Production of methanol from methane using Methanotroph

发孢甲基弯菌OB 3b（Methylosinus trichosporium OB 3b）是一种甲烷营养菌，其利用甲烷作为其唯一的碳和能量来源。最初的转化涉及甲烷单加氧酶（MMO）将甲烷转化为甲醇。本研究通过对发酵培养基和反应条件的选择，研究了甲醇积累的规律，并考察了培养基中铜离子浓度对MMO活性的影响。在高铜离子培养基上生长时，细菌的MMO活性高，但比生长速率低。为优化OB 3b发酵产甲醇的条件，考察了菌体密度、甲酸钠浓度、温度、磷酸盐缓冲液浓度、环丙醇处理条件等因素对产甲醇的影响。在10 ml样品瓶中，用9.7 mg干细胞细胞悬浮液和9.38 μ M环丙醇在2 ml 6.25 mM磷酸盐缓冲液（pH 7.0）中在20 ℃下进行环丙醇处理5分钟。通过优化反应条件，反应持续100 h，每g干电池产生的甲醇为152 mmol，比常规条件高51倍。细胞内MDH活性保持在约100 μ g/ml。在这种情况下，50%。然而，当MDH活性被环丙醇完全抑制时，没有观察到产生的甲醇。因此，在该甲醇生物合成过程中，部分甲醇可作为甲烷氧化的电子供体，反应可持续100 h以上，使甲醇产量增加。

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.