CN111041009B - 一种短链脱氢酶及其突变体与基因的制备及应用 - Google Patents

一种短链脱氢酶及其突变体与基因的制备及应用 Download PDF

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CN111041009B
CN111041009B CN201811182398.XA CN201811182398A CN111041009B CN 111041009 B CN111041009 B CN 111041009B CN 201811182398 A CN201811182398 A CN 201811182398A CN 111041009 B CN111041009 B CN 111041009B
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游松
秦斌
郭继阳
张飞霆
刘贵高
秦凤玉
张文鹤
祝天慧
唐军
闫平泽
张瑞
李衡宇
于召惠
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Abstract

本发明属于生物技术领域，涉及一种短链脱氢酶及其突变体与基因的制备及应用，具体涉及来自于巴斯德酵母(Saccharomyces pastorianus)的野生型的C＝C双键还原酶OYE1及其突变体和分别来自于发酵乳酸杆菌(Lactobacillus fermentum)、巨大芽孢杆菌(Bacillus megaterium)和枯草芽孢杆菌(Bacillus subtilis)的短链脱氢酶LfSDR1、BmSDR5和BsSDR8及它们的突变体，涉及具有立体选择性互补的C＝C双键还原酶以及C＝O双键还原酶、其基因、含有上述基因的重组表达载体的构建与制备、重组酶的表达和上述酶催化的香芹酮两步非对称还原反应、反应条件以产物二氢香芹醇的检测方法。该方法与现有的化学方法相比，具有操作简单、反应条件温和、对环境友好等优点。

Description

一种短链脱氢酶及其突变体与基因的制备及应用
技术领域
本发明涉及生物工程技术领域，涉及一种短链脱氢酶及其突变体与基因的制备及应用，具体涉及通过立体选择性互补的烯烃还原酶和短链脱氢酶作为生物催化剂，催化4R/S-香芹酮非对称还原合成立体多样性的二氢香芹醇。
背景技术
二氢香芹醇为无色至浅黄色粘稠性液体，有留兰香气，有胡椒辛辣味。不溶于水，溶于丙酮、乙醚和乙醇。二氢香芹醇是一种香料，可用作食品的人造香辛料，调制薄荷香型的香精可用于食品；调制留兰香香精，用于化妆品；也用作其他香料原料。二氢香芹醇有3个手性中心(C-1、C-2和C-4),8种立体异构体，目前市场上是以消旋体或者是非对映异构体混合物的形式出售，而光学纯的4R/S-香芹酮能够从相应的挥发油中提取出来，因此可以以4R/S-香芹酮为底物，通过高立体选择性的催化剂催化C＝C以及C＝O双键非对称还原，分别得到光学纯度高的8种立体异构的二氢香芹醇。
4R/S香芹酮拥有3个不饱和键、1个C-4位手性中心，产生下列选择性问题：(1)C＝C和C＝O官能团之间的选择性；(2)共轭的C＝C和独立的C＝C之间的选择性；(3)C＝C和C＝O还原后C-1位取代基相对于C-4位取代基的顺式与反式。如果不控制选择性，那么会产生17种氢化产物(香芹醇、二氢香芹酮或二氢香芹醇)，例如Bogel-
Figure BDA0001825316030000011
课题组在超临界二氧化碳中以钯(Pd)、铑(Rh)或钌(Ru)为催化剂(Green Chem,2011,13,2825)，Santos课题组利用人皮肤一些真菌生物转化4R/S-香芹酮得到了至少4种氢化混合物(Biocatalysis andBiotransformation,2017,1-5)，其他利用细菌(Chimie,2005,8,849–852)、真菌(J.Mol.Catal.B:Enzym,1998,5,149–154)和酵母(J.Mol.Catal.B:Enzym,2012,77,98–104.)生物转化香芹酮的报道也只能得到饱和酮/醇以及烯醇的混合物，表明全细胞中的烯烃还原酶和羰基还原酶能够竞争还原底物，而羰基还原酶还原香芹酮后的产物香芹醇，由于其C＝C双键惰性不能够被烯烃还原酶接受，导致副产物香芹醇的产生。所以采用体外酶催化的方法，串联两步还原反应，能够避免副产物的产生，并且基于酶高的化学、位置以及立体选择性的特点，其作为催化剂能够得到光学纯度高的产物。
立体选择性互补的烯烃还原酶：野生型OYE1催化香芹酮的到光学纯的二氢香芹酮的报道有许多，Stewart课题组通过对OYE1(来自于巴斯德酵母)的关键位置116位色氨酸突变发现其突变体催化4S-香芹酮还原产生构型翻转(对于C-1位甲基)的还原产物，并通过晶体结构解释了底物在酶活性中心的姿势发生了改变造成了酶的选择性的翻转(ACSCatalysis,2013,3(10),2376-2390)。
同样立体选择性互补的羰基还原酶也有一些报道，例如游松课题组通过蛋白质结构及计算机模拟辅助的方法将一些野生型的短链脱氢酶改造成能够针对卤代苯乙酮的非对称还原的、具有立体选择性互补的一些突变体(ACS Catal.,2018,8,pp 6012–6020)。
本实验室基于上述文献报道以及在此基础上通过理性设计及蛋白质定向进化提高及逆转该酶的立体选择性，使其作为催化剂催化4R/S-香芹酮还原合成立体多样性的光学纯度高的二氢香芹醇。
发明内容：
本发明目的在于解决商业可获得的二氢香芹醇是以消旋体或者是非对映异构体混合物而非单独的立体异构体的形式出售，通过理性设计及蛋白质定向进化，获得具有立体选择性互补的烯烃还原酶(或突变体)和羰基还原酶(或突变体)，非对称还原4R/S-香芹酮，获得立体多向性的二氢香芹醇。
为解决上述问题，本发明首先采取定点突变的技术，获取立体选择性互补的酶。
本发明提供一种烯烃还原酶OYE1，其氨基酸序列如SEQ ID No.2所示。
进一步地，本发明提供了一种烯烃还原酶OYE1的突变体，所述的突变体是将烯烃还原酶OYE1的116位进行突变；
或将烯烃还原酶OYE1的116位和37位同时进行突变。
为了逆转野生型OYE1对于4S-香芹酮的立体选择性，对序列表中具有如SEQ IDNo.2所示氨基酸序列进行突变，将116位的色氨酸(W)突变为具有较大空间位阻且为非极性的丙氨酸(A)，获得突变体OYE1-W116A命名为OYE1-M1(SEQ ID NO.4)。
为了逆转野生型OYE1对于4R-香芹酮的立体选择性，对序列表SEQ ID NO.2所示氨基酸序列进行突变，将116位的色氨酸(W)和37位的苏氨酸(T)同时突变成丙氨酸(A)，获得突变体OYE1-T37A/W116A命名为OYE1-M2(SEQ ID NO.6)。
为了获得立体选择性互补的羰基还原酶，首先通过基因挖掘获得来自于发酵乳酸杆菌的反Prelog规则短链脱氢酶LfSDR1(SEQ ID No.8)、来自于巨大芽孢杆菌的Prelog规则的短链脱氢酶BmSDR5(SEQ ID No.14)。和来自于枯草芽孢杆菌的Prelog规则的短链脱氢酶BsSDR8(SEQ ID No.16)。
进一步地，为了提高或逆转LfSDR1、BmSDR5或BsSDR8的立体选择性，(1)将LfSDR1的186位缬氨酸(V)突变成色氨酸(W)，获得突变体LfSDR1-V186W命名为LfSDR1-M1(SEQ IDNO.10)；(2)将LfSDR1的92位甘氨酸(G)、141位谷氨酸(E)、146位天冬氨酸(D)、186位缬氨酸(V)和206赖氨酸(K)分别突变成谷氨酸(E)、苯丙氨酸(F)、缬氨酸(V)、丙氨酸(A)和亮氨酸(L)，获得突变体LfSDR1-G92E/E141F/D146V/V186A/K206L命名为LfSDR1-M2(SEQ IDNO.12)；(3)将BsSDR8的146位苯丙氨酸(F)突变成色氨酸(W)，获得突变体BsSDR8-F146W命名为BsSDR8-M1(SEQ ID NO.18)。
本发明进一步提供了所述的短链脱氢酶及突变体催化不对称还原苯香芹酮方法。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香芹酮衍生物，之后加入定量的烯烃还原酶或突变体催化第一步还原反应产生光学纯的二氢香芹醇，GC检测反应完全后，补加定量的羰基还原酶或突变催化第二部化还原反应产生第一构型的二氢香芹醇。
具体实施方式：
实施例1立体选择性互补的酶的表达
为获得OYE1的突变体，以野生型的OYE1基因(SEQ ID NO.1)为模板，利用含有突变点的突变引物(选取突变点上下游各15-20bp的碱基，将突变点的碱基替换成突变后氨基酸的密码子作为PCR正向引物，其反向互补序列作为反向引物)，通过PCR扩增获得突变体OYE1-M1和OYE1-M2的基因。
为获取立体选择性提高的突变体，以野生型短链脱氢酶LfSDR1基因(SEQ IDNo.1)为模板，利用含有突变点的突变引物(选取突变点上下游各15-20bp的碱基，将突变点的碱基替换成突变后氨基酸的密码子作为PCR正向引物，其反向互补序列作为反向引物)，通过PCR扩增获得突变体LfSDR1-V186F与LfSDR1-V186W基因。
进一步，将突变体基因及载体质粒pET22b以内切酶EcoRⅠ与XhoⅠ进行双酶切(37℃反应4-8h)，利用普通DNA产物凝胶回收试剂盒，回收经酶切的核酸片段；以T4DNA连接酶将经过双酶切的突变基因片段及载体质粒片段连接(16℃反应2-6h)获取重组质粒pET22b-OYE1-W116A和pET22b-OYE1-W116A/T37A将重组质粒转化入E.coli Rosseta(DE3)感受态细胞中，较佳的转化方法为热激法：具体过程为45℃热激90S。将构建的重组转化体细胞经过培养表达突变体短链脱氢酶。
获取BsSDR8突变体和LfSDR1突变体的方法与上述方法相同，只是LfSDR1使用的内切酶为内切酶NdeⅠ与XhoⅠ。
实施例2(1R,2S,4R)-二氢香芹醇的合成
在1ml的反应体系中，先加入实施例1制备的烯烃还原酶OYE1 800μl和及葡萄糖脱氢酶GDH 200μl,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5mM 4R-香芹酮(助溶剂：石油醚，20％v/v)。在28℃，200rpm条件下振荡反应，反应时间为12h。反应结束后用1ml的乙酸乙酯分两次萃取，GC检测底物完全反应后，旋蒸干燥后作为第二步反应的底物(5％乙醇助溶，v/v)，第二步反应体系除酶为短链脱氢酶LfSDR1其余与第一步反应相同，反应条件为37℃，200rpm条件下振荡反应12h。反应结束后同样用1ml乙酸乙酯萃取两次，GC检测转化率>99％，单步de值>99％，总de值92.6％(由于大肠杆菌宿主细胞内源性ERs导致第一步反应产生的(1R,4R)-二氢香芹醇的de值为93％，进而导致其他构型的二氢香芹醇的产生)。
实施例3(1R,2R,4R)-二氢香芹醇的合成
除了两步反应使用的酶，该构型二氢香芹醇合成的方法、反应体系以及条件与实例二相同。两步反应所使用的酶分别为：OYE1和BmSDR5，转化率>99％，单步de值98.4％，总de值90.5％。
实施例4(1S,2R,4R)-二氢香芹醇的合成
除了两步反应使用的酶，该构型二氢香芹醇合成的方法、反应体系以及条件与实例二相同。两步反应所使用的酶分别为：OYE1-M2和BmSDR5，转化率>99％，单步de值96.7％，总de值93％。
实施例5(1S,2S,4R)-二氢香芹醇的合成
除了两步反应使用的酶，该构型二氢香芹醇合成的方法、反应体系以及条件与实例二相同。两步反应所使用的酶分别为：OYE1-M2和LfSDR1-M1，转化率>99％，单步de值97.5％，总de值94.8％。
实施例6(1R,2R,4S)-二氢香芹醇的合成
除了两步反应使用的酶，该构型二氢香芹醇合成的方法、反应体系以及条件与实例二相同。两步反应所使用的酶分别为：OYE1和LfSDR1-M2，转化率>99％，单步de值82.5％，总de值73.5％。
实施例7(1R,2S,4S)-二氢香芹醇的合成
除了两步反应使用的酶，该构型二氢香芹醇合成的方法、反应体系以及条件与实例二相同。两步反应所使用的酶分别为：OYE1和LfSDR1-M1，转化率>99％，单步de值>99％，总de值92.1％。
实施例8(1S,2S,4S)-二氢香芹醇的合成
除了两步反应使用的酶，该构型二氢香芹醇合成的方法、反应体系以及条件与实例二相同。两步反应所使用的酶分别为：OYE1-M1和LfSDR1-M1，转化率>99％，单步de值97.9％，总de值90.0％。
实施例9(1S,2R,4S)-二氢香芹醇的合成
除了两步反应使用的酶，该构型二氢香芹醇合成的方法、反应体系以及条件与实例二相同。两步反应所使用的酶分别为：OYE1-M1和BsSDR8-M1，转化率95.2％，单步de值91.3％，总de值88.9％。
实施例10气相色谱法分析底物和产物
使用非手性气相色谱(SCION 456-GC)，色谱柱
Figure BDA0001825316030000042
-1701capillary column(0.25mm×30m,0.25μm film thickness；Restek)，进样器温度为70℃，检测器温度为240℃，程序升温如下表：
Figure BDA0001825316030000041
序列表
<110> 沈阳药科大学
<120> 一种短链脱氢酶及其突变体与基因的制备及应用
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atcttcaacg ctatccacga aaaaaaatct ttcgtttggg ttcagctgtg ggttctgggt 360
tgggctgctt tcccggacaa cctggctcgt gacggtctgc gttacgactc tgcttctgac 420
aacgttttca tggacgctga acaggaagct aaagctaaaa aagctaacaa cccgcagcac 480
tctctgacca aagacgaaat caaacagtac atcaaagaat acgttcaggc tgctaaaaac 540
tctatcgctg ctggtgctga cggtgttgaa atccactctg ctaacggtta cctgctgaac 600
cagttcctgg acccgcactc taacacccgt accgacgaat acggtggttc tatcgaaaac 660
cgtgctcgtt tcaccctgga agttgttgac gctctggttg aagctatcgg tcacgaaaaa 720
gttggtctgc gtctgtctcc gtacggtgtt ttcaactcta tgtctggtgg tgctgaaacc 780
ggtatcgttg ctcagtacgc ttacgttgct ggtgaactgg aaaaacgtgc taaagctggt 840
aaacgtctgg ctttcgttca cctggttgaa ccgcgtgtta ccaacccgtt cctgaccgaa 900
ggtgaaggtg aatacgaagg tggttctaac gacttcgttt actctatctg gaaaggtccg 960
gttatccgtg ctggtaactt cgctctgcac ccggaagttg ttcgtgaaga agttaaagac 1020
aaacgtaccc tgatcggtta cggtcgtttc ttcatctcta acccggacct ggttgaccgt 1080
ctggaaaaag gtctgccgct gaacaaatac gaccgtgaca ccttctacca gatgtctgct 1140
cacggttaca tcgactaccc gacctacgaa gaagctctga aactgggttg ggacaaaaaa 1200
<210> 2
<211> 400
<212> PRT
<213> Saccharomyces pastorianus
<400> 2
Met Ser Phe Val Lys Asp Phe Lys Pro Gln Ala Leu Gly Asp Thr Asn
1 5 10 15
Leu Phe Lys Pro Ile Lys Ile Gly Asn Asn Glu Leu Leu His Arg Ala
20 25 30
Val Ile Pro Pro Leu Thr Arg Met Arg Ala Leu His Pro Gly Asn Ile
35 40 45
Pro Asn Arg Asp Trp Ala Val Glu Tyr Tyr Thr Gln Arg Ala Gln Arg
50 55 60
Pro Gly Thr Met Ile Ile Thr Glu Gly Ala Phe Ile Ser Pro Gln Ala
65 70 75 80
Gly Gly Tyr Asp Asn Ala Pro Gly Val Trp Ser Glu Glu Gln Met Val
85 90 95
Glu Trp Thr Lys Ile Phe Asn Ala Ile His Glu Lys Lys Ser Phe Val
100 105 110
Trp Val Gln Leu Trp Val Leu Gly Trp Ala Ala Phe Pro Asp Asn Leu
115 120 125
Ala Arg Asp Gly Leu Arg Tyr Asp Ser Ala Ser Asp Asn Val Phe Met
130 135 140
Asp Ala Glu Gln Glu Ala Lys Ala Lys Lys Ala Asn Asn Pro Gln His
145 150 155 160
Ser Leu Thr Lys Asp Glu Ile Lys Gln Tyr Ile Lys Glu Tyr Val Gln
165 170 175
Ala Ala Lys Asn Ser Ile Ala Ala Gly Ala Asp Gly Val Glu Ile His
180 185 190
Ser Ala Asn Gly Tyr Leu Leu Asn Gln Phe Leu Asp Pro His Ser Asn
195 200 205
Thr Arg Thr Asp Glu Tyr Gly Gly Ser Ile Glu Asn Arg Ala Arg Phe
210 215 220
Thr Leu Glu Val Val Asp Ala Leu Val Glu Ala Ile Gly His Glu Lys
225 230 235 240
Val Gly Leu Arg Leu Ser Pro Tyr Gly Val Phe Asn Ser Met Ser Gly
245 250 255
Gly Ala Glu Thr Gly Ile Val Ala Gln Tyr Ala Tyr Val Ala Gly Glu
260 265 270
Leu Glu Lys Arg Ala Lys Ala Gly Lys Arg Leu Ala Phe Val His Leu
275 280 285
Val Glu Pro Arg Val Thr Asn Pro Phe Leu Thr Glu Gly Glu Gly Glu
290 295 300
Tyr Glu Gly Gly Ser Asn Asp Phe Val Tyr Ser Ile Trp Lys Gly Pro
305 310 315 320
Val Ile Arg Ala Gly Asn Phe Ala Leu His Pro Glu Val Val Arg Glu
325 330 335
Glu Val Lys Asp Lys Arg Thr Leu Ile Gly Tyr Gly Arg Phe Phe Ile
340 345 350
Ser Asn Pro Asp Leu Val Asp Arg Leu Glu Lys Gly Leu Pro Leu Asn
355 360 365
Lys Tyr Asp Arg Asp Thr Phe Tyr Gln Met Ser Ala His Gly Tyr Ile
370 375 380
Asp Tyr Pro Thr Tyr Glu Glu Ala Leu Lys Leu Gly Trp Asp Lys Lys
385 390 395 400
<210> 3
<211> 1200
<212> DNA
<213> Saccharomyces pastorianus
<400> 3
atgtctttcg ttaaagactt caaaccgcag gctctgggtg acaccaacct gttcaaaccg 60
atcaaaatcg gtaacaacga actgctgcac cgtgctgtta tcccgccgct gacccgtatg 120
cgtgctctgc acccgggtaa catcccgaac cgtgactggg ctgttgaata ctacacccag 180
cgtgctcagc gtccgggtac catgatcatc accgaaggtg ctttcatctc tccgcaggct 240
ggtggttacg acaacgctcc gggtgtttgg tctgaagaac agatggttga atggaccaaa 300
atcttcaacg ctatccacga aaaaaaatct ttcgtttggg ttcagctggc ggttctgggt 360
tgggctgctt tcccggacaa cctggctcgt gacggtctgc gttacgactc tgcttctgac 420
aacgttttca tggacgctga acaggaagct aaagctaaaa aagctaacaa cccgcagcac 480
tctctgacca aagacgaaat caaacagtac atcaaagaat acgttcaggc tgctaaaaac 540
tctatcgctg ctggtgctga cggtgttgaa atccactctg ctaacggtta cctgctgaac 600
cagttcctgg acccgcactc taacacccgt accgacgaat acggtggttc tatcgaaaac 660
cgtgctcgtt tcaccctgga agttgttgac gctctggttg aagctatcgg tcacgaaaaa 720
gttggtctgc gtctgtctcc gtacggtgtt ttcaactcta tgtctggtgg tgctgaaacc 780
ggtatcgttg ctcagtacgc ttacgttgct ggtgaactgg aaaaacgtgc taaagctggt 840
aaacgtctgg ctttcgttca cctggttgaa ccgcgtgtta ccaacccgtt cctgaccgaa 900
ggtgaaggtg aatacgaagg tggttctaac gacttcgttt actctatctg gaaaggtccg 960
gttatccgtg ctggtaactt cgctctgcac ccggaagttg ttcgtgaaga agttaaagac 1020
aaacgtaccc tgatcggtta cggtcgtttc ttcatctcta acccggacct ggttgaccgt 1080
ctggaaaaag gtctgccgct gaacaaatac gaccgtgaca ccttctacca gatgtctgct 1140
cacggttaca tcgactaccc gacctacgaa gaagctctga aactgggttg ggacaaaaaa 1200
<210> 4
<211> 400
<212> PRT
<213> Saccharomyces pastorianus
<400> 4
Met Ser Phe Val Lys Asp Phe Lys Pro Gln Ala Leu Gly Asp Thr Asn
1 5 10 15
Leu Phe Lys Pro Ile Lys Ile Gly Asn Asn Glu Leu Leu His Arg Ala
20 25 30
Val Ile Pro Pro Leu Thr Arg Met Arg Ala Leu His Pro Gly Asn Ile
35 40 45
Pro Asn Arg Asp Trp Ala Val Glu Tyr Tyr Thr Gln Arg Ala Gln Arg
50 55 60
Pro Gly Thr Met Ile Ile Thr Glu Gly Ala Phe Ile Ser Pro Gln Ala
65 70 75 80
Gly Gly Tyr Asp Asn Ala Pro Gly Val Trp Ser Glu Glu Gln Met Val
85 90 95
Glu Trp Thr Lys Ile Phe Asn Ala Ile His Glu Lys Lys Ser Phe Val
100 105 110
Trp Val Gln Leu Ala Val Leu Gly Trp Ala Ala Phe Pro Asp Asn Leu
115 120 125
Ala Arg Asp Gly Leu Arg Tyr Asp Ser Ala Ser Asp Asn Val Phe Met
130 135 140
Asp Ala Glu Gln Glu Ala Lys Ala Lys Lys Ala Asn Asn Pro Gln His
145 150 155 160
Ser Leu Thr Lys Asp Glu Ile Lys Gln Tyr Ile Lys Glu Tyr Val Gln
165 170 175
Ala Ala Lys Asn Ser Ile Ala Ala Gly Ala Asp Gly Val Glu Ile His
180 185 190
Ser Ala Asn Gly Tyr Leu Leu Asn Gln Phe Leu Asp Pro His Ser Asn
195 200 205
Thr Arg Thr Asp Glu Tyr Gly Gly Ser Ile Glu Asn Arg Ala Arg Phe
210 215 220
Thr Leu Glu Val Val Asp Ala Leu Val Glu Ala Ile Gly His Glu Lys
225 230 235 240
Val Gly Leu Arg Leu Ser Pro Tyr Gly Val Phe Asn Ser Met Ser Gly
245 250 255
Gly Ala Glu Thr Gly Ile Val Ala Gln Tyr Ala Tyr Val Ala Gly Glu
260 265 270
Leu Glu Lys Arg Ala Lys Ala Gly Lys Arg Leu Ala Phe Val His Leu
275 280 285
Val Glu Pro Arg Val Thr Asn Pro Phe Leu Thr Glu Gly Glu Gly Glu
290 295 300
Tyr Glu Gly Gly Ser Asn Asp Phe Val Tyr Ser Ile Trp Lys Gly Pro
305 310 315 320
Val Ile Arg Ala Gly Asn Phe Ala Leu His Pro Glu Val Val Arg Glu
325 330 335
Glu Val Lys Asp Lys Arg Thr Leu Ile Gly Tyr Gly Arg Phe Phe Ile
340 345 350
Ser Asn Pro Asp Leu Val Asp Arg Leu Glu Lys Gly Leu Pro Leu Asn
355 360 365
Lys Tyr Asp Arg Asp Thr Phe Tyr Gln Met Ser Ala His Gly Tyr Ile
370 375 380
Asp Tyr Pro Thr Tyr Glu Glu Ala Leu Lys Leu Gly Trp Asp Lys Lys
385 390 395 400
<210> 5
<211> 1200
<212> DNA
<213> Saccharomyces pastorianus
<400> 5
atgtctttcg ttaaagactt caaaccgcag gctctgggtg acaccaacct gttcaaaccg 60
atcaaaatcg gtaacaacga actgctgcac cgtgctgtta tcccgccgct ggcgcgtatg 120
cgtgctctgc acccgggtaa catcccgaac cgtgactggg ctgttgaata ctacacccag 180
cgtgctcagc gtccgggtac catgatcatc accgaaggtg ctttcatctc tccgcaggct 240
ggtggttacg acaacgctcc gggtgtttgg tctgaagaac agatggttga atggaccaaa 300
atcttcaacg ctatccacga aaaaaaatct ttcgtttggg ttcagctggc ggttctgggt 360
tgggctgctt tcccggacaa cctggctcgt gacggtctgc gttacgactc tgcttctgac 420
aacgttttca tggacgctga acaggaagct aaagctaaaa aagctaacaa cccgcagcac 480
tctctgacca aagacgaaat caaacagtac atcaaagaat acgttcaggc tgctaaaaac 540
tctatcgctg ctggtgctga cggtgttgaa atccactctg ctaacggtta cctgctgaac 600
cagttcctgg acccgcactc taacacccgt accgacgaat acggtggttc tatcgaaaac 660
cgtgctcgtt tcaccctgga agttgttgac gctctggttg aagctatcgg tcacgaaaaa 720
gttggtctgc gtctgtctcc gtacggtgtt ttcaactcta tgtctggtgg tgctgaaacc 780
ggtatcgttg ctcagtacgc ttacgttgct ggtgaactgg aaaaacgtgc taaagctggt 840
aaacgtctgg ctttcgttca cctggttgaa ccgcgtgtta ccaacccgtt cctgaccgaa 900
ggtgaaggtg aatacgaagg tggttctaac gacttcgttt actctatctg gaaaggtccg 960
gttatccgtg ctggtaactt cgctctgcac ccggaagttg ttcgtgaaga agttaaagac 1020
aaacgtaccc tgatcggtta cggtcgtttc ttcatctcta acccggacct ggttgaccgt 1080
ctggaaaaag gtctgccgct gaacaaatac gaccgtgaca ccttctacca gatgtctgct 1140
cacggttaca tcgactaccc gacctacgaa gaagctctga aactgggttg ggacaaaaaa 1200
<210> 6
<211> 400
<212> PRT
<213> Saccharomyces pastorianus
<400> 6
Met Ser Phe Val Lys Asp Phe Lys Pro Gln Ala Leu Gly Asp Thr Asn
1 5 10 15
Leu Phe Lys Pro Ile Lys Ile Gly Asn Asn Glu Leu Leu His Arg Ala
20 25 30
Val Ile Pro Pro Leu Ala Arg Met Arg Ala Leu His Pro Gly Asn Ile
35 40 45
Pro Asn Arg Asp Trp Ala Val Glu Tyr Tyr Thr Gln Arg Ala Gln Arg
50 55 60
Pro Gly Thr Met Ile Ile Thr Glu Gly Ala Phe Ile Ser Pro Gln Ala
65 70 75 80
Gly Gly Tyr Asp Asn Ala Pro Gly Val Trp Ser Glu Glu Gln Met Val
85 90 95
Glu Trp Thr Lys Ile Phe Asn Ala Ile His Glu Lys Lys Ser Phe Val
100 105 110
Trp Val Gln Leu Ala Val Leu Gly Trp Ala Ala Phe Pro Asp Asn Leu
115 120 125
Ala Arg Asp Gly Leu Arg Tyr Asp Ser Ala Ser Asp Asn Val Phe Met
130 135 140
Asp Ala Glu Gln Glu Ala Lys Ala Lys Lys Ala Asn Asn Pro Gln His
145 150 155 160
Ser Leu Thr Lys Asp Glu Ile Lys Gln Tyr Ile Lys Glu Tyr Val Gln
165 170 175
Ala Ala Lys Asn Ser Ile Ala Ala Gly Ala Asp Gly Val Glu Ile His
180 185 190
Ser Ala Asn Gly Tyr Leu Leu Asn Gln Phe Leu Asp Pro His Ser Asn
195 200 205
Thr Arg Thr Asp Glu Tyr Gly Gly Ser Ile Glu Asn Arg Ala Arg Phe
210 215 220
Thr Leu Glu Val Val Asp Ala Leu Val Glu Ala Ile Gly His Glu Lys
225 230 235 240
Val Gly Leu Arg Leu Ser Pro Tyr Gly Val Phe Asn Ser Met Ser Gly
245 250 255
Gly Ala Glu Thr Gly Ile Val Ala Gln Tyr Ala Tyr Val Ala Gly Glu
260 265 270
Leu Glu Lys Arg Ala Lys Ala Gly Lys Arg Leu Ala Phe Val His Leu
275 280 285
Val Glu Pro Arg Val Thr Asn Pro Phe Leu Thr Glu Gly Glu Gly Glu
290 295 300
Tyr Glu Gly Gly Ser Asn Asp Phe Val Tyr Ser Ile Trp Lys Gly Pro
305 310 315 320
Val Ile Arg Ala Gly Asn Phe Ala Leu His Pro Glu Val Val Arg Glu
325 330 335
Glu Val Lys Asp Lys Arg Thr Leu Ile Gly Tyr Gly Arg Phe Phe Ile
340 345 350
Ser Asn Pro Asp Leu Val Asp Arg Leu Glu Lys Gly Leu Pro Leu Asn
355 360 365
Lys Tyr Asp Arg Asp Thr Phe Tyr Gln Met Ser Ala His Gly Tyr Ile
370 375 380
Asp Tyr Pro Thr Tyr Glu Glu Ala Leu Lys Leu Gly Trp Asp Lys Lys
385 390 395 400
<210> 7
<211> 744
<212> DNA
<213> Lactobacillus fermentum
<400> 7
atgggacagt ttgacaataa ggttgccttg gttaccgggg gaacgaaggg gattggctta 60
gccatcgccg agctgttttt gaaggaaggc gccaaggggg tggccttcac cggtcgtcac 120
gaagacgaag gaaaagcggt tcaagaacgc ctcggtgaac ggtctttgtt catcacccaa 180
gacgtttcca aggaagaaga ttggcaaaac gccaccaaag ccgttgttga aaaatttggg 240
cagcttgatg cgattgtcaa caacgccgga attgggactc cgctggggat cgaggaaatg 300
acgctcgatc actggaaccg cgaaatcgcc atcgatttaa cagggacgat gttaggttgc 360
aagtacgggg ttaaagcgat gaaggaacat ggtggcgcga tcgtcaacat tagttcgatc 420
gaagggatga tcggtgaccc aaccgttccg gcctacaacg ctgctaaggg gggcgtccgt 480
ctcctcacca agtcggtagc gcttgagtgt gccgaaaagg gttacgccat ccgcgtaaac 540
tcgatttacc ctggggtaat tgccacgccg ctgatcgatc acctcgatga tgcgaccaag 600
caattctaca tcgacaaaca cccaatgggc cggctgggaa agccggaaga agtggctaag 660
atggctgtct ttgttgcttc cgatggggcc tcctttagca ccggctccga gtttgttgtc 720
gatgggggct acacggcaca ataa 744
<210> 8
<211> 247
<212> PRT
<213> Lactobacillus fermentum
<400> 8
Met Gly Gln Phe Asp Asn Lys Val Ala Leu Val Thr Gly Gly Thr Lys
1 5 10 15
Gly Ile Gly Leu Ala Ile Ala Glu Leu Phe Leu Lys Glu Gly Ala Lys
20 25 30
Gly Val Ala Phe Thr Gly Arg His Glu Asp Glu Gly Lys Ala Val Gln
35 40 45
Glu Arg Leu Gly Glu Arg Ser Leu Phe Ile Thr Gln Asp Val Ser Lys
50 55 60
Glu Glu Asp Trp Gln Asn Ala Thr Lys Ala Val Val Glu Lys Phe Gly
65 70 75 80
Gln Leu Asp Ala Ile Val Asn Asn Ala Gly Ile Gly Thr Pro Leu Gly
85 90 95
Ile Glu Glu Met Thr Leu Asp His Trp Asn Arg Glu Ile Ala Ile Asp
100 105 110
Leu Thr Gly Thr Met Leu Gly Cys Lys Tyr Gly Val Lys Ala Met Lys
115 120 125
Glu His Gly Gly Ala Ile Val Asn Ile Ser Ser Ile Glu Gly Met Ile
130 135 140
Gly Asp Pro Thr Val Pro Ala Tyr Asn Ala Ala Lys Gly Gly Val Arg
145 150 155 160
Leu Leu Thr Lys Ser Val Ala Leu Glu Cys Ala Glu Lys Gly Tyr Ala
165 170 175
Ile Arg Val Asn Ser Ile Tyr Pro Gly Val Ile Ala Thr Pro Leu Ile
180 185 190
Asp His Leu Asp Asp Ala Thr Lys Gln Phe Tyr Ile Asp Lys His Pro
195 200 205
Met Gly Arg Leu Gly Lys Pro Glu Glu Val Ala Lys Met Ala Val Phe
210 215 220
Val Ala Ser Asp Gly Ala Ser Phe Ser Thr Gly Ser Glu Phe Val Val
225 230 235 240
Asp Gly Gly Tyr Thr Ala Gln
245
<210> 9
<211> 744
<212> DNA
<213> Lactobacillus fermentum
<400> 9
atgggacagt ttgacaataa ggttgccttg gttaccgggg gaacgaaggg gattggctta 60
gccatcgccg agctgttttt gaaggaaggc gccaaggggg tggccttcac cggtcgtcac 120
gaagacgaag gaaaagcggt tcaagaacgc ctcggtgaac ggtctttgtt catcacccaa 180
gacgtttcca aggaagaaga ttggcaaaac gccaccaaag ccgttgttga aaaatttggg 240
cagcttgatg cgattgtcaa caacgccgga attgggactc cgctggggat cgaggaaatg 300
acgctcgatc actggaaccg cgaaatcgcc atcgatttaa cagggacgat gttaggttgc 360
aagtacgggg ttaaagcgat gaaggaacat ggtggcgcga tcgtcaacat tagttcgatc 420
gaagggatga tcggtgaccc aaccgttccg gcctacaacg ctgctaaggg gggcgtccgt 480
ctcctcacca agtcggtagc gcttgagtgt gccgaaaagg gttacgccat ccgcgtaaac 540
tcgatttacc ctgggtggat tgccacgccg ctgatcgatc acctcgatga tgcgaccaag 600
caattctaca tcgacaaaca cccaatgggc cggctgggaa agccggaaga agtggctaag 660
atggctgtct ttgttgcttc cgatggggcc tcctttagca ccggctccga gtttgttgtc 720
gatgggggct acacggcaca ataa 744
<210> 10
<211> 247
<212> PRT
<213> Lactobacillus fermentum
<400> 10
Met Gly Gln Phe Asp Asn Lys Val Ala Leu Val Thr Gly Gly Thr Lys
1 5 10 15
Gly Ile Gly Leu Ala Ile Ala Glu Leu Phe Leu Lys Glu Gly Ala Lys
20 25 30
Gly Val Ala Phe Thr Gly Arg His Glu Asp Glu Gly Lys Ala Val Gln
35 40 45
Glu Arg Leu Gly Glu Arg Ser Leu Phe Ile Thr Gln Asp Val Ser Lys
50 55 60
Glu Glu Asp Trp Gln Asn Ala Thr Lys Ala Val Val Glu Lys Phe Gly
65 70 75 80
Gln Leu Asp Ala Ile Val Asn Asn Ala Gly Ile Gly Thr Pro Leu Gly
85 90 95
Ile Glu Glu Met Thr Leu Asp His Trp Asn Arg Glu Ile Ala Ile Asp
100 105 110
Leu Thr Gly Thr Met Leu Gly Cys Lys Tyr Gly Val Lys Ala Met Lys
115 120 125
Glu His Gly Gly Ala Ile Val Asn Ile Ser Ser Ile Glu Gly Met Ile
130 135 140
Gly Asp Pro Thr Val Pro Ala Tyr Asn Ala Ala Lys Gly Gly Val Arg
145 150 155 160
Leu Leu Thr Lys Ser Val Ala Leu Glu Cys Ala Glu Lys Gly Tyr Ala
165 170 175
Ile Arg Val Asn Ser Ile Tyr Pro Gly Trp Ile Ala Thr Pro Leu Ile
180 185 190
Asp His Leu Asp Asp Ala Thr Lys Gln Phe Tyr Ile Asp Lys His Pro
195 200 205
Met Gly Arg Leu Gly Lys Pro Glu Glu Val Ala Lys Met Ala Val Phe
210 215 220
Val Ala Ser Asp Gly Ala Ser Phe Ser Thr Gly Ser Glu Phe Val Val
225 230 235 240
Asp Gly Gly Tyr Thr Ala Gln
245
<210> 11
<211> 744
<212> DNA
<213> Lactobacillus fermentum
<400> 11
atgggacagt ttgacaataa ggttgccttg gttaccgggg gaacgaaggg gattggctta 60
gccatcgccg agctgttttt gaaggaaggc gccaaggggg tggccttcac cggtcgtcac 120
gaagacgaag gaaaagcggt tcaagaacgc ctcggtgaac ggtctttgtt catcacccaa 180
gacgtttcca aggaagaaga ttggcaaaac gccaccaaag ccgttgttga aaaatttggg 240
cagcttgatg cgattgtcaa caacgccgga attgaaactc cgctggggat cgaggaaatg 300
acgctcgatc actggaaccg cgaaatcgcc atcgatttaa cagggacgat gttaggttgc 360
aagtacgggg ttaaagcgat gaaggaacat ggtggcgcga tcgtcaacat tagttcgatc 420
tttgggatga tcggtgtgcc aaccgttccg gcctacaacg ctgctaaggg gggcgtccgt 480
ctcctcacca agtcggtagc gcttgagtgt gccgaaaagg gttacgccat ccgcgtaaac 540
tcgatttacc ctggggcgat tgccacgccg ctgatcgatc acctcgatga tgcgaccaag 600
caattctaca tcgacctgca cccaatgggc cggctgggaa agccggaaga agtggctaag 660
atggctgtct ttgttgcttc cgatggggcc tcctttagca ccggctccga gtttgttgtc 720
gatgggggct acacggcaca ataa 744
<210> 12
<211> 247
<212> PRT
<213> Lactobacillus fermentum
<400> 12
Met Gly Gln Phe Asp Asn Lys Val Ala Leu Val Thr Gly Gly Thr Lys
1 5 10 15
Gly Ile Gly Leu Ala Ile Ala Glu Leu Phe Leu Lys Glu Gly Ala Lys
20 25 30
Gly Val Ala Phe Thr Gly Arg His Glu Asp Glu Gly Lys Ala Val Gln
35 40 45
Glu Arg Leu Gly Glu Arg Ser Leu Phe Ile Thr Gln Asp Val Ser Lys
50 55 60
Glu Glu Asp Trp Gln Asn Ala Thr Lys Ala Val Val Glu Lys Phe Gly
65 70 75 80
Gln Leu Asp Ala Ile Val Asn Asn Ala Gly Ile Glu Thr Pro Leu Gly
85 90 95
Ile Glu Glu Met Thr Leu Asp His Trp Asn Arg Glu Ile Ala Ile Asp
100 105 110
Leu Thr Gly Thr Met Leu Gly Cys Lys Tyr Gly Val Lys Ala Met Lys
115 120 125
Glu His Gly Gly Ala Ile Val Asn Ile Ser Ser Ile Phe Gly Met Ile
130 135 140
Gly Val Pro Thr Val Pro Ala Tyr Asn Ala Ala Lys Gly Gly Val Arg
145 150 155 160
Leu Leu Thr Lys Ser Val Ala Leu Glu Cys Ala Glu Lys Gly Tyr Ala
165 170 175
Ile Arg Val Asn Ser Ile Tyr Pro Gly Ala Ile Ala Thr Pro Leu Ile
180 185 190
Asp His Leu Asp Asp Ala Thr Lys Gln Phe Tyr Ile Asp Leu His Pro
195 200 205
Met Gly Arg Leu Gly Lys Pro Glu Glu Val Ala Lys Met Ala Val Phe
210 215 220
Val Ala Ser Asp Gly Ala Ser Phe Ser Thr Gly Ser Glu Phe Val Val
225 230 235 240
Asp Gly Gly Tyr Thr Ala Gln
245
<210> 13
<211> 774
<212> DNA
<213> Bacillus megaterium
<400> 13
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> 14
<211> 258
<212> PRT
<213> Bacillus megaterium
<400> 14
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 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> 15
<211> 852
<212> DNA
<213> Bacillus subtilis
<400> 15
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> 16
<211> 284
<212> PRT
<213> Bacillus subtilis
<400> 16
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> 17
<211> 852
<212> DNA
<213> Bacillus subtilis
<400> 17
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 ccggatggcc 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> 18
<211> 284
<212> PRT
<213> Bacillus subtilis
<400> 18
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 Trp 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

Claims (9)

1.一种短链脱氢酶突变体，其特征在于，其氨基酸如SEQ ID No.6所示。
2.一种短链脱氢酶突变体，其特征在于，其氨基酸序列如SEQ ID No.12所示。
3.编码权利要求1或2所述的短链脱氢酶突变体的核酸，其核酸序列如SEQ ID No.5、11所示。
4.一种表达载体，该载体含有权利要求3所述的核酸且能在宿主细胞中进行表达。
5.一种宿主细胞，其含权利要求4所述的表达载体，所述的宿主组胞为大肠杆菌。
6.权利要求1或2所述的短链脱氢酶突变体在催化生物反应中的应用。
7.权利要求1或2所述的短链脱氢酶突变体在催化合成立体多样性的二氢香芹醇反应、检测立体异构的二氢香芹醇中的应用。
8.如权利要求6或7所述的应用，其特征在于，以4R/S-香芹酮为底物，通过立体选择性互补的烯烃还原酶和羰基还原酶催化其两步非对称还原，所述的烯烃还原酶如SEQ IDNo.6所示；所述的羰基还原酶如SEQ ID No.12所示。
9.一种合成立体多样性的二氢香芹醇的方法，其特征在于，以4R/S-香芹酮为底物，通过立体选择性互补的烯烃还原酶和羰基还原酶催化其两步非对称还原，所述的烯烃还原酶如权利要求1的SEQ ID No.6所示；所述的羰基还原酶如权利要求2的SEQ ID No.12所示。
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