Studies on whole cell fluorescence-based screening for epoxide hydrolases and Baeyer-Villiger monoxygenases

Abstract

Reações de biocatálise foram realizadas em microplacas (200 µL) visando a utilização de substratos fluorogênicos (100 µmol L -1 ) para prospecção rápida de epóxido hidrolases (EHs) e Baeyer-Villiger monoxigenases (BVMOs) em microrganismos (células inteiras). Um protocolo final foi alcançado para EHs, com a detecção de 3 novas fontes enzimáticas (Agrobacterium tumefaciens, Pichia stipitis, Trichosporom cutaneum). O ensaio fluorogênico para BVMO não ocorreu como esperado. A abordagem de algumas variáveis envolvidas (aeração; pH) proporcionou a detecção inédita da atividade enzimática de BVM em T. cutaneum.Biocatalysis reactions were performed on microtiter plates (200 µL) aiming at the utilization of fluorogenic substrates (100 µmol L -1 ) for rapid whole cell screening for epoxide hydrolases (EHs) and Baeyer-Villiger monoxygenases (BVMOs). A final protocol was achieved for EHs, with 3 new enzymatic sources being detected (Agrobacterium tumefaciens, Pichia stipitis, Trichosporom cutaneum). The fluorogenic assay for BVMO did not work as expected. However, an approach to possible variables involved (aeration; pH) provided the first detection of a BVMO activity in T. cutaneum.

Simultaneous Multienzymatic Screening with Fluorogenic Probes

“…This chemo-enzymatic cascade model has been widely applied by our research group and was initially published by Reymond and co-workers in their research works. 14,38 The cascade involves oxidative cleavage of the diols (caused by the action of NaIO 4 ) followed by in situ β-elimination (triggered by BSA) and release of the fluorophores. This makes it possible to simultaneously detect the two enzymatic activities in the same microplate well (Scheme 1).…”

Esterase screening using whole cells of Brazilian soil microorganisms

“…Dichloromethane was dried over CaH 2 and distilled immediately before use. 1 H NMR (300.01 MHz, CDCl 3 ) and 13 C NMR (75.50 MHz, CDCl 3 ) spectra were recorded on a Gemini 300P from Varian Instruments using (CH 3 ) 4 Si as internal reference (d 0.00). Chemical shifts d are given in ppm and coupling constants J are given in hertz.…”

“…21, No. 8, 2010 Several methodologies have been applied to screen hydrolases 11 however the fluorogenic substrates coupled to umbelliferone are stable at high temperature and over a broad range of pH-values 12 and have been reported as screening probes for epoxide hydrolases 13 and BaeyerVilliger monooxygenases, 14 using microorganism whole cells. This methodology has been applied to the screening of a great number of activities in strain collections.…”

“…This methodology has been applied to the screening of a great number of activities in strain collections. 13,14 The general principle of the method involves a sequence of steps in which a fluorescent probe is released after enzymatic transformation (Scheme 1).…”

A Fluorescence‐Based Assay for Baeyer–Villiger Monooxygenases, Hydroxylases and Lactonases

“…[20] Monitoring of the reaction over 24 h by HPLC/UV indicated the direct formation of umbelliferone from these substrates without any detectable intermediates. Al- though the intermediate lactones of the putative BaeyerVilliger reaction might be too unstable in the medium to be detectable, the presence of a possible BVMO was not be confirmed by traditional biocatalytic reactions with several ketones.…”