近日，国际著名杂志Biosensors and Bioelectronics在线刊登了中国科学院青岛生物能源与过程研究所“百人计划”入选者刘爱骅研究员领导的生物传感器团队与意大利佛罗伦萨大学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，”，研究人员开发出了新型木糖脱氢酶细菌表面展示系统，并成功将此系统应用于构建新型生物传感器。
木糖是纤维素经酶水解或化学水解的主要戊糖，可作为糖尿病、肥胖病等富贵病病人的良好食疗添加剂和食品的无热量甜味剂。开发快速、灵敏、选择性高的木糖检测手段对于监控开发纤维素乙醇等第二代生物燃料的生物过程，发展医药、营养品和食品技术等都具有重要意义。
该团队构建了基于冰核蛋白的细菌表面展示系统，将木糖脱氢酶（XDH）高效地表达在菌体表面并应用于D-木糖的高灵敏检测，同时将编码木糖脱氢酶的基因xylB与冰核蛋白N端结构域基因inaPb-N融合起来，得到质粒pTInaPbN-Xdh，转化E. coli BL21 (DE3)进行表达。该团队梁波等利用SDS-PAGE和Western Blot实验，证实了绝大多数的蛋白位于细胞外膜，外膜组分的XDH酶活占全细胞的77%，而且该蛋白对菌体的生长没有造成影响。另外，与原始菌株中胞内的木糖脱氢酶相比，该蛋白的稳定性更好。
D-木糖在菌体表面的XDH催化下，借助于辅酶烟酰胺腺嘌呤二核苷酸（NAD+）被氧化成木糖酸内酯，辅酶NAD+则被还原为还原态的烟酰胺腺嘌呤二核苷酸（NADH）。通过测定NADH在340nm处的紫外吸收峰的吸光值，可实现D-木糖的检测（如图）。此方法的线性范围为5-900 μM D-木糖。100倍过量的葡萄糖、纤维二糖、半乳糖、甘露糖、核糖、果糖、蔗糖、木糖醇、麦芽糖和10倍过量的L-阿拉伯糖对D-木糖 (100 μM) 的测定均无干扰。
此木糖脱氢酶细菌表面展示系统也可用于开发为高灵敏、高特异性的电化学木糖生物传感器 ，解决高效液相色谱、离子色谱法等传统木糖检测方法价格昂贵、分析周期长、灵敏度低等问题。
该研究得到了中科院“百人计划”和中科院工业生物技术领域基础前沿研究专项项目的资助。（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.
