Sasaki Kai, Furusawa Hiroyuki, Nagamine Kuniaki, Tokito Shizuo
Graduate School of Organic Materials Science, Yamagata University, 4-3-16 Jonan, Yonezawa, Yamagata 992-8510, Japan.
Innovative Flex Course for Frontier Organic Material Systems (iFront), Yamagata University, 4-3-16 Jonan, Yonezawa, Yamagata 992-8510, Japan.
ACS Omega. 2020 Dec 8;5(50):32844-32851. doi: 10.1021/acsomega.0c05521. eCollection 2020 Dec 22.
The application of a multienzyme cascade reaction in electrochemical biosensors has the advantage of expanding the target substrates in addition to selectivity combining multiple enzymes on an electrode. However, the multienzyme system has the drawback of inefficient substance conversion because of the time-consuming passing of intermediates between the enzymes and/or diffusional loss of the intermediates. In this study, the optimal construction of a multienzymatic film in an ammonia detection sensor was investigated using a cascade reaction of l-glutamate oxidase and l-glutamate dehydrogenase as a model sensor. Three enzymatic films were prepared: (1) a mixed film designed to have a short diffusional distance between closely located enzymes, (2) a normal-sequential layered film arranged for the correct reaction pathway, and (3) a reverse-sequential layered film as a negative control. This was followed by comparison of the conversion efficiency of ammonia to hydrogen peroxide using time-dependent potentiometric measurements of a Prussian blue electrode determining the hydrogen peroxide amount. The results indicate that the conversion efficiency of the normal-sequential layered film was the highest among the three enzymatic films. The quantitative evaluation of the intermediate conversion efficiency of the cascade reaction showed that compared to the mixed film (34%), a higher conversion efficiency of 92% was obtained in the first enzymatic reaction step. These findings will promote the use of multienzymatic cascade reaction systems not only in biosensors and bioreactors but also in various industrial fields.
多酶级联反应在电化学生物传感器中的应用，除了能在电极上组合多种酶实现选择性外，还具有扩大目标底物的优势。然而，多酶系统存在物质转化效率低的缺点，这是因为酶之间中间体的传递耗时以及中间体的扩散损失。在本研究中，以L-谷氨酸氧化酶和L-谷氨酸脱氢酶的级联反应作为模型传感器，研究了氨检测传感器中多酶膜的最佳构建。制备了三种酶膜：（1）一种设计用于使紧密相邻的酶之间扩散距离较短的混合膜；（2）一种为正确反应途径排列的正常顺序分层膜；（3）一种作为阴性对照的反向顺序分层膜。随后，通过使用普鲁士蓝电极的时间相关电位测量来确定过氧化氢量，比较氨向过氧化氢的转化效率。结果表明，正常顺序分层膜的转化效率在三种酶膜中最高。级联反应中间体转化效率的定量评估表明，与混合膜（34%）相比，在第一个酶促反应步骤中获得了更高的92%的转化效率。这些发现将促进多酶级联反应系统不仅在生物传感器和生物反应器中，而且在各种工业领域中的应用。
