AUTHOR=Zhou Hui , Han Zheng-gang , Fang Ti , Chen Yuan-yuan , Ning Shang-bo , Gan Ya-ting , Yan Da-zhong 

TITLE=Characterization of a New Cyclohexylamine Oxidase From Acinetobacter sp. YT-02

JOURNAL=Frontiers in Microbiology

VOLUME=9

YEAR=2018

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2018.02848

DOI=10.3389/fmicb.2018.02848

ISSN=1664-302X

ABSTRACT=<p>Cyclohexylamine (CHAM) is widely used in various industries, but it is harmful to human beings and the environment. <italic>Acinetobacter</italic> sp. YT-02 can degrade CHAM via cyclohexanone as an intermediate. In this study, the cyclohexylamine oxidase (CHAO) gene from <italic>Acinetobacter</italic> sp. YT-02 was cloned. Amino acid sequence alignment indicated that the cyclohexylamine oxidase (CHAO<sub>YT–02</sub>) was 48% identical to its homolog from <italic>Brevibacterium oxydans</italic> IH-35A (CHAO<sub>IH–35</sub>). The enzyme was expressed in <italic>Escherichia coli</italic> BL21 (DE3), and purified to apparent homogeneity by Ni-affinity chromatography. The purified enzyme was proposed to be a dimer of molecular mass of approximately 91 kDa. The enzyme exhibited its maximum activity at 50°C and at pH 7.0. The enzyme was thermolabile as demonstrated by loss of important percentage of its maximal activity after 30 min incubation at 50°C. Metal ions Mg<sup>2+</sup>, Co<sup>2+</sup>, and K<sup>+</sup> had certain inhibitory effect on the enzyme activity. The kinetic parameters <italic>K</italic><sub>m</sub> and <italic>V</italic><sub>max</sub> were 0.25 ± 0.02 mM and 4.3 ± 0.083 μM min<sup>−1</sup>, respectively. The biochemical properties, substrate specificities, and three-dimensional structures of CHAO<sub>YT–02</sub> and CHAO<sub>IH–35</sub> were compared. Our results are helpful to elucidate the mechanism of microbial degradation of CHAM in the strain YT-02. In addition, CHAO<sub>YT–02</sub>, as a potential biocatalyst, is promising in controlling CHAM pollution and deracemization of chiral amines.</p>