open access

https://doi.org/10.14943/doctoral.k16030

https://hdl.handle.net/2115/95481

マリアナ海溝から単離された Geobacillus kaustophilus HTA426のアルカン分解とその分子機構に関する研究

英語

2024-06-28

Hokkaido University

74

The genus Geobacillus is a Gram positive thermophilic spore-forming bacterial group, which has gained attention due to its high-temperature tolerant nature and its benefits in the industrial production of thermostable enzymes. Several Geobacillus strains have been reported to degrade hydrocarbon. In a previous study on Geobacillus thermoleovorans B23 (Boonmak et al., 2014), a broad range alkane (C11-C32) degrader, it was suggested that Geobacillus kaustophilus HTA426 can also degrade alkane. However, genome analysis of G. kaustophilus HTA426 suggested that no known alkane monooxygenase enzymes exist in the genome, e.g. AlkB-type, Alm-type, CYP-type, and Lad-type which can be found in other alkane degrading Geobacilli. I identified a candidate gene, GK2771, in the HTA426 genome that encodes an isolated ribonucleotide reductase small subunit (RNR2) located in a cluster of genes responsible for alkane degradation. Recently, GK2771 has been proposed as a member of a new protein group, R2-like ligand binding oxidase (R2lox), which resembles RNR2 class Ic but lacks ribonucleotide reductase activity. However, the function and substrate for R2lox are yet unknown. I hypothesized that it could function as an alkane monooxygenase, based on its structural high similarity to soluble methane monooxygenase (sMMO). Chapter 1, it was verified that G. kaustophilus HTA426 can degrade alkane (C11-C17) in nutrient medium LB at 60℃. Moreover, it was found to degrade wider range C10-C24 alkane in minimal medium LBM condition. Chapter 2, recombinant E. coli (pET28a-GK2771) was prepared in which GK2771 is overexpressed under the control of T7-promoter. As expected, the recombinant GK2771 crude enzyme degraded alkane and its activity was not much different between 37℃ and 60℃. This finding demonstrates that an isolated RNR2, GK2771 (GkR2loxI), is a novel alkane monooxygenase, and answers questions about the function and substrate of the R2lox protein group. Chapter 3, gk2772, a putative aldehyde dehydrogenase gene, was overexpressed in E. coli (pET28a-GK2772), purified and examined its activity. Recombinant GK2772 showed significantly higher activity against acetaldehyde at 60℃ compared to 30℃, 40℃ and 50℃ conditions. It was also suggested that GK2772 has dehydrogenase activity against fatty aldehyde, decanal C10 and C14. Chapter 4, describes general discussion including distribution of GK2771 gene homologues. This study expands our understanding of the vast diversity and new evolutionary lineage of the bacterial alkane monooxygenase family.

2024-06-28

甲第16030号

博士(環境科学)

(主査) 教授 森川 正章, 特任教授 福井 学, 教授 三輪 京子, 准教授 堀 千明

環境科学院（生物圏科学専攻）

博士論文

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