产氧克雷伯菌HP1的耐氧氢酶的表征和克隆。,Research in Microbiology

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产氧克雷伯菌HP1的耐氧氢酶的表征和克隆。
Research in Microbiology ( IF 2.5 ) Pub Date : 2010-12-29 , DOI: 10.1016/j.resmic.2010.12.003
Xiaobing Wu ₁ , Yi Liang , Qianyi Li , Juan Zhou , Minnan Long

Affiliation

纯化了来自温泉细菌产酸克雷伯菌HP1的氢化酶，发现其具有199.8 U / mg蛋白质的比活性和7.3％的产率。确定纯化的酶由六个亚基（65、33、28、23、21和18 kDa）组成，类似于来自大肠杆菌的氢化酶3，因此将其命名为Hyd3。该酶显示出显着的耐氧性。对于纯化的酶，在室温下于空气中孵育24小时后，维持50％的最大活性。克隆了氢化酶基因簇（hyc），发现该基因由hycD，hycE，hycF，hycdG，hycH和hycI基因组成。hycE和hycG基因分别编码氢化酶的大亚基和小亚基。构建了hycE基因缺失突变体ΔhycE，用于阐明hyc-操纵子在氢代谢中的功能。与野生型菌株HP1相比，该突变菌株在O（2）胁迫条件下在甲酸，丙酮酸钠和葡萄糖存在下显示出氢气产生显着减少，而在厌氧条件下检测到大量活性。这强烈表明催产克雷伯氏菌HP1独立于Hyd3携带许多加氢酶或氢代谢途径。但是，Hyd3是在O（2）应力条件下产生氢气的主要因素。催产素HP1携带许多独立于Hyd3的氢化酶或氢代谢途径。但是，Hyd3是在O（2）应力条件下产生氢气的主要因素。催产素HP1携带许多独立于Hyd3的氢化酶或氢代谢途径。但是，Hyd3是在O（2）应力条件下产生氢气的主要因素。

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Characterization and cloning of oxygen-tolerant hydrogenase from Klebsiella oxytoca HP1.

Hydrogenase from a hot spring bacterium Klebsiella oxytoca HP1 was purified and found to have a specific activity of 199.8 U/mg of protein and a yield of 7.3%. The purified enzyme was determined to consist of six subunits (65, 33, 28, 23, 21 and 18 kDa), similar to hydrogenase-3 from Escherichia coli, and therefore it was named Hyd3. The enzyme displayed remarkable oxygen tolerance. For the purified enzyme, 50% maximal activity was maintained following incubation for 24 h in air at room temperature. The hydrogenase gene cluster (hyc) was cloned and found to consist of hycD, hycE, hycF, hycdG, hycH and hycI genes. hycE and hycG genes encode for the large and small subunit of the hydrogenase, respectively. A hycE gene deletion mutant, ΔhycE, was constructed for elucidating the function of the hyc-operon in hydrogen metabolism. Compared with the wild type strain HP1, the mutant strain showed a dramatic decrease in hydrogen production in the presence of formate, sodium pyruvate and glucose under O(2)-stressed conditions, while substantial activity was detected under anaerobic conditions. This strongly suggests that K. oxytoca HP1 carries a number of hydrogenases or hydrogen metabolic pathways independently of Hyd3. However, Hyd3 is the main factor responsible for hydrogen production under O(2) stress conditions.

更新日期：2019-11-01

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