Presentation_1_Discovery and Biochemical Characterization of a Methanol Dehydrogenase From Lysinibacillus xylanilyticus.PPTX (2.1 MB)
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Presentation_1_Discovery and Biochemical Characterization of a Methanol Dehydrogenase From Lysinibacillus xylanilyticus.PPTX

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posted on 14.02.2020, 04:02 authored by Jin-Young Lee, Sung-Hyun Park, So-Hyung Oh, Jin-Ju Lee, Kil Koang Kwon, Su-Jin Kim, Minjeong Choi, Eugene Rha, Hyewon Lee, Dae-Hee Lee, Bong Hyun Sung, Soo-Jin Yeom, Seung-Goo Lee

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.

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