Simultaneous Determination of Manganese Peroxidase and Lignin Peroxidase by Capillary Electrophoresis Enzyme Assays

Sumire Kudo; Airi Harada; Hiroe Kubota; Keiko Sasaki; Takashi Kaneta

doi:10.1021/acsomega.7b00998

Simultaneous Determination of Manganese Peroxidase and Lignin Peroxidase by Capillary Electrophoresis Enzyme Assays

ACS Omega. 2017 Oct 27;2(10):7329-7333. doi: 10.1021/acsomega.7b00998. eCollection 2017 Oct 31.

Authors

Sumire Kudo¹, Airi Harada¹, Hiroe Kubota¹, Keiko Sasaki², Takashi Kaneta¹

Affiliations

¹ Department of Chemistry, Graduate School of Natural Science and Technology, Okayama University, Okayama 700-8530, Japan.
² Department of Earth Resources Engineering, Graduate School of Engineering, Kyushu University, 744 Motooka, Fukuoka 819-0395, Japan.

Abstract

Here, we developed an enzyme assay of manganese peroxidase (MnP) by capillary electrophoresis using an in-capillary reaction and applied it to a simultaneous assay of MnP and lignin peroxidase (LiP). The enzyme activity of MnP was determined from the peak area corresponding to Mn(III)-malonate produced by the plug-plug reaction between MnP and Mn(II) in a separation capillary. A background electrolyte containing 250 mM malonate buffer (pH 4.5) and 5 mM cetyltrimethylammonium bromide was employed for the separation of Mn(III)-malonate from MnP at -10 kV after a plug-plug reaction for 5 min. Although the assay permitted the determination of purified MnP, we found that both LiP and MnP have similar activities against their substrates, that is, LiP catalyzed the oxidation reaction of Mn(II) as well as MnP, whereas MnP catalyzed the oxidation reaction of veratryl alcohol which was the substrate used in the LiP assay developed previously. Thus, we proposed a method to discriminate MnP from LiP based on the difference in the activities of these enzymes to each substrate. Amounts of MnP and LiP in a mixture were successfully evaluated by the proposed method.