CN108570107B - 一种扩张蛋白和木聚糖酶融合蛋白、其编码基因和应用 - Google Patents

一种扩张蛋白和木聚糖酶融合蛋白、其编码基因和应用 Download PDF

Info

Publication number
CN108570107B
CN108570107B CN201710134735.7A CN201710134735A CN108570107B CN 108570107 B CN108570107 B CN 108570107B CN 201710134735 A CN201710134735 A CN 201710134735A CN 108570107 B CN108570107 B CN 108570107B
Authority
CN
China
Prior art keywords
gly
thr
ser
tyr
val
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
CN201710134735.7A
Other languages
English (en)
Other versions
CN108570107A (zh
Inventor
吴斌
韦利军
常思源
何冰芳
储建林
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.)
CHANGZHOU QIANHONG BIO-PHARMA CO LTD
Nanjing Tech University
Original Assignee
CHANGZHOU QIANHONG BIO-PHARMA CO LTD
Nanjing Tech University
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 CHANGZHOU QIANHONG BIO-PHARMA CO LTD, Nanjing Tech University filed Critical CHANGZHOU QIANHONG BIO-PHARMA CO LTD
Priority to CN201710134735.7A priority Critical patent/CN108570107B/zh
Publication of CN108570107A publication Critical patent/CN108570107A/zh
Application granted granted Critical
Publication of CN108570107B publication Critical patent/CN108570107B/zh
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/32—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bacillus (G)
    • 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)
    • C12N9/2477—Hemicellulases not provided in a preceding group
    • C12N9/248—Xylanases
    • 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
    • C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Landscapes

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

Abstract

本发明公开了一种扩张蛋白和木聚糖酶融合蛋白、其编码基因和应用。所述融合蛋白包括扩张蛋白EXCL和木聚糖酶XYN，EXCL位于融合蛋白的N端，XYN位于融合蛋白的C端；其中，扩张蛋白EXCL的氨基酸序列如SEQ ID NO：2所示；木聚糖酶XYN的氨基酸序列如SEQ ID NO：4所示。本发明还提供了在融合蛋白中插入连接肽的优选方式。本发明采用基因工程技术构建了扩张蛋白和木聚糖酶融合酶，酶活力相较于天然木聚糖酶提高了1.3倍，并对木质纤维素具有吸附能力。

Description

一种扩张蛋白和木聚糖酶融合蛋白、其编码基因和应用
技术领域
本发明属于基因工程技术及生物质利用领域，具体涉及对扩张蛋白和木聚糖酶融合蛋白基因及融合蛋白一体化生产木寡糖的应用。
背景技术
玉米在我国的分布面积极广，年产量达亿吨以上。玉米芯一般占玉米穗重量的20.0%~30.0%，且半纤维素含量高，是一种自然可再生资源。木寡糖或称低聚木糖，是由2~10个木糖分子以β-1,4糖苷键连接而成的低聚糖，木寡糖因较难被人体消化酶系统分解得以直接进入大肠，一方面被肠道内的益生菌如双歧杆菌和乳酸菌吸收代谢继而大量增殖；另一方面各微生物发酵产生的短链脂肪酸产物能降低肠道内的pH值，促进肠道功能、脂类代谢、钙和矿物质的吸收。与其它的功能性低聚糖如低聚果糖、低聚半乳糖、大豆低聚糖、低聚壳聚糖、低聚龙胆糖等相比，服用低聚木糖更能对有害菌进行抑制，并且使用量更少。
木寡糖常用的制备方法有化学法、酶解法、自动水解法等。与其他木寡糖制备方法相比，酶法可以有效的避免副产物以及大量单糖的产生，不需要耐高温高压的设备。目前已有多种来源的木聚糖酶利用玉米芯生产木寡糖的报道。例如，Kumar和Satyanarayana（Bioresource Technology，2015，179，382-389）报道了嗜碱芽孢杆菌TSEV1的木聚糖酶水解玉米芯可产生10.51 mg/mL木寡糖；Chapla等（Bioresource Technology，2012，115，215-221）异源表达了臭曲霉MTCC 4898的木聚糖酶，纯酶酶解玉米芯生产木寡糖，产量可达6.78mg/mL。然而，木聚糖酶通常难以作用于不溶性的木聚糖，对半纤维素的识别能力和结合能力较弱，造成产品的后期分离难度和成本的增加，严重削弱了木寡糖在工业生产中的应用前景。本发明发现，在少数真菌和细菌中存在一些在结构上类似于植物Expansin的扩张蛋白，扩张蛋白具有松散纤维素，半纤维素类材料的能力，并且对纤维素，半纤维素类底物具有吸附作用。本发明通过融合扩张蛋白来改善木聚糖酶对半纤维素的吸附和识别能力，降低了木寡糖的后期分离难度和成本。
发明内容
为了解决现有技术存在的问题，本发明的目的在于提供一种具有纤维素、半纤维素结合能力的扩张蛋白和木聚糖酶的融合蛋白。
本发明的上述目的采用如下技术方案实现：
一种扩张蛋白和木聚糖酶融合蛋白，所述融合蛋白包括扩张蛋白EXCL和木聚糖酶XYN，EXCL位于融合蛋白的N端，XYN位于融合蛋白的C端；其中，扩张蛋白EXCL的氨基酸序列如SEQ ID NO：2所示；木聚糖酶XYN的氨基酸序列如SEQ ID NO：4所示。
优选的，可在扩张蛋白EXCL和木聚糖酶XYN间插入连接肽；所述连接肽为刚性连接肽(EKKKA)n或柔性连接肽(GGGGS)n，n=1~4；
所述连接肽还可选用天然连接肽TKDSTKDIPETPSKDKHTQENG。
本发明还提供了编码上述融合蛋白的基因。其中，所述扩张蛋白EXCL的基因EXCL来自本实验室前期筛选到枯草芽孢杆菌LC，已保藏于中国典型培养物保藏中心（CCTCC），菌种保藏号CCTCC NO：M 208073；其核苷酸序列如SEQ ID NO：1所示；木聚糖酶XYN其基因XYN获取自Genbank，其Genbank登录号KX196201，其核苷酸序列如SEQ.ID.NO.3所示，可采用重叠PCR或全合成的方法来获得EXCL基因、连接肽编码基因和木聚糖酶XYN基因的融合基因。
本发明还保护含有上述编码基因的表达载体、宿主菌。其表达载体、宿主菌可采用各种常规表达载体、宿主菌，只要能将上述编码基因顺利表达。
本发明的另一目的在于提供上述融合蛋白在木寡糖生产中的应用。
本发明中具体提供了一种所述融合蛋白的应用方法，具体包括如下步骤：
步骤1融合蛋白和玉米芯的亲和吸附，向重组融合蛋白的粗酶液加入10-50g/L玉米芯,在4-40℃下搅拌50-300rpm，调节至pH 4.0-10.0，亲和吸附10-240min；
步骤2木寡糖的生产，将步骤1中吸附融合蛋白的玉米芯从粗酶液中分离出来，加入纯水后30-60℃，150-300rpm下无菌转化的反应液。反应液即为含有目标产物木寡糖的产品。
相对于现有技术，本发明具有如下优点和有益效果：
本发明的扩张蛋白-木聚糖酶融合蛋白，具有纤维素、半纤维素结合能力，能在大肠杆菌中高效表达并提高木聚糖酶比活力，并且通过连接肽的优化，酶活力比天然木聚糖酶提高了1.3倍。另外，利用底物玉米芯和融合蛋白之间的特异性吸附作用，实现了简单除去了粗酶液里大部分的杂质，然后对吸附了融合蛋白的玉米芯进行降解不需要再添加酶。同时，酶解反应上清仅含有目标产物木寡糖，有效的降低了下游产品的分离难度和成本。
附图说明
图1为重组融合蛋白的聚丙烯酰胺电泳图（M：蛋白Marker；1:木聚糖酶重组细胞破碎液；2：扩张蛋白重组细胞破碎液；3-4：融合蛋白:重组细胞破碎液）。
图2为融合蛋白和木聚糖酶的酶学性质。
图3为重组融合蛋白酶解玉米芯产生还原糖的时间曲线。
图4为融合蛋白降解产物液相色谱检测（安捷伦HPLC）图谱。
本发明的微生物分类命名为枯草芽孢杆菌（Bacillus subtilis）LC，保藏日期为2008年5月 19日，保藏单位全称为中国典型培养物保藏中心，简称CCTCC，保藏登记号为CCTCC No ：M208073。
具体实施方式
为使本发明更加容易理解，下面结合实施例对本发明做进一步的说明，但本发明要求保护的范围并不局限于实施例表述的范围。
实施例1
本实施例说明本发明扩张蛋白EXCL基因的获取方式。
本实施例说明从枯草芽孢杆菌LC获取扩张蛋白EXCL基因和木聚糖酶XYN基因的程序。
枯草芽孢杆菌LC是本实验室前期筛选获得，菌种保藏登记号为CCTCC No ：M208073。。枯草芽孢杆菌LC基因组总DNA提取采用酚-氯仿法。根据枯草芽孢杆菌168(Bacillus subtilis 168) (NCBI Reference Sequence : CM000487.1)的全基因测序结果进行分析，获得扩张蛋白EXCL基因和木聚糖酶XYN基因，根据基因序列设计EXCL和XYN扩增引物。
EXCLF序列为：GCCGCCATGGGGCATATGACGACCTGCATG
EXCLR序列为：CCGCTCGAGTTCAGGAAACTGAACATGGC
XYNF序列为：GCCGCCATGGGGCTAGCACAGACTACTGGCA
XYNR序列为：CCGCTCGAGTTACCACACTGTTACGTTAGAAC
其中，引物F下划线部分为NcoⅠ酶切位点，引物R下划线部分为XhoⅠ酶切位点。
以枯草芽孢杆菌LC的基因组为模板，进行PCR扩增。PCR体系为：2×Taq PlusMaster Mix 10 μl，引物F和R各1 μl，DNA模板1 μl和ddH2O 7 μl。PCR扩增步骤为：(1) 95℃，预变性5 min；(2) 95 ℃，变性30 s；(3) 55 ℃，退火30 s；(4) 72 ℃，延伸2 min；步骤(2)~(4)重复30次；(5) 72 ℃彻底延伸7 min，冷却至4 ℃。PCR产物经琼脂糖凝胶电泳纯化，利用琼脂糖凝胶DNA回收试剂盒回收目的条带。获得扩张蛋白EXCL基因和木聚糖酶XYN基因序列，其核苷酸序列如表中SEQ ID NO：1和SEQ ID NO：3所示。
实施例2
本实施例以融合蛋白EXCL-EK2-XYN（SEQ ID NO：7）为例，说明重组融合蛋白基因的获得，其中EK2为刚性连接肽(EKKKA)n，n=2。
以枯草芽孢杆菌LC基因组为模板，扩增含有扩张蛋白EXCL (SEQ.ID.NO.1)基因和木聚糖酶XYN（Genbank:KX196201，SEQ.ID.NO.2）基因。设计EXCL上游引物和EXCL下游引物：
EXCL上游引物：GCCGCCATGGGCGCATATGACGACCTGCATG；
EXCL下游引物：
TTTAGCCGCAGCTTCTTTTGCCGCAGCTTCTTCAGGAAACTGAACATGGC。
上游引物引入限制内切酶NcoⅠ位点，下游引物引入连接肽(EAAAK)2编码基因。PCR反应体系50 μL，其中ddH2O 20μL，10×prime star buffer mix 25 μL，枯草芽孢杆菌LC基因组1μL，EXCLF1和EXCLR1（20μM) 各2μL。设计XYN上游引物和XYN下游引物：
XYN上游引物：
GAAGCTGCGGCAAAAGAAGCTGCGGCTAAAGCTAGCACAGACTACTGGCA；
XYN下游引物：CCGCTCGAGTTACCACACTGTTACGTTAGAAC。
上游引物引入连接肽(EAAAK)2编码基因，下游引物引入限制内切酶XhoⅠ位点，划线部分显示。PCR反应体系50 μL，其中ddH2O 20 μL，10×prime star buffer mix 25 μL，枯草芽孢杆菌LC基因组1 μL，XYNF1和XYNR1（20μM) 2 μL。PCR反应参数为：95 ℃ 5 min；95℃ 30 sec，55 ℃ 30 sec，72 ℃ 60 sec，32个循环；72 ℃ 10 min。PCR产物经柱纯化后作为下一步重叠PCR的模板（图1）。重叠PCR反应体系50 μL，其中ddH2O 19 μL，10×primestar buffer MIX 25μL，EXCL和XYN PCR回收产物各1μL，EXCLF和XYNR（20μM) 2μL。PCR反应参数为：95℃ 5 min；95℃ 30 sec，55℃ 30 sec，72℃ 120 sec，32个循环；72℃ 10 min。PCR产物经柱纯化后即可获得重组融合蛋白基因。
实施例3
本实施例以融合蛋白EXCL-EK2-XYN为例，说明重组表达载体pET-EXCL-EK2-XYN的构建和重组表达转化体的制备方式。
扩张蛋白-木聚糖酶(EXCL-EK2-XYN) PCR产物经柱纯化后与质粒pET-28a (+) 在37 ℃用NcoⅠ和XhoⅠ同时过夜酶切，琼脂糖凝胶回收纯化EXCL-EK2-XYN片段及pET-28a (+)载体线性条带。接着，用T4 DNA连接酶连接这两个片段，于16 ℃连接过夜，连接产物转化大肠杆菌BL21(DE3)，并涂布于含有100 μg/mL氨苄卡纳霉素的LB培养基37 ℃过夜培养。转化子经菌落PCR验证，阳性转化子接种到液体LB培养基中用于扩大培养，培养菌液用TAKARA公司的质粒小提试剂盒提取质粒并经NcoⅠ和XhoⅠ双酶切重复鉴定，获得携带大肠杆菌表达载体pET-EXCL-EK2-XYN。
实施例4
本实施例说明产重组融合扩张蛋白-木聚糖酶工程菌BL21(DE3)摇瓶培养及重组木聚糖酶活力鉴定。
从37℃过夜培养的平板中挑取单菌落，接种到装有10 mL LB液体培养基的50 mL三角瓶中，于37℃培养至对数生长期。取0.5 mL重组菌菌液接种到装有50 mL LB液体培养基（含有终浓度100 μg/mL的氨苄卡纳霉素）的250 mL三角瓶中，在37℃，180 rpm摇床培养。培养至OD=0.6-0.8加入诱导剂IPTG（终浓度1 mM/mL。第2小时开始，每隔一段时间从三角瓶中取1 mL培养液，测定培养液的OD600和木聚糖酶活性。培养液经12000 rpm离心2 min，沉淀菌体经超声破碎，离心收集上清，即为重组融合EXCL-EK2-XYN的粗酶液。粗酶液用Ni-NTAAgarose亲和柱层析除去不带6-His标记的杂蛋白，得到了电泳纯的重组融合EXCL-EK2-XYN酶。聚丙烯酰胺凝胶电泳分析图（图1)。加蒸馏水稀释至适当的倍数，准确称取1 mL酶稀释液，加入1 mL的1 %木聚糖溶液（pH 6.5）于25 mL的比色管中，立即放置到50 ℃恒温水浴锅中反应10 min，然后立即用流水冷却，加入3 mL DNS试剂并摇匀，立即置沸水中显色反应5min，取出流水冷却，加蒸馏水稀释至刻度，摇匀。以蒸馏水代替酶液作为空白，于分光光度计测定OD540，以木糖含量为横坐标，吸光度为纵坐标绘制标准曲线。木聚糖酶酶活单位定义：在上述的测定条件下，将每分钟水解底物生成1 μmoL还原糖所需酶量定义为一个酶活单位（U/mL）。
随着培养时间的增加，大肠杆菌BL21(DE3)发酵获得的木聚糖酶活性也持续增加，在摇瓶发酵8 h后上清的酶活达到较高值220 U/mL。是Gashaw Mamo等在大肠杆菌中分泌表达酶活（14 U/mL）的15.7倍，且发酵时间仅需8 h。同目前常见的毕赤酵母发酵周期120 h相比，此枯草芽孢杆菌菌株生产重组木聚糖酶发酵周期缩短了了十分之九。
实施例5
本实施例说明融合重组EXCL-EK2-XYN酶适宜的pH、温度及稳定性。
融合重组EXCL-EK2-XYN酶最适pH测定。使用不同的pH缓冲液（5.0、6.0、7.0、8.0、9.0、10.0、11.0）分别配制1 %榉木聚糖底物溶液及融合重组酶液，然后在50 ℃下测定重组木聚糖酶在各种不同pH值下的酶活力。结果发现，N端融合扩张蛋白的融合重组EXCL-EK2-XYN酶，同无扩张蛋白的XYN比较，其pH作用范围没有明显的区别，最适pH值均为为7.0（图2）。说明整合扩张蛋白对对木聚糖酶的最适pH值和pH作用范围没有影响。重组EXCL-EK2-XYN酶最适温度测定。配制pH为7.0，1 %榉木聚糖底物溶液，将用蒸馏水适当稀释的重组木聚糖酶液在不同反应温度（40℃、50℃、60℃、70℃、80℃）下进行酶促反应，准确反应30min，测定酶活力。N端融合扩张蛋白的融合重组EXCL-EK2-XYN酶的最适作用温度仍是60 ℃（图2），同无扩张蛋白的XYN比较，最适温度也没有明显的区别。
融合重组EXCL-EK2-XYN酶的温度稳定性。将酶液置于（40℃、50℃、60℃、70℃、80℃）时保温，每隔1 h取样测酶活力，共保温测定4 h，然后在60 ℃下测定重组EXCL-EK2-XYN酶在不同时间点下的相对酶活力。保温4 h后，同无扩张蛋白的XYN比较，融合重组EXCL-EK2-XYN酶也没有明显的改变。相对酶活力在40 ℃条件下仍剩余90 %以上的酶活，在50℃-60 ℃的条件下剩余活力80 %以上的活力。这样的结果说明，融合底物扩张蛋白后，对重组EXCL-EK2-XYN酶的热稳定性没有影响。
实施例6
本实施例说明重组融合EXCL-EK2-XYN 酶吸附纤维素能力实验。
将实施例3中得到的重组融合EXCL-EK2-XYN酶和单一木聚糖酶XYN各取360 U单位，与0.5 g吸附底物（玉米芯）在pH7.0，50 mM的磷酸盐缓冲体系中混合，控制终体积为5mL。混合后将反应液置于20 ℃，150 rpm的摇床中吸附反应1 h，空白对照组用相同的条件处理，但缓冲液中不加吸附底物。然后12000 rpm离心5 min，再次测定上清中的木聚糖酶酶活和还原糖，上清中损失的酶活认为因为融合蛋白吸附到玉米芯上引起的，由此计算可吸附到玉米芯上的酶量，即吸附百分比（%）=（初始酶活-上清液酶活）/初始酶活。实验发现天然的木聚糖酶对玉米芯基本吸附能力较弱（11%），而融合重组EXCL-EK2-XYN酶对玉米芯的吸附能力达到91%，是单一木聚糖酶的8倍。结果表明，和天然木聚糖酶相比，融合EXCL-EK2-XYN酶对玉米芯具有吸附能力明显提高，可促进融合EXCL-EK2-XYN酶与玉米芯半纤维素充分接触，有效水解半纤维素。
实施例7
本实施例说明重组融合EXCL-EK2-XYN酶酶解玉米芯生产木寡糖的应用。
将实施例6中得到的已吸附重组融合EXCL-EK2-XYN酶的玉米芯经6000g离心，收集沉淀，加入pH 7.0磷酸盐缓冲重悬，离心，重复三次，加入pH 7.0磷酸盐缓冲，在水浴恒温摇床中在40 ℃温度，200 rpm条件下进行恒温无菌反应60 h。以天然木聚糖酶直接酶解玉米芯为对照。在60 h后，融合EXCL-EK2-XYN酶产生的还原糖的量达7.2 mg/mL，高于未融合底物扩张蛋白的单一木聚糖酶6.9 mg/mL（图3）。融合EXCL-EK2-XYN酶还原糖经液相测定可知，其中91%的还原糖为目标产物木寡糖组分，产量为6.6 mg/mL（图4）。
实施例8
本实施例说明9个重组融合蛋白的获得以及生产木寡糖的应用。
依据实施例2的方法，设计扩增引物，利用重叠PCR的方法获得9个重组融合蛋白，分别命名为：EXCL-XYN（SEQ ID NO：5）；EXCL-EK-XYN（SEQ ID NO：6）；EXCL-EK3-XYN（SEQ IDNO：8）；EXCL-EK4-XYN（SEQ ID NO：9）；EXCL-GS-XYN（SEQ ID NO：10）；EXCL-GS2-XYN（SEQ IDNO：11）；EXCL-GS3-XYN（SEQ ID NO：12）；EXCL-GS4-XYN（SEQ ID NO：13）；EXCL-TG-XYN（SEQID NO：14）。依据实施例3的方法，获得9个重组表达载体分别为：pET-EXCL-XYN；pET-EXCL-EK-XYN；pET-EXCL-EK3-XYN；pET-EXCL-EK4-XYN；pET-EXCL-GS-XYN；pET-EXCL-GS2-XYN；pET-EXCL-GS3-XYN；pET-EXCL-GS4-XYN；pET-EXCL-TG-XYN。依据实施例3的方法，获得9个电泳纯的重组融合蛋白。重组融合蛋白活力结果见表1。依据实施例4的方法，9个重组融合蛋白的粗酶液通过吸附、离心的过程获得玉米芯和重组融合蛋白的混合物。依据实施例5的方法，重组融合蛋白酶解玉米芯生产木寡糖，木寡糖产量见表1。
表1.重组融合蛋白活力和木寡糖的产量
融合蛋白 酶活力（U/umol） 木寡糖产量（mg/mL）
EXCL-XYN 140 4.2
EXCL-EK-XYN 189 5.6
EXCL-EK3-XYN 205 6.2
EXCL-EK4-XYN 23 0.7
EXCL-GS-XYN 192 5.7
EXCL-GS2-XYN 207 6.1
EXCL-GS3-XYN 201 6.2
EXCL-GS4-XYN） 18 0.6
EXCL-TG-XYN 199 6.2
序列表
<110> 南京工业大学
<120> 一种扩张蛋白和木聚糖酶融合蛋白、其编码基因和应用
<130> xb17030801
<160> 20
<170> PatentIn version 3.5
<210> 1
<211> 621
<212> DNA
<213> Artificial Sequence
<220>
<223> 1
<220>
<221> CDS
<222> (1)..(621)
<400> 1
gca tat gac gac ctg cat gaa ggt tat gca acg tat aca ggg tca ggt 48
Ala Tyr Asp Asp Leu His Glu Gly Tyr Ala Thr Tyr Thr Gly Ser Gly
1 5 10 15
tat tca gga gga gct ttc ctg ctg gat ccc att cct tcc gat atg gag 96
Tyr Ser Gly Gly Ala Phe Leu Leu Asp Pro Ile Pro Ser Asp Met Glu
20 25 30
att act gca ata aat ccg gcg gat ctc aat tac gga gga gta aaa gca 144
Ile Thr Ala Ile Asn Pro Ala Asp Leu Asn Tyr Gly Gly Val Lys Ala
35 40 45
gca ctt gcc ggc tct tat ttg gaa gtt gaa ggg cca aaa ggg aaa aca 192
Ala Leu Ala Gly Ser Tyr Leu Glu Val Glu Gly Pro Lys Gly Lys Thr
50 55 60
acc gta tat gtt act gat ctt tat ccc gaa ggc gct cgg gga gct ctt 240
Thr Val Tyr Val Thr Asp Leu Tyr Pro Glu Gly Ala Arg Gly Ala Leu
65 70 75 80
gat ctg tca cct aat gcc ttc cgt aaa atc ggc aat atg aaa gac gga 288
Asp Leu Ser Pro Asn Ala Phe Arg Lys Ile Gly Asn Met Lys Asp Gly
85 90 95
aaa atc aat att aaa tgg cgt gtt gtc aaa gcc cca atc acc ggc aat 336
Lys Ile Asn Ile Lys Trp Arg Val Val Lys Ala Pro Ile Thr Gly Asn
100 105 110
ttc acg tac cgg atc aaa gaa ggc agc agc agg tgg tgg gca gca atc 384
Phe Thr Tyr Arg Ile Lys Glu Gly Ser Ser Arg Trp Trp Ala Ala Ile
115 120 125
caa gtc aga aat cac aag tat cct gtt atg aaa atg gaa tat gaa aag 432
Gln Val Arg Asn His Lys Tyr Pro Val Met Lys Met Glu Tyr Glu Lys
130 135 140
gat ggt aag tgg atc aac atg gag aaa atg gac tat aac cat ttt gtg 480
Asp Gly Lys Trp Ile Asn Met Glu Lys Met Asp Tyr Asn His Phe Val
145 150 155 160
agt acg aat tta ggt aca ggc tct ctc aaa gtc aga atg act gac atc 528
Ser Thr Asn Leu Gly Thr Gly Ser Leu Lys Val Arg Met Thr Asp Ile
165 170 175
cgc gga aaa gtt gtg aaa gac acc att cca aag ctg cct gaa agc gga 576
Arg Gly Lys Val Val Lys Asp Thr Ile Pro Lys Leu Pro Glu Ser Gly
180 185 190
acg tcc aaa gcc tat aca gta ccg ggc cat gtt cag ttt cct gaa 621
Thr Ser Lys Ala Tyr Thr Val Pro Gly His Val Gln Phe Pro Glu
195 200 205
<210> 2
<211> 207
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 2
Ala Tyr Asp Asp Leu His Glu Gly Tyr Ala Thr Tyr Thr Gly Ser Gly
1 5 10 15
Tyr Ser Gly Gly Ala Phe Leu Leu Asp Pro Ile Pro Ser Asp Met Glu
20 25 30
Ile Thr Ala Ile Asn Pro Ala Asp Leu Asn Tyr Gly Gly Val Lys Ala
35 40 45
Ala Leu Ala Gly Ser Tyr Leu Glu Val Glu Gly Pro Lys Gly Lys Thr
50 55 60
Thr Val Tyr Val Thr Asp Leu Tyr Pro Glu Gly Ala Arg Gly Ala Leu
65 70 75 80
Asp Leu Ser Pro Asn Ala Phe Arg Lys Ile Gly Asn Met Lys Asp Gly
85 90 95
Lys Ile Asn Ile Lys Trp Arg Val Val Lys Ala Pro Ile Thr Gly Asn
100 105 110
Phe Thr Tyr Arg Ile Lys Glu Gly Ser Ser Arg Trp Trp Ala Ala Ile
115 120 125
Gln Val Arg Asn His Lys Tyr Pro Val Met Lys Met Glu Tyr Glu Lys
130 135 140
Asp Gly Lys Trp Ile Asn Met Glu Lys Met Asp Tyr Asn His Phe Val
145 150 155 160
Ser Thr Asn Leu Gly Thr Gly Ser Leu Lys Val Arg Met Thr Asp Ile
165 170 175
Arg Gly Lys Val Val Lys Asp Thr Ile Pro Lys Leu Pro Glu Ser Gly
180 185 190
Thr Ser Lys Ala Tyr Thr Val Pro Gly His Val Gln Phe Pro Glu
195 200 205
<210> 3
<211> 558
<212> DNA
<213> Artificial Sequence
<220>
<223> 1
<220>
<221> CDS
<222> (1)..(558)
<400> 3
gct agc aca gac tac tgg caa aat tgg act gat ggg ggc ggt ata gta 48
Ala Ser Thr Asp Tyr Trp Gln Asn Trp Thr Asp Gly Gly Gly Ile Val
1 5 10 15
aac gct gtt aat ggg tct ggc ggg aat tac agt gtt aat tgg tct aat 96
Asn Ala Val Asn Gly Ser Gly Gly Asn Tyr Ser Val Asn Trp Ser Asn
20 25 30
acc gga aat ttt gtt gtt ggt aaa ggt tgg act aca ggt tcg cca ttt 144
Thr Gly Asn Phe Val Val Gly Lys Gly Trp Thr Thr Gly Ser Pro Phe
35 40 45
agg acg ata aac tat aat gcc gga gtt tgg gca ccg aat ggc aat gga 192
Arg Thr Ile Asn Tyr Asn Ala Gly Val Trp Ala Pro Asn Gly Asn Gly
50 55 60
tat tta act tta tat ggt tgg acg aga tca cct ctc ata gaa tat tat 240
Tyr Leu Thr Leu Tyr Gly Trp Thr Arg Ser Pro Leu Ile Glu Tyr Tyr
65 70 75 80
gta gtg gat tca tgg ggt act tat aga cct act gga acg tat aaa ggt 288
Val Val Asp Ser Trp Gly Thr Tyr Arg Pro Thr Gly Thr Tyr Lys Gly
85 90 95
act gta aaa agt gat ggg ggt aca tat gac ata tat aca act aca cgt 336
Thr Val Lys Ser Asp Gly Gly Thr Tyr Asp Ile Tyr Thr Thr Thr Arg
100 105 110
tat aac gca cct tcc att gat ggc gat cgc act act ttt acg cag tac 384
Tyr Asn Ala Pro Ser Ile Asp Gly Asp Arg Thr Thr Phe Thr Gln Tyr
115 120 125
tgg agt gtt cgt cag acg aag aga cca act gga agc aac gct aca atc 432
Trp Ser Val Arg Gln Thr Lys Arg Pro Thr Gly Ser Asn Ala Thr Ile
130 135 140
act ttc agc aat cat gtg aac gca tgg aag agc cat gga atg aat ctg 480
Thr Phe Ser Asn His Val Asn Ala Trp Lys Ser His Gly Met Asn Leu
145 150 155 160
ggc agt aat tgg gct tac caa gtc atg gcg aca gaa gga tat caa agt 528
Gly Ser Asn Trp Ala Tyr Gln Val Met Ala Thr Glu Gly Tyr Gln Ser
165 170 175
agt gga agt tct aac gta aca gtg tgg taa 558
Ser Gly Ser Ser Asn Val Thr Val Trp
180 185
<210> 4
<211> 185
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 4
Ala Ser Thr Asp Tyr Trp Gln Asn Trp Thr Asp Gly Gly Gly Ile Val
1 5 10 15
Asn Ala Val Asn Gly Ser Gly Gly Asn Tyr Ser Val Asn Trp Ser Asn
20 25 30
Thr Gly Asn Phe Val Val Gly Lys Gly Trp Thr Thr Gly Ser Pro Phe
35 40 45
Arg Thr Ile Asn Tyr Asn Ala Gly Val Trp Ala Pro Asn Gly Asn Gly
50 55 60
Tyr Leu Thr Leu Tyr Gly Trp Thr Arg Ser Pro Leu Ile Glu Tyr Tyr
65 70 75 80
Val Val Asp Ser Trp Gly Thr Tyr Arg Pro Thr Gly Thr Tyr Lys Gly
85 90 95
Thr Val Lys Ser Asp Gly Gly Thr Tyr Asp Ile Tyr Thr Thr Thr Arg
100 105 110
Tyr Asn Ala Pro Ser Ile Asp Gly Asp Arg Thr Thr Phe Thr Gln Tyr
115 120 125
Trp Ser Val Arg Gln Thr Lys Arg Pro Thr Gly Ser Asn Ala Thr Ile
130 135 140
Thr Phe Ser Asn His Val Asn Ala Trp Lys Ser His Gly Met Asn Leu
145 150 155 160
Gly Ser Asn Trp Ala Tyr Gln Val Met Ala Thr Glu Gly Tyr Gln Ser
165 170 175
Ser Gly Ser Ser Asn Val Thr Val Trp
180 185
<210> 5
<211> 392
<212> PRT
<213> Artificial Sequence
<220>
<223> 1
<400> 5
Ala Tyr Asp Asp Leu His Glu Gly Tyr Ala Thr Tyr Thr Gly Ser Gly
1 5 10 15
Tyr Ser Gly Gly Ala Phe Leu Leu Asp Pro Ile Pro Ser Asp Met Glu
20 25 30
Ile Thr Ala Ile Asn Pro Ala Asp Leu Asn Tyr Gly Gly Val Lys Ala
35 40 45
Ala Leu Ala Gly Ser Tyr Leu Glu Val Glu Gly Pro Lys Gly Lys Thr
50 55 60
Thr Val Tyr Val Thr Asp Leu Tyr Pro Glu Gly Ala Arg Gly Ala Leu
65 70 75 80
Asp Leu Ser Pro Asn Ala Phe Arg Lys Ile Gly Asn Met Lys Asp Gly
85 90 95
Lys Ile Asn Ile Lys Trp Arg Val Val Lys Ala Pro Ile Thr Gly Asn
100 105 110
Phe Thr Tyr Arg Ile Lys Glu Gly Ser Ser Arg Trp Trp Ala Ala Ile
115 120 125
Gln Val Arg Asn His Lys Tyr Pro Val Met Lys Met Glu Tyr Glu Lys
130 135 140
Asp Gly Lys Trp Ile Asn Met Glu Lys Met Asp Tyr Asn His Phe Val
145 150 155 160
Ser Thr Asn Leu Gly Thr Gly Ser Leu Lys Val Arg Met Thr Asp Ile
165 170 175
Arg Gly Lys Val Val Lys Asp Thr Ile Pro Lys Leu Pro Glu Ser Gly
180 185 190
Thr Ser Lys Ala Tyr Thr Val Pro Gly His Val Gln Phe Pro Glu Ala
195 200 205
Ser Thr Asp Tyr Trp Gln Asn Trp Thr Asp Gly Gly Gly Ile Val Asn
210 215 220
Ala Val Asn Gly Ser Gly Gly Asn Tyr Ser Val Asn Trp Ser Asn Thr
225 230 235 240
Gly Asn Phe Val Val Gly Lys Gly Trp Thr Thr Gly Ser Pro Phe Arg
245 250 255
Thr Ile Asn Tyr Asn Ala Gly Val Trp Ala Pro Asn Gly Asn Gly Tyr
260 265 270
Leu Thr Leu Tyr Gly Trp Thr Arg Ser Pro Leu Ile Glu Tyr Tyr Val
275 280 285
Val Asp Ser Trp Gly Thr Tyr Arg Pro Thr Gly Thr Tyr Lys Gly Thr
290 295 300
Val Lys Ser Asp Gly Gly Thr Tyr Asp Ile Tyr Thr Thr Thr Arg Tyr
305 310 315 320
Asn Ala Pro Ser Ile Asp Gly Asp Arg Thr Thr Phe Thr Gln Tyr Trp
325 330 335
Ser Val Arg Gln Thr Lys Arg Pro Thr Gly Ser Asn Ala Thr Ile Thr
340 345 350
Phe Ser Asn His Val Asn Ala Trp Lys Ser His Gly Met Asn Leu Gly
355 360 365
Ser Asn Trp Ala Tyr Gln Val Met Ala Thr Glu Gly Tyr Gln Ser Ser
370 375 380
Gly Ser Ser Asn Val Thr Val Trp
385 390
<210> 6
<211> 397
<212> PRT
<213> Artificial Sequence
<220>
<223> 1
<400> 6
Ala Tyr Asp Asp Leu His Glu Gly Tyr Ala Thr Tyr Thr Gly Ser Gly
1 5 10 15
Tyr Ser Gly Gly Ala Phe Leu Leu Asp Pro Ile Pro Ser Asp Met Glu
20 25 30
Ile Thr Ala Ile Asn Pro Ala Asp Leu Asn Tyr Gly Gly Val Lys Ala
35 40 45
Ala Leu Ala Gly Ser Tyr Leu Glu Val Glu Gly Pro Lys Gly Lys Thr
50 55 60
Thr Val Tyr Val Thr Asp Leu Tyr Pro Glu Gly Ala Arg Gly Ala Leu
65 70 75 80
Asp Leu Ser Pro Asn Ala Phe Arg Lys Ile Gly Asn Met Lys Asp Gly
85 90 95
Lys Ile Asn Ile Lys Trp Arg Val Val Lys Ala Pro Ile Thr Gly Asn
100 105 110
Phe Thr Tyr Arg Ile Lys Glu Gly Ser Ser Arg Trp Trp Ala Ala Ile
115 120 125
Gln Val Arg Asn His Lys Tyr Pro Val Met Lys Met Glu Tyr Glu Lys
130 135 140
Asp Gly Lys Trp Ile Asn Met Glu Lys Met Asp Tyr Asn His Phe Val
145 150 155 160
Ser Thr Asn Leu Gly Thr Gly Ser Leu Lys Val Arg Met Thr Asp Ile
165 170 175
Arg Gly Lys Val Val Lys Asp Thr Ile Pro Lys Leu Pro Glu Ser Gly
180 185 190
Thr Ser Lys Ala Tyr Thr Val Pro Gly His Val Gln Phe Pro Glu Glu
195 200 205
Ala Ala Ala Lys Ala Ser Thr Asp Tyr Trp Gln Asn Trp Thr Asp Gly
210 215 220
Gly Gly Ile Val Asn Ala Val Asn Gly Ser Gly Gly Asn Tyr Ser Val
225 230 235 240
Asn Trp Ser Asn Thr Gly Asn Phe Val Val Gly Lys Gly Trp Thr Thr
245 250 255
Gly Ser Pro Phe Arg Thr Ile Asn Tyr Asn Ala Gly Val Trp Ala Pro
260 265 270
Asn Gly Asn Gly Tyr Leu Thr Leu Tyr Gly Trp Thr Arg Ser Pro Leu
275 280 285
Ile Glu Tyr Tyr Val Val Asp Ser Trp Gly Thr Tyr Arg Pro Thr Gly
290 295 300
Thr Tyr Lys Gly Thr Val Lys Ser Asp Gly Gly Thr Tyr Asp Ile Tyr
305 310 315 320
Thr Thr Thr Arg Tyr Asn Ala Pro Ser Ile Asp Gly Asp Arg Thr Thr
325 330 335
Phe Thr Gln Tyr Trp Ser Val Arg Gln Thr Lys Arg Pro Thr Gly Ser
340 345 350
Asn Ala Thr Ile Thr Phe Ser Asn His Val Asn Ala Trp Lys Ser His
355 360 365
Gly Met Asn Leu Gly Ser Asn Trp Ala Tyr Gln Val Met Ala Thr Glu
370 375 380
Gly Tyr Gln Ser Ser Gly Ser Ser Asn Val Thr Val Trp
385 390 395
<210> 7
<211> 402
<212> PRT
<213> Artificial Sequence
<220>
<223> 1
<400> 7
Ala Tyr Asp Asp Leu His Glu Gly Tyr Ala Thr Tyr Thr Gly Ser Gly
1 5 10 15
Tyr Ser Gly Gly Ala Phe Leu Leu Asp Pro Ile Pro Ser Asp Met Glu
20 25 30
Ile Thr Ala Ile Asn Pro Ala Asp Leu Asn Tyr Gly Gly Val Lys Ala
35 40 45
Ala Leu Ala Gly Ser Tyr Leu Glu Val Glu Gly Pro Lys Gly Lys Thr
50 55 60
Thr Val Tyr Val Thr Asp Leu Tyr Pro Glu Gly Ala Arg Gly Ala Leu
65 70 75 80
Asp Leu Ser Pro Asn Ala Phe Arg Lys Ile Gly Asn Met Lys Asp Gly
85 90 95
Lys Ile Asn Ile Lys Trp Arg Val Val Lys Ala Pro Ile Thr Gly Asn
100 105 110
Phe Thr Tyr Arg Ile Lys Glu Gly Ser Ser Arg Trp Trp Ala Ala Ile
115 120 125
Gln Val Arg Asn His Lys Tyr Pro Val Met Lys Met Glu Tyr Glu Lys
130 135 140
Asp Gly Lys Trp Ile Asn Met Glu Lys Met Asp Tyr Asn His Phe Val
145 150 155 160
Ser Thr Asn Leu Gly Thr Gly Ser Leu Lys Val Arg Met Thr Asp Ile
165 170 175
Arg Gly Lys Val Val Lys Asp Thr Ile Pro Lys Leu Pro Glu Ser Gly
180 185 190
Thr Ser Lys Ala Tyr Thr Val Pro Gly His Val Gln Phe Pro Glu Glu
195 200 205
Ala Ala Ala Lys Glu Ala Ala Ala Lys Ala Ser Thr Asp Tyr Trp Gln
210 215 220
Asn Trp Thr Asp Gly Gly Gly Ile Val Asn Ala Val Asn Gly Ser Gly
225 230 235 240
Gly Asn Tyr Ser Val Asn Trp Ser Asn Thr Gly Asn Phe Val Val Gly
245 250 255
Lys Gly Trp Thr Thr Gly Ser Pro Phe Arg Thr Ile Asn Tyr Asn Ala
260 265 270
Gly Val Trp Ala Pro Asn Gly Asn Gly Tyr Leu Thr Leu Tyr Gly Trp
275 280 285
Thr Arg Ser Pro Leu Ile Glu Tyr Tyr Val Val Asp Ser Trp Gly Thr
290 295 300
Tyr Arg Pro Thr Gly Thr Tyr Lys Gly Thr Val Lys Ser Asp Gly Gly
305 310 315 320
Thr Tyr Asp Ile Tyr Thr Thr Thr Arg Tyr Asn Ala Pro Ser Ile Asp
325 330 335
Gly Asp Arg Thr Thr Phe Thr Gln Tyr Trp Ser Val Arg Gln Thr Lys
340 345 350
Arg Pro Thr Gly Ser Asn Ala Thr Ile Thr Phe Ser Asn His Val Asn
355 360 365
Ala Trp Lys Ser His Gly Met Asn Leu Gly Ser Asn Trp Ala Tyr Gln
370 375 380
Val Met Ala Thr Glu Gly Tyr Gln Ser Ser Gly Ser Ser Asn Val Thr
385 390 395 400
Val Trp
<210> 8
<211> 407
<212> PRT
<213> Artificial Sequence
<220>
<223> 1
<400> 8
Ala Tyr Asp Asp Leu His Glu Gly Tyr Ala Thr Tyr Thr Gly Ser Gly
1 5 10 15
Tyr Ser Gly Gly Ala Phe Leu Leu Asp Pro Ile Pro Ser Asp Met Glu
20 25 30
Ile Thr Ala Ile Asn Pro Ala Asp Leu Asn Tyr Gly Gly Val Lys Ala
35 40 45
Ala Leu Ala Gly Ser Tyr Leu Glu Val Glu Gly Pro Lys Gly Lys Thr
50 55 60
Thr Val Tyr Val Thr Asp Leu Tyr Pro Glu Gly Ala Arg Gly Ala Leu
65 70 75 80
Asp Leu Ser Pro Asn Ala Phe Arg Lys Ile Gly Asn Met Lys Asp Gly
85 90 95
Lys Ile Asn Ile Lys Trp Arg Val Val Lys Ala Pro Ile Thr Gly Asn
100 105 110
Phe Thr Tyr Arg Ile Lys Glu Gly Ser Ser Arg Trp Trp Ala Ala Ile
115 120 125
Gln Val Arg Asn His Lys Tyr Pro Val Met Lys Met Glu Tyr Glu Lys
130 135 140
Asp Gly Lys Trp Ile Asn Met Glu Lys Met Asp Tyr Asn His Phe Val
145 150 155 160
Ser Thr Asn Leu Gly Thr Gly Ser Leu Lys Val Arg Met Thr Asp Ile
165 170 175
Arg Gly Lys Val Val Lys Asp Thr Ile Pro Lys Leu Pro Glu Ser Gly
180 185 190
Thr Ser Lys Ala Tyr Thr Val Pro Gly His Val Gln Phe Pro Glu Glu
195 200 205
Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Ala Ser
210 215 220
Thr Asp Tyr Trp Gln Asn Trp Thr Asp Gly Gly Gly Ile Val Asn Ala
225 230 235 240
Val Asn Gly Ser Gly Gly Asn Tyr Ser Val Asn Trp Ser Asn Thr Gly
245 250 255
Asn Phe Val Val Gly Lys Gly Trp Thr Thr Gly Ser Pro Phe Arg Thr
260 265 270
Ile Asn Tyr Asn Ala Gly Val Trp Ala Pro Asn Gly Asn Gly Tyr Leu
275 280 285
Thr Leu Tyr Gly Trp Thr Arg Ser Pro Leu Ile Glu Tyr Tyr Val Val
290 295 300
Asp Ser Trp Gly Thr Tyr Arg Pro Thr Gly Thr Tyr Lys Gly Thr Val
305 310 315 320
Lys Ser Asp Gly Gly Thr Tyr Asp Ile Tyr Thr Thr Thr Arg Tyr Asn
325 330 335
Ala Pro Ser Ile Asp Gly Asp Arg Thr Thr Phe Thr Gln Tyr Trp Ser
340 345 350
Val Arg Gln Thr Lys Arg Pro Thr Gly Ser Asn Ala Thr Ile Thr Phe
355 360 365
Ser Asn His Val Asn Ala Trp Lys Ser His Gly Met Asn Leu Gly Ser
370 375 380
Asn Trp Ala Tyr Gln Val Met Ala Thr Glu Gly Tyr Gln Ser Ser Gly
385 390 395 400
Ser Ser Asn Val Thr Val Trp
405
<210> 9
<211> 412
<212> PRT
<213> Artificial Sequence
<220>
<223> 1
<400> 9
Ala Tyr Asp Asp Leu His Glu Gly Tyr Ala Thr Tyr Thr Gly Ser Gly
1 5 10 15
Tyr Ser Gly Gly Ala Phe Leu Leu Asp Pro Ile Pro Ser Asp Met Glu
20 25 30
Ile Thr Ala Ile Asn Pro Ala Asp Leu Asn Tyr Gly Gly Val Lys Ala
35 40 45
Ala Leu Ala Gly Ser Tyr Leu Glu Val Glu Gly Pro Lys Gly Lys Thr
50 55 60
Thr Val Tyr Val Thr Asp Leu Tyr Pro Glu Gly Ala Arg Gly Ala Leu
65 70 75 80
Asp Leu Ser Pro Asn Ala Phe Arg Lys Ile Gly Asn Met Lys Asp Gly
85 90 95
Lys Ile Asn Ile Lys Trp Arg Val Val Lys Ala Pro Ile Thr Gly Asn
100 105 110
Phe Thr Tyr Arg Ile Lys Glu Gly Ser Ser Arg Trp Trp Ala Ala Ile
115 120 125
Gln Val Arg Asn His Lys Tyr Pro Val Met Lys Met Glu Tyr Glu Lys
130 135 140
Asp Gly Lys Trp Ile Asn Met Glu Lys Met Asp Tyr Asn His Phe Val
145 150 155 160
Ser Thr Asn Leu Gly Thr Gly Ser Leu Lys Val Arg Met Thr Asp Ile
165 170 175
Arg Gly Lys Val Val Lys Asp Thr Ile Pro Lys Leu Pro Glu Ser Gly
180 185 190
Thr Ser Lys Ala Tyr Thr Val Pro Gly His Val Gln Phe Pro Glu Glu
195 200 205
Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala
210 215 220
Ala Ala Lys Ala Ser Thr Asp Tyr Trp Gln Asn Trp Thr Asp Gly Gly
225 230 235 240
Gly Ile Val Asn Ala Val Asn Gly Ser Gly Gly Asn Tyr Ser Val Asn
245 250 255
Trp Ser Asn Thr Gly Asn Phe Val Val Gly Lys Gly Trp Thr Thr Gly
260 265 270
Ser Pro Phe Arg Thr Ile Asn Tyr Asn Ala Gly Val Trp Ala Pro Asn
275 280 285
Gly Asn Gly Tyr Leu Thr Leu Tyr Gly Trp Thr Arg Ser Pro Leu Ile
290 295 300
Glu Tyr Tyr Val Val Asp Ser Trp Gly Thr Tyr Arg Pro Thr Gly Thr
305 310 315 320
Tyr Lys Gly Thr Val Lys Ser Asp Gly Gly Thr Tyr Asp Ile Tyr Thr
325 330 335
Thr Thr Arg Tyr Asn Ala Pro Ser Ile Asp Gly Asp Arg Thr Thr Phe
340 345 350
Thr Gln Tyr Trp Ser Val Arg Gln Thr Lys Arg Pro Thr Gly Ser Asn
355 360 365
Ala Thr Ile Thr Phe Ser Asn His Val Asn Ala Trp Lys Ser His Gly
370 375 380
Met Asn Leu Gly Ser Asn Trp Ala Tyr Gln Val Met Ala Thr Glu Gly
385 390 395 400
Tyr Gln Ser Ser Gly Ser Ser Asn Val Thr Val Trp
405 410
<210> 10
<211> 397
<212> PRT
<213> Artificial Sequence
<220>
<223> 1
<400> 10
Ala Tyr Asp Asp Leu His Glu Gly Tyr Ala Thr Tyr Thr Gly Ser Gly
1 5 10 15
Tyr Ser Gly Gly Ala Phe Leu Leu Asp Pro Ile Pro Ser Asp Met Glu
20 25 30
Ile Thr Ala Ile Asn Pro Ala Asp Leu Asn Tyr Gly Gly Val Lys Ala
35 40 45
Ala Leu Ala Gly Ser Tyr Leu Glu Val Glu Gly Pro Lys Gly Lys Thr
50 55 60
Thr Val Tyr Val Thr Asp Leu Tyr Pro Glu Gly Ala Arg Gly Ala Leu
65 70 75 80
Asp Leu Ser Pro Asn Ala Phe Arg Lys Ile Gly Asn Met Lys Asp Gly
85 90 95
Lys Ile Asn Ile Lys Trp Arg Val Val Lys Ala Pro Ile Thr Gly Asn
100 105 110
Phe Thr Tyr Arg Ile Lys Glu Gly Ser Ser Arg Trp Trp Ala Ala Ile
115 120 125
Gln Val Arg Asn His Lys Tyr Pro Val Met Lys Met Glu Tyr Glu Lys
130 135 140
Asp Gly Lys Trp Ile Asn Met Glu Lys Met Asp Tyr Asn His Phe Val
145 150 155 160
Ser Thr Asn Leu Gly Thr Gly Ser Leu Lys Val Arg Met Thr Asp Ile
165 170 175
Arg Gly Lys Val Val Lys Asp Thr Ile Pro Lys Leu Pro Glu Ser Gly
180 185 190
Thr Ser Lys Ala Tyr Thr Val Pro Gly His Val Gln Phe Pro Glu Gly
195 200 205
Gly Gly Gly Ser Ala Ser Thr Asp Tyr Trp Gln Asn Trp Thr Asp Gly
210 215 220
Gly Gly Ile Val Asn Ala Val Asn Gly Ser Gly Gly Asn Tyr Ser Val
225 230 235 240
Asn Trp Ser Asn Thr Gly Asn Phe Val Val Gly Lys Gly Trp Thr Thr
245 250 255
Gly Ser Pro Phe Arg Thr Ile Asn Tyr Asn Ala Gly Val Trp Ala Pro
260 265 270
Asn Gly Asn Gly Tyr Leu Thr Leu Tyr Gly Trp Thr Arg Ser Pro Leu
275 280 285
Ile Glu Tyr Tyr Val Val Asp Ser Trp Gly Thr Tyr Arg Pro Thr Gly
290 295 300
Thr Tyr Lys Gly Thr Val Lys Ser Asp Gly Gly Thr Tyr Asp Ile Tyr
305 310 315 320
Thr Thr Thr Arg Tyr Asn Ala Pro Ser Ile Asp Gly Asp Arg Thr Thr
325 330 335
Phe Thr Gln Tyr Trp Ser Val Arg Gln Thr Lys Arg Pro Thr Gly Ser
340 345 350
Asn Ala Thr Ile Thr Phe Ser Asn His Val Asn Ala Trp Lys Ser His
355 360 365
Gly Met Asn Leu Gly Ser Asn Trp Ala Tyr Gln Val Met Ala Thr Glu
370 375 380
Gly Tyr Gln Ser Ser Gly Ser Ser Asn Val Thr Val Trp
385 390 395
<210> 11
<211> 402
<212> PRT
<213> Artificial Sequence
<220>
<223> 1
<400> 11
Ala Tyr Asp Asp Leu His Glu Gly Tyr Ala Thr Tyr Thr Gly Ser Gly
1 5 10 15
Tyr Ser Gly Gly Ala Phe Leu Leu Asp Pro Ile Pro Ser Asp Met Glu
20 25 30
Ile Thr Ala Ile Asn Pro Ala Asp Leu Asn Tyr Gly Gly Val Lys Ala
35 40 45
Ala Leu Ala Gly Ser Tyr Leu Glu Val Glu Gly Pro Lys Gly Lys Thr
50 55 60
Thr Val Tyr Val Thr Asp Leu Tyr Pro Glu Gly Ala Arg Gly Ala Leu
65 70 75 80
Asp Leu Ser Pro Asn Ala Phe Arg Lys Ile Gly Asn Met Lys Asp Gly
85 90 95
Lys Ile Asn Ile Lys Trp Arg Val Val Lys Ala Pro Ile Thr Gly Asn
100 105 110
Phe Thr Tyr Arg Ile Lys Glu Gly Ser Ser Arg Trp Trp Ala Ala Ile
115 120 125
Gln Val Arg Asn His Lys Tyr Pro Val Met Lys Met Glu Tyr Glu Lys
130 135 140
Asp Gly Lys Trp Ile Asn Met Glu Lys Met Asp Tyr Asn His Phe Val
145 150 155 160
Ser Thr Asn Leu Gly Thr Gly Ser Leu Lys Val Arg Met Thr Asp Ile
165 170 175
Arg Gly Lys Val Val Lys Asp Thr Ile Pro Lys Leu Pro Glu Ser Gly
180 185 190
Thr Ser Lys Ala Tyr Thr Val Pro Gly His Val Gln Phe Pro Glu Gly
195 200 205
Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Ser Thr Asp Tyr Trp Gln
210 215 220
Asn Trp Thr Asp Gly Gly Gly Ile Val Asn Ala Val Asn Gly Ser Gly
225 230 235 240
Gly Asn Tyr Ser Val Asn Trp Ser Asn Thr Gly Asn Phe Val Val Gly
245 250 255
Lys Gly Trp Thr Thr Gly Ser Pro Phe Arg Thr Ile Asn Tyr Asn Ala
260 265 270
Gly Val Trp Ala Pro Asn Gly Asn Gly Tyr Leu Thr Leu Tyr Gly Trp
275 280 285
Thr Arg Ser Pro Leu Ile Glu Tyr Tyr Val Val Asp Ser Trp Gly Thr
290 295 300
Tyr Arg Pro Thr Gly Thr Tyr Lys Gly Thr Val Lys Ser Asp Gly Gly
305 310 315 320
Thr Tyr Asp Ile Tyr Thr Thr Thr Arg Tyr Asn Ala Pro Ser Ile Asp
325 330 335
Gly Asp Arg Thr Thr Phe Thr Gln Tyr Trp Ser Val Arg Gln Thr Lys
340 345 350
Arg Pro Thr Gly Ser Asn Ala Thr Ile Thr Phe Ser Asn His Val Asn
355 360 365
Ala Trp Lys Ser His Gly Met Asn Leu Gly Ser Asn Trp Ala Tyr Gln
370 375 380
Val Met Ala Thr Glu Gly Tyr Gln Ser Ser Gly Ser Ser Asn Val Thr
385 390 395 400
Val Trp
<210> 12
<211> 407
<212> PRT
<213> Artificial Sequence
<220>
<223> 1
<400> 12
Ala Tyr Asp Asp Leu His Glu Gly Tyr Ala Thr Tyr Thr Gly Ser Gly
1 5 10 15
Tyr Ser Gly Gly Ala Phe Leu Leu Asp Pro Ile Pro Ser Asp Met Glu
20 25 30
Ile Thr Ala Ile Asn Pro Ala Asp Leu Asn Tyr Gly Gly Val Lys Ala
35 40 45
Ala Leu Ala Gly Ser Tyr Leu Glu Val Glu Gly Pro Lys Gly Lys Thr
50 55 60
Thr Val Tyr Val Thr Asp Leu Tyr Pro Glu Gly Ala Arg Gly Ala Leu
65 70 75 80
Asp Leu Ser Pro Asn Ala Phe Arg Lys Ile Gly Asn Met Lys Asp Gly
85 90 95
Lys Ile Asn Ile Lys Trp Arg Val Val Lys Ala Pro Ile Thr Gly Asn
100 105 110
Phe Thr Tyr Arg Ile Lys Glu Gly Ser Ser Arg Trp Trp Ala Ala Ile
115 120 125
Gln Val Arg Asn His Lys Tyr Pro Val Met Lys Met Glu Tyr Glu Lys
130 135 140
Asp Gly Lys Trp Ile Asn Met Glu Lys Met Asp Tyr Asn His Phe Val
145 150 155 160
Ser Thr Asn Leu Gly Thr Gly Ser Leu Lys Val Arg Met Thr Asp Ile
165 170 175
Arg Gly Lys Val Val Lys Asp Thr Ile Pro Lys Leu Pro Glu Ser Gly
180 185 190
Thr Ser Lys Ala Tyr Thr Val Pro Gly His Val Gln Phe Pro Glu Gly
195 200 205
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Ser
210 215 220
Thr Asp Tyr Trp Gln Asn Trp Thr Asp Gly Gly Gly Ile Val Asn Ala
225 230 235 240
Val Asn Gly Ser Gly Gly Asn Tyr Ser Val Asn Trp Ser Asn Thr Gly
245 250 255
Asn Phe Val Val Gly Lys Gly Trp Thr Thr Gly Ser Pro Phe Arg Thr
260 265 270
Ile Asn Tyr Asn Ala Gly Val Trp Ala Pro Asn Gly Asn Gly Tyr Leu
275 280 285
Thr Leu Tyr Gly Trp Thr Arg Ser Pro Leu Ile Glu Tyr Tyr Val Val
290 295 300
Asp Ser Trp Gly Thr Tyr Arg Pro Thr Gly Thr Tyr Lys Gly Thr Val
305 310 315 320
Lys Ser Asp Gly Gly Thr Tyr Asp Ile Tyr Thr Thr Thr Arg Tyr Asn
325 330 335
Ala Pro Ser Ile Asp Gly Asp Arg Thr Thr Phe Thr Gln Tyr Trp Ser
340 345 350
Val Arg Gln Thr Lys Arg Pro Thr Gly Ser Asn Ala Thr Ile Thr Phe
355 360 365
Ser Asn His Val Asn Ala Trp Lys Ser His Gly Met Asn Leu Gly Ser
370 375 380
Asn Trp Ala Tyr Gln Val Met Ala Thr Glu Gly Tyr Gln Ser Ser Gly
385 390 395 400
Ser Ser Asn Val Thr Val Trp
405
<210> 13
<211> 412
<212> PRT
<213> Artificial Sequence
<220>
<223> 1
<400> 13
Ala Tyr Asp Asp Leu His Glu Gly Tyr Ala Thr Tyr Thr Gly Ser Gly
1 5 10 15
Tyr Ser Gly Gly Ala Phe Leu Leu Asp Pro Ile Pro Ser Asp Met Glu
20 25 30
Ile Thr Ala Ile Asn Pro Ala Asp Leu Asn Tyr Gly Gly Val Lys Ala
35 40 45
Ala Leu Ala Gly Ser Tyr Leu Glu Val Glu Gly Pro Lys Gly Lys Thr
50 55 60
Thr Val Tyr Val Thr Asp Leu Tyr Pro Glu Gly Ala Arg Gly Ala Leu
65 70 75 80
Asp Leu Ser Pro Asn Ala Phe Arg Lys Ile Gly Asn Met Lys Asp Gly
85 90 95
Lys Ile Asn Ile Lys Trp Arg Val Val Lys Ala Pro Ile Thr Gly Asn
100 105 110
Phe Thr Tyr Arg Ile Lys Glu Gly Ser Ser Arg Trp Trp Ala Ala Ile
115 120 125
Gln Val Arg Asn His Lys Tyr Pro Val Met Lys Met Glu Tyr Glu Lys
130 135 140
Asp Gly Lys Trp Ile Asn Met Glu Lys Met Asp Tyr Asn His Phe Val
145 150 155 160
Ser Thr Asn Leu Gly Thr Gly Ser Leu Lys Val Arg Met Thr Asp Ile
165 170 175
Arg Gly Lys Val Val Lys Asp Thr Ile Pro Lys Leu Pro Glu Ser Gly
180 185 190
Thr Ser Lys Ala Tyr Thr Val Pro Gly His Val Gln Phe Pro Glu Gly
195 200 205
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
210 215 220
Gly Gly Ser Ala Ser Thr Asp Tyr Trp Gln Asn Trp Thr Asp Gly Gly
225 230 235 240
Gly Ile Val Asn Ala Val Asn Gly Ser Gly Gly Asn Tyr Ser Val Asn
245 250 255
Trp Ser Asn Thr Gly Asn Phe Val Val Gly Lys Gly Trp Thr Thr Gly
260 265 270
Ser Pro Phe Arg Thr Ile Asn Tyr Asn Ala Gly Val Trp Ala Pro Asn
275 280 285
Gly Asn Gly Tyr Leu Thr Leu Tyr Gly Trp Thr Arg Ser Pro Leu Ile
290 295 300
Glu Tyr Tyr Val Val Asp Ser Trp Gly Thr Tyr Arg Pro Thr Gly Thr
305 310 315 320
Tyr Lys Gly Thr Val Lys Ser Asp Gly Gly Thr Tyr Asp Ile Tyr Thr
325 330 335
Thr Thr Arg Tyr Asn Ala Pro Ser Ile Asp Gly Asp Arg Thr Thr Phe
340 345 350
Thr Gln Tyr Trp Ser Val Arg Gln Thr Lys Arg Pro Thr Gly Ser Asn
355 360 365
Ala Thr Ile Thr Phe Ser Asn His Val Asn Ala Trp Lys Ser His Gly
370 375 380
Met Asn Leu Gly Ser Asn Trp Ala Tyr Gln Val Met Ala Thr Glu Gly
385 390 395 400
Tyr Gln Ser Ser Gly Ser Ser Asn Val Thr Val Trp
405 410
<210> 14
<211> 413
<212> PRT
<213> Artificial Sequence
<220>
<223> 1
<400> 14
Ala Tyr Asp Asp Leu His Glu Gly Tyr Ala Thr Tyr Thr Gly Ser Gly
1 5 10 15
Tyr Ser Gly Gly Ala Phe Leu Leu Asp Pro Ile Pro Ser Asp Met Glu
20 25 30
Ile Thr Ala Ile Asn Pro Ala Asp Leu Asn Tyr Gly Gly Val Lys Ala
35 40 45
Ala Leu Ala Gly Ser Tyr Leu Glu Val Glu Gly Pro Lys Gly Lys Thr
50 55 60
Thr Val Tyr Val Thr Asp Leu Tyr Pro Glu Gly Ala Arg Gly Ala Leu
65 70 75 80
Asp Leu Ser Pro Asn Ala Phe Arg Lys Ile Gly Asn Met Lys Asp Gly
85 90 95
Lys Ile Asn Ile Lys Trp Arg Val Val Lys Ala Pro Ile Thr Gly Asn
100 105 110
Phe Thr Tyr Arg Ile Lys Glu Gly Ser Ser Arg Trp Trp Ala Ala Ile
115 120 125
Gln Val Arg Asn His Lys Tyr Pro Val Met Lys Met Glu Tyr Glu Lys
130 135 140
Asp Gly Lys Trp Ile Asn Met Glu Lys Met Asp Tyr Asn His Phe Val
145 150 155 160
Ser Thr Asn Leu Gly Thr Gly Ser Leu Lys Val Arg Met Thr Asp Ile
165 170 175
Arg Gly Lys Val Val Lys Asp Thr Ile Pro Lys Leu Pro Glu Ser Gly
180 185 190
Thr Ser Lys Ala Tyr Thr Val Pro Gly His Val Gln Phe Pro Glu Thr
195 200 205
Lys Asp Ser Thr Lys Asp Ile Pro Glu Thr Pro Ser Lys Asp Lys His
210 215 220
Thr Gln Glu Asn Ala Ser Thr Asp Tyr Trp Gln Asn Trp Thr Asp Gly
225 230 235 240
Gly Gly Ile Val Asn Ala Val Asn Gly Ser Gly Gly Asn Tyr Ser Val
245 250 255
Asn Trp Ser Asn Thr Gly Asn Phe Val Val Gly Lys Gly Trp Thr Thr
260 265 270
Gly Ser Pro Phe Arg Thr Ile Asn Tyr Asn Ala Gly Val Trp Ala Pro
275 280 285
Asn Gly Asn Gly Tyr Leu Thr Leu Tyr Gly Trp Thr Arg Ser Pro Leu
290 295 300
Ile Glu Tyr Tyr Val Val Asp Ser Trp Gly Thr Tyr Arg Pro Thr Gly
305 310 315 320
Thr Tyr Lys Gly Thr Val Lys Ser Asp Gly Gly Thr Tyr Asp Ile Tyr
325 330 335
Thr Thr Thr Arg Tyr Asn Ala Pro Ser Ile Asp Gly Asp Arg Thr Thr
340 345 350
Phe Thr Gln Tyr Trp Ser Val Arg Gln Thr Lys Arg Pro Thr Gly Ser
355 360 365
Asn Ala Thr Ile Thr Phe Ser Asn His Val Asn Ala Trp Lys Ser His
370 375 380
Gly Met Asn Leu Gly Ser Asn Trp Ala Tyr Gln Val Met Ala Thr Glu
385 390 395 400
Gly Tyr Gln Ser Ser Gly Ser Ser Asn Val Thr Val Trp
405 410
<210> 15
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> 1
<400> 15
gccgccatgg ggcatatgac gacctgcatg 30
<210> 16
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> 1
<400> 16
ccgctcgagt tcaggaaact gaacatggc 29
<210> 17
<211> 31
<212> DNA
<213> Artificial Sequence
<220>
<223> 1
<400> 17
gccgccatgg ggctagcaca gactactggc a 31
<210> 18
<211> 32
<212> DNA
<213> Artificial Sequence
<220>
<223> 1
<400> 18
ccgctcgagt taccacactg ttacgttaga ac 32
<210> 19
<211> 31
<212> DNA
<213> Artificial Sequence
<220>
<223> 1
<400> 19
gccgccatgg gcgcatatga cgacctgcat g 31
<210> 20
<211> 50
<212> DNA
<213> Artificial Sequence
<220>
<223> 1
<400> 20
tttagccgca gcttcttttg ccgcagcttc ttcaggaaac tgaacatggc 50

Claims (6)

1.一种扩张蛋白和木聚糖酶融合蛋白，其特征在于，所述融合蛋白由扩张蛋白EXCL、木聚糖酶XYN和连接肽组成，扩张蛋白EXCL位于融合蛋白的N端，木聚糖酶XYN位于融合蛋白的C端，扩张蛋白EXCL和木聚糖酶XYN间插入连接肽；所述连接肽为天然连接肽TKDSTKDIPETPSKDKHTQENG或刚性连接肽(EAAAK)n，n=1~3；
所述连接肽为上述刚性连接肽时，所述融合蛋白的序列选自SEQ ID NO：6、SEQ ID NO：7或SEQ ID NO：8；
所述连接肽为上述天然连接肽时，所述融合蛋白的序列如SEQ ID NO：14所示。
2.编码权利要求1所述融合蛋白的基因。
3.包含权利要求2所述融合蛋白编码基因的表达载体。
4.包含权利要求2所述融合蛋白编码基因的宿主菌。
5.权利要求1所述融合蛋白在木寡糖生产中的应用。
6.根据权利要求5所述的应用，其特征在于，采用重组融合蛋白酶解玉米芯生产木寡糖。
CN201710134735.7A 2017-03-08 2017-03-08 一种扩张蛋白和木聚糖酶融合蛋白、其编码基因和应用 Active CN108570107B (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201710134735.7A CN108570107B (zh) 2017-03-08 2017-03-08 一种扩张蛋白和木聚糖酶融合蛋白、其编码基因和应用

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201710134735.7A CN108570107B (zh) 2017-03-08 2017-03-08 一种扩张蛋白和木聚糖酶融合蛋白、其编码基因和应用

Publications (2)

Publication Number Publication Date
CN108570107A CN108570107A (zh) 2018-09-25
CN108570107B true CN108570107B (zh) 2021-12-28

Family

ID=63577125

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201710134735.7A Active CN108570107B (zh) 2017-03-08 2017-03-08 一种扩张蛋白和木聚糖酶融合蛋白、其编码基因和应用

Country Status (1)

Country Link
CN (1) CN108570107B (zh)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109400715A (zh) * 2018-11-21 2019-03-01 南京工业大学 一种固定化木聚糖酶、制备方法及其应用
CN112300291B (zh) * 2020-10-28 2022-05-06 南京工业大学 一类高比活的融合木聚糖酶突变体及其应用
CN112851750B (zh) * 2021-02-20 2022-03-22 中国标准化研究院 连接肽、含有连接肽的融合蛋白及其应用

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1834250A (zh) * 2006-03-29 2006-09-20 浙江大学 β－葡聚糖酶和木聚糖酶融合基因、构建法和用途

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1834250A (zh) * 2006-03-29 2006-09-20 浙江大学 β－葡聚糖酶和木聚糖酶融合基因、构建法和用途

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
"A fusion enzyme consisting of bacterial expansin and endoglucanase for the degradation of highly crystalline cellulose";Kazunori Nakashima et al.,;《RSC Advances》;20141231(第4期);第43815-43820页尤其是第43815页摘要部分和附图1 *
"An expansin from the marine bacterium Hahella chejuensis acts synergistically with xylanase and enhances xylan hydrolysis";Hee Jin Lee et al.,;《Bioresource Technology》;20130929;第149卷;第516-519页尤其是第516摘要部分和第517-519第2-3节 *
"GenBank: AHN14743.1 REGION: 29..213";匿名;《GenBank》;20150527;第1页 *
"NCBI Reference Sequence：WP_003231419.1 REGION:26..232";匿名;《GenBank》;20150322;第1页 *

Also Published As

Publication number Publication date
CN108570107A (zh) 2018-09-25

Similar Documents

Publication Publication Date Title
AU2007213436B2 (en) Construction of new variants of dextransucrase DSR-S by genetic engineering
CN108699549B (zh) β-半乳糖苷酶
CN112852782B (zh) 一种低温适应性改良的低温外切菊粉酶突变体MutDL121EK5及应用
CN106906236B (zh) 唾液酸酶基因重组表达载体及其构建方法，唾液酸酶及其制备方法
Saburi et al. Practical preparation of epilactose produced with cellobiose 2-epimerase from Ruminococcus albus NE1
KR100601333B1 (ko) 아가레이즈 및 그의 유전자
CN116355881B (zh) 酸耐受性提高的β-木糖苷酶突变体D395G及其应用
CN108570107B (zh) 一种扩张蛋白和木聚糖酶融合蛋白、其编码基因和应用
US10752888B2 (en) Method for enzymatically preparing highly concentrated myo-inositol
JP7344195B2 (ja) 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
CN116555233A (zh) 热不稳定的β-木糖苷酶突变体E179GD182G及其应用
Zhang et al. A novel Lactococcus lactis l-arabinose isomerase for d-tagatose production from lactose
Wang et al. Characterization of a thermostable PL-31 family alginate lyase from Paenibacillus ehimensis and its application for alginate oligosaccharides bioproduction
CN110885809A (zh) α-L-岩藻糖苷酶及其相关生物材料与应用
JP4243266B2 (ja) パエニバチルス属細菌由来のキチナーゼ及びそれをコードする遺伝子
Itoi et al. Identification of a novel endochitinase from a marine bacterium Vibrio proteolyticus strain No. 442
WO2015054947A1 (zh) 一种n-乙酰神经氨酸醛缩酶在催化合成n-乙酰神经氨酸中的应用
KR101212106B1 (ko) 신규한 베타-아가라제 및 그 용도
CN106811450B (zh) 一种双功能转糖基α-N-乙酰氨基半乳糖苷酶及其表达基因与应用
KR20120028304A (ko) 신규 단백질 및 그것을 코드하는 유전자
CN104789546A (zh) 一种脱乙酰基酶突变体及其应用
Duan et al. A GH42 β‐galactosidase from Bifidobacterium thermophilum: Biochemical characterization and synthesis of galactosyl glycerol by reverse hydrolysis
JP6993637B2 (ja) フコース含有糖鎖を特異的に切断するエンドグリコシダーゼ
CN111254153A (zh) 一种聚半乳糖醛酸酶编码基因及酶和制备与应用
CN106459941B (zh) 从链霉菌菌株hy-14生产的新型木聚糖酶

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