CN113980939A - 一种耐葡萄糖的β-葡萄糖苷酶及其表达基因和应用 - Google Patents
一种耐葡萄糖的β-葡萄糖苷酶及其表达基因和应用 Download PDFInfo
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Abstract
本发明涉及生物技术领域，尤其涉及一种耐葡萄糖的β‑葡萄糖苷酶及其表达基因和应用。本发明从红树林土壤中筛选到以葡萄糖为底物时可产β‑葡萄糖苷酶的聚多曲霉L1，通过比较转录组的方法筛选聚多曲霉L1分别以葡萄糖、微晶纤维素和纤维二糖为唯一碳源诱导时表达量相近的基因bgAs1，通过毕赤酵母表达系统进行体外表达，纯化后获得重组β‑葡萄糖苷酶BgAs1。该β‑葡萄糖苷酶具有较高的葡萄糖耐受性、盐耐受性和宽泛的底物特异性，在协同纤维素酶水解甘蔗渣上BgAs1表现出替代商业化酶Nov188的潜力，具有良好的工业应用前景。
Description
技术领域
本发明涉及生物技术领域，尤其涉及一种耐葡萄糖的β-葡萄糖苷酶及其表达基因和应用。
背景技术
纤维素作为现在地球上含量丰富的可再生能源，具有很大的开发潜力。纤维素是由β-1,4糖苷键连接葡萄糖苷而成的大分子，其作为植物多糖最主要成分，在植物结构中起到了不可或缺的作用，与半纤维素和木质素三者相互交联构成了细胞壁的主要骨架。我国是传统的农业大国，秸秆资源十分丰富，但利用率极其低下，在农业实践中常被废弃或焚烧，从而造成环境的污染。畜牧业的疾速发展增加了粮食的压力，使我国面临着人畜争粮的局面，而利用秸秆资源转换为饲料或促进饲料中纤维素等养分的吸收可以缓解这一问题。但是，秸秆及畜禽日粮中均含有纤维素等难被畜禽消化吸收物质，所以，该怎样快速高效降解纤维素成为了关键性问题，纤维素降解酶因为其降解纤维素的能力逐渐成为人们研究的热点。
纤维素生物质转化为葡萄糖由外切葡聚糖酶，内切葡聚糖酶和β-葡萄糖苷酶起协同作用以水解纤维素的β-1,4键。β-葡萄糖苷酶可以在水解过程中有效地水解纤维二糖等寡糖，从而消除了这些寡糖对上游纤维素酶的产物抑制作用，因此β-葡萄糖苷酶被认为是纤维素降解过程的关键限速酶，它决定了其余过程的性能。该转化纤维素生物质的生物转化也是生产第二代(2G)乙醇和其他化学品的绿色方式，并且它不与用于第一代生物乙醇生产的人类食物资源竞争，从而结束了有争议的粮食与燃料和使用耕地的问题。
β-葡萄糖苷酶(β-glucosidase，EC3.2.1.21)，可以催化水解β-D-葡萄糖苷键生成葡萄糖。经研究发现在一些霉菌如曲霉、木霉、青霉等，具有较高的β-葡萄糖苷酶活性。红树林分布于热带和亚热带海岸的潮间带上部，也是只生长在海滩上的森林类型，是具有重要生态意义的海岸环境。有调查表明，红树林生物资源十分丰富，包括真菌，细菌，藻类等。海南岛红树林生态面积占比达到了全国的17.9％，东寨港红树林自然保护区位于中国海南省海口市，该红树林区内有丰厚的植物凋落物，而这些植物凋落物的水解需要由土壤中的微生物产生降解纤维素的酶类参与。本发明以红树林腐殖层土壤筛选到的聚多曲霉为研究对象，其不需要特定碳源诱导产酶的特殊性质，具有较大的研究价值。
葡萄糖耐受性是β-葡萄糖苷酶的一个关键性质，具有葡萄糖高耐受性的β-葡萄糖苷酶因其独特的优势而日益在饲料工业、食品工业、生物合成以及生物燃料等领域显现出越来越重要的独特优势。在纤维素乙醇行业，具有高耐受性的β-葡萄糖苷酶也可以减弱产物葡萄糖对自身酶活的抑制。所以筛选或者改造得到高葡萄糖耐受性的β-葡萄糖苷酶对该酶的工业化应用具有重要意义。
发明内容
有鉴于此，本发明提供了一种耐葡萄糖的β-葡萄糖苷酶及其表达基因和应用，该β-葡萄糖苷酶具有较高的葡萄糖耐受性，在饲料工业、食品工业、生物合成以及生物燃料等领域具有重要的独特优势。
为了实现上述发明目的，本发明提供以下技术方案：
一种耐葡萄糖的β-葡萄糖苷酶，其具有：
(I)、如SEQ ID No.2所示的氨基酸序列；或
(II)、在SEQ ID NO:2所示的氨基酸序列中取代、缺失和/或添加一个或多个核苷酸且与(I)所述的氨基酸序列功能相同的氨基酸序列；或
(III)、与如(I)或(II)所述的氨基酸序列具有90％以上同一性的氨基酸序列。
一些实施方案中，本发明还提供了所述β-葡萄糖苷酶的制备方法，包括如下步骤：
1)、将编码所述β-葡萄糖苷酶的核酸克隆至表达载体，获得重组载体；
2)、将所述重组载体转化宿主菌，诱导表达，利用阴离子交换层析柱和镍柱纯化，获得β-葡萄糖苷酶。
本发明还提供了编码所述的β-葡萄糖苷酶的BgAs1基因。
本发明利用RNA-Seq首次从红树林源聚多曲霉L1成功克隆到差异表达的基因BgAs1，将其异源表达后鉴定为耐葡萄糖β-葡萄糖苷酶。
BgAs1基因具有I)～III)中任意一种核苷酸序列：
I)、如SEQ ID No.1所示的核苷酸序列；或
Ⅱ)、在SEQ ID NO:1所示的核苷酸序列中取代、缺失和/或添加一个或多个核苷酸且表达SEQ ID NO:2所示蛋白的核苷酸序列；或
III)、与SEQ ID NO:1所示序列互补的核苷酸序列。
本发明还提供了扩增BgAs1基因的上、下游引物bgAs1-F/bgAs1-R：
bgAs1-F：agagaggctgaagctgaattcATGACTCGGACGTTCGATATAG(SEQ ID NO:3)
bgAs1-R：gagatgagtttttgttctagaCTAAACGCCTCGCCAATACCG(SEQ ID NO:4)
其中，上下游引物的5’端引入线性化pPICZαA载体两末端同源序列，见横线所示的序列。
本发明还提供了含有所述BgAs1基因的表达模块、重组载体和宿主菌。一些实施方案中，所述重组载体的骨架为pPICZαA，导入BgAs1基因获得的重组载体命名为pPICZαA-bgAs1。一些实施方案中，所述宿主菌包括大肠杆菌、毕赤酵母。
本发明还提供了所述的β-葡萄糖苷酶、所述BgAs1基因、所述的生物材料协同纤维素酶在水解木质纤维素制备还原糖和/葡萄糖中的应用。
本发明还提供了所述的β-葡萄糖苷酶、所述BgAs1基因、所述的生物材料在商业纤维素酶利用和制备生物乙醇中的应用。
本发明还提供一种制备还原糖和/或葡萄糖的方法，以纤维素原料为底物，利用纤维素酶和本发明所述的β-葡萄糖苷酶进行酶解，获得还原糖和/或葡萄糖。
所述纤维素原料包括甘蔗渣、秸秆、糟渣、海藻中的至少一种，包括但不仅限于此。
本发明提供了β-葡萄糖苷酶及其表达基因和应用。该β-葡萄糖苷酶由聚多曲霉L1分泌，经鉴定属于GH3家族β-葡萄糖苷酶，其最适反应温度为40℃，最适pH为6，具有宽泛的底物特异性，对纤维二糖至五糖，槐糖，龙胆二糖和昆布二糖都具有一定的水解能力，尤其对大豆苷和昆布多糖具有较强的水解能力；对葡萄糖的半抑制浓度IC50大于3M，且在0-0.5M时对酶活明显有促进作用，0.4M时酶活达1.27倍；0-2.5M NaCl对BgAs1酶活均有促进作用，1M NaCl时酶活达1.35倍。在协同纤维素酶水解甘蔗渣上BgAs1表现出替代商业化酶Nov188的潜力。
附图说明
图1示BgAs1的蛋白结构与分析图；
图2示聚多曲霉L1总RNA琼脂糖凝胶电泳结果；
图3示bgAs1基因PCR产物的琼脂糖凝胶电泳；M.5000Marker，泳道1：bgAs1基因；
图4示质粒pPICZαA双酶切后琼脂糖凝胶电泳；M5000Marker，泳道1：pPICZαA，泳道2：线性化pPICZαA；
图5示重组产物转化感受态细胞过夜培养后的平板；
图6示重组质粒菌落PCR产物琼脂糖凝胶电泳；M 5000Marker；泳道1-5：bgAs1产物；
图7示酵母转化子在YPDS平板上的生长情况；
图8示Zeocin抗性梯度筛选酵母转化子；
图9示酵母转化子菌落PCR产物验证结果；M 5000Marker；1-5bgAs1产物(其中2为假阳性产物)；
图10示柱层析纯化BgAs1图谱；
图11示BgAs1的SDS-PAGE分析；M:170Kda protein Marker；泳道1：BgAs1培养基上清；泳道2：纯化的BgAs1
图12示pNP浓度标准曲线；
图13示DNS法葡萄糖标准曲线；
图14示GOD法葡萄糖标准曲线；
图15示BCA法蛋白定量标准曲线；
图16示反应pH对酶活的影响；
图17示反应温度对酶活的影响；
图18示BgAs1酶的热稳定性检测结果；
图19示BgAs1的盐耐受性检测结果；
图20示BgAs1的葡萄糖耐受性检测结果；
图21示BgAs1协同商业化纤维素酶糖化效果；(a)DNS法测定还原糖含量；(b)葡萄糖氧化酶试剂盒测定葡萄糖含量。
具体实施方式
本发明提供了一种耐葡萄糖β-葡萄糖苷酶及其表达基因和应用。本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。
本发明采用的试材皆为普通市售品，皆可于市场购得。
下面结合实施例，进一步阐述本发明：
实施例1
(一)实验方案
1.聚多曲霉菌bgl基因克隆及序列分析
1.1聚多曲霉RNA-seq测序
将聚多曲霉接种在发酵培养基中培养5天提取样品进行RNA-seq。样品名称分别标记如表，所送样品进行三次独立重复制备。
表1
1.2聚多曲霉bgAs1基因克隆及序列分析
对RNA-seq后的结果进行分析，按照基因上调表达且log2(FC)＞2.0的条件对glycoside hydrolase家族基因进行筛选，并分析筛得基因的序列情况，另经RT-PCR及Q-PCR验证其基因表达情况。
1.2.1bgl基因的引物设计
对bgAs1的核苷酸序列进行分析，在bgAs1正反向扩增引物的5’端引入线性化pPICZαA载体两末端同源序列。
表2
1.2.2聚多曲霉菌株总RNA提取与反转录
采用试剂盒来提取前期发酵得到的聚多曲霉菌株的总RNA(OMEGA公司，型号R6840-01)，具体步骤如下：
(1)使用以MCC为碳源，以尿素，Yeast extract，(NH4)2SO4为三种氮源的发酵培养基培养6d之后，用1.5mL Centrifuge tube收集菌球；将离心机的转速设置为12,000g，离心10min来收集(≤100mg)，迅速将离心得到的培养基倒进垃圾杯并将离心管置于液氮中防止RNase酶裂解RNA，在提前清理好的无酶环境中取出菌球并使用液氮研磨，提前在1.5mLCentrifuge tube中加入500μL(或600μL，根据研磨菌体的多少判断混合液的加入量)Buffer RB/β-Mercaptoethanol混合液，将研磨好的菌体马上加入混合液中，并使用漩涡振荡器充分裂解菌体；
(2)在混合液中加入500μL Phenol water和100μL2M醋酸钠(pH4.0)，使用高速漩涡振荡器均匀介质15s；
(3)在上一步的混合液中再加入200μL Trichloromethane，使用漩涡振荡器漩涡混匀15s，置于冰上10min；
(4)将1.5mL Centrifuge tube从冰盒中取出，将离心机的转速设置为12,000g，温度设为4℃，离心15min，注意移动Centrifuge tube的时候不要晃动，此刻已能看见混合液出现分层；
(5)将离心完的1.5mL Centrifuge tube取出，同时注意不要晃动，将600μL上清液转移到新的RNase-free 1.5mL Centrifuge tube中，加入600μL事先配制好的70％乙醇并用移液枪缓慢吹打混匀；
(6)把2.0mL收集管放在无酶处理过的1.5mL离心管架上，再将RNA Bindingcolumn套在2.0mL收集管上，将步骤(5)得到的1.2mL混合液轻柔加入到Binding column中，将离心机的转速设置为10000g，温度调整至室温，离心30s以结合DNA上柱，离心完成后将收集管中的滤液倒进垃圾杯并重复使用收集管；
(7)在Binding column中加入500μL RNA Wash BufferⅠ，离心机的转速设置为10,000g，温度至室温，离心30s，将收集管中的滤液倒进垃圾杯并重复使用收集管；
(8)在Binding column中加入700μL试剂盒中已用无水乙醇稀释好的RNA WashBufferⅡ至Binding column底部中央，将离心机的转速设置为10,000g，温度至室温，离心30s，将收集管中的滤液倒进垃圾杯并重复使用收集管；
(9)再次在Binding column中加入500μL RNA Wash BufferⅡ并重复步骤(8)操作；
(10)换新的收集管并将Binding column套上，将离心机的转速设置为13,000g，温度为室温，空柱离心1min以甩干Binding column中的的基质；
(11)最后将Binding column套在RNase-free处理过的1.5mL Centrifuge tube上，加入50μL DEPC-treated water至Binding column底部膜的中央，先静置1-2min，然后离心机的转速设置为13,000g，温度为室温，离心1min；
(12)琼脂糖凝胶电泳(AGE)检测RNA产物质量和浓度：配制1％琼脂糖凝胶，100V进行电泳检测提取的RNA的质量，确定样品质量较好，浓度较高时将样品分装于RNase-freeCentrifuge tube于-80℃冰箱保存。
采用反转录试剂盒(诺唯赞公司，型号R312-01/02)对提取的总RNA进行反转录，反应体系如下：
表3
用移液枪加上述试剂于RNase-free PCR管中，轻轻混匀后放置在PCR仪中65℃加热5min，反应完成后迅速置于冰上降温，静置2min。
表4
再次加入上述试剂以取出gDNA，吹打混匀后，于PCR仪中42℃反应2min。
表5
用移液枪轻轻吹打混匀，进行表6的操作。
表6
| 温度 | 时间 |
| 37℃ | 45min |
| 85℃ | 5sec |
PCR反应结束得到的产物即为cDNA，分装后用RNase-free 1.5mL离心管保存在-20℃，方便后续试验的进行。
1.2.3质粒pPICZαA的提取
1.2.3.1大肠杆菌DH5α的转化
(1)从超低温冰箱中取100μL DH5α感受态细胞，置于冰上使其缓慢融化，等待其融化后加入1μL质粒，混匀后置于冰上30min；
(2)将离心管从冰上取出马上将其放入事先预热好的42℃的PCR仪中热激45s以形成供质粒进入的缝隙，然后快速轻柔地将离心管转移到冰上静待2min(注意不要晃动离心管)；
(3)在离心管中加入700μL事先配置好的经高压灭菌过且不含Zeocin等任何抗生素的SOC液体培养基，用移液枪轻轻混匀后放于37℃，180rpm的摇床中培养1h以复苏菌种；
(4)用移液枪吸取200μL上述产物到含有25μg·mL-1ZeocinTM的LB琼脂培养基上进行涂布。
(5)将平板置于37℃培养箱中，待菌液完全吸收后，将平板倒置过夜。
1.2.3.2抽提质粒
挑取单菌落接种于100μg·mL-1Zeocin LB培养基中，200rpm，37℃培养12h至对数期，菌液作为模板进行菌落PCR反应；反应结束跑胶紫外下观察凝胶上是否有目的条带，并将验证正确的单菌落送去生工进行测序；培养测序正确的单菌落，使用E.Z.N.A.TM质粒小提试剂盒抽提质粒，操作步骤如下：
(1)培养测序正确的菌株，将培养好的菌液于室温下10,000g离心1min；
(2)倒掉上清，加入250μL已加RNA酶的试剂I后旋涡振荡，继续加入250μL试剂II，轻轻颠倒四五次；
(3)加入350μL试剂III，加完后迅速颠倒多次会发现离心管中有白色物质形成，沉淀形成后将离心管于室温下13,000g离心10min；
(4)在离心过程中将DNA mini柱子装在2mL收集管上，小心地用移液枪转移上清至结合柱中心后于室温下，13,000g离心1min，弃滤液后把柱子重新装回收集管；
(5)加入500μL HB缓冲液，离心条件同上，弃去滤液，将柱子重新装回收集管后向柱子中加入700μL洗涤液，离心条件同上，弃滤液，重复两次；
(6)将柱子装在新的收集管上，以13,000g空柱离心2min；
(7)将柱子装在1.5mL离心管后向柱子中央加入50μL洗脱液，室温静置1min，13,000g离心1min，收集质粒。
1.2.4酵母表达载体的构建
将验证测序正确的目的基因bgAs1分别用限制性内切酶EcoRI和XbaI对质粒进行双酶切，使用
One Step Cloning Kit(Vazyme)试剂盒构建酵母表达载体。
1.2.4.1线性化载体制备
使用EcoRI-HF和XbaI对pPICZαA进行双酶切，反应体系如下：
表7
PCR仪37℃反应1h，琼脂糖凝胶电泳检测酶切效果并切胶回收目标片段，使用OMEGA公司的Gel Extraction Kit试剂盒进行酶切产物的胶回收，操作步骤如下：
(1)将50μL产物全部电泳，在凝胶成像仪中切下条带放在离心管(已知重量)中，称重得出凝胶块的重量；
(2)加入同样体积的结合液，插在浮板上于55℃水浴锅中水浴7min，在水浴过程中每2min颠倒混匀；
(3)在水浴过程中取出DNA mini柱子并将其装在2mL收集管内，用移液枪将获得的混合液加入到柱子中心，室温下10,000g离心1min；
(4)将收集管中的滤液弃掉，然后将柱子装回收集管后加300μL结合液至柱子中，室温下13,000g离心1min；
(5)将滤液弃掉，将柱子装回，然后转移700μL已用Absolute ethanol稀释的SPW缓冲液到柱子中心，室温下，10,000g离心1min，弃掉滤液，再次重复上面步骤；
(6)将柱子套在新的收集管上，室温13,000xg离心2min；
(7)将柱子装在一个无酶离心管上，加入30μL洗脱液到结合柱的中心，放置在离心管架1min，13,000g离心1min洗脱DNA。琼脂糖凝胶电泳验证酶切结果。
1.2.4.2bgAs1基因的PCR扩增
插入片段用高保真酶扩增，无需考虑产物末端有无A尾，以反转录合成的cDNA为模板，用PhantaMaxSuper-FidelityDNAPolymerase(Vazyme)试剂盒对目的基因进行PCR扩增，PCR体系为：
表8
反应程序为：
表9
将产物进行琼脂糖凝胶电泳检测，剩下的产物放入-20℃保存。
1.2.4.3重组反应
重组体系：
表10
| 反应体系 | 加入体积 |
| 线性化载体 | 100ng |
| 插入片段 | 100ng |
| 5×CE II Buffer | 4μl |
| Exnase II | 2μl |
| ddH2O | To 20μl |
重组反应操作步骤如下：取无酶PCR管，将重组体系中试剂加入到PCR管中，使用移液器轻轻吸打混匀后离心，在PCR仪中37℃反应0.5h，取出后放在冰上冷却，取10μL产物加入到100μL于冰上融化的DH5α感受态细胞中，用手轻弹离心管混匀，于冰上静置30min后轻轻取出，在预热好的42℃水浴锅中热激45s后放回冰上冷却2min，冷却完成后加入700μL已灭菌SOC培养基，轻轻吹打混匀后37℃，180rpm培养1h，此时将已倒好的含博莱霉素的LLB平板也放在37℃培养箱中，培养完成后用移液枪吸取200μL菌液加在含有25μg·mL-1博莱霉素的LLB平板上，用涂布棒涂干，将平板翻过来放置于37℃过夜培养。
1.2.4.4阳性克隆的筛选及测序
对LLB平板上长出的转化子进行阳性验证，以确保bgAs1基因正确的连接到了pPICZαA质粒上，分别形pPICZαA-bgAs1，并成功导入DH5α感受态细胞中构建成阳性转化子pPICZαA-bgAs1-DH5α。验证阳性克隆采用菌落PCR的方法进行：在超净工作台用接种环挑取LLB平板上单菌落至灭菌LLB液体培养基，并于37℃控温摇床180rpm培养6h，以菌液为菌落PCR的模板，反应体系如下：
表11
反应程序为：
表12
菌落PCR鉴定为阳性的菌落，再将剩余菌液接种至含有适当抗生素的液体LLB培养基中培养过夜，进行一代测序。
2.bgAs1在毕赤酵母中的高效表达
2.1重组质粒的提取
将测序正确的bgAs1划线于LB平板上，挑取单克隆于含ZeocinLLB液体培养基中，37℃，180rpm条件下培养12h至对数期。抽提质粒，操作步骤同前。
2.2重组质粒线性化
为了提高重组质粒在毕赤酵母染色体上的整合效率，因此，需要对提取出的重组质粒pPICZαA-bgAs1进行单酶切使其线性化。通过对bgAs1的cDNA序列分析发现，序列中存在GAGCTC序列，不能使用SacⅠ(NEB)进行线性化，但其序列中均不具有GTTTAAAC，根据序列分析结果可以用PmeⅠ(NEB)对其进行线性化。
单酶切反应体系如下：
表13
PCR仪37℃反应30min，65℃加热20min后，1％琼脂糖凝胶电泳检测线性化结果，将线性化条带切胶回收。
2.3毕赤酵母酵母感受态细胞制备
毕赤酵母X-33感受态细胞制备方法如下：
(1)对实验室保藏的X-33菌种进行复苏，将甘油菌在YPD平板上划线，用封口膜封好后放置于30℃培养箱培养，等到单菌落长出来时挑取单菌落到10mL灭菌YPD培养基中，30℃，200rpm过夜培养；
(2)接种培养好的菌液按0.1％接种量到100mL灭菌YPD培养基中，30℃培养至OD600约1.5左右；
(3)培养完成后在超净工作台中取25mL菌液到50mL灭菌离心管中，于4℃，1500g下离心5分钟，将离心管上清液弃掉；
(4)加入25mL预冷的无菌水轻轻吹打将细胞悬浮，同样条件离心；
(5)加入15mL预冷无菌水重复上述操作，同样条件离心；
(6)再次加入10mL预冷无菌水重复上述操作，同样条件离心；
(7)将离心管中水倒干后加入10mL预冷的1M无菌山梨醇重复上述操作，最后离心完后小心的吸干上清液，用500μL预冷的1M山梨醇轻轻悬浮细胞混匀，按照每管80μL的量分装，供电击转化使用。
2.4重组质粒电击转化酵母感受态细胞
将线性化重组质粒与线性化空载，分别通过电击转化的方式转入到2.3制备的感受态细胞中：在超净工作台中，将20μL已线性化的重组载体加入到感受态细胞中，用移液枪吹打混匀后转入0.2cm电击转化杯(无菌，预冷)中，将其置于冰上放置5分钟，在此期间也将1M无菌山梨醇置于冰上预冷，取出电击转化杯后用吸水纸擦干再放进电击转化仪，1800V电击处理后迅速加入1mL1M预冷山梨醇，轻弹杯壁混匀倒入无菌离心管，用封口膜封好后在30℃培养箱中孵育1小时左右，提取准备好100μg·mLmL-1Zeocin的YPDS平板，放在培养箱预热，孵育完成后取200μL到平板上用涂布棒涂干，用封口膜封好后转移到30℃培养箱中过夜培养。
2.5重组菌株的筛选及鉴定
通过电转化，线性化的质粒pPICZαA-bgAs1可同源重组到Pichia pastoris X-33基因组的AOX 1位点处。为了检测bgAs1基因是否正确重组，需要对YPDS平板上长出的单菌落做菌落PCR验证。
挑取平板上单菌落接种于Zeocin抗性的液体YPD培养基中，并于30℃，180rpm中过夜培养，取1μL样品作为菌落PCR的模板，以pPICZαA采取同样操作作为空白对照。
菌落PCR引物如下:
AOX-JF：GACTGGTTCCAATTGACAAGC
AOX-JR：GCAAATGGCATTCTGACATCC
引物由Sangon合成，菌落PCR反应体系同上。
菌落PCR反应程序完成后将样品跑胶后在凝胶成像仪观察保存。
对筛选检验正确的分别含有目的基因的工程菌株pPICZαA-bgAs1-X33进行高拷贝筛选。即将测序验证正确的阳性克隆子pPICZαA-bgAs1-X33转到含200μg/mLZeocinTM的YPD琼脂培养基中，采用转化后载体扩增法(PTVA法)即每隔五天将生长状态良好的阳性克隆子分别转至含400μg·mL-1mL、800μg·mL-1mL、1200μg·mL-1mL、2000μg·mL-1mLZeocinTM的YPD琼脂培养基中以使细胞适应更高浓度的抗生素来筛选含有多个拷贝子的阳性克隆子，将筛选到的高拷贝克隆子送到Sangon测序验证。
2.6bgAs1的诱导分泌表达
在超净工作台中挑取2mg·mL-1浓度ZeocinTM平板上验证正确的pPICZαA-bgAs1-X33分别接种于装有50mL甘油培养基的250mL三角瓶中，培养二十四小时后，用100mL甲醇培养基重悬甘油培养基至OD＝1，用1L挡板摇瓶于30℃，200rpm条件下振荡培养七天，每隔一天补加1％甲醇；每天取样测定粗酶液酶活，以表达效果最好的工程菌株命名为XL1和XL2，用于后续实验。粗酶液处理如下：4℃，12000rpm离心10分钟，收集上清液，以煮沸10分钟的上清液为空白对照，采用pNPG法测定酶活性。对第7天粗酶液样品处理后留样以做SDS-PAGE蛋白电泳分析，对转入空白载体的pPICZαA菌株采取同样的操作。
3.重组酶BgAs1的分离纯化与酶学性质研究
3.1粗酶液获取及处理
重组菌株pPICZαA-bgAs1-X33在BMMY培养基经甲醇诱导6d后，分别将粗酶液和已灭菌的50mL离心管放在冰盒中预冷，将发酵液平均分装在离心管中，称重配平后，4℃12,000rpm离心10min；离心完成后收集上清，并上清置于冰上，此上清即为粗酶液；粗酶液用0.22μm滤膜抽滤，滤液用50kDa默克超滤管浓缩并用缓冲液A进行置换，4℃保存备用。
3.2阴离子交换层析
采用UNOsphereTMQ介质亲和层析；缓冲液A：柠檬酸盐缓冲液(pH7.0)；缓冲液B：高盐柠檬酸盐缓冲液(pH7.0)，进行粗酶液纯化。具体步骤如下：
(1)将泵流速设置6.0mL/分钟(288cm/hr)；
(2)用脱气低盐缓冲液A清洗交换柱2分钟；
(3)用脱气高盐缓冲液B清洗交换柱5分钟；
(4)用低盐缓冲液平衡A交换柱5分钟；
(5)将样品加入交换柱中，注意不要产生气泡；
(6)用低盐缓冲液A清洗交换柱直至λ280平稳；
(7)流速调整至1mL/min，在0-0.4M缓冲液B范围内进行线性洗脱，收集洗脱液测定酶活；
(8)用1M缓冲液B进行彻底洗脱以清洗杂蛋白；
(9)用1MNaOH对交换柱进行消毒，保持40分钟的接触时间；
(10)用1M缓冲液B重新平衡交换柱；
(11)用20％乙醇清洗交换柱，将交换柱拆下，4℃保存；
(12)将纯化成功的蛋白转移至全新的15kDa Amicon U1tra-15超滤管用HEPES(pH7.0)溶液进行置换2-3次以脱盐和浓缩。
3.3纯化蛋白含量及酶活测定
蛋白质含量通过Beyotime BCA Protein Assay Kit测定。
β-葡萄糖苷酶酶活测定采用pNPG法，操作流程如下：
将100μL 5mM pNPG溶液与同样体积的酶液(适当稀释)混合，置于50℃反应10分钟，反应完毕后立刻加入600μL 1M Na2CO3终止反应，采用煮沸处理10分钟灭活的粗酶液作为对照，于400nm波长下测定吸光度值。
3.4表达产物的SDS-PAGE分析
为验证纯化所得到目的蛋白的纯度和检测目的蛋白的分子量大小，需要对纯化后的蛋白进行聚丙烯酰胺凝胶电泳，5％浓缩胶电泳100V，10min；8％分离胶电泳120V，60min。分别取纯化后50μL体积的BgAs1蛋白样品，与10μL Protein loading buffer混合，水浴煮沸8min使蛋白变性。取20μL蛋白预混液点入蛋白凝胶电泳胶槽。电泳完成后，将聚丙烯酰胺凝胶用考马斯亮蓝在摇床染色1h，再以脱色液(乙酸:无水乙醇:ddH2O＝1:4:5)在脱色摇床上过夜脱色。
3.5重组酶BgAs1的酶学性质研究
3.5.1最适pH
为了测定不同pH的缓冲液(50mM·L-1McIlvaine buffer pH 2.2-8.0，50mM·L- 1Tris-HCl buffer pH8.0-9.0，50mM·L-1Glycine-NaOH buffer pH9.0-10.0)对BgAs1酶活力的影响，将90μL缓冲液和10μL纯化后的酶液混匀后孵育2min，加入100μL 5mM·L-1pNPG在最适温度条件下反应10min，反应完毕后加入600μL 1M Na2CO3终止反应，测定OD400nm的吸光度，每个反应三个重复，将最高酶活力定义为100％。
3.5.2最适反应温度和热稳定性
为了测定不同温度对BgAs1酶活力的影响，在相同pH条件下，取90μL缓冲液和10μL酶液混匀后加入100μL 5mM·L-1pNPG，将混合液于20-90℃(间隔为10℃)条件下进行反应10min，反应完毕后加入600μL 1M Na2CO3终止反应，测定OD400nm的吸光度。每个反应三个重复，将最高酶活力定义为100％。
在最适pH条件下，取相对酶活较高的3个温度无底物孵育10、20、30、40、50、60min后于最适温度测定剩余活性。
3.5.3底物特异性
为了测定BgAs1对不同底物的水解能力，在最适反应条件下，测定如下底物：纤维二糖、纤维三糖、纤维四糖、纤维五糖、龙胆二糖、槐糖、昆布二糖、大豆苷、pNPG、昆布多糖，每个反应三个重复，将pNPG酶活力定义为100％
酶活单位U的定义：每分钟水解pNPG底物生成1μmol pNP所需的酶量为一个U。
不同底物β-葡萄糖苷酶活性的测定方法如下(酶液煮沸10min为对照)：
pNPG：100μL适当稀释的酶液与100μL 5mM pNPG混合，最适温度下反应10min，立刻加入600μL 1M Na2CO3终止反应，测定OD400，二糖类则反应完后取100μL反应产物加入200μLGOD分析试剂，37℃反应30min后加入200μL 6M H2SO4终止反应，然后测定OD540。
大豆苷/昆布多糖：取900μL底物，最适温度下预热2min，加入100μL的适当稀释的酶液，孵育10min后，加入1.5mL DNS，煮沸5min终止反应，迅速冷却，测定OD540。
标准曲线的制定：
(1)pNP标准曲线的制定：按下表配制浓度梯度溶液，取200μL在酶标板中测定OD400。
表14
(2)DNS法葡萄糖标准曲线的制定：按下表配置浓度梯度溶液，吸取200μL混匀后在酶标板中测定OD540。
表15
| 编号 | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 |
| 10mmol/L葡萄糖(μL) | 0 | 25 | 50 | 100 | 150 | 200 | 250 | 300 | 350 |
| 柠檬酸-Na<sub>2</sub>HPO<sub>4</sub>(μL) | 1000 | 975 | 950 | 900 | 850 | 800 | 750 | 700 | 650 |
| DNS试剂(μL) | 1500 | 1500 | 1500 | 1500 | 1500 | 1500 | 1500 | 1500 | 1500 |
| 葡萄糖浓度(μmol·ml<sup>-1</sup>) | 0 | 0.1 | 0.2 | 0.4 | 0.6 | 0.8 | 1 | 1.2 | 1.4 |
(3)GOD法葡萄糖标准曲线的制定：按下表配置浓度梯度溶液，混匀后在酶标板中测定OD540。
表16
| 编号 | 1 | 2 | 3 | 4 | 5 |
| 50μg/mL葡萄糖(μL) | 0 | 20 | 40 | 60 | 80 |
| 柠檬酸-Na<sub>2</sub>HPO<sub>4</sub>(μL) | 1000 | 980 | 960 | 940 | 920 |
| GOD试剂(μL) | 2000 | 2000 | 2000 | 2000 | 2000 |
| 6MH<sub>2</sub>SO<sub>4</sub>(μL) | 2000 | 2000 | 2000 | 2000 | 2000 |
| 葡萄糖浓度(μmol·ml<sup>-1</sup>) | 0 | 0.00111 | 0.00222 | 0.00333 | 0.00444 |
3.5.4金属离子对酶活力影响
为了测定各种金属离子能否对重组酶活性产生影响，在标准反应体系中分别加入终浓度为5mM·L-1Ca2+，K+，Al3+，Ni2+，Cu2+，Mg2+，Mn2+，Co2+，Zn+，Fe3+对酶活力的影响。每个反应三个重复，以不加任何金属离子的组为100％。
3.5.5盐耐受性
在最适反应条件下，测定盐离子终浓度为0，100，200，300，400，500，1000，1500，2000，2500mM·L-1条件下对酶活力的影响。每个反应三个重复，以盐离子终浓度0组为100％。
3.5.6葡萄糖耐受性
在最适酶反应条件下，测定BgAs1在葡萄糖浓度为0，100，200，300，400，500，1000，1500，2000，2500mM·L-1条件下的酶活力。每个反应三个重复，以葡萄糖浓度为0时酶活力定义为100％。
3.5.7酶促反应动力学常数测定
以不同浓度(0.2、0.4、0.6、0.8、1、2、4mM·L-1)pNPG为底物，在酶最适反应条件下测定酶活性。测定结果根据双倒数作图法计算酶促反应动力学常数Km和Vmax。
3.6BgAs1协同纤维素酶糖化效果研究
本实验将甘蔗渣经干燥、粉碎后筛分50目间的原料，以10倍体积1％NaOH浸泡，121℃高温处理20min，将预处理的甘蔗渣过滤后用蒸馏水冲洗至pH为中性，80℃干燥至恒重，以该样品为原料进行协同糖化实验，比较现有商业化酶Novozyme、BgAs1糖化效果，试验设计如下：
称量0.2g甘蔗渣于100ml三角瓶中，加入10ml pH 6 100mM柠檬酸盐缓冲液，将样品置于40℃和120rpm的摇床中孵育10min，向各三角瓶加入相应的酶组分，每组三个平行，每种酶组合分别在0h，4h，8h，12h，24h，36h，48h，72h取出并于沸水中终止反应，水解产物经4℃，12000rpm离心10min后采用DNS法测定还原糖含量，GOD法测定葡萄糖含量。
表17
(二)实验结果
1RNA测序结果分析与检验
3.1.1测序结果
3.1.1.1RNA-seq整体质量评估
使用转录组数据比对软件TopHat2将过滤完核糖体的reads比对到参考基因组上。将CK1，CK2，CK3，T1-1，T1-2，T1-3，T2-1，T2-2，T2-3九个样本的序列同参考序列进行mapping，结果如表18，样品参考序列的mapping ratio均高于85％，因此均可以继续后续比对分析。
表18样品与参考基因组比对情况
3.1.2目的基因的筛选
木质纤维素降解酶绝大多属于CAZyme，通过RNA-seq后对聚多曲霉Aspergillussydowii L-1中CAZyme的注释分析，发现T1组和T2组均有β-葡萄糖苷酶，对差异基因比对发现，gene5910在三个组中表达量几乎一致。因此，通过RNA-seq测序得到上调表达且预测为β-葡萄糖苷酶的未知功能基因gene5910，命名为bgAs1，作为研究对象进行后续研究。
3.2bgAs1基因序列分析
对bgAs1进行序列分析，经SignalP分析表明bgAs1序列无信号肽，属于内分泌蛋白。bgAs1基因长度为2493bp(去除信号肽)，编码831个氨基酸(1个终止密码子)，预测BgAs1理论等电点pI为5.36。
通过SMART对bgAs1编码区进行蛋白质结构功能域的分析如图2-3表明，bgAs1所编码的蛋白质序列的结构域包括有29-279位点的Glyco-hydro-3功能域(Pfam:PF00933,E-value:4.6e-39)，391-536位点的PA14功能域(E-value:1.7e-6)和740-810位点的Fn3_like(E-value:2.03e-26)功能域。
通过dbCAN数据库对bgAs1进行注释，如表19所示，分别显示了bgAs1与GH3家族的一致性序列。
表19 dbCAN数据库注释
3.1bgAs1基因PCR扩增产物鉴定
提取聚多曲霉L1总RNA。取5μLRNA样品进行1％琼脂糖凝胶电泳，由图2可见RNA的完整性和纯度较高，可以进行反转录。
以转录得到的cDNA为模板，分别采用bgAs1-R和bgAs1-F进行bgAs1基因的PCR扩增，获得bgAs1的基因序列，经琼脂糖凝胶电泳检测如图3所示，bgAs1电泳条带大小约为2500bp，与其实际2493bp相近，因此认为通过PCR我们已成功克隆bgAs1基因。
3.2酵母表达载体的构建
抽提质粒后进行双酶切，对酶切产物进行胶回收及琼脂糖凝胶电泳，琼脂糖凝胶电泳结果如图4，质粒pPICZαA共有三个条带，而双酶切后产物有一条带。
重组产物转化感受态细胞后过夜培养，获得大量单克隆，如图5。
3.3转化子菌落PCR验证
挑取重组反应转化平板上若干个克隆进行菌落PCR鉴定，扩增引物使用载体pPICZαA上的通用测序引物。将菌落PCR产物进行AGE，电泳结果如图6，除4外，其它转化子均正确连接，得到重组质粒。
3.4重组表达菌株构建及多拷贝筛选
酵母转化子在YPDS平板上的生长情况如图7。对重组表达质粒pPICZαA-bgAs1和pPICZαA空质粒用PmeⅠ进行线性化单酶切，通过电击法成功将两个线性化质粒分别转入感受态细胞。
利用不同浓度的ZeocinTM平板，筛选具有多个拷贝的毕赤酵母重组子，高浓度2mg·mL-1结果如图8。
3.5重组子的菌落PCR验证
重组子在装有5mL YPD培养基的试管中于28℃恒温培养3-4天，直到长出单菌落。以酵母基因组DNA为模板，分别使用bgl1-F，3’AOX和5’AOX，3’AOX对pPICZαA-bgAs1-X33PCR进行扩增，琼脂糖凝胶电泳得到重组酵母菌，PCR验证结果见图9。
3.6bgAs1基因的诱导表达
各选取不同ZeocinTM抗性的重组子10株进行蛋白诱导表达，抗2mg·mL-1ZeocinTM水平的酵母工程菌诱导表达6d后酶活性最高，将表达bgAs1的工程菌株命名为pPICZαA-bgAs1-X33，-80℃保存。pPICZαA-bgAs1-X33在BMGY培养基中进行去抑制培养后，将菌体转移至BMMY培养基中进行发酵培养，并每隔24h加入1mL甲醇进行诱导并取上清液，用pNPG法分别测定酶活。
3.1重组酶BG2在毕赤酵母中的纯化与SDS-PAGE分析
经甲醇诱导6天后，收集BgAs1粗酶液，经离心浓缩处理后进行层析纯化，层析结果如图10所示。
纯化后，BgAs1粗酶液以及纯酶液一起进行8％的聚丙烯酰胺凝胶中电泳，通过考马斯亮蓝染色、脱色液过夜脱色后，可清晰地看到凝胶泳道有单一的蛋白条带，如图11条带大小约为80-90kDa位置，与预测大小90kDa一致，说明经过了阴离子亲和层析后，BgAs1已被纯化。
3.2重组酶BgAs1酶学性质研究
3.2.1酶活测定及蛋白定量标准曲线
3.2.1.1pNP标准曲线
标准曲线如图12。
3.2.1.2DNS法葡萄糖标准曲线
标准曲线如图13。
3.2.1.3GOD法葡萄糖标准曲线
标准曲线如图14。
3.2.1.4BCA法蛋白定量标准曲线
标准曲线如图15。
3.2.2BgAs1的最适反应pH
重组酶BgAs1酶学性质如图16所示。BgAs1最适作用pH值均为6.0，BGL1在pH5.0-9.0之间可以保持50％以上的酶活力，当pH<5.0时，随着pH的减小酶活急剧下降且在pH为2.2时失活；BgAs1在pH5.0-8.0之间可以保持50％以上的酶活力，随着pH的减小酶活急剧下降且在pH为2.2时失活。
3.2.3BgAs1的最适反应温度
如图17所示BgAs1的最适反应温度均为40℃，BgAs1在30℃-50℃均可以保持50％以上的酶活力，当温度低于30℃或高于50℃时，随着温度的变化酶活急剧下降，10℃剩余相对酶活分别为12.90％，在90℃时BgAs1剩余相对酶活为13.79％；
3.2.4BgAs1热稳定性
如图18所示，BgAs1在30℃和40℃条件下酶活相对稳定，但随温度升高，酶活力迅速下降，60℃处理60min后酶失活。
3.2.5BgAs1底物特异性测定
如表20所示，在二糖底物中，而BgAs1对各类糖苷键底物均具有相当的水解活力。在多糖底物中，BgAs1对昆布多糖的水解活力最高，其次是pNPG，且BgAs1对大豆苷有着较高的水解活力。
表20重组酶BgAs1对不同底物的水解能力
3.2.6金属离子对BgAs1活性的影响
如表21所示，Mn2+、Fe2+可以明显激活BgAs1的活性，Ni2+、Cu2+轻微抑制BgAs1的活性，其它的金属离子对BgAs1没有明显的作用。
表21金属离子对BgAs1酶活性的影响
*Values represent the mean±SD(n＝3)relative to the untreated controlsamples
3.2.7BgAs1的盐耐受性
如图19所示，BgAs1表现出高耐盐性的典型特征，在1M的NaCl溶液中，该酶的活性显著增强(135％)。在2.5MNaCl中仍有升高(116％)。
3.2.8BgAs1的葡萄糖耐受性
如图20所示，0-0.5M Glucose对重组酶BgAs1酶活有促进作用，随Glucose终浓度增加到2.5M时相对酶活可以达到71％，4M时相对酶活仍可保留47.03％。
3.2.9BgAs1酶促反应动力学常数测定
在最适反应条件下，以不同浓度的pNPG和纤维二糖为底物进行测定BgAs1酶活力，如表22所示：
表22 BgAs1反应动力学常数
3.3BgAs1协同纤维素酶糖化效果
为了评价和BgAs1在木质纤维素水解中的应用潜力，对甘蔗渣进行了酶解实验，结果如图21所示，根据图21可以看出，在不添加β-葡萄糖苷酶的情况下，糖化过程中还原糖含量在72h时最高达到132μmol，而添加Nov188后协同糖化过程中还原糖含量在72h时最高可以达到167μmol，添加重组BgAs1组72h时还原糖含量可以达到162μmol；
在不添加β-葡萄糖苷酶的情况下，糖化过程中葡萄糖含量在72h时最高达到16μmol，而添加Nov188后协同糖化过程中葡萄糖含量在72h时最高可以达到19μmol，添加BgAs1组72h时葡萄糖含量可以达到19μmol。
以上仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
序列表
<110> 海南大学
<120> 一种耐葡萄糖的β-葡萄糖苷酶及其表达基因和应用
<130> MP21018574
<160> 2
<170> SIPOSequenceListing 1.0
<210> 2
<211> 2493
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
atgactcgga cgttcgatat agaccatgtt ctggcgaaca ttagtgacca ggacaagatc 60
gcgctgcttg ccggaattga tttttggcac acgcatccaa tcccggagtt caatgtccca 120
tcggtacggg taacagatgg cccgaatggc attcgaggta ctaaattttt cgccggtgtg 180
cctgcggcat gtcttccctg cgggaccgcc ctgggagctg tttgggaccg agagctactg 240
caacgtgctg gggagcttct cggggatgag tgtatagcca agggcgcgca ttgctggctg 300
ggcccgactg taaatatgca aaggtcacca ctgggtgggc gtgggtttga gtccttctca 360
gaagacccgt acctagcagg tgttgctgcg gcttcaatga tcgttggatg tgagagtaag 420
ggcattatcg ctacagtgaa gcattttgtc ggcaacgacc aggagcatga gcgacgggct 480
gtcgatgtga ttgtgacacc tcgggcactc cgcgagatat acctacgtcc attccagatt 540
gtcgcgcgag atgccagccc tggggcgctt atgacctcgt acaacaagat caatgggaag 600
catgtcgtcg aggacaagaa gatgtatgac ctgattcgta aagagtgggg atgggacccg 660
ttggtcatga gcgattggta cgggacctat acaacgattg actcgacaaa tgcggggctg 720
gacctggaga tgcctggagt atcgagatat agggggaaat atattgagtc tgcgatacag 780
gcacgactga tcaaatcttc caccttagat gcacgagccc gcaaggtatt ggagtttgtc 840
caacgtgcaa gccgaaccaa ggtctcagag gtcgagaaag gtcgcgacta tccagacgac 900
cgggcactta tgcgtacgct ctgctcgaat agcacggttc tactgaaaaa tcagggcggt 960
atcctcccgc tcccaaagac agcgaagaaa attgcgctca tcggatcgca tatcaaatcg 1020
ccagcaatat ctggtggtgg tagtgcggcc ttgaaaccat accacatatc gagtttgtat 1080
gaagcagtac aagaggcact cccagacgca gagatcatat atgaaacggg tgctcatgcc 1140
cacaggatgc tccccgtgat tgaccgtctg ttgagcaacg ccgtgattca cttttacaac 1200
gagccggtgt ccagctcaga ccgtcaatgc ctcgggacgg agcctgttcc taccactgct 1260
tttcagttca tggactacaa acttccaggc ctgaacaggg agcttttctg gtcaacgttg 1320
attggtgact tcaactgtga cgcatctggc gtatgggatt ttgggttgac cgtgttcgga 1380
acggcaaatt tgtatataga tgacgagttg atcatcgaca acaccactaa gcaaaccaag 1440
ggaactgcat tcttcggcaa aggcacaatc gaggagactg ggtcgaagac tctagttgcc 1500
ggccaaacat acaaaattcg aatagagtac ggaagtgcca atacggccac tctgaaaaca 1560
actgggatgg tgaacttcgg cggcggtgcg gcgaacctgg gggcctctct gcgtatggat 1620
gctgaagaga tgatcgatcg agccgtgaag gcagcagggg aggctgacta cacaattctc 1680
tgcaccggac tgaacgcaga tttcgagtca gaggggtttg atagaacgca tatggacctt 1740
cctcctggta ttgacaacat gatctcgcgt gtcttggacg tggcggcaga caaaacagtt 1800
atcgtcaacc aatctgggac cccggtaaca atgccatggg tggaccaggc aaatgccatt 1860
gtacatggat ggtacggagg caatgaaaca ggacacggga ttgctgatgt cctatttggt 1920
gacgtgaatc cttcaggtaa gctctcactg tcctggccta gggatgccag acacaatcca 1980
gcatacctga attacgccag cgtcggcgga agagtacttt atggagagga tgtatatgtg 2040
ggctaccgat tttatgagaa gactggccgt gaggttttgt tcccgttcgg gtacgggctt 2100
tcttatgcaa cctttgaggt atcaccagaa gcctcagttt ccccggatgg attcacaccg 2160
gaaacccctc caactgcaac tgttcggatc aagaatatca gcgaagtagc cggtgcgcag 2220
gtgctgcaac tatacgtggg ggcccccaat agcccaacac cacgcccaca gaaagaactg 2280
caagggttcg aaaaggtatt cctgcagcca ggcgaggaga aggcagtgga tatcaagctg 2340
gataaatacg caacgagttt ctgggatgag atcgaagaca tgtggaaaag cgaggctggg 2400
gtgtatgagg tgctaattgg cacatcgagc gaagacattg ttgcgcgtgg acggttcgaa 2460
gtcggcgaga cacggtattg gcgaggcgtt tag 2493
<210> 2
<211> 830
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Met Thr Arg Thr Phe Asp Ile Asp His Val Leu Ala Asn Ile Ser Asp
1 5 10 15
Gln Asp Lys Ile Ala Leu Leu Ala Gly Ile Asp Phe Trp His Thr His
20 25 30
Pro Ile Pro Glu Phe Asn Val Pro Ser Val Arg Val Thr Asp Gly Pro
35 40 45
Asn Gly Ile Arg Gly Thr Lys Phe Phe Ala Gly Val Pro Ala Ala Cys
50 55 60
Leu Pro Cys Gly Thr Ala Leu Gly Ala Val Trp Asp Arg Glu Leu Leu
65 70 75 80
Gln Arg Ala Gly Glu Leu Leu Gly Asp Glu Cys Ile Ala Lys Gly Ala
85 90 95
His Cys Trp Leu Gly Pro Thr Val Asn Met Gln Arg Ser Pro Leu Gly
100 105 110
Gly Arg Gly Phe Glu Ser Phe Ser Glu Asp Pro Tyr Leu Ala Gly Val
115 120 125
Ala Ala Ala Ser Met Ile Val Gly Cys Glu Ser Lys Gly Ile Ile Ala
130 135 140
Thr Val Lys His Phe Val Gly Asn Asp Gln Glu His Glu Arg Arg Ala
145 150 155 160
Val Asp Val Ile Val Thr Pro Arg Ala Leu Arg Glu Ile Tyr Leu Arg
165 170 175
Pro Phe Gln Ile Val Ala Arg Asp Ala Ser Pro Gly Ala Leu Met Thr
180 185 190
Ser Tyr Asn Lys Ile Asn Gly Lys His Val Val Glu Asp Lys Lys Met
195 200 205
Tyr Asp Leu Ile Arg Lys Glu Trp Gly Trp Asp Pro Leu Val Met Ser
210 215 220
Asp Trp Tyr Gly Thr Tyr Thr Thr Ile Asp Ser Thr Asn Ala Gly Leu
225 230 235 240
Asp Leu Glu Met Pro Gly Val Ser Arg Tyr Arg Gly Lys Tyr Ile Glu
245 250 255
Ser Ala Ile Gln Ala Arg Leu Ile Lys Ser Ser Thr Leu Asp Ala Arg
260 265 270
Ala Arg Lys Val Leu Glu Phe Val Gln Arg Ala Ser Arg Thr Lys Val
275 280 285
Ser Glu Val Glu Lys Gly Arg Asp Tyr Pro Asp Asp Arg Ala Leu Met
290 295 300
Arg Thr Leu Cys Ser Asn Ser Thr Val Leu Leu Lys Asn Gln Gly Gly
305 310 315 320
Ile Leu Pro Leu Pro Lys Thr Ala Lys Lys Ile Ala Leu Ile Gly Ser
325 330 335
His Ile Lys Ser Pro Ala Ile Ser Gly Gly Gly Ser Ala Ala Leu Lys
340 345 350
Pro Tyr His Ile Ser Ser Leu Tyr Glu Ala Val Gln Glu Ala Leu Pro
355 360 365
Asp Ala Glu Ile Ile Tyr Glu Thr Gly Ala His Ala His Arg Met Leu
370 375 380
Pro Val Ile Asp Arg Leu Leu Ser Asn Ala Val Ile His Phe Tyr Asn
385 390 395 400
Glu Pro Val Ser Ser Ser Asp Arg Gln Cys Leu Gly Thr Glu Pro Val
405 410 415
Pro Thr Thr Ala Phe Gln Phe Met Asp Tyr Lys Leu Pro Gly Leu Asn
420 425 430
Arg Glu Leu Phe Trp Ser Thr Leu Ile Gly Asp Phe Asn Cys Asp Ala
435 440 445
Ser Gly Val Trp Asp Phe Gly Leu Thr Val Phe Gly Thr Ala Asn Leu
450 455 460
Tyr Ile Asp Asp Glu Leu Ile Ile Asp Asn Thr Thr Lys Gln Thr Lys
465 470 475 480
Gly Thr Ala Phe Phe Gly Lys Gly Thr Ile Glu Glu Thr Gly Ser Lys
485 490 495
Thr Leu Val Ala Gly Gln Thr Tyr Lys Ile Arg Ile Glu Tyr Gly Ser
500 505 510
Ala Asn Thr Ala Thr Leu Lys Thr Thr Gly Met Val Asn Phe Gly Gly
515 520 525
Gly Ala Ala Asn Leu Gly Ala Ser Leu Arg Met Asp Ala Glu Glu Met
530 535 540
Ile Asp Arg Ala Val Lys Ala Ala Gly Glu Ala Asp Tyr Thr Ile Leu
545 550 555 560
Cys Thr Gly Leu Asn Ala Asp Phe Glu Ser Glu Gly Phe Asp Arg Thr
565 570 575
His Met Asp Leu Pro Pro Gly Ile Asp Asn Met Ile Ser Arg Val Leu
580 585 590
Asp Val Ala Ala Asp Lys Thr Val Ile Val Asn Gln Ser Gly Thr Pro
595 600 605
Val Thr Met Pro Trp Val Asp Gln Ala Asn Ala Ile Val His Gly Trp
610 615 620
Tyr Gly Gly Asn Glu Thr Gly His Gly Ile Ala Asp Val Leu Phe Gly
625 630 635 640
Asp Val Asn Pro Ser Gly Lys Leu Ser Leu Ser Trp Pro Arg Asp Ala
645 650 655
Arg His Asn Pro Ala Tyr Leu Asn Tyr Ala Ser Val Gly Gly Arg Val
660 665 670
Leu Tyr Gly Glu Asp Val Tyr Val Gly Tyr Arg Phe Tyr Glu Lys Thr
675 680 685
Gly Arg Glu Val Leu Phe Pro Phe Gly Tyr Gly Leu Ser Tyr Ala Thr
690 695 700
Phe Glu Val Ser Pro Glu Ala Ser Val Ser Pro Asp Gly Phe Thr Pro
705 710 715 720
Glu Thr Pro Pro Thr Ala Thr Val Arg Ile Lys Asn Ile Ser Glu Val
725 730 735
Ala Gly Ala Gln Val Leu Gln Leu Tyr Val Gly Ala Pro Asn Ser Pro
740 745 750
Thr Pro Arg Pro Gln Lys Glu Leu Gln Gly Phe Glu Lys Val Phe Leu
755 760 765
Gln Pro Gly Glu Glu Lys Ala Val Asp Ile Lys Leu Asp Lys Tyr Ala
770 775 780
Thr Ser Phe Trp Asp Glu Ile Glu Asp Met Trp Lys Ser Glu Ala Gly
785 790 795 800
Val Tyr Glu Val Leu Ile Gly Thr Ser Ser Glu Asp Ile Val Ala Arg
805 810 815
Gly Arg Phe Glu Val Gly Glu Thr Arg Tyr Trp Arg Gly Val
820 825 830
Claims (10)
1.一种耐葡萄糖的β-葡萄糖苷酶，其具有：
(I)、如SEQ ID No.2所示的氨基酸序列；或
(II)、在SEQ ID NO:2所示的氨基酸序列中取代、缺失和/或添加一个或多个核苷酸且与(I)所述的氨基酸序列功能相同的氨基酸序列；或
(III)、与如(I)或(II)所述的氨基酸序列具有90％以上同一性的氨基酸序列。
2.编码权利要求1所述的β-葡萄糖苷酶的BgAs1基因。
3.根据权利要求2所述的BgAs1基因，其特征在于，其具有I)～III)中任意一种核苷酸序列：
I)、如SEQ ID No.1所示的核苷酸序列；或
II)、在SEQ ID NO:1所示的核苷酸序列中取代、缺失和/或添加一个或多个核苷酸且表达SEQ ID NO:2所示蛋白的核苷酸序列；或
III)、与SEQ ID NO:1所示序列互补的核苷酸序列。
4.含有权利要求2或3所述基因的生物材料，所述生物材料包括表达模块、重组载体和宿主菌。
5.根据权利要求5所述的生物材料，其特征在于，所述宿主菌为大肠杆菌或毕赤酵母。
6.根据权利要求5所述的生物材料，其特征在于，所述重组载体的骨架为pPICZαA。
7.权利要求1所述的β-葡萄糖苷酶、权利要求2或3所述的基因、权利要求4或5所述的生物材料协同纤维素酶在水解木质纤维素制备还原糖和/葡萄糖中的应用。
8.权利要求1所述的β-葡萄糖苷酶、权利要求2或3所述的基因、权利要求5或6所述的生物材料在制备生物乙醇中的应用。
9.一种制备还原糖和/或葡萄糖的方法，其特征在于，以纤维素原料为底物，利用纤维素酶和权利要求1所述的β-葡萄糖苷酶进行酶解，获得还原糖和/或葡萄糖。
10.根据权利要求9所述的方法，其特征在于，所述纤维素原料包括甘蔗渣、玉米秸秆、糟渣、海藻中的至少一种。
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