CN110596029B - 检测苏式β-羟基-α-氨基酸含量的方法 - Google Patents
检测苏式β-羟基-α-氨基酸含量的方法 Download PDFInfo
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Abstract
本发明公布了一种检测苏式β‑羟基‑α‑氨基酸立体异构体含量的方法，通过将脱水酶、Fe3+加入到待测体系中，根据特定紫外波长的吸光值进行定量检测，得出体系中苏式‑β‑羟基‑α‑氨基酸，从而实现β‑羟基‑α‑氨基酸立体异构体含量检测的目的，该方法不利用液相手性检测，可以高通量实现β‑羟基‑α‑氨基酸立体异构体检测。
Description
技术领域
本发明属于分析检测研究领域，尤其涉及检测苏式β-羟基-α-氨基酸含量的方法含量的方法。
背景技术
氨基酸是构成生物体蛋白的最基本物质，也是生物体内活性肽、酶和其他一些生物活性分子的重要组成成分，与生命活动有着密切的关系。大部分氨基酸均存在手性对映异构体，L-氨基酸和D-氨基酸(甘氨酸除外)，手性的氨基酸不仅与生命活动息息相关，而且可以作为合成途径中的关键中间体应用于精细化工领域。手性的β-羟基-α-氨基酸及其衍生物，例如甲砜霉素，L-苏式-3-(4-对甲砜基)苯基丝氨酸，L-苏式-苯基丝氨酸(L-threo-phenylserine)的衍生物，L-苏式-3，4-二羟基苯基丝氨酸(L-threo-DOPS)等等是一些重要的药物的中间体，β-羟基-α-氨基酸的立体异构体的含量决定了其能否作为中间体的使用。目前，手性β-羟基-α-氨基酸的检测仍然较为繁琐，在利用衍生化试剂衍生后，通过液相色谱法检测，但液相色谱方法存在着成本高、需要设备及专业人员等不足。如何快速高通量检测体系中手性L-苏式-β-羟基-α-氨基酸和L-赤式-β-羟基-α-氨基酸的含量一直未得到有效解决。
目前已知的报道中，并未发现针对苏式β-羟基-α-氨基酸含量开发一套能够定量的、高通量的检测的方法，因此开发一种苏式β-羟基-α-氨基酸立体异构体含量的方法是非常必要的。
发明内容
为了解决上述问题，本发明利用将脱水酶、Fe3+加入到待测体系中，体系中发生颜色变化，根据目测结果进行定性的检测，根据特定紫外波长的吸光值进行定量检测，得出体系中苏式-β-羟基-α-氨基酸的含量。
本发明是通过利用如下技术方案实现的：
加入脱水酶、Fe3+加入到适量待测溶液中，体系随后产生颜色，检测特定波长(640nm)的紫外吸收，得出苏式-β-羟基-α-氨基酸的含量。
本发明提供了制备脱水酶的方法。
该制备方法包括以下步骤：(1)合成脱水酶基因并构建到pET21a表达载体上，获得带有目的酶基因的重组质粒。(2)将重组质粒转入宿主菌细胞(优选大肠杆菌BL21(DE3))，获得相应的工程菌株。(3)将上述的脱水酶基因构建到pET21a表达载体上，获得带有目的酶基因的重组质粒(4)将重组质粒转入宿主菌细胞(优选大肠杆菌BL21(DE3))，获得相应的突变体工程菌株(5)将工程菌株接种至L-B培养基中，25℃培养16小时。(5)离心收集菌体，破菌留取上清液。
本发明还提供了利用脱水酶、Fe3+体系检测苏式β-羟基-α-氨基酸的方法。具体地，首先利用氨基酸的通用检测方法检测体系中总的β-羟基-α-氨基酸总量，然后通过将脱水酶、Fe3+到待测体系中，根据目测进行定性的检测，根据特定紫外波长的吸光值进行定量检测，得出体系中苏式-β-羟基-α-氨基酸。
本发明的有益效果：本发明利用脱水酶的高立体选择性的底物识别，转化苏式β-羟基-α-氨基酸为酮酸底物，在Fe3+存在下产生颜色变化，利用检测特定波长的紫外吸收，确定苏式β-羟基-α-氨基酸的含量。本方法可以同时检测多个体系，实现β-羟基-α-氨基酸的手性高通量筛选，并且操作简单方便、检测条件温和。
附图说明
图1苏式β-羟基-α-氨基酸加入脱水酶、Fe3+后的640nm处吸收的标准曲线
具体实施方式
下面的实施例进一步说明本发明的内容，但不应理解为对本发明的限制。
实施例1：脱水酶基因获得的
1.1脱水酶基因的合成及获得
脱水酶基因来源于Paraburkholderia xenovorans LB400(GenBank:AIP32383)，根据大肠杆菌密码子偏好性对该基因优化后送公司合成，密码子优化后的基因，分别在5’端添加NdeⅠ(CATATG)酶切位点、在3’端添加XhoI(CTCGAG)酶切位点，克隆至PET21a载体上。以连有RasADH基因的PET21a质粒为模板并根据RasADH基因序列设计引物进行滚环扩增，PCR的扩增反应体系为：
PCR的扩增条件为：
对PCR扩增产物采用DNA纯化试剂盒进行纯化回收。对经过DNA纯化试剂盒纯化的PCR产物采用Dpn I限制性内切酶进行酶切消化，于37℃消化1h。消化后的产物同样用DNA纯化试剂盒进行纯化回收，回收后的产物进行下步的转化。
1.2重组质粒的转化
氯化钙法制备感受态大肠杆菌细胞。
将消化纯化后的PCR产物用化学转化的方法转入大肠杆菌中，转化过程为：
(1)取10μL重组质粒于50μL大肠杆菌BL21(DE3)感受态细胞中，冰浴30min。
(2)42℃水浴热激90s，快速置于冰上1～2min。
(3)加入新鲜LB液体培养基600μL，于37℃振荡培养45～60min。
(4)取200μL菌液涂布于含有氨苄青霉素(1mg/mL)的LB固体培养基表面，
37℃培养12～16h至单菌落出现。
实施例2：脱水酶表达菌的构建及诱导表达
将消化纯化后的PCR产物用化学转化的方法转入大肠杆菌BL21(DE3)感受态细胞中后，挑取单克隆至4mL含氨苄青霉素(1mg/mL)的LB培养基中，取新鲜菌液送测序公司测序，测序结果显示正确的表达菌。配置种子液50mL，培养基为LB液体培养基(蛋白胨10g/L，酵母粉5g/L，NaCl 10g/L)，用接种环挑取基因工程菌单菌落接入培养基中，37℃，200rpm培养过夜。将过夜培养的种子液以1％的接种量转接到发酵培养基，25℃，200rpm培养20h。取发酵液5mL浓缩超声破菌后，检测脱水酶活力。
实施例3：脱水酶纯化
诱导表达后的发酵液6500rpm离心十分钟收集菌体，收集菌体用缓冲液(0.1mol/LpH 7.0磷酸钠缓冲液和0.5M NaCl)重悬洗涤一遍，采用同样方法离心缓冲液重悬，重悬液采用高压匀浆均质机进行破碎，破碎液6000rpm，4℃离心20min获得粗酶液，将获得的粗酶液采用His-Trap TM/FF亲和层析柱进行分离纯化，采用含咪唑的洗脱液(0.1M磷酸钠、0.5MNaCl、0.25M咪唑，pH 7.0)，进行梯度洗脱，收集活性部分，采用SDS-PAGE检测酶蛋白纯度。
实施例4：脱水酶、Fe3+体系检测苏式β-羟基-α-氨基酸的标准曲线的绘制
取不同浓度的苏式β-羟基-α-氨基酸加入加入纯化的脱水酶及过量的Fe3+，检测反应体系在640nm处的紫外吸收，绘制标准曲线，如附图1
实施例5：脱水酶、Fe3+体系检测苏式β-羟基-α-氨基酸含量
取含有苏式β-羟基-α-氨基酸的待测溶液，加入纯化的脱水酶及过量的Fe3+，反应体系颜色发生变化，检测在640nm出的紫外吸收，根据标准曲线，计算出溶液中苏式β-羟基-α-氨基酸的含量。
实施例6：脱水酶、Fe3+体系检测苏式β-羟基-α-氨基酸含量
取含有苏式β-羟基-α-氨基酸的待测溶液，加入脱水酶的破菌液及过量的Fe3+，反应体系颜色发生变化，检测在640nm出的紫外吸收，根据标准曲线，计算出溶液中苏式β-羟基-α-氨基酸的含量。
实施例7：脱水酶、Fe3+体系检测苏式β-羟基-α-氨基酸含量
取适量含有苏式β-羟基-α-氨基酸的待测溶液，加入脱水酶的破菌上清及过量的Fe3+，反应体系颜色发生变化，检测在640nm出的紫外吸收，根据标准曲线，计算出溶液中苏式β-羟基-α-氨基酸的含量。
序列表
<110> 中国科学院天津工业生物技术研究所
<120> 检测苏式β-羟基-α-氨基酸含量的方法
<130> 20180315
<141> 2018-06-12
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
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<213> Paraburkholderia xenovorans LB400
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Claims (2)
1.一种检测苏式β-羟基-α-氨基酸含量的方法，包括如下步骤：将脱水酶、Fe3+加入到待测溶液中，检测待测体系的紫外吸收，根据标准曲线，计算得出苏式β-羟基-α-氨基酸的含量; 其中，脱水酶的氨基酸序列分别如SEQ ID No .1所示。
2.如权利要求1所述的检测苏式β-羟基-α-氨基酸含量的方法，其特征在于，加入脱水酶、Fe3+后，反应体系颜色发生变化，根据颜色的吸光度变化实现苏式β-羟基-α-氨基酸的定量检测。
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