High-throughput screening in enzyme assays for epoxide hydrolases and monooxygenases activity detection using whole cell / Triagem de alto desempenho para detecção de atividade de epoxido-hidrolases e monooxigenases utilizando celulas integras

AUTOR(ES)
DATA DE PUBLICAÇÃO

2006

RESUMO

Biocatalysis is a multidisciplinary area, where new technologies are developing rapidly in order to offer unique options and solutions to different organic chemistry problems, complementing the classical synthetic chemistry. Facing the need imposed by progress in this area, this project aims at the implementation of high-throughput screening (HTS) methodologies allowing an easy and direct method to detect enzymatic activity using whole cells. This thesis is divided in two main topics: epoxide hydrolases (EH) and monooxygenases (MO). Each topic will report the synthesis of the fluorogenic probes used in the HTS assays and their application in preliminary evaluations with different cellular concentrations and reagents. The best experimental conditions with microbial cells were found and employed to evaluate microorganisms from Brazilian culture collections or isolated from national biomes. The fluorogenic assay results were compared with those in the literature or those obtained in our laboratory. In the EH topic, chiral probes were used, which allowed the initial velocity measurements and enantiomeric excess calculations. Additionally, the adrenaline test was applied to the epoxide hydrolases detection in microorganisms using 23 commercials substrates. This methodology associates the potential of the HTS assays with substrates of interest. Finally, the HTS outstanding results were better investigated by conventional catalysis. Therefore, using this approach we were able to provide evidences of the stereoinversion process of 1,2-octanediol catalyzed by A. niger CCT 1435 (yield >80% with 90% ee), and the kinetic resolution of 1,2-octanediol by B. pumilus CBMAI 0008, CBC 008, CBC 009 and A. niger CCT 4846. The differentiation between the monooxigenases (Baeyer-Villiger monooxygenase and hydroxilase) detected in T. cutaneum CCT 1903, R. oryzae CCT 4964, AMA 7 and the fungus CCT 5632 was also provided by conventional biocatalysis.

ASSUNTO(S)

monooxigenases high-throughput screening epoxide hydrolases triagem de alto desempenho epoxido-hidrolades

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