Triagem, aplicação e engenharia de biocatalisadores para transformações enantio e regiosseletivas / Screening, applying and engineering biocatalysts for enantio and regioselective transformations

AUTOR(ES)
FONTE

IBICT - Instituto Brasileiro de Informação em Ciência e Tecnologia

DATA DE PUBLICAÇÃO

03/06/2011

RESUMO

Biocatalysts have been widely applied in recent decades for industrial processes yielding high value products under environmentally friendly reaction conditions. The development of biocatalysts often begins with screening to identify enzymes with suitable activities followed by characterization of the enzymes chemo-, regio- and stereoselectivity or stability. However, identification of new biocatalysts does not always yield enzymes suitable for a given synthetic problem. To overcome this limitation, biocatalysts can be optimized by protein engineering using rational design or directed evolution. In this context, this thesis describes different methodologies that can be explored to obtain efficient biocatalysts for selective transformations. In Chapter I, functional screening of an 864 member metagenomic library derived from soil using a miniaturized assay based on fluorogenic substrates is described. These assays identified four clones capable of ester hydrolysis. Upon further evaluation using high value substrates, one clone, B6, was shown to display high chemo- and enantioselectivity (E>100) for propionic ester hydrolysis. Chapter II describes the study and application of secondary alcohols deracemization using Candida albicans CCT 0776 whole cells. Monitoring the reaction using phenylethanol as a substrate revealed this system furnishes the (R)-enantiomer in high yield and enantiomeric excess mediated by a cyclic process of oxidation and reduction. In summary the first step is catalyzed by a high S- selective enzyme dependent on NADP and O2 followed by a non-selective reduction catalyzed by an NADH-dependent enzyme. This whole cell biocatalyst was applied to different sec-alcohols and diols allowing the detection of the anti-Prelog products in moderate to high conversions (60 and 99%) and high enantiomeric excess (80 and 90%) within 20 to 120 hour incubation times. Finally, in Charpter III the engineering of cytochrome P450Bm3 by alanine combinatorial scanning mutagenesis followed by directed evolution was used to identify enzymes with regioselective demethylation of bulky substrates. These libraries furnished variants capable of catalyzing regioselective N-demethylation of alkaloids and diastereoselective hydroxylation of steroids in moderate yields. Taken together these studies show the variety of techniques that can be applied for development and application of biocatalysts enabling selective and efficient transformations.

ASSUNTO(S)

desracemização biocatalise metagenômica evolução dirigida biocatalysis metagenomics desracemization directed evolution

ACESSO AO ARTIGO

Documentos Relacionados