Redesigning an elusive bacterial enzyme into an efficient green catalyst
Scientists engineer the CYP107J1 enzyme from Bacillus subtilis into a more practical tool for selective oxidation chemistry
image:
The engineered CYP107J1 enzyme is driven by hydrogen peroxide instead of NAD(P)H and does not require an electron transport chain (and therefore no redox partner proteins).
view moreCredit: Professor Toshiki Furuya from Tokyo University of Science, Japan
Industrial oxidation chemistry is a cornerstone of modern manufacturing, accounting for nearly one-third of all chemical industrial processes. While essential for making pharmaceuticals, dyes, and many specialty chemicals, industrial oxidation typically relies on high-temperature, high-pressure processes involving toxic oxidizing agents. This has motivated scientists to look into cytochrome P450 monooxygenases (P450s) as a compelling alternative. These enzymes, found across virtually all living organisms, catalyze highly selective oxidation reactions at room temperature and ambient pressure, and several are already in use in pharmaceutical manufacturing. Discovering and characterizing new P450s is therefore an active area of research worldwide.
Even in one of the most extensively studied bacteria in microbiology, Bacillus subtilis strain 168, one of its eight P450 enzymes (CYP107J1) has remained functionally uncharacterized. The main reason for this is that P450 enzymes do not work alone, but instead depend on redox partner proteins called reductases that activate them by transferring electrons. In B. subtilis, the genes encoding these partner proteins are not clustered alongside the P450 genes in the genome, making it challenging to identify the natural partners of CYP107J1. Without them, scientists had to rely on partners borrowed from other organisms, which led to weak enzymatic activity and difficulties in characterizing CYP107J1.
To address this, a research team led by Professor Toshiki Furuya from the Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science (TUS), Japan, turned to a strategy that sidesteps the redox partner problem entirely. In their study, published in Volume 19, Issue 5 of Microbial Biotechnology on May 4, 2026, they characterized CYP107J1 by re-engineering it into a new form that requires no redox partners at all. Other members of the team included second-year doctoral student Hideki Kato and Assistant Professor Takafumi Hashimoto, also from TUS. This research was conducted in collaboration with the team of Dr. Stephen Bell at the University of Adelaide.
The team first confirmed that natural CYP107J1 could oxidize 4-alkylbenzoic acids (compounds consisting of a benzene ring attached to a carbon chain) when paired with substitute redox partners in Escherichia coli cells. The catalytic activity measured was, however, quite low. Thus, the researchers then introduced two targeted amino acid changes into the enzyme’s active site, converting it into a peroxygenase driven by hydrogen peroxide (H2O2). The mutations were designed rationally rather than through trial and error, as equivalent substitutions had previously conferred peroxygenase activity on a related enzyme called CYP199A4 in research by the team of collaborator Dr. Stephen Bell. Using structural modelling, the team confirmed that the corresponding residues in CYP107J1 were positioned appropriately in the active site.
This minor modification led to 28-fold higher catalytic activity toward 4-hexylbenzoic acid compared with the original enzyme with its substitute partners, without affecting selectivity for where on the substrate it places the hydroxyl group. Unexpectedly, the engineered enzyme also converted indole into indigo, a commercially important blue dye. By simply mixing the enzyme, substrate, and H2O2, the team could produce indigo at a rate that outperformed previously reported P450 peroxygenases used for the same purpose. “The method used in this study simplified the driving mechanism of the P450 reaction itself, making it effective not only for analyzing enzymes with unknown functions but also for applying them as catalysts for synthesizing useful compounds,” says Prof. Furuya.
Notably, the two-mutation engineering approach used here offers a practical template for unlocking other ‘orphan’ P450s without needing to first identify their natural redox partners. This could expand the industrial use of engineered P450 enzymes as practical biocatalysts for manufacturing pharmaceuticals, dyes, and other valuable chemicals under mild reaction conditions. Such efforts would ultimately make industrial oxidation chemistry a more sustainable activity overall.
The research team is currently working to further improve the catalytic activity of the modified CYP107J1 enzyme. Prof. Furuya also highlights that many other molecules of this kind remain to be carefully investigated and leveraged in practical applications. “Enzymes of the CYP107J subfamily are widely distributed among bacteria of the genus Bacillus. The findings from this study will facilitate further exploitation of their catalytic potential,” Prof. Furuya concludes.
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References
DOI: 10.1111/1751-7915.70369
This image shows a simplified diagram of the structure of the engineered CYP107J1 peroxygenase, alongside its catalytic process driven by hydrogen peroxide.
Credit
Professor Toshiki Furuya from Tokyo University of Science
The engineered CYP107J1 enzyme is driven by hydrogen peroxide instead of NAD(P)H and does not require an electron transport chain (and therefore no redox partner proteins).
Credit
Professor Toshiki Furuya from Tokyo University of Science, Japan
Image link: https://enviromicro-journals.onlinelibrary.wiley.com/doi/10.1111/1751-7915.70369
About The Tokyo University of Science
Tokyo University of Science (TUS) is a well-known and respected university, and the largest science-specialized private research university in Japan, with four campuses in central Tokyo and its suburbs and in Hokkaido. Established in 1881, the university has continually contributed to Japan's development in science through inculcating the love for science in researchers, technicians, and educators.
With a mission of “Creating science and technology for the harmonious development of nature, human beings, and society," TUS has undertaken a wide range of research from basic to applied science. TUS has embraced a multidisciplinary approach to research and undertaken intensive study in some of today's most vital fields. TUS is a meritocracy where the best in science is recognized and nurtured. It is the only private university in Japan that has produced a Nobel Prize winner and the only private university in Asia to produce Nobel Prize winners within the natural sciences field.
Website: https://www.tus.ac.jp/en/mediarelations/
About Professor Toshiki Furuya from Tokyo University of Science
Dr. Toshiki Furuya is a Professor at the Faculty of Science and Technology in the Department of Applied Biological Science at Tokyo University of Science, Japan. He completed his graduation and postgraduate studies from Waseda University in Tokyo, Japan. His areas of research include applied biochemistry, microbial metabolism, enzyme catalysis, bioproduction, and bioremediation. He has published more than 45 articles in reputed journals. He has won many awards, including the 24th Excellent Paper Award by the Society of Biotechnology in 2016.
Funding information
The authors have no funding information to report.
Journal
Microbial Biotechnology
Method of Research
Experimental study
Subject of Research
Cells
Article Title
Characterization of the Orphan Cytochrome P450 CYP107J1 From Bacillus subtilis Through Peroxygenase Activity Engineering
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