Discovery and Biochemical Characterization of a Methanol Dehydrogenase From Lysinibacillus xylanilyticus
| Autor: | Minjeong Choi, Soo-Jin Yeom, Eugene Rha, So-Hyung Oh, Sung-Hyun Park, Bong Hyun Sung, Hyewon Lee, Dae-Hee Lee, Jin-Young Lee, Su Jin Kim, Seung-Goo Lee, Jin Ju Lee, Kil Koang Kwon |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2020 |
| Předmět: |
0301 basic medicine
Histology Bioconversion lcsh:Biotechnology Biomedical Engineering methanol oxidation Bioengineering methanol dehydrogenase 02 engineering and technology medicine.disease_cause 03 medical and health sciences chemistry.chemical_compound lcsh:TP248.13-248.65 rational enzyme engineering medicine methylotrophy Escherichia coli chemistry.chemical_classification biology Methanol dehydrogenase Active site 021001 nanoscience & nanotechnology 030104 developmental biology Enzyme Biochemistry chemistry Lysinibacillus xylanilyticus biology.protein Specific activity Methanol NAD+ kinase 0210 nano-technology Biotechnology |
| Zdroj: | Frontiers in Bioengineering and Biotechnology, Vol 8 (2020) |
| ISSN: | 2296-4185 |
| DOI: | 10.3389/fbioe.2020.00067/full |
| Popis: | Bioconversion of C1 chemicals such as methane and methanol into higher carbon-chain chemicals has been widely studied. Methanol oxidation catalyzed by methanol dehydrogenase (Mdh) is one of the key steps in methanol utilization in bacterial methylotrophy. In bacteria, few NAD+-dependent Mdhs have been reported that convert methanol to formaldehyde. In this study, an uncharacterized Mdh gene from Lysinibacillus xylanilyticus (Lxmdh) was cloned and expressed in Escherichia coli. The maximum alcohol oxidation activity of the recombinant enzyme was observed at pH 9.5 and 55°C in the presence of 10 mM Mg2+. To improve oxidation activity, rational approach-based, site-directed mutagenesis of 16 residues in the putative active site and NAD+-binding region was performed. The mutations S101V, T141S, and A164F improved the enzyme's specific activity toward methanol compared to that of the wild-type enzyme. These mutants show a slightly higher turnover rate than that of wild-type, although their KM values were increased compared to that of wild-type. Consequently, according the kinetic results, S101, T141, and A164 positions may related to the catalytic activity in the active site for methanol dehydrogenation. It should be further studied other mutant variants with high activity for methanol. In conclusion, we characterized a new Lxmdh and its variants that may be potentially useful for the development of synthetic methylotrophy in the future. |
| Databáze: | OpenAIRE |
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