| Methanotrophs can use methane gas as carbon source for the growth of microorganism. Methanobactin (mb) is a copper-binding small peptide in methanotrophs. It can coordinate a Cu ion by its nitrogens from two oxazolone rings and its sulfurs from two enethiol groups. Mb-Cu is possibly direct associate with the particulate Methane Monooxygenase (pMMO) and plays a more direct role in pMMO loading and activity. It has been reported that Mb-Cu can increase electron flow to the activity centers of pMMO and exhibit redox activity. This suggested that mb may be used as mimic oxidoreductase. In this paper methylosinus trichosporium3011high density culture way to mb, purification method and structure characterization of mb, and the reaction dynamics research as mimic peroxide enzyme were studied.The results showed that the Methylosinus trichosporium3011can use methanol as carbon source and methanol is superior to the characteristics of methane.The methanol on-line monitored and automatic added system is established. The results showed that methanol could be substituted for methane as the carbon source. With the best addition amount of0.1%methanol intermittent added, the density of cell was1.622g dry wt L-1, and the growth rate was increased to0.043h-1relative to methane at the end of fermentation.Meantime, a longer lag time is59.6254h. And in comparison to methanol intermittent added, the method of the online monitoring methanol auto-added shortened the lag time which was37.1807h, even the period of fermentation and to accumulate methanobactin (mb) time also greatly reduced, increased the strain density which was1.772g dry wt L-1, and the density of cell the growth rate was increased to0.056h-1, the high density growth of cells were promoted by online monitoring automatic adding methanol. The macroporous resin, HP-20was used to purify mb from the spent medium by the dynamic adsorption, the results show that the volume of absorbing solution was400mL, a sample flow rate was2mL/min, the elution solution concentration of alcohol was60%. The macroporous resin dynamic adsorption get methanobactin (mb), then through supelco LC-C18solid phase extraction column and immobilized metal affinity chromatography column for further purification.The methanobactin (mb) samples were scanned by the ultraviolet-visible absorption spectrum. Mb molecules contained sulphonyl oxygen azole group, which resulted in the absorption in the394nm. After chelated copper, this absorption peak decreased. The result from fluorescence scanning Proved that sulphonyl azole ketone group could chelated with Cu (II). Result from mass spectrometry showed:the species of nuclear mass ratio (m/z)1215.696were consistent with the [Mb-2H+63Cu+]1-species of nuclear mass ratio (m/z)1215.7, which were reported by the references. The species of nuclear mass ratio (m/z)927.667were Mb-Cu without cysteine, methionine and isobutyl alcohol. The results of spectroscopy showed that mb can chelate Cu (Ⅱ).The mechanism of hydroquinone oxidation catalyzed by Mb-Cu was proposed. And a kinetic model was established. The kinetics of oxidative reaction of hydroquinone with hydrogen peroxide catalyzed by Mb-Cu were investigated by UV-vis spectrophotometer. The effect of temperature, Mb-Cu addition amount and concentration of hydrogen peroxide on the catalysis reaction were investigated. The apparent first order rate constants (kobs) was found the fastest as optimum hydroquinone concentration for5×10-4mol/L, the optimum concentration of H2O2for7×10-3mol/L, and the reaction was accord with ternary compound model.The results showed that Mb-Cu exhibited the general characteristics of the biocatalyst but had higher thermal stability than natural peroxidase.And the reaction rate for the catalytic oxidation of hydroquinone by Mb-Cu was the largest60℃, Mb-Cu can still maintain a relatively high catalytic activity. |
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