CN105907775B - 一种木聚糖酶TlXynA的突变基因TlXynA_1及其应用 - Google Patents
一种木聚糖酶TlXynA的突变基因TlXynA_1及其应用 Download PDFInfo
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Abstract
本发明公开了一种木聚糖酶TlXynA的突变基因TlXynA_1,所述基因的突变位点为R116Q和R161Q,其核苷酸序列如SEQ ID NO.1所示。本发明还公开了所述基因在制备木聚糖酶中的应用。实验证明,这种基因所编码的突变木聚糖酶活性可达到4576IU/mg,比野生型酶提高了26%;在外加盐浓度为5M时相对酶活性是野生型酶的1.60倍;在pH为4时相对酶活性是野生型酶的1.27倍。预示该突变木聚糖酶具有耐高温、耐盐的特性,在饲料、食品、造纸、医药、能源等工业生产中具有广阔的应用前景。
Description
技术领域
本发明属于基因工程领域,具体涉及一种木聚糖酶TlXynA的突变基因TlXynA_1及其应用。
背景技术
木聚糖是继纤维素后最丰富的植物细胞壁多糖,占地球上生物质资源总量的1/3。其主链骨架由木糖通过β-1,4糖苷键连接而成,而侧链包括多种官能团,如葡萄糖醛酸、阿魏酸、阿拉伯糖、乙酰基等。这种复杂的结构导致木聚糖的降解需要多种酶系共同作用。其中的关键酶是内切β-1,4-木聚糖酶,可以通过水解β-1,4-糖苷键来进行主链的降解,把木聚糖转变为木寡糖。在2008年,木寡糖已被中国卫生部批准为新功能性食品,它安全、高效、稳定,在食品、饲料、造纸、医药、能源等领域具有广阔的前景。
来自疏棉状嗜热丝孢菌(Thermomyces lanuginosus)的木聚糖酶XynA(TlXynA)是一种良好的工业用酶。它专一性地识别木聚糖底物;热稳定性强,最适温度在65℃以上,在80℃下孵育40min后仍保持60%的酶活(Damaso et al.,2003);可以耐受广泛的pH;酶活性可高达4000IU/mg(Singh et al.,2003)。TlXynA可以用不同宿主进行异源表达,例如用毕赤酵母(Pichia pastoris)异源表达时,产量可以达到1.2g/L(Mellitzer et al.,2012)。因此,TlXynA是一种理想的工业用酶,可以用作烘焙剂、纸浆漂白剂、饲料添加剂、燃料乙醇生产时的底物处理剂等。但是,在TlXynA的实际应用中,仍存在着亟待解决的问题。例如工业生产中的很多步骤往往是在高盐浓度、存在胰蛋白酶的环境中进行的,这往往会限制TlXynA将最适条件下的高酶活性发挥出来。为了扩展TlXynA的工业应用,提高其耐高温、耐盐、耐胰蛋白酶酶解等性质是一个很关键的任务,然而,经检索,还未见关于TlXynA耐盐、耐胰蛋白酶酶解等性质和其相关突变基因的报道。
发明内容
针对现有技术的不足,本发明的目的是提供一种木聚糖酶TlXynA的突变基因TlXynA_1及其应用。
本发明所述木聚糖酶TlXynA的突变基因TlXynA_1,其特征在于:所述基因的突变位点为R116Q和R161Q,其核苷酸序列如SEQ ID NO.1所示。
本发明所述木聚糖酶突变基因TlXynA_1表达的木聚糖酶,其特征在于:所述酶命名为TlXynA-R116QR161Q,其氨基酸序列如SEQ ID NO.2所示。
本发明所述木聚糖酶突变基因TlXynA_1在制备木聚糖酶TlXynA-R116QR161Q中的应用。
含有木聚糖酶突变基因TlXynA_1的重组载体pET28a-TlXynA-R116QR161Q的构建及木聚糖酶TlXynA-R116QR161Q的表达及纯化。
从NCBI数据库中获取木聚糖酶TlXynA基因,将TlXynA基因和质粒pET28a连接,获得pET28a-TlXynA质粒,其核苷酸序列如SEQ ID NO.3所示;直接对质粒进行定点突变,引入R116Q和R161Q两个突变位点;将上述突变质粒转化大肠杆菌DH5α感受态细胞,筛选并测序鉴定阳性重组载体,提取质粒,即得到含有木聚糖酶突变基因TlXynA_1的重组载体pET28a-TlXynA-R116QR161Q,其核苷酸序列如SEQ ID NO.4所示。
将前述重组载体转化大肠杆菌BL-21(DE3)感受态细胞,即获得含有重组载体的重组工程菌株。
将获得的含有重组载体的重组工程菌株接种到LB液体培养基中,于37±2℃,200±10rpm条件发酵培养至OD600=0.6-0.8;用终浓度为0.5mM的IPTG于20℃,200rpm条件培养20±2h,诱导表达得到木聚糖酶TlXynA-R116QR161Q;离心并收集菌体,破碎菌体取上清,将上清液中的蛋白用镍柱进行纯化。
本发明所述的木聚糖酶TlXynA-R116QR161Q在制备耐高温耐盐工业酶制剂中的应用。
实验证实:利用本发明所述的木聚糖酶突变基因TlXynA_1制备得到的木聚糖酶TlXynA-R116QR161Q作为饲料添加剂、造纸漂白剂、烘焙剂等工业用酶时能在更广泛的环境中保持较高活性,具体应用包括但不仅限于:在饲料工业中,此突变木聚糖酶可以作为饲料添加剂,在动物消化系统中发挥作用,增加动物对饲料的消化和吸收;在造纸工业中,此突变木聚糖酶可以用来替代氯化物进行底物漂白处理,能够改善纸张白度和质量,减少环境污染;在食品工业中,此突变木聚糖酶可以作为酿酒时采用的添加剂,降低啤酒黏度,提高澄清度,还可以获得含有特定组成的寡糖,增加饼干的口感;在能源工业中,此突变木聚糖酶可以和纤维素酶共同作用降解被酸和离子溶液处理过的木质纤维素,更高效地产生乙醇等清洁能源。预示其具有广阔的应用前景。
本发明具有的有益效果是:
(1)本发明提供的将突变木聚糖酶异源表达和纯化的方法可以有效保持目的蛋白的高表达量,方法简便实用,蛋白浓度可达到每升培养基3.5mg。
(2)本发明中突变基因TlXynA_1所编码的突变木聚糖酶TlXynA-R116QR161Q具有高酶活性,可达到4576IU/mg,相比现有野生型木聚糖酶TlXynA活性提高了26%。
(3)本发明中突变基因TlXynA_1所编码的突变木聚糖酶TlXynA-R116QR161Q具有耐盐性,在外加盐浓度为1M到5M时,其相对酶活性分别是野生型木聚糖酶TlXynA的1.09,1.26,1.28,1.35和1.60倍;上述突变木聚糖酶具有耐受低pH的能力,在pH为4和5时,其相对酶活性分别是野生型酶的1.27和1.03倍。
(4)本发明中突变基因TlXynA_1所编码的突变木聚糖酶TlXynA-R116QR161Q具有耐高温、耐盐、耐胰蛋白酶酶解的特性,在造纸、饲料、食品、医药、能源等工业领域均有广阔的应用。
附图说明
图1为本发明实施例3中TlXynA-R116QR161Q蛋白表达的SDS-PAGE结果图,其中M为Unstained Protein Marker(Thermo Scientific,MA,USA),1-7为突变木聚糖酶TlXynA-R116QR161Q。
图2为本发明实验例1中TlXynA-R116QR161Q与TlXynA在不同盐浓度下的相对酶活性图,其中WT为TlXynA,M1为TlXynA-R116QR161Q。
图3为本发明实验例1中TlXynA-R116QR161Q与TlXynA在外加盐浓度为0时的酶活性比较图,其中WT为TlXynA,M1为TlXynA-R116QR161Q。
图4为本发明实验例1中TlXynA-R116QR161Q与TlXynA在不同pH下的相对酶活性图,其中WT为TlXynA,M1为TlXynA-R116QR161Q。
具体实施方式
以下结合附图和实施例对本发明保护内容做进一步的阐述,但所述实例并不是对本发明保护内容的限制。
实施例1:
构建含有木聚糖酶TlXynA的突变基因TlXynA_1的重组载体,具体步骤如下:
(1)从NCBI数据库中获取木聚糖酶TlXynA基因,将TlXynA基因和质粒pET28a连接,获得pET28a-TlXynA质粒,其核苷酸序列如SEQ ID NO.3所示。以上述重组质粒为模板,设计突变引物,进行定点突变,引物序列如下:
PCR扩增反应体系如下:
反应程序如下:
(2)取3μL PCR产物进行琼脂糖凝胶电泳验证,留下条带清晰的反应产物。用DpnI限制酶对产物中消化野生型的质粒进行消化,确保之后步骤中的转化子均为突变型。消化体系如下表,将体系充分混合,于37℃水浴反应15min;
(3)将消化后的PCR产物进行纯化,实验步骤参照北京鼎国昌盛生物技术有限责任公司GV-High–Efficiency DNA Fragments Purification Kit;
(4)突变质粒转化大肠杆菌DH5α感受态细胞:
将10μL已纯化的上述突变质粒加入50μL大肠杆菌DH5α感受态细胞,冰浴30min;42℃热击90s;冰浴2min,加1mL液体LB,37℃摇床中培养1-1.5h;8000rpm离心2min,弃上清(留少许底部液体)。将剩余溶液涂布到含50μg/mL卡那霉素的LB平板上,37℃过夜倒置培养;次日挑取单克隆,接种在5mL含50μg/mL卡那霉素的LB培养基中,37℃200rpm过夜培养;
(5)提取质粒,实验步骤参照北京鼎国昌盛生物技术有限责任公司GV-PlasmidDNA Mini Extraction Kit;
(6)取3μL质粒进行1%琼脂糖凝胶电泳检测,留下条带清晰的质粒;
(7)取10μL质粒溶液于灭菌EP管中进行测序。比对测序结果和原始序列,确认定点突变是否成功。测序正确的质粒即为含有木聚糖酶TlXynA的突变基因TlXynA_1的重组载体pET28a-TlXynA-R116QR161Q,其核苷酸序列如SEQ ID NO.4所示。
实施例2:
构建含有上述突变基因TlXynA_1的重组工程菌,具体步骤如下:
将2μL测序正确的上述突变质粒加入50μL大肠杆菌BL-21感受态细胞,冰浴30min;42℃热击90s;冰浴2min,加入1mL液体LB,37℃摇床中培养1-1.5h;8000rpm离心2min,弃上清(留少许底部液体)。将剩余溶液涂布到含50μg/mL卡那霉素的LB平板,涂布均匀至干燥,37℃过夜倒置培养;次日挑取单克隆,接种在5mL含抗生素的LB培养基中,37℃ 200rpm过夜培养,即得到含有突变基因TlXynA_1的重组工程菌。
实施例3:
发酵表达并纯化上述突变基因TlXynA_1所编码的突变木聚糖酶TlXynA-R116QR161Q,具体步骤如下:
(1)重组蛋白的异源表达:
①取实施例2得到的含有上述突变基因TlXynA_1的重组工程菌,于5mL LB培养基中(含50μg/mL卡那霉素)37℃ 200rpm过夜培养;
②将培养过夜的菌液转接到装有300mL LB培养基的1L三角瓶中(含50μg/mL卡那霉素),37℃下培养约3h,至OD600=0.6-0.8;
③加入终浓度为0.5mM的IPTG,20℃诱导培养20h;
④8000rpm,4℃离心10min,得到菌体沉淀;
⑤用pH 8.0的NaH2PO4-NaCl缓冲液重悬菌体,将菌液置于100mL离心管中;
⑥将离心管置于冰上,超声破碎细胞,时间设置为9s ON,10s OFF,共90次;
⑦11000rpm,4℃离心30min;
⑧用0.22μm的滤头过滤上清液,把柱子的填料与滤液结合,准备进行亲和纯化。
(2)重组蛋白的纯化:
①使用GE Healthcare公司Ni Sepharose 6Fast Flow亲和柱对含有6×His tag的目的蛋白进行亲和纯化。将上一步获得的粗酶液与填料混合,于4℃转动结合2h,使填料上的镍与蛋白上的His标签充分结合;
②用pH 8.0的NaH2PO4-NaCl缓冲液配制浓度为1M的咪唑母液,然后分别稀释到5mM,20mM,60mM,100mM,200mM,用以上浓度的咪唑洗脱蛋白,分别收集至10mLEP管中;
③蛋白洗脱后镍柱再生,镍柱再生时按照如下顺序加入对应溶液:50mM EDTA→蒸馏水→1M NaCl→蒸馏水→0.1M硫酸镍→蒸馏水→70%乙醇→20%乙醇保存备用;
④将收集的酶液进行SDS-PAGE:将样品与SDS buffer按4:1混匀(样品取16μL,buffer取4μL),Marker取10μL,105℃处理10min;点样时样品点12μL,Marker点5μL。用80V电压进行电泳,待蛋白样品呈一条直线时把电压调成180V。电泳结束后考马斯亮蓝R-250染液染色30min,脱色液脱色约2h,用扫描仪扫胶观察;
⑤找出目的蛋白条带较纯的几管,加pH 6.0的Na2HPO4-柠檬酸缓冲液,4900rpm于4℃超滤,至滤出的缓冲液pH=6.0;
⑥用无菌的0.22μm滤头将超滤后的酶液过滤到灭菌的10mL离心管中,4℃保存。如要长期保存,需置于-80℃。
(3)重组蛋白的含量测定:
①将目的蛋白稀释到合适的浓度(测定时不超过标准曲线的量程);
②对照组加入0.1mL pH 6.0的Na2HPO4-柠檬酸缓冲液,实验组加入0.1mL稀释后的目的蛋白溶液。每管再分别加入1mL考马斯亮蓝染色液,摇匀。静置10min后测定OD595;
③每组三个平行,测定三次,共九组重复;
④根据标准曲线算出蛋白量和蛋白浓度。
如图1所示,SDS-PAGE电泳显示,突变木聚糖酶TlXynA-R116QR161Q的条带为25kDa左右的标准蛋白,与预期大小21.3kDa相似,和前人的研究也相符(Damaso et al.,2003)。此结果显示,本发明成功获得了目标蛋白TlXynA-R116QR161Q。
以下用实验例的方式来阐述本发明的有益效果:
实验例1:本发明中突变基因TlXynA_1所编码的突变木聚糖酶TlXynA-R116QR161Q与野生型酶TlXynA的酶学性质比较:
(1)突变木聚糖酶TlXynA-R116QR161Q和野生型酶TlXynA在不同盐浓度下的酶活性:
①将突变木聚糖酶TlXynA-R116QR161Q和野生型酶TlXynA稀释到0.0005mg/mL;
②在对照组管中加入20μL pH 6.0的Na2HPO4-柠檬酸缓冲液,实验组管中加入20μL稀释过的酶液,每管再分别加入80μL含NaCl浓度为0-5M不等的1%木聚糖底物,65℃反应10min;
③每管各加入80μL DNS,沸水浴10min;
④迅速冷却,加入820μL ddH2O,摇匀,测定OD550;
⑤每种酶做三次重复实验;
⑥根据标准曲线算出木糖量,再根据公式算出比酶活;
⑦比较两种蛋白在外加盐浓度为0时的酶活性。
突变木聚糖酶TlXynA-R116QR161Q与野生型木聚糖酶TlXynA在不同盐浓度下的酶活性如图2所示。在外加盐浓度为1M到5M时,突变木聚糖酶TlXynA-R116QR161Q的酶活性分别是野生型木聚糖酶TlXynA的1.09,1.26,1.28,1.35和1.60倍,这证明突变木聚糖酶具有较强的耐盐性。
突变木聚糖酶TlXynA-R116QR161Q与野生型木聚糖酶TlXynA在外加盐浓度为0时的酶活性如图3所示。在外加盐浓度为0时,野生型TlXynA的酶活性为3632IU/mg,而突变木聚糖酶TlXynA-R116QR161Q的酶活性为它的1.26倍,达到了4576IU/mg,这证明突变木聚糖酶酶活性很高,具有潜在的工业应用价值。
(2)突变木聚糖酶TlXynA-R116QR161Q和野生型酶TlXynA在不同pH下的酶活性:
①将突变木聚糖酶TlXynA-R116QR161Q和野生型酶TlXynA稀释到0.0005mg/mL;
②对照组加入20μL pH 6.0的Na2HPO4-柠檬酸缓冲液,实验组加入20μL稀释过的酶液,每管再分别加入80μL pH=1-6的1%木聚糖底物,65℃反应10min;
③每管各加入80μL DNS,沸水浴10min;
④迅速冷却,加入820μL ddH2O,摇匀,测定OD550;
⑤每种酶做三次重复实验;
⑥根据标准曲线算出木糖量,再根据公式算出比酶活。
突变木聚糖酶TlXynA-R116QR161Q与野生型木聚糖酶TlXynA在不同pH下的酶活性如图4所示。在pH为4和5时,突变木聚糖酶TlXynA-R116QR161Q的相对酶活性分别是野生型酶的1.27和1.03倍,这说明其耐受低pH的能力有一定的提升。
(3)突变木聚糖酶TlXynA-R116QR161Q和野生型酶TlXynA的酶动力学参数:
①配制不同浓度的木聚糖溶液,浓度分别为0.4%,0.8%,1.2%,1.6%,2%;
②根据预实验结果,将待测蛋白稀释到特定倍数;
③每种底物做三个实验组,一个对照组,在每管中加入500μL木聚糖底物;
④对照管中加入100μL pH 6.0的Na2HPO4-柠檬酸缓冲液,实验管加入100μL稀释过的酶液,65℃反应3min;
⑤每管加入400μL DNS,沸水浴10min;
⑥迅速冷却,测定OD550;
⑦每种酶做三次重复实验;
⑧将数据用米氏方程进行拟合,计算其kcat、KM和kcat/KM。
野生型酶TlXynA的kcat、KM、kcat/KM分别是5273、3.712和1421;突变木聚糖酶TlXynA-R116QR161Q的kcat、KM、kcat/KM分别是5925、3.394和1746。其kcat/KM是野生型酶的1.23倍,与酶活测定数据相符,说明本发明所涉及的突变木聚糖酶活性很高,具有潜在的工业应用价值。
上述实施例只为说明本发明的技术构思及特点,其目的在于让熟悉此项技术的人士能够了解本发明的内容并据以实施,并不能以此限制本发明的保护范围。凡根据本发明精神实质所作的等效变化或修饰,都应涵盖在本发明的保护范围之内。
Claims (4)
1.一种木聚糖酶TlXynA的突变基因TlXynA_1,其特征在于:所述基因的突变位点为R116Q和R161Q,其核苷酸序列如SEQ ID NO.1所示。
2.权利要求1所述的木聚糖酶突变基因TlXynA_1表达的木聚糖酶,其特征在于:所述酶命名为TlXynA-R116QR161Q,其氨基酸序列如SEQ ID NO.2所示。
3.权利要求1所述的木聚糖酶突变基因TlXynA_1在制备木聚糖酶TlXynA-R116QR161Q中的应用。
4.如权利要求3所述的应用,其特征在于:
①构建含所述突变基因TlXynA_1的重组载体pET28a-TlXynA-R116QR161Q,其核苷酸序列如SEQ ID NO.4所示,将其转化到大肠杆菌BL-21(DE3)中,获得重组工程菌株;
②将获得的重组工程菌株接种到LB液体培养基中,于37±2℃,200±10rpm条件发酵培养至OD600=0.6-0.8;用终浓度为0.5mM的IPTG于20℃,200rpm条件培养20±2h,诱导表达得到木聚糖酶TlXynA-R116QR161Q。
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