RESUMO
Smart biocatalysts, in which enzymes are conjugated to stimuli-responsive polymers, have gained considerable attention because of their catalytic switchability and recyclability. Although many systems have been developed, they require separate laboratory techniques for their recovery, making them unsuitable for many practical applications. To address these issues, we designed a thermomagneto-responsive biocatalyst by immobilizing an enzyme on the terminal of thermo-responsive polymer brushes tethered on magnetic nanoparticle (NP) clusters. The concept is demonstrated by a system consisting of iron oxide NPs, poly(N-isopropyl-acrylamide), and a malonyl-Coenzyme A synthetase (MatB). By using free malonate and coenzyme A (CoA), the designed catalyst exhibits adequate activity for the production of malonyl-CoA. Thanks to the use of a magnetic NP cluster, whose magnetic moment is high, this system is fully recoverable under the magnetic field at above 32 °C because of the collapse of the thermo-responsive polymer shell in the clusters. In addition, the recycled catalyst maintains moderate activity even after three cycles, and it also shows excellent catalytic switchability, that is, negligible catalytic activity at 25 °C because of the blockage of the active sites of the enzyme by the extended hydrophilic polymer chains but great catalytic activity at a temperatures above the lower critical solution temperature at which the enzymes are exposed to the reaction medium because of the thermo-responsive contraction of polymer chains. Because the azide functionality in our system can be easily functionalized depending upon our need, such catalytically switchable, fully recoverable, and recyclable multiresponsive catalytic systems can be of high relevance for other cell-free biosynthetic approaches.
Assuntos
Resinas Acrílicas/química , Proteínas de Bactérias/química , Coenzima A Ligases/química , Nanopartículas Magnéticas de Óxido de Ferro/química , Malonil Coenzima A/síntese química , Biocatálise , Enzimas Imobilizadas/química , Fenômenos Magnéticos , Estudo de Prova de Conceito , Rhizobium/enzimologia , TemperaturaRESUMO
A semi-preparative enzymic synthesis of [1-14C]malonyl-CoA from [1-14C]acetate and bicarbonate, and of [3-14C]malonyl-CoA from Na2(14)CO3 and acetate, was achieved by using chloroplasts rapidly isolated from 7-8-day-old pea shoots. Around 70% of the [1-14C]acetate was converted into malonyl-CoA in 2-3 h, and the specific radioactivity of [3-14C]malonyl-CoA synthesized in the system was 25-30 Ci/mol. Reactions were monitored and labelled products were purified by h.p.l.c.
Assuntos
Acetatos/química , Dióxido de Carbono/química , Malonil Coenzima A/síntese química , Radioisótopos de Carbono , Cloroplastos/química , Cromatografia Líquida de Alta Pressão , Endopeptidases/metabolismo , Fabaceae/química , Cinética , Malonil Coenzima A/isolamento & purificação , Malonil Coenzima A/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Plantas MedicinaisRESUMO
A method for the synthesis of [14C]malonyl-Coenzyme A starting with 10 mumol of [14C]malonate is reported. The synthesis is accomplished with yields of 48 +/- 4% (1 sigma, n = 6) using a procedure which does not require the isolation or purification of any intermediates.
