【摘要】：本研究針對(duì)谷氨酸檢測(cè)所存在的成本高昂、特異性低、穩(wěn)定性差等問(wèn)題,開(kāi)發(fā)了一種利用微生物表面展示技術(shù)進(jìn)行全細(xì)胞生物催化檢測(cè)谷氨酸含量的方法,該方法具有操作簡(jiǎn)單、成本低廉、特異性強(qiáng)、靈敏度高等優(yōu)點(diǎn)。同時(shí)該研究通過(guò)構(gòu)建不同的表面展示系統(tǒng)探究了his-tag標(biāo)簽、不同表達(dá)載體和表達(dá)菌株對(duì)蛋白可溶性表面展示的影響,為實(shí)現(xiàn)表面展示技術(shù)的實(shí)際應(yīng)用奠定了一定的理論基礎(chǔ)。外源蛋白和錨定蛋白的匹配是能否成功構(gòu)建表面展示系統(tǒng)的重要因素,本研究首先以來(lái)自于Bacillus subtilis的谷氨酸脫氫酶基因?yàn)槟康幕?以來(lái)自于Pseudomona borealis的冰核蛋白N端結(jié)構(gòu)域?yàn)殄^定蛋白構(gòu)建了表達(dá)載體。結(jié)果證明融合蛋白在大腸桿菌細(xì)胞中以包涵體形式表達(dá),說(shuō)明目的蛋白沒(méi)有成功表面展示在大腸桿菌細(xì)胞表面。本研究接著以來(lái)自于菌株Thermococcus waiotapuensis的谷氨酸脫氫酶基因?yàn)槟康幕?以冰核蛋白N端結(jié)構(gòu)域?yàn)殄^定蛋白構(gòu)建了表達(dá)載體,結(jié)果表明谷氨酸脫氫酶成功展示在大腸桿菌細(xì)胞表面,說(shuō)明冰核蛋白N端結(jié)構(gòu)域?qū)?lái)自于該菌的谷氨酸脫氫酶有很好的分泌展示功能。這是谷氨酸脫氫酶首次在大腸桿菌表面展示,為微生物表面展示技術(shù)應(yīng)用于生物傳感器、食品檢測(cè)、醫(yī)藥行業(yè)等領(lǐng)域提供了理論研究基礎(chǔ)和實(shí)際應(yīng)用生物材料。本研究主要獲得了以下幾個(gè)方面的研究成果：1.B.subtilis中谷氨酸脫氫酶表面展示系統(tǒng)的構(gòu)建首先以來(lái)自于B.subtilis的谷氨酸脫氫酶基因?yàn)槟康幕?以冰核蛋白N端結(jié)構(gòu)域?yàn)殄^定蛋白,分別構(gòu)建了表達(dá)載體pET-Inp-gldh、pET-Inp-gldhT和pACY-Inp-gldh,將其轉(zhuǎn)入大腸桿菌表達(dá)菌株。通過(guò)SDS-PAGE和酶活測(cè)定對(duì)蛋白表達(dá)進(jìn)行了定位分析,發(fā)現(xiàn)融合蛋白在大腸桿菌細(xì)胞中以無(wú)活性的包涵體形式表達(dá),說(shuō)明來(lái)源于該菌的谷氨酸脫氫酶沒(méi)有展示到到大腸桿菌細(xì)胞表面。該部分研究同時(shí)表明：來(lái)自于B.subtilis的谷氨酸脫氫酶基因的表達(dá)在有無(wú)his-tag標(biāo)簽表達(dá)的情況下均以包涵體形式被表達(dá)；表達(dá)載體pET28a (+)和pACYCDuet-1對(duì)來(lái)自于該菌的谷氨酸脫氫酶的表達(dá)效果相同,都以包涵體形式表達(dá)：質(zhì)粒pET-Inp-gldh和pACY-Inp-gldh轉(zhuǎn)入表達(dá)菌株E.coli BL21(DE3)、 E.coli transB(DE3)、E.coli transetta (DE3)、E.coli BL21 (DE3) plys后,經(jīng)誘導(dǎo)表達(dá)產(chǎn)生的融合蛋白都為包涵體,說(shuō)明以上各表達(dá)菌株對(duì)來(lái)自于該菌的谷氨酸脫氫酶表達(dá)效果一致。2. T.waiotapuensis中谷氨酸脫氫酶表面展示系統(tǒng)的構(gòu)建以T. waiotapuensis中編碼谷氨酸脫氫酶的基因?yàn)槟康幕?以冰核蛋白N端結(jié)構(gòu)域?yàn)殄^定蛋白構(gòu)建了表達(dá)載體pTInaPb-N-Gldh,將其轉(zhuǎn)入E.coli BL21(DE3)中,誘導(dǎo)表達(dá)后的菌株進(jìn)行SDS-PAGE電泳和酶活測(cè)定分析,發(fā)現(xiàn)來(lái)自于該菌的谷氨酸脫氫酶成功展示在大腸桿菌細(xì)胞表面。本研究中測(cè)得谷氨酸脫氫酶展示菌株的全細(xì)胞酶活為3.12 U/OD600,外膜組分酶活占全細(xì)胞酶活的90%：其反應(yīng)的最適溫度為70℃,最適pH為9.0。該融合蛋白在4℃條件下放置一個(gè)月之后,幾乎能保持100%的活性,具有很好的酶活穩(wěn)定性。過(guò)渡金屬離子能夠不同程度的抑制酶活,而常見(jiàn)金屬離子和陰離子對(duì)酶活幾乎沒(méi)有影響。該表面展示的谷氨酸脫氫酶能夠特異性催化L-谷氨酸,優(yōu)于目前已報(bào)道的其它谷氨酸脫氫酶；并且以NADP+為專一性輔酶,反應(yīng)產(chǎn)生的NADPH能夠在340 nm處進(jìn)行光譜測(cè)定檢測(cè)得到。本研究以表面展示谷氨酸脫氫酶的菌株為全細(xì)胞生物催化材料,用分光光度計(jì)法檢測(cè)L-谷氨酸含量的方法,具有較大的檢測(cè)范圍(10～400 μmol/L)和較低的檢測(cè)限(6 μmol/L)。用谷氨酸脫氫酶表面展示菌株進(jìn)行谷氨酸實(shí)際樣品的檢測(cè),能夠比較精確的測(cè)得實(shí)際樣品中谷氨酸的含量。結(jié)果表明,本方法成本低廉、靈敏度高、特異性強(qiáng),操作簡(jiǎn)單、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，

本文編號(hào)：2258853