CN110358755A - 酸性耐高温重组纤维素酶及其应用 - Google Patents
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Abstract
本发明公开了三种酸性耐高温重组纤维素酶及其应用,该三种重组纤维素酶的氨基酸序列分别如SEQIDNO:1‑3所示。本发明的三种重组纤维素酶在pH为4.0~5.0,60~80℃条件下对CMC‑Na酶活最高为1.540U/mg,对Avicel活力最高为0.167U/mg。本发明三种重组纤维素酶在75℃,pH为4.0~4.5条件下保温2h酶活力基本保持不变。三种重组纤维素酶可直接降解CMC‑Na、结晶纤维素、滤纸及其它天然木质纤维素如水稻秸秆和小麦秸秆,其对水稻秸秆的活力最高达0.106U/mg。本发明三种重组纤维素酶比现有的纤维素酶具有更大的优越性,可有效降解天然木质纤维素,具有潜在的工业应用价值。
Description
技术领域
本发明属于基因工程技术及生物质精炼技术领域,具体涉及三种酸性耐高温重组纤维素酶及其应用。
背景技术
木质纤维素作为一种可再生资源可用于生产生物燃料或其它高附加值产品。然而木质纤维素中的纤维素被半纤维素和木质素包裹,且以共价键连接以及纤维素自身的微纤维交联以致其很难被纤维素酶所降解,因此很难将其转化为可发酵糖。对木质纤维素进行预处理可破坏其中的氢键,去除木质素及半纤维素,增强纤维素酶的可及性。然而常规的酸碱,离子液体等预处理不仅增加了生产成本,而且易导致严重的环境污染。此外,人工合成的底物如:滤纸、结晶纤维素及CMC-Na的成分单一,结构简单,仅由不同数目的葡萄糖分子组成。因此,将滤纸、结晶纤维素及CMC-Na完全降解为葡萄糖分子仅需要内、外切葡聚糖酶和β-葡萄糖苷酶的协同作用。然而,对天然木质纤维素的完全降解需多种糖苷水解酶及非酶结合蛋白的协同作用,其中内切葡聚糖酶、碳水化合物结合结构域(CBM)和外切葡聚糖酶在此过程中起到至关重要的作用。自然界中游离的单催化域纤维素酶往往携带CBM结构域,催化结构域可通过CBM牢固吸附在底物上,提高催化效率。而内切葡聚糖酶首先随机切割非结晶区释放寡糖链,其次外切葡聚糖酶沿结晶区或非结晶区寡糖链的末端逐步切割释放纤维二糖。然而,多种游离纤维素酶混合降解木质纤维素过程中存在个别低效率酶的无效吸附问题。此外,嗜热菌Caldicellulosiruptor saccharolyticus来源的内切葡聚糖酶(Endo5-CBM28)和外切葡聚糖酶(CBM3b-Exo5)均可直接降解Avicel,可省去木质纤维素的预处理步骤,节省成本和保护环境。然而Endo5-CBM28对CMC-Na活力为8.94U/mg,然而对Avicel和FP的活力分别仅有0.0113U/mg和0.0518U/mg。此外,Endo5对Avicel无活力。
发明内容
发明目的:针对现有技术的不足存在的问题,为充分发挥木质纤维素降解酶及CBM的分子内协同作用,在Endo5、CBM及Exo5中实现融合,构建三种融合酶。本发明提供三种全新的酸性耐高温重组纤维素酶Endo5-2CBM-Exo5、Endo5-CBM3b-Exo5和Endo5-CBM28-Exo5,本发明三种重组纤维素酶均具有耐酸耐高温,活性高,可直接降解CMC-Na、结晶纤维素(Avicel)、滤纸(FP)及其它天然木质纤维素。
本发明还提供三种编码酸性耐高温重组纤维素酶的核苷酸序列以及三种酸性耐高温重组纤维素的应用。
技术方案:为了实现上述发明目的,如本发明所述酸性耐高温重组纤维素酶,所述酸性耐高温重组纤维素酶包括三种酸性耐高温重组纤维素酶,其氨基酸序列分别如SEQIDNO.1-3所示。分别命名为Endo5-2CBM-Exo5、Endo5-CBM3b-Exo5和Endo5-CBM28-Exo5。
进一步地,所述三种酸性耐高温重组纤维素酶,其DNA序列分别如SEQIDNO.4-6所示。分别为endo5-2cbm-exo5、endo5-cbm3b-exo5和endo5-cbm28-exo5。
其中,所述DNA序列SEQIDNO.4-6,分别通过下列引物(SEQ ID NO.11-16)进行扩增后拼接获得:
SEQ ID NO.11
Endo-F:5’-AACGATCCATGGCTAATACTGCGTATGAAAAGGATA-3’;
SEQ ID NO.12
Endocbm-R:5’-AACGATGAGCTCTGTAAATCTTACATTGTCTAAG-3’;
SEQ ID NO.13
Endo-R:5’-AACGATGAGCTCTTTCTGGTCGCTGCCAGG-3’;
SEQ ID NO.14
cbmExo-F:5’-AACGATGAGCTCGGGGTAACAACATCATCTC-3’;
SEQ ID NO.15
Exo-R:5’-AACGATCTCGAGTTTTGAAGCTGGAACTGGCTC-3’;
SEQ ID NO.16
Exo-F:5’-AACGATGAGCTCTTTAAGAGTGGAGCAGGGCA-3’。
本发明所述重组载体,所述重组载体克隆包括有SEQ ID NO.4-6中所任意一种DNA序列。
本发明所述重组菌,所述重组菌包含有权利要求3中的重组载体。
本发明所述酸性耐高温重组纤维素酶在酶解CMC-Na、滤纸、Avicel、和天然木质纤维素中的应用。
作为优选,所述天然木质纤维素包括水稻秸秆和小麦秸秆。
进一步地,所述酶解反应温度为60~80℃,pH为4.0~4.5,反应时间为4h。
本发明所述的重组载体在酶解CMC-Na、滤纸、Avicel、和天然木质纤维素中的应用。
本发明所述的的重组载体在酶解CMC-Na、滤纸、Avicel、和天然木质纤维素中的应用。
本发明所述三种酸性耐高温纤维素酶,氨基酸序列为SEQIDNO.1、2和3序列,由各结构域经过不同的融合方式形成具有双功能纤维素酶的融合蛋白。
本发明根据现有的酶Endo5-CBM28、Endo5、CBM3b-Exo5和Exo5与底物结合的特点(还能加点别的特征分析,我们再合成新的模板),设计了全新的基因endo5-cbm28、endo5、cbm3b-exo5和exo5基因(SEQ ID NO.7-10),并设计全新的引物对(SEQIDNO.11-16),引物和新设计的基因模板进行细致比对,有效避免了非特异的结合,再进行合成,再以全新的基因endo5-cbm28、endo5、cbm3b-exo5和exo5基因为基础,巧妙地构建全新重组基因endo5-2cbm-exo5、endo5-cbm3b-exo5和endo5-cbm28-exo5(SEQ ID NO.4-6),最后得到本发明的三种全新的酸性耐高温重组纤维素酶Endo5-2CBM-Exo5、Endo5-CBM3b-Exo5和Endo5-CBM28-Exo5。所设计全新基因和形成全新的三种酶对于底物如CMC-Na、滤纸、Avicel、和天然木质纤维素活性更好,酶解效果更优。
有益效果:与现有技术相比,本发明具有如下优点:
本发明提供的三种全新的重组纤维素酶具有耐酸耐高温,在pH为4.0~5.0,60~80℃条件对CMC-Na酶活最高,为1.540U/mg;对Avicel酶活最高为0.167U/mg;在70~80℃,pH为4.0~6.0条件下均表现出高的活力。三种重组纤维素酶在75℃,pH为4.0~4.5条件下保温2h酶活力基本保持不变。此外,三种重组纤维素酶可直接降解Avicel、FP)及其它天然木质纤维素如水稻秸秆(RS)和小麦秸秆(WS),其对水稻秸秆的活力最高达0.106U/mg。上述特性使得本发明得到的三种重组纤维素酶比现有的纤维素酶具有更大的优越性和更广的底物谱,在pH为4.0~5.0,60~80℃左右降解天然木质纤维素,活性和降解效果明显优于现有endo5-cbm28、endo5基因表达的酶Endo5-CBM28和Endo5及其他现有技术中的纤维素酶,并且本发明的重组纤维素酶易于表达,可以稳定获得,具有潜在的工业应用价值。
附图说明
图1是三种双催化域纤维素酶的融合构建方式图。
图2是三种双催化域纤维素酶Endo5-2CBM-Exo5、Endo5-CBM3b-Exo5和Endo5-CBM28-Exo5的SDS-PAGE蛋白电泳图;
图3是Endo5-2CBM-Exo5、Endo5-CBM3b-Exo5和Endo5-CBM28-Exo5活性随温度的变化结果图;
图4是Endo5-2CBM-Exo5、Endo5-CBM3b-Exo5和Endo5-CBM28-Exo5活性随pH的变化结果图;
图5是Endo5-2CBM-Exo5、Endo5-CBM3b-Exo5和Endo5-CBM28-Exo5热稳定变化结果图;
图6是Endo5-2CBM-Exo5、Endo5-CBM3b-Exo5和Endo5-CBM28-Exo5pH稳定变化结果图;
图7是Endo5-2CBM-Exo5、Endo5-CBM3b-Exo5和Endo5-CBM28-Exo5降解FP、Avicel及天然木质纤维素TLC图。
具体实施方式
下面结合实施例和附图对本发明做进一步的说明。
以下实施例中所使用的材料、试剂等,如无特殊说明,均可从商业途径获得。
实施例1
内切葡聚糖酶(Endo5)、碳水化合物结合结构域(CBM)及外切葡聚糖酶(Exo5)的基因制备。
可采用人工合成的方式制备现有技术中原始的endo5-cbm28、endo5、cbm3b-exo5和exo5基因,也可以Caldicellulosiruptor saccharolyticus的总DNA(购自美国菌种保藏中心)为模板通过PCR方式得到。
本实施例的全新的endo5-cbm28、endo5、cbm3b-exo5和exo5基因是根据酶Endo5-CBM28、Endo5、CBM3b-Exo5和Exo5与底物结合的特点,经过序列比对、预测、设计了新序列,并将新序列送至上海生工全基因合成。所设计全新基因对于底物如CMC-Na、滤纸、Avicel、和天然木质纤维素活性更好,酶解效果更优。
以全基因合成的新序列为模板,用下列全新的引物对(SEQ ID NO.11-16),分别扩增endo5-cbm28、endo5、cbm3b-exo5和exo5基因。
Endo-F:5’-AACGATCCATGGCTAATACTGCGTATGAAAAGGATA-3’;
Endocbm-R:5’-AACGATGAGCTCTGTAAATCTTACATTGTCTAAG-3’;
Endo-R:5’-AACGATGAGCTCTTTCTGGTCGCTGCCAGG-3’;
cbmExo-F:5’-AACGATGAGCTCGGGGTAACAACATCATCTC-3’;
Exo-R:5’-AACGATCTCGAGTTTTGAAGCTGGAACTGGCTC-3’;
Exo-F:5’-AACGATGAGCTCTTTAAGAGTGGAGCAGGGCA-3’。
上述引物合成时,Endo-F引入NcoI酶切位点,Endocbm-R、Endo-R、cbmExo-F和Exo-F分别引入SacI酶切位点,Exo-R引入XhoI酶切位点。
PCR反应体系:1μL的C.saccharolyticus基因组DNA,Endo-F、Endocbm-R、Endo-R、cbmExo-F、Exo-F及Exo-R分别加1μL,25μLBiouniquer2×PCRMasterMix,22μL超纯水。
PCR反应条件:94℃变性5min;94℃变性30sec,50~55℃退火30sec,72℃延伸30~120sec,30Cycles;72℃延伸10min;4℃保温。
PCR产物用1%琼脂糖凝胶电泳检测产量和特异性,并用胶回收试剂盒进行纯化(Trans,南京)。
以Endo-F和Endocbm-R为引物对扩增得到endo5-cbm28基因;以Endo-F和Endo-R为引物对扩增得到endo5基因;以cbmExo-F和Exo-R为引物对扩增得到cbm3b-exo5基因;以Exo-F和Exo-R为引物对扩增得到exo5基因。最终得到新设计的基因endo5-cbm28、endo5、cbm3b-exo5和exo5基因序列分别如SEQ ID NO.7-10。SEQ ID NO.7(endo5-cbm28),SEQ IDNO.8(endo5),SEQ ID NO.9(cbm3b-exo5),SEQ ID NO.10(exo5)。
实施例2
重组克隆、表达载体pET28a-endo5-2cbm-exo5、pET28a-endo5-cbm3b-exo5和pET28a-endo5-cbm28-exo5的构建与验证
将纯化后的PCR产物endo5-cbm28、endo5(实施例1制备)和pET28a(Novagen)分别用NcoI和SacI双酶切,琼脂糖凝胶电泳回收酶切PCR片段及载体大片段。割胶回收后的目的片段与载体,经浓缩加入8μL无菌水重悬。加入1μL10×LigaseBuffer和1μLLigase,于16℃过夜连接。连接产物转化大肠杆菌(E.coliDH5a),然后涂布于含50μg/mL卡那霉素(Kan)的LB固体培养基,37℃培养13~15h。
从转化平板上挑取多个单菌落,采用上海生工质粒提取试剂盒提取质粒。对提取的质粒进行双酶切验证后进行测序验证。测序结果显示,pET28a载体中分别***了所克隆的目的片段(endo5-cbm28和endo5大小分别为1629bp和966bp),分别得重组载体pET28a-endo5-cbm28和pET28a-endo5。接种阳性重组子,采用上海生工质粒提取试剂盒提取质粒。再将纯化后的PCR产物cbm3b-exo5、exo5(实施例1制备)及pET28a-endo5-cbm28、pET28a-endo5(实施例2制备)分别用SacI和XhoI双酶切,琼脂糖凝胶电泳回收酶切PCR片段及载体大片段。割胶回收后的目的片段cbm3b-exo5和exo5(实施例1制备)分别***pET28a-endo5-cbm28的下游;目的片段cbm3b-exo5***pET28a-endo5的下游。各片段经浓缩加入8μL无菌水重悬。加入1μL10×LigaseBuffer和1μLLigase,于16℃过夜连接。连接产物转化大肠杆菌(E.coliDH5a),然后涂布于含50μg/mL卡那霉素(Kan)的LB固体培养基,37℃培养13~15h。从转化平板上挑取多个单菌落,采用上海生工质粒提取试剂盒提取质粒。对提取的质粒进行双酶切验证后进行测序验证。测序结果显示,pET28a-endo5-cbm28载体中分别***了所克隆的目的片段(cbm3b-exo5和exo5大小分别为1998bp和1599bp);pET28a-endo5载体中***了所克隆的目的片段(cbm3b-exo5大小别为1998bp)。进而分别得到重组表达载体pET28a-endo5-2cbm-exo5、pET28a-endo5-cbm3b-exo5和pET28a-endo5-cbm28-exo5。其中endo5-2cbm-exo5、endo5-cbm3b-exo5和endo5-cbm28-exo5的DNA序列如SEQ ID NO.4、5和6所示,其翻译的蛋白(酸性耐高温重组纤维素酶)的氨基酸序列如SEQ ID NO.1、2和3所示。将这几种蛋白分别命名为Endo5-2CBM-Exo5、Endo5-CBM3b-Exo5和Endo5-CBM28-Exo5。三种双催化域纤维素酶的融合构建如图1所示。
SEQ ID NO.1(Endo5-2CBM-Exo5),SEQ ID NO.2(Endo5-CBM3b-Exo5),SEQ IDNO.3(Endo5-CBM28-Exo5),SEQ ID NO.4(endo5-2cbm-exo5),SEQ ID NO.5(endo5-cbm3b-exo5),SEQ ID NO.6(endo5-cbm28-exo5)。
实施例3
重组纤维素酶Endo5-2CBM-Exo5、Endo5-CBM3b-Exo5和Endo5-CBM28-Exo5的表达与纯化。
将重组克隆、表达载体pET28a-endo5-2cbm-exo5、pET28a-endo5-cbm3b-exo5和pET28a-endo5-cbm28-exo5(实施例2制备)化学转化至表达宿主E.coliBL21(DE3)(Novagen),获得含有重组质粒的重组菌。接种阳性重组子于5mL含有50μg/mLKan的Luria-Bertanibroth(LB)液体培养基,37℃,200rpm条件下过夜培养。转接4mL菌液于含有50μg/mLKan的500mLLB液体培养基,37℃,200rpm条件下培养菌体浓度(OD600)至0.6~0.8。加入终浓度为0.5mM的IPTG,在25℃,200rpm条件下诱导表达12~14h。将培养液在高速冷冻离心机中以4℃,10000rpm,离心8min收集菌体。加入40mL的无菌水洗涤菌体,再次以4℃,10000rpm,离心8min,弃去上清。向沉淀中加入20mL的1×bindingbuffer(0.5M的NaCl,20mM的Tris-HCl,5mM的咪唑,pH7.9),在冰水浴中通过超声波细胞破碎仪超声破碎。细胞破碎液在4℃,15000rpm,离心15min,得到含酸性耐高温重组纤维素酶的粗酶液。
粗酶液先经70℃加热预处理15min以除去热稳定性较差的杂蛋白,再过Ni-NTA亲和层析柱进行纯化(操作步骤见His-BindKits,Novagen)。纯化后酶的纯度及分子量鉴定采用SDS-PAGE进行,结果见图2,1、2和3号泳道分别是纯化过后的Endo5-2CBM-Exo5、Endo5-CBM28-Exo5和Endo5-CBM3b-Exo5,分子量分别为129kDa、114kDa和111kDa,与理论值相近。
实施例4
重组纤维素酶Endo5-2CBM-Exo5、Endo5-CBM3b-Exo5和Endo5-CBM28-Exo5的酶学特性分析。
酶活定义:在pH值4.5和80℃反应条件下,每分钟降解CMC-Na底物释放出1μmol葡萄糖所需的酶量。
(1)最适温度
利用pH值为4.5的100mM的咪唑-邻苯二甲酸氢钾缓冲液稀释实施例3制备得到的纯酶液(稀释20倍),利用稀释后的纯酶液进行活力测定。酶活测定的反应体系为150μL,包含50μL的1%CMC-Na底物,50μL的100mM的咪唑-邻苯二甲酸氢钾缓冲液,50μL的稀释后的纯酶液,反应体系的pH为4.5。将反应体系在40~90℃水浴中保温30min后,立即***冰浴终止反应。加入150μL的DNS试剂,沸水浴煮沸5min后,立即***冰浴终止反应。补水至1mL,测定520nm处的吸光度值,其中未加酶的反应管作为空白对照。实验设3次重复,计算酶活,取平均值作图。结果如图3所示,研究结果表明,Endo5-2CBM-Exo5、Endo5-CBM3b-Exo5和Endo5-CBM28-Exo5的最适酶活温度分别为80℃,60℃和80℃,其中Endo5-2CBM-Exo5在80℃条件下对CMC-Na酶活高达1.247U/mg;Endo5-CBM28-Exo5在80℃条件下对CMC-Na酶活高达0.636U/mg;Endo5-CBM3b-Exo5在80℃条件下对CMC-Na酶活高达0.320U/mg;将Endo5-2CBM-Exo5、Endo5-CBM28-Exo5、Endo5-CBM3b-Exo5在80℃条件下的酶活设置为相对活性100%,其它温度下的反应体系所测数据计算得到的活力以最高的酶活作为相对活力。在60~80℃条件下酶活力高。
(2)最适pH
酶活测定反应体系为150μL,包含50μL的1%CMC-Na底物,50μLpH4.0~8.0的100mM的咪唑-邻苯二甲酸氢钾缓冲液,50μL的稀释后的纯酶液(同上)。将反应体系分别置于最适酶活温度60或80℃水浴中保温30min后,立即***冰浴终止反应。加入150μL的DNS试剂,沸水浴煮沸5min后,立即***冰浴终止反应。补水至1mL,测定520nm处的吸光度值,其中未加酶的反应管作为空白对照。实验设3次重复,计算酶活,取平均值作图。结果如图4所示,研究结果表明,Endo5-2CBM-Exo5、Endo5-CBM3b-Exo5和Endo5-CBM28-Exo5的最适pH值分别为4.0,4.0和4.5,其中Endo5-2CBM-Exo5在80℃,pH4.0条件下对CMC-Na酶活高达1.540U/mg;Endo5-CBM28-Exo5在80℃,pH4.5条件下对CMC-Na酶活高达0.785U/mg;Endo5-CBM3b-Exo5在80℃,pH4.0条件下对CMC-Na酶活高达0.357U/mg。将Endo5-2CBM-Exo5在80℃,pH4.0条件下的酶活设置为相对活性100%,其它pH条件下的反应体系所测数据计算得到的活力以最高的酶活作为相对活力。在pH4.0~4.5条件下酶活力高。
(3)热稳定性
用pH值为4.5的100mM的咪唑-邻苯二甲酸氢钾缓冲液稀释实施4制备得到的纯酶液,利用稀释后的纯酶液进行活力测定。将稀释后的酶液在70℃,75℃,80℃,85℃,90℃水浴中保温30min,60min,90min,120min,测定残留酶活力。酶活测定反应在pH值为4.0或4.5,测定温度为60或80℃。实验设3次重复,计算酶活,取平均值作图。结果如图5所示,研究结果显示,重组酶在75℃条件下热处理2h,未见明显的活力下降,可见重组酶在75℃条件下具有好的热稳定性。
(4)pH稳定性
用pH值为4.5的100mM的咪唑-邻苯二甲酸氢钾缓冲液稀释实施4制备得到的纯酶液,利用稀释后的纯酶液进行活力测定。将稀释后的酶液在pH4.0~8.0,55℃条件下保温2h后,测定残留酶活力。酶活测定反应在60或80℃。实验设3次重复,计算酶活,取平均值作图。结果如图6所示,研究结果显示,重组酶在pH4.0~6.0条件下,55℃处理2h未见明显的活力下降,可见重组酶在pH4.0~6.0条件下具有好的稳定性。
实施例5
酸性耐高温重组纤维素酶Endo5-2CBM-Exo5、Endo5-CBM3b-Exo5和Endo5-CBM28-Exo5的应用研究。
重组纤维素酶Endo5-2CBM-Exo5、Endo5-CBM3b-Exo5和Endo5-CBM28-Exo5对FP、Avicel、RS、WS和蒸汽***的水稻秸秆(SPRS)的降解研究,具体步骤如下所述:
(1)配制纤维二糖及葡萄糖溶液,浓度均为1%;分别称取50mg的FP、Avicel、RS、WS和SPRS置于反应管,加入1mL的pH值为4.0或4.5的100mM的咪唑-邻苯二甲酸氢钾缓冲液和1mL经过适当稀释的纯酶液(稀释20倍,实施例3制备得到的纯酶液)。反应体系在60或80℃水浴摇床中,160rpm震荡4h。
(2)分别收集上述各反应管中的水解液于-20℃的冰箱中保存。
(3)配制上述水解液TLC展开剂和显色剂,展开剂成分及配比为正丁醇:乙酸:水=2:1:1;显色剂成分及配比为硫酸:甲醇=1:9。
(4)用毛细管吸取少量的1%纤维二糖、葡萄糖及上述的样品,分别点样在宽度为10cm,高度为20cm的玻璃硅胶板上。点样结束后,用吹风机吹干玻璃硅胶板后,置于含上述展开剂的层析缸中。
(5)6h后取出玻璃硅胶板,在105℃的烘箱中烘干,在通风橱将显色剂均匀喷洒在玻璃硅胶板,接着再放置105℃的烘箱中烘干。
结果如图7所示,1号泳道中的G1和G2分别代表葡萄糖和纤维二糖,2、3、4和5分别表示无酶(空白对照)、Endo5-2CBM-Exo5、Endo5-CBM3b-Exo5和Endo5-CBM28-Exo5水解上清液。研究结果显示,重组纤维素酶可有效地将FP、Avicel、RS、WS和SPRS水解为纤维二糖及葡萄糖。因此,这三种重组纤维素酶适用于水稻秸秆及小麦秸秆等原料的葡萄糖和纤维二糖生产。
(6)为进一步测定三种融合酶对CMC-Na、FP、Avicel、RS、WS和SPRS底物的降解活力,取第(2)步的各反应管中的水解液300μL。加入300μLDNS,沸水浴煮沸5min后,立即***冰浴,补水至2mL,测定520nm处的吸光度值,其中未加酶的反应管作为空白对照。实验设3次重复,计算酶活。
三种融合酶对CMC-Na、FP、Avicel、RS、WS和SPRS均表现出特异性活力。其中Endo5-2CBM-Exo5在80℃,pH4.0条件下对CMC-Na特异性活力最高,达到1.540±0.013U/mg;Endo5-CBM3b-Exo5在60℃,pH4.0条件下对FP特异性活力最高,达到0.074±0.006U/mg;Endo5-2CBM-Exo5在80℃,pH4.0条件下对Avicel特异性活力最高,达到0.167±0.008U/mg;Endo5-2CBM-Exo5在80℃,pH4.0条件下对SPRS特异性活力最高,达到0.075±0.006U/mg;Endo5-CBM3b-Exo5在60℃,pH4.0条件下对RS特异性活力最高,达到0.106±0.002U/mg;Endo5-CBM3b-Exo5在60℃,pH4.0条件下对WS特异性活力最高,达到0.078±0.008U/mg。
序列表
<110> 淮阴工学院
<120> 酸性耐高温重组纤维素酶及其应用
<160> 16
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1221
<212> PRT
<213> 人工序列(Endo5-2CBM-Exo5Artificial Sequence)
<400> 1
Met Ala Asn Thr Ala Tyr Glu Lys Asp Lys Tyr Pro His Leu Ile Gly
1 5 10 15
Asn Ser Leu Val Lys Lys Pro Ser Val Gly Ala Arg Ile Gln Leu Leu
20 25 30
Lys Asn Gln Gly Arg Lys Ile Ile Ala Asp Gln Asn Gly Glu Pro Ile
35 40 45
Gln Leu Arg Ala Met Thr Ser His Ala Leu Asn Trp Tyr Pro Asn Ile
50 55 60
Leu Asn Gln Asn Ala Tyr Ala Gly Leu Ala Gln Asp Gln Gly Leu Asn
65 70 75 80
Val Ile Arg Ile Ala Met Tyr Leu Gly Gln Gly Gly Tyr Gly Thr Gln
85 90 95
Pro Asn Val Arg Asp Lys Val Ile Glu Gly Ile Lys Ile Ala Leu Gln
100 105 110
Gln Asp Met Trp Val Leu Val Glu Trp His Ala Leu Asn Pro Ala Asp
115 120 125
Phe Asn Gly Glu Leu Tyr Lys Gly Gly Lys Asn Phe Pro Lys Glu Leu
130 135 140
Ala Asn Lys Phe Phe Asn Gln Phe His Leu Leu Tyr Glu Leu Cys Asn
145 150 155 160
Gln Pro Asp Pro Ser Asp Phe Gly Leu Thr Gln Asp Glu Gly Gly Trp
165 170 175
Arg Lys Val Arg Ala Trp Ala Gln Pro Leu Ile Arg Met Ile Arg Asn
180 185 190
Met Ala Asn Gln Asn Leu Ile Ile Ile Gly Ser Pro Asn Trp Ser Gln
195 200 205
Arg Pro Asp Phe Ala Ile Lys Asp Pro Ile Ala Asp Asp Lys Val Met
210 215 220
Tyr Ser Val His Phe Tyr Thr Gly Thr His Lys Val Asp Gly Tyr Val
225 230 235 240
Phe Glu Asn Met Lys Met Ala Ile Glu Ala Gly Val Pro Val Phe Val
245 250 255
Thr Glu Trp Gly Thr Ser Glu Ala Ser Gly Asp Gly Gly Pro Tyr Leu
260 265 270
Asp Glu Ala Asp Lys Trp Leu Glu Tyr Leu Asn Ala Asn Asn Ile Ser
275 280 285
Trp Val Asn Trp Ser Leu Thr Asn Lys Asn Glu Thr Ser Gly Ala Phe
290 295 300
Val Pro Tyr Ile Ser Gly Val Ser Gln Ala Thr Asp Leu Asp Pro Gly
305 310 315 320
Ser Asp Gln Lys Trp Asp Ile Ser Glu Leu Ser Ile Ser Gly Glu Tyr
325 330 335
Val Arg Ser Arg Ile Lys Gly Ile Pro Tyr Gln Pro Ile Glu Arg Thr
340 345 350
Leu Lys Ile Ser Gln Asp Gln Val Ala Cys Ala Pro Ile Gly Gln Pro
355 360 365
Ile Leu Pro Ser Asp Phe Glu Asp Gly Thr Arg Gln Gly Trp Asp Trp
370 375 380
Asp Gly Pro Ser Gly Val Lys Gly Ala Leu Thr Ile Glu Glu Ala Asn
385 390 395 400
Gly Ser Asn Ala Leu Ser Trp Glu Val Glu Tyr Pro Glu Lys Lys Leu
405 410 415
Gln Asp Gly Trp Ala Ser Ala Pro Arg Leu Ile Leu Arg Asn Ile Asn
420 425 430
Thr Thr Arg Gly Asp Cys Lys Tyr Leu Cys Phe Asp Phe Tyr Leu Lys
435 440 445
Pro Lys Gln Ala Thr Lys Gly Glu Leu Ala Ile Phe Leu Ala Phe Ala
450 455 460
Pro Pro Ser Leu Asn Tyr Trp Ala Gln Ala Glu Asp Ser Phe Asn Ile
465 470 475 480
Asp Leu Thr Asn Leu Ser Thr Leu Lys Lys Thr Pro Asp Asp Leu Tyr
485 490 495
Ser Phe Lys Ile Ser Phe Asp Leu Asp Lys Ile Lys Glu Gly Lys Ile
500 505 510
Ile Gly Pro Asp Thr His Leu Arg Asp Ile Ile Ile Val Val Ala Asp
515 520 525
Val Asn Ser Asp Phe Lys Gly Arg Met Tyr Leu Asp Asn Val Arg Phe
530 535 540
Thr Glu Leu Gly Val Thr Thr Ser Ser Pro Thr Pro Thr Pro Thr Pro
545 550 555 560
Thr Val Thr Val Thr Pro Thr Pro Thr Pro Thr Pro Thr Pro Thr Val
565 570 575
Thr Ala Thr Pro Thr Pro Thr Pro Thr Pro Val Ser Thr Pro Ala Thr
580 585 590
Gly Gly Gln Ile Lys Val Leu Tyr Ala Asn Lys Glu Thr Asn Ser Thr
595 600 605
Thr Asn Thr Ile Arg Pro Trp Leu Lys Val Val Asn Ser Gly Ser Ser
610 615 620
Ser Ile Asp Leu Ser Arg Val Thr Ile Arg Tyr Trp Tyr Thr Val Asp
625 630 635 640
Gly Glu Arg Ala Gln Ser Ala Val Ser Asp Trp Ala Gln Ile Gly Ala
645 650 655
Ser Asn Val Thr Phe Lys Phe Val Lys Leu Ser Ser Ser Val Ser Gly
660 665 670
Ala Asp Tyr Tyr Leu Glu Ile Gly Phe Lys Ser Gly Ala Gly Gln Leu
675 680 685
Gln Pro Gly Lys Asp Thr Gly Glu Ile Gln Ile Arg Phe Asn Lys Ser
690 695 700
Asp Trp Ser Asn Tyr Asn Gln Gly Asn Asp Trp Ser Trp Leu Gln Ser
705 710 715 720
Met Thr Ser Tyr Gly Glu Asn Glu Lys Val Thr Ala Tyr Ile Asp Gly
725 730 735
Val Leu Val Trp Gly Gln Glu Pro Ser Gly Ala Thr Pro Ala Pro Thr
740 745 750
Met Thr Val Ala Pro Thr Ala Thr Pro Thr Pro Thr Leu Ser Pro Thr
755 760 765
Val Thr Pro Thr Pro Ala Pro Thr Gln Thr Ala Ile Pro Thr Pro Thr
770 775 780
Leu Thr Pro Asn Pro Thr Pro Thr Ser Ser Ile Pro Asp Asp Thr Asn
785 790 795 800
Asp Asp Trp Leu Tyr Val Ser Gly Asn Lys Ile Val Asp Lys Asp Ala
805 810 815
Arg Phe Val Tyr Leu Thr Ala Ile Asn Tyr Phe Ala Tyr Asp Thr Ala
820 825 830
Thr Asn Leu Phe Asn Gly Ala Trp Ser Cys Asn Leu Lys Asp Thr Leu
835 840 845
Ala Glu Ile Gly Asn Lys Gly Tyr Asn Ile Ile Arg Ala Pro Leu Ser
850 855 860
Ala Glu Ile Ile Ile Asn Tyr Ser Asn Gly Leu Tyr Pro Lys Pro Asn
865 870 875 880
Ile Asn Tyr Tyr Val Asn Pro Glu Leu Glu Gly Lys Asn Ser Leu Glu
885 890 895
Val Phe Asp Ile Val Val Gln Thr Cys Lys Glu Val Gly Leu Lys Ile
900 905 910
Met Leu Asp Ile His Ser Ile Lys Thr Asp Ala Met Ala His Leu Tyr
915 920 925
Phe Val Trp Trp Asp Glu Arg Phe Ser Pro Gln Asp Pro Tyr Arg Ala
930 935 940
Cys Gln Trp Ile Thr Asn Arg Tyr Lys Asn Asp Asp Thr Ile Ile Ala
945 950 955 960
Phe Asp Leu Lys Asn Glu Pro His Gly Lys Pro Trp Gln Asp Thr Thr
965 970 975
Phe Ala Lys Trp Asp Asn Ser Thr Asp Ile Asn Asn Trp Lys Tyr Ala
980 985 990
Ala Glu Thr Cys Ala Lys Arg Ile Leu Asn Ile Asn Pro Asn Leu Leu
995 1000 1005
Ile Val Leu Glu Ala Ile Glu Gly Tyr Phe Lys Asn Asp Leu Thr Trp
1010 1015 1020
Ser Ser Lys Thr Thr Thr Asp Trp Tyr Thr Thr Trp Trp Gly Gly Asn
1025 1030 1035 1040
Leu Arg Gly Val Arg Lys Tyr Pro Ile Asn Leu Gly Lys Tyr Gln Asn
1045 1050 1055
Lys Leu Val Trp Ser Phe His Phe Tyr Ala Pro Ser Leu Tyr Glu Glu
1060 1065 1070
Pro Tyr Phe Trp Pro Gly Phe Thr Lys Glu Ser Leu Leu Gln Asp Cys
1075 1080 1085
Trp Arg Pro Asn Trp Ala Tyr Ile Met Glu Glu Asn Ile Ala Pro Leu
1090 1095 1100
Leu Ile Gly Glu Trp Gly Gly His Leu Asp Gly Ala Asp Asn Glu Lys
1105 1110 1115 1120
Trp Met Lys Tyr Leu Arg Asn Tyr Leu Leu Glu Asp His Leu His His
1125 1130 1135
Ser Phe Tyr Cys Pro Asn Ala Asn Ser Gly Asp Thr Gly Gly Leu Val
1140 1145 1150
Gly Tyr Asp Phe Thr Thr Trp Asp Glu Lys Lys Tyr Ser Phe Leu Lys
1155 1160 1165
Pro Ala Leu Trp Gln Asp Ser Gln Gly Arg Phe Val Gly Leu Asp His
1170 1175 1180
Lys Arg Pro Leu Gly Thr Asn Gly Lys Asn Ile Asn Ile Thr Thr Tyr
1185 1190 1195 1200
Tyr Asn Asn Asn Glu Pro Glu Pro Val Pro Ala Ser Lys Leu Glu His
1205 1210 1215
His His His His His
1220
<210> 2
<211> 1000
<212> PRT
<213> 人工序列(Endo5-CBM3b-Exo5Artificial Sequence)
<400> 2
Met Ala Asn Thr Ala Tyr Glu Lys Asp Lys Tyr Pro His Leu Ile Gly
1 5 10 15
Asn Ser Leu Val Lys Lys Pro Ser Val Gly Ala Arg Ile Gln Leu Leu
20 25 30
Lys Asn Gln Gly Arg Lys Ile Ile Ala Asp Gln Asn Gly Glu Pro Ile
35 40 45
Gln Leu Arg Ala Met Thr Ser His Ala Leu Asn Trp Tyr Pro Asn Ile
50 55 60
Leu Asn Gln Asn Ala Tyr Ala Gly Leu Ala Gln Asp Gln Gly Leu Asn
65 70 75 80
Val Ile Arg Ile Ala Met Tyr Leu Gly Gln Gly Gly Tyr Gly Thr Gln
85 90 95
Pro Asn Val Arg Asp Lys Val Ile Glu Gly Ile Lys Ile Ala Leu Gln
100 105 110
Gln Asp Met Trp Val Leu Val Glu Trp His Ala Leu Asn Pro Ala Asp
115 120 125
Phe Asn Gly Glu Leu Tyr Lys Gly Gly Lys Asn Phe Pro Lys Glu Leu
130 135 140
Ala Asn Lys Phe Phe Asn Gln Phe His Leu Leu Tyr Glu Leu Cys Asn
145 150 155 160
Gln Pro Asp Pro Ser Asp Phe Gly Leu Thr Gln Asp Glu Gly Gly Trp
165 170 175
Arg Lys Val Arg Ala Trp Ala Gln Pro Leu Ile Arg Met Ile Arg Asn
180 185 190
Met Ala Asn Gln Asn Leu Ile Ile Ile Gly Ser Pro Asn Trp Ser Gln
195 200 205
Arg Pro Asp Phe Ala Ile Lys Asp Pro Ile Ala Asp Asp Lys Val Met
210 215 220
Tyr Ser Val His Phe Tyr Thr Gly Thr His Lys Val Asp Gly Tyr Val
225 230 235 240
Phe Glu Asn Met Lys Met Ala Ile Glu Ala Gly Val Pro Val Phe Val
245 250 255
Thr Glu Trp Gly Thr Ser Glu Ala Ser Gly Asp Gly Gly Pro Tyr Leu
260 265 270
Asp Glu Ala Asp Lys Trp Leu Glu Tyr Leu Asn Ala Asn Asn Ile Ser
275 280 285
Trp Val Asn Trp Ser Leu Thr Asn Lys Asn Glu Thr Ser Gly Ala Phe
290 295 300
Val Pro Tyr Ile Ser Gly Val Ser Gln Ala Thr Asp Leu Asp Pro Gly
305 310 315 320
Ser Asp Gln Lys Glu Leu Gly Val Thr Thr Ser Ser Pro Thr Pro Thr
325 330 335
Pro Thr Pro Thr Val Thr Val Thr Pro Thr Pro Thr Pro Thr Pro Thr
340 345 350
Pro Thr Val Thr Ala Thr Pro Thr Pro Thr Pro Thr Pro Val Ser Thr
355 360 365
Pro Ala Thr Gly Gly Gln Ile Lys Val Leu Tyr Ala Asn Lys Glu Thr
370 375 380
Asn Ser Thr Thr Asn Thr Ile Arg Pro Trp Leu Lys Val Val Asn Ser
385 390 395 400
Gly Ser Ser Ser Ile Asp Leu Ser Arg Val Thr Ile Arg Tyr Trp Tyr
405 410 415
Thr Val Asp Gly Glu Arg Ala Gln Ser Ala Val Ser Asp Trp Ala Gln
420 425 430
Ile Gly Ala Ser Asn Val Thr Phe Lys Phe Val Lys Leu Ser Ser Ser
435 440 445
Val Ser Gly Ala Asp Tyr Tyr Leu Glu Ile Gly Phe Lys Ser Gly Ala
450 455 460
Gly Gln Leu Gln Pro Gly Lys Asp Thr Gly Glu Ile Gln Ile Arg Phe
465 470 475 480
Asn Lys Ser Asp Trp Ser Asn Tyr Asn Gln Gly Asn Asp Trp Ser Trp
485 490 495
Leu Gln Ser Met Thr Ser Tyr Gly Glu Asn Glu Lys Val Thr Ala Tyr
500 505 510
Ile Asp Gly Val Leu Val Trp Gly Gln Glu Pro Ser Gly Ala Thr Pro
515 520 525
Ala Pro Thr Met Thr Val Ala Pro Thr Ala Thr Pro Thr Pro Thr Leu
530 535 540
Ser Pro Thr Val Thr Pro Thr Pro Ala Pro Thr Gln Thr Ala Ile Pro
545 550 555 560
Thr Pro Thr Leu Thr Pro Asn Pro Thr Pro Thr Ser Ser Ile Pro Asp
565 570 575
Asp Thr Asn Asp Asp Trp Leu Tyr Val Ser Gly Asn Lys Ile Val Asp
580 585 590
Lys Asp Ala Arg Phe Val Tyr Leu Thr Ala Ile Asn Tyr Phe Ala Tyr
595 600 605
Asp Thr Ala Thr Asn Leu Phe Asn Gly Ala Trp Ser Cys Asn Leu Lys
610 615 620
Asp Thr Leu Ala Glu Ile Gly Asn Lys Gly Tyr Asn Ile Ile Arg Ala
625 630 635 640
Pro Leu Ser Ala Glu Ile Ile Ile Asn Tyr Ser Asn Gly Leu Tyr Pro
645 650 655
Lys Pro Asn Ile Asn Tyr Tyr Val Asn Pro Glu Leu Glu Gly Lys Asn
660 665 670
Ser Leu Glu Val Phe Asp Ile Val Val Gln Thr Cys Lys Glu Val Gly
675 680 685
Leu Lys Ile Met Leu Asp Ile His Ser Ile Lys Thr Asp Ala Met Ala
690 695 700
His Leu Tyr Phe Val Trp Trp Asp Glu Arg Phe Ser Pro Gln Asp Pro
705 710 715 720
Tyr Arg Ala Cys Gln Trp Ile Thr Asn Arg Tyr Lys Asn Asp Asp Thr
725 730 735
Ile Ile Ala Phe Asp Leu Lys Asn Glu Pro His Gly Lys Pro Trp Gln
740 745 750
Asp Thr Thr Phe Ala Lys Trp Asp Asn Ser Thr Asp Ile Asn Asn Trp
755 760 765
Lys Tyr Ala Ala Glu Thr Cys Ala Lys Arg Ile Leu Asn Ile Asn Pro
770 775 780
Asn Leu Leu Ile Val Leu Glu Ala Ile Glu Gly Tyr Phe Lys Asn Asp
785 790 795 800
Leu Thr Trp Ser Ser Lys Thr Thr Thr Asp Trp Tyr Thr Thr Trp Trp
805 810 815
Gly Gly Asn Leu Arg Gly Val Arg Lys Tyr Pro Ile Asn Leu Gly Lys
820 825 830
Tyr Gln Asn Lys Leu Val Trp Ser Phe His Phe Tyr Ala Pro Ser Leu
835 840 845
Tyr Glu Glu Pro Tyr Phe Trp Pro Gly Phe Thr Lys Glu Ser Leu Leu
850 855 860
Gln Asp Cys Trp Arg Pro Asn Trp Ala Tyr Ile Met Glu Glu Asn Ile
865 870 875 880
Ala Pro Leu Leu Ile Gly Glu Trp Gly Gly His Leu Asp Gly Ala Asp
885 890 895
Asn Glu Lys Trp Met Lys Tyr Leu Arg Asn Tyr Leu Leu Glu Asp His
900 905 910
Leu His His Ser Phe Tyr Cys Pro Asn Ala Asn Ser Gly Asp Thr Gly
915 920 925
Gly Leu Val Gly Tyr Asp Phe Thr Thr Trp Asp Glu Lys Lys Tyr Ser
930 935 940
Phe Leu Lys Pro Ala Leu Trp Gln Asp Ser Gln Gly Arg Phe Val Gly
945 950 955 960
Leu Asp His Lys Arg Pro Leu Gly Thr Asn Gly Lys Asn Ile Asn Ile
965 970 975
Thr Thr Tyr Tyr Asn Asn Asn Glu Pro Glu Pro Val Pro Ala Ser Lys
980 985 990
Leu Glu His His His His His His
995 1000
<210> 3
<211> 1088
<212> PRT
<213> 人工序列(Endo5-CBM28-Exo5Artificial Sequence)
<400> 3
Met Ala Asn Thr Ala Tyr Glu Lys Asp Lys Tyr Pro His Leu Ile Gly
1 5 10 15
Asn Ser Leu Val Lys Lys Pro Ser Val Gly Ala Arg Ile Gln Leu Leu
20 25 30
Lys Asn Gln Gly Arg Lys Ile Ile Ala Asp Gln Asn Gly Glu Pro Ile
35 40 45
Gln Leu Arg Ala Met Thr Ser His Ala Leu Asn Trp Tyr Pro Asn Ile
50 55 60
Leu Asn Gln Asn Ala Tyr Ala Gly Leu Ala Gln Asp Gln Gly Leu Asn
65 70 75 80
Val Ile Arg Ile Ala Met Tyr Leu Gly Gln Gly Gly Tyr Gly Thr Gln
85 90 95
Pro Asn Val Arg Asp Lys Val Ile Glu Gly Ile Lys Ile Ala Leu Gln
100 105 110
Gln Asp Met Trp Val Leu Val Glu Trp His Ala Leu Asn Pro Ala Asp
115 120 125
Phe Asn Gly Glu Leu Tyr Lys Gly Gly Lys Asn Phe Pro Lys Glu Leu
130 135 140
Ala Asn Lys Phe Phe Asn Gln Phe His Leu Leu Tyr Glu Leu Cys Asn
145 150 155 160
Gln Pro Asp Pro Ser Asp Phe Gly Leu Thr Gln Asp Glu Gly Gly Trp
165 170 175
Arg Lys Val Arg Ala Trp Ala Gln Pro Leu Ile Arg Met Ile Arg Asn
180 185 190
Met Ala Asn Gln Asn Leu Ile Ile Ile Gly Ser Pro Asn Trp Ser Gln
195 200 205
Arg Pro Asp Phe Ala Ile Lys Asp Pro Ile Ala Asp Asp Lys Val Met
210 215 220
Tyr Ser Val His Phe Tyr Thr Gly Thr His Lys Val Asp Gly Tyr Val
225 230 235 240
Phe Glu Asn Met Lys Met Ala Ile Glu Ala Gly Val Pro Val Phe Val
245 250 255
Thr Glu Trp Gly Thr Ser Glu Ala Ser Gly Asp Gly Gly Pro Tyr Leu
260 265 270
Asp Glu Ala Asp Lys Trp Leu Glu Tyr Leu Asn Ala Asn Asn Ile Ser
275 280 285
Trp Val Asn Trp Ser Leu Thr Asn Lys Asn Glu Thr Ser Gly Ala Phe
290 295 300
Val Pro Tyr Ile Ser Gly Val Ser Gln Ala Thr Asp Leu Asp Pro Gly
305 310 315 320
Ser Asp Gln Lys Trp Asp Ile Ser Glu Leu Ser Ile Ser Gly Glu Tyr
325 330 335
Val Arg Ser Arg Ile Lys Gly Ile Pro Tyr Gln Pro Ile Glu Arg Thr
340 345 350
Leu Lys Ile Ser Gln Asp Gln Val Ala Cys Ala Pro Ile Gly Gln Pro
355 360 365
Ile Leu Pro Ser Asp Phe Glu Asp Gly Thr Arg Gln Gly Trp Asp Trp
370 375 380
Asp Gly Pro Ser Gly Val Lys Gly Ala Leu Thr Ile Glu Glu Ala Asn
385 390 395 400
Gly Ser Asn Ala Leu Ser Trp Glu Val Glu Tyr Pro Glu Lys Lys Leu
405 410 415
Gln Asp Gly Trp Ala Ser Ala Pro Arg Leu Ile Leu Arg Asn Ile Asn
420 425 430
Thr Thr Arg Gly Asp Cys Lys Tyr Leu Cys Phe Asp Phe Tyr Leu Lys
435 440 445
Pro Lys Gln Ala Thr Lys Gly Glu Leu Ala Ile Phe Leu Ala Phe Ala
450 455 460
Pro Pro Ser Leu Asn Tyr Trp Ala Gln Ala Glu Asp Ser Phe Asn Ile
465 470 475 480
Asp Leu Thr Asn Leu Ser Thr Leu Lys Lys Thr Pro Asp Asp Leu Tyr
485 490 495
Ser Phe Lys Ile Ser Phe Asp Leu Asp Lys Ile Lys Glu Gly Lys Ile
500 505 510
Ile Gly Pro Asp Thr His Leu Arg Asp Ile Ile Ile Val Val Ala Asp
515 520 525
Val Asn Ser Asp Phe Lys Gly Arg Met Tyr Leu Asp Asn Val Arg Phe
530 535 540
Thr Glu Leu Phe Lys Ser Gly Ala Gly Gln Leu Gln Pro Gly Lys Asp
545 550 555 560
Thr Gly Glu Ile Gln Ile Arg Phe Asn Lys Ser Asp Trp Ser Asn Tyr
565 570 575
Asn Gln Gly Asn Asp Trp Ser Trp Leu Gln Ser Met Thr Ser Tyr Gly
580 585 590
Glu Asn Glu Lys Val Thr Ala Tyr Ile Asp Gly Val Leu Val Trp Gly
595 600 605
Gln Glu Pro Ser Gly Ala Thr Pro Ala Pro Thr Met Thr Val Ala Pro
610 615 620
Thr Ala Thr Pro Thr Pro Thr Leu Ser Pro Thr Val Thr Pro Thr Pro
625 630 635 640
Ala Pro Thr Gln Thr Ala Ile Pro Thr Pro Thr Leu Thr Pro Asn Pro
645 650 655
Thr Pro Thr Ser Ser Ile Pro Asp Asp Thr Asn Asp Asp Trp Leu Tyr
660 665 670
Val Ser Gly Asn Lys Ile Val Asp Lys Asp Ala Arg Phe Val Tyr Leu
675 680 685
Thr Ala Ile Asn Tyr Phe Ala Tyr Asp Thr Ala Thr Asn Leu Phe Asn
690 695 700
Gly Ala Trp Ser Cys Asn Leu Lys Asp Thr Leu Ala Glu Ile Gly Asn
705 710 715 720
Lys Gly Tyr Asn Ile Ile Arg Ala Pro Leu Ser Ala Glu Ile Ile Ile
725 730 735
Asn Tyr Ser Asn Gly Leu Tyr Pro Lys Pro Asn Ile Asn Tyr Tyr Val
740 745 750
Asn Pro Glu Leu Glu Gly Lys Asn Ser Leu Glu Val Phe Asp Ile Val
755 760 765
Val Gln Thr Cys Lys Glu Val Gly Leu Lys Ile Met Leu Asp Ile His
770 775 780
Ser Ile Lys Thr Asp Ala Met Ala His Leu Tyr Phe Val Trp Trp Asp
785 790 795 800
Glu Arg Phe Ser Pro Gln Asp Pro Tyr Arg Ala Cys Gln Trp Ile Thr
805 810 815
Asn Arg Tyr Lys Asn Asp Asp Thr Ile Ile Ala Phe Asp Leu Lys Asn
820 825 830
Glu Pro His Gly Lys Pro Trp Gln Asp Thr Thr Phe Ala Lys Trp Asp
835 840 845
Asn Ser Thr Asp Ile Asn Asn Trp Lys Tyr Ala Ala Glu Thr Cys Ala
850 855 860
Lys Arg Ile Leu Asn Ile Asn Pro Asn Leu Leu Ile Val Leu Glu Ala
865 870 875 880
Ile Glu Gly Tyr Phe Lys Asn Asp Leu Thr Trp Ser Ser Lys Thr Thr
885 890 895
Thr Asp Trp Tyr Thr Thr Trp Trp Gly Gly Asn Leu Arg Gly Val Arg
900 905 910
Lys Tyr Pro Ile Asn Leu Gly Lys Tyr Gln Asn Lys Leu Val Trp Ser
915 920 925
Phe His Phe Tyr Ala Pro Ser Leu Tyr Glu Glu Pro Tyr Phe Trp Pro
930 935 940
Gly Phe Thr Lys Glu Ser Leu Leu Gln Asp Cys Trp Arg Pro Asn Trp
945 950 955 960
Ala Tyr Ile Met Glu Glu Asn Ile Ala Pro Leu Leu Ile Gly Glu Trp
965 970 975
Gly Gly His Leu Asp Gly Ala Asp Asn Glu Lys Trp Met Lys Tyr Leu
980 985 990
Arg Asn Tyr Leu Leu Glu Asp His Leu His His Ser Phe Tyr Cys Pro
995 1000 1005
Asn Ala Asn Ser Gly Asp Thr Gly Gly Leu Val Gly Tyr Asp Phe Thr
1010 1015 1020
Thr Trp Asp Glu Lys Lys Tyr Ser Phe Leu Lys Pro Ala Leu Trp Gln
1025 1030 1035 1040
Asp Ser Gln Gly Arg Phe Val Gly Leu Asp His Lys Arg Pro Leu Gly
1045 1050 1055
Thr Asn Gly Lys Asn Ile Asn Ile Thr Thr Tyr Tyr Asn Asn Asn Glu
1060 1065 1070
Pro Glu Pro Val Pro Ala Ser Lys Leu Glu His His His His His His
1075 1080 1085
<210> 4
<211> 3663
<212> DNA
<213> 人工序列(endo5-2cbm-exo5Artificial Sequence)
<400> 4
atggctaata ctgcgtatga aaaggataag tatccacacc tgattggcaa cagcttggta 60
aaaaaacctt ctgtgggtgc tagaatccaa ctcctaaaaa atcagggaag aaaaattatt 120
gccgaccaaa atggagagcc tattcagctt cgtgcaatga cttctcacgc actgaattgg 180
tacccaaata tactcaacca gaatgcgtac gctggtcttg cacaggacca aggtcttaat 240
gtaattcgca ttgcaatgta tcttggacag ggtggatatg gaacacagcc taatgtaagg 300
gacaaggtca ttgaaggtat taaaattgca ctacaacagg acatgtgggt acttgtagag 360
tggcatgccc tcaatcccgc tgactttaat ggtgaactat ataaaggggg taaaaatttt 420
cctaaggagc ttgccaacaa atttttcaac cagtttcatc ttctttatga gttgtgtaat 480
caaccagatc caagtgactt tggtcttaca caagatgaag gtgggtggag gaaggtaagg 540
gcttgggctc agccactcat acgtatgatc cgtaatatgg ctaatcagaa cttaataatt 600
ataggttcgc ctaactggag ccaaagacca gattttgcga taaaagaccc aattgcagat 660
gacaaggtca tgtattctgt tcatttctat acaggtacac acaaggtaga tggatatgtg 720
tttgaaaata tgaagatggc aattgaagca ggcgttccag tatttgtaac agaatggggg 780
acaagtgagg caagcggtga tggtggacca tatcttgatg aggctgacaa atggcttgag 840
tacctcaacg caaacaatat aagctgggta aactggtcct tgacaaataa aaatgagact 900
tctggtgctt ttgtacccta cattagcggt gtgtctcaag caacagattt ggaccctggc 960
agcgaccaga aatgggatat ttcagaactt agcatctctg gtgaatatgt acgatcaaga 1020
ataaagggta ttccatatca gcctattgag agaacattga aaatttctca agatcaagtc 1080
gcgtgtgccc caattggtca acctattttg ccatctgatt ttgaagatgg tacacggcaa 1140
ggttgggact gggatggacc atctggtgtt aaaggtgctt taacaataga agaggcaaat 1200
ggttcaaatg cactttcatg ggaggttgag taccctgaaa aaaagcttca agatggttgg 1260
gcttctgctc caaggctaat tttgaggaat ataaatacaa caagaggtga ttgtaagtat 1320
ctctgctttg acttttacct aaaaccaaaa caggcaacaa aaggtgagct tgcaatcttc 1380
ctggcgtttg caccaccttc tcttaactat tgggcacagg cagaggatag ctttaatata 1440
gatttgacca atttatctac actgaagaaa acaccagatg acctttattc tttcaaaatt 1500
tcttttgatt tagataagat aaaagagggg aagattattg gacctgatac ccatctgcga 1560
gacataatca tagttgttgc agatgtaaac agtgatttta aaggtagaat gtacttagac 1620
aatgtaagat ttacagagct cggggtaaca acatcatctc cgacacctac acctacgccg 1680
acagtaacag taacaccaac tccaacaccg acaccgacac cgacagtaac agcgactcca 1740
acacctacac ctacacctgt tagcacacct gcgacaggtg ggcagataaa ggtactgtat 1800
gctaacaagg agacaaacag cacgacaaac acgataaggc catggttgaa ggtagtgaat 1860
agtggcagca gtagcataga tttgagcagg gtaacgataa ggtactggta cacggtagat 1920
ggtgaaaggg cacagagtgc ggtatcagac tgggcacaga taggagcaag caatgtaaca 1980
ttcaagtttg tgaagctgag cagtagtgta agtggagcgg attattactt ggagatagga 2040
tttaagagtg gagcagggca gttacagcct gggaaggaca caggagagat acagataagg 2100
tttaacaaga gtgactggag caattacaat caggggaatg actggtcatg gttacagagc 2160
atgacgagtt atggagagaa tgagaaggta acagcgtata tagatggagt gctggtatgg 2220
ggacaggagc cgagtggagc gacgcctgcg ccgacgatga cggttgcacc gacagcgacc 2280
ccgacaccaa ctctgagtcc gacggtaacg ccgacgccgg caccgacgca gacagcaata 2340
ccaacgccaa cattaactcc aaatccaacg ccaacatcga gtattcctga tgatacaaat 2400
gatgattggc tttatgtaag tggtaataaa atagttgata aagacgccag attcgtatac 2460
ttaacagcca ttaactactt tgcctatgac actgccacaa atctctttaa cggtgcctgg 2520
agttgtaatc tgaaagatac tcttgctgaa ataggcaata agggctacaa catcatcaga 2580
gccccactct ctgcagagat aataataaac tactcgaatg gtctatatcc aaaaccaaat 2640
ataaactact acgtaaatcc agagcttgaa ggcaaaaaca gcttggaagt atttgacata 2700
gtagtgcaaa catgtaaaga agttggtttg aaaattatgt tggatattca cagtataaaa 2760
acagacgcga tggcccatct atatttcgta tggtgggatg aacgttttag tccacaagat 2820
ccatatcgtg cgtgccagtg gattacaaat agatataaaa atgatgacac tattatagct 2880
tttgacctaa agaatgagcc acatggaaaa ccgtggcaag atacaacatt tgcaaaatgg 2940
gataattcaa cagatattaa taattggaaa tatgcagctg aaacatgtgc aaaacgtata 3000
ctgaatataa atccaaacct tctaattgta ctagaagcca ttgaaggtta tttcaagaat 3060
gacctaacat ggagttcaaa aactactact gactggtata ctacatggtg gggcggtaac 3120
ttgcgaggtg ttagaaagta tcctattaat ctgggtaaat atcaaaataa actagtatgg 3180
tcgttccatt tctacgcccc atctctatac gaagaaccgt atttttggcc agggttcaca 3240
aaagagtctt tactacaaga ttgttggcgt ccgaattggg cgtacattat ggaagaaaac 3300
attgcgccac ttctgatagg tgagtggggt ggtcatctgg atggagccga taacgaaaag 3360
tggatgaagt atttacgaaa ctatctacta gaagaccatc tacatcacag tttttattgc 3420
ccaaatgcta actcgggtga cactggagga ttggttggat atgattttac gacatgggat 3480
gagaaaaaat actcattttt gaaaccggct ctatggcaag acagtcaagg taggtttgtt 3540
ggattagatc acaaaagacc cttaggtaca aatgggaaaa acattaatat tacaacatat 3600
tacaacaata atgagcctga gccagttcca gcttcaaaac tcgagcacca ccaccaccac 3660
cac 3663
<210> 5
<211> 3000
<212> DNA
<213> 人工序列(endo5-cbm3b-exo5Artificial Sequence)
<400> 5
atggctaata ctgcgtatga aaaggataag tatccacacc tgattggcaa cagcttggta 60
aaaaaacctt ctgtgggtgc tagaatccaa ctcctaaaaa atcagggaag aaaaattatt 120
gccgaccaaa atggagagcc tattcagctt cgtgcaatga cttctcacgc actgaattgg 180
tacccaaata tactcaacca gaatgcgtac gctggtcttg cacaggacca aggtcttaat 240
gtaattcgca ttgcaatgta tcttggacag ggtggatatg gaacacagcc taatgtaagg 300
gacaaggtca ttgaaggtat taaaattgca ctacaacagg acatgtgggt acttgtagag 360
tggcatgccc tcaatcccgc tgactttaat ggtgaactat ataaaggggg taaaaatttt 420
cctaaggagc ttgccaacaa atttttcaac cagtttcatc ttctttatga gttgtgtaat 480
caaccagatc caagtgactt tggtcttaca caagatgaag gtgggtggag gaaggtaagg 540
gcttgggctc agccactcat acgtatgatc cgtaatatgg ctaatcagaa cttaataatt 600
ataggttcgc ctaactggag ccaaagacca gattttgcga taaaagaccc aattgcagat 660
gacaaggtca tgtattctgt tcatttctat acaggtacac acaaggtaga tggatatgtg 720
tttgaaaata tgaagatggc aattgaagca ggcgttccag tatttgtaac agaatggggg 780
acaagtgagg caagcggtga tggtggacca tatcttgatg aggctgacaa atggcttgag 840
tacctcaacg caaacaatat aagctgggta aactggtcct tgacaaataa aaatgagact 900
tctggtgctt ttgtacccta cattagcggt gtgtctcaag caacagattt ggaccctggc 960
agcgaccaga aagagctcgg ggtaacaaca tcatctccga cacctacacc tacgccgaca 1020
gtaacagtaa caccaactcc aacaccgaca ccgacaccga cagtaacagc gactccaaca 1080
cctacaccta cacctgttag cacacctgcg acaggtgggc agataaaggt actgtatgct 1140
aacaaggaga caaacagcac gacaaacacg ataaggccat ggttgaaggt agtgaatagt 1200
ggcagcagta gcatagattt gagcagggta acgataaggt actggtacac ggtagatggt 1260
gaaagggcac agagtgcggt atcagactgg gcacagatag gagcaagcaa tgtaacattc 1320
aagtttgtga agctgagcag tagtgtaagt ggagcggatt attacttgga gataggattt 1380
aagagtggag cagggcagtt acagcctggg aaggacacag gagagataca gataaggttt 1440
aacaagagtg actggagcaa ttacaatcag gggaatgact ggtcatggtt acagagcatg 1500
acgagttatg gagagaatga gaaggtaaca gcgtatatag atggagtgct ggtatgggga 1560
caggagccga gtggagcgac gcctgcgccg acgatgacgg ttgcaccgac agcgaccccg 1620
acaccaactc tgagtccgac ggtaacgccg acgccggcac cgacgcagac agcaatacca 1680
acgccaacat taactccaaa tccaacgcca acatcgagta ttcctgatga tacaaatgat 1740
gattggcttt atgtaagtgg taataaaata gttgataaag acgccagatt cgtatactta 1800
acagccatta actactttgc ctatgacact gccacaaatc tctttaacgg tgcctggagt 1860
tgtaatctga aagatactct tgctgaaata ggcaataagg gctacaacat catcagagcc 1920
ccactctctg cagagataat aataaactac tcgaatggtc tatatccaaa accaaatata 1980
aactactacg taaatccaga gcttgaaggc aaaaacagct tggaagtatt tgacatagta 2040
gtgcaaacat gtaaagaagt tggtttgaaa attatgttgg atattcacag tataaaaaca 2100
gacgcgatgg cccatctata tttcgtatgg tgggatgaac gttttagtcc acaagatcca 2160
tatcgtgcgt gccagtggat tacaaataga tataaaaatg atgacactat tatagctttt 2220
gacctaaaga atgagccaca tggaaaaccg tggcaagata caacatttgc aaaatgggat 2280
aattcaacag atattaataa ttggaaatat gcagctgaaa catgtgcaaa acgtatactg 2340
aatataaatc caaaccttct aattgtacta gaagccattg aaggttattt caagaatgac 2400
ctaacatgga gttcaaaaac tactactgac tggtatacta catggtgggg cggtaacttg 2460
cgaggtgtta gaaagtatcc tattaatctg ggtaaatatc aaaataaact agtatggtcg 2520
ttccatttct acgccccatc tctatacgaa gaaccgtatt tttggccagg gttcacaaaa 2580
gagtctttac tacaagattg ttggcgtccg aattgggcgt acattatgga agaaaacatt 2640
gcgccacttc tgataggtga gtggggtggt catctggatg gagccgataa cgaaaagtgg 2700
atgaagtatt tacgaaacta tctactagaa gaccatctac atcacagttt ttattgccca 2760
aatgctaact cgggtgacac tggaggattg gttggatatg attttacgac atgggatgag 2820
aaaaaatact catttttgaa accggctcta tggcaagaca gtcaaggtag gtttgttgga 2880
ttagatcaca aaagaccctt aggtacaaat gggaaaaaca ttaatattac aacatattac 2940
aacaataatg agcctgagcc agttccagct tcaaaactcg agcaccacca ccaccaccac 3000
<210> 6
<211> 3264
<212> DNA
<213> 人工序列(endo5-cbm28-exo5Artificial Sequence)
<400> 6
atggctaata ctgcgtatga aaaggataag tatccacacc tgattggcaa cagcttggta 60
aaaaaacctt ctgtgggtgc tagaatccaa ctcctaaaaa atcagggaag aaaaattatt 120
gccgaccaaa atggagagcc tattcagctt cgtgcaatga cttctcacgc actgaattgg 180
tacccaaata tactcaacca gaatgcgtac gctggtcttg cacaggacca aggtcttaat 240
gtaattcgca ttgcaatgta tcttggacag ggtggatatg gaacacagcc taatgtaagg 300
gacaaggtca ttgaaggtat taaaattgca ctacaacagg acatgtgggt acttgtagag 360
tggcatgccc tcaatcccgc tgactttaat ggtgaactat ataaaggggg taaaaatttt 420
cctaaggagc ttgccaacaa atttttcaac cagtttcatc ttctttatga gttgtgtaat 480
caaccagatc caagtgactt tggtcttaca caagatgaag gtgggtggag gaaggtaagg 540
gcttgggctc agccactcat acgtatgatc cgtaatatgg ctaatcagaa cttaataatt 600
ataggttcgc ctaactggag ccaaagacca gattttgcga taaaagaccc aattgcagat 660
gacaaggtca tgtattctgt tcatttctat acaggtacac acaaggtaga tggatatgtg 720
tttgaaaata tgaagatggc aattgaagca ggcgttccag tatttgtaac agaatggggg 780
acaagtgagg caagcggtga tggtggacca tatcttgatg aggctgacaa atggcttgag 840
tacctcaacg caaacaatat aagctgggta aactggtcct tgacaaataa aaatgagact 900
tctggtgctt ttgtacccta cattagcggt gtgtctcaag caacagattt ggaccctggc 960
agcgaccaga aatgggatat ttcagaactt agcatctctg gtgaatatgt acgatcaaga 1020
ataaagggta ttccatatca gcctattgag agaacattga aaatttctca agatcaagtc 1080
gcgtgtgccc caattggtca acctattttg ccatctgatt ttgaagatgg tacacggcaa 1140
ggttgggact gggatggacc atctggtgtt aaaggtgctt taacaataga agaggcaaat 1200
ggttcaaatg cactttcatg ggaggttgag taccctgaaa aaaagcttca agatggttgg 1260
gcttctgctc caaggctaat tttgaggaat ataaatacaa caagaggtga ttgtaagtat 1320
ctctgctttg acttttacct aaaaccaaaa caggcaacaa aaggtgagct tgcaatcttc 1380
ctggcgtttg caccaccttc tcttaactat tgggcacagg cagaggatag ctttaatata 1440
gatttgacca atttatctac actgaagaaa acaccagatg acctttattc tttcaaaatt 1500
tcttttgatt tagataagat aaaagagggg aagattattg gacctgatac ccatctgcga 1560
gacataatca tagttgttgc agatgtaaac agtgatttta aaggtagaat gtacttagac 1620
aatgtaagat ttacagagct ctttaagagt ggagcagggc agttacagcc tgggaaggac 1680
acaggagaga tacagataag gtttaacaag agtgactgga gcaattacaa tcaggggaat 1740
gactggtcat ggttacagag catgacgagt tatggagaga atgagaaggt aacagcgtat 1800
atagatggag tgctggtatg gggacaggag ccgagtggag cgacgcctgc gccgacgatg 1860
acggttgcac cgacagcgac cccgacacca actctgagtc cgacggtaac gccgacgccg 1920
gcaccgacgc agacagcaat accaacgcca acattaactc caaatccaac gccaacatcg 1980
agtattcctg atgatacaaa tgatgattgg ctttatgtaa gtggtaataa aatagttgat 2040
aaagacgcca gattcgtata cttaacagcc attaactact ttgcctatga cactgccaca 2100
aatctcttta acggtgcctg gagttgtaat ctgaaagata ctcttgctga aataggcaat 2160
aagggctaca acatcatcag agccccactc tctgcagaga taataataaa ctactcgaat 2220
ggtctatatc caaaaccaaa tataaactac tacgtaaatc cagagcttga aggcaaaaac 2280
agcttggaag tatttgacat agtagtgcaa acatgtaaag aagttggttt gaaaattatg 2340
ttggatattc acagtataaa aacagacgcg atggcccatc tatatttcgt atggtgggat 2400
gaacgtttta gtccacaaga tccatatcgt gcgtgccagt ggattacaaa tagatataaa 2460
aatgatgaca ctattatagc ttttgaccta aagaatgagc cacatggaaa accgtggcaa 2520
gatacaacat ttgcaaaatg ggataattca acagatatta ataattggaa atatgcagct 2580
gaaacatgtg caaaacgtat actgaatata aatccaaacc ttctaattgt actagaagcc 2640
attgaaggtt atttcaagaa tgacctaaca tggagttcaa aaactactac tgactggtat 2700
actacatggt ggggcggtaa cttgcgaggt gttagaaagt atcctattaa tctgggtaaa 2760
tatcaaaata aactagtatg gtcgttccat ttctacgccc catctctata cgaagaaccg 2820
tatttttggc cagggttcac aaaagagtct ttactacaag attgttggcg tccgaattgg 2880
gcgtacatta tggaagaaaa cattgcgcca cttctgatag gtgagtgggg tggtcatctg 2940
gatggagccg ataacgaaaa gtggatgaag tatttacgaa actatctact agaagaccat 3000
ctacatcaca gtttttattg cccaaatgct aactcgggtg acactggagg attggttgga 3060
tatgatttta cgacatggga tgagaaaaaa tactcatttt tgaaaccggc tctatggcaa 3120
gacagtcaag gtaggtttgt tggattagat cacaaaagac ccttaggtac aaatgggaaa 3180
aacattaata ttacaacata ttacaacaat aatgagcctg agccagttcc agcttcaaaa 3240
ctcgagcacc accaccacca ccac 3264
<210> 7
<211> 1629
<212> DNA
<213> 人工序列(endo5-cbm28Artificial Sequence)
<400> 7
aatactgcgt atgaaaagga taagtatcca cacctgattg gcaacagctt ggtaaaaaaa 60
ccttctgtgg gtgctagaat ccaactccta aaaaatcagg gaagaaaaat tattgccgac 120
caaaatggag agcctattca gcttcgtgca atgacttctc acgcactgaa ttggtaccca 180
aatatactca accagaatgc gtacgctggt cttgcacagg accaaggtct taatgtaatt 240
cgcattgcaa tgtatcttgg acagggtgga tatggaacac agcctaatgt aagggacaag 300
gtcattgaag gtattaaaat tgcactacaa caggacatgt gggtacttgt agagtggcat 360
gccctcaatc ccgctgactt taatggtgaa ctatataaag ggggtaaaaa ttttcctaag 420
gagcttgcca acaaattttt caaccagttt catcttcttt atgagttgtg taatcaacca 480
gatccaagtg actttggtct tacacaagat gaaggtgggt ggaggaaggt aagggcttgg 540
gctcagccac tcatacgtat gatccgtaat atggctaatc agaacttaat aattataggt 600
tcgcctaact ggagccaaag accagatttt gcgataaaag acccaattgc agatgacaag 660
gtcatgtatt ctgttcattt ctatacaggt acacacaagg tagatggata tgtgtttgaa 720
aatatgaaga tggcaattga agcaggcgtt ccagtatttg taacagaatg ggggacaagt 780
gaggcaagcg gtgatggtgg accatatctt gatgaggctg acaaatggct tgagtacctc 840
aacgcaaaca atataagctg ggtaaactgg tccttgacaa ataaaaatga gacttctggt 900
gcttttgtac cctacattag cggtgtgtct caagcaacag atttggaccc tggcagcgac 960
cagaaatggg atatttcaga acttagcatc tctggtgaat atgtacgatc aagaataaag 1020
ggtattccat atcagcctat tgagagaaca ttgaaaattt ctcaagatca agtcgcgtgt 1080
gccccaattg gtcaacctat tttgccatct gattttgaag atggtacacg gcaaggttgg 1140
gactgggatg gaccatctgg tgttaaaggt gctttaacaa tagaagaggc aaatggttca 1200
aatgcacttt catgggaggt tgagtaccct gaaaaaaagc ttcaagatgg ttgggcttct 1260
gctccaaggc taattttgag gaatataaat acaacaagag gtgattgtaa gtatctctgc 1320
tttgactttt acctaaaacc aaaacaggca acaaaaggtg agcttgcaat cttcctggcg 1380
tttgcaccac cttctcttaa ctattgggca caggcagagg atagctttaa tatagatttg 1440
accaatttat ctacactgaa gaaaacacca gatgaccttt attctttcaa aatttctttt 1500
gatttagata agataaaaga ggggaagatt attggacctg atacccatct gcgagacata 1560
atcatagttg ttgcagatgt aaacagtgat tttaaaggta gaatgtactt agacaatgta 1620
agatttaca 1629
<210> 8
<211> 966
<212> DNA
<213> 人工序列(endo5Artificial Sequence)
<400> 8
aatactgcgt atgaaaagga taagtatcca cacctgattg gcaacagctt ggtaaaaaaa 60
ccttctgtgg gtgctagaat ccaactccta aaaaatcagg gaagaaaaat tattgccgac 120
caaaatggag agcctattca gcttcgtgca atgacttctc acgcactgaa ttggtaccca 180
aatatactca accagaatgc gtacgctggt cttgcacagg accaaggtct taatgtaatt 240
cgcattgcaa tgtatcttgg acagggtgga tatggaacac agcctaatgt aagggacaag 300
gtcattgaag gtattaaaat tgcactacaa caggacatgt gggtacttgt agagtggcat 360
gccctcaatc ccgctgactt taatggtgaa ctatataaag ggggtaaaaa ttttcctaag 420
gagcttgcca acaaattttt caaccagttt catcttcttt atgagttgtg taatcaacca 480
gatccaagtg actttggtct tacacaagat gaaggtgggt ggaggaaggt aagggcttgg 540
gctcagccac tcatacgtat gatccgtaat atggctaatc agaacttaat aattataggt 600
tcgcctaact ggagccaaag accagatttt gcgataaaag acccaattgc agatgacaag 660
gtcatgtatt ctgttcattt ctatacaggt acacacaagg tagatggata tgtgtttgaa 720
aatatgaaga tggcaattga agcaggcgtt ccagtatttg taacagaatg ggggacaagt 780
gaggcaagcg gtgatggtgg accatatctt gatgaggctg acaaatggct tgagtacctc 840
aacgcaaaca atataagctg ggtaaactgg tccttgacaa ataaaaatga gacttctggt 900
gcttttgtac cctacattag cggtgtgtct caagcaacag atttggaccc tggcagcgac 960
cagaaa 966
<210> 9
<211> 1998
<212> DNA
<213> 人工序列(cbm3b-exo5Artificial Sequence)
<400> 9
ggggtaacaa catcatctcc gacacctaca cctacgccga cagtaacagt aacaccaact 60
ccaacaccga caccgacacc gacagtaaca gcgactccaa cacctacacc tacacctgtt 120
agcacacctg cgacaggtgg gcagataaag gtactgtatg ctaacaagga gacaaacagc 180
acgacaaaca cgataaggcc atggttgaag gtagtgaata gtggcagcag tagcatagat 240
ttgagcaggg taacgataag gtactggtac acggtagatg gtgaaagggc acagagtgcg 300
gtatcagact gggcacagat aggagcaagc aatgtaacat tcaagtttgt gaagctgagc 360
agtagtgtaa gtggagcgga ttattacttg gagataggat ttaagagtgg agcagggcag 420
ttacagcctg ggaaggacac aggagagata cagataaggt ttaacaagag tgactggagc 480
aattacaatc aggggaatga ctggtcatgg ttacagagca tgacgagtta tggagagaat 540
gagaaggtaa cagcgtatat agatggagtg ctggtatggg gacaggagcc gagtggagcg 600
acgcctgcgc cgacgatgac ggttgcaccg acagcgaccc cgacaccaac tctgagtccg 660
acggtaacgc cgacgccggc accgacgcag acagcaatac caacgccaac attaactcca 720
aatccaacgc caacatcgag tattcctgat gatacaaatg atgattggct ttatgtaagt 780
ggtaataaaa tagttgataa agacgccaga ttcgtatact taacagccat taactacttt 840
gcctatgaca ctgccacaaa tctctttaac ggtgcctgga gttgtaatct gaaagatact 900
cttgctgaaa taggcaataa gggctacaac atcatcagag ccccactctc tgcagagata 960
ataataaact actcgaatgg tctatatcca aaaccaaata taaactacta cgtaaatcca 1020
gagcttgaag gcaaaaacag cttggaagta tttgacatag tagtgcaaac atgtaaagaa 1080
gttggtttga aaattatgtt ggatattcac agtataaaaa cagacgcgat ggcccatcta 1140
tatttcgtat ggtgggatga acgttttagt ccacaagatc catatcgtgc gtgccagtgg 1200
attacaaata gatataaaaa tgatgacact attatagctt ttgacctaaa gaatgagcca 1260
catggaaaac cgtggcaaga tacaacattt gcaaaatggg ataattcaac agatattaat 1320
aattggaaat atgcagctga aacatgtgca aaacgtatac tgaatataaa tccaaacctt 1380
ctaattgtac tagaagccat tgaaggttat ttcaagaatg acctaacatg gagttcaaaa 1440
actactactg actggtatac tacatggtgg ggcggtaact tgcgaggtgt tagaaagtat 1500
cctattaatc tgggtaaata tcaaaataaa ctagtatggt cgttccattt ctacgcccca 1560
tctctatacg aagaaccgta tttttggcca gggttcacaa aagagtcttt actacaagat 1620
tgttggcgtc cgaattgggc gtacattatg gaagaaaaca ttgcgccact tctgataggt 1680
gagtggggtg gtcatctgga tggagccgat aacgaaaagt ggatgaagta tttacgaaac 1740
tatctactag aagaccatct acatcacagt ttttattgcc caaatgctaa ctcgggtgac 1800
actggaggat tggttggata tgattttacg acatgggatg agaaaaaata ctcatttttg 1860
aaaccggctc tatggcaaga cagtcaaggt aggtttgttg gattagatca caaaagaccc 1920
ttaggtacaa atgggaaaaa cattaatatt acaacatatt acaacaataa tgagcctgag 1980
ccagttccag cttcaaaa 1998
<210> 10
<211> 1599
<212> DNA
<213> 人工序列(exo5Artificial Sequence)
<400> 10
tttaagagtg gagcagggca gttacagcct gggaaggaca caggagagat acagataagg 60
tttaacaaga gtgactggag caattacaat caggggaatg actggtcatg gttacagagc 120
atgacgagtt atggagagaa tgagaaggta acagcgtata tagatggagt gctggtatgg 180
ggacaggagc cgagtggagc gacgcctgcg ccgacgatga cggttgcacc gacagcgacc 240
ccgacaccaa ctctgagtcc gacggtaacg ccgacgccgg caccgacgca gacagcaata 300
ccaacgccaa cattaactcc aaatccaacg ccaacatcga gtattcctga tgatacaaat 360
gatgattggc tttatgtaag tggtaataaa atagttgata aagacgccag attcgtatac 420
ttaacagcca ttaactactt tgcctatgac actgccacaa atctctttaa cggtgcctgg 480
agttgtaatc tgaaagatac tcttgctgaa ataggcaata agggctacaa catcatcaga 540
gccccactct ctgcagagat aataataaac tactcgaatg gtctatatcc aaaaccaaat 600
ataaactact acgtaaatcc agagcttgaa ggcaaaaaca gcttggaagt atttgacata 660
gtagtgcaaa catgtaaaga agttggtttg aaaattatgt tggatattca cagtataaaa 720
acagacgcga tggcccatct atatttcgta tggtgggatg aacgttttag tccacaagat 780
ccatatcgtg cgtgccagtg gattacaaat agatataaaa atgatgacac tattatagct 840
tttgacctaa agaatgagcc acatggaaaa ccgtggcaag atacaacatt tgcaaaatgg 900
gataattcaa cagatattaa taattggaaa tatgcagctg aaacatgtgc aaaacgtata 960
ctgaatataa atccaaacct tctaattgta ctagaagcca ttgaaggtta tttcaagaat 1020
gacctaacat ggagttcaaa aactactact gactggtata ctacatggtg gggcggtaac 1080
ttgcgaggtg ttagaaagta tcctattaat ctgggtaaat atcaaaataa actagtatgg 1140
tcgttccatt tctacgcccc atctctatac gaagaaccgt atttttggcc agggttcaca 1200
aaagagtctt tactacaaga ttgttggcgt ccgaattggg cgtacattat ggaagaaaac 1260
attgcgccac ttctgatagg tgagtggggt ggtcatctgg atggagccga taacgaaaag 1320
tggatgaagt atttacgaaa ctatctacta gaagaccatc tacatcacag tttttattgc 1380
ccaaatgcta actcgggtga cactggagga ttggttggat atgattttac gacatgggat 1440
gagaaaaaat actcattttt gaaaccggct ctatggcaag acagtcaagg taggtttgtt 1500
ggattagatc acaaaagacc cttaggtaca aatgggaaaa acattaatat tacaacatat 1560
tacaacaata atgagcctga gccagttcca gcttcaaaa 1599
<210> 11
<211> 36
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
aacgatccat ggctaatact gcgtatgaaa aggata 36
<210> 12
<211> 34
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
aacgatgagc tctgtaaatc ttacattgtc taag 34
<210> 13
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
aacgatgagc tctttctggt cgctgccagg 30
<210> 14
<211> 31
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
aacgatgagc tcggggtaac aacatcatct c 31
<210> 15
<211> 33
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
aacgatctcg agttttgaag ctggaactgg ctc 33
<210> 16
<211> 32
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
aacgatgagc tctttaagag tggagcaggg ca 32
Claims (10)
1.酸性耐高温重组纤维素酶,其特征在于,所述酸性耐高温重组纤维素酶包括三种酸性耐高温重组纤维素酶,其氨基酸序列分别如SEQ ID NO.1-3所示。
2.酸性耐高温重组纤维素酶,其特征在于,所述酸性耐高温重组纤维素酶包括三种酸性耐高温重组纤维素酶,其DNA序列分别如SEQIDNO.4-6所示。
3.根据权利要求2所述的酸性耐高温重组纤维素酶,其特征在于,所述DNA序列SEQ IDNO.4-6,分别通过下列引物SEQ ID NO.11-16进行扩增后拼接获得:
Endo-F:5’-AACGATCCATGGCTAATACTGCGTATGAAAAGGATA-3’;
Endocbm-R:5’-AACGATGAGCTCTGTAAATCTTACATTGTCTAAG-3’;
Endo-R:5’-AACGATGAGCTCTTTCTGGTCGCTGCCAGG-3’;
cbmExo-F:5’-AACGATGAGCTCGGGGTAACAACATCATCTC-3’;
Exo-R:5’-AACGATCTCGAGTTTTGAAGCTGGAACTGGCTC-3’;
Exo-F:5’-AACGATGAGCTCTTTAAGAGTGGAGCAGGGCA-3’。
4.一种重组载体,其特征在于,所述重组载体克隆包括SEQ ID NO.4-6中所任意一种DNA序列。
5.一种重组菌,其特征在于,所述重组菌包含有权利要求3中的重组载体。
6.酸性耐高温重组纤维素酶在酶解CMC-Na、滤纸、Avicel、和天然木质纤维素中的应用。
7.根据权利要求6所述的应用,其特征在于,所述天然木质纤维素包括水稻秸秆和小麦秸秆。
8.根据权利要求6所述的应用,其特征在于,所述酶解反应温度优选为60~80℃,pH为4.0~4.5,反应时间为4h。
9.权利要求4所述的重组载体在酶解CMC-Na、滤纸、Avicel、和天然木质纤维素中的应用。
10.权利要求5所述的重组菌在酶解CMC-Na、滤纸、Avicel、和天然木质纤维素中的应用。
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CN102864160A (zh) * | 2012-09-11 | 2013-01-09 | 南京林业大学 | 一种木聚糖酶基因及表达蛋白和应用 |
CN102864161A (zh) * | 2012-09-11 | 2013-01-09 | 南京林业大学 | 一种极耐热木聚糖酶基因及其表达蛋白与应用 |
CN104178472A (zh) * | 2013-05-28 | 2014-12-03 | 中国科学院青岛生物能源与过程研究所 | 降解纤维素的酶及其构建和应用 |
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CN102864160A (zh) * | 2012-09-11 | 2013-01-09 | 南京林业大学 | 一种木聚糖酶基因及表达蛋白和应用 |
CN102864161A (zh) * | 2012-09-11 | 2013-01-09 | 南京林业大学 | 一种极耐热木聚糖酶基因及其表达蛋白与应用 |
CN104178472A (zh) * | 2013-05-28 | 2014-12-03 | 中国科学院青岛生物能源与过程研究所 | 降解纤维素的酶及其构建和应用 |
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CN112501148A (zh) * | 2020-12-14 | 2021-03-16 | 黑龙江中医药大学 | 一种纤维素酶及其应用 |
CN112501148B (zh) * | 2020-12-14 | 2021-09-14 | 黑龙江中医药大学 | 一种纤维素酶及其应用 |
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