CN113293152B - 短链脱氢酶突变体及其用途 - Google Patents

短链脱氢酶突变体及其用途 Download PDF

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CN113293152B
CN113293152B CN202110804331.0A CN202110804331A CN113293152B CN 113293152 B CN113293152 B CN 113293152B CN 202110804331 A CN202110804331 A CN 202110804331A CN 113293152 B CN113293152 B CN 113293152B
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游松
秦斌
秦凤玉
张文鹤
刘亚林
祝天慧
郭继阳
张飞霆
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Abstract

本发明涉及生物技术领域，涉及短链脱氢酶突变体及其用途，该类突变体是多种短链脱氢酶的野生型出发经过突变得到的，具体涉及短链脱氢酶突变体和其制备方法以及利用短链脱氢酶突变体通过催苯乙酮衍生化合物的还原，制备有光学活性的苯乙醇衍生物的方法。

Description

短链脱氢酶突变体及其用途
本申请是沈阳药科大学2018年04月24日申请的申请号为2018103711535，名称为“短链脱氢酶突变体及其用途”的申请的分案申请。
技术领域：
本发明涉及生物技术领域，涉及短链脱氢酶突变体及其用途，该类突变体是多种短链脱氢酶的野生型出发经过突变得到的，具体涉及短链脱氢酶突变体和其制备方法以及利用短链脱氢酶突变体通过催化苯乙酮衍生化合物的还原，制备有光学活性的苯乙醇衍生物的方法。
背景技术
手性醇是众多药用活性成分的一种万用合成砌块，传统的合成手性醇的方法有手性池法、不对称合成法和拆分法等。上述三种方法都在商业化合成手性醇中有所应用，但它们却有较多缺点：如使用大量昂贵的试剂、造成大量的污染、使用极端的操作条件等，这就需要开发一种更经济的、环境友好的手性醇的合成方法。
在现在所有制备手性分子的技术中，生物催化是一种最有效且环境友好的方法(Nature 2012,485,185-194)，因为生物催化能够在温和的条件及环境温度下以极高的选择性执行复杂的化学反应。正因如此，通过全细胞或者分离的酶生物催化合成手性醇便能够克服上述的问题，其中利用酮还原酶(ketoreductases，缩写为KREDs)或者醇脱氢酶制备手性醇便是可行方法之一(Chemical Reviews 2011,111,4088-4110.Chemical Reviews2011,111,4111-4140.)。
酮还原酶或羰基还原酶(EC 1.1.1.X)能够选择性的不对称还原酮得到相应的手性醇，在还原过程中需要添加NADPH或者NADH作为辅因子。酮还原酶的底物为前手性的酮，类型比较广泛，如α酮酯、β酮酯、脂肪链酮、环状酮等。在手性药物及其中间体的合成过程中，酮还原酶正在得到广泛的应用，以调脂的他汀类药物为例，其关键中间体3，5-二羟基酸的侧链的合成需要非常苛刻的条件，而酮还原酶为其立体选择性合成提供了多种更绿色的改进路线(Angewandte Chemie International Edition 2005,44,362-365.)。日本的Daicel和Kaneka公司分别建立了利用醇脱氢酶或羰基还原酶，同时利用葡萄糖或甲酸脱氢酶来循环酶的辅因子的合成(S)-4-氯-3-羟基丁酸乙酯的生物还原系统。Kaneka公司利用重组的工程菌，可将300g/L的底物转化为产物，转化率可达96％，100％ee。Codexis公司利用醇脱氢酶/葡萄糖脱氢酶的生物还原系统建立了相似的路线，同时通过利用脱卤素酶将氯原子替换为氰基来合成(R)-4-氰基-3-羟基丁酸乙酯(HN)。此过程由Lonza公司工业化生产，并提供给辉瑞公司用于合成阿托伐他汀钙，此过程获得了2006年的由美国环保署设立的美国总统绿色化学挑战奖。BMS公司利用类似的酮还原酶，能够将6-苯甲氧基-3,5-二酮基己酸甲酯双还原直接产生有两个手性中心的(3R,5S)-二醇的手性中间体。因此能够将前体酮还原成手性醇的酮还原酶具有很大的应用前景；
而目前制约酮还原酶应用的主要问题就是天然的酮还原酶存在一定的缺陷，如活性低、选择性差、选择性与期望相反等，使其在生产实践中的应用受到一定限制，成功修饰和改造酮还原酶使其具有更加良好的性质是一个研究的热点。例如Codexis公司通过大量的突变及筛选，得到了能够用于制备阿托伐他汀(Green Chemistry 2010,12,81-86.)、孟鲁斯特(Organic Process Research&Development 2009,14(1),193-198.)、克唑替尼(Nature 2012,485,185-194)等手性药物关键中间体的酮还原酶突变体。
针对酮还原酶的选择性，自然界中大多数酮还原酶符合Prelog选择性，而反-Prelog选择性的手性醇在药物合成中具有重要的应用，因此通过蛋白质工程的策略，突变得到具有反-Prelog选择性的酮还原酶具有重要意义。目前也有一些将Prelog选择性的酮还原酶改造为反-Prelog选择性的例子，如于洪伟等通过同源酶的结构指导，将来源于恶臭假单胞菌(Pseudomonas putida ATCC 12633)的短链脱氢酶(PpYSDR)立体选择性改造为符合反Prelog规则，针对底物2,2,2-三氟-1-苯基乙酮突变体PpYSDR-M85T/W182V立体选择性由野生型的56.8(R)改变为99.5(S)(Chem.Commun.,2016,52,6284)；游松等通过蛋白质结构及计算机模拟辅助的方法将来源于光滑假丝酵母(Candida glabrata CGMCC2.234)的酮还原酶(CgKR1)改造为符合反Prelog规则的催化性质，针对底物2-氯-1-苯基乙酮突变体CgKR1-F92A立体选择性由野生型的23.1(R)改变为99.1(S)(ACS Catalysis,2016,6,6135-6140.)
发明内容：
为了克服现有技术的缺陷，本发明通过对来源于巨大芽孢杆菌及枯草芽孢杆菌的几种短链脱氢酶(Short-chain dehydrogenase/reductases，缩写为SDRs，属于酮还原酶)进行突变，改变甚至反转其不对称还原苯乙酮衍生物的选择性。
本发明通过如下技术方案实现：
本发明由野生型巨大芽孢杆菌的短链脱氢酶5(BmSDR5，SEQ ID NO.1)、短链脱氢酶11(BmSDR11，SEQ ID NO.11)、枯草芽孢杆菌的短链脱氢酶4(BsSDR4，SEQ ID NO.21)、短链脱氢酶8(BsSDR8，SEQ ID NO.25)、短链脱氢酶11(BsSDR11，SEQ ID NO.39)、短链脱氢酶13(BsSDR13，SEQ ID NO.45)通过突变得到具有改良性质的短链脱氢酶，该酶在大肠杆菌工程菌中表达，该类短链脱氢酶突变体，能够将苯乙酮衍生物还原，得到具有光学活性的苯乙醇衍生物。在本发明的实施方案中，该类短链脱氢酶酶与天然存在的短链脱氢酶野生型相比，在将底物还原为产物方面具有一个或多个改善的性质。
本发明中，该短链脱氢酶可以包括与亲本SEQ ID NO.02至少85％同一性的氨基酸序列，并且187位为芳香族氨基酸，优选苯丙氨酸(F)，如SEQ ID NO.4；或者187位为芳香族氨基酸且188位为非极性氨基酸，其中187位优选苯丙氨酸(F)，188位优选亮氨酸(L)，如SEQID NO.10。
该短链脱氢酶可以包括与亲本SEQ ID NO.12至少85％同一性的氨基酸序列，且95位为非极性氨基酸，优选丙氨酸(A)，如SEQ ID NO.14；或190位为非极性氨基酸，优选丙氨酸(A)，如SEQ ID NO.16；或205位为非极性氨基酸，优选亮氨酸(L)，如SEQ ID NO.18。或190位、205位同时为非极性氨基酸，其中190位优选丙氨酸(A)，205位优选亮氨酸(L)，如SEQ IDNO.20。
该短链脱氢酶可以包括与亲本SEQ ID NO.22至少85％同一性的氨基酸序列，且222位为芳香族氨基酸，优选苯丙氨酸(F)，如SEQ ID NO.24；
该短链脱氢酶可以包括与亲本SEQ ID NO.26至少85％同一性的氨基酸序列，且146位为芳香族氨基酸，优选色氨酸(W)，如SEQ ID NO.32。或146位为芳香族氨基酸且184位为芳香族氨基酸，其中146位优选酪氨酸(Y)，184位优选色氨酸(W)，如SEQ ID NO.34。或149位为芳香族氨基酸且184位为芳香族氨基酸，其中149位优选酪氨酸(Y)，184位优选色氨酸(W)，如SEQ ID NO.36。或149位为芳香族氨基酸，优选苯酪氨酸(Y)，如SEQ ID NO.38；
该短链脱氢酶可以包括与亲本SEQ ID NO.40至少85％同一性的氨基酸序列，且181位为芳香族氨基酸，优选苯丙氨酸(F)，如SEQ ID NO.42；或84位为非极性氨基酸，优选丙氨酸(A)，如SEQ ID NO.44；
该短链脱氢酶可以包括与亲本SEQ ID NO.46至少85％同一性的氨基酸序列，且184位为芳香族氨基酸且89位为非极性氨基酸，其中184优选苯丙氨酸(F)，89位优选丙氨酸(A)，如SEQ ID NO.48；
本发明中涉及的芳香族氨基酸为苯丙氨酸(F)、酪氨酸(Y)，色氨酸(W)，非极性氨基酸为丙氨酸(A)、缬氨酸(V)、亮氨酸(L)、异亮氨酸(I)、脯氨酸(P)、甲硫氨酸(M)。
本发明所述的短链脱氢酶突变体，其氨基酸序列优选如SEQ ID NO.4，6，8，10，14，16，18，20，24，28，30，32，34，36，38，42，44，48所示。
本发明还提供了能够编码所述的短链的核酸，所述的核酸序列如SEQ ID NO.3，5，7，9，13，15，17，19，23，27，29，31，33，35，37，41，43，47所示。
本发明的实施方式包括编码所述短链脱氢酶变体的核酸，例如与编码本发明所述短链脱氢酶变体的核酸序列具有至少85％的序列同一性。本发明的相关实施方式包括包含这些核酸的载体及包含该类载体的宿主细胞。
本发明的另一实施方式是所述的短链脱氢酶突变体不对称还原苯乙酮衍生物的方法。在pH 5-9的磷酸盐缓冲液溶液中，加入定量的用助溶剂溶解的苯乙酮衍生物底物，之后加入定量的短链脱氢酶突变体催化还原反应，该短链脱氢酶变体可以多种可行的方式如粗酶粉、酶溶液、固定化酶等多种形式存在。
所述的苯乙酮类衍生化合物为苯环或α位取代衍生物，其结构如式(II)所述，
(II)
Figure BDA0003165863660000031
其中：
R1为C1-C4烷基、卤代C1-c4烷基、卤素；R2为氢、C1-C4烷基、卤素、卤代C1-C4
烷基或硝基；
优选地，R1为Cl、Br或CH2Cl；R2为氢、F、Cl、Br。
一些典型的结构如(III)所述。
(III)
Figure BDA0003165863660000032
Figure BDA0003165863660000041
不对称还原α-氯代苯乙酮、4-F-α-氯代苯乙酮的结构式
本发明的有益技术效果：通过对野生型短链脱氢酶进行突变，改变了其对苯乙酮衍生物还原的选择性。
具体实施方式：
下面通过具体的实施方案描述本发明的短链脱氢酶突变体以及利用该酶苯乙酮衍生物的还原。除特别指明，本发明中所用的实验方案均为本领域技术人员所公知的方法。此外，实施例应理解为说明性的，而不是限制本发明。
某些术语的定义。
对映体过量定义为对映体混合物中一个异构体A比另一个异构体B多出来的量占总量的百分数，缩写为ee，公式为(A-B)/(A+B)*100％，对映体过量值用于表示一种手性化合物的光学纯度。ee值越高，光学纯度也越高。
20种氨基酸缩写如下
ASP D 天冬氨酸 Ile I 异亮氨酸
Thr T 苏氨酸 Leu L 亮氨酸
Ser S 丝氨酸 Thr T 酪氨酸
Glu E 谷氨酸 Phe F 苯丙氨酸
Pro P 脯氨酸 His H 组氨酸
Gly G 甘氨酸 Lys L 赖氨酸
Ala A 丙氨酸 Arg R 精氨酸
Cys C 半胱氨酸 Trp W 色氨酸
Val V 缬氨酸 Gln Q 谷氨酰胺
Met M 甲硫氨酸 Asn N 天冬酰胺
实施例1巨大芽孢杆菌基因组DNA的提取及野生型BmSDR5基因的克隆
巨大芽孢杆菌用LB液体培养过夜后，发酵液用1mL离心管3000r/min离心5min，弃上清收集菌体，可重复几次以得到足够量的细胞；b)菌体用足够量的DNA裂解液重悬，再加入200μL酚：氯仿：异戊醇，盖紧管盖，以防涡旋时酚漏出；c)涡旋仪高速涡旋3min；d)12000r/min离心5min，上清液(约200μL)转移至新的离心管；e)上清液加入700加入预冷的无水乙醇后，-20℃无水放置1h，12000r/min离心15min沉淀DNA，倒掉乙醇，沉淀用800乙醇，沉淀乙醇混悬，12000r/min离心15min沉淀DNA，真空干燥；f)DNA用100μL1*TE重溶。
实施例2聚合酶链式反应(PCR)
以提得巨大芽孢杆菌基因组DNA为模板进行PCR反应，反应体系如下：
Figure BDA0003165863660000051
扩增程序：94℃：10Min，(94℃：30s，45℃：30s，72℃：30s)35个循环，72℃，10min。
引物1：BmSDR5-BamHI-F：CGGGATCCGATGTTTGAAGAAAAAGTAGGGAT；
引物2：BmSDR5-XhoI-R：CCGCTCGAGCTCAAACCTAAACCCAATCC；
限制性内切酶酶切位点用下划线标出；
PCR扩增得到的DNA片段用胶回收试剂盒纯化。含有pET-22b质粒的E.coli DH5α用LB液体培养基37℃，220r/min培养过夜，质粒提取采用参照TIANprep Mini Plasmid Kit。目的片段与质粒pET-22质粒限双酶切，酶切体系如下：
Figure BDA0003165863660000052
实施例3E.coli DH5α及BL21感受态细胞的制备及转化
a)从种子培养基中取0.4mL接种于20mL LB液体培养基中培养3h；b)3000r/min，5min分两次富集2mL菌体于1.5mLEP管中，弃上清；c)加入100弃上冰冷TSS溶液，重新悬浮菌体，冰浴30min；d)加入20μL连接液(空质粒pPICZαA酶切片段、目的片段酶切片段及连接液)轻轻旋转混匀，冰浴30min；e)42℃热休克60s，冰浴2min，加入600μL LB液体培养基。37℃培养，150r/min振荡培养1h；f)分别取150μL涂布于氨苄青霉素抗性的LB平板。
实施例4突变体的构建
突变体的构建是利用突变引物和侧翼引物，重叠延伸PCR进行的。(设计的突变引物如下：)
Figure BDA0003165863660000053
PCR扩增条件：94℃：10Min，(94℃：30s，45℃：30s，72℃：30s)35个循环，72℃，10min。
所得基因片段经过纯化，按如下PCR扩增
Figure BDA0003165863660000061
所得目的片段按实施例2所述连接到载体pET-22b上、并按实施例3所述转化至大肠杆菌BL-21中。
实施例5短链脱氢酶突变体表达
将大肠杆菌(E.coli)BL21/pET-22b-SDRs，接种至含氨苄青霉素的LB培养基(PH7.0)中，37℃振荡培养过夜，按1％(v/v)的接种量接入装有100ml LB培养基的250ml三角瓶中，置37℃、180rpm的摇床振荡培养，当培养液的OD600达到0.6时，加入终浓度为0.1mM的IPTG作为诱导剂，37℃诱导12h，将培养液离心，用3ml磷酸盐缓冲溶液重悬(PH 6.0)细胞，转移至EP管中。在冰浴条件下超声破碎，低温(4℃)离心收集上清液，即为重组的短链脱氢酶的粗酶液。。
实施例6重组短链脱氢酶催化苯乙酮衍生物的不对称还原
在500μl的反应体系中，先加入实施例5制备的短链脱氢酶300μl和及葡萄糖脱氢酶100μl,然后加入终浓度为0.3g/L的NADP+和5.4g/L的葡萄糖，最后加入终浓度为15mM苯乙酮衍生物。在30℃，200rpm条件下振荡反应，反应时间为24h。反应结束后用500μl的乙酸乙酯分两次萃取，合并萃取液，旋蒸干燥后分析底物转化率和产物的ee值。结果见表2所示。
实施例7野生及突变酶对苯乙酮衍生物选择性测定结果。
Figure BDA0003165863660000062
Figure BDA0003165863660000071
a为转化率，b为ee值及选择性。
序列表
<110> 沈阳药科大学
<120> 短链脱氢酶突变体及其用途
<160> 38
<170> SIPOSequenceListing 1.0
<210> 1
<211> 774
<212> DNA
<213> 巨大芽孢杆菌(Bacillus megaterium)
<400> 1
atgtttgaag aaaaagtagg gatcattacg ggtggaacgt cggggattgg cttagctaca 60
gcggaattac tcgcaaaaga gggcatgcat atcgtgattg cttctagaaa tagtgaaaaa 120
ggagaagaag ctctttctgt attaagaaag tggtctcctc actctctttt tatcaaaacg 180
gatgtaacga atagtcaaga tgttaaaaat ctcgtgagcc aaacatactc gacgtttgga 240
aaaattgatg tgagctttaa taatgcggct aacacggagg cagcttctaa tgctacccat 300
gaattcaaag aagaagactt tgaccattta ataaacgtta cgttaaaaag tgtatggctt 360
tgtatgaaat atcagttgca ggtaatgacc aaacaaaata gcggggtcat tataaataca 420
tcgtcaatgg acgctatttt atgttcagca gggactggcg tatatgcggc tgggaaaagc 480
ggtgtgattg ctttaacgaa gtcagttgct caagaatatg gccatcaaca tataagaatt 540
aactctctgt gcccaggtgc atttcgtacg cctatgttag aagaaaggtt ttcaaatcta 600
tctgaagacg aaaaagcaaa gctcaatgaa tcctatcaaa agctaaacgc gctgggcaga 660
attggtgatc cgattgaagc cgcaaaagca gtaaaatggc tcctttctga tgatgcttcg 720
tttgttacgg gtcaaaacat tattgtagac ggtgggattg ggtttaggtt tgag 774
<210> 2
<211> 258
<212> PRT
<213> 巨大芽孢杆菌(Bacillus megaterium)
<400> 2
Met Phe Glu Glu Lys Val Gly Ile Ile Thr Gly Gly Thr Ser Gly Ile
1 5 10 15
Gly Leu Ala Thr Ala Glu Leu Leu Ala Lys Glu Gly Met His Ile Val
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Ile Ala Ser Arg Asn Ser Glu Lys Gly Glu Glu Ala Leu Ser Val Leu
35 40 45
Arg Lys Trp Ser Pro His Ser Leu Phe Ile Lys Thr Asp Val Thr Asn
50 55 60
Ser Gln Asp Val Lys Asn Leu Val Ser Gln Thr Tyr Ser Thr Phe Gly
65 70 75 80
Lys Ile Asp Val Ser Phe Asn Asn Ala Ala Asn Thr Glu Ala Ala Ser
85 90 95
Asn Ala Thr His Glu Phe Lys Glu Glu Asp Phe Asp His Leu Ile Asn
100 105 110
Val Thr Leu Lys Ser Val Trp Leu Cys Met Lys Tyr Gln Leu Gln Val
115 120 125
Met Thr Lys Gln Asn Ser Gly Val Ile Ile Asn Thr Ser Ser Met Asp
130 135 140
Ala Ile Leu Cys Ser Ala Gly Thr Gly Val Tyr Ala Ala Gly Lys Ser
145 150 155 160
Gly Val Ile Ala Leu Thr Lys Ser Val Ala Gln Glu Tyr Gly His Gln
165 170 175
His Ile Arg Ile Asn Ser Leu Cys Pro Gly Ala Phe Arg Thr Pro Met
180 185 190
Leu Glu Glu Arg Phe Ser Asn Leu Ser Glu Asp Glu Lys Ala Lys Leu
195 200 205
Asn Glu Ser Tyr Gln Lys Leu Asn Ala Leu Gly Arg Ile Gly Asp Pro
210 215 220
Ile Glu Ala Ala Lys Ala Val Lys Trp Leu Leu Ser Asp Asp Ala Ser
225 230 235 240
Phe Val Thr Gly Gln Asn Ile Ile Val Asp Gly Gly Ile Gly Phe Arg
245 250 255
Phe Glu
<210> 3
<211> 774
<212> DNA
<213> 巨大芽孢杆菌(Bacillus megaterium)
<400> 3
atgtttgaag aaaaagtagg gatcattacg ggtggaacgt cggggattgg cttagctaca 60
gcggaattac tcgcaaaaga gggcatgcat atcgtgattg cttctagaaa tagtgaaaaa 120
ggagaagaag ctctttctgt attaagaaag tggtctcctc actctctttt tatcaaaacg 180
gatgtaacga atagtcaaga tgttaaaaat ctcgtgagcc aaacatactc gacgtttgga 240
aaaattgatg tgagctttaa taatgcggct aacacggagg cagcttctaa tgctacccat 300
gaattcaaag aagaagactt tgaccattta ataaacgtta cgttaaaaag tgtatggctt 360
tgtatgaaat atcagttgca ggtaatgacc aaacaaaata gcggggtcat tataaataca 420
tcgtcaatgg acgctatttt atgttcagca gggactggcg tatatgcggc tgggaaaagc 480
ggtgtgattg ctttaacgaa gtcagttgct caagaatatg gccatcaaca tataagaatt 540
aactctctgt gcccaggttt ttttcgtacg cctatgttag aagaaaggtt ttcaaatcta 600
tctgaagacg aaaaagcaaa gctcaatgaa tcctatcaaa agctaaacgc gctgggcaga 660
attggtgatc cgattgaagc cgcaaaagca gtaaaatggc tcctttctga tgatgcttcg 720
tttgttacgg gtcaaaacat tattgtagac ggtgggattg ggtttaggtt tgag 774
<210> 4
<211> 258
<212> PRT
<213> 巨大芽孢杆菌(Bacillus megaterium)
<400> 4
Met Phe Glu Glu Lys Val Gly Ile Ile Thr Gly Gly Thr Ser Gly Ile
1 5 10 15
Gly Leu Ala Thr Ala Glu Leu Leu Ala Lys Glu Gly Met His Ile Val
20 25 30
Ile Ala Ser Arg Asn Ser Glu Lys Gly Glu Glu Ala Leu Ser Val Leu
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Arg Lys Trp Ser Pro His Ser Leu Phe Ile Lys Thr Asp Val Thr Asn
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Ser Gln Asp Val Lys Asn Leu Val Ser Gln Thr Tyr Ser Thr Phe Gly
65 70 75 80
Lys Ile Asp Val Ser Phe Asn Asn Ala Ala Asn Thr Glu Ala Ala Ser
85 90 95
Asn Ala Thr His Glu Phe Lys Glu Glu Asp Phe Asp His Leu Ile Asn
100 105 110
Val Thr Leu Lys Ser Val Trp Leu Cys Met Lys Tyr Gln Leu Gln Val
115 120 125
Met Thr Lys Gln Asn Ser Gly Val Ile Ile Asn Thr Ser Ser Met Asp
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Ala Ile Leu Cys Ser Ala Gly Thr Gly Val Tyr Ala Ala Gly Lys Ser
145 150 155 160
Gly Val Ile Ala Leu Thr Lys Ser Val Ala Gln Glu Tyr Gly His Gln
165 170 175
His Ile Arg Ile Asn Ser Leu Cys Pro Gly Phe Phe Arg Thr Pro Met
180 185 190
Leu Glu Glu Arg Phe Ser Asn Leu Ser Glu Asp Glu Lys Ala Lys Leu
195 200 205
Asn Glu Ser Tyr Gln Lys Leu Asn Ala Leu Gly Arg Ile Gly Asp Pro
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Ile Glu Ala Ala Lys Ala Val Lys Trp Leu Leu Ser Asp Asp Ala Ser
225 230 235 240
Phe Val Thr Gly Gln Asn Ile Ile Val Asp Gly Gly Ile Gly Phe Arg
245 250 255
Phe Glu
<210> 5
<211> 774
<212> DNA
<213> 巨大芽孢杆菌(Bacillus megaterium)
<400> 5
atgtttgaag aaaaagtagg gatcattacg ggtggaacgt cggggattgg cttagctaca 60
gcggaattac tcgcaaaaga gggcatgcat atcgtgattg cttctagaaa tagtgaaaaa 120
ggagaagaag ctctttctgt attaagaaag tggtctcctc actctctttt tatcaaaacg 180
gatgtaacga atagtcaaga tgttaaaaat ctcgtgagcc aaacatactc gacgtttgga 240
aaaattgatg tgagctttaa taatgcggct aacacggagg cagcttctaa tgctacccat 300
gaattcaaag aagaagactt tgaccattta ataaacgtta cgttaaaaag tgtatggctt 360
tgtatgaaat atcagttgca ggtaatgacc aaacaaaata gcggggtcat tataaataca 420
tcgtcaatgg acgctatttt atgttcagca gggactggcg tatatgcggc tgggaaaagc 480
ggtgtgattg ctttaacgaa gtcagttgct caagaatatg gccatcaaca tataagaatt 540
aactctctgt gcccaggtta ttttcgtacg cctatgttag aagaaaggtt ttcaaatcta 600
tctgaagacg aaaaagcaaa gctcaatgaa tcctatcaaa agctaaacgc gctgggcaga 660
attggtgatc cgattgaagc cgcaaaagca gtaaaatggc tcctttctga tgatgcttcg 720
tttgttacgg gtcaaaacat tattgtagac ggtgggattg ggtttaggtt tgag 774
<210> 6
<211> 258
<212> PRT
<213> 巨大芽孢杆菌(Bacillus megaterium)
<400> 6
Met Phe Glu Glu Lys Val Gly Ile Ile Thr Gly Gly Thr Ser Gly Ile
1 5 10 15
Gly Leu Ala Thr Ala Glu Leu Leu Ala Lys Glu Gly Met His Ile Val
20 25 30
Ile Ala Ser Arg Asn Ser Glu Lys Gly Glu Glu Ala Leu Ser Val Leu
35 40 45
Arg Lys Trp Ser Pro His Ser Leu Phe Ile Lys Thr Asp Val Thr Asn
50 55 60
Ser Gln Asp Val Lys Asn Leu Val Ser Gln Thr Tyr Ser Thr Phe Gly
65 70 75 80
Lys Ile Asp Val Ser Phe Asn Asn Ala Ala Asn Thr Glu Ala Ala Ser
85 90 95
Asn Ala Thr His Glu Phe Lys Glu Glu Asp Phe Asp His Leu Ile Asn
100 105 110
Val Thr Leu Lys Ser Val Trp Leu Cys Met Lys Tyr Gln Leu Gln Val
115 120 125
Met Thr Lys Gln Asn Ser Gly Val Ile Ile Asn Thr Ser Ser Met Asp
130 135 140
Ala Ile Leu Cys Ser Ala Gly Thr Gly Val Tyr Ala Ala Gly Lys Ser
145 150 155 160
Gly Val Ile Ala Leu Thr Lys Ser Val Ala Gln Glu Tyr Gly His Gln
165 170 175
His Ile Arg Ile Asn Ser Leu Cys Pro Gly Tyr Phe Arg Thr Pro Met
180 185 190
Leu Glu Glu Arg Phe Ser Asn Leu Ser Glu Asp Glu Lys Ala Lys Leu
195 200 205
Asn Glu Ser Tyr Gln Lys Leu Asn Ala Leu Gly Arg Ile Gly Asp Pro
210 215 220
Ile Glu Ala Ala Lys Ala Val Lys Trp Leu Leu Ser Asp Asp Ala Ser
225 230 235 240
Phe Val Thr Gly Gln Asn Ile Ile Val Asp Gly Gly Ile Gly Phe Arg
245 250 255
Phe Glu
<210> 7
<211> 774
<212> DNA
<213> 巨大芽孢杆菌(Bacillus megaterium)
<400> 7
atgtttgaag aaaaagtagg gatcattacg ggtggaacgt cggggattgg cttagctaca 60
gcggaattac tcgcaaaaga gggcatgcat atcgtgattg cttctagaaa tagtgaaaaa 120
ggagaagaag ctctttctgt attaagaaag tggtctcctc actctctttt tatcaaaacg 180
gatgtaacga atagtcaaga tgttaaaaat ctcgtgagcc aaacatactc gacgtttgga 240
aaaattgatg tgagctttaa taatgcggct aacacggagg cagcttctaa tgctacccat 300
gaattcaaag aagaagactt tgaccattta ataaacgtta cgttaaaaag tgtatggctt 360
tgtatgaaat atcagttgca ggtaatgacc aaacaaaata gcggggtcat tataaataca 420
tcgtcaatgg acgctatttt atgttcagca gggactggcg tatatgcggc tgggaaaagc 480
ggtgtgattg ctttaacgaa gtcagttgct caagaatatg gccatcaaca tataagaatt 540
aactctctgt gcccaggttg gtttcgtacg cctatgttag aagaaaggtt ttcaaatcta 600
tctgaagacg aaaaagcaaa gctcaatgaa tcctatcaaa agctaaacgc gctgggcaga 660
attggtgatc cgattgaagc cgcaaaagca gtaaaatggc tcctttctga tgatgcttcg 720
tttgttacgg gtcaaaacat tattgtagac ggtgggattg ggtttaggtt tgag 774
<210> 8
<211> 258
<212> PRT
<213> 巨大芽孢杆菌(Bacillus megaterium)
<400> 8
Met Phe Glu Glu Lys Val Gly Ile Ile Thr Gly Gly Thr Ser Gly Ile
1 5 10 15
Gly Leu Ala Thr Ala Glu Leu Leu Ala Lys Glu Gly Met His Ile Val
20 25 30
Ile Ala Ser Arg Asn Ser Glu Lys Gly Glu Glu Ala Leu Ser Val Leu
35 40 45
Arg Lys Trp Ser Pro His Ser Leu Phe Ile Lys Thr Asp Val Thr Asn
50 55 60
Ser Gln Asp Val Lys Asn Leu Val Ser Gln Thr Tyr Ser Thr Phe Gly
65 70 75 80
Lys Ile Asp Val Ser Phe Asn Asn Ala Ala Asn Thr Glu Ala Ala Ser
85 90 95
Asn Ala Thr His Glu Phe Lys Glu Glu Asp Phe Asp His Leu Ile Asn
100 105 110
Val Thr Leu Lys Ser Val Trp Leu Cys Met Lys Tyr Gln Leu Gln Val
115 120 125
Met Thr Lys Gln Asn Ser Gly Val Ile Ile Asn Thr Ser Ser Met Asp
130 135 140
Ala Ile Leu Cys Ser Ala Gly Thr Gly Val Tyr Ala Ala Gly Lys Ser
145 150 155 160
Gly Val Ile Ala Leu Thr Lys Ser Val Ala Gln Glu Tyr Gly His Gln
165 170 175
His Ile Arg Ile Asn Ser Leu Cys Pro Gly Trp Phe Arg Thr Pro Met
180 185 190
Leu Glu Glu Arg Phe Ser Asn Leu Ser Glu Asp Glu Lys Ala Lys Leu
195 200 205
Asn Glu Ser Tyr Gln Lys Leu Asn Ala Leu Gly Arg Ile Gly Asp Pro
210 215 220
Ile Glu Ala Ala Lys Ala Val Lys Trp Leu Leu Ser Asp Asp Ala Ser
225 230 235 240
Phe Val Thr Gly Gln Asn Ile Ile Val Asp Gly Gly Ile Gly Phe Arg
245 250 255
Phe Glu
<210> 9
<211> 774
<212> DNA
<213> 巨大芽孢杆菌(Bacillus megaterium)
<400> 9
atgtttgaag aaaaagtagg gatcattacg ggtggaacgt cggggattgg cttagctaca 60
gcggaattac tcgcaaaaga gggcatgcat atcgtgattg cttctagaaa tagtgaaaaa 120
ggagaagaag ctctttctgt attaagaaag tggtctcctc actctctttt tatcaaaacg 180
gatgtaacga atagtcaaga tgttaaaaat ctcgtgagcc aaacatactc gacgtttgga 240
aaaattgatg tgagctttaa taatgcggct aacacggagg cagcttctaa tgctacccat 300
gaattcaaag aagaagactt tgaccattta ataaacgtta cgttaaaaag tgtatggctt 360
tgtatgaaat atcagttgca ggtaatgacc aaacaaaata gcggggtcat tataaataca 420
tcgtcaatgg acgctatttt atgttcagca gggactggcg tatatgcggc tgggaaaagc 480
ggtgtgattg ctttaacgaa gtcagttgct caagaatatg gccatcaaca tataagaatt 540
aactctctgt gcccaggttt tctgcgtacg cctatgttag aagaaaggtt ttcaaatcta 600
tctgaagacg aaaaagcaaa gctcaatgaa tcctatcaaa agctaaacgc gctgggcaga 660
attggtgatc cgattgaagc cgcaaaagca gtaaaatggc tcctttctga tgatgcttcg 720
tttgttacgg gtcaaaacat tattgtagac ggtgggattg ggtttaggtt tgag 774
<210> 10
<211> 258
<212> PRT
<213> 巨大芽孢杆菌(Bacillus megaterium)
<400> 10
Met Phe Glu Glu Lys Val Gly Ile Ile Thr Gly Gly Thr Ser Gly Ile
1 5 10 15
Gly Leu Ala Thr Ala Glu Leu Leu Ala Lys Glu Gly Met His Ile Val
20 25 30
Ile Ala Ser Arg Asn Ser Glu Lys Gly Glu Glu Ala Leu Ser Val Leu
35 40 45
Arg Lys Trp Ser Pro His Ser Leu Phe Ile Lys Thr Asp Val Thr Asn
50 55 60
Ser Gln Asp Val Lys Asn Leu Val Ser Gln Thr Tyr Ser Thr Phe Gly
65 70 75 80
Lys Ile Asp Val Ser Phe Asn Asn Ala Ala Asn Thr Glu Ala Ala Ser
85 90 95
Asn Ala Thr His Glu Phe Lys Glu Glu Asp Phe Asp His Leu Ile Asn
100 105 110
Val Thr Leu Lys Ser Val Trp Leu Cys Met Lys Tyr Gln Leu Gln Val
115 120 125
Met Thr Lys Gln Asn Ser Gly Val Ile Ile Asn Thr Ser Ser Met Asp
130 135 140
Ala Ile Leu Cys Ser Ala Gly Thr Gly Val Tyr Ala Ala Gly Lys Ser
145 150 155 160
Gly Val Ile Ala Leu Thr Lys Ser Val Ala Gln Glu Tyr Gly His Gln
165 170 175
His Ile Arg Ile Asn Ser Leu Cys Pro Gly Phe Leu Arg Thr Pro Met
180 185 190
Leu Glu Glu Arg Phe Ser Asn Leu Ser Glu Asp Glu Lys Ala Lys Leu
195 200 205
Asn Glu Ser Tyr Gln Lys Leu Asn Ala Leu Gly Arg Ile Gly Asp Pro
210 215 220
Ile Glu Ala Ala Lys Ala Val Lys Trp Leu Leu Ser Asp Asp Ala Ser
225 230 235 240
Phe Val Thr Gly Gln Asn Ile Ile Val Asp Gly Gly Ile Gly Phe Arg
245 250 255
Phe Glu
<210> 11
<211> 759
<212> DNA
<213> 巨大芽孢杆菌(Bacillus megaterium)
<400> 11
atgcgttttg aagggcaaat agctgttatt acaggagctg gaagcggcat tggagaagcg 60
accgctcaga gaatggcgaa agaaggagct cgcgtcattc ttgtcgggcg aactaaagaa 120
aaactagtac aggctgcatc ggctattgac acacggtgtg aacgaaaatg cacggatatt 180
ttctcagctg atgtaacgaa agaagaagac gtcaaagagc tggttgagtt tataaaagaa 240
aagtacggac aaattcattt attaattaat aacgcaggcg gatcagttaa ttcaacaatc 300
aaagaaacga cgcttgaaca gtggaaacac gtgcaggacg tcaatttaac aagcgtattt 360
cttgtcacaa aacatttgat tcctttttta actgaagaaa taggagggaa tcgatctatt 420
gttaacatcg cttctctttc aggacataaa gctggggcac aaattcctca ctacagcgct 480
gctaaagctg gtctaattaa ctttacaaaa gcaatggcgt ttgagttagc accccacgga 540
gtcagagtaa actctgtgtc tcctggattc atagaaacac ctttaacgca gcctggactt 600
gaaaatgagc gcttcactaa ggcaatcgaa aaaaatacgg ctttaaaacg tgtaggtaaa 660
ccagatgaaa ttgcaaatgt tattgctttt gcagcgtctg aagaggcatc ttatatgacg 720
ggaagtgatt tattagtaga tggcgggtgg cttatcgtt 759
<210> 12
<211> 253
<212> PRT
<213> 巨大芽孢杆菌(Bacillus megaterium)
<400> 12
Met Arg Phe Glu Gly Gln Ile Ala Val Ile Thr Gly Ala Gly Ser Gly
1 5 10 15
Ile Gly Glu Ala Thr Ala Gln Arg Met Ala Lys Glu Gly Ala Arg Val
20 25 30
Ile Leu Val Gly Arg Thr Lys Glu Lys Leu Val Gln Ala Ala Ser Ala
35 40 45
Ile Asp Thr Arg Cys Glu Arg Lys Cys Thr Asp Ile Phe Ser Ala Asp
50 55 60
Val Thr Lys Glu Glu Asp Val Lys Glu Leu Val Glu Phe Ile Lys Glu
65 70 75 80
Lys Tyr Gly Gln Ile His Leu Leu Ile Asn Asn Ala Gly Gly Ser Val
85 90 95
Asn Ser Thr Ile Lys Glu Thr Thr Leu Glu Gln Trp Lys His Val Gln
100 105 110
Asp Val Asn Leu Thr Ser Val Phe Leu Val Thr Lys His Leu Ile Pro
115 120 125
Phe Leu Thr Glu Glu Ile Gly Gly Asn Arg Ser Ile Val Asn Ile Ala
130 135 140
Ser Leu Ser Gly His Lys Ala Gly Ala Gln Ile Pro His Tyr Ser Ala
145 150 155 160
Ala Lys Ala Gly Leu Ile Asn Phe Thr Lys Ala Met Ala Phe Glu Leu
165 170 175
Ala Pro His Gly Val Arg Val Asn Ser Val Ser Pro Gly Phe Ile Glu
180 185 190
Thr Pro Leu Thr Gln Pro Gly Leu Glu Asn Glu Arg Phe Thr Lys Ala
195 200 205
Ile Glu Lys Asn Thr Ala Leu Lys Arg Val Gly Lys Pro Asp Glu Ile
210 215 220
Ala Asn Val Ile Ala Phe Ala Ala Ser Glu Glu Ala Ser Tyr Met Thr
225 230 235 240
Gly Ser Asp Leu Leu Val Asp Gly Gly Trp Leu Ile Val
245 250
<210> 13
<211> 759
<212> DNA
<213> 巨大芽孢杆菌(Bacillus megaterium)
<400> 13
atgcgttttg aagggcaaat agctgttatt acaggagctg gaagcggcat tggagaagcg 60
accgctcaga gaatggcgaa agaaggagct cgcgtcattc ttgtcgggcg aactaaagaa 120
aaactagtac aggctgcatc ggctattgac acacggtgtg aacgaaaatg cacggatatt 180
ttctcagctg atgtaacgaa agaagaagac gtcaaagagc tggttgagtt tataaaagaa 240
aagtacggac aaattcattt attaattaat aacgcaggcg gagcagttaa ttcaacaatc 300
aaagaaacga cgcttgaaca gtggaaacac gtgcaggacg tcaatttaac aagcgtattt 360
cttgtcacaa aacatttgat tcctttttta actgaagaaa taggagggaa tcgatctatt 420
gttaacatcg cttctctttc aggacataaa gctggggcac aaattcctca ctacagcgct 480
gctaaagctg gtctaattaa ctttacaaaa gcaatggcgt ttgagttagc accccacgga 540
gtcagagtaa actctgtgtc tcctggattc atagaaacac ctttaacgca gcctggactt 600
gaaaatgagc gcttcactaa ggcaatcgaa aaaaatacgg ctttaaaacg tgtaggtaaa 660
ccagatgaaa ttgcaaatgt tattgctttt gcagcgtctg aagaggcatc ttatatgacg 720
ggaagtgatt tattagtaga tggcgggtgg cttatcgtt 759
<210> 14
<211> 253
<212> PRT
<213> 巨大芽孢杆菌(Bacillus megaterium)
<400> 14
Met Arg Phe Glu Gly Gln Ile Ala Val Ile Thr Gly Ala Gly Ser Gly
1 5 10 15
Ile Gly Glu Ala Thr Ala Gln Arg Met Ala Lys Glu Gly Ala Arg Val
20 25 30
Ile Leu Val Gly Arg Thr Lys Glu Lys Leu Val Gln Ala Ala Ser Ala
35 40 45
Ile Asp Thr Arg Cys Glu Arg Lys Cys Thr Asp Ile Phe Ser Ala Asp
50 55 60
Val Thr Lys Glu Glu Asp Val Lys Glu Leu Val Glu Phe Ile Lys Glu
65 70 75 80
Lys Tyr Gly Gln Ile His Leu Leu Ile Asn Asn Ala Gly Gly Ala Val
85 90 95
Asn Ser Thr Ile Lys Glu Thr Thr Leu Glu Gln Trp Lys His Val Gln
100 105 110
Asp Val Asn Leu Thr Ser Val Phe Leu Val Thr Lys His Leu Ile Pro
115 120 125
Phe Leu Thr Glu Glu Ile Gly Gly Asn Arg Ser Ile Val Asn Ile Ala
130 135 140
Ser Leu Ser Gly His Lys Ala Gly Ala Gln Ile Pro His Tyr Ser Ala
145 150 155 160
Ala Lys Ala Gly Leu Ile Asn Phe Thr Lys Ala Met Ala Phe Glu Leu
165 170 175
Ala Pro His Gly Val Arg Val Asn Ser Val Ser Pro Gly Phe Ile Glu
180 185 190
Thr Pro Leu Thr Gln Pro Gly Leu Glu Asn Glu Arg Phe Thr Lys Ala
195 200 205
Ile Glu Lys Asn Thr Ala Leu Lys Arg Val Gly Lys Pro Asp Glu Ile
210 215 220
Ala Asn Val Ile Ala Phe Ala Ala Ser Glu Glu Ala Ser Tyr Met Thr
225 230 235 240
Gly Ser Asp Leu Leu Val Asp Gly Gly Trp Leu Ile Val
245 250
<210> 15
<211> 759
<212> DNA
<213> 巨大芽孢杆菌(Bacillus megaterium)
<400> 15
atgcgttttg aagggcaaat agctgttatt acaggagctg gaagcggcat tggagaagcg 60
accgctcaga gaatggcgaa agaaggagct cgcgtcattc ttgtcgggcg aactaaagaa 120
aaactagtac aggctgcatc ggctattgac acacggtgtg aacgaaaatg cacggatatt 180
ttctcagctg atgtaacgaa agaagaagac gtcaaagagc tggttgagtt tataaaagaa 240
aagtacggac aaattcattt attaattaat aacgcaggcg gatcagttaa ttcaacaatc 300
aaagaaacga cgcttgaaca gtggaaacac gtgcaggacg tcaatttaac aagcgtattt 360
cttgtcacaa aacatttgat tcctttttta actgaagaaa taggagggaa tcgatctatt 420
gttaacatcg cttctctttc aggacataaa gctggggcac aaattcctca ctacagcgct 480
gctaaagctg gtctaattaa ctttacaaaa gcaatggcgt ttgagttagc accccacgga 540
gtcagagtaa actctgtgtc tcctggagcg atagaaacac ctttaacgca gcctggactt 600
gaaaatgagc gcttcactaa ggcaatcgaa aaaaatacgg ctttaaaacg tgtaggtaaa 660
ccagatgaaa ttgcaaatgt tattgctttt gcagcgtctg aagaggcatc ttatatgacg 720
ggaagtgatt tattagtaga tggcgggtgg cttatcgtt 759
<210> 16
<211> 253
<212> PRT
<213> 巨大芽孢杆菌(Bacillus megaterium)
<400> 16
Met Arg Phe Glu Gly Gln Ile Ala Val Ile Thr Gly Ala Gly Ser Gly
1 5 10 15
Ile Gly Glu Ala Thr Ala Gln Arg Met Ala Lys Glu Gly Ala Arg Val
20 25 30
Ile Leu Val Gly Arg Thr Lys Glu Lys Leu Val Gln Ala Ala Ser Ala
35 40 45
Ile Asp Thr Arg Cys Glu Arg Lys Cys Thr Asp Ile Phe Ser Ala Asp
50 55 60
Val Thr Lys Glu Glu Asp Val Lys Glu Leu Val Glu Phe Ile Lys Glu
65 70 75 80
Lys Tyr Gly Gln Ile His Leu Leu Ile Asn Asn Ala Gly Gly Ser Val
85 90 95
Asn Ser Thr Ile Lys Glu Thr Thr Leu Glu Gln Trp Lys His Val Gln
100 105 110
Asp Val Asn Leu Thr Ser Val Phe Leu Val Thr Lys His Leu Ile Pro
115 120 125
Phe Leu Thr Glu Glu Ile Gly Gly Asn Arg Ser Ile Val Asn Ile Ala
130 135 140
Ser Leu Ser Gly His Lys Ala Gly Ala Gln Ile Pro His Tyr Ser Ala
145 150 155 160
Ala Lys Ala Gly Leu Ile Asn Phe Thr Lys Ala Met Ala Phe Glu Leu
165 170 175
Ala Pro His Gly Val Arg Val Asn Ser Val Ser Pro Gly Ala Ile Glu
180 185 190
Thr Pro Leu Thr Gln Pro Gly Leu Glu Asn Glu Arg Phe Thr Lys Ala
195 200 205
Ile Glu Lys Asn Thr Ala Leu Lys Arg Val Gly Lys Pro Asp Glu Ile
210 215 220
Ala Asn Val Ile Ala Phe Ala Ala Ser Glu Glu Ala Ser Tyr Met Thr
225 230 235 240
Gly Ser Asp Leu Leu Val Asp Gly Gly Trp Leu Ile Val
245 250
<210> 17
<211> 759
<212> DNA
<213> 巨大芽孢杆菌(Bacillus megaterium)
<400> 17
atgcgttttg aagggcaaat agctgttatt acaggagctg gaagcggcat tggagaagcg 60
accgctcaga gaatggcgaa agaaggagct cgcgtcattc ttgtcgggcg aactaaagaa 120
aaactagtac aggctgcatc ggctattgac acacggtgtg aacgaaaatg cacggatatt 180
ttctcagctg atgtaacgaa agaagaagac gtcaaagagc tggttgagtt tataaaagaa 240
aagtacggac aaattcattt attaattaat aacgcaggcg gatcagttaa ttcaacaatc 300
aaagaaacga cgcttgaaca gtggaaacac gtgcaggacg tcaatttaac aagcgtattt 360
cttgtcacaa aacatttgat tcctttttta actgaagaaa taggagggaa tcgatctatt 420
gttaacatcg cttctctttc aggacataaa gctggggcac aaattcctca ctacagcgct 480
gctaaagctg gtctaattaa ctttacaaaa gcaatggcgt ttgagttagc accccacgga 540
gtcagagtaa actctgtgtc tcctggattc atagaaacac ctttaacgca gcctggactt 600
gaaaatgagc gcctcactaa ggcaatcgaa aaaaatacgg ctttaaaacg tgtaggtaaa 660
ccagatgaaa ttgcaaatgt tattgctttt gcagcgtctg aagaggcatc ttatatgacg 720
ggaagtgatt tattagtaga tggcgggtgg cttatcgtt 759
<210> 18
<211> 253
<212> PRT
<213> 巨大芽孢杆菌(Bacillus megaterium)
<400> 18
Met Arg Phe Glu Gly Gln Ile Ala Val Ile Thr Gly Ala Gly Ser Gly
1 5 10 15
Ile Gly Glu Ala Thr Ala Gln Arg Met Ala Lys Glu Gly Ala Arg Val
20 25 30
Ile Leu Val Gly Arg Thr Lys Glu Lys Leu Val Gln Ala Ala Ser Ala
35 40 45
Ile Asp Thr Arg Cys Glu Arg Lys Cys Thr Asp Ile Phe Ser Ala Asp
50 55 60
Val Thr Lys Glu Glu Asp Val Lys Glu Leu Val Glu Phe Ile Lys Glu
65 70 75 80
Lys Tyr Gly Gln Ile His Leu Leu Ile Asn Asn Ala Gly Gly Ser Val
85 90 95
Asn Ser Thr Ile Lys Glu Thr Thr Leu Glu Gln Trp Lys His Val Gln
100 105 110
Asp Val Asn Leu Thr Ser Val Phe Leu Val Thr Lys His Leu Ile Pro
115 120 125
Phe Leu Thr Glu Glu Ile Gly Gly Asn Arg Ser Ile Val Asn Ile Ala
130 135 140
Ser Leu Ser Gly His Lys Ala Gly Ala Gln Ile Pro His Tyr Ser Ala
145 150 155 160
Ala Lys Ala Gly Leu Ile Asn Phe Thr Lys Ala Met Ala Phe Glu Leu
165 170 175
Ala Pro His Gly Val Arg Val Asn Ser Val Ser Pro Gly Phe Ile Glu
180 185 190
Thr Pro Leu Thr Gln Pro Gly Leu Glu Asn Glu Arg Leu Thr Lys Ala
195 200 205
Ile Glu Lys Asn Thr Ala Leu Lys Arg Val Gly Lys Pro Asp Glu Ile
210 215 220
Ala Asn Val Ile Ala Phe Ala Ala Ser Glu Glu Ala Ser Tyr Met Thr
225 230 235 240
Gly Ser Asp Leu Leu Val Asp Gly Gly Trp Leu Ile Val
245 250
<210> 19
<211> 759
<212> DNA
<213> 巨大芽孢杆菌(Bacillus megaterium)
<400> 19
atgcgttttg aagggcaaat agctgttatt acaggagctg gaagcggcat tggagaagcg 60
accgctcaga gaatggcgaa agaaggagct cgcgtcattc ttgtcgggcg aactaaagaa 120
aaactagtac aggctgcatc ggctattgac acacggtgtg aacgaaaatg cacggatatt 180
ttctcagctg atgtaacgaa agaagaagac gtcaaagagc tggttgagtt tataaaagaa 240
aagtacggac aaattcattt attaattaat aacgcaggcg gatcagttaa ttcaacaatc 300
aaagaaacga cgcttgaaca gtggaaacac gtgcaggacg tcaatttaac aagcgtattt 360
cttgtcacaa aacatttgat tcctttttta actgaagaaa taggagggaa tcgatctatt 420
gttaacatcg cttctctttc aggacataaa gctggggcac aaattcctca ctacagcgct 480
gctaaagctg gtctaattaa ctttacaaaa gcaatggcgt ttgagttagc accccacgga 540
gtcagagtaa actctgtgtc tcctggagcg atagaaacac ctttaacgca gcctggactt 600
gaaaatgagc gcctcactaa ggcaatcgaa aaaaatacgg ctttaaaacg tgtaggtaaa 660
ccagatgaaa ttgcaaatgt tattgctttt gcagcgtctg aagaggcatc ttatatgacg 720
ggaagtgatt tattagtaga tggcgggtgg cttatcgtt 759
<210> 20
<211> 253
<212> PRT
<213> 巨大芽孢杆菌(Bacillus megaterium)
<400> 20
Met Arg Phe Glu Gly Gln Ile Ala Val Ile Thr Gly Ala Gly Ser Gly
1 5 10 15
Ile Gly Glu Ala Thr Ala Gln Arg Met Ala Lys Glu Gly Ala Arg Val
20 25 30
Ile Leu Val Gly Arg Thr Lys Glu Lys Leu Val Gln Ala Ala Ser Ala
35 40 45
Ile Asp Thr Arg Cys Glu Arg Lys Cys Thr Asp Ile Phe Ser Ala Asp
50 55 60
Val Thr Lys Glu Glu Asp Val Lys Glu Leu Val Glu Phe Ile Lys Glu
65 70 75 80
Lys Tyr Gly Gln Ile His Leu Leu Ile Asn Asn Ala Gly Gly Ser Val
85 90 95
Asn Ser Thr Ile Lys Glu Thr Thr Leu Glu Gln Trp Lys His Val Gln
100 105 110
Asp Val Asn Leu Thr Ser Val Phe Leu Val Thr Lys His Leu Ile Pro
115 120 125
Phe Leu Thr Glu Glu Ile Gly Gly Asn Arg Ser Ile Val Asn Ile Ala
130 135 140
Ser Leu Ser Gly His Lys Ala Gly Ala Gln Ile Pro His Tyr Ser Ala
145 150 155 160
Ala Lys Ala Gly Leu Ile Asn Phe Thr Lys Ala Met Ala Phe Glu Leu
165 170 175
Ala Pro His Gly Val Arg Val Asn Ser Val Ser Pro Gly Ala Ile Glu
180 185 190
Thr Pro Leu Thr Gln Pro Gly Leu Glu Asn Glu Arg Leu Thr Lys Ala
195 200 205
Ile Glu Lys Asn Thr Ala Leu Lys Arg Val Gly Lys Pro Asp Glu Ile
210 215 220
Ala Asn Val Ile Ala Phe Ala Ala Ser Glu Glu Ala Ser Tyr Met Thr
225 230 235 240
Gly Ser Asp Leu Leu Val Asp Gly Gly Trp Leu Ile Val
245 250
<210> 21
<211> 846
<212> DNA
<213> 枯草芽孢杆菌(Bacillus subtilis)
<400> 21
atggcgaatc aaaaaaagaa aacattgccg cctcagcacc agaaccagca gccgggtttt 60
gaatatctta tggatccccg tccggttttt gataagccga agatacagaa aaaattagag 120
ggcaaaaccg ctatcattac tggaggagac agcggaatcg gacgcgctgt atcggtgtta 180
ttcgcaaaag aaggggctaa tgtggtcatt gtgtatttga atgagcatca ggacgctgag 240
gagacaaagc agtatgtaga aaaggaaggg gtaaaatgcc tgctgattgc aggagatatc 300
ggagatgaag cgttttgcaa cgatgtggtc atgcaggcaa gccaagcgtt tccgtcgatt 360
gatatattgg ttaacaatgc agctgagcag catgtccagc ccagcattga aaaaatcaca 420
agccaccagc tgatcagaac cttccaaacg aacattttct ccatgtttta cttaacgaag 480
gctgtactgc ctcatttaaa aaagggaagc tctatcatta atactgcatc aattaccgcc 540
tataaaggca ataaaacgct gatcgattat tcagcgacaa aaggggcgat cgttacgttt 600
acaaggtcac tttcccagtc gcttgttcag cagggcatac gggttaatgc agtagcgcca 660
ggccccattt ggacaccgct tatcccggcc agctttgcgg caaaggatgt ggaagtgttt 720
ggttcagacg tgccgatgga acgcccggga cagccggtgg aagtggcgcc aagctattta 780
taccttgcca gtgacgattc cacctatgtc acaggacaga cgattcatgt caatggcgga 840
caattg 846
<210> 22
<211> 282
<212> PRT
<213> 枯草芽孢杆菌(Bacillus subtilis)
<400> 22
Met Ala Asn Gln Lys Lys Lys Thr Leu Pro Pro Gln His Gln Asn Gln
1 5 10 15
Gln Pro Gly Phe Glu Tyr Leu Met Asp Pro Arg Pro Val Phe Asp Lys
20 25 30
Pro Lys Ile Gln Lys Lys Leu Glu Gly Lys Thr Ala Ile Ile Thr Gly
35 40 45
Gly Asp Ser Gly Ile Gly Arg Ala Val Ser Val Leu Phe Ala Lys Glu
50 55 60
Gly Ala Asn Val Val Ile Val Tyr Leu Asn Glu His Gln Asp Ala Glu
65 70 75 80
Glu Thr Lys Gln Tyr Val Glu Lys Glu Gly Val Lys Cys Leu Leu Ile
85 90 95
Ala Gly Asp Ile Gly Asp Glu Ala Phe Cys Asn Asp Val Val Met Gln
100 105 110
Ala Ser Gln Ala Phe Pro Ser Ile Asp Ile Leu Val Asn Asn Ala Ala
115 120 125
Glu Gln His Val Gln Pro Ser Ile Glu Lys Ile Thr Ser His Gln Leu
130 135 140
Ile Arg Thr Phe Gln Thr Asn Ile Phe Ser Met Phe Tyr Leu Thr Lys
145 150 155 160
Ala Val Leu Pro His Leu Lys Lys Gly Ser Ser Ile Ile Asn Thr Ala
165 170 175
Ser Ile Thr Ala Tyr Lys Gly Asn Lys Thr Leu Ile Asp Tyr Ser Ala
180 185 190
Thr Lys Gly Ala Ile Val Thr Phe Thr Arg Ser Leu Ser Gln Ser Leu
195 200 205
Val Gln Gln Gly Ile Arg Val Asn Ala Val Ala Pro Gly Pro Ile Trp
210 215 220
Thr Pro Leu Ile Pro Ala Ser Phe Ala Ala Lys Asp Val Glu Val Phe
225 230 235 240
Gly Ser Asp Val Pro Met Glu Arg Pro Gly Gln Pro Val Glu Val Ala
245 250 255
Pro Ser Tyr Leu Tyr Leu Ala Ser Asp Asp Ser Thr Tyr Val Thr Gly
260 265 270
Gln Thr Ile His Val Asn Gly Gly Gln Leu
275 280
<210> 23
<211> 846
<212> DNA
<213> 枯草芽孢杆菌(Bacillus subtilis)
<400> 23
atggcgaatc aaaaaaagaa aacattgccg cctcagcacc agaaccagca gccgggtttt 60
gaatatctta tggatccccg tccggttttt gataagccga agatacagaa aaaattagag 120
ggcaaaaccg ctatcattac tggaggagac agcggaatcg gacgcgctgt atcggtgtta 180
ttcgcaaaag aaggggctaa tgtggtcatt gtgtatttga atgagcatca ggacgctgag 240
gagacaaagc agtatgtaga aaaggaaggg gtaaaatgcc tgctgattgc aggagatatc 300
ggagatgaag cgttttgcaa cgatgtggtc atgcaggcaa gccaagcgtt tccgtcgatt 360
gatatattgg ttaacaatgc agctgagcag catgtccagc ccagcattga aaaaatcaca 420
agccaccagc tgatcagaac cttccaaacg aacattttct ccatgtttta cttaacgaag 480
gctgtactgc ctcatttaaa aaagggaagc tctatcatta atactgcatc aattaccgcc 540
tataaaggca ataaaacgct gatcgattat tcagcgacaa aaggggcgat cgttacgttt 600
acaaggtcac tttcccagtc gcttgttcag cagggcatac gggttaatgc agtagcgcca 660
ggcttcattt ggacaccgct tatcccggcc agctttgcgg caaaggatgt ggaagtgttt 720
ggttcagacg tgccgatgga acgcccggga cagccggtgg aagtggcgcc aagctattta 780
taccttgcca gtgacgattc cacctatgtc acaggacaga cgattcatgt caatggcgga 840
caattg 846
<210> 24
<211> 282
<212> PRT
<213> 枯草芽孢杆菌(Bacillus subtilis)
<400> 24
Met Ala Asn Gln Lys Lys Lys Thr Leu Pro Pro Gln His Gln Asn Gln
1 5 10 15
Gln Pro Gly Phe Glu Tyr Leu Met Asp Pro Arg Pro Val Phe Asp Lys
20 25 30
Pro Lys Ile Gln Lys Lys Leu Glu Gly Lys Thr Ala Ile Ile Thr Gly
35 40 45
Gly Asp Ser Gly Ile Gly Arg Ala Val Ser Val Leu Phe Ala Lys Glu
50 55 60
Gly Ala Asn Val Val Ile Val Tyr Leu Asn Glu His Gln Asp Ala Glu
65 70 75 80
Glu Thr Lys Gln Tyr Val Glu Lys Glu Gly Val Lys Cys Leu Leu Ile
85 90 95
Ala Gly Asp Ile Gly Asp Glu Ala Phe Cys Asn Asp Val Val Met Gln
100 105 110
Ala Ser Gln Ala Phe Pro Ser Ile Asp Ile Leu Val Asn Asn Ala Ala
115 120 125
Glu Gln His Val Gln Pro Ser Ile Glu Lys Ile Thr Ser His Gln Leu
130 135 140
Ile Arg Thr Phe Gln Thr Asn Ile Phe Ser Met Phe Tyr Leu Thr Lys
145 150 155 160
Ala Val Leu Pro His Leu Lys Lys Gly Ser Ser Ile Ile Asn Thr Ala
165 170 175
Ser Ile Thr Ala Tyr Lys Gly Asn Lys Thr Leu Ile Asp Tyr Ser Ala
180 185 190
Thr Lys Gly Ala Ile Val Thr Phe Thr Arg Ser Leu Ser Gln Ser Leu
195 200 205
Val Gln Gln Gly Ile Arg Val Asn Ala Val Ala Pro Gly Phe Ile Trp
210 215 220
Thr Pro Leu Ile Pro Ala Ser Phe Ala Ala Lys Asp Val Glu Val Phe
225 230 235 240
Gly Ser Asp Val Pro Met Glu Arg Pro Gly Gln Pro Val Glu Val Ala
245 250 255
Pro Ser Tyr Leu Tyr Leu Ala Ser Asp Asp Ser Thr Tyr Val Thr Gly
260 265 270
Gln Thr Ile His Val Asn Gly Gly Gln Leu
275 280
<210> 25
<211> 852
<212> DNA
<213> 枯草芽孢杆菌(Bacillus subtilis)
<400> 25
atgaataaaa aaaccgcgat cgtaacagga gcgtcaagag gctttggtca gctggcagcc 60
gtaaagctcg ccaagtcatt tttcgtgatt gccacaacaa gacagcctga gaaagctgaa 120
cagcttcggg aattggccgc agcacacaat gtggctgatt ctattcacat taccgctctc 180
gacgtcacag atgagcagtc catagcctca ttcggaaaag ccattagtgc ttacgctcct 240
attgatcttc tcgtcaacaa cgccggaacg gcttacggag gatttgtcga ggatgtgcca 300
atggaacatt tcagacaaca atttgaaacg aatgtcttcg gagtgattca tgtgacaaaa 360
accgttctgc cttacataag aaagcatagt ggagcaaaga ttataaacgt gagcagcatc 420
agcggtctga ccggattccc tgcgttgtcg ccttatgcat cttccaagca tgcattggag 480
ggtttttctg agagcctgcg tgtggagctg cttccgttcg gtatccaaac cgctttgatc 540
gagccaggct catacaggac atcgatctgg tccacatcat tatcaaatta tatgtcggtg 600
cctgctgacg attcagccta tcatcaatac tataaaaaga tcctttccta cgttgaaaaa 660
aacgcagatg aaagtggaga tcctcaagag gttgccgacc tcatttatca attggcaaca 720
aaacagcacg taaagaaatt gcgatacccg atcggaaaag gcatcaagct taccttgtta 780
ttccgatcgc tttttccttg gtctgcgtgg gaatctatcc tgaagaaaaa actattcagc 840
tcatctaaat ta 852
<210> 26
<211> 284
<212> PRT
<213> 枯草芽孢杆菌(Bacillus subtilis)
<400> 26
Met Asn Lys Lys Thr Ala Ile Val Thr Gly Ala Ser Arg Gly Phe Gly
1 5 10 15
Gln Leu Ala Ala Val Lys Leu Ala Lys Ser Phe Phe Val Ile Ala Thr
20 25 30
Thr Arg Gln Pro Glu Lys Ala Glu Gln Leu Arg Glu Leu Ala Ala Ala
35 40 45
His Asn Val Ala Asp Ser Ile His Ile Thr Ala Leu Asp Val Thr Asp
50 55 60
Glu Gln Ser Ile Ala Ser Phe Gly Lys Ala Ile Ser Ala Tyr Ala Pro
65 70 75 80
Ile Asp Leu Leu Val Asn Asn Ala Gly Thr Ala Tyr Gly Gly Phe Val
85 90 95
Glu Asp Val Pro Met Glu His Phe Arg Gln Gln Phe Glu Thr Asn Val
100 105 110
Phe Gly Val Ile His Val Thr Lys Thr Val Leu Pro Tyr Ile Arg Lys
115 120 125
His Ser Gly Ala Lys Ile Ile Asn Val Ser Ser Ile Ser Gly Leu Thr
130 135 140
Gly Phe Pro Ala Leu Ser Pro Tyr Ala Ser Ser Lys His Ala Leu Glu
145 150 155 160
Gly Phe Ser Glu Ser Leu Arg Val Glu Leu Leu Pro Phe Gly Ile Gln
165 170 175
Thr Ala Leu Ile Glu Pro Gly Ser Tyr Arg Thr Ser Ile Trp Ser Thr
180 185 190
Ser Leu Ser Asn Tyr Met Ser Val Pro Ala Asp Asp Ser Ala Tyr His
195 200 205
Gln Tyr Tyr Lys Lys Ile Leu Ser Tyr Val Glu Lys Asn Ala Asp Glu
210 215 220
Ser Gly Asp Pro Gln Glu Val Ala Asp Leu Ile Tyr Gln Leu Ala Thr
225 230 235 240
Lys Gln His Val Lys Lys Leu Arg Tyr Pro Ile Gly Lys Gly Ile Lys
245 250 255
Leu Thr Leu Leu Phe Arg Ser Leu Phe Pro Trp Ser Ala Trp Glu Ser
260 265 270
Ile Leu Lys Lys Lys Leu Phe Ser Ser Ser Lys Leu
275 280
<210> 27
<211> 852
<212> DNA
<213> 枯草芽孢杆菌(Bacillus subtilis)
<400> 27
atgaataaaa aaaccgcgat cgtaacagga gcgtcaagag gctttggtca gctggcagcc 60
gtaaagctcg ccaagtcatt tttcgtgatt gccacaacaa gacagcctga gaaagctgaa 120
cagcttcggg aattggccgc agcacacaat gtggctgatt ctattcacat taccgctctc 180
gacgtcacag atgagcagtc catagcctca ttcggaaaag ccattagtgc ttacgctcct 240
attgatcttc tcgtcaacaa cgccggaacg gcttacggag gatttgtcga ggatgtgcca 300
atggaacatt tcagacaaca atttgaaacg aatgtcttcg gagtgattca tgtgacaaaa 360
accgttctgc cttacataag aaagcatagt ggagcaaaga ttataaacgt gagcagcatc 420
agcggtctga ccggattccc tgcgttgtcg ccttatgcat cttccaagca tgcattggag 480
ggtttttctg agagcctgcg tgtggagctg cttccgttcg gtatccaaac cgctttgatc 540
gagccaggct tttacaggac atcgatctgg tccacatcat tatcaaatta tatgtcggtg 600
cctgctgacg attcagccta tcatcaatac tataaaaaga tcctttccta cgttgaaaaa 660
aacgcagatg aaagtggaga tcctcaagag gttgccgacc tcatttatca attggcaaca 720
aaacagcacg taaagaaatt gcgatacccg atcggaaaag gcatcaagct taccttgtta 780
ttccgatcgc tttttccttg gtctgcgtgg gaatctatcc tgaagaaaaa actattcagc 840
tcatctaaat ta 852
<210> 28
<211> 284
<212> PRT
<213> 枯草芽孢杆菌(Bacillus subtilis)
<400> 28
Met Asn Lys Lys Thr Ala Ile Val Thr Gly Ala Ser Arg Gly Phe Gly
1 5 10 15
Gln Leu Ala Ala Val Lys Leu Ala Lys Ser Phe Phe Val Ile Ala Thr
20 25 30
Thr Arg Gln Pro Glu Lys Ala Glu Gln Leu Arg Glu Leu Ala Ala Ala
35 40 45
His Asn Val Ala Asp Ser Ile His Ile Thr Ala Leu Asp Val Thr Asp
50 55 60
Glu Gln Ser Ile Ala Ser Phe Gly Lys Ala Ile Ser Ala Tyr Ala Pro
65 70 75 80
Ile Asp Leu Leu Val Asn Asn Ala Gly Thr Ala Tyr Gly Gly Phe Val
85 90 95
Glu Asp Val Pro Met Glu His Phe Arg Gln Gln Phe Glu Thr Asn Val
100 105 110
Phe Gly Val Ile His Val Thr Lys Thr Val Leu Pro Tyr Ile Arg Lys
115 120 125
His Ser Gly Ala Lys Ile Ile Asn Val Ser Ser Ile Ser Gly Leu Thr
130 135 140
Gly Phe Pro Ala Leu Ser Pro Tyr Ala Ser Ser Lys His Ala Leu Glu
145 150 155 160
Gly Phe Ser Glu Ser Leu Arg Val Glu Leu Leu Pro Phe Gly Ile Gln
165 170 175
Thr Ala Leu Ile Glu Pro Gly Phe Tyr Arg Thr Ser Ile Trp Ser Thr
180 185 190
Ser Leu Ser Asn Tyr Met Ser Val Pro Ala Asp Asp Ser Ala Tyr His
195 200 205
Gln Tyr Tyr Lys Lys Ile Leu Ser Tyr Val Glu Lys Asn Ala Asp Glu
210 215 220
Ser Gly Asp Pro Gln Glu Val Ala Asp Leu Ile Tyr Gln Leu Ala Thr
225 230 235 240
Lys Gln His Val Lys Lys Leu Arg Tyr Pro Ile Gly Lys Gly Ile Lys
245 250 255
Leu Thr Leu Leu Phe Arg Ser Leu Phe Pro Trp Ser Ala Trp Glu Ser
260 265 270
Ile Leu Lys Lys Lys Leu Phe Ser Ser Ser Lys Leu
275 280
<210> 29
<211> 852
<212> DNA
<213> 枯草芽孢杆菌(Bacillus subtilis)
<400> 29
atgaataaaa aaaccgcgat cgtaacagga gcgtcaagag gctttggtca gctggcagcc 60
gtaaagctcg ccaagtcatt tttcgtgatt gccacaacaa gacagcctga gaaagctgaa 120
cagcttcggg aattggccgc agcacacaat gtggctgatt ctattcacat taccgctctc 180
gacgtcacag atgagcagtc catagcctca ttcggaaaag ccattagtgc ttacgctcct 240
attgatcttc tcgtcaacaa cgccggaacg gcttacggag gatttgtcga ggatgtgcca 300
atggaacatt tcagacaaca atttgaaacg aatgtcttcg gagtgattca tgtgacaaaa 360
accgttctgc cttacataag aaagcatagt ggagcaaaga ttataaacgt gagcagcatc 420
agcggtctga ccggattccc tgcgttgtcg ccttatgcat cttccaagca tgcattggag 480
ggtttttctg agagcctgcg tgtggagctg cttccgttcg gtatccaaac cgctttgatc 540
gagccaggct attacaggac atcgatctgg tccacatcat tatcaaatta tatgtcggtg 600
cctgctgacg attcagccta tcatcaatac tataaaaaga tcctttccta cgttgaaaaa 660
aacgcagatg aaagtggaga tcctcaagag gttgccgacc tcatttatca attggcaaca 720
aaacagcacg taaagaaatt gcgatacccg atcggaaaag gcatcaagct taccttgtta 780
ttccgatcgc tttttccttg gtctgcgtgg gaatctatcc tgaagaaaaa actattcagc 840
tcatctaaat ta 852
<210> 30
<211> 284
<212> PRT
<213> 枯草芽孢杆菌(Bacillus subtilis)
<400> 30
Met Asn Lys Lys Thr Ala Ile Val Thr Gly Ala Ser Arg Gly Phe Gly
1 5 10 15
Gln Leu Ala Ala Val Lys Leu Ala Lys Ser Phe Phe Val Ile Ala Thr
20 25 30
Thr Arg Gln Pro Glu Lys Ala Glu Gln Leu Arg Glu Leu Ala Ala Ala
35 40 45
His Asn Val Ala Asp Ser Ile His Ile Thr Ala Leu Asp Val Thr Asp
50 55 60
Glu Gln Ser Ile Ala Ser Phe Gly Lys Ala Ile Ser Ala Tyr Ala Pro
65 70 75 80
Ile Asp Leu Leu Val Asn Asn Ala Gly Thr Ala Tyr Gly Gly Phe Val
85 90 95
Glu Asp Val Pro Met Glu His Phe Arg Gln Gln Phe Glu Thr Asn Val
100 105 110
Phe Gly Val Ile His Val Thr Lys Thr Val Leu Pro Tyr Ile Arg Lys
115 120 125
His Ser Gly Ala Lys Ile Ile Asn Val Ser Ser Ile Ser Gly Leu Thr
130 135 140
Gly Phe Pro Ala Leu Ser Pro Tyr Ala Ser Ser Lys His Ala Leu Glu
145 150 155 160
Gly Phe Ser Glu Ser Leu Arg Val Glu Leu Leu Pro Phe Gly Ile Gln
165 170 175
Thr Ala Leu Ile Glu Pro Gly Tyr Tyr Arg Thr Ser Ile Trp Ser Thr
180 185 190
Ser Leu Ser Asn Tyr Met Ser Val Pro Ala Asp Asp Ser Ala Tyr His
195 200 205
Gln Tyr Tyr Lys Lys Ile Leu Ser Tyr Val Glu Lys Asn Ala Asp Glu
210 215 220
Ser Gly Asp Pro Gln Glu Val Ala Asp Leu Ile Tyr Gln Leu Ala Thr
225 230 235 240
Lys Gln His Val Lys Lys Leu Arg Tyr Pro Ile Gly Lys Gly Ile Lys
245 250 255
Leu Thr Leu Leu Phe Arg Ser Leu Phe Pro Trp Ser Ala Trp Glu Ser
260 265 270
Ile Leu Lys Lys Lys Leu Phe Ser Ser Ser Lys Leu
275 280
<210> 31
<211> 852
<212> DNA
<213> 枯草芽孢杆菌(Bacillus subtilis)
<400> 31
atgaataaaa aaaccgcgat cgtaacagga gcgtcaagag gctttggtca gctggcagcc 60
gtaaagctcg ccaagtcatt tttcgtgatt gccacaacaa gacagcctga gaaagctgaa 120
cagcttcggg aattggccgc agcacacaat gtggctgatt ctattcacat taccgctctc 180
gacgtcacag atgagcagtc catagcctca ttcggaaaag ccattagtgc ttacgctcct 240
attgatcttc tcgtcaacaa cgccggaacg gcttacggag gatttgtcga ggatgtgcca 300
atggaacatt tcagacaaca atttgaaacg aatgtcttcg gagtgattca tgtgacaaaa 360
accgttctgc cttacataag aaagcatagt ggagcaaaga ttataaacgt gagcagcatc 420
agcggtctga ccggattccc tgcgttgtcg ccttatgcat cttccaagca tgcattggag 480
ggtttttctg agagcctgcg tgtggagctg cttccgttcg gtatccaaac cgctttgatc 540
gagccaggct ggtacaggac atcgatctgg tccacatcat tatcaaatta tatgtcggtg 600
cctgctgacg attcagccta tcatcaatac tataaaaaga tcctttccta cgttgaaaaa 660
aacgcagatg aaagtggaga tcctcaagag gttgccgacc tcatttatca attggcaaca 720
aaacagcacg taaagaaatt gcgatacccg atcggaaaag gcatcaagct taccttgtta 780
ttccgatcgc tttttccttg gtctgcgtgg gaatctatcc tgaagaaaaa actattcagc 840
tcatctaaat ta 852
<210> 32
<211> 284
<212> PRT
<213> 枯草芽孢杆菌(Bacillus subtilis)
<400> 32
Met Asn Lys Lys Thr Ala Ile Val Thr Gly Ala Ser Arg Gly Phe Gly
1 5 10 15
Gln Leu Ala Ala Val Lys Leu Ala Lys Ser Phe Phe Val Ile Ala Thr
20 25 30
Thr Arg Gln Pro Glu Lys Ala Glu Gln Leu Arg Glu Leu Ala Ala Ala
35 40 45
His Asn Val Ala Asp Ser Ile His Ile Thr Ala Leu Asp Val Thr Asp
50 55 60
Glu Gln Ser Ile Ala Ser Phe Gly Lys Ala Ile Ser Ala Tyr Ala Pro
65 70 75 80
Ile Asp Leu Leu Val Asn Asn Ala Gly Thr Ala Tyr Gly Gly Phe Val
85 90 95
Glu Asp Val Pro Met Glu His Phe Arg Gln Gln Phe Glu Thr Asn Val
100 105 110
Phe Gly Val Ile His Val Thr Lys Thr Val Leu Pro Tyr Ile Arg Lys
115 120 125
His Ser Gly Ala Lys Ile Ile Asn Val Ser Ser Ile Ser Gly Leu Thr
130 135 140
Gly Phe Pro Ala Leu Ser Pro Tyr Ala Ser Ser Lys His Ala Leu Glu
145 150 155 160
Gly Phe Ser Glu Ser Leu Arg Val Glu Leu Leu Pro Phe Gly Ile Gln
165 170 175
Thr Ala Leu Ile Glu Pro Gly Trp Tyr Arg Thr Ser Ile Trp Ser Thr
180 185 190
Ser Leu Ser Asn Tyr Met Ser Val Pro Ala Asp Asp Ser Ala Tyr His
195 200 205
Gln Tyr Tyr Lys Lys Ile Leu Ser Tyr Val Glu Lys Asn Ala Asp Glu
210 215 220
Ser Gly Asp Pro Gln Glu Val Ala Asp Leu Ile Tyr Gln Leu Ala Thr
225 230 235 240
Lys Gln His Val Lys Lys Leu Arg Tyr Pro Ile Gly Lys Gly Ile Lys
245 250 255
Leu Thr Leu Leu Phe Arg Ser Leu Phe Pro Trp Ser Ala Trp Glu Ser
260 265 270
Ile Leu Lys Lys Lys Leu Phe Ser Ser Ser Lys Leu
275 280
<210> 33
<211> 852
<212> DNA
<213> 枯草芽孢杆菌(Bacillus subtilis)
<400> 33
atgaataaaa aaaccgcgat cgtaacagga gcgtcaagag gctttggtca gctggcagcc 60
gtaaagctcg ccaagtcatt tttcgtgatt gccacaacaa gacagcctga gaaagctgaa 120
cagcttcggg aattggccgc agcacacaat gtggctgatt ctattcacat taccgctctc 180
gacgtcacag atgagcagtc catagcctca ttcggaaaag ccattagtgc ttacgctcct 240
attgatcttc tcgtcaacaa cgccggaacg gcttacggag gatttgtcga ggatgtgcca 300
atggaacatt tcagacaaca atttgaaacg aatgtcttcg gagtgattca tgtgacaaaa 360
accgttctgc cttacataag aaagcatagt ggagcaaaga ttataaacgt gagcagcatc 420
agcggtctga ccggatatcc tgcgttgtcg ccttatgcat cttccaagca tgcattggag 480
ggtttttctg agagcctgcg tgtggagctg cttccgttcg gtatccaaac cgctttgatc 540
gagccaggct attacaggac atcgatctgg tccacatcat tatcaaatta tatgtcggtg 600
cctgctgacg attcagccta tcatcaatac tataaaaaga tcctttccta cgttgaaaaa 660
aacgcagatg aaagtggaga tcctcaagag gttgccgacc tcatttatca attggcaaca 720
aaacagcacg taaagaaatt gcgatacccg atcggaaaag gcatcaagct taccttgtta 780
ttccgatcgc tttttccttg gtctgcgtgg gaatctatcc tgaagaaaaa actattcagc 840
tcatctaaat ta 852
<210> 34
<211> 284
<212> PRT
<213> 枯草芽孢杆菌(Bacillus subtilis)
<400> 34
Met Asn Lys Lys Thr Ala Ile Val Thr Gly Ala Ser Arg Gly Phe Gly
1 5 10 15
Gln Leu Ala Ala Val Lys Leu Ala Lys Ser Phe Phe Val Ile Ala Thr
20 25 30
Thr Arg Gln Pro Glu Lys Ala Glu Gln Leu Arg Glu Leu Ala Ala Ala
35 40 45
His Asn Val Ala Asp Ser Ile His Ile Thr Ala Leu Asp Val Thr Asp
50 55 60
Glu Gln Ser Ile Ala Ser Phe Gly Lys Ala Ile Ser Ala Tyr Ala Pro
65 70 75 80
Ile Asp Leu Leu Val Asn Asn Ala Gly Thr Ala Tyr Gly Gly Phe Val
85 90 95
Glu Asp Val Pro Met Glu His Phe Arg Gln Gln Phe Glu Thr Asn Val
100 105 110
Phe Gly Val Ile His Val Thr Lys Thr Val Leu Pro Tyr Ile Arg Lys
115 120 125
His Ser Gly Ala Lys Ile Ile Asn Val Ser Ser Ile Ser Gly Leu Thr
130 135 140
Gly Tyr Pro Ala Leu Ser Pro Tyr Ala Ser Ser Lys His Ala Leu Glu
145 150 155 160
Gly Phe Ser Glu Ser Leu Arg Val Glu Leu Leu Pro Phe Gly Ile Gln
165 170 175
Thr Ala Leu Ile Glu Pro Gly Tyr Tyr Arg Thr Ser Ile Trp Ser Thr
180 185 190
Ser Leu Ser Asn Tyr Met Ser Val Pro Ala Asp Asp Ser Ala Tyr His
195 200 205
Gln Tyr Tyr Lys Lys Ile Leu Ser Tyr Val Glu Lys Asn Ala Asp Glu
210 215 220
Ser Gly Asp Pro Gln Glu Val Ala Asp Leu Ile Tyr Gln Leu Ala Thr
225 230 235 240
Lys Gln His Val Lys Lys Leu Arg Tyr Pro Ile Gly Lys Gly Ile Lys
245 250 255
Leu Thr Leu Leu Phe Arg Ser Leu Phe Pro Trp Ser Ala Trp Glu Ser
260 265 270
Ile Leu Lys Lys Lys Leu Phe Ser Ser Ser Lys Leu
275 280
<210> 35
<211> 852
<212> DNA
<213> 枯草芽孢杆菌(Bacillus subtilis)
<400> 35
atgaataaaa aaaccgcgat cgtaacagga gcgtcaagag gctttggtca gctggcagcc 60
gtaaagctcg ccaagtcatt tttcgtgatt gccacaacaa gacagcctga gaaagctgaa 120
cagcttcggg aattggccgc agcacacaat gtggctgatt ctattcacat taccgctctc 180
gacgtcacag atgagcagtc catagcctca ttcggaaaag ccattagtgc ttacgctcct 240
attgatcttc tcgtcaacaa cgccggaacg gcttacggag gatttgtcga ggatgtgcca 300
atggaacatt tcagacaaca atttgaaacg aatgtcttcg gagtgattca tgtgacaaaa 360
accgttctgc cttacataag aaagcatagt ggagcaaaga ttataaacgt gagcagcatc 420
agcggtctga ccggattccc tgcgttttcg ccttatgcat cttccaagca tgcattggag 480
ggtttttctg agagcctgcg tgtggagctg cttccgttcg gtatccaaac cgctttgatc 540
gagccaggct attacaggac atcgatctgg tccacatcat tatcaaatta tatgtcggtg 600
cctgctgacg attcagccta tcatcaatac tataaaaaga tcctttccta cgttgaaaaa 660
aacgcagatg aaagtggaga tcctcaagag gttgccgacc tcatttatca attggcaaca 720
aaacagcacg taaagaaatt gcgatacccg atcggaaaag gcatcaagct taccttgtta 780
ttccgatcgc tttttccttg gtctgcgtgg gaatctatcc tgaagaaaaa actattcagc 840
tcatctaaat ta 852
<210> 36
<211> 284
<212> PRT
<213> 枯草芽孢杆菌(Bacillus subtilis)
<400> 36
Met Asn Lys Lys Thr Ala Ile Val Thr Gly Ala Ser Arg Gly Phe Gly
1 5 10 15
Gln Leu Ala Ala Val Lys Leu Ala Lys Ser Phe Phe Val Ile Ala Thr
20 25 30
Thr Arg Gln Pro Glu Lys Ala Glu Gln Leu Arg Glu Leu Ala Ala Ala
35 40 45
His Asn Val Ala Asp Ser Ile His Ile Thr Ala Leu Asp Val Thr Asp
50 55 60
Glu Gln Ser Ile Ala Ser Phe Gly Lys Ala Ile Ser Ala Tyr Ala Pro
65 70 75 80
Ile Asp Leu Leu Val Asn Asn Ala Gly Thr Ala Tyr Gly Gly Phe Val
85 90 95
Glu Asp Val Pro Met Glu His Phe Arg Gln Gln Phe Glu Thr Asn Val
100 105 110
Phe Gly Val Ile His Val Thr Lys Thr Val Leu Pro Tyr Ile Arg Lys
115 120 125
His Ser Gly Ala Lys Ile Ile Asn Val Ser Ser Ile Ser Gly Leu Thr
130 135 140
Gly Phe Pro Ala Phe Ser Pro Tyr Ala Ser Ser Lys His Ala Leu Glu
145 150 155 160
Gly Phe Ser Glu Ser Leu Arg Val Glu Leu Leu Pro Phe Gly Ile Gln
165 170 175
Thr Ala Leu Ile Glu Pro Gly Tyr Tyr Arg Thr Ser Ile Trp Ser Thr
180 185 190
Ser Leu Ser Asn Tyr Met Ser Val Pro Ala Asp Asp Ser Ala Tyr His
195 200 205
Gln Tyr Tyr Lys Lys Ile Leu Ser Tyr Val Glu Lys Asn Ala Asp Glu
210 215 220
Ser Gly Asp Pro Gln Glu Val Ala Asp Leu Ile Tyr Gln Leu Ala Thr
225 230 235 240
Lys Gln His Val Lys Lys Leu Arg Tyr Pro Ile Gly Lys Gly Ile Lys
245 250 255
Leu Thr Leu Leu Phe Arg Ser Leu Phe Pro Trp Ser Ala Trp Glu Ser
260 265 270
Ile Leu Lys Lys Lys Leu Phe Ser Ser Ser Lys Leu
275 280
<210> 37
<211> 852
<212> DNA
<213> 枯草芽孢杆菌(Bacillus subtilis)
<400> 37
atgaataaaa aaaccgcgat cgtaacagga gcgtcaagag gctttggtca gctggcagcc 60
gtaaagctcg ccaagtcatt tttcgtgatt gccacaacaa gacagcctga gaaagctgaa 120
cagcttcggg aattggccgc agcacacaat gtggctgatt ctattcacat taccgctctc 180
gacgtcacag atgagcagtc catagcctca ttcggaaaag ccattagtgc ttacgctcct 240
attgatcttc tcgtcaacaa cgccggaacg gcttacggag gatttgtcga ggatgtgcca 300
atggaacatt tcagacaaca atttgaaacg aatgtcttcg gagtgattca tgtgacaaaa 360
accgttctgc cttacataag aaagcatagt ggagcaaaga ttataaacgt gagcagcatc 420
agcggtctga ccggattccc tgcgttttcg ccttatgcat cttccaagca tgcattggag 480
ggtttttctg agagcctgcg tgtggagctg cttccgttcg gtatccaaac cgctttgatc 540
gagccaggct catacaggac atcgatctgg tccacatcat tatcaaatta tatgtcggtg 600
cctgctgacg attcagccta tcatcaatac tataaaaaga tcctttccta cgttgaaaaa 660
aacgcagatg aaagtggaga tcctcaagag gttgccgacc tcatttatca attggcaaca 720
aaacagcacg taaagaaatt gcgatacccg atcggaaaag gcatcaagct taccttgtta 780
ttccgatcgc tttttccttg gtctgcgtgg gaatctatcc tgaagaaaaa actattcagc 840
tcatctaaat ta 852
<210> 38
<211> 284
<212> PRT
<213> 枯草芽孢杆菌(Bacillus subtilis)
<400> 38
Met Asn Lys Lys Thr Ala Ile Val Thr Gly Ala Ser Arg Gly Phe Gly
1 5 10 15
Gln Leu Ala Ala Val Lys Leu Ala Lys Ser Phe Phe Val Ile Ala Thr
20 25 30
Thr Arg Gln Pro Glu Lys Ala Glu Gln Leu Arg Glu Leu Ala Ala Ala
35 40 45
His Asn Val Ala Asp Ser Ile His Ile Thr Ala Leu Asp Val Thr Asp
50 55 60
Glu Gln Ser Ile Ala Ser Phe Gly Lys Ala Ile Ser Ala Tyr Ala Pro
65 70 75 80
Ile Asp Leu Leu Val Asn Asn Ala Gly Thr Ala Tyr Gly Gly Phe Val
85 90 95
Glu Asp Val Pro Met Glu His Phe Arg Gln Gln Phe Glu Thr Asn Val
100 105 110
Phe Gly Val Ile His Val Thr Lys Thr Val Leu Pro Tyr Ile Arg Lys
115 120 125
His Ser Gly Ala Lys Ile Ile Asn Val Ser Ser Ile Ser Gly Leu Thr
130 135 140
Gly Phe Pro Ala Phe Ser Pro Tyr Ala Ser Ser Lys His Ala Leu Glu
145 150 155 160
Gly Phe Ser Glu Ser Leu Arg Val Glu Leu Leu Pro Phe Gly Ile Gln
165 170 175
Thr Ala Leu Ile Glu Pro Gly Ser Tyr Arg Thr Ser Ile Trp Ser Thr
180 185 190
Ser Leu Ser Asn Tyr Met Ser Val Pro Ala Asp Asp Ser Ala Tyr His
195 200 205
Gln Tyr Tyr Lys Lys Ile Leu Ser Tyr Val Glu Lys Asn Ala Asp Glu
210 215 220
Ser Gly Asp Pro Gln Glu Val Ala Asp Leu Ile Tyr Gln Leu Ala Thr
225 230 235 240
Lys Gln His Val Lys Lys Leu Arg Tyr Pro Ile Gly Lys Gly Ile Lys
245 250 255
Leu Thr Leu Leu Phe Arg Ser Leu Phe Pro Trp Ser Ala Trp Glu Ser
260 265 270
Ile Leu Lys Lys Lys Leu Phe Ser Ser Ser Lys Leu
275 280

Claims (7)

1.短链脱氢酶突变体，其特征在于，其氨基酸序列如SEQ ID No. 16，18，20所示。
2.编码权利要求1所述的短链脱氢酶突变体的核酸，其核酸序列如SEQ ID No. 15，17，19所示。
3.一种表达载体，该载体含有权利要求2所述的核酸且能在宿主细胞中进行表达。
4.一种宿主细胞，其含有权利要求2所述 的核酸或权利要求3所述的表达载体。
5.如权利要求4所述的宿主细胞，其特征在于，所述的宿主细胞为大肠杆菌。
6.权利要求1所述的短链脱氢酶突变体在还原苯乙酮类衍生化合物中的应用。
7.如权利要求6所述的应用，其特征在于，所述还原苯乙酮类衍生化合物的方法为：在pH5-9的磷酸盐缓冲液溶液中，在所述的短链脱氢酶突变体的催化下，苯乙酮类衍生化合物进行还原，生成光学活性手性醇。
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