一种基因工程菌及其在制备二氢大豆苷元中的应用

文献发布时间：2023-06-19 16:11:11

技术领域

本发明涉及一种基因工程菌及其在制备二氢大豆苷元中的应用，属于生物工程技术领域。

背景技术

大豆异黄酮属于黄酮类化合物中的异黄酮类物质，主要来源于大豆，以大豆苷元和染料木素为主，因其与雌激素具有较相似的结构，在功能上也主要表现为雌激素样作用，故而又被称为“植物雌激素”。但是，其功效却不止于雌激素样作用，在增强机体免疫，预防骨质疏松等方面也有重要的作用。大豆异黄酮的生物学功效随着其逐渐被还原而增强，以大豆苷元及其代谢产物为例，生物学功能依次为：雌马酚>四氢大豆苷元>二氢大豆苷元>大豆苷元。作为大豆苷元还原的第一个代谢产物—二氢大豆苷元，其生产量直接限制了后续的进一步还原。

相较于生物法合成，化学法合成大豆苷元代谢产物涉及复杂的手性拆分问题，后续分离纯化较麻烦，难以实现大批量生产。虽然国内外对酶法制备二氢大豆苷元取得了一定的研究成果，但所获得的大豆苷元还原酶在比活方面比较低，不能满足大规模工业化生产的要求。亟需筛选鉴定出具有高催化活性的且具有大豆苷元还原酶功能的酶，以实现大豆苷元高值转化的替代方案。

发明内容

为了解决上述问题，本发明通过全细胞催化实验筛选了并鉴定出一组具有大豆苷元还原酶活性的酶，其中，氨基酸序列如SEQ ID NO.2所示的酶的催化活性高于来源于

本发明提供了一种基因工程菌，所述基因工程菌表达氨基酸序列如SEQ ID NO.2~SEQ ID NO.6任一所示的酶。

在本发明的一种实施方式中，所述基因工程菌是以枯草芽孢杆菌（

在本发明的一种实施方式中，所述基因工程菌以pET系列质粒为表达载体。

在本发明的一种实施方式中，所述基因工程菌以pET28(a)质粒为表达载体。

本发明还提供了一种全细胞催化剂，所述全细胞催化剂含有上述基因工程菌。

在本发明的一种实施方式中，所述全细胞催化剂中还含有冻干保护剂。

在本发明的一种实施方式中，所述冻干保护剂包括脱脂奶粉、蔗糖、谷氨酸钠、微晶纤维素和/或海藻糖。

本发明提供了一种核酸分子，其编码具有将大豆苷元转化为二氢大豆苷元功能的蛋白，核苷酸序列如SEQ ID NO.12~SEQ ID NO.16所示。

本发明还提供了携带上述核酸分子的表达载体。

本发明还提供了含有上述核酸分子或上述表达载体的微生物细胞。

在一种实施方式中，所述微生物细胞包括细菌细胞和真菌细胞。

本发明还提供了一种制备具备大豆苷元还原酶活性的蛋白的方法，所述方法为将上述基因工程菌接种于培养基中进行诱导培养。

在本发明的一种实施方式中，所述方法为将上述基因工程菌接种至培养基中，于35~40℃培养至OD600为0.02~0.04，加入终浓度为0.01~1 mM的IPTG，于22~25℃、125~175rpm条件下诱导表达5~15 h。

本发明还提供了一种制备二氢大豆苷元的方法，所述方法为将氨基酸序列如SEQID NO.2~SEQ ID NO.6任一所示的酶，或上述全细胞催化剂添加至含有大豆苷元的反应体系中。

在本发明的一种实施方式中，所述方法的反应条件为22~25℃、120~175 rpm。

本发明还提供了一种二氢大豆苷元的生产方法，所述方法由上述基因工程菌合成获得二氢大豆苷元。

本发明提供了氨基酸序列如SEQ ID NO.2~SEQ ID NO.6任一所示的酶，或上述基因工程菌，或上述全细胞催化剂，或上述方法在制备含有二氢大豆苷元及其下游产物的产品中的应用。

本发明提供了氨基酸序列如SEQ ID NO.2~SEQ ID NO.6任一所示的酶在制备含有二氢大豆苷元及其下游产物的产品中的应用。

本发明提供了上述基因，或上述表达载体，或上述微生物细胞在制备含有二氢大豆苷元及其下游产物的产品中的应用。

有益效果：

1、本发明通过全细胞催化实验筛选了并鉴定出一组具有催化大豆苷元还原成二氢大豆苷元活性的酶，其中氨基酸序列如SEQ ID NO.2~SEQ ID NO.6所示的酶具有大豆苷元还原酶活性，可以将大豆苷元转化为二氢大豆苷元。氨基酸序列如SEQ ID NO.2的酶的催化活性显著高于来源于

2、本发明为大豆苷元还原成二氢大豆苷元提供丰富的可选酶，也为大豆苷元的进一步还原提供更多的可用的酶元件，为促进下游高附加值产品的产量提供了可能性，推动二氢大豆苷元的产业化。

附图说明

图1为表达载体构建图；

图2为全细胞催化的色谱图；

图3为转化产物二氢大豆苷元的质谱图；

图4为全细胞催化验证催化能力的产量比较图。

具体实施方式

以下结合说明书附图和具体实施例来进一步说明本发明，但实施例并不对本发明做任何形式的限定。除非特别说明，本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。

除非特别说明，以下实施例所用试剂和材料均为市售商品或者可以通过已知方法制备。

（一）培养基及试剂

LB培养基：蛋白胨10 g/L，酵母粉5 g/L，氯化钠10 g/L。配制固体培养基还需加入18 g/L琼脂粉。

TB培养基：蛋白胨12 g/L，酵母粉24 g/L，甘油4 mL、KH

200 mM大豆苷元DMSO储备液：200 mmol大豆苷元溶解于500 mL DMSO中再定容到1L。

（二）大肠杆菌感受态细胞的制备

将大肠杆菌

感受态试剂盒采用TAKRA公司的高效感受态制备试剂盒。感受态制备过程按其说明书进行操作。

（三）表达载体的转化

1）取制备好的感受态细胞于冰上放置冻融，加入相应构建好的表达载体，冰上放置30 min。

2）将放置后的含目标载体的感受态于42℃水浴锅热激90 s，之后置于37℃摇床后培养40 min左右。

3）将感受态低速离心后去除部分上清，吹吸悬浮细胞，涂布于含相应抗性的LB平板上，进行抗性筛选，并做菌落PCR及测序验证重组转化子。

（四）HPLC测定

使用岛津高效液相色谱检测，液相检测条件为：大曹色谱柱CAPCELL PAKUG120250 mm × 4.6 mm column (particle size 5 μm)；流动相A，含有1‰甲酸的超纯水；流动相B，含有1‰甲酸的甲醇；流动相比例条件，0-2 min，5%B，2-13 min，5-100%B，13-15 min，100%B，15-18 min，100-5%B，18-20 min，5%B；流速：1 mL/min；柱温：40℃；进样量：10 μL；检测器波长：280 nm。

实施例1：表达载体构建与表达

1）表达载体构建

来源于

将来源于

将来源于.

将来源于

2）蛋白表达

将步骤（1）构建的重组质粒

将验证正确的转化子转移至含有50 mg/L硫酸卡那霉素的5 mLLB培养基中37℃过夜培养，制备种子液。将种子液以一定比例转接至含有50 mg/L硫酸卡那霉素的50 mLTB培养基中，控制初始OD

实施例2：全细胞催化验证酶的催化能力

将实施例1中重组菌株

通过全细胞催化大豆苷元以比较和验证氨基酸序列如SEQ ID NO.1所示的

结果如图2~图4所示，氨基酸序列如SEQ ID NO.2~SEQ ID NO.6所示的酶具有大豆苷元还原酶活性，可以将大豆苷元还原为二氢大豆苷元。而氨基酸序列如SEQIDNO.7-10所示的酶不具有催化大豆苷元还原为二氢大豆苷元的能力，转化率为0。

氨基酸序列如SEQ ID NO.1~SEQ ID NO.6所示的酶在加入底物后12 h表现出最大转化率，其中，以氨基酸序列如SEQIDNO.2所示的酶的产量为180.10 mg/L，转化率为58%，约为

转化率：实际生成的二氢大豆苷元摩尔数/理论完全转化生成二氢大豆苷元摩尔数*100%。

实施例3 游离酶制备二氢大豆苷元的应用

将实施例1步骤（2）中获得的菌液4000 rpm，4℃离心收集菌体，用PBS洗涤菌体两次，将所得细胞重悬在PBS(1 g细胞湿重/5 mL PBS)溶液中。利用高压均质机进行细胞破碎至澄清，作为粗酶液。向粗酶液中添加大豆苷元储液，反应制备二氢大豆苷元。

对比例1

将来源于

蛋白表达和全细胞催化能力的测定方法同实施例1和实施例2，表达SEQ ID NO.21~SEQ ID NO.25的基因工程菌的二氢大豆苷元的产量分别为30.16 mg/L，8.76 mg/L，3.24mg/L，11.02 mg/L，6.18 mg/L，转化率分别为9.7%、2.8%、1.04%、3.5%、1.99%。

虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。

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180 185 190

Ala Arg Tyr Ser Ile Glu Ser Ile Arg Glu Ile Arg Lys Asn Ile Pro

195 200 205

Glu Asp Met Pro Ile Phe Met Arg Val Pro Ala Lys Asp Asp Glu Leu

210 215 220

Pro Gly Gly Leu Ser Ile Glu Asp Val Ile Glu Phe Cys Lys Met Ala

225 230 235 240

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

245 250 255

Thr Ser Ala Met Arg Tyr Glu Val Pro Pro Leu Asp Ile Pro Arg Gly

260 265 270

Phe Asn Val Asp Asn Ala Ala Thr Ile Arg Lys Ala Thr Lys Met Pro

275 280 285

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

290 295 300

Ile Ala Glu Asp Lys Ala Asp Met Val Val Met Gly Arg Ala Gln Ile

305 310 315 320

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

325 330 335

Ile Leu Asn Cys Ile Gly Cys Asn Gln Gly Cys Tyr Glu Asn Cys Leu

340 345 350

Ile Gly Ala Pro Ile Thr Cys Leu Arg Asn Pro Ser Val Gly Lys Glu

355 360 365

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

370 375 380

Val Ile Gly Gly Gly Val Gly Gly Met Glu Ala Ala Met Met Ala His

385 390 395 400

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

405 410 415

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

420 425 430

Ala Ala Thr Ile Lys Arg Glu Lys Gln Leu Gln Arg Ala Gly Val Lys

435 440 445

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

450 455 460

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

465 470 475 480

Ile Glu Gly Val Lys Gly Asn Asn Val Tyr Asn Phe Val Asp Val Leu

485 490 495

Asn Gly Thr Lys Thr Val Glu Gly His Val Val Val Ala Gly Gly Gly

500 505 510

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

515 520 525

Val Thr Val Ile Glu Met Gln Asp Ser Val Ala Lys Asp Val Gly Ala

530 535 540

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

545 550 555 560

Thr Val Thr Asp Ser Lys Cys Val Lys Ile Thr Ser Asn Ser Val Trp

565 570 575

Val Asp Gln Lys Gly Glu Ile Lys Glu Ile Pro Cys Asp Ser Val Val

580 585 590

Leu Ala Val Gly Ser Lys Ser Asn Pro Thr Ser Trp Ile Glu Glu Tyr

595 600 605

Cys Gln Lys His Ala Val Glu Tyr Lys Val Val Gly Asp Ala Val Asn

610 615 620

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

625 630 635 640

Asn Ile Met Lys Asn Lys Tyr Tyr Pro Asn Leu Phe Lys Lys Gly Lys

645 650 655

Ile Gly Asn Val Val Leu Lys Asn Arg Ile Val Arg Asn Ser Met Gly

660 665 670

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

675 680 685

Ala Tyr Val Asp Ala Ala Glu Gly Gly Ala Gly Leu Ile Phe Met Asp

690 695 700

Asn Ala Thr Pro Val Pro Met Thr Ser Cys Gly Leu Arg Ala Asp Gly

705 710 715 720

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

725 730 735

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

740 745 750

Gly Phe Val Gly Ser Ala Asp Val Ile Gly Ala Ser Pro Ile Thr Phe

755 760 765

Glu Pro Trp Tyr Glu Ala Gly Ala Lys Leu Pro Arg Glu Leu Ser Val

770 775 780

Asp Glu Ile His Asp Leu Val Ser Lys Phe Gly Asp Ala Ala Leu Arg

785 790 795 800

Cys Arg His Ala Gly Phe Asp Val Val Glu Ile His Gly Ala Ala Gly

805 810 815

Cys Ile Pro Thr Asn Phe Leu Ser Pro His Asp Asn His Arg Thr Asp

820 825 830

Met Tyr Gly Gly Ser Leu His Asn Arg Met Arg Leu Leu Ile Glu Met

835 840 845

Val Arg Asp Met Lys Lys Lys Cys Gly Pro Asp Phe Pro Ile Gly Val

850 855 860

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

865 870 875 880

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

885 890 895

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

900 905 910

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

915 920 925

Asn Ile Pro Val Phe Val Gly His Asn Val Phe Thr Pro Asp Glu Ala

930 935 940

Glu Glu Leu Leu Lys Asn Gly Cys Gly Glu Phe Val Ala Leu Gly Arg

945 950 955 960

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

965 970 975

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

980 985 990

Arg Gly Leu Leu Asn His Thr Ala Ile His Cys Thr Val Asn Pro Gly

995 1000 1005

Leu Tyr Lys Phe Glu Arg Glu Thr Ile Asn Thr Thr Asn Pro Arg

1010 1015 1020

Asn Val Val Val Ile Gly Ala Gly Pro Ala Gly Cys Glu Ala Ala

1025 1030 1035

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

1040 1045 1050

Arg Glu Phe Gly Gly Ala Met Ile Glu Ala Ala Lys Pro Glu Asn

1055 1060 1065

Lys Lys Asn Ile Arg Arg Leu Ile Ser Tyr Tyr Lys Asp His Ile

1070 1075 1080

Asp Ala His Lys Asn Ile Thr Val Ile Asn Lys Glu Ala Thr Tyr

1085 1090 1095

Glu Glu Ile Val Ala Gly Asn Tyr Asp Val Cys Ile Ala Ala Thr

1100 1105 1110

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

1115 1120 1125

Met Val Val Asn Ala Met Asp Tyr Leu Gly Gly Glu Lys Val Asp

1130 1135 1140

Gly Asn Thr Val Val Val Ile Gly Gly Gly Ile Thr Gly Ala Glu

1145 1150 1155

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

1160 1165 1170

Glu Met Thr Asp His Phe Leu Ala Gln Lys Thr Asn Ala Cys Gln

1175 1180 1185

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

1190 1195 1200

Gly Leu Arg Leu Ile Glu Val Gly Asn His Thr Ile Val Leu Thr

1205 1210 1215

Asp Arg Tyr Gly Asn Glu Gln Asn Val Ala Cys Asp Asn Val Val

1220 1225 1230

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

1235 1240 1245

Glu Glu Glu Thr Asp Met Gln Val Phe Asn Val Gly Asp Ala Lys

1250 1255 1260

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

1265 1270 1275

Ala Lys Leu Ile His

1280

<210> 5

<211> 641

<212> PRT

<213> Sharpea porci

<400> 5

Met Lys Asn Lys Tyr Tyr Pro Asn Leu Phe Lys Lys Gly Lys Ile Gly

1 5 10 15

Asn Val Val Leu Lys Asn Arg Ile Val Arg Asn Ser Met Gly Thr Tyr

20 25 30

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

35 40 45

Val Asp Ala Ala Glu Gly Gly Ala Gly Leu Ile Phe Met Asp Asn Ala

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Trp Tyr Glu Ala Gly Ala Lys Leu Pro Arg Glu Leu Ser Val Asp Glu

130 135 140

Ile His Asp Leu Val Ser Lys Phe Gly Asp Ala Ala Leu Arg Cys Arg

145 150 155 160

His Ala Gly Phe Asp Val Val Glu Ile His Gly Ala Ala Gly Cys Ile

165 170 175

Pro Thr Asn Phe Leu Ser Pro His Asp Asn His Arg Thr Asp Met Tyr

180 185 190

Gly Gly Ser Leu His Asn Arg Met Arg Leu Leu Ile Glu Met Val Arg

195 200 205

Asp Met Lys Lys Lys Cys Gly Pro Asp Phe Pro Ile Gly Val Lys Leu

210 215 220

Ser Thr Val Asp Trp Glu Pro Glu Gly Ile Thr Leu Asp Glu Thr Ile

225 230 235 240

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

245 250 255

Met Ala Gly Thr His Ala Thr Ala Ala Met Glu Phe Leu Lys Pro Asn

260 265 270

Cys Phe Asn Gly Ala Asp Cys Lys Lys Ile Ala Asp Ala Val Asn Ile

275 280 285

Pro Val Phe Val Gly His Asn Val Phe Thr Pro Asp Glu Ala Glu Glu

290 295 300

Leu Leu Lys Asn Gly Cys Gly Glu Phe Val Ala Leu Gly Arg Ser Gln

305 310 315 320

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

325 330 335

Glu Ile Lys Pro Cys Ile Asn Cys Leu Ile Gly Cys Leu Asp Arg Gly

340 345 350

Leu Leu Asn His Thr Ala Ile His Cys Thr Val Asn Pro Gly Leu Tyr

355 360 365

Lys Phe Glu Arg Glu Thr Ile Asn Thr Thr Asn Pro Arg Asn Val Val

370 375 380

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

385 390 395 400

Asn Asn Gly His Lys Val Thr Ile Tyr Glu Lys Arg Glu Phe Gly Gly

405 410 415

Ala Met Ile Glu Ala Ala Lys Pro Glu Asn Lys Lys Asn Ile Arg Arg

420 425 430

Leu Ile Ser Tyr Tyr Lys Asp His Ile Asp Ala His Lys Asn Ile Thr

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

Gly Glu Lys Val Asp Gly Asn Thr Val Val Val Ile Gly Gly Gly Ile

500 505 510

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

515 520 525

Ile Ile Val Glu Met Thr Asp His Phe Leu Ala Gln Lys Thr Asn Ala

530 535 540

Cys Gln Ala Tyr Ser Ile Ala Ile Ser Gln Thr Asp Ile Glu Ile Lys

545 550 555 560

Thr Gly Leu Arg Leu Ile Glu Val Gly Asn His Thr Ile Val Leu Thr

565 570 575

Asp Arg Tyr Gly Asn Glu Gln Asn Val Ala Cys Asp Asn Val Val Ile

580 585 590

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

595 600 605

Glu Thr Asp Met Gln Val Phe Asn Val Gly Asp Ala Lys Arg Val Arg

610 615 620

Gln Ile Phe Asp Ala Val His Glu Gly Tyr Ile Ala Ala Lys Leu Ile

625 630 635 640

His

<210> 6

<211> 641

<212> PRT

<213> Sharpea azabuensis

<400> 6

Met Glu Asn Lys Tyr Tyr Pro Asn Leu Phe Lys Lys Gly Lys Ile Gly

1 5 10 15

Asn Val Val Leu Lys Asn Arg Ile Val Arg Asn Ser Met Gly Thr Tyr

20 25 30

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

35 40 45

Val Asp Ala Ala Glu Gly Gly Ala Gly Leu Ile Phe Met Asp Asn Ala

50 55 60

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

65 70 75 80

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

85 90 95

Ala Ala Ala Gly Met Gln Leu Ala His Pro Gly Arg Asp Ala Gly Phe

100 105 110

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

115 120 125

Trp Tyr Glu Ala Gly Ala Lys Leu Pro Arg Glu Leu Ser Val Asp Glu

130 135 140

Ile His Asp Leu Val Ser Lys Phe Gly Asp Ala Ala Leu Arg Cys Arg

145 150 155 160

His Ala Gly Phe Asp Val Val Glu Ile His Gly Ala Ala Gly Cys Ile

165 170 175

Pro Thr Asn Phe Leu Ser Pro His Asp Asn His Arg Thr Asp Met Tyr

180 185 190

Gly Gly Ser Leu His Asn Arg Met Arg Leu Leu Ile Glu Met Val Arg

195 200 205

Asp Met Lys Lys Lys Cys Gly Pro Asp Phe Pro Ile Gly Val Lys Leu

210 215 220

Ser Thr Val Asp Trp Glu Pro Glu Gly Ile Thr Leu Asp Glu Thr Ile

225 230 235 240

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

245 250 255

Met Ala Gly Thr His Ala Thr Ala Ala Met Glu Phe Leu Met Pro Asn

260 265 270

Cys Phe Asn Ala Ala Asp Cys Lys Lys Ile Ala Asp Ala Val Asn Ile

275 280 285

Pro Val Phe Val Gly His Asn Ile Phe Thr Pro Asp Glu Ala Glu Glu

290 295 300

Leu Leu Lys Asn Gly Cys Gly Glu Phe Val Ala Leu Gly Arg Ser Gln

305 310 315 320

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

325 330 335

Glu Ile Lys Pro Cys Ile Asn Cys Leu Ile Gly Cys Leu Asp Arg Gly

340 345 350

Leu Leu Asn His Thr Ala Ile His Cys Thr Val Asn Pro Gly Leu Tyr

355 360 365

Lys Phe Glu Arg Glu Thr Ile Asn Thr Thr Asn Pro Arg Asn Val Val

370 375 380

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

385 390 395 400

Asn Asn Gly His Lys Val Thr Ile Tyr Glu Lys Arg Glu Phe Gly Gly

405 410 415

Ala Met Ile Glu Ala Ala Lys Pro Glu Asn Lys Lys Asn Ile Arg Arg

420 425 430

Leu Ile Ser Tyr Tyr Lys Asp His Ile Asp Ala His Lys Asn Ile Thr

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

Gly Lys Lys Val Asp Gly Asn Thr Val Ala Val Ile Gly Gly Gly Ile

500 505 510

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

515 520 525

Ile Ile Val Glu Met Thr Asp His Phe Leu Ala Gln Lys Ser Asn Ala

530 535 540

Cys Gln Ala Tyr Ser Ile Ala Ile Ser Gln Thr Asp Ile Glu Ile Lys

545 550 555 560

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

565 570 575

Asp Arg Tyr Gly Asn Glu Gln Asn Val Ala Cys Asp Asn Val Val Ile

580 585 590

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

595 600 605

Glu Thr Asp Met Gln Val Phe Asn Val Gly Asp Ala Lys Arg Val Arg

610 615 620

Gln Ile Phe Asp Ala Val His Glu Gly Tyr Ile Ala Ala Lys Leu Ile

625 630 635 640

His

<210> 7

<211> 646

<212> PRT

<213> Catenisphaera adipataccumulans

<400> 7

Met Tyr Asn Lys Leu Phe Glu Pro Gly Lys Ile Gly Gly Leu Glu Leu

1 5 10 15

Lys Asn Arg Val Val Met Thr Ala Met Gly Cys Ser Leu Ala Glu Pro

20 25 30

Asp Gly His Pro Gly Pro Gln Met Ile Glu Tyr Tyr Lys Ala Arg Ala

35 40 45

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

50 55 60

Ala Glu Gly Ala Ser Thr Pro Cys Gln Leu Ser Val Thr Asn Leu Lys

65 70 75 80

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

85 90 95

Thr Lys Ile Leu Ile Gln Leu His His Ala Gly Asn Gln Ile Ser Ser

100 105 110

Lys Leu Ile Gly Gly Arg Gln Pro Val Ser Ala Ser Asp Val Thr Cys

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Arg Gln Tyr Cys Gly Pro Lys Phe Val Ile Ser Val Arg Met Asp Gly

210 215 220

Asp Glu Phe Ile Pro Asp Gly Leu His Leu Asp Glu Ala Val Val Val

225 230 235 240

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

245 250 255

Gly Thr Tyr Glu Ser Gly Ile Thr Ile Val Glu Pro Asn Ala Tyr Pro

260 265 270

Glu Ala Trp Pro Gln Lys Val Asn Leu Ala Lys Thr Ile Arg Glu Asn

275 280 285

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

290 295 300

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

305 310 315 320

Arg Gly Gln Leu Ala Asp Pro Glu Trp Thr Asn Lys Ala Lys Ala Gly

325 330 335

His Asp Met Arg Ile Arg Lys Cys Ile Asn Cys Met Glu Cys Phe Arg

340 345 350

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

355 360 365

Gly Arg Glu Tyr Leu Ile Gly Glu Glu His Leu Lys Lys Asp Gly Ala

370 375 380

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

385 390 395 400

Ala Leu Thr Leu Ala Lys Arg Gly Phe His Ala Val Leu Phe Glu Glu

405 410 415

His Glu His Leu Gly Gly Thr Val Asn Leu Ala Ala Val Pro Pro His

420 425 430

Lys Glu Arg Leu Leu Glu Phe Ile Asp Thr Met Ala Leu Gln Met Lys

435 440 445

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

450 455 460

Cys Gln Ala Val His Ala Glu Ala Val Phe Val Ala Thr Gly Gly Tyr

465 470 475 480

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

485 490 495

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

500 505 510

Ile Gly Gly Gly Val Thr Gly Leu Glu Thr Ala Glu Tyr Leu Ala Val

515 520 525

Asn Lys Lys Asn Lys Val Thr Val Val Glu Met Met Asp Asn Val Gly

530 535 540

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

545 550 555 560

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

565 570 575

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

580 585 590

Cys Asp Ala Val Val Leu Ala Met Gly Val Lys Pro Asp Arg Ser Leu

595 600 605

Ala Asp Asp Leu Gln Gln Ala Phe Asp Gln Val Ile Tyr Val Gly Asp

610 615 620

Cys Gln Gln Pro Gly Gln Ile Arg Glu Ala Leu His Ala Gly Tyr Asp

625 630 635 640

Lys Ala Phe Val Tyr Glu

645

<210> 8

<211> 648

<212> PRT

<213> Clostridium saccharogumia

<400> 8

Met Asn Arg Tyr Pro His Leu Phe Ser Pro Ile Lys Ile Gly Lys Gln

1 5 10 15

Val Val Lys Asn Arg Ile Phe Met Pro Pro Leu Ser Thr Asn Leu Gly

20 25 30

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

35 40 45

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

50 55 60

Pro Thr Tyr Val Tyr Leu Pro Gly Asp Met Ser Ile Tyr Asp Asp Ser

65 70 75 80

Phe Ile Glu Gly Trp Lys Lys Leu Ala Ala Ala Val His Glu Tyr Gly

85 90 95

Ala Lys Ile Leu Pro Gln Leu Phe His Pro Ala Tyr Met Ala Phe Pro

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

Ala Asp Gly Val Glu Ile His Ala Ala His Ala His Gly Leu Leu Gly

165 170 175

Gly Phe Leu Ser Pro Gln Tyr Asn Lys Arg Thr Asp Glu Tyr Gly Gly

180 185 190

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

195 200 205

Arg Lys Arg Cys Gly Glu Glu Phe Ile Ile Asp Val Arg Ile Ser Gly

210 215 220

Asp Glu Tyr Ser Asp Gly Gly Leu Asn Ile Asn Asp Met Ile Tyr Val

225 230 235 240

Ser Lys Gln Leu Glu Asn His Gly Val Asp Met Ile His Val Ser Gly

245 250 255

Gly Thr Thr Ile Ala Arg Gly Ser Ser Ile Pro Ala Ala Gly Thr Lys

260 265 270

Met Gly Ser His Ser Gln Leu Ser Ala Lys Ile Lys Lys Tyr Val Ser

275 280 285

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

290 295 300

Glu Leu Ile Ala Asn Gly Met Ala Asp Ala Cys Met Ile Gly Arg Ala

305 310 315 320

Asn Leu Cys Asp Pro Glu Phe Ser Asn Lys Ala Lys Ala Gly Lys Pro

325 330 335

Glu Asp Ile Arg Pro Cys Ile Gly Cys Leu Arg Cys Leu Asn Gly Ile

340 345 350

Met Phe Gly Lys Pro Ile Ala Cys Thr Met Asn Pro Ser Phe Ser Leu

355 360 365

Glu Asn Glu Asp Thr Ile Leu Pro Ala Asp Ile Lys Lys Lys Ile Leu

370 375 380

Val Val Gly Gly Gly Pro Ala Gly Met Glu Ala Ala Tyr Ile Ala Lys

385 390 395 400

Lys Arg Gly His Asp Val Val Leu Cys Glu Lys Asp Ser Glu Leu Gly

405 410 415

Gly Ala Leu Lys Val Ala Cys Val Pro Ile Gly Lys Gln Asp Leu Cys

420 425 430

Gln Val Ile Lys Trp Met Arg His Arg Leu Glu Lys Glu Asn Val Asn

435 440 445

Ile Gln Thr Asn Thr Asn Val Thr Leu Glu Leu Leu Lys Thr Lys Phe

450 455 460

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

465 470 475 480

Asn Ala Phe Thr Gln Phe Lys Gln Trp Met Ser Ala Asp Asp Val Leu

485 490 495

Ala Gly Arg Ala Phe Pro Gly Arg Lys Ile Val Ile Ile Gly Gly Gly

500 505 510

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

515 520 525

Arg Phe Pro Arg Asn Arg Asp Val Thr Ile Ile Glu Met Ala Gln Glu

530 535 540

Val Met Met Asn Glu Ser Gly Pro Gly Arg Ser Leu Leu Val Gln Arg

545 550 555 560

Met Ile Lys Lys Gly Val Lys Ile Leu Val Asn Thr Lys Val Ala Ala

565 570 575

Val Glu Lys Asp Lys Ile Lys Tyr Leu Gln Asn Gly Val Glu Gly Val

580 585 590

Ile Asp Asp Ala Asp Thr Leu Ile Phe Ala Cys Gly Tyr Gln Thr Asp

595 600 605

Ser Ala Phe Glu Asn Met Leu Lys Glu Leu Lys Met Ser Tyr His Leu

610 615 620

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

625 630 635 640

Ala Tyr Arg Leu Ala Arg Asp Ile

645

<210> 9

<211> 643

<212> PRT

<213> Intestinibaculum porci

<400> 9

Met Ser Tyr Thr Lys Leu Phe Glu Lys Gly Lys Ile Gly Arg Leu Glu

1 5 10 15

Leu Lys Asn Arg Ile Val Met Pro Ala Met Gly Cys Ser Leu Ala Ser

20 25 30

Ser Thr Gly Glu Ala Ser Gln Glu Met Ile Thr Tyr Tyr Ala Lys Arg

35 40 45

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

50 55 60

Asp Glu Thr Gly Ile Gly Thr Pro Asn Gln Leu Ser Val Thr Asp Leu

65 70 75 80

Lys His Ile Pro Gln Leu Thr Arg Leu Ala Glu Ala Val His Ala Tyr

85 90 95

Asn Thr Lys Ile Phe Val Gln Leu His His Pro Gly Asn Gln Thr Pro

100 105 110

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

115 120 125

Cys Ser Val Ile Gly Glu Lys Pro Arg Ala Leu Thr Thr Glu Glu Val

130 135 140

Glu Gly Leu Val Lys Lys Phe Val Phe Gly Ala Tyr Val Ala Lys Ala

145 150 155 160

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

165 170 175

Asp Gln Phe Met Ser Pro His Thr Asn Lys Arg Thr Asp Arg Tyr Gly

180 185 190

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

195 200 205

Ile Gln Ala Thr Cys Gly Pro Thr Tyr Pro Ile Ser Val Arg Met Asp

210 215 220

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

225 230 235 240

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

245 250 255

Cys Gly Thr Tyr Glu Ser Gly Tyr Thr Ile Val Glu Pro Ala Met Leu

260 265 270

Lys Glu Gly Trp Lys Ala Asp Leu Ala Arg Gln Ile Lys Ala Asn Val

275 280 285

Ser Ile Pro Val Ile Ala Val Asn Thr Ile Lys His Pro Ala Phe Ala

290 295 300

Glu Ser Leu Leu Glu Glu Asp Val Cys Asp Phe Val Gly Ile Gly Arg

305 310 315 320

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

325 330 335

Asp Leu Tyr Ile Arg Lys Cys Ile Gly Cys Leu Glu Cys Phe Arg Ile

340 345 350

Leu Asn Thr Leu Arg Pro Val Glu Cys Thr Leu Asn Pro Leu Leu Gly

355 360 365

Arg Glu Tyr His Trp Asp His Leu His Lys Thr Gly Gln His Arg Lys

370 375 380

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

385 390 395 400

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

405 410 415

Leu Gly Gly Thr Met Asn Leu Ala Ala Ile Pro Pro His Lys Glu Leu

420 425 430

Leu Gly Glu Phe Val Glu Thr Met Ala Ala Gln Val Glu Glu Ala Gly

435 440 445

Val Gln Val Val Tyr Asn Thr Lys Ala Thr Pro Glu Asp Leu Lys Glu

450 455 460

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

465 470 475 480

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

485 490 495

Lys Gly Glu His Asp Leu His Asp Gln Lys Ile Val Ile Ile Gly Gly

500 505 510

Gly Val Thr Gly Leu Glu Thr Ala Glu Thr Leu Ala Lys Asp Asn Gln

515 520 525

Val Thr Val Ile Glu Met Ala Asn Gln Val Gly Thr Thr Leu Tyr Ala

530 535 540

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

545 550 555 560

Ile Lys Thr Glu His Arg Leu Thr His Ile Glu Asp His Gln Val Tyr

565 570 575

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

580 585 590

Leu Ala Met Gly Val Lys Pro Lys Arg Glu Ala Leu Glu Asp Phe Glu

595 600 605

Lys Val Phe Asp Gln Val Ile Leu Leu Gly Asp Thr Asp His Pro Gly

610 615 620

Gln Ile Arg Glu Ala Leu His Ser Ala Tyr Asp Arg Ala Phe Val Phe

625 630 635 640

Asp Leu Gly

<210> 10

<211> 672

<212> PRT

<213> Holdemania massiliensis

<400> 10

Met Ser Asn Leu Phe Pro Ile Leu Thr Ser Pro Gly Lys Ile Gly Ser

1 5 10 15

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

20 25 30

Gly Lys Asp Gly Phe Val Thr Asp Ala Ile Val Asn Arg Tyr Ala Glu

35 40 45

Asn Ala Lys Gly Gly Thr Gly Leu Ile Ile Thr Glu Val Thr Cys Val

50 55 60

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

65 70 75 80

Asp Lys Tyr Ile Pro Gly Phe Arg Arg Ile Thr Asp Val Ile His Lys

85 90 95

Tyr Gly Ser Lys Cys Ala Ile Gln Leu Ser His Ala Gly Arg Gly Ala

100 105 110

Asn Leu Asp Val Leu His Gln Leu His Gln Pro Ala Val Ala Pro Ser

115 120 125

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

130 135 140

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

145 150 155 160

Ala Asp Ala Ala Tyr Arg Ala Lys Gln Ala Gly Tyr Asp Ala Val Glu

165 170 175

Val His Gly Ala Gly Tyr Tyr Leu Val Ala Gln Phe Phe Ser Ala Thr

180 185 190

Ala Asn Leu Arg Thr Asp Glu Tyr Gly Gly Asn Ala Phe Asn Arg Ala

195 200 205

Arg Phe Ala Cys Asn Ile Ile Arg Lys Ile Lys Ala Arg Cys Gly Ala

210 215 220

Asp Phe Pro Val Leu Ile Lys Met Asn Val Val Asp Gly Gly Leu Asn

225 230 235 240

Gly Gly Val Ser Val Met Asp Gly Leu Leu Asn Cys Tyr Leu Val Gln

245 250 255

Gln Ala Gly Ala Asp Ala Ile Glu Val Ile Ala Cys Asp Trp Ser Asp

260 265 270

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

275 280 285

Thr Phe Pro Leu Ala Gly Met Ile Arg Lys Ala Thr Ile Ala Glu Asn

290 295 300

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

305 310 315 320

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

325 330 335

Ala Leu Ala Gln Gly Leu Cys Asp Phe Ile His Met Gly Arg Gly Leu

340 345 350

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

355 360 365

Tyr Ala Arg Pro Cys Ile Gly Cys Gln Lys Cys Met Asp Asn Gln Leu

370 375 380

His His Asp Gly Glu Leu Ile Cys Ser Gly Asn Ala Val Leu Gly His

385 390 395 400

Asp Asp Asn Asp Ala Ser Leu Pro Ala Ala Lys Lys Ile Lys Asn Val

405 410 415

Met Val Val Gly Gly Gly Pro Ala Gly Ile Glu Ala Ala Arg Val Ala

420 425 430

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

435 440 445

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

450 455 460

Ala Asp Tyr Ile Pro Tyr Met Glu Val Gln Ala Glu Tyr Arg Gly Phe

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

Leu Met Gly Lys Pro Thr Gly Glu Asn Ile Val Ile Leu Gly Gly Gly

530 535 540

Ser Val Gly Cys Glu Thr Ala Glu Trp Met Leu Glu Ala Gly Lys Asn

545 550 555 560

Val Thr Ile Val Glu Met Ser Asp Gln Leu Met Gly Lys Met Val Glu

565 570 575

Thr Thr Arg Phe Ile Leu Lys Ala His Leu Arg Asp Leu Asn Cys Asn

580 585 590

Thr Leu Leu Asn Thr Arg Cys Val Glu Ile Gln Asp Gln Ala Val Val

595 600 605

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

610 615 620

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

625 630 635 640

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

645 650 655

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

660 665 670

<210> 11

<211> 1935

<212> DNA

<213> 人工序列

<400> 11

atgcagcacg ctaaataccc gcacctgttt tctaaaggta aagttggtaa agtgaccacc 60

aaaaaccgtg ttatccgtaa cagcatgggc acctacctga acgttggtaa actgtgcgat 120

gttagcgatc gtaacattaa acacgcggcg gaagcagcgg aaggtggtcc gggtatcgtg 180

ttcctggata actgcctgat cgttgatggt taccacatgg gcctggcggc gtatgacgac 240

acctacattc cgggcctgtc tatgatcgcg caggcgatgc acgatcatgg cgcggttgcg 300

ggtatgcagc tggcgcatcc gggtcgtgac atgggcttcg cgggcggcga taacgttgtt 360

gcgccgtctg ctgttctgcc ggaaatcatg attaacgcgg gtgcgaccgt tccgcgcccg 420

ctgaccatcg acgaaatcca tgaaatcgaa gaacagtatg gccaggccgc ggcgcgtgtt 480

aaacaggcgg gcttcgacat tgttgaagtg cacggtgcgt gcggctgcct gccgaccaac 540

ttcctgagcc cgcacgataa ccagcgtaac gatatctacg gcggctctct gttcaaccgc 600

cagcgtttcc tggttgaagt gatccgtgtt attaaacgtt acgtgggtcc ggatttcccg 660

gtttctgtga aactggatat ggatgattgc gaaccggatg gtatccgcct ggaagaatgc 720

atcgatacct gtcgtgttct ggaacgtgaa ggtgttgcgc tgctgaacct ggttaccgct 780

actcacgtta ctgcaaactt cagcaccagc ttctacccgt ggagctactg cgctgatatg 840

gctgctaaag ttaaagaaca ggtgcacatc ccggttatgg ttaccggcgc gatccagtct 900

ccggaagcgg ctgaaaaaat cctggctgac ggtaaagttg acttcgttgg taccgcgcgt 960

cagtgtctgg cggatcaggc ttgggttgaa aaagcacgta ccggtcatga agaagatatc 1020

cgcccgtgca tccgttgcca gatcggctgc accgatcgtg gtatcctggg ccaccacccg 1080

atcagctgcg caaccaaccc gaccctgttc cactattacg aagaactgta tccgaaagcg 1140

gaaaccccga aaaacgttgc tgtggttggc gcgggtccgg ccggttgcga agcagctctg 1200

accctgaaaa aacgtggcca caacgtggtt gttttcgaaa aacgtgaaat tggcggcacc 1260

atgatcgaag caggtgctgc ctggtacaaa tctgatatca accgtttcat cgaatactac 1320

cgtaaacagc tgaaaaaaca caacattgaa gttcgtaaac aggaagttac cccggaagat 1380

atcgcggcgg gcggtttcga tgcgtgcatc gtggcaatcg gtggtgaacc gcgtaaactg 1440

aacgtgccgg gcatcgataa accgatcgtg actgaaggta ttgatttcct gtacggctct 1500

aaaaaagttg aaggcaaaag cgctgttgtt gttggcggtg cgactaccac cgcggaaatc 1560

gcactggatc tggcggaaaa aggcatggac gttaccatcg tgaaacgtgg tactaaattc 1620

ctgaacccgg ccggctgcca gatggatatc gaatacacta ttcgtctgca ccagctgggt 1680

gtgaaactga tgaccggcta ccgcctggat tctgtgaccg acaccagcgc aattgcgatc 1740

gatcagtatg gtgaaaccgt tgaactgccg tgcgataacg ttgttatcag cgctggtttc 1800

ctgaaccgtc cgggcttcgc ggaaaaactg gaagaaatca gcgatatgga tgtttatatg 1860

gcaggtgatt gcaaaaaagt tgcggaaatc ccggacgcga cccacgcggg ttatgctgtg 1920

gcgcgtatgg tttaa 1935

<210> 12

<211> 1929

<212> DNA

<213> 人工序列

<400> 12

atgaaaaaga accagcactt cccgaaactg ttcgaacgtg gctacatcgc gggtctggaa 60

atcaaaaacc gtatcgttcg tcagccgatg ggtaccgaac tgggtaaccc ggatggttct 120

ccgtcttggg cgaccgttaa agcgtacgca gaagctgcgg acggcggtgc gggcatcgtt 180

tacatggata acgctggtgt tacccagttc caccacgttg gcctgagcat cgcgtctgat 240

ccgtatattg gtccgatgag catcctggcg aaaacgctga aacatcacgg tgctgtgccg 300

ggcctgcaga ttgtgcatcc gggtcgcgat gcagcgttcg tggcaggtga tgatctgatc 360

agtagcagcc gtgttatgtg ggaaccgtgg tacgaaaacg gcggtggtgt tccgcgcgaa 420

ctgaccatcg aagaaatcca cgaattcgtt gaagcattcg gtgatgcggc ggaacgtggt 480

cagcgtgctg gtttcgaaat tatcgatgtg cactctgctt gtggtgttct gctgagcaac 540

ttcctgtctc cgctgaacaa cacgcgtacc gacatgtacg gtggtagcct gcacaaccgt 600

atgcgtttcc tgatggaagt tatccgtaac atcaaacaga aaaccagcgt accgctgtcc 660

atccgcctta gcggttgcga cttcgaaccg ggtggtatta ccattgagga aaccatcgaa 720

gtggcgaaag cctgcgaacg tatgggcgcg gacgttatca acatcacctg gggttcccac 780

gcggaagttg tgaacgctgc gggtctgctg tccccgcacg gtgcgaacca cgttgacatg 840

gccaaacgta ttaaagacgc agtgtcaatc ccggttatgc tgtgcggtgg tatctacacc 900

ccggaaatcg gcgaacagtt gctcgaagat ggcgtgtgtg attacgtggg tatcggcaaa 960

ccggcgctgg ctgatccgtt ctgggctaaa aaagctgaag aaggccgtag cggtgacatt 1020

cgtccgtgca tcggctgcgg cgtgggttgc cacgaccgcg gcatgctttc tggtggtatg 1080

gtacagtgcg cggttaaccc gaccctgtac cagttcgacc gcgaatactt ccctaaaact 1140

gatcgtccga aaaaagttgc gattatcggc gctggtccgg cgggttctac ggctgcactg 1200

accgcggcgg aatgcggtca cgacgtgacc ctgtttgaag gccgtgaggt tggcggcgtt 1260

ctgaaagaag catccgtacc ggtgtacaaa gaagatctgg gcctgctggg taaatactat 1320

cagcgccaga tcgctaaatc taacgttaaa ctggtggaag aaaacgcaac cccggaaacc 1380

atcgcgaccg gcgacttcga cgcggttatc gtggcaaccg gtggcaaagt gcgtgaactg 1440

aacctgccgg gtctggatag cgacaacgtt atctacgcaa tggacctgat gaaacagggt 1500

tgccagctgg acgctgataa agtggtggtg gttggtggcg gtattgtggg cgcggaagct 1560

gccctgatcc tggctgaaga tttcggtaaa gatgttacca tcaccacccg tcaggataac 1620

ttcttcgtgc cgggtgtcat gggcattgca tatatgaccc gtctggcaat ggcaggtgtt 1680

aaaaccaaaa cccgtgcgaa cctggttgaa gttaaagatg gcaaaccagt attcagcacc 1740

atgaacggcc tggaaatgat ggatgtggac gcagttgttg tttctccggg cttcctgccg 1800

actagtcaga tgcgtgatga tatcgaacag attgccgacg ttgacaccta cgtaatcggt 1860

gatgcgaaag cgccgcgtct ggtgatggat gctgttcatg aaggttataa aaccgcgatt 1920

aacctgtaa 1929

<210> 13

<211> 1929

<212> DNA

<213> 人工序列

<400> 13

atgaagaaaa accagcactt cccgaaactg tttgaacgtg gttacatcgc tggtctggaa 60

atcaaaaacc gtatcgttcg tcagccgatg ggtaccgaac tgggtaaccc ggacggttct 120

ccgtcctggg cgaccgtgaa agcgtacgcg gaagctgcag atggcggtgc gggcattgtt 180

tacatggata acgccggtgt tacccagttc catcacgttg gtctgagcat cgccagcgac 240

ccgtacatcg gtccgatgag cattctggca aagaccctga aacaccacgg tgcggttccg 300

ggcctgcaga tcgtacatcc gggccgtgat gctgcctttg ttgcaggcga tgatctgatc 360

tcttcctctc gtgtgatgtg ggaaccgtgg tacgaaaacg gtggcggcgt tccgcgtgaa 420

ctgactatcg aagaaatcca cgaattcgtt gaagcgttcg gcgacgcggc tgaacgcggt 480

cagcgcgcag gcttcgaaat catcgatgtt cactccgctt gcggcgttct gctgagcaac 540

tttctgtccc cgctgaacaa cacccgtacc gatatgtacg gtggtagcct gcataaccgt 600

atgcgtttcc tgatggaagt tatccgtaac atcaaacaga aaacctctgt gccgctgtct 660

atccgcctca gcggctgcga cttcgaaccg ggtggtatca ccatcgaaga aaccattgaa 720

gttgctaaag catgcgaacg tatgggcgcg gacgtcatta acatcacctg gggcagccac 780

gccgaagtgg tgaacgcggc tggcctgctg tctccgcatg gcgctaacca cgttgatatg 840

gccaaacgca ttaaagatgc tgttagcatc ccggttatgc tgtgcggtgg tatctacact 900

ccggaaattg gtgaacaact gctggaagat ggtgtttgcg actatgtggg tattggtaaa 960

ccggccctgg ctgacccgtt ttgggcgaaa aaagctgaag aaggccgttc tggtgatatc 1020

cgcccgtgta tcggttgcgg tgttggctgc catgaccgtg gcatgctgtc tggtggtatg 1080

gtgcagtgcg cggttaaccc aaccctgtac cagttcgacc gtgaatattt tcctaaaacc 1140

gatcgtccga aaaaagttgc gatcatcggc gcaggtccgg cgggcagcgc agcggccctg 1200

accgcagcgg aatgcggcca cgatgtgacc ctgttcgaag gtcgcgaagt gggcggcgtg 1260

ctgaaagaag cgagcgtgcc ggtgtataaa gaagatctgg gcctgctggg caaatactac 1320

cagcgccaga tcgccaaatc taacgttaaa ctggttgaag aaaacgcgac tccggaaacc 1380

atcgcggcag gcgactttga tgcggtgatc attgctaccg gcggtaaagt gcgtgaactg 1440

aacctgccgg gtctggattc tgataacgtg acctacgcaa tggacctgat gaaacagggt 1500

tgccagctgg atgcagacaa agttgttgtg gttggcggcg gcattgtggg cgctgaagcc 1560

gcgctgatcc tggcggaaga tttcggtaaa gacgtgacca tcaccacccg tcaggataac 1620

ttctttgttc cgggtgtgat gggcatcgcg tatatgaccc gcctggcgat ggcaggcgtg 1680

aaaaccaaaa cccgcgcgaa cctggttgaa gtgaaagacg gcaaaccggt gttcagcacc 1740

atgaacggtc tggaaaccat ggatgttgat gcggtggtga tcagcccagg tttcctgccg 1800

accagccaga tgcgtgatga cattgaacag atcgctgacg ttgataccta tgttatcggc 1860

gacgcgaaag cgccgcgtct ggttatggat gcagttcacg aaggctataa aaccgcgatt 1920

aacctgtaa 1929

<210> 14

<211> 1800

<212> DNA

<213> 人工序列

<400> 14

atgtacgatc acctgttcag cgaaattcag atccgtggcc tgaacctgaa gaaccgtgtt 60

gtttttccgg cgatgggtag ccgcttctgt tccgatgatg gttacctgag cgatcagggt 120

atcgattact acaccgctcg cgcgaaaggc ggttgcggtc tgatcgttac cgaagcggtt 180

gcggtttgga aaccgggcag cgtgttccgt atgctgcaga tctctgacga ctcctatatt 240

gcgcaactga agaaactgac cgaatctatc cacgcagcgg gtggtaaagc gtgtatccag 300

ctgtggcagg gcggcctggc tgccagccag accccaggca gcgtgatcgt gatgccttct 360

gacctgccgc tgggcgaagg taaagtgctg ccgggtgcga gcaaagaaac cattcatgaa 420

atcgtcaaag cgttcggcga agctgcaaaa cgcgcggttg aagcaggttt cgactgcgta 480

gaattccacg cggcgcacaa ctattccccg cacagcttcc tgagcccggc gttcaaccgt 540

cgcgatgacg aatacggtgg ttctctggaa aaccgcgcac gttactctat tgaaagcatt 600

cgtgaaattc gcaaaaacat cccggaagat atgccgatct ttatgcgtgt tcctgcaaaa 660

gatgatgaac tgccgggtgg tctgtctatc gaagatgtga tcgaattctg caaaatggcg 720

aaagcggcgg gtgtggatgt tctggatgtg tcccgcggta acatcgttac ttctgccatg 780

cgttatgaag ttccgccgct ggacattccg cgtggtttca acgtggataa cgcggcaacc 840

attcgcaaag cgaccaaaat gccgacgatt gccgttggtc gtatcaacaa cgctgacctg 900

atgtacgatc acctgttcag cgaaattcag atccgtggcc tgaacctgaa gaaccgtgtt 960

gtttttccgg cgatgggtag ccgcttctgt tccgatgatg gttacctgag cgatcagggt 1020

atcgattact acaccgctcg cgcgaaaggc ggttgcggtc tgatcgttac cgaagcggtt 1080

gcggtttgga aaccgggcag cgtgttccgt atgctgcaga tctctgacga ctcctatatt 1140

gcgcaactga agaaactgac cgaatctatc cacgcagcgg gtggtaaagc gtgtatccag 1200

ctgtggcagg gcggcctggc tgccagccag accccaggca gcgtgatcgt gatgccttct 1260

gacctgccgc tgggcgaagg taaagtgctg ccgggtgcga gcaaagaaac cattcatgaa 1320

atcgtcaaag cgttcggcga agctgcaaaa cgcgcggttg aagcaggttt cgactgcgta 1380

gaattccacg cggcgcacaa ctattccccg cacagcttcc tgagcccggc gttcaaccgt 1440

cgcgatgacg aatacggtgg ttctctggaa aaccgcgcac gttactctat tgaaagcatt 1500

cgtgaaattc gcaaaaacat cccggaagat atgccgatct ttatgcgtgt tcctgcaaaa 1560

gatgatgaac tgccgggtgg tctgtctatc gaagatgtga tcgaattctg caaaatggcg 1620

aaagcggcgg gtgtggatgt tctggatgtg tcccgcggta acatcgttac ttctgccatg 1680

cgttatgaag ttccgccgct ggacattccg cgtggtttca acgtggataa cgcggcaacc 1740

attcgcaaag cgaccaaaat gccgacgatt gccgttggtc gtatcaacaa cgctgacctg 1800

<210> 15

<211> 1800

<212> DNA

<213> 人工序列

<400> 15

atggaaaaca aatactaccc gaacctgttc aaaaaaggta aaatcggcaa cgttgttctg 60

aaaaaccgta tcgttcgtaa cagcatgggc acctacctgg gtaacccgga tggcagcgtt 120

tctagccgtc agatccaggc ttatgtggat gctgcagaag gtggcgcggg cctgatcttc 180

atggataacg ctaccccggt tccgatgacc tcctgcggcc tgcgtgcaga cggcgatgag 240

ttcatcgcag gtctgagcca gctggcggat gcgctgaaaa tgcacggtgc tgctgctggc 300

atgcagctgg cgcatccggg tcgtgatgcg ggtttcgttg gttccgcgga tgttatcggc 360

gcgtccccga tcaccttcga accgtggtac gaagcgggcg cgaaactgcc gcgtgaactg 420

tctgttgacg aaattcacga tctggtttct aaattcggtg acgcagcgct gcgttgccgc 480

cacgctggct tcgacgtagt ggaaattcat ggtgccgcag gctgcatccc gaccaatttc 540

ctgtccccgc atgataacca ccgtaccgat atgtacggcg gtagcctgca caaccgcatg 600

cgtctgctga tcgaaatggt tcgcgacatg aagaaaaaat gcggcccgga tttcccgatc 660

ggcgttaaac tgtctaccgt ggactgggaa ccggaaggca tcaccctgga cgaaaccatt 720

gaagtggcga aagcgctgga aaaagaaggc gtggcttata tcaacgtaat ggctggtacc 780

cacgcgaccg cagcgatgga attcctgatg ccgaactgct tcaacgcagc ggactgcaag 840

aaaatcgctg atgcagttaa cattccggtt ttcgttggtc acaacatctt taccccggac 900

atggaaaaca aatactaccc gaacctgttc aaaaaaggta aaatcggcaa cgttgttctg 960

aaaaaccgta tcgttcgtaa cagcatgggc acctacctgg gtaacccgga tggcagcgtt 1020

tctagccgtc agatccaggc ttatgtggat gctgcagaag gtggcgcggg cctgatcttc 1080

atggataacg ctaccccggt tccgatgacc tcctgcggcc tgcgtgcaga cggcgatgag 1140

ttcatcgcag gtctgagcca gctggcggat gcgctgaaaa tgcacggtgc tgctgctggc 1200

atgcagctgg cgcatccggg tcgtgatgcg ggtttcgttg gttccgcgga tgttatcggc 1260

gcgtccccga tcaccttcga accgtggtac gaagcgggcg cgaaactgcc gcgtgaactg 1320

tctgttgacg aaattcacga tctggtttct aaattcggtg acgcagcgct gcgttgccgc 1380

cacgctggct tcgacgtagt ggaaattcat ggtgccgcag gctgcatccc gaccaatttc 1440

ctgtccccgc atgataacca ccgtaccgat atgtacggcg gtagcctgca caaccgcatg 1500

cgtctgctga tcgaaatggt tcgcgacatg aagaaaaaat gcggcccgga tttcccgatc 1560

ggcgttaaac tgtctaccgt ggactgggaa ccggaaggca tcaccctgga cgaaaccatt 1620

gaagtggcga aagcgctgga aaaagaaggc gtggcttata tcaacgtaat ggctggtacc 1680

cacgcgaccg cagcgatgga attcctgatg ccgaactgct tcaacgcagc ggactgcaag 1740

aaaatcgctg atgcagttaa cattccggtt ttcgttggtc acaacatctt taccccggac 1800

<210> 16

<211> 1800

<212> DNA

<213> 人工序列

<400> 16