谷氨酸脱氢酶在细菌表面展示系统的构建及其在谷氨酸检测中的应用
发布时间：2018-10-09 09:32
【摘要】：本研究针对谷氨酸检测所存在的成本高昂、特异性低、稳定性差等问题,开发了一种利用微生物表面展示技术进行全细胞生物催化检测谷氨酸含量的方法,该方法具有操作简单、成本低廉、特异性强、灵敏度高等优点。同时该研究通过构建不同的表面展示系统探究了his-tag标签、不同表达载体和表达菌株对蛋白可溶性表面展示的影响,为实现表面展示技术的实际应用奠定了一定的理论基础。外源蛋白和锚定蛋白的匹配是能否成功构建表面展示系统的重要因素,本研究首先以来自于Bacillus subtilis的谷氨酸脱氢酶基因为目的基因,以来自于Pseudomona borealis的冰核蛋白N端结构域为锚定蛋白构建了表达载体。结果证明融合蛋白在大肠杆菌细胞中以包涵体形式表达,说明目的蛋白没有成功表面展示在大肠杆菌细胞表面。本研究接着以来自于菌株Thermococcus waiotapuensis的谷氨酸脱氢酶基因为目的基因,以冰核蛋白N端结构域为锚定蛋白构建了表达载体,结果表明谷氨酸脱氢酶成功展示在大肠杆菌细胞表面,说明冰核蛋白N端结构域对来自于该菌的谷氨酸脱氢酶有很好的分泌展示功能。这是谷氨酸脱氢酶首次在大肠杆菌表面展示,为微生物表面展示技术应用于生物传感器、食品检测、医药行业等领域提供了理论研究基础和实际应用生物材料。本研究主要获得了以下几个方面的研究成果：1.B.subtilis中谷氨酸脱氢酶表面展示系统的构建首先以来自于B.subtilis的谷氨酸脱氢酶基因为目的基因,以冰核蛋白N端结构域为锚定蛋白,分别构建了表达载体pET-Inp-gldh、pET-Inp-gldhT和pACY-Inp-gldh,将其转入大肠杆菌表达菌株。通过SDS-PAGE和酶活测定对蛋白表达进行了定位分析,发现融合蛋白在大肠杆菌细胞中以无活性的包涵体形式表达,说明来源于该菌的谷氨酸脱氢酶没有展示到到大肠杆菌细胞表面。该部分研究同时表明：来自于B.subtilis的谷氨酸脱氢酶基因的表达在有无his-tag标签表达的情况下均以包涵体形式被表达；表达载体pET28a (+)和pACYCDuet-1对来自于该菌的谷氨酸脱氢酶的表达效果相同,都以包涵体形式表达：质粒pET-Inp-gldh和pACY-Inp-gldh转入表达菌株E.coli BL21(DE3)、 E.coli transB(DE3)、E.coli transetta (DE3)、E.coli BL21 (DE3) plys后,经诱导表达产生的融合蛋白都为包涵体,说明以上各表达菌株对来自于该菌的谷氨酸脱氢酶表达效果一致。2. T.waiotapuensis中谷氨酸脱氢酶表面展示系统的构建以T. waiotapuensis中编码谷氨酸脱氢酶的基因为目的基因,以冰核蛋白N端结构域为锚定蛋白构建了表达载体pTInaPb-N-Gldh,将其转入E.coli BL21(DE3)中,诱导表达后的菌株进行SDS-PAGE电泳和酶活测定分析,发现来自于该菌的谷氨酸脱氢酶成功展示在大肠杆菌细胞表面。本研究中测得谷氨酸脱氢酶展示菌株的全细胞酶活为3.12 U/OD600,外膜组分酶活占全细胞酶活的90%：其反应的最适温度为70℃,最适pH为9.0。该融合蛋白在4℃条件下放置一个月之后,几乎能保持100%的活性,具有很好的酶活稳定性。过渡金属离子能够不同程度的抑制酶活,而常见金属离子和阴离子对酶活几乎没有影响。该表面展示的谷氨酸脱氢酶能够特异性催化L-谷氨酸,优于目前已报道的其它谷氨酸脱氢酶；并且以NADP+为专一性辅酶,反应产生的NADPH能够在340 nm处进行光谱测定检测得到。本研究以表面展示谷氨酸脱氢酶的菌株为全细胞生物催化材料,用分光光度计法检测L-谷氨酸含量的方法,具有较大的检测范围(10～400 μmol/L)和较低的检测限(6 μmol/L)。用谷氨酸脱氢酶表面展示菌株进行谷氨酸实际样品的检测,能够比较精确的测得实际样品中谷氨酸的含量。结果表明,本方法成本低廉、灵敏度高、特异性强,操作简单、快速。
[Abstract]:Aiming at the problems of high cost, low specificity, poor stability and the like in the detection of glutamic acid, a method for detecting glutamic acid content by using a microbial surface display technology is developed, and the method has the advantages of simple operation, low cost and strong specificity, high sensitivity and the like. At the same time, the influence of his-tag tag, different expression vector and expression strain on protein soluble surface display was studied by constructing different surface display system, which laid a theoretical foundation for realizing the practical application of surface display technology. The matching of foreign protein and anchored protein is an important factor to successfully construct the surface display system. The results showed that the fusion protein expressed in E. coli cells in inclusion body, indicating that the target protein was not successfully displayed on the surface of E. coli cells. The results showed that glutamate dehydrogenase was successfully displayed on the surface of E. coli cells. It is indicated that the N-terminal domain of the nucleocapsid protein has a good secretion and display function for glutamate dehydrogenase from the strain. This is the first time that glutamate dehydrogenase is displayed on the surface of E. coli, which provides theoretical research foundation and practical application of biological materials to the field of biological sensor, food detection, medicine industry and so on. The results of this study are as follows: 1. The construction of glutamate dehydrogenase surface display system in B.subtilis is based on the gene of glutamic acid dehydrogenase from B. subtilis as the target gene, and the N terminal domain of the ice nucleoprotein is anchored protein, and the expression vectors GaP-Inp-gldh, pACY-Inp-gldhT and pACY-Inp-gldh are respectively constructed. It was transferred to E. coli expression strain. The expression of protein was analyzed by SDS-PAGE and enzyme activity assay. It was found that the fusion protein was expressed in E. coli cells with inactive inclusion bodies, indicating that the glutamate dehydrogenase derived from the strain was not shown to the surface of E. coli cells. The results showed that the expression of glutamate dehydrogenase gene from B.subtilis was expressed in inclusion body in the presence or absence of his-tag label expression, and expression vector pET28a (+) and pACYCDuet-1 expressed the same expression effect on glutamate dehydrogenase from the strain, both expressed in inclusion body form: coli BL21 (DE3), E. coli BL21 (DE3), E. coli transfer ta (DE3), E. coli BL21 (DE3) plasys, and the fusion proteins induced by induced expression were all inclusion bodies. The expression vector pTInaPb-N-Gldh was constructed with the N-terminal domain of the nucleoprotein as the target gene, and the expression vector pTInaPb-N-Gldh was constructed with the N-terminal domain of the nucleoprotein as the target gene, and transferred into E. coli BL21 (DE3). SDS-PAGE electrophoresis and enzyme activity determination were performed on the strain induced by the strain, and the glutamate dehydrogenase from the strain was found to be successfully displayed on the surface of E. coli cells. The total cellular enzyme activity of glutamate dehydrogenase demonstrated by glutamate dehydrogenase was 3.12U/ OD600, and the activity of outer membrane component was 90% of total cell enzyme activity: the optimum temperature for the reaction was 70 鈩，
本文编号：2258853
[Abstract]:Aiming at the problems of high cost, low specificity, poor stability and the like in the detection of glutamic acid, a method for detecting glutamic acid content by using a microbial surface display technology is developed, and the method has the advantages of simple operation, low cost and strong specificity, high sensitivity and the like. At the same time, the influence of his-tag tag, different expression vector and expression strain on protein soluble surface display was studied by constructing different surface display system, which laid a theoretical foundation for realizing the practical application of surface display technology. The matching of foreign protein and anchored protein is an important factor to successfully construct the surface display system. The results showed that the fusion protein expressed in E. coli cells in inclusion body, indicating that the target protein was not successfully displayed on the surface of E. coli cells. The results showed that glutamate dehydrogenase was successfully displayed on the surface of E. coli cells. It is indicated that the N-terminal domain of the nucleocapsid protein has a good secretion and display function for glutamate dehydrogenase from the strain. This is the first time that glutamate dehydrogenase is displayed on the surface of E. coli, which provides theoretical research foundation and practical application of biological materials to the field of biological sensor, food detection, medicine industry and so on. The results of this study are as follows: 1. The construction of glutamate dehydrogenase surface display system in B.subtilis is based on the gene of glutamic acid dehydrogenase from B. subtilis as the target gene, and the N terminal domain of the ice nucleoprotein is anchored protein, and the expression vectors GaP-Inp-gldh, pACY-Inp-gldhT and pACY-Inp-gldh are respectively constructed. It was transferred to E. coli expression strain. The expression of protein was analyzed by SDS-PAGE and enzyme activity assay. It was found that the fusion protein was expressed in E. coli cells with inactive inclusion bodies, indicating that the glutamate dehydrogenase derived from the strain was not shown to the surface of E. coli cells. The results showed that the expression of glutamate dehydrogenase gene from B.subtilis was expressed in inclusion body in the presence or absence of his-tag label expression, and expression vector pET28a (+) and pACYCDuet-1 expressed the same expression effect on glutamate dehydrogenase from the strain, both expressed in inclusion body form: coli BL21 (DE3), E. coli BL21 (DE3), E. coli transfer ta (DE3), E. coli BL21 (DE3) plasys, and the fusion proteins induced by induced expression were all inclusion bodies. The expression vector pTInaPb-N-Gldh was constructed with the N-terminal domain of the nucleoprotein as the target gene, and the expression vector pTInaPb-N-Gldh was constructed with the N-terminal domain of the nucleoprotein as the target gene, and transferred into E. coli BL21 (DE3). SDS-PAGE electrophoresis and enzyme activity determination were performed on the strain induced by the strain, and the glutamate dehydrogenase from the strain was found to be successfully displayed on the surface of E. coli cells. The total cellular enzyme activity of glutamate dehydrogenase demonstrated by glutamate dehydrogenase was 3.12U/ OD600, and the activity of outer membrane component was 90% of total cell enzyme activity: the optimum temperature for the reaction was 70 鈩，
本文编号：2258853
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