| Affiliation: | 1. University of Belgrade-Institute of Chemistry Technology and Metallurgy National Institute of the Republic of Serbia, Njegoševa 12, 11000 Belgrade, Serbia;2. University of Belgrade-Institute of Chemistry Technology and Metallurgy National Institute of the Republic of Serbia, Njegoševa 12, 11000 Belgrade, Serbia Department of Otorhinolaryngology Head and Neck Surgery, Grosshadern Medical Center, Ludwig-Maximilians-University, Marchioninistr. 15, 81377 Munich, Germany;3. University of Belgrade-Institute for Multidisciplinary Research, Kneza Višeslava 1, 11030 Belgrade, Serbia;4. Department of Biochemistry, University of Belgrade-Faculty of Chemistry, Studentski trg 12–16, 11000 Belgrade, Serbia |
| Abstract: | The aim of this research was to prove the function of the putative opine dehydrogenase from Desulfohalobium retbaense and to characterize the enzyme in terms of functional and kinetic parameters. A putative opine dehydrogenase was identified from a metagenomic library by a sequence-based technique search of the metagenomic library, and afterward was successfully heterologously produced in Escherichia coli. In order to examine its potential for applications in the synthesis of secondary amines, first the substrate specificity of the enzyme towards different amino donors and amino acceptors was determined. The highest affinity was observed towards small amino acids, preferentially L-alanine, and when it comes to α-keto acids, pyruvate proved to be a preferential amino acceptor. The highest activity was observed at pH 6.5 in the absence of salts. The enzyme showed remarkable stability in a wide range of experimental conditions, such as broad pH stability (from 6.0–11.0 after 30 min incubation in buffers at a certain pH), stability in the presence of NaCl up to 3.0 M for 24 h, it retained 80 % of the initial activity after 1 h incubation at 45 °C, and 65 % of the initial activity after 24 h incubation in 30 % dimethyl sulfoxide. |
