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Syncephalastrum racemosum amine oxidase with high catalytic efficiency toward ethanolamine and its application in ethanolamine determination

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Abstract

Our screening study yielded a copper amine oxidase (SrAOX) from Syncephalastrum racemosum, which showed much higher affinity and catalytic efficiency toward ethanolamine (EA) than any other amine oxidase (AOX). Following purification of the enzyme to electrophoretic homogeneity from a cell-free extract, the maximum activity toward EA was detected at pH 7.2–7.5 and 45 °C. The SrAOX complementary DNA (cDNA) was composed of a 2052-bp open reading frame encoding a 683-amino acid protein with a molecular mass of 77,162 Da. The enzyme functions as a homodimer. The deduced amino acid sequence of SrAOX showed 55.3 % identity to Rhizopus delemar AOX and contains two consensus sequences of Cu-AOX, NYDY, and HHQH, suggesting SrAOX is a type 1 Cu-AOX (i.e., a topaquinone enzyme). Structural homology modeling showed that residues 112ML113, 141FADTWG146 M158, and N318 are unique, and T144 possibly characterizes the substrate specificity of SrAOX. The recombinant enzyme (rSrAOX) was produced using Escherichia coli. Steady-state kinetic analysis of rSrAOX activity toward EA (pH 7.5 and 45 °C) gave K m and k cat values of 0.848 ± 0.009 mM and 9.11 ± 0.13 s−1, respectively. The standard curves were linear between 0.1 and 2 mM EA, and 10 μg mL−1–2.5 mg mL−1 (15 μM–3.6 mM) phosphatidylethanolamine using Streptomyces chromofuscus phospholipase D, respectively, was sufficiently sensitive for clinical use.

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Acknowledgments

We thank Associate Professor Yoshitaka Takagai of the Department of Symbiotic Systems Science and Technology, Fukushima University, for ICP-MS analysis. This work was supported in part by JSPS KAKENHI Grant Number 15K05557, a grant from the Adaptable and Seamless Technology Transfer Program through target-driven R&D, JST (grant no. AS251Z00099P), and by research grant funding from the Takahashi Industrial and Economic Research Foundation to D.S.

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Authors and Affiliations

  1. Department of Symbiotic Systems Science and Technology, Graduate School of Symbiotic Systems Science and Technology, Fukushima University, 1 Kanayagawa, Fukushima, 960-1296, Japan

    Yoshitaka Hirano, Keisuke Chonan & Daisuke Sugimori

  2. Division of Biomedical Measurements and Diagnostics, Graduate School of Biomedical Engineering, Tohoku University, 2-1 Seiryo, Aoba, Sendai, 980-8575, Japan

    Kazutaka Murayama

  3. Asahi Kasei Pharma Corp, 632-1 Mifuku, Izunokuni, Shizuoka, 410-2321, Japan

    Shin-ich Sakasegawa & Hideyuki Matsumoto

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  1. Yoshitaka Hirano
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  2. Keisuke Chonan
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  4. Shin-ich Sakasegawa
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  5. Hideyuki Matsumoto
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  6. Daisuke Sugimori
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Correspondence to Daisuke Sugimori.

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We confirm that this manuscript has not been published elsewhere and is not under consideration by another journal. This single study is not divided into several parts to increase the quantity of submissions and submitted to various journals or to one journal over time. No data have been fabricated or manipulated (including images) to support our conclusions. No data, text, or theories by others are presented as if they were the author’s own (“plagiarism”). Proper acknowledgments to other work have been given. Consent to submit has been received explicitly from all co-authors, as well as from the responsible authorities. All authors have approved the manuscript and agree with submission to Applied Microbiology and Biotechnology. Each author contributed to the article as follows. DS designed the manuscript and conceived the study. DS, SS, and HM participated in its design and coordination. YH carried out purification, characterization, and gene cloning. KC and SS carried out kinetic analysis , expression, generation of standard curve for EA and PE determination. KM carried out structure modeling studies and participated in drafting the manuscript. YH, KC, and SS drafted a part of manuscript such as figures and tables. All authors read and approved the final manuscript.

Funding

This study was funded in part by JSPS KAKENHI Grant Number 15K05557, a grant from the Adaptable and Seamless Technology Transfer Program through target-driven R&D, JST (grant no. AS251Z00099P), and by research grant funding from the Takahashi Industrial and Economic Research Foundation to D.S.

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The authors have no conflicts of interest to declare. The corresponding author, DS, certifies and declares no conflicts of interest on behalf of all authors.

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Hirano, Y., Chonan, K., Murayama, K. et al. Syncephalastrum racemosum amine oxidase with high catalytic efficiency toward ethanolamine and its application in ethanolamine determination. Appl Microbiol Biotechnol 100, 3999–4013 (2016). https://doi.org/10.1007/s00253-015-7198-5

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