Project description: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 K m and V max were 0.25 ± 0.02 mM and 4.3 ± 0.083 ?M 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.
Project description:Copper (Cu) is a heavy metal that is widely used in industry and as such wastewater from mining or industrial operations can contain high levels of Cu. Some aquatic algal species can tolerate and bioaccumulate Cu and so could play a key role in bioremediating and recovering Cu from polluted waterways. One such species is the green alga Desmodesmus sp. AARLG074. The aim of this study was to determine how Desmodesmus is able to tolerate large alterations in its external Cu and pH environment. Specifically, we set out to measure the variations in the Cu removal efficiency, growth, ultrastructure, and cellular metabolite content in the algal cells that are associated with Cu exposure and acidity. The results showed that Desmodesmus could remove up to 80% of the copper presented in Jaworski's medium after 30 min exposure. There was a decrease in the ability of Cu removal at pH 4 compared to pH 6 indicating both pH and Cu concentration affected the efficiency of Cu removal. Furthermore, Cu had an adverse effect on algal growth and caused ultrastructural changes. Metabolite fingerprinting (FT-IR and GC-MS) revealed that the polysaccharide and amino acid content were the main metabolites affected under acid and Cu exposure. Fructose, lactose and sorbose contents significantly decreased under both acidic and Cu conditions, whilst glycerol and melezitose contents significantly increased at pH 4. The pathway analysis showed that pH had the highest impact score on alanine, aspartate and glutamate metabolism whereas Cu had the highest impact on arginine and proline metabolism. Notably both Cu and pH had impact on glutathione and galactose metabolism.