利用甲硝唑及外加氧方法筛选耐氧产氢Klebsiella oxytoca HP1突变菌株

氢酶是生物制氢的关键酶, 大多数氢酶因对氧极敏感而易失活, 因此提高氢酶的氧耐受性对生物制氢有重要意义。本研究利用1%甲基磺酸乙酯对Klebsiella oxytoca HP1进行了两轮诱变, 经40 mmol/L 甲硝唑和21%氧联合处理1 h(第一轮诱变)或2 h(第二轮诱变)进行筛选。所得突变菌株经产氢测试, 结果在15%氧浓度条件下, 第一代突变菌株HP1-A15产氢活性为出发菌株Klebsiella oxytoca HP1的3.70倍, 在21%氧浓度条件下第二代突变菌株 HPA15-37产氢活性为HP1-A15菌株的2.75倍, 是出发菌株的11倍。突变菌株HP1-A15和 HPA15-37具有较好的遗传稳定性。本试验结果说明利用MNZ和外加氧的方法适用于兼性厌氧菌耐氧产氢突变菌株的筛选。

Screen of O2-tolerate Phenotype of Klebsiella oxytoca HP1 Mutants with High H2-evolving Activity by Selection with MNZ Combination O2

Hydrogenases are key enzyme for bio-hydrogen production, most of them were rapidly inactivated by oxygen. It is important to bio-hydrogen production and hydrogen application that improve the O2-tolerance of hydrogenase. In this experiment, the hydrogen producing strain Klebsiella oxytoca HP1 was treated with 1% ethyl methanesulfonate(EMS) , the mutants with high O2-toleration ability were screened with 40mmol/L metronidazole (MNZ) and 21% oxygen. The H2-evolving activity of the first generation mutant HP1-A15 was increased 3.70 times than that of the wild-type (WT) under 15% oxygen. The H2-evolving activity of the second generation mutant HPA15-37 was enhanced 11 times than that of WT under the condition of 21% oxygen. The mutants HP1-A15 and HPA15-37 had steady heredity. These results suggest that MNZ and in addition oxygen is a good way to screen of O2-tolerate phenotype of facultativeanaerobe with high H2-evolving activity.