc gtcatttgcc gaccgataaa gtgaaagacg tttacatcaa gctggaaaaa 540
gagactgacg ccgggattat cgtcagcggt gcgaaagtgg ttgccaccaa ctcggcgctg 600
actcactaca acatgattgg cttcggctcg ggttatgtga tgggcgaaaa cccggacttc 660
gcactgatgt tcgttgcgcc aatggatgcc gatggcgtga aattaatctc ccgcgcctct 720
tatgagatgg tcgcgggtgc taccggctcg ccatacgact acccgctctc cagccgcttc 780
gatgagaacg atgcgattct ggtgatggat aacgtgctga ttccatggga aaacgtgctg 840
atctaccgcg attttgatcg ctgccgtcgc tggacgatgg aaggcggttt tgcccgtatg 900
tatccgctgc aagcctgtgt gcgcctggca gtgaaattag acttcattac ggcactgctg 960
aaaaaatcac tcgaatgtac cggcaccctg gagttccgtg gtgtgcaggc cgatctcggt 1020
gaagtggtag cgtggcgcaa caccttctgg gcattgagtg actcgatgtg ttcagaagca 1080
acgccgtggg tcaacggggc ttatttaccg gatcatgccg cactgcaaac ctatcgcgta 1140
ctggcaccaa tggcctacgc gaagatcaaa aacattatcg aacgcaacgt taccagtggc 1200
ctgatctatc tcccttccag tgcccgtgac ctgaataatc cgcagatcga ccagtatctg 1260
gcgaagtatg tgcgcggttc gaacggtatg gatcacgtcc agcgcatcaa gatcctcaaa 1320
ctgatgtggg atgctattgg cagcgaattt ggtggtcgtc acgaactgta tgaaatcaac 1380
tactccggta gccaggatga gattcgcctg cagtgtctgc gccaggcaca aaactccggc 1440
aatatggaca agatgatggc gatggttgat cgctgcctgt cggaatacga ccaggacggc 1500
tggactgtgc cgcacctgca caacaacgac gatatcaaca tgctggataa gctgctgaaa 1560
taa 1563
<210> 5
<211> 1563
<212> DNA
<213> 大肠杆菌(Escherichia coli)
<400> 5
atgaaaccag aagatttccg cgccagtacc caacgtcctt tcaccgggga agagtatctg 60
aaaagcctgc aggatggtcg cgagatctat atctatggcg agcgagtgaa agacgtcacc 120
actcatccgg catttcgtaa tgcggcagcg tctgttgccc agctgtacga cgcactgcac 180
aaaccggaga tgcaggactc tctgtgttgg aacaccgaca ccggcagcgg cggctatacc 240
cataaattct tccgcgtggc gaaaagtgcc gacgacctgc gccagcaacg cgacgccatc 300
gctgagtggt cacgcctgag ctatggctgg atgggccgta ccccagacta caaagccgct 360
ttcggttgcg cactgggcgc gaatccgggc ttttacggtc agttcgagca gaacgcccgt 420
aactggtaca cccgtattca ggaaactggc ctctacttta accacgcgat tgttaaccca 480
ccgatcgatc gtcatttgcc gaccgataaa gtgaaagacg tttacatcaa gctggaaaaa 540
gagactgacg ccgggattat cgtcagcggt gcgaaagtgg ttgccaccaa ctcggcgctg 600
actcactaca acatgattgg cttcggcact atgggtgtga tgggcgaaaa cccggacttc 660
gcactgatgt tcgttgcgcc aatggatgcc gatggcgtga aattaatctc ccgcgcctct 720
tatgagatgg tcgcgggtgc taccggctcg ccatacgact acccgctctc cagccgcttc 780
gatgagaacg atgcgattct ggtgatggat aacgtgctga ttccatggga aaacgtgctg 840
atctaccgcg attttgatcg ctgccgtcgc tggacgatgg aaggcggttt tgcccgtatg 900
tatccgctgc aagcctgtgt gcgcctggca gtgaaattag acttcattac ggcactgctg 960
aaaaaatcac tcgaatgtac cggcaccctg gagttccgtg gtgtgcaggc cgatctcggt 1020
gaagtggtag cgtggcgcaa caccttctgg gcattgagtg actcgatgtg ttcagaagca 1080
acgccgtggg tcaacggggc ttatttaccg gatcatgccg cactgcaaac ctatcgcgta 1140
ctggcaccaa tggcctacgc gaagatcaaa aacattatcg aacgcaacgt taccagtggc 1200
ctgatctatc tcccttccag tgcccgtgac ctgaataatc cgcagatcga ccagtatctg 1260
gcgaagtatg tgcgcggttc gaacggtatg gatcacgtcc agcgcatcaa gatcctcaaa 1320
ctgatgtggg atgctattgg cagcgaattt ggtggtcgtc acgaactgta tgaaatcaac 1380
tactccggta gccaggatga gattcgcctg cagtgtctgc gccaggcaca aaactccggc 1440
aatatggaca agatgatggc gatggttgat cgctgcctgt cggaatacga ccaggacggc 1500
tggactgtgc cgcacctgca caacaacgac gatatcaaca tgctggataa gctgctgaaa 1560
taa 1563
<210> 6
<211> 1563
<212> DNA
<213> 大肠杆菌(Escherichia coli)
<400> 6
atgaaaccag aagatttccg cgccagtacc caacgtcctt tcaccgggga agagtatctg 60
aaaagcctgc aggatggtcg cgagatctat atctatggcg agcgagtgaa agacgtcacc 120
actcatccgg catttcgtaa tgcggcagcg tctgttgccc agctgtacga cgcactgcac 180
aaaccggaga tgcaggactc tctgtgttgg aacaccgaca ccggcagcgg cggctatacc 240
cataaattct tccgcgtggc gaaaagtgcc gacgacctgc gccagcaacg cgacgccatc 300
gctgagtggt cacgcctgag ctatggctgg atgggccgta ccccagacta caaagccgct 360
ttcggttgcg cactgggcgc gaatccgggc ttttacggtc agttcgagca gaacgcccgt 420
aactggtaca cccgtattca ggaaactggc ctctacttta accacgcgat tgttaaccca 480
ccgatcgatc gtcatttgcc gaccgataaa gtgaaagacg tttacatcaa gctggaaaaa 540
gagactgacg ccgggattat cgtcagcggt gcgaaagtgg ttgccaccaa ctcggcgctg 600
actcactaca acatgattgg cttcggctgg gaaggtgtga tgggcgaaaa cccggacttc 660
gcactgatgt tcgttgcgcc aatggatgcc gatggcgtga aattaatctc ccgcgcctct 720
tatgagatgg tcgcgggtgc taccggctcg ccatacgact acccgctctc cagccgcttc 780
gatgagaacg atgcgattct ggtgatggat aacgtgctga ttccatggga aaacgtgctg 840
atctaccgcg attttgatcg ctgccgtcgc tggacgatgg aaggcggttt tgcccgtatg 900
tatccgctgc aagcctgtgt gcgcctggca gtgaaattag acttcattac ggcactgctg 960
aaaaaatcac tcgaatgtac cggcaccctg gagttccgtg gtgtgcaggc cgatctcggt 1020
gaagtggtag cgtggcgcaa caccttctgg gcattgagtg actcgatgtg ttcagaagca 1080
acgccgtggg tcaacggggc ttatttaccg gatcatgccg cactgcaaac ctatcgcgta 1140
ctggcaccaa tggcctacgc gaagatcaaa aacattatcg aacgcaacgt taccagtggc 1200
ctgatctatc tcccttccag tgcccgtgac ctgaataatc cgcagatcga ccagtatctg 1260
gcgaagtatg tgcgcggttc gaacggtatg gatcacgtcc agcgcatcaa gatcctcaaa 1320
ctgatgtggg atgctattgg cagcgaattt ggtggtcgtc acgaactgta tgaaatcaac 1380
tactccggta gccaggatga gattcgcctg cagtgtctgc gccaggcaca aaactccggc 1440
aatatggaca agatgatggc gatggttgat cgctgcctgt cggaatacga ccaggacggc 1500
tggactgtgc cgcacctgca caacaacgac gatatcaaca tgctggataa gctgctgaaa 1560
taa 1563
<210> 7
<211> 1563
<212> DNA
<213> 大肠杆菌(Escherichia coli)
<400> 7
atgaaaccag aagatttccg cgccagtacc caacgtcctt tcaccgggga agagtatctg 60
aaaagcctgc aggatggtcg cgagatctat atctatggcg agcgagtgaa agacgtcacc 120
actcatccgg catttcgtaa tgcggcagcg tctgttgccc agctgtacga cgcactgcac 180
aaaccggaga tgcaggactc tctgtgttgg aacaccgaca ccggcagcgg cggctatacc 240
cataaattct tccgcgtggc gaaaagtgcc gacgacctgc gccagcaacg cgacgccatc 300
gctgagtggt cacgcctgag ctatggctgg atgggccgta ccccagacta caaagccgct 360
ttcggttgcg cactgggcgc gaatccgggc ttttacggtc agttcgagca gaacgcccgt 420
aactggtaca cccgtattca ggaaactggc ctctacttta accacgcgat tgttaaccca 480
ccgatcgatc gtcatttgcc gaccgataaa gtgaaagacg tttacatcaa gctggaaaaa 540
gagactgacg ccgggattat cgtcagcggt gcgaaagtgg ttgccaccaa ctcggcgctg 600
actcactaca acatgattgg cttcggcgat cctcgtgtga tgggcgaaaa cccggacttc 660
gcactgatgt tcgttgcgcc aatggatgcc gatggcgtga aattaatctc ccgcgcctct 720
tatgagatgg tcgcgggtgc taccggctcg ccatacgact acccgctctc cagccgcttc 780
gatgagaacg atgcgattct ggtgatggat aacgtgctga ttccatggga aaacgtgctg 840
atctaccgcg attttgatcg ctgccgtcgc tggacgatgg aaggcggttt tgcccgtatg 900
tatccgctgc aagcctgtgt gcgcctggca gtgaaattag acttcattac ggcactgctg 960
aaaaaatcac tcgaatgtac cggcaccctg gagttccgtg gtgtgcaggc cgatctcggt 1020
gaagtggtag cgtggcgcaa caccttctgg gcattgagtg actcgatgtg ttcagaagca 1080
acgccgtggg tcaacggggc ttatttaccg gatcatgccg cactgcaaac ctatcgcgta 1140
ctggcaccaa tggcctacgc gaagatcaaa aacattatcg aacgcaacgt taccagtggc 1200
ctgatctatc tcccttccag tgcccgtgac ctgaataatc cgcagatcga ccagtatctg 1260
gcgaagtatg tgcgcggttc gaacggtatg gatcacgtcc agcgcatcaa gatcctcaaa 1320
ctgatgtggg atgctattgg cagcgaattt ggtggtcgtc acgaactgta tgaaatcaac 1380
tactccggta gccaggatga gattcgcctg cagtgtctgc gccaggcaca aaactccggc 1440
aatatggaca agatgatggc gatggttgat cgctgcctgt cggaatacga ccaggacggc 1500
tggactgtgc cgcacctgca caacaacgac gatatcaaca tgctggataa gctgctgaaa 1560
taa 1563
<210> 8
<211> 1563
<212> DNA
<213> 大肠杆菌(Escherichia coli)
<400> 8
atgaaaccag aagatttccg cgccagtacc caacgtcctt tcaccgggga agagtatctg 60
aaaagcctgc aggatggtcg cgagatctat atctatggcg agcgagtgaa agacgtcacc 120
actcatccgg catttcgtaa tgcggcagcg tctgttgccc agctgtacga cgcactgcac 180
aaaccggaga tgcaggactc tctgtgttgg aacaccgaca ccggcagcgg cggctatacc 240
cataaattct tccgcgtggc gaaaagtgcc gacgacctgc gccagcaacg cgacgccatc 300
gctgagtggt cacgcctgag ctatggctgg atgggccgta ccccagacta caaagccgct 360
ttcggttgcg cactgggcgc gaatccgggc ttttacggtc agttcgagca gaacgcccgt 420
aactggtaca cccgtattca ggaaactggc ctctacttta accacgcgat tgttaaccca 480
ccgatcgatc gtcatttgcc gaccgataaa gtgaaagacg tttacatcaa gctggaaaaa 540
gagactgacg ccgggattat cgtcagcggt gcgaaagtgg ttgccaccaa ctcggcgctg 600
actcactaca acatgattgg cttcggctcg gcagatgtga tgggcgaaaa cccggacttc 660
gcactgatgt tcgttgcgcc aatggatgcc gatggcgtga aattaatctc ccgcgcctct 720
tatgagatgg tcgcgggtgc taccggctcg ccatacgact acccgctctc cagccgcttc 780
gatgagaacg atgcgattct ggtgatggat aacgtgctga ttccatggga aaacgtgctg 840
atctaccgcg attttgatcg ctgccgtcgc tggacgatgg aaggcggttt tgcccgtatg 900
tatccgctgc aagcctgtgt gcgcctggca gtgaaattag acttcattac ggcactgctg 960
aaaaaatcac tcgaatgtac cggcaccctg gagttccgtg gtgtgcaggc cgatctcggt 1020
gaagtggtag cgtggcgcaa caccttctgg gcattgagtg actcgatgtg ttcagaagca 1080
acgccgtggg tcaacggggc ttatttaccg gatcatgccg cactgcaaac ctatcgcgta 1140
ctggcaccaa tggcctacgc gaagatcaaa aacattatcg aacgcaacgt taccagtggc 1200
ctgatctatc tcccttccag tgcccgtgac ctgaataatc cgcagatcga ccagtatctg 1260
gcgaagtatg tgcgcggttc gaacggtatg gatcacgtcc agcgcatcaa gatcctcaaa 1320
ctgatgtggg atgctattgg cagcgaattt ggtggtcgtc acgaactgta tgaaatcaac 1380
tactccggta gccaggatga gattcgcctg cagtgtctgc gccaggcaca aaactccggc 1440
aatatggaca agatgatggc gatggttgat cgctgcctgt cggaatacga ccaggacggc 1500
tggactgtgc cgcacctgca caacaacgac gatatcaaca tgctggataa gctgctgaaa 1560
taa 1563
<210> 9
<211> 1563
<212> DNA
<213> 大肠杆菌(Escherichia coli)
<400> 9
atgaaaccag aagatttccg cgccagtacc caacgtcctt tcaccgggga agagtatctg 60
aaaagcctgc aggatggtcg cgagatctat atctatggcg agcgagtgaa agacgtcacc 120
actcatccgg catttcgtaa tgcggcagcg tctgttgccc agctgtacga cgcactgcac 180
aaaccggaga tgcaggactc tctgtgttgg aacaccgaca ccggcagcgg cggctatacc 240
cataaattct tccgcgtggc gaaaagtgcc gacgacctgc gccagcaacg cgacgccatc 300
gctgagtggt cacgcctgag ctatggctgg atgggccgta ccccagacta caaagccgct 360
ttcggttgcg cactgggcgc gaatccgggc ttttacggtc agttcgagca gaacgcccgt 420
aactggtaca cccgtattca ggaaactggc ctctacttta accacgcgat tgttaaccca 480
ccgatcgatc gtcatttgcc gaccgataaa gtgaaagacg tttacatcaa gctggaaaaa 540
gagactgacg ccgggattat cgtcagcggt gcgaaagtgg ttgccaccaa ctcggcgctg 600
actcactaca acatgattgg cttcggcact ccttatgtga tgggcgaaaa cccggacttc 660
gcactgatgt tcgttgcgcc aatggatgcc gatggcgtga aattaatctc ccgcgcctct 720
tatgagatgg tcgcgggtgc taccggctcg ccatacgact acccgctctc cagccgcttc 780
gatgagaacg atgcgattct ggtgatggat aacgtgctga ttccatggga aaacgtgctg 840
atctaccgcg attttgatcg ctgccgtcgc tggacgatgg aaggcggttt tgcccgtatg 900
tatccgctgc aagcctgtgt gcgcctggca gtgaaattag acttcattac ggcactgctg 960
aaaaaatcac tcgaatgtac cggcaccctg gagttccgtg gtgtgcaggc cgatctcggt 1020
gaagtggtag cgtggcgcaa caccttctgg gcattgagtg actcgatgtg ttcagaagca 1080
acgccgtggg tcaacggggc ttatttaccg gatcatgccg cactgcaaac ctatcgcgta 1140
ctggcaccaa tggcctacgc gaagatcaaa aacattatcg aacgcaacgt taccagtggc 1200
ctgatctatc tcccttccag tgcccgtgac ctgaataatc cgcagatcga ccagtatctg 1260
gcgaagtatg tgcgcggttc gaacggtatg gatcacgtcc agcgcatcaa gatcctcaaa 1320
ctgatgtggg atgctattgg cagcgaattt ggtggtcgtc acgaactgta tgaaatcaac 1380
tactccggta gccaggatga gattcgcctg cagtgtctgc gccaggcaca aaactccggc 1440
aatatggaca agatgatggc gatggttgat cgctgcctgt cggaatacga ccaggacggc 1500
tggactgtgc cgcacctgca caacaacgac gatatcaaca tgctggataa gctgctgaaa 1560
taa 1563
<210> 10
<211> 1563
<212> DNA
<213> 大肠杆菌(Escherichia coli)
<400> 10
atgaaaccag aagatttccg cgccagtacc caacgtcctt tcaccgggga agagtatctg 60
aaaagcctgc aggatggtcg cgagatctat atctatggcg agcgagtgaa agacgtcacc 120
actcatccgg catttcgtaa tgcggcagcg tctgttgccc agctgtacga cgcactgcac 180
aaaccggaga tgcaggactc tctgtgttgg aacaccgaca ccggcagcgg cggctatacc 240
cataaattct tccgcgtggc gaaaagtgcc gacgacctgc gccagcaacg cgacgccatc 300
gctgagtggt cacgcctgag ctatggctgg atgggccgta ccccagacta caaagccgct 360
ttcggttgcg cactgggcgc gaatccgggc ttttacggtc agttcgagca gaacgcccgt 420
aactggtaca cccgtattca ggaaactggc ctctacttta accacgcgat tgttaaccca 480
ccgatcgatc gtcatttgcc gaccgataaa gtgaaagacg tttacatcaa gctggaaaaa 540
gagactgacg ccgggattat cgtcagcggt gcgaaagtgg ttgccaccaa ctcggcgctg 600
actcactaca acatgattgg cttcggcggt cgtgatgtga tgggcgaaaa cccggacttc 660
gcactgatgt tcgttgcgcc aatggatgcc gatggcgtga aattaatctc ccgcgcctct 720
tatgagatgg tcgcgggtgc taccggctcg ccatacgact acccgctctc cagccgcttc 780
gatgagaacg atgcgattct ggtgatggat aacgtgctga ttccatggga aaacgtgctg 840
atctaccgcg attttgatcg ctgccgtcgc tggacgatgg aaggcggttt tgcccgtatg 900
tatccgctgc aagcctgtgt gcgcctggca gtgaaattag acttcattac ggcactgctg 960
aaaaaatcac tcgaatgtac cggcaccctg gagttccgtg gtgtgcaggc cgatctcggt 1020
gaagtggtag cgtggcgcaa caccttctgg gcattgagtg actcgatgtg ttcagaagca 1080
acgccgtggg tcaacggggc ttatttaccg gatcatgccg cactgcaaac ctatcgcgta 1140
ctggcaccaa tggcctacgc gaagatcaaa aacattatcg aacgcaacgt taccagtggc 1200
ctgatctatc tcccttccag tgcccgtgac ctgaataatc cgcagatcga ccagtatctg 1260
gcgaagtatg tgcgcggttc gaacggtatg gatcacgtcc agcgcatcaa gatcctcaaa 1320
ctgatgtggg atgctattgg cagcgaattt ggtggtcgtc acgaactgta tgaaatcaac 1380
tactccggta gccaggatga gattcgcctg cagtgtctgc gccaggcaca aaactccggc 1440
aatatggaca agatgatggc gatggttgat cgctgcctgt cggaatacga ccaggacggc 1500
tggactgtgc cgcacctgca caacaacgac gatatcaaca tgctggataa gctgctgaaa 1560
taa 1563
<210> 11
<211> 1563
<212> DNA
<213> 大肠杆菌(Escherichia coli)
<400> 11
atgaaaccag aagatttccg cgccagtacc caacgtcctt tcaccgggga agagtatctg 60
aaaagcctgc aggatggtcg cgagatctat atctatggcg agcgagtgaa agacgtcacc 120
actcatccgg catttcgtaa tgcggcagcg tctgttgccc agctgtacga cgcactgcac 180
aaaccggaga tgcaggactc tctgtgttgg aacaccgaca ccggcagcgg cggctatacc 240
cataaattct tccgcgtggc gaaaagtgcc gacgacctgc gccagcaacg cgacgccatc 300
gctgagtggt cacgcctgag ctatggctgg atgggccgta ccccagacta caaagccgct 360
ttcggttgcg cactgggcgc gaatccgggc ttttacggtc agttcgagca gaacgcccgt 420
aactggtaca cccgtattca ggaaactggc ctctacttta accacgcgat tgttaaccca 480
ccgatcgatc gtcatttgcc gaccgataaa gtgaaagacg tttacatcaa gctggaaaaa 540
gagactgacg ccgggattat cgtcagcggt gcgaaagtgg ttgccaccaa ctcggcgctg 600
actcactaca acatgattgg cttcggcggt ccttttgtga tgggcgaaaa cccggacttc 660
gcactgatgt tcgttgcgcc aatggatgcc gatggcgtga aattaatctc ccgcgcctct 720
tatgagatgg tcgcgggtgc taccggctcg ccatacgact acccgctctc cagccgcttc 780
gatgagaacg atgcgattct ggtgatggat aacgtgctga ttccatggga aaacgtgctg 840
atctaccgcg attttgatcg ctgccgtcgc tggacgatgg aaggcggttt tgcccgtatg 900
tatccgctgc aagcctgtgt gcgcctggca gtgaaattag acttcattac ggcactgctg 960
aaaaaatcac tcgaatgtac cggcaccctg gagttccgtg gtgtgcaggc cgatctcggt 1020
gaagtggtag cgtggcgcaa caccttctgg gcattgagtg actcgatgtg ttcagaagca 1080
acgccgtggg tcaacggggc ttatttaccg gatcatgccg cactgcaaac ctatcgcgta 1140
ctggcaccaa tggcctacgc gaagatcaaa aacattatcg aacgcaacgt taccagtggc 1200
ctgatctatc tcccttccag tgcccgtgac ctgaataatc cgcagatcga ccagtatctg 1260
gcgaagtatg tgcgcggttc gaacggtatg gatcacgtcc agcgcatcaa gatcctcaaa 1320
ctgatgtggg atgctattgg cagcgaattt ggtggtcgtc acgaactgta tgaaatcaac 1380
tactccggta gccaggatga gattcgcctg cagtgtctgc gccaggcaca aaactccggc 1440
aatatggaca agatgatggc gatggttgat cgctgcctgt cggaatacga ccaggacggc 1500
tggactgtgc cgcacctgca caacaacgac gatatcaaca tgctggataa gctgctgaaa 1560
taa 1563
<210> 12
<211> 520
<212> PRT
<213> 大肠杆菌(Escherichia coli)
<400> 12
Met Lys Pro Glu Asp Phe Arg Ala Ser Thr Gln Arg Pro Phe Thr Gly
1 5 10 15
Glu Glu Tyr Leu Lys Ser Leu Gln Asp Gly Arg Glu Ile Tyr Ile Tyr
20 25 30
Gly Glu Arg Val Lys Asp Val Thr Thr His Pro Ala Phe Arg Asn Ala
35 40 45
Ala Ala Ser Val Ala Gln Leu Tyr Asp Ala Leu His Lys Pro Glu Met
50 55 60
Gln Asp Ser Leu Cys Trp Asn Thr Asp Thr Gly Ser Gly Gly Tyr Thr
65 70 75 80
His Lys Phe Phe Arg Val Ala Lys Ser Ala Asp Asp Leu Arg Gln Gln
85 90 95
Arg Asp Ala Ile Ala Glu Trp Ser Arg Leu Ser Tyr Gly Trp Met Gly
100 105 110
Arg Thr Pro Asp Tyr Lys Ala Ala Phe Gly Cys Ala Leu Gly Ala Asn
115 120 125
Pro Gly Phe Tyr Gly Gln Phe Glu Gln Asn Ala Arg Asn Trp Tyr Thr
130 135 140
Arg Ile Gln Glu Thr Gly Leu Tyr Phe Asn His Ala Ile Val Asn Pro
145 150 155 160
Pro Ile Asp Arg His Leu Pro Thr Asp Lys Val Lys Asp Val Tyr Ile
165 170 175
Lys Leu Glu Lys Glu Thr Asp Ala Gly Ile Ile Val Ser Gly Ala Lys
180 185 190
Val Val Ala Thr Asn Ser Ala Leu Thr His Tyr Asn Met Ile Gly Phe
195 200 205
Gly Thr Leu Glu Val Met Gly Glu Asn Pro Asp Phe Ala Leu Met Phe
210 215 220
Val Ala Pro Met Asp Ala Asp Gly Val Lys Leu Ile Ser Arg Ala Ser
225 230 235 240
Tyr Glu Met Val Ala Gly Ala Thr Gly Ser Pro Tyr Asp Tyr Pro Leu
245 250 255
Ser Ser Arg Phe Asp Glu Asn Asp Ala Ile Leu Val Met Asp Asn Val
260 265 270
Leu Ile Pro Trp Glu Asn Val Leu Ile Tyr Arg Asp Phe Asp Arg Cys
275 280 285
Arg Arg Trp Thr Met Glu Gly Gly Phe Ala Arg Met Tyr Pro Leu Gln
290 295 300
Ala Cys Val Arg Leu Ala Val Lys Leu Asp Phe Ile Thr Ala Leu Leu
305 310 315 320
Lys Lys Ser Leu Glu Cys Thr Gly Thr Leu Glu Phe Arg Gly Val Gln
325 330 335
Ala Asp Leu Gly Glu Val Val Ala Trp Arg Asn Thr Phe Trp Ala Leu
340 345 350
Ser Asp Ser Met Cys Ser Glu Ala Thr Pro Trp Val Asn Gly Ala Tyr
355 360 365
Leu Pro Asp His Ala Ala Leu Gln Thr Tyr Arg Val Leu Ala Pro Met
370 375 380
Ala Tyr Ala Lys Ile Lys Asn Ile Ile Glu Arg Asn Val Thr Ser Gly
385 390 395 400
Leu Ile Tyr Leu Pro Ser Ser Ala Arg Asp Leu Asn Asn Pro Gln Ile
405 410 415
Asp Gln Tyr Leu Ala Lys Tyr Val Arg Gly Ser Asn Gly Met Asp His
420 425 430
Val Gln Arg Ile Lys Ile Leu Lys Leu Met Trp Asp Ala Ile Gly Ser
435 440 445
Glu Phe Gly Gly Arg His Glu Leu Tyr Glu Ile Asn Tyr Ser Gly Ser
450 455 460
Gln Asp Glu Ile Arg Leu Gln Cys Leu Arg Gln Ala Gln Asn Ser Gly
465 470 475 480
Asn Met Asp Lys Met Met Ala Met Val Asp Arg Cys Leu Ser Glu Tyr
485 490 495
Asp Gln Asp Gly Trp Thr Val Pro His Leu His Asn Asn Asp Asp Ile
500 505 510
Asn Met Leu Asp Lys Leu Leu Lys
515 520
<210> 13
<211> 520
<212> PRT
<213> 大肠杆菌(Escherichia coli)
<400> 13
Met Lys Pro Glu Asp Phe Arg Ala Ser Thr Gln Arg Pro Phe Thr Gly
1 5 10 15
Glu Glu Tyr Leu Lys Ser Leu Gln Asp Gly Arg Glu Ile Tyr Ile Tyr
20 25 30
Gly Glu Arg Val Lys Asp Val Thr Thr His Pro Ala Phe Arg Asn Ala
35 40 45
Ala Ala Ser Val Ala Gln Leu Tyr Asp Ala Leu His Lys Pro Glu Met
50 55 60
Gln Asp Ser Leu Cys Trp Asn Thr Asp Thr Gly Ser Gly Gly Tyr Thr
65 70 75 80
His Lys Phe Phe Arg Val Ala Lys Ser Ala Asp Asp Leu Arg Gln Gln
85 90 95
Arg Asp Ala Ile Ala Glu Trp Ser Arg Leu Ser Tyr Gly Trp Met Gly
100 105 110
Arg Thr Pro Asp Tyr Lys Ala Ala Phe Gly Cys Ala Leu Gly Ala Asn
115 120 125
Pro Gly Phe Tyr Gly Gln Phe Glu Gln Asn Ala Arg Asn Trp Tyr Thr
130 135 140
Arg Ile Gln Glu Thr Gly Leu Tyr Phe Asn His Ala Ile Val Asn Pro
145 150 155 160
Pro Ile Asp Arg His Leu Pro Thr Asp Lys Val Lys Asp Val Tyr Ile
165 170 175
Lys Leu Glu Lys Glu Thr Asp Ala Gly Ile Ile Val Ser Gly Ala Lys
180 185 190
Val Val Ala Thr Asn Ser Ala Leu Thr His Tyr Asn Met Ile Gly Phe
195 200 205
Gly Ser Gly Tyr Val Met Gly Glu Asn Pro Asp Phe Ala Leu Met Phe
210 215 220
Val Ala Pro Met Asp Ala Asp Gly Val Lys Leu Ile Ser Arg Ala Ser
225 230 235 240
Tyr Glu Met Val Ala Gly Ala Thr Gly Ser Pro Tyr Asp Tyr Pro Leu
245 250 255
Ser Ser Arg Phe Asp Glu Asn Asp Ala Ile Leu Val Met Asp Asn Val
260 265 270
Leu Ile Pro Trp Glu Asn Val Leu Ile Tyr Arg Asp Phe Asp Arg Cys
275 280 285
Arg Arg Trp Thr Met Glu Gly Gly Phe Ala Arg Met Tyr Pro Leu Gln
290 295 300
Ala Cys Val Arg Leu Ala Val Lys Leu Asp Phe Ile Thr Ala Leu Leu
305 310 315 320
Lys Lys Ser Leu Glu Cys Thr Gly Thr Leu Glu Phe Arg Gly Val Gln
325 330 335
Ala Asp Leu Gly Glu Val Val Ala Trp Arg Asn Thr Phe Trp Ala Leu
340 345 350
Ser Asp Ser Met Cys Ser Glu Ala Thr Pro Trp Val Asn Gly Ala Tyr
355 360 365
Leu Pro Asp His Ala Ala Leu Gln Thr Tyr Arg Val Leu Ala Pro Met
370 375 380
Ala Tyr Ala Lys Ile Lys Asn Ile Ile Glu Arg Asn Val Thr Ser Gly
385 390 395 400
Leu Ile Tyr Leu Pro Ser Ser Ala Arg Asp Leu Asn Asn Pro Gln Ile
405 410 415
Asp Gln Tyr Leu Ala Lys Tyr Val Arg Gly Ser Asn Gly Met Asp His
420 425 430
Val Gln Arg Ile Lys Ile Leu Lys Leu Met Trp Asp Ala Ile Gly Ser
435 440 445
Glu Phe Gly Gly Arg His Glu Leu Tyr Glu Ile Asn Tyr Ser Gly Ser
450 455 460
Gln Asp Glu Ile Arg Leu Gln Cys Leu Arg Gln Ala Gln Asn Ser Gly
465 470 475 480
Asn Met Asp Lys Met Met Ala Met Val Asp Arg Cys Leu Ser Glu Tyr
485 490 495
Asp Gln Asp Gly Trp Thr Val Pro His Leu His Asn Asn Asp Asp Ile
500 505 510
Asn Met Leu Asp Lys Leu Leu Lys
515 520
<210> 14
<211> 520
<212> PRT
<213> 大肠杆菌(Escherichia coli)
<400> 14
Met Lys Pro Glu Asp Phe Arg Ala Ser Thr Gln Arg Pro Phe Thr Gly
1 5 10 15
Glu Glu Tyr Leu Lys Ser Leu Gln Asp Gly Arg Glu Ile Tyr Ile Tyr
20 25 30
Gly Glu Arg Val Lys Asp Val Thr Thr His Pro Ala Phe Arg Asn Ala
35 40 45
Ala Ala Ser Val Ala Gln Leu Tyr Asp Ala Leu His Lys Pro Glu Met
50 55 60
Gln Asp Ser Leu Cys Trp Asn Thr Asp Thr Gly Ser Gly Gly Tyr Thr
65 70 75 80
His Lys Phe Phe Arg Val Ala Lys Ser Ala Asp Asp Leu Arg Gln Gln
85 90 95
Arg Asp Ala Ile Ala Glu Trp Ser Arg Leu Ser Tyr Gly Trp Met Gly
100 105 110
Arg Thr Pro Asp Tyr Lys Ala Ala Phe Gly Cys Ala Leu Gly Ala Asn
115 120 125
Pro Gly Phe Tyr Gly Gln Phe Glu Gln Asn Ala Arg Asn Trp Tyr Thr
130 135 140
Arg Ile Gln Glu Thr Gly Leu Tyr Phe Asn His Ala Ile Val Asn Pro
145 150 155 160
Pro Ile Asp Arg His Leu Pro Thr Asp Lys Val Lys Asp Val Tyr Ile
165 170 175
Lys Leu Glu Lys Glu Thr Asp Ala Gly Ile Ile Val Ser Gly Ala Lys
180 185 190
Val Val Ala Thr Asn Ser Ala Leu Thr His Tyr Asn Met Ile Gly Phe
195 200 205
Gly Thr Met Gly Val Met Gly Glu Asn Pro Asp Phe Ala Leu Met Phe
210 215 220
Val Ala Pro Met Asp Ala Asp Gly Val Lys Leu Ile Ser Arg Ala Ser
225 230 235 240
Tyr Glu Met Val Ala Gly Ala Thr Gly Ser Pro Tyr Asp Tyr Pro Leu
245 250 255
Ser Ser Arg Phe Asp Glu Asn Asp Ala Ile Leu Val Met Asp Asn Val
260 265 270
Leu Ile Pro Trp Glu Asn Val Leu Ile Tyr Arg Asp Phe Asp Arg Cys
275 280 285
Arg Arg Trp Thr Met Glu Gly Gly Phe Ala Arg Met Tyr Pro Leu Gln
290 295 300
Ala Cys Val Arg Leu Ala Val Lys Leu Asp Phe Ile Thr Ala Leu Leu
305 310 315 320
Lys Lys Ser Leu Glu Cys Thr Gly Thr Leu Glu Phe Arg Gly Val Gln
325 330 335
Ala Asp Leu Gly Glu Val Val Ala Trp Arg Asn Thr Phe Trp Ala Leu
340 345 350
Ser Asp Ser Met Cys Ser Glu Ala Thr Pro Trp Val Asn Gly Ala Tyr
355 360 365
Leu Pro Asp His Ala Ala Leu Gln Thr Tyr Arg Val Leu Ala Pro Met
370 375 380
Ala Tyr Ala Lys Ile Lys Asn Ile Ile Glu Arg Asn Val Thr Ser Gly
385 390 395 400
Leu Ile Tyr Leu Pro Ser Ser Ala Arg Asp Leu Asn Asn Pro Gln Ile
405 410 415
Asp Gln Tyr Leu Ala Lys Tyr Val Arg Gly Ser Asn Gly Met Asp His
420 425 430
Val Gln Arg Ile Lys Ile Leu Lys Leu Met Trp Asp Ala Ile Gly Ser
435 440 445
Glu Phe Gly Gly Arg His Glu Leu Tyr Glu Ile Asn Tyr Ser Gly Ser
450 455 460
Gln Asp Glu Ile Arg Leu Gln Cys Leu Arg Gln Ala Gln Asn Ser Gly
465 470 475 480
Asn Met Asp Lys Met Met Ala Met Val Asp Arg Cys Leu Ser Glu Tyr
485 490 495
Asp Gln Asp Gly Trp Thr Val Pro His Leu His Asn Asn Asp Asp Ile
500 505 510
Asn Met Leu Asp Lys Leu Leu Lys
515 520
<210> 15
<211> 520
<212> PRT
<213> 大肠杆菌(Escherichia coli)
<400> 15
Met Lys Pro Glu Asp Phe Arg Ala Ser Thr Gln Arg Pro Phe Thr Gly
1 5 10 15
Glu Glu Tyr Leu Lys Ser Leu Gln Asp Gly Arg Glu Ile Tyr Ile Tyr
20 25 30
Gly Glu Arg Val Lys Asp Val Thr Thr His Pro Ala Phe Arg Asn Ala
35 40 45
Ala Ala Ser Val Ala Gln Leu Tyr Asp Ala Leu His Lys Pro Glu Met
50 55 60
Gln Asp Ser Leu Cys Trp Asn Thr Asp Thr Gly Ser Gly Gly Tyr Thr
65 70 75 80
His Lys Phe Phe Arg Val Ala Lys Ser Ala Asp Asp Leu Arg Gln Gln
85 90 95
Arg Asp Ala Ile Ala Glu Trp Ser Arg Leu Ser Tyr Gly Trp Met Gly
100 105 110
Arg Thr Pro Asp Tyr Lys Ala Ala Phe Gly Cys Ala Leu Gly Ala Asn
115 120 125
Pro Gly Phe Tyr Gly Gln Phe Glu Gln Asn Ala Arg Asn Trp Tyr Thr
130 135 140
Arg Ile Gln Glu Thr Gly Leu Tyr Phe Asn His Ala Ile Val Asn Pro
145 150 155 160
Pro Ile Asp Arg His Leu Pro Thr Asp Lys Val Lys Asp Val Tyr Ile
165 170 175
Lys Leu Glu Lys Glu Thr Asp Ala Gly Ile Ile Val Ser Gly Ala Lys
180 185 190
Val Val Ala Thr Asn Ser Ala Leu Thr His Tyr Asn Met Ile Gly Phe
195 200 205
Gly Trp Glu Gly Val Met Gly Glu Asn Pro Asp Phe Ala Leu Met Phe
210 215 220
Val Ala Pro Met Asp Ala Asp Gly Val Lys Leu Ile Ser Arg Ala Ser
225 230 235 240
Tyr Glu Met Val Ala Gly Ala Thr Gly Ser Pro Tyr Asp Tyr Pro Leu
245 250 255
Ser Ser Arg Phe Asp Glu Asn Asp Ala Ile Leu Val Met Asp Asn Val
260 265 270
Leu Ile Pro Trp Glu Asn Val Leu Ile Tyr Arg Asp Phe Asp Arg Cys
275 280 285
Arg Arg Trp Thr Met Glu Gly Gly Phe Ala Arg Met Tyr Pro Leu Gln
290 295 300
Ala Cys Val Arg Leu Ala Val Lys Leu Asp Phe Ile Thr Ala Leu Leu
305 310 315 320
Lys Lys Ser Leu Glu Cys Thr Gly Thr Leu Glu Phe Arg Gly Val Gln
325 330 335
Ala Asp Leu Gly Glu Val Val Ala Trp Arg Asn Thr Phe Trp Ala Leu
340 345 350
Ser Asp Ser Met Cys Ser Glu Ala Thr Pro Trp Val Asn Gly Ala Tyr
355 360 365
Leu Pro Asp His Ala Ala Leu Gln Thr Tyr Arg Val Leu Ala Pro Met
370 375 380
Ala Tyr Ala Lys Ile Lys Asn Ile Ile Glu Arg Asn Val Thr Ser Gly
385 390 395 400
Leu Ile Tyr Leu Pro Ser Ser Ala Arg Asp Leu Asn Asn Pro Gln Ile
405 410 415
Asp Gln Tyr Leu Ala Lys Tyr Val Arg Gly Ser Asn Gly Met Asp His
420 425 430
Val Gln Arg Ile Lys Ile Leu Lys Leu Met Trp Asp Ala Ile Gly Ser
435 440 445
Glu Phe Gly Gly Arg His Glu Leu Tyr Glu Ile Asn Tyr Ser Gly Ser
450 455 460
Gln Asp Glu Ile Arg Leu Gln Cys Leu Arg Gln Ala Gln Asn Ser Gly
465 470 475 480
Asn Met Asp Lys Met Met Ala Met Val Asp Arg Cys Leu Ser Glu Tyr
485 490 495
Asp Gln Asp Gly Trp Thr Val Pro His Leu His Asn Asn Asp Asp Ile
500 505 510
Asn Met Leu Asp Lys Leu Leu Lys
515 520
<210> 16
<211> 520
<212> PRT
<213> 大肠杆菌(Escherichia coli)
<400> 16
Met Lys Pro Glu Asp Phe Arg Ala Ser Thr Gln Arg Pro Phe Thr Gly
1 5 10 15
Glu Glu Tyr Leu Lys Ser Leu Gln Asp Gly Arg Glu Ile Tyr Ile Tyr
20 25 30
Gly Glu Arg Val Lys Asp Val Thr Thr His Pro Ala Phe Arg Asn Ala
35 40 45
Ala Ala Ser Val Ala Gln Leu Tyr Asp Ala Leu His Lys Pro Glu Met
50 55 60
Gln Asp Ser Leu Cys Trp Asn Thr Asp Thr Gly Ser Gly Gly Tyr Thr
65 70 75 80
His Lys Phe Phe Arg Val Ala Lys Ser Ala Asp Asp Leu Arg Gln Gln
85 90 95
Arg Asp Ala Ile Ala Glu Trp Ser Arg Leu Ser Tyr Gly Trp Met Gly
100 105 110
Arg Thr Pro Asp Tyr Lys Ala Ala Phe Gly Cys Ala Leu Gly Ala Asn
115 120 125
Pro Gly Phe Tyr Gly Gln Phe Glu Gln Asn Ala Arg Asn Trp Tyr Thr
130 135 140
Arg Ile Gln Glu Thr Gly Leu Tyr Phe Asn His Ala Ile Val Asn Pro
145 150 155 160
Pro Ile Asp Arg His Leu Pro Thr Asp Lys Val Lys Asp Val Tyr Ile
165 170 175
Lys Leu Glu Lys Glu Thr Asp Ala Gly Ile Ile Val Ser Gly Ala Lys
180 185 190
Val Val Ala Thr Asn Ser Ala Leu Thr His Tyr Asn Met Ile Gly Phe
195 200 205
Gly Asn Pro Arg Val Met Gly Glu Asn Pro Asp Phe Ala Leu Met Phe
210 215 220
Val Ala Pro Met Asp Ala Asp Gly Val Lys Leu Ile Ser Arg Ala Ser
225 230 235 240
Tyr Glu Met Val Ala Gly Ala Thr Gly Ser Pro Tyr Asp Tyr Pro Leu
245 250 255
Ser Ser Arg Phe Asp Glu Asn Asp Ala Ile Leu Val Met Asp Asn Val
260 265 270
Leu Ile Pro Trp Glu Asn Val Leu Ile Tyr Arg Asp Phe Asp Arg Cys
275 280 285
Arg Arg Trp Thr Met Glu Gly Gly Phe Ala Arg Met Tyr Pro Leu Gln
290 295 300
Ala Cys Val Arg Leu Ala Val Lys Leu Asp Phe Ile Thr Ala Leu Leu
305 310 315 320
Lys Lys Ser Leu Glu Cys Thr Gly Thr Leu Glu Phe Arg Gly Val Gln
325 330 335
Ala Asp Leu Gly Glu Val Val Ala Trp Arg Asn Thr Phe Trp Ala Leu
340 345 350
Ser Asp Ser Met Cys Ser Glu Ala Thr Pro Trp Val Asn Gly Ala Tyr
355 360 365
Leu Pro Asp His Ala Ala Leu Gln Thr Tyr Arg Val Leu Ala Pro Met
370 375 380
Ala Tyr Ala Lys Ile Lys Asn Ile Ile Glu Arg Asn Val Thr Ser Gly
385 390 395 400
Leu Ile Tyr Leu Pro Ser Ser Ala Arg Asp Leu Asn Asn Pro Gln Ile
405 410 415
Asp Gln Tyr Leu Ala Lys Tyr Val Arg Gly Ser Asn Gly Met Asp His
420 425 430
Val Gln Arg Ile Lys Ile Leu Lys Leu Met Trp Asp Ala Ile Gly Ser
435 440 445
Glu Phe Gly Gly Arg His Glu Leu Tyr Glu Ile Asn Tyr Ser Gly Ser
450 455 460
Gln Asp Glu Ile Arg Leu Gln Cys Leu Arg Gln Ala Gln Asn Ser Gly
465 470 475 480
Asn Met Asp Lys Met Met Ala Met Val Asp Arg Cys Leu Ser Glu Tyr
485 490 495
Asp Gln Asp Gly Trp Thr Val Pro His Leu His Asn Asn Asp Asp Ile
500 505 510
Asn Met Leu Asp Lys Leu Leu Lys
515 520
<210> 17
<211> 520
<212> PRT
<213> 大肠杆菌(Escherichia coli)
<400> 17
Met Lys Pro Glu Asp Phe Arg Ala Ser Thr Gln Arg Pro Phe Thr Gly
1 5 10 15
Glu Glu Tyr Leu Lys Ser Leu Gln Asp Gly Arg Glu Ile Tyr Ile Tyr
20 25 30
Gly Glu Arg Val Lys Asp Val Thr Thr His Pro Ala Phe Arg Asn Ala
35 40 45
Ala Ala Ser Val Ala Gln Leu Tyr Asp Ala Leu His Lys Pro Glu Met
50 55 60
Gln Asp Ser Leu Cys Trp Asn Thr Asp Thr Gly Ser Gly Gly Tyr Thr
65 70 75 80
His Lys Phe Phe Arg Val Ala Lys Ser Ala Asp Asp Leu Arg Gln Gln
85 90 95
Arg Asp Ala Ile Ala Glu Trp Ser Arg Leu Ser Tyr Gly Trp Met Gly
100 105 110
Arg Thr Pro Asp Tyr Lys Ala Ala Phe Gly Cys Ala Leu Gly Ala Asn
115 120 125
Pro Gly Phe Tyr Gly Gln Phe Glu Gln Asn Ala Arg Asn Trp Tyr Thr
130 135 140
Arg Ile Gln Glu Thr Gly Leu Tyr Phe Asn His Ala Ile Val Asn Pro
145 150 155 160
Pro Ile Asp Arg His Leu Pro Thr Asp Lys Val Lys Asp Val Tyr Ile
165 170 175
Lys Leu Glu Lys Glu Thr Asp Ala Gly Ile Ile Val Ser Gly Ala Lys
180 185 190
Val Val Ala Thr Asn Ser Ala Leu Thr His Tyr Asn Met Ile Gly Phe
195 200 205
Gly Ser His Asp Val Met Gly Glu Asn Pro Asp Phe Ala Leu Met Phe
210 215 220
Val Ala Pro Met Asp Ala Asp Gly Val Lys Leu Ile Ser Arg Ala Ser
225 230 235 240
Tyr Glu Met Val Ala Gly Ala Thr Gly Ser Pro Tyr Asp Tyr Pro Leu
245 250 255
Ser Ser Arg Phe Asp Glu Asn Asp Ala Ile Leu Val Met Asp Asn Val
260 265 270
Leu Ile Pro Trp Glu Asn Val Leu Ile Tyr Arg Asp Phe Asp Arg Cys
275 280 285
Arg Arg Trp Thr Met Glu Gly Gly Phe Ala Arg Met Tyr Pro Leu Gln
290 295 300
Ala Cys Val Arg Leu Ala Val Lys Leu Asp Phe Ile Thr Ala Leu Leu
305 310 315 320
Lys Lys Ser Leu Glu Cys Thr Gly Thr Leu Glu Phe Arg Gly Val Gln
325 330 335
Ala Asp Leu Gly Glu Val Val Ala Trp Arg Asn Thr Phe Trp Ala Leu
340 345 350
Ser Asp Ser Met Cys Ser Glu Ala Thr Pro Trp Val Asn Gly Ala Tyr
355 360 365
Leu Pro Asp His Ala Ala Leu Gln Thr Tyr Arg Val Leu Ala Pro Met
370 375 380
Ala Tyr Ala Lys Ile Lys Asn Ile Ile Glu Arg Asn Val Thr Ser Gly
385 390 395 400
Leu Ile Tyr Leu Pro Ser Ser Ala Arg Asp Leu Asn Asn Pro Gln Ile
405 410 415
Asp Gln Tyr Leu Ala Lys Tyr Val Arg Gly Ser Asn Gly Met Asp His
420 425 430
Val Gln Arg Ile Lys Ile Leu Lys Leu Met Trp Asp Ala Ile Gly Ser
435 440 445
Glu Phe Gly Gly Arg His Glu Leu Tyr Glu Ile Asn Tyr Ser Gly Ser
450 455 460
Gln Asp Glu Ile Arg Leu Gln Cys Leu Arg Gln Ala Gln Asn Ser Gly
465 470 475 480
Asn Met Asp Lys Met Met Ala Met Val Asp Arg Cys Leu Ser Glu Tyr
485 490 495
Asp Gln Asp Gly Trp Thr Val Pro His Leu His Asn Asn Asp Asp Ile
500 505 510
Asn Met Leu Asp Lys Leu Leu Lys
515 520
<210> 18
<211> 520
<212> PRT
<213> 大肠杆菌(Escherichia coli)
<400> 18
Met Lys Pro Glu Asp Phe Arg Ala Ser Thr Gln Arg Pro Phe Thr Gly
1 5 10 15
Glu Glu Tyr Leu Lys Ser Leu Gln Asp Gly Arg Glu Ile Tyr Ile Tyr
20 25 30
Gly Glu Arg Val Lys Asp Val Thr Thr His Pro Ala Phe Arg Asn Ala
35 40 45
Ala Ala Ser Val Ala Gln Leu Tyr Asp Ala Leu His Lys Pro Glu Met
50 55 60
Gln Asp Ser Leu Cys Trp Asn Thr Asp Thr Gly Ser Gly Gly Tyr Thr
65 70 75 80
His Lys Phe Phe Arg Val Ala Lys Ser Ala Asp Asp Leu Arg Gln Gln
85 90 95
Arg Asp Ala Ile Ala Glu Trp Ser Arg Leu Ser Tyr Gly Trp Met Gly
100 105 110
Arg Thr Pro Asp Tyr Lys Ala Ala Phe Gly Cys Ala Leu Gly Ala Asn
115 120 125
Pro Gly Phe Tyr Gly Gln Phe Glu Gln Asn Ala Arg Asn Trp Tyr Thr
130 135 140
Arg Ile Gln Glu Thr Gly Leu Tyr Phe Asn His Ala Ile Val Asn Pro
145 150 155 160
Pro Ile Asp Arg His Leu Pro Thr Asp Lys Val Lys Asp Val Tyr Ile
165 170 175
Lys Leu Glu Lys Glu Thr Asp Ala Gly Ile Ile Val Ser Gly Ala Lys
180 185 190
Val Val Ala Thr Asn Ser Ala Leu Thr His Tyr Asn Met Ile Gly Phe
195 200 205
Gly Thr Pro Tyr Val Met Gly Glu Asn Pro Asp Phe Ala Leu Met Phe
210 215 220
Val Ala Pro Met Asp Ala Asp Gly Val Lys Leu Ile Ser Arg Ala Ser
225 230 235 240
Tyr Glu Met Val Ala Gly Ala Thr Gly Ser Pro Tyr Asp Tyr Pro Leu
245 250 255
Ser Ser Arg Phe Asp Glu Asn Asp Ala Ile Leu Val Met Asp Asn Val
260 265 270
Leu Ile Pro Trp Glu Asn Val Leu Ile Tyr Arg Asp Phe Asp Arg Cys
275 280 285
Arg Arg Trp Thr Met Glu Gly Gly Phe Ala Arg Met Tyr Pro Leu Gln
290 295 300
Ala Cys Val Arg Leu Ala Val Lys Leu Asp Phe Ile Thr Ala Leu Leu
305 310 315 320
Lys Lys Ser Leu Glu Cys Thr Gly Thr Leu Glu Phe Arg Gly Val Gln
325 330 335
Ala Asp Leu Gly Glu Val Val Ala Trp Arg Asn Thr Phe Trp Ala Leu
340 345 350
Ser Asp Ser Met Cys Ser Glu Ala Thr Pro Trp Val Asn Gly Ala Tyr
355 360 365
Leu Pro Asp His Ala Ala Leu Gln Thr Tyr Arg Val Leu Ala Pro Met
370 375 380
Ala Tyr Ala Lys Ile Lys Asn Ile Ile Glu Arg Asn Val Thr Ser Gly
385 390 395 400
Leu Ile Tyr Leu Pro Ser Ser Ala Arg Asp Leu Asn Asn Pro Gln Ile
405 410 415
Asp Gln Tyr Leu Ala Lys Tyr Val Arg Gly Ser Asn Gly Met Asp His
420 425 430
Val Gln Arg Ile Lys Ile Leu Lys Leu Met Trp Asp Ala Ile Gly Ser
435 440 445
Glu Phe Gly Gly Arg His Glu Leu Tyr Glu Ile Asn Tyr Ser Gly Ser
450 455 460
Gln Asp Glu Ile Arg Leu Gln Cys Leu Arg Gln Ala Gln Asn Ser Gly
465 470 475 480
Asn Met Asp Lys Met Met Ala Met Val Asp Arg Cys Leu Ser Glu Tyr
485 490 495
Asp Gln Asp Gly Trp Thr Val Pro His Leu His Asn Asn Asp Asp Ile
500 505 510
Asn Met Leu Asp Lys Leu Leu Lys
515 520
<210> 19
<211> 520
<212> PRT
<213> 大肠杆菌(Escherichia coli)
<400> 19
Met Lys Pro Glu Asp Phe Arg Ala Ser Thr Gln Arg Pro Phe Thr Gly
1 5 10 15
Glu Glu Tyr Leu Lys Ser Leu Gln Asp Gly Arg Glu Ile Tyr Ile Tyr
20 25 30
Gly Glu Arg Val Lys Asp Val Thr Thr His Pro Ala Phe Arg Asn Ala
35 40 45
Ala Ala Ser Val Ala Gln Leu Tyr Asp Ala Leu His Lys Pro Glu Met
50 55 60
Gln Asp Ser Leu Cys Trp Asn Thr Asp Thr Gly Ser Gly Gly Tyr Thr
65 70 75 80
His Lys Phe Phe Arg Val Ala Lys Ser Ala Asp Asp Leu Arg Gln Gln
85 90 95
Arg Asp Ala Ile Ala Glu Trp Ser Arg Leu Ser Tyr Gly Trp Met Gly
100 105 110
Arg Thr Pro Asp Tyr Lys Ala Ala Phe Gly Cys Ala Leu Gly Ala Asn
115 120 125
Pro Gly Phe Tyr Gly Gln Phe Glu Gln Asn Ala Arg Asn Trp Tyr Thr
130 135 140
Arg Ile Gln Glu Thr Gly Leu Tyr Phe Asn His Ala Ile Val Asn Pro
145 150 155 160
Pro Ile Asp Arg His Leu Pro Thr Asp Lys Val Lys Asp Val Tyr Ile
165 170 175
Lys Leu Glu Lys Glu Thr Asp Ala Gly Ile Ile Val Ser Gly Ala Lys
180 185 190
Val Val Ala Thr Asn Ser Ala Leu Thr His Tyr Asn Met Ile Gly Phe
195 200 205
Gly Gly Arg Asp Val Met Gly Glu Asn Pro Asp Phe Ala Leu Met Phe
210 215 220
Val Ala Pro Met Asp Ala Asp Gly Val Lys Leu Ile Ser Arg Ala Ser
225 230 235 240
Tyr Glu Met Val Ala Gly Ala Thr Gly Ser Pro Tyr Asp Tyr Pro Leu
245 250 255
Ser Ser Arg Phe Asp Glu Asn Asp Ala Ile Leu Val Met Asp Asn Val
260 265 270
Leu Ile Pro Trp Glu Asn Val Leu Ile Tyr Arg Asp Phe Asp Arg Cys
275 280 285
Arg Arg Trp Thr Met Glu Gly Gly Phe Ala Arg Met Tyr Pro Leu Gln
290 295 300
Ala Cys Val Arg Leu Ala Val Lys Leu Asp Phe Ile Thr Ala Leu Leu
305 310 315 320
Lys Lys Ser Leu Glu Cys Thr Gly Thr Leu Glu Phe Arg Gly Val Gln
325 330 335
Ala Asp Leu Gly Glu Val Val Ala Trp Arg Asn Thr Phe Trp Ala Leu
340 345 350
Ser Asp Ser Met Cys Ser Glu Ala Thr Pro Trp Val Asn Gly Ala Tyr
355 360 365
Leu Pro Asp His Ala Ala Leu Gln Thr Tyr Arg Val Leu Ala Pro Met
370 375 380
Ala Tyr Ala Lys Ile Lys Asn Ile Ile Glu Arg Asn Val Thr Ser Gly
385 390 395 400
Leu Ile Tyr Leu Pro Ser Ser Ala Arg Asp Leu Asn Asn Pro Gln Ile
405 410 415
Asp Gln Tyr Leu Ala Lys Tyr Val Arg Gly Ser Asn Gly Met Asp His
420 425 430
Val Gln Arg Ile Lys Ile Leu Lys Leu Met Trp Asp Ala Ile Gly Ser
435 440 445
Glu Phe Gly Gly Arg His Glu Leu Tyr Glu Ile Asn Tyr Ser Gly Ser
450 455 460
Gln Asp Glu Ile Arg Leu Gln Cys Leu Arg Gln Ala Gln Asn Ser Gly
465 470 475 480
Asn Met Asp Lys Met Met Ala Met Val Asp Arg Cys Leu Ser Glu Tyr
485 490 495
Asp Gln Asp Gly Trp Thr Val Pro His Leu His Asn Asn Asp Asp Ile
500 505 510
Asn Met Leu Asp Lys Leu Leu Lys
515 520
<210> 20
<211> 520
<212> PRT
<213> 大肠杆菌(Escherichia coli)
<400> 20
Met Lys Pro Glu Asp Phe Arg Ala Ser Thr Gln Arg Pro Phe Thr Gly
1 5 10 15
Glu Glu Tyr Leu Lys Ser Leu Gln Asp Gly Arg Glu Ile Tyr Ile Tyr
20 25 30
Gly Glu Arg Val Lys Asp Val Thr Thr His Pro Ala Phe Arg Asn Ala
35 40 45
Ala Ala Ser Val Ala Gln Leu Tyr Asp Ala Leu His Lys Pro Glu Met
50 55 60
Gln Asp Ser Leu Cys Trp Asn Thr Asp Thr Gly Ser Gly Gly Tyr Thr
65 70 75 80
His Lys Phe Phe Arg Val Ala Lys Ser Ala Asp Asp Leu Arg Gln Gln
85 90 95
Arg Asp Ala Ile Ala Glu Trp Ser Arg Leu Ser Tyr Gly Trp Met Gly
100 105 110
Arg Thr Pro Asp Tyr Lys Ala Ala Phe Gly Cys Ala Leu Gly Ala Asn
115 120 125
Pro Gly Phe Tyr Gly Gln Phe Glu Gln Asn Ala Arg Asn Trp Tyr Thr
130 135 140
Arg Ile Gln Glu Thr Gly Leu Tyr Phe Asn His Ala Ile Val Asn Pro
145 150 155 160
Pro Ile Asp Arg His Leu Pro Thr Asp Lys Val Lys Asp Val Tyr Ile
165 170 175
Lys Leu Glu Lys Glu Thr Asp Ala Gly Ile Ile Val Ser Gly Ala Lys
180 185 190
Val Val Ala Thr Asn Ser Ala Leu Thr His Tyr Asn Met Ile Gly Phe
195 200 205
Gly Gly Pro Phe Val Met Gly Glu Asn Pro Asp Phe Ala Leu Met Phe
210 215 220
Val Ala Pro Met Asp Ala Asp Gly Val Lys Leu Ile Ser Arg Ala Ser
225 230 235 240
Tyr Glu Met Val Ala Gly Ala Thr Gly Ser Pro Tyr Asp Tyr Pro Leu
245 250 255
Ser Ser Arg Phe Asp Glu Asn Asp Ala Ile Leu Val Met Asp Asn Val
260 265 270
Leu Ile Pro Trp Glu Asn Val Leu Ile Tyr Arg Asp Phe Asp Arg Cys
275 280 285
Arg Arg Trp Thr Met Glu Gly Gly Phe Ala Arg Met Tyr Pro Leu Gln
290 295 300
Ala Cys Val Arg Leu Ala Val Lys Leu Asp Phe Ile Thr Ala Leu Leu
305 310 315 320
Lys Lys Ser Leu Glu Cys Thr Gly Thr Leu Glu Phe Arg Gly Val Gln
325 330 335
Ala Asp Leu Gly Glu Val Val Ala Trp Arg Asn Thr Phe Trp Ala Leu
340 345 350
Ser Asp Ser Met Cys Ser Glu Ala Thr Pro Trp Val Asn Gly Ala Tyr
355 360 365
Leu Pro Asp His Ala Ala Leu Gln Thr Tyr Arg Val Leu Ala Pro Met
370 375 380
Ala Tyr Ala Lys Ile Lys Asn Ile Ile Glu Arg Asn Val Thr Ser Gly
385 390 395 400
Leu Ile Tyr Leu Pro Ser Ser Ala Arg Asp Leu Asn Asn Pro Gln Ile
405 410 415
Asp Gln Tyr Leu Ala Lys Tyr Val Arg Gly Ser Asn Gly Met Asp His
420 425 430
Val Gln Arg Ile Lys Ile Leu Lys Leu Met Trp Asp Ala Ile Gly Ser
435 440 445
Glu Phe Gly Gly Arg His Glu Leu Tyr Glu Ile Asn Tyr Ser Gly Ser
450 455 460
Gln Asp Glu Ile Arg Leu Gln Cys Leu Arg Gln Ala Gln Asn Ser Gly
465 470 475 480
Asn Met Asp Lys Met Met Ala Met Val Asp Arg Cys Leu Ser Glu Tyr
485 490 495
Asp Gln Asp Gly Trp Thr Val Pro His Leu His Asn Asn Asp Asp Ile
500 505 510
Asn Met Leu Asp Lys Leu Leu Lys
515 520
<210> 21
<211> 30
<212> DNA
<213> 未知(custom)
<400> 21
aaaccatggg gtttcaaaaa gttgacgcct 30
<210> 22
<211> 29
<212> DNA
<213> 未知(custom)
<400> 22
aaagcggccg cttacatcac cgcagcaaa 29
<210> 23
<211> 41
<212> DNA
<213> 未知(custom)
<400> 23
aaagctagca ggagaaacaa tatggcacct gttacaattg a 41
<210> 24
<211> 34
<212> DNA
<213> 未知(custom)
<400> 24
aaagcggccg cctatttttt atttctttta agtg 34
<210> 25
<211> 32
<212> DNA
<213> 未知(custom)
<400> 25
ggccaatcat atgaaaccag aagatttccg cg 32
<210> 26
<211> 33
<212> DNA
<213> 未知(custom)
<400> 26
aaactcgagt taaatcgcag cttccatttc cag 33

Claims (8)

1.一种HpaBC基因，它具有序列表中SEQ ID NO.1所述的核苷酸序列或SEQ ID NO.2中所述的氨基酸序列。
2.一种重组载体，它含有权利要求1所述的HpaBC基因。
3.一种HpaBC-MUT基因，它的核苷酸序列如下表任一所述或氨基酸序列如下表任一所述：
<u>突变体名称</u> <u>核苷酸序列</u> <u>氨基酸序列</u> <u>HpaBC-MUT1</u> <u>SEQ ID NO.3</u> <u>SEQ ID NO.12</u> <u>HpaBC-MUT2</u> <u>SEQ ID NO.4</u> <u>SEQ ID NO.13</u> <u>HpaBC-MUT3</u> <u>SEQ ID NO.5</u> <u>SEQ ID NO.14</u> <u>HpaBC-MUT4</u> <u>SEQ ID NO.6</u> <u>SEQ ID NO.15</u> <u>HpaBC-MUT5</u> <u>SEQ ID NO.7</u> <u>SEQ ID NO.16</u> <u>HpaBC-MUT6</u> <u>SEQ ID NO.8</u> <u>SEQ ID NO.17</u> <u>HpaBC-MUT7</u> <u>SEQ ID NO.9</u> <u>SEQ ID NO.18</u> <u>HpaBC-MUT8</u> <u>SEQ ID NO.10</u> <u>SEQ ID NO.19</u> <u>HpaBC-MUT9</u> <u>SEQ ID NO.11</u> <u>SEQ ID NO.20</u>
。
4.一种制备HpaBC-MUT基因的方法，其特征在于，包括：
利用定点饱和突变PCR的方法对hpaBC基因构建定点饱和突变文库，得到突变的PCR产物1；
将突变的PCR产物1分别NdeI和XhoI双酶切，酶切产物分别与经相同酶切的pETDuet-1-T7-TyrB-Aro10载体连接，连接产物分别转化BL21(DE3)的感受态细胞中，得到突变文库；
在无菌操作间用无菌牙签将包含着突变体的单菌落克隆挑入已分装有800ul LB培养基(胰蛋白胨10g/L，NaCl 10g/L，酵母提取物5g/L，添加抗生素卡那霉素至50μg/mL)37℃摇床培养2h后，加入诱导剂IPTG至终浓度0.4mM，同时加入酪氨酸至终浓度10mM，在30℃摇床诱导约16h；
离心、取上清液180μL至96孔酶标板，向每孔加入20μL 100mM的高碘酸钠溶液，混合均匀，静置10min，检测400nm处的吸光值，提取OD400数值比野生型高出50％及以上的重组菌。
5.一种重组载体，它含有权利要求3所述的HpaBC-MUT基因。
6.一种羟基酪醇的制备方法，其特征在于，包括：
制备大肠杆菌BL21(DE3)(pETDuet-1-T7-TyrB-Aro10-T7-HpaBC)和BL21(DE3)(pETDuet-1-T7-TyrB-Aro10-T7-HpaBCMUT)；
将大肠杆菌BL21(DE3)(pETDuet-1-T7-TyrB-Aro10-T7-HpaBC)和BL21(DE3)(pETDuet-1-T7-TyrB-Aro10-T7-HpaBCMUT)的单菌落分别接种至含有卡那霉素(终浓度为50μg/ml)的LB液体培养基中，37℃培养12h，收集发酵液；
将发酵液按照1％(体积百分含量)的接种量转接至100ml新鲜的LB液体培养基中，37℃培养至OD600达到0.6，得发酵后的LB液体培养基；
在发酵后的LB液体培养基中加入无菌的IPTG，使IPTG在培养基中的终浓度为0.4mM，在30℃进行诱导发酵；同时加入无菌的酪氨酸溶液至终浓度为10mM，16h后结束发酵。
7.根据权利要求6所述的方法，其特征在于，所述制备大肠杆菌BL21(DE3)(pETDuet-1-T7-TyrB-Aro10-T7-HpaBCMUT)的方法，包括：
(1)将序列表SEQ ID NO.3至SEQ ID NO.11的任一序列所示的DNA分子插入pET28a的NdeI和XhoI双酶切位点间得到的pETDuet-1-T7-TyrB-Aro10-T7-HpaBCMUT；
(2)再将pETDuet-1-T7-TyrB-Aro10-T7-HpaBCMUT导入BL21(DE3)的感受态细胞中，得到大肠杆菌BL21(DE3)(pETDuet-1-T7-TyrB-Aro10-T7-HpaBCMUT)。
8.根据权利要求6所述的方法，其特征在于，所述制备大肠杆菌BL21(DE3)(pETDuet-1-T7-TyrB-Aro10-T7-HpaBC)的方法，包括：
将质粒pETDuet-1-T7-TyrB-Aro10-T7-HpaBC转入BL21(DE3)的感受态细胞中，得到重组大肠杆菌BL21(DE3)(pETDuet-1-T7-TyrB-Aro10-T7-HpaBC)。
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