CN103114099B - 编码糖基水解酶家族1的β-葡萄糖苷酶基因及其应用 - Google Patents
编码糖基水解酶家族1的β-葡萄糖苷酶基因及其应用 Download PDFInfo
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Abstract
一个来自链霉菌的编码糖基水解酶家族1的β-葡萄糖苷酶的基因及其应用,其核苷酸序列如SEQ ID NO:1所示,所述基因编码的β-葡萄糖苷酶S-bgl4,其氨基酸序列如SEQ ID NO:2所示,该酶能够在分解纤维二糖中应用。
Description
技术领域
本发明涉及一个编码糖基水解酶家族1的β-葡萄糖苷酶基因及其应用,该基因编码的蛋白质可用于降解纤维二糖。
背景技术
β-葡萄糖苷酶是纤维素酶系的一种,它催化水解β-D-葡萄糖苷键,在人类、动物、植物和微生物的糖类代谢方面具有重要的价值(Ramakrishnan Anish,Mohammad Safikur Rahman,and Mala Rao,2007,Application of cellulase from analkalothermophilic Thermomonospora sp.in biopolishing of denims,Biotechnol.Bioeng.,96(1):48-56.)。根据氨基酸序列分类,人们将β-葡萄糖苷酶划分在糖苷水解酶家族1和3中。家族1中的β-葡萄糖苷酶主要来源于细菌、植物;家族3中的β-葡萄糖苷酶主要来自真菌、细菌和植物。家族1中的β-葡萄糖苷酶除有葡萄糖苷酶活性外,还有很强的半乳糖苷酶活性(HENRISSATB.,1991,A classification of glycosylhydrolases based on amino acid sequencesimilarities,Biochem,293:781-788.)。
β-葡萄糖苷酶在纤维素降解中具有重要作用(Sestelo A.B.F.,Poza M.,Villa T.C.2004,β-Glucosidase activity in a Lactobacillus plantarum wine strain.WorldJournal ofMicrobiology&Biotechnology,20:633~637.)。纤维素作为植物光合作用的主要多糖类产物,是地球上含量最丰富的碳水化合物,也是数量最大的一种可再生资源。据报道,全球每年通过光合作用产生的纤维素高达1.55×109t,其中85%尚未被人类利用(DUNLAP C,CHIANG G C.Utilization and recycle ofagriculture wastes and residues.Shuler M L.Boca Raton,Florida.USA:CRC PressInc,1980.19.)。当今,能源短缺是人类面临的重大问题之一。而对于纤维素的利用是解决能源危机、粮食短缺、环境污染的重要途径。β-葡萄糖苷酶水解纤维二糖生成两分子葡萄糖,是纤维素酶复合酶系发挥协同作的关键酶,对纤维素酶系的整体水解活性有很强的促进作用,可以通过提高纤维素酶系中β-葡萄糖苷酶的活性来改善整个纤维素酶系的功用(Bhat K.M.,Bhat S.1997,Cellulosedegrading enzymes and their potential industrial applications.Biotechnol.Adv.,15:583~620.),因此对β-葡萄糖苷酶的深入研究受到人们越来越多的关注。
除了在降解纤维素方面有着重要作用外,β-葡萄糖苷酶在其他领域也有广泛应用,特别是在医药、食品、生物转化中具有重要的应用价值(Michael E.H.,MarkF.R.,Charles E.,1999,Cellulase for commodity products from cellulosic biomass.Curr.Opin.Biotechnol.,10(4):358~364.)。在医学中,β-葡萄糖苷酶应用在某些癌症的诊断和治疗中,并获得了许多成功(邵金辉,韩金祥,等,2005,β-葡萄糖苷酶在工农医领域的应用.生命的化学,5(01):22-24.)。在水果、蔬菜、茶叶等食料和中草药中,β-葡萄糖苷酶可作为风味酶,对改良果汁风味,果酒增香和茶叶增香均有显著作用(李远华.2002,β-葡萄糖苷酶的研究进展.安徽农业大学学报.29(4):421-425.)。许多风味前体物质和药理活性成分主要以糖苷形式存在,需要β-葡萄糖苷酶这种风味酶将其释放出挥发性糖苷配基,起到增香作用(金凤燮,庄子瑜,鱼红闪,等,2009,特异的中草药配糖体苷酶微生物及其发酵与酶学特性.生物工程学报,25(12):1863-1870.)。除此之外,β-葡萄糖苷酶还具有转移葡萄糖基的活性,可用于低聚龙胆糖的生产(Yongling Qin,YunkaiZhang,Haiyan He,et al,2011,Screening and identification of a fungalβ-glucosidaseand the enzymatic synthesis of gentiooligosaccharide.Appl BiochemBiotechnol.163(8):1012-9.)、红景天苷的酶法合成(王梦亮,李万丽.2009,固定化β-葡萄糖苷酶催化合成红景天甙的研究.生物技术,19(1):68-70.)以及非离子表面活性剂烷基糖苷的合成(朱云,2007,葡萄糖酶法生物合成烷基糖苷工艺.浙江化工,38(11):3-6.)。
目前已有上百个微生物、植物及动物来源的β-葡萄糖苷酶基因得到克隆并被测序,其中许多微生物来源的β-葡萄糖苷酶基因已获得异源表达(韩笑,陈介南,王义强,等,2008,β-葡萄糖苷酶基因的克隆与表达研究进展。生物技术通报,3:8~13.)。相比之下,植物来源的β-葡萄糖苷酶活性远比微生物来源的β-葡萄糖苷酶的要低,因此,目前研究主要集中在微生物来源上(石彩蕊,王义强,陈介南,等,2011,产β-葡萄糖苷酶微生物育种研究进展,生物技术通讯,3:59-65.)。而微生物中研究的较多的是酵母和丝状真菌如木霉属(Trichoderma)、曲霉属(Aspergillus)等霉菌以及细菌中的芽孢杆菌属等(孟宪文,宋小红,陈历俊,等,2009,β-葡萄糖苷酶的研究进展.乳品加工,10:42-44.)。但木霉菌发酵产物中存在多种真菌毒素以及得到的纤维素酶活力较低,尤其是β-葡萄糖苷酶活力很低,致使纤维二糖在反应体系中积累而影响酶解效率,因而其应用范围受到限制。在产β-葡萄糖苷酶的微生物中有很多种都不止编码一种β-葡萄糖苷酶,该酶分子量变化较大,微生物中从18kD~480kD均有报道,酶学性质也可相差很远(韩笑,陈介南,王义强,2008,β-葡萄糖苷酶基因的克隆与表达研究进展。生物技术通报,3:8~13.)。
以β-葡萄糖苷酶为关键词查找国家知识产权局专利检索数据库,共出现163项发明专利。其中有30项发明专利是描述编码β-葡萄糖苷酶的基因及其应用,而其他133项是与β-葡萄糖苷酶在各种领域上的应用有关。在这30项与编码β-葡萄糖苷酶的基因及其应用相关的发明专利中的β-葡萄糖苷酶基因有8个是来自各种未培养微生物宏基因组文库的、其它22个是来自20种不同生物,有嗜热脱氨芽孢杆菌、里氏木霉、根瘤土壤杆菌、瓶霉XH8、Pyrococcus furiosus、嗜热菌、黑曲霉、端氏木霉、黑曲霉、黑翅土白蚁、Clostridium sp.WGC702、曲霉菌株、嗜热拟青霉、Dictyoglomus thermophilum DSM3960、嗜热毛壳菌、酿酒酵母、极端嗜碱菌、嗜热拟青霉、克氏内芽孢杆菌和新鞘氨醇菌;1个来自白藜芦醇。本发明获得的β-葡萄糖苷酶是自行筛选的链霉菌GX6(Streptomycessp.GX6),与基因数据库中公开的β-葡萄糖苷酶的氨基酸的同源性较低,是一个新的β-葡萄糖苷酶。
发明内容
本发明从广西南宁筛选到的链霉菌GX6的基因组中克隆到一个编码β-葡萄糖苷酶的基因,在大肠杆菌宿主细胞中表达该基因生产β-葡萄糖苷酶,并能以纤维二糖为原料降解生成葡萄糖。
本发明涉及一种编码糖基水解酶家族1的β-葡萄糖苷酶的基因S-bgl4,其核苷酸序列如SEQ ID NO:1所示,是从实验室筛选到的链霉菌GX6的基因组中克隆得到。该β-葡萄糖苷酶基因S-bg4的序列是由1278个碱基组成,含有完整的β-葡萄糖苷酶基因S-bgl4的开放阅读框(Open Reading Frame,ORF),S-bg4基因的起始密码子为ATG,终止密码子为TGA。
SEQ ID NO:2的蛋白质是基因S-bgl4编码的β-葡萄糖苷酶产物S-bgl4,由425个氨基酸组成,和S-bgl4催化功能域同源性最高的是Streptomycesambofaciens ATCC23877(生二素链霉菌ATCC23877)的putative beta-glucosidase(假定β-葡萄糖苷酶)两者的一致性=305/427(71%),相似性=345/427(80%)。
基因-Sbgl4在大肠杆菌中表达的重组产物S-bgl4能够分解纤维二糖。
本发明还涉及含有本发明基因的表达载体,及用于转化本发明基因的宿主。
本发明提供了一个编码糖基水解酶家族1的β-葡萄糖苷酶基因,该基因所编码糖基水解酶家族1的β-葡萄糖苷酶能够在分解纤维二糖中应用。
附图说明
图1是筛选含有β-葡萄糖苷酶基因S-bg4重组菌株的七叶苷选择平板图。
图2是β-葡萄糖苷酶S-bgl4纯化物的SDS-PAGE图。
图3是β-葡萄糖苷酶S-bgl4水解纤维二糖的HPLC图。
从图1了解到,含有β-葡萄糖苷酶基因S-bgl4的重组菌株能够在七叶苷筛选平板上形成黑色圈。
图2了解到,β-葡萄糖苷酶S-bgl4纯化物的分子量为46.1kDa。
图3中的A和B分别是葡萄糖和纤维二糖的标样。
从图3的C了解到,β-葡萄糖苷酶S-bgl4能将纤维二糖水解成葡萄糖。
具体实施方式
下述实施方法是为了更好的解释本发明,而不应该被解释为限制本发明的目的。
在本发明的实施例中所用到的材料包括:大肠杆菌(Escherichia coli)株系XL1-blue购自大连TaKaRa公司;表达载体pSE380购自Stratagene公司,限制性内切酶、修饰酶等试剂购自TaKaRa、MBI。
下面将通过实施例对本发明作详细描述:
1)链霉菌Streptomyces sp.GX6基因组DNA的提取
链霉菌Streptomyces sp.GX6的基因组DNA是按照BioFlux公司的细菌基因组DNA提取试剂盒Biospin Bacteria Genomic DNA Extraction Kit(目录号BSC12S1)的使用说明来提取的。
将提取好的链霉菌Streptomyces sp.GX6的基因组DNA送华大基因公司测定基因组序列。
2)链霉菌Streptomyces sp.GX6的基因组序列中编码β-葡萄糖苷酶基因序列的分析
将获得的链霉菌Streptomyces sp.GX6的基因组序列用NCBI(National Centerfor Biotechnology Information,http://www.ncbi.nlm.nih.gov)上的软件ORF fnder(http://www.ncbi.nlm.nih.gov/gorf/orfig.cgi)和Blast(http://blast.ncbi.nlm.nih.gov/Blast.cgi)进行分析。结果共获得8个与β-葡萄糖苷酶具有较高同源性的开放阅读框(Open Reading Frame,ORF),本发明只涉及其中一个ORF。
3)编码糖基水解酶家族1的β-葡萄糖苷酶基因S-bg14的核苷酸序列分析
用NCBI(National Center for BiotechnologyInformation,http://www.ncbi.nlm.nih.gov)上的软件ORF finder(http://www.ncbi.nlm.nih.gov/gorf/orfig.cgi)和Blast(http://blast.ncbi.nlm.nih.gov/Blast.cgi)对DNA序列进行分析。基因S-bgl4的开放阅读框(Open Reading Frame,ORF)由1278个核苷酸组成,序列如SEQ IDNO:1。其中,S-bgl4基因的起始密码子为ATG,终止密码子为TGA。
4)编码糖基水解酶家族1的β-葡萄糖苷酶基因S-bgl4编码的产物S-bgl4的氨基酸序列分析
β-葡萄糖苷酶基因S-bgl4编码一个含425个氨基酸的蛋白质,用DNAStar软件预测该蛋白质的理论分子量大小为46183.9道尔顿。
用简单组件结构研究工具(Simple Modular Architecture Research Tool,SMART,http://smart.embl-heidelberg.de)分析β-葡萄糖苷酶S-bgl4的组件结构,结果是自N端的第7-263位和284-410位氨基酸为家族1糖基水解酶(glycosylhydrolase)功能域、第35-192位为纤维素酶cellulase的功能域。
5)编码糖基水解酶家族1的β-葡萄糖苷酶基因S-bgl4的克隆和表达
使用上游引物5’-CACTCATGATGCACCACCACCACCACCACACGCCCCCGTTCGGGTCCGAAC-3’和下游引物5’-CATAAGCTTCTAACGCGCGGTCGGGGCACCG-3’,通过聚合酶链式反应(PCR)扩增β-葡萄糖苷酶基因S-bgl4,用限制性内切酶Pag I和Hind III酶切β-葡萄糖苷酶基因S-bgl4后,与经Nco I和Hind III酶切的表达载体pSE380进行连接。再将连接产物用CaCl2法转化到大肠杆菌XL1-blue中,涂布到含100μg/mL氨苄青霉素的LA平板上。转化得到的单菌落点板至含有100μgmL氨苄青霉素、七叶苷、柠檬酸铁铵的LA复筛培养基(每100mL复筛培养基含:七叶苷:0.2g,柠檬酸铁铵:0.5g,蛋白胨:1g,酵母粉:0.5g,NaCl:0.5g,琼脂粉:1.5g)平板上,将平板置于37℃恒温箱培养7小时,对每个转点菌落逐个滴加1μL用LB培养基稀释成1%的IPTG诱导表达,然后,将平板置于37℃恒温箱继续培养6小时。时间到后,进行氯仿熏蒸破胞,再把平板置于37°C恒温箱使表达的S-bgl4酶与七叶苷和柠檬酸铁铵反应8小时。观察复筛平板。
然后进一步提取在复筛平板能形成黑色圈的克隆子的质粒DNA,并将其命名为pSE-S-bgl4,用限制性内切酶NcoI和HindIII完全酶切pSE-S-bgl4后,进行0.8%琼脂糖凝胶电泳分析,结果pSE-S-bgl4除有一个约4.4kb的DNA片段外,还有一条大小约为975bp的DNA片段。
将含有质粒pSE-S-bgl4的重组大肠杆菌XL1-Blue菌株接种到600mL含100μg/mL氨苄青霉素的LB培养基中,37℃振荡培养,待OD600为0.4时,加入IPTG 使其终浓度为0.5mmol/L,30℃、200转诱导10小时。11000rpm离心3min,收集菌体,用4mL pH7.0100mmol/L的磷酸缓冲液重悬菌体,超声波破胞9分钟。12000rpm离心20min,取上清进行后面的蛋白质纯化。按每4mL上清液加入1mL50%的镍亲和层析胶体,在4℃用200转摇60分钟,把混合物灌注到柱子,收集流出物。加1mL冲洗缓冲液(50mmol/LNaH2PO4,300mmol/LNaCl,20mmol/L咪唑,pH8.0)到柱子里,缓慢搅拌,收集流出物。重复冲洗步骤4次。加入洗脱缓冲液(50mmol/L NaH2PO4,300mmol/L NaCl,250mmol/L咪唑,pH8.0)洗脱蛋白质。收集洗脱的蛋白质溶液,用变性的聚丙烯酰胺凝胶电泳(SDS-PAGE)验证,发现有目的大小的蛋白质条带。
6)编码糖基水解酶家族1的β-葡萄糖苷酶S-bgl4水解纤维二糖的测定
β-葡萄糖苷酶S-bgl4以纤维二糖为底物、在pH6.5的磷酸氢二钠–柠檬酸缓冲液和45℃下作用30分钟。反应时间到后,在100℃加热10分钟终止反应。S-bgl4水解纤维二糖的产物用高效液相色谱法(HPLC)进行检测。
HPLC检测的结果显示:S-bgl4能够水解纤维二糖生成葡萄糖。
HPLC条件:仪器:Agilent1100色谱仪;色谱柱:氨基柱;流动相:乙腈:水(70:30);流速:1.0mL/min;检测器:RID(折光检测器)。
S-bgl4以纤维二糖为底物的酶活力为214.78U/mg。
Claims (4)
1.一个编码糖基水解酶家族1的β-葡萄糖苷酶的基因S-bgl4,其特征在于,其核苷酸序列如SEQ ID NO:1所示。
2.根据权利要求1的基因所编码的蛋白质,其氨基酸序列如SEQ ID NO:2所示。
3.一种表达载体,其特征在于,它是含有权利要求1所述的基因。
4.一种宿主细胞,其特征在于,它是权利要求3所述表达载体转化的原核细胞或真核细胞。
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