为了探讨甲烷氧化菌素(Mb)-铜配合物(Mb-Cu)模拟过氧化物酶的可行性, 利用HP20 大孔树脂、Su-pelco LC-C₁₈ 固相萃取和固定化金属亲和层析从甲基弯菌IMV3011 中分离纯化得到Mb. 铬天青比色法显示Mb 具有铜亲和性. 通过液相色谱鄄飞行时间质谱联用仪、紫外光谱和荧光光谱对Mb 结构进行了表征. 使用Mb-Cu 配合物作为过氧化物酶模拟物, 利用紫外-可见分光光度法研究了Mb-Cu 催化过氧化氢氧化对苯二酚的动力学. 考察了体系温度、Mb-Cu 添加量及过氧化氢浓度对催化反应的影响, 发现Mb-Cu 符合生物催化剂条件影响的一般规律, 但比生物酶具有更高的热稳定性. 研究结果表明, Mb-Cu 可作为催化氧化对苯二酚的过氧化物酶模拟酶.

Methanobactin(Mb) is a copper-binding small peptide that appears to function as an agent for Cu sequestration and uptake in methanotrophs. It can coordinate a single Cu ion by its nitrogens from two oxazolone rings and its sulfurs from two enethiol groups. Mb-Cu is possibly in direct association 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 to mimic oxidoreductase. In this paper, to explore the potential peroxidase catalytic properties in this novel class of Cu binding compound, Mb from Methylosinus trichosporium IMV3011 was isolated and purified by HP20, supelco LC-C-18 and IDE-Cu immobilized metal-chelate affinity chromatography column. Chrome azurol S(CAS) calorimetric assays showed that the Mb have high Cu (H) affinities. Analysis of the Mb-Cu sample by LC/TOE MS gave a peak at m/z 1215, corresponding to an m/z 1153 Mb molecule which has lost one or two hydrogen atoms and bound one copper atom. Fluorescent and UV-Vis spectroscopy results also demonstrated that the transitions occurring when Cu(II) interacts with Mb. These results indicated that Mb has been loaded with Cu ion and Mb-Cu has been prepared. Using Mb-Cu as a mimic of peroxidase, the kinetics of oxidative reaction of hydroquinone with hydrogen peroxide catalyzed by Mb-Cu were investigated by UV-Vis spectrophotometer. The change of hydroquinone concentration was determined by monitoring the decrease of absorbance (A) at 288 nM. The apparent first order rate constants(K-abs) show that the reaction rate for the catalytic oxidation of hydroquinone increases by a factor of 4x10(2) for the Mb-Cu at 25 degrees C. The mechanism of hydroquinone oxidation catalyzed by Mb-Cu was proposed, and a kinetic model was established. The effect of temperature, Mb-Cu addition amount and concentration of hydrogen peroxide on the catalysis reaction were investigated. The results show that Mb-Cu exhibite the general characteristics of the biocatalyst but had 'higher thermal stability than that of natural peroxidase. K was found to be enhanced as temperature increased from 25 degrees C to 60 degrees C. Therefore, Mb-Cu can be used as an effective mimetic peroxidase in the process of hydroquinone oxidation.