CN114317499B - 一种酸性β-葡萄糖苷酶及其编码基因和应用 - Google Patents
一种酸性β-葡萄糖苷酶及其编码基因和应用 Download PDFInfo
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Abstract
本发明涉及生物技术领域,尤其涉及一种酸性β‑葡萄糖苷酶及其编码基因和应用。本研究利用RNA‑seq成功筛选出GH3家族β‑葡萄糖苷酶基因bgl3HB,其序列全长分别为2688bp,核苷酸序列如SEQ ID NO:1所示,氨基酸序列如SEQ ID NO:2所示。该β‑葡萄糖苷酶(简称Bgl3HB)对芳香基糖苷类底物的催化效率远高于现有技术报道的β‑葡萄糖苷酶,在较高温度条件下长时间保持较高酶活,可在50%乙醇中放置6h后酶活仍保持不变,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,尤其涉及一种酸性β-葡萄糖苷酶及其编码基因和应用。
背景技术
随着化石能源的需求量与消耗量与日俱增,化石能源储备量日益减少,生态环境的破坏逐渐加剧,全球能源危机也日益迫近。第二代生物乙醇作为生物燃料的部分替代品,也是全球最具代表性的替代能源之一,引起了人们的广泛关注。而能够利用可再生生物质资源生产生物乙醇的植物纤维质已成为近新能源近年来的研究热点。
植物生物质是世界上分布最广泛且丰富的可再生生物资源,未来可能将发挥与20世纪石油同样的作用。构成植物生物质最主要的部分是植物细胞壁,占了所有生物碳储备的90%以上。植物生物质主要构成成分是纤维素和半纤维素,若将纤维素降解为可利用的糖类物质,对解决当今世界面临的能源短缺、空气污染和饲料资源短缺等问题具有重大现实意义。
红树林(Mangrove)生长在陆地和海洋之间相对狭窄的边缘,是一种适合耐盐物种的森林且土壤偏酸性。海南岛红树林生态***存在大量植物生物质且其凋落物丰富,使得这种有机质丰富的土壤环境和独特的地理环境存在大量嗜酸、耐盐且高产纤维素酶的微生物。红树林环境的特点是养分的特殊循环和酶的相互作用,这是由于植物根系释放的氧气可以随时利用。
刘四新等(2011)发现,海南红树林土壤pH一般在3.0-7.5之间。一些研究显示,红树林土壤放线菌和真菌较多,最常见的丝状真菌是木霉菌和曲霉菌。丝状真菌产生的β-葡萄糖苷酶因其易回收和活性效价高的特性而在工业上广泛使用。近年来,由于哈茨木霉具有良好的抗生作用和重寄生作用,其研究主要表现在其能分泌抑制病原微生物增殖的挥发性或者非挥发性有毒的代谢物。关于其哈茨木霉β-葡萄糖苷酶方向的报道较少,哈茨木霉菌株很少用于生产纤维素酶,有人研究了丝状真菌从纤维素分解复合体中产酶的潜力,并观察到哈茨木霉IOC-4038在产生高水平的β-葡萄糖苷酶的同时,还能产生大量的内切葡聚糖酶;Elham发现哈茨木霉β-葡萄糖苷酶可以代替化学杀菌剂防治引起作物炭腐病的菜豆大斑病菌;Aboshosha等用紫外线照射和甲烷磺酸乙酯(EMS)处理哈茨木霉得到了高产β-葡萄糖苷酶的菌株。
在实际生产应用中,比如在酿酒过程中,耐酸的β-葡萄糖苷酶在果汁、鸡尾酒和葡萄酒中存在的糖苷前体的酶促释放芳香化合物的过程中起着关键作用。酸性β-葡萄糖苷酶在食品、饮料工业和从纤维素材料生产燃料乙醇非常重要。此外,由于市场上的大多数商品纤维素酶不具备良好的热稳定性,而β-葡萄糖苷酶又是纤维素水解过程中的限速酶,这使得热稳定β-葡萄糖苷酶的需求和市场更加宽广。首先,植物生物质酶促水解反应温度为30-50℃,热稳定的β-葡萄糖苷酶有利于酶促反应;其次,较高温度下进行酶水解反应,可以有效避免杂菌的污染,所以挖掘具有耐酸、具有热稳定性的β-葡萄糖苷酶具备广泛的商业价值和应用前景。
发明内容
有鉴于此,本发明提供了一种酸性β-葡萄糖苷酶及其编码基因和应用。该酸性β-葡萄糖苷酶(简称Bgl3HB)对芳香基糖苷类底物的催化效率远高于现有技术报道的β-葡萄糖苷酶,同时能够耐高温、嗜酸、耐受高浓度盐离子,具有想好的工业应用前景。
为了实现上述发明目的,本发明提供以下技术方案:
一种酸性β-葡萄糖苷酶,其氨基酸序列如SEQ ID NO:2所示或为SEQ ID NO:2所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的功能相同或相似的氨基酸序列。
本发明利用RNA-seq测序筛选表达量高且差异性好的β-葡萄糖苷酶基因,并在野生型毕赤酵母X33中表达,通过阴离子交换柱UNOsphereTM Q Cartridges和镍柱NuviaTMIMAC Ni-Charged分离纯化得到高活性、耐高温、嗜酸且能耐受高浓度盐离子的β-葡萄糖苷酶,其氨基酸序列如SEQ ID NO:2所示。经测定该蛋白分子大小约为90kD;以pNPG为底物测定Bgl3HB反应最适反应pH为5.0,反应最适温度为40℃。
一些实施方案中,所述酸性β-葡萄糖苷酶的制备方法,包括如下步骤:
1)、将SEQ ID NO:2所示的β-葡萄糖苷酶的编码基因克隆至表达载体,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-葡萄糖苷酶的基因,即bgl3HB基因。
一些实施方案中,所述bgl3HB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/或增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。
本发明利用RNA-seq成功筛选出GH3家族β-葡萄糖苷酶基因bgl3HB,其序列全长为2688bp,其核苷酸序列如SEQ ID NO:1,该基因来源于哈茨木霉菌株。
同时,本发明还提供了含有所述bgl3HB基因的重组载体,以及转化有所述重组载体的宿主菌。
一些实施方案中,所述宿主菌包括大肠杆菌、毕赤酵母。
另外,本发明还提供了所述的β-葡萄糖苷酶、所述的基因、所述的重组载体或所述的宿主菌在制备还原糖、葡萄糖、生物乙醇中的至少一种物质的应用。
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。
一些实施方案中,所述纤维素原料包括甘蔗渣、玉米秸秆、糟渣中的至少一种,包括但不仅限于此。
本发明提供了β-葡萄糖苷酶及其编码基因和应用。与现有技术相比,本发明β-葡萄糖苷酶对芳香基糖苷类底物的催化效率远高于现有的β-葡萄糖苷酶,在较高温度条件下长时间保持较高酶活,可在50%乙醇中放置6h后酶活仍保持不变,具有良好的应用前景。
附图说明
图1示内参基因RT-PCR扩增的熔解曲线(a)beta-tublin,(b)actin;
图2示待测基因的QT-PCR扩增的熔解曲线;
图3示Bgl3HB蛋白质结构域分析示意图;
图4示Bgl3HB dbCAN数据库注释结果;
图5示Trichoderma harzianum总RNA琼脂糖凝胶电泳;
图6示β-葡萄糖苷酶基因bgl3HB表达序列的琼脂糖凝胶电泳分析;M:分子量标准,泳道1:bgl3HB基因;
图7示重组产物pPICZαA/bgl3HB转化感受态的平板培养结果;
图8示pPICZαA/bgl3HB重组质粒的菌落PCR鉴定结果;M:分子量标准,泳道1:pPICZαA/bgl3HB重组质粒;
图9示重组质粒pPICZαA/bgl3HB毕赤酵母转化子的生长情况;
图10示pPICZαA/bgl3HB/X33毕赤酵母转化子菌落PCR鉴定结果;
图11示重组菌pPICZαA/bgl2/X33在含400μg/mL(a)、800μg/mL(b)、1200μg/mL(c)、2000μg/mL(d)Zeocin的YPD平板的生长情况;
图12示Bgl3HB柱层析纯化Bgl3HB图谱;
图13示Bgl3HB的SDS-PAGE分析结果;M:170Kda protein Marker;1:纯化后的Bgl3HB
图14示pNP浓度标准曲线;
图15示DNS法葡萄糖标准曲线;
图16示GOD法葡萄糖标准曲线;
图17示BCA法蛋白定量标准曲线;
图18示反应pH对酶活的影响;
图19示pH稳定性检测结果;
图20示反应温度对酶活的影响;
图21示热稳定性检测结果;
图22示金属离子对酶活的影响;
图23示NaCl对Bgl3HB酶活性的影响;
图24示Bgl3HB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β-葡萄糖苷酶及其编码基因和应用。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)实验方法
1、哈茨木霉RNA-seq测序
1.1样品的制备及Unigene注释
以葡萄糖(GLU)和微晶纤维素(MCC)为唯一碳源的培养基分别诱导β-葡萄糖苷酶的表达,其中GLU是对照组,MCC是处理组。对基因进行基础的四大数据库注释,GO注释,用transdecoder进行CDS预测。
1.2基因表达定量和差异表达分析
比对软件bowtie2用于比较每个样本与参考转录组的读数。此步骤用于比较表达的基因及其定量,以便对转录组进行全面评估和鉴定。
1.3目的基因的筛选及验证
利用FDR与log2FC来筛选差异基因,筛选条件为(1)FDR<0.05;(2)|log2FC|>2;(3)差异性显著。
对筛选出的基因进行序列分析,包括信号肽预测、活性结构域分析,按照所筛选基因的核酸序列设计特异性引物,通过RT-PCR及QPCR验证基因表达情况。
2、bgl3HB基因的克隆及序列分析
2.1哈茨木霉菌株总RNA提取及第一链cDNA合成
采用E.Z.N.A.TM Fungal RNA Kit试剂盒来提取前期发酵得到的哈茨木霉菌株的总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六天之后,用1.5mL离心管收集菌球;将离心机的转速设置为12000xg,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1.5mL离心管中加入500μL(或600μL,跟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***解菌体;
(2)在混合液中加入500μL Phenol water和100μL 2M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氯仿,使用漩涡振荡器漩涡混匀15s,置于冰上10min;
(4)将1.5mL离心管从冰盒中取出,12000xg,4℃,离心15min,注意移动离心管的时候不要晃动,此刻已能看见混合液出现分层;
(5)将离心完的1.5mL离心管取出,同时注意不要晃动,将600μL上清液转移到新的RNase-free 1.5mL离心管中,加入600μL事先配制好的70%乙醇并用pipettor缓慢吹打混匀;
(6)把2.0mLCollecting tube放在无酶处理过的1.5mL离心管架上,再将RNA结合柱套在2.0mL Collecting tube上,将步骤(5)得到的1.2mL混合液轻柔加入到结合柱中,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Collecting tube中的滤液倒进垃圾杯并重复使用Collecting tube;
(7)在结合柱中加入500μL RNA Wash BufferⅠ,离心机的转速设置为10000xg,温度至室温,离心30s,将Collecting tube中的滤液倒进垃圾杯并重复使用Collectingtube;
(8)在结合柱中加入700μL试剂盒中已用无水乙醇稀释好的RNA Wash BufferⅡ至结合柱底部中央,将离心机的转速设置为10000xg,温度至室温,离心30s,将Collectingtube中的滤液倒进垃圾杯并重复使用Collecting tube;
(9)在结合柱中再次加入500μL RNA Wash BufferⅡ并重复步骤(8)操作;
(10)换新的Collecting tube并将结合柱套上,将离心机的转速设置为13000xg,温度为室温,空柱离心1min以甩干结合柱中的的基质;
(11)最后将结合柱套在RNase-free处理过的1.5mL离心管上,加入50μL DEPC-treated water至结合柱底部膜的中央,先静置1-2min,然后离心机的转速设置为13000xg,温度为室温,离心1min;
(12)取少量RNA样品,用NanoPhotometer N50 Touch检测RNA质量和浓度,配制1%琼脂糖凝胶,在电压100V的条件下电泳检测提取的RNA的质量,确定样品质量较好,浓度较高时将样品分装于RNase-free离心管于-80℃冰箱保存。
采用R312-01/02试剂盒进行反转录,体系如下:
表1
RNase-free ddH2O | to 8μL |
Total RNA | 10pg-5μg |
用pipettor加上述试剂于RNase-free PCR管中,放入到预热好的PCR仪中,轻65℃加热5min,完成后静待冰上降温,静置2min。
表2
上一步的混合液 | 8μL |
5×gDNA wiper Mix | 2μL |
再次加入上述试剂以取出gDNA,用pipettor轻轻吹打混匀后,于PCR仪中42℃反应2min。
表3
上一步的混合液 | 10μL |
10×RT Mix | 2μL |
HiScript III Enzyme Mix | 2μL |
Oligo(dT)20VN | 1μL |
RNase-free ddH2O | 5μL |
用pipettor轻轻吹打混匀,进行表4操作。
表4
37℃ | 45min |
85℃ | 5s |
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。
2.2目的基因bgl3HB的扩增
对转录组的结果筛选得到表达量高且上调表达的基因bgl3HB,使用Snapgene软件分别对基因bgl3HB的核苷酸序列进行分析,再通过Primer premier5.0设计PCR扩增开放式阅读框(ORF)的引物,在序列的5’端和3’端加入EcoRI-HF、XbaI内切酶基因序列,保证bgl3HB定向连接表达载体pPICZαA;正向引物的3’端引入6His序列,以备蛋白纯化,引物设计序列如表5所示:
表5β-葡萄糖苷酶基因克隆所用引物
PCR反应体系如下(所有操作在冰上进行):
表6
反应程序如下:
表7
跑胶检测PCR产物,并在Sangon测序。剩余产物储存在-20℃保存。
2.3大肠杆菌感受态的转化
(1)从超低温冰箱中取出从Sangon购买的DH5α,于冰上静置30min后轻轻取出,等待其融化后小心加入1μL事先保好种的质粒,用pipettor轻轻吸吹混匀,置于冰上30min;
(2)将1.5mL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1.5mL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pipettor轻轻混匀后放于37℃,180rpm的摇床中培养1h以复苏菌种;
(4)用pipettor吸取200μL产物滴到含Zeocin(25μg·mL-1)的Luria-Bertani agar培养基上并涂布均匀。
(5)将干燥后的平板置于37℃培育箱中,待菌液完全吸收后,将平板倒置过夜。
2.4阳性克隆子菌落PCR验证
挑取Luria-Bertani平板上单菌落进行菌落PCR,反应体系如下:
表8
PCR扩增反应条件为:
表9
所得产物进行1%跑核酸凝胶电泳检测,无误的克隆子接入含200μg·mL-1Zeocin的Luria-Bertani液体培养基,180rpm 37℃下培养16h,为使单克隆生长至对数期。
2.5质粒pPICZαA的抽提
E.Z.N.A.TMPlasmid DNA mini Kit试剂盒抽提质粒:
(1)将试管培养的5mL菌液于室温下10000xg离心1min,弃去上清液,共离心5次;
(2)加入250μL已加RNA酶的试剂I后旋涡振荡;
(3)继续加入250μL试剂II,轻轻颠倒四五次;
(4)然后加入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;
(5)沉淀形成后将离心管于室温下13000xg离心10min;
(6)在离心过程中将DNA结合柱装在2mL收集管上,小心地用移液枪转移上清至结合柱中心膜后于室温下,13,000xg离心1min,将滤液倒进垃圾杯后把结合柱重新装回收集管;
(7)加入500μLHB缓冲液,按照上述条件离心,将滤液倒进垃圾杯;
(8)套回收集管,继续向结合柱中加入700μL DNA Wash Buffer,按照上述条件离心,将滤液倒进垃圾杯;
(9)重复上一步;
(10)将收集管装在新的收集管上,以13000xg空柱离心2min;
(11)将结合柱装在1.5mL离心管后向结合柱膜底中央加入50μL Elution Buffer,室温静置1min,13000xg离心1min,收集质粒。
2.6线性化载体制备及回收
使用EcoRI-HF和XbaI对收集的质粒pPICZαA进行双酶切,反应体系如下:
表10
37℃温浴反应6h,跑胶检测酶切产物,并采用Gel Extraction Kit试剂盒进行胶回收:
(1)跑胶后快速把目的片段切下来;
(2)将凝胶块转移至已称重1.5mL离心管中,继续称重得出凝胶块的重量。加入等体积的XP2 Binding Buffer,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结合柱并将其装在2mL收集管内;
(4)用pipettor将第三步获得的混合液加入到DNA Mini结合柱中心。离心机于室温下10,000xg离心1min。将收集管中的滤液弃掉,然后将柱子套回2mL收集管内重复使用;
(5)转移300μL XP2 Binding Buffer至柱子中,室温下,13000rpm离心1min,将滤液倒进垃圾桶;
(6)将结合柱装回收集管,转移700μL已用无水乙醇稀释的SPW Buffer到结合柱中。离心机于室温下10000xg离心1min,将滤液倒进垃圾桶;
(7)重复步骤(6);
(8)将DNA Mini结合柱套在一个新的2mL收集管上,在室温下设置离心机13,000xg离心2min甩干结合柱基质;
(9)将DNA Mini结合柱装在一个无酶1.5mL离心管上,加入30μL Elution Buffer到DNA Mini结合柱的膜底,静置1min,13000xg离心1min以完全洗脱DNA。
(10)回收质粒跑核酸凝胶电泳证实酶切成功。
2.7构建重组表达载体
采用C112构建重组载体试剂盒进行重组,用量如下:
表11
重组反应体系如下:
表12
X和Y分别为载体用量和***片段用量。将产物于37℃的条件下反应30min,立即置于冰上冷却至4℃,产物存于-20℃。
2.8重组产物转化及鉴定
(1)在冰上解冻E.coli DH5αCompetent Cells。
(2)将重组体系中试剂在PCR仪中37℃反应三十分钟,取出后放在冰上冷却。
(3)将10μL上述产物与100μL冰上融化的DH5α轻柔混匀以防细胞死亡,于冰上静置30min后轻轻取出。
(4)将产物置于提前预热的PCR仪中42℃热激45s以使重组产物能从大肠杆菌细胞的缝隙中通过,热激后立即置于冰上静待2-3min以使产物冷却,注意不要晃动以防细胞受到外力出现死亡的情况。
(5)冷却完后向离心管中加入700μL SOC液体培养基,180rpm培养1h,以复苏菌种;
(6)此时将已倒好的含Zeocin的LLuria-Bertani平板也放在37℃培育箱中。
(7)用pipettor吸取200μL已转化的感受态细胞滴在含有25μg·mL-1Zeocin的LLuria-Bertani agar培养基上,用涂布棒涂干。
(8)将平板置于培育箱中37℃培育直至菌液被平板完全吸收,将平板翻过来培养过夜。
(9)挑取重组反应转化平板上若干个克隆转进Zeocin抗性的LLuria-Bertani培养基,37℃,180rpm培育12-16h。
(10)进行菌落PCR鉴定,将PCR产物正确条带的甘油菌送去测序。
3.bgl3HB基因在毕赤酵母X33中的高效表达
3.1重组质粒提取
抽提质粒同2.5。
3.2重组质粒线性化
为了提高重组质粒在Pichia pastoris X33染色体上的连接效率、方便后续试验筛选高拷贝克隆子,对重组质粒使用内切酶PmeI和SacI进行单酶切,单酶切线性化的体系为:
表13
将PCR仪预热到37℃并反应30min,再将混合物在65℃反应20min以热失活内切酶,反应结束后跑胶检测线性化结果并回收。
3.3毕赤酵母感受态的制备
毕赤酵母X33感受态细胞具体制备方法如下:
(1)从超低温冰箱取出保藏的菌株X-33,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℃培育箱培养;
(2)将X-33单菌落接种到YPD液体培养基中,30℃过夜培养;
(3)接种(2)培养的菌液按0.1%接种量到100mL无抗YPD液体培养基中,将摇床的温度设置为30℃,转速设置为180rpm,摇至吸光度值OD600=1.5左右;
(4)加入25mL菌液,在4℃,1500xg离心5min,倒掉上清培养基收集细胞,并用25mL预冷的Sterile water冲洗离心管底部使细胞悬浮;
(5)同样条件离心,加入15mL预冷Sterile water再次冲洗离心管底部悬浮细胞;
(6)同样条件离心,加入10mL预冷Sterile water重复上述操作;
(7)同样条件离心,将水倒干后加入10mL冷的1M sorbitol重复上述操作;
(8)离心条件同上,倒去上清,最后用500μL预冷的1M sorbitol轻柔使细胞悬浮混匀并用无菌的1.5mL离心管分装,每管80μL,-80℃保存。
3.4重组质粒电击转化酵母感受态细胞
将线性化后的重组质粒与线性化后的空载电击转到X33感受态细胞中。
具体电击转化过程如下:
(1)超净工作台中,将制备好的80μL感受态细胞与20μL线性化重组载体(5-20μg)轻柔混匀,转入预冷的0.2cm电击转化杯中;
(2)于冰上5min;
(3)在此期间也将1M无菌sorbitol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,使用***推荐酵母参数进行电击处理;
(4)电击处理后立刻加入1mL已预冷的1M无菌sorbitol,轻柔混匀后转移至无菌离心管中,用封口膜封好后在30℃培育箱中孵育1h左右;
(5)提取准备好100μg·mL-1博莱霉素的YPDS平板,放在培养箱预热;取200μL转化后菌液涂布并放在30℃培育箱中,直到平板长出转化子。
3.5酵母阳性转化子的筛选及验证
以平板转化子为模板,用通用3’AOX1和5’AOX1引物进行PCR反应,并送去Sangon进行产物测序验证。pPICZαA中的AOX1通用引物扩增体系为:
表14
PCR扩增反应条件为:
表15
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扩增结束后,取10μLPCR产物进行1%跑胶电泳,从电泳条带的位置判断转化子是否为阳性克隆子,剩余产物送到生物公司进行测序验证。
3.6PTVA法筛选多拷贝
将测序验证正确的阳性克隆子pPICZαA-bgl3HB转到含200μg·mL-1Zeocin的YPDagar培养基中,采用转化后载体扩增法(PTVA法)(Sunga et al.,2010)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-1、800μg·mL-1、1200μg·mL-1、2000μg·mL- 1Zeocin的YPD agar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测序公司验证。验证正确的克隆子接种至250mLYPD液体培养基中。
3.7Bgl3HB的诱导分泌表达
用50mL BMGY培养基重悬菌体转至1000mL BMGY液体培养基中,28℃,180rpm诱导培养6d,期间每隔24h补加2%甲醇一次并检测酶活性。培养液4℃,12,000rpm的离心机中离心30min收集上清。
4.重组酶Bgl3HB的纯化
4.1样品处理
工程菌株X33-bgl3HB在BMGY培养基培养2d后转至BMMY培养基收集上清置于冰上,用0.22μm滤膜过滤除菌,所得液体用50kDa超滤管浓缩并用缓冲液A进行置换,4℃保存备用。
4.2Bio-ScaleTM Mini UNOsphereTM Q Cartridges柱层析
采用UNOsphereTM Q介质亲和层析(柱子型号:UNOsphere Q column 5mL;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·min-1(288cm·h-1);
(2)用缓冲液A清洗2min;
(3)用缓冲液B清洗5min;
(4)用缓冲液B平衡5min;
(5)上样,注意上样期间不要产生气泡,若产生气泡需用针筒将气泡排除;
(6)用缓冲液A清洗交换柱并收集穿透峰;
(1)流速调整至1mL·min-1,在1-20%缓冲液B浓度内按照5%,10%,15%,20%进行分步洗脱,期间收集不同出峰时期洗脱液,并测定其OD400nm吸光度值和SDS-PAGE变性蛋白电泳分析纯化效果;
(2)用1M缓冲液B进行彻底洗脱以清洗杂蛋白;
(3)用1M NaOH对交换柱进行消毒,保持40min的接触时间;
(4)用1M缓冲液B重新平衡交换柱;
(5)用20%乙醇清洗交换柱,将交换柱拆下,4℃保存;
(6)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。
4.3Bio-ScaleTM Mini NuviaTM IMAC Ni-Charged Cartridges柱层析
采用NuviaTM IMAC Ni-Charged介质亲和层析(柱子型号:NuviaTM IMAC column5mL进行二次纯化。具体步骤如下:
(1)用5CV的平衡缓冲液以2mL·min-1的速度平衡交换柱;
(2)以1mL·min-1的速度装载样品裂解液;
(3)用6CV的平衡缓冲液以1mL·min-1的速度洗涤交换柱;
(4)用6CV的洗涤缓冲液以2mL·min-1的速度洗涤交换柱;
(5)用10CV的洗脱缓冲液以2mL·min-1的速度洗脱纯化蛋白;
(6)用5CV的盐酸胍溶液以6mL·min-1的速度清洗交换柱;
(7)用1M NaOH对交换柱进行消毒,保持40min的接触时间;
(8)用5CV的0.1M EDTA以6mL·min-1的速度清洗交换柱以剥离金属离子;
(9)用5CV的0.1M硫酸镍溶液以6mL·min-1的速度对交换柱进行再生;
(10)用20%乙醇清洗交换柱,将交换柱拆下,4℃保存;
(11)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。
4.4纯化蛋白SDS-PAGE分析
取50μL纯化后Bgl3HB蛋白样品,与10μL protein loading buffer混合,沸水煮8min使其变性。取20μL变性混合液点入凝胶电泳胶槽。设置恒定电压为120V,时间为70min,直至条带跑到胶槽底部,采用考马斯亮蓝染液染色并根据蛋白质标记标准推断目标蛋白的分子大小。
4.5Bgl3HB的酶学特性研究
4.5.1酶活测定及蛋白定量标准曲线
4.5.1.1β-葡萄糖苷酶不同底物的活性测定
分别在不同类型糖苷配体的底物作用下测定Bgl3HB的降解能力,底物如下:纤维二、三、四、五糖、龙胆二糖、槐糖、昆布二糖、pNPG、大豆苷、昆布多糖。
β-葡萄糖苷酶活性的测定方法如下:
比活力单位U·mg-1的定义:每mg酶蛋白所含的酶活力单位。用BCA法测定酶的蛋白浓度,同时测得其β-葡萄糖酶酶活,计算出酶的比活力。
pNP糖苷类底物酶活力测定,反应步骤如下:将100μL纯化后的酶液(稀释10倍)与100μL 5mM pNPG溶液混合,最适温度下反应10min,立刻加入600μL 1M Na2CO3终止反应,以煮沸10min的酶溶液为对照,在OD400nm波长处测定吸光度值。
聚糖底物采用DNS法测定,反应步骤如下:将900μL 0.5%大豆苷/昆布多糖底物,在预热到55℃的PCR仪中反应2min,然后加入100μL的10倍稀释的酶溶液,反应10min后,加入1.5mL DNS,煮沸5min然后测定OD540nm值。
还原性寡糖类底物酶活力测定采用GOD法,测定反应步骤如下:100μL适当稀释的酶溶液与200μL葡萄糖氧化酶/过氧化物酶分析试剂,于37℃下反应30min,加入200μL 6MH2SO4终止反应,然后测定OD540nm值。
4.5.1.2标准曲线的制定
(1)pNP标准曲线制作方法参考嗜热真菌第3家族β-葡萄糖苷酶的多样性和分子改造(夏伟,2016)。
表16 pNP标准曲线的制定
(2)DNS法葡萄糖标准曲线制作方法参考嗜热真菌第3家族β-葡萄糖苷酶的多样性和分子改造(夏伟,2016)。
表17 DNS法葡萄糖标准曲线的制定
(3)GOD法葡萄糖标准曲线的制定参考嗜热真菌第3家族β-葡萄糖苷酶的多样性和分子改造(夏伟,2016)。
表18 GOD法葡萄糖标准曲线的制定
(4)BCA法蛋白标准曲线的制定:见Beyotime BCA试剂盒。
4.5.2酶活测定及数据统计所用软件
使用全波长酶标仪测定酶活,通过GraphPad Prism 9.0.0进行数据分析。
4.5.3蛋白浓度测定
纯化蛋白浓度采用Beyotime BCA试剂盒进行测定。
4.5.4反应最适pH及pH稳定性
在不同pH(pH2.2-10)条件下进行反应,测定Bgl3HB的酶比活。将90μL缓冲液和10μL纯化后的酶液混匀后加入100μL 5mM·L-1pNPG在50℃反应10min,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%。所需的不同pH值分别由下列缓冲液提供:10mM·L-1McIlvaine buffer(pH2.2-8.0)和50mM·L-1Gly-NaOH(pH9.0-10.0)。
将10μL酶液与90μL酶比活最高的3个缓冲液无底物4℃孵育10、20、30、40、50、60min后于50℃测定剩余活性。
4.5.5反应最适温度及温度稳定性
在最适pH条件下,取90μL缓冲液和10μL酶液混匀后加入100μL 5mM·L-1pNPG,将混合液分别于20,30,40,50,60,70,80,90℃条件下孵育10min,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。
4.5.6不同浓度盐离子耐受性
在最适反应条件下,测定盐离子浓度为0,200,400,600,800,1000,2000,3000,4000,5000mM·L-1条件下对酶活力的影响。取90μL盐离子溶液和10μL酶液混匀后加入100μL5mM·L-1pNPG,将混合液在最适温度下孵育10min,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%。
4.5.7不同金属离子耐受性
在最适反应条件下,分别浓度为1mM·L-1和5mM·L-1Ca2+,K+,Al3+,Ni2+,Cu2+,Mg2+,Mn2+,Co2+,Zn+,Fe3+对酶活力的影响。分别取90μL不同浓度的金属离子溶液和10μL酶液混匀后加入100μL 5mM·L-1pNPG,将混合液在最适温度下孵育10min,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%。
4.5.8底物特异性分析
分别取纯化后Bgl3HB与不同生物质底物反应。测量三次求平均值,以绝对值计算水解活性。生物质底物分别为4mM·L-1的纤维二、三、四、五糖、龙胆二糖、α-槐糖、昆布二糖、pNPG、大豆苷、昆布多糖底物,所有底物均购自上海源叶生物科技有限公司。
4.5.9酶促反应动力学常数测定
以不同浓度(1mM·L-1、2mM·L-1、4mM·L-1)上述糖溶液为底物测定酶活性。结果根据双倒数作图法计算酶促反应动力学常数Km和Vmax和酶专一性催化效率Kcat/Km。具体步骤如下:
(1)将纯化的酶液准确稀释到1mg·mL-1待用。
(2)分别将不同浓度的底物加入到上述反应体系(2.3.1.1)中,以失活酶液为空白,标准条件下测定酶活。
(3)利用双倒数法作图,公式为即可得出米氏常数Km。
4.5.10Bgl3HB协同纤维素酶糖化效果
以高温碱处理的甘蔗渣为原料进行酶促糖化试验,比较新型β-葡萄糖苷酶、通用商业化纤维素酶Celluclast 1.5L和Novozyme 188糖化效果,试验设计如下:
表19 Bgl3HB单酶糖化试验
具体步骤为:甘蔗渣在121℃的条件下用1%NaOH处理30min,ddH2O洗涤至中性,加入10mL 100mM Na2HPO4-柠檬酸缓冲液(分别为pH 5.0和pH 4.0),在100mL摇瓶中以2%(干料)的浓度混合,向各摇瓶同时加入上述酶组分,40℃,120rpm反应。每种酶组合分别在4h、12h、16h、24h、48h、72h和96h取出并用沸水终止反应,水解产物经12000rpm离心后测定其还原糖(DNS法)和葡萄糖(GOD法)含量。
(二)实验结果
1RNA-seq测序结果分析与检验
1.1原始测序数据质量分析
哈茨木霉S7两个样本转录组CK组和T1组(其中CK组是对照组,T1是试验组)测序的Bases数分别为7,0743,36,089和7,057,209,178,碱基数均大于6G。
1.2目的基因bgl3HB的筛选
将RNA-Seq测序后得到的数据按照FDR<0.05,|log2FC|>2且差异性显著的条件筛选到756个Unigenes,由于在以MCC为唯一碳源的培养基条件下可高效诱导β-葡萄糖苷酶分泌,因此通过NCBI blast将Unigenes序列比对到核酸数据库NR中筛选到5个预测可能为β-葡萄糖苷酶的基因,所选基因情况如表20所示,将差异倍数最高的Unigene0015203命名为bgl3HB,进行下一步研究。
表20差异基因比对情况
1.3bgl3HB表达验证情况
根据RNA-Seq测序预测得到的bgl3HB基因序列,设计Quantitative Real-timePCR引物(表5),以诱导培养基及对照培养基培养4天条件下提取的RNA反转录出的cDNA为模版,进行RT-PCR及Q-PCR。据不同研究发现β-tubulin和actin基因在不同条件下表达相对恒定,因此作为内参基因(孙青,2015,尉洪涛et al.,2012)。通过RT-PCR对两个内参基因的引物特异性进行了验证,结果如图1所示,熔解曲线为单一的峰,说明引物特异性较高,没有杂质干扰,可用于作为稳定的内参进行定量。
RT-PCR显示两个差异表达基因的熔融曲线如图2所示,熔融曲线为单一峰,说明该引物没有非特异性扩增。所有的数值先与内参做了ΔCt,然后分别做了2-ΔΔCt分析,以葡萄糖组为对照。RT-PCR和RNA-Seq结果显示bgl3HB表达上调且差异倍数一致。因此说明本研究基于RNA-Seq获得的基因表达数据是可靠的。
2bgl3HB基因的克隆及序列分析
2.1bgl3HB核酸序列及蛋白质结构分析
通过RNA-Seq测序得知bgl3HB的核酸分子大小分别为2388bp。对基因bgl3HB的blastx比对分析表明,它们是全新的β-葡萄糖苷酶基因,因为它们与NCBI中提交的一些序列没有较高相似性。经网站https://web.expasy.org/compute_pi/预测bgl3HB的蛋白大小和等电点分别为86.67kDa,5.35。对bgl3HB进行序列分析,经SignalP 5.0和TMHMM2.0分析表明bgl3HB序列的1-20个氨基酸残基为信号肽序列。
通过SMART对bgl3HB编码区进行蛋白质结构功能域的分析表明,bgl3HB所编码的蛋白质序列中含有Pfam Glyco-hydro-3(120-375位点,E-value:8.40e-27)功能域,PfamGlyco-hydro-3-C(414-640位点,E-value:3.70e-45)功能域和Fn3_like(694-765位点,E-value:3.46e-11)功能域,如图3所示,Glyco-hydro-3和Glyco-hydro-3-C是属于GH3家族的结构域,并参与催化作用,可能与β-葡聚糖结合,表明Bgl3HB蛋白属于糖苷水解酶第三家族,Fn3-like结构域出现在C-末端,其具体的功能还未知,可能与酶的热稳定性有关(刘春艳,2018),需要进一步探索。
再通过dbCAN来注释bgl3HB的碳水化合物活性相关酶,dbCAN数据来源主包括CAZy数据库和CAT,注释结果如图4。
2.2哈茨木霉菌株总RNA提取及第一链cDNA合成
制备完整的、纯度高的RNA是合成高质量的cDNA关键的因素。采用RNA提取试剂盒(OMEGA公司,型号R6840-01)从诱导培养4d的菌体中分离出纯度和质量较好的总RNA(如图5所示)。提取总RNA后,立即进行反转录获得cDNA存放在-20℃备用。
2.3目的基因bgl3HB的扩增
以反转录的cDNA为模板,分别以bgl3HBEF、bgl3HBER为上下游引物进行PCR,获得β-葡萄糖苷酶成熟肽的基因表达序列为2388bp,测序结果如下图。取少量产物于1%agar糖凝胶电泳进行检测,结果如图6所示。并将质粒pPICZαA转化到E.coli DH5αCompetentCells中,通过菌落PCR鉴定筛选出阳性克隆,使用EcoRI-HF和XbaI对收集的质粒pPICZαA进行双酶切。
2.4重组表达载体的构建及重组产物的转化与鉴定
采用II OneStep Cloning Kit(南京诺唯赞公司,型号C112)试剂盒进行重组反应,重组产物如图7所示,将重组产物命名为pPICZαA/bgl3HB并进行菌落PCR鉴定(如图8)同时送到生物公司测序。
3bgl3HB基因在毕赤酵母X33中的高效表达与纯化
3.1重组表达质粒转化毕赤酵母及阳性转化子的筛选和验证
将阳性转化子命名为pPICZαA/bgl3HB/X33,如图9所示。对转化子进行琼脂糖凝胶电泳验证,结果如图10所示。
3.2PTVA法筛选多拷贝及验证
重组菌pPICZαA/bgl3HB/X33在含400μg·mL-1、800μg·mL-1、1200μg·mL-1、2000μg·mL-1Zeocin的YPD平板中的生长状态如图11。结果显示,重组菌pPICZαA/bgl3HB/X33在800μg·mL-1的ZeocinYPD平板上开始有部分死亡,在1200μg·mL-1的ZeocinYPD平板上几乎有一半菌已死亡,但在2000μg·mL-1的ZeocinYPD平板上仍能够继续正常生长,将这些正常生长的单克隆投入下一步试验。
3.4重组酶Bgl3HB在毕赤酵母中的纯化
在甲醇诱导条件下发酵培养重组菌株pPICZαA/bgl3HB/X33,发现在甲醇诱导6d后,酶活性达到最高。测定Bgl3HB的蛋白量分别为0.22mg/mL;最高酶比活分别为U·mg-1。经甲醇诱导发酵6d后,收集Bgl3HB的粗酶液,50kDa AmiconU1tra-15超滤管浓缩并用缓冲液A置换掉粗酶液中的培养基,经脱气低盐缓冲液A(pH 7.0)和脱气高盐缓冲液B(pH 7.0)平衡好的UNOsphereTM Q Cartridges柱进行层析,层析结果如图12所示。纯化后的蛋白经NuviaTM IMAC Ni-Charged柱进行二次纯化,NuviaTM IMAC Ni-Charged柱可以特异性结合6His标记的蛋白,后通过高浓度的咪唑缓冲液(PBS C,pH 8.0)洗脱目的蛋白。Bgl3HB经NuviaTM IMAC Ni-Charged柱进行二次层析后,均得到单一的收集峰。
3.5纯化蛋白SDS-PAGE分析和质谱鉴定
分别配制浓度为6%的Separating glue和5%的Concentrated gum对收集到的峰值蛋白进行SDS-PAGE电泳分析,条带大小分别为95.8kDa,说明Bgl3HB蛋白正确表达并且高效收集纯化。
利用质谱,对纯化后的两个蛋白进行鉴定,发现这两个蛋白的确为目的蛋白,测序结果如图13,以保证后续实验的进行。
3.6Bgl3HB的酶学特性研究
3.6.1酶活测定及蛋白定量标准曲线
3.6.1.1 pNP标准曲线
标准曲线如图14所示。
3.6.1.2 DNS法葡萄糖标准曲线
标准曲线如图15所示。
3.6.1.3 GOD法葡萄糖标准曲线
标准曲线如图16所示。
3.6.1.4 BCA法蛋白定量标准曲线
标准曲线如图17所示。
3.6.2蛋白浓度测定
经测定,Bgl3HB的蛋白浓度分别为0.15mg·mL-1。
3.6.3反应最适pH及pH稳定性
分别配置不同pH的缓冲液体系:10mM·L-1磷酸氢二钠-柠檬酸缓冲液(pH 2-8),50mM·L-1甘氨酸-氢氧化钠缓冲液(pH 9-10)。将纯化后的Bgl3HB于不同pH值缓冲液,按照2.3.2反应体系测定酶活,试验重复三次。结果如图18所示,纯化酶的反应最适pH为4.0,当pH在2.0时仍能保持最高酶活的38%,说明该蛋白耐受酸性环境,在pH 2-6范围内能够保持大部分活性,而在pH 7.0时仅剩最高酶活的36%,表明Bgl3HB为酸性酶。
将纯化后的Bgl3HB于图19条件酸处理1h、4h、8h、12h、24h,再按照上述方式测定剩余酶活。Bgl3HB在pH 3.0条件下处理24h后仍保持55%的相对酶活,值得一提的是,在pH4.0条件下处理24h后剩余酶活保持在85%以上,说明该蛋白能一定程度上耐受极端酸性环境。
3.6.4反应最适温度及温度稳定性
将纯化后的Bgl3HB于不同的温度下(4℃-90℃),按照2.3.3的反应体系测定酶活,试验重复三次。试验结果如图20,Bgl3HB的最适反应温度分别为50℃,60℃剩余相对酶活分别为64%,70℃剩余相对酶活分别为29%,80℃剩余酶活分别为21%,90℃时剩余酶活分别为19%,海南红树林来源的哈茨木霉产生的β-葡萄糖苷酶普遍具有较高的最适反应温度且能耐受一定高温环境符合其自然生存条件。
将纯化后的Bgl3HB于图21条件分别热处理1h、4h、8h、12h、24h,再按照上述方式测定剩余酶活。试验结果如图所示,Bgl3HB在最适温度条件下酶活稳定,最适温度条件下处理24h后两个酶蛋白的相对酶活分别为72%,结果显示,哈茨木霉Bgl3HB能够在较高温度条件下长时间保持较高酶活。
3.6.5不同金属离子耐受性
分别配制1mM·L-1和5mMl·L-1Cu2+,Ca2+,Ni2+,Mg2+,K+,Al3+,Mn2+,Zn+,Fe3+,Co2+按照2.3.7的反应体系测定酶活,将空白组(不添加任何金属离子)的酶活力定义为100%,试验结果如图23,Ca2+和Mn2+极显著提高Bgl3Hb活性30.96%和29.98%
3.6.6不同浓度盐离子耐受性
将纯化后的Bgl3HB分别于不同浓度的盐离子(0-5000mM/L)按照2.3.6的反应体系测定酶活,试验重复三次,试验结果如图23。Bgl3HB相对酶活随盐离子浓度增高呈先上升后下降的趋势,在400mM·L-1盐离子浓度达到最高,在最高浓度盐离子浓度下剩余66%的酶活。Bgl3HB相对酶活随盐离子浓度增高而增高,说明盐离子对Bgl3HB分别有不同程度的促进作用。这可能是因为盐度是影响红树林湿地微生物群落结构的最重要因素(Ceccon etal.,2019,Tong et al.,2019a,Fu et al.,2019),盐度与土壤微生物的活性和多样性呈显著负相关,随着盐度和土壤渗透压的增加,一些对盐分胁迫敏感的微生物群落无法生存,故而试验从红树林土壤中分离得到的哈茨木霉可以产耐盐,甚至一定浓度的盐分对酶活性有促进作用(Wang et al.,2010,Tong et al.,2019b)。
3.6.7底物特异性测定
将Bgl3HB与不同底物进行反应,测定其对不同底物的反应活性,以高温处理后失活的酶为对照,活性单位为U·mg-1。测定结果如表所示,Bgl3HB对昆布多糖的作用效果最好,酶活分别为129.79U·mg-1;以不同键的双糖为底物时,酶的选择性大小顺序为:龙胆二糖(β-1,6键)>槐糖(β-1,2键)>海带二糖(β-1,3键)>纤维二糖(β-1,4键)>纤维戊糖(β-1,4键)>纤维四糖(β-1,4键)>纤维三糖(β-1,4键)。结果说明,Bgl3HB为底物特异性广泛的酶,并且对测定的大多数纤维素底物均有活性,并且Bgl3HB还有较强水解pNPG的能力。
表21 Bgl3HB底物特异性分析
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3.6.8酶促反应动力学常数测定
Bgl3HB对pNPG、大豆苷、昆布多糖的酶促反应动力学常数如表22所示。Bgl3HB对昆布多糖的底物亲和力和催化效率远高于其他芳基糖苷,Bgl3HB对海带多糖优异的降解能力,使得Bgl3HB在以藻类生物质为原料生产生物燃料方面具有很大的优势。
表22Bgl3HB不同底物酶反应动力学常数
3.7Bgl3HB协同纤维素酶糖化效果
为了评价Bgl3HB在实际条件下的应用价值,在pH4.0和50℃条件下,比较了Bgl3HB和商品化Nov188在纤维素原料糖化中的应用潜力。如图24所示,以蔗渣为原料,仅添加商品酶Celluclast 1.5L的空白对照组在pH4.0,50℃条件下培养96h,释放还原糖296.4μmol、葡萄糖42.9μmol(葡萄糖转化率为14.5%)。当添加12BGU的β-葡萄糖苷酶时,Celluclast1.5L与Nov188协同作用,可释放还原糖353.8μmol、葡萄糖61.5μmol(葡萄糖转化率17.4%)。Bgl3HB联合Celluclast 1.5L仅释放344.0μmol的还原糖和58.1μmol的葡萄糖(葡萄糖转化率为16.9%)。
然而,Celluclast 1.5L和Bgl3HB在添加5mMNaCl的情况下提高了协同酶糖化的性能。还原糖和可发酵葡萄糖的得率分别为380.84μmol和67.6μmol(葡萄糖转化率为17.8%)。
值得注意的是,尽管Bgl3HB的蔗渣转化率低于商品化Novozyme 188,但Bgl3HB的葡萄糖转化率高于商品化Novozyme 188。而添加5mM NaCl的Bgl3HB的蔗渣转化率高于商品化Novozyme 188,葡萄糖转化率也高于商品化Novozyme 188。这与我们研究的其他结果一致,表明NaCl可以提高Bgl3HB的活性,可能有助于提高Bgl3HB在糖化过程中的水解能力。这种效果可能是因为盐有利于酶的稳定。这种重要的相互作用将允许商业纤维素酶用较少的酶进行水解,这将降低工业应用成本。
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> 一种酸性β-葡萄糖苷酶及其编码基因和应用
<130> MP21026382
<160> 2
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actcttgcca ctctcgaact ctcgttcccc gactgcgacc atggccccct gaagaacaac 180
ctcgtctgtg actcttcggc cggatacgtc gagcgagccc aggccctcat ctccctcttc 240
accctcgagg agctgattct caacacccag aactcgggcc ccggcgtgcc tcgcctgggt 300
cttccaaact accaagtctg gaacgaggct ctgcacggct tggaccgcgc caactttgcc 360
acaaagggcg gccaatacga atgggcaacc tccttcccca tgcccatcct gtcaatggca 420
gctctcaacc gcaccctgat ccaccagatt gcggacatca tctcgaccca ggctcgagca 480
ttcagcaaca ctggccgcta cggtctcgat gtctacgccc ccaacatcaa tggcttccgt 540
agccctctct ggggccgtgg acaggagact cccggtgaag atgccaacgt gctgacctct 600
gcctacacct acgagtacat caccggtatc cagggcggtg tagaccccga gaacctcaag 660
gttgccgcca cggccaagca ctttgccggc tacgatctcg agaactacaa caaccagtct 720
cgtctgggct tcgacgccat catcacccag caggacctcg ccgagtacta cactccccag 780
ttcctcgctg cgtcgcgcta cgcaaagtct cacagcttca tgtgcgccta caactccgtc 840
aacggcgtgc ccagctgcgc caacagcttc ttcctgcaga ccctgctgag agagagctgg 900
ggcttccccg aatatggcta cgtctcgtcc gattgcgatg ccgtctacaa cgtcttcaac 960
cctcacgact acgccagcaa catgtcttca gctgctgcct cctccctgag ggccggtacc 1020
gacattgact gcggtcagac atacccatgg cacttgaacg agtcctttgt ggctggcgag 1080
gtctcccgcg gcgagatcga gcgctccgtg actcgtctgt atgccaatct cgtccagctc 1140
ggatactttg acaagaagaa cgagtaccgc tcgctcggct ggaaggacgt cgtcaagacc 1200
gatgcttgga acatttcgta tgaggctgct gtcgagggca ttgtcctgct caagaacgac 1260
ggcactctcc ctctgtccaa gaaggtcaag agcatcgccc tgatcggacc ctgggccaat 1320
gccaccaccc agatgcaggg caactacttt ggcactcctc catacctcat cagccctctc 1380
gaggctgcca agaaggctgg ctacaaggtc aactttgcgc ttggaaccga catcgccagc 1440
accagcaccg ccggctttgc caaggctatt gccgccgctg agaagtctga tgccatcatc 1500
ttcgctggtg gtatcgacaa cacggttgaa caggagggcg ctgaccgcac ggacattgct 1560
tggcccggca accagctcga cctcatcaag tcgctcagca agctcaagaa gcctctcgtc 1620
gtcctgcaga tgggcggtgg ccaggttgac tcatcttctc tcaagagcaa caagaacgtc 1680
aactcccttg tctggggtgg atatcccggc cagtctggag gtgtcgctct ctttgacatc 1740
ttgtctggca agcgtgcccc cgctggacga ttggtctcaa cccagtaccc ggccagctac 1800
gttcacgagt tcccccagaa cgacatgaac ctccgccctg atggaaagaa gaaccccgga 1860
cagacttaca tctggtacac tggcaagcct gtctaccagt ttggtgacgg tatcttctac 1920
actactttca aggagagctt gtctggcaag tccaagagcc tcaagtacaa cgttgctgaa 1980
atcattgctg gtgcccaccc tgaatacacc tacagtgagc aggttccggt cttcaccttc 2040
actgccgaga ttaagaactc tggcaagact gagtccccat actcggccat gctcttcgtc 2100
cgcacttcca acgctggtcc tgccccctac cccaacaagt ggctggttgg attcgacaga 2160
cttgccgata tcaagcctgg tcactcctct acgctcagca tccctatccc catcagcgcc 2220
cttgcccgta ccgactctct tggaaacaag attgtctacc ctggcaagta tgagctggct 2280
ctcaacactg acgagtctgt caagctggag tttgagcttg tgggcgagga ggtgatcctc 2340
gagcactggc ctctggatca gcagcagatt caggatgcca ctccataa 2388
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Met Val Asn Asn Ala Ala Leu Val Ala Ala Leu Ser Ala Leu Leu Ser
1 5 10 15
Pro Ala Leu Ala Gln Asn Asn Gln Thr Tyr Ala Asn Tyr Ser Ala Gln
20 25 30
Gly Gln Pro Asp Leu Tyr Pro Gln Thr Leu Ala Thr Leu Glu Leu Ser
35 40 45
Phe Pro Asp Cys Asp His Gly Pro Leu Lys Asn Asn Leu Val Cys Asp
50 55 60
Ser Ser Ala Gly Tyr Val Glu Arg Ala Gln Ala Leu Ile Ser Leu Phe
65 70 75 80
Thr Leu Glu Glu Leu Ile Leu Asn Thr Gln Asn Ser Gly Pro Gly Val
85 90 95
Pro Arg Leu Gly Leu Pro Asn Tyr Gln Val Trp Asn Glu Ala Leu His
100 105 110
Gly Leu Asp Arg Ala Asn Phe Ala Thr Lys Gly Gly Gln Tyr Glu Trp
115 120 125
Ala Thr Ser Phe Pro Met Pro Ile Leu Ser Met Ala Ala Leu Asn Arg
130 135 140
Thr Leu Ile His Gln Ile Ala Asp Ile Ile Ser Thr Gln Ala Arg Ala
145 150 155 160
Phe Ser Asn Thr Gly Arg Tyr Gly Leu Asp Val Tyr Ala Pro Asn Ile
165 170 175
Asn Gly Phe Arg Ser Pro Leu Trp Gly Arg Gly Gln Glu Thr Pro Gly
180 185 190
Glu Asp Ala Asn Val Leu Thr Ser Ala Tyr Thr Tyr Glu Tyr Ile Thr
195 200 205
Gly Ile Gln Gly Gly Val Asp Pro Glu Asn Leu Lys Val Ala Ala Thr
210 215 220
Ala Lys His Phe Ala Gly Tyr Asp Leu Glu Asn Tyr Asn Asn Gln Ser
225 230 235 240
Arg Leu Gly Phe Asp Ala Ile Ile Thr Gln Gln Asp Leu Ala Glu Tyr
245 250 255
Tyr Thr Pro Gln Phe Leu Ala Ala Ser Arg Tyr Ala Lys Ser His Ser
260 265 270
Phe Met Cys Ala Tyr Asn Ser Val Asn Gly Val Pro Ser Cys Ala Asn
275 280 285
Ser Phe Phe Leu Gln Thr Leu Leu Arg Glu Ser Trp Gly Phe Pro Glu
290 295 300
Tyr Gly Tyr Val Ser Ser Asp Cys Asp Ala Val Tyr Asn Val Phe Asn
305 310 315 320
Pro His Asp Tyr Ala Ser Asn Met Ser Ser Ala Ala Ala Ser Ser Leu
325 330 335
Arg Ala Gly Thr Asp Ile Asp Cys Gly Gln Thr Tyr Pro Trp His Leu
340 345 350
Asn Glu Ser Phe Val Ala Gly Glu Val Ser Arg Gly Glu Ile Glu Arg
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Ser Val Thr Arg Leu Tyr Ala Asn Leu Val Gln Leu Gly Tyr Phe Asp
370 375 380
Lys Lys Asn Glu Tyr Arg Ser Leu Gly Trp Lys Asp Val Val Lys Thr
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Tyr Phe Gly Thr Pro Pro Tyr Leu Ile Ser Pro Leu Glu Ala Ala Lys
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Thr Ser Thr Ala Gly Phe Ala Lys Ala Ile Ala Ala Ala Glu Lys Ser
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Asp Ala Ile Ile Phe Ala Gly Gly Ile Asp Asn Thr Val Glu Gln Glu
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Gly Ala Asp Arg Thr Asp Ile Ala Trp Pro Gly Asn Gln Leu Asp Leu
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Ile Lys Ser Leu Ser Lys Leu Lys Lys Pro Leu Val Val Leu Gln Met
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Gly Gly Gly Gln Val Asp Ser Ser Ser Leu Lys Ser Asn Lys Asn Val
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Asn Ser Leu Val Trp Gly Gly Tyr Pro Gly Gln Ser Gly Gly Val Ala
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Ser Thr Gln Tyr Pro Ala Ser Tyr Val His Glu Phe Pro Gln Asn Asp
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Trp Tyr Thr Gly Lys Pro Val Tyr Gln Phe Gly Asp Gly Ile Phe Tyr
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Thr Thr Phe Lys Glu Ser Leu Ser Gly Lys Ser Lys Ser Leu Lys Tyr
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Asn Val Ala Glu Ile Ile Ala Gly Ala His Pro Glu Tyr Thr Tyr Ser
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Glu Gln Val Pro Val Phe Thr Phe Thr Ala Glu Ile Lys Asn Ser Gly
675 680 685
Lys Thr Glu Ser Pro Tyr Ser Ala Met Leu Phe Val Arg Thr Ser Asn
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Ala Gly Pro Ala Pro Tyr Pro Asn Lys Trp Leu Val Gly Phe Asp Arg
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Leu Ala Asp Ile Lys Pro Gly His Ser Ser Thr Leu Ser Ile Pro Ile
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Pro Ile Ser Ala Leu Ala Arg Thr Asp Ser Leu Gly Asn Lys Ile Val
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Tyr Pro Gly Lys Tyr Glu Leu Ala Leu Asn Thr Asp Glu Ser Val Lys
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Leu Glu Phe Glu Leu Val Gly Glu Glu Val Ile Leu Glu His Trp Pro
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785 790 795
Claims (10)
1.一种酸性β-葡萄糖苷酶,其氨基酸序列如SEQ ID NO:2所示。
2.权利要求1所述的酸性β-葡萄糖苷酶的制备方法,其特征在于,包括:
1)、将权利要求1所述的酸性β-葡萄糖苷酶的编码基因克隆至表达载体,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。
3.编码权利要求1所述的酸性β-葡萄糖苷酶的基因。
4.根据权利要求3所述的基因,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/或增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。
5.含有权利要求3或4所述基因的重组载体。
6.转化有权利要求5所述重组载体的宿主菌。
7.根据权利要求6所述的宿主菌,其特征在于,所述宿主菌包括大肠杆菌、毕赤酵母。
8.权利要求1所述的酸性β-葡萄糖苷酶、权利要求3或4所述的基因、权利要求5所述的重组载体或权利要求6或7所述的宿主菌在制备还原糖、葡萄糖、生物乙醇中的至少一种物质的应用。
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所述的酸性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、糟渣中的至少一种。
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