CN112574977B - 一种低聚半乳糖生产专用酶及其制备与应用 - Google Patents

一种低聚半乳糖生产专用酶及其制备与应用 Download PDF

Info

Publication number
CN112574977B
CN112574977B CN202011051056.1A CN202011051056A CN112574977B CN 112574977 B CN112574977 B CN 112574977B CN 202011051056 A CN202011051056 A CN 202011051056A CN 112574977 B CN112574977 B CN 112574977B
Authority
CN
China
Prior art keywords
val
lys
ala
thr
gly
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN202011051056.1A
Other languages
English (en)
Other versions
CN112574977A (zh
Inventor
王正祥
牛丹丹
田康明
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Senda (Tianjin) Biotechnology Co.,Ltd.
Original Assignee
Tianjin University of Science and Technology
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Tianjin University of Science and Technology filed Critical Tianjin University of Science and Technology
Priority to CN202210851698.2A priority Critical patent/CN115927257A/zh
Priority to CN202011051056.1A priority patent/CN112574977B/zh
Priority to CN202210851915.8A priority patent/CN115927252A/zh
Priority to EP20924713.9A priority patent/EP3926043A4/en
Priority to PCT/CN2020/127693 priority patent/WO2021179652A1/zh
Publication of CN112574977A publication Critical patent/CN112574977A/zh
Priority to US17/476,433 priority patent/US11447760B2/en
Application granted granted Critical
Publication of CN112574977B publication Critical patent/CN112574977B/zh
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/75—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Bacillus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2468—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1) acting on beta-galactose-glycoside bonds, e.g. carrageenases (3.2.1.83; 3.2.1.157); beta-agarase (3.2.1.81)
    • C12N9/2471—Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/12—Disaccharides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01023—Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01108—Lactase (3.2.1.108)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/07—Bacillus
    • C12R2001/09—Bacillus circulans
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/07—Bacillus
    • C12R2001/10—Bacillus licheniformis
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
    • Y02P60/80—Food processing, e.g. use of renewable energies or variable speed drives in handling, conveying or stacking
    • Y02P60/87—Re-use of by-products of food processing for fodder production

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Virology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

本发明属于酶工程技术领域，具体涉及生成低聚半乳糖的乳糖酶及其制备和应用。本发明以两种来源的乳糖酶(来源于环状芽胞杆菌B2301的BglD305和来源于环状芽胞杆菌ATCC 31382的BglD)分子为基础进行分子进化，获得合成低聚半乳糖效率高、表达性能好的乳糖酶新酶分子；并构建乳糖酶高产菌株，能够在深层发酵时高效合成乳糖酶并将酶蛋白分子分泌到培养基中，从发酵上清中直接制备高活性的酶制剂，乳糖酶的表达水平可以达到2208U/mL，有助于降低乳糖酶的发酵制造成本、简化发酵制造工艺并提高乳糖酶酶制剂的质量。

Description

一种低聚半乳糖生产专用酶及其制备与应用
技术领域：
本发明属于酶工程技术领域，具体涉及生成低聚半乳糖的乳糖酶及其制备和应用。
背景技术：
低聚糖又称寡糖类，是指单糖分子通过糖苷键连接的、聚合度为2-10的直链或支链碳水化合物，可简单分为功能性低聚糖和普通低聚糖两大类。其中，功能性低聚糖(Functional oligosaccharides)是指由2-10个相同或不同的单糖以糖苷键聚合而成；具有糖类共有的甜味和感官特性，可直接代替蔗糖作为甜食配料，但不被人体胃酸、胃酶降解，不在小肠吸收，可到达大肠；具有促进人体内的双歧杆菌增殖等生理特性的低聚糖。在功能性低聚糖中，因单糖的异头碳原子(C1或C2)构型使其糖苷键不易被人体肠胃中的水解酶水解消化，又称为非消化性糖。
天然存在的功能性低聚糖，如人乳、牛乳、羊乳等中存在的低聚半乳糖(Galactooligosaccharides，GOS)，是一种重要的益生元，对人类健康发挥了重要作用。上世纪50年代，已有报道利用β-半乳糖苷酶催化乳糖生产低聚半乳糖的工业化技术相关研究，国际上GOS产品于1988年成功上市。
低聚半乳糖的生产，主要以乳糖为原料，利用乳糖酶的转糖基作用在乳糖水解过程中合成含一个葡萄糖或全部为半乳糖分子连接的低聚合度的寡糖，可表示为Gal-(Gal)n-Glc/Gal(n为1-4)。应用于低聚半乳糖生产的乳糖酶(β-半乳糖苷酶的一种，EC3.2.1.23)能够通过其转半乳糖基作用生成GOS，是一种在乳品工业中非常具有商业价值的酶。商业上一般选择黑曲霉(A.niger)、米曲霉(A.oryzae)、乳酸克鲁维酵母(Kluyveromyces lactis)、脆壁克鲁维酵母(K.fragilis)、罗伦特隐球菌(Cryptococcuslaurentii)和环状芽胞杆菌(Bacillus circulans)等菌株，通过深层发酵方法来制备乳糖酶产品。乳糖酶的来源和GOS的生产工艺不同，尽管在酶活组成和转苷活性上均有一定差别，但催化合成的低聚半乳糖主要以β-1,3、β-1,4、β-1,6糖苷键连接，其中以β-1,4糖苷键为主。此外，针对酶的耐高温性能，研究开发出一些新型的耐高温乳糖酶。如嗜热硫矿硫化叶菌(Sulfolobus solfataricus)、直杆糖多孢菌(Saccharopolyspora rectivirgula)、激烈火球菌(Pyrococcus furiosus)和海栖热袍菌(Thermotoga maritima)等微生物来源的乳糖酶，可以在70℃-80℃高温下催化合成GOS。现有形成商品供应的乳糖酶，一般可以乳糖为原料形成5％-50％的低聚半乳糖。环状芽胞杆菌ATCC 31382生产出的乳糖酶(如
Figure BDA0002709561960000011
)，是目前为止合成GOS能力最强的乳糖酶，这个酶在酶制剂产品中有4中不同的存在形式(Song J,Abe K,Imanaka H,Imamura K,Minoda M,Yamaguchi S,and Nakanishi K,Biosci.Biotechnol.Biochem.,2011；75,268-278)，其中，β-Gal-C和β-Gal-D被认为是最有价值用于GOS的生产。此外，从环状芽胞杆菌B2301中鉴定出的乳糖酶，具有在60℃以上的高温条件下催化乳糖形成54.5％的GOS，是目前所有报道的乳糖酶中GOS合成性能最好的乳糖酶(赵继华等，食品与发酵工业，2020)。环状芽胞杆菌B2301乳糖酶编码基因的完整读框大小为5 133bp、编码1 710个氨基酸残基、不含典型细菌信号肽序列、与已有报道的β-半乳糖苷酶的最高一致性为93.6％；此酶在60℃和pH 6.0～6.5下表现出最高催化活性，Zn2+、Fe2 +、Cu2+、EDTA和SDS对重组酶表现出不同程度的抑制作用，催化合成低聚半乳糖的Vmax为2.47g/(L·h)，Km为14.37g/L(田康明等，食品与发酵工业，2020)。
但是，目前用环状芽胞杆菌发酵生产乳糖酶十分不经济，需要很长的发酵时间，通常需要96h-200h；产酶水平也比较低，通常仅能生产出2～5U/mL的乳糖酶(赵继华等，食品与发酵工业，2020)；酶制备技术复杂，乳糖酶大部分酶活存在于细胞内，需要通过复杂的细胞破碎方法将其释放，酶产品质量也因此收到较大的影响。
将环状芽胞杆菌乳糖酶或其突变体的编码基因，在多种宿主细胞进行了表达，以了解乳糖酶的表达水平。例如：在大肠杆菌中进行表达，乳糖酶的表达水平为1～3U/mL，这种重组菌很难将合成的乳糖酶分泌到发酵液中，增加了乳糖酶酶制剂的制备难度。在巴斯德毕赤酵母(Pichiapastoris)GS115中进行表达，摇瓶发酵表达水平可以达到70U/mL，但是同样很难实现乳糖酶的分泌表达，酶的分离纯化极为困难。
发明内容：
本发明的目的是在获得优良低聚半乳糖专用酶分子的基础上，获得具有良好实现大规模发酵生产性能并具有理想乳糖酶合成与分泌能力的重组菌，由此可显著降低乳糖酶的发酵制造成本、简化乳糖酶的发酵制造工艺并显著提高乳糖酶的质量。
为了实现上述目的，本发明的技术方案之一，是提供多种乳糖酶，分别为BglD305、BglD305-C、BglD305-D、BglD、BglD-C、BglD-D、BcBG168、BcBG168-C、BcBG168-D；
其中，BglD305来自发明人自行筛选分离的环状芽胞杆菌B2301(赵继华等.高转苷活性乳糖酶快速筛选方法的建立与初步应用,食品与发酵工业，2020)，氨基酸序列如序列表SEQ ID NO.2所示；BglD305-C、BglD305-D分别为BglD305的截短序列，氨基酸序列如序列表SEQ ID NO.4和SEQ ID NO.6所示；
其中，BglD来自环状芽胞杆菌ATCC 31382，氨基酸序列如序列表SEQ ID NO.8所示；BglD-C、BglD-D分别为BglD的截短序列，氨基酸序列如序列表SEQ ID NO.10和SEQ IDNO.12所示；
其中，BcBG168为将BglD305和BglD经过DNAshuffling改造重排获得，BcBG168-C和BcBG168-D分别是在BcBG168的基础上进一步删除C-端部分氨基酸序列获得的；BcBG168、BcBG168-C和BcBG168-D的氨基酸序列如序列表SEQ ID NO.14、16和18所示。
本发明提供的技术方案之二，是含有上述乳糖酶编码基因的重组载体或重组菌株；
优选地，所述重组载体采用的表达载体包括但不限于pHY-WZX，pBL-WZX，pHY300plk，pUB110，pE194，pHT1469(MoBiTec)，pWH1520(Rygus and Hillen,1991)；
优选地，所述重组载体采用的表达载体包括但不限于pHSE-001、pHSE-002、pHSE-003、pHSE-004、pHSE-005、pHSE-006、pHSE-007、pHSE-008、pHSE-009、pHSE-010、pHSE-011、pHSE-012、pHSE-013、pHSE-014、pHSE-015、pHSE-016、pHSE-017、pHSE-018；
更优选地，所述重组载体采用的表达载体为pHSE-008质粒，是以pHY-WZX为基础表达载体的骨架，整合来源于地衣芽胞杆菌淀粉酶启动子PamyL(SEQ ID NO.20)和碱性蛋白酶aprE的信号肽SaprE(SEQ ID NO.23)所得；
优选地，所述重组菌株采用的表达宿主包括但不限于枯草芽胞杆菌、环状芽胞杆菌、巨大芽胞杆菌、短小芽胞杆菌、解淀粉芽胞杆菌、谷氨酸棒杆菌、地衣芽胞杆菌等；
更优选地，所述重组菌株采用的表达宿主为突变株BCBT0529，是在地衣芽胞杆菌CBB3008(编号CCTCC NO.M208236)的基因组上敲除aprE，vpr，wpr，lacR，lacA，lacA2，yesZ基因所得，其基因序列对应的GenBank登录号分别为：MT885340、MT885341、MT885342、MT885336、MT885337、MT885338、MT885339；
优选地，本发明提供一株高产乳糖酶的重组菌株-地衣芽胞杆菌BCBTBc168D，所述菌株是将BcBG168-D编码基因整合在pHSE-008质粒上，并在宿主细胞突变株BCBT0529中进行表达所得；采用地衣芽胞杆菌重组菌BCBTBc168D发酵制备乳糖酶BcBG168-D的表达水平可以达到2208U/mL。
本发明还提供采用上述重组菌发酵生产乳糖酶的方法：
(1)摇瓶发酵生产乳糖酶：接种重组菌至摇瓶培养基，30～45℃、120-270r/min下培养2～3天；
摇瓶培养基：酵母膏0.5～1.5％，蛋白胨1.2～3.6％，葡萄糖8～20％；pH 7.0。
(2)发酵罐发酵生产乳糖酶：将菌种按接种量为5％-10％接入发酵罐培养基；发酵过程中采用发酵温度33～45℃，控制溶氧为0.1％～20％，pH为6.0～7.8，流加30％～60％(w/w)麦芽糖浆并维持还原糖含量在0.1％-5％；发酵持续90～120h，发酵过程定时取样分析，发酵终点控制为发酵酶活增加值小于5-20U/(mL·h)。
发酵罐培养基组成为：麦芽糖浆1％～5％，棉籽粉0％～5％，玉米浆0％-4％，豆饼粉0.5～5％，硫酸铵0.1～5％，pH 6.0～8.0。
经发酵后，摇瓶发酵的乳糖酶的发酵液酶活可达25-54U/mL；发酵罐发酵的乳糖酶的发酵液酶活可达826-2208U/mL。
进一步地，发酵结束后，经板框过滤除去菌体，再经超滤系统过滤后获得酶液。
本发明还提供上述乳糖酶在生产低聚半乳糖中的应用。
有益效果：
本发明中的低聚半乳糖生产专用酶制剂为通过基因克隆与人工进化获得的、具有极高催化乳糖生成低聚半乳糖的活性的乳糖酶；本发明乳糖酶高产菌株为通过微生物菌种选育获得的重组菌，能够在深层发酵时高效合成乳糖酶并将酶蛋白分子分泌到培养基中，从发酵上清中直接制备高活性的酶制剂，应用于低聚半乳糖的高效生产。
本发明的乳糖酶高效制备方法，在本发明提供的乳糖酶高产菌和发酵工艺下，乳糖酶的表达水平可以达到2208U/mL。本发明有助于降低乳糖酶的发酵制造成本、简化发酵制造工艺并提高乳糖酶酶制剂的质量。
附图说明：
图1.蛋白酶编码基因敲除的验证图谱
泳道M为1kb分子量标准；泳道1为aprE基因删除后菌株基因组验证正确的突变菌株，PCR扩增大小1.5kb；泳道2为蛋白酶wpr基因删除后菌株基因组验证正确的突变菌株，PCR扩增大小为1.3kb；泳道3为蛋白酶vpr删除菌株基因组验证正确的突变菌株，PCR扩增大小为1.3kb；
图2.内源性乳糖酶等编码基因敲除的验证图谱
泳道M为1kb分子量标准；泳道1为基因lacR成功敲除后PCR确认的电泳图谱，大小为1.3kb；泳道2为基因lacA成功敲除后PCR确认的电泳图谱，大小为1.2kb；泳道3为基因lacA2成功敲除后PCR确认的电泳图谱，大小为1.2kb；泳道4为基因yesZ成功敲除后PCR确认的电泳图谱，大小为1.6kb；
图3.表达载体的物理图谱
a：优化后的表达载体pHES-008；b：乳糖酶表达质粒pLEBG168；
图4.乳糖酶发酵产酶曲线图；
图5.发酵液的蛋白电泳图谱
泳道M为蛋白质分子量标准；泳道1为发酵液直接电泳的结果图，箭头标记部位为发酵液中的乳糖酶；
图6.乳糖酶作用乳糖后生成的低聚半乳糖HPLC糖谱图
DP2：半乳二糖或乳糖，DP3：葡糖基半乳二糖或半乳三糖；DP4：葡糖基半乳三糖或半乳四糖；DP5：葡糖基半乳四糖或半乳五糖。
具体实施方式：
为了使本专利的目的、技术方案及优点更加清楚明白，以下结合具体实施例，对本专利进行进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本专利，并不用于限定本发明。
本发明所使用的质粒pHY-WZX为现有技术，其构建方法已公开在Niu DD,WangZX.Development of a pair of bifunctional expression vectors for Escherichiacoli and Bacillus licheniformis.J Ind Microbiol Biotechnol(2007)34:357-362.DOI 10.1007/s10295-0204-x.公众也可通过天津科技大学化工与材料学院生物催化与生物转化实验室获得。
本发明所采用的地衣芽胞杆菌(Bacillus licheniformis)CBB3008，已保藏于中国典型培养物保藏中心(简称CCTCC)，保藏日期2008年11月25日，保藏编号CCTCC NO：M208236。
本发明以两种来源的乳糖酶(来源于环状芽胞杆菌B2301的BglD305和来源于环状芽胞杆菌ATCC 31382的BglD)分子为基础进行分子进化，获得合成低聚半乳糖效率高、表达性能好的乳糖酶新酶分子(BglD305-C、BglD305-D、BglD-C、BglD-D、BcBG168、BcBG168-C、BcBG168-D)；
本发明对一种宿主细胞，其分类命名为地衣芽胞杆菌(Bacillus licheniformis)CBB3008，已保藏于中国典型培养物保藏中心，保藏编号CCTCC NO：M208236，进一步进行遗传改造，敲除其中影响乳糖酶表达的多个基因(碱性蛋白酶编码基因aprE，次要丝氨酸蛋白酶编码基因vpr，胞壁蛋白酶编码基因wpr，调控蛋白编码基因lacR，β-半乳糖苷酶编码基因lacA，β-半乳糖苷酶编码基因lacA2，β-半乳糖苷酶编码基因yesZ)，获得适合乳糖酶高效表达的新宿主菌株；
本发明构建并优化适合乳糖酶分泌表达的表达载体，为在pHY-WZX的基础上优化构建，表达载体中有引导乳糖酶高表达的优选启动子和高效介导乳糖酶分泌表达的信号肽；
本发明将环状芽胞杆菌(B.circulans)乳糖酶或其突变体(氨基酸序列2、4、6、8、10、12、14、16、18)的编码基因(对应核苷酸序列1、3、5、7、9、11、13、15、17)克隆入本发明所构建的表达载体中，遗传转化入地衣芽胞杆菌宿主菌株中获得产乳糖酶重组菌；建立并优化发酵条件与工艺，建立新型酶分离纯化与精制方法，生产出乳糖酶产品，用于GOS的工业化制造。
本发明用于乳糖酶高产新菌种的构建方法，是通过基因克隆技术在表达载体中进行克隆和获得乳糖酶的表达质粒，并转化入地衣芽胞杆菌新宿主菌中获得重组菌，实现乳糖酶的高效分泌表达；在通过重组菌优化的蛋白分泌系统高效合成乳糖酶，将合成的乳糖酶酶蛋白高效分泌到培养基中；通过发酵生产工艺，从发酵液中回收、精制乳糖酶，获得乳糖酶产品。
本发明采用的主要实验方法如下：
1、基因克隆、分子进化与表达质粒的构建
常规分子克隆操作参考文献方法(Sambrook等。Molecular Cloning：ALaboratory Manual,1989)进行。环状芽胞杆菌乳糖酶或其突变体的编码基因作为本发明中的目标基因；基础表达载体为pHY-WZX(王正祥，牛丹丹。中国发明专利，ZL200510051648；Niu&Wang.J Ind Microbiol Biotechnol,2007)。乳糖酶或其突变体的编码基因经PCR扩增后克隆入表达载体中，获得表达乳糖酶的系列重组质粒。
2、染色体DNA的提取
染色体DNA提取方法按文献(诸葛键王正祥。工业微生物实验技术手册，中国轻工业出版社，1994)进行。
3、质粒DNA的提取
质粒DNA的提取在用一定浓度的溶菌酶裂解细胞壁后使用Sigma公司的质粒小提试剂盒进行。
4、基因扩增
DNA扩增在0.2mL PCR薄壁管中进行。PCR扩增条件为：1×(95℃5min)；30×(94℃10s，58℃30s，72℃30～300s)；1×(72℃10min)。依据不同扩增长度，PCR反应的延伸温度和时间有所不同。除特别注明外，所有PCR反应皆使用Pfu DNA聚合酶进行。
5、酶分子人工进化
乳糖酶的分子进化按照文献方法(Stemmer W P C.Rapid evolution of aprotein in vitro by DNA shuffling[J].Nature,1994,370(6488):389-391.)，采用DNAshuffling进行。首先将乳糖酶基因用DNA酶进行部分酶切，通过密度梯度法回收100～200bp的片段，混合后不加引物进行基因扩增15～25个循环，然后加入两端特异性引物，对全长基因进行扩增；用PCR产物纯化试剂盒(Sigma)进行纯化，纯化后的DNA克隆入表达载体pHY-WZX中，采用CaCl2法(诸葛健王正祥编著工业微生物实验技术手册，中国轻工业出版社，1994)转化入大肠杆菌JM109中；测定乳糖酶酶活并进行比较。
6、重叠PCR
参照文献(Krishnan B R，et al.Direct and crossover PCR amplification tofacilitate Tn5supF-based sequencing of lambda phage clones.Nucleic AcidsResearch，1991，22:6177-82)进行。通用步骤为：用片段F1和片段F2的引物(P1+P2；P3+P4，引物P2和P3为反向互补的序列)介导PCR的扩增，获得基因片段；胶回收纯化扩增片段F1和F2；将纯化得到的两个片段F1和F2稀释适当倍数，以1:1摩尔比混合作为模板，以引物P1+P4介导新的PCR反应，获得全长序列。
7、地衣芽胞杆菌遗传转化
参照文献(徐敏,马骏双,王正祥。高渗透压对细菌电转化率的影响。无锡轻工大学学报，2004(04):98-100)介绍的方法进行。主要步骤如下：接种新鲜单菌落到液体LB培养基中，37℃200r/min过夜培养，转接5％菌液到新的LB培养基中继续培养至OD600为0.75-0.90。菌体冰浴10min后4℃、6000r/min离心10min收集菌体。用预冷的电转洗液(0.5mol/L山梨醇，0.5mol/L的甘露醇和10％甘油)反复洗涤细胞4次。将细胞沉淀悬浮于1mL预冷的电转洗液中，完成感受态细胞制备。取1μL质粒DNA和约100μL感受态细胞混匀，立即电击(1800v,5ms)，随即加入电转复苏液(含0.65mol/L山梨醇和0.45mol/L甘露醇的LB培养基)，37℃、160r/min复苏后涂布对应抗性的LB平板，于合适温度培养至单菌落长出。正确转化子经菌落PCR验证、质粒提取酶切、发酵验证功能等方法验证。
8、地衣芽胞杆菌特定基因的删除
参考文献方法(Cai D,et al High-level expression of nattokinase inBacillus licheniformis by manipulating signal peptide and signal peptidase.JAppl Microbiol.2016,121:704-712)进行。以地衣芽胞杆菌CCTCC NO：M208236中aprE基因删除为例的通用步骤为：以地衣芽胞杆菌基因组DNA为模板，利用apr-up1(序列30)及apr-up2(序列31)和引物apr-dn1(序列32)及apr-dn2(序列33)为引物，分别扩增上、下两个同源臂片段，得到正确大小的PCR产物后分别采用胶回收进行纯化，以胶回收产物DNA为模板进行重叠PCR，获得删除突变盒，△aprE。将该突变盒纯化后Xba I酶切后，克隆入质粒pT2ts(pT2ts是以T2(2)-ori(陈守文等，中国发明专利，ZL201310562150.7)为出发质粒，采用引物T2-1(序列28)、T2-2(序列29)进行反向扩增后，PCR产物自身环化、连接获得新质粒pT2ts)的Sma I和Xba I位点，转化大肠杆菌JM109感受态细胞，在含20μg/mL卡那霉素的LB平板进行培养，获得正确的删除质粒pT2-△aprE。按照地衣芽胞杆菌的遗传转化方法中描述的步骤，将删除质粒pT2-△aprE转化入地衣芽胞杆菌宿主细胞。经两次同源重组以后，以同源臂两侧设计引物apr-F：(序列34)和apr-R(序列35)，并以这对引物进行菌落PCR，进行转化子的验证(其他基因的删除可参考上述方法，根据删除的基因序列设计并替换引物即可)。
9、发酵试验
摇瓶发酵生产乳糖酶：在250mL三角瓶中装30mL发酵培养基(酵母膏0.5～1.5％，蛋白胨1.2～3.6％，葡萄糖8～20％；pH 7.0)，接种重组菌，30～45℃、120-270r/min下培养2～3天。
发酵罐发酵生产乳糖酶：发酵培养基组成为：麦芽糖浆1％～5％，棉籽粉0％～5％，玉米浆0％-4％，豆饼粉0.5～5％，硫酸铵0.1～5％，pH 6.0～8.0；发酵在50L～10t发酵罐中进行，接种量为5％-10％；发酵过程中采用发酵温度33～45℃，控制溶氧为0.1％～20％，pH为6.0～7.8，流加30％～60％(w/w)麦芽糖浆并维持还原糖含量在0.1％-5％；发酵持续到90～120h，发酵过程定时取样分析，发酵终点控制为发酵酶活增加值小于5-20U/(mL·h)。
10、乳糖酶酶制剂的制备方法
发酵结束后，经板框过滤除去菌体，再经超滤系统过滤后获得酶液。
11、乳糖酶酶活测定
乳糖酶的酶活测定，按照国标GB/T 33409-2016进行改进。一般过程是，反应在pH5.0和40℃下进行，以乳糖为底物。采用生物传感仪测定葡萄糖的释放量。
乳糖酶的酶活定义为：在pH 5.0和40℃下每分钟分解乳糖生成1微摩尔葡萄糖所需的酶量，定义为一个酶活力单位(U)，以U/mL或U/g表示。
12、低聚半乳糖的合成与产物分析
采用300g/L～800g/L乳糖为底物，添加5U/g～20U/g的乳糖酶，反应在50℃-70℃下进行，定时取样。反应原料和低聚半乳糖形成与含量的分析，采用HPLC法分析酶促产物特征与生成。色谱条件为：流动相为65％的乙腈，1.0mL/min流速；TSK-GEL G3000PWXL-CP(7.8mm×300mm，7μm)色谱柱，柱温25℃；蒸发光散射检测器，漂移管温度90℃，载气流速2.2mL/min。
13、其它分析方法
蛋白酶酶活测定，按照国标法(GB/T 23527-2009)进行；
基因和氨基酸序列比对，采用DNAMAN软件进行；
核苷酸序列测定采用Sanger方法进行；
序列蛋白含量按文献方法(Bradford.Anal Chem,1976)进行；
葡萄糖含量用酶电极法测定(SBA-90,山东)；
细胞密度用分光光度计(UV-2000，美国)在600nm下测定；
蛋白电泳按文献方法进行(诸葛键王正祥。工业微生物实验技术手册，中国轻工业出版社，1994)。
以下将通过具体实施例对本发明做进一步的解释说明。
实施例1：乳糖酶的分子进化
以序列表SEQ ID NO.1和SEQ ID NO.7所示的BglD305和BglD编码基因为模板，采用DNAshuffling方法对其进行分子进化。经过酶活筛选后，获得了酶活水平显著提高的乳糖酶酶分子BcBG168(核苷酸序列SEQ ID NO.13)，其氨基酸序列(氨基酸序列SEQ IDNO.14)。
通过对BglD305、BglD和BcBG168的编码基因进行不同程度的截短，并对改造序列和原序列进行高效表达，通过PCR扩增技术扩增相应的基因序列，并克隆入表达载体pHY-WZX中，获得乳糖酶表达质粒pHY-Bgl-1、pHY-Bgl-2、pHY-Bgl-3、pHY-Bgl-4、pHY-Bgl-5、pHY-Bgl-6、pHY-Bgl-7、pHY-Bgl-8、pHY-Bgl-9、pHY-Bgl-10、pHY-Bgl-11、pHY-Bgl-12。以上述地衣芽胞杆菌遗传转化方法将上述重组质粒分别转化到地衣芽胞杆菌CCTCC NO：M208236，获得相应的转化子CBB-Bgl-1、CBB-Bgl-2、CBB-Bgl-3、CBB-Bgl-4、CBB-Bgl-5、CBB-Bgl-6、CBB-Bgl-7、CBB-Bgl-8、CBB-Bgl-9、CBB-Bgl-10、CBB-Bgl-11、CBB-Bgl-12，进一步进行摇瓶发酵并进行产酶(对发酵液上清进行酶活测定)情况分析，主要内容与产酶结果见表1。
在相同条件下，BcBG168的表达酶活分别是BglD305的103.2％和BglD的109.8％。
乳糖酶的C端截短的乳糖酶在同样的表达条件下均呈现上升趋势，酶活力较原始基因序列提高40％、78％；35％、69％和31％，70％。
表1不同来源及C端截短的乳糖酶的表达效率
Figure BDA0002709561960000091
实施例2：表达宿主细胞的遗传修饰
地衣芽胞杆菌CCTCC NO：M208236中aprE基因的删除。以地衣芽胞杆菌CCTCC NO：M208236基因组DNA为模板，利用apr-up1(序列30)及apr-up2(序列31)和引物apr-dn1(序列32)及apr-dn2(序列33)为引物，分别扩增上下两个同源臂片段，大小分别为667bp和495bp。得到正确大小的PCR产物后分别采用胶回收进行纯化，以胶回收产物DNA为模板进行重叠PCR，获得大小为～1.2kb的删除突变盒△aprE。将该突变盒纯化后Xba I酶切后，克隆入质粒pT2ts的Sma I和Xba I位点，转化大肠杆菌JM109感受态细胞，在含20μg/mL卡那霉素的LB平板进行培养，获得正确的删除质粒pT2-△aprE。按照“地衣芽胞杆菌的遗传转化”方法中描述的步骤，将删除质粒pT2-△aprE转化入地衣芽胞杆菌宿主细胞CCTCC NO：M208236。经两次同源重组以后，以同源臂两侧设计引物apr-F(序列34)和apr-R(序列35)，并以这对引物进行菌落PCR验证获得正确的转化子BCBT01，正确转化子的PCR产物大小为～1.5kb(图1，泳道1)。
地衣芽胞杆菌CCTCC NO：M208236β-半乳糖苷酶编码基因lacA的删除。采用上述aprE基因删除相似的方法进行。以地衣芽胞杆菌CCTCC NO：M208236基因组DNA为模板，利用lacA-up1及lacA-up2(序列36和序列37)和引物lacA-dn1及lacA-dn2(序列38和序列39)为引物，分别扩增上下两个同源臂片段，大小分别为486bp和500bp。得到正确大小的PCR产物后分别采用胶回收进行纯化，以胶回收产物DNA为模板进行重叠PCR，获得大小为936bp的删除突变盒△lacA。将该突变盒纯化后Xba I酶切后，克隆入质粒pT2ts的Sma I和Xba I位点，转化大肠杆菌JM109感受态细胞，在含20μg/mL卡那霉素的LB平板进行培养，获得正确的删除质粒pT2-△LacA。按照“地衣芽胞杆菌的遗传转化”方法中描述的步骤，将删除质粒pT2-△LacA转化入地衣芽胞杆菌。经两次同源重组以后，以同源臂两侧引物lacA-F和lacA-R(序列40和序列41)，进行菌落PCR验证获得正确的转化子，正确转化子的PCR产物大小为～1.2kb(图2，泳道2)。
采用上述相似地方法，对地衣芽胞杆菌CCTCC NO：M208236基因组中的vpr、wpr、lacR、lacA2和yesZ基因进行不同组合的删除，其中，vpr对应同源臂引物及验证引物为序列42-47；wpr对应同源臂引物及验证引物为序列48-53；lacR对应同源臂引物及验证引物为序列54-59；lacA2对应同源臂引物及验证引物为序列60-65；yesZ对应同源臂引物及验证引物为序列66-71，获得不同缺陷型的突变株，其中突变株BCBT03-15,重新命名为BCBT0529，其遗传背景为(CBB3008，ΔaprE，Δvpr，Δwpr，ΔlacR，ΔlacA，ΔlacA2，ΔyesZ)。
实施例3敲除部分基因对宿主细胞表达的影响
(1)部分蛋白酶敲除对胞外蛋白酶酶活的影响
对实施例2获得的不同突变株进行摇瓶发酵试验，并分析其胞外蛋白酶酶活。如表2所示，在删除碱性蛋白酶编码基因aprE后，培养基中蛋白水解酶的总酶活降低了80％，进一步删除两种蛋白酶基因vpr、wpr后培养基中蛋白水解酶的总酶活力降至野生型的10％。
表2突变菌株碱性蛋白酶活力测定
Figure BDA0002709561960000101
Figure BDA0002709561960000111
(2)部分蛋白酶敲除对乳糖酶表达的影响
将实施例1中获得的产酶活力最高的重组菌CBB-Bgl-12中携带的表达质粒pHY-bgl-12转化入实施例2中获得的蛋白质水解酶基因删除的宿主菌中，获得相应的重组菌，以CCTCC NO：M208236为对照，重组菌摇瓶发酵结果见表3。删除碱性蛋白酶基因aprE后发酵液中乳糖酶酶活力明显增加，达到10.68U/mL，提高了41.08％；删除另外两个蛋白水解酶基因vpr、wpr后发酵液中乳糖酶活力进一步提高，最终达到了12.12U/mL，较出发菌株提高了60％。
表3删除蛋白酶后乳糖酶的表达水平
Figure BDA0002709561960000112
(3)部分基因敲除对乳糖酶表达的影响
宿主菌在其内源性乳糖酶相关基因突变后，在摇瓶发酵条件下的乳糖酶酶活变化如表4所示。可以看到，在删除碱性蛋白酶基因aprE和乳糖操纵子阻遏蛋白基因lacR后发酵液中乳糖酶活力有所提高，进一步删除相关内源性乳糖酶结构基因后，发酵液中乳糖酶活力低至按照现有方法测定不到。
表4内源性乳糖酶基因突变菌株乳糖酶酶活力
Figure BDA0002709561960000113
Figure BDA0002709561960000121
*n.d.:酶活未检测到
实施例4：乳糖酶的高效表达
在实施例2中获得的不同内源性乳糖酶基因删除的菌株中转入质粒pHY-bgl-12，构建重组菌并进行摇瓶发酵，测定发酵液中乳糖酶活力，结果如表5所示。宿主细胞内源性乳糖酶相关基因删除后，发现本发明的乳糖酶的表达水平大幅度提升，其中BCBT03-15，即BCBT0529作为宿主细胞时，酶活达到最高，是CCTCC NO：M208236作为宿主细胞酶活的4.47倍。
表5内源性乳糖酶删除后乳糖酶的表达水平
Figure BDA0002709561960000122
*n.d.：酶活未检测到。
在确定最优宿主细胞的基础上，进一步进行表达元件的优化，采用不同启动子与不同信号肽的组合对表达载体进行改造，提高乳糖酶的表达水平。
以质粒pHY-WZX为基础表达载体的骨架，选用3种不同的组成型启动子，其分别为Pcry(序列19，苏云金芽胞杆菌杀虫蛋白基因启动子)、PamyL(序列20，地衣芽胞杆菌淀粉酶基因启动子)、P43(序列21，枯草芽胞杆菌胞苷脱氨酶基因启动子)以及6种不同的信号肽，其分别为SamyL(序列22，地衣芽胞杆菌淀粉酶基因信号肽)、SaprE(序列23，地衣芽胞杆菌碱性蛋白酶信号肽)、SamyQ(序列24，解淀粉芽胞杆菌淀粉酶基因信号肽)、SamyE(序列25，枯草芽胞杆菌淀粉酶基因信号肽)、SnprE(序列26，地衣芽胞杆菌中性蛋白酶基因信号肽)、Schi(序列27，地衣芽胞杆菌几丁质酶基因信号肽)，以不同的组合方式替换pHY-WZX上原有的启动子和信号肽，构建成18种新的表达载体pHSE-001～018(具体见表6)，克隆入BcBG168-D编码基因序列后转化地衣芽胞杆菌宿主细胞BCBT0529，获得系列重组菌，进行摇瓶发酵并测定酶活力，结果汇总于表6。所测试的18种启动子和信号肽组合形式，皆能介导乳糖酶BcBG168-D在地衣芽胞杆菌中分泌表达。其中，来源于地衣芽胞杆菌淀粉酶启动子PamyL和碱性蛋白酶aprE的信号肽组合构成的表达质粒pHSE-008(图3a)，能够介导最高的酶表达，所获得的乳糖酶表达质粒为pLEBG168(在表达质粒pHSE-008中克隆入BcBG168-D编码基因，图3b)；所获得的菌株BCBTBc168D(在表达质粒pHSE-008中克隆入BcBG168-D编码基因序列后转化地衣芽胞杆菌宿主细胞BCBT0529)在摇瓶发酵时表达53.79U/mL的乳糖酶酶活，是野生菌株产酶水平的20倍以上，是宿主细胞遗传改造和信号肽优化前的7倍以上。
表6不同的启动子信号肽组合对乳糖酶BcBG168-D表达的影响
Figure BDA0002709561960000131
Figure BDA0002709561960000141
实施例5：50L发酵体系下的乳糖酶发酵生产工艺
将乳糖酶高产菌株BCBTBc168D在37℃培养20～40h，挑取2-3个单菌落接种于2瓶装有1000mL LB液体培养基的5L三角瓶中，37℃、230r/min，摇床培养16h作为种子液，按照5％接种量接种于装有30L发酵培养基(麦芽糖浆4％，棉籽粉2.5％，豆饼粉3.5％，硫酸铵0.5％，pH6.5)的50L全自动发酵罐中，工作发酵体积30L，过程中通过调节转速、通气量维持溶氧为0.1％～20％，发酵温度40-42℃，并控制pH 6.5±0.5，流加60％(w/w)麦芽糖浆作为碳源，并维持还原糖含量0.5-5％。定时取样分析残糖量和酶活力，发酵至120小时，酶活力增加速度低于5U/(mL·h)停止发酵，下罐并制备酶制剂。
乳糖酶的典型产酶进程曲线见图4，发酵液中酶蛋白为最主要的蛋白分子(图5)，发酵液上清中的乳糖酶酶活最高达到1131U/mL(108h时)，约为摇瓶发酵水平的21倍。
相似地，将BglD305-D和BglD-D在地衣芽胞杆菌淀粉酶启动子PamyL和碱性蛋白酶AprE的信号肽SaprE组合介导下于以pHSE-008为表达载体在地衣芽胞杆菌BCBT0529中进行表达，分别获得的乳糖酶高产菌株BCBT305D和BCBTatccD，在上述发酵条件下的乳糖酶产酶水平，分别达到820U/mL和870U/mL。
实施例6：乳糖酶的发酵生产制备
按照实施例5中50L发酵罐的工艺，相应调整操作流程后BCBTBc168D菌株在10吨发酵体系下制备乳糖酶。发酵结束后，发酵液中乳糖酶酶活达到2208U/mL。
发酵结束后，发酵液中按照1.0％加入生物絮凝剂(聚丙烯酰胺:碱式氯化铝＝8:1)，絮凝完成后添加2％的硅藻土TS-20#，然后进行板框过滤除菌。选择截留分子量为30kDa的膜材料进行超滤浓缩，40℃条件下，操作压力0.05MPa，工作2h。
超滤后添加1％苯甲酸钠，1％山梨酸钾，2％氯化钠，10％山梨醇，10％甘油作为液体剂型的稳定剂，获得液体剂型产品。
将上述液体剂型稳定剂中的10％的甘油替换为3％的乳糖和2％的硫酸钠，其他组分不变，经喷雾干燥制备固体剂型产品。
上述百分比(％)均为w/v。
实施例7：乳糖酶在低聚半乳糖制备中的应用
以浓度为600g/L的乳糖溶液为底物，用本发明制备的乳糖酶BcBG168-D催化制备低聚半乳糖，反应体系为总体积约为30L。按照20U/g底物浓度添加酶，调整pH为6.0，反应温度在65℃下于反应釜中以50r/min的搅拌速度搅拌并反应10h。
反应产物中低聚半乳糖的含量达到50％以上。图6为通过HPLC检测方法对上述所生产出的低聚半乳糖产品糖谱分析的典型结果。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本专利构思的前提下，上述各实施方式还可以做出若干变形、组合和改进，这些都属于本专利的保护范围。因此，本专利的保护范围应以权利要求为准。
SEQUENCE LISTING
<110> 天津科技大学
<120> 一种低聚半乳糖生产专用酶及其制备与应用
<130> 1
<160> 71
<170> PatentIn version 3.5
<210> 1
<211> 5133
<212> DNA
<213> 环状芽胞杆菌B2301
<400> 1
atgaaaagca cgacgagcgc cgccggcaaa agcgtcagct ataacgacgg cgaacgccgc 60
gtcaactttg aaaactggcg ctttcagcgc gaaacgaacg gcagcatcgc cggcgcccag 120
aacccgggct ttgacgacag cagctggcgc aaactgaacc tgccgcatga atggagcatc 180
gacctggact ttaacaaaaa cagcctggcc acgcatgaag gcggctatct ggacggcggc 240
atcggctgga accgcaaaac gtttacgatc ccggaaagca tgaaaggcaa acgctttagc 300
ctggacatcg acggcgtcta tatgtatagc acgacgtatc tgaacgcctt tgtcctgggc 360
acgtatccgt ttggctataa cggcgaaagc tatgacatca gcgacaaact gtataaagac 420
ggccgcgcca acgtcctggt cgtcaaagtc aacacgaacc agccgagcgg ccgctggtat 480
agcggcagca gcatctatcg caacgtctat ctgacggtca cggacccgat ccatgtcgcc 540
cgctatggca cgtttgtcac gacgccgaac ctggaaaaaa gcatcaaaga agaccgcgac 600
gccgtcaaca tcaaaacgaa aatcagcaac gccgaagcca aacaggtcaa aatcgccgac 660
aaaagcacga tctatgacgg cgccggcaac acggtccaga cggtcgaaac ggaagaaaaa 720
acggccgccg gcgccacggt cacgccgttt gaacagaaca cggtcatcaa acagccgaaa 780
ctgtggagca tcgacaaacc gtatcgctat aacctggtca cggaagtcat cgtcggcggc 840
cagacggtcg acacgtatga aacgaaattt ggcgtccgct attttaaatt tgacgaaaac 900
gaaggctata gcctgaacgg cgaatttatg aaactgcatg gcgtcagcat gcatcatgac 960
ctggccggcg gcgccgccct gacgaacgcc cgcggcgtcg aacgccagat gcagatcatg 1020
aaagacgtcg gcgtcaacgc catccgcgtc acgcataacc cggccagccc ggaactgggc 1080
ctggaatttg ccgccaacaa actgctgatc atcgaagaag cctttgacag ctgggcccag 1140
agcaaaaaac cgtatgacta tggccgcttt tttaacgcct gggccgaaca tgacatcaaa 1200
gaaatggtcg accgcggcaa aaacgaaccg gccatcatca tgtggagcat cggcaacgaa 1260
atctatgaca cgacgaacgc cgccggcgtc gaaacggccc gcaacctggt cggctgggtc 1320
aaagaaatcg acacgacgcc gcgcgccacg atcggcgaag acaaaacgcg cggcgacaac 1380
gtcacgccga tcaacagcta tatcaaagaa atctttaaca tcgtcgacgt cgtcggcctg 1440
aactatagcg aaaacaacta tgacggctat cataaacaga acccgagctg gaaactgtat 1500
ggcagcgaaa cgagcagcgc cacgcgcagc cgcggcgtct atacgcagcc gtatcagtat 1560
aaccatgaca cgaaatatgc cgacctgcag cagagcagct atgacaacga ctatgtcggc 1620
tggggccgca cggcccagga cgcctggaaa tatgaccgcg acctgaaaca tatcgccggc 1680
cagtttatct ggacgggctt tgactatatc ggcgaaccga cgccgtatta taacagctat 1740
ccggccaaaa gcagctattt tggcgccgtc gacacggccg gctttccgaa agacatcttt 1800
tattattatc agagccagtg gaaaaaagaa ccgatggtcc atctgctgcc gcattggaac 1860
tggaaagaag gcgaaaaagt ccgcgtcctg gcctatacga acgccagcaa agtcgaactg 1920
gtcctgaacg gcgaaagcct gggcaacgaa aaatatgaca acaaacagac gagctggggc 1980
gccccgtata aagaaacgaa agacggcaaa acgtatctgg aatgggccgt caaaccgctg 2040
aaaccgggca aatttgacga agccgtcgcc gaaaacggca aagtcatcgc ccgcgaccag 2100
gtcgtcacgg ccggcgaacc ggccagcgtc cgcctgacgg ccgaccgcaa agtcgtcaaa 2160
gccgacggca cggaccatag ctttatcacg gccgacatcg tcgacagcaa aggcatcgtc 2220
gtcccggacg ccgaccatct gatcacgttt aacgtcacgg gccagggcga actggccggc 2280
gtcgacaacg gcaacgccag cagcgtcgaa cgctataaag acaacaaacg caaagccttt 2340
agcggcaaag ccctggccat cgtccagagc agcaaactga gcggcaaaat cacggtccat 2400
gccagcgtcg ccggcctgag cagcgacagc acgagcgtct ttacggtcac gccggccgac 2460
catgacaaaa aaatcgtcgc cggcatcgac gacgtcaacc tgacggtcga cgtcaacgaa 2520
gccccgaaac tgccgagcga aatcaaagtc tattatagcg acgaaagcgc cgccgccaaa 2580
aacgtcacgt gggacgaagt cgacccgaaa cagtatagca cggtcggcga atttacggtc 2640
gaaggcagcg tcgaaggcac gagcctgaaa gccaaagcct ttgtcatcgt caaaggcatc 2700
gtcgccgtca aaccgtatag cacggccacg aaagtcggcg tccagccggt cctgccggaa 2760
aaagccacgc tgctgtatag cgacggcacg acgaaaggcg ccacggtcac gtgggacgaa 2820
ctgccggaag acaaactggc caaagaaggc cgctttacgg tcgaaggcag cgtcgaaggc 2880
acggacctga aagccaacgt ctatgtccgc gtcacgaacg aagtcaaaag cgtcaacatc 2940
atgctgcagg aacagggcag cgcctatccg aaactggaag ccacgtttac gaacccggcc 3000
gacaacctgc agcatctgaa cgacggcatc aaaagctata cgaacaacca ggtcaaccgc 3060
tggacgaact ggacgcgcac gccgcgcgac gccggcgaca gcatcacggt caactttggc 3120
aaaaaacatg tcatcaacaa cctggacctg tttgtcttta cggacagcgg cacggtcgtc 3180
ccggaaaaag ccgaagtcca gtattgggac ggcacggccg tcaaagacgt cgaaaacctg 3240
acgcagccga gcccgtatgt cgtcgaaaaa aacgaactga cgtttgacgc cgtcgccacg 3300
gaaaaactga aatttcatct gacgccgagc gtcaaaggca aatttctggc cctgacggaa 3360
gccgaagtct atgccgacca gatcgtcgac ggcgaaacgg ccaaactgca gagcatcacg 3420
gtcaacggca aagccctgga aggctttgac catgccaaaa aaaactatga actggtcctg 3480
ccgtatggca gcgaactgcc gaaaatcgaa gccgccgccg ccgacaacgc cacggtcacg 3540
atcctgccgg cctttagcta tccgggcacg gccaaactgt ttgtcacgag cgaagacggc 3600
aaagtcacga cggaatatag catcggcgtc agcacggaag aaccgaaact ggtcagcgcc 3660
gaactgagcg ccgacaaaac gaacgtcatg gaagacgaca tcatcgacct gaaagtcatc 3720
ggcctgtttg aaagcaaaga aaaaatcgac gtcacggaca gccagccgac gtatgaattt 3780
gaccagcaga tcatcaaaat cgaaggcaac aaactgtatg ccctggaaac gggcaacgtc 3840
aaagtcaaag tcacggtcac gtataaaggc gtcagcgtca cgacgccggc cctggaattt 3900
acgatcgcca aacatccggc cccgaaatat atcacgagcc tggaaccggt cacggtcgtc 3960
gtcaaaaaag gcgaagcccc ggaactgccg gccacggtcg tcgcccatta taaccgcggc 4020
atcccgcgcg acgtcaaagt caaatgggaa cgcatccatc cgagcaaata tcagcagctg 4080
ggcgaattta cggtcagcgg catggtcgaa ggcacggaca tcaaagccca ggccaaagtc 4140
gccgtcaaag gcgccgtcat cgtcgaagac atccgcatgg ccgtcctgct gaaacagatg 4200
ccgcagctgc cgggcaaagt cacggtctat tatagcgacg gcgccgaaga acagcgcgcc 4260
gtcaaatggg aagaaatccc gcaggaagaa ctggaaaaag tcggcgaatt taaagtcaac 4320
ggcgacgtca acggcgtcaa actgaaagcc acggccacga tccgcgtcac ggacgaagtc 4380
ggcgccgaac agaacatcag ccgcgccaaa aacggctatg aatatccgaa agccgaagcc 4440
agctttacga acgacggccc gggcagcagc gaccgcatcg aagccatcaa cgacgacgtc 4500
atcagctatg aagccaaccc gcataaccgc tggacgaact ggcagccggc cccgcgcgtc 4560
ggcgacctgg tctttacgac gtttggcgac tatgaaccga cggaatatga cgtcgacagc 4620
atggaaatcc attggtttgc cgaccatggc acgagctatc cggaacgctt tcagatcgaa 4680
tataaaagcg gcgacagctg gaaagaagtc acgagcctga aaagcgaccc ggccagcccg 4740
gccctgggca aagccaacgt ctatagcttt gaccgcgtca aaacgagcgc catccgcgtc 4800
aaaatgacgg cccaggccgg caaaagcctg gccatcacgg aactgaaagt ctttagcaaa 4860
tggccgaaag ccggcacgga accggacgtc acggacatca aagtcggcgg caaaagcatc 4920
ctggaagaat ttgaacagga aggcgaccat tatgaagcca cgatcgacgt cggcgacgcc 4980
aacgtcatgc cgaaaatcta tgtcaaagcc aaagaccaga cgagcatcag catcgtcccg 5040
gccgtcacga gcgtcagcac ggccaaagtc atcgccgaag acggcgacgg caaaaaagtc 5100
aaagtctata gcatccataa aagctataaa taa 5133
<210> 2
<211> 1710
<212> PRT
<213> 环状芽胞杆菌B2301
<400> 2
Met Lys Ser Thr Thr Ser Ala Ala Gly Lys Ser Val Ser Tyr Asn Asp
1 5 10 15
Gly Glu Arg Arg Val Asn Phe Glu Asn Trp Arg Phe Gln Arg Glu Thr
20 25 30
Asn Gly Ser Ile Ala Gly Ala Gln Asn Pro Gly Phe Asp Asp Ser Ser
35 40 45
Trp Arg Lys Leu Asn Leu Pro His Glu Trp Ser Ile Asp Leu Asp Phe
50 55 60
Asn Lys Asn Ser Leu Ala Thr His Glu Gly Gly Tyr Leu Asp Gly Gly
65 70 75 80
Ile Gly Trp Asn Arg Lys Thr Phe Thr Ile Pro Glu Ser Met Lys Gly
85 90 95
Lys Arg Phe Ser Leu Asp Ile Asp Gly Val Tyr Met Tyr Ser Thr Thr
100 105 110
Tyr Leu Asn Ala Phe Val Leu Gly Thr Tyr Pro Phe Gly Tyr Asn Gly
115 120 125
Glu Ser Tyr Asp Ile Ser Asp Lys Leu Tyr Lys Asp Gly Arg Ala Asn
130 135 140
Val Leu Val Val Lys Val Asn Thr Asn Gln Pro Ser Gly Arg Trp Tyr
145 150 155 160
Ser Gly Ser Ser Ile Tyr Arg Asn Val Tyr Leu Thr Val Thr Asp Pro
165 170 175
Ile His Val Ala Arg Tyr Gly Thr Phe Val Thr Thr Pro Asn Leu Glu
180 185 190
Lys Ser Ile Lys Glu Asp Arg Asp Ala Val Asn Ile Lys Thr Lys Ile
195 200 205
Ser Asn Ala Glu Ala Lys Gln Val Lys Ile Ala Asp Lys Ser Thr Ile
210 215 220
Tyr Asp Gly Ala Gly Asn Thr Val Gln Thr Val Glu Thr Glu Glu Lys
225 230 235 240
Thr Ala Ala Gly Ala Thr Val Thr Pro Phe Glu Gln Asn Thr Val Ile
245 250 255
Lys Gln Pro Lys Leu Trp Ser Ile Asp Lys Pro Tyr Arg Tyr Asn Leu
260 265 270
Val Thr Glu Val Ile Val Gly Gly Gln Thr Val Asp Thr Tyr Glu Thr
275 280 285
Lys Phe Gly Val Arg Tyr Phe Lys Phe Asp Glu Asn Glu Gly Tyr Ser
290 295 300
Leu Asn Gly Glu Phe Met Lys Leu His Gly Val Ser Met His His Asp
305 310 315 320
Leu Ala Gly Gly Ala Ala Leu Thr Asn Ala Arg Gly Val Glu Arg Gln
325 330 335
Met Gln Ile Met Lys Asp Val Gly Val Asn Ala Ile Arg Val Thr His
340 345 350
Asn Pro Ala Ser Pro Glu Leu Gly Leu Glu Phe Ala Ala Asn Lys Leu
355 360 365
Leu Ile Ile Glu Glu Ala Phe Asp Ser Trp Ala Gln Ser Lys Lys Pro
370 375 380
Tyr Asp Tyr Gly Arg Phe Phe Asn Ala Trp Ala Glu His Asp Ile Lys
385 390 395 400
Glu Met Val Asp Arg Gly Lys Asn Glu Pro Ala Ile Ile Met Trp Ser
405 410 415
Ile Gly Asn Glu Ile Tyr Asp Thr Thr Asn Ala Ala Gly Val Glu Thr
420 425 430
Ala Arg Asn Leu Val Gly Trp Val Lys Glu Ile Asp Thr Thr Pro Arg
435 440 445
Ala Thr Ile Gly Glu Asp Lys Thr Arg Gly Asp Asn Val Thr Pro Ile
450 455 460
Asn Ser Tyr Ile Lys Glu Ile Phe Asn Ile Val Asp Val Val Gly Leu
465 470 475 480
Asn Tyr Ser Glu Asn Asn Tyr Asp Gly Tyr His Lys Gln Asn Pro Ser
485 490 495
Trp Lys Leu Tyr Gly Ser Glu Thr Ser Ser Ala Thr Arg Ser Arg Gly
500 505 510
Val Tyr Thr Gln Pro Tyr Gln Tyr Asn His Asp Thr Lys Tyr Ala Asp
515 520 525
Leu Gln Gln Ser Ser Tyr Asp Asn Asp Tyr Val Gly Trp Gly Arg Thr
530 535 540
Ala Gln Asp Ala Trp Lys Tyr Asp Arg Asp Leu Lys His Ile Ala Gly
545 550 555 560
Gln Phe Ile Trp Thr Gly Phe Asp Tyr Ile Gly Glu Pro Thr Pro Tyr
565 570 575
Tyr Asn Ser Tyr Pro Ala Lys Ser Ser Tyr Phe Gly Ala Val Asp Thr
580 585 590
Ala Gly Phe Pro Lys Asp Ile Phe Tyr Tyr Tyr Gln Ser Gln Trp Lys
595 600 605
Lys Glu Pro Met Val His Leu Leu Pro His Trp Asn Trp Lys Glu Gly
610 615 620
Glu Lys Val Arg Val Leu Ala Tyr Thr Asn Ala Ser Lys Val Glu Leu
625 630 635 640
Val Leu Asn Gly Glu Ser Leu Gly Asn Glu Lys Tyr Asp Asn Lys Gln
645 650 655
Thr Ser Trp Gly Ala Pro Tyr Lys Glu Thr Lys Asp Gly Lys Thr Tyr
660 665 670
Leu Glu Trp Ala Val Lys Pro Leu Lys Pro Gly Lys Phe Asp Glu Ala
675 680 685
Val Ala Glu Asn Gly Lys Val Ile Ala Arg Asp Gln Val Val Thr Ala
690 695 700
Gly Glu Pro Ala Ser Val Arg Leu Thr Ala Asp Arg Lys Val Val Lys
705 710 715 720
Ala Asp Gly Thr Asp His Ser Phe Ile Thr Ala Asp Ile Val Asp Ser
725 730 735
Lys Gly Ile Val Val Pro Asp Ala Asp His Leu Ile Thr Phe Asn Val
740 745 750
Thr Gly Gln Gly Glu Leu Ala Gly Val Asp Asn Gly Asn Ala Ser Ser
755 760 765
Val Glu Arg Tyr Lys Asp Asn Lys Arg Lys Ala Phe Ser Gly Lys Ala
770 775 780
Leu Ala Ile Val Gln Ser Ser Lys Leu Ser Gly Lys Ile Thr Val His
785 790 795 800
Ala Ser Val Ala Gly Leu Ser Ser Asp Ser Thr Ser Val Phe Thr Val
805 810 815
Thr Pro Ala Asp His Asp Lys Lys Ile Val Ala Gly Ile Asp Asp Val
820 825 830
Asn Leu Thr Val Asp Val Asn Glu Ala Pro Lys Leu Pro Ser Glu Ile
835 840 845
Lys Val Tyr Tyr Ser Asp Glu Ser Ala Ala Ala Lys Asn Val Thr Trp
850 855 860
Asp Glu Val Asp Pro Lys Gln Tyr Ser Thr Val Gly Glu Phe Thr Val
865 870 875 880
Glu Gly Ser Val Glu Gly Thr Ser Leu Lys Ala Lys Ala Phe Val Ile
885 890 895
Val Lys Gly Ile Val Ala Val Lys Pro Tyr Ser Thr Ala Thr Lys Val
900 905 910
Gly Val Gln Pro Val Leu Pro Glu Lys Ala Thr Leu Leu Tyr Ser Asp
915 920 925
Gly Thr Thr Lys Gly Ala Thr Val Thr Trp Asp Glu Leu Pro Glu Asp
930 935 940
Lys Leu Ala Lys Glu Gly Arg Phe Thr Val Glu Gly Ser Val Glu Gly
945 950 955 960
Thr Asp Leu Lys Ala Asn Val Tyr Val Arg Val Thr Asn Glu Val Lys
965 970 975
Ser Val Asn Ile Met Leu Gln Glu Gln Gly Ser Ala Tyr Pro Lys Leu
980 985 990
Glu Ala Thr Phe Thr Asn Pro Ala Asp Asn Leu Gln His Leu Asn Asp
995 1000 1005
Gly Ile Lys Ser Tyr Thr Asn Asn Gln Val Asn Arg Trp Thr Asn
1010 1015 1020
Trp Thr Arg Thr Pro Arg Asp Ala Gly Asp Ser Ile Thr Val Asn
1025 1030 1035
Phe Gly Lys Lys His Val Ile Asn Asn Leu Asp Leu Phe Val Phe
1040 1045 1050
Thr Asp Ser Gly Thr Val Val Pro Glu Lys Ala Glu Val Gln Tyr
1055 1060 1065
Trp Asp Gly Thr Ala Val Lys Asp Val Glu Asn Leu Thr Gln Pro
1070 1075 1080
Ser Pro Tyr Val Val Glu Lys Asn Glu Leu Thr Phe Asp Ala Val
1085 1090 1095
Ala Thr Glu Lys Leu Lys Phe His Leu Thr Pro Ser Val Lys Gly
1100 1105 1110
Lys Phe Leu Ala Leu Thr Glu Ala Glu Val Tyr Ala Asp Gln Ile
1115 1120 1125
Val Asp Gly Glu Thr Ala Lys Leu Gln Ser Ile Thr Val Asn Gly
1130 1135 1140
Lys Ala Leu Glu Gly Phe Asp His Ala Lys Lys Asn Tyr Glu Leu
1145 1150 1155
Val Leu Pro Tyr Gly Ser Glu Leu Pro Lys Ile Glu Ala Ala Ala
1160 1165 1170
Ala Asp Asn Ala Thr Val Thr Ile Leu Pro Ala Phe Ser Tyr Pro
1175 1180 1185
Gly Thr Ala Lys Leu Phe Val Thr Ser Glu Asp Gly Lys Val Thr
1190 1195 1200
Thr Glu Tyr Ser Ile Gly Val Ser Thr Glu Glu Pro Lys Leu Val
1205 1210 1215
Ser Ala Glu Leu Ser Ala Asp Lys Thr Asn Val Met Glu Asp Asp
1220 1225 1230
Ile Ile Asp Leu Lys Val Ile Gly Leu Phe Glu Ser Lys Glu Lys
1235 1240 1245
Ile Asp Val Thr Asp Ser Gln Pro Thr Tyr Glu Phe Asp Gln Gln
1250 1255 1260
Ile Ile Lys Ile Glu Gly Asn Lys Leu Tyr Ala Leu Glu Thr Gly
1265 1270 1275
Asn Val Lys Val Lys Val Thr Val Thr Tyr Lys Gly Val Ser Val
1280 1285 1290
Thr Thr Pro Ala Leu Glu Phe Thr Ile Ala Lys His Pro Ala Pro
1295 1300 1305
Lys Tyr Ile Thr Ser Leu Glu Pro Val Thr Val Val Val Lys Lys
1310 1315 1320
Gly Glu Ala Pro Glu Leu Pro Ala Thr Val Val Ala His Tyr Asn
1325 1330 1335
Arg Gly Ile Pro Arg Asp Val Lys Val Lys Trp Glu Arg Ile His
1340 1345 1350
Pro Ser Lys Tyr Gln Gln Leu Gly Glu Phe Thr Val Ser Gly Met
1355 1360 1365
Val Glu Gly Thr Asp Ile Lys Ala Gln Ala Lys Val Ala Val Lys
1370 1375 1380
Gly Ala Val Ile Val Glu Asp Ile Arg Met Ala Val Leu Leu Lys
1385 1390 1395
Gln Met Pro Gln Leu Pro Gly Lys Val Thr Val Tyr Tyr Ser Asp
1400 1405 1410
Gly Ala Glu Glu Gln Arg Ala Val Lys Trp Glu Glu Ile Pro Gln
1415 1420 1425
Glu Glu Leu Glu Lys Val Gly Glu Phe Lys Val Asn Gly Asp Val
1430 1435 1440
Asn Gly Val Lys Leu Lys Ala Thr Ala Thr Ile Arg Val Thr Asp
1445 1450 1455
Glu Val Gly Ala Glu Gln Asn Ile Ser Arg Ala Lys Asn Gly Tyr
1460 1465 1470
Glu Tyr Pro Lys Ala Glu Ala Ser Phe Thr Asn Asp Gly Pro Gly
1475 1480 1485
Ser Ser Asp Arg Ile Glu Ala Ile Asn Asp Asp Val Ile Ser Tyr
1490 1495 1500
Glu Ala Asn Pro His Asn Arg Trp Thr Asn Trp Gln Pro Ala Pro
1505 1510 1515
Arg Val Gly Asp Leu Val Phe Thr Thr Phe Gly Asp Tyr Glu Pro
1520 1525 1530
Thr Glu Tyr Asp Val Asp Ser Met Glu Ile His Trp Phe Ala Asp
1535 1540 1545
His Gly Thr Ser Tyr Pro Glu Arg Phe Gln Ile Glu Tyr Lys Ser
1550 1555 1560
Gly Asp Ser Trp Lys Glu Val Thr Ser Leu Lys Ser Asp Pro Ala
1565 1570 1575
Ser Pro Ala Leu Gly Lys Ala Asn Val Tyr Ser Phe Asp Arg Val
1580 1585 1590
Lys Thr Ser Ala Ile Arg Val Lys Met Thr Ala Gln Ala Gly Lys
1595 1600 1605
Ser Leu Ala Ile Thr Glu Leu Lys Val Phe Ser Lys Trp Pro Lys
1610 1615 1620
Ala Gly Thr Glu Pro Asp Val Thr Asp Ile Lys Val Gly Gly Lys
1625 1630 1635
Ser Ile Leu Glu Glu Phe Glu Gln Glu Gly Asp His Tyr Glu Ala
1640 1645 1650
Thr Ile Asp Val Gly Asp Ala Asn Val Met Pro Lys Ile Tyr Val
1655 1660 1665
Lys Ala Lys Asp Gln Thr Ser Ile Ser Ile Val Pro Ala Val Thr
1670 1675 1680
Ser Val Ser Thr Ala Lys Val Ile Ala Glu Asp Gly Asp Gly Lys
1685 1690 1695
Lys Val Lys Val Tyr Ser Ile His Lys Ser Tyr Lys
1700 1705 1710
<210> 3
<211> 3672
<212> DNA
<213> 人工序列
<400> 3
atgaaaagca cgacgagcgc cgccggcaaa agcgtcagct ataacgacgg cgaacgccgc 60
gtcaactttg aaaactggcg ctttcagcgc gaaacgaacg gcagcatcgc cggcgcccag 120
aacccgggct ttgacgacag cagctggcgc aaactgaacc tgccgcatga atggagcatc 180
gacctggact ttaacaaaaa cagcctggcc acgcatgaag gcggctatct ggacggcggc 240
atcggctgga accgcaaaac gtttacgatc ccggaaagca tgaaaggcaa acgctttagc 300
ctggacatcg acggcgtcta tatgtatagc acgacgtatc tgaacgcctt tgtcctgggc 360
acgtatccgt ttggctataa cggcgaaagc tatgacatca gcgacaaact gtataaagac 420
ggccgcgcca acgtcctggt cgtcaaagtc aacacgaacc agccgagcgg ccgctggtat 480
agcggcagca gcatctatcg caacgtctat ctgacggtca cggacccgat ccatgtcgcc 540
cgctatggca cgtttgtcac gacgccgaac ctggaaaaaa gcatcaaaga agaccgcgac 600
gccgtcaaca tcaaaacgaa aatcagcaac gccgaagcca aacaggtcaa aatcgccgac 660
aaaagcacga tctatgacgg cgccggcaac acggtccaga cggtcgaaac ggaagaaaaa 720
acggccgccg gcgccacggt cacgccgttt gaacagaaca cggtcatcaa acagccgaaa 780
ctgtggagca tcgacaaacc gtatcgctat aacctggtca cggaagtcat cgtcggcggc 840
cagacggtcg acacgtatga aacgaaattt ggcgtccgct attttaaatt tgacgaaaac 900
gaaggctata gcctgaacgg cgaatttatg aaactgcatg gcgtcagcat gcatcatgac 960
ctggccggcg gcgccgccct gacgaacgcc cgcggcgtcg aacgccagat gcagatcatg 1020
aaagacgtcg gcgtcaacgc catccgcgtc acgcataacc cggccagccc ggaactgggc 1080
ctggaatttg ccgccaacaa actgctgatc atcgaagaag cctttgacag ctgggcccag 1140
agcaaaaaac cgtatgacta tggccgcttt tttaacgcct gggccgaaca tgacatcaaa 1200
gaaatggtcg accgcggcaa aaacgaaccg gccatcatca tgtggagcat cggcaacgaa 1260
atctatgaca cgacgaacgc cgccggcgtc gaaacggccc gcaacctggt cggctgggtc 1320
aaagaaatcg acacgacgcc gcgcgccacg atcggcgaag acaaaacgcg cggcgacaac 1380
gtcacgccga tcaacagcta tatcaaagaa atctttaaca tcgtcgacgt cgtcggcctg 1440
aactatagcg aaaacaacta tgacggctat cataaacaga acccgagctg gaaactgtat 1500
ggcagcgaaa cgagcagcgc cacgcgcagc cgcggcgtct atacgcagcc gtatcagtat 1560
aaccatgaca cgaaatatgc cgacctgcag cagagcagct atgacaacga ctatgtcggc 1620
tggggccgca cggcccagga cgcctggaaa tatgaccgcg acctgaaaca tatcgccggc 1680
cagtttatct ggacgggctt tgactatatc ggcgaaccga cgccgtatta taacagctat 1740
ccggccaaaa gcagctattt tggcgccgtc gacacggccg gctttccgaa agacatcttt 1800
tattattatc agagccagtg gaaaaaagaa ccgatggtcc atctgctgcc gcattggaac 1860
tggaaagaag gcgaaaaagt ccgcgtcctg gcctatacga acgccagcaa agtcgaactg 1920
gtcctgaacg gcgaaagcct gggcaacgaa aaatatgaca acaaacagac gagctggggc 1980
gccccgtata aagaaacgaa agacggcaaa acgtatctgg aatgggccgt caaaccgctg 2040
aaaccgggca aatttgacga agccgtcgcc gaaaacggca aagtcatcgc ccgcgaccag 2100
gtcgtcacgg ccggcgaacc ggccagcgtc cgcctgacgg ccgaccgcaa agtcgtcaaa 2160
gccgacggca cggaccatag ctttatcacg gccgacatcg tcgacagcaa aggcatcgtc 2220
gtcccggacg ccgaccatct gatcacgttt aacgtcacgg gccagggcga actggccggc 2280
gtcgacaacg gcaacgccag cagcgtcgaa cgctataaag acaacaaacg caaagccttt 2340
agcggcaaag ccctggccat cgtccagagc agcaaactga gcggcaaaat cacggtccat 2400
gccagcgtcg ccggcctgag cagcgacagc acgagcgtct ttacggtcac gccggccgac 2460
catgacaaaa aaatcgtcgc cggcatcgac gacgtcaacc tgacggtcga cgtcaacgaa 2520
gccccgaaac tgccgagcga aatcaaagtc tattatagcg acgaaagcgc cgccgccaaa 2580
aacgtcacgt gggacgaagt cgacccgaaa cagtatagca cggtcggcga atttacggtc 2640
gaaggcagcg tcgaaggcac gagcctgaaa gccaaagcct ttgtcatcgt caaaggcatc 2700
gtcgccgtca aaccgtatag cacggccacg aaagtcggcg tccagccggt cctgccggaa 2760
aaagccacgc tgctgtatag cgacggcacg acgaaaggcg ccacggtcac gtgggacgaa 2820
ctgccggaag acaaactggc caaagaaggc cgctttacgg tcgaaggcag cgtcgaaggc 2880
acggacctga aagccaacgt ctatgtccgc gtcacgaacg aagtcaaaag cgtcaacatc 2940
atgctgcagg aacagggcag cgcctatccg aaactggaag ccacgtttac gaacccggcc 3000
gacaacctgc agcatctgaa cgacggcatc aaaagctata cgaacaacca ggtcaaccgc 3060
tggacgaact ggacgcgcac gccgcgcgac gccggcgaca gcatcacggt caactttggc 3120
aaaaaacatg tcatcaacaa cctggacctg tttgtcttta cggacagcgg cacggtcgtc 3180
ccggaaaaag ccgaagtcca gtattgggac ggcacggccg tcaaagacgt cgaaaacctg 3240
acgcagccga gcccgtatgt cgtcgaaaaa aacgaactga cgtttgacgc cgtcgccacg 3300
gaaaaactga aatttcatct gacgccgagc gtcaaaggca aatttctggc cctgacggaa 3360
gccgaagtct atgccgacca gatcgtcgac ggcgaaacgg ccaaactgca gagcatcacg 3420
gtcaacggca aagccctgga aggctttgac catgccaaaa aaaactatga actggtcctg 3480
ccgtatggca gcgaactgcc gaaaatcgaa gccgccgccg ccgacaacgc cacggtcacg 3540
atcctgccgg cctttagcta tccgggcacg gccaaactgt ttgtcacgag cgaagacggc 3600
aaagtcacga cggaatatag catcggcgtc agcacggaag aaccgaaact ggtcagcgcc 3660
gaactgagct aa 3672
<210> 4
<211> 1223
<212> PRT
<213> 人工序列
<400> 4
Met Lys Ser Thr Thr Ser Ala Ala Gly Lys Ser Val Ser Tyr Asn Asp
1 5 10 15
Gly Glu Arg Arg Val Asn Phe Glu Asn Trp Arg Phe Gln Arg Glu Thr
20 25 30
Asn Gly Ser Ile Ala Gly Ala Gln Asn Pro Gly Phe Asp Asp Ser Ser
35 40 45
Trp Arg Lys Leu Asn Leu Pro His Glu Trp Ser Ile Asp Leu Asp Phe
50 55 60
Asn Lys Asn Ser Leu Ala Thr His Glu Gly Gly Tyr Leu Asp Gly Gly
65 70 75 80
Ile Gly Trp Asn Arg Lys Thr Phe Thr Ile Pro Glu Ser Met Lys Gly
85 90 95
Lys Arg Phe Ser Leu Asp Ile Asp Gly Val Tyr Met Tyr Ser Thr Thr
100 105 110
Tyr Leu Asn Ala Phe Val Leu Gly Thr Tyr Pro Phe Gly Tyr Asn Gly
115 120 125
Glu Ser Tyr Asp Ile Ser Asp Lys Leu Tyr Lys Asp Gly Arg Ala Asn
130 135 140
Val Leu Val Val Lys Val Asn Thr Asn Gln Pro Ser Gly Arg Trp Tyr
145 150 155 160
Ser Gly Ser Ser Ile Tyr Arg Asn Val Tyr Leu Thr Val Thr Asp Pro
165 170 175
Ile His Val Ala Arg Tyr Gly Thr Phe Val Thr Thr Pro Asn Leu Glu
180 185 190
Lys Ser Ile Lys Glu Asp Arg Asp Ala Val Asn Ile Lys Thr Lys Ile
195 200 205
Ser Asn Ala Glu Ala Lys Gln Val Lys Ile Ala Asp Lys Ser Thr Ile
210 215 220
Tyr Asp Gly Ala Gly Asn Thr Val Gln Thr Val Glu Thr Glu Glu Lys
225 230 235 240
Thr Ala Ala Gly Ala Thr Val Thr Pro Phe Glu Gln Asn Thr Val Ile
245 250 255
Lys Gln Pro Lys Leu Trp Ser Ile Asp Lys Pro Tyr Arg Tyr Asn Leu
260 265 270
Val Thr Glu Val Ile Val Gly Gly Gln Thr Val Asp Thr Tyr Glu Thr
275 280 285
Lys Phe Gly Val Arg Tyr Phe Lys Phe Asp Glu Asn Glu Gly Tyr Ser
290 295 300
Leu Asn Gly Glu Phe Met Lys Leu His Gly Val Ser Met His His Asp
305 310 315 320
Leu Ala Gly Gly Ala Ala Leu Thr Asn Ala Arg Gly Val Glu Arg Gln
325 330 335
Met Gln Ile Met Lys Asp Val Gly Val Asn Ala Ile Arg Val Thr His
340 345 350
Asn Pro Ala Ser Pro Glu Leu Gly Leu Glu Phe Ala Ala Asn Lys Leu
355 360 365
Leu Ile Ile Glu Glu Ala Phe Asp Ser Trp Ala Gln Ser Lys Lys Pro
370 375 380
Tyr Asp Tyr Gly Arg Phe Phe Asn Ala Trp Ala Glu His Asp Ile Lys
385 390 395 400
Glu Met Val Asp Arg Gly Lys Asn Glu Pro Ala Ile Ile Met Trp Ser
405 410 415
Ile Gly Asn Glu Ile Tyr Asp Thr Thr Asn Ala Ala Gly Val Glu Thr
420 425 430
Ala Arg Asn Leu Val Gly Trp Val Lys Glu Ile Asp Thr Thr Pro Arg
435 440 445
Ala Thr Ile Gly Glu Asp Lys Thr Arg Gly Asp Asn Val Thr Pro Ile
450 455 460
Asn Ser Tyr Ile Lys Glu Ile Phe Asn Ile Val Asp Val Val Gly Leu
465 470 475 480
Asn Tyr Ser Glu Asn Asn Tyr Asp Gly Tyr His Lys Gln Asn Pro Ser
485 490 495
Trp Lys Leu Tyr Gly Ser Glu Thr Ser Ser Ala Thr Arg Ser Arg Gly
500 505 510
Val Tyr Thr Gln Pro Tyr Gln Tyr Asn His Asp Thr Lys Tyr Ala Asp
515 520 525
Leu Gln Gln Ser Ser Tyr Asp Asn Asp Tyr Val Gly Trp Gly Arg Thr
530 535 540
Ala Gln Asp Ala Trp Lys Tyr Asp Arg Asp Leu Lys His Ile Ala Gly
545 550 555 560
Gln Phe Ile Trp Thr Gly Phe Asp Tyr Ile Gly Glu Pro Thr Pro Tyr
565 570 575
Tyr Asn Ser Tyr Pro Ala Lys Ser Ser Tyr Phe Gly Ala Val Asp Thr
580 585 590
Ala Gly Phe Pro Lys Asp Ile Phe Tyr Tyr Tyr Gln Ser Gln Trp Lys
595 600 605
Lys Glu Pro Met Val His Leu Leu Pro His Trp Asn Trp Lys Glu Gly
610 615 620
Glu Lys Val Arg Val Leu Ala Tyr Thr Asn Ala Ser Lys Val Glu Leu
625 630 635 640
Val Leu Asn Gly Glu Ser Leu Gly Asn Glu Lys Tyr Asp Asn Lys Gln
645 650 655
Thr Ser Trp Gly Ala Pro Tyr Lys Glu Thr Lys Asp Gly Lys Thr Tyr
660 665 670
Leu Glu Trp Ala Val Lys Pro Leu Lys Pro Gly Lys Phe Asp Glu Ala
675 680 685
Val Ala Glu Asn Gly Lys Val Ile Ala Arg Asp Gln Val Val Thr Ala
690 695 700
Gly Glu Pro Ala Ser Val Arg Leu Thr Ala Asp Arg Lys Val Val Lys
705 710 715 720
Ala Asp Gly Thr Asp His Ser Phe Ile Thr Ala Asp Ile Val Asp Ser
725 730 735
Lys Gly Ile Val Val Pro Asp Ala Asp His Leu Ile Thr Phe Asn Val
740 745 750
Thr Gly Gln Gly Glu Leu Ala Gly Val Asp Asn Gly Asn Ala Ser Ser
755 760 765
Val Glu Arg Tyr Lys Asp Asn Lys Arg Lys Ala Phe Ser Gly Lys Ala
770 775 780
Leu Ala Ile Val Gln Ser Ser Lys Leu Ser Gly Lys Ile Thr Val His
785 790 795 800
Ala Ser Val Ala Gly Leu Ser Ser Asp Ser Thr Ser Val Phe Thr Val
805 810 815
Thr Pro Ala Asp His Asp Lys Lys Ile Val Ala Gly Ile Asp Asp Val
820 825 830
Asn Leu Thr Val Asp Val Asn Glu Ala Pro Lys Leu Pro Ser Glu Ile
835 840 845
Lys Val Tyr Tyr Ser Asp Glu Ser Ala Ala Ala Lys Asn Val Thr Trp
850 855 860
Asp Glu Val Asp Pro Lys Gln Tyr Ser Thr Val Gly Glu Phe Thr Val
865 870 875 880
Glu Gly Ser Val Glu Gly Thr Ser Leu Lys Ala Lys Ala Phe Val Ile
885 890 895
Val Lys Gly Ile Val Ala Val Lys Pro Tyr Ser Thr Ala Thr Lys Val
900 905 910
Gly Val Gln Pro Val Leu Pro Glu Lys Ala Thr Leu Leu Tyr Ser Asp
915 920 925
Gly Thr Thr Lys Gly Ala Thr Val Thr Trp Asp Glu Leu Pro Glu Asp
930 935 940
Lys Leu Ala Lys Glu Gly Arg Phe Thr Val Glu Gly Ser Val Glu Gly
945 950 955 960
Thr Asp Leu Lys Ala Asn Val Tyr Val Arg Val Thr Asn Glu Val Lys
965 970 975
Ser Val Asn Ile Met Leu Gln Glu Gln Gly Ser Ala Tyr Pro Lys Leu
980 985 990
Glu Ala Thr Phe Thr Asn Pro Ala Asp Asn Leu Gln His Leu Asn Asp
995 1000 1005
Gly Ile Lys Ser Tyr Thr Asn Asn Gln Val Asn Arg Trp Thr Asn
1010 1015 1020
Trp Thr Arg Thr Pro Arg Asp Ala Gly Asp Ser Ile Thr Val Asn
1025 1030 1035
Phe Gly Lys Lys His Val Ile Asn Asn Leu Asp Leu Phe Val Phe
1040 1045 1050
Thr Asp Ser Gly Thr Val Val Pro Glu Lys Ala Glu Val Gln Tyr
1055 1060 1065
Trp Asp Gly Thr Ala Val Lys Asp Val Glu Asn Leu Thr Gln Pro
1070 1075 1080
Ser Pro Tyr Val Val Glu Lys Asn Glu Leu Thr Phe Asp Ala Val
1085 1090 1095
Ala Thr Glu Lys Leu Lys Phe His Leu Thr Pro Ser Val Lys Gly
1100 1105 1110
Lys Phe Leu Ala Leu Thr Glu Ala Glu Val Tyr Ala Asp Gln Ile
1115 1120 1125
Val Asp Gly Glu Thr Ala Lys Leu Gln Ser Ile Thr Val Asn Gly
1130 1135 1140
Lys Ala Leu Glu Gly Phe Asp His Ala Lys Lys Asn Tyr Glu Leu
1145 1150 1155
Val Leu Pro Tyr Gly Ser Glu Leu Pro Lys Ile Glu Ala Ala Ala
1160 1165 1170
Ala Asp Asn Ala Thr Val Thr Ile Leu Pro Ala Phe Ser Tyr Pro
1175 1180 1185
Gly Thr Ala Lys Leu Phe Val Thr Ser Glu Asp Gly Lys Val Thr
1190 1195 1200
Thr Glu Tyr Ser Ile Gly Val Ser Thr Glu Glu Pro Lys Leu Val
1205 1210 1215
Ser Ala Glu Leu Ser
1220
<210> 5
<211> 2436
<212> DNA
<213> 人工序列
<400> 5
atgaaaagca cgacgagcgc cgccggcaaa agcgtcagct ataacgacgg cgaacgccgc 60
gtcaactttg aaaactggcg ctttcagcgc gaaacgaacg gcagcatcgc cggcgcccag 120
aacccgggct ttgacgacag cagctggcgc aaactgaacc tgccgcatga atggagcatc 180
gacctggact ttaacaaaaa cagcctggcc acgcatgaag gcggctatct ggacggcggc 240
atcggctgga accgcaaaac gtttacgatc ccggaaagca tgaaaggcaa acgctttagc 300
ctggacatcg acggcgtcta tatgtatagc acgacgtatc tgaacgcctt tgtcctgggc 360
acgtatccgt ttggctataa cggcgaaagc tatgacatca gcgacaaact gtataaagac 420
ggccgcgcca acgtcctggt cgtcaaagtc aacacgaacc agccgagcgg ccgctggtat 480
agcggcagca gcatctatcg caacgtctat ctgacggtca cggacccgat ccatgtcgcc 540
cgctatggca cgtttgtcac gacgccgaac ctggaaaaaa gcatcaaaga agaccgcgac 600
gccgtcaaca tcaaaacgaa aatcagcaac gccgaagcca aacaggtcaa aatcgccgac 660
aaaagcacga tctatgacgg cgccggcaac acggtccaga cggtcgaaac ggaagaaaaa 720
acggccgccg gcgccacggt cacgccgttt gaacagaaca cggtcatcaa acagccgaaa 780
ctgtggagca tcgacaaacc gtatcgctat aacctggtca cggaagtcat cgtcggcggc 840
cagacggtcg acacgtatga aacgaaattt ggcgtccgct attttaaatt tgacgaaaac 900
gaaggctata gcctgaacgg cgaatttatg aaactgcatg gcgtcagcat gcatcatgac 960
ctggccggcg gcgccgccct gacgaacgcc cgcggcgtcg aacgccagat gcagatcatg 1020
aaagacgtcg gcgtcaacgc catccgcgtc acgcataacc cggccagccc ggaactgggc 1080
ctggaatttg ccgccaacaa actgctgatc atcgaagaag cctttgacag ctgggcccag 1140
agcaaaaaac cgtatgacta tggccgcttt tttaacgcct gggccgaaca tgacatcaaa 1200
gaaatggtcg accgcggcaa aaacgaaccg gccatcatca tgtggagcat cggcaacgaa 1260
atctatgaca cgacgaacgc cgccggcgtc gaaacggccc gcaacctggt cggctgggtc 1320
aaagaaatcg acacgacgcc gcgcgccacg atcggcgaag acaaaacgcg cggcgacaac 1380
gtcacgccga tcaacagcta tatcaaagaa atctttaaca tcgtcgacgt cgtcggcctg 1440
aactatagcg aaaacaacta tgacggctat cataaacaga acccgagctg gaaactgtat 1500
ggcagcgaaa cgagcagcgc cacgcgcagc cgcggcgtct atacgcagcc gtatcagtat 1560
aaccatgaca cgaaatatgc cgacctgcag cagagcagct atgacaacga ctatgtcggc 1620
tggggccgca cggcccagga cgcctggaaa tatgaccgcg acctgaaaca tatcgccggc 1680
cagtttatct ggacgggctt tgactatatc ggcgaaccga cgccgtatta taacagctat 1740
ccggccaaaa gcagctattt tggcgccgtc gacacggccg gctttccgaa agacatcttt 1800
tattattatc agagccagtg gaaaaaagaa ccgatggtcc atctgctgcc gcattggaac 1860
tggaaagaag gcgaaaaagt ccgcgtcctg gcctatacga acgccagcaa agtcgaactg 1920
gtcctgaacg gcgaaagcct gggcaacgaa aaatatgaca acaaacagac gagctggggc 1980
gccccgtata aagaaacgaa agacggcaaa acgtatctgg aatgggccgt caaaccgctg 2040
aaaccgggca aatttgacga agccgtcgcc gaaaacggca aagtcatcgc ccgcgaccag 2100
gtcgtcacgg ccggcgaacc ggccagcgtc cgcctgacgg ccgaccgcaa agtcgtcaaa 2160
gccgacggca cggaccatag ctttatcacg gccgacatcg tcgacagcaa aggcatcgtc 2220
gtcccggacg ccgaccatct gatcacgttt aacgtcacgg gccagggcga actggccggc 2280
gtcgacaacg gcaacgccag cagcgtcgaa cgctataaag acaacaaacg caaagccttt 2340
agcggcaaag ccctggccat cgtccagagc agcaaactga gcggcaaaat cacggtccat 2400
gccagcgtcg ccggcctgag cagcgacagc acgtaa 2436
<210> 6
<211> 811
<212> PRT
<213> 人工序列
<400> 6
Met Lys Ser Thr Thr Ser Ala Ala Gly Lys Ser Val Ser Tyr Asn Asp
1 5 10 15
Gly Glu Arg Arg Val Asn Phe Glu Asn Trp Arg Phe Gln Arg Glu Thr
20 25 30
Asn Gly Ser Ile Ala Gly Ala Gln Asn Pro Gly Phe Asp Asp Ser Ser
35 40 45
Trp Arg Lys Leu Asn Leu Pro His Glu Trp Ser Ile Asp Leu Asp Phe
50 55 60
Asn Lys Asn Ser Leu Ala Thr His Glu Gly Gly Tyr Leu Asp Gly Gly
65 70 75 80
Ile Gly Trp Asn Arg Lys Thr Phe Thr Ile Pro Glu Ser Met Lys Gly
85 90 95
Lys Arg Phe Ser Leu Asp Ile Asp Gly Val Tyr Met Tyr Ser Thr Thr
100 105 110
Tyr Leu Asn Ala Phe Val Leu Gly Thr Tyr Pro Phe Gly Tyr Asn Gly
115 120 125
Glu Ser Tyr Asp Ile Ser Asp Lys Leu Tyr Lys Asp Gly Arg Ala Asn
130 135 140
Val Leu Val Val Lys Val Asn Thr Asn Gln Pro Ser Gly Arg Trp Tyr
145 150 155 160
Ser Gly Ser Ser Ile Tyr Arg Asn Val Tyr Leu Thr Val Thr Asp Pro
165 170 175
Ile His Val Ala Arg Tyr Gly Thr Phe Val Thr Thr Pro Asn Leu Glu
180 185 190
Lys Ser Ile Lys Glu Asp Arg Asp Ala Val Asn Ile Lys Thr Lys Ile
195 200 205
Ser Asn Ala Glu Ala Lys Gln Val Lys Ile Ala Asp Lys Ser Thr Ile
210 215 220
Tyr Asp Gly Ala Gly Asn Thr Val Gln Thr Val Glu Thr Glu Glu Lys
225 230 235 240
Thr Ala Ala Gly Ala Thr Val Thr Pro Phe Glu Gln Asn Thr Val Ile
245 250 255
Lys Gln Pro Lys Leu Trp Ser Ile Asp Lys Pro Tyr Arg Tyr Asn Leu
260 265 270
Val Thr Glu Val Ile Val Gly Gly Gln Thr Val Asp Thr Tyr Glu Thr
275 280 285
Lys Phe Gly Val Arg Tyr Phe Lys Phe Asp Glu Asn Glu Gly Tyr Ser
290 295 300
Leu Asn Gly Glu Phe Met Lys Leu His Gly Val Ser Met His His Asp
305 310 315 320
Leu Ala Gly Gly Ala Ala Leu Thr Asn Ala Arg Gly Val Glu Arg Gln
325 330 335
Met Gln Ile Met Lys Asp Val Gly Val Asn Ala Ile Arg Val Thr His
340 345 350
Asn Pro Ala Ser Pro Glu Leu Gly Leu Glu Phe Ala Ala Asn Lys Leu
355 360 365
Leu Ile Ile Glu Glu Ala Phe Asp Ser Trp Ala Gln Ser Lys Lys Pro
370 375 380
Tyr Asp Tyr Gly Arg Phe Phe Asn Ala Trp Ala Glu His Asp Ile Lys
385 390 395 400
Glu Met Val Asp Arg Gly Lys Asn Glu Pro Ala Ile Ile Met Trp Ser
405 410 415
Ile Gly Asn Glu Ile Tyr Asp Thr Thr Asn Ala Ala Gly Val Glu Thr
420 425 430
Ala Arg Asn Leu Val Gly Trp Val Lys Glu Ile Asp Thr Thr Pro Arg
435 440 445
Ala Thr Ile Gly Glu Asp Lys Thr Arg Gly Asp Asn Val Thr Pro Ile
450 455 460
Asn Ser Tyr Ile Lys Glu Ile Phe Asn Ile Val Asp Val Val Gly Leu
465 470 475 480
Asn Tyr Ser Glu Asn Asn Tyr Asp Gly Tyr His Lys Gln Asn Pro Ser
485 490 495
Trp Lys Leu Tyr Gly Ser Glu Thr Ser Ser Ala Thr Arg Ser Arg Gly
500 505 510
Val Tyr Thr Gln Pro Tyr Gln Tyr Asn His Asp Thr Lys Tyr Ala Asp
515 520 525
Leu Gln Gln Ser Ser Tyr Asp Asn Asp Tyr Val Gly Trp Gly Arg Thr
530 535 540
Ala Gln Asp Ala Trp Lys Tyr Asp Arg Asp Leu Lys His Ile Ala Gly
545 550 555 560
Gln Phe Ile Trp Thr Gly Phe Asp Tyr Ile Gly Glu Pro Thr Pro Tyr
565 570 575
Tyr Asn Ser Tyr Pro Ala Lys Ser Ser Tyr Phe Gly Ala Val Asp Thr
580 585 590
Ala Gly Phe Pro Lys Asp Ile Phe Tyr Tyr Tyr Gln Ser Gln Trp Lys
595 600 605
Lys Glu Pro Met Val His Leu Leu Pro His Trp Asn Trp Lys Glu Gly
610 615 620
Glu Lys Val Arg Val Leu Ala Tyr Thr Asn Ala Ser Lys Val Glu Leu
625 630 635 640
Val Leu Asn Gly Glu Ser Leu Gly Asn Glu Lys Tyr Asp Asn Lys Gln
645 650 655
Thr Ser Trp Gly Ala Pro Tyr Lys Glu Thr Lys Asp Gly Lys Thr Tyr
660 665 670
Leu Glu Trp Ala Val Lys Pro Leu Lys Pro Gly Lys Phe Asp Glu Ala
675 680 685
Val Ala Glu Asn Gly Lys Val Ile Ala Arg Asp Gln Val Val Thr Ala
690 695 700
Gly Glu Pro Ala Ser Val Arg Leu Thr Ala Asp Arg Lys Val Val Lys
705 710 715 720
Ala Asp Gly Thr Asp His Ser Phe Ile Thr Ala Asp Ile Val Asp Ser
725 730 735
Lys Gly Ile Val Val Pro Asp Ala Asp His Leu Ile Thr Phe Asn Val
740 745 750
Thr Gly Gln Gly Glu Leu Ala Gly Val Asp Asn Gly Asn Ala Ser Ser
755 760 765
Val Glu Arg Tyr Lys Asp Asn Lys Arg Lys Ala Phe Ser Gly Lys Ala
770 775 780
Leu Ala Ile Val Gln Ser Ser Lys Leu Ser Gly Lys Ile Thr Val His
785 790 795 800
Ala Ser Val Ala Gly Leu Ser Ser Asp Ser Thr
805 810
<210> 7
<211> 5133
<212> DNA
<213> 环状芽胞杆菌ATCC 31382
<400> 7
atgagcaaaa cgacgagcgc cgccggcaac agcgtcagct atgacggcga acgccgcgtc 60
aactttaacg aaaactggcg ctttcagcgc gaaacgaacg gcagcatcgc cggcgcccag 120
aacccaggct ttgacgacag cagctggcgc aaactgaacc tgccgcatga ctggagcatc 180
gaactggact ttaacaaaaa cagcctggcc acgcatgaag gcggctatct ggacggcggc 240
atcggctggt atcgcaaaac gtttacgatc ccggaaagca tgaaaggcaa acgcatcagc 300
ctggactttg acggcgtcta tatgaacagc acgacgtatc tgaacggcga agtcctgggc 360
acgtatccgt ttggctataa cgcctttagc tatgacatca gcgacaaact gtataaagac 420
ggccgcgcca acgtcctggt cgtcaaagtc aacaacacgc agccgagcag ccgctggtat 480
agcggcagcg gcatctatcg caacgtctat ctgacggtca cggacccgat ccatgtcgcc 540
cgctatggca cgtttgtcac gacgccgaac ctggaaaaaa gcatcaaaga agaccgcgcc 600
gacgtcaaca tcaaaacgaa aatcagcaac gacgccgccg aagccaaaca ggtcaaaatc 660
aaaagcacga tctatgacgg cgccggcaac acggtccaga cggtcgaaac ggaagaaaaa 720
acggccgccg ccggcacggt cacgccgttt gaacagaaca cggtcatcaa acagccgaaa 780
ctgtggagca tcgacaaacc gtatcgctat aacctggtca cggaagtcat cgtcggcggc 840
cagacggtcg acacgtatga aacgaaattt ggcgtccgct attttaaatt tgacgaaaac 900
gaaggcttta gcctgaacgg cgaatatatg aaactgcatg gcgtcagcat gcatcatgac 960
ctgggcgccc tgggcgccgc cacgaacgcc cgcggcgtcg aacgccagat gcagatcatg 1020
aaagacatgg gcgtcaacgc catccgcgtc acgcataacc cggccagccc ggaactgctg 1080
gaagccgcca acaaactggg cctgtttatc atcgaagaag cctttgacag ctgggcccag 1140
agcaaaaaac cgtatgacta tggccgcttt tttaacgcct gggccgaaca tgacatcaaa 1200
gaaatggtcg accgcggcaa aaacgaaccg gccatcatca tgtggagcat cggcaacgaa 1260
atctatgaca cgacgaacgc cgccggcgtc gaaacggccc gcaacctggt cggctgggtc 1320
aaagaaatcg acacgacgcg cccgacgacg atcggcgaag acaaaacgcg cggcgacaaa 1380
gtcaacgtca cgccgatcaa cagctatatc aaagaaatct ttaacatcgt cgacgtcgtc 1440
ggcctgaact atagcgaaaa caactatgac ggctatcata aacagaaccc gagctggaaa 1500
ctgtatggca gcgaaacgag cagcgccacg cgcagccgcg gcgtctatac gcatccgtat 1560
cagtataacc agagcacgaa atatgccgac ctgcagcaga gcagctatga caacgactat 1620
gtcggctggg gccgcacggc cgaagacgcc tggaaatatg accgcgacct gaaacatatc 1680
gccggccagt ttatctggac gggctttgac tatatcggcg aaccgacgcc gtattataac 1740
agctatccgg ccaaaagcag ctattttggc gccgtcgaca cggccggctt tccgaaagac 1800
atcttttatt attatcagag ccagtggaaa aaagaaccga tggtccatct gctgccgcat 1860
tggaactgga aagaaggcga aaaagtccgc gtcctggcct atacgaacgc cagcaaagtc 1920
gaactggtcc tgaacggcga aagcctgggc gaaaaaaact atgacaacaa acagacgagc 1980
tggggcgccc cgtataaaga aacgaaagac ggcaaaacgt atctggaatg ggccgtcccg 2040
tttaaaccgg gcaaactgga agccgtcgcc aaagacgaaa acggcaaagt catcgcccgc 2100
gaccaggtcg tcacggccgg cgaaccggcc agcgtccgcc tgacggccga ccgcaaagtc 2160
gtcaaagccg acggcacgga cctgagcttt atcacggccg acatcgtcga cagcaaaggc 2220
atcgtcgtcc cggacgccga ccatctgatc acgtttaacg tcacgggcca gggcgaactg 2280
gccggcgtcg acaacggcaa cgccagcagc gtcgaacgct ataaagacaa caaacgcaaa 2340
gcctttagcg gcaaagccct ggccatcgtc cagagcagca aactgagcgg caaaatcacg 2400
gtccatgcca gcgtcgccgg cctgagcagc gacagcacga gcgtctttac ggtcacgccg 2460
gccgaccatg acaaaaaaat cgtcgccggc atcgacgacg tcaacctgac ggtcgacgtc 2520
aacgaagccc cgaaactgcc gagcgaaatc aaagtctatt atagcgacga aagcgccgcc 2580
gccaaaaacg tcacgtggga cgaagtcgac ccgaaacagt atagcacggt cggcgaattt 2640
acggtcgaag gcagcgtcga aggcacgagc ctgaaagcca aagcctttgt catcgtcaaa 2700
ggcatcgtcg ccgtcaaacc gtatagcacg gccacgaaag tcggcgtcca gccggtcctg 2760
ccggaaaaag ccacgctgct gtatagcgac ggcacgacga aaggcgccac ggtcacgtgg 2820
gacgaaatcc cggaagacaa actggccaaa gaaggccgct ttacggtcga aggcagcgtc 2880
gaaggcacgg acctgaaagc caacgtctat gtccgcgtca cgaacgaagt caaaagcgtc 2940
aacatcatgc tgcaggaaca gggcagcgcc tatccgaaac tggaagccac gtttacgaac 3000
ccggccgaca acctgcagca tctgaacgac ggcatcaaaa gctatacgaa caacccggtc 3060
aaccgctgga cgaactggac gcgcacgccg cgcgacgccg gcgacagcat cacggtcaac 3120
tttggcaaaa aacatgtcat caacaacctg gacctgtttg tctttacgga cagcggcacg 3180
gtcgtcccgg aaaaagccga agtccagtat tgggacggca cggcctggaa agacgtcgaa 3240
aacctgacgc agccgagccc gtatgtcgtc gaaaaaaacg aactgacgtt tgacgccgtc 3300
gccacggaaa aactgaaatt tcatctgacg ccgagcgtca aaggcaaatt tctggccctg 3360
acggaagccg aagtctatgc cgaccagatc gtcatgggcg aaacggccaa actgcagagc 3420
atcacggtca acggcaaagc cctggaaggc tttgaccatg ccaaaaaaaa ctatgaactg 3480
gtcctgccgt atggcagcga actgccgaaa atcgaagccg ccgccgccga caacgccacg 3540
gtcacgatcc tgccggcctt tagctatccg ggcacggcca aactgtttgt cacgagcgaa 3600
gacggcaaag tcacgacgga atatagcatc ggcgtcagca cggaagaacc gaaactggtc 3660
agcgccgaac tgagcgccga caaaacgaac gtcatggaag acgacatcat cgacctgaaa 3720
gtcatcggcc tgtttgaaag caaagaaaaa atcgacgtca cggacagcca gccgacgtat 3780
gaatttgacc agcagatcat caaaatcgaa ggcaacaaac tgtatgccct ggaaacgggc 3840
aacgtcaaag tcaaagtcac ggtcacgtat aaaggcgtca gcgtcacgac gccggccctg 3900
gaatttacga tcgccaaaaa cccggccccg aaatatatca cgagcctgga accggtcacg 3960
gtcgtcgtca aaaaaggcga agccccggaa ctgccggcca cggtcgtcgc ccattataac 4020
cgcggcatcc cgcgcgacgt caaagtcaaa tgggaacgca tcaacccgag caaatatcag 4080
cagctgggcg aatttacggt cagcggcatg gtcgaaggca cggacatcaa agcccaggcc 4140
aaagtcatcg tcaaaggcgc cgtcgccgtc gaagacatcc gcatggccgt cctgctgaaa 4200
cagatgccgc agctgccggg caaagtcacg gtctattata gcgacggcgc cgaagaacag 4260
cgcgccgtca aatgggaaga aatcccgcag gaagaactgg aaaacgtcgg cgaatttaaa 4320
gtcaaaggcg acgtcaacgg cgtcaaactg aaagccacgg ccacgatccg cgtcacggac 4380
gaagtcggcg gcgaacagaa catcagccgc gccaaaaacg gctatgaata tccgaaagcc 4440
gaagccagct ttacgaacaa cggcccaggc agcagcgacc gcatcgaagc catcaacgac 4500
gacgtcatca gctatgaagc caacccgcat aaccgctgga cgaactggca gccggtcccg 4560
cgcgccggcg actgggtcag catcacgttt ggcgactatg aaccgacgga atatgacgtc 4620
gacagcatgg aaatccattg gtttgccgac catggcacga gctatccgga acgctttcag 4680
atcgaatata aaagcggcga cagctggaaa gaagtcacga gcctgaaaag cgacccggcc 4740
agcccggccc tgggcaaagc caacgtctat agctttgacc gcgtcaaaac gagcgccatc 4800
cgcgtcaaaa tgacggccca ggccggcaaa agcctggcca tcacggaact gaaagtcttt 4860
agcaaatggc cgaaagccgg cacggaaccg gaagtcacgg acatcaaagt cggcggcaaa 4920
agcatcctgg aagactttga acagaaaggc gaccattatg aagtcacgat cgacgccggc 4980
gacgccaacg tcatgccgaa aatcaacgtc aaagccaaag accagacgag catcacgatc 5040
gtcccggccg tcacgagccc gagcacggcc aaagtcatcg ccaaaagcga agacggcaaa 5100
aaagtcaaag tctatagcat ccattataaa taa 5133
<210> 8
<211> 1710
<212> PRT
<213> 环状芽胞杆菌ATCC 31382
<400> 8
Met Ser Lys Thr Thr Ser Ala Ala Gly Asn Ser Val Ser Tyr Asp Gly
1 5 10 15
Glu Arg Arg Val Asn Phe Asn Glu Asn Trp Arg Phe Gln Arg Glu Thr
20 25 30
Asn Gly Ser Ile Ala Gly Ala Gln Asn Pro Gly Phe Asp Asp Ser Ser
35 40 45
Trp Arg Lys Leu Asn Leu Pro His Asp Trp Ser Ile Glu Leu Asp Phe
50 55 60
Asn Lys Asn Ser Leu Ala Thr His Glu Gly Gly Tyr Leu Asp Gly Gly
65 70 75 80
Ile Gly Trp Tyr Arg Lys Thr Phe Thr Ile Pro Glu Ser Met Lys Gly
85 90 95
Lys Arg Ile Ser Leu Asp Phe Asp Gly Val Tyr Met Asn Ser Thr Thr
100 105 110
Tyr Leu Asn Gly Glu Val Leu Gly Thr Tyr Pro Phe Gly Tyr Asn Ala
115 120 125
Phe Ser Tyr Asp Ile Ser Asp Lys Leu Tyr Lys Asp Gly Arg Ala Asn
130 135 140
Val Leu Val Val Lys Val Asn Asn Thr Gln Pro Ser Ser Arg Trp Tyr
145 150 155 160
Ser Gly Ser Gly Ile Tyr Arg Asn Val Tyr Leu Thr Val Thr Asp Pro
165 170 175
Ile His Val Ala Arg Tyr Gly Thr Phe Val Thr Thr Pro Asn Leu Glu
180 185 190
Lys Ser Ile Lys Glu Asp Arg Ala Asp Val Asn Ile Lys Thr Lys Ile
195 200 205
Ser Asn Asp Ala Ala Glu Ala Lys Gln Val Lys Ile Lys Ser Thr Ile
210 215 220
Tyr Asp Gly Ala Gly Asn Thr Val Gln Thr Val Glu Thr Glu Glu Lys
225 230 235 240
Thr Ala Ala Ala Gly Thr Val Thr Pro Phe Glu Gln Asn Thr Val Ile
245 250 255
Lys Gln Pro Lys Leu Trp Ser Ile Asp Lys Pro Tyr Arg Tyr Asn Leu
260 265 270
Val Thr Glu Val Ile Val Gly Gly Gln Thr Val Asp Thr Tyr Glu Thr
275 280 285
Lys Phe Gly Val Arg Tyr Phe Lys Phe Asp Glu Asn Glu Gly Phe Ser
290 295 300
Leu Asn Gly Glu Tyr Met Lys Leu His Gly Val Ser Met His His Asp
305 310 315 320
Leu Gly Ala Leu Gly Ala Ala Thr Asn Ala Arg Gly Val Glu Arg Gln
325 330 335
Met Gln Ile Met Lys Asp Met Gly Val Asn Ala Ile Arg Val Thr His
340 345 350
Asn Pro Ala Ser Pro Glu Leu Leu Glu Ala Ala Asn Lys Leu Gly Leu
355 360 365
Phe Ile Ile Glu Glu Ala Phe Asp Ser Trp Ala Gln Ser Lys Lys Pro
370 375 380
Tyr Asp Tyr Gly Arg Phe Phe Asn Ala Trp Ala Glu His Asp Ile Lys
385 390 395 400
Glu Met Val Asp Arg Gly Lys Asn Glu Pro Ala Ile Ile Met Trp Ser
405 410 415
Ile Gly Asn Glu Ile Tyr Asp Thr Thr Asn Ala Ala Gly Val Glu Thr
420 425 430
Ala Arg Asn Leu Val Gly Trp Val Lys Glu Ile Asp Thr Thr Arg Pro
435 440 445
Thr Thr Ile Gly Glu Asp Lys Thr Arg Gly Asp Lys Val Asn Val Thr
450 455 460
Pro Ile Asn Ser Tyr Ile Lys Glu Ile Phe Asn Ile Val Asp Val Val
465 470 475 480
Gly Leu Asn Tyr Ser Glu Asn Asn Tyr Asp Gly Tyr His Lys Gln Asn
485 490 495
Pro Ser Trp Lys Leu Tyr Gly Ser Glu Thr Ser Ser Ala Thr Arg Ser
500 505 510
Arg Gly Val Tyr Thr His Pro Tyr Gln Tyr Asn Gln Ser Thr Lys Tyr
515 520 525
Ala Asp Leu Gln Gln Ser Ser Tyr Asp Asn Asp Tyr Val Gly Trp Gly
530 535 540
Arg Thr Ala Glu Asp Ala Trp Lys Tyr Asp Arg Asp Leu Lys His Ile
545 550 555 560
Ala Gly Gln Phe Ile Trp Thr Gly Phe Asp Tyr Ile Gly Glu Pro Thr
565 570 575
Pro Tyr Tyr Asn Ser Tyr Pro Ala Lys Ser Ser Tyr Phe Gly Ala Val
580 585 590
Asp Thr Ala Gly Phe Pro Lys Asp Ile Phe Tyr Tyr Tyr Gln Ser Gln
595 600 605
Trp Lys Lys Glu Pro Met Val His Leu Leu Pro His Trp Asn Trp Lys
610 615 620
Glu Gly Glu Lys Val Arg Val Leu Ala Tyr Thr Asn Ala Ser Lys Val
625 630 635 640
Glu Leu Val Leu Asn Gly Glu Ser Leu Gly Glu Lys Asn Tyr Asp Asn
645 650 655
Lys Gln Thr Ser Trp Gly Ala Pro Tyr Lys Glu Thr Lys Asp Gly Lys
660 665 670
Thr Tyr Leu Glu Trp Ala Val Pro Phe Lys Pro Gly Lys Leu Glu Ala
675 680 685
Val Ala Lys Asp Glu Asn Gly Lys Val Ile Ala Arg Asp Gln Val Val
690 695 700
Thr Ala Gly Glu Pro Ala Ser Val Arg Leu Thr Ala Asp Arg Lys Val
705 710 715 720
Val Lys Ala Asp Gly Thr Asp Leu Ser Phe Ile Thr Ala Asp Ile Val
725 730 735
Asp Ser Lys Gly Ile Val Val Pro Asp Ala Asp His Leu Ile Thr Phe
740 745 750
Asn Val Thr Gly Gln Gly Glu Leu Ala Gly Val Asp Asn Gly Asn Ala
755 760 765
Ser Ser Val Glu Arg Tyr Lys Asp Asn Lys Arg Lys Ala Phe Ser Gly
770 775 780
Lys Ala Leu Ala Ile Val Gln Ser Ser Lys Leu Ser Gly Lys Ile Thr
785 790 795 800
Val His Ala Ser Val Ala Gly Leu Ser Ser Asp Ser Thr Ser Val Phe
805 810 815
Thr Val Thr Pro Ala Asp His Asp Lys Lys Ile Val Ala Gly Ile Asp
820 825 830
Asp Val Asn Leu Thr Val Asp Val Asn Glu Ala Pro Lys Leu Pro Ser
835 840 845
Glu Ile Lys Val Tyr Tyr Ser Asp Glu Ser Ala Ala Ala Lys Asn Val
850 855 860
Thr Trp Asp Glu Val Asp Pro Lys Gln Tyr Ser Thr Val Gly Glu Phe
865 870 875 880
Thr Val Glu Gly Ser Val Glu Gly Thr Ser Leu Lys Ala Lys Ala Phe
885 890 895
Val Ile Val Lys Gly Ile Val Ala Val Lys Pro Tyr Ser Thr Ala Thr
900 905 910
Lys Val Gly Val Gln Pro Val Leu Pro Glu Lys Ala Thr Leu Leu Tyr
915 920 925
Ser Asp Gly Thr Thr Lys Gly Ala Thr Val Thr Trp Asp Glu Ile Pro
930 935 940
Glu Asp Lys Leu Ala Lys Glu Gly Arg Phe Thr Val Glu Gly Ser Val
945 950 955 960
Glu Gly Thr Asp Leu Lys Ala Asn Val Tyr Val Arg Val Thr Asn Glu
965 970 975
Val Lys Ser Val Asn Ile Met Leu Gln Glu Gln Gly Ser Ala Tyr Pro
980 985 990
Lys Leu Glu Ala Thr Phe Thr Asn Pro Ala Asp Asn Leu Gln His Leu
995 1000 1005
Asn Asp Gly Ile Lys Ser Tyr Thr Asn Asn Pro Val Asn Arg Trp
1010 1015 1020
Thr Asn Trp Thr Arg Thr Pro Arg Asp Ala Gly Asp Ser Ile Thr
1025 1030 1035
Val Asn Phe Gly Lys Lys His Val Ile Asn Asn Leu Asp Leu Phe
1040 1045 1050
Val Phe Thr Asp Ser Gly Thr Val Val Pro Glu Lys Ala Glu Val
1055 1060 1065
Gln Tyr Trp Asp Gly Thr Ala Trp Lys Asp Val Glu Asn Leu Thr
1070 1075 1080
Gln Pro Ser Pro Tyr Val Val Glu Lys Asn Glu Leu Thr Phe Asp
1085 1090 1095
Ala Val Ala Thr Glu Lys Leu Lys Phe His Leu Thr Pro Ser Val
1100 1105 1110
Lys Gly Lys Phe Leu Ala Leu Thr Glu Ala Glu Val Tyr Ala Asp
1115 1120 1125
Gln Ile Val Met Gly Glu Thr Ala Lys Leu Gln Ser Ile Thr Val
1130 1135 1140
Asn Gly Lys Ala Leu Glu Gly Phe Asp His Ala Lys Lys Asn Tyr
1145 1150 1155
Glu Leu Val Leu Pro Tyr Gly Ser Glu Leu Pro Lys Ile Glu Ala
1160 1165 1170
Ala Ala Ala Asp Asn Ala Thr Val Thr Ile Leu Pro Ala Phe Ser
1175 1180 1185
Tyr Pro Gly Thr Ala Lys Leu Phe Val Thr Ser Glu Asp Gly Lys
1190 1195 1200
Val Thr Thr Glu Tyr Ser Ile Gly Val Ser Thr Glu Glu Pro Lys
1205 1210 1215
Leu Val Ser Ala Glu Leu Ser Ala Asp Lys Thr Asn Val Met Glu
1220 1225 1230
Asp Asp Ile Ile Asp Leu Lys Val Ile Gly Leu Phe Glu Ser Lys
1235 1240 1245
Glu Lys Ile Asp Val Thr Asp Ser Gln Pro Thr Tyr Glu Phe Asp
1250 1255 1260
Gln Gln Ile Ile Lys Ile Glu Gly Asn Lys Leu Tyr Ala Leu Glu
1265 1270 1275
Thr Gly Asn Val Lys Val Lys Val Thr Val Thr Tyr Lys Gly Val
1280 1285 1290
Ser Val Thr Thr Pro Ala Leu Glu Phe Thr Ile Ala Lys Asn Pro
1295 1300 1305
Ala Pro Lys Tyr Ile Thr Ser Leu Glu Pro Val Thr Val Val Val
1310 1315 1320
Lys Lys Gly Glu Ala Pro Glu Leu Pro Ala Thr Val Val Ala His
1325 1330 1335
Tyr Asn Arg Gly Ile Pro Arg Asp Val Lys Val Lys Trp Glu Arg
1340 1345 1350
Ile Asn Pro Ser Lys Tyr Gln Gln Leu Gly Glu Phe Thr Val Ser
1355 1360 1365
Gly Met Val Glu Gly Thr Asp Ile Lys Ala Gln Ala Lys Val Ile
1370 1375 1380
Val Lys Gly Ala Val Ala Val Glu Asp Ile Arg Met Ala Val Leu
1385 1390 1395
Leu Lys Gln Met Pro Gln Leu Pro Gly Lys Val Thr Val Tyr Tyr
1400 1405 1410
Ser Asp Gly Ala Glu Glu Gln Arg Ala Val Lys Trp Glu Glu Ile
1415 1420 1425
Pro Gln Glu Glu Leu Glu Asn Val Gly Glu Phe Lys Val Lys Gly
1430 1435 1440
Asp Val Asn Gly Val Lys Leu Lys Ala Thr Ala Thr Ile Arg Val
1445 1450 1455
Thr Asp Glu Val Gly Gly Glu Gln Asn Ile Ser Arg Ala Lys Asn
1460 1465 1470
Gly Tyr Glu Tyr Pro Lys Ala Glu Ala Ser Phe Thr Asn Asn Gly
1475 1480 1485
Pro Gly Ser Ser Asp Arg Ile Glu Ala Ile Asn Asp Asp Val Ile
1490 1495 1500
Ser Tyr Glu Ala Asn Pro His Asn Arg Trp Thr Asn Trp Gln Pro
1505 1510 1515
Val Pro Arg Ala Gly Asp Trp Val Ser Ile Thr Phe Gly Asp Tyr
1520 1525 1530
Glu Pro Thr Glu Tyr Asp Val Asp Ser Met Glu Ile His Trp Phe
1535 1540 1545
Ala Asp His Gly Thr Ser Tyr Pro Glu Arg Phe Gln Ile Glu Tyr
1550 1555 1560
Lys Ser Gly Asp Ser Trp Lys Glu Val Thr Ser Leu Lys Ser Asp
1565 1570 1575
Pro Ala Ser Pro Ala Leu Gly Lys Ala Asn Val Tyr Ser Phe Asp
1580 1585 1590
Arg Val Lys Thr Ser Ala Ile Arg Val Lys Met Thr Ala Gln Ala
1595 1600 1605
Gly Lys Ser Leu Ala Ile Thr Glu Leu Lys Val Phe Ser Lys Trp
1610 1615 1620
Pro Lys Ala Gly Thr Glu Pro Glu Val Thr Asp Ile Lys Val Gly
1625 1630 1635
Gly Lys Ser Ile Leu Glu Asp Phe Glu Gln Lys Gly Asp His Tyr
1640 1645 1650
Glu Val Thr Ile Asp Ala Gly Asp Ala Asn Val Met Pro Lys Ile
1655 1660 1665
Asn Val Lys Ala Lys Asp Gln Thr Ser Ile Thr Ile Val Pro Ala
1670 1675 1680
Val Thr Ser Pro Ser Thr Ala Lys Val Ile Ala Lys Ser Glu Asp
1685 1690 1695
Gly Lys Lys Val Lys Val Tyr Ser Ile His Tyr Lys
1700 1705 1710
<210> 9
<211> 3669
<212> DNA
<213> 人工序列
<400> 9
atgagcaaaa cgacgagcgc cgccggcaac agcgtcagct atgacggcga acgccgcgtc 60
aactttaacg aaaactggcg ctttcagcgc gaaacgaacg gcagcatcgc cggcgcccag 120
aacccaggct ttgacgacag cagctggcgc aaactgaacc tgccgcatga ctggagcatc 180
gaactggact ttaacaaaaa cagcctggcc acgcatgaag gcggctatct ggacggcggc 240
atcggctggt atcgcaaaac gtttacgatc ccggaaagca tgaaaggcaa acgcatcagc 300
ctggactttg acggcgtcta tatgaacagc acgacgtatc tgaacggcga agtcctgggc 360
acgtatccgt ttggctataa cgcctttagc tatgacatca gcgacaaact gtataaagac 420
ggccgcgcca acgtcctggt cgtcaaagtc aacaacacgc agccgagcag ccgctggtat 480
agcggcagcg gcatctatcg caacgtctat ctgacggtca cggacccgat ccatgtcgcc 540
cgctatggca cgtttgtcac gacgccgaac ctggaaaaaa gcatcaaaga agaccgcgcc 600
gacgtcaaca tcaaaacgaa aatcagcaac gacgccgccg aagccaaaca ggtcaaaatc 660
aaaagcacga tctatgacgg cgccggcaac acggtccaga cggtcgaaac ggaagaaaaa 720
acggccgccg ccggcacggt cacgccgttt gaacagaaca cggtcatcaa acagccgaaa 780
ctgtggagca tcgacaaacc gtatcgctat aacctggtca cggaagtcat cgtcggcggc 840
cagacggtcg acacgtatga aacgaaattt ggcgtccgct attttaaatt tgacgaaaac 900
gaaggcttta gcctgaacgg cgaatatatg aaactgcatg gcgtcagcat gcatcatgac 960
ctgggcgccc tgggcgccgc cacgaacgcc cgcggcgtcg aacgccagat gcagatcatg 1020
aaagacatgg gcgtcaacgc catccgcgtc acgcataacc cggccagccc ggaactgctg 1080
gaagccgcca acaaactggg cctgtttatc atcgaagaag cctttgacag ctgggcccag 1140
agcaaaaaac cgtatgacta tggccgcttt tttaacgcct gggccgaaca tgacatcaaa 1200
gaaatggtcg accgcggcaa aaacgaaccg gccatcatca tgtggagcat cggcaacgaa 1260
atctatgaca cgacgaacgc cgccggcgtc gaaacggccc gcaacctggt cggctgggtc 1320
aaagaaatcg acacgacgcg cccgacgacg atcggcgaag acaaaacgcg cggcgacaaa 1380
gtcaacgtca cgccgatcaa cagctatatc aaagaaatct ttaacatcgt cgacgtcgtc 1440
ggcctgaact atagcgaaaa caactatgac ggctatcata aacagaaccc gagctggaaa 1500
ctgtatggca gcgaaacgag cagcgccacg cgcagccgcg gcgtctatac gcatccgtat 1560
cagtataacc agagcacgaa atatgccgac ctgcagcaga gcagctatga caacgactat 1620
gtcggctggg gccgcacggc cgaagacgcc tggaaatatg accgcgacct gaaacatatc 1680
gccggccagt ttatctggac gggctttgac tatatcggcg aaccgacgcc gtattataac 1740
agctatccgg ccaaaagcag ctattttggc gccgtcgaca cggccggctt tccgaaagac 1800
atcttttatt attatcagag ccagtggaaa aaagaaccga tggtccatct gctgccgcat 1860
tggaactgga aagaaggcga aaaagtccgc gtcctggcct atacgaacgc cagcaaagtc 1920
gaactggtcc tgaacggcga aagcctgggc gaaaaaaact atgacaacaa acagacgagc 1980
tggggcgccc cgtataaaga aacgaaagac ggcaaaacgt atctggaatg ggccgtcccg 2040
tttaaaccgg gcaaactgga agccgtcgcc aaagacgaaa acggcaaagt catcgcccgc 2100
gaccaggtcg tcacggccgg cgaaccggcc agcgtccgcc tgacggccga ccgcaaagtc 2160
gtcaaagccg acggcacgga cctgagcttt atcacggccg acatcgtcga cagcaaaggc 2220
atcgtcgtcc cggacgccga ccatctgatc acgtttaacg tcacgggcca gggcgaactg 2280
gccggcgtcg acaacggcaa cgccagcagc gtcgaacgct ataaagacaa caaacgcaaa 2340
gcctttagcg gcaaagccct ggccatcgtc cagagcagca aactgagcgg caaaatcacg 2400
gtccatgcca gcgtcgccgg cctgagcagc gacagcacga gcgtctttac ggtcacgccg 2460
gccgaccatg acaaaaaaat cgtcgccggc atcgacgacg tcaacctgac ggtcgacgtc 2520
aacgaagccc cgaaactgcc gagcgaaatc aaagtctatt atagcgacga aagcgccgcc 2580
gccaaaaacg tcacgtggga cgaagtcgac ccgaaacagt atagcacggt cggcgaattt 2640
acggtcgaag gcagcgtcga aggcacgagc ctgaaagcca aagcctttgt catcgtcaaa 2700
ggcatcgtcg ccgtcaaacc gtatagcacg gccacgaaag tcggcgtcca gccggtcctg 2760
ccggaaaaag ccacgctgct gtatagcgac ggcacgacga aaggcgccac ggtcacgtgg 2820
gacgaaatcc cggaagacaa actggccaaa gaaggccgct ttacggtcga aggcagcgtc 2880
gaaggcacgg acctgaaagc caacgtctat gtccgcgtca cgaacgaagt caaaagcgtc 2940
aacatcatgc tgcaggaaca gggcagcgcc tatccgaaac tggaagccac gtttacgaac 3000
ccggccgaca acctgcagca tctgaacgac ggcatcaaaa gctatacgaa caacccggtc 3060
aaccgctgga cgaactggac gcgcacgccg cgcgacgccg gcgacagcat cacggtcaac 3120
tttggcaaaa aacatgtcat caacaacctg gacctgtttg tctttacgga cagcggcacg 3180
gtcgtcccgg aaaaagccga agtccagtat tgggacggca cggcctggaa agacgtcgaa 3240
aacctgacgc agccgagccc gtatgtcgtc gaaaaaaacg aactgacgtt tgacgccgtc 3300
gccacggaaa aactgaaatt tcatctgacg ccgagcgtca aaggcaaatt tctggccctg 3360
acggaagccg aagtctatgc cgaccagatc gtcatgggcg aaacggccaa actgcagagc 3420
atcacggtca acggcaaagc cctggaaggc tttgaccatg ccaaaaaaaa ctatgaactg 3480
gtcctgccgt atggcagcga actgccgaaa atcgaagccg ccgccgccga caacgccacg 3540
gtcacgatcc tgccggcctt tagctatccg ggcacggcca aactgtttgt cacgagcgaa 3600
gacggcaaag tcacgacgga atatagcatc ggcgtcagca cggaagaacc gaaactggtc 3660
agcgcctaa 3669
<210> 10
<211> 1222
<212> PRT
<213> 人工序列
<400> 10
Met Ser Lys Thr Thr Ser Ala Ala Gly Asn Ser Val Ser Tyr Asp Gly
1 5 10 15
Glu Arg Arg Val Asn Phe Asn Glu Asn Trp Arg Phe Gln Arg Glu Thr
20 25 30
Asn Gly Ser Ile Ala Gly Ala Gln Asn Pro Gly Phe Asp Asp Ser Ser
35 40 45
Trp Arg Lys Leu Asn Leu Pro His Asp Trp Ser Ile Glu Leu Asp Phe
50 55 60
Asn Lys Asn Ser Leu Ala Thr His Glu Gly Gly Tyr Leu Asp Gly Gly
65 70 75 80
Ile Gly Trp Tyr Arg Lys Thr Phe Thr Ile Pro Glu Ser Met Lys Gly
85 90 95
Lys Arg Ile Ser Leu Asp Phe Asp Gly Val Tyr Met Asn Ser Thr Thr
100 105 110
Tyr Leu Asn Gly Glu Val Leu Gly Thr Tyr Pro Phe Gly Tyr Asn Ala
115 120 125
Phe Ser Tyr Asp Ile Ser Asp Lys Leu Tyr Lys Asp Gly Arg Ala Asn
130 135 140
Val Leu Val Val Lys Val Asn Asn Thr Gln Pro Ser Ser Arg Trp Tyr
145 150 155 160
Ser Gly Ser Gly Ile Tyr Arg Asn Val Tyr Leu Thr Val Thr Asp Pro
165 170 175
Ile His Val Ala Arg Tyr Gly Thr Phe Val Thr Thr Pro Asn Leu Glu
180 185 190
Lys Ser Ile Lys Glu Asp Arg Ala Asp Val Asn Ile Lys Thr Lys Ile
195 200 205
Ser Asn Asp Ala Ala Glu Ala Lys Gln Val Lys Ile Lys Ser Thr Ile
210 215 220
Tyr Asp Gly Ala Gly Asn Thr Val Gln Thr Val Glu Thr Glu Glu Lys
225 230 235 240
Thr Ala Ala Ala Gly Thr Val Thr Pro Phe Glu Gln Asn Thr Val Ile
245 250 255
Lys Gln Pro Lys Leu Trp Ser Ile Asp Lys Pro Tyr Arg Tyr Asn Leu
260 265 270
Val Thr Glu Val Ile Val Gly Gly Gln Thr Val Asp Thr Tyr Glu Thr
275 280 285
Lys Phe Gly Val Arg Tyr Phe Lys Phe Asp Glu Asn Glu Gly Phe Ser
290 295 300
Leu Asn Gly Glu Tyr Met Lys Leu His Gly Val Ser Met His His Asp
305 310 315 320
Leu Gly Ala Leu Gly Ala Ala Thr Asn Ala Arg Gly Val Glu Arg Gln
325 330 335
Met Gln Ile Met Lys Asp Met Gly Val Asn Ala Ile Arg Val Thr His
340 345 350
Asn Pro Ala Ser Pro Glu Leu Leu Glu Ala Ala Asn Lys Leu Gly Leu
355 360 365
Phe Ile Ile Glu Glu Ala Phe Asp Ser Trp Ala Gln Ser Lys Lys Pro
370 375 380
Tyr Asp Tyr Gly Arg Phe Phe Asn Ala Trp Ala Glu His Asp Ile Lys
385 390 395 400
Glu Met Val Asp Arg Gly Lys Asn Glu Pro Ala Ile Ile Met Trp Ser
405 410 415
Ile Gly Asn Glu Ile Tyr Asp Thr Thr Asn Ala Ala Gly Val Glu Thr
420 425 430
Ala Arg Asn Leu Val Gly Trp Val Lys Glu Ile Asp Thr Thr Arg Pro
435 440 445
Thr Thr Ile Gly Glu Asp Lys Thr Arg Gly Asp Lys Val Asn Val Thr
450 455 460
Pro Ile Asn Ser Tyr Ile Lys Glu Ile Phe Asn Ile Val Asp Val Val
465 470 475 480
Gly Leu Asn Tyr Ser Glu Asn Asn Tyr Asp Gly Tyr His Lys Gln Asn
485 490 495
Pro Ser Trp Lys Leu Tyr Gly Ser Glu Thr Ser Ser Ala Thr Arg Ser
500 505 510
Arg Gly Val Tyr Thr His Pro Tyr Gln Tyr Asn Gln Ser Thr Lys Tyr
515 520 525
Ala Asp Leu Gln Gln Ser Ser Tyr Asp Asn Asp Tyr Val Gly Trp Gly
530 535 540
Arg Thr Ala Glu Asp Ala Trp Lys Tyr Asp Arg Asp Leu Lys His Ile
545 550 555 560
Ala Gly Gln Phe Ile Trp Thr Gly Phe Asp Tyr Ile Gly Glu Pro Thr
565 570 575
Pro Tyr Tyr Asn Ser Tyr Pro Ala Lys Ser Ser Tyr Phe Gly Ala Val
580 585 590
Asp Thr Ala Gly Phe Pro Lys Asp Ile Phe Tyr Tyr Tyr Gln Ser Gln
595 600 605
Trp Lys Lys Glu Pro Met Val His Leu Leu Pro His Trp Asn Trp Lys
610 615 620
Glu Gly Glu Lys Val Arg Val Leu Ala Tyr Thr Asn Ala Ser Lys Val
625 630 635 640
Glu Leu Val Leu Asn Gly Glu Ser Leu Gly Glu Lys Asn Tyr Asp Asn
645 650 655
Lys Gln Thr Ser Trp Gly Ala Pro Tyr Lys Glu Thr Lys Asp Gly Lys
660 665 670
Thr Tyr Leu Glu Trp Ala Val Pro Phe Lys Pro Gly Lys Leu Glu Ala
675 680 685
Val Ala Lys Asp Glu Asn Gly Lys Val Ile Ala Arg Asp Gln Val Val
690 695 700
Thr Ala Gly Glu Pro Ala Ser Val Arg Leu Thr Ala Asp Arg Lys Val
705 710 715 720
Val Lys Ala Asp Gly Thr Asp Leu Ser Phe Ile Thr Ala Asp Ile Val
725 730 735
Asp Ser Lys Gly Ile Val Val Pro Asp Ala Asp His Leu Ile Thr Phe
740 745 750
Asn Val Thr Gly Gln Gly Glu Leu Ala Gly Val Asp Asn Gly Asn Ala
755 760 765
Ser Ser Val Glu Arg Tyr Lys Asp Asn Lys Arg Lys Ala Phe Ser Gly
770 775 780
Lys Ala Leu Ala Ile Val Gln Ser Ser Lys Leu Ser Gly Lys Ile Thr
785 790 795 800
Val His Ala Ser Val Ala Gly Leu Ser Ser Asp Ser Thr Ser Val Phe
805 810 815
Thr Val Thr Pro Ala Asp His Asp Lys Lys Ile Val Ala Gly Ile Asp
820 825 830
Asp Val Asn Leu Thr Val Asp Val Asn Glu Ala Pro Lys Leu Pro Ser
835 840 845
Glu Ile Lys Val Tyr Tyr Ser Asp Glu Ser Ala Ala Ala Lys Asn Val
850 855 860
Thr Trp Asp Glu Val Asp Pro Lys Gln Tyr Ser Thr Val Gly Glu Phe
865 870 875 880
Thr Val Glu Gly Ser Val Glu Gly Thr Ser Leu Lys Ala Lys Ala Phe
885 890 895
Val Ile Val Lys Gly Ile Val Ala Val Lys Pro Tyr Ser Thr Ala Thr
900 905 910
Lys Val Gly Val Gln Pro Val Leu Pro Glu Lys Ala Thr Leu Leu Tyr
915 920 925
Ser Asp Gly Thr Thr Lys Gly Ala Thr Val Thr Trp Asp Glu Ile Pro
930 935 940
Glu Asp Lys Leu Ala Lys Glu Gly Arg Phe Thr Val Glu Gly Ser Val
945 950 955 960
Glu Gly Thr Asp Leu Lys Ala Asn Val Tyr Val Arg Val Thr Asn Glu
965 970 975
Val Lys Ser Val Asn Ile Met Leu Gln Glu Gln Gly Ser Ala Tyr Pro
980 985 990
Lys Leu Glu Ala Thr Phe Thr Asn Pro Ala Asp Asn Leu Gln His Leu
995 1000 1005
Asn Asp Gly Ile Lys Ser Tyr Thr Asn Asn Pro Val Asn Arg Trp
1010 1015 1020
Thr Asn Trp Thr Arg Thr Pro Arg Asp Ala Gly Asp Ser Ile Thr
1025 1030 1035
Val Asn Phe Gly Lys Lys His Val Ile Asn Asn Leu Asp Leu Phe
1040 1045 1050
Val Phe Thr Asp Ser Gly Thr Val Val Pro Glu Lys Ala Glu Val
1055 1060 1065
Gln Tyr Trp Asp Gly Thr Ala Trp Lys Asp Val Glu Asn Leu Thr
1070 1075 1080
Gln Pro Ser Pro Tyr Val Val Glu Lys Asn Glu Leu Thr Phe Asp
1085 1090 1095
Ala Val Ala Thr Glu Lys Leu Lys Phe His Leu Thr Pro Ser Val
1100 1105 1110
Lys Gly Lys Phe Leu Ala Leu Thr Glu Ala Glu Val Tyr Ala Asp
1115 1120 1125
Gln Ile Val Met Gly Glu Thr Ala Lys Leu Gln Ser Ile Thr Val
1130 1135 1140
Asn Gly Lys Ala Leu Glu Gly Phe Asp His Ala Lys Lys Asn Tyr
1145 1150 1155
Glu Leu Val Leu Pro Tyr Gly Ser Glu Leu Pro Lys Ile Glu Ala
1160 1165 1170
Ala Ala Ala Asp Asn Ala Thr Val Thr Ile Leu Pro Ala Phe Ser
1175 1180 1185
Tyr Pro Gly Thr Ala Lys Leu Phe Val Thr Ser Glu Asp Gly Lys
1190 1195 1200
Val Thr Thr Glu Tyr Ser Ile Gly Val Ser Thr Glu Glu Pro Lys
1205 1210 1215
Leu Val Ser Ala
1220
<210> 11
<211> 2463
<212> DNA
<213> 人工序列
<400> 11
atgagcaaaa cgacgagcgc cgccggcaac agcgtcagct atgacggcga acgccgcgtc 60
aactttaacg aaaactggcg ctttcagcgc gaaacgaacg gcagcatcgc cggcgcccag 120
aacccaggct ttgacgacag cagctggcgc aaactgaacc tgccgcatga ctggagcatc 180
gaactggact ttaacaaaaa cagcctggcc acgcatgaag gcggctatct ggacggcggc 240
atcggctggt atcgcaaaac gtttacgatc ccggaaagca tgaaaggcaa acgcatcagc 300
ctggactttg acggcgtcta tatgaacagc acgacgtatc tgaacggcga agtcctgggc 360
acgtatccgt ttggctataa cgcctttagc tatgacatca gcgacaaact gtataaagac 420
ggccgcgcca acgtcctggt cgtcaaagtc aacaacacgc agccgagcag ccgctggtat 480
agcggcagcg gcatctatcg caacgtctat ctgacggtca cggacccgat ccatgtcgcc 540
cgctatggca cgtttgtcac gacgccgaac ctggaaaaaa gcatcaaaga agaccgcgcc 600
gacgtcaaca tcaaaacgaa aatcagcaac gacgccgccg aagccaaaca ggtcaaaatc 660
aaaagcacga tctatgacgg cgccggcaac acggtccaga cggtcgaaac ggaagaaaaa 720
acggccgccg ccggcacggt cacgccgttt gaacagaaca cggtcatcaa acagccgaaa 780
ctgtggagca tcgacaaacc gtatcgctat aacctggtca cggaagtcat cgtcggcggc 840
cagacggtcg acacgtatga aacgaaattt ggcgtccgct attttaaatt tgacgaaaac 900
gaaggcttta gcctgaacgg cgaatatatg aaactgcatg gcgtcagcat gcatcatgac 960
ctgggcgccc tgggcgccgc cacgaacgcc cgcggcgtcg aacgccagat gcagatcatg 1020
aaagacatgg gcgtcaacgc catccgcgtc acgcataacc cggccagccc ggaactgctg 1080
gaagccgcca acaaactggg cctgtttatc atcgaagaag cctttgacag ctgggcccag 1140
agcaaaaaac cgtatgacta tggccgcttt tttaacgcct gggccgaaca tgacatcaaa 1200
gaaatggtcg accgcggcaa aaacgaaccg gccatcatca tgtggagcat cggcaacgaa 1260
atctatgaca cgacgaacgc cgccggcgtc gaaacggccc gcaacctggt cggctgggtc 1320
aaagaaatcg acacgacgcg cccgacgacg atcggcgaag acaaaacgcg cggcgacaaa 1380
gtcaacgtca cgccgatcaa cagctatatc aaagaaatct ttaacatcgt cgacgtcgtc 1440
ggcctgaact atagcgaaaa caactatgac ggctatcata aacagaaccc gagctggaaa 1500
ctgtatggca gcgaaacgag cagcgccacg cgcagccgcg gcgtctatac gcatccgtat 1560
cagtataacc agagcacgaa atatgccgac ctgcagcaga gcagctatga caacgactat 1620
gtcggctggg gccgcacggc cgaagacgcc tggaaatatg accgcgacct gaaacatatc 1680
gccggccagt ttatctggac gggctttgac tatatcggcg aaccgacgcc gtattataac 1740
agctatccgg ccaaaagcag ctattttggc gccgtcgaca cggccggctt tccgaaagac 1800
atcttttatt attatcagag ccagtggaaa aaagaaccga tggtccatct gctgccgcat 1860
tggaactgga aagaaggcga aaaagtccgc gtcctggcct atacgaacgc cagcaaagtc 1920
gaactggtcc tgaacggcga aagcctgggc gaaaaaaact atgacaacaa acagacgagc 1980
tggggcgccc cgtataaaga aacgaaagac ggcaaaacgt atctggaatg ggccgtcccg 2040
tttaaaccgg gcaaactgga agccgtcgcc aaagacgaaa acggcaaagt catcgcccgc 2100
gaccaggtcg tcacggccgg cgaaccggcc agcgtccgcc tgacggccga ccgcaaagtc 2160
gtcaaagccg acggcacgga cctgagcttt atcacggccg acatcgtcga cagcaaaggc 2220
atcgtcgtcc cggacgccga ccatctgatc acgtttaacg tcacgggcca gggcgaactg 2280
gccggcgtcg acaacggcaa cgccagcagc gtcgaacgct ataaagacaa caaacgcaaa 2340
gcctttagcg gcaaagccct ggccatcgtc cagagcagca aactgagcgg caaaatcacg 2400
gtccatgcca gcgtcgccgg cctgagcagc gacagcacga gcgtctttac ggtcacgccg 2460
taa 2463
<210> 12
<211> 820
<212> PRT
<213> 人工序列
<400> 12
Met Ser Lys Thr Thr Ser Ala Ala Gly Asn Ser Val Ser Tyr Asp Gly
1 5 10 15
Glu Arg Arg Val Asn Phe Asn Glu Asn Trp Arg Phe Gln Arg Glu Thr
20 25 30
Asn Gly Ser Ile Ala Gly Ala Gln Asn Pro Gly Phe Asp Asp Ser Ser
35 40 45
Trp Arg Lys Leu Asn Leu Pro His Asp Trp Ser Ile Glu Leu Asp Phe
50 55 60
Asn Lys Asn Ser Leu Ala Thr His Glu Gly Gly Tyr Leu Asp Gly Gly
65 70 75 80
Ile Gly Trp Tyr Arg Lys Thr Phe Thr Ile Pro Glu Ser Met Lys Gly
85 90 95
Lys Arg Ile Ser Leu Asp Phe Asp Gly Val Tyr Met Asn Ser Thr Thr
100 105 110
Tyr Leu Asn Gly Glu Val Leu Gly Thr Tyr Pro Phe Gly Tyr Asn Ala
115 120 125
Phe Ser Tyr Asp Ile Ser Asp Lys Leu Tyr Lys Asp Gly Arg Ala Asn
130 135 140
Val Leu Val Val Lys Val Asn Asn Thr Gln Pro Ser Ser Arg Trp Tyr
145 150 155 160
Ser Gly Ser Gly Ile Tyr Arg Asn Val Tyr Leu Thr Val Thr Asp Pro
165 170 175
Ile His Val Ala Arg Tyr Gly Thr Phe Val Thr Thr Pro Asn Leu Glu
180 185 190
Lys Ser Ile Lys Glu Asp Arg Ala Asp Val Asn Ile Lys Thr Lys Ile
195 200 205
Ser Asn Asp Ala Ala Glu Ala Lys Gln Val Lys Ile Lys Ser Thr Ile
210 215 220
Tyr Asp Gly Ala Gly Asn Thr Val Gln Thr Val Glu Thr Glu Glu Lys
225 230 235 240
Thr Ala Ala Ala Gly Thr Val Thr Pro Phe Glu Gln Asn Thr Val Ile
245 250 255
Lys Gln Pro Lys Leu Trp Ser Ile Asp Lys Pro Tyr Arg Tyr Asn Leu
260 265 270
Val Thr Glu Val Ile Val Gly Gly Gln Thr Val Asp Thr Tyr Glu Thr
275 280 285
Lys Phe Gly Val Arg Tyr Phe Lys Phe Asp Glu Asn Glu Gly Phe Ser
290 295 300
Leu Asn Gly Glu Tyr Met Lys Leu His Gly Val Ser Met His His Asp
305 310 315 320
Leu Gly Ala Leu Gly Ala Ala Thr Asn Ala Arg Gly Val Glu Arg Gln
325 330 335
Met Gln Ile Met Lys Asp Met Gly Val Asn Ala Ile Arg Val Thr His
340 345 350
Asn Pro Ala Ser Pro Glu Leu Leu Glu Ala Ala Asn Lys Leu Gly Leu
355 360 365
Phe Ile Ile Glu Glu Ala Phe Asp Ser Trp Ala Gln Ser Lys Lys Pro
370 375 380
Tyr Asp Tyr Gly Arg Phe Phe Asn Ala Trp Ala Glu His Asp Ile Lys
385 390 395 400
Glu Met Val Asp Arg Gly Lys Asn Glu Pro Ala Ile Ile Met Trp Ser
405 410 415
Ile Gly Asn Glu Ile Tyr Asp Thr Thr Asn Ala Ala Gly Val Glu Thr
420 425 430
Ala Arg Asn Leu Val Gly Trp Val Lys Glu Ile Asp Thr Thr Arg Pro
435 440 445
Thr Thr Ile Gly Glu Asp Lys Thr Arg Gly Asp Lys Val Asn Val Thr
450 455 460
Pro Ile Asn Ser Tyr Ile Lys Glu Ile Phe Asn Ile Val Asp Val Val
465 470 475 480
Gly Leu Asn Tyr Ser Glu Asn Asn Tyr Asp Gly Tyr His Lys Gln Asn
485 490 495
Pro Ser Trp Lys Leu Tyr Gly Ser Glu Thr Ser Ser Ala Thr Arg Ser
500 505 510
Arg Gly Val Tyr Thr His Pro Tyr Gln Tyr Asn Gln Ser Thr Lys Tyr
515 520 525
Ala Asp Leu Gln Gln Ser Ser Tyr Asp Asn Asp Tyr Val Gly Trp Gly
530 535 540
Arg Thr Ala Glu Asp Ala Trp Lys Tyr Asp Arg Asp Leu Lys His Ile
545 550 555 560
Ala Gly Gln Phe Ile Trp Thr Gly Phe Asp Tyr Ile Gly Glu Pro Thr
565 570 575
Pro Tyr Tyr Asn Ser Tyr Pro Ala Lys Ser Ser Tyr Phe Gly Ala Val
580 585 590
Asp Thr Ala Gly Phe Pro Lys Asp Ile Phe Tyr Tyr Tyr Gln Ser Gln
595 600 605
Trp Lys Lys Glu Pro Met Val His Leu Leu Pro His Trp Asn Trp Lys
610 615 620
Glu Gly Glu Lys Val Arg Val Leu Ala Tyr Thr Asn Ala Ser Lys Val
625 630 635 640
Glu Leu Val Leu Asn Gly Glu Ser Leu Gly Glu Lys Asn Tyr Asp Asn
645 650 655
Lys Gln Thr Ser Trp Gly Ala Pro Tyr Lys Glu Thr Lys Asp Gly Lys
660 665 670
Thr Tyr Leu Glu Trp Ala Val Pro Phe Lys Pro Gly Lys Leu Glu Ala
675 680 685
Val Ala Lys Asp Glu Asn Gly Lys Val Ile Ala Arg Asp Gln Val Val
690 695 700
Thr Ala Gly Glu Pro Ala Ser Val Arg Leu Thr Ala Asp Arg Lys Val
705 710 715 720
Val Lys Ala Asp Gly Thr Asp Leu Ser Phe Ile Thr Ala Asp Ile Val
725 730 735
Asp Ser Lys Gly Ile Val Val Pro Asp Ala Asp His Leu Ile Thr Phe
740 745 750
Asn Val Thr Gly Gln Gly Glu Leu Ala Gly Val Asp Asn Gly Asn Ala
755 760 765
Ser Ser Val Glu Arg Tyr Lys Asp Asn Lys Arg Lys Ala Phe Ser Gly
770 775 780
Lys Ala Leu Ala Ile Val Gln Ser Ser Lys Leu Ser Gly Lys Ile Thr
785 790 795 800
Val His Ala Ser Val Ala Gly Leu Ser Ser Asp Ser Thr Ser Val Phe
805 810 815
Thr Val Thr Pro
820
<210> 13
<211> 5132
<212> DNA
<213> 人工序列
<400> 13
atgaaaagta caaccagtgc agccggtaaa tccgttagtt ataacgacgg agagcgtaga 60
gtgaattttg agaattggcg gttccaacgc gagacgaatg gcagtattgc aggtgcgcag 120
aacccaggtt ttgatgattc atcatggaga aaacttaatc tgccgcatga atggtctatc 180
gatttagact ttaataaaaa cagtttggct acgcatgaag gaggttacct tgatggggga 240
attggctgga atagaaaaac gtttacaatt ccggagtcga tgaaagggaa gcggttttct 300
ttggactttg atggtgtcta catgaactcc actacctacc ttaacggtga agttttgggt 360
acttacccat tcggatacaa cgccttctct tacgacattt ccgacaagtt gtacaaagat 420
ggcagagcaa acgtgttggt cgtcaaagtt aacacgaatc agcctagcgg acgctggtat 480
agcggatcat caatttatag aaacgtctat cttacggtga ccgacccgat ccatgtggcg 540
agatacggca cgtttgtaac aacaccaaat ctggaaaaaa gtatcaagga agaccgggat 600
gcagttaaca ttaaaacgaa gatcagcaac gcagaagcca agcaagtcaa aatcgctatt 660
aagtctacta tctacgatgg tgctggtaac accgtccaaa ctgttgaaac cgaggaaaag 720
acagcagctg ctggtacagt cactccattc gagcagaaca ccgttattaa gcaacctaag 780
ttgtggtcca tcgacaaacc atacagatac aacttggtca ctgaagttat tgtaggaggt 840
cagacggtcg atacatatga gacgaagttt ggagtccggt acttcaaatt tgatgaaaac 900
gaaggctatt cattgaacgg tgaatttatg aaactccacg gagtctcgat gcatcacgat 960
ttggctggag gtgcggcgct tacaaatgca agaggcgtcg aaagacaaat gcaaattatg 1020
aaggatgttg gagtcaacgc gattagagtt acgcataacc ctgcttcacc agaattaggc 1080
ttagaatttg cggccaacaa attattaatc attgaggagg catttgattc ttgggcgcag 1140
tctaaaaaac cgtatgacta tggccgcttt tttaatgctt gggctgagca tgatattaag 1200
gaaatggtcg atagaggtaa aaacgaacct gcgatcataa tgtggagcat aggaaacgag 1260
atatatgaca ctactaatgc agctggagtt gagactgctc gaaatctcgt tggctgggta 1320
aaagaaatcg acacaacgcc gagagccaca attggggaag ataaaacccg aggagataat 1380
gtgacaccga ttaactcata tattaaagaa atcttcaaca ttgtggacgt cgttggtctc 1440
aactactccg aaaacaacta cgatggatac cacaagcaga acccatcttg gaagttgtac 1500
ggttccgaga ccagttccgc tactagatcc agaggtgtct acacccatcc ttaccaatac 1560
aaccatgata caaaatatgc ggatttgcag cagtcttcgt atgacaatga ttatgttgga 1620
tggggtagaa cagcacagga tgcttggaaa tatgatcgtg atcttaaaca tattgcaggt 1680
caatttattt ggacaggctt cgattatatt ggagaaccaa caccgtatta taactcatac 1740
ccggcaaaaa gctcatattt cggagcagtt gacacggcag ggttccctaa agacattttc 1800
tattattacc aaagccagtg gaaaaaagaa cctatggttc accttttgcc gcactggaac 1860
tggaaagagg gagagaaggt tcgggtattg gcgtatacca atgcgagcaa agttgaactt 1920
gtgctgaatg gagaatcact cggcaacgaa aagtatgata acaagcaaac tagttggggt 1980
gcgccgtaca aagaaacaaa agatggtaaa acatacttag aatgggcagt cccattcaag 2040
cctggaaagt tggaagctgt tgccaaggat gagaatggta aggtcattgc aagagatcaa 2100
gttgtcaccg ctggtgaacc agcctccgtt agattgactg ctgacagaaa ggtcgttaag 2160
gctgatggta ctgacttgtc gttcatcact gctgacattg tcgactccaa aggcatcgtc 2220
gtcccggacg cagatcatct gattacgttt aacgtgacag gacagggtga acttgcgggg 2280
gttgataatg gtaatgcgtc ttcagttgaa cgctacaaag ataataaaag aaaggcattt 2340
tcggggaaag cgttggccat cgtgcagagc tcaaaactga gcggcaagat aaccgtacat 2400
gcatctgtgg ctggcttaag ctccgatagc acatccgtgt tcactgtaac ccctgccgat 2460
cacgataaga aaatcgtggc cggaattgat gacgtgaatc tgacggttga tgtcaatgaa 2520
gcccctaagc ttccgagcga gattaaggtc tattactccg atgaatcagc cgcggctaaa 2580
aacgttacat gggatgaagt tgatcctaag caatatagca ccgtcggcga gtttacagtc 2640
gaagggtctg tcgaaggtac aagcttaaaa gctaaagcat tcgtaattgt taagggaatt 2700
gtcgctgtca agccgtactc gactgccaca aaggtaggag tccagccggt cttacctgaa 2760
aaagcgaccc tgctttattc agacggcaca accaaaggcg cgacagtgac atgggacgag 2820
ctgcctgagg ataagcttgc taaagagggt cggttcaccg tggaaggctc ggtagaaggc 2880
accgatttaa aagcaaatgt ctacgttcgc gtgactaacg aagttaaatc agtcaatatt 2940
atgcttcaag aacaaggatc agcttatcca aagttagagg ctacatttac gaatcctgcg 3000
gataaccttc aacatctgaa cgacggcatt aagagttata cgaataacca ggtgaaccgc 3060
tggacaaatt ggactagaac cccaagagac gctggtgaca gcatcacagt gaattttggc 3120
aagaagcacg tcattaacaa cttggacttg ttcgttttca ccgactctgg tactgtcgtt 3180
cctgagaagg ctgaagtcca gtactgggat ggtactgcct ggaaagacgt tgagaacttg 3240
actcaaccat ccccatacgt cgttgaaaag aacgagttga ccttcgatgc tgtcgctact 3300
gaaaagttga agttccactt gacaccttct gttaagggaa agttcctcgc cctgaccgaa 3360
gccgaggtgt atgcggacca gatcgttgat ggggaaacag cgaaacttca atctattacc 3420
gtgaacggca aagccttgga gggttttgat catgcaaaga aaaactatga attggtcctc 3480
ccgtacggat cggagctccc gaagatagaa gcagcggctg ccgataatgc gaccgtgaca 3540
attttgccag cattcagcta tccgggcacg gctaaattat ttgtcacgag tgaagatgga 3600
aaggtgacca ctgaatattc tatcggggta tctactgaag aaccaaaatt agtgagcgcg 3660
gaattaagtg cggataaaac gaacgttatg gaagacgata ttattgatct taaggtgatt 3720
ggtctttttg aatctaagga aaaaattgat gtgacagact cacaaccaac atacgaattt 3780
gatcagcaga ttattaagat tgaaggaaac aaactgtatg ctttggagac cggcaatgtg 3840
aaagtcaagg ttacagttac atataaaggc gtctcagtta caacgccggc tcttgagttt 3900
acaatagcta aacaccctgc gccaaaatat atcacatcac tggaacctgt tacggtggtt 3960
gttaaaaaag gtgaggcgcc agaactgcct gctactgtcg tagcgcacta taatcggggc 4020
attccgcgcg acgtaaaagt gaaatgggag cgtattcatc cttcaaaata tcaacaactg 4080
ggggagttca cagtatcagg aatggtcgaa ggtactgaca ttaaagctca agccaaagta 4140
gcggtcaaag gagcggtcat cgtagaagat attagaatgg cagttctttt gaaacagatg 4200
ccacaattac cgggaaaagt tactgtctat tactctgacg gagccgagga gcaacgggcc 4260
gttaaatggg aagaaattcc acaggaagag ctggagaaag taggagagtt caaagtcaat 4320
ggtgatgtta atggcgttaa attaaaggcc actgctacaa tacgggtgac tgacgaggtc 4380
ggagctgaac aaaacataag ccgggcgaaa aacggctatg aatacccgaa agccgaagca 4440
tcctttacga atgatggtcc gggctcctcc gatagaatcg aggccattaa cgacgacgtc 4500
atctcttacg aagccaaccc acacaacaga tggaccaact ggcaacctgt tccaagagct 4560
ggtgactggg tatccattac cttcggtgac tacgagccaa ccgaatacga cgttgactct 4620
atggagatcc actggttcgc tgaccacggt acttcctacc ctgaacgttt tcaaattgaa 4680
tataaatcag gagactcctg gaaagaagta acaagcctga aaagtgaccc agcttcccca 4740
gctttaggga aagctaatgt gtactcgttt gatcgcgtta agacgtcagc cattcgggta 4800
aaaatgacag cgcaggcagg taaaagtctg gccattaccg aacttaaagt ttttagcaaa 4860
tggccgaaag ccggaacaga acctgatgtt acagatatta aagtgggtgg aaaatccata 4920
ctggaagagt tcgaacaaga aggagaccat tatgaagcaa cgatagatgt gggcgacgcg 4980
aacgtgatgc ctaaaattta tgttaaggcg aaagaccaaa caagcatctc aatcgttccg 5040
gctgtaacga gcgtcagcac cgctaaggtg atcgcagagg acggagacgg taaaaaagtg 5100
aaggtttatt cgatccataa gagctacaat aa 5132
<210> 14
<211> 1709
<212> PRT
<213> 人工序列
<400> 14
Met Lys Ser Thr Thr Ser Ala Ala Gly Lys Ser Val Ser Tyr Asn Asp
1 5 10 15
Gly Glu Arg Arg Val Asn Phe Glu Asn Trp Arg Phe Gln Arg Glu Thr
20 25 30
Asn Gly Ser Ile Ala Gly Ala Gln Asn Pro Gly Phe Asp Asp Ser Ser
35 40 45
Trp Arg Lys Leu Asn Leu Pro His Glu Trp Ser Ile Asp Leu Asp Phe
50 55 60
Asn Lys Asn Ser Leu Ala Thr His Glu Gly Gly Tyr Leu Asp Gly Gly
65 70 75 80
Ile Gly Trp Asn Arg Lys Thr Phe Thr Ile Pro Glu Ser Met Lys Gly
85 90 95
Lys Arg Phe Ser Leu Asp Phe Asp Gly Val Tyr Met Asn Ser Thr Thr
100 105 110
Tyr Leu Asn Gly Glu Val Leu Gly Thr Tyr Pro Phe Gly Tyr Asn Ala
115 120 125
Phe Ser Tyr Asp Ile Ser Asp Lys Leu Tyr Lys Asp Gly Arg Ala Asn
130 135 140
Val Leu Val Val Lys Val Asn Thr Asn Gln Pro Ser Gly Arg Trp Tyr
145 150 155 160
Ser Gly Ser Ser Ile Tyr Arg Asn Val Tyr Leu Thr Val Thr Asp Pro
165 170 175
Ile His Val Ala Arg Tyr Gly Thr Phe Val Thr Thr Pro Asn Leu Glu
180 185 190
Lys Ser Ile Lys Glu Asp Arg Asp Ala Val Asn Ile Lys Thr Lys Ile
195 200 205
Ser Asn Ala Glu Ala Lys Gln Val Lys Ile Ala Ile Lys Ser Thr Ile
210 215 220
Tyr Asp Gly Ala Gly Asn Thr Val Gln Thr Val Glu Thr Glu Glu Lys
225 230 235 240
Thr Ala Ala Ala Gly Thr Val Thr Pro Phe Glu Gln Asn Thr Val Ile
245 250 255
Lys Gln Pro Lys Leu Trp Ser Ile Asp Lys Pro Tyr Arg Tyr Asn Leu
260 265 270
Val Thr Glu Val Ile Val Gly Gly Gln Thr Val Asp Thr Tyr Glu Thr
275 280 285
Lys Phe Gly Val Arg Tyr Phe Lys Phe Asp Glu Asn Glu Gly Tyr Ser
290 295 300
Leu Asn Gly Glu Phe Met Lys Leu His Gly Val Ser Met His His Asp
305 310 315 320
Leu Ala Gly Gly Ala Ala Leu Thr Asn Ala Arg Gly Val Glu Arg Gln
325 330 335
Met Gln Ile Met Lys Asp Val Gly Val Asn Ala Ile Arg Val Thr His
340 345 350
Asn Pro Ala Ser Pro Glu Leu Gly Leu Glu Phe Ala Ala Asn Lys Leu
355 360 365
Leu Ile Ile Glu Glu Ala Phe Asp Ser Trp Ala Gln Ser Lys Lys Pro
370 375 380
Tyr Asp Tyr Gly Arg Phe Phe Asn Ala Trp Ala Glu His Asp Ile Lys
385 390 395 400
Glu Met Val Asp Arg Gly Lys Asn Glu Pro Ala Ile Ile Met Trp Ser
405 410 415
Ile Gly Asn Glu Ile Tyr Asp Thr Thr Asn Ala Ala Gly Val Glu Thr
420 425 430
Ala Arg Asn Leu Val Gly Trp Val Lys Glu Ile Asp Thr Thr Pro Arg
435 440 445
Ala Thr Ile Gly Glu Asp Lys Thr Arg Gly Asp Asn Val Thr Pro Ile
450 455 460
Asn Ser Tyr Ile Lys Glu Ile Phe Asn Ile Val Asp Val Val Gly Leu
465 470 475 480
Asn Tyr Ser Glu Asn Asn Tyr Asp Gly Tyr His Lys Gln Asn Pro Ser
485 490 495
Trp Lys Leu Tyr Gly Ser Glu Thr Ser Ser Ala Thr Arg Ser Arg Gly
500 505 510
Val Tyr Thr His Pro Tyr Gln Tyr Asn His Asp Thr Lys Tyr Ala Asp
515 520 525
Leu Gln Gln Ser Ser Tyr Asp Asn Asp Tyr Val Gly Trp Gly Arg Thr
530 535 540
Ala Gln Asp Ala Trp Lys Tyr Asp Arg Asp Leu Lys His Ile Ala Gly
545 550 555 560
Gln Phe Ile Trp Thr Gly Phe Asp Tyr Ile Gly Glu Pro Thr Pro Tyr
565 570 575
Tyr Asn Ser Tyr Pro Ala Lys Ser Ser Tyr Phe Gly Ala Val Asp Thr
580 585 590
Ala Gly Phe Pro Lys Asp Ile Phe Tyr Tyr Tyr Gln Ser Gln Trp Lys
595 600 605
Lys Glu Pro Met Val His Leu Leu Pro His Trp Asn Trp Lys Glu Gly
610 615 620
Glu Lys Val Arg Val Leu Ala Tyr Thr Asn Ala Ser Lys Val Glu Leu
625 630 635 640
Val Leu Asn Gly Glu Ser Leu Gly Asn Glu Lys Tyr Asp Asn Lys Gln
645 650 655
Thr Ser Trp Gly Ala Pro Tyr Lys Glu Thr Lys Asp Gly Lys Thr Tyr
660 665 670
Leu Glu Trp Ala Val Pro Phe Lys Pro Gly Lys Leu Glu Ala Val Ala
675 680 685
Lys Asp Glu Asn Gly Lys Val Ile Ala Arg Asp Gln Val Val Thr Ala
690 695 700
Gly Glu Pro Ala Ser Val Arg Leu Thr Ala Asp Arg Lys Val Val Lys
705 710 715 720
Ala Asp Gly Thr Asp Leu Ser Phe Ile Thr Ala Asp Ile Val Asp Ser
725 730 735
Lys Gly Ile Val Val Pro Asp Ala Asp His Leu Ile Thr Phe Asn Val
740 745 750
Thr Gly Gln Gly Glu Leu Ala Gly Val Asp Asn Gly Asn Ala Ser Ser
755 760 765
Val Glu Arg Tyr Lys Asp Asn Lys Arg Lys Ala Phe Ser Gly Lys Ala
770 775 780
Leu Ala Ile Val Gln Ser Ser Lys Leu Ser Gly Lys Ile Thr Val His
785 790 795 800
Ala Ser Val Ala Gly Leu Ser Ser Asp Ser Thr Ser Val Phe Thr Val
805 810 815
Thr Pro Ala Asp His Asp Lys Lys Ile Val Ala Gly Ile Asp Asp Val
820 825 830
Asn Leu Thr Val Asp Val Asn Glu Ala Pro Lys Leu Pro Ser Glu Ile
835 840 845
Lys Val Tyr Tyr Ser Asp Glu Ser Ala Ala Ala Lys Asn Val Thr Trp
850 855 860
Asp Glu Val Asp Pro Lys Gln Tyr Ser Thr Val Gly Glu Phe Thr Val
865 870 875 880
Glu Gly Ser Val Glu Gly Thr Ser Leu Lys Ala Lys Ala Phe Val Ile
885 890 895
Val Lys Gly Ile Val Ala Val Lys Pro Tyr Ser Thr Ala Thr Lys Val
900 905 910
Gly Val Gln Pro Val Leu Pro Glu Lys Ala Thr Leu Leu Tyr Ser Asp
915 920 925
Gly Thr Thr Lys Gly Ala Thr Val Thr Trp Asp Glu Leu Pro Glu Asp
930 935 940
Lys Leu Ala Lys Glu Gly Arg Phe Thr Val Glu Gly Ser Val Glu Gly
945 950 955 960
Thr Asp Leu Lys Ala Asn Val Tyr Val Arg Val Thr Asn Glu Val Lys
965 970 975
Ser Val Asn Ile Met Leu Gln Glu Gln Gly Ser Ala Tyr Pro Lys Leu
980 985 990
Glu Ala Thr Phe Thr Asn Pro Ala Asp Asn Leu Gln His Leu Asn Asp
995 1000 1005
Gly Ile Lys Ser Tyr Thr Asn Asn Gln Val Asn Arg Trp Thr Asn
1010 1015 1020
Trp Thr Arg Thr Pro Arg Asp Ala Gly Asp Ser Ile Thr Val Asn
1025 1030 1035
Phe Gly Lys Lys His Val Ile Asn Asn Leu Asp Leu Phe Val Phe
1040 1045 1050
Thr Asp Ser Gly Thr Val Val Pro Glu Lys Ala Glu Val Gln Tyr
1055 1060 1065
Trp Asp Gly Thr Ala Trp Lys Asp Val Glu Asn Leu Thr Gln Pro
1070 1075 1080
Ser Pro Tyr Val Val Glu Lys Asn Glu Leu Thr Phe Asp Ala Val
1085 1090 1095
Ala Thr Glu Lys Leu Lys Phe His Leu Thr Pro Ser Val Lys Gly
1100 1105 1110
Lys Phe Leu Ala Leu Thr Glu Ala Glu Val Tyr Ala Asp Gln Ile
1115 1120 1125
Val Asp Gly Glu Thr Ala Lys Leu Gln Ser Ile Thr Val Asn Gly
1130 1135 1140
Lys Ala Leu Glu Gly Phe Asp His Ala Lys Lys Asn Tyr Glu Leu
1145 1150 1155
Val Leu Pro Tyr Gly Ser Glu Leu Pro Lys Ile Glu Ala Ala Ala
1160 1165 1170
Ala Asp Asn Ala Thr Val Thr Ile Leu Pro Ala Phe Ser Tyr Pro
1175 1180 1185
Gly Thr Ala Lys Leu Phe Val Thr Ser Glu Asp Gly Lys Val Thr
1190 1195 1200
Thr Glu Tyr Ser Ile Gly Val Ser Thr Glu Glu Pro Lys Leu Val
1205 1210 1215
Ser Ala Glu Leu Ser Ala Asp Lys Thr Asn Val Met Glu Asp Asp
1220 1225 1230
Ile Ile Asp Leu Lys Val Ile Gly Leu Phe Glu Ser Lys Glu Lys
1235 1240 1245
Ile Asp Val Thr Asp Ser Gln Pro Thr Tyr Glu Phe Asp Gln Gln
1250 1255 1260
Ile Ile Lys Ile Glu Gly Asn Lys Leu Tyr Ala Leu Glu Thr Gly
1265 1270 1275
Asn Val Lys Val Lys Val Thr Val Thr Tyr Lys Gly Val Ser Val
1280 1285 1290
Thr Thr Pro Ala Leu Glu Phe Thr Ile Ala Lys His Pro Ala Pro
1295 1300 1305
Lys Tyr Ile Thr Ser Leu Glu Pro Val Thr Val Val Val Lys Lys
1310 1315 1320
Gly Glu Ala Pro Glu Leu Pro Ala Thr Val Val Ala His Tyr Asn
1325 1330 1335
Arg Gly Ile Pro Arg Asp Val Lys Val Lys Trp Glu Arg Ile His
1340 1345 1350
Pro Ser Lys Tyr Gln Gln Leu Gly Glu Phe Thr Val Ser Gly Met
1355 1360 1365
Val Glu Gly Thr Asp Ile Lys Ala Gln Ala Lys Val Ala Val Lys
1370 1375 1380
Gly Ala Val Ile Val Glu Asp Ile Arg Met Ala Val Leu Leu Lys
1385 1390 1395
Gln Met Pro Gln Leu Pro Gly Lys Val Thr Val Tyr Tyr Ser Asp
1400 1405 1410
Gly Ala Glu Glu Gln Arg Ala Val Lys Trp Glu Glu Ile Pro Gln
1415 1420 1425
Glu Glu Leu Glu Lys Val Gly Glu Phe Lys Val Asn Gly Asp Val
1430 1435 1440
Asn Gly Val Lys Leu Lys Ala Thr Ala Thr Ile Arg Val Thr Asp
1445 1450 1455
Glu Val Gly Ala Glu Gln Asn Ile Ser Arg Ala Lys Asn Gly Tyr
1460 1465 1470
Glu Tyr Pro Lys Ala Glu Ala Ser Phe Thr Asn Asp Gly Pro Gly
1475 1480 1485
Ser Ser Asp Arg Ile Glu Ala Ile Asn Asp Asp Val Ile Ser Tyr
1490 1495 1500
Glu Ala Asn Pro His Asn Arg Trp Thr Asn Trp Gln Pro Val Pro
1505 1510 1515
Arg Ala Gly Asp Trp Val Ser Ile Thr Phe Gly Asp Tyr Glu Pro
1520 1525 1530
Thr Glu Tyr Asp Val Asp Ser Met Glu Ile His Trp Phe Ala Asp
1535 1540 1545
His Gly Thr Ser Tyr Pro Glu Arg Phe Gln Ile Glu Tyr Lys Ser
1550 1555 1560
Gly Asp Ser Trp Lys Glu Val Thr Ser Leu Lys Ser Asp Pro Ala
1565 1570 1575
Ser Pro Ala Leu Gly Lys Ala Asn Val Tyr Ser Phe Asp Arg Val
1580 1585 1590
Lys Thr Ser Ala Ile Arg Val Lys Met Thr Ala Gln Ala Gly Lys
1595 1600 1605
Ser Leu Ala Ile Thr Glu Leu Lys Val Phe Ser Lys Trp Pro Lys
1610 1615 1620
Ala Gly Thr Glu Pro Asp Val Thr Asp Ile Lys Val Gly Gly Lys
1625 1630 1635
Ser Ile Leu Glu Glu Phe Glu Gln Glu Gly Asp His Tyr Glu Ala
1640 1645 1650
Thr Ile Asp Val Gly Asp Ala Asn Val Met Pro Lys Ile Tyr Val
1655 1660 1665
Lys Ala Lys Asp Gln Thr Ser Ile Ser Ile Val Pro Ala Val Thr
1670 1675 1680
Ser Val Ser Thr Ala Lys Val Ile Ala Glu Asp Gly Asp Gly Lys
1685 1690 1695
Lys Val Lys Val Tyr Ser Ile His Lys Ser Tyr
1700 1705
<210> 15
<211> 3669
<212> DNA
<213> 人工序列
<400> 15
atgaaaagca cgacgagcgc cgccggcaaa agcgtcagct ataacgacgg cgaacgccgc 60
gtcaactttg aaaactggcg ctttcagcgc gaaacgaacg gcagcatcgc cggcgcccag 120
aacccaggct ttgacgacag cagctggcgc aaactgaacc tgccgcatga atggagcatc 180
gacctggact ttaacaaaaa cagcctggcc acgcatgaag gcggctatct ggacggcggc 240
atcggctgga accgcaaaac gtttacgatc ccggaaagca tgaaaggcaa acgctttagc 300
ctggactttg acggcgtcta tatgaacagc acgacgtatc tgaacggcga agtcctgggc 360
acgtatccgt ttggctataa cgcctttagc tatgacatca gcgacaaact gtataaagac 420
ggccgcgcca acgtcctggt cgtcaaagtc aacacgaacc agccgagcgg ccgctggtat 480
agcggcagca gcatctatcg caacgtctat ctgacggtca cggacccgat ccatgtcgcc 540
cgctatggca cgtttgtcac gacgccgaac ctggaaaaaa gcatcaaaga agaccgcgac 600
gccgtcaaca tcaaaacgaa aatcagcaac gccgaagcca aacaggtcaa aatcgccatc 660
aaaagcacga tctatgacgg cgccggcaac acggtccaga cggtcgaaac ggaagaaaaa 720
acggccgccg ccggcacggt cacgccgttt gaacagaaca cggtcatcaa acagccgaaa 780
ctgtggagca tcgacaaacc gtatcgctat aacctggtca cggaagtcat cgtcggcggc 840
cagacggtcg acacgtatga aacgaaattt ggcgtccgct attttaaatt tgacgaaaac 900
gaaggctata gcctgaacgg cgaatttatg aaactgcatg gcgtcagcat gcatcatgac 960
ctggccggcg gcgccgccct gacgaacgcc cgcggcgtcg aacgccagat gcagatcatg 1020
aaagacgtcg gcgtcaacgc catccgcgtc acgcataacc cggccagccc ggaactgggc 1080
ctggaatttg ccgccaacaa actgctgatc atcgaagaag cctttgacag ctgggcccag 1140
agcaaaaaac cgtatgacta tggccgcttt tttaacgcct gggccgaaca tgacatcaaa 1200
gaaatggtcg accgcggcaa aaacgaaccg gccatcatca tgtggagcat cggcaacgaa 1260
atctatgaca cgacgaacgc cgccggcgtc gaaacggccc gcaacctggt cggctgggtc 1320
aaagaaatcg acacgacgcc gcgcgccacg atcggcgaag acaaaacgcg cggcgacaac 1380
gtcacgccga tcaacagcta tatcaaagaa atctttaaca tcgtcgacgt cgtcggcctg 1440
aactatagcg aaaacaacta tgacggctat cataaacaga acccgagctg gaaactgtat 1500
ggcagcgaaa cgagcagcgc cacgcgcagc cgcggcgtct atacgcatcc gtatcagtat 1560
aaccatgaca cgaaatatgc cgacctgcag cagagcagct atgacaacga ctatgtcggc 1620
tggggccgca cggcccagga cgcctggaaa tatgaccgcg acctgaaaca tatcgccggc 1680
cagtttatct ggacgggctt tgactatatc ggcgaaccga cgccgtatta taacagctat 1740
ccggccaaaa gcagctattt tggcgccgtc gacacggccg gctttccgaa agacatcttt 1800
tattattatc agagccagtg gaaaaaagaa ccgatggtcc atctgctgcc gcattggaac 1860
tggaaagaag gcgaaaaagt ccgcgtcctg gcctatacga acgccagcaa agtcgaactg 1920
gtcctgaacg gcgaaagcct gggcaacgaa aaatatgaca acaaacagac gagctggggc 1980
gccccgtata aagaaacgaa agacggcaaa acgtatctgg aatgggccgt cccgtttaaa 2040
ccgggcaaac tggaagccgt cgccaaagac gaaaacggca aagtcatcgc ccgcgaccag 2100
gtcgtcacgg ccggcgaacc ggccagcgtc cgcctgacgg ccgaccgcaa agtcgtcaaa 2160
gccgacggca cggacctgag ctttatcacg gccgacatcg tcgacagcaa aggcatcgtc 2220
gtcccggacg ccgaccatct gatcacgttt aacgtcacgg gccagggcga actggccggc 2280
gtcgacaacg gcaacgccag cagcgtcgaa cgctataaag acaacaaacg caaagccttt 2340
agcggcaaag ccctggccat cgtccagagc agcaaactga gcggcaaaat cacggtccat 2400
gccagcgtcg ccggcctgag cagcgacagc acgagcgtct ttacggtcac gccggccgac 2460
catgacaaaa aaatcgtcgc cggcatcgac gacgtcaacc tgacggtcga cgtcaacgaa 2520
gccccgaaac tgccgagcga aatcaaagtc tattatagcg acgaaagcgc cgccgccaaa 2580
aacgtcacgt gggacgaagt cgacccgaaa cagtatagca cggtcggcga atttacggtc 2640
gaaggcagcg tcgaaggcac gagcctgaaa gccaaagcct ttgtcatcgt caaaggcatc 2700
gtcgccgtca aaccgtatag cacggccacg aaagtcggcg tccagccggt cctgccggaa 2760
aaagccacgc tgctgtatag cgacggcacg acgaaaggcg ccacggtcac gtgggacgaa 2820
ctgccggaag acaaactggc caaagaaggc cgctttacgg tcgaaggcag cgtcgaaggc 2880
acggacctga aagccaacgt ctatgtccgc gtcacgaacg aagtcaaaag cgtcaacatc 2940
atgctgcagg aacagggcag cgcctatccg aaactggaag ccacgtttac gaacccggcc 3000
gacaacctgc agcatctgaa cgacggcatc aaaagctata cgaacaacca ggtcaaccgc 3060
tggacgaact ggacgcgcac gccgcgcgac gccggcgaca gcatcacggt caactttggc 3120
aaaaaacatg tcatcaacaa cctggacctg tttgtcttta cggacagcgg cacggtcgtc 3180
ccggaaaaag ccgaagtcca gtattgggac ggcacggcct ggaaagacgt cgaaaacctg 3240
acgcagccga gcccgtatgt cgtcgaaaaa aacgaactga cgtttgacgc cgtcgccacg 3300
gaaaaactga aatttcatct gacgccgagc gtcaaaggca aatttctggc cctgacggaa 3360
gccgaagtct atgccgacca gatcgtcgac ggcgaaacgg ccaaactgca gagcatcacg 3420
gtcaacggca aagccctgga aggctttgac catgccaaaa aaaactatga actggtcctg 3480
ccgtatggca gcgaactgcc gaaaatcgaa gccgccgccg ccgacaacgc cacggtcacg 3540
atcctgccgg cctttagcta tccgggcacg gccaaactgt ttgtcacgag cgaagacggc 3600
aaagtcacga cggaatatag catcggcgtc agcacggaag aaccgaaact ggtcagcgcc 3660
gaactgtaa 3669
<210> 16
<211> 1222
<212> PRT
<213> 人工序列
<400> 16
Met Lys Ser Thr Thr Ser Ala Ala Gly Lys Ser Val Ser Tyr Asn Asp
1 5 10 15
Gly Glu Arg Arg Val Asn Phe Glu Asn Trp Arg Phe Gln Arg Glu Thr
20 25 30
Asn Gly Ser Ile Ala Gly Ala Gln Asn Pro Gly Phe Asp Asp Ser Ser
35 40 45
Trp Arg Lys Leu Asn Leu Pro His Glu Trp Ser Ile Asp Leu Asp Phe
50 55 60
Asn Lys Asn Ser Leu Ala Thr His Glu Gly Gly Tyr Leu Asp Gly Gly
65 70 75 80
Ile Gly Trp Asn Arg Lys Thr Phe Thr Ile Pro Glu Ser Met Lys Gly
85 90 95
Lys Arg Phe Ser Leu Asp Phe Asp Gly Val Tyr Met Asn Ser Thr Thr
100 105 110
Tyr Leu Asn Gly Glu Val Leu Gly Thr Tyr Pro Phe Gly Tyr Asn Ala
115 120 125
Phe Ser Tyr Asp Ile Ser Asp Lys Leu Tyr Lys Asp Gly Arg Ala Asn
130 135 140
Val Leu Val Val Lys Val Asn Thr Asn Gln Pro Ser Gly Arg Trp Tyr
145 150 155 160
Ser Gly Ser Ser Ile Tyr Arg Asn Val Tyr Leu Thr Val Thr Asp Pro
165 170 175
Ile His Val Ala Arg Tyr Gly Thr Phe Val Thr Thr Pro Asn Leu Glu
180 185 190
Lys Ser Ile Lys Glu Asp Arg Asp Ala Val Asn Ile Lys Thr Lys Ile
195 200 205
Ser Asn Ala Glu Ala Lys Gln Val Lys Ile Ala Ile Lys Ser Thr Ile
210 215 220
Tyr Asp Gly Ala Gly Asn Thr Val Gln Thr Val Glu Thr Glu Glu Lys
225 230 235 240
Thr Ala Ala Ala Gly Thr Val Thr Pro Phe Glu Gln Asn Thr Val Ile
245 250 255
Lys Gln Pro Lys Leu Trp Ser Ile Asp Lys Pro Tyr Arg Tyr Asn Leu
260 265 270
Val Thr Glu Val Ile Val Gly Gly Gln Thr Val Asp Thr Tyr Glu Thr
275 280 285
Lys Phe Gly Val Arg Tyr Phe Lys Phe Asp Glu Asn Glu Gly Tyr Ser
290 295 300
Leu Asn Gly Glu Phe Met Lys Leu His Gly Val Ser Met His His Asp
305 310 315 320
Leu Ala Gly Gly Ala Ala Leu Thr Asn Ala Arg Gly Val Glu Arg Gln
325 330 335
Met Gln Ile Met Lys Asp Val Gly Val Asn Ala Ile Arg Val Thr His
340 345 350
Asn Pro Ala Ser Pro Glu Leu Gly Leu Glu Phe Ala Ala Asn Lys Leu
355 360 365
Leu Ile Ile Glu Glu Ala Phe Asp Ser Trp Ala Gln Ser Lys Lys Pro
370 375 380
Tyr Asp Tyr Gly Arg Phe Phe Asn Ala Trp Ala Glu His Asp Ile Lys
385 390 395 400
Glu Met Val Asp Arg Gly Lys Asn Glu Pro Ala Ile Ile Met Trp Ser
405 410 415
Ile Gly Asn Glu Ile Tyr Asp Thr Thr Asn Ala Ala Gly Val Glu Thr
420 425 430
Ala Arg Asn Leu Val Gly Trp Val Lys Glu Ile Asp Thr Thr Pro Arg
435 440 445
Ala Thr Ile Gly Glu Asp Lys Thr Arg Gly Asp Asn Val Thr Pro Ile
450 455 460
Asn Ser Tyr Ile Lys Glu Ile Phe Asn Ile Val Asp Val Val Gly Leu
465 470 475 480
Asn Tyr Ser Glu Asn Asn Tyr Asp Gly Tyr His Lys Gln Asn Pro Ser
485 490 495
Trp Lys Leu Tyr Gly Ser Glu Thr Ser Ser Ala Thr Arg Ser Arg Gly
500 505 510
Val Tyr Thr His Pro Tyr Gln Tyr Asn His Asp Thr Lys Tyr Ala Asp
515 520 525
Leu Gln Gln Ser Ser Tyr Asp Asn Asp Tyr Val Gly Trp Gly Arg Thr
530 535 540
Ala Gln Asp Ala Trp Lys Tyr Asp Arg Asp Leu Lys His Ile Ala Gly
545 550 555 560
Gln Phe Ile Trp Thr Gly Phe Asp Tyr Ile Gly Glu Pro Thr Pro Tyr
565 570 575
Tyr Asn Ser Tyr Pro Ala Lys Ser Ser Tyr Phe Gly Ala Val Asp Thr
580 585 590
Ala Gly Phe Pro Lys Asp Ile Phe Tyr Tyr Tyr Gln Ser Gln Trp Lys
595 600 605
Lys Glu Pro Met Val His Leu Leu Pro His Trp Asn Trp Lys Glu Gly
610 615 620
Glu Lys Val Arg Val Leu Ala Tyr Thr Asn Ala Ser Lys Val Glu Leu
625 630 635 640
Val Leu Asn Gly Glu Ser Leu Gly Asn Glu Lys Tyr Asp Asn Lys Gln
645 650 655
Thr Ser Trp Gly Ala Pro Tyr Lys Glu Thr Lys Asp Gly Lys Thr Tyr
660 665 670
Leu Glu Trp Ala Val Pro Phe Lys Pro Gly Lys Leu Glu Ala Val Ala
675 680 685
Lys Asp Glu Asn Gly Lys Val Ile Ala Arg Asp Gln Val Val Thr Ala
690 695 700
Gly Glu Pro Ala Ser Val Arg Leu Thr Ala Asp Arg Lys Val Val Lys
705 710 715 720
Ala Asp Gly Thr Asp Leu Ser Phe Ile Thr Ala Asp Ile Val Asp Ser
725 730 735
Lys Gly Ile Val Val Pro Asp Ala Asp His Leu Ile Thr Phe Asn Val
740 745 750
Thr Gly Gln Gly Glu Leu Ala Gly Val Asp Asn Gly Asn Ala Ser Ser
755 760 765
Val Glu Arg Tyr Lys Asp Asn Lys Arg Lys Ala Phe Ser Gly Lys Ala
770 775 780
Leu Ala Ile Val Gln Ser Ser Lys Leu Ser Gly Lys Ile Thr Val His
785 790 795 800
Ala Ser Val Ala Gly Leu Ser Ser Asp Ser Thr Ser Val Phe Thr Val
805 810 815
Thr Pro Ala Asp His Asp Lys Lys Ile Val Ala Gly Ile Asp Asp Val
820 825 830
Asn Leu Thr Val Asp Val Asn Glu Ala Pro Lys Leu Pro Ser Glu Ile
835 840 845
Lys Val Tyr Tyr Ser Asp Glu Ser Ala Ala Ala Lys Asn Val Thr Trp
850 855 860
Asp Glu Val Asp Pro Lys Gln Tyr Ser Thr Val Gly Glu Phe Thr Val
865 870 875 880
Glu Gly Ser Val Glu Gly Thr Ser Leu Lys Ala Lys Ala Phe Val Ile
885 890 895
Val Lys Gly Ile Val Ala Val Lys Pro Tyr Ser Thr Ala Thr Lys Val
900 905 910
Gly Val Gln Pro Val Leu Pro Glu Lys Ala Thr Leu Leu Tyr Ser Asp
915 920 925
Gly Thr Thr Lys Gly Ala Thr Val Thr Trp Asp Glu Leu Pro Glu Asp
930 935 940
Lys Leu Ala Lys Glu Gly Arg Phe Thr Val Glu Gly Ser Val Glu Gly
945 950 955 960
Thr Asp Leu Lys Ala Asn Val Tyr Val Arg Val Thr Asn Glu Val Lys
965 970 975
Ser Val Asn Ile Met Leu Gln Glu Gln Gly Ser Ala Tyr Pro Lys Leu
980 985 990
Glu Ala Thr Phe Thr Asn Pro Ala Asp Asn Leu Gln His Leu Asn Asp
995 1000 1005
Gly Ile Lys Ser Tyr Thr Asn Asn Gln Val Asn Arg Trp Thr Asn
1010 1015 1020
Trp Thr Arg Thr Pro Arg Asp Ala Gly Asp Ser Ile Thr Val Asn
1025 1030 1035
Phe Gly Lys Lys His Val Ile Asn Asn Leu Asp Leu Phe Val Phe
1040 1045 1050
Thr Asp Ser Gly Thr Val Val Pro Glu Lys Ala Glu Val Gln Tyr
1055 1060 1065
Trp Asp Gly Thr Ala Trp Lys Asp Val Glu Asn Leu Thr Gln Pro
1070 1075 1080
Ser Pro Tyr Val Val Glu Lys Asn Glu Leu Thr Phe Asp Ala Val
1085 1090 1095
Ala Thr Glu Lys Leu Lys Phe His Leu Thr Pro Ser Val Lys Gly
1100 1105 1110
Lys Phe Leu Ala Leu Thr Glu Ala Glu Val Tyr Ala Asp Gln Ile
1115 1120 1125
Val Asp Gly Glu Thr Ala Lys Leu Gln Ser Ile Thr Val Asn Gly
1130 1135 1140
Lys Ala Leu Glu Gly Phe Asp His Ala Lys Lys Asn Tyr Glu Leu
1145 1150 1155
Val Leu Pro Tyr Gly Ser Glu Leu Pro Lys Ile Glu Ala Ala Ala
1160 1165 1170
Ala Asp Asn Ala Thr Val Thr Ile Leu Pro Ala Phe Ser Tyr Pro
1175 1180 1185
Gly Thr Ala Lys Leu Phe Val Thr Ser Glu Asp Gly Lys Val Thr
1190 1195 1200
Thr Glu Tyr Ser Ile Gly Val Ser Thr Glu Glu Pro Lys Leu Val
1205 1210 1215
Ser Ala Glu Leu
1220
<210> 17
<211> 2463
<212> DNA
<213> 人工序列
<400> 17
atgaaaagca cgacgagcgc cgccggcaaa agcgtcagct ataacgacgg cgaacgccgc 60
gtcaactttg aaaactggcg ctttcagcgc gaaacgaacg gcagcatcgc cggcgcccag 120
aacccaggct ttgacgacag cagctggcgc aaactgaacc tgccgcatga atggagcatc 180
gacctggact ttaacaaaaa cagcctggcc acgcatgaag gcggctatct ggacggcggc 240
atcggctgga accgcaaaac gtttacgatc ccggaaagca tgaaaggcaa acgctttagc 300
ctggactttg acggcgtcta tatgaacagc acgacgtatc tgaacggcga agtcctgggc 360
acgtatccgt ttggctataa cgcctttagc tatgacatca gcgacaaact gtataaagac 420
ggccgcgcca acgtcctggt cgtcaaagtc aacacgaacc agccgagcgg ccgctggtat 480
agcggcagca gcatctatcg caacgtctat ctgacggtca cggacccgat ccatgtcgcc 540
cgctatggca cgtttgtcac gacgccgaac ctggaaaaaa gcatcaaaga agaccgcgac 600
gccgtcaaca tcaaaacgaa aatcagcaac gccgaagcca aacaggtcaa aatcgccatc 660
aaaagcacga tctatgacgg cgccggcaac acggtccaga cggtcgaaac ggaagaaaaa 720
acggccgccg ccggcacggt cacgccgttt gaacagaaca cggtcatcaa acagccgaaa 780
ctgtggagca tcgacaaacc gtatcgctat aacctggtca cggaagtcat cgtcggcggc 840
cagacggtcg acacgtatga aacgaaattt ggcgtccgct attttaaatt tgacgaaaac 900
gaaggctata gcctgaacgg cgaatttatg aaactgcatg gcgtcagcat gcatcatgac 960
ctggccggcg gcgccgccct gacgaacgcc cgcggcgtcg aacgccagat gcagatcatg 1020
aaagacgtcg gcgtcaacgc catccgcgtc acgcataacc cggccagccc ggaactgggc 1080
ctggaatttg ccgccaacaa actgctgatc atcgaagaag cctttgacag ctgggcccag 1140
agcaaaaaac cgtatgacta tggccgcttt tttaacgcct gggccgaaca tgacatcaaa 1200
gaaatggtcg accgcggcaa aaacgaaccg gccatcatca tgtggagcat cggcaacgaa 1260
atctatgaca cgacgaacgc cgccggcgtc gaaacggccc gcaacctggt cggctgggtc 1320
aaagaaatcg acacgacgcc gcgcgccacg atcggcgaag acaaaacgcg cggcgacaac 1380
gtcacgccga tcaacagcta tatcaaagaa atctttaaca tcgtcgacgt cgtcggcctg 1440
aactatagcg aaaacaacta tgacggctat cataaacaga acccgagctg gaaactgtat 1500
ggcagcgaaa cgagcagcgc cacgcgcagc cgcggcgtct atacgcatcc gtatcagtat 1560
aaccatgaca cgaaatatgc cgacctgcag cagagcagct atgacaacga ctatgtcggc 1620
tggggccgca cggcccagga cgcctggaaa tatgaccgcg acctgaaaca tatcgccggc 1680
cagtttatct ggacgggctt tgactatatc ggcgaaccga cgccgtatta taacagctat 1740
ccggccaaaa gcagctattt tggcgccgtc gacacggccg gctttccgaa agacatcttt 1800
tattattatc agagccagtg gaaaaaagaa ccgatggtcc atctgctgcc gcattggaac 1860
tggaaagaag gcgaaaaagt ccgcgtcctg gcctatacga acgccagcaa agtcgaactg 1920
gtcctgaacg gcgaaagcct gggcaacgaa aaatatgaca acaaacagac gagctggggc 1980
gccccgtata aagaaacgaa agacggcaaa acgtatctgg aatgggccgt cccgtttaaa 2040
ccgggcaaac tggaagccgt cgccaaagac gaaaacggca aagtcatcgc ccgcgaccag 2100
gtcgtcacgg ccggcgaacc ggccagcgtc cgcctgacgg ccgaccgcaa agtcgtcaaa 2160
gccgacggca cggacctgag ctttatcacg gccgacatcg tcgacagcaa aggcatcgtc 2220
gtcccggacg ccgaccatct gatcacgttt aacgtcacgg gccagggcga actggccggc 2280
gtcgacaacg gcaacgccag cagcgtcgaa cgctataaag acaacaaacg caaagccttt 2340
agcggcaaag ccctggccat cgtccagagc agcaaactga gcggcaaaat cacggtccat 2400
gccagcgtcg ccggcctgag cagcgacagc acgagcgtct ttacggtcac gccggccgac 2460
taa 2463
<210> 18
<211> 820
<212> PRT
<213> 人工序列
<400> 18
Met Lys Ser Thr Thr Ser Ala Ala Gly Lys Ser Val Ser Tyr Asn Asp
1 5 10 15
Gly Glu Arg Arg Val Asn Phe Glu Asn Trp Arg Phe Gln Arg Glu Thr
20 25 30
Asn Gly Ser Ile Ala Gly Ala Gln Asn Pro Gly Phe Asp Asp Ser Ser
35 40 45
Trp Arg Lys Leu Asn Leu Pro His Glu Trp Ser Ile Asp Leu Asp Phe
50 55 60
Asn Lys Asn Ser Leu Ala Thr His Glu Gly Gly Tyr Leu Asp Gly Gly
65 70 75 80
Ile Gly Trp Asn Arg Lys Thr Phe Thr Ile Pro Glu Ser Met Lys Gly
85 90 95
Lys Arg Phe Ser Leu Asp Phe Asp Gly Val Tyr Met Asn Ser Thr Thr
100 105 110
Tyr Leu Asn Gly Glu Val Leu Gly Thr Tyr Pro Phe Gly Tyr Asn Ala
115 120 125
Phe Ser Tyr Asp Ile Ser Asp Lys Leu Tyr Lys Asp Gly Arg Ala Asn
130 135 140
Val Leu Val Val Lys Val Asn Thr Asn Gln Pro Ser Gly Arg Trp Tyr
145 150 155 160
Ser Gly Ser Ser Ile Tyr Arg Asn Val Tyr Leu Thr Val Thr Asp Pro
165 170 175
Ile His Val Ala Arg Tyr Gly Thr Phe Val Thr Thr Pro Asn Leu Glu
180 185 190
Lys Ser Ile Lys Glu Asp Arg Asp Ala Val Asn Ile Lys Thr Lys Ile
195 200 205
Ser Asn Ala Glu Ala Lys Gln Val Lys Ile Ala Ile Lys Ser Thr Ile
210 215 220
Tyr Asp Gly Ala Gly Asn Thr Val Gln Thr Val Glu Thr Glu Glu Lys
225 230 235 240
Thr Ala Ala Ala Gly Thr Val Thr Pro Phe Glu Gln Asn Thr Val Ile
245 250 255
Lys Gln Pro Lys Leu Trp Ser Ile Asp Lys Pro Tyr Arg Tyr Asn Leu
260 265 270
Val Thr Glu Val Ile Val Gly Gly Gln Thr Val Asp Thr Tyr Glu Thr
275 280 285
Lys Phe Gly Val Arg Tyr Phe Lys Phe Asp Glu Asn Glu Gly Tyr Ser
290 295 300
Leu Asn Gly Glu Phe Met Lys Leu His Gly Val Ser Met His His Asp
305 310 315 320
Leu Ala Gly Gly Ala Ala Leu Thr Asn Ala Arg Gly Val Glu Arg Gln
325 330 335
Met Gln Ile Met Lys Asp Val Gly Val Asn Ala Ile Arg Val Thr His
340 345 350
Asn Pro Ala Ser Pro Glu Leu Gly Leu Glu Phe Ala Ala Asn Lys Leu
355 360 365
Leu Ile Ile Glu Glu Ala Phe Asp Ser Trp Ala Gln Ser Lys Lys Pro
370 375 380
Tyr Asp Tyr Gly Arg Phe Phe Asn Ala Trp Ala Glu His Asp Ile Lys
385 390 395 400
Glu Met Val Asp Arg Gly Lys Asn Glu Pro Ala Ile Ile Met Trp Ser
405 410 415
Ile Gly Asn Glu Ile Tyr Asp Thr Thr Asn Ala Ala Gly Val Glu Thr
420 425 430
Ala Arg Asn Leu Val Gly Trp Val Lys Glu Ile Asp Thr Thr Pro Arg
435 440 445
Ala Thr Ile Gly Glu Asp Lys Thr Arg Gly Asp Asn Val Thr Pro Ile
450 455 460
Asn Ser Tyr Ile Lys Glu Ile Phe Asn Ile Val Asp Val Val Gly Leu
465 470 475 480
Asn Tyr Ser Glu Asn Asn Tyr Asp Gly Tyr His Lys Gln Asn Pro Ser
485 490 495
Trp Lys Leu Tyr Gly Ser Glu Thr Ser Ser Ala Thr Arg Ser Arg Gly
500 505 510
Val Tyr Thr His Pro Tyr Gln Tyr Asn His Asp Thr Lys Tyr Ala Asp
515 520 525
Leu Gln Gln Ser Ser Tyr Asp Asn Asp Tyr Val Gly Trp Gly Arg Thr
530 535 540
Ala Gln Asp Ala Trp Lys Tyr Asp Arg Asp Leu Lys His Ile Ala Gly
545 550 555 560
Gln Phe Ile Trp Thr Gly Phe Asp Tyr Ile Gly Glu Pro Thr Pro Tyr
565 570 575
Tyr Asn Ser Tyr Pro Ala Lys Ser Ser Tyr Phe Gly Ala Val Asp Thr
580 585 590
Ala Gly Phe Pro Lys Asp Ile Phe Tyr Tyr Tyr Gln Ser Gln Trp Lys
595 600 605
Lys Glu Pro Met Val His Leu Leu Pro His Trp Asn Trp Lys Glu Gly
610 615 620
Glu Lys Val Arg Val Leu Ala Tyr Thr Asn Ala Ser Lys Val Glu Leu
625 630 635 640
Val Leu Asn Gly Glu Ser Leu Gly Asn Glu Lys Tyr Asp Asn Lys Gln
645 650 655
Thr Ser Trp Gly Ala Pro Tyr Lys Glu Thr Lys Asp Gly Lys Thr Tyr
660 665 670
Leu Glu Trp Ala Val Pro Phe Lys Pro Gly Lys Leu Glu Ala Val Ala
675 680 685
Lys Asp Glu Asn Gly Lys Val Ile Ala Arg Asp Gln Val Val Thr Ala
690 695 700
Gly Glu Pro Ala Ser Val Arg Leu Thr Ala Asp Arg Lys Val Val Lys
705 710 715 720
Ala Asp Gly Thr Asp Leu Ser Phe Ile Thr Ala Asp Ile Val Asp Ser
725 730 735
Lys Gly Ile Val Val Pro Asp Ala Asp His Leu Ile Thr Phe Asn Val
740 745 750
Thr Gly Gln Gly Glu Leu Ala Gly Val Asp Asn Gly Asn Ala Ser Ser
755 760 765
Val Glu Arg Tyr Lys Asp Asn Lys Arg Lys Ala Phe Ser Gly Lys Ala
770 775 780
Leu Ala Ile Val Gln Ser Ser Lys Leu Ser Gly Lys Ile Thr Val His
785 790 795 800
Ala Ser Val Ala Gly Leu Ser Ser Asp Ser Thr Ser Val Phe Thr Val
805 810 815
Thr Pro Ala Asp
820
<210> 19
<211> 628
<212> DNA
<213> 苏云金芽胞杆菌
<400> 19
gaaacgtaag atgaaacctt agataaaagt gctttttttg ttgcaattga agaattatta 60
atgttaagct taattaaaga taatatcttt gaattgtaac gcccctcaaa agtaagaact 120
acaaaaaaag aatacgttat atagaaatat gtttgaacct tcttcagatt acaaatatat 180
tcggacggac tctacctcaa atgcttatct aactatagaa tgacatacaa gcacaacctt 240
gaaaatttga aaatataact accaatgaac ttgttcatgt gaattatcgc tgtatttaat 300
tttctcaatt caatatataa tatgccaata cattgttaca agtagaaatt aagacaccct 360
tgatagcctt actataccta acatgatgta gtattaaatg aatatgtaaa tatatttatg 420
ataagaagcg acttatttat aatcattaca tatttttcta ttggaatgat taagattcca 480
atagaatagt gtataaatta tttatcttga aaggagggat gcctaaaaac gaagaacatt 540
aaaaacatat atttgcaccg tctaatggat ttatgaaaaa tcattttatc agtttgaaaa 600
ttatgtatta tgataagaaa gggaggaa 628
<210> 20
<211> 180
<212> DNA
<213> 地衣芽胞杆菌
<400> 20
tttttttata ggaacattga tttgtattca ctctgccaag ttgttttgat agagtgattg 60
tgataatttt aaatgtaagc gttaacaaaa ttctccagtc ttcacatcgg tttgaaagga 120
ggaagcggaa gaatgaagta agagggattt ttgactccga agtaagtctt caaaaaatca 180
<210> 21
<211> 426
<212> DNA
<213> 枯草芽胞杆菌
<400> 21
tgtcgacgtg catgcaggcc ggggcatatg ggaaacagcg cggacggagc ggaatttcca 60
atttcatgcc gcagccgcct gcgctgttct catttgcggc ttccttgtag agctcagcat 120
tattgagtgg atgattatat tccttttgat aggtggtatg ttttcgcttg aacttttaaa 180
tacagccatt gaacatacgg ttgatttaat aactgacaaa catcaccctc ttgctaaagc 240
ggccaaggac gctgccgccg gggctgtttg cgtttttacc gtgatttcgt gtatcattgg 300
tttacttatt tttttgccaa agctgtaatg gctgaaaatt cttacattta ttttacattt 360
ttagaaatgg gcgtgaaaaa aagcgcgcga ttatgtaaaa tataaagtga tagcggtacc 420
attata 426
<210> 22
<211> 87
<212> DNA
<213> 地衣芽胞杆菌
<400> 22
atgaaacaac aaaaacggct ttacgcccga ttgctgccgc tgttatttgc gctcatcttc 60
ttgctgcctc attctgcagc agcggcg 87
<210> 23
<211> 87
<212> DNA
<213> 地衣芽胞杆菌
<400> 23
atgatgagga aaaagagttt ttggcttggg atgctgacgg ccttaatgct cgtgttcacg 60
atggccttca gcgattccgc gtctgct 87
<210> 24
<211> 93
<212> DNA
<213> 解淀粉芽胞杆菌
<400> 24
atgattcaaa aacgaaagcg gacagtttcg ttcagacttg tgcttatgtg cacgctgtta 60
tttgtcagtt tgccgattac aaaaacatca gcc 93
<210> 25
<211> 99
<212> DNA
<213> 枯草芽胞杆菌
<400> 25
atgtttgcaa aacgattcaa aacctcttta ctgccgttat tcgctggatt tttattgctg 60
tttcatttgg ttctggcagg accggcggct gcgagtgct 99
<210> 26
<211> 81
<212> DNA
<213> 地衣芽胞杆菌
<400> 26
gtgggtttag gtaagaaatt gtctgttgct gtcgctgctt cgtttatgag tttatcaatc 60
agcctgccag gtgttcaggc t 81
<210> 27
<211> 97
<212> DNA
<213> 地衣芽胞杆菌
<400> 27
atgttgatca acaaaagcaa aaagtttttc gttttttctt tcatttttgt tatgatgctg 60
agcctctcat ttgtgaatgg ggaagttgca aaagccg 97
<210> 28
<211> 33
<212> DNA
<213> 人工序列
<400> 28
ggatcccggg actagttcta gagcggccgc cac 33
<210> 29
<211> 25
<212> DNA
<213> 人工序列
<400> 29
gttaagggat gcataaactg catcc 25
<210> 30
<211> 26
<212> DNA
<213> 人工序列
<400> 30
gctctagatc aaggcaccga acgatt 26
<210> 31
<211> 40
<212> DNA
<213> 人工序列
<400> 31
ctttcgtctt ctccatttac gcgctgcaca gtttgtattt 40
<210> 32
<211> 42
<212> DNA
<213> 人工序列
<400> 32
aaatacaaac tgtgcagcgc gtaaatggag aagacgcgaa ag 42
<210> 33
<211> 26
<212> DNA
<213> 人工序列
<400> 33
cgggatccta tgcctggtgc ctgacg 26
<210> 34
<211> 26
<212> DNA
<213> 人工序列
<400> 34
gctctagatc aaggcaccga acgatt 26
<210> 35
<211> 26
<212> DNA
<213> 人工序列
<400> 35
cgggatccta tgcctggtgc ctgacg 26
<210> 36
<211> 24
<212> DNA
<213> 人工序列
<400> 36
ctgcagatta ttatgccgct atcc 24
<210> 37
<211> 50
<212> DNA
<213> 人工序列
<400> 37
tcatcctgtc tgacttgaac ggcccgggaa atgtattcgt atgggcaagc 50
<210> 38
<211> 50
<212> DNA
<213> 人工序列
<400> 38
gcttgcccat acgaatacat ttcccgggcc gttcaagtca gacaggatga 50
<210> 39
<211> 30
<212> DNA
<213> 人工序列
<400> 39
acgtctagat cttccaaagc gatgatgctc 30
<210> 40
<211> 23
<212> DNA
<213> 人工序列
<400> 40
cagatgcttg gaatgatgaa cag 23
<210> 41
<211> 21
<212> DNA
<213> 人工序列
<400> 41
gacataattg acgcctgctt c 21
<210> 42
<211> 29
<212> DNA
<213> 人工序列
<400> 42
atttctagaa gttcccgtgt aaagagttg 29
<210> 43
<211> 44
<212> DNA
<213> 人工序列
<400> 43
gcgttacaat ggatgaagcc cgggtggttt gtatccgctg aatc 44
<210> 44
<211> 44
<212> DNA
<213> 人工序列
<400> 44
gattcagcgg atacaaacca cccgggcttc atccattgta acgc 44
<210> 45
<211> 28
<212> DNA
<213> 人工序列
<400> 45
agaggatcca gattgatagc ctttgccc 28
<210> 46
<211> 20
<212> DNA
<213> 人工序列
<400> 46
cacccacttc ccattatgag 20
<210> 47
<211> 18
<212> DNA
<213> 人工序列
<400> 47
aaagcggtaa acagccag 18
<210> 48
<211> 31
<212> DNA
<213> 人工序列
<400> 48
atttctagac ggctttatcc tttgctgtta c 31
<210> 49
<211> 46
<212> DNA
<213> 人工序列
<400> 49
gaataacgaa accacaattg acccgggcgg aaacctcgct cagaac 46
<210> 50
<211> 45
<212> DNA
<213> 人工序列
<400> 50
gttctgagcg aggtttccgc ccgggtcaat gtggtttcgt tattc 45
<210> 51
<211> 29
<212> DNA
<213> 人工序列
<400> 51
agaggatcca gaagagcaaa tcaagcaac 29
<210> 52
<211> 18
<212> DNA
<213> 人工序列
<400> 52
atcatcggaa tggttgcg 18
<210> 53
<211> 22
<212> DNA
<213> 人工序列
<400> 53
actttatcgg attgcggaga tg 22
<210> 54
<211> 27
<212> DNA
<213> 人工序列
<400> 54
gctctagaat aatccgtgtg ttcgccg 27
<210> 55
<211> 39
<212> DNA
<213> 人工序列
<400> 55
tgatgatgct gactctgccc gggaaaccgt tgagatgga 39
<210> 56
<211> 39
<212> DNA
<213> 人工序列
<400> 56
tccatctcaa cggtttcccg ggcagagtca gcatcatca 39
<210> 57
<211> 20
<212> DNA
<213> 人工序列
<400> 57
ttcccagacc acgagttcct 20
<210> 58
<211> 21
<212> DNA
<213> 人工序列
<400> 58
actggtcaaa ggctaaatcg c 21
<210> 59
<211> 19
<212> DNA
<213> 人工序列
<400> 59
atccatccgc aagtcttcg 19
<210> 60
<211> 28
<212> DNA
<213> 人工序列
<400> 60
atttctagag attgatttcc gccatctc 28
<210> 61
<211> 44
<212> DNA
<213> 人工序列
<400> 61
tcccgcactc ttcaatgagc ccgggtcttc cttcttcagg ctcg 44
<210> 62
<211> 44
<212> DNA
<213> 人工序列
<400> 62
cgagcctgaa gaaggaagac ccgggctcat tgaagagtgc ggga 44
<210> 63
<211> 19
<212> DNA
<213> 人工序列
<400> 63
aacgagccaa ataacgacg 19
<210> 64
<211> 21
<212> DNA
<213> 人工序列
<400> 64
cgtcaatgaa aaagggctgt c 21
<210> 65
<211> 20
<212> DNA
<213> 人工序列
<400> 65
acggcgaaaa tacggaaaat 20
<210> 66
<211> 28
<212> DNA
<213> 人工序列
<400> 66
atttctagag ccgcttctca tcacacct 28
<210> 67
<211> 41
<212> DNA
<213> 人工序列
<400> 67
tatggtgctc tgttcttccg cattcacatg cattctttcc g 41
<210> 68
<211> 41
<212> DNA
<213> 人工序列
<400> 68
cggaaagaat gcatgtgaat gcggaagaac agagcaccat a 41
<210> 69
<211> 21
<212> DNA
<213> 人工序列
<400> 69
cggcgattca tttattctct g 21
<210> 70
<211> 23
<212> DNA
<213> 人工序列
<400> 70
atgacaatca gcatctattt ggc 23
<210> 71
<211> 20
<212> DNA
<213> 人工序列
<400> 71
cctcctttct gttcaaccga 20

Claims (9)

1.一种乳糖酶，其特征在于，所述乳糖酶氨基酸序列如序列表SEQ ID NO.18所示。
2.权利要求1所述的一种乳糖酶，其特征在于，所述乳糖酶编码基因如序列表SEQ IDNO.17所示。
3.含有权利要求2所述乳糖酶编码基因的重组载体或重组菌株。
4.如权利要求3所述的重组菌株，其特征在于，所述重组菌株采用的表达宿主为突变株BCBT0529，是在地衣芽胞杆菌CBB3008，编号CCTCC NO.M208236的基因组上敲除aprE，vpr，wpr，lacR，lacA，lacA2，yesZ基因所得；
aprE，vpr，wpr，lacR，lacA，lacA2，yesZ基因的GenBank登录号分别为：MT885340、MT885341、MT885342、MT885336、MT885337、MT885338、MT885339。
5.权利要求3所述重组菌株发酵生产乳糖酶的方法，其特征在于，摇瓶发酵生产乳糖酶：接种重组菌至摇瓶培养基，30～45℃、120-270r/min下培养2～3天；
摇瓶培养基组成：酵母膏0.5～1.5％，蛋白胨1.2～3.6％，葡萄糖8～20％；pH 7.0。
6.权利要求3所述重组菌株发酵生产乳糖酶的方法，其特征在于，发酵罐发酵生产乳糖酶：将菌种按接种量为5％-10％接入发酵罐培养基；发酵过程中采用发酵温度33～45℃，控制溶氧为0.1％～20％，pH为6.0～7.8，流加30％～60％麦芽糖浆并维持还原糖含量在0.1％-5％；发酵持续90～120h，发酵终点控制为发酵酶活增加值小于5-20U/(mL·h)；
发酵罐培养基组成为：麦芽糖浆1％～5％，棉籽粉0％～5％，玉米浆0％-4％，豆饼粉0.5～5％，硫酸铵0.1～5％，pH 6.0～8.0。
7.权利要求5或6所述重组菌株发酵生产乳糖酶的方法，其特征在于，发酵结束后，经板框过滤除去菌体，再经超滤系统过滤后获得酶液。
8.权利要求3所述重组载体或重组菌株在生产乳糖酶中的应用。
9.权利要求1或2所述乳糖酶在生产低聚半乳糖中的应用。
CN202011051056.1A 2020-09-29 2020-09-29 一种低聚半乳糖生产专用酶及其制备与应用 Active CN112574977B (zh)

Priority Applications (6)

Application Number Priority Date Filing Date Title
CN202210851698.2A CN115927257A (zh) 2020-09-29 2020-09-29 一种乳糖酶及其应用
CN202011051056.1A CN112574977B (zh) 2020-09-29 2020-09-29 一种低聚半乳糖生产专用酶及其制备与应用
CN202210851915.8A CN115927252A (zh) 2020-09-29 2020-09-29 一种乳糖酶突变体及其应用
EP20924713.9A EP3926043A4 (en) 2020-09-29 2020-11-10 SPECIAL ENZYME FOR THE PRODUCTION OF GALACTO-OLIGOSACCHARIDES, ITS PREPARATION AND USE
PCT/CN2020/127693 WO2021179652A1 (zh) 2020-09-29 2020-11-10 一种低聚半乳糖生产专用酶及其制备与应用
US17/476,433 US11447760B2 (en) 2020-09-29 2021-09-15 Special enzyme for galactooligosaccharide production as well as preparation and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202011051056.1A CN112574977B (zh) 2020-09-29 2020-09-29 一种低聚半乳糖生产专用酶及其制备与应用

Related Child Applications (2)

Application Number Title Priority Date Filing Date
CN202210851915.8A Division CN115927252A (zh) 2020-09-29 2020-09-29 一种乳糖酶突变体及其应用
CN202210851698.2A Division CN115927257A (zh) 2020-09-29 2020-09-29 一种乳糖酶及其应用

Publications (2)

Publication Number Publication Date
CN112574977A CN112574977A (zh) 2021-03-30
CN112574977B true CN112574977B (zh) 2022-07-19

Family

ID=75119712

Family Applications (3)

Application Number Title Priority Date Filing Date
CN202210851915.8A Pending CN115927252A (zh) 2020-09-29 2020-09-29 一种乳糖酶突变体及其应用
CN202210851698.2A Pending CN115927257A (zh) 2020-09-29 2020-09-29 一种乳糖酶及其应用
CN202011051056.1A Active CN112574977B (zh) 2020-09-29 2020-09-29 一种低聚半乳糖生产专用酶及其制备与应用

Family Applications Before (2)

Application Number Title Priority Date Filing Date
CN202210851915.8A Pending CN115927252A (zh) 2020-09-29 2020-09-29 一种乳糖酶突变体及其应用
CN202210851698.2A Pending CN115927257A (zh) 2020-09-29 2020-09-29 一种乳糖酶及其应用

Country Status (4)

Country Link
US (1) US11447760B2 (zh)
EP (1) EP3926043A4 (zh)
CN (3) CN115927252A (zh)
WO (1) WO2021179652A1 (zh)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111849940B (zh) * 2020-07-28 2022-03-25 量子高科(广东)生物有限公司 一种β-半乳糖苷酶的制备方法及其应用
CN114958893B (zh) * 2022-06-14 2023-08-29 中农华威生物制药(湖北)有限公司 一种乳猪高温教槽料制备所需的乳糖酶的构建方法

Family Cites Families (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9974318B2 (en) * 2009-06-05 2018-05-22 Amano Enzyme Inc. Method for producing a low-lactose milk, medicine, supplement, or galacto-oligosaccharide
KR101729048B1 (ko) * 2009-06-05 2017-04-21 아마노 엔자임 가부시키가이샤 바실러스 서쿨란스 유래의 β-갈락토시다제
CN102250856B (zh) * 2011-06-23 2013-09-18 江南大学 一种耐热β-半乳糖苷酶突变体的构建
CN104630123B (zh) * 2013-11-12 2018-09-14 华中农业大学 地衣芽胞杆菌表达宿主
CN104673769A (zh) * 2013-11-28 2015-06-03 山东省生物药物研究院 一种新型乳糖酶的制备方法及其应用
WO2016027747A1 (ja) * 2014-08-19 2016-02-25 天野エンザイム株式会社 改変型β－ガラクトシダーゼ
JP7316758B2 (ja) * 2018-03-02 2023-07-28 合同酒精株式会社 オフフレーバーのリスクを低減できるラクターゼ調製物
JP7344195B2 (ja) * 2018-04-05 2023-09-13 合同酒精株式会社 Ｐａｅｎｉｂａｃｉｌｌｕｓ ｐａｂｕｌｉ由来のガラクトオリゴ糖を製造可能な酵素、およびガラクトオリゴ糖を製造する方法
CN109182307B (zh) * 2018-10-11 2021-07-23 山东隆科特酶制剂有限公司 一种凝结芽孢杆菌液体发酵产β-半乳糖苷酶的方法

Also Published As

Publication number Publication date
US20220098563A1 (en) 2022-03-31
CN115927252A (zh) 2023-04-07
CN115927257A (zh) 2023-04-07
WO2021179652A1 (zh) 2021-09-16
US11447760B2 (en) 2022-09-20
EP3926043A1 (en) 2021-12-22
EP3926043A4 (en) 2023-02-08
CN112574977A (zh) 2021-03-30

Similar Documents

Publication Publication Date Title
Standal et al. A new gene required for cellulose production and a gene encoding cellulolytic activity in Acetobacter xylinum are colocalized with the bcs operon
EP4276171A1 (en) Bacillus subtilis genetically engineered bacterium for producing tagatose and method for preparing tagatose
CN112574977B (zh) 一种低聚半乳糖生产专用酶及其制备与应用
CN111172127A (zh) 一种蔗糖磷酸化酶在制备甘油葡萄糖苷中的应用
CN110117601B (zh) 灰树花葡聚糖合成酶、其编码基因及应用
CN112094781B (zh) 一株解淀粉芽孢杆菌及其应用
CN113234699A (zh) α-1,2-岩藻糖基转移酶及其应用
CN113151337A (zh) 一种谷氨酸棒杆菌利用EF-Tu启动子表达海藻糖合酶的方法和应用
CN110904064A (zh) 一种果糖基转移酶的基因序列及其制备方法和应用
Cho et al. Extracellular secretion of a maltogenic amylase from Lactobacillus gasseri ATCC33323 in Lactococcus lactis MG1363 and its application on the production of branched maltooligosaccharides
CN113699087B (zh) 一种转化乳糖生成乳果糖的植物乳杆菌工程菌株及其构建方法与应用
CN113564151B (zh) 一种提高ce酶结构异构催化活性的方法及其突变体
KR20020029767A (ko) 환상 뎁시펩티드 합성효소 및 그의 유전자 및 환상뎁시펩티드의 대량생산계
CN112342208B (zh) 一种普鲁兰酶突变体
WO2013173711A1 (en) Bacterial mutants with improved transformation efficiency
CN112575022A (zh) 一种体外人工脚手架蛋白介导海藻糖多酶复合体的构建方法
CN110878293A (zh) 缺失yceD基因的地衣芽胞杆菌在异源蛋白生产中的应用
CN111018957A (zh) 一种介导PGase分泌表达的信号肽及其应用
CN116949017B (zh) 一种高活性乳糖酶突变体及其应用
CN114015735B (zh) 一种蔗糖磷酸化酶和葡萄糖异构酶级联催化合成黑曲霉二糖的方法
CN116286575B (zh) 一种利用枯草芽孢杆菌高效表达生淀粉α-淀粉酶的方法
CN113881618B (zh) 一种分泌乳酪蛋白的重组枯草芽孢杆菌及其构建方法和应用
WO2024103825A1 (zh) 合成寡糖的成熟多肽序列及应用
WO2024109169A1 (zh) 合成HMOs的成熟多肽序列及应用
WO2024103822A1 (zh) 一种合成寡糖的成熟多肽

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant
TR01 Transfer of patent right
TR01 Transfer of patent right

Effective date of registration: 20221008

Address after: 605, Floor 6, B2, Animation Building, No. 126, Animation Middle Road, Zhongxin Ecological City, Binhai New Area, Tianjin, 300480

Patentee after: Senda (Tianjin) Biotechnology Co.,Ltd.

Address before: 300457 Tianjin Binhai New Area Economic and Technological Development Zone thirteenth Avenue 29

Patentee before: TIANJIN University OF SCIENCE AND TECHNOLOGY