CN103789331A - 一种高效水解大豆异黄酮糖苷的β-葡萄糖苷酶基因与应用 - Google Patents
一种高效水解大豆异黄酮糖苷的β-葡萄糖苷酶基因与应用 Download PDFInfo
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- CN103789331A CN103789331A CN201310149771.2A CN201310149771A CN103789331A CN 103789331 A CN103789331 A CN 103789331A CN 201310149771 A CN201310149771 A CN 201310149771A CN 103789331 A CN103789331 A CN 103789331A
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Abstract
本发明公开了一种新型β-葡萄糖苷酶，它的核苷酸序列如SEQIDNO.1所示。它的氨基酸序列如SEQIDNO.3所示。本发明还公开了含上述新型β-葡萄糖苷酶的表达载体，及重组β-葡萄糖苷酶及其在高效水解大豆异黄酮糖苷中的应用。该新型重组β-葡萄糖苷酶在大肠杆菌表达体系中具有高效的可溶性表达，该酶的最适反应温度和最适pH分别为45℃和pH6.0，具有良好的热稳定性和较宽的pH酶解范围。同时该酶对大豆苷、染料木苷具有高效的水解作用，水解效率为100%。
Description
技术领域
本发明涉及一种β-葡萄糖苷酶新基因，尤其涉及一种高效水解大豆异黄酮糖苷的β-葡萄糖苷酶基因及其重组酶和重组酶的应用，属于基因工程领域。
背景技术
β-葡萄糖苷酶(β-glucosidase)属于糖苷水解酶家族，又称β-D-葡萄糖苷葡萄糖水解酶(β-D-glucoside glucohydrolase；EC3.2.1.21)，一方面，该酶可以催化水解β-糖苷键，释放出葡萄糖和相应的糖苷配基。另一方面，该酶还能催化转糖苷缩合反应，合成低聚糖。研究发现，游离型的大豆异黄酮糖苷才能发挥其生物活性，β-葡萄糖苷酶在催化水解结合型糖苷产生游离型糖苷方面具有重要作用，显著提高异黄酮糖苷的水解效率，从而为工业上大批量生产高活性大豆异黄酮苷元产品提供捷径。在纤维素降解过程中，β-葡萄糖苷酶可以与其他几种纤维素降解酶协同作用，将纤维素降解为葡萄糖，后者经微生物发酵作用即可产生燃料乙醇等能源物质。在食品行业中，该酶能够利用β-葡萄糖苷酶水解风味前体物质β-糖苷以释放出风味活性物质糖苷配基，从而增强葡萄酒、果汁和茶叶等产品的香气。另外，在医药领域中，可通过检测人体血清中β-葡萄糖苷酶活性的高低来判断是否患有乳腺癌。因此，β-葡萄糖苷酶在生产有活性大豆异黄酮苷元、能源、食品及医药等领域具有重要应用价值。
大豆异黄酮主要有12种化学成分，大豆苷元、染料木素和黄豆黄素3种游离型异黄酮苷元以及它们和丙二酰基葡糖糖、乙酰基葡萄糖、葡萄糖以β-葡萄糖苷键的方式结合成的9种异黄酮糖苷组成。大豆异黄酮具有抗肿瘤、扩张血管和抗氧化等作用，同时还可预防骨质疏松症和妇女更年期综合症等疾病，因此，受到越来越多国内外消费者的青睐。研究表明，大豆苷元、染料木素和黄豆黄素3种游离型异黄酮苷元是大豆异黄酮发挥生物活性功能的主体，而占大豆异黄酮总量80％～99％的9种结合型糖苷则无生物活性。这些异黄酮糖苷只有被水解为游离型的苷元后才能被小肠壁吸收，发挥其生物活性作用，但大豆自身含有的内源性β-葡萄糖苷酶的酶活性很低，只能水解22％～29％的异黄酮糖苷，因此，在豆制品中添加高活性的β-葡萄糖苷酶以提高异黄酮糖苷水解效率在食品工业中具有重要意义。
目前，已经从不同微生物中分离出来多种β-葡萄糖苷酶，主要包括里氏木霉(Trichoderma Reesei)、黑曲霉(Aspergillus niger)、斜卧青霉(Penincillium decumbens)、土曲霉(Aspergillus terreu)、多粘性芽孢杆菌(Bacillus polymyxa)、链霉菌(Streptomyces)和肠膜明串珠菌(Leuconostoc mesenteroides)等。鉴于传统的微生物分离培养技术分离出的β-葡萄糖苷酶在酶的活性、稳定性、底物特异性等方面存在各式各样的差异，以及环境中99％的微生物是不可培养的，所以，为了适应不断发展的、不同领域的工业应用，迫切需要从自然界中挖掘和鉴定具有新特性的β-葡萄糖苷酶。
宏基因组学(Metagenomics)是一种不依赖于微生物的分离培养，直接以特定生态环境中微生物群体基因组总和作为研究对象，同时利用序列分析和功能筛选作为研究手段，构建宏基因组文库从而筛选出新的基因及生理活性物质。宏基因组技术作为一种新兴学科领域，在很大程度上促进了环境微生物资源的利用，特别是未培养微生物资源的开发利用。因此该方法为发现更多的新型β-葡萄糖苷酶提供了有效途径。到目前为止，国内外尚未有利用宏基因组技术从海底泥中获得具有高效水解大豆异黄酮糖苷性质的新型β-葡萄糖苷酶的研究报道。
发明内容
本发明的第一个目的在于提供一种β-葡萄糖苷酶的新基因。
本发明的第二个目的在于提供上述β-葡萄糖苷酶的宏基因组学克隆方法。
本发明的第三个目的在于提供上述新型β-葡萄糖苷酶的DNA表达载体。
本发明的第四个目的在于提供一种由上述表达载体转化细胞而获得的基因工程菌。
本发明的第五个目的在于提供利用上述表达载体构建的重组β-葡萄糖苷酶及其制备方法。
本发明的最后一个目的在于提供利用上述重组β-葡萄糖苷酶在高效水解大豆异黄酮糖苷中的应用。
本发明的第一个目的是通过如下技术方案来实现的：一种新型β-葡萄糖苷酶的DNA，它的氨基酸序列如SEQ ID NO.1所示。
本发明提供的上述新型β-葡萄糖苷酶，它的氨基酸序列如SEQ ID NO.3所示。
本发明的第二个目的是通过如下技术方案来实现的：一种新型β-葡萄糖苷酶的宏基因组学克隆方法，提取海底泥的总DNA并纯化，将纯化后的总DNA经Hind III酶切，连接到克隆载体pUC118上，电击转化大肠杆菌DH5α高效感受态建立宏基因组文库，通过涂布含七叶苷和柠檬酸铁铵的LB平板显色法快速筛选得到阳性克隆子，经测序和BLAST比较并设计引物，从而克隆到目的片段。
本发明的第三个目的是通过如下技术方案来实现的：一种含有上述新型β-葡萄糖苷酶的DNA的表达载体。
本发明的第四个目的是通过如下技术方案来实现的：一种基因工程菌，通过如权利要求3所述的表达载体转化寄主细胞而得。优选地，所述的宿主细胞为大肠杆菌。
本发明的第五个目的是通过如下技术方案来实现的：一种重组β-葡萄糖苷酶的制备方法，包括用上述表达载体转化表达宿主细胞，培养转化体，从培养物中获得重组β-葡萄糖苷酶。
上述重组β-葡萄糖苷酶的制备方法中，寄主细胞是大肠杆菌。
上述重组β-葡萄糖苷酶的制备方法，其具体过程为：包括用上述表达载体的目的片段经Bam HI、Hind III双酶切，与pET-28a(+)载体连接，转化至大肠杆菌BL21(DE3)，经IPTG诱导，得到高效可溶性表达。
所述的IPTG终浓度为0.1-1.3mM，诱导温度为18-37℃。
本发明提供的重组β-葡萄糖苷酶，包括用上述表达载体转化寄主细胞，培养转化体，从培养物中获得重组β-葡萄糖苷酶的方法和步骤。
本发明的最后一个目的是通过如下技术方案来实现的：本发明所述重组β-葡萄糖苷酶在高效水解异黄酮糖苷中的应用。
本发明的有益效果是：
①.本发明从海底泥样品构建好的宏基因组文库中得到一个新的β-葡萄糖苷酶的DNA序列，通过基因工程技术对其功能研究，发现该序列在大肠杆菌中高效可溶表达，经蛋白纯化及SDS-PAGE电泳，得到一条单一的蛋白条带，减去融合蛋白的大小，初步确定该β-葡萄糖苷酶的分子量约为52kDa。
②.本发明将SEQ ID NO.1所示的DNA序列克隆到原核表达载体上，转化大肠杆菌感受态细胞，通过对阳性克隆子的诱导表达得到重组蛋白，研究其酶学性质，结果如下：
(1)在大肠杆菌表达体系中，该重组蛋白具有高效可溶性表达；
(2)以对硝基苯-β-D-吡喃葡萄糖苷(ρNPG)为底物，测得该重组β-葡萄糖苷酶的最适反应温度为45℃，该酶在温度低于60℃时非常稳定，60℃保温1h后，相对酶活保持在80％以上，70℃保温10min后仍有50％以上的相对酶活。表明该酶具有良好的热稳定性；该酶的最适反应pH为6.0，在pH5.5-8.5的范围内，能够保持80％以上的相对酶活，说明其具有较宽的pH酶解范围；测定金属离子和生化试剂对酶活力影响时发现，1mM Al3+与10mMTriton X-100对重组β-葡萄糖苷酶的酶活有明显的促进作用；相反的，当反应体系中含有1mM Cu2+、10mM Tween-80时，该酶酶活受到强烈的抑制；当含稀释酶液的反应体系中分别含有10mM EDTA、1mM FeCl3、1mM CaCl2、1mM MgSO4、1mM Al2(SO4)3、1mM MnCl2、1mM ZnSO4时，该β-葡萄糖苷酶的活性没有显著改变。
[0026]③.本发明在研究重组β-葡萄糖苷酶Bgl1-11水解大豆异黄酮糖苷反应时发现，在分别含有0.5mg／mL大豆苷和染料木苷的反应体系中进行水解反应1h，经高效液相色谱(HPLC)分析，结果表明其水解速率为100％。
附图说明
图1为实施例1中重组β-葡萄糖苷酶的SDS-PAGE电泳图；
其中，M为标准蛋白分子量maker，Line1为重组蛋白全细胞粗提物，Line2为重组蛋白上清粗提物，Line3为纯化的重组蛋白；
图2为实施例3中重组蛋白水解底物ρNPG的最适温度结果折线图；
图3为实施例3中重组蛋白水解底物ρNPG的热稳定性结果折线图，其中不同符号代表不同温度，50℃(■)，55℃(●)，60℃(▲)，65℃
and70℃(◆)；
图4为实施例3中重组蛋白水解底物ρNPG的最适pH结果折线图；
图5为实施例3中重组蛋白水解底物ρNPG的pH稳定性结果折线图；
图6为重组蛋白水解大豆苷(daidzin)和染料木苷(genistin)的高效液相色谱(HPLC)分析结果图。
具体实施方式
下面结合附图和实施例对本发明做进一步说明，但不以任何形式限制本发明。
实施例1宏基因组文库的建立和阳性克隆子的获得、基因克隆与表达
1、总DNA的提取：
称取5g样品，加入13.5mL DNA提取缓冲液(0.1M Tris，0.1M EDTA-Na，0.1MNa3PO4，1.5M NaCl，1％CTAB，pH值8.0)，剧烈震荡3-5min，加入200μL溶菌酶(100mg／ml)，反复颠倒5-6次，37℃水浴30min，加入1.5mL20％SDS，65℃水浴1h(期间每隔15min上下颠倒几次)，8000r／min离心5min，取上清液，用等体积氯仿抽提2次，16000r／min离心10min，取上清，加入0.6倍体积的异丙醇，室温放置2h，20000r／min离心20min，弃上清，沉淀加5mL预冷的70％乙醇，20000r／min离心10min，收集DNA沉淀，风干，用适量TE缓冲液溶解。
2、试剂盒法纯化DNA：按照OMEGA胶回收试剂盒说明书进行。
3、宏基因组电泳检测：用1％琼脂糖凝胶电泳检测总DNA的纯度和质量。
4、酶切总DNA：用限制性内切酶Hind III部分酶切总DNA，回收2-10kb的酶切片段，方法同试剂盒法纯化DNA。
5、酶切片段的电泳检测：方法同宏基因组电泳检测。
6、酶切片段的连接：将回收得到的酶切片段和pUC118／Bam HI(BAP)载体连接过夜，向连接产物中加入0.6倍体积的异丙醇，室温沉淀1.5h，14000r／min离心20min，弃上清并向沉淀中加入70％无水乙醇洗涤2次，风干并加入适量dd H2O重溶。
7、连接产物的转化：
吸取5μL的连接产物加入100μL的大肠杆菌DH5α高效感受态中，2500V/cm(Eppdorf2510电击仪)电击1次，46℃热激6min，37℃，180rpm摇床培养45-60min，吸取30-40μL涂布于含有氨苄青霉素(100μg／mL)、七叶苷(1μg／mL)、柠檬酸铁铵(2μg／mL)和IPTG(1mM)的LB琼脂平板，37℃培养过夜。由此构建了一个库容量达40000个转化子，多样性好的宏基因组文库。
8、文库筛选和阳性克隆子的鉴定：将涂布后的平板至于37℃培养24-48h，由于β-葡萄糖苷酶可以将七叶苷水解生产七叶亭，其与铁离子反应生成黑褐色沉淀，因而显黑色的菌落即为阳性克隆子。通过筛选得到一株阳性克隆子。
从平板将阳性克隆子挑出并接种至10mL含氨苄青霉素(100μg／mL)的LB液体培养基中，37℃、220r／min摇床培养过夜，取2mL菌体进行质粒提取，对插入片段测序，将测定的序列经过NCBI的BLASTn软件分析比较，发现该DNA由1245个碱基组成，将其命名为Bgl1-11，其核苷酸序列如SEQ ID NO.1所示，该DNA编码的多肽，含414个氨基酸，其氨基酸序列如SEQ ID NO.3所示。其中SEQ ID NO.2为SEQ ID NO.1和SEQ IDNO.3的对照图。
9、基因片段的克隆：根据测序结果设计一对引物；F1和F2，引物两端插入能插入pET-28a(+)载体的Bam HI和HindIII酶切位点，引物序列如下：
F15′-TTATGGATCCATGACGGAGACGCGGGTGCCTG
F25′-TCAGAAGCTTGGTCGAGAGCGGGAGCGACGC
利用两条引物，以质粒pUC118-Bgl1-11为模板进行PCR扩增反应，PCR体系如表1所示：
表1.PCT体系
PCR反应条件：94℃5min，94℃30sec，66℃30sec，72℃1min，30个循环，72℃10min。
用胶回收试剂盒将PCR产物纯化并用Bam HI、Hind III于37℃双酶切24h，与用Bam HI、HindIII双酶切的pET-28a(+)(Invitrogen)表达载体进行连接，取5μL重组质粒转化大肠杆菌BL21(DE3)，转化液涂布含卡那霉素(50μg／mL)的LB固体培养基，37℃培养过夜，随机挑取10株单菌落接种提取质粒DNA，双酶切验证后，送交测序。
10、重组β-葡萄糖苷酶Bgl1-11粗酶液的获得及分子量检测
将重组工程菌划线至含卡那霉素(50μg／mL)的LB固体培养基中，37℃培养过夜活化，随机挑取1株重组菌接种至含卡那霉素(50μg／mL)的LB液体培养基中，37℃、220r／min摇床培养过夜，按1：100的接种量转至50mL的含卡那霉素(50μg／mL)的LB液体培养基中，当生长至OD600=0.6-0.8时加入IPTG至终浓度0.1-1.3mM，18-37℃、200r／min摇床培养12小时(OD600=1.1)，取诱导表达后的菌液1mL加入到2mL EP管中，12000rpm离心1min，收集湿细胞，用1mL50mM磷酸钠缓冲液(pH7.5)洗涤菌体两次，再重悬于1mL50mM磷酸钠缓冲液(pH7.5)中。超声破碎2min，破碎5sec间隔5sec，4℃、12000rpm离心1min，上清即为粗酶液。之后进行酶液的纯化，具体纯化方法参见His·Purification Kit(Novagen)试剂盒说明书：
(1)用10mL预冷的1×Binding Buffer悬浮从100mL培养液中收集的菌体；
(2)采用超声波破碎细胞至澄清，4℃、14000×g离心20min收集上清即得粗酶液；
(3)加入4mLHis·Bind resin至滤柱中，形成2mL的纯化柱；
(4)依次6mL无菌水洗涤，10mL1×Charge Buffer洗涤，6mL1×Binding Buffer洗涤；
(5)将上述粗酶液置于纯化柱上，除去滤液；
(6)依次用20mL1×Binding Buffer洗涤，12mL1×Wash Buffer洗涤纯化柱；
(7)最后用12mL1×Elute Buffer洗脱纯化柱中的蛋白质，即得纯化的β-葡萄糖苷酶Bgl1-11，将其加入加50％甘油并置于4℃保存备用。文中使用的稀释酶液均为β-葡萄糖苷酶Bgl1-11原液稀释100倍所得。
将获得的粗重组蛋白和纯化后的重组蛋白进行SDS-PAGE凝胶电泳(12％)将粗酶液中蛋白的各个组份分开，用考马斯亮蓝R-250染色，蛋白marker估计酶蛋白的大小，通过蛋白纯化试剂盒纯化蛋白酶蛋白，SDS-PAGE电泳得到一条单一的蛋白条带。SDS-PAGE电泳结果表明，SEQ ID NO.1所述核苷酸序列所编码的多肽在大肠杆菌BL21(DE3)中得到高效表达，且所有重组蛋白均是可溶的，无包涵体形成，初步估计重组蛋白Bgl1-11的分子量约为52kDa。(如附图1所示)
用Quantity One software(BioRad Laboratories Inc.，Hercules，CA)软件分析蛋白表达量，结果显示本发明的多肽在大肠杆菌BL21(DE3)的总可溶表达蛋白中含量高达50％。
实施例2重组β-葡萄糖苷酶Bgl1-11酶活测定
1、酶活的测定
①以ρNPG为底物的β-葡萄糖苷酶活性(U)的定义：以ρNPG为底物时，在pH6.0,45℃每分钟生成1μmolρNP所需的酶量。
②测定原理：对硝基苯-β-D-吡喃葡萄糖苷(ρNPG)为无色化合物，β-葡萄糖苷酶可将ρNPG的β-葡萄糖苷键水解，生成黄色产物对硝基苯(ρNP)，此黄色产物在OD405 nm处有吸收峰，β-葡萄糖苷酶的酶活性大小与生成产物的黄色深浅成正比，故可根据OD405 nm处的吸光度值来判断β-葡萄糖苷酶酶活性的高低。
③测定方法如下：
取25μL纯化后的Bgl1269酶液到475μL5mM的ρNPG溶液中，将反应液混匀，于45℃反应10min，反应结束后立即加入500μL10％的Na2CO3溶液，上下颠倒混匀，室温放置5min，同时以灭活的酶液做空白对照。取300μL反应液，测定OD405nm的吸光度值。
实施例3重组β-葡萄糖苷酶Bgl1-11酶学性质研究
1、重组β-葡萄糖苷酶Bgl1-11的最适反应温度和热稳定性的测定
分别测定pH6.0下不同温度条件的稀释酶液的酶活力，每个温度设三个平行，以酶活力最高者定为100％，以相对酶活力对温度作图。
将酶液分别放入不同温度的水浴中保温1h，取出酶液后置于冰上，设三个平行，同时用未经处理的样品作正对照，按照上述标准方法测定不同温度下样品的酶活力。以未处理酶液的酶活力为100％，以相对酶活力对温度作图。结果分别如附图2、3所示，β-葡萄糖苷酶Bgl1-11的最适反应温度为45℃，该酶在温度低于60℃时非常稳定，60℃保温1h后，相对酶活保持在80％以上，70℃保温10min后仍有50％以上的相对酶活，在70℃保温1h后，该酶完全失活。
2、重组β-葡萄糖苷酶Bgl1-11的最适反应pH和pH稳定性的测定
分别取不同pH的50mM的缓冲液(pH范围4.0-9.5)配置成5mM的ρNPG溶液，加入25μL稀释酶液，设置三个平行，测定45℃下各pH值条件下的酶活。以酶活力最高者定义为100％，以相对酶活力对pH作图。
将稀释酶液在不同pH的缓冲液(pH范围4.0-9.5)中37℃放置1h，各设三个平行，按上述标准方法测定不同pH下保存的β-葡萄糖苷酶Bgl1-11活性，以未处理酶液的酶活力为100％，以相对酶活力对pH作图，结果分别如附图4、5所示重组β-葡萄糖苷酶Bgl1-11的最适反应pH为6.0，在pH在pH5.5-8.5范围内比较稳定，仍保持80％以上的相对酶活。
3、金属离子和相关化学试剂对重组β-葡萄糖苷酶Bgl1-11活性的影响
在酶促反应中加入不同的金属离子和相关化学试剂，研究其对β-葡萄糖苷酶Bgl1-11活性的影响，以未加离子的稀释酶液作为对照。结果见表2：
表2.金属离子及生化试剂对重组β-葡萄糖苷酶Bgl1-11活性的影响
如表2所示，1mM Al3+与10mM Triton X-100对重组β-葡萄糖苷酶Bgl1-11的酶活有明显的促进作用；相反的，当反应体系中含有1mM Cu2+、10mM Tween-80时，该酶酶活受到强烈的抑制；当含稀释酶液的反应体系中分别含有10mM EDTA、1mM FeCl3、1mMCaCl2、1mM MgSO4、1mM Al2(SO4)3、1mM MnCl2、1mM ZnSO4时，该β-葡萄糖苷酶的活性没有显著改变。
实施例3重组β-葡萄糖苷酶Bgl1-11以大豆苷(daidzin)和染料木苷(genistin)为底物的水解反应
本研究选取大豆苷(daidzin)和染料木苷(genistin)为水解对象，用高效液相色谱仪(Agilent1200)进行定量分析，测定重组β-葡萄糖苷酶Bgl1-11对大豆苷(daidzin)和染料木苷(genistin)的水解能力。
表3HPLC检测条件
1、样品处理
取400μl纯化的稀释酶液加入到40μl浓度为0.5mg／mL的大豆苷(daidzin)和染料木苷(genistin)中，空白对照则取400μl纯化的稀释酶液加入到40μl pH6.0的100mM磷酸钠缓冲液中，40℃反应1h，然后加入500μl含有1,000ppm苯甲酸的甲醇溶液终止反应，离心菌体取上清用于HPLC以分析Bgl1-11对大豆苷(daidzin)和染料木苷(genistin)的水解情况。
检测结果如附图6所示，重组β-葡萄糖苷酶Bgl1-11对大豆苷(daidzin)和染料木苷(genistin)降解率均为100％。
综上所述，本发明SEQ ID NO.1所述的来源于海底泥的β-葡萄糖苷酶基因在大肠杆菌表达体系中高效可溶的表达重组蛋白，重组蛋白具有高效水解大豆异黄酮糖苷的作用，因此，对于工业上大批量生产高活性大豆异黄酮苷元产品具有重要的应用潜力。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围。
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ccgaggtggt tcgccgaccg aggtggctgg ctggcgcccg gagctctgga cctcttcggc 420
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cctcccccgg agggcgctga ggtgagctcg atgggctggg aggtccatcc cgacgggttc 900
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gcctccgtcg tcgccgacgg cgtcgacgtc cgcggctact tccactggtc gtcgttcgac 1080
aactacgagt ggggttccta cggaccgcgc ttcggcctca tcggcatcga ccggcaggac 1140
ggcttccgca gggtcgtgcg gccgagcgcg gtgcactacg gggacgtggc gcgcacgggc 1200
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Met Thr Glu Thr Arg Val Pro Asp Phe Pro Asp Gly Phe Leu Trp Gly
1 5 10 15
Ser Ser Thr Ala Ala His Gln Val Glu Gly Gly Asn Thr Asn Asn Asp
20 25 30
Trp Trp Ala Trp Glu His Lys Pro Gly Ser Pro Val Gln Glu Pro Ser
35 40 45
Gly Asp Gly Ile Asp His Leu His Arg Tyr Asp Ala Asp Tyr Ala Leu
50 55 60
Leu Ala Ser Leu Gly Gln Asn Ala His Arg Phe Ser Phe Glu Trp Ser
65 70 75 80
Arg Ile Glu Pro Ala Glu Gly Glu Phe Ser Gln Ala Ala Leu Asp His
85 90 95
Tyr Lys Arg Val Leu Glu Ser Gln His Arg His Gly Leu Thr Pro Phe
100 105 110
Ala Thr Leu Phe His Phe Thr Ser Pro Arg Trp Phe Ala Asp Arg Gly
115 120 125
Gly Trp Leu Ala Pro Gly Ala Leu Asp Leu Phe Gly Arg Tyr Ala Glu
130 135 140
Arg Val Ala Arg Ala Leu Gly Asp Leu Val Pro Tyr Met Gly Thr Val
145 150 155 160
Asn Glu Pro Gln Val Val Ala Leu Met Gly Tyr Leu Ala Gly Gly Phe
165 170 175
Pro Pro Gly Lys Gln Asp Leu Glu Leu Ala Arg Glu Ala Asn Arg Thr
180 185 190
Phe Ala Ala Ala His Arg Thr Ala Val Ala Ala Val Arg Ser Ala Ser
195 200 205
Ser Ser Thr Arg Val Gly Thr Cys Leu Gln Ile Pro Tyr Ile Glu Pro
210 215 220
Leu Arg Pro Asp Asp Glu Ala Asp Arg Ala Ala Ala Ala Arg Met Lys
225 230 235 240
Gly Phe Phe Gly Asp Thr His Leu Asp Asp Leu Arg Thr Ala Gly Asp
245 250 255
Ala Gly Asp Phe Val Gly Leu Gln Tyr Tyr Gly Arg Asp Leu Ile Asp
260 265 270
Ala Thr Ser Pro Ser Phe Lys Ala Pro Pro Pro Glu Gly Ala Glu Val
275 280 285
Ser Ser Met Gly Trp Glu Val His Pro Asp Gly Phe Ala Arg Val Leu
290 295 300
Arg Glu Val Ala Gln Val Gly Leu Pro Ile Ile Val Thr Glu Asn Gly
305 310 315 320
Ile Ala Thr Glu Asp Asp Ser Gln Arg Val Arg Tyr Leu Ala Gly His
325 330 335
Leu Arg Ala Leu Ala Ser Val Val Ala Asp Gly Val Asp Val Arg Gly
340 345 350
Tyr Phe His Trp Ser Ser Phe Asp Asn Tyr Glu Trp Gly Ser Tyr Gly
355 360 365
Pro Arg Phe Gly Leu Ile Gly Ile Asp Arg Gln Asp Gly Phe Arg Arg
370 375 380
Val Val Arg Pro Ser Ala Val His Tyr Gly Asp Val Ala Arg Thr Gly
385 390 395 400
Ser Leu Ala Arg Leu Ala Ala Ala Ser Leu Pro Leu Ser Thr
405 410
Claims (10)
1.一种β-葡萄糖苷酶DNA，其特征在于核苷酸序列如SEQ ID NO.1所示。
2.一种新型β-葡萄糖苷酶，其特征在于氨基酸序列如SEQ ID NO.3所示。
3.一种包含权利1所述的β-葡萄糖苷酶DNA的表达载体。
4.一种基因工程菌，其特征在于通过如权利要求3所述的表达载体转化寄主细胞而得。
5.一种重组β-葡萄糖苷酶的制备方法，其特征是：用权利要求3所述的表达载体转化寄主细胞，培养转化体，从培养物中获得重组β-葡萄糖苷酶。
6.根据权利要求5所述的重组β-葡萄糖苷酶的制备方法，其特征在于：将如权利要求3所述的表达载体的目的片段经Bam HI、Hind III双酶切，与pET-28a (+)载体连接，转化至大肠杆菌BL21，经异丙基硫代-β-D-半乳糖苷(IPTG)诱导，得到高效可溶性表达。
7.根据权利要求6所述的重组β-葡萄糖苷酶的制备方法，其特征是IPTG终浓度为0.1-1.3 mM，诱导温度为18-37℃。
8.如权利要求1所述的新型β-葡萄糖苷酶的宏基因组学克隆方法，其特征是：提取环境中的总DNA并纯化，将纯化后的总DNA经Hind Ⅲ酶切，连接pUC118载体，电击转化大肠杆菌DH5α高效感受态建立宏基因组文库，通过涂布含七叶苷和柠檬酸铁铵的LB平板显色法得到阳性克隆子，经测序和BLAST比较并设计引物，从而克隆到目的片段。
9.权利要求2所述的重组β-葡萄糖苷酶在水解大豆异黄酮糖苷中的应用。
10.如权利要求9所述的应用，其特征在于所述的应用条件为pH 5.5-8.5。
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104498455A (zh) * | 2014-12-01 | 2015-04-08 | 中国农业科学院饲料研究所 | 一种高效降解大豆异黄酮的中温中性β-葡萄糖苷酶HiBgl3B及其基因和应用 |
| CN104726432A (zh) * | 2014-12-22 | 2015-06-24 | 江苏大学 | 一种D型β-葡萄糖苷酶突变体及其表达质粒和重组菌 |
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| CN1944634A (zh) * | 2006-03-08 | 2007-04-11 | 沈阳农业大学 | 水解大豆异黄酮糖苷酶工程菌株、其构建方法及其用途 |
| CN101063158A (zh) * | 2007-05-18 | 2007-10-31 | 东北农业大学 | 一种大豆异黄酮苷元的制备方法 |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104498455A (zh) * | 2014-12-01 | 2015-04-08 | 中国农业科学院饲料研究所 | 一种高效降解大豆异黄酮的中温中性β-葡萄糖苷酶HiBgl3B及其基因和应用 |
| CN104498455B (zh) * | 2014-12-01 | 2017-08-04 | 中国农业科学院饲料研究所 | 一种高效降解大豆异黄酮的中温中性β‑葡萄糖苷酶HiBgl3B及其基因和应用 |
| CN104726432A (zh) * | 2014-12-22 | 2015-06-24 | 江苏大学 | 一种D型β-葡萄糖苷酶突变体及其表达质粒和重组菌 |
| CN104726432B (zh) * | 2014-12-22 | 2017-12-22 | 江苏大学 | 一种D型β‑葡萄糖苷酶突变体及其表达质粒和重组菌 |
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