A novel methodology is presented to investigate the organic solvent tolerability of cytochrome P450 monooxygenase BM3 (CYP BM3) mutants. A fluorescence-based continuous-flow enzyme activity detection (EAD) setup was used to screen the activity of CYP BM3 mutants in the presence of organic solvents. The methodology is based on the CYP BM3-mediated O-dealkylation of benzyloxyresorufin to form the highly fluorescent product resorufin. The assay setup not only allows detection of the formed resorufin, but it also simultaneously monitors cofactor depletion online. The EAD setup was used to test the activity of a small library of novel CYP BM3 mutants in flow-injection analysis mode in the presence of the organic modifiers methanol, acetonitrile, and isopropanol. Mutants with enhanced tolerability toward all three solvents were identified, and the EAD setup was adapted to facilitate CYP BM3 activity screening against a gradient of an organic modifier to study the behavior of the small library of CYP BM3 mutants in more detail. The simple methodology used in this study was shown to be a very powerful tool to screen for novel CYP BM3 mutants with increased tolerability toward organic solvents.
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http://dx.doi.org/10.1177/1087057115607183 | DOI Listing |
Biotechnol Lett
November 2024
School of Biological Sciences and Biotechnology, Graduate School, Chonnam National University, 77 Yongbongro, Gwangju, 61186, Republic of Korea.
The monooxygenase activity of engineered CYP102A1 on α-terpineol was investigated. CYP102A1 M850 mutant (F11Y/R47L/D68G/F81I/F87V/E143G/L188Q/E267V/H408R) showed the highest catalytic activity toward α-terpineol among the engineered mutants produced by random mutagenesis. The major product (P1) of α-terpineol, p-menth-1-ene-3,8-diol, was characterized by high-performance liquid chromatography, gas-chromatography mass spectrometry, and nuclear magnetic resonance spectroscopy.
View Article and Find Full Text PDFBiotechnol J
November 2024
MOE International Joint Research Laboratory on Synthetic Biology and Medicines, School of Biology and Biological Engineering, South China University of Technology, Guangzhou, P. R. China.
Murideoxycholic acid (MDCA), as a significant secondary bile acid derived from the metabolism of α/β-muricholic acid in rodents, is an important component in maintaining the bile acid homeostasis. However, the biosynthesis of MDCA remains a challenging task. Here, we present the development of cytochrome P450 monooxygenase CYP102A1 (P450 BM3) from Bacillus megaterium, employing semi-rational protein engineering technique.
View Article and Find Full Text PDFChembiochem
November 2024
State Key Laboratory of Bioreactor Engineering, Shanghai Collaborative Innovation Center for Biomanufacturing, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237, China.
(+)-Bicyclogermacrene is a sesquiterpene compound found in various plant essential oils and serves as a crucial precursor for multiple biologically active compounds. Many derivatives of (+)-bicyclogermacrene have been shown to exhibit valuable bioactivities. Cytochrome P450 BM3 from Bacillus megaterium can catalyze a variety of substrates and different types of oxidation reactions, making it become a powerful tool for oxidizing terpenes.
View Article and Find Full Text PDFJ Hazard Mater
December 2024
State Key Laboratory of Mariculture Biobreeding and Sustainable Goods, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Lab for Marine Drugs and Byproducts of Pilot National Lab for Marine Science and Technology, Qingdao 266071, China; Key Laboratory of Sustainable Development of Polar Fisheries, Ministry of Agriculture and Rural Affairs, Qingdao 266071, China. Electronic address:
Non-steroidal anti-inflammatory drugs (NSAIDs) are widespread pollutants in aquatic environments, posing significant risks to both ecosystems and human health due to their persistence and bioaccumulation. Effective and sustainable degradation methods are urgently required to address this environmental challenge. This study aims to design and optimize a cytochrome P450BM3-based biocatalyst for the rapid and efficient degradation of NSAIDs by direct chemical intervention and protein engineering.
View Article and Find Full Text PDFEnzyme Microb Technol
October 2024
School of Biological Sciences and Technology, Chonnam National University, 77 Yongbong-ro, Gwangju 61186, the Republic of Korea; School of Biological Sciences and Biotechnology, Graduate School, Chonnam National University, 77 Yongbong-ro, Gwangju 61186, the Republic of Korea; Institute of Synthetic Biology for Carbon Neutralization, Yongbong-ro 77, Gwangju 61186, the Republic of Korea. Electronic address:
Perillyl alcohol (POH) is a secondary metabolite of plants. POH and its derivatives are known to be effective as an anticancer treatment. In this study, oxidative derivatives of POH, which are difficult to synthesize chemically, were synthesized using the engineered bacterial cytochrome P450 BM3 (CYP102A1) as a biocatalyst.
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