Abstract
Cyclohexylamine (CHAM) is widely used in various industries, but it is harmful to human beings and the environment. Acinetobacter sp. YT-02 can degrade CHAM via cyclohexanone as an intermediate. In this study, the cyclohexylamine oxidase (CHAO) gene from Acinetobacter sp. YT-02 was cloned. Amino acid sequence alignment indicated that the cyclohexylamine oxidase (CHAOYT–02) was 48% identical to its homolog from Brevibacterium oxydans IH-35A (CHAOIH–35). The enzyme was expressed in Escherichia coli 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 Mg2+, Co2+, and K+ had certain inhibitory effect on the enzyme activity. The kinetic parameters Km and Vmax were 0.25 ± 0.02 mM and 4.3 ± 0.083 μM min−1, respectively. The biochemical properties, substrate specificities, and three-dimensional structures of CHAOYT–02 and CHAOIH–35 were compared. Our results are helpful to elucidate the mechanism of microbial degradation of CHAM in the strain YT-02. In addition, CHAOYT–02, as a potential biocatalyst, is promising in controlling CHAM pollution and deracemization of chiral amines.
Highlights
Cyclohexylamine (CHAM), an important fine chemical intermediate, is widely used in industrial manufaction of insecticides, antiseptics and plasticizers
The results indicated that CHAOYT−02 could be used for elimination of cyclohexylamine in the environment
A putative flavin adenine dinucleotide (FAD) binding consensus sequence GXGXXG was present in the N-terminus of cyclohexylamine oxidase (CHAO)
Summary
Cyclohexylamine (CHAM), an important fine chemical intermediate, is widely used in industrial manufaction of insecticides, antiseptics and plasticizers. In the process of production and usage, CHAM is released into the atmosphere, water, and soil. It was found that sodium N-cyclohexylsulfamate, which is used as a food additive and produced 100,000 tons annually in China, could be decomposed into CHAM by intestinal bacteria (Collings, 1989). CHAM is classified as a volatile organic compound that can enter human body via inhalation or skin contact. CHAM has drawn increasing attention as a weak carcinogen (Price et al, 1970; Petersen et al, 1972). To eliminate CHAM residual dispersed in the environment, certain measures have to be taken. Microbial degradation has advantages of low cost and less energy consumption and is an effective approach to eliminating CHAM pollution and residues in the environment
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