CN108384769A - 一种耐高温复合酶及其应用 - Google Patents

一种耐高温复合酶及其应用 Download PDF

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CN108384769A
CN108384769A CN201810111588.6A CN201810111588A CN108384769A CN 108384769 A CN108384769 A CN 108384769A CN 201810111588 A CN201810111588 A CN 201810111588A CN 108384769 A CN108384769 A CN 108384769A
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赵林果
李琦
裴建军
王佳宏
苏二正
吴涛
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Abstract

一种耐高温复合酶及其应用，由β‑葡萄糖苷酶与β‑木糖苷酶组成。本发明提供的复合酶专一性强，且能够完全转化黄芪甲苷，使得环黄芪醇的转化回收率提高。本发明整个工艺完成时间不超过3 h，大大缩短了环黄芪醇的制备工艺，提高了生产效率；且反应过程中不需要添加强还原剂、氧化剂及强酸等试剂，降低了环境污染；该两种酶均可以自制，生产成本大大降低，适宜于工业化生产。

Description

一种耐高温复合酶及其应用
技术领域
本发明涉及酶工程及生物医药技术领域，尤其涉及一种耐高温复合酶及其应用。
背景技术
黄芪是一味具有补气益气作用的传统中药，为黄芪属植物膜荚黄芪和蒙古黄芪的干燥根。黄芪中含有黄芪多糖、黄芪皂苷、黄芪黄酮类等主要活性成分，分别在增强机体免疫力、调节血压、保肝护肝、抗肿瘤及抗衰老等方面具有重要作用。目前，从黄芪中分离得到的三萜皂苷类化合物约有40余种，以黄芪甲苷含量最为丰富。环黄芪醇是黄芪甲苷的苷元部分，是黄芪甲苷在肠道内的主要水解代谢产物。此外，环黄芪醇是当前世界唯一已被证明的能够增加端粒酶活性的化合物，具有抗衰老明星分子之称。然而，环黄芪醇在黄芪中的含量极少，仅占干重的0.1％。因此，相对于直接从黄芪中提取环黄芪醇来说，通过水解黄芪甲苷生成环黄芪醇能够大大提高环黄芪醇的含量。
环黄芪醇制备的关键在于黄芪甲苷中糖苷键的断裂，常用的方法有：Smith降解、酸水解、微生物转化及酶解法。中国发明CN103880910B利用氧化、还原、水解、萃取、纯化等方法得到较高纯度的环黄芪醇，但步骤繁琐，操作复杂，成本较高，且反应过程中有大量甲醛生成，具有一定的污染性。申请号为201510112723.5的中国发明利用硫酸水解制备得到了环黄芪醇，但在水解过程中损失了大量的黄芪甲苷，增加了水解的成本。申请号为201610409748.6的中国发明利用微生物转化降解黄芪甲苷，虽然得到纯度较高的环黄芪醇，但转化效率仅为60％，且微生物培养周期太长，分离纯化难度大。申请号为201710319796.0的中国发明利用纤维素酶及β-葡萄糖苷酶两步酶解法转化黄芪甲苷，由于两种酶的酶解温度及pH均不相同，需要分步调节反应条件，且没有考虑利用特异性切断底物中木糖苷的β-木糖苷酶，同时整个反应过程用酶用量大、耗时长，转化底物浓度偏低，给后期纯化带来困难。
本发明筛选获得了能够有效水解黄芪甲苷上的葡萄糖残基的耐热β-葡萄糖苷酶和水解木糖残基的耐高温β-木糖苷酶，通过双酶同步转化高效制备具有抗衰老活性的环黄芪醇。本发明的技术优势有：(1)酶法转化，安全高效，酶解时间短，得率高；(2)双酶酶解反应温度匹配，最适作用温度高，底物溶解性好；(3)双酶酶解特异性强；(4)双酶催化能力及底物耐受能力强。
发明内容
解决的技术问题：本发明提供一种耐高温复合酶及其应用，本发明提供的复合酶能够同步催化黄芪甲苷制备得到环黄芪醇，酶解温度高，时间短，得率高。
技术方案：一种耐高温复合酶，由β-葡萄糖苷酶与β-木糖苷酶组成。
β-葡萄糖苷酶与β-木糖苷酶活力比为1:1-20:1。
β-葡萄糖苷酶与β-木糖苷酶活力比为5:1。
上述β-葡萄糖苷酶有来源于Dictyoglomus thermophilum DSM 3960 GH 3家族，氨基酸序列如SEQ ID NO:1所示、Thermotoga thermarum DSM 5069TGH 3家族，氨基酸序列如SEQ ID NO:2所示、Thermotoga petrophlia DSM 13995 GH1家族，氨基酸序列如SEQ IDNO:3所示、Thermotoga petrophlia DSM 13995 GH3家族，氨基酸序列如SEQ ID NO:4所示或Sulfolobus islandicus GH1家族，氨基酸序列如SEQ ID NO:5所示；所使用的β-木糖苷酶有来源于Dictyoglomus thermophilum DSM3960GH39家族，氨基酸序列如SEQ ID NO:6所示、Thermotoga thermarum DSM 5069TGH 3家族，氨基酸序列如SEQ ID NO:7所示、Thermotoga petrophlia DSM 13995 GH3家族，氨基酸序列如SEQ ID NO:8所示、Thermoanaerobacterium thermosaccharolyticum DSM571GH120家族，氨基酸序列如SEQID NO:9所示或Aspergillus niger NL-1 GH3家族，氨基酸序列如SEQ ID NO:10所示。
上述复合酶选用来源于Dictyoglomus thermophilum DSM 3960的β-葡萄糖苷酶和β-木糖苷酶基因的大肠杆菌重组菌。
上述耐高温复合酶在转化黄芪甲苷为环黄芪醇中的应用。
酶解的条件为：黄芪甲苷与β-葡萄糖苷酶和β-木糖苷酶于75℃反应3h，酶解的缓冲液为柠檬酸-磷酸氢二钠缓冲液。
上述黄芪甲苷的浓度为0.5～5g/L；β-葡萄糖苷酶的浓度为1U/mL～10U/mL；β-木糖苷酶的浓度为0.2U/mL～2U/mL。
有益效果：1.本发明第一次利用成分确定的重组β-葡萄糖苷酶和β-木糖苷酶催化黄芪甲苷制备环黄芪醇，酶法转化的摩尔转化率为94.5％。
2.本发明第一次筛选获得能够高效催化降解环黄芪醇木糖残基来源于Dictyoglomus thermophilum DSM 3960的耐热β-木糖苷酶，β-木糖苷酶的最适反应温度为75℃，该酶具有良好的耐热性能，在75℃下保温3h酶活基本保持不变。该酶适用于70℃以上高温、偏中性条件下的降解，具有潜在的工业、医药应用价值。
3.本发明筛选获得能够高效催化降解环黄芪醇葡萄糖残基来源于Dictyoglomusthermophilum DSM 3960的耐热β-葡萄糖苷酶，该β-葡萄糖苷酶的最适反应温度为90℃，具有良好的温度稳定性，催化效率高，转化效率快。
4.本发明提供双酶法联用降解黄芪甲苷制备环黄芪醇的方法，专一性强，且能够完全转化黄芪甲苷，使得环黄芪醇的转化回收率提高。本发明整个工艺完成时间不超过3h，大大缩短了环黄芪醇的制备工艺，提高了生产效率；且反应过程中不需要添加强还原剂、氧化剂及强酸等试剂，降低了环境污染；该两种酶均可以自制，生产成本大大降低，适宜于工业化生产。
附图说明
图1是β-葡萄糖苷酶Dth3和β-木糖苷酶Xln-DT转化黄芪甲苷生成环黄芪醇示意图；
图2是不同来源的β-葡萄糖苷酶和β-木糖苷酶对黄芪甲苷转化效率比较示意图；
图3是不同加酶量的β-葡萄糖苷酶和β-木糖苷酶对黄芪甲苷转化效率的影响图；
图4是β-葡萄糖苷酶Dth3和β-木糖苷酶Xln-DT的SDS-PAGE蛋白电泳图；(M：蛋白Maker；1：粗酶Dth3；2：粗酶Xln-DT；3：纯化后Dth3；4：纯化后Xln-DT)；
图5是黄芪甲苷、环黄芪醇标样HPLC谱图；
图6是葡萄糖和木糖对β-葡萄糖苷酶Dth3和β-木糖苷酶Xln-DT活力的影响图；
图7是β-葡萄糖苷酶Dth3和β-木糖苷酶Xln-DT水解黄芪甲苷转化图；(a：标样；b：反应0h；c：反应1h；d：反应3h)。
具体实施方式
本发明提供了一种复合酶酶法制备环黄芪醇的方法，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。为了使本发明的目的、技术方案及优点更加清楚明白，以下结合附图及实施例，对本发明进行进一步的详细说明。相关技术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。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。本发明提供的糖苷酶组合由β-葡萄糖苷酶和β-木糖苷酶组成；本发明实施例中，β-葡萄糖苷酶或β-木糖苷酶通过基因工程的方式制得。
其中，Dictyoglomus thermophilum DSM 3960来源的GH3家族的β-葡萄糖苷酶(氨基酸序列如SEQ ID NO:1)以大肠杆菌表达系统制备获得。表达采用的DNA序列扩增自Dictyoglomus thermophilum DSM 3960基因组DNA，扩增的引物对序列如SEQ ID NO:11～12所示。重组表达的质粒载体为pET28a；插入的酶切位点为Nde I和Not I，大肠杆菌载体为BL21(DE3)。经表达的β-葡萄糖苷酶存在于培养液中，利用His-tag标签进行纯化。
Thermotoga thermarum DSM 5069T来源的GH3家族的β-葡萄糖苷酶(氨基酸序列如SEQ ID NO:2)以大肠杆菌表达系统制备获得。表达采用的DNA序列扩增自Thermotogathermarum DSM 5069T基因组DNA，扩增的引物对序列如SEQ ID NO:13～14所示。重组表达的质粒载体为pET20b；插入的酶切位点为Nde I和Xho I，大肠杆菌载体为BL21(DE3)。经表达的β-葡萄糖苷酶存在于培养液中，利用His-tag标签进行纯化。
Thermotoga petrophila DSM 13995来源的GH1家族的β-葡萄糖苷酶(氨基酸序列如SEQ ID NO:3)以大肠杆菌表达系统制备获得。表达采用的DNA序列扩增自Thermotogapetrophila DSM 13995的基因组DNA，扩增引物对序列如SEQ ID NO:15～16所示。重组表达的质粒载体为pET20b；插入的酶切位点为Nde I和Xho I，大肠杆菌载体为JM109(DE3)。经表达的β-葡萄糖苷酶存在于培养液中，利用His-tag标签进行纯化。
Thermotoga petrophila DSM 13995来源的GH3家族的β-葡萄糖苷酶(氨基酸序列如SEQ ID NO:4)以大肠杆菌表达系统制备获得。表达采用的DNA序列扩增自Thermotogapetrophila DSM 13995的基因组DNA，扩增引物对序列如SEQ ID NO:17～18所示。重组表达的质粒载体为pET20b；插入的酶切位点为Nde I和Xho I，大肠杆菌载体为JM109(DE3)。经表达的β-葡萄糖苷酶存在于培养液中，利用His-tag标签进行纯化。
Sulfolobus islandicus来源的β-葡萄糖苷酶(氨基酸序列如SEQ ID NO:5)以大肠杆菌表达系统制备获得。表达采用的DNA序列以全基因合成方式制得；重组表达的质粒载体为pET20b，大肠杆菌载体为BL21(DE3)。经表达的β-葡萄糖苷酶存在于培养液中，利用His-tag标签进行纯化。
Dictyoglomus thermophilum DSM 3960来源的GH39家族的β-木糖苷酶(氨基酸序列如SEQ ID NO:6)以大肠杆菌表达系统制备获得。表达采用的DNA序列扩增自Dictyoglomus thermophilum DSM 3960基因组DNA，扩增的引物对序列如SEQ ID NO:19～20所示。重组表达的质粒载体为pET20b；插入的酶切位点为BamH I和Xho I，大肠杆菌载体为BL21(DE3)。经表达的β-木糖苷酶存在于培养液中，利用His-tag标签进行纯化。
Thermotoga thermarum DSM 5069T来源的GH3家族的β-木糖苷酶(氨基酸序列如SEQ ID NO:7)以大肠杆菌表达系统制备获得。表达采用的DNA序列扩增自Thermotogathermarum DSM 5069T基因组DNA，扩增的引物对序列如SEQ ID NO:21～22所示。重组表达的质粒载体为pET20b；插入的酶切位点为Nde I和Xho I，大肠杆菌载体为BL21(DE3)。经表达的β-木糖苷酶存在于培养液中，利用His-tag标签进行纯化。
Thermotoga petrophila DSM 13995来源的GH3家族的β-木糖苷酶(氨基酸序列如SEQ ID NO:8)以大肠杆菌表达系统制备获得。表达采用的DNA序列扩增自Thermotogapetrophila DSM 13995的基因组DNA，扩增引物对序列如SEQ ID NO:23～24所示。重组表达的质粒载体为pET20b；插入的酶切位点为Nco I和Xho I，大肠杆菌载体为BL21(DE3)。经表达的β-木糖苷酶存在于培养液中，利用His-tag标签进行纯化。
Thermoanaerobacterium thermosaccharolyticum DSM571来源的GH120家族的β-木糖苷酶(氨基酸序列如SEQ ID NO:9)以大肠杆菌表达系统制备获得。表达采用的DNA序列扩增自Thermoanaerobacterium thermosaccharolyticum DSM571的基因组DNA，扩增引物对序列如SEQ ID NO:25～26所示。重组表达的质粒载体为pET20b；插入的酶切位点为Nde I和Xho I，大肠杆菌载体为BL21(DE3)。经表达的β-木糖苷酶存在于培养液中，利用His-tag标签进行纯化。
Aspergillus niger NL-1来源的GH3家族的β-木糖苷酶(氨基酸序列如SEQ IDNO:10)以毕赤酵母表达系统制备获得。表达所用的DNA序列扩增自Aspergillus niger NL-1的cDNA，扩增引物对序列如SEQ ID NO:27～28所示，插入酶切位点EcoR I和Xba I。酵母载体为Pichia pastoris GS115。连接有β-木糖苷酶序列的质粒载体在转化入酵母前，以BxtXI线性化。经表达的β-木糖苷酶存在于培养液中，经透析纯化获得纯酶。
下面结合附图及具体实施例对本发明的应用原理作进一步描述。
实施例1本发明β-葡萄糖苷酶的筛选
1.1 Dictyoglomus thermophilum DSM 3960来源的β-葡萄糖苷酶基因的克隆、质粒构建及重组酶的制备
1.1.1重组质粒pET-28a-Dth3的构建
Dictyoglomus thermophilum DSM 3960基因组购于德国微生物菌种保藏中心。按照基Dictyoglomus thermophilum DSM 3960基因组(Genbank：CP001146.1)中的β-葡萄糖苷酶基因设计上下游引物：
P1:CTAGCTAGCATGAAACTCGAGTATAAAATTCC(SEQ ID NO:11)
P2:ATTTGCGGCCGC GCTATTTATTTCTTTTAATAGGTTTTCT(SEQ ID NO:12)
下划线表示酶切位点，以Dictyoglomus thermophilum DSM 3960基因组DNA为模板，用合成的引物进行PCR扩增，通过凝胶回收试剂盒对PCR产物进行纯化后得到Dictyoglomus thermophilumβ-葡萄糖苷酶基因。将以上基因序列与pET-28a分别双酶切，并分别割胶回收，浓缩后16℃连接过夜，将连接产物转化大肠杆菌Top10F’感受态细胞，转化产物涂布到含有Kana(100μg/mL)的LB固体培养基上37℃过夜培养，接种几个单菌落到含有Kana(100μg/mL)的LB液体培养基中培养8-10h后，收集菌体提取质粒，酶切验证去除空载质粒，将重组质粒进行核酸序列测定，得到正确的重组表达载体pET-28a-Dth3。
1.1.2重组β-葡萄糖苷酶的制备及纯化
将重组质粒pET-28a-Dth3转化大肠杆菌BL21(DE3)宿主菌(Novagen)，在含有Kana(100μg/mL)的LB平板(LB培养基：胰蛋白胨10g/L，酵母提取物5g/L，NaCl 5g/L，琼脂20g/L)上经过37℃培养过夜，挑转化子到200mL的LB培养基中(100μg/mL Kana)37℃，180rpm振荡培养至OD600为0.8时，加入终浓度为0.1mM异丙基β-D-硫代吡喃半乳糖苷(IPTG)诱导剂，28℃诱导培养8h，用高速冷冻离心机将培养液在4℃下，以13,000rpm离心15min，收集菌体，去上清加入无菌水，超声波破碎细胞，随后70℃热处理30min，再用Ni-NTA亲和层析柱进行纯化最终得到纯化的Dictyoglomus thermophilum DSM 3960来源的β-葡萄糖苷酶Dth3。
1.2 Thermotoga thermarum DSM 5069T来源的β-葡萄糖苷酶基因的克隆、质粒构建及重组酶的制备
1.2.1重组质粒pET-20b-BGL3T的构建
Thermotoga thermarum DSM 5069T基因组购于德国微生物菌种保藏中心。按照基Thermotoga thermarum DSM 5069T基因组中的β-葡萄糖苷酶基因设计上下游引物：
P3:CCCCATATGAAAGAGGTTAATGAAATTCTGAGCAAGCTGACCCTGGAGGAGAAAGTG(SEQ IDNO:13)
P4:CCCCTCGAGCGGCTTAAAGGTGCGCTCCTTCTCGATGCTAAATATCTTGCGCAATCTTA(SEQ IDNO:14)
下划线表示酶切位点，以Thermotoga thermarum DSM 5069T基因组为模板，用合成的引物进行PCR扩增，通过凝胶回收试剂盒对PCR产物进行纯化后得到Thermotogathermarum DSM 5069Tβ-葡萄糖苷酶基因。将以上基因序列与pET-20b分别双酶切，并分别割胶回收，浓缩后16℃连接过夜，将连接产物转化大肠杆菌Top10F’感受态细胞，转化产物涂布到含有Amp(100μg/mL)的LB固体培养基上37℃过夜培养，接种几个单菌落到含有Amp(100μg/mL)的LB液体培养基中培养8-10h后，收集菌体提取质粒，酶切验证去除空载质粒，将重组质粒进行核酸序列测定，得到正确的重组表达载体pET-20b-BGL3T。
1.2.2重组β-葡萄糖苷酶的制备及纯化
将重组质粒pET-20b-BGL3T转化大肠杆菌BL21(DE3)宿主菌(Novagen)，在含有Amp(100μg/mL)的LB平板上经过37℃培养过夜，挑转化子到200mL的LB培养基中(100μg/mLAmp)37℃，180rpm振荡培养至OD600为0.8时，加入终浓度为0.1mM异丙基β-D-硫代吡喃半乳糖苷(IPTG)诱导剂，28℃诱导培养8h，用高速冷冻离心机将培养液在4℃下，以13,000rpm离心15min，收集菌体，去上清加入无菌水，超声波破碎细胞，随后70℃热处理30min，再用Ni-NTA亲和层析柱进行纯化最终得到纯化的Thermotoga thermarum DSM 5069T来源的β-葡萄糖苷酶BGL3T。
1.3 Thermotoga petrophlia DSM 13995来源的β-葡萄糖苷酶基因的克隆、质粒构建及重组酶的制备
1.3.1重组质粒pET-20b-Tpebgl1的构建
Thermotoga petrophlia DSM 13995基因组购于德国微生物菌种保藏中心。按照基Thermotoga petrophlia DSM 13995基因组中的GH1和GH3家族的β-葡萄糖苷酶基因设计上下游引物：
P5:CCCATATGAACGTAAAAAGTTCCCTGAAG(SEQ ID NO:15)
P6:CCCTCGAGTAGAAGGTCTGACAACGAAA(SEQ ID NO:16)
下划线表示酶切位点，以Thermotoga petrophlia DSM 13995基因组为模板，用合成的引物进行PCR扩增，通过凝胶回收试剂盒对PCR产物进行纯化后得到Thermotogapetrophlia DSM 13995β-葡萄糖苷酶基因Tpebgl1。将以上基因序列与pET-20b分别双酶切，并分别割胶回收，浓缩后16℃连接过夜，将连接产物转化大肠杆菌Top10F’感受态细胞，转化产物涂布到含有Amp(100μg/mL)的LB固体培养基上37℃过夜培养，接种几个单菌落到含有Amp(100μg/mL)的LB液体培养基中培养8-10h后，收集菌体提取质粒，酶切验证去除空载质粒，将重组质粒进行核酸序列测定，得到正确的重组表达载体pET-20b-Tpebgl1。
1.3.2重组β-葡萄糖苷酶的制备及纯化
将重组质粒pET-20b-Tpebgl1转化大肠杆菌JM109(DE3)宿主菌(Novagen)，在含有Amp(100μg/mL)的LB平板上经过37℃培养过夜，挑转化子到200mL的LB培养基中(100μg/mLAmp)37℃，180rpm振荡培养至OD600为0.8时，加入终浓度为0.1mM异丙基β-D-硫代吡喃半乳糖苷(IPTG)诱导剂，28℃诱导培养8h，用高速冷冻离心机将培养液在4℃下，以13,000rpm离心15min，收集菌体，去上清加入无菌水，超声波破碎细胞，随后70℃热处理30min，再用Ni-NTA亲和层析柱进行纯化最终得到纯化的β-葡萄糖苷酶Tpebgl1。
1.4 Thermotoga petrophlia DSM 13995来源的β-葡萄糖苷酶基因的克隆、质粒构建及重组酶的制备
1.4.1重组质粒pET-20b-Tpebgl3的构建
Thermotoga petrophlia DSM 13995基因组购于德国微生物菌种保藏中心。按照基Thermotoga petrophlia DSM 13995基因组中的GH1和GH3家族的β-葡萄糖苷酶基因设计上下游引物：
P7:CCCATATGATGGAAAGATCGATGAAATCCTTT(SEQ ID NO:17)
P8:CCCTCGAGACCAAACTTAGAGAAGAGAGGGA(SEQ ID NO:18)
下划线表示酶切位点，以Thermotoga petrophlia DSM 13995基因组为模板，用合成的引物进行PCR扩增，通过凝胶回收试剂盒对PCR产物进行纯化后得到Thermotogapetrophlia DSM 13995β-葡萄糖苷酶基因Tpebgl3。将以上基因序列与pET-20b分别双酶切，并分别割胶回收，浓缩后16℃连接过夜，将连接产物转化大肠杆菌Top10F’感受态细胞，转化产物涂布到含有Amp(100μg/mL)的LB固体培养基上37℃过夜培养，接种几个单菌落到含有Amp(100μg/mL)的LB液体培养基中培养8-10h后，收集菌体提取质粒，酶切验证去除空载质粒，将重组质粒进行核酸序列测定，得到正确的重组表达载体pET-20b-Tpebgl3。
1.4.2重组β-葡萄糖苷酶的制备及纯化
将重组质粒pET-20b-Tpebgl3转化大肠杆菌JM109(DE3)宿主菌(Novagen)，在含有Amp(100μg/mL)的LB平板上经过37℃培养过夜，挑转化子到200mL的LB培养基中(100μg/mLAmp)37℃，180rpm振荡培养至OD600为0.8时，加入终浓度为0.1mM异丙基β-D-硫代吡喃半乳糖苷(IPTG)诱导剂，28℃诱导培养8h，用高速冷冻离心机将培养液在4℃下，以13,000rpm离心15min，收集菌体，去上清加入无菌水，超声波破碎细胞，随后70℃热处理30min，再用Ni-NTA亲和层析柱进行纯化最终得到纯化的β-葡萄糖苷酶Tpebgl3。
1.5 Sulfolobus islandicus来源的β-葡萄糖苷酶基因的克隆、质粒构建及重组酶的制备
1.5.1重组质粒pET-20b-Sibgl1的构建
全基因合成并通过大肠杆菌密码子偏好性优化Sulfolobus islandicus的β-葡萄糖苷酶基因，将其连接到pET-20b质粒上，获得重组质粒pET-20b-Sibgl1。
1.5.2重组β-葡萄糖苷酶的制备及纯化
将重组质粒pET-20b-Sibgl1转化大肠杆菌BL21(DE3)宿主菌(Novagen)，在含有Amp(100μg/mL)的LB平板上经过37℃培养过夜，挑转化子到200mL的LB培养基中(100μg/mLAmp)37℃，180rpm振荡培养至OD600为0.8时，加入终浓度为0.1mM异丙基β-D-硫代吡喃半乳糖苷(IPTG)诱导剂，28℃诱导培养8h，用高速冷冻离心机将培养液在4℃下，以13,000rpm离心15min，收集菌体，去上清加入无菌水，超声波破碎细胞，随后70℃热处理30min，再用Ni-NTA亲和层析柱进行纯化最终得到纯化的β-葡萄糖苷酶Sibgl1。
实施例2本发明β-木糖苷酶的筛选
2.1 Dictyoglomus thermophilum DSM3960来源的β-木糖苷酶基因的克隆、质粒构建及重组酶制备
2.1.1重组质粒pET-20b-Xln-DT的构建
Dictyoglomus thermophilum DSM 3960基因组购于德国微生物菌种保藏中心。按照基Dictyoglomus thermophilum DSM 3960基因组中的GH39家族的β-木糖苷酶基因设计上下游引物：
P9:CGCGGATCCATGAACCATATAAAGATTGAAA(SEQ ID NO:19)
P10:CCGCTCGAGATATCCACCTGGTATTTTGCTATC(SEQ ID NO:20)
下划线表示酶切位点，以Dictyoglomus thermophilum DSM 3960基因组为模板，用合成的引物进行PCR扩增，通过凝胶回收试剂盒对PCR产物进行纯化后得到Dictyoglomusthermophilum DSM 3960 β-木糖苷酶基因Xln-DT。将以上基因序列与pET-20b分别双酶切，并分别割胶回收，浓缩后16℃连接过夜，将连接产物转化大肠杆菌Top10F’感受态细胞，转化产物涂布到含有Amp(100μg/mL)的LB固体培养基上37℃过夜培养，接种几个单菌落到含有Amp(100μg/mL)的LB液体培养基中培养8-10h后，收集菌体提取质粒，酶切验证去除空载质粒，将重组质粒进行核酸序列测定，得到正确的重组表达载体pET-20b-Xln-DT。
2.1.2重组β-木糖苷酶的制备及纯化
将重组质粒pET-20b-Xln-DT转化大肠杆菌BL21(DE3)宿主菌(Novagen)，在含有Amp(100μg/mL)的LB平板上经过37℃培养过夜，挑转化子到200mL的LB培养基中(100μg/mLAmp)37℃，180rpm振荡培养至OD600为0.8时，加入终浓度为0.1mM异丙基β-D-硫代吡喃半乳糖苷(IPTG)诱导剂，28℃诱导培养8h，用高速冷冻离心机将培养液在4℃下，以13,000rpm离心15min，收集菌体，去上清加入无菌水，超声波破碎细胞，随后70℃热处理30min，再用Ni-NTA亲和层析柱进行纯化最终得到纯化的β-木糖苷酶Xln-DT。
2.2 Thermotoga thermarum DSM 5069T来源的β-木糖苷酶基因的克隆、质粒构建及重组酶制备
2.2.1重组质粒pET-20b-Tth-xynB3的构建
Thermotoga thermarum DSM 5069T基因组购于德国微生物菌种保藏中心。按照基Thermotoga thermarum DSM 5069T基因组中的GH3家族的β-木糖苷酶基因设计上下游引物：
P11:GGAATTCCATATGGATCTTTACAAGAATCCAAATGTAC(SEQ ID NO:21)
P12:CCGCTCGAGCTCGATCTTTGTATTTGTGAAGAAAAC(SEQ ID NO:22)
下划线表示酶切位点，以Thermotoga thermarum DSM 5069T基因组为模板，用合成的引物进行PCR扩增，通过凝胶回收试剂盒对PCR产物进行纯化后得到Thermotogathermarum DSM 5069Tβ-木糖苷酶基因Tth-xynB3。将以上基因序列与pET-20b分别双酶切，并分别割胶回收，浓缩后16℃连接过夜，将连接产物转化大肠杆菌Top10F’感受态细胞，转化产物涂布到含有Amp(100μg/mL)的LB固体培养基上37℃过夜培养，接种几个单菌落到含有Amp(100μg/mL)的LB液体培养基中培养8-10h后，收集菌体提取质粒，酶切验证去除空载质粒，将重组质粒进行核酸序列测定，得到正确的重组表达载体pET-20b-Tth-xynB3。
2.2.2重组β-木糖苷酶的制备及纯化
将重组质粒pET-20b-Tth-xynB3转化大肠杆菌BL21(DE3)宿主菌(Novagen)，在含有Amp(100μg/mL)的LB平板上经过37℃培养过夜，挑转化子到200mL的LB培养基中(100μg/mL Amp)37℃，180rpm振荡培养至OD600为0.8时，加入终浓度为0.1mM异丙基β-D-硫代吡喃半乳糖苷(IPTG)诱导剂，28℃诱导培养8h，用高速冷冻离心机将培养液在4℃下，以13,000rpm离心15min，收集菌体，去上清加入无菌水，超声波破碎细胞，随后70℃热处理30min，再用Ni-NTA亲和层析柱进行纯化最终得到纯化的β-木糖苷酶Tth-xynB3。
2.3 Thermotoga petrophlia DSM 13995来源的β-木糖苷酶基因的克隆、质粒构建及重组酶制备
2.3.1重组质粒pET-20b-Tpxy3的构建
Thermotoga petrophlia DSM 13995基因组购于德国微生物菌种保藏中心。按照基Thermotoga petrophlia DSM 13995基因组中的GH3家族的β-木糖苷酶基因设计上下游引物：
P13：CATGCCATGGAACTGTACAGGGATCCTTC(SEQ ID NO:23)
P14：CCGCTCGAGCTCCTCGCAGGCTTCCGTGAA(SEQ ID NO:24)
下划线表示酶切位点，以Thermotoga petrophlia DSM 13995基因组为模板，用合成的引物进行PCR扩增，通过凝胶回收试剂盒对PCR产物进行纯化后得到Thermotogapetrophlia DSM 13995β-木糖苷酶基因Tpxy3。将以上基因序列与pET-20b分别双酶切，并分别割胶回收，浓缩后16℃连接过夜，将连接产物转化大肠杆菌Top10F’感受态细胞，转化产物涂布到含有Amp(100μg/mL)的LB固体培养基上37℃过夜培养，接种几个单菌落到含有Amp(100μg/mL)的LB液体培养基中培养8-10h后，收集菌体提取质粒，酶切验证去除空载质粒，将重组质粒进行核酸序列测定，得到正确的重组表达载体pET-20b-Tpxy3。
2.3.2重组β-木糖苷酶的制备及纯化
将重组质粒pET-20b-Tpxy3转化大肠杆菌BL21(DE3)宿主菌(Novagen)，在含有Amp(100μg/mL)的LB平板上经过37℃培养过夜，挑转化子到200mL的LB培养基中(100μg/mLAmp)37℃，180rpm振荡培养至OD600为0.8时，加入终浓度为0.1mM异丙基β-D-硫代吡喃半乳糖苷(IPTG)诱导剂，28℃诱导培养8h，用高速冷冻离心机将培养液在4℃下，以13,000rpm离心15min，收集菌体，去上清加入无菌水，超声波破碎细胞，随后70℃热处理30min，再用Ni-NTA亲和层析柱进行纯化最终得到纯化的β-木糖苷酶Tpxy3。
2.4 Thermoanaerobacterium thermosaccharolyticum DSM571来源的β-木糖苷酶基因的克隆、质粒构建及重组酶制备
2.4.1重组质粒pET-20b-Tth-xyl的构建
Thermoanaerobacterium thermosaccharolyticum DSM571基因组购于德国微生物菌种保藏中心。按照基Thermoanaerobacterium thermosaccharolyticum DSM571基因组中的GH3家族的β-木糖苷酶基因设计上下游引物：
P15：CCCCATATGGAATATCATGTAGCGAA(SEQ ID NO:25)
P16：CCCCTCGAGCCAAACTTTAATATAATTATCG(SEQ ID NO:26)
下划线表示酶切位点，以Thermoanaerobacterium thermosaccharolyticumDSM571基因组为模板，用合成的引物进行PCR扩增，通过凝胶回收试剂盒对PCR产物进行纯化后得到Thermoanaerobacterium thermosaccharolyticum DSM571 β-木糖苷酶基因Tth-xyl。将以上基因序列与pET-20b分别双酶切，并分别割胶回收，浓缩后16℃连接过夜，将连接产物转化大肠杆菌Top10F’感受态细胞，转化产物涂布到含有Amp(100μg/mL)的LB固体培养基上37℃过夜培养，接种几个单菌落到含有Amp(100μg/mL)的LB液体培养基中培养8-10h后，收集菌体提取质粒，酶切验证去除空载质粒，将重组质粒进行核酸序列测定，得到正确的重组表达载体pET-20b-Tth-xyl。
2.4.2重组β-木糖苷酶的制备及纯化
将重组质粒pET-20b-Tth-xyl转化大肠杆菌BL21(DE3)宿主菌(Novagen)，在含有Amp(100μg/mL)的LB平板上经过37℃培养过夜，挑转化子到200mL的LB培养基中(100μg/mLAmp)37℃，180rpm振荡培养至OD600为0.8时，加入终浓度为0.1mM异丙基β-D-硫代吡喃半乳糖苷(IPTG)诱导剂，28℃诱导培养8h，用高速冷冻离心机将培养液在4℃下，以13,000rpm离心15min，收集菌体，去上清加入无菌水，超声波破碎细胞，随后70℃热处理30min，再用Ni-NTA亲和层析柱进行纯化最终得到纯化的β-木糖苷酶Tth-xyl。
2.5 Aspergillus niger NL-1来源的β-木糖苷酶基因的克隆、质粒构建及重组酶的制备
2.5.1 Aspergillus niger NL-1的培养
Aspergillus niger NL-1液体培养基配方为：3g/L葡萄糖，0.1g/L三水合磷酸氢二钾，0.05g/L氯化钾，0.05g/L七水合硫酸镁，0.001g/L七水合硫酸亚铁，0.02g/L硝酸钠。先用PDA培养基活化菌株，然后将活化的Aspergillus niger NL-1孢子用无菌生理盐水重悬，接种于50mL液体种子摇瓶培养基中，28℃，150rpm恒温振荡培养72h，收集菌体。
2.5.2 Aspergillus niger NL-1总RNA的提取
(1)将活化的黑曲霉菌株接种于液体培养基上，28-30℃，培养2天。
(2)过滤收集菌丝体，用无菌水洗涤3-4次至培养基完全洗净，抽干后用锡纸包好放入到液氮中冷冻。
(3)取2g菌丝体放入预冷的研钵中，加入适量液氮研磨直至菌丝体研磨成粉状。
(4)取研磨充分的菌丝体放入EP管中，加入预热到65℃的CTAB抽提液(2％巯基乙醇)，震荡混匀，65℃温浴45-60min。
(5)加入等体积的酚/氯仿/异戊醇(v:v:v＝25:24:1)混合液，混匀，12,000rpm转速下离心20-30min。
(6)吸取上层水相，用酚/氯仿/异戊醇(v:v:v＝25:24:1)混合液重复抽提。
(7)同时加入0.5倍体积冰冷的5M LiCl和0.6倍体积异丙醇，混匀，室温静置20min，12,000rpm离心10min，弃上清。
(8)加入1mL 70％乙醇，彻底悬浮沉淀，静置10min，12,000rpm离心10min，弃上清。重复一次，风干，回收沉淀。
(9)重溶于50μL含有50μg/mL的RNAase的TE缓冲液中，37℃温育1h。
(10)加入0.1倍体积的3mol/L乙酸钠(pH 5.2)和3倍体积的无水乙醇，混匀后-20℃保温1h。
(11)12,000rpm，4℃，30min离心，小心倒掉清液。
(12)加入500μL 70％乙醇，室温静置10min，4℃，12,000rpm离心30min。
(13)弃净上清液，在超净工作台上风干。
(14)加入50μL TE缓冲液重悬，-20℃保存。
2.5.3 Aspergillus niger NL-1cDNA的获得
以Aspergillus niger NL-1菌株总RNA为模板，利用逆转录合成cDNA第一链(以下各逆转录所用试剂均来自于试剂盒“PrimeScript TM1st Strand cDNA Synthesis Kit”，购自TaKaRa公司)。
在微量离心管中配制下列模板RNA/Primer反应液：
混匀后65℃下保温5min后冰上放置1min
在上述微量离心管中配制下列cDNA合成反应液：
上述反应液混匀后在50℃下保温1h，70℃下保温15min后冰上冷却，得到的反应液立即用于cDNA第二链的合成。
2.5.4 Aspergillus niger NL-1木糖苷酶基因xlnD的获得
参照NCBI上公布的黑曲霉木糖苷酶基因，进行同源性分析，根据保守序列设计黑曲霉木糖苷酶基因xlnD的上下游2条特异性引物，以提取的cDNA为模板，用合成的引物进行PCR扩增。通过凝胶回收试剂盒对PCR扩增产物进行纯化。得到Aspergillus niger NL-1来源的β-木糖苷酶基因。
P17：CCCGAATTCCAGGCCAACACCAGCTACGTC(SEQ ID NO:27)
P18：CCCTCTAGACTACTCCTTCCCCGGCCACTT(SEQ ID NO:28)
将得到Aspergillus niger NL-1来源的β-木糖苷酶基因和pPICZαA分别进行双酶切，并分别割胶回收，浓缩后16℃连接过夜，转化液涂布于含有Zencin(终浓度为25μg/mL)的LLB平板，37℃恒温倒置培养12h，挑选阳性克隆测序后，获得重组表达质粒命名为pPICZαA-xlnD。
2.5.5重组酶的制备
提取重组质粒pPICZαA-xlnD，经BxtXⅠ线性化后，采用电转化法将其导入Pichiapastoris GS115(Novagen)中，筛选阳性克隆。接种在YPD培养基中进行活化后，转入BMGY培养基中继续活化，收集OD600为2.0-3.0的菌体，转入到BMMY培养基中，置于30℃，180rpm摇床进行β-木糖苷酶的诱导表达。每隔24h按照体积比0.6％向诱导的菌液中补加无菌的甲醇，培养15天后离心取上清得粗酶液。重组蛋白的纯化：(1)向粗酶液中加终浓度为80％的硫酸铵沉淀蛋白，离心弃上清，用pH 7.5 50mM Tris-HCl缓冲液溶解沉淀的蛋白；(2)用pH 7.550mM Tris-HCl缓冲液于4℃下透析四次，每次8h，以去除盐溶液；(3)将透析后的酶液加到装好的DEAE SFF柱子中，用浓度20-300mM的NaCl梯度洗脱；(4)取合适浓度的NaCl洗脱下的酶液，用pH 6.5 10mM PB缓冲液于4℃下透析四次，每次8h，以去除盐溶液得纯酶。
实施例3 β-葡萄糖苷酶和β-木糖苷酶酶活测定
以对硝基苯酚-β-葡萄糖苷(pNP-G)为底物，水解得到的对硝基苯酚与碳酸钠发生显色反应，在405nm的波长下测定产物的吸光度。200μL反应体系包括180μL 50mM最适pH缓冲液，10μL 20mM底物，混匀预热后加入10μL稀释酶液，最适温度下反应10min，然后加入600μL 1M NaCO3终止反应，混匀后在405nm条件下酶标仪测定。同时做有酶液无底物的对照和有底物无酶液的对照。
以对硝基苯酚-β-木糖苷(pNP-X)为底物，水解得到的对硝基苯酚与碳酸钠发生显色反应，在405nm的波长下测定产物的吸光度。200μL反应体系包括180μL 50mM最适pH的缓冲液，10μL 20mM底物，混匀预热后加入10μL稀释酶液，最适温度下反应10min，然后加入600μL 1M NaCO3终止反应，混匀后在405nm条件下酶标仪测定。同时做有酶液无底物的对照和有底物无酶液的对照。
一个酶活单位(U)定义为：在最适温度和pH的条件下，水解释放1μmol对硝基酚所需的酶量。
对照标准曲线，计算酶活：
酶活(U/mL)＝c×V1/(t×V2)×N
c：由对硝基苯酚标准方程计算出的酶反应后的对硝基苯酚含量(μmol/mL)；
V1：反应体系总体积(mL)；
t：酶与底物反应时间(min)；
V2：酶反应时酶液的体积(mL)；
N：酶液稀释倍数。
实施例4 β-葡萄糖苷酶和β-木糖苷酶双酶转化黄芪甲苷制备环黄芪醇
4.1不同来源的重组酶降解黄芪甲苷催化能力的比较
4.1.1不同来源的重组β-葡萄糖苷酶降解黄芪甲苷催化能力的比较
黄芪甲苷的浓度为1g/L，以五种不同来源的β-葡萄糖苷酶在各自最适温度，最适pH为转化条件，均加入5U/mL的酶，反应30min，通过HPLC进行检测。结果表明：反应30min后，来源于Dictyoglomus thermophilum DSM 3960 GH 3家族的β-葡萄糖苷酶转化黄芪甲苷生成环黄芪醇-6-O-葡萄糖苷的摩尔转化率最高为：96％(图2a)，其中来源于GH3家族的三种β-葡萄糖苷酶Dth3、BGL3T和Tpebgl3转化效率要比GH1家族的两种β-葡萄糖苷酶Tpebgl1和Sibgl1强，说明GH3家族的β-葡萄糖苷酶能够有效的降解黄芪甲苷生成环黄芪醇-6-O-葡萄糖苷。
4.1.2不同来源的重组β-木糖苷酶降解黄芪甲苷催化能力的比较
黄芪甲苷的浓度为1g/L，以五种不同来源的β-木糖苷酶在各自最适温度，最适pH为转化条件，均加入1U/mL的纯酶，反应1h，通过HPLC进行检测。结果表明：反应1h后，仅有来源于Dictyoglomus thermophilum DSM 3960 GH39家族的β-木糖苷酶能够水解黄芪甲苷C-3位上的木糖基，说明来源于Dictyoglomus thermophilum DSM 3960 GH39家族的β-木糖苷酶能够有效水解环黄芪醇-6-O-葡萄糖苷生成环黄芪醇(图2b)。
4.1.3来源于Dictyoglomus thermophilum DSM 3960的β-葡萄糖苷酶和β-木糖苷酶加酶量对黄芪甲苷催化能力的比较
比较了不同来源的β-葡萄糖苷酶和β-木糖苷酶对降解黄芪甲苷的催化能力，从图2中可以看出，本发明选择的5种不同来源、不同家族的β-葡萄糖苷酶均能有效降解黄芪甲苷生成环黄芪醇-6-O-葡萄糖苷，且来源于Dictyoglomus thermophilum DSM 3960的GH3家族的Dth3能够在30min内迅速作用，效率最佳。而5种不同来源、不同家族的β-木糖苷酶中，仅有来源于Dictyoglomus thermophilum DSM 3960 GH39家族的β-木糖苷酶能够水解黄芪甲苷C-3位上的木糖基。因此，在双酶水解黄芪甲苷生成环黄芪醇的过程中，选用Dictyoglomus thermophilum DSM 3960 GH39家族的β-木糖苷酶和不同来源的β-葡萄糖苷酶搭配。优选在C-6位置上作用效果最佳的β-葡萄糖苷酶Dth3与β-木糖苷酶Xln-DT联用。
比较了不同的加酶量对黄芪甲苷催化能力，由图3可知，随着加酶量的增加，催化效率也随之增加，当β-葡萄糖苷酶加酶量达到5U/mL、β-木糖苷酶加酶量达到1U/mL时,其转化效率达到最大值。考虑其经济成本，故选择最少的加酶量达到最佳的转化效率。因此，在本发明中优选两种来源于Dictyoglomus thermophilum DSM 3960的耐高温的糖苷酶Dth3和Xln-DT，其加酶量的最佳搭配为:5U/mL的β-葡萄糖苷酶和1U/mL的β-木糖苷酶。
4.2 Dth3和Xln-DT的糖耐受性及底物耐受性
不同浓度的葡萄糖、木糖对β-葡萄糖苷酶Dth3和β-木糖苷酶Xln-DT酶活力的影响。随着葡萄糖和木糖浓度的增加，β-葡萄糖苷酶Dth3和β-木糖苷酶Xln-DT显示出了极好的糖耐受力。其中，木糖对于β-葡萄糖苷酶Dth3的活力没有影响；在1M葡萄糖的存在下，β-葡萄糖苷酶Dth3的相对活力仍能达到90％以上。葡萄糖对于β-木糖苷酶Xln-DT有激活作用，在1M的葡萄糖的存在下，β-木糖苷酶Xln-DT的活力提高了2.2倍。同时，β-木糖苷酶Xln-DT对于木糖的耐受性也极强，在1M的木糖存在下，β-木糖苷酶Xln-DT的相对活力能达到80％以上(图6)。由于两种酶都具有极好的糖耐受性，所以同时加入双酶的转化效率不会受葡萄糖及木糖的反馈抑制。
不同底物浓度下β-葡萄糖苷酶Dth3和β-木糖苷酶Xln-DT对黄芪甲苷的水解效率的影响。随着底物浓度的增加至10g/L时，β-葡萄糖苷酶Dth3的水解效率略有降低，但是也能达到80％以上的转化效率。而β-木糖苷酶Xln-DT对黄芪甲苷具有良好的底物耐受性，随着黄芪甲苷浓度的增加，水解效率不受影响。
4.3酶法催化转化黄芪甲苷制备环黄芪醇的工艺研究
黄芪甲苷的浓度为1g/L，pH 5.5 50mmol/L柠檬酸-磷酸氢二钠缓冲液，加入5U/mLDictyoglomus thermophilum DSM 3960的GH3家族的β-葡萄糖苷酶和1U/mL Dictyoglomusthermophilum DSM 3960 GH39家族的β-木糖苷酶，在75℃下反应3h。通过HPLC进行检测，结果表明：转化黄芪甲苷为环黄芪醇的摩尔转化率为：96.3％。
黄芪甲苷的浓度为5g/L，pH 5.5 50mmol/L柠檬酸-磷酸氢二钠缓冲液，加入5U/mLDictyoglomus thermophilum DSM 3960 GH3家族的β-葡萄糖苷酶和1U/mL Dictyoglomusthermophilum DSM 3960 GH39家族的β-木糖苷酶，于75℃下反应3h后通过HPLC进行检测。结果表明：转化黄芪甲苷生成环黄芪醇的摩尔转化率为：94.5％(图7)。
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> 南京林业大学
<120> 一种耐高温复合酶及其应用
<160> 28
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Pro Ser Lys Val His Gly Ala Ala Gly Glu Thr His Pro Ile Pro Arg
35 40 45
Leu Gly Met Pro Ala Ala Val Leu Ala Asp Gly Pro Ala Gly Leu Arg
50 55 60
Ile Asn Pro Thr Arg Glu Asn Asp Glu Lys Thr Tyr His Ala Thr Ala
65 70 75 80
Phe Pro Val Glu Thr Met Leu Ala Ser Thr Trp Asn Lys Glu Leu Leu
85 90 95
Glu Lys Val Gly Gln Ala Val Gly Glu Glu Val Arg Glu Tyr Gly Val
100 105 110
Asp Ile Leu Leu Ala Pro Ala Met Asn Ile His Arg Asn Pro Leu Cys
115 120 125
Gly Arg Asn Phe Glu Tyr Tyr Ser Glu Asp Pro Val Leu Ser Gly Glu
130 135 140
Met Ala Ala Ala Phe Val Lys Gly Val Gln Ser Gln Gly Val Gly Ala
145 150 155 160
Cys Val Lys His Phe Val Ala Asn Glu Gln Glu Thr Asn Arg Met Gln
165 170 175
Val Asp Thr Ile Val Ser Glu Arg Ala Leu Arg Glu Ile Tyr Leu Lys
180 185 190
Ala Phe Glu Ile Ala Ile Lys Lys Ala Lys Pro Trp Thr Val Met Ser
195 200 205
Ala Tyr Asn Lys Leu Asn Gly Lys Tyr Cys Ser Gln Asn Ala Trp Leu
210 215 220
Leu Lys Lys Val Leu Arg Glu Asp Trp Gly Phe Glu Gly Phe Val Met
225 230 235 240
Ser Asp Trp Tyr Ala Gly Asp Asn Pro Val Glu Gln Ile Lys Ala Gly
245 250 255
Asn Asp Leu Ile Met Pro Gly Lys Thr Tyr Gln Val Asn Phe Gln Arg
260 265 270
Lys Asp Glu Ile Glu Glu Ile Met Gln Ala Leu Lys Glu Gly Lys Leu
275 280 285
Ser Glu Asp Glu Leu Asn Glu Cys Val Arg Asn Ile Leu Asn Val Leu
290 295 300
Leu Lys Thr Pro Ser Phe Arg Lys Tyr Ser Tyr Ser Asn Lys Pro Asp
305 310 315 320
Leu Asp Ala His Ala Lys Ile Ala Tyr Glu Ala Gly Ala Glu Gly Val
325 330 335
Val Leu Leu Lys Asn Asn Asp Val Leu Pro Phe Asp Lys Thr Thr Pro
340 345 350
Phe Ala Leu Phe Gly Thr Gly Gln Ile Glu Thr Ile Lys Gly Gly Thr
355 360 365
Gly Ser Gly Asp Thr His Pro Arg Tyr Ile Val Ser Ile Leu Asp Gly
370 375 380
Ile Lys Glu Ala Gly Leu Lys Val Asp Glu Gln Leu Met Lys Arg Tyr
385 390 395 400
Glu Asp Tyr Ile Ala Lys Met Arg Glu Thr Glu Gln Tyr Lys Pro Arg
405 410 415
Ala Gly Leu Trp Gly Thr Ile Glu Lys Pro Lys Leu Pro Glu Asn Phe
420 425 430
Leu Ser Glu Asp Glu Ile Lys Val Ile Ala Glu Arg Asn Asp Val Gly
435 440 445
Ile Ile Val Ile Ser Arg Ile Ser Gly Glu Gly Tyr Asp Arg Lys Pro
450 455 460
Ile Lys Gly Asp Phe Tyr Leu Ser Asp Asp Glu Phe Glu Leu Ile Gln
465 470 475 480
Lys Val Ser Ser Gln Phe His Lys Leu Glu Lys Lys Val Leu Val Ile
485 490 495
Leu Asn Ile Gly Ser Pro Ile Glu Ile Ala Ser Trp Arg Asp Met Val
500 505 510
Asp Gly Ile Leu Leu Ile Trp Gln Ala Gly Gln Glu Thr Gly Arg Ile
515 520 525
Val Ala Asp Thr Leu Val Gly Lys Ile Asn Pro Ser Gly Lys Leu Pro
530 535 540
Thr Thr Phe Pro Lys Asp Tyr Lys Asp Ile Pro Ser Trp Asn Phe Pro
545 550 555 560
Gly Glu Pro Val Asp Asn Pro Gln Lys Val Val Tyr Glu Glu Asp Ile
565 570 575
Tyr Val Gly Tyr Arg Tyr Tyr Asp Thr Phe Asn Val Glu Pro Ala Tyr
580 585 590
Glu Phe Gly Phe Gly Leu Ser Tyr Thr Lys Phe Glu Tyr Lys Asp Leu
595 600 605
Asn Val Ser Leu Asp Gly Asp Leu Val Lys Ile Ser Tyr Val Val Thr
610 615 620
Asn Val Gly Lys Tyr Pro Gly Lys Glu Ile Ser Gln Val Tyr Val Lys
625 630 635 640
Ala Pro Lys Gly Lys Ile Asn Lys Pro Phe Gln Glu Leu Lys Ala Phe
645 650 655
His Lys Thr Arg Leu Leu Asn Pro Gly Glu Ser Glu Thr Ile Asn Leu
660 665 670
Glu Ile Pro Leu Arg Glu Leu Ala Ser Phe Val Lys Asp Glu Trp Phe
675 680 685
Val Glu Lys Gly Glu Tyr Glu Ile Arg Ile Gly Ala Ser Ser Arg Asp
690 695 700
Ile Arg Leu Arg Lys Ile Phe Ser Ile Glu Lys Glu Arg Thr Phe Lys
705 710 715 720
Pro
<210> 3
<211> 446
<212> PRT
<213> β-葡萄糖苷酶(Thermotoga petrophila DSM 13995 GH1)
<400> 3
Met Asn Val Lys Lys Phe Pro Glu Gly Phe Leu Trp Gly Val Ala Thr
1 5 10 15
Ala Ser Tyr Gln Ile Glu Gly Ser Pro Leu Ala Asp Gly Ala Gly Met
20 25 30
Ser Ile Trp His Thr Phe Ser His Thr Pro Gly Asn Val Lys Asn Gly
35 40 45
Asp Thr Gly Asp Val Ala Cys Asp His Tyr Asn Arg Trp Lys Glu Asp
50 55 60
Ile Glu Ile Ile Glu Lys Leu Gly Val Lys Ala Tyr Arg Phe Ser Ile
65 70 75 80
Ser Trp Pro Arg Ile Leu Pro Glu Gly Thr Gly Arg Val Asn Gln Lys
85 90 95
Gly Leu Asp Phe Tyr Asn Arg Ile Ile Asp Thr Leu Leu Glu Lys Gly
100 105 110
Ile Thr Pro Phe Val Thr Ile Tyr His Trp Asp Leu Pro Phe Ala Leu
115 120 125
Gln Leu Lys Gly Gly Trp Ala Asn Arg Glu Ile Ala Asp Trp Phe Ala
130 135 140
Glu Tyr Ser Arg Val Leu Phe Glu Asn Phe Gly Asp Arg Val Lys Asn
145 150 155 160
Trp Ile Thr Leu Asn Glu Pro Trp Val Val Ala Ile Val Gly His Leu
165 170 175
Tyr Gly Val His Ala Pro Gly Met Arg Asp Ile Tyr Val Ala Phe Arg
180 185 190
Ala Val His Asn Leu Leu Arg Ala His Ala Lys Ala Val Lys Val Phe
195 200 205
Arg Glu Thr Val Lys Asp Gly Lys Ile Gly Ile Val Phe Asn Asn Gly
210 215 220
Tyr Phe Glu Pro Ala Ser Glu Lys Glu Glu Asp Ile Arg Ala Ala Arg
225 230 235 240
Phe Met His Gln Phe Asn Asn Tyr Pro Leu Phe Leu Asn Pro Ile Tyr
245 250 255
Arg Gly Asp Tyr Pro Glu Leu Val Leu Glu Phe Ala Arg Glu Tyr Leu
260 265 270
Pro Glu Asn Tyr Lys Asp Asp Met Ser Glu Ile Gln Glu Lys Ile Asp
275 280 285
Phe Val Gly Leu Asn Tyr Tyr Ser Gly His Leu Val Lys Phe Asp Pro
290 295 300
Asp Ala Pro Ala Lys Val Ser Phe Val Glu Arg Asp Leu Pro Lys Thr
305 310 315 320
Ala Met Gly Trp Glu Ile Val Pro Glu Gly Ile Tyr Trp Ile Leu Lys
325 330 335
Lys Val Lys Glu Glu Tyr Asn Pro Pro Glu Val Tyr Ile Thr Glu Asn
340 345 350
Gly Ala Ala Phe Asp Asp Val Val Ser Glu Asp Gly Arg Val His Asp
355 360 365
Gln Asn Arg Ile Asp Tyr Leu Lys Ala His Ile Gly Gln Ala Trp Lys
370 375 380
Ala Ile Gln Glu Gly Val Pro Leu Lys Gly Tyr Phe Val Trp Ser Leu
385 390 395 400
Leu Asp Asn Phe Glu Trp Ala Glu Gly Tyr Ser Lys Arg Phe Gly Ile
405 410 415
Val Tyr Val Asp Tyr Ser Thr Gln Lys Arg Ile Ile Lys Asp Ser Gly
420 425 430
Tyr Trp Tyr Ser Asn Val Val Lys Ser Asn Ser Leu Glu Asp
435 440 445
<210> 4
<211> 722
<212> PRT
<213> β-葡萄糖苷酶(Thermotoga petrophila DSM 13995 GH3)
<400> 4
Met Met Gly Lys Ile Asp Glu Ile Leu Ser Gln Leu Thr Ile Glu Glu
1 5 10 15
Lys Val Lys Leu Val Val Gly Val Gly Leu Pro Gly Leu Phe Gly Asn
20 25 30
Pro His Ser Arg Val Ala Gly Ala Ala Gly Glu Thr His Pro Val Pro
35 40 45
Arg Leu Gly Ile Pro Ser Phe Val Leu Ala Asp Gly Pro Ala Gly Leu
50 55 60
Arg Ile Asn Pro Thr Arg Glu Asn Asp Glu Asn Thr Tyr Tyr Thr Thr
65 70 75 80
Ala Phe Pro Val Glu Ile Met Leu Ala Ser Thr Trp Asn Lys Asp Leu
85 90 95
Leu Glu Glu Val Gly Lys Ala Met Gly Glu Glu Val Arg Glu Tyr Gly
100 105 110
Val Asp Val Leu Leu Ala Pro Ala Met Asn Ile His Arg Asn Pro Leu
115 120 125
Cys Gly Arg Asn Phe Glu Tyr Tyr Ser Glu Asp Pro Val Leu Ser Gly
130 135 140
Glu Met Ala Ser Ala Phe Val Lys Gly Val Gln Ser Gln Gly Val Gly
145 150 155 160
Ala Cys Ile Lys His Phe Val Ala Asn Asn Gln Glu Thr Asn Arg Met
165 170 175
Val Val Asp Thr Ile Val Ser Glu Arg Ala Leu Arg Glu Ile Tyr Leu
180 185 190
Lys Gly Phe Glu Ile Ala Val Lys Lys Ala Arg Pro Trp Thr Val Met
195 200 205
Ser Ala Tyr Asn Lys Leu Asn Gly Lys Tyr Cys Ser Gln Asn Glu Trp
210 215 220
Leu Leu Lys Lys Val Leu Arg Glu Glu Trp Gly Phe Asp Gly Phe Val
225 230 235 240
Met Ser Asp Trp Tyr Ala Gly Asp Asn Pro Val Glu Gln Leu Lys Ala
245 250 255
Gly Asn Asp Met Ile Met Pro Gly Lys Ala Tyr Gln Val Asn Thr Glu
260 265 270
Arg Arg Asp Glu Ile Glu Glu Ile Met Glu Ala Leu Lys Glu Gly Arg
275 280 285
Leu Ser Glu Glu Val Leu Asn Glu Cys Val Arg Asn Ile Leu Lys Val
290 295 300
Leu Val Asn Ala Pro Ser Phe Lys Gly Tyr Arg Tyr Ser Asn Lys Pro
305 310 315 320
Asp Leu Glu Ser His Ala Lys Val Ala Tyr Glu Ala Gly Val Glu Gly
325 330 335
Val Val Leu Leu Glu Asn Asn Gly Val Leu Pro Phe Asp Glu Ser Ile
340 345 350
His Val Ala Val Phe Gly Thr Gly Gln Ile Glu Thr Ile Lys Gly Gly
355 360 365
Thr Gly Ser Gly Asp Thr His Pro Arg Tyr Thr Ile Ser Ile Leu Glu
370 375 380
Gly Ile Lys Glu Arg Asn Met Lys Phe Asp Glu Glu Leu Thr Ser Ile
385 390 395 400
Tyr Glu Asp Tyr Ile Lys Lys Met Arg Glu Thr Glu Glu Tyr Lys Pro
405 410 415
Arg Thr Asp Ser Trp Gly Thr Val Ile Lys Pro Lys Leu Pro Glu Asn
420 425 430
Phe Leu Ser Glu Lys Glu Ile Lys Lys Ala Ala Lys Lys Asn Asp Ala
435 440 445
Ala Val Val Val Ile Ser Arg Ile Ser Gly Glu Gly Tyr Asp Arg Lys
450 455 460
Pro Val Lys Gly Asp Phe Tyr Leu Ser Asp Asp Glu Leu Glu Leu Ile
465 470 475 480
Lys Thr Val Ser Arg Glu Phe His Glu Gln Gly Lys Lys Val Val Val
485 490 495
Leu Leu Asn Ile Gly Ser Pro Ile Glu Val Ala Ser Trp Arg Asp Leu
500 505 510
Val Asp Gly Ile Leu Leu Val Trp Gln Ala Gly Gln Glu Met Gly Arg
515 520 525
Ile Val Ala Asp Val Leu Val Gly Arg Val Asn Pro Ser Gly Lys Leu
530 535 540
Pro Thr Thr Phe Pro Lys Asp Tyr Ser Asp Val Pro Ser Trp Thr Phe
545 550 555 560
Pro Gly Glu Pro Lys Asp Asn Pro Gln Arg Val Val Tyr Glu Glu Asp
565 570 575
Ile Tyr Val Gly Tyr Arg Tyr Tyr Asp Thr Phe Gly Val Glu Pro Ala
580 585 590
Tyr Glu Phe Gly Tyr Gly Leu Ser Tyr Thr Lys Phe Glu Tyr Lys Asp
595 600 605
Leu Lys Ile Ala Ile Asp Gly Asp Ile Leu Arg Val Ser Tyr Thr Ile
610 615 620
Thr Asn Thr Gly Asp Arg Ala Gly Lys Glu Val Ser Gln Val Tyr Val
625 630 635 640
Lys Ala Pro Lys Gly Lys Ile Asp Lys Pro Phe Gln Glu Leu Lys Ala
645 650 655
Phe His Lys Thr Lys Leu Leu Asn Pro Gly Glu Ser Glu Lys Ile Phe
660 665 670
Leu Glu Ile Pro Leu Arg Asp Leu Ala Ser Phe Asp Gly Lys Glu Trp
675 680 685
Val Val Glu Ser Gly Glu Tyr Glu Val Arg Val Gly Ala Ser Ser Arg
690 695 700
Asp Ile Arg Leu Arg Asp Ile Phe Leu Val Glu Gly Glu Lys Arg Phe
705 710 715 720
Lys Pro
<210> 5
<211> 489
<212> PRT
<213> β-葡萄糖苷酶(Sulfolobus islandicus)
<400> 5
Met Tyr Ser Phe Pro Lys Asn Phe Arg Phe Gly Trp Ser Gln Ala Gly
1 5 10 15
Phe Gln Ser Glu Met Gly Thr Pro Gly Ser Glu Asp Pro Asn Thr Asp
20 25 30
Trp Tyr Lys Trp Val His Asp Pro Glu Asn Ile Ala Ala Gly Leu Val
35 40 45
Ser Gly Asp Leu Pro Glu Asn Gly Pro Gly Tyr Trp Gly Asn Tyr Lys
50 55 60
Thr Phe His Asp Asn Ala Gln Lys Met Gly Leu Lys Met Ala Arg Leu
65 70 75 80
Asn Val Glu Trp Ser Arg Ile Phe Pro Asn Pro Leu Pro Lys Pro Gln
85 90 95
Asn Phe Asp Glu Ser Lys Gln Asp Val Thr Glu Val Glu Ile Asn Gln
100 105 110
Asn Glu Leu Arg Arg Leu Asp Glu His Ala Asn Lys Asp Ala Leu Asn
115 120 125
His Tyr Arg Glu Ile Phe Lys Asp Leu Lys Ser Arg Gly Ile Tyr Phe
130 135 140
Ile Leu Asn Met Tyr His Trp Pro Leu Pro Ser Trp Leu His Asp Pro
145 150 155 160
Ile Arg Val Arg Arg Gly Asp Leu Ser Gly Pro Thr Gly Trp Leu Ser
165 170 175
Thr Arg Thr Val Tyr Glu Phe Ala Arg Phe Ser Ala Tyr Ile Ala Trp
180 185 190
Lys Phe Asp Asp Leu Val Asp Glu Tyr Ser Thr Met Asn Glu Pro Asn
195 200 205
Val Val Gly Gly Leu Gly Tyr Val Gly Val Lys Ser Gly Phe Pro Pro
210 215 220
Gly Tyr Leu Ser Phe Glu Leu Ser Arg Lys Ala Met Tyr Asn Ile Ile
225 230 235 240
Gln Ala His Val Arg Ala Tyr Asp Gly Ile Lys Ser Val Ser Lys Lys
245 250 255
Pro Ile Gly Ile Ile Tyr Ala Asn Ser Ser Phe Gln Pro Leu Thr Glu
260 265 270
Lys Asp Met Glu Ala Val Glu Met Ala Glu Tyr Asp Asn Arg Trp Ala
275 280 285
Phe Phe Asp Ala Ile Ile Arg Gly Glu Ile Met Lys Gly Ser Glu Lys
290 295 300
Val Val Arg Asp Asp Leu Arg Gly Arg Leu Asp Trp Ile Gly Val Asn
305 310 315 320
Tyr Tyr Thr Arg Thr Val Val Lys Lys Thr Glu Lys Gly Tyr Val Ser
325 330 335
Leu Gly Gly Tyr Gly His Gly Cys Glu Arg Asn Ser Val Ser Leu Ala
340 345 350
Gly Leu Pro Thr Ser Asp Phe Gly Trp Glu Phe Phe Pro Glu Gly Leu
355 360 365
Tyr Asp Val Leu Thr Lys Tyr Trp Asn Arg Tyr His Leu His Met Tyr
370 375 380
Val Thr Glu Asn Gly Ile Ala Asp Asp Ala Asp Tyr Gln Arg Pro Tyr
385 390 395 400
Tyr Leu Val Ser His Val Tyr Gln Val His Arg Ala Ile Asn Ser Ser
405 410 415
Ala Asp Val Arg Gly Tyr Leu His Trp Ser Leu Ala Asp Asn Tyr Glu
420 425 430
Trp Ala Ser Gly Phe Ser Met Arg Phe Gly Leu Leu Lys Val Asp Tyr
435 440 445
Gly Thr Lys Arg Leu Tyr Trp Arg Pro Ser Ala Leu Val Tyr Arg Glu
450 455 460
Ile Ala Thr Asn Gly Gly Ile Thr Asp Glu Ile Glu His Leu Asn Ser
465 470 475 480
Val Pro Pro Ile Arg Pro Leu Arg His
485
<210> 6
<211> 502
<212> PRT
<213> β-木糖苷酶(Dictyoglomus thermophilum DSM 3960)
<400> 6
Met Asn His Ile Lys Ile Glu Lys Gly Lys Tyr Val Gly Val Phe Pro
1 5 10 15
Asp Asn Trp Lys Phe Cys Val Gly Ser Gly Arg Ile Gly Leu Ala Leu
20 25 30
Gln Lys Glu Tyr Ile Asp Ala Leu Ser Phe Val Lys Arg His Ile Asp
35 40 45
Phe Lys Tyr Leu Arg Ala His Gly Leu Leu His Asp Asp Val Gly Ile
50 55 60
Tyr Arg Glu Asp Ile Val Asp Gly Lys Thr Ile Pro Phe Tyr Asn Phe
65 70 75 80
Thr Tyr Ile Asp Arg Ile Tyr Asp Ser Phe Leu Glu Ile Gly Ile Arg
85 90 95
Pro Phe Val Glu Ile Gly Phe Met Pro Ser Lys Leu Ala Ser Gly Asp
100 105 110
Gln Thr Val Phe Tyr Trp Arg Gly Asn Val Thr Pro Pro Lys Asp Tyr
115 120 125
Lys Glu Trp Glu Lys Leu Ile Lys Asn Val Val Lys His Phe Ile Asp
130 135 140
Arg Tyr Gly Glu Lys Glu Val Thr Gln Trp Pro Phe Glu Ile Trp Asn
145 150 155 160
Glu Pro Asn Leu Thr Val Phe Trp Lys Asp Ala Asn Gln Ala Glu Tyr
165 170 175
Phe Lys Leu Tyr Glu Val Thr Val Lys Ala Ile Lys Glu Val Asn Glu
180 185 190
Asn Ile Lys Val Gly Gly Pro Ala Ile Cys Gly Gly Ser Asp Tyr Trp
195 200 205
Ile Thr Asp Phe Leu Asn Phe Cys Tyr Lys Asn Asn Val Pro Val Asp
210 215 220
Phe Leu Thr Arg His Ala Tyr Thr Gly Lys Pro Pro Ile Tyr Thr Pro
225 230 235 240
His Phe Val Tyr Gln Asp Val His Pro Ile Glu Tyr Met Leu Asn Glu
245 250 255
Phe Lys Thr Val Arg Glu Met Val Lys Asn Ser Pro Phe Pro Asn Leu
260 265 270
Pro Ile His Ile Thr Glu Phe Asn Ser Ser Tyr His Pro Leu Cys Pro
275 280 285
Ile His Asp Thr Pro Phe Asn Ala Ala Tyr Leu Ala Arg Val Leu Ser
290 295 300
Glu Gly Gly Asp Tyr Val Asp Ser Phe Ser Tyr Trp Thr Phe Ser Asp
305 310 315 320
Val Phe Glu Glu Ala Asp Val Pro Arg Ser Leu Phe His Gly Gly Phe
325 330 335
Gly Leu Val Ala Phe His Asn Ile Pro Lys Pro Val Phe His Met Phe
340 345 350
Thr Phe Phe Asn Ala Met Gly Glu Lys Ile Leu Tyr Arg Asp Asp His
355 360 365
Ile Leu Ile Thr Glu Arg Glu Asp Lys Ser Val Ala Leu Ile Ala Trp
370 375 380
Asn Glu Val Met Thr Lys Glu Glu Asn Gln Glu Arg Lys Tyr Arg Ile
385 390 395 400
Glu Ile Pro Val Asp Tyr Lys Glu Val Phe Ile Lys Gln Lys Leu Ile
405 410 415
Asp Glu Glu Tyr Gly Asn Pro Trp Arg Thr Trp Ile Gln Met Gly Arg
420 425 430
Pro Arg Phe Pro Ser Lys Lys Gln Ile Glu Thr Leu Arg Glu Val Ala
435 440 445
Thr Pro Lys Val Thr Thr Phe Arg Lys Thr Val Glu Asn Gly His Ile
450 455 460
Thr Leu Glu Phe Thr Leu Gly Lys Asn Ala Val Thr Leu Phe Glu Ile
465 470 475 480
Ser Lys Val Ile Asp Glu Ser His Thr Tyr Ile Gly Leu Asp Asp Ser
485 490 495
Lys Ile Pro Gly Gly Tyr
500
<210> 7
<211> 774
<212> PRT
<213> β-木糖苷酶(Thermotoga thermarum DSM 5069T)
<400> 7
Met Asp Leu Tyr Lys Asn Pro Asn Val Pro Ala Lys Val Arg Ala Lys
1 5 10 15
Asp Leu Leu Ser Lys Met Thr Leu Glu Glu Lys Ile Ala Gln Leu Gly
20 25 30
Ser Val Trp Ser Tyr Glu Leu Leu Thr Glu Asp Gly Lys Phe Ser Val
35 40 45
Glu Lys Ala Lys Glu Ile Leu Lys His Gly Ile Gly Gln Ile Thr Arg
50 55 60
Pro Gly Gly Ala Thr Asn Phe Glu Pro Pro Glu Val Ala Lys Leu Val
65 70 75 80
Asn Glu Ile Gln Lys Phe Leu Val Glu Asn Thr Arg Leu Gly Ile Pro
85 90 95
Ala Ile Met His Glu Glu Cys Leu Ala Gly Tyr Met Gly Leu Gly Ala
100 105 110
Thr Ile Phe Pro Gln Pro Ile Gly Met Ala Ser Thr Trp Asp Pro Glu
115 120 125
Leu Val Glu Lys Ile Thr Ser Ala Ile Arg Glu Asp Leu Arg Lys Leu
130 135 140
Gly Ile Thr Gln Gly Leu Ala Pro Val Leu Asp Val Ala Arg Asp Pro
145 150 155 160
Arg Trp Gly Arg Thr Glu Glu Thr Phe Gly Glu Ser Pro Tyr Leu Val
165 170 175
Ala Lys Met Gly Val Ala Tyr Val Lys Gly Leu Gln Gly Ser Asp Ile
180 185 190
Thr Lys Gly Val Val Ala Thr Gly Lys His Phe Ala Gly Tyr Ser Ala
195 200 205
Ser Glu Gly Gly Lys Asn Trp Ala Pro Thr Asn Ile Pro Pro Arg Glu
210 215 220
Leu Arg Glu Val Phe Leu Tyr Pro Phe Glu Ala Ala Val Lys Glu Ala
225 230 235 240
Gly Leu Leu Ser Ile Met Asn Ser Tyr Ser Glu Ile Asp Gly Ile Pro
245 250 255
Cys Ala Ser Asn Arg Glu Leu Leu Thr Asp Ile Leu Arg Arg Thr Trp
260 265 270
Gly Phe Glu Gly Ile Val Val Ser Asp Tyr Phe Ala Val Asp Met Leu
275 280 285
Ala Ala Tyr His Arg Met Ala Lys Asn Lys Ala Glu Ala Ala Lys Tyr
290 295 300
Ala Leu Glu Ala Gly Ile Asp Ile Glu Leu Pro Lys Thr Asp Cys Tyr
305 310 315 320
Leu His Leu Lys Ser Leu Val Glu Asn Gly Ile Ile Ser Glu Lys Leu
325 330 335
Leu Asp Glu Ala Val Leu Arg Val Leu Thr Leu Lys Phe Leu Leu Gly
340 345 350
Leu Phe Glu Asn Pro Tyr Ala Glu Gly Gly Ser Leu Asn Asp His Asn
355 360 365
Asp Ile Ala Leu Glu Ala Ser Arg Lys Ser Ile Val Leu Leu Lys Asn
370 375 380
Asn Gly Ile Leu Pro Leu Lys Asn Asp Val Lys Ile Ala Leu Val Gly
385 390 395 400
Pro Thr Ala Asn Asp Val Arg Asn Leu Leu Gly Asp Tyr Ser Tyr Leu
405 410 415
Val His Ile Lys Thr Leu Leu Glu Asn Val Asn Ala Thr Thr Phe Asn
420 425 430
Ala Pro Lys Phe Asn Leu Lys Lys Val Glu Glu Leu Val Glu Ala His
435 440 445
Leu Lys Lys Ile Pro Ser Ile Leu Ala Glu Phe Ala Lys Arg Ala Lys
450 455 460
Glu Ile Tyr Tyr Ala Lys Gly Cys Asp Ile Thr Asp Pro Ser Arg Glu
465 470 475 480
Gly Phe Ala Glu Ala Leu Glu Ala Ala Lys Lys Ala Asp Val Val Val
485 490 495
Ala Val Val Gly Asp Arg Ser Gly Leu Thr Ile Glu Cys Thr Ser Gly
500 505 510
Glu Ser Arg Asp Met Ala Asn Leu Lys Leu Pro Gly Val Gln Glu Glu
515 520 525
Phe Ile Leu Glu Leu Thr Lys Val Gly Lys Pro Ile Val Val Val Leu
530 535 540
Val Thr Gly Arg Pro Tyr Ser Leu Lys Ala Phe Val Asn Lys Val Asn
545 550 555 560
Ala Ile Val Gln Leu Trp Leu Pro Gly Glu Thr Gly Ala Gln Ala Leu
565 570 575
Ala Glu Val Ile Phe Gly Gln Val Asn Pro Ser Gly Lys Leu Pro Ile
580 585 590
Ser Phe Pro Ala Ser Ala Gly Gln Ile Pro Val Phe His Tyr Val Lys
595 600 605
Pro Ser Gly Gly Arg Ser Cys Trp His Gly Asn Tyr Val Asp Glu Asp
610 615 620
Val Lys Pro Leu Phe Pro Phe Gly His Gly Leu Ser Tyr Thr Thr Phe
625 630 635 640
Ser Tyr Thr Asn Leu Arg Ile Glu Gln Gln Glu Ile Pro Ile Ala Gly
645 650 655
Ser Val Lys Leu Lys Val Asp Val Gln Asn Thr Gly Glu Ile Tyr Gly
660 665 670
Glu Glu Val Val Gln Leu Tyr Ile Ser Arg Glu His Ala Ser Val Thr
675 680 685
Arg Pro Val Lys Glu Leu Lys Gly Phe Ala Arg Val Lys Leu Gln Pro
690 695 700
Lys Glu Thr Lys Thr Val Val Phe Glu Ile His Thr Asp Val Leu Ala
705 710 715 720
Tyr Tyr Asp Arg Asp Met Lys Leu Val Val Glu Pro Gly Glu Tyr Lys
725 730 735
Val Leu Ile Gly Ser Ser Ala Glu Asp Ile Arg Cys Gln Gly Asn Phe
740 745 750
Lys Val Val Gly Glu Lys Arg Leu Val Gln Asp Asn Arg Val Phe Phe
755 760 765
Thr Asn Thr Lys Ile Glu
770
<210> 8
<211> 778
<212> PRT
<213> β-木糖苷酶(Thermotoga petrophlia DSM 13995)
<400> 8
Met Glu Leu Tyr Arg Asp Pro Ser Gln Pro Ile Glu Val Arg Val Arg
1 5 10 15
Asp Leu Leu Ser Arg Met Thr Leu Glu Glu Lys Ala Ala Gln Leu Gly
20 25 30
Ser Val Trp Gly Tyr Glu Leu Ile Asp Glu Arg Gly Lys Phe Ser Arg
35 40 45
Glu Lys Ala Lys Glu Leu Leu Lys Asn Gly Ile Gly Gln Val Thr Arg
50 55 60
Pro Gly Gly Ser Thr Asn Leu Glu Pro Gln Glu Ala Ala Glu Leu Val
65 70 75 80
Asn Glu Ile Gln Arg Phe Leu Val Glu Glu Thr Arg Leu Gly Ile Pro
85 90 95
Ala Met Ile His Glu Glu Cys Leu Thr Gly Tyr Met Gly Leu Gly Gly
100 105 110
Thr Asn Phe Pro Gln Ala Ile Ala Met Ala Ser Thr Trp Asp Pro Asp
115 120 125
Leu Ile Glu Lys Met Thr Thr Ala Ile Arg Glu Asp Met Arg Lys Ile
130 135 140
Gly Ala His Gln Gly Leu Ala Pro Val Leu Asp Val Ala Arg Asp Pro
145 150 155 160
Arg Trp Gly Arg Thr Glu Glu Thr Phe Gly Glu Ser Pro Tyr Leu Val
165 170 175
Ala Arg Met Gly Val Ser Tyr Val Lys Gly Leu Gln Gly Glu Asp Ile
180 185 190
Lys Lys Gly Val Val Ala Thr Val Lys His Phe Ala Gly Tyr Ser Ala
195 200 205
Ser Glu Gly Gly Lys Asn Trp Ala Pro Thr Asn Ile Pro Glu Arg Glu
210 215 220
Phe Lys Glu Val Phe Leu Phe Pro Phe Glu Ala Ala Val Lys Glu Ala
225 230 235 240
Asn Val Leu Ser Val Met Asn Ser Tyr Ser Glu Ile Asp Gly Val Pro
245 250 255
Cys Ala Ala Asn Arg Lys Leu Leu Thr Asp Ile Leu Arg Lys Asp Trp
260 265 270
Gly Phe Lys Gly Ile Val Val Ser Asp Tyr Phe Ala Val Lys Val Leu
275 280 285
Glu Asp Tyr His Arg Ile Ala Arg Asp Lys Ser Glu Ala Ala Arg Leu
290 295 300
Ala Leu Glu Ala Gly Ile Asp Val Glu Leu Pro Lys Thr Glu Cys Tyr
305 310 315 320
Gln Tyr Leu Lys Asp Leu Val Glu Lys Gly Ile Ile Ser Glu Ala Leu
325 330 335
Ile Asp Glu Ala Val Ala Arg Val Leu Arg Leu Lys Phe Met Leu Gly
340 345 350
Leu Phe Glu Asn Pro Tyr Val Glu Val Glu Lys Ala Lys Ile Glu Ser
355 360 365
His Lys Asp Ile Ala Leu Asp Ile Ala Arg Lys Ser Ile Ile Leu Leu
370 375 380
Lys Asn Asp Gly Ile Leu Pro Leu Gln Lys Asn Lys Lys Val Ala Leu
385 390 395 400
Ile Gly Pro Asn Ala Gly Glu Val Arg Asn Leu Leu Gly Asp Tyr Met
405 410 415
Tyr Leu Ala His Ile Arg Ala Leu Leu Asp Asn Ile Asp Asp Val Phe
420 425 430
Gly Asn Pro Gln Ile Pro Arg Glu Asn Tyr Glu Arg Leu Lys Lys Ser
435 440 445
Ile Glu Glu His Met Lys Ser Ile Pro Ser Val Leu Asp Ala Phe Lys
450 455 460
Glu Glu Gly Ile Glu Phe Glu Tyr Ala Lys Gly Cys Glu Val Thr Gly
465 470 475 480
Glu Asp Arg Ser Gly Phe Glu Glu Ala Ile Glu Ile Ala Lys Lys Ser
485 490 495
Asp Val Ala Ile Val Val Val Gly Asp Lys Ser Gly Leu Thr Leu Asp
500 505 510
Cys Thr Thr Gly Glu Ser Arg Asp Met Ala Asn Leu Lys Leu Pro Gly
515 520 525
Val Gln Glu Glu Leu Val Leu Glu Val Ala Lys Thr Gly Lys Pro Val
530 535 540
Val Leu Val Leu Ile Thr Gly Arg Pro Tyr Ser Leu Lys Asn Val Val
545 550 555 560
Asp Lys Val Asn Ala Ile Leu Gln Val Trp Leu Pro Gly Glu Ala Gly
565 570 575
Gly Arg Ala Ile Val Asp Ile Ile Tyr Gly Lys Val Asn Pro Ser Gly
580 585 590
Lys Leu Pro Ile Ser Phe Pro Arg Ser Ala Gly Gln Ile Pro Val Phe
595 600 605
His Tyr Val Lys Pro Ser Gly Gly Arg Ser His Trp His Gly Asp Tyr
610 615 620
Val Asp Glu Ser Thr Lys Pro Leu Phe Pro Phe Gly His Gly Leu Ser
625 630 635 640
Tyr Thr Lys Phe Glu Tyr Ser Asn Leu Arg Ile Glu Pro Lys Glu Val
645 650 655
Pro Pro Ala Gly Glu Val Val Ile Lys Val Asp Val Glu Asn Thr Gly
660 665 670
Asp Arg Asp Gly Asp Glu Val Val Gln Leu Tyr Ile Gly Arg Glu Phe
675 680 685
Ala Ser Val Thr Arg Pro Val Lys Glu Leu Lys Gly Phe Lys Arg Val
690 695 700
Ser Leu Lys Ala Lys Glu Lys Lys Thr Val Val Phe Arg Leu His Met
705 710 715 720
Asp Val Leu Ala Tyr Tyr Asp Arg Asp Met Lys Leu Val Val Glu Pro
725 730 735
Gly Glu Phe Lys Val Met Val Gly Ser Ser Ser Glu Asp Ile Arg Leu
740 745 750
Thr Gly Ser Phe Thr Val Val Gly Glu Lys Arg Glu Val Val Gly Met
755 760 765
Arg Lys Phe Phe Thr Glu Ala Cys Glu Glu
770 775
<210> 9
<211> 638
<212> PRT
<213> β-木糖苷酶(Thermoanaerobacterium thermosaccharolyticum DSM571)
<400> 9
Met Glu Tyr His Val Ala Lys Thr Gly Ser Asp Gln Gly Lys Gly Thr
1 5 10 15
Leu Lys Asp Pro Phe Leu Thr Ile Asn Lys Ala Ala Ser Val Ala Met
20 25 30
Ala Gly Asp Thr Ile Ile Val His Glu Gly Val Tyr Arg Glu Trp Val
35 40 45
Lys Pro Lys Tyr Lys Gly Leu Ser Asp Lys Arg Arg Ile Thr Tyr Lys
50 55 60
Ala Ala Glu Gly Glu Lys Val Val Ile Lys Gly Ser Glu Arg Ile Gln
65 70 75 80
Ser Trp His His Val Glu Gly Asn Val Trp Lys Cys Gln Leu Pro Asn
85 90 95
Ser Phe Phe Gly Glu Phe Asn Pro Tyr Lys Glu Glu Val Phe Gly Asp
100 105 110
Trp Leu Leu Thr Val Glu Glu Lys Lys His Leu Gly Asp Val Tyr Leu
115 120 125
Asn Gly Met Ser Phe Tyr Glu Val Thr Ser Tyr Glu Gln Leu Ile Asp
130 135 140
Pro Gln Val Arg Thr Glu Ile Ile Asp His Trp Thr Gln Lys Ile Val
145 150 155 160
Pro Val His Asn Val Glu Gln Thr Lys Tyr Val Trp Tyr Ala Glu Val
165 170 175
Asp Ser Glu Lys Thr Thr Ile Tyr Ala Asn Phe Gln Gly Ala Asp Pro
180 185 190
Asn Glu Glu Phe Val Glu Ile Asn Val Arg Arg Ser Cys Phe Tyr Pro
195 200 205
Val Glu Thr Gly Ile Asp Tyr Ile Thr Val Lys Gly Phe Glu Met Ala
210 215 220
His Ala Ala Thr Pro Trp Ala Pro Pro Thr Ala Asp Gln Pro Gly Leu
225 230 235 240
Ile Gly Pro Asn Trp Ser Lys Gly Trp Ile Ile Glu Asp Asn Ile Ile
245 250 255
His Asp Ala Lys Cys Ser Ala Ile Ser Ile Gly Lys Glu Ala Thr Thr
260 265 270
Gly His Asn Tyr Arg Ser Ile Arg Lys Asp Lys Pro Gly Tyr Gln Tyr
275 280 285
Gln Leu Glu Ala Val Phe Ser Ala Glu Arg Asn Gly Trp Ser Lys Glu
290 295 300
Lys Ile Gly Ser His Ile Ile Arg Asn Asn Thr Ile Tyr Asp Cys Gly
305 310 315 320
Gln Asn Ala Ile Val Gly His Leu Gly Cys Val Phe Ser Glu Ile Tyr
325 330 335
Asn Asn His Ile Tyr Asn Ile Ala Leu Lys Arg Glu Phe Tyr Gly His
340 345 350
Glu Ile Ala Gly Ile Lys Leu His Ala Ala Ile Asp Val Gln Ile Tyr
355 360 365
His Asn Arg Ile His Asp Cys Ser Leu Gly Leu Trp Leu Asp Trp Glu
370 375 380
Ala Gln Gly Thr Arg Val Ser Lys Asn Leu Phe Tyr Asn Asn Asn Arg
385 390 395 400
Asp Ile Phe Val Glu Val Ser His Gly Pro Tyr Val Val Asp His Asn
405 410 415
Ile Leu Ala Ser Glu Tyr Ala Ile Asp Asn Met Ser Gln Gly Gly Ala
420 425 430
Tyr Ile Asn Asn Leu Ile Ala Gly Lys Met Asn Gln Arg Lys Val Leu
435 440 445
Asn Arg Ser Thr Gln Tyr His Leu Pro His Ser Thr Lys Val Ala Gly
450 455 460
Phe Ala Phe Val Tyr Gly Gly Asp Asp Arg Phe Tyr Asn Asn Ile Phe
465 470 475 480
Ile Gly Lys Asp Gly Val Glu Asn Val Gly Thr Ser His Tyr Gln Asn
485 490 495
Tyr Thr Thr Ser Leu Glu Glu Tyr Ile Glu Lys Val Asn Ala Val Pro
500 505 510
Gly Asp Leu Gly Glu Phe Glu Arg Val Glu Gln Pro Val Tyr Ile Asn
515 520 525
Lys Asn Ala Tyr Phe Asn Gly Ala Glu Pro Phe Glu Arg Glu Lys Asp
530 535 540
Lys Leu Val Asp Arg Glu Phe Asp Pro Lys Phe Ser Ile Ile Glu Lys
545 550 555 560
Gly Asp Glu Val Tyr Leu Ser Cys Gln Leu Pro Asp Asp Phe Gly Asp
565 570 575
Ile Val Gly Asp Ile His Ser Thr Lys Thr Leu Glu Arg Val Arg Ile
580 585 590
Val Asp Ala Glu Phe Glu Ser Pro Asp Gly Lys Glu Leu Ile Leu Asp
595 600 605
Thr Asp Tyr Leu Asn Leu Lys Lys Ser Glu Ser Ser Pro Ile Gly Pro
610 615 620
Ile Thr Leu Leu Lys Lys Gly Asp Asn Tyr Ile Lys Val Trp
625 630 635
<210> 10
<211> 778
<212> PRT
<213> β-木糖苷酶(Aspergillus niger NL-1)
<400> 10
Gln Ala Asn Thr Ser Tyr Val Asp Tyr Asn Val Glu Ala Asn Pro Asp
1 5 10 15
Leu Tyr Pro Leu Cys Val Glu Thr Ile Pro Leu Ser Phe Pro Asp Cys
20 25 30
Gln Asn Gly Pro Leu Arg Ser His Leu Ile Cys Asp Glu Ser Ala Thr
35 40 45
Pro Tyr Asp Arg Ala Ala Ser Leu Ile Ser Leu Phe Thr Leu Asp Glu
50 55 60
Leu Ile Ala Asn Thr Gly Asn Thr Gly Leu Gly Val Ser Arg Leu Gly
65 70 75 80
Leu Pro Ala Tyr Gln Val Trp Ser Glu Ala Leu His Gly Leu Asp Arg
85 90 95
Ala Asn Phe Ser Asp Ser Gly Ser Tyr Asn Trp Ala Thr Ser Phe Pro
100 105 110
Gln Pro Ile Leu Thr Thr Ala Ala Leu Asn Arg Thr Leu Ile His Gln
115 120 125
Ile Ala Ser Ile Ile Ser Thr Gln Gly Arg Ala Phe Asn Asn Ala Gly
130 135 140
Arg Tyr Gly Leu Asp Val Tyr Ala Pro Asn Ile Asn Thr Phe Arg His
145 150 155 160
Pro Val Trp Gly Arg Gly Gln Glu Thr Pro Gly Glu Asp Val Ser Leu
165 170 175
Ala Ala Val Tyr Ala Tyr Glu Tyr Ile Thr Gly Ile Gln Gly Pro Asp
180 185 190
Pro Asp Ser Asn Leu Lys Leu Ala Ala Thr Ala Lys His Tyr Ala Gly
195 200 205
Tyr Asp Ile Glu Asn Trp His Asn His Ser Arg Leu Gly Asn Asp Met
210 215 220
Asn Ile Thr Gln Gln Asp Leu Ser Glu Tyr Tyr Thr Pro Gln Phe His
225 230 235 240
Val Ala Ala Arg Asp Ala Lys Val His Ser Val Met Cys Ala Tyr Asn
245 250 255
Ala Val Asn Gly Val Pro Ala Cys Ala Asp Ser Tyr Phe Leu Gln Thr
260 265 270
Leu Leu Arg Asp Thr Phe Gly Phe Val Asp His Gly Tyr Val Ser Ser
275 280 285
Asp Cys Asp Ala Ala Tyr Asn Ile Tyr Asn Pro His Gly Tyr Ala Ser
290 295 300
Ser Gln Ala Ala Ala Ala Ala Glu Ala Ile Leu Ala Gly Thr Asp Ile
305 310 315 320
Asp Cys Gly Thr Thr Tyr Gln Trp His Leu Asn Glu Ser Ile Thr Ala
325 330 335
Gly Asp Leu Ser Arg Asp Asp Ile Glu Lys Gly Val Ile Arg Leu Tyr
340 345 350
Thr Thr Leu Val Gln Ala Gly Tyr Phe Asp Ser Asn Thr Thr Lys Ala
355 360 365
Asn Asn Pro Tyr Arg Asp Leu Thr Trp Ser Asp Val Leu Glu Thr Asp
370 375 380
Ala Trp Asn Ile Ser Tyr Gln Ala Ala Thr Gln Gly Ile Val Leu Leu
385 390 395 400
Lys Asn Ser Asn Lys Val Leu Pro Leu Thr Glu Lys Ala Tyr Pro Pro
405 410 415
Ser Asn Thr Thr Val Ala Leu Ile Gly Pro Trp Ala Asn Ala Thr Thr
420 425 430
Gln Leu Leu Gly Asn Tyr Tyr Gly Asn Ala Pro Tyr Met Ile Ser Pro
435 440 445
Arg Val Ala Phe Glu Glu Ala Gly Tyr Asn Val Asn Phe Ala Glu Gly
450 455 460
Thr Gly Ile Ser Ser Thr Ser Thr Ser Gly Phe Ala Ala Ala Leu Ser
465 470 475 480
Ala Ala Gln Ser Ala Asp Val Ile Ile Tyr Ala Gly Gly Ile Asp Asn
485 490 495
Thr Leu Glu Ala Glu Ala Leu Asp Arg Glu Ser Ile Ala Trp Pro Gly
500 505 510
Asn Gln Leu Asp Leu Ile Gln Lys Leu Ala Ser Ser Ala Gly Ser Lys
515 520 525
Pro Leu Ile Val Leu Gln Met Gly Gly Gly Gln Val Asp Ser Ser Ser
530 535 540
Leu Lys Asn Asn Thr Asn Val Ser Ala Leu Leu Trp Gly Gly Tyr Pro
545 550 555 560
Gly Gln Ser Gly Gly Phe Ala Leu Arg Asp Ile Ile Thr Gly Lys Lys
565 570 575
Asn Pro Ala Gly Arg Leu Val Thr Thr Gln Tyr Pro Ala Ser Tyr Ala
580 585 590
Glu Glu Phe Pro Ala Thr Asp Met Asn Leu Arg Pro Glu Gly Asp Asn
595 600 605
Pro Gly Gln Thr Tyr Lys Trp Tyr Thr Gly Glu Ala Val Tyr Glu Phe
610 615 620
Gly His Gly Leu Phe Tyr Thr Thr Phe Ala Glu Ser Ser Ser Asn Thr
625 630 635 640
Thr Thr Arg Glu Ile Lys Leu Asn Ile Gln Asp Ile Leu Ser Gln Thr
645 650 655
His Glu Asp Leu Ala Ser Ile Thr Gln Leu Pro Val Leu Asn Phe Thr
660 665 670
Ala Asn Ile Lys Asn Thr Gly Lys Val Glu Ser Asp Tyr Thr Ala Met
675 680 685
Val Phe Ala Asn Thr Ser Asp Ala Gly Pro Ala Pro Tyr Pro Val Lys
690 695 700
Trp Leu Val Gly Trp Asp Arg Leu Gly Glu Val Lys Val Gly Glu Thr
705 710 715 720
Arg Glu Leu Arg Val Pro Val Glu Val Gly Ser Phe Ala Arg Val Asn
725 730 735
Glu Asp Gly Asp Trp Val Leu Phe Pro Gly Thr Phe Glu Leu Ala Leu
740 745 750
Ile Leu Glu Arg Lys Val Arg Val Lys Val Val Leu Ser Gly Glu Glu
755 760 765
Glu Val Val Leu Lys Trp Pro Gly Lys Glu
770 775
<210> 11
<211> 32
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
ctagctagca tgaaactcga gtataaaatt cc 32
<210> 12
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
atttgcggcc gcgctattta tttcttttaa taggttttct 40
<210> 13
<211> 57
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
ccccatatga aagaggttaa tgaaattctg agcaagctga ccctggagga gaaagtg 57
<210> 14
<211> 59
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
cccctcgagc ggcttaaagg tgcgctcctt ctcgatgcta aatatcttgc gcaatctta 59
<210> 15
<211> 29
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
cccatatgaa cgtaaaaagt tccctgaag 29
<210> 16
<211> 28
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
ccctcgagta gaaggtctga caacgaaa 28
<210> 17
<211> 32
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
cccatatgat ggaaagatcg atgaaatcct tt 32
<210> 18
<211> 31
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
ccctcgagac caaacttaga gaagagaggg a 31
<210> 19
<211> 31
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 19
cgcggatcca tgaaccatat aaagattgaa a 31
<210> 20
<211> 33
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 20
ccgctcgaga tatccacctg gtattttgct atc 33
<210> 21
<211> 38
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 21
ggaattccat atggatcttt acaagaatcc aaatgtac 38
<210> 22
<211> 36
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 22
ccgctcgagc tcgatctttg tatttgtgaa gaaaac 36
<210> 23
<211> 29
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 23
catgccatgg aactgtacag ggatccttc 29
<210> 24
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 24
ccgctcgagc tcctcgcagg cttccgtgaa 30
<210> 25
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 25
ccccatatgg aatatcatgt agcgaa 26
<210> 26
<211> 31
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 26
cccctcgagc caaactttaa tataattatc g 31
<210> 27
<211> 29
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 27
ccgaattcca ggccaacacc agctacgtc 29
<210> 28
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 28
ccctctagac tactccttcc ccggccactt 30

Claims (8)

1.一种耐高温复合酶，其特征在于由β-葡萄糖苷酶与β-木糖苷酶组成。
2.根据权利要求1所述耐高温复合酶，其特征在于β-葡萄糖苷酶与β-木糖苷酶活力比为1:1-20:1。
3.根据权利要求2所述耐高温复合酶，其特征在于β-葡萄糖苷酶与β-木糖苷酶活力比为5:1。
4.根据权利要求1所述耐高温复合酶，其特征在于所述β-葡萄糖苷酶有来源于Dictyoglomus thermophilum DSM 3960 GH 3家族，氨基酸序列如SEQ ID NO:1所示、Thermotoga thermarum DSM 5069T GH 3家族，氨基酸序列如SEQ ID NO:2所示、Thermotoga petrophlia DSM 13995 GH1家族，氨基酸序列如SEQ ID NO:3所示、Thermotoga petrophlia DSM 13995 GH3家族，氨基酸序列如SEQ ID NO:4所示或Sulfolobus islandicus GH1家族，氨基酸序列如SEQ ID NO:5所示；所使用的β-木糖苷酶有来源于Dictyoglomus thermophilum DSM3960 GH39家族，氨基酸序列如SEQ ID NO:6所示、Thermotoga thermarum DSM 5069T GH 3家族，氨基酸序列如SEQ ID NO:7所示、Thermotoga petrophlia DSM 13995 GH3家族，氨基酸序列如SEQ ID NO:8所示、Thermoanaerobacterium thermosaccharolyticum DSM571 GH120家族，氨基酸序列如SEQID NO:9所示或Aspergillus niger NL-1 GH3家族，氨基酸序列如SEQ ID NO:10所示。
5.根据权利要求1所述耐高温复合酶，其特征在于所述复合酶选用来源于Dictyoglomus thermophilum DSM 3960的β-葡萄糖苷酶和β-木糖苷酶基因的大肠杆菌重组菌。
6.权利要求1-5任一项所述耐高温复合酶在转化黄芪甲苷为环黄芪醇中的应用。
7.根据权利要求6所述的应用，其特征在于酶解的条件为：黄芪甲苷与β-葡萄糖苷酶和β-木糖苷酶于75℃反应3 h，酶解的缓冲液为柠檬酸-磷酸氢二钠缓冲液。
8.根据权利要求6所述的应用，其特征在于所述黄芪甲苷的浓度为0.5~5 g/L；β-葡萄糖苷酶的浓度为1 U/mL ~10 U/mL；β-木糖苷酶的浓度为0.2 U/mL ~2 U/mL。
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CN111893158A (zh) * 2020-08-14 2020-11-06 威海百合生物技术股份有限公司 一种利用双酶复合转化黄芪甲苷制备环黄芪醇的方法
CN111849959B (zh) * 2020-08-14 2023-10-27 威海百合生物技术股份有限公司 一种利用共固定双酶催化黄芪甲苷制备环黄芪醇的方法
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