J. Technology and Education, Vol.23, No.2, pp.57-61 (2016)

研究論文

 

好熱菌Rubrobacter xylanophilus 由来D-アミノ酸オキシダーゼ

を用いたＤ-バリンセンサーの構築

 

高山 勝己^*1，坂元 知里¹，高橋 祥司²，阿部 勝正², 廣部 綾乃¹，越桐 武児¹

¹福井工業高等専門学校物質工学科（〒916-8507福井県鯖江市下司町）

²長岡技術科学大学生物機能工学専攻（〒940-2188新潟県長岡市上富岡町）

*takayama@fukui-nct.ac.jp

 

Construction of a D-valine sensor using D-amino acid oxidase

of Rubrobacter xylanophilus

 

Katsumi TAKAYAMA^*1, Chisato SAKAMOTO¹, Shouji TAKAHASHI²,

Katsumasa ABE², Ayano HIROBE¹, and Takeji KOSHIGIRI¹

 

¹Department of Chemistry & Biology Engineering, Fukui National College of Technology

(Geshi, Sabae 916-8507, Japan)

²Department of Bioengineering, Nagaoka University of Technology

(Kamitomioka, Nagaoka 940-2188, Japan)

 

(Received September 20, 2016; Accepted October 20, 2016)

 

Abstract

A biosensor for D-valine was constructed by using D-amino acid oxidase of Rubrobacter xylanophilus (RxDAO) as a biocatalyst. This enzymatic reaction consists of enzymatic and nonenzymatic reactions. During the enzymatic reaction process, an imino acid is produced that is accompanied by oxygen consumption and hydrogen peroxide formation. In this study, RxDAO was trapped on horseradish peroxidase and an osmium polymer-modified carbon paste electrode (RxDAO-HRP-Os CPE). The detection limit of RxDAO-HRP-Os CPE was about 5 μM for D-valine. The response of RxDAO-HRP-Os CPE to amino acids corresponded with the substrate selectivity of RxDAO without D-tyrosine. The micromolar concentration level of D-valine can be detected with the coexistence of 1 mM L-valine. The linear relationship of the calibration curve was between 80 μM and 1 mM. The response was maintained over 2 weeks. RxDAO was not deactivated at 50℃.

 

Keywords:  D-valine, Rubrobacter xylanophilus, D-amino acid oxidase, biosensor, HRP osmium polymer