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Transient kinetic studies of novel decarboxylases by stopped-flow absorption spectroscopy

Grant number: 21/08141-9
Support Opportunities:Scholarships abroad - Research Internship - Post-doctor
Effective date (Start): October 04, 2021
Effective date (End): August 03, 2022
Field of knowledge:Engineering - Chemical Engineering - Chemical Technology
Principal Investigator:Antonio Maria Francisco Luiz Jose Bonomi
Grantee:Letícia Leandro Rade
Supervisor: Thomas Michael Makris
Host Institution: Centro Nacional de Pesquisa em Energia e Materiais (CNPEM). Ministério da Ciência, Tecnologia e Inovações (Brasil). Campinas , SP, Brazil
Research place: North Carolina State University (NC State), United States  
Associated to the scholarship:18/04897-9 - Production of biodiesel and renewable hydrocarbons via enzymatic route: from the enzyme synthesis to the economic evaluation of the integrated process, BP.PD


After the discovery of the P450 decarboxylase enzyme CYP152 OleTJE in 2011, with the unique property of decarboxylating fatty acids using hydrogen peroxide as a cofactor and producing 1-alkenes as the main product, the scientific and technological interest in this family of enzymes has vastly increased. It is well known that alkenes have an economic appeal, especially in the biofuels field since they have similar chemical and physical properties to conventional fossil fuels, with no oxygen in their composition. Considering that peroxygenases have the potential activity of decarboxylating and hydroxylating fatty acids, the elucidation of the intriguing mechanistic involved in the decarboxylation preferential from OleTJE is still a challenge. Thus, the elucidation of decarboxylase structures and kinetics studies of intermediaries (Compound I and Compound II) involved in the catalytic cycle consist of relevant information to better understand those enzymes' functionality and reactional mechanisms. So far, OleTJE is the only decarboxylase with kinetics parameters of Compound I and Compound II, measured by the stopped-flow technique, available in the literature. Dr. Zanphorlin's research group from LNBR recently discovered a new decarboxylase (OleTRN, patent number BR 10 2020 016004 4), which has low sequence similarity to OleTJE. This new decarboxylase will be applied in the alkenes production process, proposed in the post-doctorate project. In this project, the alkenes will be produced by a two steps process: hydrolysis of vegetable oils followed by decarboxylation of the fatty acids. For that purpose, OleTRN was thoroughly characterized, and its structure was elucidated. Additionally, structure-guided mutations were proposed to understand the mechanism of the enzyme and obtain higher catalytic activities. Then, it was observed that some mutations presented different specificity and chemoselectivity by varying the chain-length of the substrates, which is extremely interesting from a biotechnological view as those mutations could diversify the applications. In this context, the present exchange research project proposes transient kinetic studies for OleTRN and its mutants, and new UndA decarboxylases, by the stopped-flow technique, available at Dr. Makris's laboratory at the North Carolina State University. The biochemical characterization could be extremely relevant for a better understanding of the catalytic cycle of OleTRN and the selection of the more active mutation for the application on the alkene production route proposed in the post-doctorate project. Then, the project would have contributions not only in terms of application but also in terms of fundamental research. (AU)

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