CN113667662A

CN113667662A - 高性能淀粉脱支酶嵌合体菌种构建及其生产方法和应用

Info

Publication number: CN113667662A
Application number: CN202110756019.9A
Authority: CN
Inventors: 范岩; 牟洋
Original assignee: Nanjing Newfield Technology Co ltd
Current assignee: Nanjing Newfield Technology Co ltd
Priority date: 2021-07-05
Filing date: 2021-07-05
Publication date: 2021-11-19

Abstract

本发明公开了高性能淀粉脱支酶嵌合体菌种构建及其生产方法和应用。所述高性能淀粉脱支酶嵌合体由两个亲代普鲁兰酶氨基酸序列的有效嵌合得到，且所述高性能淀粉脱支酶嵌合体与亲代普鲁兰酶氨基酸序列同源性不低于95％；所述高性能淀粉脱支酶嵌合体是由Bacillus acidopullulyticus的亲代普鲁兰酶的N‑末端1‑476氨基酸残基和Bacillus deramificans的亲代普鲁兰酶C‑末端465‑976氨基酸残基嵌合而获得的。嵌合普鲁兰酶具有较高的糖化速率，这个嵌合普鲁兰酶同时还具有比亲本普鲁兰酶更高的耐热性和热稳定性，适用于糖化反应和淀粉液化。

Description

高性能淀粉脱支酶嵌合体菌种构建及其生产方法和应用

技术领域

本发明涉及淀粉脱支酶技术领域，尤其涉及高性能淀粉脱支酶嵌合体菌种构建及其生产方法和应用。

背景技术

对于现有的普鲁兰酶来说，存在糖化效率低、在酸性高温条件下的酶活低等缺点。因此，为适应工业化生产的需要，亟需解决普鲁兰酶的糖化效率和在酸性高温条件下(特别是当温度达到60℃及pH值低于4.5时)的酶活问题。

发明内容

基于背景技术存在的技术问题，本发明提出了高性能淀粉脱支酶嵌合体菌种构建及其生产方法和应用，与来自芽孢杆菌的母普鲁兰酶相比提高了糖化效率，能适应工业化生产的需求，本发明的嵌合普鲁兰酶的酶活及其他性质与亲本相比获得了提高。

本发明提出的高性能淀粉脱支酶嵌合体，所述高性能淀粉脱支酶嵌合体由两个亲代普鲁兰酶氨基酸序列的有效嵌合得到，且所述高性能淀粉脱支酶嵌合体与亲代普鲁兰酶氨基酸序列同源性不低于95％。

优选地，两所述亲本普鲁兰酶为Bacillus acidopullulyticus普鲁兰酶和/或Bacillus deramificans普鲁兰酶。

优选地，所述高性能淀粉脱支酶嵌合体是由Bacillus acidopullulyticus的亲代普鲁兰酶的N-末端1-476氨基酸残基和Bacillus deramificans的亲代普鲁兰酶C-末端465-976氨基酸残基嵌合而获得的。

本发明提出的编码上述高性能淀粉脱支酶嵌合体的表达载体，由一个合成的多聚核苷酸组成。

优选地，所述表达载体包括由一启动子序列、一核糖体结合位点、高性能淀粉脱支酶嵌合体编码基因序列、一终止子序列组成的表达组件。

优选地，所述表达载体还包括用于指导高性能淀粉脱支酶嵌合体分泌的信号序列，信号序列位于密码子的上游。

本发明提出的重组的微生物宿主细胞，包含上述所述的表达载体。

优选地，所述表达载体的高性能淀粉脱支酶嵌合体编码基因序列被整合到重组宿主细胞的染色体上。

本发明提出的生产高性能淀粉脱支酶嵌合体的方法，方法步骤如下：

S1：培养上述的重组的微生物宿主细胞，用于表达高性能淀粉脱支酶嵌合体；

S2：在培养的重组的微生物宿主细胞或其上清液中得到高性能淀粉脱支酶嵌合体。

本发明提出的组合物，所述组合物包括上述的高性能淀粉脱支酶嵌合体。

优选地，所述组合物还包括淀粉酶。

本发明提出的上述组合物在催化碳水化合物糖化中的应用。

优选地，所述碳水化合物至少含有一个α-1,6糖苷键。

本发明提出的催化碳水化合物的方法，采用碳水化合物和上述的组合物进行反应。

优选地，所述碳水化合物为淀粉，支链淀粉，右旋糖苷，麦芽糊精，普鲁兰糖或肝糖。

优选地，所述反应的条件为：pH为4.0-4.5、温度60-64℃。

有益效果：

本发明的高性能淀粉脱支酶嵌合体由两个亲代普鲁兰酶氨基酸序列的有效嵌合得到，具有更好的pH耐受性、耐热性和热稳定性。此外，本发明中的嵌合普鲁兰酶与黑曲霉来源的糖化酶混合成复合糖化酶，体现出优良的性能，且便于生产操作，可以满足大多数客户利用淀粉原料生产葡萄糖(或及其衍生产品)的需要。在糖化上，在0.300PU/gDS剂量情况下，亲本普鲁兰酶24h的葡萄糖产量94.5％，而本发明制备的高性能淀粉脱支酶嵌合体则在0.150PU/gDS剂量情况下24h的葡萄糖产量即可达到94.6％，从而说明本申请制备的高性能淀粉脱支酶嵌合体在糖化上的应用效果得到显著地提升。

附图说明

图1为本发明提出的pYF-tsDE载体的示意图；

图2为本发明提出的pUC57-KS-erm载体的示意图；

图3为本发明提出的pYF-tsINT-pul载体的示意图。

具体实施方式

(1)pYF-tsDE质粒的构建

pYF-tsDE的示意图如图1所示，其以南京擎科生物科技有限公司合成的pUC57-KS-erm为原料制备，具体方法参照CN107532155A。

(2)蛋白酶缺陷型枯草芽孢杆菌菌株的构建

本发明以枯草芽孢杆菌作为宿主细胞，经处理后直接作为PCR反应的模板，以下引物由Genscript合成，分别用于PCR反应扩增侧翼序列Apr,Npr和SpoIIAC：

扩增Apr基因上游序列的引物为：

pksb-Apr_czF1：GGTATCGATA AGCTTCCTGC AGATCTCTCA GGAGCATTTAACCT

pksb-Apr_R1：GCACCTACTG CAATAGTAAG GAACAGATTG CGCAT

扩增Apr基因下游序列的引物为：

pksb-Apr_F2：ATGCGCAATC TGTTCCTTAC TATTGCAGTA GGTGC

pksb-Apr_czR2：AATATGGCGG CCGCGAATTC AGATCTCTAA TGCTGTCTCG CGT

扩增Npr基因上游序列的引物为：

pksb-Npr_czF1：GGTATCGATA AGCTTCCTGC AGATCTCATC TTCCCCTTGAT

pksb-Npr_R1：CAGTCTTCTG TATCGTTACG CTTTTAATTC GGCT

扩增Npr基因下游序列的引物为：

pksb-Npr_F2：AGCCGAATTAAAAGCGTAAC GATACAGAAG ACTG

pksb-Npr_czR2：TATGGCGGCC GCGAATTCAG ATCTCCTGGC CAGGAGAATC T

扩增SpoIIAC：基因上游序列的引物为：

pksb-Spo_czF1：GGTATCGATA AGCTTCCTGC AGGAACAATC TGAACAGCAG GCACTC

pksb-Spo_R1：TTGTCAAACCATTTTTCTTC GCCCGATGCA GCCGATCTG

扩增SpoIIAC基因下游序列的引物为：

pksb-Spo_F2：CAGATCGGCT GCATCGGGCG AAGAAAAATG GTTTGACAA

pksb-Spo_czR2：ATATGGCGGC CGCGAATTCA GATCTGTTCA TGATGGCAAGACAC

反应条件参照CN107532155A的记载，反应结束后对扩增产物进行检测和纯化。

为了得到在设计位点进行基因替换的菌株，将几个筛选出的单克隆接种到2YT培养基中，30℃培养5-7天(每2天继代一次)。将对红霉素敏感的菌株进行PCR筛选(见SEQ IDNO.7、8、9)。在1％的脱脂牛奶平板上进行蛋白酶缺陷表型验证。

(3)普鲁兰酶生产菌株的构建

普鲁兰酶表达框主要包括：启动子序列(SeqID:NO 10)、核糖体结合位点(Seq ID:NO 11)、嵌合普鲁兰编码基因和终止序列(Seq ID:NO 12)。此外，本发明还在普鲁兰酶编码基因启动子的上游插入一个从枯草芽孢杆菌中筛选的强的天然信号序列(Seq ID:NO 13)，用于增强普鲁兰酶的分泌效率。用assembly master mix试剂盒(New England Biolabs)将完整的普鲁兰酶表达框插入线性化的pYF-tsDE中的BglII位点，并将得到的整合质粒命名为pYF-tsINT-puI(图3)。

(4)普鲁兰酶生产的摇瓶发酵

发酵方法参照CN107532155A，并对发酵后的产物进行SDS-PAGE分析，确定嵌合普鲁兰酶的有效分泌。

(5)普鲁兰酶分步补料发酵工艺

将步骤(3)中得到的-80℃冷冻保存的基因工程芽孢杆菌菌株划线与琼脂斜面上，37℃过夜恢复培养。具体的补料发酵工艺参照CN107532155A。

(6)普鲁兰酶活力测定

酶活测定参照CN107532155A的记载。

(7)嵌合普鲁兰酶的应用

以下所有结果都是基于嵌合的普鲁兰酶(SEQ ID NO.6)，这个嵌合的普鲁兰酶是由Bacillus acidopullulyticus的亲代普鲁兰酶的N-末端1-470氨基酸残基和Bacillusderamificans的亲代普鲁兰酶C-末端469-928氨基酸残基嵌合而获得的。嵌合普鲁兰酶的氨基酸序列的同源性至少达到95％，如95％，96％，97％，98％，99％或者100％等。

嵌合普鲁兰酶糖化测试条件：31％干物质(DS)、充分混匀、pH用50％(w/v)NaOH调节至4.3。分别加入0.3、0.15PU/gDS的普鲁兰酶和葡糖淀粉酶(混合浓度：0.225GU/gDS)和空白标记水。原未经改造的普鲁兰酶(来自Bacillus deramificans，0.300PU/gDS)加入一个摇瓶作为对照组。结果见表1。

表1：在pH4.3时，截短普鲁兰酶和亲本普鲁兰酶糖化应用比较

由表1可知，在添加量减少一半的酶剂量下(0.150PU/gDS)，与亲本葡萄糖产量(94.5-96.4％)相比，嵌合的普鲁兰酶保持甚至增加了葡萄糖产量(94.6-97.8％)，这证实了嵌合普鲁兰酶不仅不会影响普鲁兰酶的活力。重要的是，在48h内达到葡萄糖产量97.8％，说明在实际应用中，嵌合普鲁兰酶只需要较低的剂量就可以带来很好的生产性能，可以帮助客户降低酶制剂使用成本。此外，在开始反应的24h内，嵌合普鲁兰酶催化的糖化作用速率要高于亲本普鲁兰酶(结果未列)。综上所述，嵌合普鲁兰酶造成了更好的结构稳定性和更高的酶活力。

其次，我们通过在低pH条件下的糖化实验测定了嵌合的普鲁兰酶对pH的耐受性。糖化反应条件同上所述，pH为4.0。结果见表2。

表2：在pH4.0时，截短普鲁兰酶和亲本普鲁兰酶糖化应用比较

如表2所示，在pH4.0的条件下，在反应至48h，亲本普鲁兰酶的葡萄糖产量仅为96.1％，低于淀粉工业中所必需的葡萄糖产量最小限度的百分比(96.5％)。而对于嵌合普鲁兰酶来说，其在酸性条件pH4.0的条件下显示出更高的催化活力，在反应时间为48h时，嵌合普鲁兰酶的最终的葡萄糖产量可达97.2％(表2)。

另外，对嵌合普鲁兰酶和亲本普鲁兰酶的耐热性和热稳定性进行测定，结果见表3。

表3：不同温度下的嵌合普鲁兰酶和亲本普鲁兰酶糖化应用比较(pH4.3)

由表3可知，在64℃高温条件下，嵌合普鲁兰酶能够显著的保持持续的较高葡萄糖产量，也即嵌合普鲁兰酶的耐热性和热稳定性相比于亲本普鲁兰酶有显著提高。

最后，还对嵌合普鲁兰酶的糖化反应进行了研究。通过50％(w/v)NaOH调节pH至5.2，其余反应条件均参照CN107532155A的记载。结果见表4。

表4：嵌合普鲁兰酶和原普鲁兰酶麦芽糖产率的应用比较

如表4所示，与原普鲁兰酶比较，嵌合普鲁兰酶在同样的条件下表现更好。麦芽糖产量明显高于亲本普鲁兰酶，这说明嵌合普鲁兰酶在麦芽糖工业生产的催化活度得到了较大程度的改善。

综上所述，依照本发明中的实验结果，嵌合的普鲁兰酶比亲本普鲁兰酶有更好的pH耐受性，耐热性和热稳定性。本发明中嵌合的普鲁兰酶在一定条件下也体现出了一定优势。因此，本发明中的嵌合普鲁兰酶可以应用于淀粉原料的糖化作用过程，尤其是在淀粉加工工业中。

序列表

<110> 南京纽田科技有限公司

<120> 高性能淀粉脱支酶嵌合体菌种构建及其生产方法和应用

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gataacggta catggaaact tcaggtttct ggtaatcttg aaaactggta ttatctatat 1140

caagtcacag tgaatgggac aacacaaacg gcagttgatc catatgcgcg tgctatttct 1200

gtcaatgcaa cacgcggtat gattgtggac ctaaaagcta ccgatcctgc agggtggcag 1260

ggagatcatg aacagacacc tgcgaatcca gtagatgaag tgatttatga agcgcatgta 1320

cgcgattttt cgattgatgc taattcaggt atgaaaaata aagggaagta tttagcgttt 1380

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tttgacttaa tggctctttt aggaaaagac acgatggcaa aaatatcaaa cgagctgcat 1920

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Asp Ser Thr Ser Thr Lys Val Ile Val His Tyr His Arg Phe Asp Ser

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Asn Tyr Thr Asn Trp Asp Val Trp Met Trp Pro Tyr Gln Pro Val Asn

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Gly Asn Gly Ala Ala Tyr Gln Phe Thr Gly Thr Asn Asp Asp Phe Gly

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Ala Val Ala Asp Thr Gln Val Pro Gly Asp Asn Thr Gln Val Gly Leu

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Ile Val Arg Lys Asn Asp Trp Ser Glu Lys Asn Thr Pro Asn Asp Leu

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

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

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

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Leu Ser Met Pro Met Thr Leu Ala Asp Ala Ala Ser Gly Phe Thr Val

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

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Gly Ala Ala Asn Asn Trp Ser Pro Asp Asp Asp His Thr Leu Leu Lys

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

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Val Leu Asn Leu Pro Arg Tyr Asp Tyr Ser Gly Asn Asp Leu Gly Asn

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Val Tyr Ser Lys Asp Ala Thr Ser Phe Arg Val Trp Ala Pro Thr Ala

325 330 335

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

340 345 350

Lys Gln Leu Glu Met Gln Lys Ser Asp Asn Gly Thr Trp Lys Leu Gln

355 360 365

Val Ser Gly Asn Leu Glu Asn Trp Tyr Tyr Leu Tyr Gln Val Thr Val

370 375 380

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

385 390 395 400

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

405 410 415

Ala Gly Trp Gln Gly Asp His Glu Gln Thr Pro Ala Asn Pro Val Asp

420 425 430

Glu Val Ile Tyr Glu Ala His Val Arg Asp Phe Ser Ile Asp Ala Asn

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

Ile Asp Glu Thr Gln Pro Asp Thr Tyr Asn Trp Gly Tyr Asp Pro Arg

500 505 510

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

515 520 525

Ala Arg Ile Thr Glu Leu Lys Gln Leu Ile Gln Ser Leu His Gln Gln

530 535 540

Arg Ile Gly Val Asn Met Asp Val Val Tyr Asn His Thr Phe Asp Val

545 550 555 560

Met Val Ser Asp Phe Asp Lys Ile Val Pro Gln Tyr Tyr Tyr Arg Thr

565 570 575

Asp Ser Asn Gly Asn Tyr Thr Asn Gly Ser Gly Cys Gly Asn Glu Phe

580 585 590

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

595 600 605

Tyr Trp Val Asn Glu Tyr His Val Asp Gly Phe Arg Phe Asp Leu Met

610 615 620

Ala Leu Leu Gly Lys Asp Thr Met Ala Lys Ile Ser Asn Glu Leu His

625 630 635 640

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

645 650 655

Thr Ser Gly Leu Ser Ser Asp Gln Leu Val Thr Lys Gly Gln Gln Lys

660 665 670

Gly Leu Gly Ile Gly Val Phe Asn Asp Asn Ile Arg Asn Gly Leu Asp

675 680 685

Gly Asn Val Phe Asp Lys Thr Ala Gln Gly Phe Ala Thr Gly Asp Pro

690 695 700

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

705 710 715 720

Phe Thr Ser Ala Pro Ser Glu Thr Ile Asn Tyr Val Thr Ser His Asp

725 730 735

Asn Met Thr Leu Trp Asp Lys Ile Leu Ala Ser Asn Pro Ser Asp Thr

740 745 750

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

755 760 765

Thr Ser Gln Gly Val Pro Phe Met Gln Gly Gly Glu Glu Met Leu Arg

770 775 780

Thr Lys Gly Gly Asn Asp Asn Ser Tyr Asn Ala Gly Asp Ser Val Asn

785 790 795 800

Gln Phe Asp Trp Ser Arg Lys Ala Gln Phe Lys Asp Val Phe Asp Tyr

805 810 815

Phe Ser Ser Met Ile His Leu Arg Asn Gln His Pro Ala Phe Arg Met

820 825 830

Thr Thr Ala Asp Gln Ile Lys Gln Asn Leu Thr Phe Leu Glu Ser Pro

835 840 845

Thr Asn Thr Val Ala Phe Glu Leu Lys Asn Tyr Ala Asn His Asp Thr

850 855 860

Trp Lys Asn Ile Ile Val Met Tyr Asn Pro Asn Lys Thr Ser Gln Thr

865 870 875 880

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

885 890 895

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

900 905 910

Ala Ile Ser Thr Leu Ile Leu Lys Gln

915 920

<210> 3

<211> 2787

<212> DNA

<213> Bacillus deramificans(脱支芽孢杆菌)

<400> 3

gatgggaaca cgacaacgat cattgtccac tattttcgcc ctgctggtga ttatcaacct 60

tggagtctat ggatgtggcc aaaagacgga ggtggggctg aatacgattt caatcaaccg 120

gctgactctt ttggagctgt tgcaagtgct gatattccag gaaacccaag tcaggtagga 180

attatcgttc gcactcaaga ttggaccaaa gatgtgagcg ctgaccgcta catagattta 240

agcaaaggaa atgaggtgtg gcttgtagaa ggaaacagcc aaatttttta taatgaaaaa 300

gatgctgagg atgcagctaa acccgctgta agcaacgctt atttagatgc ttcaaaccag 360

gtgctggtta aacttagcca gccgttaact cttggggaag gcgcaagcgg ctttacggtt 420

catgacgaca cagcaaataa ggatattcca gtgacatctg tgaaggatgc aagtcttggt 480

caagatgtaa ccgctgtttt ggcaggtacc ttccaacata tttttggagg ttccgattgg 540

gcacctgata atcacagtac tttattaaaa aaggtgacta acaatctcta tcaattctca 600

ggagatcttc ctgaaggaaa ctaccaatat aaagtggctt taaatgatag ctggaataat 660

ccgagttacc catctgacaa cattaattta acagtccctg ccggcggtgc acacgtcact 720

ttttcgtata ttccgtccac tcatgcagtc tatgacacaa ttaataatcc taatgcggat 780

ttacaagtag aaagcggggt taaaacggat ctcgtgacgg ttactctagg ggaagatcca 840

gatgtgagcc atactctgtc cattcaaaca gatggctatc aggcaaagca ggtgatacct 900

cgtaatgtgc ttaattcatc acagtactac tattcaggag atgatcttgg gaatacctat 960

acacagaaag caacaacctt taaagtctgg gcaccaactt ctactcaagt aaatgttctt 1020

ctttatgaca gtgcaacggg ttctgtaaca aaaatcgtac ctatgacggc atcgggccat 1080

ggtgtgtggg aagcaacggt taatcaaaac cttgaaaatt ggtattacat gtatgaggta 1140

acaggccaag gctctacccg aacggctgtt gatccttatg caactgcgat tgcaccaaat 1200

ggaacgagag gcatgattgt ggacctggct aaaacagatc ctgctggctg gaacagtgat 1260

aaacatatta cgccaaagaa tatagaagat gaggtcatct atgaaatgga tgtccgtgac 1320

ttttccattg accctaattc gggtatgaaa aataaaggga agtatttggc tcttacagaa 1380

aaaggaacaa agggccctga caacgtaaag acggggatag attccttaaa acaacttggg 1440

attactcatg ttcagcttat gcctgttttc gcatctaaca gtgtcgatga aactgatcca 1500

acccaagata attggggtta tgaccctcgc aactatgatg ttcctgaagg gcagtatgct 1560

acaaatgcga atggtaatgc tcgtataaaa gagtttaagg aaatggttct ttcactccat 1620

cgtgaacaca ttggggttaa catggatgtt gtctataatc atacctttgc cacgcaaatc 1680

tctgacttcg ataaaattgt accagaatat tattaccgta cggatgatgc aggtaattat 1740

accaacggat caggtactgg aaatgaaatt gcagccgaaa ggccaatggt tcaaaaattt 1800

attattgatt cccttaagta ttgggtcaat gagtatcata ttgacggctt ccgttttgac 1860

ttaatggcgc tgcttggaaa agacacgatg tccaaagctg cctcggagct tcatgctatt 1920

aatccaggaa ttgcacttta cggtgagcca tggacgggtg gaacctctgc actgccagat 1980

gatcagcttc tgacaaaagg agctcaaaaa ggcatgggag tagcggtgtt taatgacaat 2040

ttacgaaacg cgttggacgg caatgtcttt gattcttccg ctcaaggttt tgcgacaggt 2100

gcaacaggct taactgatgc aattaagaat ggcgttgagg ggagtattaa tgactttacc 2160

tcttcaccag gtgagacaat taactatgtc acaagtcatg ataactacac cctttgggac 2220

aaaatagccc taagcaatcc taatgattcc gaagcggatc ggattaaaat ggatgaactc 2280

gcacaagcag ttgttatgac ctcacaaggc gttccattca tgcaaggcgg ggaagaaatg 2340

cttcgtacaa aaggcggcaa cgacaatagt tataatgcag gcgatgcggt caatgagttt 2400

gattggagca ggaaagctca atatccagat gttttcaact attatagcgg gctaatccac 2460

cttcgtcttg atcacccagc cttccgcatg acgacagcta atgaaatcaa tagccacctc 2520

caattcctaa atagtccaga gaacacagtg gcctatgaat taactgatca tgttaataaa 2580

gacaaatggg gaaatatcat tgttgtttat aacccaaata aaactgtagc aaccatcaat 2640

ttgccgagcg ggaaatgggc aatcaatgct acgagcggta aggtaggaga atccaccctt 2700

ggtcaagcag agggaagtgt ccaagtacca ggtatatcta tgatgatcct tcatcaagag 2760

gtaagcccag accacggtaa aaagtaa 2787

<210> 4

<211> 928

<212> PRT

<213> Bacillus deramificans(脱支芽孢杆菌)

<400> 4

Asp Gly Asn Thr Thr Thr Ile Ile Val His Tyr Phe Arg Pro Ala Gly

1 5 10 15

Asp Tyr Gln Pro Trp Ser Leu Trp Met Trp Pro Lys Asp Gly Gly Gly

20 25 30

Ala Glu Tyr Asp Phe Asn Gln Pro Ala Asp Ser Phe Gly Ala Val Ala

35 40 45

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

50 55 60

Thr Gln Asp Trp Thr Lys Asp Val Ser Ala Asp Arg Tyr Ile Asp Leu

65 70 75 80

Ser Lys Gly Asn Glu Val Trp Leu Val Glu Gly Asn Ser Gln Ile Phe

85 90 95

Tyr Asn Glu Lys Asp Ala Glu Asp Ala Ala Lys Pro Ala Val Ser Asn

100 105 110

Ala Tyr Leu Asp Ala Ser Asn Gln Val Leu Val Lys Leu Ser Gln Pro

115 120 125

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

130 135 140

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

145 150 155 160

Gln Asp Val Thr Ala Val Leu Ala Gly Thr Phe Gln His Ile Phe Gly

165 170 175

Gly Ser Asp Trp Ala Pro Asp Asn His Ser Thr Leu Leu Lys Lys Val

180 185 190

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

195 200 205

Gln Tyr Lys Val Ala Leu Asn Asp Ser Trp Asn Asn Pro Ser Tyr Pro

210 215 220

Ser Asp Asn Ile Asn Leu Thr Val Pro Ala Gly Gly Ala His Val Thr

225 230 235 240

Phe Ser Tyr Ile Pro Ser Thr His Ala Val Tyr Asp Thr Ile Asn Asn

245 250 255

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

260 265 270

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

275 280 285

Gln Thr Asp Gly Tyr Gln Ala Lys Gln Val Ile Pro Arg Asn Val Leu

290 295 300

Asn Ser Ser Gln Tyr Tyr Tyr Ser Gly Asp Asp Leu Gly Asn Thr Tyr

305 310 315 320

Thr Gln Lys Ala Thr Thr Phe Lys Val Trp Ala Pro Thr Ser Thr Gln

325 330 335

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

340 345 350

Val Pro Met Thr Ala Ser Gly His Gly Val Trp Glu Ala Thr Val Asn

355 360 365

Gln Asn Leu Glu Asn Trp Tyr Tyr Met Tyr Glu Val Thr Gly Gln Gly

370 375 380

Ser Thr Arg Thr Ala Val Asp Pro Tyr Ala Thr Ala Ile Ala Pro Asn

385 390 395 400

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

405 410 415

Trp Asn Ser Asp Lys His Ile Thr Pro Lys Asn Ile Glu Asp Glu Val

420 425 430

Ile Tyr Glu Met Asp Val Arg Asp Phe Ser Ile Asp Pro Asn Ser Gly

435 440 445

Met Lys Asn Lys Gly Lys Tyr Leu Ala Leu Thr Glu Lys Gly Thr Lys

450 455 460

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

465 470 475 480

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

485 490 495

Glu Thr Asp Pro Thr Gln Asp Asn Trp Gly Tyr Asp Pro Arg Asn Tyr

500 505 510

Asp Val Pro Glu Gly Gln Tyr Ala Thr Asn Ala Asn Gly Asn Ala Arg

515 520 525

Ile Lys Glu Phe Lys Glu Met Val Leu Ser Leu His Arg Glu His Ile

530 535 540

Gly Val Asn Met Asp Val Val Tyr Asn His Thr Phe Ala Thr Gln Ile

545 550 555 560

Ser Asp Phe Asp Lys Ile Val Pro Glu Tyr Tyr Tyr Arg Thr Asp Asp

565 570 575

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

580 585 590

Glu Arg Pro Met Val Gln Lys Phe Ile Ile Asp Ser Leu Lys Tyr Trp

595 600 605

Val Asn Glu Tyr His Ile Asp Gly Phe Arg Phe Asp Leu Met Ala Leu

610 615 620

Leu Gly Lys Asp Thr Met Ser Lys Ala Ala Ser Glu Leu His Ala Ile

625 630 635 640

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

645 650 655

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

660 665 670

Gly Val Ala Val Phe Asn Asp Asn Leu Arg Asn Ala Leu Asp Gly Asn

675 680 685

Val Phe Asp Ser Ser Ala Gln Gly Phe Ala Thr Gly Ala Thr Gly Leu

690 695 700

Thr Asp Ala Ile Lys Asn Gly Val Glu Gly Ser Ile Asn Asp Phe Thr

705 710 715 720

Ser Ser Pro Gly Glu Thr Ile Asn Tyr Val Thr Ser His Asp Asn Tyr

725 730 735

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

740 745 750

Asp Arg Ile Lys Met Asp Glu Leu Ala Gln Ala Val Val Met Thr Ser

755 760 765

Gln Gly Val Pro Phe Met Gln Gly Gly Glu Glu Met Leu Arg Thr Lys

770 775 780

Gly Gly Asn Asp Asn Ser Tyr Asn Ala Gly Asp Ala Val Asn Glu Phe

785 790 795 800

Asp Trp Ser Arg Lys Ala Gln Tyr Pro Asp Val Phe Asn Tyr Tyr Ser

805 810 815

Gly Leu Ile His Leu Arg Leu Asp His Pro Ala Phe Arg Met Thr Thr

820 825 830

Ala Asn Glu Ile Asn Ser His Leu Gln Phe Leu Asn Ser Pro Glu Asn

835 840 845

Thr Val Ala Tyr Glu Leu Thr Asp His Val Asn Lys Asp Lys Trp Gly

850 855 860

Asn Ile Ile Val Val Tyr Asn Pro Asn Lys Thr Val Ala Thr Ile Asn

865 870 875 880

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

885 890 895

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

900 905 910

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

915 920 925

<210> 5

<211> 2793

<212> DNA

<213> Debranching enzyme chimera(脱支酶嵌合体)

<400> 5

gattctactt cgactaaagt tattgttcat tatcatcgtt ttgattccaa ctatacgaat 60

tgggacgtct ggatgtggcc ttatcagcct gttaatggta atggagcagc ttaccaattc 120

actggtacaa atgatgattt tggcgctgtt gcagatacgc aagtgcctgg agataataca 180

caagttggtt tgattgttcg taaaaatgat tggagcgaga aaaatacacc aaacgatctc 240

catattgacc ttgcaaaagg ccatgaagta tggattgtac aaggggatcc aactatttat 300

tacaatctga gcgacgcaca ggctgccgca ataccatctg tttcaaatgc ctatcttgat 360

gatgaaaaaa cagtactagc aaagctaagt atgccgatga cgctggcgga tgctgcaagc 420

ggctttacgg ttatagataa aaccacaggt gaaaaaatcc ctgtcacctc tgctgtatcc 480

gcaaatccgg taactgccgt tcttgttgga gatttacaac aggctttggg agcagcgaat 540

aattggtcac cagatgatga tcacacactg ctaaaaaaga taaatccaaa cctttaccaa 600

ttatcgggga cacttccagc tggtacatac caatataaga tagccttgga ccattcttgg 660

aatacctcct atccaggtaa caatgtaagt cttactgttc ctcagggagg ggaaaaggtt 720

acctttacct atattccatc taccaaccag gtattcgata gcgtcaatca tcctaaccaa 780

gcattcccta catcctcagc aggggtccag acaaatttag tccaattgac tttagcgagt 840

gcaccggatg tcacccataa tttagatgta gcagcagacg gttacaaagc gcacaatatt 900

ttaccaagga atgttttaaa tctgccgcgg tatgattata gtggaaatga tttgggtaat 960

gtttattcaa aggatgcaac atccttccgg gtatgggctc caacagcttc gaatgtccag 1020

ttgcttttat acaatagtga gaaaggttca ataactaaac agcttgaaat gcaaaagagt 1080

gataacggta catggaaact tcaggtttct ggtaatcttg aaaactggta ttatctatat 1140

caagtcacag tgaatgggac aacacaaacg gcagttgatc catatgcgcg tgctatttct 1200

gtcaatgcaa cacgcggtat gattgtggac ctaaaagcta ccgatcctgc agggtggcag 1260

ggagatcatg aacagacacc tgcgaatcca gtagatgaag tgatttatga agcgcatgta 1320

cgcgattttt cgattgatgc taattcaggt atgaaaaata aagggaagta tttagcgttt 1380

acagagcatg gaacaaaagg accggatcat gtaaagacgg ggatagattc cttaaaacaa 1440

cttgggatta ctcatgttca gcttatgcct gttttcgcat ctaacagtgt cgatgaaact 1500

gatccaaccc aagataattg gggttatgac cctcgcaact atgatgttcc tgaagggcag 1560

tatgctacaa atgcgaatgg taatgctcgt ataaaagagt ttaaggaaat ggttctttca 1620

ctccatcgtg aacacattgg ggttaacatg gatgttgtct ataatcatac ctttgccacg 1680

caaatctctg acttcgataa aattgtacca gaatattatt accgtacgga tgatgcaggt 1740

aattatacca acggatcagg tactggaaat gaaattgcag ccgaaaggcc aatggttcaa 1800

aaatttatta ttgattccct taagtattgg gtcaatgagt atcatattga cggcttccgt 1860

tttgacttaa tggcgctgct tggaaaagac acgatgtcca aagctgcctc ggagcttcat 1920

gctattaatc caggaattgc actttacggt gagccatgga cgggtggaac ctctgcactg 1980

ccagatgatc agcttctgac aaaaggagct caaaaaggca tgggagtagc ggtgtttaat 2040

gacaatttac gaaacgcgtt ggacggcaat gtctttgatt cttccgctca aggttttgcg 2100

acaggtgcaa caggcttaac tgatgcaatt aagaatggcg ttgaggggag tattaatgac 2160

tttacctctt caccaggtga gacaattaac tatgtcacaa gtcatgataa ctacaccctt 2220

tgggacaaaa tagccctaag caatcctaat gattccgaag cggatcggat taaaatggat 2280

gaactcgcac aagcagttgt tatgacctca caaggcgttc cattcatgca aggcggggaa 2340

gaaatgcttc gtacaaaagg cggcaacgac aatagttata atgcaggcga tgcggtcaat 2400

gagtttgatt ggagcaggaa agctcaatat ccagatgttt tcaactatta tagcgggcta 2460

atccaccttc gtcttgatca cccagccttc cgcatgacga cagctaatga aatcaatagc 2520

cacctccaat tcctaaatag tccagagaac acagtggcct atgaattaac tgatcatgtt 2580

aataaagaca aatggggaaa tatcattgtt gtttataacc caaataaaac tgtagcaacc 2640

atcaatttgc cgagcgggaa atgggcaatc aatgctacga gcggtaaggt aggagaatcc 2700

acccttggtc aagcagaggg aagtgtccaa gtaccaggta tatctatgat gatccttcat 2760

caagaggtaa gcccagacca cggtaaaaag taa 2793

<210> 6

<211> 930

<212> PRT

<213> Debranching enzyme chimera(脱支酶嵌合体)

<400> 6

Asp Ser Thr Ser Thr Lys Val Ile Val His Tyr His Arg Phe Asp Ser

1 5 10 15

Asn Tyr Thr Asn Trp Asp Val Trp Met Trp Pro Tyr Gln Pro Val Asn

20 25 30

Gly Asn Gly Ala Ala Tyr Gln Phe Thr Gly Thr Asn Asp Asp Phe Gly

35 40 45

Ala Val Ala Asp Thr Gln Val Pro Gly Asp Asn Thr Gln Val Gly Leu

50 55 60

Ile Val Arg Lys Asn Asp Trp Ser Glu Lys Asn Thr Pro Asn Asp Leu

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Leu Ser Met Pro Met Thr Leu Ala Asp Ala Ala Ser Gly Phe Thr Val

130 135 140

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

145 150 155 160

Ala Asn Pro Val Thr Ala Val Leu Val Gly Asp Leu Gln Gln Ala Leu

165 170 175

Gly Ala Ala Asn Asn Trp Ser Pro Asp Asp Asp His Thr Leu Leu Lys

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

Thr Phe Thr Tyr Ile Pro Ser Thr Asn Gln Val Phe Asp Ser Val Asn

245 250 255

His Pro Asn Gln Ala Phe Pro Thr Ser Ser Ala Gly Val Gln Thr Asn

260 265 270

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

275 280 285

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

290 295 300

Val Leu Asn Leu Pro Arg Tyr Asp Tyr Ser Gly Asn Asp Leu Gly Asn

305 310 315 320

Val Tyr Ser Lys Asp Ala Thr Ser Phe Arg Val Trp Ala Pro Thr Ala

325 330 335

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

340 345 350

Lys Gln Leu Glu Met Gln Lys Ser Asp Asn Gly Thr Trp Lys Leu Gln

355 360 365

Val Ser Gly Asn Leu Glu Asn Trp Tyr Tyr Leu Tyr Gln Val Thr Val

370 375 380

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

385 390 395 400

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

405 410 415

Ala Gly Trp Gln Gly Asp His Glu Gln Thr Pro Ala Asn Pro Val Asp

420 425 430

Glu Val Ile Tyr Glu Ala His Val Arg Asp Phe Ser Ile Asp Ala Asn

435 440 445

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

450 455 460

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

465 470 475 480

Leu Gly Ile Thr His Val Gln Leu Met Pro Val Phe Ala Ser Asn Ser

485 490 495

Val Asp Glu Thr Asp Pro Thr Gln Asp Asn Trp Gly Tyr Asp Pro Arg

500 505 510

Asn Tyr Asp Val Pro Glu Gly Gln Tyr Ala Thr Asn Ala Asn Gly Asn

515 520 525

Ala Arg Ile Lys Glu Phe Lys Glu Met Val Leu Ser Leu His Arg Glu

530 535 540

His Ile Gly Val Asn Met Asp Val Val Tyr Asn His Thr Phe Ala Thr

545 550 555 560

Gln Ile Ser Asp Phe Asp Lys Ile Val Pro Glu Tyr Tyr Tyr Arg Thr

565 570 575

Asp Asp Ala Gly Asn Tyr Thr Asn Gly Ser Gly Thr Gly Asn Glu Ile

580 585 590

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

595 600 605

Tyr Trp Val Asn Glu Tyr His Ile Asp Gly Phe Arg Phe Asp Leu Met

610 615 620

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

625 630 635 640

Ala Ile Asn Pro Gly Ile Ala Leu Tyr Gly Glu Pro Trp Thr Gly Gly

645 650 655

Thr Ser Ala Leu Pro Asp Asp Gln Leu Leu Thr Lys Gly Ala Gln Lys

660 665 670

Gly Met Gly Val Ala Val Phe Asn Asp Asn Leu Arg Asn Ala Leu Asp

675 680 685

Gly Asn Val Phe Asp Ser Ser Ala Gln Gly Phe Ala Thr Gly Ala Thr

690 695 700

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

705 710 715 720

Phe Thr Ser Ser Pro Gly Glu Thr Ile Asn Tyr Val Thr Ser His Asp

725 730 735

Asn Tyr Thr Leu Trp Asp Lys Ile Ala Leu Ser Asn Pro Asn Asp Ser

740 745 750

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

755 760 765

Thr Ser Gln Gly Val Pro Phe Met Gln Gly Gly Glu Glu Met Leu Arg

770 775 780

Thr Lys Gly Gly Asn Asp Asn Ser Tyr Asn Ala Gly Asp Ala Val Asn

785 790 795 800

Glu Phe Asp Trp Ser Arg Lys Ala Gln Tyr Pro Asp Val Phe Asn Tyr

805 810 815

Tyr Ser Gly Leu Ile His Leu Arg Leu Asp His Pro Ala Phe Arg Met

820 825 830

Thr Thr Ala Asn Glu Ile Asn Ser His Leu Gln Phe Leu Asn Ser Pro

835 840 845

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

850 855 860

Trp Gly Asn Ile Ile Val Val Tyr Asn Pro Asn Lys Thr Val Ala Thr

865 870 875 880

Ile Asn Leu Pro Ser Gly Lys Trp Ala Ile Asn Ala Thr Ser Gly Lys

885 890 895

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

900 905 910

Gly Ile Ser Met Met Ile Leu His Gln Glu Val Ser Pro Asp His Gly

915 920 925

Lys Lys

930

<210> 7

<211> 1590

<212> DNA

<213> Bacillus subtilis(枯草芽孢杆菌切出AprE基因后的序列)

<400> 7

ttttttcatt ctatcccttt tctgtaaagt ttatttttca gaatactttt atcatcatgc 60

tttgaaaaaa tatcacgata atatccattg ttctcacgga agcacacgca ggtcatttga 120

acgaattttt tcgacaggaa tttgccggga ctcaggagca tttaacctaa aaaagcatga 180

catttcagca taatgaacat ttactcatgt ctattttcgt tcttttctgt atgaaaatag 240

ttatttcgag tctctacgga aatagcgaga gatgatatac ctaaatagag ataaaatcat 300

ctcaaaaaaa tgggtctact aaaatattat tccatctatt acaataaatt cacagaatag 360

tcttttaagt aagtctactc tgaatttttt taaaaggaga gggtaaagag tgagaagcaa 420

aaaattgtgg atcagcttgt tgtttgcgtt aacgttaatc tttacgatgg cgttcagcaa 480

catgtctgcg caggctgccg gaaaaagcag tacagaaaag aaatacattg tcggatttaa 540

acagacaatg agtgccatga gttccgccaa gaaaaaggat gttatttctg aaaaaggcgg 600

aaaggttcaa aagcaattta agtatgttaa cgcggccgca gcaacattgg atgaaaaagc 660

tgtaaaagaa ttgaaaaaag atccgagcgt tgcatatgtg gaagaagatc atattgcaca 720

tgaatatgcg caatctgttc ctactattgc agtaggtgcg gtaaacagca gcaaccaaag 780

agcttcattc tccagcgcag gttctgagct tgatgtgatg gctcctggcg tgtccatcca 840

aagcacactt cctggaggca cttacggcgc ttataacgga acgtccatgg cgactcctca 900

cgttgccgga gcagcagcgt taattctttc taagcacccg acttggacaa acgcgcaagt 960

ccgtgatcgt ttagaaagca ctgcaacata tcttggaaac tctttctact atggaaaagg 1020

gttaatcaac gtacaagcag ctgcacaata atagtaaaaa gaagcaggtt cctccatacc 1080

tgcttctttt tatttgtcag catcctgatg ttccggcgca ttctcttctt tctccgcatg 1140

ttgaatccgt tccatgatcg acggatggct gcctctgaaa atcttcacaa gcaccggagg 1200

atcaacctgg ctcagccccg tcacggccaa atcctgaaac gttttaacag cggcttctct 1260

gttctctgtc aactcgatcc catactggtc agccttattc tcctgataac gcgagacagc 1320

attagaaaaa ggcgtaaccg caaagctcaa aacagaaaac aaaagcaata acagcggaag 1380

tgccgcaaga tcatgccgcc cttctaaatg aaacatgctg cgggttaggc gaaccgtccg 1440

cttgtaaagc ttatcaatga cataaaatcc ggcgagcgac acgagcaaat agccagccag 1500

accgatgtaa acgtgcttca tgacataatg gcccatttcg tggcccataa taaacagaat 1560

ttctgaatcg tcaagtttgt tcagcgtcgt 1590

<210> 8

<211> 1705

<212> DNA

<213> Bacillus subtilis(枯草芽孢杆菌切出spoIIAC基因后的序列)

<400> 8

gctcggggct tggcgttatt ttaggaagat acaagcaaat taagcaaatt ggcggagaaa 60

tggttgtttg cgctatctct cctgcggtga agcgattgtt tgatatgtcg ggtctgttta 120

aaattatccg atttgaacaa tctgaacagc aggcactcct gacactgggg gtggcatcat 180

gaaaaatgaa atgcaccttg agttttctgc cctcagtcag aatgaatcgt tcgcccgtgt 240

gacagttgct tcatttatag ctcagctgga cccgacaatg gatgaactga ctgaaatcaa 300

aacagtcgtg tcagaggctg tcacgaatgc gattatccat ggatatgaag agaactgtga 360

agggaaagtt tacatttcag tgacgctgga agatcatgtc gtatatatga ctattcgtga 420

tgaaggctta ggcattacag atcttgaaga agcccgtcag cctctattta cgactaagcc 480

tgagcttgag cgctctggaa tgggctttac cattatggaa aatttcatgg atgatgtcag 540

tatcgattca tcgcctgaaa tgggaacaac gattcgctta acaaagcact tatcaaaaag 600

caaagcgctt tgtaattaag gagatttgtt atggatgtgg aggttaagaa aaacggcaaa 660

aacgctcagc tgaaggatca tgaagtaaag gaattaatca aacaaagcca aaatggcgac 720

cagcaggcaa gagacctcct catagaaaaa aacatgcgtc ttgtttggtc tgtcgtacag 780

cggtttttaa acagaggata tgagcctgac gatctcttcc agatcggctg catcgggcga 840

agaaaaatgg tttgacaaaa ttgcgctgaa agaagcgatc agcgatttgg aggaaaggga 900

aaaactaatc gtctatctca gatattataa agaccagaca cagtccgagg tggctgagcg 960

gctcgggatc tctcaggtgc aggtttccag gcttgaaaag aaaatattaa aacagatcaa 1020

ggttcaaatg gatcatacgg atggctagtc tgcagtgcag gctagctttt ttgtgcaaaa 1080

gcgtggtaat ttatggtctt ttcgagcgga tgaatgagaa caaaatcgaa ccacatacta 1140

catatataac caccgaaaga tggtgatcaa tgatggaacg acgaatattt atccggcttc 1200

gccaccgagt gctggcacat ccaggggata ttattaccgt tggagatgcc gcgcaaatag 1260

aagggcagct tcagctgaaa aagaaacttt cggctatgcc gctttatcag gtgagcgaaa 1320

aagataaaaa tatcgtaatt ctggatatca tacaagtcct cagagccatt catttacaag 1380

acccgacaat tgatgttcaa accgtaggcg gagcagaaac cattgttgaa attcagtatc 1440

gaaagcgaaa tttatcaacg gttctattta tcggtgtctg gctgcttctg tttattggat 1500

cgtgtcttgc catcatgaac tttcatgagg atgtaagcat gagagatgtt catatcgcac 1560

tatatgaaat cataaccgga gagaggaatg actatccata tttgcttcaa atcccataca 1620

gcatcggttt gggactgggg atgatcgtgt tttttaacca catatttaaa aagcgcctaa 1680

atgaagagcc cagcccgctg gaggt 1705

<210> 9

<211> 1596

<212> DNA

<213> Bacillus subtilis(枯草芽孢杆菌切出spoIIAC基因后的序列)

<400> 9

ttgtctgctt aatataaaat aacgttcgaa atgcaataca taatgactga ataactccaa 60

cacgaacaac aatcctttac ttcttattaa ggcctcattc ggttagacag cggacttttc 120

aaaaagtttc aagatgaaac aaaaatatct catcttcccc ttgatatgta aaaaacataa 180

ctcttgaatg aaccaccaca tgacacttga ctcatcttga tattattcaa caaaaacaaa 240

cacaggacaa tactatcaat tttgtctagt tatgttagtt tttgttgagt attccagaat 300

gctagtttaa tataacaata taaagttttc agtattttca aaaaggggga tttattgtgg 360

gtttaggtaa gaaattgtct gttgctgtcg ctgcttcgtt tatgagttta tcaatcagcc 420

tgccaggtgt tcaggctgct gaaggtcatc agcttaaaga gaatcaaaca aatttcctct 480

ccaaaaacgc gattgcgcaa tcagaactct ctgcaccaaa tgacaaggct gtcaagcagt 540

ttttgaaaaa gaacagcaac atttttaaag gtgacccttc caaaaggctg aagcttgttg 600

aaagcacgac tgatgccctt ggatacaagc actttcgata tgcgcctgtc gttaacggag 660

tgccaattaa agattcgcaa gtgatcgttc acgtcgataa atccgataat gtctatgcgg 720

tcaatggtga attacacaat caatctgctg caaaaacaga taacagccaa aaagtctctt 780

ctgaaaaagc gctggcactc gctttcaaag ctatcggcaa atcaccagac gctgtttcta 840

acggagcggc caaaaacagc aataaagccg aattaaaagc gtaacgatac agaagactgg 900

gacatcggtg aagacattac ggtcagccag cctgctcttc gcagcctgtc caaccctaca 960

aaatacaacc agcctgacaa ttacgccaat taccgaaacc ttccaaacac agatgaaggc 1020

gattatggcg gtgtacacac aaacagcgga attccaaaca aagccgctta caacaccatc 1080

acaaaacttg gtgtatctaa atcacagcaa atctattacc gtgcgttaac aacgtacctc 1140

acgccttctt ccacgttcaa agatgccaag gcagctctca ttcagtctgc ccgtgacctc 1200

tacggctcaa ctgatgccgc taaagttgaa gcagcctgga atgctgttgg attgtaatat 1260

taggaaaagc ctgagatccc tcaggctttt attgttacat atcttgattt ctctctcagc 1320

tgaaacgacg aaaagatgct gccatgagac agaaaaccgc tcctgatttg cataaagagg 1380

gatgcagccg caagtgcgca ttttataaaa gctaatgatt cagtccacat aattgataga 1440

cgaattctgc tacaggtcac gtggctatgt gaaggatcgc gcgtccagtt aagagcaaaa 1500

acattgacaa aaaaatttat ttatgctaaa atttactatt aatatatttg tatgtataat 1560

aagattctcc tggccagggg aatcttattt tttgtg 1596

<210> 10

<211> 800

<212> DNA

<213> Promoter(启动子序列)

<400> 10

tggctgaaga agtggatcga ttgtttgaga aaagaagaag accataaaaa taccttgtct 60

gtcatcagac agggtatttt ttatgctgtc cagactgtcc gctgtgtaaa aaataggaat 120

aaaggggggt tgacattatt ttactgatat gtataatata atttgtataa gaaaatgaga 180

gctctcgaaa cgtaagatga aaccttagat aaaagtgctt tttttgttgc aattgaagaa 240

ttattaatgt taagcttaat taaagataat atctttgaat tgtaacgccc ctcaaaagta 300

agaactacaa aaaaagaata cgttatatag aaatatgttt gaaccttctt cagattacaa 360

atatattcgg acggactcta cctcaaatgc ttatctaact atagaatgac atacaagcac 420

aaccttgaaa atttgaaaat ataactacca atgaacttgt tcatgtgaat tatcgctgta 480

tttaattttc tcaattcaat atataatatg ccaatacatt gttacaagta gaaattaaga 540

cacccttgat agccttacta tacctaacat gatgtagtat taaatgaata tgtaaatata 600

tttatgataa gaagcgactt atttataatc attacatatt tttctattgg aatgattaag 660

attccaatag aatagtgtat aaattattta tcttgaaagg agggatgcct aaaaacgaag 720

aacattaaaa acatatattt gcaccgtcta atggatttat gaaaaatcat tttatcagtt 780

tgaaaattat gtattatgat 800

<210> 11

<211> 7

<212> DNA

<213> Ribosome binding site(核糖体结合位点序列)

<400> 11

aagaaag 7

<210> 12

<211> 227

<212> DNA

<213> Terminator(终止序列)

<400> 12

tcaataataa taacgctgtg tgctttaagc acacagcgtt ttttagtgtg tatgaatcga 60

gatcctgagc gccggtcgct accattacca gttggtctgg tgtcaaaaat aataataacc 120

gggcaggcca tgtctgcccg tatttcgcgt aaggaaatcc attatgtact atttcgatca 180

gaccagtttt taatttgtgt gtttccatgt gtccagtttg gcgcgcc 227

<210> 13

<211> 99

<212> DNA

<213> Signal peptide(信号肽序列)

<400> 13

atgttgatca acaaaagcaa aaagtttttc gttttttctt tcatttttgt tatgatgctg 60

agcctctcat ttgtgaatgg ggaagttgca aaagccgca 99

Claims

1.高性能淀粉脱支酶嵌合体，其特征在于，所述高性能淀粉脱支酶嵌合体由两个亲代普鲁兰酶氨基酸序列的有效嵌合得到，且所述高性能淀粉脱支酶嵌合体与亲代普鲁兰酶氨基酸序列同源性不低于95％；

优选地，两所述亲本普鲁兰酶为Bacillus acidopullulyticus普鲁兰酶和/或Bacillus deramificans普鲁兰酶；

2.编码权利要求1所述的高性能淀粉脱支酶嵌合体的表达载体，其特征在于，由一个合成的多聚核苷酸组成；

优选地，所述表达载体包括由一启动子序列、一核糖体结合位点、高性能淀粉脱支酶嵌合体编码基因序列、一终止子序列组成的表达组件；

3.一种重组的微生物宿主细胞，其特征在于，包含如权利要求2所述的表达载体；

4.一种生产高性能淀粉脱支酶嵌合体的方法，其特征在于，方法步骤如下：

S1：培养如权利要求3所述的重组的微生物宿主细胞，用于表达高性能淀粉脱支酶嵌合体；

5.一种组合物，其特征在于，所述组合物包括如权利要求1所述的高性能淀粉脱支酶嵌合体；

优选地，所述组合物还包括淀粉酶。

6.一种如权利要求5所述的组合物在催化碳水化合物糖化中的应用。

7.根据权利要求6所述的组合物在催化碳水化合物糖化中的应用，其特征在于，所述碳水化合物至少含有一个α-1,6糖苷键。

8.一种催化碳水化合物的方法，其特征在于：采用碳水化合物和如权利要求5所述的组合物进行反应。

9.根据权利要求7所述的催化碳水化合物的方法，其特征在于，所述碳水化合物为淀粉，支链淀粉，右旋糖苷，麦芽糊精，普鲁兰糖或肝糖。

10.根据权利要求7所述的催化碳水化合物的方法，其特征在于，所述反应的条件为：pH为4.0-4.5、温度60-64℃。