CN104232605B - 一种木糖苷酶Xyl_S及其编码基因与应用 - Google Patents
一种木糖苷酶Xyl_S及其编码基因与应用 Download PDFInfo
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- CN104232605B CN104232605B CN201310251300.2A CN201310251300A CN104232605B CN 104232605 B CN104232605 B CN 104232605B CN 201310251300 A CN201310251300 A CN 201310251300A CN 104232605 B CN104232605 B CN 104232605B
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- xylosidase
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Abstract
一种木糖苷酶Xyl_S及其编码基因与应用,该木糖苷酶的氨基酸序列如SEQ ID NO.2所示,其核苷酸编码序列如SEQ ID NO.1所示。该酶可以采用基因工程方法或者人工合成方法制备,具有β-木糖苷水解酶和β-葡萄糖苷酶双重活性,可以特异性地水解相应的木糖苷键和葡萄糖苷键,可广泛用于化工、食品、生物能源、医药工业方面等应用领域。
Description
技术领域
本发明属于生物工程领域,具体涉及一种木糖苷酶Xyl_S及其编码基因与应用。
背景技术
β-木糖苷酶是一种外切酶,以外切方式从非还原性末端水解木二糖及木二糖以上的低聚木糖,水解产物为木糖。它是木聚糖降解的关键酶之一,有着重要的工业应用价值。在能源工业中,工农业废弃物中的木聚糖可被木聚糖酶系转化为木糖,而木糖又可被细菌及真菌转化成酒精等有价值的燃料;在医药行业中,木聚糖酶系水解特定底物可产生在医药行业具有重要应用价值的中间转化产物。例如,Patel等人利用Moraxella.spβ-木糖苷酶水解10-去乙酰紫杉醇木糖苷的7位木糖残基,得到重要的中间产物10-去乙酰紫杉醇,为紫杉醇的合成开辟了一条崭新的途径(US005700669A;EP0668360B1)。
纤维化纤维微细菌是一种能够高效利用纤维素、半纤维素的放线菌,其可以产生多种水解酶,如Shi-HsiangShen等人(TheJournalofBiologicalChemistry,1991,266(2):1058-1063)从此菌株发酵液上清中分离得到了β-1,3-葡萄糖苷酶,并对其进行了克隆和外源表达;PetraTiels等人(NatureBiotechnology2012,30:1225-1231)对此菌株所产生的5个甘露糖苷酶进行了克隆和外源表达。但目前为止,并没有获得其中木糖苷酶的报道。我们在前期工作中分离到一株纤维化纤维微细菌(Cellulosimicrobiumcellulans)菌株F16(CCTCCM2013201),通过深入研究发现其培养液上清能够分泌一种木糖苷酶,该酶具有β-木糖苷水解酶和β-葡萄糖苷酶水解酶双重活性,可以特异性水解相应的木糖苷键和葡萄糖苷键,同时具有广泛的底物特异性,可水解多种木糖苷底物和葡萄糖苷底物,例如木寡糖、4-硝基苯基-β-D-吡喃木糖苷(pNP-β-Xyl)、4-硝基苯基-β-D-吡喃葡萄糖苷(pNP-β-Glu)、7-木糖苷紫杉烷、黄芪甲苷,以及人参皂苷Rb1、Rb2、Re等。这些优良的水解性质使其有可能具有非常高的工业利用价值。因此,如何大量获得该酶或含有该酶的菌已经成为制约其工业利用的关键问题。
发明内容
本发明的目的是提供一种木糖苷酶Xyl_S及其编码基因与应用,本发明通过基因表达后大量产生具有广泛底物特异性的木糖苷酶Xyl_S,用以水解木糖苷化合物生成木糖及相应的苷元,亦可用于水解葡萄糖苷化合物生成葡萄糖及相应的苷元。
本发明提供了一种木糖苷酶Xyl_S,其氨基酸序列包含如序列表中SEQIDNO.2所示序列的至少1642个氨基酸序列;优选SEQIDNO.2示序列的第24个氨基酸残基到第1151个氨基酸残基在内的至少1128个氨基酸序列,进一步优选包括SEQIDNO.2所示序列的第87个氨基酸残基到第1119个氨基酸残基在内的至少1033个氨基酸序列。
本发明提供了一种木糖苷酶Xyl_S的编码基因,该基因的核苷酸序列具有SEQIDNO.1所示的核苷酸序列的至少4929个核苷酸;优选具有SEQIDNO.1所示的核苷酸序列的第72个核苷酸到第3453个核苷酸在内的至少3372个核苷酸;进一步优选具有SEQIDNO.1所示的核苷酸序列的第261个核苷酸到第3357个核苷酸在内的至少3099个核苷酸。
本发明提供了一种含有所述编码基因的重组载体,该重组载体是大肠杆菌表达载体、酿酒酵母表达载体、毕赤酵母表达载体、枯草芽孢杆菌表达载体、乳酸菌表达载体、丝状真菌表达载体中的任意一种。
本发明提供了一种含有所述重组载体的重组细胞株,该重组细胞株的宿主细胞是大肠杆菌、酿酒酵母、毕赤酵母、枯草芽孢杆菌、乳酸菌、丝状真菌的任意一种。该重组细胞株可以表达木糖苷酶Xyl_S,既可以胞内表达,也可以分泌到细胞外。
本发明所涉及的基因是通过DNA重组技术从纤维化纤维微细菌(Cellulosimicrobiumcellulans)菌株F16(CCTCCM2013201)的染色体中克隆,并经连接在pColdIV表达载体后在大肠杆菌中过量表达。经过量表达的木糖苷酶Xyl_S既可以分泌到细胞外,也可以在细胞内表达。其在SDS-PAGE上呈现的分子量约为140KDa,其最佳反应pH为7.5,有效作用的pH范围为5.5~9.5,并且在20~50℃均具备良好的催化活性。
由于密码子的简并性,所有与SEQIDNO.1同源性低至约60%的简并序列也能编码出SEQIDNO.2所述的序列。另外,任何基因通过DNA重组技术可以改变其序列,从而产生各种不同的突变体。这些突变体所表达的蛋白质通常具有类似的性质。当基因或蛋白质序列达到一定的同源性时,它们所表达的蛋白质的性质随同源性增加而更为相似。本发明涉及的基因及其产物也具有相同的特点。当同源性达到80%以上时,类似基因所表达的蛋白质将会具有催化木糖苷化合物水解为木糖和苷元的性质。
由于上述技术方案的使用,本发明的有益效果是:利用本发明的基因进行表达,获得木糖苷酶Xyl_S或含该酶的宿主细胞,能有效地催化木糖苷化合物以及葡萄糖苷化合物的水解,可应用于各种工业,例如用于生物质转化,如用于由包含生物质的纤维素生产燃料乙醇的过程中,用于饲料组合物中,提高动物对粗纤维的利用率,或用于面包制造面团中,还可以用于医药中间体的获得,如水解7-木糖-10-去乙酰紫杉醇获得10-去乙酰紫杉醇,同时水解黄芪甲苷IV上的β-木糖苷键和β-葡萄糖苷键从而获得相应苷元,水解掉人参皂苷Rb1、Rb2或Re上的木糖苷和葡萄糖苷得到相应人参皂苷CK、CompoundY、PPD等等。
本发明提供的木糖苷酶Xyl_S或重组细胞株应用于医药,生物,农业,能源等领域,将木糖苷化合物(或葡萄糖苷化合物)中的糖苷键水解,获得相应的苷元。
附图说明
图1:聚丙烯酰胺凝胶电泳分析分离出纯的木糖苷酶Xyl_S,其中,MUX为4-甲基伞形酮酰-β-D-吡喃木糖苷,其糖苷键经水解后经365nm激发光激发后出现荧光,从而实现对各个组分中β-木糖苷酶的监测和酶谱分析;左图:2%-15%梯度NativePAGE,考染;中图:2%-15%梯度NativePAGE,MUX染色;右图:10%SDS-PAGE,考染;样品1—BSAferrintin;2—发酵液上清;3—多酶复合物;4—木糖苷酶Xyl_S;
图2:纯酶催化pNP-β-D-Xyl水解,底物浓度对应相应初始反应速率的米氏曲线;
图3:纯酶催化pNP-β-D-Glu水解,底物浓度对应相应初始反应速率的米氏曲线;
图4:琼脂糖电泳检测PCR产物,其中,M为标准品,λ噬菌体HindIII降解物;1为克隆出的Xyl_S基因;
图5:纯化后的重组酶Xyl_S;
图6:Xyl_S纯酶转化纯度为99%的10DAXT结果,其中,10DAXT为7-木糖-10-去乙酰基紫杉醇,10DAT为10-去乙酰基紫杉醇;
图7:Xyl_S纯酶转化7-木糖紫杉烷混合物的结果,其中,10DAXT为7-木糖-10-去乙酰基紫杉醇,10DAT为10-去乙酰基紫杉醇,10DAXC为7-木糖基-10-去乙酰三尖杉宁碱,10DAC为10-去乙酰三尖杉宁碱,10DAXTC为7-木糖-10-去乙酰基紫杉醇C,10DATC为10-去乙酰基紫杉醇C;
图8:水解产物的质谱图,其中,A为10-去乙酰三尖杉宁碱,B为10-去乙酰基紫杉醇;
图9:纯酶转化黄芪甲苷IV生成环黄芪醇TLC图。
具体实施方式
下面的实施例将对本发明予以进一步的说明,但并不因此而限制本发明。
下述实施例中,如无特殊说明,均为常规方法。
本发明所依赖的遗传资源是纤维化纤维微细菌(Cellulosimicrobiumcellulans)菌株F16,保藏号为CCTCCM2013201,保存于中国典型培养物保藏中心。
实施例1:糖苷酶的纯化及其活性亚基的分离
1、纤维化纤维微细菌(Cellulosimicrobiumcellulans)菌株F16的培养
从培养好的菌种斜面(木聚糖培养基,含1.5%琼脂)挑取约1cm2见方的菌苔,接种到100ml无菌的麦麸液体培养基中(木聚糖培养基成分:木聚糖2%,酵母膏0.2%,蛋白胨0.2%,K2HPO40.1%),30℃、160rpm摇瓶培养2d。
2、木糖苷酶Xyl_S的分离纯化
10000g/min,离心2min后收集上清即为粗酶液。以对硝基苯基-β-D-木糖苷(pNP-Xyl)作为特异性生色底物对有β-木糖苷酶活性的蛋白进行跟踪。一个酶单位定义为在30℃,pH7.5,以pNP-Xyl为底物,1h内催化产生1μmol对硝基酚所需要的酶量。依次经过硫酸铵沉淀,收集20%-40%阶段的沉淀组分;ToyopearlDEAE650M离子交换柱,收集0.5~0.7mol/LNaCl阶段洗脱组分;SephacrylS-200HR凝胶过滤层析柱,收集分子量在30kDa~200kDa之间的活性组分;将上述活性组分过Source15Q柱分离,收集比活性最高的酶活组分,即得纯的木糖苷酶Xyl_S(图1)。
实施例2:木糖苷酶Xyl_S编码基因的克隆
以纤维化纤维微细菌(Cellulosimicrobiumcellulans)菌株F16基因组DNA为模板,PCR扩增木糖苷酶Xyl_S的编码基因4929bp(见图4),克隆至pMD-T载体上,测序验证结果显示,与SEQIDNO.1所示序列一致。PCR反应体系以及反应条件见下表:
实施例3:木糖苷酶Xyl_S在大肠杆菌中的重组表达
以实施例2所得载体为模板,PCR扩增目的基因CDS区4929bp,片段两侧添加NdeI/BamHI酶切位点,终止密码子TGA前添加6*His标签,克隆至pColdIV表达载体中,挑选2个阳性克隆质粒进行测序验证,结果显示序列无误。即得相应重组载体。PCR反应体系,反应条件及所用引物见下表:
将上述重组载体取1μl转入大肠杆菌BL21感受态细胞中,使用LB/抗生素Amp(100μg/ml)平板,50ul转化液涂布,37℃O/N培养。ControlpColdIV进行同样操作。
分别挑取单菌落至50mlLB/Amp(100μg/ml)培养基中,37℃O/N培养,至OD600值约为0.6,15℃15min,添加100mMIPTG50ul(final1mMIPTG)进行诱导,15℃培养22hr。收集菌体细胞,加入10ml的PBS重悬后进行超声波破碎,对菌体破碎液进行离心分离(12000rpm,5min)。
His-TrapNi亲和层析柱纯化目标蛋白:以磷酸缓冲液(pH7.5,50mM,含20mM咪唑),1ml/min平衡层析柱,随后以0.5ml/min将菌体破碎液上清上样,同样的平衡液继续洗柱,至OD280降到基线,以磷酸缓冲液(pH7.5,50mM,含150mM咪唑),1ml/min回收目标蛋白,结果见图5。
实施例4:木糖苷酶Xyl_S的关键序列在大肠杆菌中的重组表达
以实施例2所得载体为模板,PCR扩增目的基因CDS区的第261个核苷酸到第3357个核苷酸共3099bp,片段两侧添加NdeI/BamHI酶切位点,终止密码子TGA前添加6*His标签,克隆至pColdIV表达载体中,挑选2个阳性克隆质粒进行测序验证,结果显示序列无误。即得相应重组载体。
将上述重组载体取1μl转入大肠杆菌BL21感受态细胞中,使用LB/抗生素Amp(100μg/ml)平板,50ul转化液涂布,37℃O/N培养。ControlpColdIV进行同样操作。
分别挑取单菌落至50mlLB/Amp(100μg/ml)培养基中,37℃O/N培养,至OD600值约为0.6,15℃15min,添加100mMIPTG50ul(final1mMIPTG)进行诱导,15℃培养22hr。收集菌体细胞,加入10ml的PBS重悬后进行超声波破碎,对菌体破碎液进行离心分离(12000rpm,5min)。
His-TrapNi亲和层析柱纯化目标蛋白:以磷酸缓冲液(pH7.5,50mM,含20mM咪唑),1ml/min平衡层析柱,随后以0.5ml/min将菌体破碎液上清上样,同样的平衡液继续洗柱,至OD280降到基线,以磷酸缓冲液(pH7.5,50mM,含150mM咪唑),1ml/min回收目标蛋白,结果见图5。
实施例5:纯酶水解不同糖苷类底物的特异性实验
1、底物特异性检测
选取木糖苷、葡萄糖苷、果糖苷、甘露糖苷、岩藻糖苷、半乳糖苷和纤维二糖苷等多种生色底物,均用50mM的Tris-HCl缓冲液配制成5mM、pH7.5的溶液,以30μl实施例1所得纯酶+150μlbuffer+10μl20mMCaCl2+10μl底物的反应体系,30℃条件下在96孔板中反应,BioTekhyrbidReader酶标仪监测405nm下的吸光值。
结果显示,纯的木糖苷酶Xyl_S仍具有高的β-木糖苷和β-葡萄糖苷的水解活力,同时也有一定的β-纤维二糖苷的水解能力(表1)。
表1底物与酶活力
注:—代表未检测到。
2、木糖苷酶Xyl_S催化pNP-β-D-Xyl与pNP-β-D-Glu水解的反应动力学分析
在一个96孔板内,配制以下反应体系:每孔总体积200μl,其中实施例3的完整序列重组酶和实施例4的关键序列重组酶均为30μl,底物与缓冲液的加入量为达到以下9个底物浓度:5μM,10μM,20μM,50μM,100μM,200μM,500μM,1000μM,2000μM。以上每个浓度下做一个平行样,两底物pNP-β-D-Xyl与pNP-β-D-Glu均是如此,共占用72个孔,并以pNP标准品制作标准曲线。
使用BioTekhyrbidReader酶标仪的实时监测功能,设定每1min测定一次每个孔内反应体系在405nm下的吸光值,共测定61次,以此数据计算出每个底物浓度下的初始反应速率,做出底物浓度-反应速率的米氏曲线,求出对应的Km和Vmax值(图2,图3)。
实验结果表明,木糖苷酶Xyl_S对pNP-β-D-Xyl的结合能力大于pNP-β-D-Glu(前者Km值小于后者),但对二者却有着相同的转化常数Kcat(即最大反应速率与酶浓度的比值,Vmax/[E])。也即,木糖苷酶Xyl_S对β-木糖苷化合物和β-葡萄糖苷化合物有着相同的转化能力。同时,只表达关键区域后,酶对底物的亲和力略有下降,但仍具有相同活力,能够很好地水解底物(表2)。
表2酶促反应动力学参数
实施例6:木糖苷酶Xyl_S在毕赤酵母中的重组表达
将实施例2所得Xyl_S基因的CDS区域通过PCR方法在其5’端、3’端分别加上XhoI、XbaI酶切位点,连接到重组表达质粒pPICZα中(加入分泌表达信号肽和组氨酸标签),测序验证序列的正确性。将验证无误的重组载体电转化法转入巴斯德毕赤酵母中,甲醇诱导表达。离心去菌体,上清作为粗酶液,采用与实施例4同样的亲和层析柱分离纯化重组蛋白。
实施例7:木糖苷酶Xyl_S在枯草芽孢杆菌中的重组表达
将实施例2所得Xyl_S基因的CDS区域通过PCR方法在其5’端、3’端分别加上BamHI、HindIII酶切位点,连接到pP43NMK穿梭表达载体中(添加了组氨酸标签),测序验证序列的正确性,将验证无误的重组载体转入枯草芽孢杆菌B.subtilis1A752S中。离心去菌体,上清作为粗酶液,采用与实施例4同样的亲和层析柱分离纯化重组蛋白。
实施例8:木糖苷酶Xyl_S的应用
1、纯酶水解7-木糖-10-去乙酰紫杉醇(10DAXT)
将纯度为99%的10DAXT以10mg/ml的终浓度溶解于甲醇之中,以200μl的反应体系进行水解反应:其中来自实施例3的纯酶30μl+150μlTris盐酸缓冲液(50mM,pH7.5)+10μlCaCl2+10μl底物。在35℃下反应15min后,加入200μl甲醇终止反应,HPLC-UV方法检测转化率,检测结果见图6。
具体检测方法:色谱柱:KromasilC18色谱柱(200mm×4.6mmi.d.,5μm);流动相:乙腈:水(V/V),具体梯度:0-15min,30→70;15-22min,10%→10%;22-30min,70%→70%;柱温40℃;检测波长:227nm;流速:1.0ml/min;进样量:20μl。
若想将体系中全部的10DAXT均转化为10DAT,可继续反应~105min即可。
2、纯酶水解7-木糖紫杉烷混合物
以7mg/ml的终浓度溶解7-木糖紫杉烷混合物于甲醇之中,反应体系,反应条件及检测方法均与上例中水解99%纯度的10DAXT基本相同,不同的是反应时间改为30min,检测结果见图7。
此外,通过UPLC-PDA-MS法对其水解前后的分子量进行了质谱分析,具体方法如下:KromasilC18色谱柱(200mm×4.6mmi.d.,5μm)。Tedia公司色谱纯乙腈,Millipore公司超纯水。质谱用ESI接口离子源,氮气为夹套气和吹扫气,夹套气压力40psi,辅助气20a.u.,源电压4.0kV,毛细管温度200℃,雾化器温度325℃。检测结果见图8。
3、纯酶水解黄芪甲苷IV
以5mg/ml的终浓度溶解黄芪甲苷IV纯品于甲醇之中,以200μl的反应体系进行水解反应:其中来自实施例3的纯酶30μl+150μlTris盐酸缓冲液(50mM,pH7.5)+10μlCaCl2+10μl底物。在35℃下反应15min后,检测方法采用TLC法进行(图9)。
Claims (9)
1.一种木糖苷酶Xyl_S,其特征在于:其氨基酸序列如序列表中SEQIDNO.2所示序列的1642个氨基酸序列。
2.一种木糖苷酶Xyl_S的编码基因,其特征在于:该基因的核苷酸序列为SEQIDNO.1所示的核苷酸序列的4929个核苷酸。
3.一种含有权利要求2所述编码基因的重组载体。
4.按照权利要求3所述的重组载体,其特征在于:该重组载体是大肠杆菌表达载体、酿酒酵母表达载体、毕赤酵母表达载体、枯草芽孢杆菌表达载体、乳酸菌表达载体、丝状真菌表达载体中的任意一种。
5.一种含有权利要求3所述重组载体的重组细胞株。
6.按照权利要求5所述的重组细胞株,其特征在于:该重组细胞株的宿主细胞是大肠杆菌、酿酒酵母、毕赤酵母、枯草芽孢杆菌、乳酸菌、丝状真菌的任意一种。
7.按照权利要求5所述的重组细胞株,其特征在于:该重组细胞株表达木糖苷酶Xyl_S,既能够胞内表达,也能够分泌到细胞外。
8.一种应用,其特征在于:权利要求1所述木糖苷酶Xyl_S或权利要求5所述重组细胞株应用于医药,生物,农业,能源领域,将β-木糖苷化合物中的β-糖苷键水解,获得相应的苷元。
9.一种应用,其特征在于:权利要求1所述木糖苷酶Xyl_S或权利要求5所述重组细胞株应用于医药,生物,农业,能源领域,将β-葡萄糖苷化合物中的β-糖苷键水解,获得相应的苷元。
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