CN117210442B - 一种用于修饰人参皂苷的β-糖苷酶SS-BGL突变体及其应用 - Google Patents
一种用于修饰人参皂苷的β-糖苷酶SS-BGL突变体及其应用 Download PDFInfo
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Abstract
本发明属于基因工程技术领域，具体涉及一种用于修饰人参皂苷的β‑糖苷酶SS‑BGL突变体及其应用。所述β‑糖苷酶SS‑BGL突变体是将氨基酸序列如SEQ ID NO.1所示的β‑糖苷酶SS‑BGL第128位和第302位的天冬酰胺突变为天冬氨酸。本发明所得的β‑糖苷酶SS‑BGL突变体提高了天然β‑糖苷酶SS‑BGL在极高温下的热稳定性，更利于SS‑BGL在人参皂苷制备中的应用。
Description
技术领域
本发明属于基因工程技术领域，具体涉及一种用于修饰人参皂苷的β-糖苷酶SS-BGL突变体及其应用。
本发明是申请号为202310625368.6申请案的分案申请，申请日为2023年05月30日。
背景技术
近年来，作为人参主要药效部位之一的皂苷类成分因其广泛的抗癌活性受到关注，研究发现，人参皂苷的次级代谢产物“稀有人参皂苷”显示有更强的抗肿瘤活性，可通过体外总皂苷降解获得。人参皂苷化合物K[20-o-二葡糖基-20(S)-原苯二醇；CK]是人参皂苷的主要脱糖基化代谢物，它已经显示了多种有趣的生物活性，包括抗癌、抗糖尿病、抗炎、抗过敏、抗血管生成、抗衰老、神经保护和肝保护作用。但其制备的困难极大的限制了CK的进一步应用，为了提高CK的产量，目前已发展多种方法用于CK的制取。
人参皂苷的结构修饰，主要在于对特定位点的糖基进行水解。糖苷酶是最常见的工业酶之一，它负责糖基键的水解裂解，在食品工业中被用作乳制品中乳糖水解的催化剂。来自嗜热古菌Sulfolobus solfataricus(生长环境为87℃，pH3.0)的β-糖苷酶(SS-BGL，氨基酸序列如SEQ ID NO.1所示)，由于其广泛的水解活性，包括β-D-葡萄糖苷酶、β-D-半乳糖糖苷酶、β-D-羟基葡萄糖苷酶和α-葡萄糖苷酶活性，已被证实是一种高效的以糖基化PPD型人参皂苷为底物的CK产生酶。SS-BGL以人参皂苷Rb1为底物制备稀有人参皂苷CK的水解路径为：Rb1→Rd→F2→CK。高温意味着高反应速率和较低的污染，嗜热糖苷酶在工业应用中有着绝佳的优势。研究显示，SS-BGL(最适温度85℃，最适pH5.5)在高于其最适温度时稳定性会极速降低，这大大限制了SS-BGL的进一步应用。
发明内容
为解决上述技术问题，本发明提供一种热稳定性提高的β-糖苷酶SS-BGL突变体及其应用。
第一方面，本发明提供一种用于修饰人参皂苷的β-糖苷酶SS-BGL突变体，所述β-糖苷酶SS-BGL突变体是将氨基酸序列如SEQ ID NO.1所示的β-糖苷酶SS-BGL第128位和第302位的天冬酰胺突变为天冬氨酸。
第二方面，本发明提供所述β-糖苷酶SS-BGL突变体在制备人参皂苷中的应用。
第三方面，本发明提供一种编码所述β-糖苷酶SS-BGL突变体的基因。
第四方面，本发明提供一种携带所述基因的重组表达载体。
进一步的，所述重组表达载体以pET-24a(+)载体作为原始表达载体。
第五方面，本发明提供一种由所述重组表达载体转化得到的基因工程菌。
进一步的，所述基因工程菌以大肠杆菌BL21(DE3)为宿主。
本发明具有如下有益效果：
1)本发明在天然β-糖苷酶SS-BGL的基础上，通过理性设计，结合定点突变生物技术改造β-糖苷酶SS-BGL分子结构，分析了突变后残基对酶热稳定性的影响，并最终获得了稳定性和皂苷转化活性同时提高的组合突变菌株(突变菌Q96E/N97D/N302D和Q96E/N97D/N128D/N302D)。
2)天然β-糖苷酶SS-BGL的Tm值为89.62℃，本发明提供的β-糖苷酶SS-BGL突变体Q96E/N97D/N302D的Tm值达到102.23℃，比天然β-糖苷酶SS-BGL的Tm值升高了12.61℃；突变体Q96E/N97D/N128D/N302D的Tm值达到103.89℃，比天然β-糖苷酶SS-BGL的Tm值升高了14.27℃。
3)本发明提供的β-糖苷酶SS-BGL突变体在热稳定性显著提高的同时皂苷的转化活性也大幅提升。其中，β-糖苷酶SS-BGL突变体Q96E/N97D/N302D和Q96E/N97D/N128D/N302D在95℃下皂苷转化的相对酶活分别为160.83％、115.95％。
4)本发明所得的β-糖苷酶SS-BGL突变体提高了天然β-糖苷酶SS-BGL在极高温下的热稳定性，更利于SS-BGL在人参皂苷制备中的应用。
附图说明
图1为WT、N128D/N302D、Q96E/N97D/N302D和Q96E/N97D/N128D/N302D纯蛋白溶液经十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)分析后的条带；
图2为野生型β-糖苷酶SS-BGL(a)及β-糖苷酶SS-BGL突变体N128D/N302D(d)、Q96E/N97D/N302D(c)和Q96E/N97D/N128D/N302D(b)在95℃条件下孵育不同时间的酶活性变化测试结果；
图3为野生型β-糖苷酶SS-BGL(a)及β-糖苷酶SS-BGL突变体N128D/N302D(d)、Q96E/N97D/N302D(c)和Q96E/N97D/N128D/N302D(b)在不同温度下的酶活测试结果。
具体实施方式
下面结合附图和具体实施例对本发明进行详细说明，但不应理解为本发明的限制。如未特殊说明，下述实施例中所用的技术手段为本领域技术人员所熟知的常规手段，下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
下述实施例中，涉及的培养基及配方如下：
LB液体培养基：10g/L蛋白胨、5g/L酵母粉、10g/L NaCl。
LB固体培养基：在LB液体培养基的基础上添加2％琼脂。
下述实施例中所涉及的检测方法如下：
β-糖苷酶SS-BGL酶活测定方法：
1)通过检测对硝基苯基-β-D-吡喃葡萄糖苷(pNPG)中对硝基苯酚的释放量测试酶活。100μL 1.0mg/ml的酶液加600μL 4mM pNPG溶液，在85℃下反应10min，加入200μL 1mM碳酸钠溶液终止反应。对硝基苯酚的释放量通过测试405nm下的吸光度获得。酶的活性定义为每分钟释放1μmol的对硝基苯酚所需要的酶量。所有的实验均进行三次平行实验。
2)以5mg/ml Rb1为底物，加入1ml 1.0mg/ml溶于50mM柠檬酸/磷酸盐缓冲溶液的酶液，95℃反应1h后加入等体积甲醇终止反应，超声30min，用0.45μm的无菌滤头过滤。CK的生成通过高效液相色谱(HPLC)监测。酶活定义：定义在95℃、pH 5.5的条件下，每分钟转化lμmol人参皂苷Rb1所需的酶量，为一个酶活单位U。
实施例1：构建含β-糖苷酶SS-BGL突变体的重组质粒
(1)含有野生型的β-糖苷酶SS-BGL的重组质粒的构建
化学合成核苷酸序列如SEQ ID NO.2所示的野生型的β-糖苷酶SS-BGL的基因序列，与pET-24a(+)载体采用BamHI酶和EcoRI酶酶切后连接，制备得到重组载体pET-24a(+)-SS-BGL。
(2)含有突变体的重组载体的获得
利用全质粒PCR技术，将步骤(1)制备得到的重组载体pET-24a(+)-SS-BGL为模板进行定点突变，获得含有突变基因的重组质粒：
pET-24a(+)-SS-BGL-Q96E；
pET-24a(+)-SS-BGL-N97D；
pET-24a(+)-SS-BGL-N128D；
pET-24a(+)-SS-BGL-N302D；
pET-24a(+)-SS-BGL-N128D/N302D；
pET-24a(+)-SS-BGL-Q96E/N97D/N302D
pET-24a(+)-SS-BGL-Q96E/N97D/N128D/N302D。
设计的引物序列如下：
Q96E_F(SEQ ID NO.3):GCGCCCGGAGAATTTTGATGAAAGCAAACAGGATG；
Q96E_R(SEQ ID NO.4):TCAAAATTCTCCGGGCGCGGCAGCGGATTCGGAAA；
N97D_F(SEQ ID NO.5):GCGCCCGCAGGATTTTGATGAAAGCAAACAGGATG；
N97D_R(SEQ ID NO.6):TCAAAATCCTCCGGGCGCGGCAGCGGATTCGGAAA；
N128D_F(SEQ ID NO.7):TGCACTGGACCATTATCGTGAAATCTTTAAAGATC；
N128D_R(SEQ ID NO.8):CGATAATGGTCCAGTGCATCTTTATTTGCATATTC；
N302D_F(SEQ ID NO.9):CCCGTGGTGATGAAAAAATTGTTCGTGATGATCTG；
N302D_R(SEQ ID NO.10):TTTTTTCATCACCACGGGTAATTTCACCACGAATG；
其中，PCR扩增程序设定为：首先，95℃预变性30s；然后进入30个循环：95℃变性15s，60℃退火15s，72℃延伸7min，4℃保温。PCR产物用1.0％的琼脂糖凝胶电泳进行检测。
将最终扩增片段用DpnI酶在37℃下作用1h用于去除模板，然后使用2×HieffClone Enzyme Premix，在50℃下进行20min重组反应。最后将重组混合物转化到E.coliDH5α感受态细胞中，转化液涂布含卡那霉素(50μg/ml)LB固体培养基上，提取质粒并测序，测序工作由上海生工完成。
实施例2：β-糖苷酶SS-BGL突变体的表达、分离、纯化
(1)构建含重组质粒的基因工程菌
将实施例1得到的重组质粒转化到E.coli BL21(DE3)感受态细胞中，分别制备得到基因工程菌：
E.coli/pET-24a(+)-SS-BGL-Q96E；
E.coli/pET-24a(+)-SS-BGL-N97D；
E.coli/pET-24a(+)-SS-BGL-N128D；
E.coli/pET-24a(+)-SS-BGL-N302D；
E.coli/pET-24a(+)-SS-BGL-N128D/N302D；
E.coli/pET-24a(+)-SS-BGL-Q96E/N97D/N302D；
E.coli/pET-24a(+)-SS-BGL-Q96E/N97D/N128D/N302D。
(2)β-糖苷酶SS-BGL突变体的诱导表达
分别将步骤(1)制备得到的基因工程菌接种至10ml含有50μg/ml卡那霉素的LB液体培养基中，在37℃、220rpm下培养过夜，制备得到种子液；将制备得到的种子液按0.5％(v/v)的接种量转接至100ml含有50μg/ml卡那霉素的LB液体培养基中，在37℃，220rpm下培养至OD600为0.6时，加入终浓度为1mM的IPTG，在16℃条件下继续培养16h，得到发酵液；将制备得到的发酵液在10000rpm、4℃条件下离心处理5min得到细胞菌体，用50mM柠檬酸/磷酸盐缓冲液重悬。用超声破碎仪在冰浴条件下处理重悬后的细胞10min，离心10min(10000rpm，4℃)，取上清液，得到粗酶液。
(3)β-糖苷酶SS-BGL突变体的分离纯化
粗酶液在80℃下加热30min后，用0.45μm的无菌过滤膜过滤，滤液即为纯化的酶液，蛋白浓度使用BCA蛋白浓度试剂盒进行测定。
分别将得到的纯酶液经十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)分析，如图1所示。结果显示：在60KDa处有明显的条带，证明β-糖苷酶SS-BGL得到表达。
(4)β-糖苷酶SS-BGL突变体的热稳定性研究
将得到的纯酶在95℃恒温真空干燥烘箱中孵育30min后，取出测定残余酶活，以未经高温处理的纯酶液的酶活为空白对照，得到的残余酶活的百分比。测试结果见表1。
表1 95℃热处理30min残余酶活
由表1可以看出，单突变体均保留高于野生型酶的活性，将以上单点突变进行优势叠加获得双突变体N128D/N302D、三突变体Q96E/N97D/N302D以及四突变体Q96E/N97D/N128D/N302D。重复步骤(2)、(3)，将以上多突变体获得的纯酶置于95℃下2h测残余活性。结果如表2所示。
表2多突变体在95℃下热处理2h残余酶活
由表2可知，多突变体经95℃孵育2h后的残余活性除了N128D/N302D低于野生酶，三突变体Q96E/N97D/N302D和四突变体Q96E/N97D/N128D/N302D在高温处理后仍保留较高活性。
(5)测试突变对酶活的影响
步骤(3)制备得到的纯酶以pNPG为底物进行初始相对酶活测定，分别检测步骤(3)制备得到的含有野生型SS-BGL、Q96E(SEQ ID NO.11)、N97D(SEQ ID NO.12)、N128D(SEQ IDNO.13)、N302D(SEQ ID NO.14)、N128D/N302D(SEQ ID NO.15)、Q96E/N97D/302D(SEQ IDNO.16)、Q96E/N97D/N128D/N302D(SEQ ID NO.17)的纯酶液，结果如表3所示。
表3pNPG为底物测试相对酶活
实施例3：β-糖苷酶SS-BGL组合突变体酶学性质测试
1、热稳定性
分别取实施例2步骤(3)制备得到的含有野生型SS-BGL的纯酶液，含有N128D/N302D的纯酶液、含有Q96E/N97D/N302D的纯酶液和含有Q96E/N97D/N128D/N302D的纯酶液，置于95℃恒温真空烘箱中，一定时间间隔取样，使用pNPG法测定其残余酶活，比较其热稳定性，测试结果见图2。酶的活力随时间增长不断下降，多突变体除了N128D/N302D组合热稳定性低于WT以外，Q96E/N97D/N302D和Q96E/N97D/N128D/N302D热稳定性均高于野生型，其半衰期分别为野生型SS-BGL的2.5、3.3倍。
2、最适温度
在25℃-95℃温度范围内测试酶活，结果如图3所示，突变体N128D/N302D的最适温度为85℃，与野生型一致；Q96E/N97D/N302D和Q96E/N97D/N128D/N302D则在95℃显示更高活性。
3、动力学参数
取100μL浓度为1.0mg/ml的纯酶液分别加入2、4、6、8、10、12、14、16和18mM pNPG溶液，于最适温度下反应10min，然后加入等体积的碳酸钠终止反应，进行吸光度检测。结果见表4。
表4动力学参数表
4、皂苷转化
取1.0mg/ml的纯酶液1ml，称取5.0mg/ml Rb1在95℃下反应1h，加入等体积甲醇终止反应，超声30min，用0.45μL滤膜过滤后用HPLC检测CK的生成。结果见表5。
表5皂苷转化相对活性
尽管已描述了本发明的优选实施例，但本领域内的技术人员一旦得知了基本创造性概念，则可对这些实施例作出另外的变更和修改。所以，所附权利要求意欲解释为包括优选实施例以及落入本发明范围的所有变更和修改。
显然，本领域的技术人员可以对本发明进行各种改动和变型而不脱离本发明的精神和范围。这样，倘若本发明的这些修改和变型属于本发明权利要求及其等同技术的范围之内，则本发明也意图包含这些改动和变型在内。
Claims (7)
1.一种用于修饰人参皂苷的β-糖苷酶SS-BGL突变体，其特征在于，所述β-糖苷酶SS-BGL突变体是将氨基酸序列如SEQ ID NO.1所示的β-糖苷酶SS-BGL第128位和第302位的天冬酰胺突变为天冬氨酸。
2.权利要求1所述β-糖苷酶SS-BGL突变体在制备人参皂苷中的应用。
3.一种编码权利要求1所述β-糖苷酶SS-BGL突变体的基因。
4.一种携带权利要求3所述基因的重组表达载体。
5.如权利要求4所述的重组表达载体，其特征在于，所述重组表达载体以pET-24a(+)载体作为原始表达载体。
6.一种由权利要求4所述重组表达载体转化得到的基因工程菌。
7.如权利要求6所述的基因工程菌，其特征在于，所述基因工程菌以大肠杆菌BL21(DE3)为宿主。
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| CN104232671A (zh) * | 2013-06-20 | 2014-12-24 | 中国科学院微生物研究所 | 筛选具有将大量存在的人参皂苷转化为稀有人参皂苷的能力的菌株的方法 |
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