Content Measurement, Biosynthesis And Structure Characterization Of Methanobactin

Methanotrophs and methane monooxygenase (MMO) have great potential for application as biocatalyst, and had been received the attentions of many research institutions. Methanobactin (mb) was a small, copper binding peptide produced by methanotrophic bacteria. Mb had received much attention for its role in the physiology and activity of Methanotrophs. Unfortunately, Isolation, purification and function study of these molecules has been challenging. It is needed for seeking a method to detect its content. In addition, the basic structure and composition of mb that produced by different growing Methanotrophs cultures also had differences. The purpose of this paper was focus on content measurement, biosynthesis and structure characterization of mb.The main contents and results are summarized as follows:According to the copper-binding characteristic of mb, a rapid and efficient protocol for detection mb system of liquid state was established. The results showed that the optimum conditions for the detection was,2mL of chrome azurol S (CAS),1mL of Cu2+,4mL of dyeing assistant agent, and0.5mL of buffer solution. The mixture was incubated for10min at room temperature. The content of mb was detected by UV-visible spectrophotometer with the605nm wavelength. The developed method exhibited simple, rapid, accurate and practical, thus providing the basis for the mb fermentation production.The production of mb during growth of Methylosinus trichosporium3011(IMV3011), Methylosinus trichosporium OB3b(OB3b), Methylococcus capsulatus3021(IMV3021), Methylomonas sp.GYJ3(GYJ3) on methane and methanol was determined by split nitrate mineral salts(NMS)/CAS-Cu plates. The mb was found to be accumulated in the spent medium of four kinds of Methanotrophs under copper-limited condition. The mb production ability of methanol-grown cells were obviously higher than that of methane-grown cells.The results showed that with an increase concentration of copper from OμM to1.5μM, the mb yield of unit cell weight were accumulated to the maximum amount on methane and methanol medium at the copper concentration of OμM. When the Cu2+concentration is1.5μM the whole amount of mb of methanol is higher than that of methane. With the concentration of the copper change from OμM to1.5μM mb production variation of methane is greater than that of methanol.Consistent with the role in copper uptake, mb from IMV3011can stimulate particulate Methane Monooxygenase (pMMO) activity of all Methanotrophs in the presence of copper. The results suggested that Methanotrophs may take delivery of copper from mb released by other species and have not the genus specificity. In this paper, the mb was purified based on solid phase extraction (SPE). The purification conditions for solid phase extraction were established.The characterization of mb was determined by HPLC-MS, UV/Vis and fluorescence spectra. The results showed that the calculated exact mass for this structure of mb from IMV3011was very likely to846.1441. and its molecular formula may be C30H38N8O13S4.