CN116376880A - 一种β-葡萄糖苷酶、表达载体、宿主细胞和应用 - Google Patents
一种β-葡萄糖苷酶、表达载体、宿主细胞和应用 Download PDFInfo
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- CN116376880A CN116376880A CN202310632274.1A CN202310632274A CN116376880A CN 116376880 A CN116376880 A CN 116376880A CN 202310632274 A CN202310632274 A CN 202310632274A CN 116376880 A CN116376880 A CN 116376880A
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- glucosidase
- baohuoside
- baicalein
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Abstract
本发明属于分子生物学领域,涉及酶法提取,特别是指一种β‑葡萄糖苷酶、表达载体、宿主细胞和应用。本发明从奶牛粪便宏基因组文库中克隆到了一个β‑葡萄糖苷酶基因,将该β‑葡萄糖苷酶的基因克隆到大肠杆菌表达载体上,获得可异源表达该酶的大肠杆菌重组菌株,该菌株异源表达制备的β‑葡萄糖苷酶在适宜条件下不仅能高效转化淫羊藿苷为宝藿苷I,还可高效转化黄芩苷为黄芩素。转化淫羊藿苷为宝藿苷I的转化率最高可达100%,转化黄芩苷为黄芩素的转化率最高可达98%,证明了本申请的β‑葡萄糖苷酶cdw11实现了同时高效率制备宝藿苷I和黄芩素的目的。
Description
技术领域
本发明属于分子生物学领域,涉及酶法提取,特别是指一种β-葡萄糖苷酶、表达载体、宿主细胞和应用。
背景技术
β⁃葡萄糖苷酶(β⁃glucosidase,EC 3.2.1.21),又称β-D-葡萄糖糖苷水解酶,是一类纤维素酶,能够从含糖化合物中催化水解末端的非还原性β⁃D⁃糖苷键,释放出β-D-葡萄糖及相应的单糖、寡糖或复合糖。β-葡萄糖苷酶根据氨基酸和结构相似性分属于GH1、GH2、GH3、GH5、GH9、GH30和GH116家族,被广泛用于食品、药品、酿酒等行业。β-葡糖苷酶还可以催化合成烷基糖苷,烷基糖苷可用作治疗剂、诊断工具、益生菌的生长促进剂和表面活性剂等。酶法反应条件温和、产物纯、易分离等优点而逐渐被人们选择。因此β-葡糖苷酶在合成糖苷及天然产物的改造中都发挥着极其重要的作用。
淫羊藿的主要活性成分为总黄酮(Total flavons of Epimedium,TFE),是在C-8位有异戊烯基的黄酮苷类,如多糖苷类化合物朝藿定A、朝藿定B、朝藿定C;低糖苷类化合物淫羊藿苷、宝藿苷I以及苷元淫羊藿素。其中淫羊藿苷和朝藿定C具有天然丰度。淫羊藿苷(Icariin,ICA)是淫羊藿中含量最高的黄酮类成分,是其生物活性指标,具有多种药理作用,但口服利用率较低。淫羊霍次苷II(icariside II, ICS-II )又名宝藿苷I,结构式:,是淫羊藿苷、朝藿定A、朝藿定B、朝藿定C等多种淫羊藿黄酮化合物的主要代谢产物,同时也是吸收入血的主要成分之一。代谢和药代动力学研究表明,淫羊藿苷在体内被肠道菌群代谢,并以宝藿苷I的形式被吸收。ICA II还具有抗炎、抗骨质疏松、和抗癌活性,并且研究表明它通常比ICA更有效。同时淫羊藿苷与宝藿苷I两者之间仅相差一个葡萄糖,通过有效的手段选择性的切除淫羊藿苷上的葡萄糖残基,将获得低含量高附加值的产物宝藿苷I。生物酶法因其选择性强、副产物少、转化率高以及反应条件温和等优点逐渐被应用于天然产物的改性增效。张振海等发表的β-葡萄糖苷酶酶解淫羊霍苷制备宝藿苷I的工艺研究中,公开了利用β-葡萄糖酶酶解淫羊藿制备宝藿苷I的方案;而不同生物中克隆的β-葡萄糖苷酶酶的功能不同。
发明内容
为解决上述技术问题,本发明提出一种β-葡萄糖苷酶、表达载体、宿主细胞和应用,提供了一种可同时将淫羊藿苷转化为宝藿苷I、黄芩苷转化为黄芩素得方案。
本发明的技术方案是这样实现的:
一种β-葡萄糖苷酶,其对对硝基苯酚的比活力为854 U/mg。
其基因的核苷酸序列如SEQ ID No.1所示。
一种重组载体,含有如SEQ ID No.1所示的核苷酸序列。
一种宿主细胞,含有上述的β-葡萄糖苷酶;或者含上述的重组载体。
一种同时制备宝藿苷I和黄芩素的方法,,步骤为:
(1)将SEQ ID NO:1所示核苷酸序列重组、构建到pET-28a(+)载体中;再转化到大肠杆菌菌株中,得到表达株;
(2)表达株在LB液体培养基中培养,并加入0.2 mM的IPTG诱导;发酵完毕,超声破碎,离心取上清,得可溶性的β-葡萄糖苷酶采用Ni-NTA亲和层析柱进行蛋白纯化得到较纯的β-葡萄糖苷酶,并对β-葡萄糖苷酶最适温度和最适pH进行测定;
(3)将淫羊藿药材和黄芩药材烘干、粉碎过60目筛后,经含弱碱甲醇等有机溶剂回流提取三次,合并三次提取液过滤,滤液浓缩干燥成粉末即得混合提取物;
(4)将步骤(3)所得混合提取物添加于β-葡萄糖苷酶的水溶液中,于合适的酶反应温度和pH,加热上述混合物,过滤收集滤液;
(5)将步骤(4)所得滤液加入乙酸乙酯将反应产物萃取出来,获得宝藿苷I和黄芩素。
本发明具有以下有益效果:
1、本发明将序列表中SEQ ID NO:1的核苷酸序列利用pET-28a(+)载体和大肠杆菌表达菌株实现了一种新的β-葡萄糖苷酶的表达,得到大量β-葡萄糖苷酶蛋白,并将其用于同时转化淫羊藿苷为宝藿苷I、黄芩苷转化为黄芩素;通过本发明所提供的蛋白制备方法得到的β-葡萄糖苷酶蛋白稳定性好、具有很强的生物活性,pH 6.5,45℃下的比活力为9500U/mg蛋白,能够高效分解淫羊藿苷及黄芩苷的β-葡萄糖苷键,高效获得更有效的成分—宝藿苷I、黄芩素,转化率高达100%,而且有利于规模化生产。
2、该β-葡萄糖苷酶基因是一种新发现的基因,目前还没有对其进行研究开发的报道。因此,通过表达纯化方法高效生产β-葡萄糖苷酶,加入该β-葡萄糖苷酶,能高效裂解淫羊藿苷的β-葡萄糖苷键,获得更有效的活性成分:宝藿苷I和黄芩素。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1为本发明所述β-葡萄糖苷酶转化淫羊藿苷制备宝藿苷I的水解线路图
图2为β-葡萄糖苷酶基因的表达产物的SDS-PAGE图。其中,泳道1为含空载质粒pEASY-E2的大肠杆菌细胞裂解液;泳道2为超声后的破碎液;泳道3为破碎液上清;泳道4为破碎液沉淀;泳道5为镍柱流出液;泳道M为蛋白Marker;泳道6为纯化后蛋白的电泳结果(由上到下的条带依次代表100、70、55、40、35、25、15 kDa),目的蛋白在图中由箭头指出。
图3为β-葡萄糖苷酶蛋白在不同温度下的酶活力曲线,45℃为β-葡萄糖苷酶最适反应温度。
图4为β-葡萄糖苷酶蛋白在不同pH下的酶活力曲线;pH 4.0-6.0范围内选择NaAc-HAc缓冲液,pH 6.0-8.0范围内选择NaH2PO4-Na2HPO4缓冲液,pH 8.0-10.0选择Glycine-NaOH缓冲液进行测定。最适pH为6.5。
图5为薄层色谱法检测酶解产物结果;其中a为淫羊藿苷、宝藿苷I标准品混合液;b为酶液与淫羊藿苷提取物的反应产物。结果表明反应产物为宝藿苷I。
图6为薄层色谱法检测酶解产物结果;其中a为黄芩苷、黄芩素标准品混合液;b为酶液与黄芩苷提取物的反应产物。结果表明反应产物为黄芩素。
具体实施方式
下面将结合本发明实施例,对本发明的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有付出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
实验仪器、材料和试剂
1、实验仪器: 电子天平、真空干燥箱、恒温水浴锅、粉碎机、离心机、PCR仪
2、菌株与载体、试剂
大肠杆菌菌株Top10、BL21(DE3)、载体pET-28a(+)均购买于购自安徽吐露港生物科技有限公司;无水乙醇、蒸馏水、淫羊藿、pNPG,购自美仑生物。
3、酶与试剂盒
高保真酶、质粒提取试剂盒、胶回收试剂盒购自南京诺唯赞生物科技股份有限公司,重组酶购自安徽吐露港生物科技有限公司,DNeasy PowerSoil Pro Kit (Qiagen,Germany)。
4、培养基及试剂配制
(1)100mL液体LB培养基:胰蛋白胨1 g、氯化钠1g、酵母浸粉0.5 g、超纯水100 mL,高压灭菌,室温保存;
(2)LB/Kana平板:NaCl 2 g,胰蛋白胨2 g,酵母浸粉1 g,超纯水200 mL,琼脂粉4g,高压灭菌后,冷却至70℃以下,加入200 μL浓度为50 mg/mL的卡那霉素(Kanamycin),充分混匀后倒板,4℃避光保存;
(3) 50×TAE琼脂糖凝胶电泳缓冲液:Tris 121 g,冰乙酸28.6 mL,0.5 mol/LEDTA (pH8.0)50 mL,加蒸馏水定容至500 mL,室温保存;
(4)50 mg/mL卡那霉素保存液:卡那霉素0.5 g,加蒸馏水溶解并定容至10 mL,分装后于-20℃保存;
(5) 5×SDS-PAGE上样缓冲液:1M Tris-HCl (pH 6.8) 1.25 mL,SDS 0.5 g,BPB25 mg,甘油2.5 mL,加去离子水溶解后定容至5 mL,分装(约500 μL每份)后于室温保存,使用每份加入25 μL β-巯基乙醇混匀;
(6) 5×SDS-PAGE电泳缓冲液:Tris 15.1 g,甘氨酸94 g,SDS 5 g,加入约800 mL去离子水,充分搅拌溶解后定容至1 L,室温保存;
实施例1
一种新发现β-葡萄糖苷酶编码基因的获得:
Illumina高通量测序技术获得奶牛粪便微生物群落短序列的获得和拼接:
取0.25g奶牛粪便样品,利用DNeasy PowerSoil Pro Kit (Qiagen, Germany)试剂盒进行DNA提取。之后,利用Illumina高通量技术对提取的奶牛粪便DNA进行高通量测序,共获得429,806,922个序列。宏基因组测序数据分析基本分析流程包括:原始数据质量评估、质量控制、去除宿主序列(需提供明确宿主信息)、短读长(shortreads)的种群结构分析以及功能结构分析、序列拼装、基因预测、构建基因集合以及功能注释,基因组重构等。最终共得到2.2 M大于1 kb的contigs,并对他们的基因进行了注释。其中一个含有完整的GH1家族基因cdw11,该基因长1359bp。利用该基因设计引物:
F:ATGAGCTTCAAGAAAGGCTTTACCT
R:ATTGCTGTGATCTTCGATCCAAGA
利用上述引物对奶牛粪便DNA进行扩增,扩增的DNA聚合酶为Vazyme Phanta®Max Super-Fidelity DNA Polymerase,最终得到该目的基因序列如SEQ ID No.1所示。
实施例2
实施例1中的β-葡萄糖苷酶编码基因在大肠杆菌中的表达
1、重组质粒的构建
通过PCR以奶牛粪便DNA为模板克隆β-葡萄糖苷酶酶基因cdw11的ORF编码基因(SEQ ID No.1),所用正向引物为SEQ ID No.2:5’GCCTGGTGCCGCGCGGCAGCATGAGCTTCAAGAAAGGCTTTACCT,其5’端添加了与pET-28a(+)载体的重组序列:GCCTGGTGCCGCGCGGCAGC;反向引物为SEQ ID No.3:5’GGTGCTCGAGTGCGGCCGCATTGCTGTGATCTTCGATCCAAGA,其5’端添了与pET-28a(+)载体的重组序列:GGTGCTCGAGTGCGGCCGC。
将PCR产物纯化后的DNA片段和载体pET-28a(+)用一步法同源重组酶ClonExpressII One Step Cloning试剂盒,得到重组表达载体pET-28a(+)-cdw11。将上述构建好的质粒pET-28a(+)-cdw11转化入大肠杆菌BL21菌株中 ,在含有 卡那霉素的LB平板上长出菌落,菌落PCR鉴定阳性克隆,经测序后,验证结果正确。
2、重组蛋白的表达
接种大肠杆菌cdw11克隆至100 mL含100 μL50mg/mL 卡那霉素的LB培养基中,37℃,200 rpm培养至OD600为0.6-0.8。冷却后,加入IPTG至终浓度为1mM,于20℃,200 rpm继续培养16个小时,离心收集菌体。用Binding Buffer(NaH2PO4 50 mM,NaCl 300 mM,pH7.4)悬浮收集的菌体,超声波破碎细胞后的裂解液即为粗酶液。粗酶液在12000g离心20分钟,收集裂解液上清,即为所需的粗酶液。采用Ni-NTA(常州天地人和生物科技有限公司)纯化蛋白质,纯化后的蛋白质浓缩在10 kDa超滤管(Merck, Millipore, USA)中。
3、用20 μL蛋白进行蛋白SDS-PAGE电泳检测,如图2所示,泳道1为含空载质粒pEASY-E2 的大肠杆菌细胞裂解液;泳道2为超声后的破碎液;泳道3为破碎液上清;泳道4为破碎液沉淀;泳道5为镍柱流出液;泳道M为蛋白Marker;泳道6为纯化后蛋白的电泳结果(由上到下的条带依次代表100、70、55、40、35、25、15 kDa),目的蛋白在图中由箭头指出。
实施例3
本申请的β-葡萄糖苷酶的重组蛋白酶学性质的分析
β-葡萄糖苷酶的酶活测定采用pNPG法,具体操作如下:
1、对硝基苯酚标准曲线的绘制
10 mL蒸馏水中充分溶解15 mg对硝基苯酚(pNP)即可得到浓度为10 mM的pNP溶液,置于4℃冰箱中储存备用。依次加入1 mM的pNP溶液,pH为7.0的 磷酸盐缓冲液和0.5 M的Na2CO3。混合均匀后,取200 μL置于96孔板中检测405 nm波长下的吸光值,以pNP物质的量为横坐标,吸光值为纵坐标,绘制标准曲线。
2、标准酶活测定
反应总体积为200 μL,依次加入170 μL磷酸盐缓冲液(20 mM pH 7.0),20 μL浓度为10 mM的pNPG,10 μL适当浓度的酶液,37 ℃下进行反应。加入酶液后立即计时反应5min,然后立即加入50 μL 0.5 M的Na2CO3终止反应。取 200 μL产物于405 nm波长下测定吸光值,根据标准曲线计算酶活力。
比活力单位的定义:每毫克蛋白质所含的酶活力(U/mg)。
结果表明,cdw11对pNPG最适pH为6.5,45℃下的比活力为9500 U/mg蛋白。
3、cdw11最适温度测定
在pH7.4条件下,在温度范围为20-60℃之间,按如前所述标准酶活测定方法步骤测定。
结果如图3所示,cdw11的最适温度为45℃,故以此温度下的酶活力为100%,换算各个温度下的相对酶活力。
4、cdw11最适pH测定
分别利用不同的缓冲液测定不同pH范围内重组β-葡萄糖苷酶的最适pH和pH稳 定性。pH 4.0-6.0范围内选择Na Ac-HAc缓冲液,pH 6.0-8.0范围内选择Na H2PO4Na2HPO4缓冲液,pH 8.0-10.0选择Glycine-NaOH缓冲液进行测定。反应体系为200 μL,分别加入170 μL不同pH的缓冲液,20 μL 5 mM pNPG和10 μL稀释一定倍数的纯 酶液,在37 °C下反应5 min后立即加入50 μL 0.5 M Na2CO3终止反应。取200 μL置于96孔板中在405 nm波长下测定吸光值。吸光值最高的一组缓冲液所代表的pH为最适pH,将该条件下的酶活力定义为100%,其他条件下的酶活力与该组pH进行比较。
结果如图4所示,cdw11最适pH为6.5。
应用例1
利用本申请的β-葡萄糖苷酶cdw11在转化淫羊藿苷为宝藿苷I、转化黄芩苷为黄芩素的应用,包括以下步骤:
(1)原材料预处理
将淫羊藿药材及黄芩药材烘干、粉碎过60目筛后,取药材1 Kg加入6升经含弱碱甲醇回流过滤,收集滤液。合并三次滤液,浓缩干燥即得淫羊藿苷、黄芩苷提取物。
(2)酶处理
将步骤(1)所得提取物按固液比添加8倍量的本发明制得的β-葡萄糖苷酶cdw11制剂的水溶液,调pH到6.5,将反应物混匀,45℃放置3小时,搅拌3小时。
(3)萃取
将步骤(2)所得混合液加入其二分之一体积乙酸乙酯进行反应产物的萃取。
(4)点样
将上述萃取产物点样于硅胶板,在乙酸乙酯:甲酸:氯仿:水(10:1:1:1)的展开剂中进行展开,再喷以10%硫酸乙醇溶液显色。
(5)HPLC检测
淫羊藿苷和宝藿苷I、黄芩苷和黄芩素的HPLC检测条件为:Agilent C18 5μm×250×4.6mm色谱柱,波长UV 270nm,流动相为90%乙腈-水溶液,流速1.0ml/min,温度25℃。
应用例2
利用本申请的β-葡萄糖苷酶cdw11在转化淫羊藿苷为宝藿苷I、转化黄芩苷为黄芩素的应用,包括以下步骤:
(1)原材料预处理
将淫羊藿药材及黄芩药材烘干、粉碎过60目筛后,取药材1 Kg加入3升经含弱碱甲醇回流过滤,收集滤液。合并三次滤液,浓缩干燥即得淫羊藿苷、黄芩苷提取物。
(2)酶处理
将步骤(1)所得提取物按固液比添加8倍量的本发明制得的β-葡萄糖苷酶cdw11制剂的水溶液,调pH到6.5,将反应物混匀,45℃放置3小时,搅拌3小时。
(3)萃取
将步骤(2)所得混合液加入其二分之一体积乙酸乙酯进行反应产物的萃取。
(4)薄层色谱法点样
将上述萃取产物点样于硅胶板,在乙酸乙酯:甲酸:氯仿:水(10:1:1:1)的展开剂中进行展开,再喷以10%硫酸乙醇溶液显色。
(5)HPLC检测
淫羊藿苷和宝藿苷I、黄芩苷和黄芩素的HPLC检测条件为:Agilent C18 5μm×250×4.6mm色谱柱,波长UV270nm,流动相为90%乙腈-水溶液,流速1.0ml/min,温度25℃。
对比例1
本对比例提供一种酶法在淫羊藿苷转化为宝藿苷I、黄芩苷转化为黄芩素的应用,与应用例1的区别在于:将β-葡萄糖苷酶cdw11改为纤维素酶,其他步骤均相同。
对比例2
本对比例提供一种酶法在淫羊藿苷转化为宝藿苷I、黄芩苷转化为黄芩素的应用,与应用例2的区别在于:将β-葡萄糖苷酶cdw11改为蜗牛酶,其他步骤均相同。
应用例1、2与对比例1、2所得的检测结果见下表:
由上表可知:采用β-葡萄糖苷酶在最适条件下转化淫羊藿苷、黄芩苷,应用例工艺均能转化淫羊藿苷为宝藿苷I的转化率最高可达100%,转化黄芩苷为黄芩素的转化率最高可达98%,证明了本申请的β-葡萄糖苷酶cdw11实现了同时高效率制备宝藿苷I和黄芩素的目的;而对比例转化率明显较低,表明相比于纤维素酶、蜗牛酶,本申请的β-葡萄糖苷酶cdw11对淫羊藿苷、黄芩苷的酶解效率最好。
以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (10)
1.一种β-葡萄糖苷酶,其对对硝基苯酚的比活力为854 U/mg。
2.根据权利要求1所述的β-葡萄糖苷酶,其基因的核苷酸序列如SEQ ID No.1所示。
3.一种重组载体,其特征在于:含有如SEQ ID No.1所示的核苷酸序列。
4.一种宿主细胞,其特征在于:含有权利要求1或2所述的β-葡萄糖苷酶;或者含有权利要求3所述的重组载体。
5.一种同时制备宝藿苷I和黄芩素的方法,其特征在于:所述方法是利用权利要求1或2所述的β-葡萄糖苷酶基因或者权利要求3所述的重组载体或者权利要求4所述的宿主细胞实现的。
6.根据权利要求5所述的同时制备宝藿苷I和黄芩素的方法,其特征在于,步骤为:
(1)构建含有β-葡萄糖苷酶基因的重组载体,转化至大肠杆菌中获得表达株;
(2)表达株经培养、IPTG诱导、发酵、超声破碎后离心取上清,得可溶性的β-葡萄糖苷酶,再经蛋白纯化得纯的β-葡萄糖苷酶;
(3)将淫羊藿药材和黄芩药材烘干、粉碎、回流提取后,合并滤液,浓缩干燥得混合提取物;
(4)将混合提取物加入步骤(2)的β-葡萄糖苷酶的水溶液中,经酶解反应后,加热过滤并收集滤液;
(5)向步骤(4)的滤液中加入乙酸乙酯进行萃取,获得宝藿苷I和黄芩素。
7.根据权利要求6所述的同时制备宝藿苷I和黄芩素的方法,其特征在于:所述步骤(1)中β-葡萄糖苷酶基因的核苷酸序列如SEQ ID No.1所示。
8.根据权利要求6所述的同时制备宝藿苷I和黄芩素的方法,其特征在于:所述步骤(2)中IPTG的浓度为0.2 mM;蛋白纯化采用Ni-NTA亲和层析柱进行。
9.根据权利要求6所述的同时制备宝藿苷I和黄芩素的方法,其特征在于:所述步骤(3)中回流提取采用含弱碱甲醇的有机溶剂。
10.根据权利要求6所述的同时制备宝藿苷I和黄芩素的方法,其特征在于:所述步骤(4)中酶解反应的pH为6.5,温度为45℃,β-葡萄糖苷酶的水溶液的质量浓度为55 g/L,混合提取物与β-葡萄糖苷酶的水溶液的固液比为1:8。
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