RESUMO
Open tubular capillary enzyme reactors were studied for rapid protein digestion and possible on-line integration into a CE/ESI/MS system. The need to minimize the time of the analyte molecules to diffuse towards the surface immobilized enzyme and to maximize the surface-to-volume (S/V) ratio of the open tubular reactors dictated the use of very narrow bore capillaries. Extremely small protein amounts (atto-femtomoles loaded) could be digested with enzymes immobilized directly on the inside wall of a 10 microm I.D. capillary. Covalently immobilized L-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK)-trypsin and pepsin A were tested for the surface immobilization. The enzymatic activity was characterized in the flow-through mode with on-line coupling to electrospray ionization-time of flight-mass spectrometer (ESI/TOF-MS) under a range of protein concentrations, buffer pH's, temperatures and reaction times. The optimized reactors were tested as the nanospray needles for fast identification of proteins using CE-ESI/TOF-MS.
Assuntos
Eletroforese Capilar/métodos , Enzimas Imobilizadas/síntese química , Espectrometria de Massas por Ionização por Electrospray/métodos , Materiais Revestidos Biocompatíveis , Eletrólitos , Eletroforese em Microchip , Enzimas Imobilizadas/classificação , Estudos de Viabilidade , Microquímica , Sistemas On-Line , Pepsina A/metabolismo , Proteínas/análise , Proteínas/química , Sensibilidade e Especificidade , Análise de Sequência de Proteína/métodos , Tripsina/metabolismoRESUMO
Membrane bioreactors are being increasingly used in enzymatic catalysed transformations. However, the application of enzymatic-based treatment systems in the environmental field is rather unusual. The aim of this paper is to overview the application of enzymatic membrane reactors to wastewater treatment, more specifically to dye decolourisation. Firstly, the basic aspects such as different configurations of enzymatic reactors, advantages and disadvantages associated to their utilisation are revised as well as the application of this technology to wastewater treatment. Secondly, dye decolourisation by white-rot fungi and their oxidative enzymes are discussed, presenting an overall view from for in vivo and in vitro systems. Finally, dye decolourisation by manganese peroxidase in an enzymatic membrane reactor in continuous operation is presented.
Assuntos
Reatores Biológicos , Corantes/química , Enzimas Imobilizadas/química , Membranas Artificiais , Peroxidases/química , Purificação da Água/métodos , Compostos Azo/química , Compostos Azo/isolamento & purificação , Compostos Azo/metabolismo , Basidiomycota/metabolismo , Benzenossulfonatos/química , Benzenossulfonatos/isolamento & purificação , Benzenossulfonatos/metabolismo , Biodegradação Ambiental , Cor , Corantes/isolamento & purificação , Corantes/metabolismo , Ativação Enzimática , Enzimas Imobilizadas/classificação , Enzimas Imobilizadas/metabolismo , Desenho de Equipamento , Resíduos Industriais , Especificidade por Substrato , Poluentes Químicos da Água/isolamento & purificaçãoRESUMO
Although numerous reactions have been performed using lipases and related enzymes (e.g. esterases and phospholipases), it is still a challenge to identify the most suitable biocatalyst and best reaction conditions for an efficient application. Frequently used methods such as immobilization and optimization of the reaction medium cannot be transferred from one reaction system or substrate to another. However, in the past few years, rational protein design and directed evolution have emerged as efficient alternative methods to optimize biocatalytic reactions.
