Production of methanol from methane using Methanotroph

甲基三磷菌OB3b （OB3b）是一种利用甲烷作为碳和能量的唯一来源的甲烷化菌。最初的转化是通过甲烷单加氧酶（MMO）将甲烷转化为甲醇。本研究通过选择培养物和反应条件对甲醇的富集进行了研究。考察了培养基中铜浓度对MMO活性的影响。OB3b在高铜离子培养基上生长时，MMO活性高，但比生长率低。培养基中铜的最佳浓度为1.25 muM。为优化OB3b制甲醇工艺，研究了细胞密度、甲酸钠浓度、温度、磷酸盐缓冲液浓度和环丙醇处理条件对反应的影响。将9.7 mg干细胞悬浮液和9.38 muM环丙醇溶于2 ml 6.25 mM磷酸盐缓冲液（pH 7.0）中，置于10 ml样品瓶中，在20℃下处理5分钟。通过优化反应条件，反应持续100 h，生产的甲醇为152 mmol / g，是常规条件下的51倍。在此条件下，细胞中的MDH活性保持在50%左右。然而，当MDH活性被环丙醇完全抑制时，没有观察到生成的甲醇。因此，在甲醇生物合成过程中，产生的部分甲醇可以作为甲烷氧化的电子供体，反应可以持续100 h以上，从而使产生的甲醇增加。

Methylosinus trichosporium OB3b (OB3b) is a methanotroph that utilizes methane as their sole source of carbon and energy. The initial transformation involves the conversion of methane into methanol by methane monooxygenase (MMO). In this study, methanol accumulation by selecting the culture and the reaction conditions was studied.The effect of copper concentration in medium on the MMO activity was investigated. When OB3b was grown on the medium containing high copper ion, the MMO activity was high, however, the specific growth rate was low. The optimimum concentration of copper in the medium was 1.25 muM.To optimize the methanol production by OB3b, the effects of cell density, sodium formate concentration, temperature, phosphate buffer concentration, and cyclopropanol treatment condition, on the reaction were studied. The cyclopropanol treatment was carried out with 9.7 mg of dry-cell cell suspension and 9.38 muM cyclopropanol in 2 ml of 6.25 mM phosphate buffer (pH 7.0) in a 10 ml sample bottle for 5 min at 20。C.By optimizing the reaction condition, the reaction continued for 100 h and the produced methanol was 152 mmol per g of dry-cell, which is 51-fold higher than that of the conventional condition. The MDH activity in the cells retained ca. 50% under this condition. However, when the MDH activity was completely inhibited by cyclopropanol, the produced methanol was not observed. So a part of the produced methanol may serve as the electron donor for methane oxidation in this methanol biosynthesis, the reaction may continue for over 100 h, so that the produced methanol increased.