Lenz Oliver, Lauterbach Lars, Frielingsdorf Stefan
Department of Chemistry, Technische Universität Berlin, Berlin, Germany.
Department of Chemistry, Technische Universität Berlin, Berlin, Germany.
Methods Enzymol. 2018;613:117-151. doi: 10.1016/bs.mie.2018.10.008. Epub 2018 Nov 24.
Dioxygen-tolerant [NiFe]-hydrogenases are defined by their ability to catalyze the reaction, H⇌2H+2e even in the presence of O. Catalytic and probably also noncatalytic mechanisms protect their active sites from being inactivated by reactive oxygen species, which makes them attractive subjects of investigation from both fundamental and applied perspectives. Prominent representatives of the O-tolerant [NiFe]-hydrogenases have been isolated from the chemolithoautotrophic model organism Ralstonia eutropha H16, which can thrive in a simple mineral medium supplemented with the gases H, O, and CO. In this chapter, we describe methods for cultivation and genetic manipulation of R. eutropha, both of which are prerequisites for the reproducible manufacturing of high-quality hydrogenase preparations. The purification procedures for two different O-tolerant [NiFe]-hydrogenases from R. eutropha are described in detail, as well as the corresponding biochemical procedures used for the determination of the catalytic properties of these sophisticated enzymes.
耐氧[NiFe]氢化酶的定义是，即使在有氧存在的情况下，它们也能够催化反应H⇌2H⁺ + 2e⁻。催化机制以及可能的非催化机制保护其活性位点不被活性氧灭活，这使得它们从基础和应用的角度来看都是具有吸引力的研究对象。耐氧[NiFe]氢化酶的突出代表已从化能自养模式生物嗜糖假单胞菌H16中分离出来，该生物可以在补充了H₂、O₂和CO₂气体的简单矿物培养基中生长。在本章中，我们描述了嗜糖假单胞菌的培养方法和基因操作方法，这两者都是可重复生产高质量氢化酶制剂的先决条件。详细描述了从嗜糖假单胞菌中纯化两种不同耐氧[NiFe]氢化酶的程序，以及用于测定这些复杂酶催化特性的相应生化程序。
