基于酵母表面酶共展示全细胞的新型电化学顺序酶生物传感器的构筑研究

The sequence biosensors can be used to measure substances which are hard to be determined with single-enzyme-based biosensors. However, critical issues exist in development of sequential enzyme elctrodes, such as difficulities in controlling the ratio of co-immobilized enzymes and thereafter the sequential catalytic kinetics, insensitivity, and poor reproducibility during enzyme membrane preparation. In this project, taking inosine biosensor as a model, a novel sequential enzyme electrode will be constructed using yeast-surface co-displaying bienzyme whole-cell as a new molecular recognition element. Via Coh-Dock and Z-Fc protein interactions, the yeast surface can co-display purine nucleoside phosphorylase (PNP) and xanthine oxidase (XOD) at certain proportions. The position of PNP and XOD in the whole-cell is observed to determine the distribution of these bienzyme proteins in the same cell surface, and to clarify the spatial relationship between them. The enzymatic catalytic kinetics, stability and substrate specificity of the bienzyme-displaying whole-cell catalyst will be investigated to identify whether the enzymatic properties are affected by the co-displaying mechanism. Then an inosine sensor will be fabricated by immobilization of bienzyme-displayed yeast whole-cell on Pt electrode, which is expected stable,reproducible and excellent in performance. This work will establish a novel strategy for sequential enzyme electrode and overcome the common problems were experienced for traditional bienzyme electrodes. This proposed approach would not only illuminate new idea to solve the low efficiency of sequential enzymatic reactions, but also pave ways to design other new sequential enzyme electrodes. Furthermore, our study would provide new concept for construction of whole-cell reactors.

顺序酶传感器能实现单酶传感器难以进行的组分检测，但是共固定化酶的比例和顺序催化动力学不可控、灵敏度低、酶膜的互换性差是限制其应用的关键问题。本项目以肌苷传感器为研究对象，拟选择酵母表面双酶共展示全细胞构筑顺序酶传感器。利用Coh-Dock和Z-Fc这两组蛋白之间的相互作用将嘌呤核苷磷酸化酶（PNP）和黄嘌呤氧化酶（XOD）按照比例共展示在酵母表面。对全细胞进行PNP和XOD的定位观测，确定这两个酶蛋白在同一个细胞表面的分布情况，阐明它们之间的空间位置关系。评估共展示酶的催化反应动力学、储存稳定性和底物特异性，揭示不同的共展示机制对于酶学性质的影响。利用最优化的双酶全细胞催化剂构建稳定、灵敏、互换性好的顺序酶电极，旨在解决共固定化顺序酶电极存在的难题。本研究提出了构建顺序酶传感器的新思路，将为解决其他顺序酶反应的低效率，研制其他顺序酶电极奠定基础，也为构筑全细胞生物反应器提供了新的模式方法。

顺序酶传感器能实现单酶传感器难以进行的组分检测，但存在共固定化酶的比例和顺序催化动力学不可控、灵敏度低等问题。本项目以淀粉顺序酶传感器为研究对象，将分别来源于扣囊复膜酵母的糖化酶（GA）编码基因和黑曲霉的葡萄糖氧化酶（GOx）编码基因与具有不同特异性的dockerin结构域DocC和DocT编码基因进行融合，利用毕赤酵母分别分泌表达融合蛋白GA-DocC和GOx-DocT；利用a凝集素锚定蛋白将具有不同特异性的支架蛋白cohesin结构域CohC和CohT按照可控的比例共展示在酿酒酵母细胞的表面；将GA-DocC和GOx-DocT 融合蛋白与展示有支架蛋白的细胞进行孵育，通过cohesin-dockerin的特异性作用将GA和GOx按可控比例、高效、稳定地共展示在酵母细胞表面，并保持其相对独立的空间结构。由于GOx-DocT 融合蛋白的分子量远大于GA-DocC 融合蛋白，因此先组装GA-DocC后组装GOx-DocT所得到的酵母表面共展示全细胞的整体反应速率最高。与游离的顺序酶复合体和随机共展示全细胞相比，酵母表面共展示全细胞（GA:GOx=1:1）的总反应速率分别提高了0.8倍和1.6倍，说明该体系很好地控制了顺序酶的相对位置和顺序，从而显著提高了整体反应速率。通过调整CohC和CohT结构域的比例，构筑了GA和GOx按可控比例共展示的酵母细胞。当酵母表面共展示GA和GOx (2:1)时，全细胞具有最佳的催化效率和储存稳定性。然后利用聚丙烯酸分散的还原氧化石墨烯（RGO）分别与游离GA和GOx混合液、展示不同比例GA和GOx的酵母全细胞（yeast–GA&GOx(x:1, x=1,2,3)）涂布在玻碳电极（GCE）表面制作不同的修饰电极，按比例共展示全细胞修饰电极检测淀粉的响应时间、灵敏度均优于非共展示的体系。对同等浓度的淀粉，yeast–GA&GOx(2:1)/RGO/GCE传感器具有最大电流响应。进一步优化条件，利用yeast–GA&GOx(2:1)全细胞催化剂构建了稳定、灵敏的顺序酶传感器，并利用酵母表面单展示GOx的全细胞葡萄糖传感器（yeast–GOx/RGO/GCE），实现了实际样品中葡萄糖和淀粉的检测。本研究提出了构建顺序酶传感器的新思路，也为解决其他顺序酶反应的低效率，研制其他顺序酶传感器提供了新的方法。