近日,，國際著名雜志Biosensors and Bioelectronics在線刊登了中國科學(xué)院青島生物能源與過程研究所“百人計劃”入選者劉愛驊研究員領(lǐng)導(dǎo)的生物傳感器團隊與意大利佛羅倫薩大學(xué)M. Mascini教授合作的最新研究成果“A Selective and Sensitive D-Xylose Electrochemical Biosensor Based on Xylose Dehydrogenase Displayed on the Surface of Bacteria and Multi-Walled Carbon Nanotubes Modified Electrode,，”,，研究人員開發(fā)出了新型木糖脫氫酶細(xì)菌表面展示系統(tǒng)，并成功將此系統(tǒng)應(yīng)用于構(gòu)建新型生物傳感器,。
木糖是纖維素經(jīng)酶水解或化學(xué)水解的主要戊糖，可作為糖尿病、肥胖病等富貴病病人的良好食療添加劑和食品的無熱量甜味劑,。開發(fā)快速、靈敏,、選擇性高的木糖檢測手段對于監(jiān)控開發(fā)纖維素乙醇等第二代生物燃料的生物過程,，發(fā)展醫(yī)藥、營養(yǎng)品和食品技術(shù)等都具有重要意義,。
該團隊構(gòu)建了基于冰核蛋白的細(xì)菌表面展示系統(tǒng),，將木糖脫氫酶（XDH）高效地表達在菌體表面并應(yīng)用于D-木糖的高靈敏檢測，同時將編碼木糖脫氫酶的基因xylB與冰核蛋白N端結(jié)構(gòu)域基因inaPb-N融合起來,，得到質(zhì)粒pTInaPbN-Xdh,，轉(zhuǎn)化E. coli BL21 (DE3)進行表達。該團隊梁波等利用SDS-PAGE和Western Blot實驗,，證實了絕大多數(shù)的蛋白位于細(xì)胞外膜,，外膜組分的XDH酶活占全細(xì)胞的77%，而且該蛋白對菌體的生長沒有造成影響,。另外,，與原始菌株中胞內(nèi)的木糖脫氫酶相比，該蛋白的穩(wěn)定性更好,。
D-木糖在菌體表面的XDH催化下,，借助于輔酶煙酰胺腺嘌呤二核苷酸（NAD+）被氧化成木糖酸內(nèi)酯，輔酶NAD+則被還原為還原態(tài)的煙酰胺腺嘌呤二核苷酸（NADH）,。通過測定NADH在340nm處的紫外吸收峰的吸光值,，可實現(xiàn)D-木糖的檢測（如圖）。此方法的線性范圍為5-900 μM D-木糖,。100倍過量的葡萄糖,、纖維二糖、半乳糖,、甘露糖,、核糖、果糖,、蔗糖,、木糖醇、麥芽糖和10倍過量的L-阿拉伯糖對D-木糖 (100 μM) 的測定均無干擾。
此木糖脫氫酶細(xì)菌表面展示系統(tǒng)也可用于開發(fā)為高靈敏,、高特異性的電化學(xué)木糖生物傳感器 ,，解決高效液相色譜、離子色譜法等傳統(tǒng)木糖檢測方法價格昂貴,、分析周期長,、靈敏度低等問題。
該研究得到了中科院“百人計劃”和中科院工業(yè)生物技術(shù)領(lǐng)域基礎(chǔ)前沿研究專項項目的資助,。（生物谷Bioon.com）
doi:10.1016/j.bios.2011.12.027
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A Selective and Sensitive D-Xylose Electrochemical Biosensor Based on Xylose Dehydrogenase Displayed on the Surface of Bacteria and Multi-Walled Carbon Nanotubes Modified Electrode
Liang Lia, b, 1, Bo Lianga, 1, Jianguo Shic, Feng Lib, Marco Mascinid, , Aihua Liua,
A novel Nafion/bacteria-displaying xylose dehydrogenase(XDH)/multi-walled carbon nanotubes (MWNTs) composite film-modified electrode was fabricated and applied for the sensitive and selective determination of D-xylose (INS 967), where the XDH-displayed bacteria(XDH-bacteria) was prepared using a newly identified ice nucleation protein from Pseudomonas borealis DL7 as an anchoring motif. The XDH-displayed bacteria can be used directly, eliminating further enzyme-extraction and purification, thus greatly improved the stability of the enzyme. The optimal conditions for the construction of biosensor were established: homogeneous Nafion-MWNTs composite dispersion (10 μL) was cast onto the inverted glassy carbon electrode, followed by casting 10-μL of XDH-bacteria aqueous solution to stand overnight to dry, then a 5-μL of Nafion solution (0.05 wt%) is syringed to the electrode surface. The bacteria-displaying XDH could catalyze the oxidization of xylose to xylonolactone with coenzyme NAD+ in 0.1 M PBS buffer (pH7.4), where NAD+(nicotinamide adenine dinucleotide) is reduced to NADH (the reduced form of nicotinamide adenine dinucleotide). The resultant NADH is further electrocatalytically oxidized by MWNTs on the electrode, resulting in an obvious oxidation peak around 0.50 V (vs. Ag/AgCl). In contrast, the bacteria-XDH-only modified electrode showed oxidation peak at higher potential of 0.7 V and less sensitivity. Therefore, the electrode/MWNTs/bacteria-XDH/Nafion exhibited good analytical performance such as long-term stability, a wide dynamic range of 0.6-100 μM and a low detection limit of 0.5 μM D-xylose (S/N = 3). No interference was observed in the presence of 300-fold excess of other saccharides including D-glucose, D-fructose, D-maltose, D-galactose, D-mannose, D-sucrose, and D-cellbiose as well as 60-fold excess of L-arabinose. The proposed microbial biosensor is stable, specific, sensitive, reproducible, simple, rapid and cost-effective, which holds great potential in real applications.
