基于细菌表面共展示顺序酶的生物传感器的制备及其在麦芽糖检测中的应用

Sequential enzyme biosensor is an analytical device through combination of several related enzymes to catalyze serial reactions sequentially and coordinately, which could measure substances that are hard to be done with single-enzyme based biosensors. Thus the sequential enzyme biosensor can find important application in analysis. However, to date, the sequential enzyme biosensors experienced low sensitivity and poor reproducibility due to the random distribution of the sequential enzymes on the electrode by chemical crosslinking or entrapment method. To challenge this, in this project the maltose biosensor will be developed as an example. Sequential enzymes of both glucoamylase (GA) and glucose dehydrogenase (GDH) will be co-displayed on the bacterial surface through ice nuclear protein and Cohesin-Dockerin protein interaction, which are located on the cell surface proportionally and directionally. The position of GA and GDH on the entire-cell will be determined to judge the distribution of these bi-enzyme proteins on the same cell surface, and to clarify the spatial relationship between them. The enzymatic catalytic kinetics, stability of the bienzyme-displaying entire-cell catalyst will be investigated to identify whether the enzymatic properties are affected by the co-displaying mechanism. Subsequently, sequential enzyme electrode will be prepared on basis of bacterial surface co-displayed GA/GDH, aiming at creating novel biosensing interface. Further, the entire-cell biocatalyst based sequential enzyme electrochemical biosensor will be developed through optimizing experiment conditions. The analytical performance of the biosensor including sensitivity, stability and reproducibility will be studied. Finally, the proposed biosensor will be applied to detect maltose in beer. Due to avoiding the loss of enzyme activity caused by chemical cross-linking, it is anticipated that GA/GDH-co-displayed bacteria based biosensor will be superior to traditional bi-enzyme biosensor. Thus, this work may pave a new way to prepare sequential enzyme biosensor through the interdisciplinary study.

顺序酶生物传感器指多种相关酶顺序、协调地完成一系列催化反应，可实现单酶传感器难以实现的检测，在分析检测领域具有重要应用。但目前制约其发展和应用的瓶颈在于化学交联法或包埋法共固定化酶在电极上呈随机分布、重现性差、灵敏度低。本项目以麦芽糖传感器为研究对象，拟采用细菌表面糖化酶(GA)和葡萄糖脱氢酶(GDH)共展示全细胞构筑顺序酶电极。利用冰核蛋白锚定系统与Cohesin-dockerin蛋白结合反应，旨在实现GA和GDH细菌表面按比例、空间位置可控的展示。对全细胞进行定位观测，确定两种酶蛋白在同一细胞表面的分布。探讨共展示酶的催化反应动力学和储存稳定性。利用获得的高性能共展示菌体创新传感界面；进一步优化实验条件，制备全细胞顺序酶电化学生物传感器，实现麦芽糖的高灵敏检测并用于啤酒中麦芽糖的分析，旨在提高传感器的灵敏度、稳定性和重现性。本项目提出了通过化学与生物的交叉研究，构建顺序酶电极的新思路。

电化学生物传感器具有灵敏度高、检测响应快和便携等优点，在分析检测领域用途广泛。其中顺序酶生物传感器由相关酶顺序、协调地完成一系列催化反应，可实现单酶传感器难以实现的检测。为进一步改善化学交联法或包埋法共固定化酶在电极上呈随机分布、重现性差和灵敏度低的问题，本项目以麦芽糖传感器为研究对象，通过微生物表面展示技术，制备了细菌表面糖化酶(GA)和葡萄糖脱氢酶(GDH)按比例共展示顺序酶修饰电极，实现了灵敏度高响应快的麦芽糖检测。. 首先将冰核蛋白(INP)编码插入pET-28a载体中，得到pTInaPbN载体。将两种Cohesin蛋白结构域基因按照不同的比例插入编码pTInaPbN载体多克隆位点中，得到INP在细胞表面展示Cohesin结构域支架蛋白的表达载体。经过转化、筛选、诱导表达等步骤，最终得到按比例共展示GA和GDH的全细胞。通过测定米氏常数、最大反应速率等，获得顺序酶反应动力学最优的共展示全细胞。. 然后将得到的按比例共展示顺序酶全细胞通过化学交联法固定到纳米材料修饰的电极表面，测试不同展示比例的全细胞对麦芽糖的检测效果。并以未经表面展示的游离酶和单展示共混的双酶修饰电极作为对照，通过对麦芽糖检测范围、检测限、响应速度和灵敏度的考察，确认了按比例共展示顺序酶全细胞能够通过提高酶的整体协同催化作用，实现麦芽糖的快速、高灵敏度检测。当GA和GDH展示比例3:1时，制备的修饰电极表现出最佳的麦芽糖检测线性范围0.02-32 mM，最低检出限为0.01 mM (信噪比为3)。并且所制备的传感器具有良好的稳定性和重现性，能够用于直接分析啤酒、果汁等液体样品中的麦芽糖含量，无需预处理，检测结果准确。