해수에서 분리한 신규메탄올 산화세균 Methylophaga aminisulfidivorans MPT 유래 methanol dehydrogenase와 MxaJ 단백질의 상호관계 및 trimethylamine biosensor 개발

Abstract

A new restricted facultatively methylotrophic marine strain MPT (KCTC 12909T= VKM B-2441T = JCM 14647T) possessing the ribulose monophosphate pathway of C1-carbon compound assimilation was isolated from a seawater sample obtained from Mokpo, South Korea. The isolate was aerobic, Gram-negative, asporogenous, and was non-motile short rod. It grew well on methanol, methylated amines, dimethylsulfide and dimethylsulfoxide. Optimal growth occurred with 3% NaCl at 30oC and pH 7.0. Fructose was utilized as a multicarbon source. Growth factors were not required and vitamin B12 did not stimulate growth. Its cellular fatty acid profile consists primarily of straight-chain saturated C16:0 and unsaturated C16:1 acids. The major ubiquinone is Q-8. The dominant phospholipids are phosphatidylethanolamine and phosphatidylglycerol. The DNA G + C content is 44.9 mol% (Tm). Based on 16S rRNA gene sequence analysis and DNA-DNA relatedness (25 ~ 41%) with the type strains of marine methylotrophs belonging to the genus Methylophaga, we proposed the isolate MPT (= KCTC 12909 = VKM B-2441 = JCM 14647) be assigned as Methylophaga aminisulfidivorans sp. nov. Methylophaga aminisulfidivorans MPT, a restricted facultative marine methylotrophic bacterium, was able to utilize methanol as a sole carbon and energy source, and possessed a methanol dehydrogenase (MDH) that is a key enzyme in the process of methanol oxidation. During purification of MDH, two types of MDH (MDH I and II) were obtained in the cell free extracts from MPT cells grown on methanol. When analyzed by SDS-PAGE and ESI-FT ICR MS, MDH I was confirmed to consist of two subunits of with molecular masses of ~66 and ~10 kDa, respectively, in a form of ?2?2. By contrast, MDH I and MDH II contained an additional ~30 kDa protein, designated ?, in a form of ?2?2? and ?2?2?2, respectively. MDH II showed 1.5?2.0 times higher activity than MDH I. Based on these observations and experimental data, it seems that the original MDH conformation is ?2?2?2 within MPT, and subunit ? keeps MDH in an active form, and/or makes MDH easily bind to the substrate, methanol. Methylophaga aminisulfidivorans MPT could be grown aerobically on trimethylamine (TMA) as a source of carbon and energy. The trimethylamine dehydrogenase (TMADH) could catalyze the oxidative demethylation of TMA. The active TMADH from MPT was purified 12.3 fold through three purification steps. We detected the trimethylamine using an electrochemical biosensor with the electron-conducting redox polymers, Osmium and Ferrocene-imidazole complexes were electrode posited on the thiol-modified gold electrodes. As TMA was catalyzed by TMADH, the electrical signal obtained from a biosensor was monitored at 0.3 V versus Ag/AgCl. The produced electrons were transferred to the electrode via Ferrocene-imidazole complexes. The detection limit of TMA biosensor was 0.125 mM. The intensity of catalytic current almost linearly increased depending on the TMA concentrations.