Alcohol dehydrogenases (ADHs) are a class of key enzymes responsible for the oxidation of alkyl alcohols in the aerobic alkane metabolic pathway. Currently, the degradation mechanisms of short- and medium-chain alkanes are commonly reported, while those of long-chain alkanes have received less attention. In this work, a putative long-chain ADH was screened from Acinetobacter venetianus RAG-1 via RNA-seq with n-octacosane (C28) as the sole carbon source. Conserved sequence analysis revealed that it is a group III (Fe-containing/activated) ADH, which is widespread in the genus Acinetobacter. The deletion of adhA led to a significant reduction in the degradation of C28. AdhA exhibited optimal oxidative activity at pH 8.0 and 50 °C with NAD+ as coenzyme, while showing better tolerability to chemical reagents. Enzyme activity assay showed that AdhA owed the oxidative activity to a wide range of substrates including alkyl alcohols (C1-C32) and some isomeric alcohols, such as isopropanol, isobutanol, isoamyl alcohol, and propanetriol, and could reduce the alkyl aldehyde (C1-C12). Meanwhile, the binding of AdhA to different alkyl alcohols was mediated by different amino acids. AdhA is an ADH with an extremely broad substrate utilization range and excellent biochemical characteristics. These results provided important insights in the subsequent investigation of long-chain alkane degradation and petroleum pollution bioremediation.
Keywords: Acinetobacter venetianus; Long-chain alcohol dehydrogenase; Long-chain alkane degradation.
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