Maeng J H, Sakai Y, Tani Y, Kato N
Department of Agricultural Chemistry, Kyoto University, Japan.
J Bacteriol. 1996 Jul;178(13):3695-700. doi: 10.1128/jb.178.13.3695-3700.1996.
In the Finnerty pathway for n-alkane, oxidation in Acinetobacter sp., n-alkanes are postulated to be attacked by a dioxygenase and the product, n-alkyl hydroperoxide, is further metabolized to the corresponding aldehyde via the peroxy acid [W. R. Finnerty, P. 184-188, in A. H. Applewhite (ed.), Proceedings of the World Conference on Biotechnology for the Fats and Oil Industry, 1988]. However, no biochemical evidence regarding the first-step reaction is available. In this study, we found a novel n-alkane-oxidizing enzyme that requires only molecular oxygen, i.e., not NAD(P)H, in our isolate, Acinetobacter sp. strain M-1, and purified it to apparent homogeneity by gel electrophoresis. The purified enzyme is a homodimeric protein with a molecular mass of 134 kDa, contains 1 mol of flavin adenine dinucleotide per mol of subunit, and requires CU2+ for its activity. The enzyme uses n-alkanes ranging in length from 10 to 30 carbon atoms and is also active toward n-alkenes (C12 to C20) and some aromatic compounds with substituted alkyl groups but not toward a branched alkane, alcohol, or aldehyde. Transient accumulation of n-alkyl hydroperoxide was detected in the course of the reaction, and no oxygen radical scavengers affected the enzyme activity. From these properties, the enzyme is most probably a dioxygenase that catalyzes the introduction of two atoms of oxygen to the substrate, leading to the formation of the corresponding n-alkyl hydroperoxide. The enzymatic evidence strongly supports the existence of an n-alkane oxidation pathway, which is initiated by a dioxygenase reaction, in Acinetobacter spp.
在不动杆菌属中n - 烷烃的芬纳蒂途径中，据推测n - 烷烃会被双加氧酶攻击，产物n - 烷基过氧化氢会通过过氧酸进一步代谢为相应的醛[W. R. 芬纳蒂，第184 - 188页，载于A. H. 阿普尔怀特（编），《油脂工业生物技术世界会议论文集》，1988年]。然而，尚无关于第一步反应的生化证据。在本研究中，我们在我们的分离株不动杆菌属菌株M - 1中发现了一种仅需分子氧（即不需要NAD(P)H）的新型n - 烷烃氧化酶，并通过凝胶电泳将其纯化至表观均一性。纯化后的酶是一种分子量为134 kDa的同二聚体蛋白，每摩尔亚基含有1摩尔黄素腺嘌呤二核苷酸，其活性需要Cu2 + 。该酶可作用于碳原子数在10至30之间的n - 烷烃，对n - 烯烃（C12至C20）以及一些带有取代烷基的芳香化合物也有活性，但对支链烷烃、醇或醛无活性。在反应过程中检测到n - 烷基过氧化氢的瞬时积累，且没有氧自由基清除剂影响该酶的活性。基于这些特性，该酶很可能是一种双加氧酶，催化将两个氧原子引入底物，从而形成相应的n - 烷基过氧化氢。这些酶学证据有力地支持了不动杆菌属中存在由双加氧酶反应引发的n - 烷烃氧化途径。
