Chemo-enzymatic cascade reactions of flavin-dependent monooxygenase and firefly luciferase for detection of halogenated and nitro phenols

Pratchaya Watthaisong1, Aisaraphon Phintha2, Vinutsada Pongsupasa1,Panu Pimviriyakul1, Yoshihiro Ohmiya3,Pimchai Chaiyen1*

1School of Biomolecular Science and Engineering, Vidyasirimedhi Institute of Science and Technology (VISTEC), Rayong21210, Thailand.

2Department of Biochemistry/Center for Excellence in Protein and Enzyme Technology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand.

3National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki, Japan.

E-mail: pimchai_chaiyen@vistec.ac.th

Halogenated and nitro phenols are used widely as agro- and industrial chemicals. Their accumulations in environment are seriously concerned because they are highly toxic. Their usage and environmental contamination are regulated by United State Environmental Protection Agency (US-EPA). Our group has studied enzymatic reactions of flavin-dependent monooxygenase (HadA) which is able of catalyzing dehalogenation and denitration of 4-halogenated and 4-nitro phenols to generate p-benzoquinone. As p-benzoquinone from the HadA reactions can react with D-cysteine to generate D-luciferinwhich is a substrate for firefly luciferase, we have developed a novel chemo-enzymatic method for detection of p-halogenated and p-nitro-phenols. Ratios of p-benzoquinone and D-cysteine were varied and monitored absorption (330 nm) and fluorescence (Em 530 nm) spectrum changes to obtain the best condition for D-luciferin synthesis. We obtained the best ratio of D-luciferin synthesis as 1:4 of p-benzoquinone:D-cysteine. We coupled chemo-enzymatic detection to detect the conversion of p-nitrophenol to D-luciferin using LC-ESI-qTOF techniques. The results showed that not only D- luciferin could be formed but a6-benzothiazole-2-carbaldehyde was also detected. However, this novel chemo-enzymatic reactions can be coupled with the reaction of firefly luciferase to generate light, indicating that this method can be used to detect halogenated and nitro-phenols.